3.3.1. β-Substituted Ethylamines
Two members of this family, 2-aminoethanol and ethylenediamine, capable of forming five-member rings for developing an intramolecular H-bond were already investigated in Subsection 3.2.1. Further members of the family with some having aromatic rings in the β-position which allow for H+…π interactions with protonated species will be surveyed here (Figure 8). Indeed, these ethylamines belong to the group of the extremely important neurotransmitters, which overwhelmingly adopt the amino N-protonated form at pH = 7.4, where they are involved in biological signal-transduction processes. Nonetheless, neurotransmitters maintain some small percent of the neutral form even at this pH, and create another zero-net-charge species, the zwitterionic structure up to 7.2% in total for the two forms for molecules b-e in Figure 8 [123,192]. These protonation states [193], more abundant at higher pH, will also be discussed below.
For histamine, formation of an intramolecular H-bond between the ethylamine side chain and the N1 nitrogen of the imidazole ring is feasible both in the neutral and the protonated forms. In cases of norepinephrine and epinephrine, an OH group, as another β-substituent is also found in the molecule. For these two latter, O–H…N and N–H+…O bridges can be formed in five-member rings for the neutral and protonated species, respectively. Furthermore, an N(amine)–H… π or an N(amine)–H+…π intramolecular H-bond is always possible for each molecule in Figure 8. The methods applied to the gas-phase and in-solution structure analyses are summarized in Table 2.
Histamine (2-(1H-imidazol-4-yl)ethanamine). Details regarding the challenges posed by the tautomerization of this molecule were provided recently [22]. This review remains focused on aspects associated with the intramolecular H-bond formation. The rotational spectrum of gas-phase histamine was recorded by Vogelsanger et al. [194]. Four major conformations were identified, all of which are stabilized by intramolecular H-bonds involving a gauche ethylamine side-chain. These conformers encompass the N1H–N3H tautomerization for the imidazole ring and both H-bond donor and acceptor properties of the imidazole as well as of the amino group. For protonated histamine, two major gas-phase isomers were detected by Lagutschenkov et al., in the IR spectrum [195]. The more stable one is protonated on the ring and a N1(ring)-H+…N(amine) H-bond is formed. This structure is more stable by a few kJ/mol than the N1…+HN(amine) state formed by proton jump from the ring to the amino group. Both protonation forms can preferably create H-bonding within a gauche ethylamine side-chain conformation.
Figure 8  Neurotransmitters portrayed in the neutral form: (a) Histamine; (b) Tyramine; (c) Dopamine; (d) Norepinephrine (R=H), Epinephrine (R=CH3); and (e) Serotonin.   In this paper, Lagutschenkov et al., provided an excellent overview of the present status of histamine-structure research. The histamine problem is threefold: Protonation state, prevailing tautomer and conformations. If the neutral and the monoprotonated structures are separately investigated, the problem becomes twofold. The amine pKa is 9.75–9.80, so neutral histamine must be the major species in aqueous solution when it is dissolved in pure water. The solute becomes protonated only to a very small degree under such conditions, but then becomes the prevalent species at pH = 7.4.
ijms-15-19562-t002_Table 2 Table 2  Comprehensive summary of the applied experimental and theoretical methods for the conformational/tautomeric equilibria of neurotransmitters in aqueous solution a.   a In cases, when more than one quantum mechanical methods were used, the level producing the best result is indicated. The “+” sign indicates that ZPE/Gth was calculated; b Reference [57]; c Reference [59]; d Reference [52].   Calculations to date show no consensus regarding the structure of the neutral histamine in aqueous solution. Karpińska et al., [196] using the continuum solvent SCRF method at the MP2/6-31G//MP2/6-31G level found the N3H tautomer as the most stable without an intramolecular H-bond. The next stable structure is higher in energy by only 0.8 kJ/mol, where the ring proton of the N1H tautomer is the H-bond donor to the amino group of the gauche ethylamine side chain. Nagy et al., [198] found the N3H tautomer with predominantly (83%) trans ethylamine side-chain conformation as the most stable structure on the basis of MP2/6-311++G**//HF/6-31G* + MC/FEP calculations. This structure prevents the formation of an intramolecular H-bond in an aqueous solution. The second most stable form, N3H/gauche ethylamine chain, is present at 12%.
In contrast to the above results, Ramirez et al., [199] predicted the N3H/gauche side-chain conformation to be predominant at the PCM/B3LYP/6-311G** level. Raczyńska et al., [200] performed theoretical calculations utilizing the PCM approach at the HF, MP2, and B3LYP levels with basis sets up to 6-311++G**. The prediction is a gauche-trans equilibrium for the neutral N1H tautomer in aqueous solution. Forti et al., [201] published recently a paper describing a multilevel strategy for the exploration of the conformational flexibility of small molecules. The predicted trans/gauche ratio strongly depends on the applied basis set, changing from 64/36 to 48/52 when the B3LYP/6-31G*, MP2/aug-cc-pvdz and MP2/aug-cc-pvtz theoretical levels are considered. On the other hand, the N1/N3 ratio was predicted consistently as 48:52. In summary, structure predictions for the neutral histamine in aqueous solution have not been able to form a consensus.
The agreement is much better regarding the monoprotonated species [196,198,200,202]. All these calculations favor the N3H tautomer in combination with a protonated ethylamine side-chain. When the side-chain conformation was also investigated, the predominant gauche structure forms an intramolecular N1…+HN(amine) H-bond. Thus the stability order differs from that in the gas phase where this species is only the second most stable structure.
An experimental conformational analysis in aqueous solution was performed by Kraszni et al. [197]. These authors determined the position-specific standard 1H NMR coupling constants for the gauche and trans conformers. This study helped predict the population of the trans conformer to be 41%, 38%, and 50% in neutral, monocationic and dicationic forms, respectively.
Tyramine (4-(2-aminoethyl)phenol). A good summary of recent gas-phase experiments for studying the structure of ethylamine derivatives with β-phenol and catechol substituents was provided by Ishiuchi et al. [218]. For tyramine, the gas-phase conformations were determined by Melandri and Maris [203] using a free-jet microwave study. The authors found four structures where the side-chain adopts the C(ring)–C–C–N gauche conformation and where an N–H…π H-bond may stabilize the structure. The four structures differ in the relative rotational positions of the –NH2 and the OH groups. MP2/6-31G* calculations predicted an energy range of 1 kJ/mol for these conformers. With a trans side-chain, the lowest relative energy is 5 kJ/mol.
Nagy et al., [123,204] studied tyramine theoretically in the gas-phase and in aqueous solution. Although the conformer energies of the neutral form with gauche and trans chains hardly differed as calculated at the B3LYP/6-31G* level, the free energy at T = 298 K was lower by 1.8 kJ/mol for the trans structure, in contrast to experimental data. Tyramine may form an N–H…π intramolecular H-bond in the case of the gauche side-chain conformation. The B3LYP method is known to fail accounting for dispersion interactions. On this basis, one may think that the lack of considering dispersion interactions in [123] led to the prediction of the absence of an intramolecular H-bond, which would have been stabilized by favorable and remarkable dispersion interactions otherwise. Not accounting for dispersion interactions may be the major the reasons for the trans preference, because these kinds of interactions are more important between the NH2 group and the ring than their role in stabilizing an intramolecular H-bond. The distance between the amino group and the ring is smaller in the gauche than in the trans conformation; consequently a missed account of dispersion contributions to the conformer stabilizing energy terms would more sensitively affect the gauche than the trans form. Indeed, MP2/6-31G* calculations by Melandri and Maris above, where the dispersion interactions are considered, clearly indicate the gauche preference.
In aqueous solution at physiological pH 7.4, the protonated form is present at about 99% [204]. For this structure, the only stabilization possible is when the –CH2–CH2–NH3+ chain bends above the aromatic ring, forming an N–H+…π intramolecular H-bond. Also in the solution are two zero-net charge forms, zwitterionic (zw) and neutral (neu), in a total population slightly more than 1%. The zw: neu ratio rapidly decreases from 10.72 to 2.45 in aqueous solution when the temperature is raised from 14 to 37 °C [123]. Nonetheless, the zw form must be the prevalent zero-net-charge structure when tyramine dissolves in pure water at room temperature. Theoretical calculations at the PCM/MP2/6-31G*//B3LYP/6-31G* level found the neu form prevalent. In contrast, using the MC/FEP method, the zw form is the stable overall neutral tautomer, although the relative free energy is much exaggerated in comparison with the derivable experimental value [123].
Dopamine (4-(2-Aminoethyl)benzene-1,2-diol). Cabezas et al., [205] found experimentally seven conformers for the gas-phase (neutral) dopamine. All structures maintain an O–H…O H-bond on the benzene ring. The ethylamine side-chain has a gauche C(ring)–C–C–N conformation. The seven structures come into existence with N–H…π interactions in different rotational positions of the –NH2 group relative to the O–H…O bond. Lagutschenkov et al. [208] recorded the gas-phase IR spectrum for protonated dopamine. Not surprisingly, the protonated side-chain in the most stable conformers adopts the gauche arrangement as defined above for the neutral form and bends above the aromatic ring. This interaction corresponds to a N–H+…π intramolecular H-bond. The authors performed B3LYP and MP2 calculations using the cc-pvdz basis set and concluded that the H-bond on the ring is of O(3)–H…O(4) type in the two lowest energy structures, separated only by 0.1 kJ/mol in free energy.
Other theoretical studies in the literature also found a H-bonded 3-OH/4-OH substructure, nearly coplanar with the ring both in the gas phase and aqueous solution, see, e.g., [123,206,209]. Šolmajer et al., [207] studied the protonation process experimentally for several neurotransmitters, including dopamine. The protonated form stably exists up to about pH = 8 and then the proton is gradually lost in the pH range of 8–10. Less than 5% of the amines remain protonated above pH = 11.5. The ethylamine side-chain may adopt three main conformations along the C(ring)–C–C–N path. Two, nearly equal-energy gauche and one trans conformations are stable, with different rotational positions for the neutral –NH2. An intramolecular H-bond in the form of N–H…π or N–H+…π is possible only in the gauche conformation of the side-chain.
Both the neutral and a zwitterionic zero-net-charge structures are present in aqueous solution, where one of the hydroxy protons jumps to the –NH2 group or (more likely) the zwitterion gets formed by water catalysis. Although the population of the zero-net-charge form is pH dependent and is present in a total of only about 3% at pH = 7.4, the neu:zw ratio of about 10 must be constant in any aqueous solution [204]. By performing MC/FEP solvent-effect calculations, Nagy et al., [123] predicted that the proton comes from the 4-OH group in the zwitterion, still maintaining the O(3)–H…O(4) intramolacular bond on the benzene ring. The related conformation of the ethylamine side-chain is preferably trans. For the protonated dopamine, the ratio of the gauche (G) and trans (T) side-chains in aqueous solution was calculated by Nagy et al., [209] theoretically, using HF/6-31G* relative internal energies and MC/FEP simulations for estimating the solvent effect. The predicted G:T ratio of at least 75:25 is somewhat comparable with the experimentally found value of 58:42 at pH = 7 [207].
Norepinephrine (4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol), is a derivative of the 2NH2-ethanol with a 3,4-dihydroxyphenyl substituent at C1 of the ethane chain. The gas-phase structure of the neutral norepinephrine (with older name: Noradrenaline) was studied by Snoek et al. [210]. The authors found that almost the entire population of jet-cooled noradrenaline adopts a conformation with extended ethanolamine side-chain allowing for a H-bond between the side-chain OH and the amino group, as well as between the phenolic hydroxyls.
The prevailing structure is the protonated form in physiological systems, 92.8% at pH = 7.4 [192]. An N–H+…O intramolecular H-bond is favorable, which can exist in one of the C(ring)–C–C–N gauche conformations and in the trans form. In the other gauche conformation, where the formation of the N–H+…O bond is prevented because of the local O–C–C–N trans arrangement, the possible conformer-stabilizing effect through the N-H+… π interaction should be emphasized. The two phenolic OH groups form a hydrogen bond like in the gas phase, but probably only on the basis of the distance criterion, since Mandado et al., [7] did not find a (3, −1) BCP for 1,2-dihydroxybenzene.
The equilibrium conformer fractions were calculated at the ab initio and DFT levels using the PCM continuum solvent approach and the FEP method in MC simulations [192]. The method applied for calculating the relative internal free energies affect the final conclusions. Overall, the internally bound OCCN conformers were found to dominate the composition in fair agreement with experimental findings at pH = 7 [207].
Using the PCM method, Alagona and Ghio [211] studied the conformer population for neutral and protonated norepinephrine in aqueous solution at the HF/6-31G* and MP2/6-31G*//HF/6-31G* levels. The calculated T fraction regarding the C(ring)–C–C–N torsion of the neutral form is 61%–72%, whereas the trans form was populated experimentally by about 59% (pH = 11.5). The T:G ratio for the protonated species was calculated as 89:11 and 44:56 at the HF and MP2 levels, respectively, in comparison with the experimental composition of 65:35 at pH = 7.0 [207].
Epinephrine ((R)-4-(1-Hydroxy-2-(methylamino)ethyl)benzene-1,2-diol). Epinephrine (with its older name, adrenaline) is the N-methyl derivative of norepinephrine. Its gas-phase structure was studied by a combination of mass-selected ultraviolet and infrared holeburn spectroscopy [212]. The identified conformation has an extended side-chain structure with an intramolecular O–H…N H-bond. The authors also identified experimentally the H-bonded substructure for the two phenolic OH groups.
Epinephrine is only slightly soluble in water and alcohol, but is readily soluble in aqueous solution of mineral acids. At pH = 7.4 only the protonated form, 94.8% [204], should be the subject of theoretical calculations. Alagona and Ghio [213] studied the conformational equilibrium for protonated adrenaline in aqueous solution at the DFT and ab initio MP2 levels and using the IEF-PCM solvation approximation. The C(ring)–C–C–N trans arrangement (corresponding to a gauche OCCN arrangement) was found as the most stable conformation allowing for the O…+H–N H-bond but preventing the H+…π interaction with the catechol ring. The two OH groups on the benzene ring maintain the O–H…O H-bond in aqueous solution.
The OCCN trans conformation was found to be only 10% in the experimental composition at pH = 9 for norepinephrine [207]. The corresponding value for ephedrine is 13%. Ephedrine is structurally related to epinephrine, bearing an N-methyl group, as well, but missing phenolic hydroxy groups and with an additional methyl group on the ethyl chain. Overall, the H-bond pattern for epinephrine is expected to be similar to that for norepinephrine.
Serotonin (5-Hydroxytryptamine). LeGreve et al., [214] investigated the gas-phase serotonin conformers using different spectroscopic methods. They identified eight neutral serotonin conformers including the side-chain both in C(ring)–C–C–N gauche and anti (trans) conformations, and two main rotational positions for the OH group. The most populated conformation is Gpy(out)/anti OH, where the gauche side-chain is on the pyrrole side of indole, one of the NH2 hydrogens points toward the pyrrole nitrogen, and the indole (N)H is in anti position with respect to the hydroxy hydrogen. Thus the intramolecular H-bond is basically of an N–H…π type.
Lagutschenkov et al., [215] recorded the gas-phase IR spectrum of protonated serotonin. They found a gauche conformation for the protonated ethylamine side-chain rotated toward and above the phenolic ring of the indole moiety. In this position, N–H+…π, cation…π interaction stabilizes the structure. The preference of this conformation was supported by B3LYP and MP2 calculations.
Nagy et al., [192] calculated the distribution of the zero-net-charge forms, neutral (neu) and zwitterionic (zw), for serotonin at pH = 7.4. In that solution, the protonated form is present at 99.7%, and the zero-net-charge form is present only at about 0.3%. However, since the determined neu:zw ratio of about 1.2 is pH independent, if serotonin is still non-protonated when dissolved in aqueous solution, the above neu:zw ratio should hold for the major zero-net-charge protonation state.
Alagona and coworkers studied the conformational equilibrium for the protonated serotonin in aqueous solution [216,217]. According to the covalent structure of the molecule, the only possible intramolecular H-bond is of N–H+… π type, similar to that for tyramine and dopamine (not considering the stably maintained O-H…O bond for the latter.) For such systems, the correct prediction of the gauche-trans conformational equilibrium for the side-chain is crucial. For histamine, norepinephrine and epinephrine, the side-chain conformation is probably more effectively dictated by the possible formation of N–H+…N and N–H+…O H-bonds.
The serotonin study in [216] shows almost all computational difficulties emerging throughout the conformational analysis for solutes. The obtained relative internal energy results depend on the level of theory used during the calculations. Contributions of the relative thermal corrections to the total relative conformational free energies are critical. It has also revealed that the solvation method, thus whether the relative solvation free energy was calculated at the PCM level or in a MC/FEP process, has an effect on the final results. Should the counterion be allowed to freely move in the solution in MC simulations, or a fixed solute-counterion separation is acceptable for expediting the FEP calculations? Are atomic charges more preferable from CHELPG or RESP fit to the in-solution MEP?
Due to the listed problems, the results from the above study were not conclusive. Different combinations of the relative free energy components, calculated on the basis of the IEF-PCM/B3LYP/6-31G* and IEF-PCM/MP2-6-31G*//B3LYP/6-31G* levels for the internal terms and using MC/FEP relative solvation free energies, could lead to the preference for either the trans or gauche side-chain conformations. In the absence of experimental data, the “best” choice was not clear. Nonetheless, all calculations predicted an observable equilibrium between gauche and trans side-chain conformations, since the total relative free energies were within a range of about 4 kJ/mol.
By performing an IEF-PCM analysis for the solution phase, Alagona and Ghio [217] studied the protonated serotonine conformations in the gas phase and in water solvent. The potential curve at MP2/6-31G* level in the gas phase for the hydroxy hydrogen rotation shows that the hydrogen atom is syn to the indole (N)H. The IEF-PCM/MP2/6-31G*//MP2/6-31G* free energy in solution is more negative by 5.3 kJ/mol for the gauche conformer with the –NH3+ group rather away (G1) than toward (G2) the indole ring. The trans form is higher in free energy by 1.5 kJ/mol than G2. These calculations predict the overwhelming presence of the two gauche conformers in aqueous solution in comparison with the trans structure.
In conclusion, the intramolecular H-bond in aqueous solution is generally maintained either in the form of NH+…X (X = O, N) or through NH+…π interactions for neurotransmitters with a protonated amino group. For the neutral structures in this phase, the theoretical calculations have led to different conclusions. The neutral form is prevalent generally at pH > 9, where all studied neurotransmitters possessing at least one phenolic OH can adopt also the zwitterionic form in aqueous solution. The pH independent neu:zw ratio is largely varying at T = 298 K, from about 0.2 for tyramine to 1.2 for serotonine and to about 10 for dopamine.