Multicomponent reactions (MCRs) are fundamentally different from two-component reactions in several aspects. Among the MCRs, those with isocyanides have developed into popular organic-chemical reactions in the pharmaceutical industry for the preparation of compound libraries of low-molecular druglike compounds. With a small set of starting materials, very large libraries can be built up within a short time, which can then be used for research on medicinal substances. Due to the intensive research of the last few years, many new backbone types have become accessible. MCRs are also increasingly being employed in the total synthesis of natural products. MCRs and especially MCRs with isocyanides offer many opportunities to attain new reactions and basic structures. However, this requires that the chemist learns the “language” of MCRs, something that this review wishes to stimulate.

Multicomponent Reactions with Isocyanides.

Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.

https://www.future-science.com/doi/pdf/10.2144/04375RV01

Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.

Antisera were raised against γ-aminobutyric acid (GABA) or glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde. After purification, these antisera reacted strongly with fixed GABA or Glu, but not significantly with other amino acids fixed with glutaraldehyde to brain macromolecules. The antisera were used to demonstrate the distributions of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) in parts of the perfusion-fixed mouse and rat brain, including the olfactory bulb, cerebral neocortex, thalamus, basal ganglia, lower brain stem, and cerebellum.



The level of GABA-LI varied widely among brain regions, thus it was very high in the globus pallidus and substantia nigra and low in the bulk of the thalamus. The GABA antisera labeled nonpyramidal neurons of the neocortex, most cells of the reticular nucleus of the thalamus, medium-sized cells of the caudatoputamen, and stellate, basket, Golgi, and Purkinje cells of the cerebellum. The distribution of GABA-LI closely matched that of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), as revealed in immunocytochemical studies by others. However, the GABA antisera seem to be better suited than GAD antisera for demonstrating putative GABA-ergic axons. The results suggest that GABA-LI, as displayed by the present method, is a good marker of neurons thought to use GABA as a transmitter.



Glutamate-like immunoreactivity was much more evenly distributed among regions than GABA-LI, but was particularly low in globus pallidus and substantia nigra and high in the cerebral cortex. Mitral cells of the olfactory bulb, pyramidal neocortical cells, and other cells assumed to use Glu or aspartate as transmitter were stained for Glu-LI, but so also were neurons that are thought to use other transmitters, such as cells in the substantia nigra pars compacta, in the dorsal raphe nucleus, and in the brain stem motor nuclei. The Glu antisera seem to reveal the “transmitter pool” as well as the “metabolic pool” of Glu in perfusion-fixed material.



This report shows that it is possible by means of immunocytochemistry to display reliably the tissue contents of GABA and Glu in material that has been fixed by perfusion with glutaraldehyde.

Glutamate- and GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique.

https://ris.utwente.nl/ws/files/6862443/Olde_Damink96cross.pdf

Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some Physiology Blut ist ein ganz besondrer Saft1

The products of interaction of glutaraldehyde with 6-aminohexanoic acid and with α-N-acetyl-lysine have been separated and partially purified; studies of the most highly purified fraction are indicative of the formation of 1,3,4,5-tetrasubstituted pyridinium salts, such as (II), analogous to desmosine. It is suggested that similar structures are present in the cross-linked products formed by the action of glutaraldehyde on proteins.

The nature of the cross-linking of proteins by glutaraldehyde. Part I. Interaction of glutaraldehyde with the amino-groups of 6-aminohexanoic acid and of α-N-acetyl-lysine

FOLDED AND EXTENDED STRUCTURES OF HOMOOLIGOPEPTIDES FROM α,α‐DIALKYLATED GLYCINES. A CONFORMATIONAL ENERGY COMPUTATION AND X‐RAY DIFFRACTION STUDY

Folded and extended structures of homooligopeptides from .alpha.,.alpha.-dialkylated .alpha.-amino acids. An infrared absorption and proton nuclear magnetic resonance study

A series of compounds structurally related to adipokinetic hormone, the decapeptide neurohormone < Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, have been prepared by synthesis and by enzymic cleavages of synthetic hormone. Their relative agonist activities in mobilising lipids over a fixed time interval (1 h) in locusts were assessed. The similar time courses for lipid release shown by two of the peptide analogues and adipokinetic hormone suggest that the analogues and the hormone are transported to the receptors on the fat body cells, and are also degraded, at similar rates. Consequently, the analogue activities can be correlated with the structural requirements of the locust fat body hormone receptors. The requirements for activity demonstrated in this study are as follows. Residues 1–8 from the N-terminus are necessary to elicit some activity (20%). Residues 5 and 7 in the octapeptide can be changed without affecting activity but l-pyroglutamic acid as the N-terminal residue is necessary for maximum activity both in the octapeptide and the decapeptide. Full activity is achieved only by adding the dipeptide glycyl threonine amide to the active octapeptide ‘core’. In the decapeptide, residues cannot be interchanged to the same extent as in the octapeptide without reducing activity. The peptide probably has to be uncharged. Inactive analogues of seven residues or less do not interfere in the hormone-receptor interaction.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1978.tb20913.x

Structure‐Activity Relationships for the Lipid‐Mobilising Action of Locust Adipokinetic Hormone

1-(5-Amino-5-carboxypentyl)pyridinium chloride (3) and 1-(5-amino-5-carboxypentyl)-3-[1-(5-amino-5-carboxypentyl)-2-piperidyl]pyridinium chloride, anabilysine (4), have been isolated from acid hydrolysates of glutaraldehyde-treated ovalbumin and their structures confirmed by synthesis. It is concluded that the strong chromophore at ca. 265 nm shown by glutaraldehyde-treated proteins is due to the formation of such quaternary pyridinium compounds and that anabilysine residues are important in the cross-linking of proteins by glutaraldehyde.

The nature of the cross-linking of proteins by glutaraldehyde. Part 2. The formation of quaternary pyridinium compounds by the action of glutaraldehyde on proteins and the identification of a 3-(2-piperidyl)-pyridinium derivative, anabilysine, as a cross-linking entity

Paul M. Hardy

Papers

The nature of the cross-linking of proteins by glutaraldehyde. Part I. Interaction of glutaraldehyde with the amino-groups of 6-aminohexanoic acid and of α-N-acetyl-lysine

FOLDED AND EXTENDED STRUCTURES OF HOMOOLIGOPEPTIDES FROM α,α‐DIALKYLATED GLYCINES. A CONFORMATIONAL ENERGY COMPUTATION AND X‐RAY DIFFRACTION STUDY

Folded and extended structures of homooligopeptides from .alpha.,.alpha.-dialkylated .alpha.-amino acids. An infrared absorption and proton nuclear magnetic resonance study

Structure‐Activity Relationships for the Lipid‐Mobilising Action of Locust Adipokinetic Hormone

The nature of the cross-linking of proteins by glutaraldehyde. Part 2. The formation of quaternary pyridinium compounds by the action of glutaraldehyde on proteins and the identification of a 3-(2-piperidyl)-pyridinium derivative, anabilysine, as a cross-linking entity