CA2434685A1

CA2434685A1 - Novel approach to design glycopeptides based on o-specific polysaccharide of shigella flexneri serotype 2a

Info

Publication number: CA2434685A1
Application number: CA002434685A
Authority: CA
Inventors: Laurence Mulard; Armelle Phalipon; Philippe Sansonetti; Francoise Baleux; Frederic Belot; Cyrille Grandjean
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-07-04
Filing date: 2003-07-04
Publication date: 2005-01-04

Abstract

Both intestinal secretory IgA (SIgA) and serum IgG specific for the O-antigen (O-Ag), the polysaccharide part of the bacterial lipopolysaccharide (LPS) are elicited upon Shigella infection, the causative agent of bacillary dysentery. We have addressed here the protective role of the anti-LPS IgG response, using the marine model of pulmonary infection. Upon intraperitoneal (i.p.) immunization writh killed Shigella flexneri 2a bacteria, mice were shown to elicit a serum, but not a local, anti-LPS IgG response that conferred only partial protection against intranasal (i.n.) challenge with the homologous virulent strain.
However. mice intranasally administered with, prior to in challenge, an anti-LPS IgG
polyclonal serum from i.p. immunized mice, showed a significant antibody dose-dependent decrease of the lung-bacterial load in comparison to mice that received preimmune serum. Using marine monoclonal antibodies (mAbs) of the G isotype (mIgG) representative of the different IgG
subclasses and specific for serotype-specific determinants on the O-Ag, we showed that each IgG subclass exhibited a similar serotype-specific protective capacity, with significant reduction of the lung-bacterial load and of subsequent inflammation and tissue destruction. In contrast, different subclasses of mIgG specific for the invasins IpaB or IpaC
did riot confer protection. In conclusion, the IgG-mediated systemic response to serotype-specific determinants contributes to protection against homologous Shigella infection, if the effectors are present locally at the time of mucosal infection.

Description

LMPPZo Btockwise approach to fragments of the O-specific polysaccharide of Sltigella Jlexneri serotype 2a: Convergent synthesis of a decasaccharide representative of a dimer of the branched repeating unit' This paper discloses the synthesis of a decasaccharide corresponding to two consecutive repeating units of the 0-Ag of S Jlex~eri 2a, based on the condensation of two key pentasaccharide irztermsdiates. Several routes to these two building blocks were investigated, involving either blockwise strategies or a lutear one. The latter was the preferred one based on yields of condensation and the number of steps.

LMPPIO.fheo-bt'e~ct-dacaOMc Bloclcwise approach to fragments of the O-specific polysaccharide of Shigella flexneti serotype Za: Convergent synthesis of a decpsaccharide representative of a dimer of the branched repeating unite ABSTRACT
Introduction i Shigellosis or bacillary dysentery is a worldwide disease, occurring in humans only, caused by organisms of the genus Shigella. Responsible for an estimated 200 million cases annually.
Shigella is increasingly resistant to antimicrobial drugs, ZShigellosis is a priority target as defined by the World Health Organization since this disease is a major cause of mortality in developing countries: especially among children under S years of age and in the immunoeotnpromised population. 3Although no vaccine is yet available against shigellosis, several programs targeting the eradi6ation of this bacterial infection are under development, with emphasis on vaccination strategies involving either live attenuated strains of Shigella4 or acellular vaccines based on lipopolysa.ccharide (LPS) antigens and derivatives thereof. SOf particular interest in the later approach is the design of glycoconjugate vaccines based on the use of detoxified LPS. Indeed, there is evidence that natural and experimental infections with Shigella confer type-specific immunityb which points to the O-specific polysaccharide (O-SP) moiety of the LPS as the target antigen of the host's protective immune response to infection. Besides, data show that significant levels of pre-existing antibodies specific for the O-SF corzelate with a diminished attack rate of shigellosis.' Furthermore, it was recently demonstrated in field trials that protein conjugates of i LMPP10-then-~evet~ceaOMe detoxified ~PS provided protection to human volunteers against infections caused by S: sonnei.g As was particularly emphasized in the case of S rlysenteriae type l, conjugates incorporating oIigosa~eeh~rid.e fragments of the native bacterial polysaccharides may be even more immunoge~ic than their counterparts made of the detoxified LPS.9 Of most concern amongst the different species of Shigella, is S flexneri serotype 2a, the prevalent '~fective agent responsible for the endemic form ofthe disease.t° Indeed, major efforts from diffqrent laboratories including the development of conventional polysaccharide-protein conjugates,l' aim at the development of a vaccine against the disease associated with this particular I serotype. In parallel, a program aimed at the design of chemically defined glycoconj~gate vaccines based on the use of synthetic fragments of the O-SP of S flexneri 2a, is under development in this laboratory. We adopted a rational approach, involving a preliminary study of the interaction between the bacterial O-SP and homologous protective monoclonal antibodies.
A B E C D

2)-a-L-~hap-(1--~2)-a-L-Rhap-(la3)-[a-D-Glcp-(1-~4)]-a-L-Rhap-(1 >3)-[i-D-GIcNAcp(1-~~
The 0-Sl~ of S fIexneri 2a is a heteropolysa.ccharide defined by the pentasaccharide repeating unit Liiv~ It features a linear tetrasaccharide backbone, which is comtt~n to alI S flexneri 0-antigens end comprises a N acetyl glucosannine and three rhamnose residues, together with an a-D-glucopyranose residue branched at position 4 of one of the rhamnoses.
Besides the known methyl g~ycoside of the EC disaccharide;t~,ls a set of di- to pentasaccharidesl6'Lg and more recently Vin. octasaccharidel9 representative of fragments of S. flexneri 2a 0-SP have been synthesi2~d recently. The use of these compounds as molecular probes for mapping at the molecular level the binding characteristics of a set of protective antibodies against S flexneri 2a infection~indicated that access to larger oligosaccharides would help the characterization of the carbohydrate antigenic determinants. For this purpose, methodologies allowing a straightforward access to!5,~1'exneri 2a oligosaccharides of larger size are under study in this laboratory. We now report the synthesis of the first decasaccharide in the series, namely the D'A'B'(E')C'DtI.B(E)C
fragment; which was prepared as its methyl glycoside (1).
z LMPP10-theo~Etcyct~decnoMe Re9ults and discussion Considerir4g its dimeric nature, a convergent synthetic strategy to the target 1 was considered.
Indeed, re'trosynthetic analysis, supported by previous work in the feld,~9''Z
indicated that disconnections at the C-D linkage, thus based on two DAB(E)C branched pentasaccharides corresponding to a frame-shifted repeating unit I, would be the most advantageous (Scheme 1).
Such a strategy would involve a pentasaccharide acceptor easily derived from the known methyl glycoside ZI' or from the corresponding N-acetylated analogue 3 and a pentasaccharide donor bearing a ~-0-acyl protecting group at the reducing residue (C) in order to direct glycosylation towards the desired stereoehemistry. Depending on the nature of the 2-N acyl group in residue D, the latter could derive from the allyl glycosides 4 or 5. Besides, bearing in mind that the major drawbacks of the linear synthesis of pentasaccharide Z reported so far" dealt with the selective deblocking of key hydroxyl groups to allow further chain elongation, we describe herein various attempts at a convergent synthesis of the fully protected DAB(E)C
pentasaccharide as its methyl (2, 3) or allyl (4, 5) glycosides- Precedents concerning a related serotype of S. flexneri have indicated that disconnection at the D-A linkage should be avoided. zt.zZ
However to our knowledge, disconnection at the B-C or A-)i3 linkages was never attempted in the series. Both routes were considered in the :following study. The nature of the repeating unit I indicated that any blockwise synthesis involving such linkages would rely on donors lacking any participating group at position 2 of the reducing residue, thus the relevance of t's strategy may be questioned.
Nevertheless, although (3-glycoside formation was observed occasionally,'3 the good a-sterooselectivity reported on several occasions in the literature for glycosylation reactions based on mannopyranosyl'~~'3 and derivatives such as perosaminylz6~' fef KOVACI or rhamnopyranosyl donors that were either glycosylated at C-2,Zg or blocked at this position with a non partidipating group,~9 encouraged the evaluation of the above mentioned block strategies. To follow up:the work developed thus far in the S. Jlexneri 2a series, emphasis was placed on the use of the use of trichloroaeetimidate (TCA) chemistry,3o Stt~ategy based on the disconnection at the A-B linkage (Scheme l, rouge a):
Such 3 strategy involves the coupling of suitable DA donors to an appropriate B(E)C acceptor.
Takzng into account the glycosylation chemistry, two sets of disaccharide building blocks (6, 7, 8), easily obtained 'from known monosaccharide precursors which were readily available by standard

3 LMPP1~-then-tirevet-deceOMc As shown previously in the construction of the DA intermediate 17, the 7~~
ttichloroacetyl trichloroacetimidate 16 appears to be a highly suitable prECUrsor to residue D
when involved in the formation of the ~i-GIcNAc Linkage at the poorly reactive 2,~ position.
Indeed, reaction of 16 with the acceptor 24 in 1,2-dichloroethane in the presence of TMSOTf went smoothly and gave the trisaccharide 26 in 96% yield, However, conversion of the N-trichloroacetyl gxoup to the N
acetyl derivative 27 was rather Less successful as the desired trisaccharide was obtained in only 42 % yield when treated under conditions that had previously been used in the case of a related oligosaccharide (sodium methoxide, ht3N, followed by re-N, 0-acetylation).t~
This result led us to reconsider the protection pattern of the glucosamine donor. The N
tetrachlorophthalimide gzoup has been proposed as an alternative to overcome problems associated with the widely spread phthalimic~o procedure when introducing a 2-acetamido-2,deoxy-ji-D-glucopyranosidic linkage.°' Thus, the IV tetrachlorophthalimide trichloroacetimidate donor 25 was selected as an alternative.
It was prepared as described from commercially available D-glucosamine,°2 apart from in the final imidate formation step, where we found the use of potassium carbonate as base to be more satisfactory than DBU. GlyeosyIation of 24 with 25 in the presence of TMSOTf resulted itt the trisaccharide 28 in 65% yield. The tetrachlorophthaloyl group was then removed by the action of ethylenediamine, and subsequent re-A' 0-aeetylation gave the trisaccharide 27 in 6S% yield. The latter was next converted into the donor 13 in two steps, analogous to those described for the preparation of 6 from 17. Indeed, de-O-allylation of 27 cleanly gave the hemiacetal 29 (83°/), which was then activated into the required trichIoroacetimidate (94%), It is worth mentioning that although they involve a different D precursor, both strategies give access to the intermediate 2'7 in closely related yields, 40 and 42%, respectively.
Initial attempts to form the pentasaccharide 5 from 13 and the previously described acceptor llis in the presence of TMSOTf as promoter were rather unsuccessful, resulting in at best 17% of the desired product, accompanied by decomposition of the donor into the hemiacetal 29 (75%).
Using BF3.OEt2 as the promoter in place of TMSOTf, reaction of l I with 13 at mom temperature provided 5 in 44% yield, with the acceptor 11 and hemiacetal 29 also recovered in 54 and 29'/°
yield, res~ectiveIy. We considered that the poor reactivity of the acceptor was responsible for these results, as the "C NMR of 13, showing several distorted signals (notably ???), suggests that there is considerable steric hindrance around the position 3c. For that matter, the 2~-0-acetylated disaccharide I2 was considered as an alternate aceepfor.
Analogously to the L.~tPPI O-thco-brevet-decaOMe preparation of 11, it was obtained from the known diol 30 through regioscIective opening of the intermediate orthoester. However, coupling of the potentially less hindered acceptor 12 and the trisaccharitle donor 13 resulted, at best, in the isolation of the condensation product 31 in 42%
yield (not described).
The modest yield of 1 obtained by this route made the alteznative reaction path (Scheme 4) worth investigating, despite the mere numerous synthetic steps required. Indeed, it was found rather appealing twhen eva.l.uated independently in a closely related series (unpublished results). By this route, a tetrasaccharidc acceptor can be formed from two disaccharide building blocks (EC and AB), and coupled with an appropriate monosaccharide donor as precursor to D.
Considering that selective deprotection of the ~A hydroxyl group would occur in the course of the synthesis, glycosylation attempts were limited to the 2-D-benzoylated acceptor 11. The disaccharide donor necessary for this path could be derived from the building block 24, already in hand. The choice of temporary protecting group at position 2A was deternined by our experience of the stepwise synthesis of the corresponding methyl pentasaccharide," where we noted that an acetate group at this position may not be fully orthogonal to the benzoate located at position 20. The chosen group had also to support removal of the anomeric allyl group and the subsequent conversion to the trichloroaeetimidate. At first, a chloroacetate group was anticipated to fulfil these requirements.
Thus, the disaccharide 24 was treated with chloroacetio anhydride and pyridine to give the derivative 32 (57%). Anomeric deprotection to give the hemiacetal 33 (B4%) and subsequent trichloroacetimidate activation of the latter into the donor 34 (83%) were performed in the same way as before. Coupling of 11 with 34, carried out in the presence of TMSOTf at -40°C, yielded a complex mixture of products. When the temperature was lowered to -60°C, the condensation product 3$ could be isolated in Z2% yield. The a-stereoselectivity of the glycosylation was ascertained from the value of the 'Jc,H coupling constant at C-1$ which was XX
Hz.43''~(A
faire ?) Alternative donor protection was attempted. Treatrr~nt of 24 with p-methoxybenryl chloride and sodium hydride gave the fully protected derivative 35 (97%), which was cleanly converted into the trichloroacetimidate donor 37 (82%) in two steps involving the hemiacetal intermediate 36 (73%). GIycosylation of 1I with 37 in the presence of TMSOTf at -40°C gave the desired tetrasaccharide 39 in 44% yield. Again, the s~tereochemistry of the newly created linkage vvas ascertained based on the IJc,H heteronuelear coupling constants.
When the temperature was lowered to -GO°C, the yield of 39 fell to 34% anti a second major product 40 LMPPlO-theo-brevet-dccaOMe (21%) was observed in the mixture. Indeed. examination of the NMR spectra of this product revealed that the pMeOBn group had been lost. That 40 was the acceptor required for the next step brought the. estimated yield of condensation to 55%. Nevertheless, the overall outcome of this blockwise strategy did not match our ea-pectations, and this route was abandoned.
Linear strategy to the fully protected pentasaccharide d.~ As preliminary studies have demonstrated, rapid access to suitable building blocks allowing the synthesis of higher-order oligosaccharides representative of fragments of the 0-SP of S. flex~eri 2a remains a challenge.
Major conclusions drawn from our studies favour the design of a linear synthesis of the target 4.
Indeed, when put together with our previous work, such as the synthesis of tetrasaccharide 41 (95%)" or that of trisaccharide 42 (97%),~e all the above described attempted couplings outlined the loss of e~ciency of glycosylation reactions involving rhamnopyranosyl donors glyeosylated at position 2 in comparison to those involving the corresponding acetylated donor. Thus, matching the linear strategy of the methyl pentasaceharide 2 described previously,t' a synthesis of 4, based on donors bearing a participating group at 0-2, was designed.
Three key building blocks were selected. These were the readily accessible )EC disaccharide acceptor 11 benzoylated at C-2 as required for the final condensation step leading to the fully protected decasaccharide intermediate; the rhamnopyranosyl trichloroacetimidate 22, which serves as a precursor to residues ~1 and B, and bears a both temporary and participating group at position 2; and the trichloroacetamide glueosatninyl donor 16 as a precursor to residue D. As stated above, coupling of 11 and 22 gave 42 in high yield. As observed in the methyl glycoside series,l' de-D
acetylation using MeONa or methanolic HCl was poorly selective. Although, guanidinelguanidinium nitrate was proposed as a mild and selective O-deacetylation reagent compatible with the presence of benzoyl protecting groups,'S none of the conditions tested prevented partial debenzoylation leading to diol 43, as confirmed from mass spectroscopy and NMR analysis (ct Aone-Laure). The required alcohol 10 was readily obtained in an acceptable yield of 84% yield by a five-day acid catalysed methanolysis, using HBFs in diethyl ether/tnet'hanol,l'~'6 of the fully protected intermediate 42 (Scheme 5).
Repeating this two-step process using 10 as the acceptor and 22 as the donor resulted first in the intermediate 44 (90%), and next ~in the tetrasaccharide acceptor d0 (84%). Glycosylation of the latter with 16 gave the fully protected pentasaccharide 4 in high yield (98%), thus confirming that the combinatuon of the LMPP l0-then-brevet~decaOMc trichloroachtamide participating group and the trichloroacetimidate activation mode in 16 results in a potent donor to be used as a precursor to residue D in the S flexnert series, where low-reactive glycosyl acceptors are concerned. Following the above described procedure, selective anomeric deprotection of 4 furnished the hemiacetal 45 wluch was smoothly converted to the trichloroacetimidate donor 46 (66% from 4). From these data, the linear synthesis of 4, truly benefiting from the use of 2z as a common precursor to residue A and B, appears as a reasonable alterriative.to the block syntheses which were evaluated in parallel.
Synthesfr of the targel decasacchnride l: Having a peniasaccharide donor in hand, focus was next placed on the synthesis of an appropriate pentasaccharide acceptor. In our recent descxiption of the convergent synthesis of the B'(E')C'DAB(E)C oetasaccharide,'9 the pentasaccharide 48, bearing a 4p,6p-O-isopropylidene protecting group, was found a most convenient acceptor which encouraged its selection in the present work. Briefly, 48 was prepared in two steps from the known Z. Thus, mild transesterification of 2 under Zemplen conditions allowed the selective removal of the acetyl groups to give triol 47, which was converted to the required acceptor 48 (72% from 2) upon subsequent treatment with 2,2-dimethoxypropane. Relying on previous optimisation of the glycosylation step, i9 the condensation of 48 and 46 was performed in the presence of a catalytic amount of triftic acid. However, probably due to the closely related nature of the donor and acceptor, the reaction resulted in an inseparable mixture of the fully protected 49 and the hemiacetal 4S resulting from partial hydrolysis of the donor. Most conveniently, acidic hydrolysis of the mixture, allowing the selective removal of the isopropylidene group in 49, gave the intermediate diol 50 in a satisfactory yield of 72% for the two steps.
According to the deprotectlon strategy used for the preparation of the closely related octasaccharide,t9 diol SO was engaged in a controlled de-O-acylation process upon treatment with hot methanolic sodium methoxide. However, partial cleavage of the trichloroacetyl moiety, leading to an inseparable mixture, was observed which prevented further use of this strategy. Indeed, it was assumed that besides being isolated and therefore resistant to Zempl~n transacetylation conditions,a~~9 the 2c-O-benzoyl groups were most probably highly hindered which contributed to their slow deprotection. Alternatively, 50 was submitted to an efficient two-step in-house process involving fizst, hydrogenolysis under acidic conditions which allowed the removal of the benzyl groups and second, basic hydrochlorination which resulted in the conversion of the N
trichloroacetyl groups LMPP10-tl~eo-brc~~et-deeaoMc into the required N acetyl ones, thus affording 51. Subsequent transESterification gave the final target l in 37% yield from S0.
CONCLUSION
The decasaccharide 1, corresponding to two consecutive repeating units of the O-Ag of S.
Jlexneri 2a was synthesized successfully based on the condensation of two key pentasaccharide intermediates, the donor 46 and acceptor 48. Several routes to those two building blocks were investigated, involving either blockwise strategies or a linear one. The latter was the preferred one based on yields of condensation and the number of steps.
ACKNOWL,EDt~EMENTS
The authors thank Pr. P.7. Sansonetti who is a scholar of the Howard Hughes Medical Institute fior his key input in the project. The authors are grateful to J. Ughetto-Monfrin (unite de Chimie Organique, Institut Pasteur) for retarding all the NMR spectra. The authors thank the CANAi.'~I
and the Fondation pour la Recherche Medicate (predoctoral fellowship to C.
C.), the Bourses Mrs Frank Howard Foundation for its postdoctoral fellowship to K. W. and fnancial support, as well as the Bourses Roux foundation (postdoctoral fellowship to F. B.).

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LMPPtUa~pbrcvctdeceOhlr General rriethods Optical rotations were measured for CHCh solutions at 25°C, expect where indicated otherwise; with a Perkin-Elmer automatic polarimeter, Model 241 MC. TLC were performed on precoated slides of Silica Gel 60 F25~ (Merck). Detection was effected when applicable, with UV light, andlor by charring in 5% sulfuric acid in ethanol.
Preparative chromatography was performed by Elution from columns of Silica Gel 60 (particle size 0.040-0.063 mm). For all compounds the NMR spectra were recorded at 25°C for solutions in CDCl3, on a Broker AVI 400 spectometer (400 MHz for 'H, 100 MHz for "C).
External references : for solutions in CDCIa, TMS (0.00 ppm for both 'H and "C). Proton-signal assignements were made by first-order analysis of the spectra, as well as analysis of 2D
'H-'H correlation maps (COSY) and selective TOCSY experiments. Of the two magnetically non-equivalent geminal protons at C-6, the one resonating at lower field is denoted H-6a and the one at higher field is denoted H-6b. The "C NMR assignments were supported by 2D'3C-'H correlations maps (HETCOR). Interchangeable assignments are marked with an asterisk in the listing of signal assignments. Sugar residues in oligosaccharides are serially lettered according to the lettering of the repeating unit of the O-SP and identified by a subscript in the fisting of'signal assignments. Fast atom bombardment mass spectra (FAB-MS) were recorded in the positive-ion mode using dithioerythridol/dithio-L-threitol (4 :I, MB) as the matrix, in the presence of NaI, and Xenon as the gas. Anhydrous DCM, I,2-DCE and Et20, sold on molecular sieves were used as such 4 ~ powder molecular sieves was kept at 100°C and activated before use by pumping under heating at 250°C.
Phenyl (3,4,6-tri.O-acetyl-2-deoxy-2-trichloroaeetaraido-/3-n-gtucopyranosyt)-(la2)-(3,4-di~O~benzyt-1-thin-a-trrhamnopyranoside) (8). A mixture of alcohol 15 (0.12 g, 0.27 mmol) and imidate 16 (0.245 g, 0.41 mmol) in anhydrous DCM ( 10 mL) was stirred for 1 S
min under dry ar. After cooling at 0°C, Me3Si0Tf (28 p,L) was added dropwise and the mixture was stirred for 0.5 h. Triethylamine (60 pL) was added anrl the mixture was concentrated. The residue was eluted from a column of silica gel with 4:1 cyclohexane-EtOAc to give 8 (227 mg, 97 %) as a colorless foam; [aJD -G3' (c 1; CHCl3). 1H NMR
(CDCl3): o 7.10-7.40 (m, 15H, Ph}, &.73 (d, 1H, J2,~.,= 8.5 Hz, NHb), 5.47 (d, 1H, J,,2 =
1.2 Hz, H-1~), t~t~pioG~Ptrev~.e~or,~

5.07 (dd, 1H, Jz,~ = J,,4 = 10.0 Ha., H-3n), 4.99 {dd, 1H, J~,S = 10.0 Hz, H-4n), 4.80-4.SS (m 4H, CH,Ph), 4.52 (d, 1H, J~,Z = 8.2 H2, H-lp), 4.13-3.95 (m, 2H, Js,s = 5.3 Hz, J6,,5~ = 12.2 Ha., H-GaD, 6ba), 4.I0 {m, IH, J~,S = 9.5 H~., Js,b = G.1 Hz, H-Sb), 4.00 (dd, IH, Jz,3 = 3.0 Hz, H-2A), 3.99 (m, IH, H-2D), 3.77 (dd, IH, J3,a = 9.4 Hz, H-3A), 3.50 (m, IH, H-Sp), 3.39 (dd, 1H, H-4,;), 1.90, 1.93, 1.95 (3s, 9H, OAc), 1.23 (d, 3H, H-6A). '3C NMR
(CDCl3):8 1'11.1, 170.9, 169.6, 162.1 (C=0), 127-138 {Ph), 102.1 (C-1D), 92.7 (CCl3), 87.4 (C-lA), 81.3 (C-4A); 80.5 {C-3A), 79.I (C-2,~), 76.4, 74.1 (CH2Ph?, 72.4 (C-SD), 72.4 (C-3p), 69.8 (C-5"), 68.?
(C-4o), G2.3 {G-6n), SG.2 (C-2D); 21.0, 20.9, 20.8 (3C, OAc), 18.1 {C-6n).
FARMS of CAOH4~C13N0iZS (M, 867), m1z 890 ([M+Na]"). Anal. Calcd for C~oH4aC~N0,zS : C, 55.27 ;
H,S.lO;N, 1.61. FoundC,55.1G;H,5.18;N, 1.68.
Allyl (3,4,6-tri-p-acetyl-2-deoay-Z-trichloroacetamido-ø-D-gtucopyraoosyl)-(1--~Z)-(3,4-di-O-benzyl-a-trrhamnopyranoside) (17). A mixture of alcohol 14 (1.86 g, 4.86 moral) and imidate 16 (3.85 g, G.47 mmol) in anhydrous CH3CN (80 mL) was stirred for 15 min under dry Ar. After cooling at 0°C, Me3Si0Tf (46 uL) was added dropwise and the mixture was stirred for 0.5 h. Triethylamine {150 uL) was added and tile mixture was concentrated.
The residue was eluted from a column of silica gel with 7:3 eyclohexane-EtOAe to give 17 (4.0 g, 99 %) as a white solid; [a]n -3° (e 1, CHC13). 1H NMR (CDC)3):b 7.18-7.32 (m, IOH, Ph), 6.70 (d, 1H, Ji,~ = 8.4 Hz, NHD); 5.78-5.82 (m, 1H, All), 5.05-5.20 (m, 2H, All), 5.00 (tn, 2H, JZ,3 = J~., _ Ja.$ = 9.5 Hz, H-3D, 40), 4.45-4.75 (m, 4H, CH,Ph), 4.76 (d, IH, J,,~ = 1.1 Hz, H-1~), 4.60 (d, 1H, J~,z = 8.5 Hz, H-lo); 4.05-4.15 (m, 2H, J5,6 = 4.8 Hz, Jba.6b = I2.2 Hz, H-6ao, 6bD), 3.98 (m, I H, H-2o), 3.90 (m, 2H, Alt), 3.86 (dd, 1 H, JZ., = 3.2 Hz, H-2A), 3.81 (dd, 1 H, J3,4 = 9.5 Hz, H-3A), 3.G2 (m, 1H, J4.5 = 9.5 Hz, Js,s = G.1 Hz, H-5,,), 3.50 (m, 1H, H-SD), 3.32 (dd, 1H, H-4,,), 1.93, 1.97, 2.02 (3 s, 9H, OAc), 1.24 (d, 3H, H-6A). "C NMR (CDC13):b 171.0, 170.9, 169.6, I62.I (C=O), 117.1-138.5 {Ph, All), 101.8 (C-lo), 98.5 (C-1"), 92.6 (CCl,), 8I.4 (C-4A), 80.4 (C-3~), 7?.I (C-2,,), 75.9, 74.1 (CHZPh), 72.7 {C-3p), 72.5 (C-Sp), GB.G (C-4D), 68.3 (C-5~), 68.1 (All), G2.3 (C-6n), SG.1 (C-2n), 2i.1, 20.9, Z0.9 (OAcj, 18.2 (C-6A). FABMS of C~,Ha,CI,NOj; {M, 815), m/a 838 ([VI+Na]"). Anal. Calcd for C37H,sdCI3NO~3: C, 54.39 ; H, 5.43 ; N, 1.71. Found C, 54.29 ; H, 5.45 ; N: 1.72.
(3,4,6~tri-p-acet3rl-Z-deoxy-2-trichloroacetamido-[i-D-glucopyranoFyl)-(1->2)-(3,4-di-O-bearyl-a-t..rhamnopyranose) (18). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium LMPPtObdrbrcYctti°_noMe hexafluorophosphate (120 mg, 140 umol) was dissolved tetzahydrofuran (10 mL), and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stzeam. A solution of 17 (1.46 g, 1.75 mmol) in tetrahydrofuran (20 mL) was degassed and added. The mixture was stirred at rt overnight. The mixture was concentrated.
The residue was taken up in acetone (27 mL.), and water (3 mL) was added.
Mercuric bromide (949 mg, 2.63 mmol) and mercuric oxide (761 mg. 3.5 mmol) were added to the mixture, protected from light. The mixture was stizxed For 2 h at rt, then concentrated. The residue was taken up in CHzCIz and washed three times with sat. aq. KI, then with brine.
The organic phase was dried and concentrated. The residue was purified by column chromatography (Cyclohexane-AcOEt 4:I) to give 18 (1.13 g, 81 %) as a white foam. [a]n +4° (c 1. CHCl3), 1H NMR (CDCl3):8 7.05-7.35 (m, 10H, Ph), 6.74 (d, 1H, J2,~ = 8.5 Hz, NHn), 5.10 (d, 1H, J,,z = 1.1 Hz, H-1,,), 5.02 (m, 2H, Jz,~ = J3,4 = J,,,s = 9.5 Hz, H-3p, 4D), 4.50-4.80 (m, 4H, CHzPh), 4.61 (d, IH, Jl,i = 8.5 Hz, H-lp), 4.08-4.15 (m., 2H, Js,b = 4.5 Hz, Jsa,6b = 12.3 Hz, H-6an, Gbn), 4.00 (m, 1H, H-2p), 3.90 (dd, 1H. Ja.s = 3.3 Hz, H-2~), 3.86 (dd, 1H, J,,a = 9.5 Hz, H-3,,), 3.85 (m, 1H, J4_5 = 9.5 HZ, Js,~ = 6.2 Hz, H-5~), 3.50 (m, 1H, H-5n), 3.30 (dd, IH, H-4A), 2.85 (d, 1H, J ,.oH = 3.5 Hz, OH), 1.94, 1.97, 2.02 (3s, 9H, OAc), I.23 (d, 3H, H-GA). "C
NMR (CDCl3):8 171.1, 170.0, 169.6, 162.1 (C=O), 127.1-138.5 (Ph), 101.7 (C-In), 94.I (C-lA), 92.6 (CCL), 81.4 (C-4A), 79.9 (C-2A), 77.3 (C-3"), 75.9, 74.1 (CHzPh), 72.7 (C-3p), 72.5 (C-Sn), 68.6 (C-4n), 68.4 (C-5~), 62.2 (C-6n), SG.I (C-2D), 21.1, 21.0, 20,9 (OAc), 18.3 (C-6A), FABMS Of C3dHsOCI~NO,3 {VI, 775), m1z 789 ([M~-Na]+) ; Anal. Calcd for C,4H~oC~N0,3: C, 52.55 ; H, 5.19 ; N, 1.80. Found G, 52.48 ; H, 5.37 ; N, 1.67.
(3,4,6-tti-O-acetyl-2-deoYy-2-trichloroaeetamido-[3-n-glucopyrsnosyl}-(I-~2}-3,4.di-O-benzyl-a-r,-rhamnopyranose trichloroacetimidate (6). The hemiacetal 18 (539 mg, 0.68 mmol) was dissolved in CHzCIz (50 mL), placed under azgon and cooled to 0°C.
Trichloroacetonitrile (0.6 mT..., 6.8 rnmol), then DBU (10 ~tL, 70 p.mol) were added. The mixtuze~ was stirred at 0°C for 30 mm. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was eluted from a column of silica gel with 7:3 eyclohekane-EtOAC and 0.2 % of Et3N to give 6 (498 mg, 78 %) as a colourless foam; [a]D -18° (e 1, CHCl3). iH NMR (CDCI3):8 8.48 (s, IH, N=H), 7,15-7.40 (tn, lOH, Ph), 6.75 (d, IH, JZ.NH = 8.5 Hz, NHo), 6.18 (d, 1H, Jl,z = 1.1 Hz, H-1~), 5.15 (dd, 1H, Jz., = J3.a = 9.5 Hz, Lt~FI Oexpbrevecaecav~~.~

H-3D), 5.07 (dd, IH, J4,s = 9.5 Hz, H-4p), 4.50-4.82 (n~, 4H, CI~zPh), 4.62 (d, 1H, J~,z = 8.5 Hz, H-ln), 4.03-4.20 (m, 2H, J5,6 = 4.5 Hz, Jsy,sb = 12.3 H~~, H-6aD, 6ba), 3.98 (m, 1H, H-2D), 3.85 (m, 1H, Ja,s = 9.5 Hz, J5,5 = 6.2 H~, H~5A), 3.84 (dd, IH, Jz,3 = 3.3 Hz, H-2"), 3.83 (dd, IH, J~,A = 9.5 Hz, H-3,,), 3.55 (m, 1H, H-Sp), 3.45 (dd, 1H, H-4A), 1.93, 1.96, 1.98 (3s, 9H, OAc), 1.23 (d, 3H, H-6A). 13C NMR (CDC13):8 171.1, 170.0, 169.6, I62.I (C=Oj, I27.2-138.4 (Phj, 101.7 (C-lo), 97.2 (C-lA), 92,6 (CCI~), 80.5 (C-4A), 79.I (C-3A), 76.2 (C-2x), 7G.2, 74.1 (CHzPh), 74.4 (C-3n), 74.1 (C-Sn), 71.3 (C-5A), 68.6 (C-4D), 62.3 (C-6D), 56.3 (C-2nj, 21.1, 21.0, 20.9 (3C, OAc), 18.2 (C-6A). Anat. Calcd for C36I3,,oCI6NZO13: C, 46.93 ; H, 4.38 ; N, 3.04. Found C, 46.93 ; H, 4.52 ; N, 2.85.
Al~y1 (2-acetamido-3,4,5-tri-O-acetyl-Z-deory-~3-n-glucopyranosyl)-(1-~2)-(3,4-di-0-bcnryl-a;-1.-rhamnopyranoside) (19). A mixture ofthe protected disaccharide 17 (3.0 g, 3.61 mmol) in MeOH {50 mL) was cold to 0°C and treated by NHS gaz overnight.
The solution was concentrated and the residue (2.02 g) was dissolved again in MeOH (50 mI,) and treated by AczO (3.98 mL,, 3G.1 mol). The solution was stirred for 2 h and then concentrated. The residue was eluted from a column of silica gel with 95:5 DCM-EtOAC to give the intermediate trial which was dissoh~ed in Pyridine (5 mL), cold to 0°C and treated by AciO
(2.4 mL). The mixture was stirred overnight and concentrated. The residue was eluted from a column of silica gel with3:2 cyclohexane-fitOAC to give I9 (2.3 g, 90 %) was obtained as a colourless foam [
a]p -12° (c 1. CHCI;). ~H NMR (CDC13):d 7.18-7.32 (m, lOH, Ph), 5.70-5.80 (m, 1H, All), 5.40 (d, 1H, J2,~ = 8.1 Hz, NH), 5.10-5.20 (m, ZH, All), 4.9G (dd, 1H, J,,, =
J4,~ = 9.5 Hz, H-4D), 4.90 (dd, 1H, Jz,3 = 9.5 Hz, H-3p), 4.52-4.76 (m, 4H, CHzPh), 4.80 (d, 1H, J,,z = 1.2 Hz, H-I,,), 4.46 (d, 1H, Ji,2 = 8.5 Hz, H-In), 4.OZ-4.I0 (m, 2H, Js,b = 4.7 Hz, J~a,6b = I I.2 Hz, H-6aD, Gbo), 3.92 (m, 1H, H-2D), 3.87 (m, 2H, All), 3.86 (dd, IH, Jz,s = 3.5 Hz, H-2A), 3.82 (dd, IH, J3,a = 9.5 Hz. H-3n), 3.62 (m, 1H, Ja,s = 9.5 Hz, Js,s = 6.2 Hz, H-5A), 3.52 (m, 1H, H-5o), 3.30 (dd, 1H, H-4A), 1.92, I.94, 1.98 (3 s, 9H, OAc), 1.26 (d, 3H, H-G~). ~'C
NMR (CDC13):8 171.1, 171.0, 170.3, 169.6 (C=O), I 17-138 (Ph, All), 103.4 (C-1D), 98.5 (C-lA), 8I.3 (C-4A), 80.4 (C-3A), 78.5 (C-2,,), 75.9, 73.9 (CHzPh), 73.6 (C-3n), 72.4 (C-5p), 68.7 (C-4p), 68.2 (C-5,~, b8.1 (All), 62.5 (C-6p), 54.5 (C-2D), 23.4 (AcNH), 21.2, 21.1, 21.0 (OAc), 18.1 (C-G0.).
FABMS of C3,Ha,N0,3 (M, 713.3), rrt~'~ 736.2 ([M+Na]') Anal. Ca>cd for Ca~He,N0~3: C, 62.26 ; H, 6.G4 ; N, 1.96. Found C, d2.I2 ; H, G.79 ; N, 1.87.

(2-acetam ido-3,4,d-tri-0-acetyl-2-deoay-[i-n-glucopyranosy 1)-(1-~2)-(3,4-di-O-benryl-a-L-rhamnopyranose) (20). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (10 mg, 12 ~mol) was dissolved tetrahydrofuran (10 mL), and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream A solution of 19 (830 mg, 1.16 mmol) in tetrahydrofuran (40 mL) was degassed and added. The mixture was stirred at rt overnight. The mixture was concentrated.
The residua was taken up in acetone (90 mL), and water (10 mL) was added.
Mercuric chloride (475 mg; 1.75 mmol) and mercuric oxide (504 mg, 2.32 mmnl) were added to the mixture, protected 'from light. The mixture was stirred for 2 h at rt, then concentrated. The residue was taken up in CHZCI? and washed three times W th sat. a.q. KI, then with brine. The organic phase was dried and concentrated. The residue was purified by column chromatography (Cyclohexane-AcOEt 3:7) to give 20 (541 mg, 69 %) as a white foam; [aJp +16° (c 1, CHCl3).~H NMR (CDC)3):cS 7.05-7.35 (m, IOH, Ph), 5.50 (d, 1H, Jz,rrEt = 8.2 Hz, NHD), 5.22 (d, IH, Ji,z = 1.1 Hz, H-lA), 5.06 (dd, 1H, J3,4 = Ja,s = 9.5 Hz, H-4D), 5.00 (dd, 1H, Jz,~ ~ 9.5 Hz, H-3D), 4.60-4.85 (m, 4H, CHZPh), 4.56 (d, 1H, J,,~ = 7.0 Hz, H-1D), 4.13-4.22 (m, 2H, J5,6 = 4.5 Hz, J6a,Eb = 12.3 Hz, H-6ao, Gbo), 4.03 (m, IH, H-2D), 4.00 (m, IH, J4,s = 9.5 Hz, J5,6 = 6.2 Hz, H~5e,), 3.96 (dd, IH, J2,3 = 3.3 Hz, H-2A), 3.90 (dd, IH, .1~,4 = 9.5 Hz, H-3A), 3.60 (m, 1H, H-SD), 3.48 (d, IH, J,,ox = 3.5 Hz, OH), 3.40 (dd, IH, H-4A), 2.01, 2.03, 2.08 (3s, 9H, OAc), 1.65 (s, 3H, AcNH), 1.30 (d, 3H, H-6~). "C NMR (CDCl3):8 171.2, 171.0, 170.4, 169.6 (C=0), 128.0-138.2 (Ph), 103.3 (C-1D), 94.1 (C-la,), 81.4 (C-4"), '19.9 (C-2,,), 78.7 (C-3"), 75.8, 73.9 (CHzPh), 73.6 (C-3n), 72.4 (C-5o), 68.7 (C-4p), 68.2 (G5~), 62.4 (C-6p), 54.5 (C-2p), 23.3 (AcNH); 21.1, 21.0, 21.0 (3C, OAc), 18.3 (C-6A). FMS of C34HmNO,~ (M, 673.2), nzl6 696.3 ([M+Na)T) Anal. Calcd for C~,H4a'Iv'0,,: C, 60.61 ; H, 6.43 ; N. 2.08. Found C, G0.4G ; H, 6.61 ; N, 1.95.
(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-~-n-glucopyranosyl)-(1 ~2)-3,4-di-O-bcuxyl-a-L-rhamnopyranoge trichloroacetimidate (7). The hemiacetal 20 (541 mg, 0.80 mmol) was dissolved in CHaCh (20 mL), placed under argon and cooled to 0°C.
Trichloroacetonitrile (0.810 mL, 8 mtnol), then DBLT (10 ~tL, 80 pmol) were added. The mixture was stirred at 0°C
for I h. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was eluted from a column of silica gel with I : I cyclohexane-EtOAC
and 0.2 % of Et3N

to give 7 (5G0 mg, 86 %) as a colourless foam; [a]p +2° (c l, CHCIa).
'H NMR (CDCI3):8 8.5G (s, 1H, N-H), 7.20-7.50 (m, lOH, Ph), G.29 (d, 1H, J~,2 = 1.3 Hz, H-lA), 5.50 (d, 1H, J2.,,.H = 8.3 Hz, NHD), 5.17 (dd, 1H, J2,3 = J;,A = 9.5 Hz, H-3D), 5.09 (dd, 1H, Ja,s = 9.5 Hz, H-4D), 4.60-4.85 (m, 4H, CHzPh), 4.68 (d, 1 H, .I,,2 = 8.0 Hz, H-1D), 4.10-4.22 (m, 2H, Js,b = 5.0 Hz, J6a.Gb .= 12.2 Hz, H-Gao, Gbn), 4.00 (m, 1H, H-2p), 3.99 (dd, 1H, Jz,s =
3.5 Hz, H-2A), 3.90 (m, 1H, J4,s = 9.G Hz, Js,s = 6.2 Hz, H-5A), 3.89 (dd, 1H, J3,s = 9.5 Hz, H-3w), 3.62 (m, IH, H-Sp), 3.50 (dd, IH, H-4A), 1.98, 2.00, 2.02 (3s, 9H, OAc), 1.65 (s, 3H, AcNH), 1.32 (d, 3H, H-6w), ~'C NMR (CDC13):5 171.2, 171.0, 170.4, 1G9.G (C=0), 160.5 (C=NH), 128-138 (Ph), 103.3 (C-1D), 97.3 (C-lA), 91.4 (CC13), 80.3 (C-4w), 79.9 (C-3"), 77.5 (C-2w), 7G.0, 73.8 (2C, CH2Ph), 73.1 (C-3o), 72.2 (C-5D), 71.1 (C-5A), 68.8 (C-4D), 62.5 (C-6o), 54.8 (C-2D), 23.3 (AcNH), 21.4, 21.1, 21.0 (3C, OAcj, 18.4 (C-GA). Anal. Calcd for C36H4;C13Na0,3: C, 52.85 ;
H, 5.30 ; N. 3.42. Found C, 52.85 ; H, 5.22 ; N, 3.47.
Altyl (Z-0-acetyl-3,4-di-O-benzy!-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-0-henryl-a-L-rhamnopyranoside) (23). The acceptor Z1 (1.78 g, 4.65 mmol) and the trichloroacetitnidate donor 2Z (2.96 g. 5.58 rnmol) were dissolved in anhydrous ether (100 mL). The mixture was placed under argon and cooled to -55°C. TMSOTf (335 ~L, 1.8G mmol) was added dropwise.
The mixture was stirred at -55°C to -20°C over 3 h.
Triethylamine (0.75 mL) was added, and the mixture was allowed to warm to rt. The mixture was concentrated. The residue was purified by column chromatography (solvent x, 80 :20) to give 23 as a colourless syrup (3.21 g, 92 %) ; [a]D -1G° {c 0.55, CHC13). ~H NMR (CDC13):8 7.30-7.42 (m, 20H, Ph), 5.82-5.92 (m, 1H, All), 5.G2 (dd, 1H, Jl,z = 1.G Hz, J,_, = 3.2 Hz, H-2,~), 5.20-5.32 (m, 2H, All), 5.07 (d, 1H, H-lA), 4.82 (d, IH, J,,Z = 1.0 Hz, H~1B), 4.G0-4.95 (m, 8H, CH2Ph), 4.15-4.20 (m, 1H, All), 4.09 (d, IH, Jz,3 = 3.0 Hz, H~2B), 4.05 (dd, 1H. J~,n = 9.4 Hz, H-3A), 3.95-4.05 (m, 1H, Allj, 3.9G (dd, 1H, J;,q = 9.5 Hz, H-3a), 3.89 (m, 1H, Ja.s = 9.5 Hz, J5,6 =
6.3 Hz, H-51), 3.7G
{dd, 1H, J,,s = 9.5 Hz, Js,b = 6.2 Hz, H-5s), 3.52 (m, 1H, H-4B), 3.50 (m, IH, H-4,,), 2.18 (s, 3H, OAc), 1.39 (d, 3H, H-6,,), 1.36 (d, 3H, H-6H). ~3C NMR (CDCI,):S 170.8 (C=0), 117.1-138.4 (Ph, All), 99.5 (C-lA), 98.4 (C-18), 80.5 (2C, C-4w, 4H), 80.0 (C-3H), 78.1 (C-3~), 75.8, 75.7 (CHZPh), 74.9 (C-2B), 72.5, 72.2 (CHZPh), G9.3 (C-2~), 68.6 (C-SA), G8,4 (C-5B), G8.0 (All), 21.5 (OAc), 18.4, 18.2 (2C, C-6~, Ga). CI-MS for C,sHszOio (M = 752) rrJz 770 [M +
NH4]'. Anal. Calcd, for CasHs20~o : C, 71.79 ; H, G.9G. Found C, 70.95 ; H, 7.01.

FAH-MS for CS,H580~o (M = 830.4) mlz 853.5 [M -~ Na]''. An3l. Calcd. for CS,HsaO,o ; C, 73.71;H,7.03.FoundC,73.57;H,7.21.
(3,4-di-D-penzyl-2-0-paramethoxy benzyl-a-L-rbamnopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rbamnopyranose) (36). I,5-Cyclooctadiene-bis(methyldiphenylphosph.ine)iridium hexafluorophosphate (50 mg, GO p.mol) was dissolved tetrahydrofuran (G mL), and the resulting rid solution was degassed in an argon stream. Hydrogen was then bubbled through the solutidn, causing the colour to change to yellow. The solution was then degassed again in an argon istream. A solution of 35 (4.23 g, 5.09 tntnol) in tetrahydrofuzan (24 mL) was degassed end added. The mixture was stirred at rt overnight. The ,mixture was concentrated.
The residue was taken up in acetone (45 mL), and watEr (5 mL) was added.
Mercuric chloride (2.07 g, 7.63 mmol) and mercuric oxide (2.2 g, 10.2 mmol) were added to the mixture, protected. from light. The rni?tture was stirred for 2 h at rt, then concentrated. The residue was taken up in CHZCIz and washed three times with sat, sq. KI, then with brine, Ths organuc phase was dr'u'd and concentrated. The residue was purified by column chromatography (CyclohE~Cane-AcOEt 4:l) to give 36 (2.97 g, 73 %) as a white foam; [cz]D
+8° (c 1, CHC>3).
'H NMR'(CDC13):8 7.25-7.40 (m, 20H, Ph), 6.73-7.18 (~ 4H, Ph), S.I2 (d, IH, J,,z < 1.0 Hz, H-lA), Sa05 (d, IH, J1,2 < 1.0 Hz, H-IH), 4.40-4.80 (m, lOH, PhCHi), 4.08 (dd, 1H, Jz~ = 3.0 Hz, H-2~), 3.80-3.90 (m, 2H, J3,4 = Ja,s = 9.5 Hz, Js,b = 6.1 Hz, H-38, 5$), 3.78-3.80 (m, 2H, J~,~ = 3.1: Hz, J4,s = 9.4 Hz, Js,° = G.1 Hz, H-2A, 5A), 3.73 (m, 1H, J3,4 = 9.4 Hz, H-3A), 3.72 (s, 3H, OCH3), 3.60 (dd, IH, H-4,,), 3.33 (dd, 1H, H-4H), 1.34 (d, 3H, H-Gn), 1.24 (d, 3H, H-6a).
''C NM~t (CDCI,):cS 113.2-129.8 (Ph), 99.1 (C-lA), 93.8 (C-1H), 80.7 (C-4,,), 80.3 (C-4a), 79.7 (C 3H), 79.2 (C-3p), 75.5, 75.4, 72.6, 72.5, 72.4, (PhCH~), 74.2 (C-2"), 74.1 (C-2g), 68.5 (C-5,~, 68.1 (C-5H), 55.3 (OCH3), I 8,1 (2C, C-6~, 6H). FAB-MS for C.aHs<Ot° (M = 790.4) m/z 813.4 [M + i~a]+. Anal. Calcd. for C~gHs4O,G : C, 72.89 ; H, G.BB. Found C, 72.86 ;
H, &.98:
(3,4.diO.benxyl-Z-O-paramethoxybenzyl-a-t.-rhamnopyranosyl)-(1-->2)-3,4-di-O~
benzyl-,or.-z- rhamnopyranose trichlorogcetimidate (37). The herniacetal 36 (2.1 g, 2.6G
mrnol) v~~as dissolved in CHzC)z (20 mL), placed under argon and cooled to 0°C.
Trichloroacetonitrile (2.7 mL, 2G mmol), then DBU (40 ~,L, 0.2G mmol) were added. The mixture was stirred at 0°C for 30 min. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was eluted from a column of silica gel with 8:2 cyclohexane-EtOAC and 0.2 % EtzN to give 37 (2.03 g, 82 %) as a colourless foam; [a]a -I O°
(c 1, CHCl3). 'H NMR (CDC13):S 8.50 (s, 1H, C=NH), 7.05-7.25 (m, 20H, Ph), 6.62-?.OS (m, 4H, Ph), 6.08 (d, 1H, Jm < 1.0 Hz, H-la), 5.10 (d, 1H, J~,z < 1.0 Hz, H-1,,), 4.40-4.80 (m, IOH, PhCHa), 4.10 (dd, IH, Jz,3 = 3.0 Hz, H-2B), 3.80-3.90 (m, 4H, H-38, 2", 3A, SA), 3.72-3.80 (m. 1H, H-5H), 3.72 (s, 3H, OCH~j, 3.G3 (dd, 1H, J3.e = Je,s = 9.5 Hz, H-4,,), 3.42 (dd, IH, J,,e =~Ja,S = 9.5 Hz, H-4a), 1.30 (d, 3H, H-6s), 1.25 (d, 3H, H-6,~). '3C
NMR (CDCIJ):S
161.1 (C=NH), 113.4-129.5 (Ph), 99.6 (C-1"), 97.0 (C-19), 80.6 (C-4"), 79.6 (C-4H). 79.3 (2C, C-3~;. 3H), 75.7, 75.5, 72.8, 72.3, 72.0, (PhCH~), 74.4 (C-2A), 72.6 (C-28), 71.1 (C-S,J, G8.9 (C-5g), 55.3 (OCHy), 18.1 (2C, C-6A, 6H). Anal. Calcd, for C.c,H54C13NO,o : C, 64,21 ; H, 5.82; N, 1.50. Found C, 64.67 ; H, G.O1; N, 1.28.
Aryl (3;4-di-0-benzyl-2-O-chloroacetyl-a-z-rhamnopyranosy!)-(1-32)-(3,d-di-O-benzyl-a-~rhamnopyranoside) (32). To a mi~rture of 24 (3.8 g, 5.35 mrnol) in pyridine (40 mL) was added chloroactie anhydride (1.83 g, 10.7 mmol) at 0°C. The solution was stirred overnight at 0°C. MeOH (10 mL) was added and the mixture was concentrated. The residue was eluted from a oolumn of silica gel with 95:5 cyclohexane-acetone to give 32 (2.4 g, 57 %) as a colorless. syrup; [a]ø -15° (c l, CHC~). 'H NMR (CDCI3):8 7.15-7.30 (m, 20H, Ph), 5.71-5,81 (m, 1H, All), 5.49 (dd, 1H, J,,Z = 1.7 Hz, Jz,3 = 3.2 Hz, H-2A). 5.08-5.20 (m, 2H, All), 4.90 (d, 1H, H-I~), 4.50-4.84 (m, 8H, PhCHa), 4.65 (d, 1H, J,,2 < 1.0 Hz, H-Ie), 3.85-4,04 (m, 2H, All), 4.02 (m, 2H, CHaCI), 3.93 (dd, 1 H, Jz j = 3.0 Hz, H-2H), 3.88 (dd, 1H, J~,d = 9.5 Hz, H-3n), 3.81 (dd, 1 H, J3,4 = 9.5 Hz, H-38), 3.62 (m, 1 H, J,,S = 9.0 Hz, Js,s =
6.1 Hz, H-58), 3.73 (m, 1H, J4,s = 9.5 Hz, Js,B = 6.2 Hz, H-5,,), 3.34 (dd, 1H, H-48), 3.30 (dd, IH, H-4,,), 1.22 (d, 3H, H-6A), 1.21 (d, 3H, H-Gs). "C NMR (CDC13):8 166.9 (C=O), 117.2-138.5 (Ph, All), 99.2 (C-1"), 98.2 (C-IB), 80.4 (C-4.,), 80.3 (C-38), 80.2 (C-4a), 77.9 (C-3A), 75.8. 75.7. 72.6, 72.4 (PhCHz), 74.9 (C-2B), 71.2 (C-2A), 68.G (C-5A), 68.4 (C-58), 68.0 (All), 41.3 (CHZCI), 18.3 (2C, C-6A, 6B). FABMS of C45Hs,C10,o (M, 786.3), m/z 809.3 ([M+Na]'). Anal.
Calcd for CrsHsiClO,o: C, 68.65 ; H, 6.53. Found C, 68.51 ; H, 6.67.
(3,4-di-O-benzyl-Z-0-chloroacetyl-a-t~-rhamnopyranosyl)-(1~2)-(3,4-di-0-bcnzyl-a![i-L-rhamnopyranose) (33). 1,5~Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexaflu.orophosphate (40 mg, 46 psnol) was dissolved tetrahydrofuran (7 mL), and the I
i i resulting red solution was degassed in an argon stream Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream. A solution of 32 (2.39 g, 3.04 mmoI) in tetrahydrofuran (I8 mL) was degassed end added. The mixture was stirred at rt overnight. The mixture was concentrated.
The residue was taken up in acetone (30 mL), and water (S mL) was added.
Mercuric chloride (1.24 g, 4.56 mmol) and mercuric oxide (1.3 g, 6.08 mmol) were added to the mixture, protected from light. The mixture was stirred for 2 h at rt, then concentrated. The residue was taken up in CHzCh and washed three times with sat, aq. KI, then with brine.
The organic phase was dried and concentrated. The residue was purified by column chromatography (Cyclohexane-AcOEt 4:1) to give 33 (1.91 g, 84 %) as a white foam. [a]p -2° (r 1, CHCIa). 'H
NMR (CDCl3):8 7.10-7.40 (m, 20H, Ph), 5.49 (dd, 1H, J,,z = 1.7 Hz, JZ,3 = 3.2 Hz, H-2"), 4.99 (d, 1H, J,,z < 1.0 Hz, H-18), 4.90 (d, 1H, H-l,,), 4.45-4.85 (m, 8H, PhCHa), 4.01 (m, 2H, CHzCI), 3.93 (dd, 1H, Jz,3 = 3.0 Hz, H-Z$), 3.90 (dd, 1H, J3,, = 9.3 Hz, H-3~), 3.84 (dd, 1H, .7,,4 = 9.0 Hz, H-3B), 3.81 (m, 1H, J4,s = 9.0 Hz, JS,~ = 6.2 Hz, H-SH), 3.72 (m, 1H, J4,s = 9.5 Hz, J~,6 = 6.2 Hz, H-5A), 3.33 (dd, 1H, H-4H), 3.30 (dd, 1H, H-4A), 2.81 (d, 1H, Jz,oH = 3.4 Hz, OH), 1.22 (d, 3H, H-6,,), 1.20 (d, 3H, H~68). "C NMR (CDCI3):8 167.0 (C=O), 127.2-138.5 (Ph), 99.1 (C-1"), 93.9 (C-1H), 80.3 (C-48), 80.2 (C-4A), 79.7 (C-3H), 77.8 (C-3A), 75.8, 75.7, 72.G, 72.4 (PhCHz), 75.0 (C-2a), 71.1 (C-2A), 68.6 (C-S"), 68.4 (C-SH), 41.3 (CHiCI), 18.1 (2C, C-6A, 6B). FARMS of C,,Ha,CIO,o (M, 74b.3), m/~ 769.3 ([M+Na]*).
Anal. Calcd for CaiHd,ClO,o: C, 67.51 ; H, 6.34. Found C, 67.46 ; H, 6.39.
(3,4-di-D-benzyl-2-O-chloroacety!-a-Irrhamnopyranosy!)-(1->2)-3,4-di-0-6enry1-a-L-rhamnopyranose trichloroscetimidate (34). The hemiacetal 33 (1.80 g, 2.41 mmol) was dissolved in CH~CIz (25 mL), placed under argon and cooled to 0°C.
Trichloroacetonitrile (2.4 mL, 24 mmol), then DBU (3S uL, 0.24 mmol) were added. The mixture was stirred at 0°C for 40 min. The. mixture was concentrated and toluene was co-evaporated from the residue. The residue was eluted from a column of silica gel W th 4: I cyclohexane~EtOAC and 0.2 % Et3N to give 34 (1.78 g, 83 %j as a colourless foam; [a]o -12° (c 1, CHC13). ~H
Tv'NLR (CDCI,):S 8.60 (s, 1H, C=NH), 7.30-7.S0 (nt, 20H, Ph), 6.21 (d, lI~i, J,,2 = 1.8 Hz, H-1H), 5.63 (dd, 1H, J,Z =
1.5 Hz, Ja.3 = 3.2 Hz, H-2"), 5.07 (d, 1H, H-1"), 4.65-5.00 (m, 8H, PhCH2), 4.19 (m, 2H, CH~CI), 4.09 (dd, 1H, Jz,, = 3.2 Hz, H-28), 4.04 (dd, 1H, J~,e = 9.0 Hz, H-3B), 3.95 (m, 3H, H-3~, 5A, 58), 3.58 (dd, IH, H-4A), 3.48 (dd, 1H, H-4g), 1.39 (m, 6H, H-6A, 68). ~'C NMR

(CDC~):o 167.1 (C=O), 160.7 (C=N), 127.0-138.3 (Ph), 99.4 (C-lA). 97.5 (C-1H), 91.4 (CC13), 80.1 (C-4$), 84.0 (C-4A), 79.2 (C-3A), 77.9 (C-38), 75.9, 75.8, 73.0, 72.6 (PhCH2), 73.7 (C-2a), 71.4 (C-2,~, 71.2, 68.9 (2C, C-5A, 5g), 41.3 (CH2C1), 18.4, 18.2 (2C, C-6,,, 6H).
Anal. Calcd for C,aHq~CI.,NOIO: C, 59.27 ; H, 5.31 ; N, 1.57. Found C, 59.09 ;
H, 5.49 ; N, 1.53.
Altyl (3,4,6-tri-O-acetyl-2-deo~y-2-trichloroacetamido-~3-D-glucopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1--i2)-3,4-di-0-benzyl-a-L-rbamnopyranoslde (26).
1,2-Dichloroethane (35 mL) was added to the trichloroacetimidate donor 16 (2.49 g, 4.20 mmol), the acceptor 24 (2.48 g, 3.50 mewl) anrl 4A-MS powder (4 g). The mixture was stirred for 1.5 h at rt under argon. The mixture was cooled to -20°C
and TMSOTf (230 ~L, I.26 mmol) was added. The temperature was allowed to rise to 0°C over 1 h, and the mixture was stirred for an additional 2 h at this temperature. Trietl~ylamine (0.5 mL) was added and the mixture was allowed to warns to rt. The mixture was diluted with CHZC12 and filtered. The .filtrate was concentrated. The residue was purified by column chromatography with 3 :1 cyclohexane-AcOEt to give 26 (3.83 g, 96 %) as a colourless amorphous solid ;
[a]D --6° (c 0.5, CHCI,). 'H NMR (CDCIj) : b 7.28-7.52 (m, 20H, Ph), 6.83 (d, 1H, J1,NX =
8.4 Hz, NH), 5.85 (m, 1H, AII), 5.09-5.26 (m, 4H, H-3D, 40, A11), 4.98 (d, 1H, J~,~ = I.4 Hz, H-IA), 4.58-4.98 (m, lOH, H-1H, lp, CHZPh), 4.08 (m, 4H, H-2", 2p, 6aD, All), 3.91 (m, 5H, H-2B, 3A, 3H, 6bp, All), 3.79 (m, 2H, H-SA, 5B), 3.45 (m, 3H, H~4n, 4H, 5D), 1.97, 2.02, 2.04 (3s, 9H, OAc), 1.30 (m, GH, H-6A, 6H). "C NMR (CDC~) : 0 170.6, 170.3, 169.1, 163.2, 161.6 (C=0), 138.4-117.1 (Ph, All), 101.3 (C-1D), 100.9 (C-1~), 97.6 (C-1B), 92.0 (CCt~), 80.9, 80.4 (2C, C-4A, 4g), 79.1, 79.0 (2C, C-3,,, 3B), 77.3 (C-2A), 76.5 (C-2B), 75.4, 75.2, 73.6 (CHzPh), 72.2 (C-3p), 71.9 (C-Sp), 71.6 (CH~Ph), 68.2 (C-5B*), 67.8 (C-4D), 67.5 (C-5~*), 67.5 (CHzO), G1.3 (C-6D), 55.7 (C-2D), 20.5 (OAC), 17.9, 17.7 (2C, C-6A, 6s). FAB-MS for Cs~H~sCbNO"
(M = 1141.3) miz 1166.3, 1164.3 [M + Na]+. Anal. Calcd. for CS~H66C1~N0~,: C, 59.87 ; H, 5.82 ; N. 1.22. Found C, 59.87 ; H, 5.92 : N, 1.16.
Attyl (3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-p-D-glucopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1->2)-3,4-di-O-benzyl-a-L-rbamnopyranoside (Z8), Anhydrous ether (30 mh) and CHzCIz (15 mL) were added to the trichloroacetimidate donor 25 (3.34 g, 4.66 mmol), the acceptor 24 (2.20 g, 3.I0 mmol). The mixture was cooled to 0°C and TMSOTf (85 pL, 0.46d mmol) was added dropwise. The mixture was stirred at 0°C for 1 h, then at rt for 3 h. Triethylamine (1 mL) was added and the mixture was stirred for min., then concentrated. The mixture was taken up in ether and the resulting precipitate was filtered o~ The filtrate was concentrated. The residue was purified by column chromatography with 7:3 cyclohexane-AcOEt to give 28 (2.57 g, 65 %) as a colourless amorphous solid ; [a]o +22° (c 1, CHC~). 'H NMR (CDCl3) : 8 7.16-7.42 (m, 20H, Ph), 5.91 (dd, 1H, H-3p}, 5.81 (m, 1H, All), 5.10-5.24 (m, 4H, H-lp, 4p, All), 4.93 (s, 1H, H-1"), 4.53-4.81 (m, SH, H-1H, CH~Ph), 4.23-4.45 (m, 5H, H-2D, CHZPhj, 4.05 (m, 2H, H-Gap, All), 3.58-3.91 (m, 8H, H-2A, 28, 3A, 3B; SA, 5a, 6bD, All), 3.38 (m, IH, H-So), 3.13-3.21 (m, 2H, H-4A, 4H), 2.00, 2.02, 2.05 (3s, 9H, OAc), 1.24 (m, GH, H-GA, 6H). "C NMR ~ 170.4, 169.3 (C=O), 117.1-138.4 (Ph, All), 101.1 (C-lA), 99.9 (C-lo), 97.7 (C-1B), 80.6 (2C, C-4,,, 4g), 78.9, 79.7 (2C, C-3~, 3B), 78.2 (C-2A), 76.3 (C-2B), 75.2, 75.1, 72.6, 71.3 (CHZPh), 71.2 (C-5p), 70.1 (C-3D), 68.4 (C-5s*), 68.4 (C-4D), 67.6 (C-5A*), 67.6 (All), 61.3 (C-6D), 55.4 (C-2D), 20.6 (OAc), 18.0, 17.6 (2C, C-6~, 6g). FAB-MS for C63H~SChNO~A (M = 1263.3) mlz 1288.4, 1286.4 [M +
Na]".
Anal. Calcd. for C6~H65C)aNO~g : C, 59.77 ; H, 5.17 ; N, 1.11. Found C, 60.19 ; H, 5.53 ; N, 1.18.
Allyl (2-acetamido-2-deoxy-~3-D-glucopyranosyl)-(1--~2)-(3,4-di-0-benzyl-a-L-chamnopyranosyl)-(1 >2)-3,4-di-O-benzyl-a,-C,-rhamnopyranoside (27). The trisaceharide 26 (1.71 g, 1.50 mmol) was dissolved in MeOH (20 mL). A 1M solution of sodium methoxide in methanol (9 mL) and triEthylamine (5 mL) were added, and the mixture was stirred at rt for 18 h_ The mixture was cooled to 0°C and acetic anhydride was added dropwise until the pH
reached 6. A further portion of acetic anhydride (0.4 tnL) was added, and the murturc was stirred at n for 30 min. The mixture was concentrated, and toluene was co-evaporated from the residue. The residue was purified by column chromatography with 95 :5 DCM-MeOH to give 27 (623 mg, 45 %) as a colourless amorphous solid ; [a]o -16° (c 0.5, CHCIz). 'H NMR
(CDC13) : 8 7.24-7.48 (m, 20H, Ph), 6.79 (d, IH, NH), 5.73 (m, 1H, All), 5.12 (m, 3H, H-la, All), 4.52-4.86 (m, 9H, H-1B, CHzPh), 4.34 (d, 1H, H-lp), 3.79-4.08 (m, 6H, H-2A, 28, 3A, 3H, AIl), 3.53-3.74 (m, 3H, H-5a, 5B, 6ap), 3.24-3.45 (m, GH, H-2p, 3D, 4A, 4H, 4T,, Gbp), 3.20 (m, 1H, H-SD), 1.46 (s, 3H, OAc), 1.24 (m, 6H, H-G~, 6B). '3C NMR G 173.6 (C=0), 117.3-137.4 (Ph, All), 103.2 (C-lo), 100.3 (C-1~), 97.9 (C-1H}, 81.3, 80.4 (2C, C-4~, 4H), 79.9 (2C, C-3A, 3B), 79.9 (C-2$*), 78.9 (C-3o), 75.7 (C-So), 75.6 75.3, 74.5 (CHaPh), 73.6 (C-2,,*), 72.5

12 (CHiPh), 71.9 (C-4D), G8.2. 68.0 (2C, C-5", 5H), 67.7 (CHiO), 62.5 (C-6n), 58.8 (C-2p), 22.3 (OAc), 18.0,17.8 (2C, C-6", 6s). FAB-MS for CsvHs3NO~a (M = 913.4) m/z 936.6 [M + Na]''.
Anal. Calcd, for CS;H6~NO;a.H~O: C, 65.72 ; H, 7.03 ; N, 1.50. Found C, 65.34 ; H, 7.03 ; N, I.55.
Altyl (2-acetamido-3,4,6-tti-O-acetyl-Z-deoxy-[i-D-glucopyranosyl)-(i--32)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1 >2)-3,4-di-O-benzyl-a-L-rhamnopyranoside (27).
(a) Pyridine (5 mL) was added to 27a (502 mg, 0.55 mmol) and the mixture was cooled to 0°C.
Acetic anhydride (3 mL) was added. The mixture was stirred at rt for 18 h. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was taken up in CHzC>~ and washed successively with 5% aq HCl and saturated aq NaHC03. The organic phase was dried and concentzated to give 27 (538 mg, 94 %) as a colourless foam.
(b) Tetrahydrofuran (3 mL) and ethanol (3.3 mL) were added to 28 (384 mg, 0.303 mmol).
Ethylenedia.mine (90 ~L, I.3G mmnl) was added and the mia.-turE was heated at 55°C for 4 h.
The mixture was alloyed to cool to rt. Acetic anhydride ( 1.0 mL) was added, and the mixture was stirred at rt for 1.5 h. The mixture was concentrated. The residue was taken up in pyridine (5 mL) and the mixture was cooled to 0°C. Acetic anhydridE (2.5 mL) was added. The mixture was stirred at rt for 18 h. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was taken up in CH~C1~, which caused the formation of a whit precipitate. The mi.~cture was filtered through a plug of silica geL eluting with 7:3 Cyclohexane-acetone. The filtrate was concentrated to give 27 (215 mg, 68 %) as a colourless foam ; [a]n -7° (c 0.5, CHCL). ~H NMR (CDCb) : 0 7.24-7.48 (m, 20H, Ph), 5.84 (m, 1H, All), 5.53 (d, 1H, NH), 5.19 (m, 2H, Ah), 5.03 (dd, IH, H-4n), 4.98 (m, 2H, H-1", 3D), 4.54-4.95 (m, IOH, H-I H, 10, CHzPh), 4.07 (m, 4H, H-2A, 2p, 6aD, All), 3.88 (m, 5H, H-28, 3A, 38, 6bp, AIl), 3.79, 3.68 {2m, 2H, H-SA, 5$), 3.42 (m, 3H, H-4A, 4a, 5n), 2.02, 2.01, 1.97, 1.G4 (4s, 12H, OAcj, 1.30 (m, 6H, H-6", 6B). t'C NMR (CDCI3) S 170.7, 170.4, 169.9, 169.1 (C=0), 117.1-138.5 (Ph, All), 102.9 (C-la), 101.2 (C-1,,), 97.7 (C-la), 81.0, 80.5 (2C, C-4A, 4B), 79.5, 79.1 {2C, C-3A, 3s), 78.2 (C-2A), 76.1 (C-2B), 75.5, 75.2, 73.6 (CH2Ph), 73.3 (C-3D), 71.9 (C-5D), 71.7 (CH2Ph), 68.3 (C-5A''), 68.0 (C-4D), 67.G (C-5H'"), 67.6 (CHzO), 61.6 (C-GD), 54.1 (C-2p), 22.9 (AcNH), 20.6 (OAc), 18.0, 17.7 (2C, C-6", 6B). FAB-MS for Cs,Hb9N0;, (M =
1039.5) rn/z 1062.4 [M + Na]+. Anal. Calcd. for Cs,Hs9N0": C, 65.82 ; H, 6.69 ; N, 1.35. Found C, 65.29 ; H, 6.82 ; N, 1.29.

13 (Z-acetamid o-3,4,6-tri-O-acety )-2-deoxy-[i-D-glucopyranosyl)-(I ~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1~2)-3,4-di-O-benzyl-a!(3-L-rhamnopyranose (29). I,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hcxafluorophosphate (30 mg, 35 pmol) was dissolved tetrahydrofuran (5 mL), and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream, A solution of Z7 (805 mg, 0.775 mmol) in tetrahydrofuran (10 mL) was degassed and added. The mixture was stirred at rt overnight. The mixture was concentrated. The residua was taken up in acetone (15 mL), and water (1.5 mL) was added. Mercuric chloride (315 mg, 1.16 mmol) and mercuric oxide (335 mg, 1.55 mmol) were added to the mixture, protected from light. The mixture was stirred for I h at rt, then concentrated. The residue was taken up in CHzCIz and washed three times with sat, aq. KI, then with brine. The organic phase was dried and concentrated. The residue was purified by column chromatography with 4 :6 AcOEt-cyelohexane to give Z9 {645 mg, 83 %) as a white foam. The'H NMR spectra showed the a :~3 ratio to be 3.3 :1 ;
[a]D +3° (c 0.5, CHC13). 'H NMR (CDCIa) a-anomer : 8 7.30-7.47 (m, 20H, Ph), 5.53 (d, 1H, NH), 5.17 (d, 1H, J,,2 = 1.9 Hz, H-1H), 5.08 (m, IH, H-4D), 5.03 (d, 1H, J,,~ = 1.5 Hz, H-lA), 4.99 (m, IH, H-3o), 4.62-4.92 (m, 8H, CFI~Ph), 4.60 (d, 1H, J,,z = 8.4 Hz, H-ID), 4.01-4.18 (m, 3H, H-2A, 20, 6aD), 3.90-3.97 (m, SH, H-2H, 3A, 3H, 5A*, 6bD), 3.83 (m, 1H, H-SH*), 3.37-3.45 (m, 3H, H-4A, 4H, Sp), 2.04, 2.03, 1.99, 1.68 (4s, 12H, OAc, AcNH), 1.32 {m. 6H, H-6A, 6H). "C
NMR (CDC13) S 170.7, 170.4, 169.9, 169.1 (C=O), 129.3-138.5 (Ph), 103.3 (C-I~), 101.6 (C-In), 93.9 (C-1H), 81.5, 80.8 (2C, C-4", 4B), 79.9, 78.9 (2C, C-3", 3H), 78.6 (C-2a), 76.8 (C-2H), 76.0, 75.5, 74.0 (CH~Ph), 73.7 (C-3o), 72.4 (C-5o), 72.2 (CHZPh), 68.7 (C-SA*), 68.5 (C-4D), 68.2 (C-58*), 62.0 (C-6D), 54.6 (C-2D), 23.4 (AcNH), 21.1 (OAc), 18.5, 18.1 (2C, C-6~, 6H). FAB-MS for C~H65N0,~ (M = 999.4) mlr. 1022.5 [M + Na]+. Anal. Calcd. for CsaH6sN0» : C, 64.85 ; H, 6.55 ; N. 1.40. Found C, 64.55 ; H, 7.16 ; N, I.IS.
(2-acetamido-3,4,6-tri-O-acetyl-Z-deo~y-[i-D-glucopyranosyl)-(1--~2)-(3,4-di-O-bcnzy 1-ac~
Irrhamnopyranosyl)-(1-~2)-3,4-di-O-benzyl-a/(3-L-rhamnopyranosyl trichloroacetimidate (13). The hemiacetal 29 (595 mg, 0.59 mmol) was dissolved in CH2Clz (IO mL), placed udder argon and cooled to 0°C. Trichloroacetonitrile (0.6 ml:,, 6 mmol), then DBU (10 pL, 59 ~rmol) were added. The mixture was stirred at 0°C for 20 min., then at rt for

14 20 min. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was purified Iry flash chromatography with 1:1 cyclohexane-AcOEt and 0.2 % of Et3N
to give 13 (634 mg, 94 %) as a colourless foam. The'H NMR spectra showed the a :~3 ratio to be 10 :1. [a)D -20° (c 1, CHC13). 'H NVIR (CDCl3) a-anomer : E 8.47 (s, IH, C=NH), 7.20-7.38 (m, 20H, Ph), 6.10 (d, 1H, J,,z = 1.3 Hz, H-lg), 5.40 (d, 1H, NH), 5,01 (m, 1H, H-4D), 4.95 (d, 1H, Jl,z = 1.2 Hz, H-1"), 4.89 (m, 1H, H-3o), 4.55-4,85 (tn, 9H, H-1D, CHlPh), 4.07 (dd, 1H, H-6ap), 4.03 (m, 1H, H-2A), 3.97 (m, IH, H-2a), 3.91 (dd, 1H, H-6bo), 3.7I-3,85 (m, 5H, H-2B, 3A, 3B, 5A, SH), 3.31-3.45 (m, 3H, H-4A, 4s, 5n), 1.58, 1.91. 1.9G, 1.99 (4s, I2H, OAc, AcNH), 1.26 (m. 6H, H-6A, 6H). '3C NIvIR (CDCl3) 0 171.1, 170.9, 170.3, 169.6 (C=0), 160.6 (C=NH), 128.1-138.6 (Ph), 103.3 (C-lp), 101.6 (C-lA), 96.9 (C-1g), 91.3 (CC~), 81.4.
80.2 (2C, C-4", 4H), 79.9, 78.5 (2C, C-3~, 3B), 78.3 (C-2A), 75.9 (CHzPh), 75.0 (C-28), 73.7 (CHZPh), 73.7 (C-3o), 72.4 (CHzPh), 72.4 (C-5n), 71.0, 69.0 (2C, C-SA, 5H), 68.5 (C-4D), G2.1 (C-6o), 54.6 (C-2n), 23.4 (AcNI-1), 21.1 (OAc), 18.5.18.0 (2C, C-6A, 6B).
Anal. Calcd. for CssH65C~Nz0": C, 58.77 ; H, 5.72 ; N, 2.45. Found C, 58.78 ; H, 5.83; N, 2.45.
Allyl (2-acetamido-3,4,b-tri-O-acetyl-2-deo~y-]i-D-glucopyranosyl)-(1 >Z)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(i-~3}-(2,3,4,6-tetra-O-benryl-a-D-glucopy ranosy 1-(1-->4)-]-2-O-benzoyl-a-L-rhamnopyranoside (5}. Anhydrous ether (5 mL) was added to the donor 13 (500 mg, 0.437 mmol) and the acceptor 11 (242 mg, 0.29 mmol) and powdered 4~-MS. The mixture was placed under argon and cooled to 0°C. Boron trifluoride etherate (415 p.L, 3.27 mmol) was added. The mixture was stirred at 0°C for 1 h, then at rt for 18 h. The mixture was diluted with CHzCIz and triethylamine ( 1 mL) was added. The mixture was filtered through a pad of Celite and the filtrate was concentrated. The residue was purified by column chromatography with 3:2 cyclohexane-AcOEt to give, in order, the acceptor 11 (132 mg, 54 %), 5 (231 mg, 44 %) and the hemiacetal 29 (129 mg, 29 %). The desired pentasaccharide S was obtained as a colourless foam ; (a]D +10° (c 1, CHCl3). 'H NMR (CDC)3): & 7.09-8.02 (gin, 45H, Ph), 5.92 (m 1 H, Atl), 5.65 (d, 1H, NH), 5.37 (m,IH, H-2c), 5.19 (m, 2H, All), 5.13 (bs, 1H, H-l~), 4.35-4.96 (m, 15H, H-1B, lc, 1D, ls, 2e, 3n, 4D, CHZPh), 4.17 (n~, 2H, H-2A, All), 3.8?-4.04 (m, 8H, H-2D, 3A, 3c, 3E, 5A, SE, ban, All), 3.63-3.81 (m, 7H, H-3B, 4c, 4E, Sc, Gas, 6bE, 6bn), 3.59 (m 1H, H-58), 3.43 (tn, 3H, H-2E, 4~, 50), 3.28 (t, 1H, H-48), 1.66, 1.71, 1.99, 2.01 (4s, 12H, OAc, AcNH), 1.34 (m, 6H, H-6A, 6c), 1.00 (d, 3H, H-6g).'3C NMR (CDCl3): b 170.5, 170.0, 169.3, 165.8, 163.5 (C=O), 117.6-138.7 (Ph, Allj, 102.7 (C-lp), 100.8 (2C, C-1,,, 1H), 98.1 (GIE}, 95.9 (C-lc), 81.8 (C-3E), 81.2 (2C, C-2E, 4A), 80.0 (C-4B), 79.7 (2C, C-3A, 3c}, ?8.2 (C-3a), 77,7 (C-2,,), 77.3 (2C, C-4~, 4E), 75.G, 75.4, 74.9 (CHZPh), 74.3 (C-2$), 73.8 (CHZPh), ?3.7 (C-3p), 72.8 (CH,.Ph), 72.3 (C-2c), 72.1 (C-Sc), 7I.5 (C-5E}, 70.2 (CHzPh), G8.5 (C-SE), G8.4 (C-SA, CHz4), 68.2 (C-4D), 6?.9 (C-GE}, 67.4 (C-Sc), G1.8 (C-GD}, 54.3 (C-2D), 23.1 (AcNH), 20.7, 20.6, 20.4 (OAc}, 18.6 (C-6A), 18.0 (C-6C), 17.8 (C-GH). FAB-MS for C,o4Hi~,NOa~ (M = 1812.1) m/z 1836.2, 1835.2 [M + Na]t. Anal. Calcd. for C,o~H»,NOz~: C, 68.90 ; H, G.50 ; N, 0.77. Found C, G8.G4 ; H, 6.6G ; N, I.OS.
Allyl (3,4-di-O-benryl-2-O-chloroaceCyl-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1 >3)-[2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1~4)-]-benzoyl-a-G-rhamnopyranoside (38). A mixture of alcohol 11 (212 mg, 0.255 mmol) and imidate 34 (270 mg, 0.33 mmol) in anhydrous EtiO (4 mL) was stirred fnr IS min under dry Ar. After cooling at -60°C, Me3Si0Tf (30 ~L, 0.166 mmol) was added dropwise and the mixture was stirred overnight and allowed to reach rt. Triethylamine ( 120 ~,L) was added and the mixture was concentrated. The residue was eluted from a column of silica gel with 7:1 cyclohe-EtOAc to give 38 (8G mg, 22 %) as a foam; [a]D +5° (c 1, CHC13). ~H NMR
(CDC13):8 6.95-8.00 (m, 45H, Ph), 5.80-6.00 (m, 1H, All), 5.56 (dd, 1H, H-2A), 5.40 (dd, IH, J~,z < 1.0 Hz, Jz,3 = 3.0 Hz, H-2c), 5.20-5.37 (m, 2H, All), 5.08 (d, 1H, J,,Z
= 3.2 Hz, H~ls), 5.04 (d, 1H, J,,~ < 1.0 Hz, H-lAj, 5.00 (d, 1H, J~,Z < 1.0 Hz, H-18), 4.99 (d, 1H, H-lc), 4.30-4.90 (m; I6H, CHZPh), 4.35 (dd; IH, J2,3 -- 3.0 Hz, H-2a), 4.14 (dd, 1H, J3,a = 9.5 Hz, H-3c), 4.03 (dd, 1 H, Jz,s = J~,4 = 10.0 Hz, H-3E), 3.90-4.20 (m, 2H, All), 3.75-4.00 (m, 4H, CIAc, H-6aE, 6bs), 3.96 (dd, I H, H-3"), 3.95 (m, 1 H, H-SA), 3.95 (dd, 1 H, H-Se), 3.83 (dd, 1 H, H-4~), 3.80 (m 1H, H-Sc), 3.72 (dd, 1H, H-4~, 3.64 (dd, 1H, H-3a), 3.60 (m, 1H, H-SH), 3.52 (dd, 1H, H-2g), 3.39 (dd, 1H, H-4A), 3.30 (dd, 1H, H-48), 1.35 (d, IH, H-6A), 1.30 (d, 1H, H-6c), 1.00 (d, 1H, H-6H). 1'C NMR (CDC13):S 166.1, 165.? (C=O), 117.0-133.4 (Ph), 100.9 (C-1H), 98.9 (C-lAj, 97.8 (C-lE), 96.0 (C-1~), 8I.8 (C-3E), 80.9 (C-2E), 79.9 (G4n), 79.6 (C-48). 79.6 (C-3c), 78.9 (C-3g), 78.0 (C-4c), 77.5 (C-4E), 77.3 (C-3A), 75.6, 75.3, 75.0, 74.7, 73.9, 73.5, 72.8, 70.9, (G'HZPh, All), 74.9 (C-29), 72.5 (C-2~), 71.2 (C-5E), 70.9 (C-2"), G8.8 (C-5~), G8.5 (C-6~), 68.3 (C-5,,), G7.5 (C-5c), 40.9 (CIAc), 18.8 (C-6,,), 18.2 (C-dc), 17.8 (C-68). FAB-MS
for C91H~9CIO~o (M =1558.6) m/z 1581.7 [M + Na]+. Anal Calcd, for C9aH99C1Ozo : C, 70.82 ;
H, 6.40. Found C, 70.67 ; H, 6.58.

Allyl '(3,4-di-O-benzyl-2-0 pmetho~ybenzyl-a-L-rhamnopyranosyl)-(1-->2)-(3,4-di-0-benryl-a-L-rhamnopyranosyl)-(1->3)-[2,3,4,6-tetra-0-benzyl-a-D-glucopyranosyl-(1~
4)-]-Z-0-benzoyl-a-L-rhamnopyranoside (39). A mixture of alcohol 11 (125 mg, 0.15 mmol) and 4~ molecular sieves in anhydrous EtzO (3 mL) was stirred for 45 min under dry Ar.
After cooling at -40°C, Me3Si0Tf (20 ~.L, 0.112 mmol) was added dropwise. A solution of the donor 37 (210 mg, 0.225 mmo!) in anhydrous EtzO (2 mL) was added dropwise to the solution of the acceptor during I h. The mixture was stirred for 3 h at -40°C.
Triethylamine (100 uL) was added and the mixture was filtered and concentrated. The residue was eluted from a column of silica gel with 85:15 cyclohexane-EtOAe to give 39 ( 107 mg, 44 %) as a foam; [a]D
+12° (c 1; CHC~). 1H NMR (CDC~):8 7.1-8.1 (m, 45H, Ph), 6.50-7.00 (m, 4H, CHzPhOMe), 5.70-5.90 (m, IH, All), 5.32 (dd, 1H, J,a = 1_G, Jz,3 --- 3.1 Hz, H-2c), 5.10-5.25 (m, 2H, All), 5.05 (d, IH, H-lB), 4.98 (d, 1H, J~~ = 3.2 Hz, H-Ifi), 4.85 (m, 2H, H-lA, Ic), 4.20-4.80 (m, 18H, CHzPh), 3.90-4.20 (m, 2H, All), 3.00-4.20 (m, 20H, H-2A, 28, ZF, 3,,, 3H, 3c, 3E, 4A, 4B, 4c, 4E, 5A, 5B, Sc, 5E, 6aE, Gbs, 0CH3), 0.82-I.30 (3 d, 9H, H-G,,, 6H, Gc).
~3C NMR (CDC)3):8 166.3 (C=O), 118.2-138.5 (Ph, All), 99.5, 99.3 (2C, C-IA, lg), 98.4 (C-IE), 96.4 (C-lc), 82.3, 81.4, 81.1. 80.5, 80.3, 79.5, 78.2, 77.6 (8C, C-2E, 3," 3B, 3c, 3E, 4~" 4H, 4c), 76.0, 75.5, 75.3, 74.9, 74.3, 73.3, 72.3. 71.8, 71.6, (CH~Ph), 72.5 (C-ZC), 72.0 (C-4E), 69.2, 69.0, 68.9 (3C, C-SA> Se, 5~), 68.8, 68.6 (All, C-GE), 67.8 (C-5E), 55.5 (OCH3), 19.0, 18.8, 18.4 (3C, C-6~, G8, 6c). FAB-MS for C9BH,osOzo (M - 1603.8) mlz 1626.6 [M + Na]+. Correct elem.
analysis could not be obtain for this compound.
Altyl (2-O-acetyl-3,4-di-O-benTyl-ac-I~rhamnupyranosyl)-(1-->3)-[2,3,4,b-tetra-O-benzyl-a-o-glucopyranosyl-(1-->4)]-2-0-benzoyl-a-L-rhamnopyranoside (42).
A mixture of alcohol 11 (G.5 g, 7.8 mmol) and imidate 2Z (G.5 g, I2.2 mmol) in anhydrous EtzO (86 mL) was stirred far 15 min under dry Ar. After cooling at -50°C, MejSiOTf (560 p.
L, 3.1 mmol) was added dropwise and the mi,~cture was stirred and allowed to rt overnight.
TriEthylamine (I.1 mL) was added and the mixture was concentrated. The residue was eluted from a column of silica gel with 6;1 cyclohexane-EtO.~c to give 42 (8.0 g, 84 %) as a colorless foam; [aJo +2I° (c I, CHC13). ~H NMR (CDC13):8 7.1-8.2 (m, 35H, Ph), 5.95 (m, 1H, All), 5.72 (dd, I H, J,,z = 1.0, Jz,3 = 3.1 Hz, H-2B), 5.44 (dd, 1 H, J,,z = 1.6 Hz, Jz,3 = 3.1 Hz, H-2e), 5.30 (m, 2H, All), 5.07 (d, 1H, J,,z = 3.05 Hz, H-lE), 5.05 (d, 1H, H-1H), 4.95 (d, 1H, Jt,z =

r.........~ _.

1.6 Hz, H-lc), 4.35-4.90 (m 12H, CHZPh}, 4.00-4.20 (m, 2H, All), 4.20 (dd, 1H, J3,4 = 8.5 Hz, H-3c); 4.05 (dd, 1H, Jz,3 = 9-7, J3 4 = 10.0 Hz, H-3E}; 3.80-3.90 (m, 2H, H-GaE, 6bE), 3.82 (m, 1H, Js.s = 6.0 Hz, H-5c), 3.80 (m, 2H, H-4~, 5E), 3.76 {m, 1H, H-4c), 3.75 (dd, 1H, J3,a =
8.SHz, H-3H), 3.69 (m, 1H, J4,s = 8.5, J5,6 = 6.1 Hz, H-5B), 3.53 (dd, 1H, H-2E), 3.35 (dd, IH, H-4B), 2.15 (s, 3H, OAc), 1.40 (d, 3H, H-6~), 1.01 (d, 3H, H-6B}. "C NMR
(CDCI3):8 170.3, 166.1 (C=0), 118.2-138.6 (Fh, All), 99.7 (C-1a), 98.6 (C-IE), 96.4 (C-1~), 82.2 (C-3E), 81.7 (C-2E), 80.2 (C-4H), 80.I (C-3~), 78.0 (C-4c), 77.8 (C-3H), 75.9; 75.4, 75.2, 74.3, 73.3, 70.9 (6C, Cl-IaPh), 72.5 (C-2c), 72.0 (C-4F), 69.0 (C-Sc), G9.0 (C-5$), 68.9 (2C, All, C-2a), 68.0 (C-6E), 67.8 (C-5E), 21.1 {OAC), 19.0 (C-Gc), 18.1 (C-GB), FARMS of C,lH~eO~s (M, 1198.5), m/z 1221.4 ((M+Na~+).
Altyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(la3)-[2,3,4,6-tetra-O-benzyi-a-D-glucopyranosyl-(1~4)J-Z-O-benzoyl-a-L-rhamnopyranoside (10). A mixture of the trisaccharide 42 (8.0 g, 6.5 mmol) in MeOH (128 mL) was treated with 5.7 mL of HBF4/ExzO
at rt. The solution was stirred during 4 days. Et3N was added until neutralization and concentrated. The residue was diluted with DCM, washed with satd aq NaHC03 and water.
The organic layer was dried on MgSO,, filtered and concentrated. The residue was eluted from a column of silica gel with 15:1 toluene-AcOEt to give 10 (6.31 g, 84 %) as a foam; [a]p +14°
(c 1, CHC~);'H NMR (CDCIj):& 7.05-8.10 (m, 35H, Ph), 5.82 (m, IH, All), 5.25 (dd, IH, Jl.a = 1.7 Hz, Jz,~ - 3.1 Hz, H-2c), 5.19 (m. 2H, All), 5.00 (d, 1H, J~,Z = 3.1 Hz, H-Ir), 4.87 (d, IH, Jt,a = 1.8 Hz, H-IB), 4.81 (d, IH, H-lc), 4.35-4.90 (m, 12H, CHzPh), 4.00-4.20 (m, 2H, All), 4.10 (dd, 1H, J~,,= 8.5 Hz, H-3c), 4.09 (dd, 1H, Ja.3 = 3.2 Hz, H-2s), 3.95 (m, IH, Ja,s =
9.5 Hz, H~5E), 3.92 (dd, 1H, Jz,~ = 9.5 Hz, J3,~ - 9,5 Hz, H-3s), 3.78 (m, 1H, J5,6 = 6.0 Hz, H-5c), 3.70 (m, 1H, H-4~}, 3.58-3.62 (m, 2H, H-GaE, bbfi), 3.59 (m, 1H. J4,5 =
9.0 Hz, J5,6 = 6.2 Hz, H-5g), 3.54 (dd, I H, H-4E}, 3.48 (dd, 1H, J;,4 = 8.5 Hz, H-3a), 3.45 (dd, 1 H, H-2~j, 3.31 (dd, I H, H-4B), 2.68 (d, I H, Jz,o,~ = 2.3 Hz, 0-H), 1.29 (d, 3H, H-6c), 1.09 (d, 3H, H-6H). ''~C
NMR (CDC13):d 166.2 (C=O), 118.2-137.5 (Ph, AII), 103.1 (C-18), 98.5 (C-la), 96.6 (GIc), 82.1 (C-3E), 81.4 (C-2a), 80.4 (G-4H), 79.7 (C-3H), 79.4 (C-4c), 78.9 (C-3c}, 78.1 (C-4E), 76.0, 75.5, 74.5, 74.2, 73.6, 72.1 (CHaPh), 73.7 (C-2c), G8.9 (C-6E), G8.8 (C-5B), 68.7 (All, C-5s), 68.1 (C-5c), 19.1 (G-6c), 18.2 (C-6a). FABMS of C~oH»O~s (M, 1156.5), m/z I179.5 ([M+Na]'). Anal Calcd for C,oH~60,s: C, 72.64; H, 6.62. Found C, 72.49; H, 6.80.

Altyl (2-0-acetyl-3,4-di-O~benzyt-a-t,-rhamnopyranosyl)-(1 >2)-(3,4-dl-O-benzyl-oc-L~
rhamnopyranosyl)-(1 >3)-j2,3,4,6-tetra-O-benzyt-a-D-glucopyranosyl-(1~4)]-2-0-benzoyl-a-i,-rhamnopyranoside (44).
A mixture of alcohol 18 (5.2 g, 4.49 mmol), imidate 2 (3.58 g, 6.74 mtrwt) and 4A molecular sieves in anhydrous Et20 (117 mL) was stirred for 1 h under dry ar. After cooling at -30°C, Me~SiOTf (580 EtL, 3.2 mmolj was added dropwise and the mi~.-ture was stirred and allowed to rt overnight. Triethylamine {1.2 mL) was added and the mixture was filtered and concentrated.
The residue was eluted from a column of silica gel with 9:1 cyclohexane-EtOAc to give 44 (G.1G g, 90 %); (a]D +13° (c 1, CHC)3). ~H NMR (CDC13):8 7,00-8.10 (m, 45H, Ph), 5.82 (m, 1 H, All), 5 .45 (dd, 1 H, J,,2 = 1.5 Hz, Js,~ = 2.5 Hz, H-2~), 5.29 (dd, 1 H, Jl,z = I . 5 Hz, J23 = 2.5 Hz, H-2C), 5.19 (m, 2H, AII), 4.9? (d, 1H, J,,~ = 3.2 Hz, H-lE), 4.95 (d, 1H, H-lA), 4.91 (d, 1H, Jt,z = 1.G Hz, H-Ie), 4.84 (d, 1H, H-lc), 4.35-4.90 (m, IGH, CHZPh), 4.29 (dd, 1H, Jz,3 =
2.6 Hz, H-2a), 4.00-4.10 (m, 2H, All), 4.02 (dd, 1 H, J~.4 = 8.5 Hz, H-3c), 3.90 (m, ZH, J~,3 =
Ja.4 = J4,s = 9.5 Hz, H-3E, Ss), 3.85 (m, 2H, J,,, = 9.3 Hz, J4,s = 9.5 Hz, H-3", SA), 3.72 (m, 2H, JS,h= 6.0 Hz, H-4c, 5c), 3.G2-3.6G (m, ZH, H-6aE, 6b~, 3.61 (dd, 1H, H-4E), 3.54 (dd, 1H, ,h,4 = 9.4 Hz, H-3Bj, 3.45 (dd, IH, J~,a = 9.5 Hz, J5,6 = 6.1 Hz, H-5B), 3.39 (dd, 1H, H-2s). 3.34 (dd, 1H, H-4,~, 3.21 (dd, 1H, H-4$), 1.89 (s, 3H, OAcj, 1.26 {2d, GH, H-G~, Gcj, 0.89 (d, 3H, H-6B). "C NMR (CDCl3):8 170.2, 166.1 (2C, C=0), 118.1-138.4 (Ph, Allj, 101.3 (C-18), 99.8 (C-lA), 98.2 (C-lE), 96.4 (C-lc), 82,Z (C-3s), 81.4 (C-2s), 80.G (C-4A), 80.5 (C-3~), 80.1 (C-4g), 79.3 (C-3ej, 78.5 (C-4~). 78.1 (C-3A)> 78.0 (C-4E), 76.0, 75.9, 75.7, 75.2, 74.3, 73.3, 72.1, 71.1 (GH~Ph), 75.2 (C-2a), 72.9 (C-2o), 71.7 (C-5E), 69.5 (C-2p), 69.2 (2C, C-5,,, 5$), 68.9 (Atl, C-2H), 68.9 (C-GE), 67.9 (C-5C), 21.4 (OAc), 19.1 (C-GA), 18.7 (C-Gcj, 18.1 (C-GH).
FABMS of C9oHiooOzo (M, 1524.7), m/z 1547.8 ([M+Na]+). Anal. Calcd for C9~H,ooO~u: C, 72.42; H, 6.61. Found C, 72.31; H, 6.75.
Allyt (3,4-di-O-bcnzyl-a-~rhamnopyranosyl)-(1--~2)-(3,4-di-O-benzyi-a-t,-rhamnopyranosyl}-(1-33)-[2,3,4,6-tetra-O-benzyl-a-n-glacopyranosyl-(1-~4))-2-0-benzoyl-a-z-rhamnopyranoside (40), A mixture of 44 (6.0 g, 3.93 mmol) in MeOH (200 mL) was treated with 10 mI, of H8F4lEt~0 at rt. The solution was stirred during 5 days. Et3N was added until neutralization and concentrated. The residue was diluted with DCM, washed with satd aq NaHC03 and water.
I
The organic layer was dried on MgS04, filtered and concentrated. The residue was eluted from i a column of silica gel with 6:1 cyclohexane-AcOEt to give 40 (5.0 g, 84 %) as a colorless foam; [a]p +12° {c 1, CHCI~).'H NhiR (CDCh):o 7.00-8.00 (m, 45H, Ph), 5.83 (m, 1H, All), 5.29 (dd, 1H, J~,2= 1.8 Hz, J2,3= 2.9 Hz, H-2c), 5.19 (m, 2H, Alt), 4.99 {d, 1H, Jl~= 1.4 Hz, H-1~), 4.97 (d, 1H, J ,,~= 3.3 Hz, H-lE), 4.94 (d, 1H, J,,a,= 1.7 Hz, H-18), 4.83 (d, 1H, H-lc), 4.35-4.90 (m, 16H, CHzPh), 4.30 (dd, 1H, J,"3 = 2.7 Hz, H-Zg), 4.00-4.I0 (m.
2H, Ah), 4.02 (dd, IH, Ja,3= 3.5 Hz, J3,q= 8.5 Hz, H-3c), 3.98 (dd, IH, H-2A), 3.91-3.95 (m, 3H, H-SE, 6aE, Garb, 3.90 (dd, 1H, J,,, = 9.5 Hz, J3,q = 9.4 Hz, H-3E), 3.73-3.82 (m, 4H, H-3A, 5", 4c, 5c), 3.GG (dd, 1H, J4,s = 9.6 Hz, H-4E), 3.53 (dd, IH, J~,4 = 9.5 Hz, H-3B), 3.48 (m IH, J4,s = 9.5 ~~ Js,s= 5.1 Hz, H-SH), 3.40.3.44 (m, 2H, H-4A, 2E), 3.17 (dd, IH, H-4B), 2.18 (d, IH, JZ.os°
Z,0 Hz, 0-H), 1.26 (d, 3H, H-6c), 1.25 (d, 3H, H-6~), 0.90 (d, 3H, H-6H). "C
NMR (CDC13):
~ 1G6.2 (C=0), 118.0-138.3 (Ph, All). 101.5 (C-18), 101.4 (C-IA), 98.2 (C-lE), 96.4 (C-Ic), 82.2 {C-3s), 8I.4 (C-2~, 80.G (C-4A), 80.3 (C-48), 79.9 (2C, C-3c, 3~), 79.2 (C-3H), 78.3 (C-4c), 78.0 (C-4E), 75.9, 75.6, 75.5, 74.8, 74.2, 73.5, 72.4, 71.0 (CHzPh), 75.3 (C-2B), 72.9 (C-2c), 71.6 {C-2A), 69.2, G9.I, GB.G, 68.3, 67.9 (SC, C-5", 5a, Sc, SE, 6E), 68.9 (All), 19.1 (C-6c), 18.6 {C-6A), 18.1 (C-6,~). FABMS of C9°H9g019 (VI, 1482.7), rrrlz 1505.8 ([M+Na]').
Anal. Calcd for CgpH98019~2H20: C, 71.12; H, 6.77. Found C, 71.21; H, 6.78.
Altyl (3,4,6-tri-O-acetyl-2-deoxy-Z-tric6loroacctamido-[3-D-glucopyranosyl)-(1--~2)-(3,4-di-O-benzyl-a-t-rhamnopyranosyn-(1-~2)-(3,4-di-O-bcnzyl-oc-lfrhamnopyranosyl)-(1-->
3)-[2,3,4,6-tetra-0-benzyl-a-n-giueopyranosyl-(1--~4)]-2~O-benzoyl-a-L-rhamnapyranoxidt (4). A mixture of alcohol 10 (5.0 g, 3.37 mnwl), imidate 16 (3,0 g, 5.04 mmol) algid 4A molecular sieves in anhydrous DCM (120 mL) was stirred for I h under dry Ar.
After cooling at 0°C, Me3SiOTf (240 uL, 1.32 mmol) was added dropwise and the mixture was stirred for 2.5 h while coming back to rt. Et3N (800 p.L) was added, and the mi~cture was filtered and concentrated. The residue was eluted from a column of silica gel with 4:1 to 2:1 cycbhExane-EtOAc to give X4 (G.27 g, 98 %); [a]D +1.5° (c l, CHCIa). 'H
NMR (CDCl3):c5 7.00-8.00 (m, 45H, Ph), 6.68 (d, 1H, J2,~= 8.5 Hz, N-HD), 5.82 (m, 1H, All), 5.29 (dd, 1H, J,,z = 1.0 Hz, J~,~ = 2.3 Hz, H-2c), 5.19 (m, 2H, All), 5.00 (d, 1H, J~,~ =
1.0 Hz, H-la), 4.96 (dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 10.5 Hz, H-3D), 4.88 (d, 1H, J,,z = 3.3 Hz, H-lc), 4.85 (d, 1H, H-lc), 4.82 (d, IH, J,2= I.7 Hz, H-IB), 4.81 (dd, 1H, Je,s=10.0 Hz, H-4D), 4.72 (d, 1H, J~,~=
8.G Ha, H-ID), 4.35-4,90 (m, 1GH, CHZPh), 4.38 (m, 1H, H-2g), 4.00~4.10 (m, 2H, All), 4.05 (dd, 1 H. Ja,~ = 2.7 Hz, H-2A), 3.95 (dd, 1H, Ja,j = 3.5 Hz f3,4 = 8.5 Hz, H-3c), 3.90 (m, 2H, H-Se, 4E), 3.82-3.8G (m, 2H, H-ban, 6ho), 3.70-3.84 (m, 6H, H-3E, GaP, 6bs, 3,,, SA, 2D), 3.68 (m;
1 H, H-Sc), 3.61 (dd, 1 H, Jd,$ = 9.0 Hz, H-4o), 3.SG (dd, I H, J3,4 = 9.5 Hz, H-3H), 3.47 (rn, 1 H, Ja,s = 9.5 Hz, Js,b = 6. I Hz, H-58), 3.33-3.35 (m, 3H, H-4~, Sp, 2F), 3.17 (dd, 1H, H-4g), 1.98, 2.00, 2.02 (3s, 9H, OA.c), 1.24 (d, 3H, J5,6= G.0 Hz, H-G,,), 1.23 {d, 3H, Js.s= 5.9 Hz, H-6c), 0.90 (d, .3H, H-6g). '3C NMR (CDCl3):& 170.9, 170.7, 1G9.G, 166.1, 162.1 (C=0), 118.1-138.3 (Ph, AII), IOLS (C-lD}, 102.4 (C-ls), 101.1 (C-IA), 98.5 (C-IE), 9G.4 (C-lc), 92.G
(CC)3), 82.1 (C-3e), 81.7 (C-3c}, 8L6 (C-2s), 80.4 (C-4s), 80.1 (C-3,,), 79.1 (C-4c), 78,5 (C-3a), 77.9 (C-4,~), 77.G {C-4E), 76.4 (C-2,,), 7G.1, 75.8, 75.4, 74.7, 74.3, 74.2, 73.2, ?0.4 (CHZPh), 74.9 (C-2H), 72.9 (C-3D), 72.7 (C-2G): 72.5 (C-So), 71.9 (C-5E), G8.4 (C-G~, 68.8 (A!1), G8.9, 68.7, 68.5, 67.7 {4C, C-4c, S", 5B, Sc), G2.2 (C-6p), 56.2 (C-2D), 20.9, 20.9, 20.7 (3C, OAc), 19.0 (C-GA), 18.5 (C-G~), 18.2 (C-GH). FABMS of C,o4H"4C1,N0~~ (Vf, 1916.4):
m/z I938.9 [M+Na]+. Anal. Calcd for CIO4H, iaCIJNOz?: C, 65.18 ; H, 6.00 ; N, 0.73. Found C, 64.95 ; H, 6.17 ; N, 0.76.
(2,3,4-tri-O-acetyl-a-deoxy-Z-trichloroncetamido-~i-D-glucopyranosy I)-(1-->2)-(3,4-di-O-benzyl-a-C,-rhamnopyranosyl)-(1 >2)-(3,4-di-O-benzyl-a-z-rhamnopyranosyl)-(1--~3)-(Z,3,4,6-tetra-O-benzyl-a-D-glucapyranosyl-(1-~4)]-2-O-benzoyl-a-crrhamnopyranosyl trichloroacetlmidate (46). Compound 4 {3.S g, 1.8 mmvl) was dissolved in anhydrous THF
(35 mL). The solution was degassed and placed under Ar. 1,5-Cyclooctadiene-bis(methyldiphenylphosphine}iridium hcxafluorophosphate (81 mg) was added, and the solution was degassed again. The catalyst was activated by passing over a stream of hydrogen until the solution has turned yellow. The reaction mixture was degassed again and stirred under an Ar atmosphere, then concentrated to dryness. The residue was dissolved in acetone (15 mL). then water (3 mL), mercuric chloride (490 mg) anti mercuric oxide (420 mg) were added successively, 'The mixture protected from light was stirred at rt for 2 h and acetone was evaporated. The resulting suspensinnwas taken up in DCM, washed twzce with 50%
aq KI, water and satd aq NaCI, dried and concentrated. The residue was eluted from a column of silica gel with 2:1 petroleum ether-EtOAe to give the corresponding hemiacetal 45.
Trichloroacetonitrile (G.5 mL) and DBU (97 p.L) were added to a solution of the residue in anhydrous diehloromethane (33 mL) at 0°C. After 1 h, the mixture was concentrated. The residue was eluted from a column of silica geI with 5:2 cyclohexane-EtOAC and 0.2 % Et3N to give 46 (2.48 g, 6G °!°); [a]~ -~4° (e 1, CHCu). 'H NMR
(CDCl3):S 8.71 (s, 1H, N=H), 7.00-l,rvW uvcvy-u.- ...

8.00 (m, 45H, Ph), 6.80 (d, IH, Jz,~,~., = 8,2 Hz, NHp), G.37 (d, 1H, J,,z =
2.6 Hz., H-lc), 5.59 (dd, 1H, Jz,; = 3.0 Hz. H-2c), 5.10 (d, 1H, Ji,z = I.0 Hz, H-lA); 5.05 (dd, I
H, H-3D), 4.98-5.00 (m, 2H, H-lE, ls), 4.97 (dd, 1H, H-4n), 4.00-5.00 (m I9H, 8 CHaPh, H-3c, 2A, 2$), 3.20-4.00 (m, 17H, H-2E, 3E, 4E, 5E, Gas. 6bE, 4c, Sc, 3B, 4a, 5s~ 3~, 4n, $na Sa~ 6ao, 6bn), 1.80, 2.02, 2.03 (3s, 9H, OAc), 1.39, 1.32 and I .00 (3d, 9H, H-6~" 6s, 6c). i3C NvLR (CDC~):8 169.7, 169.5, 168.3, 164.5, 160.9 (C=0, C=N), 126.2-137,5 (Ph), lOI.G (C-1D), 101.3 (2C, C-1~, ls), 98.7 (C-IE), 94.8 (C-lc), 91.3 (CCI~), 82.1, 81.5, 80.4, 80.1, 78.4, 77.9, 77.6, 76.5 (IOC, C-2A, 2E, 3,,, 3B, 3c, 3E, 4A, 4B, 4c, 4E), 76.0, 75.9, 75.5, 74.9, 74.3, 73.3 (CH2Ph), 72.9, 72.6, 71.9, 70.9, 70.d, 69.I, 68.8, 68.5 (9C, C-2B, 2c, 3p, 4D, SA, 5a, Sc, SD, 5~), 68.3, 62.1 (2C, C-6D, 6E), 56.2 (C-2D), 21.0, 20.9, 20.8 (3 OAc), 19.1, 18.3, 18.1 (3C, C-6,,, 6B, 6c).
Anal. Calcd for C103H110C~N2O27 C: 61.22, H: 5.49, N: 1.39. Found C: 6I.24. H. 5.50, N: 1.21.
Methyl (3,4,6-tri-O-acetyl-Z-deo~y-Z-trichioroacetamido-[i-n-glucopyranosyl)-(1--~Z)-(3,4-di-O-benryl-a-z-rhamnopyranosyl)-(1--~2)-(3,4.di-O-benzyl-a-z.-rhamnopyranosyl)-(1-a3)-[Z,3,4,6-tetr8-O-benzyl-a-n-glucopyrenosyl-(1-~4)]-2-O-benzoyf-a-L-rhamnopyranosyl)-(1-~3)-(Z-dcory-4,6-O-isopropyliden~2-tricttloroacetamido-j3-n-glucopyranosyl)-(1-i2)-(3,4-di-O-benzyl-a-L-rhamnapyranosyl)-(1--~2)-(3,4-di-D-bcnzyl-a-L-rhamnopyranosyl)-(1-~3)-[2,3,d,6-tetra-O-benzyl-a-D-glucopyranosyl-(1-~4)]-benzoyl-a-Irrbamnopyranoside (49), A mixture of 46 (154 mg, 76 p.mol) and 48 (92 mg, 51 ~.tnal). 4A molecular sier,~es and dry 1,2-DGE (3 mL), was stirred for 1 h, then cooled to -35°C. Triflic acid (6 ~.L) was added. The stirred mixture was allowed to reach 10°C in 2.5 h.
Et3N (25 ~.L) was added and the mixture was filtered. After evaporation, the residue was eluted from a column of silica gel with 2:1 eyclohexane-EtOAC and 0.5 % of Et3N to give 49 which could not be obtained as pure material at this stage, and was directly engaged in the next reaction.
Methyl (3,4,6-tri-O-acetyl-Z-deoay-Z-trichloroacetamido-[i~D-glucopyranosy~-(1-~2)-(3,4-di-O-benzyl-a-z-rhamnopyranosyl)-(I--~Z)-(3,4-di-D-benzyl-a-L-rhamnopyranosyn-(1--~3)-[2,3,4,6-tetra-O-benryl-a-v-glucopyt~tnosyl-(1-~4)]-(z-O-benzoy 1-a-L-rhamnopyranosyt)-(1-~3)-(Z-deoxy-2-trichioroacetamido-[i-D-glucopyranosyl)-(1-~3)-(3,4-di-O-benzyl-oG-L-rhamnopy~nosyn-(1-~2)-(3,4-di-O-benzyl-a-z-rhamaopyranosy ~-(1-~3)-[Z,3,4,6-tetra-O-bcnzyl-a-n-glucopyranosyl-(x-~4)]-2-O-benzoyl-a-L-LMYltvcay~um. _.._ rhamnopyranoside (50). To a solution of the residue 49 (I86 mg, 51 ~mol) in DCM (3 mL) was added dropwise, at 0°C, a solution of TFA (0.5 mL) and water (0.5 mL). The mixture was stirred for 3 h, then concentrated by co-evaporation with water then toluene.
The residue was eluted from a column of silica gel with 2:1 to 1:1 petroleum ether-EtOAC to give 50 (134 mg, 72 %, 2 steps); [a]p +G° (c l, CHCl3).'H NMR (CDCI~); 8 7.10-8.05 (m, 90H, Ph), 6.82-6.8G
(2d, 2H, Jz,~ = 8.0 Hz, J~,r,~.~ = 8.5 Hz, NHo, NHb~}. S.I9-5.35 (m, 2H, H-2c, 2c~), 5.20, S_OS
(2s, 2H, H-lA, 1"~), 5.05 (dd, 1H, H-3p~), 4.99-4.80 (m, 9H, H-18, la~, lc, Ic~, 1D, 1D-, IE, Is~, 4D~), 4.30-4.80 (m, 32H, OCHzPh), 3.15-4.10 (m, 44H, H-2A, 2A~, 2g, 28., 2D, 2D~. 2E, 2E~, 3w, 3AV 3B> 3s', 3c, 3c~, 30> 3s: 3s~: 4a~ 4w, 4s~ 4av 4ct 4cv 4p~ 4s~ 4EV Sn~ Sav ~s. Ssv Sc, Scv SD, SD~, SE, SE~. 6ao, 6bo, Gao~, 6bD~, 6aa, 6bs, 6aE~, 6br~), 3.42 (3H, s, OMe), 2.02. 2.04, 2.08 (9H, 3s, OAc}, 1.40-0.96 (I8H, m, H-6A, G,,~, 6H, 68~, Gc, 6c.). 13C ~ (CDCIs) : S
171.5, 170.9, 170.8, 169.6, 166.2, 162.4, 162.1 (C=O), 127.2-139.5 (Ph), 10I.9, IOI.G, 101.5, 101.3, 99.2, 98.8, 98.2 (lOC, C-lA, 1,~~, la, la~, Ic, lc~, 1D, 1D~, lE, IE,), 92.7, 92.6 (2C, CCl3}, 82.1, 81.8, 81.7, 80.5, 80.3, 80.1, 79.3, 77.9, 77.8, 73.0, 72.6, 72.5, 72Ø 69.4, 69.0, 68.9, 67.4, (39C, C-2n~ 2w. 2s, 2a~, 2c, 2r, 2s, 2s~, 3n~ 3av 3s~ 3av 3c~ 3cv 3D~ 3D°~ 3c, 3ev 4A, 4nv 4s~ 4ae 4c~ 4ce 40, 4D~, 4Ea 4E~, Sn, SA', Ss~ Sav 5c, 5c~, SD, 5p', 5E, Sa~, Gp-), 76,0, 75.9. 74.8, ?4.3, 73.6, 73.2.
68.6 (CHzPh), 62.3, 62.2, 60.7 (3C, C-Gp, GF, 6E~), SS_5, SG.2 (3C, C-2D, 2D,, OCH~), 20.97, 20.94, 20.77 (OAc), 19.01, 18.72, 18.62, 18.15, 17.90 (GC. C-Ga, 6,,~, 6H, 6g~, 6c, 6~~).
FABMS for Cy9,H~,aCIaNzOso (M, 3622.5), mla 3645.3 [M+Na]+. Anal. Calcd for C~97H114C~Ny050 C: 65.32, H: 5.95, N: 0.77. Found C: 65.20, H: 6.03, N: 0.78.
Methyl (2-acetamido-2-deoxy-[3-D-glucapyranosyl)-(I >2)-(a-L-rhamnopyrano9yl)-(1-~
2)-(a-L-rhamnopyranosyl)-(1-->3)-[a-n-glucopyranosyl-(1~4)J-(a-L-rhamnopyranosyl)-(Z-~3)-(2-acetamido-2-deo~,y-(i-n-glucopyranosyl)-(1--~2)-(a-z-rhamnopyratnosyl)-(1-->
Z)-(a-L-rhamnopyrvnosyl)-(r-~3)-[a-D-glucopyraaosyi-(1 >4))-a-t,-rhamnopyranosidc (1). A solution of 50 (183 mg, 50 ~.mol), in EtOH (3 mL), EtOAc (0.3 mL}, 1M
HCI (100 p.L) was hydrogenated in the presence of Pd/C (2~0 mg) for 72 h at rt_ The mixture was filtered and concentrated. A solution of the residue in MeOH (4 mL) and Et3N (200 uL) was hydrogenated in the presence of PdIC (200 mg) for 24 h at rt. The mixture was altered and concentrated. A solution of the residue (50 mg, 25 p.mol) in MeOH (3 mL) and DCM (0.5 mL}
was treated by MeONs until pH=10. The mixture was stirred overnight at 55°C. After cooling at rt, IR 120 (H") was added until neutral pH, and the solution was filtered and concentrated, v,.,. , , .,...,. _. _ _ then was eluted from a column of C-18 with waterJCH3GN and freeze-dried to afford amorphous I (30 mg, 37 %), [aJp -1° (c 1, Hz0). 'H NMR (D20): 8 S.i3 (2d, 2H, J,,z =
3.5Hz, H-lE, IE~), 4.75, 4.95, 5.05 (m, 5H, H-lA, 1a, lA~, IB~, lc-), 4.62~4.64 (2d, 2H, J,,i= 7.0 Hz, J~,I= 8.0 Hz, H-la, ID~), 4.58 (d, 1H, J,,z= 2.2 Hz, H-lc), 3.20-4.10 (m, S1H, H-2A, 2A~, 2e, 2a', 2c, 2cv 2n, 2av 2E, 2EV 3a, 3nv 3a, 3e', 3c, acv 3n, 3v~, 3e, 3e°, 4A, 4w, 4s, 4BV 4c, 4cv 4D, 4av 4E~ 4EV 5A, 5nr sa, SH~, 5c, 5cv 5D, Sov 5a, SE', 6aD, 61~. 6aw, 6tfi~, 6aE, 6bE, 6aE~, 61~~, OCH3), 1.97, 1.99 (2s, 6H, 2 AcNH), 1.I5-I.33 (6d, 18H, J5,6 = 6.3Hz, H-6A, 68, 6c, 6n', 6sv 6c-). "C NMR (D~0): 8 175.2, 174.7 (C=0), 103.1 (2C, C-1D~, Ia), 102.6; 101.7, 101.3, 100.8 (6G, C-lA, 1B, lc, lA~, la~, lc,), 98.0 (2C, C-lE, lE~), 81.6, 79.7, 79.G, 79.1, 76.2,, 76.1, 73.9, 73.0, 72.7, 72.6, 72.5, 72.2. 72.1, 71.6, 70.1, 70.0, 69.7, 69.0, 68.5 (38C, C-2A, 2A., 2a, 2H~~ 2c~ 2c~, 2F, 2F.~, 3n, 3w, 3B, 38~~ 3c, acv 3a, 3a°, 3g, 3s~, 4n, 4w. 48, 4s~, 4c, 4c~, 4D, 4av 4e, 4E~, 5A, SA~, 5a, SB., 5c, 5c., 5p, Sp~, 5E, 5a.), 60.9 (4C, C-6E, GE~. 6a, 6p~), 56.20, SG.00, 55.31 (3C, C-2p, 2a~, OCH,), 22.7, 22.6 (2C, AcNH), 18.3, I8.1, 17.2, 17.1, 16.95, 16.90 (6C, C-6A, 68, 6c, 6,,', 6H', 6c~)- HR~1LS: calculated for CssH~ioNaO<s+Na:
1661.6278. Found 1661.6277.

'_r v =O
O
N ~P ~..~ N O
O Oco O o' D_D~ ~ ~ z O z o m C7 C7 ~ ~ O ~ O O O~ =O o '~ ~ O = O
O Z
O O O
w ~ O_ c~a p O z Z ~O ~ O
m r C7 V O cv ~ O
O coo ~ ~ m O
co M
p d N
O
N
'r ~ WO

WO
C1 cp O ~ ~1 ~1 s O
O ~ II II Q ~ O
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r L, M~,Pl l Synthesis of a tetra- and two pentaaaccharide fragments of the O-specific polysaccharide of Sfiigella Jtexneri serotype 2a This paper discloses the synthesis of a methyl glycoside of the repeating unit I of the S
~lexneri Za O-SP. together with that of a corresponding pentasaccharide and a tetrasaccharide.
All the methyl glycosides of the di- to pentasaccharides obtained by circular permutation of the monosaccharide residues partaking in the linear backbone of I, and comprising the EC
portion. are now available. Their binding to a set of available protective IgG
antibodies is reported elsewhere LM?Pll~tAeo~brcvet-pent~OMe cA o24s4ss5 zoos-o~-o4 Synthesis of a tetra- and two pentasaccharide fragments of the O-specific polysaccharide of Shigella Jlexneri serotype 2a INTRODUCTION
Shigellosis, also known as bacillary dysentery, is a major enteric disease which accounts for some 165 million annual episodes, among which 1.1 million deaths, occurring mostly in developing countries. {Kotloff, 1999 #147} Young children and immunocompromised individuals are the main victims. Occurrence of the disease is seen as a correlate of sanitary conditions, and those are not likely to improve rapidly in areas at risk. The financial status of the populations in which shigellosis stands in its endemic or epidemic forms, as well as the emerging resistance to antimicrobial drugs, {Khan, 1985 #3G7} *
; Salam, 1991 #368} * {Ashkenazi, 1995 #328} * {Iversen, 1998 #357} * {Iwalol''un, 2001 #358} * limit the impact of the latter. Some 15 years ago, vaccination was defined as a priority by the WHO in its program on enteric. diseases.REF However, there is still no license vaccine against this bacterial infection although intensive research is ongoing in the field.{Hale, 1995 #102}(voir si ref reeente PS) Shigellae are Gram negative bacteria. As for other bacterial pathogens, their lipopolysaccharide (LPS) is an important virulence factor. It is also a major target of the host's protective immunity against infection. Indeed, data from infected patients indicated that circulating anti-LPS antibodies were strong markers of acquired immunity.
{Cohen, 1988 #329} {Cohen, 1991 #52} It was also demonstrated in a marine model that the presence locally, preliminary to infection, of a secretory antibody of isotype A
specific for an epitope located on the O-specific polysaccharide (O-SP) moiety of the LPS of Shigella,~lexrteri Sa, prevented any host's homologous infection.{Pha(ipon, 1995 #228) Importantly, field trial of an investigaiional Shigella sonnei 0-SP conjugate, which was shown to induce anti-LpS secrewry IgAs, thus suggesting mucosal stimulation,{CohEn, 1996 #360; demonstrated 7S%
efficacy.{Cohen, 199? #54}
Shigella flexneri 2a is the prevalent serotype in dEVeloping countries, where it is responsible for the endemic form of the disease. Based on the early hypothesis that a critical LMPP77-~1~0'D~OVM~p~tItBOMC ~ 02434685 2003-07-04 level of serum IgG antibodies specific for the 0-specific polysaccharide (0-SP) moiety of the LPS Was sufficient to confer protection against homologous infections,{Robbins, 1992 #256} {Robbins, 1994 #2S7} several S. ,~lexneri 2a 0-SP-protein conjugates were designed.
They were found safe and immunogenic in both. adults and children.{Ashkenazi, #362} {Passwell, 2001 #220}
Allowing a better cornrol of the various structural parameters possibly involved in the immunogenicity of glycoconjugate vaccines, oligosaccharide-protein conjugates were proposed as alternatives to polysaccharide.-protein conjugate vaccines against bacteria. {Pozsgay, 2000 #247} Indeed, such constructs were found immunogenic on several occasions, including examples whereby the oligosaceharide portion was made of one repeating unit only.~Benaissa-Trouw, 2001 #363}{Mawas, 2002 #364} We reasoned that glycoconjugates incorporating chemically synthesized oligosaeeharides, appropriately selected for their ability to mimic the native O-SP in terms of both antigenicity and solution conformation, may offEr an alternative to the S ,~lexneri 2a O-SP-protein conjugates currently under study, Our approach relies on a rational basis. Indeed, in order to select the best oligosaccharide mimic, we have undertaken the characterization of the antigenic determinants of S. j<lexneri 2a 0-SP recognized by serotype-specific protective monoclonal antibodies. The synthesis of a panel of methyl glycoside oligosaccharides representative of fragments of S.
flexneri 2a 0-SP was thus undertaken to be used as probes in the study of antibody recognition.
A B E C D
2)-a-L-Rhap-(1--~2)-a-L-Rhap-(1-~3)-[a-D-Glcp-(I-~4)J-a-L-Rhap-(1~3)-[i~D-GIcNAcp(1 ~
The 0~SP of S ,flexnerf 2a is a heteropolysaccharide defined by the pentasaccharide repeating unit T.{Simnwns, 1971 #88; Lindberg, 1991 #46} It features a linear tetrasaccharide backbone, which is common to all f. ,~lexneri O-antigens and comprises a N
acetyl glucosamine and three rhamnose residues, together with an a-D-glucopyranose residue branched at position 4 of one of the rhamnoses. We have already reported on the synthesis of the methyl glycosides of various fragments of the 0-SP, including the known EC
i i disaccharide, {Henry, 1974 #224; Lipkind, 1987 #223 } f Mulard, 2000 #52}
the ECD (Mulard, 2000 #52} and B(E)C{Mulard, 2000 #52} trisaccharides, the ECDA{Segat, 2002 #225} and AB(E)L{Costachel, 2000 #I01} tetrasaccharides, the B(E)CDA{Segat, 2002 #225}
and DAB(E)C {Costachel, 2000 #101 } pentasa,ceharides and more recently the B(E)CDAB(E)C
octasaccharide.{Belot, 2002 #314} In the following, we report on the synthesis of the LMPPt 1~the°-b«VCt~pCfl~lOM~ ~ 02434685 2003-07-04 ECDAH, AB(E)CD pentasaeeharides as well on that of the B(E)CD tetrasaccharide as their methyl glycosides, 1, a and 3, respectively.
RESULTS AND DISCUSSION
Analysis of the targets shows that all the glycosylation reactions to set up involve 1,2-trans glycosidie linkages except for that of the E-C junction which is 1,2-cis.
Consequently, the syntheses described herein rely on key EC disaccharide building blocks as well as on appropriate A, H and D monosaccharide precursors.
Synthesis of the linear ECDAB-OMe pentasaccharide (l): Based on earlier findings in the series which have demonstrated that the C-D linkage was an appropriate disconnection site,{Segat, 2002 #283} a blockwise synthesis of 1 was designed (Scheme 1). It is based on the glycosylation of the known EC trichloroacetimidate donor XX,{Mulard, 2000 #19G}
obtained in three steps (69%) from the key intermediate XX,{Segat, 2002 #283}
and the DAB
trisaccharide acceptor XX. The latter was obtained by the stepv~~ise condensation of known monosaccharide precursors, readily available by selective protection, deprotection and activation sequences. Thus, TMSOTf catalysed condensation of the rhamnopyranoside acceptor XX{Pozsgay, 1987 #241 } with the trichloroacetimidate donor XX
{Castro-Palomino, 1996 #47} in diethyl ether to give the fully protected rhamnobioside XX, and subsequent de-O-acetylation under Zempl~n conditions gave the AB disaccharide acceptor XX in XX%
overall yield, which compares favourably with the previously described preparation. {Pozsgay, 1987 #, 241 } Conventional glycosylation o.f XX with the known glucosaminyl bromide,{Debenham, 1995 #63}chosen as the precursor to residue D, under base-deficient conditions in order to avoid orthoester formation, smoothly afforded the fully protected DAB trisaccharide (XX%). Removal of 'the tetrachlorophtalimide and concomitant deacetylation by action of XXX in XXX, followed by N acetylation furnished the triol XX
(XX%), which was next protected at positions 4D and 6o by regioselective intxoduction of an isopropylidene acetal upon reaction with 2,2-dimethoxypropane under acid-catalysis (XX%).
(mentiohner produit vert fluo) Indeed, data previously obtained when synthESizing shorter fragments in the series outlined the interest of using 4,G-D-isopropylidene-glucosami,nyl intermediates instead of the more common benrylidene analogs.{Mulard, 2000 #196} Once the two key building blocks made available, their condensation was performed in dichloromethane in the presence of a catalytic amount of TMSOTf to give the fully protected pentasaccharide XX (XX%). Conventional stepwise deprotection involving (i) acidic LMPPII-thco~brevtt~pentaOMe cA o24s4ss5 zoos-o~-o4 hydrolysis of the isopropylidene acetal using 90% aq TFA to give diol XX
(XX%), (ii) conversion of the latter into the corresponding tetraol XX under Zempl6n conditions (XX%), and (iii) final hydrogenolysis of the benzyl protecting groups, gave the linear pentasaecharide I in XX% yield.
Synthesis ojthe AB(E)C,D perttasaccharide 2 acrd of the B(E)CD tetrasaccharide 3 (Scheme 2). For reasons mentioned above, compound XX,{Mulard, 2000 #196} protected at its 4 and 6 hydroxyl groups by an isvpropylidene acetal was the precursor of choice for residue D. In the past, inuoduction of residue B at position 3~ was performed on a 2~-O-benzoylated EC
acceptor resulting from the regioselective acidic hydrolysis of the corresponding 2,3-orthoester intermediate.{Costachel, 2000 #55} ~Segat, 2002 #283} It rapidly occurred to us that opening of the required phenyl orthoester was not compatible with the presence of an isopropy'lidene a.cetal. For that reason, the trichloroacetimidate donor XX, suitably benzoylated at 7.c and orthogonally protected by a chloroacetyl group at position 3c was used as the EC building block instead of the previously mentioned XX. The choice of protecting group at position 2 of the rhamnosyl precursor to residue B was again crucial in the synthesis of 2. Indeed, most of our previous work in the series relied on the use of the known 2-0-acetyl rhamnopyranosyl donor XX, {Castro-Palomino, 1996 #47} as an appropriate precursor to residue B. In the reported syntheses.{Costachel. 2000 #55} selective 2a~0-deacetylation the presence of a 2c-O-benzoate was best performed by treatment with methanolic IIBF4.OEtz for 8ve days, Clearly, such deacetylation conditions are not compatible with the presence of isopropylidene group on the m~olecuIe either. To overcome this limitation, the corresponding 2-0-chloroacetyl rhamnopyranosyl trichloroacetimidate XX was selected as an alternate donor. The latter could indeed also serve as an appropriate precursor to residue A.
Regioselective conversion of diol XX into its 2-D-benzoylated counterpart XX
was performed as described. {Segat, 2002 #283; Treatment of the latter with chloroacetic anhydride and pyridine gave the orthogonally protected XX (XX%), which was smoothly de-0-allylated to yield the corresponding hemiacetal XX (XX%) by a two-step process, involving (i) iridium (I)-promoted isomerisation{Oltvoort, 1981 #216} of the allyl glycoside and (ii) subsequent hydrolysis in the presence of iodine.(Re~ ef Nacira) The selected trichloroacetimidate leaving group was successfully introduced by treatment of XX with trichloroacetonltrile in the presence of DBU, which resulted in the formation of XX (3CY%). TMSOTf mediated glycosylation of donor XX and acceptor XX furnished the fully protected ECD
trisaccharide (XX%), which was as expected readily converted to the required acceptor XX
upon selective LMPP1 i.theo.brwa-pa°taOMa deblocking of the chloroacetyl protecting group with thiourea (XX%). Following the two-step protocol described above for the preparation of XX, the known allyl rhamnopyranoside XX, { Westerduin, 1988 #348 ) bearing a 2-O-choloacetyl protecting group, was converted to the hemia~cetal XX (XX%). Next; treatment of the latter with trichloroacetonitrile and a slight amount of DBU ga~~e donor XX in an acceptable yield of XX%. Glycosylation of the ECD
acceptor XX and the B donor XX was attempted under various conditions of solvent and catalyst. Whatever the conditions, hardly separable mixtures of compounds were obtained.
When using TMSOTf as the promoter and X.XX as the solvent, the expected tetrasaccharide XX was indeed formed, although it was often markedly contaminated with glycosylation intermediates such as the. silylated XX as well as the. orthoester side-product XX, as suggested from mass spectroscopy analysis and NMR data. In fact, the nature of the latter was fully ascertained at the next step in the synthesis. Indeed, treatment of a miuture of the condensation products XX and supposedly XX resulted in the expected tetrasaccharide acceptor XX contaminated by the trisaccharide a.eceptor XX, whereas the corresponding ~3B-tetrasaccharide isomer could not be detected at this stage, which indicated that the corresponding ehloroacetylated XX was not part of the initial mixture.
Formation of the starting XX during the dechloroacetylation step is not unexpected as it may be explained by intramolecular rearrangement Leading to expulsion of the B residue, following dechlorination in the presence of XXX. Starting from XX, the isolated yield of the tetzasaccharide acceptor XX was XX%, which encouraged us to reconsider the use of the 2-O-acetyl analogue XX as a precursor to residues B and A in the synthesis of 2.
A suivre ...
CONCLUSION
The synthesis of the methyl glycoside (2) of the reputing unit I of the S
~l'exrreri 2a O-SP, togethez with that of the corresponding pentasaccharide 1 and tetrasaeeharide 3 were described. All the methyl glycosides of the di- to pentasaccharides obtained by circular permutation of the monosaccharide residues partaking in the linear backbone of I, and comprising the EC portion, are now available in the laboratory. Their binding to a set of available protective IgG antibodies will be reported elsewhere.

LMPP1 I-exp-brevet-pentaOMe EXPERIMENTAL

General Methods. General experimental methods not referred to in this section were as described previously.(REF) TLC on precoated slides of Silica Gel 60 FZSa (Merck) was performed with solvent mixtures of appropriately adjusted polarity consisting of A, dichloromethane-methanol; B, cyclohexane-ethyl acetate, C, cyclohexane-diethyl ether, D, water-acetonitrile, E, iso-propanol-ammonia-water. F, cyclohexane-diethyl ether-ethyl acetate.
Detection was effected when applicable, with UV light, and/or by charring with orcinol (3S
mM) in 4N aq HZS04. In the NMR spectra, of the two magnetically non-equivalent geminal protons at C-6, the one resonating at lower field is denoted H-6a and the one at higher field is denoted H-6b. Interchangeable assignments in the 13C NMR spectra are marked with an asterisk in listing of signal assignments. Sugar residues in oligosaccharides are serially lettered according to the lettering of the repeating unit of the O-SP and identified by a subscript in listing of signal assignments. Low-resolution mass spectra were obtained by Either chemical ionisation (CIMS) using NH3 as the ionising gas, by electrospray mass spectrometry (ESMS), or by fist atom bombardment mass spectrometry (FARMS).
Methyl (2-acetamido-2-deoay-4,6-O-isopropylidene-(i-D-glucopyranosyl)-(1-~2)-(3,4.~di-O-benzyi-ot-L-rhamnopyranocyl)-(1~2)-(3,4-dr-0-benzyl-a-L-rhamnopyranoside (XX).
2,2-dimethoxypropane (4.9 mL. 39.8 mmol) and para-toluenesulfonic acid (18 mg, 95 pmol) were added to a solution of the triol XX (964 mg, 1.09 mmol) in acetone (3 mL) and the mixture was stined at rt for lh. Et3N was added, and volatiles were evaporated. Column chromatography of the residu~ (solvent D, 99:1) gave the acceptor XX as a white solid (969 mg, 96%) which could be crystallized from AcOEt:iPrzO; mp XX°C; [a]D XX (c 1.0); NMR: ~ H, 8 7.45-7.31 (m, 20H, Ph), 6,98 (d, 1 H, JrrE.t,i = 2.4 Hz, NH), G.37 (bs, 1 H, OH), 5.07 (d, 1 H, Jm =
1.9 Hz, H-I,~, 4.90 (d, 1 H, J = 10.8 Hz, OCHZ), 4.85 (d, 1H, J = 10.1 Hz, OCH~), 4.84 (d, 1 H, J = 10.8 Hz, OCHZ), 4.76 (d, 1 H, OCHZ), 4.69 (d, 1 H, OCHz), 4.68 (s, 2H, OCHZ), 4.G5 (d, 1 H, OCH?), 4.61 (d, 1 H, Ji,~ = 1.6 Hz, H-1 H), 4.48 (d, 1 H,1~ ~ = 8.3 Hz, H- l o), 4.09 (dd, 1 H, H-2,~, 4.01 (dd, 1 H, J2,3 = 3.2, J3,4 = 9.4 Hz, H-3,~, 3.91 (dd, 1H, H-28), 3.89-3.84 (m, 2H, J5,6 = 6.3, J4,s =
9.4,1i~,3~ = 3.3, J3,,d~ = 9.4 Hz, H-5~,, 3H), 3.68 (dq, partially overlapped, Js,b = 6.2, Ja,s = 9.5 Hz, H-S$), 3.66-3.58 (m,. 4H, H-6aD, 6bn, 20, 4D), 3.44 (pt, 1H, H-4,~, 3.41 (pt, 1H, H-4$), 3.32 (s, 3H, OCH3), 3.16 (m, 1H, H-SD), 1.60 (s, 3H, C(0)CH3), 1.54, 1.48 (2s, 6H, C(CH3jZ), 1.35 (d, 6H, H-6A, 6B);
13C, S 173.9 (CO), 138.8-128.0 (Ph), 103.7 (C-lv), 101.3 (C-1~, 100.3 (C(CH3)2), 100.2 (C-1B), 81.9 (C-4,~, 80.8 (C-4H), 80.5 (C-3,~, 79.7 (C-3g), 79.4 (C-2,,), 76.2 (OCHZ), 76.0 (C-2H), 75.6. 75.I (2C, OCHZ), 74.4 (C-4D), 74.4 (C-3D), 72.6 (OCHZ), 68.6 (C-5~, 68.0, 67.9 (2C, C-Sg, SD), 62.2 (C-GD), 60.6 (C-2o), 55.1 (OCH3), 29.5 (C(CH3)2), 22.7 (C(0)CH3), 19.4 (C(CH3)2), 18.5, 18.2 (2C, C-6,~ 6B).
FABMS for C52H65N014 (Vi, 92'7.44) ml~ 950.5 [M+Na]+.
Anal. Calcd for C52H65N014~ C, 67.30; H, 7.06; N, 1.~1%. Found: C, 67.15; H, 7.24; N, LMPP11-exp-brevet~penmOMe 1.44%.

Methyl (2,3,4,6-Tetra-0-benzyl-a-D-glucopyranosyl)-(1-~4)-(2,3-di-O-benzoyl-a-L-rhamaopyranosyl)-(1-~3}-(2-acetamido-2-deoaty-4,6-D-isopropylidcnc-[i-D-glucopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1-3Z)-(3,4-di-O-benzyl-a-1.-rhamnopyranoside (XX). Activated powdered 4A molecular sieves were added to a solution of the trisaccharide acceptor XX (202 mg, 0.22 mmol) and the disaccharide donor XX (263 mg, 0.25 mmol) in anhydrous CHzC)z (5 mL} and the suspension was stirred for 30 min at -IS°C.
Trifluoromethanesulfonic acid (7 pL, 34 ~,mol) was added and the mixture was stirred for 2 h while the bath temperature was slowly coming back to 10°C. TLC (solvent D, 49:1) showed that no XX remained. Et3N was added and after 30 min, the suspension was filtered through s pad of Celite. Concentration of the filtrate and chromatography of the residue (solvent B, 9:1 -~ 17:5) gave the fully protected pentasaceharide XX (330 mg, 84%) as a white foam;
[a]D XX (c 1.0);
NMR: tH, 8 8.07-6.96 (m, SOH, Ph), 5.82 (d, 1H, JN~= 7.4 Hz, NH), 5.63 (dd, 1H, J2,3= 3.5, J3,a = 9.5 Hz, H-3o), 5.43 (dd, 1H, 3»= 1.6 Hz, H-2o), 5.09 (bs, IH, H-18), 5.02 (d, 1H, J1,Z= 3.4 Hz, H~IE}, 4.99 (d, 1H, J1,Z = 8.3 Hz, H-lo), 4.95 (d, 1H, J1~, = 1.1 Hz, H-1~), 4.94-4.63 (m, 13H, OCHZ), 4.63 (s, 1H, H-1,~, 4.37 (d, 1H, J = 11.0 Hz. OCH2), 4.29 (dq, 1H, J4,s= 9.5, Js,b= 6.2 Hz, H-5~), 4.25 (d, 1 H, J = 9.5 Hz, OCHZ), 4.23 (pt, I H, J3,4 = J4,s = 9.5 Hz, H-3D), 4.01 (m, 1 H, H-2H), 3.97-3.86 (m, 5H, H-3>;, 2A, 3fi, 4~, OCHZ), 3.82 (m, 1H, H-3A, 5a), 3.71-3.57 (m, 7H, H-SD, 4E, 5,,, 4p, 4E, 6aE, 6bE), 3.54-3.41 (m, 3H, H-2E, 4B, 2p) 3.38-3.31 (m, 2H, H-4A, 6aD), 3.31 (s, 3H, OCH3), 3.17 (m, 1H, H-5E), 3.08 (d, 1H, J6~,6b° 10.1 Hz, H-61~), 1.84 (s, 3H, NHAc), 1.46 (s, 3H, C(CH3)z), 1.45 (d, 3H, J5,6 = 5.9 Hz, H-6~), 1.35 (m, 6H, Js,6 = 5.9 Hz, H-6B, C(CH3)z), 1.31 (d, 3H, Js.b= 6.2 Hz, H-GA); 13C, 8 171.7, 165.9, 165.8 (3C, CO), 138.9-127.9 (Ph), 102.3 (C-IX, J =
167 Hz), 101.5 (C-1x, J = 170 Hz), 100.3 (C.lx, J = 170 Hz), 99.8 (C(CH3)2), 99.6 (C-lx, J = 172 Hz), 98.2 (C-lx, J =172 Hz), 82.0 (C-X~, 81.2 (C-Xg), 80.9 (C-X~, 80.7 (C-Xe), 79.7, 79.3 (3C, OCHz), 78.1 (C-X~, 77.8, 77,4, 75.5, 7I,8 (SC, OCHZ), 71.7 (C-XB), 71.6 (C-X), 68.8 (C-X), 68.0 (C-6E), 67.6 (C-XD), 62.5 (C-do), 58.9 (C-2o), 55.0 (OCH3), 29.5 (C(CH3)2), 23.8 (C(O)CH3), 19.8 (C(CH3)Z), 18.6 (C-Go), 18.5 (C-G,~. 18.3 (C~6$). XXMS for Cio6HmNOzs (M, 1803.79) m/z XXX
[M+H]*.
Anal. Calcd for CI~HIi~NOzs: C, 70.53; H, 6.53; N, 0.78%. Found: C, XXXX; H, XXX;
N, XXX%.
Methyl (2,3,4,6-Tetra-0-benxyl-a-D-glucopyranosyl)-(1--~4)-(2,3-di-0-benzoyl-a-L-rhamnopyranosyl)-(1-->3)-(2-acetamido-2-deoxy-[i-D-8lucopyrano9y~-(1 >2)-(3,4-di-0-benzyl-a-L-r6amnopyranosyl)-(1-~2)-3,4-di-0-benryl-a-L-rhamnopyranoside (XX).
90% aq TFA (750 ~.L) was added at 0°C to a solution of the fully protected XX
(588 mg, 326 p.mol) in CH2Cl2 (6.7 mL) and the mixture was stirred at this temperature for 1 h. TLC
(solvent B, 1.5:1) showed that no XX remained. Volatiles were evaporated by repeated addition of toluene.
Chromatography of the residue (solvent B, 4;1 --> 1:1) gave XX (544 mg, 95%) as a white foam;
z LMPP 1 I-erp-brevet-pentaOMc [aJD X?C° (c 1.0); NMR: tH, b 8.06-7.06 (m, 50H, Ph), 5.82 (d, 1H, J~,z= 7.1 Hz, NH), 5.65 (dd, IH, Ja,3 = 3.8, J3,a = 9.0 Hz, H-3c), 5.53 (m, 1H, H-2c), 5.34 (s, IH, H-IBj, 5.04 (d, iH, J,,~ = 8.3 Hz, H-lo), 5.00 (m, 2H, H-lo, lF), 4.97-4.63 (m, 13H, OCHz), 4.48 (bs, IH, H-1,~, 4.40 (d, IH, J
= 8.4 Hz. OCHZ), 4.29 (d, 1 H, J = 8.0 Hz, OCHz), 4.28-4.21 (m, 2H. H-3D, 5~), 4.10 (m. 1 H, H-2,~, 4.04 (m, IH, H-2H), 3.99 (d, 1H, OCHZ), 3.95-3.89 (m, 3H, H-3H, 3E, 4c), 3.87 (dd, 1H, J2,3=
2.7, J3,a = 9.7 Hz, H-3,~, 3.81-3.64 (m, SH, H-5E, 5H, GaD, 4E, 5"), 3.54 (dd, 1 H, J~,2 = 3.2, J2,3 = 9.7 Hz, H-2E), 3.51 (pt, 1H, J3,a = Ja,s = 9.5 Hz, H-4H), 3.45-3.37 (m, 4H, H-4A, 4p, 6aE; 2p), 3.33 (m, 5H, H-5o, GbD, OCH3}, 3.12 (d, 1H, Jse,6b= 10.6 Hz, H-GbE), 2.28 (bs, 1H, OH),1.97 (bs, 1H, OH), 1.84 (s, 3H, NHAc), 1.54 (d, 3H. Js,fi= G.1 Hz, H-G~j, 1.37 (m GH, H-GB, GA);
~3C, 8 171.5, 165.8, IGS.G (3C, CO), 138.8-127.9 (Ph), 101.6 (C-1D), 100.8 (C-IH), 100.5 (C-1,~, 100.1 (C-lE~), 99.9 (C-lc*), 84.9 (C-3p), 82.1 (C-3E), 80.9, 80.7, 80.6, 80.5 (4C, C-4B, 3A, 4,,, 2E), 79.7 (C-4~), 79.3 (C-38), 77.8 (2C, C-2B, 4E), 76.0, 75.9 (2C, OCH2), 75.8 (C-5D), 75.d, 75.I, 74.6, 73.7, 73.1 (5C, OCHZ), 72.8 (C-2,~. 72.6 (OCHz), 71.8 (C-5E), 71.6 (C-4D), 71.3 (C-3c), 71.1 (C-2o), 69.4 (C-5~), 68.8 (C-5B), 68.3 (C-5A), 68.1 (C-6E}, 63.0 (C-6D), 57.6 (C-2D), 55.0 (OCH3), 23.8 (M-iAc), 18.8 (C-do}, 18.6, 18.5 (2C, C-6,v 6H). XXNiS for C~o3H1~3N025 (M, 1763.76) m/zX30i [M+HJ+.
Anal. Calcd for C,o3H1t3N023: C. XX; H, XX; N, XX%. Found: C. ~;XXX; H, XXX;
N, XXX%.
Methyl (2,3,4,6-Tetra-0-benzyl-a-A-glucopyranosyl)-(1-->4)-a-L-rhamnopyranosyl-(1-~3)-(2-acctamido-2-deoxy-[3-D-glucopyranosyl)-(I-~2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1 >2)-3,4-di-0-benzyl-a-L-rhamnopyranoside (XX). 1M
Methanolic sodium methoxide was added to a solution of XX (277 mg, 157 lrmol) in a 1:1 mixture of CH2C12 and MeOH (6 mL) until the pH was 10. The mixture was stirred overnight at rt and neutralized with Amberlite IR-120 (H+). The crude material was chromatographed (solvent D, 49:1) to give XX (211 mg, 86%) as a white foam; [aJp XX° (c 1.0); NMR; ~H, 68.07-7.06 (m, 50H, Ph), 5.82 (d, IH. JHH,1= 7.1 Hz, NIA, S. 65 (dd, IN, J~,3 = 3.8, J3,a = 9.0 Hz, l~ 3c), 5.53 (dd, IH, J,,1 = 1.6 Hz, H-2~), S. 34 (s, 1 H, H I ~, 5. 04 (d, 1 H, J~,1= 8. 3 Hz, H 1 D), 5. 00 (m, 2H, H 1 c, I ~, 9. 97 4. 63 (m, 13H, OCH~, 4. 48 (Us, I H, H I,~, 4. 40 (d, 1 H, J = 8. 4 Hz, OCH,~, 4. 29 (d, I H, J = 8. 0 Hz, OCH~), 4. 28-4. 21 (m, 2H, H 3a Sc), 4.10 (m, ll~ H 2,~ j, 4. 04 (m, ll~ l~
2B), 3. 99 (d, 1 H, OCHZ), 3. 95-3.89 (m, 3I~ H 3r, 3B, 4c), 3.87 (dd, I H, J1,3 = 2. 7, J3,., = 9. 4 Hz, H 3,~, 3.81-3. 64 (m, 5H, H
5B, 5E, 6ao, 5,,, 9,~, 3. 54 (dd, I H, J1,3 = 3. 2, J,~,a = 9. 7 Hz. H 2E), 3.51 (pt, 1 H, J4,s = Jj,d = 9.5 Hz, l~ 4B), 3.45-3.37 (nr, 4H. H 4A, 4D. 6aE, 2D), 3.33 (m, 5H, H So, 6bD, OCHj), 3.12 (d, III J6a.ch =
10. 6 Hz, H 6b,~ 2. 28 (bs, I H, 01~, 1. 97 (bs, I H, Oll), 1. 8~ (s, 3H, NHAc), 1.53 (d, 3H, J5,6 = 6.1 Ice, H 6~, 1.37 (m, 6H, H Ge, b,~; '3C, 0171. 5, 1 GS. 8, 165. 6 (3C, CO), 138. 8-127.9 (Ph), 101. d (C-1 ~, 100. 8 (C-18), l 00. S (Gl,~, 100. l, 99. 9 (ZC, C-I E. 1 c), 84. 9 (C-3D), 82.1 (C 3~, 80. 9, 80. 7, 80. 6, 80.5, 79. 7 (Sc, C-dg, 3A, 4A, 2E, 4C) (3C, OCHZ), 78. I (C-X,~, 7? 8, 77.4, 75. 5, i I. 8 (SC, OCH~j, 71. 7 (C-X~, 71.6 (C ~, 68.8 (C ~, 68. 0 (C-6~, 67. 6 (C-XQj, 62.5 (C-5D), 58.9 (C-2D), 55.0 (OCH,~, 29.5 (C(CH3)~, 23.8 (C(O)CH~), 19.8 (C(CHa),~, 18.6 (C-d), 18.5 (C-6,~,), 18.3 LhlPPI t ~exp-brevet-pent~OMe (C-d~.'XXMS for Ct03HI13N~25 (M. 1763.76) m1z XXX [M+H]+.
Anal. Calcd for C103HI13N~25~ C. XX; H, XX; N, XX%. Found: C, XXXX; H, XXX; N, XXX%.
Methyl arD-Glueopyranosyl-(1~4)-a-L-rhamnopyrsnosyl-(1~3)-2-acctamido-2-deoxy-p-D-glucopyranosyl-(1-~2)-a-L-rhamnopyranosyl-(1->,2)-a-L-rhamnopyranoside (1). The bensylaled tetrasaccharide 23 (484 mg, 394 lemol) was dissolved in a mixture of methanol (IO mL) and AcOH (1 mL), treated with 10% Pd-C catalyst (200 mgj, and the suspension was stirred overnight at rt. TLC (solvent D, 3:2) showed that the starting material had been transformed into a mare polar product. The suspension was filtered on a pad of Celite. The filtrate was concentrated and coevaporated repeatedly with cyclohexane. Reverse phase chromatography of the residue (solvent F, 100: 0 -~ 49: I), followed by freeze-drying, gave the target tetrasaccharide 1 as an amorphous powder (230 mg, 85%); (aJo +3° (c 1.0, water); NMR: t H, d 5. 04 (d, 1 H, Jt, ~ = 3. 8 Hz. H I ,~, 4. 87 (bs, 1 H, H
I C), 4. 84 (bs, ll~ H
I,~, 4. 7d (d, overlapped, 11~ H I D), 4.10 (dq, I H, J~, g = 9. S Hz, H SCj, 4. 01 (m, I H, H-2~, 4.00 (m, ll~ H S~J, 3.92 (dd, IH, J6a,6b = 12.0, J5,6a = 1.8 Hz, H dap), 3.87-3.73 (m, 7H. H
3C, 3,i, Gag. 6bE, Zp, 2C, 6bD), 3. 73-3. dl (m. 3H, N 3g, 3D, 5~, 3.59-3.43 (m, SH, H 2~, 4D, 9C, Sp, 4~, 3.39 (s, 31~ OCHj), 3.32 (pt, IH, Jj,4 = J4,5 = 9.6 Hz, H ~4,~, 2.07 (s, 31~
C(0)CH3), 1.32 (c~ 3H, J,5,6 = 6.2 Ha, H dC), and 1.28 (d, 3H, J5,6 = d.2 Hz, H d,~; ~jC, d 175. 3 (C(O)), I 02. 7 (C-1 D, J = I d3 Hz), 102. 0 (C-I C, J = 170 H;), 100. 5 (2C, C-I,q, 1 ~, J =
170 Hz), 82.3 (C-3D), 81.8 (C-4C), 79.3 (C-2A), 7ti.7 (C-4~, 73. d (C-3~, 73.1 (C-4~, 72. 6 (C-S~, 72.4 (C-2~, 71.8 (C-2~, 70. 7 (C-3,~, 70. l (C-SD), 69. 7 (C-3C), 69.3 (C-5,~, 69.2 (C-dD), 68.9 (C-SCJ, 61.4 (C-6p), 60.9 (C-d,~, 56.4 (C 2p), 55.6 (OCHj), 23.0 (C(O)CH3), 17.5 (C-d,~, and 17.3 (C-d~. FABMSfor CzTH.tyNOlg (ELI, 689.3) mlz 712.2 (M+NcrJ+.
3,4-Di-O-benzyl-2-0-chloroacetyl-a-L-rhamnopyranose (XX). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (lr(I), 25 mgj was dissolved in dry THF (5 mL) and the resulting red solution was degassed in an argon stream.
Hydrogen was then bubbled tluough the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream A solution of XX (3.28 g, 7.12 mmol) in THF (30 mL) was degassed and added. The miarture was stirred overnight at rt, and a solution of iodine (3.G
g, 14.2 mmol) in a mixture of THF (70 mL) and water (20 mL) was added. The mixture was stirred at rt for 1 h, then concentrated. The residue was taken up in CHZC12 and washed twice with 5% aq NaHSOa. The organic phase was dried and concentrated. The residue was purified by column chromatography (solvent B, X:X) to give XX (2.53 g, 85%) as a slightly yellow . , (aJD +25° (c L0); IH NMR: S 7.40-7.28 (m, IOH, Ph), 5.57 (bd, 0.2H, H-2[i), 5.45 (dd, O.8H, Jt,Z - 2.0 Hz, H-2a), 5.13 (bd, 0.8H, H-la), 4.92 (d, 1H, J = 10.9 Hz, OCHZa, OCHZ/3), 4.79 (d, 0.2H, J = 11.2 Hz, OCH2(3), 4.74 (d, 1 H, J = 11.2 Hz, OCHZa; H-1 [3), 4.65 (d, 0.8H, OCH2a), 4.64 (d, 0.2H, OCHZp), 4.58 (d, 0.8H, OCHza), 4.54 (d, 0.2H, OCHz(i), LMPPII-cxp~brcvn~penL~OMe ~ o24s4ss5 zoos-o~-o4 3H, CH2C1, H-5c, 3c), 3.84 (m, 1H, H-5E), 3.78-3.74 (m, 2H, H-6aE, 4E), 3.70 (pt, 1H, Ja,s =
J3 4 = 9.3 Hz, H-4c), 3.58-3.54 (m, 2H, H-6b~, 2E), and i .50 (d, 3H, J5,6 =
6.2 Hz, H-6o); '3C
NMR: 6167.0 (C=O, CIAc), 166.0 (C=O, Bz), 139.1-128.0 (Ph, All), 118.5 (All), 99.5 (C-1E), 96.8 (C-lc), 81.9 (C-3fi), 81.0 (C-2E), 79.7 (C-4c), 77.7 (C-4e), 76.0, 75.4, 74.1, 73.8 (4C, OCHZ), 73.5 (C-3c), 71.8 (C-5E), 70.9 (C-2c), 68.8 (OCHypli), 68.1 (C-6E), 67.7 (C-Sc), 41.5 (CHZCI), and 18.6 (C-dc); FAB-MS far C52H55012 (M, 906.5) mlz 929.3 [M+Na]*.
Anal. Calcd for C52H5sC1O12: C, 68.83; H, 6.11%. Found: C, 68.74; H, 6.19%.
(2,3,4,6-Tetra-D-benzyl-a-D-glucopyranosyl)-(1-~4)-2-O-benzoyl-3-0-chloroacetyl-a,/[3-L-rh9mnopyranose (XX). A solution of XX (7.21 g, 7.95 mmol) in THF (80 mL) containing activated iridium complex (GO mg) was treated as describd for the preparation of XX. The mixture was stirred at rt for 3 h, at which point a solution of iodine (4.0 g,

15.7 mmol) in a mixture of THF (90 mL) and water (24 mL) was added. The mixture was stirred at rt for 30 min, then concentrated. The residue was taken up in CHzCIZ and washed twice with 5%
aq NaHSOd, then with brine. The organic phase was dried and concentrated. The residue was purified by column chromatography (solvent B, 4:1) to give XX (6.7 g, 97%) as a slightly yellow foam, (aJD +ZS° (c I. O); 1H NMR: 8 8.10~7.09 (m 25H, Fh), 5.47 (dd, 1 H, J2,3 = 3.5, J3,a = 9.3 Hz, H-30), 5.41 (bs, 1H, H-2c), 5.03 (bs, IH, H-lc), 4.94 (d, IH, J = 10.9 Hz, OCHa), 4.87 (d, 1H, J~,~
= 3.4 Hz, H-lE), 4.85 (d, 1H, OCHz), 4.80 (m, 2H, OCH2), 4.64 (m, 2H, OCH~), 4.45 (d, 1H, J=
10.7 Hz. OCHi), 4.41 (d, 1 H, J = 12.1 Hz, OCHZ), 4.16 (dq, 1 H, J4,5 = 9.3 Hz, H-5c), 4.09 (d, 1H, J = IS.G Hz, CHzCI). 3.96 (d, 1H, CHiCI), 3.93 (pt, 1H, H-3E), 3,83 (m, 1H, H-5e), 3,77-3.68 (m, 2H, H-4E, 6aE), 3.65 (pt, 1H, H-4c), 3.54 (m, 2H, H-6bE, 2E), and 1.48 (d, 3H, J5,6= 6.2 Hz, H-6c); ~~C NMR: E167.0 (C=0, CIAc), 166.0 (C=O, Bz), 139.1-127.9 (Ph), 99.5 (C-la), 92.3 (C-lc), 81.9 (C-3E), 81.0 (C-2s), 79.9 (C-4c), 77.6 (C-4E), 76.0, 75.6, 74.2, 74.1 (4C, OCHZ), 72.1 (C-3c), 71.7 (C-4E), 71.1 (C-2c), 68.0 (C-GE), 67.5 (C-Sc), 41.5 (CHZCI), and 18.9 (C-6c); FAB-MS for C49HstC1Oli (M, 866.3) m/z 889.3 [M+Na]+.
Anal. Calcd for Ca9H3iC101Z: C, 67.85; H, 5.93%. Found: C, 67.72; H, 6.00%.
(2,3,4,6-tetra-O-6enzyl-a-D-glucopyranosyl)-(1 >4)-2-O-benzoyl~3-0-chtoroscetyl-a-L-rhamnopyranosyl trichloroacetimidate (XX). Trichloroacetonitrile (I.1 mL, 10.9 mmol) and DBU (17 p.L) were added to a solution of the hemiacetal XX (950 mg, 1.09 mmol) in dry DCM
(8 mL), and the mixture was stirred at 0°C for 1.5 h. Toluene was added, and volatiles were evaporated. Th,e residue was purified by flash chromatography (solvent B, 3:2 containing 0.1%
Et3N) to give XX (930 mg. 84%) as a ~XX~Cx: Further elution gave some remaining starting material XX (13G mg, 14°l). ~aJD +25° (c 1.0); 1H NMR: S 8.7G
(s, 1H, NH), 8.12-7.17 (m, 25H, Ph), 6.34 (d, IH, Jl,z= 1.5 Hz, H-lc), 5.67 (dd, 1H, H-2c), 5.54 (dd, 1H, JZ,~= 3.4, J3,a= 8.8 Hz, H-3C), 4.98 (d, 1 H, OCHZ), 4.88 (d, 1 H, Jls = 3.4 H-1 E), 4.84 (d, 1 H, J = 11,1 Hz, OCHz), (d, 0.8H, OCH2a), 4.64 (d, LMPPII-exp-brevd~penmOMc ~ o24s4ss5 zoos-o~-o4 4,82 (d, 1H, J = 11.2 Hz, OCHz), 4.65 (d, IH, OCHZ), 4.G2 (d, 1H, OCHz), 4.4d (d, 1H, J = 11.4 Hz, OCHz), 4.41 (d, 1H, J=11.8 Hz, OCHZ), 4.14 (dq, 1H, Ja,s = 9.5 Hz, H-5c), 4.11 (d, 1H, J =
I5.5 Hz, CHZCI), 3.98 (d, 1H, CHZCI), 3.94 {pt, 1H, H-3E), 3.83-3.71 (m, 4H, H-SE, 6aE, 4E, 4c), 3.56-3.51 (m, 2H, H-6bE, 2a), and 1.51 (d, 3H, J5,6 = 6.2 Hz, H-Gc); t3C NMR:
O I G7.1 (C=0, CIAc), 165.7 (C=0, Bz), IG9.6 (C=NH), 139.0-127.9 (Ph), 99.9 (C-IE), 95.2 (C-lc), 82.1 (C-3E), 80.9 (C-2~, 79.0 (C-4o), 77.6 (C-4E), 76.0, 75.6, 74.2, 73.8 (4C, OCHz), 73.0 (C-3c), 71.9 (C-5E), 70_7 (C-5c), 69.2 (C-2c), 68.0 (C-6E), 67.7 (C-Sc), 41.4 (CHZCI), and 18.6 (C-6c).
Anal. Calcd for CslHstClaNOIZ: C, 60.54; H, 5.08; N, 1.38%. Found: C, 60.49;
H, 5.01;
N, 1.34%.
Methyl (Z,3,4,6-tetra-O-beazyl-a-D-glucopyranosy()-(1-->4)-(2-0-benzoyl-3-O-chloroacetyLa-L-rhamnopyranosyl)-(1--~3)-2-acetamido-2-deoxy-3,4-O-isopropylidene-~i-D-glucopyranoside (XI~. The acceptor XX (500 mg, 1.82 mmol) was dissolved in DCM
(5.5 mL) and 4A-MS (300 mg) were added. The mixture was cooled to -60°C
and stirred for 15 min. TMSOTf (35 ~L, mmol) and a solution of the disaccharide donor XX (2.39 g, 2.3G
mm~ol) in DC~f (7.5 mh) were added. The mixture was stirred for 45 min while the cooling bath was coming back to rt, and for more 3 h at rt. The mixture was then heated at 65°C for 1 h 30 min. EtjN was added and the mixture was stirred at rt for 20 min, then diluted with CHzCIz and filtered through a pad of Celite. The filtrate was concentrated and purified by column chromatography (solvent B, 85:15 -> l:l) to give XX (1.64 g, 80%) as a '.
(a~D +25° (c 3.0); tH NMR: S 8.06-6,93 (m, 25H, Ph), b.18 (d, 1H, JNH.z = 7.3 Hz, NHD).
5.40 (dd, 1H, Jz,3= 3.5 Hz, H-3c), 5.38 (bs, 1H, H-Zc), 4.98 (d, IH, JS,~= 8.3 Hz, H-1D), 4.94 (bs. IH, H-Ic), 4.94 (d, 1H, OCHZ), 4.93 (d, IH, Jl,~= 3.4 Hz, H-IE), 4.83 (d, 2H, J = 10.7 Hz, OCHi), 4.81 (d, 1H, J = 10.6 Hz; OCHz), 4.67 (d, 1H, J = 11.7 Ha, OCHZ), 4.62 (d, 1H; J =
11.4 Hz, OCHa), 4.47 (m, 3H, H-3D, OCHz), 4.22 (dq, 1H,14,s= 9.4, Js,s= 6.2 Hz, H-Sc), 4.10 (d, 1H, J = 15.5 Hz, CHzCI), 3.96 (m, 2H, H-Gap, CH2C1), 3.91 (pt, IH, H-3E), 3.82 {m, 2H, H-SE, 6bD), 3.72 (m, 3H, H-GaE, 4E, 4c), 3.G2 (pt, 1H, J~,4 = Js,s = 9.4 Hz, H-4D), 3.55 (m, 2H;
H-6bE, 2~), 3.SI (s, 3H, OMe), 3.41 (m, 1H, H-5D), 3.15 (m, 1H, H-2D), 2.04 (s, 3H, NHAc), I.51 (s, 3H, CMez), I.42 (m, 6H, H-6c; CMea), and I.51 (d, 3H, JS,6 = G.2 Hz, H-6C); t3C
NMR: 8 171.8 (C=O, NHAc), 167.3 (C=O, CIAc), 166.1 (C=O, Bz), 139.0-128.0 (Ph), IOI.I
(C-ID, JcH < 164 Hz~, 99.9 (CMez), 99.4 (C-lE, 1cH > 165 Hz), 98.2 (C-Ic, JcH
= 172 Hz), 81.8 (C-3E), 80.9 (C-2~, 79.0 (C-4c"'), 77.7 (C-4E*), 76.7 (C-3D), 75.9, 75.3, ?4.2, 73.9 (4C, OCHz), 73.7 (C-4D), 73.4 (C-3c), 71.9 (C-5E), 71.2 (C-2c), 68.2 (C-G~), G7.8 (C-Sc), 67.4 (C-SD), 62.7 (C-do); 59.6 (C-2D), 57.6 (OMe), 41.5 (CHzCI), 29.5 (CMe2), 27.3 (NHAc), 19.7 (CMez), and 18.G (C-6c); FAB-MS for C6tH~oCINOt~ (M, I 123.4) m/z 1146.5 [M+Na]+.
Anal. Calcd fvr CsiH~oCINOt~: C, 65.15; H, 6.27; N, 1.25%. Found: C, 65.13; H, 6.23;
N, 1.22%.

GMPP I 1-exp-brcva-penmOMc Methyt (2,3,4,6-tetra-O-benzyha-D-gtucopyranosyl)-(1-->4)-(2-O-bepzoyl-a-L-rhnmaopyranoayl)-(1-~3)-Z-acetamido-2-deoxy-3,4-O-isopropylidene-p-D-glucopyranoside (XX). To a solution of the fully protected XX (1.40 g, 1.25 mewl) in a mixture of methanol (18 mL) and pyridine (18 mL) was added thiourea (951 mg, 12.5 mmol). The mixture was stirred at 65°C for 5 h at which time no TLC (solvent D, 4:1) that no starting material remained.
E~~aporation of the volatiles and co-evaporation of petroleum ether form the residue resulted in a crude solid which was taken up in a minimum of methanol. A large excess of DCM
was added and the mixture was left to stand at 0°C for 1 h. The precipitate was ftltzated on a pad of Celite and the filtrated was concentrated. Column chromatography of the residue (solvent C, 4:1) gave the trisaccharide acceptor XX (1.28 g, 97%) as a XXXX. ~aJD +ZS° (c 1.0J; tH NMR: & 8.10-6.96 (m, 25H, Ph), 6.09 (d, 1H, J~Z -- 7.9 Hz, NHD), 5.26 (dd, 1H, J~,~ = 1.6, JZ,3 = 3.4 Hz, H-2~), 4.97 (m, 3H, H-1~, lE, OCHZ), 4.86 (m, 3H, H-1D, OCH~), 4.81 (d, IH, OCHZ), 4.72 (d, 1H, OCl-h), 4.58 (d, 1H, J =12.2 Hz, OCHi), 4.51 (d, 1H, 3 = 10.9 Hz, OCH2), 4.48 (d, 1H, J = 12.2 Hz, OCHZ), 4.23 (pt, 1H; JZ,~ = J3,d = 9.4 Hz, H-3D), 4. I 8-4.10 (m, ZH, H-So, Ss), 4.OG-3.95 (m, 3H, H-3c, 3E, 6aD), 3.80 (pt, 1H, Js,sb= Js°,sb= 10.4 Hz, H-6bn), 3.GG
(m, 2H, H-6aE, 6bE), 3.G2 (dd, 1 H, Jz,3 = 9.8, Jl s = 4.1 Hi, H-2E), 3.59 (pt, 1H, J3,4 = Ja.s = 8.9 Hz, H-4E), 3.55 (pt, 1H, J3,4 =
J4,s = 9.2 Hz, H-4p), 3.51 (pt, 1 H, J3,a = Ja,s = 9.3 Hz, H-4~), 3.49 (s, 3H, OGH~}, 2.22 (s, 3H, NHAc), 1.90 (bs, 1H, OH), 1.49 (s, 3H, CMe2), 1.43 (s, 3H, CMez), and 1.40 (s, 3H, J5,6 = 6.2 Hz, H-6c); 13C NMR: b171.8, 166.6 (2C, C=O), 138.9-128.1 (Ph), 101.6 (C-1D), 99.8 (CMe2), 98.6 (C-IE*), 98.3 (C-to*), 85.4 (C-4c), 82.0 (C-3s), 80.4 (C-2fi), 78.2 (C-4E), 77.1 (C-3d), 75.9, 75.5, 74.2, 73,9 (4C, OCH2}, 73.6 (C-4D*), 73.5 (C-2~*), 7I.7 (C-SE}, 69.0 (C-6E), 68.3 (C-3c), G7.S (C-SD), 66.9 (C-Sc), 62.7 (C-6n), 58.9 (C-2o), 57.5 (OMe), 29.5 (CMeZ), 24.0 (NHAc), 19.7 (CMe2), and 18.2 (C-6~); FAB-MS for Cs9H69N016 (M, 1047,5) mla 1070.4 jM+Na)+.
Anal. Calcd for C7pH~6016: C, 67.61; H, 6.64; N, 1.34%. Found: C, 67,46; H, 6.78; N, 1.24%.
Methyl (3,4-Di-0-benzyl-2-D-cbloroacetyl-a-L-rhamnopyranosyl)-(1-~3)-j(2,3,4,G-tetra-O-benzyl-a-D-glucopyranosyi-{1-~4))-(2-O-benzoyl-3-O-chioroacetyl-a-L-rhamnopyranosyl)-(1~3~-2-xcetamido-2-dcoxy-3,4-0-isopropylideae-(3-n-glucopyranoside (XX). The trisaccharide acceptor XX (GI5 mg, 0.58 mmol) was dissolved in EtzO (10 mL) and the solution was cooled to -60°C. TMSOTf (32 pL) and donor XX (497 mg, 0.88 mmol) in EtZO (12 mL) were added, and the mixture was stired for 1 h while the bath was slowly coming back to -20°C.
The mixture was stirred for 4 h at this temperature, then at 0°C
overnight. More XX (SO mg, 88 pmol) was added, and the mixture was stirred at rt for 3 h more at 0°C.
Et3N was added, and the mixture was concentrated. Column chromatography of the residue (solvent B, 9:1 --; 1:I) gave the orthoester XX (44 mg, 5%) then the fully protected XX (445 mg, 52%}
contaminated with the trimethylsilyl side product XX (Ji;~UXX: XX:XX) together with a mixture of XX and XX (65 mg, 8°~a), and the starting XX (27 mg, 4%). Compound XX (alpha) had ('aJp +25° (e 1.0J; 1H

LMPP l 1-~cp-brevet-pentaOMc NMR b 8.07-7.12 (m, 3 SH, Ph), 5.9G (d, 1 H, J~,2 = 7.9 Hz, NHo), 5.82 (m, 1 H, H-2c), 5.33 (dd, IH, J2,3= 3.2 Hz, H-2B), 5.07 (m, 1H, JlZ= 3.2 Hz, H-lE), 5.05 (d, 1H, J~,2=
1.7 Hz, H-lc), 4.98 (d, IH, OCHZ), 4.97 (bs, 1H, H-1B), 4.91-4.78 (m, 5H, H~lp, OCH~), 4.64 (d, 1H, J = 11.6 Hz, OCHz), 4.60-4.45 (m, 5H, OCH~, 4.36 (d, 1 H, J = 11.9 Hz, OCHi), 4.26 (pt, 1 H, JZ,3 = J3.a = 9.5 Hz, H-3D), 4.17 {dd, 1H, Jz,3 = 3.4 Hz, H-3B), 4.16 (d, 1H, J = 15.1 H~~
CHZCI), 4.11 (d, 1H, CHzCI), 4.10 (dq, 1H, Ja,s= 9.1, Js,s= 6.3 Hz, H-58), 4.06 (m, IH, H-5E), 4.00 (pt, 1H, J3,a= Jz,;=
9.4 Hz, H-3F), 3.97 (dd, IH, J5,6~= 5.3, J6a,6b= 10.8 Hz, 6aa), 3.89 (m, 1H, H-6aE), 3.88-3.68 (m, 4H, H-6bE, 6bD, 4g, 3c), 3.G7 (m, 1H, H-Sc), 3.58 (pt, 1H, J3,a = Ja,s ° 9.4 Hz, H-4D), 3.52 (dd, 1H, Ji,2= 3.3, JZ,3= 9.8 Hz, H-2E), 3.49 (s, 3H, OCH3), 3.39 (m, 1H, H-5D), 3.30 (nn, 2H, H-2v, 4c), 2.12 (s, 3H, NHAc), I.52 (s, 3H, CMez), 1.42 {s, 3H, CMe2), 1.33 (d, 3H, J,,6 = 6.2 Hz, H-6X), and 0.96 (s, 3H, J5,6 = 6.2 Hz, H-6X): '3C NMR: 0171.9 (C=O, NHAc), 167.0 (C=O, CHZCI), 166.3 (C=O, Bz), 138.8-128.0 (Ph), 101.4 (C-1D,1cx = 164 Hz), 99.9 (CMe2}, 99.3 (C-lc, Jcx = 168 Hz), 98.3 {C-lE, J~ = 168 Hz), 97.9 (C-1H, Jcx = 171 Hz), 82.1 (C-3E), 81.8 (C-2E), 80.4 (bs, G3B), 84.0 (C-4c), 78.8 (bs, C-4E*), 78.3 (C-4B*), 77.7 (C-3c*), 76.9 (C-3D), 75.9, 75.5, 75.3, 74.3 (4C, OCHz), 73.4 (C-4o), 73.2 (OCHz), 72.7 (C-2B), 72.1 (C-5E), 69.1 (C-5c), 67.7(C-SDI), 67.6 (C-5B"'), 62.7 (C-6D), 59.1 (C-2p), 57.5 (OMe), 41.4 (CHzCI), 29.5 (CMez}, 24.0 (NHAc), 19.7 (CMez), I8.8 (C-6~), and 18.2 (C-6~; FAB-MS for C8~H9zNC1021 (M, 1449.5) m/z 1472.7 [vI+Na]T.
Anal. Calcd for CBtH92NClOz,: C, 67.05; H, G.39; N, 0.97%. Found: C, 66.21; H, 6.46;
1.01%.
Compound XX (ortltoester) had (aJp +25° (c 1.0); 'H NMR: 8 8.07-7.I5 (m, 35H, Ph), 5.47 (d, 1 H, Juu,z = 7.4 Hz, NHD), 5.45 (bs, 1 H, H-2c), 5.42 (d, 1 H, JI,Z = 2.3 Hz, H-1 B), 5.24 (d, 1 H, J~,z= 3.4 Hz, H-lE), 4.94 (d, 1H, Jt,z= 8.2 Hz, H-1D), 4.91-4.82 {m, 7H, H-lc, OCHZ), 4.80 (d, 1H, J = 11 Hz, OCHz), 4.75 {d, 1H, J = 11.6 Hz, OCHz), 4.68 (dd, 1H, J~,i=
2.4, Jz,3 = 4.0 Hz, H-2H), 4.65-4.4? (m, 4H, OCHZ), 4.44-4.32 (m, 4H, H-5E, 3D, 3c, OCHz), 4.15 (nn, 1H, H-5c), 4.05 (pt, 1H, JZ,3 = J3,., = 9.5 Hz, H-3E), 4.03 (pt, 1H, J3,4 = J.~,~ = 9.4 Hz, H-4C), 3.94 (dd, 1H, Js,s, = 5.3, Js,,6b= 10.7 Hz, H-6ap), 3.83-3.75 (m, 4H, H-6aE, 6bo, CHZCI), 3.74-3.70 (m, 3H, H-4E, GE, 3H), 3.65 (dd, IH, J1,2= 3.4, JZ,~= 9.4 Hz, H-2E), 3.48 (pt, 2H, H-4B, 4D}, 3.46 (s, 3H, OCH3), 3.38 (m, 1H, H-5D), 3.22 (dq, 1H, J4,~ = 9.5, JS 6 = G.2 Hz, H-SB), 2.88 (m, 1H, H-2D), I.90 (s, 3H, NHAc), 1.42 (s, 3H, CMez), 1.36 (s, 6H, CMe2, H-6~}, and 1.30 (s, 3H, JS,s= 6.3 Hz, H-68); ~3C NMR: X171.8 (C=O, NHAc), 166.4 (C=0, Bz), 139.1-122.5 (Ph), 101.0 (C-ID, JcH =165 Hz), 99.7 (CMea), 98.3 (C-lc, JcH =172 Hz), 97.8 (bs, C-1 E, Jcx =
170 Hz), 97.5 (C-18, JcH = 176 Hz), 82.2 {C-3E), 80.7 (G-2~), 79.3 (bs, C-4H), 78.8 (C-3B), 78.1 (bs, C-4E), 77.3 (C-2H), 76.2 (bs, C-3~), 75.8, 75.6, 74.9, 74.6, 73.9 (6C, C-4~, OCHZ), 73.5 {2C, C-4D,2c), 71.4 (OCHz), 71.0 (C-3o), 70.7 (2C, C-SF, SH), G9.0 (C-5c), 68.8 (C-6E), 67.2 (C-Sp}, 62.5 (C-6p), 60.0 (C-2D), 57.6 (OMe), 46.9 (CHzCI), 29.5 (CMeZ), 23.9 (NHAc), 19.7 (CMez), 19.0 (C-Gg), and 18.4 (C-6~); FAB-MS for CelH9zNC10zt (M, 1449.5) rrt~a 1472.7 [M+Na]+.

LMPPI 1-exp~brevet-pcntnOMe Anal. Calcd for Cg~II92NCIO~~~I-.T2O: C, 66.23; H, 6.34; N, 0.96%. Found: C, 66,11; H, 6.62; N. 0.85%.
1~'olr tentative dcrblocage orthoester seul ou biers mcrlange alorthoesler issu du couplage data des proportions eonnues au depart.(6~4 il-12 par ex) Methyl (2-O-Acetyl-3,4-di-0-benryl-oc-L-rhamnopyranosyl)-(1--~3}-j(2,3,4,6-tetra-O-benzyl-a-n-glucopyranosyl-(1 >4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1 >3)-2-acetamido-2-deoxy-3,4-0-isopropylldene-[3-D-glucopytanoside (XX). The trisaccharide acceptor XX (500 mg, 0.47 mm~ol) was dissolved in DCM (5 mL) and the solution was cooled to -40°C. TMSOTf (21 ~L) and donor XX (328 mg, 0.62 mmol) were added and the mixture was left under stirring while the bath was slowly conning back to rt. Afier S
h, more XX (50 mg, 94 ~Imol) was added and the mixture was stirred at rt for 1 h more at rt.
Et3N was added and the mixture was concentrated. Column chromatography of the residue (solvent B, 4:1 --l:l) gave the fully protected XX (484 rrtg, 72%) slightly contaminated with the corresponding trimethylsityl side-product XX. The XX:XX ratio was estimated to be XX:XX from the 'H
NMR spectrum R. II~IV c? ai FAB-MS for CglHg3NOz1 (M, 1415) m~'z XXXX [M+Na]+. Yoir si presence silyl Anal. Calcd for CgIH93NOzIH20: C, 68.69; H, 6.57; N, 0.98%. Found: C, 67.64;
H, G.G7; N, 0.88%.
Methyl (3,4-Di-O-benzyl-a-L-rhamnopyranosyl)-(1--~3)-j(2,3,4,6-tetra-0-benzyl-a-D-gtucopyranosyl-(1--~4)j-(2-O~benzoyt-a-L-rhamnopyranosyl)-(1--~3)-2-acetamido-deoxy-3,4-O-isopropylidene-~-D-glucopyrsnoside (XX). (a) Thiourea (362 mg, 4.76 tnmol) was added to an unseparable mixture of XX and XX (689 mg, 0.48 mmol) in MeOHlpyridine (1/1, 16 mL), and the mixture was heated overnight at 65°C. Volatiles were evaporated, and the solid residue thus obtained was taken up in the minimum of MeOH. DCM was added, and the suspension was left standing at 0°C for 1 h The precipitate was filtrated on a pad of Celite, and the filtrate was concentrated. Column chromatography of the residue (solvent B, 9:1 -» 1:1) gave the trisaccharide acceptor XX (107 mg, 22%) as the first elating product.
Further elution gave the tetrasaccharide acceptor (419 mg, 63%) together with a mixture of XX and XX (G6 mg).
(b) The monoacetytated XX (52.3 mg, 37 letrwl) was dissolved in a mixture of EtOH (10 mL) and DCM (100 IIL). A freshly prepared 0.4M ethanolic solution of guanidine (92 pL, 37 pmolj was added anrl the mixture was stirred at tt overnight. VoLatiles were evaporated, and the residue taken up in DCM was washed with water. The organic phase was dried and concentrated.
Column chromatography of the crude product gave XX (42 mg, 83%). Compound XX
had LMPP11~exp-taevet~p~rttaOMc RMN ~ faire FAH-MS for C7gHg~NOZO (M, 1373) m~z 1396.5 [M+Na]+.
Anal. Calcd for C~9H91NOao~ 0.5 H20: C, 68.56; H, 6.65; N, 1.01 %. Found: C, 58.53; H, 6.71; N, 1.01%.
Methyl (2-0-Acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1~3)-[(2,3,4,6-tetra-benzyl-a-n-glucopyranosyi-(1~4))-(2-0-benzoyl-3-O-chloroacetyl-a.L-rhamnopyranosyl)-(1~3)-2-acetamido-2-deoay-3,4-O-isopropylidene-p-D-glucopyranoside (XX). 4A Molecular sieves and TMSOTf (16 ~L) were added to a solution of the tetrasaccharide acceptor XX (406 mg, 0.29 mmol) in EtzO (10 mL), and the mixture was stirred at -60°C for 30 min. The donor XX (234 mg, 0.44 moral) in DCM (7 mL) was added, and the mixture was stizred for 1 h while the bath temperature was reaching rt. After a further 1 h at this temperature, more XX (50 mg, 94 Etrrwl) was added, and the mixture was stirred for 1 h before Et3N was added. Filtration through a pad of Celite and evaporation of the volatiles gave a residue which was column chromatographed twice (solvent B, 4:1; then solvent D, 17:3) to give XX (262 mg, 52%); (aJp +25° (c 1.0); LH NMR: &
8.07-7.13 (m, 4SH, Ph), 6.03 (bs, 1H, NHo), 5.59 (bs, 1H, H-2,~, 5.35 (bs, iH, H-2c), 5.16 (bs, 1H, H-lE), 5.13 (bs, 1H, H-lA), 5.06 (bs, 1H, H-1B), 5.02-4.97 (m, 4H, H-1~, lc, OCH~), 4.91~4.50 (m, 12H, OCHz), 4.44-4.32 (m, 4H, H-Ze, 3D, OCHi), 4.20-3.96 (m, 7H, H-5F, 5A, 3c, 3E, 6aD, 5c, 3~, 3.87-3.68 (m, 6H, H-4g, dar, Gba, 6bD, 40, 3s), 3.64-3.47 (m, 7H, H-5H, 4D, 2E, 4A, OCH3), 3.42 (m, 1 H, H-Sp), 3.34 (pt, 1 H, J3,4 = Ja,s = 9.3 Hz, H-4a), 3,17 (m, 1 H, H-ZD), 2.13 (s, 3H, Iv'HAc), 1.49 (s, 3H, CMez), 1.43 (s, 6H, CMe2, H-6c), 1.33 (d, 3H, Js,b = 6.I Hz, H-6,~, and 1.01 (s, 3H, Js,s= 5.8 Hz, H-6H); 13C NMR: cS i71.9 (C=O, NHAc), 170.3 (C=0, Ac), 166.3 (C=0, Bz), 139.2-127.6 (Ph), 101.5 (bs, C-la, JcH = 171 Hz), 101.2 (C-1D, JcH = 163 Hz), 99.8 (CMe2), 99.7 (C-I A, JcH = 171 Hz), 97.9 (2C, C-lE, lC, JcH =172, 1G9 Hz), 82.4 (C-3E), 82.1 (C-2E), 80.5 (C-4A), 80.2 (C-3c), 80.1 (C-4$), 79,4 (C-3$*), 78.1 (2C, C-4~*, 3,~, 78.0 (C-4c), 76.6 (C-3D), 75.9. 75.8, 75.4 (3C, OCHZ), 74.8 (2C, C-2B, OCH?), 73.5 (C-4D), 73.4 (OCFii), 73.2 (C-2c), 72.1 (OCHZ), 71.8 (C-5A), 71.2 (OCHZ), 69.4 (C-2,~, G9.2 (C-5B), 68.9 (C-6~, 68.7 (C-5c), 67.8 (C-SE), 67.5 (C-SD), 62.7 (C-6p), 59.G (C-2p), 57.G (OIvte), 29.5 (CMe2), 24.0 (NHAc), 21.4 (O Ac), 19.7 (CMe2), 19.1 (C-6~), 18.8 (C-6c), and 18.2 (C-6B);
FAB-MS for CLOIHtISNOzs (M. 1741.7) m/z 1765.9 [M+Na]''.
Anal. Calcd for C,alHl isNO~s: C, 69.60; H, 6.65; N, 0.80%. Found: C, 69.56;
H, 6.75; N, 0.73%.
I Methyl arL-rhamnopyranosyi-(1-->3)-[(2,3,4,d-tetra-0-benzyl-a-n-glucopyranosyl-(1~4))-a-L-rhl~mnopyranosyl-(1--~3)-2-scetamido-2-dcoay-[3-D-gtucopyranoside (XX).
50% aq TFA (1 mL) was added at 0°C to a solution of the fully- protected pentasaccharide XX (155 mg, j 89 ~mol) dissolved in DCM (4 mL,). After 1 h at this temperature, volatiles were evaporated.

LMPPII~GXp-bftwtt-pClIrHOMC CA 02434685 2003-07-04 The residue was taken up in O.SM methanolic sodium methoxide (8 mL) and the miscture was heated overnight at 55°C. Neutralisation with Dowex X8 (Hi'), evaporation of the volatiles, and colunnn chromatography of the residue gave XX (171 mg, 98%). Compound XX (111 mg, 81 ~mol) was dissolved in a mixture of ethanol (13 mL) and ethyl acetate (2.G mL) containing 1N
aq HCl (130 ~L). Palladium on charcoal (130 mg) was added and the suspension was stirred under a hydxogen atmosphere for 2 h. Filtration of the catalyst and reverse phase chromatography gave the target pentasaccharide (60 mg, 88%) as a slightly yellow foam. RP-HPLC purification (solvent XX, XXX) followed by freeze-drying gave pure XX (36 mg).
RMN
FIRMS (MALDI) Calcd for C33HS~NOi3 + Na: 858.3219. Found: 858.3089.

LMPPI 1-Sheme-brevet-pecCsOMc OTCA OMe OM8 OAC
OMe BnOe ~ 0 Bnbe O Ac0'~~"B~
BnOMe 0 Bn0 pAc Bn0 o Ac0 NPhtCla 8n0 pH ~ Bn0 a o ~~ -....
Bn OR R
At H
R3 ~ Ra Re Ac Ac Ac 08n Bn0 p Me O
Bzo pBz OBn Ben~~~ OAIf Bno o Me p R O
HO~ pH

LMPP11~Shemc-btevet~pen~eOMe OH ~0-~~0' ~OMe OAiI
HO ~0 0 H~_5~0Me HO~Ac Me 0 NHAc HO--_~
HO
Me O + 0 Ogn O OH ~ g BnO~ OTCA ~ +
H~ Bn0 0 Me 0 O O8n CIAcO Ogz E~O~ '~ g B O~OTO~t HO pH OTCA ~Oan Bn0 Me O
Bn0 pAc L PPl2 Preparation of chemically defined gtycopeptides as potential synthetic conaugate vaccines against Shigella flexneri secotype 2a disease.
This paper discloses the synthesis of three fully synthetic glycopeptides incorporating a tri-, tetra-, and pentasaccharide haptens representative of fragments of the 0-Ag of S flexi~erl serotype 2a covalently 1'ed to the PADRE-sequence, which acts as a universal T
cell epitope is reported. The carbohydrate haptens were selected based on a preliminary study of the recognition of synthetic oligosaccharides with homologous protective antibodies. They were synthesized following a common block strategy, in a form allowing their coupling by chemical ligation onto a maleimido-activated PADRE. Evaluation of the immunogenicity of the conjugates in mice is ongoing.

LMPP12-thco-ixevdgp Preparation of chemically defined glycopeptides as potential Synthetic conjugate vaccines against Shigella Jlexneri serotype 2a discascl Abstract INTRODUCTION
Since the discovery of Shigella dysereteriae type 1 (Shiga's bacillus) more than a century ago, 'shigellosis or bacillary dysentery has long been known as a serious infectious disease, occurring in humans only. 3In a recent survey of the litezature published between 19GG and 1977. 4the number of episodes of shigellosis occurring annually throughout the world was estimated to be 164.7 million, of which 163.2 nnillion were in developing countries. Up to 1.1 million annual deaths were associated to shigellosis during the same period. Of the four species of Shigellae, Shigella Jlexneri is the major responsible of the endemic form of the disease, with serotype 2a being the most prevalent. The critical importance of the development of a vaccine against Shigellae infections was first outlined in 1987. SDue to increasing resistance of all groups of Shigellae to antibiotics, hit remained a high priority as stated by the World Health Organization ten years later. 'In the meantime, several experimental vaccines have gone through field evaluation, 8'1°but there are yet no licensed vaccines for shigellosis.
Shigella's lipopolysaecharide (LPS) is a major surface antigen of the bacterium. The corresponding 0-specific polysaccharide domain (0-SP) is both an essential virulence factor LMPPi 2~tbco~brevetgp and the target of the infected host's protective immune response. it.iZlndeed, using the pulmonary marine model for shigellosis, it was recently demonstrated that secretory IgA
specific for the O-SP of S. flexneri serotype Sa were protective against an homogolous infection when present locally prior to the challenge. i313ased on the former hypothesis that serum IgG anti-LPS antibodies may confer specific protection against shigellosis, '4several polysaccharide-proteine conjugates, targeting either Shigella sonnei, S.
dysenteriae 1 or S.
flexneri serotype 2a, were evaluated in humans. lo,isln the case of S sontrei, recent field trials ahowed Bobbins and co-workers to demonstrate the efficacy of a vaccine made of the corresponding detoxified LPS covalently linked to recombinant exoprotein A.
i6Conversion of polysaccharide T-independent antigens to T-depend ones through their covalent attachment to a carrier protein had a tremendous impact in the field of ba.eterial wecines.
Several such neoglycoconjugate vaccines are currently in use against Haemophih~s influeniae, '~Neisseria meningitidis, igand Streptococcus pneumoniae. i9These polysaccharide-protein conjugate vaccines are highly complex structures, whose immunogenicity depends of several parameters amongst which the length and nature of the saceharide component as well as its loading on the protein. It is reasonably admitted that control of these parameters is somewhat difficult when dealing with polysaccharides purified from bacterial cell cultures. As recent progress in carbohydrate synthesis allows access to complex saccharides, it was suggested that the use of welt-defined synthetic oligosaccharides may show a better control, and consequently the optimisation, of these parameters. Indeed, available data on S, dysenteriae type 1 indicate that neoglyeacanjugates incorporating di-, tri- or tetramers of the O-SP repeating unit were mare immunogenic than a detoxified LPS-human serum albumin conjugate of reference.
z°Besides, recent reports demonstrate that short oligosa;ccharides comprising one repeating unit or less may be immunogenic in animal models. zi.2zAnother critical parameter in the design of neoglycoconjugate vaccines is the carrier protein. As potential applications for these vaccines are expanding, the need for new carrier proteins licensed for human uee is growing. 23That synthetic peptides representing immunodominant T-cell epitopes could act as carriers in polysaccharide and oligosaccharide conjugates has been suggested, ~~and latter on demonstrated. Zs,zeBesides, the use of T-cell epitopes offer several advantages, including potential access to well-defined conjugates with no risk of epitopic suppression, as the latter phenomenon appeared as a major drawback of protein carriers. 2~-3oPolypeptides containing multiple T-cell epitopes have been generated in order to address the extensive polymorphism of HLA molecules. ~i.3ZIn other strategies, universal T-helper epitopes compatible with human use have been characterized, for example from tetanus toxoid, 33or engineered such as the pan LMPP12~theo~brevetgp HLA DR-binding epitope (PADRE). 34Recently, covalent attachment of the human nnilk oligosaceharide, facto-N fucopentose II, to PADRE resulted in a linear glycopeptide of comparable immunogenicity to that of a glycoconjugate employing HSA as the carrier. 3s Based on these converging data, we focused on the development of well-defined neoglycopeptides as an alternative to polysaccharide~proteine conjugate vaccines targeting infections caused by S. flexneri 2a. The target neoglycopeptides were constructed by covalently linking a short peptide, serving as a T-helper epitope, to appropriate carbohydrate haptens, serving as B epitopes mimicking the S. "~texneri 2a 0-Ag. Our approach is based on rational bases involving a preliminary study of the interaction between the bacterial O-SP and homologous protective monoclonal antibodies, which helped to define the carbohydrate haptens.
RESULTS AND DISCUSSIO\T
A S E C D
2)-a-L-Rhap-( 1 ~2}-a-L-Rhap-( 1 ~3 )-(a-D-Glcp-( 1->4)]-a-L-Rh,a~p-( 1-33)-~i-D-GIcNficp( 1 ~
I
The O-SP of S flexneri 2a is a heteropolysaccharide defined by the pentasaccharide repeating unit I. 3w~It features a linear tetrasaccharide backbone. which is common to all S. flexneri O-antigens and comprises a N-acetyl glucosaminc (D) and three rhamnose residues (A, B, C}
The specificity of the serotype is associated to the a-D-glucopyranose residue linked to position 4 of rharnnose C. Besides the known methy~1 glycoside of the EC
disaccharide, 38°39a set of di- to pentasaccharides corresponding to frame-shifted fragments of the repeating unit I, °o-~3an octasaccharide4~ and more recently a decasaccharide45 representative of fragments of S.
Jlexneri 2a 0-SP have been synthesized in this laboratory. Based on the use of these compounds as molecular probes for mapping at the molecular level the binding characteristics of a set of protective nwnoelonal antibodies against S. flexneri 2a infection, ~6fragments ECD, B(E)CD and AB(E)CD were selected as haptens that will act as B-epitopes in the conjugates.
Three fully synthetic linear neoglycopeptides 1, 2 and 3, corresponding to haptens ECD, B(E)CD, and AB(E)CD, respectively, were synthesized according to a strategy built up on the concept of chemoseiective ligation which allows the selective one-point attachment of the free B and T epitopes in aqueous media. All conjugates involve the peptide PADRE as the universal T-cell epitope.

LMPPLZ-theo-brcretgP
Scheme 1:

Retrosynthetic analysis o~"the saccharidic haptens (Scheme 1): Analysis of S,'lexr~eri 2a 0-SP suggests that, due to the 1,2-cis glyeosidic linkage involved, construction of the EC
disaccharide is probably the most demanding. Besides, prior work in this laboratory has shown that the C-D glycosidic linkage was an appropriate disconnection site when dealing with the blockwise synthesis of oligosaccharide fragments of S flexrteri 0-2a SP. 4o,a2,a~These observations supported the design of a synthetic strategy common to all three targets.
Basically, it relies on (i) the condensation of an EC (4), 4~H(E)C (5) 42or A.B(E)C (6) donor to a D acceptor (7), functionalized at the anomeric position with an azidoethyl spacer; (ii) elongation of the spacer with introduction of a masked thiol group to allow its coupling onto a PADRE peptide derivatized by a maleimido group on a C-terminal Lysine (8). The carbohydrate synthesis relies on the trichloroacetimidate methodologya~ and the use of known building blocks whenever possible.
Scheme 2:
Synthesis of the aminoethyl LcCD building block IS (Scheme 2): The now easily accessible disaccharide donor 4, '~zwith a benzoyI participating gxoup at position 20, was used as the precursor to the EC moiety in the construction of 1. It was prepared, as described, ~~in 5 steps and 45% overall yield from 2,3,4,6-tetra-O-henry)-~i-D-glucopyranosyl trichloroacetinudate (9) dg'a9and ally) 2,3-O-isopropylidene-a-D-rhamnopyranoside (IO) s°through Lhe key intermediate diet 1I (69% from IO). Introduction of the azidoethyl spacer on a glucosaminyl intermediate was performed according to a known procedures) by coupling of azidoethanolsz onto the oxazoline53 12 to give the triaeetate I3. sl,s'4We have shown on several occasions in the S Jlexneri series, that regioselective protection of the 4- and 6-OH
groups of precursors to residue D with an isopropylidene acetal was appropriate, especially when such precursors are involved in a blockwise synthesis based on the disconnection at the C-D
linkage. ~~,44Thus, Zemplbn deacetylation of I3 gave the trio) I4 which was converted to the key acceptor 7 (81% from 13) upon reaction with 2,2-dimethoxypropane under acid catalysis.
V~hen the latter was glycosylated with the donor 4 in the presence of BF3.OEtz in dichloromethane, the fully protected trisaccharide IS was isolated in S8% yield together with the diet 16 (30%), resulting from partial loss of the isopmpylidene acetal. When 4 and 7 were glycosylated in the presence of a catalytic amount of TMSOTf, no side-reaction was observed, and the condensation product 15 was obtained in 86% yield. Quantitative conversion of 15 into I6 LMPP E 2-thco-brrvetgp was more conveniently achieved by acidic hydrolysis of the former with 95% aq TFA
Zemplen debenzoylation of 16 gave the tetraol 17 (94%) which was subsequently transformed into the aminoethyl-armed trisaccharide 18 (69%) by hydrogena.tinn in the presence of palladium-on-charcoal (Pd/C) and 1N aq HCl to convert the formed amine to its hydrochloride salt. Indeed, others have pointed out that hydrogenoiysis using Pd/C in the presence of a free amine was sluggish and low-yielding. ss-s~In order to prevent any side-reaction at a latter stage of the synthesis, isolation of pure 18 was performed by reversed-phase HPLC (ItP-HPLC).
Scheme 3:
Synthesis of tlae aminoethyl B(Lr')CD building block 25 (Scheme 3): The known rhamnopyranosyl tricholoracetimidate 20, SRacetylated at its 2-, 3-, and 4-OH
groups thus acting as a chain terminator, was chosen as the precursor to residue C.
Benzoylation of diol 11 to give 19 was performed by regioselective opening of the cyclic orthoester intermediate as described. ~lGlyeosylati~on of the latter by donor 20, with activation by a catalytic amount of TMSOTf proceeded smoothly in EtZO to yield the fully protected trisaccharide 21 (89%), which was de-O-allylated into the hemiacetal 22 (80%) following a two step process involving (i) iridium(I)-catalysed isomerisation of the allyl glycoside to the prop-1-enyl glyCOSldes9 and (ii) subsequent hydrolysis. so,s°The selected trichloroacetimidate leaving group was introduced by treatment of 22 with trichloroacetonitrile in the presence of a catalytic amount of DBU, which resulted in the formation of S (99%).
Condensation of the latter with acceptor 7 was performed in CHZC1Z in the presence of a catalytic amount of trifluoromethanesulfonic acid (TfbH) to give the required tetrasaccharide 23 (76%). Acidic hydrolysis of the latter using 95% aq TFA gave the intermediate diol 24 in 95%
yield.
Deacylation of the resulting diol under Zemplen conditions followed by debenzylation and concomitant conversion of the azide into the corresponding amine to give the key aminoethyl-armed tetrasaccharide 25 (77%) was performed by treatment of 24 with hydrogen in the presence of Pd/C under acidic conditions. Again, compound 25 vt~as purified by Rl'-HLPG
before elongation ofthe spacer or conjugation.
Scheme 4:
Synthesis of !he aminoethyl AB(E)CD building block 37 (Scheme 4); The synthesis of 37 is based on the condensation of acceptor 7 and donor 6, which resulted from the selective deallylation and anomeric activation of the key intermediate tetrasaccharide 33. The latter ws LMPP12~theo-Mevetgp obtained according to two routes following either a block strategy (route 1) based on the condensation of an AB disaccharide donor (30) and the EC disaccharide acceptor

16, or a linear strategy (route 2) involving the stepwise elongation of 16. The construction of the donor 30 was based on the use of the known allyl rhamnopyranoside 26, 6~having permanent protecting groups at position 3 and 4, as the precursor to residue B, and the trichloroacetimidate chain terminator Z7, 62acting as a precursor to residue A. Condensation of the two entities in the presence of a catalytic amount of TMSOTf resulted in the fully protected 28 (96%), which was selectively de-O-allylated into 29 (84%) according to the protocol described above for the preparation of 22. Subsequent treatment of 29 with trichloroacetonitrile and a catalytic amount of DBU gave the required 30 (96%).
Glyeosylation of 16 with the latter under TMSOTf promotion afforded the fully protected I tetrasaccharide 34 in 55% yield. No (3-anomer was detected. The stcreochemical outcome of this glycosylation step involving a rhamnosyl donor glycosylated at C-2, thus lacking any participating group at this position is not without precedent. Related examples involving rhamnopyranosyl donors may be found in the synthesis of oligosaccharides representative of the capsular polysaccharide of the ~i-hemolytic Streptococcus Group A, ~3or of the O-Ag of Serratia marcescens 0186" as well as in our own wroxk on S ~lexneri seroty~pe 2a. A'Route 1 was considered initially in order to prevent extensive consumption of the EC
disaccharide il.
C3iven the relatively low yield of coupling of 16 and 30, route 2 was considered as well. Of all precursors to 34, only that to residue B, namely the donor and potential acceptor 31, differed from those used in routs 1. Conventional glycosylation of disaccharide 16 and 31 and subsequent selective deacetylation using methanolic IdBF4, gave the acceptor 32 in 70% yield from 16. 45The trisaccharide 32 was glycosylated vvith trichloroacetimidate 27 in as analogous fashion to its glycosylation with 30, yielding 34 (92%). Deallylation of this key intermediate, as described above fox the preparation of 22, gave the corresponding hemiacetal 35 (94%) which was converted into the required trichloroacetixnidate 6 ($8%) upon treatment with triehloroacetonitrile and DBU. Condensation of donor 6 with the glucosaminyl acceptor 7 was performed under promotion by TfOFi or TMSOTf, which resulted in the fully protected pentasaccharide 35 in 62% and 80% yield, respectively. Following the process described for the preparation of 25, compound 35 was submitted to acetolysis (97%) and subsequent ZemplEn deacylation to give the partially debloeked 36 (87%), which was next converted to the aminoethyl-spacer pentasaccharide 37 upon treatment with hydrogen in the presence of Pd/C. Final RP-HPLC purification resulted in the isolation of 37 in 53% yield.

LMPPllwlteo-httroetgp Scheme 5:

Synthesis of the target neoglycopeptides 1-3 (Scheme S): In all casES, chemoselective ligation of the B and T epitopes was achieved through coupling of the carbohydrate haptens pre-functionalized wish a thiol function and a maleimido group properly introduced at the C
terminus of the T helper peptide. Such a strategy was chosen i.n order to exploit the high reactivity and specificity of thiol groups towards the maleimide functionality, dswhich shows specific and high-yielding modification of the former in the presence of other nucleophiles.
slit was used previously under various forms in the coupling of carbohydrate haptens to either proteins6~~6g or peptides. 2s,saTo our knowledge, in all the reported cases the maleimide functionality was introduced onto the carbohydrate hapten. On the contrary, our strategy relies on the introduction of this activating group on the T helper peptide. The immunogenicity of various maleimide-derived coupling reagents was evaluated in a model system.
69Based on the reported data, 694-(N maleimido)-n-butanoyl was selected as the linker, and incorporated by coval~nt linkage to the side chain amino group of a Lysine residue added at the C-terminus of the PADRE sequence (PADRE-Lys). It is worth mentioning that the strategy described herein somewhat differs from that described by others when demonstrating the usefulness of PADRE
in the construction of immunogenic neoglycopeptides. 3s The Lysine-modified PADRE (8) was assembled using standard Fmoc chemistry for solid-phase peptide synthesis. ~°Standard side chain protecting groups were used, except. fo.r that of the C-ternzinal Lysine side chain which was protected by the 1-(4,4-dimethyl-?.,b-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) group. "Indeed, this orthogonal protecting group strategy allows specific introduction of the maleimide group on the C~terminat Lysine, upon selective cleavage of the ivDde by hydrazine, The thiol functionality was intzoduced onto the carbohydrate haptens as a masked fhiol function (acetylthioester), which is easily generated in situ during the conjugation process Thus, reaction of IS, 25, and 27 with S-acetylthioglycolie acid pentafluorophenyl ester (SAMA-offp) resulted in the site-selective elongation of their aminoethyl spacer via a thioacetyl acetamido linker.
Derivati7.ation could be monitored by RP-HPLC with detection at 215 nm. Under these conditions, the required thioacetyl-armed intermediates, 38, 39 and 40 were isolated in 53%, 74%, and 7S% yield, respectively. Their structure was confirmed based on MS and NMR analysis.
Conjugation of the carbohydrate haptens to the maleimido activated PADRE-Lys (8) was run in phosphate buffer at pH G.0 in presence of hydroxylamine'Zand monitored by RP-HPLC.
Lastly, RP-LMPP12-tho°-brevet~p HPLC purification gave the target neoglyeopeptides 1, 2, and 3 as single products, which identity was assessed based on MS analysis, in yields of 58%, 48% and 46%, respectively.
CONCLUSION
The synthesis of three fully synthetic glycopeptides incorporating a trl-, tetra-, and pentasaccharide haptens representative of fragments of the 0-Ag of S flexneri serotype 2a covalently linked to the PADRE-sequence, which acts as a universal T cell epitope is reported. The carbohydrate haptens were selected based an a preliminary study of the recognition of synthetic oligosaccharides with homologous protective antibodies. They were synthesized following a common block strategy, in a form allowing their coupling by chemical ligation onto a maleimido-activated PADRE. Evaluation of the immunogenicity of the conjugates in mice is ongoing.
ACKNO WLEDGEMENTS
The authors are grateful to J. Ughetto-Monfrin (Unite de Chimie Organique, Institut Pasteur) for recording all the NMR spectra. The authors thank the Bourses Mrs Frank Howard Foundatian for the postdoctora! fellowship awarded to K. W., and the Institut Pasteur for its financial support (grant no. PTR 99 j.
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LMPP 12-then-brevetgp

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LMPP 12exp-brevet-gp General Methods. General experimental methods not referred to in this section were as described previously.(REF) TLC on precoated slides of Silica Gel GO Fzsa (Merck) was performed with solvent mixtures of appropriately adjusted polarity consisting of A, dichloromethane-methanol; B, cyclohexanc-ethyl acetate, G, cyclohexane-diethyl ether, D, toluene-acetone. Detection was effected when applicable, with UV light, and/or by charring with orcinol (35 ttLM) in 4N aq HzSOa. NMR Spectra were measured in CDC13 unless stated otherwise. In the NMR spectra, of the two magnetically non-equivalent geminal protons at C-6, the one resonating at lower field is denoted H-da and the one at higher field is dcnr~ted H-6b.
Interchangeable assignments in the 1'C NMR spectra are marked with an asterisk in listing of signal assignments. Sugar residues in oligosaccharides arc serially lettered according to the lettering of the repeating unit of the O-SP and identified by a subscript in listing of signal assignments. Low-resolution mass spectra were obtained by either chemical ionisation (CIMS}
using NHj as the ionising gas, by electrospray mass spectrometry ($SMS), or by fast atom bombardment mass spectrometry (FABMS), High-resolution mass spectra were obtained by MALDI-MS.
Solid phase peptide synthesis was performed using standard Fmoc chemistry protocols on a Pioneer peptide synthesiser (AppliedBiosystem). Fmoc-Lys{iv-Dde)-OH, Fmoc-Cha-OH, Fmoc-D-Ala-OH, Fmoe-eAhx-OH and Boc-D-Ala-OH were purchased from Noval3iochem (VWR). All others reagents and amino acids were purchased from Applied Biosystem-2-A~idoethyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-~-D-glucopyranoside (7).
Camphorsulfonic acid (200 mg, 0.9 mmol) was added to a solution of triol 14 (1.31 g, 4.52 mmol) in a mixture of DMF (4 mL) and 2,2-dimethoxypropane (4 mL). After 3 h at rt, low boiling point solo~ents were evaporated under reduced pressure and more 2,2-dimethoxypropane (2 mL, 15.8 mmol) was added. The mixture was stirred for 2h at rt, Et3N
was added; and the mixture was concentrated. The crude product was purified by column chromatography (solvent A, 19:1) to give 7 as a white solid (1.21 g, 81%), [a]p -89.8; 'H
NMR: b 6.15 (d, 1H, J = 5.9 Hz, NH), 4.70 (d, 1H, Jt,2 = 8.3 Hz, H-1), 4.05 (m, 1H, OCHZ), 3.97-3.89 (m, 2H, H-6a, 3), 3.79 (pt, 1H, Js,6b = 3s~,sb = 10.5 Hz, H-Gb), 3.70 (m, 1H, OCHz), 3.62-3.46 (m, 3H, H-2, 4, OCHZ), 3.35-3.ZG (m, 2H, H-5, CH2N3), 2.05 (s, 3H, Ac), I.52 (s, 3H, C(CH3)z), 1.44 (s, 3H, C(CH3~); "C NMR: 8 100.9 (C-1), 74.3 {C-4), 81.8 (C-3), 68.6 (OCHZ), 67,3 (C-5), 62.0 (C-G}, 58.7 {C-2), 50.7 (CHzN3), 29.0 (C(CH3)Z), 23.G
(CH3C0), 19.1 (C(CH3)x). CIMS for C,3H~I~406 (330) mla 331 [M+H]+. Anal. Calcd. for Cs~H~aN40'~~O.SHZO: C, 46.OI; H, 6.83; N, 16.51. Found C, 46.37; H, G.69; N, 16.4.6.
2-Azidoethyl (2,3,4,6-Tetra-O-bearyl-a-D-glucopyranosy>)-(1--;~4)-(2,3-di-0-be~nzoyl~a-L-rhamnopyranosyl)-(I >3)-2-acetamido-Z-deo~,y-4,6-0-isopropylidene-(3-D-LvIPPl2cxp-brevet-gp glucapyranoslde (15). (a) The disaccharide donor 4 (1.425 g, 1.37 mmol) and the acceptor 7 (377 mg, 1.14 mmol} with 4A-MS (2 g) were pla;eed under argon and CHzCl2 (15 mL) was addod. The mixture was stirred for 1 h at rt, then cooled to -40°C. A
solution of BF3.OEtz (0.5 mL, 4.11 mmol) in CHZCh (5 mL;) was added dropwzse. The mix-hue was stirred at -40°C to -15°C over 3 h. Triethylaminc (2.5 mL) was added and the mixture stirred for ZO min. The mixture was filtered through a pad of Celite, and the filtrate was concentrated. The mixture was purified by column chromatogaphy (solvent A. 2:3) to give 15 (803 mg, 58%) as a colourless foam. Further elution (solvent B, 9:1) gave 16 (395 mg, 30%) as a colourless foam.
(b) 4 A Molecular sieves (560 mg) were added to a solution of donor 4 (565 mg, 0.54 mmol) arid acceptor 7 (150 mg, 0.45 mmol) in DCM (3 mL) and the suspension was stirred for 15 min-40°C. Triflic acid (1G pL) was added and the mixture was stirred for 3h at rt once the cooling bath had reached rt. Et3N was added and after 15 min, the mixture v~~as filtered through a pad of Celite. Volatiles were evaporated and the residue was column chromatographed (solvent B, 9:1) to give 15 (475 mg, 87%). [oc]n +87.7 (c 0.32);'H NM1Z: 8 6.99-8.07 (m, 30H, Ph), 6.21 (d, 1H, NH), 5.58 (dd, 1H, H-3~), 5.44 (m, 1H, H-2c), 5.13 (d, 1H, Jl,z = 8.3 Hz, H-lp), 5.02 (d, 1H, J,,z = 3.4 Hz, H-lE), 4.97 (d, 1H, Jl,z = 1.5 Hz, H-lc), 4.64-4.90 (m, SH, CHzPh), 4.45 (t, 1H, H-3n), 4.27 (m, 3H, H-Sc. CHzPh), 3.79-4.05 (m, 7H, H-3E, 4c, So, Gap, 6bD, CHzO, CH2Ph), 3.60-3.76 (rrt, 4H, H-4D, 4E, 5E, CHzO), 3.37-3.51 (m, 3H, H-2s, 5n, CHzN3), 3.16-3.34 (tn, 3H, H-2n, GaE, CHzN3), 3.04 (d, 1H, H-6bE), 2.01 (s, 3H, CH3C=0), 1.43 (s, 6H, (CH3)zC), 1.36 (d, 3H, H-6c); 13C NMR: cS 171.7, 165.6, 163.4 (C=O), 127.3-138.6 (Ph), 99.6 (C-1D), 99.1 (C-lE), 97.7 (C-lc), 91.9 ((CH3)zG~, 81.4 (C-3E), 80.3 (C-2E), 79.4 (C-4~), 77.1 (C-4p), 76.0 (C-3d), 75.3, 74.6, 73.9, 73.2 (4C, CH2Ph), 73.1 (C-4E), 71.2 (2C, C-2o, 3c), 71.1 (C-5E), 68.6 (CH20), 67.5 (C-Sc), 67.4 (C-6a), 67.1 (C-5D), 62.1 (C-6D), 59.0 (C-2D), 50.5 (CH2N3), 28.9 ((CH3)zC), 23.4 (CH3C0), 19.2 ((CH3)zC), 18.1 (C-G~). FAB-MS for C6~H~4NdOu (1206) m/z 1229 [M+Na]+.
Anal. Calcd. for C6~H7~N401~: C, 60.41; H, 5.66; N, 4.82. Found: C, 60.36; H.
5.69; N, 4.78.
Z-Azidoethyl (2,3,4,G-Tetra-O-benzyl-a-D-glucopyranosyl)-(1~4)-(Z,3-di-O-benzoyl-a-L-rbamnopyranosyl)-(1~3)-Z-acetamido-Z-deo~y-p-D-gtueopyranoside (16). Compound (95 mg, 79 umol) was dissolved in 80% aq AcOH (2.5 mI,), and the mixture was heated at GO°C for 1 h. After cooling to rt and repeated co-evaporation with toluene, the crude residue was column chromatographed (solvent B, 1:4 -~ 0:1) to give 16 (80 mg, 87%) as a white foam. [a]D +91.5 (c 0.18); tH NMR: & 6.99-8.02 (m, 30H, Ph), 6.10 (d, 1H, NH), 5.60 (dd, 1H, H-3c), 5.52 (m, 1H, H-2c), 5.20 (d, 1H, J,~ = 8.3 Hz, H-1D), 5.00 (d, 1H, Jl~ = 1.9 Hz. H-L~lPPt2exp-brcva-gp Ic), 4.95 (d, 1H, Jt,z = 3.4 H~, H-lE), 4.63-4.89 (m, 5H, 5 CHzPh), 4.47 (pt, 1H, H-3D), 4.25 (d, 1H, CHzPh), 4.19 (m, 2H, H-5c, CH2Ph), 4.06 (rn, IH, CHzO), 3.87 (m, 5H, H-3E, 4c, 6aD, Gbn, CHzPh), 3.58-3.74 (m, 4H, H-4E, 5n, 5E, CHzO), 3.50 (m, 3H, H-2E, 4D, CH~N3), 3.29 (m, 2H, H-6aF, CHzN3), 3.04 (d, 2H, H-2p, 6bE), 2.02 (s, 3H, CH3C0), 1.SI (d, 3H, H-6c);
'3C NMR: 8 171.5, 165.6, 165.2 (3C, C=0), 127.3-138.6 (Ph), 99.G (C-lc), 99.5 (C-lF), 99.0 (C-ln), 83.4 (C-3n), 81.6 (C-3E): 80.1 (C-2E), 79.2 (C-4c), 77.2 (C-4F), 75.5 (CH~h), 75.1 (C-4p), 74.7, 74.0, 73.2 (3C, CHlPh), 71.3 (C-5o*)> 70.9 (C-5$*), 70.8 (C-3c), 70.4 (C-2c), 69.0 (C-Sc), 68.8 (CH20), 67.5 (C-GE), 67.6 (C-GD), 57.9 (C-2d), 50.5 (CHZNa);
23.4 (CH3C0), 18.2 (C-Gc). FAH-MS for C~yH~GN4Ol7 (11GG) m/z 1185 [M+Na]+.
Anal. Calcd. for C6~H~oNaO~,~HzO: C, 64.85; H, 6.12; 1~', 4.73. Found: C, 64.71; H, 6.01; N, 4.83.
2-Azidocthyl (2,3,4,6-Tetra-0-benzyi-a-D-glucopyranosyi)-(1--~4)-a-L-rhamnopyrnnosyl-(1--~3)-2-acetamido-2-deo~.y-[i-D-glucopyranoside (17). An ice cold solution of95% aq TFA (1.5 mL) in CHzCl2 (13.5 mL) was added to the trisaccharide 15 (730 mg, 0.60 mmol). The mixture was kept at 0°C for 15 min, then diluted with toluene and concentrated. Toluene was co-evaporated from the residue. The residue was dissolved in MeOH (20 mL), and a 1M solution of sodium methoxide in MeOH (1.5 mL) was added. Ths mi.~cture was left to stand at rt for 3 h. The mixture was neutralised with Amberlite IR-120 (H~ resin and filtered. The filtrate was concentrated. The mixture was purified by column chromatography (solvent A, 9:1) to give 17 (S48 mg, 94%) as a colourless foam (a]n +9.7 (c 0.48, MeOH); 1H NMR: 8 7.13-7.31 (m, 8H, Ph), 5.99 (d, 1H, NH), 4.79-4.97 (m, 7H, H-lc, lo, lE, CHZPh), 4.35-4.74 (m, 4H, CHZPh), 3.91-4.10 (m, 7H, H-2c, 3n, 3E, 5c, 5E, ban, CH?O), 3.80 (m, 2H, H-3E: Gbp), 3.73 (m, 1H, CH20), 3.40-3.63 (m, 8H, H-2E, 4c, 4n, 4E, 5p, 6aE, 6bE, CHzN3), 3.27 (m, 2H, H-2D, CH2N3), 1.99 (s, 3H, CH3C0), 1.41 (d, 3H, H-dc); 13C
NMR 8 170.7 (C=O), 127.6-138.4 (Ph), 101.2 (C-1~), 99.7 (C-lFj, 99.0 (C-ln), 84.7 (C-4c), 84.3 (C-3o), 81.5 (C-3E), 79.G (C-2F), 77.6 (C-4D*), 75.G (CHzPh), 75.3 (C-4s*), 74.9, 73.5, 73.4 (3C, CH2Ph), 71.2 (C-SE), 70.8 (C-5c), 70.8 (C-5n), G9.4 (C-3c), 68.6 (C-6E), 68.4 (CH20), 67.6 (C-2c), G2.G (C-6n), 5G.4 (C-2n), 50.5 (CHzN3), 23.5 (CH3C0), 17.6 (C-6c).
FAB-MS for CsoHszNaO~s (958) rn/z 981 [M+Na)~'.
Anal. Calcd. for C5oH62N,~O~s.HZO : C, 61.46; H, G.GO; N, 5.73. Found: C, 61.41; H, 6.61; N, 5.97.

LMPP I ~exp-brevd~gp 2-Aminoethyl a-D-Glucopyranosyl-(1-~4)-a-L-rhamnopyrnnosyl-(1--~3)-2-acetamido-2~
deo~,y-[i-D-glucopyranoside (I8). The trisaccharide I7 (368 mg, 0.38 mmol) was dissolved in a mixture of EtOH (10 mL) and EtOAc (1 mL). A 1N solution of aqueous HCI
(0.77 mL) was added. The mixture was stirred under hydrogen in the presence of 10% Pd/C
(400 mg) for 24 h. The mixture was diluted with water and filtered. The filtrate was concentrated, then lyophilised. The residue was dissolved in a solution of NaHC03 (75 mg) in water (1 mL) and purified by passing first through a column of C~8 silica (eluting with water), then through a column of Sephadex Gio (eluting with water) to give, after lyophilisation, 18 (151 mg, 69%).
HPLC (230 nm): Rt 4,09 min (Kromasil 5 p.m C18 100 A 4.Gx250 mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mLlmin flow rate).'H NMR (Dz0): 8 5.03 (d, 1H, Jl,z = 3.8 Hz, H-1~), 4.84 (bs, 1H, H-lc), 4.58 (d, 1H, Jt,z = 8.5 H~ H-1D), 4.10 (m, IH, H-Sc), 3.98 (m, 3H, H-5E, 6I~, CHzO), 3.79 (m, 6H, H-?c, 2D, 3p, 6aE, 6bE, CHzO), 3.68 (pt, 1H, H-3E), 3.42-3.G0 (m, 6H, H-2E, 30, 4c, 4D, 4E, SD), 3.03 (m, 2H, CHZNHz), 2.OG (s, 3H, CH3G0), 1.31 (d, 3H, H-Gc); '3C S 175.2 (C=O), 101.9 (C-lc), 101.0 (C-lo), 100.3 (C-lE), 82.4 (C-3B), 81.G (C-4c), 76.5 (C-2E), 73.3 (C-3E), 72.4 (C-56), ?2.1 (C-4D), 71.G (C-2c), 69.9 (C-4E), G9.5 (C-3c), G9.0 (C-Sp), G8.7 (C-5c), 68.7 (CHzO), 61.2 (C-6D), G0.7 (C-GE), 55.8 (C-2D), 40.3 (CHzNHz), 22.7 (CH3C0), 17.3 (C-Gc).
Electrospray-MS for CzzHaoNz0ls (572) »~/z 573 [M+H]f. Manqae 6anel3~; 2x30 HRMS (MALDI] Calcd for CZZHaoNsOis+Na: 595.2326. Found: ~;XXXX.
Altyl (2,3,4-tri-0-acetyl-a-L-rhamnopyrranosyl)-(1-r3)-[(2,3,4,6-tetra-0-benzyl-a-D-glucopyranoayl)-(1-->4)]-2-0-ben~.oyl-a-L-rhamnopyranoside (21). TMSOTf (100 ~.L) was added to a solution of donor 20 (2.5 g, 5.78 mmol) and acceptor 19 (4.0 g, 4.80 mmol) in EtzO
(40 mL) at -SO°C. The mixture was stirred for 2.5 h, at which time the cooling bath had reached rt. Et3N was added and after 15 min, volatiles were evaporated. Column chromatography (solvent C, 4:1) of the crude product ga~~e the folly protected 21 (4.74 g, 89%) as a white solid. 'H NMR: 8 8.00-6.90 (m, 25H, Ph), 5.92 (m, 1H, CH=), 5.53 (dd, 1H, H-28), 5.40-5.20 (m, 4H, H-lE, 20, CHz=), 5.18 (dd, 1H, Jz,~ = 3.2, J3,4 =
IO.X Hz, H-31s), 5.10 (d, IH, H-1H), 5.00-4.40 (m, 10H, H-4B, lc, OCHz), 4,30-4.00 (m, SH, H-3s, 3c, SE, OCHz), 4.00-3.50 (m, 7H, H-2r, 4F, 6aE, 6bE, Sa, So, 4c), I.90 (s, 3H, Ac), 1.G0 (s, 3H, Ac), 1.22 (s, 3H. Ac), 1.20 (d, 3H, J5,6 = X.X Hz, H-Gc), 0.80 (d, 3H, JS,~ = X.X Hz, H-Gn);
"C NMR: b 16.89-16.55, 1GG.1 (4C, C=O), 133.4-127.3 (Ph), 117.5 (=CHz), 9.8 (C-1B), 96.9 (C-lE), 95,7 (C-lc), 81.4 (C-3E), 80.7 (C-2~), 7.3 (C-3c), 77.7 (C-4E), 77.5 (C-4c), 75.6-72.6 (4C, OCHzfh), 72.7 (C-2c), 7.9 (2C, C-5E, 48), G.0 (C-2H), 68.7 (C-GE), 68.6 (C-3H), 68.2 (OCHz), LMPPI2exp~brevct-Rp ~1 02434685 2003-07-04 G7.2 (C-Sc), 6G.8 (C-5a), 20.7 20.2 {3C, C(0)CH3), 18.5 (C-6c), 16.8 (C-6B).
CI-MS for C62H70018 (1102) m/z 1125 [M+Na]+.
Anal. Calcd. for C62H~o~~s: C, 67.50; H, G.40. Found: C, 67.51; H, 6.52.
(2,3,4-Tri-0-acetyl-a-L-rhamnopyranosyl)-(1->3)-((2,3,4,G-tetra-0-benzyl-a-D-glucopyranosyl)-{1---~4)]-2-O-benzoyl-a-L-rhamnopyranose (22). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (33 mg) was dissolved in THF
(10 mL) and the resulting red solution was degassed in an argon stream Hydrogen was then bubbled through the solution, until the colour had changed to yellow. The solution was then degassed again in an argon stream. A solution of 21 (4.59 g, 4.1 G mmol) in THF (30 mL) was degassed and added. The mixture was stirred at rt overnight, then concentrated. The residue was taken up in a mixture of acetone (10:1, 44 mL). Mercuric bromide (1.78 g, $.32 mmol) and mercuric oxide (1.69 g, G.24 mmol) were added to the mixture, which was protected from light. The suspension was stirred at rt for 1 h, then concentrated. The residue was taken up in CH2Clz and washed three times with sat aq KI, then with brine. The organic phase was dried and concentrated. The residue was purified by column chromatography (solvent B, 3:1 ) to give 22 (3.52 g, 80%) as a colourless foam; [a]D -~-17.7; ~H NMR: 0 7.15 (m, 25H, Ph), 5.50 (dd, 1H, H-2H), 5.30-5.27 (m, 2H, H-lo, H-Zc), 5,23 (d, 1H, Jl~ = 3.3 Hz, H-lE), 5.18 (dd, 1H, 1i,3 = 3.2, J3,4 = 10.0 Hz, H-3n), 5.10 (d, 1H, J~a = 1.2 Hz, H-la), 5.00-4.35 (m, 9H, H-4B, OCH2), 4.28 (dd, IH, Jz,3 = 3.2, J3,4 = 8.G Hz, H-3o), 4.20-4.00 (m, 3H, H-3~, 5s, Sc), 3.80-3.50 (m, GH, H-2s; 6aE, 6bs, 5H; 4E, 40), 3.05 (d, 1H, JoH,, = 4.0 Hz, OH), 2.09, 1.81, 1.44 (3s, 9H, CH3C=0), 1.37 (d, 3H, J5,6 = 6.2 Hz, H-Gc), 0.95 (d, 3H, J5,6 = 6.2 Hz, H-6B); 13C NMR:
0169.9-169.6, I6G.2 {4C, C=O), 138.9-127.5 (Ph), 99.8 (C-1B), 97.3 (C-le), 91.3 (C-lc), 81.7 (C-3~, 80.7 (C-2E), 78.8 (C-3c), 78.1-78.0 (2C, C-4E, 4c), 7G.b, 75.5 (2C, CHzPh), 74.9 (2C, C-2E, CHzPh), 73.8 (CHzPh), 73.3 (2C, C-4B, 5E), 72.9 (C-2B), 71.2 (2C, C-3a, GE), 67.5 (C-5c), G7.1 (C-5a), 21.0-20.6 (3C, CH3C=0), 18.9 (C-6c), 17.1 (C-6B). FAB-MS for C59Hss0is (1042) »t/z 1085 [M+NaJ+.
Anal. Calcd. for G59H66~18'H2~~ C, 65.54; H, G.34. Found: C, 65.68; H, 6.41.
(2,3,4-Tri-0-acetyl-o~-L-rhamnopyranasyl)-(1-~3)-((2,3,4,6-terra-0-benzyl-a-n-glucopyranosyt)-(1-~4)]-2-O-benzoyl-a-L-rhamnopyranose trichloroacetimidate (S).
DBU (100 ~.L) was added at 0°C to a solution of the hemiacetal 22 (3.8 g, 3.58 mmol) in DCM (40 mL) containing trichloroacetonitrile (4 mL). The mixture was stirred for 30 min at 0°C, and volatiles were evaporated. Flash chromatography (solvent B, 7;3 + 0.2% Et3i~ of the etude material gave the donor S (3.9 g, 90%) as a white solid; [a]D +2.8 (c ?);
h'MR ???
Anal. Calcd. for C61H6sC13NOts: C, 60.67; H, S.SI; N, 1.16. Found: C, 60.53;
H, 5.48; N, 1.38.

LMPPIZB%p-brevet-gp C~1 02434685 2003-07-04 2-Azidoethyl (2,3,d-Tri-O-acetyl-a-L-rhamnopyranosyl)-(1-~3)-[(2,3,4,6-tetra-D-benzyl-a-D-glucopyranosyl)-(1-~4)]-(Z-0-benzoyl-a-L-rhamnopy ranosyl)-(Z-~3)-2-acetamido-2-dco~y-4,6-O~isopropylidene-~-p-glucopyrnnoside (23). The trisaccharide donor 5 (1.86 g, 1.54 mmol) and the acceptor 7 (712 mg, 2.16 mmol) were dissolved in 1,2-dichloroethane (15 mL) and 4~-MS (2 g) were added. The mixture was stirred at rt for I h. The mixture was cooled to 0°C and triflic acid (34 p>r, 0.385 mmol) was added. The mixture was stinted at 0°C
for 30 min, then at rt for 30 min. The mixture was then heated at GS°C
for 1 h. The mi~rture was allowed to cool, Et3N (0.5 mL) was added, and the mixture was stirred at rt for 20 min.
The mixture was diluted with CI-IzClz and filtered through a pad of Celite.
The filtrate was concentrated and purified by column chromatography (solvent B, 1:1) to give 23 (1.61 g, 76%). 1H NMR: b 7.90-G.90 (m, ZSH, Ph), 5.92 (d, IH, J = 7.5 Hz, NH), 5.53 (dd, 1H, Jl,a=
1.8 Hz, H-2a), 5.29 (d, 1H, H-lE), 5.19 (m, 2H, H-2x, 3H), 5.09 (m, 2H, H-lx, 1D), 4.97 (bs, 1H, H-IB), 4.9G-4.70 (m, 9H, CHaPh, H-4a), 4.54-4.41 (m, H, CHaPh), 4.34 (pt, 1H, J3,4 = J4,s = 9.3 Hz, H-3D), 4.19-3.89 (m, 6H, H-3~, 5x, 5E, 3E, 6aD, OCHz), 3.79-3.60 (m 5H, H-6ba, 4~, 5H, 2E, OCHa), 3.SG-3.33 (m, 4H, H-5n, 4E, 4n, CH2N3), 3.27-3.12 (m, 2H, GHaN3, H-2D), 2.10, 2.09 (2s, 6H, C(CHa)a), 1.78 (s, 3H, OAc), 1.73 (s, 3H, NHAc), 1.42, 1.35 (2s, 6H, OAc), 1.30 (d, 3H, J5,6 = 6.2 Hz, H-Go), 0.90 (d, 3H, Js,6 = 6.2 Hz, H-6H);
~3C NMR: 8 171.4, 169.7. 169.6, 169.5, 166.0 (5C, C=0), 138.7-127.2 (Ph), 99.8, 99.7 (C-lp, lc), 97.1 (C-1B), 96.4 (C-lp), 81.5 (C-3E), 81.1 (C-2E), 79.5 (bs, C-3c), 77.9 (C-4o), 77.0 (bs, C-4x), 75.4 (C-3,~), 75.3, 74.7, 73.6 (3C, CHzPh), 73.0, 72.9 (2C, C-2c, 4E), 72.9 (CHaPh), 71.2 (C-Sa), 71.1 (C-48), 69.9 (C-2$), 69.2 (C-6E), G8.8 (C-3H), 68.7 (OCHz), 67.2, 67.1 (3C, C-5~, 5H, 5p), 62.2 (C-6D), 59.0 (C-2p), 50.G (CHaN3), 29.0, 23.4 (2C, C(CH3)a), 20.9, 20.4 (3C, OAc), 19.0 (NHAc), 18.4 (C-6x),17.0 (C-GB). FAB-MS for ClZHa6N4023 (1374) m/~ 1397 [M+Na)+.
Anal. Calcd. for C~zHBsNa0z3: C, 62.87; H, 6.30; N, 4.07. Found: C, ?P; I~, ??; N, P?.
Z-Azidocthyl (2,3,4-Trl-0-acetyl-a-L-rhamnopyranosyl)-(Ia3)-[(2,3,4,6-tetra-O-benryl-a-D-glucopyranosyl)-(I~4))-(Z-O-bcnzayl-a-L-rhamnopyranosyl)-(1-~3)-2-acetamido-2-deoxy-[i-D-glucopyranoside (24). 50% aq TFA (I.3 mL) was added to a solution of the fully protected tetrasaccharide 23 (210 mg, 11 I pmol) in DCM (6 mL). The mixture was stirred at 0°C for 1 h. Volatiles were evaporated and toluene was co-evaporated from the residue.
Column chromatography (solvent B, 7:3 -~ 1:1) of the crude product gave 24 (195 mg. 95%).
[a)D-6.9 (c 0.5, MeOH);
NMR?
FAB-MS for C(,9HgaNqOZ3 (1334) m/z 1357_5.
Anal. Calcd. for C69HgaN4021'H20~ C, 60.43; H, 6.32; Iv', 4.09. Found: C, 60.56; 6.22, 3.92.

C.MPPI2cxp-brtvet-Sp 2-Aminocthyl a-L-Rhamnopyranosyl-(1-->3)-(a-D-glucopyranosyi-(1-~4)J-o~.-L-rhamnopyranosyl-(1--~3)-2-acetamido-2-deogy-~-D-glueopyranoside (25). An ice cold solution of 95% aqueous trifluoroace~tic acid (2.4 mL) in CHZCIz (21.6 mL) was added to the te2rasaccharide 23 (1.93 g, 1.40 mmol). The mikrture ws kept at 0°C for 5 min., then diluted with toluene and concentrated. Toluene was co-evaporated from the residue. The residue was dissolved in MeOH (6S mL), and a 1M solution of sodium methoxide in MeOH (3 mL) was added. The mixture was left to stand at rt for 18 h. then neutralised with Amberlite IR-120 (F~ resin, and filtered. The filtrate was concentrated, and the residue was purified by column chromatography (solvent B. 9:1) to give 24 (1.38 g, 89°/) as a colourless foam The tetrasaccharirie 24 (1.38 g, I.25 mmol) was dissolved in a mixture of EtOH (35 mL) and EtOAc (3.5 mL). A 1N solution of aq HC1 (2.S mL) was added. The mixture was stirred under hydrogen in the presence of 10% Pd/C (1.5 g) for 72 h, then diluted with water and filtered.
The filtrate was concentrated, then lyophilised. The residue was dissolved in a solution of 5%
aqueous NaHC03 and purified by passing first through a column of Cts silica (eluting with water), then through a column of Sephadex Glo (eluting with water) to give, after lyophilisation, 25 (693 mg, 77%). HPLC (234 nm): Rt 4.78 min (Kromasil S pm 4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mL/min flow rate). ~H NMR (Dz0): 8 5.10 (d, IH, J,,Z = 3.7 Hz, H-lE), 4.89 (d, IH, J,,Z = 1.1 Hz, H-1B), 4.73 (d, 1H, Ji,~ = 1.0 Hz, H-lc), 4.50 (d, 1H, Jl,z = 8.6 Hz, H-lo), 4.08 (m, IH, H-5~), 3.96 (m, IH, H-2H), 3.9I (m, ZH, H-GaD, CHZO), 3.68-3.88 (m, 12H, H-2~, 2p, 3H, 3c, 4$, 4~, 5s, SE, 6bn, 6aE, 6bE, CHaO), 3.59 (pt, 1H, H-3E), 3.52 (pt, 1H, H-3D), 3.33-3.48 (m, 4H, H-2E, 4D, 4E, 5D), 3.OI (m, 2H, CH2NH2), 1.99 (s, 3H, CH3C=O), 1.28 (d, 3H, H-6c), 1.18 (d, 3H, H-GH); 13C ~ 174.8 (C=O), 103.2 (C-1$), 101.4 (C-lo), 100.9 (C-ID), 98.G (C-lE), 81.9 (C-3p), 79.0 (C-4g), 76.6 (C-4~), 76.3 (C-2E), 72.9 (C-3fi), 72.3 (C-5E), ?2.3 (C-4D), 71.8 (C-3~), 71.1 (C-2~), 70.5 (C-2B, 3B), 69.7 (C-4H), 69.5 (C-4E), G9.2 (C-Sp), 68.8 (2C, C-SB, 50), 67.9 (CHZO), 61.0 (C-6D), 60.8 (C-6~), 55.5 (C-2D), 40.0 (CHZNHZ), 22.6 (CH3C=O), 18.0 (C-6~). I 7.0 (C-6B). XXMS for CZBHSONZ019 (718) mlz 741 [M + NaJ+.
HRUIS (MALDI) Calcd for C2sH5oNZO19: 741.2905. Found: XXX?i.
Altyl (2,3,4-Tri-0-benxoyl-a-L-rhamnopyranosyi)-(1-+2)-3,4-di-0-benzyl-a-L-rhamnopyraboside (28j. TM50Tf (11 uL, 59 wmol) was added to a solution of the rhamnoside 26 (2.26 g, 5.88 mmol) and the trichloroacetimidate 27 (4.23 g, 6.82 mmol) in LMPPI2exp-bteve!-gp anhydrous EtzO (G0 mL) at --70°C. The reaction mixture was stirred For 8 h while the cooling bath was slowly coming back to rt. Et3N (100 pL) was added, and the mixture was stirred at rt for 15 man. Solvents were evaporated, and the crude material was purified by column chromatography (solvent B, 49:1 --~ 9:1), to givE 28 as a white foam (4,78 g, 9G%). 1H NMR:
8 8.17-7.12 (m, 25H, Ph), 5.97-5.85 {m, 3H, H-2~, 3A, CH=), 5.67 (pt, 1H, J3,4 = 9.6 Hz, H-4A), 5.34-5.19 (m, 3H, H-1,," CHZ=), S.OI (d, 1 H, J = 9.0 Hz, CHzPh), 4.92 (d, 1 H, J~,2 = 1.3 ' Hz, 1-I-1 g), 4.82-4, 74 (m, 2H, CHZPh), 4. 71 (d, 1 H, J = 11. 8 Hz, OCHz), 4.31 (dq, I H, Ja,s =
9.7 Hz. H-5,~, 4.21 (m, 1H, OCHz), 4.10 (dd, 1H, H-2B), 4.02 (m, 1H, OCHz), 3.97 (dd, 1H, Jz,3 = 3.0, J3,4 = 9.2 Hz, H-3H), 3.82 (dq, 1 H, Ja,s = 9.4 Hz, H-5$), 3.71 (pt, IH, H-4$), 1.43 (d, 3H, Js,6 = G.1 Hz, H-6B), 1.37 (d, 3H, Js,6 = G.2 Hz, H-G,~}; ~3C NMR: &
166.3, 165.9, 165.7 (3C, C=O), 139.0-127.9 (CH=, Ph), 117.8 (CHz=), 99.9 {C-1,~, 98.3 (C-IB), 80.6 (C-48), 80.2 (C-3a), 76.5 (C-2$), 76.0, 72.9 (2C, CHZPh), 72.3 (C-4,~, 71.0 (C-ZA*), 70.4 (C-3A*), 68.7 (C-SH), 68.1 (OGHz), G7.5 (C-5,~, 18.4 (C-GB), I8.1 (C-6,~. FAB-MS far Csol-lso0~a (M =
842.3) m/z 8G5.1 [M+Na]'.
Anat. Calcd for CsoHso0J2: C, 71.21; H, 5.98. Found C, XX; X, XX~IG
(2,3,4-tri-O-Benzoyl-a-L-rhamnopyraaosyt)-(1-~2)-3,4-di-O-beezyt-a-t~-rhamnopyranose (29). 1,5-Cyclooctadiene-bis(methyldiphenylphosphinejiridium hekafluorophosphate (25 mg) was dissolved in THF (10 mL) and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, until the colour had changed to yellow. The solution was then degassed again in an argon stream. A
solution of 28 (4.71 g, 5.59 mmol) in THF (40 mL) was degassed and added. The mixture was stirred at rt overnight, then concentrated. The residue was taken up in acetone (350 mL) and i water (82 mL). Mercuric bromide (3.23 g, 8.9G mmol) and mercuric oxide (2.64 g, 12.3 mtnol) were added to the mixture, which was protected from light. The suspension was stirred at rt fox 1 h, then concentrated. The residue was taken up in CHiCIz and washed three times with sat aq KI, then with brine. The organic phase was dried and concentrated.
The residue was purified by column chromatography (solvent B, 3:1) to give 29 (3.87 g, 84%) as a i colourless foam ~H NMR: 8 8.15-7.12 (m, 25H, Ph), 5.94-5.88 (m, 3H, H-2A, 3A, CH=), 5.70 (pt, 1H, J3,4= 9.7 Hz, H-4,~, 5.31 (dd, 1H, Jl.ort=3.0 Hz, H-1H), 5.28 (bs, 1H, H-1,~, 4.98 (d, 1H, J = I1.0 Hz, CHzPh), 4.82-4.G8 (m, 3H, CHZPh), 4.31 (dq, 1H, J4,5= 9.8 Hz, H-5~, 4.13 (dd, 1H, Jl,z = 2.1 Hz, H-2B), 4.06-3.99 (m, 2H, H-3B, Sa), 3.72 (pt, 1 H, J3,4 = Jd,s = 4.4 Hz, H-48), 2.79 (bs, 1H, OH-1B), 1.41 (d, 3H, Js,6= 6.2 Hz, H-GB), 1.37 (d, 3H, JS,~= 6.3 Hz, H-6,~;
i LM~PI~C?t~-hICVE(-~1 "C NMR: s 166.2, 165,9, 165.7 (3C, C=0), 138.9-127.9 (Ph), 99.7 (C-1,~, 94.2 (C-1H), 80.5 (C-4g), 79.6 (C-3a), 7'7.6 (C-2B), 76.5, ?2.5 (2C, CI~Ph), 72.3 (C-4~, 7I-0 (C-2A*), 70.4 (C-3A~), 68.8 {C-SH), 67.6 (GSA), 18.5 (C-6s*), 18.1 (C-6a*). FAB-MS for Ca~H4sOu (M =
802.3) »r/z 825.1 [M+Na]+.
Anal. Calcd. for C4~H,s011: C, 70.31; H, 5.78. Found C, XX; H, XXX.
i {2,3,4-Tri-O-bcnzoyl-a-L-rhamnopyranosyl)-(1-->2)-3,4-di-O-benzyl-a-Lr rhamnopyranosyl Trlchloroacetimidate {30). The hemiacetal 29 (3.77 g, 4.71 mmol) was dissolved in CHzCIZ (15 mL) and the solution was cooled to 0°G.
TrichloroacetonitriIe (2.5 mL) was added, then DBU (200 pL). The mixture was stirred at rt for 2 h Toluene was added, and co-evaporated twice from the residue. The crude material was purified by flash chromato~aphy (solvent B, 4:1 + 0.1% Et3N) to give 30 as a white foam (4.29 g, 96°.0). Some hydrolyzed material 29 (121 mg, 3%) was eluted next. The trichloroa,cetimidate 30, isolated i as an alb mixture had 1H NMR (a anomer): 8 8.62 (s, 1H, NH), 8.20-7.18 (m, 25H, Ph), 6.31 (s. 1 H, H- I e), 5.94 (dd, 1 H,11,~ = 1.6 Hz, H-2,~, 5. 89 (dd, 1 H, Ja,3 =
3.4, 33,4 = 9.9 Hz, H-3,~, 5.71 (pt, IH, H-4,~, 5.27 (bs, 1H, H-1,~, 5.02 (d, 1H, J = 14.8 Hz, CH2Ph), 4.84 (d, 1H, T =
11.9 Hz, CH~h), 4.79 (d, 1H, CHzPh), 4.72 (d, 1H, CH~h), 4.3G (dq, IH, J4,5=
9.8 Hz, H-' S~, 4.13 (dd, IH, H-2B), 4.03-3.97 (m, 2H, H-3s, 5B), 3.80 (pt, 1H, J3,a =
9.5 Hz, H-4B), 1.45 (d, 3H, Js,s = 6.1 Hz, H-6H), 1.40 (d, 3H, Is,s= G.2 Hz, H-6,J; 13C NMR (a an~omer): b 166.2, IG5.9, 165.7 (3C, C=O), 160.8 (C=NH), 138.6-128.2 (Ph), 99.9 (C-1~, 97.2 (C-18), 91.4 (CCI~), 79.9 (C-4B), 79.1 (C-3$), 76.2 (CH2Ph), 74.9 (C-2B), 73.3 (CH2Ph), 72.1 (C-4B), 71.7 ' (C-Ss), 71.0 (C-2,~, 70.2 (C-3.~, 6?.8 (C-5~, 18.4 (C-6H),18.0 (C-6,~.
Ana6 Calcd, for Ca9HasC13NO,a: C, 62.13; H, 4.89; N, 1.48. Found C, XX; H, XXX, N, X.XX.
I Aryl (2,3,4-Tri-0-bcnzoyl-arrL-rhamnopyranosyl)-(1--~2)-(3,4-di-0-benzyl-arL-rhamnopyranosyl)-(1-~3)-[(2,3,4,6-tetra-0-benzyl-a-D-glueopyranosyl)-(la4)]~2-benzoyl-a-L-rhamnopyranoside (33). (a) The acceptor 16 (465 mg, 0.56 mmol) was dissolved in ether (3 mL). The solution was cooled to -GO°C and TMSOTf (GS ~L, 0.3G
mmol) was added. The donor 30 (G90 mg, 0.73 mmol) was dissolved in ether (6 mL) and added to the acceptor solution in two portions with an interval of 30 min. The mixture was stirred at -GO°C to -30°C over 2 h. Et3N (100 1rL) was added.
The mixture was concentrated LMPP1 aexp-brevet-gp and the residue was purified by column chromatography (solvent B; 7:1) to give 33 (501 mg, 55°/'0).
(b} A solution of the donor 27 (1.41 g, 2.25 mmol) and the acceptor 32 (1.07 g, 1.79 mmol} in anhydrous EtzQ (88 mL) was cooled to --60°C. TMSOTf (63 ~L} was added, and the mixture was stirred at -60°C to -20°C over 2.5 h. Et3N was added (I00 pL). The mixture was concentrated and the residue was purified by column chromatography (solvent D, 49.1) to give 33 (2.66 g, 92%); (a]p +74.1 (c 0.5); 'H R'MR: S 7.06-8.11 (m, 50H, Ph), 5.88-6.05 (m, 3H, H-2~, 3,,, CH = ), 5.71 (t, 1H, H-4,~. 5.51 (dd, 1H, H-2~), 5.22-5.41 (m, 3H, H-1,4, CHz =
), 5.14 (d, 1H, l~,z = 0.9 Hz, H-1B), 5.10 (d, 1H, J1,2 = 3.Z H~~ H-lp), 4.97 (bs, 1H, H-lo), 4.35-5.00 (m, 14H, H-28, Sa, 12 x CHZPh), 3.98-4.19 (m, SH, H-3C, 3r, 5E, OCHZ), 3.43-3.87 (m, 9H, H-2p, 38, 4a, 40, 4x, SB, 50, 6E, 6',~, 1.44 (d, 3H, H-G,~, 1.40 (d, 3H, H-6o), 1.13 (d, 3H, H-6B); 13C NMR: 8 165.9, 165.4, 165.1 (C=O), I27.1-138.7 (CH=, Ph), 117.8 (CHz=), 101.3 (C-1H), 99.d (C-1,,~, 97.9 (C-lE), 96.1 (C-lo), 81.9 (C-3E), 81.0 (C-2E), 80,1 (C-3o}, 79.8 (C~4aj, 78.9 (C-3~), 77.9 (C-4~), 77.4 (C-4E), 75.9 (C-2B), 75.G, 75.0, 74.9, 73.9, 72.9 (CHzPh), 72.4 (C-2o), 71.9 (C-4,~, 71.2 (C-SE), 70.9 (CHzPh), 70.7 (C-2,,*), 70.0 (C~3,,'"), 69.2 (C-5$), 68.5 (OCHz), 68.1 (C-6E), 67.6 (C-So), 67.2 (C-5,~, 18.8 (C-d,~, 18.1 (C-6c), 17.8 (C-GB). FAB-MS for Cg7H9gO?~ (1614) rrllz 1637 [M+Na]-.
Anal. Calcd, for C9~H9gOzz: C, 72.10; H, 6.11. Found: C, 71.75; H, G.27.
(2,3,4-Tri-O-benzoyl-a-L-rhamnopyraaosy!)-(1-->2)-(3,4-di-O-bcnryl-a-L-rhamnopyranosylj-(1~3)-((2,3,4,6-tetra-O-benryl-a-D-glucopyranosyi)-(1-~4)]-(2-O-benzoyl-ocl(i-L-rhamnopyranose (34). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (12.5 mg) was dissolved in THF
(5 mL) and Lhe resulting red solution was degassed in an argon stream.
Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream. A solution of 33 (1.138 g, 0.70 mmol) in THF (15 mL) was degassed and added. The mixture was stirred at rt overnight. The miucture was concentrated. The residue was taken up in acetone (7 mL) and water (0.7 mL}.
Mercuric chloride (285 mg, 1.05 rnmol) and mercuric oxide (303 mg, I.4 mmol) were added to the mixture, which was protected from light. The mixture was stirred at rt for 1 h, then concentrated. The residue was taken up in CHzCIz and washed three times with sat. aq. RI, then with brine. The organic phase was dried anti concentrated. The residue was purified by column chromatography (solvent B, 7:3) to give 34 (992 mg, 90%) as a colourless foam jH
to LMPPI2cxp.brevct~gp NMR: $ 7.05-8.16 (m, SOH, Ph), 5.88-5.93 (m, 2H, H-2~. 3A), 5.73 (pt, IH, H-4,~, 5.55 (m, 1 H, H-2c), 5.3 7 (6s, 1 H, H-1,~, 5.28 (bs, 1 H, H-1 c), 5.14 (bs, 1 H, H-1 H), 5.07 (d, 1 H, ,I ~ ,z =
3.1 Hz, H-lE}, 4.78-4.99 (m, 6H, CHzPh), 4.31-4.68 (m, 8H, H-2a, 5A, CHzPh), 4.24 (dd, 1H, H-3~), 3.99-4.09 (m, 3H, H-3E, Sc, SE), 3.82 (pt, 1H, H-4c), 3.57-3.76 (m, 5H, H-3a, 4E, 5B, 6aE, .6bE), 3.48 (dd, IH, H-2s), 3,17 (d, 1H, OH), 1.43 (d, GH, H-G", Gc), 1.14 (d, 3H, H-6H) "C NMR: b 166.0, 165.6, 165.2 (4C, C=O), 127.2-138.9 (Ph), 101.1 (C-18), 99.7 (C-1,~, 98.1 (C-IE), 9L6 (C-Ic), 81.9 (C-3H), 81,1 (C-2E), 79.9 (C-4B): 79.4 (G3c), 78.9 (C-3$), 78.3 (C-4c), 77.6 (C-4E), 76.1 (C~28), 75.8, 75.3, 75.1, 74.0, 73.1 (XXC, CH~T'h), 72.7 (C-2c), 72.1 (C-4~, 71.4 (C-5E), 71.1 (CH2Ph), 70.8 (C-2A*), 70.2 (C-3A*), 69.4 (C-5B), 68.3 (C-6E), 67.7 (C-5c), 67.3 (C-5,~, 19.0 (C-6,~, 18.2 (C-6c), 17.9 (C-6B). FAB-MS for Cg4H94072 (1$74) m1z 1597 [M+Na]t.
Anal. Calcd. fnr CgqH~sOI2: C, 71.65; H, 6.01. Found: C, 71.48; H, 6.17.
(2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl)-(1--~2)-(3,4-di-O-benzyi-a-L-rhamnopyranosyl)-(1-~3)-((2,3,4,6-tetra-O-benzyl-a-n-glueopyranosyl)-(1-34)]-(2-O-benzoyl-a![i-L-rhamnopyranosyl trichloroacetimidate (35). The he.miacetal 34 (412 mg, 0.26 mmol) was dissolved in CHzCIz (5 mL) and the solution ws cooled to 0°C.
Trichloroacetonitrile (0.26 mL) was added, then DBU (4 ~L). The mixture was stirred at 0°C
for 1 h. The mixture was concentrated and toluene was co-evaporated from the residue. The residue was purified by flash chromatography (solvern B, 4:1 + 0.1% Et3N) to give 34 (393 mg, 88%). 'H NMR: & 8.74 (s, 1H, NH), 7.03-8.10 (m, 50H, Ph), 6.42 (d, 1H, J~,z = 2.3 Hz, H-Ic), 5.87 (m, 2H, H-2~, 3a). 5.67 (m 2H, H-2c, 4~. 5.30 (bs, 1H, H-l~, 5,14 (bs, 1H, H-1H), 5.08 (d, 1H, J,,2 = 3.1 Hz, H-lE), 4.74-4.98 (m, Gl-I, CH~'h}, 4.23-4.69 (m, 9H, H-2H, 3c, 5," CHzPh), 3.88-4.07 (m, 3H, H-3E, 5H, 5E), 3.57-3.74 (m, 7H, H-2c, 4g, 4c, 4E, Sc, Gae, 6GE), 3.50 (dd, IH, H-3H), 1.38 (d, 6H, H-6A, 6H), 1.07 (d, 3H, H-bc); 1'C NMR: 8 165.9, 165.5, 165.4, 165.1 (4C, C=O), 160.1 (C=NH), 127.2-138.7 {Ph), 101.2 (C-Ix), 99.7 (C-1,~, 98.3 (C-lE), 94.3 (C-lc), 90.9 (CCl3), 81.7 (C-3E), 80.9 (C-2E), 79.6 (C-3c, 4B), 78.5 (C-3a), 77.2 {C-4c), 77.5 (C-4E), 75.9 (C-2H), 75.6, 75.1, ?5.0, 74.0, 72.9 (CH~h), 71.8 (C-2c), 71.3 (C-4~,).
71.0 (CHzPtt), 70.7 (C-5E): 70.5 (C-2~*), 70.3 (C-3~*), 70.0 (C-SB), 69.5 (C-5c), 67.9 (C-6~), 67.2 (C-5,~, 18.7 (C-GA), 17.8 (C-6c), 17.7 (C-6g).
Anal. Calcd. for Cg6Hg~C13NO2z: C, 67.03; H, 5.5I; N, 0.81. Found: C, 63.14, H, 5.14; N, 1.00.

LMPPI2expbcevet~gp CA 02434685 2003-07-04 2-Azidoethy! (2,3,4-Tri-O-benzoyt-a-L-rhamaopyranosyl)-(1-a2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1-->3)-[(2,3,4,6-tetra-O-benry!-a-D-8lucopyranosyl)-(1~4)]-(2-O-benzoyl-ac-L-rhamnopyranosyI}-(la3)-2-xcetamido-2-deoxy-4,6-O-isopropylidene-[3-D-glucopyranoside (35). (a) The tetrasaccharide donor 6 (500 mg, 0.29 mmol) and the acceptor 7 (140 mg, 0.42 mmol) were dissolved in I,2-dichloroethane (5 mL) and 4A-MS
(d00 mg) were added. The mixture was stirred at rt for 2 h. The mixture was cooled to 0°C and triflic acid (7 ~L, 0.072 mmol) was added. The mixture was stirred at 0°C to rt over 1 h 30 min. The mixture was then heated at 65°C for 1 h 30 min. The mixture was allowed to cool, Et3N (0.5 mL) was added, and the mixture was stirred at rt for 20 min. The mixture was diluted with CHZC~ and filtered through a pad of Celite. The filtrate was concentrated and purified by column chromatography (solvent B, 4:3) to give 35 (340 mg, G2%).
(b) The tetrasaecharide donor 6 (250 mg, 145 ltmol) and the acceptor 7 (G7 mg, 204 pawl) were dissolved in DCM {1.5 mL) and 4~1-MS (200 mg) were added. The mixture was stirred at -40°C For 30 min and triflic acid (5 p.L) was added. The. mixture was stirred at rt over 3 h, triethylamine was added, and the mixture was stirred at rt for IS min. The mixture was diluted with CHzCIz and filtered through a pad of Celite. The filtrate was concentrated and purified by column chromatography (solvent B, 9:1 -~ 1:1) to give 35 (219 mg, 80%).
[a)n +29.0 (c 0,25, MeOH); 'H NMR: b 7.04-8.06 (m, 50H, Ph), 6.24 (d, 1 H, NH), 5.90 (m, 2H, H-2A, 3~, 5.70 (t, 1H, H-4~, 5.42 (m, 1H, H-2c), 5.35 (bs, 1H, H-lA), 5.13 (m 3H, H-1B, lo, lr), 4.77-5.00 (m, 5H, H-lo, CH2Ph), 4.29-4.66 (m, 11H, H-2B, 3n, 5~. CHzPh), 3.80-4.11 (m, 6H, H-3c, 3s~ Sc. 5E, 6aD~ CHiO), 3.45-3.78 (m, 12H, H-2E, 3e~ 4e~ 4c~ 4p~ 4p~ 5B, So~ dbn. 6aF, Gbe CHZO), 3.39 (m, 1H, CHaN3), 3.23 (m, 2H, H-2D, CH2N3), 2.13 (s, 3H, CH3C0), 1.43 (d, 9H, H-6A, (CH3)2C), 1.29 (d, 3H, H-Gc), 1.1 I (d, 3H, H-6g); "C NMR; S 171.8, 1 G5.9, 165.5, 155.0, 163.5 {SC, C=O), 127.1-138.7 (Ph), 101.3 (C-Ie), 99.8 (C-ID), 99.3 (C-I,~, 97.7 (C-Ic), 97.6 (C-1~, 91.8 (C{CH3)~J, 81.6 (C-3E), 81.0 (C-2a), 80.0 (C-3c), 79.7 (C-4D), 78.9 (C-48), 77.5 (C-3H, 4c), 76.4 (G3D), 75.6 (C-28), 75.5, 74.9, 74.8, 73.8, 73.0 (SC, CHzPh), 72.9 (C-4s), 72.7 (G-2c), 71.8 (C-4,~, 71.3 (C-SE), 71.0 (CH~Ph), 70.6 (C-2A*), 70.0 (C-3A*), 69.3 (C-5B), 68.6 (OCH2), 68.3 (C-6s), 67.5 (C-Sc), 67.3 (C-5,~, 67.1 (G5p), 62.2 (C-6D), 58.9 (C-2n), 50.6 (CHzN3), 29.1 (CH3C), 23.6 (CH3C=O), 19.2 (CH3C), 18.6 (C-6A), 18.0 (C-6c), 17.6 (G6H). FA.B-MS for C'o~H' 14N40a~ (1886) nt/z 1909 [M + Na]+.
Anat. Calcd. for CIO~W laNaOi~: C, 68.07, Fi, 6.09; N, Z.97. Found: contlent du CCL3CN

LMPPI2exp-brevet-&p 2-Aminoethyl a-L-Rharnnopyranosyl-(1-->2)-a,-L-rhamnopyranosyl-(I--~3)-[a-b-gtacopyranoeyl)-(1->4)~-a-L-rhamnopyranosyl-(1 >3)-2-acetamido-2-deny-[i-b-glucopyranoside (37). An ice cold solution of 95% aq TFA (I mL) in CHZCIz {9 mL) was added to the pentasaccharide 35 (645 mg, 0.34 mmol). The mixture was kept at 0°C for 10 min, then diluted with toluene and concentrated. Toluene was co-evaporated from the residue.
The residue was dissolved in MeOH (20 mL), and a iM solution of methanolic sodium methoxide (3.5 mL) was added. The mixture was stirred at 50°C for 18 h.
The mixture was neutralised with Amberlite IR-120 (I-l~ resin and filtered. The filtrate was concentrated. The mixture was purified by column chromatography (solvent A, 9:I) to give 36 (374 mg, 77%) as a colourless foam The crude pentasaccharide 36 (3G0 mg, 0.25 mrrwl) was dissolved in a mixture ofEtOH (IO mL) and EtOAc (1 mL). A IN solution of aq HCl (0.5 mL) was added.
The mixture was stinted under hydrogen in the presence of 10% PdIC (400 mg) for 18 h. The mixture was diluted with water and filtered. The filtrate was concentrated, then lyophilised.
The residue was dissolved in a solution of NaHC03 (75 mg) in water (1 mL) and purifed by passing fast through a column of Cte silica (eluting with water), then through a column of Sephadex Glo (eluting with water) to give, after lyophilisation, 37 ( 138 mg, 64%). HPLC (230 nor): Rt 5.87 min (Kromasil 5 pm C18 I00 A 4.6xZ50 mm analytical column, using a 0-20%
linear gradient over 20 min of CI-~CN in O,O1M aq TFA at I mL/min flow rate).
~H NMR
(Di0): S 5.15 (d, 1H, J,s = 3.7 Hz, H-IE), 5.00 (bs, 1H, H-1,~, 4.92 (d, 1H, J1,2 = I.1 Hz, H-1H), 4.7G (bs, IH, H-lc), 4.53 (d, 1H, Jl,z = 8.6 Hz, H-lp), 4.10 (m, 1H, H-5c), 4.03 (m, 2H, H-2,~, 2g), 4.01 (m, 3H, H-4A, 4H, CHZO), 3.83-3.88 (m, 7H, H-2c, 2D; 3,~, GaD, Gbn, 6aE:
CHZO), 3.69~3.76 (m, 7H, H-3a, 30, 3E, 40, 5,~, SB, 6ba), 3.52 (pt, 1H, H-3D), 3.33-3.54 (m, SH, H-2E, 4n, 4a, So. SE), 3.09 (m, 2H, CHZNHZ), 1.98 (s, 3H, CH3C=0), 1.28 (d, 3H, H-Gc), 1.22 (tn, 6H, H-6A, GB); 13C Iv'MR (Di0): ~ I75.3 (C=0), 103.4 (C-1H j, I O
I.9 (C-1 ~, 101.4 (C-lc, ID), 98.4 (C-lE), 82.3 (C-3D), 80.2 (C-2H), 79.9, 76.7 (C-2a), 72.9, 72.4, 72.4, 72.2, j 71.8, 71.6, 70.5, 70.4, 70.I, 70.0, 69.7, 69.6, 69.4, 68.7, 66.7 (???"?CHsO), 61.0 (2C, C-6o, 6E), 55.5 (C-2D), 39.9 (CHzNHz), 22.6 (GH~C=O), 18.2 (C-. Gc), 17.2 (C-6,~, I7.0 (C-GH). MS
for HRMS (MALDI) Calcd foz C34HHONzOz.3+H; 86S.36G5. Found: 865.3499.
Maleimido activated PADRE Lys (8).
Starting from 0.1 mm41 of Fmoc Pal Peg Ps resin, amino acids (0.4 mmol) were incorporated using HATU/DIEA (0.4 moral) activation. The N-terminal D-Ala was incorporated as Boe-D-Ala-OH. After completion of the chain elongation, the resin was treated three times with hydrazine nwnohydrate (2% solution in DMF, 2S mL/g of peptide resin) for 3min, which LMPPlZexp~brcvet-gp allowed the selective deblocking of the Dde protecting group. To a solution of maleimide butyric acid (183 mg, 1.0 mmol) in DCM (2 mL) was added DCC (103 mg, 0.5 mmol). After stirring for 10 min, the suspension was filtered, arid the filtrate was added to the drained peptide resin. DIEA (17 ~L, 0.5 mmol) was added. After 30 min, the peptide resin was washed with DMF (100 mL), MeOH (100 rnL), and dried under vacuum. After (95/2.5/2.5) cleavage (10 mL/g of resin, 1.5 h), the crude peptide (157 mg) was dissolved in 16 mL of 15% CH;CN in 0,08% aq TFA, and purified by reverse phase Medium Pressure Liquid Chromatography (MPLC) on a Nucleoprep 20 yn C18 100 ~ column, using a 15-75%
linear gradient of CH3CN in 0,08% aq TFA over 60 min at 25 mLlmin flow rate (2.14 nm detection) to give 8 (107 mg, 61%). HPLC (214 nm): Rt 13_4 min (94% pure, Nucleosil 5 um C18 300 A analytical column, using a 15-45% linear gradient over 20 min of CH3CN in 0,08% aq TFA at 25 mLlmin flow rate). Positive ion ESMS Calcd for Cs3H~39NZZ0,9:
1759.18. Found: 1758.83 (SD: 0.40).
(S-Acetylthio methyl)carbonylaminoethyl a-D-Glucopyranosy I-(1--~4)-a-L-rhamnopyranosyl-(1--~3)-2-acetsmido-2-deoxy-(i-D-glucopyranoside (38). The trisaccharide 18 (58 mg, 0.1 mmol) was dissolved in DMF (I mL). SAMA-Pfp (33 mg, 0.11 mmol) was added, and the mixture was left to stand at rt for 40 min. Toluene was added and the mixture was concentrated. Ether was added to the residue. The resulting precipitate was collected and purified by passing through a column. of Cts silica (solvent D, garadiont) to give 38 (36 mg, 53%). HPLC (230 nm): Rt 13.74 min (99% pure, Isromasil 5 pm C1$ 100 A
4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in 0,01M aq TFA at 1 mL/min flow rate). t3C NMR (Dz0): S 200.3 (SC=0), 175.2, 171.9 (NC=O), 102.1 (C-1~), 101.2 (C-ln), 100.5 (C-lE), 82.7 (C-3n), 81.8 (C-4~.), 76.8 (C-2F), 73.6 (C-3s), 72.6 (C-5E), 72.4 (C-4D), 71.8 (C-2c), 70.2 (C-4E), 69.7 (C-3c), 69.4 (C-SD), 68.9 (C-Sc), 68.9 (CHiO), 61.6 (C-6D), 60.9 (C-6fi), 56.1 (C-2D), 40.6 (CH2NH), 33.7 (CHZS), 30.4 (CH3C(O)S), 23.0 (CH3C(O)N), 17.5 (C-6c). ES-MS for C26H,~N2O17S (688) trtlz (M+H]+.
HRMS (MALDn Calcd for C~6H.,4N~01~5 +Na: 7I1.ZZ58. Found: XXXXX.
(S-Acetylthiomethyl)carbonytaminoethyl a-L-Rhamnopyraaosyl-(1-~3)-[a-D-glucopyra nosyl-(1-~4)]-ci-L-rhamno py raoosyl-( 1~3)-2-aceta mido-2-deoxy-[3-D-giueopyranoside (39). A solution of SAMA-Pfp (16.7 mg, 40 p.mo1) in acetonitrile (150 pL) was added to the tetrasaccharide 25 (20 mg, 28.8 umol) in O.1M phosphate buffer {pH 7.4, d00 pL). The mixture was stirred at n for 45 min and purified by RP-HPLC to give 39 (17 LMPPI2expbrcvet~~

mg, 74%). HPLC (230 run); Rt 13.63 min (98% pure, Kromasii 5 ~m C18 100 e~
4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M
aq TFA at 1 mL/min flow rate). 'H NMR (Da0): 8 5.10 (d, 1H, Ji,z --- 3.7 Hz, H-IE), 4.91 (d, IH, J~,Z =
0.8 Hz, H-Ie), 4.73 (bs, 1H, H-Ic), 4.45 (d, 1H. J1,2 = 8.5 Hz, H-lD), 4.09 (m, 1H, H-5c), 3.97 (m, 1H, H-2s), 3.87 (m, 4H, H-2c, 3c, GaD, CHzO), 3.67.-3.78 (m, 8H, H-2n, 3H, 4c, 5B, GbD, Gas, GbE, 1 x CHaO), 3.60 (m, 3H, H-3E, CHzS), 3,48 (pt, 1H, H-3D), 3.39-3.46 (m, GH, H.2E, 4s, 4D, 4E, 5n, 5E), 3.33 (m, 2H, CHzNH2), 2.35 (s, 3H, CH3C(O)S), I.98 (s, 3H, CH3C(O)N), 1.27 (d, 3H, H-Gc), 1.23 (d, 3H, H-6B): ~3C NMR (Dz0): ~S 199.8 (SC=O), 174.5, 171.3 (NC(O)), 103.2 (C-18), 101.4 (C-lc), 100.9 (C-lv), 98.6 (C-IE), 82,0 {C-3D), 79.0 (C-4B), 76.6 (C-4c), 76.3 (C-2E), 72.9 (C-3~), 72.3 (C-5E): 72.2 (C-4D), 71.8 {C-3c), 71.0 (C-2c), 70.5 (C-28, 38), 69.7 (C-4H), 69.5 (C-4E), 69.1 (C-5c, 5D), 68.8 (C-5g), 68.7 (CH?0), 61.1 (C-GD), 60.7 (C-6E), 55.5 (C-2D), 40.1 (CHlNH), 33.2 (CHzS), 29.9 (CH3C(O)S), 22.6 (CH3C(O)N), 17.9 (C-6c), 16.9 (C-da). MS for C3z~i54N20zrS (834) nrlz 857 [Ni + Na]+, HRMS-MALDI Calcd for C3aHsaNzOziS+Na: 857.?-838. Found: 857.2576.
(S-Acetylthiomethyl)carbonylaminoethyl a-L-Rhampopyranosyl-(1--~Z)-a-L-rhamnopyranosyl~(1-~3)-[c~A-glacopyranosyl)-(1~4)]-a-L-rhamnopyranosyl-(1 >3)-Z-acetamldo-Z-deoxy-[i-D-glueopyranoside (40). The pentasaccharide 37 (G.4 mg, 7.4 itmol) was dissolved in O.1M phosphate buffer (pH 7.4, I.0 mL). SAMA-Pfp (6.6 mg, 22 ~mol) was added, and the mixture was stirred at rt for 5 h. More SAMA-Pfp (G.6 mg, 22 pmol) was added end the mixture was stirred for I h more at rt. RP-HPLC of the mixture gave 40 (5.4 mg, 75%). HPLC (230 nm): Rt 13.86 min (100% pure, Kromasil 5 gm C18 100 ~
4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mL/min Ilow rate). ~H NMR (DZO): b 5,13 (d, 1H, J~,2 = 3.7 Hz, H-lE), 4.98 (bs, IH, H-I,~, 4.90 (bs, 1H, H-1B), 4.74 (bs, iH, H-lc), 4.47 (d, 1H, J,~ = 8.5 Hz, H-1D), 4.09 (m, 1H, H-5c), 4.00 (m, 2H, H-2A, 28), 3.79-3.85 (m, 8H, H-2c, 2p, 3a, 4A, 4B, 6aD, Gbn, CHzO), 3.65-3.74 (m, 9Fi, H-3g, 30, 3E, 40, 5A, 58, 6aE, 6bE, CH20), 3.60 (m, 2H, CHZS), 3.53 (pt, 1H, H-3D), 3.13-3.49 (m, 7H, H-2E, 4D, 4F, 5n, 5E, CHz~IH), 2.35 (s, 3H, CH3C=OS), 1.99. (s, 3H, CHIC=ON), 1.28 (d, 3H, H-Gc), I.20 (m, 6H, H-6,~" GB); 13C NMR (DzO): 8 199.9 (SC=O), 174.5, 171.4 (NC=O), 102.8 (C-1H), 101.7 (C-lA), 101.4 (C-1~), 100,9 (C-1D), 97.9 (C-lE), 82.0 (C-3D), 79.7 (C-28), 79.0, 76.3, 72.9, 72.4, ?2.2, 71.8, 71,0, 70.5, 69.7, 69.5, 69.1, 68.8, 68.5 (XXXX, CHaO), 61.1 (2C, C-6p, 6~, 60.7 (C-6E), 55.6 (C-2n), 40.1 (CHZNH), 33.2 LMPPlzexp-brcvct~gp CA 02434685 2003-07-04 (CHZS), 29.9 (CHIC=OS), 22.7 (CH3C=ON), 18.2 (C-Go), 17.2 (C-G~, 17.0 (C-GB).
HRIvIS
(MALDI) Calcd for C38H6aNZOa55+Na: 1003.3417. Found: 1003.3426.
PADRE (thiomethy~carbonylaminoethyl a-b-glucopyranosyl-(1-->4)-a-L-rhamnopyranosyl-(1-~3)-2-aeetamido-2~deozy-~-D-glucopyranoside (1). Compound (5.0 mg. 7.3 pmol) was dissolved in watez (500 p,L) and added to a solution of PADRE-Mal {10 mg, 5.68 ptnol) in a mixture of water (900 ItL), acetonitrile (100 pL) and O.1M phosphate buffer (pH 6.0, 1 mL). 117 ~tL of a solution of hydroxylamine hydrochloride (139 mg/mL) in O.1M phosphate buffer (pH 6.0) was added and the mixture was stirred for 1 h-RP-HPLC
purification gave the pure glycopeptide 1 ($.5 mg, 62%). HPLC (230 nm): Rt 10.40 min (100% pure, Kromasil 5 pm C18 100 A 4.6x250 mm anal~2ical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mL/min flow rate). ESMS
Calcd for C~ogHIg,NZ343ss: 2405.85. Found: 2405.52.
PADRE (thiomethyl)earbonylaminoethyl a-L-rhamnopyranosy!-(I-~3)-[a~D-glucopyranosy!)-(1~4)]-a-L-rhamnopyranosyl-(1 >3)-2-acetamido-2-dco~y-[3-n-glucopyranoside (2). Compound 39 (4.9 mg, 5.8 pmol) was dissolved in water (500 p.L) and added to a solution of PADRE-Mal (13 mg, 7.4 p,mol) in a mixture of water (1 mL), acetonitrile (200 ~L) and O.SM phosphate buffer (pH 5.7, 1.?_ mL). 117 pL of a solution of hydroxylaxnine hydrochloride (139 mg/mL) in O.SM phosphate buffer (pH 5.7) was added, and the mixture was stirred for 1 h. 1tP-HPLC purification gave the pure glycopeptude 2 (G.7 mg, 48%). HPLC (230 nm): Rt 11.60 min (100°fo pure, Kromasil 5 Itm C18 100 A 4.6x250 mm analytical column, using a 20-50% linear gradient over 20 min of CH3CN in 0,01M aq TFA at 1 mL/min flow rate). ESIvfS Calcd for C125H~gIN730395~ 2552. Found:
2551.90.
PADRE (thiomethyl)carbonylaminoethyl a-L-Rhamnopyranosyl-(1~2)-a-L-rhamnopyranosyl-(1-~3)-[oc-D-glucopyranosyl)-(1-~4)]-a-L-rharnnopyranosyl-(1-~3)-2-acetamido-2-deoxy-[i-D-glucopyranoside (3). Compound 40 (5.59 mg, 5.7 pmol) was dissolved in water (500 uL) and added to a solution of PADRE-Mal (12.6 mg. 7.2 umol) in a mixture of water (1 mL), acetonitrile (200 ~,L), which had been previously diluted with 0.5M
phosphate buffer (pH 5.7, 1.2 mL). A solution of hydroxylamine hydrochloride (139 mg/mL) in 0.5M phosphate buffer (pH 5.7,117 p.L) was added and the mixture was stirred for 1 h. RP-HPLC purification gave the pure glycopeptide 3 (7.1 mg, 4G%). HPLC (230 nm):
Rt 10.33 LMPPI2exp-brevet-gp CA °2434685 2003-07-04 min (100% pare, Kromasil 5 ltm C18 100 A 4.Gx250 mm analytical column, using a 20-50%
linear gradient over 20 min of CH3CN in O.O1M aq TFA at 1 mL/min flow rate).
ESMS Calcd for CiZiHzuiN~30a3S: 2698. Found: 2698.09.

LMPPI2.sehema6revet-gp CA 02434685 2003-07-04 T epilope r PADRE-Lys 8 ep'rtope r aKXVAAWTLKAAaZ-rvN
OH CONHz O
HOO O p H b~p~ ,~,S I p NNAc NH 1N.~--NH R
Ma p 1 o H
R O 2 a-L-Rha a-L-Rha-(1..~ 2)-a-L-Rha DAn s~gdrto~ OTCA
Bn'oOMe O p O
RO aZ + HO~O~N~
NNAc Bn0 MA O + ~.~"NH
R BZ Ac0 MA O BnO~ O
Ac0 p~ O
B20 Me O PqORE.L
B
08z LMPP1Z-s<hertu-brtva-gp /OAc OR°
ACQ 0 R°0 ~ 0 ~c0~ R30 ' y O~~N9 . R . ._R° Rc NHAc t2 ~ ~ 13 ac Ac ac ~ 14 ~ H ~ H H
OAII ~.OBn ~OBn v 7 H IPr HO MATO a BflO ~1 ~ QTCA B p~ OR' BnO~ Bn,O I0 Me To \0 Bn0 a 1 I All H cc 4 , TC~1I Bz alp -~r~ OH OOH
08n a BnO~ R p~Q~./~Na Q Ho~ H O~O.~-wIJH
AnO~ NHAC H~-~~ NHac Bn0 p Me , O HO O Me Ro OR ~ R R4 RB H0~ off p ~ 1~5 ~ Bz . - ~iPr ~ 18 16 Bz H H

LMPPI2.uheme-6revctgp CA 02434685 2003-07-04 oTCA Ogn 06n Ac0 M-.~/Bn0 p B8 D ~ OAII AcO~ pAC 6n0 en0 Me~p DR
eno Me Q 20 O
HO OR ~ Ac0 ~'le O 08z b 21 All ° Aco c ~ x2 H
OAc 5 TCA ~ a/j3 h ~ 1t H
19 Bz r w3 R Rz Ra R~
Ac 8z iPr 24 Ac ~ 8z ~ H H

~''~NHZ

LMPPIZ-~oherr~~b~evet.gp OH OH O
OH ~~-Q ~ ~' ~
HHp O H 0~~~NHz HO ~0 O HO-0~ ,~.,,SAc HO ~~NH'AC NH
HO Mo O
Ho Me O
RO
OH
R R
1& H 38 H
25 a.-L~Rha 39 u-L-Rhe 37 ot-L-Rha-('~~.,2)~a-t.-Rha do o.-~-Rh2-(1-.-2)-o.-t_-Rha OH O O
OH HO O
H ~ p NHA ~NH~,r N~ NH
r~0 O Mo ~ f1 I .MJ~. ~ H RO OH 0 PADRE-Ly9 PADRE-Cy4 i H
2 ~ a-l-Rha 3 a-~-Rha-(1...,.2)-a~~.Rha pADfiE-~ys-NHt Sold phase peptide synthexis (Fmoc chemiBetty) Fmoc PsI Pog P9 te6sn LM_ PP13 Synthesis of a pentasaccharide building black of the O-specific polysaccharide of SlrigellaJlexneri serotype 2a.
This paper disclosed the preparation of oligo- or polysaccharides made of two repeating units.
The inventors reasoned that it would best rely on the use of a pre-functionalized building block, representative of the repeating unit of the 0-Ag, or of a frame-shifred sequence thereof, and susceptible to act either as a donor and potential accEptor, or as an acceptor and potential donor. The synthesis of such a key synthetic intermediate is described, together with its conversion in the form of either a donor or an acceptor.

LMPP 13 ~thco~brevet-peritablock Synthesis of a pentasaecharide building block of tl~e 0-specific po>;ysaecharide of ShigellaJhxneri serotypc 2a~~~
Abstract INTRODUCTION
Shigellosis or bacillary dysentery is a sexious infectious disease, responsible for some 200 million episodes annually, mostly in children and immunocompromised individuals living int areas were sanitary coruiitions are insufficient. ~Z~ Of the four species of Shigellae, Shigella ffexneri is the major responsible of the endemic form of the.
disease, with serotype 2a being the most prevalent, Due to increasing resistance of all groups of Shigellae to antibiotics, X31 the development of a vaccine against shigellosis is of high priority as stated by the World Health Organization in its program against enteric diseases. ~4t However, there are yet no licensed vaccines for shigellosis, Shigella's Iipopolysaccharide ()rPS) is a major surface antigen of the bacterium. The corresponding 0-antigen (O-Ag) is both an essential virulence. factor and the target of the infected host's protective immune response. ~5' 6~ Based on the former hypothesis that serum 1gG anti-LPS antibodies may confer specific protection against shigellosis, ~~
several polysaccharide-proteine conjugates, targeting either $higella sonnei, Slaigella dysenteriae 1 or S flexneri serotype 2a, were evaluated in humans. ~g~ 9~ Tn the case of S.
sonnei, recent field trials allowed Robbins and co-workers to demonstrate the efficacy of a vaccine made of the corresponding detoxified LPS covalently linked to recombinant exoprotein A.
~y°t Even though efficient, polysaccharide-protein conjugate vaccines remain highly complex structures, whose immunogenicity depends on several parameters amongst which the length and nature LMPP13-then-6ievet-pcntablock of the saccharide component as well as its loading on the protein- It is reasonably admitted that the standardization of these parameters is somewhat difficult when dealing with polysaccharides purified from bacterial cell cultures. That short oligosaccharides were immunogetiic when conjugated onto a protein carrier was demonstrated on several occasions.
~~ ~~ It may be assumed that the use of well-defined synthetic oligosaccharides would allow a better control, and consequently the optimisation, of the above mentioned parameters. Indeed, available data on S. dysenteriae type 1 indicate that neoglycoconjugates incorporating di-, tri-or tetramers of the O-Ag repeating writ were more immunvgenic than a detoxified I,PS-human serum albumin conjugate of reference. t~~~ Others have shown that conjugates incorporating oligosaccharides comprising one repeating unit or smaller fragments were immunogenic in mice. U3, ia~
Along this line, we recently prepared three neoglycoproteins as potential semi-synthetic vaccines against Shigella Jlexneri 2a infection. These incorporated short oligosaccharide haptens, representative either of part or of the whole repeating unit of the 0-Ag of S ./lexneri serotype 2a. Preliminary data indicate that two out ofthe three conjugates are immunogenic in mice.(Phalipon et al, unpublished results) However, parallel studies on the recognition of synthetic fragments of the O-Ag by protective homologous monoclonal antibodies suggested that sequences comprising more than one repeating unit of the O-Ag were more antigenic, thus probably better mimicking the natural polysaccharide. t"~ It is anticipated that better mimics of the O-SP would lead to conjugates of higher immunogenicity. Thus, the preparation of oligo~ or polysaccharidest'61 made of two repeating units or more was considered. We reasoned that it would best rely on the use of a pre-functionalized building block, representative of the repeating unit of the 0-Ag, or of a frame~shifted sequence thereof, and susceptible to act either as a donor and potential acceptor, or as an acceptor and potential donor. The synthesis of such a key synthetic intermediate is described in the following, together with its conversion in the form of either a donor or an acceptor.
RESULTS AND DISCUSSION
A B E C D
2)-a-L-Rhap-(1->2)-a-L-Rh~-(1~3)-[a-D-Glcp-(1 >4)]-a-L-Rhep-(1--~3)-[3-D-GIcNAcp(1~
I

LMPP13-then-brovet.pcntablock The O-SP of S flexneri 2a is a branched heteropolysaccharide defined by the pentasaccharide repeating unit I. tl~~ tsl It features a linear tetrasaccharide backbone, which is common to all S.
flexneri 0~antigens and comprises a N acetyl glucosamine (D) and three rhamnose residues (A, B, C). The specificity of the serotype is associated to the a-D-glucopyranose residue linked to position 4 of rhamnose C.
As part of a study of the mapping at the molecular level of the binding of protective monoclonal antibodies to S. fTexrleri 2a 0-antigen, a set of of di- to pentasaccharides corresponding to frame-shifted fragments of the repeating unit h ti9-zz) an octasaccharide~31 and more recently a deeasaccharide~~°1 have been synthesized in this laboratory. The latter, namely D'A'S'(E')C'DAB(E)C, was synthesized as its methyl glycoside by condensing a chain terminator pentasaccharide donor and a methyl glycoside pentasaccharide acceptor. In the following, the key intermediate is the DAH(E)C pentasaccharide 1, which is protected in an orthogonal fashion at position 0-3o with an acetyl group and at the reducing end by an allyl group. At this stage, the acetamido function is already present at position 2p. Compound 1 may be converted to the corresponding alcohol 2, which acts as an acceptor and a masked donor, or to the trichloroacetimidate 3 which acts as an acceptor allowing subsequent chain elongation at the non-reducing end (Scheme 1 ). Previous work in the laboratory has shown that in order to construct the DAB(E)C sequence, the linear approach involving stepwise elongation at the non-reducing end, was more suitable than the blockwise one.
D-glucosamine uhit(D). In order w limit the number of steps at the pentasaccharide level, we reasoned that an appropriate precursor to residue D should have (i) permanent protecting groups at positions 4 and 6, (ii) a participating group at position 2 and (iii) an orthogonal protecting group at position 3, allowing easy cleavage. As they allow a wide range of protecting group manipulations previously to ultimate activation, thioglycosides are highly convenient masked donors. Recently, two sets of non-malodorous thioglycosyl donors have been proposed~ZS~Ref??, among which the thiododecanyl moiety was selected.
Thus, the known peracetylated trichloroacetamide XX~~6~ was reacted with dodecanthiol in the presence of BF3.OEt2 to give thioglycoside XX in high yield (97%). Zemplen deacetylation cleanly afforded the corresponding triol XX, which was selectively protected at position 4 and 6 upon reaction with 2,2-dimethaxypropane (8~% from XX). Indeed, previous observations in the series have demonstrated that 4.6~O-isopropylidene-D-glucosaminyl derivatives were highly LMPP13-rheo-b~evct~pentablock suitable precursors to residue D, t19, z3~ Next, conventional acetylation of XX gave the required donor thioglycoside XX.
G-Rhamnose units (A, B): Previous work in the series was mostly based on the use of the 2-O-acetyl trichloroacetimidate rhamrtopyranosyl donor XX. ~°. 24~
Condensation yields were excellem. However, the acetyl protecting group not being fully orthogonal to the benzoyl one, the weak point of the strategy resides in the de-O-acetylation step which, in fact, is required twice. The levulinate on the contrary is fully orthogonal to either beniyl or allyl ethers, and to benzoates. The 2~O-levulinoyl trichloroacetimidate donor XX was thus evaluated as an alternative to XX. It was prepared from the known allyl rhamnopyranoside XXtZ'~ in three steps. Indeed, treatment of XX with levulinic acid gave the fully protected XX
(XX%, ALGlGL), deallylation of which proceeded in two steps based on (i) isomerisation of the ailyl group into the propen-1-yI ether using an iridium complex, lasl and (2) subsequent oxidative cleavage of the latter to give the hemiacetal XX {XX%, ALGlGL). tz91 Reaction of the latter with trichloroacetonitrile un the presence of 1,8-diazabicyclo[5.4.OJundec-7-ene (DBU) resulted in the required donor XX (XX%, L.A GIGL). OnE should note that several routes to the known XX have been described including opening of the intermediate 2,3-O-benzylidene derivativet~'lor regioselective benzylation of the corresponding 2,3-diol via the stannylidene intermediate.(ref~) Alternatively, XX could be prepared from the orthoester XX, readily available from acetobromorhamnose XX upon reaction with allylic alcohol in the presence of lutidine (XX% from L-rhamnose, lI~IP et ???). Deacetylation of XX in methanolic ammoniac gave diol XX, which was next benzylated into the 1,2-orthoacetate XX (XX% from XX~P et ???). Isomerisation of the latter to the corresponding glycoside in the presence of TVfSOT~
analogously to that described in the mannose series, t3°' a O gave the firlly protected XX (XX%, GL, ~ together with the p-anomer XX (XX%, GL. MP). Zemplen deacctylation of the former gave XX quantitatively. Besides, XX is a convenient intermediate to the acetylated donor XX.
Synthesis of the pentasaccharide 1: The known allyl glycoside XX, acting as an EC acceptor, temporarily protected at the anomeric position and having a participating group at position Z.c, was prepared as described in 63% yield from allyl 2,3-0-ispropylidene-a-L-rhamnopyranoside. tZll Its condensation with the trichloroacetimidate donor XX, performed in the presence of a catalytic amount of TMSOT'~ afforded the fully protected trisaccharide XX
(XX%, ALG reproduire), and subsequently the knoum H(E)C acceptor XX~Z~1 upon selective LMPP13-theo~brcvet~p~tablock removal of the O-levulinoyl group v4~ith hydrazine hydrate (XX%, ALG
reproduire). Starting from XX, this two-step process was repeated to give first the fully protectEd XX (XX%), then the known AS(E)C acceptor XX~2'~~ in XX% yield. According to this strategy, XX
was obtained in XX% overall yield from the key disaccharide XX, which compares favourably with the 6Z% yield obtained in the previously described strategy involving the 2-O-acetylated trichloroacetimidate donor XX. ~z"~ Besides, considering that selective deblocking at positions 2B and ZA was completed in overnight runs instead of the 5 days required for each corresponding chemoseleetive O-deacetylation steps, the use of the 2-O-levulinoyl donor appeared as a suitable alternative to that of XX, although its preparation, may be somewhat lower-yielding (XX'~ instead of XX% from XX, ALG/GL). Using a mixture of NIS
and triflic acid as the promoter, condensation of the tetrasaeeharide acceptor XX with the thioglycoside donor XX gave the key intermediate XX in 58% yield. Although alternative conditions in terms of promoters and solvents (not described) were tested, this rather low yield could not be improved. Radical dechlorination of XX using Bu3SnH and a catalytic amount of AIBN
readily afforded the corresponding acetamido key intermediate 1 (74%).
(attention schema On one hand, compound 1 may be efficiently converted to the acceptor building block 2 under Zempl~n conditions. On the other hand, it was smoothly deallylated into the hemiacetal XX, following a two-step process as described above. Next, treatment of XX with trichloroacetonitrile and DBU allowed its conversion to the building block 3 (82% from XX).
ACKNO WL.EDGEMENTS
The authors ate grateful to J. Ughetto-Monfrin (Unity de Chimie Organique, Institut Pasteur) for recording all the NMR spectra. The authors thank the Bourses Mrs Frank Howard Foundation for the postdoctoral fellowship awarded to K. W., and the Institut Pasteur for its financial support (grant no. PTR 99).
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LNlPP l3-e~ep-brevet~pentablock General methods Optical rotations were measured for CHC13 solutions at 25°C, expect where indicated otherwise, with a Perkin-Eliner automatic polarimeter, Model 241 MC. TLC were performed on precoated slides of Silica Crel GO Fz<,~ (Merck). Detection was effected when applicable, with UV light, and/or by charring in 5% sulfuric acid in ethanol.
Preparative chromatography was performed by elution from columns of Silica Gel 60 (particle size 0.040-0.063 mm). For all compounds the NMR spectra were recorded at 2~°C for solutions in CDCl3, on a Broker AM 400 spectometer (400 MHz for 'H, 100 MHz for ''C).
External refotences : for solutions in CDCl3, TMS (0.00 ppm for both'H and '3C). Proton-signal assignements were mane by first-order analysis of the spectra, as well as analysis of 2D
'H-'H correlation maps (COSY) and selective TOCSY experiments. Of the two magnetically non-equivalent geminal protons at C-6, the one resonating at lower field is denoted H-6a and the one at higher field is denoted H-Gb. The '3C N'vIR assignments were supported by 2D "C-'H correlations maps (HETCOR). Tnterchangeable assignments are marked with an asterisk in the listing of signal assignments. Sugar residues in oligosaccharides are serially lettered according to the lettering of the repeating unit of the 0-SP and identified by a subscript in the listing of signal assignments. Fast atom bombardment mass spectra (FAB-MS) were recorded in the positive-ion mode using dithioery~thridol/dithio-L-thrEitol (4 :1, MB) as the matrix, in the presence of NaI, anal Xenon as the gas, Anhydrous DCM, 1,2-DCE and Et20, sold on molecular sieves were used as such. 4 ~ powder molecular sieves vvas kept at 100°C and activated before use by pumping under heating at 250°C.
Dodecyl 3,4,6-tri-O-acetyl-2-dcoxy-1-thio-2-trichloroacetamido-[i-D-glucopyranoside (5).
A mixture of the peracetylated 4 (6.2 g, 12.5 pmol) and dodeeanthiol (2.5 mL, 94 Nmol), 4A
molecular sieves and dry 1,2-DCE (90 mL) was stirred for 1 h then cooled to 0°C. BF3.Et~0 (1.57 mI,, 12.5 ~mol) was added. The stirred mixture was allowed to reach rt in 2h30. Et3N
was added until neutral pH and the mixture filtered. After evaporation, the residue was eluted from a column of silina gel with 2:1 cyclohexane-EtOAc to give 5 as a white solid (7.5 g, 93 %); [a]p -20° (c l, CHCIs). 'H NMR (CDC)J);8 G.82 (d, 1H, ,IZ,Nh = 9.2 Hz, NH), 5.31 (dd, LMPP13~dcPbrcvet-pentablock 1H, JZ,3 = 9.9 Hz, J3,a ' 9.6 Hz, H-3), 5.15 (dd, 1H, J,,,s = 9.6 Hz, H-4), 4.68 (d, 1H, JI,Z =
10.3 Hz, H-1), 4.28 (dd, 1H, Js,So = S.0 Hz, Jg~fib = 12.3 Hz, H-Ga), 4.17 (dd, 1H, JS,eb = 2.3 Hz, H-6b), 4.11 (dd, 1H, H-2), 3.75 (m, 1H, H-5), 2.70 (m, 2H, SCHZ(CH~)loCH3). 2.10, 2.05, 2.04 (3s, 9H, OAc), 1.G5-1.20 (m, 20H, SCHZ(CH~),oCH3), 0.90 (t, 3H, SCHz(CH~),oCH3). '3C NMR (CDCl3):o 171.0, 170.7, 169.3 (C=0), 161.9 (C=OCC1,), 92.3 (CC13), 84.2 (C-1), 76.5 (C-5), 73.4 (C-3), 68.6 (C-4j, G2.6 (C-6), 55,2 (C-2), 32.3, 30.6, 30.0-29.1, 14.5 (S(CHz)iICH,), 21.1, 21.0, 20.9 (OAc). FABMS of Cz6H4~C13NOeS
(M, 635.0), m/z 658.1 [vI+Na]''. Anal. Calcd for Cz~H4zC13NOeS, C: 49.17, H: 6.67, N: 2.21.
Found C: 49.16, H: 6.71, N: 2.13.
Dodecyl 2-deoxy-4,6-O-isopropylidene-1-thio-2-trichloroacetamido-~-D-glucopyranoside (7?.
A mixture of 5 (5 g, 7.87 mmol) in MeOH (15 mL) was deacetylazed by MeONa overnight.
The solution was neutralized by 1R 120 (H+) and Filtrated. After concentration in ~~acuo, the residue 6 was treated by 2,2-dimethoxypropane (70 mL, 546 mtnol) and APTS (148 mg, 0.94 mmol) in DMF (20 mL). After stirring overnight, the mixture was neutralized with Et;N and concentrated. The residue was eluted from a column of silica gel with 3:1 cyclohexane-EtOAc to give 7 as a white solid (3.45 g, 80 %); [a]p -35° (c 1, CHC)r).
1H NMR (CDCL):8 6.92 (d, 1H, .I,,~ = 8.0 Hz, NH), 4.77 (d, 1H, J,,2 = 10.4 Hz, H-1), 3.98 (m 1H, Ja,3 = Ja.a = 9.2 Hz, H-3), 3.88 (dd, 1H, Js,c,q = 5.4 Hz, J~,,Eh =
10.8 Hz, H-6aj, 3.70 (dd, 1H, J5.6b = 0.5 Hz, H-db), 3.63 (tn, 1H, H-2), 3.53 (dd, 1H, J~,S = 9.2 Hz, H-4), 3.29 (m, 1H, H-5). 2.98 {s, 1H, OH), 2.G0 (m, 2H, SCH~(CHi),oCH3), 1.60-1.10 (m, 20H, SCHa(CHi)loCHl), 1.45, 1.35 (2 s, 6H, C(CH3)Z), 0.80 (t, 3H, SCH2(CHZ)loCHa).
s3C NMR
(CDC13):8 IG2,5 (C=OCC13), 100.3 (C(CH3)i), 92.8 (CC13), 84.0 (C-1), 74.6 (C-4), 72.3 (C-3), 71.7 (C-5), 62.2 (C-G), 58.3 (C-2), 29.3, 19.5 (C(CH3)z), 32.3, 30.8, 30.1-29.5, 29.1, 14.5 ?-LMPPI3~cpbrcvet-pent~block (SCH,z(CHs),oCHs). FABMS Of Cz3HtoC13NO5S (M, 548.9), m/z 5'72_2 [~I+NaJ'.
Anah Calcd for CZ,H4oCl~NOsS, C: 50.32, H: 7.34, N: 2.55. Found C. 50.30, H: 7.40, N:
2.36.
Dodeeyl 3-D-acetyl-2-deoay-4,6-O-isopropylidene-1-thio-2-trichlotroacetamido-[i-D-glucopyranoside (8).
A mixture of 7 (I.07 g, 1.94 mmol) in pyridine (10 mL) was cooled to 0°C. Ac~O (5 mL) was added and the solution was allowed to reach rt in 2 h. The mixture was then concentrated and the pyridine coevaporated with toluene. The residue was eluted from a column of silica gel with 6:1 cyclohexane-EtOAc with 0.2% of Et3N to give 8 as a white solid (1.12 g, 97 %), [a]D

-62° (c 1, CHCIa) 'H NMR (CDC13):b 7.51 (d, IH, Jz,~c = 9.7 Hz, NH); 5.40 (dd, 1H, Jz,3 = J3.< =
I0.0 Hz, H-3), 4.62 (d, 1H, Ji,z = 10.4 Hz, H-1), 4.20 (m, 1H, H-2), 4.01 (dd, 1H, ,Isss =
5.2 Hz, J6a,6b = 10.7 Hz, H-6a), 3.84 (dd, 1 H, J,,s = 9.7 Hz, H-4), 3.70 (m, 2H, H-5, H-6b), 2.68 (m, 2H, SCHz(Cl~),oCH3), 2.09 (s, 3H, OAc), 1.60-1.20 (m. 20H, SCHz(CHz),oCHa). 1.52, 1.38 (2 s, 6H, C(CH3)2), 0.90 (t, 3H, SCH2(CHz)~oCH,). ~3C NMR (CDCl3):8 171.4 (C=OCH3), 161.8 (C=OCCl3), 99.5 (C(CH,)z), 92.3 (CCl3), 84.6 (C-1), 73.6 (C-3), 72.0 (C-4), 71.9 (C-5), 42.2 (C-6), 55.0 (C-2), 29.1, 19.3 (C(CH3)z). 32.3, 30.7, 30.0-29.0, 14.5 (SCHz(CHz),oCH3).
FABMS of CzSH,,iCI31~065 (M, 591.0), m/z G14.1 [M+Na]+. Anal. Calcd for CzsH4zC13N06S, C: 50.80, H: 7.16, N: 2.37. Found C: 50.67, H: 7.32, N: 2.24.
3,4-Di-O-acetyl-1,Z-O-allyloxyethylidcne-(i-L-r6amnopyranose (12). A mixture of L-rhamnose mnnohydrate (50 g, 274 mmol) in pyridine (4I0 mL) was cooled to 0°C. AczO (I70 mL) was added and the solution was allowed to reach rt overni.ght. MeOH (100 mL) was added and the solution concentrated. The resulting suspension was taken up in DCM, washed with water, satd aq NaHC03, water, and satd aq NaCI, successively. The organic layer was LMPP13-cx~brovet.pentabloclc dried and concentrated to give the crude peracetylated rhamnose (quart,) as a slightly yellow oil. A solution of latter (21.1 S g, 63.7 mmol) in acetic acid (38 mL) and acetic anhydride (6.7 mL) was treated by a 33% solution of HBr in AcOH (8G mL), then stirred for 15 h at rt. The mixture was concentrated by repeated coevaporation with cyclohexane. The resulting suspension was taken up in DCM, washed with satd aq NaHC03 and water. The orgatuc layer was dried and concentrated to give 11 (quart.) as a brown oil. A solution of the crude il (22.29 g) in anhydrous 2,6-lutidine (37 mL) was treated by AllOH (9.6 mL, 142 mmol) at rt.
The solution was stirred overnight, then filtered and the solids were washed W
th EtOAc. The liquid layer was concentrated anri the residuE was taken up in DCM, washed with 1M HCl cold solution, water and satd aq NaCI. The organic layer was dried and concentrated by coevaporation with toluene. Chromatography of the crude residue (toluene:acetone, 49:1 containing 0.1% Et3N) gave orthoester 12 (18.5 g, 88%) as a slightly yellow oil which crystallized on standing. An analytical sample was recristallized from isopropyl ether:petroleum ether; mp XX°C, [a)o -XX° (c 1, CHCl3); 1H N1~IR (CDC13):8 5.88 (m, 1H, AIl), 5.42 (d, 1H, J,,Z = 2.3 Hz, H-1), 5.25-5.40 (m, 2H, All), 5.10 (dd, 1H, J 2,3 = 3.3 Hz, H-3), 5.05 (dd, 1H, J4,s = 6.3 Hz, H-4), 4.60 (dd, 1H, H-2), 4.05 (m, 2H, All), 3.50 (qd, 1H, J5,6 =- 6.2 Hz, H-5), 2.12, 2.OG (2s, 6H, OAc), 1.7G (s, 3H, CHs), 1.23 (d, 3H, H-G); '3C NMR
(CDCI;): 8171.4 (C=OCHj), 1 bl. 8 (C=OCCI3), 99. S (C(CH;)a), 92.3 (CCI3), 84. 6 (C-1), 73. 6 (C-3), 72. 0 (C-4), 71.9 (GS), 62.2 (C G), 55.0 (G2), 29.1, 19.3 (C(CH3)~, 31.3, 30.7, 30.0-29.0, 14.5 (SCHi(CH~~oCH,~. FABMS of C2sH~ZCl3NOrS' (A~ 591.0) tnlz 614.1 (M+NaJ'. Anal.
Calcd for C~sH4aCISN06S, C: 50.80, H.' 7.16, N.' 2.37. Found C.' SO. G7, H: 7.32, N.' 2.24.
3,4-Di-0-benzyl-1,2-0-altyloxyethylidene-p-L-rhamnopyranose (14). A solution of the crude peraeetylated rhamnose (9.0 g, 27 mmol) was processed as described for the preparation i of 12. A solution of the crude lZ thus obtained in MeOH (GS mL) was cooled to 0°C and LMPPI3.exp-brovn-pent3block ~ 02434685 2003-07-04 treated with NI-13 until saturation. The solution was stirred for 6 h at rt, then concentrated by eo-evaporation with toluene to give 13. Column chromatography (DCM:M~eOH, 49:1) gave pure 13 as a white solid. 'H NMR (CDC13):S 5.75 (m, 1H, All), 5.22 (d, 1H, H-1), 5.00-5.10 (m, 2H, All), 4.60 (dd, 1H, H-2), 4.30 (d, 1H, H-3), 3,80 (m, 2H, All), 3.50 (m, 1H, H-5), 3.20 (t, 1H, H-4), 1.80 (s, 3H, CH3), 1,20 (d, 3H, J5,6 = 6.2 Hz, H-6).
A solution of crude 13 in anhydrous DMF {90 mL) was cooled to 0°C. NaH
(4.32 g, 108 rnrrwl) was added in 30 min then BnBr {8.5 mL, 71 mmol) was added dropwise at 0°C. The solution was stirred overnight at rt, then MeOH (20 mL) was added dropwise at 0°C. The solution was allowed to reach rt in 2 h, then concentrated. The residue was taken up in DCM, washed with satd aq NaHCO, until neutral pH, water and satd aq NaCI. The organic layer was dried and concentrated, After evaporation, the residue was eluted from a column of silica geI
with 9:1 cycIohexane-EtOAc and 0.2 % of Et3N to give I4 as a white solid (8 g, 70%).
Crystallization of an analytical sample from isopropyl ether:petroleum ether gave 13 as white crystals; mp XX°C, [a]p XX° (c I, CHC13);'H NMR (CDC~):S 7.35 (m, IOH, Ph), 5.90 (m, 1H, All), 5.30 (d, 1H, Jr,2 = 2.2 Hz, H-1), 5.28-5.43 (m, 2H, All), 4.95-4.65 (m, 4H, CH~Ph), 4.40 (dd, 1 H, Jz a = 4.0 Hz, H-2), 4.10 {m, 2H, All), 3.70 (d, 1 H, J;,d =
9.0 Hz, H-3), 3.50 (t, 1H, J,,s = 9.0 Hz, H-4), 3.35 (m, 1H, Js,6 = 6.2 Hi, H-S), 1.77 (s, 3H, CHI), 1.33 (d, 3H, H-6); ''C NMR (CDCl3): ~S 171.4 (C=OCH~, 161.8 (C=OCCl3), 99. S (C(CH;j~, 92.3 (CCI~, 84.6 (C-1), 73.6 (C-3), 72.0 (C-4), 71.9 (C-S), 62.2 (C-6), SS.O (C-2), 29.1, 19.3 (C(CH3)~, 32.3, 30.7, 30.0-29,0, 14.5 (SCH~(CH~taCH3). FABMS of CiJH.,IChNOsS (M, 591.0) mlz 614.1 jM+NaJ~. Anal. Calcd for C~sH41C13N06S, C.' 50.80, H.~ 7.16, N: 2.37.
FouNd C:
50.67, H.' i.32, N.' 2.24 Altyl 2-O-acetyl 3,4-Di-0-benzyl-~i-L-rhamnopyranoside (15). A mixture of the ort)zoester LMPP13-cxp-b~avct-pentabiock H-5), 3.43 (pt, 1H, H-4), 2.80 (m, 4H, levy. 2.19 (s, 3H, Ac), 1.37 (d, 3H, H-G).i3C NMR
(CDCI3): b 124.0-125.1 (Ph), 1 I 8.0 (All ), 97.0 (C-1), 80.2 (C-4), 78.5 (C-3), 75.2 (CHZPh), 72:0 (CHZPh), 70.2 (C-2), 68.5 (All ), G8.3 (C-5), 38.5 (Lev), 31.5 (Ae), 28,5 (lev), 20.1 (C~
G).
3,d-Di-O-benzyt-2-0-Icvulinoyl-a-L-rhamnopyranose (18). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium, hexafluorophosphate (25 mg, 20 p,mol) was dissolved THF (? mL), and the resulting red solution was degassed in an argon stream.
Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream A solution of 17 (1.4 g, 3.12 mmol) in tetrshydrofuran (? mL) was degassed and added. The mixture was stirred at rt overnnght, then concentrated to dryness. The residue was dissolved in a solution of Iz (1.37 g, 5.4 mmol) in 30 mL of THF/Hz0 (15;4). The mixture was stirred at rt for 1 h and THF .vas evaporated.
The resulting suspension was taken up in DCM, washed twice tenth water, said aq NaHS03, water, satd aq NaHC03, water and said aq NaCI, successively. The organic layer was dried and concentrated.
The residua was eluted from a column of silica gel with 7:3 to 6:4 cyclohexane-EtOAc to give the corresponding hemiacetal 18 (1.3 g, 93 %). 'H NMR (CDC13): 'H b 7.3-7.4 (m, IOH, Ph), 5.40 (dq, 1H, J,,z = 1.8, Jz,3 = 3.4 Hz, H-2 ), 4.93 (d, 1H, CHzPh), 4.78 (d, 1H, J,,z = 1.6 Hz, H-1), 4.78 (d, 1H, CHZPh), 4.G3 (d, 1H, CHZPh), 4.51 (d, IH, CHzPh), 3.99 (m, IH, .I3_4 = 9.5 Hz, H-3), 3.78 (dq, 1H, Ja,s = 9.5, Js.s = G.2 Hz, H-5), 3.43 (pt, 1H, H-4), 2.80 (m, 4H, lev ), Z.I9 (s, 3H, Ac), I.37 (d, 3H, H-6).
3,4-Di-O-benzyt-2-O-ievutinoyl-a-L-rhamnopyranosyl trichloroaeetimidate (19).
Trichloroacetonitrile (1.3 mL. 13 mmol) and DBU (51 pL, 0.3 mrrwl) wire added to a solution ofthe residue I8 (1.0 g, 2.3 rrunol) in anhydrous DCM (G mL) at 0°C.
After 2 h, the mixture LhIpPI 3eapbrevct-pcntablock was concentrated. The residue was eluted from a column of silica gel W th 3:1 eyelohexane-EtOAc and 0.2 % Bt3N to give I9 as a white foam (1.0 g, 95 %); [a]D XX°
(c I, CHCl3). 'H
NMR (CDC.I~): 'H s 8.G7 (s, 1H, NH ). 7.3-7.4 (m, IOH, Ph), 6.19 (d, 1H, Jt.2 = 1.9 Hz, H-1), 5.48 (dd,, lH, J,,i = 2.0, Jz,3 = 3.3 Hz, H-2 ), 4.95 (d, 1H, CHaPh), 4.73 (d, 1H, CHaPh), 4.66 (d, 1H, CHZPh), 4.58 (d; 1H, CHzPh), 4.51 (d, 1H, CHzPh), 4.00 (dd, 1H, J3,a =
9.5 Hz, H-3), 3.95 (dq, 1H, J4,s = 9.G, Js,b -- 6.3 Hz, H-5), 3.52 (pt, 1H, H-4), 2.80 (m, 4H, levy, 2.20 (s, 3H, Ac), 1.36 (d, 3H, H-6).
Altyl (2-O-levulinoyl-3,4-di-O-beniyl-a-z-rhamttopyranosyl)-(1-~3)-[2,3,4,6-tetra-0-benryl-a-D-glucopyranosyl-(1 >4)]-2-O-benzoyl-a-trrhamnopyranoside (22j. A
mixture of alcohol 21 (300 mg, 0.3G mmol) and imidate 19 (320 mg, 0.54 mmol) in anhydrous EtzO
(20 mh) was stirred for 15 min under dry Ar. A$er cooling at -75°C, Me3Si0Tf (13 p,L, 70 pmoi) was added dropwise and the mixture was stirred 3 h. Trietlrylamine (G0 11L) was added and the mixture was concentrated. The residue w-as eluted from a column of silica gel with 9:1 cyclohexane-EtOAc to give 22 (440 mg, 92 %) as a colorless foam; [a]D
XX° (c 1, CHC13).'H
NMR (CDC13):8 7.1-8.1 (m, 35H, Ph), 5.95 (m, 11-l, All), 5.73 (dd, 1H, .1,,2 =
2.2, Ji,3 = 2.3 Hz, H-2B), 5.43 (dd, 1H, JI,2 - 2.0 Hz, JZ,3 = 3.0 Hz, H-2~), 5.30 (m, 2H, All), 5.08 (d,1H, JI,~
= 3.2 Ha, H-ls), 5.03 (d, IH, Ji,z =1.7 Hz, H-lg), 4.97 (d, 1H, J~,~ = I .9 Hz, H-Ic), 4.30-5.00 (m, 12H, CHZPh), 4.20 (m, 2H, All, H-3c), 4.05 (m, 3H, All, H-3E, SE), 3.98 (m, 1H, H-6aE), 3.81 (m. SH, H-3H, 4c, 4~, Sc, GE), 3.G9 (dq, 1H, J~,s = 9.3, Js.6 = 6.0 Hz. H-58), 3.52 (dd, 1H, Ji,l = 9.7 Hz, H-2E), 3.29 (dd, 1H, J3 4 = J4,s = 9.4 Hz, H-4H), 2.71 (m, 4H, Levy, (s, 3H, Ac), 1.40 (d, 3H, H-6c), 1.01 (d, 3H, H-6a).
Aliyl (3,4-dl-0-benryl-a-1.-rhsmnopyrnnosyl)-(I~3)-[2,3,4,6-tetra-0-ben2yl-a-D-glucopyranosyl-(1--~4)]-2-O-benzoyl-a-L-rhamnopyrsmoside (23). The trisa.ccharide 22 LJvfPPJ 3-agr.6rtwct-pcnusblack (200 mg, 0.16 mmoI) was treated wJJth 0.4 mL of a solution 1 M of hydrazine (I00 nng) diluted in a mixture of pyridine (1.6 mL) and acetic acid (0.4 tnL) at rt. The solution was stirred during 20 min. Acetone (1.2 mL) was added and the solution was concentrated.
The residue was eluted from a column of silica gel with 98.5:1.5 Diehlorometharre-AcOEt to give 23 (174 mg, 92 %) as a foam; [a]p +14° (c 1, CHC13); 'H NMR (CDCI,):o 7.05-8.10 (m, 35H, Ph), 5.82 (m, 1 H, All), 5.25 (dd, 1 H, JJ,z = 1.7 Hz, Jz,3 = 3.1 Hz, H-2~), 5.19 (m, 2H, All), 5.00 (d, 1H, J~,z= 3.1 Hz, H-lE), 4.87 (d, 1H, J,,2= 1.8 Hz, H-IH), 4.81 (d, IH, H-Ic), 4.35-4.90 (m, 12H, CI~ZPh), 4.00-4.20 (m, 2H, All), 4.10 (dd, 1H, J;,4 = 8.5 Hz, H-3c), 4.09 (dd, IH, Jz,~ _ 3.2 Hz, H-2B), 3.95 (m, I H, J4,s = 9_5 Hz, H-5E), 3.92 (dd, 1 H> Ja,3 = 9.5 Hz, J3,, = 9.5 Hz, H-3E), 3.78 (m, 1H, Js,a = 6.0 Hz, H-5~), 3.70 (m, 1H, H-4c), 3.58~3.62 (m, 2H, H-6aE, 6bE), 3.59 (m, 1H, J~,s = 9.0 Hz, -Is,s = 6.2 Hz, H-Se), 3.54 (dd, 1H, H-4E), 3.48 (dd, IH, J3,d = 8.5 Hz, H-3H), 3.45 (dd, 1H, H-2fi), 3.31 (dd, 1H, H-4H), 2.68 (d, 1H, JZ,oH=2.3 Hz, O-H), 1.29 (d, 3H, H-G~), 1.09 (d, 3H, H-6B). "C NMR (CDCu):8 166.2 (C=0), 118.2-137.5 (Ph, All), 103.1 (C-1B), 98.5 (C-ls), 96.6 (C-lc), 82.1 (C-3E), 81.4 (C-2E), 80.4 (C-48j, 79.7 (C-3B), 79.4 (C.4~), 78.9 (C~3~), 78.1 (C-4E), 76.0, 75.5, 74.5, 74.2, 73.6, 72.1 (CHZPh), 73.7 (C-2c), 68.9 (C-6s), 68.8 (C-5e), 68.7 (All, C-5E), 68.1 (C-5c), 19.1 (C-6c), 18.2 (C-68). FABMS
of C~oH~60,s (M, 1156.5), mlz 1179.5 ([M+Na]+). Anal. Calcd for C~oH,EO,s: C, 72.64; H, 6.62. Found C, 72.49; H, 6.80.
Ally! (3-0-acetyl-4,G-0-isopropylidcne-.2-trichloroacetamido-2-deoxy-[3-n-glucopyranosyt)-(1-~2)-(3,4-di-D-benzyl-a.-~rhamnopyranosyl)-(1--~Z)-(3,4-di-O-benzyt-a-r.-rhamnopyranosyl)-(1-~3)-[2,3,4,G-tetrn~D-benzyl-a-n-glucopyranosyl-(1-~4)]-2-D-benzoy!-a-L-rbamnopyranoside (Z6). A mixture of the donor 8 (294 mg, 357 ~tmol) and the acceptor 25 (313 mg, 211 pmol), 4~1 molecular sieves and dry DCM (4 mL) was stirred for LMPP13-cxp~brevebprntn6lock 1.5 h then cooled to -15°C. NIS (94 mg, 0.42 mmol) and Triflic acid (8 N.L, 0.1 mmol) were successively added. The stirred mixture was allowed to reach 0°C in 1.5 h. Et3N (25 pL) was added and the mixture filtered. After evaporation, the residue was eluted from a column of silica 8el with G:1 cyclohexane-EtOAc and 0.5 % of Et3N to give 26 as a white foam (232 mg, 58 %); [oc]p -2° (c l, CHCl3); 'H NMR (CDCIs): 'H cS 7.04~8.00 (m, 45H, Ph), 6.81 (d, IH, J2_~ = 9.0 Hx, NHD), 5.82 (m, 1H, All), 5.30 (dd, 1H, J,~ = 1.0, JZ,~ = 3.0 Hz, H-2c), 5.10-S.Z3 (m, 2H, All), 4.96 (bs, 1H, H-1,,), 4.91 (d, IH, Jl,~ = 3.1 I-Iz, H-IE), 4.87 (d, 1H, J,_i =
1.6 Hz, H-IB), 4.84 (bs, IH, H-lc), 4.79 (dd, 1H,.T~,3 =J3,a = lO.OHz, H-3p), 4.35 (d, 1H, H-ln), 4.34 (dd, IH, H-Zg), 4.20-4.80 (m, 1GH, CH2Ph), 4.00 (dd, IH, H-2A), 3.90 (dd, 1H, H-2D), 2.90-4.10 (m, 22H, All, H-2~,, 3~, 3g, 3c, 3F, 4A, 4H, 4c, 4D, 4E, SA, 58, Sc, 5~, SE, Gac, Gbu, Gas, GbE), 1.93 (s, 3H, OAC), I.Z-0.9 (m, 15H, C(CH3)i, H-6A, GH, 6c). "C NMR
(CDCI..,):8 170.7, 165.5, 161.7 (C~O), 138.4-117.3 (Ph, All), 101.7 (C-1D), 100.8 (C-1H), 100.6 (C-IA}, 99.5 (C(CH3)2), 97.9 (C-IE), 95.7 (C-lc), 92.0 (CC)3), 82.2, 81.7, 81.6, 80.3, 79.9, 78.8, 77.9, 77.9, 76.6, 76.0, 75.8, 75.4, 75.1, 74.7, 74.3, 74.1, 73.3, 72.8, 72.6, 71.9, 71.5, 70.8, 69.0, d8.8, 68_5, 68.0, 67.8, 62.0, SG.7 (C-2D), 28.6 (C(CH,)~), 21.3 (OAc), 19.4 (C(CH3)~), 19.0, 18.5, 18.4 (3C, C-GA, 6B, Gc). FABMS of C1a31i,~aCl3NOis (M, 1872.3), m/z 1894.6 [vI+Na]+.
Anal. Calcd for C,°3H~~aCI3NOZ5, C: 66.07, H: 6.14, N: 0.75. Found C:
66.08, H: 6.09, I~':
a.81.
Atly1 (2-acetamido-4,G-O-isopropylidene-Z-deoxy-~-D-glucopyranoayl)-(Z-~Z)-(3,4-di-O-benzyl-a-t,-rhamnopyranosyl)-(Z.-~2)-(3,4-di-0-benry1-a-L-rhamnopyraaosyt)-(1-a3)-(2,3,4,6-tetra-0-benzyt-a-D-glucopyranosyl-(1--~4)-)-2-D-benzoyl-a-L-rhamnopyranoside (2). The pentasaccharide X (2.65 g, 1.47 mmol) was dissolved in MeOH {20 mL).
MeONa was added until pH=10. The mixture was stirred for 25 min then treated by IR
120 (H') until LMPP t 7-exp~breveipcutsblak gel with 2:1 Cyclohexane-AcOEt and 0.2 % of Et3N to give 1 as a white foam (1.99 g, 94 %);
[cc)b +1° (e I, CHC13).
(b) A mixture of26 (144 mg, 0.06 mmol), Bu3SnH (O.t mL, 0.37 mmol) and AIBN
(10 mg) in dry toluene (3 mL) was stirred for 1 h at rt under a stream of dry Ar, then was heated for 1.5 h at 90°C, cooled and concentrated. The residue was eluted from a column of silica gel with 2:1 cyatohexane-1?tOAc and 0.2 % of Et3N to give 1 (100 mg, 74 %). 'H NMR
(CDC13): 5 6.95-8.40 (m, 45H, Ph), 5.82 (m, 1H, All), 5.46 (d, 1H, Jz,uH = 8.0 Hz. NHti), 5.29 (dd, 1H, J~.z = 1.0, Ja,3 = 3.0 Hz, H~2c), 5.1 I-5.25 (m, 2H, All), 5.00 (bs, 1H, H-lA), 4.90 (d, 1H, J~.z =
3.1 Hz, H-IE), 4.85 (d, 1H, J,,z = 1.6 Hz, H-1B), 4.83 (bs, 1H, H-1~), 4.70 (dd, 1H, Jz,3 =J3,, _ 10.0 Hz, H-3o), 4.44 (d, 1H, H-1D), 4.34 (dd, IH, H-28), 4.20-4.80 (m, 1GH, CHzPh), 4.02 (dd, 1H, H-2A), 3.37 (dd, 1H, H-2E), 2.90-4.10 (m; 21H, All, H-2o, 3A, 3B, 3c, 3E, 4A, 4B, 4c, 4p, 4F, 5,,, SH, Sc, SD, 5E, GaD, 6bi,, 6a~, Gbs), 1.92 (s, 3H, OAc), 1.57 (s, 3H, AcNH), 1.27-0,90 (m, 15H, C(CH3)Z, H-6", 6a, Gc). t'C a 171.3, 170.3, 166.2 (C=0). 138.7-117.9 (Ph, All), 103.9 (C-1D), 101.5 (C-lg), 101.4 (C-lA), 99.9 (C(CH3)?), 98.5 (C-ls), 96.3 (C-Ic), 82.1, 81.7, 81.6, 80.3, 80.1, 78.8, 78.1, 77.8, 76.0, 75.8, 75.3, 75.1, 74,7, 74,2, 73.6, 73.3, 72.7, 71.9, 71.4, 70.8, 69.0, 68.8, 68,7, 68.4, 68,1, 67.8, 62.1, 55.0 (C-2D), 30.0 (C(C'F~,)~), 23.5 (AcNH), 21.6 (OAc), 19.2 (C(CH3)2), 19.0, 18.3, 18.2 (3C, C-6A, GB, 60). FAB-ViS for Cio3H,17N025 (M = 1769.0) m!z 1791.9 [M + Na)+. Anal. Calcd. for C,o3Ht,7N025 : C, 69.93 ;
H, 6.67 : N, 0.79. Found C, 69.77; H, 6.84; N, 0.72.
(2-acetamido-3-O-acetyl-4,6-0-isopropylideno-l-deoxy-[3-D-glucopyranosyl)-(1-->2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyt)-(1~2)-(3,4-di-0-ben2yl-n-z-rhamnopyrsnosyl)-(1-->
3)-[2,3,4,6-tetra-O-6enzy I-a-D-glucopyranosyl-(1 >4)-]-Z-O-beozoy I-a-L-rhamnopyranosyl trichloroacctimidate (3). 1,5-Cyclooctadiene-bis(methyldiphsnylphosphine)iridium hexafluorophosphate (50 mg, 58 pmol) was dissolved LMPP13-Schcmes-brc~wPcnG~block OBn BnBOCi ~ ORS
Bn0 0 Me O
0 OBz Bn0 Me 0 Bn0 O
' OO O 0~ OBn Rs~~ Me OBn NHAc R' R3 All Ac 2 All H
3 TCA Ac L.htPPl3~Schcme,;-hrcvet-pentabtock OAc ~A ~~S~OAc ~~~_~~S(CNZ)WH3 NHC(O)CCf3 NHC(0)CCI3 R
6 Ac O t --r R30 R
NHC(0}CCi3 r ~ S(CNZ)~tCH3 H
g 5(CHg}oCH3 Ac g OH Ac OTCA Ac Synthesis of spacer-armed hexa-, deca-, and pentasaccharide haptens representative of the O-spccitic polysaccharide of Sh~gella Jtexneri serotype Za1 This paper disclosES total synthesis of fully defined aligomeric repeating unit glycosides mimicking the branched bacterial O-5Ps in the S. flexneri series. The strategy disclosed herein gives access to extended fragments of the O-SP of S flexneri serotype 2a in a spacer-armed form suitable for irnmunological studies. Indeed, amounts required for the synthesis of fully synthetic oIigosaccharide conjugates as potential vaccines targeting S
flexneri 2a infection were made available LMPPI dthco-brc..ct-synlong~

Synthesis of apa~er-armed hexa-, deca-, and pentasaccharide haptens representative of the O-specific polysaccharide of fhigello fl'exneri aerotype 2a1 Abstract IN~'RODUGTION
Shigellosis or bacillary dysentery is a serious infectious disease, responsible for some 200 million episodes annually, mostly in children and immunocompramised individuals living in areas were sanitary conditions are insuffrcient. 1 Of the four species of Shigellae, Shigella flexneri is the major responsible of the endemic form ojthe disease, with serotype 2a being the most prevalent. Due to increasing resistance of all groups of Shigellae to antibiotics, j the development of a vaccine against shigellosis is of high priority as stated by the World Health Organization in its program against enteric diseases. ' However, there are yet no licensed vaccitres for shigellosis.
As for other Gram negative bacteria, Shigella's Iipopolysaccharide (LPS) is a major surface antigen of the bacterium. The corresponding 0-specific polysaccharide (O-SPj, a polymer of less than 3Q lcDa, defines the serogroup and serotype of the bacteria. Besides, it is both an essential virulence factor and the target of the infected host's protective immune response. s'6 However, O-SPs are T-cell independent antigens, 7v which are not immunogenic by themselves. Nevertheless, benefiting from the successful conversion of bacterial capsular polysaccharides from T-independent antigens bo T-dependent ones through their covalent coupling to a protein carrier, it was shown that 0-SPs could be fumed into immunogens.
Indeed, based on the former hypothesis that serum IgG anti-LPS antibodies may confer LMPPI4th~o-6revd~synlongc specific protection against shigellosis, 9 several polysaccharide-proteine conjugates, targeting either Shigella sonnet, Shigella dysenteriae 1 or S flexneri serotype 2a, Were shown to be safe and immunogenic in humans. lo.n In the case of S, sonnet, recent field trials allowed ).B.
Robbins and co-workers to demonstrate the efI'tcacy of a vaccine made of the corresponding detoxified LPS covalently linked to recombinant exoprotein A, ~Z Even though e~cient, polysaccharide-protein conjugate vaccines remain highly complex structures, whose immunogenicity depends of several parameters amongst which, the length and nature of the saccharide component as well as its loading on the protein. It is reasonably admitted that control of these parameters, and indeed standardization, are somewhat difficult when dealing with polysaccharides purified from bacterial cell cultures, or fragments thereof resulting from their partial hydrolysis. Mixture are often obtained, which may become a real drawback in terms of analysis of the products, particularly when multivalent wccines are needed, as in the case of shagellosis. It may be assumed that the use of well-defined synthetic oiigosaccharides suitable for single-point attachment on to the carrier would allow a better control, and consequently the optimisation, of the above mentioned parameters. That low molecular weight oligosaceharides mimicking antigenic determinants were immunogenic when conjugated onto a protein carrier was demonstrated in the late 30s, l3,la and since then exploited;on several occasions. is Indeed, available data on S dysenteriae type I indicate that neoglycoconjugates incorporating di-, tri- or tetramers of the 0-SP repeating unit were more immunogenic than a detoxified LPS-human serum albumin conjugate of refierence.
16 In the case of heteropolysaccharides, oligosaccharides made of at least two contiguous repeating units were originally considered to be necessary for the corresponding oligosaccharide-protein conjugates to induce anti-polysaccharide antibodies. ~~ However, more recent data demonstrated that neoglycoproteins incorporating oiigosaccharides comprising one repeating unit or smaller fragments were immunogenic in mice. ~B'1' Along this line, we recently reported the synthesis of three fully synthetic glycopeptides as potential vaccines against Shigella Jlexneri 2a infection. a° These incorporated short oIigosaccharidc haptens, representative either of part or of the whole repeating unit of the 0-SP of S.
flexneri serotype 2a. Preliminary data indicate that two out of the three conjugates are immunogenie in mice.(Phalipon et al, unpublished results) Besides, we found that the corresponding neoglycoproteins consisting of the oligosaccharides covalently linked to tetanus toxoid via single-point attachment were also immunogenic in mice.(Phalipon et at, unpublished results) Parallel studies on the recognition of synthetic fragments of the 0-SP by protective homologous monoclonal antibodies suggested that sequences larger than one repeating unit LMPPl4theo-bravo-synlongs were more antigenic, thus probably better mimicking the natural polysaccharide than shorter ones. 21 Indeed, it is anticipated that better mimics of the O-SP, in terms o~
both antigEnicity and conformation, would Iead to conjugates of higher immunogenicity. For that reason, the preparation of oligo- or polysaccharides22 made of two repeating units or more, in a form suitable for conjugation onto a caxtier, was undertaken.
RESULTS AND DISCUSSION
A B E C D
2)-a-L-Rhap-(1-a2)-a-L-Rhap-(1-~3)-[a-D-Gtcp-(1-~4)]-a-L-Rhap-(1 >3)-(i-D-GIcNAcp(1-3 I
The 0-SP of S flexneri 2a is a branched h.eteropolysaccharide deFmed by the pentasaecharide repeating unit I. 23,ara It features a linear tetrasa~ccharide backbone, which is common to all S.
,flexneri 0-SPs and comprises a N acetyl glucosamine (D) and three rhamnose residues (A, B, C). The spec~city of the serotype is associated to the a-D-glucopyranose residue linked to position 4 of rharnnose C.
Evaluation of the antigenicity of a panel of di- to pentasaccharides representative of frame-shifted fragments of I, had pointed out that the ECD portion was the minimal sequence required for binding, and that the B(E)C ramification had a great impact on the recognition process. zs Hosed on theses data, we described recently the synthesis of the ECD, B(E)CD
and AB(E)CD fragments functionalized with an aminoethyl spacer at their reducing end, and demonstxated that the later could serve as a suitable anchoring point.
Z° As stated above, subsequent work outlined the impact of chain elongation on the recognition process. Taking both sets of data into account, we report herein on the synthesis of the 2-ami.noethyl glycosides of a deco- (1) and a pentadecasaccharide (2), corresponding to sequences [AB(E)CD}Z and [AB(E)CD]s, respectively. The corresponding D'AB(E)CD
hexasaccharide (3) was used as a model Considering the target 1 and 2, a disconnection at the D-A linkage would appear most appropriate. However, others have shown that such a disconnection strategy was not suitable even when involving di- or trisaccharide building blocks, z6,Z~ and this route was avoided.
More recently, disconnections at the A-B, B-C and C-D linkages were evaluated in this laboratory when synthesizing successfully the methyl glycoside of the frame~shifted decasaccharide D'A'B'(E')C'DAB(E)C by condensing a chain terminator pentasaccharide LMPPt4theo-brcvn-synlongs donor and a methyl glycoside pentasaccharide acceptor. Z& It was demonstrated on that occasion that disconnection at the C-D linkage was indeed appropriate for the construction of large fragments of the S. flexneri 2a 0-SP. Based on oar experience in the field, a blockwise strategy to targets 1 and Z, implicating a DAB(E)C potential acceptor acting as a donor, an AB(E)C tetrasaccharide donor, and the recently disclosed acceptor XX~°
as a precursor to the spacer-armed D residue (Scheme 1). Although permanent blocking of OH-4D and OH-Gn with an isopropylidene acetal may appear somewhat unusual, this choice was a key feature of the strategy. It was based on former observations in the methyl glycoside series, demonstrating that its use could overcome some of the known drawbacks of the cozresponding benzylidene acetal, Z9''° including its poor solubility. Compound XX was readily obtained from the known triacetate XX~1 (81%), by transesterification and subsequent treatment with 2,2-dimethoxypropane.
S~nrhesfs of the hexasaccharide 3 (Scheme 2): In a preliminary study towards the target 3, the DAS(E)C building block bearing the required a,cetamido function at position 2D
was used as the donor. It was obtained 5rom the recently described precursor XX. ig Indeed, reductive free-radical deehlorination of XX using Bu3SnH in the presence of catalytic AIHN
allowed the conversion of the N trichloroacetyl moiety into N acetyl, to give XX (G8%). The latter was converted to the hemiacetal XX following a two-step process including Iridium complex promoted isomerisation of the aIlyl rr>Qiety into the propen-1-yl, 3Z
and hydrolysis of the latter upon treatment with aqueous iodine. 33 Subsequent reaction of XX
with trichloroacetonitrile in the presence of catalytic 1,8-diazabicyclo[5.4.0)undec-7-ene (DgU) cleanly gave the trichloroacetimidate donor XX (85% from XX). Previous glycosidation attempts in the series indicated that when run at low temperature or room temperature, reactions using the D acceptor XX occasionally resulted in a rather poor yield of the condensation product. This was tentatively explained by the still rather poor solubility of the acceptor XX. When using I,2-dichloroethane (DCE) as the solvent, the condensation could be performed at higher temperature, which proved rewarding. Indeed, optimized coupling conditions relied on the concomitant use of a catalytic amount of triflic acid in the presence of 4th molecular sieves as the promoter and DCE as the solvent, while the condensation was perfiormed at 80°C. The fully protected hexasaccharide XX was isolated in a satisfactory 78%
yield. That the hemiacetal XX, resulting from the hydrolysis of the excess donor could be recovered was of great advantage is one considers scaling up the process (not described).
Acidic hydrolysis of the isopropylidene acctal smoothly converted XX into the corresponding LMPPI4theo~brevei-synlongs dioI XX (94°/a). Resistance of isolated benzoyl groups to Zempldn transesterification has been reported, 3a.ys Ii was also observed previously in the series, upon attempted removal of a benzoyl group located at position 2~. 2$ Again; the 2~-O-benzoyl group in XX
was particularly resistant to Zempl6n de-O-acylation, and in that case, successful transesterification required a week. In that case, heating was avoided in order to prevent any potential migration of the aryl group which would lead to the N deacylated product.
Conversion of the hexaol XX into the target 3 was successfully accomplished upon concomitant hydrogenolysis of the remaining benzyl protecting group and reduction of the azido moiety into the corresponding amine. As observed earlier, ~°~~
the latter was best performed under acidic conditions. The target 3 was isolated in 77% yield after reverse-phase chromatography.
Synthesis of the decasaccharide f (5clzeme 3): Having the fully protected hexasaccharide XX in hands, we reasoned that a convenient access to I could involve the condensation of an AB(E)C tetrasaccharide donor and a DAB(E)CD hexasaccharide acceptor prepared from XX. Preparation of the former was conveniently achieved from the previously described tetrasaccharide XX. 2g Removal of the anomeric allyl protecting group involved a two-step process as described above for the preparation of XX. The hemiacetal was readily converted into the trichtoroaeetimidate donor XX, which was isolated in an unoptimized yield of 56% over the two steps. Taking advantage of the stability of the 2~-O-benzoyl group under Zempl~n conditions, selective chemical modification at the D residue of XX was anticipated to give easy access to the selected acceptor XX. Indeed, transesterification of the acetyl groups in XX gave the expected triol XX, which was further regioselectively protected at the 4D and 6o hydzoxyl groups when treated with 2,2-dimethoxypropane. However, the key acceptor XX was isolated in 50% yield only. Condensation of the latter and XX
was performed in DCE using triflie acid as the promoter. One may note that although the condensation involves the construction of the C-D linkage, thus somewhat resembling the preparation of the hexasaccharide XX, heating was not required and the glycosylation went smoothly at low temperature to give the fully protected decasaccharide XX
(82%). Acidic hydrolysis of the acetals gave the tetraol XX (?5%). 'I~ansesterif catioa of the aryl groups was best performed by overnight heating of XX in methanolic sodium methoxide.
Final hydrogenolysis of the benzyl groups and concomitant conversion of the azido group into the corresponding amine gave the target 1 (71% from XX).

LMPPI4thco-brevet-synlonas Synthesis of the pentadecasacel:aride Z: If the synthesis of 2 was to mimic that of I, the transformation of the non reducing 3,4,6-tri-O-acetyl D residue into the corresponding 4,G-0-isopropylidene one was to be performed twice. Considering that besides being rather low, the yield of the transformation of XX into XX was also poorly reproducible, considerable loss of two costly intermediate, namely first the hexasaccharide XX, then the undeeasaccharide XX, was to be expected. The use of a pre-funetionalized DAB(E)C
building block, that could act both as a donor and an acceptor based on appropriate orthogonal protection, was considered as an amactive alternative. Such an intermediate (XX) was recently prepared in the laboratory by condensation of an AB(E)C
tetrasaccharide acceptor ~(XX) 3a to a fully functionalized D thioglycoside donor (XX), and subsequent free-radical conversion of the N trichloroacetyl into the corresponding aeetamide (Scheme 4). 3a Since the condensation of XX and XX was somewhat low-yielding, another route to XX is disclosed herein. Intakes advantage of the high-yielding condensation of the tetrasaccharide acceptor XX with the known trichloroacetimidate donor XX, 39 giving access to the fully protected XX
(98%), za and subsequently to th:e corresponding acetamido derivative XX as described above.
Controlled de-0-acetylation of XX under zemplGn conditions gave the triol XX, which was next converted to the corresponding alcohol XX upon reaction with 2,2-dimethoxypropane ($1% from XX). Conventional acetylation at position 3D then gave the key intermediate XX
(94%). Transformation of the latter into the trichloroacetimidate donor XX
(82%) was performed as described for the preparation of XX via the hemiacetal intermediate XX.
The rather satisfactory yields obtained all along the synthesis of the building block XX
allowed the targeting of larger sequences. Indeed, when the newly formed pentasaccharide donor XX and the spacer-armed D acceptor XX were heated in DCE in the presence of triflic acid and 4A molecular sieves as described for the preparation of XX, the condensation product was isolated in 78%. The resistance of the two isapropylidene acetals to the harsh acidic conditions of the glycosidation reaction is noteworthy. Selective deacetylation at the 3-OH of the non reducing residue, then gave the D'AB(E}CD acceptor XX in a yield of 76%, confirming indeed than this route to XX was more appropriate than that described above. This two-step glycosidationldeacetylation process was repeated. However, whereas the above mentioned glycosidatinns required heating, condensation of the hexasaecharide acceptor XX
and the pentasaccharide donor XX in the presence of triflic acid was run at Iow temperature.
Under such conditions, the fully protected undecasaceharide XX was isolated In an excellent yield of 90%. Zempl~n transesterification at the non reducing 3p-OH of the latter proved as efficient, and gave the required acceptor XX (91%j. Condensation of this key intermediate LMPPI4theo-brevet-9yalonge with the tetrasaccharide trichloroacetimidate donor XX was again perfornled at low temperature, using triflic acid as the promoter. The fully protected pentadecasaecharide XX
was isolated in a satisfactory yield of 82%. Conversion of XX to the target 2 was performed according to the stepwise sequence described for the preparation of 3. Acidic hydrolysis of the isopropylidene groups afforded the hexaol XX (83%). Again, running the transesteriftcation step at high temperature allowed to overcome the resistance of benzoyl groups to Zemplen conditions. Conventional hydrogenolysis of the intermediate XX, finally gave the pentadecasaccharide hapten 3 (65% from XX).
CONCLUSION
The synthesis of the 0-SP of S. flex»eri Y by way of polycondensation of a tritylated cyanoethylidene tetrasaccharide was reported by others. 4° However, this is to our knowledge the first report on the fatal synthesis of fully defined oligomeric repeating unit glycosides mimicking the branched bacterial O-SPs in the S, flexr:eri series. The strategy disclosed herein gives access to extended fragments of the O-SP of S. flexneri serotype 2a in a spacer-armed form suitable for immunological studies. Indeed, amounts required for the synthesis of fully synthetic oligosaccharide conjugates as potential vaccines targeting S
flexneri 2a infection were made available. The preparation of such conjugates is in progress in the laboratory.
ACKNOWLEDGEMENTS
The authors are grateful to J. Ughetto-Monfrin (Unitd de Chimie Organique, Institut Pasteur) for recording all the N1IR spectra. The authors thank the Bourses Roux Foundation for the postdoctoral fellowship awarded to F. B., and the Institut Pasteur for its financial support (grant no. PTR 99).
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(2) Kotloff, K. L.; Winickoff, J. P.; Ivanoff, B.; Clemens, J. D.; Swerdlow, D. L.;
Sansonetti, P. J.; Adak, G. K, Levine, M. M. Bull. y~HO 1999, 77, 651-666.

LMPPL4theo~brcvet~synlongs (3) Ashkenazi, S.; May-Zahav, M.; Sulkes, J.; Samna, Z. Antimicrob. Agents Chemother. 1995, 39, 8 i 9-823.
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Immunol: 1993, I51, 2419.
(8) Ishioka, G. Y.; Lamont, A. G.; Thomson, D.; Bulbow, N.; Gaeta, F. C. A.;
Sette, A.; Grey, H. M. J. Imrntrnol. 1992, 14~, 2446.
(9) Robbins, J. B.; Chu, C.; Schneerson, R. Clin. Infect. Dis. 1992,15, 346-3GI.
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Infect. Immun. 1993, dl, 3678-3687, (11) Passweil. J. H.; Harlev, E.; Ashketuuizzi, S.; Chu, C.; Miron, D.; Ramon, R.;
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Hate, T. L.; Sadoff, J. C.; Pavliovka, D.; Schneerson, R; Robbins, J. B. The Lancet 1997, 349, 155-159.
(13) Goebel, W. F. J. Exp. Med 1940, 72, 33.
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(15) Pozsgay, V. In Adv. Carbohydr. Chem. Biochcm.; Horton, D., Ed.; Academic Press: San Diego, 2000; Vol. 56, pp 153-199.
(1G) Pozsgay, V.; Chu, C.; Panell, L.; Wolfe, J.; Robbins, J. B.; Schneerson, R
Proc. Natl. Aced Sci. USA 1999, 96, S I 94-5197.
(17) Peeters, C. C. A. M.; Lagerman, P. R.: Weers, O. d.; Ooemn, L. A.;
Hoogerhout, P.; Beurret, M.; Poolmat~, J. T. In Vaccine Protocols; Robinson, A.; Fatter, G., Wiblin, C., Eds.; Humarta Press Inc.: Totowa N, J., 1996, pp 1 I 1-133.
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Phalipon, A. in preparation 2003.
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L;~iPPl4.e~cQ6revet.syatongg ~1 02434685 2003-07-04 CH3C=O), 1.65 (s, 3H, GH3C=ONH), 1.32 (d, 3H, Js,s = 6.1 Hz, H-6,J, 1.30 (d, 3H, Js,6 = 6.0 Hz, H-6c), 0.97 (d, 3H, Js,6 = 6.0 Hz, H-6B). "C NMR (CDC13):8 171.1, 170.8, 170.2, 169.6, 166.2 (SC, C=0), 138.2-118.5 (Ph, All), 103.1 (C-1D), 101.4 (C-1H), 101.2 (C-1,~), 98.5 (C-IE), 96.4 (C-1~), 82.2 (C-3E), 81.7 (C-2E), 81.7 (C-4A), 80.4 (C-4B), 80.2 (C-3c), 79.0 (C-3"), 78.6 (C-3$), 78.1 (C-2A), 77.8 (C-4c), 77.G (C-4E), 76.0, 75.8, 75.4, 74.7, 74.3, 74.2, 73.3, 70.5 (8C, CHaJ?h), 74.9 (C-2H), 72.7 (C-2~), 72.G (C-3D), 71.9 (2C, C-SE, SD), 69.1 (C-5H), 68.9 (2C, All, C-SA), 68.3 (C-6E), 67.8 (C-Sc), 62.3 (C-6p), 54.6 (C-2Dj, 23.5 (1C, NHC=OCH3), 21.1, 21Ø 20.8 (3C, C=OCH~), 19.0 (C-Gc), 18.4 (C-6,,), 18.2 (C-6B).
FABMS of GtoaHImNOz, (M, 1913.1), m/z 1936.2 [M+Na]*. Anal. Calcd. for ClodH1"NO2~
C, 68.90 ; H, 6.50 ; N, 0.77. Found C, 68.64 ; H, 6.66 ; N, I .05.

LMPPI4.exp~btevct~s~'nlongs C~";H~ ~~Chi~tOs.
E~cntt Me.s~ 1914,66 Mol. Wt. 1917,35 H 5,94, Ci 5,55; N l.dG; O 77,13 we; C:61.33%. H.G.IOSi, ; i: I a5?.
AIphaD-r10°, c=1, CHCI3 (2-aeetamido-3,4,6-tri-0-acetyl-2-deoxy-(3-D-glueopyranosyl)-(Z-~2)-(3,4-di-O-benzyl-a-L-rham>nopyraaosy!)-(1->2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1-~3)-[2,3,4,6-tetra-0-benzyl-a-D-glucopyranosyl-(1-~4))-2-0-beazoyt-a-L-rhamnopyraaosyl trichloroacetimidate (X).
1,5-Cyclnoctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (25 mg, 29 p.
mol) was dissolved tetrahydrofuran (5 mL), and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stzeam. A solution of 7 (I.0 g, 0.55 mmol) in tetrahydrofuran (10 mL) was degassed and added, The mixture was stirred at rt overnight, then concentrated to dryness. Ths residue was dissolved in acetone (5 mL), then water (1 mL), mercuric chloride (140 mg) and mercuric oxide (120 mg) were added successively. The mixture protected from Iight was stirred at rt for 2 h and acetone was evaporated. The resulting suspension was taken up in DCM, washed twice with 50% aq KI, water and satd aq NaCI, dried and concentrated. The residue was eluted from a column of silica gel with 2:1 petroleum ether-EtOAc to give the corresponding hemiacetal.
Trichloroacetonitrile (2.5 mL) and DBU (37 uL) wcre added to a solution of the residue in L1~P14txp-brevet-synlon6s anhydrous dichlorom~ethane (12.5 mL) at 0°C. ARer 1 h, the mixture was concentrated. The residue was eluted from a column of silica gel with S:4 cyclohexane-EtOAc and 0.2 °!o Et3N to give X as a white foam (0.9 g, 8S °!°); [a]D +10° (c 1, CHC13).
'H NMR (CDCh):8 8.70 (s, IH, C=NH), 8.00-7.00 (m, 4SH, Ph), 6.36 (d, IH, J,,~
= 2.6 Hz, H-lc), 5.59 (m 2H, N-HD, H~2~), 5.13 (d, IH, J,,Z = 1.0 Hz, H-I,,), S.O1-4.98 (m, ZH, H-le, IH), 4.92 (dd, 1H, H-3p), 4.90 (dd, 1H, H-4p), 4.G8 (d, 1H, H-1D), 5.00-4.02 (m, 19H, 8 CHZPh, H-3c, 2,,, 2$), 4.01 (dd, 1H, H-2E), 4.00-3.20 (m, 16H, H~3E, 4E, SF, GaE, Gbe., 4~, 5c, 38, 4s, SB, 3,,, 4~, 5~, SD, ban, 6bD), 2.02, 2.00, 1.75, 1.65 (4s, 12H, C=OCH3), 1.40, 1.32 and 1.00 (3d, 9H, H-G~, 6s, Gc). "C NMR (partial) (CDCIz):8 170.2, 1b9.9, 169.3, 168.7, 164.9 (GC, C=O, C=N), 103.2 (C-1D), 101.4 (2C, C-lA, 1H), 99.0 (C-lE), 94.8 (C-lc), 21.1, 20.9, 20.8 (3C, CH3C=0), I9.1, 18.2 (3C, C-GA, GB, G~). FABMS of Clo3H»3C13Ni02, (M, 1917.4), mla 1930.9 [M+Na]''. Anal. Calcd. for C,o3H~ nClzNiOa~ : C. 64.52 ; H, 5.94 ;
N, 1.46. Found C,64.47;H,5.99;N, 1.45.

LMPP i Atopbrevet-synlongs ~OBn ~o-~

9n0~ O
An0 B~
~

~~

0 OAz ~.,~ CuaHmN~~3z e~

ei,o p Exact Mose:
2AR3,R9 T~.~ Mol. W,.' 295,29 en0~ C 6 5,66; K 6;43, :~: 3,3b;
O 14,55 tr°uot: C:G5.37, H 6.51, N 3.18 AIphaD='G 3°, C=1, CHC13 2-A~eidoet6yl (2-acetamido-3,4,6-trl-O-acetyl-2-dcoxy-(i-n-glucopyranosyl)-(1~2)-(3,4-di-0-benzyl-a-L-rhamnopyranosy 1)-(1~2)-(3,4-dl-O-beazyl-a-irrhamnopyranosyt)-(1--~3)-[2,3,4,6-tetra-O-beaxyl-a-v-glucopyranosyl-(1-~4)j-(2-0-ben~.oyl-a-1.-rhamaopyranosyl)-(1->3)-2-acetamido-2-deoxy-4,6-D-isopropylidene-[i-n-glucopyranosidc (X).
A mixture of alcohol X (110 mg, 330 wnwl), imidate X (720 mg, 376 ~mol) and 4tl molecular sieves in anhydrous DCE (6 mL) was stirred for 1 h under dry Ar. After cooling at 0°C, Tfl~H
(16 ~L; 180 ~mol) was added dropwise and the mixture was stirred at 80°C for 2.5 h.
Triett>sylamirte (60 ~L} was added and the mixture was filtered and concentrated. The residue was eluted from a column of silica gel with 3:4 cyclohexane-EtOAc and Et3N
(0.2 %) to give X as a colorless oil (540 mg, 78 %); [a]D +6.5° (c 1, CHCl3).
'H Ni~iR (CDCI3):8 8.00-7.00 (m, 45H, Ph), 5.95 (d, 1H, J2,~ = 7.1 Hz, NHDj, S.SI (d, 1H, J,.~., = 8.1 Hz, NHS), 5.20 (dd, 1H, Jl,~ = 1.7 Hz, J~,, = 3.0 Hz, H-2c), 5.08 (d, 1H, J1,2 = 1.0 H2, H-IA), 5.05 (d, 1H, J1,2 = 8.3 Hz, H-1D), 4.93 (d, 1H, JI,2 = 3.1 Hz, H-lE), 4.87 (d, 1H, J,,2 = 1.0 Hz, H-lg}, 4.82 (d, 1H, JI,2 = 1,7 Hz, H-lc), 4.80 (dd, 1H, J3,~
=J4,s = I0.0 Hz. H-4~), 4.76 (dd, 1H, Ja,~ = 9.5 Hz, H-3o), 4.75-4.30 (m, 16H, CH2Ph), 4.57 (d, 1H, J,,~ -- 7.8 LMPPl4~xx~brevet-iynlongs Hz, H-la~), 4.35 (dd, 1H, H-2a), 4.30 {dd, 1H, JZ,3 = 10.0 Hz, J3,a = 9.6 Hz;
H-3D), 4.02 (dd, 1H, JZ,~ = 2.0 Hz, H-2"), 4.00-3.60 (m, IGH, H-6ao, GbD, 3E, 4~, SE, 6aE, 61~, 3c, 4~, 5c, 3B, 3", 5~, 2a~, 6ao~, 6bo~), 3.48 (m, 1 H, J~,S = 9.5 Hz, H-SB), 3.46 (dd, 1 H. H-4p), 3.40 (m, 1H, H-5D), 3.36 (dd, 1H, H-2E), 3.35, 3.19 (m, 4H, OCH~CHzNz), 3.30 (dd, IH, H-4A), 3.I9 (dd, 1H, J3,4 = 9.5 Hz, H-4B), 3.17 (m, IH, H-SD), 3.02 (m, IH, H-2D), 1.90-1.60 (6s, 18H, CH,C=0), 1.33, 1.26 {2s, GH, C(CH3)2), 1.27 (d, 1H, J5,6 = 6.2 Hz, H-G,,), 1.18 (d; 3H, J5,6 = 6.1 Hz, H-6c), 0.90 (d, 3H, Js,s = 6.lHz, H-6H). '3C NMR (CDCh):S 172.1, 171.1, 170.8, 170.1, 169.6, 166.2 (6C, C=0), 139.2-127.1 (Ph), 103.05 (C-1D.), 101.6 (C-1H), 101.0 (C-lA), 140.0 {C-1D), 98.1 (C-lE), 97.8 (C-1~), 82.0 (C-2E), 81.7, 81.5, 80.2, 78.6, 78.4. 77.9, 77.9 ($C, C-3s, 4E, 3c, 4c, 3s, 4~, 3n, 4,~), 77.8 (C-Z,,), 76.0, 74.6 (2C, C-3a, 3a~), 74.0 (C-Ze), 73.4 (C-4a), 73.3 (C-2c), 72.2, 71.9 (2C, C-So, So~), 68.9, 68.8, 67.7 (3C, C-SA, 5B, SE), 68.6 (C-4a~), 68.5 (C-6E), 67.5 (C-5~), 62.6, 62.2 (2C, C-6o, 6n~), 59.7 (C~2p), 54.6 (C-2D~), 51.0 (CH~N~), 29.5 (C(CH3)2), 23.9, 23.5, 21.1, 20.9, 20.7 (C=OCH3), 19.6 (C(CH3)z), 18.9 (C-G~), 18.4 (C-6n), 18.2 (C-6B). FARMS of C"~Hi3~N~03~ (M, 2085.3), m/z 2107.9 [M+Na]' L~l4Qp-~1C112~8yhl0flg9 C~1 02434685 2003-07-04 N, ~mNI:nNsou E :nu Mass: 2oaJ,eG
Mol. Wr.:20aS,2J
C 65,19: H 6,J6; N 3 42, 0 25,03 AlphaD=t9', c= I, CHCI3 2-Azidoethyl (2,3,4-tri-D-acetyl-2-deoxy-2-acetamido-~i-D-glucopyranosyl)-(1--~2)-(3,4-di-O-benryi-a-L-rhamnopyraaosyl)-(1-~2)-(3,4-di.0-benzyl-a-L-rhamnopyranosyl)-(1-~3)-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1-~4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1-~3)-2-acetamido-2-deoxy-[i-D-glucopyrnnoside (X).
To a solution of X (503 mg, 241 ~.mDl) in AcOH (6 mL) was added dropwise, water (1.5 mL) at rt. The mixture was stirred for 1 h at 60°C then concentrated by successive coevaporation with water and toluene. The residue was eluted from a column of silica gel with 1:4 Cyclohexane-EtOAe to give X as a white foam (463 mg, 94 %); [aJD +9° (c l, CHCh).
1H NMR (CDCI3):cS 8.00-7.00 (m 45H, Ph), 5.70 (d, 1H, NHD), 5.46 (d, 1H, Jz,r~., = 8.0 Hz, NFiD~), 5.25 (dd, 1H, H-2~), 5.05 (d, 1H, J ,,2 = 8.4 Hz, H-1D), S.Oa (d, 1H, Jl,z = 1.0 Hz, H-l,,), 4.86 (m, 3H, H-lc, 3n', 40~), 4.84 (m, 2H, H-la, lE), 4.56 (d, 1H, H-ln~)~ 4.40 (dd, 1H, H-3E), 4.35 (dd, 1H, H-2$), 4.15 (dd, 1H, H-3D), 4.80-4.00 (m, 1GH, C.H2Ph), 4.03 (dd, IH, H-2a), 4.00-3.00 (m, 26H, H-4D, SD, 6aD, 6bn, 2E, 4E, Se~ 6a6, 6be, 3c. 4c, Sc, 3a, 4a, SH~ 3a~ 4n~
SA, 2D~, SD., 6aD~, 6tro~, OCH2CH2NJ), 2,99 (m, IH, H-2p), 1.85-1.60 (Ss, 1SH, CHJC=O), 1.25 and 0.85 (3d, 9H, H-6A, 6H, 6c). 13C NMR (partial) (CDC1J):S 171.6, 171.4, 170.8, 170.1, 169.6 (C=0), 140,0-I27.I (Ph), 103.I (C-1~), 101.2 (C-l,,), 99.G (2C, C-IE, 1B), 99.4 (C-1D), 99.0 (C-1~), 23.8, 23.5 (2C, NHC=OCH3), 21.1, 20.9, 20.8 (3 CHJC=0), 19.1.
18.5, 18.2 (C-LMPPI4~t~6t~~atttynlongs 6~, 6s, 6~). FABMS of C~ nHiz9Ns03a (M, 2045.2 j, m/z 2067.9 [M+Na]''. Anal.
Calcd for C",H,i9N503z C: 65.19, H: 6.36, N: 3.42. Found C: 65.12, H: 6.51. N: 3.41.

L1~P14.ex~brNCt~synlcng~

Ho---1 o~
~

HNO ~~ NN

~

~,~, ,, 0.

~ tow C ,H N O
Ho Ho Exact Mass 1067 Mol. Wt.: Io68,U324 ~
Ho H C 47,23: H 6.89;
N 3,93; O 41,94 ~
HO

'NLH
~O

2-Aminoethyl (2-deoxy-2-acetamido-ji-D-glucopyranosyI)-(1-~Z)-(a-L-rhamnopyranosyl)-(1~2)-( a-L-rhamnopyranosyt)-(I-~3)-[a-D-glucopyrauosyl-(1-~4)j-( a-L-rhamnopyranosyt)-(1--r3)-2-acetamido-2-deoxy-[i-D-glueopyranoaide (X).
A mixture of X (207 mg, 101 utnol) in MeOH (5 mL) was treated by MeONa until pH=9. The mixture was stirred 1 week at rt. IR 120 (Fib was added until neutral pH and the solution was filtered and concentrated. The residue was eluted from a column of silica gel with 20:1 to 15:1 DCM-MeOH to give an am~oiphous residue. A solution of this residue in EtOH
(2.2 mL), EtOAc (220 ~,L), IM HC1 (I72 il.L, 2 eq) was hydrogenated in the presence of PdIC (180 mg) for 72 h at rt. The trlikrture was filtered and concentrated , then was eluted from a column of C-18 with wated and freeze-dried to afford amorphous X (81 mg. 77 %); [a]D -10° (c 1, Hz0).
'H NMR partial (D20):8 5.12 (d, 1H, Jt,? = 3.4 Hz; H-ls), 5.07 (d, 1H, J~,2 =
1.0 Hz, H-1R~), 4.94 (d, 1H, J,,Z = 1.0 Hz, H-1Rh"), 4.75 (d, 1H, J,,Z = 1.0 Hz, H-1R~), 4.63 (d, 1H, JI,Z = 8.35 Hz, H-lGlcNx), 4.54 (d, 1H, J,,i = 8.3 Hz, H-Ic,°,,u), 1.98 and I.96 (2s, 6H, 2 C'H3C=ONH), 1.28-1.20 (m, 9H, H-6,,, 68, 6~). "C NMR partial (Dz0):8 175.2, 174.8 (C=O), 103.1 (C-lp~), 101.6, 101.4 (3C, C-lA, 18, 1~) 100.8 (C-ID), 97.9 (C-lE), 56.2, 55.4 (2C, C-2p, 2a), 22.7, 22.6 (2 NHC=OCH3), 18.2, 17.2, 17.0 (3C, C-6", dB, 6~). HRMS: calculated for C4aH~3N30ie+Na: 1090.4278. Found 1090.428&.

LtvtPP 14-exp-brevet ;rynlongs 8z ~~nIHtISN~:a Exact Masb. 1725.7845 Mol, l~'t.~ 1726,9861 70,24; H 6.71; N 0,81; 0 22,23 Aityl (2-acetamido-4,6-0-isopropylidene-Z-deoxy-~-n-glucopyranoayl)-(I~2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1~2)-(3,d-di-0-benzyl-a-L-rhamnopyranosyl)-(1~3)-[2~,4,6-tetra-O-benzyl-a-la-glucopyranosyl-(1--~4)-]~2-0-benzoyl-a~L-rhamnopyranoaide (X).
The pentasaccharide X (2.65 g, 1.47 mmol) was dissolved in MeOH (20 mL). MeONa was added until pH=10. The mixture was stinted for 25 min, then treated by IR 120 (H'') until neutral pH. The solution was filtered and concentrated. The residue was eluted from a column of silica gei with 9 :1 DCM-MeOH to give the expected triol which was then treated by 2,2-dimethoxypropane (11 mL,, 0.1 mot) and APTS (20 mg, 0.17 mmolj in DMF (20 mL) overnight. Et3N was added and the solution evaporated. The residue was eluted from a column of silica gel with 1:1 Cyclohexane-AcOEt and 0.2 % of Et3N to give X as a white foam (2.05 g, 81 % from X); [a)D +3° (c 1, CHCl3).
NMR (CDC~) :'H b 6.98-8.00 (m 45H, Ph), 6.17 (bs, 1H, NHD), 5.82 (m, 1H, All), 5.30 (dd, 1H, J1,2 = 1,0, J~,3 = 3.0 Hx, H-2~), 5.11-5.25 (m, 2H, All), 5.06 {bs, 1H, H-I"), 4.92 (d, 1H, J1,~ = 3.I Hz, H-lE), 4.88 (d, 1H, J1.2 = 1.6 Hz, H-IB), 4.84 (bs, 1H, H-lo), 4.35 (d, 1H, H-1D), 4.34 (dd, 1H, H-2H), 4.20-4.80 (m, 16H, CHZPh), 4.05 (dd, 1H, H-2,,~, 3.36 (dd, 1H, H-2e)t 2.90-4.10 (m, 22H, All, H-2D, 3n, 3>j, 3c. 30~ 3E~ 4n~ 4s~ 4c~ 4v. 4E, Sn. Sa, Sc~ SD~ Sa, 6aD, LMPPl4~ex~brrvct-sy~lengs 6bp, GaE, 6b~, 1.5 (s, 3H, FIcNH), 1.2-0.9 (m, 15H, C(CH3)2, H-6A, 6H, 6c). "C
8 ; 172.7 (C=0), 164.9 (C--0), 137.7-116.? (Ph, All), 102.3 (C-1D), 100.2 (C-1g), 100.0 (C-I,,), 98.9 (G(CH,)z), 97.2 (C-lE), 95.1 (C-1~), 82.1, 82.0, 81.8, 81.6, 80.6, 80.3, 79.0, 78.8, 78.3, 77.8, 77.6, 75.7, 75.6, 75.0, 74.3, 72.8, 71.8, 71.6, 70.8, 70.3, 69.0, 68.5, 67.8, 67.4, 61.9, 60.8, 60.5, 29.4 (C(GH,)2), 22.7 (AcNEi), 19.0 (C(C'H3)z), 18.9, 18.4, 18.2 (3C, C-6,,, 68, Gc). FAB-MS for C,orHrtsNOZa (M = 1726.9) »r/a 1749.7 [M + Naj'. Anal. Calcd. fnr CrotHt rsNOza.H?O : C, 69.52 ; H, 6.76 ; N, 0.80. Found C, 69.59; H 6.71 ; N, 0.57.

LMPP la.cspbrevet-eynlanga i O°n °8 0 enc 1 ~,..,,~

0~ IOBz .~ 0~
BnO
Ctc:Hi i~NO~s Exact Mss: 1767,7915 Mol. Wt.'. 1759,0225 C 69,93: H 6,67; N 4.79; O 22,61 Attyt (2-acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-[i-D-glucopyraaosyl)-(1-32)-(3,4-di-O-benzyl-a-L-rhamnopyrunosyl)-(1->2)-(3,4-dl-O-benzyl-a-L-rhamnopyranosyl)-(l.-33)-[2,3,4,6-tetra-O-benzyl-a-D-gluoopyranosyl-(1-~4)-)-2-O-benzoy 1-a-t.-rhamno.pyranoside (X).
a) A mixture of X (2.05 g, 1.19 mmol) in Pyridine (GO mL) was cooled to 0°C. AcaO (20 mL) was added and the solution was stirred 2,5 h. The solution was concentrated and coevaporated with toluene. The residue was elutEd from a column of silica geI with 2:1 Cyclohexane-AcOEt and 0,2 % of Et3\T to give X as a white foam (1.99 g, 94 %); [a]D +I°
(c I, CHCIJ).
b) A mixture of X (144 mg, 0.06 mmol), Bu3SnH (0.1 mL, 0.37 rnmol) and AIBN
(10 mg) in dry toluene (3 mL) was stirred for 1 h at rt under a stream of dry Ar, then was heated for I.5 h at 90°C, cooled and concentrated. The residue was eluted from a column of silica gel with 2:1 cyclohexane-BtOAc and 0.2 % of Et3N to give X (100 mg, 74 %).
NMR (CDC13) : IH 5 6.95-8.00 (m, 4~H, Ph), 5.82 (m, 1H, All), 5.46 (d, 1H, Jz,NH = 8.0 Hz, NHD), 5,29 (dd, 1H, Jt,2 =' 1.0, Jz ~ = 3.0 Hz, H-2c), 5.11-5.25 (m, 2H, All), 5.00 (bs, 1H, H-lA), 4.90 (d, 1H, J,,2 = 3.1 Hz, H-lE), 4.85 (d, 1H, Jl,z = 1.6 Hz, H-18), 4.83 (bs, 1H, H-lc), 4.70 (dd, 1 H, Jz.3 = .13,4 = 10.0 Hz, H-3 p), 4,44 (d, 1 H, H-1 a), 4. 34 (dd, 1 H, H-2B), 4.20-4. SO
(m, 16H, CHzPh), 4.02 (dd, 1H, H-2A), 3.37 (dd, 1H, H-2s), 2.90-4.10 (m, 21H, All, H-2D, 3A, 3a, 3c, 3E, 4A, 4H, 4c, 4a, 4E, 5.,, 58, Sc, SD, 5fi, 6aD, 6ba, GaE, GbE), 1.92 (s, 3H, OAe), 1,57 (s, LMPP l4~exp.breve4synlongc 3H, AcNH), 1.27-0.90 (m, 15H, C(CH3)Z, H-6A, 6g, Gc). '3C o 171.3, 170.3, 1GG.2 (C=0), 138.7-117.9 (Ph, All), 103.9 (C-1D), 101.5 (C-1B), 101.4 (C-1~), 99.9 (C(CH3)~), 98.5 (C-IE), 96.3 (C-lc), 82.1, 81.7, 81.6, 80.3, 80.1, 78.8, 78.1, 77.8, 76.0, 75.8, 75.3, 75.1, 74.7, 74.2, 73.6, 73.3, 72.7, 71.9, 71.4, 70.8, 69.0, 68.8, 68.7, 58.4, 68.1, 67.8, 62.1, 55.0 (C-2o), 30.0 (C(CH3)z), 23.5 (AcNH), 21.6 (OAc), 19,2 (C(CH3)a), 19.0, 18.3, 18.2 (3C, C-dA, 6H, 6~).
FAB-MS for C,olH"~NO25 (M = 1769.0) mlz 1791.9 [M + Na]+. Anal. Calcd. for C,o3H,l,NO~s : C, 69.93 ; H, 6.67 ; N, 0.79. Found C, 69.77; H, 6.84; N, 0.72.

LMPPIS-cxp~6revet.rynlengs NH
~OBn ~
--~-~ p~ 0'r \CCia BBnp Bn0 o ~_~J ClntxtuCl~NaOts ~J~/ Exsct Mass: 1870,6698 Mol. Wt 187?.3~:2 ~ C 65,A0; H 6,08; CI 5,68; N L50; 0 11,35 (Z-acetamido-3-O-acetyl-4,6-O-itoprapylideec-Z-deozy-(3-D-glucopyranosyl)-(1-->2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1~~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1--~
3)-jZ,3,4,6-tetra-0-benzyl-a-D-glucopyranosyi-(1--~4)-]-2-O-benzoyl-ac-L-rhamnopyranosyl ttichlaroacetimidate (X).
1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (50 mg, 58 a mol) was dissolved tetrahydrofuran (10 mL), and the resulting red solution was degassed in an argon stream Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream, A solution of X (1.8 g, 1.02 mmol) in tetrahydrofuran (ZO mL) was degassed and added. The mixture was stirred at rt overnight then concentrated to dryness. The residue was dissolved in acetone (9 mL), then water (2 mL), mercuric chloride (236 mg) and mercuric oxide (200 mg) were added successively. The mixture protected from light was stirred at rt for 2 h and acetone was evaporated. The resulting suspension was taken up in DCM, washed twice with 50% aq K!, water and Satd aq NaCI, dried and concentrated. The residue was eluted from a column of silica gel with 3:2 Cyclohexane-AcOEt and 0.2 % Et3N to give the corresponding hemiacetal.
Trichloroacetonitrile (2.4 mL) and DBU (72 uL) were added to a solution of the residue in anhydrous dichloromethane (24 mL) at 0°C. After 1 h, the mixture was concentrated. The LI~ffP! 4.cxp-brevet-synlon~

residue vas eluted tom a column of silica gel with 3;2 Cyclohexane-AcOEt and 0.2 % Et3N to give X as a colorless oil (1.58 g, 82 %); (a)a +2° (c 1, CHCI3).
NMR (CDC13) : 'H s 8.GZ (s, 1H, C=NH), 6.95-8.00 (rn, 45H, Ph), 6.24 (d, 1H, Jl,i = 2.6 Hz, H-lc), 5.48 (dd, IH, J2,3 = 3.0 Hz, H-2e), 5.41 (d, 1H, J,,N~r = 8.4 Hz, NHp), 4.99 (bs, 1H, H-lA), 4.92 {d, 1H, Jt,~ = 3.2 Hz, H-lE), 4.88 (d, 1H, Jl,z = 1.6 Hz, H-lg), 4.G9 (dd, IH, Jz,3 =
J3.a = 10.0 Hz, H-3n), 4.44 (d, 1H, H-1D), 4.34 (dd, 1H, H-2H), 4.20-4.80 (m, 16H, CHZPh), 4.02 (dd, 1H, H-2~), 3.38 (dd, 1H, H-2E), 2.90-4.10 (m, 19H, H-2D, 3", 38, 3~, 3x, 4A, 4H, 4~, 4D, 4E, 5,,, 5B, Sc, Sp, 5E, 6aD, 6bn, 6a~, 6b~), 1.95 (s, 3H, OAc), 1.55 (s, 3H, AcNH), 1.30-0.85 {m, 15H, C(CH3)z, H-6", 6B, 6c). "C b 172.4, 171.4, 166.9 (C=O), 140.2-128.9 (Ph), 104.2 (C-1D), 101,4 (2C, C-IA, 1$), 101.1 (C(CH3h), 98.0 (C-1~), 94.8 (C-lc), 92.4 (CC13), 82.1, 81.5, 80.2, 80.1, 78.6, 78.1, 77.8. 77.6, 76.0, 75.8, 75.5, 75.0, 74,3, 74.2, 73.5 (C-3o), 73.4, 71.9, 71.4, 71.0, 70.5, 69.2, G8.8, 68.3, 68.1, 62.1, 54.9 (C-2D), 29.3 (C(CH3)i), 23.4 (AcNi-I), 21.4 (OAc), 19.2 (C{CH,)z), 19.0, 18.2, 18.1 (3C, C-GA, 6B, 6C). FAB-MS for C~ozHmCl3Naaas (M = 1873.3) m/z 1896.3 [M + Na]'". Anal. Calcd. for C,ozH~
oCl,No02s : C, 65.40 ; H, 6.08 ; N, 1.50. Found C, 65.26; H, 6.02; N, 1.31.

LMPP l4erpbcevet~synl°t~a a lsHi33Ns0:°
t Mass; 2039.9035 ',. Wt.: 2041,2808 ~ G,57; N 3.43; 0 23,51 2-Azidoethyl (2-acetamido-3-O-acetyl-2-deoxy-4,6-D-isopropylidene-(i-D-glucopyranosyl)-(1~2)-(3,4-di-0-benzyl-a-L-r6amnopyranosyl)-(1~2)-(3,4-di-0-benzyl-a-trrhamnopyranosyl)-(1~3)-(2,3,4,6-tetra-O-benzyl-a-u-glucopyranosyl-(1~4)j-(2-O-benzoyl-a-z-rhamnopyranosyl)-(1 >3)-2-acctamido-2-deoxy-4,6-O-fsopropylidene-(3-n-glucopyranoside (X).
A mixture of donor X (745 mg, 0.4 ~l) and acceptor X (170 mg, 0.51 mmol), 4 ~
molecular sieves and dry 1,2-DCE (12 mL), was stirred for 1 h then cooled to 0°C. Triflic acid (25 NL) was added. The stirred mixture was allowed to reach rt in 10 min then stirred again for 2.5 h at 75°C. After cooling to rt, Et,N (I00 p.L) was added and the mixture filtered. After evaporation, the residue was eluted from a column of silica gel with 1:2 Cyclohexane-AcOEt and 0.2 % Et3N to give X as a white foam (615 m;, 76 %); [a]o +0° (c 1, CHCI,).
NMR (CDCI,) : iH 8 6.95-7.90 (m, 45H; Ph), 6.02 (d, 1H, Ji,N,.i = 7.1 Hz, NHa), 5.4G (d, 1H, J2,NH " 8.6 Hz, NHD~), 5.20 (dd, IH, J,_Z = 1.0, J2,; = 3.0 Hz, H-2c), 5.03 (d, 1H, J,,, = 8.1 Hz, H-1D), 5.02 (bs, IH, H-1,~), 4.92 (d, 1H, Jl,~ = 3.1 Hz, H-lE), 4.85 (d, 1H.
J,,Z = 1.6 Hz, H-1B), 4.82 (bs, 1H, H-lc), 4.70 (dd. 1H, H-3o~), 4.44 {d, 1H, H-1D-), 4.30 (dd, 1H, H-2B), 4.20-4.$0 (m, 16H, CI~Ph), 3.99 (dd, 1H, H-2,,), 3.37 (dd, 1H, H-2E), 2.90-3.95 (m, 29H, H-2D, 2nv 3~, 3s, 3c, 3ti, 3e. 4A, 48, 4c, 4n, 4D~, 4E, Sn. Sp. Sc, 5D, SD~, $e, Gao, 6bo, GW, Glfi', 6aE, 6bE, OCHZCHZN3), 2.00 (s, 3H, AcNH), 1.92 (s, 3H, OAC), I .57 (s, 3H, AcNH), 1.27-0.90 (m, L;~3F I 4.exp-brevet~syTlongs 21H, 2 C(CH3)z, H-6A, 6~, 6c). 13C o 172.1, 171.5, 170.3, 1GG.2 (C=O), 139.0-127,7 (Ph), 103.9 (C-1D.), 101.7 (C-1H), 101.2 (C-I~), 100.0 (C-la), 99.9, 99.8 (2C, C(CH3)z), 98.3 (C-lE), 97.8 (C-lc), 82.0, 81.7, 81.5, 80.8, 80.2. 80.1, 78.9, 78.6, 78.0, 77.9, 76.0, 75.9, 75.8, 75.3, 74:8, 74.6, 74.2, ?4.0, 73.6, 73.5, 73.4, 73.0, 71.9, 71.4, 70.8, 69.1, 69.0, 68.8, 68.6, 68.0, 67.7, 67.6, 62.6, 62.1, 60.8, 59.7 (C-2p), 55.0 (C-2D~), S 1.1 (0(CH~)~N~), 29.5 (C(CH3)Z), 29.3 (C(CHa)z), 23.9 (AcNH), 23.5 (AcNH), 21.3 (OAc), 19.7 (C(CH3)~), 19.2 (C(CH3)2), 18.8, 18.4, 18.2 (3C, C-6,,, 6$, 6~). FAB-MS for C113Hu3NsO3o (M =
2041.3) mla 2064.2 [M + Naj+. Anal. Calcd, for C"3H,~3NsO,o : C, 66.49 ; H, 6.57 ; N, 3.43. Found C, 65.93; H, 6.57; N, 2.61.

LMPPId-apbre et-oynlongs C:nHmVsOtq tSact t.4acs: 1997.19 Mal. Wt.: 199924 C 68.fiB: H 6.60: N 3.50; 0 23,21 AIphwD=~t °, c t, CHCt?
2-Azidoethyl (2-acetamido-2-deoxy-4,6-D-isopropylidene-~-n-glucopyranosyt)-(1~2)-(3,4-di-D-benzyl-a-L-rhamnopyranosyi)-(1-~2)-(3,4-di-0-benzyi-a-trrhamnopyranosyt)-(1-->3}-(2,3,4,6-tetra-0-benzyl-ot-D-glucopyranosyl~(1--~4)]-(2-0-benzoyl-a-tJ-rhamnopyranosyl)-(1->3)-2-acetamido-Z~deoxy-4,6-O-isopropylidene-(3-n-glucopyranoside (X).
a) The hexasaccharide X (G15 mg, 0.30 mmol) was dissolved in MeOH (8 mL).
MeONa was added until pH=9. The mixture was stirred for 3 h then treated by IR I20 (H') until neutral pH.
The solution was filtered and concentrated. The residue was eluted fiom a column of silica gel with 25:1 DCM-MeOH and 0.2 % of 1?t?N to give X as a white foam (590 mg. 97 %); [a]ti +1° (c l, CHCh).
b) To a mixture of X (770 mg, 370 ~mol) in MeOH (S mL) vras added MeONa until pH=9.
The solution was stirred for 4U min, Amberlite IR 120 (Hf) was added until neutral pH and the mixture was filtered and concentrated. The residue was eluted fxom a column of silica gel with 20:1 DCM-MeOH and Et,N to give a residue which was dissolved in DMF (2 mL).
The mixture was treated by 2-methoxypropene (200 pL, 2.1 mmol) and CSA (20 mg) at rt. After I
h, more 2-methoxypropene (200 pL) was added and the mixture was stirred 1 h.
Et?N (IGO

LMPPl4.c~cp~bm~ea~aynlongs ~L) was added and the solution was concentrated. The residue was eluted from a column of silica gel with 2:3 toluene-EtOAc and Et3N (0.2 %) to give X (400 mg, 54%).
~H NMR (CDCl3):o 8.00-7.00 (m, 45H, Ph), 6.10 (d, 1H, NHp~), 6.05 (d, 1H, .h,~= 7.4 Hz, NHo), 5.20 (dd, 1H, Jt,~ = 1.7 Hz, J2,3 = 3.0 Hz, H-2c), 5.10 (d, 1H, J,,z =
1.OHz, H-In), 4.99 (d, 1H, Jt,~ = 8.3 Hz, H-lnj, 4,96 (d, 1H, J,3 = 3.2 Hz, H-le), 4.90 (d, 1H, J,,a = I.0 Hz, H-1B), 4.86 (d, 1H, J,,z= 1.0 Hz, H-ic), 4.52 (d, 1H, J~,2= 7.5 Hz, H-lp~), 4.37 (dd, 1H, H-2a), 4.Z2 .(dd, 1H, H-3o), 4.02 (dd, 1H, H-2A), 4.80-4.00 (m, 16H, C~IzPh), 4.00-2.95 (m 30H, H-2D, 4D~ Sn, 6an~ ~bn~ 2E, 3s~ 4E~ Ss~ 6aE~ dbe., 3c~ 4c, Sc, 3a, 4B~ SH, 3A~
4n~ Sn~ 2D~, 3n~~ 4DV Snv 6a~, bb~, OCHzCHaN3), 2.00-0.92 (6s, 3d, 27H, 2 CH3C=0, 2 C(CH,)Z, H-6A, 68, Gc). ~jC
NMR (CDCI3) partial: cS 173.9, 172.1, 166.3 (C=0), 140.0-125.0 (Ph), 103.6 (C-lp), 101.7 (C-1$), 101.2 (C-IA), 100.2 (C(CH3),), 100.2 (C-1D), 99.9 (C(CH,3)Z), 98.2 (C-lE); 97.8 (C-lc), 29.4, 29.3, 23.9, 22.8, 19.6, 19.2, 18.9., 18.4, 18.2 (C-6A, 6B, ~c, 2 CH3C=0, 2 C(CH3)z).
FAB-MS for C,nH,3iNs0~9 (M = 1999.2) m/z 2021.8 [M + Na]'. Anal. Calcd. for Ct~~H~31~'SO~9 : C, 66.68 ; H, 6.60 ; N, 3,50. Found C, 66.63 ; H, 6.78 ; ~', 3.32.

CMPPI4ccpbceve~synlangs cl, C91 H96C ~)N~Z4 Exact Mass; I627,56 Met. Wt.: 1630.09 ? 5,94; Cl 6,5?.; h 0.86; 0 19.6'3 tphaD=T22' C=1. CHCf3 (2-0-acetyl-3,4-di-O-benzyl-a-L-r6amnopyrnnosyl)-(1 >2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1->3)-(2,3,4,6-tetra-O-benry1-a-D-glacopyranosyt-(1-~4))-2-O-benzoyi-a-L-rhamnopyranosyl trichloroacetimidate (X).
1,5-Cyclooctadiene-bis{rnethyldiphenylphosphine)iridium hexafluorophosphate (80 mg, 93 ~
mol) was dissolved tetrahydrofuran (10 rnL), and the resulting red solution was degassed in an argon stream. Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution leas then degassed again in an argon stream. A
solution of X (2.55 g, 1.G7 mmol) in te~trahydrofuran (20 mL) was degassEd and added. The mixture was stirred at rt overnight then concentrated to dryness. The residue was dissolved in acetone (15 mL), then water (3 mL), mercuric chloride (380 mg) and mercuric oxide (320 mg) were added successively. The mixture protected from light was stirred at rt for 2 h and acetone was evaporated. The resulting suspension was taken up in DCM, washed twice writh 50% aq KI, water and satd aq NaCI, dried and concentrated. The residue was eluted from a column of silica gel with 3:1 petroleum ether-EtOAc to give the corresponding hemiacetaL
Trichloroacetonitrile (2.0 mL) and DBU (25 uL) were added to a solution of the residue in anhydrous dichloromethane (15 mL) at 0°C. After I h, the mixture was concentrated. The residue was eluted from a column of silica gel with 3:1 petroleum ether-EtOAc and 0.2 LMPPI4~ecp~brcvet-synlon~

EtSN to give X as a white foam (1.5 g, 56 %); [a]D +22° (c 1, CHC13).
'H NMR (CDC13):b 8.72 (s, 1H, C=NH), 8.00-7.00 (m, 45H, Ph), 6.39 (d, 1H, Ji,a = 2.5 H2, H-lc), 5.60 (dd, 1H, JZ,~ = 3.0 Hz, H-2~), 5.58 (dd, 1H, Jl,z = 1.7 Hz, Jz,3 =
3.0 Hz, H-2a), 5.12 (d, 1H, J~,z = 3.2 H~. H-lE), 5.08 (m, 2H, H-ln, Is), 5.00-4.00 (m, 16H, CHzPh), 4.20 (dd, 1H, H-3c), 4.05 (dd, IH, H-3E), 4.00-3.35 (m, I4H, H-2E, 4E, SE, GaE, 6bE, 4c, 5c, 2B, 3a, 4B, SB, 3~, 4," S,J, 2.05 (s, 3H, C=OCHj), 1.42, 1.36 and 1.00 (3d, 9H, H-6n, 6s, 6c). '3C
NMR (CDCl3):b 170.3, 165.8 (G=0), 138-127 (Ph), 99.9 (zC, C-lA, 1B), 98.5 (C-lE), 94.7 (C-lc), 82.1, 81.2, 80.4, 80.0, 79.1. 78.1, 78.0, 75.2, 71.7, 71.2, 70.7, 69.5, 69.4, 68.? (16C, C-2n, 3n, 4n, 5", 2e~ 3s. 4B. 5a, 2c~ 3c. 4c~ ~c~ 2s, 3s. 4a. 5E), 76.0, 75.7, 75.5. 75.1, 74.3, 73.3, ?2.2, 71:2 (8C, PhCI-ia), 68.5 (C-6F), 21.4 (C=OGH~), 19.2, 18.5, 18.1 (C-6A, 6B, 6c). Anal.
Calcd. for C9,Hg6C~NO20 : C, 67.05 ; H, 5.94 ; N, 0.86. Found C, 66.44 ; H, 6.21 ; N, 0.93.

LMPPI4~cp~brc~ct-synlongs N~

~cot1»s~:Qat ;t Mt9s. 3464,53 .l. Wt.:3466,93 6,54; N ~,a2; 0 22,15 , CHCt3 2-Azidoethyl (2-O-acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1 >2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1-a3)-[2,3,4,b-tetra-D-benzyl-a-D-glucopyranosyl-(1-~4)J-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1--~3)-(2-acetamido-Z-deoxy-4,6-0-isopropylidene-[i-D-glucopyranosyl)-(1~2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1--~2)-(3,4-di-O-benzyl-a-L-rhamaopyranosyl)-(1-~3)-[Z,3,4,6-tetra-O-benryl-a-D-gtucopyranosyl-(1-~4)]-(2-O-benzoyl-a-L-rhamnopyranosylj-(1--~3r2-acetamido-2-deory-4,6-0-isopropylidene-[i-D-glucopyranoside (X).
A mixture of alcohol X (110 mg, 55 umol), imidate X (179 mg, I 10 1t1n41) and 4A molecular sieves in anhydrous DCE (2.5 mL,) was stirred for 1 h under dry Ar. After cooling at -35°C, TfDH (5 uL, SD ~.m~ol) was added dropwise and the mixture was stirred for 2.5 h and allowed to reach 10°C. Et3N (25 p.L) was added and tile mixture was filtered and concentrated. The residue was eluted from a column of silica gel with 4:1 to 3:1 toluene-EtOAc and Et3N (0.2 °.'°) to give X as a white foam ( 158 mg. 82 %); [a]D +18° (c I, CHC~).

LW(pPl4s~cøbrcve4symlongs rH NMR (CDCis):& 8.00-6.90 (90H, m, Ph), 5.90 (d, 1H, Ji,,.~ = 7.0 Hz, N-HD), 5.58 (d, 1H, Jz.uf, = 7.5 Hz, N-HD.), 5.45, 5.22 (m, 2H, J,,2 = 1.0 Hz, Jz,a = 2.0 Hz, H-2c, 2c~), 5.12 (dd, 1 H, H-2,,~), 5.I 1 (d, 1H, Ji,z = 8.3 Hz, H-ID), 5.05 (d, IH, Jl,~ = 1.0 Hz, H-lA), 5.01 (d, 1H, J,,~
3.2 Hz, H-lE), 4.96 (d, 1H, J,,~ = 1.0 Hz, H-Ic), 4.94 (m, 2H, H-lp, 1H), 4.86 (d, IH, H-1B), 4,82 (d, 1H, H-lc), 4.72 (d, 1H, H-1~,), 4.70 (d, 1H, H-lA~), 4.90-4.20 (m, 36H, 16 OCXiPh, H-2s, 2B', 3n, 3rr): 4.00-2.90 (m, 45H, H-2D, 4n, SD, Gao, 6UD, 3c, 4c~ 5c, 2a, 3s, 4s, 5s, Gas.
6be, 3a. 4e~ Sa. 2n, 3n: 4~, 5w: 2n~: 4a~ Sn~: 6a~. Gba, 3c~, 4c~, Sc~, 2s., 3s~, 4E~, SE~, Ga~~, GbB~, 3g', 4H., SB~, 3A~, 4"~, 5"., OCHICHzN~), 2.00 (s, 3H, AcNH), 1,88 (s, 3H, OAc), 1.86 (s, 3H, AcNH), 1,40-0.82 (m, 30H, G H-6R,~, 2 C(CH3)Z). "C NMR (partial) (CACI~):8 172.1, 171.4, 170.2, 166.2, 165.9 (C=O), 102.7 (C-1B~), 101.6, 101.2 (2C, C-1H, 1B~), 101.1 (C-ln), 99.8 (C-1D), 99.7 (C-Ic), 98.2 (2C, C-lE, 1,,~), 97.2 (ZC, C-tc, IE), 63.3, 62.6 (2C, C-6E, 6E'), 60.0, 57.8 (2C, C-2o, 2D~). S 1.0 (OCH2CHZNl), 29.5, 29.4 (2C, C(CH3)Z), 24.0 (2C, 2 AcNH), 21.3 (Ac0), I9.6, 19.5 (2C, C(CH~)2), 19.I, 18.9, 18.8, 18.5, 18.2, 18.1 (6C, C-6A, 68, 6c, 6n~, 68~, 6~~). FABMS of CzooHnsNsOaa (M, 3446.9), nr/z 3489.5 ([M+Na]'). Anal. Calcd for C2uoHiuNsOae +SHiO, C: 67.47, H: 6.65, N: I.96. Found C: 67.40, H: 6.57, N:
1.72.

LMPP 14-exPbmcMynt°n~
Ho !pH HO

NOp HO , ~,.,~,,~
0..
_0' OOH

HO
Hp H
OH HO~O, Oa~W i:N30,s Exact MQ$F; 1667.66dG
Mol, Wt ~ 1668,5966 C 47.St: H 6.93: N 2.52; 0 43,15 2-Aminoethyl (a-L-rhamnopyranosyl)-(1~2)-(a-L-rhamnopyranosyl)-(1~3)-[a-D-glucopyranosyl-(1-~4)]-(oc-L-rhamaopyranosyt)-(1-~3)-(Z-acetamido-Z-deoxy-[i-D-gtucopyranosyl)-(1-~2)-(a-L-rhamnopyranosyl)-(I~2)-(a-L-rhamnopyranosyt)-(1--~3)-[a-D-glucopyranosyl-(1--1~4)]-(a-L-rhamnopyranosy I)-(1-~3)-2-acetamid o-2-deoxy-[i-D-glucopyranoside (X)-A mixture of X (130 mg, 38 p,nwl) in MeOH (4 mL) was treated by MeONa until pH=9. The mixture was stirred 1 h ai rt, then heated at 55°C and, stirred overnight. After cooling at rt, IR
120 (H') was added until neutral pH and the solution was filtered and concentrated. The residue was eluted from a column of silica gel with 25:1 to 20:1 DCM-MeOH to give an amorphous residue. A solution of this residue in EtOH (1.5 mL), EtOAc (150 pL), 1M HCl (66 ~L, 2 eq) was hydrogenated in the presence of PdIC (100 mg) for 72 h at rt. The mixture Llvg'Pl4~xpbrevec-syniongs was filtered and concentrated , then was eluted from a column of C-18 with wated and freeze-dried to afford amorphous X as a white foam (41 mg, ?1 %); (a]D -7° (c 1, HBO). 'H NMR
partial (D~0):8 4.90 (m, 2H, J1,2 = 3.S Hz, 2 H-lE), 4.82 (bs, 1H, H-1R,,~, 4.76 (bs, 1H, H-1R,~), 4.72 (bs, 1H, H-IRh~, 4.67 (bs, IH, H-lpr,), 4.52 (bs, 1H, H-IAh°), 4.51 (bs, 1H, H-lp~, 4,41 (d, 1H, J 1,2 = 8.6 Hz, H-louhu), 4.29 (d, IH, JI,Z = 8.6 Hz, H-l~~cua~), 1.77 (s, 6H, 2 CH3C=ONH), I.15-0.96 (m, 18H, H-6Rh,). 1'C NMR partial (D,0):S 174.8, 174.7 (C=O), 102.9 (C-IRS), 102.6 (C-lQ,tNa~), 101,8 (2C, Z C-lRha), 101.6 (C-1R~), 101.4 (C-lRha), 101.3 (C-lah4), 100,8 (C-IotcNzc), 97.9 (2C, 2 C-Ipso), 56.0, 56.4 (2 C, 2 Co~cN~), 22.7, 22,6 (2 NHC=OCH3), 18.2, 17.2, 17.0, 16.9 (bC, 6 C-6R~,). HRMS: calculated for C~sH~,3NsO4s+Na:
1690.6544. Found 1690.6537.

LMPPI4.cx~bmet~synlongs ~o oen o~
eBnD ~ ~ Ns Bn0 OBI
Bn0 8~

~z i ~ Htaa'-'le~s~
Exact Mass: 3707,6426 Mot. Wt:3710,l878 3,3I: H 6.57: N 2,27; 0 22,86 2-Azidoethyl (Z-aeetamido-3-O-acetyl-2-deoxy-4,6-O-isopropylidene-[i-n-glucopyranosyl)-(1--~Z)-(3,4-di-0-benzyl-a-L-rhamnopyranosyI)-(1-~2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1--~3)-[2,3,4,6-tetra-D-benzyl-a-n-giucopyranosy I-(1~4)]-(Z-O-benzoyl-a-L-rhamnopyranosyl)-(1 >3)-(Z-acetamido-Z-deoxy-4,6-O-Isopropylidene-~i-n-glucopyrano9yl)-(1-~Z)-(3,4-di-O-benzyl-oc-z-rhantnopyranosyl)-(1 >Z)-(3,4-di-O-benzyt-a.-c,-rhamnapyranosyl)-(I >3)-[2,3,4,6-tetra-O-benzyl-a-D.glucopyranosyl-(1--~4)j-(2-O-benzoyl-a-cr-rhamaopyranosy I)-(1-~3)-Z-acetumido-Z-deo~y-4,6-O-isopropylidene-~-n-glucopyranoside (X).
A mixture of donor X (835 mg. 0.44 mmol) and acceptor X (590 mg, 0.3 mm.ol), 4 ~
molecular sieves and dry 1,2~DCE (12 mL), was stirred for 1 h then cooled to -30°C. Triflic acid (35 1cL) was added, The stirred mixture was allowed to reach 5°C
in 2.5 h. Et)N (150 pL) was added and the mixture filtered. After evaporation, the residue was eluted from a column of Lfv~P I4.rxp.breve4synlongs silica gel with 1:2 Cyclohexane-AcOEt and 0.2 % >;t3N to give X as a white foam (990 mg, 90 %); [a]D +10° (c 1, CHC13).
'H NMR (CDCL,): partial b 6.95-7.90 (m, 90H, Ph), 5.98 (d, 1H, Jz,N,~ = 6.9 Hz, NHD), S.GO
(d, 1H, JZ,~ = 7.5 Hz, NHc), 5.45 (d, 1H, Jz,~ = 8.5 Hz, NHp), 5.22 (dd, IH, ,T~,Z = 1.0, Jz.a =
3.0 Hz, H-2c), 5.13 (dd, 1H, J,,z = 1.0, J2,3 = 3.0 Hz, H-2c), 5.08 (d, 1H, J~,z = 8.3 Hz, H-lp), 5.07 (bs, 1 H, H-1 "), 5.04 (bs, 1 H, H-1,,), 4.97 (d, 1 H, J,,z = 3.0 Hz, H-1 E), 4.94 (d, 1 H, J,,i =
3.0 Hz, H-lE), 4.90 (bs, 1H, H-Is), 4.86 (bs, 1H, H-I$), 4.82 (bs, 1H, H-lc), 4.73 (d, 1H, H-lp), 4.70 (bs, 1H, H-lc), 4.43 (d, 1H, H-1D), 4.20-4.80 (m IGH, CH2Ph), 2.00, 1.85, 1.58 (3s, 9H, AcNH), 1.95 (s, 3H, OAc), 1.37-0.85 (m, 36H, 3 C(CH3)z, 2H-GA, 2H-6H,. .
2H-6~). '3C
NMR (CDC13) partial: S 171.7, 170.8, 169.8, 165.8, 1b5.4 (C=0), 139.0-127.7 (Ph), I03.9 (C-1D), 102.8 (C-1D), 101.5 (2C, C-lg), 101.3 (C-1A), 101.1 (C-lA), 100.0 (C-1D), 99.5, 99.3 (3 C(CH3h), 98.3 (C-1fi), 98.1 (2C, C-lc, lE), 97.8 (C-lc), 82.0, 81.7, 81.6, 81.4, 80.3, 80.2, 80.1, 79.5, 79.2, 78.9, 78.7, 78.4, 78.1, 77.9, 77.8, 77.6, 76.0, 75.8, 75.3, 75.2, 74.7, 74.4, 74.1, 74.0, 73.6, 73.5, 73.4, 73.3, ?3.0, 72.7, 71.9, 71.4, 70.9, 70.8, 69.1, 69.0, 68.9, 68.7, 68.6, GB.S, 68.1, 67.8, 67.7, 67.5, 62.6, 62.3, 62.1, 60.8, 59.9 (C-2D), 57.9 (C-2D), 55.0 (C-2D), 51.1 (0(CH1)~N3), 29.5, 29.4, Z9.3 (3 C(CHz)Z), 24.0, 23.9, 23.5 (3 AcNH), 21.3 (OAc), 19.7, 19.6, 19.2 (3 C(CH?)z), 18.9, 18.8, 18.6, 18.5. 18.2, 18.1 (GC, 2 C-6A, 6H, Gc). FAB-MS
for Cz~,Hz~zNsOs3 (M ' 3710.2) m/z 3733.3 [M + Na]". Anal. Calcd. for Cz,lHzazNsOss : C, 68.31 ; H, 6.57 ; N, 2.27. Found C, 68. i 7; H, 6.74; N, 2.12.

LMPPI4exp6tevehsynloBgs Ha ~Bz ~OBn Ba ° Bn0 ' Nrl Cao9H2.eoNsOs2 Exacs Mass: 3665,6320 Mol. Wt.: 3668,1511 C 68,43; H 6,.9; N 2,29; O 22.68 2-Azidoethyl (2-acetamido-2-deozy-4,6-O-isoprapylidenc-ji-D-glucopyranosyi)-(1-->2)-(3,4-di-O-benzyl-a-L-rbamnopyrsrnosyt)-(1->2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1-~3)-[2,3,4,6-tetra-D-bevzyt-a-n-gtucopyranosyl-(1->4)1-(Z-0-benzoyl-a-it rhamnopyranosyl}~(1 >3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-(3-D-glucopyranosyl)-(1-~~)-(3,4-di-0-benzyl-a-trrhamnopyranasyl)-(1->2)-(3,4-di-0-benzyl~
a-1,-rhamnopyranosyl)-(1 >3)-j2,3,4,6-tetra-0-bcnzyl-a-n-gIucopyranosyl-(1->4)~-(Z-0-benzoyl-a-L-rhantnopy ranosyl)-(1-~3)-2-acetamido-2-deoxy-4,G-D-esopropylidene-[3-n-glucopyranoside (X).
The undecasaccharide X (990 mg, 0.27 mmol) was dissolved in MeOH (30 mL).
MeONa was added until pH=9. The mixture was stirred for 3 h then treated by IR 120 (H") until neutral pH.
The solution was filtexed and concentrated. The residue was eluted from a column of silica gel with 1:1 toluene-AcOEt and 0.2 % of Et3N to give X as a white foam (900 mg, 91 %); [a]D
+15° (c I, CHCl3).

L~Pl4cap-bm~t-synlonga 'H NMR (CDC)'): partial 0 6.95-8,00 (m, 90H, Ph), 6.19 (bs, 1H, NHD), 5.96 (d, IH, Jz,~H =
6.8 Hz, NHo), 5.57 (d, 1H, JZ,uH = 6.8 Hz. NHp), 5.22 (dd, 1H, H-Z~), 5.13 (dd, 1H, H-2c), 5.10 (d, 1H, H-lv), 5.07 (bs, 1H, H-la), 5.04 (bs, 1H, H-la), 4.96 (d, 1H, H-lE), 4.94 (d, 1H, H-le), 4.85 (bs, 1H, H-1g), 4.84 (bs, 1H, H-1H), 4.82 (bs, 1H, H-l~), 4.70 (d, 1H, H-1~), 4.67 (bs, 1H, H-1D), 4.44 (d, 1H, H-1D), 4.20-4.80 (m, 16H, CHzPh), 2.00, 1.85, 1,58 (3s, 9H, AcNH), 1.37-0.80 (m, 36H, 3 C(CH3)z, 2H-6a, 2H-6B, 2H-6~). ~3C NMR (CDCl3) partial: 8 172.8, 170.9, 170.3, 165.1, 164.7 (C=O), 139.0-127.7 (Ph), 103.5 (C-1D), 103.1 (C-lp), 101.5 (2C, C-lB), 101.2 (C-la), 101.1 (C-lA), 99.9 (C-1D), 99.0, 98.8, 98.7 (3 C(CH~)Z), 98,3 (C-lE), 98.I (2C, C-1~, lE), 97.8 (C-1~), 82.1, 82.0, 81.9, 81.7, 81.6, 81.5, 80.6, 80.3, 80.2, 80.1, 79.7, 79.1, 78.9, 78.5, 77.9, 77.6, 75.7, 74.9, 74.6, 74.3, 73.3, 73.0, 72.7, 71.9, 71.8, 69.1, 68.9, 68.7, 68.5, 68.0, 67.8, 67.7, 67.6, 67.5, 62.6, 62.3, 61.9, 60.5, 59.9 (C-2D), 57.4 (C-2o), 55.0 (C-2b), 51.0 (O(CH~)1N3), 29.51, 29.47, 29.3 (3 C(CH,)~): 24.0, 23.9, 22.7 (3 AcNH), 19.7, 19.6, 19.3 (3 C(CH3)z), 19.0, 18.9, 18.G, 18,5, 18.2, 18.1 (6C, 2 C-6a, 68, 6c). FAB-MS
for C=o9HaeoNsOsa (M = 3668.1) mla 3690.8 [M + Na]~. Anal. Calcd. for C2[,H24~'6Os3 : C, 68.43 ; H, 6.59 ; N, 2.29. Found C, 68.28; H, 6.72; N, 2.11.

IHfPPI4.ap-6~evet.synionga C~1 02434685 2003-07-04 :2,12 2-Azidoethyl (2-O-acetyl-3,4-di-0-benzyl-a-tr-rhamnopyranosyl)-(I-a2)-(3,4-di-benzyl-a-z-rbamnopyranosyl)-(1->,3)-[2,3,4,6-tetra-O-benzyl-a-n-gtucopyranosyl-(1-->
4)]-(2-0-benzoyl-a-~t,-rb amnopy ranosyl)-(1-~3)-(2-acetam ido-2-deoxy-4,6-0-isopropylidene-[3-o-glucopyranosyl)-{1->2)-(3,4-di-O-bcnryl-a-L-rhamnopyranosyl)-(1 2)-(3,4-di-0-benzyl-a-irrhamnopyranosyl)-(1~3)-[2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1-ad)]-(2-O-benxoyl-a-r~rhamnopyranosyl)-(1-~3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-[i-D-glucopyranosyl)-(1->2)-(3,4-di-O-benzyl-a-~-rbamnopyranosyl)-(1->Z)-(3,4-di-O-benzy 1-a-z-rhamnopyranosyl)-( 1 ~3)-[2,3,4,6-tetra-LMPPI4tx~b~evetsyal°n~s O-beazyl_a-n-glucopyranosyl-(1-~4)]-(1-0-benzoyl-a-z-rhamnopyranosyt)-(1-~3)-2-acetamido-2-deoay-4,6-O-isopropylidene-[i-n-glucopyranoside (X).
A mixture of donor X (377 mg, 0.230 mmol) and acceptor X (427 rz>8, 0.115 mmol), 4 t~
molecular sieves and dry 1,2-DCE (10 mL), was stirred for 1 h then cooled to -30°C. Triflic acid (20 p.L) was added. The stirred mixture was allowed to reach 5°C
in 2.5 h. Et;N (150 p.L) was added and the mixture filtered. After evaporation, the residue was eluted from a column of silica gel with 3:1 toluene-AcOEt and 0.2 % Et;N to give X as a foam (490 mg, 82 %); [a]p +20° (c 1, CHC13).
'H NMR (CDC~): partial 8 6.90-8.00 (m, 135H, Ph), 5.95 (d, 1H, J?,~ = 6.6 Hz, NHD), S.GO
(d, 1 H, Jz.~, = 8.0 Hz, NHc), 5.59 (d, 1 H, J1,~, = 7.5 Hz, NHD), 5.44 (dd, I
H, H-2c), 5.22 (dd, 1H, H-2c), 5.10 (dd, 1H, H-2~), 2.20 (s, 3H, OAc), 2.00, 1.85, 1.84 (3s, 9H, AcNH), 1.40-0.80 (m, 45H, 3 C(CH~)~, 3H-6A, 3H-6g, 3H-Gc). "C NMR (CDC13) partial: b 173.2, 172.6, 172_5, 171.3, 167.4, 167.0, 166.9 (C=O), 140.2-126.8 (Ph), 102.8, 102.7, 101.5, 101.3, 101.1, 99.9, 99.8, 98.1, 97.8, 82.0, 81.7, 81.5, 81.4, 80.2, 80.1, 79.6, ?9.4, 78.9, 78.6, 78.0, 77.9, 77.6, 75.5, 73.4, 73.3, 73.0, 72.8, 71.9, 71.G, 69.4, G9.1, G9.0, G8.G, 67.8, 67.7, 67.6, 67.5, 62.6, 62.3, 60.0, 57.9, 57.7, 51.0 (OCHZCHZN3), 30.5 (3C, C(G'Fi3)z), 25.0, 22.4 (3C, AcNH), 22.9 (OAc), 20.7. 20.6, 20.2 (3C, C(CHy), 20.0, 19.9, 19.8, 19.7, 19.6, 19.3, 19.2.
19.1 (9C, 3 C-6A, 6H, 6~). FAB-MS for CzggH;3qN6O7, (M = 5135.8) m!z 5159.3 [M
+ Na]'.
Anal. Calcd. for C298H;;aN6O7t : C, 69.69 ; H, 6.55 ; N, 1.64. Found C, 69.74;
H, G.72; N, 1.49.

LMPPI4~x~bm~errynlon8.c 8n0 B~

J~
~1 Z
H
~~

eno 771 o 2 Exaa I~i s e Mel. Wt.: 5015,6468 C 69.21; H 6,47;
N 1,68; 0 22,65 2-Azidoethyl (2-0-acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1~2)-(3,4-di-D-benzyl-a-t-rhamnopyranosyl)-(1->3)-[~,3,4,G-tetra-O-benryl-a-n-glucopyranosyl-(1-~
4)]-(2-O-ben~oyl-a-z-rhamnopyranosyl)-(1 >3)-(Z-acetamido-2-deoxy-a-n-glucopyranosyl)-(1->2)-(3,4-di-0-benzyl-a-i.-rhamnopyranosyt)-(1-~2)-(3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1->3)-[2,3,4,G-tetra-O-benzyl-a-n-glucopyranosyl-(1->4)]-(Z-0-benzoyl-a-L-rbamnopyranosy I)-(1-~3)-(2-acetamido-2-deoay-[3-n-gtucopyranosyl)-(1~
2)-(3,4-di-O-benryi-a-irrhamnopyranosyl)-(1 >2j-(3,4-di-0-benzyl-a-ir-rhamnopy ranosyl)-(1-~3)-[2,3,4,G-tetra-0-benzyl-a-n-glucopyranosyl-(1~4)]-(2-O-benxoyl-a-z-rhamnopyranosyl)-(1--~3)-2-acetamido-2-deo~y-[i-D-glucopyraaoside (X).
To a solution ofthe pentadecasaccharide X (480 mg, 93 pmol) in DCM (14 mL) was added dropwise at 0°C, a solution of TFA (1.75 mL) and water (1.75 mL). The mik~ture was stirred for 3 h then concentrated by coevaporation with successively water and toluene. The residue LMPPI4.exp.brevet-syntongg was eluted from a column of silica gel with 1:1 toluene-AcOEt to give X as a white foam (390 mg, 83 %); [a]D +12° (c l, CHCl3).
FAB-MS for Czs9H3a2ycCm (M = 5015.6) m/z 5037.2 [M + lvTajy.
Anal. Calcd. for C2pgH322~6W1~81~0: C, 67.27 ; H, G.60 ; 1~', 1.63. Found C, 67.31; H, 6.45;
N~ 1.64.

LA?PPl4~x~brcvacynlon~
HO
Hi HO

H
C98H1 bGV4~67 HO Exact Mass: 2470,9706 ~

H Mol. Wt.: 2472,3534 "

C 47,61; H 6,77;
N 2,27; O 4336 NO O
OH

O

Ho N
HO

H
HO

Hd H

Z-Aminoethyt (a-1.-rhamnopyranosyt)-(I >2)-(a-z-r6amnopyranosyt)-{I~3)-[a-n-glucopyranosyt-(1 >4)]-(a-c,-rhamnopyranosyl)-(1->3)-(2-acetamido-2-deoxy-(3-D-gtucopyranosyt)-(I-32)-(a-t,-rhamnopyranosyl)-(1-->2)-(a.-L~rhamnopyranosyl)-(1-33)-a-n-glucapyranosyl-(1-~4)]-(a-L-rhamnapyranosyl)-(I-~3)-(2-acetamido-2-deoxy-~-n-gtucopyranosyt)-(I~2)-(a-~rrhamnopyranosyl)-(I-~2)-(a-z-rhamnopyranosyt)-(1-~3)-[
a-v-glucopyranosyl-(I-~4)]-(a-z-rhamnopyranosyl)-(1-~3)-2-acetamido-2-deo~cy-[3-D-glucopyranoside (X).
A solution of the partially deprotected pentadecasaccharide X (390 mg, 77 ltmol) in MeOH
(10 mL) was treated by MeONa until pI-I=10. The mixture was stirred overnight at 55°C. After cooling at rt, IR 120 (H~ was added until neutral pH and the solution was filtered and concentrated, then was eluted from a column of silica gel with 20:1 DCM-MeOH
to give the benrylated residue (252 mg). A solution of this residue in EtOH (3 mL), AcOEt (250 p.L) and LMPP t4txp.brcvel~synlongc 1M HCl (106 wL) was hydrogenated in the presence of Pd/C (300 mg) for 43 h at rt. The mixture was filtered and concentrated, then was eluted from a column of C-18 with water/CH;CN and freeze-dried to afford amorphous X (127 mg, GS %): [aJD -5° (c 1, H~0).
'H NMR (D2~): partial 8 5.13 (m, 3H, 3 H-lE), 5.07 (m, 2H, H-lRhn)~ 4.99 (bs, 1H, H-lRha)~
4.95 (m, 2H, H-lRh~), 4.90 (m, IH, H-IRU), 4.75 (m, 3H, H-1R~,), 4.63 (d, 2H, J,,2 = 8.5 Hz, 2 H-1Q), 4.51 (d, 1H, Jl,s = 8.5 Hz, H-1D), 2.00 (s, 9H, 3 AcNH), L30-1.18 (m, 27H, 3H-6", 3H-68, 3H-6~). "C NMR (CDCh): 8 174.8, 174.7 (3C, C=O), 102.9, 102.6, 101.7, 101.3, 100.8, 97.9, 81.8, 81.7, 79.6, 79.0, 76.3, 76.2, 73.0, 72.7, 72.4, 72.1, 71.6, 70.5, 70.1, 70.0, 69.7, 69.6, 69.4, 68.7, 68.6, 66.0, 61Ø 56.0, 55.4, 39.8, 22.7, 22.6 (AcNH), 18.2, 17.2, 17.0, 16.9 (9C, 3 C-6h, 6s, dc). FAB~MS for C98H166N4067 (M = 2470.9706) tnla 2493.9660[M +
Na]'.

LMPPIb-schemca-brcv~-s't~loogs ~OBn cJ'"~y~yO.J'Na o --~'0 eng p NHAc 9nOOMe XX a_~gz OR~
Bn~ $n4' o O Q Rdg ~ O./~R~
R~ ~ NHAc OBn ~'O O ~ ROpM-~
O O O~pBBn O OR' 9no~ , p~! _ 0 BnO-'~"'1 OR d ~"~~~t OR
Bn0 O RR ~ Me R 00 NHAc~OR
~ R
a0 O a~~09n ~ 0 Ra R" NHAc OBn Me 0 RO R
R ORs AC OR Rap O
R~0 ~ ta~ Ac O
M° 1~..' R~ R2 Rs Rd.~-R
M O ORS gn N3 Bx Ac -- iPr -RO R~ a Bn~ Ns 6z Ac H H
gn N3 H H H H
..s ~~na '~ H NH2 ~ ~ H M

LMPP14-nchcmcs~brcvet-synlongs OBn Bn0 O OR' Bn0 Bno 0 Me O Rt R3 R4 Rs O OBz a All H H H

Me O f All H - iPr -BnO

9 All Ac - iPr Bn0 -O OH Ac - iPr -~ORG ~ OTCA Ac - iPr -Bn )Bn N HAc OBn \O B6 O O O-~e~0~N3 O O NHAc HO~~~O~N3 Bn0 0 Me O
NHAc o OBz BnOMe O R
Bn0 O a ~ Ac N
O O O
O O Me OBn OBn NHAc LMPPId.srhemes-Ixevet~synlong9 OR
OAc RO--~~S(CHz)~~CH3 ~ ~ ~ Ac p RO H
Ac0 ~~OAC NHC(O)CC13 NHC(O)CCI3 RCS( z)11 \NHC(O)CCI3 O6n Dan R Bn0 ~ O OAfI
O OAII ( H BnO~ Me O
Ac Bn< OBz Bn0 M~
B Of Bn0 O
~O O p OSn Me OBn Ac R~
CC13C(O)NH
H

LMPP (4-schcmes~brcva-4yn(ongs <OB n OBn /OAc B~O~ 3 R~
BAnO O oAU ~O \ O
Bn0 Me 0 Ac0 B°OO'~~~c 0 O CChC(0)NH TCA " OBz ~Bz Bn0 Me O Ma 0 Bn0 ""' B Bn0 O R~ R~
d Bn0 Mo O ~OAC O a AA C(0)CCIs 0.
Bn0 R Ac0 t~~OB~ f All Ac a OR c ~ ~ AcO~a OBn g H Ac an TCA Ac a!p ORe OBn R°O 0 OH
Bn0 0 ~O~ OH
Bn0 O NHAc Ns HO-~S~ O~ HO~O~
Bn0 0 Me O H~ 0 NHAc NHa t R~ H'O O M~0 Bn0 Me j H
O Bn0 O HO M-~../~~
HO 'NHAc~Ns OR3 HO
RaO~p OBn ~OH~
Rs0 Me Bn HO~O
NHAC -Ra Rs Ra Re HO O/ Me H
NHAc OH
OAe p Bz Ac - iPr AeA 0 O O~Ns Bt Ac H H
NHAc 9 H H H H

LMPPl4.~brcvctaynlongt 'OBn p p~
BBnp Bn0 p ° OBa An0 Bn0 C,o,H",N0~7 Exac~M~ss; tett,78 Bn0 Bnp Moh Wc: 1813,03 Acp~ C 88,90, H 6,50; N 0,77, U 23,9J
u~-p~.,\~~~J// AIphaD-+3°, C'1, CHC13 ~VH
y~p~0 Allyl (2-acetamido-3,4,6-tri-D-acetyl-2-deoxy-[3-D-glucopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1 >2)-(3,4-di-O-bcnzyl-ac-L-rhamnopyranosyl)-(1--1,3)-[2,3,4,6-tetra-D-benzyl-a-D-glucopyranosyl-(1-r4)]-2-D-benzoyl-a-L-rhamuopyranoside {X).
A mixture of X (3.14 g, 1.6 mmol), Bu3SnH (2.5 rnL, 9.3 rnnwlj and AIBN (240 mg) in dry toluene (40 mL) was stirred for 30 min at rt under a stream of dry Ar, then was heated for 1 h at 100°C, cooled and concentrated. The residue was eluted from a column of silica gel with 3:2 petroleum ether-EtOAc to give X as a white foam (2.0 g, G8 %); [a]p +3°
(c 1, CHC13).
1H Ni~IR (CDC13):8 8,00-7,00 (m, 45H, Ph), 5.82 (m, 1H, All), 5.58 (d, 1H, J2,NH = 8.0 Hz; N-HD), 5.35 (dd, 1 H, J,,2 = I .0 Ha, J2,3 = 2.3 Hz, H-2c), 5.19 (m, ZH, All), 5. I 0 (d. I H, J,,2 = 1.0 Hz, H-lA), 4.92 (dd, 1H, J2,3 = 10.5 Hz, J3,4 = 10.5 Hz, H-3D), 4,92 (d, 1H, J,,2 = 3.3 Hz, H-1 E), 4.90 (d, 1 H, Jt.2 = 1.7 Hz, H-18), 4, 89 (d, 1 H, H-1 ~), 4.88 (dd, 1 H, J4,s = 10.0 Hz, H-4D), 4.62 (d, 1H, J,,2 = 8.5 Hz, H-1D), 4.90-4.35 (m, IGH, CH2Ph), 4.40 (m, 1H, H-28), 4,10-4.00 (m, 2H, All), 4.08 (dd, 1H, J2,3 = 2.4 Hz, H-2A), 4.02 (dd, 1H, H-3c), 3.91 (m, 1H, H-2D), 3.90-3.70 (m, 11H, H-4~, 5~, 3A, 5A, 6aD, 6bD, 3~, 4E, SE, 6aE, 6bE), 3.61 (dd, 1H, J3,a = 9.S Hz, H-38), 3.55 (m, 1H, H-5B), 3.41-3.40 (tn, 3H, H-4A, 5D, 2E), 3.47 (m, 1H, J4,s = 9.5 H~, JS,s =
6.1 Hz, H-5B), 3.35-3.33 (m, 3H, H-4,,, 5D, 2E), 3.25 (dd, 1H, H-48), 1.95, 1.70 (3s, 9H, LMPP14-cxp~brrva~ynlongs ~1 02434685 2003-07-04 CH3C=0), 1.65 (s, 3H, CH3C=ONH}, 1.32 (d, 3H, .~5,~ = 6.1 Hz, H-GA), 1.30 (d, 3H, J5,6 = 6.0 Hz, H-6~), 0.97 (d, 3H. J;,b = 6.0 Hz, H-6H). '3C NMR (CDCI,):8 171.1, 170.8, 170.2, 169.6, 166.2 (5C, C=0}, 138.2-118.5 (Ph, All), 103.1 (C-1D), 101.4 (C-lB), 101.2 (C-1~), 98.5 (C-lE), 96.4 (C-l~), 82.2 (C-3E), 81.7 (C-2E), 81.7 (C-4A), 80.4 (C-4B), 80.2 (C-3~), 79.0 (C-3A), 78.6 (C-3B), 78.1 (C-2A), 77.8 (C-4~), 77.6 (C-4E), 76.0, 75.8, 75.4, 74.7, 74.3, 74.2, 73.3, 70.5 (8C, C'~hPh), 74.9 (C-2s), 72.7 (C-2o), 72.G (C-3D), 71.9 (2C, C-Se, 5n), G9.1 (C-5B), 68.9 (2C, All, C-5,,), G8.3 (C-6E), 67,8 (C-5~), 62.3 (C-6o}, 54.6 (C-2D), 23.5 (1C, NHC=OCHz), 21.1, 21.0, 20.8 (3C, C=OCH3), 19.0 (C-G~), 18.4 (C-6A), 18.2 (C-Gs).
FARMS of C,o4Hi1~N0~~ (M, 1913.1), m/z 1936.2 [M~-Na]'. Anal. Calcd. for C,naH,ml'TOz, C, 68.90 ; H, 6.50 ; N, 0.77. Found C, 68.64 ; H, 6.G6 ; N, 1.05.

LMPPl4<xp-bcevet-synlongs Cc o~Ht uChN~Oz?
EaAtt',~ta~,5: 1914,86 Mol. Wc: 1917,35 H 5,95: CI 5,55; N 1 a6; O 7.2.59 rc: C:61.3&°~. H fi.10!~, Id:Ls54o AlDhanxlD', c-1, CHCt3 (Z-acetamido-3,4,6-tri-0-acetyl-Z-deoay-~3-D-glucopyranosyl)-(1-~2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1--~2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1->3)-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1 >4)J-Z-0-benzoyl-a-L~rhamnopyranosyl trichloroacetimidate (X).
1,5-Cyclooctadiene~bis(methyldiphenylphosphine)zridium hexafluorophosphate (25 mg, 29 1l mol) was dissolved tetrahydrofuran (5 mL), and the resulting zed solution was degassed in an argon stream Hydrogen was then bubbled through the solution, causing the colour to change to yellow. The solution was then degassed again in an argon stream A solution of 7 (1.0 g, 0.55 mrnol) in tetrahydrofitran ( 10 rnL) was degassed and added. The mixture was stirred at rt overnight, then concentrated to dryness. The residue was dissolved in acetone (5 mL), then water (1 mL), mercuric chloride (140 mg) and mercuric oxide (120 mg) were added successively. The mixture protected from light was stirred at rt for 2 h and acetone was evaporated. The resulting suspension was taken up in DCM, washed twice with 50% aq ~:I, water and sold aq NaCI, dried and concentrated. Th>r residue was eluted from a column of silica gel with 2;1 petroleum ether-EtOAc to give the corresponding hemiacetal.
Trichloroacetonitrile (2.5 mL) and DBU (37 p.L) were added to a solution of the residue in LMPPl4.c:cp.brcvec-eynlongs anhydrous dichloromethane (12.5 ml.) at 0°C. Aver I h, the mixture was concentrated. The residue was eluted from a column of silica gel with 5:4 cyclohexane-EtOAc and 0.2 % Et3N to give X as a white foam (0.9 g, 85 %); (a)o +10° (c I, CHC13).
~H NMR (CDC~):8 8.70 (s, IH, C=NH), 8.00-7.00 (m, 45H, Ph), G.3G (d, 1H, J,,~
= 2.6 Hz, H~Ic), 5.59 (m, 2H, N-Ho, H-2c), 5.13 (d, 1H, Ji,2 = I.0 Hz, H-l,,), 5.01-4.98 (m, 2H, H-lr, 18), 4.92 (dd, 1H, H-3p), 4.90 (dd, 1H, H-4D), 4.68 (d, 1H, H-ID), 5.00-4.02 (m, 19H, 8 CHZPh, H-3c, 2A, 28), 4.01 (dd, 1H, H-2E), 4.00-3.20 (m, 1GH, H-3E, 4E, 5~, GaE, GbG, 4c, 5c, 3e, 4H, Sg, 3A, 4,,, 5A, Sn, 6aD, 6th), 2.02, 2.00, 1.75, 1.G5 (4s, 12H, C=OCH3), 1.40, 1.32 and 1.00 (3d, 9H, H-6~. 6B, 6~). "C NMR (partial) (CDC13):cS 170.2, 1 G9.9, 1 G9.3, 168.7, 164.9 (GC, C=O, C=N). 103.2 (C-ID), 101.4 (2C, C-1,,, 1H), 99.0 (C-lE), 94.8 (C-I~), 21,1, 20.9, Z0.8 (3C, CH3C=O), 19.1, 18.2 (3C, C-6,,, 5g, G~). FABMS of C~o3Hi,3C13Nz02~
(M, 1917.4), mfz 1930.9 [M+NaJ'. Anal. CaIcd. for C,o3H"3CI3Nz027 : C, 64.52 : H, 5.94 ; N, 1.46. Found C, 64.47 ; H, 5.99 ; N, 1.45.

8~i~elln flcsenri Sa ICS-ELISA d'Iobi6itCoe AntigEnicitE des Oligosaecharldes SynthEtiques (ICso - ELISA d'inhibition) Shigella Jl'exrrerl Sa La reconnaissance oligosaccharideslanticorps a etC waluee pas ELISA (Enzyme-Linked Immunosorbent Assay) d'inhibition, qui sans dormer acres ~ une constants d'aflinite absolue », permet de comparer aisement et rapidement un grand hombre de ligands. Il apparait que le rEsidu glucose E E5t indispensable a la reconnaissance quelque soil 1'anticorps etudie. En outre, les constantes d'inhibition (ICSO) les plus favorables sont obtenues pour !es pentasaccharides. Les donn6es disponibles indiquent que - les sequences CDA(E)B-OMs et DA(E)BC-OMs sont les mieux reconnues par les IgA TS
et IgA C5, aver des ICso de fordre de 25 mM.
- le trisaccharide A{E)B-OMs (ICso = 1,3 ~, correspondant au site de ramification, semble definir to structure minimale necessaire ~ la reconnaissance par fIgG C20.
Mais, le pentasaccharide DA(E)BC-OMs (ICso = 25 ~Imol) correspond ~ la sequence la mieux reconnue parmi celles testes.
Ces resultats correlent parfaitement aver les donnees d'analyse conformationnelle. Le pentasaccharide DA(E)BC-OMc est, a ce niveau d'etude, le plus representatif de fantig6nicite du PS de S. flexrleri sErotype Sa. D'autre part, la difference d'afI'mite poux Ies oligosaccharides synthetiques observes entry IgG serique et IgA secretoires (sIgA) est remarquable. Par analOgie aver le mode du reconnaissance des antigenes polymeriques par Ies immunoglobulines d'isotype M, fhypothese a ete emise que. le caractere dimerique des slgA
impliquait un phenomene d'avidit6 compensant Ia faibIe affinite intrinsCque des sites do reconnaissance. Les donnees quant a la reconnaissance sIgA:LPS valident cells hy~poth~se.
L'etude des complexes sIgA/oligosaccharides en solution a ete affinee par RMN
a (aide de deux techniques particulierement petformantes et compl~mentaires (i) Ies exp6ziences de NOE-transferes et (ii) les experiences de transfert de saturation (STD-RMN), compatibles aver la faible afFmite des sIgA pour les pentasaccharides.
Les donnees de M.-J. Clement (These Mars 2003, Unite de Resonance MagnCtique Nucleaire des Biomol6cules), obtenues sur les sequences DA(E)BC-OMs et CDA(E}B-OMs, indiquent que Ies pentasaccharides en interaction aver les IgA conservent la conformation moyenne qu'ils adoptent sons leur forrne fibre. II apparait egatement que les atomes d'hydrogene port6s par le glucose E et le rhamnose B sont tons en interaction directs aver les anticorgs. Daps leer ensemble, les resultats sont en faveur dune contribution directs a (interaction de ehacun des residus eomposant les pentasaccharides et sous-entendent que les slgA accommodent un bpitope relativement large. En accord aver les donnees de TCso obtenues pour fIgG C20, yes resultats confirment Egalement que le site de ramification A(E)H

Shigtllo Jlexneri 9a iCs. - ELISA d'ladihiNoa represents to sequence critique pour la reconnaissance du PS de S. ,~lexneri Sa par les anticorps monocl.onaux protecteurs.
ShigellaJlexneri Za Comme en ssrie S. flexneri ssrotyPe Sa, la reconnaissance oligosa.ccharides/anticorps a tits evaluee par EL1SA d'inhibition. Quatre des cinq IgG evaluss semblent avoir un eomportement tres proche en terms de reconnaissance. du LPS et different de celui du quatrieme, l'IgG F22-4, plus aff"m. Cependant quelque soit I'anticorps pris en consideration, le residu D-glucose E
et le residu N acetyl-b-glucosarnine D semblEnt tous deux iruiispensables ~ la reconnaissance.
Ainsi, la sequence minimale reconnue par I'IgG F22-4 est le trisacoharide lineaire ECD-OMe. Dune importance moindre puisque non reconnue en tent que tells ~. la concentration de 1 mM (absence de reconnaissance du pentasaccharide DAB(E)C-OMs par exemplej, la ramification B(E)C joue sgalement un role critique. En comparaison, la contribution du residu A a la reconnaissance semble faible si celui-ci est introduit ~
fextremite reductrice des sequences ECD-OMs et B(E)CD-OMs.
Comme illustre pour 1'octa- et le deeasaccharide. 1'slongation de la chains ~.1'extrCmitd r6ductrice par introduction de la. sEquence B(E)C ameliore grossiLrement la reconnaissance pour quatre des anticoips drn facteur 5. Il est vraisemblable que la contribution impliquee est de type confotmationnel et non pas interaction directs. En revanche, anti amelioration d'un facteur 30 cst observes pour 1'IgG A-2, rl est possible que tie demier aecommode. un epitope plus long. La comparaison des donnses obtenues pour 1'octa- et le pentasaccharide indique quc dens 1'ensemble, la contribution do la sequence DA est minims, voice negative daps le cas de I'TgG F22-4.
En permettant la couverture complete des sequences obtenues par permutation circulaire des residue composant 1'unite repetitive de 1' Ag-0 de S. flexneri. 2a, 1'Evaluation des deux pentasaccharides ECDAB-OMs et AB(E)CD-OMs apportera des elements d'information comptementaires a notre etude. A ce stale, nos resultats suggerent que quatre des cinq IgGs disponibles reconnaissent, avec anti affinite plus ou moms bonne, anti sequence minimale commune (B(E)CD) qui peat titre qualifies d'u epitope immunodominant ». Dens tous tee cas, 1'elongation de la chains ameliore Ia reconnaissance. Cette observation suggere que lee anticorps reconna.issent un spitope presents de faron multiple le long du polymers (17 unites rspstitive.s en tnnyenne) et non pas unique en bout de chains.

AntigEnicitE des olioecelurides synthetiques (IC,o~EL1SA d'inhibitioa) Antigenieit~ des oIiosaccharides synthefiques (ICsa-ELISA d'inhibition) Shigella Jlexneri 2a L'etude de la reconnaissance, en ELISA d'inhibition, de 1'ensemble des di-, tri-, tetra-et pentasa.ccharides obtenus par permutation circulaire des r~sidus composant 1'unite repetitive (AB(E')CD) de. fAg-O de S flexneri 2a, et specifiques de ce s~rotype, par cinq anticorps monoclonaux d'isoptype G, sprscifique de S. flex~eri 2a et protecteurs a et~
completee (Annexe 3). Deux groupes d'anticorps peuvent titre distingues. Tout d'abord F22-4, anti IgGI qui est le soul anticorps ~ reconnaitre le.s oligosaccharides lineaires E'CD, E'CDA et E'CDAB. Pour les quatre autres anticorps, le residu B est requis pour observer anti inhibition a anti concentration en ligand inferieure ~ 1 mM. Ainsi, la sequence B(E')CD
apparait comme la sequence minimale permeYtant d'observer anti inhibition ~
anti concentration en ligand inferieure ~ 1 mM quelque snit 1'anticozps. Comparee a la contribution des residus B, E, C et D, (influence du rGsidu A sur la reconnaissance, evaluGe par elongation du fragment B(E')CD a son extrem3te reduetrice ou nan rt;ductrice, semble relativement minim~e. Toutefois, les pentasaccharides AS(E')CD et B(E')CDA
sorer les sequences les mieux reconnue.s par (ensemble des anticorps.
Les hapt~nes selectionnes pour les etudes d'immunogc;nicite sorer les stsquences E'CD, B(E')CD et AB(E')CD qui reprEsente (unite reptstitive de 1'antigene 0 (Ag-0).
Afire d'analyser la contribution de residus additionnels dens la.
reconnaissance de la sequence minimale par les anticorps, 1'octasaccharide B(E')CDAB(E')C et Le decasaccharide DAB(E')CDAB(E')C ont ere synthetises. En moyenne, 1'elongation a 1'extrEmite reductrice de B(E')CDA par B(E')C pour eonduire a 1'octasaccharide ameliore legeremcnt la reconnaissance. Il en est de nc»me de 1'elongation de 1'octasaccharide ~ son extremite non reductrice par Ia sEquence DA pour conduire au dr'casaccharide. Le cumin des deux contributions correspond ~ un gain en reconnaissance superieur ~ un log pour 1'ensemble des anticoxps t:xcepte 1'IgG FZZ-4. Pour ce dernier, la sequence la mieux reconnue correspond a 1'octasa:echaride. L'influence de la longueur de la chauie est done manifesto, elle doit titre prise en compte duns les etudes d'immunogenicitE.

Proro°ole-IC50-S~JexSa INHIBITION ELISA
PROTOCOLE
Obtention of IgGC20 Mice were immunized intraperitoneally with lOg killed S. ,~exneri Sa bacteria, Those developing an anti-S. flexneri Sa LPS antibody response were used for the.
obtention of monoclonal antibodies as previously described (Kohler and Milstein). The hybridoma obtained were tested for the production of a monoclonal antibody specifte for S. ,~I'exrreri Sa LPS by ELISA using purified LPS from different S flexneri serotypes. Only monoclonal IgG
recognizing exclusively LPS Sa were selected, Inhibition ELISE.
First of all, a standard curve with IgGC20 was established. Different concentrations of the anti~dy was incubated at 4°C overnight and then incubated on microtiter plates coated with purified Shigella flexneri Sa LPS at a concentration of S~g/ml in carbonate buffer at pH 9.6, and previously incubated with PBSBSA 1% for 30 min at 4°C. After washing with PBS-Tween 20 (0.05%), alkaline phosphatase-conjugated anti-mouse IgG was added at a dilution of 1:5000 (Sigma Chemical C0.) for I h at 37°C. After washing with PBS-Tween 20 (0,05%), the substrate was added (I2 mg of p-nitrophenylphosphate in 1.2 ml of Tris, HCl buffer ph 8.8 and 10.8 ml ofNaCl SM). Once the color developped, the plate was read at 405 nm (Dinatech MR 4000 microplate reader). A standard curve OD= f(antibody concentration) was fitted to the quadratic equation Y= aX2+bX+c where Y is the OD and X is the antibody concentration. Correlation factor (r2) of 0.99 were routinely obtained.
Then, the amount of oligosaccharides giving 50% inhibition of IgGC20 binding to LPS
(IC50) was then determined as follows. IgGC20 at a given concentration (chosen as the minimal concentration of antibody which gives the ma~nal OD on the standard curve) was incubated overnight at 4°C with various concentrations of each of the oligosaccharides to be tested, in PBSBSA 1%. Measurement of unbound IgGC20 was performed as described above fNHIHITION ELISA

INHIBITION ELISA
Shigella flexr2eri serotype Sa IgG C20 / synthetic oligosaccharides Only, those oligosaccharides which were recognized with an IC50 bElow 1 mmol/L
are listed.
A(E)B > I OOOfnM pas d'~cart type A(E)BC 208 +/- I08 uM
A(E)BCD 389 +I- 84 uM
DA(E)B 242 +/- 25 uM
DA(E)BC 39 +/- 19 uM
CDA(E)B 268, 5 +/- 180 uM

Serum IgG pspiu~brevet The serum immunoglobulin G-mediated response to serolype-specific determinants of ShigellaJlrxneri lipopotysaccharide profecta against experimental shigellosis 5ctumIgOpapier~brc~ct ~1 02434685 2003-07-04 Introduction Shigellosis is a major cause of infant morbidity and mortality in developing countries but an increasing number of cases in industrialized countries has been recently reported (33).
Shigella, the causative agent of bacillary dysentery, invades the human colonic epithelial cells by manipulating processes that control the host cytoskeletal dynamics (8).
Host response to bacterial infection is characterised by the development of an acute inflammation which is responsible for the destruction of the colonic mucosa and accounts for the symptoms observed at the early stage of the disease (53). Acquired humoral immunity induced upon primary infection confers protection against re-infection, although the duration of the disease~induced immunity seems to be limited. Antibody-mediated protection is species- and serotype-specific, pointing out LPS as the major protective antigen (19, 22, 38).
In fact, species and sexotypes among a given species are defined by the structure of the repeated saccharide unit that forms the 0-Ag polysaccharide part of LPS (35).
Other bacterial antigens, as for example the invasins IpaB and IpaC, are recognized by sera from convalescent patients (18, 45, 46, G3), but their contribution to protective immunity is poorly documented.
Both intestinal SIgA and serum IgG directed against the O-Ag are elicited (13, 28, 31, 69). However, the respective protective roles of local and systemic humoral immunity remain unclear. The ineffectiveness of parenterally injected inactivated whole-cell vaccines in inducing protection, despite the high level of anti-LPS serum IgG antibodies raised, has led to the belief that serum antibodies do not confer protection (21, 25). However, several indirect pieces of evidence suggest that anti-O-Ag serum IgG may confer protection during natural infection. A correlation was found between the level of anti-LPS IgG
antibodies and resistance to shigellosis among Israeli soldiers (14, 15), and an inverse relationship exists between the age of incidence of shigellosis and the presence of IgG antibodiES
to Shigella Serum IgG papicr~brc~n LPS (47, 63). In addition, a detoxified LPS-based conjugate vaccine administered parenterally and eliciting mainly, if not only, serum antibodies has bean shown to induca protective immunity ( 1 G j.
The use of experimental models of shigellosis has allowed the study of; at least in part, Shigella specific humoral immunity. The rabbit ileal loop model has been used to assess SIgA-mediated antibody response (31, 32), and more recently, the mouse model of pulmonary infection has been developed (51, GG). Following i.n. administration of bacteria, mice develop an acute broncho-pneumonia leading to massive destruction of the lung tissues.
This response mimics the acute inflammation developed in intestinal tissues in the course of shigellosis.
This model has been used to assess the immunogenicity and protective capacity of different Shigella vaccine candidates, either live attenuated strains administered i.n., or subunit vaccines administered parenterally (3, 34, 36, GS). Using this model, we have demonstrated that the IgA-mediated immune response specific for a serotype-specific determinant is sufficient to confer protection, (51), with an improved protective capacity of the IgA when bound to secretory component (52). In the current study, using the same experimental model and specific polyclonal serum or mIgG, we have addressed the protective role of serum IgG
recognizing serotype-specific LPS determinants or peptide epitopes on the invasins IpaB and IpaC.

6crum 1gG pnpirnbrrvet ~1 02434685 2003-07-04 Materials and Methods Bacteria! strai»s M90T, an invasive isolate of S ,Jlexneri serotype Sa, an,d 454, an invasive isolate of S. flexneri setotype 2a, were the virulent strains of reference. For i.n. infection, bacteria were. routinely grown on Luria Bertoni agar plates at 37°C. They were recovered from plates and bacterial dilutions were performed in 0.9% NaCI with the consideration that, for an optical density of 1 at 600 nm, the bacterial concentration was 5 x 108 c.fu.lml. Killed bacteria for systemic immunizations were prepared from bacterial cultures at stationary phase, diluted to S x 108 c.f.u. /ml in 0.9% NaCI, and then incubated at 100°C for lh. They were then kept at -20°C in aliquots.
Production and characterization of mAbs specific for S. , fTexneri LPS
BALHIc mice were immunized i.p. with 10' c.f.u. of killed S. jlexneri Sa or S, flexneri Za bacteria three times at 3 week-intervals. Mice eliciting the highest anti-LPS
antibody response were given an intravenous booster injection 3 days before being sacrificed for splenic B cell fusion according to Kohler and Mitstein (30). PTybridoma culture supernatants were screened for antibody production by ELISA using purified S, flexneri Sa or 2a LPS. We selected only the hybridoma cells secreting mlgG reacting specifically with LPS homologous to the strain used far immunization, i. e. recognizing serotype-specific determinants on the LPS 0-Ag.
Those selected were then cloned by limiting dilution, and injected i. p, into histocompa,tible mice. for ascitis production. mlgG were precipitated with 50% ammonium sulfate from ascitis fluid, centrifuged, and dialysed against PBS before being purified using ion-exchange chromatography as previously described (2, 50). The avidity of anti-LPS mIgG
was determined as follows: various concentrations of LPS were incubated in solution overnight at 4°C with a defined amount of a given mlgG until equilibrium was reached. Each mixture was SaumI~(3(18Pt2T-b~°V2t CA 02434685 2003-07-04 then transferred to a microtiter plate previously coated with homologous purified LPS. Bound antibodies were detected by using peroxidase-conjugated anti-mouse immunoglobulins specific for IgG subclasses. ICso was defined as the concentration of LPS
required to inhibit 50% of mlgG binding, Aclive arid passive imrnureiEation ojitrice xo obtain polyclonal serum, mice were immunized i.p. with 5 x 10' killed bacteria, three times at 3 week-intervals. After bleeding, anti-LPS antibody titer in the polyelonal sera was measured by ELISA, as described below, and those ranging from low (1/4,000) to high liter (1164,000) were used fox i.n. passive transfer. Purified mAbs (20 or 2 fig) w~ete also administered intranasally. All i.n. administrations were performed using a volume of 20pI and mice previously anesthesized via the intramuscular route with 50p1 of a mixture of 12.5%
ketamine (Merial , Lyon, France) and 12.5°,'o acepromazine (~~etoquinol, Lure, France). Each experiment was performed using I O mice per group and was repeated three times.
Proleclion experiments Intranasal challenge was performed using either 109 live virulent bacteria when protection was assessed by mortality assay or 108 bacteria when protection was assessed by measurement of the lung-bacterial load. Naive. mice were used as controls in each experiment.
Mice immunized i.p. were challenged i.n. with virulent bacteria, 3 weeks after the Last immunization. Mice passively transferred i.n. with polyelonal sera or with purified mAbs were challenged I h after administration of the mt~.bs. Measurement of lung-bacterial load was performed at 24h post infection as follows. Mice were sacrificed by cervical dislocation and lungs were removed « en bloc » and ground in 10 ml sterile PHS (Ultra Turrax T25 apparatus, Janke and Kunkel IKA Labortechnik GmbH, Staufen, Germany). Dilutions were then plated on Trypticase Soy Broth plates for c.fu. enumeration.
I

Serum IgG papia-brcva ELISA
Hybridoma culture supernatants were tested by ELISA for the presence of anti-LPS
antibodies as previously described (2, SO) except that LPS purified according to Westphal (67) was used at a concentration of 5pg/ml in PBS. As secondary antibodies, anti-mouse IgG-or IgM- or IgA-alkaline phosphatase-Labeled conjugate (Sigma) were used at a dilution of 1:5,000. To measure the anti-LPS antibody titer in polyclonal serum, biotin~labeled Abs to IgG and its different subclasses (IgGl,-Za, -2b, -3) (Pharmingen) aiui avidity conjugated with alkaline phosphatase (Sigm ) were used at a dilution of 1:5,000. Antibody titers were defined as the last dilution of the sample giving an OD at least twice that of the control.
,~isdopathologicat studies Mice were anesthesized, their trachea catheterized, and 4% formality injected in order to fill the bronchoalveolar space. Lungs were then removed and fixed in 4% formality before being processed for histopathologieal studies. Ten-micrometer paraffin sections were stained with Hematoxiline and Eosin (HE), and observed with a BX50 Olympus microscope (Olympus Optical, Europa, GmbH, Hamburg, Germany).
Slaifsfical analysis Significant differences were compared using the Student's test. Probability values < 0.05 were considered significant.

$G'fllml~G, Jl9PlMWV~ CA 02434685 2003-07-04 Results 1) Protection conferred upon systemic immunization or lntranasal administration of specific immune serum.
Firstly, to address the role of the systemic anti-LPS IgG antibody response in protection against the mueosal infection, we assessed the protection conferred against i.n.
challenge with a lethal dose of S. flexneri 2a bacteria in mice immunized i.p.
with the homologous killed bacteria. Antibodies induced upon such an immunization were mainly anti-LPS IgG antibodies (data not shown) with all the IgG subclasses similarly elicited (Figure 1 A). No mucosal response was elicited, as reflected by the absence of anti-LPS
antibody response detectable in the bronchoalveolar lavage of immunized mice.
Only 40% of the immunized mice survived the. i.n. challenge, whereas I00% of naive mice succumbed. The low efficacy of systemic immunisation in inducing protection could be due to either the inability of anti-LPS IgG to be protective or the absence of the protective antibodies (or their presence but in insufficient amount) in the mucosal compartment at the time of i.n. challenge.
We, therefore, tested whether the anti-LPS IgG antibodies may confer protection if present locally prior to mucosal challenge. Polyclonal sera exhibiting different anti-LPS
antibody titers were intranasally administered to naive mice 1 h prior to i.n.
infection with a sublethal dose of S. flexr~eri 2a bacteria. Protection was assessed by the reduction of the lung-bacterial load in comparison to control mice and mice receiving preimmune serum. In contrast to control mice and mice receiving preimmune serum, naive mice receiving anti~LPS IgG
serum showed a significant decrease of the lung-bacterial load. The reduction was dependent on the amount of anti-LPS IgG antibodies administered as reflected by the anti-LPS antibody titer of the immune serum used for passive transfer. Thus, the highest reduction was obtained with serum having the highest anti-LPS antibody titer ( I/G4,000) (Figure 1 B, c) (p=5 x 10-6 in comparison to mice receiving preimmune serum). However, in mice receiving immune serum Serum IgC papist-hrcvZ
with lower anti-LPS antibody titer (1/16,000 and 114,000) (Fig. IB, a and b), even if less efficient, the decrease of the bacterial load was still significant in comparison to mice receiving preinunune serum (p = 0, 027 and 0, 015, respectively).
These results demonstrated that, if present locally at the time of rnucosal challenge, the anti-LPS IgG antibodies were protective, thus limiting bacterial invasion.
2) Protective capacity of mAbs specific for S, flexrzeri Za serotype determinants and representative of the different IgG subclasses Depending of the infecting strain, different subclasses of IgG specific for LPS are induced followizig natural Slzigella infection (28). To test whether the different anti-LPS IgG
subclasses exhibit similar protective capacity, marine mIgG specific for serotype determinants on the O-Ag and, representative of each of the four marine IgG subclasses were obtained, We selected 5 mIgG specific for S. Jlexoeri 2a LPS : mIgG F22 (IgGl), mlgG D15 (IgGl), mIgG
A2 (IgG2a), mIgG E4 (IgG2b) and mIgG C1 (IgG3). The avidity of each mIgG for LPS, defined by ICSo, ranged from 2 to 20 ng/ml. To analyse the protective capacity of the selected mAbs, naive mice were administered i.n, with each of the purified mIgG prior to i.n.
challenge with a S. fJexneri sublethal dose. Upon challenge, lung-bacterial load in mice passively administered with 20 ~g of Each of the mIgG specific for S. flexneri 2a LPS was significantly reduced in comparison to mice receiving PBS (Fig. 2A). Upon passive transfer using 2pg ofmlgG, only mIgG D15, A2 and E4 were shown to significantly reduce the lung-bacterial load in comparison to control mice, but with much less efficiency than that observed using 20p,g (Fig. 2A), As shown in Figure 2H, reduction of lung-bacterial load in mice receiving 20 p.g of mIgG was accompanied by a reduction of inflammation and therefore of subsequent tissue destruction. In comparison to control mice showing an acute broncho-alveolitis with diffuse and intense polymorphonuclear cell infiltration (Figure 2H, a, h) Setuml&Crpapia-bcnvet ~1 02434685 2003-07-04 associated with tissular dissemination of bacteria (Figure 2B, c), only restricted areas of inflammation were observed, essentially at the infra- and peribronchial level (Figure 2B, d, e), where bacteria localized (Figure ZB,,~. Following passive administration with 2~g of mIgG, inflammation resembled that of the control mice with a similar pattern of PMN
infiltration and tissue destruction, in a,eeordance with the very loin, if any, reduction in lung-bactezial load (data not shown).
3) Serotype-specific protection induced by the anti-LPS mlgG
Antibodies specific for epitopes common to several serotypes of a given species as well as serotype-specific antibodies are elicited upon natural or experimental infection (58, 64).
However, the serotype-specific protection observed following natwal or experimental infection suggests that the antibodies directed against serotype determinants play a major protective role (19, 38). For instance, mIgA specific for S. flexneri serotype Sa has been shown to protect only against homologous challenge (51). We, therefore, tested whether the protection observed with the anti-LPS mIgG obtained in this study was also sErotype-specific.
Mice passively administered with 20 ug of m>;gG C1 specific for S. J).~2xneri 2a were protected against homologous challenge, but not upon heterologous challenge with S
Jlexneri Sa bacteria (Fig. 3A). Similarly, mice receiving 20 ~g of mIgG C20, a mAb specific for S
flexrreri serotype Sa and, of the same isotype than mIgG C1, i.e. IgG3, showed a significant reduction of lung-bacterial load upon i.n. challenge with S flexneri Sa, but not with S. flexneri 2a (Figure 3A). In mice protected against homologous challenge, inflammation was dramatically reduced with a slight infra- and peribronchial PNfhl infiltrate remaining present (Figure 3B, b and r). In contrast, in mice not protected upon heterologous challenge (Figure 3B, a and c~, inflammation and tissue destruction were similar to those observed in control mice (Figure 2B, a and b).

sCfvln (gG pspier~brevet C~1 02434685 2003-07-04 4) Protective capacity of mIgG specific for S. fl'exneri invasins The invasins IpaH and IpaC are essential to the expression of the Shigella invasive phenotype (39). Moreover, they are targets for the humoral response since antibodies specific for both proteins are detected in sera ofpatients convalescent from shigellosis (18, 45, 46, 63).
To assess whether the anti-invasin antibody response may contribute to protection, in addition to the anti-LPS anfibody response, we used mlgG recognizing different epitopes on IpaB or IpaC (z, 50). Whatever the dose used, in contrast to mIgG C20, no reduction in Iung-bacterial load was measured upon challenge in mice treated with mIgG H16 and mIgG H4 recognizing distinct epitopes in the central region of IpaB or with mlgG J22 and mlgG K24 recognizing the N~ and the C-termini domain of IpaC, respectively (Figure 5). Protection was also not observed upon combining anti-IpaB and anti-IpaC mIgG (data not shown.

Seism I~Cr paDier-brevet Dixcussion To date, the respective roles of local and systemic humoral immune responses specific for LPS 0-Ag in protection against Shigella infection remained unclear, although this question is crucial for the design of accurate vaccine candidates, Indirect evidence has suggested a protective role for anti-LPS IgG (14, 15, 1G, 34, 47, 63). We demonstrate here for the first time, using poiyctonal serum and specific mAbs, that the systemic IgG-mediated response specific for serotype determinants carried by LPS 0-Ag confers protection against mucosal infection. if present locally at the time of bacterial challenge.
LPS has been recognised for a long time as the major protective antigen (19, 22, 38).
However, the question of the protective role of the antibody response to bacterial proteins remains unanswered. Among the proteins recognized by sera from patients convalescent from shigellosis, IpaB and IpaC, the invasins involved in the entry of bacteria into enterocytes, are two major antigens. Only indirect evidence suggested that the systemic response to these two virulence factors was not essential for protection ( 1 G). We show here that mlgG specific for IpaB or IpaC are not protective despite the fact that they are directed against epitopes located in different regions of these proteins (2, 50) and that they have been shown to interfere with their functional properties in in vitro studies (4, 40). The most likely explanation is that these invasins, that are secreted through the type III secretion apparatus, are injected straight into the host cell, upon contact of the bacterium with the cell membrane (G, 41).
Therefore, there is probably very limited access, if any, for specific antibodies to interact with their targets.
Although not tested. it is unlikely that the local SIgA-mediated response to these proteins will be protective.
In the past, several sets of mAbs of M or G isotype specific for Shigella species have been produced. They are directed against the 0-Ag of S sonnet (1 ), of S.
dysenteriae ( 20, 56, 60) and, of S. flexneri (9, 10 1 l, 24, 26, GO). However, as the goal Was to develop diagnostic. tests Scrum IgG papler-breve~

for Shigella identification (12, 27), their protective properties have not been investigated.
Except for a few (42, 43), the sequence of the VH and VL genes is unknown.
Similarly, the oligosaccharide determinants they recognize have not been characterized, except for 2 mAbs specific for S dysenteriae 1 (43, 49). Thus, for a better understanding of carbohydrate antigenlantibady interactions, we are currently characterising the fine specificities of recognition between the mAbs obtained in this study and the 0-Ag they recognize.
To obtain tnIgG, hybridoma cells were selected, upon cell fusion, on the basis of their secretion of mAb recognizing determinants specific for the S. flexneri serotype used for immunization, i.e. serotype 2a and serotype Sa, respectively. During the screening, we observed that most of the hybridoma cells tested (about 90%) were secreting serotype-specific mAbs. This result slightly differs from previous reports showing the obtention of mAbs directed to determinants common to several S. flexneri serotypes including 2a and Sa (11, 24), However, it may be explained by recent new insights on bacterial 0-Ag conformation. For instance, in the case of S. dyser~teriae l, the a-z-I2hap-(1-->2)-a-D-Galp disaccharide represents the major antigenic epitope on the O-Ag. Interestingly, in the proposed conformational model of S. dysenteriae 1 O-Ag, which is a left-handed helical structure, the galactose residues protrude radially at the helix surface, therefore resulting in a pronounced exposure of both the galactose and the adjacent rhamnose of each repeating unit (44). A
similar result has been obtained in our hands with the O-Ag of S. .Jlexneri Sa. In that case, the branched glueosyl residue specifying this serotype and required for recognition by serotype-specific antibodies (L. Mulard and A. Phalipon, persona! communication) points out of the surface of the helix, which exhibits a right~handed three-fold helical structure (M. J. Clement and M. Delepierre, personal communication ). Therefore, we may reasonably hypothesize that these peculiar sugar residues repeatedly exposed at the O-Ag surface, and therefore at the Scrum IgG papier-brevet bacterial surface, preferentially trigger B cell receptor-mediated recognition, thus leading to the induction of a predominant anti-serotype specific antibody response.
In humans, depending on the infecting strain, different subclasses of IgG
specific fox LPS
are induced following natural Shigella infection (28). For instance, S
flexneri 2a and S.
dysenxeriae 1 preferentially induce IgG2, whereas S. .sonnei mainly induces IgGI. Similarly, upon vaccination with glyeoeonjugate vaccines using detoxified LPS from S, flexneri 2a and S. sonnei, the same pattern is obsen~ed, IgG2 and IgGI, respectively. These antibodies may confer protection by different pathways involving or not the complement cascade. In the present study, all the different marine IgG subclasses were shown to be protective, suggesting that depending on the subclass, different mechanisms may be involved in IgG-mediated protection. Whereas antibody-dependant cellular cytotoxicity (ADCC) has been reported for Shigella-specific secretary IgA and lymphocytes from the gut-associated lymphoid tissues (61), Shigella IgG-mediated ADCC occurs in vitro with splenic T cells but not with T
lymphocytes from the GALT (G2). Further studies using mice deficient for T
cells or for proteins of the complement cascade will be required to analyze the IgG-mediated protective mechanisms in viva.
The protective role of the serotype-specific antibody response has been firstly emphasized in a study using a m~orwclonal dimeric IgA (mIgA) specific for a S. flexneri serotype Sa determinant (51). Here, we demonstrate that mIgGs specific for S, flexneri serotype 2a or serotype Sa also confer serotype-specific protection. It seems that whatever the antibody isotype and the bacterial strain, the serotype-specific antibody response is protective against homologous bacterial challenge. It should be noted that using the same amount of mlgA and mIgG specific for S. flexneri Sa, both exhibiting a similar ICs for LPS, reduction in lung-bacterial load was much more efficient with mIgA. Actually, in contrast to mIgG, protection was observed in the presence of 2pg of mIgA. The dijcrepancy between the two isotypes may Serum 7gG pnpier.brevet be duE to the dimericlpolymeric (dlp) form of mIgA, which mimicks the IgA
response at the mucosal surface. In contrast to monomeric IgG, interaction of d/p IgA
exhibiting at least four antigen-binding sites with a specific determinant highly repeated on the bacterial O-Ag surface may lead to the formation of aggregates that are efficiently removed by local physical mechanisms (17). Also, quantitative assessment of IgG and IgA subclass producing cells in the rectal mucosa during shigellosis in humans has revealed the predominance of the IgA
response. The IgG response which is about 50 times lower than the IgA response is mainly IgG2 and correlates with the presence of specific IgG2 in serum. This correlation suggests that the majority of the Shigella specific serum antibodies are derived from the rectal mucosa (29). Together, these results suggest that in the situation where both local and systemic anti-LPS antibody responses are induced, as for example upon natural infection, the local SIgA-mediated response will be the major protective response, with the IgG-mediated response possibly contributing to a lesser extent to local protection.
On the other hand, our data suggest that in the absence of local SIgA-mediated response, as for example upon vaccination via the systemic route using glycoconjugate vaccines, the systemic anti-O-Ag response induced is effective in protecting against homologous Slaigella infection, if the effectors are present locally. Previous reports have shown that serwn IgGs may protect from gastrointestinal infections (7, 54).
Therefore, it should be admitted that serum IgG efficiently gain access to the intestinal barrier in ozder to prevent bacterial invasion and dissemination. How IgG crosses the epithelial barrielr to function in mucosal immunity remains unclear. One possible pathway is passive transudatinn fxom serum to intestinal secretions (5, 37, 67). After its passage of the intestinal barrier through M cells and its interaction with resident macrophages and epithelial cells, Slaigella initiates an inflammatory response leading to infiltration of the infected tissues with polymorphonuclear cells (53). We may therefore reasonably envision that specific serum IgGs IS

Serum IgG papier-brevet transudate to the intestinal tissue during this inflammatory process that occurs very soon after bacterial transloeation. Another explanation could be the involvement of the FcRn receptor in IgG transport. FcIW was firstly identified as the Fc receptor responsible for transferring maternal IgGs from mother's milk across the intestinal EC of the neonatal gut of rodents.
Much evidence supports the concept that FcRn is ubiquitously expressed in adult tissues and plays a role in IgG homeostasis, dealing with IgG half life (23). It has been recently reported that this receptor is expressed by enterocytes in human adults and mediates transcytosis of IgG in both direction across the intestinal epithelial mnnolayer (57). Further investigation is required to improve our Imowledge on the role played by Fcltn in IgG-mediated protection of the intestinal barrier against enteropathogens. Nevertheless, the existence of such a pathway already enlarges the current view of the humoral response at mucosal surfaces.
To conclude, our data are in favor of the hypothetical concept proposed by Robbins et al. stating that protection against bacterial enteric diseases may be conferred by serum IgG
antibodies to the 0-Ag of their bacterial LPS (59). The demonstration of the protective role of anti-LPS IgG-mediated systemic response against Shigella infection supports vaccine approaches based on detoxified LPS/protein glycoconjugate vaccines administered parenteTally (47). In addition, the serotype-specific protection suggests that, upon their characterisation, the protective serotype-specific determinants for prevalent Shigella strains could be suitably combined in order to develop a multivalent synthetic vaccine for parenteral vaccination, since promising results have been recently obtained with synthetic oligosaccharides as immunogenic conjugates (55).

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molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp. Glycobiology. 11:945-55.
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Ashkenazi. 1995. Shigella lipopolysaccharide antibodies in pediatric populations. Pediatr.
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Schneerson, J. B. Robbins, end C. P. J. Gisudemans. 1993. Binding of the 0-antigen of Shigella dysenteriae type 1 and ?.6 related synthetic fragments to a monoclonal IgM
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52. Phalipon, A., A. Cardona, J. P. Kraehenbubl, L. Edelman, P. J. Sansonetti, and B.
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Serum IgG papia-brcvd 54. Pier, G. B., G. Meluleni, and J. B, Goldberg. 1995. Clearance of Pseudomonas aeruginosa from the marine gastrointestinal tract is effectively mediated by O-antigen-specific circulating antibodies. Infect. Immun. G3 : 2818-2825.
S5. Pozsgay, V, C. Chu , L. Pannetl , J. Wolfe, .1. B. Robbins , and R.
Schneerson . 1999.
Protein conjugates of synthetic saccharides elicit higher LevEls of serum IgG
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SG. Qatri, F., T. Azim, A. Hossain, A. Chowdhury, and M. J. Albert. 1994.
Monoclonal antibodies specific for Shigella dysenteriae serotype 13. Production, eharacteri7ation, and diagnostic application. Diagn. Microbiol. Infect. Dis. 18 : 145-149.
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transcytosis and reclycling by FeRn e~cpressed in MDCK cells reveals ligand-induced redistribution.
EMBO J. 21 : 590-GO1.
58. Rasolofo-Razanamparany, V., A. M. Cassel-B~raud, J. Roux, P. J.
Sansonetti, and A.
Phalipon. 2001. Predominance of serotype-specific mucosal antibody response in Shigella flexneri-infected humans living in an area of endemicity. Infect. Immun. 69 :
5230-5234.
59. Robbing, J. B., C. Chu, and R. Schneersan. 1992. Hypothesis for vaccine development protective immunity to enteric diseases caused by non typhoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the 0-specific polysaccharide of their lipopolysaccharide. Clin. Infect. Dis. 15 : 346-361.
60. Suzuki, K., and T. Takeda. 1989. Monoclonal atibodies against the surface antigens of Shigella flexneri serotype lb and Shigella dysenteriae serotype 1. Microbiol.
Immunol. 33:
897-906.
I

sClt71711gG pepicr-brc~~d CA 02434685 2003-07-04 61, Tagliabue, A ., L. Necioni, L. Viiia, D. F. Keren, G. H. Lowest, and D.
Boragchi. 1983.
Antibody-dependent cell-mediated antibacterial activity of intestinal lymphocytes with secretory IgA. Nature 306 : 184-186.
62. Tagliabue, A., D. Boraschi, L. Villa, D. F. Keren, G, H. Lawell, R.
Rappuoli, and L.
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63. Van de Verg, L., D. A. Herrington, J. Baslego, A. A. Lindberg, and M. M.
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1992. Age-specific prevalencE of serum antibodies to the invasion pIasmid and polysaccharide of Shigella species in Chilean and north-americsn populations.
1. Infect. Dis.
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64. Van de Verg, L. L., N. O. Bendiuk, K. kotlotT, M. M. Marsh, J. L. Ruckert, J. L.
Puryear, D. N. Taylor, and A. B. Hartman. 1996. Cross-reactivity of Shigella Jlexneri serotype 2a 0 antigen antibodies following immunization or infection. Vaccine 14 : 1062-1068.
i

65. Vecino, W. H., P. M. Morin, R. Agha, W. R. Jaeobs, and G. J. Fennelly.
2002.
Mucosal DNA vaccination with highly attenuted Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV.
Immunol. Lett.
82 : 197-204.

66. Voino-Yasenetsky, M. V., and M. K. Voino-Yasenetskaya. 1961. Experimental pneumonia caused by bacteria of the shigella group. Acts. Morpho. XI : 440.

67. Wernet, P., H. Breu, J. Knop, and D. Rowley. 1971. Antibacterial action of specific IgA
and transport of IgM, IgA and IgG from serum into the small intestine. J.
Infect. Dis. 124 i 223-226.

68. Westphal, O,, and J. Jana. 1965. Bacterial lipopolysaccharides :
extraction with phenol-water and further application of the procedures. Methods Carbohydr. Chem. 5 :
83-91.

Serum IgG papicr-brcrct

69. Winsor, D. K., J. J. Mathewson, and H. L. DuPont. 1988. Comparison of serum and fecal antibody responses of patients with naturally acquired Shigella sonnei infection. J.
Infect. Dis. 158 : 1108-1112.

Serum 7 ~0 p~pierbrcvet Legends of figures Figure 1 : Protection conferred by immune serum specific for S.jdexnert 2a LPS
intranasa113~ administered prior to i.n. challenge.
A) Serum IgG subclasses elicited in mice upon i.p, immunization with killed S.
Jlexrreri 2a bacteria. - represents the mean value of the antibody titer (n=10 mice).
B) Protection assessed by reduction of lung-bacterial load in mice receiving anti-S.flexneri 2a LPS immune serum raised upon i.p, immunization, lh prior to i.tt. challenge with a sublethal dose of S, flexneri 2a bacteria. a, b, c, correspond to immune sera exhibiting an anti-S
,flexneri 2a LPS IgG antibody titer of 1/4,000, 1/16,000 and 1/64,000, respectively. Standard deviatifln is indicated (n-I O mice per group).
Figure 2 : Protection conferred by different subclasses of mIgG specific for S. Jlexneri 2a serotype determinants.
A : mice receiving intranasally 20pg and 2pg of purified mIgG, respectively, lh prior to i .n.
challenge with a sublethal dose of S flexneri 2a. Lung-bacterial load was expressed using arbitrary units with 100 corresponding to the bacterial count in lungs of control mice.
Standard deviations are represented (n=10 mice per group).
B : Histopathological study of mouse Iungs. Upper row : control mice. Lower row : mice receiving mIgG. HE staining : a and d magnification x 40 ; b and a magnification x 100.
Itnmunostaining using an anti-LPS antibody specific for S. ~lexrreri serot3~pe 2a : c and f magnification x100.
Figure 3: Serotype-specific protection conferred by the anti-LPS mIgG.
A : Mice were receiving i.n. 20Ng of each of the purified mIgG, C20 and C1, lh prior to i.n.
challenge with a sublethal dose of S. ,/lexneri serotype 2a (.A) or serotype Sa (B) bacteria.
I

....,...,~ ag.n y.nym.n-mvrw Lung-bacterial load was expressed using arbitrary units with 100 corresponding to the bacterial count in lungs of contzol mice. Standard deviations are represented (n=10 mice per group).
H : Histopathological study of mouse lungs. a and b : mice receiving mIgGC20 specific for S.
~lexneri serotype Sa and challenged with S. flexneri serotype 2a and 5a, respectively, c and d mice receivir~ mIgGC1 specific for S flexneri 2a prior to challenge with S.
~exneri serotype 2a and Sa, respectively. HE staining, magnification x 100.
Figure 4 : Protection conferred by mIgG specific .for S. flexrreri IpaS or IpaC invasins.
Mice were receiving i.n. 20wg of each of the purified mIgG, H4, H16, J22, KZ4, and C20, lh prior to i.n. challenge with a sublethal dose of S flexneri semtype 5a. Lung-bacterial load was expressed using arbitrary units with 100 corresponding to the bacterial count in lungs of control mice. Standard deviations are represented (n=10 mice per group j.

Serum Ig0 papia-brevet Acknowledgements : We thank Nicole blusher and Michel Huerte (Unite de reeherche et d'expertise Histoteclmologie et Pathologie, Institut Pasteur) for their unvaluable work in histology, Verronique Cadet (Hybridolab, Institut Pasteur) for her help in mAbs production, and Josette Arondel for the mice experiments she did just before getting retraited. We also thank Isabel Fernandez and Maria Mavris for careful reading of the manuscript.
P. J. S. is a Howard Hughes A~fedical Institute scholar, ,.~........n. w u..: a , cpu~U~~CS
PREPARATION OF THE OLIGOSACCHARIDE-TETANUS TOXOID CONJUGATES
CGS0303-8-3: Tetanus toxoid-ECD conjugate CGS0303-8-4: Tetanus toxoid-B(E)CD conjugate CGS0303-8-5: Tetanus toxoid- AB(E)CD conjugate EXPERIMENTAL SECTION
General procedures. N (y-maleimidobutiryloxy) sulfosuccinimide ester (sulfo-GMBS) was purchased from Pierce. Tetanus toxoid (TTY (MW 150 kDa) (batch n°FA
045644), was purchased from Aventis Pasteur (Marcy fEtoile, France), and stored at 4°C in a 39.4 mg.mL't solution.
Dialyses were performed with Slide-A-Lyzer~ Dialysis Cassettes (Pierce).
Derivatization of the tetanu9 toxoid Protocol A
To a solution of tetanus toxoid (12 mg, 304 p,L, 0.08 mole) diluted in PBS 0.1 M, pH 7.3 (29G p,L), was added N (y-maleimidobutiryloxy) sulfosuccinimide ester (GMBS) (3 x 1.53 mg, 3 x 29 pL of an 60 mg.mL-' solution in CH3CN, 3 x 50 equiv), in three portions every 40 minutes. The pH of the reaction mixture was controlled (indicator paper) and maintained at ?-7.5 by addition of aq NaOH O.SM. Following an additional reaction period of 40 minutes, the crude reaction mixture was dialy2ed against 3 x 2 L ofphosphate buffer 0.1 M, pH 6.0 at 4°C
to eliminate excess reagent.
Preparation and characterization of the conjugates Following dialysis, maleimide activated-TT in. phosphate buffer O.1M solution was divided into three portions which were fi~th~z reacted with reacted S-acetylthioacetylated-tri-, tetra-and penta-saccharide from Shige~Ia flexneri 2a antigen-O in a I:12 molar ratio, respectively.
Reaction mixtures were buffered at a 0.5 M concentration by addition of phosphate buffer 1M, pH 6Ø Then, NHzOH, HCl (7.5 pL of a 2 M solution in phosphate buffer 1 M, pH G), was added to the different mixtures and the couplings were carried out for 2 h at room teperature. The conjugated products were dialyzed against 3 x 2 L of PBS 0.05 M, pH ?.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL~6B column (1 I

Synthesis ofihe TT C°I~U~7~CS ~1 02434685 2003-07-04 m x 164 mm) (Pharma.cia .Biotech), using PBS 0.05 M, pH 7.4 as eluent at a flow rate of 0.2 mL.miri ~, with detection by measuring the optical density at 280 nm and the refractive index.
The appropriate fractions were pooled and concentrated over a dialysis tubing-visking size 1-8132" membrane. The conjugates were stored at 4°C in the presence of thimerosal (0.1 mg.mL'i) and assessed for their total carbohydrate and protein content.
Hexose concentrations were measured by a colorimetric method based on the anthrone reaction, using pmLPS as a standard.
Protein concentrations were measured by the Lowry's spectrophotometric method, using HSA
as a standard and/o total acidic hydrolysis (6N HCl at X°C for 20 h), using norleucine as an internal standard.
Determination of hexoses with anthrone Reagents: The reagents are as follows Stock sulfuric acid. Add 750 mL of concentrated sulfuric acid to 250 mL of distilled water and cool the solution to 4°C.
Anthrone reagent. Dissolve 1.5 g of anthrone in 100 mL of ethyl acetate and cool the solution to 4°C.
Standard pmLPS O1 Inaba solution: Prepare a solution at a concentration of $
mg.mL't in water. Prepare serial dilutions of 800 to 25 p,Mol of pmLPS O1 Inaba standard solution in water.
Procedr~re:
Prepare serial dilutions of 800 to 25 pMol of pmLPS O1 Inaba standard solution in water (1 mL) in screw-threaded tubes. Prepare similarly a reagent blank containing 1 mL
water and control reagents containing a known amount of pmLPS O1 Inaba or glucose in 1 mL water.
Prepare samples and make up to 1 mL if necessary by adding water. Cool all tubes in ice-water.
To each tube, add ~ mL of the concentrated HzSOs and 0.5 mL of the anthronc solutions. Heat the tubes at 100°C, caps unscrewed for 3 minutes and then caps screwed for 7 minutes. After exactly 10 minutes, return the tubes to an ice-bath and when cool measure the absorbances in ' a spectrophotometer (Seconam 5.750I), at a wavelength of 625 nm. The quantity of carbohydrate in the unknown samples can be read off from the standard curve prepared with the standard solution samples and the blank Determination of~rotein content ~Lowry~
i Reagents: The reagents are as follows Synthesis of the TT conjuggtcs Stock solution A. Add 1 g of sodium carbonate to 50 mL of 0.1 N aqueous sodium hydroxide Stock solution B. Mix 1 mL of a stock solution of 1°/a (w/v) aqueous cupper(II) sulfate with 1 mL of a stock solution of 2% (w/v) aqueous sodium tartrate;
Stock solution AB: Mix 1 mL of the stock solution B with 50 mL of the stock solution A-Standard BSA solution: Prepare a solution at a concentration of 1 mg.mL-I in water.
Folin reagent Procedure:
Prepare a blank and serial dilutions of BSA by adding 0, 10, 20, 30, 40 or 50 p.L of standard BSA solution to 1 mL of stock solution AB in clean disposable tubes. Complete to 1.2 mL by adding water. Prepare similarly sample dilutiotls by adding 200 p,L of the sample solutions to 1 mL of stock solution AB.
Incubate for 10 minutes at room temperature and add 100 pL of folin reagent in each tube.
Incubate for 20 minutes at room temperature and measure the absorbance in a spectrophotometer (Seconam 5.750I), at a wavelength of 660 nm. The quantity of protein in the unknown samples can be read off from the standard curve prepared with the standard solution samples and the blank.
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Claims

CLAIMS, 1) A glycopeptide comprising an immunogenic carrier compound congugated to a synthetic oligosaccharide derived from the O-specific polysaccharide of Shigella flexneri selected among the group consisting of:
{ABC}n (BCD}n {CDA}n (DAB}n {B(E)C}n ((E)CD}n {AB(E)C}n {R(E)CD}n {(E)CDA}n (DAB(E)C}n {B(E)CDA}n {(E)CDAB}n {AB(E)CD}n (B(E)CDAB(E)C}n {DAB(E)CDAB(E)C}n wherein A is an alphaLRhap-(1,2) residue B is an alphaRhap-(1,3) residue C is an alphaLRhap-(1,3) residue E is an [alphaDGlcp-(1,4)] residue D is a betaDGIcNacp-(1, residue and wherein n is an integer comprised between 1 and 10 and preferably between 2 and 6.

2) A glycoconjugate according to the claim 1 wherein the synthetic olygosaccharide is a derived Omethyl derivative.

3) A glycoconjugate according to the claim 1 wherein the immunogenic carrier compound is selected among an immunogenic protein, an immunogenic peptide or a derivative thereof.

4) A glycoconjugate according to claim 3, wherein the immunogenic carrier is the peptide PADRE.

5) A glycoconjugate according to claim 3, wherein the immunogenic carrier compound is the Tetanus toxine.

6) A glycoconjugate according to anyone claims 1 to 5 wherein the oligosaccharide is directly coupled to the immunogenic carrier compound.

7) A glycoconjugate according to anyone claims 1 to 5 wherein the oligosaccharide is coupled to the immunogenic carrier compound via an arm spacer.

8) A glycoconjugate according to claim 7 wherein the arm spacer is an alanine derivative.

10) A glycoconjugate according to the claim 1 wherein the synthetic olygosaccharide is a selected among the hexasaccharide, the decasaccharide and the pentasaccharide depicted in Figure 1 11) Composition useful to induce an immune response against Shigella comprising an efficient amount of a glycoconjugate according to any claims 1 to 8.
Obviously also methods to obtain the oligosaccharides, the oligosaccharide derivatives to be conjugated to the immunogenic carrier and the glycopeptides must be claimed.