Der Methylsulfonyläthyloxycarbonyl‐Rest als reversible Aminoschutzgruppe für Insulin

Abstract

Der Methylsulfonylathyloxycarbonyl(Msc-)Rest ' I la& sich spezifisch in die Aminogruppen von A I-Glycin und B29-Lysin des lnsulins einfuhren. Er erhoht die Loslichkeit des lnsulins in polaren Losungsmitteln. Diese Eigenschaft erleichtert die Reinigung von Msc,-Insulin durch Verteilungschromatographie. Die Abspaltung der Schutzgruppen gelingt durch kurzes Einwirken von verd. Natronlauge bei 0°C ohne bedeutende Schadigung des Molekiils. Nach chromatographischer Reinigung ist die Kristallisationsneigung des zuriickgebildeten lnsulins gegeniiber nativem Material nicht verandert. Die biologische Wirkung bleibt voll erhalten. The Methylsulfonylethyloxycnrboayl Residue as a Reversible Amino Protecting Group for IasulinThe methylsulfonylethyloxycarbonyl(Msc) residue is introduced specifically into the amino groups of A 1 glycine and B29 lysine of insulin. It increases the solubility of insulin in polar solvents. This property facilitates the purification of Msc,-insulin by partition chromatography. Deprotection is accomplished by short treatment with dilute NaOH at O' C, without noticable damage of the molecule. Compared with native material, the tendency of the deprotected insulin to crystallize, following chromatographic purification, had not changed. The recovered insulin exhibits full biological activity.Die unterschiedliche Reaktivitat der 3 Aminogruppen des Insulins kommt darin zum Ausdruck, daD Isothiocyanate bevorzugt an der Aminogruppe des B I -Phenylalanins angreifen ' ), wahrend unter geeigneten Bedingungen Reagentien wie ten-Butyloxycarbonyl-azid uberwiegend mit den Aminogruppen von A 1-Glycin und B29-Lysin reagieren4). Als Folge dieses Verhaltens entstand ein Bediirfnis nach siiurestabilen Schutzgruppen, welche im Gegensatz zur Boc-Gruppe beim sukzessiven Abbau der B-Kette nach Edman 5 , stabil bleiben. I ) Abkiirzungen: Msc = Methylsulfonylathyloxycarbonyl-, PIC = Phenylthiocarbamoyl-, Tfa = Trifluoracetyl-. Boc = rerr-Butyloxycarbonyl-, -ONSu = N-Hydroxysuccinimidester.

Insulin structure and function

“…Strategies for selective N-terminal modification of A and B chains are based upon the differential reactivity of the three primary amines. Specifically, the relatively greater reactivity of the A1 and B29 primary amines enables the preparation of the A1, B29 bis-protected insulin intermediates using either the Boc 34 or methylsulfonylethoxycarbonyl (Msc) 35 protecting groups. This approach permits selective removal and replacement of the B-chain N-terminus with nonnative residues ( Figure 4 a methionine-based protecting group at B1 and B29 to carry out modifications to the N-terminus of the A-chain followed by methionine cleavage with cyanogen bromide.…”

Synthesis and Characterization of New N-Terminal Bchain Modified Human Insulin Analogues

“…The scheme 1 illustrates synthetic routes used in this work. To ensure regioselectivity within the synthesis of insulin analogues the amino functions of glycine A1 and Lysine B29 side chains were selectively blocked with the protecting group (Msc) according to Geiger [19]. This reaction proceeds in a basic medium and therefore reaction was stopped by acidification with glacial acetic acid.…”

“…To a solution of 1.00 g (172.2 µmol) zinc-free insulin dissolved in 70 ml Dimethylsulfoxide (DMSO) and 0.7 ml Triethylamine, a solution of 91.4 mg (344.4 µmol) Msc-ONSu in 14 ml DMSO is added drop-wise at room temperature [19]. After 20 min the reaction was stopped by the addition of 2 ml glacial acetic acid.…”

Enhancing the T R Transition of Insulin by Helix-Promoting Sequence Modifications at the N-Terminal B-Chain

“…As a first step the amino functions of GlyA1 and LysB29 of human insulin were selectively protected by reacting insulin with MscONSu in DMSO according to Geiger et al (1975). By-products were removed by gel filtration and cation exchange chromatography.…”