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

Summary

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Abstract

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Introduction

Unnatural amino acids (UAA's) bearing side chains that vary from those of the twenty naturally occurring amino acids found in nature have found utility in a wide-range of applications. Synthesis of these UAA's, however, can be difficult depending on the structure of the side-chains and the stereochemistry of the amino acid backbone. C-H bond activation of glycine in the context of a nickel Schiff-base complex has been used to produce a variety of amino acid derivatives including α,β-diamino acids¹ and UAA's bearing fluorinated² or heterocyclic side-chains.³

After addition of unnatural side-chains, functionalized UAA's are typically removed from the Schiff-base complex by reflux in hydrochloric acid⁴ and are subsequently isolated using ion-exchange chromatography. While generally efficient, this protocol generates amino acids that may be unsuitable for use in solid-phase peptide synthesis (SPPS). The nature of SPPS requires the presence of acid-labile side-chain protecting groups and the strongly acidic nature of typical Ni-Schiff-base decomposition conditions prevents isolation of UAA's with these protecting groups intact. To our knowledge, only one alternative decomposition method has been reported: use of ethylenediaminetetraacetic acid (EDTA) and hydrazine at elevated temperatures,⁵ conditions that themselves may not be suitable for some side-chain protecting groups such as phthalimides.

Figure 1: Synthesis of Ni-PBP-Gly from Ni²⁺, PBP, and Glycine (Gly). Please click here to view a larger version of this figure.

Herein, we report a method for hydrolysis of a Ni-Schiff-base complex, Ni-PBP-Gly (Figure 1). This complex, derived from Ni²⁺, glycine, and pyridine-2-carboxylic acid(2-benzoyl-phenyl)-amide (PBP),⁶ has been demonstrated to be a useful platform for synthesis of a variety of UAA's and is easily accessible using a two-step synthetic route.⁷ Synthesis of this complex is literature-precedented in high yield.⁶ Our results described below demonstrate the applicability of hydrolysis conditions utilizing EDTA at mildly acidic to neutral pH conditions appropriate for use with UAA's bearing acid-labile side-chain protecting groups. Following hydrolysis, the resulting aqueous solution can be isolated and subjected immediately to standard Fmoc protection conditions to afford an Fmoc-protected amino acid (Figure 2).

Figure 2: Hydrolysis and Fmoc-protection of an Amino Acid Isolated from Ni-PBP-Gly. Reaction Conditions: i. EDTA (12 equiv), pH 4.5; ii. Ethyl acetate wash and adjustment to pH 7; iii. Fmoc-OSu (1 equiv), NaHCO₃ (2 equiv). Please click here to view a larger version of this figure.

Protocol

1. Hydrolysis of Ni-Schiff-Base Complex

1. Dissolve 1 mmol of the Ni-PBP-Schiff-base complex in 40 mL of N,N-dimethylformamide (DMF) with stirring in a 250 mL round bottom flask at room temperature.
2. Add 60 mL of 0.2 M aqueous EDTA solution, pH 4.5.
3. Using a magnetic stir bar and stir plate, stir the combined solution overnight. As the Schiff-base complex is hydrolyzed, the color will shift from a deep red to white.
4. After completion of the reaction as indicated by the absence of any red coloring, transfer the reaction to a 250 mL separatory funnel.
5. Add 50 mL dichloromethane, cap the separatory funnel, and mix. Drain the organic wash into a waste beaker. Repeat this process three times to remove PBP and any residual Ni-PBP-Schiff-base complex. Collect the remaining aqueous layer in a 250 mL round bottom flask.

2. Fmoc Protection of Hydrolyzed Amino Acid

1. Adjust the isolated aqueous layer from step 1.5 described above to pH 7 using solid sodium bicarbonate. Check pH periodically using pH test strips.
2. Add 168 mg sodium bicarbonate (2.00 mmol, 2 equiv) to the solution and stir using a magnetic stir bar and stir plate.
3. Dissolve 337 mg Fmoc N-hydroxysuccinimide ester (1.00 mmol, 1 equiv) in a minimal amount of dioxane (roughly 4 or 5 mL) in a 10 mL vial. Transfer this solution to the aqueous solution and stir overnight.
4. After allowing the reaction to stir overnight, acidify the resulting solution to pH 2 with 1 M hydrochloric acid. Check pH periodically using pH test strips.
5. Transfer the reaction to a 250 mL separatory funnel and add 50 mL ethyl acetate. Cap the separatory funnel, mix well, and collect the organic layer in a 250 mL Erlenmeyer flask. Repeat this process two additional times, combining the organic extracts. Dry the combined organic extracts with roughly 3 g of magnesium sulfate.
6. Concentrate the combined organic extracts using a rotary evaporator to afford the crude Fmoc-protected amino acid.

Results

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.

Discussion

The protocol described above is useful in its ability to facilitate the isolation of an amino acid backbone from a Ni-Schiff-base complex under mild pH conditions and subsequent Fmoc protection of this isolated amino acid through two critical steps. The first step involves stirring a DMF/water solution containing EDTA to facilitate release of the amino acid from the complex. Residual complex or organic byproducts can easily be removed with extraction. The second step of this protocol involves an Fmoc protection of the am...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ni-PBP-Gly			Synthesized from published protocol
DMF	Fisher	D119-4
EDTA	Fisher	S311-100
Dichloromethane	Acros	AC610050040
Sodium Bicarbonate	Fisher	S233-500
Fmoc-OSu	Chem-Impex	"00147"
Dioxane	Fisher	D111-500
Hydrochloric Acid	Fisher	A144-500
Ethyl Acetate	Acros	AC610060040
Magnesium Sulfate	Fisher	M65-500
ZEOPrep 60ECO Silica Gel	ZEOChem
Hexanes	Fisher	3200250.650.443
Chromatography Column
pH Test Strips
Rotary Evaporator
250 mL Separatory Funnel
250 mL Round Bottom Flask
Stir Bar
Stir Plate