Semisynthesis and analysis of lipophilically modified unfractionated and low molecular mass heparins.
Unfractionated heparin and LMMH were substituted with different lipophilic organic compounds. Specifically endpoint attached (LMMH-tyramine and LMMH-tyramine-FITC) and nonspecifically substituted heparins (acylated heparins, and LMMH-biotin and LMMH-cholesterol hemisuccinate) were obtained. The lipophilically substituted heparins were analysed by HPSEC and showed different retention times, high peak purity, different UV/VIS absorbances, and areas under the absorbance time curve. The determination of the average molecular mass Mn, Mm, and Mz and the polydispersity P was performed by PAGE. The substituted heparins showed an increase in their molecular mass Mm, ranging from 2.9 to 129.7% unfractionated heparin and 3.9 to 224.0% (LMMH) compared with the parent compounds (unfractionated heparin and LMMH). The anticoagulant activity was measured by anti-Factor Xa. Lipophilically modified heparin had an aXa activity ranging from 52 to 168 U/mg (unfractionated) and 60 to 108 U/mg (LMMH) and antithrombin activity ranging from 31 to 270 U/mg (unfractionated) and 5 to 15 U/mg (LMMH). The thrombin generation inhibition assay demonstrated an effective anticoagulant potency of the modified compounds. They were neutralized by different amounts (1.1 to 4.1, w/w) of protamin. 1H NMR spectroscopy revealed the specific endpoint attachment of tyramine to LMMH and FITC to LMMH-tyramine. The lipophilically modified heparins showed intact anticoagulant properties and are now used for pharmacokinetic investigations.
Malsch
R
R
1st Department of Medicine, Medical University Clinic Mannheim, Germany.
Harenberg
J
J
Guerrini
M
M
Torri
G
G
Casu
B
B
Heene
D L
DL
D000215:Acylation; D000586:Amination; D001710:Biotin; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D016650:Fluorescein-5-isothiocyanate; D006493:Heparin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006495:Heparin, Low-Molecular-Weight / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D013917:Thrombin / Q000096:biosynthesis; D014439:Tyramine
D065427:Factor Xa Inhibitors; D006495:Heparin, Low-Molecular-Weight; D001710:Biotin; D006493:Heparin; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine