Rapid Flow‐Based Peptide Synthesis

Abstract

A flow-based solid phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 minutes under automatic control, or every three minutes under manual control, is described. This is accomplished by passing a stream of reagent through a heat exchanger, into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable the continuous delivery of heated solvents and reagents to the solid support at high flow rate, maintaining a maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to traditional batch methods, and, in all cases, the desired material was readily purifiable via RP-HPLC. The application of this method to the synthesis of the 113 residue B. amyloliquefaciens RNase and the 130 residue pE59 DARPin is described in the accompanying manuscript.

Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope

“…For the sake of rapid troubleshooting, we opted to utilize Pentelute’s thermally heated rapid flow-based peptide synthesis platform. 18 In this method, deblock, wash or activated amino acid solutions travel through a heated reactor containing SPPS resin after first being pumped through a narrow stainless-steel coil that is also submerged in the heating bath. This flow setup results in rapid heating of reagents seconds prior to contact with the resin, and ensures a continuous and fresh supply of reagents that have been exposed only to brief heating throughout the coupling reaction.…”

“…18 HO-Trityl ChemMatrix ® resin was loaded with alanine by 1) chlorination with acetyl chloride, treatment with Fmoc-Ala-OH/diisopropylethylamine followed by Fmoc removal. Loading was measured at 0.32 mequiv/g, as assessed by UV absorption of the Fmoc cleavage product.…”

Synthesis of l‐ and d‐Ubiquitin by One‐Pot Ligation and Metal‐Free Desulfurization

“…[26] Unfortunately,w ea nd others found that Thz was unstable under the hydrazide oxidation conditions, and Pentelute's group reported that an unproductive nitric oxide adduct might form. [27] Althoughs everalN -terminal Cys protecting groups have been reported to replace Thz, some of them are unstablet oT CEP (a routine reductant in NCL), [28,29] whereas others requirel ong multistep syntheses. [30,31] Luckily, we noticed that trifluoroacetyl (Tfa) protected primaryo rs econdary amines could be mildly deprotected by treatment with aqueous base.…”

Harnessing the Versatility of Continuous-Flow Processes: Selective and Efficient Reactions

“…More recently, a CF‐based peptide synthesis reactor was constructed that contained a heat exchanger 73. The passage of heated solvent through the resin‐filled column allowed the rapid synthesis of peptides.…”