Molecular demultiplexer as a terminator automaton

Abstract

Molecular logic gates are expected to play an important role on the way to information processing therapeutic agents, especially considering the wide variety of physical and chemical responses that they can elicit in response to the inputs applied. Here, we show that a 1:2 demultiplexer based on a Zn²⁺-terpyridine-Bodipy conjugate with a quenched fluorescent emission, is efficient in photosensitized singlet oxygen generation as inferred from trap compound experiments and cell culture data. However, once the singlet oxygen generated by photosensitization triggers apoptotic response, the Zn²⁺ complex then interacts with the exposed phosphatidylserine lipids in the external leaflet of the membrane bilayer, autonomously switching off singlet oxygen generation, and simultaneously switching on a bright emission response. This is the confirmatory signal of the cancer cell death by the action of molecular automaton and the confinement of unintended damage by excessive singlet oxygen production.

Fig. 1

DEMUX combinatorial circuit and structures of relevant Bodipy compounds. a DEMUX circuit with standard logic gate symbols. b Isosceles trapezoid is a common symbol for the MUX/DEMUX circuits. I is the data input, S is the select or address input, and O1 and O2 are two different outputs. c The truth table for a 1:2 DEMUX circuit is on the right. d Structures of the parent Bodipy compound B-1, and meso-pyridyl/bipyridyl substituted derivatives, together with their respective fluorescence and intersystem crossing quantum efficiencies are demonstrated. Protonation of both B-2 and B-3 result in the pyridinium cation, which switches on the intramolecular charge transfer process. Coordination of Zn²⁺ ions or quaternization at the pyridine nitrogen (B-4) results in the same photophysical consequences

Fig. 2

The structures of the molecular automata T-1 and T-2 and the Jablonski diagram depicting processes involved. a L indicates solvent molecules as ligands, water, or acetonitrile. Bodipy and the terpyridyl planes are orthogonal to each other due to the presence of 1,7-dimethyl substitution of the Bodipy chromophore. Double mTEG (methoxytriethyleneglycol) substitution at the boron center enhances water solubility. b Thicker green arrow is absorption, the other green arrows indicate radiative relaxations. Black-dashed arrows are non-radiative relaxation processes. Blue arrows indicate transitions between various excited states. CTS to T₁ transition is particularly enhanced because of the orthogonal geometry of the terpyridyl-Bodipy diad. Energy levels were experimentally estimated using the spectral data (S₁, CTS) in analogy to previous literature, or based on the phosphorescence data for similar Bodipy compounds (T₁)

Fig. 3

Operation of the molecular automata as evidenced by spectroscopic and cell culture data. a MTT assay data: Green open circles correspond to percent death of K562 cells under irradiation at 522 nm for 12 h followed by continued incubation in dark for another 12 h. Solid black circles correspond to cells kept under identical conditions of incubation with the agent, but in dark. Positive control (dashes at 100% line) corresponds to cells incubated in DMSO-growth medium mixture (50/50, v/v). b The truth table with the data and switch inputs, and the corresponding outputs clearly identified, and the particular set of conditions valid under the light irradiation conditions on power-up were highlighted. c Switch input 0 (no added phosphate in the model system or lack of phosphatidylserine (PS) in the external leaflet of the cell membrane in the cell cultures) selects singlet oxygen as the primary output. d The model compound T-2, which is the Zn²⁺ complex of the meso-terpyridyl-bodipy compound, has a very low fluorescence emission intensity in acetonitrile. e The addition of phosphate ions results in a very sharp increase in emission intensity. The low emission intensity is due to the availability of a charge transfer state (CTS) resulting in enhanced intersystem crossing, which in turn leads to efficient generation of singlet oxygen. f The decrease in the absorbance at 411 nm, in an oxygen saturated ethanol solution of selective singlet oxygen trap DPBF (50 µM) in the presence of 2.0 µM T-2 and under irradiation with 522 nm green LED light source (solid blue squares). The singlet oxygen quantum yield (ϕ_Δ) of T-2 is 0.11. The addition of phosphate (red open circles) destabilizes the CTS state, blocking access to the triplet manifold. The absorbance data presented is the net absorbance values obtained by subtracting any background decrease in the probe absorbance due to light alone

Fig. 4

T-1 signals apoptosis by switching to diagnostic mode. a PE-Annexin labels most of the cells incubated with T-1 under light irradiation (Annexin V (+) region of the green area). b Green channel: cells incubated with T-1 under irradiation. c Green channel: cells incubated with T-1 in the dark. d The 2D plot for both green and red channels: T-1 and PE-Annexin V (a specific apoptosis marker) stain the same kind of cells with a large (86%) agreement: 70% of the irradiated cells were co-stained with both T-1 and PE-Annexin V, indicating only apoptotic cells are fluorescently labeled with T-1. e The truth table with input, the switch and the outputs clearly identified, and the particular set of conditions were highlighted. f Switch input (appearance of phosphatidylserine in the external leaflet of the cell membrane) selects fluorescence emission as the primary output. g Cells treated with PE-Annexin V and the T-1, and kept in dark, show no signs of morphological change, and the cellular membranes are not stained with either one of the agents. h, i Cells were incubated with T-1, irradiated with the LED light source, then treated with PE-Annexin V. The two agents (T-1 and PE-Annexin V, green and red, respectively) label the same regions in the cells undergoing apoptosis. (scale bar, 10 μm)

Fig. 5

T-1 induces apoptosis and then switches to diagnostic mode and fluorescently tags apoptotic cells. Blue polar heads represent phosphatidylcholine and sphingomyelins, whereas yellow, pink and purple heads represent phosphatidylserine, phosphatidylinositol and other negatively charged lipids. Apoptosis is accompanied by a loss of membrane asymmetry