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

Glenn D. Wrighta, Chao-Yi Yaoa, Thomas S. Moodyb and A. Prasanna de Silvaa
aSchool of Chemistry and Chemical Engineering, Queen's University, Belfast BT9 5AG, Northern Ireland, UK. E-mail: a.desilva@qub.ac.uk
bAlmac Sciences, Craigavon BT63 5QD, Northern Ireland, UK

Received 18th January 2020 , Accepted 5th May 2020

First published on 5th May 2020


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+).


Molecular logic-based computation1–4 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 pKa 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.14 1's pH-dependent fluorescence intensity (IF) is analysed according to eqn (1)14 to give pKa = 3.6. The fluorescence quantum yields are ΦFmax = 0.97 and ΦFmin = 0.02.

 
log[(IFmaxIF)/(IFIFmin)] = pH − pKa (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 pKa = 6.7. The fluorescence quantum yields of 2 are ΦFmax = 0.48 and ΦFmin = 0.01. The lower ΦFmax 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.


image file: d0cc00478b-s1.tif
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 1–8 via steady-state and time-dependent fluorescence spectroscopya
Gate pKa Φmax Φmin KM (10−5 M) Vmax (10−9 M s−1)
a In water[thin space (1/6-em)]:[thin space (1/6-em)]methanol (4[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v), except for 7 and 8 where water[thin space (1/6-em)]:[thin space (1/6-em)]DMSO (1[thin space (1/6-em)]:[thin space (1/6-em)]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 5.8c 0.18,c 0.16d 0.09c 0.22c 0.75c
8 8.8c 0.15,c 0.12d 0.06c


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.


image file: d0cc00478b-f1.tif
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 λexc = 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 KM of 1.9 × 10−5 M and Vmax 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 = [dIF/dt]·(1)t=0/{[(ΦF2/ΦF1) − 1]·IFt=0} (2)
 
1/V = [(KM/Vmax)/(1)] + 1/Vmax (3)


image file: d0cc00478b-f2.tif
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[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) (λexc = 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) ΦF of 1 (black) and 2 (orange) as a function of pH. (c) Pictorial version of truth table showing ΦF values extracted from (a). Output threshold chosen at ΦF = 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 pKa determination. pKa 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 pKa 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, KM values are not very different, i.e. 1.1 × 10−5 and 1.6 × 10−5 M, for 3 and 5 respectively. However, Vmax values differ significantly, i.e. 7.5 × 10−9 and 9.7 × 10−8 M s−1, as a result of CALB's enantioselectivity.


image file: d0cc00478b-f3.tif
Fig. 3 (a) Fluorescence spectra of 7.3 × 10−6 M 3 under stimulation with H+ and CALB inputs for 30 min in water[thin space (1/6-em)]:[thin space (1/6-em)]methanol (4[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) (λexc = 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) ΦF of 5 (black) and 6 (orange)as a function of pH. (d) Pictorial version of truth table showing ΦF 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 ΦF = 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 pKa = 5.8 (at 1.0 M Na+) in water[thin space (1/6-em)]:[thin space (1/6-em)]DMSO (1[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v). 8, the hydrolysis product of 7, has pKa = 8.8 under the same conditions. The log[thin space (1/6-em)]βNa+ 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 KM = 2.2 × 10−6 M and Vmax = 7.5 × 10−10 M s−1. This proof of principle study does not examine selectivity issues with respect to other metal ions.


image file: d0cc00478b-f4.tif
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[thin space (1/6-em)]:[thin space (1/6-em)]DMSO (1[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) (λexc = 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) ΦF 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 ΦF values extracted from (a). Output threshold chosen at ΦF = 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, OCH2C[H with combining low line]3, J = 7 Hz), 2.44 (s, 3H, NC[H with combining low line]3), 3.38 (s, 2H, ArC[H with combining low line]2N), 3.84 (s, 2H, NC[H with combining low line]2CO), 3.89 (s, 3H, OC[H with combining low line]3), 4.21 (q, 2H, OC[H with combining low line]2CH3, J = 7 Hz), 6.35 (s, 1H, C[H with combining low line]CO), 6.83 (m, 1H, Ar[H with combining low line]), 6.87 (m, 1H, Ar[H with combining low line]), 7.90 (d, 2H, Ar[H with combining low line], 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(C[H with combining low line]3)H, J = 7 Hz), 1.86 (s, 1H, N[H with combining low line]), 3.44 (q, 1H, C(CH3)[H with combining low line], J = 7 Hz), 3.77 (s, 3H, CO2C[H with combining low line]3), 3.87 (s, 3H, OC[H with combining low line]3), 3.89 (dd, 2H, C[H with combining low line]2NH, J = 16, 107 Hz), 6.43 (s, 1H, C[H with combining low line]CO), 6.82 (m, 1H, Ar[H with combining low line]), 6.86 (m, 1H, Ar[H with combining low line]), 7.58 (d, 1H, Ar[H with combining low line], 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(C[H with combining low line]3)H, J = 7 Hz), 1.86 (s, 1H, N[H with combining low line]), 3.44 (q, 1H, C(CH3)[H with combining low line], J = 7 Hz), 3.77 (s, 3H, CO2C[H with combining low line]3), 3.87 (s, 3H, OC[H with combining low line]3), 3.89 (dd, 2H, C[H with combining low line]2NH, J = 16, 107 Hz), 6.43 (s, 1H, C[H with combining low line]CO), 6.82 (m, 1H, Ar[H with combining low line]), 6.86 (m, 1H, Ar[H with combining low line]), 7.58 (d, 1H, Ar[H with combining low line], 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, CH2C[H with combining low line]3, J = 7 Hz), 2.49 (s, 3H, NC[H with combining low line]3), 3.43 (s, 2H, NC[H with combining low line]2), 3.50–3.90 (m, 16H, C[H with combining low line]2O), 4.19 (q, 2H, OC[H with combining low line]2CH3, J = 7 Hz), 4.73 (2H, s, AnthC[H with combining low line]2N), 4.88 (s, 2H, AnthC[H with combining low line]2Ar), 6.46–6.57 (3H, m, Ar[H with combining low line]), 7.48 (m, 4H, Anth[H with combining low line]), 8.19 (d, 2H, Anth[H with combining low line], J = 9 Hz), 8.61 (d, 2H, Anth[H with combining low line], 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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