The work herein reported was inspired by the simple consideration that, in spite of the biological relevance of sialic acid (Sia), no analytical method has been reported to permit a suitable qualitative and quantitative mapping of the components of this acidic carbohydrate family. Indeed, the inspection of the literature reports, showed that the existing methods suffer of different drawback deriving, in some cases, from a methodological fault; the authors did not dispose of authentic reference standards and of suitable inner standards for the quantitative detection of sialic acids. Thus, the many lists of sialic acids derivatives with different substituens at the alcoholic groups (that can be differently O-acylated, O-alkilated, O-sulfate, etc), which have been formed free or linked to carbohydrates chains of glycolipids and glycoproteins, have not the validity of an absolute demonstration of their presence in various biological natural districts, from bacteria to humans. Moreover, in the best cases, the quantitative detection of Sias is not absolute but refers to a reciprocal relative ratio. Our attention was directed to differently functionalized Sias derivatives from N-acetyl-neuraminic acid (Neu5Ac) (1) and N-glycolil-neuraminic acid (Neu5Gc) (2) and, in particular, to the 1,7 lactone of Neu5Ac (4). This lactone have never been isolated in pure form and in sufficient amounts to be identified beyond any reasonable doubt . The supposed presence of Neu5Ac1,7L (4) and of Neu5Gc1,7L (5) in biological media has still to be demonstrated too. It is, moreover reported that the Neu5Ac1,7L (4), present in the Bufo bufo mucins, is a ligand of human interleukin 4 and is well described as a component of sialoglycoproteins present in the membrane of red blood cells (RBCs) in Polycythemia Vera (PV), a malignant disorder of haematopoietic stem cells In this case the authors, who did not isolate the lactone, identified it, as the 4,8,9-tri-heptafluorobutyrril derivative, applying a method associating gas chromatography and electron mass spectrometry to the samples obtained by acidic hydrolysis of the RBCs. Successful results of the work, here described are: • the first synthesis of the 1,7 lactones of Neu5Ac (4) and of Neu5Gc (5), and their isotopologues (8) and (9); • the set-up of suitable analytical method for the analyses of these lactones by HPLC-MS methodology, which does not require any functionalization; • a collection of evidences that impose to reject as demonstrated the presence of the lactone (4) in PV red blood cells. Finally we could explain the mistake that deceived the authors who did not sufficiently demonstrated the presence of 1,7 lactone. In fact, we can suggest a possible artefactual formation of 1,7-lactones during derivatization of Sias. Moreover, some accessory results obtained in the last part of our work impose to reconsider the analytical work reported by the same authors, and others, who used their methodology in the analysis of acylated amido sugars or acylated aminoacids.
|Titolo:||First synthesis of authentic 1,7-lactones of sialic acids and new reliable protocol for the analysis of these free and functionalized acids, allow to reject the reported identification of 1,7-lactones in the membrane of policythemia vera red blood cells since obtained by an inaccurate procedure|
|Data di pubblicazione:||17-dic-2009|
|Parole Chiave:||sialic acid ; 1,7-lactone ; polycythemia vera ; GC/MS|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||First synthesis of authentic 1,7-lactones of sialic acids and new reliable protocol for the analysis of these free and functionalized acids, allow to reject the reported identification of 1,7-lactones in the membrane of policythemia vera red blood cells since obtained by an inaccurate procedure ; tutor: M. Anastasia. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE BIOMEDICHE, CHIRURGICHE ED ODONTOIATRICHE, DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2009 Dec 17. ((22. ciclo, Anno Accademico 2008/2009.|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|