The Organic Chemistry of Isotopic Labelling

James R Hanson

• Publisher: Royal Society of Chemistry
• Year: 2019
• Print ISBN:
  9781849731881
• Edition: 1
• eBook ISBN:
  9781788018180
• Topic: Physical Sciences
• Subtopic: Chemistry
• Index: Chemistry

The chemical synthesis of isotopically labelled compounds is a pre-requisite for many chemical, biochemical and medicinal investigations. The constraints imposed by the requirements for regiospecific labelling and, in some instances, the time-scale of the synthesis often lead to quite different synthetic strategies to those that are used for the unlabelled material. Whilst there are many specialist papers, reviews and long books devoted to particular isotopes, there is no currently available short introductory book devoted to the organic chemistry of isotopic labelling. The aim of this book is to introduce research workers to a variety of methods that have been used to achieve these synthetic labelling objectives before exploring a particular method in detail. It covers a number of different isotopes and the methods that have been used to introduce them into organic compounds. Labelling methods also provide useful undergraduate teaching examples of modern synthetic reactions and their stereochemical consequences using relatively simple substrates. The book will therefore have a wider appeal than just those involved in using isotopes in research such as environmental and pharmaceutical chemists as well as organic chemists.

CHAPTER 1

The Discovery and Detection of Isotopes

1.1 INTRODUCTION

Labelled compounds in which a specific atom has been replaced by an isotope have played a major role in elucidating reaction mechanisms in chemistry, in establishing metabolic pathways in biochemistry and in imaging organs in medicine. This book is concerned with the organic chemistry that has been used in the synthesis of isotopically labelled compounds employed in these studies.

The isotopes of an element have a nucleus with the same number of protons but vary in the number of neutrons that are present. Each isotope of an element has the same atomic number but a different atomic mass. Since the different isotopes of the same element have the same number of protons and electrons, their overall chemical properties are virtually identical, although there may be subtle differences associated with reaction rates. However, the physical properties of isotopes can differ sufficiently to allow their presence to be detected. Some isotopes such as deuterium and carbon-13 are stable whereas others such as tritium and carbon-14 are unstable and undergo radioactive decay. Since isotopes can be detected by differences in their physical properties, they can act as tracers for the origin and fate of particular atoms within a molecule in the course of a reaction. The application of isotopic tracers in chemistry, biochemistry and medicine has led to the solution of many problems of wide interest.

1.2 THE DISCOVERY OF ISOTOPES

The discovery, isolation and utilization of isotopes took place throughout the twentieth century. As their availability and the methods for their detection became more refined, so the nature of the problems that could be examined by isotopic methods changed. After the discovery of X-rays by Röntgen in 1895, the first physical observations of radioactivity were reported in 1896 by Becquerel. In 1898, studies by Marie Curie and her husband Pierre Curie on the uranium ore pitchblende led to the isolation of the radioactive elements polonium, a congener of bismuth, and radium, which was obtained from the barium fraction. Further radioactive elements were then discovered associated with thorium.

Investigations by Rutherford in 1899 into the nature of the radiation emanating from these materials and its response to magnetic fields revealed the presence of α- and β-rays. The more penetrating γ-rays ware detected by the Curies in 1900 in the radiation from radium atoms. The distinction between α-, β- and γ-rays was not only in their deflection by electromagnetic fields but also in terms of their penetrating power. At this time radiation was detected not just by photographic plates but also by the flashes produced by a phosphor, zinc sulfide, a precursor of modern scintillation counting. Early versions of the Geiger counter, which relied on the ionization of a gas permitting an electric discharge, were introduced in 1908. The Wilson cloud chamber was introduced in 1911 and this revealed the track of an α-particle and β-radiation by the condensation of supersaturated moist air on an ionized gas. Many of the physical foundations for the detection of radioactivity were laid before the First World War.

The ideas of radioactive disintegration and radioactive decay followed from the work of Rutherford, Soddy and Becquerel in the early years of the twentieth century. The identification of disintegration or decay series with a non-radioactive end product then followed. The end product is typically lead. A consequence of lead being produced from different decay series was that the measured atomic weight and isotopic distribution of lead vary from one source to another. This has been used in the modern environmental monitoring of the source of lead contamination.

The construction of these decay series led to the term isotope being coined by Margaret Todd and Frederick Soddy in 1913 to denote atoms of the same element having different nuclear masses. Many of the radioactive isotopes that were detected at this time were of the heavier elements.

A precursor of the mass spectrometer, which revealed the presence of isotopes of lighter elements, was described by Thomson in 1912. A gas was ionized in a cathode ray tube and passed through electric and magnetic fields. The ions were deflected and collected on a photographic plate. When neon was examined in this tube, two spots appeared, which were identified as neon-20 and neon-22. In 1913, and then after the First World War, Aston was able to show that these isotopes could be fractionated by careful distillation and by diffusion through a clay pipe, thus paving the way for later diffusion methods of separating isotopes.

The development of the mass spectrometer by Aston from 1919 through the 1920s, and with various modifications by Dempster and Bainbridge in terms of focusing methods and by Nier in 1940 on the accelerator beam, provided a valuable instrument for the detection of isotopes. The number of known isotopes increased rapidly through the 1920s. Other investigations during the 1920s by Mulliken provided evidence for the existence of a number of isotopes of the light elements leading to the detection of species such as ¹⁶O–¹⁸O, ¹⁵N–¹⁶O and ¹³C–¹⁶O.

Developments during the 1930s in the preparation of isotopes laid the foundations for their application as tracers. Methods were found for the enrichment of stable isotopes and the creation of radioactive isotopes of the lighter elements that were of interest to the organic chemist and biochemist. Many of the first examples are found in the 1930s in which isotopic tracers were used in the elucidation of organic reaction mechanisms and in the delineation of biochemical pathways.

Although the existence of deuterium was predicted by Rutherford in 1920, the isotope was not detected by Urey until 1932. In 1923 a suggestion had been made by Kendall and Crittenden that isotopes might be separated by electrolysis, and in 1933 Lewis found that the electrolysis of water led to the concentration of deuterium oxide. A number of other methods of separation were explored at this time, including the distillation of water, the adsorption of hydrogen or water on charcoal, the diffusion of hydrogen through palladium and the reaction of water or acid with metals. However, over the next few years, systematic studies on the electrolysis of water led to the commercial availability of deuterium oxide (heavy water) and to many applications of this isotope.

Exploitation of equilibrium isotope effects in exchange reactions by Urey during the 1930s permitted the enrichment of a number of isotopes in useful quantities. This was applied to the enrichment of nitrogen-15 using the exchange reaction between ammonia gas and ammonium nitrate solution:

The ammonia gas was passed up a column down which a solution of ammonium nitrate was percolating. The nitrogen-15 was concentrated in the ammonium nitrate. Modifications to the construction of the column eventually gave a system which allowed an enrichment to 60% nitrogen-15. The system was modified in 1940 to enrich carbon-13 by using the exchange reaction between gaseous hydrogen cyanide and a solution of sodium cyanide; the reaction was carried out in the presence of sodium sulfite to prevent the polymerization of hydrogen cyanide:

In this case the carbon-13 was concentrated in the gas. It was possible to obtain an enrichment of 25%. Much higher enrichments have been obtained later by other methods.

Water sufficiently enriched in oxygen-18 for chemical studies was produced by Ingold and Watson in 1937 by careful fractional distillation. More recent methods for obtaining these isotopes are described later in the book when the individual isotopes are discussed.

The foundations for the artificial disintegration of elements and the eventual creation of artificial radioactivity were laid in 1919. Rutherford bombarded nitrogen with α-particles and obtained fast-moving protons by the reaction

These were detected by a zinc sulfide phosphor with different sheets of mica being placed between the source and the detector. A number of developments were reported in 1932 which paved the way for the discovery of artificial radioactivity. Up to that time, nuclear disintegration had been induced by naturally produced α-particles. Cockroft and Walton reasoned that the smaller hydrogen nuclei had a better chance of penetrating the atom than the α-particle. They ionized hydrogen by electron impact and accelerated the ions across a potential drop. Bombardment of a target such as lithium led to nuclear disintegration with the formation of pairs of helium nuclei:

In 1932, Lawrence and Livingston designed the cyclotron as a means of accelerating ions using an alternating current and a magnetic field. Ions were repetitively accelerated through the alternating electric field to give them sufficient energy to overcome the electrical repulsion of the nucleus.

Two other discoveries in 1932 were of the positive electron known as the positron and the neutron. The latter was discovered by Chadwick as a consequence of its effect on other elements such as lithium. A commonly used source of neutrons became the bombardment of beryllium atoms by α-particles produced by the decay of radium:

Because of their lack of charge, neutrons have a greater likelihood of penetrating the atom compared with α-particles. High-energy neutrons tend to produce nuclear disintegration. However, if the neutron energy is moderated by passage through water or paraffin wax, the neutron may be captured by a nucleus to produce a nuclear reaction.

In 1933, Madame I. Curie-Joliot and her husband F. Joliot were studying the effect of bombarding boron, magnesium and aluminium with α-particles. They found that positrons were emitted in addition to protons and neutrons. In 1934, they reported that the positron emission continued after the activating radiation had been removed. The radioactive substances producing this secondary radiation were separated from their parents and shown to be the source of the positrons. Thus irradiation of boron nitride gave a radioactive isotope of nitrogen whereas aluminium gave radioactive phosphorus:

These reports stimulated many further studies and by the end of 1935 it was estimated that over 100 new radioisotopes had been discovered. Although many of these were short-lived curiosities, there were a number of considerable importance, such as carbon-11. By the mid-1930s, many of the physical phenomena had been reported which were to underpin the development of the organic chemistry of isotopic labelling.

1.3 EARLY APPLICATIONS OF ISOTOPICALLY LABELLED COMPOUNDS

The isolation in the 1930s of both stable isotopes and radioisotopes of elements found in organic compounds soon led to their application to biochemical and chemical problems. Comparison of the contents of the Biochemical Journal and the Journal of Biological Chemistry between the late 1930s and 1940s and a decade earlier reveals the impact of isotopic methods. Prior to this time, many of the metabolic relationships between compounds had been proposed as a result of feeding large amounts of a putative precursor to a living system, with the inherent dangers of perturbing pathways that such a strategy involves. In 1935, Schoenheimer and Rittenberg reported the catalytic deuteriation of linoleic acid to stearic acid and its incorporation by rats into fats. They also prepared a deuteriated sample of the sterol 5β-cholestan-3-one (coprostan-3-one) and used it to show that it was an intermediate in the biological conversion of cholesterol to coprostanol. The presence of deuterium in the metabolites was established by combustion and the use of micro-density methods to determine the density of the water that was produced. In 1937, the same group prepared amino acids labelled with nitrogen-15. The first experiments used [¹⁵N]glycine and [¹⁵N]hippuric acid and showed that they were absorbed by rats. Subsequent experiments with [¹⁵N]amino acids were used in a form of dilution analysis to detect the amounts of various amino acids in protein hydrolysates such as that of α-lactoglobulin. The ¹⁵N:¹⁴N ratio was measured mass spectrometrically in the ammonia obtained by a micro-Kjeldahl digestion of the amino acid. One of the earliest double-labelling experiments, which was reported in 1940, examined the metabolism of the unnatural amino acid d-leucine to its natural l-enantiomer. The labelled sample contained deuterium in the chain and nitrogen-15 in the amino group. The deuterium was substantially retained in the l-leucine that was produced whereas the nitrogen-15 was lost. The utiliz...

Table of contents

• Cover
• Title Page
• Copyright Page
• Preface
• Contents
• An Introduction to the Organic Chemistry of Isotopic Labelling
• Chapter 1 The Discovery and Detection of Isotopes
• Chapter 2 Labelling Compounds with Carbon-13 and Carbon-14
• Chapter 3 Labelling with Deuterium and Tritium
• Chapter 4 Stereochemical Aspects of Labelling with Hydrogen Isotopes
• Chapter 5 The Synthesis of Labelled Amino Acids
• Chapter 6 The Labelling of Some Compounds of Pharmaceutical Interest
• Chapter 7 Labelling Compounds with the Stable Isotopes of Nitrogen and Oxygen
• Chapter 8 Labelling with Isotopes of Phosphorus, Sulfur and the Halogens
• Chapter 9 Labelling Organic Compounds for Diagnostic Imaging
• Conclution
• Further Reading
• Glossary
• Appendix
• Subject Index