In chemistry, a crossover experiment is a method used to study the mechanism of a chemical reaction. In a crossover experiment, two similar but distinguishable reactants simultaneously undergo a reaction as part of the same reaction mixture. The products formed will either correspond directly to one of the two reactants (non-crossover products) or will include components of both reactants (crossover products). The aim of a crossover experiment is to determine whether or not a reaction process involves a stage where the components of each reactant have an opportunity to exchange with each other.
The results of crossover experiments are often straightforward to analyze, making them one of the most useful and most frequently applied methods of mechanistic study. In organic chemistry, crossover experiments are most often used to distinguish between intramolecular and intermolecular reactions.Carroll, Felix A.; Perspectives on Structure and Mechanism in Organic Chemistry; Brooks/Cole Publishing, Pacific Grove, CA, 1998.Brüncker, Reinhard; Advanced Organic Chemistry: Reaction Mechanisms; Academic Press, San Diego, 2002. Inorganic and organometallic chemists rely heavily on crossover experiments, and in particular isotopic labeling experiments, for support or contradiction of proposed mechanisms.Crabtree, Robert H.; The Organometallic Chemistry of the Transition Metals; Third Ed. Wiley, NY, 2001. When the mechanism being investigated is more complicated than an intra- or intermolecular substitution or rearrangement, crossover experiment design can itself become a challenging question.Edwards, John O., Ed; Progress in Inorganic Chemistry: Inorganic Reaction Mechanisms; vol. 17, 1972. A well-designed crossover experiment can lead to conclusions about a mechanism that would otherwise be impossible to make. Many mechanistic studies include both crossover experiments and measurements of rate and kinetic isotope effects.
Purpose
Crossover experiments allow for experimental study of a reaction mechanism. Mechanistic studies are of interest to theoretical and experimental chemists for a variety of reasons including prediction of stereochemical outcomes, optimization of reaction conditions for rate and selectivity, and design of improved catalysts for better turnover number, robustness, etc.Eldik, R. Chem. Rev. 2005, 105, 1917. Since a mechanism cannot be directly observed or determined solely based on the reactants or products, mechanisms are challenging to study experimentally. Only a handful of experimental methods are capable of providing information about the mechanism of a reaction, including crossover experiments, studies of the kinetic isotope effect, and rate variations by substituent. The crossover experiment has the advantage of being conceptually straightforward and relatively easy to design, carry out, and interpret. In modern mechanistic studies, crossover experiments and KIE studies are commonly used in conjunction with computational methods.Crabtree, R. H.; Dalton Trans., 2013, 42, 4104.
Theory
The concept underlying the crossover experiment is a basic one: provided that the labeling method chosen does not affect the way a reaction proceeds, a shift in the labeling as observed in the products can be attributed to the reaction mechanism. The most important limitation in crossover experiment design is therefore that the labeling not affect the reaction mechanism itself.
It can be difficult to know whether or not the changes made to reactants for a crossover experiment will affect the mechanism by which the reaction proceeds. This is particularly true since the aim of the crossover experiment is to provide insight into the mechanism that would allow these types of predictions. There is always the possibility that a label will alter the course of the reaction.
In practice, crossover experiments aim to use the least change possible between the usual conditions of the reaction being studied and the conditions of the crossover experiment. This principle favors isotopic labeling, since changing the isotope of one atom in a molecule is the smallest change that can be both easily enacted and traced in the reaction. If the isotope is placed in the molecule at a position directly involved in the mechanism of the reaction, a kinetic isotope effect is expected. This can be used to study aspects of the mechanism independently or alongside a crossover experiment. The kinetic isotope effect is a change in the rate of reaction based on the change in isotope, not a change in the mechanism of the reaction itself, so isotopic labeling generally satisfies the requirements for a valid crossover experiment. In crossover experiments that do not use isotopic labeling, addition or subtraction of a methyl substituent at a position not involved in any proposed mechanism for the reaction is typically expected to give a valid crossover experiment.
Design
In designing a crossover experiment the first task is to propose possible mechanisms for the reaction being studied. Based on these possible mechanisms, the goal is to determine either a traditional crossover experiment or an isotope scrambling experiment that will enable the researcher to distinguish between the two or more possible mechanisms. Often many methods of mechanistic study will have to be employed to support or discount all of the mechanisms proposed. However, in some cases a crossover experiment alone will be able to distinguish between the main possibilities, for example in the case of intramolecular vs. intermolecular organic reaction mechanisms.
The mechanism of the thermal Claisen rearrangement has been studied by crossover experiment and serves as an excellent example of how to apply this technique. Before the mechanism was determined, it was proposed that the reaction could proceed via an intermolecular or intramolecular route.
Looking at these two proposed mechanisms, it is clear that a crossover experiment will be suitable for distinguishing between them, as is generally the case for inter- and intramolecular mechanisms. The next step in crossover experiment design is to propose labeled reactants. For a non-isotopic labeling method the smallest perturbation to the system will be by addition of a methyl group at an unreactive position.
Predicting the products given by each mechanism will show whether or not a given crossover experiment design can distinguish between the mechanisms in question. This is particularly relevant when employing an isotopic label. It is possible that labeling at one position could distinguish between only two of several possible mechanisms, while placing the isotopic label at a different position could distinguish between three potential mechanisms or provide insight into transition states or intermediates, etc. After the interpretational value is established it is relevant to consider the practical aspects, such as whether or not the synthesis of the proposed reactant is possible, and how easy or difficult it is to distinguish the predicted products for each proposed mechanism and starting materials.
For the Claisen rearrangement, labeling by addition of a single methyl group produces an under-labeled system. The resulting crossover experiment would not be useful as a mechanistic study since the products of an intermolecular or intramolecular mechanism are identical.
To have a sufficiently labeled system, both “halves” of the molecule that would separate in an intermolecular mechanism must be labeled. This is known as a doubly labeled system, and is generally the requirement for a crossover experiment. Predicting the products of each mechanism then shows that the crossover products are distinct from the non-crossover products. Once this has been established and the products predicted, the experiment can be carried out and the products characterized. When isotopic labeling is used the products are often more varied and the distribution of the label more convoluted. In this case it is also important to explicitly predict the relative amounts of the label expected to appear at each position depending on the mechanism.
When the crossover experiment on the Claisen rearrangement is carried out only non-crossover products are observed. Based on this the mechanism is determined to be intramolecular, as depicted in the standard arrow-pushing mechanism for this rearrangement.
Isotopic labeling experiment
An isotopic labeling experiment is an experiment used in mechanistic study that employs isotopes as labels and traces these labels in the products. Isotopic labeling experiments are commonly considered to be a type of crossover experiment. However, there are far more possibilities for the manner of labeling and potential products in an isotopic labeling experiment than in a traditional crossover experiment. The classification of an isotopic labeling experiment as a crossover experiment is based on the similar underlying concept, goal, and design principles in the two experiments rather than on direct similarity. An isotopic labeling experiment can be designed to be directly analogous to a traditional crossover experiment, but there are many additional ways of carrying out isotopic labeling experiments.
Although isotopic labeling experiments have the advantage of using the smallest perturbation to the reaction system, they are limited by the possibility of isotopic exchange with solvent or other species present in the reaction mixture. If the isotopic label exchanges with another isotope of the same atom in the solvent, the results of an isotopic labeling experiment are unusable. This limits the use of deuterium labeling at certain positions in protic solvents, for example. However, this exchange can be useful when investigating interaction with the solvent of a reaction, since isotopic labeling can detect this interaction.
Isotopic labeling experiments have been carried out on the thermal Claisen rearrangement. When the terminal carbon is labeled with 14C, there is only one product, with the isotopic label appearing at the benzylic position. Since the expected product of an intermolecular mechanism is not observed the conclusion matches that of the traditional crossover experiment.
Characterization
A major advantage of the crossover experiment is that the results of the experiment are obtained by direct characterization of the product. The techniques involved are therefore those already familiar to the experimental chemist. Mass spectrometry and NMR spectroscopy are the two most common ways of determining the products and their relative ratios. NMR spectroscopy is particularly useful for isotopic labeling studies that use isotopes of hydrogen or carbon.
IR spectroscopy can be useful in specialized situations, such as when 13CO was used to probe the mechanism of alkyl insertion into metal-carbon monoxide bonds to form metal-acyl complexes. Tracking the 13CO in the products was accomplished using IR spectroscopy because the greater mass of 13C compared to 12C produces a distinctive shift of the ν(CO) stretching frequency to lower energy.
Interpretation
The products that are expected from any given mechanism are determined during the design of the crossover experiment. This can be quite complicated to establish, but it makes for straightforward interpretation of the results. If the observed products match those predicted by a given mechanism, then it is reasonable to conclude that mechanism is operating in the reaction. If the results do not match any expected distribution, it is necessary to consider alternate mechanisms and/or the possibility that the labeling has affected the way the reaction proceeds.
For crossover experiments used to distinguish between intermolecular and intramolecular reactions, the absence of crossover products is less conclusive than the presence of crossover products. This is because solvent cage effects could be masking an intermolecular mechanism.