Enzymatic and Non-Enzymatic Crosslinks Found in Collagen and Elastin and Their Chemical Synthesis Cite This: Org

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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 properties. 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 collagen 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

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– 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 methods for the separ- frameworks as heterogeneous ation and quantitation of col- Jakob Gaar catalysts for the Heck reaction. Rafea Naffa lagen crosslinks and advanced Since 2016 he is pursuing his glycation end products using PhD degree under the supervision of Prof. Brimble at the LC-MS. He has published more than 15 peer-reviewed papers University of Auckland in New Zealand. His research interests are since 2017 including 5 method papers. Currently, he is studying the synthesis of advanced glycation endproducts and their chemo- the extraction, purification and characterization of collagen from selective incorporation into biomimetic peptides. different animal species for several applications.

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Collagen enzymatic crosslinks Collagen non-enzymatic crosslinks

After removing the propeptide ends, the resulting tropocolla- Over its lifetime, collagen undergoes further modification by gen molecules spontaneously assemble into collagen fibrils sugar or sugar metabolites, with often undesired side- which are then stabilised by the formation of covalent cross- effects.33,34 The adventitious reaction of specific lysine and linking catalysed by lysyl oxidases (LOXs).26,27 LOXs are arginine residues on collagen with sugars occurs naturally copper-dependent amine oxidase with five disulfide bridges during ageing and results in the formation of non-enzymatic and require lysine tyrosylquinone (LTQ), ascorbic acid, Cu2+ crosslinks (Fig. 1).35 These are known as the Advanced 28 and O2 as cofactors. The telopeptidyl lysine 1 and hydroxyly- Glycation End-products (AGEs) and are linked to the decreas- sine 3 residues are oxidized into allysine 2 and hydroxyallysine ing mechanical strength of collagen fibers with age.36,37 The 4 respectively. The degree and types of enzymatic crosslinks formation of AGEs involves a complex series of non-enzymatic depend on the location and relative amount of lysine 1 and spontaneous condensation and rearrangement modifications hydroxylysine 3 in the helical and telopeptide domains. resulting in a wide-ranging group of non-enzymatic cross- During collagen synthesis, several types of crosslinks are links.38 Since their discovery, AGEs have been frequently formed between tropocollagen molecules developed within the linked to several diseases including cardiovascular diseases,39 fibrils thus defining the mechanical properties of the tissue nephropathy,40 cancer,41 retinopathy42 and Alzheimer’s.43 (Fig. 1). It has been shown that the formation of several covalent chemical bonds between collagen molecules is essential for the stabilization of collagen fibrils.29 Altered or insuffi- Elastin cient biosynthesis of collagen crosslinks leads to several diseases including bone fragility,30,31 skin hyperelasticity, and Elastin, similar to collagen, is a major component of the ECM cardiovascular diseases.18,32 that is a highly crosslinked protein polymer primarily known The crosslinking profiles of collagen in skin, bone, tendon for its elastic properties. The biosynthetic pathway of elastin is and cartilage are different, due to the differences in the similar to collagen where it starts intracellularly by the for- hydroxylation of lysine at both telopeptide and helical cross- mation of a soluble tropoelastin monomer which is then 44,45 linking sites.12 This suggests that the different types of secreted into the ECM. Unlike collagen, elastin can stretch collagen crosslinks are tissue specific rather than species to twice its length and recoil to its original shape after being specific.17,24 stretched. This property is essential for the biomechanical function of skin, blood vessels, lung, and ligaments providing elasticity and tensile strength.46 Although one third of the amino acids in elastin are glycine which is similar to that of Margaret Brimble is the Director collagen, elastin contains 75% nonpolar and uncharged of Medicinal Chemistry and a amino acids with a high proportion of valine and alanine and 47 Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. Distinguished Professor at the it contains no hydroxylysine, histidine or methionine. The University of Auckland where her interaction between the hydrophobic domains of tropoelastin research program focuses on the results in the formation of aggregates, which are further stabil- synthesis of bioactive natural ised by crosslinks contributing to the unique mechanical 48 products, antimicrobial peptides recoiling function of elastin. The extensive crosslinking of and peptidomimetics. She has elastin results in an extremely slow protein turnover that is 49–51 published 500 papers, 80 estimated to take more than 70 years. Analogous to col- reviews, holds 50 patents, won lagen, crosslinks are crucial for the biophysical properties of the 2012 RSNZ Rutherford elastin and the dysfunction of these complex modifications 52 53 Medal, the 2010 RSC Natural can lead to atherosclerosis, cystic fibrosis, and other 54 Margaret Brimble Products Award and the 2007 elastin-driven diseases. L’Oreal-UNESCO Women in Science laureate in Materials Science for Asia-Pacific. In 2018 she became a Fellow of the Royal Society (FRS) and in 2019 she was Biosynthesis pathways and chemical awarded a Damehood (DNZM) and admitted into the American synthesis of collagen and elastin Chemical Society Medicinal Chemistry Hall of Fame. She is Past- crosslinks President of IUPAC Organic and Biomolecular Division III, Past- President of the International Society of Heterocyclic Chemistry Over the past century, crosslinks found in collagen and elastin and an Associate Editor for Organic Letters. She co-founded the have become an interesting target for total organic synthesis. spin-out company SapVax using new technology to develop self- Due to the complex nature of crosslinks in collagen and adjuvanting cancer vaccines and discovered the drug trofinetide elastin and their sensitivity and susceptibility to degrade (NNZ2566) for Neuren Pharmaceuticals that is in phase 3 clinical during the extensive extraction procedures (including acidifica- trials for the neurogenerative disorder Rett Syndrome. tion and reduction), results of these isolation experiments

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Fig. 1 Biosynthesis of collagen and its enzymatic and non-enzymatic crosslinks. In the nucleus, collagen gene’s DNA sequence is transcribed to make an RNA molecule which is then translated into polyproline-II peptide chains in the ribosome. Several post-translational modifications (PTM) occur intracellularly in the endoplasmic reticulum (ER) including hydroxylation of proline and lysine into hydroxyproline and hydroxylysine respectively, and glycosylation of specific hydroxylysines by adding one or two sugar moieties. Three PP-II chains are then folded from (C-toN-) direction to produce a procollagen triple helix molecule with large non-helical end-peptides. The procollagen is transported into the extracellular matrix (ECM) before being attacked by several metalloproteases catalysing the formation of tropocollagen. The biosynthesis of collagen crosslinks is initiated by lysyl oxidases (LOXs) which catalyses the oxidative deamination of telopeptidyl lysine and hydroxylysine into allysine and hydroxyallysine respectively. These aldehydes then spontaneously react with other aldehyde, lysine and hydroxylysine residues driving the self-assembly collagen into fibrils. Ageing and glycation of the collagen fibrils resulted in the formation of mature crosslinks and advanced glycation end products.

have to be treated with care. Therefore, it is useful to chemi- field offers a wide variety of interesting targets for the develop- cally synthesize the proposed chemical structures that are ment of new synthetic methods and techniques. expected to be present in collagen and elastin to compare them to those isolated experimentally. Furthermore, the total organic synthesis of these structures provides sufficient pure Enzymatic collagen crosslinks material for use as internal standards, for further structural investigation or to enable the introduction of selective modifi- Lysine crosslink formation cations such as increased side-chain size.55 While the isolation Enzymatic collagen crosslinks are derived from lysine 1 that studies and the investigation of the effects of collagen cross- has been enzymatically modified to either allysine 2, hydroxyly- links are still ongoing, total organic synthesis of newly discov- sine 3, or hydroxyallysine 4. The amines available in these resi- ered molecules has become rare in recent years. Thus, this dues can facilitate nucleophilic attack onto aldehydes to form

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imine containing crosslinks. These are known as immature The organic synthesis of the reduced derivatives of the divalent crosslinks which then can mature into tri-valent or stereoisomers of the immature crosslinks HLNL 21 and tetra-valent crosslinks with other amino acids particularly his- DHLNL 22 was undertaken by Anastasia et al. in 2005 tidine. A variety of these crosslinks and references to their (Fig. 5).70 At this time, the stereochemistry of naturally occur- respective syntheses (where available) are depicted in Fig. 2. ring HLNL 21 and DHLNL 22 (nor the imine versions) was not Immature crosslinks like dehydro-lysinonorleucine 5 confirmed and a stereoselective method for the synthesis of all (deH-LNL), dehydro-hydroxylysinonorleucine 6 and 8 possible stereoisomers was required as the previous synthesis (deH-HLNL), and deyhdro-dihydroxylysinonorleucine 7 from Bailey only resulted racemic mixtures.71 (deH-DHLNL) were established as the initial crosslinks for Williams’ glycine template methodology was utilized for lysine-derived crosslinks, building the foundation for many the introduction of the desired stereochemistry at the other more complex crosslinks (Fig. 3). α-position of the amino acids.72 Stereoselective preparation of While the overall structure, localization in different tissues 21a, 22a, and 22b was achieved in a simple two step synthesis and isolation was published by Tanzer56 and Bailey,57 the from 23a and 23b, which was previously described by the same stereochemistry of the inherent alcohol groups was only group.73 The final compounds were obtained by removal of the reported for HLNL isolated from skin.21 The types of immature protecting groups. The overall yield of around 25–30% over 4 crosslinks are determined by the rate of hydroxylation of the steps from the starting epoxide afforded sufficient material for lysine in the telopeptide and the juxtaposed helical crosslinking further testing and modification of the desired target. sites.58 When the rate of hydroxylation of lysine in the telopeptide domain is very low, aldimine formation becomes predomi- Pyrrole containing crosslinks nant as reported for skin and rat tail tendon.58 However, two Pyrrole containing crosslinks are the trifunctional maturation reaction pathways are possible which involves telopeptidyl ally- products of ketoamines containing one pyrrole moiety, and sine 2 and either helical hydroxylysine (as collagen in skin) or were firstly postulated after the observation of a pink colour lysine (as in elastin) resulting in the formation of either when peptides isolated from connective tissues were reacted – deH-HLNL 6 and 8 or deH-LNL 5, respectively (Fig. 3).59 61 Both with Ehrlich’s reagent (p-dimethylaminobenzaldehyde).74,75 crosslinks have been shown to be essential for the formation of The original structures of deoxypyrrololine (d-Prl) 11 and pyr- mature crosslinks.60,61 DeH-HLNL 6 and 8 includes two com- rololine (Prl) 12 were proposed based on a postulated biosyn- pounds which are either derived from allysine 2 and hydroxyly- thesis which included the formation of a 1,3,4-trisubstituted sine 3, or hydroxyallysine 4 and lysine 1, respectively. pyrrole cross-link by condensation of a ketoamine with an Another type of immature crosslink is ketoamine, which is allysine.76,77 While further investigations confirmed the exist- found mainly in cartilage as most of lysine in the telopeptide ence of a pyrrole containing crosslink, the substitution of the domain is hydroxylated into hydroxylysine 2.12 These cross- pyrrolic core remained controversial. The original structures links have been also found in bone collagen, where ∼50% of d-Prl 11 and Prl 12 were later investigated by reacting collagen lysine 1 in the telopeptides is hydroxylated.58 Although peptides with biotinylated Ehrlich reagents, which identified Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. deH-HLNL 8 is classified as an aldimine, it can spontaneously the formed pyrrole containing structures in collagen.78 undergo an Amadori rearrangement to produce lysine-keto- However, based on mechanistic studies, Eyre et al. proposed a norleucine 19 (LKNL). In similar fashion the telopeptidyl different core-structure and substitution on the pyrrole func- deH-DHLNL 7 undergoes rearrangement to produce hydroxyly- tionality resulting in the revised structures lysylpyrroline 9 and sine-keto-norleucine 18 (HLKNL).62,63 Ketoamines are found hydroxyl-lysylpyrroline 10 (Fig. 2).18 Isolation and characteriz- predominately in connective tissues that bear large mechanical ation of these crosslinks remains difficult due to the instability loads such as bone, cartilage and tendon64 however, they were of the pyrrole moiety to acids or alkaline hydrolysis.79 recently detected in skin.65 Organic synthesis of d-Prl 11 was undertaken to further All divalent immature crosslinks are both acid and heat investigate these challenging crosslinks and the first total syn- labile due to the presence of the Schiff base double bond. thesis of d-Prl 11 was published by Adamczyk et al. in 1999 They are also reducible and can therefore be isotopically (Fig. 6).80 This synthesis utilized benzyl isocyanoacetate in the labelled using reducing agents such as tritiated sodium boro- key step to form the pyrrole core-structure 28 from 27.81 hydride which was the first method used for the discovery of Alkylation, followed by global deprotection and decarboxyl- the collagen crosslinks.66,67 The two aldimine crosslinks ation at the 2-position of the pyrrole ring yielded the desired deH-HLNL 5&7, deH-LNL 5, and deH-DHLNL 7 can be d-Prl 11. This is the only synthesis reported for pyrrole contain- reduced by sodium borohydride to form the more stable lysi- ing crosslinks, and further studies are required to confirm the nonorleucine 20 (LNL), hydroxylysinonorleucine 21 (HLNL), structure or the possible application of this type of lysine- and dihydroxylysinonorleucine 22 (DHLNL) respectively derived crosslink. (Fig. 4).56,62,68,227,228 Reduction also increases the stability of the amine towards hydrolysis in comparison to the imine. The Pyridinium-salt containing crosslinks standard procedures for the analysis of crosslinks in collagen Fluorescent crosslinks are an attractive target for isolation

requires the reduction of any imine with NaBH4 therefore the studies, because they can be separated and purified using synthetic, amine derivatives can be used as internal standards.69 RP-HPLC with fluorescence detectors without solely relying on

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Fig. 2 The chemical structures of collagen and elastin crosslinks.

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Fig. 3 Biosynthetic pathway of enzymatic lysine crosslinks. The t-Lysald represents telopeptidyl lysine oxidised into allysine, hel-Hyl represents the helical hydroxylysine and hel-Lys represents the helical lysine.

use as biomarkers for osteoporosis,86 metabolic bone dis- eases87 and osteogenesis imperfecta.88 An extensive review was published by M. Anastasia et al.89 focusing on the synthesis and the application of Pyr 13. While Pyr 13 is commonly found in bone, cartilage and tendon but is rare in skin, concentration of Dpyr 14 is minimal in cartilage and tendon.38,90 Pyr and Dpyr are destroyed upon exposure to UV radiation but due to the lack of

an imine moiety deuteration experiments using NaBD4 is not possible.91 Their formation is associated with a high number of hydroxylated lysine residues in the telopeptide domain found in cartilage and bone, where a high concentration of these crosslinks are detected.12 Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. The first postulated scheme of their formation was proposed by Fujimoto et al., involving two allysine 2 and a hydroxylysine for the formation of Pyr 13.92 Later a mechanism of their formation was proposed by Eyre and Oguchi who reported that two keto-amines react together by releasing hydroxylysine (Fig. 7).93 A second mechanism, based on the condensation of a keto-amine and hydroxyallysine 4 was proposed by Robins and Duncan.94 Although the two mechanisms are similar, they diﬀer in the way the collagen molecules are linked. According to Eyre’s mechanism, pyridinium crosslinks ’ Fig. 4 Chemical reduction of imine-based crosslinks using sodium connect three collagen molecules while Robin s mechanism

borohydride (NaBH4). supports a linkage between two collagen molecules. The latter was supported by Light and Bailey who reported that Pyr 13 crosslinked two type I collagen molecules.95 mass spectrometry.82,83 A common motif for this group of The first stereoselective syntheses of Pyr 13 and Dpyr 14 crosslinks are the pyridinium salt structures. The two most were published by Adamczyk et al. in order to provide clean well investigated structures are pyridinoline (Pyr) 13 and deoxy- material for the development of immunoassays for osteoporo- pyridinoline (Dpyr) 14 (Fig. 2). sis.55 While an earlier synthesis was published by Waelchli Pyr 13 and Dpyr 14 are two fluorescent pyridinium cross- et al. the lack of stereoselectivity for the introduction of the links that were isolated and identified by Fujimoto et al. (1977) hydroxyl functionality of Pyr 13 made this synthesis undesir- and Ogawa et al. (1982), respectively.84,85 Both structures able for the preparation of material to use as an internal stan- consist of trivalent crosslinks that have been widely investi- dard.96 A modular synthesis of Dpyr 14 was published in 1999, gated for their controversial formation mechanism and their which involves initial assembly of the pyridine ring and then

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Fig. 5 Synthesis of reduced 5-hydroxylsinonorleucine 21a and 5,5’-dihydroxylysinonorleucine 22a, 22b and 22c stereoisomers Anastasia et al.70 Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM.

Fig. 6 Total synthesis of deoxypyrrololine 11.80

eﬀects alkylation of the pyridine to obtain the protected Histidine-containing crosslinks 97 version of the desired crosslink (Fig. 8). There are two crosslinks that contain histidine as part of their Improvements for the stereoselective preparation of these structure: histidinohydroxylysinonorleucine (HHL) 15 and his- intriguing crosslinks were published in 2002 by Anastasia tidinohydroxymerodesmosine (HHMD) 16. Both HHL 15 and et al. employing a more eﬃcient route to yield Pyr 13 and DPyr HHMD 16 are only described as protein modifications and to 98 14 from the starting structure 34 (Fig. 9). In the same report, date no total organic synthesis has been reported.101 synthesis of 34 was accomplished following a procedure pub- Furthermore, previous NMR studies have shown that HHMD lished earlier that accessed this versatile core structure in five 16 appears to contain a mixture of R- and S-configurations at steps.99 Since then, several syntheses of native Pyr 13 and DPyr the stereocenter connecting the histidine and lysine residues 14 have been published including work on unnatural homol- while HHL 15 appears to have one configuration (Fig. 2, ogues by Anastasia et al. which were proposed as internal stan- asterix).102 The mechanism of formation and stereochemistry dards for the detection of pyridinolic crosslinks.100 of these crosslinks has not been fully confirmed to date and

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Fig. 7 Proposed pathways of pyridinoline 13 by (A) Eyre and Oguchi93 and (B) Robins and Duncan.94 Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM.

Fig. 8 First synthesis of deoxypyridinoline 14.97

remains controversial.90,103,104 Contrary findings have been C-telopeptide first reacts with hydroxylysine 3 producing a reported for HHMD 16, as it has been shown by Robins and divalent HLNL 21 crosslink which then reacts with histidine to Bailey to be a possible artefact during the reduction process form HHL 15.104,111,112 Yamauchi et al. confirmed that the – which was later challenged by Bernstein and Mechanic.105 107 imidazole C-2 of histidine is linked to C-6 of norleucine, which HHL 15 is a trivalent crosslink found mainly in skin and in is in turn linked to the C-6 amino group of histidine.104 some tendons at much lower concentrations.59,69,108 Its pro- Eyre et al.113,114 (2019) have recently reported that HHL 15 duction starts at birth with an initial rapid increase in concen- is a laboratory artefact rather than a natural cross-linking tration, followed by a more gradual increase with ageing.109 structure however Yamauchi et al.115 (2019) have challenged Stiﬀness of skin has been linked to a decrease in the concen- this finding. While no definite answer has been provided, tration of HLNL 21 crosslinks and a concomitant increase in recent reports showed the presence of HHL 15 in alpaca skin HHL 15 crosslinks.109 Furthermore, it has been reported that although its collagen lacks the histidine in the C-telopeptide UV radiation has no eﬀect on the HHL 15 structure.110 The which was reported by Eyre et al. to be essential for its current proposed biosynthesis is that allysine 2 from the formation.113,116

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Fig. 9 Improved synthesis of pyridininoline 13 and deoxypyridinoline 14 by Anastasia et al.98

The second histidine-containing crosslink is HHMD 16 on this condensation reaction, several immature intermediate which consists of four amino acid residues making it a crosslinks have been reported including dehydromerodesmo- tetravalent crosslink.117 As mentioned previously, the biosyn- sine 38, merodesmosine 39 (Fig. 10). thesis of HHMD 16 is controversial with one hypothesis In formation experiments deyhdromerodesmosine 38 could proposing the involvement of histidine, hydroxylysine 3 and be identified as precursor for the tetravalent desmosine 46.125 the Aldol condensation product (ACP) 37.118,119 However, Early work from Suyama described a cyclic trimeric precur- another mechanism is thought to involve the reaction of aldol- sor, which was called 1-cyclopen-1-one crosslink 41. Acid histidine 17 with hydroxylysine 3.103 The presence of HHMD hydrolysis, which is required to investigate crosslinks in col- 16 in vivo is debatable, despite it having been isolated, lagen, converts this immature crosslink into the dimeric identified and quantified in diﬀerent tissues by several crosslink cyclopentenosine 40.126 While the precursor is a tri- – Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. researchers.62,102,120 124 meric crosslink, a free alpha amine function undergoes ring- Although both HHL 15 and HHMD 16 crosslinks were closure resulting in a dimeric crosslink. Besides publications hypothesized to be laboratory artefacts,113 these findings have reporting its isolation and characterisation, no further been criticized.114,115 Whether, HHL 15 and HHMD 16 are research involving this crosslink has been described. Two com- only artefacts found in mass-spectrometry, or are indeed a pounds oxodesmosine 42 and Isooxodesmosine 43 were also natural collagen crosslink remains uncertain. Recently, HHMD reported in elastin however they are structurally similar to 16 has been isotopically labelled in vivo then measured by cyclopentenosine 40.127,128 LC-MS which confirmed the presence of one double bond. This suggests that HHMD 16 is susceptible to degradation at Pyridine containing crosslinks low pH.123 At this point no total organic synthesis of either of It has been proposed that dehydromerodesmosine 38 these crosslinks has been published to further understand the and ACP 37 are potent precursors for more multivalent nature of these intriguing histidine-containing crosslinks. crosslinks.125,129,130 The structures of desmopyridine 44, and isodesmopyridine 45 were determined by isolation of the crosslinks from bovine ligamentum nuchae elastin (Fig. 10).131 Elastin crosslinks Investigations of the impact of desmopyridine 44 and isodesmopyridine 45 on collagen are not available but the organic Immature crosslinks synthesis for desmopyridine 44 has been synthesised by Usuki Similar to collagen, LOX oxidatively deaminates specific lysine et al. (Fig. 12).132 residues to form aldehyde groups which then undergo Schiﬀ base formation to form immature crosslinks or undergo aldol Pyridinium-salt crosslinks condensation with another allysine 1 residue forming allysine Partridge et al. first described two pyridinium-containing aldol (ACP) 37.45 This aldol-condensation plays a vital role in elastin crosslinks and Thomas et al. isolated and named them the formation of several crosslinks found in elastin.46 Based as desmosine 46 and isodesmosine 47 in 1963 (Fig. 10).133,134

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Fig. 10 Chemical structures of crosslinks found in elastin.

These are the only examples of isolated and characterized starting material and ineﬃcient coupling during the synthesis tetravalent fluorescent pyridinium salt crosslinks, which are lead to the development of an improved synthesis. only found in elastin.135 This improved synthesis yielded desmosine 46 in 6 steps in Thebiosynthesisofbothdesmosine46 and isodesmosine 47 an overall yield of 15% (Fig. 11).144 Chemo- and regioselective was postulated by Anwar et al., who proposed that deH-LNL 5 Sonogashira and Negishi cross couplings were utilized as key reacts with ACP 37 to produce these stable elastin crosslinks.136 transformations from starting material 48. Selective alkylation After its isolation and characterization, desmosine 46 was of the pyridine ring with 51, was achieved under reflux in 145 extensively studied as a biomarker for chronic obstructive pul- MeNO2. Reduction and deprotection of 50 was later – monary disease (COPD)137 140 – a disease that is the fourth reported to be an eﬃcient pathway to yield the trivalent cross- leading cause of death – and it occurs due to the irreversible link desmopyridine 44.132 Additionally, deuterated versions for degradation of elastin in the lung tissue. This results in the further testing as biomarkers for COPD have been reported.146 release of elastin-specific crosslinks which can then be The latest improvement in the organic synthesis of these intri- detected in the blood and urine of patients.135,141,142 guing crosslinks was reported by Usuki et al., who utilized a Since its discovery, several total syntheses of desmosine 46 biomimetic Chichibabin reaction to yield desmosine 46 and have been reported including the first total synthesis by Usuki isodesmosine 47 in small amounts using a two-step synthesis et al. in 2012.143 Challenges in the large scale preparation of the (Fig. 12).147

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Fig. 11 Total synthesis of desmosine 46 and desmopyridine 44 by Usuki et al.144

Fig. 12 Biomimetic Chichibabin reaction towards isodesmosine 47 Usuki et al.147

Other amino acid containing divalent crosslinks noalanine 58 was first described as a collagen crosslink in cal- Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. cified bone collagen (Fig. 13).155 Since its discovery as a col- Crosslinks are a multifaceted group of molecules found in collagen crosslink it has been discovered as a naturally occurring lagen and elastin, and previously described examples were crosslink in mineral binding phosphoproteins156 and the grouped as enzymatic crosslinks based on enzymatic lysine cyclopeptide theonellamine B.157 Successful stereoselective modifications. The following group of crosslinks includes less syntheses of this crosslink have been reported by several defined examples of this intriguing class of protein modifi- groups.158,159 cations (Fig. 13). Arginine. Recently, Eyre et al. reported a new crosslink in ε γ Glutamine. In keratin, fibrin and fibrillin, ( -glutamyl)- cartilage formed by the spontaneous oxidation of a ketoamine lysine crosslinks 55 are synthesized by transglutaminase with arginine to produce arginoline 57 (Fig. 13).160 This cross- 148–150 (Fig. 13). Collagen can also be stabilized by transgluta- link has yet to be isolated and purified from cartilage due to ε γ minase through the formation of ( -glutamyl)-lysine links its acid sensitivity. No chemical synthesis has been published 151,152 between lysine and glutamine residues. Because of the to date which would help to support the preliminary findings high glutamine concentration in collagen, such crosslinks reported by Eyre et al. could be important. The best described glutamine-based cross- Tyrosine. Three tyrosine containing crosslinks including 153 link is ε(γ-glutamyl)-lysine 55. dityrosine 59,trityrosine60, isotrityrosine 61 were isolated and Phenylalanine. A phenylalanine containing unusual collagen characterized from cuticle collagen in which isotrityrosine 61 crosslink is found in marine mussels (Fig. 13).154 It is formed was shown to have an ether linkage (Fig. 13).161,162 If exposed to by the reaction of oxidized 3,4-dihydroxyphenylalanine (DOPA histidine the crosslink reacts with histidine residues or metal o-quinone) 56 and lysine residues as a result of the activity of ions to form a more complex crosslink, but the exact mecha- – catechol oxidase. nism is still unknown.163 167 Recently, isotrityrosine crosslink Histidine and alanine. Histidine has been already described 61 has gained more interest due to its appearance in resilin, a as a potential partner for crosslinking lysine residues in HHL type of elastin, and its possible application in biomaterial 15 and HHMD 16. The alanine containing crosslink histidi- research.168,169 Dityrosine 59 was firstly synthesised by Gros and

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Fig. 13 Chemical structures of other amino acid containing crosslinks.

Sizer by enzymatic peroxidation of tyrosine.170 The formation of of AGE products is still much lower than those containing this crosslink has been found to be promoted by oxidising crosslinks formed by the activity of lysyl oxidases.12 agents as well as ultraviolet and γ-radiation.171 Since its discov- Collagen is known to have a long half-life, with specific

Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. ery this crosslink was utilized as biomarker for several diseases types of collagen in flexible tendon tissue having an estimated including Alzheimer’sdisease172 and Parkinson’sdisease.173 Of life-time of above 200 years.182 This makes collagen prone to particular interest there are efficient analytical methods to accumulation of undesired glycation modifications over time determine dityrosine 59 crosslinks in pathologically relevant and thus it is an attractive target for studying the influence of proteins or peptides like amyloid-beta peptide.174 AGE modifications on biological and structural properties.183 Crosslinking via glycation is uncontrolled and alters the structural and biological properties of the native peptide. Several Glycation studies have demonstrated that loss of structural integrity or changes in biochemical behaviour can contribute to diabetes- 184 185 Lysine and arginine are the two major residues involved in related failure of tissue, arteriosclerosis and oxidative 186 Maillard reactions which drive the formation of AGE products. stress. Initially a sugar reacts with a lysine residue to form a Schiff base which then undergoes an Amadori rearrangement to Fluorescent non-enzymatic crosslinks (AGEs) produce the Amadori product.175 Subsequent reactions of the AGEs are known for being a loosely grouped class of molecules Amadori products can yield a variety of AGEs including cross- and over time several approaches have been undertaken to linking AGEs if further lysine or arginine residues are present further classify different AGEs. The first common classification – (Fig. 14).176 178 is into single-site and crosslinking AGEs. The latter are diami- Maillard reaction generates several reactive intermediates nodicarboxylic acids, derived from two amino acid residues, that then undergo several oxidation reactions, or non-oxidative predominantly lysine or arginine, reacting with sugars or sugar rearrangements, resulting in the formation of many crosslinks metabolite intermediates.187 These AGEs are of particular between protein molecules.179,180 Not surprisingly, the rate of interest due to their ability to crosslink two or more protein glycation is accelerated in the presence of high glucose con- chains together, effecting the structural and biological pro- centrations and radiation.181 In spite of this, the concentration perties of the modified proteins. Distinguishing between the

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Fig. 14 Maillard reaction for the formation of Advanced Glycation Endproducts (AGEs).

fluorescent properties is an excellent way to further diﬀeren- lished a shorter and higher yielding synthesis of pentosidine tiate between diﬀerent kinds of AGEs. Like non-fluorescent 62, in which formation of the imidazo[4,5-b]pyridine core AGEs, fluorescent crosslinking AGEs result in an irreversible made use of a key reaction developed by their group modification, derived from the reaction between glycating (Fig. 16).201 agents with at least two amino acid residues, resulting in an Protection of 2-chloro-3-amino-pyridine 67 with the elec- elaborate π-electron system allowing absorption and emission tron donating group dimethoxybenzaldehyde (DMB) enabled of light. Fluorescent crosslinking AGEs have become increas- successful execution of the previously reported ring-closure

ingly more important, due to their valuable application as reaction using Pd2(dba)3*CHCl3 and the Me4t-BuXPhos potential biomarkers for systemic diseases,188peripheral artery ligand.202 Ornithine fragment 69 was obtained from com- – disease,189,190 kidney disease,40,191 and diabetes.192 194 A list of mercially available Boc-Orn(Cbz)-OH, while lysine fragment fluorescent crosslinking AGEs and their respective chemical 71 was obtained following a short route reported by syntheses (if available) is shown in Fig. 15. Adamczyk.55 Final deprotection of the protected pentosidine While single-site non-fluorescent modification AGEs are 72 yielded the desired crosslink pentosidine 62. With an Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. among the most abundantly found, the presence of fluo- overall yield of 30.1% in 10 steps, at the longest route, this rescent crosslinking AGEs, enables the identification and synthetic pathway is very eﬃcienttoobtainthisfluorescent quantification of these AGEs by measuring their absorbance crosslink. and emission.195 This often has the advantage of allowing non-invasive methods, like skin-auto fluorescence,196 or fluo- Vesperlysine and crossline 66 rescence quenching197 to be used. While this is a simple yet The vesperlysine-type AGEs were first described by Nakamura powerful tool to ascertain accumulation of AGEs in patients, and co-workers in 1997 by incubating bovine serum albumin standardized procedures are required to obtain reliable in glucose.203 After acidic hydrolysis of the modified protein, results.198 HPLC purification resulted in the identification of three diﬀerent AGEs – vesperlysine A, B, and C – and structural Pentosidine 62 determination via nuclear magnetic resonance (NMR) revealed Pentosidine 62 is the most well-known and studied cross- three individual structures. Much like pentosidine 62,itwas linking AGE that exhibits fluorescent properties. Monnier and proposed that the three AGEs were glycoxidation products of co-workers199 first discovered pentosidine 62 as a crosslinked two lysine residues with two or more sugar molecules. This modification of extracellular proteins by extraction from was then confirmed by identifying the same products in the human dura mater via acid hydrolysis and extensive HPLC reaction between lysine with both ascorbic acid and short purification. The first total synthesis was published in 1999, by chain sugars. Later Monnier and co-workers identified vesper- Shioiri et al.200 enabling access pentosidine 62 without lysine A 63 as a crosslink present in the human lens protein complex HPLC. Unfortunately, while Shioiri managed to from the elderly.204 obtain the core structure in relatively few steps, the necessary A similar but less well described AGE is crossline 66, which amino acid fragments to access the sidechains equipped with was first described by Kondo and co-workers.205 Crossline dis- a thiocyanate- or an iodo-functionality, required a laborious plays similar fluorescence characteristics to natural in vivo and low-yielding synthesis. In 2012 Clark and co-workers pub- fluorophores and has been widely investigated as a biomarker

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Fig. 15 Fluorescent crosslinking AGEs. Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM.

Fig. 16 Total synthesis of pentosidine 62 by Clark et al.201

– for diabetic retinopathy205 and nephropathy.206 208 While it Non-fluorescent AGE crosslinks has often been referenced for its spectroscopic properties research looking into pathways for formation of crossline has Non-fluorescent crosslinking AGE, much like the fluorescent suggested that it is not formed in vivo.209 crosslinks, comprise two, or more amino acids but lack an elab- The chemical synthesis of vesperlysine-type AGEs and cross- orate π-system to absorb and emit light. Their exposure to line has not previously been reported. diﬀerent proteins leads to a wide variety of diﬀerent crosslinking

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Fig. 17 Non-ﬂuorescent crosslinking.

AGEs. Several non-fluorescent crosslinking AGEs and their riboses or glucosone.210 Previous work by Baynes et al. utilized respective chemical syntheses (if available) are shown in Fig. 17. hippuryllysine, allowing a simple purification of the obtained hippuryllysine AGEs GOLD and MOLD.211 Utilizing the direct GOLD 73 and MOLD 74 modification of the ω-amine, on suﬃciently protected lysine, a Prominent representatives of the non-fluorescent crosslinking high yielding synthesis of Boc-protected MOLD was reported AGEs are the glyoxal lysine-dimer 73 (GOLD), methylglyoxal by Esposito and coworkers.212 lysine-dimer 74 (MOLD), and the well-studied and described Based on this protocol and work from Lederer et al.213 a glucosepane 75. simple two step synthesis for the preparation of Fmoc-pro- GOLD 73 and MOLD 74 are typically derived from the reac- tected GOLD 73 and MOLD 74 was published by Brimble et al., tion of two lysine-residues with glyoxal or methyl-glyoxal, which aﬀords the AGEs in relatively high yields and avoids respectively. While they are the main products from these two laborious HPLC purification (Fig. 18).214 distinct glycation reagents, formation of the crosslinks can be After incubation of Fmoc-Lys-OH, with methylglyoxal or observed when other glycation reagents are utilized like glyoxal to access GOLD and MOLD respectively, the reaction

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Fig. 18 Synthesis of GOLD 73 and MOLD 74 by Brimble et al.214

mixture can be directly purified by column chromatography, GOLA 76 and MOLA 77 allowing simple and scalable synthesis of these protected AGE α GOLA 76 was identified as amide containing AGE crosslink, crosslinks. Simple deprotection of the N -amine protecting which is formed under similar conditions to GOLD 73.221,222 215 groups, yields GOLD 73 and MOLD 74 in quantitative yields. Any glycation reagents that can form glyoxal can lead to the formation of this crosslink. The impact GOLA 76 ad MOLA 77 Glucosepane 75 on diseases has not been described in the literature. Recently Quantification of the above-mentioned Maillard-derived cross- Glomb and coworkers described the first synthesis of MOLA links in senescent human extracellular matrix, lead to the con- 77 which was based on their own previous work with GOLA clusion that concentration of these crosslinks is elevated in 76.223 The obtained crosslink was identified and quantified 216 elderly patients and is increased in diabetic patients. While in vitro and in vivo and was correlated to tendon stiffness in rat all crosslink concentrations are elevated glucosepane 75 was tail-collagen.223 Similar to the previous findings with GOLA 76, found to be the major crosslink. good correlation was found for the pathway of formation Glucosepane 75 was first discovered by Lederer et al. by between MOLA 77 and MOLD 74. 217 incubating bovine serum albumin (BSA) with D-glucose. This unusual AGE crosslink has a rare 7-membered ring and Histidino-threosidine 78 Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. constitutes a mixture of four diastereomers. While other AGEs have been shown to be stable under acid hydrolysis, glucose- Histidino-Threosidine His-Thre 78 was the first histidine containing AGE crosslink isolated and characterised by Monnier pane 75 is unstable under conventional hydrolysis conditions, 224 and exhaustive mild enzymatic digestion of protein sources is et al. by incubating lysine, histidine and D-threose. The con- required to obtain the single molecule.216,218,219 Furthermore, centration of D-threose required to form this crosslink was sig- glucosepane 75 exhibits absorption of only short wavelengths, nificantly higher than the physiologically relevant concen- making HPLC purification coupled with absorption spec- tration, making it unlikely that this crosslink is formed in vivo. troscopy ineffective for analysis. Lederer described the formation of glucosepane 75 and was GODIC 79, MODIC 80, DOGDIC 81 and DOPDIC 82, able to isolate it in his experiments. Unfortunately, the stereo- Pentosinane 83 selective synthesis of single isomers was not achievable using GODIC 79 and MODIC 80 were first described as a Bovine this unselective incubation method. The first stereoselective Serum Albumin (BSA) incubation modification and were syn- synthesis of glucosepane 75 was published by Spiegel et al. in thesized in the same work in a simple but low yielding syn- 2015 proceeding in an overall yield of 12% in a total of eight thesis and used as internal standards for LC-MS (Fig. 20).225 steps starting from commercially available starting materials Synthesis of DOGDIC 81, DOPDIC 82 and pentosinane 83 (Fig. 19).220 was carried out by Lederer et al.,226 as part of investigations While the synthesis requires the preparation of starting into the pathways for several AGE crosslinks. Isolation of pen- material 87 it offers an elegant way to obtain all possible tosinane 93 and subsequent deuteration experiments, con- stereoisomers of glucosepane 75. This makes the synthesis firmed this crosslink as the precursor to the fluorescent cross- α α particularly interesting for the utilization of the obtained AGEs link pentosidine 62. Incubation of N -Boc-lysine 92 and N - as immunoreagents. It is expected that the synthesis and avail- Boc-arginine 93 with different sugars, purification by RP-HPLC ability of glucosepane 75 will further boost the understanding and deprotection of the protecting groups lead to the isolation of how this complex AGE contributes to metabolic diseases. of all crosslinks in various quantities. Unfortunately, this

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Fig. 19 Chemical synthesis of glucosepane 75.220

enzymatic processes. The cross links in these proteins have Published on 04 August 2020. Downloaded 10/10/2021 2:57:08 AM. been linked to several diseases including cancer metastasis, osteoporosis, cardiovascular diseases and osteoarthritis. The chemistry of these crosslinks has been known since the 1960s however many of them have not been isolated and their structures have yet to be determined by NMR spectroscopy. Therefore, organic synthesis offers a versatile way to prove the existence of crosslinks in vivo and can facilitate structural determination. This review summarizes the biosynthesis and chemical synthesis (if available) of more than 30 enzymatic and non-enzymatic crosslinks found in collagen and elastin to date. The intriguing process of the biosynthesis of enzymatic crosslinks is described then the challenges and gaps in the analysis and Fig. 20 Synthesis of GODIC 79 and MODIC 80. determination of their chemical structures are presented. The enzymatic crosslinks have been classified based on their chem- method does not enable a stereoselective synthesis of DOGDIC istry while the AGEs were divided into fluorescent and non- 81 and DOPDIC 82, which to date has not been achieved. fluorescent crosslinks to ease navigation through this review. Identification of such crosslinks has been a subject of research for a long time and to date crucial information, like Conclusion stereochemistry, still remains unavailable for some examples. Of note, instability towards acid hydrolysis is often noted as a The biosynthesis of collagen and elastin crosslinks is a reason for difficulties encountered when attempting to obtain complex process which involves several enzymatic and non- specific crosslinked structures in sufficient quantities or

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