5 α-Amino Acids In Water: A Review of VCD and ROA Spectra
89
terion (from the german Zwitter, meaning hybrid). the maximum concentration of
the stable zwitterion is characteristic of each individual amino acid and occurs at the
isoelectric point I, which is determined by the formula pI = ½( pK 1 + pK 2 ). Although,
at the isoelectric point, the population of the zwitterion is maximum, the presence
of the other amino acid forms is not always negligible. however, in water, the nonionised neutral form of amino acids is a very minor species.
O
O
NH 3 +
O
H
O
O
O
NH 3 +
O
O
O
O
NH 2
O
O
O
OH
NH 3 +
O
H
O
Scheme 2 Four forms of glutamic acid considered in protolytic equilibria in water.
A change in ph from the isoelectric point to more basic or more acidic conditions increases the population of anions or cations, respectively. moreover, if an
additional dissociating functional group is present in the amino acid molecule, it
gives rise to the formation of more complex equilibria. on the other hand, if an additional dissociating group is absent, the pI value of the proteinogenic α-amino acid
at 25 °C varies between 5.4 and 6.3 (table 5.1). Below or above the ph determined
by pK 1 and pK 2 (table 5.1), the cation or anion are the prevailing forms, respectively. Although the dissociation constants of the amino groups in the α position are
only slightly different from that of ammonia ( pK b = 9.24), this is not the case with
the juxtaposition of the carboxylic group with that of acetic acid. the presence of
the α-amine group causes the pK a to sharply increase to ca. 4.75. In amino acids
bearing an R substituent with ionisable groups, the double cations (histidine, lysine,
and arginine) or double anions (aspartic acid, glutamic acid, but also cysteine and
tyrosine, table 5.1) can prevail at their specific ph levels. For example, in a recent
study on the protolytic equilibria of L-glutamine and L-glutamic acid, three forms
of glutamine and four forms of glutamic acid were found to be present in the ph
range from 0 to 12 (Scheme 2) [36]. Similarly, four forms of aspartic acid were
considered to occur at equilibrium in acetonitrile [37].
5.3.2 Solubility of Amino Acids in Water
measuring amino acids in water requires the formulation of adequate solutions; however, this is very difficult for many of them. Although α-amino acids in their native
conditions are dissolved in various physiological media, their solubilities in water
(table 5.1) range from very large (proline, glycine, and alanine) through moderate
(valine, threonine, isoleucine, asparagine, and glutamine) to low or very low (aspartic
and glutamic acids, lysine, and tyrosine). A change in the surrounding acidity or basicity increases the solubility of the ionic forms of amino acids. Also, the presence of
other water soluble molecules brings about an increase in solubility. Nevertheless, for
quite a number of amino acids, vibrational spectroscopy measurements in water are
very challenging and require tricks [38]. Because of their high polarity, amino acids
are insoluble in inert and slightly polar solvents and barely soluble in ethanol [34].
89
terion (from the german Zwitter, meaning hybrid). the maximum concentration of
the stable zwitterion is characteristic of each individual amino acid and occurs at the
isoelectric point I, which is determined by the formula pI = ½( pK 1 + pK 2 ). Although,
at the isoelectric point, the population of the zwitterion is maximum, the presence
of the other amino acid forms is not always negligible. however, in water, the nonionised neutral form of amino acids is a very minor species.
O
O
NH 3 +
O
H
O
O
O
NH 3 +
O
O
O
O
NH 2
O
O
O
OH
NH 3 +
O
H
O
Scheme 2 Four forms of glutamic acid considered in protolytic equilibria in water.
A change in ph from the isoelectric point to more basic or more acidic conditions increases the population of anions or cations, respectively. moreover, if an
additional dissociating functional group is present in the amino acid molecule, it
gives rise to the formation of more complex equilibria. on the other hand, if an additional dissociating group is absent, the pI value of the proteinogenic α-amino acid
at 25 °C varies between 5.4 and 6.3 (table 5.1). Below or above the ph determined
by pK 1 and pK 2 (table 5.1), the cation or anion are the prevailing forms, respectively. Although the dissociation constants of the amino groups in the α position are
only slightly different from that of ammonia ( pK b = 9.24), this is not the case with
the juxtaposition of the carboxylic group with that of acetic acid. the presence of
the α-amine group causes the pK a to sharply increase to ca. 4.75. In amino acids
bearing an R substituent with ionisable groups, the double cations (histidine, lysine,
and arginine) or double anions (aspartic acid, glutamic acid, but also cysteine and
tyrosine, table 5.1) can prevail at their specific ph levels. For example, in a recent
study on the protolytic equilibria of L-glutamine and L-glutamic acid, three forms
of glutamine and four forms of glutamic acid were found to be present in the ph
range from 0 to 12 (Scheme 2) [36]. Similarly, four forms of aspartic acid were
considered to occur at equilibrium in acetonitrile [37].
5.3.2 Solubility of Amino Acids in Water
measuring amino acids in water requires the formulation of adequate solutions; however, this is very difficult for many of them. Although α-amino acids in their native
conditions are dissolved in various physiological media, their solubilities in water
(table 5.1) range from very large (proline, glycine, and alanine) through moderate
(valine, threonine, isoleucine, asparagine, and glutamine) to low or very low (aspartic
and glutamic acids, lysine, and tyrosine). A change in the surrounding acidity or basicity increases the solubility of the ionic forms of amino acids. Also, the presence of
other water soluble molecules brings about an increase in solubility. Nevertheless, for
quite a number of amino acids, vibrational spectroscopy measurements in water are
very challenging and require tricks [38]. Because of their high polarity, amino acids
are insoluble in inert and slightly polar solvents and barely soluble in ethanol [34].