Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate-protein interactions in microtiter plates

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PAPER

Synthesis of amine-functionalized heparin oligosaccharides for the

investigation of carbohydrate–protein interactions in microtiter plates†

Susana Maza, Giuseppe Macchione, Rafael Ojeda, Javier López-Prados, Jesús Angulo, José L. de Paz* and

Pedro M. Nieto*

Received 20th September 2011, Accepted 19th December 2011

DOI: 10.1039/c2ob06607f

The synthesis of well-deﬁned oligosaccharides is crucial for the establishment of structure–activity

relationships for speciﬁc sequences of heparin, contributing to the understanding of the biological role of

this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal

functional group that allows selective conjugation of the probes and expands their use as chemical tools in

glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides

using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to

produce efﬁciently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl

spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps,

avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were

immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the

basic ﬁbroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the

amount of sugar required for attachment to the solid support. Using this approach we quantiﬁed heparin-

protein interactions, and surface dissociation constants for the synthetic heparin derivatives were

determined.

homogeneous GAG oligosaccharides from natural sources, due

to the structural complexity of these molecules.

Introduction

Glycosaminoglycans (GAGs) are natural, linear polysaccharides

that in general present a high level of sulfation. This family of

molecules includes heparin, heparan sulfate, and chondroitin

sulfate, among others. GAGs are biosynthesized in the Golgi

apparatus of eukaryotic cells through sequential action of a series

of enzymes. This natural process results in GAG chains with a

high level of structural diversity. The heterogeneity of GAG

polysaccharides enables their binding to a wide range of

proteins.^1–5As a consequence of these interactions, GAGs

inﬂuence many biological processes such as inﬂammation,

angiogenesis and blood coagulation.⁶However, the GAG struc-

tural requirements for binding and mediating biological activity

have been only established for a few cases, the most studied

example being the interaction of antithrombin with heparin.⁷In

many other cases, the encoded information contained in speciﬁc

GAG sequences is still poorly understood. This fact can be

explained by the difﬁculties to obtain well-deﬁned,

In this context, chemical synthesis appears as an alternative,

powerful tool to access well-deﬁned GAG oligosaccharides^8,9

for the determination of structure–activity relationships and the

correlation of speciﬁc sequences and sulfation patterns with

protein binding and biological activity.^10,11Furthermore, chemi-

cal synthesis can provide unnatural GAG sequences and ana-

logues, as potential tools to modulate GAG-protein interactions.

It is highly desirable that synthesized structures contain an

orthogonal functional group, with unique reactivity, for further

selective conjugation of the probes.¹²The incorporation of such

a group, usually at the reducing end of the oligosaccharide,

opens the way for a wide variety of valuable experiments in gly-

cobiology. For example, orthogonally functionalized sugars can

be selectively immobilized on glass surfaces to create carbo-

hydrate microarrays for high-throughput screening of inter-

actions,^13,14or selectively conjugated to multivalent scaffolds to

generate heparin/GAG mimetics.^15–17The protecting group that

masks this orthogonal functionality should be compatible with

the complex protecting-group strategy required for the synthesis

of heparin oligosaccharides.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ),

Centro de Investigaciones Cientíﬁcas Isla de La Cartuja, CSIC and

Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

E-mail: jlpaz@iiq.csic.es; Fax: +34 954 460565

†Electronic supplementary information (ESI) available: NMR spectra of

compounds 3–24. See DOI: 10.1039/c2ob06607f

Since the pioneering synthesis of the antithrombin III binding

heparin pentasaccharide,¹⁸several synthetic approaches for

heparin have been described,^19–31including polymer-supported

strategies,^32–35preactivation-based one-pot synthesis,³⁶and

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reactivity-based one-pot synthesis.³⁷Despite all these signiﬁcant

advances in the ﬁeld, the synthesis of these molecules is still a

challenging task. Various difﬁculties are associated with these

syntheses due to the complex structure of heparin and the great

diversity of native sequences. A careful design of an appropriate

set of protecting groups is required for the introduction of sulfate

groups on selected hydroxyl and amino functions and for the

stereoselective construction of glycosidic bonds. These protect-

ing groups can have a profound effect on the efﬁciency of glyco-

sylation reactions that sometimes is not easily predictable. On

the other hand, deprotection/sulfation steps may require more

effort and time than the assembly of the protected intermediates.

These ﬁnal steps, particularly sulfation reactions, on valuable

and elaborated compounds, often lead to a signiﬁcant decrease

in isolated overall yield.

We report here the preparation of a series of heparin oligosac-

charides (one di-, one tetra- and two hexamers) that contain the

GlcNSO₃(6-OSO₃)-IdoA(2-OSO₃) repeating unit of the major

sequence of heparin. An aminoethyl spacer was placed at the

reducing end of the structures. A careful optimization of the reac-

tion conditions was necessary for the efﬁcient glycosylation of

building blocks containing an N-benzyloxycarbonyl-protected

amino spacer. The use of microwave irradiation facilitated O-

and N-sulfation steps, reducing reaction times and increasing iso-

lated yields.³⁸The reported experimental protocol adds valuable

knowledge to the existing data on heparin-like oligosaccharide

synthesis. Furthermore, the synthesized oligosaccharides were

immobilized in appropriate microtiter plates by using the anome-

ric amino group. The resulting heparin arrays were employed to

measure, qualitative- and quantitatively, sugar-protein inter-

actions. We demonstrate the utility of this system by studying the

carbohydrate afﬁnity of a well-characterized heparin-binding

protein, the basic ﬁbroblast growth factor FGF-2 that is impli-

cated in angiogenesis, cell growth and differentiation.

Scheme 1 Synthesis of disaccharide building blocks.

experiments on a model monosaccharide, we found that the use

of N-(benzyl)benzyloxycarbonyl amino spacer did not provide

the target compound (Scheme 2). Final hydrogenolysis over

Pd(OH)₂proceeded sluggishly and gave a complex mixture of

compounds. Interestingly, Boons and co-workers reported a

similar difﬁculty in the one-step hydrogenation of a heparin

hexasaccharide containing benzyl ethers and a N-(benzyl)

benzyloxycarbamate, although they could solve this problem by

using a two-step procedure involving hydrogenation over Pd/C

and then over Pd(OH)₂.²¹

On the other hand, hydrogenolysis of monosaccharide 6 gave

cleanly the fully deprotected compound 7. For this reason, we

decided to employ N-benzyloxycarbonyl aminoethanol to

protect the reducing end of our synthetic structures. Thus, 4 was

efﬁciently transformed into acceptor 9 (Scheme 1).

We then investigated the glycosylation between 4 and 9 under

standard conditions (Table 1, entry 1) using catalytic TMSOTf

as promoter at 0 °C, but no target tetrasaccharide was isolated.

The acceptor 9 and the hydrolyzed donor 3 were recovered from

the reaction mixture. We found that addition of further quantities

of promoter (up to 30 mol% with respect to the donor) was

necessary to obtain tetrasaccharide 10 in good yield (Table 1,

entry 4). Glycal 11 was detected as side product in the exper-

iments carried out with large amounts of TMSOTf (Table 1,

entries 2–4). Treatment of tetrasaccharide 10 with hydrazine

monohydrate in a pyridine/acetic acid solution followed by

Results and discussion

Synthesis of amine-functionalized heparin oligosaccharides

For the synthesis of these orthogonally functionalized oligosac-

charides, we followed a stereoselective n+2 modular approach

that utilized disaccharide 4 as a key building block (Scheme 1).

4 was prepared by glycosylation of trichloroacetimidate 1²⁶and

diol 2³⁹followed by pivaloylation at position 2, desilylation and

anomeric activation, according to the synthetic strategy devel-

oped by Martín-Lomas and co-workers.⁴⁰Interestingly, lactone 5

was systematically detected in the desilylation and trichloroaceti-

midation reactions.⁴¹This side product resulted from intramole-

cular transesteriﬁcation of the methyl ester group with the free

anomeric hydroxyl group and could be totally removed by silica

gel chromatography at a later stage. Then, we introduced a pro-

tected aminoethyl spacer at the anomeric position. The choice of

the amino protecting group is a crucial point of the synthetic

scheme. The yields of glycosylation reactions are signiﬁcantly

lower when using benzyloxycarbonyl amino linkers as reported

by Seeberger and co-workers²⁰and recently indicated by Boons

and co-workers.²¹An alternative N-(benzyl)benzyloxycarbonyl-

amino linker has been successfully considered to avoid the deac-

tivating effect of the –NH group. However, in preliminary

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an alternative approach was considered to install a permanent

benzyl group at position 4 of the non-reducing end of the oligo-

saccharides involving glycosidation with capped disaccharide 18

(Scheme 5). Coupling of monosaccharide 16^19,26and diol 2, fol-

lowed by pivaloylation at position 2, desilylation and trichloro-

acetimidate activation gave 18. Then, glycosylation of 9 with 18

afforded tetrasaccharide 19 in moderate yield (Scheme 5). A con-

siderable amount (45%) of unreacted starting material was recov-

ered from the reaction mixture. Surprisingly, analogous coupling

between 12 and 18 failed to provide the corresponding hexasac-

charide 20 under the reaction conditions previously optimized

for the preparation of similar oligosaccharides (Table 2, entry 1).

This reaction was also unsuccessful at room temperature, using

the inverse procedure⁴⁴and lowering the amount of TMSOTf

(Table 2, entries 2–4). In all cases, the acceptor was partly recov-

ered and only traces of the desired hexasaccharide were detected

in the crude mixtures by mass spectrometry.

Poor yields of glycosylations involving NH-benzyloxycarbo-

nyl containing sugars can be associated with those obtained with

N-acetylglucosamine derivatives.

Bonnaffé and co-workers reported the failure of a glycosyla-

tion between a 2-azidoglucose trichloroacetimidate disaccharide

donor and a range of disaccharide acceptors containing an N-

acetyl group.⁴⁵They demonstrated that the presence of the

NHAc group, even at a position remote from the reactive OH

acceptor, inhibits the coupling. The low reactivity of the 4-

hydroxy group of N-acetylglucosamine acceptors and the poor

yields associated with glycosylations at this position have long

been known. Several explanations have been proposed for this

general trend in preparative carbohydrate chemistry. It has been

demonstrated that the participation of the NHAc group in intra-

and intermolecular hydrogen bonds decrease the reactivity of the

OH group.⁴⁶Alternatively, poor yields of couplings of N-acetyl

containing carbohydrates also result from the formation of stable

glycosyl imidate side products.⁴⁷These intermediates, proposed

as the kinetic products of a glycosylation reaction, derive from

the nucleophilic attack of the oxygen of the NHAc group to the

oxocarbenium cation. Auzanneau and co-workers reported suc-

cessful glycosylations at OH-4 of N-acetylglucosamine using

an excess (2 equiv.) of a mild Lewis acid such as BF₃·Et₂O at

elevated temperatures.^48–50They hypothesized that 1 equiv. of

Scheme 2 Previous experiments with glucosamine monosaccharides.

coupling with 4 afforded hexasaccharide 13 that was sub-

sequently delevulinated (→14, Scheme 3).

With the oligosaccharides 9, 12 and 14 at hand, we initially

planned the benzylation reaction of OH-4 under neutral con-

ditions in order to introduce the required permanent protecting

group at this position of the non-reducing end. Treatment of 9

with BnBr and silver oxide in DMF gave the fully protected dis-

accharide 15 although in low 25% yield (Scheme 4).⁴²Starting

material and two side products, one resulting from the N-benzy-

lation of the carbamate linker and a second one derived from

H-5 iduronic acid elimination reaction⁴³with concomitant clea-

vage of the GlcN-IdoA glycosidic bond, were detected in the

reaction mixture by mass spectrometry and NMR. Attempts of

consuming 9 with longer reaction times resulted in an increase

of byproduct formation and lower yields of target 15. Therefore,

Table 1 Reaction conditions for the glycosylation between 4 and 9

Entry

Ratio 4 : 9 (equiv.)

TMSOTf (equiv.)

T (°C)

Recovered 9 (%)

Isolated yield (%)

1

2

3

4

1.3 : 1

1.5 : 1

0.05

0.26

0.45

0

25

0

93

43

50

23

—

27

25

74

0

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Scheme 3 Synthesis of hexasaccharide intermediate 14.

the yield (Table 2, entries 6–7). As long as sugar derivatives

withstand treatment with multiple equivalents of Lewis acid,

these conditions seem an attractive and practical alternative to

couple unreactive building blocks containing N-benzyloxycarbo-

nyl groups.

Scheme 4 Benzylation of disaccharide 9.

After preparing the fully protected oligosaccharides 15, 19

and 20, we carried out the deprotection and sulfation steps to

produce the ﬁnal amine-terminated probes. The use of micro-

wave irradiation greatly facilitated O-sulfations, avoiding exper-

imental problems associated with this chemical transformation

such as long reaction times and poor isolated yields.³⁸

Treatment of 15 with lithium hydroperoxide and then NaOH

hydrolysed acyl and methyl ester groups. Then, O-sulfation

using SO₃·Me₃N in DMF at 100 °C under microwave heating

gave the corresponding di-O-sulfated disaccharide in excellent

yield. Reduction of the azido group using Staudinger conditions

BF₃·Et₂O interacts noncovalently with the nucleophilic N-acetyl

group and the second one promotes coupling. We decided to

employ these conditions for the coupling between 12 and 18. We

envisioned that the excess of BF₃·Et₂O would reduce the nucleo-

philicity of the N-benzyloxycarbonyl group. Gratifyingly, coup-

ling between 12 and 18 with 1.5 equiv. of BF₃·Et₂O at room

temperature gave the target hexasaccharide 20 in acceptable

yield (Table 2, entry 5). A larger excess of donor or the appli-

cation of the inverse procedure⁴⁴did not signiﬁcantly improve

Scheme 5 Synthesis of tetrasaccharide derivative 19.

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Table 2 Reaction conditions for the glycosylation between 18 and 12

Entry

Ratio 18 : 12 (equiv.)

Promoter/equiv.

T (°C)

Recovered 12 (%)

Isolated yield (%)

1

2

3

4

5

6

7

1.5 : 1

1.6 : 1

2.5 : 1

1.6 : 1

TMSOTf/0.45

TMSOTf/0.15

TMSOTf/0.45

BF₃·Et₂O/1.5

BF₃·Et₂O/1.75

BF₃·Et₂O/1.5

0

25

0

25

71

99

67

27

39

60

—

a

—

a

52

61

19^a

^aInverse procedure

followed by N-sulfation using SO₃·Py, and hydrogenolysis to

remove benzyl and carbamate groups yielded the desired disac-

charide 21 in excellent 45% isolated yield from 15 (Scheme 6),

averaging 85% yield per step.

N-sulfated intermediate in excellent yield after simple gel ﬁl-

tration. To the best of our knowledge, this is the ﬁrst example of

microwave-assisted sulfation of an amine group. These results

highlight the potential of microwaves to overcome practical pro-

blems presented during sulfation steps. Final hydrogenation

afforded deprotected hexasaccharide 23. Similarly, the deprotec-

tion/sulfation sequence was carried out on fully protected 20 to

give target hexasaccharide 24 (Scheme 7).

Starting from tetrasaccharide 19, saponiﬁcation followed by

microwave-assisted O-sulfation, Staudinger reduction, N-sulfa-

tion and hydrogenolysis afforded tetrasaccharide 22 in 36% yield

over ﬁve steps (Scheme 6). We also performed the deprotection

and sulfation of hexasaccharide 14 to obtain a non-natural

heparin-like oligosaccharide that contains an additional O-sulfate

group at position 4 of the non-reducing terminus, not presented

in heparin. The removal of the methoxycarbonyl and acyl groups

was performed on 14 with lithium hydroperoxide and then

NaOH (Scheme 7). The resulting saponiﬁed hexasaccharide was

O-sulfated by treatment with SO₃·Me₃N complex under micro-

wave irradiation and submitted to Staudinger reduction. At this

point, conventional N-sulfation with SO₃·Py complex in a

mixture of Et₃N/pyridine at room temperature afforded a

complex mixture of partially sulfated hexasaccharides. Fortu-

nately, the same reaction proceeded smoothly with microwave

heating, using SO₃·Me₃N in DMF/Et₃N at 60 °C, to afford the

Binding studies in microtiter plates

The amine-functionalized heparin-like oligosaccharides 21–24

were immobilized in Nunc Immobilizer Amino™ microtiter

plates. Monosaccharide 7 was used as negative control. We

employed 384-well plates to minimize the volume of sugar sol-

utions required for immobilization. Oligosaccharides were dis-

solved in sodium bicarbonate buffer (50 mM, pH 9.6) and

incubated overnight at room temperature in the microtiter plate

wells. During optimization of this protocol it was found that the

addition of hexadecyltrimethylammonium bromide in the immo-

Scheme 6 Synthesis of disaccharide 21 and tetrasaccharide 22. Reaction conditions: a) LiOH, H₂O₂, THF; NaOH, MeOH; b) SO₃·Me₃N, DMF,

100 °C, MW; c) Me₃P, THF, NaOH; d) SO₃·Py, Py, Et₃N; e) H₂, Pd(OH)₂, H₂O/MeOH, 21, 45%; 22, 36%, ﬁve steps.

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Scheme 7 Synthesis of hexasaccharides 23 and 24. Reaction conditions: a) LiOH, H₂O₂, THF; NaOH, MeOH; b) SO₃·Me₃N, DMF, 100 °C, MW;

c) Me₃P, THF, NaOH; d) SO₃·Me₃N, DMF, Et₃N, 60 °C, MW; e) H₂, Pd(OH)₂, H₂O/MeOH, 23, 30%; 24, 13%, ﬁve steps.

bilization buffer, at 100-fold higher concentration than the carbo-

hydrate, greatly reduced the sugar concentration required for

efﬁcient attachment. The use of this ammonium salt has been

previously reported to improve the efﬁciency of the attachment

of heparin oligosaccharides to SPR gold chips.^15,51We hypoth-

esized that electrostatic repulsion between highly sulfated oligo-

saccharides that could hamper their coupling to a solid support is

reduced in the presence of positively charged micelles, facilitat-

ing the immobilization of these compounds in the wells. In this

way, as low as 10 μM concentration of sugar in immobilization

Fig. 1 shows a one-step qualitative analysis using only one

protein concentration. This experiment gives information to

differentiate compounds that interact strongly (22, 23, 24),

weakly (21) or not at all (7) with FGF-2. However, classiﬁcation

of relative binding strengths of hexasaccharides 23 and 24 and

tetrasaccharide 22 from this experiment can be inaccurate.

Ranking of relative binding afﬁnities requires the quantiﬁcation

of the interactions by using different protein concentrations.^55,56

Thus, oligosaccharides 22, 23 and 24 were incubated with 9 con-

centrations of FGF-2, ranging from 581 to 3 nM. For each sugar,

average ﬂuorescence intensities of ﬁve replicate wells were

plotted against protein concentration (Fig. 2). Assuming that the

system reached equilibrium during incubation, the curves were

analyzed as Langmuir isotherms to determine surface dis-

sociation constants (K_{D, surf}).⁵⁵The best ﬁt was obtained with a

one-binding site model. All the K_{D, surf}values were similar and

indicated oligosaccharide binding to FGF-2 in the nanomolar

range. The K_{D, surf}values for hexasaccharides 24 and 23 were

close, 32 4 and 44 7 nM respectively, suggesting that the

presence of a non natural sulfate group at position 4 of the non

reducing end of 23 did not signiﬁcantly affect binding. A K_{D, surf}

value of 68 11 nM was determined for 22, indicating a slight

reduction in afﬁnity compared to the hexasaccharides. These

values were consistent with previous measurements of heparin-

FGF2 binding afﬁnities.⁵⁷These results show that the apparent

binding strength of heparin–protein interactions can be quantitat-

ively analyzed by this method with small amounts of carbo-

hydrate and protein and using standard microplate equipment.

buffer containing

1 mM of hexadecyltrimethylammonium

bromide resulted in adequate attachment to study binding events

in the wells.

After immobilization and subsequent quenching with etha-

nolamine, the binding of the attached compounds to a model

heparin-binding protein, FGF-2, was studied. The binding assay

involved incubation with the protein, followed by incubation

with anti-FGF polyclonal and ﬂuorescently labelled secondary

antibodies. Bound protein was detected by using a standard ﬂu-

orescence microplate reader. No binding of the antibodies to

wells without FGF-2 was observed. Fluorescence signals at

hexa- and tetrasaccharide positions (Fig. 1) were observed.

FGF-2 bound to disaccharide 21 with the lowest afﬁnity while

no interaction was detected for disulfated monosaccharide 7.

These results are in good agreement with the minimal structural

requirements of GAGs to bind FGF-2.^20,52–54Interestingly, the

short length of the C-2 amino spacer did not negatively inﬂuence

sugar recognition.

Conclusions

In summary, we have reported several practical improvements

that facilitate the production of amine-functionalized heparin-

like oligosaccharides. We have demonstrated that the difﬁculties

encountered in glycosylations involving N-benzyloxycarbonyl

containing building blocks can be overcome by the use of an

excess of BF₃·Et₂O. These couplings are often associated with

poor yields due to the nucleophilicity of the NH group. We have

shown that the application of these reaction conditions can solve

this problem as long as donor and acceptor are stable under these

harsh conditions. On the other hand, microwave heating

increased the yield and efﬁciency of O- and N-sulfations. The

use of microwave irradiation avoided some problems related to

the complete sulfation of oligosaccharides containing multiple

Fig. 1 Binding of synthetic heparin-like oligosaccharides to FGF-2.

Sugar-coated wells were incubated with 291 nM protein. For each oligo-

saccharide, ﬂuorescence signals are the average of ﬁve replicate wells

and the error bars show the standard deviations for these measurements.

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(1 : 9), with cerium(^IV) sulfate (10 g), phosphomolybdic acid

(13 g), sulfuric acid (60 mL) solution in water (1 L) or with an-

isaldehyde solution (anisaldehyde (25 mL) with sulfuric acid

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

heating at over 200 °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

1

with a Perkin-Elmer 341 polarimeter. H- and ¹³C-NMR spectra

were acquired on Bruker DPX-300, and DRX-500 spec-

trometers. Unit A refers to the reducing end monosaccharide in

the NMR data. Electrospray mass spectra (ESI-MS) were carried

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

High resolution mass spectra (HR MS) were carried out by the

Mass Spectrometry Service, CITIUS, Universidad de Sevilla.

ESI-MS of ﬁnal compounds 21, 22, 23 and 24 were obtained

with a QSTAR mass spectrometer (Applied Biosystems) at SIdI,

Universidad Autónoma de Madrid. Microwave-based sulfation

reactions were performed using a Biotage Initiator Eight synthe-

sizer in sealed reaction vessels.

Binding assays in microtiter plates

Oligosaccharides 21–24 and monosaccharide 7 were dissolved in

sodium bicarbonate buffer (50 mM, pH 9.6) containing hexa-

decyltrimethylammonium bromide (1 mM) to afford 10 μM

sugar solutions. These solutions were transferred to the wells of

Nunc Immobilizer Amino™ 384 microtiter plates (from Thermo

Scientiﬁc) (20 μL per well). Blank wells were incubated with

100 mM ethanolamine in sodium phosphate buffer (50 mM, pH

9.6) containing hexadecyltrimethylammonium bromide (1 mM)

(80 μL per well). All samples were performed in replicates of

ﬁve. The plate was shaken overnight at room temperature and the

wells were emptied and washed with water. All wells were then

blocked by 1 h incubation with 100 mM ethanolamine in

sodium phosphate buffer and washed with water. 20 μL of a

recombinant human basic Fibroblast Growth Factor solution

(FGF-2, from Peprotech, 5 μg mL⁻¹in PBS containing 1%

BSA) were added to each well. The microplate was incubated

with gentle agitation at room temperature for 1 h and washed

with PBS containing 1% Tween 20 and 0.1% BSA, and water.

Rabbit anti-human FGF-2 antibody (from Peprotech, 5 μg mL⁻¹

in PBS containing 1% BSA, 20 μL per well) was added to the

wells. The plate was shaken for 1 h and washed with PBS con-

taining 1% Tween 20 and 0.1% BSA, and water. The primary

anti-FGF2 antibody was detected by using Alexa Fluor 488

labelled anti-rabbit IgG (from Invitrogen, 20 μg mL⁻¹in PBS

containing 1% BSA, 20 μL per well). The plate was incubated

with shaking in the dark for 1 h and then washed with PBS con-

taining 1% Tween 20 and 0.1% BSA, and water.

Fig. 2 Binding curves for oligosaccharides 22, 23 and 24. Average ﬂu-

orescence intensities of ﬁve replicates were plotted against FGF-2 con-

centrations. The K_{D, surf}values were obtained by ﬁtting the curves to a

Langmuir isotherm for a one-site model of interaction.

OH and NH₂groups. Moreover, we have developed an approach

to study interactions between these synthetic oligosaccharides

and proteins in microtiter plates. Highly negatively charged

sugars were efﬁciently attached to the solid surface with the help

of positively charged hexadecyltrimethylammonium bromide.

The binding of the immobilized oligosaccharides to FGF-2 was

analyzed qualitative- and quantitatively by using standard micro-

plate equipment, with minimal consumption of sugar (0.2 nmol

per well) and protein (pmol per well). The presented approach

provides useful information for the preparation of heparin oligo-

saccharides and the evaluation of their interactions with proteins

that are crucial aspects to improve our understanding of the bio-

logical role of heparin at the molecular level.

The ﬂuorescence was read at 535 nm using a TRIAD multi-

mode microplate reader (from Dynex). The blank well measure-

ments were subtracted from all values.

Experimental

General procedures

Determination of surface dissociation constants (K_{D, surf})

Thin layer chromatography (TLC) analyses were performed on

silica gel 60 F₂₅₄precoated on aluminium plates (Merck) and the

compounds were detected by staining with sulfuric acid/ethanol

Oligosaccharides 22–24 were attached to Nunc Immobilizer

Amino™ 384 microplates as described before. The wells were

incubated with 9 different concentrations of FGF-2, ranging

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from 581 to 3 nM. All concentrations and samples were per-

formed in ﬁve replicates. The bound protein was detected by

using the corresponding primary and secondary antibodies as

above. Data were ﬁtted to a Langmuir equation, assuming a one-

site model of the interaction.

CH₂(OBn)), 4.58 (d, 1H, J_4,5= 3.3 Hz, H-5A), 3.80 (m, 4H,

COOMe, OH), 3.40 (dd, 1H, J_2,3= 10.0 Hz, H-2B), 1.29 (s, 9H,

(CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃) (selected data for

minor anomer): δ 128.7–127.8 (Ar-CH), 98.6 (C-1B), 92.7

(C-1A), 75.0–74.7 (CH₂(OBn)), 73.6 (C-5A), 70.3 (C-2A), 52.8

(COOMe), 39.2 (C(Piv)), 27.4 ((CH₃)₃(Piv)), 20.9 (CH₃(Ac));

HR MS: m/z: calcd for C₃₉H₄₉N₃O₁₅Na: 822.3061; found:

822.3064 [M +Na]⁺.

Methyl 4-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-

levulinoyl-α-^D-glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-α,β-L-

idopyranosuronate (3)

O-(Methyl 4-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-

levulinoyl-α-^D-glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-α,β-L-

idopyranosyluronate) trichloroacetimidate (4)

TMSOTf (126 μL, 0.70 mmol) was added under an argon atmos-

phere at −30 °C to a mixture of 1 (10.1 g, 17.4 mmol) and 2

(15.2 g, 34.5 mmol) in dry CH₂Cl₂(625 mL). After stirring for

30 min at −30 °C, the reaction mixture was neutralized with

Et₃N and concentrated to dryness. The residue was separated by

ﬂash column chromatography (hexane/EtOAc 2 : 1) to obtain

unreacted acceptor (7.2 g, 47%) and fractions containing the

desired disaccharide; these were combined, concentrated, and

dissolved in Py (100 mL). Pivaloyl chloride (40 mL) and DMAP

(cat.) were added and the solution was stirred at room tempera-

ture. After 36 h, the mixture was diluted with CH₂Cl₂, washed

with 1 ^MHCl aqueous solution, saturated NaHCO₃aqueous sol-

ution and H₂O, dried (MgSO₄), ﬁltered, and concentrated in

vacuo. The residue was puriﬁed by ﬂash column chromato-

graphy (hexane–EtOAc 8 : 1 → 3 : 1) to yield the corresponding

2-O-pivaloylated disaccharide (7.6 g, 46%; 2 steps).

An excess of (HF)_n·Py (7.6 mL) was added at −15 °C to a sol-

ution of this disaccharide (1.52 g, 1.61 mmol) in dry THF

(40 mL). The reaction was warmed to room temperature and

stirred for 28 h under an argon atmosphere. The mixture was

diluted with CH₂Cl₂and washed with H₂O and saturated

NaHCO₃solution until neutral pH. The organic layers were

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

was puriﬁed by column chromatography (hexane–EtOAc 1 : 1)

to afford 3 (880 mg, 68%) as a mixture of α/β anomers. TLC

(hexane–EtOAc 1 : 1) R_f0.18; ¹H-NMR (300 MHz, CDCl₃)

(data for major anomer): δ 7.38–7.28 (m, 10H, Ar), 5.36 (dd,

1H, J_1,2= 4.4 Hz, J_1,OH= 8.6 Hz, H-1A), 5.06 (m, 1H, H-4B),

5.04 (d, 1H, J_1,2= 3.5 Hz, H-1B), 4.88 (m, 1H, H-2A), 4.80 (m,

K₂CO₃(83 mg, 0.61 mmol) and trichloroacetonitrile (0.82 mL,

8.25 mmol) were added at room temperature to a solution of 3

(440 mg, 0.55 mmol) in dry CH₂Cl₂(5 mL). After stirring for

6 h at room temperature, the reaction mixture was ﬁltered

through a pad of Celite and concentrated to dryness. The residue

was puriﬁed by column chromatography (hexane-EtOAc 2 : 1 +

1% Et₃N) to yield 4 (460 mg, 88%). TLC (hexane-EtOAc 1 : 1)

R_f0.44, 0.51 (α and β anomers);¹H-NMR (300 MHz, CDCl₃)

(data for major anomer): δ 8.72 (s, 1H, NH), 7.36–7.25 (m, 10H,

Ar), 6.42 (d, 1H, J_1,2= 2.1 Hz, H-1A), 5.20 (bt, 1H, H-2A),

5.10 (bt, 1H, J_3,4= J_4,5= 9.6 Hz, H-4B), 5.04 (d, 1H, J_1,2= 3.5

Hz, H-1B), 4.92 (d, 1H, J_4,5= 3.0 Hz, H-5A), 4.86–4.63 (m,

4H, CH₂(OBn)), 4.25–4.03 (m, 4H, H-4A, H-6aB, H-6bB,

H-3A), 4.01 (m, 1H, H-5B), 3.90 (t, 1H, J_2,3= J_3,4= 9.2 Hz,

H-3B), 3.80 (s, 3H, COOMe), 3.40 (dd, 1H, H-2B), 2.70 (m,

2H, CH₂(Lev)), 2.56–2.37 (m, 2H, CH₂(Lev)), 2.17 (s, 3H,

CH₃(Lev)), 2.04 (s, 3H, CH₃(Ac)), 1.28 (s, 9H, (CH₃)₃(Piv));

¹³C-NMR (75 MHz, CDCl₃) (data for major anomer): δ 206.2

(CO(Lev)), 177.6, 171.6, 170.8, 168.8, (CO(Lev, Ac, Piv,

COOMe)), 160.3 (CvNH), 137.5, 137.2 (Ar-C), 128.6–127.7

(Ar-CH), 98.6 (C-1B), 95.6 (C-1A), 90.8 (CCl₃), 77.5 (C-3B),

74.7, 73.3 (CH₂(OBn)), 74.2 (C-4A), 74.1 (C-3A), 70.0 (C-4B),

69.8 (C-5A), 69.2 (C-5B), 67.2 (C-2A), 63.0 (C-2B), 61.5

(C-6B), 52.5 (COOMe), 39.0 (C(Piv)), 37.9(CH₂(Lev)), 29.9

(CH₃(Lev)), 27.9 (CH₂(Lev)), 27.3 ((CH₃)₃(Piv)), 20.7

(CH₃(Ac)); ¹H-NMR (300 MHz, CDCl₃) (selected data for

minor anomer): δ 8.66 (s, 1H, NH), 7.36–7.25 (m, 10H, Ar),

6.35 (d, 1H, J_1,2= 2.7 Hz, H-1A), 5.24 (dd, 1H, J_2,3= 5.6 Hz,

H-2A), 5.00 (d, 1H, J_1,2= 3.7 Hz, H-1B), 4.86–4.63 (m, 5H,

H-5A, CH₂(OBn)), 4.33 (t, 1H, H-3A), 3.79 (s, 3H, COOMe),

3.44 (dd, 1H, H-2B), 2.70 (m, 2H, CH₂(Lev)), 2.56–2.37 (m,

2H, CH₂(Lev)), 2.15 (s, 3H, CH₃(Lev)), 2.05 (s, 3H, CH₃(Ac)),

1.25 (s, 9H, (CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃) (selected

data for minor anomer): δ 99.5 (C-1B), 94.8 (C-1A), 74.6

(C-3A), 68.1 (C-2A), 63.3 (C-2B), 52.3 (COOMe), 39.1

(C(Piv)), 37.9 (CH₂(Lev)), 29.9 (CH₃(Lev)), 27.9 (CH₂(Lev)),

27.3 ((CH₃)₃(Piv)), 20.7 (CH₃(Ac)); ESI MS: m/z: calcd for

C₄₁H₄₉Cl₃N₄O₁₅Na: 965.2; found: 965.7 [M + Na]⁺.

1H, H-5A), 4.80–4.62 (m, 4H, CH₂(OBn)), 4.19 (dd, 1H, J_5,6a

=

4.5 Hz, J_6a,6b= 12.5 Hz, H-6aB), 4.10 (m, 2H, H-3A, H-4A),

4.09 (dd, 1H, J_5,6b= 2.2 Hz, H-6bB), 4.00 (ddd, 1H, J_4,5= 10.3

Hz, H-5B), 3.86 (t, 1H, J_2,3= J_3,4= 10.2 Hz, H-3B), 3.80 (s,

3H, COOMe), 3.52 (d, 1H, OH), 3.37 (dd, 1H, H-2B), 2.70 (t,

2H, CH₂(Lev)), 2.56–2.37 (m, 2H, CH₂(Lev)), 2.16 (s, 3H,

CH₃(Lev)), 2.06 (s, 3H, CH₃(Ac)), 1.25 (s, 9H, (CH₃)₃(Piv));

¹³C-NMR (75 MHz, CDCl₃) (data for major anomer): δ 206.2

(CO(Lev)), 178.5, 171.6, 170.8, 169.6 (CO(Lev, Ac, Piv,

COOMe)), 137.5, 137.1 (Ar-C), 128.7–128.1 (Ar-CH), 99.0

(C-1B), 93.7 (C-1A), 77.4 (C-3B), 75.6 (C-4A or C-3A), 74.8,

74.7 (CH₂(OBn)), 74.1 (C-4A or C-3A), 71.3 (C-2A), 70.2

(C-4B), 69.8 (C-5A), 69.2 (C-5B), 63.0 (C-2B), 61.8 (C-6B),

52.5 (COOMe), 39.1 (C(Piv)), 37.9 (CH₂(Lev)), 29.9

(CH₃(Lev)), 28.0 (CH₂(Lev)), 27.3 ((CH₃)₃(Piv)), 20.9

(CH₃(Ac)); ¹H-NMR (300 MHz, CDCl₃) (selected data for

(4-O-(6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-

α-^D-glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-β-L-idopyranosyl)-

6,1-lactone (5)

minor anomer): δ 7.38–7.28 (m, 10H, Ar), 5.22 (dd, 1H, J_1,2

=

2.4 Hz, J_1,OH= 9.9 Hz, H-1A), 4.97 (d, 1H, J_1,2= 3.8 Hz,

H-1B), 4.94 (dd, 1H, J_2,3= 4.9 Hz, H-2A), 4.80–4.62 (m, 4H,

TLC (hexane-EtOAc 1 : 1) R_f0.57; ¹H-NMR (300 MHz,

CDCl₃): δ 7.37–7.24 (m, 10H, Ar), 5.86 (d, 1H, J_1,2= 2.1 Hz,

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H-1A), 5.14 (d, 1H, J_1,2= 3.9 Hz, H-1B), 5.09 (dd, 1H, J_3,4

=

solution in Py/AcOH 3 : 2) was added. After stirring at room

temperature for 2 h, the reaction mixture was quenched with

acetone (1.5 mL). The mixture was diluted with CH₂Cl₂and

washed with H₂O. The organic layer was dried (MgSO₄), ﬁltered

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (hexane–EtOAc 1 : 1) to yield 9 (204 mg,

91%). TLC (hexane–EtOAc 1 : 1) R_f0.5; [α]²_D⁰+1 (c 1.2,

9.3 Hz, J_4,5= 10.2 Hz, H-4B), 5.01 (d, 1H, CH₂(OBn)), 4.96

(dt, 1H, J_2,3= 6.0 Hz, J_2,4= 1.8 Hz, H-2A), 4.76 (m, 2H,

CH₂(OBn)), 4.73 (m, 2H, H-4A, H-5A), 4.66 (d, 1H,

CH₂(OBn)), 4.40 (ddd, 1H, J_5,6a= 5.1 Hz, J_5,6b= 2.1 Hz,

H-5B), 4.23 (dd, 1H, J_6a,6b= 12.3 Hz, H-6aB), 4.13 (dd, 1H,

H-6bB), 3.95 (m, 2H, H-3A, H-3B), 3.60 (dd, 1H, J_2,3= 10.2

Hz, H-2B), 2.71 (t, 2H, CH₂(Lev)), 2.50 (m, 2H, CH₂(Lev)),

2.17 (s, 3H, CH₃(Lev)), 2.05 (s, 3H, CH₃(Ac)), 1.24 (s, 9H,

(CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃): δ 206.2 (CO(Lev)),

177.8, 171.7, 170.5, 169.4 (CO(Ac, Lev, Piv, lactone)), 137.6,

137.5, 128.6, 128.2, 128.1, 127.8 (Ar), 100.6 (C-1B), 100.3

(C-1A), 79.4 (C-3A), 77.9 (C-3B), 75.9, 75.4 (CH₂(OBn)), 73.5

(C-2A), 70.9 (C-4A, C-5A), 70.5 (C-4B), 69.1 (C-5B), 63.5

(C-2B), 62.4 (C-6B), 39.0 (C(Piv)), 37.9 (CH₂(Lev)), 29.9

(CH₃(Lev)), 28.0 (CH₂(Lev)), 27.1 ((CH₃)₃(Piv)), 20.9

(CH₃(Ac)); ESI MS: m/z: calcd for C₃₈H₄₅N₃O₁₄Na: 790.3;

found: 790.2 [M + Na]⁺.

1

CH₂Cl₂); H-NMR (300 MHz, CDCl₃): δ 7.42–7.29 (m, 15 H,

Ar), 5.19 (bt, 1H, NH), 5.08 (m, 3H, H-1A, CH₂(Z)), 5.03 (d,

1H, J_1,2= 3.3 Hz, H-1B), 4.95 (t, 1H, J_1,2= J_2,3= 4.5 Hz,

H-2A), 4.85 (m, 2H, CH₂(OBn)), 4.75 (m, 2H, CH₂(OBn)), 4.72

(d, 1H, J_4,5= 4.2 Hz, H-5A), 4.55 (dd, 1H, J_5,6a= 3.6 Hz, J_6a,6b

= 12.6 Hz, H-6aB), 4.17 (dd, 1H, J_5,6b= 2.0 Hz, H-6bB), 4.12

(t, 1H, H-4A), 3.97 (t, 1H, J_3,4= 5.1 Hz, H-3A), 3.81 (m, 2H,

H-5B, CH₂-O), 3.77 (s, 3H, COOMe), 3.72 (dd, 1H, J_2,3= 10.2

Hz, J_3,4= 9.0 Hz, H-3B), 3.63 (m, 1H, CH₂-O), 3.47 (m, 1H,

H-4B), 3.41 (m, 2H, CH₂-N), 3.23 (dd, 1H, H-2B), 2.09 (s, 3H,

CH₃(Ac)), 1.21 (s, 9H, (CH₃)₃(Piv)); ¹³C-NMR (75 MHz,

CDCl₃): δ 177.7, 172.1, 169.8 (CO(Ac, Piv, COOMe)), 156.5

(CO(Z)), 137.9–136.6 (Ar-C), 128.8–127.7 (Ar-CH), 99.1

(C-1A), 98.6 (C-1B), 78.9 (C-3B), 75.6 (C-3A), 75.3

(CH₂(OBn)), 74.0 (C-4A), 73.3 (CH₂(OBn)), 71.2 (C-5B), 70.6

(C-4B), 70.1 (C-2A), 70.0 (C-5A), 68.4 (CH₂-O), 66.8

(CH₂(Z)), 62.8 (C-2B, C-6B), 52.5 (COOMe), 40.9 (CH₂-N),

38.9 (C(Piv)), 27.2 ((CH₃)₃(Piv)), 20.9 (CH₃(Ac)); HR MS: m/z:

calcd for C₄₄H₅₄N₄O₁₅Na: 901.3483; found: 901.3514

[M + Na]⁺.

Methyl (N-benzyloxycarbonyl-2-aminoethyl 4-O-(6-O-acetyl-2-

azido-3-O-benzyl-2-deoxy-4-O-levulinoyl-α-^D-glucopyranosyl)-3-

O-benzyl-2-O-pivaloyl-α-^L-idopyranoside) uronate (8)

TMSOTf (170 μL of a 0.092 M solution in dry CH₂Cl₂) was

added under an argon atmosphere at 0 °C to a mixture of 4

(280 mg, 0.30 mmol) and benzyl N-(2-hydroxyethyl)-carbamate

(116 mg, 0.593 mmol) in dry CH₂Cl₂(1.5 mL). After stirring

for 15 min at 0 °C, the reaction mixture was neutralized with

Et₃N and concentrated to dryness. The residue was puriﬁed by

column chromatography (toluene–EtOAc 3 : 2) to afford 8

(224 mg, 77%). TLC (toluene–EtOAc 3 : 2) R_f0.3; ¹H-NMR

(300 MHz, CDCl₃): δ 7.36–7.28 (m, 15 H, Ar), 5.17 (bt, 1H,

NH), 5.08 (m, 5H, H-1A, H-1B, H-4B, CH₂(Z)), 4.95 (t, 1H,

J_1,2= J_2,3= 4.5 Hz, H-2A), 4.79–4.70 (m, 4H, CH₂(OBn),

H-5A (4.73)), 4.65 (d, 1H, CH₂(OBn)), 4.19 (dd, 1H, J_5,6a= 4.2

Hz, J_6a,6b= 12.6 Hz, H-6aB), 4.14 (t, 1H, J_3,4= J_4,5= 4.8 Hz,

H-4A), 4.08 (dd, 1H, J_5,6b= 2.1 Hz, H-6bB), 4.00 (ddd, 1H, J_4,5

N-Benzyloxycarbonyl-2-aminoethyl (6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-4-O-levulinoyl-α-^D-glucopyranosyl)-(1 → 4)-

(methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 →

4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-

(1 → 4)-methyl 3-O-benzyl-2-O-pivaloyl-α-^L-

idopyranosiduronate (10)

TMSOTf (305 μL of a 0.18 M solution in dry CH₂Cl₂) was

added under an argon atmosphere at 0 °C to a mixture of 9

(107 mg, 0.122 mmol) and 4 (172 mg, 0.183 mmol) in dry

CH₂Cl₂(1.2 mL). After stirring for 20 min at 0 °C, the reaction

mixture was neutralized with Et₃N and concentrated to dryness.

The residue was puriﬁed by column chromatography (toluene–

EtOAc 4 : 1 → 2 : 1) to afford 10 (150 mg, 74%), unreacted 9

(25 mg, 23%) and 11 (42 mg, 0.054 mmol). TLC (hexane–

EtOAc 2 : 3) R_f0.57; [α]²_D⁰+8 (c 0.9, CH₂Cl₂); ¹H-NMR

(500 MHz, CDCl₃): δ 7.34–7.25 (m, 25H, Ar), 5.26 (d, 1H, J_1,2

= 5.0 Hz, H-1C), 5.16 (bt, 1H, NH), 5.11 (d, 1H, J_1,2= 4.5 Hz,

= 10.2 Hz, H-5B), 3.96 (t, 1H, H-3A), 3.87 (t, 1H, J_2,3= J_3,4

=

10.2 Hz, H-3B), 3.78 (m, 4H, COOMe, CH₂-O), 3.63 (m, 1H,

CH₂-O), 3.41 (m, 2H, CH₂-N), 3.35 (dd, 1H, J_1,2= 3.6 Hz,

H-2B), 2.70 (t, 2H, CH₂(Lev)), 2.46 (m, 2H, CH₂(Lev)), 2.16 (s,

3H, CH₃(Lev)), 2.04 (s, 3H, CH₃(Ac)), 1.22 (s, 9H,

(CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃): δ 206.2 (CO(Lev)),

177.6, 171.6, 170.7, 169.7 (CO(Lev, Ac, Piv, COOMe)), 156.5

(CO(Z)), 137.5–136.6 (Ar-C), 128.6–127.7 (Ar-CH), 99.2

(C-1A), 98.3 (C-1B), 77.4 (C-3B), 75.4 (C-3A), 74.8

(CH₂(OBn)), 74.1 (C-4A), 73.3 (CH₂(OBn)), 70.2 (CH₂(Z)),

70.0 (C-2A), 69.7 (C-5A), 69.1 (C-5B), 68.3 (CH₂-O), 66.9

(C-4B), 62.9 (C-2B), 61.8 (C-6B), 52.5 (COOMe), 40.9 (CH₂-

N), 38.9 (C(Piv)), 37.9 (CH₂(Lev)), 29.8 (CH₃(Lev)), 27.9

(CH₂(Lev)), 27.2 ((CH₃)₃(Piv)), 20.9 (CH₃(Ac)); HR MS: m/z:

calcd for C₄₉H₆₀N₄O₁₇Na: 999.3851; found: 999.3880 [M + Na]⁺.

H-1A), 5.10–5.06 (m, 3H, H-4D, CH₂(Z)), 5.05 (d, 1H, J_1,2

=

3.6 Hz, H-1D), 5.03 (d, 1H, J_1,2= 3.6 Hz, H-1B), 4.98 (t, 1H,

J_2,3= 5.0 Hz, H-2C), 4.94–4.92 (m, 2H, H-2A, CH₂(OBn)),

4.80–4.63 (m, 9H, CH₂(OBn), H-5A (4.67), H-5C (4.65)),

4.36–4.30 (m, 2H, H-6aB, H-6bB), 4.16 (dd, 1H, J_5,6a= 4.0 Hz,

J_6a,6b= 12.5 Hz, H-6aD), 4.11 (t, 1H, J_3,4= J_4,5= 5.0 Hz,

H-4A), 4.07 (t, 1H, J_3,4= J_4,5= 5.0 Hz, H-4C), 4.02 (dd, 1H,

J_5,6b= 2.0 Hz, H-6bD), 3.96–3.93 (m, 5H, H-3A, H-3C, H-4B,

H-5B, H-5D), 3.84–3.80 (m, 2H, H-3D, CH₂-O), 3.75–3.70 (m,

4H, H-3B, COOMe), 3.64–3.59 (m, 4H, COOMe, CH₂-O), 3.40

(m, 2H, CH₂-N), 3.35 (dd, 1H, J_2,3= 10.3 Hz, H-2D), 3.29 (dd,

1H, J_2,3= 10.3 Hz, H-2B), 2.74–2.66 (m, 2H, CH₂(Lev)),

Methyl (N-benzyloxycarbonyl-2-aminoethyl 4-O-(6-O-acetyl-2-

azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-3-O-benzyl-2-O-

pivaloyl-α-^L-idopyranoside) uronate (9)

Compound 8 (250 mg, 0.26 mmol) was dissolved in CH₂Cl₂

(1.7 mL) and hydrazine monohydrate (1.0 mL of a 0.5

M

2154 | Org. Biomol. Chem., 2012, 10, 2146–2163

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2.52–2.36 (m, 2H, CH₂(Lev)), 2.16 (s, 3H, CH₃(Lev)), 2.11,

1.99 (2 s, 6H, CH₃(Ac)), 1.22, 1.21 (2 s, 18H, (CH₃)₃(Piv));

¹³C-NMR (125 MHz, CDCl₃): δ 206.1 (CO(Lev)), 177.6, 177.3,

171.5, 170.7, 170.6, 169.8, 169.5 (CO(Lev, Ac, Piv, COOMe)),

156.4 (CO(Z)), 137.8–136.6 (Ar-C), 129.1–127.5 (Ar-CH), 99.1

(C-1A), 98.4 (C-1D), 98.1 (C-1B), 98.0 (C-1C), 78.1 (C-3B),

77.4 (C-3D), 76.0 (C-3C), 75.9 (C-3A), 75.0 (C-4B,

CH₂(OBn)), 74.7 (CH₂(OBn)), 74.0 (C-4C), 73.8 (C-4A), 73.7

(CH₂(OBn)), 73.5 (CH₂(OBn)), 70.5 (C-2C), 70.4 (C-2A,

C-5C), 70.3 (C-5A), 70.0 (CH₂(Z)), 69.8 (C-5B), 69.0 (C-5D),

68.4 (CH₂-O), 66.8 (C-4D), 63.0 (C-2B), 62.9 (C-2D), 62.1

(C-6B), 61.6 (C-6D), 52.4, 52.1 (COOMe), 40.9 (CH₂-N), 38.9

(C(Piv)), 37.8 (CH₂(Lev)), 29.8 (CH₃(Lev)), 27.9 (CH₂(Lev)),

27.2, 27.1 ((CH₃)₃(Piv)), 20.9, 20.8 (CH₃(Ac)); HR MS: m/z:

calcd for C₈₃H₁₀₁N₇O₂₉Na: 1682.6541; found: 1682.6498

[M + Na]⁺.

(d, 1H, J_1,2= 4.7 Hz, H-1A), 5.09 (m, 2H, CH₂(Z)), 5.04–5.00

(m, 2H, H-1D, H-1B), 4.99–4.96 (m, 1H, H-2C), 4.95–4.91 (m,

2H, H-2A, CH₂(OBn)), 4.89–4.82 (m, 2H, CH₂(OBn)),

4.81–4.66 (m, 6H, CH₂(OBn), H-5A (4.67)), 4.60 (d, 1H, J_4,5

=

4.9 Hz, H-5C), 4.51 (dd, 1H, J_5,6a= 3.2 Hz, J_6a,6b= 12.5 Hz,

H-6aD), 4.32 (m, 2H, H-6aB, H-6bB), 4.13–4.06 (m, 3H, H-4A,

H-4C, H-6bD), 3.99–3.93 (m, 4H, H-3A, H-3C, H-4B, H-5B),

3.85–3.76 (m, 2H, H-5D, CH₂-O), 3.76–3.71 (m, 4H, H-3D,

COOMe), 3.68 (dd, 1H, J_2,3= 10.2 Hz, J_3,4= 8.7 Hz, H-3B),

3.64–3.57 (m, 4H, COOMe, CH₂-O), 3.48–3.36 (m, 3H, H-4D,

CH₂-N), 3.29 (dd, 1H, J_1,2= 3.6 Hz, J_2,3= 10.2 Hz, H-2D),

3.23 (dd, 1H, J_1,2= 3.6 Hz, H-2B), 2.90 (m, 1H, OH), 2.11,

2.06 (2 s, 6H, CH₃(Ac)), 1.22, 1.19 (2 s, 18H, (CH₃)₃(Piv));

¹³C-NMR (75 MHz, CDCl₃): δ 177.6, 177.3, 172.0, 170.9,

169.9, 169.7 (CO(Ac, Piv, COOMe)), 156.5 (CO(Z)),

137.9–136.7 (Ar-C), 128.8–127.6 (Ar-CH), 99.2, 99.0, (C-1A,

C-1B or C-1D), 98.1, 98.0 (C-1C, C-1B or C-1D), 78.7 (C-3B),

77.9 (C-3D), 76.0 (C-3C, C-3A), 75.0 (C-4B, CH₂(OBn)), 74.0,

73.7 (C-4C, C-4A), 73.5 (CH₂(OBn)), 71.0 (C-5D), 70.8 (C-2C,

C-5C), 70.4 (C-2A, C-4D), 70.2 (C-5A), 69.7 (C-5B), 68.2

(CH₂-O), 66.7 (CH₂(Z)), 62.8 (C-2B, C-2D), 62.4 (C-6D), 62.0

(C-6B), 52.2, 51.8 (COOMe), 40.8 (CH₂-N), 39.0 (C(Piv)),

27.3, 27.2 ((CH₃)₃(Piv)), 20.9 (CH₃(Ac)); HR MS: m/z: calcd

for C₇₈H₉₅N₇O₂₇Na: 1584.6174; found: 1584.6115 [M + Na]⁺.

Methyl 4-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-

levulinoyl-α-^D-glucopyranosyl)-1,5-anhydro-3-O-benzyl-2-O-

pivaloyl-^L-xylo-hex-1-enitoluronate (11)

TLC (toluene–EtOAc 2 : 1) R_f0.33; [α]²_D⁰+6 (c 1.0, CHCl₃);

¹H-NMR (500 MHz, CDCl₃): δ 7.38–7.25 (m, 10H, Ar), 6.79 (s,

1H, H-1A), 5.07 (dd, 1H, J_3,4= J_4,5= 9.7 Hz, H-4B), 4.98 (d,

1H, J_1,2= 3.3 Hz, H-1B), 4.79 (d, 1H, CH₂(OBn)), 4.67 (bs, 2H,

CH₂(OBn)), 4.65 (d, 1H, CH₂(OBn)), 4.58 (bs, 1H, H-5A), 4.35

(m, 1H, H-4A), 4.19 (dd, 1H, J_5,6a= 4.0 Hz, J_6a,6b= 12.1 Hz,

H-6aB), 4.19 (bd, 1H, J_3,4= 1.9 Hz, H-3A), 4.09 (dd, 1H, J_5,6b

= 2.1 Hz, H-6bB), 3.96 (dd, 1H, J_2,3= 9.8 Hz, H-3B), 3.83 (m,

4H, H-5B, COOMe), 3.35 (dd, 1H, H-2B), 2.72–2.29 (m, 4H,

CH₂(Lev)), 2.15 (s, 3H, CH₃(Lev)), 2.06 (s, 3H, CH₃(Ac)), 1.24

(s, 9H, (CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃) (Signiﬁcant

data from HMQC experiment): δ 138.6 (C-1A), 97.6 (C-1B),

77.0 (C-3B), 74.5 (CH₂(OBn)), 73.2 (C-4A), 72.0 (C-5A), 71.9

(CH₂(OBn)), 69.6 (C-4B, C-3A), 69.0 (C-5B), 62.7 (C-2B),

61.4 (C-6B), 52.5 (COOMe), 38.2 (CH₂(Lev)), 29.5

(CH₃(Lev)), 28.4 (CH₂(Lev)), 26.9 ((CH₃)₃(Piv)), 20.7

(CH₃(Ac)); ESI MS: m/z: calcd for C₃₉H₄₇N₃O₁₄Na: 804.3;

N-Benzyloxycarbonyl-2-aminoethyl (6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-4-O-levulinoyl-α-^D-glucopyranosyl)-(1 → 4)-

(methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 →

4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-

(1 → 4)-(methyl 3-O-benzyl-2-O-pivaloyl-α-^L-

idopyranosyluronate)-(1 → 4)-(6-O-acetyl-2-azido-3-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-methyl 3-O-benzyl-2-O-

pivaloyl-α-^L-idopyranosiduronate (13)

TMSOTf (157 μL of a 0.18 M solution in dry CH₂Cl₂) was

added under an argon atmosphere at 0 °C to a mixture of 12

(135 mg, 0.086 mmol) and 4 (133 mg, 0.14 mmol) in dry

CH₂Cl₂(1.0 mL). After stirring for 20 min at 0 °C, the reaction

mixture was neutralized with Et₃N and concentrated to dryness.

The residue was puriﬁed by column chromatography (hexane–

EtOAc 2 : 1 → 3 : 2) to afford 13 (123 mg, 61%), unreacted 12

(38 mg, 28%) and 11 (30 mg, 0.039 mmol). TLC (hexane–

EtOAc 1 : 1) R_f0.22; [α]²_D⁰+5 (c 1.1, CH₂Cl₂); ¹H-NMR

(500 MHz, CDCl₃): δ 7.37–7.26 (m, 35 H, Ar), 5.37 (d, 1H, J_1,2

= 6.1 Hz, H-1E), 5.28 (d, 1H, J_1,2= 5.1 Hz, H-1C), 5.16 (bt,

1H, NH), 5.12 (d, 1H, J_1,2= 4.7 Hz, H-1A), 5.10-5.08 (m, 3H,

H-4F, CH₂(Z)), 5.06–5.03 (m, 3H, H-1B, H-1D, H-1F), 5.00 (t,

1H, J_2,3= 6.4 Hz, H-2E), 4.99 (t, 1H, J_2,3= 5.5 Hz, H-2C),

4.95–4.90 (m, 3H, H-2A, CH₂(OBn)), 4.85–4.64 (m, 11H,

CH₂(OBn), H-5A (4.65)), 4.62 (d, 1H, J_4,5= 4.5 Hz, H-5C),

4.50 (d, 1H, J_4,5= 5.7 Hz, H-5E), 4.35–4.27 (m, 4H, H-6aB,

found: 804.1 [M

+

Na]⁺; HR MS: m/z: calcd for

C₃₉H₄₇N₃O₁₄Na: 804.2956; found: 804.2982 [M + Na]⁺.

N-Benzyloxycarbonyl-2-aminoethyl (6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-(methyl 3-O-

benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 → 4)-(6-O-

acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-

methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosiduronate (12)

Compound 10 (110 mg, 0.066 mmol) was dissolved in CH₂Cl₂

(1.0 mL) and hydrazine monohydrate (0.26 mL of a 0.5 M sol-

ution in Py/AcOH 3 : 2) was added. After stirring at room temp-

erature for 2 h, the reaction mixture was quenched with acetone

(0.5 mL). The mixture was diluted with CH₂Cl₂and washed

with H₂O. The organic layer was dried (MgSO₄), ﬁltered and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (toluene–EtOAc 5 : 2) to yield 12 (85 mg,

82%). TLC (hexane–EtOAc 2 : 3) R_f0.63; [α]²_D⁰−2 (c 1.0,

CHCl₃); ¹H-NMR (300 MHz, CDCl₃): δ 7.41–7.28 (m, 25H,

Ar), 5.29 (d, 1H, J_1,2= 4.6 Hz, H-1C), 5.14 (bt, 1H, NH), 5.11

H-6bB, H-6aD, H-6bD), 4.16 (dd, 1H, J_5,6a= 4.2 Hz, J_6a,6b

=

12.6 Hz, H-6aF), 4.11 (dd,1H, J_3,4= J_4,5= 5.4 Hz, H-4A), 4.07

(dd,1H, J_3,4= J_4,5= 5.4 Hz, H-4C), 4.03 (m, 1H, H-4E), 4.01

(dd, 1H, J_5,6b= 2.1 Hz, H-6bF), 3.96–3.88 (m, 8H, H-3A,

H-3C, H-3E, H-4B, H-4D, H-5B, H-5D, H-5F), 3.84–3.78 (m,

2H, H-3F, CH₂-O), 3.75 (s, 3H, COOMe), 3.71 (dd, 1H, J_2,3

=

10.4 Hz, J_3,4= 8.4 Hz, H-3B or H-3D), 3.63 (dd, 1H, J_2,3= 10.3

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Hz, J_3,4= 8.7 Hz, H-3B or H-3D), 3.61 (m, 4H, COOMe, CH₂-

O), 3.60 (s, 3H, COOMe), 3.44–3.34 (m, 2H, CH₂-N), 3.36 (dd,

1H, J_2,3= 10.2 Hz, H-2F), 3.32–3.28 (2dd, 2H, H-2B and

H-2D), 2.74–2.65 (m, 2H, CH₂(Lev)), 2.53–2.35 (m, 2H,

CH₂(Lev)), 2.16 (s, 3H, CH₃(Lev)), 2.11, 2.08, 1.99 (3 s, 9H,

CH₃(Ac)), 1.21, 1.19 (3 s, 27H, (CH₃)₃(Piv)); ¹³C-NMR

(125 MHz, CDCl₃): δ 206.2 (CO(Lev)), 177.6, 177.3, 177.2,

170.5–169.6 (CO(Lev, Ac, Piv, COOMe)), 156.6 (CO(Z)),

137.9–136.7 (Ar-C), 128.6–127.3 (Ar-CH), 99.2 (C-1A), 98.4,

98.3, 98.1 (C-1B, C-1D, C-1F), 97.9 (C-1C), 97.7 (C-1E), 78.1,

77.9 (C-3B, C-3D), 77.5 (C-3F), 77.0–75.8 (C-3A, C-3C,

C-3E), 75.2 (CH₂(OBn)), 74.9 (C-4B, C-4D), 74.8 (CH₂(OBn)),

74.3, 73.8 (CH₂(OBn)), 74.2 (C-4C), 74.0 (C-4E), 73.6 (C-4A),

71.5 (C-2E, C-5E), 71.1 (C-2C), 70.8 (C-5C), 70.6 (C-2A), 70.5

(C-5A), 69.8 (C-5B, C-5D, CH₂(Z)), 69.0 (C-5F), 68.5 (CH₂-O),

66.9 (C-4F), 63.0 (C-2B, C-2D), 62.9 (C-2F), 62.0 (C-6B,

C-6D), 61.7 (C-6F), 52.5, 52.1 (COOMe), 40.9 (CH₂-N), 39.0

(C(Piv)), 37.9 (CH₂(Lev)), 29.9 (CH₃(Lev)), 27.9 (CH₂(Lev))

27.3, 27.2 ((CH₃)₃(Piv)), 20.9, 20.8 (CH₃(Ac)); ESI MS: m/z:

calcd for C₁₁₇H₁₄₂N₁₀O₄₁Na: 2365.9; found: 2366.4 [M + Na]⁺.

C-3E), 75.3 (CH₂(OBn), C-4B or C-4D), 75.0 (C-4B or C-4D),

74.9 (CH₂(OBn)), 74.4, 73.9 (C-4A or C-4C or C-4E), 73.8

(CH₂(OBn)), 73.6 (C-4A or C-4C or C-4E, CH₂(OBn)), 71.6

(C-5E, C-2C or C-2E), 71.3 (C-2C or C-2E), 71.2 (C-5C, C-5F),

70.5 (C-2A, C-5A, C-4F), 69.8 (C-5B, C-5D), 68.5 (CH₂-O),

66.8 (CH₂(Z)), 62.9 (C-2B, C-2D, C-2F), 62.6 (C-6F), 62.0

(C-6B, C-6D), 52.5, 52.1 (COOMe), 40.9 (CH₂-N), 39.0

(C(Piv)), 27.3, 27.2 ((CH₃)₃(Piv)), 20.9 (CH₃(Ac)); ESI MS: m/z:

calcd for C₁₁₂H₁₃₆N₁₀O₃₉Na: 2267.9; found: 2268.3 [M + Na]⁺.

Methyl (N-benzyloxycarbonyl-2-aminoethyl 4-O-(6-O-acetyl-2-

azido-3,4-di-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-3-O-benzyl-

2-O-pivaloyl-α-^L-idopyranoside) uronate (15)

Benzyl bromide (40 μL, 0.34 mmol) was added under an argon

atmosphere at 0 °C to a solution of 9 (30 mg, 0.034 mmol) and

freshly prepared Ag₂O (18 mg, 0.079 mmol) in dry DMF

(0.5 mL). After stirring for 8 h at room temperature, the solution

was ﬁltered through Celite, diluted with EtOAc and washed with

H₂O. The organic layer was dried (MgSO₄), ﬁltered and concen-

trated in vacuo. The residue was puriﬁed by column chromato-

graphy (hexane–EtOAc 1 : 0 → 2 : 1) to afford 15 (8 mg, 25%)

and unreacted 9 (8 mg, 27%). TLC (hexane–EtOAc 3 : 2) R_f0.4;

[α]²_D⁰+8 (c 1.0, CHCl₃); ¹H-NMR (300 MHz, CDCl₃): δ

7.39–7.25 (m, 20H, Ar), 5.18 (bt, 1H, NH), 5.09 (m, 3H, H-1A,

N-Benzyloxycarbonyl-2-aminoethyl (6-O-acetyl-2-azido-3-O-

benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-(methyl 3-O-

benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 → 4)-(6-O-

acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-

(methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 →

4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-

(1 → 4)-methyl 3-O-benzyl-2-O-pivaloyl-α-^L-

CH₂(Z)), 5.05 (d, 1H, J_1,2= 3.6 Hz, H-1B), 4.95 (t, 1H, J_1,2

=

J_2,3= 4.8 Hz, H-2A), 4.85 (d, 1H, CH₂(OBn)), 4.83 (bs, 2H,

CH₂(OBn)), 4.75 (m, 2H, CH₂(OBn)), 4.72 (d, 1H, J_4,5= 4.4

Hz, H-5A), 4.59 (d, 1H, CH₂(OBn)), 4.32 (dd, 1H, J_5,6a= 1.9

Hz, J_6a,6b= 12.1 Hz, H-6aB), 4.18 (dd, 1H, J_5,6b= 4.2 Hz,

H-6bB), 4.14 (t,1H, J_3,4= 4.5 Hz, H-4A), 3.98 (t, 1H, H-3A),

3.94 (m, 1H, H-5B), 3.87 (dd, 1H, J_2,3= 10.3 Hz, J_3,4= 8.9 Hz,

H-3B), 3.81 (m, 1H, CH₂-O), 3.78 (s, 3H, COOMe), 3.63 (m,

1H, CH₂-O), 3.52 (dd, 1H, J_4,5= 9.9 Hz, H-4B), 3.40 (m, 2H,

CH₂-N), 3.28 (dd, 1H, H-2B), 2.01 (s, 3H, CH₃(Ac)), 1.20 (s,

9H, (CH₃)₃(Piv)); ¹³C-NMR (125 MHz, CDCl₃): δ 177.6, 170.6,

169.7 (CO(Ac, Piv, COOMe)), 156.5 (CO(Z)), 137.8–136.6 (Ar-

C), 128.7–127.6 (Ar-CH), 99.2 (C-1A), 98.3 (C-1B), 80.0

(C-3B), 77.8 (C-4B), 75.8 (C-3A), 75.5, 74.9 (CH₂(OBn)), 73.8

(C-4A), 73.4 (CH₂(OBn)), 70.4, 70.1 (C-2A, C-5A, C-5B), 68.5

(CH₂-O), 66.8 (CH₂(Z)), 63.4 (C-2B), 62.6 (C-6B), 52.5

(COOMe), 40.9 (CH₂-N), 38.9 (C(Piv)), 27.2 ((CH₃)₃(Piv)),

20.9 (CH₃(Ac)); HR MS: m/z: calcd for C₅₁H₆₀N₄O₁₅Na:

991.3953; found: 991.3984 [M + Na]⁺.

idopyranosiduronate (14)

Compound 13 (119 mg, 0.051 mmol) was dissolved in CH₂Cl₂

(1.0 mL) and hydrazine monohydrate (0.197 mL of a 0.5 M sol-

ution in Py/AcOH 3 : 2) was added. After stirring at room temp-

erature for 2 h, the reaction mixture was quenched with acetone

(0.5 mL). The mixture was diluted with CH₂Cl₂and washed

with H₂O. The organic layer was dried (MgSO₄), ﬁltered and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (hexane–EtOAc 3 : 2) to yield 14 (100 mg,

1

87%). TLC (hexane–EtOAc 1 : 1) R_f0.33; H-NMR (500 MHz,

CDCl₃): δ 7.40–7.25 (m, 35 H, Ar), 5.38 (d, 1H, J_1,2= 6.1 Hz,

H-1E), 5.31 (d, 1H, J_1,2= 5.5 Hz, H-1C), 5.16 (bt, 1H, NH),

5.12 (d, 1H, J_1,2= 4.8 Hz, H-1A), 5.12 (m, 2H, CH₂(Z)),

5.04–5.02 (m, 3H, H-1B, H-1D, H-1F), 5.02–4.98 (m, 2H,

H-2E, H-2C), 4.96–4.92 (m, 3H, H-2A,CH₂(OBn)), 4.87–4.68

(m, 10H, CH₂(OBn)), 4.65 (d, 1H, J_4,5= 4.6 Hz, H-5A), 4.58

(d, 1H, J_4,5= 5.1 Hz, H-5C), 4.53–4.48 (m, 2H, H-6aF, H-5E),

4.36–4.26 (m, 4H, H-6aB, H-6bB, H-6aD, H-6bD), 4.13–4.02

(m, 4H, H-6bF, H-4A, H-4C, H-4E), 3.99–3.87 (m, 8H, H-3A,

H-3C, H-3E, H-4B, H-4D, H-5B, H-5D, H-5F), 3.84–3.56 (m,

14H, H-3F, H-3B, H-3D, CH₂-O, COOMe (3.79, 3.62)),

3.47–3.33 (m, 3H, H-4F, CH₂-N), 3.33–3.22 (3dd, 3H, H-2F,

H-2B, H-2D), 2.93 (bd, 1H, OH), 2.11, 2.07, 2.06 (3 s, 9H,

CH₃(Ac)), 1.20, 1.19 (3 s, 27H, (CH₃)₃(Piv)); ¹³C-NMR

(125 MHz, CDCl₃): δ 177.6, 177.3, 177.2, 172.0, 170.9, 170.8,

170.0, 169.9, 169.7 (CO(Ac, Piv, COOMe)), 156.5 (CO(Z)),

137.9–136.6 (Ar-C), 128.8–127.2 (Ar-CH), 99.1 (C-1A), 99.0,

98.3, 98.0 (C-1B, C-1D, C-1F), 97.8 (C-1C), 97.7 (C-1E), 79.0

(C-3F), 78.1, 77.9 (C-3B, C-3D), 76.9–76.4 (C-3A, C-3C,

Methyl 4-O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-

glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-α,β-^L-

idopyranosuronate (17)

TMSOTf (22 μL of a 0.18 M solution in dry CH₂Cl₂) was added

to a cooled (0 °C) solution of 2 (1.21 g, 2.75 mmol) in dry

CH₂Cl₂(30 mL) under an argon atmosphere. While the reaction

was stirred, a solution of 16 (1.31 g, 2.29 mmol) in dry CH₂Cl₂

(10 mL) was added dropwise. After 20 min at 0 °C, the mixture

was neutralized with Et₃N and concentrated in vacuo. The

residue was separated by ﬂash column chromatography (toluene/

EtOAc 12 : 1) to obtain the desired α (1 → 4) disaccharide. This

compound was dissolved in Py (15 mL). Pivaloyl chloride

2156 | Org. Biomol. Chem., 2012, 10, 2146–2163

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(5 mL) and DMAP (cat.) were added and the solution was stirred

at room temperature. After 24 h, the mixture was diluted with

CH₂Cl₂, washed with 1 M HCl aqueous solution, saturated

NaHCO₃aqueous solution and H₂O, dried (MgSO₄), ﬁltered,

and concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography (toluene/EtOAc 49 : 1) to yield the

desired 2-O-pivaloylated disaccharide (1.1 g, 47%, 2 steps).

TLC (toluene–EtOAc 12 : 1) R_f0.43; ¹H-NMR (500 MHz,

CDCl₃): δ 7.39-7.25 (m, 15H, Ar), 5.04 (bs, 2H, H-1A, H-2A),

4.94 (d, 1H, J_1,2= 3.5 Hz, H-1B), 4.85–4.58 (m, 6H,

CH₂(OBn)), 4.42 (bd, 1H, H-5A), 4.35 (dd, 1H, J_5,6a= 1.9 Hz,

J_6a,6b= 12.3 Hz, H-6aB), 4.20 (dd, 1H, J_5,6b= 2.8 Hz, H-6bB),

4.09–4.04 (m, 2H, H-5B, H-4A), 4.01–3.97 (m, 2H, H-3A,

H-3B), 3.74 (s, 3H, COOMe), 3.57 (t, 1H, J_4,5= 9.5 Hz, H-4B),

3.28 (dd, 1H, J_2,3= 10.3 Hz, H-2B), 2.00 (s, 3H, CH₃(Ac)),

1.63 (hp, 1H, CH(CH₃)₂), 1.27 (s, 9H, C(CH₃)₃), 0.88–0.85

(12H, CH(CH₃)₂, C(CH₃)₂), 0.20, 0.15 (2 s, 6H, Si(CH₃)₂);

¹³C-NMR (125 MHz, CDCl₃) (Signiﬁcant data from HMQC

experiment): δ 98.8 (C-1B), 94.5 (C-1A), 80.2 (C-3B), 77.5

(C-4B), 75.6 (C-3A), 75.4 (CH₂(OBn)), 74.7 (CH₂(OBn)), 74.4

(C-4A), 73.4 (C-5A), 73.0 (CH₂(OBn)), 69.8 (C-5B), 67.7

(C-2A), 63.5 (C-2B), 62.3 (C-6B), 52.0 (COOMe); ESI MS: m/z:

calcd for C₄₉H₆₇N₃O₁₃SiNa: 956.4; found: 956.0 [M + Na]⁺.

To a solution of this 2-O-pivaloylated disaccharide (0.8 g,

0.86 mmol) at –15 °C in dry THF (23 mL), an excess of

(HF)_n·Py (2.4 mL) was added. The reaction was warmed to 0 °C

and stirred for 28 h under an argon atmosphere. Then, the reac-

tion was stirred at room temperature until complete disappear-

ance of starting material. The mixture was diluted with CH₂Cl₂

and washed with H₂O and saturated NaHCO₃solution until

neutral pH. The organic layer was dried (MgSO₄), ﬁltered, and

concentrated in vacuo to give 17 (615 mg, 91%). TLC (hexane–

EtOAc 2 : 1) R_f0.29; ¹H-NMR (300 MHz, CDCl₃) (data for

(63 mg, 80 μmol) in dry CH₂Cl₂(1 mL). After stirring for 6 h at

room temperature, the reaction mixture was ﬁltered through a

pad of Celite and concentrated to dryness. The residue was

puriﬁed by column chromatography (hexane–EtOAc 3 : 1 + 1%

Et₃N) to yield 18 (70 mg, 94%). TLC (hexane–EtOAc 2 : 1) R_f

1

0.49, 0.59 (α and β anomers); H-NMR (300 MHz, CDCl₃) (for

major anomer): δ 8.71 (s, 1H, NH), 7.36–7.25 (m, 15H, Ar),

6.44 (d, 1H, J_1,2= 2.7 Hz, H-1A), 5.20 (t, 1H, J_2,3= 3.3 Hz,

H-2A), 5.03 (d, 1H, J_1,2= 3.5 Hz, H-1B), 4.91 (d, 1H, J_4,5= 3.2

Hz, H-5A), 4.87–4.58 (m, 6H, CH₂(OBn)), 4.35 (dd, 1H, J_5,6a

=

2.0 Hz, J_6a,6b= 12.1 Hz, H-6aB), 4.23 (t, 1H, J_3,4= 3.8 Hz,

H-4A), 4.18 (dd, 1H, J_5,6b= 3.7 Hz, H-6bB), 4.07 (t, 1H,

H-3A), 3.95 (ddd, 1H, H-5B), 3.90 (dd, 1H, J_2,3= 10.3 Hz, J_3,4

= 8.8 Hz, H-3B), 3.80 (s, 3H, COOMe), 3.54 (dd, 1H, J_4,5= 9.9

Hz, H-4B), 3.33 (dd, 1H, H-2B), 2.01 (s, 3H, CH₃(Ac)), 1.26 (s,

9H, (CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃) (selected data for

major anomer from HSQC experiment): δ 98.4 (C-1B), 95.5

(C-1A), 80.1 (C-3B), 77.5 (C-4B), 75.3, 74.8 (CH₂(OBn)), 74.2

(C-3A), 73.7 (C-4A), 72.8 (CH₂(OBn)), 70.1 (C-5A, C-5B),

67.6 (C-2A), 63.3 (C-2B), 62.4 (C-6B), 52.4 (COOMe), 27.1

((CH₃)₃(Piv)), 20.7 (CH₃(Ac)); ¹H-NMR (300 MHz, CDCl₃)

(selected data for minor anomer): δ 8.64 (s, 1H, NH), 7.36–7.25

(m, 15H, Ar), 6.38 (d, 1H, J_1,2= 2.7 Hz, H-1A), 5.21 (m, 1H,

H-2A), 5.00 (d, 1H, J_1,2= 3.9 Hz, H-1B), 4.87–4.58 (m, 7H,

CH₂(OBn), H-5A (4.66)), 4.40–4.29 (m, 2H, H-3A, H-6aB),

4.25–4.04 (m, 2H, H-6bB, H-4A), 3.99–3.82 (m, 2H, H-3B,

H-5B), 3.78 (s, 3H, COOMe), 3.52 (m, 1H, H-4B), 3.38 (dd,

1H, J_2,3= 10.3 Hz, H-2B), 2.01 (s, 3H, CH₃(Ac)), 1.26 (s, 9H,

(CH₃)₃(Piv)); ¹³C-NMR (75 MHz, CDCl₃) (selected data for

minor anomer from HSQC experiment): δ 99.3 (C-1B), 94.8

(C-1A), 80.1 (C-3B), 77.5 (C-4B), 76.1 (C-4A), 74.6 (C-3A),

70.1 (C-5B), 68.5 (C-2A), 63.6 (C-2B), 52.4 (COOMe), 27.1

((CH₃)₃(Piv)), 20.7 (CH₃(Ac)); ESI MS: m/z: calcd for

C₄₃H₄₉Cl₃N₄O₁₃Na: 957.2; found: 956.7 [M + Na]⁺.

major anomer): δ 7.37–7.25 (m, 15H, Ar), 5.38 (bd, 1H, J_1,2

=

4.8 Hz, H-1A), 5.06 (d, 1H, J_1,2= 3.6 Hz, H-1B), 4.89–4.78 (m,

7H, H-2A, H-5A, CH₂(OBn)),4.6 (m, 1H, CH₂(OBn)), 4.33 (dd,

1H, J_5,6a= 2.1 Hz, J_6a,6b= 12.0 Hz, H-6aB), 4.21–4.10 (m, 3H,

H-6bB, H-3A, H-4A), 3.95 (ddd, 1H, J_4,5= 10.0 Hz, J_5,6b= 4.1

Hz,H-5B), 3.85 (dd, 1H, J_2,3= 10.2 Hz, J_3,4= 8.8 Hz, H-3B),

3.80 (s, 3H, COOMe), 3.51 (dd, 1H, H-4B), 3.30 (dd, 1H,

H-2B), 2.02 (s, 3H, CH₃(Ac)), 1.24 (s, 9H, (CH₃)₃(Piv));

¹³C-NMR (75 MHz, CDCl₃) (selected data for major anomer

from HMQC experiment): δ 99.0 (C-1B), 93.6 (C-1A), 80.0

(C-3B), 77.7 (C-4B), 75.4, 74.1 (C-3A, C-4A), 70.1 (C-5A),

70.0 (C-5B), 63.3 (C-2B), 62.4 (C-6B), 52.4 (COOMe), 27.2

((CH₃)₃(Piv)), 20.8 (CH₃(Ac)); ¹H-NMR (300 MHz, CDCl₃)

(selected data for minor anomer): δ 7.37–7.25 (m, 15H, Ar),

5.23 (bd, 1H, H-1A), 4.99 (d, 1H, J_1,2= 3.6 Hz, H-1B),

4.94–4.57 (m, 8H, H-2A, H-5A, CH₂(OBn)), 3.80 (s, 3H,

COOMe), 3.34 (dd, 1H, H-2B), 2.02 (s, 3H, CH₃(Ac)), 1.24 (s,

9H, (CH₃)₃(Piv)); HR MS: m/z: calcd for C₄₁H₄₉N₃O₁₃Na:

814.3163; found: 814.3159 [M + Na]⁺.

N-Benzyloxycarbonyl-2-aminoethyl O-(6-O-acetyl-2-azido-3,4-

di-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl 3-

O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(6-

O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 →

4)-methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosiduronate (19)

TMSOTf (127 μL of a 0.18 M solution in dry CH₂Cl₂) was

added under an argon atmosphere at 0 °C to a mixture of 9

(44 mg, 0.05 mmol) and 18 (70 mg, 0.075 mmol) in dry CH₂Cl₂

(1.0 mL). After stirring for 20 min at 0 °C, the reaction mixture

was neutralized with Et₃N and concentrated to dryness. The

residue was puriﬁed by column chromatography (toluene–

EtOAc 5 : 1) to afford 19 (40 mg, 48%) and unreacted 9 (20 mg,

45%). TLC (hexane–EtOAc 2 : 1) R_f0.26; [α]²_D⁰+10 (c 1.2,

CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 7.37–7.23 (m, 30H,

Ar), 5.31 (d, 1H, J_1,2= 5.2 Hz, H-1C), 5.16 (bt, 1H, NH), 5.11

(d, 1H, J_1,2= 4.7 Hz, H-1A), 5.09 (bs, 2H, CH₂(Z)), 5.05 (d,

1H, J_1,2= 3.5 Hz, H-1D), 5.02 (d, 1H, J_1,2= 3.5 Hz, H-1B),

4.99 (t, 1H, J_2,3= 5.6 Hz, H-2C), 4.96–4.92 (m, 2H, H-2A,

O-(Methyl 4-O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-

glucopyranosyl)-3-O-benzyl-2-O-pivaloyl-α,β-^L-

idopyranosyluronate) trichloroacetimidate (18)

CH₂(OBn)), 4.86–4.68 (m, 8H, CH₂(OBn)), 4.67 (d, 1H, J_4,5

=

4.6 Hz, H-5A), 4.62 (d, 1H, J_4,5= 5.0 Hz, H-5C), 4.59 (d, 1H,

CH₂(OBn)), 4.36–4.29 (m, 2H, H-6aB, H-6bB), 4.26 (dd, 1H,

K₂CO₃(13 mg, 96 μmol) and trichloroacetonitrile (119 μL,

1.19 mmol) were added at room temperature to a solution of 17

J_5,6a= 1.8 Hz, J_6a,6b= 12.2 Hz, H-6aD), 4.16 (dd, 1H, J_5,6b

=

This journal is © The Royal Society of Chemistry 2012

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3.4 Hz, H-6bD), 4.11 (t, 1H, J_3,4= 5.2 Hz, H-4A), 4.08 (t, 1H,

J_3,4= 5.3 Hz, H-4C), 3.97–3.90 (m, 5H, H-3A, H-3C, H-4B,

H-5B, H-5D), 3.84–3.80 (m, 2H, H-3D, CH₂-O), 3.75–3.70 (m,

4H, H-3B, COOMe), 3.64–3.58 (m, 4H, COOMe, CH₂-O), 3.54

(t, 1H, J_3,4= J_4,5= 9.3 Hz, H-4D), 3.39 (m, 2H, CH₂-N), 3.29

(m, 2H, H-2D, H-2B), 2.10, 1.96 (2 s, 6H, CH₃(Ac)), 1.21, 1.19

(2 s, 18H, (CH₃)₃(Piv)); ¹³C-NMR (125 MHz, CDCl₃) (Signiﬁ-

cant data from HSQC experiment): δ 99.0 (C-1A), 98.4 (C-1D),

97.9 (C-1B), 97.8 (C-1C), 79.9 (C-3D), 77.9 (C-3B), 77.4

(C-4D), 75.9 (C-3C, C-3A), 75.3, 75.0 (CH₂(OBn)), 74.9

(C-4B), 74.7, 73.8 (CH₂(OBn)), 73.7 (C-4C, C-4A), 73.3

(CH₂(OBn)), 70.8 (C-2C), 70.7 (C-5C), 70.2 (C-2A), 70.1

(C-5A), 69.9 (C-5B), 69.7 (C-5D), 68.2 (CH₂-O), 66.6

(CH₂(Z)), 63.0 (C-2B, C-2D), 62.3 (C-6D), 61.9 (C-6B), 52.2,

51.9 (COOMe), 40.8 (CH₂-N), 27.1 ((CH₃)₃(Piv)), 20.7

(CH₃(Ac)); HR MS: m/z: calcd for C₈₅H₁₀₁N₇O₂₇Na:

1674.6643; found: 1674.6697 [M + Na]⁺.

66.7 (CH₂(Z)), 62.9 (C-2B, C-2D, C-2F), 62.2 (C-6F), 61.8

(C-6B, C-6D), 52.3–52.0 (COOMe), 40.9 (CH₂-N), 27.0

((CH₃)₃(Piv)), 20.8 (CH₃(Ac)); ESI MS: m/z: calcd for

C

₁₁₉H₁₄₂N₁₀O₃₉Na: 2357.9; found: 2357.7 [M + Na]⁺.

2-Aminoethyl 4-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-D-

glucopyranosyl)-2-O-sulfo-α-^L-idopyranosiduronic acid (21)

H₂O₂(30%, 1.8 mL) and a solution of LiOH (0.7 M, 1.1 mL)

were added at −5 °C to a solution of 15 (43 mg, 44 μmol) in

THF (4.3 mL). After stirring for 20 h at room temperature,

MeOH (4.3 mL) and an aqueous solution of NaOH (4 ^M,

1.1 mL) were added. After stirring for 20–24 h at room tempera-

ture, the reaction mixture was neutralized with a 4 ^Msolution of

HCl and then diluted with CH₂Cl₂(40 mL) and washed with

H₂O (20 mL). The organic phase was extracted with a solution

of Na₂SO₃(10%), dried (MgSO₄), ﬁltered, and concentrated to

give N-benzyloxycarbonyl-2-aminoethyl 4-O-(2-azido-3,4-di-O-

benzyl-2-deoxy-α-^D-glucopyranosyl)-3-O-benzyl-α-L-idopyrano-

siduronic acid (30 mg, 82%). ESI MS: m/z: calcd for

C₄₃H₄₇N₄O₁₃: 827.3; found: 826.8 [M − H]⁻.

This compound (12 mg, 14 μmol), sulfur trioxide–trimethyla-

mine complex (20 mg, 145 μmol) and a magnetic stirrer bar

were placed in a 5 mL microwave reaction vial and ﬁtted with a

septum, which was then pierced with a needle. The closed vial

was then evacuated under high vacuum and left to dry for 12 h.

Argon was let in, dry DMF (1.0 mL) was added and the reaction

mixture was subjected to microwave radiation for 15 min at

100 °C (80 W average power). The reaction vessel was cooled

under a stream of nitrogen and quenched with Et₃N (200 μL).

MeOH (1 mL) and CH₂Cl₂(1 mL) were added, and the solution

was layered on the top of a Sephadex LH-20 chromatography

column which was eluted with CH₂Cl₂/MeOH (1 : 1) to obtain

N-Benzyloxycarbonyl-2-aminoethyl O-(6-O-acetyl-2-azido-3,4-

di-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl 3-

O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(6-

O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-glucopyranosyl)-(1 →

4)-O-(methyl 3-O-benzyl-2-O-pivaloyl-α-^L-idopyranosyluronate)-

(1 → 4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-^D-

glucopyranosyl)-(1 → 4)-methyl 3-O-benzyl-2-O-pivaloyl-α-^L-

idopyranosiduronate (20)

BF₃·Et₂O (145 μL of a 0.2 M solution in dry CH₂Cl₂) was added

under an argon atmosphere at room temperature to a mixture of

12 (30 mg, 19 μmol) and 18 (29 mg, 31 μmol) in dry CH₂Cl₂

(1.0 mL). After stirring for 30 min, the reaction mixture was neu-

tralized with Et₃N and concentrated to dryness. The residue was

puriﬁed by column chromatography (toluene–EtOAc 5 : 1 →

1 : 1) to afford 20 (23 mg, 52%), and unreacted 12 (8 mg, 27%).

TLC (toluene–EtOAc 4 : 1) R_f0.38; [α]²_D⁰+4 (c 1.1, CHCl₃);

¹H-NMR (500 MHz, CDCl₃): δ 7.39–7.25 (m, 40H, Ar), 5.37

(d, 1H, J_1,2= 6.0 Hz, H-1E), 5.31 (d, 1H, J_1,2= 5.4 Hz, H-1C),

5.15 (bt, 1H, NH), 5.12 (d, 1H, J_1,2= 4.8 Hz, H-1A), 5.09 (bs,

2H, CH₂(Z)), 5.06–4.97 (m, 5H, H-1B, H-1D, H-1F, H-2C,

H-2E), 4.96–4.90 (m, 3H, H-2A, CH₂(OBn)), 4.87–4.67 (m,

11H, CH₂(OBn)), 4.65 (d, 1H, J_4,5= 4.6 Hz, H-5A), 4.61–4.56

(m, 2H, H-5C, CH₂(OBn)), 4.49 (d, 1H, J_4,5= 5.6 Hz, H-5E),

4.34–4.24 (m, 5H, H-6aB, H-6bB, H-6aD, H-6bD, H-6aF), 4.15

(dd, 1H, J_5,6= 3.3 Hz, J_6a,6b= 12.2 Hz, H-6bF), 4.11 (t, 1H, J_3,4

= 5.3 Hz, H-4A), 4.08 (t, 1H, J_3,4= 5.7 Hz, H-4C), 4.03 (t, 1H,

J_3,4= 6.2 Hz, H-4E), 3.97–3.86 (m, 8H, H-3A, H-3C, H-3E,

H-4B, H-4D, H-5B, H-5D, H-5F), 3.84–3.78 (m, 2H, H-3F,

CH₂-O), 3.75–3.69 (m, 4H, H-3B or H-3D, COOMe), 3.66–3.58

(m, 8H, H-3B or H-3D, COOMe (3.64, 3.61), CH₂-O), 3.53 (t,

1H, J_3,4= J_4,5= 9.4 Hz, H-4F), 3.39 (m, 2H, CH₂-N), 3.32–3.27

(m, 3H, H-2B, H-2D, H-2F), 2.11, 2.07, 1.96 (3 s, 9H,

CH₃(Ac)), 1.19 (bs, 27H, (CH₃)₃(Piv)); ¹³C-NMR (125 MHz,

CDCl₃) (Signiﬁcant data from HSQC experiment): δ 99.0

(C-1A), 98.1 (C-1B, C-1D, C-1F), 97.7 (C-1C), 97.5 (C-1E),

80.0 (C-3F), 79.9, 77.7 (C-3B, C-3D), 77.4 (C-4F), 76.8–73.5

(C-3A, C-3C, C-3E, C-4B, C-4D, C-4A, C-4C, C-4E,

CH₂(OBn)), 71.5 (C-2E, C-5E), 71.0 (C-2C, C-5C), 70.4

(C-2A), 70.3 (C-5A), 69.8 (C-5B, C-5D, C-5F), 68.4 (CH₂-O),

N-benzyloxycarbonyl-2-aminoethyl

4-O-(2-azido-3,4-di-O-

benzyl-2-deoxy-6-O-sulfo-α-^D-glucopyranosyl)-3-O-benzyl-2-

O-sulfo-α-^L-idopyranosiduronic acid as triethylammonium salt

(17 mg, 91%). TLC (EtOAc-Py-H₂O-AcOH 10 : 5 : 3 : 1) R_f

1

0.51; H-NMR (500 MHz, MeOD): δ 7.43–7.25 (m, 20 H, Ar),

5.18 (bs, 1H, H-1A), 5.06 (m, 3H, H-1B, CH₂(Z)), 4.90–4.65

(m, 7H, H-5A (4.74), CH₂(OBn)), 4.49 (bs, 1H, H-2A), 4.33

(dd, 1H, J_5,6a= 2.8 Hz, J_6a,6b= 10.7 Hz, H-6aB), 4.25 (bt, 1H,

H-3A), 4.20 (dd, 1H, J_5,6b= 1.9 Hz, H-6bB), 4.14 (bt, 1H,

H-4A), 3.97–3.93 (m, 2H, H-5B, H-3B), 3.80 (m, 1H, CH₂-O),

3.66 (t, 1H, J_3,4= J_4,5= 9.4 Hz, H-4B), 3.59 (m, 1H, CH₂-O),

3.32 (m, 3H, H-2B, CH₂-N), 3.19 (q, Et₃NH⁺), 1.32 (t, Et₃NH⁺);

¹³C-NMR (125 MHz, MeOD) (Signiﬁcant data from HSQC

experiment): δ 99.4 (C-1A), 96.5 (C-1B), 79.7 (C-3B), 77.6

(C-4B), 72.8 (C-3A), 71.9 (C-4A), 71.7 (C-2A), 70.2(C-5B),

67.1 (C-5A), 66.8 (CH₂-O), 65.5 (C-6B), 63.4 (C-2B); ESI MS:

m/z: calcd for C₄₃H₄₇N₄O₁₉S₂: 987.2; found: 986.7 [M + 2H]⁻;

calcd for C₆₁H₉₄N₇O₁₉S₂: 1292.6; found: 1292.1 [M + 3Et₃NH

+ H]⁺.

This compound (45 mg, 35 μmol) was dissolved in THF

(4.5 mL) and treated with a 0.1 ^Maqueous solution of NaOH

(1.5 mL). Then, a solution of Me₃P in THF (139 μL of a 1 M sol-

ution) was added and the reaction was stirred for 3 h. The reac-

tion mixture was neutralized with a 0.1 ^Msolution of HCl

and concentrated to afford N-benzyloxycarbonyl-2-aminoethyl

2158 | Org. Biomol. Chem., 2012, 10, 2146–2163

This journal is © The Royal Society of Chemistry 2012

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4-O-(2-amino-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-α-D-glucopyr-

anosyl)-3-O-benzyl-2-O-sulfo-α-^L-idopyranosiduronic acid as

sodium salt. TLC (EtOAc-Py-H₂O-AcOH 10 : 5 : 3 : 1) R_f0.61;

ESI MS: m/z: calcd for C₄₃H₄₈N₂O₁₉S₂Na: 983.2; found: 982.7

[M + Na + H]⁻; calcd for C₄₃H₄₈N₂O₁₉S₂: 480.1; found: 479.7

[M + H]²⁻; calcd for C₄₃H₄₉N₂O₁₉S₂: 961.2; found: 960.7

[M + 2H]⁻.

Triethylamine (0.68 mL) and then sulfur trioxide-pyridine

complex (56 mg, 350 μmol) were added to a solution of this dis-

accharide (35 μmol) in anhydrous pyridine (3.0 mL). After stir-

ring for 4 h at room temperature under an argon atmosphere, the

reaction mixture was puriﬁed by Sephadex LH-20 chromato-

graphy (CH₂Cl₂/MeOH 1 : 1) and RP-18 chromatography

(10 m^MAcOH-Et₃N (pH 7.0)/MeOH 70 : 30 → 30 : 70) to give

for C₁₄H₂₄N₂O₂₀S₃Na: 659.00; found: 658.99 [M+2H+Na]⁻;

calcd for C₁₄H₂₅N₂O₂₀S₃: 637.01; found: 637.01 [M + 3H]⁻.

2-Aminoethyl O-(2-deoxy-2-sulfamido-6-O-sulfo-α-D-

glucopyranosyl)-(1 → 4)-O-(2-O-sulfo-α-^L-idopyranosyluronic

acid)-(1 → 4)-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-^D-

glucopyranosyl)-(1 → 4)-2-O-sulfo-α-^L-idopyranosiduronic acid

(22)

H₂O₂(30%, 0.44 mL) and a solution of LiOH (0.7 M, 0.27 mL)

were added at −5 °C to a solution of 19 (18 mg, 11 μmol) in

THF (1.2 mL). After stirring for 20 h at room temperature,

MeOH (1.2 mL) and a solution of NaOH (4 ^M, 0.28 mL) were

added. After stirring for 20–24 h at room temperature, the reac-

tion mixture was neutralized with a 4 ^Msolution of HCl and then

diluted with CH₂Cl₂(20 mL) and washed with H₂O (10 mL).

The organic phase was extracted with a solution of Na₂SO₃

(10%), dried (MgSO₄), ﬁltered, and concentrated to give N-ben-

N-benzyloxycarbonyl-2-aminoethyl

4-O-(3,4-di-O-benzyl-2-

deoxy-2-sulfamido-6-O-sulfo-α-^D-glucopyranosyl)-3-O-benzyl-2-

O-sulfo-α-^L-idopyranosiduronic acid as triethylammonium salt.

The corresponding sodium salt was obtained by elution from a

column of Dowex 50WX4-Na⁺with MeOH/H₂O 9 : 1. TLC

(EtOAc-Py-H₂O-AcOH 10 : 5 : 3 : 1) R_f0.29; ¹H-NMR

(500 MHz, MeOD) (data for triethylammonium salt): δ 7.46-

7.21 (m, 20H, Ar), 5.45 (d, 1H, J_1,2= 3.2 Hz, H-1B), 5.22 (bs,

1H, H-1A), 5.14 (d, 1H, CH₂(OBn)), 5.03 (m, 2H, CH₂(Z)),

4.88-4.73 (m, 6H, H-5A (4.74), CH₂(OBn)), 4.55 (m, 2H, H-2A,

H-3A), 4.36 (dd, 1H, J_5,6a= 2.1 Hz, J_6a,6b= 10.7 Hz, H-6aB),

4.24 (dd, 1H, J_5,6b= 1.8 Hz, H-6bB), 4.21 (bs, 1H, H-4A), 3.92

(bd, 1H, H-5B), 3.80 (dd, 1H, J_2,3= 10.4 Hz, J_3,4= 9.4 Hz,

H-3B), 3.76 (m, 1H, CH₂-O), 3.62 (t, 1H, J_4,5= 9.4 Hz, H-4B),

3.55 (m, 1H, CH₂-O), 3.44 (dd, 1H, H-2B), 3.35 (m, 2H,

CH₂-N), 3.19 (q, Et₃NH⁺), 1.30 (t, Et₃NH⁺); ¹³C-NMR

(125 MHz, MeOD) (Signiﬁcant data from HSQC experiment): δ

102.1 (C-1B), 100.5 (C-1A), 81.2 (C-3B), 78.5 (C-4B), 78.0

(C-4A), 76.1 (C-3A, CH₂(OBn)), 75.7 (CH₂(OBn)), 73.0

(CH₂(OBn)), 72.6 (C-2A), 71.1 (C-5B), 67.9 (CH₂-O), 67.7

(C-5A), 67.2 (CH₂(Z)), 67.0 (C-6B), 60.3 (C-2B); ESI MS: m/z:

calcd for C₄₃H₄₆N₂O₂₂S₃NaK: 550.1; found: 550.7 [M + Na +

zyloxycarbonyl-2-aminoethyl

O-(2-azido-3,4-di-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(3-O-benzyl-α-L-idopyra-

nosyluronic acid)-(1 → 4)-O-(2-azido-3-O-benzyl-2-deoxy-α-^D-

glucopyranosyl)-(1

→

4)-3-O-benzyl-α-^L-idopyranosiduronic

acid (12 mg, 80%). ESI MS: m/z: calcd for C₆₉H₇₆N₇O₂₃:

1370.5; found: 1369.9 [M − H]⁻; calcd for C₆₉H₇₄N₇O₂₃Na₄:

1460.4; found: 1459.8 [M − 3H + 4Na]⁺.

This compound (12 mg, 8.7 μmol) and sulfur trioxide–tri-

methylamine complex (24 mg, 174 μmol) were dissolved in dry

DMF (1.0 mL) and heated at 100 °C for 30 min using micro-

wave radiation as described above. The reaction vessel was

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

were added. The solution was layered on the top of a Sephadex

LH-20 chromatography column which was eluted with CH₂Cl₂/

MeOH (1 : 1) to obtain N-benzyloxycarbonyl-2-aminoethyl O-

(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-α-^D-glucopyrano-

syl)-(1 → 4)-O-(3-O-benzyl-2-O-sulfo-α-^L-idopyranosyluronic

K]²⁻

.

acid)-(1 → 4)-O-(2-azido-3-O-benzyl-2-deoxy-6-O-sulfo-α-^D-

A solution of this compound (35 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 over Celite

and concentrated. The residue was puriﬁed by Sephadex G-25

chromatography (H₂O/MeOH 9 : 1) to give 21 after lyophilisa-

tion (15.3 mg, 60% from N-benzyloxycarbonyl-2-aminoethyl 4-

O-(2-azido-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-α-^D-glucopyra-

glucopyranosyl)-(1 → 4)-3-O-benzyl-2-O-sulfo-α-^L-idopyranosi-

duronic acid as triethylammonium salt (19 mg, 95%). The corre-

sponding sodium salt was obtained by elution from a column of

Dowex 50WX4-Na⁺with MeOH/H₂O 3 : 1. TLC (EtOAc–Py–

H₂O–AcOH 10 : 5 : 3 : 1) R_f0.44;¹H-NMR (500 MHz, MeOD):

δ 7.46–7.17 (m, 30H, Ar), 5.49 (bs, 1H, H-1C), 5.22 (bs, 1H,

H-1A), 5.10 (d, 1H, J_1,2= 3.7 Hz, H-1B), 5.06 (bs, 2H,

nosyl)-3-O-benzyl-2-O-sulfo-α-^L-idopyranosiduronic acid,

3

CH₂(Z)), 5.01 (d, 1H, J_1,2= 3.7 Hz, H-1D), 5.00 (d, 1H, J_4,5=

steps; 45% from 15, 5 steps, 85% average yield per step).

¹H-NMR (500 MHz, D₂O): δ 5.46 (d, 1H, J_1,2= 3.5 Hz, H-1B),

5.15 (d, 1H, J_1,2= 3.4 Hz, H-1A), 4.55 (d, 1H, J_4,5= 2.9 Hz,

H-5A), 4.36 (dd, 1H, J_5,6a= 3.1 Hz, J_6a,6b= 11.2 Hz, H-6aB),

4.33 (dd, 1H, J_2,3= 6.5 Hz, H-2A), 4.23 (m, 2H, H-3A, H-6bB),

4.14 (t, 1H, J_3,4= 3.3 Hz, H-4A), 4.04 (m, 1H, CH₂-O), 4.00

(dt, 1H, J_4,5= 9.8 Hz, H-5B), 3.82 (m, 1H, CH₂-O), 3.64 (t, 1H,

J_2,3= J_3,4= 9.7 Hz, H-3B), 3.57 (t, 1H, J_4,5= 9.7 Hz, H-4B),

3.26 (m, 3H, H-2B, CH₂-N); ¹³C-NMR (125 MHz, D₂O) (Sig-

niﬁcant data from HMQC experiment): δ 99.3 (C-1A), 96.4

(C-1B), 76.7 (C-2A), 75.7 (C-4A), 70.8 (C-3B), 69.7 (C-5B),

69.3 (C-5A), 69.2 (C-3A), 69.1 (C-4B), 66.5 (C-6B), 64.1 (CH₂-

O), 57.7 (C-2B), 39.2 (CH₂-N); ESI MS: m/z: calcd for

C₁₄H₂₃N₂O₂₀S₃Na₂: 680.98; found: 680.97 [M+H+2Na]⁻; calcd

1.5 Hz, H-5C), 4.90–4.64 (m, 10H, CH₂(OBn), H-5A (4.83)),

4.60 (bs, 1H, H-2C), 4.52 (bs, 1H, H-2A), 4.42 (d, 1H,

CH₂(OBn)), 4.35–4.31 (m, 2H, H-6aB, H-6aD), 4.27–4.22 (m,

3H, H-3A, H-3C, H-6bB), 4.16 (bs, 1H, H-4A), 4.12 (bd, 1H,

H-6bD), 4.05 (t, 1H, J_3,4= J_4,5= 9.6 Hz, H-4B), 3.97 (m, 2H,

H-4C, H-5B), 3.85 (m, 2H, H-3D, CH₂-O), 3.74 (m, 2H, H-3B,

H-5D), 3.67–3.60 (m, 2H, H-4D, CH₂-O), 3.47 (dd, 1H, J_2,3

=

10.3 Hz, H-2D), 3.37 (m, 2H, CH₂-N), 3.29 (dd, 1H, J_2,3= 10.3

Hz, H-2B);¹³C-NMR (125 MHz, MeOD) (Signiﬁcant data from

HSQC experiment): δ 100.6 (C-1A), 98.2 (C-1C), 96.6 (C-1D),

96.3 (C-1B), 81.5 (C-3D), 79.6 (C-3B), 78.5 (C-4D), 76.2, 76.1,

75.6, 73.1, 73.1 (CH₂(OBn)), 72.4 (C-3A), 72.1 (C-2A, C-4B),

72.0 (C-4C), 71.7 (C-3C, C-4A), 71.3 (C-5B), 71.2 (C-5D),

70.7 (C-2C), 68.2 (CH₂-O), 67.8 (C-5A), 67.3 (C-5C),

Org. Biomol. Chem., 2012, 10, 2146–2163 | 2159

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67.2 (CH₂(Z)), 67.1 (C-6B), 66.6 (C-6D), 65.3 (C-2B), 65.0

(C-2D), 41.4 (CH₂-N); ESI MS: m/z: calcd for

C₆₉H₇₃N₇O₃₅S₄K₂: 882.6; found: 882.4 [M+2H+2 K]²⁻; calcd

A solution of this compound (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 over Celite and

concentrated. The residue was puriﬁed by Sephadex G-25

chromatography (H₂O/MeOH 9 : 1) to give 22 after lyophilisa-

tion (3.2 mg, 47% from the O-sulfated intermediate, 3 steps;

for C₇₅H₉₀N₈O₃₅S₄: 895.2; found: 894.9 [M + 3H + Et₃NH]²⁻

;

calcd for C₆₉H₇₁N₇O₃₅S₄K₂Na₂: 904.6; found: 904.9 [M + 2K +

2Na]²⁻

.

1

This compound (9 mg, 5 μmol) was dissolved in THF

(1.6 mL) and treated with a 0.1 ^Maqueous solution of NaOH

(0.43 mL). Then, a solution of Me₃P in THF (40 μL of a 1 M sol-

ution) was added and the reaction was stirred for 3 h. The reac-

tion mixture was neutralized with a 0.1 ^Msolution of HCl and

concentrated to afford N-benzyloxycarbonyl-2-aminoethyl O-(2-

amino-3,4-di-O-benzyl-2-deoxy-6-O-sulfo-α-^D-glucopyranosyl)-

(1 → 4)-O-(3-O-benzyl-2-O-sulfo-α-^L-idopyranosyluronic acid)-

(1 → 4)-O-(2-amino-3-O-benzyl-2-deoxy-6-O-sulfo-α-^D-gluco-

pyranosyl)-(1 → 4)-3-O-benzyl-2-O-sulfo-α-^L-idopyranosiduro-

nic acid as sodium salt. TLC (EtOAc–Py–H₂O–AcOH

10 : 5 : 3 : 1) R_f0.43; ESI MS: m/z: calcd for C₆₉H₇₉N₃O₃₅S₄:

818.7; found: 818.1 [M + 4H]²⁻; calcd for C₆₉H₇₅N₃O₃₅S₄Na₄:

36% from 19, 5 steps, 82% average yield per step). H-NMR

(500 MHz, D₂O): δ 5.47 (d, 1H, J_1,2= 3.6 Hz, H-1B), 5.39 (d,

1H, J_1,2= 3.5 Hz, H-1D), 5.21 (bd, 1H, H-1C), 5.08 (d, 1H, J_1,2

= 4.1 Hz, H-1A), 4.82 (bd, 1H, H-5C), 4.53 (d, 1H, J_4,5= 3.2

Hz, H-5A), 4.40–4.25 (m, 5H, H-2C (4.34), H-2A (4.31),

H-6aB, H-6aD, H-6bB or H-6bD), 4.21–4.16 (m, 3H, H-3C,

H-3A, H-6bB or H-6bD), 4.14 (t, 1H, J_3,4= J_4,5= 3.8 Hz,

H-4A), 4.08 (bt, 1H, H-4C), 4.05–3.97 (m, 3H, H-5B, H-5D,

CH₂-O), 3.81 (m, 1H, CH₂-O), 3.76 (t, 1H, J_3,4= J_4,5= 9.5 Hz,

H-4B), 3.67–3.61 (m, 2H, H-3B, H-3D), 3.56 (t, 1H, J_3,4= J_4,5

= 9.6 Hz, H-4D), 3.29–3.21 (m, 4H, H-2B, H-2D, CH₂-

N);¹³C-NMR (125 MHz, D₂O) (Signiﬁcant data from HSQC

experiment): δ 99.6 (C-1A), 99.1 (C-1C), 97.2 (C-1D), 95.9

(C-1B), 77.5 (C-2A), 76.1 (C-4B, C-4C), 75.9 (C-4A), 75.4

(C-2C), 69.9 (C-5A, C-3A, C-5D), 69.3 (C-3B, C-3D), 69.1

(C-5B, C-4D), 68.9 (C-5C), 68.5 (C-3C), 66.5, 66.3 (C-6B,

C-6D), 64.4 (CH₂-O), 58.0 (C-2B), 57.8 (C-2D), 39.4 (CH₂-N);

ESI MS: m/z: calcd for C₂₆H₃₈N₃O₃₉S₆Na₅: 661.45; found:

661.44 [M + 5Na + H]²⁻; calcd for C₂₆H₃₉N₃O₃₉S₆Na₄: 650.46;

found: 650.45 [M + 4Na + 2H]²⁻; calcd for C₂₆H₄₀N₃O₃₉S₆Na₃:

862.6; found: 862.0 [M + 4Na]²⁻

.

Triethylamine (110 μL) and then sulfur trioxide-pyridine

complex (16 mg, 98 μmol) were added to a solution of this com-

pound (5 μmol) in anhydrous pyridine (0.6 mL). After stirring

for 6 h at room temperature under an argon atmosphere, the reac-

tion mixture was puriﬁed by Sephadex LH-20 chromatography

(CH₂Cl₂/MeOH 1 : 1) and RP-18 chromatography (10 mM

AcOH–Et₃N (pH 7.0)/MeOH 90 : 10 → 0 : 100) to give N-ben-

639.47; found: 639.46 [M + 3Na + 3H]²⁻

.

zyloxycarbonyl-2-aminoethyl

sulfamido-6-O-sulfo-α-^D-glucopyranosyl)-(1

O-(3,4-di-O-benzyl-2-deoxy-2-

4)-O-(3-O-

→

benzyl-2-O-sulfo-α-^L-idopyranosyluronic acid)-(1 → 4)-O-(3-O-

benzyl-2-deoxy-2-sulfamido-6-O-sulfo-α-^D-glucopyranosyl)-(1

2-Aminoethyl O-(2-deoxy-2-sulfamido-4,6-di-O-sulfo-α-D-

glucopyranosyl)-(1 → 4)-O-(2-O-sulfo-α-^L-idopyranosyluronic

acid)-(1 → 4)-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-^D-

glucopyranosyl)-(1 → 4)-O-(2-O-sulfo-α-^L-idopyranosyluronic

acid)-(1 → 4)-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-^D-

glucopyranosyl)-(1 → 4)-2-O-sulfo-α-^L-idopyranosiduronic acid

(23)

→

4)-3-O-benzyl-2-O-sulfo-α-^L-idopyranosiduronic acid as

triethylammonium salt. The corresponding sodium salt was

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

MeOH–H₂O 9 : 1. TLC (EtOAc–Py–H₂O–AcOH 5 : 5 : 3 : 1) R_f

0.57;¹H-NMR (500 MHz, MeOD): δ 7.52–7.05 (m, 30H, Ar),

5.99 (bs, 1H, H-1C), 5.36 (d, 1H, J_1,2= 3.6 Hz, H-1D),

5.32–5.26 (m, 4H, H-1A (5.32), H-1B (5.30), CH₂(OBn)), 5.03

(m, 2H, CH₂(Z)), 4.93 (d, 1H, CH₂(OBn)), 4.80 (bs, 1H, H-5C),

4.77–4.71 (m, 6H, H-2A (4.74), H-2C (4.73), CH₂(OBn)), 4.68

(d, 1H, CH₂(OBn)), 4.61 (bs, 1H, H-5A), 4.57 (d, 1H,

CH₂(OBn)), 4.51–4.47 (m, 2H, H-3A (4.50), CH₂(OBn)), 4.41

(m, 1H, H-5B), 4.37–4.32 (m, 3H, H-3C (4.36), H-6aD,

H-6bD), 4.20–4.06 (m, 6H, H-4C (4.18), H-6aB (4.16), H-4A

(4.13), H-6bB (4.11), H-5D (4.09), H-3B (4.08)), 3.88 (t, 1H,

J_3,4= J_4,5= 10.4 Hz, H-4B), 3.85 (t, 1H, J_2,3= J_3,4= 9.8 Hz,

H-3D), 3.73 (m, 1H, CH₂-O), 3.62–3.50 (m, 4H, H-4D (3.60),

H-2B (3.56), H-2D (3.53), CH₂-O), 3.31 (m, 2H, CH₂-

N);¹³C-NMR (125 MHz, MeOD) (Signiﬁcant data from HSQC

experiment): δ 100.1 (C-1D), 100.0 (C-1A), 97.2 (C-1B), 94.4

(C-1C), 81.4 (C-3D), 78.8 (C-4D), 77.1 (C-4B), 76.4 (C-4C),

76.2, 76.0 (CH₂(OBn)), 75.9 (C-3B), 75.5 (C-3C), 75.2

(CH₂(OBn)), 74.6 (C-4A), 72.9 (C-2A), 72.8, 72.6 (CH₂(OBn)),

71.7 (C-3A), 71.5 (C-2C), 70.8 (C-5D), 69.6 (C-5C), 68.8

(C-5A), 68.6 (C-5B), 68.4 (C-6B), 68.0 (CH₂-O), 67.6 (C-6D),

67.3 (CH₂(Z)), 60.2 (C-2B), 59.7 (C-2D), 41.3 (CH₂-N); ESI

MS: m/z: calcd for C₆₉H₇₃N₃O₄₁S₆Na₆: 964.5; found: 964.5

[M + 6Na]²⁻; calcd for C₆₉H₇₃N₃O₄₁S₆Na₁₀: 1010.6; found:

1010.6 [M + 10Na]²⁺.

H₂O₂(30%, 1.4 mL) and a solution of LiOH (0.7 M, 0.84 mL)

were added at −5 °C to a solution of 14 (76 mg, 34 μmol) in

THF (3.8 mL). After stirring for 20 h at room temperature,

MeOH (3.8 mL) and a solution of NaOH (4 ^M, 0.87 mL) were

added. After stirring for 20–24 h at room temperature, the reac-

tion mixture was neutralized with a 4 ^Msolution of HCl and then

diluted with CH₂Cl₂(80 mL) and washed with H₂O (40 mL).

The organic phase was extracted with a solution of Na₂SO₃

(10%), dried (MgSO₄), ﬁltered, and concentrated to give the

desired saponiﬁed hexasaccharide (60 mg, 97%). ESI MS: m/z:

calcd for C₈₈H₉₆N₁₀O₃₃Na₃: 1889.6; found: 1889.5 [M + 3Na −

4H]⁻; calcd for C₈₈H₉₉N₁₀O₃₃: 1823.6; found: 1823.5

[M − H]⁺.

This compound (12 mg, 6.7 μmol) and sulfur trioxide–tri-

methylamine complex (33 mg, 230 μmol) were dissolved in dry

DMF (1.0 mL) and heated for 30 min at 100 °C using micro-

waves as described above. The reaction vessel was cooled and

Et₃N (150 μL), MeOH (1 mL) and CH₂Cl₂(1 mL) were added.

The solution was layered on the top of a Sephadex LH-20

chromatography column which was eluted with CH₂Cl₂–MeOH

(1 : 1) to obtain the desired hepta O-sulfated hexasaccharide as

triethylammonium salt. The corresponding sodium salt was

2160 | Org. Biomol. Chem., 2012, 10, 2146–2163

View Article Online

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

H-2A (4.53), H-5B, H-5D, H-6aF (4.52), H-6bF (4.45),

CH₂(OBn)), 4.35–4.03 (m, 15H, H-4C (4.27), H-4E (4.24),

H-4F (4.24), H-4A (4.20), H-3C (4.20), H-3A (4.13), H-3E

(4.13), H-3B, H-3D, H-3F, H-5F (4.05), H-6aB, H-6bB, H-6aD,

H-6bD), 3.95 (t, 1H, H-4B or H-4D), 3.80 (m, 1H, CH₂-O), 3.74

(t, 1H, H-4B or H-4D), 3.61–3.52 (m, 4H, CH₂-O, H-2B, H-2D,

H-2F), 3.33 (m, 2H, CH₂-N); ¹³C-NMR (125 MHz, MeOD)

(Signiﬁcant data from HSQC experiment): δ 99.5 (C-1A), 96.8

(C-1B, C-1D, C-1F), 94.3 (C-1C), 94.2 (C-1E), 77.6 (C-4B or

C-4D), 77.4 (C-4F), 77.1 (C-4B or C-4D), 75.4, 75.2, 74.9

(CH₂(OBn)), 74.3 (C-3A, C-3E), 73.6–70.8 (C-3C, C-3B, C-3D,

C-3F, C-4A, C-4C (73.2), C-4E (71.8), C-2A (71.8), C-2C

(72.4), C-2E (71.2), C-5B, C-5D, CH₂(OBn)), 69.6 (C-5C), 69.4

(C-5F), 69.1 (C-5E), 68.7 (C-5A), 68.1 (C-6B or C-6D), 67.9

(C-6F), 67.7 (C-6B or C-6D), 67.3 (CH₂(Z)), 67.2 (CH₂-O),

60.1 (C-2B, C-2D, C-2F), 42.0 (CH₂-N); ESI MS: m/z: calcd for

MeOH–H₂O 3 : 1. TLC (EtOAc–Py–H₂O–AcOH 5 : 5 : 3 : 1) R_f

1

0.42; H-NMR (500 MHz, MeOD): δ 7.52–7.18 (m, 35H, Ar),

5.53 (bs, 1H, H-1E), 5.47 (bs, 1H, H-1C), 5.22 (bs, 1H, H-1A),

5.21 (d, 1H, CH₂(OBn)), 5.12 (d, 1H, J_1,2= 3.7 Hz, H-1B or

H-1D), 5.09 (d, 1H, J_1,2= 3.8 Hz, H-1B or H-1D), 5.07 (bs, 3H,

H-1F, CH₂(Z)), 5.04 (bs, 1H, H-5C), 5.03 (bs, 1H, H-5E),

4.90–4.79 (m, 3H, CH₂(OBn), H-5A (4.82)), 4.77–4.59 (m, 7H,

CH₂(OBn), H-2E (4.62)), 4.53 (bs, 2H, H-2C, H-2A), 4.50 (m,

2H, CH₂(OBn), H-6aF), 4.49–4.42 (m, 2H, CH₂(OBn)),

4.36–4.15 (m, 11H, H-4F, H-6aB, H-6aD, H-3E, H-3C, H-3A,

H-6bB, H-6bD, H-6bF, H-4A, H-4C), 4.07 (m, 2H, H-4B,

H-4D), 4.03 (m, 1H, H-4E), 4.00 (m, 1H, H-5B or H-5D), 3.94

(m, 1H, H-5F), 3.90–3.73 (m, 4H, H-3F, CH₂-O, H-5B or H-5D,

H-3B or H-3D), 3.71–3.60 (m, 2H, H-3B or H-3D, CH₂-O),

3.45–3.39 (m, 2H, H-2F, H-2B or H-2D), 3.37 (m, 2H, CH₂-N),

3.31 (m, 1H, H-2B or H-2D);¹³C-NMR (125 MHz, MeOD)

(Signiﬁcant data from HSQC experiment): δ 100.1 (C-1A), 98.6

(C-1E), 98.5 (C-1C), 96.8, 96.1 (C-1B, C-1D), 95.8 (C-1F), 80.2

(C-3F), 80.1, 79.7 (C-3B, C-3D), 78.0 (C-4F), 76.7, 76.5, 76.1,

73.5, 73.3 (CH₂(OBn)), 72.9 (C-3A or C-3C), 72.7 (C-2A), 72.3

(C-4B, C-4D), 72.1 (C-4A), 71.5 (C-4E, C-2C), 71.4 (C-3E,

C-3A or C-3C), 71.3, 71.2 (C-5B, C-5D), 71.1 (C-4C, C-5F),

70.9 (C-2E), 68.3 (CH₂-O), 68.1 (C-5A), 67.8 (C-6F), 67.5

(C-5C, C-5E), 67.4 (CH₂(Z), C-6B, C-6D), 65.9, 65.3 (C-2B,

C-2D), 64.5 (C-2F), 41.5 (CH₂-N); ESI MS: m/z: calcd for

C

₁₃₀H₂₁₂N₁₁O₆₃S₁₀Na: 1639.0; found: 1639.2 [M + 7Et₃NH +

Na + 7H]²⁺; calcd for C₁₄₂H₂₄₃N₁₃O₆₃S₁₀: 1729.2; found:

1729.7 [M + 9Et₃NH + 6H]²⁺.

A solution of this hexasaccharide (6.7 μmol, as sodium salt)

in H₂O/MeOH (1.8 mL/0.2 mL) was hydrogenated in the pres-

ence of Pd(OH)₂. After 24 h, the suspension was ﬁltered over

Celite and concentrated. The residue was puriﬁed by Sephadex

G-25 chromatography (H₂O–MeOH 9 : 1) to give 23 after lyo-

philisation (4.4 mg, 31% from the saponiﬁed intermediate, 4

steps; 30% from 14, 5 steps, 79% average yield per step).

¹H-NMR (500 MHz, D₂O): δ 5.50–5.47 (m, 2H, H-1F, H-1B or

H-1D), 5.43 (d, 1H, J_1,2= 3.4 Hz, H-1B or H-1D), 5.27–5.21

(m, 2H, H-1C, H-1E), 5.12 (d, 1H, J_1,2= 3.8 Hz, H-1A), 4.84

(m, 2H, H-5C, H-5E), 4.56 (d, 1H, J_4,5= 3.1 Hz, H-5A),

4.48–4.26 (m, 10H, H-2A, H-2C, H-2E, H-6aB, H-6aD, H-6aF,

H-6bB, H-6bD, H-6bF, H-4F), 4.24–4.19 (m, 3H, H-3A, H-3C,

H-3E), 4.18–4.10 (m, 4H, H-4A, H-4C, H-4E, H-5F), 4.09–4.02

(m, 3H, H-5B, H-5D, CH₂–O), 3.87–3.76 (m, 4H, CH₂-O,

H-4B, H-4D, H-3F), 3.74–3.64 (m, 2H, H-3B, H-3D), 3.36 (dd,

1H, J_1,2= 3.3 Hz, J_2,3= 10.8 Hz, H-2F), 3.33–3.21 (m, 4H,

H-2B, H-2D, CH₂-N); ¹³C-NMR (125 MHz, D₂O) (Signiﬁcant

data from HSQC experiment): δ 99.6 (C-1A), 99.2 (C-1C,

C-1E), 96.4 (C-1B or C-1D), 95.9 (C-1F, C-1B or C-1D), 77.1

(C-2A, C-4F), 75.9 (C-4B, C-4D, C-4A, C-4C, C-4E, C-2C,

C-2E), 69.9 (C-5A), 69.4 (C-3A, C-3C, C-3E, C-3B, C-3D,

C-3F), 69.2 (C-5C, C-5E), 69.0 (C-5B, C-5D), 68.1 (C-5F), 66.4

(C-6B, C-6D, C-6F), 64.4 (CH₂-O), 57.8 (C-2B, C-2D), 57.4

(C-2F), 39.1 (CH₂-N); ESI MS: m/z: calcd for

C

₁₄₂H₂₃₇N₁₉O₅₄S₇: 1648.2; found: 1648.3 [M + 9Et₃NH +

3H]²⁺.

This hexasaccharide (6.7 μmol) was dissolved in THF

(2.4 mL) and treated with a 0.1 ^Maqueous solution of NaOH

(0.96 mL). Then, a solution of Me₃P in THF (88 μL of a 1 M sol-

ution) was added and the reaction was stirred for 6 h. The reac-

tion mixture was neutralized with a 0.1 ^Msolution of HCl and

concentrated. MeOH (1 mL), CH₂Cl₂(1 mL) and Et₃N (250 μL)

were added, and the solution was layered on the top of a Sepha-

dex LH-20 chromatography column which was eluted with

CH₂Cl₂–MeOH (1 : 1) to obtain the corresponding intermediate.

TLC (EtOAc–Py–H₂O–AcOH 8 : 5 : 3 : 1) R_f0.56; ESI MS: m/z:

calcd for C₈₈H₉₇N₄O₅₄S₇Na₁₀: 2527.2; found: 2527.2 [M +

10Na + H]⁺; calcd for C₈₈H₉₇N₄O₅₄S₇Na₈:2481.2; found:

2481.0 [M + 8Na + H]⁻.

This hexasaccharide (6.7 μmol) and sulfur trioxide–trimethy-

lamine complex (31 mg, 219 μmol) were dissolved in dry DMF

(2.0 mL) in a 5 mL microwave reaction vial under an argon

atmosphere. Et₃N (250 μL) was added and the reaction mixture

was subjected to microwave radiation for 30 min at 60 °C (20 W

average power). The reaction vessel was cooled under a stream

of nitrogen. The reaction mixture was puriﬁed by Sephadex

LH-20 chromatography (CH₂Cl₂–MeOH 1 : 1) and RP-18

chromatography (10 m^MAcOH-Et₃N (pH 7.0)/Acetonitrile

95 : 5 → 50 : 50) to give the desired tri N-sulfated hexasaccharide

as triethylammonium salt. The corresponding sodium salt was

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

MeOH–H₂O 3 : 1. TLC (EtOAc–Py–H₂O–AcOH 6 : 5 : 3 : 1) R_f

C₃₈H₅₁N₄O₆₁S₁₀Na₁₀: 696.24; found: 696.22 [M + 10Na]³⁻

.

2-Aminoethyl O-(2-deoxy-2-sulfamido-6-O-sulfo-α-D-

glucopyranosyl)-(1 → 4)-O-(2-O-sulfo-α-^L-idopyranosyluronic

acid)-(1 → 4)-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-^D-

glucopyranosyl)-(1 → 4)-O-(2-O-sulfo-α-^L-idopyranosyluronic

acid)-(1 → 4)-O-(2-deoxy-2-sulfamido-6-O-sulfo-α-^D-

glucopyranosyl)-(1 → 4)-2-O-sulfo-α-^L-idopyranosiduronic acid

(24)

1

0.21; H-NMR (500 MHz, MeOD): δ 7.59–7.06 (m, 35H, Ar),

6.05 (bs, 1H, H-1E), 6.01 (bs, 1H, H-1C), 5.35–5.30 (m, 6H,

H-1A, H-1B, H-1D, H-1F, CH₂(OBn)), 5.05 (m, 2H, CH₂(Z)),

4.96–4.80 (m, 6H, H-5E (4.92), H-5C (4.81), CH₂(OBn)),

4.77–4.42 (m, 14H, H-5A (4.69), H-2E (4.67), H-2C (4.64),

H₂O₂(30%, 0.64 mL) and a solution of LiOH (0.7 M, 0.38 mL)

were added at −5 °C to a solution of 20 (36 mg, 15 μmol) in

THF (1.7 mL). After stirring for 20 h at room temperature,

MeOH (1.7 mL) and a solution of NaOH (4 ^M, 0.39 mL) were

Org. Biomol. Chem., 2012, 10, 2146–2163 | 2161

View Article Online

added. After stirring for 20–24 h at room temperature, the reac-

tion mixture was neutralized with a 4 ^Msolution of HCl and then

diluted with CH₂Cl₂(20 mL) and washed with H₂O (10 mL).

The organic phase was extracted with a solution of Na₂SO₃

(10%), dried (MgSO₄), and concentrated to give the desired

saponiﬁed intermediate. ESI MS: m/z: calcd for

C₉₅H₁₀₂N₁₀O₃₃Na₃: 1979.6; found: 1979.5 [M + 3Na − 4H]⁻.

This compound (15 μmol) and sulfur trioxide–trimethylamine

complex (64 mg, 462 μmol) were dissolved in dry DMF

(2.0 mL) and heated for 30 min at 100 °C using microwaves as

described above. The reaction vessel was cooled and Et₃N

(150 μL), MeOH (1 mL) and CH₂Cl₂(1 mL) were added. The

solution was layered on the top of a Sephadex LH-20 chromato-

graphy column which was eluted with CH₂Cl₂–MeOH (1 : 1) to

give the desired hexa O-sulfated hexasaccharide as triethylam-

monium salt. The corresponding sodium salt was obtained by

elution from a column of Dowex 50WX4-Na⁺with MeOH–H₂O

3 : 1 (30 mg, 75% from 20, 2 steps). TLC (EtOAc–Py–

H₂O–AcOH 8 : 5 : 3 : 1) R_f0.24;¹H-NMR (500 MHz, MeOD): δ

7.42–7.16 (m, 40H, Ar), 5.93 (bs, 1H, H-1E), 5.82 (bs, 1H,

H-1C), 5.23 − 5.10 (m, 4H, H-1F (5.21), H-1B or H-1D, H-1A

(5.16), CH₂(OBn)), 5.09—4.88 (m, 9H, H-1B or H-1D, CH₂(Z),

CH₂(OBn)), 4.84 − 4.50 (m, 13H, H-5E, H-5C, H-2E (4.70),

H-2C (4.65), H-5A (4.61), H-2A (4.52), CH₂(OBn)), 4.41–3.98

(m, 16H, H-3E (4.39), H-6aB, H-6aD, H-6aF, H-3C (4.33),

H-6bB, H-6bD, H-6bF, H-3A (4.27), H-4C, H-4E (4.11), H-4A

(4.09), H-4B, H-4D, H-5B, H-5D), 3.89 (m, 1H, H-3F),

3.81–3.46 (m, 9H, H-3B, H-3D, CH₂-O, H-2F (3.65), H-5F

(3.64), H-4F (3.63), H-2B, H-2D), 3.36 (m, 2H, CH₂-

N);¹³C-NMR (125 MHz, MeOD) (Signiﬁcant data from HSQC

experiment): δ 100.6 (C-1A), 95.4 (C-1C), 94.9 (C-1E), 94.7,

93.8 (C-1B, C-1D), 93.7 (C-1F), 82.5 (C-3F), 79.4 (C-4F), 78.8,

78.7 (C-3B, C-3D), 76.0, 75.9, 75.6, 74.8 (CH₂(OBn)), 72.6

(C-4A), 72.5, 72.4 (CH₂(OBn)), 71.3 (C-2C), 71.2 (C-2A), 71.1

(C-3A), 70.8–69.5 (C-4E, C-5F, C-2E, C-3E, C-3C, C-4C,

C-4B, C-4D, C-5B, C-5D), 69.1 (C-5C, C-5E), 68.2 (C-5A),

67.6 (CH₂-O), 67.2 (CH₂(Z), 66.7 (C-6B, C-6D, C-6F), 65.7

(C-2F), 65.2 (C-2B, C-2D), 41.3 (CH₂-N); ESI MS: m/z: calcd

Dowex 50WX4-Na⁺with MeOH–H₂O 3 : 1. TLC (EtOAc–Py–

H₂O–AcOH 26 : 25:15 : 5) R_f0.26.

A solution of this compound (5.8 μmol, as sodium salt) in

H₂O–MeOH (1.35 mL/0.15 mL) was hydrogenated in the pres-

ence of Pd(OH)₂. After 24 h, the suspension was ﬁltered over

Celite and concentrated. The residue was puriﬁed by Sephadex

G-25 chromatography (H₂O/MeOH 9 : 1) to give 24 after lyophi-

lisation (2.0 mg, 17% from the O-sulfated intermediate, 3 steps;

1

13% from 20, 5 steps, 66% average yield per step). H-NMR

(500 MHz, D₂O): δ 5.48 (bd, 1H, H-1B or H-1D), 5.44 (bd, 1H,

H-1F), 5.39 (bs, 1H, H-1B or H-1D), 5.34 (bs, 1H, H-1C or

H-1E), 5.25 (bs, 1H, H-1C or H-1E), 5.11 (bd, 1H, H-1A),

4.90–4.83 (m, 2H, H-5E, H-5C), 4.55 (bd, 1H, H-5A),

4.46–3.99 (m, 19H, H-6aB, H-6aD, H-6aF, H-6bB, H-6bD,

H-6bF, H-2E, H-2C, H-2A (4.32), H-3A, H-3C, H-3E, H-4A

(4.15), H-4C, H-4E, H-5B, H-5D, CH₂-O (4.03), H-5F (4.01)),

3.87–3.77 (m, 3H, H-4B, H-4D, CH₂-O (3.82)), 3.75–3.63 (m,

3H, H-3B, H-3D, H-3F (3.66)), 3.58 (t, 1H, J_3,4= J_4,5= 9.6 Hz,

H-4F), 3.33–3.18 (m, 5H, H-2B, H-2D, H-2F, CH₂-N

(3.24));¹³C-NMR (125 MHz, D₂O) (Signiﬁcant data from HSQC

experiment): δ 99.5 (C-1A), 99.2, 98.9 (C-1C, C-1E), 96.9

(C-1B or C-1D), 96.7 (C-1F), 95.9 (C-1B or C-1D), 77.7

(C-2A), 76.1 (C-4B or C-4D), 75.9 (C-4B or C-4D, C-4A,

C-4C, C-4E, C-2C or C-2E), 75.2 (C-2C or C-2E), 70.9 (C-3F),

69.9 (C-3A, C-5A), 69.7 (C-5F), 69.5 (C-5C, C-5E, C-3B,

C-3D), 69.2 (C-4F), 69.1 (C-5B, C-5D), 68.7, 68.5 (C-3C,

C-3E), 66.4, 66.3 (C-6B, C-6D, C-6F), 64.5 (CH₂-O), 57.8

(C-2B, C-2D, C-2F), 39.3 (CH₂-N). ESI MS: m/z: calcd for

C₃₈H₅₂N₄O₅₈S₉Na₉: 662.26; found: 662.24 [M + 9Na]³⁻; calcd

for C₃₈H₅₃N₄O₅₈S₉Na₈: 654.94; found: 654.92 [M + 8Na + H]³⁻

calcd for C₃₈H₅₄N₄O₅₈S₉Na₇: 647.61; found: 647.59 [M + 7Na

+ 2H]³⁻

;

.

Acknowledgements

We thank the CSIC (Grant 201180E021), the Spanish Ministry

of Science and Innovation (Grant CTQ2009-07168), Junta de

Andalucía (Grant P07-FQM-02969, and “Incentivo a Proyecto

Internacional”) and the European Union (FEDER support and

Marie Curie Reintegration Grant) for ﬁnancial support. J. A.

acknowledges ﬁnancial support from the MICINN through the

Ramón y Cajal program.

for C₉₅H₉₇N₁₀O₅₁S₆Na₇: 1273.1; found: 1272.1 [M + 7Na]²⁻

.

This O-sulfated hexasaccharide (15 mg, 5.8 μmol) was dis-

solved in THF (2.0 mL) and treated with a 0.1 ^Maqueous sol-

ution of NaOH (0.77 mL). Then, a solution of Me₃P in THF

(70 μL of a 1 ^Msolution) was added and the reaction was stirred

for 6 h. The reaction mixture was neutralized with a 0.1 ^Msol-

ution of HCl and concentrated to obtain the desired amino con-

taining compound. TLC (EtOAc–Py–H₂O–AcOH 10 : 5 : 3 : 1)

R_f0.34; ESI MS: m/z: calcd for C₉₅H₁₀₃N₄O₅₁S₆Na₈: 2491.3;

found: 2491.1 [M + 8Na]⁻; calcd for C₉₅H₁₀₃N₄O₅₁S₆Na₇:

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Article Doi

Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate-protein interactions in microtiter plates

DOI: 10.1039/c2ob06607f

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Full text of DOI:10.1039/c2ob06607f

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