This protocol describes the synthesis and application of PODS, a new reagent for the site-specific bioconjugation of proteins and peptides. This technology represents a marked improvement over traditional maleimide-thiol-based bioconjugations. The biggest advantage of this procedure is the simplicity with which the reagent is made.
The method allows the reagent to be synthesized in virtually any laboratory. In a 10-milliliter round-bottom flask, dissolve 100 milligrams of the aminophenyl oxadiazole thiol in three milliliters of methanol. To the solution, add 360 microliters of diisopropylethylamine and a magnetic stir bar.
Seal the flask with a rubber stopper, and stir the solution for 10 minutes at room temperature. Using a one-milliliter glass syringe, poke a hole through the rubber stopper and quickly add 32 microliters of iodomethane to the mixture. Allow the mixture to react for 45 minutes at room temperature.
In a 25-milliliter round-bottom flask, dissolve 387 milligrams of the boc-protected carboxylic acid in 10 milliliters of dichloromethane. To the solution, add 480 microliters of diisopropylethylamine, 264 milligrams of EDCI, 200 milligrams of the aniline, and a magnetic stir bar. Seal the flask with a glass stopper, and allow the reaction to stir for five days at room temperature.
Once the reaction is complete, transfer the reaction mixture to a separatory funnel, and wash three times with five milliliters of one-molar hydrochloric acid. Collect the organic phase and transfer it to the separatory funnel. Then wash the organic phase with one-molar sodium carbonate followed by water.
Next, collect the organic phase and add magnesium sulfate to remove any traces of water. Then filter the mixture using a medium glass frit. Using a rotary evaporator, remove the volatile solvents under reduced pressure to afford an off-white solid.
Following this, redissolve the isolated solid in 10 milliliters of ethyl acetate. Then precipitate the product via the gradual addition of 30 milliliters of cyclohexane. Filter the solution using a medium glass frit to obtain the carbamate product as a white powder.
In a 10-milliliter round-bottom flask, dissolve 30 milligrams of the carbamate in four milliliters of dichloromethane. Slowly add 49 milligrams of 70%m-chloroperbenzoic acid and a magnetic stir bar to the mixture, and seal the flask with a glass stopper. Stir the solution overnight at room temperature, ultimately yielding a yellow mixture.
On the following day, transfer the mixture to a separatory funnel. Then wash the mixture with 0.1-molar sodium hydroxide followed by water. Collect the organic phase and add magnesium sulfate to remove any traces of water.
Then filter the mixture using a medium glass frit. In a 25-milliliter round-bottom flask, dissolve 30 milligrams of the carbamate in two milliliters of dichloromethane. Add a magnetic stir bar to the mixture.
And 400 microliters of trifluoroacetic acid and seal the flask with a glass stopper. Then stir the reaction mixture at room temperature for three hours. Once the reaction is complete, remove the volatiles under reduced pressure at room temperature using a rotary evaporator to obtain an oily residue.
Dissolve the oily residue in seven milliliters of water. And transfer to a separatory funnel. Wash the aqueous solution three times with four milliliters of ethyl acetate.
In a 1.5-milliliter microcentrifuge tube, dissolve 10 milligrams of PODS and 300 microliters of dimethyl sulfoxide. Then add 26 microliters of N, N-diisopropylethylamine to the solution. Dissolve 15.2 milligrams of DOTA NCS in 100 microliters of dimethyl sulfoxide, and combine the solution with the PODS solution.
Seal the microcentrifuge tube, and allow the reaction to incubate overnight at room temperature. Next, dilute 61 microliters of a trastuzumab stock solution with 859 microliters of PBS in a low-protein-binding 1.5-milliliter microcentrifuge tube. To this mixture, add 6.7 microliters of a freshly-made 10-millimolar solution of TCEP in water.
Add 73 microliters of a one-milligram-per-milliliter PODS-DOTA solution to the reaction mixture. Finally, seal the microcentrifuge tube and incubate the solution for two hours at room temperature. The PODS synthesis is robust and reliable.
Deproteination and substitution of the aminophenyl oxadiazole thiol afforded thioether one in quantitative yield. Ligation of thioether one and the boc-protected carboxylic acid yielded compound two in 55%yield. Oxidation afforded compound three in 90%yield.
And removal of the bocs-protecting group yielded PODS in 98%yield. The identity of each product was confirmed via proton NMR, carbon NMR, and high-resolution MS.An isothiocyanate-bearing variant of the chelator DOTA was coupled to the pendant amine of PODS to obtain the bifunctional chelator in 75%yield. The identity of the product was confirmed via proton NMR, carbon NMR, and high-resolution MS.Site-selective bioconjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab is shown here.
The disulfide linkages of the antibody's hinge region were selectively reduced, and the antibody was then incubated with PODS-DOTA to afford the DOTA-bearing immunoconjugate an 80%yield. MALDI-TOF analysis revealed a degree of labeling of 1.8 DOTA per antibody. Which remains consistent across a range of human, humanized, and chimeric IgG1 antibodies.
However, the same conditions produce immunoconjugates with a degree of labeling of 1.5 for murine IgG1 antibodies. Due to the light sensitivity of this compound, it's important to keep all reactions in foil-covered vessels. This will increase the overall yield of the product.
While this protocol describes the synthesis of PODS-DOTA, a similar procedure can be applied to a wide range of potential cargoes, including fluorophores, toxins, and other chelators. This method will facilitate the construction of well-defined, homogenous, and highly-stable immunoconjugates that can be used in a wide variety of fields. It's important to remember that the iodomethane added in step one of this experiment can have harmful effects, and should therefore be used in a fume hood.
