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

We hypothesized that removal of the Ni²⁺ from the Ni-PBP-Gly complex could allow an efficient aqueous hydrolysis of the Schiff-base without the need for harsh pH conditions. As EDTA is an inexpensive and well-studied chelating agent,¹⁰ we hypothesized that addition of EDTA to a solution of Ni-PBP-Gly would facilitate chelation of Ni²⁺ ions, thereby promoting hydrolysis of the complex.

To test our theory that EDTA alone could effectively promote hydrolysis of the complex, we subjected a solution of Ni-PBP-Gly in DMF at room temperature to increasing equivalents of EDTA in pH 4.5 aqueous solution, the unadjusted pH of aqueous EDTA disodium salt solution. Progress of the hydrolysis reaction can be monitored by the observing the color of the solution; unreacted Ni-PBP-Gly in solution displays a deep red color while PBP isolated from this complex is white. We tracked the progress of the hydrolysis reaction by monitoring the color change of solution from red to white (Table 1). Reactions involving fewer than eight equivalents showed some transition of coloring from red to white, but the reaction was incomplete in each case. No color change was evident for conditions without EDTA.

The effect of pH on hydrolysis was similarly assessed. Subjection of the Ni-PBP-Gly complex to hydrolysis conditions using 12 equivalents of EDTA overnight with varying pH at room temperature showed successful hydrolysis of the complex occurs under pH conditions ranging from 4.5 to 7.5. This demonstrates the flexibility of EDTA hydrolysis; pH can be increased to account for more acid-sensitive side-chain protecting groups.

Table 1: Ni-PBP-Gly Hydrolysis Conditions.

To verify that hydrolysis was complete using the most efficient condition, 7, we extracted the reaction three times with dichloromethane, combined and dried the organic layers and analyzed the resulting residue using nuclear magnetic resonance spectroscopy (NMR). The NMR spectrum showed no evidence of Ni-PBP-Gly in the sample; the only resonances found in the spectrum matched literature reports of PBP, suggesting complete hydrolysis of the complex.

We examined the feasibility of our optimal hydrolysis conditions with amino acids containing a range of side-chain protecting groups. Samples of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu(tBu)-OH),Fmoc-O–tert-butyl-L-threonine (Fmoc-Thr(tBu)-OH),Fmoc-O–tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), andN_(in)-Boc-N_α-Fmoc-L-tryptophan (Fmoc-Trp(Boc)-OH) were dissolved in DMF and subjected to hydrolysis at room temperature using 12 equivalents of EDTA at pH 4.5. After being allowed to stir overnight, the solutions were extracted with dichloromethane and analyzed by NMR. A representative NMR is included for Fmoc-Glu(tBu)-OH (Figure 3). In each case, spectral data showed full retention of side-chain protecting groups.^11,12,13

Figure 3: Representative NMR of an Fmoc-monomer Bearing an Acid-labile Side-chain Protecting Group after Subjection to Hydrolysis Conditions.
The acid-labile side-chain protecting group is highlighted with a blue box. Rounded integration values for the proton signals of this compound are included in parentheses. Please click here to view a larger version of this figure.

After hydrolysis and organic extraction, the remaining aqueous layer contains the free amino acid along with residual EDTA and Ni²⁺ ions. To ensure that the presence of these substances would not interfere with subsequent Fmoc protection of the free amino acid, we performed a test reaction by dissolving glycine in an aqueous solutions containing EDTA and Ni²⁺ ions with concentrations similar to hydrolysis entry 7. We then subjected this solution to a standard aqueous Fmoc protection.⁸ After reaction completion and work-up, we determined the reaction afforded a 25% yield of Fmoc-protected glycine. This percent yield is not surprising considering the dilute concentration of the reaction and suggests that the presence of Ni²⁺ ions and EDTA do not interfere with a standard Fmoc protection reaction.

With evidence supporting that each individual component of our methodology (hydrolysis, isolation of free amino acid, and Fmoc protection) is feasible, we performed a proof-of-concept experiment using Ni-PBP-Gly. We assessed the viability of the hydrolysis conditions described above and subsequent Fmoc protection using a standard protocol⁸ to afford an Fmoc-protected amino acid in reasonable yield. To a stirred solution of Ni-PBP-Gly (404 mg, 0.971 mmol, 1 equiv) in 40 mL DMF at room temperature was added 0.2 M EDTA solution at pH 4.5 (65 mL, 13 mmol, 13 equiv). The reaction was allowed to stir overnight and then washed four times with dichloromethane. The aqueous layer was then adjusted to pH 7 using solid sodium bicarbonate. To the aqueous layer was added sodium bicarbonate (163 mg, 1.93 mmol, 2 equiv) and Fmoc-OSu (327 mg, 0.971 mmol, 1 equiv) dissolved in a minimal amount of dioxane. The reaction was allowed to stir overnight and then acidified with 1 M hydrochloric acid and extracted three times with ethyl acetate. The organic extracts were combined, washed six times with brine, concentrated, dried with magnesium sulfate, and dried under vacuum to afford a mixture of unreacted Fmoc-OSu and Fmoc-Gly-OH (160 mg, 0.540 mmol, 54% yield). Spectral data matched previously published spectra for Fmoc-Gly-OH.⁹ This final experiment indicates that it is feasible to isolate free glycine from the Ni-PBP-Gly complex and carry it forward to its Fmoc-protected form.