Identification of aggregation breakers for bevacizumab (Avastin®) self-association through similarity searching and interaction studies.

Aggregation is a common challenge in the optimization of therapeutic antibody formulations. Since initial self-association of two monomers is typically a reversible process, the aim of this study is to identify different excipients that are able to shift this equilibrium to the monomeric state. The hypothesis is that a specific interaction between excipient and antibody may hinder two monomers from approaching each other, based on previous work in which dexamethasone phosphate showed the ability to partially reverse formed aggregates of the monoclonal IgG1 antibody bevacizumab back into monomers. The current study focuses on the selection of therapeutically inactive compounds with similar properties. Adenosine monophosphate, adenosine triphosphate, sucrose-6-phosphate and guanosine monophosphate were selected in silico through similarity searching and docking. All four compounds were predicted to bind to a protein-protein interaction hotspot on the Fc region of bevacizumab and thereby breaking dimer formation. The predictions were supported in vitro: An interaction between AMP and bevacizumab with a dissociation constant of 9.59±0.15 mM was observed by microscale thermophoresis. The stability of the antibody at elevated temperature (40 °C) in a 51 mM phosphate buffer pH 7 was investigated in presence and absence of the excipients. Quantification of the different aggregation species by asymmetrical flow field-flow fractionation and size exclusion chromatography demonstrates that all four excipients are able to partially overcome the initial self-association of bevacizumab monomers.

Diastereoselective assembly of pentanuclear circular helicates.

Reaction of a ligand which contains two N-donor and O-donor tridentate domains separated by a 1,3-phenylene spacer unit with Zn(2+) ions results in a pentanuclear circular helicate [Zn(5)(L)(5)](10+) and this structure persists in both the solid and solution state. The formation of this high nuclearity species is governed by unfavourable steric interactions between the phenyl units which destabilize the simple linear helicate. Incorporation of enantiopure units within the ligand strand controls the diastereoselectivity with up to 80% d.e.

Controlling the formation of metallosupramolecular assemblies by metal ionic radii.

The formation of either dinuclear double-stranded or pentanuclear circular helicates from a ligand containing two tridentate domains separated by a phenylene unit can be controlled by inter-ligand steric interactions which themselves are governed by the size of the metal ion.

Coumarin-based luminescent ligand that forms helicates with dicationic metal ions.

The potentially hexadentate ligand L, which contains two terminal coumarin fluorophores, forms dinuclear double-stranded helicates with dicationic metal ions, giving species of the generic form [M(2)L(2)](4+). In solution the free ligand was fluorescent with emission attributed to the coumarin fluorophores (lambda(em) = 437 nm). The luminescent properties of the corresponding dimetallic helicates complexes were examined and revealed that the Zn(2+) complex demonstrates enhanced emission when compared to the parent ligand, whereas Co(2+), Cu(2+), Cd(2+) and Hg(2+) induce varying degrees of fluorescence quenching. In particular, comparative luminescence measurements at 77 K and room temperature showed that the quenching mechanism for [Cu(2)L(2)](4+) can be attributed to a photoinduced electron transfer. ESI-MS selectivity studies showed that in the presence of a mixture of metal dications no preference for any one metal ion was observed.

Metal-specific allosteric activation and deactivation of a diamine.

The reaction of a potentially tetradentate bis(pyridyl-thiazole) ligand with acetone is allosterically activated upon complexation with Cd(II) but deactivated by reaction with Cu(I), demonstrating metal-specific allosteric controlled reactivity.

Allosteric deprogramming of a trinuclear heterometallic helicate.

Two multidentate ditopic ligands L1 and L2 which contain both N-donor and crown ether units have been synthesised. The potentially octadentate ligand L1 forms a trinuclear heterometallic double helicate with Cu(I) and Zn(II) ([Zn2Cu(L1)2](5+)), whereas L2 forms a tetranuclear heterometallic double helicate with the same metal ions ([Zn2Cu2(L2)2](6+)). Both species have been characterised by (1)H NMR, ESI-MS and single crystal X-ray crystallography. Reaction of [Zn2Cu2(L2)2](6+) with Ba(2+) results in the coordination of the crown ether units giving the simple barium coordinated species [Zn2Cu2(L2)2Ba2](10+). However, reaction of [Zn2Cu(L1)2](5+) with Ba(2+) deprograms the ligand and results in the formation of a mixture of species.

This facile interconversion of dinuclear double helicates and side-by-side species: a reprogrammable ligand with potential sensor applications.

The ligand L1 forms a dinuclear double helicate with Cu+ but upon addition of Ba2+ to the system a side-by-side species is formed both in solution and in the solid state; in the presence of Na+ both the helicate and the side-by-side species are formed in roughly equal amounts in solution.

Allosteric pitch length tuning of a dinuclear double helicate.

Self assembly of the ditopic ligand L1 with Cu2+ gives the dinuclear double helicate [Cu2(L1)2]4+, which can further coordinate s-block cations. This coordination alters the helicate pitch to a variety of different lengths depending on the size and charge of the guest cation.

Reactions of the dianion [1,1,1-(CO)3-2-Ph-closo-1,2-MnCB9H9]2- with transition-metal cations: facile insertion and then extrusion of cluster vertexes.

The manganacarborane dianion in [N(PPh(3))(2)][NEt(4)][1,1,1-(CO)(3)-2-Ph-closo-1,2-MnCB(9)H(9)] (1b) reacts with cationic transition metal-ligand fragments to give products in which the electrophilic metal groups (M') are exo-polyhedrally attached to the {closo-1,2-MnCB(9)} cage system via three-center two-electron B-H --> M' linkages and generally also by Mn-M' bonds. With {Cu(PPh(3))}(+), the Cu-Mn-Cu trimetallic species [1,6-{Cu(PPh(3))}-1,7-{Cu(PPh(3))}-6,7-(mu-H)(2)-1,1,1-(CO)(3)-2-Ph-closo-1,2-MnCB(9)H(7)] (3a) is formed, whereas reactions with {M'(dppe)}(2+) (M' = Ni, Pd; dppe = Ph(2)PCH(2)CH(2)PPh(2)) give [1,3-{Ni(dppe)}-3-(mu-H)-1,1,1-(CO)(3)-2-Ph-closo-1,2-MnCB(9)H(8)] (5a) and [1,3,6-{Pd(dppe)}-3,6-(mu-H)(2)-1,1,1-(CO)(3)-2-Ph-closo-1,2-MnCB(9)H(7)] (5b), both of which contain M'-Mn bonds. The latter reaction with M' = Pt affords [3,6-{Pt(dppe)}-3,6-(mu-H)(2)-1,1,1-(CO)(3)-2-Ph-closo-1,2-MnCB(9)H(7)] (6), which lacks a Pt-Mn connectivity. Compound 6 itself spontaneously converts to [1-Ph-2,2,2-(CO)(3)-8,8-(dppe)-hypercloso-8,2,1-PtMnCB(9)H(9)] (7b) and thence to [3,6,7-{Mn(CO)(3)}-3,7-(mu-H)(2)-1-Ph-6,6-(dppe)-closo-6,1-PtCB(8)H(6)] (8). This sequence occurs via initial insertion of the {Pt(dppe)} unit and then extrusion of {Mn(CO)(3)} and one {BH} vertex. In the presence of alcohols ROH, compound 6 is transformed to the 7-OR substituted analogues of 7b. X-ray diffraction studies were essential in elucidating the structures encountered in compounds 5-8 and hence in understanding their behavior.

Anion control of the formation of geometric isomers in a triple helical array.

The amide-substituted unsymmetrical ligand L(1) upon coordination to Co(2+) forms the dinuclear triple helicate [Co(2)(L(1))(3)](4+) which exists as both possible C(1) and C(3) symmetric geometric isomers in solution, however, upon addition of nitrate ions, the formation of an anion binding cavity is favoured and hence the sole product is the C(3) symmetric isomer in which all three amide substituents are hydrogen bonded to the nitrate anion.

Anion control of ligand self-recognition in a triple helical array.

Self-assembly of the ligand L(1) with Co(2+) into a dinuclear triple helicate [Co(2)(L(1))(3)](4+) produces an anion binding pocket which encapsulates perchlorate anions. Furthermore we demonstrate how this ability to bind anions can control ligand self-recognition properties.

The seventeen- and eighteen-electron metallacarbaboranes [1,1,1-(CO)3-2-Ph-closo-1,2-MnCB9H9]n- (n = 1, 2): a structurally characterized, redox-related pair.

An eleven-vertex manganese-monocarbaborane dianion, upon one-electron oxidation, gives a stable radical monoanion in which the unpaired electron is delocalized over the cluster.