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Conjugated Polyimine Dynamers as Phase-Sensitive Membrane Probes
SAKAI, Naomi, MATILE, Stefan
Abstract
In this report, dynamic polyimines are introduced as multifunctional sensors of lipid bilayer phases. Under mildly acidic conditions, self-condensation of push–pull amino formyl fluorenes into polyimines occurs in solid- or liquid-ordered phases but not in liquid-disordered phases of vesicular membranes. The obtained conjugated polymers are characterized by a progressive red shift of the absorption maxima, the appearance of exciton-coupled circular dichroism (CD) bands, and fluorescence quenching. These characteristics allow multiple modes of detection of membrane phases, which are known to change under membrane tension.
SAKAI, Naomi, MATILE, Stefan. Conjugated Polyimine Dynamers as Phase-Sensitive
Membrane Probes. Journal of the American Chemical Society, 2018, vol. 140, no. 36, p.
11438-11443
DOI : 10.1021/jacs.8b06668
Available at:
http://archive-ouverte.unige.ch/unige:107782
Disclaimer: layout of this document may differ from the published version.
Conjugated Polyimine Dynamers as Phase-Sensitive Membrane Probes Naomi Sakai* and Stefan Matile*
Department of Organic Chemistry, University of Geneva, CH-1211 Geneva 4, Switzerland
ABSTRACT: In this report, dynamic polyimines are introduced as multifunctional sensors of lipid bi- layer phases. Under mildly acidic conditions, self-condensation of push-pull amino formyl fluorenes into polyimines occurs in solid- or liquid-ordered phases but not in liquid-disordered phases of vesicular mem- branes. The obtained conjugated polymers are characterized by a progressive red shift of the absorption maxima, the appearance of exciton-coupled CD bands, and fluorescence quenching. These characteristics allow multiple modes of detection of membrane phases, which are known to change under membrane tension.
INTRODUCTION
“Dynamers,”1 i.e., polymers composed of dynamic covalent bonds,2 are hyper-responsive by nature be- cause the sensitivity of these bonds to their environment is amplified. Such properties are ideally suited for broad range of applications, most notably self-healing materials,3 degradable, thus non-toxic intracel- lular delivery vehicles4 and repairable, thus highly ordered porous frameworks5 and surface architectures.6 Among dynamic polymers, conjugated polyimines stand out as ideal scaffolds for sensor applications because 1) they are particularly responsive due to the high reversibility of imine bonds in water, thus their formation often requires peculiar conditions such as the presence of templates or aggregates, while 2) the resulting conjugated polymers are easily characterizable by the length dependent optical or electrochem- ical properties.7,8 Equipped further with the signal amplification capabilities of conjugated polymers,9 polyimines were indeed shown to be thermo-, acido- and electrochromic and respond to the presence of various analytes, such as nitro derivatives and peptide nanotubes.7,8
In our quest towards the optical detection of forces in biomembranes,10 dynamic polyimines attracted our interest (Figure 1). Although membrane tension is believed to play important roles in regulating many biological processes,11 only a few optical probes exist so far.12 Our current best probe was inspired by nature, and functions by conformational changes in the ground state.10 Different mechanisms account for other membrane probes, e.g., molecular rotors13 and solvatochromism14 to detect viscosity and hydration level, respectively. Toward the sensing of membrane tension, our objective was to develop a new mode of detection of membrane phases, as they were shown to change dramatically upon application of mem- brane tension.15,10c Moreover, membrane phases themselves are a topic of importance because the exist- ence and significance of membrane nanodomains or "rafts" are still under debate.16
We envisioned that the formation of polyimines would reflect the local environment of monomer units.
In a dense ordered phase of lipid bilayer membranes, monomer units should segregate from lipids, result- ing in the high local concentration to favor the formation of polyimines, while in disordered or stretched phase of the membranes, lowered local concentration and increased hydration would promote the hydrol- ysis into monomeric form (Figure 1). Mechano- or strain sensitivity of imine bonds17 could further con- tribute to the latter process. The formation and the degradation of conjugated polyimines should be easily detectable by the length dependent absorption and emission maxima of the polymer. Also, freezing of the polymer conformation in an ordered phase of lipid bilayer membranes could result in elongation of the conjugation length, leading to the red shift of absorption, and diminution of vibrational deactivation of the excited state, thus increase in fluorescence, in a way similar to some conjugated polymer based mech- anophores9c,18 or molecular rotors.13 In other words, polyimines have the potential to sense membrane phases by the two most common mechanisms of mechanophores: Conformational change,18,19 and bond cleavage.20
Figure 1. Schematic description of dynamic polyimines as phase-sensitive probes in lipid bilayer mem- branes. Cyan and dark gray curves attached on the probes represent hydrophilic and hydrophobic chains, respectively. See Figure 2a for the complete chemical structure.
In this report, we describe the design, synthesis and evaluation of conjugated polyimine dynamers as phase-sensitive membrane probes. Their characterization in organic solvents covers GPC, MALDI mass spectrometry, and absorption and fluorescence spectroscopy. The same polyimines form selectively in ordered phases of lipid bilayer membranes under weakly acidic conditions and provide access to the op- tical detection of different phases of lipid bilayer membranes.
RESULTS AND DISCUSSION
Design and Synthesis. The design of monomer 1m was based on the previously reported highly fluores- cent polyfluorene-imines (Figure 2).7 The self-condensation characteristic for this type of monomer was considered advantageous over commonly used hetero-condensation between diamines and dialdehydes, as the latter requires a precise control of the concentrations of two components, which is difficult to achieve in lipid bilayer membranes. The push-pull character acquired by the attachment of amine and aldehyde groups made the optical properties of the monomers sensitive to their environment.14 Alkyl and ethylene glycol chains attached to the fluorene core provided the amphiphilicity needed for delivery and positioning of monomer/polymer near the membrane-water interface (Figure 1).
Monomer 1m was easily accessible from commercially available 2,7-dibromofluorene (Scheme S1).
The details of synthesis can be found in the Supporting Information.21
N N N
H2N CHO
1 1m
Dynamic Polymerization in Organic Solvents. Upon removal of the amine-protecting Boc group under acidic conditions, monomer 1m polymerized spontaneously. MALDI-TOF MS showed a charac- teristic polymer pattern with multiple peaks separated by the mass of a dehydrated monomer unit (Figure 2a). GPC in THF further corroborated the formation of polymers with a broad distribution of molecular weight up to ~30 kDa (n ≈ 67), according to the polystyrene molecular-weight standards (Figure 2b, top;
Figure S2). Consistent with the dynamic nature of imines, the addition of a small amount of TFA caused the disappearance of the broad polymer peak to give a sharp peak at Rt ≈ 13.5 min (Figure 2b, bottom).
The formed species could be identified as the monomer 1m by comparison to LC-MS analysis. As ex- pected for the conjugated polymer, the absorption maxima gradually red-shifted by increasing polymer length until reaching to a plateau at lmax ≈ 420 nm above the estimated molecular weight of ~2 kDa (Figures 2c, d). Such saturation behavior at n ≈ 5 is typical for oligomeric compounds.8a,22
Phase-Sensitive Polymerization in Lipid Bilayer Membranes. Dynamic polymerization was eval- uated first in DPPC LUVs. Partitioning of 1/1m into the Ld membranes of DPPC LUVs at 50 °C resulted in the appearance of a distinct absorption band in a lipid-concentration dependent manner (Figure 3a, solid red line, Figure S9g). Without LUVs, only a weak feature dominated by scattering was observed probably due to the precipitation of the probe in the buffer (Figure S9g). The absorption maxima found at 375 nm between pH 4 and 7 and 355 nm below pH 4 were consistent with the presence of neutral and protonated monomer 1m in Ld bilayers, respectively (Figure 3b, ○). Upon lipid phase transition to the So
state by cooling of the mixture to 25 °C, the characteristic red-shifted polymer absorption band appeared between pH 3 and 6 (Figure 3a, solid blue line; Figure 3b, ●; Figure S6). The observed pH dependence was consistent with the known reactivity of imines, which are in dynamic equilibrium between the con- densed and the hydrolyzed form in weak acid but poorly reactive in neutral and hydrolyzed in strongly acidic conditions.8b Thus, these results suggested that the dynamic formation of polyimine 1 is responsive to the phase of bilayer membranes. Indeed, the blue-shifted monomer absorption was consistently ob- served above the phase-transition temperature Tm = 41 °C of DPPC, while below Tm the absorption
Figure 2. (a) MALDI-TOF MS of polymer 1. (b) GPC profiles of polymer 1 in THF before (top) and after the addition of TFA (bottom), detected at l = 390 nm. Molecular weights were estimated by com- paring the retention times to those of polystyrene calibration standards (Figure S1). Note, small underes- timates of molecular weights are expected for amine-containing compounds. (c) Normalized absorption spectra of 1 eluted at Rt = 10.5 to 13.5 min (0.5 min intervals, red to blue). (d) Dependence of lmax on the molecular weight estimated from GPC.
maxima gradually red-shifted in a temperature-dependent manner (Figure 3a). These changes were highly reproducible and reversible, with the transition always occurring at 40.5 ± 0.5 °C (Figure S6h). Nearly unaffected Tm supported the non-disruptive nature of 1/1m in DPPC bilayers. Regardless of pH or
b)
c)
a) N
O
O
O H O
H n
1 1m: n = 1
n = 1 2 3
4
5 6
7 8 9 10 11 12 13
m/z 100 30 10 3 1 0.3 kDa
λmax(nm) d)
1000 2000 3000 4000 5000 6000
9 10 11 12 13 14 15 16
t (min)
350 400 450 500 0.1 1 10
λ (nm) Mw (kDa)
420
400
380
360
temperature, polymer 1 failed to form in DOPC LUVs, which remain in Ld state at > –17 °C (Figure 3b,
■, £; Figure S7). Contrarily, polymer 1 stayed intact even at 50 °C in SM/CL 1:1 vesicles, which remain
in Lo phase also at this elevated temperature (Figure 3b, ▲, r; Figure S8).23
Supported by examples in the literature for the aggregation induced imine formation,8b-h,24 we ration- alized these results by the phase separation and aggregation of monomer 1m in ordered (So and Lo) bi- layers. The dependence of polyimine formation on the probe-to-lipid molar ratio demonstrated that the polymerization occurs within the confinement of lipid bilayers (Figure 3c, ●, ○, and ▲; Figure S9). On the other hand, good solubility, thus low local concentration of 1m seemed to disfavor the polyimine for- mation in Ld bilayers (Figure 3c, ■). The concentration21 dependence profile in mixed Ld-So phases of DOPC/DPPC LUVs15c was nearly identical to that in DOPC LUVs and supported that monomer 1m is better soluble in Ld than in So membranes (Figure 3c, ▼). Interestingly, the blue-shifted monomer ab- sorption was never observed in the Lo phase of SM/CL LUVs (Figure 3c, ▲). Increasing scattering at lower concentrations of 1 implied that monomer 1m poorly partitions into the Lo phase membrane and thus precipitates in the buffer. In the mixed Ld-Lo phases of DOPC/SM/CL LUVs,23 the extent of polyi- mine formation was in between that in DOPC and SM/CL LUVs (Figure 3c, ◆). Nearly equal distribution of 1/1m in both phases was supported by the good match between the experimental and simulated spectra obtained by mixing the spectra in Lo and Ld phases in ~1:1 ratio (Figure S9h).
Chiral Recognition of Lipid Bilayer Membranes. The two enantiomers of push-pull fluorenes could be obtained by a chiral separation of Boc-protected 1m, followed by deprotection (Figure S1). CD spectra of the two enantiomers of 1m in Ld phase of DPPC LUVs at 50 °C gave nondescript nearly symmetrical CD signals (Figure 3d, red dashed lines). However, the CD signals of the polymers in So phase of DPPC LUVs at 25 °C were not symmetrical (Figure 3d, blue lines). Bisignate CD Cotton effects observed with one of the enantiomers (Figure 3d, solid line) implied that the CD signals comprise of the intrinsic and exciton-coupled CD signatures,25 which are dependent and independent of the monomer chirality, respec- tively.
Figure 3. (a) Absorption spectra of 1 (3 µM) in DPPC LUVs (0.15 mM) at 50 °C (red solid line) and every 1 °C from 45 to 25 °C (magenta dashed to blue solid, bold lines every 5 °C) at pH 5. (b) pH dependence of absorption maxima of 1 in DPPC (●, ○), DOPC (■, £) or SM/CL LUVs (▲, r) at 25 °C (●, ■, ▲) or 50 °C (○, £, r). (c) Dependence of lmax on the probe-to-lipid molar ratio for 1 in DPPC (●, ○), DOPC (■), SM/CL (▲), DOPC/DPPC (▼) and DOPC/SM/CL (◆) at pH 5. Data were obtained by varying the concentration of 1 (filled symbols) or lipids (empty symbols). (d) CD spectra of the enan- tiomers of 1 (15 µM) in DPPC LUVs (0.3 mM) at 50 °C (red lines) or 25 °C (blue lines) at pH 5.
Exciton coupling reflects the chiral twist between the monomer units.25 The different CD signature obtained for the two enantiomers of 1 implied that this twist between monomers in polyimine 1 is dictated by the chirality of DPPC. The small amplitudes of the bisignate exciton coupled CD signals in So DPPC were consistent with a relatively planar conformation of dynamer 1. Although intensely studied, success- ful examples for the chiral recognition of lipid bilayer membranes are rare and generally considered as challenging to obtain, particularly with regard to So membranes.26
Fluorescence Properties in Lipid Bilayer Membranes. In various solvents with/without acid/base, we observed fluorescence only upon excitation of the monomer unit at 370 nm, and not of the polymer 1
d)
Δε(M–1cm–1) –2 –1 0 1 2
λ (nm)
350 400 450 500
λmax (nm)
2 3 4 5
350 375 400 425
a) pH
b)
λmax (nm)
c1/cL (mol%) c)
0.5 1 3 5
360 380 400 420
6 7
A (AU)
0 0.1
at 420 nm (Figure S3-S5). These results were surprising given the high fluorescence reported for the similar polymer,7 but consistent with the poor fluorescence found with many aromatic aldimines.8a,27 As a result, polyimine formation in LUVs could be detected as the quenching of monomer fluorescence (Fig- ures 4, S10). The partitioning of the probe 1 at low concentration in LUVs was evidenced by the large fluorescence enhancement (Figure 4a, blue and red compared to green lines). The excitation spectra measured in LUVs matched well with the monomer absorption, demonstrating the fluorescence to arise from the monomer 1m (Figure 4a, dashed lines).
Figure 4. (a) Fluorescence excitation (dashed lines, for fluorescence emission at 450 nm) and emission spectra (solid lines, excitation at 370 nm) of 1 (0.25 µM) in buffer (pH 5, green), DOPC (red) or DPPC LUVs (blue) at 25 °C. (b) Dependence of fluorescence intensity at 500 nm on the probe-to-lipid molar ratio for 1 in DOPC (■), DPPC (●), SM/CL (▲) and DOPC/SM/CL LUVs (◆), or without LUVs (x, measured at the same concentrations of 1). (c) Confocal laser scanning microscopy image, (d) corre- sponding bright field image and (e) the overlay of 1/1m (1 mol%) in DOPC/SM/CL GUVs. Excitation laser was at 405 nm and detection at 450-540 nm.
400 500 600 0 1 2 3
e) λ (nm)
a) b)
c1/cL (mol%)
F (cps/µA)
c) d)
0 1 106
10 µm
In DOPC LUVs at pH 5, emission intensity increased linearly with the concentration of 1 until rela- tively high levels of ~2 mol% (Figure 4b, ■). These results corroborated the findings from absorption spectroscopy and indicated that 1 remains in monomeric form in Ld DOPC membranes at <2 mol%. The estimated quantum yield of 54% was comparable to the values reported for similar push-pull fluorene- based probes in DOPC membranes.14b
With 1 in So DPPC membranes, the increase of emission intensity deviated from linearity at a much lower probe-to-lipid ratio of ~0.7 mol% (Figure 4b, ●). This behavior indicated the onset of polymeriza- tion at this low concentration. Indeed, the absorption maximum observed under the same conditions was
~10 nm red-shifted compared to that in DOPC LUVs (Figure 3c).
Below these critical polymerization concentrations (cpc), distinct fluorescence spectra were obtained for 1m in DOPC and DPPC LUVs (Figure 4a). The sharp and nearly symmetrical excitation and emission peaks obtained in DPPC membranes were similar to those in EtOAc and implied the presence of the chromophore near the water-membrane interface (Figure 4a, blue lines). In DOPC membranes, however, unusual double peaks appeared in the emission spectrum (Figure 4a, red solid line). This split was ob- served independent of the concentration (below cpc), pH and the identity of the lipid (i.e., DPPC at 50
°C), and absent in similar push-pull fluorene-based membrane probes.14b We thus attributed the split peaks to chromophores residing in deeper and more hydrophobic or shallower and more polar environments within bilayer membranes. This hypothesis was supported by the different degrees of quenching of the two peaks by 5- and 12-doxyl labelled PC (Figure S11). Parallax analysis gave distances of 5.7 and 8.6 Å from the center of bilayers to the fluorophores emitting at 460 and 530 nm, respectively.28
Poor partitioning of monomeric 1m into Lo SM/CL membranes, as postulated above, was confirmed by negligible fluorescence intensities, very similar to those in membrane-free buffer (Figure 4b, ▲ vs x). In the mixed Lo-Ld phases of DOPC/SM/CL LUVs, the emission spectra resembled those in DOPC LUVs, but the intensity stopped growing already at ~0.7 mol% 1m, indicating the facilitated formation of non- fluorescent polymer in the Lo phase (Figure 4b, ◆). Indeed, incorporated in Lo-Ld phase separated GUVs
composed of the same lipid mixture, the fluorescence of 1m was observed exclusively from one of the phases (Figure 4c-e). Based on the results with LUVs, the fluorescent phase can be safely assigned to Ld
phase, while the polyimine formation in Lo phase resulted in fluorescence quenching. Further support of the probe location was obtained by using GUVs co-stained with 16:0 Liss Rhod PE fluorophore, which is known to label Ld phase selectively (Figure S13).29 The perfect overlap in the obtained images demon- strated that both fluorophores accumulate in the same Ld domains of the mixed membranes. However, the presence of polymer 1 in Lo domains could not be confirmed by color bright field imaging even at higher concentrations, due probably to much lower sensitivity.
Membrane Tension. The ability of polyimines to sense the membrane tension was evaluated using LUVs under osmotic stress (Figure 5). In this commonly used model, transmembrane osmolarity differ- ence is applied to swell or shrink the vesicles, which results in stretching or bending of the membrane.
Such stresses have been shown to alter many physical properties of bilayer membranes, including the fluidity,12e hydration level,12b dipole potential12c and lipid domain formation behavior.15,10c
Upon exposure of 1/1m in DOPC or DOPC/SM/CL LUVs to osmotic stress, the fluorescence intensity of the red-shifted band at 530 nm remained nearly constant (Figure S12). Thus, osmotic stress was unable to affect the degree of polymerization as anticipated. However, the intensity of the blue-shifted band at 460 nm was dependent on the osmolarity of the media (Figure 5). Similar results were reported with Laurdan,12b and indicated that the push-pull fluorene monomer 1m relocates within bilayer in response to swelling or shrinking of the vesicles under hypo- or hyper-osmotic stress, respectively. This interpretation was supported by the absence of such spectral changes when osmolarities of both intra and extra vesicular buffers were varied together, thus without the osmotic stress (Figure S12c). The physical origin of this behavior is unknown but unlikely to be the reorganization of lipid domains by osmotic stress, as compa- rable results were obtained using single phase DOPC and phase-separated DOPC/SM/CL LUVs (Figure 5b).
Figure 5. (a) Normalized emission spectra of 1 (0.7 µM) in DOPC LUVs under iso- (green), hyper- (red) and hypo-osmotic conditions (blue). (b) The dependence of the fluorescence ratio F460/F530 of 1 (0.7 µM) in DOPC (●) or DOPC/SM/CL LUVs (■) to the osmolarity of the media. Plotted are the average values
± error from three independent experiments.
CONCLUSIONS
In summary, this study introduces amphiphilic polyimines as multifunctional optical sensors of the organ- ization of lipid bilayer membranes. The formation of dynamic polyimines was responsive to the phase of bilayer membranes and resulted in bathochromic shifts of absorption maxima, the appearance of exciton- coupled CD signals, and quenching of fluorescence. Selective fluorescence labeling of Ld phases was evidenced by imaging phase-separated GUVs containing 1/1m. The fluorescent monomer was found to be able to sense the osmotic stress applied to vesicles. These promising results leave a lot to wish for.
The high-energy excitation of monomeric fluorophores can probably be circumvented by using two-pho- ton excitation, as demonstrated for similar push-pull fluorene fluorophores.14f More importantly, fluores- cent polyimines would provide access to multicolor imaging of membrane phases. This challenge could presumably be met based on various strategies available in the literature.8a,27 Besides membrane phases, more traditional mechanochromic applications could also be envisaged using dynamic polyimines em- bedded in plastic films.19,20,30 On-off fluorescence upon dynamic de-/polymerization would be beneficial in these applications for the easy detection of defects. Studies along these lines are ongoing.
b)
F460 / F530
Hypo Hyper
F (relative)
a)
λ (nm)
400 500 600 100 1000
Π (mOsM)
0.0 0.5 1.0
0.8 0.9
ASSOCIATED CONTENT Supporting Information
Detailed experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Authors
[email protected], [email protected] ORCID
Naomi Sakai: 0000-0002-9460-1944 Stefan Matile: 0000-0002-8537-8349 Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the NMR, the MS and the Bioimaging Platforms for services, and the University of Geneva, the Swiss National Centre of Competence in Research (NCCR) Chemical Biology, the NCCR Molecular Systems Engineering and the Swiss NSF for financial support.
ABBREVIATIONS
CD, circular dichroism; CL, cholesterol; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC, 1,2- dipalmitoyl-sn-glycero-3-phosphocholine; GPC, gel permeation chromatography; GUVs, giant unilamel- lar vesicles; Ld, liquid disordered; Lo, liquid ordered; LUVs, large unilamellar vesicles; SM, sphingomy- elin; So, solid ordered; TFA, trifluoroacetic acid.
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18 TOC
O
CHO H2N
360 nm Ld ICD
420 nm Lo ECCD
N N
O 3
5
3 5
