Anita M. Jansson

Azido Acids in a Novel Method of Solid-Phase Peptide Synthesis.

Azido acids were produced from α-branched acids by α-bromination with NBS followed by substitution with sodium azide and the products were used in a novel method of solid-phase synthesis. The azido acids were transformed into the highly activated acid chlorides and used synthesis of extremely hindered peptides containing up to four successive diphenyl glycine or Aib residues. By reaction of the genetically encoded amino acids with TfN3 and then SOCl2 they were transformed into α-azido acid chlorides used in solid-phase peptide synthesis without racemization.

The active ester N-fmoc-3-O-[Ac4-α-D-Manp-(1→2)-Ac3-α-D-Manp-1-]=thre-O-Pfp as a building block in solid-phase synthesis of an o-linked dimannosyl glycopeptide.

Abstract The active ester N α -Fmoc-3-O-[Ac 4 -α-D-Manp-(1-→2)-Ac 3 -α-D-Manp-1-]-Thr-O-Pfp ( 6 ) was synthesized by direct condensation of Ac 4 -α-D-Manp-(1→2)-Ac 3 -α-D-Manp-Br ( 4 ) with Fmoc-Thr-O-Pfp ( 5 ) and used as a building block in an automated continuous-flow solid-phase synthesis of an O-glycosylated heptadeca amino acid fragment of the insulin-like growth factor 1 (IGF-1).

Silyl protection in the solid-phase synthesis of N-linked glycopeptides. Preparation of glycosylated fluorogenic substrates for subtilisins

The trimethylsilyl (TMS) group was used for protection of the hydroxy groups of three disaccharide 1-amino-alditols and of the glycosylamines of glucose, maltotriose and maltoheptose. The per-O-trimethylsilylated derivatives were coupled with N alpha-Fmoc-Asp(Cl)-OPfp 7 to give six glycosylated building blocks for the solid-phase synthesis of N-linked glycopeptides. Building block 8 was used in the synthesis of five internally quenched fluorescent substrates which were studied by enzymatic hydrolysis with savinase, a subtilisin-type enzyme.

Synergistic Amylomaltase and Branching Enzyme Catalysis to Suppress Cassava Starch Digestibility.

Starch provides our main dietary caloric intake and over-consumption of starch-containing foods results in escalating life-style disease including diabetes. By increasing the content of α-1,6 branch points in starch, digestibility by human amylolytic enzymes is expected to be retarded. Aiming at generating a soluble and slowly digestible starch by increasing the content and changing the relative positioning of the branch points in the starch molecules, we treated cassava starch with amylomaltase (AM) and branching enzyme (BE). We performed a detailed molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility. Step-by-step enzyme catalysis was the most efficient treatment, and it generated branch structures even more extreme than those of glycogen. All AM- and BE-treated samples showed increased resistance to degradation by porcine pancreatic α-amylase and glucoamylase as compared to cassava starch. The amylolytic products showed chain lengths and branching patterns similar to the products obtained from glycogen. Our data demonstrate that combinatorial enzyme catalysis provides a strategy to generate potential novel soluble α-glucan ingredients with low dietary digestibility assets.

Solid-phase synthesis and characterization of O-dimannosylated heptadecapeptide analogues of human insulin-like growth factor 1 (IGF-1)

N α -Fmoc-3-O-[Ac4-α-D-Manp-(1→2)-Ac3-α-D-Manp-1-]-Thr-OPfp, 7, and Nα-Fmoc-3-O-[Ac4-α-D-Manp-(1→2)-Ac3-α-D-Manp-1-]-Ser-OPfp, 8, were prepared and used as building blocks in automated continuous-flow solid-phase glycopeptide synthesis of two O-glycosylated heptadecapeptide analogues of human insulin-like growth factor 1. The corresponding non-glycosylated fragments were also prepared, and comparative NMR studies regarding the influence of the sugar moiety on the peptide backbone showed only limited effects due to glycosylation.

Structure of branching enzyme- and amylomaltase modified starch produced from well-defined amylose to amylopectin substrates

Thermostable branching enzyme (BE, EC 2.4.1.18) from Rhodothermus obamensis in combination with amylomaltase (AM, EC 2.4.1.25) from Thermus thermophilus was used to modify starch structure exploring potentials to extensively increase the number of branch points in starch. Amylose is an important constituent in starch and the effect of amylose on enzyme catalysis was investigated using amylose-only barley starch (AO) and waxy maize starch (WX) in well-defined ratios. All products were analysed for amylopectin chain length distribution, α-1,6 glucosidic linkages content, molar mass distribution and digestibility by using rat intestinal α-glucosidases. For each enzyme treatment series, increased AO content resulted in a higher rate of α-1,6 glucosidic linkage formation but as an effect of the very low initial branching of the AO, the final content of α-1,6 glucosidic linkages was slightly lower as compared to the high amylopectin substrates. However, an increase specifically in short chains was produced at high AO levels. The molar mass distribution for the enzyme treated samples was lower as compared with substrate WX and AO, indicating the presence of hydrolytic activity as well as cyclisation of the substrate. For all samples, increased amylose substrate showed decreased α- and β-amylolysis. Surprisingly, hydrolysis with rat intestinal α-glucosidases was higher with increasing α-1,6 glucosidic linkage content and decreasing M¯w indicating that steric hindrance towards the α-glucosidases was directed by the molar mass rather that the branching density of the glucan per se. Our data demonstrate that a higher amylose content in the substrate starch efficiently produces α-1,6 glucosidic linkages and that the present of amylose generates a higher M¯w and more resistant product than amylopectin. The combination of BE→AM→BE provided somewhat more resistant α-glucan products as compared to BE alone.

Solid-phase glycopeptide synthesis of tyrosine-glycosylated glycogenin fragments as substrates for glucosylation by glycogenin

The four building blocks Nα-Fmoc-Tyr(Bz4-α-D-Glc)-OPfp 1, Nα-Fmoc-Tyr(Bz4-β-D-Glc)-OPfp 2, Nα-Fmoc-Tyr[Bz4-α-D-Glc(1→4)-Bz3-α-D-Glc]-OPfp 3 and Nα-Fmoc-Tyr[Ac4-α-D-Glc(1→4)-Ac3-β-D-Glc]-OPfp 4 were used in continuous-flow solid-phase synthesis of four 25-amino-acid-long glycopeptides 5–8 related to glycogenin. The unglycosylated peptide 9 was synthesized as well, and the five glycogenin fragments were tested as acceptors for glucosyl transfer by recombinant glycogenin.

An enzyme-linked immunosorbent assay for the detection of diacetyl (2,3-butanedione)

Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.

Synthesis of 1,2-phenylenediamine capturing molecule for the detection of diacetyl

Here we describe the design of 1,2-phenylenediamine capturing molecule and the synthesis steps necessary for its preparation. The designed 1,2-phenylenediamine derivative is able to capture diacetyl in solution, as shown by ESIMS, forming a chemical adduct, 1-4-quinoxaline. The methyl esters of diacetyl-adduct (DAA) and pentanedione-adduct (PDA) are incorporated to the lysines in BSA and the conjugate used for antibody screening and selection. In the research article is described an enzyme-linked immunosorbent assay developed to detect and quantify diacetyl in complex media.