Volume 7 | Number 18 | 21 September 2020 ORGANIC CHEMISTRY FRONTIERS rsc.li/frontiers-organic ORGANIC CHEMISTRY FRONTIERS View Article Online REVIEW View Journal | View Issue Enzymatic and non-enzymatic crosslinks found in collagen and elastin and their chemical synthesis Cite this: Org. Chem. Front., 2020, 7, 2789 Jakob Gaar, a,b Rafea Naffac and Margaret Brimble *a,b Collagen and elastin are the most abundant structural proteins in animals and play an integral biological and structural role in the extracellular matrix. The biosynthesis and maturation of collagen and elastin occurs via multi-step intracellular and extracellular processes including the formation of several covalent crosslinks to stabilise their structure, confer thermal stability and provide biochemical properties to tissues. There are two major groups of crosslinks based on their formation pathways, enzymatic and non- enzymatic. The biosynthesis of enzymatic crosslinks starts with the enzymatic oxidation of lysine or hydroxylysine residues into aldehydes. These aldehdyes undergo a series of spontaneous condensation reactions with lysine, hydroxylysine or other aldehdye residues to form immature covalent crosslinks which are further matured via poorly understood mechanisms into multivalent crosslinks. While enzymatic crosslinks make up the majority of protein–protein crosslinks, the non-enzymatic unselective glycation of lysine residues via the Maillard reaction results in the formation of Advanced Glycation Endproducts (AGEs). These latter biosynthesis pathways are not fully understood as they are produced by a series of oxidative reactions between carbohydrates and collagen via Amadori rearrangements. Both covalent crosslinks and AGEs appear to correlate with several diseases such as skin and bone disorders, cancer metastasis, diabetes, Alzheimer’s and cardiovascular diseases. Although several crosslinks are isolated, purified and described in collagen and elastin, only a few of them are chemically synthesized. Chemical synthesis plays an essential and important role in research providing pure crosslinks as reference materials and enabling the discovery of compounds to understand the biosynthesis of crosslinks and their pro- perties. Synthetic crosslinks are crucial to verify the structures of collagen and elastin crosslinks where only a handful of structures have been determined by NMR spectroscopy and many other structures have Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. Received 22nd May 2020, only been predicted using mass spectrometry. This makes crosslinks and AGEs an interesting target for Accepted 4th August 2020 organic synthesis to produce sufficient quantities of material to enable studies on their biological signifi- DOI: 10.1039/d0qo00624f cance and determine their absolute stereochemistry. The biological and chemical synthesis of both enzy- rsc.li/frontiers-organic matic and non-enzymatic crosslinks are extensively described in this review. Collagen part in cell adhesion, migration and proliferation as well as providing strength.3 The most common type is the fibrillar Collagen is integral for the structure of the extracellular matrix type I collagen (Col-I) contributing about 90% of total collagen (ECM) and therefore vital for living organisms like mammals.1 content in the body. Col-I has an average size of 3000 amino Collagen is expressed throughout all organs and tissues acid residues and is involved in the formation of the structural making it the main component of connective tissues in the network of tissues including skin, tendons, bones, cornea and body.2 In vertebrates, up to 28 different types of collagen are the vascular system.4 known, most of them interact with other ECM proteins to There are significant differences between the amino acid form supramolecular network architecture which play a vital number and sequence, structure, and the role of different col- lagen types, however all share a common feature of at least one triple helical domain.5 This domain is formed by three helical aSchool of Chemical Sciences, The University of Auckland, 23 Symonds Street, polyproline type II (PP-II) chains tightly packed into a right- Auckland Central 1010, New Zealand handed triple helix which consists of a characteristic repeating bThe Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, amino acid motif (X -Y -Gly)n. Glycine occupies every third Private Bag 92019, Auckland 1010, New Zealand AA AA cNew Zealand Leather and Shoe Research Association, 69 Dairy Farm Road, position in the sequence which fit into the centre of the triple Palmerston North, New Zealand helix, therefore larger residues are not tolerated. Even minor This journal is © the Partner Organisations 2020 Org. Chem. Front.,2020,7, 2789–2814 | 2789 View Article Online Review Organic Chemistry Frontiers – substitutions, such as alanine, result in an alteration of the fairly consistent across different species (7% to 14%).12 16 The 6 conformation of the triple helix. The XAA and YAA positions hydroxylation of lysine in collagen type I shows profound are more variable in their allocation and are located on the differences between different tissues and even between the surface, with a high percentage of proline and trans-4-hydroxy- helical and telopeptide domains.12,13,17 Furthermore, only proline which allows the formation of the PP-II helix and pro- 50% of lysine is hydroxylated in bone collagen compared with vides conformational stability via interchain hydrogen bonds. 0% in skin type I collagen and 100% in type II collagen of Other amino acids in these positions include cationic residues cartilage.18 lysine or arginine, the anionic residues glutamic or aspartic The newly formed hydroxylysines can then be utilized as acid which drive the electrostatic interactions. More impor- an anchor point for O-linked glycosylation.11 This glycosyla- tantly, hydroxylysine is crucial for the collagen glycosylation tion occurs either with a galactose or glucose-galactose mole- and the formation of stable, covalent bonds for stabilizing the cule attached to the 5R-hydroxy group on the lysine residue.19 supramolecular structures.7 Although the 5R-configuration has been known since 1955,20 The biosynthesis of collagen has been widely investigated recent reports show the existence of both the 5R and 5S con- and is driven by the abundant nature of collagen in living figuration of the hydroxylysine moiety,21 The composition organisms and the central role it plays in cell differentiation and the frequency of these glycosylation events varies for and its functions through its interactions with ECM.8,9 As with different collagen types and tissue, and the impact for col- many other proteins, the synthesis of collagen is complicated lagen function has not been fully understood.22 Other PTMs and occurs via multi-step intracellular and extracellular pro- include disulfide bond formation, prolyl cis–trans isomeriza- cesses (Fig. 1). tion and trimerization of three PP-II chains, followed by The intracellular synthesis starts by the transcription from directional (from C-toN-) folding into a procollagen triple DNA to messenger RNA within the nucleus then translation of helix molecule flanked at each end by large non-helical pep- the PP-II polypeptide chains in the ribosome (Fig. 1). The PP-II tides. The three PP-II chains identify the type of procollagen polypeptide chains then undergo a variety of post-translational molecule whether all identical (homotrimer) or different modification (PTM) in the endoplasmic reticulum (ER), (heterotrimer). The most commonly found collagen type-I trafficking, aggregation and several enzymatic processes to (Col-I) is a heterotrimer which consists of two α1(I) and one produce a procollagen molecule which is exported into the α2(II) chains. ECM for fibrillogenesis (Fig. 1). Trafficking of the procollagen molecule from inside the The PTM of PP-II polypeptide chains involves the hydroxy- fibroblast into the ECM by Golgi is still controversial and not lation of proline and lysine residues to give hydroxyproline and well explained.23,24 When exported into the ECM, the large C- hydroxylysine (Fig. 1, middle). Such PTM requires both prolyl and N-terminals of procollagen are cleaved off by a group of and lysyl hydroxylases (PHX and LHX) and several cofactors metalloproteinases producing a helical tropocollagen molecule including 2-oxyglutarate (2-OG), oxygen, ferrous iron, and with a short and non-helical telopeptide region (Fig. 1). Several ascorbic acid (vitamin C).10,11 It has been previously shown types of crosslinks are then formed via enzymatic and non- Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. that the hydroxylysine content in different collagen types can enzymatic pathways. The formation of a crosslinking profile vary from 15% to 90%, unlike hydroxyproline levels which are varies during maturation, and ageing of collagen.25 Jakob Gaar was born in Rafea Naffa is a scientist at the Germany in 1990. He received Leather and Shoe Research his Master’s degree in organic Association of New Zealand and inorganic chemistry from based in Palmerston North. He the Ludwig-Maximillian’s finished his PhD in Biochemistry University in Munich in 2016. from School of Fundamental His Master’s thesis was con- Sciences at Massey University in ducted at Stockholm University 2017. His research focus is the under supervision of analysis of collagen in skins and Prof. B. Martín-Matutés hides where he developed several researching metal–organic analytical