A synthetic trivalent hapten that aggregates anti-2,4-DNP IgG into bicyclic trimers

Abstract

This paper describes the synthesis of the trivalent hapten molecule 1, containing three 2,4-dinitrophenyl (2,4-DNP) groups, and the use of this molecule to aggregate three molecules of anti-2,4-DNP IgG into a complex with 3:2 stoichiometry (IgG312). The equilibrium product IgG312 was generated in approximately 90% yield upon mixing IgG and 1; during incubation, thermodynamically unstable, high-molecular-weight aggregates (>104 nm in diameter) form first and convert subsequently to IgG312. The thermodynamics and the kinetics of the formation of aggregates were studied using size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). An analytical model based on multiple species in equilibrium was developed and used to interpret the SE-HPLC data. The aggregate IgG312 was more stable thermodynamically and kinetically than monomeric aggregates of this IgG with monomeric derivatives of 2,4-DNP; this stability suggests potential applications of these aggregates in biotechnology.

Scheme 1

The structure proposed for IgG₃1₂.

Scheme 2

Only the Fab regions (PDB ID# 1A0Q) from three IgG molecules (for clarity) are shown as they are superimposed against a single trivalent hapten molecule 1 to estimate the dimensions potentially available in an IgG₃1₂ aggregate. The Fab region of an IgG antibody at its widest point is ∼ 5 nm. Each one of the EG₈ linkers connecting the antigens to the core of the antigen molecule can extend to ∼ 3.2 nm in length. In a fully extended form of the EG₈ linkers, the Fab regions could adopt the relative distances shown in figure.

Figure 1

SE-HPLC chromatograms of complexes of IgG^DNP and 1 (at [1] = 0.0−1.2 μM and [IgG^DNP] = 0.6 μM). The graph at the top calibrates the column against proteins with relevant molecular weights.

Figure 2

Mole fraction (lines) produced by fitting the equilibrium model described in the text to the data (markers) from SE-HPLC experiments ([IgG^DNP] was kept constant 0.6 μM). The error bars are from peak integrations of four separate experiments; each datum is the mean of these measurements and the error bars show the maximum deviation.

Figure 3

Diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 °C) with [1]:[IgG] = 2:3, as measured by DLS. A is IgG^DNP alone, for B-E incubation intervals are shown on the plot.

Figure 4

AUC equilibrium experiment of 0.10 μM anti-DNP IgG incubated with 0.067 μM 1 at 6K rpm as observed at 230 nm at 25 °C. The hollow circles are experimental data and the line is the fit for a single ideal species. The expected molecular weight is ∼ 450 ± 12 kDa and the calculated molecular weight is 464 ± 35 kDa.

Scheme 3

Synthesis of 1.