Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?

Omar Luna,Fanny Guzmán,Johana Gomez,Fernando Albericio,Sergio Marshall,Constanza Cárdenas

doi:10.3390/molecules21111542

Abstract

The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.

Highlights

The 9-fluorenylmethoxycarbonyl (Fmoc) strategy is the most used strategy in solid phase peptide synthesis (SPPS) and remains valid even forty years after its implementation [1], thanks to the constant development and improvement in reagents and strategies for the different steps [2,3,4,5]. it is commonly understood that coupling is the most demanding reaction in the whole synthetic process, the α-amino deprotection step is crucial in order to secure the quality of the target peptide
Results of high-performance liquid chromatography (HPLC) and mass spectrometry (electrospray ionization (ESI)-MS) for crude synthetic peptides are summarized in Figure 1 and Table 3
Solvents and other chemicals used for high-performance liquid chromatography (HPLC), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and circular dichroism (CD) spectroscopy analyses were of HPLC reagent grade and purchased from Merck

Summary

Introduction

The 9-fluorenylmethoxycarbonyl (Fmoc) strategy is the most used strategy in solid phase peptide synthesis (SPPS) and remains valid even forty years after its implementation [1], thanks to the constant development and improvement in reagents and strategies for the different steps [2,3,4,5]. it is commonly understood that coupling is the most demanding reaction in the whole synthetic process, the α-amino deprotection step is crucial in order to secure the quality of the target peptide. Contrary to tert-butoxycarbonyl (Boc) chemistry, where the α-amino deprotection is carried out in trifluoroacetic acid (TFA), which is the best solvent/reagent to disaggregate the peptide chain, Molecules 2016, 21, 1542; doi:10.3390/molecules21111542 www.mdpi.com/journal/molecules. Fmoc removal is carried out in N,N-dimethylformamide (DMF), which is a worse solvent to disrupt the interchain aggregation, very out often by the presence of(DMF), the ownwhich. Fmoc removal in solidvery phase peptide synthesis (SPPS) proceeds through a two-step disrupt the group interchain aggregation, often favored by the presence of the own Fmoc group [6]. Proceedsring through two-step base, preferably a secondary amine, the subsequent β-elimination that yields a highlybyreactive mechanism: the removal of the acidicand proton at the 9-position of the fluorene ring system a mild dibenzofulvene intermediate is subsequent immediatelyβ-elimination trapped by the secondary amine reactive to form base, preferably (DBF).

Methods

Results

Discussion

Conclusion