Structural Change of Neurotransmitter Amine with CO₂ in Water: Formation of Covalently Bound Carbamic Acid

Abstract

Structural transformation changes the activity of biological reactions. Neurotransmitter aralkylamines, such as phenethylamine, tyramine, dopamine, tryptamine, serotonin, and histamine, absorb aerial CO₂, and heteronuclear multiple bond connectivity (HMBC) correlations between the carbon derived from CO₂ and the α-hydrogen of the several amines were confirmed in the D₂O solution. The isolation of methyl carbamate from phenethylamine and CO₂ in water with TMSCHN₂ also supported the formation of covalently bound carbamic acid in the amine aqueous solution containing CO₂. Therefore, it is suggested that CO₂ produced in the body would react with neurotransmitter amines to form covalently bound carbamic acid, which might affect biological reactions.

Introduction

Low molecular weight compounds, which are bioactive substances, exhibit a variety of activities depending on their structure. For instance, ingestion of methanol (CH₃OH) causes blindness,^1–3) while 1-carbon homologated ethanol (CH₃CH₂OH) causes drunkenness. Also, the catecholamine, noradrenaline, works as a neurotransmitter in the brain.^4,5) However, adrenaline (N-methylated noradrenaline) does not act in the brain but plays a major role in other parts of the body, such as the heart.⁶⁾ Thus, the structure of a compound has a significant impact on its physiological effects.

Amine functional groups exist ubiquitously in living organisms, such as in amino acids and the catecholamine neurotransmitters. The concentration of exhaled CO₂ is about 4%, and thus CO₂ is constantly being produced as a metabolite in the body.⁷⁾ The reaction of an amine with CO₂ in water is shown in Fig. 1.^8–25) Carbamic acid A is formed by a basic amine and CO₂ via neutralization, and is further neutralized by another amine to form neutralized carbamic acid B. Ammonium hydrogen carbonate C is derived from carbamic acid A and water. Reactions of neutralized carbamic acid B with water, and ammonium hydrogen carbonate C with amine, give a common ammonium carbonate D. Recent efforts in our laboratory focus on CO₂ capture with amines, and we found that several aralkylamines selectively reacted with CO₂ in water without hydration.^26–29) Many amines in neurotransmitters, such as catecholamines, are classified as aralkylamines. We hypothesized that these neurotransmitter aralkylamines would selectively react with CO₂ in the body to form covalent carbamic acids A and B. Although this structural change is reversible, it is suggested that the structural change upon covalent bond formation is accompanied by a significant change in biological activity. Herein, we report the reactivity of neurotransmitter aralkylamines 1a–f with CO₂ in water and the formation of covalently bound carbamic acid.

Fig. 1. Reaction of Neurotransmitter Aralkylamines with CO₂ in Water

Results and Discussion

The concentration of CO₂ in air is about 400 ppm (= 0.04 vol%), and the CO₂ concentration in exhaled air is about 4 vol%. Therefore, in the in vivo environment involving neurotransmitters, CO₂ concentrations are estimated to be at least greater than 0.04 vol%. At the beginning of this work, we focused on the aerial CO₂ absorption of neurotransmitter aralkylamines 1a–f in water. A mixture of an amine (1 mmol) in water (10 mL) and a CO₂ densitometer was placed in a desiccator (5.0 L), which was then sealed, and the CO₂ concentration was measured over time. The results are shown in Fig. 2. Phenethylamine (1a) showed a drop in CO₂ concentration from 409 to 0 ppm after 2 h. CO₂ absorption of 1a in a larger sealed box has previously been reported.²⁶⁾ When tyramine (1b) was used, the CO₂ concentration was reduced to 99 ppm after 2.5 h. In the case of dopamine (1c), which has a hydroxy group at the 3-position, the CO₂ concentration decreased to 218 ppm at 2.5 h. The secondary amine adrenaline (1d) did not cause a drop in CO₂ concentration. Tryptamine (1e), which has an indole skeleton, absorbed atmospheric CO₂ as well as tyramine (1b), reaching 87 ppm after 2.5 h. Serotonin (1f) with a hydroxyl group at the 5-position further reduced the CO₂ concentration, reaching 23 ppm after 2.5 h. Histamine (1g), which contains an imidazole skeleton, also efficiently absorbed CO₂, with a value of 28 ppm after 2.5 h.

Fig. 2. Aerial CO₂ Absorption of Neurotransmitter Aralkylamines 1a–g in Water

Next, NMR measurements in a CO₂ environment were performed to confirm the formation of a covalent bond by the reaction of CO₂ with an amine. CO₂ gas was added to the D₂O solution of phenethylamine (1a), and ¹³C-NMR spectra were measured over time (Fig. 3). After 10 s, a sharp peak at 165 ppm was detected. After 40 s, the peak at 161 ppm became the primary signal instead of that at 165 ppm. If the amine reacts with CO₂ to form a covalent bond, yielding a carbamic acid derivative, a heteronuclear multiple bond connectivity (HMBC) correlation between the carbon derived from CO₂ and the α-hydrogen of the amine should be observed. Indeed, when HMBC measurement was performed on the sample after 10 s, a correlation was observed between the ¹³C-NMR peak at 165 ppm and the ¹H-NMR peak at 3.1 ppm, which corresponds to the α-hydrogen of the amine. On the other hand, in the case of the sample at 40 s, no HMBC correlation of the ¹³C-NMR peak at 161 ppm was detected.

Fig. 3. NMR Spectra of Phenethylamine (1a) in D₂O upon Addition of CO₂

The change in products over time with CO₂ addition was considered as follows (Fig. 4). In the initial stage of CO₂ addition at low CO₂ concentration, the ratio of amine to CO₂ would be 1 : 0.5, forming a neutralized carbamic acid B or ammonium hydrogen carbonate D. It is known that aralkylamines selectively absorb CO₂ in air without hydration. Therefore, HMBC correlation of a neutralized carbamic acid B would be observed in a D₂O solution of phenethylamine (1a) 20 s after adding CO₂. At high CO₂ concentration, the ratio of amine to CO₂ should change to 1 : 1, forming acidic carbamic acid A and ammonium hydrogen carbonate C. In the D₂O solution of phenethylamine (1a) 40 s after adding CO₂, ionic ammonium hydrogen carbonate C might be the main product formed due to the hydration and acidity, which would have no HMBC correlation.

Fig. 4. Reaction of Amine and CO₂ in Water

Based on the result of phenethylamine (1a), the reaction of tyramine (1b) in D₂O upon CO₂ addition was also investigated (Fig. 5). After 20 s, an HMBC correlation between the ¹³C-NMR peak at 165.20 ppm and the ¹H-NMR peak at 3.26 ppm was detected. In the case of dopamine (1c), an HMBC correlation between the ¹³C-NMR peak at 165.24 ppm and the ¹H-NMR peak at 3.18 ppm was also detected at 20 s. In the examinations of adrenaline (1d), no HMBC correlation was observed. In this case, the bulky secondary amine might prevent the formation of a covalent bond between the amine and CO₂.

Fig. 5. NMR Spectra of Tyramine (1b) and Dopamine (1c) in D₂O upon CO₂ Addition

Next, aralkylamines containing heterocycles in D₂O upon CO₂ addition were measured by HMBC (Fig. 6). Although tryptamine (1e) and serotonin (1f), which include an indole skeleton, did not show the HMBC correlations, possibly due to low solubility or instability under D₂O, HMBC correlations of histamine (1g) were confirmed between the carbon (¹³C-NMR 165.23 ppm) derived from CO₂ and the α-position hydrogen (¹H-NMR 3.25 ppm) of the amine upon addition of CO₂ after 10 s.

Fig. 6. NMR Spectra of Histamine (1g) in D₂O upon CO₂ Addition

Finally, methylation of phenethylamine (1a) in water and CO₂ was investigated.^30,31) After CO₂ gas was added to the aqueous phenethylamine (1a) solution for 20 s, the resulting mixture was poured into a solution of TMSCHN₂ in MeOH to afford the desired methyl carbamate (2a) in 10% yield (Fig. 7). This result suggests the formation of carbamic acid in the reaction.

Fig. 7. Methylation of Phenethylamine (1a) in Water and CO₂

Conclusion

In summary, neurotransmitter aralkylamines bearing primary amines absorbed CO₂ under low CO₂ concentration conditions (air). HMBC NMR results suggested that the corresponding amine(s)/CO₂ complexes formed covalent carbamic acid intermediates even in aqueous solution. The fact that methyl carbamate was obtained from methylation in a phenethylamine aqueous solution upon addition of CO₂ gas also suggested covalent bond formation between the amine and CO₂. These results indicate that neurotransmitter aralkylamines would react with CO₂ to form covalent bond-type carbamic acid intermediates even in the body, and the resulting carbamic acid intermediates might cause other biological reactions different from the original actions. Further exploration of the interactions between neurotransmitter aralkylamines and CO₂ is ongoing.

Acknowledgments

We are thankful for the support from Panasonic and the Takeda Science Foundation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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