Backbone Amide Linker (BAL) Strategy forN^α-9-Fluorenylmethoxycarbonyl (Fmoc) Solid-Phase Synthesis of Unprotected Peptidep-Nitroanilides and Thioesters¹

Abstract

A novel and general backbone amide linker (BAL) strategy has been devised for preparation of C-terminal modified peptides containing hindered, unreactive, and/or sensitive moieties, in concert with N(alpha)()-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis protocols. This strategy comprises (i) start of peptide synthesis by anchoring the penultimate residue, with its carboxyl group orthogonally protected, through the backbone nitrogen, (ii) continuation with standard protocols for peptide chain elongation in the C --> N direction, (iii) selective orthogonal removal of the carboxyl protecting group, (iv) solid-phase activation of the pendant carboxyl and coupling with the desired C-terminal residue, and (v) final cleavage/deprotection to release the free peptide product into solution. To illustrate this approach, several model peptide p-nitroanilides and thioesters have been prepared in excellent yields and purities, with minimal racemization. Such compounds are very difficult to prepare by standard Fmoc chemistry, including the BAL strategy as originally envisaged.

The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

“…Although most of the synthetic peptides have either a carboxylic acid or a primary caboxyamide group as the C-terminal functionality, there are other C-terminal modified peptides of potential interest in the production of therapeutic agents (Camarero et al, 2000, Gazal et al, 2005, enzymatic substrates (Alsina et al, 1999, Kwon et al, 2004, cyclic peptides (Camarero and Muir, 1997, Zhang and Tam, 1997, Camarero et al, 1998b, Shao et al, 1998, Camarero and Muir, 1999 or intermediates for the chemical engineering of proteins Mitchell, 2005, Muralidharan andMuir, 2006). Several resin-linker systems are now available which allow the generation of C-terminal functionalities such as carboxylic acids, carboxyamides, thioesters, esters, hydrazides and hydroxamic acids (Alsina and Albericio, 2003).…”

Synthesis of Proteins by Native Chemical Ligation Using Fmoc‐Based Chemistry

“…Alsina et al [37] reported the introduction of the α-thioester group at the end of the synthesis of the target peptide. Key to their approach was the use of the backbone amide linker (BAL) [38,39] which was employed to generate medium sized peptide thioesters using an Fmoc-based strategy.…”

“…This problem was minimized by reacting the activated carboxylic group with a preformed amino acid thioester residue [37]. Under these conditions epimerization of the penultimate residue was reduced to 2%.…”

A Designed Well‐Folded Monomeric Four‐Helix Bundle Protein Prepared by Fmoc Solid‐Phase Peptide Synthesis and Native Chemical Ligation

“…[37] The allyl is an orthogonal protecting group on the a-carboxy group, which is simply and selectively removed after completion of the sequential solid-phase peptide synthesis. Glycine-benzyl thioester is then coupled to the resulting free a-carboxy group using standard coupling reagents; [38] thereafter, the desired peptide-thioester is released from the resin through standard TFA-cleavage procedures.…”

“…The C-terminal allyloxycarbonyl group was selectively removed by treatment with tetrakis(triphenylphosphine)palladium (0) The C-terminal thioester of GTD-(1,1') was prepared by coupling GlySBzl (4 equiv) with PyBop (4 equiv), HOBt (4 equiv), and DIPEA (8 equiv) in DMF (6 mL per g of polymer) for 1 h. Gly-SBzl was synthesized as previously described. [38] GTD-(1,1')-SBzl was deprotected and cleaved from the resin by stirring with a mixture of TFA/TIS/H 2 O (95:2.5:2.5, v/v; 15 mL per g of resin) for 2 h at room temperature. After removing the resin by filtration the peptide was precipitated and washed three times with cold diethyl ether.…”