Efficient and stereocontrolled construction of homo-and heterogeneously 4-and 6-sulfated biotinylated chondroitin oligomers

FULL PAPER

DOI: 10.1002/ejoc.201100298

Efficient and Stereocontrolled Construction of Homo- and Heterogeneously

4- and 6-Sulfated Biotinylated Chondroitin Oligomers^[‡]

Aude Vibert,^[a]Chrystel Lopin-Bon,^[a]and Jean-Claude Jacquinet*^[a]

Keywords: Carbohydrates / Glycosylation / Oligosaccharides / Biotinylated sugars / Chondroitin sulfate

An efficient and stereocontrolled construction of homo- and

heterogeneously 4- and 6-sulfated biotinylated chondroitin

oligomers has been investigated and is reported for the first

time. This procedure allowed the preparation of oligomeric

chondroitin derivatives with sulfate groups at specific posi-

tions; these should be useful probes for the study of the start-

ing point of the polymerization reaction as well as the sub-

strate specificity of sulfotransferases involved in the biosyn-

thesis of chondroitin sulfate chains.

was observed in the latter. Polymerizing activity requires

concomitant expression of a chondroitin polymerizing fac-

tor (ChPf),^[4]and it has been very recently demonstrated

that 4-O-sulfation on the nonreducing GalNAc residue en-

hanced the elongation of CS chains by ChSy-1 and ChPf.^[5]

This finding suggests that sulfation and polymerization of

CS chains are strictly regulated. However, the minimal

structure from which polymerization begins is still un-

known.

Cartilage contains the largest amount of CS of all body

tissues and is efficiently biosynthesized in the Golgi appara-

tus of chondrocytes.^[6]In normal cartilage, aggrecan, the

major CS polymer, is composed of various proportions of

4- and 6-sulfated disaccharide repeating units. A defect in

the biosynthesis of CS chains in cartilage leads to osteoar-

thritis, which is a severe degenerative pathology with in-

creasing socio-economic impact in terms of cost and dis-

ability. To date, no therapy is available and there is a need

to better understand the mechanism of polymerization of

CS chains in cartilage and to further identify pharmacologi-

cal targets to design new drugs.

We previously reported a step-economical process for the

chemical synthesis of size-defined chondroitin oligomers by

semisynthesis starting from a commercially available natu-

ral CS polymer,^[7]and the application of this strategy for

the stereocontrolled construction of non-sulfated and bio-

tinylated chondroitin oligomers,^[8]as well as those of all

known variants of CS.^[9]Within a program devoted to the

study of the biosynthetic pathways of CS chains in relation

to osteoarthritis, we report herein an efficient and stereo-

controlled construction of homo- and heterogeneously 4-

and 6-sulfated chondroitin oligomers as their biotinylated

conjugates, which are useful molecules as probes for study-

ing the initiation of the polymerization of CS chains and

the role of sulfotransferases involved in CS chain construc-

tion in cartilage.

Introduction

Chondroitin sulfate (CS) belongs to the family of glycos-

aminoglycans (GAGs); a class of complex, linear, poly-

anionic polymers widely distributed on the surface of the

cells and within the extracellular matrix. As is the case for

most of the other GAGs (heparin, heparan, and dermatan

sulfate), CS is attached to an l-serine residue of the core

protein to form the CS proteoglycan (CSPG). After the

construction of a common tetrasaccharide linkage region

[β-d-GlcpA-(1Ǟ3)-β-d-Galp-(1Ǟ3)-β-d-Galp-(1Ǟ4)-β-d-

Xylp-(1ǞOSer)] between the CS chain and the core protein,

repeating disaccharide units of N-acetyl-d-galactosamine

(GalNAc) and d-glucuronic acid (GlcA) arranged in the se-

quence [Ǟ4)β-d-GlcpA-(1Ǟ3)-β-d-GalpNAc-(1Ǟ]_nand

with sulfate groups at various positions are synthesized.^[1]

CSPGs vary considerably in size, number of GAG chains

on the core protein, and degree of sulfation. They also store

a considerable amount of information and exhibit a large

variety of biological functions.^[2]Biosynthesis of a specific

CS chain is initiated by the transfer of a GalNAc residue

to the tetrasaccharide linker, followed by the addition of

GlcA and GalNAc units in iterative chain elongation steps.

Several specific sulfotransferases modify the CS backbone,

resulting in structural diversity. In the last decade, several

chondroitin synthases (ChSy) with both glucuronyl- and N-

acetylgalactosaminyltransferase activities have been ex-

pressed and cloned,^[3]but no chondroitin polymerization

[‡] From Polymer to Size-Defined Oligomers, 3. Part 2: Ref.^[9]

[a] Institut de Chimie Organique et Analytique, UMR 6005

Université d’Orléans et CNRS, Université dЈOrléans,

B. P. 6759, 45067 Orléans Cedex, France

Fax: +33-2-38417281

E-mail: Jean-Claude.Jacquinet@univ-orleans.fr

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201100298.

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(Δδ = 1.1 ppm) of the signal for GalNAc 4-H in 7 and those

(Δδ = 0.5 ppm) of the signals for GalNAc 6-Ha,6b in 8.^[11]

Deprotection of 7 and 8 was then achieved through a two-

step saponification process used for uronic ester containing

structures with lithium hydroperoxide^[12]and sodium hy-

droxide to afford the sodium salts 9 (71%) and 10 (66%),

Results and Discussion

Synthesis of the 4- and 6-Sulfated Biotinylated

Disaccharide Derivatives

In this context, the preparation of 4- and 6-sulfated bio-

tinylated disaccharide derivatives was first studied. To this

end, we took advantage of the availability of the disaccha-

ride derivative 1 (Scheme 1), which was easily prepared on

a multigram scale in eight steps from natural CS polymer.^[8]

The N-trichloroacetyl group, a powerful stereocontrolling

auxiliary for the synthesis of 1,2-trans-2-amino-2-deoxygly-

cosides,^[10]was systematically employed along this work for

the preparation of β-galactosaminides. The protective

group pattern in 1 allowed fast access to O-4 and O-6 at

the GalNAc moiety and further possible biotinylation at

the reducing end. The benzylidene acetal in 1 was hy-

drolyzed to afford the diol 2 in good yield. To avoid tedious

protection–deprotection steps for the preparation of key in-

termediates 3 and 4, in which hydroxy groups were further

O-sulfonated, we applied a one-pot process similar to those

still reported for the preparation of CS –K and L oligo-

mers.^[9]Diol 2 was thus treated with trimethyl orthobenzo-

ate and a catalytic amount of CSA in dichloromethane to

give the intermediate 4,6-orthoester, which upon mild acid

hydrolysis afforded the 6-O-benzoyl (3) and the 4-O-benzoyl

(4) derivatives in a 1:1 ratio and in excellent yield. Radical

reduction^[10]of 3 and 4 with tri-n-butylstannane afforded

1

respectively. The H and ¹³C NMR spectra for 9 and 10

showed high purity and were in full agreement with the ex-

pected structures. Finally, catalytic hydrogenation of 9 and

10 with 10% palladium on carbon in water gave the inter-

mediate corresponding amines, which were further treated

with N-hydroxysuccinimidoyl 6-(biotinylamido)hexanoate^[8]

and triethylamine in DMF/water to afford, for the first

time, after purification on Sephadex LH-20 in water, the

target 4- (11) and 6-sulfated (12) biotinylated conjugates,

respectively, in good yields. The structure and chemical pu-

1

rity of 11 and 12 were readily established through H and

¹³C NMR spectroscopy (see the Supporting Information).

It should be noted that the intermediate amines obtained

by hydrogenation of 9 and 10 were versatile derivatives that

could be substituted by other groups, such as fluorophores,

proteins, dendrimers, and microarrays.

Synthesis of the Homo- and Heterogeneously 4- and 6-

Sulfated Biotinylated Trisaccharide Derivatives

For the construction of homo- and heterogeneously 4-

smoothly the acetamides 5 (79%) and 6 (88%), respectively, and 6-sulfated trisaccharide derivatives, suitably protected

which were further treated with sulfur trioxide/trimeth- common disaccharide acceptors were first required. They

ylamine complex in N,N-dimethylformamide (DMF) to could be prepared from 1 through selective protections on

provide, after ion exchange with a [Na⁺] resin, the sulfated the GlcA unit to allow further elongation at the non-reduc-

derivatives 7 (92%) and 8 (82%), respectively. Comparison ing end. However, they were more efficiently prepared from

1

of the H NMR spectra of 7 and 8 and those of their re- disaccharide derivative 13 (Scheme 2), which was easily

spective precursors showed the expected downfield shifts available in 12 steps from the natural polymer,^[8]and start-

Scheme 1. Access to 4- and 6-sulfated biotinylated chondroitin disaccharide derivatives. a) 3:1 trifluoroacetic acid (TFA)/H₂O, CH₂Cl₂,

0 °C, 1 h, 87%; b) PhC(OMe)₃, 10-camphorsulfonic acid (CSA), CH₂Cl₂, 40 min; then 80% AcOH, 15 min, 45% for 3, 45% for 4;

c) Bu₃SnH, 2,2Ј-azobis(isobutyronitrile) (AIBN), benzene/N,N-dimethylacetamide (DMAC), 80 °C, 6 h, 79% for 5, 88% for 6;

d) Me₃N·SO₃, DMF, 60 °C; then SP C-25 [Na⁺] resin, 92% for 7, 82% for 8; e) LiOH/H₂O₂, THF/H₂O, –10 °CǞroom temp., 16 h; then

4 m NaOH, MeOH, 8 h, 71% for 9, 66% for 10; f) H₂, Pd/C, H₂O; then N-hydroxysuccinimidoyl 6-(biotinylamido)hexanoate, Et₃N,

DMF/H₂O, 1 h, 50 % for 11, 68% for 12. TCA = trichloroacetyl, Z = benzyloxycarbonyl.

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Construction of Chondroitin Oligomers

Scheme 2. Preparation of disaccharide glycosyl donors and acceptors. a) 3:1 TFA/H₂O, CH₂Cl₂, 0 °C, 1 h, 84%; b) PhC(OMe)₃, CSA,

CH₂Cl₂, 40 min; then 80% AcOH, 15 min, 35% for 15, 37% for 16; c) (ClAc)₂O, CH₂Cl₂/pyridine, 0 °C, 30 min, 90% for 17, 87%

for 18; d) 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), CH₂Cl₂/MeOH, 24 h; then Cl₃CCN, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),

CH₂Cl₂, 30 min, 66% for 19, 65% for 20; e) 2-(benzyloxycarbonylamino)ethanol, trimethylsilyl triflate (TMSOTf), CH₂Cl₂, 30 min, 80%

for 21, 70% for 22; f) hydrazine acetate, pyridine, 2 min, 78% for 23, 77% for 24. Bz = benzoyl, Lev = levulinoyl, NAP = 2-naphthylmethyl.

ing from a sequence similar to that achieved above could agreement with the newly established 1,2-trans-linkages. Se-

be applied. In 13, the NAP group^[15]was conveniently used lective delevulinoylation of 21 and 22 with hydrazine acet-

as a temporary protection at the anomeric center of the d- ate^[14]gave the alcohols 23 (78%) and 24 (77%), respec-

galactosamine units. To this end, the benzylidene acetal in tively, which were now ready for further elongation at the

13 was hydrolyzed to give the crystalline diol 14 (84%), non-reducing end.

which upon treatment with trimethyl orthobenzoate, as de-

The construction of homogeneously sulfated trisaccha-

scribed above, afforded the 6-O-benzoyl (15) and the 4-O- ride species required a suitably protected and activated de-

benzoyl (16) derivatives in a 1:1 ratio and in good yield. rivative of d-galactosamine in which the substitution

The free hydroxy groups in 15 and 16 were temporarily pro- pattern was orthogonal to those designed in the disaccha-

tected through conventional monochloroacetylation to af- ride acceptors 23 and 24. To this end, the known triol 25,

ford 17 (90%) and 18 (87%), respectively. However, com- easily prepared from d-glucosamine hydrochloride,^[16]was

pounds 17 and 18 could be obtained independently (not used as a starting material (Scheme 3).

described in the Exp. Section), that is, for 17, by selective

Treatment of 25 with 2-methoxypropene and CSA under

benzoylation of diol 14 with benzoyl cyanide (93%) fol- kinetic control gave the 4,6-O-isopropylidene derivative 26

lowed by chloroacetylation (96%) and, for 18, by selective as the major product in 83% yield. Benzoylation of 26 gave

chloroacetylation with chloroacetic anhydride at –20 °C quantitatively the 3-benzoate 27, which upon mild acid hy-

(72%) followed by benzoylation (90%).

drolysis afforded the diol 28 in good yield. Surprisingly,

Glycosides 17 and 18 were then transformed into the cor- when diol 28 was treated with trimethyl orthobenzoate as

responding glycosyl trichloroacetimidates^[13]through oxi- described above, the 6-benzoate 29 and the 4-benzoate 30

dative removal of the 2-naphthylmethyl group with DDQ were obtained, respectively, in a 3:1 ratio. In addition, when

followed by imidoylation with trichloroacetonitrile and the transient orthoester was not isolated, but aqueous

DBU in dichloromethane to afford the α-imidates 19 and methanol was directly added to the mixture, a reversed

20, respectively, in 66 and 65% overall yield, respectively; selectivity (≈ 1:3) was observed. These results showed that

1

the H NMR spectra of which showed characteristic cou- this technique, if applicable and reproducible on disaccha-

pling constants (J_1,2= 3.5 Hz) for 1,2-cis species. These ver- ride derivatives in which the O-3 of the GalNac moiety was

satile imidates not only serve for the preparation of com- substituted by a bulky glucuronate residue, was less efficient

mon acceptors, but also as donors for the construction of on monomeric units in the d-galacto series. However,

homogeneously sulfated tetrasaccharide derivatives. A cou- alcohols 29 and 30 could be easily obtained from diol 28,

pling reaction of 2-(benzyloxycarbonylamino)ethanol with and were then temporarily protected through chloroacetyl-

imidates 19 and 20 in dichloromethane under catalysis by ation to afford esters 31 (88%) and 32 (80%), respectively.

TMSOTf afforded the β-glycosides 21 (80%) and 22 (70%), The glycosides 31 and 32 were transformed into imidates

respectively, as single stereoisomers. The coupling constants through oxidative removal of their 4-methoxyphenyl (MP)

1

(J_1,2≈ 8.0 Hz) observed in their H NMR spectra were in glycosides with CAN, followed by imidoylation, as de-

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Scheme 3. Preparation of monomeric glycosyl donors. a) 2-methoxypropene, CSA, DMF, 2 h, 83%; b) PhCOCl, CH₂Cl₂/pyridine, 0 °C,

30 min, quantitative; c) 80% AcOH, 80 °C, 40 min, 85%; d) PhC(OMe)₃, CSA, CH₂Cl₂; then 80% AcOH, 74% for 29, 25% for 30; or

as previously then MeOH/H₂O, 15 min, 25% for 29, 68% for 30; e) (ClAc)₂O, CH₂Cl₂/pyridine, 0 °C, 30 min, 88% for 31, 80% for 32;

f) cerium(IV) diammonium hexanitrate (CAN), toluene/MeCN/H₂O, 30 min; then Cl₃CCN, DBU, CH₂Cl₂, 30 min, 60% overall for 33

and 34.

scribed above, to afford the α-imidates 33 and 34, respec- reported above, to afford the acetamides 39 (81%) and 40

tively, in 60% overall yield. Coupling reactions of alcohol (71%), respectively. Sulfation of 39 and 40, as described

23 with imidate 33 and of alcohol 24 with imidate 34 under above, afforded the disulfates 41 (85%) and 42 (80%),

catalysis by TMSOTf (Scheme 4) gave the trisaccharide de- respectively; the ¹H NMR spectra of which were in full

rivatives 35 (66%) and 36 (52%), respectively; the ¹H NMR agreement with the expected structures and showed the rel-

spectra of which showed characteristic coupling constants evant downfield shifts due to sulfation. Saponification of

(J_1,2= 8.0 Hz) for 1,2-trans-linked species. Noticeable is the 40 and 41, as described above, afforded the sodium salts

absence of 1,2-cis-linked species in these and further re- 43 (82%) and 44 (77%), respectively, which upon catalytic

ported reactions using 2-deoxy-2-trichloroacetamido deriv- hydrogenation and biotinylation, as described above, gave

atives.^[10]Selective removal of the chloroacetate esters in 35 the target molecules 45 (90%) and 46 (70%), respectively.

1

and 36 was achieved by treatment with thiourea in pyridine/ The H and ¹³C NMR spectra for 45 and 46 showed high

ethanol to provide the diols 37 (73%) and 38 (85%), respec- purity and were fully consistent with the expected struc-

tively, which were then submitted to radical reduction, as tures.

Scheme 4. Access to homogeneously sulfated biotinylated trisaccharide derivatives. a) TMSOTf, CH₂Cl₂, 30 min, 66% for 35, 52% for

36; b) thiourea, pyridine/EtOH, 80 °C, 2 h, 73% for 37, 85% for 38; c) Bu₃SnH, AIBN, benzene/(DMAC), 80 °C, 6 h, 81% for 39, 71%

for 40; d) Me₃N·SO₃, DMF, 60 °C; then LH-20; then Sephadex SP C-25 [Na⁺] resin, 85% for 41, 80% for 42; e) LiOH/H₂O₂, THF/H₂O,

–10 °CǞroom temp., 16 h; then 4 m NaOH, MeOH, 8 h, 82% for 43, 77% for 44; f) H₂, Pd/C, H₂O; then N-hydroxysuccinimidoyl 6-

(biotinylamido)hexanoate, Et₃N, DMF/H₂O, 1 h, 90 % for 45, 70% for 46.

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Construction of Chondroitin Oligomers

For the synthesis of the heterogeneously sulfated trisac- derivatives 60 (55%) and 61 (63%), respectively; the ¹H

charide derivatives, that is, non-sulfated on the non-reduc- NMR spectra of which showed the four expected 1,2-trans

ing d-GalNAc unit, the known acetylated imidate 47^[16]was linkages (J_1,2= 7.5–8.0 Hz). Selective dechloroacetylation

selected (Scheme 5). Coupling reactions of imidate 47 with of 60 and 61 afforded the diols 62 (84%) and 63 (77%),

alcohols 23 and 24 under catalysis by TMSOTf afforded the respectively, which were submitted to radical reduction to

trisaccharide derivatives 48 (65%) and 49 (59%), respec- provide the acetamides 64 (79%) and 65 (68%), respectively,

tively; the ¹H NMR spectra of which were in full agreement which upon sulfation, as described above, gave the disulf-

with the expected structures. Starting from 48 and 49, a ates 66 (81%) and 67 (67%); the ¹H NMR spectra of which

similar sequence to those reported above was then achieved were in full agreement with the expected structures. Saponi-

as follows: Selective dechloroacetylation of 48 and 49 gave fication of 66 and 67 afforded the sodium salts 68 (84%)

alcohols 50 (67%) and 51 (86%), respectively, the N-tri- and 69 (71%), respectively, which after catalytic hydrogena-

chloroacetyl groups of which were reduced, as described tion and biotinylation, as described above, provided the tar-

1

above, to afford the acetamides 52 (70%) and 53 (77%), get molecules 70 and 71, respectively, in good yield; the H

respectively. Sulfation of 52 and 53 gave the monosulfates and ¹³C NMR spectra of which showed high purity.

1

54 (70%) and 55 (75%), respectively; the H NMR spectra

of which showed the expected downfield shifts due to sulf-

ation. Saponification of 54 and 55 gave the sodium salts 56

(70%) and 57 (72%), respectively. Catalytic hydrogenation

followed by biotinylation, as described above, afforded the

target molecules 58 and 59, respectively, in good yields. The

¹H NMR spectrum for 58 (4-sulfate) showed a typical

downfield shift (Δδ ≈ 1 ppm) for the signal for 4-H on the

reducing GalNAc unit compared with those on the non-

reducing GalNAc unit, and for 59 (6-sulfate), an average

downfield shift (Δδ ≈ 0.5 ppm) for the signals for 6-Ha,6b

on the reducing GalNAc unit compared with those on the

non-reducing GalNAc unit.

For the synthesis of the heterogeneously 4- and 6-sulfated

biotinylated tetrasaccharide derivatives, as discussed above,

a fully acetylated and activated disaccharide derivative was

required. The known disaccharide imidate 72,^[7]easily pre-

pared in seven steps from the natural polymer, fulfils these

requirements (Scheme 7). The coupling reaction of imidate

72 with alcohols 23 and 24, as described above, gave the

tetrasaccharide derivatives 73 (62%) and 74 (67%), respec-

1

tively; the structures of which were evident from their H

NMR spectra. Selective dechloroacetylation of 73 and 74

afforded the diols 75 (82%) and 76 (86%), respectively,

which were submitted to radical reduction to provide the

acetamides 77 (63%) and 78 (63%), respectively, which

upon sulfation, as described above, gave the disulfates 79

1

(84%) and 80 (78%); the H NMR spectra of which were

in fully consistent with the expected structures. Saponifica-

tion of 79 and 80 afforded the sodium salts 81 (75%) and

82 (91%), respectively, which after catalytic hydrogenation

Synthesis of the Homo- and Heterogeneously 4- and 6-

Sulfated Biotinylated Tetrasaccharide Derivatives

For the construction of the homogeneously 4- and 6-sulf- and biotinylation, as described above, provided the target

1

ated biotinylated tetrasaccharide derivatives, donor and ac- molecules 83 and 84, respectively, in good yields. Their H

ceptor blocks were ready to use. Coupling reactions of im- NMR spectra showed the same features as those reported

idate 19 with alcohol 23, and of imidate 20 with alcohol 24 for the heterogeneously sulfated trisaccharide derivatives.

(Scheme 6), as described above, afforded the tetrasaccharide For all target biotinylated molecules, the ¹³C NMR spectra

Scheme 5. Access to heterogeneously sulfated biotinylated trisaccharide derivatives. a) TMSOTf, CH₂Cl₂, 30 min, 65% for 48, 59% for

49; b) thiourea, pyridine/EtOH, 80 °C, 2 h, 67% for 50, 86% for 51; c) Bu₃SnH, AIBN, benzene/DMAC, 80 °C, 6 h, 70% for 52, 77%

for 53; d) Me₃N·SO₃, DMF, 60 °C; then LH-20; then Sephadex SP C-25 [Na⁺] resin, 70% for 54, 75% for 55; e) LiOH/H₂O₂, THF/H₂O,

–10 °CǞroom temp., 16 h; then 4 m NaOH, MeOH, 8 h, 70% for 56, 72% for 57; f) H₂, Pd/C, H₂O; then N-hydroxysuccinimidoyl 6-

(biotinylamido)hexanoate, Et₃N, DMF/H₂O, 1 h, 56 % for 58, 70% for 59.

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Scheme 6. Access to homogeneously sulfated biotinylated tetrasaccharide derivatives. a) TMSOTf, CH₂Cl₂, 2 h, 55% for 60, 63% for 61;

b) thiourea, pyridine/EtOH, 80 °C, 2 h, 84% for 62, 77% for 63; c) Bu₃SnH, AIBN, benzene/DMAC, 80 °C, 6 h, 79% for 64, 68% for

65; d) Me₃N·SO₃, DMF, 60 °C; then LH-20; then Sephadex SP C-25 [Na⁺] resin, 81% for 66, 67% for 67; e) LiOH/H₂O₂, THF/H₂O,

–10 °CǞroom temp., 16 h; then 4 m NaOH, MeOH, 8 h, 84% for 68, 71% for 69; f) H₂, Pd/C, H₂O; then N-hydroxysuccinimidoyl 6-

(biotinylamido)hexanoate, Et₃N, DMF/H₂O, 1 h, 89 % for 70, 77% for 71.

(see the Supporting Information) clearly showed the down-

field shifts (Δδ ≈ 9 ppm) for the signals for GalNAc C-4 in

4-sulfated GalNAc units, and those (Δδ ≈ 7 ppm) for the

Conclusions

An efficient and stereocontrolled approach for the con-

signals of GalNAc C-6 in 6-sulfated GalNAc units com- struction of homo- and heterogeneously 4- and 6-sulfated

pared with those of the non-sulfated units. biotinylated chondroitin oligomers is reported herein. This

Scheme 7. Access to heterogeneously sulfated biotinylated tetrasaccharide derivatives. a) TMSOTf, CH₂Cl₂, 30 min, 62% for 73, 67% for

74; b) thiourea, pyridine/EtOH, 80 °C, 2 h, 82% for 75, 86% for 76; c) Bu₃SnH, AIBN, benzene/DMAC, 80 °C, 6 h, 63% for 77 and 78;

d) Me₃N·SO₃, DMF, 60 °C; then LH-20; then Sephadex SP C-25 [Na⁺] resin, 84% for 79, 78% for 80; e) LiOH/H₂O₂, THF/H₂O,

–10 °CǞroom temp., 16 h; then 4 m NaOH, MeOH, 8 h, 75% for 81, 91% for 82; f) H₂, Pd/C, H₂O; then N-hydroxysuccinimidoyl 6-

(biotinylamido)hexanoate, Et₃N, DMF/H₂O, 1 h, 88 % for 83, 87% for 84.

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Construction of Chondroitin Oligomers

(2.7 mL) and toluene (1.3 mL), and the mixture was stirred for

40 min. Triethylamine (212 μL, 1.5 mmol) was then added and the

mixture was diluted with CH₂Cl₂, washed with a saturated aqueous

solution of NaHCO₃and water, dried (MgSO₄), and concentrated.

A solution of the residue in 80% AcOH (5 mL) was stirred for

15 min at room temp. then was diluted with water, and concen-

trated. Flash silica chromatography (EtOAc/petroleum ether,

1:1Ǟ3:2) gave first the 6-benzoate 3 (144 mg, 45%). Next eluted

(EtOAc/petroleum ether, 3:1) was the amorphous 4-benzoate 4

(144 mg, 45%).

reproducible procedure allowed the preparation of reason-

able amounts of chemically pure substances that were fully

characterized through ¹H and ¹³C NMR spectroscopy. This

collection of potential probes, which are in great demand,

is now available for studying the polymerization reaction in

CS chains and the substrate specificity of sulfotransferases

involved in their biosynthesis. All of these structures are

currently being investigated in biological assays, the results

of which will be reported elsewhere in due course.

Data for 3: M.p. 115–116 °C (from EtOAc/petroleum ether). [α]²_D²

= –5 (c = 1 in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.03–

7.08 (m, 11 H, GalN NH, arom. H), 5.33 [m, 1 H, NHC(O)O],

Experimental Section

5.25–5.16 (m, 2 H, GlcA 3-H, 4-H), 5.07 (dd, J_1,2= 8.0, J_2,3

9.0 Hz, 1 H, GlcA 2-H), 5.03 (s, 2 H, CH₂-Ph), 4.93 (d, J_1,2

=

General Methods: Solvents were dried by standard methods and

molecular sieves were activated prior to use by heating for 4 h at

500 °C. Melting points were determined in capillary tubes with a

Büchi apparatus. Optical rotations were measured at 20–25 °C with

a Perkin–Elmer 341 polarimeter. ¹H and ¹³C NMR spectra were

recorded at 25 °C with a Bruker Advance II 400 instrument, with

Me₄Si as the internal standard, unless otherwise stated. Assign-

ments were based on homo- and heteronuclear correlations by

using the supplier’s software. In the NMR spectroscopic data for

oligomers, GlcA^Iand GalN^I(with Roman numeral superscripts)

refer to d-glucuronic acid and d-galactosamine residues, respec-

tively, in the reducing disaccharide unit. Low-resolution mass spec-

tra were obtained on a Perkin–Elmer SCIEX API 300 spectrometer

operating in the ion-spray mode or on a Micromass Quattro Ul-

tima spectrometer equipped with a Z-spray ionization source op-

erating in the negative mode. HRMS were obtained from the Cen-

tre Regional de Mesure Physique (Université Blaise Pascal, Cler-

mont-Ferrand, France). Flash silica chromatography was per-

formed on Silica gel 60 (0.040–0.063 mm, Merck, Darmstadt). The

reactions were monitored by TLC on coated aluminum sheets (sil-

ica gel 60 GF₂₅₄, Merck) and spots were detected under UV light

and by charring with a 95:5 mixture of ethanol and sulfuric acid.

Elemental analyses were carried out at the Service Central de

Microanalyse du CNRS (Vernaison, France).

8.0 Hz, 1 H, GalN 1-H), 4.80 (d, 1 H, GlcA 1-H), 4.61 (dd, J_5,6a

= 5.0, J_6a,6b= 11.5 Hz, 1 H, GalN 6-Ha), 4.57 (dd, J_5,6b= 7.0 Hz,

1 H, GalN 6-Hb), 4.46 (dd, J_3,4= 2.0, J_2,3= 11.0 Hz, 1 H, GalN

3-H), 4.23 (ddd, J_4,5Ͻ 1.0, J_4,OH= 2.0 Hz, 1 H, GalN 4-H), 4.08

(d, J_4,5= 9.5 Hz, 1 H, GlcA 5-H), 3.96–3.80 (m, 2 H, GalN 5-H,

CH₂-O), 3.79 (m, 1 H, GalN 2-H), 3.71 (s, 3 H, COOCH₃), 3.68

(m, 1 H, CH₂-O), 3.31 (d, 1 H, OH), 3.45–3.33 (m, 2 H, CH₂-N),

2.01, 2.00, 1.99 [3 s, 9 H ppm, OC(O)CH₃] ppm. MS: m/z: 938 [M

+ NH₄]⁺(for ³⁵Cl). C₃₈H₄₃Cl₃N₂O₁₈(922.12): calcd. C 49.50, H

4.70, N 3.04; found C 49.71, H 4.57, N 3.02.

Data for 4: [α]²_D²= +28.5 (c = 1 in chloroform). ¹H NMR

(400 MHz, CDCl₃): δ = 8.04–7.16 (m, 11 H, GalN NH, arom. H),

5.62 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 5.35 [m, 1 H,

NHC(O)O], 5.13–5.00 (m, 4 H, GlcA 3-H, 4-H, CH₂-Ph), 5.07 (dd,

J_1,2= 8.0, J_2,3= 9.0 Hz, 1 H, GlcA 2-H), 4.93 (d, J_1,2= 8.0 Hz, 1

H, GalN 1-H), 4.80 (d, 1 H, GlcA 1-H), 4.59 (dd, J_2,3= 11.0 Hz,

1 H, GalN 3-H), 4.05 (d, J_4,5= 9.5 Hz, 1 H, GlcA 5-H), 4.03 (m,

1 H, GalN 2-H), 3.85 (m, 1 H, CH₂-O), 4.72–3.62 (m, 2 H, GalN

6-Ha, CH₂-O), 3.79 (m, 1 H, GalN 5-H), 3.74 (s, 3 H, COOCH₃),

3.56–3.41 (m, 2 H, GalN 6-Hb, OH), 3.39–3.31 (m, 2 H, CH₂-N),

1.99, 1.97, 1.93 [3 s, 9 H, OC(O)CH₃] ppm. MS: m/z: 938 [M +

NH₄]⁺(for ³⁵Cl). C₃₈H₄₃Cl₃N₂O₁₈(922.12): calcd. C 49.50, H 4.70,

N 3.04; found C 49.55, H 4.71, N 2.97.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-deoxy-2-trichloroacetamido-β- -ga- General Procedure for the Reduction of a 2-Trichloroacetamido

lactopyranoside (2): A mixture of TFA/water (3:1, 1.8 mL) was Group: Argon was bubbled for 1 h at room temp. through a solu-

added at 0 °C to a solution of 1^[8](516 mg, 0.57 mmol) in CH₂Cl₂tion of the trichloroacetamide (0.2 mmol), Bu₃SnH (0.28 mL,

D-

D

(5 mL). The reaction mixture was stirred for 1 h at 0 °C, then was

diluted with CH₂Cl₂, washed with a saturated aqueous solution of

NaHCO₃and water, dried (MgSO₄), and concentrated. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the crystalline

1 mmol), and AIBN (50 mg) in benzene/DMAC (1:1, 5 mL), and

the mixture was stirred for 2 h at 80 °C. Solutions of Bu₃SnH

(0.17 mL, 0.6 mmol) and AIBN (20 mg) in DMAC (1 mL) were

then added after 2 and 4 h, and stirring was resumed for 2 h at

diol 2 (404 mg, 87%); m.p. 115–116 °C (from EtOAc/petroleum 80 °C. The mixture was then cooled and concentrated, and the resi-

ether). [α]²_D²= –5 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 7.68–7.62 (m, 5 H, arom. H), 5.51 (dd, J_3,4= J_4,5

due was stirred at 0 °C with diethyl ether/petroleum ether (1:1,

10 mL). The solids were filtered off and washed with diethyl ether.

Flash silica chromatography afforded the corresponding acetamide.

=

9.5 Hz, 1 H, GlcA 4-H), 5.51 (dd, J_2,3= 9.5 Hz, 1 H, GlcA 3-H),

5.37 (s, 2 H, CH₂-Ph), 5.31 (dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H),

4.94 (d, 1 H, GlcA 1-H), 4.85 (d, J_1,2= 8.0 Hz, 1 H, GalN 1-H),

4.47 (d, 1 H, GlcA 5-H), 4.38–4.29 (m, 3 H, GalN 2-H, 3-H, 4-H),

4.20–4.03 (m, 3 H, GalN 6-Ha, 6-Hb, CH₂-O), 4.07 (s, 3 H, CO-

OCH₃), 3.93 (m, 1 H, CH₂-O), 3.84 (m, 1 H, GalN 5-H), 3.66–3.60

(m, 2 H, CH₂-N), 2.37, 2.35, 2.33 [3 s, 9 H, OC(O)CH₃] ppm. MS:

m/z: 834 [M + NH₄]⁺(for ³⁵Cl). C₃₁H₃₉Cl₃N₂O₁₇(818.01): calcd.

C 45.62, H 4.81, N 3.42; found C 45.64, H 4.67, N 3.32.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benzoyl-2-deoxy-β-

-galactopyranoside (5): The product was obtained from 3 (186 mg,

D-

D

0.20 mmol) by using the general procedure described above. Flash

silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded the crystal-

line acetamide 5 (130 mg, 79%); m.p. 237–239 °C (from EtOAc).

[α]²_D²= +1 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

1

= 8.02–7.11 (m, 10 H, arom. H), 6.19 (d, J_2,NH= 6.5 Hz, 1 H,

GalN NH), 5.46 [m, 1 H, NHC(O)O], 5.26 (dd, J_2,3= J_3,4= 9.0 Hz,

1 H, GlcA 3-H), 5.19 (dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 5.08–

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl- (3) and 4-O-Benzoyl-2-

deoxy-2-trichloroacetamido-β-

D-

D-galactopyranoside (4): Trimethyl 5.01 (m, 3 H, GlcA 2-H, CH₂-Ph), 4.97 (d, J_1,2= 8.0 Hz, 1 H,

orthobenzoate (0.35 mL, 2 mmol) was added to a solution of 2

(270 mg, 0.33 mmol) and CSA (70 mg) in anhydrous CH₂Cl₂

GlcA 1-H), 4.77 (d, J_1,2= 8.0 Hz, 1 H, GalN 1-H), 4.59 (dd, J_5,6a

= 5.0, J_6a,6b= 11.5 Hz, 1 H, GalN 6-Ha), 4.07 (dd, J_5,6b= 7.0 Hz,

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4189

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

1 H, GalN 6-Hb), 4.52 (m, 1 H, GalN 3-H), 4.14 (ddd, J_4,5Ͻ 1.0,

J_4,OHϽ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 4.08 (d, J_4,5= 9.5 Hz,

1 H, GlcA 5-H), 3.89 (m, 1 H, GalN 5-H), 3.82 (m, 1 H, CH₂-O),

5.23 (dd, J_2,3= J_3,4= 9.5, J_1,2= 8.0 Hz, 1 H, GlcA 3-H), 5.08 (s,

2 H, CH₂-Ph), 5.04 (dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 4.92 (d, 1

H, GlcA 1-H), 4.78 (dd, 1 H, GlcA 2-H), 4.52 (d, J_1,2= 8.0 Hz, 1

3.68 (m, 1 H, CH₂-O), 3.66 (s, 3 H, COOCH₃), 3.49–3.26 (m, 2 H, H, GalN 1-H), 4.21 (d, 1 H, GlcA 5-H), 4.25–4.19 (m, 1 H, GalN

GalN 2-H, CH₂-N), 3.13 (d, 1 H, OH), 2.02, 2.01 [2s, 9 H, OC- 2-H), 4.17 (dd, J_5,6a= 4.0, J_6a,6b= 11.0 Hz, 1 H, GalN 6-Ha), 4.11

(O)CH₃], 1.93 [s,

3

H, NHC(O)CH₃] ppm. MS: m/z: 819

(dd, J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.07 (dd, J_5,6b= 8.0 Hz, 1 H,

[M + H]⁺, 836 [M + NH₄]⁺. C₃₈H₄₆N₂O₁₈(818.78): calcd. C 55.74, GalN 6-Hb), 3.96–3.87 (m, 2 H, GalN 5-H, CH₂-O), 3.69 (s, 3 H,

H 5.66, N 3.04; found C 55.59, H 5.67, N 3.34.

COOCH₃), 3.63 (m, 1 H, CH₂-O), 3.35–3.28 (m, 2 H, CH₂-N),

2.02, 1.98, 1.94 [3 s, 12 H, OC(O)CH₃, NHC(O)CH₃] ppm. HRMS:

m/z: calcd. for C₃₈H₄₅N₂O₂₁S [M – Na]^–: 897.2236; found: 897.228.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-D-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benzoyl-2-deoxy-β-

-galactopyranoside (6): The product was obtained from 4 (193 mg,

D

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

D-gluco-

0.21 mmol) by using the general procedure described above. Flash

silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded the acet-

amide 6 (150 mg, 88%) as a white powder. [α]²_D²= +25 (c = 1 in

pyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-β-D-

galactopyranoside (9): A solution of ester 7 (98 mg, 0.11 mmol) in

THF (2 mL) was treated at –10 °C with a freshly prepared solution

chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.02–7.11 (m, 10 of hydrogen peroxide (30 wt.-% solution in water, 265 μL) and

H, arom. H), 6.10 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.54 (dd,

LiOH (1 m, 530 μL), and the mixture was stirred for 1 h at –10 °C

J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 5.40 [m, 1 H, NHC- and 16 h at room temp. then was cooled to 0 °C. Methanol

(O)O], 5.16 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H), 5.09 (dd, J_4,5

= 9.0 Hz, 1 H, GlcA 4-H), 5.07 (s, 2 H, CH₂-Ph), 4.95 (d, J_1,2

(1.5 mL) and sodium hydroxide (4 m, 0.53 mL) were added and the

mixture was stirred for 8 h at room temp. The solution was concen-

=

8.5 Hz, 1 H, GalN 1-H), 4.92 (dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H), trated and water (10 mL) was added. The mixture was treated with

4.78 (d, 1 H, GlcA 1-H), 4.63 (dd, J_2,3= 10.0 Hz, 1 H, GalN 3-

H), 4.02 (d, 1 H, GlcA 5-H), 3.85 (m, 1 H, CH₂-O), 3.77 (m, 1 H,

GalN 5-H), 3.72–3.58 (m, 3 H, GalN 2-H, 6-Ha, CH₂-O), 3.62 (s,

Amberlite IR-120 [H⁺] resin to pH 3 (pH meter control), then was

filtered, and concentrated. The residue was washed twice with

EtOH and was then dissolved in water (5 mL). The pH of the solu-

3 H, COOCH₃), 3.48 (m, 1 H, GalN 6-Hb), 3.42–3.31 (m, 2 H, tion was brought to 8 (pHmeter control) with 0.1 m NaOH. The

CH₂-N), 3.22 (m, 1 H, OH), 2.02, 1.98, 1.94 [3 s, 12 H,

OC(O)CH₃, NHC(O)CH₃] ppm. MS: m/z: 819 [M + H]⁺, 836 [M

+ NH₄]⁺. C₃₈H₄₆N₂O₁₈(818.78): calcd. C 55.74, H 5.66, N 3.42;

found C 55.74, H 5.76, N 3.37.

solution was eluted from a column (3ϫ80 cm) of Sephadex LH-20

with water and lyophilized to afford the sodium salt 9 (55 mg, 71%)

as a white powder. R_f= 0.35 (EtOAc/MeOH/water, 4:2:1). [α]²_D²

=

1

–22 (c = 1 in water). H NMR (400 MHz, D₂O, internal HOD, δ_H

= 4.79): δ = 7.45 (s, 5 H, arom. H), 5.13 (s, 2 H, CH₂-Ph), 4.79

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-

(dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 4.52 (d, J_1,2

=

acetyl-β-

D-glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benzoyl-

7.0 Hz, 1 H, GalN 1-H), 4.48 (d, J_1,2= 8.0 Hz, 1 H, GlcA 1-H),

4.10–4.01 (m, 2 H, GalN 2-H, 3-H), 3.93 (m, 1 H, CH₂-O), 3.85–

3.75 (m, 3 H, GalN 5-H, 6-Ha, 6-Hb), 3.68 (d, J_4,5= 9.5 Hz, 1 H,

GlcA 5-H), 3.71 (m, 1 H, CH₂-O), 3.55 (dd, J_3,4= 9.0 Hz, 1 H,

GlcA 4-H), 3.51 (dd, J_2,3= 9.0 Hz, 1 H, GlcA 3-H), 3.37 (dd, 1 H,

GlcA 2-H), 3.35–3.31 (m, 2 H, CH₂-N), 1.98 [s, 3 H, NHC(O)-

CH₃] ppm. HRMS: m/z: calcd. for C₂₄H₃₃N₂O₁₇S [M – 2Na +

H]^–: 653.1500; found: 653.1486.

2-deoxy-4-O-sulfonato-β-

D

-galactopyranoside (7): mixture of

A

alcohol 5 (106 mg, 0.12 mmol) and sulfur trioxide/trimethylamine

complex (80 mg, 0.57 mmol) in anhydrous DMF (1 mL) was stirred

overnight at 60 °C, then was cooled. Methanol (0.5 mL) was added

and the mixture was concentrated. Flash silica chromatography

(CH₂Cl₂/MeOH, 10:1), followed by ion exchange on a column

(1.5ϫ30 cm) of Sephadex SP C-25 [Na⁺] (CH₂Cl₂/MeOH/water,

9:5:1) afforded the 4-sulfate 7 (110 mg, 92%) as a white powder.

[α]²_D²= –2 (c = 1 in chloroform/methanol, 9:1). ¹H NMR (400 MHz,

CD₃OD/CDCl₃, 3:1): δ = 8.02–7.11 (m, 10 H, arom. H), 5.32 (dd,

J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA 3-H), 5.22 (dd, J_4,5= 10.0 Hz, 1 H,

GlcA 4-H), 5.11 (dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H), 5.05 (s, 2 H,

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

pyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-β-

-galactopyranoside (10): From ester 8 (100 mg, 0.11 mmol) as de-

scribed for the preparation of 9 to afford the sodium salt 10 (50 mg,

D-gluco-

D

CH₂-Ph), 4.93 (dd, J_4,5= 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 4.86 66%) as a white powder. R_f= 0.35 (EtOAc/MeOH/water, 4:2:1).

1

(d, 1 H, GlcA 1-H), 4.70 (dd, J_5,6a= 4.0, J_6a,6b= 11.0 Hz, 1 H,

GalN 6-Ha), 4.63 (dd, J_5,6b= 7.0 Hz, 1 H, GalN 6-Hb), 4.57 (d,

[α]²_D²= –13.5 (c = 1 in water). H NMR (400 MHz, D₂O, internal

HOD, δ_H= 4.79): δ = 7.46 (s, 5 H, arom. H), 5.14 (s, 2 H, CH₂-

J_1,2= 8.0 Hz, 1 H, GalN 1-H), 4.21 (d, 1 H, GlcA 5-H), 4.12 (dd, Ph), 4.51 (d, J_1,2= 8.0 Hz, 1 H, GlcA 1-H), 4.48 (d, J_1,2= 8.5 Hz,

J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.95 (m, 1 H, GalN 5-H), 3.89 (m, 1 H, GalN 1-H), 4.26–4.17 (m, 3 H, GalN 4-H, 6-Ha, 6-Hb), 4.02

1 H, GalN 2-H), 3.80 (m, 1 H, CH₂-O), 3.72 (s, 3 H, COOCH₃),

(dd, J_2,3= 11.0 Hz, 1 H, GalN 2-H), 3.93 (m, 1 H, CH₂-O), 3.8

3.63 (m, 1 H, CH₂-O), 3.30 (m, 2 H, CH₂-N), 2.07, 2.04, 2.01 [3 s, (m, 1 H, GalN 5-H), 3.83 (dd, 1 H, GalN 3-H), 3.72 (d, J_4,5

=

9 H, OC(O)CH₃], 1.96 [s, 3 H, NHC(O)CH₃]. HRMS: m/z: calcd.

9.5 Hz, 1 H, GlcA 5-H), 3.73 (m, 1 H, CH₂-O), 3.53–3.46 (m, 2 H,

for C₃₈H₄₇N₂O₂₁S [M – Na + 2H]⁺: 899.2392; found: 899.2381.

GlcA 3-H, 4-H), 3.38–3.31 (m, 3 H, GlcA 2-H, CH₂-N), 1.97 [s, 3

H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₂₄H₃₃N₂O₁₇S [M –

2Na + H]^–: 653.1500; found: 653.1501.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-

acetyl-β-

D-glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benzoyl-

2-deoxy-6-O-sulfonato-β-

D

-galactopyranoside (8): The product was

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-

β- -galactopyranoside (11): A solution of 9 (23 mg, 33 μmol) in

water (2 mL) was hydrogenolyzed in the presence of 10% Pd/C cat-

alyst (25 mg) for 20 h at room temp. The catalyst was removed by

filtration through a Celite pad and the filtrate was concentrated to

give the intermediate amine. A solution of the amine (18 mg,

32 μmol) and N-hydroxysuccinimidoyl 6-(biotinylamido)hexanoate

(58 mg, 0.13 mmol) in DMF (675 μL), NEt₃(75 μL), and water

D-

obtained by using the same procedure as that used for 7 from

alcohol 6 (117 mg, 0.14 mmol) and sulfur trioxide/trimethylamine

complex (44 mg, 0.32 mmol) in anhydrous DMF (1 mL) for 4 h at

60 °C. Flash silica chromatography (CH₂Cl₂/MeOH, 8:1), followed

by ion exchange, as described above, afforded the 6-sulfate 8

(107 mg, 82%) as a white powder. [α]²_D²= +17.5 (c = 1 in methanol).

¹H NMR (400 MHz, CD₃OD/CDCl₃, 3:1): δ = 8.02–7.08 (m, 10

H, arom. H), 5.67 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H),

D

4190

www.eurjoc.org

Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

(750 μL) was stirred for 1 h at room temp. then was concentrated.

The resulting solid was washed with absolute EtOH, and the resi-

due was eluted from a column (3ϫ80 cm) of Sephadex LH-20 with

water and lyophilized to afford the biotinylated disaccharide 11

(15 mg, 50%) as a white powder. R_f= 0.40 (EtOAc/MeOH/water,

197–198 °C (from EtOH); [α]²_D²= +18 (c = 1 in chloroform). ¹H

NMR (400 MHz, CDCl₃): δ = 7.89–7.34 (m, 17 H, arom. H), 6.81

(d, J_NH,2= 7.0 Hz, 1 H, GalN NH), 5.64 (dd, J_2,3= J_3,4= 9.0 Hz,

1 H, GlcA 3-H), 5.49–5.42 (m, 2 H, GlcA 2-H, 4-H), 5.09 (d, J_1,2

= 8.0 Hz, 1 H, GalN 1-H), 5.00 (d, J_1,2= 7.0 Hz, 1 H, GlcA 1-H),

4.88 (ABq, 2 H, CH₂-Ar), 4.61 (dd, J_3,4= 3.0, J_2,3= 11.5 Hz, 1 H,

GalN 3-H), 4.26 (m, 1 H, GalN 4-H), 4.28 (d, J_4,5= 9.5 Hz, 1 H,

1

4:2:1). [α]²_D²= +4 (c = 1 in water). H NMR (400 MHz, D₂O, in-

ternal HOD, δ_H= 4.79): δ = 4.81 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1

H, GalN 4-H), 4.63 (ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 GlcA 5-H), 4.03 (dd, J_5,6a= 6.5, J_6a,6b= 11.5 Hz, 1 H, GalN 6-

H, H-c), 4.55 (d, J_1,2= 8.0 Hz, 1 H, GalN 1-H), 4.50 (d, J_1,2Ha), 3.87 (dd, J_5,6a= 4.5 Hz, 1 H, GalN 6-Hb), 3.77 (s, 3 H, CO-

=

8.0 Hz, 1 H, GlcA 1-H), 4.44 (dd, J_b,e= 5.0 Hz, 1 H, H-b), 4.09– OCH₃), 3.75–3.63 (m, 2 H, GalN 2-H, 5-H, 6-OH), 3.21 (m, 1 H,

4.06 (m, 2 H, GalN 2-H, 3-H), 3.95 (m, 1 H, CH₂-r), 3.85–3.78 (m, 4-OH), 2.62–2.37 (m, 4 H, CH₂CO), 2.05 (s, 3 H, COCH₃) ppm.

3 H, GalN 5-H, 6-Ha, 6-Hb), 3.75 (m, 1 H, CH₂-r), 3.69 (d, J_4,5

=

MS: m/z: 982 [M + Na]⁺(for ³⁵Cl). C₅₂H₄₈Cl₃NO₁₇(1065.31):

9.5 Hz, 1 H, GlcA 5-H), 3.55 (dd, J_3,4= 9.0 Hz, 1 H, GlcA 4-H),

3.48 (d, J_2,3= 9.0 Hz, 1 H, 3-H GlcA), 3.42–3.33 (m, 4 H, GlcA

2-H, H-e, CH₂-q), 3.20 (dd, J_k,kЈ= J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.02

(dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.81 (dd, 1 H, H-d), 2.29–2.23 (m,

4 H, CH₂-i, CH₂-o), 2.04 [s, 3 H, NHC(O)CH₃], 1.80–1.30 (m, 12

H, CH₂-f, CH₂-g, CH₂-h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR

(100MHz, D₂O, internal acetone, δ_C= 30.83): δ = 177.59–175.26

(4 C, GlcA C-6, C=O), 165.99 (1 C, C-a), 103.83 (1 C, GlcA C-1),

101.79 (1 C, GalN C-1), 77.03 (1 C, GalN C-4), 76.99 (1 C, GlcA

C-5), 75.74 (1 C, GlcA C-3), 75.31 (1 C, GalN C-3), 75.10 (1 C,

GalN C-5), 73.12 (1 C, GlcA C-2), 72.42 (1 C, GlcA C-4), 68.87

(1 C, C-r), 62.71 (1 C, C-b), 61.66 (1 C, GalN C-6), 60.87 (1 C, C-

c), 56.01 (1 C, C-e), 52.40 (1 C, GalN C-2), 40.32 (1 C, C-d), 39.97,

39.70 (2 C, C-k, C-q), 36.26, 36.14 (2 C, C-i, C-o), 28.62, 28.45,

28.30, 26.18, 25.83, 25.61 (6 C, C-f, C-g, C-h, C-l, C-m, C-n), 22.92

[1 C, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₃₂H₅₂N₅O₁₇S₂

[M – 2Na + H]^–: 858.2749; found: 858.2752.

calcd. C 56.23, H 4.61, N 1.46; found C 56.20, H 4.42, N 1.29.

2-Naphthylmethyl O-(Methyl-2,3-di-O-benzoyl-4-O-levulinoyl-β-

glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl- (15) and 4-O-benzoyl-

2-deoxy-2-trichloroacetamido-β- -galactopyranoside (16): From

D-

D

diol 14 (863 mg, 0.90 mmol) as described for the preparation of 3

and 4. Flash silica chromatography (EtOAc/petroleum ether,

1:1Ǟ3:2) gave first the crystalline 6-benzoate 15 (333 mg, 35%).

Next eluted (EtOAc/petroleum ether, 3:1) was the amorphous 4-

benzoate 16 (357mg, 37%).

Data for 15: M.p. 183 °C (from EtOAc/petroleum ether). [α]²_D²

=

+15 (c = 1 in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.11–

7.22 (m, 22 H, arom. H), 6.80 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH),

5.69 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H), 5.52–5.45 (m, 2 H,

GlcA 2-H, 4-H), 5.03 (d, J_1,2= 8.5 Hz, 1 H, GalN 1-H), 5.02 (d,

J_1,2= 7.5 Hz, 1 H, GlcA 1-H), 4.86 (ABq, 2 H, CH₂-Ar), 4.67–4.60

(m, 2 H, GalN 6a-H, 6b-H), 4.62 (dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1

H, GalN 3-H), 4.32 (ddd, J_4,5Ͻ 1.0, J_4,OH= 2.0 Hz, 1 H, GalN

4-H), 4.28 (d, J_4,5= 9.5 Hz, 1 H, GlcA 5-H), 3.95–3.90 (m, 1 H,

GalN 5-H), 3.72 (s, 3 H, COOCH₃), 3.70–3.59 (m, 1 H, GalN 2-

H), 3.23 (d, 1 H, OH), 2.65–2.32 (m, 4 H, CH₂CO), 2.01 (s, 3 H,

COCH₃) ppm. MS: m/z: 1086 [M + Na]⁺(for ³⁵Cl). C₅₂H₄₈Cl₃NO₁₇

(1065.31): calcd. C 58.63, H 4.54, N 1.31; found C 58.73, H 4.44,

N 1.33.

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-

β- -galactopyranoside (12): From 10 (22 mg, 32 μmol) as described

D-

D

for the preparation of 11 to afford the biotinylated disaccharide 12

(20 mg, 68%) as a white powder. R_f= 0.40 (EtOAc/MeOH/water,

4:2:1). [α]²_D²= +10 (c = 1 in water). ¹H NMR (400 MHz, D₂O,

internal HOD, δ_H= 4.79): δ = 4.63 (ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c

= 8.0 Hz, 1 H, H-c), 4.55 (d, J_1,2= 8.0 Hz, 1 H, GalN 1-H), 4.53

(d, J_1,2= 8.0 Hz, 1 H, GlcA 1-H), 4.44 (dd, J_b,e= 5.0 Hz, 1 H, H-

b), 4.22 (d, J_5,6a= 4.5, J_6a,6b= 11.0 Hz, 1 H, GalN 6-Ha), 4.30 (m,

1 H, GalN 4-H), 4.26 (dd, J_5,6b= 7.5 Hz, 1 H, GalN 6-Hb), 4.04

(dd, J_2,3= 11.0 Hz, 1 H, GalN 2-H), 3.96–3.89 (m, 2 H, GalN 5-

H, CH₂-r), 3.87 (dd, 1 H, GalN 3-H), 3.78 (m, 1 H, CH₂-r), 3.71

(d, J_4,5= 9.5 Hz, 1 H, GlcA 5-H), 3.53–3.47 (m, 2 H, GlcA 3-H,

Data for 16: [α]²_D²= +29 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.09–7.22 (m, 22 H, arom. H), 6.89 (d, J_2,NH= 7.0 Hz,

1 H, GalN NH), 5.69 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-

H), 5.58 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA 3-H), 5.36 (d, J_4,5

=

10.0 Hz, 1 H, GlcA 4-H), 5.32 (dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H),

4.14 (d, J_1,2= 8.0 Hz, 1 H, GalN 1-H), 4.96 (d, 1 H, GlcA 1-H),

4.85 (ABq, 2 H, CH₂-Ar), 4.85(dd, J_3,4= 3.5, J_2,3= 11.0 Hz, 1 H,

GalN 3-H), 4.17 (d, 1 H, GlcA 5-H), 3.88–3.69 (m, 3 H, GalN 2-

H, 5-H, 6a-H), 3.74 (s, 3 H, COOCH₃), 3.55 (dd, J_5,6b= 8.5, J_6a,6b

= 11.5 Hz, 1 H, GalN 6b-H), 3.41 (m, 1 H, OH), 2.61–2.12 (m, 4

H, CH₂CO), 1.99 (s, 3 H, COCH₃) ppm. MS: m/z: 1086 [M +

Na]⁺(for ³⁵Cl). C₅₂H₄₈Cl₃NO₁₇(1065.31): calcd. C 58.63, H 4.54,

N 1.31; found C 58.74, H 4.47, N 1.24.

4-H), 3.40–3.31 (m, 4 H, GlcA 2-H, H-e, CH₂-q), 3.20 (dd, J_k,kЈ

=

J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.01 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ),

2.80 (dd, 1 H, H-d), 2.30–2.22 (m, 4 H, CH₂-i, CH₂-o), 2.03 [s, 3

H, NHC(O)CH₃], 1.80–1.30 (m, 12 H, CH₂-f, CH₂-g, CH₂-h, CH₂-

l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal ace-

tone, δ_C= 30.83): δ = 177.61–175.35 (4 C, GlcA C-6, C=O), 165.99

(1 C, C-a), 104.70 (1 C, GlcA C-1), 101.94 (1 C, GalN C-1), 80.56

(1 C, GalN C-3), 76.78 (1 C, GlcA C-5), 75.99, 72.44 (2 C, GlcA

C-3, C-4), 73.35, 73.32 (2 C, GalN C-5, GlcA C-2), 69.03 (1 C, C-

r), 68.40 (1 C, GalN C-6), 68.19 (1 C, GalN C-4), 62.74 (1 C, C-

b), 60.91 (1 C, C-c), 56.03 (1 C, C-e), 51.73 (1 C, GalN C-2), 40.36

(1 C, C-d), 39.98, 39.74 (2 C, C-k, C-q), 36.29, 36.20 (2 C, C-i, C-

o), 28.65, 28.50, 28.34, 26.21, 25.89, 25.64 (6 C, C-f, C-g, C-h, C-

l, C-m, C-n), 22.94 [1 C, NHC(O)CH₃] ppm. HRMS: m/z: calcd.

for C₃₂H₅₂N₅O₁₇S₂[M – 2Na + H]^–: 858.2749; found: 858.2737.

2-Naphthylmethyl O-(Methyl-2,3-di-O-benzoyl-4-O-levulinoyl-β-

glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-4-O-chloroacetyl-2-

deoxy-2-trichloroacetamido-β- -galactopyranoside (17): Freshly

D-

D

recrystallized chloroacetic anhydride (150 mg, 0.88 mmol) was

added at 0 °C to a solution of alcohol 15 (467 mg, 0.44 mmol) in

anhydrous CH₂Cl₂(5 mL) and pyridine (1.25 mL), and the mixture

was stirred for 30 min at 0 °C. Crushed ice was then added and the

mixture was diluted with CH₂Cl₂; washed with water, a saturated

aqueous solution of NaHCO₃and water; dried (MgSO₄); and con-

centrated. Flash silica chromatography (CH₂Cl₂/acetone, 19:1) af-

forded the crystalline 4-chloroacetate 17 (448 mg, 90%); m.p. 186–

187 °C (from EtOAc/petroleum ether); [α]²_D²= +12 (c = 1 in chloro-

form). ¹H NMR (400 MHz, CDCl₃): δ = 8.12–7.34 (m, 22 H, arom.

H), 6.73 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.71 (dd, J_4,5Ͻ 1.0,

2-Naphthylmethyl O-(Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-deoxy-2-trichloroacetamido-β-

D

-

D

galactopyranoside (14): From disaccharide 13^[8](1.67 g, 1.59 mmol)

as described for the preparation of 2. Recrystallization of the resi-

due from hot EtOH afforded crystalline diol 14 (1.29 g, 84%); m.p.

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4191

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

J_3,4= 3.5 Hz, 1 H, GalN 4-H), 5.59 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H,

GlcA 3-H), 5.46 (dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 5.33 (dd, J_1,2

19. Flash silica chromatography [CH₂Cl₂/acetone (19:1) containing

0.1% of NEt₃] afforded the α-imidate 20 (1.40 g, 65%) as a white

= 7.5 Hz, 1 H, GlcA 2-H), 5.06 (d, J_1,2= 8.5 Hz, 1 H, GalN 1-H), powder; m.p. 135–137 °C (from diethyl ether). [α]²_D²= +111 (c = 1

4.85 (d, 1 H, GlcA 1-H), 4.84 (ABq, 2 H, CH₂-Ar), 4.82 (dd, J_2,3

= 11.0 Hz, 1 H, GalN 3-H), 4.57 (dd, J_5,6a= 6.5, J_6a,6b= 11.5 Hz, C=NH), 8.02–7.34 (m, 15 H, arom. H), 6.95 (d, J_2,NH= 7.5 Hz, 1

in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.75 (s, 1 H,

1 H, GalN 6a-H), 4.38 (dd, J_5,6b= 6.5 Hz, 1 H, GalN 6b-H), 4.26

(ABq, 2 H, CH₂Cl), 4.13 (d, 1 H, GlcA 5-H), 4.09 (m, 1 H, GalN

5-H), 3.69 (s, 3 H, COOCH₃), 3.58 (m, 1 H, GalN 2-H), 2.60–2.35

(m, 4 H, CH₂CO), 2.02 (s, 3 H, COCH₃) ppm. MS: m/z: 1162 [M

+ Na]⁺(for ³⁵Cl). C₅₄H₄₉Cl₄NO₁₈(1141.79): calcd. C 56.81, H

4.33, N 1.23; found C 56.87, H 4.21, N 1.29.

H, GalN NH), 6.69 (d, J_1,2= 3.5 Hz, 1 H, GalN 1-H), 5.86 (dd,

J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 5.63 (dd, J_2,3= J_3,4

=

9.5 Hz, 1 H, GlcA 3-H), 5.44 (dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H),

5.38 (dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 5.15 (d, 1 H, GlcA 1-H),

4.77 (ddd, J_2,3= 11.0 Hz, 1 H, GalN 2-H), 4.53 (dd, 1 H, GalN 3-

H), 4.51 (m, 1 H, GalN 5-H), 4.35 (dd, J_5,6a= 5.5, J_6a,6b= 11.5 Hz,

1 H, GalN 6a-H), 4.24 (d, 1 H, GlcA 5-H), 4.16 (dd, J_5,6b= 6.0 Hz,

1 H, GalN 6b-H), 4.02 (s, 2 H, CH₂Cl), 3.68 (s, 3 H, COOCH₃),

2.69–2.31 (m, 4 H, CH₂CO), 2.03 (s, 3 H, COCH₃) ppm. MS: m/z:

1021 [M – Cl₃CC(NH) + Na]⁺(for ³⁵Cl). C₄₅H₄₁Cl₇N₂O₁₈

(1145.99): calcd. C 47.16, H 3.61, N 2.44; found C 47.30, H 3.68,

N 2.36.

2-Naphthylmethyl O-(Methyl-2,3-di-O-benzoyl-4-O-levulinoyl-β-

glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-6-O-chloroacetyl-2-

deoxy-2-trichloroacetamido-β- -galactopyranoside (18): From

D-

D

alcohol 16 (200 mg, 0.19 mmol) as described for the preparation

of 17. Flash silica chromatography (CH₂Cl₂/acetone, 19:1Ǟ17:1)

afforded the amorphous 6-chloroacetate 18 (187 mg, 87%). [α]²_D²

=

+39 (c = 1 in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.06–

7.22 (m, 22 H, arom. H), 6.97 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH),

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

levulinoyl-β-D-glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-4-O-chlo-

roacetyl-2-deoxy-2-trichloroacetamido-β-

D

-galactopyranoside (21):

5.76 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 5.57 (dd, J_2,3

=

A mixture of imidate 19 (160 mg, 0.14 mmol), 2-benzyloxycarbony-

laminoethanol (49 mg, 0.25 mmol), and 4 Å powdered molecular

sieves (0.1 g) in anhydrous CH₂Cl₂(1.6 mL) was stirred for 30 min

at room temp. under dry argon. A solution of TMSOTf in toluene

(1 m, 41 μL) was added and the mixture was stirred for 30 min,

then was quenched with NEt₃(9 μL, 0.12 mmol), filtered, and con-

centrated. Flash silica chromatography [CH₂Cl₂/acetone

(12:1Ǟ10:1) containing 0.1% of NEt₃] afforded the glycoside 21

(133 mg, 80%) as a white powder; m.p. 122–123 °C (from EtOAc/

petroleum ether). [α]²_D²= +37 (c = 1 in chloroform). ¹H NMR

(400 MHz, CDCl₃): δ = 8.05–7.28 (m, 20 H, arom. H), 6.82, (d,

J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.70 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz,

1 H, GalN 4-H), 5.62 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA 3-H),

5.48 (dd, J_4,5= 10.0 Hz, 1 H, GlcA 4-H), 5.34 (dd, J_1,2= 8.0 Hz,

1 H, GlcA 2-H), 5.10 [m, 1 H, NHC(O)O], 5.03 (s, 2 H, CH₂-Ph),

4.98 (d, J_1,2= 8.5 Hz, 1 H, GalN 1-H), 4.90 (d, 1 H, GlcA 1-H),

4.78 (dd, J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.46 (dd, J_5,6a= 7.0,

J_6a,6b= 11.5 Hz, 1 H, GalN 6a-H), 4.34 (dd, J_5,6b= 6.0 Hz, 1 H,

GalN 6b-H), 4.24 (ABq, 2 H, CH₂Cl), 4.16 (d, 1 H, GlcA 5-H),

4.05 (m, 1 H, GalN 5-H), 3.82 (m, 1 H, CH₂-O), 3.71 (s, 3 H,

COOCH₃), 3.64 (m, 1 H, GalN 2-H), 3.50 (m, 1 H, CH₂-O), 3.41–

3.25 (m, 2 H, CH₂-N), 2.65–2.62 (m, 4 H, CH₂CO), 2.04 (s, 3 H,

COCH₃) p pm . M S: m/ z: 11 94 [M + N H₄]⁺(for ^{3 5}Cl).

C₅₃H₅₂Cl₄N₂O₂₀(1178.81): calcd. C 54.00, H 4.45, N 2.38; found

C 53.94, H 4.45, N 2.38.

J_3,4= 9.5 Hz, 1 H, GlcA 3-H), 5.41 (dd, J_4,5= 9.5 Hz, 1 H, GlcA

4-H), 5.32 (dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H), 5.11 (d, J_1,2= 8.0 Hz,

1 H, GalN 1-H), 4.96 (d, 1 H, GlcA 1-H), 4.84 (ABq, 2 H, CH₂-

Ar), 4.74 (dd, J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.39 (dd, J_5,6a= 5.0,

J_6a,6b= 11.5 Hz, 1 H, GalN 6a-H), 4.38 (dd, J_5,6b= 7.0 Hz, 1 H,

GalN 6b-H), 4.17 (d, 1 H, GlcA 5-H), 4.06 (s, 2 H, CH₂Cl), 4.02

(m, 1 H, GalN 5-H), 3.86 (ddd, 1 H, GalN 2-H), 3.74 (s, 3 H,

COOCH₃), 2.60–2.31 (m, 4 H, CH₂CO), 1.97 (s, 3 H, COCH₃)

ppm. MS: m/z: 1162 [M + Na]⁺(for ³⁵Cl). C₅₄H₄₉Cl₄NO₁₈

(1141.79): calcd. C 56.81, H 4.33, N 1.23; found C 56.72, H 4.43,

N 1.25.

O-(Methyl-2,3-di-O-benzoyl-4-O-levulinoyl-β-

ate)-(1Ǟ3)-6-O-benzoyl-4-O-chloroacetyl-2-deoxy-2-trichloro-

acetamido-1-O-trichloroacetimidoyl-α- -galactopyranose (19): A

D-glucopyranosyluron-

D

mixture of glycoside 17 (201 mg, 0.18 mmol) and DDQ (119 mg,

0.53 mmol) in CH₂Cl₂/MeOH (9:1; 2 mL) was stirred for 24 h at

room temp. then was diluted with CH₂Cl₂, washed twice with a

saturated aqueous solution of NaHCO₃and water, dried (MgSO₄),

and concentrated. Flash silica chromatography (CH₂Cl₂/acetone,

19:1Ǟ9:1) afforded the corresponding free hemiacetal as a pale yel-

low powder. A mixture of the hemiacetal (146 mg, 0.15 mmol), tri-

chloroacetonitrile (115 μL, 1.46 mmol), and DBU (30 μL, 20 μmol)

in CH₂Cl₂(1.5 mL) was stirred for 30 min at room temp. then was

concentrated. Flash silica chromatography [CH₂Cl₂/acetone (19:1)

containing 0.1% of NEt₃] afforded the α-imidate 19 (110 mg, 66%)

as a white powder. [α]²_D²= +78 (c = 1 in chloroform). ¹H NMR

(400 MHz, CDCl₃): δ = 8.79 (s, 1 H, C=NH), 7.92–7.31 (m, 15 H,

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

levulinoyl-β-D-glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-6-O-chlo-

roacetyl-2-deoxy-2-trichloroacetamido-β-

D

-galactopyranoside (22):

arom. H), 6.79 (d, J_2,NH= 8.0 Hz, 1 H, GalN NH), 6.56 (d, J_1,2

=

From imidate 20 (722 mg, 0.63 mmol) as described for the prepara-

tion of 21. Flash silica chromatography [CH₂Cl₂/acetone (8:1Ǟ7:1)

containing 0.1% of NEt₃] afforded the glycoside 22 (523 mg, 70%)

3.5 Hz, 1 H, GalN 1-H), 5.82 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H,

GalN 4-H), 5.68 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA 3-H), 5.44

(dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 5.42 (dd, J_1,2= 7.5 Hz, 1 H,

GlcA 2-H), 5.12 (d, 1 H, GlcA 1-H), 4.66 (ddd, J_2,3= 11.0 Hz, 1

H, GalN 2-H), 4.53 (m, 1 H, GalN 5-H), 4.43 (dd, 1 H, GalN 3-

H), 4.35–4.37 (m, 2 H, GalN 6a-H, 6b-H) 4.25 (d, 1 H, GlcA 5-

H), 4.26 (ABq, 2 H, CH₂Cl), 3.76 (s, 3 H, COOCH₃), 2.68–2.33

(m, 4 H, CH₂CO), 2.03 (s, 3 H, COCH₃) ppm. MS: m/z: 1021 [M –

Cl₃CC(NH) + Na]⁺(for ³⁵Cl). C₄₅H₄₁Cl₇N₂O₁₈(1145.99): calcd. C

47.16, H 3.61, N 2.44; found C 47.30, H 3.74, N 2.32.

1

as a white powder. [α]²_D²= +54.5 (c = 1 in chloroform). H NMR

(400 MHz, CDCl₃): δ = 7.98–7.18 (m, 20 H, arom. H), 6.91, (d,

J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.68 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz,

1 H, GalN 4-H), 5.54 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA 3-H),

5.35 (dd, J_4,5= 10.0 Hz, 1 H, GlcA 4-H), 5.25 (dd, J_1,2= 7.5 Hz,

1 H, GlcA 2-H), 5.13 [m, 1 H, NHC(O)O], 4.98 (s, 2 H, CH₂-Ph),

4.96 (d, J_1,2= 8.5 Hz, 1 H, GalN 1-H), 4.94 (d, 1 H, GlcA 1-H),

4.74 (dd, J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.32 (dd, J_5,6a= 5.0,

J_6a,6b= 11.5 Hz, 1 H, GalN 6a-H), 4.17 (d, 1 H, GlcA 5-H), 4.13

(dd, J_5,6b= 6.0 Hz, 1 H, GalN 6b-H), 3.98 (s, 2 H, CH₂Cl), 3.96

O-(Methyl-2,3-di-O-benzoyl-4-O-levulinoyl-β-

ate)-(1Ǟ3)-4-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-trichloro-

acetamido-1-O-trichloroacetimidoyl-α-

glycoside 18 (2.3 g, 2.23 mmol) as described for the preparation of

D-glucopyranosyluron-

D

-galactopyranose (20): From (m, 1 H, GalN 5-H), 3.80 (m, 1 H, CH₂-O), 3.72 (s, 3 H, CO-

OCH₃), 3.72 (m, 1 H, GalN 2-H), 3.63 (m, 1 H, CH₂-O), 3.37–3.25

4192

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Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

(m, 2 H, CH₂-N), 2.60–2.23 (m, 4 H, CH₂CO), 1.96 (s, 3 H,

COCH₃) p pm . M S: m/z: 11 94 [M + N H₄]⁺(for ^{3 5}Cl).

C₅₃H₅₂Cl₄N₂O₂₀(1178.81): calcd. C 54.00, H 4.45, N 2.38; found

C 53.75, H 4.53, N 2.48.

from hot EtOAc afforded the acetal 26 (3.44 g, 83%); m.p. 187–

188 °C. [α]²_D²= –20 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 7.01–6.78 (m, 4 H, arom. H), 6.89 (d, J_NH,2= 7.5 Hz,

1 H, NH), 5.16 (d, J_1,2= 8.5 Hz, 1 H, 1-H), 4.23 (dd, J_4,5Ͻ 1.0,

J_3,4= 4.0 Hz, 1 H, 4-H), 4.18 (ddd, J_3,OH= 10.5, J_2,3= 11.0 Hz, 1

H, 3-H), 4.11 (dd, J_6a,5= 3.0, J_6a,6b= 13.0 Hz, 1 H, 6a-H), 4.04–

3.98 (m, 2 H, 2-H, 6b-H), 3.79 (s, 3 H, OCH₃), 3.51–3.50 (m, 1 H,

5-H), 3.62 (d, 1 H, OH), 1.50 [s, 6 H, C(CH₃)₂] ppm. MS: m/z: 487

[M + NH₄]⁺(for ³⁵Cl). C₁₈H₂₂Cl₃NO₇(470.73): calcd. C 45.93, H

4.71, N 2.98; found C 46.05, H 4.62, N 3.02.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-β-

glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-4-O-chloroacetyl-2-

deoxy-2-trichloroacetamido-β- -galactopyranoside (23): A freshly

D-

D

prepared mixture of pyridine/AcOH/hydrazine hydrate (6:4:0.5;

2.6 mL) was added to a solution of ester 21 (150 mg, 0.13 mmol)

in pyridine (1.5 mL) and the mixture was stirred for 2 min at room

temp. then was diluted with CH₂Cl₂; washed with water, a saturated

aqueous solution of NaHCO₃, and water; dried (MgSO₄); and con-

centrated. Flash silica chromatography (CH₂Cl₂/acetone, 6:1) af-

forded the alcohol 23 (107 mg, 78%) as a white powder; m.p. 123–

124 °C (from diethyl ether/petroleum ether); [α]²_D²= +46 (c = 1 in

chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.05–7.23 (m, 20

H, arom. H), 6.82 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.71 (dd,

4-Methoxyphenyl 3-O-Benzoyl-2-deoxy-4,6-O-isopropylidene-2-tri-

chloroacetamido-β-D-galactopyranoside (27): Benzoyl chloride

(1.7 mL, 0.015 mmol) was added at 0 °C to a solution of alcohol

26 (3.49 g, 7.71 mmol) in CH₂Cl₂(46 mL) and pyridine (9 mL) and

the mixture was stirred for 30 min at 0 °C. Methanol (2.3 mL) was

added and the solution was diluted with CH₂Cl₂; washed with

water, a saturated aqueous solution of NaHCO₃, and water; dried

(MgSO₄); and concentrated to give the ester 27 (4.21g, 99%) as a

white solid; m.p. 203–204 °C (from EtOAc/petroleum); [α]²_D²= +53

J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN 4-H), 5.49 (dd, J_2,3= J_3,4

=

9.5 Hz, 1 H, GlcA 3-H), 5.29 (dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H),

1

(c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ = 8.04–6.78

5.09 [m, 1 H, NHC(O)O], 5.02 (s, 2 H, CH₂-Ph), 4.99 (d, J_1,2

8.5 Hz, 1 H, GalN 1-H), 4.88 (d, 1 H, GlcA 1-H), 4.75 (dd, J_2,3

=

(m, 9 H, arom. H), 6.83 (d, J_NH,2= 8.0 Hz, 1 H, NH), 5.52 (dd,

J_3,4= 3.5, J_2,3= 11.0 Hz, 1 H, 3-H), 5.22 (d, J_1,2= 8.5 Hz, 1 H, 1-

H), 4.60 (ddd, 1 H, 2-H), 4.50 (dd, J_4,5Ͻ 1.0 Hz, 1 H, 4-H), 4.14

11.0 Hz, 1 H, GalN 3-H), 4.45 (dd, J_5,6a= 7.0, J_6a,6b= 11.5 Hz, 1

H, GalN 6a-H), 4.36 (dd, J_5,6b= 6.0 Hz, 1 H, GalN 6b-H), 4.28–

4.21 (ABq, 2 H, CH₂Cl), 5.48 (dd, J_4,OH= 3.0, J_4,5= 10.0 Hz, 1

H, GlcA 4-H), 4.07 (m, 1 H, GalN 5-H), 4.04 (d, 1 H, GlcA 5-H),

3.85 (m, 1 H, CH₂-O), 3.71 (s, 3 H, COOCH₃), 3.61 (m, 1 H, CH₂-

O), 3.50 (m, 1 H, GalN 2-H), 3.39 (d, 1 H, 4-OH), 3.39–3.23 (m,

2 H, CH₂-N) ppm. MS: m/z: 1096 [M + NH₄]⁺(for ³⁵Cl).

C₄₈H₄₆Cl₄N₂O₁₈(1080.71): calcd. C 53.35, H 4.29, N 2.59; found

C 53.30, H 4.25, N 2.61.

(dd, J_5,6a= 2.0, J_6a,6b= 12.5 Hz, 1 H, 6a-H), 4.05 (dd, J_5,6b

=

1.5 Hz, 1 H, 6b-H), 3.76 (s, 3 H, OCH₃), 3.61 (m, 1 H, 5-H), 1.51,

1.43 [2s, 6 H ppm, C(CH₃)₂] ppm. MS: m/z: 591 [M + NH₄]⁺(for

³⁵Cl). C₂₅H₂₆Cl₃NO₈(574.84): calcd. C 52.24, H 4.56, N 2.44;

found C 52.54, H 4.66, N 2.41.

4-Methoxyphenyl 3-O-Benzoyl-2-deoxy-2-trichloroacetamido-β-D-

galactopyranoside (28): A solution of acetal 27 (4.21 g, 7.32 mmol)

in 80% AcOH (30 mL) was stirred for 40 min at 80 °C, then was

cooled. Water was added and the solution was concentrated.

Recrystallization of the residue from hot 2-propanol afforded the

diol 28 (3.34g, 85%); m.p. 193–194 °C. [α]²_D²= +73 (c = 1 in meth-

anol). ¹H NMR (400 MHz, CD₃OD): δ = 8.05–6.83 (m, 9 H, arom.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-β-

glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-6-O-chloroacetyl-2-

deoxy-2-trichloroacetamido-β- -galactopyranoside (24): From ester

D-

D

22 (199 mg, 0.17 mmol) as described for the preparation of 23.

Flash silica chromatography (CH₂Cl₂/acetone, 8:1) afforded the

alcohol 24 (140 mg, 77 %) as a white powder; m.p. 158–159 °C

(from EtOAc/petroleum ether); [α]²_D²= +46 (c = 1 in chloroform).

¹H NMR (400 MHz, CDCl₃): δ = 8.04–7.24 (m, 20 H, arom. H),

6.93 (d, J_2,NH= 7.0 Hz, 1 H, GalN NH), 5.75 (dd, J_4,5Ͻ 1.0, J_3,4

= 3.0 Hz, 1 H, GalN 4-H), 5.44 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA

H), 5.33 (dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1 H, 3-H), 5.20 (d, J_1,2

=

8.5 Hz, 1 H, 1-H), 4.70 (dd, 1 H, 2-H), 4.28 (dd, J_4,5Ͻ 1.0 Hz, 1

H, 4-H), 3.87–3.80 (m, 3 H, 5-H, 6a-H, 6b-H), 3.74 (s, 3 H, OCH₃)

ppm. MS: m/z: 551 [M + NH₄]⁺(for ³⁵Cl). C₂₂H₂₂Cl₃NO₈(534.78):

calcd. C 49.41, H 4.15, N 2.62; found C 49.48, H 4.07, N 2.61.

4-Methoxyphenyl 3,6-Di-O-benzoyl- (29) and 3,4-Di-O-benzoyl-2-

3-H), 5.35 (dd, J_4,5= 10.0 Hz, 1 H, GlcA 4-H), 5.26 (dd, J_1,2

=

deoxy-2-trichloroacetamido-β-D-galactopyranoside (30): Procedure

7.5 Hz, 1 H, GlcA 2-H), 5.16 [m, 1 H, NHC(O)O], 5.05 (s, 2 H,

CH₂-Ph), 4.96 (d, J_1,2= 8.5 Hz, 1 H, GalN 1-H), 4.95 (d, 1 H,

GlcA 1-H), 4.78 (dd, J_2,3= 11.0 Hz, 1 H, GalN 3-H), 4.39 (dd,

A (with neutralization followed by hydrolysis): Diol 28 (500 mg,

0.94 mmol) was treated as described for the preparation of 3 and

4. Flash silica chromatography (CH₂Cl₂/acetone, 19:1) afforded

first the 3,6-dibenzoate 29 (440 mg, 74%). Next eluted (CH₂Cl₂/

acetone, 12:1) was the 3,4-dibenzoate 30 (158 mg, 25%).

J_5,6a= 5.0, J_6a,6b= 11.5 Hz, 1 H, GalN 6a-H), 4.19 (dd, J_5,6b

=

6.0 Hz, 1 H, GalN 6b-H), 4.11 (d, 1 H, GlcA 5-H), 4.02 (s, 2 H,

CH₂Cl), 4.02 (m, 1 H, GalN 5-H), 3.84 (m, 1 H, CH₂-O), 3.85 (s,

3 H, COOCH₃), 3.73 (m, 1 H, GalN 2-H), 3.66 (m, 1 H, CH₂-O),

3.45–3.27 (m, 3 H, CH₂-N, 4-OH) ppm. MS: m/z: 1096 [M +

NH₄]⁺(for ³⁵Cl). C₄₈H₄₆Cl₄N₂O₁₈(1080.71): calcd. C 53.35, H

4.29, N 2.59; found C 53.25, H 4.17, N 2.65.

Procedure B (without neutralization): Diol 28 (500 mg, 0.94 mmol)

was treated as described for the preparation of 3 and 4, but the

reaction was not quenched with NEt₃. A mixture of MeOH/water

(1:1, 2 mL) was directly added, and the mixture was stirred for

15 min at room temp. then was concentrated. Flash silica

chromatography (CH₂Cl₂/acetone, 19:1Ǟ12:1) afforded, respec-

tively, the 3,6-dibenzoate 29 (156 mg, 25%) and the 3,4-dibenzoate

30 (408 mg, 68%).

4-Methoxyphenyl 2-Deoxy-4,6-O-isopropylidene-2-trichloroacet-

amido-β-D-galactopyranoside (26): Aliquots of 2-methoxypropene

(0.42 mL, 4.4 mmol, 6 additions) were added every 20 min to a

solution of 25^[16](3.80 g, 8.82 mmol) and CSA (60 mg) in DMF

(40 mL). Triethylamine (0.11 mL) was added and the solution was Data for 29: M.p. 243–244 °C (from EtOAc/petroleum ether).

1

concentrated. The residue was redissolved in CH₂Cl₂; washed with

a saturated aqueous solution of NaHCO₃, a saturated aqueous

solution of NaCl, and water; dried (MgSO₄); and concentrated.

The solid residue was then redissolved in CH₂Cl₂/MeOH (2:1,

50 mL) and stirred with Amberlite IR-120 [H⁺] resin (≈ 1 mL) for

2 min at room temp., filtered, and concentrated. Recrystallization

[α]²_D²= +34 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

= 8.42–6.73 (m, 15 H, arom. H, NH), 5.49 (dd, J_3,4= 3.0, J_2,3

=

11.0 Hz, 1 H, 3-H), 5.41 (d, J_1,2= 8.5 Hz, 1 H, 1-H), 5.03 (d, J_OH,4

= 5.5 Hz, 1 H, HO-4), 4.86 (ddd, 1 H, 2-H), 4.70 (dd, J_5,6a= 8.0,

J_6a,6b= 11.0 Hz, 1 H, 6a-H), 4.61 (dd, J_5,6b= 4.5 Hz, 1 H, 6b-H),

4.51 (ddd, J_4,5Ͻ 1.0 Hz, 1 H, 4-H), 4.32 (m, 1 H, 5-H), 3.71 (s, 3 H,

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4193

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

OCH₃) ppm. MS: m/z: 655 [M + NH₄]⁺(for ³⁵Cl). C₂₉H₂₆Cl₃NO₉3,4-Di-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-trichloroacetamido-1-

FULL PAPER

(638.88): calcd. C 54.52, H 4.10, N 2.19; found C 54.23, H 4.02, N

2.21.

O-trichloroacetimidoyl-α-D-galactopyranose (34): From glycoside 32

(518 mg, 0.81 mmol) as described for the preparation of 33. Flash

silica chromatography [CH₂Cl₂/acetone (29:1) containing 0.1% of

NEt₃] afforded the α-imidate 34 (339 mg, 60% from 32) as a white

Data for 30: M.p. 196–197 °C (from EtOAc/petroleum ether).

1

[α]²_D²= +114 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃):

1

powder. [α]²_D²= +123 (c = 1 in chloroform). H NMR (400 MHz,

δ = 8.14–6.80 (m, 15 H, arom. H, NH), 5.83 (dd, J_4,5Ͻ 1.0, J_3,4

=

CDCl₃): δ = 8.91 (s, 1 H, C=NH), 8.13–7.32 (m, 10 H, arom. H),

7.14 (d, J_NH,2= 9.0 Hz, 1 H, NH), 6.71 (d, J_1,2= 3.5 Hz, 1 H, 1-

3.0 Hz, 1 H, 4-H), 5.77 (dd, J_2,3= 11.0 Hz, 1 H, 3-H), 5.31 (d, J_1,2

= 8.5 Hz, 1 H, 1-H), 4.81 (ddd, 1 H, 2-H), 4.11 (m, 1 H, 5-H), 3.86

(m, 1 H, 6a-H), 3.76 (s, 3 H, OCH₃), 3.72 (m, 1 H, 6b-H), 2.91 (m,

1 H, HO-6) ppm. MS: m/z: 655 [M + NH₄]⁺(for ^{3 5}Cl).

C₂₉H₂₆Cl₃NO₉(638.88): calcd. C 54.52, H 4.10, N 2.19; found C

54.40, H 3.94, N 2.17.

H), 5.98 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, 4-H), 5.80 (dd, J_2,3

=

11.0 Hz, 1 H, 3-H), 4.98 (ddd, 1 H, 2-H), 4.65 (m, 1 H, 5-H), 4.39–

4.35 (m, 2 H, 6a-H, 6b-H), 4.00 (s, 2 H, CH₂Cl) ppm. MS: m/z:

624 [M – Cl₃CC(NH) + Na]⁺(for ³⁵Cl). C₂₆H₂₁Cl₇N₂O₉(753.63):

calcd. C 41.44, H 2.81, N 3.72; found C 41.81, H 2.85, N 3.67.

4-Methoxyphenyl 3,6-Di-O-benzoyl-4-O-chloroacetyl-2-deoxy-2-tri-

chloroacetamido-β-D-galactopyranoside (31): From alcohol 29

(430 mg, 0.67 mmol) as described for the preparation of 17. Flash

silica chromatography (CH₂Cl₂/acetone, 19:1) afforded the 4-chlo-

roacetate 31 (422 mg, 88%); m.p. 139–140 °C (from EtOAc/petro-

leum ether). [α]²_D²= –1.5 (c = 1 in chloroform). ¹H NMR (400 MHz,

2-(Benzyloxycarbonylamino)ethyl O-(3,6-Di-O-benzoyl-4-O-chlo-

roacetyl-2-deoxy-2-trichloroacetamido-β-

(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-

6-O-benzoyl-4-O-chloroacetyl-2-deoxy-2-trichloroacetamido-β- -ga-

D-galactopyranosyl)-

D

lactopyranoside (35): A mixture of imidate 33 (283 mg, 0.38 mmol)

and alcohol 23 (226 mg, 0.21 mmol) was treated as described for

the preparation of 21. Flash silica chromatography [CH₂Cl₂/ace-

tone (29:1) containing 0.1% of NEt₃] afforded the trisaccharide 35

(232 mg, 66%) as a white powder. [α]²_D²= –10 (c = 1 in chloroform).

¹H NMR (400 MHz, CDCl₃): δ = 8.08–7.27 (m, 30 H, arom. H),

6.96 (d, J_NH,2= 9.0 Hz, 1 H, GalN^IINH), 6.81 (d, J_NH,2= 7.0 Hz,

1 H, GalN^INH), 5.70 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^I

4-H), 5.65 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H), 5.48 (dd, J_4,5

Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.36 (dd, J_2,3= 11.0 Hz, 1

H, GalN^II3-H), 5.31 (dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H), 5.10 [m,

1 H, NHC(O)O], 5.06 (d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 5.03 (s,

2 H, CH₂-Ph), 5.02 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 4.89 (d, 1

H, GlcA 1-H), 4.37 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.45 (dd,

J_5,6a= 7.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.36 (m, 1 H, GalN^I

6b-H), 4.35 (dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 4.27 (ABq, 2 H,

CH₂Cl), 4.23 (m, 1 H, GalN^II2-H), 4.14 (d, 1 H, GlcA 5-H), 4.06

(m, 1 H, GalN^I5-H), 4.00 (m, 1 H, GalN^II5-H), 3.96 (ABq, 2 H,

CDCl₃): δ = 8.05–6.67 (m, 15 H, arom. H, NH), 5.79 (dd, J_3,4

=

3.0, J_2,3= 11.0 Hz, 1 H, 3-H), 5.79 (dd, J_4,5Ͻ 1.0 Hz, 1 H, 4-H),

5.37 (d, J_1,2= 8.5 Hz, 1 H, 1-H), 4.63–4.54 (m, 2 H, 2-H, 6a-H),

4.46 (dd, J_5,6a= 6.0, J_6a,6b= 11.0 Hz, 1 H, 6b-H), 4.35 (m, 1 H, 5-

H), 4.22 (ABq, 2 H, CH₂Cl), 3.72 (s, 3 H, OCH₃) ppm. MS: m/z:

731 [M + NH₄]⁺(for ³⁵Cl). C₃₁H₂₇Cl₄NO₁₀(715.37): calcd. C

52.05, H 3.80, N 1.96; found C 51.75, H 3.92, N 1.82.

4-Methoxyphenyl 3,4-Di-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-tri-

chloroacetamido-β-D-galactopyranoside (32): From alcohol 30

(500 mg, 0.78 mmol) as described for the preparation of 17. Flash

silica chromatography (CH₂Cl₂/acetone, 19:1) afforded the 6-chlo-

roacetate 32 (450 mg, 80%); m.p. 139–140 °C (from EtOAc/petro-

leum ether); [α]²_D²= +72 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.13–6.81 (m, 15 H, arom. H, NH), 5.88 (dd, J_4,5

Ͻ

1.0, J_3,4= 3.5 Hz, 1 H, 4-H), 5.85 (dd, J_2,3= 11.0 Hz, 1 H, 3-H),

5.34 (d, J_1,2= 8.5 Hz, 1 H, 1-H), 4.66 (ddd, 1 H, 2-H), 4.46 (dd,

J_5,6a= 8.5, J_6a,6b= 11 Hz, 1 H, 6a-H), 4.46 (dd, J_5,6a= 5.0 Hz, 1

H, 6b-H), 4.38 (m, 1 H, 5-H), 4.04 (s, 2 H, CH₂Cl), 3.77 (s, 3 H,

OCH₃) ppm. MS: m/z: 731 [M + NH₄]⁺(for ³⁵Cl). C₃₁H₂₇Cl₄NO₁₀

(715.37): calcd. C 52.05, H 3.80, N 1.96; found C 52.18, H 3.60, N

1.94.

CH₂Cl), 3.84 (m, 1 H, CH₂-O), 3.74 (dd, J_5,6a= 6.0, J_6a,6b

=

11.0 Hz, 1 H, GalN^II6a-H), 3.67 (s, 3 H, COOCH₃), 3.67–3.61 (m,

1 H, CH₂-O), 3.51 (m, 1 H, GalN^I2-H), 3.45 (dd, J_5,6b= 8.0 Hz,

1 H, GalN^II6b-H), 3.38–3.26 (m, 2 H, CH₂-N) ppm. MS: m/z:

1685 [M + NH₄]⁺(for ³⁵Cl). C₇₂H₆₅Cl₈N₃O₂₆(1671.93): calcd. C

51.72, H 3.92, N 2.51; found C 51.49, H 3.93, N 2.47.

3,6-Di-O-benzoyl-4-O-chloroacetyl-2-deoxy-2-trichloroacetamido-1-

O-trichloroacetimidoyl-α-

D-galactopyranose (33): A solution of gly-

2-(Benzyloxycarbonylamino)ethyl O-(3,4-Di-O-benzoyl-6-O-chlo-

coside 31 (580 mg, 0.81 mmol) in toluene/MeCN/water (1:1.5:1,

30 mL) was stirred with CAN (2.22 g, 4.05 mmol) for 30 min at

room temp. The mixture was then diluted with CH₂Cl₂, washed

roacetyl-2-deoxy-2-trichloroacetamido-β-

(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-

4-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-trichloroacetamido-β- -ga-

D-galactopyranosyl)-

D

with a saturated aqueous solution of NaCl and water, dried lactopyranoside (36): From imidate 34 (251 mg, 0.33 mmol) and

(MgSO₄), and concentrated. Flash silica chromatography (CH₂Cl₂/

acetone, 19:1) afforded the corresponding free hemiacetal (358 mg,

72%) as an orange powder. A mixture of the hemiacetal (358 mg,

0.59 mmol), trichloroacetonitrile (589 μL, 5.88 mmol), and DBU in

toluene (1 m, 88 μL) in dry CH₂Cl₂(3.6 mL) was stirred for 30 min

at room temp. then was concentrated. Flash silica chromatography

[CH₂Cl₂/acetone (29:1) containing 0.1% of NEt₃] afforded the α-

imidate 33 (367 mg, 60% from 31) as a white powder. [α]²_D²= +41

alcohol 24 (240 mg, 0.22 mmol) as described for the preparation of

21. Flash silica chromatography [CH₂Cl₂/acetone (19:1) containing

0.1% of NEt₃] afforded the trisaccharide 36 (192 mg, 52%) as a

white powder; m.p. 128 °C (from 2-propanol); [α]²_D²= +27 (c = 1 in

chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.11–7.16 (m, 30

H, arom. H), 6.99 (d, J_NH,2= 9.0 Hz, 1 H, GalN^IINH), 6.95 (d,

J_NH,2= 7.0 Hz, 1 H, GalN^INH), 5.78 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz,

1 H, GalN^I4-H), 5.63 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H),

5.56 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.39 (dd, J_2,3

1

(c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ = 8.87 (s, 1

H, C=NH), 7.99–7.13 (m, 10 H, arom. H), 7.14 (d, J_NH,2= 9.0 Hz, = 11.0 Hz, 1 H, GalN^II3-H), 5.27 (dd, J_1,2= 7.0 Hz, 1 H, GlcA

1 H, NH), 6.64 (d, J_1,2= 3.5 Hz, 1 H, 1-H), 5.89 (dd, J_4,5Ͻ 1.0,

J_3,4= 3.0 Hz, 1 H, 4-H), 5.76 (dd, J_2,3= 11.0 Hz, 1 H, 3-H), 4.90

2-H), 5.16 [m, 1 H, NHC(O)O], 5.07 (d, J_1,2= 8.5 Hz, 2 H, 2 GalN

1-H), 5.06 (s, 2 H, CH₂-Ph), 4.98 (d, 1 H, GlcA 1-H), 4.82 (dd, J_2,3

= 11.0 Hz, 1 H, GalN^I3-H), 4.43 (dd, J_5,6a= 4.0, J_6a,6b= 11.5 Hz,

(ddd, 1 H, 2-H), 4.66 (m, 1 H, 5-H), 4.54 (dd, J_5,6a= 6.5, J_6a,6b

=

11.0 Hz, 1 H, 6a-H), 4.42 (dd, J_5,6b= 7.0 Hz, 1 H, 6b-H), 4.24 1 H, GalN^I6a-H), 4.37 (ddd, 1 H, GalN^II2-H), 4.32 (dd, J_4,5

=

(ABq, 2 H, CH₂Cl) ppm. MS: m/z: 629 [M – Cl₃CC(NH) + Na]⁺9.0 Hz, 1 H, GlcA 4-H), 4.18 (m, 1 H, GalN^I6b-H), 4.18 (d, 1 H,

(for ³⁵Cl). C₂₆H₂₁Cl₇N₂O₉(753.63): calcd. C 41.44, H 2.81, N 3.72;

found C 41.36, H 2.72, N 3.80.

GlcA 5-H), 4.03 (m, 1 H, GalN^I5-H), 4.00 (s, 2 H, CH₂Cl), 4.02

(s, 2 H, CH₂Cl), 3.90 (s, 3 H, COOCH₃), 3.89–3.84 (m, 2 H, GalN^II

4194

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Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

5-H, CH₂-O), 3.72–3.65 (m, 2 H, GalN^I2-H, CH₂-O), 3.43–3.35 benzoyl-2-deoxy-β-

D-galactopyranoside (39): From bis-tri-

(m, 2 H, CH₂-N), 3.36 (dd, J_5,6a= 7.0, J_6a,6b= 11.0 Hz, 1 H, Gal-

chloroacetamide 37 (200 mg, 130 μmol) as described in the general

procedure using a twofold amount of reagents. Flash silica

N^II6a-H), 3.28 (dd, J_5,6b= 6.0 Hz, 1 H, GalN^II6b-H) ppm. MS:

m/z: 1685 [M + NH₄]⁺(for ³⁵Cl). C₇₂H₆₅Cl₈N₃O₂₆(1671.93): calcd. chromatography (CH₂Cl₂/MeOH, 16:1) afforded the bis-acetamide

C 51.72, H 3.92, N 2.51; found C 51.43, H 4.09, N 2.50.

39 (139 mg, 81 %) as a white powder; m.p. 212–215 °C (from

EtOAc/petroleum ether). [α]²_D²= +16 (c = 1 in chloroform). ¹H

NMR (400 MHz, CDCl₃): δ = 8.13–7.12 (m, 30 H, arom. H), 5.83

(d, J_NH,2= 9.0 Hz, 1 H, GalN^IINH), 5.71 (dd, J_2,3= J_3,4= 9.0 Hz,

1 H, GlcA 3-H), 5.51 (dd, J_NH,2= 7.0 Hz, 1 H, GalN^INH), 5.45

(dd, J_1,2= 8.0 Hz, 1 H, GlcA 2-H), 5.30 [m, 1 H, NHC(O)O], 5.19

2-(Benzyloxycarbonylamino)ethyl O-(3,6-Di-O-benzoyl-2-deoxy-2-

trichloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-2-deoxy-

2-trichloroacetamido-β- -galactopyranoside (37): A mixture of ester

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

D

35 (178 mg, 0.11 mmol) and thiourea (32 mg, 0.43 mmol) in pyr-

idine/EtOH (1:1, 2.4 mL) was stirred for 2 h at 80 °C, then was

cooled and concentrated. A solution of the residue in CH₂Cl₂was

washed with water, a saturated aqueous solution of NaCl, and

water; dried (MgSO₄); and concentrated. Flash silica chromatog-

raphy (CH₂Cl₂/acetone, 8:1) afforded the diol 37 (117 mg, 73%) as

a white powder; m.p. 176–177 °C (from 2-propanol). [α]²_D²= +19 (c

= 1 in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.03–7.12

(m, 31 H, GalN^IINH, arom. H), 7.02 (d, J_NH,2= 7.0, 1 H, GalN^I

NH), 5.53 (dd, J_2,3= 8.5, J_3,4= 9.0, 1 H, GlcA 3-H), 5.43 (dd, J_1,2

= 7.0, 1 H, GlcA 2-H), 5.26 (dd, J_3,4= 3.0, J_2,3= 11.0, 1 H, GalN^II

3-H), 5.25 [m, 1 H, NHC(O)O], 5.07 (d, J_1,2= 8.0, 1 H, GalN^II1-

(dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 5.01 (d, J_1,2

=

8.0 Hz, 1 H, GalN^I1-H), 5.00 (s, 2 H, CH₂-Ph), 4.97 (d, 1 H, GlcA

1-H), 4.85 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.61 (dd, J_5,6a= 5.0,

J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.58–4.49 (m, 2 H, GalN^I3-

H, 6b-H), 4.39 (dd, J_4,5= 10.0 Hz, 1 H, GlcA 4-H), 4.29 (d, J_4,5

=

10.0 Hz, 1 H, GlcA 5-H), 4.25 (m, 1 H, GalN^II2-H), 4.18 (ddd,

J_OH,4= J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^I4-H), 3.99 (ddd, J_4,5

Ͻ 1.0, J_OH,4= 6.0 Hz, 1 H, GalN^II4-H), 3.98–3.91 (m, 2 H, 2

GalN 5-H), 3.83–3.63 (m, 4 H, GalN^II6a-H, 6b-H, CH₂-O), 3.74

(s, 3 H, COOCH₃), 3.38–3.27 (m, 3 H, GalN^I2-H, CH₂-N), 2.79

(d, 1 H, GalN^I4-OH), 2.43 (d, 1 H, GalN^II4-OH), 1.85, 1.26 [2 s,

6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₆₈H₆₉N₃O₂₄Na₂

[M + 2Na]²⁺: 678.7033; found: 678.7025.

H), 5.03 (d, 1 H, GlcA 1-H), 4.99 (s, 2 H, CH₂-Ph), 4.94 (d, J_1,2

=

8.0, 1 H, GalN^I1-H), 4.65 (dd, J_5,6a= 5.0, J_6a,6b= 11.5, 1 H,

GalN^I6a-H), 4.55 (dd, J_5,6b= 5.0, 1 H, GalN^I6b-H), 4.52–4.42

(m, 2 H, GalN^I3-H, GalN^II2-H), 4.35 (dd, J_4,5= 9.0, 1 H, GlcA

4-H), 4.31 (ddd, J_4,5Ͻ 1.0, J_3,4= 3.0, J_OH,4= 6.0, 1 H, GalN^I4-

H), 4.23 (d, 1 H, GlcA 5-H), 4.13 (ddd, J_OH,4= J_4,5Ͻ 1.0, 1 H,

GalN^II4-H), 3.96–3.89 (m, 2 H, 2 GalN 5-H), 3.89–3.52 (m, 5 H,

GalN^I2-H, GalN^II6a-H, 6b-H, CH₂-O), 3.67 (s, 3 H, COOCH₃),

3.42–3.22 (m, 2 H, CH₂-N), 3.20 (d, 1 H, GalN^II4-OH), 3.09 (d,

J_OH,4= 6.0 Hz, 1 H, GalN^I4-OH) ppm. MS: m/z: 1533 [M +

NH₄]⁺(for ³⁵Cl). C₆₈H₆₃Cl₆N₃O₂₄(1518.97): calcd. C 53.77, H

4.18, N 2.77; found C 53.94, H 3.79, N 2.80.

2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,4-di-O-benz-

oyl-2-deoxy-β-

β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benzoyl-

2-deoxy-β- -galactopyranoside (40): From 38 (99 mg, 59 μmol) as

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-

D

described in the general procedure using a twofold amount of rea-

gents. Flash silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded

the bis-acetamide 40 (92 mg, 71%) as a white powder; m.p. 148–

150 °C (from EtOAc/petroleum ether). [α]²_D²= +38 (c = 1 in chloro-

form). ¹H NMR (400 MHz, CDCl₃): δ = 8.06–7.12 (m, 30 H, arom.

H), 5.97 (m, 1 H, GalN^IINH), 5.79 (m, 1 H, GalN^INH), 5.71 (dd,

J_4,5Ͻ 1.0, J_3,4= 2.0 Hz, 1 H, GalN^I4-H), 5.61 (dd, J_2,3= J_3,4

=

2-(Benzyloxycarbonylamino)ethyl O-(3,4-Di-O-benzoyl-2-deoxy-2-

8.5 Hz, 1 H, GlcA 3-H), 5.45 (dd, J_4,5Ͻ 1.0, J_3,4= 2.0 Hz, 1 H,

GalN^II4-H), 5.36 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 5.31 (dd,

J_1,2= 7.0 Hz, 1 H, GlcA 2-H), 5.03 (s, 2 H, CH₂-Ph), 5.02 [m, 1

H, NHC(O)O], 5.02 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.98 (d, 1

H, GlcA 1-H), 4.97 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.72 (m,

1 H, GalN^I3-H), 4.32–4.26 (m, 2 H, GalN^II2-H, GlcA 4-H), 4.22

(d, J_4,5= 10.0 Hz, 1 H, GlcA 5-H), 3.80 (s, 3 H, COOCH₃), 3.79–

3.22 (m, 10 H, GalN^I2-H, 5-H, 6a-H, 6b-H, 6-OH, GalN^II5-H,

trichloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-2-deoxy-

2-trichloroacetamido-β- -galactopyranoside (38): From ester 36

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

D

(99 mg, 59 μmol) as described for the preparation of 37. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the diol 38

(76 mg, 85%) as a white powder; m.p. 147–149 °C (from 2-propa-

1

nol/petroleum ether); [α]²_D²= +31 (c = 1 in chloroform). H NMR

(400 MHz, CDCl₃): δ = 8.10–6.95 (m, 30 H, arom. H), 6.96 (d,

J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 6.92 (d, J_NH,2= 7.0 Hz, 1 H,

GalN^INH), 5.77 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^I4-H),

5.59 (dd, J_2,3= J_3,4= 8.5 Hz, 1 H, GlcA 3-H), 5.46 (dd, J_4,5Ͻ 1.0,

J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.35 (dd, J_2,3= 11.0 Hz, 1 H,

GalN^II3-H), 5.31 (dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H), 5.15 [m, 1

H, NHC(O)O], 5.05 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 5.03 (s, 2

H, CH₂-Ph), 5.00 (d, 1 H, GlcA 1-H), 4.99 (d, J_1,2= 7.5 Hz, 1 H,

GalN^II1-H), 4.80 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.47 (ddd,

1 H, GalN^II2-H), 4.22 (dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 4.16

(d, 1 H, GlcA 5-H), 3.88 (s, 3 H, COOCH₃), 3.89–3.62 (m, 6 H,

GalN^I2-H, 5-H, 6a-H, GalN^II6-OH, CH₂-O), 3.51 (m, 1 H, GalN^I

6b-H), 3.42–3.31 (m, 2 H, CH₂-N), 3.35 (d, J_OH,6= 4.5 Hz, 1 H,

GalN^I6-OH), 2.75 (ddd, J_OH,6a= 5.0, J_5,6a= 7.0, J_6a,6b= 12.0 Hz,

1 H, GalN^II6a-H), 2.51 (ddd, J_OH,6b= 5.0, J_5,6b= 7.0 Hz, 1 H,

GalN^II6b-H), 2.14 (m, 1 H, GalN^II5-H) ppm. MS: m/z: 1533 [M

+ NH₄]⁺(for ³⁵Cl). C₆₈H₆₃Cl₆N₃O₂₄(1518.97): calcd. C 53.77, H

4.18, N 2.77; found C 54.03, H 3.86, N 2.78.

CH₂-O, CH₂-N), 2.85 (ddd, J_OH,6a= 5.0, J_5,6a= 7.0, J_6a,6b

=

12.0 Hz, 1 H, GalN^II6a-H), 2.64 (ddd, J_OH,6b= 5.0, J_5,6b= 7.0 Hz,

1 H, GalN^II6b-H), 2.39 (d, 1 H, GalN^II6-OH), 1.83, 1.32 [2 s, 6

H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₆₈H₆₉N₃O₂₄Na₂

[M + 2Na]²⁺: 678.7033; found: 678.7030.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium 2-acetamido-

3,6-di-O-benzoyl-2-deoxy-4-sulfonato-β-

(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acet-

amido-6-O-benzoyl-2-deoxy-4-sulfonato-β- -galactopyranoside (41):

D-galactopyranosyl)-(1Ǟ4)-

D

From diol 39 (79 mg, 60 μmol) and sulfur trioxide/trimethylamine

complex (140 mg, 1 mmol) as described for the preparation of 7.

The mixture was eluted from a column (3ϫ80 cm) of Sephadex

LH-20 with 1:1 CH₂Cl₂/MeOH, then submitted to ion-exchange as

described to afford the disulfate 41 (77 mg, 85%) as a white pow-

der. [α]²_D²= +12 (c = 1 in methanol). ¹H NMR (400 MHz, CD₃OD):

δ = 8.15–7.19 (m, 30 H, arom. H), 5.79 (dd, J_2,3= J_3,4= 8.0 Hz, 1

H, GlcA 3-H), 5.49 (dd, J_1,2= 7.0 Hz, 1 H, GlcA 2-H), 5.22–5.11

(m, 3 H, GalN^II3-H, 4-H, GlcA 1-H), 5.00 (s, 2 H, CH₂-Ph), 5.00–

4.98 (m, 1 H, GalN^II1-H), 4.91 (m, 1 H, GalN^I4-H), 4.82 (m, 1

H, GlcA 4-H), 4.69–4.61 (m, 2 H, GalN^I6a-H, 6b-H), 4.51 (m, 1

H, GalN^I1-H), 4.43 (d, J_4,5= 9.5 Hz, 1 H, GlcA 5-H), 4.39–4.31

2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,6-di-O-benz-

oyl-2-deoxy-β-

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-

D

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4195

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

(m, 2 H, GalN^II2-H, 6a-H), 4.12–3.98 (m, 3 H, GalN^I2-H, 3-H, Sodium 2-[6-(Biotinylamido)hexanoylamino]ethyl O-(Sodium 2-acet-

5-H), 3.75 (s, 3 H, COOCH₃), 3.70–3.52 (m, 4 H, GalN^II5-H, 6b-

amido-2-deoxy-4-O-sulfonato-β- -galactopyranosyl)-(1Ǟ4)-(sodium

H, CH₂-O), 3.22–3.15 (m, 2 H, CH₂-N), 1.89, 1.45 [2s, 6 H, β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-

sulfonato-β- -galactopyranoside (45): From 43 (40 mg, 40 μmol) as

D

NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₆₈H₆₇N₃O₃₀S₂[M –

D

2Na]^2–: 734.6625; found: 734.6617.

described for the preparation of 11 to afford the biotinylated trisac-

charide 45 (43 mg, 90%) as a white powder. R_f= 0.50 (EtOAc/

MeOH/water, 3:2:2). [α]²_D²= +6.5 (c = 1 in water). ¹H NMR

(400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ = 4.76 (br. s, 1 H,

GalN^I4-H), 4.70 (d, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 4.64 (ddd,

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium 2-acetamido-

3,4-di-O-benzoyl-2-deoxy-6-O-sulfonato-β-

(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-

2-acetamido-4-O-benzoyl-2-deoxy-6-O-sulfonato-β- -galacto-

D-galactopyranosyl)-

D

J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 H, H-c), 4.55 (d, J_1,2

=

pyranoside (42): From diol 40 (85 mg, 64 μmol) and sulfur trioxide/

trimethylamine complex (45 mg, 0.32 mmol) as described for the

preparation of 41 to afford the disulfate 42 (79 mg, 80%) as a white

powder. [α]²_D²= –15 (c = 1 in methanol). ¹H NMR (400 MHz,

CD₃OD): δ = 8.13–7.17 (m, 30 H, arom. H), 5.74 (m, 1 H, GalN^I

4-H), 5.71 (m, 1 H, GlcA 3-H), 5.63 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz,

1 H, GalN^II4-H), 5.37 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 5.24

(dd, J_1,2= 8.0, J_2,3= 9.0 Hz, 1 H, GlcA 2-H), 5.18 (d, 1 H, GlcA

1-H), 5.02 (ABq, 2 H, CH₂-Ph), 4.76 (d, J_1,2= 8.0 Hz, 1 H, GalN^II

1-H), 4.51 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.36–4.31 (m, 2 H,

GalN^I3-H, GlcA 4-H), 4.28 (dd, J_5,6a= 3.0, J_6a,6b= 11.0 Hz, 1 H,

GalN^I6a-H), 4.20–4.14 (m, 2 H, GalN^II2-H, GlcA 5-H), 4.13–

4.08 (m, 2 H, GalN^I2-H, 5-H), 4.00 (m, 1 H, GalN^II5-H), 3.94 (s,

3 H, COOCH₃), 3.96–3.83 (m, 2 H, GalN^I6b-H, CH₂-O), 3.62 (dd,

J_5,6a= 6.0, J_6a,6b= 11.0 Hz, 1 H, GalN^II6a-H), 3.58 (m, 1 H, CH₂-

O), 3.36 (m, 1 H, GalN^II6b-H), 3.30–3.25 (m, 2 H, CH₂-N), 1.85,

1.26 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₆₈H₆₇N₃O₃₀S₂[M – 2Na]^2–: 734.6625; found: 734.6643.

8.0 Hz, 2 H, 2 GalN 1-H), 4.52 (d, J_1,2= 8.0 Hz, 1 H, GlcA 1-H),

4.44 (dd, J_b,e= 5.0 Hz, 1 H, H-b), 4.10–4.04 (m, 2 H, GalN^I2-H,

3-H), 3.96 (m, 1 H, CH₂-O), 3.92–3.86 (m, 2 H, GalN^II2-H, 3-H),

3.88–3.78 (m, 8 H, GlcA 4-H, 5-H; 2 GalN 5-H, 4GalN 6-H), 3.76

(m, 1 H, CH₂-O), 3.62 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H),

3.42 (dd, 1 H, GlcA 2-H), 3.40–3.34 (m, 3 H, CH₂-N, H-e), 3.22–

3.18 (m, 2 H, CH₂N-k), 3.03 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.81

(dd, 1 H, H-d), 2.30–2.24 (m, 4 H, CH₂-i, CH₂-o), 2.09, 2.04 [2 s,

6 H, NHC(O)CH₃], 1.81–1.30 (m, 12 H, CH₂-f, CH₂-g, CH₂-h,

CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal

acetone, δ_C= 30. 83): δ = 177.49, 177.14, 175.65, 175.35, 174.95 (5

C, GlcA C-6, C=O), 165.89 (1 C, C-a), 102.25 (1 C, GlcA C-1),

101.97, 101.66 (2 C, 2 GalN C-1), 80.98 (2 C, GalN^IC-3, GlcA C-

4), 76.95, 76.89 (2 C, 2 GalN C-4), 75.19, 75.07 (2 C, 2 GalN C-

5), 75.00 (1 C, GlcA C-5), 72.61 (2 C, GalN^IIC-3, GlcA C-3), 70.51

(1 C, GlcA C-2), 68.83 (1 C, CH₂-O), 62.67 (1 C, C-b), 61.56 (2 C,

2 GalN C-6), 60.83 (1 C, C-c), 55.98 (1 C, C-e), 53.18, 52.24 (2 C,

2 GalN C-2), 40.30 (1 C, C-d), 39.65, 39.60 (2 C, C-k, C-q), 36.22,

36.11 (2 C, C-i, C-o), 28.60, 28.45, 28.23, 26.16, 25.82, 25.57 (6 C,

C-f, C-g, C-h, C-l, C-m, C-n), 23.03, 22.87 [2 C, NHC(O)CH₃]

ppm. HRMS: m/z: calcd. for C₄₀H₆₄N₆O₂₆S₃[M – 3Na + H]^2–:

570.1516; found: 570.1521.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium 2-acetamido-

2-deoxy-4-O-sulfonato-β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-

β- -galactopyranoside (43): From ester 41 (80 mg, 53 μmol) as de-

D-galactopyranosyl)-(1Ǟ4)-(sodium β-D-

D

scribed for the preparation of 9 to give the disodium salt 43 (44 mg,

82%) as a white powder. R_f= 0.35 (EtOAc/MeOH/water, 4:2:1).

[α]²_D²= –15 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

H₂O, δ_H= 4. 79): δ = 7.45–7.35 (m, 5 H, arom. H), 5.12 (s, 2 H,

CH2Ph), 4.72 (br. s, 1 H, GalN^I4-H), 4.68 (d, J_3,4= 3.0 Hz, 1 H,

GalN^II4-H), 4.55 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.51 (d, J_1,2

= 8.0 Hz, 1 H, GalN^I1-H), 4.47 (d, J_1,2= 7.5 Hz 1 H, GlcA 1-H),

4.03–3.98 (m, 2 H, GalN^I2-H, 3-H), 3.92 (m, 1 H, CH₂-O), 3.90–

3.85 (m, 2 H, GalN^II2-H, 3-H), 3.84–3.76 (m, 7 H, GlcA 4-H, 2

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium 2-acet-

amido-2-deoxy-6-O-sulfonato-β-

β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-

sulfonato-β- -galactopyranoside (46): From 44 (17 mg, 17 μmol) as

D-galactopyranosyl)-(1Ǟ4)-(sodium

D

described for the preparation of 11 to afford the biotinylated trisac-

charide 46 (14 mg, 70%) as a white powder. R_f= 0.50 (EtOAc/

MeOH/water, 3:2:2). [α]²_D²= +15 (c = 1 in water); ¹H NMR

(400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ = 4.62 (ddd, J_c,d

Ͻ

GalN 5-H, 4GalN 6-H), 3.70 (m, 1 H, CH₂-O), 3.67 (d, J_4,5

=

1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 H, H-c), 4.54–4.50 (m; 3 H, 2 GalN

1-H, GlcA 1-H), 4.45 (dd, J_b,e= 5.0 Hz, 1 H, H-b), 4.28–4.20 (m,

4 H, 2 GalN 6a-H, 6b-H), 4.19 (dd, J_4,5Ͻ 1.0, J_3,4= 2.5 Hz, 1 H,

GalN^I4-H), 4.03 (dd, J_1,2= 8.5, J_2,3= 11.0 Hz, 1 H, GalN^I2-H),

4.01 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 3.99–3.89 (m,

4 H, GalN^II2-H, 2 GalN 5-H, CH₂-r), 3.86 (dd, J_2,3= 11.0 Hz, 1

H, GalN^I3-H), 3.80–3.69 (m, 4 H, GalN^II3-H, GlcA 4-H, 5-H,

CH₂-r), 3.60 (dd, J_3,4= 9.0, J_2,3= 8.0 Hz, 1 H, GlcA 3-H), 3.40–

3.30 (m, 4 H, GlcA 2-H, H-e, CH₂-q), 3.18 (dd, J_k,kЈ= J_k,l= 7.0 Hz,

2 H, CH₂-k), 3.01 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.79 (dd, 1 H,

8.0 Hz, 1 H, GlcA 5-H), 3.57 (t, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-

H), 3.38 (dd, 1 H, GlcA 2-H), 3.35–3.30 (m, 2 H, CH₂-N), 2.04,

1.98 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₃₂H₄₅N₃O₂₅S₂[M – 3Na + H]^2–: 467.5892; found: 467.5889.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium 2-acetamido-

2-deoxy-6-O-sulfonato-β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-

β- -galactopyranoside (44): From ester 42 (45 mg, 30 μmol) as de-

scribed for the preparation of 9 to give the disodium salt 44 (23 mg,

D-galactopyranosyl)-(1Ǟ4)-(sodium β-D-

D

77%) as a white powder. R_f= 0.35 (EtOAc/MeOH/water, 4:2:1). H-d), 2.30–2.24 (m, 4 H, CH₂-i, CH₂-o), 2.06, 2.02 [2 s, 6 H,

[α]²_D²= –2.5 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

H₂O, δ_H= 4. 79): δ = 7.46 (s, 5 H, arom. H), 5.14 (s, 2 H, CH₂-

Ph), 4.52 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 4.51 (d, J_1,2= 8.0 Hz,

1 H, GlcA 1-H), 4.48 (d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 4.25–

NHC(O)CH₃], 1.79–1.30 (m, 12 H, CH₂-f, CH₂-g, CH₂-h, CH₂-l,

CH₂-m,CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal acetone,

δ_C= 30. 83): δ = 177.56, 177.23, 175.65, 175.34, 174.98 (5 C, GlcA

C-6, C=O), 165.95 (1 C, C-a), 104.88 (1 C, GlcA C-1), 102.13,

101.95 (2 C, 2 GalN C-1), 81.57, 80.51 (2 C, GalN^IC-3, GlcA C-

4.18 (m, 4 H, 2 GalN 6a-H, 6b-H), 4.16 (dd, J_4,5Ͻ 1.0, J_3,4

=

3.0 Hz, 1 H, GalN^II4-H), 4.12–3.89 (m, 5 H, GalN^I2-H, 4-H, 5- 4), 77.08, 74.47, 73.31, 73.30, 73.02,71.51 (6 C, GalN^IC-3, 2 GalN

H, GalN^II2-H, CH₂-O), 3.84 (m, 1 H,GalN^II5-H), 3.82 (dd, J_2,3C-5, GlcA C-2, C-3, C-5), 68.02 (1 C, C-r), 68.41, 67.59 (2 C, 2

= 11.0 Hz, 1 H, GalN^II3-H), 3.77–3.70 (m, 4 H, GalN^I3-H, GlcA GalN C-6), 68.28, 68.05 (2 C, 2 GalN C-4), 62.74 (1 C, C-b), 60.90

4-H, 5-H, CH₂-O), 3.59 (d, J_2,3= 8.5, J_3,4= 9.0 Hz, 1 H, GlcA 3-

H), 3.39 (dd, 1 H, GlcA 2-H), 3.36–3.30 (m, 2 H, CH₂-N), 2.07,

1.97 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₃₂H₄₅N₃O₂₅S₂[M – 3Na + H]^2–: 467.5892; found: 467.5902.

(1 C, C-c), 56.04 (1 C, C-e), 52.81, 51.69 (2 C, 2 GalN C-2), 40.37

(1 C, C-d), 39.98, 39.74 (2 C, C-k, C-q), 36.29, 36.20 (2 C, C-i, C-

o), 28.66, 28.52, 28.35, 26.23, 25.91, 25.65 (6 C, C-f, C-g, C-h, C-

l, C-m, C-n), 23.16, 22.93 [2 C, NHC(O)CH₃] ppm. HRMS: m/z:

4196

www.eurjoc.org

Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

calcd. for C₄₀H₆₄N₆O₂₆S₃[M – 3Na + H]^2–: 570.1516; found:

570.1531.

silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded the alcohol

50 (124 mg, 67%) as a white powder; m.p. 138–140 °C (from 2-

propanol). [α]²_D²= +12 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.05–7.23 (m, 20 H, arom. H), 7.03 (d, J_NH,2= 7.0 Hz,

1 H, GalN^INH), 6.93 (d, J_NH,2= 8.5 Hz, 1 H, GalN^IINH), 5.63

2-(Benzyloxycarbonylamino)ethyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-

trichloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-4-O-

chloroacetyl-2-deoxy-2-trichloroacetamido-β- -galactopyranoside

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

D

(dd, J_3,4= 8.0, J_2,3= 9.0 Hz, 1 H, GlcA 3-H), 5.43 (dd, J_1,2

=

D

7.0 Hz, 1 H, GlcA 2-H), 5.23 [m, 1 H, NHC(O)O], 5.16–5.08 (m,

2 H, GalN^II3-H, 4-H), 5.02 (d, 1 H, GlcA 1-H), 4.99 (s, 2 H, CH₂-

Ph), 4.95 (d, J_1,2= 8.5 Hz, 2 H, 2 GalN 1-H, 1-H), 4.64 (dd, J_5,6a

= 5.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.55 (dd, J_5,6b= 7.0 Hz,

1 H, GalN^I6b-H), 4.52 (dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1 H, GalN^I

(48): A mixture of imidate 47^[16](248 mg, 0.42 mmol) and alcohol

23 (300 mg, 0.28 mmol) was treated as described for the prepara-

tion of 23. Flash silica chromatography [CH₂Cl₂/acetone (12:1)

containing 0.1% of NEt₃] afforded the trisaccharide 48 (276 mg,

65 %) as a yellow powder; m.p. 130–132 °C (from 2-propanol).

3-H), 4.38 (dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 4.29 (ddd, J_OH,4

=

[α]²_D²= +4 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

1

J_4,5Ͻ 1.0 Hz, 1 H, GalN^I4-H), 4.21 (d, 1 H, GlcA 5-H), 4.01

(ddd, 1 H, GalN^II2-H), 3.91 (m, 1 H, GalN^I5-H), 3.84 (m, 1 H,

CH₂-O), 3.70 (s, 3 H, COOCH₃), 3.72–3.54 (m, 4 H, GalN^I2-H,

GalN^II5-H, CH₂-O), 3.37–3.23 (m, 4 H, GalN^II6a-H, 6b-H, CH₂-

N), 3.22 (d, 1 H, OH), 1.96, 1.94, 1.93 [3 s, 9 H, OC(O)CH₃] ppm.

= 8.06–7.22 (m, 20 H, arom. H), 6.84 (d, J_NH,2= 9.0 Hz, 1 H,

GalN^IINH), 6.81 (d, J_NH,2= 7.0 Hz, 1 H, GalN^INH), 5.59 (dd,

J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^I4-H), 5.58 (dd, J_2,3= J_3,4

=

9.0 Hz, 1 H, GlcA 3-H), 5.29 (dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H),

5.13 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.10 [m, 1 H,

NHC(O)O], 5.07 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 5.02 (s, 2

H, CH₂-Ph), 5.01 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 4.88 (d, J_1,2

= 7.5 Hz, 1 H, GalN^II1-H), 4.92 (d, 1 H, GlcA 1-H), 4.73 (dd, J_2,3

= 11.0 Hz, 1 H, GalN^I3-H), 4.44 (dd, J_5,6a= 7.0, J_6a,6b= 11.5 Hz,

1 H, GalN^I6a-H), 4.34 (dd, J_5,6b= 5.5 Hz, 1 H, GalN^I6b-H), 4.25

(ABq, 2 H, CH₂Cl), 4.23 (dd, J_4,5= 9.5 Hz, 1 H, GlcA 4-H), 4.08

(d, 1 H, GlcA 5-H), 4.05 (m, 1 H, GalN^I5-H), 4.00 (m, 1 H, GalN^II

2-H), 3.84 (m, 1 H, CH₂-O), 3.68 (m, 1 H, GalN^II5-H) 3.65 (s, 3

H, COOCH₃), 3.62 (m, 1 H, CH₂-O), 3.50 (m, 1 H, GalN^I2-H),

3.39–3.23 (m, 4 H, GalN^II6a-H, 6b-H, CH₂-N), 1.97, 1.95, 1.93 [3

s, 9 H, OC(O)CH₃] ppm. MS: m/z: 1527 [M + NH₄]⁺(for ³⁵Cl).

C₆₂H₆₂Cl₇N₃O₂₆(1513.35): calcd. C 49.21, H 4.13, N 2.78; found

C 49.43, H 3.98, N 2.82.

HRMS: m/z: calcd. for C₆₀H₆₁Cl₆N₃O₂₅Na₂[M + 2Na]^2+–

:

739.5760; found: 739.5734.

2-(Benzyloxycarbonylamino)ethyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-

trichloroacetamido-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-2-deoxy-

2-trichloroacetamido-β- -galactopyranoside (51): From ester 49

D

(186 mg, 0.12 mmol) as described for the preparation of 37. Flash

silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded the alcohol

51 (153 mg, 86%) as a white powder. [α]²_D²= +15 (c = 1 in chloro-

form). ¹H NMR (400 MHz, CDCl₃): δ = 8.12–7.23 (m, 20 H, arom.

H), 6.83 (d, J_NH,2= 7.0 Hz, 1 H, GalN^INH), 6.74 (d, J_NH,2

=

9.0 Hz, 1 H, GalN^IINH), 5.77 (dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H,

GalN^I4-H), 5.58 (dd, J_3,4= J_2,3= 9.0 Hz, 1 H, GlcA 3-H), 5.26

(dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H), 5.13 [m, 1 H, NHC(O)O], 5.12

(dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.06 (d, J_1,2

=

2-(Benzyloxycarbonylamino)ethyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-

8.5 Hz, 1 H, GalN^I1-H), 5.03 (s, 2 H, CH₂-Ph), 5.02 (m, 1 H,

GalN^II3-H), 4.97 (d, 1 H, GlcA 1-H), 4.87 (d, J_1,2= 8.5 Hz, 1 H,

GalN^II1-H), 4.83 (dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1 H, GalN^I3-H),

4.18 (dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 4.12 (d, 1 H, GlcA 5-H),

4.02 (ddd, 1 H, GalN^II2-H), 3.87 (s, 3 H, COOCH₃), 3.88–3.81

(m, 2 H, GalN^I5-H, CH₂-O), 3.72–3.61 (m, 4 H, GalN^I2-H, 6a-

H, GalN^II5-H, CH₂-O), 3.51 (m, 1 H, GalN^I6b-H), 3.39–3.32 (m,

2 H, CH₂-N), 3.37 (d, J_OH,6= 2.0 Hz, 1 H, OH), 3.19–3.16 (m, 2

H, GalN^II6a-H, 6b-H), 1.96, 1.93, 1.92 [3 s, 9 H, OC(O)CH₃] ppm.

trichloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-6-O-

chloroacetyl-2-deoxy-2-trichloroacetamido-β- -galactopyranoside

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

D

(49): From imidate 47 (99 mg, 0.17 mmol) and alcohol 24 (120 mg,

0.11 mmol) as described for the preparation of 23. Flash silica

chromatography [CH₂Cl₂/acetone (12:1Ǟ10:1) containing 0.1% of

NEt₃] afforded the trisaccharide 49 (98 mg, 59%) as a white pow-

der; m.p. 129–130 °C (from 2-propanol/petroleum ether). [α]²_D²

=

+18 (c = 1 in chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.08–

7.24 (m, 20 H, arom. H), 6.94 (d, J_NH,2= 7.0 Hz, 1 H, GalN^INH),

6.84 (d, J_NH,2= 8.0 Hz, 1 H, GalN^IINH), 5.77 (dd, J_4,5Ͻ 1.0, J_3,4

= 3.0 Hz, 1 H, GalN^I4-H), 5.55 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H,

GlcA 3-H), 5.23 (dd, J_1,2= 7.0 Hz, 1 H, GlcA 2-H), 5.16 [m, 1 H,

NHC(O)O], 5.13 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H),

5.11 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 5.05 (s, 2 H, CH₂-Ph),

HRMS: m/z: calcd. for C₆₀H₆₁Cl₆N₃O₂₅Na₂[M + 2Na]²⁺

:

739.5760; found: 739.5749.

2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,4,6-tri-O-acet-

yl-2-deoxy-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-

β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benzoyl-2-

deoxy-β- -galactopyranoside (52): From 50 (112 mg, 780 μmol) as

D

5.03 (m, 1 H, GalN^I1-H), 4.98 (d, 1 H, GlcA 1-H), 4.89 (d, J_1,2

=

described for the preparation of 39. Flash silica chromatography

(CH₂Cl₂/MeOH, 19:1) afforded the bis-acetamide 52 (67 mg, 70%)

as a white powder; m.p. 146–149 °C (from EtOAc/petroleum ether).

8.5 Hz, 1 H, GalN^II1-H), 4.79 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-

H), 4.42 (dd, J_5,6a= 4.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.25

(dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 4.17 (dd, J_5,6b= 8.0 Hz, 1 H,

GalN^I6b-H), 4.13 (d, 1 H, GlcA 5-H), 4.08–4.01 (m, 2 H, GalN^I

5-H, GalN^II2-H), 4.00 (s, 2 H, CH₂Cl), 3.87 (m, 2 H, CH₂-O),

3.86 (s, 3 H, COOCH₃), 3.75–3.61 (m, 2 H, GalN^I2-H, GalN^II5-

1

[α]²_D²= +17 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

= 8.05–7.23 (m, 20 H, arom. H), 5.74 (d, J_NH,2= 8.5 Hz, 1 H,

GalN^IINH), 5.66 (d, J_NH,2= 8.5 Hz, 1 H, GalN^INH), 5.64 (dd,

J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA 3-H), 5.42 (dd, J_1,2= 7.5 Hz, 1 H,

H), 3.42–3.32 (m, 2 H, CH₂-N), 3.26 (dd, J_5,6a= 7.5, J_6a,6b

=

GlcA 2-H), 5.35 [m, 1 H, NHC(O)O], 5.19 (dd, J_3,4= 3.0, J_2,3

=

11.5 Hz, 1 H, GalN^II6a-H) 3.16 (dd, J_5,6b= 6.0 Hz, GalN^II6b-

H), 1.95, 1.94 [2 s, 9 H, OC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₆₂H₆₂Cl₇N₃O₂₆Na₂[M + 2Na]²⁺: 777.5618; found: 777.5593.

11.0 Hz, 1 H, GalN^II3-H), 5.10 (m, 1 H, GalN^II4-H), 5.01 (s, 2

H, CH₂-Ph), 5.00 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.94 (d, 1 H,

GlcA 1-H), 4.76 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.62 (dd, J_5,6a

= 5.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.52 (dd, J_5,6b= 7.0 Hz,

2-(Benzyloxycarbonylamino)ethyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-

trichloroacetamido-β-

benzoyl-β-

trichloroacetamido-β-

(196 mg, 0.13 mmol) as described for the preparation of 37. Flash

D =

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O- 1 H, GalN^I6b-H), 4.52 (m, 1 H, GalN^I3-H), 4.38 (dd, J_4,5

D

-glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-2-deoxy-2- 9.0 Hz, 1 H, GlcA 4-H), 4.23 (d, 1 H, GlcA 5-H), 4.18 (m, 1 H,

D

-galactopyranoside (50): From ester 48

GalN^I4-H), 3.93–3.75 (m, 3 H, GalN^I5-H, GalN^II2-H, CH₂-O),

3.72 (s, 3 H, COOCH₃), 3.64 (m, 1 H, CH₂-O), 3.56 (m, 1 H, Gal-

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4197

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

N^II5-H), 3.42–3.25 (m, 5 H, GalN^I2-H, GalN^II6a-H, 6b-H, OH, 8.5 Hz, 1 H, GalN^II1-H), 4.46 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H),

CH₂-N), 1.95, 1.93, 1.92, 1.89 [4 s, 12 H, OC(O)CH₃, NHC(O)- 4.27 (dd, J_4,5= 9.0 Hz, 1 H, GlcA 4-H), 4.23–4.04 (m, 5 H, GalN^I

CH₃], 1.28 [s, 3 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

2-H, 3-H, 6a-H, 6b-H, GlcA 5-H), 3.90 (s, 3 H, COOCH₃), 3.90–

3.81 (m, 3 H, GalN^I5-H, GalN^II2-H, CH₂-O), 3.62–3.56 (m, 2 H,

GalN^II5-H, CH₂-O), 3.44 (dd, J_5,6a= 8.0, J_6a,6b= 11.0 Hz, 1 H,

GalN^II6a-H), 3.29–3.24 (m, 2 H, CH₂-N), 3.23 (dd, J_5,6b= 5.5 Hz,

1 H, GalN^II6b-H), 1.92, 1.90, 1.88, 1.87 [4 s, 12 H, OC(O)CH₃,

NHC(O)CH₃], 1.26 [s, 3 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd.

for C₆₀H₆₆N₃O₂₈SNa₃[M + 2Na]²⁺: 688.6623; found: 688.6641.

C₆₀H₆₇N₃O₂₅Na₂[M + 2Na]²⁺: 637.6930; found: 637.6938.

2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,4,6-tri-O-acet-

yl-2-deoxy-β-

β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benzoyl-2-

deoxy-β- -galactopyranoside (53): From 51 (153 mg, 0.1 mmol) as

described for the preparation of 39. Flash silica chromatography

(CH₂Cl₂/MeOH, 19:1) afforded the bis-acetamide 53 (96 mg, 77%) Sodium 2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-2-deoxy-

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-

D

as a white powder; m.p. 156–160 °C (from EtOAc/petroleum ether).

β-

D

-galactopyranosyl)-(1Ǟ4)-(sodium β-

D

-glucopyranosyluronate)-

-galactopyranoside

[α]²_D²= +20 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-β-

D

1

= 8.05–7.23 (m, 20 H, arom. H), 5.81 (d, J_NH,2= 8.5 Hz, 1 H,

(56): From ester 54 (37 mg, 28 μmol) as described for the prepara-

tion of 9 to afford the sodium salt 56 (17.5 mg, 70%) as a white

GalN^IINH), 5.75–5.71 (m, 2 H, GalN^INH, 4-H), 5.56 (dd, J_2,3

=

J_3,4= 8.5 Hz, 1 H, GlcA 3-H), 5.37 (dd, J_1,2= 7.0 Hz, 1 H, GlcA

powder. R_f= 0.45 (EtOAc/MeOH/water, 4:2:1). [α]²_D²= –11 (c = 1

2-H), 5.33 [m, 1 H, NHC(O)O], 5.09–5.01 (m, 5 H, GalN^I1-H, in water). H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ

1

GalN^II3-H, 4-H, CH₂-Ph), 4.97 (d, 1 H, GlcA 1-H), 4.76 (m, 1 H, = 7.44 (s, 5 H, arom. H), 5.12 (s, 2 H, CH₂-Ph), 4.72 (dd, J_3,4

=

GalN^I3-H), 4.59 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.28 (dd, J_4,5

J_4,5Ͻ 1.0 Hz, 1 H, GalN^I4-H), 4.50 (d, J_1,2= 8.0 Hz, 1 H, GalN^I

1-H), 4.48 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.44 (d, J_1,2

= 9.5 Hz, 1 H, GlcA 4-H), 4.17 (d, 1 H, GlcA 5-H), 3.89–3.77 (m,

=

3 H, GalN^I2-H, 5-H, CH₂-O), 3.82 (s, 3 H, COOCH₃), 3.69–3.62 7.5 Hz, 1 H, GlcA 1-H), 4.05–3.99 (m, 2 H, GalN^I2-H, 3-H), 3.92

(m, 2 H, GalN^I6a-H, CH₂-O), 3.53–3.40 (m, 3 H, GalN^I6b-H, (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 3.92–3.65 (m, 12

GalN^II2-H, 5-H), 3.47–3.22 (m, 5 H, GalN^II6a-H, 6b-H, OH, H, GalN^II2-H, 3-H, 2 GalN 5-H, 6a-H, 6b-H, GlcA 4-H, 5-H,

CH₂-N), 2.08 [s, 3 H, NHC(O)CH₃], 1.95, 1.94, 1.92, 1.89 [3 s, 9 CH₂-O), 3.56 (d, J_2,3= 9.0, J_3,4= 9.5 Hz, 1 H, GlcA 3-H), 3.40

H, OC(O)CH₃], 1.35 [s, 3 H, NHC(O)CH₃] ppm. HRMS: m/z: (dd, 1 H, GlcA 2-H), 3.34–3.29 (m, 2 H, CH₂-N), 2.05, 1.95 [2 s,

calcd. for C₆₀H₆₇N₃O₂₅Na₂[M + 2Na]²⁺: 637.6930; found:

637.6944.

6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₃₂H₄₅N₃O₂₂S

[M – 2Na]^2–: 427.6108; found: 427.6121.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,4,6-tri-

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-2-deoxy-

O-acetyl-2-deoxy-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-

benzoyl-2-deoxy-4-O-sulfonato-β- -galactopyranoside (54): A mix-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

β-

D

-galactopyranosyl)-(1Ǟ4)-(sodium β-

D

-glucopyranosyluronate)-

-galactopyranoside

D

(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-β-

(57): From ester 55 (75 mg, 56 μmol) as described for the prepara-

tion of 9 to afford the sodium salt 57 (36 mg, 72%) as a white

D

ture of alcohol 52 (50 mg, 41 μmol) and sulfur trioxide/trimeth-

ylamine complex (14 mg, 0.10 mmol) in anhydrous DMF (0.5 mL) powder. R_f= 0.50 (EtOAc/MeOH/water, 4:2:1). [α]²_D²= –1.5 (c = 1

1

was stirred for 4 d at 65 °C, then was treated as described for the

preparation of 41 to afford the 4-sulfate 54 (38 mg, 70%) as a white

powder. [α]²_D²= +13 (c = 1 in methanol). ¹H NMR (400 MHz,

in water). H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ

= 7.46 (s, 5 H, arom. H), 5.14 (s, 2 H, CH₂-Ph), 4.52 (d, J_1,2

8.5 Hz, 1 H, GlcA 1-H), 4.48 (d, J_1,2= 9.0 Hz, 2 H, 2 GalN 1-H),

4.25–4.18 (m, 2 H, GalN^I6a-H, 6b-H), 4.16 (dd, J_3,4= J_4,5

1.0 Hz, 1 H, GalN^I4-H), 4.01 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I2-

=

CD₃OD): δ = 8.12–7.21 (m, 20 H, arom. H), 5.63 (dd, J_2,3= J_3,4

=

Ͻ

7.0 Hz, 1 H, GlcA 3-H), 5.41 (dd, J_1,2= 6.5 Hz, 1 H, GlcA 2-H),

5.20 (m, 1 H, GalN^I4-H), 5.18 (d, 1 H, GlcA 1-H), 5.10 (dd, J_4,5H), 3.97–3.68 (m, 12 H, GalN^II2-H, 4-H, 6a-H, 6b-H, 2 GalN 3-

Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.00 (s, 2 H, CH₂-Ph), 4.95

(dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 4.92 (m, 1 H, GlcA 4-H),

H, 5-H, GlcA 4-H, 5-H, CH₂-O), 3.58 (d, J_2,3= 9.0, J_3,4= 9.5 Hz,

1 H, GlcA 3-H), 3.40 (dd, 1 H, GlcA 2-H), 3.36–3.33 (m, 2 H,

4.86 (d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 4.72 (dd, J_5,6a= 4.5, J_6a,6bCH₂-N), 2.07, 1.96 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z:

= 12 Hz, 1 H, GalN^I6a-H), 4.63 (dd, J_5,6b= 8.0 Hz, 1 H, GalN^Icalcd. for C₃₂H₄₅N₃O₂₂S [M – 2Na]^2–: 427.6108; found: 427.6121.

6b-H), 4.52 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.31 (d, J_4,5

=

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(2-Acetamido-

2-deoxy-β- -galactopyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyl-

uronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-β- -galactopyr-

10.0 Hz, 1 H, GlcA 5-H), 4.12–3.98 (m, 3 H, GalN^I2-H, 3-H, 5-

H), 3.93 (dd, 1 H, GalN^II2-H), 3.80 (s, 3 H, COOCH₃), 3.76 (m,

1 H, CH₂-O), 3.62–3.53 (m, 3 H, GalN^II5-H, 6a-H, CH₂-O), 3.34

(m, 1 H, GalN^II6b-H), 3.26–3.21 (m, 2 H, CH₂-N), 1.95, 1.94,

1.89, 1.88 [4 s, 12 H, OC(O)CH₃, NHC(O)CH₃], 1.52 [s, 3 H,

NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₆₀H₆₆N₃O₂₈SNa₃[M

+ 2Na]²⁺: 688.6623; found: 688.6627.

D

anoside (58): From 56 (10 mg, 15 μmol) as described for the prepa-

ration of 11 to afford the biotinylated trisaccharide 58 (14 mg,

56%) as a white powder. R_f= 0.45 (EtOAc/MeOH/water, 3:2:2).

[α]²_D²= –11 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

H₂O, δ_H= 4. 79): δ = 4.75 (dd, J_4,5Ͻ 1.0, J_3,4= 1.5 Hz, 1 H, GalN^I

4-H), 4.63 (ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 H, H-c),

4.55 (d, J_1,2= 8.0 Hz, 1 H, GlcA 1-H), 4.49 (d, J_1,2= 8.5 Hz, 1 H,

GalN^II1-H), 4.48 (d, J_1,2= 7.5 Hz, 1 H, GalN^I1-H), 4.44 (dd, J_b,e

= 5.0 Hz, 1 H, H-b), 4.05–4.03 (m, 2 H, GalN^I2-H, 3-H), 3.96–

3.66 (m, 13 H, GalN^II2-H, 3-H, 4-H, 2 GalN 5-H, 6a-H, 6b-H,

GlcA 4-H, 5-H, CH₂-r), 3.58 (dd, J_3,4= J_2,3= 9.0 Hz, 1 H, GlcA

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(2-Acetamido-3,4,6-tri-

O-acetyl-2-deoxy-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-

benzoyl-2-deoxy-6-O-sulfonato-β- -galactopyranoside (55): From

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

D

alcohol 53 (92 mg, 0.37 mmol) as described for the preparation of

54 to afford the 6-sulfate 55 (75 mg, 75 %) as a white powder.

[α]²_D²= +2 (c = 1 in methanol). H NMR (400 MHz, CD₃OD): δ =

3-H), 3.44–3.33 (m, 4 H, GlcA 2-H, H-e, CH₂-q), 3.19 (dd, J_k,kЈ=

1

8.08–7.26 (m, 20 H, arom. H), 5.63 (m, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1

H, GalN^I4-H), 5.58 (dd, J_2,3= J_3,4= 8.5 Hz, 1 H, GlcA 3-H), 5.15

(dd, J_1,2= 7.5 Hz, 1 H, GlcA 2-H), 5.13 (d, 1 H, GlcA 1-H), 5.06

J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.02 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ),

2.80 (dd, 1 H, H-d), 2.30–2.24 (m, 4 H, CH₂-i, CH₂-o), 2.06, 2.03

[2 s, 6 H, NHC(O)CH₃], 1.76–1.30 (m, 12 H, CH₂-f, CH₂-g, CH₂-

(dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.01 (s, 2 H, CH₂- h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal

Ph), 4.93 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 4.58 (d, J_1,2

=

acetone, δ_C= 30. 83): δ = 177.63, 177.28, 175.68, 175.33, 175.15 (5

4198

www.eurjoc.org

Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

C, GlcA C-6, C=O), 166.02 (1 C, C-a), 104.22 (1 C, GlcA C-1),

101.87, 101.79 (2 C, 2 GalN C-1), 80.64, (1 C, GlcA C-4), 77.34,

77.07, 75.84, 75.80, 75.12, 74.19, 72.88, 71.74, 68.42 (8 C, 2 GalN

C-3, C-4, C-5, GlcA C-2, C-3, C-5), 68.90 (1 C, C-r), 62.76 (1 C,

C-b), 61.67, 61.63 (2 C, 2 GalN C-6), 60.92 (1 C, C-c), 56.06 (1 C,

(dd, J_4,5= 10.0 Hz, 1 H, GlcA^I4-H), 4.33 (dd, J_5,6b= 7.5 Hz, 1 H,

GalN^I6b-H), 4.21 (ABq, 2 H, CH₂Cl), 4.11 (d, 1 H, GlcA^II5-H),

4.01 (m, 1 H, GalN^I5-H), 3.98 (d, 1 H, GlcA^I5-H), 3.91 (ABq, 2

H, CH₂Cl), 3.89 (m, 1 H, GalN^II5-H), 3.75–3.70 (m, 2 H, GalN^II

6a-H, CH₂-O), 3.67, 3.62 (2 s, 6 H, COOCH₃), 3.53 (dd, J_5,6b

=

C-e), 52.94, 52.34 (2 C, 2 GalN C-2), 40.37 (1 C, C-d), 39.91, 39.74 7.0, J_6a,6b= 11.5 Hz, 1 H, GalN^II6b-H), 3.50–3.35 (m, 4 H, 2

(2 C, C-k, C-q), 36.30, 36.18 (2 C, C-i, C-o), 28.67, 28.50, 28.34, GalN 2-H, CH₂-N), 2.71–2.39 (m, 4 H, CH₂CO), 2.00 (s, 3 H,

26.22, 25.88, 25.66 (6 C, C-f, C-g, C-h, C-l, C-m, C-n), 23.15, 22.94 COCH₃) p pm . M S: m/ z: 20 77 [M + N H₄]⁺(for ^{3 5}Cl).

[2 C, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₄₀H₆₄N₆O₂₃S₂

C₉₁H₈₃Cl₈N₃O₃₅(2062.28): calcd. C 52.95, H 4.15, N 2.04; found

C 52.82, H 4.11, N 2.04.

[M – 2Na]^2–: 530.1732; found: 530.1738.

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(2-Acetamido-

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

2-deoxy-β-

D

-galactopyranosyl)-(1Ǟ4)-(sodium β-

D

-glucopyranosyl-

-galactopyr-

levulinoyl-β-

chloroacetyl-2-deoxy-2-trichloroacetamido-β-

(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-

4-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-trichloroacetamido-β- -ga-

lactopyranoside (61): From imidate 20 (207 mg, 0.18 mmol) and

alcohol 24 (150 mg, 0.14 mmol) for 2 h as described for the prepa-

ration of 21. Flash silica chromatography [CH₂Cl₂/acetone (12:1)

D

-glucopyranosyluronate)-(1Ǟ3)-(4-O-benzoyl-6-O-

uronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-β-

D

-galactopyranosyl)-

anoside (59): From 57 (14 mg, 19 μmol) as described for the prepa-

ration of 11 to afford the biotinylated trisaccharide 59 (20 mg,

70%) as a white powder. R_f= 0.50 (EtOAc/MeOH/water, 3:2:2).

[α]²_D²= +18 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

H₂O, δ_H= 4. 79): δ = 4.63 (ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz,

D

1 H, H-c), 4.52 (d, J_1,2= 8.0 Hz, 2 H, GalN^I1-H, GlcA 1-H), 4.48 containing 0.1% of NEt₃] afforded the tetrasaccharide 61 (181 mg,

(d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 4.44 (dd, J_b,e= 5.0 Hz, 1 H,

H-b), 4.26–4.21 (m, 2 H, GalN^I6-H), 4.18 (dd, J_4,5Ͻ 1.0, J_3,4

1.5 Hz, 1 H, GalN^I4-H), 4.03 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I2-

63%) as a white powder; m.p. 131–132 °C (from 2-propanol/petro-

leum ether). [α]²_D²= +33 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.07–7.22 (m, 33 H, arom. H), 7.04 (d, J_NH,2= 7.0 Hz,

=

H), 3.97–3.68 (m, 11 H, GalN^II2-H, 4-H, 6a-H, 6b-H, 2 GalN 3- 1 H, GalN^INH), 6.91 (d, J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 6.90–

H, 5-H, GlcA 4-H, 5-H, CH₂-r), 3.59 (dd, J_3,4= J_2,3= 9.0 Hz, 1

H, GlcA 3-H), 3.40–3.32 (m, 4 H, GlcA 2-H, H-e, CH₂-q), 3.19

(dd, J_k,kЈ= J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.02 (dd, J_d,dЈ= 13.0 Hz, 1

H, H-dЈ), 2.81 (dd, 1 H, H-d), 2.30–2.23 (m, 4 H, CH₂-i, CH₂-o),

2.07, 2.03 [2 s, 6 H, NHC(O)CH₃], 1.80–1.33 (m, 12 H, CH₂-f,

6.85 (m, 2 H, arom. H), 5.71 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H,

GalN^I4-H), 5.53 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^II3-H), 5.49

(dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.46 (dd, J_2,3

=

9.5, J_3,4= 10.0 Hz, 1 H, GlcA^I3-H), 5.34 (dd, J_4,5= 10.0 Hz, 1 H,

GlcA^II4-H), 5.26–5.18 [m, 3 H, 2 GlcA 2-H, NHC(O)O], 5.09 (d,

CH₂-g, CH₂-h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 5.05 (s, 2 H, CH₂-Ph), 5.02 (d,

D₂O, internal acetone, δ_C= 30. 83): δ = 177.60, 177.27, 175.70,

175.36, 175.26 (5 C, GlcA C-6, C=O), 165.98 (1 C, C-a), 104.89 (1

C, GlcA C-1), 101.93, 101.65 (2 C, 2 GalN C-1), 80.56, 80.23 (2 C,

J_1,2= 8 Hz, 1 H, GalN^I1-H), 4.94, 4.88 (2 d, J_1,2= 7.5 Hz, 2 H,

2 GlcA 1-H), 4.75 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.67 (dd,

J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 4.39 (dd, J_5,6a= 4.0, J_6a,6b

=

GalN^IC-3, GlcA C-4), 77.05, 75.88, 74.37, 73.30, 73.12, 11.5 Hz, 1 H, GalN^I6a-H), 4.29 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^I

71.77,68.45, 68.27 (8 C, GalN^IIC-3, 2 GalN C-4, C-5, GlcA C-2,

C-3, C-5), 69.03 (1 C, C-r), 68.37 (1 C, GalN^IC-6), 62.75 (1 C, C-

4-H), 4.14 (dd, J_5,6b= 7.5 Hz, 1 H, GalN^I6b-H), 4.12 (d, 1 H,

GlcA^II5-H), 4.07 (s, 2 H, CH₂Cl), 4.06 (d, 1 H, GlcA^I5-H), 4.00

b), 61.70 (1 C, GalN^IIC-6), 60.91 (1 C, C-c), 56.04 (1 C, C-e), (s, 2 H, CH₂Cl), 3.99 (m, 1 H, GalN^I5-H), 3.84, 3.74 (2 s, 6 H,

52.98, 51.69 (2 C, 2 GalN C-2), 40.37 (1 C, C-d), 39.99, 39.74 (2 COOCH₃), 3.86–3.77 (m, 2 H, GalN^II5-H, CH₂-O), 3.72–3.59 (m,

C, C-k, C-q), 36.29, 36.20 (2 C, C-i, C-o), 28.65, 28.50, 28.34, 26.22,

25.89, 25.65 (6 C, C-f, C-g, C-h, C-l, C-m, C-n), 23.14, 22.93 [2 C,

NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₄₀H₆₄N₆O₂₃S₂[M –

2Na]^2–: 530.1732; found: 530.1755.

3 H, 2 GalN 2-H, CH₂-O), 3.49 (dd, J_5,6a= 6.0, J_6a,6b= 11.5 Hz,

1 H, GalN^II6a-H), 3.45–3.30 (m, 2 H, CH₂-N), 3.13 (dd, J_5,6b

=

7.0 Hz, 1 H, GalN^II6b-H), 2.62–2.31 (m, 4 H, CH₂CO), 2.00 (s, 3

H, COCH₃) ppm. MS: m/z: 1927 [M – 2 ClCH₂C(O) + Na]⁺(for

³⁵Cl). C₉₁H₈₃Cl₈N₃O₃₅(2062.28): calcd. C 52.95, H 4.15, N 2.04;

found C 52.78, H 3.95, N 2.02.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

levulinoyl-β-

chloroacetyl-2-deoxy-2-trichloroacetamido-β-

(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β-

6-O-benzoyl-4-O-chloroacetyl-2-deoxy-2-trichloroacetamido-β-

lactopyranoside (60): From imidate 19 (287 mg, 250 μmol) and

alcohol 23 (210 mg, 195 μmol) for 2 h as described for the prepara-

tion of 21. Flash silica chromatography [CH₂Cl₂/acetone (12:1)

containing 0.1% of NEt₃] afforded the tetrasaccharide 60 (212 mg,

D

-glucopyranosyluronate)-(1Ǟ3)-(6-O-benzoyl-4-O-

D

-galactopyranosyl)-

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

D

-glucopyranosyluronate)-(1Ǟ3)- levulinoyl-β-

-ga- 2-trichloroacetamido-β-

O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-2-deoxy-

2-trichloroacetamido-β- -galactopyranoside (62): From 60 (109 mg,

D-glucopyranosyluronate)-(1Ǟ3)-(6-O-benzoyl-2-deoxy-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-

D

53 μmol) as described for the preparation of 37. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the diol 62

55 %) as a white powder; m.p. 131–132 °C (from 2-propanol). (85 mg, 84%) as a white powder; m.p. 135–136 °C (from 2-propa-

1

[α]²_D²= +24 (c = 1 in chloroform). H NMR (400 MHz, CDCl₃): δ

= 8.11–7.22 (m, 35 H, arom. H), 6.80 (d, J_NH,2= 7.0 Hz, 1 H,

GalN^INH), 6.78 (d, J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 5.62 (dd,

nol). [α]²_D²= +38 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.07–7.13 (m, 35 H, arom. H), 6.82 (d, J_NH,2= 7.0 Hz,

1 H, GalN^INH), 6.78 (d, J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 5.62

(dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^II3-H), 5.58 (dd, J_3,4= 8.0, J_2,3

= 9.0 Hz, 1 H, GlcA^I3-H), 5.44 (dd, J_4,5= 9.5 Hz, 1 H, GlcA^II4-

J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^I4-H), 5.57 (dd, J_2,3= J_3,4

=

9.5 Hz, 1 H, GlcA^II3-H), 5.49 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H,

GalN^II4-H), 5.47 (dd, J_2,3= J_3,4= 10.0 Hz, 1 H, GlcA^I3-H), 5.42

H), 5.42 (dd, J_1,2= 7.0 Hz, 1 H, GlcA^II2-H), 5.35 (dd, J_1,2

=

(dd, J_4,5= 10.0 Hz, 1 H, GlcA^II4-H), 5.28–5.22 (m, 2 H, 2 GlcA 7.0 Hz, 1 H, GlcA^I2-H), 5.20 [m, 1 H, NHC(O)O], 5.11 (d, J_1,2

2-H), 5.08 [m, 1 H, NHC(O)O], 5.07 (d, J_1,2= 8.0 Hz, 1 H, GalN^II8.0 Hz, 1 H, GalN^I1-H), 5.01 (s, 2 H, CH₂-Ph), 4.98, 4.95 (2 d, 2

1-H), 5.03 (s, 2 H, CH₂-Ph), 5.01 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1- H, 2 GlcA 1-H), 4.92 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.62 (dd,

H), 4.84, 4.78 (2 d, J_1,2= 7.5 Hz, 2 H, 2 GalN 1-H), 4.73 (dd, J_2,3

= 11.0 Hz, 1 H, GalN^I3-H), 4.63 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II7.0 Hz, 1 H, GalN^I6b-H), 4.48 (dd, J_3,4= 3.5, J_2,3= 11.0 Hz, 1

3-H), 4.43 (dd, J_5,6a= 4.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.36 H, GalN^I3-H), 4.43 (dd, J_3,4= 3.0, J_2,3= 11.0 Hz, 1 H, GalN^II3-

J_5,6a= 5.5, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-H), 4.55 (dd, J_5,6b

=

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4199

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

H), 4.41 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^I4-H), 4.21 (d, 1 H, GlcA^II

benzoyl-2-deoxy-β-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

5-H), 4.20 (m, 1 H, GalN^I4-H), 4.17 (d, 1 H, GlcA^I5-H), 4.11 (m, benzoyl-β-

D

-glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benz-

-galactopyranoside (65): From 63 (133 mg, 70 μmol)

1 H, GalN^II4-H), 4.09 (dd, J_5,6a= 5.0, J_6a,6b= 11.5.0 Hz, 1 H, oyl-2-deoxy-β-

D

GalN^II6a-H), 3.89–3.53 (m, 7 H, 2 GalN 2-H, 5-H, GalN^II6b-H, as described in the general procedure using a twofold amount of

CH₂-O), 3.68, 3.60 (2 s, 6 H, COOCH₃), 3.42–3.27 (m, 3 H, GalN^Ireagents. Flash silica chromatography (CH₂Cl₂/MeOH, 19:1) af-

4-OH, CH₂-N), 2.75 (d, J_1,2= 3.5 Hz, 1 H, GalN^II4-OH), 2.68– forded the bis-acetamide 65 (80 mg, 68%) as a white powder; m.p.

2.38 (m, 4 H, CH₂CO), 2.04 (s, 3 H, COCH₃) ppm. MS: m/z: 1925 143–144 °C (from EtOAc/petroleum ether). [α]²_D²= +15 (c = 1 in

[M + NH₄]⁺(for ³⁵Cl). C₈₇H₈₃Cl₆N₃O₃₃(1911.33): calcd. C 54.67, chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.02–6.88 (m, 35

H 4.38, N 2.20; found C 54.44, H 4.28, N 2.16.

H, arom. H), 5.56 (dd, J_2,3= J_3,4= 10.0 Hz, 1 H, GlcA^I3-H), 5.51

(dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H, GalN^I4-H), 5.40 (dd, J_2,3= J_3,4

= 9.5 Hz, 1 H, GlcA^II3-H), 5.41 (m, 1 H, GalN^INH), 5.33–5.23

(m, 5 H, GalN^IINH, 4-H, GlcA^I4^I-H^I, 2 GlcA 2-H), 5.15 [m, 1

H, NHC(O)O], 5.08 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 5.03 (s, 2

H, CH₂-Ph), 4.85–4.72 (m, 5 H, GalN^II1-H, 2 GalN 3-H, 2 GlcA

1-H), 4.06 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^II4-H), 4.03 (d, 1 H,

GlcA^II5^IV-H), 3.96 (d, J_4,5= 10.0 Hz, 1 H, GlcA^I5-H), 3.38 (s, 6

H, COOCH₃), 3.82–3.57 (m, 4 H, GalN^I5-H, OH, O-CH₂)_,3.48–

3.27 (m, 6 H, GalN^I2-H, 6a-H, 6b-H, GalN^II5-H, CH₂-N), 3.10

(m, 1 H, GalN^II2-H), 2.86–2.72 (m, 2 H, GalN^II6a-H, OH), 2.56–

2.31 (m, 4 H, CH₂CO), 2.20–2.12 (m, 1 H, GalN^II6b-H), 1.99 (s,

3 H, COCH₃), 1.29, 1.17 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS:

m/z: calcd. for C₈₇H₈₉N₃O₃₃Na₂[M + 2Na]²⁺: 874.7587; found:

874.7590.

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

levulinoyl-β-

2-trichloroacetamido-β-

O-benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-2-deoxy-

2-trichloroacetamido-β- -galactopyranoside (63): From 61 (115 mg,

D-glucopyranosyluronate)-(1Ǟ3)-(4-O-benzoyl-2-deoxy-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-

D

56 μmol) as described for the preparation of 37. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the diol 63

(82 mg, 77%) as a white powder; m.p. 146–148 °C (from 2-propa-

nol). [α]²_D²= +26 (c = 1 in chloroform). ¹H NMR (400 MHz,

CDCl₃): δ = 8.07–7.13 (m, 33 H, arom. H), 6.83 (d, J_NH,2= 7.0 Hz,

1 H, GalN^INH), 6.82–6.77 (m, 2 H, arom. H), 6.72 (d, J_NH,2

=

7.0 Hz, 1 H, GalN^IINH), 5.68 (dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H,

GalN^I4-H), 5.52 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^II3-H), 5.42

(dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^I3-H), 5.38 (dd, J_4,5Ͻ 1.0, J_3,4

= 3.0 Hz, 1 H, GalN^II4-H), 5.29 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^II

4-H), 5.27–5.20 (m, 2 H, 2 GlcA 2-H), 5.11 [m, 1 H, NHC(O)O],

5.05 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 5.05–5.01 (m, 3 H, GalN^I

1-H, CH₂-Ph), 4.91, 4.86 (2 d, J_1,2= 7.5 Hz, 2 H, 2 GlcA 1-H),

4.76 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.58 (dd, J_2,3= 11.0 Hz,

1 H, GalN^II3-H), 4.20 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^I4-H), 4.09

(d, 1 H, GlcA^II5-H), 4.00 (d, 1 H, GlcA^I5-H), 3.82, 3.71 (2 s, 6

H, COOCH₃), 3.83–3.42 (m, 8 H, GalN^I6a-H, 6b-H, 2 GalN 2-

H, 5-H, CH₂-O), 3.38–3.31 (m, 3 H, OH, CH₂-N), 2.89–2.79 (m, 2

H, GalN^II6a-H, OH), 2.56–2.31 (m, 4 H, CH₂CO), 2.27–2.20 (m,

1 H, GalN^II6b-H), 2.00 (s, 3 H, COCH₃) ppm. MS: m/z: 1925 [M

+ NH₄]⁺(for ³⁵Cl). HRMS: m/z: calcd. for C₈₇H₈₃Cl₆N₃O₃₃Na₂

[M + 2Na]²⁺: 976.6418; found: 976.6444.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benz-

oyl-4-O-levulinoyl-β-

acetamido-6-O-benzoyl-2-deoxy-4-O-sulfonato-β-

yl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-

(1Ǟ3)-2-acetamido-6-O-benzoyl-2-deoxy-4-O-sulfonato-β- -galac-

D

-glucopyranosyluronate)-(1Ǟ3)-(sodium 2-

D

-galactopyranos-

D

topyranoside (66): From 64 (115 mg, 68 μmol) as described for the

preparation of 41 to afford the disulfate 66 (105 mg, 81%) as a

white powder. R_f= 0.40 (CH₂Cl₂/MeOH, 9:1). [α]²_D²= +41 (c = 1

in 3:1 MeOH/chloroform). ¹H NMR (400 MHz, 3:1 CD₃OD/

CDCl₃): δ = 8.07–7.18 (m, 35 H, arom. H), 5.96 (dd, J_2,3= J_3,4

=

9.5 Hz, 1 H, GlcA^II3-H), 5.59 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^I

3-H), 5.12–5.07 (m, 2 H, GlcA^II2-H, 4-H), 5.48 (dd, J_1,2= 7.5 Hz,

1 H, GlcA^I2-H), 5.34 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II

4-H), 5.25 (d, 1 H, GlcA^I1-H), 5.22 (d, J_1,2= 7.5 Hz, 1 H, GlcA^II

1-H), 5.15–5.08 (m, 4 H, GalN^I4-H, GlcA^II4-H, CH₂Ph), 4.97 (d,

J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.75–4.65 (m, 2 H, GalN^I6a-H,

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O-

levulinoyl-β-

benzoyl-2-deoxy-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benz-

oyl-2-deoxy-β- -galactopyranoside (64): From 62 (184 mg, 96 μmol)

D

-glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-6-O-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

6b-H), 4.63 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.48 (d, J_4,5

=

D

10.0 Hz, 1 H, GlcA^II5-H), 4.40 (d, J_4,5= 10.0 Hz, 1 H, GlcA^I5-

H), 4.32–4.24 (m, 2 H, GalN^I5-H, GalN^II3-H), 4.14–4.05 (m, 3

H, GalN^I2-H, GalN^II2-H, 6a-H), 3.92–3.87 (m, 2 H, GalN^I3-H,

CH₂-O), 3.73, 3.68 (2 s, 6 H, COOCH₃), 3.70–3.62 (m, 3 H, 2

GalN^II6-H, CH₂-O), 3.48 (m, 1 H, GalNII 5-H), 3.30 (m, 2 H,

CH₂-N), 2.75–2.38 (m, 4 H, CH₂CO), 2.09 (s, 3 H, COCH₃), 1.54,

1.39 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₈₇H₈₇N₃O₃₉S₂[M – 2Na]^2–: 930.7179; found: 930.7227.

D

as described in the general procedure using a twofold amount of

reagents. Flash silica chromatography (CH₂Cl₂/MeOH, 19:1) af-

forded the bis-acetamide 64 (130 mg, 79%) as a white powder; m.p.

226–228 °C (from MeOH). [α]²_D²= +39 (c = 1 in 3:1 MeOH/chloro-

form). ¹H NMR (400 MHz, 3:1 CD₃OD/CDCl₃): δ = 8.20–7.40 (m,

35 H, arom. H), 5.94 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^II3-H),

5.80 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^I3-H), 5.61 (dd, J_4,5

=

10.0 Hz, 1 H, GlcA^II4-H), 5.58–5.50 (m, 2 H, 2 GlcA 2-H), 5.23,

5.22 (2 d, J_1,2= 7.5 Hz, 2 H, 2 GlcA 1-H), 5.20 (s, 2 H, CH₂Ph),

4.91 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.71 (d, J_1,2= 8.0 Hz, 1

H, GalN^II1-H), 470–4.65 (m, 2 H, GalN^I6a-H, 6b-H), 4.56–4.50

(m, 2 H, GlcA^I4-H, 5-H), 4.45 (d, 1 H, GlcA^II5-H), 4.38 (dd, J_2,3

= 10.0, J_3,4= 3.0 Hz, 1 H, GalN^II3-H), 4.33 (dd, J_4,5Ͻ 1.0 Hz, 1

H, GalN^II4-H), 4.30–4.20 (m, 3 H, GalN^I3-H, 4-H, GalN^II6a-

H), 4.05–3.95 (m, 4 H, GalN^I2-H, 5-H, GalN^II6b-H, CH₂-O),

3.85, 3.83 (2 s, 6 H, 2COOCH₃), 3.30–3.20 (m, 3 H, GalN^II2-H,

5-H, CH₂-O), 3.48–3.42 (m, 2 H, CH₂-N), 2.80–2.51 (m, 4 H,

CH₂CO), 2.21 (s, 3 H, COCH₃), 1.50, 1.46 [2 s, 6 H, NHC-

(O)CH₃] ppm. HRMS: m/z: calcd. for C_{8 7}H₈₉N₃O_{3 3}Na₂

[M + 2Na]²⁺: 874.7587; found: 874.7592.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benz-

oyl-4-O-levulinoyl-β-

acetamido-4-O-benzoyl-2-deoxy-6-O-sulfonato-β-

yl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β- -glucopyranosyluronate)-

(1Ǟ3)-2-acetamido-4-O-benzoyl-2-deoxy-6-O-sulfonato-β- -galac-

D

-glucopyranosyluronate)-(1Ǟ3)-(sodium 2-

D

-galactopyranos-

D

topyranoside (67): From 65 (60 mg, 350 μmol) as described for the

preparation of 41 to afford the disulfate 67 (40 mg, 67%) as a white

1

powder. [α]²_D²= +11 (c = 1 in 1:1 methanol/chloroform). H NMR

(400 MHz, 3:1 CD₃OD/CDCl₃): δ = 8.07–6.97 (m, 35 H, arom. H),

5.69 (dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H, GalN^I4-H), 5.68 (dd, J_2,3

=

J_3,4= 9.5 Hz, 1 H, GlcA^II3-H), 5.59 (m, 1 H, GlcA^I3-H), 5.54

(dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.28 (dd, J_4,5

10.0 Hz, 1 H, GlcA^II4-H), 5.20 (dd, J_1,2= 7.5 Hz, 2 H, 2 GlcA 2-

=

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3-di-O-benzoyl-4-O- H), 5.08, 5.07 (2 d, 2 H, 2 GlcA 1-H), 5.02 (ABq, 2 H, CH₂-Ph),

levulinoyl-β- -glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-4-O-

D

4.53 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.47 (d, J_1,2= 8.0 Hz, 1

4200

www.eurjoc.org

Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

H, GalN^II1-H), 4.34 (d, J_4,5= 10.0 Hz, 1 H, GlcA^II5-H), 4.29– 1 H, H-b), 4.10–4.04 (m, 4 H, 2 GalN 2-H, 2 GalN 3-H), 3.94 (m,

4.22 (m, 3 H, GalN^I6a-H, GlcA^I4-H, 5-H), 4.22 (dd, J_2,3

=

1 H, CH₂-O), 3.88–3.75 (m, 7 H, GlcA^I4-H, 2 GalN 5-H, 4GalN

11.0 Hz, 1 H, GalN^II3-H), 4.15–4.10 (m, 2 H, GalN^I2-H, 3-H), 6-H), 3.74 (m, 1 H, CH₂-O), 3.68, 3.67 (2 d, J_4,5= 10.0 Hz, 2 H, 2

4.07 (m, 1 H, GalN^I5-H), 3.92 (dd, J_5,6b= 8.0, J_6a,6b= 11.0 Hz, 1 GlcA 5-H), 3.60 (dd, J_2,3= 8.5, J_3,4= 9.0 Hz, 1 H, GlcA^I3-H),

H, GalN^I6b-H), 3.88–3.73 (m, 2 H, GalN^II5-H, CH₂-O), 3.83, 3.55 (dd, J_3,4= 9.0 Hz, 1 H, GlcA^II4-H), 3.49 (dd, 1 H, GlcA^II3-

3.73 (2 s, 6 H, COOCH₃), 3.72 (m, 1 H, GalN^II2-H), 3.62–3.48 H), 3.41 (dd, 1 H, GlcA^I2-H), 3.40–3.32 (m, 4 H, GlcA^II2-H, H-

(m, 2 H, GalN^II6a-H, CH₂-O), 3.32–3.25 (m, 3 H, GalN^II6b-H, e, CH₂-N), 3.20 (dd, J_k,kЈ= J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.03 (dd,

CH₂-N), 2.62–2.31 (m, 4 H, CH₂CO), 1.99 (s, 3 H, COCH₃), 1.28,

1.22 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₈₇H₈₇N₃O₃₉S₂[M – 2Na]^2–: 930.7179; found: 930.7227.

J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.81 (dd, 1 H, H-d), 2.30 (m, 4 H,

2CH₂CO), 2.06, 2.03 [2 s, 6 H, NHC(O)CH₃], 1.81–1.28 (m, 12

H, CH₂-f, CH₂-g, CH₂-h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR

(100MHz, D₂O, internal acetone, δ_C= 30. 83): δ = 177.30, 176.94,

175.31, 175.04 (6 C, 2 GlcA C-6, C=O), 165.68 (1 C, C-a), 104.05,

103.94 (2 C, 2 GlcA C-1), 101.64, 101.49 (2 C, 2 GalN C-1), 80.98

(1 C, GlcA^IC-4), 77.22, 76.98 (2 C, 2 GalN C-3), 76.92, 76.89 (2

C, 2 GalN C-4), 75.71, 75.64, 75.61, 75.13, 74.96, 74.09, 73.06,

72.66 (9 C, 2 GalN C-5, 2 GlcA C-2, C-3, C-5, GlcA^IIC-4), 68.77

(1 C, CH₂-O), 62.61 (1 C, C-b), 61.53 (2 C, 2 GalN C-6), 60.77 (1

C, C-c), 55.92 (1 C, C-e), 52.18, 52.11 (2 C, 2 GalN C-2), 40.24 (1

C, C-d), 39.59, 39.48 (2 C, CH₂-N, C-k), 36.16, 36.05 (2 C, C-i, C-

o), 28.54, 28.38, 28.22, 26.10, 25.75, 25.51 (6 C, C-f, C-g, C-h, C-

l, C-m, C-n), 23.08, 22.82 [2 C, NHC(O)CH₃] ppm. HRMS: m/z:

calcd. for C₄₆H₇₂N₆O₃₂S₃[M – 4Na + 2H]^2–: 658.1677; found:

658.1681.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

anosyluronate)-(1Ǟ3)-(sodium 2-acetamido-2-deoxy-4-O-sulfonato-

β- -galactopyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyluronate)-

(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-β- -galactopyranoside

D-glucopyr-

D

(68): From 66 (104 mg, 54 μmol) as described for the preparation

of 9 to afford the sodium salt 68 (55 mg, 84%) as a white powder.

R_f= 0.45 (EtOAc/MeOH/water, 3:2:1). [α]²_D²= –29 (c = 1 in water).

¹H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ = 7.45–

7.35 (m, 5 H, arom. H), 5.10 (s, 2 H, CH₂Ph), 4.77, 4.72 (2dd, J_3,4

= 2.0, J_4,5Ͻ 1.0 Hz, 2 H, 2 GalN 4-H), 4.54, 4.48 (2 d, J_1,2

=

8.0 Hz, 2 H, 2 GalN 1-H), 4.46 (d, J_1,2= 7.5 Hz, 2 H, 2 GlcA 1-

H), 4.08–3.98 (m, 4 H, 2 GalN 2-H, 2 GalN 3-H), 3.90 (m, 1 H,

CH₂-O), 3.84–3.74 (m, 7 H, GlcA^I4-H, 2 GalN 5-H, 4GalN 6-H),

3.70–3.62 (m, 3 H, GlcA^I5-H, GlcA^II5-H, CH₂-O), 3.57 (dd, J_2,3

=

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-

glucopyranosyluronate)-(1Ǟ3)-(sodium 2-acetamido-2-deoxy-6-O-

sulfonato-β- -galactopyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyl-

uronate)-(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-β- -galactopyr-

D-

J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 3.52 (dd, J_3,4= J_4,5= 9.0 Hz, 1 H,

GlcA^II4-H), 3.45 (dd, J_2,3= 9.0 Hz, 1 H, GlcA^II3-H), 3.37 (dd,

J_2,3= 9.0 Hz, 1 H, GlcA^I2-H), 3.34–3.27 (m, 3 H, GlcA^II2-H,

CH₂-N), 2.04, 1.96 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z:

calcd. for C₃₈H₅₃N₃O₃₁S₂[M – 4Na + 2H]^2–: 555.6052; found:

555.6063.

D

anoside (71): From 69 (15 mg, 12 μmol) as described for the prepa-

ration of 11 to afford the biotinylated tetrasaccharide 71 (13 mg,

77%) as a white powder. R_f= 0.50 (EtOAc/MeOH/water, 3:2:2).

[α]²_D²= –3.5 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

H₂O, δ_H= 4. 79): δ = 4.62 (ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz,

1 H, H-c), 4.56 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 4.52, 4.50 (2 d,

J_1,2= 8.0 Hz, 2 H, 2 GlcA 1-H), 4.51 (d, J_1,2= 8.5 Hz, 1 H, GalN^II

1-H), 4.45 (dd, J_b,e= 5.0 Hz, 1 H, H-b), 4.28–4.17 (m, 6 H, 2 GalN

4-H, 6a-H, 6b-H), 4.03, 4.02 (2 dd, J_2,3= 11.0 Hz, 2 H, 2 GalN 2-

H, 2-H), 3.96–3.66 (m, 9 H, 2 GalN 3-H, 5-H, GlcA^I4-H, 2 GlcA

5-H, CH₂-r), 3.60 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 3.50–

3.47 (m, 2 H, GlcA^II3-H, 4-H), 3.42–3.31 (m, 5 H, 2 GlcA 2-H,

H-e, CH₂-q), 3.19 (dd, J_k,kЈ= J_k,l= 7.0 Hz, 2 H, CH₂-k), 3.01 (dd,

J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.80 (dd, 1 H, H-d), 2.29–2.23 (m, 4

H,CH₂-i, CH₂-o), 2.03, 2.02 [2 s, 6 H, NHC(O)CH₃], 1.81–1.32 (m,

12 H, CH₂-f, CH₂-g, CH₂-h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C

NMR (100MHz, D₂O, internal acetone, δ_C= 30. 83): δ = 177.62,

177.30 (6 C, 2 GlcA C-6, C=O), 165.97 (1 C, C-a), 104.86, 104.75

(2 C, 2 GlcA C-1), 101.86, 100.60 (2 C, 2 GalN C-1), 81.75, 80.53,

80.47 (3 C, 2 GalN C-3, GlcA^IC-4), 76.98, 76.67, 75.89, 74.45,

73.30, 73.21, 73.19, 72.95, 72.91 (9 C, 2 GalN C-5, GlcA^IIC-4, 2

GlcA C-2, C-3, C-5), 68.96 (1 C, C-r), 68.33, 68.09 (2 C, 2 GalN

C-6), 68.19, 67.98 (2 C, 2 GalN C-4), 62.67 (1 C, C-b), 60.84 (1 C,

C-c), 55.96 (1 C, C-e), 51.60, 51.48 (2 C, 2 GalN C-2), 40.28 (1 C,

C-d), 39.90, 39.66 (2 C, C-k, C-q), 36.21, 36.12 (2 C, C-i, C-o),

28.57, 28.42, 28.26, 26.14, 25.81, 25.56 (6 C, C-f, C-g, C-h, C-l, C-

m, C-n), 23.13, 22.85 [2 C, NHC(O)CH₃] ppm. HRMS: m/z: calcd.

for C₄₆H₇₂N₆O₃₂S₃[M – 4Na + 2H]^2–: 658.1677; found: 658.1689.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

anosyluronate)-(1Ǟ3)-(sodium 2-acetamido-2-deoxy-6-O-sulfonato-

β- -galactopyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyluronate)-

(1Ǟ3)-2-acetamido-2-deoxy-6-O-sulfonato-β- -galactopyranoside

D-glucopyr-

D

(69): From 67 (38 mg, 20 μmol) as described for the preparation of

9 to afford the sodium salt 69 (17 mg, 71%) as a white powder. R_f

= 0.45 (EtOAc/MeOH/water, 3:2:1). [α]²_D²= –7.5 (c = 1 in water).

¹H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ = 7.45 (s,

5 H, arom. H), 5.14 (s, 2 H, CH₂-Ph), 4.56 (d, J_1,2= 8.5 Hz, 1 H,

GalN^I1-H), 4.52, 4.51 (2 d, J_1,2= 8.0 Hz, 2 H, 2 GlcA 1-H), 4.48

(d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 4.23 (dd, J_4,5Ͻ 1.0, J_4,3

=

3.0 Hz, 1 H, GalN^I4-H), 4.26–4.15 (m, 4 H, 2 GalN 6a-H, 6b-H),

4.17 (dd, J_4,5Ͻ 1.0, J_4,3= 3.0 Hz, 1 H, GalN^II4-H), 4.16–3.95 (m,

3 H, 2 GalN 2-H, 1GalN 5-H), 3.92 (m, 1 H, CH₂-O), 3.88–3.69

(m, 7 H, GalN^I3-H, GalN^II3-H, 1GalN 5-H, GlcA^I4-H, 5-H,

GlcA^II5-H, CH₂-O), 3.61 (dd, J_2,3= 8.5, J_3,4= 9.0 Hz, 1 H, GlcA^I

3-H), 3.51–3.47 (m, 2 H, GlcA^II3-H, 4-H), 3.42–3.31 (m, 4 H, 2

GlcA 2-H, CH₂-N), 2.04, 1.96 [2 s, 6 H, NHC(O)CH₃] ppm.

HRMS: m/z: calcd. for C₃₈H₅₃N₃O₃₁S₂[M – 4Na + 2H]^2–

:

555.6052; found: 555.6058.

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-

D

-

glucopyranosyluronate)-(1Ǟ3)-(sodium 2-acetamido-2-deoxy-4-O-

sulfonato-β- -galactopyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyl-

uronate)-(1Ǟ3)-2-acetamido-2-deoxy-4-O-sulfonato-β- -galactopyr-

D

anoside (70): From 68 (48 mg, 40 μmol) as described for the prepa-

ration of 11 to afford the biotinylated tetrasaccharide 70 (50 mg,

89%) as a white powder. R_f= 0.45 (EtOAc/MeOH/water, 3:2:2).

[α]²_D²= –12 (c = 1 in water). ¹H NMR (400 MHz, D₂O, internal

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-(4,6-di-O-acetyl-2-deoxy-2-tri-

chloroacetamido-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-4-O-

-galactopyranoside

D-

D

H₂O, δ_H= 4. 79): δ = 4.82, 4.77 (2 dd, J_3,4= 2.0, J_4,5Ͻ 1.0 Hz, 2 chloroacetyl-2-deoxy-2-trichloroacetamido-β-

D

H, 2 GalN 4-H), 4.65 (m, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 (73): From imidate 72^[7](241 mg, 0.27 mmol) and alcohol 23

H, H-c), 4.58, 4.56 (2 d, J_1,2= 8.0 Hz, 2 H, 2 GalN 1-H), 4.51, (200 mg, 0.18 mmol) as described for the preparation of 21. Flash

4.49 (2 d, J_1,2= 7.5 Hz, 2 H, 2 GlcA 1-H), 4.44 (m, J_b,e= 5.0 Hz,

silica chromatography [CH₂Cl₂/acetone (12:1) containing 0.1% of

4201

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

NEt₃] afforded the tetrasaccharide 73 (204 mg, 62%) as a white

powder; m.p. 138–140 °C (from 2-propanol). [α]²_D²= +12 (c = 1 in

(164 mg, 82%) as a white powder; m.p. 143–146 °C (from 2-propa-

nol). [α]²_D²= +16 (c = 1 in chloroform). ¹H NMR (400 MHz,

chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.12–7.31 (m, 20 CDCl₃): δ = 8.16–7.21 (m, 20 H, arom. H), 7.13 (d, J_NH,2= 7.0 Hz,

H, arom. H), 6.82 (d, J_NH,2= 7.5 Hz, 1 H, GalN^INH), 6.81 (d, 1 H, GalN^INH), 7.07 (d, J_NH,2= 7.5 Hz, 1 H, GalN^IINH), 5.61

J_NH,2= 9.0 Hz, 1 H, GalN^IINH), 5.67 (dd, J_4,5Ͻ 1.0, J_3,4

3.5 Hz, 1 H, GalN^I4-H), 5.54 (dd, J_2,3= 9.0, J_3,4= 9.5 Hz, 1 H,

=

(dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 5.42 (dd, J_1,2= 7.0 Hz,

1 H, GlcA^I2-H), 5.29 [m, 1 H, NHC(O)O], 5.22 (dd, J_4,5Ͻ 1.0,

GlcA^I3-H), 5.29 (dd, J_1,2= 7.5 Hz, 1 H, GlcA^I2-H), 5.22 (dd, J_4,5J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.15 (dd, J_3,4= 7.5, J_4,5= 9.0 Hz,

Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.18 (dd, J_3,4= 9.0, J_4,5

9.5 Hz, 1 H, GlcA^II4-H), 5.10 (dd, J_2,3= 9.0 Hz, 1 H, GlcA^II3-

=

1 H, GlcA^II4-H), 5.11 (dd, J_2,3= 9.0 Hz, 1 H, GlcA^II3-H), 5.03

(d, 1 H, GlcA^I1-H), 5.00 (s, 2 H, CH₂-Ph), 4.99 (d, J_1,2= 8.5 Hz,

H), 5.08 [m, 1 H, NHC(O)O], 5.03 (s, 2 H, CH₂-Ph), 5.01 (d, J_1,21 H, GalN^II1-H), 4.92 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.87

= 8.5 Hz, 1 H, GalN^I1-H), 4.95 (d, J_1,2= 8.5 Hz, 1 H, GalN^II1- (dd, J_1,2= 8.0 Hz, 1 H, GlcA^II2-H), 4.64 (dd, J_5,6a= 5.5, J_6a,6b

=

H), 4.86 (d, 1 H, GlcA^I1-H), 4.85 (dd, J_1,2= 7.5 Hz, 1 H, GlcA^II

2-H), 4.74 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.60 (d, 1 H,

GlcA^II1-H), 4.44 (dd, J_5,6a= 7.0, J_6a,6b= 11.5 Hz, 1 H, GalN^I6a-

11.0 Hz, 1 H, GalN^I6a-H), 4.63 (d, 1 H, GlcA^II1-H), 4.53 (dd,

J_5,6b= 7.0 Hz, 1 H, GalN^I6b-H), 4.50 (dd, J_2,3= 11.0 Hz, 1 H,

GalN^I3-H), 4.38 (dd, J_4,5= 9.0 Hz, 1 H, GlcA^I4-H), 4.35 (dd, J_2,3

H), 4.36 (dd, J_5,6b= 6.0 Hz, 1 H, GalN^I6b-H), 4.34 (dd, J_4,5

=

= 11.0 Hz, 1 H, GalN^II3-H), 4.26 (ddd, J_OH,4= J_4,5Ͻ 1.0, J_3,4

=

9.5 Hz, 1 H, GlcA^I4-H), 4.26 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3- 3.5 Hz, 1 H, GalN^I4-H), 4.20 (d, 1 H, GlcA^I5-H), 3.95 (d, 1 H,

H), 4.25 (ABq, 2 H, CH₂Cl), 4.06 (d, 1 H, GlcA^I5-H), 4.05 (m, 1

GlcA^II5-H), 3.91 (m, 1 H, GalN^I5-H), 3.83 (m, 1 H, CH₂-O),

H, GalN^I5-H), 3.93 (d, 1 H, GlcA^II5-H), 3.83 (m, 1 H, CH₂-O), 3.71, 3.69 (2 s, 6 H, COOCH₃), 3.72–3.60 (m, 3 H, GalN^II5-H, 2

3.73, 3.68 (2 s, 6 H, COOCH₃), 3.70–3.59 (m, 3 H, GalN^II2-H, 5- GalN 2-H, CH₂-O), 3.44 (dd, J_5,6a= 6.5, J_6a,6b= 11.0 Hz, 1 H,

H, CH₂-O), 3.47 (m, 1 H, GalN^I2-H), 3.43 (dd, J_5,6a= 6.0, J_6a,6b

= 11.5 Hz, 1 H, GalN^II6a-H), 3.39–3.26 (m, 2 H, CH₂-N), 3.23

GalN^II6a-H), 3.39–3.31 (m, 2 H, CH₂-N), 3.44 (dd, J_5,6b= 7.0 Hz,

1 H, GalN^II6b-H), 3.21 (d, 1 H, OH), 2.02, 2.01, 1.98, 1.97,

(dd, J_5,6b= 6.0 Hz, 1 H, GalN^II6b-H), 2.06, 2.03, 1.98, 1.97, 1.83 1.83 (5 s, 15 H, OCOCH₃) ppm. HRMS: m/z: calcd. for

[5 s, 15 H, OC(O)CH₃] ppm. MS: m/z: 1801 [M + NH₄]⁺(for ³⁵Cl). C₇₁H₇₅Cl₆N₃O₃₃Na₂[M + 2Na]²⁺: 876.6105; found: 876.6108.

C₇₃H₇₆Cl₇N₃O₃₄(1787.57): calcd. C 49.05, H 4.29, N 2.35; found

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-D-

C 48.83, H 4.17, N 2.35.

glucopyranosyluronate)-(1Ǟ3)-(4,6-di-O-acetyl-2-deoxy-2-tri-

chloroacetamido-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

-glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-2-deoxy-2-

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O- trichloroacetamido-β- -galactopyranoside (76): From 74 (554 mg,

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

D-

D

glucopyranosyluronate)-(1Ǟ3)-(4,6-di-O-acetyl-2-deoxy-2- benzoyl-β-

trichloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-4-O-benzoyl-6-O-

chloroacetyl-2-deoxy-2-trichloroacetamido-β- -galactopyranoside

D

0.31 mmol) as described for the preparation of 37. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the alcohol 76

D

(74): From imidate 72 (301 mg, 0.35 mmol) and alcohol 24 (454 mg, 86%) as a white powder. [α]²_D²= +24 (c = 1 in chloroform).

(250 mg, 0.23 mmol) as described for the preparation of 21. Flash

silica chromatography [CH₂Cl₂/acetone (10:1) containing 0.1% of

NEt₃] afforded the tetrasaccharide 74 (278 mg, 67%) as a white

powder; m.p. 130–131 °C (from 2-propanol). [α]²_D²= +25 (c = 1 in

¹H NMR (400 MHz, CDCl₃): δ = 8.15–7.20 (m, 20 H, arom. H),

6.82 (d, J_NH,2= 7.0 Hz, 1 H, GalN^INH), 6.75 (d, J_NH,2= 8.0 Hz,

1 H, GalN^IINH), 5.72 (dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H, GalN^I

4-H), 5.53 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^I3-H), 5.25 (dd, J_1,2

chloroform). ¹H NMR (400 MHz, CDCl₃): δ = 8.15–7.30 (m, 20 = 8.0 Hz, 1 H, GlcA^I2-H), 5.18 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1

H, arom. H), 6.86 (d, J_NH,2= 7.0 Hz, 1 H, GalN^INH), 6.81 (d, H, GalN^II4-H), 5.14–5.10 (m, 2 H, GlcA^II3-H, 4-H), 5.12 [m, 1

J_NH,2= 7.5 Hz, 1 H, GalN^IINH), 5.73 (dd, J_4,5Ͻ 1.0, J_3,4

=

H, NHC(O)O], 5.04 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 5.03 (s, 2

3.5 Hz, 1 H, GalN^I4-H), 5.52 (dd, J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^IH, CH₂-Ph), 4.93 (d, 1 H, GlcA^I1-H), 4.91 (d, J_1,2= 8.5 Hz, 1 H,

3-H), 5.23 (dd, J_1,2= 7.5 Hz, 1 H, GlcA^I2-H), 5.22 (dd, J_4,5Ͻ 1.0, GalN^II1-H), 4.84 (dd, J_1,2= 8.0, J_2,3= 9.0 Hz, 1 H, GlcA^II2-H),

J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 5.15 (dd, J_3,4= 9.0, J_4,5= 9.0 Hz, 4.79 (dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.58 (d, 1 H, GlcA^II1-

1 H, GlcA^II4-H), 5.11 [m, 1 H, NHC(O)O], 5.10 (dd, J_2,3= 9.0 Hz, H), 4.23–4.17 (m, 2 H, GalN^II3-H, GlcA^I4-H), 4.07 (d, J_4,5

=

1 H, GlcA^II3-H), 5.06 (s, 2 H, CH₂-Ph), 5.05 (d, J_1,2= 7.5 Hz, 1 10.0 Hz, 1 H, GlcA^I5-H), 3.92 (d, J_4,5= 9.5 Hz, 1 H, GlcA^II5-H),

H, GalN^I1-H), 4.90 (d, 1 H, GlcA^I1-H), 4.89 (d, J_1,2= 8.5 Hz, 1 3.86, 3.71 (2 s, 6 H, COOCH₃), 3.85 (m, 1 H, CH₂-O), 3.81 (m, 1

H, GalN^II1-H), 4.85 (dd, J_1,2= 7.5 Hz, 1 H, GlcA^II2-H), 4.79 H, GalN^I5-H), 3.72–3.60 (m, 5 H, GalN^I6a-H, GalN^II5-H, 2

(dd, J_2,3= 11.0 Hz, 1 H, GalN^I3-H), 4.59 (d, 1 H, GlcA^II1-H),

4.41 (dd, J_5,6a= 4.0, J_6a,6b= 12.0 Hz, 1 H, GalN^I6a-H), 4.29–4.25

GalN 2-H, CH₂-O), 3.50 (m, 1 H, GalN^I6b-H), 3.40–3.32 (m, 3 H,

GalN^II6a-H, OH, CH₂-N), 3.14 (dd, J_5,6b= 6.5, J_6a,6b= 11.5 Hz, 1

(m, 2 H, GalN^II3-H, GlcA^I4-H), 4.16 (dd, J_5,6b= 7.5 Hz, 1 H, H, GalN^II6b-H), 2.02, 1.98, 1.97, 1.96, 1.82 (5 s, 15 H, OCOCH₃)

GalN^I6b-H), 4.08 (d, J_4,5= 10.0 Hz, 1 H, GlcA^I5-H), 4.03 (m, 1

ppm. HRMS: m/z: calcd. for C₇₁H₇₅Cl₆N₃O₃₃Na₂[M + 2Na]²⁺

:

H, GalN^I5-H), 4.02 (ABq, 2 H, CH₂Cl), 3.93 (d, 1 H, GlcA^II5- 876.6105; found: 876.6135.

H), 3.85 (m, 1 H, CH₂-O), 3.88, 3.68 (2 s, 6 H, COOCH₃), 3.65–

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

D-

3.59 (m, 4 H, 2 GalN 2-H, 5-H, CH₂-O), 3.38 (dd, J_5,6a= 7.0, J_6a,6b

= 12.0 Hz, 1 H, GalN^II6a-H), 3.39–3.36 (m, 2 H, CH₂-N), 3.18

(dd, J_5,6b= 6.5 Hz, 1 H, GalN^II6b-H), 2.03, 2.00, 1.98, 1.96, 1.82

[ 5 s, 1 5 H , O C ( O ) CH ₃] p p m . H R M S : m /z : c a l c d . fo r

C₇₃H₇₆Cl₇N₃O₃₄Na₂[M + 2Na]²⁺: 914.5963; found: 914.5965.

glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-4,6-di-O-acetyl-2-

deoxy-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benzoyl-2-deoxy-β-

-galactopyranoside (77): From 75 (150 mg, 90 μmol) as described

D

D-

D

in the general procedure using a twofold amount of reagents. Flash

silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded the bis-acet-

amide 77 (83 mg, 63%) as a white powder; m.p. 158–161 °C (from

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-(4,6-di-O-acetyl-2-deoxy-2-tri-

chloroacetamido-β-

benzoyl-β- -glucopyranosyluronate)-(1Ǟ3)-6-O-benzoyl-2-deoxy-2-

trichloroacetamido-β- -galactopyranoside (75): From 73 (210 mg,

D-

D

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O- 2-propanol). [α]²_D²= +22 (c = 1 in chloroform). ¹H NMR

D

(400 MHz, CDCl₃): δ = 8.09–7.21 (m, 20 H, arom. H), 5.90 (d,

J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 5.63 (d, J_NH,2= 6.5 Hz, 1 H,

GalN^INH), 5.58 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 5.41

(dd, J_1,2= 7.5 Hz, 1 H, GlcA^I2-H), 5.30 [m, 1 H, NHC(O)O],

D

0.12 mmol) as described for the preparation of 37. Flash silica

chromatography (CH₂Cl₂/MeOH, 19:1) afforded the alcohol 75

4202

www.eurjoc.org

Eur. J. Org. Chem. 2011, 4183–4204

Construction of Chondroitin Oligomers

5.16–5.09 (m, 3 H, GalN^II4-H, GlcA^II3-H, 4-H), 4.99 (s, 2 H,

H, CH₂-N), 2.04, 1.97, 1.96, 1.95, 1.92 [5 s, 18 H, OC(O)CH₃,

NHC(O)CH₃], 1.52 [s, 3 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd.

CH₂-Ph), 4.96 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H), 4.92 (d, 1 H,

GlcA^I1-H), 4.87 (d, J_1,2= 8.0 Hz, 1 H, GalN^II1-H), 4.87 (m, 1 for C₇₁H₈₀N₃O₃₆SNa₃[M + 2Na]²⁺: 825.6968; found: 825.6967.

H, GlcA^II2-H), 4.60 (d, J_1,2= 7.5 Hz, 1 H, GlcA^II1-H), 4.60–4.55

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-

(m, 2 H, GalN^I3-H, 6a-H), 4.54–4.48 (m, 2 H, GalN^I6b-H, GalN^II

acetyl-β-

acetyl-2-desoxy-β-

benzoyl-6-O-sulfonato-β-

amido-6-O-benzoyl-2-desoxy-β-

D

-glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-4,6-di-O-

-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-

-glucopyranosyluronate)-(1Ǟ3)-2-acet-

-galactopyranoside (80): From 78

3-H), 4.35 (dd, J_4,5= 9.0 Hz, 1 H, GlcA^I4-H), 4.18 (d, 1 H, GlcA^I

5-H), 4.14 (m, 1 H, GalN^I4-H), 3.94 (d, J_4,5= 9.5 Hz, 1 H, GlcA^II

5-H), 3.87 (m, 1 H, GalN^I5-H), 3.75 (m, 1 H, CH₂-O), 3.73, 3.71

(2 s, 6 H, COOCH₃), 3.63–3.55 (m, 2 H, GalN^II5-H, CH₂-O), 3.48

(dd, J_5,6a= 7.0, J_6a,6b= 11.5 Hz 1 H, GalN^II6a-H), 3.32–3.15 (m,

5 H, GalN^II6b-H, 2 GalN 2-H, CH₂-N), 3.91 (d, J_OH,4Ͻ 1.0 Hz,

1 H, OH), 2.01, 2.00, 1.98, 1.97, 1.92 [5 s, 15 H, OC(O)CH₃], 1.77,

1.27 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₇₁H₈₁N₃O₃₃Na₂[M + 2Na]²⁺: 774.7274; found: 774.7280.

D

(248 mg, 0.17 mmol) as described for the preparation of 41 to af-

ford the 6-sulfate 80 (206 mg, 78%) as a white powder. [α]²_D²= +4

1

(c = 1 in methanol). H NMR (400 MHz, CD₃OD): δ = 8.11–7.22

(m, 20 H, arom. H), 5.75 (dd, J_4,5Ͻ 1.0, J_3,4= 2.5 Hz, 1 H, GalN^I

4-H), 5.57 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 5.25 (dd,

J_2,3= J_3,4= 9.5 Hz, 1 H, GlcA^II3-H), 5.19 (dd, J_1,2= 7.5 Hz, 1 H,

GlcA^I2-H), 5.13 (d, 1 H, GlcA^I1-H), 5.12 (m, 1 H, GalN^II4-H),

5.03 (dd, J_4,5= 10.0 Hz, 1 H, GlcA^II4-H), 5.02 (s, 2 H, CH₂-Ph),

4.87–4.78 (m, 2 H, GlcA^II1-H, 2-H), 4.50 (d, J_1,2= 8.0 Hz, 1 H,

GalN^I1-H), 4.44 (d, J_1,2= 8.5 Hz, 1 H, GalN^II1-H), 4.29 (dd,

J_5,6a= 3.5, J_6a,6b= 11.0 Hz, 1 H, GalN^I6a-H), 4.23, 4.22 (2 d, J_4,5

= 10.0 Hz, 2 H, GlcA^I5-H, GlcA^II5-H), 4.18–4.15 (m, 3 H, GalN^I

2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-acetyl-β-

glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-4,6-di-O-acetyl-2-

deoxy-β- -galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benzoyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-acetamido-4-O-benzoyl-2-deoxy-β-

-galactopyranoside (78): From 76 (455 mg, 0.27 mmol) as de-

D-

D

D-

D

scribed in the general procedure using a twofold amount of rea-

gents. Flash silica chromatography (CH₂Cl₂/MeOH, 19:1) afforded

the bis-acetamide 78 (248 mg, 63%) as a white powder; m.p. 148–

151 °C (from EtOAc/petroleum ether). [α]²_D²= +24 (c = 1 in chloro-

form). ¹H NMR (400 MHz, CDCl₃): δ = 8.15–7.20 (m, 20 H, arom.

2-H, 3-H, GlcA^I4-H), 4.10 (m, 1 H, GalN^I5-H), 4.01 (dd, J_2,3

=

11.0, J_3,4= 2.5 Hz, 1 H, GalN^II3-H), 3.97, 3.67 (2 s, 6 H, CO-

OCH₃), 3.96–3.84 (m, 2 H, GalN^I6b-H, CH₂-O), 3.72 (m, 1 H,

GalN^II2-H), 3.60–3.56 (m, 2 H, GalN^II5-H, CH₂-O), 3.48 (dd,

J_5,6a= 6.0, J_6a,6b= 11.0 Hz, 1 H, GalN^II6a-H), 3.29 (m, 1 H, CH₂-

N), 3.06 (dd, J_5,6b= 6.5 Hz, 1 H, GalN^II6b-H), 2.02, 2.01, 1.99,

1.96, 1.94 [5 s, 15 H, OC(O)CH₃], 1.82, 1.25 [2 s, 6 H, NHC-

(O)CH₃] ppm. HRMS: m/z: calcd. for C₇₁H₈₀N₃O₃₆SNa₃[M +

2Na]²⁺: 825.6968; found: 825.6960.

H), 5.83 (d, J_NH,2= 7.0 Hz, 1 H, GalN^IINH), 5.61 (d, J_NH,2

=

7.0 Hz, 1 H, GalN^INH), 5.58 (dd, J_4,5Ͻ 1.0, J_3,4= 3.5 Hz, 1 H,

GalN^I4-H), 5.53 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 5.30

(dd, J_1,2= 8.0 Hz, 1 H, GlcA^I2-H), 5.26 [m, 1 H, NHC(O)O],

5.15–5.13 (m, 2 H, GlcA^II3-H, 4-H), 5.10 (dd, J_4,5Ͻ 1.0, J_3,4

=

3.0 Hz, 1 H, GalN^II4-H), 5.05 (d, J_1,2= 8.5 Hz, 1 H, GalN^I1-H),

5.03 (s, 2 H, CH₂-Ph), 4.89 (d, 1 H, GlcA^I1-H), 4.87 (dd, J_1,2

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

anosyluronate)-(1Ǟ3)-(2-acetamido-2-deoxy-β- -galactopyranosyl)-

(1Ǟ4)-(sodium β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-

deoxy-4-O-sulfonato-β- -galactopyranoside (81): From 79 (65 mg,

D-glucopyr-

=

8.0, J_2,3= 9.0 Hz, 1 H, GlcA^II2-H), 4.73 (d, J_1,2= 8.0 Hz, 1 H,

GalN^II1-H), 4.70 (m, 1 H, GalN^I3-H), 4.59 (d, 1 H, GlcA^II1-H),

4.50 (dd, J_2,3= 11.0 Hz, 1 H, GalN^II3-H), 4.17 (dd, J_4,5= 9.5 Hz,

1 H, GlcA^I4-H), 4.08 (d, 1 H, GlcA^I5-H), 3.94 (d, J_4,5= 10.0 Hz,

1 H, GlcA^II5-H), 3.82–3.77 (m, 2 H, GalN^I5-H, CH₂-O), 3.78,

3.70 (2 s, 6 H, COOCH₃), 3.69–3.61 (m, 2 H, GalN^I6a-H, CH₂-

O), 3.50–3.19 (m, 7 H, GalN^I2-H, GalN^I6b-H, GalN^II5-H, Gal-

N^II6a-H, OH, CH₂-N), 3.16–3.11 (m, 2 H, GalN^II2-H, GalN^II

6b-H), 2.02, 1.99, 1.97, 1.93, 1.91 [5 s, 15 H, OC(O)CH₃], 1.78,

1.23 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₇₁H₈₁N₃O₃₃Na₂[M + 2Na]²⁺: 774.7274; found: 774.7285.

D

40 μmol) as described for the preparation of 9 to afford the sodium

salt 81 (33 mg, 75%) as a white powder. R_f= 0.50 (EtOAc/MeOH/

1

water, 3:2:2). [α]²_D²= –15.5 (c = 1 in water). H NMR (400 MHz,

D₂O, internal H₂O, δ_H= 4. 79): δ = 7.42 (s, 5 H, arom. H), 5.14

(s, 2 H, CH₂-Ph), 5.74 (dd, J_4,5Ͻ 1.0, J_3,4= 1.5 Hz, 1 H, GalN^I

4-H), 4.53, 4.50 (2 d, J_1,2= 8.5 Hz, 2 H, 2 GalN 1-H), 4.50 (d, J_1,2

= 8.0 Hz, 1 H, GlcA^II1-H), 4.48 (d, J_1,2= 7.5 Hz, 1 H, GlcA^I1-

H), 4.18 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H), 4.05–4.00

(m, 3 H, GalN^I3-H, 2 GalN 2-H), 3.93 (m, 1 H, CH₂-O), 3.84–

3.66 (m, 11 H, GalN^II3-H, 2 GalN 5-H, 6a-H, 6b-H, GlcA^I4-H,

2 GlcA 5-H, CH₂-O), 3.58 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-

H), 3.51–3.47 (m, 2 H, GlcA^II3-H, 4-H), 3.40 (dd, J_1,2= 7.0 Hz,

1 H, GlcA^I2-H), 3.35–3.30 (m, 3 H, GlcA^II2-H, CH₂-N), 2.04,

1.97 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: calcd. for

C₃₈H₅₃N₃O₂₈S [M – 3Na + H]^2–: 515.6274; found: 515.6287.

Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Methyl 2,3,4-tri-O-

acetyl-β-

acetyl-2-deoxy-β-

oyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-acetamido-6-O-benzoyl-2-

deoxy-4-O-sulfonato-β- -galactopyranoside (79): From 77 (75 mg,

D

-glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-4,6-di-O-

D-galactopyranosyl)-(1Ǟ4)-(methyl 2,3-di-O-benz-

D

50 μmol) as described for the preparation of 41 to afford the 4-

sulfate 79 (67 mg, 84%) as a white powder. [α]²_D²= +23 (c = 1 in

methanol). ¹H NMR (400 MHz, CD₃OD): δ = 8.09–7.21 (m, 20 H, Sodium 2-(Benzyloxycarbonylamino)ethyl O-(Sodium β-

D

-glucopyr-

-galactopyranosyl)-

-glucopyranosyluronate)-(1Ǟ3)-2-acetamido-2-

1 H, GlcA^II3-H), 5.24 (dd, J_4,5Ͻ 1.0, J_3,4= 1.5 Hz, 1 H, GalN^Ideoxy-6-O-sulfonato-β-

-galactopyranoside (82): From 80 (114 mg,

4-H), 5.18 (d, 1 H, GlcA^I1-H), 5.17 (m, 1 H, GalN^II4-H), 5.03

70 μmol) as described for the preparation of 9 to afford the sodium

arom. H), 5.61 (dd, J_2,3= 7.0, J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 5.58 anosyluronate)-(1Ǟ3)-(2-acetamido-2-deoxy-β-

(dd, J_1,2= 7.0 Hz, 1 H, GlcA^I2-H), 5.33 (dd, J_2,3= J_3,4= 8.0 Hz,

(1Ǟ4)-(sodium β-

D

(d, J_4,5= 10.0 Hz, 1 H, GlcA^II4-H), 5.01 (s, 2 H, CH₂-Ph), 4.99 salt 82 (71 mg, 91%) as a white powder. R_f= 0.50 (EtOAc/MeOH/

(m, 1 H, GlcA^I4-H), 4.86–4.75 (m, 4 H, GalN^I6a-H, GalN^II1-H, water, 3:2:2). [α]²_D²= –18 (c = 1 in water). ¹H NMR (400 MHz,

GlcA^II1-H, 2-H), 4.65 (dd, J_5,6b= 8.0, J_6a,6b= 11.0 Hz, 1 H, GalN^ID₂O, internal H₂O, δ_H= 4. 79): δ = 7.45 (s, 5 H, arom. H), 5.13

6b-H), 4.55 (d, J_1,2= 8.0 Hz, 1 H, GalN^I1-H), 4.32 (d, J_4,5

=

(s, 2 H, CH₂-Ph), 4.53, 4.49 (2 d, J_1,2= 8.5 Hz, 2 H, 2 GalN 1-H),

4.51, 4.50 (2 d, J_1,2= 8.0 Hz, 2 H, 2 GlcA 1-H), 4.22–4.19 (m, 2

H, GalN^I6a-H, 6b-H), 4.18, 4.16 (2 dd, J_4,5Ͻ 1.0, J_3,4= 2.8 Hz,

2 H, 2 GalN 4-H), 4.05–3.99 (m, 2 H, 2 GalN 2-H), 3.93 (m, 1 H,

10.0 Hz, 1 H, GlcA^I5-H), 4.23 (d, 1 H, GlcA^II5-H), 4.15 (dd, J_2,3

= 11.0 Hz, 1 H, GalN^I3-H), 4.08–4.02 (m, 2 H, GalN^I2-H, 5-H),

3.93–3.90 (m, 2 H, GalN^II2-H, 3-H), 3.81, 3.66 (2 s, 6 H, CO-

OCH₃), 3.72 (m, 1 H, CH₂-O), 3.61–3.57 (m, 2 H, GalN^II6a-H, CH₂-O), 3.84–3.68 (m, 10 H, GalN^II6a-H, 6b-H, 2 GalN 3-H, 5-

CH₂-O), 3.42 (m, 1 H, GalN^II5-H), 3.27–3.11 (m, 3 H, GalN^II6b-

H, GlcA^I4-H, 2 GlcA 5-H, CH₂-O), 3.60 (dd, J_2,3= J_3,4= 9.0 Hz,

Eur. J. Org. Chem. 2011, 4183–4204

www.eurjoc.org

4203

A. Vibert, C. Lopin-Bon, J.-C. Jacquinet

FULL PAPER

1 H, GlcA^I3-H), 3.51–3.49 (m, 2 H, GlcA^II3-H, 4-H), 3.40 (dd, acetone, δ_C= 30.83): δ = 177.58, 177.24, 175.21, 175.35 (6 C, 2

J_1,2= 8.0 Hz, 1 H, GlcA^I2-H), 3.35–3.31 (m, 3 H, GlcA^II2-H,

GlcA C-6, C=O), 165.97 (1 C, C-a), 104.88, 104.77 (2 C, 2 GlcA

CH₂-N), 2.05, 1.97 [2 s, 6 H, NHC(O)CH₃] ppm. HRMS: m/z: C-1), 101.92, 101.49 (2 C, 2 GalN C-1), 80.98, 80.55, 80.36 (3 C, 2

calcd. for C₃₈H₅₃N₃O₂₈S [M – 3Na + H]^2–: 515.6268; found: GalN C-3, GlcA^IC-4), 77.04, 76.72, 75.95, 73.29 (4 C, 2 GalN C-

515.6266.

5, 2 GlcA C-5), 75.63, 72.44 (2 C, GlcA^IIC-3, C-4), 74.38 (1 C,

GlcA^IC-3), 73.37, 73.07 (2 C, 2 GlcA C-2), 69.02 (1 C, C-r), 68.36,

68.32 (2 C, 2 GalN C-4), 68.27 (1 C, GalN^IC-6), 62.74 (1 C, C-b),

61.73 (1 C, GalN^IIC-6), 60.89 (1 C, C-c), 56.03 (1 C, C-e), 51.67

(2 C, 2 GalN C-2), 40.36 (1 C, C-d), 39.97, 39.73 (2 C, C-k, C-q),

36.29, 36.19 (2 C, C-i, C-o), 28.65, 28.51, 28.34, 26.22, 25.89, 25.64

(6 C, C-f, C-g, C-h, C-l, C-m, C-n), 23.17, 22.92 [2 C, NHC-

(O)CH₃] ppm. HRMS: m/z: calcd. for C₄₆H₇₂N₆O₂₉S₂[M – 3Na +

H]^2–: 618.1893; found: 618.1903.

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-D-

glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-2-deoxy-β-

pyranosyl)-(1Ǟ4)-(sodium β- -glucopyranosyluronate)-(1Ǟ3)-2-

acetamido-2-deoxy-4-O-sulfonato-β- -galactopyranoside (83): From

D-galacto-

D

81 (19 mg, 17 μmol) as described for the preparation of 11 to afford

the biotinylated tetrasaccharide 83 (20 mg, 88%) as a white powder.

R_f= 0.50 (EtOAc/MeOH/water, 3:2:2). [α]²_D²= –8.5 (c = 0.70 in

1

water). H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ =

4.78 (dd, J_4,5Ͻ 1.0, J_3,4= 1.5 Hz, 1 H, GalN^I4-H), 4.63 (ddd, J_c,d

Ͻ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 H, H-c), 4.53, 4.51 (2 d, J_1,2

=

Acknowledgments

8.5 Hz, 2 H, 2 GalN 1-H), 4.50 (d, J_1,2= 8.0 Hz, 1 H, GlcA^II1-

H), 4.49 (d, J_1,2= 7.5 Hz, 1 H, GlcA^I1-H), 4.44 (dd, J_b,e= 5.0 Hz,

1 H, H-b), 4.18 (dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz, 1 H, GalN^II4-H),

4.06–3.99 (m, 3 H, GalN^I3-H, 2 GalN 2-H), 3.94 (m, 1 H, CH₂-

r), 3.85–3.65 (m, 10 H, GalN^II3-H, 2 GalN 5-H, 6a-H, 6b-H,

GlcA^I4-H, 5-H, CH₂-r), 3.68 (d, J_4,5= 9.5 Hz, 1 H, GlcA^II5-H),

3.60 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 3.51–3.46 (m, 2 H,

GlcA^II3-H, 4-H), 3.41 (d, J_2,3= 9.5 Hz, 1 H, GlcA^I2-H), 3.41–

This research was supported by the Agence Nationale de la Recher-

che (programmes ANR-05-BLAN “GT-GAG” and ANR-09-

BLAN “GlycoCart”. The authors are also grateful for support by

the Ministère de l’Enseignement Supérieur et de la Recherche

through a studentship to A. V., and to H. Blin for helpful technical

assistance.

3.30 (m, 4 H, GlcA^II2-H, H-e, CH₂-q), 3.19 (dd, J_k,kЈ= J_k,l

=

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7.0 Hz, 2 H, CH₂-k), 3.01 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ), 2.80

(dd, 1 H, H-d), 2.29–2.23 (m, 4 H, CH₂-i, CH₂-o), 2.04, 2.03 [2 s,

6 H, NHC(O)CH₃], 1.81–1.32 (m, 12 H, CH₂-f, CH₂-g, CH₂-h,

CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal

acetone, δ_C= 30. 83): δ = 177.62, 177.30 (6 C, 2 GlcA C-6, C=O),

165.97 (1 C, C-a), 104.80, 104.18 (2 C, 2 GlcA C-1), 101.80, 101.70

(2 C, 2 GalN C-1), 81.05, 80.81 (2 C, GalN^IIC-3, GlcA^IC-4),

77.37, 77.05, 76.77, 75.96, 75.73, 75.61, 75.13, 74.23, 73.39, 72.87,

72.47 (11 C, GalN^IC-3, C-4, 2 GalN C-5, GlcA^IIC-4, 2 GlcA C-

2, C-3, C-5), 68.92 (1 C, C-r), 68.30 (1 C, GalN^IIC-4), 62.76 (1 C,

C-b), 61.69 (1 C, 2 GalN C-6), 60.92 (1 C, C-c), 56.05 (1 C, C-e),

52.34, 51.67 (2 C, 2 GalN C-2), 40.37 (1 C, C-d), 39.91, 39.74 (2

C, C-k, C-q), 36.30, 36.19 (2 C, C-i, C-o), 28.67, 28.49, 28.34, 26.22,

25.87, 25.66 (6 C, C-f, C-g, C-h, C-l, C-m, C-n), 23.19, 22.95 [2 C,

NHC(O)CH₃] ppm. HRMS: m/z: calcd. for C₄₆H₇₂N₆O₂₉S₂[M –

3Na + H]^2–: 618.1898; found: 618.1902.

Sodium 2-(6-(Biotinylamido)hexanoyl)aminoethyl O-(Sodium β-

glucopyranosyluronate)-(1Ǟ3)-(2-acetamido-2-deoxy-β- -galacto-

pyranosyl)-(1Ǟ4)-(sodium β-

acetamido-2-deoxy-6-O-sulfonato-β-

D-

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15, 9561–9578.

D-galactopyranoside (84): From

82 (36 mg, 33 μmol) as described for the preparation of 11 to afford

the biotinylated tetrasaccharide 84 (37 mg, 87%) as a white powder.

R_f= 0.50 (EtOAc/MeOH/water, 3:2:2). [α]²_D²= –2.5 (c = 1 in water).

¹H NMR (400 MHz, D₂O, internal H₂O, δ_H= 4. 79): δ = 4.63

(ddd, J_c,dϽ 1.0, J_c,dЈ= 5.0, J_b,c= 8.0 Hz, 1 H, H-c), 4.53, 4.52 (2

d, J_1,2= 8.5 Hz, 2 H, 2 GalN 1-H), 4.50, 4.49 (2 d, J_1,2= 8.0 Hz,

1 H, 2 GlcA 1-H), 4.44 (dd, J_b,e= 5.0 Hz, 1 H, H-b), 4.24–4.21

(m, 2 H, GalN^I6a-H, 6b-H), 4.18 (2 dd, J_4,5Ͻ 1.0, J_3,4= 3.0 Hz,

2 H, 2 GalN 4-H), 4.06–3.99 (m, 2 H, 2 GalN 2-H), 3.94 (m, 1 H,

CH₂-r), 3.90–3.70 (m, 9 H, GalN^II6a-H, 6b-H, 2 GalN 3-H, 5 Gl-

HcA^I4-H, 5-H, CH₂-r), 3.70 (d, J_4,5= 9.5 Hz, 1 H, GlcA^II5-H),

3.60 (dd, J_2,3= J_3,4= 9.0 Hz, 1 H, GlcA^I3-H), 3.51–3.48 (m, 2 H,

GlcA^II3-H, 4-H), 3.41–3.33 (m, 5 H, 2 GlcA 2-H, H-e, CH₂-q),

3.22–3.20 (m, 2 H, CH₂-k), 3.01 (dd, J_d,dЈ= 13.0 Hz, 1 H, H-dЈ),

2.80 (dd, 1 H, H-d), 2.29–2.25 (m, 4 H, CH₂-i, CH₂-o), 2.04, 2.02

[2 s, 6 H, NHC(O)CH₃], 1.70–1.28 (m, 12 H, CH₂-f, CH₂-g, CH₂-

h, CH₂-l, CH₂-m, CH₂-n) ppm. ¹³C NMR (100MHz, D₂O, internal

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8207–8228.

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Ed. Engl. 1986, 25, 212–235.

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1980, 21, 381–382.

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Received: March 3, 2011

Published Online: May 27, 2011

4204

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Eur. J. Org. Chem. 2011, 4183–4204

Article Doi

Efficient and stereocontrolled construction of homo-and heterogeneously 4-and 6-sulfated biotinylated chondroitin oligomers

DOI: 10.1002/ejoc.201100298

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