Liquid Phase Peptide Synthesis via One-Pot Nanostar Sieving (PEPSTAR)

Abstract

Herein, a one-pot liquid phase peptide synthesis featuring iterative addition of amino acids to a "nanostar" support, with organic solvent nanofiltration (OSN) for isolation of the growing peptide after each synthesis cycle is reported. A cycle consists of coupling, Fmoc removal, then sieving out of the reaction by-products via nanofiltration in a reactor-separator, or synthesizer apparatus where no phase or material transfers are required between cycles. The three-armed and monodisperse nanostar facilitates both efficient nanofiltration and real-time reaction monitoring of each process cycle. This enabled the synthesis of peptides more efficiently while retaining the full benefits of liquid phase synthesis. PEPSTAR was validated initially with the synthesis of enkephalin-like model penta- and decapeptides, then octreotate amide and finally octreotate. The crude purities compared favorably to vendor produced samples from solid phase synthesis.

Keywords: liquid phase peptide synthesis; membranes; organic solvent nanofiltration (OSN); peptides.

Figure 1

Key features of PEPSTAR: Activated Fmoc‐amino acid building blocks couple to chain termini of H‐peptide‐nanostar; piperidine quenches excess amino acid and removes Fmoc; diafiltration washes all reaction by‐products through a membrane; purified H‐peptide‐nanostar is retained in the synthesizer, ready to repeat the cycle. Every process is analyzed in real time by UHPLC‐MS.

Scheme 1

Synthesis of nanostars and chain extension cycle: a) Tris‐hydroxy terminated octagol‐nanostar 1 is converted to Wang‐type, 4, and Rink, 6, nanostar; the Wang anchor is loaded by slow esterification with the first AA before entering the synthesizer. b) Using the Fmoc strategy, peptide‐nanostars are grown in a three‐step cycle of coupling, Fmoc removal, and diafiltration, then removed from the synthesizer for global deprotection.

Figure 2

Variation in reaction rates for a dimer‐nanostar: a) The intermediates of peptide‐nanostar chain extension. b) The variation in the average rates of coupling to form Fmoc‐AA‐Phe‐OMe and of Fmoc removal with solvent polarity; tested with AA=Val, Leu, Glu(Trt), Asp(tBu), Trp(Boc), Arg(Pbf), Ala. As a general trend, solubility of H‐dipeptides increases with solvent polarity. c–e) Variation in concentration with time of 1‐arm 13 and 2‐arm 14 intermediates, and Fmoc‐dipeptide‐nanostar 9, respectively, from different substrate concentrations; red 1 wt %, blue 2 wt % and yellow 5 wt % starting concentration of H‐Thr‐nanostar 8. NB the drop in relative absorbance for 2 wt % after 125 mins in (e) was most likely caused by sample preparation errors, high dilution was necessary in order to quench the reaction prior to the UHPLC‐MS analysis.

Figure 3

Synthesizer design and separation performance: a) Rejection of peptide‐nanostars 8 and 10, and reaction by‐products 11 and 12, by candidate membranes. b) Modelling the retention and purity of dipeptide‐nanostar 10 in single stage or two‐stage membrane separators containing PBI_2005(1); the early dip in the two‐stage yield curve is a result of redistribution of a small proportion of 10 from stage 1 to stage 2. Diavolume is the ratio of cumulative volume of wash solvent introduced to the synthesizer at any given time of diafiltration per volume of stage 1 (DV=Wash solvent volume / stage 1 volume), 1 DV=200 mL. c) Schematic of synthesizer layout, with picture of the synthesizer setup shown in Figure S8. d) Purification of H‐dipeptide‐nanostar 10 in the synthesizer, from the largest by‐products H‐Asn(Trt)‐Pip 11 and DBF‐Pip 12; to the 2‐arm by‐product from diketopeparazine (DKP) can be detected, although this does not affect final peptide purity.

Figure 4

Comparison of the synthesis of model peptides by PEPSTAR and SPPS: a) Synthesis of enkephalin‐like model peptides. b) UHPLC chromatograms for the chain extension of H‐dipeptide‐nanostar, exhibiting the 1‐arm and 2‐arm chain extended intermediates, and Fmoc removal. c) Rise of H‐peptide‐nanostar rejection with peptide length; NB the error bars for stage 2 rejection (not shown) are large, due to low concentrations and corresponding variation in peak integrals above noise. d & e) Comparison of the crude purities of pentapeptide 17 and decapeptide 19 prepared by vendor SPPS or by PEPSTAR.

Figure 5

Comparison of the synthesis of linear octreotate by PEPSTAR: a & b) Synthesis of linear octreotate amide and acid. c & d) Comparison of the crude purities of SPPS vendor peptides with crude octreotate amide 21 and octeotate acid 23, respectively, prepared by PEPSTAR. e) Comparison of Process Mass Intensity (PMI) and costs of materials for different methods of synthesis for octreotate amide 21.