Molecular Logic Gates Derived from Fluorescent Natural Products

Abstract

Fluorescent natural products have been a source of fascination for centuries. In this review, we highlight natural products and natural product derivatives as optical logic-based molecules. The early beginnings of the field of molecular logic-based computation are introduced and followed with literature examples of logic gates from fluorescent natural products. The intention is to arouse the curiosity of readers to go out and discover more fluorescent natural products with intrinsic logic properties.

Introduction

The field of molecular logic celebrated its 30th birthday at the beginning of July 2023. ¹ The story goes that the roots of molecularlogic-based computation began in Colombo, Sri Lanka and flowered in Belfast, Northern Ireland, and then spread across the globe.^2–5 Supported by detailed world maps, over 1260 laboratories worldwide are documented to have contributed to the development of molecular logic concepts and examples.^2–12 A Web of Science search using the topic keyword molecular logic gates reveals over 2800 papers dating back from 1994. Considering the volume of published papers, it is rather surprising that of all the reported examples of molecular logic gates, relatively few are derived from natural products. It was mentioned en passant that the anti-malarial quinine, and its cinchona relatives, have the hallmarks of fluorescent pH indicators, recognizable by a fluorophore-spacer-receptor modular system consistent with the photoinduced electron transfer (PET) paradigm.^2,7,13,14 A few laboratories have independently come to the realization, for various reasons, that natural products are a valuable source of fluorescent probes.^15–22 By one estimate, it is believed that Nature has engineered more than 300 intrinsically fluorescent molecules. ¹⁹ An obvious advantage of tapping into this natural reserve is the time saved from reducing or eliminating synthetic effort. Forthcoming in this review, we will highlight fluorescent natural products and related semi-synthetic derivatives with logic-based computational capabilities.^23–32

An Introduction to Molecular Logic Gate Operators

Chemical sensors are devices with an equilibrium measured as a continuous sigmoidal response curve. However, the 2 extremes of the sigmoidal curve have a plateau, which can be analyzed in an off-on or on-off fashion. By setting a defined threshold, the chemical sensors are converted to logic gates able to encode data in binary code as either high (binary 1) or low (binary 0). ²⁷ Hence, the 2 plateaux of the sigmoidal curve cater to the 2 binary digital states.

The off-on and on-off chemical sensors then become YES and NOT logic gates. Generally, the equilibria examined by chemical sensors involve a binding interaction; however, non-binding and physical inputs, such as temperature and pressure, are also useful for specific applications. ¹² Within the field of molecular logic, fluorescence, or some other related form of luminescence, has customarily been the output signal.^23,24,27

Tables 1 and 2 present the truth tables for single-input and many 2-input logic gates, respectively. A YES gate, the basis for a switch, is pictorially presented by a triangle. A NOT gate is represented as a triangle with an open circle at the right tip (or as a solid white dot). PASS 0 and PASS 1 logic functions are trivial from the point of view that the output is independent of the input. However, representing PASS 0 and PASS 1 symbolically requires the integration of 2 logic gate types. PASS 0 is an integration of a NOT (white dot) and an AND gate. PASS 1 is an integration of a NOT (white dot) and an OR gate.

Table 1.

The Truth Tables for Single-Input Single-Output Logic Gates and the Representative Logic Gate Symbols.

Table 2.

The Truth Tables for 8 Double-Input Single-Output Logic Gates and the Representative Logic Gate Symbols.

There are 16 two-input logic gates of which, arguably, the most common 8 are shown in Table 2. The AND logic function is selective and representative of cooperativity. An output of 1 only occurs when both inputs are 1. The NAND gate has the opposite output pattern of 0 only when both inputs A and B are 1. The reader will notice that the NAND gate is an integration of an AND gate plus a NOT gate. The OR logic function represents non-selectivity. An OR gate gives an output of 0 only when both inputs are 0, otherwise the output is 1. The inverse of the OR gate is the NOR gate, which only gives an output of 1 when both inputs are 0. Once again, the NOR logic gate is an integration of an OR gate plus a NOT gate. The (exclusive OR) XOR gate is specific for giving an output of 1 in the case when only 1 of the 2 inputs are present. The XNOR gate is the inverse of XOR with an additional NOT gate giving an output of 1 in the case when both two inputs are the same value. The INH (INHIBIT) logic function is a non-cooperative type of gate that combines the integration of AND and NOT functions, but in an arrangement different from a NAND gate. Only an input of 1 from input B (Table 2) results in an output of 1. However, unlike previous examples, the NOT function operates on just 1 input (in Table 2, input A). The IMP (IMPLICATION) logic function, as the inverse of the INH gate, gives an output of 0 only in the case of a 1 from input B. The IMP gate can be considered an INH gate with an additional NOT gate, like the comparison between an AND gate versus a NAND gate. However, according to De Morgan's theorem a more simplified yet equivalent logic symbol is shown (Table 2) based on an OR gate and only 1 NOT gate at input B rather than an AND gate with 2 NOT gates, 1 at input A, and the other at the output (not shown). From these basic logic gate functions, more complex logic operations consisting of 3 or more inputs can be designed.

In the forthcoming discussion, Boolean logic systems are demonstrated with fluorescent natural products where chemicals are the inputs and emission the output. The reader is encouraged to refer frequently to Tables 1 and 2 as they make their way through the review.

Examples of Fluorescent Natural Product Logic Gates

The macrocyclic diphthalate ester 1 (Figure 1) was isolated from the exuviae of the scorpion Liocheles australasiae. ³³ Before this discovery, the compound was thought to be a synthetic creation. ³⁴ Excitation of the crude extract at 270 nm provides a blue emission about 450 nm. Emission is also observed at 330 nm on excitation at 300 nm. It is speculated that the compound may play a role to protect DNA from solar UV radiation or against parasitic infections. The mass spectrum of the compound shows evidence for complexation with ion peaks at m/z 514 ([M + NH₄]⁺), 519 ([M + Na]⁺), and 535 ([M + K]⁺) in positive mode. This natural macrocyclic 1 brings to mind the fluorescence quenching of dibenzo-18-crown-6 ether in the presence of K⁺ and I⁻, an early example of a NAND logic gate. ³⁵

Figure 1.

Molecules present in scorpions and plants.

Coumarins (benzopyran-2-one) have a broad utility in biological and pharmaceutical applications. ³⁶ The scorpion Mesobuthus martensii is strongly fluorescent under UV light due to the presence of umbelliferone 2 (7-hydroxycoumarin) and 4-methylumbelliferone 3 in the exoskeleton. ³⁷ Coumarins exhibit a large Stokes shift associated to an internal charge transfer (ICT) mechanism, which makes them desirable as a fluorophore. Many can be readily synthesized in an undergraduate laboratory. ³⁸ The phenolic proton within hydroxylated coumarins is suited for exploitation as fluorescent pH and metal ion indicators. ³⁹ Coumarins were exploited by Majumdar within emissive cascade logic systems.^40,41

Coumestrol 4 has 2 phenolic hydrogen atoms with emission at 440 nm.^42,43 It is used in fluorescence assays as a binder to the estrogen receptor (ER). Consideration of 4 as the input, an emission increase is observed on binding to the ER, which would be construed as YES logic. Displacement of 4 from the receptor site by competitive binding by bisphenol A and tamoxifen renders a decrease in fluorescence, which is 1-input NOT logic. The presence of 2 acidic phenolic protons provides an opportunity for the realization of other logic properties.

Reconfigurable logic gates by Pérez-Inestrosa are based on the benzylisoquinoline alkaloid, papaverine 5 (Figure 2).^44,45 The isoquinoline-N-oxide-methylene-arene systems 6 and 7 have PET and ICT mechanisms and a dual locally excited and charge transfer state. Logic gate 6 has 2 protonation sites and 2 charge transfer states. Excitation at < 330 nm demonstrates H⁺, OH⁻-driven INHIBIT logic while observing the emission of the broad locally excited (LE) band at 381 nm. ⁴⁴ The second emitting channel at 500 nm occurs on excitation > 360 nm in agreement with an XOR gate. Emission is not observed when the N-oxide is protonated due to PET from the phenoxyether across the methylene spacer to the isoquinoline or when the phenol is unprotonated. ⁴⁴ From the 2 output channels at 381 nm and 500 nm, a fluorescent half-subtractor operator was demonstrated.

Figure 2.

Isoquinoline-N-oxide-methylene-arene logic gates 6 and 7 inspired from papaverine 5.

The sister molecule 7 has an electron-rich benzo-15-crown-5 ether as the source of the PET. ⁴⁵ Complexation occurs with Zn²⁺ or H⁺ at the N-oxide. The benzocrown readily binds K⁺ and Ba²⁺. Under the ideal conditions, excitation at 400 nm provides an output at 550 nm. PET from the benzocrown to the positively charged acceptor occurs on protonation of the oxime. Gate 7 is multi-functional and reconfigurable as an H⁺-enabled; K⁺-disabled INHIBIT logic gate; an H⁺, K⁺, Ba²⁺ OR-INHIBIT logic gate; and as a H⁺, Zn²⁺, K⁺, NOR-OR logic gate.

Berberine 8 is a charged isoquinoline alkaloid (Figure 3) with various medicinal properties. ⁴⁶ It is used as a light-up fluorescent ligand to monitor pH-driven conformational conversion of intercalated-motif DNA rich in cytosine. ⁴⁷ As a fluorescence probe, 8 is capable of generating a turn-on response to an i-motif structure involving CC+ base pairs. The INHIBIT logic gate is the response to H⁺ and Ag⁺. Ag⁺ stabilizes the cytosine-cytosine mismatch by a C-Ag⁺-C interaction and H⁺ causes folding into the i-motif structure via hemi-protonated base pairs. The DNA complex is pH-insensitive between pH 5.0 and 8.0, so the fluorescence enhancement between berberine and i-motif structures is formed in an acidic solution < pH 5.0.

Figure 3.

Examples of natural products used as components in logic systems.

Curcumin 9 is used as a flavoring and coloring agent and has anti-inflammatory and anticancer properties. ⁴⁸ The fluorescence is green in 5:1 DMSO/H₂O on excitation at 420 nm. ⁴⁹ The emission turns off in the presence of hydroxide due to deprotonation of the phenolic hydrogens, or on addition of Hg²⁺—the outcome is 2-input NOR logic. ⁵⁰

Molecule 10 is a logic gate derivative of curcumin synthesized from benzoyl acetone and 4-hydroxybenzaldehyde. ⁵¹ Sensing is demonstrated for citrulline, a product of the urea cycle for the elimination of ammonia as urea. Addition of citrulline in DMSO/PBS buffer causes the emission at 571 nm to decrease, a typical NOT logic gate response. Rather interestingly, the fluorescent color changes from orange-red to black under 365 nm light, while many other related biomolecules have no effect.

Calcimycin 11 is an antibiotic produced by fermentation of the bacteria Streptomyces chartreusensis. Developed into a fiber optic ion sensor, ⁵² it is a divalent metal ionophore used for transporting such ions across cell membranes, and for in situ calibration of Mg²⁺ or Ca²⁺ fluorescent indicators to equilibrate the intracellular and extracellular metal ion concentrations. Bright fluorescence is observed at 430 nm in TRIS/HCl buffer (pH 7). The presence of either Mg²⁺ or Ca²⁺ causes significant quenching of the fluorescence according to 2-input NOR logic. High H⁺ levels greater than pH 5 also causing quenching on protonation of the carboxylate. Viewed from a logic perspective, 11 emulates 3-input NOR logic. ⁵³

Anabasine is a natural product from the tree tobacco plant consisting of pyridine attached to piperidine. Molecules 12 and 13 (Figure 4) exploit this alkaloid as a dual-proton receptor for fluorescent off–on–off switching with decreasing pH.^54,55 The fluorophore within 12 is a methylumbelliferone derivative of 3. Connected these 2 components made 12 the first PET logic gate engineered from 2 natural products. A semi-natural product analog 13 has anthracene as the fluorophore. Even though the 2 logic gates have the same modular receptors, the pK_as are different at 6.8 and 4.6 in 1 : 1 methanol-water. The electron-withdrawing tendency of the carbonyl group in coumarin brings about this change in pK_a values. The second pK_as of the pyridine nitrogen are 2.8 and 1.8 for 12 and 13, respectively. The 2 different types of nitrogen atoms contribute towards ternary off-on-off logic. ⁵⁶ The addition of a Na⁺-capturing benzo-crown ether at the opposite side of anthracene within 14 introduces an enable-disable switching function, and the first fluorescent switch with 3 PET processes modulated by 3 binding events. ⁵⁴ Subsequent 3-input molecular devices would be exemplified by a reconfigurable 2Na⁺, H⁺-driven/Cs⁺, H⁺-driven AND logic gate and a lab-on-a-molecule for detecting a congregation of 3 cations, Na⁺, H⁺ and Zn²⁺.^57,58

Figure 4.

Off-on-off logic gates based on natural product components.

Canthin-5,6-dione is a shared feature within the fluorescent natural products amarastelline 15 and nigakinone 16 (Figure 5). ⁵⁹ Compound 17 shows an off-on-off ternary logic fluorescence response with increasing pH on sequential addition of acid. ⁶⁰ Emission is observed between pH 7 and pH 10. The parent compound 15 showed moderate fluorescence in chloroform with 2 emission maxima at 432 nm and 616 nm with Stokes shifts up to 258 nm. In phosphate buffer (100 mM, pH 7.4), 17 has a wavelength and quantum yield of 503 nm and 0.081. The compound is brightly fluorescent under 365 nm UV light in many solvents and stains the cytoplasm of HeLa cancer cells. ⁶¹

Figure 5.

Off-on-off logic gates based on canthin-5,6-dione.

The natural product andrographolide was used as a platform for the synthesis 18 (Figure 6) via a terminal alkyne, and subsequently by Cu(I) catalyzed azide-alkyne cycloaddition. ⁶² Irradiated at 343 nm, an intense excimer emission is observed at 482 nm, while a weaker monomer emission appears around 370–400 nm. The emission decreases on addition of Fe³⁺, but not in the presence of other tested metal ions. Tested as a Fe³⁺, EDTA-driven logic gate, the emission is low only on the addition of Fe³⁺. EDTA has no immediate effect on the molecule and chelates Fe³⁺. Deoxycholic acid is the scaffold used for the attachment of 2 coumarins in 19 by click chemistry. ⁶³ The logic gate turns off towards Cu²⁺ and turns on with H₂PO₄⁻ or an amino acid with a [19·Cu²⁺] complex. The Boolean operation in both cases is that of IMPLICATION logic. ⁶⁴

Figure 6.

Natural products as a platform for fluorescent logic gates.

The fascinating blue emission of quinine 20 and quinidine 21 (Figure 7) has long been noted in the primary literature. ⁶⁵ The fluorophore-spacer-receptor model is easily recognizable.^13,14 Now, consider the nitrogen atom of the quinoline as a receptor too and the cinchona alkaloids consist of a receptor₁–fluorophore–spacer–receptor₂ format. ⁶⁶ The nitrogen atom of the 6-methoxyquinoline is receptor₁ and nitrogen atom of the quinuclidine ring is receptor₂ separated by a hydroxylated ethylene spacer. The emission from the diastereomeric pair is bright at pH 2 with a broad peak at 450 nm and a fluorescence quantum yield of 0.55 in water. Halides quench the fluorescence, hence the reason why fluorescence quantum yield measurements with 21 are performed in aqueous sulfuric acid. It is noteworthy to mention that chloride quenching of 6-methoxyquinoline derivatives is a method exploited for measuring blood analyte levels in blood and tears.^67,68 Now combine the possible photophysical responses of the cinchona alkaloids 20–23 to chloride and acid and the outcome are H⁺, Cl⁻-driven INHIBIT logic gates.^69,70 Extend the number of inputs to Br⁻ and I⁻ and the molecules demonstrate combinatorial OR-INHIBIT logic. Furthermore, in aprotic polar solvents such as DMSO the emission is off as the lowest transition is n → π*. In water, the lowest transition is π → π*. Hence, solvent polarity is another possible input. ⁷¹

Figure 7.

The cinchona alkaloids.

The cinchona alkaloids have a vinyl moiety, which can be exploited for polymerization. Quinidine was polymerized with methoxypolyethylene glycol, and quinine and cinchonine with chitosan for use as heterogenous catalysts.^72,73 We prepared a quinidine copolymer by free radical polymerization using an acrylamide subunit. Both the monomer 20 and copolymer 24 (Figure 8) have identical photophysical properties confirming the conservation of modularity. However, the copolymer does have a noticeably lower pK_a than the monomer. A distinct advantage of this approach is the progression towards supramolecular applications. The first copolymer INHIBIT logic gate was a T, H⁺-driven INHIBIT logic gate (T = temperature) with H⁺ as the disabling input. ⁷⁴ Our copolymer 24 emulates H⁺, Cl⁻-driven INHIBIT logic with Cl⁻ as the disabling input. ⁶⁹

Figure 8.

Copolymers of quinidine 24 and quinine 25.

Similarly, copolymers of quinine and quinidine were recently proposed as circularly polarized organic afterglow materials and applied as components to encryption and anti-counterfeiting devices. ⁷⁵ The green afterglow of the films lasts on the order of seconds. The different phosphorescent color, lifetime, and light was used to demonstrate an excitation-dependent encryption application with 254 nm and 365 nm light. The study brings to mind the triple emissive carbon dots prepared from m-phenylenediamine and poly(vinyl alcohol) that are used as an advanced anti-counterfeiting measure in 100 Renminbi banknotes. ⁷⁶

Quinine 21 was copolymerized with trimethylammonium chloride to give cationic polymer 25 (Figure 8). Van Bruggen presented a quinine copolymer as a “trackable delivery systems” for gene therapies, and beneficially the copolymer showed increased drug resistance compared to monomeric quinine. ⁷⁷ The quinuclidine moiety interacts with DNA by electrostatics and intercalation. The observable blue fluorescence was used to directly monitor the DNA-copolymer interaction. Potentiometric titrations of quinine and the 2-hydroxyethyl acetate provide pK_as of 8.5 for the monomer and 6.8 for the copolymer. The lower value for the copolymer due to an electrostatic contribution from the polycation nature. Natural product polymers with chitosan were reported by Schneider as chemomechanical logic gates.^78–80 Future prototypes incorporating luminescent properties are envisioned.

Cytochrome c 26 (Figure 9) is a water-soluble protein associated with the inner membrane of the mitochondria. It contains an iron metal center embedded within a porphyrin and a single tryptophan residue 27, Trp59. Trp59 is normally within close proximity to the heme group, which consequently results in efficient intramolecular fluorescence quenching by energy transfer. Unfolding of the protein by inputs acid, base and urea induces reversibly unfolding of the polypeptide chain and increases the separation distance between the 2 chromophores. By careful selection of 2 of the listed denaturing agents AND, OR (1 : 1 MeOH/H₂O), and XOR are observed from emission at 350 nm. ⁸¹

Figure 9.

The heme subunit of cytochrome c 26, amino acid tryptophan 27, photochrome flindersine 28 and synthetic porphyrins 29–31.

Gentili proposed logic gates from the intermolecular association of flindersine 28 (Figure 9), bovine serum albumin (BSA), and tryptophan. ⁸² Flindersine is a naturally occurring photochrome found in plants of the genus Haplophyllum, while BSA is a mammalian protein, and tryptophan 27, an amino acid. BSA and tryptophan have emissions that overlap with the absorption of 28 allowing for an energy transfer pathway. Thus, NOT logic occurs when BSA is excited at 275 nm in the presence of 28. Temperature (T) is examined as an input and causes an emission decrease in a NOT logic fashion. The net result is that BSA functions as a 28, T-driven NOR logic gate. The thermal back reaction of FL takes about 40 min allowing for resetting capability. The trio of natural products was also examined within the context of fuzzy logic.

Porphyrin-based logic gates 29–31 (Figure 9) have been demonstrated readily in absorbance mode as INHIBIT and XOR gates in DMF, water, and DMSO/water, respectively, using H⁺ and OH⁻ as the inputs.^83–85 Consideration of INHIBIT and XOR logic in parallel at 2 different output channels yields a half-subtractor.^44,83,85,86 Temoporfin [5,10,15,20-tetrakis(3-hydroxyphenyl) chlorin functions as an OR gate for coordinating various metal ions each with a distinctive color. ⁸⁷ Photoactivated at 652 nm, it is used in photodynamic therapy under the brand name Foscan for the treatment of epidermoid carcinoma. An ion-pair digital comparator for F⁻ and K⁺ functions in various optical modes (absorbance, transmittance and fluorescence) as a reconfigurable logic gate. ⁸⁸ INHIBIT and XOR logic gates were interpreted by modulating charge transfer and aggregation chelation quenching switching. Furthermore, porphyrins have been used as key building blocks within all-optical logic gates, as AND and INHIBIT gates, and as an 1 : 2 demultiplexer and keypad lock.^89–92

Conclusions and Perspectives

Molecular logic as a field has required researchers to “[think] outside the box,” and to consider the adage “old molecules, new concepts.”^93,94 Fluorescent natural products are old molecules, which we believe are an untapped sources for new concepts in the field of molecular-logic based computation. The first molecular logic gate was designed and engineered in an organic laboratory, and ever since, so have many thousands of molecular logic gates.^1,8–11,27 However, we hint that many more examples of molecular logic gates derived from Nature are waiting to be discovered. The cinchona alkaloids and quinine were readily available as INHIBIT logic gates before the first molecular proof of an INHIBIT gate.^70,95 The antibiotic calcimycin, a medicinal natural product repurposed for fluorescent imaging and quantification of the alkaline earth metals Ca²⁺ and Mg²⁺ in cells, was demonstrated as a 2-input NOR logic gate before the molecular proof of a NOR gate. ⁵⁰ These fluorescent natural products were known to chemists before the birth of molecular logic, and even before the introduction of mathematical logic by George Boole.^1,96

We conclude by suggesting some strategies for the development of natural product-derived fluorescent logic gates:

The synthetically convenient option is to find fluorescent natural products that can be studied directly by recognizing an architectural design built into the molecule. With the cinchona alkaloids we recognized a receptor₁–fluorophore–spacer–receptor₂ format.

A semi-synthetic approach is to use the fluorescent core of a natural product and perform additional chemical transformations. The isoquinoline-N-oxide-methylene-arene logic gates based on papaverine are an example.

Synthesize molecular logic gates from readily available natural product components. The coupling of a coumarin derivative with anabasine provided a ternary off-on-off logic gate.

We envision that the screening of natural products from a molecular logic viewpoint should result in the discovery of other unnoticed fluorescent switches and logic gates.