Synthetic heparin and heparan sulfate: probes in defining biological functions

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ScienceDirect Synthetic heparin and heparan sulfate: probes in defining biological functions Ching-Ting Tsai1,2, Medel Manuel L Zulueta1,3 and Shang-Cheng Hung1 Heparin and heparan sulfate are glycosaminoglycans that modulate numerous biological processes. The desire to capture the structural diversity responsible for their functions provides notable issues during synthesis, including siteselective sulfonation, stereoselective glycosylation and the sheer number of probable targets at hand. With current advances in synthetic approaches, carbohydrate chemists generate these complex targets by chemical and enzymatic methods. Fondaparinux and a number of polysaccharides have been synthesized to probe anticoagulation and other biological functions. Moreover, a trove of structural information could be obtained by many analytical methods, which provide hints to the potential protein-binding sequences within the sugar chain. Further structure–activity relationship studies help unveil the secrets of the heparin/heparan sulfate code, providing potential candidates for drug development. Addresses 1 Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road, Taipei 115, Taiwan 2 Department of Chemistry, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan 3 Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines Corresponding author: Tsai, Ching-Ting ([email protected])

Current Opinion in Chemical Biology 2017, 40:152–159 This review comes from a themed issue on Synthetic biomolecules Edited by Peter H Seeberger and Beate Koksch

progression [2–5]. Structurally, they both contain alternating 1!4-linked a-D-glucosamine (GlcN) and either b-D-glucuronic acid (GlcA) or a-L-iduronic acid (IdoA) residues (Figure 1). Heparin is a common anticoagulant drug used to treat and counteract clots in the veins, arteries or lungs [6]. Although containing similar disaccharide units as HS, heparin carries more sulfate/sulfamate groups on average and exhibits a relatively more homogeneous functional group pattern on the sugar chain. Heparin is found in granules of mast cells while HS is ubiquitously located on the cell surface and the extracellular matrix. As with the differences in their location and sulfonation levels, heparin and HS each hold distinct functions in organisms [7]. By virtue of their structural diversity, heparin and HS play pivotal roles in cell physiology and diseases, prompting the critical study of their structure–activity relationships. Many aspects of the synthetic acquisition of heparin and HS have been summarized previously [8,9–12]. This review provides a brief discussion outlining the more recent advancement, including chemical and chemoenzymatic approaches, of heparin/HS synthesis. Given the progressive importance of the therapeutic application of heparin/HS derivatives, anticoagulation and other biological functions of heparin-related molecules are also addressed. Also, approaches toward the identification of rare sequence for specific protein interaction are summarized, which offer several promising ways to find potential targeted sequences for further applications.

http://dx.doi.org/10.1016/j.cbpa.2017.09.012

Chemical approaches

1367-5931/ã 2017 Elsevier Ltd. All rights reserved.

Chemical synthesis has the merit of defining many natural and even unnatural oligosaccharide sequences and guaranteeing substance homogeneity. To assemble oligosaccharides in an effective way, regular disaccharide units have been used to form longer skeletons. Given the nature of the GlcN and the uronic acid (UA) repeating units, chemists are inclined to synthesize heparin/HS with either UA–GlcN or GlcN–UA disaccharide precursor. Strategies have been progressively developed and refined in the past few decades [8,10,11]. Various aspects of the synthesis have been addressed and continuously being improved with two main issues being prominent. Stereochemical control in glycosidic bond formation is the first issue. While 1,2-trans glycosylation can be accomplished by neighboring group participation of ester group positioned at C2 of the uronic acid precursor, 1,2-cis

Introduction Proteoglycans are a family of multifunctional biomolecules ubiquitously present in extracellular matrices and cell surfaces. They comprise a core protein with at least one covalently attached glycosaminoglycan — a polydisperse linear polysaccharide with repeating disaccharide structure [1]. Heparin and heparan sulfate (HS) are types of glycosaminoglycans that are crucially involved in various biological processes, including regulation of anticoagulation, inflammation, angiogenesis and cancer Current Opinion in Chemical Biology 2017, 40:152–159

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Synthetic heparin and heparan sulfate: probes in defining biological functions Tsai, Zulueta and Hung 153

Figure 1

OH O O HO

–

OR3 O

O2C O

NH O Ac HO

O

OR

4

–

NH

O

R

R2

1

m

OH O

O2C O

O HO

Variable sequence

OH

O HO

–

O2C

NH O Ac HO n

HO O

OH O

O

HO

OH O

OH

OH

Major component

HN O

O OH HO

O

OR3 O

–

O HO

O2C NH O Ac HO

O OR4

O O

NH R2

Variable sequence R1 = H, Ac or CO3–, R2 = H or CO3– R3 = H or CO3–, R4 = H or CO3–

OCO3– O

R

1

m

O HO

–

Ac

NH

Tetrasaccharide bridge

OCO3– O

O2C

O NH HO

O

OH

Heparan sulfate proteoglycan

OH O

O

O

O

HO

O – O3C

NH –

O3C

–

O2C OH O O HO

O –

Core protein

O3CO

Antithrombin III-binding sequence

OCO3– O NH –

O3C

–

O2C OH O O HO

O –

O3CO

OCO3– O NH O –

O3C

n

Major component

Heparin Current Opinion in Chemical Biology

General structures of heparan sulfate as a proteoglycan component and heparin.

glycosylation involving the GlcN derivative is quite difficult. The nonparticipating C2-azido group is often utilized in this case, relying mainly on the anomeric effect. Other steric effect and remote group participation were also used to help increase preference for a-glucosaminylation. Another critical point is the regioselective protection of the hydroxy and amino groups to enable sitespecific sulfonation (or acetylation, if targeting an acetamido group). Temporary protection such as ester groups may be chosen to mask alcohols that would be sulfonated [13] and other more robust groups act as permanent protecting groups, which could be removed at the later stage of the synthesis to unveil the free hydroxyls. With maturing retrosynthetic tactics and protection strategies, longer sugars have been achieved. Iterative convergent approaches [14–19,20] are usually incorporated during the synthesis to obtain long oligo-/polysaccharides, even allowing access to gram-scale amounts of heparanoid dodecasaccharide mimetics [16]. Because disaccharide iterative coupling may suffer from lengthy preparation process and possible cumulative isomerism, tetrasaccharide iterative coupling strategy provides a relatively better way to access longer sugar chains. Gardiner et al. accomplished the assembly of heparin-based oligosaccharide backbones extending up to 40-mer by tetrasaccharide block-wise iterative approach (Figure 2) [20]. In dealing with the number of functional groups of such a big molecule, technical issues are expected to be encountered in the final transformation steps. Nevertheless, the same group successfully transformed a 20-mer derivative www.sciencedirect.com

into the fully functionalized heparin-like chain. Such long sugars can mimic certain behaviors of natural heparin, and thus, allow further understanding of its biological functions. Still, fundamental challenges remain associated with the functional group conversions in long chain derivatives, particularly about keeping track of the complete formations of the desired carboxylate and sulfate or sulfamate moieties. To understand the role of glycoproteins in physiology, synthetic glycopeptides provide a relatively simple way to define their structures [21]. Although the synthesis of glycopeptides bearing N-glycans and mucin-type O-glycans has been widely studied, the preparation of proteoglycans, let alone glycopeptides based on it, is rarely reported. This is mainly because of the difficulties in glycosylation, peptide elongation and deprotection brought about by the complexity of glycosaminoglycans as well as the specific tetrasaccharide that act as bridge to the core protein [22]. The first synthesis of glycopeptides derived from syndecan-1 with one attached sugar chain has been achieved by Huang et al. through careful choice of linkage formations and deprotection sequence [23]. In combination with partial deprotection strategy, the same group also assembled glycopeptides based on syndecan-3, bearing two HS tetrasaccharide chains each linked to the peptide by a tetrasaccharide bridge [22,24]. The presence of two glycan chains provided more complexity that compromised stability toward some final deprotection procedures. Although there are still plenty of improvements needed, these works built a solid foundation for Current Opinion in Chemical Biology 2017, 40:152–159

154 Synthetic biomolecules

Figure 2

OBn O MeO2C H

N3O

O BnO

OBn O O BnO

OBn O MeO2C N3 O

BzO n = 0, 1, 2, 3, 4, 5, 6 OBn O MeO2C TCAO BnO

N3

OBn O

OBn O MeO2C

N3 O

O BnO

BzO

OBn O SPh

N3 O

OBn OMe O 6

n

BzO

OBn O O BnO

O

OBn O MeO2C

MeOH, pyridine

BzO

BzO NIS, AgOTf

OBn O MeO2C TCA

O BnO

N3 O

OBn O O BnO

OBn O MeO2C N3 O

BzO

OBn O MeO2C OBn O

BzO

N3O

O BnO

OBn OMe O

BzO

6

n+1

n = 0, 1, 2, 3, 4, 5, 6 Using n = 1 (20-mer) 1. LiOH, 48% 2. SO3•NMe3, dimethylformamide, 90% 3. H2, Pd(OH)2, 89% 4. SO3•pyridine, H2O OH OH

–O C 2

O –O S 3

O HO

NH –O S 3

O

O

OH O

OBn O O HO

–O SO 3

–O C 2

NH –O S 3

O

–O SO 3

OH O

O HO

–O C 2

NH –O S 3

O

OH OMe O

–O SO 3

6

2 Current Opinion in Chemical Biology

Iterative convergent synthesis of long-chain heparin/heparan sulfate. Abbreviations: Bn, benzyl; Bz, benzoyl; NIS, N-iodosuccinimide; TCA, trichloroacetyl; Tf, trifluoromethanesulfonyl.

targeting more complex HS glycopeptide/proteoglycan structures. Several glycomimics were also reported as potential alternatives to the typically linear heparin/HS. Taking advantage of multivalent interaction to help biomolecular recognition [25], single-entity tetravalent glycomimics capped with different sugar sequence up to tetrasaccharides were prepared [26]. In this case, the sugar component was attached through its amino-terminated linker to the N-hydroxysuccinimide-activated tetramer dendritic core. Also, tailored glycopolymers, which were generated by ring-opening metathesis polymerization (ROMP), Current Opinion in Chemical Biology 2017, 40:152–159

displayed the ability of modulating the activity of several heparin-related proteins including those related to anticoagulation [27,28].

Chemoenzymatic approaches Chemoenzymatic synthesis features a highly stereoselective, site-selective and mostly protecting group-free method in polysaccharide production. Compared to chemical synthesis, chemoenzymatic method provides a relatively simple way to produce long-chain heparinbased and HS-based sugars [29]. The substrate for heparin or HS synthesis may come from Escherichia coliexpressed glycosaminoglycans or bacterial heparosan, www.sciencedirect.com

Synthetic heparin and heparan sulfate: probes in defining biological functions Tsai, Zulueta and Hung 155

which could be subjected to enzymatic modification to possess particular recurring structural patterns. However, ensuring a single composition for the heparosan starting material and uniform transformations of the multiple enzyme modification sites are difficult. Thus, the usually heterogeneous product may put restrictions on further studies looking at the precise relationship of structure and function. There are also major hurdles in the generation of irregular sequences, which requires differentiation of similar sites on identical sugar units present in the chain. Using synthetic molecules as starting material help obviate the multiple components issue. Several classes of enzymes are involved in the chemoenzymatic acquisition of heparin/HS [9,29]. To construct the sugar backbone, the bacterial glycosyltransferases KfiA and PmHS2 allow the alternating transfer of GlcN and GlcA residues, respectively, from their uridine diphosphate forms extending toward the nonreducing end. C5-epimerase is used to epimerize GlcA toward IdoA in either a reversible and irreversible manner. Different sulfotransferases, each type occurring in different isoforms, are in charge of site-selective sulfonation [30]. The inherent considerations here are the proper design of starting materials and the meticulous arrangement of these enzymatic transformations. One pot chemoenzymatic synthesis was also developed, which enabled the preparation of complex heparin/HS polysaccharides from heparosan [31]. To achieve maximum synthetic efficiency, chemical methods are incorporated in the enzymatic synthesis. The synthesis of diazoacetyl-functionalized HS oligomer [32], preparation of biotinylated heparosan hexasaccharide in a one-pot fashion [33] and divergent enzymatic Osulfonation of hexasaccharides from the same backbone [34] demonstrate the flexibility of this incorporation. In the first two examples, the sugar chains were elongated by bacterial glycosyltransferases starting from anomerically functionalized GlcA residues. Moreover, the N-trifluoroacetylated GlcN is incorporated to the growing chain to help ensure selective N-sulfonation after a base-mediated deprotection and also direct an irreversible C5-epimerization of a downstream GlcA. To boost the purification process, fluorous-tagging of sugars, which aided purification in chemical synthesis [35], is also applied to chemoenzymatic synthesis of HS with the fluorous group incorporated in the reducing end GlcN residue prior to enzymatic chain elongation [36].

Anticoagulation Heparin is a natural anticoagulant that is widely used for the treatment of venous and arterial thromboembolism. It acts through the allosteric activation of antithrombin III, a serine protease inhibitor that subsequently inhibits the activity of the coagulation cascade enzymes factor Xa and factor IIa. A problem with pharmaceutical heparin is the www.sciencedirect.com

risk of heparin-induced thrombocytopenia due to complexation with platelet factor 4 [6] and the susceptibility to adulteration along the manufacturing supply chain [37]. Fondaparinux, a synthetic 3-O-sulfonated pentasaccharide based on the antithrombin III binding site within heparin, can selectively inhibit factor Xa, bypassing the thrombocytopenia problem [38]. Nevertheless, it lacks antidotes and is inadequate for treating renal-impaired patients. Implementation of strict regulations and use of independent analytical methodologies help guarantee heparin safety and reduce the possibility of adulteration [37]. Because heparin-related compounds are front and center of the anticoagulant issue, many chemists aimed for well-assured, highly specific and low side effect heparin-related anticoagulants. The identification of the active domain of heparin led to fondaparinux, originally synthesized in about 55 steps [39]. Arixtra, its brand name, has been off-patent since 2002, but faced slow competition due to the difficulty in fondaparinux preparation. Many chemists endeavored to develop novel synthetic methods that are amenable to produce this drug in large scale. A reduction of the synthetic steps was achieved starting from D-glucose and cellobiose and mainly using benzoyl, acetyl and benzyl protecting groups [40]. Another synthetic route, proposed by Hung et al., showed improved yield and reduced the collective number of steps to 32 from commercially available starting materials. In this case, common intermediates were employed to prepare three different GlcN residues, and a number of tandem reactions were implemented [41] (Figure 3a). Other groups have also reported practical and relatively efficient methods to prepare fondaparinux [42,43]. Besides, an alternative strategy — hydrogenation–sulfonation– deprotection hydrogenation — was demonstrated, which is also as effective as the traditional sulfonation–hydrogenation– sulfonation process [44]. Several fondaparinux-related compounds were also prepared. Homogeneous short-chain heparins carrying the required antithrombin III-binding pentasaccharide sequence, chemoenzymatically synthesized in 10 and 12 steps, showed similar outstanding anticoagulant activity to fondaparinux [45]. Homogeneous low-molecular-weight heparins can also be prepared in a similar manner (Figure 3b). The resulting dodecasaccharide showed better chance of treating renal-impaired patients, and their sensitivity to protamine neutralization allowed control of overdosage [46]. Similarly to deal with the overdosage problem, fondaparinux-like biotin conjugates were synthesized in anticipation that the biotin tag can be captured by streptavidin in biological settings [33]. The design and synthesis of heparin ROMP glycopolymers shows similar anticoagulation activity to heparin but minimized the risks associated with heparin-induced thrombocytopenia [27]. Moreover, HS sequences containing rare 2-O-sulfonated Current Opinion in Chemical Biology 2017, 40:152–159

156 Synthetic biomolecules

Figure 3

(a)

(b) AcO AcO

O O

OAc O

OH O

OAc

AcO

O O BnO

HO HO HCl•H2N

O O

3 steps Ph

OH O

O

STol

OBz

OH

O OH

–O

O NAPO HO

O

NAPO PBBO

O

N3 O O

OBz

Bn

OBn O STol

LevO

Lev

O2C HO HO

O O

O2 C

O O OH HO

O

N3 O O

NAPO PBBO

OTBDPS O O

OBz

Bn

N3

O

O LevO

Lev

OTBDPS O

HO HO

–

O2C O HO NH HO HO – O3S

O HO

O

NH

O –

–

O3S

O3S

–

O2 C

O –

O3SO

–

O2C

O NH HO Ac

O

OPNP

OH

–

O3 S

O2C

O –

OH OH O O HO

O3SO

O

–

O2C NH O HO – O3S 3

O

OPNP

OH

O

–

O

O OH HO

NH –

O3 S

O2C

O –

OH OH O O HO

O3SO

O

–

O2C NH O HO – O3S 4

O

OPNP

OH

–

OSO3 O

–

–

OSO3 O

–

O2 C NH O HO – O3C 3

1. 6-O-Sulfotransferase isoform 1 and isoform 3, PAPS 2. 3-O-Sulfotransferase isoform 1, PAPS

9 Steps –

OSO3 O

NH

N3 OMe

O PBB

–

OH

O

OBn O O

O

1. KfiA, UDP-GlcNAc 2. C5-epimerase, 2-O-sulfotransferase, PAPS OH

OAc

O3SO

O

N3 OMe

HO PBBO

OAc

OH O O HO

O –

OTBDPS O

+

NIS, TfOH OTBDPS O

O3S

OH

O2C

OH –

O NH HO – O3S

O OH HO

OAc

N3 O

O

–

OH

2 steps

OTBDPS O O

NH

1. LiOH 2. N-Sulfotransferase, PAPS 3. PmHS2, UDP-GlcA

N3 STol

7 steps

OAc

O OH HO

OTBDPS O

–

OTBDPS O

O

O NH HO TFA

HO HO

–

O

2C

5 steps

5 steps HO BnO BzO

OH

OH O O HO

OSO3 O

NH –

HO HO OMe

O3S

OSO3 O

–

O2C O NH HO Ac

O

O

NH OH O – – O3S O3S

–

O2C

O –

O3SO

OH O O HO

OSO3 O

–

O2C NH O HO – O3S 4

O

OPNP

OH

Current Opinion in Chemical Biology

Preparation of anticoagulants. (a) Chemical synthesis of fondaparinux. (b) Chemoenzymatic synthesis of a dodecasaccharide carrying the antithrombin-binding sequence. Abbreviations: Ac, acetyl; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Lev, levulinyl; NAP, 2naphthylmethyl; PAPS, 30 -phosphoadenosine-50 -phosphosulfate; PBB, p-bromobenzyl; PNP, p-nitrophenyl; TBDPS, tert-butyldiphenylsilyl; TFA, trifluoroacetyl; Tol, 4-tolyl; UDP, uridine diphosphate.

glucuronic acid residues were found through computation and modeling, and later through bioassay of chemoenzymatically modified heparosan, to possess the ability to activate antithrombin III [47]. These structures also directly inhibited factor IIa but not factor Xa, suggesting a possible new mechanism of action that is worthy of further exploration.

Synthetic heparin/HS and other protein interactions Rare and exotic sequences with heparin/HS that specifically bind to important heparin-binding proteins are ideal targets in the development of therapeutic agents because of their slim chance of binding with other proteins, minimizing side effects. The identification of these sequences is an intense area of study for heparin-/HSbased drug development. With the aid of numerous novel analytical techniques and various assays, the possibility of polysaccharide binding sequences can be determined by the revealed structure information (Figure 4). X-ray analysis of protein complex and the substrate analogue is the classical method to decipher residues that take part in Current Opinion in Chemical Biology 2017, 40:152–159

substrate recognition and these clues offer great assistance to design the optimal sugar ligand. NMR-based methods have also helped decipher some key information in binding, such as the orientation of a synthetic 13Clabeled heparin as it provides assistance to the growth of amyloid-b fibrils associated with Alzheimer’s disease [48]. Although the complexity of HS placed some limitations on sequencing, Boons et al. demonstrated another elegant approach, which integrated sugar separation by protein affinity and chemical derivatization workflow with the mass spectrometry and computational platform. The combination of these methods suggested the structure of a specific irregular octasaccharide that binds the protein Robo1, and the synthetic target corroborated the results [49]. Conversely, the genetic algorithm-based computational docking by Desai et al. narrowed the possible HS active sequence and pointed out a novel mechanism of anticoagulation [47]. On the basis of previous biological studies, chemists started to prepare libraries of oligosaccharides to engage in the interaction with a number of proteins and help www.sciencedirect.com

Synthetic heparin and heparan sulfate: probes in defining biological functions Tsai, Zulueta and Hung 157

Figure 4

Potential sequence

Computer docking simulation

Chemical synthesis Chemoenzymatic synthesis

I

m/z X-ray analysis MS flow system Current Opinion in Chemical Biology

Array of methods used in identification of rare heparin/heparan sulfate sequences that are targeted for synthesis.

identify the optimal binding structure [50]. The ability of these sugar libraries in emulating the natural molecule has been limited by the huge number of possible structures present in the sugar chain and the synthetic capacities and skills of independent laboratories. Growth factors, particularly those involved in angiogenesis, are one of the important groups that required HS binding. Screening of 48 synthetic disaccharides obtained by divergent transformations identified three compounds containing N-sulfonated GlcN and 2-O-sulfonated IdoA that associated and were characterized in their bound form with fibroblast growth factor 2 [51]. Synthetic heparin-like octasaccharides to dodecasaccharides indicated the importance of 6-O-sulfonation in modulating the activity of angiogenic growth factors [15,19]. Chemokines are also another critical groups involved in HS binding that relates to angiogenesis, metastasis and inflammation. Gardiner et al. showed that installation of a single 6-Osulfonate group on the nonreducing end leads to a switch of inhibition between CXCL8 and CXCL12 [18]. The oligomerization process and the associated functions of CCL3 and CCL5 were also found to be highly modulated by HS interaction [52]. Screening of the prepared HS with other proteins also indicated some intriguing results. A number of 6-O-sulfonated octasaccharides to dodecasaccharides behaved as effective inhibitors to target the b-secretase BACE-1 [17], which strongly participates in the key step of amyloid plaque formation in Alzheimer’s disease. Also, a library of trisaccharides to tetrasaccharides was found as effective substrates to heparanase [53]. www.sciencedirect.com

Because heparanase is closely associated with tumor growth and metastasis, understanding of the specific binding sequences may shed light on the future design of potential inhibitors.

Conclusions Benefiting from the steady development of chemical and chemoenzymatic synthesis, long-chain heparin/HS and some irregular sequences are already being made available. In hope of carrying out the synthesis in an efficient, convenient and economical way, the concept of one-pot method and iterative convergent strategy have been integrated into the synthesis. Synthetic targets can be determined on the basis of several analytical methods including X-ray crystallography, mass-spectrometry platform and computer simulation. Certain reliable protocols have been established to fulfill the synthesis of various heparin-related compounds. Thus, addressing the relationship between specific sugar sequences and proteins have assisted in understanding the role of heparin/HSbased compounds in anticoagulation and other biological functions. These studies should lay the groundwork for the development of potential therapeutic agents.

Acknowledgements This work was supported by the Ministry of Science and Technology of Taiwan (MOST 104-0210-01-09-02, 104-2628-M-001-001 and 105-0210-0113-01) and Academia Sinica. Current Opinion in Chemical Biology 2017, 40:152–159

158 Synthetic biomolecules

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