Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds

Abstract

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Chemical synthesis of proteins in solution

“…48 After 24 h, a single component was formed, which was isolated by reverse phase HPLC (RP-HPLC), followed by ion-exchange HPLC. 58 As can be seen from the figure, the principal product was formed as a single component and the yield of the highly purified product was more than 25% of the starting precursor molecule. 48 These experiences were then incorporated into the protocol used for the folding reaction of the precursor molecule of MK and the folding process subsequently analyzed by HPLC as shown in Figure 1.…”

“…The principle on which our strategy is based was successfully applied to the total synthesis of proteins such as human angiogenin (a 123-residue protein having 3 disulfide bonds), 48 human midkine (MK, a 121residue protein having 5 intramolecular disulfide bonds) 58 and human pleiotrophin (PTN, a 136-residue protein having 5 intramolecular disulfide bonds). 59 Human angiogenin has a homologous structure similar to that of bovine pancreatic ribonuclease A, although it has three disulfide bonds.…”

Synthesis Concepts for Peptides and Proteins

“…A mixture of chloroform (CHL) and 2,2,2-trifluoroethanol (TFE) have been used for the solution synthesis of, e.g., midkine, a 121-aa protein [36], and human pleiotrophin, a 136-aa protein [37]. A mixture of chloroform (CHL) and 2,2,2-trifluoroethanol (TFE) have been used for the solution synthesis of, e.g., midkine, a 121-aa protein [36], and human pleiotrophin, a 136-aa protein [37].…”

Synthetic Approaches for Total Chemical Synthesis of Proteins and Protein-Like Macromolecules of Branched Architecture

“…The problem of low segment solubility is addressed either by backbone protection strategy [49,125,126] or by choosing a suitable solvent mixture [119,127,128]. Obviously a careful selection of the side chain protecting groups can greatly improve the solubility at the individual protected fragments [129].…”

“…8) [135][136][137][138]. Despite the limitations described above, the convergent approach using fully protected segments has been applied in solution or in combination with solid phase in the synthesis of many proteins and macromolecules especially of branched architecture [116,118,126,128,130,134,[140][141][142][143][144][145]. The intermediate iminophosphorane rearranges to form an amidophosphonium salt, which hydrolyses to yield the amide bond and a phosphine oxide (Fig.…”