On Aromaticity of the Aromatic α-Amino Acids and Tuning of the NICS Indices to Find the Aromaticity Order

The NICS aromaticity indices of the rings in flexible phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and histidine (His) chiral molecules were analyzed. These molecules have several dozens of conformers, and their rings are slightly non-planar. Therefore, the population-averaged NICSpav index was defined, and the NICS scans had to be performed with respect to planes found by the least-squares routine. A rule differentiating an obverse and a reverse ring face in aromatic amino acids was formulated. The NICS scan minima corresponding to the obverse and reverse face were unequal, which prompted us to use the term ring face aromaticity/ring face tropicity. It appeared that for Phe, Trp, Tyr, and His, the reverse face has always had higher ring face aromaticity/ring face tropicity than the obverse one. Despite the NICS modifications, uncertainty about the amino acid aromaticity order remained. This motivated us to use the integral INICS index newly proposed by Stanger as well. Then, the following sequence was obtained: Trp(phenyl) > Phe > Trp(pyrrole) > His > Tyr. The juxtaposition of the INICS indices of amino acids with that of some model rings revealed a fair transferability of the values. Finally, analysis of the substituent effect on INICS demonstrated that the aromaticity of Tyr is the lowest due to the strength of the OH group π-electron-donating effect able to perturb enough the ring charge distribution and its magnetic aromaticity. The NICS calculations were executed using the ARONICS program written within the project.

1 Figure S1. The NICS ZZ scans for aromatic rings in the Phe conformers in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S3 2 Figure S2. The NICS ZZ scans for aromatic rings in the Tyr conformers in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S4 3 Figure S3. The NICS ZZ scans for aromatic pyrrole rings in the Trp conformers in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S5 4 Figure S4. The NICS ZZ scans for aromatic phenyl rings in the Trp conformers in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S6 5 Figure S5. The NICS ZZ scans for aromatic rings in conformers of the His(N ε H) tautomer in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S7 6 Figure S6. The NICS ZZ scans for aromatic rings in conformers of the His(N δ H) tautomer in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level. S8 7 Figure S7. The NICS ZZ scans for aromatic rings in conformers of the His(N δ H) and His(N ε H) tautomers and their mixture His in the gas phase estimated based on (a) total energies and (b) Gibbs free energies calculated at the B3LYP/D3/aug-cc-pVTZ level. In the mixture the total content of the His(N δ H) tautomer is only 1.9 %, therefore black and red lines representing total population and His(N ε H) tautomers overlap.

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8 Figure S8. The population averaged aromaticity indices of rings in the Phe, Tyr, Trp, and His aromatic amino acids (light gray) and selected 6-membered (black) and 5-membered rings (dark gray): (a) and (b) indices taken in points NICS ZZ (min rev ) pav and NICS ZZ (0) pav , respectively. Calculations were done at the B3LYP/D3/aug-cc-pVTZ level. S10 9 Figure S9. The NICS ZZ scans for a series of benzenes monosubstituted with groups of different σ-and π-electron activity. S11 10 Figure S10. The linear correlations between differences of the integral aromaticity indices of rings in a series of benzenes monosubstituted with groups of different σ-and π-electron activity, Δ(INICS ZZ (tot))= INICS ZZ (benzene) -INICS ZZ (substituted benzene), and analogous differences for indices taken in points: (a) NICS ZZ (1) and (b) NICS ZZ (0). Calculations were done at the B3LYP/D3/aug-cc-pVTZ level. Notice that the greater the value of difference the smaller the NICSZZ aromaticity.

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11 Table S1. Energy differences (kcal/mol) and population (%, according to total energies) of the most frequent three conformers in vacuum calculated at the B3LYP/D3/aug-cc-pVTZ level. Data for all conformers found is gathered in S1-S4 of ESI.

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12 Table S2. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the Phe conformers assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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13 Table S3. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the Tyr conformers assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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14 Table S4. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the Trp conformers assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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15 Table S5. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the conformers of the His(N ε H) tautomer assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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16 Table S6. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the conformers of the His(N δ H) tautomer assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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17 Table S7. Total energy, Gibbs free energy and zero-point energy (hartree) and populations determined based on first two kinds of enegy (%) for the conformers of the His(N δ H) and His(N ε H) tautomers assuming T = 298.15 K. Calculations were performed at the B3LYP/D3/aug-cc-pVTZ level.

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18 Table S8. Different NICS aromaticity indices of rings in Phe, Tyr, Trp, and His aromatic amino acids and their three the most populated conformers calculated at the B3LYP/D3/aug-cc-pVTZ level. min, max, and s denote the minimum and maximum value of the population and the standard deviation. Population of the three most frequent conformers were taken according to total energies, T = 298.15 K, or Gibbs free energies, T=298.15 K.

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19    Figure S3. The NICS ZZ scans for aromatic pyrrole rings in the Trp conformers in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level.   Figure S5. The NICS ZZ scans for aromatic rings in conformers of the His(N ε H) tautomer in the gas phase calculated at the B3LYP/D3/aug-cc-pVTZ level.  Figure S7. The NICS ZZ scans for aromatic rings in conformers of the His(N δ H) and His(N ε H) tautomers and their mixture His in the gas phase estimated based on (a) total energies and (b) Gibbs free energies calculated at the B3LYP/D3/aug-cc-pVTZ level. In the mixture the total content of the His(N δ H) tautomer is only 1.9 %, therefore black and red lines representing total population and His(N ε H) tautomers overlap.    (1) and (b) NICS ZZ (0). Calculations were done at the B3LYP/D3/augcc-pVTZ level. Notice that the greater the value of difference the smaller the NICSZZ aromaticity. S13 Table S1. Energy differences (kcal/mol) and population (%, according to total energies) of the most frequent three conformers in vacuum calculated at the B3LYP/D3/aug-cc-pVTZ level. Data for all conformers found is gathered in S1-S4 of ESI.