In complex molecules (e. g., in organic compounds), it is important to know the posi­tion of the labeling atom, and in some cases, such a compound has to be synthesized where the labeling atom is in a desired position. In this latter case, the position of the radioactive atom also has to be determined because the chemical reactions initiated by the radiation (Szilard—Chalmers reactions, discussed in Section 6.4) can influence its position.

When the binding energy of the bond between the labeling atom and the neigh­boring atom is relatively low, an exchange reaction can take place between the differently labeled species. For example, when labeling diiodine methane with radioactive iodine isotope (e. g., 131I), three types of molecules are formed:

CH2I2 + CH2131I232CH2I131I (8.9)

The equilibrium constant of the process is:

The equilibrium constant determines the ratio of the differently labeled molecules.

When the binding energy is high, the position of the labeling atom cannot always be changed. For example, in the case of 14C-labeled acetic acid, the carbon atoms of the methyl and carboxyl groups cannot change their positions; therefore, several labeled molecules can be produced. Specifically labeled compounds are synthetized when the labeling atom is at a well-defined position of the molecule. In the case of acetic acid, the radioactive isotope (14C) can be in the methyl group (14CH3COOH) or the carboxyl group (CH314COOH). When every carbon atom is labeled (in acetic acid, this means two labeling 14C isotopes, 14CH314COOH), the molecule is universally labeled. A compound is generally labeled when the labeling atoms are statistically positioned; every labeled atom has the same specific activity, independent of the position in the molecule. In the case of acetic acid, half of the 14C atoms are in the methyl group, the other half are in the carboxyl group, and the substance is the mixture of 14CH3COOH and CH314COOH in 1:1. The different types of specifically labeled compounds can be produced only when the chemical bonds are strong enough. If not, an exchange can take place between the different labeled molecules, which always results in a generally labeled compound.

The preparation of the different types of the labeled compounds demands suitable labeled reagents and synthetic procedures. Generally labeled organic com­pounds can be prepared from 14C-labeled carbon dioxide or acetylene. When acety­lene contains only one labeled carbon atom (14CCH2, discussed in Section 8.6), generally labeled acetaldehyde can be produced by reacting labeled acetylene with water:

14CCH2 1 H2O! 14CH3CHO 1 CH314CHO (8.11)

From the generally labeled acetaldehyde, specifically labeled acetic acid can be produced in the following reactions. At first, acetaldehyde is transformed into methanol and carbon dioxide:

14CH3CHO ! 14CH3OH 1 CO2

CH314CHO ! CH3OH 114CO2 (8.12)

The carbon dioxide is eliminated, so methanol is obtained in which the ratio of the labeled (14CH3OH) and nonlabeled (CH3OH) molecules is 1:1. By carboxylation of methanol with the Grignard reagent in the presence of inactive carbon dioxide,

specifically labeled acetic acid can be obtained, and only the carbon atoms in the methyl groups are labeled (14CH3COOH):

Since half of the methanol molecules are not labeled, half of the acetic acid molecules are also not labeled, and the acetic acid is not carrier-free. The specific activity (radioactivity per mass of carbon) of the acetic acid will be half of the ini­tial, generally labeled acetic acid, since the half of the radioactive carbon atoms was previously lost as 14CO2.

Acetic acid labeled in the carboxyl group can also be prepared. For this, inactive methanol is carboxylated with labeled carbon dioxide (14CO2) in the same reaction (Eq. (8.13)). Universally labeled acetic acid can be synthetized from universally labeled acetylene (14C2H2).

The position of the labeling atom in the organic molecules can be determined by stepwise decomposition reactions. Some possible ways are as follows:

• Schmidt decomposition of carboxylic acids:

(8.14)

• Decarboxylation with copper chromite in boiling kinoline.

• Iodoform reaction, which cuts the bond of the methyl group next CO or CHOH:

R — CHOH — COOH CHI3 1 R—1 (COOH)2 (8.15)

• Oxidation of amino acids with ninhydrin:

ninhydrin

R — CHNH2 — COOH——————- y—— ! R — CHO 1NH3 1 CO2 (8.16)

The position of the labeling atom can be determined by the separation and radio­activity measurements of the products. The side reactions and the isotope exchange in the products, however, may present some difficulties.

The position of the labeling atom also can be determined using biological pro­cesses. For instance, the formation of carboxylic acid from carbohydrates (e. g., malonic acid, citric acid, or tartaric acid) can be mentioned. For example, tartaric acid can be dehydrogenized by tartaric acid dehydrogenase enzyme; the product of the reaction, fumarate, is then oxidized by potassium permanganate. Another possi­bility for determining the position of the labeled atoms in carbohydrates is through their decomposition by acetic acid bacteria, which decompose the carbohydrates to carbon dioxide, acetic acid, and propionic acid. By the separation and activity measurements of the products, the position of the labeled atom can be determined approximately.

Some plants and animals can synthesize labeled compounds. Canna indica can produce generally labeled carbohydrate from 14CO2; pigeons produce uric acid from 14C-labeled nutrients.

The number and position of the labeling atoms must be included in the nomen­clature of the labeled compounds. The International Union of Pure and Applied Chemistry (IUPAC) has recommendations for the formulas and names of the labeled compounds:

The formula of an isotopically substituted compound is written in the usual way except that appropriate nuclides symbols are used. When different isotopes of the same element are present in the same position, common usage is to write their sym­bols in order of increasing mass number.

The name of an isotopically substituted compound is formed by inserting in parentheses the nuclide symbol(s), preceded by any necessary locant(s), letters, and/or numerals, before the name or preferably before the denomination of that part of the compound that is isotopically substituted. Immediately after the paren­theses there is neither space nor hyphen, except that when the name, or a part of a name, includes a preceding locant, a hyphen is inserted. When polysubstitution is possible, the number of atoms substituted is always specified as a right subscript to the atomic symbol(s), even in case of monosubstitution.

Some examples are listed in Table 8.2.