current medicinal chemistry, 1993-2031 1993 recent ... · 1994 current medicinal chemistry, 2003,...

Current Medicinal Chemistry, 2003, 10, 1993-2031 1993

0929-8673/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd.

Fig. (1). Generalized structure of the major forms of proteoglycans.

Recent Chemical and Enzymatic Approaches to the Synthesis ofGlycosaminoglycan Oligosaccharides

Nathalie A. Karst and Robert J. Linhardt*

Division of Medicinal and Natural Products Chemistry, Department of Chemistry and Department of Chemicaland Biochemical Engineering, University of Iowa, Iowa City, Iowa, 52242, USA

Abstract: Glycosaminoglycans, highly charged polycarboxylated, polysulfated polysaccharides, are an

important class of therapeutic agents and investigational drug candidates. Heparin has been widely used as a

clinical anticoagulant for over 60 years. Low molecular weight heparins have begun to displace heparin and

recently a synthetic heparin pentasaccharide was approved for clinical use in Europe. In addition to heparin

(and the related heparan sulfate glycosaminoglycan), dermatan sulfate, chondroitin sulfate, hyaluronan and

their derivatives are all in various stages of clinical evaluation. This review focuses on the chemical and

chemoenzymatic synthesis of glycosaminoglycan oligosaccharides. Recent advances in functional group

protection chemistry, conversion of D-gluco to L-ido or D-galacto configurations, glycosylation reactions and

the preparation and use of novel starting materials in acidic oligosaccharide synthesis are discussed.

Keywords: Glycosaminoglycans, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate,oligosaccharide synthesis, enzymatic synthesis.

I. INTRODUCTION

I.1. Glycosaminoglycans

Glycosaminoglycans (GAGs) are polyanionic, linear,microheterogeneous polysaccharides that are often covalentlylinked to a protein core, called a proteoglycan (PG) “Fig.(1)”. PGs are found in the membranes of all animal cells,intracellularly in secretory granules of selected cells or

extracellularly in the matrix, where they display a widevariety of biological functions. Although the core protein ofPGs is also essential, many events mediated by PGs arebelieved to result from their GAG chains. With thediscovery of the anticoagulant activity of heparin, interest in

*Address correspondance to this author at the Division of Medicinal andNatural Products Chemistry, Department of Chemistry and Department ofChemical and Biochemical Engineering, University of Iowa, Iowa City,Iowa, 52242, USA; Fax: 319-335-6634; E-mail: [email protected]

the other members of the GAG family has increased; thusheparan sulfate (HS), hyaluronic acid (HA), chondroitinsulfate (CS) and dermatan sulfate (DS) became a focus ofnumerous investigations. The complexity of GAG polymersmakes the elucidation of their structure difficult and thestructural basis for many of their important biologicalactivities still remain unclear. Preparation of pure GAGoligosaccharides required for structural determination, hasinvolved many years of research by carbohydrate chemists

around the world. Since only few reviews [1,2] have beenpublished on this subject, the aim of this paper is to give aglobal view and the major recent advances in the chemicaland enzymatic synthesis of GAG oligosaccharides.

I.2. Synthetic Methodology for Oligosaccharides

Common strategies in carbohydrate synthesis rely onefficient protecting group manipulations. GAGoligosaccharide synthesis also requires stereoselective

1994 Current Medicinal Chemistry, 2003, Vol. 10, No. 19 Karst and Linhardt

Fig. (2). Glycosylation reaction features. The three major features of glycosyl donor and acceptor are labelled.

Table 1. Activation of the Anomeric Position Used in the Syntheses of Various GAGs(References for the Syntheses can be FoundAll Through Out the Review)

C-1 activation

Uronic Acid Hexosamine

GAG D-Glc, L-Ido/ D-GlcA, L-IdoA D-GlcN, D-GalN

HA -OC(NH)CCl3, -SOPh -OC(NH)CCl3, -Br

CS -OC(NH)CCl3, -Br -OC(NH)CCl3

DS -OC(NH)CCl3, n-pentenyl-Br, -Cl, -SPh

-OC(NH)CCl3

HS/HP -OC(NH)CCl3, -Br -OC(NH)CCl3, -Cl, -F

Table 2. Substituents at the C-2 Position of Donor Units Used in the Syntheses of Various GAGs

C-2 protection

Uronic Acid Hexosamine

GAG D-Glc, L-Ido/ D-GlcA, L-IdoA D-GlcN, D-GalN

HA MBz, Ac, Bz, Piv N-Phth NH-TCA, NH-Troc, N3

CS Ac, Bz, MBz NH-TCA, N3

DS Ac, Bz, Piv NH-TCA, N3

HS/HP Ac, Bz N3

glycosylation steps “Fig. (2)”. A high yield and highstereoselectivity in the coupling step are critical features ofefficient oligosaccharide synthesis. Several factors caninfluence the stereoselectivity (α/β ratio) in glycosylations,among the most important are the nature of the leavinggroup at the anomeric position and the substituent at C-2position of the glycosyl donor.

Glycosyl Donor Activation

Thioglycosides, n-pentenyl glycosides, bromides,fluorides and sulfoxides have been used in GAG synthesis.Nevertheless trichloroacetimidate donors, introduced bySchmidt [3], remain the most commonly encounteredactivation groups for forming glycosyl linkages to bothhexosamine and uronic acid residues (Table 1).

Substituent at C-2 Position of the Donor

Formation of a 1,2-trans glycosidic linkage is a majorrequirement in HA, CS, DS synthesis. A participating groupat the C-2 position of the donor unit is commonly used todirect the stereoselectivity. Thus, the C-2 of uronic acidderivatives are generally protected as esters (Table 2).Moreover, since donors with N-acetyl-D-GlcN are poor

glycosyl donors, various alternative amino group protectionshave been investigated. N -Phthaloyl (N -Phth), N H -trichloroethoxycarbonyl (NH-Troc) and NH-trichloroacetyl(NH-TCA) derivatives afford efficient glycosylation donorsdirecting the formation of 1,2-t rans - 2 - a m i n o - 2 -deoxyglycosides. The non-participating azido grouprepresents an excellent latent functionality for an aminogroup and has been widely used in all type of GAG

Synthesis of Glycosaminoglycan Oligosaccharides Current Medicinal Chemistry, 2003, Vol. 10, No. 19 1995

Table 3. Structures of Different Synthetic Hyaluronic Acid Oligosaccharides

Oligosaccharide structure

1992 βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc-OMP [11]

1993 βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcNA-OMP [12]

1994 βGlcA(1→3) βGlcNAc-OMP [13, 14]

βGlcA(1→3) βGlcNAc(1→4) βGlcA –OMP

βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc-OMP

βGlcNAc(1→4) βGlcA-OMP

βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA-OMP

1994 βGlcA(1→3) βGlcNAc-OMP [15]

1996 βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA-OMe [16]

βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA-OMe

βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA-OMe

1998 βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc-OMP [17]

βGlcA(1→3) βGlcNAc(1→4) βGlcA(1→3) βGlcNAc(1→4) ) βGlcA(1→3) βGlcNAc-OMP

2000 βGlcA(1→3) ) βGlcNAc(1→4) βGlcA-OMe [18]

βGlcNAc(1→4) βGlcA(1→3) βGlcNAc-OMe

synthesis. A major limitation to its use is the additionalcosts associated with this starting material. In contrast, N-Phth and NH-TCA derivatives can be readily obtained frominexpensive D-glucosamine hydrochloride and theirtransformation into the final desired N-acetyl hexosaminecontaining product is usually straightforward. Cleavage ofthe N-Phth requires basic conditions and is directly followedby a N-acetylation step while transformation of NH-TCAinto N-acetyl is achieved through radical reduction underneutral conditions.

Substituent at the Reducing End

The nature of the substituent at the reducing end of theoligosaccharide is another important feature in GAGsynthesis. Since anomerization significantly complicatesproduct identification by NMR spectroscopy, few synthesesare reported in which a free hemiacetal is found at thereducing end of the target molecule. The C-1 at the reducingend is often protected with 4-methoxyphenyl ether (MP) tofacilitate selective removal for subsequent glycosylation andchain elongation. Since MP group is large, it does notaccurately mimic the native oligosaccharide, and smaller O-methyl group at C-1 represents better structural mimic and isalso useful marker for NMR studies.

II. HYALURONIC ACID SYNTHESIS

Hyaluronic acid (HA), first isolated from bovine vitreousbody of eye [4], is a linear, non-sulfated, extracellularpolymer of 2-acetamido-2-deoxy-D-glucose and D-glucuronicacid disaccharide repeating units [4)-β-D-GlcpA-(1→3)-β-D-GlcpNAc-(1→]n. “Fig. (3)” HA is a major component ofsoft connective tissues and is biosynthesized by animal cellsand certain bacterial strains [5], at the inner side of plasma

membranes by a membrane-bound HA-synthase and thenextruded to the cell surface [6]. The biological roles of HAare highly diversified and dependent on its chain length.High molecular weight HA demonstrates anti-angiogenicproperties [7] whereas HA oligosaccharides with 4-25 (3-10)disaccharide units stimulate endothelial cell proliferation andmigration and induce angiogenesis in the chickchorioallantoic membrane (CAM) assay [8]. The medicalapplications of HA are closely related to its high waterbinding capacity and viscoelasticity. HA is used as asurgical aid in the treatment of osteoarthritis, in cataractsurgery or as a template for nerve regeneration [9].

Fig. (3). Structure of hyaluronic acid.

Several HA oligosaccharides, having different lengthsand modified sequences, have been synthesized over the pastdecade (Table 3). Two different approaches toward HAsynthesis can be differentiated. The presence of neighboringuronic acid ester group decreases the yield of theglycosylation step in galacturonic acid containingoligosaccharides [10]. To avoid this complication in thesynthesis of glucuronic acid (GlcA) derivatives, synthesis ofHA analogs containing glucose (Glc) was carried out and theGlc residues were converted into GlcA after construction ofthe oligosaccharide backbone. The subsequent developmentof more powerful glycosyl donors has facilitated the directcoupling between D-GlcA and D-GlcN derivatives.

Using the first approach, Ogawa and coworkers [11]reported the synthesis of HA tetrasaccharide 1 having thesequence: GlcA-GlcN-GlcA-GlcN-OMP “Fig. (4)”. The


Fig. (4). Synthesis of a D-GlcA terminated HA tetrasaccharide.

Fig. (5). Synthesis of a D-GlcN terminated HA tetrasaccharide.

synthesis was achieved in a stepwise manner by addition ofsuitably designed monosaccharides. Starting from β-D-Glcpentaacetate and D-GlcN hydrochloride, intermediates 2, 3, 4and 5 were prepared in good yields. The use ofallyloxycarbonyl group at both C-4 of D-Glc and C-3 of D-GlcN allowed selective deprotection required for chainelongation using glycosylation reactions based on thetrichloroacetimidate donors. Good yield of 87% for the di-(1.3 eq of donor), 88% for the tri- (2.5 eq of donor), 87% for

the tetrasaccharide (5 eq of donor) were reported. Once thetetrasaccharide skeleton 7 was formed, oxidation of D-Glcresidues into D-GlcA was accomplished by Swern oxidationfollowed by sodium hypochlorite treatment (86%).Simultaneous deprotection of both ester and N -Phthfollowed by selective N-acetylation afforded the desiredcompound 1 (82%).

Ogawa and coworkers also reported the synthesis of thereverse sequence of the tetrasaccharide GlcN-GlcA-GlcN-


Fig. (6). Synthesis of larger HA tetrasaccharide.

GlcA-OMP 8 using a ‘2+2’ glycosylation strategy [12] “Fig.(5)”. Starting from known monosaccharides 9, 10 and 11,disaccharide donor 12 and acceptor 13 were prepared. The‘2+2’ glycosylation reaction afforded the expectedtetrasaccharide in 81% yield (2.5 eq of donor). O -Decacetylation and oxidation of the two primary hydroxylgroups (76%) was followed by final deprotection to affordtetrasaccharide 8.

Following of preliminary investigations [13,14],Vliegenthart and coworkers described the synthesis of HApenta- 14 and hexasaccharide 15 and their 6-O-sulfo analogs16 and 17 , having a N -acetyl-β-D-glucosamine at thereducing end [17] “Fig. (6)”. Their studies demonstrated thatoxidation of Glc into GlcA could be achieved on largermolecules in the last stage of the synthesis. Thepentasaccharide backbone was obtained by glycosylation oftrisaccharide acceptor 19 with disaccharide donor 18 (81%).Coupling of pentasaccharide acceptor 20 with the knowntrichloroacetimidate 5 afforded the hexasaccharide backbone(62%). Oxidation of the two partially deprotected derivatives21 and 22 was studied. In the case of these largeroligosaccharides, classical Swern oxidation followed byoxidation with sodium hypochlorite gave unsatisfactoryresults. A successful alternative involved oxidation withpyridinium dichromate/acetic anhydride in dichloromethaneand afforded the expected penta- and hexasaccharidederivatives in 70% and 58% yield, respectively. Startingfrom the common intermediates 21 and 22, 6-O-sulfonation

was performed to prepare the two O-sulfo group containingHA analogs 16 and 17.

In all these syntheses, the D-GlcA residue was obtainedby selective oxidation at C-6 of the corresponding D-glucose(D-Glc) residue after construction of the oligosaccharidebackbone. A more straightforward strategy was developed byBlatter and Jacquinet in 1996 for the synthesis of tetra- 23,hexa- 24 and octasaccharide 25 having a methyl β-D-GlcAresidue at the reducing end [16] “Fig. (7)”. In this case, theoligosaccharide backbone was obtained by direct couplingbetween D-GlcA and D-GlcN derivatives. Starting from1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronate, the D-GlcAacceptor 27 was obtained in good yield. The 2-deoxy-2-trichloroacetamido-β-D-glucopyranose imidate 26 waschosen as a donor for the coupling reaction affording thedesired β-disaccharide in 89% yield. Oxidative removal ofMP followed by activation afforded the pivotaltrichloroacetimidate donor 28, which could also be used as aprecursor to the disaccharide acceptor 29 . A ‘2+2’glycosylation reaction between 2 8 and 2 9 gavetetrasaccharide 30 in 87% yield. The chloroacetyl group atC-3 of the D-GlcN residue was then selectively deprotectedfor further chain extension at the non-reducing end. Thushexa- 31 and octasaccharide 32 were obtained by second andthird coupling with imidate 28 (1.5 and 1.7 eq respectively)both in 93% yield. Complete deprotection afforded the HAtetra-, hexa- and octasaccharides.


Fig. (7). Alternarive approach for the synthesis of HA oligosaccharides using D-GlcA donor.

Fig. (8). Synthesis of HA trisaccharide using NH-TROC protection.


Fig. (9). Enzymatic synthesis of HA with regeneration of sugar nucleotides.

A final chemical synthesis of HA was recently describedin 2000 by Petillo and coworkers [18] “Fig. (8)”. Two HAtrisaccharides, with GlcA-GlcN-GlcA-OMe 33 and GlcN-GlcA-GlcN-OMe 34 were synthesized by sequential additionof monomers 35 → 40 through a combination of phenylsulfoxide and trichloroacetimidate activated glycosylationreactions. This was the first reported use of N H -trichloroethoxycarbonyl participating group at C-2 in GAGoligosaccharide synthesis. Interestingly, NH-Troc wassensitive to Zemplén conditions and was unexpectedlytransformed to the corresponding methyl carbamate.Alternative methods for ester deprotection, usingguanidinium nitrate, and cleavage of the methyl carbamatewere developed to overcome this problem.

While many chemical syntheses of HA oligosaccharideshave been reported, only a few examples of enzymaticsynthesis have been described. There are two differentapproaches toward the synthesis of HA. In 1995, the in vitrosynthesis of HA was described using a HA synthase andUDP-GlcNAc and UDP-GlcA as substrates [19] “Fig. (9)”.Because of a low yield (20%) and of the high price of thesugar nucleotides, a system for the in situ regeneration ofUDP-GlcNAc and UDP-GlcA was developed. This approachallowed the preparation of multimilligram quantities ofsynthetic HA with an average molecular weight of 5.5 ×105.

In a second approach, Kobayashi and coworkers reportedthe first hyaluronidase-based synthesis of HA [20] “Fig.(10)”. The oxazoline 43 was chemically synthesized and

used as a substrate for the testicular hyaluronidase affordingtwo HA products with molecular weight of 1.74 ×104 and1.35×104.

III. CHONDROITIN SULFATE SYNTHESIS

Chondroitin sulfate (CS) GAGs occur in many tissues asside chains of CS-PGs. CSs are found in various bodyfluids, at the cell surface or in extracellular matrix orintracellularly in secretory granules. [21] These linearcopolymers consist of 2-acetamido-2-deoxy-D-galactose andD-glucuronic acid residues, [4)-β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→]n, and contain, on average, one O-sulfo groupper disaccharide unit “Fig. (1 1 )”. CSs aremicroheterogeneous polymers that display various sulfationpatterns. The major variants contain the 4 or 6-O-sulfogroups in D-GalN residues and are designated as CS-A andCS-C, respectively. Oversulfated CSs are characterized bythe presence of disulfated units such as CS-D (6,2’-di-O-sulfo), CS-E (4,6- di-O-sulfo) and CS-K (4,3’- di-O-sulfo).These subtle differences in sulfation pattern result insignificantly different biological properties. CS-A and C, themost abundant types, have been extensively studied for theirroles in cell-cell recognition [22], osteoarthritis [23],inhibition of complement factor Clq [24], and the activationof AT-III mediated anticoagulant activity [25]. Oversulfatedvariants have been less well studied but recently CS-D wasshown to play an important role in brain development [26]


Fig. (10). Postulated mecanism for hyaluronidase catalysed polymerisation of HA monomer.

Table 4. Structures of Different Synthetic Chondroitin Sulfate Oligosaccbarides


1989 βGlcA(1→3)βGalNAc4S-OMe [28]

1990 βGalNAc4S(1→4)βGlcA-OMe βGlcA(1→3) βGalNAc4S-OMe [29]

βGalNAc6S(1→4)βGlcA-OMe βGlcA(1→3) βGalNAc6S-OMe

1995 βGalNAc4S(1→4)βGlcA-OMP βGalNAc4S(1→4) βGlcA(1→3) βGalNAc4S(1→4)βGlcA-OMP [30]

1995 βGlcA(1→3)βGalNAc4S(1→4) βGlcA-OMe [31]

1998 βGalNAc(1→4)βGlcA-OMP βGlcA(1→3) βGalNAc(1→4) βGlcA-OMP [32]

βGalNAc4S(1→4)βGlcA-OMP βGlcA(1→3) βGalNAc4S(1→4) βGlcA-OMP

βGalNAc6S(1→4)βGlcA-OMP βGlcA(1→3) βGalNAc6S(1→4) βGlcA-OMP

βGalNAc4,6S(1→4)βGlcA-OMP βGlcA(1→3) βGalNAc4,6S(1→4) βGlcA-OMP

and to exhibit a neurite outgrowth promoting activity towardembryonic rat mesencephalic and hypocampal neurons [27].

Fig. (11). Structure of chondroitin sulfate.

The presence of O -sulfo esters on the CS GAGsignificantly complicates its synthesis. Sulfo groups are

sensitive to acid and base, limit the solubility of CSoligosaccharides in standard synthetic solvents and posedifficulties in separation and analysis. Thus, these groups aregenerally introduced at the last stage of the synthesis andrequire the efficient manipulation of orthogonal protectinggroups.

Besides the presence of the O-sulfo esters, CS differsfrom HA in that it contains D-GalN instead of D-GlcN in itsrepeating structure. Since D-GalN is a rare, somewhat moreexpensive sugar, two main ways have been developed for itspreparation, the azidonitration of D-galactal and theinversion of configuration at C-4 of D-GlcN derivatives. Thenature of the protection at C-2 position of D-GalN moetiesis closely related to the choice of the starting material. Thenon-participating C-2 azido group is favored when startingfrom D-galactal whereas the participating 2-trichloroacetamido is used when starting from D-GlcN.


(Table 4). contd.....


βGalNAc(1→4) βGlcA(1→3) βGalNAc(1→4) βGlcA-OMP

βGalNAc4S(1→4) βGlcA(1→3) βGalNAc4S(1→4)βGlcA-OMP

βGalNAc6S(1→4) ) βGlcA(1→3) βGalNAc6S(1→4)βGlcA-OMP

βGalNAc4,6S(1→4) ) βGlcA(1→3) βGalNAc4,6S(1→4)βGlcA-OMP

1998 βGlcA(1→3)βGalNAc6S-OH βGlcA(1→3)βGalNAc4S-OH [33]

1999 βGlcA(1→3)βGalNAc4S-OH βGlcA(1→3)βGalNAc6S-OH [34]

βGlcA(1→3)βGalNAc4,6S-OH βGlcA2S(1→3)βGalNAc-OH

βGlcA2S(1→3)βGalNAc4S-OH βGlcA2S(1→3)βGalNAc6S-OH

βGlcA(1→3)βGalNAc4,6S-OH

2000 βGlcA(1→3) βGalNAc4S(1→4) βGlcA(1→3)βGalNAc4S(1→4)βGlcA-OMe [35]

βGlcA(1→3) βGalNAc6S(1→4) βGlcA(1→3)βGalNAc6S(1→4)βGlcA-OMe

2000 βGlcA(1→3) βGalNAc4S(1→4) βGlcA(1→3)βGalNAc4S-OH [36]

βGlcA(1→3) βGalNAc6S(1→4) βGlcA(1→3)βGalNAc6S-OH

βGlcA(1→3) βGalNAc4S(1→4) βGlcA(1→3)βGalNAc4S(1→4) βGlcA(1→3)βGalNAc4S-OH

βGlcA(1→3) βGalNAc6S(1→4) βGlcA(1→3)βGalNAc6S(1→4) βGlcA(1→3)βGalNAc6S-OH

2000 βGlcA2S(1→3)βGalNAc6S-OH βGlcA2S(1→3)βGalNAc6S-OMe [37]

2002 βGlcA2S(1→3)βGalNAc6S(1→4) βGlcA2S(1→3)βGalNAc6S-OMe [38]

βGlcA2S(1→3)βGalNAc6S(1→4) βGlcA2S(1→3)βGalNAc6S(1→4)βGlcA2S(1→3)βGalNAc6S-OMe

Fig. (12). Synthesis of CS A trisaccharide.

The syntheses of a number of CS-A and CS-Coligosaccharides have been reported since 1989 (Table 4). In1995, Coutant and Jacquinet described the syntheses ofchondroitin trisaccharide GlcA-GalN(4S)-GlcA-OMe 44through the inversion of configuration at C-4 of the centralGlcNAc unit of the HA trisaccharide [31] “Fig. (12)”. TheHA trisaccharide derivative was obtained by stepwiseconstruction of the sugar skeleton, using D-GlcNthioglycoside 45 and D-GlcA trichloroacetimidate 47 asdonors for the synthesis. A good yield (90% for di- and 92%for trisaccharide coupling) of 1,2-trans-glycosides wasobtained in each cases. The crucial inversion of configurationat C-4 was achieved on trisaccharide 49 by treatment withtriflic anhydride followed by nucleophilic displacement ofthe intermediate triflate by tetrabutylammonium nitrite(87%). After reduction of the trichloroacetamido group, 4-O-sulfonation and saponification afforded the expectedtrisaccharide 44 in good yield. This same strategy was laterextended to the synthesis of CS 4S and 6S tetra- andhexasaccharides [36].

In 1998, Jacquinet and coworkers reported a multigramsynthesis of both 4-O-sulfo 50 and 6-O-sulfo 51 reducingdisaccharides having a GlcA-GalN motif [33] “Fig. (13)”. Inaddition to the preparation of large quantities of thesedisaccharides, regioselective O-sulfonation of the 4,6-diol atC-6 was demonstrated. Glycosylation of D-GalN acceptor 53with bromide donor 52 afforded the expected disaccharide(70%), which after hydrolysis of the benzylidene acetal gavethe common intermediate diol 54. Treatment of 54 withbenzoyl cyanide allowed selective protection of the primary


Fig. (13). Synthesis of the 4-O and 6-O-sulfo CS disaccharides.

Fig. (14). Synthesis of various CS trisaccharides from a common disaccharide precursor.

alcohol and the free hydroxyl group was then 4-O-sulfonated(90%). Alternatively, direct treatment of 54 with sulfurtrioxide- trimethylamine complex (3 eq) in DMF at 50°C for6 h afforded 6-O-sulfo derivative 56 in 90% yield. Less than2% of the 4,6-disulfated disaccharide was observed.Complete deprotection afforded target molecules 50 and 51.

Tamura and coworkers reported the syntheses of CS 4-O-sulfo di-, and tetrasacccharide as MP glycosides [30]. Later,systematic syntheses of CS di-, tri-, and tetrasacccharideshaving 4-O-sulfo, 6-O-sulfo mono, and 4,6 di-O-sulfogroups were carried out [32] and relied on the common keydisaccharide precursor 66 “Fig. (14)”. Disaccharide 66 was


Fig. (15). Synthesis of CS A and C pentasaccharides.

well designed for chain elongation and contains selectivelyremovable MP glycoside and 3-O-Lev protecting group atthe reducing and non-reducing end, respectively. Thisallowed its ready conversion into the correspondingdisaccharide donor 71 and acceptor 68. Access to the CS 4-O-sulfo disaccharide was accomplished by regioselectivereductive opening of the benzylidene acetal, followed by 4-O-sulfonation. Surprisingly, attempts to regioselectivelyform the 6-O-sulfo derivative from the 4,6-diol failed,giving a mixture (65% of 6-S, 11% of 4-S and 7% of 4,6-S)of mono- and di-O-sulfo products that were difficult toresolve. Instead, compound 67 was acetylated at the 4-position and the 6-hydroxyl group exposed byhydrogenolysis was sulfonated to afford the desired 6-O-sulfo disaccharide, after saponification. The 4,6-di-O-sulfoderivative was prepared from 67 after hydrogenolysis,sulfonation and deprotection. The trisaccharide skeleton wasobtained in only 39% yield by coupling acceptor 68 andmethyl glucuronate trichloroacetimidate 69. Strategies forthe synthesis of both 4-O-sulfo and 6-O-sulfo derivativeswere similar to those previously described. Interestingly

attempts to synthesize the disulfonated derivative throughdirect sulfonation of the 4,6-diol did not succeed and 6 daysreaction were necessary to afford the compound starting fromthe 4-O-sulfo trisaccharide. The tetrasaccharide skeleton wasprepared by a ‘2+2’ coupling of donor 71 and acceptor 68.Similar transformations afforded the 4-O-sulfo derivative andin this particular case, regioselective sulfonation of the 4,6-diol afforded the expected 6-O-sulfo derivative in 78% yield.

Recently, Bélot and Jacquinet reported the synthesis ofpentasaccharides of CS-A 72 and CS-C 73 with the GlcA-GalN-GlcA-GalN-GlcA-OMe sequence [35] “Fig. (15)”. Thepentasaccharide backbone was obtained by stepwise additionof suitably designed monomers 74, 75, 76 and knowndisaccharide 77. The glycosylation steps proceeded in goodyields: GlcA + GalN-GlcA, 84%; GalN + GlcA-GalN-GlcA,78% and GlcA + GalN-GlcA-GalN-GlcA, 55%.Alternatively, the tetrasaccharide backbone was prepared by‘2+2’ coupling in 50% yield. The pentasaccharide tetraol 81was then used as a common intermediate. Selective 6-O-sulfonation followed by saponification gave the 6-sulfonatedderivative. Furthermore, after selective 6-O-benzoylation, O-


Fig. (16). Combinatorial approach toward CS disaccharides.

sulfonation and deprotection afforded the 4-O-sulfonatedtarget.

While many syntheses of CS-A and C oligosaccharideshave been described, disulfonated variants of CS have drawnless attention. Tamura and coworkers, for example, describedthe synthesis of CS-E (4,6-di-O-sulfo) in 1998 [32].

A combinatorial approach for the synthesis of a CSdisaccharide library was proposed by Lubineau and Bonnaféin 1999 [34], and resulted in the preparation of eightdifferent sulfonated GlcA-GalN “Fig. (16)”. The disaccharideskeleton 92 was first prepared by glycosylation of D-GlcNacceptor 91 with D-glucose donor 90. Epimerization at theC-4 position of D-GlcN moiety was achieved using Swernoxidation followed by K-selectride reduction, affordingdisaccharide 92 in 88% yield. The common intermediate 92contains orthogonal protecting groups that wereindependently manipulated to prepare of the eight sulfonateddisaccharides in 13 steps. Interestingly, O-sulfo esters werefound to be efficient hydroxyl protecting groups in thiscombinatorial approach. Indeed these O-sulfo groups werestable to basic and low-temperature acidic conditions andwithstood Swern oxidation of D-Glc to D-GlcA.

CS-D (2’,6-di-O-sulfo) oligosaccharides have also beenrecently synthesized. The synthesis of GlcA-GalN-OMe and

GlcA-GalN-OH disaccharides, reported by Karst andJacquinet [37], were further elaborated to prepare thecorresponding tetra- and hexasaccharide methyl glycosides,94 and 95 [38] “Fig. (17)”. A common key disaccharideintermediate 98 was used iteratively to afford the tetra- andhexasaccharide skeletons. Preparation of the disaccharidebackbone was achieved by glycosylation of D-GalN acceptor97 with D-GlcA imidate 96 (71% yield). The commondisaccharide building block 98 was transformed to acceptor99 by dechloroacetylation and then glycosylated. Both ‘2+2’and ‘2+4’ coupling proceeded in a stereoselective mannergiving the corresponding tetra- and hexasaccharide β-derivatives 100 and 101 in 44% and 46% yield, respectively.In this reaction sequence the 2’-O-sulfonation reactions weresluggish, requiring large excess of reagent to go tocompletion.

Enzymatic reconstruction of CS oligosaccharides using atransglycosylation reaction catalyzed by testicularhyaluronidase was first reported in 1995 [39]. Testicularhyaluronidase is an endo-β-N-acetylhexosaminidase thatnormally hydrolyzes internal glycosidic linkages in both HAand CS. This enzyme can also catalyze transglycosylationand hydrolysis. Previous studies [40] have elucidated theglycosyl transfer mechanism showing that hyaluronidasetransferred one disaccharide, GlcA-GlcN from the non-


Fig. (17). Synthesis of CS D tetra- and hexasaccharides.

Fig. (18). Transglycosylation reaction by testicular hyaluronidase.

reducing terminal of HA oligosaccharide donor to anotherHA oligosaccharide acceptor “Fig. (18)”. Knowing that theenzyme hydrolyzed both HA and CS, the authors examinedthe feasibility of reconstructing various oligosaccharides ofGAGs using the transglycosylation reaction. The use of afluorogenic reagent, 2-amino-pyridine (PA) at the reducingend of the oligosaccharides allowed the analysis of thereaction products by ion spray mass spectroscopy and theoptimal conditions for the transglycosylation were found tobe pH 7.0 at 37°C for 1 hour. Chondroitin, chondroitin 4Sand 6S (but not chondroitin 4S), all showed good efficiency

in transglycosylation when used as donors or PA-acceptors.Moreover, the authors studied the possibility of usingdisulfonated chondroitin disaccharide units (2’,6S and 4,6S)as substrates for the chain reconstruction. They observed thatwhile these disaccharides could not be used as donors, it waspossible to transfer other sugar chains to oligosaccharidescontaining these sequences. As a result of their experiments,a library of natural and unnatural hybrids oligosaccharides,containing about fifty kinds of chimeric type CS hexa- oroctasaccharides, was obtained [41] (Table 5).


Table 5. Enzymatic Reconstruction of Chondroitin Sulfate-Example of Hybrid Oligosaccharides Contained in this Library[41].(A) Non Sulfated and Monosulfated Disaccharide Units; (B) Disulfated Disaccharide Units. (C) Iduronic AcidContaining Disaccharide Units

Oligosaccharides structure

(A) GlcA-GalN(4S)-GlcA-GalN-GlcA-GalN-GlcA-GalN-PA

GlcA-GalN(6S)-GlcA-GalN(6S) GlcA-GalN-GlcA-GalN- GlcA-GalN-PA

GlcA-GalN(6S)-GlcA-GalN(4S) GlcA-GalN-GlcA-GalN- GlcA-GalN-PA

GlcA-GalN-GlcA-GalN(4S) GlcA-GalN(4S)-GlcA-GalN(4S)- GlcA-GalN(4S)-PA

GlcA-GalN(6S)-GlcA-GalN-GlcA-GalN(4S)-GlcA-GalN(4S)- GlcA-GalN(4S)-PA

GlcA-GalN-GlcA-GalN(4S)-GlcA-GalN(6S)-GlcA-GalN(6S)-GlcA-GalN(6S)-PA

GlcA-GalN(4S)-GlcA-GalN(6S)-GlcA-GalN(6S)-GlcA-GalN(6S)- GlcA-GalN(6S)-PA

(B) GlcA-GalN-GlcA(2S) GalN(6S)-GlcA-GalN(4S) GlcA(2S) GalN(6S)- PA

GlcA-GalN(6S)- GlcA-GalN(6S)- GlcA(2S)-GalN(6S)- GlcA-GalN(4S)- GlcA(2S)-GalN(6S)-PA

GlcA-GalN(4S)-GlcA-GalN(4,6S)-GlcA-GalN(4,6S) GlcA-GalN(4,6S)- PA

GlcA-GalN(6S)- GlcA-GalN(6S)- GlcA-GalN(4,6S)-GlcA-GalN(4,6S) GlcA-GalN(6S)- PA

(C) IdoA-GalN-GlcA-GalN(4S)- GlcA-GalN(4S)- GlcA-GalN(4S)-PA

IdoA-GalN- GlcA-GalN(4,6S)- GlcA-GalN(4,6S)- GlcA-GalN(6S)-PA

IdoA-GalN- IdoA-GalN- GlcA(2S)- GalN(6S)- GlcA-GalN(4S)- GlcA(2S)- GalN(6S)-PA

Table 6. Structures of Different Synthetic Dermatan Sulfate Oligosaccharides


1989 αIdoA(1→4)βGalNAc4S-OMe [60]

1994 αIdoA2S(1→4)βGalNAc4S(1→4) αIdoA2S(1→4)βGalNAc4S-OiPr [61]

1994 αIdoA2S(1→4)βGalNAc4S(1→4) αIdoA2S(1→4)βGalNAc4S(1→4) αIdoA2S(1→4)βGalNAc4S-OH [62]

1997 αIdoA2S(1→4)βGalNAc4S(1→4) αIdoA2S(1→4)βGalNAc4S(1→4) αIdoA2S(1→4)βGalNAc4S-OMe [63]

1998 αIdoA2S(1→4)βGalNAc4S-OH [64]

2000 βGalNAc4S(1→4) αIdoA2S-OH [59]

βGalNAc4S(1→4) αIdoA2S-OMe

2002 αIdoA2S(1→4)βGalNAc(1→4)αIdoA2S-OMe [65]

αIdoA2S(1→4)βGalNAc4S(1→4)αIdoA2S-OMe

βGalNAc4S(1→4) αIdoA2S(1→4) βGalNAc4S-OMe

IV. DERMATAN SULFATE SYNTHESIS

Dermatan sulfate (DS) is found in a wide variety ofanimal tissues and was first isolated from pig skin. It is amicroheterogeneous linear copolymer composed primarily ofdisaccharide repeating unit of L-iduronic acid (L-IdoA) and2-acetamido-2-deoxy-D-galactose, namely [4)α-L-IdopA-(1→4)-β-D-GalpNAc-(1→] “Fig. (19)”. Depending on itsorigin, DS displays a variety of different sulfation patternsand a range of L-IdoA/D-GlcA ratios. The D-GalN residuesin DS are commonly sulfated at C-4, but 6-sulfo and 4,6-disulfo sequences have also been isolated [42-44]. The L-IdoA residues may also be sulfated at the C-2 position. Themost studied biological properties of DS are itsanticoagulant [45] and antithrombotic [46] activities, as itdisplays lower hemorrhagic effects than heparin [47]. Otherinvolvment of DS in biological processes have been studied

including its possible anti-oncogenic role [48], its regulationof hepatocyte growth factor activity [49] and its effect as amediator of FGF-2 responsiveness [50].

Fig. (19). Structure of dermatan sulfate.

As a member of the galactosaminoglycan family, DSshares with CS, the presence of D-GalN units in itsskeleton. The nature of the uronic acid distinguishes DSfrom CS. The presence of L-IdoA, the C-5 epimer of D-


Fig. (20). Glycosylation of 3,4-diol vs 3-OH D-GalN with L-IdoA donors.

Table 7. Utility of L-IdoA Glycosyl Donors

L-IdoA donor D-GalN acceptor αααα-linked disaccharide ββββ-linked disaccharide

pentenyl (108) (112) (85%) _ [66]

imidate (109) (112) 86% _ [66]

fluoride (110) 112) degradation _ [66]

thioglycoside (111) (112) degradation _ [66]

imidate(114) (115) 63% 27% [64]

imidate(114) (116) 52% 22% [64]

chloride(113) (115) _ 70% [64]

chloride(113) (116) _ 60% [64]

Fig. (21). L-IdoA as donors in glycosylation.

GlcA, complicates the syntheses of DS. Indeed, L-IdoA is arare sugar, which is not commercially available and theefficient preparation of L-Ido derivatives is usually the firstconcern in the syntheses of DS. A number of differentapproaches have been developed for its preparation during

the past decade. Many of these techniques give direct accessto L-IdoA derivatives, for example: radical reduction of 5-bromo uronate [51], functionalization of ∆4-uronic acidspecies [52], stereoselective addition on D-xylo-dialdose [53]or epimerization of D-GlcA derivatives [54,55]. A number ofL-Ido derivatives have been prepared throughdiastereoselective hydroboration of exo-glucals [56,57],however, for large scale synthesis, the older procedures forthe preparation of L-idose (L-Ido ) are still used. Thus, themost commonly applied method for DS and heparin (HP)syntheses remains the intramolecular nucleophilicsubstitution at C-5 starting from 3-O -benzyl -1 ,2 -isopropylidene-α -D-glucofuranose. This approach, firstdescribed by van Boeckel [58], was recently slightlymodified by Barroca and Jacquinet [59].

Several syntheses of DS oligosaccharides have beenreported over the past decade (Table 6). The first synthesis ofa IdoA-GalN(4S)-OMe disaccharide of DS was described in1989 by Marra and coworkers [60] “Fig. (20)”. In theirsynthesis D-GalN 3,4-diol 102 was examined as an acceptorbut failed to give the desired product, instead forming the1,2-orthoacetate 104. When L-IdoA derivatives 103 and 105were used as donors, the trichloroacetimidate 105 was foundto give better yield (68%) than the corresponding bromide103 (54%) for the glycosylation of acceptor 106.


Fig. (22). Synthesis of DS hexasaccharide utilizing L-Ido donors.

The investigation of the reactivity of various L-IdoAglycoside donors was later an object of study of Sinaÿ andcoworkers [66] “Fig. (21)”. Comparison was made betweenthioglycosides, trichloroacetimidate, n-pentenyl and fluoridedonors. Trichloroacetimidate and n-pentenyl were proven tobe efficient L-IdoA glycosyl donors, whereas thioglycosidesand fluoride failed to give satisfactory results for the

coupling step (Table 7). Similar data were reported byRochepeau-Jobron and Jacquinet in 1998 [64], in their studyfor the synthesis of a disaccharide unit of DS “Fig. (21)”.They observed that when the glycosylations were performedusing L-IdoA chloride donors, the β−linked disaccharideswere exclusively obtained, despite the presence of aparticipating group at C-2 position. In contrast, when L-IdoA


Fig. (23). Synthesis of DS hexasaccharide methyl glycoside.

trichloroacetimidate derivative was used, the expected α-linked disaccharide was formed together with β−linkeddisaccharide as a minor product (Table 7). Although L -IdoAderivatives can be used as glycosyl donors in the synthesisof DS oligosaccharides, they are considered poor acceptorswhen glycosylated with D-GalN donors. Therefore, in thesynthesis of longer oligosaccharides, L-Ido derivatives areoften used as acceptors and subsequently oxidized to thecorresponding L-IdoA residues.

This strategy is illustrated in the syntheses ofhexasaccharides containing IdoA(2S)-GalN(4S), described byGoto and Ogawa in 1994 [62] and Sinaÿ and coworkers in1997 [63]. The synthesis of hexasaccharide 117 carried outby Goto and coworkers utilized successive addition of acommon disaccharide 118 “Fig. (22)”. Disaccharide 118 wasobtained in 83% yield from the glycosylation of L-Idose 120

with tricholoroacetimidate 119. Successive '2+1' and '2+3'glycosylation reactions using trichloroacetimidate 118afforded 86% and 87% yields, respectively. Elaboration ofthe hexasaccharide backbone resulted from glycosylation ofacceptor 123 with imidate 124. Swern oxidation of the L-Idounits of hexasaccharide 125 followed by treatment withsodium hypochlorite and after esterification afforded theexpected uronate derivative in 39% overall yield. Aftersaponification, 2’ and 4-O-sulfonation were performed andthe O-sulfo derivative was deprotected to give the targethexasaccharide 117 in 50% (3 steps).

In 1997, Sinaÿ and coworkers reported a new syntheticroute toward the hexasaccharide methyl glycoside 126 [63]“Fig. (23)”. Their strategy relied on three disaccharidebuilding blocks 127, 128 and 129 that were successivelyglycosylated in ‘2+2’ and ‘2+4’ reactions. The reducing end


Fig. (24). Structure of proposed α-nitrilium intermediate directing β-glycosylation in the absence of a participating group at C-2.

Fig. (25). Synthesis of various sulfoforms of DS trisaccharide methyl glycosides.

127 and central 128 disaccharides both contained a L-Idoresidue to facilitate the glycosylation. These were obtainedfrom the common L-Ido thioglycoside donor 130 byglycosylation of suitably designed D-GalN acceptors 131and 132. The terminal building block 129 which contained aL-IdoA residue, was prepared by glycosylation of acceptor131 with L-IdoA n-pentenyl glycoside 133. Despite thepresence of the non-participating azido group at C-2position, ‘2+2’ coupling step of 127 and 128 gave the β-linked tetrasaccharide in 51% yield. No trace of the α−anomer was observed in the reaction. This surprisingstereoselectivity has been explained by the formation of anintermediate α-nitrilium ion in the acetonitrile solvent “Fig.(24)”. The same β selectivity was observed for the ‘2+4’

coupling, affording the expected hexasaccharide in 42%yield. Oxidation of the L-Ido units at tetra- andhexasaccharide stages used Swern oxidation and Dess Martinreagent, respectively.

In 2000, Barroca and Jacquinet reported the synthesis ofDS disaccharide GalN(4S)-IdoA(2S) and its methylglycoside [59]. Their study demonstrated that relativelyunreactive 4-OH L-IdoA acceptors can afford good yields of1,2-trans disaccharide when 2-deoxy-2-trichloroacetamido-D-GalN trichloroacetimidate derivatives are used as donors. Asan application of this new approach they investigated thesynthesis of larger oligosaccharides and in 2002 reportedvarious sulfoforms of DS trisaccharides [65] “Fig. (25)”. In


Fig. (26). Structure of a glycosaminoglycan hexasaccharide analog of DS.

Fig. (27). Heparin pentasaccharide containing residues “DEFGH” binding to ATIII and potent analog 153.

their syntheses, a pair of key disaccharides 139 and 140 wasprepared by glycosylation of D-GalN acceptors 142 and 143with L-Ido trichloroacetimidate 141. It is interesting to notethat the regioselective oxidation of disaccharides containingthe 4,6-Ido diol residue using TEMPO followed byesterification afforded the methyl esters in 63 and 66% yield.The L− IdoA acceptor 1 4 4 was glycosylated withdisaccharide donor 140 to afford the 1,2-trans trisaccharide145 in 61% yield. Since pivalate esters are more stable underbasic conditions than acetate, it was possible to differentiallysulfonate, affording IdoA(2S)-GalN(0S)-IdoA(2S) 136 andIdoA(2S)-GalN(4S)-IdoA(2S) 137. Intermediates 139 and140 were converted to their β-D-methyl glycosides 146, 147and used as glycosyl acceptors with the D-GalNtrichloroacetimidate 148 affording the expected trisaccharidesin 80 and 75% yield. While the pivalate ester showed greaterstability to saponification conditions, the correspondingsulfoform was not investigated. Ester groups werecompletely removed and the sulfonated trisasaccharideGalN(4S)-IdoA(2S)-GalN(4S) 138 was obtained. Finally, todemonstrate the efficiency of their D-GalN donor, a ‘2+2’coupling reaction was performed that afforded the expectedtetrasaccharide in 63% yield.

In 1994, the synthesis of a glycosaminoglycanhexasaccharide analog of DS was reported by van Boeckeland coworkers [67] “Fig. (26)”. Using a disaccharide

building block strategy and thioglycoside glycosylationstrategy, the hexasaccharide backbone was obtained andsulfonated to give 151, which bound heparin cofactor II withhigh affinity “Fig. (26)”.

Only a few enzymatic syntheses of DS oligosaccharideshave been reported. Takagaki and Ishida synthesized hybridscontaining L-IdoA- D-GalN disaccharides using testicularhyaluronidase [41] (Table 5). Although testicularhyaluronidase failed to use disaccharide units derived fromDS, L-IdoA- D-GalN4S, the desulfated disaccharide L-IdoA-D-GalN could be transferred to oligosaccharides with D-GlcA at their non-reducing end. The regeneration of sulfogroups was not investigated and remains a major problem ofthis type of synthesis.

V. HEPARIN AND HEPARAN SULFATESYNTHESIS

Heparin (HP) and heparan sulfate (HS) are linear,heterogeneously sulfated, anionic polysaccharides composedof alternating α -(1→4) linked L-IdoA or D-GlcA and D-GlcN units. These polysaccharides also contain minoramounts of unsubstituted D-GlcN and show a considerablesequence heterogeneity, giving rise to very complexstructures.


Fig. (28). Heparin tetra- and hexasaccharide methyl glycosides based on the regular repeat unit of HP.

HS is widely distributed in different kinds of animalcells and tissues, whereas HP is primarily found in thegranules of mast cells.

The biological activities of HS and HP PGs mainlyresult from the specific interaction of their GAG chains withheparin-binding proteins [68]. At this time more than 100HP binding proteins have been identified. The most famousexample is the interaction of HP with antithrombin III(ATIII), which is responsible of its anticoagulant activitymaking HP and its derivatives the most commonly usedclinical anticoagulants. HP and HS have also been studiedfor their crucial roles in regulation of many otherphysiological processes such as hemostasis, growth factorsactivity, cell-adhesion and enzyme regulation [20].

Its significant medical potential makes HP the moststudied GAG and numerous syntheses of HPoligosaccharides have been reported over the past 20 years.Until recently nearly all the syntheses have focused onpentasaccharide 152 [69], known to bind specifically to ATIII “Fig. (27)”. A review article by van Boeckel and Petitouin 1993 [70] gives a complete overview of the synthesis of

various HP ATIII-binding pentasaccharide analogs. Thesestudies have resulted in a better knowledge of HP structure-activity relationships and preparation of potent, simplifiedHP oligosaccharides analogs such as pentasaccharide 153[71], where the N-sulfo groups are replaced by O-sulfogroups and hydroxyl groups are replaced by O-methylgroups.

Synthesis of IdoA(2S)-GlcNS(4S) Oligosaccharides fromthe Regular Repeating Region of HP

In 1999, Sinaÿ and coworkers reported the synthesis ofHP tetra- 154 and hexasaccharide 155 methyl glycosides [72]“Fig. (28)”. Their strategy relied on the preparation of threedisaccharide building blocks designed as a seeding block156, an elongation block 157 and a capping block 158. Thelevulinoyl group, which can be selectively deprotected forchain elongation, was called the elongation group. The p-methoxybenzyl group was designed as a stopping group andwas also used to replace the levulinoyl protection in thetetrasaccharide synthesis. Trichloroacetimidate donors wereused in glycosylations, giving products in good yield (82%


Fig. (29). Versatile disaccharide building blocks for HP tetrasaccharide synthesis.

for 159 and 80% for 160). No trace of the β-anomer wasdetected making this elongation strategy quite valuable.Once the tetra- and hexasaccharide skeletons were formed,standard deprotection and O- and N-sulfonation affordedtarget compounds 154 and 155.

In 1999, Lay and coworkers reported the synthesis of HPdisaccharides having a D-GlcN unit at the non-reducing end[73]. Their strategy later extended to the preparation of HP-like tetrasaccharides having different sulfation pattern at theC-6 position [74] “Fig. (29)”. This synthesis was based onthe preparation of versatile orthogonally protecteddisaccharides 166→→→→ 169, which allowed the selectivesulfonation of the C-6 position of each D-GlcN residues.Disaccharide 164 was converted to trichloroacetimidatedonor 167 and to two disaccharide acceptors 166 and 168with selective benzyl and acetyl protection at the C-6position. Lipase P catalyzed selective C-6-transacetylationusing vinyl acetate, affording an 85% yield. Disaccharidedonor 169 was prepared from the known 165. Formation of

the tetrasaccharides proceeded in average to good yield (50%to 63%) and was followed by O -deacetylation, O -sulfonation, removal of the protecting groups and N -sulfonation to afford the three target tetrasaccharides.

Recently, Martin-Lomas and coworkers reported thesyntheses of hexa- 170 and octasaccharide 171 correspondingto the HP regular region. [75] “Fig. (30)”. Their approachwas similar to the one developed by Sinaÿ in 1999. Threedifferent disaccharide building blocks 172, 173 and 174 werebuilt and used in ‘n+2’ glycosylation reactions. Elongationblock 172 possessed a 4,6-benzylidene acetal, which couldbe hydrolyzed to allow selective 6-O-benzoylation andfurther elongation step at the 4 position of D-GlcN. For thepreparation of disaccharides 172 and 174, the 2,4-diol 176was used as acceptor in the glycosylation step. The goodregioselectivity observed in both cases, could be explainedby the steric hindrance at the 2-OH from the neighboringdimethylthexylsilyl group. All glycosylation reactions gavegood to moderate yields (172+173, 79%, 174+178, 58%,


Fig. (30). Synthesis of a hexa- and octasaccharide corresponding to the heparin regular region.

172+178, 52%, 172+hexa, 60%) and large amounts ofunreacted acceptor could be recovered. Classicaldeprotection, sulfonation and purification sequence resultedin target molecules 170 and 171.

Suda and coworkers reported the synthesis of varioussulfonated di- and trisaccharide analogs of HP regular regionfor studies of HP binding to platelets [76].

Selectively protected disaccharides having IdoA-GlcNand GlcN-IdoA sequences were first synthesized “Fig. (31)”.This group had previously reported [77] the synthesis of HPdisaccharide GlcNS6S-IdoA2S using thioglycoside D-GlcNas a donor and 2,4-diol L-IdoA as acceptor. Thestereocontrol was good but only a moderate yield (40%) ofthe expected α-(1,4) disaccharide was obtained along withthe α-(1,2) (13%) and α-(1,2) α-(1,4) trisaccharide (14%).The yield of GlcN+IdoA coupling was improved usingtrichloroacetimidate donor 184 (79%). Transesterification ofacetate protection, followed by O-sulfonation and afterdeprotection, selective N -sulfonation afforded targetcompound 179. Disaccharides with IdoA-GlcN sequencewere prepared by coupling of D-GlcN acceptors 188 and 189with L-Ido trichloroacetimidate 187. Transesterificationfollowed by formation of the 4,6-isopropylidene on the L-Ido unit allowed differentiation at the 6-position of D-GlcN.TEMPO oxidation of L-Ido residue of 4,6-diol disaccharides

into L-IdoA gave good yields. Disaccharide 190 was thenconverted in the usual manner into target disaccharide 180.Acceptors 190 and 191 were also glycosylated withtrichloroacetimidate donors 192 and 193 and after the usualtransformations, three trisaccharide analogs 181, 182 and183, having different sulfonation patterns, were obtained.

Based on their observation that the number of GlcNS6S-IdoA2S sequences within HP increased binding to platelets,Suda and coworkers focused their attention on thepreparation of cluster analogs containing more than onedisaccharide unit. They reported the synthesis of threeoligomers having two or three disaccharide units linkedthrough an amide linkage [78] “Fig. (3 2 )”. Twodisaccharides building blocks were first synthesized usingtrichloroacetimidate 202 and 204 as donors in theglycosylation step. The allyl group at the C-4 position of D-GlcN was oxidized to a carboxymethyl group to afford thekey disaccharide 200. Condensation of two and three unitsof 200 to a ethylenediamine linker, using pentafluorophenyldiphenylphosphinate (FDPP) gave the expected oligomers ingood yield (58% for n=1 and 63% for n=2). Targetmolecules 197 and 198 were prepared by deprotection andsulfonation. Removal of N-Fmoc group gave access todisaccharide 201 which was coupled with 200 using FDPPand after transformations afforded oligomer 199.


Fig. (31). Synthesis of trisaccharides from the regular region of HP.

Fig. (32). Synthesis of a disaccharide dimer for use as a HP tetrasaccharide analog.


Fig. (33). Synthesis of branched HP oligomer analogs using reductive amination.

Fig. (34). Synthesis of HP/HS disaccharide analogs involving selective TEMPO oxidation of D-Glc and L-Ido 4,6-diol.

The synthesis of larger branched oligomer analogs wasrecently reported [79] using a coupling strategy based onreductive amination “Fig. (33).” A common trisaccharidederivative 209 having a D-Glc residue at the reducing endwas synthesized and submitted to reductive amination withselected amine linkers to give target compounds 206, 207and 208.

Synthesis of HP/HS Analogs Containing D-GlcAResidues

Various modifications of HP sequence have been studiedto understand HP binding with fibroblast growth factor(FGF) [80]. Replacement of the L-IdoA residue present inHP regular sequence with D-GlcA has been reported by anumber of groups.


Fig. (35). Synthesis of GlcA containing HP/HS di- and trisaccharides.

In 1994, Flitsch and coworkers reported the synthesis ofvarious sulfated disaccharides having a Glc-IdoA and Glc-GlcA sequences where the N-sulfo groups were replaced byO-sulfo groups [81] “Fig. (34)”. Selective oxidation of D-Glc and L-Ido 4,6-diol into D-GlcA and L-IdoA usingTEMPO was reported here for the first time. A non-participating benzyl group was introduced at C-2 position ofD-Glc trichloroacetimidate to ensure the α -selectivity.Glycosylation of acceptors 214 , 215 and 216 withtrichloroacetimidates 217 and 218 proceeded in moderateyields (41-53%) affording the expected α - l i nkeddisaccharides 219→222. Hydrogenolysis, O-sulfonation andcomplete deprotection lead to target molecules 210→213.

The synthesis of HP/HS di- and trisaccharide 223→229was reported in 1995 by Nilsson and coworkers [82] “Fig.(35)”. Uronyl bromides 230 and 231 were used as donors inglycosylation steps with common acceptor 232 affording thecorresponding disaccharides in moderate to good yield (71%for 233 and 55% for 234). For the synthesis of the reversesequence D-GlcN- D-GlcA, D-GlcA acceptor 236 wascoupled with chloride 235. Despite the presence of the azidogroup at C-2 position of D-GlcN, the stereoselectivity waspoor, but α− and β-anomers 237 and 238 could be separated

after regioselective opening of benzylidene acetal and wererecovered in 55% and 25% yield, respectively. Disaccharideacceptor 237 was glycosylated with uronate 230 affordingthe expected trisaccharide in 50% yield. Target molecules227→229 were obtained after standard deprotection, N-acetylation or N-sulfonation.

Westman and Nilsson also reported the synthesis ofHP/HS di-, tri- and tetrasaccharide analogs in which D-GlcNresidues were replaced with D-Glc [83] “Fig. (36)”. Theirstrategy took advantage of the pre-existing α -glycosidiclinkage present in maltose, which was used as startingmaterial for their synthesis. Starting from the knownmaltose derivatives 242 and 243, the 6-position could bedifferentiated using tert-butyl dimethylsilyl protection andlater oxidized to give disaccharide intermediates 246 and247. Deprotection of 246 afforded the target disaccharide239. After regioselective ring opening of the benzylideneacetal in 246, disaccharide acceptor 248 was obtained andglycosylated with the known D-GlcA bromide 230 andthioglycoside disaccharide 2 4 7 to give tri- andtetrasaccharide in 46% and 74% yield, respectively.Hydrolysis of the tert-butyl ester followed by saponificationand hydrogenolysis afforded the expected compounds 240


Fig. (36). Synthesis of HP/HS di-, tri- and tetrasaccharide analogs from maltose.

and 241, which were tested with FGF in competitivebinding assays.

Studies of HP binding to bFGF (FGF-2) have shownthat pentasaccharide containing two N-sulfo and at least oneO-sulfo groups could correspond to the binding site [80,84].The nature of two of the three uronic sugars present in theHS binding sequence was examined by Sinaÿ and coworkers“Fig. (37)”. They synthesized the 4 possible bFGF-bindingpentasaccharides 2 5 1 → 254 containing differentcombinations of D-GlcA and L-IdoA [85,86]. Fivedisaccharides building blocks 256, 257 and 258→260 wereused. Two were designed for chain elongation with aselectively removable levulinoyl group at C-4 position ofthe uronic acid. The reducing end L-IdoA unit 255 containeda temporary allyl protection at C-2 for subsequent O -sulfonation. The ‘2+1’ and ‘2+3’ glycosylation reactionswere performed using trichloroacetimidate donors with goodyield and selectivity (256+255, 84%; 258+255 , 75%,259+262, 64%; 260+262, 64%; 260+261, 54%; 257+261,68%). Deallylation was followed by saponification, O-sulfonation, hydrogenolysis and selective N-sulfonationafforded the targets for bFGF binding studies.

Synthesis of HP/HS Mimetics as New AntithromboticsDrugs

Studies on synthetic analogs of the HP ATIII bindingpentasaccharide have lead to the elaboration of potent

molecules, such as the chemically more accessiblecompound 153, where hydroxyl groups are methylated andN -sulfo groups replaced by O -sulfo groups. With thestructure responsible for HP’s anticoagulant activity in hand,researchers focused on the preparation of synthetic agentsthat displayed both anticogulant and antithromboticactivities. HP’s antithrombotic activity results from theformation of a ternary complex between heparin, ATIII andthrombin and HP oligosaccharides having at least sixteensugar units are necessary to form this complex [87]. The HPregion that binds to thrombin is called thrombin bindingdomain and corresponds to the regular HP sequence IdoA2S-GlcNS6S. Since the interaction of HP with thrombin wasfound to be non-specific, other sulfated linearoligosaccharides might also be used as the thrombin bindingdomain [88].

Petitou and coworkers reported the synthesis of hexa,deca- to eicosasaccharide 263→269 IdoA-Glc HP analogs[89,90] “Fig. (38)”. Hydroxyl groups were alkylated and N-sulfo groups replaced with O-sulfo groups to simplify thesynthesis. The key disaccharide intermediate 270 wasobtained after glycosylation of acceptor 271 with L-Idothioglycoside 272 and subsequent transformations.Acetolysis of the methyl glycoside, followed by anomericdeacetylation and activation afforded the correspondingtrichloroacetimidate donor 273. In addition, acceptor 274was obtained after selective deprotection of the levulinoylgroup. Glycosylation reaction of 273 with 272 afforded the


Fig. (37). Synthesis of HP/HS pentasaccharides for bFGF binding studies.

expected α-linked tetrasaccharide (54%), which, followingcleavage of the levulinoyl group, gave acceptor 275, whichwas coupled with 273. The process was reiterated untileicosasaccharide was obtained. The yield observed in thisiterative process was good (60%) and no β-coupled productswere detected. Once the skeleton was formed,oligosaccharides were submitted to hydrogenolysis,saponification and sulfonation to afford the correspondingtargets. The same article described the synthesis of twohexasaccharides 276 and 277 containing GlcA residues fromthree disaccharides building blocks (278, 279 and 280).Interestingly, while IdoA-Glc donor always afforded goodyields and stereoselectivity, even for larger oligosaccharides,Petitou and coworkers observed that ‘2+4’ glycosylation

reaction involving GlcA-Glc donor afforded a mixture of α−and β-anomers (39% α and 8% β for 276 and 63% α and7% β for 277).

New approaches toward HP mimetics have involved thepreparation of oligosaccharides containing a modified ATIIIbinding pentasaccharide linked to a regular HP sequencethrough an oligosaccharide, or a non-carbohydrate spacer.Van Boeckel and coworkers have reported the syntheses ofvarious glycoconjugate mimics such as the pentasaccharide-oligonucleotide conjugate 281 [91] or conjugate 282 with aflexible polyethylene glycol spacer [92] “Fig. (39)”. In 1998,this group described the synthesis of glycoconjugate 283[93] where a fully methylated maltose oligosaccharide waschosen as a linker between modified pentasaccharide DEFGH


Fig. (38). Synthesis of HP antithrombotic oligosaccharides containing L-IdoA or D-GlcA.

and a polysulfated tetrasaccharide. The spacer was preparedfrom the known glucose and maltose derivatives 284 and285 . After glycosylation, the benzoyl protection wasreplaced by methyl groups and hydrolysis of the benzylideneacetal was followed by selective 6-O-benzoylation to affordtrisaccharide acceptor 286. Iteration of this sequence resultedin chain elongation giving nonasaccharide 287, which,following transformation, afforded donor 288. Glycosylationof known tetrasaccharide acceptor 2 8 9 withtrichloroacetimidate 288 afforded the expected 13-mer as amixture of anomers (α:β=85:15) in 63% yield. Removal ofthe levulinoyl group afforded the corresponding acceptor290, which was coupled with the maltotriose thioglycoside2 9 1 in 62% yield. Hydrogenolysis followed bysaponification and subsequent sulfonation afforded the targetglycoconjugate 283.

Petitou and coworkers reported the synthesis ofnonadesaccharide 292 [94]. The design of 292 involved

reasoning similar to that used in the design of 283. Amodified ATIII pentasaccharide DEFGH was linked througha neutral hexasaccharide to a modified, sulfated sugar “Fig.(4 0 )”. In this case, the structure of the terminalhexasaccharide was chosen to mimic the charge densitypresent on HP regular sequence to simplify the synthesis.Glc2S6S units were selected to replace IdoA2S andGlcNS6S residues. The galacto epoxide 293 was chosen asstarting material for the synthesis of the Glc containingdonor 294 and acceptor 295. Glycosylation of 295 with 294afforded a mixture of anomers (α:β 3:2) despite the presenceof non-participating benzyl group at C-2 of the donor.Subsequent transformations of α -disaccharide provideddisaccharide acceptor 296 and donor 297. Iterative couplingbetween 296 and 297 afforded hexasaccharide 298 .Replacement of 4-methoxyphenyl by acetyl followed byactivation of the anomeric position provided thetrichloroacetimidate donor 300 , corresponding to the


husain

husain


(Fig. 39). contd.....

Fig. (39). Structure of HP antithrombotic mimetics with various linkers.

terminal region. In addition, direct activation of 298 affordeddonor 299, which was used to glycosylate acceptor 301affording heptasaccharide 302. Acetyl protection was nextconverted to methyl groups and p-methoxybenzoyl ester wasselected to replace the silyl protection, since this ester favorsα-selectivity in the glycosylation. Condensation of donorheptasaccharide 302, corresponding to the linkage region,with tetrasaccharide 303 afforded the expected α-anomer in56% yield. Selective MP deprotection followed bycondensation of 304 with donor 300 provided thenonasaccharide skeleton in good yield (70%).Hydrogenolysis and saponification were followed bysulfonation to afford the target molecule 292.

As part of their research Petitou and coworkers alsoreported the synthesis of analogs 305→→→→307 [95] “Fig. (41)”.Since the preparation of the non-sulfated linker alwaysrequired a multi-step synthesis, a common sequencecontaining lower charge density was designed to mimic the

thrombin binding domain and the linkage region. Differentoligosaccharides having 16, 18 or 20 Glc6S units per chainwere chosen for this purpose. The synthetic strategy reliedon the preparation of a common dodecasaccharide donor 308that was used to be condensed with various oligosaccharideacceptors (309, 310) containing ATIII binding domainpentasacchar ide (DEFGH). Dodecasacchar idetrichloroacetimidate donor was obtained by successive ‘4+4’and ‘4+8’ glycosylations. Thioglycoside 311 was used asdonor and afforded good yield (80% and 71%) andstereoselectivity during the elongation. Only a small amountof β-anomer (3%) was isolated after the ‘4+8’ coupling.Acceptors 309 and 310 were prepared in good yield byglycosylation of known EFGH tetrasaccharide 303 with di-and tetrasaccharide trichloroacetimidates 313 and 314.Glycosylation of acceptors 309 and 310 with donor 308yielded the expected hexa-, octa- and eicosasaccharide in44%, 48% and 51%, respectively. A standard deprotectionsequence afforded target molecules 305→307.


Fig. (40). HP antithrombotic mimetic with Glc linker and Glc(2S)(6S) thrombin binding domain.

Three glycoconjugates 315, 316 and 317 were designedbased on similar criteria to those previously described [96]“Fig. (42)”. To simplify the synthesis, no oligosaccharidelinker was used and a disulfated oligosaccharide designed tomimic the charge density of HP’s regular region was chosenfor elongation at the non-reducing end of the ATIII bindingpentasaccharide (DEFGH). Two building blocks 324 and

326 were used for preparation of penta-, hepta- andnonasaccharide skeletons from a common disaccharide allylglycoside intermediate 321. Construction of the thrombinbinding domain was achieved by ‘2+2’ and ‘4+4’glycosylation reactions using trichloroacetimidate 320.Vinyl glycoside 319 was first tested as donor in the ‘2+2’glycosylation step but since only moderate yield (44%) was


Fig. (41). HP antithrombotic mimetic with Glc(6S) thrombin binding domain.

obtained it was converted to trichloroacetimidate to affordthe desired tetrasaccharide in 80% yield. Since attempts tocouple levulinoylated monosaccharide 325 with knowntetrasaccharide 303 only afforded poor yields (27%) of

desired product, trisaccharide 326 was prepared for theformation of heptasaccharide 327 in 64% yield. Differencesin reactivity of mono- and trisaccharide were ascribed to theproximity of the levulinoyl group to the anomeric center of


monosaccharide 325 in 303. Heptasaccharide acceptor 327was then glycosylated with trichloroacetimidate donor 324affording the pentadecasaccharide skeleton. Subsequentglycosylations of acceptor 328 with additional donors 320and 322 afforded the corresponding heptadeca- andnonadecasaccharide. Standard deprotection and sulfonationchemistry provided the desired compounds 315→317.

Alternative Approaches Towards the Synthesis ofHP/HS

The high structural diversity of HP/HS represents amajor challenge for all chemists undertaking their synthesis.Thus, the preparation of defined HP/HS oligosaccharidesrequires the synthesis of tailor made monomers. Boons andHaller developed a ‘modular approach for HS synthesis’ toprepare all combinations of monosaccharides building blocks[97] “Fig. (43)”. Their approach consisted of the preparationof the nineteen different disaccharides found in HP/HS.Oxidation of C-6 position of glucosides or idosides into D-GlcA or L-IdoA was planned after oligosaccharide formationand sulfonation to overcome problems related to the poorglycosyl-donating properties of L-IdoA and C-5epimerization of uronic acids. The synthesis of di- 329 andtrisaccharide 330 demonstrated the feasibility of this

approach. Glycosylation reaction of acceptor 331 with vinylglycoside 332 gave moderate yield (40%) of a 3:1 mixture ofα- and β-anomers. Trichloroacetimidate 333 and fluoride334 prepared from 332, afforded 78% of a 3:1 mixture of α-and β-anomers and exclusively α-derivative 335 in modestyield (50%), respectively. Saponification of 335 followed byconversion of azido into acetamido, O-sulfonation andhydrogenolysis gave 336. Oxidation of the C-6 positionusing TEMPO afforded the expected disaccharide in 62%yield. The pH was maintained below 10 during the reactionto prevent the loss of O-sulfo groups. Removal of TBDPSprotection required HF.pyridine, since tetrabutylammoniumfluoride in THF or HF in acetonitrile resulted in loss of theO-sulfo esters. After removal of the MPM protection, thedisaccharide acceptor was glycosylated with vinyl glycoside337 to afford the expected trisaccharide in 75% yield, whichwas deprotected using standard conditions. This strategy wasnext applied to the synthesis of a D-GlcA-D-GlcN(3S)disaccharide 339 [97] “Fig. (44)”. Disaccharide buildingblock 340 was obtained by glycosylation of acceptor 341with donors 342 and 343 (56% for thioglycoside, 72% fortrichloroacetimidate). The levulinoyl group was thenremoved, azido converted into acetamido, and O-sulfonationwas performed. Surprisingly, anomerization was observedduring the sulfonation step. After hydrogenolysis, acetatedeprotection under Zemplén conditions lead to partial


(Fig. 42). contd.....

Fig. (42). HP antithrombotic mimetic with Glc(2S)(6S) thrombin binding domain.

cleavage of the O-sulfo ester. An alternative enzymaticdeacetylation using Pseumomonas lipase type B afforded theexpected compound in 78% yield. Oxidation and TBDPSremoval were then performed in usual manner to afford 339.

The introduction of the α- glycosidic linkage between D-GlcN and uronic acid residues is most commonly controlledusing a non participating azido group at the C-2 position ofGlcN in HP oligosaccharides synthesis. However, selectivityvaries and β-anomer formation can be observed. Based on

the observation that IdoA acceptor gives only α- derivativeswhen coupled with GlcN, Seeberger and coworkers designeda new strategy [99]. The conformation of the GlcA acceptoris locked in a 1C4 conformation giving selective preparationof α - product “Fig. (45)”. The 1,2-acetal groups wereintroduced on GlcA and IdoA monomers 353 and 354 inmoderate to good yield and the different disaccharidesacceptors 344→347 were used in glycosylation reaction withvarious fluorides or trichloroacetimidates. All reactionproceeded with good yield and good selectivity, only α -


Fig. (43). Synthesis of unsulfated trisaccharide and 2-O-sulfonated D-GlcA disaccharide using modular approach.

disaccharides were obtained without any trace of of β-anomer(Table 8).

Fig. (44). Synthesis of 3-O-sulfonated D-GlcN disaccharideusing modular approach.

Table 8. Glycosylation Using Locked Uronic Acid Acceptors[99]

Acceptor Donor Yield (%)

344 348 80

344 349 86

344 350 83

344 351 92

344 352 77

345 348 79

345 349 80

345 350 82

346 349 91

346 351 90

347 351 88


Fig. (45). Locked uronic acids acceptors for 1,2-cis linkage formation.

Fig. (46). Chemoenzymatic approach toward heparin synthesis.

In 1996, Linhardt and coworkers proposed achemoenzymatic approach to HP oligosaccharides [100]“Fig. (46)”. Enzymatic depolymerization of HP usingheparin lyase afforded unsaturated sulfated disaccharide 355,

which could be used as starting material. Their strategyrelied on the use of sulfate esters as temporary protectinggroups during the synthesis. The first steps of the synthesisshowed that acylation and removal of anomeric acetyl of HP


disaccharide could be achieved without lost of sulfate ester.Successful functionalization of the ∆4-uronate into D-GlcAor L-IdoA has been reported by the same group [52] andcould be applied here for the preparation of various buildingblocks that could lead to HP oligosaccharides.

VI. FUTURE AND PROSPECTIVE IN GAGOLIGOSACCHARIDES SYNTHESIS

Many groups were involved in GAG oligosaccharidesynthesis over the last past decade and allowed considerableprogress in GAG synthesis and towards understandingbiological activities. Recent advances in carbohydratechemistry have made GAG oligosaccharides more accessiblewhile still posing a significant synthetic challenge. Novelapproaches toward the synthesis of larger and more complexGAG oligosaccharides are required. Solid phase synthesis ofGAG oligosaccharides is also currently under activeinvestigation [101], and a library of glucosamine donors hasrecently been synthesized and evaluated [102] by Seebergerand coworkers. Enzymatic and chemoenzymatic approachesare also an area of growing interest. Mutant enzymes of HAand CS synthase fom Pasteurella multocida studied by DeAngelis and coworkers [103,104] are able to transfer singlesugars (GlcN or GlcA or GalN) to short oligosaccharideacceptors and could be used for GAG chain elongation. Casuand coworkers have been able to generate GAGs with HP/HSlike–sequence using chemoenzymatic modifications of acapsular bacterial polysaccharide obtained from Escherichiacoli K5 [105]. Due to their structural complexity, synthesisof every different type of GAG oligosaccharide stillrepresents as a daunting task. However, the recentinvestigations reviewed above should allow faster progress,despite the great difficulties involved in GAG synthesis, andresult in an improved understanding of their biological roles.

ABBREVIATIONS

GAG = Glycosaminoglycan

PG = Proteoglycan

HS = Heparan sulfate

HP = Heparin

HA = Hyaluronic acid

CS = Chondroitin sulfate

DS = Dermatan sulfate

Glc = Glucose

GlcA = Glucuronic acid

Ido = Idose

IdoA = Iduronic acid

GlcN = Glucosamine

GalN = Galactosamine

Phth = Phtaloyl

Troc = Trichloroethoxycarbonyl

TCA = Trichloroacetyl

CAM = Chick chorioallantoic membrane

MP = 4-methoxyphenyl

OMe = O-methyl

UDP = Uridine diphospate

ATIII = Antithrombin III

Lev = Levulinoyl

PA = 2-amino-pyridine

FGF = Fibroblast growth factor

TEMPO = 2,2,6,6-tetramethyl-1-piperinidyloxy

Ac = Acetyl

AOC = Allyloxycarbonyl

MBz = p-methoxybenzoyl

Me = Methyl

Ph = Phenyl

Bz = Benzoyl

ClAc = Chloroacetyl

MPM = p-methoxybenzyl

Bn = Benzyl

UTP = Uridine triphosphate

NAD = Nicotinamide adenine dinucleotide

NADH = Nicotinamide adenine dinucleotide (reduced form)

PPi = Inorganic pyrophosphate

Pi = Inorganic orthophosphate

TBDMS = Tert-butyldimethylsilyl

Piv = Pivaloyl

All = Allyl

iPr = Isopropyl

DTS = Dimethylthexylsilyl (thexyl= 1,1,2-trimethylproplyl)

Z = Carbobenzyloxy

But = Tert-butyl

SE = Trimethylsilyl ethyl

TBDPS = Tert-butyldiphenylsilyl

DMF = N,N-dimethylformamide

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