Cross-linking intra-/intermolecular reactions

In either dilute or concentrated solutions, additional reactions occur that result in both intra- and intermolecular cross-linking of proteins. There is little direct chemical information from such techniques as nuclear magnetic resonance spectroscopy or mass spectrometry to detail the precise nature of these cross-links.5,6... 

The lathyrogenic compounds can therefore be seen to inhibit both intra-and intermolecular cross-linking. This would indicate that either the intramolecular linking is an essential prerequisite for intermolecular cross-linking, or, more likely, that both processes occur by a very similar reaction or sequence of reactions. Further studies with different lathyrogens and compounds able to reverse their action may well give an insight into the nature of the cross-links themselves. 

The visible absorption spectrum of a solution containing a known concentration of nitrated protein is measured in a solution buffered at pH 9.0, and the absorbance at the maximum (near 428 nm) used to calculate the nitrotyrosine content ( 428nm for the nitrophenoxide ion is 4200). The tyrosine and nitrotyrosine content of the modified protein should also be determined by amino acid analysis. If the sum of these values does not add up to the tyrosine content of the unmodified protein, intra- or intermolecular cross-linking may have occurred. The amino acid analysis may also reveal whether other side-reactions have taken place. Particular attention should be paid to the half-cystine, cysteine, methionine, histidine and tryptophan contents of the modified proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate offers a rapid and highly sensitive way of detecting products of intermolecular cross-linking. Such products are readily removed by gel filtration. 

Figure 1 The common solvents n-hexane and methyl n-butyl methane are converted by co-1 hydroxy lation and oxidation to the ultimate toxicant, 2,5-hexanedione (2,5-HD). 2,5-HD reacts with lysyl e-amines of proteins (black rectangle) to form pyrrolylated proteins, which undergo intra- and intermolecular cross-linking reactions, including dimer formation.

Fradet and co-workers reported on the thermal ROP of y-carboxyethyl- s-caprolactam and y-aminoethyl- s-caprolac-tam (compare Scheme 7). Both monomers were polymerized in bulk at 250 °C. In both cases, the authors observed that monomer conversion was limited and did not exceed a plateau value of 0.53 (after a reaction time of 3 h) or 0.57 (after 30 min) for y-carboxyethyl- s-caprolactam and y-aminoethyl- s-caprolactam, respectively. The limiting monomer conversion was ascribed to ring-chain equilibria in both cases. The polymerizations could be accelerated by the addition of polyamidation catalysts, such as phosphorous and hypo-phosphoric acids, but no change of the maximum monomer conversion was observed. In a control experiment, 4-aminoethyl-1,7-heptanedioic acid was polymerized via thermal polymerization however, this only resulted in a low molecular mass compound. This was attributed to the much faster rate of the intra- versus the intermolecular amidation reaction. Cross-linked material was obtained, when both monomers were heated for a prolonged time, and loss of NH3 was observed, which was ascribed to amidine formation and deamination. 

For an example of the application of Radziszewski reaction for intra- and intermolecular polymer coupling, see K.-S. Krannig, D. Esposito, M. Antonietti, Macromolecules 2014, 47, 2350-2353. Highly efficient transfer of amino groups to imidazolium entities for polymer coupling and cross-linking. 

The remarkable intra-intermolecular polymerization in which a monomer with a functionality of four gives linear, soluble, high molecular weight polymer is shown schematically in Eq. (Int.-6). In both reactions, Eqs. (lnt.-5) and (Int.-6), the polyfunctionality of the monomers that might lead to cross-linked products is not operative because other factors prevailing in the monomers determine a different reaction course. That this situation can occur with many different types of polyfunctional reactants is amply illustrated by the examples in this book. 

---