Small Molecule Catalyst for Peptide Synthesis

Abstract

Peptide synthesis is a highly optimized process that has led to the production of new classes of therapeutics and materials. The process of peptide synthesis is straightforward: commercially available, orthogonally protected amino acids can be linked on the solid phase using highly efficient coupling agents. However, the simplicity of peptide synthesis masks a significant drawback of the current method: it is highly wasteful and utilizes a solvent that is facing restrictions on its use. A catalyst that allows solid phase synthesis of peptides in benign solvents without requirement for excess reagents and protected amino acids would have a significant impact. Here, we describe the development of a small molecule catalyst for peptide synthesis. The catalyst design incorporates redox recycling of diselenide and phosphine with air as the ultimate oxidant and phenylsilane as the ultimate reductant. The catalyst affords efficient coupling of amino acids in the solution and solid phase. Significantly, the catalyst functions with acetonitrile, bypassing the need for DMF. The current effort builds on mechanistic analysis of reaction rates and intermediates in our prior work which led to a hydrogen bonding catalyst: [Handoko; ; Panigrahi, N. R.; Arora, P. S. J. Am. Chem. Soc. 2022, 144, 3637-3643]. Here, we significantly simplified earlier designs to afford an easily accessible small molecule catalyst.

1

(A) Our previous work on sustainable peptide synthesis showed that a combination of urea diselenide catalyst 1a and phosphetane oxide 2-[O] can lead to catalytic formation of the amide bond. This dual catalytic system utilizes phenylsilane as the terminal reductant and molecular oxygen as the terminal oxidant to drive the catalytic cycle. (B) ³¹P NMR analysis of key intermediates reveals that the intermolecular reaction between phosphetane 2 and diselenide 1b to generate the key selenophosphonium intermediate 3 is sluggish, requiring approximately 4 h for completion. (C) In the prior work, the selenophosphonium intermediate was generated via an intermolecular reaction between phosphetane 2 and a diaryldiselenide. (D) In the current effort, we developed a small molecule organocatalyst for peptide synthesis wherein the selenophosphonium intermediate is generated through an intramolecular reaction.

2

(A) Proposed catalytic cycle for the redesigned organocatalyst for amide bond synthesis. The proposed cycle begins with the reduction of phosphetane oxide to generate II which is predicted to undergo an intramolecular reaction to generate selenophosphonium intermediate III. The subsequent reaction of carboxylic acid with III would lead to the formation of selenoester V via an acyl transfer step involving intermediate IV. Condensation of the selenoester with an amine facilitates amide bond formation, releasing selenol VI which is then oxidized under aerobic conditions to regenerate catalyst I, completing the catalytic cycle. (B) Preliminary comparison of the catalytic efficiency of the intermolecular catalytic system (1c + 2-[O]) and intramolecular catalytic system 3a and 3b for catalytic amide bond formation.

3

(A) Overview of two distinct synthetic routes developed for phosphetane oxide-tethered aryldiselenide catalysts. (B) Structures of catalysts 3a–3k. These analogs were compared for their efficiency for catalytic amidation for a model reaction between toluic acid 4 and benzylamine 5 to yield 6. Reaction conditions: 4 (50 μmol), 5 (65 μmol), 3a–3k (20 mol %, 10 μmol), PhSiH₃ (2 equiv, 100 μmol) in CH₃CN (1 mL) at 80 °C under air. The %product formation was assessed by HPLC using an internal control (Supporting Information).

4

We used a suite of NMR active isotopes (¹H, ¹⁹F, ³¹P, and ⁷⁷Se) to assess the proposed mechanism. Carboxylic acid (p-fluorobenzoic acid) was chosen to follow product formation by ¹⁹F NMR for these studies. (A) Analogs 3l and 3m were used to confirm that a leaving group on selenium is required for the initiation of the catalytic cycle. Comparison of the performance of 3g under O₂ versus N₂ environments supports the hypothesis that air reoxidation of selenol is needed for the reaction cycle. (B) We simultaneously monitored the ¹⁹F (left) and ³¹P (right) NMRs to identify the selenophosphonium intermediates 3g-III and 3g-VI. (C) Intermediates 14, 3g-III, 3g-VI, and product 15 were identified by NMR.

5

Computational analysis of the impact of ring substituents on the catalyst performance. (A, B) Transition states leading to selenophosphonium adduct III and selenoester V were calculated. Free energies were calculated at the DLPNO–CCSD­(T)/def2-TZVPP//r²SCAN-3c-SMD­(MeCN) level.

1. Catalytic Synthesis of Dipeptides

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Catalytic solid phase peptide synthesis using 3g. HPLC chromatograms (λ 220 nm) of crude peptides (A) Fmoc-KCGFG-NH₂ (17a), (B) Ac-LWFGA-NH₂ (17b), (C) Ac-QCFVAYKCGFG-NH₂ (17c) obtained from catalytic SPPS, and (D) 17c synthesized using standard Fmoc solid phase peptide synthesis (SPPS) using 5 equiv each of HBTU and Fmoc-amino acid per coupling. Detailed reaction conditions and HPLC purification protocols are included in the Supporting Information.