Chemoenzymatic synthesis of ultralow and low-molecular weight heparins

Journal Pre-proof Chemoenzymatic synthesis of ultralow and low-molecular weight heparins

Ting Wang, Li Liu, Josef Voglmeir PII:

S1570-9639(19)30187-6

DOI:

https://doi.org/10.1016/j.bbapap.2019.140301

Reference:

BBAPAP 140301

To appear in:

BBA - Proteins and Proteomics

Received date:

31 July 2019

Revised date:

14 October 2019

Accepted date:

15 October 2019

Please cite this article as: T. Wang, L. Liu and J. Voglmeir, Chemoenzymatic synthesis of ultralow and low-molecular weight heparins, BBA - Proteins and Proteomics(2019), https://doi.org/10.1016/j.bbapap.2019.140301

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© 2019 Published by Elsevier.

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Chemoenzymatic synthesis of ultralow and low-molecular weight heparins Ting Wang, Li Liu, and Josef Voglmeir* [email protected] or lichen.liu@ njau.edu.cn Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and

Corresponding author.

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*

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Technology, Nanjing Agricultural University, China

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Abstract:

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Heparin is a naturally occurring glycosaminoglycan isolated from animal tissues and is medically used

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as an anticoagulant drug. Adulteration attempts of isolated heparin with chondroitin sulfate in the past

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resulted in great safety concerns. Also, increasing demands on batch-to-batch homogeneity for better evaluation and control of its pharmacodynamic and pharmacokinetic properties kindled the

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development of synthetic routes for the production of heparin and its derivatives. The discovery of enzymes involved in glycosaminoglycan biosynthesis and their application in chemoenzymatic synthesis makes it feasible to generate low molecular weight heparins (LMWHs) and ultra-low molecular weight heparins (ULMWHs). Understanding the scope and limitations of these enzymes currently used in the production of synthetic heparins will help to achieve more defined heparins with controlled medicative properties. Here, we summarized the recent advances in the chemoenzymatic synthesis of LMW/ULMW heparins. Keywords: synthetic heparin, LMWHs, ULMWHs, chemoenzymatic synthesis, oligosaccharide biosynthesis 1

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1. A brief introduction of heparins Heparins are highly sulfated polysaccharides and are commonly isolated from animal tissues. In a clinical context, heparin is often used as a synonym for unfractionated heparin (UFH) and is used for more than 80 years as an anticoagulant [1, 2]. UFH is a natural product extracted from porcine intestine

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with an average molecular weight (MWavg) of 15 kDa [1]. In the 1980s, the second generation of

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heparins classified as low-molecular-weight heparins (LMWHs) such as enoxaparin, dalteparin or tinzaparin were approved for clinical use [3, 4]. LMWHs with MWavg ranging from 3000 to 6500 Da

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are derived from unfractionated heparin with chemical hydrolysis, enzymatic digestion or physical

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ultra-filtration [5]. However, even sharing the similar sugar composition, the pharmacodynamic and

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pharmacokinetic properties of these LMWHs are significantly different from each other due to the

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heterogeneity caused either by the origin of the UFH or by using different methods during the depolymerization process [5-8]. For both UFH and LMWHs, reports confirmed adulteration attempts

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by adding chondroitin sulfate (a polysaccharide consisting of similar sugar monomers but very different physiological functions), which led to severe side effects [9]. In 2002, the first example of the third generation heparins, the ultra-low-molecular-weight heparin (ULMWHs, MWavg < 3000 Da), fondaparinux (a chemically synthesized sulfated pentasaccharide), was approved for medical use with highly consistent and controllable pharmaceutical parameters [10, 11]. Although the reported synthesis yields of fondaparinux were low (0.1% ~ 1%), and only little progress has been achieved during these years for improving these yields, these works are still considered as milestones in synthetic carbohydrate synthesis [12-14]. Given the complexity of the chemical synthesis of heparins at industrial scales, the depolymerization of UFH is still the major technique applied in the production of 2

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LMWHs or ULMWHs. For example, semuloparin, another ULMWH, is produced by the depolymerization of heparin using a selective β-elimination reaction [6, 15]. The use of enzymes for synthesizing ULMWHs was first demonstrated in 2011, with two homogenous and bioactive heptasaccharides with overall yields of 35% were generated in milligram scale [16]. Overcoming the limitations of low yields of the chemical synthesis and the heterogeneity of depolymerized UFH,

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enzymatic and chemoenzymatic synthesis of ULMWHs became a vibrant research area. The next

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chapters are going to discuss the influence of the molecular composition of heparins on their pharmacodynamic and pharmacokinetic properties and summarize the recent advances in the

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chemoenzymatic synthesis of LMW/ULMW heparins.

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2. Effects of structures on pharmacodynamics and pharmacokinetics of heparins

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Heparin contains a disaccharide repeating unit, which consists itself of partly sulfated glucosamine

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(GlcN), glucuronic acid (GlcA) or iduronic acid (IdoA) monomers. The anticoagulant activities of heparins are strictly dependent on the binding of a unique pentasaccharide recognition motif,

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GlcNAc6S-GlcA-GlcNS3S±6S-IdoA2S-GlcNS6S to anti-thrombin III, which is an inhibitory protein of both coagulation factor Xa (FXa) and thrombin (factor IIa, FIIa) [17, 18]. As illustrated in Fig. 1, the binding of this carbohydrate motif or called anti-thrombin III binding domain (AT binding domain) triggers a conformational change of anti-thrombin III, which leads to a complex formation between heparin, anti-thrombin III and FXa, and thereby results in blocking the coagulation cascade by the inhibition of FXa activity (anti-FXa). An extension of this recognition motif at the non-reducing end enhances the binding of anti-thrombin III to thrombin (FIIa), and therefore inhibits the activity of FIIa (anti-FIIa), and directly blocks the conversion of soluble fibrinogen to insoluble fibrin strands, which leads to anti-coagulation and simultaneously increases the risk of bleeding and other side effects [1]. 3

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Hence, a high ratio of anti-FXa to anti-FIIa is considered to be associated with higher efficiency and results in lower risks of bleeding complications [19]. Heterogeneity of LMWHs is caused both from the initial starting materials and from variations in the production methods and results in variable anti-FXa/anti-FIIa ratios among different commercial heparins [6, 20, 21]. The lowest anti-FXa/anti-FIIa ratio observed in UFH is about 1 [19]. Another concerning side effect of heparins

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is heparin-induced thrombocytopenia (HIT), which was observed with a lower incidence in LMWHs

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and ULMWHs compared to UFH [22]. A dodecasaccharide motif containing repeated trisulfated units (IdoA2S-GlcNS6S) of heparin binds to platelet factor IV (PF4) and consequentially causes

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aggregation and the loss of platelets [23, 24]. Anionic heparin polymers could also bind to protamine,

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a poly-cationic peptide, which results in the neutralization of the anticoagulant effect of UFH. The

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interaction between heparin and protamine was reported to depend strongly on the molecular weight

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and degree of sulfation of the heparin [25]. The anticoagulant activity of ULMWHs and LMWHs could not or only partly be neutralized by protamine [26]. The structural features of heparins, including the

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degree of polymerization and modification pattern also influences their pharmacokinetic properties such as metabolism and clearance. Stabilin-2 and its homolog Stabilin-1 are scavenger receptors on endothelial liver cells, and responsible for the clearance of UFH and parts of LMWHs [27]. For effective recognition, Stabilin-1 and 2 require at least a heparin nonasaccharide with 3-O-sulfation on its glucosamine units [27]. Results from mouse models showed that a heparin derivative with these 3-O-sulfations exhibited a significant accumulation in the liver within 3 hours and retained in the blood with a half-life of 90 minutes [28]. ULMWHs like fondaparinux, and semuloparin and some isolated LMWH fractions were reported to be primarily cleared via the kidneys and having comparatively longer plasma half-lifes [29]. The difference between pharmacodynamics and pharmacokinetics 4

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properties caused by compositional features of heparins determines their different applications in patients [30]. UFH has a short plasma half-life, high risk of bleeding but could be neutralized by protamine, therefore it is usually applied in continuously monitored medical procedures such as surgery. LMWHs and ULMWHs are more suitable for long time patient treatments or even prophylactic

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treatment (i.e. venous thromboembolism) through subcutaneous administration [31].

Figure 1. The anticoagulant mechanism of heparin and its derivatives. The binding of the terminal pentasaccharide highlighted in red to antithrombin triggers the conformational change of antithrombin and leads to the formation of an inhibitory complex of antithrombin-thrombin with anti-FIIa activity or antithrombin-FXa with anti-FXa activity. The binding of thrombin needs an extension of five repeated disaccharide units (in blue) at the non-reducing end of heparin. FXa, coagulation factor Xa; FIIa, thrombin or coagulation factor IIa.

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3. Chemoenzymatic synthesis of LMW/ULMW heparins Chemoenzymatic synthesis strategies of LMWHs/ULMWHs generally mimic the heparin biosynthesis, which can be divided into three phases including initiation, polymerization, and modification [17]. As illustrated in Fig. 2, the heparin biosynthesis is initiated by the formation of a tetrasaccharide linker (Xyl-Gal-Gal-GlcA) to a serine residue of the core protein and elongated with repeating GlcNAc-GlcA

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disaccharide units by exostosin glycosyltransferase 1 and 2 (EXT 1 and EXT 2) [17, 32]. Then, the

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acetyl groups of the GlcNAc units were partly removed by N-deacetylase/N-sulfotransferase (NDST), which also consecutively catalyzes the N-sulfation of the resulting GlcN units [33, 34]. Furthermore,

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C5-epimerase (C5-epi) converts GlcA into IdoA [35]. Then both of these sugar units can be sulfated by

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2-O-sulfotransferase (2-OST). Additional sulfations at the 6-position of GlcN are catalyzed by

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6-O-sulfotransferase (6-OST) [36]. The most important sugar modification of heparin for anti-thrombin

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III binding is the sulfation of the 3-position of GlcN by 3-O-sulfotransferase (3-OST) [37]. Most of these biosynthetic enzymes can be actively expressed and recombinantly produced on large scale and

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have been successfully applied as biocatalysts in heparin synthesis [38, 39]. However, the efficient recombinant production of NDST and EXT in active form using bacterial expression hosts is still challenging [40, 41]. An elegant strategy to overcome the lack of recombinant NDST as a biocatalyst was to perform the required N-deacetylation/N-sulfation step by chemical means in an initial phase of the ULMWHs synthesis, and then to perform enzymatic epimerization and O-sulfation steps [42].

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Figure 2. Biosynthesis of heparin in mammals. Heparin biosynthesis includes the elongation of a tetrasaccharide linker by EXT1 and EXT2, partially N-deacetylation/N-sulfation with NDST, glycosyltransferase;

NDST,

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epimerization of GlcA into IdoA by C5-epi, and O-sulfations with 2-, 3-, and 6-OST. EXT, exostosin N-deacetylase/N-sulfotransferase;

C5-epi,

C5-epimerase;

2-OST,

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2-O-sulfotransferase; 6-OST, 6-O-sulfotransferase; 3-OST, 3-O-sulfotransferase.

3.1. Chemoenzymatic synthesis of heparins by modification of heparosan Heparosan is a glycosaminoglycan with repeating GlcNAc-GlcA disaccharide units and is synthesized by bacteria such as E. coli K5, Pasteurella multicida or Bacillus subtilis as a capsule polysaccharide [43]. The close similarity to heparin and the high yields obtained from bacterial fermentations make heparosan a promising starting material for heparin synthesis. Metabolic engineering strategies based on the biosynthesis pathway of heparosan in microorganism (as illustrated in Fig. 3) are commonly applied to improve the yield of heparosan by fermentation. For example, Leroux et al. showed that an

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E. coli strain with an overexpressed GlcA transferase (KfiC), GlcNAc transferase (KfiA) and UDP-glucose dehydrogenase (KfiD) yielded up to 1.5 g/L of heparosan in fed-batch fermentation [43]. The groups of Chen and Gao improved the yields of heparosan in B. subtilis strain to 2.37 g/L and 5.82 g/L by optimizing the synthetic pathway of UDP-precursors [44, 45]. Huang et al. showed that the deletion of the waaR gene in E. coli, which encodes an α1,2 glucosyltransferase involved in the

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synthesis of a lipopolysaccharide, could also increase the yield of heparosan to around 3 g/L [46].

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Wang et al. reported that a fed-batch fermentation with addition glucose and oxygen could even

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produce heparosan at 15 g/L [47].

Figure 3. Biosynthesis pathway of heparosan in E. coli K5. Fructose-6-P, fructose-6-phosphate; Glucose-1-P,

glucose-1-phosphate;

Glucosamine-6-phosphate;

Glucose-6-P,

glucose-6-phosphate;

N-acetylglucosamine-1-P,

Glucosamine-6-P,

N-acetylglucosamine-1-phosphate;

N-acetylglucosamine-6-P, N-acetylglucosamine-6-phosphate; PPi, pyrophosphoric acid.

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As shown in Fig. 4, heparosan could be modified into heparin and heparan sulfate using biotransformations by including enzymes performing N-deacetylation/N-sulfation, O-sulfation, epimerization and depolymerization reactions [48, 49]. However, to improve the overall yield, the limiting biotransformation steps such as the N-deacetylation/N-sulfation, O-sulfation and depolymerization reactions could also be replaced by chemical methods [50, 51]. Different research

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groups applied this strategy to generate modified heparosan with anticoagulant activity, but further

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pharmaceutical properties of these products have not been reported yet [52, 53].

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Journal Pre-proof Figure 4. Chemoenzymatic synthesis of heparins by modification of heparosan. The biosynthesis pathway of heparin could be reproduced in vitro by modifying heparosan with recombinant enzymes. NDST, N-deacetylase/N-sulfotransferase; C5-epi, C5-epimerase; 2-OST, 2-O-sulfotransferase; 6-OST, 6-O-sulfotransferase; 3-OST, 3-O-sulfotransferase.

N-deacetylation/N-sulfation by NDST and chemical methods Four human NDST isoforms were identified which are distributed in various tissues and cells, but so

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far, only the isoforms NDST-1 and 2 have been applied for the enzymatic synthesis of heparin [33].

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Although NDSTs were actively expressed in recombinant form in yeast or insect cells, low expression levels limit the applicability of recombinant NDSTs as biocatalysts [41, 54]. For example, Kuberan et

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al. expressed NDST-2 in a baculovirus expression and successfully performed an enzymatic

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N-deacetylation/N-sulfation reaction of heparosan in microgram scale [55]. Sarıbaş et al. successfully

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expressed rat NDST-1 in Saccharomyces cerevisiae and demonstrated its activity of the partially

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purified enzyme on a series of heparan sulfate derivatives [41]. In several works, NDST was reported to be replaced by chemical N-deacetylation and N-sulfation steps. For instance, the groups of

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Rosenberg and Linhardt both reported that sequential treatments with NaOH for the deacetylation step and with trimethylamine-sulfur trioxide for the N-sulfation step [52, 56]. The ratio of N-sulfation to N-acetylation of the obtained heparin derivatives could be controlled by the chemical reaction time to obtain comparable N-sulfation levels as in therapeutic UFH [56]. Furthermore, the NaOH treatment step of N-deacetylation led to a significant decrease in the molecular weight of the starting material, which contributed to the depolymerization of the synthesized heparin derivatives [56, 57]. Epimerization The conversion of GlcA to IdoA by C5-epimerase is a key step in the synthesis of heparin derivatives. C5-epimerase was first purified from bovine liver and subsequently expressed in recombinant form 10

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using a baculovirus expression system [58]. It was also reported to be actively and solubly expressed in E. coli, which facilitated its use as a biocatalyst in heparin synthesis [59]. C5-epimerase homologous derived from other species were able to catalyze similar epimerization reactions, such as RED-C5-epimerase, a C5-epimerase from the marine bacterium Bermanella marisrubri [38]. Compared to mouse C5-epimerase, RED-C5-epimerase showed less selectivity to substrates with or

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without N-sulfation, but only exhibited a poor conversion efficiency of 10%. Another peculiar

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recombinant 5-epimerase originating from the snail Achatina fulica could only epimerize GlcA to IdoA in non-sulfated substrates like heparosan and desulfated N-acetylated heparin [60].

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Human C5-epimerase was initially believed to have a reversible activity and could convert GlcA to

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IdoA and vice versa [59, 61]. However, experimental evidence showed that this reversibility strictly

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depends on the positioning of the N-acetylation and O-sulfation of the heparin oligomer [62-64]. As

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shown in Fig. 5A, the mode of action of C5-epimerase is as follows: the recognition of a GlcNS at -1 position of epimerization site (EPS) allows C5-epimerase bind to the substrate, whereas a GlcNAc at

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the same -1 recognition site will prevent the binding of the enzyme and therefore no epimerization reaction can be initiated; The recognition of a GlcNS, a GlcN or unoccupied position at -3 of the EPS will allow a reversible epimerization, while a GlcNAc at the same position prevents the reversibility of the epimerization of GlcA to IdoA; Further modification of IdoA at the EPS with 2-O-sulfations also prevents the reverse epimerization and leads to an accumulation of sulfated IdoA in heparins [62-64]. A recent assessment of C5-epimerase binding to heparin oligomers containing GlcA, IdoA, and GlcNS was performed using real-time NMR spectroscopy [65]. The results confirmed the importance of the GlcNS residues at -1 and +1 position of the EPS and that 2-O-sulfo IdoA and or 6-O-sulfo/3-O-sulfo

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GlcN at -2 to +1 position of the EPS would prevent the epimerization reaction of the ligand substrate

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[65].

Figure 5. Mode of action of C5-epimerase and regeneration system of PAPS. (A) Scheme of the

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recognition pattern and reversibility of C5-epimerase. (B) Chemoenzymatic N-sulfation scheme of heparin with PAPS regeneration system catalyzed by arylsulfotransferase. C5-epi, C5-epimerase; NDST, N-deacetylase/N-sulfotransferase; NST, N-sulfotransferase; OST, O-sulfotransferase; PAPS,

O-sulfation

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3’-phosphoadenosine 5’-phosphosulfate; PAP, 3’-phophoadenosine-5’-phosphate.

O-sulfation reactions on heparins include 2-O-sulfation of IdoA and GlcA, 6-O-sulfation of GlcNAc and GlcNS, and 3-O-sulfation of GlcNAc and GlcNS residues. Several works reported that O-sulfation prevents the enzymatic activity of NDST and C5-epimerase, thus biocatalytic O-sulfations are usually performed after N-sulfation and epimerization [66, 67]. O-sulfation by chemical means may result in the unspecific O-sulfation of unintended positions such as the C3 hydroxy group of IdoA, and decreasing the anticoagulant activity [51]. An elegant strategy of an initial per-O-sulfation and consecutive solvolytic desulfation step which removes 3-O-sulfate groups of IdoA selectively and

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preserves the 2-O-sulfo groups of IdoA was proposed by Lindahl et al. in the synthesis of LMWHs analog neoheparin [53]. Despite neoheparin presented a comparable anti-FXa activity to that of commercial LMWHs, the 3-O-sulfation pattern of GlcA in neoheparin differed to that of natural heparin. To avoid these selectivity problems of the chemical synthesis, fully enzymatic sulfation strategies are more desirable. All the required 2-O-, 3-O- and 6-O-sulfotransferases for generating

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functional heparin derivatives were successfully expressed in E. coli and applied in the specific

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O-sulfation of K5 heparosan [52]. 2-OST catalyzes the 2-O-sulfation of IdoA and 2-O-sulfation to a lesser extent of GlcA. Although the lacking of 2-O-sulfation has little effect on the anticoagulant

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activity, this reaction is still usually carried out simultaneously with C5-epimerization to increase the

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ratio of sulfated IdoA to GlcA [49, 52]. It was also found that 6-O-sulfation of a GlcN residue adjacent

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to IdoA could hinder the 2-O-sulfation of IdoA, and therefore the 6-O-sulfation step is usually

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performed after the epimerization/2-O-sulfation reaction steps [68]. 6-O-sulfation is a critical modification for the anticoagulant activity in heparins. Oligosaccharide

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substrates just bearing 6-O-sulfation and 3-O-sulfation could still have partial inhibitory activities against thrombin and factor Xa, whereas those only bearing 2-O-sulfation and 3-O-sulfation fully lost the anticoagulant activity [69]. 6-O-sulfation also has other important biological functions like anti-inflammation and regulation of the fibroblast growth factor mediated signaling pathway. Although it is proven that regioselective 6-O-sulfation could be achieved by chemical methods (as demonstrated in the synthesis of keratan sulfate), generally a biocatalytic approach using 6-O-sulfotransferease is recommended in the chemoenzymatic synthesis of heparan sulfates because of high conversion efficiency and the mild reaction conditions [70]. 6-O-sulfotransferases are used to transfer sulfo groups at a C6 position of GlcNS or GlcNAc residues. Three isoforms of 6-O-sulfotransferases were identified 13

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from either human or mouse, including 6-OST-1, 6-OST-2 and 6-OST-3. They all show similar substrate specificity pattern, which was supported by their crystal structures [36, 71]. For example, the work by Kuberan et al. indicated that both 6-OST-1 and 6-OST-2 modified K5 heparosan at the same position from the data of disaccharide analysis with heparinases [52]. Later it was shown by Smeds et al. that 6-OST isoforms still had minor differences in their selectivity for polysaccharides, as 6-OST-1

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was more selective for heparosan substrates without 2-O-sulfation [72]. Chen et al. also found a

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reduced proportion of 6-O-sulfation on GlcNS residues and decreased anticoagulant activity in the synthesis of active heparin since just 6-OST-1 was used [69]. Therefore, a combination of 6-OST

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6-OST-3 for the 6-O-sulfation approach [73].

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isoforms is recommended to catalyze 6-O-sulfation. For example, Liu et al. chose 6-OST-1 and

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The 3-O-sulfation on GlcNS linked to GlcA/IdoA is critical for the binding of antithrombin III and

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prevents the processing of pro-fibrin into fibrin [74]. The different 3-OST isoforms (6 in humans) exhibit different substrate specificities. For example, 3-OST-1 catalyzes the 3-O-sulfation to GlcNS±6S

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linked to GlcA/IdoA at the non-reducing end, which is the final modification in the synthesis of the AT binding domain motif, and therefore endows the anticoagulant activity [75]; 3-OST-2, 3-OST-3A, 3-OST-3B, 3-OST-4, and 3-OST-6 transfer sulfo groups to GlcNS±6S next to IdoA2S at the non-reducing end, which poses a binding motif for the glycoprotein D of the herpes simplex virus type 1 [76, 77]; whereas 3-OST-5 can sulfate motifs of GlcNS±6S linked to either IdoA2S or GlcA/IdoA [76, 78]. Hence, the recombinant 3-OST-1 alone (to generate AT-binding domain), or combined with 3-OST-5 (to generate extra 3-O-sulfation), was used in the chemoenzymatic synthesis of heparin in different studies [69, 79]. The comparison of dodecasaccharide heparan sulfates with non, single and double 3-O-sulfation showed that only the latter two had anti-coagulant activities [28, 80]. Although 14

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both heparan sulfates with or without 3-O-sulfation could bind to human Stabilin-2 and exhibited similar endocytosis activities, only those bearing 3-O-sulfation accumulated in the liver and exhibited a longer serum half-life [28]. Interestingly, a recent pharmacokinetic work in mice with kidney failure showed that the clearance of these dodecasaccharides with 3-O-sulfation was impaired [81]. Renal clearance seems to be the major mode for the removal of LMWHs with 3-O-sulfation. 3-O-sulfation

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also contributes to the binding between heparin and its antidote protamine. Xu et al. found that after

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3-O-sulfation, a dodecasaccharidic heparan sulfate showed an increased sensitivity to protamine neutralization and presented reversible anticoagulant activity [80, 81].

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PAPS regeneration system

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3’-Phosphoadenosine 5’-phosphosulfate (PAPS) is an activated donor substrate required for

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sulfotransferase catalyzed N-sulfation or O-sulfation reactions [16, 82]. However, the high cost of

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PAPS and inhibiting effects of the reaction by-product 3’-phophoadenosine-5’-phosphate (PAP) are two critical limitations of enzymatic sulfation reactions. Hence, recycling strategies of PAPS using

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cofactor regeneration are necessary for practical applications (Fig. 5B). Burkart et al. developed a PAPS regeneration system using the arylsulfotransferase IV using inexpensive p-nitrophenyl sulfate as donor substrate [83], which was further improved by Xiong et al. by immobilizing arylsulfotransferase IV to a solid support for more effective PAPS cofactor recycling [84]. Depolymerization The molecular weight of K5 heparosan (>20 kDa) is much higher than heparin. After the chemical N-deacetylation/N-sulfation treatment, the molecular weight could be reduced to around 10 kDa [56]. To obtain the smaller molecular weights which are typical for LMWHs or ULMWHs, depolymerization step is required. For example, Lindahl et al. applied a controlled depolymerization 15

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step with nitrous acid and successfully obtained LMWHs of about 8000 Da in size with anticoagulant activity [53]. However, using a chemical depolymerization approach for heparosan after the final O-sulfation step may lead to changes in this modification pattern and destroy the anti-thrombin III binding motif. Therefore, chemical depolymerization steps are usually performed as a first step on the underivatized heparosan prior to any other modification procedures. Besides the traditional

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degradation using acid or alkaline treatment, Higashi et al. reported a photochemical method catalyzed

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by titanium dioxide to generate oligosaccharides from E. coli K5 heparosan of about 8000 Da in size [85]. This strategy was also applied in the depolymerization of UFH to generate LMWHs by the same

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group [86]. Enzymatic depolymerization is an alternative strategy with high selectivity and mild

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reaction conditions. Heparinases from Flavobacterium heparinum are widely applied in the

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depolymerization of both heparosan and heparin [87]. All three isoforms of heparinases could

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hydrolyze β1-4 linkages between hexosamines and uronic acids but with subtle differences in their substrate specificities. Heparinase I (HepI) cleaves primarily heparins at the GlcNS3S6S-IdoA2S site

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[88]. Heparinase III (HepIII) acts only on heparin and heparan sulfate containing the disaccharide consensus sequence GlcNS±6S/GlcNAc±6S-IdoA/GlcA. Heparinase II (HepII) is able to degrade heparin and heparan sulfate at the consensus sequence GlcNS±3S6S-IdoA±2S [89, 90]. Wu et al. reported that a combination of HepIII and HepI can produce LMWHs which molecular weight comparable to the standards of the European Pharmacopoeia [91]. Kuberan et al. applied HepI to depolymerize N-deacetyl- and N-sulfo K5 heparosan to generate hexasaccharide products [55]. However, during the depolymerization of heparosan with heparinases, unsaturated urinate moieties at the non-reducing end of the hydrolysis products such as 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acids, are generated. These undesired residues have to be removed before or after further modification 16

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steps. Su et al. used a recombinant glucuronidase from Pedobacter heparinus to remove unsaturated bonds from different products of heparin or heparosan digested with HepI, HepII or HepIII [92], and Masuko et al. applied ozonolysis for the removal of unsaturated residues from heparin or heparosan [93].

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3.2. De-novo synthesis of heparins from carbohydrate primers

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Although a comparable anti-coagulant activity was observed in heparosan-derived heparin analogs or LMWHs, the heterogeneity of products and the consequential diversity of pharmacodynamic and

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pharmacokinetic properties are inevitable due to the size heterogeneity of heparosan and the incomplete

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enzymatic epimerization and sulfation reactions. Hence, more attention was paid on another strategy

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which starts from monosaccharide or oligosaccharide primers [82]. Using this strategy, the synthesis of

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homogenous heparan sulfates, ULMWHs or LMWHs of defined size and designed structure is feasible [48, 73, 94]. However, a precise and concerted step-by-step regiment of different enzymes is required

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to obtain the desired heparin oligomer. Attempts of using one-pot reaction multi-enzyme setups usually result in a mixture of ULMWH derivatives of different molecular weights [79]. Synthesis of size-defined ULMW/LMW heparins Xu et al. first designed an enzymatic method to synthesize the oligosaccharide backbone using the disaccharide glucuronic acid-anhydromannitol (GlcA-AnMan) as a carbohydrate primer (Fig. 6A) [16]. Two glycosyltransferases were involved in the polymerization step, GlcN-acetylglucosaminyl transferase from E. coli K5 (KfiA) and heparosan synthase 2 from Pasteurella multocida (PmHS2). The primers were elongated by the alternating action of these two enzymes using UDP-N-trifluoroacetylglucosamine (UDP-GlcNTFA) and UDP-glucuronic acid (UDP-GlcA) as donor 17

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sugars and resulted in the synthesis of a repeating disaccharide unit (-α1,4GlcNTFAβ1,4GlcA-). The incorporation of GlcNTFA can be then easily de-N-acetylated under mild chemical reaction conditions to obtain the free amine. After two cycles of elongation with KfiA and PmHS2, an extra GlcNAc was added by KfiA to form the AT binding domain as illustrated in Fig. 6A. Besides UDP-GlcNTFA, also UDP-2-azido-2-deoxyglucose (UDP-GlcN3) and UDP-N-acetyl-6-azido-6-deoxyl-glucosamine

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(UDP-GlcNAc6N3) could be introduced to the backbone of heparins by PmHS2, which could be used

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for selective N-deacetylation reactions [95]. After chemical N-deacetylation, the N-sulfation was catalyzed by recombinant NST, a truncated form of NDST which only maintains N-sulfotransferase

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activity and is efficiently expressed in E. coli [16]. Using the similar strategy as illustrated in Fig. 6B,

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the group of Xu et al. also synthesized a series of LMWHs with both an anti-thrombin III binding motif

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and a thrombin binding domain using GlcA-AnMan as priming sugar [96]. The results showed that at

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least a nondeca oligosaccharide motif consisting of five repeating disaccharides is necessary for the thrombin binding affinity. Based on the current understanding of the structure-activity relationship of

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heparin derivatives, the same group also synthesized homogeneous LMWHs, which combine the pharmacological properties of high anti-FXa/anti-FIIa ratio, clearance from the liver, and reversible neutralization activity by protamine [80]. As illustrated in Fig. 6C, the modified synthesis strategy included repeated N-deacetylation/N-sulfation, and elongation and epimerization steps, leading to a backbone of defined oligomerization (12-mers). This length allowed good binding affinities to Stablin-2 and protamine but not to thrombin [80]. Experiments in mice indicated that a tested dodecasaccharide containing 17 sulfo groups was cleared by the liver rather than through the kidneys.

18

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Figure 6. Chemoenzymatic synthesis of size defined heparins from carbohydrate primers. (A) Synthetic scheme for ULMWHs with anti-FXa activity. (B) Synthetic scheme for LMWHs with both anti-FXa and anti-FIIa activities. (C) Synthetic scheme for LMWHs with reversible coagulant activity. KfiA, GlcN-acetylglucosaminyl transferase from E. coli K5; PmHS2, heparosan synthase 2 from Pasteurella multocida; C5-epi, C5-epimerase; NST, N-sulfotransferase; OST, O-sulfotransferase; AT binding domain, anti-thrombin III binding domain. 19

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One-pot backbone synthesis As illustrated in Fig. 7, an improved one-pot method was developed by Chandarajoti et al. in order to simplify the overall chemoenzymatic synthesis [79]. Instead of using a combination of PmHS2 and KfiA, only PmHS2 was used for the synthesis of the disaccharide repeating unit. Interestingly, this enzyme was found to possess a functional duality of both α1,4 N-acetyl-glucosaminyltransferase and

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β1,4 glucuronyltransferase activity [95]. A detailed analysis of the substrate specificity showed that PmHS2 exhibited a broader tolerance than KfiA towards C6’/C4’-derivatives of UDP-GlcNAc,

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UDP-GlcA and UDP-GalNAc [97, 98]. Using optimized processing conditions and a defined ratio of

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primer, UDP-GlcNTFA, and UDP-GlcA (1:4:4), heparin oligosaccharides were synthesized by a

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one-pot strategy resulting in a degree of polymerization (dp) between 8-16, which is comparable to the

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commercial LMWHs oligomerization range (dp = 8-20) [79]. These so-called narrow-size distribution

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LMWHs showed higher anti-Xa activity than the LMWH oligomer Enoxaparin [79].

Figure 7. One-pot chemoenzymatic synthesis of LMWHs from oligosaccharide primer. KfiA, GlcN-acetylglucosaminyl transferase from E. coli K5; PmHS2, heparosan synthase 2 from Pasteurella multocida; C5-epi, C5-epimerase; NST, N-sulfotransferase; OST, O-sulfotransferase; AT binding domain, anti-thrombin III binding domain. 20

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Carbohydrate primers used in the synthesis of heparins and heparan sulfates The chemoenzymatic synthesis of heparin and heparan sulfates requires mono- or oligosaccharide acceptors as primer substrates. PmHS2 and KfiA were shown to have a broad tolerance towards saccharides of different length and modification. For example, GlcA-AnMan or pNP-GlcA were

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applied in the synthesis of LMWHs [16, 96]. Also, longer primers, such as the tetrasaccharide

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GlcA-GlcNAc-GlcA-AnMan, the hexasaccharide GlcA-GlcNAc-GlcA-GlcNAc-GlcA-AnMan or the octasaccharide GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-AnMan were used as substrates for

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PmHS2 in the enzymatic elongation of the heparin backbone [79]. Functional groups on the reducing

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end of the primers (like pNP) can be used to facilitate the detection and further purification of the

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obtained reaction products. Primers containing azido tags (i.e. GlcA-AnMan-N3) were successfully

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used as acceptor substrates for KfiA [99]. In a recent report, Zhang et al. constructed a library of 66 defined heparan sulfates or heparin oligosaccharides using azide-tagged disaccharides as primers [100].

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Cai et al. reported that tert-butyl dicarbonate (FBoc) tagged primers were also good choices for the chemo-enzymatic synthesis of heparan sulfate due to the simple purification procedure [101].

Synthesis of donor substrates Nucleotide sugars are essential building blocks for the chemoenzymatic synthesis of heparins, and limitations of their availability in large scale also restricts the enzymatic de novo synthesis of heparins. Especially the synthesis of N-acyl derivatives of UDP-GlcNAc which can introduce natural modification or unnatural modification to heparins is challenging at larger scales. Masuko et al. found that GlcNAc-1-phosphate and GlcNTFA-1-phosphate could be converted to the corresponding UDP 21

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sugars by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from E. coli with more than 70% conversion rate [102]. In order to synthesize UDP-GlcNAc derivatives directly from the monosaccharide, enzymatic cascade usually consisting of a sugar kinase, a nucleotide sugar transferase and an inorganic pyrophosphatase (which remove inhibitory pyrophosphate from the reaction mixture) were developed. Chen et al. achieved the synthesis of UDP-GlcNAc derivatives including

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UDP-GlcNTFA, UDP-GlcN3 and UDP-GlcNAc6N3 with yields of 97%, 54% and 72%, respectively,

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using recombinant N-acetylhexosamine-1-kinase from Bifidobacterium longum (BlNahK), N-acetylglucosamine-1-phosphate uridylyltransferase from Pasteurella multocida (PmGlmU) and

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inorganic pyrophosphatase from P. multocida (PmPpA) [103]. Li et al. also employed a similar

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reaction system but with GlmU and PpA originated from E. coli K12 MG1655 and successfully

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synthesized UDP-GlcNAc and UDP-GlcNTFA with the scales around 50 g/L [104]. Guan et al. found

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that UDP-N-acetyl-hexosamine pyrophosphorylase from humans (AGX1) is a good alternative to GlmU for its broad specificities towards C-2,4,6 modified GlcNAc- and GalNAc-1-phosphate [105].

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Fischöder et al. found that recombinant AGX1 could synthesize UDP-GlcNAc and UDP-GalNAc with high space-time-yields of 9.9 g/L*h and 19.4 g/L*h, respectively [106]. UDP-GlcA could be synthesized either by a de novo synthesis pathway using UDP-glucose as starting material or via the salvage pathway using glucuronic acid as the starting substrate. In the de novo pathway, UDP-glucose can be converted to UDP-GlcA by UDP-glucose 6-dehydrogenase (UGD). For example, Wei et al. reported a bacterial UDP-glucose 6 dehydrogenase (GbUGD) originating from Granulibacter bethesdensis was able to perform this reaction with high efficiency [107]. Duan et al. and Gu et al. identified thermostable UDP-glucose dehydrogenases allowing them to perform this transformation at elevated temperatures [108, 109]. In the salvage pathway, GlcA is first converted to 22

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GlcA-1-phosphate by the action of a glucuronokinase, and then activated by a UDP-sugar pyrophosphorylase to UDP-GlcA. Guo et al. used AtGlcAK (glucuronokinase from Arabidopsis thaliana), AtUSP (UDP-sugar pyrophosphorylase from Arabidopsis thaliana) and EcPpA (pyrophosphatase from E. coli) to convert GlcA to UDP-GlcA in 100 mg scale [110]. The enzymatic synthesis of UDP-sugars and their derivatives using enzymatic cascades could be

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further combined with the respective glycosyltransferases required for the elongation of the

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oligosaccharide backbone of heparins. Chen et al. used a one-pot four-enzyme GlcNAc activating and transferase system consisting of NahK, PmGlmU, PmPpA, and PmHS2 for the synthesis of a 2AA

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(2-aminoantranile) labeled GlcNAc-GlcA disaccharide [111]. The same group also produced

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2AA-GlcA-GlcNAc-GlcA using a three-enzyme strategy consisting of EcGalU, PmUGD, and PmHS2

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4. Conclusion

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[111].

Substantial progress in developing chemoenzymatic approaches of heparin synthesis for commercial

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applications was made within the last decade. It has been shown that ULMWHs and LMWHs could be enzymatically synthesized with a defined structure, and have comparable or even better medicative properties than isolated heparins. Although some heparin structures were described to be readily synthesized in milligram to gram scale in laboratories, improvements are still required for the synthesis in pilot or industrial scale [81, 112]. The two major synthesis strategies have different advantages and disadvantages: The de-novo synthesis of heparins from carbohydrate primers could generate homogenous oligosaccharides with designed structure, but the whole synthesis route usually involves dozens of steps. The synthesis by modification of heparosan needs fewer steps but is difficult to obtain homogenous products. How to balance the two strategies and develop methods more suitable for 23

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industrial-scale synthesis will be the emphasis for the chemoenzymatic synthesis of heparin and heparan sulfates in the upcoming years. Due to the limitation of synthesis scale, only little comprehensive data from preclinical evaluations of these synthetic heparins are available. Furthermore, some missing pieces to the puzzle of the exact effects of sulfation on pharmacodynamics and pharmacokinetics of heparins, such as the modes of clearance, still have to be clarified. In conclusion,

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these designed ULMWHs and LMWHs show promising future applications as drugs with improved

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activities and lower side effects when compared with currently available heparins. Still, more efforts are needed regarding the scale-up and shorter synthesis routes, especially for the synthesis of highly

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homogenous heparins.

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Funding

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This work was supported by the Fundamental Research Funds for the Central Universities (grant numbers KYZ201824 to T. W.), the National Natural Science Foundation of China (NSFC) 31401648

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(to T.W. and L.L), 31471703, 31671854, 31871793 and 31871754 (to JV and LL), and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

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Journal Pre-proof

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Highlights: • Recent advances in the chemo-enzymatic synthesis of low molecular weight heparins. • Description of structure/fuction mechanisms of the synthesized heparin derivatives. • Comparison of de-novo heparin oligomer synthesis and heparan-derived oligomers.

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