Fluorescent molecular logic gates based on photoinduced electron transfer (PET) driven by a combination of atomic and biomolecular inputs

Abstract

Molecular AND logic gates 1, 3, 5 and 7, which are designed according to principles of photoinduced electron transfer (PET) switching, respond to co-existing Candida antarctica lipase B and H⁺ (and Na⁺).

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Molecular logic-based computation^1–4 requires gates to process and store information. Besides its ability to operate in biocompatible micrometric spaces, the diversity of information available to molecular logic distinguishes it from its semiconductor cousin which employs voltage information only. For instance, the inputs feeding gates can take the form of physical entities (e.g. light dose,⁵ temperature⁶), chemical species (e.g. atomic,¹ molecular⁷) and biochemical species (e.g. nucleotides,⁸ enzymes³). However, there are hardly any examples of combined atomic and enzyme inputs in the literature, if at all.

Fluorescent PET switches grew out of the sensing literature.⁹ Although atomic inputs were present from the beginning,¹⁰ protein inputs were incorporated only recently.¹¹ Even these covered only some receptor- and transport-proteins.§ A way to incorporate hydrolase enzymes was described by Ojida et al.¹² and us.¹³ Here, a fluorescent PET system based on a ‘fluorophore-spacer-amine’ format relied on the upward shift of the amine's pK_a value by ∼2 pH units upon hydrolyzing a neighbouring ester into a carboxylate anion. Now we show how such systems can serve as fluorescent molecular logic gates driven by atomic ions, H⁺ and Na⁺, and a hydrolase enzyme, Candida antarctica lipase B (CALB).

Logic gate 1¶ is synthesized by nucleophilic substitution of 4-bromomethyl-7-methoxycoumarin with sarcosine ethyl ester. 1 is a typical fluorescent PET ‘off–on’ switch of the ‘fluorophore-spacer–receptor’ format⁸ with H⁺ being the input. By itself, the 7-methoxycoumarin fluorophore has no significant interaction with H⁺ in the pH range of our experiments since it lacks a suitable receptor.¹⁴1's pH-dependent fluorescence intensity (I_F) is analysed according to eqn (1)¹⁴ to give pK_a = 3.6. The fluorescence quantum yields are Φ_Fmax = 0.97 and Φ_Fmin = 0.02.

log[(I_Fmax − I_F)/(I_F − I_Fmin)] = pH − pK_a (1)

The hydrolysis product of 1, which is 2 (shown in Scheme 1 as the carboxylate form owing to the operational pH range of 6–10 for gate tests), is tested similarly and yields pK_a = 6.7. The fluorescence quantum yields of 2 are Φ_Fmax = 0.48 and Φ_Fmin = 0.01. The lower Φ_Fmax value of 2 (cf.1) is due to the three-atom linker folding the carboxylate unit over the fluorophore¹⁵ to allow intramolecular interaction in the excited state.¹⁶ All these parameters for all compounds studied are collected in Table 1.

Scheme 1 Structures of logic gates 1, 3, 5 and 7 and their hydrolysis products 2, 4, 6 and 8 respectively.

Table 1 Parameters obtained for 1–8via steady-state and time-dependent fluorescence spectroscopy^a

Gate	pKa	Φ max	Φ min	K M (10^−5 M)	V max (10^−9 M s^−1)
a In water : methanol (4 : 1, v/v), except for 7 and 8 where water : DMSO (1 : 1, v/v) is used. b Not determined, but all these values are expected to be close to those of 6. c At 1.0 M Na^+. d At ∼0.0 M Na^+.
1	3.6	0.97	0.02	1.9	0.50
2	6.7	0.48	0.01	—	—
3	4.5	0.56	0.05	1.1	7.5
4	—^b	—^b	—^b	—	—
5	4.5	0.57	0.07	1.6	97
6	7.1	0.20	0.01	—	—
7	5.8^c	0.18,^c 0.16^d	0.09^c	0.22^c	0.75^c
8	8.8^c	0.15,^c 0.12^d	0.06^c	—	—

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1 was subjected to an enzyme screen to see which enzyme would hydrolyze 1 most efficiently to 2 at pH 7 (Fig. 1). CALB was found to be the most efficient, which is gratifying since CALB is known¹⁷ to hydrolyze a variety of esters over a wide pH range. Additionally, it was found that the hydrolysis reaction could be most conveniently followed by the fluorescence emission signal (Fig. 1). Thus we realize that 1 becomes a ‘fluorophore–spacer₁–receptor–spacer₂–enzyme substrate’ system.

Fig. 1 The influence of various enzymes on the % yields of the hydrolysis product of 1, i.e.2, and their corresponding fluorescence output after exposure for 12 h at pH 7. Total fluorescence intensity monitoring conducted with λ_exc = 326 nm. Enzyme numbers are shown in red. Enzymes: 1. Candida cylindracea lipase C1, 2. Candida cylindracea lipase C2, 3. Rhizopus oryzae lipase, 4. Achromobacter spp. lipase, 5. Alcaligenes spp. lipase, 6. Pseudomonas cepacia lipase, 7. Pseudomonas stutzeri lipase, 8. Rhizopus spp. lipase, 9. Rhizopus niveus lipase, 10. Aspergillus niger lipase, 11. Alcaligenes spp. lipase, 12. Pseudomonas cepacia lipase P2, 13. Mucor javanicus lipase, 14. Penicillium camambertii lipase, 15. Rhizopus oryzae lipase, 16. Rhizopus niveus protease, 17. Bacillus stearothermophilus protease, 18. Aspergillus oryzae protease, 19. Aspergillus melleus protease, 20. Bacillus subtilis protease, 21. Aspergillus spp. aminoacylase, 22. Penicillium fluoroscens lipase, 23. Candida antarctica B lipase, 24. Mucor meihei lipase, 25. Candida antarctica A lipase, 26. Fiscus spp. ficin, 27. Bromeliaceae bromelain, 28. Carica papaya papain, 29. Candida antarctica (immob) lipase B, 30. Porcine pancrease, type II, 31. Bacillus lentus protease, 32. Bacillus lentus (immob) protease, 33. Bacillus lincheniformis protease, 34. Bacillus lincheniformis (immob) protease, 35. Porcine pancrease grade II, 36. Pig liver esterase, 37. Penicillin acylase. 38. Alpha amylase. Data point 39 is pure 2.

The fluorescence spectra of 1 at pH 6 and 10 with/without CALB exposure for 30 min are shown in Fig. 2a. The AND logic response of the fluorescence signal is clear since both H⁺ and CALB are needed to elicit a ‘high’ fluorescence response from 1. When exposed to CALB at pH 6, 1 gradually hydrolyzes to 2 and shows a gradual increase in fluorescence intensity. The 1–CALB interaction is governed by a Michaelis constant K_M of 1.9 × 10⁻⁵ M and V_max value of 5.0 × 10⁻¹⁰ M s⁻¹. These values are obtained by applying eqn (2) to convert the rate of change of fluorescence intensity into the rate of change of the product 2 concentration, followed by the application of eqn (3).¹⁸

V = d(2)/dt = [dI_F/dt]·(1)_t=0/{[(Φ_F2/Φ_F1) − 1]·I_Ft=0} (2)

1/V = [(K_M/V_max)/(1)] + 1/V_max (3)

Fig. 2 (a) Fluorescence spectra of 7.3 × 10⁻⁶ M 1 under stimulation with H⁺ and CALB inputs for 30 min in water:methanol (4 : 1, v/v) (λ_exc = 326 nm) at 20 °C where ‘high’ and ‘low’ H⁺ correspond to pH 6 and 10 respectively and ‘high’ and ‘low’ CALB correspond to 7.1 × 10⁻⁶ M and 0 M respectively. Input states (H⁺, CALB) of (0,0), (1,0), (0,1) and (1,1) are shown in blue, red, green and purple respectively. The ‘high’ level of H⁺ input, 10⁻⁶ M, is chosen from the pH value at which maximum discrimination of the fluorescence signals between 1 and 2 is seen in (b). (b) Φ_F of 1 (black) and 2 (orange) as a function of pH. (c) Pictorial version of truth table showing Φ_F values extracted from (a). Output threshold chosen at Φ_F = 0.13.

It is to be noted that the enzyme reaction is irreversible under our experimental conditions so that the logic device is suitable only for single-use situations. Such single-use situations are commonly present in the medical diagnostics sphere, as seen with two¹⁹- or three²⁰ (Scheme 1).

Owing to the diversity available in inputs, outputs, power supplies and devices within molecular logic, several routes to reconfigurability have become available.² However, we are not aware of any cases in the primary literature where logic is reconfigured by changing molecular configuration. Since the enantiodiscrimination of enzymes is well-established, we now have an opportunity to present such an approach.

The enantiomeric pair of logic gates 3¶ and 5¶ arise from a synthesis analogous to that of 1. The hydrolysis products of these are 4 and 6 respectively, although only 6 was available for pK_a determination. pK_a values are measured for these compounds with the aid of eqn (1), as done for 1 and 2. The values obtained for 3, 5 and 6 are 4.5, 4.5 and 7.1 respectively. The pK_a value of 4 is expected to be the same as that found for its opposite enantiomer 6, i.e. 7.1.

As seen in Fig. 3a, the fluorescence spectra of 3 at pH 6 and 10 with/without CALB exposure for 30 min correspond to a PASS 0 logic action. On the other hand, Fig. 3b shows an AND logic action for 5. 3 and 5 differ in the configuration of the functional groups around the asymmetric carbon. Thus, logic reconfiguring is achieved by changing the molecular configuration of the device. At pH 6, K_M values are not very different, i.e. 1.1 × 10⁻⁵ and 1.6 × 10⁻⁵ M, for 3 and 5 respectively. However, V_max values differ significantly, i.e. 7.5 × 10⁻⁹ and 9.7 × 10⁻⁸ M s⁻¹, as a result of CALB's enantioselectivity.

Fig. 3 (a) Fluorescence spectra of 7.3 × 10⁻⁶ M 3 under stimulation with H⁺ and CALB inputs for 30 min in water : methanol (4 : 1, v/v) (λ_exc = 326 nm). ‘High’ and ‘low’ input levels are as in Fig. 2. Input states are coloured as in Fig. 2. The ‘high’ level of H⁺ input, 10⁻⁶ M, is chosen from the pH value at which maximum discrimination of the fluorescence signals between 5 and 6 is seen in (c). (b) Same as in (a) but for 5 instead of 3. (c) Φ_F of 5 (black) and 6 (orange)as a function of pH. (d) Pictorial version of truth table showing Φ_F values extracted from (a) and (b). Left-hand bars of each pair correspond to 3 whereas right-hand bars correspond to 5. Output threshold chosen at Φ_F = 0.13.

We have explored the modularity of our design by building a prototype 3-input AND gate 7¶ of the ‘receptor₁–spacer₁–fluorophore–spacer₂–receptor₂–spacer₃–enzyme substrate’ format, which is driven by CALB, H⁺ and Na⁺. The sensitivity of logic gate 7's fluorescence to Na⁺, cf. that of 1, arises from the new benzo-18-crown-6 ether functional group within 7.

7 is synthesized by reacting a known anthracene-crown ether conjugate²¹ with sarcosine ethyl ester. Its AND logic behaviour is shown in Fig. 4a and c. Its pK_a = 5.8 (at 1.0 M Na⁺) in water : DMSO (1 : 1, v/v). 8, the hydrolysis product of 7, has pK_a = 8.8 under the same conditions. The log β_Na⁺ values for 7 and 8 are 0.7 and 0.8 respectively (at pH 4.5). The 7–CALB interaction is characterized at pH 7 and at 1.0 M Na⁺ by K_M = 2.2 × 10⁻⁶ M and V_max = 7.5 × 10⁻¹⁰ M s⁻¹. This proof of principle study does not examine selectivity issues with respect to other metal ions.

Fig. 4 (a) Fluorescence spectra of 7.3 × 10⁻⁶ M 7 under stimulation with H⁺, Na⁺ and CALB inputs for 30 min in water : DMSO (1 : 1, v/v) (λ_exc = 378 nm). ‘High’ and ‘low’ input levels for H⁺ and CALB are as in Fig. 2, except that ‘high’ H⁺ is 10⁻⁷ M, with ‘high’ and ‘low’ Na⁺ levels being chosen as 1.0 and 0 M respectively. Input states are coloured as in Fig. 1 for ‘high’ Na⁺, while those with ‘low’ Na⁺ are shown in the same colours but with 50% transparency. The ‘high’ level of H⁺ input, 10⁻⁷ M, is chosen from the pH value at which maximum discrimination of the fluorescence signals between 7 and 8 is seen in (b). (b) Φ_F of 7 (black) and 8 (orange) as a function of pH in the presence of 1.0 M Na⁺. (c) Pictorial version of truth table showing Φ_F values extracted from (a). Output threshold chosen at Φ_F = 0.13.

We conclude that the fluorescent PET sensing/switching design is a useful starting point for constructing tailored molecular logic systems which employ mixed inputs from the chemical and biological spheres, especially when the latter concerns a hydrolase enzyme. Such systems are unique when compared with previously developed AND and other logic gates.^2,3 This approach also allows demonstration of logic reconfiguring by changing the molecular configuration of the logic device.

We acknowledge the Department of Employment and Learning, Northern Ireland and T. J. Lively for support and help.

Note added in proof: Fluorescent PET probes for some oxidoreductase proteins are also available.§²²

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnotes

† In honour of Professor Eric Anslyn's 60th Birthday.

‡ Electronic supplementary information (ESI) available: Synthesis procedures and characterization details for all compounds. See DOI: 10.1039/d0cc00478b

§ See note added in proof.

¶ 1; ¹H NMR (CDCl₃): δ 1.30 (t, 3H, OCH₂CH̲₃, J = 7 Hz), 2.44 (s, 3H, NCH̲₃), 3.38 (s, 2H, ArCH̲₂N), 3.84 (s, 2H, NCH̲₂CO), 3.89 (s, 3H, OCH̲₃), 4.21 (q, 2H, OCH̲₂CH₃, J = 7 Hz), 6.35 (s, 1H, CH̲CO), 6.83 (m, 1H, ArH̲), 6.87 (m, 1H, ArH̲), 7.90 (d, 2H, ArH̲, J = 9 Hz). ¹³C NMR (CDCl₃): δ 41.1, 41.5, 56.8, 58.5, 100.9, 111.8, 112.7, 112.8, 126.8, 151.9, 156.0, 162.2, 163.7, 172.0. MS(ES): 306.1341 [M + H⁺]. Calculated m/z for C₁₆H₂₀NO₅⁺, 306.1355.

3; ¹H NMR (CDCl₃): δ 1.38 (d, 3H, C(CH̲₃)H, J = 7 Hz), 1.86 (s, 1H, NH̲), 3.44 (q, 1H, C(CH₃)H̲, J = 7 Hz), 3.77 (s, 3H, CO₂CH̲₃), 3.87 (s, 3H, OCH̲₃), 3.89 (dd, 2H, CH̲₂NH, J = 16, 107 Hz), 6.43 (s, 1H, CH̲CO), 6.82 (m, 1H, ArH̲), 6.86 (m, 1H, ArH̲), 7.58 (d, 1H, ArH̲, 9 Hz). ¹³C NMR (CDCl₃): δ 17.4, 45.8, 50.1, 53.8, 54.4, 99.1, 108.5, 110.1, 110.4, 123.2, 151.6, 153.6, 159.6, 160.7, 174.0. MS(ES): 292.1170 [M + H⁺]. Calculated m/z for C₁₅H₁₈NO₅⁺, 292.1185.

5; ¹H NMR (CDCl₃): δ 1.38 (d, 3H, C(CH̲₃)H, J = 7 Hz), 1.86 (s, 1H, NH̲), 3.44 (q, 1H, C(CH₃)H̲, J = 7 Hz), 3.77 (s, 3H, CO₂CH̲₃), 3.87 (s, 3H, OCH̲₃), 3.89 (dd, 2H, CH̲₂NH, J = 16, 107 Hz), 6.43 (s, 1H, CH̲CO), 6.82 (m, 1H, ArH̲), 6.86 (m, 1H, ArH̲), 7.58 (d, 1H, ArH̲, 9 Hz). ¹³C NMR (CDCl₃): δ 17.4, 45.8, 50.1, 53.9, 54.4, 99.1, 108.6, 110.1, 110.4, 123.2, 151.5, 153.7, 159.6, 160.7, 174.0. MS(ES): 292.1182 [M + H⁺]. Calculated m/z for C₁₅H₁₈NO₅⁺, 292.1185.

7; ¹H NMR (CDCl₃): δ 1.29 (t, 3H, CH₂CH̲₃, J = 7 Hz), 2.49 (s, 3H, NCH̲₃), 3.43 (s, 2H, NCH̲₂), 3.50–3.90 (m, 16H, CH̲₂O), 4.19 (q, 2H, OCH̲₂CH₃, J = 7 Hz), 4.73 (2H, s, AnthCH̲₂N), 4.88 (s, 2H, AnthCH̲₂Ar), 6.46–6.57 (3H, m, ArH̲), 7.48 (m, 4H, AnthH̲), 8.19 (d, 2H, AnthH̲, J = 9 Hz), 8.61 (d, 2H, AnthH̲, J = 9 Hz). ¹³C NMR (CDCl₃): δ 14.7, 35.0, 45.1, 55.0, 60.6, 69.4, 70.0, 70.9, 71.4, 114.3, 114.9, 117.4, 121.2, 122.5, 124.0, 125.8, 127.3, 129.0, 140.9, 141.4, 171.7. MS(ES): 588.2994 [M + H⁺]. Calculated m/z for C₃₅H₄₂NO₇⁺, 588.2961.

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