Chiral figure-eight molecular scaffold for fluorescent probe development

Abstract

Targeted fluorescent molecular probes are useful for cell microscopy, diagnostics, and biological imaging. An emerging discovery paradigm is to screen libraries of fluorescent molecules and identify hit compounds with interesting targeting properties. However, a current limitation with this approach is the lack of fluorescent molecular scaffolds that can produce libraries of probe candidates with three dimensional globular shape, chiral centers, and constrained conformation. This study evaluated a new probe scaffold called squaraine figure-eight (SF8), a self-threaded molecular architecture that is comprised of an encapsulated deep-red fluorescent squaraine dye, surrounding tetralactam macrocycle, and peripheral loops. Easy synthetic variation of the loops produced four chiral isomeric SF8 probes, with the same log P values. Cell microscopy showed that subtle changes in the loop structure led to significant differences in intracellular targeting. Most notably, a comparison of enantiomeric probes revealed a large difference in mitochondrial accumulation, very likely due to differences in affinity for a chiral biomarker within the organelle. A tangible outcome of the research is a probe candidate that can be: (a) developed further as a bright and photostable, deep-red fluorescent probe for mitochondrial imaging, and (b) used as a molecular tool to identify the mitochondrial biomarker for selective targeting. It will be straightforward to expand the SF8 probe chemical space and produce structurally diverse probe libraries with high potential for selective targeting of a wide range of biomarkers.

Introduction

One way to discover new fluorescent molecular probes with novel intracellular targeting capabilities is to screen libraries of candidate molecules and identify hit compounds with promising targeting properties.^{1,2,3,4,5,6,7,8,9,10,11,12} Almost exclusively, the screening has examined libraries of achiral fluorescent dyes with predominantly flat chemical structures which means that only a limited amount of three dimensional structural space is explored. The related field of drug discovery has addressed this limited molecular space sampling problem by developing synthetic methodologies that produce three dimensional molecular architectures with constrained conformations.^13,14,15,16 These synthetic methods produce libraries of drug candidates with relatively rigid shapes and large surface areas that enable multiple favorable contacts with a biomarker without suffering a large entropic penalty.^17,18 Small molecules that possess three dimensional shapes and chiral centers are attractive as drug candidates because they are more likely to produce a unique binding interface that can only be recognized by a specific biomarker surface and thus they are less likely to exhibit undesirable off-target association.^19,20 Similarly, it is reasonable to expect that expanding the molecular space of fluorescent probe libraries will improve the chances of discovering new classes of probe/biomarker pairs. However, there is presently a lack of synthetic methods that can produce libraries of fluorescent molecules with three dimensional molecular architecture, chiral centers, and constrained conformations. A literature search uncovers a scattered collection of studies using chiral luminescent metal coordination complexes as probes for cell microcopy.^{21,22,23,24,25,26} But currently there are virtually no reports of chiral organic dye scaffolds with globular, three dimensional shapes that can be synthetically converted into libraries of fluorescent molecular probes.²⁷

Recently, we reported a potential breakthrough by developing a synthetic method that transforms a mechanically bonded squaraine rotaxane (SR) molecule into a single, covalently connected fluorescent molecule that we call squaraine figure-eight (SF8).²⁸ The self-threaded architecture of an SF8 molecule is reminiscent of lasso peptides, but with a fluorophore as the encapsulated component.^29,30 Shown in Scheme 1A is the chemical structure of the organic-soluble prototype compound, SF8(C6)₂, whose structure is illustrated as three colored-coded sections; encapsulated squaraine dye (blue); surrounding tetralactam macrocycle (red), and two identical hexyl-containing (C6) loops (green). We also synthesized some water-soluble homologues with charged and highly polar peptidyl loops and observed excellent fluorescent imaging properties, including protease resistance. The X-ray crystal structure of SF8(C6)₂ (Scheme 1A) reflects the globular structure that is inherent to SF8 molecules and also highlights two key intramolecular interactions: (a) hydrogen bonding between the two oxygen atoms in the encapsulated squaraine and the amide NH residues in the surrounding tetralactam, and (b) aromatic stacking of the two tetralactam 1,4-phenylene rings over both faces of the uncharged squaraine C₄O₂ core which has zwitterionic character. Fluorescence imaging studies of the initial set of SF8 molecular probes showed that they exhibit bright deep-red fluorescence that is well-suited for multicolor imaging of cells, thick biological samples, or living subjects.²⁸

Scheme 1.

Chemical structures of SF8 molecules used in this study. (A) Previously reported achiral prototype SF8(C6)₂ along with an X-ray crystal structure highlighting the self-threaded molecular architecture. (B) Four uncharged chiral isomers that were evaluated for differences in intracellular targeting. Color-coded within each single molecule are three sections; encapsulated squaraine dye (blue); surrounding tetralactam macrocycle (red), and peripheral loops (green).

The goal of this present study was to assess the potential for the SF8 architecture as a synthetic scaffold in library preparation and screening to find probe candidates with interesting intracellular targeting properties. In addition to synthetic feasibility, we wanted to determine if the intracellular targeting of fluorescent SF8 probes was sensitive to subtle structural changes in molecular chirality or loop flexibility. We report the synthesis and intracellular cell targeting capabilities of the four chiral SF8 compounds shown in Scheme 1B. Each compound has two chiral centers associated with two phenylalanine residues and the four compounds are structurally interrelated as isomers. Specifically, the uncyclized SR pair SR(azido-F)₂ and SR(azido-f)₂ are enantiomers and so are the looped SF8 pair SF8(F)₂ and SF8(f)₂. In addition, each pair of SR or SF8 compounds with the same chirality (e.g., SF8(f)₂ and SR(azido-f)₂) are related as constitutional isomers. These four uncharged, isomeric probes were designed to have the same cell permeation capability and differ only in chirality or structural rigidity. We find that the four probes exhibit a marked difference in intracellular targeting which is likely due to a change in affinity for a chiral biomarker at the target site. The results suggest that screening of fluorescent SF8 probe libraries has great promise as a method to discover new biomarkers for targeted intracellular fluorescence imaging.

Results and discussion

Synthesis and Molecular Structure

The general synthetic method to make the compounds in Scheme 1B has been previously described.²⁸ Briefly, the SR precursors, SR(azido-F)₂ and SR(azido-f)₂, were converted into SF8 probes, SF8(F)₂ and SF8(f)₂, in 42 and 55% isolated yield, respectively, by conducting a double macrocyclization reaction using copper catalyzed azide/alkyne cycloaddition (CuAAC) conditions. The structure and high purity of each compound was confirmed by standard spectroscopic methods. Synthetic conversion of an uncyclized SR isomer to its closed loop SF8 counterpart produces informative changes in ¹H NMR spectral patterns that reflect differences in molecular motion. The ¹H NMR spectra in Figure 1 highlight two sets of information. First, there is line broadening at higher temperature due to two internal molecular motions that have been previously documented.^28,31 One motion is hindered rotation of the two 1,4-phenylene units in the tetralactam macrocycle (which exchanges protons f and f’) and the other is hindered rotation of the squaraine aryl rings (which exchanges proton pairs h/h’ and g/g’) (Figure 1A). Analysis of variable temperature NMR spectra for SF8(F)₂ determined the free energy of activation for phenylene spinning 63.2 kJ.mol⁻¹ (Figure S1–S2), which is very close to 64.4 kJ.mol⁻¹ for the same phenylene spinning process in SF8(C6)₂ and corresponds to a rate constant of ~200 s⁻¹ at room temperature.²⁸

Figure 1.

(A) Partial ¹H NMR spectra (500 MHz, 10% CD₃OD in CDCl₃) of SF8(f)₂ at two temperatures. (B) Circular dichroism of SF8(F)₂ and SF8(f)₂.

The ¹H NMR spectra in Figure 1 also provide evidence that the peripheral loop regions of the SF8 molecules are conformationally constrained. For example, the diastereotopic methylene proton pairs (b,b’) and (r,r’) within SF8(f)₂ (Figure 1A) exhibit much greater chemical shift inequivalence than the equivalent methylene proton pairs within uncyclized isomer SF8(azido-f)₂. Another diagnostic NMR feature exhibited by closed loop SF8 structures (SF8(F)₂ and SF8(f)₂) is the separate signals for bridgehead protons c and c’ (Figure 1A) which reflects inequivalent chemical shielding of the front and back faces of the molecule. Independent support for this picture of limited loop flexibility was gained by acquiring CD spectra of SF8(F)₂ and SF8(f)₂. As shown in Figure 1B, the CD spectra exhibit strong Cotton effects of opposite sign consistent with conformationally constrained loops and strong electronic communication between the chiral center in each loop and the encapsulated squaraine fluorophore. In contrast, CD spectra of uncyclized SR analogues do not exhibit observable Cotton effects.

The absorption and fluorescence emission wavelengths of the four isomeric probes (Table 1) are all very close to 646 nm and 662 nm, respectively. This corresponds to the standard Cy5 filer set that is commonly available on most microscopes, imaging stations, and flow cytometers. The insensitivity of fluorescence brightness to structural variation of the loops in the SF8 probes implies a constant microenvironment surrounding the encapsulated squaraine fluorochrome. This means that probe fluorescence brightness in cell microscopy experiments does not change with intracellular location, a technical attribute that facilitates quantitative analysis of the cell micrographs. A series of tests that exposed the SF8 probes with various common intracellular analytes found no loss of absorption signal (Figure S4) and MTT assays showed no evidence of cell toxicity at probe concentrations used for cell microscopy studies (Figure S5).

Table 1.

Photophysical data for SF8 and SR molecules.

Compound	λabs (nm)	λem (nm)	Logε[a]	Φf[b]
SF8(C6)2	645	660	5.49±0.10	0.64±0.05
SF8(F)2	646	662	5.49±0.10	0.73±0.05
SF8(f)2	646	662	5.38±0.10	0.80±0.05
SR(azido-F)2	647	664	5.56±0.10	0.89±0.05
SR(azido-f)2	647	664	5.42±0.10	0.86±0.05

^[a]ε is molar absorptivity.

^[b]In CHCl₃, measurements of quantum yield (Φ_f) relative to 4,4-[bis(N,N-dimethylamino)-phenyl]squaraine (Φ_f = 0.70).

Cell Microscopy

Separate samples of HT-1080 human fibrosarcoma cells were incubated with each fluorescent probe (1 μM) for 30 minutes. Epifluorescent cell micrographs were acquired, and the mean deep-red fluorescence intensity was quantified for each condition. Large differences were observed in the extent of probe uptake by the cells. Notably, cell uptake of the cyclized SF8(F)₂ was 8 times higher than its uncyclized analogue SR(azido-F)₂ (Figure 2A–2C), and cell uptake of SF8(f)₂ was 13 times higher than its uncyclized analogue SR(azido-F)₂ (Figure 2B–2D). This large difference in cell accumulation is unlikely due to a difference in cell permeation by membrane diffusion because all four isomers are uncharged and exhibit the same fluorescence brightness (Table 1) and log P value of −0.5 (Figure S3).

Figure 2.

Probe uptake by HT-1080 cells. (A,B) Representative epifluorescence cell micrographs with (C,D) calculated mean fluorescence intensity showing probe uptake by the cells after a 30 min incubation with each probe (1 μM). Red fluorescence shows probe; blue fluorescence shows Hoechst nuclear stain. Scale bar = 30 μm. Asterisks represent **** p<0.0001

Not only was there a large disparity in the extent of intracellular accumulation there was also an obvious difference in the intracellular location of the probes. Inspection of the fluorescent micrographs suggested significant levels of probe accumulation in the mitochondria and lysosomes. To confirm this hypothesis, a series of multicolor fluorescence colocalization studies were conducted using either MitoTracker Green FM or LysoTracker Red DND-99 to label the mitochondria and lysosomes, respectively. All microscopy data for achiral SF8(C6)₂ is shown in Figure S6 (lysosome and mitochondria colocalization experiments), whereas the data for the four chiral isomers are shown in Figure 3 (mitochondria) and Figure 4 (lysosome) with a listing of Pearson’s correlation coefficients in Figure 5. Analysis of these data sets leads to three major observations. (1) Achiral control SF8 probe SF8(C6)₂ (structure lacks the phenylalanine residues) does not localize in either the mitochondria and lysosomes; the colocalization micrographs in Figure S4 produce Pearson’s correlation coefficients below 0.1 which is considered negligible colocalization.³² (2) The looped SF8 probes $SF8{\left(F\right)}_{2_2}$ and SF8(f)₂ exhibit much higher colocalization in the lysosomes than their uncyclized SR isomers SR(azido-F)₂ and SR(azido-f)₂, respectively (Figure 5). There is also higher colocalization of the looped SF8 probes in the mitochondria but the difference is smaller. (3) The enantiomeric probes SF8(F)₂ and SF8(f)₂ accumulate in the lysosome to similar extents but there is a large difference in the degree of mitochondrial accumulation (Figure 5). The uncyclized enantiomers, SR(azido-F)₂ and SR(azido-f)₂, exhibit the same trend.

Figure 3.

Localization of fluorescent probes within cell mitochondria. HT-1080 cells were incubated with probes (1 μM) for 30 min and co-stained with MitoTracker Green FM (100 nM) for 15 min. Red fluorescence shows squaraine probes; green fluorescence shows MitoTracker Green FM; yellow fluorescence shows colocalization. Scale bar = 30 μm.

Figure 4.

Localization of probes within cell lysosomes. HT-1080 cells were incubated with probe (1 μM) for 30 min and co-stained with LysoTracker Red DND-99 (100 nM) for 15 min. Red fluorescence shows squaraine probe; yellow fluorescence shows LysoTracker Red DND-99; orange fluorescence shows colocalization. Scale bar = 30 μm.

Figure 5.

Pearson’s correlation coefficient for probe localization within cell organelles. HT-1080 cells were incubated with probes (1 μM) for 30 min and co-stained with either with 100 nM MitoTracker Green FM or 100 nM LysoTracker Red DND-99 for 15 min. Asterisks represent ** p<0.01, *** p<0.001, and **** p<0.0001.

Since the four chiral probes are uncharged isomers, with the same log P values of −0.5, we can assume that they can diffuse in and out of cells at equal rates and that they have the same affinity for achiral intracellular lipophilic sites such as the interior of the cell bilayer membranes. Based on this assumption, we make the following assertions. (1) The higher cell uptake of the two closed-loop SF8 probes (SF8(F)₂ and SF8(f)₂) compared to their uncyclized SR analogues (SR(azido-F)₂ and SR(azido-f)₂) is due to increased retention of the SF8 probes within the mitochondria and lysosomes. The SR analogues are weakly retained and thus they more readily diffuse out of the cells and into the extracellular media where they are washed away. (2) The large difference in mitochondria accumulation between probe enantiomers reflects a difference in probe affinity for an unknown chiral biomarker within the mitochondria (probably a protein).³³ The enantiomeric probes form diastereomeric probe/biomarker complexes with different stabilities; the probes with two R stereocenters (i.e., SF8(f)₂ and SR(azido-f)₂) have higher biomarker affinities than their enantiomers with two S stereocenters (i.e., SF8(F)₂ and SR(azido-F)₂). The much higher cell uptake of SF8(f)₂ compared to SR(azido-f)₂ implies that the more rigid SF8(f)₂ has higher biomarker affinity than flexible SR(azido-f)₂.

The high selectivity of SF8(f)₂ and SR(azido-f)₂ for the mitochondria is unusual because they are uncharged molecules and structurally quite different to the usual mitochondria seeking molecules which are cationic and hydrophobic.^34,35 The much higher cell uptake of SF8(f)₂ makes it the lead candidate for follow-up studies where there are two potential research directions. One future goal is elucidating the identity of the putative mitochondrial biomarker that is targeted by SF8(f)₂. The central role played by the mitochondrial function in normal cell operation and the correlation of mitochondrial disfunction with various diseases such as cancer³⁶ and type-2 diabetes³⁷ makes the search for new probe/biomarker pairs a significant future goal. A range of standard biomarker discovery methods can be pursued using SF8(f)₂ as molecular bait to tag or capture the biomarker. These methods might employ modified versions of SF8(f)₂ with reactive groups that enable covalent labelling of the biomarker.^38,39,40,41 A second and related future goal is optimization of SF8(f)₂ as selective deep-red fluorescent probe for highly selective mitochondrial imaging. Even without knowing the biomarker identity, it should be possible to increase the targeting effectiveness of SF8(f)₂ by incorporating substructures into the SF8 scaffold (such as hydrophobic cations) that are known to increase probe partitioning within the mitochondria.^34,35 We have previously shown that peptide bonds within SF8 loops are protease resistant.²⁸ This attractive stability feature is further amplified here since the two chiral phenylalanine centers within SF8(f)₂ are the unnatural R (or D) configuration which is not recognized by most proteases.

Conclusions

Squaraine figure-eight (SF8) probes display excellent deep-red, fluorescent cell imaging capabilities with high chemical and photostability, along with protease resistance.²⁸ The squaraine dye is encapsulated within the self-threaded architecture; thus, SF8 probes are especially attractive for intracellular imaging because the squaraine fluorescence properties do not change with variation of the peripheral loops or with changes in the probe microenvironment. Easy synthetic variation of the probe loops by standard solid phase peptide synthesis technology makes the SF8 scaffold a convenient choice for preparation of fluorescent probe libraries. Moreover, the NMR and CD data in Figure 1 shows that the conformation of the probe loops are constrained, and the overall molecular shape is globular and chiral. The microscopy data (Figures 2 – 5) confirm our central hypothesis that subtle changes in the loop regions of fluorescent SF8 probes can lead to significant differences intracellular targeting most likely because the probes exhibit different affinities for intracellular targets. A tangible outcome of this study is the identification of SF8(f)₂ as a lead candidate for development as a novel fluorescent probe with selective affinity for a chiral biomarker within the mitochondria. In terms of screening methodology, we find that comparative imaging of probe enantiomers is a simple but powerful way to identify new chiral intracellular biomarker target(s) for molecular imaging. We have already shown that the peptide loops in an SF8 probe can be synthetically extended,²⁸ and thus we can easily expand the SF8 probe chemical space to produce larger, structurally diverse fluorescent probe libraries.^42,43 The two loops associated with the figure eight topology suggest an alluring path towards bifunctional SF8 molecules that can act like molecular glue (inducers of proximity) to target unique pairs of biomarkers.⁴⁴ Additional studies of deep-red fluorescent peptidyl SF8 probes are underway and the results will be reported in due course.

Experimental

General Materials

All chemicals and solvents were purchased as reagent grade and used without further purification unless otherwise noted. Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel 60-F254 plates, visualized by ultraviolet (254, 365 nm). NMR spectra (¹H, ¹³C, ¹H-¹H COSY) were recorded on Bruker AVANCE III HD 400 or 500 MHz spectrometer at 25 °C. Chemical shift was presented in ppm and referenced by residual solvent peak. High-resolution mass spectrometry (HRMS) was performed using a Bruker micro-TOP II spectrometer. Absorption and fluorescence spectra were collected on Evolution 201 UV/Vis Spectrometer with Thermo Insight software, and Horiba Fluoromax4 Fluorometer with FluoroEssence software, respectively. Measurements were conducted at room temperature with quartz cuvette (1 mL, 10 mm path length). Column chromatography was performed on Biotage using silica gel (silicaFlash P60 from SILICYCLE).

Probe Synthesis

Compound synthesis and characterization are described in the Electronic Supplementary Information.

Log P Measurements

Probe stock solutions were made at 1 mM in DMSO. Each SF8 probe (10 μM) was then added to a biphasic mixture composed of octanol/water in a volume ratio of 1:1 and vortexed for 30 seconds. Samples were centrifuged at 1000 g for 4 minutes to rapidly separate the phases and then photographed under ambient light. Each phase was transferred to a 96-well plate, and the absorbance at 655 nm was measured. All measurements were performed in triplicate. Log P was calculated using the following formula: log P = log(A_octanol/A_water).

Circular Dichroism

Circular dichroism spectra were collected with a Jasco J-1500 circular dichroism spectrophotometer with 1 nm data pitch, 1 nm bandwidth, and 100 nm/min scanning speed at 24 °C. For each measurement, 10 μL of the respective stock solution (1 mM in DMSO) was transferred into a quartz cuvette (10 mm, 1 mL) and diluted with 700 μL deionized water to give a final concentration of 14.1 μM.

Cell Culture

All cells were cultured and maintained in a humidified incubator at 37 °C under 5% CO₂. The cell medium (EMEM), was purchased from American Type Culture Collection (ATCC). Fetal bovine serum was purchased from Atlanta Biologicals, and all other supplies were purchased from Sigma Aldrich. The HT-1080 (ATCC CCL-121) human fibrosarcoma cells were maintained in EMEM medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, and 1% penicillin/streptomycin.

Cell Toxicity

HT-1080 cells were cultured in EMEM medium (supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, and 1% penicillin/streptomycin) at 37 °C and 5% CO₂ in a humidified incubator. Cells were seeded into 96-microwell plates and grown to 80% confluency. The medium was removed and replaced with solution of (A) SF8(C6)₂, (B) SF8(F)₂, or (C) SF8(f)₂ at various concentrations in EMEM medium. After 6 hours at 37 °C and 5% CO₂ in a humidified incubator, the surrounding solution was removed and replaced with EMEM medium containing [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT,1.1 mM). After a 4-hour incubation at 37 °C and 5% CO₂, SDS-DMSO detergent solution was added, the samples incubated overnight, and the absorbance of each well was measured at 590nm. The readings for each probe were normalized to untreated cells. All measurements were made in triplicate.

Fluorescence Cell Microscopy

HT-1080 cells were seeded into 8-well chambered slides (Lab-Tek, Nunc, USA) and were grown to 70% confluency. The cells were incubated with 1 μM SF8 probe in media for 30 min at 37 °C. The cells were washed three times with phosphate buffered saline and co-stained with either 100 nM MitoTracker Green FM (Thermo Fisher Scientific) or 100 nM LysoTracker Red DND-99 (Thermo Fisher Scientific) for 15 min in opti-MEM at 37 °C. Cells were then washed two times with phosphate buffered saline and incubated with 3 μM Hoechst 33342 for 10 min at room temperature. The live cells were washed two additional times and imaged on a Zeiss Axiovert 100 TV epifluorescence microscope equipped with a UV filter (ex: 387/11, em: 447/60), FITC filter (ex: 485/20, em: 524/24), TxRed filter (ex: 562/40, em: 624/40), and Cy5.5 filter (ex: 655/40, em: 716/40). Image processing for each micrograph was then conducted using ImageJ2 software with a 10-pixel rolling ball radius background subtraction.

Statistical Analysis

Colocalization experiments were analyzed by applying a colocalization threshold program (JaCoP) using ImageJ2 software to determine the Pearson’s correlation coefficient. Averages and SEM were plotted in GraphPad Prism. For determining the mean fluorescence intensity of cell micrographs, the average mean fluorescence intensity was calculated using ImageJ2 software and 20 randomly generated 25 × 25-pixel extra-nuclear regions of interest. The averages and SEM were plotted in GraphPad Prism.