Planarizable push-pull probes : overtwisted flipper mechanophores

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Article

Reference

Planarizable push-pull probes : overtwisted flipper mechanophores

MACCHIONE, Mariano, et al.

Abstract

Planarizable push–pull fluorescent probes, also referred to as flipper probes, have been introduced as conceptually innovative mechanophores that report on forces in their local environment in lipid bilayer membranes. The best flipper probes respond to a change from liquid disordered to solid ordered membranes with a red shift in excitation of 50–90 nm. A simultaneous increase in fluorescence lifetime and negligible background fluorescence from the aqueous phase qualifies these fluorescent probes for meaningful imaging in live cells.

Here, we report that the replacement of methyl with isobutyl substituents along the scaffold of a dithienothiophene dimer strongly reduces fluorescence intensity but increases solvatochromism slightly. These trends imply that the large substituents in “leucine flippers”

hinder the planarization in the first excited state to result in twisted intramolecular charge transfer (TICT). As a result of this overtwisting, the leucine flippers form interesting fluorescent micelles in water but fail to respond to changes in membrane order. These dramatic changes in function provide one of the [...]

MACCHIONE, Mariano, et al . Planarizable push-pull probes : overtwisted flipper mechanophores. Chempluschem , 2017, vol. 82, no. 7, p. 1062-1066

DOI : 10.1002/cplu.201600634

Available at:

http://archive-ouverte.unige.ch/unige:95494

Disclaimer: layout of this document may differ from the published version.

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Planarizable Push–Pull Probes: Overtwisted Flipper Mechanophores

Mariano Macchione, Nicolas Chuard, Naomi Sakai, and Stefan Matile*

^[a]

Introduction

The concept of planarizable push–pull probes^[1] has been in- spired by the change of color of lobsters during cooking.^[2]The idea is to combine the planarization and the polarization of twisted fluorophores in the ground state (Figure 1a).^[1] Their planarization by mechanical or other forces exerted by the sur- rounding environment should then result in large shifts in excitation, whereas emission from the planar first excited state^[3]

should be mechano-insensitive. This mechanism is distinct from and somewhat complementary to solvatochromism, twisted intramolecular charge transfer (TICT), photoinduced electron transfer (PET), and excited-state intramolecular proton transfer (ESIPT) of other membrane probes and related systems, which mostly report in emission.^[4]

The currently best flipper probe 1,^[5]the result of quite extensive optimization,^[5–7] is composed of two dithienothiophene (DTT)^[8] monomers (Figure 1b). These “fluorescent flippers” are bright enough to keep emitting when twisted out of conjugation and have a large enough surface for extensive mechanical interactions with the environment. The push–pull system is established with accepting “sulfone” and donating

“sulfide” bridges in the DTT^[6]and further supported by cyano acceptors and intriguing chalcogen-bonding thenyl ether

donors.^[5]The headgroup further contains an essential triazole to prevent elimination^[5]and a carboxylate to produce an overall amphiphilic probe. This is essential to deliver and orient flipper probes in membranes.^[6]

Deplanarization^[9,10]of the DTT dimer1is achieved by chalcogen-bond repulsion^[5]between methyl groups and endocyclic sulfur atoms. The deplanarization of conjugated oligomers like 1in the ground state is reported by a blue shift of the absorption or excitation maxima. The subsequent planarization of the twisted mechanophore in confined space results in the corre- sponding red shift in excitation.^[5–7]Consistent with mechanical planarization in the ground state, increasingly red-shifted excitation was found for flipper probe1in lipid bilayer membranes of increasing order. Such mechanosensitivity was of interest to image so far invisible forces in biological systems.^[11]However, in single crystals, face-to-face p-stacked mechanophores1 are Planarizable push–pull fluorescent probes, also referred to as

flipper probes, have been introduced as conceptually innovative mechanophores that report on forces in their local environment in lipid bilayer membranes. The best flipper probes respond to a change from liquid disordered to solid ordered membranes with a red shift in excitation of 50–90 nm. A simultaneous increase in fluorescence lifetime and negligible background fluorescence from the aqueous phase qualifies these fluorescent probes for meaningful imaging in live cells. Here, we report that the replacement of methyl with isobutyl substituents along the scaffold of a dithienothiophene dimer

strongly reduces fluorescence intensity but increases solvatochromism slightly. These trends imply that the large substituents in “leucine flippers” hinder the planarization in the first excited state to result in twisted intramolecular charge transfer (TICT). As a result of this overtwisting, the leucine flippers form interesting fluorescent micelles in water but fail to respond to changes in membrane order. These dramatic changes in function provide one of the most impressive illustrations for the hypersensitivity of fluorescent membrane probes toward small changes in their structure.

Figure 1.a) The concept of planarizable push–pull probes together with b) the structure of the currently best flipper probe1and the “leucine flipper”2introduced here.

[a]M. Macchione, N. Chuard, Dr. N. Sakai, Prof. S. Matile Department of Organic Chemistry, University of Geneva CH-1211 Geneva (Switzerland)

E-mail: stefan.matile@unige.ch

Homepage: www.unige.ch/sciences/chiorg/matile/

Supporting information (including experimental details) and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10.1002/cplu.201600634.

This article is part of the “Novel Aromatics“ Special Issue. To view the complete issue, visit: https://doi.org/10.1002/cplu.v82.7.

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already fully planarized.^[7] This observation suggested that increasing twisting could perhaps further increase the mechanosensitivity of flipper probe1. Here, we report that this is unlike- ly. Already the replacement of methyl by isobutyl groups, an overall small change, is shown to afford with “leucine flipper”

2 a fluorophore that has lost all functional relevance. This quite spectacular response, from best to worst, provides a won- derful illustration for the high sensitivity of fluorescent membrane probes, mechanophores, and beyond, to small changes of their structure.

Results and Discussion

The synthesis of leucine flipper2required strategic reconsider- ation because the convenient traditional approach from meth- ylthiophene was not applicable.^[6,8] Tetrabromothiophene 3 was selected as the starting point instead (Scheme 1).^[12]A cas- cade condensation with aldehyde4and ethyl 2-thioacetate (5) afforded the new DTT core 6 with the two desired isobutyl substituents in good yield.^[12,13]Ester hydrolysis followed by de- carboxylation liberated theorthopositions in DTT7 for further elaboration along the routes developed for the original probe 1.^[5,6]The first step was a formylation. The resulting aldehyde8 was reduced, and protection of the resulting thenyl alcohol with a silyl group led to9. To access the accepting flipper, the same aldehyde 8 was converted into nitrile 10. Bromination with NBS readily gave DTT11, which was oxidized withmCPBA to DTT12with a sulfone instead of a sulfide bridge. The electron-rich DTT9 and the electron-poor DTT12were then con- nected by Stille coupling. The resulting twisted dimer 13was obtained in good yield. Removal of the silyl protecting group and reaction of thenyl alcohol14with bromide15gave alkyne 16, ready for cycloaddition with azide17to yield leucine flip- per2. Detailed procedures and full characterization of all new compounds can be found in the Supporting Information.^[14]

This synthetic approach provides an alternative route to flipper probes in general. It will be of use also to produce probes

such as flipper1on a larger scale because it is more cost effective.

In toluene, the absorption maximum of the hydrophobic precursor 13 of leucine flipper 2, was at labs=403 nm (Fig- ure 2a). This wasDlabs=@10 nm blue-shifted compared to18,

the previously reported^[5,6]“methyl” homolog of13. Moreover, the maximum of13extended more toward the blue, whereas the maximum of 18 showed a distinct shoulder toward the red. The differences supported that in solution, the ground state of leucine flipper2is more deplanarized than that of flip- per1. Consistent with previous observations,^[6] the absorption spectra showed little dependence on solvent polarity, and excitation and absorption spectra were nearly identical.

The emission maximum of the hydrophobic, not amphiphilic pre-leucine flipper 13 in toluene was at lem=562 nm (Fig- ure 2b, solid, blue). The slight red shift Dlem= +6 nm compared to the original18supports that the blue shift in absorption of 13 originates from ground-state deplanarization and not from unrelated effects.^[3]

A red shift in the emission of13compared to the less twisted original18was observed also in more polar solvents such Figure 2.Normalized a) absorption and b) emission spectra of hydrophobic precursors of2(i.e.,13, solid blue line) and1(i.e.,18, dashed green line)^[5]

in toluene, and emission of13(solid cyan line) and18(dotted light-green line)^[5]in EtOAc.

Scheme 1.a) 1.4, BuLi, THF,@788C, 2 h, 68%; 2. Na2Cr2O7, H2SO4, H2O, acetone, rt, 16 h, 72%; 3.5, NaOEt, EtOH, reflux, 2 h, 86%;^[10]b) 1. LiOH, EtOH, reflux, 16 h, 95 %; 2. Ag2CO3, AcOH, DMSO, 1208C, 16 h, 73%;^[10]c) POCl3, DMF, 08C to 508C, 2 h, 91%; d) 1. NaBH4, DMF, 708C, 2 h; 2. TBDPSCl, imidazole, DMF, rt, 12 h, 87 %; e) NaN3, TfOH, MeCN, 708C, 1 h, 86%; f) NBS, CH2Cl2, rt, 12 h, 94%; g)mCPBA, CHCl3, rt, 24 h, 66%; h) 1. LDA, Bu3SnCl, THF,@788C to rt, 1 h;

2. Pd(Ph3)4, toluene, 708C, 24 h, 82%; i) TBAF, AcOH, THF, rt, 30 min, 94%; j) NaH, THF, 08C to rt, 16 h, 36%; k) CuSO4·5H2O, sodium ascorbate, TBTA, CH2Cl2/ H2O 4:1, rt, 4 h, 97%. TBDPSCl=tert-butyldiphenylsilyl chloride, TfOH=trifluoromethanesulfonic acid, NBS=N-bromosuccinimide,mCPBA=meta-chloroper- benzoic acid, LDA=lithium diisopropylamide, TBAF=tetra-n-butylammonium fluoride, TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

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as EtOAc (Figure 2b, solid cyan vs. dotted green spectra, Fig- ure S1). The spectral shift in emission on going from hexane to DMSO was calculated to be 3778 cm^@1 for leucine flipper 13 and 3147 cm^@1 for original 18. Quantitative Lippert analysis^[6]

of their Stokes shifts as a function of solvent polarity gave the apparent difference in dipole moments of the ground and the excited states (Figure S2). The obtainedDmL=14.8 D of leucine flipper 13 clearly exceeded the DmL=13.6 D of original 18 (Figure 3).

Using Rhodamine 6G as an established standard, we found that the fluorescence quantum yieldf=4.4% obtained for leucine flipper13in EtOAc was clearly below thef=35% of the original18(Figure 3). Both the decreasing quantum yield and the increasing positive solvatochromism with the increasing twist of the push–pull fluorophore implied the emergence of twisted excited states. With the original flipper probe1, planarization of the excited state upon intramolecular charge transfer (ICT) has been confirmed experimentally by time-resolved fluorescence emission spectroscopy.^[3] The planar first excited state of push–pull chromophores is characterized by bright fluorescence, high quantum yields, and long fluorescence life- times. However, several twistable push–pull chromophores have been shown not to relax into planar ICT but into twisted excited states with fully decoupled aromatic rings that are per- pendicular to each other.^[4,10] These TICT states are characterized by strong solvatochromism due to full charge separation and poor fluorescence due to non-radiative decay. The here identified trends with flipper probes thus suggest that with increasing “pretwisting” in the ground state, exited state relaxa- tion into planar ICT states becomes increasingly unfavorable and TICT states start to dominate (Figure 3). This interpretation that “pretwisting” in the ground state enables TICT is consistent with previously reported results with simpler model systems.^[10]

One of the hallmarks of the mechanosensitive membrane probe 1 is complete fluorescence quenching in water (Fig- ure 4a, solid line).^[5,6]This is essential for measurements in cells and model membranes without disturbance of background fluorescence. In sharp contrast, leucine flipper 2 showed in- tense fluorescence in water (Figure 4b, solid line). In water,

both amphiphiles are expected to self-assemble into micelles.

In micellar 1, fluorescence quenching presumably originates from face-to-facepstacking^[7]of the mechanophores. The pre- served fluorescence of micellar2suggested that the bulky isobutyl groups along the scaffold preventpstacking.

Fluorescence quenching upon self-assembly as seen with mechanophore 1 is a general phenomenon. Fluorescent micelles, other nanoparticles, and also solids have attracted recent attention for this reason.^[15,16] For the fluorescent micelles formed by the twisted push–pull amphiphile2in water, a fluorescent quantum yield off=4.2% was measured. Using Rhodamine 6G as an established standard, we found an un- changedf=4.4 % for13in EtOAc.

A standard experiment to determine aggregation-induced emission (AIE)^[16] was used to obtain deeper insight into the formation of fluorescent micelles. As expected, the fluorescence intensity of2 increased nonlinearly with the increasing percentage of water in MeOH/water mixtures (Figure 5a, filled circles). The formation of fluorescent micelles around 60%

water in MeOH coincided with a blue shift of the excitation maximum by Dlex=@8 nm (Figure 5a, empty circles). Al- though the precise origin of this small shift will remain un- known, it certainly demonstrated that the micelle formation does not cause planarization of the overtwisted leucine flippers 2. The concentration dependence in buffer was linear, suggesting that the critical micelle concentration (cmc) is below 250 nm (Figure 5b). Fluorescent micelles of2 could be observed directly as small (at resolution limit), fairly homogenous spheres in fluorescent micrographs (Figure 5c).

Partitioning into membranes was measured with large unila- mellar vesicles (LUVs) composed of dipalmitoylphosphatidyl- choline (DPPC) at 558C, that is, well above the temperature of the transition into the liquid-disordered (Ld) phase at 418C. Not emitting in water, the partitioning of the original probe1into Figure 3.Decreasing fluorescence quantum yieldfin EtOAc and increasing

positive solvatochromismDmLsuggests that the “pretwisting” of flipper probes in the ground state increasingly favors twisted and disfavors planar excited states.

Figure 4.Scaled, not normalized fluorescence emission spectra of1^[5](a) and 2(b) in buffer (solid line) and in LdDPPC LUVs (dashed line; 558C), and excitation spectra of1^[5](c) and2(d) in SoDPPC LUVs (solid line; 258C) and Ld

DPPC LUVs (dashed line; 558C, 10 mmTris, 100 mmNaCl, pH 7.4).

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LdDPPC membranes caused the known dramatic fluorescence recovery (Figure 4a).^[5]Once again, the behavior of leucine flipper 2 could not be more different: The strong emission in water did not change much in the presence of LdDPPC membranes except for general contributions from scattering (Fig- ure 4b).

The red shift in excitationDlex>0 plus intensityISo/ILd>1 in response to cooling upon transition from Ldinto solid-ordered (So) DPPC membranes are the characteristics of operational flipper probes (Figure 4c).^[5,6] Flipper 1, the current best, fea- tures Dlex= +50–90 nm and ISo/ILd=2.7, the latter being also reflected in an increase in fluorescence lifetime tLo/tLd=2.1, tLo=5.8 ns (Figure 4c).^[5]The new leucine flipper 2 simply did not respond to the phase transition from Ld into So DPPC membranes (Figure 4d). The contrast between the two could not be stronger: the properties of 2 could not be worse, all mechanosensitivity of1was completely lost. With fluorescence being unresponsive also to partitioning (Figure 4b), the possi- bility that leucine flipper 2 did not enter the Ld DPPC membranes and remained in micellar form in water instead could not be excluded. However, the preparation of DPPC LUVs in the presence of leucine flipper2gave the same result. Similar, but clearly less pronounced loss in mechanosensitivity upon overtwisting was observed previously during twistome screen- ing in the quaterthiophene series.^[17]

Conclusion

In summary, we report that the replacement of methyl groups by isobutyl groups along the DTT dimer scaffold completely destroys the activity of fluorescent flipper probes. The best flipper probes: 1) show a red shift in excitation and an increase in intensity and lifetime upon transition from Ldto Somembranes and 2) do not fluoresce in water (Figure 4a,c). Presumably due to overtwisting of the push–pull fluorophore (Figure 2), the new “leucine flippers” 1) do not respond to the transition from Ld to So membranes and 2) fluoresce strongly in water (Fig- ure 4b,d). The observed formation of small, spherical, and quite homogenous fluorescent nanoparticles with overtwisted push–pull amphiphiles 2 (Figure 5) is of interest because the self-assembly of fluorescent amphiphiles in water usually results in complete quenching. With regard to spectroscopic

properties in solution, the consequences of overtwisting the flipper probe 2 in the ground state are reduced quantum yields and increased solvatochromism, that is an increasing preference for twisted rather than planar ICT excited states (Figure 3).

This quite spectacular response in function to an overall small change in structure nicely illustrates that the discovery of operational mechanosensitive membrane probes such as flip- per1 that operate by a combination of planarization and polarization in the ground state is far from trivial. In other words, the here reported results testify to the importance of molecular precision in probe design and generate appreciation for the excellent performance of flipper probe 1 by contrast. More- over, besides producing a completely useless twisted push–

pull probe, we also introduce a new synthetic approach to fluorescent flippers that, being more cost effective, will be essential for the production of operational flipper probes 1 on a larger scale.

Acknowledgements

We thank S. Soleimanpour for contributions to synthesis, the NMR, the Mass Spectrometry and the Bioimaging platforms for services, and the University of Geneva, the Swiss National Centre of Competence in Research (NCCR) Chemical Biology, the Swiss NCCR Molecular Systems Engineering, and the Swiss NSF for fi- nancial support.

Conflict of interest

The authors declare no conflict of interest.

Keywords: conformation analysis · fluorescent probes · membranes·mechanophores·push–pull chromophores

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Manuscript received: December 16, 2016 Revised manuscript received: February 4, 2017 Version of record online: February 16, 2017