Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

The overall goal of this procedure is to conveniently isolate unnatural amino acids from a nickel shift base complex, and subsequently Fmoc protect these amino acids. This method can help answer key questions in the organic chemistry field, such as how to more easily synthesize unnatural amino acids. The main advantage of this technique is that it allows for isolation of amino acids with acid labile side chain protecting groups from a nickel shift base complex.

Individuals new to this method will struggle because there is a limited solubility of the nickel complex in organic solvents. DMF is the most appropriate solvent because it can resolve the complex and is miscible with water. We first had the idea for this method when we received insight from our inorganic professor, Dr.Boron about nickel and metal chelating agents.

To begin this procedure add 40 milliliters of DMF to a 250 milliliter round bottom flask. Dissolve one millimole of the nickel PBB shift base complex and the DMF with stirring at room temperature. Next add 60 milliliters of 0.2 molar aqueous EDTA solution at pH 4.5.

Using a magnetic stir bar and stir plate, stir the solution over night. After the reaction is complete transfer the solution to a 250 milliliter separatory funnel. Add 50 milliliters of DCM.

Then cap the separatory funnel and mix. Drain the organic wash into a waste beaker. Repeat the process of adding DCM mixing and removing the organic wash three times.

After this collect the remaining aqueous layer in a 250 milliliter round bottom flask. First use solid sodium bicarbonate to adjust the pH to the isolated aqueous layer to pH seven. Next add 168 milligrams sodium bicarbonate.

Stir the solution using the magnetic stir bar and stir plate. It's critical to adjust the pH to seven for adding two equivalents of sodium bicarbonate. This is important because the solution needs to be slightly basic to facilitate the Fmoc protection.

In a 10 milliliter vial dissolve 337 milligrams Fmoc and hydroxysuccinimide ester and five milliliters of dioxane. Transfer this mixture to the aqueous solution and stir over night. The next day use one molar hydrochloric acid to acidify the solution to pH two.

After this transfer the reaction to a 250 milliliter separatory funnel. Add 50 milliliters FO-acetate. It is important that the pH does not drop below two and that the step is completed quickly.

Failure to do so could result in the loss of side chain protecting groups. Cap the separatory funnel and mix well. Then collect the organic layer in a 250 milliliter Erlenmeyer flask.

Repeat the process of adding ethyl acetate, mixing and collecting the organic layer two additional times, combining the organic extracts. Next dry the combined organic extracts with approximately three grams of magnesium sulfate. Using a rotary evaporator, concentrate the combined organic extracts to afford the crude Fmoc protected amino acid.

In this study an amino acid back bone is isolated from nickel shift base complex under mild pH conditions and then subsequently undergoes Fmoc protection through two critical steps. In the fist step a DMF water solution containing EDTA is stirred to facilitate the release of the amino acid from the complex. After the released amino acid is isolated and extracted it is subjected to Fmoc protection conditions affording an Fmoc protected amino acid.

The progress of the hydrolysis reaction is tracked by monitoring the color change of the solution from red to white. Reactions involving fewer than eight equivalents of EDTA show some color transition but are always incomplete. While reactions without EDTA show no color change at all.

Therefore, at least eight equivalents of EDTA are needed to bring the reaction to completion. The effective pH on the hydrolysis reaction is then assessed. Successful hydrolysis is seen under pH conditions ranging from 4.5 to 7.5 at room temperature with over night stirring.

Which demonstrates the flexibility of EDTA hydrolysis. The feasibility of these hydrolysis conditions is then tested using a range of amino acids with differing side chain protecting groups. In each case the side chain protecting groups are fully retained as evidence by proton NMR.

Once mastered this technique can be done in 48 hours if it is performed properly. While attempting this procedure it's important to remember to adhere to the specified pH level described throughout. Following this procedure other methods like hydrolysis of other amino acids with acid labile side chain protecting groups can be performed in order to answer additional questions like how useful this technique can be across a variety of substrates.

After its development this technique will pave the way for researchers in the field of organic synthesis to explore the synthesis of unnatural amino acids with acid labile side chain protecting groups. After watching this video you should have a good understanding of how to isolate amino acids from a nickel PPB shift base complex using reaction conditions with mild pH ranges. Don't forget that working with organic solvents specifically DMF, can be extremely hazardous and precautions such as use of personal protective equipment should always be taken while performing this procedure.