Insights on Chemical Crosslinking Strategies for Proteins

Abstract

Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical methods, along with the corresponding crosslinking reactions/conditions, material properties and significant applications.

Keywords: biomaterials; chemical crosslinkers; drug delivery; protein crosslinking; protein materials.

Scheme 1

Functional group targets in proteins/peptides that are susceptible to physical, chemical or enzymatic modifications.

Figure 1

(a) Genipin-amino group monomer formation by reaction of genipin with N-terminal or side chain amine group of a lysine residue in a protein like collagen. (b) The reaction of the genipin-amino group monomer with the amine group of either N-terminus/side chain of lysine resulting in a non-cytotoxic crosslinked form of intermolecularly crosslinked collagen proteins.

Figure 2

Scheme showing the first reaction (1) where nucleophilic substitution occurs between carboxyl groups in citric acid and the nucleophilic amine groups in one protein (protein 1); (2) second reaction showing more than one carboxyl groups of citric acid reacting with two proteins (protein 1 and 2) which could result in either intra/inter-crosslinking; reaction (3) showing all the three carboxyl groups of citric acid involved in crosslinking three proteins (proteins 1, 2 and 3). Nucleophilic substitution occurs by the attack of a partial negative charge on the nitrogen of NH₂ on the partial positive charge on COO⁻, resulting in amide bond formation only in increased pH conditions.

Figure 3

Reaction showing polymerization of nordihydroguairetic acid-bisquinone, initiated by autooxidation of the two catechols at neutral/alkaline pH conditions forming quinones, that associated together to form aryloxy free radical resulting in bisquinone crosslinks forming large network of crosslinked bisquinone polymer with collagen fibrils embedded without any crosslinks with amino acid side chains of collagen.

Figure 4

Illustration of collagen triple helix structure (PDB ID: 1BKV) crosslinked with procyanidins by hydrogen bond formation.

Figure 5

Mechanism of crosslinking of proteins/peptides by tannic acid.

Figure 6

Scheme showing the reactions of EDC (1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide) with carboxylates of aspartate/glutamate amino acids in collagen: (1) Attack of carboxylate oxygen of protein on electrophilic carbon of EDC; (2) An intermediate O-acylisourea reacts with the adjacent amine group of a lysine residue, forming a peptide bond; (3) O-acylisourea being unstable hydrolyses or forms comparatively stable N-acylisourea; (4) Side reaction with the addition of the positively charged group and removal of the negatively charged carboxyl group is shown; (5) The intermediate O-acylisourea reacts with sulpho-NHS (N-hydroxysuccinimide) to form another intermediate with more stability that results in high yields of favorable peptide bonds; (6) one more side reaction at the amide nitrogen of the successive residue forming cyclic imide.

Figure 7

Reactions showing 1,4-butanediol diglycidyl (BDDGE) ether mediated crosslinking of collagen proteins under (a) basic and (b) acidic conditions. (a) In basic conditions, nucleophilic attack occurs at the less substituted alkane of epoxide BDDGE with the amine group of a lysine residue in collagen, (b) In acidic conditions, nucleophilic attack occurs at the more substituted alkane of epoxide BDDGE with the carboxyl group of aspartate/glutamate residues in collagen.

Figure 8

Reaction showing the formation of pullulan dialdehyde on oxidation of pullulan with sodium periodate, followed by crosslinking with gelatin at 40 °C.

Figure 9

The reaction of N-hydroxysulfosuccinimide and aryl sulfonyl fluoride (NHSF), a multitargeting crosslinker, in which the succinimide being highly electrophilic, links with the lysine amino side chain of a protein and places the weakly reactive aryl sulphonyl fluoride with the adjacent protein’s nucleophilic residues (Nu), by sulfur-fluoride exchange (SuFEx).

Figure 10

Crosslinking of two proteins of close proximity by a heterobifunctional cross-linker, NHQM, with an NHS ester and a photocaged ortho-Quinone Methides (o-QM), where (1) NHS ester rapidly reacts with amine groups of lysine amino acids of proteins, followed by (2) exposure to UV (365 nm) activating the more reactive o-QM that (3) reacts with multiple nucleophilic (Nu) amino acids.

Figure 11

Mechanism of crosslinking proteins in the presence of tetrakis (hydroxymethyl) phosphonium chloride.

Figure 12

A peptide-small protein-based crosslinker, SpyTag-SpyCatcher, forms an isopeptide bond by the reaction of the 117th residue (aspartic acid-D) of SpyTag and 31st residue (lysine-K) of SpyCatcher (GenBank: AFD50637.1).

Figure 13

Mechanism of thiol-specific protein crosslinking using maleimide as crosslinker.

Figure 14

Immobilization of enzymes following glutaraldehyde crosslinking mechanism.