Fmoc-Removal with Pyrrolidine Expands the Available Solvent Space in Green Solid-Phase Peptide Synthesis

Abstract

Green binary solvent mixtures with a polarity and viscosity close to that of DMF perform similarly in solid-phase peptide synthesis (SPPS). However, while coupling reactions readily proceed in solvents of significantly lower polarity than that of DMF, a high solvent polarity is essential for Fmoc-removal using piperidine, which limits the options for green SPPS solvents. Herein, we report our efforts to expand the available solvent polarity space for green SPPS. We identified pyrrolidine as an efficient base to enable Fmoc-removal in less polar solvent mixtures that also favor coupling reactions, such as dimethyl sulfoxide/ethyl acetate (1:9) and N-butylpyrrolidone/1,3-dioxolane (2:8 and 4:6). Employing less polar binary solvent mixtures in combination with pyrrolidine gave crude peptide purities comparable to or better than for DMF with piperidine in the SPPS of challenging peptide targets. An evaluation of base-dependent side reactions such as diketopiperazine (DKP) and aspartimide formation showed increased side-product formation when using pyrrolidine on DKP- and aspartimide-prone sequences. However, the scaled-up syntheses (5 and 7.5 mmol, respectively) of the peptide therapeutics dasiglucagon (29-mer) and bivalirudin (20-mer) gave good crude peptide purities and purity profiles amenable to SPPS optimization. Pyrrolidine therefore represents a useful alternative to piperidine for Fmoc-removal in an expanded solvent space for green SPPS.

Sustainability in peptide chemistry: current synthesis and purification technologies and future challenges

“…Analogous to piperidine, several secondary amines, namely piperazine, 100 morpholine, 95 a ,101 methyl-piperidines, 4-methylpiperazine, 102 and pyrrolidine 103 have been described to efficiently remove the Fmoc moiety, taking advantage of both basicity for the first step and nucleophilicity for DBF trapping (see Fig. 11).…”

Predicting the Success of Fmoc-Based Peptide Synthesis

“…The conditions of Fmoc chemistry are milder as compared to the more senior Boc chemistry, which led to a shift in the field in the late 1990s, and Fmoc became the chemistry of choice in a majority of peptide laboratories . However, the solvents commonly used in Fmoc chemistry, such as DMF, NMP, and dichloromethane (DCM), are known to be hazardous and considerable research has been done in recent years to identify less toxic solvents as alternatives. − It is currently unclear how PepSySco will perform on peptides produced with synthesis chemistries other than the standard Fmoc chemistry, and the effectiveness of PepSySco might be limited to peptides synthesized with this specific methodology. We plan to evaluate PepSySco on peptide sets that were synthesized using other methods when they become available to us in the future.…”

Environmentally Sensible Organocatalytic Fmoc/t-Bu Solid-Phase Peptide Synthesis

“…The Fmoc/ t -Bu variant of Merrifield’s solid-phase peptide synthesis (SPPS) is the prevalent strategy for efficient and scalable peptide synthesis, although poor atom economy and reliance on hazardous reagents and solvents hamper its sustainability, prompting substantial efforts aimed at Fmoc/ t -Bu SPPS greening . Most notably and in keeping with the above KRAs, several greener solvents ,, and solvent mixtures ,, were reported as replacements for the standard SPPS solvent DMF . On the other hand, little attention has been paid to the use of catalysis in Fmoc/ t -Bu SPPS which currently requires stoichiometric amounts of various coupling agents and large amounts of amine bases, typically piperidine (Pip), for Fmoc deprotections (Figure a, i).…”

“…With regard to conditions for Fmoc deprotections, several alternatives to the classical Pip in DMF have been developed including piperazine, 4-methylpiperidine (4-MP), DBU, and t -butyl amine . With increasing focus on SPPS greening, ,− bases compatible with greener solvents were reported including 4-MP, ,,, NaOH, DBU, 3-(diethylamino)­propylamine, morpholine, and pyrrolidine, although to our knowledge, in SPPS, Fmoc deprotections using catalytic base amounts have not been reported. Nevertheless, as solution-phase Fmoc deprotections using catalytic amounts of DBU have been reported, we reasoned that base-catalyzed Fmoc deprotections could be developed for SPPS as well (Figure b, base catalytic cycle).…”

“…We imagined that using an Fmoc removal base catalytically would not only be beneficial for atom economy but also enable significant reduction of solvent washing as well as minimization of side reactions promoted by the large amounts of strong bases customarily used in SPPS. Here we demonstrate the feasibility of organocatalytic Fmoc/ t -Bu SPPS in an environmentally sensible solvent (ESS), which is not only a green solvent suitable for use in SPPS ,− but also easily amenable to recycling, enabling replacement of hazardous peptide synthesis solvents while also drastically reducing solvent consumption . As a first step, we examined catalytic peptide couplings, and to test the different protocols under adequately challenging conditions, we used (i) a sterically hindered AA (Fmoc-Val-OH) and (ii) minimal excess of AA vs resin (1.1 equiv).…”