Homework #3 Isotopic Labeling and Crossover experiments
1. The NIH mechanism of enzymatic aryl hydroxylation (named because it was discovered at the
National Institute of Health) was elucidated with an isotopic labeling of tritium in the place of a proton.
The enzyme generates “HO+” which is a strong electrophile, able to react in an Electrophilic Aromatic
Substitution. This experiment was conducted so that the rate could be monitored by how fast the
tritium was displaced, but instead, a puzzling result occurred.
A. Draw the expected EAS mechanism for this enzymatic reaction:
B. Give a mechanism consistent with the actual enzymatic result:
C. The following substrate is different than the one above because only about 5% of the product retains
tritium; most of the tritium is lost (like part A.) Explain mechanistically.
2. What do the following isotope labeling experiments say about substitution of phosphate
monoesters?
3. Cyclopentadiene forms Diels-Alder adducts when stored. Propose an experiment to test whether
these adducts are formed irreversibly, or if they exist in a rapid equilibrium that lies toward the adduct.