Martin
Wilke
a,
Christian
Brand
ab,
Josefin
Wilke
a and
Michael
Schmitt
*a
aHeinrich-Heine-Universität, Institut für Physikalische Chemie I, D-40225 Düsseldorf, Germany. E-mail: mschmitt@hhu.de; Fax: +49 211 8112179; Tel: +49 0211 8112100
bFaculty of Physics, University of Vienna, VCQ, QuNaBioS, Boltzmanngasse 5, A-1090 Vienna, Austria
First published on 26th April 2016
The 5-hydroxytryptamine receptors (5HTn) are optimized for 5-hydrotryptamine molecules, resulting in a significantly enhanced psychoactive response compared with the 4-, 6-, 7-isomers. This is despite their relatively similar energetic stabilities, excited state lifetimes and emission characteristics. In this work we investigate the conformational space of serotonin (5-hydroxytryptamine) using a combination of rotationally resolved electronic spectroscopy and ab initio calculations. The geometries of the four most abundant conformers are assigned from their molecular parameters in the electronic ground and excited state. We find a conformer-dependent competition between two polar groups trying to establish a hydrogen bond with the same H-atom in the most stable conformer of serotonin. The result explains some remarkable deviations with respect to the conformational space of the closely related neurotransmitter tryptamine. Based on the comparison to other 5-substituted indoles we propose to generalize this finding to explain the conformational preferences of indole-based neurotransmitters.
A suitable nomenclature for the conformational space of serotonin is depicted in Fig. 1.5 For all experimentally observed conformers, the ethyl amino side chain is perpendicular to the aromatic plane which results in three possible conformations of the NH2 group: gauche to the pyrrole ring (Gpy), gauche position to the phenyl moiety (Gph) or pointing away from the chromophore (Anti). For both gauche conformations the lone pair (LP) of the NH2 group can point up, out, or in. The in conformers, however, are much higher in energy and will be neglected in this study. For the Anti conformers the LP can point up or to either one of the rings: phenyl (ph) or pyrrole (py). Finally, the orientation of the OH-group is denoted with anti when it is anti-parallel to the NH bond of the pyrrole ring, or syn- for the other option. In the end a set of 14 possible conformers exists which is shown in the online ESI.†
An experimental investigation of the conformational space of the protonated form was performed by Lagutschenkov et al.6 Both the bare5 and the singly hydrated neutral serotonin7 were studied with vibrational resolution in the group of Zwier. They found eight conformers which they divided into a group of five transitions labeled as A to E, blue-shifted by around 200 cm−1 from the second group of three transitions labeled as F to H.5 The lowest energy conformation was stated to be the Gpy(out)/anti conformer like in the closely related neurotransmitter tryptamine which essentially is serotonin without the hydroxyl group. Later, this assignment was corrected by Cabezas et al.8 who established the Gph(out)/anti conformer to be the global minimum based on rotational spectroscopy. This is in agreement with theoretical predictions from Srivastava and Singh9 who performed a comprehensive computational study on serotonin at various levels of theory. Also Van Mourik and Emson investigated the conformational space of serotonin and tryptamine theoretically.10
Apart from the conformational analysis the study of LeGreve et al. pointed to some intriguing irregularities in the vibronic spectrum of serotonin.5 The energetic gap between the syn- and the anti-conformer with respect to the hydroxyl group is relatively constant at 230 cm−1, close to the respective value for 5-hydroxindole.11,12 However, for the Gph(out) conformers the energetic gap is increased by 30 cm−1. This is very surprising as the only difference is a rotation about a single OH-bond by 180°. Furthermore, the nitrogen atom of the ethyl amino side chain is 510 pm away from the oxygen atom of this group, making a direct interaction unlikely. So far, this effect has been tentatively explained in the literature by electronic5,9 or dipole–dipole interactions.6,7 Additionally, the most stable conformer of serotonin does not coincide with the energetically lowest one of tryptamine. Thus, the question arises which kind of mechanism leads to this remarkable stabilization and irregularity for the Gph(out)/anti conformer of serotonin.
In the present contribution, we use a combination of rotationally resolved electronic spectroscopy and high-level quantum mechanical calculations to investigate the conformational space of serotonin and compare it to that of tryptamine. Tryptamine itself has been studied at rotational13 and vibronic resolution14 as well as at partial15,16 and full17–19 rovibronic resolution. Seven different conformers were identified and assigned based on their rotational constants and vibrational spectra. Based on the comparison of the existing data on serotonin derivatives,20–22 we propose the formation of intramolecular hydrogen bonds to be responsible for the conformational preference for many indole-based neurotransmitter. The results give new and important insights into the stabilization mechanisms of this complex class of molecules.
Conformer | A′′/MHz | B′′/MHz | C′′/MHz | A′/MHz | B′/MHz | C′/MHz | θ/° | ϕ/° |
---|---|---|---|---|---|---|---|---|
Gpy(out)/anti | 1294.3 | 578.1 | 440.8 | 1295.8 | 574.6 | 438.8 | +27 | 76 |
Gpy(out)/syn | 1288.8 | 580.9 | 441.8 | 1300.1 | 574.0 | 438.8 | +35 | 75 |
Gpy(up)/anti | 1276.5 | 580.1 | 441.3 | 1293.4 | 570.9 | 437.1 | +26 | 76 |
Gpy(up)/syn | 1275.5 | 581.1 | 441.6 | 1303.1 | 568.3 | 436.2 | +34 | 75 |
Gph(out)/anti | 1164.4 | 663.9 | 445.0 | 1167.7 | 657.9 | 453.1 | +23 | 78 |
Gph(out)/syn | 1165.0 | 662.1 | 454.8 | 1172.9 | 654.4 | 452.9 | +31 | 76 |
Gph(up)/anti | 1184.0 | 640.5 | 454.2 | 1177.9 | 640.2 | 452.8 | +24 | 76 |
Gph(up)/syn | 1179.3 | 644.3 | 455.4 | 1176.8 | 643.0 | 454.1 | +33 | 75 |
Anti(ph)/anti | 1178.3 | 562.1 | 394.3 | 1171.9 | 563.5 | 395.1 | +12 | 80 |
Anti(ph)/syn | 1175.1 | 562.7 | 394.4 | 1177.7 | 561.3 | 394.7 | +20 | 80 |
Anti(py)/anti | 1183.4 | 556.5 | 392.4 | 1180.7 | 557.3 | 393.0 | +13 | 80 |
Anti(py)/syn | 1177.3 | 560.3 | 393.5 | 1179.9 | 558.8 | 393.7 | +21 | 80 |
Anti(up)/anti | 1177.7 | 557.3 | 392.6 | 1175.9 | 558.0 | 393.4 | +13 | 80 |
Anti(up)/syn | 1175.2 | 559.2 | 393.4 | 1178.9 | 557.5 | 393.8 | +21 | 79 |
The orientation of the transition dipole moment (TDM) given by its angles θ with the inertial a-axis and ϕ with the c-axis (cf.Fig. 2), provides additional information to distinguish the conformers. The respective values for all 14 conformers are also listed in Table 1. While ϕ is quite similar for all conformers and no systematic differences can be observed, the changes of θ are a reliable measure to distinguish between syn- and anti-conformers: For the anti-conformers θ is always smaller by 8° than those of the respective syn counterparts.
In the case of tryptamine it has been shown that the most stable ground state conformer is Gpy(out) followed by Gpy(up) and Gph(out).40–42 Also for serotonin the Gpy(out)/anti conformer is the most stable ground state conformer according to density functional theory (DFT).5,9 Using functionals including dispersion interaction9 or changing to perturbation theory5,9 results in a change of the relative energy order, in agreement with the experimental findings from Cabezas et al.8 who found the Gph(out)/anti conformation to be the most stable one. The relative ground state energies of the seven most stable tryptamine conformers43 are compared to those of the 14 serotonin conformers9 in Table 2.
Tryptamine | Serotonin | ||||
---|---|---|---|---|---|
MP2/6-311++G(d,p)43 | MP2/6-311++G(d,p)9 | CC2/cc-pVTZ | |||
anti-5OH | syn-5OH | anti-5OH | syn-5OH | ||
Gpy(out) | 0.0 | 8.4 | 122.8 | 155.6 | 270.4 |
Gpy(up) | 125.6 | 93.7 | 243.4 | 158.1 | 285.1 |
Gph(out) | 136.4 | 0.0 | 375.3 | 0.0 | 330.5 |
Gph(up) | 304.3 | 352.9 | 482.3 | 252.9 | 351.5 |
Anti(py) | 482.3 | 524.3 | 629.6 | 710.4 | 790.8 |
Anti(ph) | 475.0 | 460.6 | 695.7 | 622.9 | 804.1 |
Anti(up) | 499.8 | 527.4 | 656.2 | 626.4 | 747.4 |
A | Gpy(out)/anti | B | Gpy(up)/anti | C | Gph(out)/anti | F | Gpy(out)/syn | |
---|---|---|---|---|---|---|---|---|
A′′/MHz | 1286.50(1) | 1294.3 | 1267.28(1) | 1276.5 | 1163.12(1) | 1164.4 | 1281.86(1) | 1288.8 |
B′′/MHz | 571.74(1) | 578.1 | 574.75(1) | 580.1 | 650.60(1) | 663.9 | 573.92(1) | 580.9 |
C′′/MHz | 435.63(1) | 440.8 | 436.11(1) | 441.3 | 450.06(1) | 455.0 | 436.39(1) | 441.8 |
ΔI′′/amu Å2 | −116.66 | −118.15 | −119.27 | −121.74 | −88.37 | −84.65 | −116.75 | −118.40 |
A′/MHz | 1286.82(1) | 1295.8 | 1269.06(1) | 1293.4 | 1164.19(1) | 1167.7 | 1287.31(1) | 1300.1 |
B′/MHz | 567.46(1) | 574.6 | 570.14(1) | 570.9 | 644.10(1) | 657.9 | 567.85(1) | 574.0 |
C′/MHz | 433.20(1) | 438.8 | 433.89(1) | 437.1 | 447.13(1) | 453.1 | 433.59(1) | 438.8 |
ΔI′/amu Å2 | −116.71 | −117.74 | −119.87 | −119.57 | −88.45 | −85.58 | −116.99 | −117.45 |
ΔA/MHz | +0.32(1) | +1.5 | +1.78(1) | +16.9 | +1.07 | +3.3 | +5.45 | +11.3 |
ΔB/MHz | −4.28(1) | −3.5 | −4.61(1) | −9.2 | −6.50 | −6.0 | −6.07 | −6.9 |
ΔC/MHz | −2.43(1) | −2.0 | −2.22(1) | −4.2 | −2.93 | −1.9 | −2.80 | −3.0 |
θ/° | ±38.83 | +27 | ±44.84 | +26 | ±34.60 | +23 | ±46.52 | +35 |
ϕ/° | 69.65 | 76 | 70.53 | 76 | 73.93 | 78 | 65.29 | 75 |
ν 0/cm−1 | 32571.60 | 33029 | 32535.23 | 33007 | 32532.30 | 33023 | 32341.26 | 32653 |
τ/ns | 6 | — | 5 | — | 9 | — | >12 | — |
The experimental parameters (rotational constants of the electronic ground (A′′, B′′, C′′) and excited state (A′, B′, C′), the inertial defect of both states (ΔI), the center frequencies of the respective bands (ν0) and the orientation of the TDM) of the four conformers are given in Table 3 and are compared to the results of the ab initio calculations. The assignments will be discussed in the next section.
LeGreve et al.5 assigned the group of transitions F–H to the syn-conformers based on the origin shifts in 5-hydroxyindole (5OHI). If we follow this argumentation, bands A and B are members of the Gpy/anti family while band F belongs to the Gpy/syn family. The shift of the electronic origins between band A and F amounts to 230.34 cm−1, which is very close to the shift of the respective syn- and anti-conformers in 5OHI (230.91 cm−1).12 Regarding the rotational constants, a difference of +4.64 MHz in the A′′, −2.18 MHz in the B′′ and −0.76 MHz in the C′′ constant is observed experimentally between band A and F. For the anti- and syn-conformers of Gpy(out) (Gpy(up)) a change of +5.5 (+1.0) MHz in the A′′, −2.8 (−1.0) MHZ in the B′′ and −1.0 (−0.3) MHz in the C′′ constant is calculated from the ab initio optimized geometries. Hence, we assign the band A to Gpy(out)/anti and the band F to the respective syn-conformer. A confirmation of this assignment is given by the orientations of the transition dipole moments for the different conformers. It was mentioned that the CC2/cc-pVTZ calculations predict an increase of 8° in the angle θ when we go from a anti to a syn-conformation. A shift by the same amount can be observed between the experimentally determined angles of serotonin A and F. An additional affirmation can be extracted from the excited state lifetimes. In 5OHI it increases from 7.5 to 10 ns when going from the anti- to the syn-conformer.12 In the same manner the excited state lifetime increases from serotonin A (6 ns) to serotonin F (>12 ns).
For band B we know that it belongs to the Gpy/anti family which has three members. Gpy(out)/anti is already assigned to band A and Gpy(in)/anti is excluded by its high energy. Thus, the only possible assignment for band B is to the Gpy(up)/anti conformer. This is supported by the differences between the experimental rotational constants of the B conformer and the ab initio calculated Gpy(up)/anti rotational constants (ΔA = +9.2 MHz, ΔB = +5.3 MHz, ΔC = +5.2 MHz) which are very similar to the respective differences of the A conformer (ΔA = +7.8 MHz, ΔB = +6.4 MHz, ΔC = +5.2 MHz). A comparable deviation between theory and experiment can be expected for members of the same family. For the syn-conformers the deviations are higher, supporting the assignment that the grouping into syn- and anti-conformers is correct.
In Table 4 the least squares of the differences of the rotational constants of the A, B, and C conformers with the respective calculated values of anti-conformers of Gpy(out) and (up) and Gph(out) and (up) are determined. The A band shows the lowest χ2 value and thus the best agreement with Gpy(out)/anti. The B band has its best least square with Gpy(up)/anti and the C band shows the best agreement with Gph(out)/anti.
Conformer | Gpy(out)/anti | Gpy(up)/anti | Gph(out)/anti | Gph(up)/anti |
---|---|---|---|---|
A | 504 | 29629 | 3561892 | 2327121 |
B | 52801 | 1541 | 2832778 | 1739439 |
C | 3258502 | 2625435 | 11406 | 71777 |
An independent argument for this assignment arises from the comparison of the experimental ground state rotational constants in Table 3 with those from microwave spectroscopy.8 Cabezas et al. identified three conformers on the basis of the nuclear quadrupole constants as Gpy(out)/anti, Gpy(up)/anti and Gph(out)/anti. Since the rotational constants show an excellent agreement with our ground state values, the aforementioned assignment is confirmed unambiguously.
Fig. 4 Relative energies of the 14 most stable conformers of serotonin according to the CC2/cc-pVTZ calculations. All energies are zero-point corrected and given in cm−1. The letters refer to the designation of LeGreve et al.5 |
In order to answer this question we compare the conformational spaces of serotonin and tryptamine on the same level of theory, as shown in Fig. 5. The respective relative energies are given in Table 2. Most of the conformers follow a common trend. The relative energy of a conformer is comparable in tryptamine and the respective anti(OH)-conformation. Rotating the OH-bond to the syn-conformation increases the relative energy by around 120 cm−1. Two conformers do not follow this trend: Anti(ph) and Gph(out). Judging from the distance between the NH2 group and the chromophore this points to a long-range effect between the OH and the NH2 group for Anti(ph). However, the most pronounced effect is observed for Gph(out) where the anti-conformation is stabilized significantly while the syn-conformer is destabilized by a comparable energy.
In Fig. 6 we take a detailed look at the structures of the Gpy and Gph conformers of tryptamine and serotonin. In tryptamine the energetic ordering can be explained by the π–H distances which are increasing in the same way as the conformers are shifting towards higher energies. This confirms the explanation of a hydrogen bond formation between the amino hydrogen and the pyrrole (Gpy) or phenyl (Gph) π cloud as given by Carney et al.14 Addition of the hydroxyl group changes these distances only slightly, so that another effect must be responsible for the modulation of the excitation energy of the Gph(out) conformers.
A possible candidate for the additional stabilization is the interaction of the amino LP and an adjacent hydrogen atom at the indole chromophore (H2 at C2 for the Gpy conformers and H4 at C4 for the Gph conformers, cf.Fig. 1). For the Gpy(up) and Gph(up) conformers of serotonin this can be excluded since the amino LP points away from the hydrogen atom. Also for Gpy(out) no effect is expected, because the distances do not change in comparison to tryptamine. For Gph(out)/anti, however, the amino LP is oriented towards the hydrogen atom H4 and the distance shortens from 288 pm to 283 pm by the addition of an hydroxyl group in anti orientation. In the case of the respective syn-conformer the LP of both the oxygen and the nitrogen atom are oriented towards H4. Hence, a competition of these LP to form a hydrogen bond or an electrostatic repulsion of both lone pairs seems to be a likely explanation for the destabilization of the syn-conformer and an increase of the amino LP hydrogen distance.
In this context one might ask why the additional stabilization through a hydrogen bond between the amino LP and H4 does not happen for the Gph(out) conformer in tryptamine. The answer for this can be given based on the natural charges in the indole chromophore as shown in Fig. 7.
In the pyrrole ring no or small changes between the natural charges of the tryptamine and serotonin conformers can be observed. This is different for the phenyl ring. Here, a pronounced increase in the electron density at C4 and C6 occurs, while C7 and especially C5 become more positive. The main difference between the syn- and anti-conformers of serotonin is the charge distribution at the carbon atoms C4 and C6: the electron density depends on the orientation of the lone pair and is either −0.24 or −0.27. When the lone pair is pointing towards the respective atom, the negative charge is decreased. This trend is in agreement with the results from Srivastava and Singh9 and Oeltermann et al.12
We propose that the increase of electron density at the C4 atom increases the polarity of the CH-bond which activates it for hydrogen bond formation. In tryptamine this activation is not possible. In turn, we assign the leading contribution of the stabilization of the Gph(out)/anti conformer to an intramolecular hydrogen-bond between the amino nitrogen LP and H4-atom at the phenyl ring. In general, a red shift of the C4–H4 stretch frequency due to a hyper-conjugative interaction between the lone pair of the nitrogen and the anti-bonding CH orbital is expected. The calculations, however, predict a blueshift of around 12 cm−1 between the CH vibrations of Gph(out)/anti and the other anti-conformers with Gpy, Gph and Anti conformation.9 It can be explained by the concept of “improper” hydrogen bonds.44 Following the argumentation of Joseph and Jemmis45 hydrogen bonds can be decomposed into long-range and short-range interactions. In the former, the electron density of hydrogen bond acceptor leads to a polarization of the covalent bond. The resulting increase in the electrostatic interaction between C and H leads to a shortened bond length and in a blueshift of the stretch vibration. When the acceptor comes closer the hyperconjugative interaction leads in most cases to the familiar redshift. Mo et al. have computed the change in CH bond length for the system F3C–H⋯NH3 which may provide a guideline for the discussed interaction.46 They found that for donor–acceptor distances larger than 210–240 pm the CH bond length is decreased. As in the present case the distance for the hydrogen bond is around 280 pm it is likely that the electrostatic interaction is dominating, leading to the predicted blueshift.
In case of the Gph(out)/syn-conformer we postulate a destabilization mechanism which arises from an intramolecular competition between the amino nitrogen and oxygen lone pair for the hydrogen bond with the H4-atom. This is backed by the computed C4–H4 stretch frequencies for the Gph(out)/syn and the other syn-conformers with Gpy, Gph and Anti conformation which show a similar blue shift of around 9 cm−1,9 close to our MP2 values.
A closely related system is mexamine (5-methoxytryptamine). The exchange of the hydroxyl group by an methoxy group leads to a decrease in the total number of observed conformers.20 Like in serotonin, also for 5-methoxytryptamine theoretical calculations predict the Gph(out)/anti as lowest energy conformer in the ground state. Additionally, the calculated energy gap between syn and anti increases for the Gph(out) by 200 cm−1 which is in good agreement with the values presented here for serotonin. Hence, the same effect is expected to be responsible for the shape of the mexamine conformational space as well. The same trend was observed for 5-methoxy N-acetyl tryptophan methyl amide, where only one conformer is observed in the IR-UV holeburn spectrum.22 This is assigned to a structure in which one of the LP of an oxygen atom is pointing towards H4. Also for the 5-methoxy substituted N-acetyltryptamine (melatonin)21 the anti-conformers are selectively stabilized. For the two most abundant conformers a hydrogen bond is formed between H4 and the N-acetylamino side chain: either with the LP of the oxygen or the nitrogen atom. Thus, the stabilization of anti-conformers via oxygen atoms connected to position 5 in the indole chromophore seems to be a general effect in these class of neurotransmitters.
Since the anti-conformers are always more stable in the ground state and the syn-conformers in the excited state (cf.Fig. 4) a stabilization of Gph(out)/anti and a destabilization of Gph(out)/syn leads to an increase of the syn–anti gap in the ground state and a decrease in the excited state. This might be the reason for the larger experimental difference in the excitation energies between both Gph(out) conformers.
The present results illustrate that even substituents which do not interact directly can communicate via competitive interactions and have a large influence on the shape of the conformational space. We could show that for various 5-substituted tryptamines (5-hydroxytryptamine, 5-methoxytryptamine, N-acetyl-5-methoxytryptamine, 5-methoxy N-acetyl tryptophan methyl amide) the hydrogen bond between the flexible side chain and H4 is a central motif which governs the most stable structure that can be experimentally observed.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp02130a |
This journal is © the Owner Societies 2016 |