Chemical Synthesis of Glycosaminoglycans - Chemical Reviews (ACS

Review pubs.acs.org/CR

Chemical Synthesis of Glycosaminoglycans Marco Mende,† Christin Bednarek,† Mirella Wawryszyn,† Paul Sauter,† Moritz B. Biskup,‡ Ute Schepers,§ and Stefan Bras̈ e*,†,§ †

Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany Division 2Informatics, Economics and Society, Karlsruhe Institute of Technology (KIT), Kaiserstraße 12, D-76131 Karlsruhe, Germany § Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany ‡

ABSTRACT: Glycosaminoglycans (GAGs) as one major part of the glycocalyx are involved in many essential biological cell processes, as well as in many courses of diseases. Because of the potential therapeutic application of GAG polymers, fragments, and also derivatives toward different diseases (e.g., heparin derivatives against Alzheimer’s disease), there is a continual growing demand for new chemical syntheses, which suffice the high claim to stereoselectivity and chemoselectivity. This Review summarizes the progress of chemical syntheses of GAGs over the last 10 years. For each class of the glycosaminoglycanshyaluronan (HA), heparan sulfate/heparin (HS/HP), chondroitin/dermatan sulfate (CS/DS), and keratan sulfate (KS)mainly novel glycosylation strategies, elongation sequences, and protecting group patterns are discussed, but also (semi)automated syntheses, enzymatic approaches, and functionalizations of synthesized or isolated GAGs are considered.

CONTENTS 1. Introduction 1.1. Hyaluronan 1.2. Heparan Sulfate/Heparin 1.3. Chondroitin Sulfate/Dermatan Sulfate 1.4. Keratan Sulfate 1.5. Complexity of GAGs 1.6. Challenges in Identification and Synthesis of GAGs 2. General Considerations 2.1. General Building Block Design 2.2. Elongation StrategiesBlock Glycosylation versus Extension by Disaccharide Units 2.2.1. Solid- and Solution-Phase Combinatorial Chemistry and (Semi)automated SynthesesGeneral Remarks 2.3. Glycosylation Sequence and Activation Systems 3. Chemical Syntheses of Glycosaminoglycans from Monosaccharides 3.1. Chemical Syntheses of Hyaluronan 3.1.1. Synthesis of Building Blocks for Hyaluronans 3.1.2. Commonly Used Building Block.s 3.1.3. Building Block SynthesisNovel Glycosylation StrategiesDeprotection Sequences 3.1.4. (Semi)automated and Combinatorial Syntheses 3.1.5. Syntheses of Hyaluronan Conjugates and Other Related Structures © 2016 American Chemical Society

3.2. Chemical Syntheses of Heparin/Heparan Sulfate 3.2.1. Commonly Used Building Blocks 3.2.2. Building Block SynthesisNovel Glycosylation StrategiesDeprotection and Sulfation Sequences 3.2.3. (Semi)automated Synthesis and Microarrays 3.2.4. Syntheses of Heparan Conjugates and Other Related Structures 3.3. Chemical Syntheses of Chondroitin/Dermatan Sulfate 3.3.1. Commonly Used Building Blocks 3.3.2. Building Block SynthesisNovel Glycosylation StrategiesDeprotection and Sulfation Sequences 3.3.3. (Semi)automated Syntheses 3.3.4. Syntheses of Chondroitin Conjugates and Other Related Structures 3.4. Chemical Syntheses of Keratan Sulfate 3.4.1. Commonly Used Building Blocks 3.4.2. Building Block SynthesisNovel Glycosylation StrategiesDeprotection and Sulfation Sequences 3.5. Enzymatic Oligo/Polymerizations of Glycosaminoglycans 3.5.1. Hyaluronan 3.5.2. Heparin/Heparan Sulfate

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8215 Received: January 5, 2016 Published: July 13, 2016

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DOI: 10.1021/acs.chemrev.6b00010 Chem. Rev. 2016, 116, 8193−8255

Chemical Reviews 3.5.3. Chondroitin/Dermatan Sulfate 3.5.4. Keratan Sulfate 4. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments Dedication Abbreviations References

Review

proteoglycans (PGs)12 (e.g., HP, HS, KS, CS, and DS)10 or as the oligosaccharide chain itself (HA).13 PGs are a large heterogeneous family of macromolecules consisting of a central, membrane-spanning or lipid-bound protein backbone to which one or more unbranched GAG chains are covalently linked. These structures are ubiquitously present mainly in the glycocalyx of the cell surface as well as in the extracellular matrix. It is known that their composition varies according to cells types, tissue, and even species and that they are of enormous importance for cell division, morphogenesis, and interactions with the matrix, with other carbohydrates, or with other cells.14−21 In addition, some studies investigated the influence of dietary GAGs on human health, especially on the intestine and effects toward inflammation.22 Additionally, mono- or oligosaccharides can be key components of biologically active compounds and are of great importance for detoxification during metabolism.23 In eukaryotes the biosynthesis19,24 of PGs takes place in the endoplasmic reticulum and the Golgi apparatus. Eventually, they are secreted to plasma membrane, where they are exposed toward the extracellular space. An exception is HA, which is exclusively synthesized at the plasma membrane by a membrane-bound HA-synthase.25,26 The GAG chains are added to a core protein via a GAG−protein linker tetrasaccharide (GlcAβ(1→3)Galβ(1→3)Galβ(1→4)Xylβ(1→O)Ser).19 Several sugar chains can be coupled to different sites of the core protein. Subsequently, the polymerization of the disaccharide chains takes place. Key enzymes in this process are the so-called glycosyltransferases and glycosidases.27 To exert their biological function, GAGs are further processed, i.e., by epimerization (GlcA to IdoA), deacetylation, and/or sulfation (N- and/or O-sulfation).19 Due to these various modification possibilities, a high variability of individual GAG structures is generated. This microheterogeneity represents the unique characteristic of glycan structures as the most informationdense biopolymers. The particular modification pattern, the number of attached GAG chains, and the destination of a specific proteoglycan are dependent on the origin of the cell or the respective tissues and even its health.19 In contrast to the well-understood principles in glycoprotein biosynthesis, glycan biosynthesis is not template-driven and consequently cannot be easily predicted.24,28

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1. INTRODUCTION Over the past few decades, more and more attention was paid to glycostructures of cell surfaces or of the extracellular matrix (ECM), as they play an important role in cell recognition and signaling processes. One class of these oligosaccharide structures are glycosaminoglycans (GAGs), which comprise linear polydisperse heteropolysaccharides of high molecular weight up to several million Dalton.1−3 GAGs consist of up to 1000 repetitive disaccharide units, each containing one acetamido sugar residue and one uronic acid except keratan, which is composed of acetamido sugar-galactose disaccharides. In natural systems these compounds usually contain partially O-sulfated and N-acetylated, as well as partially N-deacetylated and N-sulfated, saccharides. This implies the polyanionic character of GAG structures4 and consequently their great potential for interaction with positively charged moieties like plasma proteins, growth factors, cytokines, or amino acids.5,6 Common monosaccharides that occur as building blocks in oligosaccharides of higher animals are shown in Figure 1.7,8 Figure 1 depicts an overall accepted symbolic system for the representation of these monosaccharides. Depending on the composition of the disaccharide unit of each GAG, they are classified in the following four classes: (I) hyaluronan (HA), (II) heparin (HP)/heparan sulfate (HS), (III) chondroitin sulfate (CS)/dermatan sulfate (DS), and (IV) keratan sulfate (KS) (Figure 2).3,10 GAGs represent a key component of the human glycome, which is defined as “the entire set of glycans in an organism, which may range from mono- to polysaccharides, either free or linked to aglycon moieties such as proteins or lipids”.11 Therefore, GAGs occur either as the constituents of so-called

Figure 1. Common monosaccharides found in higher animals and their symbolic nomenclature.7−9 8194


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Figure 2. Composition of the disaccharide units of the four GAG classes: (I) HA, (II) HP/HS, (III) CS/DS with DS having IdoA, and (IV) KS and their potential sulfation sites.

Figure 5. Disaccharide subunit found in CSs 19. Repetitive unit [4)-βGlcA-(1→3)-β-GalNAc-1→] motive with several sulfation patterns.

Figure 3. AT-binding pentasaccharide sequence.139,171

Figure 6. Substructure of keratan sulfate I−III 20: repetitive N-acetyl lactosamine subunit, 3Gal-β-(1,4)-GlcNAcβ1, sulfated at the C6 of hexose moieties.

1.1. Hyaluronan

In contrast to other GAGs, hyaluronan (HA) exists solely protein-free,26 contains long oligosaccharide chains with a molecular mass of 1−10 million Da and an extended length of 2−20 μm,29 and is exclusively nonsulfated.16 The repeating disaccharide unit of HA is composed of GlcAβ(1→3) glycosidically linked to GlcNAc. Each of the disaccharide subunits are connected via β(1→4) glycosidic linkages (Figure 2).3,30,31

Figure 4. Structures of fondaparinux (17) and idraparinux (18).171 8195


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Figure 7. Corneal KS I.

Table 1. Types of CS19 CS type

major disaccharides

other disaccharides

CS-A CS-B (dermatan sulfate) CS-C CS-D CS-E

GlcA−GalNAc4S IdoA−GalNAc4S GlcA−GalNAc6S GlcA2S−GalNAc6S GlcA−GalNAc4S6S

GlcA−GalNAc/GlcA2S−GalNAc IdoA2S−GalNAc4S/GlcA3S−GalNAc IdoA−GalNAc4S6S/GlcA3S−GalNAc4S IdoA2S−GalNAc4S6S/GlcA3S−GalNAc4S6S IdoA2S−GalNAc/GlcA3S−GalNAc6S

Table 2. Number of Constitutional Isomers of Glycans, Which Considerably Exceeds Comparable Isomers of Peptides254 monomers

composition

oligopeptides

oligosaccharides

2 3 4 5

AA/AB AAA/ABC AAAA/ABCD AAAAA/ABCDE

1/2 1/6 1/24 1/120

11/20 120/720 1 424/34 560 17 872/2 144 640

Scheme 2. Strategy for the Convergent Synthesis of Glycosaminoglycans: An Example for Keratan Sulfate

Scheme 1. Strategy for the Stepwise Synthesis of Glycosaminoglycans: An Example for Keratan Sulfate

Scheme 3. Strategy for the Solid-Phase Synthesis of Glycosaminoglycans: An Example for Keratan Sulfate

As already stated above, in contrast to the other GAGs, which are synthesized at the Golgi apparatus, it is formed at the inner plasma membrane by multipass transmembrane proteins called hyaluronic acid synthases (HAS1, HAS2, or HAS3), which mainly differ in their catalytic activities and the size of the synthesized HA. While HAS1 makes medium molecular weight (MMW) HA to long, high molecular weight (HMW) HA (200−2000 kDa) and HAS2 is mainly synthesizing (HMW) HA (>2000 kDa), HAS3 is responsible for the synthesis of low molecular weight (LMW) chains (80% of the GlcNAc residues are N-deacetylated and N-sulfated, and >70% of the glucuronic acid is converted to iduronic acid.143 In contrast, the relatively small set of HSPGs (∼17) is either present in the ECM, such as agrin, perlecan, and type XVIII collagen, or cell membrane bound (105−106 molecules/cell surface).140,144 The integration into the membrane appears either through integral 8200


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Scheme 14. Synthesis of the Unprotected Hyaluronan Hexamer 88: (a) Et2O, MS 4 Å, rt, 1 h, Then −65 °C, AgOTf, Et2O, 5 min, Then p-TolSCl, 5 min, Then 80, CH2Cl2, TTBP, −65 °C to rt, 1.5 h, 75%; (b) Et2O, CH2Cl2, MS 4 Å, rt, 1 h, Then −65 °C, AgOTf, Et2O, 10 min, Then p-TolSCl, 5 min, −65 °C to rt, 1.5 h, 88%; (c) Pyridine, HF·Pyridine, 0 °C, 48 h, 92% 84, and 87% 85; (d) Et2O, MS 4 Å, rt, 1 h, Then −65 °C, AgOTf, Et2O, 10 min, Then p-TolSCl, 5 min, then 84, CH2Cl2, TTBP, −65 °C to rt, 1.5 h, 75%; (e) Et2O, MS 4 Å, rt, 1 h, Then −65 °C, AgOTf, Et2O, 10 min, Then p-TolSCl, 5 min, Then 85, CH2Cl2, TTBP, −65 °C to rt, 1.5 h, 57%; (f) CAN, MeCN, H2O, 0 °C to rt, 1 h, 74%; (g) (i) TEMPO, NaOCl, NaBr, TBABr, NaHCO3, CH2Cl2, H2O, 0 °C to rt, 1 h, Then tert-BuOH, THF, H2O, NaClO2, 2-Methyl-2-butene, NaH2PO4; (ii) CH2Cl2, Et2O, PhCHN2, rt, 2.5 h; (h) (i) H2, Pd(OH)2, THF, MeOH, AcOH, rt, 48 h; (ii) 33% MeNH2, rt, 72 h; (iii) MeOH, Ac2O, NEt3, 0 °C, 2.5 h, Then Amberlite IR-120, 30 min, 44% (3 Steps)

However, most common are a sulfation on C4 and C6 of GalNAc and C2 and C3 of the uronic acid.19 Chondroitin sulfates (CSs) are copolymers made up of dimeric subunits that are composed of D-glucuronic (D-GlcA) or rarely iduronic acid (L-IdoA) (i.e., DS) and are β(1→3)linked to N-acetyl-D-galactosamine (D-GalNAc) that is β(1→ 4)-linked to the first monosaccharide in turn so the disaccharide consists of a repeating unit [4)-β-GlcA-(1→3)-βGalNAc-1→]. CS contains, on average, one or two sulfate groups per disaccharide unit but several variants, the so-called sulfo form or also sulfo code (Figure 5). Nevertheless, the sense of the natural heterogeneity is not entirely understood.213−215 During polymerization the disaccharide chondrosin2-amino2-deoxy-3-O-β-D-glucopyranuronosyl-D-galactoseundergoes sulfation at various positions. Therefore, the sulfated GAG consists of a more or less polyanionic character, due to the negatively charged sulfate groups and the number of chain length.214,215 The CS chains vary from a range of 10 to 200 repeating disaccharide units that are linked to carrier proteins through a tetrasaccharide moiety.215 The tetramer is covalently bound to an L-serine residue to from a proteoglycan, and it is mainly found in the extracellular matrix of a wide variety of tissues but is also present in cell surfaces.215

CS variants can be divided into two main classes: those bearing sulfate group(s) on the D-GlcA or on both D-GalNAc and D-GlcA moieties simultaneously. The sulfo code gives rise to various crucial biological functions that were approved by a large variety of interactions between CS chains and growth factors, cytokines, and adhesion molecules as reported by Jacquinet et al.213 According to Despras et al., the sulfation pattern encodes information decipherable by positively charged domains of protein receptors.215 As the grade of sulfation encodes for many negative charges, the sulfo code might deliver information on the binding to positively charged domains of protein receptors.215 However, deciphering the respective interaction sites is still the biggest obstacle due to the limited access to enough pure CS material containing homogeneous sulfation pattern for biological studies.213 Modern glycoscience is dependent on new tools for structure−function relationships; studies are required to provide the evidence that CS glycosaminoglycans encode functional information in a sequence-specific manner.216 So far, microarrays of CS oligosaccharides are used to measure the interactions with biomolecules.214 Previously distinct torsion angles for the sulfation pattern could be determined by performing molecular dynamics simulations showing that CSs 8201


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plays a potential role in diverse tissues and is mostly found in two principal forms, keratan sulfate I (KS I) and keratan sulfate II (KS II) (Figure 6). Structurally it consists of a repetitive N-acetyl lactosamine subunit, meaning glycosidically β(1→3)linked sulfated 3Gal-β-(1,4)-GlcNAcβ1 unit (Figure 2).3,245 Consequently, its structure differs from the other GAG classes as its uronic acid moiety is replaced by a neutral D-galactose unit. KSs are relatively small and range from 5 up to 30 disaccharides. Sulfation of KS occurs exclusively at the 6-OH on either the 3,246 D-galactose or on the N-acetyl-D-glucosamine residue. KS occurs in two main forms, KS I and KS II. They differ in their attachment to the core protein: KS I is linked via an N-glycosidic linkage to L-asparagine residues.247 The linkage region of KS I derives from a complex type of N-linked glycan precursor as depicted for the corneal KS I (Figure 7).3,245,248 Like the other GAGs, KS chains are attached to cell-surface or extracellular matrix proteins including lumican, keratocan, mimecan, fibromodulin, PRELP, osteoadherin, and aggrecan. In comparison, KS II is attached O-glycosidically to L-serine or 249 L-threonine. KS I and II also differ in their tissue distribution. KS I is found in the cornea, where it was discovered and identified in 1939,245 and in the embryonic liver and lung.245,250 It is mainly responsible for tissue hydration. KS II, however, is predominantly found in bone, cartilage, and skin.3,248,249 An additional and less frequently occurring KS type was found that possesses O-glycosidic linkages of human to L-serine residues. This is considered as KS III245 and is mainly found in proteoglycans from brain.251 For more details on KS, see papers by Funderburgh245 and Quantock et al.252

Scheme 15. Synthesis of the Monosaccharide Building Blocks for the Preparation of the Hyaluronan Hexamer 100: (a) NaOAc, Ac2O, 140 °C; (b) (i) 4-Methoxyphenol, BF3· OEt2, CH2Cl2, 0 °C, 30 min, Then rt, 2 h; (ii) NaOMe, MeOH, rt, 16 h; (iii) Benzaldehyde Dimethyl Acetal, pTsOH, DMF, 70 °C, 1 h; (c) (i) BzCl, Pyridine, CH2Cl2, 0 °C to rt, 16 h; (ii) 80% AcOH, CHCl3, 80 °C, 16 h, 78% (3 Steps); (d) LevOH, 2-Chloro-1-methylpyridinium Iodide, DABCO, 1,2-Dichloroethane, CHCl3, rt, 30 min, 90%; (e) (i) Ac2O, Pyridine, rt, 16 h; (ii) CAN, MeCN, H2O, 0 °C, 30 min; (iii) Cl3CCN, DBU, CH2Cl2, 0 °C, 2 h, 70%; (f) (i) Phthalic Anhydride, NaOH (aq.); (ii) Ac2O, Pyridine; (g) (i) 4-Methoxyphenol, BF3·OEt2, CH2Cl2, 40 °C, 1 h; (ii) NaOMe, MeOH, rt, 3 h; (h) Benzaldehyde Dimethyl Acetal, p-TsOH, DMF, 70 °C, 1.5 h, 63% (3 Steps); (i) (Mca)2O, Pyridine, CH2Cl2, 0 °C, 40 min, 85%; (j) (i) CAN, MeCN, H2O, 0 °C, 40 min; (ii) Cl3CCN, DBU, CH2Cl2, 0 °C, 2 h, 56%; (k) 6-Azido-1-hexanol, TMSOTf, CH2Cl2, MS 4 Å, −30 to 0 °C, 1 h, 92%; (l) Thiourea, 2,6-Lutidine, MeOH, CH2Cl2, 40 °C, 4 h, 83%

1.5. Complexity of GAGs

It is important to note that at least a linear sequence of 5−6 monosaccharides is required for recognition processes with binding affinities (Kd (pentasaccharides) = 10−6−10−8 M) that are up to 3 orders of magnitude higher than those of mono- or trisaccharides (Kd (mono- to trisaccharides) = 10−3−10−6 M). Thus, multivalent interactions of PGs bearing more than one GAG chain are of great importance. This explains why glycoproteins and proteoglycans usually present repetitive epitopes on their carbohydrate chains and glycosphingolipids are closely associated in clusters or patches.17 Considering all possible combinations of the monosaccharides that comprise the GAG classes shown in Figure 2, it is possible to generate 3981 pentasaccharides (HA = 2, HP/HS = 2916, CS/DS = 999, and KS = 64). However, as the mammalian glycome comprises further monosaccharides (Figure 1), the total number of theoretical pentasaccharides exceeds 7 000.11 Furthermore, the distinct heterogeneity of all these glycan structures is considerably extended by the variability of sulfation patterns and potential epimerizations (for a novel method to elucidate epimers by mass spectrometry, see paper by Hofmann et al.253). In contrast to other biomacromolecules like DNA or proteins, which are linear polymers, glycan structures exhibit branches as well as anomeric configurations and therefore a much higher complexity than peptides and DNA (Table 2). The high complexity of these biostructures is further enhanced by its highly dynamic constituents. Possible interactions of GAGs with proteins or other structures are strongly dependent on the respective microenvironment, i.e., pH value, type of cations, and their concentration.151,255−258 In addition, cells independently regulate their environment with GAG structures and thus the physicochemical properties on the cell surface. This continuous metabolic turnover enables cells to rapidly adapt to the given microenvironment.29

develop unique electrostatic and van der Waals surfaces for interaction with proteins.216 Moreover, a fluorescence polarization competition assay was employed by Maza et al.216 to evaluate the interactions between the CS tetrasaccharide and FGF-2 (basic fibroblast growth factor) to analyze the binding.217 In addition, microarray-bound CS tetrasaccharides were probed with biological samples to elucidate their binding properties.216,218,219The sulfation pattern of CSs gives rise to various biological functions. For example, the involvement in cell−cell recognition processes,220 anti-inflammatory effects,221−223 and brain development166,224−227 is discussed. Besides, the potential of CS as a therapeutic agent against osteoarthritis is depicted by Lamari228 and others.229−237 Furthermore, a large variety of interactions between CS chains and growth factors, cytokines, and adhesion molecules are reported by Jacquinet et al.213 However, further biological functions are explored for DS: antioncogenic238 and antithrombotic239 potential as well as growth factor regulations.240,241 For reviews on CS and DS, see papers by Volpi242 and others.243,244 1.4. Keratan Sulfate

The last family of GAGs are the keratan sulfates (KSs). KS was identified in 1939 by Suzuki in extracts of cornea, and in the 1950s Karl Meyer had characterized this tissue matrix as a linear polymer of repeating disaccharide subunits.245 Keratan sulfate 8202


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Scheme 16. Synthesis of the Amino-Functionalized Hyaluronan Hexamer 100: (a) TMSOTf, CH2Cl2, MS 4 Å, −40 to 0 °C, 2.5 h, 85% 94, and 70% 99; (b) Thiourea, 2,6-Lutidine, MeOH, CH2Cl2, 40 °C, 4 h, 83% 95, and 65% 98 (2 Steps); (c) (i) CAN, MeCN, H2O, 0 °C, 40 min; (ii) Cl3CCN, DBU, CH2Cl2, 0 °C, 2 h, 67% 96, and 57% 97 (2 Steps); (d) (i) 80% AcOH, 80 °C, 5 h; (ii) Ac2O, Pyridine, rt, 16 h, 78%; (e) N2H4·AcOH, EtOH/Toluene (2:1), rt, 3 h, 89%; (f) PDC/Ac2O, CH2Cl2, rt, 8 h, 67%; (g) (i) Ethylenediamine, 1-Butanol, 90 °C, 8 h; (ii) Ac2O, Pyridine, rt, 16 h; (iii) 1 M LiOH (aq.), THF, 0 °C to rt, 20 h, 78%; (h) 10% Pd/C, NaBH4, 0.05 M NaOH (aq.), H2O, rt, 5 h, 85%

Figure 10. Automated synthesis of oligosaccharides: application in the preparation of glycoarrays and glycoconjugates.290,324

1.6. Challenges in Identification and Synthesis of GAGs

glycocalyx in atherosclerosis-susceptible arteries. In comparison, in arteries with low risk of atherosclerosis, the thickness of the glycocalyx layer is ∼400 nm.259 Moreover, a significant degradation was shown in capillaries of diabetes mellitus type I patients.260,261

A change in the glycocalyx or the entire glycome of a cell or a tissue is often associated with various diseases. An example of such a pathological change is the altered, thinner (∼100 nm) 8203


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Table 3. Examples of Glycosaminoglycan Oligosaccharide Synthesis (Also Covering Earlier Syntheses) glycosaminoglycan type

fully unprotected synthetic oligosaccharides (number of monosaccharides)

(partially) protected synthetic oligosaccharides (number of monosaccharides)

semisynthetic oligosaccharides (number of monosaccharides)

2384 2395 4217

2,340,385 4,291,309,385−390 6,385 8,318,385 10,391 15341 6,330 12,396−398 2,213,219,377,400−409 2,366−372,380,411−454

2,392 4−52,393 3−17394 6399 14410

hyaluronan heparin/heparan sulfatea chondroitin/dermatana keratana a

Also unsulfated oligosaccharides.

Scheme 17. Synthesis of the Azido-Functionalized Unprotected Hyaluronan Heptamer 107: (a) Ph2SO, Tf2O, MS 3 Å, CH2Cl2, −60 to 0 °C, 3 h, 70%; (b) Ph2SO, Tf2O, 3-Azido-1-propanol, MS 3 Å, CH2Cl2, −78 to 0 °C, 4 h, 94%; (c) N2H4·H2O, Pyridine, AcOH, rt, 15 min, 85% 103, 43% 104 (2 Steps), and 89% 105 (2 Steps); (d) 41, NIS, TfOH, MS 3 Å, CH2Cl2, 0 °C to rt, 2 h, 61%; (e) (i) 3HF/NEt3, THF, rt, 2 h; (ii) KOH, THF, H2O, rt, 4 d, 59%; (f) (i) Ac2O, MeOH, rt, 4 h; (ii) LiOH, H2O, rt, 2 h, 78%

Figure 11. Typical building block design for hyaluronic acid oligosaccharide synthesis.

The trigger in this case is probably a hyperglycemia, which results in an increased superoxide anion formation that eventually leads to the damage of the glycocalyx. Infusion and administration of N-acetylcysteine as an antioxidant can prevent this degradation.262 Furthermore, glycan composition of tumor cells shows quantitative changes in GAG synthesis compared to healthy cells.263,264 Besides, tumor and normal cells differ in branching degrees,265 sialylation of N-glycans,266 and an enhanced expression of short-chain mucin-type O-glycans. However, these differences are tumor-type dependent.17 In this context, cell surface carbohydrates on tumor cells can be used as markers to generate antibodies, which can be used to target anticancer drugs and thus increase their efficacy. In general, diseases of glycosylation are known in nearly all glycosylation pathways. The clinical features associated with these alterations reflect the multiple contributions of glycans in human physiology. Changes in the binding patterns of carbohydrate-specific antibodies or lectins or in the molecular weight of known glycoproteins can be used to identify such glycan-dependent diseases. However, the identification and characterization of diseases associated with alterations of glycosylation still remains challenging as the tremendous structural complexity of glycans makes it quite difficult to predict the biological importance of individual structures.17 Some tools for functional studies of glycostructures have already been developed or are currently in the developmental and optimization phase. Genetic approaches are still quite limited as the glycan structures are posttranslation modifications that can even be modified after presentation to the cell surface. Therefore, genetic approaches can mainly be used to decode the genes encoding glycosyltransferases and glycosidases or other enzymes such as epimerases and offer the possibility to unravel at least partially altered glycosylation and thus the roles of specific glycans.24,267 Biochemical methods, which are utilized, are often based on the use of lectins or antibodies. However, one drawback is their intrinsic binding specificity that makes it difficult to detect the slightest changes in glycan structures.24 Therefore, the methods of choice are generally of a chemical nature.268 One of the methods to detect and analyze GAGs in a spatiotemporal manner is based on the bioorthogonal labeling of sugars. Non-native sugars bioorthogonally labeled are metabolized by the cells, UDP and GDP activated, and incorporated into their glycocalyx.269 Eventually, chemoselective labeling of the bioorthogonal group allows for the labeling of the respective modified glycostructure.24 Many

different bioorthogonal sugars such as azide- or alkyne-modified ManNAc, GlcNAc, GalNAc, or fucose270 and sialic acid271 have been shown to be metabolized and incorporated into the glycostructures of cells. Probing for the GAGs is usually performed by classical click reactions such as Staudinger ligation272 with phosphines or Cu(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC)273 or strain-promoted 1,3-dipolar cycloaddition (SPAAC274) using the corresponding alkyne or the azide.24 Isolated GAGs or PGs can then be characterized in mass spectrometry experiments. In addition synthetic carbohydrates were used for direct interaction studies. Thus, both enzymes with catalytic activity, like glycosidases275 and enzymes with specific affinity, for example, lectins, can be detected. Furthermore, the biological functions of glycans can be directly analyzed by glycoprotein 8204


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Scheme 18. Synthesis of the Protected Key Hyaluronan Disaccharide 109: (a) (i) NaH, BnBr, DMF, 90 °C, 2 h; (ii) 0.1 M H2SO4, 90 °C, 2 h; (iii) NaOAc, Ac2O, 110 °C, 4 h, 87%; (b) BF3·OEt2, EtSH, MS 4 Å, CH2Cl2, rt, 3 h, 53%; (c) NaOMe, MeOH, CH2Cl2, rt, 1 h, 99%; (d) pMethoxybenzaldehyde Dimethyl Acetal, p-TsOH, MeCN, 40 °C, 3 h, 82%; (e) BzCl, CH2Cl2, Pyridine, rt, 16 h, 90%; (f) NaCNBH3, MS 4 Å, DMF, TFA, rt, 20 h, 88%; (g) DIC, DMAP, LevOH, CH2Cl2, rt, 18 h, 88%; (h) Anisaldehyde, 1 M NaOH (aq.), 0 °C, 1 h, 82%; (i) Ac2O, DMAP, Pyridine, rt, 16 h, 84%; (j) 5 M HCl (aq.), Acetone, 60 °C, 10 min, 91%; (k) TcaCl, NEt3, CH2Cl2, 30 min, 82%; (l) (i) HBr in AcOH, CH2Cl2, rt, 16 h; (ii) InCl3, AllylOH, MS 4 Å, CH2Cl2, rt, 16 h, 75%; (m) Conc. NH3 (aq.), MeOH, rt, 16 h, 94%; (n) Benzaldehyde Dimethyl Acetal, p-TsOH, MeCN, 40 °C, 1 h, 91%; (o) DIC, DMAP, LevOH, CH2Cl2, rt, 18 h, 99%; (p) Et3SiH, cat. TFAA, TFA, CH2Cl2, 0 °C to rt, 20 h, 77%; (q) Ac2O, DMAP, CH2Cl2, rt, 1 h, 92%; (r) N2H4·AcOH, MeOH, CH2Cl2, rt, 1 h, 96%; (s) (i) NIS, TMSOTf, CH2Cl2, MS 4 Å, −30 °C, 1 h; (ii) CAN, MeCN, H2O, rt, 1 h, 72%; (t) (i) H5IO6, CrO3, MeCN, H2O, 0 °C, 1.5 h; (ii) CH2N2, Et2O, CH2Cl2, rt, 62%

Scheme 19. Synthesis of the Partially Deprotected Hyaluronan Tetramer 113: (a) PdCl2, NaOAc, AcOH, H2O, rt, 12 h, 79%; (b) Cl3CCN, DBU, CH2Cl2, 0 °C, 2 h, 52%; (c) N2H4·AcOH, MeOH, CH2Cl2, rt, 2 h, 90%; (d) TMSOTf, CH2Cl2, 0 °C, 1.5 h, 51%; (e) N2H4·AcOH, MeOH, CH2Cl2, rt, 2 h, 91%; (f) 1,4-Dioxane, Zn/AcOH, rt, 15%; (g) (i) PdCl2, AcOH, H2O, NaOAc, rt, 12 h; (ii) Cat. Pd(OH)2, H2, MeOH, rt, 16 h, 37% (2 Steps)

Scheme 20. Synthesis of Acceptor Building Blocks 114 and 115: (a) (i) NaOMe, MeOH, rt, 45 min; (ii) TcaCl, NEt3, rt, 16 h; (b) Ac2O, Pyridine, rt, 2 days, 66% (2 Steps); (c) p-TolSH, BF3·OEt2, CH2Cl2, rt, 16 h, 90%; (d) NaOMe, MeOH, CH2Cl2, rt, 2 h; (e) Benzaldehyde Dimethyl Acetal, CSA, Toluene, 80 °C, 1 h, 70% (2 Steps); (f) HBr in AcOH, rt, 6 h; (g) Ag2CO3, CaSO4, MeOH, rt, 1 day, 80% (2 Steps); (h) Benzaldehyde Dimethyl Acetal, CSA, Toluene, 80 °C, 1 h, 70% synthesis. This can be done by means of convergent or fragment assembly.276−282 Likewise microarrays can serve as a high-throughput tool for the identification of specific roles of glycans.283−287 So far, microarrays based on GAGs have already been used for the profiling of sulfation patterns during growth factor binding.216,288 It is of great relevance to have as many and diverse carbohydrates available for these microarrays. The use of solid-phase assays for the specific analysis of glycoconjugates is described in more detail by Ziouti et al.289 Other methods for mapping specific functions of glycans are described by Werz and Seeberger.290 An alternative to solidphase bound oligosaccharides for probing the interaction with biological materials is the use of 13C-labeled structures, which was shown, e.g., for derivatives of HA by Rigol et al.291 Other investigations were carried out by modeling and NMR investigations, i.e., the interaction of HA292 and HP293 with their targets, that showed that the distinct stereochemistry of these carbohydrates played a pivotal role in their high affinity. Nevertheless, despite these manifold methods to study the specific functions of carbohydrates, in particular the GAGs, their extraordinary microheterogeneity still considerably complicates the analysis and elucidation of the molecular basis of

biological processes. Although oligosaccharides, respectively GAGs, are ubiquitous in nature and in living organisms, it is extremely difficult to obtain them in high purity and quantity. This has led to tremendous scientific efforts for the synthesis of GAG structures. The main synthetic focus in the last 10 years has been in accessing chemically defined GAGs and their non-natural derivatives in sufficient purity and quantity for further biological or medicinal studies. Here the strategies seem to follow three major approaches: (i) (chemo)enzymatic approaches/semisynthesis starting from purified GAGs obtained from natural sources,294−299 (ii) chemical functionalization of isolated GAG fragments, or (iii) total synthesis of GAG fragments, generally starting from monosaccharidic building blocks. This Review will intensively focus on the two latter approaches. 8205


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Scheme 21. Synthesis of the Unprotected Hyaluronan Decamer 123: (a) (i) p-TolSCl, AgOTf, Et2O, MS 4 Å, −78 °C, 1 h; (ii) 114, TTBP, CH2Cl2, MeCN, −78 to −10 °C, 2 h, 80%; (b) DDQ, CH2Cl2, Na2CO3 (aq.), 0 °C to rt, 2 h; (c) PDC, DMF, MS 4 Å, rt, 16 h; (d) PhCHN2, CH2Cl2, rt, 3 h, 65% (3 Steps); (e) HF·Pyridine, rt, 1 day, 90% 117, 48% 118 (5 Steps), and 90% 121; (f) (i) 115, p-TolSCl, AgOTf, Et2O, CH2Cl2, MeCN, MS 4 Å, −78 °C, 1 h; (ii) TTBP, −78 to −10 °C, 2 h; (g) (i) p-TolSCl, AgOTf, Et2O, MS 4 Å, −78 °C, 1 h; (ii) 117, TMSOTf, CH2Cl2, MeCN, −78 to −10 °C, 2 h, 82%; (h) (i) CH2Cl2, MeCN, MS AW-300, −78 °C, 1 h; (ii) p-TolSCl, AgOTf, Et2O, TMSOTf, −78 to −10 °C, 2 h, 71% 120, and 77% 122; (i) KOH, H2O, THF, rt, 5 weeks; (j) Ac2O, MeOH, NEt3, rt, 2 days; (k) Pd(OH)2, H2, AcOH, MeOH, THF, rt, 3 days, 28% (4 Steps)

Scheme 22. Synthesis of the Merrifield Resin-Based Linker 127: (a) mCPBA, K2HPO4, CH2Cl2, H2O, 0 °C; (b) H5IO6, H2O, Dioxane, 0 °C; (c) AllylMgBr (1 M in Et2O), THF, 0 °C, 8% (3 Steps); (d) (i) KOt-Bu, 18-crown-6, TBAI, THF, 3 days; (ii) KOMe, 1 day, 80%

One has to meet several challenges during GAG synthesis. The synthesis of the complex linear and branched oligosaccharide structure from the diverse monosaccharides with biological function strongly relies on the correct stereochemistry. Therefore, protecting group strategies are key features of glycan synthesis as they both protect and confer specific reactivity to each given derivative.300 Glycosyl donors301 containing an anomeric leaving group as well as glycosyl acceptors with a free hydroxyl group are typically used. Examples for such glycosyl donors are thioglycosides, halides, and trichloroacetimidates. In particular, these are easy

to prepare in large amounts and are chemically stable before activation.302 The so-called de novo synthesis is based on the synthesis of individual monosaccharides by using smaller substrates and C−C bond formation.303 A second strategy is the synthesis of the required protected monosaccharides from unprotected, commercially available monosaccharides. Subsequently, the respective monosaccharide building blocks can be assembled in a linear fashion to form oligosaccharide structures, for example, starting from the reducing end. A further possibility is to first create larger saccharide blocks and to connect them in a 8206


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Scheme 23. Solid-Phase Synthesis of Unprotected Hyaluronan Tetramer 132: (a) TfOH, CH2Cl2, 0 °C, 30 min; (b) N2H4·AcOH, Pyridine, AcOH, 40 °C, 10 min; (c) 42, TfOH, CH2Cl2, 0 °C, 30 min; (d) Grubbs 2nd-Generation Catalyst, Trichloroacetamide, CH2Cl2, 3 h, 73% (5 Steps); (e) (i) 3HF/NEt3, THF, 30 min, 83%; (ii) 0.5 M KOH, THF, 4 days, 52%; (iii) Ac2O, THF, H2O, 4 h, 91%

Scheme 24. Preparation of the Hyaluronan Disaccharide Repeating Unit 42 for the Automated Solid-Phase Synthesis: (a) TcaCl, NEt3, MeOH, 0 °C to rt, 6 days; (b) (tBu)2Si(OTf)2, Pyridine, DMF, −40 °C, 1 h, 86% (2 Steps); (c) Cs2CO3, ClC(NPh)CF3, Acetone, 0 °C, 1 h, 98%; (d) LevOH, DIC, DMAP, CH2Cl2, 0 °C, 2.5 h, 82%; (e) TfOH, CH2Cl2, 0 °C, 1 h, 78%; (f) NBS, Acetone, H2O, rt, 3 h, 75%; (g) Cs2CO3, ClC(NPh)CF3, Acetone, 0 °C, 16 h, 75%

added subsequently. The advantage is that donor and acceptor may hold the same leaving group, but at the same time a reduced amount of protection groups at the acceptor are necessary.307,308 Further optimization of the glycan synthesis is possible through a combination of one-pot and preactivation strategy. For general and earlier publications concerning the synthesis of glycans, specifically GAG, see the following citations: refs 3, 135,303, and 309−315. The scope of this Review is to report on the new and innovative advances in chemical syntheses of GAGs and on their functionalization.

2. GENERAL CONSIDERATIONS In this section some topics are discussed that all glycosaminoglycan syntheses have in common. This includes general building block design (section 2.1), elongation strategies (section 2.2), and elongation methods (glycosylations) (section 2.3). 2.1. General Building Block Design

Besides the general challenges of oligosaccharide synthesis (stereochemistry of the glycosidic bonds, orthogonal protecting groups, etc.), glycosaminoglycan chemistry faces three additional challengesthe carboxylate functionality of the uronic acid moiety, the amino function of the neutral sugar moiety, and the sulfation pattern of the oligosaccharide. For the carboxylate moiety, oxidation methods have been developed to oxidize the corresponding C6-alcohol at a late stage (see, e.g., Scheme 14). The amino group is mostly taken through the entire synthesissometimes azide groups have been used as surrogates (see, e.g., Scheme 53). Most of the building blocks can be prepared from readily available sugar base materials. However, selected syntheses are also based on de novo sugar chemistry.316 The usual oligosaccharide protecting groups are used: acyl (e.g., Lev, Ac, Bz, monochloroacetyl (Mca)), Bn,317 PMB, and silyl (TBDPS and TBS) to protect hydroxy groups; Me, allyl, p-methoxyphenyl (MP), (dimethyl)thexylsilyl (TDS), and naphthylmethyl (NAP) for the protection of anomeric hydroxy groups; benzylidene acetals and silylene acetals for diols; Me

following step.17,303 Finally, the generated saccharide structures can be functionalized further and thus possibly possess altered functions compared to the initial structure. Because these syntheses are often very time-consuming, it is the aim of many chemical research groups to accelerate them considerably and to develop more practical and more efficient synthetic strategies to defined oligosaccharides. One possibility is the so-called one-pot strategy304 that was also summarized by Wang et al. in a review article.305 The method is based on the simultaneous presence of glycosyl donor building blocks that possess different reactivities due to a multistep activation of their leaving groups. This sequential activation ultimately leads to the controlled formation of glycosidic bonds. The strategy therefore represents an extension and facilitation of the linear synthesis. An overview of current research in this area is provided by Kaeothip and Demchenko.306 Another optimization is the preactivation strategy. In this case, the donor is transferred to the active intermediate without the simultaneous presence of the acceptor. The latter is 8207


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Scheme 25. Automated Solid-Phase Synthesis of the Unprotected Pentadecamer 138: (a) TfOH, CH2Cl2; (b) N2H4·AcOH, Pyridine, AcOH; (c) 42, TfOH, CH2Cl2; (d) Grubbs 1st-Generation Catalyst, Trichloroacetamide, Ethylene, CH2Cl2, rt, 16 h; (e) 3HF/NEt3, THF, rt, 2.5 h, 18%; (f) KOH, H2O, THF, rt, 3.5 days; (g) Ac2O, NaHCO3, H2O, THF, rt, 1 h

Scheme 27. Attachment of Protected D-Glucosamine 143 and Protected D-Glucuronic Acid 145 to the PEG Acylsulfonamide Linker 142: (a) TMSOTf, CH2Cl2, rt, 1 h

Scheme 28. Synthesis of the PEG-Modified Disaccharide 147: (a) TMSOTf, CH2Cl2, rt, 1 h

2,2,2-trichloroethoxycarbonyl (Troc), phthaloyl (Phth), and monochloroacetyl (Mca) for amines. Fluorinated protecting groups are used for fluorous chemistry (see section 2.2.1). Typical monosaccharidic building blocks for glycosaminoglycans are shown in Figure 8 and Figure 9. 2.2. Elongation StrategiesBlock Glycosylation versus Extension by Disaccharide Units

Scheme 26. Synthesis of the Polyethylene Glycol (PEG) Acylsulfonamide Linker. (a) 4-Sulfamoyl−Benzoic Acid, DIC, HOBt, CH2Cl2, rt, 16 h; (b) 2-O-Acetyl−Glycolic Anhydride, DIPEA, DMAP, CH2Cl2, rt, 24 h; (c) NaOMe, MeOH, rt, 12 h

The assembly of oligosaccharides with a chain length over four monomers requires careful planning of the connections.319 In principal, there are two major strategies feasibleboth being realized in glycosaminoglycan syntheses. The first one is the classical stepwise approach: each building block (or the disaccharide unit) is appended to the growing glycan chain. In principle, both parts can act as an acceptor or as the donor. Because one can drive the reaction to higher conversion using the smaller unit in excess, the yields of each single step are generally higher (Scheme 1). In the case of solid-phase syntheses (see also section 2.2.1) or liquidphase combinatorial chemistry, the acceptor is immobilized and the growing chain is prolonged by donor units (see, e.g., Scheme 23). The second approach is a convergent one in which similarly sized units are preassembled. Although this approach requires (at least in principle) less steps, the main disadvantage is the need to activate highly precious starting materials that cannot be recovered when used in excess (Scheme 2). The decision to use either one of the described strategies (including iterative extensions) usually depends on a multitude of factors, e.g., literature precedent, sourcing of starting materials, and individual expertise. A clear preference for a

and Bn for the protection of uronic acids as their ester derivatives; and Cbz, trichloroacetyl (Tca), trifluoroacetyl (Tfa), 8208


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Scheme 29. Synthesis of Hyaluronan−Cholesterol Conjugates: (a) p-TsCl, Pyridine, rt, 16 h; (b) TEG, Dioxane, 101 °C, 5 h; (c) Hyaluronan, H2O, DMF, EDC, NHS, 24 h

Scheme 31. Synthesis of the Butanal-Functionalized Hyaluronan Disaccharide 160: (a) CdCO3, H2O, MeCN, 75 °C, 4 h, 75%; (b) Cl3CCN, 1,2-Dichloroethane, DBU, −10 to 0 °C, 1 h, 60%; (c) (i) TfN3, MeOH, DMAP, rt, 18 h; (ii) Ac2O, Pyridine, 0 °C, 10 h, 75%; (d) N2H4·AcOH, DMF, 0 °C to rt, 45 min, 70%; (e) K2CO3, Cl3CCN, CH2Cl2, rt, 48 h, 74%; (f) (i) TMSOTf, 4-Penten-1-ol, CH2Cl2, MS 4 Å, 0 °C, 1 h; (ii) NaOMe, MeOH, 0 °C to rt, 6 h, 80%; (g) CSA, THF, Benzaldehyde Dimethyl Acetal, 70 °C, 6 h, 75%; (h) TMSOTf, CH2Cl2, 0 °C to rt, 3.5 h, 78%; (i) AcSH, rt, 24 h, 70%; (j) TFA/H2O (2:1), CH2Cl2, 0 °C, 1 h, 86%; (k) 3 M NaOH (aq.), MeOH/H2O (9:1), rt, 2 h, 86%; (l) O3, −78 °C, Then Me2S, −78 °C to rt, 24 h, 85%

Scheme 30. Synthesis of the Hyaluronan Conjugate 155: (a) BF3·Et2O, Toluene, MS 4 Å, 0 °C, 2 h, 83%; (b) Zn, Ac2O, AcOH, THF, rt, 4 h, 88%; (c) DDQ, CH2Cl2/MeOH (4:1), rt, 12 h, 89%; (d) LHMDS, [(BnO)2P]2O, THF, −78 to 0 °C, 3 h, 81%; (e) H2, Pd/C, EtOAc/MeOH (1:1), NEt3, rt, 9 h, 90%; (f) (i) UMP-morpholidate, 1H-Tetrazole, DMF/ Pyridine (3:1), rt, 2 days; (ii) 3 M NaOH (aq.), MeOH, rt, 10 h; (iii) RP-HPLC, Gel-Filtration Column HPLC, 45%

efforts290 and especially for glycosaminoglycans. However, due to the large potential of such compounds for biochemical investigations, an increasing number of publications emerged also about glycosaminoglycan chemistry (see, e.g., Scheme 25).43−46,287,320−323 However, unlike peptide and nucleic acid chemistry, in the case of carbohydrate chemistry more sophisticated building blocks are needed, which are (so far) not commercially available (for a reasonable price). The challenges are the optimization of solid supports, the linker,323 the attachment/direction of the glycan, and glycosylation reaction as well as the cleavage cocktails for product liberation. It turned out that metathetic cleavage is very useful.323 Scheme 3 shows the general strategy for a glycosylation sequence on solid phase, and Figure 10 shows an automated oligosaccharide synthesis setup. In addition to the synthesis on solid supports, this technique has also been applied for the preparation of microarrays.322,325 Besides polymeric, insoluble supports, two other techniques should be mentioned as they are examples in glycosaminoglycan chemistry. Highly fluorinated compounds are highly soluble in fluorous solvents (and quite insoluble in normal organic solvents); thus, extraction of compounds and/or reagents with these solvents facilitates workup and purifications. In addition, cartridges with fluorinated resins can be used for purification (FluoroFlash, FSPE). These techniquesthe so-called fluorous chemistryhave also found

single strategy is not encountered; usually linear approaches to di- to pentasaccharides are encountered, and these larger building blocks are used in late-stage assembly. While the chemical opportunities are indeed limitless, resource availability still dictates the accessible chemical space. 2.2.1. Solid- and Solution-Phase Combinatorial Chemistry and (Semi)automated SynthesesGeneral Remarks. Despite the successful automated syntheses of other biopolymers, similar techniques have not yet been fully established for oligosaccharides in general despite recent 8209


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Scheme 32. Synthesis of the Dendritic Hyaluronan Dimer 162 and the Tetramer 163: (a) MeOH, rt, 1 h, Then NaCNBH3, rt, 16 h, 32% 162, and 15% 163

Scheme 33. Synthesis of Fluorescein-Labeled Hyaluronan Oligomers: (A) Fluorescein Hydrazide, DMSO/Phosphate Buffer (1:1) pH = 5.5, 30 °C, 92% 166, and 95% 167

applications in the synthesis of glycosaminoglycans (see, e.g., Scheme 54).326−333 In addition to this solution-phase approach, PEG-based solution-phase combinatorial chemistry has been explored. The soluble PEG conjugate can be used for traditional chemistry in most of the common solvents and is then precipitated with diethyl ether. Although this method works with small molecules, peptides, and other bioactive entities, glycosaminoglycan syntheses with PEG supports are seldom realized despite select successful examples.321,334,335

Scheme 34. Synthesis of L-Iduronic Acid Donor Building Blocks for the Synthesis of Heparin/Heparan Sulfate Oligosaccharides: (a) (i) Tf2O, Pyridine; (ii) NaOPiv, DMF; (b) NEt3, MeOH, 0 °C; (c) 90% TFA, rt, 3 h; (d) Imidazole, TDSCl, MeCN, −20 °C, 35% (2 Steps)

2.3. Glycosylation Sequence and Activation Systems

The assembly of the building blocks to intermediates and finally the complete target oligosaccharide requires careful choice of glycosylation methods as the building blocks are often sensitive to harsh reaction conditions, and the glycosidic bond has to be constructed in a stereospecific manner. For numerous applications (see, e.g., Scheme 9 and Scheme 81), the so-called Schmidt glycosylation using trichloroacetimidates has been used.336−338 This method gives not only highly stereoselective coupling reactions, it also provides stable intermediates that can be chromatographed and stored. The 8210


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Scheme 35. De Novo Synthesis of L-Iduronic Acid Donor Building Blocks for the Synthesis of Heparin/Heparan Sulfate Oligosaccharides: (a) (i) Anhydrous CuSO4, Acetone, H2SO4, rt, 24 h; (ii) 0.1 M HCl, MeOH, 40 °C, 1.5 h, 98%; (b) TrCl, Pyridine, rt, 12 h, Quant.; (c) BnBr, NaH, TBAI (Cat.), DMF, rt, 12 h, Quant.; (d) ZnBr2, EtSH, CH2Cl2, 0 °C, 75 min, 91%; (e) p-TsOH, Acetone, rt, 12 h, 93%; (f) PivCl, DMAP, CH2Cl2, 0 °C, 1 h, Quant.; (g) AcOH/H2O, 50 °C, 3 h, Quant.; (h) TrCl, Pyridine, rt, 24 h, 90%; (i) LevOH, DIC, DMAP, CH2Cl2, rt, 4 h, Quant.; (j) Tes, TFA, CH2Cl2, 0 °C, 5 min, 90%; (k) SO3·Pyridine, DMSO, DIPEA, 0 °C, 15 min, 94%; (l) (i) TMSCN, MgBr2· OEt2, CH2Cl2, 0 °C; (ii) N2H4·AcOH, CH2Cl2, MeOH, rt, 12 h, 82%; (m) AcCl, MeOH, Toluene, rt, 20 h, 70%; (n) (i) LevOH, DIC, DMAP, CH2Cl2, 0 °C, 2.5 h; (ii) BTI, NaHCO3 (aq.), MeCN, rt, 30 min, then TFA, 50 °C, 30 min; (iii) CCl3CN, DBU, CH2Cl2, 77% (3 Steps); (o) (i) LevOH, DIC, DMAP, CH2Cl2, 0 °C, 2.5 h; (ii) NIS, CH2Cl2, rt, 15 min, 80% (2 Steps)

Scheme 36. Synthesis of Heparin-like Trisaccharides for Conformational Studies of Iduronate: (a) TMSOTf, CH2Cl2, MS 4 Å, rt, 75−91%

chloride (Scheme 7).340−344 Both methods provide similar selectivity.345 In addition to this, glycosyl bromides (Figure 14)214 or thioglycosides (see, e.g., Scheme 4)346−356 are successfully employed in glycosylation reactions. For the activation of thioglycosides, a combination of NIS and TMSOTf (preferred method) or a mixture of silver triflate and p-TolSCl is used (see, e.g., Scheme 14 and Scheme 21), and for the glycosyl bromides, the famous Koenigs−Knorr glycosylation357 using silver carbonate (see, e.g., Scheme 20) or other promoters like indium(III) chloride (see, e.g., Scheme 18) can be utilized. Glycosyl fluorides358 have also been explored with success. The Ph2SO/Tf2O method also found use in some of the glycosylation reactions (see, e.g., Schemes 6, 12, 17, 41, and 80).309,346,359 Another method is the pentenyl ether method (see, e.g., Scheme 37).287,320,322,360−364

activation is usually achieved with Lewis acids such as BF3·OEt2 (see, e.g., Scheme 9 and Scheme 81) or TMSOTf (see, e.g., Scheme 16 and Scheme 19). A variation of this method used N-phenyltrifluoroacetimidates (Scheme 23),339 which are accessible via the corresponding N-phenyl imidoyl

Figure 12. Compilation of common orthogonally protected donor and acceptor building blocks for the synthesis of heparin/heparan sulfate oligosaccharides. 8211


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Scheme 37. Synthesis of the Protected Heparin Sulfate Tetrasaccharide 215: (a) Tf2O, CH2Cl2, −78 to −35 to 3 °C, 32%; (b) Pyridine, AcOH, N2H4·H2O, rt, 69%; (c) TMSOTf, CH2Cl2, −25 to −10 °C, 26%

Scheme 38. Synthesis of a Heparin-Related Tetrasaccharide 225 via Iduronamide 217: (a) HCl 30% aq., 72%; (b) Ac2O, DMAP, CH2Cl2, 66%; (c) Isopentyl Nitrite, AcOH, 69%; (d) DMF·DMA, CH2Cl2, 84%; (e) TMSOTf, CH2Cl2, 67% 222, 61% 225; (f) BnNH2, Et2O, 54%; (g) CCl3CN, DBU, CH2Cl2, 86%; (h) DDQ, CH2Cl2/H2O, 85%

(Chemo)enzymatic synthesis of GAGs also offer ample opportunities. Two strategies have routinely been pursued. In the first strategy, the native substrates (UDP sugars) have been linked via enzymes like hyaluronidase. This reaction can be used for the synthesis of different building blocks (lactosamine365 via galactosidases;366−370 others371,372) as well as for full GAGs like hyaluronan,373,374 heparan,327,375,376 and chondroitin.377 This method is described in section 3.5. In the second strategy, unnatural substrates like oxazolines170 have been used as donor (see, e.g., Schemes 66, 82, and 86)213,214,378 for hyaluronidase- and chitinase-catalyzed379,380 glycosidations. Oxazolines are strained substrates that release the natural N-acetyl moiety upon oligo- and polymerization. The combinations of two-enzyme,381,382 threeenzyme,383 and four-enzyme systems have also been explored.

Liberation of the target compounds and the purification of the polyanionic target molecules still is a major challenge in the chemical preparation of GAGs, and this liberation is usually optimized individually. Thus, the developments achieved in this area are treated in depth.

3. CHEMICAL SYNTHESES OF GLYCOSAMINOGLYCANS FROM MONOSACCHARIDES In this section the synthetic advances in preparing GAGs achieved within the last 10 years are reviewed (for earlier syntheses, see section 1.6, section 2.3, and Table 3). This part is subdivided along the structurally similar, major types of glycosaminoglycans (HA, HP/HS, CS/DS, and KS). As the access to the monosaccharidic building blocks or the disaccharidic repeating units is still the time-limiting step all GAG syntheses have to tackle, huge effort has been undertaken to optimize these; the discussion of different building block approaches unique to the discussed glycosaminoglycan class will be prominently featured in this part. The majority of all glycosaminoglycan syntheses follow classical glycosylation protocols; these already have been presented in section 2.3. Thus, only novel or unusual glycosylation methodologies will be presented in detail.

3.1. Chemical Syntheses of Hyaluronan

As elucidated earlier, the synthesis of hyaluronan requires the assembly of building blocks. 3.1.1. Synthesis of Building Blocks for Hyaluronans. A major challenge in the construction of hyaluronan is the installation of the sensitive glucuronic acid, which is in most cases installed via the neutral hexose and subsequent oxidation. A mild and efficient method for the oxidation of primary alcohols to the corresponding carboxylic acid applying TEMPO, NaOCl, and NaClO2 was established by Huang et al. This variant and related variants using TEMPO and [bis(acetoxy)iodo]benzene (BAIB) are perfectly suitable for the creation of the carboxylic acid functionality at the protected oligomers at late stage, due to its high tolerance toward many functional groups (see also Schemes 4, 5, 6, 14, 37, 60, and 71).388 8212


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Scheme 39. Fluorous-Supported Synthesis of the Heparan Sulfate Tetramer 229: (a) TfOH, MS 4 Å, CH2Cl2, −20 °C, 15 min, 72%; (b) N2H4·AcOH, CH2Cl2/MeOH (1:1), rt, 2 h, 86%; (c) SO3·Pyridine, DMF, rt, 5 h, 75%; (d) LiOH, H2O2, THF, rt, 18 h, 82%; (e) PMe3, NaOH, THF, rt, 5 h, 78%; (f) SO3·Pyridine, NEt3, NaOH, MeOH, CF3CH2OH, 0 °C to rt, 1 h, 87%; (g) (i) Pd/C, H2, AcOH, MeOH/H2O (1:1), rt, 12 h; (ii) Pd(OH)2/C, H2, AcOH, H2O, rt, 12 h, 85% (2 Steps)

Scheme 40. Mix Synthesis of a Small Heparan Sulfate Library 240−242: (a) TMSOTf, CH2Cl2, −30 °C, 3 h; (b) K2CO3, MeOH, rt, 18 h; (c) H2, Pd/BaSO4, 0.1 M Pyridine, MeOH/ THF 2:1, 24 h; (d) SO3·Pyridine, Pyridine, 24 h, rt, Then 16 h, 50 °C; (e) LiOH, H2O2, n-BuOH/THF 1:1, 0 °C, 3 h, Then KOH, rt, 57 h, (5 Steps) 20% 237, 22% 238, 4% 239; (f) H2, Pd(OH)2/C, 100 mM Phosphate Buffer pH = 7.0/ t-BuOH 6:4, Quant. 240 and 241, 73% 242

The conformationally fixed thioglycoside donor 27 was designed by Furukawa et al. starting from compound 25 (Scheme 4). By using di-tert-butylsilylbis(trifluoromethanesulfonate), a silylene acetal can be formed with free hydroxyl groups at the C2- and C4-position achieving the stereochemically disfavored but highly reactive inverted chair conformation of the pyranose ring, where every group is in the axial position. Because of the high steric hindrance due to the tert-butyl groups, nucleophiles are only able to attack on the upper side of the molecule, leading to strict β-selectivity in glycosylation reactions.455 3.1.2. Commonly Used Building Block.s. For the synthesis of hyaluronic acid oligomers, there are plenty of suitable orthogonally protected building blocks for liquid- and solid-phase chemistry and also for (semi)automated preparations. Therefore, Figure 11 should just illustrate how the protecting group pattern is often chosen in hyaluronic acid syntheses.318 Concerning the structure of the protecting groups, there is a huge variety of alterations. In the following sections there are many more examples of the building block design. 3.1.3. Building Block SynthesisNovel Glycosylation StrategiesDeprotection Sequences. The disaccharide of hyaluronan2-(acetylamino)-2-deoxy-3-O-β-D-glucopyranuronosyl-β-D-glucopyranosehas been the subject of a number of earlier attempts (see section 3). Furukawa et al. described an excellent glycosylation method for the β(1→3) glycosidic linkage. Here, conformationally constrained, highly reactive phenyl 2,4-O-acetyl-3,6-lactone-1-thio-β-D-glucopyranosides

(30) are used, which are easily synthesized from compound 28, obtaining near-quantitative yields and high β-stereoselectivity (Scheme 5).456 A synthesis of unprotected hyaluronan dimers 39 and 40 (Scheme 6) and tetramers 46 and 47 (Scheme 7) including di-tert-butylsilylene protecting groups was reported by Gold et al. where the reducing end of the oligomer bears a fluorescent 4-methylumbelliferryl group (MUF) for potential monitoring of hyaluronidase activities. The hydroxyl group in the C4-position at the nonreducing end is removed or methylated to avoid transglycosylation reactions.340 A special method to access unprotected hyaluronan oligomers was established by Ferrer Lopez et al. The synthesis starts from a commercially available chondroitin polymer that was degraded into its disaccharide subunits and then converted 8213


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Scheme 41. Synthesis of the Protected Heparin-Like Pentasaccharide 251: (a) Ph2SO, Tf2O, TTBP, CH2Cl2, −40 °C to rt, 91% 244, 51% 249; (b) BSP, Tf2O, CH2Cl2, −60 °C to rt, 76% 246, 53% 251; (c) (i) 10% TFA in Ac2O, 0 °C to rt; (ii) 6% Piperidine in THF, 92%

into the fully protected hyaluronan disaccharide repeating unit 51 (Scheme 8). The key step is the epimerization of the hydroxyl group in the C4-position at the D-galactosamine subunit on molecule 49 by activation and subsequent SN2-reaction to obtain a D-glucosamine moiety and, after acetylation, the protected hyaluronan disaccharide 50. With compound 51 in hand, unprotected hyaluronan di-, tri-, tetra-, and pentamers are easily accessible. The synthesis of the tetramer is depicted in Scheme 9.405,409 Rigol et al. synthesized the unprotected hyaluronan tetramer 62 with an allyl group at the reducing end, starting from three different monosaccharide building blocks (Scheme 10). For biophysical studies as well as for use in surface modifications, the group prepared disaccharide 60, incorporating a 13C-labeled glucuronic acid moiety in good yields. The methodology presented in Scheme 10 can also be used for the synthesis of higher oligosaccharides.291 The synthesis of an unprotected hyaluronan tetramer 68 containing a p-methoxyphenyl group at the reducing end was reported by Macchione et al. The synthesis starts with the three monosaccharide building blocks shown in Scheme 11, following a preglycosylation oxidation strategy. To simplify purification of intermediates, the group also explored the preparation of a hyaluronan trimer using a fluorous-assisted approach (see section 2.2.1). The fluorine side-chain was connected to the carboxylic acid functionality via an esterification reaction (not shown in Scheme 11).326 Furthermore, a combination of stepwise glycosylation reactions and one-pot strategies leading to the unprotected hyaluronan pentamer 76, bearing an azidopropyl group at the reducing end, was investigated by Dinkelaar et al. For the glycosylation sequences they applied Ph2SO/Tf2O as the activator system for the thiophenyl glycoside donors. The synthesis and the deprotection steps are depicted in Scheme 12.309

An efficient one-pot strategy for the synthesis of hyaluronan oligomers was developed by Huang and co-workers. The synthesis is based on an iterative preactivation method using thiotolyl glycosides as donors, which makes it easy to fashion oligosaccharides of up to six monosaccharide units in length in one vessel without any purification of intermediate oligosaccharides. Additionally this method proved to be very efficient, because the glycosylation sequences require only near-stoichiometric amounts of the building blocks in each stage. The most suitable building blocks for the preactivation procedure routinely contain a silyl protecting group to increase their solubility in diethyl ether. The preparation of the thioglycoside donor 79 is presented in Scheme 13, and that of the unprotected hyaluronan hexamer 88 is presented in Scheme 14.390 Gu et al. synthesized a fully unprotected hyaluronan hexamer bearing an amino functionality at the reducing end to form tetanus toxoid and human serum albumin conjugates linked via a squarate derivative. The preparation of the four monosaccharide building blocks needed for the hexamer synthesis 100 (Scheme 16) is depicted in Scheme 15. The conjugates can be used as disease markers by monitoring hyaluronan levels and potential vaccines against Group A Streptococcus infections.457 Dinkelaar et al. established an efficient synthesis toward unprotected hyaluronan tri-, penta-, and heptamers bearing an azidopropyl group at the reducing end, by using a combination of chemoselective and one-pot condensation strategies, last applying different activation methods. The route for the preparation of the fully protected hyaluronan heptamer 106 using thiophenyl glycosides is shown in Scheme 17 as well as the deprotection sequence to the desired all OH-free 107.352 A modular multigram synthesis of protected hyaluronan dimers up to octamers was established by Virlouvet et al. To get access to hyaluronan oligomers, they utilized the protected disaccharide 109 as a key intermediate, prepared from 8214


Chemical Reviews

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Scheme 42. Synthesis of the Heparin Pentasaccharide 261: (a) NIS, TfOH, CH2Cl2, −45 °C to rt, 89% 253, 96% 257; (b) (i) HF·Pyridine, THF; (ii) TEMPO, KBr, NaOCl, CH2Cl2, H2O, Then MeI, KHCO3, DMF; (iii) N2H4/AcOH/Pyridine, 45% 256, 77% 258; (c) (i) NIS, TfOH, CH2Cl2, −45 °C to rt; (ii) NIS, TfOH, CH2Cl2, −45 °C to rt, 20%; (d) (i) LiOOH, THF; (ii) SO3·NEt3, DMF; (iii) H2, Pd/C; (iv) SO3·Pyridine, H2O, 33%

1,2:5,6-di-O-isopropylidene-α-D -glucofuranose (108) and D-glucosamine hydrochloride (89) (Scheme 18). The fundamental coupling reaction to higher oligomers and the partial deprotection sequence is exemplarily shown in Scheme 19 in accessing the tetramer.318 The group around Lu et al. reported on a preactivation-based chemoselective glycosylation strategy to assemble a hyaluronan decamer. Therein they used three different disaccharide building blocks that were accessed from the monosaccharides 114 and 115 (Scheme 20). Initially they synthesized the tetramer 119, then the hexamer 121, and finally, via glycosylation reaction between those two oligosaccharide fragments under the action of a Lewis acid promoter, the unprotected decamer 123 (Scheme 21). TMSOTf seemed to be the best Lewis acid for their glycosylation methodology, as it prevents the side reaction to the formation of an oxazoline species

at the reducing end, which ultimately stops the saccharide coupling.391 3.1.4. (Semi)automated and Combinatorial Syntheses. As mentioned in section 2.2.1, despite the successful automated syntheses of other biopolymers, similar techniques have not been fully established for hyaluronan. However, recently, two remarkable solid-phase synthesis approaches to HA have been presented, showing the potential for hyaluronan syntheses. de Jong et al. designed a metathesis-cleavable linker starting from 1,4-cyclohexadiene (124) for solid-phase hyaluronan elongation sequences (Scheme 22). Regarding its acid and base stability, the linker-Merrifield resin system adapts well for oligosaccharide preparations. By using resin 127 the group was able to form several fully unprotected hyaluronan oligomers carrying a cyclopentene group at the reducing end after liberation from the solid support by metathesis−cleavage (Scheme 23).323 8215


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Scheme 43. Total Synthesis of the Antithromboembolic Drug Fondaparinux (17) by Lin et al.: (a) TMSOTf, Toluene, MS 4 Å, −20 °C, 30 min, 95%; (b) (i) Ac2O, TESOTf, 0 °C, 30 min; (ii) NH3, THF/MeOH (7:3), 0 °C, 30 min; (iii) CCl3CN, K2CO3, CH2Cl2, rt, 68% (3 Steps); (c) NIS, AgOTf, MS 4 Å, −15 °C, 30 min, 85%; (d) 0.2 M HCl/MeOH, 0 °C, 90%; (e) TEMPO, Ca(ClO)2, KBr, Aliquat 336, NaHCO3−Na2CO3 Buffer, CH2Cl2, CH2N2, 0 °C, 1 h, 83% (2 Steps); (f) TMSOTf, CH2Cl2, MS 4 Å, −20 °C, 30 min, 78%; (g) LiOH, H2O2, THF, −5 °C, 6 h, Then NaOH, 6 h, 65%; (h) SO3·TMA, DMF, 50 °C, 10 h, 91%; (i) 10% Pd/C, H2, MeOH, H2O/t-BuOH, rt, 10 h, 98%; (j) SO3·Pyridine, NaHCO3, H2O, 8 h, 65%

The micelles release the drugs when they enter areas of the body or cells were hyaluronidase enzymes are effective because of the hyaluronan degradation. The synthesis of such conjugates is shown in Scheme 29.127 The first chemical synthesis of an unprotected hyaluronan dimer attached to uridine diphosphate (UDP), the starting point of HA synthesis in vivo, was investigated by Wei et al. and is presented in Scheme 30. The hyaluronan conjugate 155 was then used to elucidate the catalytic mechanism of hyaluronic acid synthases.458 Rele et al. reported on a synthesis of dimeric and tetrameric glycodendrimers as hyaluronan mimetics for the investigation of receptor interactions. To do this, the hyaluronan disaccharide unit 160 bearing a butanal group at the reducing end was prepared from D-glucosamine hydrochloride (89) and acetobromo-α-D-glucuronic acid methyl ester (156) (Scheme 31) and subsequently attached to the diaminodiamide aromatic scaffold 161 via reductive amination (Scheme 32). The reductive amination led to a 2.1:1 mixture of the dimer 162 and the tetramer 163.459 To study the biomolecular intermolecular interactions of hyaluronan oligomers in solution, Maza et al. synthesized the fluorescein-functionalized hyaluronan dimer 166 and tetramer 167 to allow fluorescent polarization experiments (Scheme 33). The starting compounds for the attachment were provided by enzymatic degradation of native hyaluronan polysaccharides.217

A completely automated solid-phase construction of unprotected hyaluronan oligosaccharides up to a pentadecamer was developed by Walvoort et al. The elongation process was realized with monosaccharide 133 and disaccharide 42 (Scheme 24) on a metathesis-cleavable Merrifield resin-linker setup. The automatized cycle for the pentadecamer 138 preparation is shown in Scheme 25.341 In addition to the work reported regarding solid-phase syntheses of GAGs, de Paz et al. explored a hyaluronan oligosaccharide synthesis relying on a polyethylene glycol (PEG) polymer linked to the oligosaccharide chain by an acylsulfonamide group. The preparation of the linker system is presented in Scheme 26. The acylsulfonamide group was used as a linker as it enables simultaneous cleavage of the saccharide chain from the polymer and allows orthogonal functionalization, making it suitable for interesting (bio)conjugates. The group successfully attached two protected monosaccharides to the PEG acylsulfonamide linker 142 (Scheme 27). Starting from compound 144, an azide-functionalized tetraethylene glycol monoamine was connected to the saccharide residue by cleaving the sulfonamide group, but further elongation attempts failed. However, it was shown that it is possible to perform oligosaccharide synthesis on a PEG-polymer, avoiding the fragile acylsulfonamide linker (Scheme 28).334 3.1.5. Syntheses of Hyaluronan Conjugates and Other Related Structures. The group of Deng et al. synthesized a hyaluronan-based drug-delivery system where lipophilic cholesterol is attached to hydrophilic hyaluronan to afford amphiphilic conjugates. These conjugates are able to self-assemble into stable micelles in which drugs can be temporally stored.

3.2. Chemical Syntheses of Heparin/Heparan Sulfate

Low-molecular-weight heparins (LMWHs) are carbohydratebased anticoagulants clinically used to treat thrombotic and 8216


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Scheme 44. Total Synthesis of the Antithromboembolic Drug Fondaparinux (17) by Chang et al.: (a) NIS, TfOH, CH2Cl2, −60 to −40 °C, 3 h, 70%; (b) NaOMe, MeOH, rt, 4 h, 92%; (c) (i) LevOH, EDC, DMAP, CH2Cl2, 0 °C, 16 h; (ii) DDQ, H2O, rt, 4 h, 70%; (d) (i) NIS, TfOH, CH2Cl2, −60 to −40 °C, 3 h; (ii) TFA, H2O, rt, 1 h, 58%; (e) Ac2O, NEt3, CH2Cl2, 0 °C to rt, 16 h, 82%; (f) (i) NIS, TfOH, CH2Cl2, −78 to −20 °C, 4 h; (ii) TMSOTf, Ac2O, −20 °C, 8 h, 65%; (g) TMSSTol, ZnI2, CH2Cl2, rt, 1 h, 85%; (h) NIS, TfOH, CH2Cl2, −20 to 0 °C, 4 h, 61%; (i) Mg(OMe)2, MeOH, CH2Cl2, rt, 16 h, 83%; (j) (i) TEMPO, BAIB, CH2Cl2, H2O, rt, 24 h; (ii) N2H4, Pyridine, AcOH, 0 °C, 2 h; (k) (i) CH2N2, Et2O, CH2Cl2, rt, 16 h; (ii) HF·Pyridine, Pyridine, rt, 3 days, 60% (2 Steps); (l) SO3·NEt3, DMF, 60 °C, 73%; (m) LiOH, H2O2, THF, 37 °C, 3 days, 71%; (n) (i) Pd(OH)2, H2, Phosphate Buffer (pH = 7), MeOH, rt, 2 days, 80%; (ii) SO3·Pyridine, NaOH, H2O, rt, 2 days, 81%

glycosylated in good yields when compared to commonly used uronic acid donors. They successfully used these donors for the preparation of heparan sulfate oligosaccharides.395 Figure 12 shows a compilation of common, orthogonally protected donor and acceptor building blocks for the synthesis of heparin/heparan sulfate oligosaccharides.349,395,462,465,466 3.2.2. Building Block SynthesisNovel Glycosylation StrategiesDeprotection and Sulfation Sequences. In contrast to hyaluronan, heparan sulfate exhibits distinct sulfation patterns, thus requiring appropriate synthetic approaches. The natural sulfation is catalyzed by enzymes (sulfotransferases),467,328 and these can be used also to decorate chemically prepared heparan. The challenge for heparin synthesis is the distinct sulfation patterns (mediated by sulfatases468) that require sophisticated protecting group strategies to allow the preparation of different, defined species. Sulfated heparans have been the subject of various investigations producing heparans with chain lengths from 2469,470 up to 40 units.471−473 Tatai et al. developed a new activation method for thioglycosides using dimethyl disulfide−triflic anhydride (DMTST). The method was successfully applied in the synthesis of various disaccharides in good yields and short reaction times. This could also be utilized to prepare heparin-like disaccharides in excellent yields.350

other disorders. Therefore, various strategies have been published to synthesize defined heparans. Some of the molecules have trivial names, such as mucosin, which is 2-amino-2-deoxy4-O-β-D-glucopyranuronosyl-α-D-glucopyranose.460,461 3.2.1. Commonly Used Building Blocks. Ke et al. developed a convenient route for the preparation of iduronic acid building blocks from 1,2-O-isopropylidene-6,3-glucuronolactone. These building blocks were used for the synthesis of heparin-like disaccharides and one trisaccharide with different sulfation patterns. This provides an effective synthetic access to heparin-like oligosaccharides. The synthesis of different 462,463 L-iduronic acid building blocks is depicted in Scheme 34. Adibekian et al. developed a route for the de novo synthesis of D-glucuronic and L-iduronic acid donor building blocks starting from D-xylose, which is available commercially in bulk quantities. Key steps of the synthesis were a chelationcontrolled Mukaiyama-type aldol addition for accessing the D-glucuronic acid and a chelation-controlled cyanation for obtaining the L-iduronic acid derivative. They demonstrated the synthetic utility of these building blocks by preparing a heparin disaccharide bearing an amino linker at the reducing end. The preparation of the L-iduronic acid moieties is shown in Scheme 35.316,464 Dhamale et al. developed a series of efficient glucuronic acid donors with PivOAc esters at the C2-position. These donors 8217


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Scheme 45. Synthesis of Fondaparinux Sodium Starting from the Commercially Available Heparin-Like Pentasaccharides 277: (a) SO3·NEt3, DMF, 50 °C, 92%; (b) 5% Pd/C, H2, MeOH/THF (3:1), NEt3, rt, 30 min, 90%; (c) PtO2, NH4HCO2, MeOH/ H2O (1:1), rt, 2.5 h, 60%; (d) SO3·Pyridine, H2O, Na2CO3, NaOH, rt, 80%; (e) SO3·NEt3, DMF, 55 °C, 89%; (f) Pd black, NH4HCO2, MeOH/H2O (1:1), 50 °C, 30 min, 55%

Scheme 46. Synthesis of the Heparan Sulfate Hexasaccharide 287: (a) TMSOTf, − 20 to 5 °C, MS 4 Å, 64% 284, 65% 286; (b) NEt3, CH2Cl2, 82%; (c) N2H4·AcOH, Toluene/EtOH, 90%; (d) SO3·Pyridine, DMF; (e) (i) NEt3, DMF; (ii) LiOH, H2O2, THF; (iii) 4 M NaOH, MeOH, 58% (3 Steps); (f) PMe3, THF, NaOH, 65%; (g) SO3·Pyridine, MeOH, NEt3, 0.1 M NaOH, 50%; (h) (i) Pd/C, H2, MeOH/H2O; (ii) Pd(OH)2/C, H2, H2O, 67%

8218


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Scheme 47. One-Pot Preactivation Synthesis of HeparinLike Hexasaccharide 296: (a) HF·Pyridine, Pyridine, rt, 92% 289, 89% (2 Steps) 291, 86% 293; (b) BnBr, Ag2O, MS 4 Å, rt, 94% 290, 85% 294; (c) pTolSCl/AgOTf, CH2Cl2/Et2O, N-Benzyl-N-benzyloxycarbonyl-3-amino Propanol, −78 to −10 °C; (d) (i) 294, AgOTf; (ii) pTolSCl, −78 °C, 5 min; (iii) 289, TTBP, −78 to −10 °C, 120 min; (iv) 295, −78 °C, 5 min; (v) pTolSCl, −78 to −10 °C, 62%

Scheme 48. Heparin Oligosaccharide Synthesis with Preinstalled Sulfate Esters: (a) Mg(OMe)2, MeOH/CH2Cl2, 0 °C, 82%; (b) 299, 1,2-Dimethylimidazole, CH2Cl2, 35 °C 80%; (c) (i) DDQ, H2O/CH2Cl2; (ii) Levulinic Acid, EDC, DMAP, 74%; (d) AgOTf, pTolsSCl, CH2Cl2/MeCN, −78 °C, 82%; (e) TMSOTf, CH2Cl2, −20 °C, 84%; (f) 300, AgOTf, pTolsSCl, CH2Cl2/MeCN, −78 to 0 °C, 70%

The synthesis of two heparan sulfate trisaccharides representing the respective minimal binding motif for FGF-1 and FGF-2 was successfully undertaken by Tatai and Fuegedi. Starting with their established synthesis of iduronic thioglycosides, they prepared orthogonally protected iduronic acid building blocks, as acceptor and donor, respectively. The required glucosamine building block was synthesized in good yields and coupled highly stereoselectively with the iduronic acceptor by the activation method with dimethylsulfonium triflate (DMTST). After deprotection of the temporary chloroacetyl protecting group, the obtained disaccharide acceptor was coupled under similar conditions with the iduronic acid thioglycoside in good yield, affording the desired compounds. The design of the protecting group pattern allowed for two different deprotection sulfation sequences, which gave trisaccharides resembling both the FGF-1 and FGF-2 minimal binding motifs.475 Munoz-Garcia et al. investigated the conformational equilibrium of iduronate in heparin-like trisaccharides with different substituents on the neighboring ring. Therefore, they prepared several heparin trisaccharides with different sulfation patterns. Known disaccharide donor 201 was coupled with four different glucosamine acceptors (202−205), and the resulting trisaccharides were subjected to different deprotection sulfation

Mensah et al. developed a new α-selective glycosylation with N-benzylidene glucosamine trichloroacetimidates.336−338 They used substoichiometric amounts of a nickel catalyst for the α-selective glycosylation of glucosamine donors with a wide range of mono- and disaccharide acceptors. The α-selectivity proved to be unaffected by the nature of the protecting groups of the acceptor. However, the N-benzylidene residue was crucial for retaining the excellent stereoselectivity. By this method several heparin disaccharides could be prepared in good yields and superior stereoselectivity.474 Tatai and Fuegedi described efficient preparations of idose and iduronic acid thioglycosides. Starting from 1,2:5,6-di-Oisopropylidene-α-D-glucofuranose (108), which was readily transformed to the L-ido epoxide, they prepared a 1,6-anhydroidopyranose derivative. Thiolysis with consecutive acetylation afforded the desired thioglycoside with full α-selectivity. Standard manipulations gave idopyranosyl glycosyl donor with orthogonal protection at position 6 for the later conversion to the uronic acid. This was achieved with pyridinium dichromate in tert-butanol to afford the corresponding ester. The glycosyl donors were successfully applied in glycosylation reactions to heparin-related trisaccharides.475 8219


Chemical Reviews

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Scheme 49. Synthesis of a Sulfated Heparin-like Hexasaccharide 317: (a) TMSOTf, CH2Cl2, 76%; (b) NaBH3CN, HCl/Et2O, THF, 90%; (c) TMSOTf, CH2Cl2, 59% (18% 306 Recovered); (d) N2H4·AcOH, CH2Cl2, 93%; (e) Ag2O, DMF, BnBr, 75%; (f) EtSH, p-TsOH (Cat.), 84%; (g) BzCN, NEt3 (Cat.), MeCN, −40 °C, 99%; (h) TMSOTf, CH2Cl2, 52% (44% 312 Recovered); (i) H2O2, LiOH (Aq.), THF, KOH (Aq.), MeOH, 96%; (j) SO3·Pyridine, Pyridine, 50%; (k) H2, Pd/C, MeOH, H2O, SO3·Pyridine, H2O, pH = 9.5, 93%

sequences to afford eight sulfated heparin trisaccharides (four are shown in Scheme 36). Those were analyzed at several temperatures via NMR experiments.465 The synthesis of the tetrameric heparin sulfate 215 was successfully conducted by Bindschadler et al. They used a (2 + 2)-glycosylation approach. Several donors were used for the coupling of disaccharide 212 with N,N-diacetyl glucosamine acceptor 211; however, sulfoxide 210 proved to be the most effective one. After levulinate deprotection, tetrasaccharide 215 was synthesized with the Schmidt donor336−338 214, which was previously prepared by the same working group (Scheme 37).364 The synthesis of two tetrameric candidate epitopes against 10E4 monoclonal antibodies was conducted by Hamza et al. In the convergent approach, an acyl migration of an α-acetate was used for differentiation of the two amino groups. The glycosylation of a dimeric Schmidt donor336−338 with either glucuronic or iduronic acid containing disaccharide acceptors furnished the tetrasaccharides in good yields and stereoselectivities.476

A tetrasaccharide analogue of the minimum substrate of heparanase was prepared by Chen et al. Their convergent approach relied on a (2 + 2)-glycosylation strategy. The respective disaccharides were synthesized starting from glucosamine and glucose and oxidized to the corresponding uronic acid derivatives after successful glycosylation in excellent yields. The glycosylation to the tetrasaccharide was conducted in mediocre yield and moderate stereoselectivity. After deprotection and sulfation, the minimum substrate analogue of heparanase was successfully obtained.477 A convergent (2 + 2)-glycosylation approach for the synthesis of a heparan sulfate tetrasaccharide antigen associated with prion diseases was described by Daragics and Fuegedi. The challenge of the differentiation of the two amino moieties in such oligosaccharides was overcome by direct use of an acetylated glucosamine acceptor at the reducing end. The required disaccharide donor was prepared by glycosylation of a glucose donor and subsequent oxidation to the uronic acid. By careful choice of donor and promoter, they were able to overcome the inherent low reactivity of N-acetyl acceptors 8220


Chemical Reviews

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Scheme 50. Exemplary Synthesis of a Heparin Hexasaccharide 329: (a) (i) TMSOTf (Cat.), CH2Cl2, −20 °C; (ii) N2H4·H2O, Pyridine/AcOH, CH2Cl2, 56%; (b) TMSOTf (Cat.), CH2Cl2, −20 °C, MS 4 Å, 70%; (c) N2H4·H2O, Pyridine/AcOH, CH2Cl2, 89%; (d) TMSOTf (Cat.), CH2Cl2, −20 °C, MS 4 Å, 82%; (e) N2H4·H2O, Pyridine/AcOH, CH2Cl2, 86%; (f) TMSOTf (Cat.), CH2Cl2, −20 °C, MS 4 Å, 80%; (g) LiOH, H2O2, KOH, MeOH, 63%; (h) PMe3, THF, NaOH, 83%; (i) SO3·Pyridine, NEt3, MeOH, NaOH, Quant.; (j) H2, Pd/C, 97%

to yield only the desired α-linked tetrasaccharide in good yield.354 A novel route to heparin-like di- and tetrasaccharides via iduronamides was developed by Hansen et al. Their approach based on their previously reported large-scale synthesis of cyanohydrin 216, which could readily be converted to iduronic acid acceptor 220 via acidic hydrolysis, selective 1,2-diacetylation, and amide hydrolysis by isopentyl nitrite. The iduronic acid 220 served as the starting material for the preparation of several thioglycosides as well as acceptor for the generation of disaccharide 222, which could be iteratively elongated to the corresponding tetrasaccharide 225 (Scheme 38).356 The synthesis of a tetrasaccharide substrate for human heparanase was conducted by Takeda et al. Starting from a common disaccharide, consisting of glucose and 1,6-anhydroglucosamine, they prepared a disaccharide chloro donor and acceptor with the anhydro moiety still intact. After α-selective glycosylation, the tetrasaccharide was subjected to acetolysis and transformed to the trichloroacetimidate for coupling with n-octanol. The synthesis was completed by O-sulfation, global

deprotection, and subsequent N-sulfation, during which an unexpected 3-O-sulfation occurred.478 Zong et al. established a fluorous-supported modular synthesis of the heparan sulfate tetramer 229, which is shown in Scheme 39. Because of the perfluorodecyl group at the reducing end of the saccharides, the polar compounds can be purified easily by fluorous solid-phase extraction (FSPE). The fluorous chain also made it possible to repeat the glycosylation reactions as required to drive them to completion. Using the same methodology, the group also synthesized a heparan sulfate tetramer bearing an acetyl group at the amine functionality instead of the SO3− group.329 Dilhas et al. presented a mix synthesis with charge-based demixing of heparin sulfate libraries. For their combinatorial synthesis of heparin sulfate fragments, they prepared a set of disaccharide building block acceptors (231−233). Those were mixed in an equimolar ratio and glycosylated with donor 230 prepared from a similar precursor. Glycosylation with iduronate building blocks proceeded in quantitative yield and full stereoselectivity regarding the formed glycosidic bonds, whereas the glucuronate derivative only led to poor yields and lower 8221


Chemical Reviews

Review

Scheme 51. Synthesis of Oligosaccharide Backbone 335 for Preparation of Glycopeptide Syndecan-1: (a) AgOTf, pTolSCl, MS 4 Å, −78 °C, Then 331, TTBP, −78 to 0 °C, 93%; (b) AgOTf, pTolSCl, MS 4 Å, −78 °C, Then 332, TTBP, −78 to 0 °C, 83%; (c) (i) HF·Pyridine; (ii) TEMPO, BAIB, CH2Cl2/H2O/t-BuOH (4:1:4); (iii) MeI, K2CO3, DMF, 84%; (d) Peptide Coupling with Preformed Peptide [Syndecan-1 Has SGSGAGALPETGG]; Deprotection

Scheme 52. Synthesis of Heparin Analogues: (a) TMSOTf, MS 4 Å, CH2Cl2, −70 to 0 °C, 84%; (b) DDQ, CH2Cl2/H2O (18:1), rt, 92% 340, 93% 344; (c) Ac2O, Cu(OTf)2, 0 °C, 99% 341; (d) (i) NH3, MeOH/THF (1:4), 0 °C; (ii) CCl3CN, K2CO3, 90% 342; (e) 340, TMSOTf, MS 4 Å, CH2Cl2, −40 to 0 °C, 98%; (f) 344, TMSOTf, MS 4 Å, CH2Cl2, −40 to 0 °C, 84%; (g) NaOMe, CH2Cl2/MeOH (1:1), 92%; (h) TEMPO, BAIB, CH2Cl2, 86%; (i) (i) TBAF, THF, 50 °C, Then LiOH; (ii) SO3·NEt3, DMF, 60 °C, 62%

widely due to the increasing charge of the respective tetrasaccharides. After demixing, a small heparin sulfate library (240− 242) was obtained via parallel debenzylation (Scheme 40).479

stereoselectivity. After exclusion chromatography the obtained mixture of tetrasaccharides (234−236) was deprotected and sulfated and then separated via HPLC. The retention times differed 8222


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Review

Scheme 53. Synthesis of a 3-O-Sulfonated Heparan Sulfate Octasaccharide 360: (a) DDQ, CH2Cl2/H2O, 87%; (b) Lev2O, NEt3, DMAP, CH2Cl2, 95% 348, 81% (2 Steps) 350; (c) Cu(OTf)2, Ac2O, 95%; (d) TMSSTol, ZnI2, CH2Cl2, 92%; (e) Thioacetic Acid, Pyridine, CHCl3, 87%; (f) Isoprenyl Acetate, p-TsOH, 65 °C, 78%; (g) (i) NH3 in MeOH/THF; (ii) CCl3CN, K2CO3, CH2Cl2, 90%; (h) 340, NIS, TfOH, CH2Cl2, −78 to −20 °C, 91% 358; (i) (i) N2H4, AcOH, Pyridine, 93%; (ii) NaH, BnBr, CH2Cl2/DMF, 75% (2 Steps); (j) TMSOTf, CH2Cl2, −40 °C, 70%; (k) N2H4, AcOH, Pyridine, 85%; (l) (i) Cu(OTf)2, Ac2O, 88%; (ii) TMSSTol, ZnI2, CH2Cl2, 95%; (m) 350, NIS, TfOH, CH2Cl2, −78 to −20 °C, 91%; (n) N2H4, AcOH, Pyridine, CH2Cl2, 81%; (o) (i) NaOMe, MeOH/CH2Cl2, 85%; (ii) TEMPO, BAIB, CH2Cl2/H2O, 80%; (p) (i) DDQ, CH2Cl2/H2O, 71%; (ii) LiOH, THF; (iii) CH2N2, CH2Cl2, Et2O, 81%; (q) SO3·NEt3, DMF, 60 °C, 76%; (r) (i) HF·Pyridine, Pyridine, 83%; (ii) LiOH, H2O2, H2O, THF, MeOH, 37 °C, 74%; (s) 1,3-Propanedithiol, NEt3, Pyridine/H2O, 50 °C, 81%; (t) (i) SO3· Pyridine, NaOH, NEt3, MeOH; (ii) Pd(OH)2/C, H2, Phosphate Buffer pH = 7, 47%

α(1→4) or β(1→4) glycosidic linkage in excellent yields. They used this methodology for the synthesis of the protected pentasaccharide 251 (Scheme 41).347 A one-pot synthetic strategy for assembly of a heparin-like pentasaccharide was presented by Polat and Wong. They

Codee et al. described a modular approach for the synthesis of heparin and heparin sulfate fragments, by using a linear glycosylation strategy (Scheme 41). They successfully applied 1-hydroxyl glucosamine donors 243 and 1-thiophenyl acceptors (200, 248) for the stereoselective formation of the respective 8223


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Review

Scheme 54. Synthesis of a Heparin Dodecasaccharide: (a) (Mca)2O, Pyridine, 94%; (b) NBS, H2O, 74%; (c) CCl3CN, Cs2CO3; (d) TMSOTf, MS 4 Å, CH2Cl2, −40 °C, 65%; (e) NIS, TfOH, MS 4 Å, −20 °C, 63%; (f) Thiourea, 2,6-Lutidine, 96% 366; (g) NIS, TfOH, MS 4 Å, CH2Cl2, −10 °C, 69%; (h) 365, NIS, TfOH, MS 4 Å, CH2Cl2, −10 °C, 68%

Scheme 55. Reaction Cycle for the Synthesis of a Protected Heparin-Related Dodecamer: (a) NIS, AgOTf, MS 4 Å, CH2Cl2, 0 °C, 2 h, 88% 375, 94% 376, 80% 377, 91% 378, and 90% 379; (b) Pyridine, MeOH, 50 °C, 4−7 h, 94% 370, 94% 371, 92% 372, and 86% 373

prepared idopyranosyl thioglycoside 254, glucopyranosyl thioglycoside 191 building blocks, and azidoglucosyl acceptors 252 and 255 with well-defined reactivity. After successful application of the reactivity-based glycosylation to the respective disaccharides (253 and 257) with excellent yields and stereoselectivity, they extended this methodology to the synthesis of a tetra- and pentasaccharide 260 (Scheme 42). The formation of the uronic acids was conducted after glycosylation by oxidation of the primary hydroxyl groups to avoid the use of the electron-deficient uronic acid glycosyl donors, which perform poorly in this kind of glycosylation reaction. Deprotection and sulfation provided the desired heparin pentasaccharide 261.349 A total synthesis toward the antithromboembolic heparinrelated pentasaccharide fondaparinux (17) was created by Lin et al. The synthesis follows a linear route with entirely 36 steps,

Figure 13. Variety of synthesized heparan sulfate oligomers.

with an overall yield of ∼0.017%. However, the preparation utilizes only D-glucose (1) and cellobiose (262) as starting materials (Scheme 43). The main advantage of this strategy is the use of only benzyl-, acetyl-, and benzoyl-protection groups, which facilitates the deprotection sequence.480 Another total synthesis of the antithromboembolic drug fondaparinux (17) was created by Chang et al. The synthesis 8224


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Scheme 56. Solid-Phase Synthesis of Heparan Sulfate Trisaccharide 385: (a) NIS, TMSOTf; (b) N2H4·AcOH, CH2Cl2; (c) TMSOTf, CH2Cl2; (d) NIS, TMSOTf, CH2Cl2; (e) NaOMe, MeOH

Scheme 57. Synthesis of the Heparin Glycopolymer 390: (a) TMSOTf, CH2Cl2, −30 °C, 1 h, 85%; (b) (i) LiOH (Aq.), H2O2, THF, rt, 12 h; (ii) MeOH, NaOH, rt, 12 h, 82% (2 Steps); (c) SO3·TMA, DMF, 55 °C, 2 days, 71%; (d) PMe3, NaOH, THF, rt, 16 h; (e) SO3·Pyridine, Pyridine, NEt3, rt, 24 h, 54% (2 Steps); (f) Grubbs 3rd-Generation Catalyst, MeOH, 1,2-Dichloroethane, 55 °C, 2 h; (g) Pd(OH)2/C, H2, MeOH, Phosphate Buffer pH = 7.4, rt, 2 days, 63−92%

takes place in 32 collective steps, reaching an overall yield of 0.63% over 22 linear steps from D-glucosamine hydrochloride (89). Starting from the three abundant available monosaccharides D-glucosamine hydrochloride (89), D-glucose (1), and 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (108), the five required protected monosaccharide building blocks are readily accessible. Scheme 44 illustrates the entire total synthesis.481 Additionally, there are two more total syntheses of the antithromboembolic drug fondaparinux (17) by Li et al.482 and Dai et al.483 The syntheses follow the same route as shown above in Scheme 43, just using different starting materials. A different approach for the synthesis of fondaparinux sodium was established by Manikowski et al. Starting from the commercially available heparin-like pentasaccharides 277, they prepared the desired fondaparinux sodium by reversing the common O-sulfation−hydrogenation−N-sulfation sequence to a hydrogenation−sulfation−hydrogenation sequence (Scheme 45). The involved one-pot N- and O-sulfation was achieved by increasing the reaction temperature and employing a large excess of sulfur trioxide triethylamine complex.484 Koziol et al. created a very similar route leading to fondaparinux sodium. For the simultaneous removal of O-benzyl, N-carboxybenzyl protection groups and the reduction of azide groups to the corresponding amine, the group used transfer hydrogenation conditions and ammonium formate as a hydrogen source. The reaction generally proceeds in

Chemical Synthesis of Glycosaminoglycans - Chemical Reviews (ACS

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