Radiolabeling and Isotopic Markers - Introduction

This article addresses radiolabeling and isotopic labeling in the context of organic synthesis. In general, this labeling involves the application of known synthetic methods to target molecules in which at least one atom (or a statistical portion thereof) is present as an isotope other than its naturally most abundant one. Molecules that contain such an isotope are referred to being labeled because such isotopically distinct atoms serve to mark the molecule (or a fragment thereof) for later detection by various means.

All elements can exist as two or more isotopes that differ in the number of neutrons in the nucleus. Some isotopes are stable indefinitely, while others are unstable (radioactive). Radioactive isotopes decay with a defined half-life, and primarily through release of helium nuclei (α particles), electrons or positrons (β particles), and γ radiation. The ready detection of this emitted radiation, even on a very small scale, underlies the utility and high sensitivity of the radioactive label.

This article focuses on the isotopes primarily of interest to organic chemists, which include the non-metal Main Group elements. The isotopes of metals within the Main Group, as well as alkali metals, alkaline earths, transition metals, lanthanides and actinides have many important applications in medicine, geology, inorganic chemistry, but are outside the scope of this discussion. The nuclei considered here are listed in the table below.

Minor Main-group Isotopes

Family	Nucleus	Natural abundance	Half-life
Hydrogen	2H	0.0156%	stable
	3H	trace	4500±8 d
Carbon	11C	~	20 m
	13C	1.1%	stable
	14C	<10-12	5,730 y
Pnictides	13N	~	9.97 m
	15N	0.36%	stable
	32P	~	14.2 d
Chalcogens	15O	~	122 s
	17O	0.038%	stable
	18O	0.21%	stable
	33S	0.76%	stable
	34S	4.3%	stable
	35S	~	87 d
	36S	0.02%	stable
Halogens	18F	~	110 m
	36Cl	700 × 10-15	3.01 × 105 y
	76Br		16.2 h
	123I		13.2 h
	124I		100.3 h
	125I		59.4 d
	131I		8.02 d

d: days; m: minutes; s: seconds; y: years

The person generally credited with developing the radiotracer technique is the Hungarian radiochemist György Hevesy (George de Hevesy), who was awarded the 1943 Nobel Prize in Chemistry for this achievement. His initial published experiments nearly a century ago involved the use of ²¹⁰Pb (radium-D) and ²¹²Pb to study the chemistry of lead, and later to conduct tracer studies in plants.

The analysis methods used in the present time are significantly more sophisticated. Once prepared, labeled compounds meet a variety of fates, but the goal is ultimately to detect the labeled molecule, fragment or metabolite. The differences between isotopes in terms of chemical behavior are nearly negligible for all elements but hydrogen, and in fact this is the reason isotopic labeling is used. The presence of the label is usually assumed to exert no effect on the physical or chemistry of the molecule, and it thus serves as a label or marker that allows normal chemical or biochemical processes to be monitored without causing any interference. When a new labeled molecule is prepared based on a biologically active compound, it is necessary to verify that it behaves substantially the same as the parent, by determining that the probe is active in vitro to ensure that attachment of the label has not impaired the biological activity.

For a radiolabeled compound, the minimal levels of radioactivity in the environment provide a sufficiently low background against which it can be detected. Additionally, since the amount of radiation released by the radiolabeled molecule is independent of structure, accurate quantitation of the parent molecule and any degradation or metabolic products can be done without the use of reference standards so long as the radioactive atom is still attached.³⁾ Only very small amounts of radiolabel are required for analysis and detection, while the health hazards, cumbersomeness in handling, and costs increase with the amount of label present. For this reason, many researchers dilute the label by adding unlabeled or “cold” compound as a carrier.

Given that the isotopically-enriched precursors employed in preparing these labeled compounds are more expensive, and that the use of radioactive compounds can entail a risk of contamination or damage to health, the synthetic methods employed should optimally be highly efficient (high yield) and simple, and the labeled component is best introduced as late in the synthesis route as possible. In practice, however, yield becomes secondary to expediency, reflected in the speed of conversion or minimization of waste.

Thus, the rapidity of the procedure is particularly important in the case of radioisotopes with relatively short half-lives such as ¹¹C (half-life: 20 min) and ¹⁸F (half-life: 110 min). The use of radioisotopes with even shorter half-lives, such as ¹³N (half-life: 9.97 min) and ¹⁵O (half-life: 2 min), in synthesis procedures is generally not practical, although these do find applications when used in simple inorganic forms such as cyanide, ammonia, water, oxygen gas and carbon monoxide and dioxide. When using these relatively short lived radioisotopes, yield is often sacrificed for rapidity, as the objective is to have the final product sterile, pyrogen-free and in a physiological vehicle within three half-lives.

General approaches used to promote efficiency include the use of one-pot reactions, micro-reactors, ultrasound, microwaves,tt and solid support-linked reactants and reagents.¹⁾ This area is the subject of a recent review.²⁾ When it is not possible to introduce a radiolabel with a short half-life as the final step in a sequence, any remaining steps should likewise proceed rapidly and cleanly. An example is the use of “click chemistry” to incorporate a label into a radiopharmaceutical.¹⁹⁾ The application of click chemistry to ¹⁸F- and ¹¹C- containing intermediates has been covered in a recent review,³⁵⁾ and this method can also be adapted to parallel synthesis.

Finally, it was mentioned above that isotopes differ little in terms of chemical behavior except for hydrogen. This is related to the fact that the change in nuclear mass is substantial in the series of hydrogen (¹H) → deuterium (²H) → tritium (³H), and the differences in the zero point energy of bonds to these hydrogen isotopes are significant and affect rate of any reaction in which that bond is broken or formed. More detail about this kinetic isotope effect is outside the scope of this article, but it is sufficient to note that when a molecule is labeled with a hydrogen isotope at the position that undergoes reaction (bond cleavage and/or formation), differences in chemical behavior will be evident.

Nomenclature of Radiolabels

The standard way to indicate a labeled compound is to prefix the name of the compound with the isotope designation in square brackets. For example, deuterium oxide (D₂O) would be [²H]H₂O by this convention. The trivial labels D for deuterium and T for tritium are still used quite commonly, though.

In many cases, the yield for the step in which the radiolabel is introduced is described in terms of the “radiochemical yield” (RY): The yield of a radiochemical separation expressed as a fraction of the activity originally present [IUPAC]. Essentially, this means that the radiolabeling agent is the limiting reagent in the reaction.

Some preparations of radiolabeling agents or final radiolabeled compounds are characterized as “no carrier added” (NCA or n.c.a.); this means a preparation of a radioactive isotope which is essentially free from stable isotopes of the element in question [IUPAC].

Some researchers present a radiochemical yield that is qualified by the expression “c.f.d.”, which means “corrected for decay”. This means that the expected decay over the synthesis cycle is factored into the yield to give a more optimistic representation.

Compounds labeled with non-radioactive isotopes are referred to as SIL (stable isotope-labeled) compounds.

Purpose of Radiolabeling

Isotopic labeling is used to monitor the fate of a molecule or a fragment thereof through the use of detection methods that specifically distinguish the isotope used against a natural abundance background. There are many applications for the specific labeling of molecules with radioactive or stable isotopes. The technique can be used to prepare substrates for the study of reaction mechanism, in either an artificial or a biological medium. It can also be used to trace the movement of a molecule, or its degradation or metabolic product, in vivo, in vitro, or in the environment. In the medical field, a variety of imaging techniques have been developed that rely on materials labeled with radioactive isotopes. The use of radiolabeled compounds is also of critical importance in the drug development process for use as radioligands in lead discovery, as metabolic tracers in development, and in phase IV clinical studies.³⁾ They play a similar role in the compound development process for crop protection chemicals, being used in metabolic investigations and environmental fate studies.