Chemical approaches to the sulfation of small molecules: current progress and future directions

Sulfur has been recognised as one of the earliest known elements for its therapeutic properties by the ancient Greeks [1]. It is one of the essential components for all living organisms, occurring in the amino acids, methionine and cysteine, and in the vitamins, thiamine and biotin, amongst others [1,2]. Approximately, 250 drugs bearing sulfur-containing functional groups are approved by the FDA, including for hypertension, diabetes mellitus, bacterial infections, migraine, cardiovascular diseases (CVD), neurological disorders, cancer, and human immunodeficiency virus (HIV) (Figure 1) [1–4].

Sulfur is the tenth most abundant element in nature, accounting for approximately 0.03% to 0.06% of the earth’s crust by weight [1]. The sulfur atom exists in multiple oxidation forms, ranging from -2 to +6. Sulfur is present predominantly in the +6-valance state as the sulfate form in the earth's atmosphere.

Sulfur trioxide (SO₃) is a key precursor to sulfate and present in oleum, a solution of sulfur trioxide (25–65%) in sulfuric acid (H₂SO₄) [5]. Moreover, sulfur trioxide is a Lewis acid and engages in reactions with Lewis bases such as trimethylamine (Me₃N), triethylamine (Et₃N), dioxane, and pyridine (Py). These reactions result in the formation of sulfur trioxide adducts, which are employed in the sulfation process of various organic substrates, leading to the formation of organosulfate esters (Figure 2) [6].

Sulfation is an important conjugation reaction during the phase II metabolism of xenobiotics [7]. The sulfation process which mainly occurs in the liver, increases the hydrophilicity of metabolites, facilitating their elimination from the body [8]. Sulfate conjugation usually results in reducing the biological activity of the metabolite [9] but not always. Sulfation can increase the therapeutic activity of certain drugs including minoxidil sulfate, the active form of minoxidil used for the treatment of hypertension and hair loss [10,11]. The sulfation reaction is catalysed by a group of enzymes called sulfotransferases (SULTs), which facilitate the transfer of the sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the universal sulfate donor, to a nucleophilic site of an acceptor molecule such as phenol (Scheme 1) [12–14].

Since the sulfation of the antiseptic, phenol, was discovered in the urine of a patient in 1876 by Eugen Baumann [15] – the role of sulfated small molecules in man has evolved from a detoxification step to critical signalling mechanism. Thus, methods to access these important sulfated biomolecules are critical to understanding the intricacies of the sulfate lifecycle.

Organic O-sulfates and N-sulfamates have several crucial biological applications, ranging from the metabolism of xenobiotics to the downstream signalling of steroidal sulfates in pathological conditions [16]. Anti-coagulant, anti-viral, anti-inflammatory, immunomodulatory, and anti-tumour properties have been associated with sulfated polysaccharides, flavonoids, steroids, and proteins. Heparin and heparan sulfate are examples of glycosaminoglycans (GAGs) that contain sulfate groups which promote ionic molecular interactions with protein ligands and binding at the cellular surface [17]. The incorporation of polar, hydrophilic sulfate groups onto drug-like molecules has facilitated the investigation of novel sulfated biomolecules as potential new therapies [18]. However, the chemical synthesis and purification of sulfated compounds are challenging when one or more sulfate groups are present, due to both anionic crowding, lack of regioselectivity, and poor solubility in organic solvents [19,20]. Successfully sulfated compounds remain sensitive to both acidic and high temperature reaction environments [21]. As a result, the sulfation reaction is often the last step in a synthetic process, which restricts further potential chemical modifications [22].

Given the growing interest in sulfation and the significant biological functions of sulfated molecules, several synthetic approaches to sulfate oxygen, nitrogen, oxime, and phosphate functionalities have been developed. This Essay will explore the most commonly encountered and latest chemical sulfation approaches that have been applied to a wide range of small molecules since the last major review in 2010 [23].

Sulfuric acid (H₂SO₄) has been employed for the sulfation of (cyclo)alkenes at modest temperature and pressure resulting in (cyclo)alkyl sulfates [6] and the sulfation of polysaccharides and flavonoids [24,25]. The high reactivity of sulfuric acid can be modified via the sulfamic acid (H₂NSO₃H) reagent, which has been used for the sulfation of saturated alcohols, carbohydrate, and flavonoids including the naturally occurring triterpenoid betulin. Betulin is found in the bark of birch trees and possesses anti-viral, anti-inflammatory, anti-oxidant, and anti-coagulant properties [26]. Betulin was sulfated using the sulfamic acid method in the presence of a urea catalyst and alternatively DMF or 1,4-dioxane as solvent. Dual sulfation of betulin was achieved and isolated as the ammonium, sodium, and potassium salts (Scheme 2).

An alternative approach utilises chlorosulfonic acid, which has been applied for the sulfation of polysaccharides, phenolic acids, and flavonoids amongst others. Agarose sulfate, an example of a sulfated seaweed polysaccharide which was proposed to have an anti-coagulant activity comparable to heparin. Youping and co-workers have reported the formation of agarose sulfate using the chlorosulfonic acid protocol [27]. Agarose polysaccharide was dissolved in formamide solvent followed by the addition of chlorosulfonic acid/pyridine solution and stirred for 4 h at 65°C affording agarose sulfate (Scheme 3).

A modified version of chlorosulfonic acid-pyridine procedure was implemented using chlorosulfonic acid followed by cation exchange with tetrabutylammonium hydrogensulfate (Bu₄NHSO₄) [28,29]. The sulfation reaction of different organic molecules such as phenols, benzyl alcohols, anilines, and benzylamines has been accomplished using this modified protocol (Scheme 4).

The sulfation reaction using sulfur trioxide amine/amide complexes is the most used method for alcohol or phenol groups in carbohydrates, flavonoids, steroids, proteins, and related scaffolds. Ball and co-workers have successfully used a sulfur trioxide trimethylamine complex (Me₃N·SO₃) and a lipophilic cation-exchange to access Avibactam®, a sulfate containing β-lactamase inhibitor [30]. They reported a simultaneous one-pot deprotection and sulfation reaction of a hydroxylamine intermediate using the Me₃N·SO₃ complex. The resulting intermediate was ion exchanged with tetrabutylammonium acetate (TBAOAc) making it more lipophilic and therefore facilitates the extraction of the organosulfate intermediate into DCM. Finally, a lipophilic sodium salt exchange reagent, sodium-2-ethyl-hexanoate (NEH) was added, affording Avibactam® in 90% yield as the sodium salt (Scheme 5).

The Me₃N·SO₃ complex was reported for the site selective sulfation of polysaccharides including pyranoside scaffold [31]. The site selective sulfation of pyranoside scaffolds was carried out under catalytic conditions using diarylborinic acids [32,33]. This method led to the sulfation of different hydroxyl group positions including cis-1,2-diol and 1,3-diol pyranoside derivatives. β-Thioglycoside pyranoside was selected as a model for the site selective sulfation using Me₃N·SO₃ and the addition of diarylborinic acid and DIPEA was critical to improve the isolated yield (Scheme 6) [31].

The Me₃N·SO₃ complex has also found use in the preparation of sulfamates which contain polar functional groups that are significant to a wide range of biological functions, including viral infection and protein–protein interactions. The sulfamation reaction of benzylamine derivatives were carried out using Me₃N·SO₃ complex under a low reaction temperature (30–60°C) affording the corresponding trimethylammonium cation [34]. This was subsequently exchanged with a more lipophilic counterion, tributylamine, then exchanged with a sodium salt (NEH) leading to the formation of sulfamates (Scheme 7) and employed in related examples [35].

The use of Me₃N·SO₃ complex was also reported in the sulfation of resveratrol, a naturally occurring polyphenolic that is found in peanuts, grapes, and berries [36]. It was reported that resveratrol has gained more interest due its important biological applications such as anti-inflammatory, anti-cancer, and anti-oxidant activity. The sulfation reaction of resveratrol was carried out using Me₃N·SO₃ complex in the presence of base (Et₃N) at reflux affording the potassium salt of sulfated resveratrol (Scheme 8).

A summary of the scope and range of sulfated molecules achievable with Me₃NSO₃ complex are listed in Table 1.

An alternative sulfur trioxide amine complex is triethylamine SO₃ complex (Et₃N·SO₃), which was also used for the sulfation of a wide range of small organic molecules such as flavonoids, polysaccharides, proteins, and steroids. For instance, the sulfation of polyphenolic flavonoids such as protocatechuic acid (PCA), quercetin, and catechin were reported using Et₃N·SO₃ complex [37]. PCA sulfate lower the production of interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (VCAM-1), pro-inflammatory cytokine genes that are linked to cardiovascular diseases [38]. Gutierrez-Zetina and co-workers have described the mono-sulfation reaction of PCA using superstoichiometric equivalents of Et₃N·SO₃ (10-fold) at 40°C for 3 h. As a result, two compounds were isolated as PCA-3-sulfate and PCA-4-sulfate, respectively (Scheme 9) [37].

The mono-sulfate analogues of quercetin and catechin were also prepared following the mono-sulfation reaction procedure used with PCA employing Et₃N·SO₃ as a source of the sulfate group [39]. Correia-da-Silva and co-workers have investigated the synthesis of persulfated compounds which can be used as an anticoagulant and antiplatelet agents for the treatment of thrombosis [40]. Sulfation of different polyphenolic molecules such as gallic acid, 4-methyl 7-hydroxycoumarin 7-ß-D-glucopyranoside, and ascorbic acid was afforded using Et₃N·SO₃ (2–8 eq./OH group) in dimethylacetamide (DMA) at 65°C for 24 h reaction duration. A summary of the scope and range of sulfated molecules achievable with Et₃NSO₃ complex are listed in Table 2.

During the writing of this Essay, single crystal X-ray crystallography confirmed that triethylamine-sulfur trioxide complex also exists as triethylsulfoammonium betaine (TESAB) not a dative bond adduct [41].

A novel sulfating reagent was recently developed by Gill and co-workers, tributylsulfoammonium betaine (TBSAB) (Scheme 10) [42].

TBSAB provides a simplified purification and isolation of sulfated molecules due to the greater lipophilicity profile of the corresponding tributylammonium intermediate (log₁₀P = 4.01) [43]. The scope of the TBSAB reagent is demonstrated in Scheme 11.

TBSAB was used for the sulfation and sulfamation of a wide range of benzyl alcohols, benzylamines, amino acids, and carbohydrates [44,45]. TBSAB has also been used for the chemoselective sulfation of steroids affording the corresponding sulfated steroid molecules such as, estrone sulfate, pregnenolone sulfate, and pregnanediol sulfate [46]. TBSAB has also found use as a reagent to install aniline N-sulfamates and ylideneamino sulfates prior to their intermolecular rearrangement [47,48]. A summary of the scope and range of sulfated molecules achievable with Bu₃NSO₃ complex are listed in Table 3.

Alternative sulfur trioxide complexes involving Py·SO₃ and DMF·SO₃ were used for the sulfation of polysaccharides and polyphenolic flavonoids [49]. Sun and co-workers reported the installation of sulfates and sulfamate moieties onto protected heparan sulfate oligosaccharide derivatives using Py·SO₃ complex. The preparation of a sulfated and sulfamated tetrasaccharide substrate was achieved despite multiple reaction steps and the use of several protecting groups which added complexity to the sulfation process (Figure 3) [50]. Xu and co-workers have reported the use of an efficient microwave-assisted approach for the simultaneous O,N-sulfation of heparin and heparan sulfate saccharides despite the multi-step reaction synthesis including protection/deprotection steps. In this approach, both Et₃N·SO₃ and Py·SO₃ sulfating agents were employed in a solvent mixture of Et₃N/pyridine at between 55–100°C for 15–45 min. This approach was appropriate to install sulfate/sulfamate moieties onto mono-, di-, tri- and tetra-saccharides in a short reaction time and good to excellent isolated yields (Scheme 12) [51].

Cellulose sulfate is an example of sulfated polysaccharide which has been studied for its potential biological and pharmaceutical applications such as anti-coagulant, anti-microbial, anti-oxidant, and used in drug delivery systems. Richter and co-workers have described the regioselective sulfation of the trimethylsilyl cellulose (TMSC) using sulfur trioxide complexes including Py·SO₃, DMF·SO₃, and Et₃N·SO₃ [52]. The resulting intermediate was treated with sodium hydroxide yielding the final desired sodium cellulose sulfate as the sodium salt (Scheme 13).

Malins and co-workers have reported the use of Py·SO₃ and DMF·SO₃ complexes for the preparation of sulfated xylooligosaccharides that could be a promising therapeutic agent similar to the known exemplar, pentosan polysulfate (PPS) [53]. Pentosan polysulfate is a semi-synthetic polysulfated xylan that is related to glycosaminoglycans (GAGs) containing β-1→4-linked xylooligosaccharides and was approved for the treatment of interstitial cystitis (inflammation of bladder) (Figure 4) [54].

An initial attempt for the sulfation of xylooligosaccharides was examined on different substrates such as xylose, methyl-β-xyloside, and xylobiose used Py·SO₃ [53]. Unfortunately, the β-1→4 linkage of xylan derivatives was cleaved by the nucleophilic addition of pyridine (Scheme 14) [55].

Due to the previous unsuccessful attempt with Py·SO₃, DMF·SO₃ complex was used for the sulfation of xylobiose [53]. The sulfation of xylobiose with DMF·SO₃ afforded xylobiose hexasulfate and xylobiose pentasulfate, a reduced sugar, which was formed via hydrolysis of the hexasulfate product (Scheme 15).

Malins. has also reported an in situ synthesis of DMF·SO₃ complex using the strategy of the addition of methyl chlorosulfate to DMF at 0°C. This reaction is exothermic leading to the formation of methylchloride as a side product [53]. This protocol was initially investigated on the methyl-α-glucoside following the optimised conditions, affording the methyl-α-glucoside tetrasulfate in 47% isolated yield. This protocol was further explored on a wide range of small molecules including amino acids, disaccharide, steroids, and acid-sensitive substrates (Scheme 16).

During the writing of this Essay, Yue and co-workers have reported an innovative strategy for the O-sulfation using dimethyl sulfate (DMS) and diisopropyl sulfate (DPS) [56]. This strategy requires tetrabutylammonium bisulfate (Bu₄NHSO₄) [57] to activate the dialkyl sulfates, which facilitates the sulfation of a wide range of functional groups, including alcohols, phenols, and oxime containing substrates. Furthermore, tetrabutylammonium bisulfate improves the solubility of the sulfated products in organic solvent. This investigation was initiated with the reaction of 3-phthalimido-1-propanol and DMS affording the tetrabutylammonium 3-phthalimido-1-propanol sulfate in 84% isolated yield (Scheme 17).

Recently, early stage O-sulfation reaction between aryl fluorosulfates and silyl ethers (Scheme 18) was reported leading to the formation of sulfuric acid diesters, which are subsequently deprotected to the target sulfates via a hydrogenolysis step [58]. This strategy was applied to a wide range of small molecules such as monosaccharides, disaccharides, an amino acid, and a steroid by Liu and co-workers (Figure 5) [58].

Despite the effectiveness of direct sulfation methods, the formation of complex sulfated molecules may be hampered by the poor solubility of sulfated molecules in organic solvents, stability issues, and purification challenges of sulfated molecules [23]. As a result, there was a growing interest in developing protection/deprotection strategy which involves the incorporation of sulfate group(s) in a masked form into the target scaffold followed by a deprotection step affording the final sulfate molecules [23,59]. Simpson and co-workers have used alkyl protecting groups such as isobutyl (iBu) and neopentyl (nP) groups for the preparation of sulfate monoesters [59]. In this method, phenol or alcohol containing substrates were initially treated with a strong base such as sodium hydride (NaH) or sodium hexamethyldisilazide (NaHMDS) at −75°C followed by the addition of isobutyl or neopentyl chlorosulfate affording the desired protected sulfate monoesters. Subsequent deprotection step using sodium azide or sodium iodide affording the desired sulfates in most cases (Scheme 19) [59].

Sulfation is one of the most important modifications that occur to a wide range of small biomolecules including polysaccharides, proteins, flavonoids, and steroids. The incorporation of a sulfate moiety to an appropriate substrate, results in an increase of the substrate’s hydrophilicity and therefore facilitate its elimination from the body. However, sulfated scaffolds such as polysaccharides, proteins, flavonoids, and steroids have significant biological and pharmacological roles such as cell signalling, modulation of immune and inflammation response, anti-coagulation, anti-atherosclerosis, and anti-adhesive properties. It is imperative that sulfated molecules can be easily prepared to further extend the biological understanding of the role of sulfate in the body. An emerging area of importance for analytical chemistry but outside the scope of this Essay is enzymatic modification for the synthesis of low molecular weight heparins, protein and carbohydrate sulfation [60–63].

This Essay summarised the most encountered chemical sulfation methods of small molecules (Figure 6). Traditional sulfation reactions have relied on sulfuric acid derivatives. Sulfation reaction using sulfur trioxide amine/amide complexes was the most used method for alcoholic or phenolic groups in carbohydrates, steroids, proteins, and aliphatic or alicyclic scaffolds. Despite the effectiveness of these methods, they suffer from some issues such as multiple-purification steps reactions, toxicity issues (e.g. pyridine contamination), purification challenges, stoichiometric excess of reagents which leads to increase of a reaction cost, and intrinsic stability issues of both the reagent and product.

Recent advances (Figure 7) including SuFEx, the in situ reagent approach and TBSAB show the widespread appeal of novel sulfating approaches that will enable a larger exploration of the field in the years to come by simplifying the purification and isolation to access bespoke sulfated small molecules.

• Sulfation of small molecules is implicated in critical biological signalling cascades and a key phase II drug metabolism step.

• Methods to prepare sulfated molecules to enable biological study are both limited and have practical disadvantages to isolate tractable quantities of the desired sulfate.

• Emerging methods that build upon the early work of amine-sulfur trioxide complexes (e.g. tributylsulfoammonium betaine, TBSAB), in situ preparation of reactive sulfating complexes, and SuFEx methodology are gaining traction in the sulfation community.

Tributylsulfoammonium betaine (TBSAB) is commercially available developed by the lead author's research group.

The authors thank King Khalid University, Saudi Arabia (JAA) for providing his PhD scholarship and supporting his studies.

J.A.A. and A.M.J. drafted and revised the manuscript.

DMA

dimethylacetamide

DMS

dimethyl sulfate

DPS

diisopropyl sulfate

NaH

sodium hydride

NaHMDS

sodium hexamethyldisilazide

PPS

pentosan polysulfate

TBSAB

tributylsulfoammonium betaine

TMSC

trimethylsilyl cellulose