Synthesis of chondroitin sulfate oligosaccharides using N-(tetrachlorophthaloyl)- and N-(trifluoroacetyl)galactosamine building blocks

FULL PAPER

DOI: 10.1002/ejoc.201402222

Synthesis of Chondroitin Sulfate Oligosaccharides Using

N-(Tetrachlorophthaloyl)- and N-(Trifluoroacetyl)galactosamine

Building Blocks

Giuseppe Macchione,^[a][‡]Susana Maza,^[a][‡]M. Mar Kayser,^[a]José L. de Paz,*^[a]and

Pedro M. Nieto*^[a]

Keywords: Carbohydrates / Oligosaccharides / Glycosylation / Glycosaminoglycans / Protecting groups

We have explored synthetic routes for the preparation of

chondroitin sulfate (CS) oligosaccharides based on the use of

N-tetrachlorophthaloyl- (N-TCP) and N-trifluoroacetyl-sub-

stituted (N-TFA) galactosamine building blocks. Using N-

TCP units, we carried out the total synthesis of two CS disac-

charides, demonstrating the compatibility of TCP protection

with the final deprotection/sulfation steps. However, an at-

tempted 2+2 coupling of N-TCP-containing disaccharides

for the synthesis of CS tetrasaccharides failed. In contrast, a

synthetic route using N-TFA galactosamine units efficiently

led to biologically relevant CS-like oligosaccharides. The N-

TFA groups could easily be removed at the end of the synthe-

sis, and microwave irradiation greatly facilitated the sulfation

reactions. The utility of this approach is illustrated with the

total synthesis of two CS-like tetrasaccharides with different

sulfate distribution patterns. Finally, we used a fluorescence

polarization assay to estimate the relative abilities of the syn-

thesized compounds to inhibit the interaction between FGF-

2 and heparin.

Introduction

Chondroitin sulfate (CS) is a linear sulfated polysac-

charide that belongs to the glycosaminoglycan family.^[1]CS

is made up of repeating of GlcA-β(1Ǟ3)-GalNAc-β(1Ǟ4)

disaccharide units (GlcA = d-glucuronic acid, GalNAc =

N-acetyl-d-galactosamine). This repeating unit can be sulf-

Figure 1. Some of the typical disaccharide units found in CS.

ated at various positions, giving rise to CS polymer chains tein receptors.^[4]For example, CS-E binds to several hepa-

with different sulfation patterns and a high level of struc- rin-binding growth factors and chemokines,^[5,6]and so plays

tural diversity. CS chains are classified into different types important roles in the central nervous system. Recent stud-

based on the distribution pattern of the sulfate groups, al- ies indicated that a CS-E tetrasaccharide interacts with mid-

though a combination of different patterns is often found kine, a growth factor that participates in the development

in CS samples. For instance, CS-A predominantly contains and repair of neural tissues.^[7]This interaction requires a

one sulfate at position 4 of the GalNAc unit, CS-C has a specific arrangement of sulfate groups, since other oligosac-

sulfate group at position 6 of the GalNAc, and CS-E has charides with different sulfation patterns bind significantly

two sulfates at positions 4 and 6 of the GalNAc residue more weakly (or not at all) to midkine. A different class of

(Figure 1).

CS, CS-A, has an important role in pregnancy-associated

CS is involved in important biological processes such as malaria.^[8]This subtype of CS binds to Plasmodium falcipa-

central nervous system development^[2]and malaria infec- rum erythrocyte membrane protein 1 (PfEMP1), and this

tion.^[3]The participation of CS in these processes is due to binding event is a critical step in the adhesion of P. falci-

the action of certain oligosaccharide sequences, with par- parum infected red blood cells to the placenta.

ticular sulfate distributions, which interact with several pro-

Due to the heterogeneity of the polymer, structurally de-

fined CS oligosaccharides are not easily obtained in signifi-

cant amounts from natural sources. Therefore, well-defined

synthetic oligosaccharides are required for the study of CS–

protein interactions at the molecular level, the establish-

ment of structure–activity relationships, and the evaluation

of the biological activities and potential therapeutic appli-

cations of this type of compounds. Despite recent advances

in the synthesis of CS oligosaccharides and their ana-

[a] Glycosystems Laboratory, Instituto de Investigaciones

Químicas (IIQ), Centro de Investigaciones Científicas Isla de

La Cartuja, CSIC and Universidad de Sevilla,

Americo Vespucio, 49, 41092 Sevilla, Spain

E-mail: jlpaz@iiq.csic.es

http://ww w .iiq.csic.es/en/glycosystem-laboratory

[‡] These authors contributed equally to this work

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201402222.

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Synthesis of Chondroitin Sulfate Oligosaccharides

logues,^[7,9–19]new approaches to the preparation of these benzylidenation, compound 1. Donor 2 was prepared from

molecules are still required. A well-designed protecting 1 by levulination at position 3, followed by oxidative re-

group strategy is required to obtain the right configurations moval of the 4-methoxyphenyl group with cerium(IV) am-

for the glycosidic bonds and the correct positions for the monium nitrate (CAN) and trichloroacetimidate activation.

sulfate groups. The choice of protection for the amino

group^[20]of the GalNAc units is an important point in this

design. Most reported syntheses of CS oligomers have used

N-(trichloroacetyl)galactosamine building blocks.^[21]How-

ever, the use of this type of units presents some limitations.

The formation of stable trichlorooxazoline by-products has

been reported in glycosidation reactions of 2-deoxy-2-tri-

chloroacetamido donors.^[22–24]Additionally, the final trans-

formation of the 2-trichloroacetamide to the desired 2-acet-

amido group can be problematic. The basic hydrolysis of

Scheme 1. Reagents and conditions: a) 4-methoxyphenol, BF₃·Et₂O,

CH₂Cl₂; b) NaOMe, MeOH, 0 °C; c) PhCH(OMe)₂, pTsOH,

multiple trichloroacetamide groups requires very long reac-

tion times,^[22]and the alternative reduction method [using

tributylstannane/AIBN (azobisisobutyronitrile) or Zn/ace-

tic acid] leads, in some cases, to significant amounts of

mono- and dichloroacetamide intermediates.^[25–27]For these

reasons, we decided to explore the use of new galactosamine

monomers that contain a N-tetrachlorophthaloyl (N-

TCP)^[28,29]or a N-trifluroacetyl (N-TFA)^[30–32]protecting

group for the synthesis of CS oligosaccharides.

We envisioned that the TCP group would be a good al-

ternative for 2-amino protection because this group avoids

the formation of stable oxazolines, while strongly favouring

the formation of 1,2-trans glycosidic linkages.^[20,33]More-

over, cleavage of the N-TCP moiety does not require the

harsh reaction conditions needed for the removal of the

analogous N-phthaloyl group.^[34]On the other hand, we en-

visioned that the N-TFA group could facilitate the final de-

protection steps, since it can be removed under milder con-

ditions than the trichloroacetyl group. The N-TFA group

also ensures high β selectivities in glycosidation reac-

tions.^[35,36]Below, we describe the results obtained with

both types of building block. As a first goal, we considered

the synthesis of CS tetrasaccharides, since it has been dem-

onstrated that tetramers are long enough to interact with

several proteins and show biological activity.^[7,9,37,38]

CH₃CN, 50% (3 steps, from 3); d) Lev₂O, DMAP [4-(dimeth-

ylamino)pyridine], CH₂Cl₂, 92%; e) CAN, toluene/CH₃CN/H₂O,

0 °C, 84%; f) Cl₃CCN, DBU (1,8-diazabicycloundec-7-ene),

CH₂Cl₂, 91%; MP = 4-methoxyphenyl, Lev = levulinoyl, TCA =

trichloroacetimidoyl.

Next, we studied the utility of 1 and 2 in glycosylation

reactions with glucuronic acid moieties to give fully pro-

tected CS disaccharide precursors (Scheme 2). Glucuronic

acid trichloroacetimidate 8^[41,42]was coupled with acceptor

1 to give disaccharide 9 in good yield. We tested different

reaction conditions, and the best results were obtained with

BF₃·Et₂O as catalyst and toluene as solvent. On the other

hand, donor 2 was coupled to acceptor 10^[43]to give disac-

charide 11, with the alternative monosaccharide sequence

GalNAc-GlcA. To introduce an amine-terminated linker at

the reducing end of the chain to allow further conjugation

of the final molecules, 11 was transformed into trichloro-

acetimidate 13. Glycosylation between 13 and 5-(benzyloxy-

carbonylamino)-1-pentanol gave the desired disaccharide

(i.e., 14) in good yield.

Results and Discussion

N-TCP Galactosamine Building Blocks for the Synthesis of

CS Oligosaccharides

We prepared GalNAc monomers 1 and 2 (Scheme 1),

which can be used as glycosyl acceptor and donor, respec-

tively, in coupling reactions with glucuronic acid derivatives

to give CS-like disaccharides. The 4,6-O-benzylidene acetal

allows the regioselective sulfation of these positions at the

end of the synthesis (see below). Tetraacetate 3^[39]was con-

verted into 4-methoxyphenyl glycoside 4 by treatment with

4-methoxyphenol and BF₃·Et₂O. De-O-acetylation of 4 is

not trivial: the N-TCP group is base-sensitive, and the for-

mation of methyl glycoside by-products has been observed

during the acid-catalysed hydrolysis of a similar glucos-

amine derivative.^[40]Therefore, de-O-acetylation was carried

Scheme 2. Reagents and conditions: a) BF₃·Et₂O, toluene, 54% +

27% recovered 1; b) TMSOTf (trimethylsilyl trifluoromethanesulf-

onate), CH₂Cl₂, 58% + 16% recovered 10; c) CAN, CH₂Cl₂/

CH₃CN/H₂O, 84%; d) Cl₃CCN, K₂CO₃, CH₂Cl₂, 95%; e) 5-(benz-

yloxycarbonylamino)-1-pentanol, TMSOTf, CH₂Cl₂, 67%; Z =

out under strictly controlled basic conditions to give, after benzyloxycarbonyl.

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Scheme 3. Reagents and conditions: a) TFA, CH₂Cl₂, 0 °C, 99% b) SO₃·Me₃N (10 equiv.), DMF, 100 °C, 40 min, MW, 83%; c) SO₃·Me₃N

(2 equiv.), DMF, 50 °C, 30 min, MW, 84%; d) H₂O₂, LiOH, THF; NaOH, MeOH, 86% for 19, 89% for 23; e) NH₂(CH₂)₂NH₂, DMF,

100 °C, 90 min, MW; f) Ac₂O, Et₃N, MeOH, 75% for 21, 88% for 25 (2 steps, from 19 and 23, respectively); g) H₂, Pd(OH)₂, H₂O/

MeOH, quantitative for 15, quantitative for 16.

Having achieved the synthesis of these fully protected di- charide could not be isolated. In an alternative approach,

saccharides, we then checked the compatibility of the N- we tried to prepare a (GalNAc-GlcA)₂tetrasaccharide from

TCP group with the deprotection and sulfation steps re- compound 11. Compound 11 was similarly converted into

quired for the synthesis of final deprotected CS oligomers. the corresponding acceptor and donor disaccharides, but

Thus, starting from 14, we successfully prepared disaccha- the 2+2 condensation of these units failed again. In all

rides 15 and 16, which correspond to the sulfation patterns these glycosylation trials, most of the starting glycosyl ac-

of CS-E and CS-C, respectively (Scheme 3). The benzyl- ceptor was recovered from the reaction mixture.

idene acetal of 14 was removed using TFA (trifluoroacetic

acid), and the resulting diol was extensively sulfated to give

Synthesis of CS Oligomers Using N-TFA-Protected

Galactosamine Units

18. Here, the reaction time was significantly decreased

(40 min) using microwave heating (MW).^[44]The next step

was the basic hydrolysis of the ester groups, which occurred

For the synthesis of biologically relevant CS tetrasac-

with concomitant partial hydrolysis of the N-TCP moiety charides, we turned our attention to N-TFA-protected

to give 19. The amide bond in 19 was cleaved by treatment GalNAc units. We have previously reported the preparation

with ethylenediamine in DMF. This reaction took only and use of this type of building blocks for the synthesis of

90 min using microwave heating.^[45,46]Finally, the amine a hybrid chondroitin/dermatan sulfate oligosaccharide,^[43]

group of 20 was selectively acetylated, and the resulting de- containing both GlcA and l-iduronic acid (IdoA). Mono-

rivative 21 was hydrogenated to give CS-E disaccharide 15 saccharide 26, containing

a 4,6-O-di-tert-butylsilylene

in good yield. For the preparation of CS-C dimer 16, diol group, acted as an excellent glycosyl acceptor in a coupling

17 was heated at 50 °C under microwave irradiation in the reaction with poorly reactive GlcA trichloroacetimidate 27

presence of SO₃·NMe₃complex (2 equiv.). The mixture was to generate key intermediate 28 (Scheme 4).^[43]

stirred for 30 min, and 6-O-sulfated compound 22 was ob-

Here, we expand the utility of this N-TFA monomer to

tained in high yield. When we carried out the selective 6- the preparation of a CS tetrasaccharide sequence, contain-

OH sulfation with conventional heating in an oil bath, the ing exclusively GlcA (Scheme 5). For this purpose, we

reaction time was much longer, and the yield was lower, planned a 2+2 condensation of appropriate disaccharide

due to the recovery of some unsulfated starting material. derivatives. First, 28 was transformed into a suitable donor

Saponification, followed by amide bond cleavage, N-acet- containing acyl groups at positions 4 and 6 of the GalNAc

ylation, and hydrogenolysis gave final disaccharide 16.

moiety in order to selectively obtain the β(1Ǟ4) glycosidic

Next, we attempted the synthesis of longer CS sequences bond. Thus, compound 28 was treated with (HF)_n·Py and

by a 2+2 coupling of disaccharide units. For this purpose, then with levulinic anhydride and DMAP to give disac-

disaccharide 9 was transformed into the corresponding charide 29. Trichloroacetimidate 31 was obtained by

glycosyl acceptor (by delevulination) and donor (by cleav- cleavage of the anomeric 4-methoxyphenyl group followed

age of the anomeric 4-methoxyphenyl group and trichloro- by treatment with trichloroacetonitrile and DBU. Coupling

acetimidate formation). Unfortunately, the 2+2 glycosyl- between 31 and 32^[43]gave tetrasaccharide 33 in good yield.

ation failed, and the desired (GlcA-GalNAc)₂tetrasac- Importantly, 31 was prepared immediately before its use in

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Synthesis of Chondroitin Sulfate Oligosaccharides

Scheme 4. Synthesis of key disaccharide 28 using N-TFA unit 26.

the glycosidation reaction, since we observed partial de-

composition of this trichloroacetimidate after extended

storage, even at low temperature (–28 °C/4 °C range). Tetra-

saccharide 33 has a versatile protecting group distribution

that paves the way for the preparation of CS sequences with

different sulfation motifs. We decided to prepare, for the

first time, unnatural oversulfated compound 38, since we

have found that analogous IdoA-containing tetrasac-

charides (Figure 3, see below) are able to interact with

FGF-2 (basic fibroblast growth factor), a heparin-binding

protein involved in angiogenesis. Thus, removal of the silyl-

ene group was followed by basic hydrolysis and selective N-

acetylation to give 36. Treatment with SO₃·NMe₃complex

at 100 °C using microwave irradiation^[47]gave hepta-O-sulf-

ated tetrasaccharide 37. The introduction of the sulfate

Scheme 5. Reagents and conditions: a) (HF)_n·Py, THF, 0 °C;

Lev₂O, Py, DMAP, 83%; b) CAN, CH₂Cl₂/CH₃CN/H₂O, 81 %;

c) Cl₃CCN, DBU, CH₂Cl₂, 70%; d) TMSOTf, CH₂Cl₂, 0 °C, 71%;

e) (HF)_n·Py, THF, 0 °C, 75%; f) LiOH, H₂O₂, THF; NaOH,

MeOH; g) Ac₂O, MeOH, Et₃N, 90% (2 steps, from 34);

h) SO₃·Me₃N, DMF, 100 °C, MW, 2 h, 56%; i) H₂, Pd(OH)₂, H₂O/

MeOH, 97%.

1

groups at the desired positions was confirmed by H and

¹³C NMR spectroscopic data, which showed typical down-

field shifts for the sulfated positions (Tables 1 and 2). Fi-

nally, hydrogenolysis of 37 yielded the fully deprotected CS

tetramer 38. NMR spectroscopic analysis confirmed the

1

structure of 38. The H and ¹³C NMR spectroscopic data

Table 2. ¹³C NMR chemical shifts [ppm] for sulfated positions of

compounds 37 and 38, and the corresponding nonsulfated posi-

tions of 36.

are in full agreement with those reported for similar CS

derivatives.

As mentioned before, 38 is an oversulfated CS sequence

with a sulfation pattern not found in nature. To demon-

strate that N-TFA building blocks can be also used for the

preparation of oligosaccharides with naturally occurring

sulfation patterns, we synthesized, for the first time, tetra-

saccharide 50 (Scheme 6). This compound contains sulfate

groups at positions 4 and 6 of the GalNAc units, the sulf-

ation pattern of biologically relevant CS-E. Moreover, 50

C-4A

C-6A

61.1

69.3–68.6 80.0

69.0–68.7 80.7

C-2B C-4C

C-6C

61.1

69.3–68.6 80.0

69.0–68.7 80.7

C-2D C-4D

36^[a]67.6

37^[b]76.9

38^[b]77.0

72.7

67.6

76.9

77.0

73.0

–

[c]

77.2

79.0

[a] Sodium salt, in [D₄]methanol. [b] Sodium salt, in D₂O. [c] Not

determined.

has a IdoA unit at the nonreducing end. It has been re- copolymers, has critical roles in the development of the cen-

ported that the presence of IdoA (instead of GlcA) in CS tral nervous system.^[2,48]Oligosaccharide sequences con-

–

chains, giving rise to hybrid chondroitin/dermatan sulfate taining GlcA/IdoA-GalNAc (4,6-OSO₃) interact with

1

Table 1. H NMR Chemical shifts [ppm] for sulfated positions of compounds 37 and 38, and the corresponding nonsulfated positions of

36.

4A-H

6A-H

2B-H

4C-H

6C-H

2D-H

4D-H

36^[a]

37^[b]

38^[b]

4.19–4.12

4.96

4.95

3.86–3.56

4.36–4.10

4.36–4.21

3.46

4.41

4.28–4.21

4.19–4.12

4.96

4.93

3.86–3.56

4.36–4.10

4.36–4.21

3.41

4.41

4.28–4.21

3.72–3.56

4.93–4.85

4.51

[a] Sodium salt, in [D₄]methanol. [b] Sodium salt, in D₂O.

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l- and P-selectins and several chemokines and heparin-

Tetrasaccharide 50 was obtained from disaccharides

binding growth factors.^[6,49]The chemical synthesis of these 39^[43]and 32 (Scheme 6). The protecting groups of 39 were

structures, containing both GlcA and IdoA, will contribute rearranged to produce the CS-E sulfation pattern at the end

to determine the role of hybrid sequences in biological pro- of the synthesis. Delevulination followed by pivaloylation

cesses, including stem-cell proliferation, neurogenesis, and gave compound 40 (Scheme 7). The silylene group was then

neural network formation.

exchanged for two levulinoyl groups (Ǟ41). This allows se-

lective deprotection and subsequent sulfation at positions 4

and 6 of the GalNAc moiety, and, at the same time, favours

the correct stereochemistry for the glycosidic bond in the

2+2 condensation. The formation of trichloroacetimidate

43 was then achieved by oxidative removal of the anomeric

4-methoxyphenyl group with CAN followed by treatment

with trichloroacetonitrile and DBU. Fully protected tetra-

mer 44 was prepared by glycosylation of 43 and 32. The

protecting group distribution of 44 can lead, among other

outcomes, to a CS-E sulfation pattern. Treatment with

hydrazine monohydrate and then with (HF)_n·Py complex

gave tetraol 46. The released hydroxy groups were sulfated

in 30 min, using microwave heating, to give 47. The ¹H

NMR spectrum of 47 showed the expected downfield shifts

for 4-H GalNAc (δ = 4.14–3.93 ppm in 46; δ = 4.85–

4.80 ppm in 47) and 6a,b-H GalNAc (δ = 3.83–3.48 ppm in

46; δ = 4.52–4.25 in 47). The ¹³C NMR spectrum of 47 also

Scheme 6. Building blocks required for the synthesis of tetrasac-

charide 50.

Scheme 7. Reagents and conditions: a) NH₂NH₂·H₂O, Py/AcOH, CH₂Cl₂; PivCl, DMAP, Py, 87%; b) (HF)_n·Py, THF, 0 °C; Lev₂O, Py,

DMAP, 81%; c) CAN, CH₂Cl₂/CH₃CN/H₂O, 70%; d) Cl₃CCN, DBU, CH₂Cl₂, 78%; e) 32, TMSOTf, CH₂Cl₂, 0 °C, 53%;

f) NH₂NH₂·H₂O, Py/AcOH, CH₂Cl₂, 87%; g) (HF)_n·Py, THF, 0 °C, 97%; h) SO₃·Me₃N, DMF, 100 °C, MW, 30 min, quantitative;

i) LiOH, H₂O₂, THF; NaOH, MeOH; j) Ac₂O, MeOH, Et₃N, 86% (2 steps, from 47); k) H₂, Pd(OH)₂, H₂O/MeOH, quantitative.

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Synthesis of Chondroitin Sulfate Oligosaccharides

showed the expected downfield shifts of the signals for C-4

GalNAc (δ = 68.6 ppm in 46; δ = 75.4–75.1 ppm in 47) and

C-6 GalNAc (δ = 62.7–62.5 ppm in 46; δ = 68.0–67.5 ppm

in 47). Hydrolysis of ester and amide functionalities fol-

lowed by N-acetylation and hydrogenolysis gave tetrasac-

charide 50 in high yield. The structure of this compound

was confirmed by NMR spectroscopy (COSY, TOCSY, and

1

HSQC experiments) and mass spectrometry. The H and

¹³C NMR chemical shifts are in good agreement with those

published for similar oligosaccharides.

Figure 2. Competition assay to compare the relative inhibitory po-

tencies of the synthetic CS-like oligosaccharides. The graphic pres-

ents (in grey) the polarization values obtained from wells contain-

ing inhibitor (25 μm), FGF-2 (73 nm), and fluorescent heparin-like

probe (10 nm). The composition of the control wells (in white) is

described in the main text. All the measurements are the average

of three replicate wells, and the error bars show the standard devia-

tions for these measurements.

Fluorescence Polarization Competition Assay

FGF-2 is a heparin-binding protein^[50,51]that also recog-

nizes CS sequences.^[5]We finally screened the interactions

between FGF-2 and the synthetic water-soluble CS oligo-

saccharides (15, 16, 38, and 50, and dibenzylated tetrasac-

charide precursors 37 and 49). For this purpose, we used a

fluorescence polarization competition assay, previously de-

veloped in our group.^[43]This assay measures the relative

ability of the synthetic CS oligomers to inhibit the interac-

tion between FGF-2 and a fluorescently labelled heparin

probe. Briefly, the fluorescence polarization of samples con-

taining fixed concentrations of protein and fluorescent

probe were measured in the presence of the different CS

oligosaccharides (Figure 2). The binding of a CS oligomer

to FGF-2 displaces the fluorescent probe from its complex

with the protein, resulting in a decrease of the polarization

value. In this experiment we included two control samples

(in white, Figure 2). The first one (on the left) contained

only the fluorescent probe, and indicates the expected value

for 100% inhibition. The second one (on the right) con-

tained FGF-2 and fluorescent probe, without inhibitor, and

indicates the expected value for 0% inhibition. As shown in

Figure 2, at a 25 μm inhibitor concentration, disaccharides

15 and 16 and tetrasaccharides 49 and 50 showed only low

inhibitory activity, while compounds 37 and 38 were able

to inhibit 47 and 63%, respectively, of the interaction. How-

ever, the inhibitory activities of oversulfated compounds 37

and 38 were lower than those obtained with previously syn-

thesized IdoA-containing tetrasaccharides 51 and 52 (Fig-

ure 3).^[43]In summary, these data indicate that oversulfated,

unnatural tetrasaccharides (i.e., 37–38 and 51–52) show

stronger inhibitory abilities that those tetrasaccharides with

natural sulfation patterns (i.e., 49 and 50). These results

also suggest that the presence of an IdoA unit, instead of a

GlcA, at the nonreducing end of oversulfated structures, as

in compounds 51 and 52, can increase the relative binding

affinities of the synthetic unnatural CS oligosaccharides to

FGF-2. The interaction between FGF-2 and heparin is cru-

cial for tumor growth and angiogenesis. Therefore, the discov-

ery of compounds that inhibit the FGF-2/heparin interaction

is an area of great interest.^[52,53]Although the tested com-

pounds showed modest activities, this experiment gives some

interesting data on structural features beneficial for the inhibi-

tion of heparin binding to FGF-2. Moreover, this assay illus-

trates the potential of our fluorescence polarization method

for the fast comparison of relative inhibitory activities.

Figure 3. Structures of IdoA-containing tetrasaccharides 51 and

52.

Conclusions

We have explored and compared the utility of two dif-

ferently protected galactosamine units for the preparation

of CS oligosaccharides. Although N-TCP protection was

useful at the disaccharide level, we were not able to synthe-

size longer sequences with this type of unit. In contrast, we

have showed that a strategy based on N-TFA building

blocks can be used to synthesize biologically relevant CS

oligosaccharides in good yield. The efficiency of this ap-

proach was exemplified by the synthesis of two new CS-

like tetrasaccharides, with different sulfation patterns and

uronic acid distributions. The N-TFA groups led to the ster-

eoselective formation of 1,2-trans glycosidic bonds, and

they could be easily removed at the end of the synthesis.

Microwave irradiation facilitated the sulfation reactions. Fi-

nally, we compared the abilities of the synthetic CS oligo-

saccharides to inhibit heparin binding to FGF-2, and we

obtained some interesting information about structural

features that influence inhibitory activity.

Experimental Section

General Procedures: Thin-layer chromatography (TLC) analysis

was carried out on silica gel 60 F₂₅₄precoated on aluminium plates

(Merck), and the compounds were detected by staining with sulf-

uric acid/ethanol (1:9), with cerium(IV) sulfate (10 g)/phospho-

molybdic acid (13 g)/sulfuric acid (60 mL) solution in water (1 L),

or with anisaldehyde solution [anisaldehyde (25 mL) with sulfuric

acid (25 mL), ethanol (450 mL), and acetic acid (1 mL)], followed

by heating at Ͼ200 °C. Column chromatography was carried out

on silica gel 60 (0.2–0.5 mm, 0.2–0.063 mm or 0.040–0.015 mm;

Merck). Optical rotations were determined with a Perkin–Elmer

341 polarimeter. ¹H and ¹³C NMR spectra were acquired on

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Bruker DPX-300, Avance III-400, and DRX-500 spectrometers.

Unit A refers to the reducing-end monosaccharide in the NMR

spectroscopic data. Electrospray mass spectra (MS (ESI)) were re-

corded with an Esquire 6000 ESI-Ion Trap from Bruker Daltonics.

High-resolution mass spectra (HRMS) were carried out by CITIUS

(Universidad de Sevilla), CCiT (Universitat de Barcelona), and

SIdI (Universidad Autónoma de Madrid). Microwave-based sulf-

ation reactions were carried out using a Biotage Initiator Eight

synthesizer in sealed reaction vessels.

67.8 (C-3), 67.0 (C-5), 55.7 [Me(OMP)], 55.4 (C-2) ppm. HRMS:

calcd. for C₂₈H₂₁Cl₄NO₈Na [M + Na]⁺661.9919; found 661.9899.

4-Methoxyphenyl 4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-tetra-

chlorophthalimido-β-D-galactopyranoside (6): Lev₂O preparation:

LevOH (2.0 mL, 19.1 mmol) was added to a solution of 1,3-dicy-

clohexylcarbodiimide (1.97 g, 9.54 mmol) in CH₂Cl₂(16 mL) at

0 °C. The mixture was stirred for 5 min at room temperature, then

it was cooled and filtered, and the urea precipitate was washed

with additional CH₂Cl₂(2.7 mL), to give a Lev₂O solution (0.51 m;

4-Methoxyphenyl 4,6-O-Benzylidene-2-deoxy-2-tetrachlorophthal- 18.7 mL).

imido-β- -galactopyranoside (1): BF₃·Et₂O (731 μL, 5.82 mmol)

D

Lev₂O (0.51 m solution in CH₂Cl₂; 16.4 mL) was added at room

temperature to a mixture of 1 (2.16 g, 3.37 mmol) and DMAP

(61.1 mg, 0.51 mmol). The mixture was stirred for 1 h 30 min, then

it was diluted with CH₂Cl₂, and washed with saturated aqueous

NaHCO₃and H₂O. The organic phase was dried (MgSO₄), filtered,

and concentrated to dryness. The residue was purified by column

chromatography (toluene/EtOAc, 9:1) to give 6 (2.29 g, 92%). TLC

was added to a mixture of 3 (1.79 g, 2.91 mmol) and 4-meth-

oxyphenol (723 mg, 5.82 mmol) in dry CH₂Cl₂(8.0 mL) under an

argon atmosphere at 0 °C. The temperature was gradually raised

to room temperature over 2 h, and the mixture was stirred for 24 h.

Then it was diluted with CH₂Cl₂(40 mL), and washed with H₂O,

saturated aqueous NaHCO₃, and H₂O. The organic phase was

dried (MgSO₄) and concentrated to dryness. The residue was puri-

fied by column chromatography (hexane/EtOAc, 3:1) to give 4

(toluene/EtOAc, 6:1): R_f= 0.31. [α]²_D⁰= +14 (c = 1.0, CHCl₃). H

1

NMR (300 MHz, CDCl₃): δ = 7.60–7.55 (m, 2 H, Ar), 7.42–7.38

(1.63 g). TLC (toluene/acetone, 3:2): R_f

=

0.44. ¹H NMR

(m, 3 H, Ar), 6.90 (m, 2 H, Ar), 6.74 (m, 2 H, Ar), 5.82 (d, J_1,2

=

(300 MHz, CDCl₃): δ = 6.86 (m, 2 H, Ar), 6.75 (m, 2 H, Ar), 5.79

(d, J_1,2= 8.5 Hz, 1 H, 1-H), 5.78 (dd, J_2,3= 11.3, J_3,4= 3.1 Hz, 1

H, 3-H), 5.53 (d, 1 H, 4-H), 4.79 (dd, 1 H, 2-H), 4.28–4.10 (m, 3

H, 5-H, 6a-H, 6b-H), 3.74 [s, 3 H, Me(OMP)], 2.23, 2.06, 1.91 (3

s, 9 H, OAc) ppm. MS (ESI): calcd. for C₂₇H₂₃Cl₄NO₁₁Na [M +

Na]⁺700.0; found 700.1.

8.5 Hz, 1 H, 1-H), 5.80 (dd, J_2,3= 11.4, J_3,4= 3.6 Hz, 1 H, 3-H),

5.58 (s, 1 H, Ph-CH), 5.00 (dd, 1 H, 2-H), 4.45 (br. d, 1 H, 4-H),

4.37 (dd, J_5,6a= 1.4, J_6a,6b= 12.5 Hz, 1 H, 6a-H), 4.12 (dd, J_5,6b

=

1.4 Hz, 1 H, 6b-H), 3.73 [s, 3 H, Me(OMP)], 3.72 (m, 1 H, 5-H),

2.76–2.39 [m, 4 H, CH₂(Lev)], 1.93 [s, 3 H, CH₃(Lev)] ppm. ¹³C

NMR (75 MHz, CDCl₃): δ = 206.4 [CO(Lev)], 172.0 [CO(Lev)],

164.0, 163.0 (2 CO), 155.8, 150.5, 140.3, 140.2, 137.6, 130.1, 129.9

(Ar-C), 129.3, 128.4 (Ar-CH), 127.7, 127.4 (Ar-C), 126.5, 119.3,

114.5 (Ar-CH), 101.1 (Ph-CH), 97.6 (C-1), 73.0 (C-4), 69.1 (C-6),

68.7 (C-3), 66.9 (C-5), 55.7 [Me(OMP)], 51.7 (C-2), 37.9

[CH₂(Lev)], 29.6 [CH₃(Lev)], 28.2 [CH₂(Lev)] ppm. HRMS: calcd.

for C₃₃H₂₇Cl₄NO₁₀Na [M + Na]⁺760.0287; found 760.0278.

Compound 4 (1.63 g, 2.4 mmol) was dissolved in MeOH (186 mL),

and NaOMe (1 m in MeOH; 3.2 mL) was added. The mixture was

stirred at 0 °C for 10 min, and then AcOH (15.5 mL) was added.

The mixture was stirred at room temperature for 10 min, and then

it was coconcentrated twice with toluene. The residue was filtered

through a silica column (toluene/acetone, 2:1) to give 5 (855 mg)

and a mixture of partially deacetylated compounds (348 mg). The

partially deprotected products were dissolved in MeOH (40 mL)

and treated with additional NaOMe (1 m in MeOH; 0.68 mL) at

0 °C. After stirring for 7 min at 0 °C, AcOH (3.3 mL) was added,

and the mixture was concentrated. The residue was purified by col-

umn chromatography to give additional 5 (197 mg). TLC (toluene/

acetone, 2:1): R_f= 0.29. ¹H NMR (300 MHz, CDCl₃): δ = 6.87 (m,

2 H, Ar), 6.74 (m, 2 H, Ar), 5.62 (d, J_1,2= 8.4 Hz, 1 H, 1-H), 4.61

(br. t, 1 H, 2-H), 4.46 (dd, J_2,3= 11.0, J_3,4= 3.0 Hz, 1 H, 3-H),

4.03 (d, 1 H, 4-H), 3.84–3.75 (m, 3 H, 5-H, 6a-H, 6b-H), 3.69 [s, 3

H, Me(OMP)] ppm. MS (ESI): calcd. for C₂₁H₁₇Cl₄NO₈Na [M +

Na]⁺574.0; found 574.1.

4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-tetrachlorophthalimido-

α,β-D-galactopyranose (7): CAN (6.65 g, 11.9 mmol) was added at

0 °C to a solution of 6 (2.2 g, 3.0 mmol) in toluene/MeCN/H₂O

(1:6:1; 104 mL). The mixture was stirred for 1 h at 0 °C, then it

was diluted with EtOAc, and washed with H₂O, saturated aqueous

NaHCO₃, and H₂O. The organic phase was dried (MgSO₄), fil-

tered, and concentrated to dryness. The residue was purified by

column chromatography (toluene/EtOAc, 4:1) to give 7 (1.60 g,

84%) as a mixture of α/β anomers (2:10). TLC (toluene/EtOAc,

4:1): R_f= 0.23 (for a mixture of α/β anomers). ¹H NMR (300 MHz,

CDCl₃, data for β anomer): δ = 7.55–7.48 (m, 2 H, Ar), 7.39–7.33

(m, 3 H, Ar), 5.77 (dd, J_2,3= 11.3, J_3,4= 3.5 Hz, 1 H, 3-H), 5.54

(s, 1 H, Ph-CH), 5.47 (d, J_1,2= 8.3 Hz, 1 H, 1-H), 4.67 (dd, 1 H,

2-H), 4.40 (br. d, 1 H, 4-H), 4.34 (dd, J_5,6a= 1.4, J_6a,6b= 12.6 Hz,

1 H, 6a-H), 4.08 (dd, J_5,6b= 1.4 Hz, 1 H, 6b-H), 3.84 (br. s, 1 H,

OH), 3.67 (m, 1 H, 5-H), 2.69–2.35 [m, 4 H, CH₂(Lev)], 1.91 [s, 3

H, CH₃(Lev)] ppm. ¹³C NMR (75 MHz, CDCl₃, data for β an-

omer): δ = 206.5 [CO(Lev)], 171.9 [CO(Lev)], 163.8, 163.5 (2 CO),

140.1, 137.5, 129.9, 129.8 (Ar-C), 129.3, 128.3 (Ar-CH), 127.6,

Compound 5 (1.052 g, 1.90 mmol) was dissolved in dry MeCN

(19 mL). Benzaldehyde dimethyl acetal (0.43 mL, 2.85 mmol) and

pTsOH (0.5 m solution in dry MeCN; 188 μL) were added, and the

mixture was stirred for 45 min. Then it was diluted with EtOAc

(200 mL), and washed with saturated aqueous NaHCO₃and H₂O.

The organic phase was dried (MgSO₄) and concentrated to dryness.

The residue was purified by column chromatography (toluene/

EtOAc, 8:1) to give 1 (930 mg, 50% over three steps from 3). TLC 127.5 (Ar-C), 126.3 (Ar-CH), 100.9 (Ph-CH), 92.8 (C-1), 72.9 (C-

1

(toluene/EtOAc, 8:1): R_f= 0.27. [α]²_D⁰= +11 (c = 1.0, CHCl₃). H

NMR (300 MHz, CDCl₃): δ = 7.55 (m, 2 H, Ar), 7.40 (m, 3 H,

Ar), 6.91 (m, 2 H, Ar), 6.75 (m, 2 H, Ar), 5.75 (d, J_1,2= 8.3 Hz, 1

4), 69.2 (C-6), 68.5 (C-3), 66.9 (C-5), 53.5 (C-2), 37.9 [CH₂(Lev)],

29.6 [CH₃(Lev)], 28.2 [CH₂(Lev)] ppm. ¹H NMR (300 MHz,

CDCl₃, data for α anomer): δ = 7.55–7.48 (m, 2 H, Ar), 7.39–7.33

H, 1-H), 5.61 (s, 1 H, Ph-CH), 4.69 (br. t, 1 H, 2-H), 4.51 (dd, J_2,3(m, 3 H, Ar), 6.33 (dd, J_2,3= 12.1, J_3,4= 3.4 Hz, 1 H, 3-H), 5.57

= 10.9, J_3,4= 3.1 Hz, 1 H, 3-H), 4.38 (d, J_6a,6b= 12.4 Hz, 1 H, 6a-

H), 4.33 (br. d, 1 H, 4-H), 4.13 (d, 1 H, 6b-H), 3.73 [s, 3 H, Me(-

OMP)], 3.70 (s, 1 H, 5-H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

(s, 1 H, Ph-CH), 5.50 (m, 1 H, 1-H), 5.04 (dd, J_1,2= 3.1 Hz, 1 H,

2-H), 4.57 (br. d, 1 H, 4-H), 4.26 (br. d, J_6a,6b= 12.2 Hz, 1 H, 6a-

H), 4.14–4.07 (m, 2 H, 5-H, 6b-H), 4.00 (br. s, 1 H, OH), 2.69–2.35

164.2, 163.6 (2 CO), 155.8, 150.6, 140.5, 140.4, 137.3, 130.1, 129.8 [m, 4 H, CH₂(Lev)], 1.97 [s, 3 H, CH₃(Lev)] ppm. ¹³C NMR

(Ar-C), 129.6, 128.5 (Ar-CH), 127.6, 127.3 (Ar-C), 126.6, 119.3, (75 MHz, CDCl₃, significant data for α anomer): δ = 93.0 (C-1),

114.5 (Ar-CH), 101.7 (Ph-CH), 97.6 (C-1), 74.9 (C-4), 69.2 (C-6), 73.3 (C-4), 69.5 (C-6), 65.5 (C-3), 62.6 (C-5), 51.3 (C-2) ppm.

3874

www.eurjoc.org

Eur. J. Org. Chem. 2014, 3868–3884

Synthesis of Chondroitin Sulfate Oligosaccharides

HRMS: calcd. for C₂₆H₂₁Cl₄NO₉Na [M + Na]⁺653.9868; found

653.9876.

(d, J_1,2= 8.4 Hz, 1 H, 1B-H), 5.20 [d, 1 H, CH₂(Bn)], 5.04 [d, 1 H,

CH₂(Bn)], 4.99 [d, 1 H, CH₂(Bn)], 4.91 (d, 1 H, 1A-H), 4.81 [d, 1

H, CH₂(Bn)], 4.70 (dd, 1 H, 2B-H), 4.36 (t, J_3,4= J_4,5= 9.0 Hz, 1

H, 4A-H), 4.31 (d, 1 H, 4B-H), 4.17 (d, J_6a,6b= 12.4 Hz, 1 H, 6aB-

H), 3.95–3.89 (m, 2 H, 3A-H, 5A-H), 3.86 (d, 1 H, 6bB-H), 3.69

[s, 3 H, Me(OMP)], 3.19 (br. s, 1 H, 5B-H), 2.69–2.32 [m, 4 H,

CH₂(Lev)], 1.92 [s, 3 H, CH₃(Lev)] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 206.3 [CO(Lev)], 171.9, 167.3, 165.1, 164.2, 163.4

[CO(Lev, N-TCP, COOBn, Bz)], 155.8, 151.0, 140.0, 139.8, 138.3,

137.7, 135.1 (Ar-C), 133.3 (Ar-CH), 130.0, 129.8, 129.7, 129.5,

129.1, 128.7, 128.5, 128.2, 128.1, 127.9, 127.6, 127.3, 126.4 (Ar-C,

Ar-CH), 119.1, 114.5 (Ar-CH), 101.1, 101.0 (PhCH, C-1A), 97.9

(C-1B), 80.2 (C-3A or C-5A), 77.8 (C-4A), 74.7 (C-3A or C-5A),

74.5 [CH₂(Bn)], 73.0 (C-2A), 72.7 (C-4B), 68.9 (C-6B), 68.3 (C-3B),

67.5 [CH₂(Bn)], 66.4 (C-5B), 55.7 [Me(OMP)], 52.0 (C-2B), 37.9

[CH₂(Lev)], 29.6 [CH₃(Lev)], 28.2 [CH₂(Lev)] ppm. HRMS: calcd.

for C₆₀H₅₁Cl₄NO₁₇Na [M + Na]⁺1220.1809; found 1220.1812.

O-(4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-tetrachlorophthal-

imido-β-D-galactopyranosyl) Trichloroacetimidate (2): Trichloro-

acetonitrile (5.3 mL, 54 mmol) and catalytic DBU (0.11 m solution

in dry CH₂Cl₂; 96 μL) were added to a solution of 7 (680 mg,

1.07 mmol) in dry CH₂Cl₂(15 mL). The mixture was stirred for 7 h

at room temperature, then it was concentrated to dryness. The resi-

due was purified by flash chromatography (toluene/EtOAc, 5:1 +

1% Et₃N) to give 2 (763 mg, 91%). TLC (toluene/EtOAc, 3:1): R_f

1

= 0.40. H NMR (300 MHz, CDCl₃): δ = 8.64 (s, 1 H, NH), 7.57

(m, 2 H, Ar), 7.41 (m, 3 H, Ar), 6.54 (d, J_1,2= 8.9 Hz, 1 H, 1-H),

5.90 (dd, J_2,3= 11.4, J_3,4= 3.6 Hz, 1 H, 3-H), 5.59 (s, 1 H, Ph-

CH), 5.05 (dd, 1 H, 2-H), 4.49 (d, 1 H, 4-H), 4.43 (d, J_6a,6b

=

12.4 Hz, 1 H, 6a-H), 4.14 (d, 1 H, 6b-H), 3.87 (br. s, 1 H, 5-H),

2.71–2.41 [m, 4 H, CH₂(Lev)], 1.93 [s, 3 H, CH₃(Lev)] ppm. MS

(ESI): calcd. for C₂₈H₂₁Cl₇N₂O₉Na [M + Na]⁺796.9; found 797.0.

Benzyl [2-O-Benzoyl-3-O-benzyl-4-O-(4,6-O-benzylidene-2-deoxy-3-

O-levulinoyl-2-tetrachlorophthalimido-β-D-galactopyranosyl)-α,β-D-

4-Methoxyphenyl 3-O-(Methyl 2,3-Di-O-benzoyl-4-O-levulinoyl-β-

D-glucopyranosyluronate)-4,6-O-benzylidene-2-deoxy-2-tetrachloro-

glucopyranose]uronate (12): A solution of CAN (79 mg, 0.14 mmol)

in H₂O (0.3 mL) was added to a solution of 11 (42 mg, 0.035 mmol)

in CH₂Cl₂/MeCN (1:2; 2.7 mL). The mixture was vigorously stirred

for 1 h at room temperature, then it was diluted with EtOAc, and

washed with H₂O, saturated aqueous NaHCO₃, and H₂O. The or-

ganic phase was dried (MgSO₄), filtered, and concentrated to dry-

ness. The residue was purified by column chromatography

(CH₂Cl₂/MeOH, 80:1) to give 12 (40 mg, 84%) as a mixture of α/

phthalimido-β- -galactopyranoside (9): BF₃·Et₂O (0.26 m solution

D

in dry toluene; 326 μL) was added under an argon atmosphere at

room temperature to a mixture of 1 (154 mg, 0.24 mmol) and 8

(475 mg, 0.72 mmol) containing freshly activated 4 Å molecular si-

eves in dry toluene (2.5 mL). The mixture was stirred for 45 min at

room temperature, then it was neutralized with Et₃N, and concen-

trated to dryness. The residue was purified by column chromatog-

raphy (toluene/acetone, 8:1) to give 9 (148 mg, 54%), and unreacted

acceptor (41 mg, 27 %). TLC (toluene/acetone, 3:1): R_f= 0.48.

[α]²_D⁰= +18 (c = 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ =

7.76–7.09 (m, 15 H, Ar), 6.79 (m, 2 H, Ar), 6.68 (m, 2 H, Ar), 5.64

(t, J_2,3= J_3,4= 9.5 Hz, 1 H, 3B-H), 5.59 (s, 1 H, PhCH), 5.49 (d,

J_1,2= 8.3 Hz, 1 H, 1A-H), 5.41 (t, J_4,5= 9.6 Hz, 1 H, 4B-H), 5.34

(dd, J_1,2= 7.6 Hz, 1 H, 2B-H), 5.07 (d, 1 H, 1B-H), 4.99 (dd, J_2,3

= 11.3 Hz, 1 H, 2A-H), 4.82 (dd, J_3,4= 3.2 Hz, 1 H, 3A-H), 4.63

(br. d, 1 H, 4A-H), 4.36 (br. d, J_6a,6b= 12.2 Hz, 1 H, 6aA-H), 4.24

(d, 1 H, 5B-H), 4.13 (br. d, 1 H, 6bA-H), 3.72, 3.69 [2 s, 6 H,

COOMe, Me(OMP)], 2.66–2.30 [m, 4 H, CH₂(Lev)], 2.03 [m, 3 H,

CH₃(Lev)] ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.3, 167.0,

165.6, 164.4, 164.1, 162.0 [CO(Lev, N-TCP, COOMe, Bz], 155.6,

150.5, 140.3, 140.0, 137.7 (Ar-C), 133.5, 133.1, 129.9–125.4, 119.0,

114.4 (Ar-CH, Ar-C), 101.7 (C-1B), 100.9 (PhCH), 97.7 (C-1A),

76.2 (C-3A), 75.1 (C-4A), 72.3 (C-5B), 72.2 (C-3B), 71.9 (C-2B),

69.4 (C-4B), 69.0 (C-6A), 66.9 (C-5A), 55.5, 52.9 [COOMe, Me-

(OMP)], 52.0 (C-2A), 37.4 [CH₂(Lev)], 29.5 [CH₃(Lev)], 27.6

[CH₂(Lev)] ppm. HRMS: calcd. for C₅₄H₄₅Cl₄NO₁₈Na [M + Na]⁺

1158.1288; found 1158.1327.

1

β anomers (2:1). TLC (CH₂Cl₂/MeOH, 80:1): R_f= 0.23, 0.27. H

NMR [300 MHz, CDCl₃, for major anomer (α)]: δ = 7.94 (m, 2 H,

Ar), 7.58–7.07 (m, 18 H, Ar), 5.69 (dd, J_2,3= 11.3, J_3,4= 3.6 Hz,

1 H, 3B-H), 5.49–5.45 (m, 3 H, PhCH, 1B-H, 1A-H), 5.19 [d, 1 H,

CH₂(Bn)], 5.11–5.00 [m, 3 H, CH₂(Bn), 2A-H], 4.85 [d, 1 H,

CH₂(Bn)], 4.69 (m, 1 H, 2B-H), 4.39 (d, J_4,5= 9.0 Hz, 1 H, 5A-H),

4.29–4.08 (m, 4 H, 4B-H, 3A-H, 4A-H, 6aB-H), 3.80 (d, J_6a,6b

=

12.0 Hz, 1 H, 6bB-H), 3.10 (s, 1 H, 5B-H), 2.68–2.37 [m, 4 H,

CH₂(Lev)], 1.91 [s, 3 H, CH₃(Lev)] ppm. ¹³C NMR [75 MHz,

CDCl₃, for major anomer (α)]: δ = 206.3 [CO(Lev)], 171.9, 168.5,

165.9, 164.2, 163.5 [CO(Lev, N-TCP, COOBn, Bz)], 140.0, 139.8,

138.3, 137.7, 135.2 (Ar-C), 133.4 (Ar-CH), 130.0–126.5 (Ar-C, Ar-

CH), 101.0 (PhCH), 98.0 (C-1B), 90.7 (C-1A), 77.8, 77.4 (C-3A,

C-4A), 74.8 [CH₂(Bn)], 73.2 (C-2A), 72.8 (C-4B), 70.5 (C-5A), 68.9

(C-6B), 68.5 (C-3B), 67.5 [CH₂(Bn)], 66.4 (C-5B), 52.1 (C-2B), 37.9

[CH₂(Lev)], 29.6 [CH₃(Lev)], 28.2 [CH₂(Lev)] ppm. ¹H NMR

[300 MHz, CDCl₃, for minor anomer (β)]: δ = 7.94 (m, 2 H, Ar),

7.58–7.07 (m, 18 H, Ar), 5.71 (dd, J_2,3= 11.4, J_3,4= 3.6 Hz, 1 H,

3B-H), 5.49–5.45 (m, 1 H, PhCH), 5.39 (d, J_1,2= 8.4 Hz, 1 H, 1B-

H), 5.21 [d, 1 H, CH₂(Bn)], 5.11–5.00 [m, 3 H, CH₂(Bn), 2A-H],

4.83 [d, 1 H, CH₂(Bn)], 4.72–4.66 (m, 2 H, 1A-H, 2B-H), 4.29–4.08

(m, 3 H, 4B-H, 4A-H, 6aB-H), 3.97–3.78 (m, 3 H, 3A-H, 5A-H,

6bB-H), 3.12 (s, 1 H, 5B-H), 2.68–2.37 [m, 4 H, CH₂(Lev)], 1.91

[s, 3 H, CH₃(Lev)] ppm. ¹³C NMR [75 MHz, CDCl₃, selected data

for minor anomer (β)]: δ = 101.0 (PhCH), 97.8 (C-1B), 96.3 (C-

1A), 79.9 (C-3A), 77.8 (C-4A), 75.4 (C-2A), 74.8 [CH₂(Bn)], 74.7

(C-5A), 73.2 (C-4B), 68.9 (C-6B), 68.3 (C-3B), 67.7 [CH₂(Bn)], 66.4

(C-5B), 52.0 (C-2B), 37.9 [CH₂(Lev)], 29.6 [CH₃(Lev)], 28.2

[CH₂(Lev)] ppm. HRMS: calcd. for C₅₃H₄₅Cl₄NO₁₆Na [M + Na]⁺

1114.1390; found 1114.1425.

Benzyl [4-Methoxyphenyl 2-O-Benzoyl-3-O-benzyl-4-O-(4,6-O-

benzylidene-2-deoxy-3-O-levulinoyl-2-tetrachlorophthalimido-β-

D-ga-

lactopyranosyl)-β- -glucopyranoside]uronate (11): Donor 2 (655 mg,

D

0.842 mmol) and acceptor 10 (274 mg, 0.468 mmol) were dissolved

in dry CH₂Cl₂(5 mL) in the presence of freshly activated 4 Å mo-

lecular sieves. The mixture was stirred for 10 min at room tempera-

ture, then TMSOTf (0.18 m solution in dry CH₂Cl₂; 229 μL) was

added under an argon atmosphere. The mixture was stirred for

9 min, then it was neutralized with Et₃N and concentrated to dry-

ness. The residue was purified by column chromatography (toluene/

EtOAc, 9:1) to give 11 (325 mg, 58%), and unreacted 10 (45 mg,

16%). TLC (toluene/EtOAc, 4:1): R_f= 0.43. [α]²_D⁰= –9 (c = 1.0,

CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃): δ = 7.94 (m, 2 H, Ar), 7.56

(m, 1 H, Ar), 7.48–7.15 (m, 17 H, Ar), 6.77 (m, 2 H, Ar), 6.64 (m,

O-[Benzyl 2-O-Benzoyl-3-O-benzyl-4-O-(4,6-O-benzylidene-2-de-

oxy-3-O-levulinoyl-2-tetrachlorophthalimido-β-

α,β- -glucopyranosyluronate] Trichloroacetimidate (13): Trichloro-

acetonitrile (1.57 mL, 15.8 mmol) and K₂CO₃(47 mg, 0.35 mmol)

D-galactopyranosyl)-

D

2 H, Ar), 5.75 (dd, J_2,3= 11.4, J_3,4= 3.6 Hz, 1 H, 3B-H), 5.48 (s, were added to 12 (345 mg, 315 μmol) in dry CH₂Cl₂(8 mL) under

1 H, PhCH), 5.46 (dd, J_1,2= 7.4, J_2,3= 8.6 Hz, 1 H, 2A-H), 5.38 an argon atmosphere. The mixture was stirred at room temperature

Eur. J. Org. Chem. 2014, 3868–3884

www.eurjoc.org

3875

G. Macchione, S. Maza, M. Mar Kayser, J. L. de Paz, P. M. Nieto

FULL PAPER

for 15 h, then it was filtered and concentrated in vacuo to give 13

(370 mg, 95%) as an α/β mixture (75:100). TLC (toluene/acetone,

8:1): R_f= 0.40. ¹H NMR [300 MHz, CDCl₃, for major anomer

(β)]: δ = 8.53 (s, 1 H, NH), 7.91 (m, 2 H, Ar), 7.58–7.09 (m, 18 H,

Ar), 6.00 (d, J_1,2= 6.1 Hz, 1 H, 1A-H), 5.69 (dd, J_2,3= 11.3, J_3,4

= 3.6 Hz, 1 H, 3B-H), 5.52–5.43 (m, 3 H, PhCH, 1B-H, 2A-H),

68.3 (C-3B), 67.5 [CH₂(Bn)], 66.6 [CH₂(Z)], 66.3 (C-5B), 52.1 (C-

2B), 40.9 (CH₂-N), 37.9 [CH₂(Lev)], 29.6 (CH₂), 29.4 [CH₃(Lev)],

28.9 (CH₂), 28.1 [CH₂(Lev)], 23.1 (CH₂) ppm. HRMS: calcd. for

C₆₆H₆₂Cl₄N₂O₁₈Na [M + Na]⁺1333.2649; found 1333.2604.

Benzyl [N-Benzyloxycarbonyl-5-aminopentyl 2-O-Benzoyl-3-O-

benzyl-4-O-(2-deoxy-3-O-levulinoyl-2-tetrachlorophthalimido-β-

D-ga-

5.19–4.87 [m, 4 H, CH₂(Bn)], 4.70 (m, 1 H, 2B-H), 4.48 (dd, J_3,4

=

lactopyranosyl)-β- -glucopyranoside] Uronate (17): TFA (100 μL)

D

8.0, J_4,5= 9.8 Hz, 1 H, 4A-H), 4.34–4.20 (m, 1 H, 4B-H), 4.16–

4.08 (m, 2 H, 5A-H, 6aB-H), 4.00 (dd, J_2,3= 6.5 Hz, 1 H, 3A-H),

3.84 (d, J_6a,6b= 11.5 Hz, 1 H, 6bB-H), 3.13 (s, 1 H, 5B-H), 2.69–

2.32 [m, 4 H, CH₂(Lev)], 1.92 [s, 3 H, CH₃(Lev)] ppm. ¹³C NMR

[75 MHz, CDCl₃, for major anomer (β)]: δ = 206.2 [CO(Lev)],

171.9, 167.4, 164.9, 164.2, 163.4 [CO(Lev, N-TCP, COOBn, Bz)],

160.8 (C=NH), 140.0, 139.8, 138.2, 137.7, 135.0 (Ar-C), 133.5 (Ar-

CH), 129.9–126.4 (Ar-C, Ar-CH), 100.9 (PhCH), 98.2 (C-1B), 95.5

(C-1A), 90.5 (CCl₃), 80.5 (C-3A), 77.4 (C-4A), 74.4 (C-5A), 73.7

[CH₂(Bn)], 72.7 (C-4B), 71.4 (C-2A), 68.9 (C-6B), 68.5 (C-3B), 67.5

[CH₂(Bn)], 66.5 (C-5B), 52.0 (C-2B), 37.9 [CH₂(Lev)], 29.6

[CH₃(Lev)], 28.2 [CH₂(Lev)] ppm. ¹H NMR [300 MHz, CDCl₃, for

minor anomer (α)]: δ = 8.50 (s, 1 H, NH), 7.91 (m, 2 H, Ar), 7.58–

7.09 (m, 18 H, Ar), 6.56 (d, J_1,2= 3.6 Hz, 1 H, 1A-H), 5.70 (dd,

J_2,3= 11.4, J_3,4= 3.8 Hz, 1 H, 3B-H), 5.52–5.43 (m, 2 H, PhCH,

1B-H), 5.34 (dd, J_2,3= 9.3 Hz, 1 H, 2A-H), 5.21–4.82 [m, 4 H,

CH₂(Bn)], 4.70 (m, 1 H, 2B-H), 4.34–4.08 (m, 5 H, 3A-H, 4A-H,

5A-H, 4B-H, 6aB-H), 3.80 (d, J_6a,6b= 11.6 Hz, 1 H, 6bB-H), 3.11

(s, 1 H, 5B-H), 2.69–2.32 [m, 4 H, CH₂(Lev)], 1.92 [s, 3 H,

CH₃(Lev)] ppm. ¹³C NMR [75 MHz, CDCl₃, selected data for

minor anomer (α)]: δ = 160.4 (C=NH), 129.9–126.4 (Ar-C, Ar-

CH), 100.9 (PhCH), 98.4 (C-1B), 93.4 (C-1A), 79.9, 77.9, 75.0 (C-

3A, C-4A, C-5A), 73.5 [CH₂(Bn)], 72.7 (C-4B), 71.9 (C-2A), 68.9

(C-6B), 68.5 (C-3B), 67.6 [CH₂(Bn)], 66.4 (C-5B), 52.1 (C-2B) ppm.

HRMS: calcd. for C₅₅H₄₅Cl₇N₂O₁₆Na [M + Na]⁺1257.0486; found

1257.0470.

was added to a solution of 14 (25 mg, 19 μmol) in CH₂Cl₂(1 mL)

at 0 °C. The solution was stirred for 2 h at 0 °C, then it was diluted

with CH₂Cl₂, and washed with saturated NaHCO₃aqueous solu-

tion and brine. The organic layer was dried (MgSO₄), filtered, and

concentrated in vacuo. The residue was purified by column

chromatography (toluene/acetone, 7:2) to give 17 (23 mg, 99%).

TLC (toluene/acetone, 7:2): R_f= 0.14. [α]²_D⁰= –12 (c = 1.0, CHCl₃).

¹H NMR (300 MHz, CDCl₃): δ = 7.97 (m, 2 H, Ar), 7.55 (m, 1 H,

Ar), 7.43–7.13 (m, 17 H, Ar), 5.59 (dd, J_2,3= 11.3, J_3,4= 3.1 Hz,

1 H, 3B-H), 5.28–5.13 [m, 4 H, 1B-H, 2A-H, CH₂(Bn)], 5.06 [m, 2

H, CH₂(Z)], 4.85 [d, 1 H, CH₂(Bn)], 4.66–4.55 [m, 3 H, CH₂(Bn),

2B-H, NH], 4.48 (d, J_1,2= 7.4 Hz, 1 H, 1A-H), 4.25 (t, J_3,4= J_4,5

= 9.3 Hz, 1 H, 4A-H), 4.13 (br. d, 1 H, 4B-H), 3.87 (d, 1 H, 5A-

H), 3.82 (t, J_2,3= 8.9 Hz, 1 H, 3A-H), 3.73 (m, 1 H, CH₂-O), 3.62

(m, 2 H, 6aB-H, 6bB-H), 3.34 (m, 2 H, CH₂-O, 5B-H), 2.91 (m, 2

H, CH₂-N), 2.66, 2.40 [2 m, 4 H, CH₂(Lev)], 2.03 [s, 3 H,

CH₃(Lev)], 1.49–1.11 [m, 6 H, (CH₂)₃] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 207.4 [CO(Lev)], 171.8, 167.6, 165.1, 164.1, 163.3

[CO(Lev, N-TCP, COOBn, Bz)], 156.4 [CO(Z)], 140.0, 139.8, 137.9,

136.8, 135.1 (Ar-C), 133.4 (Ar-CH), 129.8, 129.7, 129.6, 128.7,

128.6, 128.5, 128.3, 128.2, 128.0, 127.6, 127.3 (Ar-C, Ar-CH), 101.4

(C-1A), 97.0 (C-1B), 79.3 (C-3A), 76.9 (C-4A), 74.9 (C-5A), 73.7

(C-5B), 73.5 [CH₂(Bn)], 72.5 (C-2A), 70.1 (C-3B), 69.7 (CH₂-O),

67.4 [CH₂(Bn)], 67.2 (C-4B), 66.5 [CH₂(Z)], 62.7 (C-6B), 51.7 (C-

2B), 40.8 (CH₂-N), 38.0 [CH₂(Lev)], 29.5 [CH₂, CH₃(Lev)], 28.8

(CH₂), 28.1 [CH₂(Lev)], 22.9 (CH₂) ppm. HRMS: calcd. for

C₅₉H₅₈Cl₄N₂O₁₈Na [M + Na]⁺1245.2336; found 1245.2332.

Benzyl [N-Benzyloxycarbonyl-5-aminopentyl 2-O-Benzoyl-3-O-

benzyl-4-O-(4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-tetrachloro- Benzyl [N-Benzyloxycarbonyl-5-aminopentyl 2-O-Benzoyl-3-O-

phthalimido-β- -galactopyranosyl)-β- -glucopyranoside] Uronate benzyl-4-O-(2-deoxy-3-O-levulinoyl-4,6-di-O-sulfo-2-tetrachloro-

phthalimido-β- -galactopyranosyl)-β- -glucopyranoside] Uronate

D

(14): Donor 13 (184 mg, 0.15 mmol) and benzyl N-(5-hydroxy-

pentyl) carbamate (107 mg, 0.45 mmol) were dissolved in dry

CH₂Cl₂(1 mL) in the presence of freshly activated 4 Å molecular

sieves. The mixture was stirred for 45 min at room temperature,

then TMSOTf (0.092 m solution in dry CH₂Cl₂; 162 μL) was added

under an argon atmosphere. The mixture was stirred for 15 min,

then it was neutralized with Et₃N and concentrated to dryness. The

residue was purified by column chromatography (toluene/acetone,

10:1) to give 14 (130 mg, 67%). TLC (toluene/EtOAc, 3:1): R_f=

D

(18): Compound 17 (13 mg, 11 μmol) and sulfur trioxide–trimeth-

ylamine complex (15 mg, 0.11 mmol) were dissolved in dry DMF

(1.0 mL), and the mixture was heated at 100 °C for 40 min using

microwave irradiation (28 W average power). The reaction vessel

was cooled, and Et₃N (150 μL), MeOH (1 mL), and CH₂Cl₂(1 mL)

were added. The solution was loaded onto a Sephadex LH-20

chromatography column, which was eluted with CH₂Cl₂/MeOH

(1:1) to give 18 as its triethylammonium salt (14 mg, 83%). TLC

1

0.44. [α]²_D⁰= –5 (c = 1.0, CHCl₃). H NMR (300 MHz, CDCl₃): δ

(CH₂Cl₂/MeOH, 9:2): R_f= 0.40. H NMR (300 MHz, CD₃OD): δ

= 7.92 (m, 2 H, Ar), 7.53 (m, 1 H, Ar), 7.48–7.02 (m, 22 H, Ar),

5.71 (dd, J_2,3= 11.5, J_3,4= 3.5 Hz, 1 H, 3B-H), 5.46 (s, 1 H, PhCH),

5.36 (d, J_1,2= 8.4 Hz, 1 H, 1B-H), 5.21 [m, 2 H, 2A-H, CH₂(Bn)],

= 7.98 (m, 2 H, Ar), 7.60 (m, 1 H, Ar), 7.50–7.05 (m, 17 H, Ar),

5.77 (dd, J_2,3= 11.6, J_3,4= 3.3 Hz, 1 H, 3B-H), 5.35 (d, J_1,2

=

8.3 Hz, 1 H, 1B-H), 5.29 [d, 1 H, CH₂(Bn)], 5.17 [d, 1 H, CH₂(Bn)],

5.05 [m, 3 H, CH₂(Z), CH₂(Bn)], 4.94 [d, 1 H, CH₂(Bn)], 4.79 [d, 5.06 (t, J_1,2= 8.0, J_2,3= 8.7 Hz, 1 H, 2A-H), 5.02 [m, 2 H, CH₂(Z)],

1 H, CH₂(Bn)], 4.68 (dd, 1 H, 2B-H), 4.55 (br. t, 1 H, NH), 4.44 4.96 [d, 1 H, CH₂(Bn)], 4.86 (br. d, 1 H, 4B-H), 4.68 (d, 1 H, 1A-

(d, J_1,2= 7.5 Hz, 1 H, 1A-H), 4.26 (br. d, 1 H, 4B-H), 4.24 (t, J_3,4H), 4.56 [m, 2 H, CH₂(Bn), 2B-H], 4.45 (dd, J_5,6a= 3.4, J_6a,6b

= J_4,5= 9.5 Hz, 1 H, 4A-H), 4.13 (d, J_6a,6b= 12.1 Hz, 1 H, 6aB- 11.9 Hz, 1 H, 6aB-H), 4.22 (m, 2 H, 6bB-H, 4A-H), 4.11 (m, 2 H,

=

H), 3.88–3.80 (m, 3 H, 3A-H, 5A-H, 6bB-H), 3.70, 3.30 (2 m, 2 H,

CH₂-O), 3.10 (br. s, 1 H, 5B-H), 2.89 (m, 2 H, CH₂-N), 2.69–2.32

[m, 4 H, CH₂(Lev)], 1.92 [s, 3 H, CH₃(Lev)], 1.49–1.06 [m, 6 H,

5A-H, 5B-H), 4.00 (t, J_3,4= 9.2 Hz, 1 H, 3A-H), 3.69 (m, 1 H,

CH₂-O), 3.42 (m, 1 H, CH₂-O), 3.19 (q, 12 H, Et₃NH⁺), 2.82 [m,

3 H, CH₂-N, CH₂(Lev)], 2.60 [dt, 1 H, CH₂(Lev)], 2.39 [m, 2 H,

(CH₂)₃] ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 206.3 [CO(Lev)], CH₂(Lev)], 1.92 [s, 3 H, CH₃(Lev)], 1.44–1.08 [m, 24 H, (CH₂)₃,

171.9, 167.7, 165.1, 164.2, 163.5 [CO(Lev, N-TCP, COOBn, Bz)],

156.4 [CO(Z)], 140.0, 139.8, 138.4, 137.7, 136.8 (Ar-C), 133.3 (Ar-

CH), 129.9, 129.8, 129.4, 129.1, 128.7, 128.6, 128.5, 128.4, 128.2,

Et₃NH⁺] ppm. ¹³C NMR (75 MHz, CD₃OD, Significant data from

HSQC experiment): δ = 102.2 (C-1A), 97.8 (C-1B), 79.8 (C-3A),

77.8 (C-4A), 74.9 (C-5A), 74.5 (C-5B), 74.2 [CH₂(Bn)], 73.7 (C-

127.9, 127.4, 127.3, 126.4 (Ar-C, Ar-CH), 101.4 (C-1A), 100.9 2A), 72.7 (C-4B), 70.6 (CH₂-O), 68.5 (C-3B), 68.4 (C-6B), 68.1

(PhCH), 97.9 (C-1B), 80.4 (C-3A), 78.0 (C-4A), 74.7 (C-5A), 74.3

[CH₂(Bn)], 66.9 [CH₂(Z)], 52.9 (C-2B), 47.4 (Et₃NH⁺), 41.3 (CH₂-

[CH₂(Bn)], 73.2 (C-2A), 72.7 (C-4B), 69.8 (CH₂-O), 68.9 (C-6B), N), 38.1 [CH₂(Lev)], 29.8 (CH₂), 29.6 (CH₂), 29.1 [CH₃(Lev)], 28.8

3876

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Eur. J. Org. Chem. 2014, 3868–3884

Synthesis of Chondroitin Sulfate Oligosaccharides

[CH₂(Lev)], 23.5 (CH₂), 8.7 (Et₃NH⁺) ppm. MS (ESI): calcd. for

(C-1B), 83.8 (C-3A), 79.8 (C-4A), 78.0 (C-5A), 76.8 (C-4B), 75.6

C₅₉H₅₆Cl₄N₂O₂₄S₂Na [M + Na]^–1403.1; found 1402.7. HRMS: [CH₂(Bn)], 74.0 (C-5B, C-2A), 73.5 (C-3B), 70.4 (CH₂-O), 67.8 (C-

calcd. for C₅₉H₅₆Cl₄N₂O₂₄S₂[M]^2–690.0715; found 690.0709.

6B), 67.0 [CH₂(Z)], 54.4 (C-2B), 41.5 (CH₂-N), 30.4, 30.0, 23.8

[(CH₂)₃], 23.0 (NAc) ppm. MS (ESI): calcd. for C₃₄H₄₄N₂O₂₀S₂[M

+ H]^2–432.1; found 431.8. HRMS: calcd. for C₃₄H₄₄N₂O₂₀S₂[M

+ H]^2–432.0969; found 432.0956.

N-Benzyloxycarbonyl-5-aminopentyl 4-O-(2-Acetamido-2-deoxy-

4,6-di-O-sulfo-β-D-galactopyranosyl)-3-O-benzyl-β-D-glucopyranos-

iduronic Acid (21): H₂O₂(30%; 0.52 mL) and an aqueous solution

of LiOH (0.7 m; 0.32 mL) were added at –5 °C to a solution of 18

(21 mg, 13 μmol) in THF (1.4 mL). The mixture was stirred for

20 h at room temperature, then MeOH (1.4 mL) and NaOH (4 m

aq.; 0.33 mL) were added. The mixture was stirred for 24 h at room

temperature, then it was neutralized with Amberlite IR-120 (H⁺)

resin, filtered, and concentrated. The residue was dissolved in

CH₂Cl₂/MeOH/Et₃N (1.0 mL/1.0 mL/0.1 mL) and purified by Se-

phadex LH-20 chromatography (CH₂Cl₂/MeOH, 1:1) to give com-

pound 19 as its triethylammonium salt (16 mg, 86%). TLC (EtOAc/

pyridine/H₂O/AcOH, 9:5:3:1): R_f= 0.27. ¹H NMR (300 MHz,

CD₃OD): δ = 7.59–7.19 (m, 10 H, Ar), 5.06 [m, 3 H, CH₂(Z),

CH₂(Bn)], 4.79 (br. d, 1 H, 4B-H), 4.72 [d, 1 H, CH₂(Bn)], 4.62 (d,

J_1,2= 8.3 Hz, 1 H, 1B-H), 4.45 (dd, J_5,6a= 3.9, J_6a,6b= 11.8 Hz, 1

H, 6aB-H), 4.41 (d, J_1,2= 8.0 Hz, 1 H, 1A-H), 4.22 (m, 2 H, 2B-

H, 6bB-H), 4.06 (br. t, 1 H, 4A-H), 3.96 (m, 1 H, 5B-H), 3.86 (m,

1 H, CH₂-O), 3.77 (dd, J_2,3= 10.8, J_3,4= 2.9 Hz, 1 H, 3B-H), 3.58

(m, 3 H, CH₂-O, 3A-H, 5A-H), 3.37 (t, J_2,3= 8.0 Hz, 1 H, 2A-H),

3.19 (q, 24 H, Et₃NH⁺), 3.11 (m, 2 H, CH₂-N), 1.66–1.38 [3 m, 6

H, (CH₂)₃], 1.29 (t, 36 H, Et₃NH⁺) ppm. ¹³C NMR (75 MHz,

CD₃OD, Significant data from HSQC experiment): δ = 129.5,

128.9, 128.7, 128.3 (Ar-CH), 104.0 (C-1A), 100.8 (C-1B), 83.7 (C-

3A), 78.9 (C-4A), 76.8 (C-4B), 75.4 [CH₂(Bn)], 74.5 (C-5B), 74.0

(C-2A), 72.8 (C-3B), 70.5 (CH₂-O), 68.6 (C-6B), 67.0 [CH₂(Z)],

55.3 (C-2B), 47.6 (Et₃NH⁺), 41.3 (CH₂-N), 30.1, 30.0, 23.8

[(CH₂)₃], 8.9 (Et₃NH⁺) ppm. MS (ESI): calcd. for

C₄₀H₄₃Cl₄N₂O₂₂S₂[M + 3H]^–1107.1; found 1106.7.

5-Aminopentyl

lactopyranosyl)-β-

4-O-(2-Acetamido-2-deoxy-4,6-di-O-sulfo-β-

D-glucopyranosiduronic Acid (15): A solution of

D-ga-

21 (7.9 mg, 8.5 μmol, as sodium salt) in H₂O/MeOH (2.7 mL/

0.3 mL) was hydrogenated in the presence of Pd(OH)₂. After 24 h,

the suspension was filtered through Celite, and the filtrate was con-

centrated. The residue was purified by Sephadex G-10 chromatog-

raphy (H₂O/MeOH, 9:1) to give 15 as its sodium salt after lyophili-

zation (6.0 mg, quantitative; 53% over six steps from 14, 90%

1

average yield per step). H NMR (500 MHz, D₂O): δ = 4.72 (br. d,

J_3,4= 2.1 Hz, 1 H, 4B-H), 4.58 (d, J_1,2= 7.6 Hz, 1 H, 1B-H), 4.47

(d, J_1,2= 8.1 Hz, 1 H, 1A-H), 4.32 (dd, J_5,6a= 3.3, J_6a,6b= 11.3 Hz,

1 H, 6aB-H), 4.25 (dd, J_5,6b= 8.7 Hz, 1 H, 6bB-H), 4.12 (dd, 1 H,

5B-H), 3.94–3.87 (m, 3 H, 2B-H, 3B-H, CH₂-O), 3.77 (m, 3 H, 4A-

H, 5A-H, CH₂-O), 3.62 (t, J_2,3= J_3,4= 9.2 Hz, 1 H, 3A-H), 3.35

(t, 1 H, 2A-H), 3.00 (t, 2 H, CH₂-N), 2.05 (s, 3 H, NAc), 1.67, 1.46

[2 m, 6 H, (CH₂)₃] ppm. ¹³C NMR (125.5 MHz, D₂O, Significant

data from HSQC experiment): δ = 102.8 (C-1A), 102.2 (C-1B), 82.3

(C-4A), 77.2 (C-5A), 76.0 (C-4B), 74.9 (C-3A), 73.1 (C-2A), 72.9

(C-5B), 70.6 (CH₂-O), 70.5 (C-3B), 68.3 (C-6B), 53.0 (C-2B), 39.9

(CH₂-N), 28.6, 26.8 (CH₂, CH₂), 23.1 (NAc), 22.5 (CH₂) ppm. MS

(ESI): calcd. for C₁₉H₃₃N₂O₁₈S₂[M + 2H]^–641.1; found 640.8.

HRMS: calcd. for C₁₉H₃₃N₂O₁₈S₂[M + 2H]^–641.1188; found

641.1170.

Benzyl [N-Benzyloxycarbonyl-5-aminopentyl 2-O-Benzoyl-3-O-

benzyl-4-O-(2-deoxy-3-O-levulinoyl-6-O-sulfo-2-tetrachlorophthal-

imido-β-D-galactopyranosyl)-β-D-glucopyranoside] Uronate (22):

Ethylene diamine (76 μL, 1.1 mmol) was added to a solution of 19

(16 mg, 11 μmol) in dry DMF (1.3 mL) under an argon atmo-

sphere, and the reaction mixture was subjected to microwave irradi-

ation for 90 min at 100 °C. The reaction vessel was cooled under a

stream of nitrogen, and the mixture was concentrated to dryness.

The residue was purified by Sephadex LH-20 chromatography

(CH₂Cl₂/MeOH, 1:1) to give 20. TLC (EtOAc/pyridine/H₂O/

AcOH, 9:5:3:1): R_f= 0.38. MS (ESI): calcd. for C₃₂H₄₂N₂O₁₉S₂[M

+ H]^2–411.1; found 410.8.

Compound 17 (27 mg, 22 μmol) and sulfur trioxide–trimeth-

ylamine complex (6.2 mg, 44 μmol) were dissolved in dry DMF

(3.0 mL), and the mixture was heated at 50 °C for 30 min using

microwave irradiation (15 W average power). The reaction vessel

was cooled, and Et₃N (150 μL), MeOH (1 mL), and CH₂Cl₂(1 mL)

were added. The solution was purified by Sephadex LH-20

chromatography (CH₂Cl₂/MeOH, 1:1) and silica gel column

chromatography (CH₂Cl₂/MeOH, 12:1 + 1% Et₃N) to give 22 as

its triethylammonium salt (26 mg, 84 %). TLC (CH₂Cl₂/MeOH,

1

Triethylamine (0.18 m solution in dry MeOH; 207 μL) and acetic

anhydride (5 μL, 0.05 mmol) were added to a cooled solution of 20

(11 μmol) in dry MeOH (2.3 mL). The mixture was stirred for 2 h

at 0 °C, then additional triethylamine (0.18 m solution in dry

MeOH; 207 μL) and acetic anhydride (5 μL, 0.05 mmol) were

added, and the reaction mixture was stirred for 1 h at room tem-

perature. The mixture was purified by Sephadex LH-20 chromatog-

raphy (CH₂Cl₂/MeOH, 1:1). The residue was converted into the

sodium salt by elution from a column of Dowex 50WX4-Na⁺with

MeOH/H₂O, 9:1 to give 21 (7.9 mg, 75% from 19). TLC (EtOAc/

10:1): R_f= 0.41. H NMR (300 MHz, CD₃OD): δ = 7.97 (m, 2 H,

Ar), 7.60 (m, 1 H, Ar), 7.47 (m, 2 H, Ar), 7.39–7.26 (m, 10 H, Ar),

7.13–7.03 (m, 5 H, Ar), 5.66 (dd, J_2,3= 11.4, J_3,4= 3.2 Hz, 1 H,

3B-H), 5.42 (d, J_1,2= 8.5 Hz, 1 H, 1B-H), 5.30 [d, 1 H, CH₂(Bn)],

5.14 [d, 1 H, CH₂(Bn)], 5.08 (t, J_1,2= 7.8, J_2,3= 8.6 Hz, 1 H, 2A-

H), 5.02 [m, 2 H, CH₂(Z)], 4.89 [d, 1 H, CH₂(Bn)], 4.68 (d, 1 H,

1A-H), 4.59 (dd, 1 H, 2B-H), 4.55 [d, 1 H, CH₂(Bn)], 4.23–4.08

(m, 5 H, 4A-H, 4B-H, 5A-H, 6aB-H, 6bB-H), 3.99 (t, 1 H, 3A-H),

3.81 (br. t, 1 H, 5B-H), 3.69 (m, 1 H, CH₂-O), 3.40 (m, 1 H, CH₂-

O), 3.18 (q, 6 H, Et₃NH⁺), 2.82 (m, 2 H, CH₂-N), 2.65 [m, 2 H,

pyridine/H₂O/AcOH, 9:5:3:1): R_f= 0.28. ¹H NMR (500 MHz, CH₂(Lev)], 2.38 [m, 2 H, CH₂(Lev)], 1.89 [s, 3 H, CH₃(Lev)], 1.44–

CD₃OD): δ = 7.54–7.19 (m, 10 H, Ar), 5.04 [m, 3 H, CH₂(Z), 1.10 [m, 15 H, (CH₂)₃, Et₃NH⁺] ppm. ¹³C NMR (75 MHz,

CH₂(Bn)], 4.78 (br. s, 1 H, 4B-H), 4.68 [m, 2 H, CH₂(Bn), 1B-H],

4.38 (dd, J_5,6a= 5.0, J_6a,6b= 11.3 Hz, 1 H, 6aB-H), 4.28 (d, J_1,2

CD₃OD, Significant data from HSQC experiment): δ = 134.4,

130.6, 129.4, 129.2, 129.0, 128.6, 128.5 (Ar-CH), 102.2 (C-1A), 97.9

(C-1B), 79.8 (C-3A), 77.7 (C-4A, C-5A), 75.3 (C-4B), 73.8 [C-5B,

CH₂(Bn)], 73.7 (C-2A), 70.8 (C-3B), 70.6 (CH₂-O), 68.1 [CH₂(Bn)],

=

7.3 Hz, 1 H, 1A-H), 4.16 (dd, J_5,6b= 6.3 Hz, 1 H, 6bB-H), 4.05 (t,

J_1,2= J_2,3= 9.5 Hz, 1 H, 2B-H), 3.99 (t, J_3,4= J_4,5= 9.1 Hz, 1 H,

4A-H), 3.96 (m, 1 H, 5B-H), 3.86 (m, 1 H, CH₂-O), 3.66 (m, 2 H, 67.1 [CH₂(Z)], 66.2 (C-6B), 53.1 (C-2B), 47.7 (Et₃NH⁺), 41.2 (CH₂-

5A-H, 3B-H), 3.52 (m, 1 H, CH₂-O), 3.44 (br. t, 1 H, 3A-H), 3.38

(br. t, 1 H, 2A-H), 3.11 (t, 2 H, CH₂-N), 2.04 (s, 3 H, NAc), 1.62,

N), 38.2 [CH₂(Lev)], 30.3 (CH₂), 29.9 (CH₂), 29.1 [CH₃(Lev)], 28.7

[CH₂(Lev)], 23.8 (CH₂), 9.0 (Et₃NH⁺) ppm. MS (ESI): calcd. for

1.51, 1.40 [3 m, 6 H, (CH₂)₃] ppm. ¹³C NMR (125.5 MHz, CD₃OD, C₅₉H₅₇Cl₄N₂O₂₁S [M]^–1301.2; found 1301.0. HRMS: calcd. for

Significant data from HSQC experiment): δ = 104.1 (C-1A), 101.5

C₅₉H₅₇Cl₄N₂O₂₁S [M]^–1301.1934; found 1301.2029.

Eur. J. Org. Chem. 2014, 3868–3884

www.eurjoc.org

3877

G. Macchione, S. Maza, M. Mar Kayser, J. L. de Paz, P. M. Nieto

+ H]^–785.2; found 785.1. HRMS: calcd. for C₃₄H₄₄N₂O₁₇S [M]^2–

FULL PAPER

N-Benzyloxycarbonyl-5-aminopentyl 4-O-(2-Acetamido-2-deoxy-6-

O-sulfo-β-D-galactopyranosyl)-3-O-benzyl-β-D-glucopyranosiduronic 392.1185; found 392.1180.

Acid (25): H₂O₂(30%; 0.73 mL) and an aqueous solution of LiOH

(0.7 m; 0.45 mL) were added at –5 °C to a solution of 22 (26 mg,

18 μmol) in THF (2.0 mL). The mixture was stirred for 20 h at

room temperature, then MeOH (2.0 mL) and NaOH (4 m aq.;

0.46 mL) were added. The mixture was stirred for 24 h at room

temperature, then the reaction mixture was neutralized with Am-

berlite IR-120 (H⁺) resin, filtered, and concentrated. The residue

was dissolved in CH₂Cl₂/MeOH/Et₃N (1.0 mL/1.0 mL/0.1 mL) and

purified by Sephadex LH-20 chromatography (CH₂Cl₂/MeOH, 1:1)

to give compound 23 as its triethylammonium salt (22 mg, 89%).

TLC (EtOAc/pyridine/H₂O/AcOH, 9:5:3:1): R_f= 0.34. ¹H NMR

(300 MHz, CD₃OD): δ = 7.54 (m, 2 H, Ar), 7.35–7.20 (m, 8 H,

Ar), 5.06 [m, 2 H, CH₂(Z)], 5.04 [d, 1 H, CH₂(Bn)], 4.72 [d, 1 H,

CH₂(Bn)], 4.68 (d, J_1,2= 8.8 Hz, 1 H, 1B-H), 4.36 (d, J_1,2= 7.6 Hz,

1 H, 1A-H), 4.29 (dd, J_5,6a= 7.4, J_6a,6b= 10.5 Hz, 1 H, 6aB-H),

4.23 (br. t, 1 H, 2B-H), 4.15 (dd, J_5,6b= 5.8 Hz, 1 H, 6bB-H), 4.07

(t, J_3,4= J_4,5= 8.4 Hz, 1 H, 4A-H), 3.99–3.94 (m, 2 H, 4B-H, 5A-

H), 3.85 (m, 1 H, CH₂-O), 3.76 (m, 1 H, 5B-H), 3.67 (m, 1 H, 3B-

H), 3.54 (m, 2 H, CH₂-O, 3A-H), 3.38 (t, J_2,3= 8.0 Hz, 1 H, 2A-

H), 3.18 (q, 18 H, Et₃NH⁺), 3.11 (m, 2 H, CH₂-N), 1.66–1.35 [3

m, 6 H, (CH₂)₃], 1.29 (t, 27 H, Et₃NH⁺) ppm. ¹³C NMR (75 MHz,

CD₃OD, Significant data from HSQC experiment): δ = 129.4,

128.9, 128.4 (Ar-CH), 103.9 (C-1A), 100.7 (C-1B), 83.8 (C-3A),

78.7 (C-4A), 77.0 (C-5A), 75.4 [CH₂(Bn)], 74.6 (C-5B), 74.2 (C-

2A), 74.0 (C-3B), 70.6 (CH₂-O), 68.5 (C-4B), 67.1 [CH₂(Z)], 66.7

(C-6B), 55.0 (C-2B), 47.4 (Et₃NH⁺), 41.5 (CH₂-N), 30.3, 30.1, 23.8

[ ( C H ₂) ₃] , 8 . 9 ( E t ₃N H ⁺) p p m . M S ( E S I ) : c a l c d . f o r

C₄₀H₄₂Cl₄KN₂O₁₉S [M + H + K]^–1065.0; found 1065.0.

5-Aminopentyl 4-O-(2-Acetamido-2-deoxy-6-O-sulfo-β-

D-galacto-

pyranosyl)-β- -glucopyranosiduronic Acid (16): A solution of 25

D

(10 mg, 12 μmol, as sodium salt) in H₂O/MeOH (3.6 mL/0.4 mL)

was hydrogenated in the presence of Pd(OH)₂. After 24 h, the sus-

pension was filtered through Celite, and the filtrate was concen-

trated. The residue was purified by Sephadex G-10 chromatography

(H₂O/MeOH, 9:1) to give 16 as its sodium salt after lyophilization

(7.4 mg, quantitative; 66% over five steps from 17, 92% average

yield per step). ¹H NMR (500 MHz, D₂O): δ = 4.53 (d, J_1,2

=

8.5 Hz, 1 H, 1B-H), 4.48 (d, J_1,2= 8.1 Hz, 1 H, 1A-H), 4.25 (m, 2

H, 6aB-H, 6bB-H), 4.00 (m, 2 H, 4B-H, 5B-H), 3.92 (m, 2 H, 2B-

H, CH₂-O), 3.74 (m, 4 H, 3B-H, 4A-H, 5A-H, CH₂-O), 3.63 (t, J_2,3

= J_3,4= 8.7 Hz, 1 H, 3A-H), 3.36 (t, 1 H, 2A-H), 3.00 (t, 2 H,

CH₂-N), 2.06 (s, 3 H, NAc), 1.68, 1.47 [2 m, 6 H, (CH₂)₃] ppm.

¹³C NMR (125.5 MHz, D₂O, Significant data from HSQC experi-

ment): δ = 102.7 (C-1A), 102.0 (C-1B), 81.6 (C-4A), 77.1 (C-5A),

74.6 (C-3A), 73.2 (C-5B), 73.1 (C-2A), 71.2 (C-3B), 70.6 (CH₂-O),

67.9 (C-4B), 67.6 (C-6B), 52.5 (C-2B), 39.8 (CH₂-N), 28.5, 26.9

(CH₂, CH₂), 23.0 (NAc), 22.4 (CH₂) ppm. MS (ESI): calcd. for

C₁₉H₃₃N₂O₁₅S [M + H]^–561.1613; found 561.0. HRMS: calcd. for

C₁₉H₃₃N₂O₁₅S [M + H]^–561.1619; found 561.1599.

4-Methoxyphenyl 3-O-(Benzyl 2-O-Benzoyl-3-O-benzyl-4-O-

levulinoyl-β-

2-trifluoroacetamido-β-

D

-glucopyranosyluronate)-2-deoxy-4,6-di-O-levulinoyl-

-galactopyranoside (29): An excess of (HF)_n·

D

Py (5.5 mL) was added to a solution of 28 (1.23 g, 1.14 mmol) in

dry THF (25 mL) at 0 °C under an argon atmosphere. After 24 h

at 0 °C, the mixture was diluted with CH₂Cl₂and washed with H₂O

and saturated NaHCO₃solution until neutral pH. The organic

layer was dried (MgSO₄), filtered, and concentrated in vacuo to

give the corresponding diol, which was used in the next step with-

out further purification [TLC (toluene/EtOAc, 1:4): R_f= 0.46].

Ethylene diamine (111 μL, 1.65 mmol) was added to a solution of

23 (22 mg, 16 μmol) in dry DMF (1.0 mL) under an argon atmo-

sphere, and the reaction mixture was subjected to microwave irradi-

ation for 90 min at 100 °C. The reaction vessel was cooled, and the

mixture was concentrated to dryness. The residue was purified by

Sephadex LH-20 chromatography (CH₂Cl₂/MeOH, 1:1) to give 24.

TLC (EtOAc/pyridine/H₂O/AcOH, 12:5:3:1): R_f= 0.23. MS (ESI):

calcd. for C₃₂H₄₃N₂O₁₆S [M + H]^–743.2; found 743.2.

LevOH (2.32 mL, 22.8 mmol) was added to a solution of 1,3-di-

cyclohexylcarbodiimide (2.35 g, 11.4 mmol) in CH₂Cl₂(20 mL) at

0 °C. The mixture was stirred for 5 min at room temperature, then

the mixture was cooled, filtered, and concentrated to give levulinic

anhydride quantitatively (Lev₂O, 11.4 mmol).

Triethylamine (0.36 m solution in dry MeOH; 150 μL) and acetic

anhydride (7.8 μL, 83 μmol) were added to a cooled solution of 24

(16 μmol) in dry MeOH (1.5 mL). The mixture was stirred for 3 h

at room temperature, then additional Et₃N (0.2 mL) was added,

and the reaction mixture was purified by Sephadex LH-20

chromatography (CH₂Cl₂/MeOH, 1:1). The residue was converted

into the sodium salt by elution from a column of Dowex 50WX4-

A solution of the diol (1.14 mmol) in dry pyridine (30 mL) was

added to a flask containing Lev₂O (11.4 mmol) and DMAP

(140 mg, 1.14 mmol) under an argon atmosphere. The mixture was

stirred for 5 h at room temperature, then the reaction mixture was

diluted with CH₂Cl₂and washed with HCl (1 m aq.), saturated

aqueous NaHCO₃, and H₂O. The organic layers were dried

(MgSO₄), filtered, and concentrated in vacuo. The residue was

Na⁺with MeOH/H₂O, 9:1 to give 25 (12 mg, 88% over two steps purified by column chromatography (toluene/EtOAc, 2:1) to give

from 23). TLC (EtOAc/pyridine/H₂O/AcOH, 12:5:3:1, two elu-

29 (950 mg, 83%). TLC (toluene/EtOAc, 2:1): R_f= 0.37. [α]²_D⁰

=

tions): R_f= 0.41. ¹H NMR (400 MHz, CD₃OD): δ = 7.50–7.20 (m,

+20 (c = 1.0, CHCl₃). H NMR (500 MHz, CDCl₃): δ = 7.94 (m,

1

10 H, Ar), 5.06 [s, 2 H, CH₂(Z)], 5.02 [d, 1 H, CH₂(Bn)], 4.69 [d, 2 H, Ar), 7.59 (m, 1 H, Ar), 7.44 (m, 2 H, Ar), 7.39–7.30 (m, 5 H,

1 H, CH₂(Bn)], 4.62 (d, J_1,2= 8.3 Hz, 1 H, 1B-H), 4.28 (d, J_1,2

=

Ar), 7.13–7.07 (m, 5 H, Ar), 6.89 (m, 2 H, Ar), 6.77 (m, 2 H, Ar),

6.9 Hz, 1 H, 1A-H), 4.25 (dd, J_5,6a= 8.2, J_6a,6b= 10.2 Hz, 1 H,

6.69 (br. s, 1 H, NH), 5.47 (d, J_3,4= 2.6 Hz, 1 H, 4A-H), 5.32 [pt

6aB-H), 4.10 (dd, J_5,6b= 5.4 Hz, 1 H, 6bB-H), 4.03–3.93 (m, 2 H, (pseudotriplet), 1 H, 4B-H], 5.28 (pt, 1 H, 2B-H), 5.19–5.11 [m, 3

2B-H, 4A-H), 3.91 (br. s, 1 H, 4B-H), 3.86 (m, 1 H, CH₂-O), 3.74– H, 1A-H and CH₂(Bn)], 4.79 (d, J_1,2= 7.5 Hz, 1 H, 1B-H), 4.61–

3.65 (m, 2 H, 5B-H, 5A-H), 3.52 (m, 2 H, 3B-H, CH₂-O), 3.44 (t, 4.52 [m, 3 H, 3A-H and CH₂(Bn)], 4.15–4.09 (m, 2 H, 2 6A-H),

J_2,3= J_3,4= 8.9 Hz, 1 H, 3A-H), 3.39 (t, 1 H, 2A-H), 3.11 (t, 2 H,

4.07 (d, J_4,5= 9.6 Hz, 1 H, 5B-H), 3.89–3.80 (m, 3 H, 5A-H, 3B-

CH₂-N), 2.06 (s, 3 H, NAc), 1.62, 1.51, 1.41 [3 m, 6 H, (CH₂)₃] H and 2A-H), 3.75 (s, 3 H, OCH₃), 2.80–2.20 [m, 12 H, 3 OC-

ppm. ¹³C NMR (100 MHz, CD₃OD, Significant data from HSQC

experiment): δ = 128.9–128.1 (Ar-CH), 103.9 (C-1A), 101.2 (C-1B),

O(CH₂)₂], 2.15 (s, 3 H, COCH₃), 2.14 (s, 3 H, COCH₃), 2.10 (s, 3

H, COCH₃) ppm. ¹³C NMR (125 MHz, CDCl₃): δ = 206.84

83.8 (C-3A), 79.7 (C-4A), 77.7 (C-5A), 75.2 [CH₂(Bn)], 74.5 (C- [CO(Lev)], 206.81 [CO(Lev)], 206.1 [CO(Lev)], 172.3 (CO), 171.5

3B), 73.9 (C-2A), 73.8 (C-5B), 70.2 (CH₂-O), 68.2 (C-4B), 66.9 (CO), 171.2 (CO), 166.9 (CO), 165.0 (CO), 157.6 (q, J_C,F= 38.7 Hz,

[CH₂(Z)], 66.2 (C-6B), 54.4 (C-2B), 41.3 (CH₂-N), 30.1, 29.8, 23.6 COCF₃), 155.9 (Ar), 151.0 (Ar), 137.4–114.3 (Ar-C and Ar-CH),

[(CH₂)₃], 22.6 (NAc) ppm. MS (ESI): calcd. for C₃₄H₄₅N₂O₁₇S [M 115.5 (q, J_C,F= 289.0 Hz, COCF₃), 100.1 (C-1B), 98.9 (C-1A), 79.2

3878

www.eurjoc.org

Eur. J. Org. Chem. 2014, 3868–3884

Synthesis of Chondroitin Sulfate Oligosaccharides

(C-3B), 74.2 [CH₂(Bn)], 73.2 (C-3A), 72.8 (C-5B), 72.6 (C-2B), 71.6 O-Benzoyl-3-O-benzyl-β-

(C-5A), 70.8 (C-4B), 68.7 (C-4A), 67.9 [CH₂(Bn)], 62.3 (C-6A), oxy-4,6-O-di-tert-butylsilylene-2-trifluoroacetamido-β-

D

-glucopyranosyluronate)-(1Ǟ3)-2-de-

-galactopyr-

anoside (33): Donor 31 (143 mg, 0.12 mmol) and acceptor 32

D

55.7 (OCH₃), 54.6 (C-2A), 38.2 [OCO(CH₂)₂], 38.0 [OCO(CH₂)₂],

37.7 [OCO(CH₂)₂], 29.9 (COCH₃), 29.8 (2 COCH₃), 28.0 [OC-

O(CH₂)₂], 27.9 [OCO(CH₂)₂], 27.8 [OCO(CH₂)₂] ppm. HRMS:

calcd. for C₅₇H₆₀F₃NNaO₂₀[M + Na]⁺1158.3558; found

1158.3596.

(67 mg, 0.07 mmol) were coevaporated with toluene, concentrated

in vacuo and dissolved in dry CH₂Cl₂(3 mL) in the presence of

freshly activated 4 Å molecular sieves. The mixture was stirred for

10 min at 0 °C, then TMSOTf (0.09 m solution in dry CH₂Cl₂;

264 μL) was added under an argon atmosphere. The mixture was

stirred for 30 min at 0 °C, then it was neutralized with Et₃N, fil-

tered, and concentrated to dryness. The residue was purified by

column chromatography (toluene/acetone, 5:1) to give 33 (97 mg,

71%). TLC (toluene/EtOAc, 2:1): R_f= 0.34. [α]²_D⁰= +11 (c = 1.0,

3-O-(Benzyl 2-O-Benzoyl-3-O-benzyl-4-O-levulinoyl-β-

anosyluronate)-2-deoxy-4,6-di-O-levulinoyl-2-trifluoroacetamido-

α,β- -galactopyranose (30): CAN (0.44 m solution in H₂O; 7.6 mL)

D-glucopyr-

D

was added to a solution of 29 (950 mg, 0.84 mmol) in CH₂Cl₂/

MeCN (1:2; 22.8 mL), and the mixture was stirred vigorously for

1 h at room temperature. It was then diluted with EtOAc, and

washed with H₂O, saturated aqueous NaHCO₃, and H₂O. The or-

ganic phase was dried (MgSO₄), filtered, and concentrated to dry-

ness. The residue was purified by column chromatography (toluene/

acetone, 3:1) to give 30 (695 mg, 81%) as a mixture of α/β anomers.

TLC (toluene/acetone, 3:2): R_f= 0.44 and 0.31. ¹H NMR

(500 MHz, CDCl₃, data for α anomer): δ = 7.94 (m, 2 H, Ar), 7.57

(m, 1 H, Ar), 7.44–7.32 (m, 7 H, Ar), 7.15–7.05 (m, 5 H, Ar), 6.69

(d, J_2,NH= 8.7 Hz, 1 H, NH), 5.39 (d, J_3,4= 2.3 Hz, 1 H, 4A-H),

1

CHCl₃). H NMR (500 MHz, CDCl₃): δ = 7.96 (m, 2 H, Ar), 7.91

(m, 2 H, Ar), 7.57 (m, 2 H, Ar), 7.46–7.29 (m, 14 H, Ar), 7.15–

7.08 (m, 10 H, Ar), 6.90 (m, 2 H, Ar), 6.84 (d, J_2,NH= 6.9 Hz, 1

H, NH), 6.78 (m, 2 H, Ar), 6.52 (d, J_2,NH= 8.7 Hz, 1 H, NH),

5.37–5.33 (m, 2 H, 1A-H and 4D-H), 5.27–5.15 [m, 8 H, 1B-H, 2B-

H, 4C-H, 2D-H and 2 CH₂(Bn)], 4.78 [d, J_gem= 11.1 Hz, 1 H,

CH₂(Bn)], 4.73 (d, J_1,2= 7.4 Hz, 1 H, 1D-H), 4.61–4.52 [m, 3 H,

4A-H and CH₂(Bn)], 4.46 [d, J_gem= 11.1 Hz, 1 H, CH₂(Bn)], 4.33

(dd, J_2,3= 11.1, J_3,4= 1.9 Hz, 1 H, 3A-H), 4.15–4.08 (m, 3 H, 1C-

H, 5D-H and 6A-H), 4.05–4.02 (m, 2 H, 4B-H and 6A-H), 3.96–

3.81 (m, 6 H, 2A-H, 2C-H, 2 6C-H 5B-H and 3D-H), 3.75 (s, 3 H,

OCH₃), 3.69 (m, 1 H, 3C-H), 3.63 (m, 1 H, 3B-H), 3.34 (m, 2 H,

5A-H and 5C-H), 2.75–2.21 [m, 12 H, 3 OCO(CH₂)₂], 2.18 (s, 3 H,

COCH₃), 2.11 (s, 3 H, COCH₃), 2.07 (s, 3 H, COCH₃), 1.04 [s, 9

H, C(CH₃)₃], 0.99 [s, 9 H, C(CH₃)₃] ppm. ¹³C NMR (125 MHz,

CDCl₃): δ = 207.1 [CO(Lev)], 206.6 [CO(Lev)], 206.0 [CO(Lev)],

172.2 (CO), 171.4 (CO), 171.2 (CO), 168.6–164.9 (4 CO), 157.6 (2

q, 2 COCF₃), 156.0–114.5 (Ar-C and Ar-CH), 117.9 (2 q, 2

COCF₃), 100.2 (C-1B), 99.8, 99.7 (C-1C and C-1D), 99.3 (C-1A),

80.1 (C-3B), 79.2 (C-3D), 77.8 (C-4B), 75.4 (C-3A), 75.1 [CH₂(Bn)],

74.3, 74.2, 74.1, 73.2, 73.0 [C-3C, C-5B, CH₂(Bn), C-4A, C-5D],

72.5, 72.4 [2 C: C-2B or C-4C or C-2D or CH₂(Bn)], 71.4, 71.1 (C-

5A and C-5C), 70.9 (C-4D), 68.2, 68.1, 67.9 [3 C: CH₂(Bn) or C-

2B or C-4C or C-2D], 67.0 (C-6A), 61.5 (C-6C), 55.7 (OCH₃), 54.0,

53.0 (C-2A and C-2C), 38.1 [OCO(CH₂)₂], 38.0 [OCO(CH₂)₂], 37.7

[OCO(CH₂)₂], 29.9 (COCH₃), 29.8 (COCH₃), 29.7 (COCH₃), 28.1

[OCO(CH₂)₂], 27.9 [OCO(CH₂)₂], 27.7 [OCO(CH₂)₂], 27.6, 27.5,

27.5 [C(CH₃)₃], 23.3, 20.8 [C(CH₃)₃] ppm. HRMS: calcd. for

C₁₀₀H₁₁₀N₂O₃₂F₆NaSi [M + Na]⁺2015.6607; found 2015.6607.

5.31–5.25 (m, 3 H, 1A-H, 2B-H and 4B-H), 5.19–5.11 [2 d, J_gem

=

11.9 Hz, 2 H, CH₂(Bn)], 4.81 (d, J_1,2= 7.7 Hz, 1 H, 1B-H), 4.55 [2

d, J_gem= 11.6 Hz, 2 H, CH₂(Bn)], 4.40–4.32 (m, 2 H, 2A-H and

5A-H), 4.27–4.20 (m, 2 H, 6aA-H and 3A-H), 4.05 (d, J_4,5

=

9.7 Hz, 1 H, 5B-H), 3.94 (dd, J_5,6b= 8.8, J_6a,6b= 11.4 Hz, 1 H,

6bA-H), 3.82 (pt, 1 H, 3B-H), 2.84–2.21 [m, 12 H, 3 OCO(CH₂)₂],

2.16 (s, 3 H, COCH₃), 2.10 (s, 3 H, COCH₃), 2.09 (s, 3 H, COCH₃)

ppm. ¹³C NMR (125 MHz, CDCl₃, data for α anomer): δ = 208.8

[CO(Lev)], 206.9 [CO(Lev)], 206.0 [CO(Lev)], 172.2 (CO), 171.8

(CO), 171.2 (CO), 166.8 (CO), 165.0 (CO), 157.1 (q, COCF₃),

137.3–127.8 (Ar-C and Ar-CH), 115.7 (q, COCF₃), 99.6 (C-1B),

91.4 (C-1A), 79.3 (C-3B), 74.2 [CH₂(Bn)], 73.0 (C-5B), 72.2 (C-2B),

72.0 (C-3A), 71.0 (C-4B),68.9 (C-4A), 68.0 [CH₂(Bn)], 67.0 (C-5A),

63.0 (C-6A), 50.1 (C-2A), 38.5 [OCO(CH₂)₂], 38.1 [OCO(CH₂)₂],

37.7 [OCO(CH₂)₂], 30.0 (COCH₃), 29.9 (COCH₃), 29.8 (COCH₃),

28.3 [OCO(CH₂)₂], 28.0 [OCO(CH₂)₂], 27.7 [OCO(CH₂)₂] ppm.

HRMS: calcd. for C₅₀H₅₄NO₁₉NaF₃[M + Na]⁺1052.3140; found

1052.3175.

O-[3-O-(Benzyl 2-O-Benzoyl-3-O-benzyl-4-O-levulinoyl-β-

pyranosyluronate)-2-deoxy-4,6-di-O-levulinoyl-2-trifluoroacetamido-

α,β- -galactopyranosyl] Trichloroacetimidate (31): Compound 30

D-gluco-

4-Methoxyphenyl O-(3-O-Benzyl-2,4-di-O-sulfo-β-

syluronic Acid)-(1Ǟ3)-O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-β-

galactopyranosyl)-(1Ǟ4)-O-(3-O-benzyl-2-O-sulfo-β- -glucopyrano-

syluronic Acid)-(1Ǟ3)-2-acetamido-2-deoxy-4,6-di-O-sulfo-β- -ga-

D-glucopyrano-

D

D-

(120 mg, 0.11 mmol) was dissolved in dry CH₂Cl₂(3 mL), and

Cl₃CCN (234 μL, 2.3 mmol) and DBU (0.066 m solution in

CH₂Cl₂; 261 μL) were added. The mixture was stirred at room tem-

perature for 5 h, then it was concentrated in vacuo. Flash

chromatography on silica gel (toluene/acetone, 5:1 + 1 % Et₃N)

gave 31 (95 mg, 70%) as an α/β mixture. TLC (toluene/acetone,

D

lactopyranoside (37): An excess of (HF)_n·Py (102 μL, 3.8 mmol) was

added at 0 °C under an argon atmosphere to a solution of 33

(39 mg, 0.02 mmol) in dry THF (1.0 mL). After 24 h at 0 °C, the

mixture was diluted with CH₂Cl₂, and then washed with H₂O and

saturated NaHCO₃solution until it reached neutral pH. The or-

ganic layer was dried (MgSO₄), filtered, and concentrated in vacuo.

The residue was purified by column chromatography (toluene/

EtOAc, 2:3) to give 34 (27 mg, 75%). TLC (toluene/EtOAc, 1:2):

R_f= 0.36. ¹H NMR (400 MHz, CDCl₃): δ = 7.96 (m, 2 H, Ar),

7.86 (m, 2 H, Ar), 7.56 (m, 2 H, Ar), 7.46–7.33 (m, 14 H, Ar),

7.13–7.06 (m, 10 H, Ar), 6.86 (m, 2 H, Ar), 6.74 (m, 2 H, Ar),

5.34–5.11 [m, 9 H, 4D-H, 2D-H, 1A-H, H2B, 4C-H and CH₂(Bn)],

4.75 [m, 3 H, 1B-H, 1D-H and CH₂(Bn)], 4.60–4.49 [m, 4 H, 1C-

H and CH₂(Bn)], 4.38 (br. s, 1 H, 3A-H), 4.15 (br. s, 1 H, 4B-H),

4.08–3.97 (m, 4 H, 4A-H, 5D-H, 5B-H and 3C-H), 3.90 (m, 3 H,

2C-H and 2 6A-H or 6C-H), 3.82 (m, 3 H, 2A-H, 3D-H and 6A-

H or 6C-H), 3.72 (m, 4 H, 3B-H and OCH₃), 3.56 (m, 3 H, 5A-H,

5C-H and 6A-H or 6C-H), 2.74–2.17 [m, 12 H, 3 OCO(CH₂)₂],

1

3:2): R_f= 0.65 and 0.48. H NMR (500 MHz, CDCl₃, data for α

anomer): δ = 8.71 [s, 1 H, NH(TCA)], 7.96 (m, 2 H, Ar), 7.59 (m,

1 H, Ar), 7.44 (m, 2 H, Ar), 7.37 (m, 5 H, Ar), 7.13–7.05 (m, 5 H,

Ar), 7.00 [br. d, J_NH,2= 7.3 Hz, 1 H, NH(TFA)], 6.54 (d, J_1,2

=

3.4 Hz, 1 H, 1 H, 1A-H), 5.55 (br. d, 1 H, 4A-H), 5.35 (pt, 1 H,

2B-H), 5.23 (pt, 1 H, 4B-H), 5.18–5.06 [2 d, J_gem= 12.1 Hz, 2 H,

CH₂(Bn)], 4.93 (d, J_1,2= 7.9 Hz, 1 H, 1B-H), 4.62–4.51 [m, 3 H,

2A-H and CH₂(Bn)], 4.35 (dd, J_3,4= 2.9, J_2,3= 11.0 Hz, 1 H, 3A-

H), 4.28 (m, 1 H, 5A-H), 4.12–4.04 (m, 3 H, 5B-H and 2 6A-H),

3.85 (pt, 1 H, 3B-H), 2.74–2.16 [m, 12 H, 3 OCO(CH₂)₂], 2.14 (s,

3 H, COCH₃), 2.10 (s, 3 H, COCH₃), 2.03 (s, 3 H, COCH₃) ppm.

4-Methoxyphenyl O-(Benzyl 2-O-Benzoyl-3-O-benzyl-4-O-levulin-

oyl-β-D-glucopyranosyluronate)-(1Ǟ3)-O-(2-deoxy-4,6-di-O-levulin-

oyl-2-trifluoroacetamido-β-

D

-galactopyranosyl)-(1Ǟ4)-O-(benzyl 2-

Eur. J. Org. Chem. 2014, 3868–3884

www.eurjoc.org

3879

G. Macchione, S. Maza, M. Mar Kayser, J. L. de Paz, P. M. Nieto

FULL PAPER

2.12 (s, 3 H, COCH₃), 2.10 (s, 3 H, COCH₃), 2.06 (s, 3 H, COCH₃) CH₂(Bn)], 4.80–4.71 [m, 4 H, 1C-H, 1D-H or 1B-H, and CH₂(Bn)],

ppm. ¹³C NMR (100 MHz, CDCl₃, selected data from HSQC ex-

periment): δ = 100.1 (C-1B), 99.7 (C-1D), 99.6 (C-1C), 98.7 (C-

1A), 79.3 (C-3B), 79.1 (C-3D), 77.5 (C-3A), 77.3 (C-4B), 74.5

4.41 (m, 2 H, 2B-H and 2D-H), 4.36–4.23 (m, 5 H, 3D-H or 5D-

H, 3A-H and 3 6A-H or 6C-H), 4.20 (m, 2 H, 3D-H or 5D-H, and

5A-H or 5C-H), 4.15 (m, 2 H, 4B-H and 3C-H), 4.10 (m, 1 H, 6A-

[CH₂(Bn)], 74.1 [CH₂(Bn)], 74.1 (C-5B or C-5D), 74.0 (C-5A or C- H or 6C-H), 3.99 (m, 2 H, 2C-H and 2A-H), 3.93 (m, 1 H, 5A-H

5C), 73.9 (C-3C), 72.7 (C-5B or C-5D), 72.4 (C-2B, C-4C), 71.0

or 5C-H), 3.88 (pt, 1 H, 3B-H), 3.83 (s, 3 H, OCH₃), 3.81 (m, 1

(C-5A or C-5C), 70.7 (C-4D), 68.3 (C-4A), 68.0 [C-2D, 2 CH₂(Bn)], H, 5B-H), 2.09 (NHCOCH₃), 2.08 (NHCOCH₃) ppm. ¹³C NMR

62.6 (C-6A or C-6C), 61.7 (C-6A or C-6C), 55.7 (COCH₃), 54.1 (125 MHz, D₂O, 40 °C, selected data from HSQC experiment): δ =

(C-2A), 53.2 (C-2C) ppm. HRMS: calcd. for C₉₂H₉₄F₆N₂Na₂O₃₂

130.6–116.4 (Ar), 103.8 (C-1D or C-1B), 102.4 (C-1B or C-1D),

101.6 (C-1A), 101.4 (C-1C), 81.6 (C-3B), 80.0 (C-2B and 2D), 80.0

(C-5A or C-5C or C-3D or C-5D) 79.5 (C-3D or C-5D), 78.7 (C-

5B), 78.6 (C-3C and C-4B), 77.9 (C-3A), 77.2 (C-4D), 76.9 (C-4A

and C-4C), 75.1 [CH₂(Bn)], 74.2 [CH₂(Bn)], 74.1 (C-5A or C-5C

or C-3D or C-5D), 73.4 (C-5A or C-5C), 69.3 (C-6A or C-6C),

68.6 (C-6A or C-6C), 57.2 (OCH₃), 53.9 (C-2A and C-2C), 23.9 (2

NHCOCH₃) ppm.

[M + 2Na]²⁺949.2739; found 949.2735.

H₂O₂(30%; 0.26 mL) and a solution of LiOH (0.7 m; 0.16 mL)

were added at –5 °C to a solution of 34 (12 mg, 6.5 μmol) in THF

(1.0 mL). The mixture was stirred for 24 h at room temperature,

then MeOH (1 mL) and a solution of NaOH (4 m; 0.33 mL) were

added. The mixture was stirred for 3 d at room temperature, then

it was neutralized with Amberlite IR-120 (H⁺) resin, filtered, and

concentrated to give 35. MS (ESI): calcd. for C₄₅H₅₆N₂NaO₂₂[M

+ Na]^–999.3; found 999.4.

4-Methoxyphenyl O-(2,4-Di-O-sulfo-β-

Acid)-(1Ǟ3)-O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-β-

pyranosyl)-(1Ǟ4)-O-(2-O-sulfo-β- -glucopyranosyluronic Acid)-

(1Ǟ3)-2-acetamido-2-deoxy-4,6-di-O-sulfo-β- -galactopyranoside

D

-glucopyranosyluronic

D

-galacto-

D

Et₃N (12 μL, 85 μmol) and Ac₂O (12 μL, 129 μmol) were added to

a cooled (0 °C) solution of 35 (6.5 μmol) in dry MeOH (2.0 mL).

The mixture was stirred for 3 h at room temperature, then triethyl-

amine (0.3 mL) was added, and the mixture was coevaporated with

toluene and methanol. The residue was purified by Sephadex LH-

20 chromatography (MeOH/CH₂Cl₂, 1:1) to give 36 as its trieth-

ylammonium salt. The sodium salt of 36 (6.2 mg, 90%) was ob-

tained by treatment with Amberlite IR-120 (H⁺) resin in MeOH

(pH ca. 3), followed by filtration, treatment with NaOH (0.04 m;

D

(38): A solution of 37 (4.0 mg, 2.5 μmol, sodium salt) in H₂O/

MeOH (3.6 mL/0.4 mL) was hydrogenated at 1.5 bar pressure in

the presence of Pd(OH)₂(12 mg). After 24 h, the suspension was

filtered through Celite, and the filtrate was concentrated to give 38

after lyophilization (3.4 mg, 97%). ¹H NMR (500 MHz, D₂O, data

for sodium salt): δ = 7.10 (m, 2 H, Ar), 6.99 (m, 2 H, Ar), 5.22 (d,

J_1,2= 8.5 Hz, 1 H, 1A-H), 4.95 (d, J_3,4= 2.6 Hz, 1 H, 4A-H), 4.93

(d, J_3,4= 2.4 Hz, 1 H, 4C-H), 4.77 (m, 2 H, 1C-H and 1D-H), 4.71

(d, J_1,2= 7.9 Hz, 1 H, 1B-H), 4.51 (pt, 1 H, 4D-H), 4.36–4.31 (m,

2 H, 2 6A-H or 6C-H), 4.28–4.21 (m, 6 H, 2 6A-H or 6C-H, 5A-

H or 5C-H, 2D-H, 3A-H, and 2B-H), 4.16–4.08 (m, 3 H, 3C-H,

1

pH ca. 7), and concentration. H NMR (500 MHz, [D₄]methanol,

data for sodium salt): δ = 7.58 (m, 2 H, Ar), 7.44 (m, 2 H, Ar),

7.34–7.20 (m, 6 H, Ar), 6.98 (m, 2 H, Ar), 6.82 (m, 2 H, Ar), 5.05

[d, J_gem= 10.3 Hz, 1 H, CH₂(Bn)], 4.93 (d, J_1,2= 8.5 Hz, 1 H, 1A-

H), 4.88 [m, 2 H, CH₂(Bn)], 4.72 [d, J_gem= 10.3 Hz, 1 H, CH₂(Bn)],

4.62 (d, J_1,2= 8.5 Hz, 1 H, 1C-H), 4.43 (m, 2 H, 1B-H and 1D-H),

4.27 (dd, J_1,2= 8.5, J_2,3= 10.7 Hz, 1 H, 2A-H), 4.19–4.12 (m, 3 H,

2C-H, 4C-H and 4A-H), 3.96 (m, 1 H, 4B-H), 3.86–3.76 (m, 4 H,

3A-H and 3 6A-H or 6C-H), 3.74 (s, 3 H, OCH₃), 3.72–3.56 (m, 6

H, 5B-H, 6A-H or 6C-H, 3C-H, 5A-H or 5C-H, 4D-H, and 5D-

H), 3.51 (m, 1 H, 5A-H or 5C-H), 3.46 (m, 2 H, 2B-H and 3B-H),

3.41 (m, 2 H, 2D-H and 3D-H), 2.05 (s, 3 H, NHCOCH₃), 2.05 (s, 3

H, NHCOCH₃) ppm. ¹³C NMR (100 MHz, [D₄]methanol, selected

data from HSQC experiment): δ = 128.5–113.7 (Ar), 104.2 (C-1B

and C-1D), 100.6 (C-1A), 100.0 (C-1C), 84.1 (C-3D), 82.6 (C-3C),

82.5 (C-3B), 80.6 (C-3A), 77.6 (C-4B), 76.4 (C-5B), 75.8 (C-5A or

5A-H or 5C-H, and 2A-H), 4.03 (pt, 1 H, 3D-H), 3.98 (d, J_4,5

=

8.5 Hz, 1 H, 5D-H), 3.93 (m, 2 H, 4B-H and 2C-H), 3.85–3.81 (m,

4 H, 3B-H and OCH₃), 3.72 (d, J_4,5= 9.6 Hz, 1 H, 5B-H), 2.07

(NHCOCH₃), 2.06 (NHCOCH₃) ppm. ¹³C NMR (125 MHz, D₂O,

selected data from HSQC experiment): δ = 119.6 (Ar), 116.2 (Ar),

103.8 (C-1B), 102.9 (C-1D), 102.6 (C-1C), 101.7 (C-1A), 81.5 (C-

4B), 80.7 (C-2B and C-2D), 79.0 (C-4D), 77.9 (C-3A), 77.8 (C-5B),

77.5 (C-3C and C-5D), 77.0 (C-4A and C-4C), 74.4 (C-3B), 74.2

(C-3D), 73.9 (C-5A or C-5C), 73.5 (C-5A or C-5C), 69.0 (C-6A or

C-6C), 68.7 (C-6A or C-6C), 56.8 (OCH₃), 53.3 (C-2A), 23.7 (2

NHCOCH₃) ppm. MS (ESI): calcd. for C₃₅H₄₁N₂O₄₅S₇Na₇[M +

7Na]^2–796.9; found 796.7.

C-5C), 75.2 (2 C: C-4D or C-5D or C-5A or C-5C), 75.0 [CH₂(Bn)], 4-Methoxyphenyl 3-O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

74.0 [CH₂(Bn)], 73.0 (C-2D), 72.7 (C-2B), 72.0 (C-4D or C-5D or idopyranosyluronate)-2-deoxy-4,6-O-di-tert-butylsilylene-2-trifluoro-

C-5A or C-5C), 67.6 (C-4A and C-4C), 61.1 (C-6A and C-6C), acetamido-β- -galactopyranoside (40): Compound 39 (427 mg,

L-

D

54.4 (COCH₃), 51.5 (C-2C), 51.2 (C-2A), 22.1 (NHCOCH₃), 21.6 0.428 mmol) was dissolved in CH₂Cl₂(8.5 mL), and hydrazine

(NHCOCH₃) ppm. HRMS: calcd. for C₄₉H₆₀N₂O₂₄[M]^2–monohydrate (0.5 m solution in pyridine/AcOH, 3:2; 3.42 mL) was

530.1773; found 530.1775.

added. The mixture was stirred at room temperature for 3 h, the

reaction mixture was quenched with acetone (5.0 mL). The mixture

was diluted with CH₂Cl₂and washed with HCl (1 m aq.), saturated

aqueous NaHCO₃, and H₂O. The organic layer was dried (MgSO₄),

filtered, and concentrated in vacuo to give the corresponding diol,

which was used in the next step without further purification [TLC

(toluene/EtOAc, 1:2): R_f= 0.20].

Compound 36 (6 mg, 5.4 μmol) and sulfur trioxide–trimethylamine

complex (26 mg, 0.19 mmol) were dissolved in dry DMF (1.5 mL),

and the mixture was heated at 100 °C for 2 h using microwave irra-

diation (20 W average power). The reaction vessel was cooled, and

Et₃N (150 μL) and MeOH (1 mL) were added. The solution was

loaded onto a Sephadex LH-20 chromatography column, which

was eluted with MeOH to give 37 as its triethylammonium salt.

The residue was converted into the sodium salt by elution from a

column of Dowex 50WX4-Na⁺with MeOH/H₂O, 9:1 (5 mg, 56%).

¹H NMR (500 MHz, D₂O, 40 °C, data for sodium salt): δ = 7.62

This diol was dissolved in pyridine (9 mL). Pivaloyl chloride

(2.5 mL) and DMAP (21 mg, 0.17 mmol) were added, and the solu-

tion was stirred at room temperature. After 45 h, the mixture was

diluted with CH₂Cl₂, washed with HCl (1 m aq.), saturated aqueous

(m, 2 H, Ar), 7.50–7.36 (m, 8 H, Ar), 7.12 (m, 2 H, Ar), 7.00 (m, NaHCO₃, and H₂O, dried (MgSO₄), filtered, and concentrated in

2 H, Ar), 5.31 (d, J_1,2= 8.5 Hz, 1 H, 1A-H), 4.96 (m, 2 H, 4A-H vacuo. The residue was purified by flash column chromatography

and 4C-H), 4.93–4.85 [m, 4 H, 1B-H or 1D-H, 4D-H and

(toluene/EtOAc, 6:1) to give 40 (363 mg, 87% over two steps). TLC

3880

www.eurjoc.org

Eur. J. Org. Chem. 2014, 3868–3884

Synthesis of Chondroitin Sulfate Oligosaccharides

1

(toluene/EtOAc, 4:1): R_f= 0.40. [α]²_D⁰= –19 (c = 1.0, CHCl₃). H

COCF₃), 155.9, 151.1, 137.6 (Ar-C), 128.0, 127.9, 119.0 (Ar-CH),

NMR (400 MHz, CDCl₃): δ = 7.39–7.26 (m, 5 H, Ar), 6.94 (m, 2 115.7 (q, J_C,F= 288.2 Hz, COCF₃), 114.7 (Ar-CH), 100.9 (C-1B),

H, Ar), 6.79 (m, 2 H, Ar), 6.66 (d, J_2,NH= 7.2 Hz, 1 H, NH), 5.31

(d, J_1,2= 8.4 Hz, 1 H, 1A-H), 5.29 (m, 1 H, 4B-H), 5.15 (d, J_4,5

99.2 (C-1A), 75.4 (C-3A), 74.4 (C-3B), 72.6 [CH₂(Bn)], 71.4 (C-

5A), 68.6 (C-4A), 68.0 (C-2B), 67.8 (C-4B), 67.1 (C-5B), 61.9 (C-

=

2.0 Hz, 1 H, 5B-H), 5.04 (br. s, 1 H, 1B-H), 4.86 (m, 1 H, 2B-H), 6A), 55.7 [COOMe or Me(OMP)], 54.7 (C-2A), 52.4 [COOMe or

4.79 [2 d, 2 H, CH₂(Bn)], 4.53 (d, J_3,4= 2.8 Hz, 1 H, 4A-H), 4.39

(dd, J_2,3= 11.0 Hz, 1 H, 3A-H), 4.20 (m, 2 H, 6aA-H, 6bA-H),

Me(OMP)], 39.0, 38.7 [C(CH₃)₃(Piv)], 38.0, 37.9 [CH₂(Lev)], 29.9,

29.7 [CH₃(Lev)], 27.9, 27.8 [CH₂(Lev)], 27.2, 27.0 [C(CH₃)₃(Piv)]

3.98 (dt, 1 H, 2A-H), 3.76 [s, 3 H, Me(OMP) or COOMe], 3.74 [m, ppm. HRMS: calcd. for C₄₉H₆₂F₃NO₁₉Na [M + Na]⁺1048.3766;

4 H, 3B-H, Me(OMP) or COOMe], 3.47 (br. s, 1 H, 5A-H), 1.19,

1.17, 1.08, 0.97 [4 s, 36 H, C(CH₃)₃] ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 177.9, 177.6 [CO(Piv)], 169.1 (COOMe), 157.9 (q, J_C,F

= 37.2 Hz, COCF₃), 156.0, 151.2, 137.9 (Ar C), 128.5, 127.7, 127.5,

120.0 (Ar-CH), 115.6 (q, J_C,F= 288.4 Hz, COCF₃), 114.6 (Ar-CH),

101.4 (C-1B), 99.2 (C-1A), 78.8 (C-3A), 74.0 (C-3B), 72.7 (C-4A),

72.5 [CH₂(Bn)], 71.3 (C-5A), 67.7 (C-4B), 67.5 (C-2B), 67.0 (C-

6A), 66.9 (C-5B), 55.7 [COOMe or Me(OMP)], 54.1 (C-2A), 52.4

[COOMe or Me(OMP)], 39.1, 38.8 [C(CH₃)₃, Piv], 27.8, 27.4, 27.3,

27.1 [C(CH₃)₃], 23.3, 21.0 [C(CH₃)₃] ppm. HRMS: calcd. for

C₄₇H₆₆F₃NO₁₅SiNa [M + Na]⁺992.4052; found 992.4087.

found 1048.3790.

3-O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

ate)-2-deoxy-4,6-di-O-levulinoyl-2-trifluoroacetamido-α,β-

L

-idopyranosyluron-

-galacto-

D

pyranose (42): CAN (0.63 m solution in H₂O; 1.6 mL) was added

to a solution of 41 (346 mg, 0.337 mmol) in CH₂Cl₂/MeCN (1:2;

15 mL). The mixture was stirred for 1 h 30 min at room tempera-

ture, then it was diluted with EtOAc, and washed with H₂O, satu-

rated aqueous NaHCO₃, and H₂O. The organic phase was dried

(MgSO₄), filtered, and concentrated to dryness. The residue was

purified by column chromatography (toluene/acetone, 7:2) to give

42 (218 mg, 70%) as a mixture of α/β anomers. TLC (toluene/acet-

1

one, 7:2): R_f= 0.20 and 0.11. H NMR (300 MHz, CDCl₃) (data

4-Methoxyphenyl 3-O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

idopyranosyluronate)-2-deoxy-4,6-di-O-levulinoyl-2-trifluoroacet-

amido-β- -galactopyranoside (41): An excess of (HF)_n·Py (1.85 mL,

L-

for α anomer): δ = 7.40–7.26 (m, 5 H, Ar), 6.65 (d, J_2,NH= 9.5 Hz,

1 H, NH), 5.35 (m, 2 H, 4A-H, 1A-H), 5.20 (br. t, 1 H, 4B-H),

5.02 (br. s, 1 H, 1B-H), 4.80 (d, J_4,5= 2.4 Hz, 1 H, 5B-H), 4.76 (m,

1 H, 2B-H), 4.74–4.62 [2 d, 2 H, CH₂(Bn)], 4.53 (dt, J_1,2= 3.5, J_2,3

= 10.6 Hz, 1 H, 2A-H), 4.34 (dd, J_5,6a= 3.7, J_5,6b= 8.8 Hz, 1 H,

D

71.1 mmol) was added to a solution of 40 (363 mg, 0.374 mmol) in

dry THF (8.0 mL) at 0 °C under an argon atmosphere. After 24 h

at 0 °C, the mixture was diluted with CH₂Cl₂and washed with H₂O

and saturated aq. NaHCO₃until it reached neutral pH. The or-

ganic layers were dried (MgSO₄), filtered, and concentrated in

vacuo to give the corresponding diol (290 mg), which was used for

the next step without further purification [TLC (toluene/EtOAc,

1:6): R_f= 0.45].

5A-H), 4.22 (dd, J_6a,6b= 11.5 Hz, 1 H, 6aA-H), 4.10 (dd, J_3,4

=

3.1 Hz, 1 H, 3A-H), 3.95 (dd, 1 H, 6bA-H), 3.77 (s, 3 H, COOMe),

3.70 (br. t, 1 H, 3B-H), 2.82–2.26 [m, 8 H, CH₂(Lev)], 2.19, 2.05 [2

s, 6 H, CH₃(Lev)], 1.18, 1.15 [2 s, 18 H, (CH₃)₃(Piv)] ppm. ¹³C

NMR (75 MHz, CDCl₃, data for α anomer): δ = 208.5, 206.7

[CO(Lev)], 177.8, 177.6, 172.3 [CO(Lev, Piv)], 169.0 (COOMe),

157.7 (q, J_C,F= 37.2 Hz, COCF₃), 137.5 (Ar-C), 128.5, 128.1, 128.0

(Ar-CH), 115.9 (q, J_C,F= 288.0 Hz, COCF₃), 101.1 (C-1B), 91.7

(C-1A), 75.5 (C-3A), 73.7 (C-3B), 72.4 [CH₂(Bn)], 69.4 (C-4A),

67.9 (C-4B), 67.5 (C-2B), 67.0 (C-5B), 66.9 (C-5A), 62.7 (C-6A),

52.4 (COOMe), 50.2 (C-2A), 39.0, 38.7 [C(CH₃)₃(Piv)], 38.3, 37.9

[CH₂(Lev)], 29.9, 29.7 [CH₃(Lev)], 28.1, 27.7 [CH₂(Lev)], 27.2, 27.1

[C(CH₃)₃(Piv)] ppm. HRMS: calcd. for C₄₂H₅₆F₃NO₁₈Na [M +

Na]⁺942.3347; found 942.3311.

LevOH (0.72 mL, 7.0 mmol) was added at 0 °C to a solution of

1,3-dicyclohexylcarbodiimide (721 mg, 3.5 mmol) in CH₂Cl₂

(6 mL). The mixture was stirred for 5 min at room temperature,

then it was cooled, filtered, and concentrated to give levulinic anhy-

dride quantitatively (Lev₂O, 3.5 mmol).

A solution of the diol (290 mg) in dry pyridine (10 mL) was added

to a flask containing Lev₂O (3.5 mmol) and DMAP (44 mg,

0.35 mmol) under an argon atmosphere. The mixture was stirred

for 26 h at room temperature, then it was diluted with CH₂Cl₂and

washed with HCl (1 m aqueous), saturated aqueous NaHCO₃, and

brine. The organic layers were dried (MgSO₄), filtered, and concen-

trated in vacuo. To complete the reaction, this residue was redis-

solved in dry pyridine (10 mL), and this solution was added to a

flask containing additional Lev₂O (3.5 mmol) and DMAP (44 mg,

0.35 mmol) under an argon atmosphere. The mixture was stirred

for 26 h at room temperature, then it was diluted with CH₂Cl₂and

washed with HCl (1 m aq.), saturated aqueous NaHCO₃, and brine.

The organic layers were dried (MgSO₄), filtered, and concentrated

in vacuo. The residue was purified by column chromatography (tol-

O-[3-O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

uronate)-2-deoxy-4,6-di-O-levulinoyl-2-trifluoroacetamido-α,β-

L

-idopyranosyl-

-ga-

D

lactopyranosyl] Trichloroacetimidate (43): Trichloroacetonitrile

(162 μL, 1.6 mmol) and catalytic DBU (0.084 m solution in dry

CH₂Cl₂; 80 μL) were added to a solution of 42 (60 mg, 65 μmol)

in dry CH₂Cl₂(1.3 mL). The mixture was stirred for 10 h at room

temperature, then it was concentrated to dryness. The residue was

purified by flash chromatography (toluene/acetone, 5:1 + 1% Et₃N)

to give 43 (54 mg, 78%) as a mixture of α/β anomers. TLC (toluene/

acetone, 3:1): R_f= 0.45 (for α anomer). ¹H NMR (400 MHz,

CDCl₃, data for α anomer): δ = 8.81 [s, 1 H, NH(TCA)], 7.35–7.26

(m, 5 H, Ar), 6.78 [d, J_2,NH= 8.5 Hz, 1 H, NH(TFA)], 6.47 (d, J_1,2

uene/EtOAc, 2:1) to give 41 (311 mg, 81%). TLC (toluene/EtOAc,

1

1:1): R_f= 0.30. H NMR (300 MHz, CDCl₃): δ = 7.36–7.26 (m, 5 = 3.5 Hz, 1 H, 1A-H), 5.52 (br. d, 1 H, 4A-H), 5.24 (br. t, 1 H, 4B-

H, Ar), 6.95 (m, 2 H, Ar), 6.79 (m, 3 H, NH, Ar), 5.41 (d, J_3,4

3.2 Hz, 1 H, 4A-H), 5.26 (m, 2 H, 4B-H, 1A-H), 4.98 (br. s, 1 H, 4.75–4.67 [m, 4 H, 2B-H, 2A-H, CH₂(Bn)], 4.29 (t, J_5,6a= J_5,6b

1B-H), 4.86 (d, J_4,5= 2.4 Hz, 1 H, 5B-H), 4.83 (br. t, 1 H, 2B-H), 6.5 Hz, 1 H, 5A-H), 4.23 (dd, J_2,3= 11.0, J_3,4= 3.1 Hz, 1 H, 3A-

4.71 [m, 2 H, CH₂(Bn)], 4.50 (dd, J_2,3= 10.9, J_3,4= 3.3 Hz, 1 H, H), 4.08 (m, 2 H, 6aA-H, 6bA-H), 3.77 (s, 3 H, COOMe), 3.72 (br.

3A-H), 4.16 (dd, J_5,6a= 7.4, J_6a,6b= 11.4 Hz, 1 H, 6aA-H), 4.09 t, 1 H, 3B-H), 2.71–2.17 [m, 8 H, CH₂(Lev)], 2.16, 2.08 [2 s, 6

(dd, J_5,6a= 5.8 Hz, 1 H, 6bA-H), 3.98 (m, 1 H, 2A-H), 3.93 (br. t,

H, CH₃(Lev)], 1.18, 1.14 [2 s, 18 H, (CH₃)₃(Piv)] ppm. ¹³C NMR

1 H, 5A-H), 3.76 [s, 6 H, Me(OMP), COOMe], 3.69 (br. t, 1 H, (100 MHz, CDCl₃, data for α anomer): δ = 206.5, 206.1 [CO(Lev)],

=

H), 5.11 (br. s, 1 H, 1B-H), 4.85 (d, J_4,5= 2.6 Hz, 1 H, 5B-H),

=

3B-H), 2.72–2.23 [m, 8 H, CH₂(Lev)], 2.16, 2.08 [2 s, 6 H,

178.0, 177.7, 172.2, 172.0 [CO(Lev, Piv)], 168.9 (COOMe), 160.3

CH₃(Lev)], 1.18, 1.14 [2 s, 18 H, CH₃(Piv)] ppm. ¹³C NMR (C=NH), 157.9 (q, J_C,F= 37.9 Hz, COCF₃), 137.3 (Ar-C), 128.6,

(75 MHz, CDCl₃): δ = 206.8, 206.3 [CO(Lev)], 177.8, 177.5, 172.2, 128.1, 128.0 (Ar-CH), 115.7 (q, J_C,F= 289.0 Hz, COCF₃), 101.3

172.1 [CO(Lev, Piv)], 168.9 (COOMe), 158.0 (q, J_C,F= 37.3 Hz,

(C-1B), 94.8 (C-1A), 90.7 (CCl₃), 75.2 (C-3A), 73.9 (C-3B), 72.7

Eur. J. Org. Chem. 2014, 3868–3884

www.eurjoc.org

3881

G. Macchione, S. Maza, M. Mar Kayser, J. L. de Paz, P. M. Nieto

FULL PAPER

[CH₂(Bn)], 70.0 (C-5A), 68.9 (C-2B), 68.4 (C-4A), 67.6 (C-4B), 67.5 aqueous NaHCO₃, and H₂O. The organic layer was dried (MgSO₄),

(C-5B), 61.8 (C-6A), 52.5 (COOMe), 49.9 (C-2A), 39.1, 38.8 filtered, and concentrated in vacuo. The residue was purified by

[C(CH₃)₃(Piv)], 37.9 [CH₂(Lev)], 29.9, 29.7 [CH₃(Lev)], 27.9, 27.8 flash column chromatography (toluene/EtOAc, 6:1) to give 45

[CH₂(Lev)], 27.2, 27.1 [C(CH₃)₃(Piv)] ppm. MS (ESI): calcd. for

(82 mg, 87%). TLC (toluene/EtOAc, 3:2): R_f= 0.32. [α]²_D⁰= –7 (c

= 1.0, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.97 (d, 2 H, Ar),

7.57 (t, 1 H, Ar), 7.46–7.33 (m, 12 H, Ar), 7.13 (m, 5 H, Ar), 6.91

(m, 2 H, Ar), 6.80 (m, 3 H, Ar, NH), 6.52 (d, J_2,NH= 8.4 Hz, 1 H,

NH), 5.38 (d, J_1,2= 8.3 Hz, 1 H, 1A-H), 5.30–5.20 [m, 5 H, 1B-H,

C₄₄H₅₆Cl₃F₃N₂O₁₈Na [M + Na]⁺1085.2; found 1085.5.

4-Methoxyphenyl O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

pyranosyluronate)-(1Ǟ3)-O-(2-deoxy-4,6-di-O-levulinoyl-2-tri-

fluoroacetamido-β- -galactopyranosyl)-(1Ǟ4)-O-(benzyl 2-O-Benz-

oyl-3-O-benzyl-β- -glucopyranosyluronate)-(1Ǟ3)-2-deoxy-4,6-O-

di-tert-butylsilylene-2-trifluoroacetamido-β- -galactopyranoside

L-ido-

D

2B-H, CH₂(Bn), 4D-H], 5.08 (br. d, 1 H, 1D-H), 4.94 (d, J_4,5

=

D

2.7 Hz, 1 H, 5D-H), 4.86 [d, 1 H, CH₂(Bn)], 4.85 (br. d, 1 H, 2D-

H), 4.79–4.68 [2 d, 2 H, CH₂(Bn)], 4.58 (br. s, 1 H, 4A-H), 4.54 [d,

1 H, CH₂(Bn)], 4.38 (br. d, J_2,3= 11.1 Hz, 1 H, 3A-H), 4.27 (d,

D

(44): Donor 43 (168 mg, 0.158 mmol) and acceptor 32 (103 mg,

0.105 mmol) were dissolved in dry CH₂Cl₂(3.0 mL) in the presence

of freshly activated 4 Å molecular sieves. The mixture was stirred

for 30 min at 0 °C, then TMSOTf (0.092 m solution in dry CH₂Cl₂;

343 μL) was added under an argon atmosphere. The mixture was

stirred for 30 min at 0 °C, then it was neutralized with Et₃N, fil-

tered, and concentrated to dryness. The residue was purified by

column chromatography (toluene/acetone, 7:1) to give 44 (106 mg,

53%). TLC (toluene/acetone, 3:1): R_f= 0.55. [α]²_D⁰= –2 (c = 1.0,

J_1,2= 8.5 Hz, 1 H, 1C-H), 4.24 (m, 1 H, 2C-H), 4.11 (d, J_6a,6b

=

12.3 Hz, 1 H, 6aA-H), 4.05 (t, J_3,4= J_4,5= 9.5 Hz, 1 H, 4B-H),

3.99 (m, 1 H, 6bA-H), 3.98–3.91 (m, 3 H, 5B-H, 2A-H, 4C-H),

3.76–3.75 [2 s, 6 H, COOMe, Me(OMP)], 3.70 (m, 2 H, 3D-H, 3B-

H), 3.56–3.45 (m, 3 H, 6aC-H, 6bC-H, 3C-H), 3.32 (m, 1 H, 5A-

H), 3.12 (m, 1 H, 5C-H), 1.20, 1.18, 1.06, 1.02 [4 s, 36 H,

C(CH₃)₃] ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 177.6, 177.4,

169.0, 168.7, 165.1 [CO(Piv), CO(Bz), COOBn, COOMe], 157.9 (q,

J_C,F= 36.9 Hz, COCF₃), 157.7 (q, J_C,F= 36.7 Hz, COCF₃), 156.0,

151.1, 137.7, 137.2, 134.7 (Ar-C), 133.5, 129.9, 129.5, 129.4, 129.2,

129.1, 128.6, 128.5, 128.3, 128.1, 128.0, 127.8, 120.3 (Ar-C, Ar-

CH), 116.0 (q, J_C,F= 288.3 Hz, COCF₃), 115.5 (q, J_C,F= 288.3 Hz,

COCF₃), 114.6 (Ar-CH), 100.4 (C-1B), 100.2 (C-1C), 99.6 (C-1D),

99.3 (C-1A), 80.6 (C-3B), 79.0 (C-3C), 78.0 (C-4B), 75.8 [CH₂(Bn)],

75.5 (C-3A), 74.6 (C-5C), 74.1 (C-5B), 73.7 (C-3D), 73.3 (C-4A),

72.6 (C-2B), 72.4 [CH₂(Bn)], 71.4 (C-5A), 68.3 [CH₂(Bn), C-4C],

68.2 (C-2D), 67.8 (C-5D), 67.0 (C-4D, C-6A), 62.3 (C-6C), 55.7

[COOMe or Me(OMP)], 54.1 (C-2A), 52.5 [COOMe or Me(OMP)],

52.3 (C-2C), 39.1, 38.8 [C(CH₃)₃(Piv)], 27.6, 27.3, 27.1 [C(CH₃)₃],

2 3 . 4 , 2 0. 8 [ C( CH₃)₃(DBSi)] ppm. HRMS: calcd. for

C₈₂H₁₀₀F₆N₂O₂₇SiNa [M + Na]⁺1709.6085; found 1709.6031.

1

CHCl₃). H NMR (400 MHz, CDCl₃): δ = 7.92 (d, 2 H, Ar), 7.56

(t, 1 H, Ar), 7.49–7.22 (m, 12 H, Ar), 7.07 (m, 5 H, Ar), 6.95 (d,

J_2,NH= 6.5 Hz, 1 H, NH), 6.90 (m, 2 H, Ar), 6.78 (m, 2 H, Ar),

6.68 (d, J_2,NH= 8.9 Hz, 1 H, NH), 5.37 (d, J_1,2= 8.2 Hz, 1 H, 1A-

H), 5.30 (d, J_1,2= 7.9 Hz, 1 H, 1B-H), 5.28 [m, 2 H, CH₂(Bn)],

5.24 (m, 1 H, 4D-H), 5.21 (t, 1 H, 2B-H), 5.17 (d, J_3,4= 3.1 Hz, 1

H, 4C-H), 4.95 (br. s, 1 H, 1D-H), 4.91 (d, J_4,5= 2.2 Hz, 1 H, 5D-

H), 4.84 [d, 1 H, CH₂(Bn)], 4.77 (br. s, 1 H, 2D-H), 4.72–4.66 [2 d,

2 H, CH₂(Bn)], 4.57 (br. s, 1 H, 4A-H), 4.51 [d, 1 H, CH₂(Bn)],

4.36 (br. d, J_2,3= 11.3 Hz, 1 H, 3A-H), 4.24 (d, J_1,2= 8.5 Hz, 1 H,

1C-H), 4.17–3.98 (m, 4 H, 2C-H, 6aA-H, 4B-H, 6bA-H), 3.97–3.93

(m, 2 H, 2A-H, 5B-H), 3.87 (m, 2 H, 6aC-H, 6bC-H), 3.78, 3.75 [2

s, 6 H, Me(OMP), COOMe], 3.68 (m, 2 H, 3B-H, 3D-H), 3.57 (dd,

J_2,3= 10.5 Hz, 1 H, 3C-H), 3.45 (t, J_5,6a= J_5,6b= 6.5 Hz, 1 H, 5C-

H), 3.32 (m, 1 H, 5A-H), 2.68 [m, 2 H, CH₂(Lev)], 2.48–2.16 [m,

8 H, CH₂(Lev), CH₃(Lev) (2.17)], 2.10–2.04 [m, 1 H, CH₂(Lev)],

1.96 [s, 3 H, CH₃(Lev)], 1.20, 1.18, 1.05, 1.01 [4 s, 36 H, C(CH₃)₃]

ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.9, 206.1 [CO(Lev)],

177.9, 177.3, 172.2, 172.0, 169.1, 168.9, 165.1 [CO(Lev), CO(Piv),

CO(Bz), COOBn, COOMe], 158.2 (q, J_C,F= 37.1 Hz, COCF₃),

157.7 (q, J_C,F= 36.8 Hz, COCF₃), 156.0, 151.1, 137.8, 137.6, 134.6

(Ar-C), 133.4, 129.9, 129.7, 129.5, 129.4, 129.3, 128.5, 128.2, 128.0,

127.7, 120.3 (Ar-C, Ar-CH), 116.0 (q, J_C,F= 288.3 Hz, COCF₃),

115.5 (q, J_C,F= 288.6 Hz, COCF₃), 114.6 (Ar-CH), 100.4 (C-1D),

100.2, 100.1 (C-1B, C-1C), 99.3 (C-1A), 80.2 (C-3B), 78.3 (C-4B),

76.5 (C-3C), 75.3 [CH₂(Bn)], 75.2 (C-3A), 74.2 (C-3D), 74.1 (C-

5B), 73.4 (C-4A), 72.5 [CH₂(Bn)], 72.4 (C-2B), 71.4 (C-5A), 71.2

(C-5C), 68.5 [CH₂(Bn)], 68.1 (C-4C), 67.8 (C-4D, C-2D), 67.6 (C-

6A), 67.0 (C-5D), 61.4 (C-6C), 55.7 [COOMe or Me(OMP)], 54.1

(C-2A), 53.3 (C-2C), 52.4 [COOMe or Me(OMP)], 39.1, 38.7

[C(CH₃)₃(Piv)], 38.0, 37.8 [CH₂(Lev)], 29.9, 29.6 [CH₃(Lev)], 28.0,

27.7 [CH₂(Lev)], 27.6, 27.3, 27.1 [C(CH₃)₃], 23.4, 20.8 [C(CH₃)₃

(DBSi)] ppm. HRMS: calcd. for C₉₂H₁₁₂F₆N₂O₃₁SiNa [M + Na]⁺

1905.6820; found 1905.6755.

4-Methoxyphenyl O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

pyranosyluronate)-(1Ǟ3)-O-(2-deoxy-2-trifluoroacetamido-β-

lactopyranosyl)-(1Ǟ4)-O-(benzyl 2-O-Benzoyl-3-O-benzyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-deoxy-2-trifluoroacetamido-β- -ga-

L-ido-

D

-ga-

D

-

D

lactopyranoside (46): An excess of (HF)_n·Py (223 μL, 8.6 mmol) was

added to a solution of 45 (75 mg, 0.044 mmol) in dry THF

(4.0 mL) at 0 °C under an argon atmosphere. After 24 h at 0 °C,

the mixture was diluted with CH₂Cl₂, and then washed with H₂O

and saturated NaHCO₃solution until it reached neutral pH. The

organic layers were dried (MgSO₄), filtered, and concentrated in

vacuo to give 46 (67 mg, 97%). TLC (toluene/EtOAc, 1:2): R_f=

1

0.20. [α]²_D⁰= –3 (c = 1.0, CHCl₃). H NMR (400 MHz, CDCl₃): δ

= 7.85 (d, 2 H, Ar), 7.56 (t, 1 H, Ar), 7.44–7.26 (m, 13 H, Ar, NH),

7.10–7.03 (m, 5 H, Ar), 6.83 (m, 2 H, Ar), 6.72 (m, 2 H, Ar), 6.64

(m, 1 H, NH), 5.37 (m, 1 H, 1A-H), 5.29 (m, 1 H, 2B-H), 5.22 [m,

3 H, 4D-H, CH₂(Bn)], 5.06 (br. d, 1 H, 1D-H), 4.93 (d, J_4,5

=

2.8 Hz, 1 H, 5D-H), 4.87 [d, 1 H, CH₂(Bn)], 4.84 (m, 1 H, 2D-H),

4.81 (d, J_1,2= 7.4 Hz, 1 H, 1B-H), 4.77–4.56 [3 d, 3 H, CH₂(Bn)],

4.48 (m, 2 H, 1C-H, 3A-H), 4.31 (t, J_3,4= J_4,5= 8.8 Hz, 1 H, 4B-

H), 4.16 (m, 1 H, 2C-H), 4.14 (br. s, 1 H, 4A-H), 4.06 (br. d, 1 H,

5B-H), 3.93 (br. d, J_3,4= 2.6 Hz, 1 H, 4C-H), 3.88 (m, 1 H, 2A-

4-Methoxyphenyl O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

pyranosyluronate)-(1Ǟ3)-O-(2-deoxy-2-trifluoroacetamido-β-

lactopyranosyl)-(1Ǟ4)-O-(benzyl 2-O-Benzoyl-3-O-benzyl-β-

glucopyranosyluronate)-(1Ǟ3)-2-deoxy-4,6-O-di-tert-butylsilylene-

2-trifluoroacetamido-β- -galactopyranoside (45): Compound 44

L-ido-

D

-ga- H), 3.83–3.70 [m, 9 H, 6aA-H, 3B-H, 3D-H, Me(OMP), COOMe],

D

-

3.59 (m, 4 H, 6bA-H, 6aC-H, 3C-H, 5A-H), 3.48 (dd, J_5,6b= 2.8,

J_6a,6b= 12.1 Hz, 1 H, 6bC-H), 3.26 (m, 1 H, 5C-H), 2.29 (m, 4 H,

OH), 1.19, 1.18 [2 s, 18 H, C(CH₃)₃] ppm. ¹³C NMR (100 MHz,

CDCl₃, selected data from HSQC experiment): δ = 133.7, 130.2,

129.7–127.6, 119.1, 114.7 (Ar-CH), 101.7 (C-1B), 100.1 (C-1C),

99.6 (C-1D), 98.7 (C-1A), 80.5 (C-3B), 78.8 (C-3C), 78.6 (C-3A),

77.4 (C-4B), 75.9 [CH₂(Bn)], 75.2 (C-5C), 74.5 (C-5A), 74.2 (C-

5B), 74.1 (C-3D), 72.8 (C-2B), 72.7 [CH₂(Bn)], 68.8 [CH₂(Bn)], 68.6

D

(105 mg, 0.056 mmol) was dissolved in CH₂Cl₂(1.5 mL), and hy-

drazine monohydrate (0.5 m solution in pyridine/AcOH, 3:2;

0.45 mL) was added. The mixture was stirred at room temperature

for 3 h, then it was quenched with acetone (0.7 mL). The mixture

was diluted with CH₂Cl₂and washed with HCl (1 m aq.), saturated

3882

www.eurjoc.org

Eur. J. Org. Chem. 2014, 3868–3884

Synthesis of Chondroitin Sulfate Oligosaccharides

(C-4A, C-4C, C-2D), 68.0 (C-5D), 67.6 (C-4D), 62.7 (C-6C), 62.5 white solid was then eluted through a column of Dowex 50WX4-

(C-6A), 55.9 [COOMe or Me(OMP)], 54.5 (C-2A), 52.8 [C-2C, CO-

OMe or Me(OMP)], 27.5 [C(CH₃)₃] ppm. HRMS: calcd. for

C₇₄H₈₄F₆N₂O₂₇Na [M + Na]⁺1569.5063; found 1569.5101.

Na⁺(H₂O/MeOH, 9:1) to give 49 as its sodium salt (6 mg, 86%)

after lyophilization. TLC (EtOAc/pyridine/H₂O/AcOH, 6:5:3:1): R_f

= 0.17. H NMR (500 MHz, D₂O): δ = 7.57–7.36 (m, 10 H, Ar),

1

7.09 (d, 2 H, Ar), 6.97 (d, 2 H, Ar), 5.03 (d, J_1,2= 8.5 Hz, 1 H,

1A-H), 4.96 [d, J = 10.5 Hz, 1 H, CH₂(Bn)], 4.85 (d, J_3,4= 2.5 Hz,

1 H, 4A-H), 4.84–4.77 [m, 6 H, CH₂(Bn), 5D-H (4.83), 1D-H

(4.80), 4C-H (4.76)], 4.70 (d, J_1,2= 8.0 Hz, 1 H, 1C-H), 4.55 (d,

J_1,2= 7.5 Hz, 1 H, 1B-H), 4.35–4.29 (m, 2 H, 2A-H, 6A-H or 6C-

H), 4.26–4.19 (m, 3 H, 5A-H or 5C-H, 2 6A-H or 6C-H), 4.16 (dd,

1 H, 3A-H), 4.14–4.04 (m, 4 H, 6A-H or 6C-H, 4D-H, 2C-H, 4B-

H), 3.99 (m, 1 H, 5A-H or 5C-H), 3.95 (m, 1 H, 3C-H), 3.81 [s, 3

H, Me(OMP)], 3.76 (d, J_4,5= 9.5 Hz, 1 H, 5B-H), 3.67–3.53 (m, 4

H, 2D-H, 3B-H, 2B-H, 3D-H), 2.04, 2.03 (2 s, 6 H, NHAc) ppm.

¹³C NMR (125 MHz, D₂O, selected data from HSQC experiment):

δ = 130.1–116.1 (Ar-CH), 105.3 (C-1D), 104.9 (C-1B), 101.6 (C-

1A), 101.3 (C-1C), 82.9 (C-3D), 82.4 (C-3B), 78.4 (C-4B), 77.8 (C-

5B), 77.6 (C-4C, C-3C), 76.9 (C-4A), 75.9 (C-3A), 74.7, 74.4

[CH₂(Bn)], 73.8–73.4 (C-5A, C-5C, C-4D), 72.6 (C-2B), 71.8 (C-

2D), 68.9, 68.5 (C-6A, C-6C), 56.8 [Me(OMP)], 52.9, 52.6 (C-2A,

C-2C), 23.6 (NAc) ppm. MS (ESI): calcd. for C₄₉H₅₈N₂O₃₆S₄Na₃

[M + 3Na + 2H]^–1447.1; found 1447.0.

4-Methoxyphenyl O-(Methyl 3-O-Benzyl-2,4-di-O-pivaloyl-α-

pyranosyluronate)-(1Ǟ3)-O-(2-deoxy-4,6-di-O-sulfo-2-trifluoroacet-

L-ido-

amido-β-

benzyl-β-

D

-galactopyranosyl)-(1Ǟ4)-O-(benzyl 2-O-Benzoyl-3-O-

D-glucopyranosyluronate)-(1Ǟ3)-2-deoxy-4,6-di-O-sulfo-2-

trifluoroacetamido-β-D-galactopyranoside (47): Compound 46

(16 mg, 10 μmol) and sulfur trioxide–trimethylamine complex

(58 mg, 0.41 mmol) were dissolved in dry DMF (1.5 mL), and the

mixture was heated at 100 °C for 30 min using microwave irradia-

tion (23 W average power). The reaction vessel was cooled, and

Et₃N (300 μL), MeOH (1 mL), and CH₂Cl₂(1 mL) were added.

The solution was loaded onto a Sephadex LH-20 chromatography

column, which was eluted with CH₂Cl₂/MeOH (1:1) to give 47 as

its triethylammonium salt (23 mg, quantitative). TLC (EtOAc/Py/

H₂O/AcOH 12:5:3:1): R_f= 0.14. ¹H NMR (500 MHz, CD₃OD/

CDCl₃, 7:1): δ = 7.94 (d, 2 H, Ar), 7.62 (t, 1 H, Ar), 7.54–7.05 (m,

17 H, Ar), 6.97 (d, 2 H, Ar), 6.82 (d, 2 H, Ar), 5.68 (d, J_4,5

2.7 Hz, 1 H, 5D-H), 5.43 [d, J = 11.8 Hz, 1 H, CH₂(Bn)], 5.34 (dd,

1 H, 4D-H), 5.26–5.22 [m, 2 H, 2B-H, CH₂(Bn)], 5.05 (d, J_1,2

=

4-Methoxyphenyl O-(α-

acetamido-2-deoxy-4,6-di-O-sulfo-β-

(β- -glucopyranosyluronic Acid)-(1Ǟ3)-2-acetamido-2-deoxy-4,6-di-

O-sulfo-β- -galactopyranoside (50): A solution of 49 (5.8 mg,

3.8 μmol, sodium salt) in H₂O/MeOH (4.5 mL/0.5 mL) was hydro-

genated in the presence of Pd(OH)₂(12 mg). After 24 h, the suspen-

sion was filtered through Celite, concentrated, and lyophilized to

give 50 as its sodium salt (5.1 mg, quantitative). ¹H NMR

(500 MHz, D₂O): δ = 7.09 (d, 2 H, Ar), 6.98 (d, 2 H, Ar), 5.02 (d,

J_1,2= 8.5 Hz, 1 H, 1A-H), 4.85 (d, J_3,4= 2.6 Hz, 1 H, 4A-H), 4.83

L

-Idopyranosyluronic Acid)-(1Ǟ3)-O-(2-

2.4 Hz, 1 H, 1D-H), 5.02–4.97 [m, 2 H, 2D-H, CH₂(Bn)], 4.90 (d,

J_1,2= 7.8 Hz, 1 H, 1B-H), 4.85–4.80 (m, 3 H, 1A-H, 4A-H, 4C-H),

4.76 (d, J_1,2= 8.4 Hz, 1 H, 1C-H), 4.70 [m, 2 H, CH₂(Bn)], 4.52–

4.43 [m, 3 H, CH₂(Bn), 2 6A-H or 6C-H], 4.40–4.35 (m, 2 H, 4B-

H, 6A-H or 6C-H), 4.33–4.25 (m, 2 H, 6A-H or 6C-H, 2C-H),

4.20–4.08 (m, 3 H, 2A-H, 3A-H, 5B-H), 4.01 (dd, J_2,3= 11.0, J_3,4

= 3.0 Hz, 1 H, 3C-H), 3.97–3.94 (m, 2 H, 5A-H, 5C-H), 3.85 (dd,

1 H, 3B-H), 3.78–3.74 [2 s, 6 H, Me(OMP), COOMe], 3.61 (dd, 1

H, 3D-H), 3.16 (q, 24 H, Et₃NH⁺), 1.28 (t, 36 H, Et₃NH⁺), 1.18,

1.14 [2 s, 18 H, C(CH₃)₃] ppm. ¹³C NMR (125 MHz, CD₃OD/

CDCl₃, 7:1, selected data from HSQC experiment): δ = 128.3–114.1

(Ar-CH), 101.6 (C-1B), 101.1 (C-1D), 100.7 (C-1C), 99.3 (C-1A),

79.9 (C-3B), 76.9 (C-3A), 75.8 (C-4B), 75.4–75.1 (C-4A, C-4C, C-

3C), 74.9 (C-3D), 74.6–74.5 [C-5B, CH₂(Bn)], 73.4 (C-5A, C-5C),

72.6 (C-2B), 71.4 [CH₂(Bn)], 69.2 (C-4D), 68.8 (C-2D), 68.0–67.5

[CH₂(Bn), C-6C, C-6A, C-5D], 54.6 [COOMe or Me(OMP)], 53.0

(C-2A), 51.8 (C-2C), 51.5 [COOMe or Me(OMP)], 26.3 [C(CH₃)₃]

ppm. MS (ESI): calcd. for C₇₄H₈₁F₆N₂O₃₉S₄Na [M + Na + H]^2–

943.2; found 943.3.

D-galactopyranosyl)-(1Ǟ4)-O-

D

(d, J_1,2= 4.5 Hz, 1 H, 1D-H), 4.76 (d, 1 H, 4C-H), 4.67 (d, J_1,2

=

8.1 Hz, 1 H, 1C-H), 4.63 (d, J_4,5= 3.6 Hz, 1 H, 5D-H), 4.53 (d,

J_1,2= 7.8 Hz, 1 H, 1B-H), 4.34–4.21 [m, 6 H, 2A-H (4.31), 2 6A-

H, 2 6C-H, 5A-H or 5C-H (4.23)], 4.17–4.15 (m, 2 H, 5A-H or 5C-

H, 3A-H), 4.09–4.02 (m, 2 H, 2C-H, 3C-H), 3.93 (dd, 1 H, 4D-H),

3.81 [s, 3 H, Me(OMP)], 3.78 (dd, 1 H, 4B-H), 3.69 (d, J_4,5

=

9.7 Hz, 1 H, 5B-H), 3.63–3.59 (m, 2 H, 3B-H, 3D-H), 3.49 (dd, J_2,3

= 7.5 Hz, 1 H, 2D-H), 3.44 (dd, 1 H, 2B-H), 2.05, 2.03 (2 s, 6 H,

NHAc) ppm. ¹³C NMR (125 MHz, D₂O, selected data from HSQC

experiment): δ = 119.6–116.2 (Ar), 104.7 (C-1B), 104.1 (C-1D),

102.6 (C-1C), 101.8 (C-1A), 83.4 (C-4B), 77.8 (C-5B), 77.0 (C-4C,

C-4A), 76.5 (C-3C), 76.0 (C-3A), 75.0 (C-3B or C-3D), 73.7, 73.5

(C-5A, C-5C), 73.4 (C-3B or C-3D), 73.1 (C-2B), 72.7 (C-4D), 72.3

(C-5D), 71.4 (C-2D), 68.9–68.8 (C-6A, C-6C), 56.9 [Me(OMP)],

52.7 (C-2A, C-2C), 23.5 (NAc) ppm. MS (ESI): calcd. for

C₃₅H₄₆N₂O₃₆S₄Na₃[M + 3Na + 2H]^–1267.0; found 1266.9.

4-Methoxyphenyl O-(3-O-Benzyl-α-

(1Ǟ3)-O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-β-

syl)-(1Ǟ4)-O-(3-O-benzyl-β- -glucopyranosyluronic Acid)-(1Ǟ3)-2-

acetamido-2-deoxy-4,6-di-O-sulfo-β- -galactopyranoside (49): H₂O₂

L

-idopyranosyluronic Acid)-

D

-galactopyrano-

D

(30%; 0.19 mL) and an aqueous solution of LiOH (0.7 m; 0.12 mL)

were added at 0 °C to a solution of 47 (11 mg, 4.8 μmol) in THF

(0.5 mL). The mixture was stirred for 24 h at room temperature,

then MeOH (1.0 mL), H₂O (0.3 mL), and NaOH (4 m aq.;

0.24 mL) were added. The mixture was stirred for 72 h at room

temperature, then it was neutralized with Amberlite IR-120 (H⁺)

resin, filtered, and concentrated to give 48 {MS (ESI): calcd. for

C₄₅H₅₄N₂O₃₄S₄Na₂: 670.0; found 669.9 [M + 2Na + 2H]^2–}.

Fluorescence Polarization Assay: Fluorescence polarization mea-

surements were performed in 384-well microplates (black poly-

styrene, nontreated, Corning) using a TRIAD multimode reader

(Dynex). The fluorescent probe (a fluorescent heparin-like hexasac-

charide) and inhibitors were dissolved in PBS buffer (phosphate-

buffered saline; 10 mm, pH 7.4). Recombinant human FGF-2

(Peprotech) was dissolved in PBS buffer (10 mm, pH 7.4) contain-

ing BSA (bovine serum albumin; 1%). For the inhibition assay,

probe solution (10 μL) and protein (20 μL) at fixed concentration

(40 nm and 145 nm, respectively) were mixed with inhibitor solution

(10 μL; 100 μm). The total sample volume in each well was 40 μL.

Therefore, all measurements were done in PBS + 0.5% BSA, and

the final concentrations of inhibitor, fluorescent probe and FGF-2

in each well were 25 μm, 10 nm, and 73 nm, respectively. After stir-

ring for 5 min in the dark, fluorescence polarization was recorded.

Triethylamine (9 μL, 64 μmol) and acetic anhydride (9 μL, 97 μmol)

were added to a cooled (0 °C) solution of 48 in dry MeOH

(1.5 mL). The mixture was stirred for 2 h at room temp., then Et₃N

(200 μL) was added, and the mixture was concentrated to dryness.

The residue was purified by Sephadex LH-20 chromatography

(MeOH) to give 49.

This compound was then dissolved in H₂O (2 mL), and Amberlite

IR-120 H⁺resin was added (pH = 3.3). The mixture was filtered,

treated with NaOH (0.04 m aq.; pH = 7.5) and lyophilized. The

Eur. J. Org. Chem. 2014, 3868–3884

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3883

G. Macchione, S. Maza, M. Mar Kayser, J. L. de Paz, P. M. Nieto

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Received: March 7, 2014

Published Online: May 19, 2014

3884

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Synthesis of chondroitin sulfate oligosaccharides using N-(tetrachlorophthaloyl)- and N-(trifluoroacetyl)galactosamine building blocks

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