[0001 ] The invention relates to the field of synthetic chemistry. More particularly, this invention relates to a method of N- or O-sulfation of a compound.

BACKGROUND TO THE INVENTION

[0002] Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0003] Sulfation reactions have been used for the synthesis of a wide variety of sulfated materials including sulfated surfactants, sulfated glycosaminoglycans (GAG) and GAG mimetics as well as sulfated polysaccharides generally, including Fondaparinux Sodium and Pentosan Sulfate. A number of sulfated compounds are undergoing clinical trials for the treatment of cancers, including heterogeneous phosphomannopentaose sulfate, as described in WO 1996/033726, and homogeneous sulfated polyglucoside, as described in WO 2009/049370. The preparation of ursodeoxycholic acid di-sodium 3,7-disulfate is also described in WO 2004/092193.

[0004] The O-sulfation of hydroxyl groups of sulfation precursors generally involves the use of a sulfur trioxide trimethylamine complex (SO ₃ .TMA) or sulfur trioxide triethylamine complex (SO ₃ .TEA) in DMF and heating of the reaction mixtures at 50-60 °C (Al-Horani et al., Tetrahedron 2010, 66 (16), 2907-2918). Alternatively, O-sulfation with a sulfur trioxide pyridine complex (S0 ₃ .Py) using either pyridine or DMF as a solvent has been used in some cases.

[0005] Direct O-sulfation under microwave-assisted conditions have been described by Maza et al, Tetrahedron Lett. 2011 , 52 (3), 441 -443 and in WO2012035188. Per-O-sulfation of multiple hydroxyl groups or partial-O- sulfation of protected precursors usually require very long reaction times of hours to days, an excess of the sulfating reagent and extremely dry conditions. For subsequent work-up and purification procedures, most manufacturing processes include the often difficult removal of solvent, dialysis, and even size- exclusion chromatography (SEC). Additionally, either incomplete sulfation or post-reaction treatments can result in sub-optimal yields of the final product (Usov et al., Carbohydr. Res. 1971 , 18 (2), 336-338; Polat et al., J. Am. Soc. Chem. 2007, 129 (42), 12795-12800; Yu et al., Eur. J. Org. Chem. 2002, 37 (10), 783-791 ).

[0006] Further, WO 2015/01 1519 describes a process for the production of Fondaparinux sodium and discloses a sulfation step using a sulfur trioxide- amine complex, such as SO3.TMA or SO3.TEA. This is stated to be a preferred approach to the use of SOsPyr which led to impurities including desulfation products. The sulfation step occurs in the presence of DMAc.

[0007] Therefore, both conventional and microwave-assisted sulfation procedures have demonstrated a number of potential disadvantages including unpredictable reaction times at different scales, uncontrolled reversible desulfation, sub-optimal yield, high usage of sulfating reagent and tedious purification. For example, microwave-assisted sulfation approaches generally rely on a higher temperature reaction which has been shown to destabilize both O- and /V-sulfate functionalities on sulfated molecules. Such reversible desulfation side-reactions occurring at high temperature (100 °C) under microwave irradiation are neither desirable nor reliable for large-scale work.

[0008] A further approach is described in W02003020735, which discloses the sulfation of low molecular weight saccharides derived from glycosaminoglycans using a sulphating reagent within a dipolar aprotic solvent selected from pyridine, pyridine-DMF, and pyridine-DMSO. This method is said to provide a more efficient and high-yielding process when compared to prior art methods (Petitou et al., U.S. Patent No. 5,013,724) which require a conversion of the glycosaminoglycan into an organic amine salt before sulfation. Nonetheless, it still suffers from the requirement for tedious work up and solvent removal procedures.

[0009] It would therefore be desirable to provide for an improved method of sulfation which avoids or reduces the impact of at least one of the drawbacks of the prior art.

SUMMARY OF INVENTION

[0010] According to a first aspect of the invention, there is provided a method of N- or O-sulfation of a compound, including the steps of:

(a) dissolving the compound in a co-solvent system wherein, once dissolved, the co-solvent system comprises of at least one participating component and at least one non-participating component; and

(b) contacting the compound with a sulfating reagent; to thereby sulfate the compound.

[001 1 ] Suitably, the compound comprises at least one of a nitrogen- containing functional group or an oxygen-containing functional group.

[0012] In one embodiment, the nitrogen-containing functional group is selected from an amine, amide, sulfonamide, imine and /V-oxide. It is highly preferred that the nitrogen-containing functional group is an amine.

[0013] Suitably, the nitrogen-containing functional group is an -Nhh group.

[0014] Preferably, the oxygen-containing functional group is hydroxyl.

[0015] Most preferably, the compound displays one or more free hydroxyl groups for sulfation.

[0016] In certain embodiments, the at least one participating component may be an electron donor molecule and/or a polar aprotic compound. [0017] In embodiments, the at least one participating component may be selected from the group consisting of heterocycles, formamides, phosphoramides, sulfoxides, anilides, and ethers.

[0018] Suitably, when the at least one participating component is a heterocycle it is selected from oxygen and/or nitrogen-containing five to seven- membered heterocycles which may optionally be substituted or fused with one or more further five to seven-membered rings which may themselves be heterocyclic, carbocyclic, aryl or heteroaryl.

[0019] In one embodiment, the at least one participating component may be selected from the group consisting of A/,A/-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), morpholine, /V-formylmorpholine, /V-formylpiperidine, N- methylformanilide, 1 -formylpyrrolidine, 2-bromopyridine, and hexamethylphosphoramide (HMPA).

[0020] In certain embodiments, the at least one non-participating component may be a non-polar aprotic compound.

[0021 ] In embodiments, the at least one non-participating component may be selected from the group consisting of haloalkyl, nitrile, nitroalkyl, heterocyclic, alcohol, aqueous alcohol, water, ester and optionally substituted aryl

[0022] In one embodiment, the at least one non-participating component may be selected from the group consisting of 1 ,2-dichloroethane (DCE), dichloromethane (DCM), chlorobenzene, toluene, methanol, ethanol, water, methanol/water blends, acetonitrile, acetone, nitromethane, tetrahydrofuran, chloroform, 1 ,2,3,4-tetrahydro-naphthalene, trans- 1 ,2-dichloroethylene, c/s-1 ,2- dichloroethylene, xylenes, butyl acetate and ethyl acetate.

[0023] It will be understood that the at least one participating component and at least one non-participating component are both present when sulfation of the compound occurs. [0024] The method may further include the step of precipitating the N- or O- sulfated compound from the co-solvent system.

[0025] The precipitating may be triggered by addition of further amounts or non-participating solvent which may be the same or different to the non participating solvent used in the sulfation reaction.

[0026] The N- or O-sulfated compound may be present as an amine salt at the point of precipitation from the co-solvent system. That is, the sulfated compound of step (b) may be precipitated from the co-solvent system as an amine salt.

[0027] According to a second aspect of the invention there is provided an N- or O-sulfated compound when synthesised by the method of the first aspect.

[0028] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections as appropriate.

[0029] Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures wherein :

[0031 ] FIG 1 A is a graphical representation demonstrating S-N bond dissociation of a SO3.TMA complex in the presence of A/,A/-dimethylformamide as a participating component;

[0032] FIG 1 B shows real-time ¹ FI NMR detection of dissociation of the S-N bond demonstrated in FIG 1 A; a mixture of S03.Me3N (6.95 mg, 50 mitioI) and DMF (1 1 .5 mI_, 150 mitioI) with CD ₃ CN (0.5 ml_) in 5 mm NMR tube was sonicated for 1 min and subsequently measured in ¹ FI NMR at 50 ^° C. For the control experiment, S03.Me3N remained intact in the absence of DMF at 50 ^° C for 24 h;

[0033] FIG 2A is a graphical scheme regarding a postulated mechanism for O-sulfation according to the present invention;

[0034] FIG 2B is a general scheme postulating the role of the participating/non-participating components in the present sulfation method as applied to /V-sulfation; and

[0035] FIG 3 shows ¹ FI-NMR monitoring of sulfation of methyl a-D-glucoside performed in neat DMF at 60 ^° C.; the experimental procedure was to place SO ₃ .TMA (1 .5 eq per OFI, 3.6 mmol, 500 mg) and glycoside (129 mg, 0.6 mmol) in 2-necked round flask, the flask was charged with Ar balloon and degassed for 3 times after which time 30 ml_ of anhydrous DMF was added into the flask and the resulting reaction was heated at 60 ^° C and monitored via ¹ FI NMR at different time courses.

DETAILED DESCRIPTION OF THE DRAWINGS

[0036] The present invention is predicated, at least in part, on the finding that the desulfation of sulfated product in a reaction mixture may be reduced, the work up procedure may be greatly simplified and the overall yield of product may be improved by the use of a co-solvent system comprising a participating component and a non-participating component during the sulfation reaction. Without wishing to be bound by theory, it is believed that the participating component, for example DMF as is commonly used as solvent in a standard sulfation reaction, interacts with the sulfating reagent to improve reactivity and help achieve the sulfated product and then solubilises this product. Flowever, DMF can be difficult, expensive and time-consuming to remove in the subsequent work up. Further, especially under heating to remove the DMF or during extended reaction times, it has been found that significant levels of desulfation can occur. The present invention employs the use of a non participating component which is postulated to assist in actively removing or sequestering the participating component away from the sulfated product. This has benefits in decreasing the likelihood or extent of desulfation of the sulfated product and, importantly, can result in precipitation of the sulfated product thereby allowing for simple collection.

[0037] This theory is graphically represented in FIG 2A, and the role of the participating and non-participating components is further set out in in relation to /V-sulfation in FIG 2B, which demonstrates the sulfation of a generic hydroxyl- containing organic compound (R-OFI) using SO3.W (where W could be, for example, TMA) with e.g. DMF (represented as X in FIG 2A) as the participating component and e.g. DCE (represented as Y in FIG 2A) as the non-participating component. In FIG 2B it can be seen that the non-participating component may be, for example, MeOH and water. The postulated mechanism involves the replacement of the trimethylamine portion (W) of the sulfating reagent with DMF (X) to form a more reactive sulfating agent (as perFIG 2A). This reacts with the R-OH group to form the sulfated product as shown in FIG 2A. It is the presence of the non-participating component, such as DCE, which, due to its miscibility with the participating component, sequesters the participating component and encourages the sulfated product to precipitate out of the co-solvent system. In the example shown, the sulfated product precipitates out as the amine salt.

[0038] As used herein, the term “participating component’ refers to a component of a co-solvent system which is actively involved in the sulfation reaction. Specifically, the term is used for a component of the co-solvent system which forms a complex with the SO3 component of the sulfating reagent during the sulfation reaction to form a more reactive sulfating intermediate. For example, in the reaction shown in FIG 2A the participating component forms a complex with the SO3 component of the SO3.TMA sulfating reagent during the sulfation reaction. Whether or not a given molecule is acting as a participating component can be ascertained in the following manner. FIG 1 B indicates a reliable way in which the continuing dissociation of the S-N bond (generically indicated in FIGs 2A and 2B as S-W) of the sulfating complex in the presence of the participating component can be monitored. The breaking of this bond and the action of the participating component within this is a key part of the sulfation reaction. This reaction and monitoring thereof allows a person skilled in the art to run a simple test to ascertain if, indeed, they have a compound or solvent which is acting as a participating component in a sulfation reaction. The participating component may or may not be acting as a bulk solvent in that it may be present only in catalytic amounts in the co-solvent system.

[0039] As used herein, the term“non-participating component’ refers to a component of a co-solvent system which is not actively involved in the sulfation reaction perse. Specifically, the term is used for a component of the co-solvent system which does not form a complex with the SO3 component of the sulfating reagent during the sulfation reaction to form a more reactive sulfating intermediate. The converse of the approach set out for testing whether or not a component of the co-solvent system may be used to identify whether or not a component is a non-participating solvent. That is, the same test may be used with a negative result indicating a non-participating component. The ability to precipitate the sulfated product also indicates the presence of a successful non participating component.

[0040] As used herein, the term “polar aprotic compound” refers to a compound possessing sufficient polarity and, more importantly, sufficient electron-donating capability to act as a participating component and react with the sulfating reagent to form an intermediate, as described herein. In certain embodiments, the polar aprotic compound may be a polar aprotic solvent although it need not always be so. For example, A/,A/-diphenylformamidine may be capable of acting as a polar aprotic compound and a participating component even though it is solid at room temperature and so may not be considered to be a traditional solvent. Whether or not a polar aprotic compound has sufficient electron-donating capability to act as a participating component can be determined experimentally using the testing procedure described herein. [0041 ] According to a first aspect of the invention, there is provided a method of N- or O-sulfation of a compound, including the steps of:

(a) dissolving the compound in a co-solvent system wherein, once dissolved, the co-solvent system comprises of at least one participating component and at least one non-participating component; and

(b) contacting the compound with a sulfating reagent; to thereby sulfate the compound.

[0042] Suitably, the compound comprises at least one of a nitrogen- containing functional group or an oxygen-containing functional group.

[0043] In one embodiment, the nitrogen-containing functional group is selected from an amine, amide, sulfonamide, imine and /V-oxide. It is highly preferred that the nitrogen-containing functional group is an amine.

[0044] Suitably, the nitrogen-containing functional group is an -Nhh group.

[0045] Preferably, the oxygen-containing functional group is hydroxyl.

[0046] The compound may be modified to present the oxygen-containing or nitrogen-containing functional groups. Further, the compound may have such groups but presented in a form whereby they are not available for sulfation, for example protected with a suitable protecting group. The relevant group may be removed by conventional chemical reactions prior to being exposed to the sulfation reagent and so all such compounds may be suitable for use in the present method.

[0047] Most preferably, the compound displays one or more free hydroxyl groups for O-sulfation thereof. It will be appreciated that the compound may initially have the hydroxyl group protected but this protecting group may be removed prior to the sulfation. [0048] In certain embodiments, the compound may be selected from the group consisting of a carbohydrate, a carboxylic acid, an alkanol, a haloalkanol, an aldehyde, a phenol or other hydroxyl-substituted aryl, a sulfonic acid, a hydroxyl-substituted heteroaryl, a hydroxyl-substituted heterocycle, a thioalkanol, a diol, triol or polyol, and a sterol.

[0049] The co-solvent system may comprise one or more additional solvents or components beyond the at least one participating component and at least one non-participating component. For example, the at least one participating component may, in certain embodiments, be two, three or four participating components. Further, the non-participating component may, in certain embodiments, be two, three or four non-participating components.

[0050] In an alternative embodiment, the co-solvent system may comprise only one participating component.

[0051 ] In a further embodiment, the co-solvent system may comprise only one participating component and either one or two non-participating components.

[0052] In certain embodiments, the at least one participating component may be an electron donor molecule and/or a polar aprotic compound.

[0053] In certain embodiments, the at least one participating component may be an aprotic dipolar compound.

[0054] In embodiments, the at least one participating component may be selected from the group consisting of heterocycles, formamides, phosphoramides, sulfoxides, anilides, and ethers.

[0055] Suitably, when the at least one participating component is a heterocycle it is selected from oxygen and/or nitrogen-containing five to seven- membered heterocycles which may optionally be substituted or fused with one or more further five to seven-membered rings which may themselves be heterocyclic, carbocyclic, aryl or heteroaryl. [0056] In one embodiment, the at least one participating component may be selected from the group consisting of A/,A/-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), morpholine, /V-formylmorpholine, /V-formylpiperidine, N- methylformanilide, 1 -formylpyrrolidine, 2-bromopyridine, and hexamethylphosphoramide (HMPA).

[0057] In embodiments, the at least one participating component may be a component or solvent also associated with the sulfating reagent. For example, the at least one participating component may be pyridine. Such solvents or components can be associated with the sulfating reagent in the form S0 ₃ .Pyr (Pyr = pyridine). In this embodiment, the co-solvent system comprises the pyridine as the at least one participating component with the non-participating component also present, for example as DCE.

[0058] In alternative embodiments, the participating component may not be a component of the sulfating reagent. That is, the participating component will be added as an additional and separate component to any equivalent component of the sulfating reagent. This separation may be particularly so if the reaction is to be microwave assisted.

[0059] In /V-sulfation reactions, the participating component may, in certain embodiments, be considered to be the /V-sulfation precursor itself. Flence, once the /V-sulfation precursor compound is dissolved the co-solvent system is formed comprising of both the participating and non-participating components.

[0060] In embodiments, the at least one non-participating component may be selected from the group consisting of haloalkyl, nitrile, alcohol, aqueous alcohol, water, nitroalkyl, heterocyclic, ketone, optionally substituted aryl and ester.

[0061 ] In one embodiment, the at least one non-participating component may be selected from the group consisting of 1 ,2-dichloroethane (DCE), dichloromethane (DCM), chlorobenzene, toluene, methanol, ethanol, water, methanol/water blends, acetonitrile, acetone, nitromethane, tetrahydrofuran, chloroform, 1 ,2,3,4-tetrahydro-naphthalene, trans- 1 ,2-dichloroethylene, c/s-1 ,2- dichloroethylene, xylenes, butyl acetate and ethyl acetate.

[0062] In embodiments, the at least one non-participating component may be two or more components selected from the groups already described. For example, in some reactions a combination of non-participating components may be useful such as, for example, ethyl acetate and acetonitrile.

[0063] When the reaction is an /V-sulfation reaction then, in certain embodiments, it may be preferred that the non-participating component is an alcohol or aqueous alcohol. Methanol and water or blends thereof may be particularly preferred. For example, methanol and water may act as non participating components which enable the solubilizing of all reactants and which compromise the selectivity between the NFh and OFI functionalities. Therefore, in one embodiment, the non-participating component for /V-sulfation may be selected from polar aprotic molecules including, but not limited to, alcohols, including methanol, and water. Flowever, the non-participating component for /V-sulfation may not be limited to polar protic solvents and may, in certain embodiments, be selected from non-polar aprotic solvents including DCE, DCM, Acetonitrile, EtOAc and the like.

[0064] The sulfation may, in one embodiment, be a selective /V-sulfation in the presence of hydroxyl groups on the compound. The nitrogen of, for example, a free amino group is more nucleophilic than an oxygen of a hydroxyl and so the amine nitrogen can be selectively sulfated. Conditions of either one or both of the participating component and non-participating component may be manipulated to favour the /V-sulfation.

[0065] When carrying out an /V-sulfation it may be preferable to further include a base. The base may be an amine base, a carbonate base or a bicarbonate base. Preferred bases may include trialkylamines, such as triethylamine, and sodium and/or potassium carbonates and bicarbonates. In one embodiment, the base is an additional component in the reaction mixture. That is, in embodiments, neither the participating component nor the non participating component is an alkylamine bases, particularly not a trialkylamine.

[0066] The base may act as an acid scavenger to neutralize the increasing acidity derived from the generated acid due to the potential volatilization of the W component during the course of the reaction. If the basicity of the base is stronger than W, then the W+H cation would be replaced by H+base as the more stable salt. Therefore, the base may be selected from any suitable inorganic or organic base including but not limited to triethylamine and sodium and potassium carbonates and bicarbonates.

[0067] In certain embodiments, the participating and/or non-participating component is not a base. Particularly, the non-participating component may not be an amine base.

[0068] In embodiments, the non-participating component may not be selected from heterocyclic amine bases, such as pyridine.

[0069] In embodiments, the non-participating component is not an alkylamine, including TEA and TMA. While such an amine may assist in the breaking of the key S-W bond (preferably an S-N bond) of the sulfating reagent an analogous reagent is formed and so O-sulfation is not achieved.

[0070] The method may further include the step of precipitating the N- or O- sulfated compound from the co-solvent system. This step is linked to the level of miscibility of the at least one participating component and at least one non participating component, as discussed further below. Further non-participating component may be added to the post-reaction mixture to achieve precipitation. That further non-participating component may be the same as or different to the non-participating component present during the sulfation reaction.

[0071 ] The N- or O-sulfated compound may be present as an amine salt or pyridinium at the point of precipitation from the co-solvent system. That is, the sulfated compound of step (b) may be precipitated from the co-solvent system as an amine or pyridinium salt. [0072] It will be appreciated that the precipitation of the N- or O-sulfated compound may be achieved in a number of ways such as, for example, by variation of the alkyl chain length associated with the amine functionality which can allow for tailoring of precipitation based upon the polarity of the co-solvent system.

[0073] In certain embodiments, the step of precipitating the N- or O-sulfated compound from the co-solvent system may not require the addition of a further solvent which is not already part of the co-solvent system in which the sulfation reaction occurred. That is, the precipitation step does not necessarily require the addition of a further solvent to crash out the sulfated product. It will be appreciated, however, that in some embodiments it may be desirable or efficient to add additional post-reaction non-participating component to improve the yield of product by encouraging precipitation of product.

[0074] Suitably, the at least one participating component and at least one non-participating component are at least partially miscible.

[0075] Preferably, the at least one participating component and at least one non-participating component are miscible. They will suitably be miscible such that they can be mixed, in all concentrations, to form a homogeneous solution. Miscibility can be ascertained or predicted from one of many solvent miscibility tables available in the literature or can be simply tested by an experiment mixing the two components in a variety of ratios and miscibility visibly determined. Representative solvent miscibility tables may be found at https://erowid.org/archive/rhodium/pdf/solvent.miscibility.p df and https://www.csustan.edu/sites/default/files/groups/Chemistry /Drake/documents/ solvent_miscibility_table.pdf).

[0076] The benefit of a high level of miscibility between the at least one participating component and at least one non-participating component is that the at least one non-participating component is better able to sequester the at least one participating component which makes it more likely that the sulfated reaction product will precipitate from the reaction. While not an essential step in the present method, distinct advantages in operation are achieved by the precipitation of the reaction product.

[0077] The method may therefore also include the step of collecting the solid sulfated product. The collecting may be by simple filtration. This is a significant advantage compared with attempting to extract the sulfated product from a significant volume of DMF, as occurs in prior art sulfation approaches. Apart from the cost and time savings, there is a significant risk of desulfation of the sulfated product during neutralisation and DMF removal in prior art approaches. The present method therefore assists greatly in preserving the sulfated product. FIG 3 demonstrates the desulfation side reaction which occurs with extended heating times in convention reaction approaches. The present approach and, particularly, employing precipitation of the product can greatly reduce or avoid this issue.

[0078] In one embodiment, the method does not include the step of column chromatography for removal of solvent, such as DMF.

[0079] In certain embodiments, the ratio of the at least one participating component to the at least one non-participating component in the co-solvent system is between 10:1 to 0.1 :1000 including between 10:1 to 0.1 :500, 10:1 to 0.1 :250, 10:1 to 0.1 :100, 10:1 to 0.1 :50, 10:1 to 0.1 :25, 5:1 to 0.1 :500, 5:1 to 0.1 :250, 5:1 to 0.1 :100, 5:1 to 0.1 :50, 5:1 to 0.1 :25, 1 :1 to 0.1 :500, 1 :1 to 0.1 :250, 1 :1 to 0.1 :100, 1 :1 to 0.1 :50, 1 :1 to 0.1 :25, 10:1 to 1 :500, 10:1 to 1 :250, 10:1 to 1 :1 00, 10:1 to 1 :50, 10:1 to 1 :25, 5:1 to 1 :500, 5:1 to 1 :250, 5:1 to 1 :100, 5:1 to 1 :50, 5:1 to 1 :25, 1 :1 to 1 :500, 1 :1 to 1 :250, 1 :1 to 1 :100, 1 :1 to 1 :50, and 1 :1 to 1 :25.

[0080] It has been found that adjusting the ratio of the at least one participating component to the at least one non-participating component in the co-solvent system can influence the course of the reaction and can reduce the incidence of unwanted side reactions as well as assisting in optimising reaction yield. The optimal ratio may vary depending on the substrate to be sulfated but can be identified by simultaneously running a number of reactions, preferably on a small scale, varying only that ratio. This routine testing will then indicate a preferred range of ratio of the at least one participating component to the at least one non-participating component in the co-solvent system which can be applied to scaled up reactions.

[0081 ] As discussed previously, in certain embodiments the participating component may be formed during the reaction by the breaking of the S-W bond (as per FIG 2A, typically an S-N bond) of the sulfating reagent with the W component effectively becoming the participating component. For example, S0 ₃ .Pyr will release pyridine to act as a participating component under certain conditions.

[0082] The sulfating reagent may be selected from those which are commonly used in sulfation reactions and particularly those applicable to sulfation of carbohydrates. The sulfating reagent may be of formula SO ₃ .W wherein W may be selected from, but is not limited to, formamide, sulfoxide, phosphoramide, dioxane, pyridine and alkylamine. Specifically, W may be selected from TMA, TEA, Pyr, DMF and 1 ,4-Dioxane. W may be any electron- donating molecule or species. Examples of suitable sulfating reagents may be selected from, but are not limited to, S03.Pyr and SO3.NR1 R2R3 which latter general reagent class includes where each R is independently selected from C1 -C6 alkyl and the formula includes sulfating reagents such as SO ₃ .TMA and SO ₃ .TEA.

[0083] In one embodiment, the method may further include the step of treating the sulfated product with a base. This may be useful to convert the sulfated product salt form into a more desirable form. For example, if the sulfating reagent is S0 ₃ .Pyr then the sulfated product will be a pyridine salt. It may be desirable to convert this into a more preferred salt which can be achieved by addition of a suitable base such as NaOFI, NaFIC0 ₃ , TMA and TEA, for example. [0084] The method of the first aspect may include heating of the reaction mixture comprising the co-solvent system, the compound to be sulfated and the sulfating reagent. The heating may be the typical application of heat e.g. via an oil bath, heating mantle or the like. Microwave heating may be acceptable in some circumstances but is not preferred as it can lead to significant desulfation of the product.

[0085] Therefore, in one embodiment, the method of the first aspect does not involve microwave heating. That is, the sulfation reaction mixture is not contacted with microwave radiation or does not require microwave radiation for the sulfation reaction to be substantially complete. Microwave radiation may be useful in some N-sulfation reactions to replace multiple sulfation cycles or reduce the need for such repetition but it is an advantage of the present inventive method that such microwave radiation is not required to achieve sulfation in excellent yield.

[0086] In one embodiment, the reaction mixture is heated at between 50°C to 140°C. Preferably, the reaction mixture is heated at between 60°C to 130°C, including between 70°C to 120°C, 75°C to 1 10°C and 80°C to 100°C. The choice of optimal temperature may depend on, for example, the proportion of non-participating solvent in that highly-sulfated products with a higher proportion of non-participating solvent in the reaction are more tolerant to high temperatures. The choice of temperature is therefore a balance between sufficient heating to spur the reaction and a temperature which does not increase desulfation.

[0087] In certain embodiments, it may be preferred to remove excess participating component, base or some other solvent component by co evaporation with an unreactive solvent. The unreactive solvent will be one which does not react with the sulfated product to cause desulfation. Chlorobenzene and toluene are examples of such unreactive solvents with chlorobenzene being preferred. Such solvents may be chosen based upon their boiling points being close to the solvent component to be removed. [0088] In some embodiments, the method may include the step of selecting the conjugate base forming part of the sulfating reagent. For example, trimethylamine is volatile and can easily escape the reaction mixture thereby increasing the acidity of the mixture which increases the risk of desulfation of the sulfated product. It may therefore be desirable to select a conjugate base which is less volatile to thereby avoid the issue of a lowering of pH.

[0089] According to a second aspect of the invention there is provided an N- or O-sulfated compound when synthesised by the method of the first aspect.

[0090] The N- or O-sulfated compound will clearly be a sulfated analogue of the compound classes set out for the first aspect.

[0091 ] In one embodiment, the N- or O-sulfated compound may be a persulfated compound.

[0092] The present method may present one or more advantages over prior art approaches (typically heating the substrate in DMF with the sulfating reagent) which may be selected from (i) improved yield; (ii) higher degree of sulfation of product; (iii) simpler work up procedure to obtain final sulfated product; (iii) reduced reaction time; (iv) reduced solvent usage; (v) reduced overall synthesis/manufacturing cost; (vi) purer sulfated product due to less partially sulfated products; (vii) improved precipitation or aggregation of solid sulfated product when compared with a similar reaction employing only the participating component and not the non-participating component; and (viii) reduced product degradation and/or desulfation.

EXPERIMENTAL

Example 1

O-Sulfation Approach - Formation in co-solvent system ( DMF/CHsCN )

[0093] A mixture of 4-T olyl 6-0-acetyl-2-0-benzoyl-3-0-benzyl-1 -thio- -D- glucopyranoside (65.5 mg, 0.5 mmol), DMF (575 mI_, 7.5 mmol) and SO ₃ .TMA (347.5 mg, 2.5 mmol) in CH ₃ CN (25 ml_) was heated at 60 °C for 2 h. After heating, the reaction mixture became homogeneous. TLC (EtOAc/MeOH, 4/1 v/v): F?t 0.42. Upon cooling, the resulting solution was co-evaporated with chlorobenzene (10 ml_) at 42 °C under reduced pressure. DCM (20 ml_) was added into the white mixture and the product dissolved as much as possible. After filtration through sinter funnel, the DCM filtrate was concentrated and subjected to silica gel squat column chromatography (EtOAc/Hex, 1 /1 v/v; EtOAc; EtOAc/MeOH, 4/1 v/v). The layer of mixed EtOAc/MeOH was concentrated to give the product (288 mg, 0.475 mmol, 95.0%). ¹ H NMR (400 MHz, CD ₃ CN) d 8.03 - 7.96 (m, 2H, Ar-H), 7.70 - 7.63 (m, 1 H, Ar-H), 7.59 - 7.45 (m, 2H, Ar-H), 7.32 (dd, J = 10.2, 3.7 Hz, 2H, Ar-H), 7.16 - 7.02 (m, 7H, Ar-H), 5.1 1 (dd, J = 10.0, 9.3 Hz, 1 H, H-2), 5.03 (d, J= 10.9 Hz, 1 H, OCH ₂ Ph), 4.93 (d, J= 12.4 Hz, 1 H, H-1 ), 4.70 - 4.61 (dd, J= 12.4, 2.4 Hz, 1 H, H-6), 4.54 (d, J = 10.9 Hz, 1 H, OCZ-fePh), 4.36 (dd, J= 9.9, 9.1 Hz, 1 H, H-4), 4.22 (dd, J = 12.4, 8.0 Hz, 1 H, H-6’), 3.91 (dd, J = 9.1 Hz, 9.1 Hz, 1 H, H-3), 3.75 (ddd, J = 10.0, 7.9, 2.0 Hz, 1 H, H-5), 3.05 (s, 9H, H ⁺ N(C H ₃ ) ₃ ), 2.29 (s, 3H), 2.03 (s, 3H). ¹³ C NMR (101 MHz, CD ₃ CN) d 172.0 (OCOR), 166.3 (OCOR), 139.9, 139.2, 134.8, 133.0, 132.8, 131 .1 , 131 .0, 130.9, 130.8, 130.0, 129.6, 129.4, 129.3, 129.1 , 128.7 (aromatic), 87.1 (C-1 ), 83.3 (C-3), 78.5 (C-5), 77.1 (C-4), 76.0 (OCH ₂ Ph), 73.2 (C-2), 65.2 (C-6), 50.0 (CH ₃ ), 21 .5 (CH ₃ ), 21 .4 (CH ₃ ). LRMS (ESI) calcd for C ₂₉ H ₃ oN ₃ Oi ₂ S ₂ [M-H]- m/z -601 .1 , found -601 .1 .

Example 2 Test Method to Confirm Appropriate Participating/Non-Participating Component in Sulfation Reaction

[0094] Step 1 : A mixture of 4-Tolyl 6-0-acetyl-2-0-benzoyl-3-0-benzyl-1 - thio- -D-glucopyranoside (52 mg, 100 mitioI), SO ₃ .TMA (69.5 mg, 500 mitioI) and additive (300 mmol) in anhydrous 1 ,2-dicholoroethane (DCE, 1 ml_) was heated under Ar at 80 °C for 30 min. Upon cooling, 100 mI_ of reaction mixture withdrawn from each reaction was directly concentrated in vacuum for 30 min to furnish the crude products dissolved in CDCI ₃ for measurement of conversion in ¹ H NMR. These results are recorded in Table 1.

Table 1. Investigation towards participating components in O-sulfation reaction

a- Sulfation reaction in CD3CN is monitored from ¹ H NMR.

b- The conversion is determined by ¹ H NMR.

[0095] Step 2: A mixture of 4-Tolyl 6-0-acetyl-2-0-benzoyl-3-0-benzyl-1 - thio- -D-glucopyranoside (26 mg, 50 mitioI), SO ₃ .TMA (35 mg, 250 mitioI) and in anhydrous solvent (2 ml_) was heated under Ar at 60 °C for 5 h. Upon cooling, 100 mI_ of reaction mixtures withdrawn from each reaction was directly concentrated in vacuum for 30 min to furnish the crude products dissolved in CDCI ₃ for measurement of conversion in ¹ H NMR. These results are recorded in Table 2.

Table 2. Investigation towards participating solvent in O-sulfation reaction

b- The conversion is determined by ¹ H NMR.

Example 3

Synthesis of mCBS in co-solvent system (DMF/DCE)

[0096] A mixture of compound A-02 (84.0 g, 236 mmol), SO ₃ .TMA (367.4 g, 2.64 mol, 1 1 .2 equiv), anhydrous DMF (3140 ml_) and anhydrous DCE (767 ml_) was degassed under Ar for three times and heated at 80-90 °C for 2 h. (Reaction monitoring: After heating for 10 min, the creamy mixture turned to be a clear solution. After 30 min, the solution became cloudy again. After 50 min, the aggregated solid was observed on the surface of flask.). Upon cooling, the resulting mixture was moved to the cold room (~5 °C) and settled overnight which allows the solid to completely aggregate from the solvent. The complete conversion from compound A-02 into P-02 was confirmed by ¹ H-NMR. Decant the solution into the drain. The crude solid was filtered and washed with DCM for a couple of times. The resulting solid was dissolved in de-ionized water and directly subjected to ion-exchange column [Na form of DOWEX 50Wx8: 3 kg of resin (H ⁺ form) was pre-packed in glass gravity column, regenerated by elution of 1 M NaOH (~ 6 L) and neutralized with de-ionized water (~12 L)]. The collected fractions were concentrated to yield the final sulfated cellobioside (mCBS) P-02 (232.1 g, 92.0% yield) as the glassy solid. [OC] ²⁵ D -3.0 (c 5.0, H ₂ 0); ¹ H NMR (400 MHz, D ₂ 0) d 4.87 (d, J = 7.2 Hz, 1 H), 4.84 (d, J = 5.6 Hz, 1 H), 4.68 (dd, J= 6.8 Hz, 1 H), 4.67 (dd, J= 7.4 Hz, 1 H), 4.58 (dd, J= 1 1 .1 , 3.2 Hz, 1 H), 4.48 (dd, J= 8.0 Hz, 1 H), 4.42 (dd, J= 1 1 .2, 4.4 Hz, 1 H), 4.41 (dd, J = 6.0 Hz, 1 H), 4.37 (dd, J= 6.4 Hz, 1 H), 4.35 (dd, J= 8.4, 3.2 Hz, 1 H), 4.22 (dd, J 10.8, 7.2 Hz, 1 H), 4.20 (dd, J 6.8 Hz, 1 H), 4.09 - 4.02 (m, 2H), 3.57 (s, 3H). ; ¹³ C NMR (101 MHz, D ₂ 0) d 101 .0 (CH), 100.00 (CH), 77.7 (CH), 77.5 (CH), 77.4 (CH), 77.2 (CH), 74.4 (CH), 73.8 (CH), 73.5 (CH), 73.1 (CH), 67.7 (CH ₂ ), 66.7 (CH ₂ ), 57.0 (CH ₃ ). ¹ H NMR spectroscopic data are in agreement with published values [J. Carbohydr. Chem. 2001 , 20 (7-8), 549-560]. HRMS (ESI) calcd for CI ₃ H ₂₂ 0 ₃₂ S ₇ [M-2H] ² m/z -456.9070, found -456.9076. Note: 1 . SO3. TMA complex is purchased from Aldrich and directly used for sulfation reaction without any pre-treatment. 2. DMF and DCE in anhydrous grades are purchased from Aldrich.

Example 4

Optimization of co-solvent ratios for synthesis of mCBS (DMF/DCE)

[0097] The mixture of compound A-02 (1 .0 equiv.), SO ₃ .TMA (x equiv.) in anhydrous DMF/DCE (v/v) with the specified concentration was degassed under Ar for three times and heated at T°C in the different time course. Upon cooling, the aggregation of sulfation product was determined by eyes observation and sulfation conversion of mCBS with trimethyammonium salt was confirmed by ¹ H-NMR. The results are recorded in Table 3. A comparison of yield and procedures between the approach of the present invention and a prior art approach is detailed in Table 4.

Table 3. Optimizing the ratio of co-solvent for synthesis of per-O-sulfated b- methyl cellobioside (mCBS)

a- In the preliminary study to optimize ratios of co-solvent systems, the sulfation level of product was determined by ¹ H NMR

b- In Entry 6, it requires conventional work-up and purification procedures.

c- The experiment of Entry 8 was repeated for three times before conducting large scale synthesis.

d- The experimental procedure for Entry 9 and 10 is detailed in Example 3.

Table 4. Comparison between prior art approach and the method of the first aspect for the preparation of mCBS

Example 5

Synthesis of oer-O-sulfated Methyl q-D-glucoside

in co-solvent system (DMF/DCE)

[0098] A mixture of methyl a-D-glucoside (232 mg, 1 .2 mmol), SO3.TMA (1 .33 g, 9.6 mmol, 2.0 eq per OH), anhydrous DMF (10 ml_) and anhydrous DCE (10 ml_) was degassed under Ar for three times and heated at 90 °C for 15 min. Reaction monitoring: After heating for 10 min, the creamy mixture turned to a clear solution. After 15 min, the solution became cloudy again. The aggregated solid was observed on the surface of flask. Upon cooling, the resulting mixture was moved to the cold room (~5 °C) and settled overnight which allows the solid to completely aggregate from the solvent. Decant the solution into the drain. The crude solid was filtered and washed with DCM for a couple of times. The resulting solid was dissolved in de-ionized water and directly subject to Na form of DOWEX 50Wx8 ion-exchange column. The collected fractions were lyophilized to yield the sulfated methyl a-D-glucoside as the glassy solid (687 mg, 95.3% yield). ¹ H NMR (400 MHz, D ₂ 0) d 5.20 (d, J = 3.6 Hz, 1 H), 4.70 - 4.60 (m, 2H), 4.44 (dd, J= 9.8, 3.6 Hz, 1 H), 4.36 - 4.31 (m, 1 H), 4.22 - 4.1 1 (m, 2H), 3.50 (s, 3H). ¹³ C NMR (101 MHz, D ₂ 0) d 96.3 (CH), 75.2 (CH), 74.5 (CH), 73.8 (CH), 68.0 (CH), 66.9 (CH ₂ ), 54.8 (CH ₃ ). ¹ H and ¹³ C NMR spectroscopic data are in agreement with published values [J. Org. Chem. 2006, 71 (19), 7473-7476]

Example 6

Synthesis of oer-O-sulfated Methyl q-D-glucoside in non-participating solvent (DCE only)

[0099] Methyl glycoside (194 mg, 1 .0 mmol), S0 ₃ .Pyr (2.5 eq per OH, 10 mmol, 1 .59 g) and anhydrous 1 ,2-dichloroethane (DCE, 20 ml_) were stirred at 100 °C for 10 mins. Upon cooling, decant the solvent and add another portion of DCM to wash the crude residue. The remaining solid was dried under vacuum for 30 min. The resulting solid was dissolved in de-ionized water and directly subject to Na form of DOWEX 50Wx8 ion-exchange column. The collected fractions were lyophilized to yield the sulfated methyl a-D-glucoside as a glassy solid (563 mg, 93.5%). This indicates that pyridine released from the sulfating reagent can act as the participating component and so this does not necessarily need to be added separately at the start of the reaction.

Example 7

O-Sulfation Approach - Performing with catalytic participating component ( DMF/DCE )

[00100] To a stirred solution of the lactone A-04 (4.8 mg, 7 mitioI), N,N- dimethylformamide (DMF, 0.052 mI_, 0.7 mitioI) in 1 ,2-dichloroethane (DCE, 1 ml_) was added SO3.TMA complex (9.7 mg, 70 mitioI). The reaction vessel was subject to microwave irradiation at 100 °C for 1 h. The reaction was monitored on TLC (EtOAc/MeOH, 93/7 v/v, fit 0.41 ). The resulting reaction mixture was directly subject to silica gel column chromatography (EtOAc; EtOAc/MeOH, 19/1 v/v; EtOAc/MeOH, 9/1 v/v). The fraction of EtOAc/MeOH (9/1 v/v) was concentrated to yield the product P-04 (5.1 mg, 93.7% yield). ¹ H NMR (300 MHz, MeOD) d 7.80 - 7.76 (m, 4H), 7.53 - 7.35 (m, 6H), 7.35 - 7.21 (m, 7H), 5.21 (d, J = 3.7 Hz, 1 H), 5.10 - 4.97 (m, 2H), 4.94 - 4.76 (m, 5H), 4.73 (d, J = 2.6 Hz, 2H), 4.57 (d, J 3.2 Hz, 1 H), 4.43 (dd, J 1 1 .0, 2.8 Hz, 1 H), 4.38 - 4.26 (m, 2H), 3.96 - 3.74 (m, 4H), 3.54 - 3.48 (m, 1 H), 3.49 (s, 3H). ¹³ C NMR (75 MHz, MeOD) d 169.9 (C), 139.5 (C), 138.8 (C), 137.0 (C), 134.8 (C), 134.5 (C), 129.4 (CH), 129.3 (CH), 129.2 (CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 127.9 (CH), 127.3 (CH), 127.0 (CH), 126.9 (CH), 100.8 (CH), 99.5 (CH), 81 .1 (CH), 81 .0 (CH), 80.7 (CH), 79.0 (CH), 76.1 (CH ₂ ), 76.1 (CH ₂ ), 73.6 (CH), 73.0 (CH ₂ ), 72.0 (CH), 70.9 (CH), 66.9 (CH ₂ ), 64.6 (CH), 57.1 (CH ₃ ). LRMS (ESI) calcd for C38H39N3O13S [M-H]- m/z -776.2, found -776.5.

Example 8

O-Sulfation Approach - Performing in co-solvent system ( DMF/DCE )

[00101 ] To a mixture of tetrasaccharide A-05 (75 mg, 54.5 mitioI), sulfur trioxide trimethylamine complex (SO3.TMA, 15 eq., 5 eq./per OH, 817.5 mitioI, 1 13 mg) and 1 ,2-dichloroethane (DCE, 3.0 ml_) was added L/,/V-dimethyl - formamide (DMF, 360 mI_) under Argon. The resulting mixture was heated at 80 °C for 15 min. Upon cooling, add another portion of SO3.TMA complex (4.0 eq., 1 .34 eq./per OH, 216 mitioI, 30 mg). The mixture was heated at 80 °C for another 15 min. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as a white solid with trimethylammonium salt P-05 (71 .5 mg, 73.3% yield). ¹ H NMR (300 MHz, MeOD) d 8.32 - 8.21 (m, 2H), 7.87 - 7.78 (m, 4H), 7.71 - 7.41 (m, 7H), 7.39 - 7.20 (m, 13H), 7.18 - 7.04 (m, 5H), 5.47 (d, J = 3.7 Hz, 1 H), 5.35 - 5.24 (m, 2H), 5.16 (d, J= 10.5 Hz, 1 H), 5.07 - 4.91 (m, 4H), 4.90 - 4.69 (m, 3H), 4.71 - 4.50 (m, 4H), 4.50 - 4.35 (m, 2H), 4.33 - 4.24 (m, 1 H), 4.23 - 4.15 (m, 4H), 4.02 - 3.86 (m, 5H), 3.83 - 3.62 (m, 3H), 3.50 (dd, J= 10.4, 3.7 Hz, 1 H), 3.47 - 3.38 (m, 1 H), 3.28 (s, 3H), 3.19 (dd, J = 10.4, 3.6 Hz, 1 H), 3.02 (dd, J = 10.9,

5.6 Hz, 1 H), 2.87 - 2.77 (m, 28H). ¹³ C NMR (75 MHz, MeOD) d 171 .2 (C),

166.6 (C), 139.8 (C), 139.5(C), 139.2 (C), 139.1 (C), 137.2 (C), 134.9 (C), 134.7 (CH), 134.4 (C), 131 .2 (CH), 130.6 (C), 130.1 (CH), 129.4 (CH), 129.3 (CH),

129.3 (CH), 129.2 (CH), 129.1 (CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.0 (CH), 127.5 (CH), 127.1 (CH), 127.0 (CH), 101 .9 (CH), 101 .7 (CH), 99.3 (CH), 96.8 (CH), 83.8 (CH), 81 .2 (CH), 79.1 (CH),

78.6 (CH), 78.1 (CH), 77.9 (CH), 76.4 (CH ₂ ), 76.1 (CH ₂ ), 76.0 (CH ₂ ), 75.2 (CH),

73.3 (CH), 73.2 (CH ₂ ), 73.0 (CH), 71 .7 (CH), 71 .6 (CH), 71 .2 (CH), 68.4 (CH), 66.8 (CH ₂ ), 65.7 (CH ₂ ), 64.9 (CH), 63.6 (CH), 56.2 (CH ₃ ), 52.5 (CH ₃ ), 45.6 (CH ₃ ). LRMS (ESI) calcd for C ₇₂ H ₇₆ N ₆ 0 ₃ I S ₃ [M-3H] ³ - m/z -537.8, found -538.0; C ₇₂ H ₇₆ N ₆ 0 ₃ I S ₃ [M-3H-Na] ² m/z -818.2, found -818.1 .

Example 9

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

[00102] To a mixture of disaccharide A-06 (134 mg, 120 mitioI), sulfur trioxide trimethylamine complex (S0 ₃ .TMA, 5 eq., 5 eq./per OH, 0.60 mmol, 84 mg) and 1 ,2-dichloroethane (DCE, 10 ml_) was added A/,A/-dimethylformamide (DMF, 0.378 ml_) under Argon. The resulting mixture was heated at 90 °C for 10 min. Upon cooling, add another portion of S0 ₃ .TMA complex (5.0 eq./per OH, 0.60 mmol, 84 mg). The mixture was heated at 90 °C for another 60 min. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as a white solid with the trimethylammonium salt (1 18 mg, 78.4% yield). ¹ H NMR (400 MHz, MeOD) d 7.96 (s, 2H), 7.80 - 7.68 (m, 4H), 7.59 - 7.43 (m, 2H), 7.43 - 7.30 (m, 2H), 7.30 - 7.10 (m, 13H), 7.12 - 6.89 (m, 7H), 5.36 (d, J= 3.5 Hz, 1 H), 5.17 (br, 1 H), 5.05 - 4.96 (m, 3H), 4.94 - 4.64 (m, 5H), 4.59 (s, 2H), 4.46 (d, J= 8.4 Hz, 1 H), 4.38 - 4.09 (m, 7H), 3.86 - 3.72 (m, 2H), 3.70 (s, 3H), 3.52 - 3.26 (m, 1 H), 3.19 - 3.00 (m, 2H), 2.81 (s, 9H), 1 .82 (s, 3H), 1 .68 - 1 .47 (m, 2H); ¹³ C NMR (101 MHz, MeOD) d 173.4 (C), 173.30 (C), 170.78 (C), 166.8 (C), 139.7(C), 138.3 (C), 137.0 (C), 134.8 (C), 134.5 (C), 134.3 (CH), 130.7 (CH), 130.5 (C), 129.8 (CH), 129.5 (CH), 129.4 (CH), 129.3 (CH), 129.1 (CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 1 28.7 (CH), 128.6 (CH), 128.5 (CH), 127.9 (CH), 127.3 (CH), 127.1 (CH), 127.0 (CH), 102.0 (CH), 99.0 (CH), 83.2 (CH), 81 .3 (CH), 78.8 (CH), 76.1 (CH ₂ ), 76.0 (CH ₂ ), 75.5 (CH ₂ ), 75.6 (CH), 75.3 (CH), 75.0 (CH), 71 .8 (CH), 68.5 (CH ₂ ), 68.2 (CH ₂ ), 66.8 (CH ₂ ), 54.0 (CH), 53.6 (CH), 51 .8 (CH ₂ ), 45.5 (CH ₃ ), 23.0 (CH ₃ ). LRMS (ESI) calcd for C ₆₅ H ₆ sN ₂ 0i ₈ S [M-H]- m/z -1 195.4, found -1 196.0.

Example 10

O-Sulfation Approach - Performing in co-solvent system ( DMF/DCE )

[00103] To a mixture of pentasaccharide A-07 (530 mg, 357 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 25 eq., 5 eq./per OH, 8.925 mmol, 1 .24 g) and 1 ,2-dichloroethane (DCE, 24 ml_) was added A/,A/-dimethyl - formamide (DMF, 6 ml_) under Argon. The resulting mixture was heated at 80 °C for 15 min. Upon cooling, add another portion of SO ₃ .TMA complex (12.5 eq., 2.5eq./per OH, 4.462 mmol, 0.62 g). The mixture was heated at 80 °C for another 15 min. Upon cooling, the mixture was co-evaporated with chlorobenzene (15 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as a white solid as the trimethylammonium salt P-07 (656 mg, 84.3% yield). ¹ H NMR (400 MHz, MeOD) d 7.46 (d, J = 6.8 Hz, 2H), 7.42 - 7.16 (m, 23H), 7.16 - 7.09 (m, 4H), 7.08 - 7.00 (m, 1 H), 5.48 (d, J = 3.8 Hz, 1 H), 5.40 (s, 1 H), 5.25 (d, J= 3.9 Hz, 1 H), 5.02 - 4.77 (m, 6H), 4.77 - 4.56 (m, 6H), 4.57 - 4.50 (m, 1 H), 4.49 - 4.25 (m, 6H), 4.20 - 4.12 (m, 2H), 4.13 - 4.05 (m, 2H), 4.04 - 3.92 (m, 4H), 3.91 - 3.85 (m, 3H), 3.84 - 3.80(m, 1 H), 3.79 - 3.48 (m, 5H), 3.45 (s, 3H), 3.37 - 3.32 (m, 2H), 2.90 (s, 45H). ¹³ C NMR (101 MHz, MeOD) d 172.3 (C), 140.1 (C), 140.0 (C), 139.7 (C), 139.5 (C), 139.1 (C), 139.0 (C), 129.9 (CH), 129.7 (CH), 129.5 (CH), 129.4 (CH), 129.3 (CH), 129.3 (CH), 129.2 (CH), 129.1 (CH), 128.9 (CH), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 102.8 (CH), 100.0 (CH), 99.2 (CH), 99.1 (CH), 95.0 (CH), 85.2 (CH), 83.5 (CH), 81 .1 (CH), 80.0 (CH), 79.1 (CH), 78.6 (CH), 76.6 (CH), 76.1 (CH ₂ ), 76.0 (CH ₂ ), 75.9 (CH ₂ ), 75.9 (CH ₂ ), 74.4 (CH), 73.5 (CH ₂ ), 73.1 (CH), 71 .5 (CH), 71 .4 (CH), 71 .4 (CH), 71 .3 (CH), 71 .1 (CH), 70.2 (CH), 67.9 (CH), 67.2 (CH ₂ ), 66.8 (CH ₂ ), 66.5 (CH ₂ ), 65.0 (CH), 64.7 (CH), 64.6 (CH), 55.7 (CH ₃ ), 45.8 (CH ₃ ). ¹ H and ¹³ C NMR spectroscopic data are in agreement with published values [ChemMedChem 2014, 9 (5), 1071 -1080]

Example 11

O-Sulfation Approach - Performing in co-solvent system (DMF/EtOAc)

[00104] To a mixture of Fmoc-Ser-OH A-08 (327 mg, 1 mmol), sulfur trioxide trimethylamine complex (S0 ₃ .TMA, 3 eq., 3.0 mmol, 417 mg) and ethylacetate (EtOAc, 10 ml_) was added A/,A/-dimethylformamide (DMF, 9 eq., 9.0 mmol, 0.69 ml_). The resulting mixture was heated at 80 °C for 25 min under Ar. Upon cooling, the mixture was co-evaporated with chlorobenzene (20 ml_) for 3 cycles to give the crude product which was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product P-08 was obtained as an amorphous solid (415 mg, 0.891 mmol, 89.1 % yield). ¹ H NMR (400 MHz, MeOD) d 7.78 (d, J= 7.5 Hz, 2H), 7.68 (dd, J= 7.2, 4.1 Hz, 2H), 7.38 (dd, J = 7.4, 7.4 Hz, 2H), 7.32 (ddd, J= 7.4, 3.0, 1 .1 Hz, 2H), 4.50 (t, 3.6 Hz, 1 H), 4.42 - 4.27 (m, 4H), 4.24 (d, J= 7.1 Hz, 1 H), 2.86 (s, 9H); ¹³ C NMR (101 MHz, MeOD) d 172.7 (C), 158.5 (C), 145.2 (C), 145.1 (C), 142.5 (C), 128.8 (CH), 128.2 (CH), 126.4 (CH), 126.3 (CH), 120.9 (CH), 68.3 (CH ₂ ), 68.2 (CH ₂ ), 55.3 (CH), 48.0 (CH), 45.5 (CH ₃ ). LRMS (ESI) calcd for CI ₈ HI ₇ N0 ₈ S [M-H] - m/z -406.1 , found - 406.0.

Example 12

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

H ⁺ N(CH ₃ ) ₃

,oso ₃

H ⁺ N(CH ₃ ) ₃

A-09 P-09

[00105] To a mixture of A-09 (134 mg, 1 mmol), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 9 eq., 9.0 mmol, 1 .25 g) and 1 ,2-dichloroethane (DCE, 10 ml_) was added A/,A/-dimethylformamide (DMF, 96 eq., 96.0 mmol, 7.4 ml_). The resulting mixture was heated at 80 °C for 25 min under Ar until the reaction mixture become homogenous. Upon cooling, the mixture was co-evaporated with chlorobenzene (20 ml_) for 3 cycles to give the crude product which was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as an amorphous solid P-09 (473 mg, 0.858 mmol, 85.8% yield). ¹ H NMR (400 MHz, MeOD) d 4.52 - 4.44 (m, 1 H)„ 4.17 (d, J= 4.4 Hz, 2H), 4.01 (t, J= 6.4 Hz, 2H), 2.93 (s, 27H), 1 .86 - 1 .64 (m, 4H), 1 .64 - 1 .48 (m, 2H); ¹³ C NMR (101 MHz, MeOD) d 77.7 (CH), 69.4 (CH ₂ ), 68.9 (CH ₂ ), 45.6 (CH ₃ ), 32.1 (CH ₂ ), 30.3 (CH ₂ ), 22.5 (CH ₂ ). LRMS (ESI) calcd for CI ₈ HI ₇ N0 ₈ S [M-H]- m/z -373.0, found -372.8.

Example 13 O-Sulfation Approach - Performing in co-solvent system (DMSO/Toiuene)

[00106] To a mixture of A-10 (32 mg, 0.2 mmol), sulfur trioxide trimethylamine complex (SO3.TMA, 10 eq., 2.0 mmol, 278 mg) and toluene (2 ml_) was added dimethylsulfoxide (DMSO, 10 eq., 2.0 mmol, 0.142 ml_). The resulting mixture was heated at 90 °C for 60 min under Ar. Upon cooling, the mixture was quenched by NaOMe (2 ml_, 1 M in MeOH) at ice bath and followed by co-evaporation with chlorobenzene (5 ml_) for 3 cycles to give the crude product which was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as an amorphous solid P-10 (67 mg, 0.153 mmol, 76.5% yield). ¹ H NMR (400 MHz, MeOD) d 8.24 (d, J = 8.1 Hz, 1 H), 7.85 - 7.78 (m, 1 H), 7.65 (d, J = 2.0 Hz, 1 H), 7.61 (d, J = 2.2 Hz, 1 H), 7.52 - 7.43 (m, 2H); ¹³ C NMR (101 MHz, MeOD) d 151 .2 (C), 150.0 (C), 136.0 (C), 128.3 (CH), 128.1 (CH), 126.4 (C), 126.2 (CH), 123.8 (CH), 1 15.5 (CH), 1 14.0 (CH). LRMS (ESI) calcd for CIOH ₈ 0 ₈ S2 [M-2H+Na] ^_ m/z -341 .0, found -340.8.

Example 14

O-Sulfation Approach - Performing in co-solvent system in (HMPA/DCE) [00107] To a mixture of A-11 (131 mg, 0.33 mmol), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 8 eq., 2.64 mmol, 366 mg) and1 ,2- dichloroethane (DCE, 5 ml_) was added hexamethylphosphoramide (HMPA, 100 eq., 33.3 mmol, 5.79 ml_). The resulting mixture was heated at 90 °C for 3 h under Ar. Upon cooling, the mixture was co-evaporated with chlorobenzene (10 ml_) for 3 cycles to give the crude product which was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product was obtained as an amorphous solid P-11 (172 mg, 0.257 mmol, 77.1 % yield). ¹ H NMR (400 MHz, MeOD) d 4.67 (s, 1 H), 4.36 - 4.18 (m, 1 H), 2.93 (s, 18H), 2.43 - 2.15 (m, 3H), 2.00 - 1 .69 (m, 10H), 1 .66 - 1 .55 (m, 2H), 1 .55 - 1 .23 (m, 8H), 1 .23 - 1 .08 (m, 2H), 1 .06 (d, J= 6.5 Hz, 3H), 1 .03 - 0.96 (m, 1 H), 0.95 (s, 3H), 0.77 (s, 3H); ¹³ C NMR (101 MHz, MeOD) d 177.9 (C), 82.7 (CH), 80.4 (CH), 49.8 (CH), 47.7 (CH), 47.2 (C) , 45.5 (CH ₃ ), 43.5 (CH), 37.1 (CH), 36.7 (CH), 36.4 (CH ₂ ), 35.2 (C), 34.9 (CH) , 34.6 (CH ₂ ), 32.1 (CH ₂ ), 32.0 (CH ₂ ), 28.8 (CH ₂ ), 28.6 (CH ₂ ), 28.2 (CH ₂ ), 27.4 (CH ₂ ), 25.9 (CH ₂ ), 24.8 (CH ₂ ), 23.5 (CH ₃ ) , 18.1 (CH ₃ ), 12.9 (CH ₃ ). LRMS (ESI) calcd for C ₂₄ H ₄₀ OI ₀ S ₂ [M-H] m/z -551 .2, found -551 .4.

Example 15

O-Sulfation Approach - Performing in co-solvent system ( DMF/Toluene )

[00108] To a mixture of A-12 (254 mg, 1 mmol), sulfur trioxide trimethylamine complex (S0 ₃ .TMA, 18 eq., 18.0 mmol, 2.51 g) and toluene (5 ml_) was added A/,A/-dimethylformamide (DMF, 200 eq., 200.0 mmol, 15.4 ml_). The resulting mixture was heated at 80 °C for 50 min under Ar until precipitation was observed in the flask. Upon cooling, add dichloromethane (50 ml_) into the mixture and more precipitation was formed with vigorous stirring. The final slurry was filtered through sinter funnel to obtain the crude product which was further purified through Sephadex LH-20 size exclusion chromatography to furnish an amorphous solid P-12 (1 .02 g, 0.938 mmol, 93.8% yield). ¹ H NMR (400 MHz, D ₂ 0) d 4.14 (s, 12H), 3.60 (s, 4H), 2.94 (s, 54H); ¹³ C NMR (101 MHz, D ₂ 0) d 68.8 (CH ₂ ), 66.6 (CH ₂ ), 43.3 (C). LRMS (ESI) calcd for C10H22O25S6 [M-H] m/z - 732.9, found -732.7.

Example 16

Microwave-assisted N-Sulfation Approach for Synthesis of Fondaoarinux

[00109] To a mixture of pentasaccharide A-13 (120 mg, 84.5 mitioI), MeOH/hhO (v/v, 1/1 , 6 mL/6 ml_), triethylamine (TEA, 10 eq./per Nhh, 30 eq., 2.535 mmol, 352 mI_) was added sulfur trioxide trimethylamine complex (SO ₃ .TMA, 24 eq., 8 eq./per NH2, 2.028 mmol, 282 mg). The resulting mixture was subject to microwave irradiation at 80 °C for 10 min. Upon cooling, add another portion of triethylamine (TEA, 5 eq./per NH2. 15 eg, 1 .267 mmol, 176 uU and SOa.TMA complex (12 eg, 4 eq./per NH2. 1 .014 mmol, 142 mg). The resulting mixture was heated under microwave irradiation at 80 °C for 10 min. Repeat the procedure underlined as above for another 2 cycles. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to give the crude product which was dissolved in water and passed through an ion- exchange column of DOWEX 50W-X8 (Na form). The collected fractions were concentrated to give the product mixture as sodium salt which was further purified through Sephadex G25. The collected fractions were lyophilized to furnish the product P-13 as a white solid with sodium salt (121 mg, 82.9 % yield). ¹ H NMR (600 MHz, D ₂ 0) d 5.63 (d, J = 3.7 Hz, 1 H), 5.55 (d, J= 3.4 Hz, 1 H), 5.17 (d, J = 4.1 Hz, 1 H), 5.03 (d, J = 3.6 Hz, 1 H), 4.73 (d, J= 2.8 Hz, 1 H), 4.62 (d, J = 7.5 Hz, 1 H), 4.51 - 4.47 (m, 1 H), 4.44 - 4.38 (m, 2H), 4.38 - 4.33 (m, 4H), 4.33 - 4.25 (m, 2H), 4.21 - 4.13 (m, 4H), 4.00 - 3.94 (m, 2H), 3.93 - 3.87 (m, 1 H), 3.87 - 3.82 (m, 2H), 3.80 - 3.75 (m, 2H), 3.69 - 3.55 (m, 3H), 3.47 - 3.39 (m, 2H), 3.42 (s, 3H), 3.31 - 3.24 (m, 2H). ¹³ C NMR (151 MHz, D ₂ 0) d 178.0 (C), 176.6 (C), 103.7 (CH), 102.3 (CH), 100.8 (CH), 100.2 (CH), 98.6 (CH), 80.3 (CH), 79.8 (CH), 79.5 (CH), 79.0 (CH), 78.8 (CH), 78.7 (CH), 75.5 (CH), 75.4 (CH), 73.8 (CH), 73.4 (CH), 73.1 (CH), 72.4 (CH), 72.3 (CH), 72.2 (CH), 71 .6 (CH), 71 .2 (CH), 69.4 (CH ₂ ), 68.9 (CH ₂ ), 68.6 (CH ₂ ), 60.6 (CH), 60.3(CH) , 59.3 (CH), 58.0 (CH3). ¹ H and ¹³ C NMR spectroscopic data are in agreement with published values [ChemMedChem 2014, 9 (5), 1071 -1080].

[001 10] Table 5, below, indicates the results from optimisation of conditions for selective /V-sulfation in the presence of a free hydroxyl. It is an advantage of the present method that the choice of participating and non-participating component can be tailored to favour such a selective approach.

Example 17

Optimal Conditions for Chemoselective N-Sulfation

Table 5. Results from optimisation of conditions for selective /V-sulfation in the presence of a free hydroxyl. Sulfating Temp Yield of

Entry Base Solvent (v/v) Time

reagent ³ (°C) P-14b (%) ^b

1 SOs.NMes EtsN DCE rt 24 h NR

2 SC>3.NMe3 none MeOH rt 24 h NR

3 SC>3.py EteN MeOH rt 24 h - ^c

4 SOs.NMes EtsN DMF/DCE (1 /9) 80 °C ^d 16 h NR

5 SOs.NMes EtsN MeOH/DCE (1 /9) 80 °C ^d 16 h NR

4 SOs.NMes NaOH MeOH 60 °C ^e 16 h

5 SOs.NMes NH ₄ HCOs MeOH 60 °C ^e 16 h 68

6 SOs.NMes NH ₄ OH MeOH 60 °C ^e 16 h 59

7 SOs.NMes NaHCOs MeOH 60 °C ^e 16 h 92

8 SOs.NMes EtsN MeOH rt 24 h 95

9 SOs.NMes EtsN MeOH/HsO (1 /1 ) rt 24 h 80

10 SOs.NMes EtsN MeOH/HsO (1 /1 ) 80 °C ^d 20 min 95

1 1 SOs.NMes EtsN H ₂ 0 rt 24 h 51

12 SOs.NMes EtsN H ₂ 0 80 °C ^d 20 min 92 ^f

³ All reactions were performed in 0.1 M of solution by sequential mixing starting material A-14 (0.1 mmol, 1 .0 equiv.), sulfating reagent (3.0 equiv.), base (3.0 equiv.) and solvent. ^b Isolated yield intractable mixtures. ^d Microwave-assisted heating. ^e Conventional heating. 'This reaction is repeated in 1 .0 mmol of scale.

[001 1 1 ] To the mixture of glucosamine hydrochloride A-14 (215 mg, 1 .0 mmol), sulfur trioxide trimethylamine complex (SO3.TMA, 3.0 eq., 417 mg, 3.0 mmol) was added H2O (10 ml_) and triethylamine (TEA, 3.0 eq., 417 mI_, 3.0 mmol). The resulting mixture was heated under microwave irradiation at 80 °C for 20 min. The reaction was monitored by TLC. ( R of product is around 0.36 in IPA/MeOH/h O = 5/2/1 , starting material is around 0.10). The reaction was quenched with 1 M NaHC0 ₃ (3 ml_) and the pH was maintained at 9.5. After stirring for 10 min, the reaction mixture was concentrated with toluene. The final solid residue was dissolved in water, filtered with a pad of cotton and passed through an ion-exchange column of DOWEX 50W-X8 (Na form). The collected fractions were concentrated to give the product mixture as sodium salt which was further purified through Sephadex G25. The combined fractions was lyophilized to furnish the /V-sulfated product P-14b as sodium form (258 mg, 91 .8% yield). ¹ H NMR (400 MHz, D ₂ 0) d 5.47 (d, J= 3.5 Hz, 0.6H), 4.72 (d, J = 8.3 Hz, 0.4H), 4.02 - 3.73 (m, 3H), 3.56 - 3.42 (m, 2H), 3.24 (dd, J= 10.3, 3.6 Hz, 0.6H), 3.01 (dd, J = 10.0, 8.3 Hz, 0.4H). ¹³ C NMR (100 MHz, D ₂ 0) d 95.7, 91 .4, 75.9, 74.8, 71 .5, 71 .3, 70.2, 70.0, 61 .1 , 60.9, 60.8, 58.1 . ¹ H and ¹³ C NMR spectroscopic data are in agreement with published values [Chem. Comm. 2010, 46 (33), 6066-6068]

Example 18

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

[001 12] To a mixture of A-15 (20 mg, 7.09 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 40 eq., 10 eq./per OH, 0.283 mmol, 39.4 mg) and 1 ,2-dichloroethane (DCE, 3 ml_) was added A/,A/-dimethylformamide (DMF, 0.15 ml_) under Argon. The resulting mixture was heated at 80 °C for 15 min. Upon cooling, add another portion of SO ₃ .TMA complex (20 eq., 5.0 eq./per OH, 0.142 mmol, 19.7 mg). The mixture was heated at 80 °C for another 15 min. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product P-15 was obtained as a white solid with trimethyllammonium salt (21 .8 mg, 98.1 % yield). ¹ H NMR (600 MHz, MeOD) d 8.29 - 8.21 (m, 5H), 8.03 - 7.97 (m, 4H), 7.84 - 7.77 (m, 4H), 7.71 - 7.52 (m, 10H), 7.52 - 7.40 (m, 6H), 7.39 - 7.29 (m, 6H), 7.29 - 7.18 (m, 6H), 7.16 - 6.96 (m, 13H), 5.36 - 4.94 (m, 8H), 4.70 - 4.44 (m, 18H), 4.28 - 4.08 (m, 8H), 4.05 - 3.73 (m, 14H), 3.71 - 3.76 (m, 4H), 3.64 (s, 3H), 3.57 - 3.52 (m, 4H), 3.37 (s, 3H), 3.22 (s, 3H), 2.98 (s, 3H), 2.96 (s, 3H), 2.86 (s, 27H); d ¹³ C NMR (150 MHz, MeOD) d 170.13, 169.95, 169.76, 166.67, 166.59, 166.56, 166.50, 139.52, 139.45, 139.44, 139.24, 138.98, 138.91 , 137.27, 134.89,

134.78, 134.62, 134.46, 131 .30, 131 .27, 130.91 , 130.73, 130.54, 130.08,

130.06, 129.85, 129.81 , 129.72, 129.71 , 129.30, 129.27, 129.24, 129.21 ,

129.19, 129.14, 129.03, 129.01 , 128.92, 128.90, 128.86, 128.81 , 128.70,

128.63, 128.60, 127.73, 127.29, 127.00, 126.85, 103.08, 101 .80, 99.54, 98.91 ,

98.75, 83.48, 83.21 , 83.1 1 , 81 .1 1 , 79.16, 78.98, 78.83, 78.72, 78.24, 77.1 1 ,

77.03, 76.68, 76.46, 76.41 , 76.31 , 76.15, 76.10, 75.95, 75.29, 75.04, 74.96,

74.76, 72.00, 71 .32, 71 .19, 66.50, 65.49, 65.42, 65.33, 65.04, 64.09, 63.98,

57.35, 53.45, 52.95, 52.72, 49.57, 49.57, 49.23, 49.08, 48.93, 48.78, 45.49 ;

LRMS (ESI) calcd for Ci ₄₈ Hi ₅₂ Ni ₂ 0 ₅₇ S ₄ [M-4H] ^4_ m/z -783.2, found -783.5.

Example 19

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

[001 13] To a mixture of A-16 (88 mg, 59 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 20 eq., 10 eq./per OH, 1 .18 mmol, 164 mg) and 1 ,2- dichloroethane (DCE, 12 ml_) was added A/,A/-dimethylformamide (DMF, 0.82 ml_) under Argon. The resulting mixture was heated at 90 °C for 10 min. Upon cooling, add another portion of SO ₃ .TMA complex (10 eq., 5.0 eq./per OH, 0.59 mmol, 82 mg). The mixture was heated at 90 °C for another 10 min. Upon cooling, the mixture was co-evaporated with chlorobenzene (10 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product P-16 was obtained as a white solid with trimethylammonium salt (78 mg, 80.1 % yield). ¹ H NMR (400 MHz, MeOD) d 8.28 - 8.24 (m, 4H), 8.03 - 7.99 (m, 4H), 7.84 - 7.76 (m, 8H), 7.70 - 7.39 (m, 10H), 7.36 - 7.19 (m, 5H), 7.16— 7.03 (m, 6H), 5.35 - 5.23 (m, 5H), 5.15 - 5.01 (m, 5H), 5.01 - 4.77 (m, 7H), 4.64 - 4.52 (m, 6H), 4.45 - 4.37 (m, 3H), 4.31 - 4.22 (m, 3H), 4.21 - 4.14 (m, 3H), 4.07 - 3.83 (m, 10H), 3.65 (s, 1 H), 3.58 - 3.51 (m, 2H) 3.37 (s, 3H), 3.21 (s, 3H), 2.87 (s, 18H); ¹³ C NMR (100 MHz, MeOD) d 170.20, 169.94, 166.67, 166.61 , 139.51 , 139.44, 139.22, 138.89, 137.25, 134.90, 134.77, 1 34.66,

134.45, 131 .26, 130.88, 130.74, 130.65, 130.06, 129.82, 129.73, 129.33,

129.29, 129.28, 129.25, 129.17, 129.1 1 , 129.02, 128.97, 128.96, 128.93,

128.90, 128.74, 128.68, 128.65, 128.62, 128.59, 127.73, 127.26, 127.05,

126.91 , 103.10, 101 .72, 99.46, 98.86, 83.21 , 83.16, 81 .07, 79.00, 78.64, 78.07, 76.91 , 76.66, 76.31 , 76.14, 76.08, 76.03, 75.93, 75.18, 74.85, 74.81 , 71 .97, 71 .28, 66.51 , 65.40, 64.99, 63.92, 57.39, 53.44, 52.93, 52.90, 45.48 ; LRMS (ESI) calcd for C ₈₀ H ₈₂ N ₆ O ₂₉ S ₂ [M-2H] ² m/z -826.2, found -826.3.

Example 20

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

[001 14] To a mixture of A-17 (20 mg, 9.74 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 40 eq., 10 eq./per OH, 0.39 mmol, 54 mg) and 1 ,2-dichloroethane (DCE, 3 ml_) was added A/,A/-dimethylformamide (DMF, 0.18 ml_) under Argon. The resulting mixture was heated at 90 °C for 10 min. Upon cooling, add another portion of SO ₃ .TMA complex (20 eq., 5.0 eq./per OH, 0.19 mmol, 27 mg). The mixture was heated at 90 °C for another 10 min. Upon cooling, the mixture was co -evaporated with chlorobenzene (5 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex LH-20 size exclusion chromatography. The resulting pure product P-17 was obtained as a white solid with trimethylammonium salt (22 mg, 95.2% yield). ¹ H NMR (600 MHz, MeOD) d 8.31 - 8.20 (m, 4H), 7.89 - 7.76 (m, 4H), 7.74 - 7.40 (m, 10H), 7.40 - 7.14 (m, 9H), 7.16 - 6.98 (m, 6H), 5.35 (d, J = 3.0 Hz, 1 H), 5.32 (d, J = 2.4 Hz, 1 H), 5.36 - 5.09 (m, 6H), 5.08 - 4.75 (m, 10H), 4.64 - 4.50 (m, 6H), 4.46 - 4.22 (m, 4H), 4.19 - 4.09 (m, 3H), 4.03 - 3.97 (m, 2H), 3.95 - 3.74 (m, 6H), 3.72 - 3.60 (m, 2H), 3.64 (s, 3H), 3.57 - 3.52 (m, 2H), 3.42 - 3.25 (m, 4H), 3.34 (s, 3H), 3.27 (s, 3H), 3.20 - 3.14 (m, 2H), 2.95 (s, 3H), 2.80 (s, 36H); d ¹³ C NMR (150 MHz, MeOD) d 171 .26,

167.97, 166.62, 139.60, 139.40, 139.03, 137.23, 134.93, 134.88, 134.77,

134.45, 131 .29, 130.10, 129.83, 129.67, 129.44, 129.32, 129.28, 129.20,

129.19, 129.14, 129.01 , 128.97, 128.86, 128.79, 128.73, 128.63, 127.75,

127.29, 127.01 , 126.87, 101 .98, 101 .85, 101 .81 , 99.57, 98.74, 96.72, 83.49, 83.18, 81 .1 1 , 78.95, 78.82, 78.66, 78.24, 76.97, 76.71 , 76.44, 76.17, 76.09, 75.98, 75.33, 75.00, 74.81 , 74.49, 73.38, 72.98, 72.00, 71 .56, 71 .20, 71 .1 1 , 68.32, 66.50, 65.56, 65.51 , 65.03, 64.08, 63.67, 56.21 , 53.49, 52.71 , 52.52, 45.60; LRMS (ESI) calcd for CiozHnsNgO^ [M-4H] ⁴ m/z -470.3, found -470.2.

Example 21

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

To a mixture of A-18 (42.3 mg, 150 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 30 eq., 6 eq./per OH, 4.5 mmol, 625 mg) and 1 ,2- dichloroethane (DCE, 2 ml_) was added A/,A/-dimethylformamide (DMF, 3 ml_) under Argon. The resulting mixture was heated at 60 °C for 16 h. Upon cooling, the mixture was co -evaporated with chlorobenzene (5 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex G-25 size exclusion chromatography. The resulting pure product was obtained as a white solid with trimethyllammonium salt which was dissolved in de-ionized water and directly subjected to ion-exchange column (Na form of DOWEX 50Wx8). The combined fractions were lyophilized to yield the final sulfated product P-18 as sodium salt (88 mg, 65.6% yield). ¹ H NMR (400 MHz, D ₂ 0) d 5.99 (d, J = 3.3 Hz, 0.4H, H-1 b), 5.84 (s, 0.6H, H-1 a), 5.23 (s, 0.6H, H-1 '), 5.16 (s, 0.4H, H-1 '), 4.92 - 4.48 (m, 5.2H), 4.45 (dd, J= 9.3, 3.4 Hz, 0.4H), 4.35 (d, J = 1 1 .8 Hz, 0.6H, H-5a), 4.10 - 3.85 (m, 3.2H); ¹³ C NMR (100 MHz, D ₂ 0) d 99.69 (C-1 '), 97.63 (C-1 '), 95.53, 95.37 (C-1 b), 75.65 (C-1 a), 74.95, 74.51 , 74.38, 73.67, 72.39, 71 .88, 71 .43, 71 .29, 71 .21 , 71 .08, 70.81 , 60.85 (C-6), 59.14 (C-6), 58.59 (C-6), 58.47 (C-6); HRMS: Intensity of signals was decreased dramatically due to serious decomposition. Only de-sulfated species were found in spectrums of HRMS. HRMS (ESI) calculated for CI OHI ₈ 0 ₂₇ S ₆ [M-1 xS0 ³ -5H+3Na] ² m/z -372.9047, found -372.9050; [M-2xS0 ³ - 4H+2Na] ² m/z -321 .9353, found -321 .9360.

Example 22

O-Sulfation Approach - Performing in co-solvent system (DMF/DCE)

[001 15]

To a mixture of A-19 (28 mg, 94.5 mitioI), sulfur trioxide trimethylamine complex (SO ₃ .TMA, 25 eq., 5 eq./per OH, 2.36 mmol, 328 mg) and 1 ,2-dichloroethane (DCE, 1 .4 mL) was added A/,A/-dimethylformamide (DMF, 2.1 mL) under Argon. The resulting mixture was heated at 70 °C for 1 h. Upon cooling, the mixture was co-evaporated with chlorobenzene (8 ml_) for 3 cycles to remove DMF under reduced pressure. This crude residue was further purified through Sephadex G-25 size exclusion chromatography. The resulting pure product was obtained as a white solid with trimethylammonium salt which was dissolved in de-ionized water and directly subjected to ion-exchange column (Na form of DOWEX 50Wx8). The combined fractions were lyophilized to yield the final sulfated product P-19 as sodium salt (36 mg, 47.3% yield). ¹ H NMR (400 MHz, D ₂ 0) d 5.18 (s, 1 H, H-1 '), 4.90 (s, 1 H , H-1 ), 4.85 - 4.81 (m, 2H, H-3', H-3), 4.58 - 4.54 (m, 1 H, H-4'), 4.54 - 4.49 (m, 2H, H-5a', H-2'), 4.41 (s, 1 H, H-2), 4.13 (dd, J= 13.2, 2.0 Hz, 1 H, H-5a), 3.96 -3.80 (m, 3H, H-4, H-5b', H-5b), 3.46 (s, 3H); ¹³ C NMR (100 MHz, D ₂ 0) d 99.49 (C-V), 98.54 (C-1 ), 74.15 (C-4), 72.34 (C-2'), 71 .82 (C-2), 71 .80 (C-3), 71 .40 (C-4'), 71 .16 (C-3'), 58.55 (C-5'), 58.00 (C-5), 55.55 (OCH3); HRMS: calculated for CI I H ₂ O0 ₂₄ SS [M-5H+3Na] ^-2 m/z - 379.9125, found -379.9140.

Example 23

Microwave-assisted N-Sulfation Approach for Synthesis of Hexasaccharide

[001 16] To a mixture of A-20 (1 1 .5 mg, 7.57 mitioI), MeOH/H ₂ 0 (v/v, 1 /1 , 1 mL/1 ml_), triethylamine (TEA, 10 eq./per NH ₂ , 30 eq., 0.23 mmol, 32 mI_) was added sulfur trioxide trimethylamine complex (SO ₃ .TMA, 24 eq., 8 eq./per NH ₂ , 0.182 mmol, 25.2 mg). The resulting mixture was subject to microwave irradiation at 80 °C for 10 min. Upon cooling, add another portion of triethylamine (TEA, 5 eq./per NH _2, 15 eg, 0.1 15 mmol, 16 uU and SO3 MA complex (12 eg, 4 eg./per NH _2, 0.091 mmol, 12.6 mg). The resulting mixture was heated under microwave irradiation at 80 °C for 10 min. Repeat the procedure underlined as above for another 2 cycles. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to give the crude product which was dissolved in water and passed through an ion-exchange column of DOWEX 50W-X8 (Na form). The collected fractions were concentrated to give the product mixture as sodium salt which was further purified through Sephadex G25. The collected fractions were lyophilized to furnish the product P-20 as a white solid with sodium salt (6.2 mg, 51.4 % yield). ¹ H NMR (600 MHz, D ₂ 0) ¹ H NMR (600 MHz, D ₂ 0) d 5.67 (d, J= 4.0 Hz, 1 H, C-1 A), 5.67 (d, J= 4.0 Hz, 1 H, C-1 c), 5.39 (d, J= 3.6 Hz, 1 H, C-1 _E ), 5.10 (d, J= 1.6 Hz, 1 H, C-1 _F ), 4.66 (d, J= 7.8 Hz, 1 H, C-1 _{B o} r _D ), 4.66 (d, J= 7.8 Hz, 1 H, C-1 B or D), 4.55 - 4.48 (m, 3H), 4.41 (dd, J = 1 1.1 Hz, J = 2.3 Hz, 1 H), 4.30 - 4.18 (m, 5H), 4.15 - 4.10 (m, 1 H), 4.10 - 4.05 (m, 2H), 3.96 - 3.76 (m, 11 H), 3.73 (appt, J= 9.9 Hz, 1 H), 3.67 (appt, J= 9.7 Hz, 1 H), 3.62 (dd, J= 10.7 Hz, J = 8.4 Hz, 1 H), 3.46 (s, 3H), 3.43 (dd, J = 9.5 Hz, J = 7.9 Hz, 1 H), 4.36 - 4.32 (m, 2H), 3.30 (dd, J = 10.1 Hz, J = 3.7 Hz, 1 H); ¹³ C NMR (150 MHz, D ₂ 0) d 175.07, 174.81 , 101.78 (C-1 B/D), 99.63 (C-1 _F ), 97.51 (C-1 _Aor c), 97.18 (C-1 _Aor c), 96.83 (C-1 E), 77.04, 76.79, 76.65, 76.59, 76.41 , 76.28, 75.89, 75.85, 75.70, 75.16, 72.79, 72.73, 71.07, 69.72, 69.35, 69.30, 68.93, 68.79, 68.57, 68.06, 67.80, 66.25, 65.86, 65.74, 57.91 , 57.47, 55.33; HRMS (ESI) calcd for C37H61 N3O52S7 [M-10H+8Na] ² - m/z -888.4332, found -888.4316; [M-8H+6Na] ² - m/z -866.4513, found -866.4500; [M-5H+3Na] ² - m/z -833.4784, found - 833.4763; [M-10H+7Na] ³ - m/z -584.6256, found -584.6244.

Example 24

Microwave-assisted N-Sulfation Approach for Synthesis of Hexasaccharide

[001 17] To a mixture of A-21 (9.9 mg, 7.0 mitioI), MeOH/hhO (v/v, 1 /1 , 1 ml_/1 ml_), triethylamine (TEA, 10 eq./per Nhh, 30 eq., 0.23 mmol, 32 mI_) was added sulfur trioxide trimethylamine complex (SO3.TMA, 24 eq., 8 eq./per NH2, 0.168 mmol, 23.3 mg). The resulting mixture was subject to microwave irradiation at 80 °C for 10 min. Upon cooling, add another portion of triethylamine (TEA, 5 eq./per NH2. 15 eg, 0.105 mmol, 15 uU and SOa.TMA complex (12 eg, 4 eq./per NH2. 0.084 mmol, 1 1 .6 mg). The resulting mixture was heated under microwave irradiation at 80 °C for 10 min. Repeat the procedure underlined as above for another 2 cycles. Upon cooling, the mixture was co-evaporated with chlorobenzene (5 ml_) for 3 cycles to give the crude product which was dissolved in water and passed through an ion-exchange column of DOWEX 50W-X8 (Na form). The collected fractions were concentrated to give the product mixture as sodium salt which was further purified through Sephadex G25. The collected fractions were lyophilized to furnish the product P-21 as a white solid with sodium salt (8.2 mg, 68.0 % yield). ¹ H NMR (600 MHz, D2O) d 5.81 - 5.74 (m, 3H, H-1 _A , H-1 c, H-1 E), 4.74 (d, J= 7.9 Hz, 2H, H-1 _B , H-1 _D ), 4.63 - 4.57 (m, 2H), 4.55 (d, J= 8.1 Hz, 1 H, H-1 _F ), 4.50 (dd, J= 1 1 .1 Hz, J= 2.1 Hz, 1 H), 4.36 - 4.27 (m, 3H), 4.19 - 4.1 1 (m, 2H), 4.06 - 3.85 (m, 10H), 3.84 - 3.78 (m, 4H), 3.77 - 3.71 (m, 2H), 3.69 (s, 3H), 3.47 - 3.37 (m, 4H), 3.46 - 3.42 (m, 2H), 3.40 (dd, J= 10.2 Hz, J= 3.7 Hz, 1 H); ¹³ C NMR (150 MHz, D ₂ 0) d 175.07, 175.02, 103.09 (C-1 _F ), 101 .85 (C- D), 1 01 .79 (C-1 _B or _D ), 97.51 (C-1 AorCorE), 97.14 (C-1 A or c or E) , 96.91 (C-1 A or c or E), 77.13, 76.78, 76.56, 76.42, 76.37, 76.34, 76.24, 75.89, 75.83, 72.78, 72.54, 71 .07, 69.72, 69.36, 68.92, 68.56, 68.50, 66.24, 65.74, 57.91 , 57.44, 57.37, 57.15; HRMS (ESI) calcd for C37H61 N ₃ 0 ₄₉ S ₆ [M-5H+3Na] ² m/z -793.5000, found -793.4976; [M-2H] ² m/z - 760.5271 , found -760.5254; [M-9H+6Na] ³ - m/z -550.6460, found -550. 6453. Example 25

O-Sulfation Approach - Post-Sulfation Precipitation (DMF/DCM)

[001 18] The mixture of D-Melezitose monohydrate A-22 (18.07 g, 34.6 mmol), SO ₃ .TMA (264.5 g, 1 .903 mol, 5 eq./per OH, 55 eq.) and anhydrous DMF (1 142 ml_) was degassed under Ar for three times and heated at 70 °C for 18 h. Upon cooling, 500 ml_ of anhydrous DCM was added into the resulting mixture. The vigorous stirring with DCM enforces more precipitation of solid out of reaction solution. The precipitate formed as trimethylammonium salt was filtered off by sinter funnel, washed with 2 cycles of DCM (200 ml_) and dried over vacuum to yield the crude product. The resulting solid was dissolved in de ionized water and directly subjected to ion-exchange column Na form of DOWEX 50Wx8 and further purified through SEC Sephadex G25. The collected fractions were lyophilized to furnish the product P-22 as a white solid with sodium salt (36.9 g, 65.7%). ¹ H NMR (400 MHz, D ₂ 0) d 5.89 (d, J= 3.8 Hz, 1 H, H-1 ^* ), 5.76 (d, J = 3.7 Hz, 1 H, H-1 ^* ), 4.90 - 4.83 (m, 2H)), 4.82 - 4.72 (m, 3H), 4.64 - 4.37 (m, 1 1 H), 4.29 (s, 2H, H-1 'a, H-1 'b), 4.14 (d, J= 9.9 Hz, 1 H, H- 3'); ¹³ C NMR (100 MHz, D ₂ 0) d 102.62 (C-2'), 95.91 (C-1 ^* ), 89.40 (C-1 ^* ), 79.50, 78.92, 77.88, 75.81 , 75.42, 74.39, 74.1 1 , 73.84, 73.41 , 69.38 (C-6'), 68.83 (C- 3'), 68.79, 66.25 (C-6 ^* ), 65.97 (C-6 ^* ), 65.82 (C-1 '). Note ^* : Highly congested proton NMR spectra are intractable to detailed analysis and assignment; HRMS: calculated for Ci ₈ H ₃₂ 0 _4g Sn [M-1 1 H+9Na] ^_ m/z -789.7579, found - 789.7553. [001 19] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00120] The use of the terms“a” and“an” and“the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,”“having,”“including,” and“containing” are to be construed as open-ended terms (i.e. , meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00121 ] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by those of ordinary skill in the art to which this invention belongs.

[00122] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. It is expected that skilled artisans will employ such variations as appropriate and it is considered within the scope and spirit of the present invention for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

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