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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.

SUBMITTER: Bilgicer B

PROVIDER: S-EPMC2535943 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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

Bilgiçer Basar B Moustakas Demetri T DT Whitesides George M GM

Journal of the American Chemical Society 20070228 12

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 th ...[more]

PMID: 17326636

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Project description:Background2,4-Dinitrophenol (2,4-DNP) is an effective but highly dangerous fat burner, not licensed for human consumption. Death cases reported for 2,4-DNP overdose, particularly among young adults, have raised concerns about the ineffective regulatory control, lack of education and risks associated with impurity, and the unknown concentration of 2,4-DNP purchased on the Internet.MethodsUsing a sequential mixed method design and based on a hypothetical scenario as if 2,4-DNP was a licensed pharmaceutical drug, first we conducted a qualitative study to explore what product attributes people consider when buying a weight-loss aid. Focus group interviews with six females and three males (mean age = 21.6 ± 1.8 years) were audiorecorded, transcribed verbatim, and subjected to thematic analysis. Sixteen attributes were identified for the Best-Worst Scale (BWS) in the quantitative survey with 106 participants (64% female, mean age = 27.1 ± 11.9 years), focusing on 2,4-DNP. Demographics, weight satisfaction, and risk for eating disorder data were collected.ResultsIn contrast to experienced users such as bodybuilders, our study participants approached 2,4-DNP cautiously. Attributes of 2,4-DNP as a hypothetical weight-loss drug comprised a range of desirable and avoidable features. Of the 16 selected attributes, BWS suggested that long-term side effects were the most and branding was the least important attribute. Effectiveness and short-term side effects were also essential. Those in the >25 year group showed least concerns for legality. Neutral BWS scores for cost, treatment, degree of lifestyle changes required, and specificity required for the hypothetical weight-loss drug to be effective were likely caused by disagreement about their importance among the participants, not indifference.ConclusionWith advances in research, 2,4-DNP as a pharmaceutical drug in the future for treating neurodegenerative diseases and potentially for weight loss is not inconceivable. Caution is warranted for interpreting the BWS scores. Owing to the difference in what data represent at individual vs. population levels, with pooled data, the method correctly identifies attributes by which most people are satisfied but misrepresents attributes that are individually very important but not universally agreed. Whilst this may be an advantage in marketing applications, it limits the utility of BWS as a research tool.

Project description:Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvβ3 or α5β1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvβ3 or α5β1, compared to monocyclic or linear RGD peptides, with a slight preference to αvβ3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.

Dataset Information

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

Publications

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

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