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.

Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.

/pdf/glutaraldehyde-in-bio-catalysts-design-a-useful-crosslinker-15gbs09ph3.pdf

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

Glutaraldehyde is commonly used to control physical and biological properties of collagen structure by means of intramolecular and/or intermolecular crosslinking of collagen molecules. Solubility, antigenicity, and biodegradation of naturally occurring or reconstituted collagenous matrices are effectively reduced by glutaraldehyde treatment. Adverse biological reactions to glutaraldehyde have been limited to infrequent contact dermatitis and to biocidal effects which are exploited in chemical sterilization media. In the present study of glutaraldehyde-tanned collagen sponge, the presence of glutaraldehyde was correlated with cytotoxic effects upon fibroblasts in tissue culture and foreign body giant cell reaction to bioimplants of the sponge. Fibroblast growth in tissue culture is 99% inhibited at media concentrations of 3.0 ppm glutaraldehyde. Extracts of glutaraldehyde collagen sponge in aqueous media at pH 7 and 4.5 yielded 6 micrograms and 65 micrograms glutaraldehyde per gram of collagen sponge, respectively. The yield increased tenfold at pH 4.5. Observations indicate that leaching of the glutaraldehyde from glutaraldehyde-tanned collagen sponge is sufficient to produce potentially adverse cellular effects both in vivo and in vitro.

Biological effects of residual glutaraldehyde in glutaraldehyde‐tanned collagen biomaterials

Chemically recyclable polymers: a circular economy approach to sustainability

A new resist system is described which undergoes spontaneous relief image formation. The resist is formulated from end capped poly(phthaladehyde), PPA, and a cationic photoinitiator such as a diaryliodonium or triarylsulfonium metal halide. The extreme sensitivity of the resist is the result of designing for chemical amplification. The desired amplification results from the fact that photolysis of the sensitizer generates acid which catalyzes main chain cleavage of the polyaldehyde. The uncapped polymer is thermodynamically unstable with respect to reversion to monomer at room temperature so a single acid catalyzed scission results in complete depolymerization to volatile monomer. A single radiochemical event is thereby amplified in the sense that it produces an enormous number of subsequent chemical transformations. PPA/onium salt resist films are so sensitive that exposure to low doses of e-beam, X-ray or ultraviolet radiation results in complete self development without post-exposure processing of any kind. The exposed area simply vaporizes.

Chemical amplification in the design of dry developing resist materials

Progress in polymer science, japan, volume 1.

Polymerization of aromatic aldehydes. II. Cationic cyclopolymerization of phthalaldehyde

Polymerization of Aromatic Aldehydes. III. The Cyclopolymerization of Phthaldehyde and the Structure of the Polymer

Vinylferrocene was shown to undergo readily cationic polymerization by FRIEDEL-CRAFTS and other cationic catalysts. The polymers obtained possessed rather low molecular weights, the highest being 3500. In spite of the high reactivity of the ferrocene nucleus towards electrophilic substitution, no mode of propagation other than vinyl addition was detected when the infrared spectra of polymers prepared by cationic and free radical reactions were compared. The electronic spectra of polyvinylferrocene and its oxidized form were similar to those of ferrocene and ferricinium ion, respectively. In the cationie copolymerization with styrene, the latter was incorporated into the polymer only when the styrene content in the monomer feed exceeded 90%. The copolymerization with vinyl isobutyl ether (M2) by BF3OEt2 in toluene at 0°C gave the reactivity ratios: r1 = 0.1 ± 0.1, r2 = 9.7 ± 1.0. The high cationic reactivity of vinylferrocene is consistent with the stability of the α-ferrocenylearbonium ion inferred from solvolysis and other reactions.



Eswurde gezeigt, das sich Vinylferrocen durch FRIEDEL-CRAFTS- und andere kationische Katalysatoren leicht polymerisieren last. Die erhaltenen Polymeren hatten jedoch nur maximale Molekulargewichte von 3500. Trotz der hohen Reaktivitat des Ferrocenkerns gegenuber einer elektrophilen Substitution wurde keine andere Wachstumsreaktion gefunden als die Vinyladdition, wie man durch Vergleich der IR-Spektren der durch kationische Polymerisation erhaltenen Polymeren mit den durch Radikalpolynierisation erhaltenen feststellen konnte. Die Elektronenspektren des Polyvinylferrocens und seiner oxydierten Form gleichen denen des Ferrocens bzw. des Ferriciniumions. Bei der kationischen Copolymerisation mit Styrol wird dieses nur dann in nennenswerter Menge eingebaut, wenn der Styrolgehalt in der Monomerenmischung 90% ubersteliat. Die Copolymerisation mit Vinylisobutylather (M2) durch BF3O(C2H5)2 in Toluol bei 0°C ergab die Copolymerisations-parameter: r1 = 0,1 ± 0,1, r2 = 9,7 ± 1,0. Die hohe kationische Reaktivitat des Vinylferrocens steht in Ubereinstimmung mit der aus der Solvolyse und anderen Reaktionen abgeleiteten Stabilitat des α-Ferrocenylearboniumions.

Chuji Aso

Papers

Progress in polymer science, japan, volume 1.

Polymerization of aromatic aldehydes. II. Cationic cyclopolymerization of phthalaldehyde

Polymerization of Aromatic Aldehydes. III. The Cyclopolymerization of Phthaldehyde and the Structure of the Polymer

Cationic polymerization and copolymerization of vinylferrocene

Imidazole Catalyses in Aqueous Systems. I. The Enzyme-like Catalysis in the Hydrolysis of a Phenyl Ester by Imidazole-Containing Copolymers