Selective Dual Protection Of 4R-amino Proline And The Application In Collagen Model Peptide

Objective:Proline is one of the common canonical amino acids which plays an important role in protein stucture. For convenient connection of labeling molecules to large biomoleculars for tracking various biological activities, derivatives with specific side chains of proline were introduced to large moleculars as replacement of proline. Amino proline is a commonly used proline derivative whose side chain is amino group, and amino and imino groups should be efficiently and conveniently protected for application. Collagen contains large amounts of hydroxy proline which is very similar to amino proline in structure, therefore double-protected 4-amino-proline was synthesized as an intermediate for introducing small labeling molecules into collagen model peptides, and changes of small labling molecules after replacing hydroxy proline with amino proline were measured in order to reveal the dynamic kinetics of the structure change of collagen in vitro.Methods:A non-canonical amino acid (2S,4R)-amino-proline whose amino and imino group were protected by benzyloxycarbonyl (CBZ), and carboxyl group by benzyl (Bn) temporarily was used. The second protecting group tert-Butoxycarbonyl (Boc) was added to amino group because of the specific structure difference of amino and imino group. Then CBZ and Bn were deprotected by hydrogenation. Finally, imino group was protected by 9-Fluorenylmethyloxycarbonyl (Fmoc). Then a selective dual-protection (2S, 4R)-amino-proline were obtained by the totally new strategy above and then it was characterized by ESI-MS,1H-NMR,13C-NMR and specific rotation. The dual-protected (2S,4R)-amino-proline was introduced into Yaa position of collagen model 23 peptide [Ac-(Gly-Pro-Hyp)3-Gly-Pro-Yaa-(Gly-Pro-Hyp)3-Gly-Gly-NH2] which was synthesized by solid-phase peptide synthesis. Then the amidation reaction was used to conjunct the free radical and fluorescein molecule with the free amino group of collagen model peptide to create free radical or fluorescein labeled collagen model peptide, as to study the kinetic process of folding and unfolding of collagen triple helix through spectroscopy. Results andConclusion:Synthesis of Fmoc-(2S,4R)-Amp(Boc)-OH reaches a total yield of 47.23% via five-steps. Compared to other methods utilizing hydroxyproline as substrate, this new route has several advantages as higher yield, efficiency and much more convenience. In our study, (2S,4R)-amino-proline were introduced into the 23 peptide collagen model successfully, and 3-carboxy nitroxyl radical and 5-Carboxyfluorescein were conjuncted to the peptide via the exposed 4-NH2.