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. 2024 May 10;7(6):1839–1846. doi: 10.1021/acsptsci.4c00058

Deuteration-Driven Photopharmacology: Deuterium-Labeled AzoCholine for Controlling Alpha 7 Nicotinic Acetylcholine Receptors

Xingye Yang †,‡, Xin Zhou †, Xiaojun Qin ‡, Dong Liang †, Xuhui Dong †, Huimin Ji †, Siman Wen ‡, Lupei Du †, Minyong Li †,§,*
PMCID: PMC11184602  PMID: 38898952

Abstract

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Photopharmacology is a powerful approach to investigate biological processes and overcomes the common therapeutic challenges in drug development. Enhancing the photopharmacology properties of photoswitches contributes to extend their applications. Deuteration, a tiny structural modification, makes it possible to improve the photopharmacology and photophysical properties of prototype compounds, avoiding extra complex chemical changes or constructing multicomponent systems. In this work, we developed a series of D-labeled azobenzenes to expand the azobenzene photoswitchable library and introduced the D-labeled azobenzene unit into the photoagonist of α7 nicotinic acetylcholine receptors (α7 nAChRs) to investigate the effects of deuteration in photopharmacology. Spectral data indicated that deuteration maintained most of the photophysical properties of azobenzenes. The D-labeled photoagonist exhibited good control of the activity of α7 nAChRs than the prototype photoagonist. These results confirmed that deuteration is a promising strategy to improve the photopharmacological properties.

Keywords: deuteration, photopharmacology, photoswitch, α7 nicotinic acetylcholine receptors, d9-AzoCholine, azobenzene

Since the discovery of deuterium in the 1930s,1 the deuterium isotope effect (DIE) has attracted researchers’ attention. DIE is attributed to the large difference in the atomic mass and nuclear spin between protium and deuterium. Numerous physiological processes would be altered with deuteration, such as proliferation,2 apoptosis,3 and metabolism.4 Based on DIE, deuterium substitution can effectively change metabolic stability, efficacy, and distribution patterns of parent molecules.5,6 The low toxicity of deuterated compounds attracts medicinal chemists to develop deuterated probes and new chemical entities7,8 for tracking in a broad range of chemical and biological disciplines and applying in pharmacology and medicinal chemistry areas,9,10 such as deuterated tyramine,11 avadomide,12 paroxetine,13 methylxanthine,14 and ivacaftor.15 Among multitudinous deuterated compounds, deutetrabenazine16 has been approved by FDA, and donafenib,17 VV116,18 and deucravacitinib19 have been approved by CFDA.

Photoswitchable molecules have been widely used to regulate pharmacological activities,20 investigate mechanisms21 and modulate functions of materials22 via photoisomerization. In photopharmacology, the characteristic of ultrahigh spatiotemporal resolution makes it possible to precisely regulate the pharmacological activity of bioactive molecules.23−27 Azobenzenes, spiropyrans, diarylethenes, and stilbene-like compounds are typically photoswitchable units.28 Among them, azobenzene photoswitches are broadly applied in photopharmacology with their structural stability, modifiability, and photomanipulation. Chemists and medicinal chemists are committed to enhancing isomerization yield, rate, biocompatibility, and antiphotodegradation to improve the characteristics of azobenzene.29−34 Supramolecular approach, which relied on a combination of a macrocyclic host and a photosensitizer to selectively bind and sensitize E-azobenzenes for isomerization,35ortho fluoro/chloro/bromo/methoxy/dimethylamino substitution,36,37 tetra-ortho-fluoro-azobenzene derivatives,38 bridged azobenzene,39 and azoheteroarene22 have been developed and applied in vitro and in vivo. These approaches may indeed improve the photophysical characteristics of azobenzenes, like red-shifting photoisomerization wavelength or increasing photo-induced ratios and rates. However, inappropriate modifications may lead to decreased optical activities or even deactivation. The deuterium strategy is a minor modification, which scarcely affects the molecular structure. It could be applied to enhance the pharmacological properties of bioactive molecules5 and photophysics properties of fluorophores,40−42 photoswitches,43 and luminophores.44 Trauner and co-workers45 have confirmed that the activity of α7 nicotinic acetylcholine receptors (α7 nAChRs) could be regulated by photoligands. In this work, we developed a deuterated photoagonist and applied it to optically control the activity of α7 nAChRs in cells and behaviors of nematodes to investigate the effects of deuterium substitution on photophysical and photopharmacological properties of photoswitches.

Results and Discussion

First, we designed and synthesized five forms of deuterated azobenzenes to investigate their photophysical properties (Scheme 1). In this work, we developed a novel and simple deuterated method without complex catalytic agents46 or conditions.47,48 Silver oxide catalyzed and oxidized the C–B bond and formed the C–D bond using potassium phosphate as the base in the mixed solvents of ethanol-d6 and D2O at ambient temperature. Following this synthetic method, the monodeuterium was introduced at the ortho site of aniline (Scheme S1). d7-Aniline was obtained from commercial sources. The general synthetic route of azobenzene is shown in Scheme 1a. Aniline was oxidized by oxone in CH2Cl2/H2O, forming nitrosobenzene compounds. Another aniline coupled with nitrosobenzene compounds by the Mills reaction obtained azobenzene photoswitches. The synthesized details are shown in the Supporting Information.

Scheme 1. (a) General Synthetic Route of Azobenzene and Deuterated Azobenzenes; (b) The Structure of Azobenzene and Five Forms of Deuterated Azobenzenes.

Scheme 1

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Reaction conditions: (1) oxone, DCM/H2O, r.t, yield was calculated as 100% without purification. (2) CH3COOH, r.t, 72.57 (3), 33.04 (4), 40.49 (5), 78.94 (6), 26.63 (7), and 67.07% (8).

We then examined the photophysical properties of deuterated azobenzenes. The UV–vis spectroscopic data showed that thermal state isomers of deuterated azobenzenes were trans isomers. The trans isomers were photoinduced to cis isomers upon 365 nm irradiation, reaching 365 nm photostationary state (PSS) (Figures 1a and S1–S6). The cis isomers reverted to trans isomer upon 415, 455, and 530 nm irradiation reaching 415, 455, and 530 nm PSS. 415 nm PSS was found to produce a higher fraction of trans isomer than 455 and 530 PSS. Therefore, the optimal isomerization wavelengths were 365 and 415 nm (Figures 1a and S1–S6). The UV–vis spectroscopic data are shown in Table 1. It was discovered that the trans to cis photoisomerization rates of deuterated azobenzenes were strongly dependent on the polarity of solvents (Figure S7a and Table 1).

Figure 1.

Figure 1

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(a) UV–vis spectra of azobenzene and deuterated azobenzenes (100 μM in acetonitrile). The structures of the azobenzenes were inserted in each spectrum. The spectrum respectively presented dark PSS (blue lines), 365 nm PSS (red lines), and 415 nm PSS (green lines) under dark, 365, and 415 nm irradiation for reaching PSS. 365 and 415 nm wavelengths were optimal wavelengths for photoinduced isomerization. (b) The cis/trans ratios of azobenzene and deuterated azobenzenes (0.2 M in MeOH-d4) at 365 nm PSS (left) and 415 nm PSS (right) were measured by NMR. n = 3, data were shown as mean ± SEM, two-tailed unpaired t-test, and no significant (P > 0.05) were observed between 3 and 4–8. Advanced Four-Channel LED Driver DC4104 (THORLABS) equipped with 365 and 415 nm LEDs, the optical densities were 0.753 and 0.736 W/cm2 under experimental conditions, respectively.

Table 1. UV–Vis Spectral Data of Azobenzene and Deuterated Azobenzenesa.

compd π → π*
 n →π*
 t1/2
  λmax (trans) (nm) λmax (cis) (nm) Δλπ→π* (nm) λmax (trans) (nm) λmax (cis) (nm) Δλn→π* (nm) t1/2trans→cis (s) t1/2cis→trans (s)
3a 319 316 3 448 440 8 109.4 26.4
3b 317 314 3 435 429 6 111.4 27.4
3c 317 317 0 436 436 0 57.7 25.7
4a 319 316 3 448 440 8 96.2 17.3
4b 317 313 4 442 431 11 78.6 31.0
4c 317 315 2 437 430 7 62.9 23.2
5a 319 316 3 443 437 6 102.2 23.5
5b 316 314 2 442 431 11 71.3 20.5
5c 317 317 0 434 434 0 69.7 23.4
6a 319 316 3 448 439 9 127.3 27.2
6b 317 313 4 442 431 11 63.7 25.3
6c 317 317 0 435 429 6 62.4 27.3
7a 319 308 11 443 437 11 107.0 24.9
7b 317 313 4 443 431 12 91.3 25.4
7c 317 314 3 443 438 5 59.8 21.1
8a 318 316 2 448 438 10 89.3 20.8
8b 316 313 3 443 431 12 75.0 23.1
8c 315 314 1 440 436 4 68.8 21.4
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a

The spectral data of compounds 3–8 (100 μM) was performed in CCl4a, MeOHb and CH3CNc. Dark PSS (trans isomers) were converted into 365 nm PSS (cis isomers) upon 365 nm irradiation, and 365 nm PSS (cis isomers) reverted to 415 nm PSS (trans isomers) upon 415 nm irradiation. λmax: the maximum absorption wavelength at corresponding PSS. Δλπ→π*: the difference between λmax (trans) and λmax (cis) at the π → π* band. Δλn→π*: the difference between λmax (trans) and λmax (cis) at the n → π* band. t1/2: photoisomerization half-lives. trans → cis: trans isomer to cis isomer. cis → trans: cis isomer to trans isomer. The optical densities of 365 and 415 nm LED lights (THORLABS) were set as 0.753 and 0.736 W/cm2.

The dependence was not observed for cis to trans (Figure S7b and Table 1). It may be attributed to a dipolar transition state of switching process.49,50 The photoisomerization rates of deuterated azobenzene were obviously increased in MeOH (Figure S7a,b). Due to the larger separation of n–π*, the higher isomerization ratios are found.36,37,51,52 The π–π* and n–π* separations of compounds 3–8 are intuitively present in Figure S8 and Table 1. The cis/trans ratios, analysis referred to the peak point, showed that photoisomerization ratios were also dependent on the polarity of solvents (Figure S7c,d).53 The n–π* separations of 6–8 were slightly large relative to classic azobenzene in CCl4, MeOH, and CH3CN, which indicated that photoisomerization ratios of multiple deuterium substitutions were a minor improvement (Figure S8). To accurately represent photoisomerization ratios, NMR was used to character quantify trans and cis. NMR data confirmed that cis/trans ratios of deuterated azobenzenes have an increase tendency upon 365 nm irradiation (Figures 1b and S9–S14). According to the suggestions by Bunce and Zerner,53ortho deuterium substitution lowered n–π* energy of the cis isomer, which increased cis fraction at trans to cis with light. The shorter C–D bond length and reduced vibrations of C–D may also contribute to raise cis fraction upon 365 nm irradiation.54−56 Therefore, we inferred that more deuterium substitution numbers were profitable for increasing photoisomerization ratios. However, even if all numbers of hydrogen of azobenzene were substituted by deuterium, no significant improvement of cis proportion was observed at 365 and 415 nm PSS. The thermal isomerization kinetics cis to trans of 3–8 were tracked by recording UV–vis at 60 °C (Figure S15). The results showed that deuterium substitution slightly shortened the thermal half-lives of azobenzene. The UV–vis and NMR data indicated that deuterium substitution, the most conservative modification of azobenzene, maintained most of the photophysical properties of azobenzene.

Based on DIE, we envisaged that introducing a deuterated azobenzene unit into photoligands enabled the enhancement of their photopharmacological properties. Considering the effects of deuteration on the photophysical behavior of azobenzene, the difficulty for preparing deuterated compounds, and uncertain deuteration position for DIE, the azobenzene unit of the photoligand was replaced with total deuterium substitution azobenzene. We focused our attention on photoagonists of nAChRs. Disorders of nAChRs may cause mental illnesses, inflammation, cytokine storm, or lung cell carcinoma. Developing a photocontrolled nAChRs agonist is instrumental in distinguishing the unique biophysical and pharmacological properties of nAChRs. Several kinds of photochromic agents possessed good photopharmacological properties for manipulating nAChRs, such as DitIMI,57 MAACh,58 MAHoCh,58 azocuroniums,59 4FAB,60AzoCholine,45 and BisQ.61−63 Among them, the structure of AzoCholine, which has been used to optically control α7 nAChRs, is prone to be deuterated.45 Hence, we designed and synthesized D-labeled AzoCholine, termed d9-AzoCholine, to evaluate DIE in photopharmacology.

The synthetic route of d9-AzoCholine was similar to that of AzoCholine (Scheme 2), and d7-aniline (13) was oxidized by oxone in CH2Cl2/H2O obtaining d5-nitrosobenzene (14). d7-4-aminophenol (15) coupled with 14 in AcOH affording d9-azophenol (16). N,N-Dimethylaminoethyl chloride hydrochloride (11) reacted with 16 getting d9-tertiary amine (17). Methyl iodide methylated 17 giving d9-AzoCholine.

Scheme 2. Synthetic Route of d9-AzoCholine.

Scheme 2

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Reaction conditions: (1) oxone, DCM/H2O, r.t, (1) oxone, DCM/H2O, r.t, yield was calculated as 100% without purification. (2) CH3COOH, r.t, 36.39%. (3) K2CO3, DMF, KI, 80 °C, 62.85%. (4) THF, r.t, 35.33%.

We then evaluated the photoisomerization behaviors of d9-AzoCholine by UV–vis, HPLC, and NMR. Upon 365 nm irradiation, the trans-d9-AzoCholine were isomerized into cis-, and cis was returned into trans at 415, 455, or 530 nm light (Figures 2a,b, S16, and S17). We observed that UV–vis spectroscopic curves were overlapping upon 415 and 455 nm irradiation, and the conversion rate of 455 nm PSS was faster than that of 530 nm in HEPES buffer. Considering deep tissue penetration and facilitate photoswitchable ligands, 365 and 455 nm light were used for further evaluation. The UV–vis spectral data showed that deuterium substitution increased the photoisomerization rate, both trans to cis and cis to trans in organic and aqueous solutions (Table 2). NMR data showed that deuterium modification had no significant effect on improving photoisomerization ratios, which was consistent with no change of n–π* separations (Figures 2c–e, S18, S19 and Table 2). HPLC data were consistent with UV–vis and NMR data (Figures 2f,g and S20). After several cycles of irradiation, both AzoCholine and d9-AzoCholine showed no photobleaching (Figure S21). In the dark, the thermal relaxation of cis-d9-AzoCholine (365 nm PSS) was significantly shorter than that of cis-AzoCholine (Figure S22, 2.74 ± 0.08 vs 2.10 ± 0.05 h). The results indicated that deuterium substitution has no effect on photoisomerization ratios, ratios, and photostability of AzoCholine.

Figure 2.

Figure 2

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Photophysical properties of AzoCholine and d9-AzoCholine. (a,b) UV–vis spectrum of AzoCholine (a) and d9-AzoCholine (b) upon 365 and 455 nm irradiation reaching 365 nm PSS and 455 nm PSS (100 μM in acetonitrile). (c,d) The representative NMR spectrum of AzoCholine (c, 0.2 M in DMSO-d6) and d9-AzoCholine (d, 0.2 M in DMSO-d6) at dark PSS, 365 nm PSS and 455 nm PSS. Dark PSS was achieved without light and wrapped in tinfoil. 365 nm PSS was prepared by irradiating dark PSS under 365 nm light for 10 min. 455 nm PSS was prepared by irradiating 365 nm PSS under 455 nm light for 10 min. (e). Statistical NMR analysis of AzoCholine and d9-AzoCholine at 365 nm and 455 nm PSS. n = 3, data were shown as mean ± SEM, two-tailed unpaired t test was performed for statistical analysis, and no significant was found. (f,g). The representative HPLC spectrum of AzoCholine (f) and d9-AzoCholine (g) (200 μM in 10 mM HEPES, pH = 7.4) at dark PSS, 365 nm PSS, and 455 nm PSS. The optical densities of 365 nm and 455 nm LED lights (THORLABS) were set as 0.753 and 0.736 W/cm2.

Table 2. UV–Vis Spectral Data of AzoCholine and d9-AzoCholinea.

compd π → π*
 n →π*
 t1/2
  λmax (trans) (nm) λmax (cis) (nm) Δλπ→π* (nm) λmax (trans) (nm) λmax (cis) (nm) Δλn→π* (nm) t1/2trans→ is (s) t1/2trans→cis (s)
AzoCholinea 340 298 42 432 432 0 10.1 10.0
AzoCholineb 344 307 37   428   10.8 10.3
d9-AzoCholinea 340 298 42 432 432 0 9.4 9.5
d9-AzoCholineb 342 305 37   426   9.3 9.8
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a

The spectral data of AzoCholine and d9-AzoCholine (100 μM) was performed in acetonitrilea and 10 mM pH = 7.4 HEPES bufferb. Dark PSS (trans isomers) were converted into 365 nm PSS (cis isomers) upon 365 nm irradiation, and 365 nm PSS (cis isomers) reverted to 455 nm PSS (trans isomers) upon 455 nm irradiation. The optical densities of 365 and 455 nm LED lights (THORLABS) were set as 0.753 and 0.736 W/cm2.

Subsequently, we carried out the evaluation of photopharmacological characteristics in cells. The cytotoxicity assays of AzoCholine and d9-AzoCholine were investigated in HEK293 cells. There was no difference in cytotoxicity between AzoCholine and d9-AzoCholine, which revealed the safety of deuterium modification (Figure S23, AzoCholine, IC50 = 171 ± 9.30 μM vs d9-AzoCholine IC50 = 168 ± 3.85 μM). We went on to evaluate the optically controlled activity on α7 nAChRs. In this work, fluorescence Ca2+ indicator Fluo-4 AM was used to measure the Ca2+ signal evoked by d9-AzoCholine in α7/GlyR HEK293 cells.64 The Ca2+ fluxes were calculated by deducting the response of blank control groups and normalizing to the maximum response of d9-AzoCholine. According to photophysical properties of AzoCholine and d9-AzoCholine, 365 and 455 nm light were used for photoinduced conversion. It has been assessed and confirmed that the photoinduced wavelengths had no effect on the Ca2+ signal (Figure S24a). Ca2+ flux experiments demonstrated that d9-AzoCholine was a potent photoagonist for α7 nAChRs, deuteration improved the photopharmacological properties of AzoCholine (Figures 3 and S24). Each PSS of d9-AzoCholine exhibited a higher efficacy on α7 nAChRs than that of AzoCholine (Figure S24b). It was notable that d9-AzoCholine at dark PSS, an almost absolute trans isomer, showed the highest efficacy (Figure S24b). The maximal response (ΔCa2+max) of d9-AzoCholine was 3.39 times that of AzoCholine at dark PSS (Figure 3b, ΔCa2+max-trans-d9-AzoCholine = 105 ± 4.61%, ΔCa2+max-cis-d9-AzoCholine = 71.4 ± 2.00%, ΔCa2+max-trans-AzoCholine = 31.9 ± 3.94%, and ΔCa2+max-cis-AzoCholine = 15.0 ± 1.80%). Upon 365 nm irradiation, trans to cis, ΔCa2+max and EC50 values significantly changed (Figures 3b and S24b,c, EC50-trans-d9-AzoCholine = 0.69 ± 0.023 mM, EC50-trans-AzoCholine = 1.59 ± 0.57 mM). The potency of 365 PSS recovered upon 455 nm irradiation (Figure S24b,c). Compared with dark PSS, 455 nm PSS shifted the activity rightward and downward. It may be due to the isomerization of cis to trans being less than 100% upon 455 nm irradiation. The cell assays indicated that deuteration significantly enhanced the activity and modulability of AzoCholine on α7 nAChRs. It suggested that deuteration improved the pharmacological properties of the prototype compound.

Figure 3.

Figure 3

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Light-induced AzoCholine and d9-AzoCholine controlled the activity of α7 nAChRs. (a) AzoCholine and d9-AzoCholine were used as photoagonists to regulate Ca2+ signal under light, and Fluo-4 AM was used to measure dose–response curves in HEK293 cells transiently transfected with α7/GlyR-YFP. (b) Significant difference analysis of Ca2+ peak evoked by AzoCholine and d9-AzoCholine. n = 3, data were shown as mean ± SEM, two-tailed unpaired t-test, NS was no significant, **P < 0.01, ***P < 0.001. 365 nm was acquired by illuminating 365 nm light (0.753 W/cm2) to reach PSS.

We also explored the effect of d9-AzoCholine on light-dependent behaviors in Caenorhabditis elegans. In M9 buffer, the nematode C. elegans swam with a dorsoventrally alternating c-shaped body posture. Trauner et al.45 confirmed that trans-AzoCholine significantly inhibited the thrashing frequency of lite-1 mutation type nematodes and had no effect on wild-type nematodes. In these assays, the behaviors were observed in M9 buffer containing AzoCholine (500 μM) or d9-AzoCholine (500 μM); thrashing movements were recorded under 365 and 455 nm pulse irradiation, 30 s interval for 180 s. The thrashing frequency was counted per 10 s. Upon 365 nm irradiation, no difference was observed between AzoCholine and d9-AzoCholine. While espousing to 455 nm light, the thrashing frequency was significantly suppressed (Figure 4). The inhibitory effect at 455 nm irradiation reached its maximum for 20 s and then restored and completely recovered under 365 nm illumination. d9-AzoCholine still maintained good inhibitory effects after the triple cycle. Notably, d9-AzoCholine exhibited a more significant inhibitory effect than that of AzoCholine under 455 nm light. Both AzoCholine and d9-AzoCholine had no effect on wild-type nematodes (Figure S25). The results suggested that deuteration enhanced the photopharmacological activity of AzoCholine and verified the promise of deuterated photoswitches.

Figure 4.

Figure 4

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Light-induced C. elegans thrashing movements. (a) The curves of the number of thrashes times per 10 s of lite-1 mutant nematodes (KG1180) in M9 buffer (black, Control), with AzoCholine (500 μM, blue, AzoCholine) and d9-AzoCholine (500 μM, red, d9-AzoCholine) under pulsing 365/455 nm irradiation. (b) Histogram of a during an illuminated cycle (365 nm for 30 s, 455 nm for 30 s). n = 6, data were shown as mean ± SEM, double-tailed unpaired t-test, *P < 0.05, **P < 0.01, and ***P < 0.001. The optical densities of 365 and 455 nm LED lights (THORLABS) were set as 0.753 and 0.736 W/cm2.

In this work, we introduced deuterium into azobenzene to investigate the effect of deuteration in photopharmacology. We first prepared a series of basic deuterated azobenzenes and examined their photophysical properties. Deuterium substitution had no significant effect on most of the photophysical properties of azobenzene photoswitches. Afterward, we developed a deuterated photo agonist d9-AzoCholine for optically controlling α7 nAChRs. The d9-AzoCholine maintained most of the photophysical properties of AzoCholine. In photopharmacology, d9-AzoCholine exhibited a higher sensitivity and stronger response than AzoCholine on α7 nAChRs. In addition, we validated that deuterium substitution significantly increased the photocontrolled activity of AzoCholine on the behavers of C. elegans. The results indicated that d9-AzoCholine was a more valuable photoswitchable tool to manipulate the activity of the α7 nAChRs. The deuteration approach could be applied to develop more effective photoagonists to investigate the details of receptor activation processes and treat diseases related to α7 nAChRs, such as schizophrenia, Alzheimer’s disease, neurologic diseases, and inflammatory disorders. In summary, we provided a strategy to construct neophotoswitches. Although, the mechanism of properties of deuterated azobenzene was undefined. It is certain that deuteration brings opportunities for the photopharmacology area.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (82173667, 82273892, 82360706, and 82361138572), the Taishan Scholar Program at Shandong Province, the Shandong Natural Science Foundation (ZR2022LSW013), and the Foundation for Innovative Research Groups of State Key Laboratory of Microbial Technology (WZCX2021-03).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00058.

  • Details of synthesized deuterated azobenzenes, spectral data of the compounds, procedures and data of photophysical experiments, calcium flow assays, and C. elegans behavior tests (PDF)

Author Contributions

X.Y., X.Z., and X.Q. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt4c00058_si_001.pdf (3.6MB, pdf)

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