We present the further development and fine-tuning of an efficient, economic (≤3 equivalents of activated amino acid ), and environmentally friendly (6 mL organic waste/cycle) procedure for peptide synthesis using a fast amino acid coupling reaction (1.7 min). The designed setup can assist the synthesis of highly pure (>80%) raw materials even for long (up to 30 aa.) and/or difficult sequences. The significant reduction of the coupling time by using the effective PyAOP and N,N′-diisopropylcarbodiimide/ HOBt coupling reagents was achieved, and virtually racemization-free (L-His/D-His <1−5%) peptides can now be synthesized, even at higher column temperature: T = 70 °C. The purity of the product and the efficiency of the synthesis were evaluated using different solid phase supports and protocols. We successfully completed the synthesis of "difficult" sequences [e.g., insulin B-chain and cecropin A(1−7)-melittin(2−9) hybrid peptide] wasting only a fraction of the organic solvents compared to other methods, furthering peptide chemistry toward a greener approach.

Pseudoproline derivatives are incorporated into polypeptides as aggregation disrupters during synthesis, from which the unmodified side chains can be recovered during the final step (resin cleavage). However, direct coupling to pseudoproline is challenging and thus dipeptides of pseudoproline, Fmoc-Xaa-Yaa(Ψ Me,Me pro)-OH, are traditionally introduced, which requires the entire library of Fmoc-Xaa-Thr(Ψ Me,Me pro)-OH in stock, making this approach expensive. Here, we show how the in situ acylation of the incorporated H-Thr(ΨPro)− can be done successful in flow peptide chemistry, with almost all proteinogenic amino acids (Xaa or X). The Xaa-Thr(ΨPro) amide bond was established with a reagent excess of three to five equivalents, with better than 75% efficiency for most Xaa and 70% in the case of Met. Only for Asp was the efficiency too low (8%) to support the direct use of pseudoproline derivatives.

Two ring size β-Sugar Amino Acids, βSAAs, Fmoc-RibAFU(ip)-OH and Fmoc-GlcAPU(Me,Bn)-OH, as Lego-elements are introduced to make α/β-chimera peptides by flow-based solid phase peptide synthesis (SPPS). Their synthesis alongside selected αamino acids, αAA, is fine-tuned. The recently published 50 % TFA cleavage protocol of tBu protected shorter (Ser, Asp) and larger aromatic (Tyr, Trp), with bulky side chain protected Arg (Pbf) and Gln(Trt) residues was probed. We found that this milder condition is sufficient to successfully remove both the 1,2-O-isopropylidene from RibAFU(ip) and tBu, Pbf and Trt from the other αAA residues, but to preserve the 2,3-di-O-benzyl protection of GlcAPU(Me,Bn). Note that O-benzyl groups can be subsequently cleaved by HF or catalytic hydrogenation. Tuned protocols allow the efficient synthesis of 16-mer penetratin analogues via continuous flow conditions incorporating either RibAFU(ip) or GlcAPU(Me,Bn) βSAAs. Both acid concentration (50 %/95 %) and type (TFA/HF) allow a versatile protecting group removal and thus, to fine-tune the hydrophilicity and aromaticity of the above building blocks in chimera constructs.