Sensitive and specific enzyme immunoassays for antigenic trisaccharide from Bacillus anthracis spores

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Sensitive and speciﬁc enzyme immunoassays for antigenic trisaccharide from

Bacillus anthracis spores

Sandrine G. Y. Dhe´nin,^aVincent Moreau,*^aMarie-Claire Nevers,^bChristophe Cre´minon^band

Florence Djeda¨ıni-Pilard*^a

Received 20th July 2009, Accepted 16th September 2009

First published as an Advance Article on the web 19th October 2009

DOI: 10.1039/b914534f

A straightforward synthesis of an anthrose-containing trisaccharide derived from Bacillus anthracis was

achieved. Antibodies raised against this hapten provide a highly sensitive enzyme immunoassay with a

detection limit of 8.5 pmol mL^-1. By investigating the speciﬁcity of the antibodies obtained using

different mono-, di- and trisaccharide synthetic analogues, we demonstrated that the epitope was

mainly made up of the methyl group at C-5, the butamido group at C-4 and the hydroxyl at C-3 of the

anthrose unit, the other parts of the trisaccharide appearing little involved in the recognition.

by antibodies elicited by B. anthracis spores.^3b,4bMoreover, it

Introduction

has been shown that monoclonal antibodies generated from

Bacillus anthracis, a spore-forming Gram positive bacteria, is the

mice immunised with tetrasaccharide 1-KLH conjugates are able

to bind speciﬁcally to B. anthracis spores.^4aRecently, we have

causative agent of anthrax. The immunodominant glycoprotein

BclA, which covers the surface of the spores, bears a tetrasac-

charide 1 containing a unique terminal sugar at the non-reducing

end, the so-called anthrose (Fig. 1). Since its structure has been

reported^3cthe synthesis of a derivative of anthrose 2: when

attached to a carrier protein, this very small hapten induced a

speciﬁc and strong immune response in rabbits.

elucidated,¹and the suggestion that this characteristic antigen

The main goal of our research is the production of antibodies

may be an interesting target for the development of a vaccine or

directed against fragments of tetrasaccharide 1 and their use in the

antibody-based detection system, several studies have reported the

development of an antibody-based detection system. In this com-

synthesis²of this sugar or of one of its related fragments³and/or

petitive context, we wish to design the minimal hapten to be both

their use in developing immunological tools.^3b,4

It has been clearly demonstrated that the synthetic tetrasac-

charide 1 and its related trisaccharide fragment are recognised

easily synthesised and allowing the production of a strong speciﬁc

and sensitive immune response for further detecting anthrax

spores. We report here the synthesis of an anthrose-containing

trisaccharide 29-KLH glycoconjugate to produce speciﬁc rabbit

^aLaboratoire des Glucides, UMR-CNRS 6219, Institut de Chimie de

Picardie, Universite´ de Picardie Jules Verne, 33 rue St Leu, F-80039, Amiens,

France; Fax: +33 03 22 82 75 62; Tel: +33 03 22 82 75 62. E-mail:

ﬂorence.pilard@u-picardie.fr, vincent.moreau@u-picardie.fr

polyclonal antibodies. A structure-activity relationship study was

performed using several mono-, di- and trisaccharide synthetic

analogues to check the speciﬁcity and sensitivity of the assay and

to characterise the optimum synthetic target via a competitive

enzyme immunoassay (EIA).

^bCEA, iBiTecS, SPI, Laboratoire de Recherche en Immunoanalyse, F-91191,

Gif-sur-Yvette, France

Fig. 1 Structures of the Bacillus anthracis antigenic tetrasaccharide 1, the trisaccharide 29 used for immunization and antibody production after ligation

to a protein carrier, and analogues 2,^3c27, 28, 37, 41, 50 and 55 used for immunoassay experiments (A, B, C letters located below pyranosidic rings refer

to each unit and are used to indicate their associated protons and carbons in the NMR data).

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appeared to be more regioselective than the traditional Garegg-

Samuelsson procedure⁸but failed to give a high yield on a large

scale. We thought that protection at O-4 would allow use of

the Garegg-Samuelsson procedure. We thus chose to proceed via

a reductive ring opening of the 4,6-O-benzylidene derivative 6.

As expected, use of BH₃.THF and dibutylboron triﬂate⁹gave

the p-methylphenyl 2-O-acetyl-3-O-benzoyl-4-O-benzyl-1-thio-b-

D-galactopyranoside 7 in 81% yield. Iodination of compound

7 using Garegg-Samuelsson’s conditions (triphenylphosphine/

imidazole/iodine in toluene at 60 ^◦C) afforded 8 in good yield

(70%) even when carried out on a large scale. Although cat-

alytic hydrogenolysis of 8 induced dehalogenation in high yield,

the benzyl group at O-4 appeared totally unreactive under all

hydrogenolysis conditions (high pressure and excess of catalyst)

attempted. Finally, using the BF₃.OEt₂-NaI reagent system,¹⁰we

were able to release the hydroxyl at O-4 while ensuring the integrity

of the tolyl group at the anomeric position. Compound 9 was thus

obtained in 77% yield in two steps from 8 without puriﬁcation of

the 4-O-benzyl-6-deoxygalactopyranoside intermediate.

Results and discussion

Even if Bacillus anthracis is not the only pathogen with potential

use for biological warfare, the development of methods allowing

rapid and speciﬁc detection and diagnosis remains of interest. In

this context, the production of speciﬁc antibodies to further design

immunomethods requires the identiﬁcation of speciﬁc immuno-

genic target(s). Steinman^4band co-workers have demonstrated that

the anthrose and anthrose-containing disaccharide are marginally

reactive while the anthrose-containing tetrasaccharide is highly

reactive against antibodies elicited by the native spores of Bacillus

anthracis. In this case, the antibody binding reactivity appears

to directly correlate with the size of the saccharides. Moreover,

Boons and co-workers have reported that the terminal 3-methyl-

butyryl function is an important antigenic component of the

immunological recognition between synthetic trisaccharide and

spore-recognising antisera.^3bIn the case of polyclonal antibodies

directed against the anthroside unit alone, we showed that the

linker used for the synthesis of glycoconjugate is not involved in

the recognition, while the C-4 butamido substituent of anthrose is

strictly necessary for antibody recognition. Finally, modiﬁcation

at C-3 of anthrose strongly decreases recognition.^3c

Subsequent reaction of 9 with triﬂic anhydride followed by

nucleophilic displacement of the resulting 4-O-triﬂate in 10 with

azide¹¹gave the expected thioglycoside 11 in 85% yield in 2 steps

(Scheme 1).

To evaluate the importance of the size of the saccharide hapten,

antibodies were raised against anthrose-containing trisaccharide

29 using the protocol previously used to produce antibodies

against anthrose derivative 2.^3c

Since the introduction of the methoxy group and reduction of

primary hydroxyl groups at C-2 and C-6, respectively, are key

steps in synthesis, the inﬂuence of these functional groups on

antibody binding was evaluated by analysing the recognition of

the derivatives 37, 41, 49 and 55 displayed in Fig. 1.

The speciﬁcity of the resulting antibodies for the rhamnose

moiety was determined using two rhamnoside analogues 31 and

33 as competitor in enzymatic immunoassays.

Scheme 1 Reagents and conditions: (a) BH₃(1 M in THF), Bu₂BOTf (1 M

in CH₂Cl₂), 0 ^◦C, 5 h, 81%; (b) PPh₃, imidazole, I₂, toluene, 60 ^◦C, 4 h,

70%; (c) DMF, Pd/C, H₂1 atm., NaHCO₃, 5 h then CH₃CN, BF₃.OEt₂,

NaI, 7 h, 77%.

Synthesis of thioglycoside 11

The reported syntheses of the tetrasaccharide 1 and parts thereof

differ only slightly except for the de novo asymmetric approach

starting from 2-acetylfuran described by Guo and O’Doherty.^2d

These former approaches differ mainly in the choice of the

starting monosaccharide, the protecting groups and the leaving

group used to prepare the intermediate glycoside donor needed

to obtain the anthrose moiety. In the most lengthy strategy,

D-mannose was used.^2cTwo required the use of rare and expensive

D-fucose.^2a,3bWe^3cand others (Crich^2eand Kovac^3d) published

almost simultaneously a synthesis starting from D-galactose which

compares well with that starting from D-fucose.

Thus, thioglycoside 11 was prepared following our previously

described strategy^3cstarting from the known p-methylphenyl

1-thio-b-D-galactopyranoside 3.⁵Reaction of 3 with benzaldehyde

dimethyl acetal provided the 4,6-O-benzylidene derivative 4.⁶

Selective benzoylation at O-3 was performed with benzoyl cyanide

in the presence of triethylamine as a base at -70 ^◦C in high yield

(89%) followed by further O-acetylation at O-2.

Synthesis of acceptor 20

Both acceptor 15 and donor 18 were obtained from the key

intermediate 14, namely p-methylphenyl 2-O-benzoyl-4-O-benzyl-

1-thio-a-L-rhamnopyranoside, which was prepared in six steps

from L-rhamnose (Scheme 2).

The known fully acetylated p-methylphenyl 1-thio-L-

rhamnopyranoside¹²12 was obtained as an a/b mixture in two

steps according to the procedure reported by Wong¹³and co-

workers in 80% yield. 12 was deacetylated by Zemple`n methanoly-

sis to give 13¹⁴which was then reacted with triethyl orthobenzoate

in the presence of camphor-10-sulfonic acid as a catalyst. The 2,3-

orthoester intermediate thus obtained was benzylated at O-4 in the

same pot. The cyclic orthoester was then opened in the presence

of aq. 2 M HCl, according to the procedure described by Field,¹⁵

affording the expected p-methylphenyl 2-O-benzoyl-4-O-benzyl-1-

thio-a-L-rhamnopyranoside 14 (76% in 3 steps) and its b analogue

(15%).

At this stage, our ﬁrst approach, involving acid-catalysed

O-debenzylidenation of 6 and regioselective iodination of the

resulting diol at the primary hydroxyl, was slightly modiﬁed. Iod-

ination in the presence of N-iodosaccharine-triphenylphosphine⁷

Glycosylation of benzyl N-(2-hydroxyethyl) of NIS/TfOH

carbamate with compound 14, without previous protection of the

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Scheme 2 Reagents and conditions: (a) MeONa (1 M in MeOH), MeOH, 15 h, 98%; (b) triethyl orthobenzoate, CSA, DMF, 3 h; (c) NaH, BnBr, 15 h;

(d) aq. 2 M HCl, 76% in 3 steps. (e) NIS, TfOH, CH₂Cl₂, benzyl N-(-2-hydroxyethyl)carbamate, 1 h 30, 58%; (f) chloroacetyl chloride, CH₂Cl₂, pyridine,

3 h, 98%; (g) NBS, acetone, 0 ^◦C to rt, 1 h 20, 91%; (h) CCl₃CN, DBU, 0 ^◦C to rt, 82%; (i) 15, TMSOTf, CH₂Cl₂, 30 min., 90%; (j) thiourea, NaHCO₃,

TBAI, THF, 55 ^◦C, 80%.

remaining hydroxyl group, was achieved in the presence to give

the acceptor 15 in 58% yield.

1,2,3-triazolo-(4,5-b)-pyridin-1-ylmethylene]-N-methylmethan-

aminium hexaﬂuorophosphate N-oxide (HATU) gave 27 in 85%

overall yield (2 steps). Finally, debenzoylation using base-catalysed

methanolysis conditions followed by catalytic hydrogenolysis of

the benzyl groups and of the carbamate group gave the target

spacer arm equipped trisaccharide derivative 29 (Scheme 3).

Donor 18 was prepared in three steps from 14. The remain-

ing hydroxy group at O-3 was ﬁrst chloroacetylated in classic

manner in high yield (91%). Cleavage of the thiotoluene group

in the presence of NBS followed by subsequent reaction with

trichloroacetonitrile under DBU catalysis gave the donor 18.

The ﬁrst attempt at glycosylation of acceptor 15 was per-

formed with the thioglycoside donor 16 using standard conditions

(NIS/TfOH). The expected disaccharide 19 was obtained in

relatively low yield (54%). On the other hand, use of the rhamnosyl

trichloroacetimidate donor 18 afforded 19 in high yield (90%, 67%

from 16 over 3 steps). Next, the chloroacetyl group was selectively

removed by treatment with thiourea giving the dirhamnosyl

acceptor 20 (80%) (Scheme 2).

Synthesis of the competitors

Speciﬁcity of the produced antibodies was evaluated using several

competitors in EIA: among them, two analogues of the rhamno-

side part 31 and 33 as well as two monosaccharides 37 and 50

and two trisaccharides 41 and 55 with a modiﬁed anthrose moiety

were prepared.

Both rhamnosyl derivatives 31 and 33 were obtained in a

deacylation step followed by a catalytic hydrogenolysis step from

15 and 19, respectively.

Synthesis of the targeted trisaccharide 29

Both competitors 37 and 41, devoid of the characteristic

methoxy group of anthrose, were prepared from the already de-

scribed N-benzyloxyaminoethyl-b-D-glucopyranoside derivative

34^3cand the trisaccharide 23, respectively (Scheme 3). Reduction

of the azide, peptide-type coupling of 3-hydroxy-3-methylbutyric

acid followed by base-catalysed methanolysis gave the expected

2-hydroxy anthrose analogues 37 and 40. Treatment of 40 by

catalytic hydrogenolysis of the benzyl groups and of the carbamate

group afforded the target trisaccharide derivative 41.

As shown by our work and previous reports, synthesis of the

anthropyranoside moiety is quite long and tedious starting from D-

galactose as well as from the expensive D-fucose. We thus decided

to study the inﬂuence of the methyl group at C-5 and the possibility

of replacing it by a hydroxymethyl group. Synthesis of competitors

50 and 55 was undertaken (Scheme 4).

Glycosylation of the disaccharide 20 with the thioglycoside donor

11 in the presence of NIS and triﬂic acid as catalyst failed to

give the expected trisaccharide. Once again, we chose to use the

trichloroacetimidate method,¹⁶thioglycoside 11 being converted

into its trichloroacetimidate analogue 22 in two steps as described

above in the case of compound 18.

Condensation of 20 with donor 22 in the presence of a catalytic

amount of TMSOTf afforded the trisaccharide 23. At this stage, we

were not able to remove all the unreacted acceptor and the next step

was performedonpartiallypuriﬁed trisaccharide 23. Selective2-O-

deacylation of 23 was performed by acid-catalysed methanolysis¹⁷

to afford 24 in 50% yield from 20. The characteristic methoxy

group of anthrose residue was then introduced by reaction of

24 with diazomethane in the presence of BF₃-Et₂O affording the

expected derivative 25 in 40% yield.

Acetolysis of the known methyl 2,3,6-tri-O-benzoyl-4-azido-

4-deoxy-a-D-glucopyranoside¹⁹42 and selective removal of the

resulting 1-O-acetyl derivative 43 by treatment with hydrazine

acetate gave glucopyranose derivative 44²⁰in 67% yield in 2 steps.

Subsequent reaction of 44 with trichloroacetonitrile under DBU

catalysis gave the donor 45.

Next, reduction of the azide in 25 was achieved with sodium

borohydride in the presence of nickel chloride to ensure integrity

of the carbamate group as previously described by Alpe and

Oscarson,¹⁸affording amine 26. Treatment of 26 with 3-hydroxy-

3-methylbutyric acid in the presence of N-[(dimethylamino)-1H-

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Scheme 3 Reagents and conditions: (a) NBS, acetone, 0 ^◦C to rt, 2 h 20 min, 90%; (b) CCl₃CN, DBU, 0 ^◦C, 2 h, 84%; (c) TMSOTf, CH₂Cl₂, 45 min; (d)

CH₃COCl, MeOH, CH₂Cl₂, 0 ^◦C to rt, 6 days, 50% in two steps; (e) CH₂N₂, BF₃OEt₂, CH₂Cl₂, 0 ^◦C, 40%; (f) NaBH₄, NiCl₂.6H₂O, EtOH, CH₂Cl₂; (g)

HO(CH₃)₂CCH₂CO₂H, HATU, DIPEA, CH₂Cl₂, 18 h, 85% (27), 62% (36), 53% (39) in two steps; (h) MeONa (1 M in MeOH), MeOH, 15 h, 76% (37);

(i) Pd(OH)₂, AcOH, H₂, tBuOH, 5 h to 20 h, 40% (29), 50% (41) in two steps.

Scheme 4 Reagents and conditions: (a) Ac₂O, AcOH, H₂SO₄, 50 ^◦C, 24 h, 74%; (b) hydrazine acetate, DMF, 4 h, 91%; (c) CCl₃CN, DBU, 0 ^◦C, 1 h, 87%;

(d) 20, TMSOTf, CH₂Cl₂, 1 h; (e) TMSOTf, CH₂Cl₂, benzyl N-(-2-hydroxyethyl)carbamate, 0 ^◦C, 1 h, 73%; (f) NaBH₄, NiCl₂.6H₂O, EtOH, CH₂Cl₂; (g)

HO(CH₃)₂CCH₂CO₂H, HATU, DIPEA, CH₂Cl₂, 18 h and 3 days respectively, 60% (48), 47% (53) in two steps; (h) MeONa (1 M in MeOH), MeOH,

18 h; (i) Pd(OH)₂, AcOH, H₂, tBuOH, 20 h, 72% (50), 44% (55) in two steps.

Then, condensation of benzyl N-(2-hydroxyethyl)carbamate or

dirhamnosyl acceptor 20 with 45 was performed in the presence of

a catalytic amount of TMSOTf to afford the expected b-linked gly-

coside 46 and the trisaccharide 51, respectively. Reduction of the

azide, peptide-type coupling of 3-hydroxy-3-methylbutyric acid,

base-catalysed methanolysis and ﬁnally catalytic hydrogenolysis

of the benzyl groups and of the carbamate group afforded the

target competitors 50 and 55.

rabbits were immunised and given booster injections every two

months with 29-KLH conjugate. Antibody titre was measured

by EIA using compound 29 labeled with acetylcholinesterase

(AChE) as tracer. The latter was prepared in two steps by

ﬁrst deriving the trisaccharide 29 via reaction with S-acetyl

thioglycolic acid N-succinimidyl ester before covalently coupling

the introduced thiol moiety to AChE through a succinimidyl 4-

(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

This procedure ensures full immunoreactivity of the enzymatic

tracer 29-AChE and avoids the possible involvement of the spacer

arm in the recognition by antibodies. A titre above 1/1,000,000

was measured from the ﬁrst booster injection onwards, showing

the strong immunogenicity of the 29-KLH immunogen (Fig. 2).

Using the 29-AChE tracer, we determined the optimal dilution

of antisera for the assay and plotted standard curves using 29

as reference. Antibodies raising against 29 provided a detection

Synthesis of a trisaccharide-KLH conjugate and investigation of

the immune response

The trisaccharide-KLH conjugate was prepared according to

our previously described procedure^3cusing glutaraldehyde to

ensure coupling between the amino groups of the protein car-

rier and the trisaccharide 29. To induce antibody production,

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Fig. 2 Structure of 29-KLH immunogen and of 29-AChE tracer.

Table 1 Relative cross-reactivity observed with 2, 27, 28, 31, 33, 37, 41,

50, and 55 taking 29 as reference and using the glycoconjugate 29-AchE

as tracer

and 50, 2 exhibited an almost good cross-reactivity, revealing that

this part of the immunogen is critical for the antibody recognition.

On the other hand, the better recognition of 29 in comparison

with 2 could be explained by a size effect. It is also noteworthy

that the lack of the C2 methyl only slightly modiﬁed the binding

(37 vs 2), while the addition of a hydroxyl group to C6 strongly

decreased recognition (50 vs 37). Considering the different trisac-

charides analysed, although the absence of recognition of the fully

protected trisaccharide 27 was expected, the partially deprotected

derivative 28, exposing the anthrose residue and the C2 hydroxyl

functions of the rhamnose, presented almost the same reactivity as

compound 29.

competitor

IC50 (ng mL^-1)^a

IC50 (pmol mL^-1)^a

CR (%)^b

29

31

33

37

41

50

55

27

28

2

5.2

—

37.2

1.9

2600

128

—

8.48

—

84.45

3.17

8065

208.25

—

100

—

9.5

260

0.1

3.9

—

9.8

15.9

10.57

49.62

80

16

Next, we evaluated what kind of modiﬁcation of the an-

throse unit itself could be tolerated leading to a potentially more

accessible synthesis of hapten. We^3cand others^3bhave already

observed that the C-4 butamido substituent of anthrose is strictly

necessary for antibody recognition. Moreover, we know that the

latter is dramatically decreased by a modiﬁcation at C-3.^3cData

published by Boons^3bindicate that the methyl ether at C-2 is

not critical for the antispore antibody binding. The full cross-

reactivity exhibited by the trisaccharide 41 lacking a methoxy

group at C-2 (3.2 pmol mL^-1vs 8.5 pmol mL^-1for 29) corroborates

this observation in the case of antibodies elicited by the synthetic

29-KLH glycoconjugate. On the other hand, the poor recognition

of 50 and 55 bearing a hydroxy group at C-6 indicates that

the 6-deoxy feature of the anthrose residue has to be con-

served to preserve the immunogenic potential of future synthetic

analogues.

Our results showed that the presence of speciﬁc substituents at

positions C-3, C-4 and C-6 of the anthrose unit is dramatic for

antibody recognition and for the immune response. On the other

hand, the methyl ether at C-2 seems not to be necessary. Finally,

while the overall size of the trisaccharide appears to play a role

in the recognition phenomenon, the rhamnoside part is poorly

immunogenic.

^aIC50: concentration of the competitor required to inhibit 50%

of the 29-AchE tracer/antibody binding. ^bCross reactivity (CR) =

IC50(29)/IC50(competitor) with IC50 in pmol mL^-1.

limit of 8.5 pmol mL^-1, similar to the previous assay developed for

monosaccharide 2 (8.7 pmol mL^-1).^3cHowever, the higher immune

titre seems to indicate a stronger immunogenic potency for the

29-KLH immunogen.

Taking compound 29 as reference, different standard curves

were plotted using analogues 31, 33, 37, 41, 50, 55 and two

intermediates of the synthesis (27 and 28) as competitor to better

analyse the speciﬁcity of the antibodies. The previously synthesised

hapten 2 was also evaluated (Fig. 1).^3c

The results are summarised in Table 1. Antigen 29 presented an

IC50 value (concentration of the competitor required to inhibit

50% of the tracer/antibody binding) of 5.2 ng mL^-1.

As already observed by Steinman,^4bthe rhamnoside derivatives

31 and 33 were not able to bind the antibodies, demonstrating

that the rhamnoside part was only negligibly involved in the

recognition by the antibodies. By comparing the IC50 obtained

with the different monomeric anthrose derivatives, namely 2, 37

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specialised microtitration equipment a washer (ELX 405, Bio-Tek

Instruments) using automatic plate washer and automatic plate-

reader (Multiskan, Thermo Life Sciences).

Experimental section

General remarks

All reactions were monitored by TLC on Kieselgel 60 F254 (E.

Merck). Detection was achieved by charring with vanillin. Silica

gel (E. Merck, 240-400 mesh) was used for chromatography.

Solutions were concentrated under reduced pressure. Optical

rotation was measured with a JASCO DIP-370 digital polarimeter,

using a sodium lamp (l = 589 nm) at 20 ^◦C. All NMR experiments

were performed at 300.13 and 500.13 MHz using Bruker DMX300

and DRX500 spectrometers equipped with a Z-gradient unit for

pulsed-ﬁeld gradient spectroscopy. Assignments were performed

by stepwise identiﬁcation using COSY, successive RELAY, HSQC

and HMBC experiments using standard pulse programs from

the Brucker library. Chemical shifts are given relative to external

TMS with calibration involving the residual solvent signals. When

D₂O was used, TMS was used as internal standard reference in

a previous ¹³C NMR experiment performed in the same experi-

mental conditions. The length of the 90^◦pulse was approximately

7 ms (¹H NMR) and 10 ms (¹³C NMR). 1D NMR data spectra

were collected using 16 K data points. 2D experiments were

run using 1K data points and 512 time increments. The phase-

sensitive (TTPI) sequence was used and processing resulted in a

1K*1K (real-real) matrix. A 45^◦ﬂip angle (3.5 ms) and a total

recovery time of 5 s were used to ensure complete relaxation of the

protons and quantitative measurements. The digital integration of

the transformed spectra was performed after polynomial baseline

correction.

Low-resolution ESI mass spectra were obtained on a hybrid

quadrupole/time-of-ﬂight (Q-TOF) instrument, equipped with

a pneumatically assisted electrospray (Z-spray) ion source (Mi-

cromass). High-resolution mass spectra were recorded in positive

mode on a ZabSpec TOF (Micromass, UK) tandem hydrid mass

spectrometer with EBETOF geometry. The compounds were

individually dissolved in 1:1 water-MeCN at a concentration of

10 mg cm^-3and then infused into_◦the electrospray ion source at a

ﬂow rate of 10 mm³min^-1at 60 C. The mass spectrometer was

operated at 4 kV whilst scanning the magnet at a typical range

of 4000-100 Da. The mass spectra were collected as continuum

proﬁle data. Accurate mass measurement was achieved using

polyethylene glycol as internal reference with a resolving power

set to a minimum of 10 000 (10% valley).

p-Methylphenyl 2-O-acetyl-3-O-benzoyl-4-O-benzyl-1-thio-

b-D-galactopyranoside (7)

p-Methylphenyl

2-O-acetyl-3-O-benzoyl-4,6-O-benzylidene-1-

thio-b-D-galactopyranoside (6)^3c(5.34 g, 10.3 mmol) was

dissolved in a solution of BH₃(1 M in THF, 30 mL) and cooled at

0 ^◦C. A solution of dibutylboron triﬂate (1 M in CH₂Cl₂, 10 mL)

was added and the reaction mixture stirred for 5 h. Methanol

(around 1 mL with care until the gaseous emission ceased) was

added. The solvents were removed under reduced pressure. The

residue was dissolved in CH₂Cl₂and washed with water. The

organic layer was dried over Na₂SO₄, ﬁltered and concentrated.

The crude product was puriﬁed by ﬂash chromatography (7.5:2.5

cyclohexane-EtOAc) to give 7 (4.37 g, 81%), [a]_D+34 (c 0.72,

CHCl₃); d_H(300 MHz, CDCl₃) 8.04-7.13 (m, 14H, Ar), 5.65

(t, 1H, J_1,2= J_2,310 Hz, H-2), 5.24 (dd, 1H, J_3,43 Hz, H-3),

4.76 (d, 1 H, H-1), 4.75 (d, 1H, J 11.5 Hz, OCH₂Ar), 4.50 (d, 1

H, OCH₂Ar), 4.14 (d, 1H, H-4), 3.95-3.86 (m, 1H, H-6a), 3.73

(t, 1H, J_5,65.5 Hz, H-5), 3.66-3.57 (m, 1H, H-6b), 2.37 (s, 3H,

SC₆H₄CH₃), 2.04 (s, 3H, CH₃CO); d_C(75 MHz, CDCl₃) 169.4

(CH₃CO), 165.8 (OCOAr), 137.9-127.5 (Ar), 86.5 (C-1), 78.8

(C-5), 75.9 (C-3), 74.5 (OCH₂Ar), 73.6 (C-4), 67.8 (C-2), 61.8

(C-6), 21.1 (SC₆H₄CH₃), 20.8 (CH₃CO); HRMS (ES⁺) Calcd. for

C₂₉H₃₀NaO₇S (MNa⁺) 545.1610. Found 545.1614.

p-Methylphenyl 2-O-acetyl-3-O-benzoyl-4-O-benzyl-6-deoxy-6-

iodo-1-thio-b-D-galactopyranoside (8)

To compound 7 (4.37 g, 8.37 mmol) in toluene (200 mL) was

added successively at room temperature triphenylphosphine (3.3 g,

12.6 mmol), imidazole (870 mg, 12.8 mmol) and iodine (3.17 mg,

12.5 mmol). The reaction mixture was stirred at 60 ^◦C for 4 h.

The reaction mixture was cooled, saturated aq. NaHCO₃(70 mL)

was added and the mixture was stirred for 5 min. Iodine was

added in portions until the red color persists. Excess iodine was

removed by the addition of saturated aq Na₂S₂O₃(15 mL). The

mixture was diluted with ethyl acetate. Then the organic layer was

separated and extracted with brine, dried over Na₂SO₄, ﬁltered and

concentrated. The residue was puriﬁed by ﬂash chromatography

(9:1 cyclohexane-EtOAc) to give 8 (3.71 g, 70%), [a]_D+49 (c 1.6,

CHCl₃); d_H(300 MHz, CDCl₃) 8.09-7.22 (m, 14H, Ar), 5.60 (t, 1H,

J_1,2= J_2,310.0 Hz, H-2), 5.27 (dd, H, J_3,43 Hz, H-3), 4.85 (d, 1H,

J 11.5 Hz, OCH₂Ar), 4.77 (d, 1H, H-1), 4.60 (d, 1H, OCH₂Ar),

4.41 (d, 1H, H-4), 3.86 (t, 1H, J_5,67 Hz, H-5), 3.42-3.29 (m, 2H,

H-6a, H-6b), 2.45, (s, 3H, SC₆H₄CH₃), 2.05 (s, 3H, CH₃CO); d_C

(75 MHz, CDCl₃) 169.4 (CH₃CO), 165.7 (OCOAr), 138.1-127.7

(Ar), 86.6 (C-1), 79.0 (C-5), 75.8 (C-3), 75.2 (OCH₂Ar), 74.50 (C-

4), 67.3 (C-2), 21.1 (SC₆H₄CH₃), 20.7 (CH₃CO), 1.6 (C-6); HRMS

(ES⁺) Calcd. for C₂₉H₂₉INaO₆S (MNa⁺) 655.0627. Found 655.064.

Elemental analyses were performed at the Service de Microanal-

yse of the Universite´ de Champagne-Ardennes in Reims, France.

The samples were previously dried under reduced pressure for

one week.

Acetylcholinesterase (AChE, EC 3.1.1.7) from the electric

organs of the electric eel Electrophorus electricus, was puriﬁed

by afﬁnity chromatography as reported.²¹The modiﬁed Ellman’s

reagent was a solution of acetylthiocholine iodide (enzyme

substrate) and 5,5-dithiobis-2-nitrobenzoic acid (chromogen) in

phosphate buffer (pH 7.4). All reagents used for the EIA were

diluted in the following buffer (EIA buffer): 0.1 M potassium

phosphate (pH 7.4) containing 0.15 M NaCl, 0.1% bovine serum

albumin (BSA) and 0.01% sodium azide. The washing buffer was

10 mM phosphate (pH 7.4) containing 0.05% Tween 20. Solid

phase EIA was performed in 96-well microtitre plates (immuno-

plate Maxisorb with certiﬁcate, Nunc, Roskilde Denmark) with as

p-Methylphenyl 2-O-acetyl-3-O-benzoyl-1-thio-b-

D-fucopyranoside (9)

To compound 8 (1.6 g, 2.53 mmol) in dry DMF (80 mL) was added

20% palladium-on-charcoal catalyst (650 mg), and NaHCO₃

(688 mg, 8.19 mmol). The reaction mixture was stirred for 5 h

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in a hydrogen atmosphere. The catalyst was ﬁltered on celite

521 and the ﬁltrate was concentrated. The residue was dissolved

in ethyl acetate. The organic layer was washed with water and

brine, dried over Na₂SO₄, ﬁltered and concentrated. The residue

was dissolved in CH₃CN (10 mL) and NaI (3.79 g, 25.3 mmol)

and BF₃.OEt₂(3.2 mL, 25.3 mmol) were added. The reaction

mixture was stirred at rt for 7 h, then diluted with CH₂Cl₂.

The organic solution was washed with aq. Na₂S₂O₃and brine,

dried over Na₂SO₄, ﬁltered and concentrated. Puriﬁcation by ﬂash

chromatography (4:1 cyclohexane-EtOAc) afford 9 (810 mg, 77%)

as a white powder, [a]_D+25 (c 0.15, CHCl₃); d_H(300 MHz, CDCl₃)

8.03 (d, 2H, J 8 Hz, SC₆H₄CH₃), 7.60-7.41 (m, 5H, OCOAr), 7.16

(d, 2H, SC₆H₄CH₃), 5.44 (t, 1H, J_1,2= J_2,310 Hz, H-2), 5.15 (dd,

1H, J_3,43 Hz, H-3), 4.70 (d, 1H, H-1), 4.06 (broad s, 1H, H-4),

3.84 (q, 1H, J_5,66.5 Hz, H-5), 2.36 (s, 3H, SC₆H₄CH₃), 2.02 (s, 3H,

CH₃CO), 1.39 (d, 3H, H-6); d_C(75 MHz, CDCl₃) 169.5 (CH₃CO),

165.8 (OCOAr), 139.0-128.0 (Ar), 86.3 (C-1), 75.9 (C-3), 74.6 (C-

5), 70.0 (C-4), 67.3 (C-2), 21.1 (SC₆H₄CH₃), 20.8 (CH₃CO), 16.4

(C-6); HRMS (ES⁺) Calcd. for C₂₂H₂₄O₆Na³²S (MNa⁺) 439.1191.

Found 439.1211. Found: C, 63.08; H, 5.92. C₂₂H₂₄O₆S requires C,

63.44; H, 5.81%.

a syrup from which was separated by ﬂash chromatography (4:1

cyclohexane-EtOAc) the desired compound 14 (3.92 g, 76%) and

its b-analogue (0.59 g, 15%). Data for 14: [a]_D-23 (c 1.15, CHCl₃);

d_H(300 MHz, CDCl₃) 8.23 (d, 2H, J 8.2 Hz, SC₆H₄CH₃), 7.67-

7.38 (m, 10 H, Ar), 7.22 (d, 2H, SC₆H₄CH₃), 5.83 (dd, 1H, J_1,2

1.3 Hz, J_2,33.2 Hz, H-2), 5.71 (d, 1H, H-1), 5.11 (d, 1H, J 11.2 Hz,

OCH₂C₆H₅), 4.91 (d, 1H, OCH₂C₆H₅), 4.51 (dd, 1H, J_4,59.3 Hz,

J_5,66.2 Hz, H-5), 4.43-4.39 (m, 1H, H-3), 3.79 (t, 1H, J_3,4= J_4,5

9.3 Hz, H-4), 3.49 (d, 1H, J 4.7 Hz, OH), 2.42 (s, 3H, SC₆H₄CH₃),

1.58 (d, 3H, H-6); d_C(75 MHz, CDCl₃) 165.9 (OCOAr), 137.9-

127.5 (Ar), 85.9 (C-1), 81.1 (C-4), 74.7 (C-2, OCH₂Ar), 70.6 (C-3),

68.4 (C-5), 20.7 (SC₆H₄CH₃), 17.7 (C-6); HRMS (ES⁺) Calcd.

for C₂₇H₂₈NaO₅³²S (MNa⁺) 487.1555. Found 487.1548. Found: C,

69.22; H, 6.03. C₂₇H₂₈O₅S requires C, 69.80; H, 6.07%.

N-Benzyloxycarbonylaminoethyl 2-O-benzoyl-4-O-benzyl-a-

L-rhamnopyranoside (15)

A

solution of 14 (2 g, 4.31 mmol) and benzyl N-(2-

hydroxyethyl)carbamate²²(6.72 g, 34.48 mmol) in dry CH₂Cl₂

◦

˚

(130 mL) containing molecular sieves 4 A (1 g) was cooled to 0 C.

N-Iodosuccinimide (1.94 g, 8.62 mmol) and triﬂuoromethane-

sulfonic acid (765 mL, 8.62 mmol) were added. The reaction

mixture was stirred at room temperature for 1 h 30 min, then

quenched by addition of Et₃N. The molecular sieves were removed

by ﬁltration. The organic layer was washed with saturated aq

Na₂S₂O₃, water, dried over Na₂SO₄, ﬁltered and concentrated. The

residue was puriﬁed by ﬂash chromatography (4:1 cyclohexane-

EtOAc) to give 15 (1.34 g, 58%) as an oil, [a]_D-11 (c 0.18,

CHCl₃); d_H(300 MHz, CDCl₃) 8.11-7.33 (m, 10H, Ar), 5.39

(dd, 1H, J_1,21.6 Hz, J_2,33.4 Hz, H-2), 5.28 (t, 1H, J 4.9 Hz,

NH), 5.15 (broad s, 2H, NHCOOCH₂Ar), 4.93-4.89 (m, 2H,

OCH₂Ar, H-1), 4.78 (d, 1H, J 11.1 Hz, OCH₂Ar), 4.25 (dd, 1H,

J_3,49.4 Hz, H-3), 3.87-3.72 (m, 2H, CH₂CH₂NH, H-5), 3.57-

3.49 (m, 2H, CH₂CH₂NH, H-4), 3.46-3.42 (m, 2H, CH₂CH₂NH),

1.42 (d, 3H, J_5,66.2 Hz, H-6); ¹³C NMR: d_C(75 MHz, CDCl₃)

166.2 (OCOAr), 156.3 (NHCO), 138.0-127.9 (Ar), 97.5 (C-1), 81.4

(C-4), 75.2 (OCH₂Ar), 73.1 (C-2), 70.3 (C-3), 67.7 (C-5), 66.9

(CH₂CH₂NH), 66.8 (NHCOOCH₂Ar), 40.8 (CH₂CH₂NH), 18.1

(C-6); HRMS (ES⁺) Calcd. for C₃₀H₃₃NNaO₈(MNa⁺) 558.2104.

Found 558.2107. Found: C, 67.02; H, 6.21; N, 2.51. C₃₀H₃₃NO₈

requires C, 67.28; H, 6.21; N 2.62%.

p-Methylphenyl 1-thio-L-rhamnopyranoside (13)

To a suspension of p-methylphenyl 2,3,4-tri-O-acetyl-1-thio-L-

rhamnopyranoside 12¹²(20.82 g, 52.58 mmol) in methanol

(300 mL) was added dropwise at 0 ^◦C a solution of sodium

methoxide (26 mL, 1 M in MeOH). The reaction mixture was

stirred at room temperature for 15 h. An acidic resin (Amberlite

IR 120 H⁺) was added to neutralise MeONa. The resin was ﬁltered

and the solvent removed to afford derivative 13 (13.90 g, 98%) as

a white powder, d_H(300 MHz, CDCl₃) 7.31 (d, 2H, J 8.1 Hz,

SC₆H₄CH₃), 7.03 (d, 2H, SC₆H₄CH₃), 5.47 (broad s, 1H, J_1a,2a

<

0.5 Hz, H-1a), 4.78 (broad s, 1H, J_1b,2b< 0.5 Hz, H-1b), 4.28-

4.27 (m, 2H, H-2a, H-2b), 4.26-4.21 (m, 1H, H-5a), 3.87 (dd,

2H, J_2a,3a= J_2b,3b2.6 Hz, J_3a,4a= J_3b,4b9.4 Hz, H-3a, H-3b), 3.66-

3.57 (m, 2H, H-4a, H-4b), 3.32-3.28 (m, 1H, H-5b), 2.30 (s, 3H,

SC₆H₄CH₃), 1.41 (d, 3H, J_5b,6b6.0 Hz, H-6b), 1.35 (d, 3H, J_5a,6a

6.1 Hz, H-6a); d_C(75 MHz, CDCl₃) 137.3-129.7 (SC₆H₄CH₃), 88.2

(C-1a), 87.4 (C-1b), 76.6 (C-5b), 74.8 (C-4b), 73.1 (C-4a), 72.5 (C-

2a, C-2b), 72.1 (C-3a, C-3b), 69.3 (C-5a), 21.0 (SC₆H₄CH₃), 17.9

(C-6b), 17.5 (C-6a); LRMS (ES+) Calcd. for C₁₃H₁₈NaO₄S (M

Na)⁺293. Found 293.

p-Methylphenyl 2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-1-

thio-a-L-rhamnopyranoside (16)

p-Methylphenyl 2-O-benzoyl-4-O-benzyl-1-thio-

a-L-rhamnopyranoside (14)

To a solution of 14 (3.66 g, 7.89 mmol) in CH₂Cl₂(65 mL)

containing pyridine (3.3 mL) was added dropwise at 0 ^◦C a

solution of chloroacetyl chloride (2.5 mL, 31.56 mmol) in CH₂Cl₂

(25 mL). The reaction mixture was stirred at room temperature for

3 h, then stopped by careful addition at 0 ^◦C of methanol (20 mL).

The solution was evaporated and the residue was dissolved in

CH₂Cl₂. The solution was successively washed with aq. KHSO₄,

saturated aq. NaHCO₃and water. The organic layer was dried over

Na₂SO₄, ﬁltered and concentrated. The residue was puriﬁed by

ﬂash chromatography (4:1 cyclohexane-EtOAc) to give 16 (4.17 g,

98%) as a brown oil, [a]_D-53 (c 0.33, CHCl₃); d_H(300 MHz,

CDCl₃) 8.13 (d, 2H, J 8.2 Hz, SC₆H₄CH₃), 7.71-7.30 (m, 10H,

Ar), 7.20 (d, 2H, SC₆H₄CH₃), 5.86 (dd, 1H, J_1,21.7 Hz, J_2,3

To a suspension of derivative 13 (3 g, 11.1 mmol) in dry DMF

(100 mL) were added triethyl orthobenzoate (4 mL, 17.8 mmol)

and camphor-10-sulfonic acid (516 mg, 2.22 mmol). The reaction

mixture was stirred for 3 h at room temperature. The solution was

neutralised with triethylamine (1.5 mL) and cooled to 0 ^◦C. NaH

(800 mg, 33.33 mmol) was added followed by dropwise addition

of benzyl bromide (2.4 mL, 20 mmol). The reaction mixture was

stirred for 15 h at room temperature, then methanol (30 mL)

was slowly added. The solution was evaporated and the residue

was dissolved with ethyl acetate. The solution was successively

washed with water and twice with 2 M HCl. The organic layer

was dried over Na₂SO₄, ﬁltered and concentrated in vacuo to give

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3.2 Hz, H-2), 5.56 (dd, 1H, J_3,49.7 Hz, H-3), 5.53 (d, 1H,

H-1), 4.83 (d, 1H, J 11.3 Hz, OCH₂Ar), 4.78 (d, 1H, OCH₂Ar),

4.54-4.44 (m, 1H, H-5), 4.00 (d, 1H, J 14.9 Hz, COCH₂Cl), 3.92

(d, 1H, COCH₂Cl), 3.80 (t, 1H, J_3,4= J_4,59.7 Hz, H-4), 2.39 (s, 3H,

SC₆H₄CH₃), 1.50 (d, 3H, J_5,66.2 Hz, H-6); d_C(75 MHz, CDCl₃)

166.3, 165.4 (OCOAr, COCH₂Cl), 138.1-127.8 (Ar), 85.9 (C-1),

78.7 (C-4), 75.2 (OCH₂Ar), 74.1 (C-3), 71.8 (C-2), 69.1 (C-5), 40.5

(COCH₂Cl), 21.0 (SC₆H₄CH₃), 17.9 (C-6); HRMS (ES⁺) Calcd.

for C₂₉H₂₉³⁵ClNaO₆³²S (MNa⁺) 563.1271. Found 563.1285. Found:

C, 63.84; H, 5.31. C₂₉H₂₉ClO₆S requires C, 64.38; H, 5.40%.

puriﬁed by ﬂash chromatography (4:1 cyclohexane-EtOAc) to give

19 (2.11 g, 90%) as a white powder, [a]_D+30 (c 0.33, CHCl₃); d_H

(300 MHz, CDCl₃) 8.18-7.16 (m, 25H, Ar), 5.65 (dd, 1H, J_1,2

1.9 Hz, J_2,33.2 Hz, H-2B), 5.47-5.43 (m, 2H, H-3B, H-2C), 5.22

(d, 1H, H-1B), 5.16 (broad s, 3H, NHCOOCH₂Ar, NH), 5.03 (d,

1H, J 10.8 Hz, OCH₂Ar), 4.95 (d, 1H, J_1,21.7 Hz, H-1C), 4.78 (d,

1H, J 10.8 Hz, OCH₂Ar), 4.64 (d, 1H, J 11.6 Hz, OCH₂Ar),

4.59 (d, 1H, J 11.6 Hz, OCH₂Ar), 4.30 (dd, 1H, J_2,33.4 Hz,

J_3,49.2 Hz, H-3C), 4.00-3.77 (m, 5H, H-5C, H-5B, CH₂CH₂NH,

COCH₂Cl), 3.70 (t, 1H, J_3,4= J_4,59.3 Hz, H-4C), 3.64-3.55 (m,

2H, H-4B, CH₂CH₂NH), 3.50-3.43 (m, 2H, CH₂CH₂NH), 1.39

(d, 3H, J_5,66.1 Hz, H-6C), 1.26 (d, 3H, J_5,66.2 Hz, H-6B); d_C

(75 MHz, CDCl₃) 166.3, 166.0 (OCOAr), 165.4 (COCH₂Cl), 156.3

(NHCOOCH₂Ar), 137.7-127.4 (Ar), 99.6 (C-1B), 97.1 (C-1C),

79.8 (C-4C), 78.9 (C-3C), 78.0 (C-4B), 75.8, 74.3 (OCH₂Ar), 73.5,

72.5 (C-3B, C-2C), 70.4 (C-2B), 68.6 (C-5C), 68.0 (C-5B), 67.1

(CH₂CH₂NH), 66.8 (NHCOOCH₂Ar), 40.8 (CH₂CH₂NH), 40.6

(COCH₂Cl), 18.1 (C-6C), 17.8 (C-6B); HRMS (ES⁺) Calcd. for

C₅₂H₅₄³⁵ClNNaO₁₄(MNa⁺) 974.3131. Found 974.3132. Found: C,

65.11; H, 5.66; N, 1.52. C₅₂H₅₄ClNO₁₄requires C, 65.57; H, 5.71;

N 1.47%.

2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-L-rhamnopyranose (17)

◦

N-Bromosuccinimide (1.73 g, 9.72 mmol) was added at 0 C to

a solution of thioglycoside 16 (3.50 g, 6.48 mmol) in acetone

(180 mL). The reaction mixture was stirred at 0 ^◦C for 20 min., then

at room temperature for 1 h. Ammonium chloride (500 mg) was

added. After 10 min of stirring, the mixture was diluted with ethyl

acetate (100 mL) and washed with water (80 mL). The organic layer

was dried over Na₂SO₄, ﬁltered and concentrated. The residue

was puriﬁed by ﬂash chromatography (4:1 cyclohexane-EtOAc)

to give 17 (2.57 g, 91%) as a colourless oil, d_C(75 MHz, CDCl₃)

166.4, 165.7 (OCOC₆H₅, OCOCH₂Cl), 137.7-127.8 (OCOC₆H₅,

OCH₂C₆H₅), 92.4 (C-1b), 92.0 (C-1a), 78.5 (C-4a, C-2b), 77.8

(C-4b), 75.7, 75.3 (OCH₂C₆H₅), 73.6 (C-3a), 71.8 (C-5b), 71.2 (C-

3b), 70.8 (C-2a), 67.7 (C-5a, C-5b), 40.6, 40.5 (OCOCH₂Cl), 18.0

(C-6a, C-6b); HRMS (ES⁺) Calcd. for C₂₂H₂₃³⁵ClNaO₇(MNa⁺)

457.1030. Found 457.1022.

N-Benzyloxycarbonylaminoethyl 2-O-benzoyl-4-O-benzyl-a-L-

rhamnopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-a-L-

rhamnopyranoside (20)

To a solution of derivative 19 (720 mg, 0.76 mmol) in THF (35 mL)

were added thiourea (115 mg, 1.51 mmol), NaHCO₃(144 mg,

1.51 mmol) and a catalytic amount of TBAI (14 mg, 38 mmol).

The solution was warmed to 55 ^◦C and stirred for 12 h. The

reaction mixture was then cooled to room temperature, diluted

with CH₂Cl₂(40 mL) and washed with water. The organic layer

was dried over Na₂SO₄, ﬁltered and concentrated. The residue

was puriﬁed by ﬂash chromatography (4:1 cyclohexane-EtOAc)

to give 20 (530 mg, 80%) as a white powder, [a]_D+31 (c 0.17,

CHCl₃); d_H(300 MHz, CDCl₃) 8.14-7.26 (m, 25H, Ar), 5.45 (dd,

1H, J_1,21.7 Hz, J_2,33.4 Hz, H-2B), 5.43 (dd, 1H, J_1,21.9 Hz, J_2,3

3.3 Hz, H-2C), 5.24 (d, 1H, H-1B), 5.19-5.17 (m, 1H, NH), 5.15

(broad s, 2H, NHCOOCH₂Ar), 4.98 (d, 1H, J 10.8 Hz, OCH₂Ar),

4.92 (d, 1H, H-1C), 4.76-4.72 (m, 2H, OCH₂Ar), 4.68 (d, 1H, J

11.4 Hz, OCH₂Ar), 4.29 (dd, 1H, J_3,49.3 Hz, H-3C), 4.17-4.12

(m, 1H, H-3B), 3.92-3.84 (m, 1H, H-5B), 3.83-3.78 (m, 2H, H-5C,

CH₂CH₂NH), 3.67 (t, 1H, J_3,4= J_4,59.3 Hz, H-4C), 3.60-3.54

(m, 1H, CH₂CH₂NH), 3.50-3.44 (m, 3H, H-4B, CH₂CH₂NH),

2.19 (d, 1H, OH), 1.37 (d, 3H, J_5,66.1 Hz, H-6C), 1.30 (d, 3H,

J_5,66.2 Hz, H-6B); d_C(75 MHz, CDCl₃) 165.9, 165.8 (OCOAr),

156.3 (NHCOOCH₂Ar), 138.2-127.7 (Ar), 99.5 (C-1B), 97.2 (C-

1C), 81.1 (C-4B), 80.2 (C-4C), 77.6 (C-3C), 75.7, 74.0 (OCH₂Ar),

73.1 (C-2B), 72.6 (C-2C), 69.8 (C-3B), 68.3 (C-5B), 68.0 (C-5C),

67.1 (CH₂CH₂NH), 66.7 (NHCOOCH₂Ar), 40.8 (CH₂CH₂NH),

18.0 (C-6C), 17.9 (C-6B); HRMS (ES⁺) Calcd. for C₅₀H₅₃NNaO₁₃

(MNa⁺) 898.3415. Found 898.3429. Found: C, 68.22; H, 5.90; N,

1.57. C₅₀H₅₃NO₁₃requires C, 68.58; H, 6.10; N, 1.60%.

2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-a-L-rhamnopyranosyl

trichloroacetimidate (18)

To a solution of 17 (2.57 g, 5.91 mmol) in dry CH₂Cl₂(15 mL)

were added at 0 ^◦C trichloroacetonitrile (12.4 mL, 0.12 mol)

and DBU (222 mL, 1.5 mmol). After 1 h of stirring at room

temperature, solvent was removed. The residue was puriﬁed

by ﬂash chromatography (4:1 cyclohexane-EtOAc, Et₃N 2%) to

afford the donor 18 (2.81 g, 82%), [a]_D+9 (c 0.33, CHCl₃); d_H

(300 MHz, CDCl₃) 8.78 (s, 1H, NH), 8.15-7.34 (m, 10H, Ar), 6.38

(d, 1H, J_1,22.0 Hz, H-1), 5.78 (dd, 1H, J_2,33.3 Hz, H-2), 5.57

(dd, 1H, J_3,49.8 Hz, H-3), 4.79 (d, 1H, J 11.2 Hz, OCH₂Ar),

4.73 (d, 1H, OCH₂Ar), 4.22-4.15 (m, 1H, H-5), 3.99 (d, 1H, J

15.0 Hz, COCH₂Cl), 3.90 (d, 1H, COCH₂Cl), 3.78 (t, 1H, J_3,4

=

J

_4,59.8 Hz, H-4), 1.50 (d, 3H, J_5,66.2 Hz, H-6); d_C(75 MHz, CDCl₃)

166.3, 165.4 (OCOAr, OCOCH₂Cl), 160.1 (OCNHCCl₃), 137.4-

128.0 (Ar), 94.8 (C-1), 77.8 (C-4), 75.4 (OCH₂Ar), 73.6 (C-3),

70.7 (C-5), 68.7 (C-2), 40.5 (COCH₂Cl), 18.0 (C-6); HRMS (ES⁺)

Calcd. for C₂₄H₂₃³⁵Cl₄NaO₇(MNa⁺) 600.0126. Found 600.0126.

N-Benzyloxycarbonylaminoethyl 2-O-benzoyl-4-O-benzyl-3-

O-chloroacetyl-a-L-rhamnopyranosyl-(1→3)-2-O-benzoyl-

4-O-benzyl-a-L-rhamnopyranoside (19)

TMSOTf (223 mL, 1.23 mmol) was added under argon at -40 ^◦C

to a solution of donor 18 (1.71 g, 2.95 mmol) and acceptor 15

(1.31 g, 2.46 mmol) in dry CH₂Cl₂(70 mL) containing molecular

2-O-Acetyl-4-azido-3-O-benzoyl-4,6-dideoxy-D-glucopyranose

(21)

˚

sieves 4 A (500 mg). After 30 min of stirring, the solution was

neutralised with Et₃N. The molecular sieves were removed by

ﬁltration. The reaction mixture was washed with water (30 mL),

dried over Na₂SO₄, ﬁltered and concentrated. The residue was

N-Bromosuccinimide (640 mg, 3.6 mmol) was added at 0 ^◦C to a

solution of thioglycoside 11 (1.06 g, 2.4 mmol) in acetone (85 mL).

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The reaction mixture was stirred at 0 ^◦C for 20 min, then at room

temperature for 2 h. Ammonium chloride (500 mg) was added.

After 10 min of stirring, the mixture was diluted with ethyl acetate

(50 mL) and washed with water. The organic layer was dried over

Na₂SO₄, ﬁltered and concentrated. The residue was puriﬁed by

ﬂash chromatography (1:1 cyclohexane-EtOAc) to give 21 (723 mg,

90%) as a colourless oil, d_H(300 MHz, CDCl₃) 8.13-7.38 (m, 5H,

Ar), 5.78 (t, 1H, J_2,3= J_3,410.0 Hz, H-3a), 5.47-5.41 (m, 2H,

H-1a, H-4b), 5.10-5.04 (m, 2H, H-2a, H-2b), 4.87-4.85 (m, 2H,

H-1b, H-3b), 4.16-4.06 (m, 1H, H-5a), 3.58-3.43 (m, 1H, H-5b),

3.38 (t, 1H, J_4,510.0 Hz, H-4a), 1.96 (s, 3H, CH₃CO), 1.42 (d, 3H,

J_5,65.8 Hz, H-6b), 1.36 (d, 3H, J_5,66.2 Hz, H-6a); d_C(75 MHz,

CDCl₃) 170.5 (CH₃CO), 165.6 (OCOAr), 133.5-128.3 (Ar), 94.6

(C-1b), 89.9 (C-1a), 73.4 (C-4b), 73.1 (C-2b, C-3b), 71.5 (C-2a),

70.7 (C-3a, C-5b), 66.1 (C-4a), 65.6 (C-5a), 20.3 (CH₃CO), 17.9

(C-6a, C-6b); HRMS (ES⁺) Calcd. for C₁₅H₁₇N₃NaO₆(MNa⁺)

358.1015. Found 358.1021. Found: C, 53.15; H, 5.11; N, 12.49.

C₁₅H₁₇N₃O₆requires C, 53.73; H, 5.11; N, 12.53%.

[a]_D+3 (c 0.1, CHCl₃); d_H(500 MHz, CDCl₃) 8.25-7.31 (m, 30H,

Ar), 5.56-5.55 (m, 1H, H-2C), 5.54-5.53 (m, 1H, H-2B), 5.34 (d,

1H, J_1,2< 1 Hz, H-1B), 5.27 (t, 2H, J_2,3= J_3,49.7 Hz, H-3A,

NH), 5.22 (broad s, 2H, NHCOOCH₂Ar), 5.15-5.12 (m, 2H, H-

2A, OCH₂Ar), 4.98 (d, 1H, J_1,2< 1 Hz, H-1C), 4.86 (d, 1H, J

11.4 Hz, OCH₂Ar), 4.83 (d, 1H, J 10.9 Hz, OCH₂Ar), 4.64 (d, 1H,

J 11.4 Hz, OCH₂Ar), 4.56 (d, 1H, J_1,27.9 Hz, H-1A), 4.40 (dd, 1H,

J

_2,33.1 Hz, J_3,49.3 Hz, H-3C), 4.22 (dd, 1H, J_2,33.1 Hz, J_3,49.3 Hz,

H-3B), 3.95-3.88 (m, 3H, H-5B, H-5C, CH₂CH₂NH), 3.76 (t, 1H,

J_3,4= J_4,59.3 Hz, H-4C), 3.65-3.60 (m, 2H, H-4B, CH₂CH₂NH),

3.57-3.48 (m, 2H, CH₂CH₂NH), 3.26 (t, 1H, J_3,4= J_4,59.7 Hz,

H-4A), 3.09-3.07 (m, 1H, H-5A), 1.69 (s, 3H, CH₃CO), 1.46 (d,

3H, J_5,66.2 Hz, H-6C), 1.24 (d, 3H, J_5,66.1 Hz, H-6B), 1.14 (d,

3H, J_5,66.1 Hz, H-6A); d_C(125 MHz, CDCl₃) 169.3 (CH₃CO),

165.8, 165.6 (OCOAr), 156.3 (NHCOOCH₂Ar), 138.2-127.3 (Ar),

100.5 (C-1A), 98.6 (C-1B), 97.3 (C-1C), 80.1 (C-4C), 79.0 (C-

4B), 78.7 (C-3B), 77.3 (C-3C), 75.4, 74.3 (OCH₂Ar), 73.8 (C-3A),

72.3 (C-2B, C-2C), 71.7 (C-2A), 70.5 (C-5A), 68.6 (C-5B), 68.0

(C-5C), 67.2 (CH₂CH₂NH), 66.7 (NHCOOCH₂Ar), 65.5 (C-4A),

40.8 (CH₂CH₂NH), 20.2 (CH₃CO), 18.0 (C-6C), 17.8 (C-6A, C-

6B); HRMS (ES⁺) Calcd. for C₆₅H₆₈N₄NaO₁₈(MNa⁺) 1215.4426.

Found 1215.4399. Found: C, 64.43; H, 5.74; N, 4.51. C₆₅H₆₈N₄O₁₈

requires C, 65.43; H, 5.74; N 4.7%.

2-O-Acetyl-4-azido-3-O-benzoyl-4,6-dideoxy-D-glucopyranosyl

trichloroacetimidate (22)

To a solution of 21 (723 mg, 2.16 mmol) in dry CH₂Cl₂(5 mL)

were added at 0 ^◦C trichloroacetonitrile (4.5 mL, 45.3 mmol) and

DBU (81 mL, 0.54 mmol). After 2 h of stirring at 0 ^◦C, solvent was

removed. The residue was puriﬁed by ﬂash chromatography (4:1

cyclohexane-EtOAc, Et₃N 2%) to afford the donor 22 (865 mg,

84%), d_H(300 MHz, CDCl₃) 8.74 (broad s, 1H, NH-b), 8.67

(broad s, 1H, NH-a), 8.09-7.40 (m, 5H, Ar), 6.50 (d, 1H, J_1,2

N-Benzyloxycarbonylaminoethyl 4-azido-3-O-benzoyl-4,6-

dideoxy-b-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-a-D-

rhamnopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-a-D-

rhamnopyranoside (24)

3.6 Hz, H-1a), 5.90 (d, 1H, J_1,28.0 Hz, H-1b), 5.80 (t, 1H, J_2,3

=

A freshly prepared solution of acetyl chloride (1.9 mL, 26.3 mmol)

in methanol (48 mL) was added dropwise at 0 ^◦C to a solution of

crude compound 23 (920 mg) in CH₂Cl₂(36 mL). The reaction

mixture was stirred at room temperature for 6 days, then poured

into saturated aq. NaHCO₃. The organic layer was washed with

water, dried over Na₂SO₄, ﬁltered and concentrated. The residue

was puriﬁed by ﬂash chromatography (4:1 cyclohexane-EtOAc) to

give 24 (307 mg, 50% from 20) as a white powder, [a]_D+17 (c 0.05,

CHCl₃); d_H(500 MHz, CDCl₃) 8.15-7.03 (m, 30H, Ar), 5.52-5.50

(m, 1H, H-2B), 5.48-5.46 (m, 1H, H-2C), 5.24 (d, 1H, J_1,2< 1 Hz,

J

_3,410.1 Hz, H-3a), 5.48 (t, 1H, J_2,3= J_3,49.6 Hz, H-3b), 5.38 (dd,

1H, H-2b), 5.25 (dd, 1H, H-2a), 4.07-3.97 (m, 1H, H-5a), 3.72-

3.63 (m, 1H, H-5b), 3.52-3.42 (m, 2H, H-4a, H-4b), 1.92 (s, 3H,

CH₃CO), 1.46 (d, 3H, J_5,66.1 Hz, H-6b), 1.42 (d, 3H, J_5,66.2 Hz,

H-6a); d_C(75 MHz, CDCl₃) 169.9 (CH₃CO), 165.3 (OCOAr),

160.8 (OCNHCCl₃), 133.5-128.5 (Ar), 95.3 (C-1b), 93.1 (C-1a),

73.5 (C-3b), 71.8 (C-5b), 70.4 (C-2b, C-3a), 70.0 (C-2a), 68.9 (C-

5a), 65.7 (C-4a), 65.5 (C-4b), 20.3 (CH₃CO), 18.2 (C-6a, C-6b);

LRMS (ES⁺) Calcd. for C₁₇H₁₇Cl₃NNaO₆(MNa⁺) 501. Found

501.

H-1B), 5.17-5.14 (m, 3H, NHCOOCH₂Ar, NH), 5.09 (t, 1H, J_2,3

=

J

_3,49.6 Hz, H-3A), 5.05 (d, 1H, J 10.9 Hz, OCH₂Ar), 4.90 (d, 1H,

_1,2< 1 Hz, H-1C), 4.85 (d, 1H, J 11.0 Hz, OCH₂Ar), 4.73 (d, 1H,

N-Benzyloxycarbonylaminoethyl 2-O-acetyl-4-azido-3-O-benzoyl-

4,6-dideoxy-b-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-

benzyl-a-D-rhamnopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-

a-D-rhamnopyranoside (23)

J 10.8 Hz, OCH₂Ar), 4.64 (d, 1H, J 11.0 Hz, OCH₂Ar), 4.34-4.30

(m, 2H, H-1A, H-3C), 4.21 (dd, 1H, J_2,33.0 Hz, J_3,49.4 Hz, H-3B),

3.92-3.88 (m, 1H, H-5B), 3.83-3.79 (m, 2H, H-5C, CH₂CH₂NH),

3.67 (t, 1H, J_3,4= J_4,59.3 Hz, H-4C), 3.63-3.58 (m, 2H, H-4B,

CH₂CH₂NH), 3.57-3.42 (m, 3H, H-2A, CH₂CH₂NH), 3.15 (t, 1H,

J_3,4= J_4,59.6 Hz, H-4A), 3.03-2.88 (m, 1H, H-5A), 2.68 (broad s,

1H, OH), 1.37 (d, 3H, J_5,66.0 Hz, H-6C), 1.22 (d, 3H, J_5,66.0 Hz,

H-6B), 1.07 (d, 3H, J_5,66.0 Hz, H-6A); d_C(125 MHz, CDCl₃) 166.8,

166.2, 166.1 (OCOAr), 156.8 (NHCOOCH₂Ar), 138.5-128.1 (Ar),

103.6 (C-1A), 99.3 (C-1B), 97.8 (C-1C), 80.5 (C-4C), 80.2 (C-4B),

78.2 (C-3B), 78.1 (C-3C), 76.4 (C-3A), 75.9, 75.1 (OCH₂Ar), 73.6

(C-2A), 73.0 (C-2B), 72.8 (C-2C), 70.9 (C-5A), 69.2 (C-5B), 68.5

(C-5C), 67.7 (CH₂CH₂NH), 67.2 (NHCOOCH₂Ar), 66.1 (C-4A),

41.3 (CH₂CH₂NH), 18.5, 18.4 (C-6A, C-6B, C-6C); HRMS (ES⁺)

Calcd. for C₆₃H₆₆N₄NaO₁₇(MNa⁺) 1173.4321. Found 1173.4292.

Found: C, 65.47; H, 5.75; N, 4.82. C₆₃H₆₆N₄O₁₇requires C, 65.73;

H, 5.78; N, 4.87%.

TMSOTf (73 mL, 0.4 mmol) was added under argon at -70 ^◦C

to a solution of donor 22 (383 mg, 0.8 mmol) and acceptor 20

(465 mg, 0.53 mmol) in dry CH₂Cl₂(20 mL) containing molecular

˚

sieves 4 A (500 mg). After 45 min of stirring, the solution was

neutralised with Et₃N. The molecular sieves were removed by

ﬁltration. The reaction mixture was washed with water (15 mL),

dried over Na₂SO₄, ﬁltered and concentrated. The residue was

puriﬁed by ﬂash chromatography (9:1 cyclohexane-EtOAc) to give

a mixture (920 mg) of the expected trisaccharide 23 and some

unreacted acceptor 20 which was used in the next step without

further puriﬁcation. A sample was puriﬁed by high performance

liquid chromatography (Prevail C₁₈5 mm (4.6 ¥ 250 mm), H₂O-

MeOH 6:4 to 0:1 in 30 min.) to afford pure 23 as a white powder,

5192 | Org. Biomol. Chem., 2009, 7, 5184–5199

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N-Benzyloxycarbonylaminoethyl 4-azido-3-O-benzoyl-4,6-

dideoxy-2-O-methyl-b-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-

O-benzyl-a-D-rhamnopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-

a-D-rhamnopyranoside (25)

puriﬁed by ﬂash chromatography (3:2 cyclohexane-EtOAc) to give

27 (48 mg, 85%) as a colourless oil, [a]_D= +52 (c 0.05, CHCl₃);

d_H(300 MHz, CDCl₃) 8.14-7.27 (m, 30H, Ar), 5.79 (d, 1H, J_NH,H4

9.4 Hz, NHCO), 5.54 (dd, 1H, J_1,21.9 Hz, J_2,33.1 Hz, H-2B),

5.45 (dd, 1H, J_1,21.8 Hz, J_2,33.0 Hz, H-2C), 5.27 (d, 1H, H-

1B), 5.18-5.15 (m, 3H, NHCOOCH₂Ar, NHCOOCH₂Ar), 5.07

(d, 1H, J 10.8 Hz, OCH₂Ar), 4.97 (t, 1H, J_2,3= J_3,49.6 Hz, H-

3A), 4.91-4.87 (m, 2H, H-1C, OCH₂Ar), 4.72 (d, 1H, J 10.8 Hz,

OCH₂Ar), 4.62 (d, 1H, J 10.8 Hz, OCH₂Ar), 4.53 (d, 1H, J_1,2

7.7 Hz, H-1A), 4.32 (dd, 1H, J_2,33.0 Hz, J_3,49.2 Hz, H-3C), 4.27

(dd, 1H, J_2,33.1 Hz, J_3,49.4 Hz, H-3B), 4.00-3.77 (m, 4H, H-4A,

H-5C, H-5B, CH₂CH₂NH), 3.73-3.60 (m, 2H, H-4B, H-4C), 3.58-

3.52 (m, 1H, CH₂CH₂NH), 3.47-3.42 (m, 2H, CH₂CH₂NH), 3.37

(s, 3H, OCH₃), 3.25 (dd, 1H, H-2A), 3.02-2.94 (m, 1H, H-5A),

2.21 (d, 1H, J 14.7 Hz, NHCOCH₂C(CH₃)₂OH), 2.13 (d, 1H,

NHCOCH₂C(CH₃)₂OH), 1.71 (broad s, 1H, OH), 1.35 (d, 3H,

J_5,66.1 Hz, H-6C), 1.23 (d, 3H, J_5,66.1 Hz, H-6B), 1.10 (s, 3H,

NHCOCH₂C(CH₃)₂OH), 0.98 (s, 3H, NHCOCH₂C(CH₃)₂OH),

0.91 (d, 3H, J_5,66.2 Hz, H-6A); d_C(75 MHz, CDCl₃) 172.3

(NHCO), 167.2, 165.8, 165.6 (OCOAr), 156.3 (NHCOOCH₂Ar),

138.2-127.6 (Ar), 102.8 (C-1A), 99.2 (C-1B), 97.2 (C-1C), 81.5

(C-2A), 80.3 (C-4B), 79.8 (C-4C), 78.1 (C-3C), 76.4 (C-3B), 75.5

(OCH₂Ar), 75.0 (C-3A), 74.3 (OCH₂Ar), 72.9 (C-2B), 72.5 (C-2C),

71.3 (C-5A), 69.2 (NHCOCH₂C(CH₃)₂OH), 68.6 (C-5B), 68.0 (C-

5C), 67.2 (CH₂CH₂NH), 66.8 (NHCOOCH₂C₆H₅), 60.7 (OCH₃),

54.6 (C-4A), 47.9 (NHCOCH₂C(CH₃)₂OH), 40.8 (CH₂CH₂NH),

29.1, 28.9 (NHCOCH₂C(CH₃)₂OH), 18.0 (C-6C), 17.9 (C-6B),

17.5 (C-6A); HRMS (ES⁺) Calcd. for C₆₉H₇₈N₂NaO₁₉(MNa⁺)

1261.5096. Found 1261.5105.

Afreshly preparedsolution of diazomethane²³was added dropwise

◦

at 0 C to a solution of 24 (80 mg, 0.7 mmol) in CH₂Cl₂(3 mL)

in the presence of BF₃.OEt₂(3 mL, 0.21 mmol) until the yellow

colour persisted. The reaction mixture was stirred at 0 ^◦C until

TLC showed complete consumption of starting material and was

then allowed to reach room temperature. The white precipitate

was ﬁltered and the ﬁltrate washed with saturated aq. NaHCO₃

(6 mL) and water (6 mL). The organic layer was dried over

Na₂SO₄, ﬁltered and concentrated. The residue was puriﬁed by

ﬂash chromatography (4:1 cyclohexane-EtOAc) to give 25 (32 mg,

40%) as a colourless oil, [a]_D+33 (c 0.06, CHCl₃); d_H(300 MHz,

CDCl₃) 8.16-7.23 (m, 30H, Ar), 5.56 (dd, 1H, J_1,21.9 Hz, J_2,3

3.2 Hz, H-2B), 5.51 (dd, 1H, J_1,21.8 Hz, J_2,33.2 Hz, H-2C), 5.27

(d, 1H, H-1B), 5.23-5.16 (m, 4H, H-3A, NHCOOCH₂Ar, NH),

5.06 (d, 1H, J 10.7 Hz, OCH₂Ar), 4.92 (d, 1H, H-1C), 4.88 (d,

1H, J 10.8 Hz, OCH₂Ar), 4.73 (d, 1H, J 10.7 Hz, OCH₂Ar),

4.63 (d, 1H, J 10.8 Hz, OCH₂Ar), 4.57 (d, 1H, J_1,27.8 Hz, H-

1A), 4.33 (dd, 1H, J_2,33.2 Hz, J_3,49.3 Hz, H-3C), 4.27 (dd, 1H,

J_2,33.2 Hz, J_3,49.4 Hz, H-3B), 3.94-3.77 (m, 3H, H-5B, H-5C,

CH₂CH₂NH), 3.69 (t, 1H, J_3,4= J_4,59.4 Hz, H-4C), 3.66-3.58 (m,

2H, H-4B, CH₂CH₂NH), 3.50-3.38 (m, 2H, CH₂CH₂NH), 3.35

(s, 3H, OCH₃), 3.20-3.11 (m, 2H, H-2A, H-4A), 3.08-2.99 (m, 1H,

H-5A), 1.37 (d, 3H, J_5,66.1 Hz, H-6C), 1.25 (d, 3H, J_5,66.2 Hz,

H-6B), 1.02 (d, 3H, J_5,65.9 Hz, H-6A); d_C(75 MHz, CDCl₃) 165.8,

165.6, 165.5 (OCOAr), 156.3 (NHCOOCH₂Ar), 137.7-127.7 (Ar),

102.7 (C-1A), 99.1 (C-1B), 97.2 (C-1C), 81.9 (C-2A), 80.2 (C-4B),

79.9 (C-4C), 77.9 (C-3C), 76.0 (C-3B), 75.6 (OCH₂Ar), 74.7 (C-

3A), 74.3 (OCH₂Ar), 73.0 (C-2B), 72.4 (C-2C), 70.2 (C-5A), 68.6

(C-5B), 68.0 (C-5C), 67.2 (CH₂CH₂NH), 66.5 (NHCOOCH₂Ar),

65.9 (C-4A), 60.6 (OCH₃), 40.8 (CH₂CH₂NH), 18.0 (C6-C), 17.9

(C-6A, C-6B); HRMS (ES⁺) Calcd. for C₆₄H₆₈N₄NaO₁₇(MNa⁺)

1187.4477. Found 1187.4490. Found: C, 65.32; H, 5.81; N, 4.73.

C₆₄H₆₈N₄O₁₇requires C, 65.97; H, 5.88; N, 4.81%.

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl-(1→3)-4-

O-benzyl-a-D-rhamnopyranosyl-(1→3)-4-O-benzyl-a-D-

rhamnopyranoside (28)

A solution of sodium methoxide (1 mL, 1 M in MeOH) was

added dropwise at 0 ^◦C to a solution of 27 (39 mg, 31 mmol)

in MeOH (15 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺) was

added to neutralise MeONa. The resin was ﬁltered and the solvent

removed. The residue was then puriﬁed by ﬂash chromatography

(2:3 cyclohexane-EtOAc) to give 28 (23 mg, 80%) as a colourless

oil, [a]_D+13 (c 0.07, CHCl₃); d_H(300 MHz, CDCl₃) 7.76-7.32

(m, 15H, Ar), 6.05 (d, 1H, J_NH,H48.6 Hz, NHCO), 5.17 (d,

1H, J_1,2< 1 Hz, H-1B), 5.15-5.13 (m, 3H, NHCOOCH₂Ar,

NHCOOCH₂Ar), 4.92 (d, 1H, J 10.6 Hz, OCH₂Ar), 4.80 (d, 1H, J

10.7 Hz, OCH₂Ar), 4.77 (d, 1H, J_1,2< 1 Hz, H-1C), 4.66-4.59 (m,

2H, OCH₂Ar), 4.53 (d, 1H, J_1,27.8 Hz, H-1A), 4.17-4.13 (m, 1H,

H-2B), 4.07 (dd, 1H, J_2,33.2 Hz, J_3,48.7 Hz, H-3B), 4.02-4.00 (m,

1H, H-2C), 3.98-3.90 (m, 2H, H-3C, H-5C), 3.77-3.65 (m, 3H, H-

4A, H-5B, CH₂CH₂NH), 3.62 (s, 3H, OCH₃), 3.60-3.33 (m, 8H,

H-4B, H-3A, H-3C, H-4C, CH₂CH₂NH, H-5A, CH₂CH₂NH),

3.11 (t, 1H, J_1,2= J_2,37.8 Hz, H-2A), 3.00-2.98 (m, 1H, OH-B),

2.39 (s, 2H, NHCOCH₂C(CH₃)₂OH), 1.38 (d, 3H, J_5,66.3 Hz,

H-6C), 1.34-1.27 (m, 9H, H-6B, NHCOCH₂C(CH₃)₂OH), 1.16

(d, 3H, J_5,66.1 Hz, H-6A); d_C(75 MHz, CDCl₃) 172.9 (NHCO),

156.3 (NHCOOCH₂Ar), 138.0-127.8 (Ar), 103.0 (C-1A), 101.3 (C-

1B), 99.4 (C-1C), 83.8 (C-2A), 80.6 (C-3B), 80.0 (C-3C), 79.8 (C-

4B, C-4C), 75.4, 74.9 (OCH₂Ar), 74.6 (C-3A), 70.7 (C-2B, C-2C,

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-3-O-benzoyl-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl-

(1→3)-2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranosyl-(1→3)-

2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranoside (27)

To a solution of 25 (53 mg, 45 mmol) in a mixture of dry CH₂Cl₂

(1.5 mL) and EtOH (5 mL) was added NaBH₄(4 mg, 90 mmol)

and a catalytic amount of NiCl₂.6H₂O. The reaction mixture

was stirred at room temperature for 1 h and concentrated. The

residue was dissolved in CH₂Cl₂(5 mL). The organic layer was

washed with water (3 mL) and brine (3 mL), dried over Na₂SO₄,

ﬁltered and concentrated. Amine 26 (51 mg) was used in the

next step without puriﬁcation. To a solution of compound 26 (51

mg) in dry CH₂Cl₂(5 mL) was added dropwise in the following

order: 3-hydroxy-3-methylbutanoic acid (8.5 mL, 67 mmol), HATU

(26 mg, 68 mmol) then DIPEA (12 mL, 68 mmol). The reaction

mixture was stirred at room temperature under argon for 18 h.

The residue was diluted with CH₂Cl₂(5 mL). The organic layer

was washed with saturated aq. NaHCO₃(5 mL), water (5 mL),

dried over Na₂SO₄, ﬁltered and concentrated. The residue was

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C-5A), 69.9 (NHCOCH₂C(CH₃)₂OH), 68.3 (C-5C), 67.7 (C-5B),

66.9 (CH₂CH₂NH), 66.8 (NHCOOCH₂Ar), 60.9 (OCH₃), 56.8

(C-4A), 48.6 (NHCOCH₂C(CH₃)₂OH), 40.8 (CH₂CH₂NH), 29.8,

29.2 (NHCOCH₂C(CH₃)₂OH), 18.0 (C-6A), 17.8 (C-6B, C-6C);

HRMS (ES⁺) Calcd. for C₄₈H₆₆N₂NaO₁₆(MNa⁺) 949.4310. Found

949.4296.

1H, H-1), 4.71 (d, 1H, OCH₂Ar), 3.93-3.92 (m, 2H, H-2, H-3),

3.76-3.67 (m, 2H, H-5, OH), 3.50-3.34 (m, 5H, H-4, CH₂CH₂NH,

CH₂CH₂NH), 3.09 (broad s, 1H, OH), 1.35 (d, 3H, J_5,66.2 Hz,

H-6); d_C(75 MHz, CDCl₃) 156.4 (NHCOOCH₂Ar), 138.2-127.8

(Ar), 99.4 (C-1), 81.3 (C-4), 74.9 (OCH₂Ar), 71.3, 70.9 (C-2, C-

3), 67.4 (C-5), 66.7 (NHCOOCH₂Ar), 66.5 (CH₂CH₂NH), 40.6

(CH₂CH₂NH), 17.9 (C-6); HRMS (ES⁺) Calcd. for C₂₃H₂₉NNaO₁₁

(MNa⁺) 454.1842. Found 454.1847. Found: C, 64.40; H, 6.80; N,

3.33. C₂₃H₂₉NO₇requires C, 64.02; H, 6.77; N, 3.25%.

2-Aminoethyl 4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-2-O-

methyl-b-D-glucopyranosyl-(1→3)-a-D-rhamnopyranosyl-(1→3)-

a-D-rhamnopyranoside (29)

2-Aminoethyl a-L-rhamnopyranoside (31)

To a solution of 28 (23 mg, 25 mmol) in tBuOH (2.3 mL) was

added AcOH (1.9 mL) and a catalytic amount of Pd(OH)₂. The

reaction mixture was stirred at room temperature under hydrogen.

The reaction was monitored by mass spectrometry. After 5 h, the

reaction was complete. The catalyst was ﬁltered over celite and

the ﬁltrate was concentrated. The residue was dissolved in water

and washed with CHCl₃. The organic layer was extracted twice

with water. The aq. layers were combined and concentrated. The

residue was puriﬁed on a resin cation exchanger (Bio-Rad, AG

MP-50 Resin, analytical grade 100-200 mesh). A glass column

(400 mm ¥ 15 mm) was packed with the resin (10 mL) which was

washed with water and then acidiﬁed to pH 1 with a solution

of HCl (1 N). An acidiﬁed aq. solution of the residue (1 mL,

pH 1) was added on the top of the column. The resin was then

washed with water (2 ¥ 10 mL) and 29 was eluted with a solution

of ammonia 10% (3 ¥ 10 mL). After lyophilisation, compound

29 (6 mg, 40%) was obtained as a white solid, [a]_D+ 45 (c 0.05,

H₂O); d_H(500 MHz, D₂O) 4.97 (d, 1H, J_1,2< 1 Hz, H-1B), 4.74

(d, 1H, J_1,2< 1 Hz, H-1C), 4.68 (d, 1H, J_1,28.3 Hz, H-1A),

4.23-4.22 (m, 1H, H-2B), 4.00-3.99 (m, 1H, H-2C), 3.95-

3.92 (m, 2H, H-3B, CH₂CH₂NH), 3.84-3.78 (m, 2H, H-3C, H-

5C), 3.70-3.63 (m, 2H, H-5C, CH₂CH₂NH), 3.58 (s, 3H, OCH₃),

3.56-3.47 (m, 5H, H-4B, H-3A, H-4A, H-5A, H-4C), 3.08 (d,

1H, J_1,2= J_2,38.3 Hz, H-2A), 3.05-2.98 (m, 2H, CH₂CH₂NH),

2.41 (s, 2H, NHCOCH₂C(CH₃)₂OH), 1.25-1.23 (m, 12H, H-6B,

H-6C, NHCOCH₂C(CH₃)₂OH), 1.17 (d, 3H, J_5,66.0 Hz, H-6A);

d_C(75 MHz, D₂O) 174.1 (NHCO), 103.6 (C-1A), 102.3 (C-1B),

99.8 (C-1C), 83.3 (C-2A), 79.5 (C-3B), 78.5 (C-3C), 72.8 (C-3A),

71.1 (C-4B, C-4C), 70.8 (C-5A), 70.2 (NHCOCH₂C(CH₃)₂OH),

69.7 (C-2B, C-2C), 69.3 (C-5C), 69.0 (C-5B), 63.4 (CH₂CH₂NH),

60.0 (OCH₃), 56.6 (C-4A), 48.9 (NHCOCH₂C(CH₃)₂OH), 39.1

(CH₂CH₂NH), 28.3, 28.1 (NHCOCH₂C(CH₃)₂OH), 17.0 (C-6A),

16.6 (C-6B, C-6C); HRMS (ES⁺) Calcd. for C₂₆H₄₈N₂NaO₁₄

(MNa⁺) 635.3003. Found 635.3011.

AcOH (30 mL) and a catalytic amount of Pd(OH)₂were added

to a solution of 30 (495 mg, 1.14 mmol) in tBuOH (40 mL). The

reaction mixture was stirred at room temperature under hydrogen.

The reaction was monitored by mass spectrometry. After 12 h, the

reaction was complete. The catalyst was ﬁltered over celite and

the ﬁltrate was concentrated. The residue was dissolved in water

(30 mL) and washed with CHCl₃(15 mL). The organic layer was

extracted with water (5 ¥ 10 mL). The aq. layers were combined

and concentrated. The residue was puriﬁed by high performance

liquid chromatography (Prevail C₁₈5 mm (4.6 ¥ 250 mm), H₂O-

MeOH 1:0 to 0:1 in 30 min.) to afford 31 (159 mg, 67%) as a white

powder, [a]_D-8 (c 0.16, H₂O); d_H(300 MHz, D₂O) 4.74 (broad s,

1H, H-1), 3.91-3.88 (m, 1H, H-2), 3.72 (dd, 2H, J_2,33.3 Hz, J_3,4

9.7 Hz, H-3, CH₂CH₂NH), 3.51-3.44 (m, 1H, CH₂CH₂NH), 3.38

(t, 1H, J_3,4= J_4,59.7 Hz, H-4), 3.17 (t, 1H, J 5.5 Hz, NH₂), 2.84

(broad s, 2H, CH₂CH₂NH), 1.23 (d, 3H, J_5,66.2 Hz, H-6); d_C

(75 MHz, D₂O) 99.9 (C-1), 72.0 (C-4), 70.2 (C-2), 70.0 (C-3), 68.6

(C-5), 68.0 (CH₂CH₂NH), 39.8 (CH₂CH₂NH), 16.6 (C-6); HRMS

(ES⁺) Calcd. for C₈H₁₈NO₅(MH⁺) 208.1185. Found 208.1186.

Found: C, 42.03; H, 8.24; N, 5.85. C₈H₁₇NO₅requires C, 42.66; H,

8.50; N, 5.85%.

N-Benzyloxycarbonylaminoethyl 4-O-benzyl-a-L-rhamno-

pyranosyl-(1→3)-4-O-benzyl-a-L-rhamnopyranoside (32)

A solution of sodium methoxide (1.5 mL, 1 M in MeOH) was

◦

added dropwise at 0 C to a solution of 19 (320 mg, 0.37 mmol)

in MeOH (10 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺) was

added to neutralise MeONa. The resin was ﬁltered and the solvent

removed. The residue was then puriﬁed by ﬂash chromatography

(3:2 cyclohexane-EtOAc) to give 32 (200 mg, 82%) as a white

powder, [a]_D-52 (c 0.18, CHCl₃); d_H(300 MHz, CDCl₃) 7.40-

7.30 (m, 10H, Ar), 5.23-5.21 (m, 1H, NH), 5.14 (broad s, 2H,

NHCOOCH₂Ar), 5.11 (d, 1H, J < 1 Hz, H-1B), 4.81 (d, 1H, J

11.2 Hz, OCH₂Ar), 4.75-4.60 (m, 4H, H-1C, OCH₂Ar), 3.99-3.86

(m, 4H, H-2B, H-3B, H-2C, H-5C), 3.75-3.69 (m, 3H, H-5B, H-

3C, CH₂CH₂NH), 3.51-3.38 (m, 5H, H-4B, H-4C, CH₂CH₂NH,

CH₂CH₂NH), 2.82, 2.76, 2.68 (3 broad s, 3H, OH), 1.38 (d, 3H,

J_5,66.2 Hz, H-6B or H-6C), 1.31 (d, 3H, J_5,66.2 Hz, H-6B or

H-6C); d_C(75 MHz, CDCl₃)156.3 (NHCOOCH₂Ar), 138.1-127.8

(Ar), 101.2 (C-1B), 99.3 (C-1C), 81.3, 80.2 (C-4B, C-4C), 79.2 (C-

2B or C-2C), 75.4, 75.1 (OCH₂Ar), 71.3, 70.7 (C-2B or C-2C,

C-3B, C-5C), 68.2, 67.8 (C-5B, C-3C), 66.7 (CH₂CH₂NH,

NHCOOCH₂Ar), 40.6 (CH₂CH₂NH), 18.0, 17.7 (C-6B, C-6C);

HRMS (ES⁺) Calcd. for C₃₆H₄₅NNaO₁₁(MNa⁺) 690.2890. Found

N-Benzyloxycarbonylaminoethyl 4-O-benzyl-a-L-

rhamnopyranoside (30)

A solution of sodium methoxide (3.7 mL, 1 M in MeOH) was

added dropwise at 0 ^◦C to a solution of 15 (1 g, 1.87 mmol)

in MeOH (50 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺) was

added to neutralise MeONa. The resin was ﬁltered and the solvent

removed. The residue was then puriﬁed by ﬂash chromatography

(3:2 cyclohexane-EtOAc) to give 30 (572 mg, 71%) as a white

powder, [a]_D-29 (c 0.16, CHCl₃); d_H(300 MHz, CDCl₃, 300 MHz)

7.39-7.30 (m, 10H, Ar), 5.33-5.31 (m, 1H, NH), 5.13 (broad s, 2H,

NHCOOCH₂Ar), 4.83 (d, 1H, J 11.2 Hz, OCH₂Ar), 4.77 (broad s,

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690.2889. Found: C, 64.29; H, 6.72; N, 2.16. C₃₆H₄₅NO₁₁requires

C, 64.75; H, 6.79; N, 2.10%.

d_C(75 MHz, CDCl₃) 173.0 (NHCO), 169.9 (CH₃CO), 166.4

(OCOAr), 156.6 (NHCOOCH₂Ar), 136.4-121.1 (Ar), 100.6

(C-1), 72.8 (C-3), 71.8 (C-2), 70.8 (C-5), 69.1 (CH₂CH₂NH),

68.8 (NHCOCH₂C(CH₃)₂OH), 66.3 (NHCOOCH₂Ar), 54.4 (C-

4), 47.3 (NHCOCH₂C(CH₃)₂OH), 40.5 (CH₂CH₂NH), 28.8, 28.6

(NHCOCH₂C(CH₃)₂OH), 20.2 (CH₃CO), 17.0 (C-6); LRMS

(ES⁺) Calcd. for C₃₀H₃₈N₂NaO₁₀(MNa⁺) 609. Found 609.

2-Aminoethyl a-L-rhamnopyranosyl-(1→3)-a-L-rhamnopyranoside

(33)

AcOH (11 mL) and a catalytic amount of Pd(OH)₂were added

to a solution of 32 (145 mg, 0.22 mmol) in tBuOH (16 mL). The

reaction mixture was stirred at room temperature under hydrogen.

The reaction was monitored by mass spectrometry. After 12 h, the

reaction was complete. The catalyst was ﬁltered over celite and

the ﬁltrate was concentrated. The residue was dissolved in water

(15 mL) and washed with CHCl₃(7 mL). The organic layer was

extracted with water (5 ¥ 5 mL). The aq. layers were combined and

concentrated. The residue was puriﬁed on a resin cation exchanger

(Bio-Rad, AG MP-50 Resin, analytical grade 100-200 mesh). After

lyophilisation, compound 33 (50 mg, 65%) was obtained as a white

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-4,6-dideoxy-b-D-glucopyranoside (37)

A solution of sodium methoxide (320 mL, 1 M in MeOH)

was added dropwise at 0 ^◦C to a solution of 36 (52 mg,

88 mmol) in MeOH (8 mL). The reaction mixture was stirred

at room temperature for 18 h. An acidic resin (Amberlite IR

120 H⁺) was added to neutralise MeONa. The resin was ﬁltered

and the solvent removed to give 37 (30 mg, 76%) as a white

powder, [a]_D= -5 (c 0.04, CHCl₃); d_H(300 MHz, MeOD) 7.39-

7.32 (m, 5H, Ar), 5.12 (broad s, H, NHCOOCH₂Ar), 4.27 (d,

1H, J_1,27.7 Hz, H-1), 3.92-3.86 (m, 1H, CH₂CH₂NH), 3.69-

3.58 (m, 2H, H-4, CH₂CH₂NH), 3.49-3.39 (m, 4H, H-3, H-5,

CH₂CH₂NH), 3.26 (dd, 1H, J_2,39.0 Hz, H-2), 2.41 (d, 2H, J 2.8 Hz,

NHCOCH₂C(CH₃)₂OH), 1.32, 1.31 (2 s, 6H, NHCOCH₂C-

(CH₃)₂OH), 1.24 (d, 3H, J_5,66.1 Hz, H-6); d_C(75 MHz, MeOD)

175.4.0 (NHCO), 156.6 (NHCOOCH₂Ar), 131.6-120.9 (Ar), 105.2

(C-1), 76.7 (C-2), 76.2, 73.2 (C-3, C-5), 71.5 (NHCOCH₂C-

(CH₃)₂OH), 70.8 (CH₂CH₂NH), 68.4 (NHCOOCH₂Ar), 59 (C-

4), 49.9 (NHCOCH₂C(CH₃)₂OH), 42.8 (CH₂CH₂NH), 30.5, 30.4

(NHCOCH₂C(CH₃)₂OH), 19.3 (C-6); HRMS (ES⁺) Calcd. for

C₂₁H₃₂N₂NaO₈(MNa⁺) 463.2056. Found 463.2036.

solid, [a]_D-66 (c 0.06, H₂O); d_H(300 MHz, D₂O) 5.04 (d, 1H, J_1,2

<

1 Hz, H1-B), 4.79 (broad s, 1H, H-1C), 4.09-4.04 (m, 2H, H-2B, H-

2C), 3.87-3.73 (m, 5H, H-3B, H-5B, H-3C, H-5C, CH₂CH₂NH),

3.57-3.44 (m, 3H, H-B, H-4C, CH₂CH₂NH), 3.25 (t, 2H, J 5.3 Hz,

NH₂), 3.02-3.00 (m, 2H, CH₂CH₂NH), 1.31-1.25 (m, 6H, H-6B,

H-6C); d_C(75 MHz, D₂O) 102.5 (C-1B), 99.8 (C-1C), 78.4 (C-3B),

72.0, 71.3 (C-4B, C-4C), 70.1, 69.9 (C-2B, C-2C, C-3C), 69.1, 68.8

(C-5B, C-5C), 66.9 (CH₂CH₂NH), 39.6 (CH₂CH₂NH), 16.6 (C-

6B, C-6C); HRMS (ES⁺) Calcd. for C₁₄H₂₈NO₉(MH⁺) 354.1764.

Found 354.1769. Found: C, 36.79; H, 8.44; N, 3.74. C₁₄H₂₇NO₉

requires C, 36.44; H, 8.52; N, 3.04%.

N-Benzyloxycarbonylaminoethyl 2-O-acetyl-4-(3-hydroxy-3-

methylbutamido)-3-O-benzoyl-4,6-dideoxy-b-D-glucopyranoside

(36)

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-2-O-acetyl-3-O-benzoyl-4,6-dideoxy-b-D-glucopyranosyl-

(1→3)-2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranosyl-(1→3)-

2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranoside (39)

NaBH₄(32 mg, 0.84 mmol) and a catalytic amount of NiCl₂.6H₂O

were added to a solution of 34^3c(215 mg, 0.42 mmol) in a mixture of

dry CH₂Cl₂(10 mL) and EtOH (40 mL). The reaction mixture was

stirred at room temperature for 1 h and concentrated. The residue

was dissolved in CH₂Cl₂(30 mL). The organic layer was washed

with water (10 mL) and brine (10 mL), dried over Na₂SO₄, ﬁltered

and concentrated. Amine 35 (203 mg, 0.42 mmol) was used in the

next step without puriﬁcation. To a solution of compound 35 (203

mg) in dry CH₂Cl₂(30 mL) was added dropwise in the following

order: 3-hydroxy-3-methylbutanoic acid (79 mL, 0.62 mmol),

HATU (240 mg, 0.63 mmol) then DIPEA (108 mL, 0.63 mmol).

The reaction mixture was stirred at room temperature under argon

for 18 h. The residue was diluted with CH₂Cl₂(10 mL). The organic

layer was washed with saturated aq NaHCO₃(10 mL), water

(10 mL), dried over Na₂SO₄, ﬁltered and concentrated. The residue

was puriﬁed by ﬂash chromatography (1:1 cyclohexane-EtOAc) to

give 36 (152 mg, 62%) as a white powder, [a]_D= +18 (c 1.8, CHCl₃);

d_H(300 MHz, CDCl₃) 7.91-7.10 (m, 10H, Ar), 6.26 (d, 1H, J_NH,H4

10.1 Hz, NHCO), 5.55-5.53 (m, 1H, NHCOOCH₂Ar), 5.35 (t,

1H, J_2,3= J_3,410.1 Hz, H-3), 5.04 (m, 3H, H-2, NHCOOCH₂Ar),

NaBH₄(15 mg, 0.4 mmol) and a catalytic amount of NiCl₂.6H₂O

were added to a solution of 23 (146 mg, 0.12 mmol) in a mixture

of dry CH₂Cl₂(2 mL) and EtOH (9 mL). The reaction mixture

was stirred at room temperature for 3 h 30 min and concentrated.

The residue was dissolved in CH₂Cl₂and washed with water and

brine, then dried over Na₂SO₄, ﬁltered and concentrated. Amine

38 (131 mg) was used in the next step without puriﬁcation. To a

solution of compound 38 (131 mg) in dry CH₂Cl₂(2.5 mL) was

added in the following order: 3-hydroxy-3-methylbutanoic acid

(30 mL, 236 mmol), HATU (90 mg, 236 mmol) then DIPEA (40 mL,

236 mmol). The reaction mixture was stirred at room temperature

under argon for 18 h. The residue was diluted with CH₂Cl₂and

the organic solution was washed twice with saturated aq NaHCO₃,

then with water, dried over Na₂SO₄, ﬁltered and concentrated. The

residue was puriﬁed by ﬂash chromatography (3:2 cyclohexane-

EtOAc) to give 39 (82 mg, 53%) as a colourless oil, [a]_D= +10

(c 0.77, CHCl₃); d_H(300 MHz, CDCl₃) 8.11-7.17 (m, 30H, Ar),

6.01 (d, 1H, J_NH,H49 Hz, NHCO), 5.43-5.38 (m, 2H, H-2B,

H-2C), 5.22 (broad s, 2H, H-1B, NHCOOCH₂Ar), 5.12-5.01 (m,

5H, H-2A, H-3A, OCH₂Ar, NHCOOCH₂Ar), 4.85 (broad s, 1H,

H-1C), 4.75 (d, 1H, J 12 Hz, OCH₂Ar), 4.75 (d, 1H, J 12 Hz,

OCH₂Ar), 4.69 (d, 1H, J 12 Hz, OCH₂Ar), 4.51 (d, 1H, J 12 Hz,

OCH₂Ar), 4.43 (d, 1H, J_1,29 Hz, H-1A), 4.28 (dd, 1H, J_2,33 Hz,

4.49 (d, 1H, J_1,27.9 Hz, H-1), 4.05 (q, 1H, J_3,4= J_4,5

=

J_NH,H410.1 Hz, H-4), 3.83-3.78 (m, 1H, CH₂CH₂NH), 3.68-3.61

(m, 2H, H-5, CH₂CH₂NH), 3.33-3.31 (m, 1H, CH₂CH₂NH),

2.22 (d, 1H, J 14.6 Hz, NHCOCH₂C(CH₃)₂OH), 2.13 (d, 1H,

NHCOCH₂C(CH₃)₂OH), 1.81 (s, 3H, CH₃CO), 1.22 (d, 3H,

J_5,65.8 Hz, H-6), 1.03, 0.92 (2 s, 6H, NHCOCH₂C(CH₃)₂OH);

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J_3,49 Hz, H-3B or H-3C), 4.10 (dd, 1H, J_2,33 Hz, J_3,49 Hz, H-

3B or H-3C), 3.92-3.30 (m, 9H, H-4A, H-4B, H-4C, H-5B, H-5C,

CH₂CH₂NH), 3.03-2.94 (m, 1H, H-5A), 2.22 (d, 1H, J 15 Hz,

NHCOCH₂C(CH₃)₂OH), 2.14 (d, 1H, NHCOCH₂C(CH₃)₂OH),

1.53 (s, 3H, CH₃CO), 1.32 (d, 3H, J_5,66 Hz, H-6B or H6-

C), 1.16 (d, 3H, J_5,66 Hz, H-6B or H-6C), 1.10 (s, 3H,

NHCOCH₂C(CH₃)₂OH), 1.01 (s, 3H, NHCOCH₂C(CH₃)₂OH),

0.90 (d, 3H, J_5,66 Hz, H-6A); d_C(75 MHz, CDCl₃) 172.1

(NHCO), 169.1 (CH₃CO), 166.9, 165.8, 165.7 (OCOC₆H₅), 156.3

(NHCOOCH₂Ar), 138.0-127.2 (Ar), 100.5 (C-1A), 98.7 (C-1B),

97.2 (C-1C), 79.9, 79.0 (C-4B, C-4C), 78.7, 77.7 (C-3B, C-3C), 75.3

(OCH₂Ar), 74.2 (OCH₂Ar), 73.3 (C-3A), 72.3, 72.1 (C-2B, C-2C),

71.5 (C-2A), 71.3 (C-5A), 69.2 (NHCOCH₂C(CH₃)₂OH), 68.5,

67.9 (C-5B, C-5C), 67.1 (CH₂CH₂NH), 66.7 (NHCOOCH₂Ar),

54.5 (C-4A), 47.9 (NHCOCH₂C(CH₃)₂OH), 40.8 (CH₂CH₂NH),

29.1, 29.0 (NHCOCH₂C(CH₃)₂OH), 20.2 (CH₃CO), 18.0 (C-6B

or C-6C), 17.7 (C-6B or C-6C), 17.3 (C-6A); HRMS (ES⁺) Calcd.

for C₇₀H₇₈N₂NaO₂₀(MNa⁺) 1289.5046. Found 1289.5012.

H-3a, H-1b), 5.73 (t, 1H, J_2,3= J_3,49 Hz, H-3b), 5.52 (dd, 1H,

J_1,28 Hz, H-2b), 5.41 (dd, 1H, H-2a), 4.72-4.60 (m, 4H, 2H-

6a, 2H-6b), 4.17-4.11 (m, 2H, H-5a, H-4b), 4.01-3.95 (m, 2H,

H-4a, H-5b), 2.16 (s, 3H, CH₃CO); d_C(75 MHz, CDCl₃) 168.58

(CH₃CO), 166.01, 165.56, 165.33 (OCOAr), 133.56-128.47 (Ar),

91.84 (C-1b), 89.24 (C-1a), 73.60 (C-3b), 73.43 (C-5b), 70.80 (C-

3a), 70.68 (C-2b), 70.60 (C-5a), 70.11 (C-2a), 62.89 (C-6b), 62.76

(C-6a), 60.41 (C-4a, C-4b), 20,78 (CH₃CO); HRMS (ES⁺) Calcd.

for C₂₉H₂₅N₃NaO₉(MNa⁺) 582.1489. Found 582.1473. Found: C,

62.44; H, 4.69; N, 7.42. C₂₉H₂₅N₃O₉requires C, 62.25; H, 4.50; N

7.51%.

4-Azido-2,3,6-tri-O-benzoyl-4-deoxy-D-glucopyranose (44)

Hydrazine acetate (450 mg, 4.9 mmol) was added to a solution of

derivative 43 (2.57 g, 4.6 mmol) in DMF (15 mL). The reaction

mixture was stirred for 4 hours and then diluted in ethyl acetate

(50 mL). The organic layer was washed with brine (2 ¥ 50 mL)

and with water (50 mL). The organic layer was dried over Na₂SO₄,

ﬁltered and concentrated to afford crude 44 (2.17 g, 91%) as an a/b

(7.5:2.5) mixture which was used in the next step without further

puriﬁcation, d_H(300 MHz, CDCl₃) 8.09-7.24 (m, 15H, Ar), 6.06

(t, 1H, J_2,3= J_3,410 Hz, H-3a), 5.74 (t, 1H, J_2,3= J_3,49.8 Hz,

H-3b), 5.68 (d, 1H, J_1,23.5 Hz, H-1a), 5.29 (dd, 1H, J_1,28 Hz,

H-2b), 5.22 (dd, 1H, H-2a), 4.97 (d, 1H, H-1b), 4.75-4.55 (m, 4H,

2H-6a, 2H-6b), 4.34-4.28 (m, 1H, H-5a), 3.90 (t, 1H, J_4,510 Hz,

H-4b), 3.82-3.78 (m, 1H, H-5a); d_C(75 MHz, CDCl₃) 166.34,

165.94, 165.65 (OCOAr), 133.43-128.43 (Ar), 95.91 (C-1b), 90.49

(C-1a), 74.11 (C-2b), 72.88 (C-3b, C-5b), 72.08 (C-2a), 70.71 (C-

3a), 69.09 (C-5a), 63.22 (C-6a, C-6b), 60.95 (C-4a, C-4b); LRMS

(ES⁺) Calcd. for C₂₇H₂₃N₃NaO₈(MNa⁺) 540. Found 540.

2-Aminoethyl 4-(3-hydroxy-3-methylbutamido)-6-deoxy-b-

D-glucopyranosyl-(1→3)-a-D-rhamnopyranosyl-(1→3)-a-D-

rhamnopyranoside (41)

A solution of sodium methoxide (0.6 mL, 1 M in MeOH) was

added dropwise at 0 ^◦C to a solution of 39 (75 mg, 59 mmol)

in MeOH (2 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺)

was added to neutralise MeONa. The resin was ﬁltered and the

solvent removed. AcOH (3 mL) and a catalytic amount of Pd(OH)₂

were added to a solution of crude 40 in tBuOH (6 mL). The

reaction mixture was stirred at room temperature under hydrogen.

The reaction was monitored by mass spectrometry. After 20 h,

the reaction was complete. The catalyst was ﬁltered over celite

and the ﬁltrate was concentrated. The residue was puriﬁed on

a resin cation exchanger (Bio-Rad, AG MP-50 Resin, analytical

grade 100-200 mesh). After lyophilisation, compound 41 (18 mg,

50%) was obtained as a white solid, [a]_D-58 (c 0.45, H₂O); d_C

(75 MHz, D₂O) 174.0 (NHCO), 103.5 (C-1A), 102.2 (C-1B), 99.8

(C-1C), 79.5, 78.4, 74.0, 73.4, 71.9, 71.2, 71.0, 69.8, 68.9 (C-2A,

C-3A, C-5A, C-2B, C-3B, C-4B, C-5B, C-2C, C-3C, C-4C,

C-5C, NHCOCH₂C(CH₃)₂OH), 65.9 (CH₂CH₂NH), 56.5 (C-4A),

48.9 (NHCOCH₂C(CH₃)₂OH), 39.5 (CH₂CH₂NH), 28.3, 28.1

(NHCOCH₂C(CH₃)₂OH), 17.08, 16.6, 16.5 (C-6A, C-6B, C-6C);

HRMS (ES⁺) Calcd. for C₂₅H₄₇N₂O₁₄(MH⁺) 599.3027. Found

599.3030.

4-Azido-2,3,6-tri-O-benzoyl-4-deoxy-a-D-glucopyranosyl

trichloroacetimidate (45)

Trichloroacetonitrile (5 mL) and DBU (80 mL) were added to a

solution of compound 44 (1.08 g, 2 mmol) in dry CH₂Cl₂(5 mL).

After 1 h of stirring at 0 ^◦C, solvent was removed. The residue was

puriﬁed by ﬂash chromatography (7.5:2.5 cyclohexane-EtOAc,

Et₃N 2%) to afford the donor 45 (1.2 g, 87%), d_H(300 MHz,

CDCl₃) 8.60 (s, 1H, NH), 8.09-7.34 (m, 15H, Ar), 6.73 (d, 1H,

J_1,23.6 Hz, H-1), 6.09 (t, 1H, J_2,3= J_3,410 Hz, H-3), 5.48 (dd,

1H, H-2), 4.68-4.66 (m, 2H, H-6a, H-6b), 4.29-4.24 (m, 1H, H-5),

4.01 (t, 1H, J_4,510 Hz, H-4); d_C(75 MHz, CDCl₃) 165.99, 165.40

(OCOAr), 160.42 (OCNHCCl₃), 133.59-128.43 (Ar), 93.14 (C-1),

70.95 (C-5), 70.74 (C-3), 70.47 (C-2), 62.77 (C-6), 60.26 (C-4);

LRMS (ES⁺) Calcd. for C₂₉H₂₃Cl₃N₃NaO₈(MNa⁺) 683. Found

683.

1-O-Acetyl-4-azido-2,3,6-tri-O-benzoyl-4-deoxy-D-glucopyranose

(43)

N-Benzyloxycarbonylaminoethyl 4-azido-2,3,6-tri-O-benzoyl-4-

deoxy -b-D-glucopyranoside (46)

A solution of the known methyl glucoside derivative 42¹⁹(4,8 g,

9 mmol) in a mixture Ac₂O (38 mL), AcOH (11 mL) and H₂SO₄

(280 mL) was stirred at 50 ^◦C for 24 hours. The reaction mixture was

concentrated and the residue dissolved in ethyl acetate (50 mL).

The organic layer was washed with saturated aq. NaHCO₃(50 mL)

and brine (50 mL). The organic layer was dried over Na₂SO₄,

ﬁltered and concentrated. The residue was puriﬁed by ﬂash

chromatography (4:1 cyclohexane-EtOAc) to give 43 (3.74 g, 74%)

as an a/b (9:1) mixture, d_H(300 MHz, CDCl₃) 8.10-7.35 (m, 15H,

Ar), 6.54 (d, 1H, J_1,23.6 Hz, H-1a), 5.98 (t, 2H, J_2,3= J_3,410 Hz,

TMSOTf (130 mL, 0.71 mmol) was added at 0 ^◦C to a solution of

45 (1.2 g, 1.8 mmol) and benzyl N-(2-hydroxyethyl)carbamate²¹

(1.1 g, 5.64 mmol) in dry CH₂Cl₂(12 mL). The reaction mixture

was stirred at 0 ^◦C for 1 h, then quenched by addition of

Et₃N. The reaction mixture was concentrated. The residue was

puriﬁed by ﬂash chromatography (7.5:2.5 cyclohexane-EtOAc) to

give 46 (920 mg, 73%), [a]_D+66 (c 3.7, CHCl₃); d_H(300 MHz,

CDCl₃) 8.12-7.29 (m, 20H, Ar), 5.68 (t, 1H, J_2,3= J_3,49.8 Hz,

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H-3), 5.41 (dd, 1H, J_1,27.9 Hz, H-2), 5.20 (broad s, 1H,

NH), 5.05 (d, 1 H, J 12 Hz, NHCOOCH₂Ar), 4.96 (d, 1 H,

NHCOOCH₂Ar), 4.80 (dd, 1H, J_5,62.3 Hz, J_6,612.2 Hz H-

6a), 4.74 (d, 1H, H-1), 4.62 (dd, 1H, J_5,64.5 Hz, H-6b),

3.98-3.90 (m, 3H, H-4, CH₂CH₂NH), 3.85-3.70 (m, 1H, H-5),

3.40-3.35 (m, 2H, CH₂CH₂NH); d_C(75 MHz, CDCl₃) 166.06,

165.54, 165.29 (OCOAr), 156.26 (NHCOOCH₂Ar), 133.49-127.98

(Ar), 101.34 (C-1), 73.46 (C-3), 72.66 (C-5), 71.78 (C-2), 69.55

(CH₂CH₂NH), 66.51 (NHCOOCH₂Ar), 63.07 (C-6), 60.60.74 (C-

4), 40.80 (CH₂CH₂NH); HRMS (ES⁺) Calcd. for C₃₇H₃₄N₄NaO₁₀

(MNa⁺) 717.2173. Found 717.2175.

of Pd(OH)₂were added to a solution of crude 49 in tBuOH

(12 mL). The reaction mixture was stirred at room temperature

under hydrogen. The reaction was monitored by mass spec-

trometry. After 20 h, the reaction was complete. The catalyst

was ﬁltered over celite and the ﬁltrate was concentrated. The

residue was puriﬁed on a resin cation exchanger as described

for 29. After lyophilisation, compound 50 was obtained as a

white powder (42 mg, 72%), [a]_D-15 (c 0.5, H₂O); d_C(75 MHz,

D₂O) 174.3 (NHCO), 102 (C-1), 74.9, 73.5, 73.3 (C-2, C-3, C-5),

71.9 (NHCOCH₂C(CH₃)₂OH), 65.8 (CH₂CH₂NH), 60.8 (C-6),

51.4 (C-4), 48.8 (NHCOCH₂C(CH₃)₂OH), 39.5 (CH₂CH₂NH),

28.3, 28.1 (NHCOCH₂C(CH₃)₂OH); HRMS (ES⁺) Calcd. for

C₁₃H₂₇N₂O₇(MH⁺) 323.1818. Found 323.1817.

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-2,3,6-tri-O-benzoyl-4-deoxy-b-D-glucopyranoside (48)

N-Benzyloxycarbonylaminoethyl 4-azido-2,3,6-tri-O-benzoyl-4-

deoxy-b-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-a-D-

rhamnopyranosyl-(1→3)-2-O-benzoyl-4-O-benzyl-a-D-

rhamnopyranoside (51)

NaBH₄(95 mg, 2.5 mmol) and a catalytic amount of NiCl₂.6H₂O

were added to a solution of 46 (880 mg, 1.26 mmol) in a mixture

of dry CH₂Cl₂(18 mL) and EtOH (90 mL). The reaction mixture

was stirred at room temperature for 1 h and concentrated. The

residue was dissolved in CH₂Cl₂. The organic layer was washed

with water and brine, dried over Na₂SO₄, ﬁltered and concentrated.

Amine 47 (709 mg) was used in the next step without puriﬁcation.

To a solution of compound 47 (709 mg 1.06 mmol) in dry

CH₂Cl₂(25 mL) was added dropwise in the following order:

3-hydroxy-3-methylbutanoic acid (293 mL, 2.3 mmol), HATU

(582 mg, 1.53 mmol) then DIPEA (253 mL, 1.46 mmol). The

reaction mixture was stirred at room temperature under argon

for 18 h. The residue was diluted with CH₂Cl₂. The organic layer

was washed twice with saturated aq. NaHCO₃, water, dried over

Na₂SO₄, ﬁltered and concentrated. The residue was puriﬁed by

ﬂash chromatography (2:3 cyclohexane-EtOAc) to give 48 (575 mg,

60%), [a]_D= +57 (c 0.705, CHCl₃); d_H(300 MHz, CDCl₃) 8.12-

7.26 (m, 20H, Ar), 6.61 (d, 1H, J_NH,H49.2 Hz, NHCO), 5.69 (t,

1H, J_2,3= J_3,410.1 Hz, H-3), 5.47 (dd, 1H, J_1,27.9 Hz, H-2),

5.25-5.21 (m, 1H, NHCOOCH₂Ar), 5.04 (d, 1H, J 12.3 Hz,

NHCOOCH₂Ar), 4.94 (d, 1H, NHCOOCH₂Ar), 4.77 (d, 1H, H-

1), 4.74 (dd, 1H, J_5,62 Hz, J_6,612.3 Hz, H-6a), 4.51-4.41 (m, 2H,

H-4, H-6b), 4.03 (ddd, 1H, J_5,6b6.3 Hz, H-5), 3.94-3.88 (m, 1H,

CH₂CH₂NH), 3.74-3.66 (m, 1H, CH₂CH₂NH), 3.41-3.34 (m, 2H,

CH₂CH₂NH), 2.28 (d, 2H, J 1.8 Hz, NHCOCH₂C(CH₃)₂OH),

1.55 (s, 1H, OH), 1.13, 1.09 (2 s, 6H, NHCOCH₂C(CH₃)₂OH); d_C

(75 MHz, CDCl₃) 172.4 (NHCO), 166.8, 166.4, 165.2 (OCOAr),

156.3 (NHCO), 133.6-127.9 (Ar), 101.1 (C-1), 73.69 (C-5), 72.6

(C-3), 71.9 (C-2), 69.4 (CH₂CH₂NH), 66.5 (NHCOOCH₂Ar),

63.7 (C-6), 50.7 (C-4), 48.3 (NHCOCH₂C(CH₃)₂OH), 40.8

(CH₂CH₂NH), 29.2 (NHCOCH₂C(CH₃)₂OH); HRMS (ES⁺)

Calcd. for C₄₂H₄₄N₂NaO₁₂(MNa⁺) 791.2792. Found 791.2781.

Found: C, 65.29; H, 5.72; N, 3.96. C₄₂H₄₄N₂O₁₂requires C, 65.61;

H, 5.77; N 3.6%.

TMSOTf (73 mL, 0.4 mmol) was added under argon at -50 ^◦C

to a solution of donor 45 (479 mg, 0.72 mmol) and acceptor 20

(500 mg, 0.57 mmol) in dry CH₂Cl₂(25 mL) containing molecular

˚

sieves 4 A (600 mg). After 60 min of stirring, the reaction mixture

was allowed to reach room temperature for overnight additional

stirring. The solution was neutralised with Et₃N and the molecular

sieves were removed by ﬁltration. The residue was puriﬁed by ﬂash

chromatography (7:3 cyclohexane-EtOAc) to give a mixture (838

mg) of the expected trisaccharide 51 and some unreacted acceptor

20 which was used in the next step without further puriﬁcation. A

pure fraction was used for the following characterisation of 51, [a]_D

+38 (c 1.2, CHCl₃); d_H(500 MHz, CDCl₃) 8.16-6.91 (m, 40H, Ar),

5.48-5.44 (m, 3H, H-3A, H-2B, H-2C), 5.38 (dd, 1H, J_1,27.7 Hz,

J_2,39.8 Hz, H-2A), 5.29 (t, 1H, J 5.6 Hz, NHCOO), 5.20 (d, 1H,

J

_1,21.6 Hz, H-1B), 5.14 (d, 1H, J 12.3 Hz, NHCOOCH₂Ar), 5.11

(d, 1H, NHCOOCH₂Ar), 5.02 (d, 1H, J 11 Hz, OCH₂Ar), 4.89

(d, 1H, J_1,21.3 Hz, H-1C), 4.73 (d, 1H, OCH₂Ar), 4.62 (d, 1H, H-

1A), 4.54 (d, 1H, J 11.6 Hz, OCH₂Ar), 4.36-4.30 (m, 3H, H-6Aa,

H-3C, OCH₂Ar), 4.23 (dd, 1H, J_5,61.9 Hz, J_6a,6b12.2 Hz, H-6Ab),

4.18 (dd, 1H, J_2,33.4 Hz, J_3,49.3 Hz, H-3B), 3.86-3.78 (m, 3H, H-

5B, H-5C, OCH₂CH₂NHZ), 3.76 (t, 1H, J_3,4= J_4,59.3 Hz, H-4A),

3.66 (t, 1H, J_3,4= J_4,59.4 Hz, H-4C), 3.56 (m, 1H, OCH₂CH₂NH),

3.49-3.37 (m, 3H, H-4B, CH₂CH₂NH), 3.05 (m, 1H, H-5A), 1.39

(d, 3H, J_5,66.1 Hz, H-6C), 1.01 (d, 3H, J_5,66.1 Hz, H-6B); d_C

(125 MHz, CDCl₃) 165.8, 165.7, 165,6, 165.5, 165,2 (OCOAr),

156.3 (NHCOO), 137.9-127.2 (Ar), 101.2 (C-1A), 98.1 (C-1B),

97.3 (C-1C), 80.1 (C-4C), 79.1 (C-3B), 79.0 (C-4B), 76.9 (C-3C),

75.3, 74.4 (OCH₂Ar), 73.5, 72.2, 72.0 (C-3A, C-5A, C-2B, C-2C),

71.7 (C-2A), 68.5 (C-5C), 67.9 (C-5B), 67.1 (CH₂CH₂NH), 66.7

(NHCOOCH₂Ar), 62.3 (C-6A), 64.0 (C-4A), 40.8 (CH₂CH₂NH),

17.9 (C-6C), 17.7 (C-6B); HRMS (ES⁺) Calcd. for C₇₇H₇₄N₄NaO₂₀

(MNa⁺) 1397.4794. Found 1397.4733.

2-Aminoethyl 4-(3-hydroxy-3-methylbutamido)-4-deoxy-b-D-

glucopyranoside (50)

N-Benzyloxycarbonylaminoethyl 4-(3-hydroxy-3-methylbut-

amido)-2,3,6-tri-O-benzoyl-4-deoxy-b-D-glucopyranosyl-(1→3)-

2-O-benzoyl-4-O-benzyl-a-D-rhamnopyranosyl-(1→3)-2-

O-benzoyl-4-O-benzyl-a-D-rhamnopyranoside (53)

A solution of sodium methoxide (1 mL, 1 M in MeOH) was

added dropwise at 0 C to a solution of 48 (138 mg, 0.18 mmol)

in MeOH (9 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺)

was added to neutralise MeONa. The resin was ﬁltered and

the solvent removed. AcOH (10 mL) and a catalytic amount

◦

NaBH₄(50 mg, 1.3 mmol) and a catalytic amount of NiCl₂.6H₂O

were added to a solution of 51 (885 mg, 0.64 mmol) in a mixture of

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dry CH₂Cl₂(10 mL) and EtOH (45 mL). The reaction mixture was

stirred at room temperature for 2 h, then additional NaBH₄(50 mg,

1.3 mmol) and a catalytic amount of NiCl₂.6H₂O were added.

The reaction mixture was stirred overnight and concentrated.

The residue was dissolved in CH₂Cl₂. The organic layer was

washed with water and brine, dried over Na₂SO₄, ﬁltered and

concentrated. Amine 52 (642 mg) was used in the next step without

puriﬁcation. To a solution of compound 52 (642 mg) in dry CH₂Cl₂

(10 mL) were added dropwise in the following order: 3-hydroxy-

3-methylbutanoic acid (132 mL, 1.05 mmol), HATU (399 mg,

1.05 mmol) then DIPEA (180 mL, 1.05 mmol). The reaction mix-

ture was stirred at room temperature under argon for 3 days. The

residue was diluted with CH₂Cl₂. The organic layer was washed

with saturated aq. NaHCO₃, water, dried over Na₂SO₄, ﬁltered and

concentrated. The residue was puriﬁed by ﬂash chromatography

(3:2 cyclohexane-EtOAc) to give 53 (442 mg, 47%), [a]_D= +21

(c 0.675, CHCl₃); d_H(300 MHz, CDCl₃) 8.16-6.91 (m, 40 H, Ar),

6.15 (d, 1H, J_NH,H49 Hz, NHCO), 5.57-5.06 (m, 9H, H-2A, H-3A,

H-1B, H-2B, H-2C, NHCOOCH₂Ar, OCH₂Ar, NHCOO), 4.91

(broad s, 1H, H-1C), 4.74-4.70 (m, 2H, H-1A, OCH₂Ar), 4.57 (d,

1H, J 12 Hz, OCH₂Ar), 4.39-4.16 (m, 5H, H-4A, H-6Aa, H-3B,

H-3C, OCH₂Ar), 3.86-3.35 (m, 10H, H-6Ab, H-4B, H-5B, H-4C,

H-5C, CH₂CH₂NH), 2.18 (broad s, 2H, NHCOCH₂C(CH₃)₂OH),

1.44 (s, 3H, NHCOCH₂C(CH₃)₂OH), 1.36 (d, 3H, J_5,66.1 Hz,

H-6B or H-6C), 1.08 (s, 3H, NHCOCH₂C(CH₃)₂OH), 1.00 (d,

3H, J_5,66.1 Hz, H-6B or H-6C); d_C(75 MHz, CDCl₃) 172.0

(NHCO), 166.6, 166.2, 165.7, 165.6, 165.0 (OCOAr), 156.3

(NHCOOCH₂Ar), 138.1-127.2 (Ar), 101.2 (C-1A), 98.4 (C-1B),

97.2 (C-1C), 80.1, 79.0 (C-4B, C-4C), 78.9, 77.4 (C-3C, C-3B),

75.2, 74.2 (OCH₂Ar), 72.7, 72.6, 72.3, 72.1, 71.9 (C-2A, C-

3A, C-5A, C-2B, C-2C), 69.3 (NHCOCH₂C(CH₃)₂OH), 68.4,

67.8 (C-5B, C-5C), 67.1 (CH₂CH₂NH), 66.7 (NHCOOCH₂Ar),

62.4 (C-6A), 50.2 (C-4A), 48.1 (NHCOCH₂C(CH₃)₂OH), 40.7

(CH₂CH₂NH), 29.2, 29.1 (NHCOCH₂C(CH₃)₂OH), 18.0, 17.7

(C-6B, C-6C); HRMS (ES⁺) Calcd. for C₈₂H₈₄N₂NaO₂₂(MNa⁺)

1471.5413. Found 1471.5450. Found: C, 67.82; H, 6.41; N, 1.97.

C₈₂H₈₄N₂O₂₂requires C, 67.94; H, 5.84; N 1.93%.

NHCOCH₂C(CH₃)₂OH), 66.8 (CH₂CH₂NH), 60.9 (C-6A), 51.4

(C-4A), 48.8 (NHCOCH₂C(CH₃)₂OH), 39.6 (CH₂CH₂NH), 28.3,

28.1 (NHCOCH₂C(CH₃)₂OH), 16.6 (C-6B, C-6C); HRMS (ES⁺)

Calcd. for C₂₅H₄₇N₂O₁₅(MH⁺) 615.2977. Found 615.2979.

Antiserum production

For the production of antibodies, the trisaccharide derivative 29

was covalently linked to KLH (keyhole limpet haemocyanin) using

glutaraldehyde, which cross-links primary amino groups of the

carrier protein to the spacer arm of the monosaccharide analogue.

Derivative 29 (3.54 mg, 5.8 mmol) dissolved in 50% MeOH was

reacted with KLH (20 mg, 0.29 mmol) and glutaraldehyde (25 mL,

25% in water) in phosphate buffer (0.1 M, 5 mL, pH 7.4). The

reaction mixture was stirred overnight at 4 ^◦C in the dark before

aliquoting and freezing at -20 ^◦C.

Rabbits (Blanc de Bousact, Evic, France) are housed under

normal conditions in accredited animal husbandry, receiving tap

water and food given ad libitum. All experiments are performed

according to the European Community rules of animal care and

are supervised by authorized researchers (permissions delivered

by the French Veterinary Authorities). Rabbits were immunised

and given booster injections every two months with immunogen

(1 mg in complete Freund’s adjuvant) in multiple subcutaneous

injections according to the procedure described by Vaitukaitis.²⁴

Rabbits were bled from the central ear artery before the ﬁrst

immunisation (serum S₀), then on a weekly basis after each booster

(serums S₁, S₂,. . .). The sera were kept at 4 ^◦C in the presence of

sodium azide (0.01% ﬁnal concentration).

Preparation of the enzymatic tracer

The tracer was obtained by covalently coupling derivative 29 to

AChE using the procedure previously described for haptens²⁵and

proteins.²⁶In the ﬁrst step, a thiol group was introduced into

the hapten 29 by reaction of its primary amino group with

N-succinimidyl-S-acetyl thioacetate (SATA). Brieﬂy, a solution

of SATA (1.15 mg, 5 mmol) and derivative 29 (0.31 mg, 0.5 mmol)

in a borate buffer (130 mL, 0.1 M, pH 8.5) was stirred for 1 h at

20 ^◦C. The thiolated derivative was puriﬁed using a Sep-Pak-C18

cartridge (Waters, Milford, USA) before deprotecting the thiol

function in the presence of hydroxylamine. The enzyme conjugate

was obtained by mixing the thiolated derivative 29 (0.1 nmol) with

AChE-SMCC (0.1 nmol) prepared as previously described²⁴for

18 h at 4 ^◦C. The enzyme conjugate was puriﬁed by molecular

sieve chromatography on a Bio-Gel A 1.5 M column (90 ¥ 1.5 cm

2-Aminoethyl 4-(3-hydroxy-3-methylbutamido)-4-deoxy-b-D-

glucopyranosyl-(1→3)-a-D-rhamnopyranosyl-(1→3)-a-D-

rhamnopyranoside (55)

A solution of sodium methoxide (1.6 mL, 1 M in MeOH) was

◦

added dropwise at 0 C to a solution of 53 (227 mg, 0.15 mmol)

in MeOH (5 mL). The reaction mixture was stirred at room

temperature for 18 h. An acidic resin (Amberlite IR 120 H⁺) was

added to neutralise MeONa. The resin was ﬁltered and the solvent

removed. AcOH (8 mL) and a catalytic amount of Pd(OH)₂were

added to a solution of crude 54 in tBuOH (15 mL). The reaction

mixture was stirred at room temperature under hydrogen. The

reaction was monitored by mass spectrometry. After 20 h, the

reaction was complete. The catalyst was ﬁltered over celite and

the ﬁltrate was concentrated. The residue was puriﬁed on a resin

cation exchanger (Bio-Rad, AG MP-50 Resin, analytical grade

100-200 mesh). After lyophilisation, compound 55 (44 mg, 44%)

was obtained as a white solid, [a]_D-48 (c 0.78, H₂O); d_C(75 MHz,

D₂O) 174.2 (NHCO), 103.6 (C-1A), 102.2 (C-1B), 99.8 (C-1C),

80.0, 78.4, 74.8, 73.8, 73.3, 71.3, 71.0, 70.1, 69.8, 69.0, (C-2A, C-

3A, C-5A, C-2B, C-3B, C-4B, C-5B, C-2C, C-3C, C-4C, C-5C,

◦

Bio-Rad) eluted with EIA buffer and was stored at -20 C until

use.

Competitive EIA procedure

Competitive EIA was performed in 96-well microtitre plates

coated with mouse monoclonal anti-rabbit immunoglobulin an-

tibodies in order to ensure separation between the free and

bound moieties of the enzymatic tracer during the immunolog-

ical reaction. The assay was performed in a total volume of

150 mL, each reagent (enzymatic tracer, diluted rabbit antisera

and standard (derivatives 2, 27, 28, 29, 31, 33, 37, 41, 50, 55))

being added in a 50 mL volume. The optimal working dilution of

antiserum was previously determined by serial dilution. After 18 h

5198 | Org. Biomol. Chem., 2009, 7, 5184–5199

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◦

of immunological reaction at 4 C, the plates were washed with

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the washing buffer and AChE substrate (200 mL, Ellman’s reagent)

was added to each well. After 1 or 2 h of gentle shaking in the dark

at room temperature, the absorbance of each well was measured

at 414 nm. The results were expressed in terms of (B/B₀) ¥ 100

as function of concentration (logarithmic scale) where B and B₀

represent bound enzymatic activity in the presence and absence

of the competitor (antigen or analogues), respectively. A linear

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Sensitive and specific enzyme immunoassays for antigenic trisaccharide from Bacillus anthracis spores

DOI: 10.1039/b914534f

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