Characterization of the Hydrolysis Mechanism of Polyalternating Alginate in Weak Acid and Assignment of the Resulting MG-Oligosaccharides by NMR Spectroscopy and ESI−Mass Spectrometry

Abstract

Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.

In situ Synthesis of Magnetic Iron Oxide Nanoparticles in Thermally Responsive Alginate‐Poly(N‐isopropylacrylamide) Semi‐Interpenetrating Polymer Networks

“…However, the peaks located at 1 093 and 1 028 cm À1 disappear in the spectra that correspond to the samples with iron oxide particles (spectra d and e in Figure 2). This result points to the hydrolysis of alginate as a consequence of the acidic medium provided by the iron chloride solutions, [21] which mainly affects to the C-O and the C-O-C bonds (both alcoholic and ether groups). Moreover, the band located at 947 cm À1 in the spectrum that corresponds to pure alginate, assigned to the a 1 !…”

“…In the case of FeAlg-PNiPAAm (3c), which contains the highest amount of iron oxide, the pore size greatly decreases as it can be seen in Figure 3d. We hypothesize that the hydrolysis of the alginate chains to give rise to oligomers [21] results in the collapse of the gel network. The DSC of the hydrogels under investigation is displayed in Figure 4a, which illustrates their LCST behavior.…”

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

“…Since M and G are epimers of each other, the alginate polymer is synthesised as polymannuronic acid and epimerisation of individual residues within the polymannuronic acid chain to guluronic acids is carried out by a family of epimerases (Haug & Larsen, 1971a, b;. This enzymatic epimerisation results in varied patterns of the sequences GMG and GG being generated within the polymannuronic acid, with different organisms having different epimerases that make alginates specific to each species Hartmann, Holm, Johansen, Skjåk-Braek, & Stokke, 2002;Høidal, Ertesvåg, Skjåk-Braek, Stokke, & Valla, 1999;Holtan, Zhang, Strand, & Skjåk-Braek, 2006). …”

Design of a Bioartificial Pancreas

“…Both the composition and block structure vary in different types of alginate, but the length of the G blocks is the main structural feature that contributes to gelation because the diaxially linked G residues form cavities that function as binding sites for ions (16). As already mentioned, alginate gels easily in the presence of divalent cations, such as Ca 2+ , Sr 2+ and Ba 2+ , which interact with Na + ions from the G –monomers in the polymer chains to form ionic bridges between adjacent polymers (16,17). Among the possible cross-linking cations for alginate microbeads, Ba 2+ and Ca 2+ have been the most researched in cell microencapsulation studies.…”