Ein neues bifunktionelles Reagens zur intramolekularen Vernetzung von Insulin

Abstract

Zusammenfassung: Bis-[2-(succinimidooxycarbonyloxy)äthyl]sulfon [SO 2 (Eoc-ONSu) 2 ] reagiert mit Insulin in IN NaHC0 3 /Dimetbvlformamid in 20-bis 35proz. Ausbeute zuN aAl ,N eB29 -2,2'-Sulfonylbis(äthoxycarbonyl)insulin [S0 2 (Eoc) 2 -Insulin], das durch Verteilungschromatographie gereinigt werden kann. Aufspaltung der Disulfidbrücken und Reoxidation in hoher Verdünnung gibt etwa 60% Insulinaktivität zurück. Die Abspaltung des Vernetzungsreagens gelingt mit 0.5N NaOH bei 0 °C innerhalb weniger Sekunden und führt zu biologisch voll wirksamem Insulin. Summary: Reaction of bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone[S0 2 (Eoc-ONSu) 2 ]with insulin in IN NaHCO 3 /dimethylformamide forms J /V a!A1 , J /V reB29 -2,2'-sulfonylbis(ethoxycarbonyl)insulin [S0 2 (Eoc) 2 -insulin] in 20 -35% yield. The product can be purified by partition chromatography. After cleavage of the disulfide bridges, reoxidation in very dilute solution reconstitutes about 60% of the original insulin activity. Cleavage of the crosslinking moiety can be achieved with 0.5N NaOH at 0 °C in only a few seconds, rendering a biologically fully active insulin.

Biomimetic Synthesis of Lispro Insulin via a Chemically Synthesized “Mini-Proinsulin” Prepared by Oxime-Forming Ligation

“…Construct 1 was prepared by oxime-forming ligation (Figure S1, for synthesis see Supporting Information). Under standard redox conditions, the oxime-linked polypeptide 1 folded and formed disulfides in relatively good yield (Figure 2A), which agrees with the reported high-yield refolding of N αA1 , N ε B29 -suberoylinsulin, 15 N αA1 , N ε B29 -2,2′-sulfonylbis(ethoxycarbonyl)insulin 16 and N αA1 , N ε B29 -carbonylbis-(methionyl)insulin. 17 When folded 1 was treated with trypsin (after solid phase extraction and lyophilization), the Arg residue at the ε -amino group of Lys B28 was retained (Figure S2, Supporting Information), indicating that as expected the amide bond between Arg and N ε -Lys B28 was not cleaved by the trypsin and thus 1 did not lead to native insulin.…”

“…We designed and made three mini-proinsulins using oxime-forming ligation in the chemical tether regions. Mini-proinsulin precursor 1 , the design of which was inspired by the works of Wollmer and Brandenburg et al, 15 Obermeier and Geiger, 16 and Busse and Carpenter, 17 folded cleanly and in relatively good yield, but enzymatic cleavage necessarily gave an insuln with a residual Arg on the side chain of Lys B28 . Mini-proinsulin precursor 2 , in which the chemical tether joined the N-terminal residues of the A-and B-chains, did not fold properly.…”

“…14 In principle, it would be possible to use total chemical synthesis to make a similar mini-proinsulin polypeptide construct. But, there is no absolute requirement for a linear polypeptide chain precursor molecule containing only peptide bonds: Wollmer and Brandenburg et al, 15 Obermeier and Geiger, 16 and Busse and Carpenter 17 independently reported efficient folding of intermediates in which the insulin A and B chains were covalently linked by artificial suberoyl, 15 sulfonylbis(ethoxycarbonyl), 16 or carboxyl(bismethionine) 17 chemical tethers between the epsilon amino group of Lys B29 and the N-terminal residue of the A chain.…”

Facile folding of insulin variants bearing a prosthetic C-peptide prepared by α-ketoacid-hydroxylamine (KAHA) ligation

“…have designed a cleavable chemical linker that can mimic the function of the C-peptide of proinsulin and efficiently fold the reduced single-chain insulin into its native structure. 14 Although a very promising as a strategy for folding synthetic insulin, this linker was employed only on isolated bovine insulin and – to the best of our knowledge – has not been used for the chemical synthesis insulin.…”

Neue Bifunktionelle Brückenfunktionen zur A1‐B29‐Verklammerung der beiden Insulinketten

“…Die verwendeten Klammerreagenzien wurden nach Vorschriften aus den Arbeiten von LOSSE und bzw. GEIGER und Mitarb [14]. hergestellt.…”