Development of Semisynthetic, Regioselective Pathways for

Jun 24, 2019 - Department of Experimental Medicine, Section of Biotechnology,. University of Campania “Luigi Vanvitelli”,. via de Crecchio 7, I-80...
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Article Cite This: Biomacromolecules 2019, 20, 3021−3030

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Development of Semisynthetic, Regioselective Pathways for Accessing the Missing Sulfation Patterns of Chondroitin Sulfate Giulia Vessella,† Serena Traboni,† Donatella Cimini,‡ Alfonso Iadonisi,† Chiara Schiraldi,‡ and Emiliano Bedini*,† †

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Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126 Napoli, Italy ‡ Department of Experimental Medicine, Section of Biotechnology, University of Campania “Luigi Vanvitelli”, via de Crecchio 7, I-80138 Napoli, Italy S Supporting Information *

ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan playing several biological functions, which seem to be encoded through its sulfation pattern. This “sulfation code” is still to be deciphered. One of the barriers to this goal is the difficulty in achieving structurally well-defined CS polysaccharides since extraction from natural sources often leads to complex heterogeneous structures. Instead, an approach relying on chemical modification of a microbially sourced unsulfated chondroitin can allow access to semisynthetic CS polysaccharides with a well-defined sulfation pattern. We report herein some new, suitably developed chemical strategies affording CSs with unprecedented sulfation patterns, carrying a single sulfate group regioselectively placed at either C-2 or C-3 position of the glucuronic acid residues or at both sites. In this way, all the possible variants of CS sulfation patterns can be now accessed. This will allow more detailed and complete structure−activity relationship investigations of CS biological functions and applications.



INTRODUCTION

Decoration of chondroitin with sulfate groups seems to be a result of evolution in order to let CS play key roles in some processes typical of higher animals (e.g., central nervous system development).2 Indeed, simple and evolutionary more ancient organisms such as bacteria and nematodes possess only or mostly unsulfated chondroitin,3 whereas from arthropods up to mammals, chondroitin can be found exclusively in one or more of its sulfated variants. In particular, terrestrial animals possess almost exclusively CSs with sulfate groups located at the position C-4 and/or C-6 of GalNAc units, whereas marine species usually display additional sulfate groups on GlcA residues at C-24,5 or C-36−8 or, very rarely, at both C-2 and C3 positions.9 However, it is worth noting that the distribution of sulfate groups on CS depends not only on animal species but also on tissue source and, in higher animals, on physiopathological conditions such as aging, inflammation, and tumor formation. Moreover, sulfate group distribution along the CS backbone seems to encode specific functional information. Even if in the last years, researchers started to unveil the role played by such a “sulfation code” in some cases,10−15 it is still very far from being deciphered. One of the barriers is the difficulty in achieving pure and structurally welldefined CS oligo- and polysaccharides. Indeed, extraction from natural sources often leads to complex structures, showing two

Proteoglycans are complex glycoconjugates involved in a variety of biological events, such as extracellular matrix assembly, signaling pathway regulation, tissue growth and development, cell proliferation, adhesion, and motility.1 From a structural point of view, they are composed of a protein core and highly negatively charged polysaccharide chains, termed glycosaminoglycans (GAGs). One of the most important and widespread GAGs is chondroitin sulfate (CS), composed of glucuronic acid (GlcA) and 2-acetamido-2-deoxy-galactose (Nacetyl-galactosamine, GalNAc) residues linked together through alternating β-1 → 3 and β-1 → 4 glycosidic linkages. The resulting → 4)-β-GlcA-(1 → 3)-β-GalNAc-(1 → disaccharide repeating unit can be sulfated to various extents (Figure 1 and Table 1).

Received: April 29, 2019 Revised: June 13, 2019 Published: June 24, 2019

Figure 1. CS repeating unit (see Table 1 for sulfation patterns found in nature up to now). © 2019 American Chemical Society

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Biomacromolecules Table 1. Disaccharide Sulfation Patterns of Natural and Semisynthetic CSs Obtained up to Now nonsulfated monosulfated

disulfated

trisulfated

persulfated

CS

R4 GalNAc

R6 GalNAc

R2′ GlcA

R3′ GlcA

natural sourceda,b

semisynthetica,c,d

0 A C U Ve B D E K L R M S S T

H SO3− H H H SO3− H SO3− SO3− H H SO3− SO3− H SO3− SO3−

H H SO3− H H H SO3− SO3− H SO3− H SO3− H SO3− SO3− SO3−

H H H SO3− H SO3− SO3− H H H SO3− H SO3− SO3− SO3− SO3−

H H H H SO3− H H H SO3− SO3− SO3− SO3− SO3− SO3− H SO3−

yes yes yes

yes yes

yes yes yes yes yes

yes yes yes yes

yes yes yes yes

yes yes yes yes

a

Disaccharide sulfation patterns present exclusively or in combination with other ones along the polysaccharide backbone. bSee ref 34. cSynthetic oligosaccharides (degree of polymerization = DP ≤ 10) are not considered here. dSee refs 21 and 23. eLetter V to indicate this unprecedented sulfation pattern is proposed in this work for the first time.

limitations, in continuation with our research on regioselective derivatization of microbially sourced chondroitin,26,27,32,33 we have hypothesized that we might access the semisynthesis of CS polysaccharides with well-defined sulfation patterns displaying one or two sulfate groups placed exclusively on GlcA residues. The aim is to fill the gap of access to the missing sulfation patterns of natural and semisynthetic CSs obtained up to now (CS-R, CS-U, and CS-V in Table 1).

or even more differently sulfated variants of the disaccharide repeating unit distributed along the same CS polysaccharide chain. Nonetheless, an alternative approach to pure, welldefined species such as the chemical synthesis of CS oligosaccharides is a demanding task as well,16−19 especially because long chain species are required as bioactivity largely depends on chain length and spatial conformation. During this decade, two alternative approaches toward welldefined CS species emerged, which are (chemo)-enzymatic synthesis20−22 and semisynthesis. In particular, the latter relies upon the structural manipulation of natural polysaccharides through single regioselective reactions or multistep procedures to afford polysaccharides with a modified, and often a more well-defined, sulfation pattern.23 Such approach allowed the obtainment of a number of semisynthetic CS polysaccharides starting not only from already sulfated animal-sourced CSs21,24,25 but also from unsulfated chondroitin derived from microbial sources.26,27 Noteworthy, in the latter case, ethical and regulatory guidelines pushing to avoid animal-derived products are satisfied. In Table 1, the possible variants of the sulfation pattern in CS disaccharide repeating units are listed, together with their occurrence in natural and semisynthetic CS polysaccharides. Most of the semisynthetic efforts have been focused on regioselective sulfation of the position 4 and/or 6 of GalNAc residues.21 The reason is that CS polysaccharides with A, C, and E patterns, found in terrestrial animal species, have been extensively investigated for biomedical applications such as osteoarthritis treatment28 and regeneration of central nervous system functionality after a damage.10,29,30 Instead, only few semisynthetic studies targeted sulfation of GlcA residues although such rarer CS sulfation patterns could encode important regulatory information.31 For example, CS possessing K subunits with 3-O-sulfonated GlcA units displayed a neurite outgrowth activity comparable to CS-E species.8 Nonetheless, its scarce natural availability, limited to few marine species, together with its presence within polysaccharide chains showing also other sulfation variants, hampers detailed structure−activity relationship investigations and subsequent biomedical applications. To overcome these



EXPERIMENTAL SECTION

General Methods. Commercial grade reagents and solvents were used without further purification, except where differently indicated. The term “deionized water” refers to water purified by a Millipore Milli-Q gradient system. Dialyses were conducted on a Spectra/Por 3.5 kDa cut-off membranes at 4 °C. Freeze-drying was performed with a 5Pascal Lio 5P 4K freeze dryer. Centrifugations were performed with an Eppendorf centrifuge 5804R instrument at 4 °C (4600 g, 5 min). NMR spectra were recorded on a Bruker DRX-600 (1H: 600 MHz, 13C: 150 MHz) instrument equipped with a cryo-probe, in D2O (acetone as the internal standard, 1H: (CH3)2CO at δ 2.22 ppm; 13C: (CH3)2CO at δ 31.5 ppm) or DMSO-d6 (1H: CHD2SOCD3 at δ 2.49 ppm; 13C: CD3SOCD3 at δ 39.5 ppm). Standard Bruker software was used for all the experiments. Gradient-selected COSY and TOCSY experiments were performed using spectral widths of either 6000 Hz in both dimensions, using data sets of 2048 × 256 points. The TOCSY mixing time was set to 120 ms. HSQC-DEPT experiments were measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C domain, using data sets of 2048 × 512 points and typically 60 increments. As for HSQCTOCSY and HMBC, data sets of 2048 × 256 points were used, with 120 increments, and the mixing time for HSQC-TOCSY was set to 120 ms. A Viscotek instrument (Malvern) was used to determine molecular mass data. General Procedure for Silylation. Polysaccharide 135 (67.5 mg, 0.140 mmol repeating unit) was dissolved in DMF (2.5 mL), which was freshly dried over 4 Å molecular sieves and treated with tbutyldimethylsilyl chloride (TBDMSCl, 106 mg, 0,703 mmol), thexyldimethylsilyl chloride (TDMSCl, 125 mg, 0.702 mmol), or triisopropylsilyl chloride (TIPSCl, 135 mg, 0.701 mmol). Imidazole (143 mg, 2.104 mmol) was then added. The resulting yellowish solution was stirred at 50 °C (35 °C in the case of reaction with TDMSCl) overnight, then cooled to r.t., and treated with diisopropyl ether (12 mL). The obtained precipitate was collected by 3022

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mL). The obtained white precipitate was collected by centrifugation and dried under vacuum overnight. Crude derivative 5 (39.2 mg, 88.3% weight yield) was obtained as a white powder. Determination of Molecular Mass. The molecular weight analyses of the CS samples were performed by a high-performance size-exclusion chromatographic system (Viscotek, Malvern, Italy), equipped with an integrated gel permeation chromatography system (GPCmax VE 2001,Viscotek, Malvern, Italy) and a triple detector array module (TDA 305,Viscotek, Malvern, Italy) including a refractive index detector (RI), a four-bridge viscosimeter (VIS), and a laser detector (LS) made of a right-angle light scattering (RALS) detector and a low-angle light scattering (LALS) one. Two gelpermeation columns (TSK-GEL GMPWXL, 7.8 × 30.0 cm, Tosoh Bioscience, Italy), equipped with a guard column (TSK-GEL GMPWXL, 6.0 × 4.0 cm, Tosoh Bioscience, Italy), were set in series to perform the analyses. An OmniSEC software (Viscotek, Malvern, Italy) program was used for the acquisition and analysis of the data. The analytical method was recently extensively described.36 The refractive index increment (dn dc−1) used for the samples was referred to the literature value for CS: 0.147 mL g−1.37

centrifugation and then dried under vacuum overnight. Crude derivative 2-i (135 mg, 200% weight yield) was obtained as a yellowish amorphous solid [2-ii (160 mg, 237% weight yield) as a yellowish oil; 2-iii (52.5 mg, 77.8% weight yield) as a white amorphous solid]. General Procedure for Sulfation and Global Deprotection. Polysaccharide 135 (or 2-i−v, 4, and 5) (36.9 mg) was dissolved in DMF (1.0 mL), which was freshly dried over 4 Å molecular sieves and then treated with a 0.90 M solution of pyridine−sulfur trioxide complex in freshly dried DMF (1.7 mL). After overnight of stirring at 50 °C, a saturated NaCl solution in acetone (4 mL) was added at r.t. The obtained yellowish precipitate was collected by centrifugation and then suspended in deionized water (5.0 mL). The acid mixture (pH ≈ 2) was heated to 50 °C and stirred for 2 h to give a yellowish solution that was cooled to r.t. and then treated with a 4 M NaOH solution to adjust pH to 12. The solution was stirred at r.t. overnight, and then 1 M HCl was added until neutralization. Dialysis and subsequent freeze-drying yielded polysaccharide CS-vi (or CS-i− v,viii) (32.5 mg, 88% weight yield) as a white amorphous solid. To obtain CS-vii, an additional alkaline hydrolytic reaction was performed by treating a solution of the obtained solid (22.4 mg in 1.9 mL of H2O) first with 2:1 v/v aqueous 1 M LiOH−30% H2O2 (1.4 mL, pH ≈ 10) at r.t. overnight and then with 4 M NaOH solution to adjust pH to 12. The obtained solution was stirred at r.t. overnight, then neutralized with 1 M HCl, and finally, treated with 1 M NaCl (2.9 mL). After 1 h of stirring at r.t., the solution was dialyzed and subsequently freeze-dried to yield polysaccharide CS-vii (11.7 mg) as a white amorphous solid. Derivative 2-iv. Polysaccharide 135 (69.3 mg, 0.144 mmol repeating unit) was suspended in 3:1 v/v CH3CN−DMF (3.7 mL), which was freshly dried over 4 Å molecular sieves, treated with acetic anhydride (Ac2O, 29.9 μL, 0.317 mmol), and heated to 40 °C. To the yellowish suspension, a solution (232 μL, 86.4 μmol) of tetra-nbutylammonium acetate (TBAOAc, 56.1 mg) in freshly dried CH3CN (500 μL) was finally added. The mixture was stirred at 40 °C for 44 h, then cooled to r.t., and treated with diisopropyl ether (15 mL). The obtained precipitate was collected by centrifugation and then dried under vacuum overnight. Crude derivative 2-iv (57.5 mg, 83% weight yield) was obtained as a white amorphous solid. Derivative 2-v. A mixture of polysaccharide 135 (40.1 mg, 83.4 μmol repeating unit) and benzoic anhydride (Bz2O, 20.7 mg, 91.7 μmol) was suspended under an Ar atmosphere in 3:1 v/v CH3CN− DMF (1.3 mL), which was freshly dried over 4 Å molecular sieves. The mixture was then treated with N,N-diisopropylethylamine (DIPEA, 17.4 μL, 100 μmol). After 90 min of stirring at r.t., the mixture was heated to 45 °C and stirred for additional 24 h, then cooled to r.t., and treated with diisopropyl ether (8 mL). The obtained precipitate was collected by centrifugation and then dried under vacuum overnight. Crude derivative 2-v (39.5 mg, 99% weight yield) was obtained as a white amorphous solid. Derivative 4. Polysaccharide 326 (114 mg, 0.242 mmol repeating unit) was dissolved under an Ar atmosphere in DMF (5.0 mL), which was freshly dried over 4 Å molecular sieves. The solution was treated with Bz2O (1.64 g, 7.26 mmol) and then heated to 85 °C. After 26 h of stirring, it was cooled to r.t. and treated with pyridine (4.3 mL) and 4-(dimethylamino)pyridine (DMAP, 59.1 mg, 0.484 mmol). The yellowish solution was stirred at r.t. for 68 h and then treated with methanol (4.3 mL) and sodium acetate (NaOAc, 29.8 mg, 0.363 mmol). After 26 h of stirring, it was concentrated by rotoevaporation to approximately 10 mL in volume and then treated with diisopropyl ether (25 mL). The obtained white precipitate was collected by centrifugation, then dissolved in DMSO (4 mL), and precipitated again with 2:1 v/v acetone−diisopropyl ether (18 mL). The white solid was collected by centrifugation and dried under vacuum overnight to give crude derivative 4 (152 mg, 133% weight yield). Derivative 5. Polysaccharide 326 (44.4 mg, 94.7 μmol repeating unit) was dissolved under an Ar atmosphere in DMF (2.0 mL), which was freshly dried over 4 Å molecular sieves. The solution was treated with Bz2O (642 mg, 2.84 mmol) and then heated to 85 °C. After 26 h of stirring, it was cooled to r.t. and treated with diisopropyl ether (12



RESULTS AND DISCUSSION Access to semisynthetic CS-U and CS-V polysaccharides, bearing a single sulfate group per disaccharide repeating unit at the GlcA O-2 and O-3 sites, respectively, required the regioselective protection of either OH-2 or OH-3 of the GlcA 2,3-diol. The difference in reactivity between such hydroxyls is subtler than between those at positions C-4 and C-6 of GalNAc units. Indeed, the latter diol is composed of a primary and secondary, axially-oriented alcohol, with the former hydroxyl much more nucleophilic toward a regioselective sulfation24,38 or protection.39 On the contrary, the 2,3O-β-gluco-configured diol of CS presents two secondary hydroxyls, both displaying an equatorial orientation and also both flanked by equatorial glycosidic substituents. When we started our research, to the best of our knowledge, no regioselective reactions giving a clear distinction between O-2 and O-3 hydroxyls in chondroitin or CS had been reported yet. Therefore, we looked at cellulose chemistry, as its → 4)-β-Glc(1 → repeating unit resembles quite well the β-1 → 4-linked GlcA residue of chondroitin. Regioselective modification of cellulose has been investigated for decades.40 Apart the most obvious differentiation between O-6 and O-2,3 positions, a marked regioselective protection of O-2 (together with O-6) alcohol with respect to O-3 could be achieved by silylation with bulky TDMSCl under homogeneous conditions in LiCl/ dimethylacetamide and pyridine.41 We decided to investigate whether a regioselective silylation could be achieved on the chondroitin backbone too. To this aim, Escherichia coli O5:K4:H4 sourced chondroitin42 was converted into its methyl ester derivative and then protected at GalNAc 4,6diols with benzylidene rings through a known procedure to give polysaccharide 135 that was then submitted to a reaction with differently hindered trialkylsilyl chlorides (TBDMSCl, TDMSCl, and TIPSCl; Scheme 1). Silyl ether installation regioselectivity on the polysaccharide could not be easily evidenced by NMR since no significant shift is usually observed for the 1H or 13C atom at the silylated position with respect to the unreacted one. Therefore, polysaccharides 2-i−iii were recovered from the reaction mixture by precipitation and then directly subjected to sulfation of the free alcohols with SO3·pyridine complex in DMF, followed by global deprotection under acid and then alkaline hydrolytic conditions to give, after purification by dialysis, semisynthetic CS-i−iii. 3023

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in order to provide a standard for the unprecedented CS-R polysaccharide showing exclusively 2,3-disulfated GlcA residues. CS-vi 2D-NMR spectra were first studied. The anomeric CH region of the DEPT-HSQC spectrum displayed two signals at δH,C 5.04/102.4 and 4.65/104.7 ppm (Figure 2). The latter could be attributed to a nonsulfated GalNAc unit by means of a HSQC-TOCSY spectrum showing a correlation with the CH signal at δH,C 4.06/52.2 ppm, attributable to GalNAc CH-2, which in turn was connected to typical signals of a nonsulfated GalNAc unit (see the Supporting Information for full NMR signal assignments).46 The former anomeric signal (δH,C 5.04/102.4 ppm) could be correlated to a signal at δH,C 4.47/79.0 ppm, which in turn was connected to the one at δH,C 5.12/77.0 ppm. The high downfield shift of all these three signals with respect to the ones reported for nonsulfated GlcA units in CS46 was indicative of a 2,3-disulfate decoration as expected for CS-vi (CS-R). Finally, minor signals clearly attributable to the GalNAc6S unit (δH,C 4.28/67.8 and 4.07/ 73.5 ppm for CH2-6 and CH-5, respectively) could be found in the DEPT-HSQC spectrum. Their presence was hypothetically related to a slow cleavage of a minor amount of the benzylidene rings of 1 catalyzed by the Lewis acidity of SO3 during the sulfation reaction. The ratio between GalNAc and GalNAc6S units in CS-vi was estimated to be 91:9 by HSQCDEPT 2D-NMR integration of the CH2-6 signals relative to the two kind of residues, assuming that they displayed similar 1 JCH coupling constants and that a difference of around 5−8 Hz from the experimental set value did not cause a substantial variation of the integrated peak volumes.47,48 With the complete assignment of 1H and 13C NMR signals of the CS-R standard in hand, the study of CS-i−v NMR spectra could be then pursued. They furnished more complicated DEPT-HSQC spectra; in fact, more than two signals were displayed in the anomeric region. In particular, CS-i showed anomeric signals at δH/C 5.04/102.4, 4.82/102.9, and 4.70/105.1 ppm, that, by the help of HSQC-TOCSY, COSY, and TOCSY correlations (see Figure 3 for DEPTHSQC and HSQC-TOCSY superimposition), could be assigned to GlcA2,3S, GlcA2S, and GlcA3S, respectively. The relative integration of the well-resolved signals assigned to CH3 of these three residues in the DEPT-HSQC spectrum indicated a slight preponderance of GlcA3S units over the other two monosaccharides along the CS-i polymer chain (Table 2). Under the highly realistic hypothesis that the sulfation reaction is not able to discriminate between the hydroxyls at positions 2 and 3 of GlcA units, this could be correlated with a slight preference of 2-O- versus 3-O-protection in the TBDMS installation reaction, as GlcA2S, GlcA3S, and Glc2,3S residues should derive from 3-O-, 2-O-, and not protected units in synthetic intermediate 2-i, respectively. The 2-O/3-O-regioselectivity could be similarly estimated also for TDMS protection of 1 to 2-ii, as the resulting polysaccharide CS-ii showed a DEPT-HSQC NMR spectrum very similar to the CS-i one (see the Supporting Information). An evident hindrance effect was observed in the case of the bulkiest TIPS group. Indeed, the DEPT-HSQC spectrum of CS-iii polysaccharide was very similar to the CS-R standard one (see the Supporting Information), displaying almost exclusively densities related to 2,3-disulfated GlcA units that should derive from nonprotected residues in 2-iii. A DSTIPS equal or slightly higher than zero could be therefore estimated

Scheme 1. Multistep Transformation of Microbial-Sourced Chondroitin into CS Polysaccharide through GlcA 2,3-Diol Regioselective Protection by Silylation or Acylation

A differentiation of O-2 and O-3 reactivity on GlcA residues was pursued not only through silyl ether protection but also by acylation reactions, conducted under conditions very recently shown to give a good regioselectivity on diols and polyols of monosaccharides and glycoconjugates, including the selective acylation of the alcohol at the position 3 of β-configured Glc glycosides.43,44 In particular, a hydrogen-bond driven regioselective acetylation or benzoylation was attempted on polysaccharide 1, by treating it with Ac2O and TBAOAc43 or Bz2O and DIPEA,44 respectively (Scheme 1). After purification of the intermediate polysaccharides 2-iv and 2-v by precipitation from the crude reaction mixture, sulfation and global deprotection gave CS-iv and -v. These polysaccharides were purified by dialysis and then subjected, as well as CS-i− iii, to deep structural characterization. To elucidate the sulfation pattern distribution, a standard, combined enzymatic-HPLC analysis 45 was not possible due to the degradation of CS disaccharide units containing a GlcA3S residue by chondroitinase ABC after its enzymatic release from the reducing end of the cleaved polysaccharide chain.6 Therefore, an extensive 2D-NMR spectroscopy investigation was performed. To help the structural study, a further semisynthetic CS (CS-vi, Scheme 1) was obtained by direct sulfation of polysaccharide 1, followed by a global deprotection 3024

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Figure 2. 1H, DEPT-HSQC (black and gray, respectively), and HSQC-TOCSY (red) NMR spectra (600 MHz, D2O, 298 K, zoom) of CS-vi ( CS-R) (densities enclosed in the highlighted areas of HSQC-DEPT spectrum were integrated for GalNAc/GalNAc6S ratio estimation; only some of the HSQC-TOCSY correlations are highlighted with dotted lines).

Figure 3. 1H, DEPT-HSQC (black and gray, respectively), and HSQC-TOCSY (red) NMR spectra (600 MHz, D2O, 298 K, zoom) of CS-i (densities enclosed in the highlighted areas of the HSQC-DEPT spectrum were integrated for GlcA2S/GlcA3S/GlcA2,3S and GalNAc/GalNAc6S ratio estimation; only some of the HSQC-TOCSY correlations are highlighted with dotted lines)

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Biomacromolecules Table 2. Yield and Structural Features of Semisynthetic CS-i−vi sample CS-i CS-ii CS-iii CS-iv CS-v CS-vi

GlcA protecting group

% Weight yield (from 1)

% GlcA2Sa

% GlcA3Sb

% GlcA2,3Sc

% GlcAd

GalNAc/GalNAc6S ratioe

2-O/3-O protection regioselectivity for 2-i−vf

TBDMS TDMS TIPS Ac Bz

69% 35% 44% 82% 66% 88%

26% 24% n.d.g 25% 17% n.d.g

43% 32% n.d.g 34% 6% n.d.g

31% 44% 100% 28% 77% 100%

n.d.g n.d.g n.d.g 13% n.d.g n.d.g

78:22 87:13 88:12 89:11 91:9 91:9

1.7 1.3 1.0 1.2 0.4

a

Estimated as the percentage ratio between the integral of GlcA2S CH-3 peak volume and the sum of GlcA2S, GlcA3S, GlcA2,3S, and GlcA CH-3 peak volumes in the DEPT-HSQC spectrum. bEstimated as the percentage ratio between the integral of GlcA3S CH-3 peak volume and the sum of GlcA2S, GlcA3S, GlcA2,3S, and GlcA CH-3 peak volumes in the DEPT-HSQC spectrum. cEstimated as the percentage ratio between the integral of GlcA2,3S CH-3 peak volume and the sum of GlcA2S, GlcA3S, GlcA2,3S, and GlcA CH-3 peak volumes in the DEPT-HSQC spectrum. d Estimated as the percentage ratio between the integral of GlcA CH-3 peak volume and the sum of GlcA2S, GlcA3S, GlcA2,3S, and GlcA CH-3 peak volumes in the DEPT-HSQC spectrum. eEstimated as the ratio between the integral of GalNAc and GalNAc6S CH2-6 peak volumes in the DEPT-HSQC spectrum. fEstimated as [%GlcA3S + %GlcA)]/[%GlcA2S + %GlcA] taking into account that GlcA2S, GlcA3S, and GlcA residues derive from a 3-O-, 2-O-, and 2,3-di-O-protected unit, respectively. gNot detected.

means of COSY and TOCSY, as expected by the 2-Obenzoylation. By employing the same 3,6-lactonization reaction, but without the following benzoylation and lactone cleavages, it was possible to obtain derivative 5, possessing a single hydroxyl at the position 2 of GlcA residues. Also in this case, the 1H and DEPT-HSQC NMR spectra displayed a homogeneous polysaccharide with well-resolved signals (see Figure 4b and the Supporting Information for full NMR signal assignments) that could be assigned to the corresponding atoms through COSY and TOCSY spectra. Furthermore, in the HMBC spectrum, a correlation between GlcA CH-3 signal at δH/C 4.76/73.1 ppm and a CO signal at δC 170.5 ppm (see the Supporting Information) clearly demonstrated the presence of the 3,6-lactone ring on GlcA residues. The two polysaccharide derivatives 4 and 5, having a single hydroxyl per disaccharide repeating unit at positions 2 and 3 of the GlcA residue, respectively, were subjected to sulfation under standard conditions with SO3·pyridine complex in DMF and then to a global deprotection under acidic and then alkaline hydrolytic conditions (Scheme 2). Concerning the last step, an overnight treatment with an aqueous NaOH solution (pH = 12) allowed a complete de-O-acylation in the case of 5 to CS-viii conversion, whereas Bz esters at O-2 proved to be rather recalcitrant to hydrolysis as after the same alkaline treatment a large amount of 2-O-acylated GlcA units still remained unaffected (data not shown). A longer reaction time gave no increase in the extent of de-O-acylation, whereas performing the reaction at a higher temperature (40 °C instead of r.t.) caused extensive polysaccharide depolymerization reasonably due to α,β-eliminative cleavage of 1 → 4 glycosidic bonds, with an almost total loss of the sample after dialysis purification. Finally, employment of an aqueous solution of lithium hydroperoxide (obtained in situ from LiOH and H2O2) followed by a treatment with aqueous NaOH at r.t. overnight,52 allowed complete debenzoylation without a significant depolymerization and gave semisynthetic CS-vii after purification by dialysis. Structural characterization of CSvii and CS-viii by 2D-NMR analysis and comparison with data obtained for CS-i−vi confirmed the presence of a single sulfate group per disaccharide repeating unit at the GlcA O-2 position in CS-viii and O-3 in CS-vii (see Figure 5 and the Supporting Information for full NMR signal assignments). These sulfation patterns, to the best of our knowledge, unprecedented in both natural and semisynthetic CS polysaccharides, are indicated as CS-U and CS-V, respectively. The presence of minor signals

for the semisynthetic intermediate 2-iii. Actually, the CS-iii 1 H-NMR spectrum clearly showed the presence of a small signal at δ 1.09 ppm (see the Supporting Information), attributable to the six methyl groups of the residual TIPS group. Its presence is reasonably due to the much higher stability reported for such silyl ethers with respect to TBDMS one under both acidic and alkaline hydrolytic conditions.49 A relative integration of this peak with the one at δ 2.05 ppm, assigned to the methyl group of GalNAc N-acetyl, returned a DSTIPS = 0.06 for CS-iii (similarly, a residual DSTDMS = 0.02 could be assigned for CS-ii by a careful analysis of its 1H-NMR spectrum, see the Supporting Information). As for CS-iv and CS-v polysaccharides obtained from partially acylated derivatives 2-iv and 2-v, respectively (Scheme 1), a low regioselectivity of protection could be inferred also in this case. In particular, together with GlcA2S, GlcA3S, and GlcA2,3S as for CS-i−iii and -v, also the presence of unsulfated GlcA residues along the polymer could be detected for CS-iv (Table 2). This is in agreement with the possibility to have also 2,3-diprotected (di-O-acetylated) GlcA residues in 2iv due to a minor hindrance of Ac esters with respect to silyl ethers and Bz esters. During the development of this work, a paper appeared in the literature reporting that the regioselective installation of a Bz ester at the position O-2 of the two or three GlcA residues in partially protected tetra- or hexasaccharide chondroitin derivatives, respectively, through a 3,6-lactonization/2-Obenzoylation/lactone opening one-pot sequence.50 Since protection of one of the two hydroxyls of GlcA 2,3-diol at the polysaccharide level by the methods described above occurred in all cases with an unsatisfying regioselectivity, we decided to test whether the 3,6-lactonization procedure could be extended from the tetra- and hexasaccharide cases to the polysaccharide structure. Therefore, chondroitin derivative 326 was treated with benzoic anhydride in DMF at 85 °C51 to form a 3,6-lactone on GlcA residues, followed by one-pot addition first of DMAP and pyridine to allow 2-O-benzoylation and then of sodium acetate and methanol to perform lactone opening and obtain derivative 4 with a free hydroxyl restored at the position 3 (Scheme 2). 1 H and DEPT-HSQC NMR spectra clearly showed a homogeneous structure for derivative 4 (see Figure 4a and the Supporting Information for full NMR signal assignments), with well-resolved signals and a single downfield-shifted carbinol signal, that could be assigned to CH-2 of GlcA residues by 3026

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Scheme 2. Multistep Transformation of Microbial-Sourced Chondroitin into CS Polysaccharides through GlcA Regioselective Protection by 3,6-Lactonization

chondroitin (38 kDa), even if the polydispersity was found to be not increased. The decrease in Mw was hypothesized to be due mainly to acid-mediated reactions (benzylidene ring installation26 and cleavage and, in case of CS-R, also methyl esterification35) and only in a minor amount to the final alkaline hydrolytic step too. To test this hypothesis, derivative 3 was directly converted back to starting chondroitin, without any further derivatization, by benzylidene ring cleavage under the same acid hydrolysis conditions employed at the end of the entire semisynthetic strategies (pH ≈ 2, 50 °C, 2 h). A molecular weight value very similar to CS-vi−viii was found (Mw = 10.1 ± 0.2 kDa, Mw/Mn = 1.33 ± 0.04), thus confirming our hypothesis. Anyway, it is worth noting that, in addition to the demonstrated chain shortening, spatial conformation changes related to the presence of sulfate groups in CS-vi−

related to unsulfated GlcA units, presumably due to a nonexhaustive sulfation, was also detected and estimated to count for 15% of total GlcA residues in CS-viii, as the percentage ratio between the integral of GlcA CH-2 peak volume and the sum of GlcA2S and GlcA CH-2 peak volumes in the DEPT-HSQC spectrum. The polymeric nature of CS-vi−viii, namely, the CS polysaccharides with unprecedented sulfation patterns R, U, and V, was clear from the fact that these semisynthetic products were all retained inside a 3500 Da cut-off dialysis bag. Evaluation of their weight-averaged molecular mass (Mw) and polydispersity was made by high-performance size-exclusion chromatography combined with a triple detector array (HPSEC-TDA)36,53,54 (Table 3). The three semisynthetic CS polysaccharides each showed a Mw value that was rather lower (7−9 kDa) with respect to starting microbially sourced 3027

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Figure 4. 1H and DEPT-HSQC NMR spectra (600 MHz, DMSO-d6, 298 K, zoom) of (a) 4 and (b) 5.

Figure 5. 1H and DEPT-HSQC NMR spectra (600 MHz, D2O, 298 K, zoom) of (a) CS-vii (CS-V) and (b) CS-viii (CS-U) (densities enclosed in the highlighted areas of the HSQC-DEPT spectrum were integrated for estimation of GlcA and GlcA2S percentage amounts).

The semisynthetic strategies here developed for regioselective sulfation of a single GlcA position were based both on GlcA 3,6-O-lactonization as key regioselective step to give a chondroitin derivative showing a single hydroxyl group at position 2 of GlcA units or, after two one-pot additional steps, at the position 3 of the same residues. Interestingly, these derivatives could be used in the near future also as polysaccharide glycosyl acceptors in glycosylation reactions with suitably protected L-fucosyl donors in order to access semisynthetic fucosylated chondroitin sulfate (fCS),55 polysaccharides currently extracted from sea cucumbers showing very promising bioactivities, with a much higher regiocontrol of the fucosyl branching with respect to the first semisynthetic fCS polysaccharides recently reported by us.35,56 Work is in progress to this aim and will be published as soon as possible elsewhere. Furthermore, the key lactonization reaction here developed on chondroitin will be investigated in the near future on other polysaccharides carrying uronic acid units (e.g., hyaluronic acid, alginate, and pectin) for exploring applications and limits of its employment as a new tool for tailored structural modifications of such important biomacromolecules.

Table 3. Molecular Mass Data of Semisynthetic CS-R, CS-U, and CS-V sample E. coli chondroitin CS-vi (CS-R) CS-vii (CS-V) CS-viii (CS-U)

Mw (kDa)a 38.0 9.7 7.3 8.2

± ± ± ±

1.0 0.1 0.2 0.9

Mw/Mna,b 1.34 1.31 1.31 1.34

± ± ± ±

0.04 0.02 0.12 0.05

a

Mw = weight-averaged molecular mass. bMn = number-averaged molecular mass.

viii could also have a role in the molecular weight measurement performed by HP-SEC-TDA.



CONCLUSIONS In this work, we have demonstrated the possibility to develop semisynthetic strategies for the chemical transformation of E. coli O5:K4:H4 sourced chondroitin into CS polysaccharides with unprecedented sulfation patterns carrying a single sulfate group regioselectively placed at either O-2 or O-3 position of the GlcA residues or at both sites. This has allowed us to complete the panel of CS sulfation patterns, by adding the unprecedented CS-R, CS-U, and CS-V ones. Now, all the possible variants can be accessed, through extractive and/or semisynthetic methods, thus allowing more detailed and complete structure−activity relationship investigations of CS biological functions and biomedical applications.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00590. 3028

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Copies of 1D- and 2D-NMR spectra and complete chemical shift data assignments for all the semisynthesized CS polysaccharides (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +39(0)81674153. Fax: +39(0)81674393. ORCID

Emiliano Bedini: 0000-0003-4923-3756 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR, FFABR 2017, “Fondo per il finanziamento delle attività base di ricerca”). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Elisabetta Cassese for the technical assistance with HP-SEC-TDA experiments. Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) is acknowledged for funding (FFABR 2017, “Fondo per il finanziamento delle attività base di ricerca”).



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