Uses and methods for in SITU generating PAPS from cyclo-PAPS using a cell free extract

Abstract

The present disclosure relates to uses and methods implementing a cell free extract (CFE) having an activity for converting adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) into 3'-phosphoadenosine-5'-phosphosulfate (PAPS) useful for sulfating a substrate.

Description

[TITLE]USES AND METHODS FOR IN SITU GENERATING PAPS FROM CYCLO-PAPS USING A CELL FREE EXTRACT[TECHNICAL FIELD]

[0001] The present disclosure relates to a new route, i.e. an alternative and cost-effective process to in situ produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS) starting from adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS). The present disclosure further relates to cell free extracts (CFE) able to convert cyclo-PAPS into PAPS. Also, the present disclosure relates to methods for sulfation of substrates and for synthesizing sulfated glycosaminoglycans or oligosaccharides compounds based thereon.[TECHNICAL BACKGROUND]

[0002] Sulfation is a conjugation process involved in numerous biological processes including synthesis of proteins, peptides or glycosaminoglycans (GAGs), detoxification, hormone regulation, molecular recognition, cell signaling, or viral entry into cells.

[0003] The sulfation reaction needs a sulfotransferase (SULT) enzyme as a catalyst and a co-substrate as a sulfuryl (or sulfo group or sulfate group) donor. PAPS dependent Sulfotransferases (SULTS) are a family of enzymes that transfer the sulfate group from PAPS onto usually a hydroxyl or amine group of a target substrate. For eukaryotes, sulfotransferases are PAPS dependent and use 3'-phosphoadenosine 5'- phosphosulfate (PAPS) as sulfate donor.

[0004] Among the sulfonated glycosaminoglycans (GAGs) issued from sulfation processes, are heparan sulfate (HS) and heparin. Those GAGs are closely related sulfated polysaccharides consisting of repeating disaccharide units of glucuronic acid or iduronic acid linked to glucosamine and involved in a number of important biological and pharmacological activities.

[0005] HS is a component of the cell surface and extracellular matrix and is involved in a wide range of physiologic and pathophysiologic functions, such as blood coagulation and viral infection (Esko and Selleck (2002) Annu. Rev. Biochem. 71 , 435-471 ; Liu and Thorp (2002) Med. Res. Rev. 22, 1 -25). It is a highly charged mixture of polysaccharides comprising 1— >4-linked glucosamine and glucuronic / iduronic acid units that contain both N- and O-sulfo groups.

[0006] Heparin, which is more sulfated than heparan sulfate, is found primarily intracellularly in the granules of mast cells and is a commonly used anticoagulant drug. Three injectable forms of heparinoids can be found on the market: unfractionated heparins (UFH) (mean MWavg-17000 Da); low molecular weight heparins (mean MWavg-6000 Da); and the synthetic pentasaccharide (MW 1728.0 Da). UF heparin is used in surgery and kidney dialysis due to its relatively short half-life while LMW heparins and the synthetic pentasaccharide are intended for preventing venous thrombosis among high-risk patients or after surgery, for the treatment of deep vein thrombosis and pulmonary embolism.

[0007] In the human body, HS and heparin (HP) are biosynthesized in the endoplasmic reticulum (ER) and the Golgi compartments. Glycosyltransferase enzymes catalyze the alternating addition of II DP-activated p-D-glucuronic acid (GlcA) and N- acetylglucosamine (GIcNAc) residues to generate a polysaccharide chain, which is then modified by / V-deacetylase, C5-epimerase and sulfotransferase enzymes. / V- deacetylase / / V-sulfotransferases (NDST) replace / V-acetyl groups with an / V-sulfo group, and C5-epimerase and O-sulfotransferases (OSTs) work together to convert GlcA into a-L- iduronic acid (IdoA), and then into ldoA2S (addition of a 2-O-sulfo group). D-glucosamine residues are then modified by 6-O-sulfotransferases (6OSTs), followed by 3-O- sulfotransferases (3OSTs). Tissue specific expression of different enzyme isoforms finetunes the synthesis of HP and HS to produce different structures, allowing adaptation of function to the local cellular environment (Fu et al., Adv Drug DelivRev. 2016;97:237-249).

[0008] Application of biosynthetic enzymes for generating bioengineered GAGs like heparin and oligosaccharides with desired biological activities is now possible with the successful expression of recombinant heparin biosynthetic enzymes (Fu et al., Adv Drug Deliv Rev. 2016;97:237-249).

[0009] In the bioprocesses developed for synthesizing HS and heparin, OSTs act on / V-sulfoheparosan in the presence of the cofactor 3'-phosphoadenosine-5'- phosphosulfate (PAPS) (Fu et al., Adv Drug Deliv Rev. 2016;97:237-249). 3- Phosphoadenosine-5'-phosphosulfate (PAPS) is a derivative of adenosine monophosphate that is phosphorylated at the 3' position and has a sulfate group attached to the 5' phosphate. It is the most common coenzyme involved in sulfotransferase reactions.

[0010] However, PAPS which is a universal sulfate donor and source of sulfate for a lot of sulfotransferases is a highly expensive molecule that has been an obstacle to the large-scale production of enzymatically sulfated products. In the case ofbioengineered heparin manufacturing, the required use of purified enzymes is also a major cost contributor to the overall cost of goods.

[0011] PAPS is commercially available but only at small scale and at high prices depending on the supplier: from 2500 to 101 000 k€ / kg (see e.g. the product commercialized by Sigma, Glycan Therapeutics and Yamasa). It appears that fully enzymatic or chemo-enzymatic processes are commonly used for its production.

[0012] As previously described e.g. in US 4,169,011 , a chemo-enzymatic process is as shown below:

[0013] First and in the chemical part of the process, adenosine or 5’-AMP is converted to cyclo-PAPS. Then, cyclo-PAPS is converted to PAPS by opening the cyclic 2’,3’-phosphate through a nucleophilic addition of water. At this stage, both PAPS and iso- PAPS can be obtained, wherein PAPS is the most active sulfate donor:

[0014] In order to be specific and to obtain only PAPS, this reaction has almost exclusively been described using a selective phosphatase. To the best knowledge of the inventors, all examples in the literature of this reaction describe the use of Ribonuclease T2 (RNase T2). Although this enzyme allows a very selective ring-opening, it is very expensive to use and its cost is the principal contributor to the costs associated with PAPS production for chemo-enzymatic approaches like illustrated above.

[0015] As an example, the ribonuclease T2 (RNase T2) may be the recombinant enzyme from Aspergillus oryzae sold by Creative Enzymes® or Worthington biochemical®.

[0016] Therefore, there is a need to optimize the conversion of cyclo-PAPS into PAPS.

[0017] There is a need to have new material usable in bioprocess to convert cyclo-PAPS into PAPS.

[0018] There is a need to have new source of phosphatase activity to convert cyclo-PAPS into PAPS.

[0019] There is a need to have a new reagent for sulfate donation with good activity and lower price.

[0020] There is a need to have methods for sulfation of substrate with lower cost and / or improved yield.

[0021] There is a need to have methods for sulfation of oligo and polysaccharides, N-sulfated heparosan, heparosan, heparin, or heparan sulfate with lower cost and / or improved yield.

[0022] There is a need to have a method to produce a bioengineered heparin with lower cost and / or improved yield.

[0023] There is a need to have methods for sulfation of substrate or for production of bioengineered of heparin which are simplified.

[0024] The present has for purpose to meet all or part of these needs.[SUMMARY]

[0025] According to one of its objects, the present disclosure relates to the use of a cell free extract (CFE) to convert adenosine 2',3'-cyclic phosphate 5'- phosphosulfate (cyclo-PAPS) into 3’-phosphoadenosine-5’-phosphosulfate (PAPS).

[0026] As shown in the Examples illustrating the present disclosure, the inventors have surprisingly found that cell free extracts, especially those obtained from host cells used to produce enzymes involved in sulfation reactions (e.g. sulfotransferases), are able to convert cyclo-PAPS into PAPS, in an efficient manner. Besides, the sulfation of the substrate can take place simultaneously and in the same reaction medium as the conversion of cyclo-PAPS into PAPS.

[0027] In other words, the present disclosure makes it possible to use cyclo- PAPS as a primary sulfate donor which is in situ converted to PAPS by the activity of a cell free extract as disclosed herein. Said conversion can take place simultaneously to the use of the PAPS so generated in sulfation reactions, without the need to isolate PAPS. Besides, in view of the low price of cyclo-PAPS compared to PAPS, the combination of cyclo-PAPS and a cell free extract as disclosed herein offers a valuable alternative solution.

[0028] According to one of its objects, the present disclosure relates to the use of adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) as a reagent in sulfotransferase catalyzed reactions. Such a reagent constitutes a less expensive alternative to PAPS.

[0029] According to one of its objects, the present disclosure relates to a method for performing sulfotransferase catalyzed reactions using adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) as a reagent.

[0030] According to one of its objects, the present disclosure relates to the use of adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) as a primary sulfate donor in sulfotransferase catalyzed reactions. Such a reagent constitutes a less expensive alternative to PAPS.

[0031] According to one of its objects, the present disclosure relates to a method for performing sulfotransferase catalyzed reactions using adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) as a primary sulfate donor .

[0032] According to one of its objects, the present disclosure relates to the use of cyclo-PAPS and a cell free extract, as an alternative to PAPS, in sulfotransferase catalyzed reactions.

[0033] According to one of its objects, the present disclosure relates to a method for performing sulfotransferase catalyzed reactions using cyclo-PAPS and a cell free extract, as an alternative to PAPS.

[0034] The cell free extracts (CFE) as disclosed herein can be advantageously used in sulfation bioprocess systems for generating PAPS and optionally providing enzymes involved in sulfation.

[0035] The present disclosure advantageously provides a source of PAPS and enzymes involved in sulfation, at low cost and high yield, allowing the large-scale synthesis of sulfated substrates such as glycosaminoglycans (GAGs) including heparin.

[0036] Furthermore, the present disclosure provides cell free extracts able to convert cyclo-PAPS into PAPS which can be easily obtained.

[0037] The present disclosure advantageously provides methods for obtaining sulfated substrates, such as heparin, at high-yield and low cost, allowing an efficient industrial scale-up.

[0038] According to one of its objects, the present disclosure relates to the use of a cell free extract (CFE) for generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS).

[0039] According to one of its objects, the present disclosure relates to a method for generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) using a cell free extract (CFE)

[0040] According to one of its objects, the present disclosure relates to the use of a CFE as disclosed herein for in situ generating PAPS from cyclo-PAPS.

[0041] According to one of its objects, the present disclosure relates to a method for in situ generating PAPS from cyclo-PAPS using a CFE as disclosed herein.

[0042] According to one of its objects, the present disclosure relates to the use of cyclo-PAPS and a cell free extract (CFE) as disclosed herein for in situ generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS).

[0043] According to one of its objects, the present disclosure relates to a method for in situ generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) using cyclo- PAPS and a cell free extract (CFE) as disclosed herein.

[0044] According to one of its objects, the present disclosure relates to a method of preparing PAPS comprising incubating cyclo-PAPS with a cell free extract (CFE) in a reaction medium.

[0045] According to one of its objects, the present disclosure relates to a method of in situ preparing PAPS comprising incubating cyclo-PAPS with at least a cell free extract (CFE) as disclosed herein in a reaction medium.

[0046] A cell free extract as disclosed herein may contain at least one enzyme able to convert cyclo-PAPS into PAPS.

[0047] A cell free extract (CFE) as disclosed herein may contain native enzyme(s) able to convert cyclo-PAPS into PAPS, i.e. endogenously produced by the cell used to prepare the extract.

[0048] A cell free extract as disclosed herein may be obtained from any suitable cell, in particular a host cell which can be a prokaryotic or eukaryotic cell, usually a bacterial, yeast, plant, and animal cell.

[0049] In some embodiments, the cell free extract as disclosed herein is from Escherichia coli. In some embodiments, the cell used to prepare the cell free extract (CFE) as disclosed herein, for example E. coli, endogenously produces at least one enzyme selected in the group consisting of: 2’,3’-cyclic-nucleotide 2’-phosphodiesterase / 3’- nucleotidase (cpdB), acid phosphatase (aphA) and 3’(2’),5’-biphosphate nucleotidase (cysQ).

[0050] In some embodiments, the cell free extract as disclosed herein is from Aspergillus oryzae. In some embodiments, the cell used to prepare the cell free extract (CFE) as disclosed herein endogenously produces a ribonuclease T2 (RNase T2), for example the A. oryzae RNase T2.

[0051] In some embodiments, the cells used to prepare the cell free extract (CFE) as disclosed herein also produces, endogenously or recombinantly, other enzymes of interest which are retrieved in the CFE. In some embodiments, said enzymes are enzymes using PAPS as a sulfate donor, in particular sulfotransferases.

[0052] In some embodiments, a cell free extract as disclosed herein may further contain at least an enzyme involved in sulfating a substrate.

[0053] A reaction medium adapted for a cell free extract (CFE) as disclosed herein may allow conversion of cyclo-PAPS into PAPS but may also be adapted to any further enzymatic activity of interest contained in the CFE.

[0054] A reaction medium as disclosed herein may have a pH from 6 to 8, optionally a neutral pH, e.g. a pH of 7. In some embodiments, it may comprise a buffer, optionally a 4 Morpholine-Ethane Sulfonic acid (MES) buffer, e.g. MES buffer at a concentration of 50 mM.

[0055] In some embodiments, the reaction medium as disclosed herein may also contain the substrate(s) for the enzyme(s) of interest contained in the cell free extract.

[0056] The concentration of cyclo-PAPS in the reaction medium may be from 0.1 mM to 1M or from 0.1 mM to 100 mM, e.g. 10 mM.

[0057] In some embodiments, cyclo-PAPS which is the primary sulfate donor in said reactions may be provided as a salt, e.g. a metal salt or an amine salt. Examples of metal salt include lithium salt or sodium salt. An amine salt may be formed between cyclo-PAPS and an amine compound. Such an amine compound may have one or more amino or imino groups, may be a secondary amine, optionally having a C1 -C10 hydrocarbon chain, a tertiary amine, optionally having a C1-C7 hydrocarbon chain, a polyamine or an amino acid. Said amine compound can be selected in the group consisting of: diethylamine, piperidine, trimethylamine, triethylamine, triethanolamine, putrescine, polyethyleneimine, and L-histidine. It may be in the form of a triethylamine ammonium salt.

[0058] In some embodiment, cyclo-PAPS to be used in the methods of the present disclosure may be in the form of a triethylammonium salt (TEA).

[0059] In some embodiments, the PAPS so produced is not isolated nor purified from the reaction medium but is directly consumed by the sulfation reaction (s).

[0060] According to one of its objects, the present disclosure relates to a reaction medium containing a cell free extract as disclosed herein and PAPS that is formed in situ from cyclo-PAPS.

[0061] According to one of its objects, the present disclosure relates to a method for sulfating a substrate, comprising incubating in a reaction medium cyclo-PAPS, at least one cell free extract as disclosed herein, the substrate to be sulfated and an enzyme involved in the sulfation of the substrate.

[0062] According to one of its objects, the present disclosure relates to a method for sulfating a substrate, comprising at least a step of contacting said substrate to be sulfated with:

[0063] at least an enzyme involved in the sulfation of the substrate, optionally a sulfotransferase

[0064] a sulfo group donor comprising PAPS prepared according to the method as disclosed above, i.e. a cell free extract as disclosed herein and cyclo-PAPS

[0065] in conditions suitable for the conversion of cyclo-PAPS into PAPS and for a transfer of the sulfo group from the sulfo group donor (i.e. PAPS) to said substrate.

[0066] In uses or methods as disclosed herein, the enzyme involved in sulfating a substrate may be selected in the group consisting of: SULT1A1 , SULT1A2, SULT1A3, SULT1 B1 , SULT1C1 , SULT1C2, SULT1 E1 , SULT2A1 , heparan sulfate D- glucosaminyl 3-O-ST-3a and b, heparan sulfate D-glucosaminyl 3-O-ST-1 , heparan sulfate iduronic acid 2-O-ST, heparan sulfate D-glucosaminyl 6-O-ST, tyrosylprotein ST, N- acetylglucosamine 6-O-ST, chondroitin sulfate / V-galactosamine 4-O-ST-1 , 2, and 3, chondroitin sulfate / V-galactosamine 6-O-ST, HNK-1 glucuronic acid 3-O-ST, N-acetylgalactosamine 4-O-ST, dermatan sulfate / V-galactosamine 4-O-ST, keratan sulfate galactose 6-O-ST and combinations thereof.

[0067] In some embodiments, the cell free extract as disclosed herein may contain at least one enzyme selected in the group consisting of: a heparan sulfate 3-0- sulfotransferase, a heparan sulfate 2-O-sulfotransferase, a heparan sulfate 6-0- sulfotransferase, a C5-epimerase and combinations thereof.

[0068] In some embodiments, the enzyme(s) may be recombinantly expressed by the cells serving for the preparation of the cell free extract as disclosed herein.

[0069] In some embodiments, in case of multiple enzyme(s), they may be provided in the same cell free extract as disclosed herein or in distinct cell free extracts which may be mixed or added successively in the reaction medium.

[0070] In uses or methods as disclosed herein, a substrate may be sulfated with one or a plurality of sulfotransferases to carry out a plurality of sulfations.

[0071] In uses or methods as disclosed herein, a plurality of sulfations may be carried out concomitantly or sequentially.

[0072] In uses or methods as disclosed herein a step of converting cyclo- PAPS into PAPS may be carried out concomitantly with the sulfation. In some embodiments, the step of sulfation and the step of converting cyclo-PAPS into PAPS may be carried out concomitantly in a same reaction mixture, optionally using a single cell free extract.

[0073] In uses or methods as disclosed herein a substrate may be selected in a group comprising synthetic oligosaccharides such as NS-octasaccharide, (aryl) p- Nitrophenol, NS-heparosan, NS.2S-epi-heparosan, NS.2S.6S-epi-heparosan and heparin, optionally porcine lung heparin and bovine intestinal heparin.

[0074] In uses or methods as disclosed herein, a substrate may be selected in the group comprising phenols, catecholamines, thyroid hormones, aryl hydroxylamines, hydroxysteroids, CCR-5, PSGL-1 , sialyl Lewis, chondroitin sulfate, keratan sulfate, HNK-1 sulfate, saccharide receptor, units on lutropin, thyrotropin, pro-opiomelanocortin and dermatan sulfate.

[0075] According to one of its objects, the present disclosure relates to a method for sulfating a substrate or for preparing a heparin product comprising a step of generating PAPS from cyclo-PAPS using a cell free extract as disclosed herein, optionally according to a method as disclosed above.[DESCRIPTION OF THE FIGURES]

[0076] Figure 1 represents chromatograms of samples of cyclo-PAPS:

[0077] (Figure 1A) in the presence of purified 6-OST

[0078] (Figure 1B) upon addition of a cell free extract (CFE) containing 6- OST

[0079] (Figure 1C) 6 hours after addition of a cell free extract (CFE) containing 6-OST.

[0080] Figure 2 represents the kinetics of 2-0 sulfotransferase reactions on NS-octasaccharide with PNP tag using:

[0081] - CFE 2-OST + cyclo-PAPS (•)

[0082] - purified 2-OST + cyclo-PAPS (A)

[0083] - purified 2-OST + PAPS (■)

[0084] Figure 3 represents the kinetics of the 6-0 sulfotransferase reactions on NS-octasaccharide with PNP tag using:

[0085] - CFE 6-OST + cyclo-PAPS (•)

[0086] - purified 6-OST + cyclo-PAPS (A)

[0087] - purified 6-OST + PAPS (■)

[0088] Figure 4 represents the kinetics of the 3-0 sulfotransferase reactions on NS-octasaccharide with PNP tag using:

[0089] - CFE 3-OST + cyclo-PAPS (•)

[0090] - purified 3-OST + cyclo-PAPS (A)

[0091] - purified 3-OST + PAPS (■)

[0092] Figure 5 represents the kinetics of the 3-0 sulfotransferase reactions on porcine lung heparin using:

[0093] - CFE 3-OST + cyclo-PAPS (■)

[0094] - purified 3-OST + cyclo-PAPS (A)

[0095] - purified 3-OST + PAPS Li (•)[DETAILED DESCRIPTION]Definitions

[0096] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. “A” and “an” mean “at least one”, unless the content clearly dictates otherwise.

[0097] The terms “about” or “approximately” as used herein refer to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term “about” refers to ±10% of a given value. However, whenever the value in question refers to an indivisible object, such as a molecule or other object that would lose its identity once subdivided, then “about” refers to ±1 of the indivisible object.

[0098] Within the disclosure, the expression “sulfotransferase” (noted SULT or ST) intends to refer to an enzyme that catalyzes the sulfate conjugation of a product.

[0099] Within the disclosure, the expression “phosphatase” intends to refer to an enzyme belonging to a subcategory of hydrolases, that uses water to cleave a phosphoric acid ester into a phosphate ion and an alcohol. Phosphatases are also able to use cyclic phosphates such as cyclo-PAPS as a substrate, thereby generating a phosphoric acid ester instead of a phosphate ion.

[0100] Within the disclosure, the expression “phosphatase activity” intends to refer to the catalytic activity of a phosphatase opening (e.g. through a nucleophilic addition of water) the cyclic 2’, 3’-phosphate on cyclo-PAPS to generate PAPS. Said activity may result in the production of PAPS (3'-phosphoadenosine 5'-phosphosulfate; CAS number: 482-67-7), possibly iso-PAPS (2'-phosphoadenosine 5'-phosphosulfate), and the disappearance of cyclo-PAPS (adenosine 2',3'-cyclic phosphate 5'-phosphosulfate; CAS number: 62230-90-4).

[0101] Within the disclosure, the expression “cell free extract” (“cell extract” or “cell free lysate” or “lysate”) intends to refer to the cell soluble fraction obtained after lysis of the cell. Cell lysis can be performed by any means known in the art such as thermal, chemical (e.g. lysis buffer), mechanical (e.g. by high-pressure homogenization, mechanical grinding, mechanical lysis using glass beads, ultrasonic disintegration, repeated freeze-thaw cycles or osmotic shock) or enzymatic (using endogenous or exogenous enzyme) lysis. During lysis, cell membranes are destabilized, disintegrated and / or destroyed, thereby liberating the internal content of the cells in its native state. Cellmembranes can be removed, e.g. by centrifugation or micro-filtration. A cell free extract can be in the form of a powder if it is freeze-dried or spray-dried, optionally a fine hydrosoluble powder, or as a liquid, possibly a concentrated liquid.

[0102] It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of” aspects and embodiments. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of” implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of” implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “comprising only”, “consisting of” or “consisting essentially of”.

[0103] The expression “enhanced activity” with regard to an enzyme intends to mean that the enzyme has a catalytic activity, or a thermal stability or a structure stability which is enhanced compared to a reference enzyme.

[0104] Within the disclosure, the expression "isolated" with regard to a compound or entity, such as an enzyme, refers to this compound or entity in an environment different from the one in which the compound or entity may naturally occur. "Isolated" is meant to include compound or entity in samples which are substantially enriched for this compound or entity and / or in which this compound or entity is partially or substantially purified. In some cases, an isolated compound or entity (e.g. a protein; a nucleic acid; a recombinant vector) is purified, e.g. it is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99%, pure.

[0105] Within the disclosure, the expression “non-naturally occurring” as used herein with regard to a nucleic acid, a peptide, polypeptide, or protein refers to any nucleic acid, peptide, polypeptide, or protein which are not found in nature.

[0106] Within the disclosure, the expression “mutant” as used herein with regard to a peptide, polypeptide, or protein refers to any peptide, polypeptide, or protein comprising at least one amino acid mutation. “Amino acid mutation” and “mutation” are used interchangeably and intend to refer to a substitution, a deletion, or an insertion of an aminoacid, as compared to a wild-type, or naturally occurring, counterpart. In particular, a mutant peptide, polypeptide, or protein may comprise at least one amino acid substitution.

[0107] A “recombinant protein” as used herein intends to refer to a protein produced with a recombinant DNA. A “recombinant DNA” refers to a genetically- engineered DNA molecule formed by splicing fragments of DNA from different sources or from another part of the same source, and then introduced into the recipient (host) cell. For example, a recombinant protein may be produced by inserting the corresponding coding nucleic acid in a plasmid vector and delivering the vector in a host cell suitable for the expression of the protein.

[0108] Within the disclosure, the term “significantly” used with respect to change intends to mean that the observe change is noticeable and / or it has a statistic meaning.

[0109] Within the disclosure, the term “substantially” used in conjunction with a feature of the disclosure intends to define a set of embodiments related to this feature which are largely but not wholly similar to this feature. The difference between the set of embodiments related to the given feature and the given feature is such that in the set of embodiments, the nature and function of the given feature is not materially affected.

[0110] Within the disclosure, the expression “substantially the same or greater than” used to qualify the catalytic activity of a given enzyme with respect to the catalytic activity of a reference enzyme intends to define (i) that the catalytic activity of both enzymes, when measured with same protocol and conditions, are not significantly different or (ii) that the catalytic activity of the given enzyme is significantly above the catalytic activity of the reference enzyme, when both measured with same protocol and conditions.

[0111] The terms “sulfation” “sulfonation” and “sulfurylation” are used interchangeably and herein refer to a transfer of a sulfonate or sulfuryl group from one molecule to another.

[0112] The term “in situ" means “in the reaction mixture”, i.e. in the reaction medium wherein the CFE and cyclo PAPS are incubated (without isolating PAPS from said medium).

[0113] The terms “heparin product” and “heparin compound” means any of various glycosaminoglycans which are structurally or pharmacologically related to heparin.

[0114] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0115] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0116] The list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0117] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0118] All lists of items, such as, for example, lists of ingredients, are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ the list of items “and combinations and mixtures thereof.”

[0119] Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g. those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.Cell free extract

[0120] In some embodiments, a cell free extract may be obtained from cells by lysis of said cells. In some embodiments, the cultured cells are suspended in a so-called lysing buffer (e.g. 20 mM TRIS, 200 mM NaCI pH 7,5) and submitted to high-pressure homogenization (e.g. at 29000 PSI). After centrifugation (e.g. at 25000 g for 1 h at 4 °C), the soluble fraction of the cells comprising the internal content thereof but deprived of cell membranes and debris may be further filtrated, e.g. by microfiltration over 0.2 .m membrane. In some embodiments, the cell free extracts as disclosed herein are in liquid form. Afterwards, they may be stored as such, e.g. using a preservative such as glycerol for storage at -80 °C. Alternatively, the cell free extracts as disclosed herein are dried or lyophilized to be stored as a paste or a powder and reconstituted in liquid form before running the reaction.

[0121] In the context of the present disclosure, the cells serving for the preparation of a cell free extract (CFE) as disclosed herein have an activity able to convert cyclo-PAPS into PAPS. In some embodiments, this activity is endogenous, i.e. due to the presence in the cells of enzymes having said phosphatase activity.

[0122] In some embodiments, the cells have endogenous enzymes able to convert cyclo-PAPS into-PAPS.

[0123] According to the present disclosure, E. coli cells may be used to prepare a cell free extract as disclosed above.

[0124] In some embodiments, enzymes involved in the conversion of cyclo- PAPS into PAPS are 2’,3’-cyclic-nucleotide 2’-phosphodiesterase / 3’-nucleotidase (cpdB), acid phosphatase (aphA) and / or 3’(2’),5’-biphosphate nucleotidase (cysQ).

[0125] In some embodiments, the 2’,3’-cyclic-nucleotide 2’- phosphodiesterase / 3’-nucleotidase may be encoded by the E. coli cpdB gene, the acid phosphatase may be encoded by the E. coli aphA gene and the 3’(2’),5’-biphosphate nucleotidase may be encoded by the E. coli cysQ gene. As known in the art, equivalent genes can be screened in any other cell.

[0126] According to the present disclosure, A. niger cells may be used to prepare a cell free extract as disclosed above.

[0127] In some embodiments, the enzyme involved in the conversion of cyclo-PAPS into PAPS is a ribonuclease T2 (RNase T2).

[0128] In some embodiments, the ribonuclease T2 (RNase T2) may be encoded by the A niger genome. As known in the art, equivalent genes can be screened in any other cell.

[0129] In some embodiment, a cell free extract (CFE) as disclosed herein is further able to degrade PAP, the product resulting from the donation of the sulfate by PAPS, in particular into 5’-AMP. As known in the art, PAP may be an inhibitor of sulfotransferases.

[0130] In the context of the present disclosure, the cells serving for the preparation of a cell free extract (CFE) as disclosed herein may be host cells for (over)expressing / producing recombinant enzymes. In some embodiments, said recombinant enzymes are involved in sulfation reactions.

[0131] As known in the art, host cells for protein production are classically prokaryotic or eukaryotic cell, usually bacterial, yeast, plant, and animal cells.

[0132] In some embodiments, the cell free extract having the phosphatase activity able to convert cyclo-PAPS into PAPS and the cell free extract having the enzymatic activity involved in sulfation reactions are distinct and possibly originate from different cell types. In practice, they can be mixed in the reaction medium as disclosed herein. As an example, a cell free extract, obtained from E. coli cells and able to concert cyclo-PAPS into PAPS, may be used in combination with a cell free extract, obtained from P. pastori cells recombinantly producing the enzymatic activity involved in sulfation reactions.Recombinant expression

[0133] The enzymes of interest in the frame of the present disclosure, i.e. involved in sulfation reactions, can be produced by any suitable method, including recombinant and non-recombinant methods.

[0134] Where an enzyme of interest is produced using recombinant techniques, the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial, yeast, plant, and animal cells. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected to provide for stable expression of the introduced enzyme encoding nucleic acid. The enzyme encoding nucleic acid can be provided as an inheritable episomal element (e.g. plasmid) or can be genomically integrated.

[0135] In some embodiments the hosts are filamentous fungi or yeast strains. In some embodiments, hosts are bacterial strains. Non-limiting examples of hostcells which may be used to produce a cell free extract as disclosed herein are: Escherichia coli, Pichia pastori, Saccharomyces cerevisiae, Bacillus sp., Aspergillus sp., Yarrowia lipolytica, Kluyveromyces lactis. An appropriate strain of Escherichia coli is e.g. E. Coli Origami B (DE3) (Novagen) or E. coli Shuffle T7 express (NEB).

[0136] The present disclosure provides nucleic acids, including isolated or recombinant nucleic acids, that comprise a nucleotide sequence encoding enzymes of interest. In some embodiments, the present disclosure provides a nucleic acid (or nucleotide sequence) encoding said enzyme. In some embodiments, the nucleotide sequence is operably linked to a transcriptional control element, e.g. a promoter.

[0137] In some instances, a nucleic acid comprising a nucleotide sequence encoding an enzyme of interest may be present in an expression vector. In some embodiments, the present disclosure provides a recombinant expression vector comprising a nucleic acid encoding an enzyme of interest. The present disclosure provides a recombinant expression vector (e.g. an isolated recombinant expression vector) that comprises a nucleotide sequence encoding an enzyme of interest.

[0138] In some embodiments, the nucleotide sequence encoding an enzyme of interest is operably linked to a transcriptional control element, e.g. a promoter. The promoter is in some cases constitutive. The promoter is in some cases inducible. In some cases, the promoter is suitable for use (e.g. active) in a prokaryotic host cell. In some cases, the promoter is suitable for use (e.g. active) in a eukaryotic host cell.

[0139] Suitable vectors for transferring encoding nucleic acid can vary in composition.

[0140] Integrative vectors can be conditionally replicative or suicide plasmids, bacteriophages, and the like. The constructs can include various elements, including for example, promoters, selectable genetic markers (e.g. genes conferring resistance to antibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)), origin of replication (to promote replication in a host cell, e.g. a bacterial host cell), and the like. The choice of vector will depend upon a variety of factors such as the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.

[0141] In one example, the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g. in both E. coli and N. meningitidis). One example of such a "shuttle vector" is the plasmid pFPIO (Pagotto et al. (2000) Gene 244: 13-19).

[0142] Constructs (recombinant vectors) can be prepared by, for example, inserting a polynucleotide of interest into a construct backbone, typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence, while sitespecific recombination can be accomplished through use of sequences that facilitate sitespecific recombination (e.g. cre-lox, att sites, etc.). Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence.

[0143] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. Vectors are amply described in numerous publications well known to those in the art, including, e.g. Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expression of the nucleic acids encoding the protein of interest, may provide for propagating the subject nucleic acids, or both.

[0144] Examples of vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pBR322, plIC 19 / 18, plIC 118, 119 and the M13 mp series of vectors may be used. pET21 is also an expression vector that may be used. Bacteriophage vectors may include AgtIO, Agtl I, Agtl8-23, AZAP / R and the EMBL series of bacteriophage vectors. Further vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

[0145] For expression of a protein of interest, an expression cassette may be employed. Thus, the present disclosure provides a recombinant expression vector comprising a subject nucleic acid. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to the enzyme of interest or may be derived from exogenous sources. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g. T7, and the like).

[0146] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0147] Any of a number of suitable host cells can be used. In general, the protein of interest described herein may be expressed in prokaryotes or eukaryotes, e.g. bacteria such as Escherichia coli in accordance with conventional techniques. Thus, the present disclosure further provides an in vitro host cell, which comprises a nucleic acid encoding an enzyme of interest. Host cells for production (including large scale production) of a protein of interest can be selected from any of a variety of available host cells.

[0148] Examples of host cells for expression include those of a prokaryotic or eukaryotic unicellular organism, such as bacteria (e.g. Escherichia coli strains), yeast (e.g. Saccharomyces cerevisiae, Pichia spp., and the like), and may include host cells originally derived from a higher organism such as insects, vertebrates, e.g. mammals. Suitable bacteria include but are not limited to BL21 Competent E. coli, BL21(DE3) Competent E. coli, NEB Express Competent E. col, NEB Express Iq Competent E. coli, T7 Express Competent E. coli, T7 Express Iq Competent E. coli, T7 Express lysY Competent E. coli, T7 Express lysY / lq Competent E. coli, T7 Express Crystal Competent E. coli, SHuffle Express Competent E. coli, SHuffle T7 Express Competent E. coli, SHuffle T7 Express lysY Competent E. coli, SHuffle T7 Competent E. coli, NiCo21 (DE3) Competent E. coli, Lemo21 (DE3) Competent E. coli. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g. American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.ATCC Nos. CRL9618, CCL61 , CRL9096), 293 cells (e.g. ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g. ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.). In some cases, bacterial host cells and yeast host cells are of particular interest for production of the protein of interest.

[0149] In the frame of the present disclosure, the use of cell free extracts (CFE) containing the enzymes of interest, in particular those involved in sulfation reactions, avoid purifying said enzymes. Whereas the use of CFE is drastically cheaper than the use of purified enzymes, the skilled person would have been reluctant in using CFE which are complex extracts susceptible to inhibit the sensitive sulfotransferase activity.Kits

[0150] In some embodiments, the disclosure relates to a kit for preparing PAPS.

[0151] A kit for preparing PAPS may comprise at least:

[0152] one cell free extract as disclosed herein in a first container; and

[0153] cyclo-PAPS in a second container.

[0154] A kit as disclosed herein may contain more than one cell free extract, all mixed in the same container or each being packaged in a separate container.

[0155] A kit as disclosed herein may be used for converting cyclo-PAPS into PAPS.

[0156] The kit may further comprise instructions for preparing PAPS.

[0157] A kit may further contain a buffer suitable for the catalytic phosphatase activity of the cell free extract. The buffer may be packaged with the cell free extract(s) as disclosed herein or may be packaged in a separate container. A suitable buffer for the reaction may be, for example, a 4 Morpholine-Ethane Sulfonic acid (MES) buffer. A suitable pH is about 7. In some embodiments, the buffer is supplemented with additives such as a solubilizing agent and / or surfactant (e.g. polysorbate 80), a salt (e.g. NaCI or CaCh) and / or a reducing agent (e.g. tris(2-carboxy-ethyl)phosphine or TCEP).

[0158] In some embodiments, the kit may further comprise at least one enzyme. Said enzyme can be in the form of a purified enzyme or contained in a cell free extract, possibly the same as the cell free extract having the ability to convert cyclo-PAPS into PAPS as disclosed above. In some embodiments, such an enzyme may be involved insulfation reactions. In some embodiments, such an enzyme may use PAPS as a sulfate donor. In some embodiments, such an enzyme may be a sulfotransferase. In some embodiments, such an enzyme may be involved in heparin synthesis, for example a C5- epimerase or an O-sulfotransferase (OST) enzyme, such as 2-OST, 3-OST and 6-OST. When a kit contains two or more enzymes, each enzyme may be packaged in separate container.

[0159] In some embodiments, the disclosure relates to a kit for sulfating a substrate.

[0160] A kit as disclosed herein may be used for sulfating a polysaccharide. A kit as disclosed herein may be used for synthesizing a sulfated substrate. A kit as disclosed herein may be used for synthesizing heparin or heparin products.

[0161] The kit may further comprise instructions for sulfating a substrate, for example a polysaccharide. The instructions may concern the synthesis of heparin or heparin products.

[0162] A kit may further contain a buffer suitable for the catalytic activity of the further enzyme(s), for example a sulfotransferase. The buffer may be packaged with a sulfotransferase as disclosed herein or may be packaged in a separate container. A suitable buffer may be, for example, TRIS-buffer, sodium phosphate buffer, and potassium phosphate buffer. A suitable pH is from 6.0 to 7.5, and around 7.0.Catalytic activity and screening methods

[0163] A cell free extract as disclosed herein may have a cyclic phosphate opening activity for converting cyclo-PAPS into PAPS.

[0164] Such activity may be detected and measured according to any known method in the art. In some embodiments, the activity of the cell free extract is measured by chromatography, e.g. by high-performance liquid chromatography (HPLC) as disclosed in the examples, which allows to differentiate cyclo-PAPS and PAPS. In some embodiments, the method allows measuring the amount of PAPS released (or produced) and / or the amount of cyclo-PAPS consumed. As a reference, a ribonuclease T2 (RNase T2), for example the Aspergillus oryzae RNase T2, can be used.

[0165] In some embodiments, a method of screening and / or selecting a cell free extract able to convert cyclo-PAPS into PAPS may comprise the steps of:

[0166] a) contacting a cell free extract with a sufficient amount of cyclo- PAPS, in a suitable buffer,

[0167] b) acquiring a measure representative of PAPS produced at step a).

[0168] A measure representative of PAPS produced during the reaction may be obtained by a measure of the surface of the pic corresponding to PAPS on a chromatogram, for example using a HPLC system according to manufacturer’s recommendations. The obtained measure may be expressed in arbitrary Unit of surface.

[0169] A suitable buffer for the reaction may be a 4 Morpholine-Ethane Sulfonic acid (MES) buffer at pH 7.

[0170] A suitable temperature of reaction may be about 37°C.

[0171] The acquisition of the measure may be carried out at 30, 60 or 90 minutes after initiation of the reaction, for example 60 minutes after initiation of the reaction. The values should be in the linear part of the kinetic graph to be able to measure the initial rate.

[0172] The detection of a rate or an amount of formation of PAPS at step b) may be carried out directly by measuring the amount of metabolite PAPS, indirectly by measuring the amount of the product to be transformed cyclo-PAPS.Uses and methods for preparing PAPS from cyclo-PAPS

[0173] In some embodiments, a cell free extract as disclosed herein may be used for preparing PAPS from cyclo-PAPS.

[0174] In some embodiments, the disclosure relates to a method for preparing PAPS comprising at least a step of contacting or incubating cyclo-PAPS with a cell free extract as disclosed herein. In some embodiments, contacting comprises incubating cyclo-PAPS with a cell free extract as disclosed herein in a reaction medium.

[0175] In some embodiments, the incubation conditions are adapted to the catalytic phosphatase activity of the cell free extract, i.e. to allow selective opening, of, cyclic 2’, 3’-phosphate on cyclo-PAPS to generate PAPS.

[0176] In some embodiments, the incubation conditions are also adapted to the activity of the further enzyme(s), especially enzymes involved in sulfation reactions, contained in the reaction medium in the form of a cell free extract.

[0177] In some embodiments, the reaction medium may comprise a buffer. In some embodiments, a buffer may be a 4 Morpholine-Ethane Sulfonic acid (MES) buffer.

[0178] In some embodiments, the buffer is at a concentration from 10 mM to 200 mM, e.g. 50 mM.

[0179] In some embodiments, the pH of the reaction medium is adapted according to the optimal pH for the activity of the cell free extracts and enzymes as disclosed herein. In some embodiments, the pH of the reaction medium may be from 6.0 to 8.0. In some embodiments, the reaction medium may be 7.0.

[0180] In some embodiments, the concentration of the cell free extract as disclosed herein in the reaction medium is adapted to allow an efficient and specific conversion of cyclo-PAPS into PAPS. The adequate concentration, which may also depend on other factors such as the cyclo-PAPS concentration and the incubation time, can be easily determined by the skilled person, by monitoring the generation of PAPS using a method (e.g. HPLC) as disclosed above. In some embodiments, the concentration of the cell free extract in the reaction medium may be adapted to the enzymatic activity involved in sulfation reactions so that the quantity of PAPS to be used as a sulfate donor by said enzymes(s) is sufficient.

[0181] In some embodiments, the incubation temperature is adapted according to the optimal temperature for the activity of the cell free extract and the enzymes as disclosed herein. In some embodiments, the incubation temperature may be between 15°C and 40°C, e.g. room temperature (RT) such as 25°C or 37°C.

[0182] In some embodiments, the incubation time may be between 1 hour and several days. The adequate time, which may also depend on other factors such as the cell free extract concentration, can be easily determined by the skilled person, by monitoring the generation of PAPS using a method (e.g. HPLC) as disclosed above. In some embodiments, the incubation time may be inferior to 24 hours, e.g. 10 hours. In some embodiments, the incubation time is sufficient to ensure the sulfation of the substrate present in the reaction medium by the enzymes contained in the cell free extract.

[0183] The incubation between a cell free extract (CFE) as disclosed herein and its substrate (cyclo-PAPS and optionally the substrate to be sulfated) may be stopped after an appropriate time, i.e. several minutes, several hours or even several days, optionally between 1 hour and 48 hours, e.g. not after 24 hours. It can be stopped by inactivation of the enzymes, e.g. by heat inactivation, acidification of the reaction medium or by addition of a denaturating solvent such as methanol. The reaction can also be stopped by removal of the enzyme by, for example, ultrafiltration.

[0184] In some embodiments, the concentration of the substrate(s) to be sulfated in the reaction medium may be from 0.1 g / L to 50 g / L, e.g. 10 g / L.

[0185] In some embodiments, the reaction may be stopped by any method known in the art, e.g. by inactivating or removing the cell free extract or the enzyme.

[0186] According to the present disclosure, there is no need to isolate or purify PAPS since it can be directly consumed in the sulfation reactions, e.g. by having the enzymes involved in the sulfation reactions in the reaction medium. Said enzyme(s) can be provided in a purified form or as a cell free extract. In some embodiments, the enzymes are provided in a cell free extract, optionally in the cell free extract having the phosphatase activity to convert cyclo-PAPS into PAPS.Uses and methods for preparing PAPS from adenosine or 5’ -AMP

[0187] In some embodiments, a cell free extract as disclosed herein may be used in a method for preparing PAPS from adenosine or 5’-AMP.

[0188] In some embodiments, a method of preparing PAPS from adenosine or 5’-AMP may comprise at least the steps of:

[0189] a) Phosphorylation of adenosine or 5’-AMP into PAP and iso-PAP, using a phosphorylation agent

[0190] b) Conversion of PAP and iso-PAP into cyclo-PAPS

[0191] c) Conversion of cyclo-PAPS into PAPS using a method as disclosed above.

[0192] In step a), adenosine or 5’-AMP is phosphorylated to yield a mixture of PAP and iso-PAP. In some embodiments, phosphorylation may be performed using different phosphorylating agents such as P2CI4O3 (Haas, T. M. et a / Chem. Commun. 2019, 55, 5339-5342), POCI3 (Collier, A. etal. Org. Biomol. Chem. 2006, 4, 4526-4532) or sodium cyclo-triphosphate (P3m) (Inoue, H., Phosphorus Research Bulletin 1995, 5, 137-142). In some embodiments, this step may result in the synthesis of PAP / iso-PAP sodium salt.

[0193] In step b), the PAP / iso-PAP mixture is transformed to cyclo-PAPS. In some embodiments, this may be done in one step using Et3N-SO3 (Cherniak, R. J. Biol. Chem. 1964, 239(9), 2986-2990). In other embodiments, this may be done in two steps in which first cyclo-PAP is formed by using DCC (Dicyclohexylcarbodiimide) followed by sulfonation using Et3N-SO3 (Sekura, R. Methods in Enzymology, 1981 , 77, 413-415). In some embodiments, this step may result in an aqueous cyclo-PAPS solution.Uses and methods for sulfating a substrateSulfation

[0194] In some embodiments, the disclosure relates to the use of PAPS prepared with the method as disclosed above for sulfating a substrate.

[0195] In some embodiments, the disclosure relates to a method of sulfating a substrate comprising at least a step of contacting the substrate to be sulfated with a) a sulfotransferase and b) a sulfo group donor comprising PAPS prepared with the method as disclosed above, in conditions suitable for a transfer of the sulfo group from the sulfo group donor to said substrate.

[0196] In some embodiments, the disclosure relates to a method of sulfating a substrate comprising at least a step of contacting the substrate to be sulfated with a) a sulfotransferase, b) cyclo-PAPS and c) a cell free extract as disclosed herein, in conditions suitable for a conversion of cyclo-PAPS into PAPS and the transfer of the sulfo group from PAPS to said substrate.

[0197] The uses or methods of the disclosure may be for synthesizing heparin.

[0198] The method may further comprise a step of retrieving the sulfated substrate.

[0199] A substrate to be sulfated may be selected in a group comprising, a polysaccharide, an oligosaccharide, an heparan, an heparosan, an heparosan sulfate, or a sulfated heparin.

[0200] The disclosure relates to a method for obtaining a sulfated substrate by sulfating a substrate with at least one sulfotransferase and PAPS, said method including at least one step of converting cyclo-PAPS into PAPS by contacting said cyclo-PAPS with a cell free extract as disclosed herein.

[0201] According to a specific embodiment, the step of converting cyclo- PAPS into PAPS is simultaneous to the step of sulfation.

[0202] According to another embodiment, the step of converting cyclo-PAPS into PAPS and the step of sulfation are sequential. According to one embodiment, the step of converting cyclo-PAPS into PAPS precedes the step of sulfation. In practice, the cell free extract responsible for the conversion of cyclo-PAPS into PAPS may be first incubated with cyclo-PAPS and a further cell free extract containing the enzymes involved in sulfation reactions is added in the reaction medium.

[0203] The methods may further comprise a step of recovering the so- formed sulfated substrate.

[0204] A method as disclosed herein may be for synthesizing a heparin.

[0205] Sulfation of a substrate may be carried out using various sulfotransferases including an O-sulfotransferase (OST) enzyme, such as for example 2- OST, 3-OST, 3-OST-1 , 3-OST-3, 6-OST, 6-OST-1 , 6-OST3, or a N-sulfotransferase such as NDST1 , NDST2.

[0206] Step of sulfation of a substrate and step of converting cyclo-PAPS in PAPS may be carried out sequentially or simultaneously in a one-pot reaction.

[0207] In some embodiments, step of sulfation of a substrate of the methods disclosed herein may comprise a plurality of sub-steps ai), 82), as), ... , during which the substrate may undergo successive enzymatically catalyzed reactions. Those reactions may be sulfation at different positions within the substrate, carried out by different sulfotransferases using PAPS as sulfo donor group. The different sulfotransferases may be, for example, different OSTs. In some embodiments, the different sulfotransferases are provided by different cell free extracts.

[0208] In some embodiments, step of sulfation of a substrate may comprise a plurality of simultaneous or sequential sub-steps ai), 82), as), ... , an), and wherein at least two sub-steps comprise each a sulfation catalyzed by a sulfotransferase using PAPS as a sulfo group donor to obtain PAP and a sulfated substrate.

[0209] An aspect of the disclosure is directed to a method for sulfation of a polysaccharide substrate. The method can be of a type wherein the sulfation of a polysaccharide substrate is catalyzed by a sulfotransferase, such as one or more OSTs, with a conversion of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to adenosine 3', 5'- diphosphate (PAP). The sulfation process can be coupled with the PAPS preparation using cyclo-PAPS and a cell free extract as disclosed herein.Sulfo group donor

[0210] The sulfo group donor may be PAPS generated by the in situ conversion of cyclo-PAPS using a cell free extract as disclosed herein.Sulfotransferases and substrates

[0211] As known in the art, there are two classes of sulfotransferases (SLILTs): cytosolic and membrane-associated SLILTs. Cytosolic SLILTs sulfonate small endogenous and exogenous compounds, such as hormones, bioamines, drugs, and various xenobiotic agents. Membrane-associated SLILTs, many of which are implicated incrucial biological processes, sulfonate larger biomolecules, such as carbohydrates and proteins.

[0212] Examples of human cytosolic sulfotransferases and their cognate substrates are listed below:

[0213] The SULT1A1 enzyme has phenols as substrates.

[0214] The SULT1A2 enzyme has phenols (low affinity) as substrates.

[0215] The SULT1A3 enzyme has catecholamines as substrates.

[0216] The SULT1 B1 enzyme has thyroid hormones as substrates.

[0217] The SULT1C1 and SULT1C2 enzymes have aryl hydroxylamines as substrates.

[0218] The SULT1 E1 enzyme has estrogens as substrates.

[0219] The SULT2A1 and SULT1A1 enzymes have hydroxysteroids as substrates.

[0220] Examples of representative substrates are: pNP, 2-napthol, dopamine, tyramine, minoxidol, 3,3’-T2, T3, PNP, T4, rT3, N-OH-2AFF, estradiol, DHEA, androstenediol, pregnenolone, 17a-pregnenolone.

[0221] Examples of membrane-associated sulfotransferases and their cognate substrates are listed below:

[0222] The heparan sulfate D-glucosaminyl 3-O-ST-3a and b, the heparan sulfate D-glucosaminyl 3-O-ST-1, the heparan sulfate iduronic acid 2-O-ST, the heparan sulfate D-glucosaminyl 6-O-ST have heparan and heparin as substrates.

[0223] The tyrosylprotein ST has CCR-5 and PSGL-1 as substrates.

[0224] The / V-acetylglucosamine 6-O-ST has sialyl Lewis as substrates.

[0225] The chondroitin sulfate / V-galactosamine 4-O-ST-1 , 2, and 3 have chondroitin sulfate as substrate.

[0226] The chondroitin sulfate / V-galactosamine 6-O-ST has chondroitin sulfate and keratan sulfate as substrates.

[0227] The HNK-1 glucuronic acid 3-O-ST has HNK-1 sulfate as substrate.

[0228] The / V-acetylgalactosamine 4-O-ST has saccharide receptor, units on lutropin, thyrotropin, pro-opiomelanocortin as substrates.

[0229] The dermatan sulfate / V-galactosamine 4-O-ST has dermatan sulfate as substrate.

[0230] The keratan sulfate galactose 6-O-ST has keratan sulfate as substrate.

[0231] The substrate may be selected in a group comprising a polysaccharide, an oligosaccharide, an heparan, an heparosan sulfate, an heparosan, a chemically desulfated N-sulfated (CDSNS) heparin, a glycosaminoglycan (GAG), an heparan sulfate or a sulfated heparin.

[0232] In a method for sulfating a substrate where a cell free extract as disclosed herein is used to convert cyclo-PAPS in PAPS, a substrate to be sulfated may be selected in a group comprising a polysaccharide, an oligosaccharide, an heparan, an heparosan, a chemically desulfated N-sulfated (CDSNS) heparin, a glycosaminoglycan (GAG), an heparan sulfate or a sulfated heparin.

[0233] A polysaccharide substrate may be partially sulfated prior to reaction mixture incubation. In some embodiments, the sulfated polysaccharide is a glycosaminoglycan (GAG), such as for example a heparan sulfate (HS). In some embodiments, the sulfated polysaccharide is an HS that is an anticoagulant-active HS, an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, a herpes simplex virus envelope glycoprotein D-binding HS or has a combination of these properties.

[0234] In some embodiments, a substrate to be sulfated may undergo further to sulfation at least one additional enzymatically catalyzed reaction. This or these additional reaction(s) may be carried out before or after the sulfation.

[0235] A substrate to be sulfated may be a polysaccharide substrate previously N,O-desulfated and re-N-sulfated polysaccharide, such as for example a chemically desulfated N-sulfated (CDSNS) heparin. For example, a polysaccharide, such as CDSNS, can be reacted with a particular OST in presence of PAPS to produce a sulfated polysaccharide intermediate product that can then be reacted subsequently with a different OST in presence PAPS to further sulfate the polysaccharide at different locations. This sequential process of reacting the polysaccharide substrate with different OSTs can be continued until a final polysaccharide is produced exhibiting desired biological activities.

[0236] The sulfation methods disclosed herein allows producing a multitude of sulfated polysaccharides, such as heparan sulfate molecules having varied biological activities by selecting appropriate sulfotransferases and by sequentially controlling the addition of those sulfotransferases to the reaction system to facilitate appropriate timing ofsulfation of the polysaccharide. For example, heparan sulfate having specific biological activities which can be synthesized includes anticoagulant heparan sulfate, heparin, fibroblast growth factor-2-binding activity, herpes simplex virus glycoprotein D (gD)-binding HS, and fibroblast growth factor 2 (FGF2) receptor-binding HS. Only two or three enzymatic steps are required for the synthesis of each of these biologically active heparan sulfate molecules. Thus, the methods disclosed herein, because of the effective and low cost PAPS generating system, provide efficient and effective methods for the large-scale synthesis of a wide range of heparan sulfate with specific activities.

[0237] In some embodiments, the sulfated polysaccharide substrate can be a glycosaminoglycan (GAG). GAGs are the most abundant heteropolysaccharides in the body. These molecules are long unbranched polysaccharides containing a repeating disaccharide unit. The disaccharide units can contain either of two modified sugars: N- acetylgalactosamine (GalNAc) or N-acetylglucosamine (GIcNAc) and a uronic acid such as glucuronate or iduronate. GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution. Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides passageways between cells, allowing for cell migration. The specific GAGs of physiological significance are hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate (including heparin), and keratan sulfate. Thus, in some embodiments, the sulfated polysaccharide product is a HS. In some embodiments, the sulfated polysaccharide product is an anticoagulant-active HS, an antithrombin-binding HS, an FGF-binding HS, and an HSV gD-binding HS.Heparin synthesis

[0238] In some embodiments, the presently disclosed subject matter provides a method of synthesizing a heparin compound.

[0239] A method for synthesizing heparin may comprise obtaining a sulfated heparin precursor by sulfating the heparin precursor with at least one sulfotransferase and PAPS, said method including at least one step of converting cyclo-PAPS into PAPS by contacting said cyclo-PAPS with at least a cell free extract as disclosed herein.

[0240] Heparosan may be used as the polysaccharide raw material for the method for synthetizing heparin of the present disclosure. Heparosan is a polysaccharide composed of repetitive structures of a disaccharide composed of a glucuronic acid (GlcA) residue and an N-acetyl-D-glucosamine (GIcNAc) residue [->4)-p-D-GlcA-(1 ->4)-a-D-GlcNAc-(1 ^]. Heparosan can be produced, for example, by a fermentation method utilizing a bacterium having an ability to produce heparosan.

[0241] In some embodiments, the method disclosed herein for synthetizing heparin may use heparosan as heparin precursor. Heparin may be produced by subjecting5 heparosan as a starting material to different steps comprising depolymerization, partial N- deacetylation, N-sulfation, C5-epimerization, 2-O-sulfation, 6-O-sulfation, 3-O-sulfation. Heparin may be produced by subjecting heparosan to one, several or all of these steps or a combination of some of these steps or all of these steps. The method for producing heparin may further comprise a depolymerization step. The implementation order of the steps in the heparin production process is not particularly limited, so long as heparin having desired properties can be obtained.

[0242] In the method disclosed herein for synthetizing heparin these steps can be performed chemically or enzymatically or can be a combination between chemically and enzymatically performed steps. An enzymatic step may be, for example, a N-sulfation,15 a 2-O-sulfation enzymatic step, a 3-O-sulfation enzymatic step, a 6-O-sulfation enzymatic step or a succession or a combination of these steps. Such an enzymatic step can be performed by using, for example, a N-sulfotransferase enzyme such as NDST1 or NDST2, an O-sulfotransferase (OST) enzyme such as, for example, 2-OST, 3-OST, 3-OST-1 , 3- OST-3, 6-OST, 6-OST-1 , 6-OST-3. An enzymatic step for synthetizing heparin according to20 the method disclosed herein may be performed by more than one sulfotransferase or by a combination between one or more sulfotransferases and another enzyme, for example, a 2-OST and a C5 epimerase. The method as disclosed herein comprises at least one step with one sulfotransferase, cyclo-PAPS and a cell free extract as disclosed herein.

[0243] A method for synthesizing heparin may comprise further additional enzymatically catalyzed reactions, e.g. an epimerization using a C5 epimerase.Characterization of heparin products

[0244] The nomenclature and structural symbols used in the present disclosure in relation to the characterization of heparin and heparin products are shown in Table 1 below.30

[0245] TABLE 1 : NOMENCLATURE AND STRUCTURAL SYMBOLSNomenclatureHexUA = Uronic acid IdoA = L-iduronic acidGlcA = D-glucuronic acid AHexUA = 4,5-unsaturated uronic acidGlcN = D-glucosamine ManN= D-MannosamineGlcNS1 6'anllydro=N-sulfated D-glucosamine with 1.6-anhydro ring ManNS1"""1'1'11" = N-sulfated D-Mannosamine with 1.6-anhydro ringNAc = \'-acetvl 2S = 2-O-sulfateNS = jV-sulfate 6S = 6-O-sulfateS = 3-O-sulfate aMan = 2,5-anhydro- D-MannoseGalA = D-galacturonic acid aMan-s = 2,5-anhydro- D-Mannose tagged with sulfanilic acid w / w = weight / weightStructural SymbolsIVaid = IdoA-GlcNAc IVagiu= GlcA-GlcNAcIVsld= IdoA-GlcNS IVsgiu = GlcA-GlcNSIIald= IdoA-GlcNAc(6S) IIagiu= GlcA-GlcNAc(6S) maid = IdoA(2S)-GlcNAc IVsgai= GalA-GlcNSIIsld= IdoA-GlcN(NS,6S) 11 sgiu= GlcA-GlcN (NS,6S)IIISM = IdoA(2S)-GlcNS HIsgiu= GlcA(2S)-GlcNS laid = IdoA(2S)-GlcNAc(6S) 11 sg;li= GalA-GlcN(NS,6S)Isid = IdoA(2S)-GlcN(NS,6S) Isgiu = GlcA(2S) -GlcN(NS,6S)Ilsgiu = GlcA-GlcN(NS,3S,6S) IIISM = IdoA(2S)-GlcN(NS,3S)Isld= IdoA(2S)-GlcN(NS,3S,6S) Isgiu = GlcA(2S)-GlcN(NS,3S,6S)IVsMan= IdoA-aMan IVs ^‘ln=GlcA-aMan lls^ln= IdoA-aMan(6S) IIs^“ = GlcA-aMan(6S)IIIs^an= IdoA(2S)-aMan IIIs^“ = IdoA(2S)-aManIVs±n= GlcA-aMan(3S) HIs^™ = IdoA(2S)-aMan(3S)Hs^n= GlcA-aMan(3S,6S) Is*}™ = IdoA(2S)-aMan(3S,6S)Is^an= GlcA(2S)-aMan(3S,6S)AlVa = AHexUA-GlcNAc AIVs = AHexUA-GlcNSAl Vsepi= AHexUA-ManNS Alla = AHexUA-GlcNAc(6S)Allla = AHexUA(2S)-GlcNAc Alls = AHexUA-GlcN(NS,6S)AIIIs = AHexUA(2S)-GlcNS AIIs^1= AHexUA-ManNS(6S)All I sc|:l= AHexUA(2S)-ManNS Afei e-anhydro = AHexUA(2S)-GlcNS1 6-alllydroAla = AHexUA(2S)-GlcNAc(6S) AIs = AHexUA(2S)-GlcN(NS,6S)AIsepi= AHexUA(2S)-ManNS(6S) AIs-Isid1 6'anllydro= AHexUA(2S)-GlcN(NS,6S)-IdoA(2S)-GlcNS1 6‘ anhy*0+AHexUA(2S)-GlcN(NS,6S)-IdoA(2S)-ManNS1 6'™llydroAlls = AHexUA-GlcN(NS,3S,6S) AIIIs = AHexUA(2S)-GlcN(NS,3S)AIs = AHexUA(2S)-GlcN(NS,3S,6S) IVsgai = GalA-GlcNSAIs-IdoAl'2S_) = AHexUA(2S)-GlcN(NS,6S)-IdoA(2S)Ilsgai = GalA-GlcN(NS,6S) Ulsid = IdoA(2S)-GlcNSIlsgiu = GlcA-GlcN (NS,6S) Isld= IdoA(2S)-GlcN(NS,6S)IVsgiu = GlcA-GlcNS fed = IdoA(2S)-GlcN(NS,3S,6S) fegiu = GlcA(2S)-GlcN(NS,3S,6S) IIISM = IdoA(2S)-GlcN(NS,3S)Ilsgiu = GlcA-GlcN(NS,3S,6S) IVseiu= GlcA-GlcN(NS,3S)When underlined, the symbol corresponds to the 3S GlcN derivative

[0246] Heparin may be digested into distinctive di-, tri- and tetra-saccharide building blocks groups and analysis performed by liquid chromatography -ultraviolet spectrometry (LC-LIV) method (see e.g. P. Mourier et al., Analytical Chemistry Research 3 5 (2015) 46-53).

[0247] As illustrated in the examples, this hydrolysis can be obtained enzymatically; with a mixture of three heparin lyases (heparinases I, II and III) or chemically with nitrous acid (HNO2). In some embodiments, the building blocks so obtained may be tagged, e.g. by sulfanilic acid as described by P. Mourier (2020, Molecules, 25(23), 5553).10

[0248] In particular, enzymatic digestion gives rise mainly to the following disaccharides and tetrasaccharides (see Table 1 above):

[0249] AlVa: [AUA-GIcNAc]

[0250] AIVs: [AUA-GIcNS]

[0251] Alla: [AUA-GlcNAc6S]15

[0252] Al I la: [AUA2S-GlcNAc]

[0253] Aills: [AUA2S-GlcNS]

[0254] Alls: [AUA-GlcNS6S]

[0255] Ala: [AUA2S-GlcNAc6S]

[0256] Als: [AUA2S-GlcNS6S]

[0257] AllaIVSgi,,

[0258] AllallSgiu

[0259] AlallSgiu

[0260] AlsllSgiu

[0261] AUA corresponds to 4-deoxy-a-L-threo-hex-4-enopyranosyl uronic acid; GlcN corresponds to D-glucosamine; Ac corresponds to acetyl and S corresponds to sulfo.

[0262] Based on this analytical method, the following structural features can be measured:

[0263] Sulfate / Carboxylate (S / C) ratio

[0265] Where x corresponds to building blocks, n^S03and n£00Hare respectively the numbers of sulfates and carboxyl.

[0266] % N-acetylated glucosamines

[0267] Acetyls % = 100

[0268] Where x corresponds to building blocks and n^00His the number of carboxyl; i corresponds to acetylated building blocks (AlVa, Alla, Allla, Ala, AllaIVSgi, ,. AllallSgiu, AlallSgiu).

[0269] % 2-OS y ■ A xn?~0H

[0270] % 2-OS = 100 - (2 - 0 / 7)% = 100 - 100 x ^^COOH

[0271] Where x corresponds to building blocks; i corresponds to building blocks containing 2-OH and nf~0His the number of 2-OH: one for AlVa, AIVsgai, AIVs, Alla, AllSgal, Alls, IlSglu, Alls, GIC(NS.6S)-HSg|„. AlsllSgiu, Alai ISglu 30d tWO for AllaIVSgi,,, AllallSgiu,

[0274] Where x corresponds to building blocks and n00His the number of carboxyl; i corresponds to building blocks containing one 6-OH (AlVa, AIVsgai, Al Vs, Al I la, Aills, AHIs, AllaIVSgh, Glc(NS.3S)).

[0275] % 3-OS

[0276] % 3-OS = 3 - OS % = 100

[0277] Where x corresponds to building blocks and n00His the number of carboxyl; i corresponds to 3-0 sulfated building blocks (Glc(NS,3S), Glc(NS,3S,6S), I lsai„, Als, Alls, Aills, Glc(NS,6S)-IISgiu, Al lalVsaiu, Allal lsai„, Alai lsaiu, Alsl lsaiu).

[0278] The present disclosure will be further understood from the following non-limiting examples. The following examples are provided to describe in detail some of the representative, presently preferred methods and materials of the present disclosure. These examples are provided for purposes of illustration of the inventive concepts and are not intended to limit the scope of the present disclosure as defined by the appended claims.[EXAMPLES]Example 1 : Preparation of an Escherichia coli Cell Free Extract (CFE) overexpressing enzymesMaterials & MethodsStrains, enzymes and plasmids

[0279] The strains, enzymes and plasmids used in the examples are shown in Table 2 below:

[0280] TABLE 2: STRAINS, ENZYMES AND PLASMIDS

[0281] The coding sequences of the enzymes were cloned in the corresponding plasmids and introduced in the corresponding hosts.Culture conditions

[0282] The host cells were grown in an appropriate culture medium according to the provider’s instructions, supplemented with the inducers and selective markers as indicated in Table 2, leading to the maintenance of the plasmids and the expression of the enzymes.Extract preparation

[0283] The cultured cells so harvested were submitted to the following protocol:

[0284] - Suspension of wet cells in 4 volumes of lysing buffer (20 mM TRIS,200 mM NaCI pH 7,5)

[0285] - Lysis in high-pressure homogenizer at 29000 psi

[0286] - Centrifugation at 25000 g for 1 h at 4 °C

[0287] - Microfiltration over 0.2 .m membrane

[0288] - Addition of glycerol to obtain 15 wt% glycerol solution

[0289] - Dispatch in different bottles / tubes

[0290] - Storage at -80 °C

[0291] The cell free extract (CFE) so obtained contains the overexpressed enzymes as well as the native enzymes of the host cells.

[0292] CFE’s were used as such in reactions without determination of their activity. The required CFE volume to reach the desired conversion was determined by performing an use-test.Enzyme purification

[0293] When needed, the overexpressed enzymes fused to a tag are easily purified by affinity chromatography using the appropriate column: in case the protein is fused to a His tag, it can be purified using a His tag column; when the protein is a MBP (Maltose Binding Protein) protein fusion, it can be purified using an amylose resin.Example 2: Identification of an activity in the CFE able to convert cyclo-PAPS into PAPS

[0294] To investigate the reactive species in the reaction mixtures containing purified enzymes or CFE’s, 10 mM of cyclo-PAPS was allowed to stand for 6h in the presence of purified 6-OST or 6-OST CFE. The mixtures were analyzed by HPLC to evaluate the fate of cyclo-PAPS in the two cases.Materials & Methods

[0295] The experimental set-up is shown in Table 3 below:

[0296] TABLE 3: REACTION SET-UP

[0297] Cyclo-PAPS was stirred in 1 .5 mL Eppendorf tubes at 700 rpm in the presence of MES-buffer and NaCI at 37°C for 6h in in Eppendorf Thermomixer C. Purified enzyme or CFE was added and after 0, 1 , 2, 4 and 6h, 50 pL samples were taken and treated at 95 °C for 5 min. After centrifugation at 10000 rpm for 5 min, 20 pL of supernatant was diluted with 180 pL of MES buffer and the analyzed by IPC / HPLC.HPLC analysis

[0298] The chromatographic conditions are as shown in Table 4 below:

[0299] TABLE 4: CHROMATOGRAPHIC CONDITIONS

[0300] The HPLC chromatograms of the samples of cyclo-PAPS in the presence of purified 6-OST enzyme is illustrated in Figure 1A. No significant conversion of cyclo-PAPS (major peak) occurs.

[0301] However, upon addition of the 6-OST CFE (Figure 1B), it can be clearly seen that almost immediately cyclo-PAPS is converted into PAPS and that PAPS itself remains stable even after 6 hours (Figure 1C).

[0302] It can be concluded that cyclo-PAPS can be converted to PAPS by native enzymes that are present in the Cell Free Extract from E. coli. Cyclo-PAPS could then be used as a sulfate donor in biocatalytic sulfation reactions, with the possibility to convert it in situ, in the presence of a cell free extract possibly containing further enzymes involved in said sulfation reactions.Example 3: Use of cyclo-PAPS as a sulfate donor in the presence of CFE for the sulfation of synthetic Oligosaccharides substrates

[0303] In order to develop a reliable and rapid test to assess 2-OST, 6-OST and 3-OST activity in cell lysate, alternative model substrates have been used. These substrates are synthetic NS-octasaccharides with a PNP tag, commercialized by Glycan Therapeutics, which have a 300 nm maximal absorption.

[0304] Each enzymatic block has a different substrate copying the sulphated product of the previous block. Their reactions were followed by HPLC with UV detector to directly assess their activity in CFE.3.1 / 2-O-sulfotransferaseMaterials & MethodsSubstrate

[0305] The 2-O-sulfation reaction on the used substrate (GT24-PP-011 ; NS- octasaccharide with PNP tag) is detailed below:Reaction setup

[0306] The samples composition is detailed in Table 5 below:TABLE 5: REACTION SET-UP

[0307] The reagents were added in the order of the table to 0.5 mL Eppendorf tubes in order to reach a final volume of 210 pL. Before adding the substrate, the solution was stirred at 37°C and 700 rpm for 10 minutes. The reaction started upon addition of the substrate and was conducted at 37°C and 700 rpm for 2 hours. 40 pL samples were collected every 30 minutes and diluted in 160 pL of demi-water. They were heated at 95°C, 700 rpm for 5 minutes to deactivate the enzymes, and centrifuged. The supernatants were collected.Analysis method

[0308] The analysis method is described in Table 6 below: TABLE 6: ANALYTICAL METHOD FOR 2-OST ACTIVITY TESTResults

[0309] The chromatograms are integrated manually, with a reject area of 1.0, and the data are collected in the Excel sheet. The relative amount of each product and the total 2-O-sulfation yield are calculated with the following formulas:

[0310] O / Oi=ASruemas(sAui)m

[0311] %2 — 0 — sulfation = %mono+ 2 x %di+ 3 x %tri

[0312] With i standing for each type of product: mono-sulfated, di-sulfated and tri-sulfated.

[0313] The kinetics of the reactions are shown in Figure 2.

[0314] The kinetics in Figure 2 show the substrate GT24-PP-011 was sulfated in samples 1 and 3 (CFE 2-OST with cyclo-PAPS and purified 2-OST with PAPS) as expected. It was observed that the slope of sample 1 is 4 times higher than the slope of sample 3, meaning that the purified 2-OST are 4 times more active than the CFE 2-OST. This could be explained by a concentration of the enzyme during the purification.

[0315] However, it was not sulfated in sample 3 (purified 2-OST with cyclo- PAPS). This results shows the need of E.coli native enzymes contained in the CFE to transform cyclo-PAPS into PAPS.

[0316] Thus, it was demonstrated that the synthetic substrate GT24-PP-011 can be 2-O-sulfated. Moreover, cyclo-PAPS can be used as a sulfate donor for this reaction only in presence of CFE 2-OST.3.2 / 6-O-sulfotransferaseMaterials & MethodsSubstrate

[0317] The 6-O-sulfation reaction on the used substrate (GT24-PP-019; NS- octasaccharide with PNP tag) is detailed below:Reaction setup

[0318] The samples composition is detailed in Table 7 below:

[0319] TABLE 7: REACTION SET-UP

[0320] The reagents were added in the order of the table to 0.5 mLEppendorf tubes in order to reach a final volume of 210 pL. Before adding the substrate, the solution was stirred at 37°C and 700 rpm for 10 minutes. The reaction started upon addition of the substrate and was conducted at 37°C and 700 rpm for 20 minutes. 40 pLsamples were collected every 4 minutes and diluted in 160 pL of demi-water. They were heated at 95°C, 700 rpm for 5 minutes to deactivate the enzymes, and centrifuged. The supernatants were collected.Analysis method

[0321] The slight differences versus the conditions of Table 6 are shown in Table 8 below:

[0322] TABLE 8: ANALYTICAL METHOD FOR 6-OST ACTIVITY TEST

[0323] The chromatograms are integrated manually, with a reject area of 1.0, and the data are collected in the Excel sheet. The relative amount of each product and the total 6-O-sulfation yield are calculated with the following formulas:

[0324] %t = ASruemas(sAui)m

[0325] %6 — 0 — sulfation = %mono+ 2 x %di+ 3 x %tri

[0326] With i standing for each type of product: mono-sulfated, di-sulfated and tri-sulfated.

[0327] The kinetics of the reactions are shown in Figure 3.

[0328] The kinetics in Figure 3 show that the substrate GT24-PP-019 was sulfated in samples 1 and 3 (CFE 6-OST with cyclo-PAPS and purified 6-OST with PAPS). It was observed that the purified 6-OST with PAPS sample’s slope is 5 times higher than the CFE 6-OST with cyclo-PAPS sample’s one. This corresponds to the enzyme dilution in the samples (1:1000 for CFE 6-OST while the purified 6-OST was only diluted by 200).

[0329] The substrate was lightly sulfated in sample 2 (purified 6-OST with cyclo-PAPS) because cyclo-PAPS contains a small amount of PAPS. This indicates that the cyclo-PAPS was not transformed into PAPS by the purified enzymes.

[0330] Thus, it was demonstrated that the synthetic substrate GT24-PP-019 can be 6-O-sulfated. Moreover, cyclo-PAPS can be used as a sulfate donor for this reaction only in presence of CFE 6-OST.3.3 / 3-O-sulfotransferaseMaterials & MethodsSubstrate

[0331] The 3-O-sulfation reaction on the used substrate (GT24-PP-022; NS- octasaccharide with PNP tag) is detailed below:Reaction setup

[0332] The samples composition is detailed in Table 9 below:

[0333] TABLE 9: REACTION SET-UP

[0334] The reagents were added in the order of the table to 0.5 mL Eppendorf tubes in order to reach a final volume of 210 pL. Before adding the substrate, the solution was stirred at 37°C and 700 rpm for 10 minutes. The reaction started upon addition of the substrate and was conducted at 37°C and 700 rpm for 20 minutes. 40 pL samples were collected every 4 minutes and diluted in 160 pL of demi-water. They were heated at 95°C, 700 rpm for 5 minutes to deactivate the enzymes, and centrifuged. The supernatants were collected.Analysis method

[0335] The slight differences versus the conditions of Table 6 are shown in Table 10 below:

[0336] TABLE 10: ANALYTICAL METHOD FOR 3-OST ACTIVITY TEST

[0337] The chromatograms are integrated manually, with a reject area of 1.0, and the data are collected in the Excel sheet. The relative amount of each product and the total 3-O-sulfation yield are calculated with the following formulas:

[0338] O / Oi=Sum A°Areas sum

[0339] %3 — 0 — sulfation = %mono+ 2 x %di+ 3 x %tri

[0340] With i standing for each type of product: mono-sulfated, di-sulfated and tri-sulfated.

[0341] The kinetics of the reactions are shown in Figure 4.

[0342] The kinetics in Figure 4 show that the substrate GT24-PP-022 was sulfated in samples 1 and 3 (CFE 3-OST with cyclo-PAPS and purified 3-OST with PAPS). It was observed that the purified 3-OST with PAPS sample’s slope is 7 times higher than the CFE 3-OST with cyclo-PAPS sample’s one, meaning the purified 3-OST are more active than the CFE 3-OST.

[0343] The substrate was lightly sulfated in sample 2 (purified 3-OST with cyclo-PAPS) because cyclo-PAPS contains a small amount of PAPS. This indicates that the cyclo-PAPS was not transformed into PAPS by the purified enzymes and confirms the need of E. coli native enzymes to do so.

[0344] Thus, it was demonstrated that the synthetic substrate GT24-PP-022 can be 3-O-sulfated. Moreover, cyclo-PAPS can be used as a sulfate donor for this reaction only in presence of CFE 3-OST.Conclusions

[0345] Synthetic oligosaccharides with defined configuration are good substrates for sulfation using cyclo-PAPS and OST CFE’s.

[0346] It has been clearly shown that cyclo-PAPS itself is not an active sulfate donor that can be directly used with O-sulfotransferases. However, in the presence of native enzymes that are present in E. coli CFE’s, cyclo-PAPS is transformed to PAPS which is the normal sulfate donor for these enzymes.Example 4: Use of cyclo-PAPS as a sulfate donor in the presence of CFE for the sulfation of Porcine Lung Heparin

[0347] The porcine lung heparin is low in 3-O-sulfation. The goal is to increase this ratio with the use of CFE 3-OST and cyclo-PAPS.Materials & MethodsReaction setup

[0348] The samples composition is detailed in Table 11 below:TABLE 11 : REACTION SET-UP

[0349] The reagents were added in the order of the table to 2 mL Eppendorf tubes in order to reach a final volume of 1 .0 mL. Before adding the enzymes, the solutions’ pH were adjusted to pH 7.0, and they were stirred at 37°C and 700 rpm for 10 minutes. The reaction started upon addition of the enzymes and was conducted at 37° C and 700 rpm for 24 hours. 100 pL samples were collected after 2, 4, 7.3 and 24 hours. They were heated at 95°C, 700 rpm for 5 minutes to deactivate the enzymes, and centrifuged. The supernatants were collected.Analysis method

[0350] The samples were diluted with 100 mM Potassium Phosphate buffer pH 7.0 to reach a 0.2 g / L substrate concentration. 75 pL of this solution was mixed with 10 pL of 100 mM Potassium Phosphate buffer pH 7.0, 10 pL of heparinase I (0.4 lU / mL), 10 pL of heparinase II (0.4 lU / mL) and 10 pL of heparinase III (0.4 lU / mL). The solutions were stirred at 25°C and 700 rpm for 18 hours in an Eppendorf thermomixer C. The building blocks were analyzed by HPLC as disclosed by P. Mourier et al. (Analytical Chemistry Research 3 (2015) 46-53) and explained above. The limit of quantification of this method is 0.1% w / w.Results

[0351] The kinetics of the reactions are shown in Figure 5.

[0352] The kinetics demonstrate that purified enzymes cannot conduct the sulfation reaction with cyclo-PAPS. On the contrary, the use of PAPS Li with purified enzymes resulted in a 8.1% 3-O-sulfated product. The assay using cyclo-PAPS with CFE’s (8.9% of 3-O-sulfation) has similar kinetics than the PAPS Li and purified enzymes assay.

[0353] These results confirm the hypothesis that cyclo-PAPS can be used for sulfation reaction only with CFE’s and not with purified enzymes.

[0354] The building block analysis after enzymatic digestion followed by sulfanilic tagging is summarized in in Table 12 below:

[0355] TABLE 12: BUILDING BLOCK ANALYSIS AFTER ENZYMEDIGESTION AND SULFANILIC TAGGING

[0356] As seen in the kinetics graph (Figure 5), the second assay (purified enzymes and cyclo-PAPS) did not give any reaction, whereas the two other assays reached 8.9% and 8.1% of 3-O-sulfation. At the end of the day, it is possible to generate a modified porcine heparin from lung origin which is comparable or even higher to the reference porcine mucosal heparin in terms of 3-O-sulfatation (8.7% versus 4.9%). It then illustrates the possibility to give activity to a non active heparin, by a single enzymatic reaction.Example 5: Use of cyclo-PAPS as a sulfate donor in the presence of CFE for the sulfation of Bovine Intestinal Heparin

[0357] The main structural specificity of Bovine Intestinal Heparin (BIH) is its low 6-0 sulfation (=60-65%) when compared to PMH (Porcine Mucosa Heparin). It gives to this heparin a considerable structural diversity and most of all, it explains its rather low activity (anti-FI la around 100 Ul / mg and ratio anti-FXa I anti-Flla close to 1).

[0358] This is why there is a high interest to “correct” the deficiencies of BI H by adequate enzymatic sulfation. The first and most important step will be obviously a 6OST in order to increase the 6-0 sulfation approximately up to the level of PMH. The secondenzymatic step will be 3-0 sulfation, but logically, the first 6-0 sulfation should be sufficient to increase significantly the activity.5.1 / BLOCK 3 (6-OST)

[0359] The BIH is low in 6-0-sulfation. The goal is to increase this ratio with the use of CFE 6-OST and cyclo-PAPS.

[0360] The sample composition is detailed in Table 13 below:

[0361] TABLE 13: REACTION SET-UP

[0362] The sample has been treated as in example 4.

[0363] The analysis of the samples is summarized in in Table 14 below:

[0364] TABLE 14: BUILDING BLOCK ANALYSIS AFTER ENZYMEDIGESTION AND SULFANILIC TAGGING, AND ANTICOAGULANTACTIVITIES MEASUREMENT (USP method)5.2 / BLOCK 4 (3-OST)

[0365] The impact of 3-O-sulfation on 6-0 sulfated BIH has been tested using a CFE 3-OST and cyclo-PAPS.

[0366] The sample composition is detailed in Table 15 below:

[0367] TABLE 15: REACTION SET-UP

[0368] The sample has been treated as in example 4.

[0369] The analysis of the samples is summarized in Table 16 below:

[0370] TABLE 16: BUILDING BLOCK ANALYSIS AFTER ENZYME DIGESTION AND SULFANILIC TAGGING, AND ANTICOAGULANT ACTIVITIES MEASUREMENT (USP method)

[0371] These data conform that the degree of 6-0 sulfation as well as the degree of 3-0 sulfation of a bovine intestinal heparin (BIH) can be modified using a CFE expressing the proper enzyme and cyclo-PAPS.Example 6: Use of cyclo-PAPS as a sulfate donor in the presence of CFE for Bioengineered Heparin synthesis6.1 / BLOCK 2 (2-OST+C5-epimerase)

[0372] Heparosan was submitted to depolymerization / deacylation and N- sulfonation, as known in the art.

[0373] The NS-heparosan so prepared was then submitted to enzymatic reactions with 2-OST and C5-epimerase in the presence of cyclo-PAPS as a sulfate donor.Materials & MethodsReaction setup

[0374] The sample composition is detailed in Table 17 below:

[0375] TABLE 17: REACTION SET-UP

[0376] After 24h of reaction, the 2-O-sulfation degree was below the targeted 70%. Therefore, after treating the reaction mixture as in example 4, the obtained product was allowed to react under the conditions shown in Table 18 for 31 h.

[0377] TABLE 18: REACTION SET-UPAnalysis method

[0378] The sample has been treated as in example 4.Results

[0379] The building block analysis after enzymatic digestion is summarized in in Table 19 below:

[0380] TABLE 19: BUILDING BLOCK ANALYSIS AFTER ENZYMATICDIGESTION6.2 / BLOCK 3 (6-OST)

[0381] The NS.2S-epimerized-heparosan so prepared was then submitted to enzymatic reaction with 6-OST in the presence of cyclo-PAPS as a sulfate donor.

[0382] The sample composition is detailed in Table 20 below:

[0383] TABLE 20: REACTION SET-UP

[0384] The building block analysis after enzymatic digestion is summarized in in Table 21 below:

[0385] TABLE 21 : BUILDING BLOCK ANALYSIS AFTER ENZYMATICDIGESTION

[0386] The sample analysis, e.g. by enzymatic digestion, confirmed the obtention of a NS.2S.6S-epi-heparosan having a degree of 6-O-sulfation in the expected ranges (from 80 % to 95%).6.3 / BLOCK 4 (3-OST)

[0387] The NS.2S.6S-epi-heparosan so prepared was then submitted to enzymatic reactions with 3-OST in the presence of cyclo-PAPS as a sulfate donor.Reaction setup

[0388] The sample composition is detailed in Table 22 below:

[0389] TABLE 22: REACTION SET-UP

[0390] The NS.2S.6S.3S-epi-Heparosan so obtained was analyzed after enzymatic digestion followed by sulfanilic tagging, as disclosed above. The results are depicted in Table 23 below:

[0391] TABLE 23: BUILDING BLOCK ANALYSIS AFTER ENZYME DIGESTION AND SULFANILIC TAGGING

[0392] The NS.2S.6S.3S-epimerized-Heparosan so obtained was submitted to purification procedures well known in the art.

[0395] These data show that the bioengineered heparin so obtained has the main characteristics of the porcine mucosa heparin, at least in terms of anti-coagulant activity anti-FXa and anti-FI la.6.5 / LMWH Bioengineered Heparin sourced production

[0396] The heparin so obtained was submitted to the enoxaparin synthesis process well known in the art (see e.g. US5,389,618).

[0397] The resulting product, i.e. low molecular weight heparin (LWMH), was analysed for its main characteristics (molecular weight and anti-coagulant activity) as shown in Table 25, as well as for its building block composition after enzymatic digestion as shown in Table 26 below.

[0398] TABLE 25: COMPARISON WITH ENOXAPARIN SPECIFICATIONS

[0399] TABLE 26: BUILDING BLOCK ANALYSIS AFTER ENZYMEDIGESTION AND SULFANILIC TAGGING

[0400] These data show that the product so obtained using cyclo-PAPS and CFE has a comparable analytical profile as enoxaparin and meets the LMWH targets.[REFERENCES]Cherniak R, Davidson EA. Synthesis of Adenylyl Sulfate and Adenylyl Sulfate 3'- Phosphate. J. Biol. Chem. 1964 ;239(9) :2986-2990Collier A, Wagner G. A facile two-step synthesis of 8-arylated guanosine mono- and triphosphates (8-aryl GXPs). Org. Biomol. Chem. 2006;4:4526-4532Esko JD, Selleck SB. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem. 2002;71 :435-471. doi: 10.1146 / annurev. biochem .71.110601.135458Fu L, Suflita M, Linhardt RJ. Bioengineered heparins and heparan sulfates. Adv Drug Deliv Rev. 2016;97:237-249. doi:10.1016 / j.addr.2015.11.002Haas TM, Ebensperger P, Eisenbeis VB, Nopper C, Durr T, Jork N, Steck N, Jessen-Trefzer C, Jessen HJ. Magic spot nucleotides: tunable target-specific chemoenzymatic synthesis. Chem. Commun. 2019;55:5339-5342Inoue H, Baba Y, Tsuhako M. Phosphorylation of Adenosine 5’-monophosphate (5’-AMP) with cyclo-triphosphate and purification of the phosphorylated products. Phosphorus Research Bulletin. 1995;5:137-142Liu J, Thorp SC. Cell surface heparan sulfate and its roles in assisting viral infections. Med Res Rev. 2002;22(1):1-25. doi: 10.1002 / med.1026Mourier P. Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid. Molecules 2020;25(23):5553Mourier P, Anger P, Martinez C, Herman F, Viskov C. Quantitative Compositional Analysis of Heparin Using Exhaustive Heparinase Digestion and Strong Anion Exchange Chromatography. Analytical Chemistry Research 2015;3:46-53Pagotto F, Salimnia H, Dillon JAR, Totten PA. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication. Gene 2000;244:13-19Sekura, R.

[0053] Adenosine 3'-phosphate 5'-phosphosulfate. Methods in Enzymology 1981 ;77:413-415US 4,169,011 Facile synthesis of 3’-phosphoadenosine 5’-phosphosulfate (PAPS)US 5,389,618 Mixtures of particular LMW heparinic polysaccharides for the prophylaxis / treatment of acute thrombotic events

Claims

1. [CLAIMS]1. Use of adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) as a primary sulfate donor in sulfotransferase catalyzed reactions.

2. Use of cyclo-PAPS and a cell free extract for in situ generating 3'- phosphoadenosine-5'-phosphosulfate (PAPS).

3. Use of a cell free extract for in situ generating 3'-phosphoadenosine-5'- phosphosulfate (PAPS) from adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo- PAPS).

4. A method of in situ preparing PAPS comprising incubating cyclo-PAPS with at least one cell free extract in a reaction medium.

5. A method of sulfating a substrate comprising incubating in a reaction medium cyclo-PAPS, at least one cell free extract, the substrate to be sulfated and an enzyme involved in the sulfation of the substrate.

6. Use according to claims 2 to 3 or a method according to claims 4 to 5, wherein the cell free extract contains at least one enzyme able to convert cyclo-PAPS into PAPS.

7. Use according to claim 6 or a method according to claim 6, wherein the cell free extract is from a prokaryotic or eukaryotic cell, optionally a bacterial, yeast, plant, or animal cell.

8. Use according to claim 7 or a method according to claim 7, wherein the cell endogenously produces at least one enzyme able to convert cyclo-PAPS into PAPS.

9. Use according to claim 8 or a method according to claim 8, wherein the cell is Escherichia coli and endogenously produces at least one enzyme selected in the group consisting of: 2’,3’-cyclic-nucleotide 2’-phosphodiesterase / 3’-nucleotidase (cpdB), acid phosphatase (aphA) and 3’(2’),5’-biphosphate nucleotidase (cysQ).

10. Use according to any of claims 2 to 3 and 6 to 9 or a method according to any of claims 4 to 9, wherein the cell free extract further contains at least an enzyme involved in the sulfation of a substrate.

11. Use according to claim 10 or a method according to claim 10, wherein the enzyme involved in the sulfation of a substrate is a sulfotransferase, optionally selected in the group consisting of SULT1A1 , SULT1A2, SULT1A3, SULT1 B1 , SULT1C1 , SULT1C2, SULT1 E1 , SULT2A1 , heparan sulfate D-glucosaminyl 3-O-ST-3a and b, heparan sulfate D- glucosaminyl 3-O-ST-1 , heparan sulfate iduronic acid 2-O-ST, heparan sulfate D-glucosaminyl 6-O-ST, tyrosylprotein ST, / V-acetylglucosamine 6-O-ST, chondroitin sulfate / V-galactosamine 4-O-ST-1 , 2, and 3, chondroitin sulfate / V-galactosamine 6-O-ST, HNK-1 glucuronic acid 3-0- ST, / V-acetylgalactosamine 4-O-ST, dermatan sulfate / V-galactosamine 4-O-ST, keratan sulfate galactose 6-O-ST and combinations thereof.

12. Use according to claim 10 or 11 , or a method according to claim 10 or 11 , wherein the enzyme is involved in heparin production, optionally selected in the group consisting of a heparan sulfate 3-O-sulfotransferase, a heparan sulfate 2-O-sulfotransferase, a heparan sulfate 6-O-sulfotransferase, a C5-epimerase and combinations thereof.

13. Use according to any of claims 10 to 12 or a method according to any of claims 10 to 12, wherein the enzyme is recombinantly expressed by the cell.

14. A method according to any of claims 5 to 13, wherein the substrate is chosen in the group consisting of synthetic oligosaccharides such as NS-octasaccharide, (aryl) p- Nitrophenol, heparosan, NS-heparosan, NS.2S-epimerized-heparosan, NS.2S.6S- epimerized-heparosan and heparin, optionally porcine lung heparin and bovine intestinal heparin.

15. A method according to any of claims 5 to 13, wherein the substrate is selected in the group consisting of phenols, catecholamines, thyroid hormones, aryl hydroxylamines, hydroxysteroids, CCR-5, PSGL-1 , sialyl Lewis, chondroitin sulfate, keratan sulfate, HNK-1 sulfate, saccharide receptor, units on lutropin, thyrotropin, pro-opiomelanocortin and dermatan sulfate.

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