Carbohydrate Research 386 (2014) 33–40

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Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Preparation of synthetic oligosaccharide-conjugatesof poly-b-(1?6)-N-acetyl glucosamine

0008-6215/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carres.2013.12.022

⇑ Corresponding author. Tel.: +36 52 512 900; fax: +36 52 512 744.E-mail address: fekete.aniko@science.unideb.hu (A. Fekete).

Anikó Fekete a,⇑, Dániel Eszenyi c, Mihály Herczeg a, Vince Pozsgay b, Anikó Borbás c

a Department of Organic Chemistry, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungaryb National Institute of Child Health and Human Development, National Institutes of Health, 31 Center Dr., MSC 2423 Bethesda, MD 20892-2423, United Statesc Department of Pharmaceutical Chemistry, Medical and Health Center, University of Debrecen, PO Box 70, H-4010 Debrecen, Hungary

a r t i c l e i n f o

Article history:Received 25 October 2013Received in revised form 23 December 2013Accepted 27 December 2013Available online 8 January 2014

Keywords:Carbohydrate chemical synthesisPoly-b-(1?6)-N-acetyl glucosamineReductive aminationGlycoconjugate

a b s t r a c t

Staphylococcus aureus and Staphylococcus epidermidis are prominent bacterial pathogens of nosocomialinfections. Both microorganisms colonize medical devices by forming adherent biofilms.Poly-b-D-(1?6)-N-acetyl-glucosamine (PNAG) is a surface polysaccharide antigen which was found onboth S. aureus and S. epidermidis. Animal studies have proved that PNAG can elicit antibodies which pro-tect against staphylococcal infections. We have presented the synthesis of di-, tetra- and hexasaccharidefragments of PNAG with formyl-heptyl aglycone and their attachment to bovine serum albumin (BSA) byreductive amination.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

In the last few decades Staphylococcus aureus and Staphylococcusepidermidis have become the most dominant microorganisms thatcause nosocomial infections in association with indwelling medicaldevices like intravascular catheters, pacemakers, prosthetic heartvalves, peritoneal dialysis catheters and prosthetic joints, resultingin life-threatening illnesses. Both pathogens colonize biomaterialsin adherent biofilms composed of multilayered cell clustersembedded in a slime matrix, which make the organisms moreresistant to antibiotics.1–3 The emergence of antibiotic resistanceamong staphylococcal isolates has made the prevention of staphy-lococcal infections by immunization more important.4 A majorcomponent of the S. aureus5 and S. epidermidis6 biofilm matrix isa poly-b-D-(1?6)-N-acetyl-glucosamine (PNAG) surface polysac-charide antigen. Some animal studies have established that puri-fied PNAG can elicit protective immunity against both species,suggesting that PNAG is a vaccine candidate for many clinicallyimportant strains of staphylococci.5,7

The use of vaccines based on carbohydrates against bacterialinfections has become widespread by now. Heidelberger and Averyreported in their early work that a ‘soluble specific substance’isolated from pneumococci was immunogenic in animals.8 Thissubstance consisted of mostly polysaccharides, namely capsularpolysaccharides of pneumococci possessing serotype-specificity.Francis and Tillett demonstrated that purified pneumococcal

capsular polysaccharides elicited anti-polysaccharide antibodiesin humans.9 First, McLeod et al. used these polysaccharides as vac-cines.10 However, the effective application of antibiotics againstbacterial pathogens postponed the development of polysaccharidevaccines. Increasing occurrence of antibiotic resistance has re-sulted in renewed interest for prevention by vaccination in the late1960s. Then several polysaccharide vaccines have been developedand licensed. Despite their success in adults, this type of vaccineshas limitations. They are poorly immunogenic in infants underthe age of two years old and elderly because polysaccharides areT cell independent antigens. In 1931 Avery and Goebel describedthat covalent attachment of capsular polysaccharides to proteinsincreased their immunogenicity.11 Since glycoproteins are T celldependent antigens therefore glycoconjugate vaccines are effectivein both young children and elderly. The recognition, that lowmolecular weight fragments of capsular polysaccharides termedhaptens in the form of protein conjugates can produce polysaccha-ride-specific antibodies, resulted in the development of oligosac-charide-conjugate vaccines.12 Oligosaccharides obtained bychemical synthesis are homogeneous and their attachment to pro-tein affords well-defined glycoconjugates, as well as their use ashaptens makes possible the investigation of the influence of chainlength and oligosaccharide loading of the protein into glycoconju-gate on the immune response.13

Pier and his co-workers have conjugated purified PNAG andN-deacetylated derivative of PNGA termed dPNAG (degree ofN-acetylation �15%) to the carrier protein diphtheria toxoid (DT)and used them to immunize animals. They have found both conju-gates were very immunogenic in mice and rabbits. Antibodies

34 A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40

raised to the conjugates in rabbits mediated the opsonic killing ofvarious staphylococcal strains, but the specificity of the opsonickilling was primarily to dPNGA. Passive immunization of mice withanti-dPGNA-DT rabbit sera showed significant levels of clearanceof S. aureus from blood, whereas PNGA-specific antibodies wereineffective at clearing S. aureus.14

We planned to realize the synthesis of di-, tetra-, hexa- andoctasaccharide fragments of PNAG bearing a formyl heptyl agly-cone and their attachment to bovine serum albumin (BSA) byreductive amination to investigate the immunological propertiesof synthetic oligosaccharide-conjugates of PNAG. Meanwhile,Nifantiev and Pier, as well as their co-workers have synthesizedpenta- and nonasaccharide fragments of both poly-b-D-(1?6)-glu-cosamine and poly-b-D-(1?6)-N-acetyl-glucosamine. These oligo-saccharides were conjugated to tetanus toxoid and used toimmunize animals. They have found the N-acetylated oligosaccha-ride-conjugates elicited high titres of nonopsonic antibodies inmice, whereas the non-N-acetylated oligosaccharide-conjugateselicited highly active opsonic antibodies in mice and rabbits. Inaddition, they have realized that the antibodies arising from latterspecies showed excellent passive protective efficacy against S. aur-eus skin infections and Escherichia coli peritonitis.15 Investigation ofthe oligosaccharide-BSA conjugates of PNAG that we synthesized isexpected to provide further information for the development ofvaccine against staphylococcal infections.

2. Results and discussion

Several syntheses of b-D-1,6-linked N-acetyl-glucosamine oligo-saccharides have been published to date.16–21 Earlier we have de-scribed the synthesis of b-(1-6)-linked N-acetyl-glucosamineoligosaccharides series with phenylthio aglycone (from disaccha-ride to pentasaccharide) and their application to investigate thesubstrate specificity of Dispersin B enzyme. For the synthesis ofoligomers, 1+2, 2+2, 1+4 block syntheses were applied, using phe-nyl 1-thioglycosides as glycosyl acceptors and bromo-sugars asglycosyl donors.22

A similar approach was used to prepare hexasaccharide 4 andoctasaccharide 7. The formation of 1,2-trans interglycosidic bondhas been ensured by 2-phtalimido protecting group; during thesynthesis chloroacetyl group was used to protect temporarily the6-hydroxy group and acetate esters were applied as permanentprotecting groups. After bromination of disaccharide 122 with bro-mine at room temperature the obtained bromo sugar 2 was cou-pled without purification with tetrasaccharide acceptor 322 usingAgOTf promotion to give the hexasaccharide 4 in 49% yield.(Scheme 1).

Tetrasaccharide 522 was also converted into glycosyl bromidewhose coupling with tetrasaccharide acceptor 3 afforded octasac-charide 7 in 39% yield. (Scheme 2).

Subsequently, the synthesized phenyl 1-thioglycosides 1, 5, 4and 7 were reacted with 7-(1,3-dioxane-2-yl)-heptane-1-ol (8)23

to yield the corresponding spacer-containing oligosaccharides 9,10, 11 and 12 (Scheme 3).

Based on previous experiments, deblocking of the fully pro-tected oligosaccharides 9–11 was performed by simultaneousdephthaloylation and deacetylation with ethylenediamine in etha-nol, then the obtained amines were acetylated with acetic anhy-dride and pyridine to give 13, 14 and 15 in 84%, 66% and 46%overall yields, respectively. We performed peracetylation after theremoval of the phthaloyl groups since dephthaloylation resultedin a rather complex reaction mixture and isolation of the fully acet-ylated derivatives was easier by silica gel column chromatographythan the selectively N-acetylated derivatives. Unfortunately, in the

case of octasaccharide 12 the removal of the phthaloyl groups wasunsuccessful. Finally, de-O-acetylation of 13, 14 and 15 by Zempléntransesterification gave spacer-containing b(1-6)-linked GlcNAcoligosaccharides 16, 17 and 18 (Scheme 3).

Before the conjugation step, the acetal protecting group of theaglycone was removed with acid hydrolysis, then aldehydes 19,20 and 21 were linked to the lysine e-amino groups of BSA to formthe corresponding Schiff bases, which were subsequently reducedwith sodium cyanoborohydride to give the stable synthetic oligo-saccharide conjugates 22a, 22b, 23a, 23b and 24a (Scheme 4).Molecular mass determination of the synthesized glycoproteinswas achieved by MALDI-TOF mass spectrometry.

To reach different densities of oligosaccharides on the proteintwo different molar ratios of oligosaccharide to BSA were appliedin the case of disaccharide 19 and tetrasaccharide 20. Hence, fiveoligosaccharide conjugates were synthesized (22a, 22b, 23a, 23band 24) by means of the conjugation methods, possessing differentnumber of sugar residues as shown in Table 1.

In conclusion, we have synthesized di-, tetra- and hexasaccha-ride fragments of PNAG with formyl-heptyl aglycone (16, 17 and18) and they have been covalently attached to BSA by reductiveamination. Five oligosaccharide conjugates have been synthesized(22a, 22b, 23a, 23b and 24) which makes possible the investigationof the influence of oligosaccharide chain length and oligosaccha-ride density on the protein for the immune response.

3. Experimental

Optical rotations were measured at room temperature with aPerkin–Elmer 241 automatic polarimeter in CHCl3. TLC was per-formed on Kieselgel 60 F254 (Merck) with detection by charringwith 50% aqueous sulphuric acid. Column chromatography wasperformed on Silica gel 60 (Merck 63–200 mesh). The 1H(360 MHz and 400 MHz) and 13C NMR (90.54 MHz and 128 MHz)spectra were recorded with Bruker AM-360 and Bruker DRX-400spectrometers. Internal references: TMS (0.000 ppm for 1H), CDCl3

(77.00 ppm for 13C for organic solutions). The 1H and 13C NMRassignments have been established from 1D NMR spectra and theproton-signal assignments were supported by analysis of two-dimensional 1H–1H correlation spectra (COSY), as well as the car-bon-signal assignments by two-dimensional 13C–1H correlationmaps (HETCOR). MALDI-TOF MS analyses of oligosaccharides werecarried out in the positive reflectron mode using a BIFLEX III massspectrometer with saturated 2,4,6-trihydroxy-acetofenon in aceto-nitrile as matrix. MALDI-TOF MS analyses of the glycoproteinswere carried out in positive linear mode using a BIFLEX III massspectrometer (Bruker) with delayed-ion extraction. External cali-bration was applied using bovine serum albumin (6–8 mg/mL in0.1% aq trifluoroacetic acid, TFA). TA solution was prepared by dis-solving 0.1% TFA in a mixture of 2:1 acetonitrile–water. 10 lL sam-ple solution (6–8 mg/mL in 0.1% aq TFA), 25 lL matrix solution(saturated 3,5-dimethoxy-4-hydroxycinnamic acid in TA) and15 lL TA solution were mixed and 0.5 lL was applied to the targetplate and allowed to dry at room temperature before analysis.

3.1. Typical procedure A for glycosylation reaction I

To a solution of starting material (0.29 mmol) in dry CH2Cl2

(10 mL) bromine (19 lL, 0.35 mmol) was added at room tempera-ture. After stirring for 2 h the reaction mixture was concentrated.Dry toluene was added to and evaporated from the residue. To asolution of crude bromo sugar (0.29 mmol) and acceptor(0.24 mmol) in dry CH2Cl2 (10 mL) were added collidine (46 lL,0.35 mmol) and 4 Å molecular sieves. After stirring for 30 min at

OO

AcOAcO

NPhthO

OAcOAcO

NPhthO

OAcOAcO

NPhthO

OAcOAcO

NPhth

SPh

7

5iO

ClAcOAcOAcO

NPhthO

OAcOAcO

NPhthO

OAcOAcO

NPhthO

OAcOAcO

NPhthBr

6

ii3

ClAc

2

OClAcOAcOAcO

NPhthO

OAcOAcO

NPhth OO

AcOAcO

NPhthO

OAcOAcO

NPhth

SPh

Scheme 2. Reagents and conditions: (i) Br2, CH2Cl2, rt, 0.5 h, (ii) AgOTf; collidine, CH2Cl2, �74 �C ? rt, 24 h, 39% for two steps.

1OHO

AcOAcO

NPhthO

OAcOAcO

NPhth

OClAcOAcOAcO

NPhthO

OAcOAcO

NPhth

SPh

i

2

OClAcOAcOAcO

NPhthO

OAcOAcO

NPhthBr

ii

OO

AcOAcO

NPhthO

OAcOAcO

NPhth

SPh

OAcOAcO

NPhthO

OAcOAcO

NPhth OO

AcOAcO

NPhthO

OAcOAcO

NPhth

SPh

3

4

OClAcOAcOAcO

NPhthO

OAcOAcO

NPhth

O

Scheme 1. Reagents and conditions: (i) Br2, CH2Cl2, rt, 0.5 h, (ii) AgOTf; collidine, CH2Cl2, �74 �C ? rt, 24 h, 49% for two steps.

A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40 35

room temperature the mixture was cooled to �74 �C and silver tri-flate (121 mg, 0.43 mmol) dissolved in toluene was added. Thenthe reaction mixture was left to attain room temperature overnight. The mixture was diluted with CH2Cl2, and filtered throughCelite. The filtrate was washed with 10% aq Na2S2O3 and water,dried and concentrated. The crude product was purified by silicacolumn chromatography.

3.2. Typical procedure B for glycosylation reaction II

To a solution of starting material (0.21 mmol) and 7-(1,3-dioxan-2-yl)-heptanol 1023 (65 mg, 0.32 mmol) in dry CH2Cl2

(5 ml) were added 4 Å molecular sieves. After stirring for 30 minat room temperature the mixture was cooled to �20 �Cand the solution of NIS (58 mg, 0.25 mmol) and TfOH (7 lL,

O

NPhth

OAcOAcO

n

SPh

ClAc

O

HO(CH2)7HC

O

i

O

NPhth

OAcOAcO

n

ClAc OO(CH2)7HC

O

1 n=25 n=44 n=6

8

O

NHAc

OHOHO

n

H O

O(CH2)7HC

O

16 n=217 n=418 n=6

O

NHAc

OAcOAcO

n

Ac OO(CH2)7HC

O

7 n=8

9 n=210 n=411 n=612 n=8

13 n=214 n=415 n=6

ii

iii

Scheme 3. Reagents and conditions: (i) NIS/TfOH; CH2Cl2, THF, 0 �C ? rt, 24 h, 62%, 58%, 56% and 35% (9, 10, 11 and 12); (ii) (a) ethylenediamine, EtOH, 70 �C, 24 h; (b) Ac2O,Py, rt, 24 h, 84%, 66% and 46% for two steps (13, 14 and 15); (iii) NaOMe, MeOH, 93%, 84% and 88% (16, 17 and 18).

O

NHAc

OHOHO

n

H OO(CH2)7HC

O16 n=217 n=418 n=6

O

NHAc

OHOHO

n

H OO(CH2)7C

19 n=220 n=421 n=6

H

22a, 22b n=223a, 23b n=424a n=6

O

NHAc

OHOHO

O(CH2)8 NH BSA

m

H

n

iii

Scheme 4. Reagents and conditions: (i) 80% AcOH, 50 �C, 24 h, (ii) (a) BSA, phosphate buffer (0.2 M, pH = 7), rt, 2 h; (b) NaCNBH3; rt, 48 h.

Table 1Synthetic oligosaccharide-conjugates of PNGA

Oligosaccharide Hapten excess/NH2 group on BSA Glycoconjugate Mass by MALDI (Da) Hapten loading on BSA (mol/mol)

19 5 equiv 22a 81,410 2719 10 equiv 22b 98,893 5920 5 equiv 23a 75,973 1020 10 equiv 23b 94,030 2821 5 equiv 24 75,445 6

36 A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40

0.08 mmol) in dry THF (500 lL) was added dropwise. Then thereaction mixture was left to attain room temperature, stirred fortwo days and neutralized with pyridine (50 lL). The mixture wasdiluted with CH2Cl2, extracted with aqueous 10% Na2S2O3 andwater, then dried and concentrated. The crude product was puri-fied by silica column chromatography.

3.3. Typical procedure C for deacylation and peracetylation ofthe obtained amine

To a solution of starting material (0.08 mmol) in ethanol(15 mL) was added ethylene diamine (5 equiv per phthaloyl groupof starting material). After stirring for one day at refluxtemperature the mixture was concentrated. The residue was dis-solved in pyridine (10 mL) and acetic anhydride (5 mL) was addedto. After stirring for one day at room temperature the mixturewas concentrated and then co-evaporated with toluene. The res-idue was diluted with CH2Cl2, washed with water, dried and con-centrated. The crude product was purified by silica columnchromatography.

3.4. Typical procedure D for de-O-acetylation

To a solution of starting material (0.06 mmol) in methanol(15 mL) catalytic amount of NaOMe (pH �8) was added. After stir-ring for one day at room temperature the mixture was diluted withwater, neutralized with Amberlite IR-120 H+ ion-exchange resin,filtered and concentrated. The residue was liophylized.

3.5. Typical procedure E for conjugation of oligosaccharides toBSA

A solution of starting material (0.06 mmol) in 80% aq acetic acid(4 mL) was stirred at 50 �C for one day then the mixture was con-centrated. The residue was dissolved in phosphate buffer (2 mL,0.2 M, pH = 7.0) and 13 mg (0.2 lmol) bovine serum albumin wasadded to the solution and the mixture was stirred for one hour.Then 7.5 mg (0.12 mmol) sodium cyanoborohydride was added.After stirring for two days at room temperature the resulting gly-coprotein was dialysed (membrane cut off 3500 Da) against waterand lyophilized.

A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40 37

3.6. Typical procedure F for conjugation of oligosaccharides toBSA

A solution of starting material (0.1 mmol) in 80% aq acetic acid(4 mL) was stirred at 50 �C for one day then the mixture was con-centrated. The residue was dissolved in phosphate buffer (2 mL,0.2 M, pH = 7.0) and 10.9 mg (0.17 lmol) bovine serum albuminwas added to the solution and the mixture was stirred for 1 h. Then6.3 mg (0.1 mmol) sodium cyanoborohydride was added. Afterstirring for two days at room temperature the resulting glycopro-tein was dialysed (membrane cut off 3500 Da) against water andlyophilized.

3.7. Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-phtha-limido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside (4)

Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-phthalimi-do-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalim-ido-1-thio-b-D-glucopyranoside (1)22 (268 mg, 0.29 mmol) wasreacted with bromine and then the obtained bromo sugar 2 wascoupled with phenyl 3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside (3)22 according to typical procedure A.The crude product was purified by silica column chromatography(CH2Cl2–acetone 98:2) to yield 4 (284 mg, 49%) as a colourless syr-up. [a]D +33.91 (c 0.68, CHCl3); 1H NMR (CDCl3, 400 MHz) d 8.01–7.53 (m, 24H, H aromatic), 7.33–7.17 (m, 5H, H aromatic), 5.82 (t,J 9.5 Hz, 1H), 5.74–5.44 (m, 7H), 5.37–5.25 (m, 4H), 5.21 (t, J9.5 Hz, 1H), 4.95 (t, J 9.5, 1H), 4.88–4.74 (m, 4H), 4.52–4.33 (m,3H), 4.30–4.08 (m, 7H), 4.25 (s, 2H, COCH2Cl)), 4.02–3.95 (m, 1H),3.94–3.35 (m, 15H), 2.26, 2.06, 1.96, 1.95, 1.93, 1.87, 1.78 and1.74 (8s, 36H, 12 COCH3); 13C NMR (CDCl3, 128 MHz) d 169.73,169.34, 169.20, 167.49, 167.02 and 166.59 (CO), 134.30–121.02 (Caromatic), 97.91, 97.71, 97.55, 97.46 and 97.34 (C-1I, C-1II, C-1III,C-1IV, C-1V), 82.26 (C-1), 72.77, 72.69, 72.49, 71.69, 71.38, 70.54,70.46, 70.41, 69.46, 69.38, 69.30, 68.82 and 68.15 (C-3, C-3I, C-3II,C-3III, C-3IV, C-3V, C-4, C-4I, C-4II, C-4III, C-4IV, C-4V, C-5, C-5I, C-5II,C-5III, C-5IV, C-5V), 67.69, 67.41, 67.10 and 63.35 (C-6, C-6I, C-6II,C-6III, C-6IV, C-6V), 54.24 and 53.20 (C-2, C-2I, C-2II, C-2III, C-2IV, C-2V), 40.59 (COCH2Cl), 20.56, 20.42, 20.33 and 20.17 (12 COCH3).MALDI-TOF MS calcd for: C116H109ClN6O49SNa, 2459.55 [M+Na]+.Found: 2460.19 [M+Na]+.

3.8. Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-phtha-limido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-de-oxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside (7)

Phenyl 3,4-di-O-acetyl-6-O-chloroacetyl-2-deoxy-2-phthalimi-do-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phtha-limido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phtha-limido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside (5)22 (440 mg, 0.26 mmol)

was reacted with bromine and then the obtained bromo sugar 6was coupled with acceptor 3 according to typical procedure A.The crude product was purified by silica column chromatography(CH2Cl2–acetone 95:5) to yield 7 (273 mg, 39%) as a colourless syr-up. [a]D +34.55 (c 0.52, CHCl3); 1H NMR (CDCl3, 400 MHz) d 7.97–7.50 (m, 32H), 7.30–7.14 (m, 4H), 5.78 (t, J 10 Hz, 1H), 5.69–5.48(m, 7H), 5.46 (d, J 10.5 Hz, 1H), 5.39–5.09 (m, 6H), 4.92 (t, J9.5 Hz, 1H), 4.85–4.71 (m, 4H), 4.52–4.27 (m, 3H), 4.28–4.05 (m,6H), 4.22 (s, 2H, COCH2Cl), 4.00–3.31 (m, 20H), 2.03, 1.91, 1.90,1.84, 1.75 and 1.71 (16s, 48H, 16 COCH3); 13C NMR (CDCl3,128 MHz) d 169.76, 169.39, 169.26, 167.55 and 167.08 (CO),134.26–123.38 (C aromatic), 97.98, 97.77, 97.64, 97.50 and 97.40(C-1I, C-1II, C-1III, C-1IV, C-1V, C-1VI, C-1VII), 82.33 (C-1), 72.82,72.70, 72.50, 71.75, 71.43, 69.55, 69.47, 69.37, 69.12, 68.88 and68.74 (C-3, C-3I, C-3II, C-3III, C-3IV, C-3V, C-3VI, C-3VII, C-4, C-4I, C-4II, C-4III, C-4IV, C-4V, C-4VI, C-4VII, C-5, C-5I, C-5II, C-5III, C-5IV, C-5V,C-5VI, C-5VII), 68.21, 67.82, 67.50, 67.35, 67.13 and 63.41 (C-6, C-6I, C-6II, C-6III, C-6IV, C-6V, C-6VI, C-6VII), 54.30 and 53.26 (C-2, C-2I,C-2II, C-2III, C-2IV, C-2V, C-2VI, C-2VII), 40.63 (COCH2Cl), 20.47,20.38 and 20.22 (12 COCH3). MALDI-TOF MS calcd for: C152H143-

ClN8O65SNa, 3209.74 [M+Na]+. Found: 3210.19 [M+Na]+.

3.9. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4-di-O-acetyl-6-O-chloro-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranoside (9)

Compound 1 (200 mg, 0.21 mmol) was coupled with 7-(1,3-di-oxan-2-yl)-heptanol (8)23 according to typical procedure B. Thecrude product was purified by silica column chromatography(CH2Cl2–acetone 95:5) to yield 9 (134 g, 62%) as a colourless syrup.[a]D +2.7 (c 0.31, CHCl3); 1H NMR (CDCl3, 360 MHz) d 7.88–7.79 (m,4H, H aromatic), 7.76–7.69 (m, 4H, H aromatic), 5.78 (dd, J30 ,40

= 9 Hz, 1H, H-30), 5.68 (dd, J3,4 9 Hz, 1H, H-3), 5.48 (d, J10 ,20 8.5 Hz,1H, H-10), 5.17 (d, J1,2 8.5 Hz, 1H, H-1), 5.15 (t, J40 ,50 9 Hz, 1H, H-40),4.88 (t, J4,5 9 Hz, 1H, H-4), 4.46–4.40 (m, 2H, H-20, H acetalic),4.36–4.29 (m, 2H, H-6a0, H-6b0), 4.20 (s, 2H, COCH2Cl), 4.16 (dd,J2,3 10.5 Hz, 1H, H-2), 4.09 (dd, J 10.5, 5 Hz, 2H), 3.93–3.87 (m, 1H,H-5), 3.86 (dd, J6a,6b 10.5 Hz, J5,6a 2 Hz, 1H, H-6a), 3.79–3.70 (m,3H, H-50, CH2), 3.65 (dd, J5,6b 7 Hz, 1H, H-6b), 3.49–3.41 (m, 1H,CHa), 3.20–3.10 (m, 1H, CHb), 2.15–1.99 (m, 1H, CHa0), 2.04, 1.96,1.86, 1.80 (4s, 12H, 4 COCH3),1.48–1.41 (m, 2H, CH2), 1.37–1.30(m, 1H, CHb0), 1.28–1.10 (m, 4H, 2 CH2), 1.04–0.82 (m, 6H, 3 CH2);

13C NMR (CDCl3, 90.54) d 170.07, 169.53 and 167.19 (CO),134.24, 131.43, 131.36, 123.59 and 123.50 (C aromatic), 102.33(C acetalic), 97.99 (C-10), 97.72 (C-1), 73.01 (C-5), 71.79 (C-50),70.63 (C-30), 70.59 (C-3), 69.78 (C-40), 69.55 (CH2), 68.79 (C-4),68.46 (C-6), 66.86 (2CH2) 63.42 (C-60), 54.61 (C-20), 54.44 (C-2),40.72 (ClCH2CO), 35.16, 29.24, 29.09, 29.00, 25.85, 25.66 and23.77 (7 CH2), 20.60, 20.58 and 20.41 (4 COCH3). MALDI-TOF MScalcd for: C49H57ClN2O20Na, 1051.31 [M+Na]+. Found: 1051.47[M+Na]+.

3.10. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4-di-O-acetyl-6-O-chloro-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyrano-side (10)

Compound 5 (220 mg, 0.13 mmol) was coupled with 7-(1,3-di-oxan-2-yl)-heptanol (8)23 according to typical procedure B. Thecrude product was purified by silica column chromatography(CH2Cl2–acetone 95:5) to yield 10 (136 mg, 58%) as a colourlesssyrup.

38 A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40

[a]D +2.8 (c 0.27, CHCl3); 1H NMR (CDCl3, 400 Hz) 8.11–7.78 (m,16H, H aromatic), 5.94 (t, J 9.5 Hz, 1H), 5.83–5.70 (m, 3H), 5.65 (d, J8.5 Hz, 1H), 5.46 (d, J 8.5 Hz, 1H), 5.41 (d, J 8.5 Hz, 1H), 5.33 (t, J9.5 Hz, 1H), 5.26 (d, J 8.5 Hz, 1H), 5.06 (t, J 9.5 Hz, 1H), 4.99–4.89(m, 2H), 4.66–4.46 (m, 4H), 4.37 (s, 2H, COCH2Cl), 4.35–4.19 (m,4H), 4.14–4.07 (m, 1H), 4.06–4.00 (m, 1H), 3.95–3.53 (m, 12H),3.33–3.25 (m, 1H), 2.19, 2.09, 2.08, 2.06, 2.00, 1.94, 1.92 and 1.91(8s, 24H, COCH3), 1.77–1.64 (m, 1H), 1.61–1.22 (m, 8H), 1.17–0.91 (m, 5H); 13C NMR (CDCl3, 128 MHz) d ppm 170.00, 169.92,169.49, 169.45, 169.38, 169.28 and 167.16 (9 CO), 134.42–123.40(C aromatic), 102.22 (C acetalic), 97.92, 97.54 and 97.49 (C-1,C-1I, C-1II, C-1III), 72.85, 72.67, 71.76, 70.58, 70.53, 70.50 and69.29 (C-3, C-3I, C-3II, C-3III, C-4, C-4I, C-4II, C-4III, C-5, C-5I, C-5II,C-5III), 68.74, 68.16, 67.57 and 63.43 (C-6, C-6I, C-6II, C-6III),54.53, 54.35 and 54.25 (C-2, C-2I, C-2II, C-2III), 40.71 (COCH2Cl),69.56, 67.84, 35.07, 29.15, 28.93, 25.75, 25.59, 25.51 and 23.68(10 CH2), 20.54, 20.49, 20.44 and 20.31 (8 COCH3). MALDI-TOFMS calcd for: C85H91ClN4O36Na, 1801.50 [M+Na]+. Found:1801.65 [M+Na]+.

3.11. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4-di-O-acetyl-6-O-chloro-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyrano-syl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-gluco-pyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranoside (11)

Compound 4 (293 mg, 0.12 mmol) was coupled with 7-(1,3-di-oxan-2-yl)-heptanol (8)23 according to typical procedure B. Thecrude product was purified by silica column chromatography (CH2-

Cl2–acetone 93:7) to yield 11 (170 mg, 56%) as a colourless syrup.[a]D +35.37 (c 0.23, CHCl3); 1H NMR (CDCl3, 400 Hz) d 8.00–7.63(m, 24H, H aromatic), 5.81 (dd, J 10, 9.5 Hz, 1H), 5.72–5.55 (m,5H), 5.53 (d, J 8.5 Hz, 1H), 5.37–5.17 (m, 5H), 5.12 (d, J 8.5 Hz,1H), 4.95 (t, J 9.5 Hz, 1H), 4.88–4.74 (m, 4H), 4.51–4.36 (m, 4H),4.26 (s, 2H, ClCH2CO), 4.29–4.06 (m, 8H), 4.02–3.95 (m, 1H),3.94–3.88 (m, 1H), 3.85–3.69 (m, 8H), 3.68–3.33 (m, 11H), 3.18–3.09 (m, 1H), 2.07, 1.95, 1.94, 1.88, 1.80, 1.79 and 1.78 (7s, 24H,8 CH3CO),1.48–1.10 (m, 8H), 1.03–0.77 (m, 7H); 13C NMR (CDCl3,128 Hz) d 169.81, 169.75, 169.70, 169.33, 169.19 and 167.01(CO), 134.28-123.75 (C aromatic), 102.08 (C acetalic), 97.92,97.56 and 97.45 (C-1, C-1I, C-1II, C-1III, C-1IV, C-1V), 72.78, 72.52,71.70, 70.47, 70.41 and 69.11 (C-3, C-3I, C-3II, C-3III, C-3IV, C-3V,C-4, C-4I, C-4II, C-4III, C-4IV, C-4V, C-5, C-5I, C-5II, C-5III, C-5IV, C-5V), 68.70, 68.13, 67.73, 67.47, 67.07 and 63.36 (C-6, C-6I, C-6II,C-6III, C-6IV, C-6V), 54.44 and 54.24 (C-2, C-2I, C-2II, C-2III, C-2IV,C-2V), 40.58 (COCH2Cl), 69.46, 66.63, 34.95, 29.03, 28.85, 28.79,25.65, 25.46 and 23.55 (CH2), 20.41, 20.32 and 20.16 (12 COCH3).MALDI-TOF MS calcd for: C121H125ClN6O52Na, 2551.61 [M+Na]+.Found: 2551.91 [M+Na]+.

3.12. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4-di-O-acetyl-6-O-chloro-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyrano-syl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyr-anosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-gluco-pyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranoside (12)

Compound 7 (260 mg, 0.08 mmol) was coupled with 7-(1,3-di-oxan-2-yl)-heptanol (8)23 according to typical procedure B. The

crude product was purified by silica column chromatography(CH2Cl2–acetone 95:5) to yield 12 (94 mg, 35%) as a colourless syr-up. [a]D +53.11 (c 0.10, CHCl3); 1H NMR (CDCl3, 400 MHz) d 7.99–7.63 (m, 32H), 5.79 (dd, J 10.5 Hz, J 9.5 Hz, 1H), 5.70–5.54 (m, 8H),5.51 (d, J 8.5 Hz, 1H), 5.36–5.15 (m, 9H), 5.10 (d, J 8.5 Hz, 1H), 4.92(t, J 9.5 Hz, 1H), 4.87–4.70 (m, 7H), 4.48–4.34 (m, 4H), 4.23 (s, 2H,COCH2Cl), 4.28–4.03 (m, 10H), 4.00–3.93 (m, 1H), 3.89 (d, J 9.5 Hz,1H), 3.83–3.64 (m, 10H), 3.63–3.29 (m, 16H), 3.17–3.07 (m, 1H),2.04,1.93, 1.85, 1.78, 1.77, 1.76 and 1.75 (16s, 48H, 16 COCH3),1.46–1.38 (m, 2H), 1.37–1.06 (m, 7H), 0.98–0.78 (m, 6H); 13CNMR (CDCl3, 128 MHz) d 169.94, 169.88, 169.82, 169.45, 169.35,167.54, 167.43 and 167.15 (CO), 134.33-123.39 (C aromatic),102.22 (C acetalic), 98.04, 97.70, 97.57 and 97.48 (C-1I, C-1II,C-1III, C-1IV, C-1V, C-1VI, C-1VII), 72.88, 72.56, 71.81, 70.60, 70.49,69.63, 69.55, 69.45 and 68.80 (C-3, C-3I, C-3II, C-3III, C-3IV, C-3V,C-3VI, C-3VII, C-4, C-4I, C-4II, C-4III, C-4IV, C-4V, C-4VI, C-4VII, C-5,C-5I, C-5II, C-5III, C-5IV, C-5V, C-5VI, C-5VII), 68.27, 67.90, 67.58,67.42, 67.15 and 63.47 (C-6, C-6I, C-6II, C-6III, C-6IV, C-6V, C-6VI,C-6VII), 54.56 and 54.35 (C-2, C-2I, C-2II, C-2III, C-2IV, C-2V, C-2VI,C-2VII), 40.69 (COCH2Cl), 69.25, 66.76, 35.08, 29.15, 28.98, 28.92,25.77, 25.59 and 23.67 (CH2), 20.53, 20.45 and 20.29 (12 COCH3).MALDI-TOF MS calcd for: C157H159ClN8O68Na, 3301.88 [M+Na]+.Found: 3302.12 [M+Na]+.

3.13. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranoside (13)

Compound 9 (85 mg, 0.08 mmol) was treated with ethylenediamine and then the obtained amine was peracetylated accordingto typical procedure C. The crude product was purified by silica col-umn chromatography (CH2Cl2–acetone 7:3) to yield 13 (55 mg,84%) as a colourless syrup. [a]D �1.33 (c 0.23, CHCl3); 1H NMR(CDCl3, 360 MHz) d 6.01 (d, J = 8.5 Hz, 1H, NH), 5.79 (d, J = 8.5 Hz,1H, NH), 5.33 (t, J20 ,30 9.5 Hz, J30 ,40 9.5 Hz, 1H, H-30), 5.19 (t, J2,3

10 Hz, J 3,4 10 Hz,1H, H-3), 5.07 (t, J4,5 10 Hz, 1H, H-4), 5.00 (t,J40 ,50 9.5 Hz, 1H, H-40), 4.67 (d, J10 ,20 8.5 Hz, 1H, H-10), 4.53 (d, J1,2

8.5 Hz, 1H, H-1), 4.51 (t, J = 5 Hz, 1H, acetal-CH), 4.27 (dd, J6a0 ,6b0

12 Hz, J50 ,6a0 5 Hz, 1H, H-6a0), 4.15–4.06 (m, 3H, H-6b0, CH2), 4.06–3.94 (m, 2H, H-2, H-6a), 3.91–3.60 (m, 6H, CHa, CH2, H-20, H-5,H-50), 3.51–3.39 (m, 2H, H-6b, CHb), 2.09, 2.04, 2.03, 2.02, 2.01,1.95, 1.93 (7s, 21H, 7 COCH3), 1.63–1.47 (m, 4H, 2 CH2), 1.46–1.14 (m, 10H, 5 CH2); 13C NMR (CDCl3, 90.54): d 170.64, 170.55,170.40, 170.28, 169.85 and 169.25 (7 CO), 102.28 (C acetalic),100.99 (C-1), 100.26 (C-10) 72.73 (C-3), 72.46 (C-3, C-50), 71.76(C-5), 69.13 (C-40), 68.43 (C-4), 67.84 (C-6), 61.91 (C-60), 54.47(C-20), 53.93 (C-2), 69.32, 66.72, 34.97, 29.10, 28.85, 25.69, 25.41,23.59 (10 CH2), 23.04, 22.96, 20.60, 20.54 and 20.47 (7 COCH3).MALDI-TOF MS calcd for: C37H58N2O18Na, 841.36 [M+Na]+. Found:841.47 [M+Na]+.

3.14. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranoside (14)

Compound 10 (68 mg, 0.038 mmol) was treated with ethylenediamine and then the obtained amine was peracetylated accordingto typical procedure C. The crude product was purified by silica col-umn chromatography (CH2Cl2–methanol 95:5) to yield 14 (35 mg,66%) as a colourless syrup. [a]D�0.5 (c 0.13, CHCl3); 1H NMR (CDCl3,360 MHz) d 7.11 (d, J 9 Hz, 1H, NH), 6.96 (d, J 8.5 Hz, 1H, NH), (d, J8.5 Hz, 1H, NH), 6.52 (d, J 9 Hz, 1H, NH), 5.58–5.39 (m, 2H), 5.34(t, J 10 Hz, 1H), 5.24 (t, J 10 Hz, 1H), 5.18–4.81 (m, 5H), 4.74 (d, J8.5 Hz, 1H), 4.63 (t, J 5 Hz, 1H), 4.59 (d, J 8.5 Hz, 1H), 4.51 (dd, J

A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40 39

12 Hz, J 6 Hz, 1H), 4.35–4.08 (m, 7H), 4.00–3.75 (m, 11H), 3.65–3.55(m, 1H), 2.27, 2.23, 2.20, 2.18, 2.17, 2.16, 2.13, 2.09, 2.07 and 2.06(11s, 33H, COCH3), 1.73–1.58 (m, 4H), 1.54–1.36 (m, 9H); 13CNMR (CDCl3, 90.54) d ppm 171.14, 170.91, 170.66, 170.22, 170.15,170.07, 169.53 and 169.45 (13 COCH3), 102.47 (C acetalic),102.96, 102.14, 101.05 and 100.42 (C-1, C-1I, C-1II, C-1III), 73.50,73.46, 72.60, 72.55, 72.22, 72.16, 72.12, 71.60, 70.65, 70.46, 69.67and 69.25 (C-3, C-3I, C-3II, C-3III, C-4, C-4I, C-4II, C-4III, C-5, C-5I,C-5II, C-5III), 70.02, 69.49 and 62.59 (C-6, C-6I, C-6II, C-6III), 55.41,54.70 and 54.22 (C-2, C-2I, C-2II, C-2III), 71.24, 66.79, 35.12, 29.31,29.12, 25.84, 25.66, 23.82 (10 CH2), 23.10, 22.92, 20.71, 20.63 and20.53 (13 CH3CO). MALDI-TOF MS calcd for: C61H92N4O32Na,1415.56 [M+Na]+. Found: 1415.56 [M+Na]+.

3.15. [7-(1,3-Dioxan-2-yl)-heptyl] 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-3,4-di-O-acetyl-2-acetamido-2-deoxy-b-D-glucopyranoside (15)

Compound 11 (155 mg, 0.06 mmol) was treated with ethylenediamine and then the obtained amine was peracetylated accordingto typical procedure C. The crude product was purified by silica col-umn chromatography (CH2Cl2–methanol 9:1) to yield 15 (56 mg,46%) as a colourless syrup. [a]D �15.93 (c 0.11, CHCl3);1H NMR(CDCl3, 400 MHz) d 7.94 (d, J 7.77 Hz, 2H, NH), 7.81 (d, J 9.57 Hz,2H, NH), 6.99 (br s, 1H, NH), 5.83 (br s, 1H, NH), 5.46 (t, J 9 Hz,1H), 5.38–5.13 (m, 5H), 5.12–4.71 (m, 9H), 4.70–4.53 (m, 10H),4.50 (t, J 5.17 Hz, 1H), 4.46–3.68 (m, 27H), 3.64–3.35 (m, 7H),2.22, 2.16, 2.12, 2.10, 2.09, 2.06, 2.04, 2.03, 1.99, 1.98, 1.97, 1.95and 1.94 (13s, 57H, 19 CH3CO), 1.67–1.19 (m, 15H); 13C NMR(CDCl3, 128 MHz) d 171.96, 171.14, 171.06, 170.96, 170.91,170.48, 170.38, 170.30, 170.18, 170.07, 169.88, 169.79, 169.56and 169.29 (CO), 102.37 (C acetalic), 104.46, 104.11, 103.18,103.00, 101.88 and 100.22 (C-1, C-1I, C-1II, C-1III, C-1IV, C-1V),74.11, 73.71, 72.60, 72.31, 72.11, 71.76, 71.70, 71.36, 70.21,70.07, 69.84 and 69.24 (C-3, C-3I, C-3II, C-3III, C-3IV, C-3V, C-4,C-4I, C-4II, C-4III, C-4IV, C-4V, C-5, C-5I, C-5II, C-5III, C-5IV, C-5V),72.46, 69.39 and 62.97 (C-6, C-6I, C-6II, C-6III, C-6IV, C-6V), 54.59,54.45, 54.33, 54.25 and 53.92 (C-2, C-2I, C-2II, C-2III, C-2IV, C-2V),72.46, 66.81, 35.20, 29.57, 29.49, 28.97, 25.94, 25.83 and 24.00(10 CH2), 23.40, 23.20, 23.07, 22.93, 21.16, 20.92, 20.77 and20.61 (COCH3). MALDI-TOF MS calcd for: C85H126N6O46Na,1989.76 [M+Na]+. Found: 1989.91 [M+Na]+.

3.16. [7-(1,3-Dioxan-2-yl)-heptyl] 2-acetamido-2-deoxy-b-D-glu-copyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranoside(16)

Compound 13 (50 mg, 0.06 mmol) was treated with NaOMeaccording to typical procedure D. The crude product was liophy-lized to give 16 (35 mg, 93%) as a white solid. [a]D +5.6 (c 0.16,H2O); 1H NMR (D2O+D3OD, 360 MHz) d 4.76 (t, J 5.5 Hz, 1H, Hacetalic), 4.59 (d, J1,2 8.5 Hz, 1H, H-1), 4.52 (d, J1020 8.5 Hz, 1H, H-10), 4.25 (d, J6a,6b 11 Hz, J5,6a 1.5 Hz, 1H, H-6a), 4.14 (dd, J 11 Hz, J5 Hz, 2H, CH2), 4.01–3.87 (m, 4H, H-6a0, CHa, CH2), 3.83–3.74 (m,3H, H-2, H-6b, H-6b0), 3.69 (dd, J10 ,20 10 Hz, 1H, H-20), 3.63–3.54(m, 4H, H-3, H-30, H-5, H-50), 3.53–3.48 (m, 2H, H-4, CHb), 3.43 (t,J 9 Hz, 1H, H-40), 2.04 and 2.03 (2s, 6H, 2 COCH3), 2.19–1.89 (m,1H, CHa), 1.67–1.50 (m, 4H, 2 CH2), 1.45–1.29 (m, 9H, CHb, 4CH2); 13C NMR (D2O+D3OD, 90.54 MHz): d 175.35 and 175.27 (2CO), 103.53 (C acetalic), 102.48 (C-1), 102.03 (C-1’) 76.90 (C-3),75.60, 74.89 and 74.79 (C-5’, C-3’, C-4), 71.11 (C-5), 70.97 (C-4’),69.66 (C-6), 61.74 (C-6’), 56.62 (C-2), 56.51 (C-2’), 71.25, 67.96,

35.20, 29.54, 29.26, 26.18, 26.01, 24.14 (10 CH2), 23.25 and 23.19(2 COCH3). MALDI-TOF MS calcd for: C27H48N2O13Na, 631.30[M+Na]+. Found: 631.33 [M+Na]+.

3.17. [7-(1,3-Dioxan-2-yl)-heptyl] 2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranoside (17)

Compound 14 (30 mg, 0.07 mmol) was treated with NaOMeaccording to typical procedure D. The crude product was liophy-lized to give 17 (18 mg, 84%) as a white solid. [a]D �2.1 (c 0.13in H2O); 1H NMR (DMSO+D3OD, 400 MHz) 4.62 (t, J = 4.89,4.89 Hz, 1H), 4.55–4.41 (m, 3H), 4.37 (d, J = 7.92 Hz, 1H), 4.20–4.05 (m, 5H), 3.92–3.77 (m, 6H), 3.76–3.30 (m, 29H), 3.28–3.14(m, 1H), 2.02–1.96 (m, 1H), 1.97, 1.96 and 1.93 (4s, 12H), 1.64–1.51 (m, 4H), 1.50–1.31 (m, 9H); 13C NMR (DMSO+D3OD,128 MHz) d 170.42, 170.34, 170.11 and 169.99 (4 COCH3), 102.41(C acetalic), 103.01, 102.84, 102.63 and 101.88 (C-1, C-1I, C-1II,C-1III), 77.98, 76.69, 76.60, 76.52, 76.17, 75.19, 75.06, 71.87,71.79 and 71.61 (C-3, C-3I, C-3II, C-3III, C-4, C-4I, C-4II, C-4III, C-5,C-5I, C-5II, C-5III), 69.96, 69.84 and 62.05 (C-6, C-6I, C-6II, C-6III),56.60, 56.51, 56.42 and 56.36 (C-2, C-2I, C-2II, C-2III), 69.96, 67.00,35.71, 29.98, 29.78, 26.47, 26.38 and 24.48 (10 CH2), 24.12, 24.07and 24.01 (4 CH3CO). MALDI-TOF MS calcd for: C43H74N4O23Na,1037.46 [M+Na]+. Found: 1037.50 [M+Na]+.

3.18. [7-(1,3-Dioxan-2-yl)-heptyl] 2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acet-amido-2-deoxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-de-oxy-b-D-glucopyranosyl-(1?6)-2-acetamido-2-deoxy-b-D-gluco-pyranoside (18)

Compound 15 (50 mg, 0.07 mmol) was treated with NaOMeaccording to typical procedure D. The crude product was liophy-lized to give 18 (31 mg, 88%) as a white solid. 1H NMR (DMSO) d4.55–4.43 (m, 3H), 4.42–4.20 (m, 8H), 4.12–3.92 (m, 10H), 3.76–3.63 (m, 5H), 3.62–3.39 (m, 13H), 3.38–2.97 (m, 22H), 1.98–1.86(m, 1H), 1.87 and 1.83 (2s, 18H), 1.50–1.37 (m, 1H), 1.36–1.14(m, 9H); 13C NMR (DMSO+D3OD, 400 MHz) d 171.23, 171.18,171.12, 170.98 and 170.90 (6CO), 102.76, 102.73, 102.68, 102.60and 102.50 (C-1, C-1I, C-1II, C-1III, C-1IV, C-1V), 102.43 (C acetalic),77.61, 76.30, 76.17, 75.95, 75.85, 75.71, 74.95, 74.89, 74.79,71.50, 71.38 and 71.29 (C-3, C-3I, C-3II, C-3III, C-3IV, C-3V, C-4,C-4I, C-4II, C-4III, C-4IV, C-4V, C-5, C-5I, C-5II, C-5III, C-5IV, C-5V),69.77, 69.35, 69.31, 69.27 and 61.88 (C-6, C-6I, C-6II, C-6III, C-6IV,C-6V), 56.38 (C-2, C-2I, C-2II, C-2III, C-2IV, C-2V), 69.91, 67.04,35.60, 29.85, 29.64, 28.97, 26.35 and 24.38 (10 CH2), 23.88, 23.85and 23.77 (6COCH3). MALDI-TOF MS calcd for: C59H100N6O33Na,1443.62 [M+Na]+. Found: 1443.62 [M+Na]+.

3.19. BSA-conjugate (22a)

Oligosaccharide 16 (36 mg, 0.06 mmol) was treated with 80% aqacetic acid, then the obtained aldehyde was attached to BSA andpurified according to typical procedure E yielding 7 mg of com-pound 22a. MALDI-TOF MS: m/z 81410 [M]+.

3.20. BSA-conjugate (22b)

Oligosaccharide 16 (60 mg, 0.1 mmol) was treated with 80% aqacetic acid, then the obtained aldehyde was attached to BSA andpurified according to typical procedure F yielding 12.5 mg of com-pound 22b. MALDI-TOF MS: m/z 98893 [M]+.

40 A. Fekete et al. / Carbohydrate Research 386 (2014) 33–40

3.21. BSA-conjugate (23a)

Oligosaccharide 17 (25 mg, 0.025 mmol) was treated with 80%aq acetic acid, then the obtained aldehyde was attached to BSAand purified according to typical procedure E yielding 7.3 mg ofcompound 23a. MALDI-TOF MS: m/z 75973 [M]+.

3.22. BSA-conjugate (23b)

Oligosaccharide 17 (41 mg, 0.04 mmol) was treated with 80% aqacetic acid and then the obtained aldehyde was attached to BSAaccording to typical procedure F. The resulting glycoprotein wasdialysed against water and lyophilized to yield 6.3 mg of com-pound 23b. MALDI-TOF MS: m/z 94030 [M]+.

3.23. BSA-conjugate (24)

Oligosaccharide 18 (35 mg, 0.025 mmol) was treated with 80%aq acetic acid, then the obtained aldehyde was attached to BSAand purified according to typical procedure E yielding 8 mg of com-pound 24. MALDI-TOF MS: m/z 75445 [M]+.

Acknowledgements

This work was supported by the Hungarian Scientific ResearchFund (Project No. PD73064).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.carres.2013.12.022.

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