Chloroformate resin 12 was reacted with L-leucine N'-Boc-hydrazide and triethylamine in chloroform, then capped on treatment with anhydrous dialkylamine. The Boc protection was removed using 4 M HCl in dioxane and the resulting hydrazide 23 was treated with n-butyl nitrite in tetrahydrofuran (THF) at -30 °C to provide acyl azide 24. Notably, THF gave more reproducible results than dimethylformamide (DMF). After a sequence of couplings of acyl azides with amino N'-Boc-hydrazides in THF, acid mediated Boc removal and nitrite activation, the protected peptide was terminated using H-Val-Ot-Bu. The final peptide, H-Leu- Ala-Gly-Val-OH (10) was cleaved from the resin using HBr in TFA and purified on a column of Dowex 50-X4 resin eluting with a pyridine acetate buffer at pH 4.0. In concluding the study, the authors stated that the "overall yields are presently marginal and we do not recommend this new approach as a general replacement for solid phase peptide synthesis. However, it may be a valuable supplement to peptide synthesis…". Subsequently, the Mukaiyama laboratory reported N-C-SPPS using thiopyridyl ester activation (Scheme 4). In a one-pot process, activation of N-Cbz-leucine with 2,2'-dipyridyl disulfide (PySSPy) and triphenylphosphine gave the corresponding N-Cbz-amino thiopyridyl ester which reacted with ethyl glycinate to provide the corresponding dipeptide Cbz-Leu-Gly-OEt in a 96:4 enantiomeric ratio. The thiopyridyl ester activation strategy was applied in preparations of luteinizing hormone-releasing hormone (LHRH) and adrenocorticotropin-(1-24) by N-C-SPPS. The final peptides were claimed to exhibit comparable activity as the native peptides. Scheme 4. Application of thiopyridyl esters in N-C-SPPS