Increasingly twisted push–pull oligothiophenes and their planarization in confined space

Abstract

A series of systematically deplanarized push–pull oligothiophenes is designed and synthesized to determine the perfect twist for maximal spectroscopic response to their planarization within lipid bilayer membranes. Weak deplanarization naturally gives weak shifts, but strong deplanarization also gives weak shifts because planarization becomes impossible. Intermediate deplanarization turns out to be ideal. The shifts found in response to chromophore planarization are not as dramatic as with lobsters during cooking but sufficient to discriminate solid-ordered and liquid-disordered membranes with the naked eye.

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Introduction

In nature, the combination of chromophore planarization and polarization occurs in processes ranging from the chemistry of vision to the pigmentation of lobsters.¹ Increased conjugation and thus improved communication between the polarizing groups are responsible for a dramatic red shift upon flattening of these chromophores. With fluorescent probes, the same coupled process is poorly explored,^1,2 although fluorophore planarization³ and polarization⁴ as isolated phenomena have received extensive attention, and the twisting of the push–pull fluorophore in the excited state is known in the context of molecular rotors.⁵ Recently, we have introduced the [111]-trimethyl-quaterthiophene 1 as a planarizable push–pull probe (Fig. 1).¹ Decreasing the fluidity of lipid bilayer membranes caused bathochromic shifts in the excitation but not the emission spectra, suggesting that [111]-probe 1 can be planarized in the ground state but is already planar in the excited state. Here, we report that [201]-trimethyl-quaterthiophene 2 gives highly twisted probes that can be planarized in both ground and excited states, whereas [221]-pentamethyl-quaterthiophene 3 is already too twisted for planarization in confined space.

Fig. 1 Systematic deplanarization of the original [111]-oligothiophene amphiphile 1 leads to [201]-probe 2 and [221]-3. Numbers in brackets indicate the number of methyl groups on β,β′-positions of each thiophene–thiophene bond.

Results and discussion

In [111]-trimethyl-quaterthiophene 1, the push–pull system is created with a methoxy donor and a cyano acceptor, and partial chromophore deplanarization is achieved with one methyl group in β-position per α,α′-ring junction.¹ The probe further contains a positive charge, attached in situ by oxime formation, to ensure its delivery to and orientation in lipid bilayer membranes. To systematically increase the twist of the oligothiophene scaffold, we first envisioned probe 2, the [201]-regioisomer of probe 1 with two proximal methyl substituents to cause a strong twist between rings A and B. The [221]-pentamethyl-quaterthiophene 3 with A–B and B–C twists was considered next to achieve full deplanarization.

The new oligothiophenes were synthesized in strict analogy to the previously reported probes.^1,6 In brief, the carboxylic acid 4 was subjected to esterification under microwave conditions, and the resulting methyl ester 5 was regioselectively iodinated in the α-position with N-iodosuccinimide (NIS) (Scheme 1). Suzuki–Miyaura cross-coupling between the iodinated thiophene 6 with boronate ester 7 yielded the twisted bithiophene 8. Iodination with NIS gave the halide compound 9 for Suzuki–Miyaura cross-coupling with the regioisomeric boronic ester 10. The obtained terthiophene 11 was regioselectively iodinated, and the resulting product 12⁶ was subjected to Suzuki–Miyaura cross-coupling with the donor terminus 13. The ester group of the quaterthiophene 14 was reduced to the corresponding primary alcohol, which was then oxidized under mild conditions with Dess–Martin periodinane (DMP) to afford aldehyde 15. The strong cyano acceptor terminus was installed by Knoevenagel condensation with the cyanoamide 16, which was readily accessible from ester 17 and amine 18. Deprotection of acetal 19 followed by covalent capture of the guanidinium moiety 20 gave amphiphile 2. The preparation of amphiphiles in situ by oxime or hydrazone formation has been introduced recently to secure synthetic access to membrane-based differential odorant sensors, biosensors, fluorescent membrane probes and siRNA transfection agents.^1,7 This dynamic covalent approach is important because the resulting amphiphiles are often problematic to purify and characterize due to self-assembly into micelles and reversed micelles in most solvents at already relatively low concentrations. Nevertheless, oxime 2 has been purified and characterized by RP-HPLC, ¹H and ¹³C NMR spectroscopy, and ESI-MS (Fig. S4†). The same synthetic route was followed to prepare the fully twisted probe 3 (Fig. S5†).

Scheme 1 Reagents and conditions: (a) MeOH, H₂SO₄, μW, 100 °C, 15 min, 71%; (b) NIS, CHCl₃, AcOH, rt, 16 h, 73%; (c) Pd(PPh₃)₄, CsF, DMF, 80 °C, 16 h, 77%; (d) NIS, CHCl₃, AcOH, rt, 16 h, 51%; (e) Pd(PPh₃)₄, CsF, DMF, 80 °C, 16 h, 74%; (f) NIS, CHCl₃, AcOH, rt, 16 h, quantitative; (g) Pd(PPh₃)₄, CsF, DMF, 80 °C, 16 h, 71%; (h) 1. DIBAL, CH₂Cl₂, −78 °C, 2. DMP, CH₂Cl₂, rt, 20 min, 57%; (i) neat, rt, 48 h, 92%; (j) piperidine, MeCN, 70 °C, 3 h, 82%; (k) 1. TsOH·H₂O, CH₂Cl₂, rt, 10 min, 55%, 2. DMSO, 60 °C, 10 min, 80%.

In the excitation spectra in DMF, the intermediately twisted [201]-isomer 2 showed a maximum at λ_ex ∼ 390 nm (Fig. 2, black, solid; Table 1). This was Δλ_ex = −71 nm blue-shifted from the weakly twisted [111]-probe 1 at λ_ex = 461 nm (Table 1). The strongly twisted [221]-oligothiophene 3 shifted further to λ_ex = 371 nm (Δλ_ex = −90 nm, Fig. S1,† black, solid; Table 1). Increasing deplanarization in the ground state was thus correctly reflected in the increasing blue shifts of the excitation maxima.

Fig. 2 Excitation (solid) and emission spectra (dashed) of 2 in DMF (black) and DPPC LUVs at 55 °C (gold) and 25 °C (blue), with an indication of excitation (a–c and e–g) and emission maxima (d) of 1 (a–d) and 3 (e–g) in DPPC LUVs at 55 °C (b, d and f) and 25 °C (c,d and g), and in DMF (a and e).

Table 1 Excitation and emission data in DMF^a

Cpd^b	λex ^c (nm)	Δλex ^d (nm)	λem ^e (nm)	Δλem ^f (nm)
a Compare Fig. 2, S1, measured at 25 °C.b Compounds, see Fig. 1.c Excitation maximum, in wavelength (in nanometers).d Shift of the excitation maximum compared to 1.e Emission maximum.f Shift of the emission maximum compared to 1.g Data from ref. 1.
1^g	461	—	736	—
2	390	−71	569	−167
3	371	−90	572	−164

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In comparison to the original maximum of [111]-probe 1, the emission maxima of both [201]-probe 2 and [221]-probe 3 were blue shifted by Δλ_em ∼ −165 nm to λ_em ∼ 570 nm (Fig. 2, black, dotted, Table 1). This result demonstrated that strongly deplanarized push–pull probes remain twisted also in the excited state. This finding was very important because it suggested that the planarization of the new probes should be detectable not only in the excitation but also in the emission spectra.

The planarizability of push–pull probes 2 and 3 was tested in DPPC LUVs (large unilamellar vesicles composed of dipalmitoyl phosphatidylcholine). At room temperature, DPPC membranes are in solid-ordered (S_o) phase. At 41 °C, they undergo a phase transition to liquid-disordered (L_d) phase. Consistent with their planarization by a confining environment, the excitation spectrum of the weakly twisted [111]-probe 1 showed a red shift of +20 nm upon cooling from L_d to S_o DPPC LUVs (Fig. 2b and c; Table 2).¹ The emission maximum at 650 nm was insensitive to membrane fluidity because in the excited state, the fluorophore is already planar (Fig. 2d, Table 2).¹

Table 2 Excitation and emission data in DPPC LUVs^a

Cpd^b	λex ^c (nm) 55 °C	λex ^d (nm) 25 °C	Δλex ^e (nm)	λem ^f (nm) 55 °C	λem ^g (nm) 25 °C	Δλem ^h (nm)
a Compare Fig. 2, S1, DPPC = dipalmitoyl phosphatidylcholine, LUVs = large unilamellar vesicles.b Compounds, see Fig. 1.c Excitation maximum at 55 °C, in wavelength (in nanometers).d Excitation maximum at 25 °C.e Shift of excitation maximum upon cooling from 55 °C to 25 °C.f Emission maximum at 55 °C.g Emission maximum at 25 °C.h Shift of the emission maximum upon cooling from 55 °C to 25 °C.i Data from ref. 1.
1^i	467	487	+20	650	650	0
2	449	483	+34	562	600	+38
3	413	423	+10	590	582	−8

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The excitation maximum of the intermediately twisted [201]-probe 2 in L_d DPPC LUVs was at λ_ex = 449 nm (Fig. 2, gold, solid; Table 2). The blue shift Δλ_ex = −18 nm compared to the [111]-probe 1 demonstrated that [201]-probe 2 is more twisted in L_d phase. In S_o DPPC LUVs at 25 °C, the excitation maxima of both probes were at λ_ex ∼ 485 nm. This suggested that the ground state of both probes is fully planarized in this highly confining solid-ordered environment. More ground-state twisting in L_d phase with identical planarization in S_o phase naturally meant that the [201]-probe 2 is more sensitive to L_d–S_o transition than the [111]-probe 1. The bathochromic shift in response to pressure from the surrounding membrane increased from Δλ_ex = +20 nm for 1 to Δλ_ex = +34 nm for 2 (Table 2). Considering the experimental inaccuracy, it can be said that with optimized deplanarization, the sensitivity of the fluorescent membrane probes almost doubled (compare below for DOPC).

The emission maximum of probe 2 in L_d DPPC LUVs was at λ_em = 562 nm (Fig. 2, gold, dashed; Table 2). The strong hypsochromic shift of Δλ_em = −88 nm compared to [111]-probe 1 suggested that the excited state of [201]-probe 2 remains partially twisted in L_d DPPC LUVs. Unlike the phase-insensitive emission of probe 1, a bathochromic shift of Δλ_em = +38 nm was observed with 2 in response to L_d–S_o transition of DPPC LUVs. These results suggested that also the excited state of [201]-probe 2 is deplanarized in L_d DPPC LUVs and provided corroborative evidence that the L_d–S_o transition can be sensed not only by ground-state but also by excited-state planarization.

The strongly twisted, weakly fluorescent [221]-probe 3 showed little difference in excitation and emission spectra in response to changes in membrane structure. The maximum in the excitation spectrum remained strongly blue-shifted compared to the maxima of probes 1 and 2, suggesting that the ground state of the probe 3 is too twisted to be planarized in either L_d or S_o DPPC membranes (Fig. 2f and g, Table 2, Fig. S1†). The same is true for the excited state (Table 2, Fig. S1†).

Control experiments were performed in dioleoyl phosphatidylcholine (DOPC) LUVs. These membranes remain in L_d phase at 25 °C and at 55 °C. In DOPC LUVs at 55 °C, the fluorescence of the original [111]-probe 1 was almost the same as that in organic solvents or L_d DPPC LUVs and showed very little red shift upon cooling (λ_ex = 466 nm, Δλ_ex = +2 nm, Table 3).¹ The excitation maximum of the hypersensitive [201]-probe 2 was at λ_ex = 436 nm under identical conditions, that is, Δλ_ex = −30 nm compared to 1 and Δλ_ex = −13 nm compared to 2 in L_d DPPC LUVs (Tables 2 and 3, Fig. S2†). These overall small shifts suggested that the ground state of 2 remains partially twisted also in “hot” DOPC LUVs.

Table 3 Excitation and emission data in DOPC LUVs^a

Cpd^b	λex ^c (nm) 55 °C	λex ^d (nm) 25 °C	Δλex ^e (nm)	λem ^f (nm) 55 °C	λem ^g (nm) 25 °C	Δλem ^h (nm)
a Compare Fig. S2 and S3, DOPC = dioleoyl phosphatidylcholine, LUVs = large unilamellar vesicles.b Compounds, see Fig. 1.c Excitation maximum at 55 °C, in wavelength (in nanometers).d Excitation maximum at 25 °C.e Shift of excitation maximum upon cooling from 55 °C to 25 °C.f Emission maximum at 55 °C.g Emission maximum at 25 °C.h Shift of the emission maximum upon cooling from 55 °C to 25 °C.i Data from ref. 1.
1^i	466	468	+2	650	650	0
2	436	446	+10	557	557	0
3	367	367	0	607	595	−12

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The Δλ_ex = +10 nm found upon cooling placed the excitation of 2 in “cold” DOPC LUVs together with that in L_d DPPC LUVs. This small shift could originate from partial ground-state planarization, although thermochromic effects cannot be excluded. In any case, the overall hypsochromic position and poor sensitivity of the excitation spectrum of 2 in DOPC LUVs provided excellent general support that the large Δλ_ex = +34 nm found for L_d–S_o transition in DPPC LUVs originates indeed from planarization of 2 in the ground state. Comparison of DOPC-LUV and DPPC-LUV samples at 25 °C demonstrated that the Δλ_ex = +37 nm for planarization of [201]-probe 2 in S_o lipid bilayer membranes is sufficient to be seen with the “naked eye” (compare TOC graphic). This colorimetric detection of the nature of lipid bilayer membranes with the naked eye was not observed with the poorly twisted original [111]-probe 1¹ and is thus unique for the almost perfect twist in the new [201]-probe 2.

The sensitivity of the best probe 2 to liquid-ordered (L_o) phase was explored in the biologically relevant SM–CL LUVs (SM: sphingomyelin, CL: cholesterol; SM–CL = 3 : 1). Excitation was found at λ_ex = 488 nm, which is slightly red-shifted from that in S_o DPPC LUVs (Δλ_ex = +5 nm, Fig. 3, blue, solid). The same trend was observed for emission, which maximized at λ_em = 619 nm (Δλ_em = +19 nm, Fig. 3, blue, dotted). This suggested that further planarization of probe 2 might occur in L_o phase in both ground and excited states compared to that in S_o phase, although the effect is rather small.

Fig. 3 Excitation (solid) and emission spectra (dashed) of 2 in DOPC (gold), SM–CL (blue), and DOPC–SM–CL LUVs (grey) at 25 °C (blue). Maxima are indicated in nanometers.

Fluorescent properties of probe 2 were also measured in DOPC–SM–CL LUVs under conditions where L_o and L_d phases coexist in the same vesicle (DOPC–SM–CL = 1 : 1 : 1). The excitation maxima moved to λ_ex = 466 nm, which is nearly halfway between excitation in pure L_o and L_d phases (Fig. 3, grey, solid). This finding suggested that probe 2 partitions into both phases and can, in principle, report their coexistence in heterogeneous vesicles simultaneously. The emission in DOPC–SM–CL LUVs was roughly the same as in L_o phase (Fig. 3, grey, dashed). This difference in the behavior of the excitation spectra could possibly be explained by fluorescence resonance energy transfer from the twisted fluorophore in L_d to the flattened fluorophore in L_o phase.

Conclusions

In summary, ground- and excited-state planarization of intermediately twisted push–pull oligothiophenes in L_o, S_o, but not L_d lipid bilayer membranes causes significant red shifts in both excitation and emission spectra. With the best planarizable push–pull probe, L_d and S_o membranes can be discriminated with the naked eye (although the shifts are not yet as spectacular as the ones observed when cooking lobsters). In sharp contrast, highly twisted push–pull oligothiophenes resist planarization under the same conditions. These results provide systematic insights into the deplanarization of oligothiophenes as well as a solid basis for further fine-tuning and applications towards the sensing of more demanding, biologically relevant characteristics such as membrane tension.

Acknowledgements

We thank the NMR and the Sciences Mass Spectrometry (SMS) platforms for their services, and the University of Geneva, the European Research Council (ERC Advanced Investigator), the National Centre of Competence in Research (NCCR) Chemical Biology and the Swiss NSF for financial support.

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Footnote

† Electronic supplementary information (ESI) available: Detailed procedures and results for all reported experiments. See DOI: 10.1039/c3ob41706a

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