title: Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters.
abstract: Although the biomimetic dimetal complex [LGa2(OH)2(H2O)2](3+) [L = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate] provides efficient protection against phosphate loss in phosphopeptides upon collision-induced dissociation tandem mass spectrometry (CID MS/MS), the underlying mechanism remains unknown. Here, we explored the mechanism in detail and investigated the selective binding to phosphate groups in solution. Dimetal complexes containing combinations of Ga(3+), In(3+), Fe(3+), Co(3+), Zn(2+), Cu(2+), and V(2+) were reacted with HPO4(2-), phosphoserine, and a phosphopeptide (FQpSEEQQQTEDELQDK, abbreviated "βcas") and studied with isothermal titration calorimetry (ITC), CID MS/MS, and density functional theory (DFT). Ka for HPO4(2-) binding scaled with the metal charge and was 35-fold larger for [LGa2(OH)2(H2O)2](3+) (3.08 ± 0.31 × 10(6) M(-1)) than for [LZn2(HCOO)2](+). CID MS/MS of [LGa2(βcas)](n+) revealed protection against phosphate detachment (<3% of the total ion intensity). Phosphate detachment from βcas was 22-40% and increased to 42-71% when bound to dimetal complexes of lower charge than {LGa2}(5+). CID data suggests that facile metal-phosphate dissociation is associated with proton transfer from the intermediate oxazoline ring formed in the phosphopeptide to the metal-phosphate complex. The observed phosphate stabilization was attributed to a significant reduction in the gas-phase basicity (GB) of the phosphate group when bound to {LGa2}(5+)/{LIn2}(5+) complex cores. Absence of proton transfer results in formation of an ion-zwitterion intermediate with a greater dissociation threshold. This hypothesis is supported by DFT calculations for [LGa2(PO4)](2+), [LGaZn(PO4)](+), [LZn2(PO4)], and 2,4-dimethyl-3-oxazoline showing that [LGa2(PO4)](2+) is the only compound with a substantial lower GB (321 kJ/mol less) than 2,4-dimethyl-3-oxazoline.
authors.affiliation: †Department of Biochemistry and Molecular Biology, and ‡Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230, Odense M, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Svane
authors.affiliation: †Department of Biochemistry and Molecular Biology, and ‡Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230, Odense M, Denmark.
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.affiliation: †Department of Biochemistry and Molecular Biology, and ‡Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230, Odense M, Denmark.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: McKenzie
authors.affiliation: †Department of Biochemistry and Molecular Biology, and ‡Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230, Odense M, Denmark.
authors.forename: Frank
authors.initials: F
authors.lastname: Kjeldsen
mesh_terms: D000595:Amino Acid Sequence; D040761:Biomimetic Materials / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D056831:Coordination Complexes / Q000737:chemistry / Q000378:metabolism; D004952:Esters; D005740:Gases / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D056831:Coordination Complexes; D004952:Esters; D005740:Gases; D008670:Metals; D010710:Phosphates; D010748:Phosphopeptides; D010768:Phosphoserine