Properties Of Nucleic Acids

Chemical Properties

Deamination Reaction. This reaction is the most important one with regard to production of the seasoning; it converts the amino bond to a hydroxyl bond on the purine base via nitrous acid, which may occur at any stage in nucleotides, nucleosides, and bases. Inosinic acid is produced by this process from adenylic acid (19).

Acylation Reaction. The hydroxyl bond on the sugar site of nucleosides has the ability of acylation. The order of the reaction ability is as follows: 5'-site > 3'-site > 2'-site.

When reacted with POCl3 a product containing a mixture of three kinds of monophosphate esters was obtained; although the ratio of these products changes with the moleratio of water, no selective condition for 5'-phosphate was found.

Hydrolysis by Acid or Alkali. Both pyrimidine bases and purine bases are relatively resistant to the attack of acid or alkali under ordinary conditions, except that those with an amino group have a tendency to convert into a hydroxyl group when reacted with acid. Any AT-glycoside bond of a nucleoside is relatively stable under alkali conditions, but not under acid conditions. The AT-glycoside of pyrimidine-riboside is, contrarily, stable under acid conditions, which is related to the existence of the double bond between the 4- and 5-positions of the pyrimidine, for when this double bond is cleaved by an addition of hydrogen or bromide, N-glycoside is easily hydrolyzed with acid. The jV-glycoside of a purine-riboside is easily hydrolyzed by acid as in the case of ordinary Af-glycoside, but in the case of reaction of nitrous acid to adenosine or guanosine, no change in the site of the AT-glycoside bond occurs, these two are converted to inosine or xanthosine, respectively. On the other hand, the JV-glycoside of purine-deoxyriboside is extremely unstable under the reaction of nitrous acid.

In acid or alkali, the phosphate bond, conversely to the iV-glycoside bond, is easily hydrolyzed by alkali and comparatively resistant to acid. Hence, when nucleotide was treated with alkali, both nucleoside and phosphoric acid were produced; with acid, on the other hand, base and pentose phosphoric acid ester were produced. But under conditions in the vicinity of pH 4, it is reported that the cleavage of a P = 0 bond occurred by a quite different mechanism.

DNA is stable with alkali but is unstable with acid, as mentioned earlier; the purine base is released by a cleavage of the iV-glycoside bond of purine-nucleotide sites. RNA is easily hydrolyzed both with alkali and with acid; when in 1 N alkali solution for one day at room temperature, RNA decomposed to the mixture of mononucleotide and 2'-and 3'-nucleotide. When in 1 N HC1 for 1 h at 100°C, RNA decomposed qualitatively into base, phosphoric acid, and ribose. Nevertheless, it is impossible to obtain 5'-nucleo-tides by using a reaction of the acid or alkali. For this purpose, an enzymatic hydrolysis method is utilized to obtain 5'-nucleotide from RNA.

Physical Properties

Tautomerism of the Bases. The pyrimidine base has two OH bonds that exist tautomerically (20), in the enol and lactam forms, respectively. Both in the solid state and in solution, pyrimidine is found to exist in the lactam form, as found from the IR spectrum (solid state) and the UV absorption spectrum (in solution) compared with relative compounds. Hydrogen in the NH = bond, which is located at the 3-position of pyrimidine, reacted with pentose, which constitutes a nucleoside. The ionic form of these pyrimidine bases changes with changing pH or solvent as cationic form (NH+ at the 3-position of the ring), anionic form (= 0 at the 4-position, changeable to an -OH" bond), which shows the same amphoteric substance as amino acids. A purine base has both OH" = and NH2 = bonds in the ring and shows the same both tautomerism and amphoteric character that a pyrimidine base does—lactam form for oxypurine (inosine, guanine) and amino form for aminopurine (adenine).

Nucleoside is also an amphoteric substance. No substantial change of physical properties is observed, except for the optical rotation caused by the induction of pentose, which changes with changes in pH in solution.

Localization of the Attachment and Detachment of Protons in Nucleosides and Nucleotides. It is natural to expect that a proton would be attached to those atoms in the molecule on which the highest net negative charge due to n electrons is concentrated, to the nitrogen atoms forming the two c bonds. The possible sites of protonation (shown inside the circle in Fig. 9) in the predominant tautomeric forms of the bases are N3 in cytosine; Nl, N3, and N7 in adenine; N3 and N7 in guanine and hypoxanthine; and N7 in xanthine. From the theoretical calculation, which takes into account

Adenine

Guanine

Hypoxanthine

Xanthine

Figure 9. Chemical structure of various nucleosides; R denotes a hydrogen atom or various radicals.

Cytosine

Adenine

Guanine

Hypoxanthine

Xanthine

Figure 9. Chemical structure of various nucleosides; R denotes a hydrogen atom or various radicals.

the lone pair electrons, the position of the attachment of the proton is predicted to be the N1 position of adenine and the N7 of guanine and hypoxanthine. The results of X-ray structural analysis confirm the correctness of these conclusions regarding the site of proton attachment to the basis moieties of crystalline protonated bases, nucleosides, and nucleotides. On the basis of the UV spectra of bases, the location of protons in the ionized compounds can be established precisely.

Arguments in favor of protonation of adenosine at N1 and of guanosine at N7, as well as evidence of the protonation of cytidine at N3, at least in nonaqueous solvents, are given by the results of NMR spectroscopy (21).

Ionization Constants of the Bases of Nucleic Acids. Ionization constants are expressed in the form pKa = — log^.

Some of the bases of nucleic acids possess strong basic properties and are protonated in a weakly acidic medium but are deprotonated only in a strongly alkaline medium; other bases are weak acids and, although they form anions in a weakly alkaline medium, they are protonated only in a strongly acid medium. The first group includes cytosine and adenine; the values of pKa are associated with

The second group includes thymine and uracil; the values of pKa are

Hypoxanthine, xanthine, and guanine show an intermediate position; they are protonated at comparatively high pH values for these compounds in accordance with equation 1 and are deprotonated in accordance with equation 2 at fairly low pH values in the alkaline region. Accordingly, their acid-base properties are described by two values of pKa. Phosphate groups in nucleotides exert an appreciable influence on the pKa value of the base. Values of the corresponding ionization constants pKa for base, nucleosides, and nucleotides are given in Table 11.

The reason for the increase in pK'a in series observed— nucleoside < nucleoside-2', and 3'-phosphates < nucleo side^'-phosphate—is evidently interaction of the ionized base with the phosphate group of the nucleotide.

It can be concluded from the conformation of ribose and deoxyribose that the possibility of such interaction is particularly great in the case of nucleoside-5'-phosphates, the phosphates groups of which may be in spatial proximity to the ring of the base. On dissociation of uracil derivatives, interaction between the negative charge on the ionized base and the negative charges of the phosphate group must lead to a decrease in the stability of the ionized form of the base and the shift of equilibrium toward the neutral form; pK'a must increase in this case (22). These pK'a values are utilized for the separation of these bases by ion exchange chromatography or electrophoresis.

Characteristics of Inosinic Acid and Guanylic Acid

Regarding 5'-nucleotides, both inosinic acid and guanylic acid are used as umami seasoning. The characteristics of these substances are summarized here (23).

Inosinic Acid. The common forms are 5'-inosinic acid, inosine 5'-phosphate, and inosine 5'-monophosphoric acid (5'-IMP). Molecular formula: C10H13O8N4P; molecular weight 348.2; C: 34.9 (%), H: 3.76 (%), O: 36.76 (%), N: 16.09 (%), P: 8.90 (%).

5'-IMP, which was the first nucleotide discovered by von Liebig in 1847, was isolated from the broth extracted from muscle as a salt of barium. It is said that von Liebig recognized that this inosinic acid related to the taste of the meat extract broth. In 1913, 5'-inosinic acid histidine salt was isolated from the extract of dried bonito as a umami component by Kodama. Later, the contribution of histidine to the umami taste was denied.

Inosinic acid, which is produced by a conversion of 5'-AMP through enzymatic deamination reaction, is found in the muscle of any kind of animals. In the metabolic pathway of nucleotides of microorganisms, 5'-IMP is produced first and then is converted to 5'-AMP by an amination reaction, and then proceed to 5'-XMP by oxidation with the aid of NAD; it then goes to 5'-GMP by a second amination reaction. Hence, 5'-IMP in microorganisms was observed when any enzymatic reactions in the pathway were blocked.

Table 11. pKâ Values for Bases, Deoxyribonucleosides, and Ribonucleosides

Nucleotides

Nucleotides

Table 11. pKâ Values for Bases, Deoxyribonucleosides, and Ribonucleosides

Derivatives

Base

Deoxyribonucleosides

Ribonucleosides

2'-Phosphate

2 ' (3 ' )-Phosphate

3'-Phosphate

5'-Phosphate

2',3'-Cyclic phosphate

Uracil

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