Schaefer, Csaszar, and Allen have applied the focal point method towards predicting the energies and structures of cysteine.¹ This very high level method refines the structures that can be used to compare against those observed by Alonso² in his laser ablation molecular beam Fourier transform microwave spectroscopy experiment (see this post). They performed a broad conformation search, initially examining some 66,664 structures. These reduced to 71 unique conformations at MP2/cc-pvTZ. The lowest 11 energy structures were further optimized at MP2(FC)/aug-cc-pV(T+d)Z. The four lowest energy conformations are shown in Figure 1 along with their relative energies.

I (0.0)	II (4.79)
III (5.81)	IV (5.95)

Figure 1. MP2(FC)/aug-cc-pV(T+d)Z optimized geometries and focal point relative energies (kJ mol^-1) of the four lowest energy conformers of cysteine.¹

The three lowest energy structures found here match up with the lowest two structures found by Alonso and the energy differences are also quite comparable: 4.79 kJ and 5.81 mol^-1 with the focal point method 3.89 and 5.38 kJ mol^-1 with MP4/6-311++G(d,p)// MP2/6-311++G(d,p). So the identification of the cysteine conformers made by Alonso remains on firm ground.

References

(1) Wilke, J. J.; Lind, M. C.; Schaefer, H. F.; Csaszar, A. G.; Allen, W. D., "Conformers of Gaseous Cysteine," J. Chem. Theory Comput. 2009, DOI: 10.1021/ct900005c.

(2) Sanz, M. E.; Blanco, S.; López, J. C.; Alonso, J. L., "Rotational Probes of Six Conformers of Neutral Cysteine," Angew. Chem. Int. Ed. 2008, 4, 6216-6220, DOI: 10.1002/anie.200801337

InChIs

Cysteine:
InChI=1/C3H7NO2S/c4-2(1-7)3(5)6/h2,7H,1,4H2,(H,5,6)/t2-/m0/s1
InChIKey: XUJNEKJLAYXESH-REOHCLBHBU

Molecular structures can differ depending on phase, particularly between the gas and solution phase. Kass has looked at the protonation of 4-aminobenzoic acid. In water, the amino is its most basic site, but what is it in the gas phase? The computed relative energies of the protonation sites are listed in Table 1. If one corrects the B3LYP values for their errors in predicting the proton affinity of aniline and benzoic acid, the carbonyl oxygen is predicted to be the most basic site by 5.0 kcal mol^-1, in nice accord with the G3 prediction of 4.1 kcal mol^-1. Clearly, the structure depends on the medium.

Table 1. Computed relative proton affinities (kcal mol^-1) of 4-aminobenzoic acid.

Electrospray of 4-aminobenzoic acid from 3:1 methanol/water and 1:1 acetonitrile/water solutions gave different CID spectra. H/D exchange confirmed that electrospray from the emthanol/water solution gave the oxygen protonated species while that from the acetonitrile/water solution gave the ammonium species.

References

(1) Tian, Z.; Kass, S. R., “Gas-Phase versus Liquid-Phase Structures by Electrospray Ionization Mass Spectrometry,” Angew. Chem. Int. Ed., 2009, 48, 1321-1323, DOI: 10.1002/anie.200805392.

InChIs

4-aminobenzoic acid: InChI=1/C7H7NO2/c8-6-3-1-5(2-4-6)7(9)10/h1-4H,8H2,(H,9,10)/f/h9H
InChIKey=ALYNCZNDIQEVRV-BGGKNDAXCD

Following on their prediction that the thiol of cysteine¹ is more acidic than the carboxylic acid group (see this post), Kass has examined the acidity of tyrosine 1.² Which is more acidic: the hydroxyl (leading to the phenoxide 2) or the carboxyl (leading to the carboxylate 3) proton?

Kass optimized the structures of tyrosine and its two possible conjugate bases at B3LYP/aug-cc-pVDZ, shown in Figure 1, and also computed their energies at G3B3. 2 is predicted to be 0.2 kcal mol^-1 lower in energy than 3 at B3LYP and slightly more stable at G3B3 (0.5 kcal mol^-1). However, both computational methods underestimate the acidity of acetic acid more than that of phenol. When the deprotonation energies are corrected for this error, the phenolic proton is predicted to be 0.4 kcal mol^-1 more acidic than the carboxylate proton at B3LYP and 0.9 kcal mol^-1 more acidic at G3B3.

1
2	3

Figure 1. B3LYP/aug-cc-pVDZ optimized structures of tyrosine 1 and its two conjugate bases 2 and 3.²

Gas phase experiments indicate that deprotonation of tyrosine leads to a 70:30 mixture of the phenoxide to carboxylate anions. The computations are in nice agreement with this experiment. (A Boltzmann weighting of the computed lowest energy conformers makes only a small difference to the distribution relative to using simply the single lowest energy conformer.) This demonstrates once again the important role of solvent, since only the carboxylate anion is seen in aqueous solution.

References

(1) Tian, Z.; Pawlow, A.; Poutsma, J. C.; Kass, S. R., "Are Carboxyl Groups the Most Acidic Sites in Amino Acids? Gas-Phase Acidity, H/D Exchange Experiments, and Computations on Cysteine and Its Conjugate Base," J. Am. Chem. Soc., 2007, 129, 5403-5407, DOI: 10.1021/ja0666194.

(2) Tian, Z.; Wang, X.-B.; Wang, L.-S.; Kass, S. R., "Are Carboxyl Groups the Most Acidic Sites in Amino Acids? Gas-Phase Acidities, Photoelectron Spectra, and Computations on Tyrosine, p-Hydroxybenzoic Acid, and Their Conjugate Bases," J. Am. Chem. Soc., 2009, 131, 1174-1181, DOI: 10.1021/ja807982k.

InChIs

1: InChI=1/C9H11NO3/c10-8(9(12)13)5-6-1-3-7(11)4-2-6/h1-4,8,11H,5,10H2,(H,12,13)/t8-/m0/s1/f/h12H
InChIKey=OUYCCCASQSFEME-QAXLLPJCDY

2: InChI=1/C9H11NO3/c10-8(9(12)13)5-6-1-3-7(11)4-2-6/h1-4,8,11H,5,10H2,(H,12,13)/p-1/t8-/m0/s1/fC9H10NO3/q-1
InChIKey=OUYCCCASQSFEME-HVHKCMLZDU

3: InChI=1/C9H11NO3/c10-8(9(12)13)5-6-1-3-7(11)4-2-6/h1-4,8,11H,5,10H2,(H,12,13)/p-1/t8-/m0/s1/fC9H10NO3/h11h,12H/q-1
InChIKey=OUYCCCASQSFEME-XGYCJDCADS

The gas phase structure of the amino acids is in their canonical or neutral form, while their aqueous solution phase structure is zwitterionic. An interesting question is how many water molecules are needed to make the zwitterionic structure more energetically favorable than the neutral form. For glycine, it appears that seven water molecules are needed to make the zwitterion the favorable tautomer.^1,2

Arginine, on the other hand, appears to require only one water molecule to make the zwitterion lower in energy than the neutral form.³ The B3LYP/6-311++G** structures of the lowest energy neutral (1N) and zwitterion (1Z) cluster with one water are shown in Figure 1. The zwitterion is 1.68 kcal mol^-1 lower in energy. What makes this zwitterion so favorable is that the protonation occurs on the guanidine group, not on the amine group. The guanidine group is more basic than the amine. Further, the water can accept a proton from both nitrogens of the guanidine and donate a proton to the carboxylate group.

1N (1.68)

1Z (0.0)

Figure 1. B3LYP/6-311++G** structures and relative energies (kcal mol^-1) of the lowest energy arginine neutral (1N) and zwitterion (1Z) cluster with one water.³

References

(1) Aikens, C. M.; Gordon, M. S., "Incremental Solvation of Nonionized and Zwitterionic Glycine," J. Am. Chem. Soc., 2006, 128, 12835-12850, DOI: 10.1021/ja062842p.

(2) Bachrach, S. M., "Microsolvation of Glycine: A DFT Study," j. Phys. Chem. A, 2008, 112, 3722-3730, DOI: 10.1021/jp711048c.

(3) Im, S.; Jang, S.-W.; Lee, S.; Lee, Y.; Kim, B., "Arginine Zwitterion is More Stable than the Canonical Form when Solvated by a Water Molecule," J. Phys. Chem. A, 2008, 112, 9767-9770, DOI: 10.1021/jp801933y.

InChIs

1: InChI=1/C6H14N4O2/c7-4(5(11)12)2-1-3-10-6(8)9/h4H,1-3,7H2,(H,11,12)(H4,8,9,10)/f/h8,10-11H,9H2
InChIKey=ODKSFYDXXFIFQN-MYOKTFMPCK

Alonso and coworkers have developed the technique of laser ablation molecular beam Fourier transform microwave spectroscopy to detect biomolecules. In a recent paper¹ they determined the structure of the glycine:one water complex – it is of the neutral configuration. They have now examined the conformations of cysteine². The presence of the thiol side group adds considerable complexity to the problem due to the many conformations possible.

The experiment detected six conformers. Determining the structures responsible for each set of signals was made possible by comparing the experimental results with those determined by computation. Alonso computed 11 low energy conformations of cysteine at MP2/6-311++G(d,p). Then comparing the computed rotational constants and ¹⁴N nuclear quadrupole coupling tensor components with the experiment, they were able to match up all six experimental conformers with computed structures. The experimental and computed constants for the three most abundant structures are listed in Table 1. The geometries of all six conformers are drawn in Figure 1.

Table 1.Experimental and computed spectroscopic constants (MHz) for the three most abundant conformers of cysteine.²

^aRelative energy in cm^-1 computed at MP4/6-311++G(d,p)// MP2/6-311++G(d,p).

IIb (0.0)	Ia (450)
Ib (325)	IIa (527)
IIIβc (765)	IIIβb (585)

Table 1. Optimized structures of the six observed conformers of cysteine. Relative energies in cm^-1 computed at MP4/6-311++G(d,p)//MP2/6-311++G(d,p). (Note – the geometries shown were optimized at PBE1PBE/6-311+G(d,p) since they MP2 structures are not available!)

This study demonstrates the nice complementary manner in which computation and experiment can work together in structure determination.

References

(1) Alonso, J. L.; Cocinero, E. J.; Lesarri, A.; Sanz, M. E.; López, J. C., "The Glycine-Water
Complex," Angew. Chem. Int. Ed. 2006, 45, 3471-3474, DOI: 10.1002/anie.200600342

(2) Sanz, M. E.; Blanco, S.; López, J. C.; Alonso, J. L., "Rotational Probes of Six Conformers of Neutral Cysteine," Angew. Chem. Int. Ed. 2008, DOI: 10.1002/anie.200801337

InChI

Cysteine: InChI=1/C3H7NO2S/c4-2(1-7)3(5)6/h2,7H,1,4H2,(H,5,6)/t2-/m0/s1
InChIKey: XUJNEKJLAYXESH-REOHCLBHBU

Poutsma has followed up on the work he reported earlier in collaboration with Kass concerning the gas-phase acidity of the amino acids.¹ Their previous work reported on cysteine,² with the unusual result that the thiol group is more acidic than the carboxylic acid group. (I blogged on this a previous post.) Now, he reports the experimental and DFT acidities of all 20 amino acids, shown in Table 1. The experiments were done using the kinetic method in a quadrupole ion trap with electrospray ionization. The computations were performed at B3LYP/6-311++G**//B3LYP/6-31+G*, following some MM searching to identify low-lying conformations. The computed acidities were obtained relative to acetic acid, i.e. R-CH₂COOH + OAc^- → R-CH₂COO^- +HOAc.

Table 1. Relative acidities (kJ mol^-1) of the amino acids¹

The computed values are in very good agreement with the experimental values. The amino acids are ordered in increasing acidity in Table 1. The order predicted by experiment and DFT are quite close, and the disagreements are well within the error bar of the experiment.

Similar to the result for cysteine, tyrosine also displays unusual acidity. The alcohol proton is more acidic than the carboxylic acid proton. The structures of tyrosine, and its two conjugate
bases, one from loss of the phenolic proton and the other from loss of the carboxylic acid proton are shown in Figure 1. The stability of the tyrosine conjugate base from loss of the phenolic
hydrogen arises from both the stability of phenoxide and the internal hydrogen bond from the carboxylic acid proton to the amine. This is different that in the cysteine case, the thiolate anion is stabilized by an internal hydrogen bond from the carboylic acid group (see Figure 2c here).

tyrosine
Tyrosine conjugate base (loss of phenolic hydrogen)	Tyrosine conjugate base (loss of carboxylate hydrogen)

Figure 1. B3LYP/6-31G* optimized structures of tyrosine and its conjugate bases.¹

References

(1) Jones, C. M.; Bernier, M.; Carson, E.; Colyer, K. E.; Metz, R.; Pawlow, A.; Wischow, E. D.; Webb, I.; Andriole, E. J.; Poutsma, J. C., "Gas-Phase Acidities of the 20 Protein Amino Acids," Int. J. Mass Spectrom. 2007, 267, 54-62, DOI: 10.1016/j.ijms.2007.02.018.

(2) Tian, Z.; Pawlow, A.; Poutsma, J. C.; Kass, S. R., "Are Carboxyl Groups the Most Acidic Sites in Amino Acids? Gas-Phase Acidity, H/D Exchange Experiments, and Computations on Cysteine and Its Conjugate Base," J. Am. Chem. Soc. 2007, 129, 5403-5407, DOI: 10.1021/ja0666194.

InChI

Tyrosine: InChI=1/C9H11NO3/c10-8(9(12)13)5-6-1-3-7(11)4-2-6/h1-4,8,11H,5,10H2,(H,12,13)/t8-/m0/s1

Kass has once again uncovered a simple system that challenges our notions of basic chemical concepts. It is a well accepted notion that the most acidic proton of all of the amino acids is the carboxylic acid one. However, acidities are strongly influenced by the solvent, and the absence of solvent in the gas phase can dramatically alter things.

Kass and co-workers examined the gas-phase acidity of cysteine with computational and
experimental techniques.¹ The lowest energy conformer of cysteine is 1a, characterized by having three intramolecular hydrogen bonds (Figure 1). The next lowest conformer, 1b, has only two intramolecular hydrogen bonds and is 1.5 kcal mol^-1 higher in energy at G3B3.

1a xyz	1b xyz

Figure 1. B3LYP/aug-cc-pVDZ optimized structures of cysteine 1.¹

They optimized a number of different configurations of the conjugate base of cysteine: two conformers from the loss of the carboxylate proton (2a and 2b), two conformers from the loss of the thiol proton (2c and 2d), and one conformer from the loss of the thiol proton of the zwitterion (2e). These structures are shown in Figure 2 along with their relative energies. All of these structures possess two intramolecular hydrogen bonds.

2a (3.1) xyz	2b (3.4) xyz
2c (0.0) xyz	2d (5.1) xyz
2e (10.1) xyz

Figure 2. B3LYP/aug-cc-pVDZ optimized structures of the conjugate base of cysteine 2. Relative energies (kcal mol^-1) in parenthesis computed at G3B3.¹

The gas phase acidity of carboxylic acids is greater than thiols; the deprotonation energy of propanoic acid (CH₃CH₂CO₂H) is 347.7 kcal mol^-1 at G3B3 (347.2 expt.²), about 6 kcal mol^-1 less than that of ethanethiol (CH₂CH₂SH: 355.0 at G3B3 and 354.2 expt.²). However, the computations indicate that 2c is the lowest energy structure of deprotonated cysteine, and 2c comes about by loss of the thiol proton! Te lowest energy cysteine conjugate base from loss of the carboxylate proton is 1a, which is 3.1 kcal mol^-1 higher in energy. Apparently, the hydrogen bonding network in 2c is quite favorable, able to make up for the inherent favorability of a carboxylate over a thiolate anion.

The G3B3 computed deprotonation energy of cysteine is 333.3 kcal mol^-1 (for removal of the thiol proton). Kass determined the deprotonation energy of cysteine using a kinetic and a thermodynamic method. The kinetic method gives a value of 332.9 ± 3.3 kcal mol^-1­, while the thermodynamic method gives 334.4 ± 3.3 kcal mol^-1­. These are in fine agreement with the computed value.

This study ably demonstrates the dramatic role that solvent can play in determining molecular properties. Kass titled the article “Are carboxyl groups the most acidic sites in amino acids?” and answers with “no” – in the gas phase the thiol group is more acidic. He ends the article with an indication that the alcohol of tyrosine may be competitive in acidity with its carboxylic group, too.

References

(1) Tian, Z.; Pawlow, A.; Poutsma, J. C.; Kass, S. R., "Are Carboxyl Groups the Most Acidic Sites in Amino Acids? Gas-Phase Acidity, H/D Exchange Experiments, and Computations on Cysteine and Its Conjugate Base," J. Am. Chem. Soc., 2007, 129, 5403-5407, DOI: 10.1021/ja0666194.

(2) NIST, NIST Chemistry WebBook, 2005, http://webbook.nist.gov/.

InChIs

1: InChI=1/C3H7NO2S/c4-2(1-7)3(5)6/h2,7H,1,4H2,(H,5,6)