Accepted Manuscript Synthesis of the tetrasaccharide outer core fragment of Burkholderia multivorans lipooligosaccharide Marcello Ziaco, Cristina De Castro, Alba Silipo, Maria Michela Corsaro, Antonio Molinaro, Alfonso Iadonisi, Rosa Lanzetta, Michelangelo Parrilli, Emiliano Bedini PII: DOI: Reference:

S0008-6215(14)00179-7 http://dx.doi.org/10.1016/j.carres.2014.04.018 CAR 6735

To appear in:

Carbohydrate Research

Received Date: Revised Date: Accepted Date:

21 March 2014 22 April 2014 25 April 2014

Please cite this article as: Ziaco, M., Castro, C.D., Silipo, A., Corsaro, M.M., Molinaro, A., Iadonisi, A., Lanzetta, R., Parrilli, M., Bedini, E., Synthesis of the tetrasaccharide outer core fragment of Burkholderia multivorans lipooligosaccharide, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.04.018

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Synthesis of the tetrasaccharide outer core fragment of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Burkholderia multivorans lipooligosaccharide

Marcello Ziaco, Cristina De Castro, Alba Silipo, Maria Michela Corsaro, Antonio Molinaro, Alfonso Iadonisi, Rosa Lanzetta, Michelangelo Parrilli, Emiliano Bedini* Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S.Angelo, Via Cintia 4, 80126 Napoli, Italy

Abstract The first synthesis of the outer core fragment of Burkholderia multivorans lipooligosaccharide [D-Glc-(1→3)--D-GalNAc-(1→3)--D-GalNAc-(1→3)-L-Rha]

as -allyl tetrasaccharide was

accomplished. The glycosylations involving GalNAc units were in-depth studied testing them under several conditions. This allowed the building of both the - and the -configured glycosidic bond by employing the same GalNAc glycosyl donor, thus considerably shortening the total number of synthetic steps. The target tetrasaccharide was synthesized with an allyl aglycone to allow its future conjugation with an immunogenic protein en route to the development of a synthetic neoglycoconjugate vaccine against the Burkholderia cepacia pathogens.

Keywords:

oligosaccharide,

glycosylation,

N-acetyl-galactosamine,

glucose,

rhamnose,

Burkholderia cepacia.

1. Introduction Cystic fibrosis (CF) is a genetically inherited disease caused by a mutation that alters host pulmonary defences, allowing colonization from a variety of opportunistic bacteria. These include Pseudomonas aeruginosa, that is the most common species infecting CF patients,1 Burkholderia

*

Corresponding author: Tel. +39-(0)81674153; Fax +39-(0)81674393; e-mail: [email protected]

1

cepacia complex (Bcc), a group of closely related Gram-negative bacteria comprising at least 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

seventeen species, and several others. Bcc is responsible for the most dangerous infections in CF,2 which are characterized by a high transmissibility, a marked resistance to antibiotic treatment3 and antimicrobial peptides.4 As result, the health decline of Bcc infected CF patients is more rapid than that caused by P. aeruginosa infections. Furthermore, among bacteria colonizing CF patients, only the Bcc is able to determine an acute and almost invariably fatal respiratory illness, named „cepacia syndrome‟.5 A key obstacle to the treatment of these pathogens, is the natural selection of multipleantibiotic resistant strains, therefore the development of convenient vaccines represents a desirable resource to prevent infection and to increase life expectancy and/or quality of CF patients. Among the many types of vaccines developed in the last two decades, those carbohydrates-based were safe and effective against several human pathogens, such as Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae. The main improvement on traditional carbohydrate vaccines, is given by the conjugation of the carbohydrate antigen - per se unable to promote immunological memory - to a carrier, that is an immunogenic protein, through a suitable linker.6 Lipopolysaccharides (LPSs) - the major virulence factors of Gram-negative bacteria - are glycolipid macromolecules covering about 75% of the bacterial outer membrane surface. From a structural point of view, they are divided into three chemically and genetically distinct regions. The first one, termed lipid A, is anchored to the lipid external membrane of bacteria and represents the toxic part of LPSs. It is elongated with an oligosaccharide, named core and further divided into an outer and an inner core. In turn, the outer core is elongated with a polysaccharide region (O-chain, O-antigen), that is responsible for the serological classification of the bacterial strains.7 Bacterial surface carbohydrates as the O-chain region of LPSs are often useful sources of sugar antigens for vaccine candidates against the pathogens from which they arise from. For instance, a glycoconjugate vaccine candidate against Pseudomonas aeruginosa - the most common pathogen that infects CF patients - was built conjugating the whole O-chain to exotoxin A.8 Preclinical studies in mice demonstrated that it conferred significant protection against P. aeruginosa infection. 2

Furthermore, 10-years long phase II and III clinical studies revealed that the vaccine candidate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

promoted the opsonophagocytic killing of P. aeruginosa by human neutrophils and protected from chronic infection stimulating cell-mediated immunity.9 The synthesis of the O-chain repeating unit of the LPS of a Burkholderia cepacia strain isolated from CF patients was also reported, en route to a potential synthetic glycoconjugate vaccine.10 Some bacteria possess no O-chain in their LPS structure and the outer core results as the saccharide portion most exposed toward the external environment. In these cases, it is usually thought that the core can play some roles in place of the O-chain. For this reason, the synthesis of outer core oligosaccharide

fragments

of

P.

aeruginosa

lipooligosaccharide

(LOS)11

as

well

as

neoglycoconjugates containing Bcc inner core epitopes12 were very recently reported. In the last decade, our group and others determined the structure of surface carbohydrates from pathogenic bacterial strains isolated from CF patients with chronic Bcc infections belonging to several species.13 Interestingly, the three most prevalent clinical Bcc species – B. cenocepacia, B. multivorans and B. vietnamiensis – all exposed very similar saccharide structures on their external membrane (Figure 1).14-17 En route to the development of a synthetic neoglycoconjugate vaccine against Bcc, the obtainment of these oligosaccharide structures in a pure form was the first step. To this aim, very recently we reported the first synthesis of the trisaccharide outer core fragment of B. vietnamiensis LOS, carrying an allyl aglycone as handle for the conjugation with an immunogenic protein (1, Figure 2).18 In this work we present the first total synthesis of the -allyl glycoside of the tetrasaccharide outer core fragment of B.multivorans (2).

2. Results and discussion The target tetrasaccharide possesses two 2-acetamido-2-deoxy-galactose (GalNAc) residues with different stereochemistry at the anomeric position. Several methods were developed for the stereoselective glycosylation of 2-amino-2-deoxyglycosides.19 Usually, different GalNAc building blocks are employed for the obtainment of - or -glycosidic linkages, respectively. Amido-, 3

carbamato- or imido- protecting group at the nitrogen atom, that are capable of efficient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

participation via acyloxonium ion, are frequently employed for the synthesis of -anomers, whereas -stereoselectivity is often obtained with 2-azido-2-deoxy-Gal glycosyl donors. To minimize the number of monosaccharide building blocks necessary for the synthesis of 2, we tested an alternative approach using a unique GalNAc donor for the obtainment of both - and -glycosidic linkages of the target. Both - and -stereoselective glycosylations have been reported for 2-azido-2-deoxyGal20-22 and 2-deoxy-2,3-oxazolidinone-Gal donors.23-25 Since several protocols are known for the straightforward synthesis of orthogonally protected 2-azido-2-deoxy-Gal building blocks from diverse commercially available starting compounds,26,27 we embarked in the synthesis of a suitably protected GalN3 galactosyl donor. Three known steps converted commercially available 3,4,6-tri-Oacetyl-galactal into selenoglycoside 3,28 that was then protected at O-3 position with a methoxycarbonyl group (Scheme 1). This protecting group was chosen because it could be installed and removed under mild conditions with very high chemoselectivity in the presence of several other protecting groups, including esters. Indeed, by treating 3 with methyl chloroformate and N,N,N′,N′tetramethylethylenediamine (TMEDA) in CH2Cl2,29 carbonate 4 could be obtained pure in 99% yield after a simple extractive work-up. Hydrolysis of the selenoglycoside with Nbromosuccinimide (NBS) in aqueous THF furnished hemiacetal 5 in 83% yield. Installation at the anomeric position of a -trichloroacetimidate was possible by treatment of 5 with Cl3CCN and 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) in CH2Cl2. The obtained GalN3 donor 6 (82%) was used for glycosylation with acceptor 7, that was synthesized in turn from L-rhamnose through a known procedure.30 A low-substrate-concentration reaction condition in nitrile solvents was firstly tested for the glycosylation. Although it has been recently employed for -glycosylations of 4,6-Obenzylidene-protected GalN3 thioglycosides,22,31 no disaccharide product could be obtained with trichloroacetimidate donor 6 (Table 1, entry 1). Some examples in the literature report the glycosylation of 2-azido-2-deoxyglycosyl--trichloroacetimidates under SN2-like conditions.20,32-36

4

Therefore, the coupling between 6 and 7 was attempted at low temperature (-85°C/-75°C) in a 3:2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

v/v CH2Cl2-n-hexane solution with BF3•OEt2 as catalyst. -Disaccharide 8 (Scheme 2) was obtained exclusively and in low yield (19%, entry 2). The more reactive -configured GalN3 donor 6 was then synthesized in 84% yield from hemiacetal 5 with Cl3CCN and K2CO3 in CH2Cl2 and coupled with acceptor 7 under TMSOTf catalysis in a 3:2 v/v CH2Cl2-n-hexane solution at -85°C/75°C (entry 3). -Disaccharide 8 expected under this conditions from a SN2-like attack of 7 on a -configured glycosyl triflate intermediate,20 was not isolated, whereas -configured disaccharide 8 was obtained exclusively. This could be ascribed to a torsional disarming effect of the 4,6-Obenzylidene ring37 on donors 6and/or to a possible remote participation of the carbonate group at position O-3,20 both favouring -stereoselectivity. Furthermore, the stereochemical outcome of the glycosylation reactions involving GalN3 donors have been already reported to be rather dependent on acceptor structure,38 with low reactive species – such as 7 due to the benzoate esters adjacent to the hydroxyl group - giving -products predominantly.21 By glycosylating 6 in nitrile solvent (EtCN) at low temperature (-80°C/-70°C), a 7:3 / mixture of 8 could be obtained in modest yield (entry 4). By increasing reaction temperature and switching from propionitrile to acetonitrile, glycosylation yield was considerably increased, with -disaccharide slightly predominating over the  (entry 6). To avoid any hypothetical -directing effect by participation of the carbonate group at position O-3 of 6, a new GalN3 donor (11) was designed with an ether protecting group at that position (Scheme 1). In particular, a 2-naphtylmethyl (NAP) protecting group was chosen, because it could be orthogonally cleaved in the presence of benzylidene and allyl groups. Compound 11 was synthesized in three steps from 3. Etherification with NAPBr and NaH in DMF gave 9 (86%), that was converted in turn into hemiacetal 10 (83%) by hydrolysis of the selenoglycoside with NBS in aqueous THF. Reaction of 10 with Cl3CCN and K2CO3 in CH2Cl2 furnished glycosyl donor 11 (68%), that was then coupled with acceptor 7 by activation with

5

TMSOTf in acetonitrile (Table 1, entry 7) to give disaccharide product 12 with a slight 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

improvement in yield but no / ratio increase with respect to donor 6. Even if the yield of the desired -disaccharides 8 and 12 in the glycosylation step was only moderate, they could be obtained as pure anomers by silica-gel chromatography, thus allowing us to proceed with the synthesis. Selective cleavage of methoxycarbonyl group in 8 to disaccharide acceptor 13 was conducted with the procedure developed by us, employing lithium iodide and acetic acid in refluxing pyridine.39 Alcohol 13 was obtained in almost quantitative yield (98%) without any cleavage of the benzoate esters (Scheme 2). The same alcohol could be synthesized in 91% yield from 12 by oxidative cleavage of NAP ether with DDQ in 4:1 v/v CH2Cl2-MeOH. The glycosylation of disaccharide acceptor 13 with GalN3 donor 11 to give trisaccharide 14 with a configuration at the newly formed glycosidic bond, was firstly attempted in 1:1 v/v CH2Cl2-1,2dimethoxyethane (DME)40 (Table 1, entry 8). Glycosylations of 4,6-O-benzylidene-protected GalN3 donors in CH2Cl2-ethereal solvent mixtures have been reported to proceed in high yield and stereoselectivity,41,42 nonetheless no coupling product could be isolated in this case. By conducting the reaction in neat CH2Cl2, the desired -trisaccharide 14 was obtained in good yield (65%, entry 9). A slight yield decrease was observed by lowering the reaction temperature from -35 to -75°C (entry 10). By substituting GalN3 donor 11 with 6, a very similar behaviour was observed (entry 11). Trisaccharides 14 and 15 were then converted into acceptor 16 by treatment with DDQ in 4:1 v/v CH2Cl2-MeOH (68%) or lithium iodide and AcOH in refluxing pyridine (84%), respectively. Coupling of 16 with per-O-benzoyl glucose trichloroacetimidate 1743 under TMSOTf catalysis in CH2Cl2 at 0°C furnished tetrasaccharide 18 in 67% yield as the sole -anomer. Conversion of 18 into the target tetrasaccharide 2 was accomplished in four steps. Firstly, the reduction of azido groups was attempted by Staudinger reaction with PPh3 in 5:1 v/v THF-H2O at 50°C, that afforded a complex mixture of products. Better results were obtained by reaction with Zn/Cu in 3:2:1 v/v/v

6

THF-Ac2O-AcOH, that allowed the reduction of the two azido groups to amines and its 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

concomitant acetylation to 19 (50%). Benzylidene cleavage was then performed under mild acid hydrolysis conditions by employing 90% v/v aqueous AcOH at 50°C. Since the desired tetraol 20 could not be obtained in pure form by silica-gel chromatography, it was peracetylated with Ac2O in pyridine to give derivative 21 that could be purified smoothly (70% over two steps from 19). Finally, a Zemplèn transesterification afforded pure target 2 in 91% yield (HSQC-DEPT NMR spectrum in Figure 3).

3. Conclusion The first synthesis of the -allyl glycosideof -Glc-(1→3)--GalNAc-(1→3)--GalNAc(1→3)-L-Rha - the tetrasaccharide fragment of the outer core region of Burkholderia cepacia pv. multivorans LOS - was here reported. The synthetic strategy was carefully designed to minimize the number of steps. In particular, the reaction conditions for the glycosylations involving GalN units were in-depth studied, in order to use a single building block for the synthesis of both the - and the -configured glycosidic bond. The allyl aglycone of the target tetrasaccharide will allow the conjugation with an immunogenic protein in order to study its antigenic properties toward the development of a synthetic neoglycoconjugate vaccine candidate against Burkholderia cepacia pathogens.

4. Experimental General methods.  1H and 13C NMR spectra were recorded on Bruker DRX-400 (1H NMR: 400 MHz,

13

C NMR: 100 MHz), Varian INOVA 500 (1H NMR: 500 MHz,

instruments or on a Bruker-600 DRX (1H NMR: 600 MHz,

13

13

C NMR: 125 MHz)

C NMR: 150 MHz) instrument

equipped with a cryo probe, in CDCl3 (internal standard, for 1H: CHCl3 at  7.26 ppm; for

13

CDCl3 at  77.0 ppm) or in D2O (internal standard, for 1H: (CH3)2CO at  2.22 ppm; for

13

7

C: C:

(CH3)2CO at  30.9 ppm). Positive MALDI-MS spectra were recorded on a Applied Biosystem 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Voyager DE-PRO MALDI-TOF mass spectrometer in the positive mode: compounds were dissolved in CH3CN at a concentration of 0.1 mg/mL and one microliter of these solutions were mixed with one microliter of a 20 mg/mL solution of 2,5-dihydroxybenzoic acid in 7:3 CH3CN/H2O. Optical rotations were measured on a JASCO P-1010 polarimeter. Elemental analysis were performed on a Carlo Erba 1108 instrument. Centrifugations were performed with an Eppendorf Centrifuge 5804 R instrument. Freeze-dryings were performed with a 5Pascal Lio 5P 4K freeze dryer. Analytical thin layer chromatographies (TLCs) were performed on aluminium plates precoated with Merck Silica Gel 60 F254 as the adsorbent. The plates were developed with 10% H2SO4 ethanolic solution and then heating to 130°C. Flash column chromatographies were performed on Kieselgel 60 (63-200 mesh).

Phenyl

2-azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl-1-seleno--D-

galactopyranoside (4)  A solution of 328 (1.053 g, 2.436 mmol) in CH2Cl2 (14 mL) was cooled to 0°C and treated with TMEDA (551 L, 3.654 mmol) and methyl chloroformate (376 L, 4.872 mmol). The formation of a white precipitate was observed. The mixture was stirred at 0°C for 1h, then diluted with CH2Cl2 (120 mL) and washed with brine (120 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give pure 4 (1.178 g, 99%) as a white foam. 1H NMR (500 MHz, CDCl3): 7.59-7.28 (m, 10H, H-Ar), 6.12 (d, 1H, J1,2 5.0 Hz, 1H), 5.58 (s, 1H, CHPh), 4.92 (dd, 1H, J3,2 11.0 Hz, J3,4 3.5 Hz, H-3), 4.56 (d, 1H, J4,3 3.5 Hz, H-4), 4.53 (dd, 1H, J2,3 11.0 Hz, J2,1 5.0 Hz, H-2), 4.17-4.06 (m, 3H, H-5, H-6a, H-6b), 3.86 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3):  154.7 (C=O), 134.0, 133.9 (2 Cipso), 129.2-126.2 (C-Ar), 100.8 (CHPh), 84.6 (C1), 75.4, 72.7, 68.9, 64.8, 58.5, 55.3 (C-2, C-3, C-4, C-5, C-6, OCH3). MALDI TOF-MS for C21H21N3O6Se (m/z): Mr (calcd) 491.06, Mr (found) 514.28 (M+Na)+. Anal calcd: C. 51.44, H 4.32, N, 8.57; found C 51.20, H 4.46, N 8.45.

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl-D-galactopyranose

(5)



Compound 4 (1.151 g, 2.344 mmol) was dissolved in 4:1 v/v THF-H2O (10 mL) and then treated with NBS (834 mg, 4.69 mmol). The orange solution was stirred at 0°C for 90 min, then diluted with CH2Cl2 (120 mL) and washed with 1:1 v/v 1M NaHCO3-10% Na2S2O3 (120 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (2:1 to 1:1 v/v n-hexane-ethyl acetate) to afford 5 (648 mg, 83%, / = 2.5:1) as a white foam. 1H NMR (400 MHz, CDCl3): 7.52-7.37 (m, 5H + 5H, H-Ar), 5.57 (s, 1H, CHPh), 5.56 (s, 1H, CHPh), 5.50 (t, 1H, J1,2=J1,OH 3.6 Hz, H-1), 5.20 (dd, 1H, J3,2 11.2 Hz, J3,4 3.6 Hz, H-3), 4.67 (t, 1H, J1,2=J1,OH 7.6 Hz, H-1), 4.57 (dd, 1H, J3,2 10.8 Hz, J3,4 3.6 Hz, H-3), 4.52 (d, 1H, J4,3 3.6 Hz, H-4), 4.38 (d, 1H, J4,3 3.6 Hz, H-4), 4.32 (dd, 1H, Jgem 12.4 Hz, J6a,5 1.6 Hz, H-6a), 4.25 (dd, 1H, Jgem 12.4 Hz, J6a,5 1.2 Hz, H-6a), 4.08 (dd, 1H, Jgem 12.4 Hz, J6a,5 1.6 Hz, H-6b), 4.06-4.01 (m, 1H + 1H, H-2, H-6b), 3.99 (bs, 1H, H-5), 3.92 (dd, 1H, J2,3 10.8 Hz, J2,1 7.6 Hz, H-2), 3.86 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.81 (bd, 1H, J5,6b 7.2 Hz, H-5), 3.49 (bs, 1H, OH), 3.21 (d, 1H, JOH,1 3.6 Hz, OH);

13

C NMR (100

MHz, CDCl3):  155.0 (C=O), 137.3 (Cipso), 129.1, 128.2, 126.1 (C-Ar), 100.7, 99.5 (CHPh), 96.5, 92.7 (C-1), 75.4, 73.2, 72.8, 72.4, 69.1, 68.9, 66.4, 62.5, 61.9, 57.9, 55.4, 55.3 (C-2, C-3, C-4, C-5, C-6, OCH3). MALDI TOF-MS for C15H17N3O7 (m/z): Mr (calcd) 351.11, Mr (found) 374.39 (M+Na)+. Anal calcd: C. 51.28, H 4.88, N, 11.96; found C 51.04, H 4.95, N 11.80.

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl--D-galactopyranosyl trichloroacetimidate (6)  A solution of 5 (194 mg, 0.580 mmol) in CH2Cl2 (4.2 mL) was treated with Cl3CCN (867 L, 8.70 mmol) and then with a 0.44 M solution of DBU in CH2Cl2 (400 L, 0.176 mmol). After 80 min stirring at rt, the obtained brownish solution was diluted with toluene (2 mL) and concentrated to give a syrup that was subjected to flash chromatography 9

(7:1:0.005 to 3:1:0.005 v/v/v n-hexane-ethyl acetate-triethylamine) to afford 6 (228 mg, 82%) as a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

white foam. []D +160.1 (c 1.0, CH2Cl2); 1H NMR (600 MHz, CDCl3): 8.77 (s, 1H, NH), 7.517.37 (m, 5H, H-Ar), 6.61 (d, 1H, J1,2 3.0 Hz, H-1), 5.58 (s, 1H, CHPh), 5.19 (dd, 1H, J3,2 11.4 Hz, J3,4 3.6 Hz, H-3), 4.63 (d, 1H, J4,3 3.6 Hz, H-4), 4.34-4.31 (m, 2H, H-2, H-6a), 4.07 (dd, 1H, J6b,5 13.2 Hz, Jgem 1.8 Hz, H-6b), 3.99 (bs, 1H, H-5), 3.86 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3):

 160.5 (C=N), 154.8 (C=O), 137.1 (Cipso), 129.2, 128.2, 126.1 (C-Ar), 100.7 (CHPh), 95.2 (C-1), 73.1, 72.6, 68.7, 64.8, 57.0, 55.4 (C-2, C-3, C-4, C-5, C-6, OCH3). MALDI TOF-MS for C17H17Cl3N4O7 (m/z): unstable. Anal calcd: C. 41.19, H 3.46, N, 11.30; found C 40.98, H 3.55, N 11.18.

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl--D-galactopyranosyl trichloroacetimidate (6)  A solution of 5 (151 mg, 0.431 mmol) in CH2Cl2 (5.1 mL) was treated with Cl3CCN (215 L, 2.15 mmol) and then with K2CO3 (179 mg, 1.29 mmol). After 4.5 h stirring at rt, the suspension was diluted with toluene (10 mL), filtered on a Celite pad and concentrated to give a syrup that was subjected to flash chromatography (6:1:0.005 to 2:1:0.005 v/v/v n-hexane-ethyl acetate-triethylamine) to afford 6 (172 mg, 84%) as a white foam. []D +44.8 (c 1.8, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.78 (s, 1H, NH), 7.55-7.39 (m, 5H, H-Ar), 5.73 (d, 1H, J1,2 8.4 Hz, H-1), 5.57 (s, 1H, CHPh), 4.69 (dd, 1H, J3,2 10.8 Hz, J3,4 3.6 Hz, H-3), 4.47 (d, 1H, J4,3 3.6 Hz, H-4), 4.37 (dd, 1H, Jgem 12.4 Hz, J6a,5 0.8 Hz, H-6a), 4.22 (dd, 1H, J2,3 10.8 Hz, J2,1 8.4 Hz, H-2), 4.07 (dd, 1H, Jgem 12.4 Hz, J6b,5 1.6 Hz, H-6b), 3.87 (s, 3H, OCH3), 3.68 (bs, 1H, H-5); C NMR (100 MHz, CDCl3):  161.2 (C=N), 154.9 (C=O), 137.2 (Cipso), 129.2, 128.1, 126.2 (C-

13

Ar), 100.8 (CHPh), 97.0 (C-1), 75.4, 72.1, 68.6, 66.9, 60.0, 55.3 (C-2, C-3, C-4, C-5, C-6, OCH3). MALDI TOF-MS for C17H17Cl3N4O7 (m/z): unstable. Anal calcd: C. 41.19, H 3.46, N, 11.30; found C 41.02, H 3.54, N 11.15.

10

Phenyl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2-azido-4,6-O-benzylidene-2-deoxy-

3-O-(2-naphthalenylmethyl)-1-seleno--D-

galactopyranoside (9)  A solution of 3 (1.809 g, 4.19 mmol) in DMF (12.0 mL) was cooled to 0°C and treated with NAPBr (1.157 mg, 5.23 mmol) and NaH (60% dispersion in mineral oil, 201 mg, 8.38 mmol). The mixture was gradually heated to rt and stirred for 1.5 h. It was then diluted with CH2Cl2 (200 mL) and washed with H2O (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated and coevaporated eight times with toluene (50 mL). The obtained residue was subjected to flash chromatography (8:1 to 1:1 v/v n-hexane-ethyl acetate) to afford pure 9 (2.050 g, 86%) as a white powder. []D +153 (c 0.6, CH2Cl2); 1H NMR (600 MHz, CDCl3): 7.85-7.26 (m, 17H, H-Ar), 6.07 (d, 1H, J1,2 4.8 Hz, H-1), 5.48 (s, 1H, CHPh), 4.97 (d, 1H, Jgem 12.0 Hz, OCHH-naphthyl), 4.91 (d, 1H, Jgem 12.0 Hz, OCHH-naphthyl), 4.46 (dd, 1H, J2,3 10.2 Hz, J2,1 4.8 Hz, H-2), 4.24 (d, 1H, J4,3 3.2 Hz, H-4), 4.11 (dd, 1H, Jgem 13.8 Hz, J6a,5 1.2 Hz, H6a), 4.03-3.98 (m, 2H, H-5, H-6b), 3.83 (dd, 1H, J3,2 10.2 Hz, J3,4 3.2 Hz, H-3);

13

C NMR (100

MHz, CDCl3):  137.3, 135.0, 134.3, 133.1, 132.9 (5 Cipso), 133.7-125.5 (C-Ar), 100.9 (CHPh), 85.4 (C-1), 77.5, 72.6, 71.5, 69.0, 65.0, 59.8 (C-2, C-3, C-4, C-5, C-6, OCH2-naphthyl). MALDI TOF-MS for C30H27N3O4Se (m/z): Mr (calcd) 573.12, Mr (found) 596.00 (M+Na)+. Anal calcd: C. 62.94, H 4.75, N, 7.34; found C 62.78, H 4.85, N 7.22.

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthalenylmethyl)-D-galactopyranose (10)  Compound 9 (100 mg, 0.152 mmol) was dissolved in 4:1 v/v THF-H2O (2.0 mL) and cooled to 0°C. The solution was treated with NBS (49.0 mg, 0.275 mmol) and stirred at 0°C for 1.5 h. It was then diluted with CH2Cl2 (50 mL) and washed with 1:1 v/v 1M NaHCO3-10% Na2S2O3 (50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (3:1 to 1:1 v/v n-hexane-ethyl acetate) to afford pure 10 (67.3 mg, 89%, / = 2.5:1) as a brownish waxy solid. 1H NMR (400 MHz, CDCl3): 7.88-7.36 (m, 12H + 12H, H-Ar), 5.46 (s, 1H, CHPh), 5.45 (s, 1H, CHPh), 5.38 (d, 1H, J1,2 2.8 Hz, H-1),

11

4.96-4.88 (m, 2H + 2HOCH2-naphthyl), 4.48 (d, 1H, J1,2 8.4 Hz, H-1), 4.25 (d, 1H, Jgem 12.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Hz, H-6a), 4.18-3.87 (m, 5H + 3H, H-2, H-2, H-3, H-4, H-4, H-6a, H-6b, H-6b), 3.75 (bs, 1H, H-5), 3.41 (dd, 1H, J3,2 10.4 Hz, J3,4 3.6 Hz, H-3), 3.23 (bs, 1H, H-5);

13

C

NMR (100 MHz, CDCl3):  137.5, 135.3, 133.0 (Cipso), 129.0-125.6 (C-Ar), 100.8 (CHPh), 96.2, 92.5 (C-1), 77.9, 74.4, 73.0, 72.2, 71.7, 71.4, 69.3, 69.0, 66.5, 63.6, 62.5, 59.3 (C-2, C-3, C-4, C-5, C-6, OCH2-naphthyl). MALDI TOF-MS for C24H23N3O5 (m/z): Mr (calcd) 433.16, Mr (found) 456.03 (M+Na)+. Anal calcd: C. 66.50, H 5.35, N, 9.69; found C 66.65, H 5.49, N 9.50.

2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthalenylmethyl)--D-galactopyranosyl trichloroacetimidate (11)  A solution of 10 (959 mg, 2.215 mmol) in CH2Cl2 (25 mL) was treated with Cl3CCN (1.11 mL, 11.07 mmol) and then with K2CO3 (917 mg, 6.645 mmol). After 6 h stirring at rt, the suspension was diluted with toluene (20 mL), filtered on a Celite pad and concentrated to give a syrup that was subjected to flash chromatography (7:1:0.005 to 6:1:0.005 v/v/v toluene-ethyl acetate-triethylamine) to afford 11 (872 mg, 68%) as a yellowish oil. []D +33.4 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.72 (s, 1H, NH), 7.90-7.41 (m, 12H, H-Ar), 5.62 (d, 1H, J1,2 8.8 Hz, H-1), 5.51 (s, 1H, CHPh), 4.97 (s, 2H, OCH2-naphthyl), 4.34 (d, 1H, Jgem 12.4 Hz, H-6a), 4.21 (t, 1H, J2,3=J2,1 8.8 Hz, H-2), 4.17 (d, 1H, J4,3 3.6 Hz, H-4), 3.99 (d, 1H, Jgem 12.4 Hz, H-6b), 3.57 (dd, 1H, J3,2 8.8 Hz, J3,4 3.6 Hz, H-3), 3.48 (bs, 1H, H-5);

13

C NMR (100

MHz, CDCl3):  161.4 (C=N), 137.4, 134.9, 133.0 (Cipso), 129.1-125.6 (C-Ar), 101.0 (CHPh), 96.9 (C-1), 77.8, 72.1, 71.8, 68.7, 67.3, 61.5 (C-2, C-3, C-4, C-5, C-6, OCH2-naphthyl). MALDI TOFMS for C26H23Cl3N4O5 (m/z): unstable. Anal calcd: C. 54.04, H 4.01, N, 9.70; found C 53.89, H 4.14, N 9.58.

Allyl

(2-azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl-D-galactopyranosyl)-(1→3)-

2,4-di-O-benzoyl--L-rhamnopyranoside (8)  A mixture of 7 (22.9 mg, 55.6 mol) and 6

12

(39.9 mg, 83.4 mol) was coevaporated three times with dry toluene (1 mL). The residue was dried, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

mixed with freshly activated AW-300 4Å molecular sieves and then suspended under argon atmosphere in CH3CN (1.4 mL). The mixture was stirred at -40°C for 10 min. A 0.42 M TMSOTf solution in CH3CN (19.9 L, 8.3 mol) was then added. The mixture was stirred for 1.5 h at -40°C and then quenched by adding one drop of Et3N, filtered over a Celite pad and concentrated. Flash chromatography (5:1 to 2:1 v/v n-hexane-ethyl acetate) afforded, as first eluted compound, 8(12.6 mg, 31%) as a yellowish oil. []D +123 (c 0.4, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.15-7.29 (m, 15H, H-Ar), 5.96 (m, 1H, OCH2CH=CH2), 5.58 (dd, 1H, J2,3 3.2 Hz, J2,1 2.0 Hz, H-2A), 5.51 (t, 1H, J4,3=J4,5 10.0 Hz, H-4A), 5.35 (dd, 1H, Jvic 17.2 Hz, Jgem 1.6 Hz, trans OCH2CH=CHH), 5.27 (dd, 1H, Jvic 10.4 Hz, Jgem 1.6 Hz, cis OCH2CH=CHH), 5.25 (s, 1H, CHPh), 5.20 (d, 1H, J1,2 3.6 Hz, H-1B), 5.02 (d, 1H, J1,2 2.0 Hz, H-1A), 4.75 (dd, 1H, J3,2 11.2 Hz, J3,4 3.6 Hz, H-3B), 4.38 (dd, 1H, J3,4 10.0 Hz, J3,2 3.2 Hz, H-3A), 4.24 (dd, 1H, Jgem 12.8 Hz, Jvic 5.2 Hz, OCHHCH=CH2), 4.13-4.06 (m, 2H, H-5A, OCHHCH=CH2), 3.97 (d, 1H, J4,3 3.2 Hz, H-4B), 3.86 (dd, 1H, J2,3 11.2, J2,1 3.6 Hz, H-2B), 3.78 (dd, 1H, Jgem 12.4 Hz, J6a,5 0.8 Hz, H-6aB), 3.72 (s, 3H, OCH3), 3.39 (bs, 1H, H-5B), 3.37 (dd, 1H, Jgem 12.4 Hz, J6b,5 1.6 Hz, H-6bB), 1.34 (d, 3H, J6,5 6.0 Hz, H-6A);

13

C NMR (100

MHz, CDCl3):  166.0, 165.3 (2 COPh), 154.4 (CO2CH3), 137.2 (Cipso), 133.6-126.3 (OCH2CH=CH2, C-Ar), 117.9 (OCH2CH=CH2), 100.5 (CHPh), 96.8, 96.2 (C-1A, C-1B), 73.6, 72.8, 72.7, 72.6, 68.8, 68.7, 68.5, 66.5, 62.5, 57.2, 55.0 (C-2A, C-2B, C-3A, C-3B, C-4A, C-4B, C-5A, C-5B, C-6B, OCH2CH=CH2, COCH3), 17.6 (C-6A). MALDI TOF-MS for C38H39N3O13 (m/z): Mr (calcd) 745.25, Mr (found) 768.08 (M+Na)+. Anal calcd: C. 61.20, H 5.27, N, 5.63; found C 61.09, H 5.29, N 5.59. As second eluted compound, 8 (15.6 mg, 39%) was obtained as a yellowish oil. []D +28.6 (c 1.1, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.14-7.14 (m, 15H, H-Ar), 5.98 (m, 1H, OCH2CH=CH2), 5.63 (t, 1H, J4,3=J4,5 9.9 Hz, H-4A), 5.59 (dd, 1H, J2,3 3.6 Hz, J2,1 1.6 Hz, H-2A), 5.37 (dd, 1H, Jvic 17.2 Hz, Jgem 1.4 Hz, trans OCH2CH=CHH), 5.35 (s, 1H, CHPh), 5.28 (dd, 1H, Jvic 10.3 Hz, Jgem 1.4

13

Hz, cis OCH2CH=CHH), 5.04 (d, 1H, J1,2 1.3 Hz, H-1A), 4.48 (d, 1H, J1,2 8.0 Hz, H-1B), 4.46 (dd, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1H, J3,4 9.7 Hz, J3,2 3.7 Hz, H-3A), 4.30-4.22 (m, 4H, H-3B, H-4B, H-6aB, OCHHCH=CH2), 4.114.01 (m, 2H, H-5A, OCHHCH=CH2), 3.91 (dd, 1H, Jgem 11.0 Hz, J6a,5 1.3 Hz, H-6bB), 3.73-3.69 (m, 4H, H-2B, OCH3), 3.39 (bs, 1H, H-5B), 1.29 (d, 3H, J6,5 6.3 Hz, H-6A);

13

C NMR (100 MHz,

CDCl3):  166.1, 165.6 (2 COPh), 154.8 (CO2CH3), 137.3 (Cipso), 133.5-126.3 (OCH2CH=CH2, CAr), 118.1 (OCH2CH=CH2), 102.7, 100.9, 96.6 (C-1A, C-1B, CHPh), 76.2, 75.5, 73.1, 72.3, 72.1, 68.6, 68.5, 66.6, 66.0, 60.5, 55.1 (C-2A, C-2B, C-3A, C-3B, C-4A, C-4B, C-5A, C-5B, C-6B, OCH2CH=CH2, COCH3), 17.7 (C-6A). MALDI TOF-MS for C38H39N3O13 (m/z): Mr (calcd) 745.25, Mr (found) 768.17 (M+Na)+. Anal calcd: C. 61.20, H 5.27, N, 5.63; found C 61.11, H 5.33, N 5.57.

Allyl

[2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthalenylmethyl)-D-galactopyranosyl]-

(1→3)-2,4-di-O-benzoyl--L-rhamnopyranoside (12)  A mixture of 7 (21.3 mg, 51.6 mol) and 11 (52.0 mg, 90.3 mol) was coevaporated three times with dry toluene (1 mL). The residue was dried, mixed with freshly activated AW-300 4Å molecular sieves and then suspended under argon atmosphere in CH3CN (1.6 mL). The mixture was stirred at -40°C for 10 min. A 0.42 M TMSOTf solution in CH3CN (22.2 L, 9.3 mol) was then added. The mixture was stirred for 1.5 h at -40°C and then quenched by adding one drop of Et3N, filtered over a Celite pad and concentrated. Flash chromatography (5:1 to 2:1 v/v n-hexane-ethyl acetate) afforded, as first eluted compound, 12(16.4 mg, 38%) as a yellowish oil. []D +38.1 (c 1.6, CH2Cl2); 1H NMR (600 MHz, CDCl3):

8.19-7.26 (m, 22H, H-Ar), 5.96 (m, 1H, OCH2CH=CH2), 5.60 (bs, 1H, H-2A), 5.50 (t, 1H, J4,3=J4,5 9.8 Hz, H-4A), 5.34 (dd, 1H, Jvic 17.2 Hz, Jgem 1.2 Hz, trans OCH2CH=CHH), 5.27 (dd, 1H, Jvic 10.3 Hz, Jgem 1.2 Hz, cis OCH2CH=CHH), 5.13 (bs, 2H, H-1B, CHPh), 5.03 (bs, 1H, J1,2 3.6 Hz, H-1A), 4.67 (d, 1H, Jgem 12.2 Hz, OCHH-naphthyl), 4.51 (d, 1H, Jgem 12.2 Hz, OCHH-naphthyl), 4.36 (dd, 1H, J3,4 9.7 Hz, J3,2 3.2 Hz, H-3A), 4.25 (dd, 1H, Jgem 12.8 Hz, Jvic 5.1 Hz, OCHHCH=CH2), 4.104.06 (m, 2H, H-5A, OCHHCH=CH2), 3.83 (dd, 1H, J2,3 10.5 Hz, J2,1 3.2 Hz, H-2B), 3.73-3.68 (m,

14

2H, H-3B, H-6aB), 3.60 (bs, 1H, H-4B), 3.28 (bs, 1H, H-5B), 3.27 (d, 1H, Jgem 11.0 Hz, H-6bB), 1.32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(d, 3H, J6,5 6.2 Hz, H-6A);

13

C NMR (100 MHz, CDCl3):  166.0, 165.3 (2 COPh), 137.4 (Cipso),

133.6-125.5 (OCH2CH=CH2, C-Ar), 118.1 (OCH2CH=CH2), 100.7 (CHPh), 96.9, 96.7 (C-1A, C1B), 75.0, 73.7, 73.1, 73.0, 71.9, 69.1, 68.7, 68.6, 66.4, 63.0, 58.6 (C-2A, C-2B, C-3A, C-3B, C-4A, C4B, C-5A, C-5B, C-6B, OCH2CH=CH2, OCH2-naphthyl), 17.6 (C-6A). MALDI TOF-MS for C47H45N3O11 (m/z): Mr (calcd) 827.31, Mr (found) 850.49 (M+Na)+. Anal calcd: C. 68.19, H 5.48, N, 5.08; found C 68.00, H 5.57, N 4.99. As second eluted compound, 12(17.6 mg, 41%) was obtained as a yellowish waxy solid. []D +51.4 (c 1.0, CH2Cl2); 1H NMR (600 MHz, CDCl3): 8.15-7.14 (m, 22H, H-Ar), 5.96 (m, 1H, OCH2CH=CH2), 5.61 (t, 1H, J4,3=J4,5 9.8 Hz, H-4A), 5.56 (dd, 1H, J2,3 3.6 Hz, J2,1 1.8 Hz, H-2A), 5.36 (dd, 1H, Jvic 17.0 Hz, Jgem 1.6 Hz, trans OCH2CH=CHH), 5.27 (s, 1H, CHPh), 5.25 (dd, 1H, Jvic 10.3 Hz, Jgem 1.6 Hz, cis OCH2CH=CHH), 5.03 (d, 1H, J2,1 1.8 Hz, H-1A), 4.74 (d, 1H, Jgem 12.6 Hz, OCHH-naphthyl), 4.68 (d, 1H, Jgem 12.6 Hz, OCHH-naphthyl), 4.39 (dd, 1H, J3,4 9.8 Hz, J3,2 3.6 Hz, H-3A), 4.32 (d, 1H, J1,2 7.9 Hz, H-1B), 4.22-4.19 (m, 2H, H-6aB, OCHHCH=CH2), 4.10-4.06 (m, 2H, H-5A, OCHHCH=CH2), 3.92 (d, 1H, J4,3 3.4 Hz, H-4B), 3.81 (dd, 1H, Jgem 12.1 Hz, J6b,5 1.7 Hz, H-6bB), 3.67 (dd, 1H, J2,3 10.2 Hz, J2,1 7.9 Hz, H-2B), 3.18 (bs, 1H, H-5B), 3.13 (dd, 1H, J3,2 10.2 Hz, J3,4 3.4 Hz, H-3B), 1.32 (d, 3H, J6,5 6.3 Hz, H-6A); 13C NMR (100 MHz, CDCl3):  166.0, 165.6 (2 COPh), 137.5, 135.1 (Cipso), 133.4-125.5 (OCH2CH=CH2, C-Ar), 117.9 (OCH2CH=CH2), 102.5, 101.0, 96.5 (C-1A, C-1B, CHPh), 77.7, 76.2, 73.0, 72.2, 72.0, 71.3, 68.6, 68.5, 66.5, 66.2, 62.0 (C-2A, C-2B, C-3A, C-3B, C-4A, C-4B, C-5A, C-5B, C-6B, OCH2CH=CH2, OCH2-naphthyl), 17.6 (C-6A). MALDI TOF-MS for C47H45N3O11 (m/z): Mr (calcd) 827.31, Mr (found) 850.40 (M+Na)+. Anal calcd: C. 68.19, H 5.48, N, 5.08; found C 67.98, H 5.55, N 5.01.

Allyl (2-azido-4,6-O-benzylidene-2-deoxy--D-galactopyranosyl)-(1→3)-2,4-di-O-benzoyl--Lrhamnopyranoside (13)  From 8 : Pyridine (500 L) and acetic acid (7.0 L) were added to a

15

mixture of 8(17.0 mg, 23.3 mol) and LiI (9.4 mg, 70.0 mol). The mixture was heated to reflux 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(120°C) and stirred for 2 h. It was then diluted with CH2Cl2 (30 mL) and washed with water (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (3:1 to 1:2 v/v petroleum ether-ethyl acetate) to afford 13(15.8 mg, 98%) as a white foam.  From 12A solution of 12(281 mg, 0.340 mmol) in CH2Cl2 (14.6 mL) was treated with water (1.6 mL) and then with DDQ (116 mg, 0.510 mmol). The mixture was vigorously stirred at rt overnight. It was then diluted with CH2Cl2 (200 mL) and washed with saturated aqueous NaHCO3 (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (3:1 to 1:2 v/v petroleum ether-ethyl acetate) to afford 13(241 mg, 91%) as a white foam. []D +48.0 (c 1.0, CH2Cl2);

1

H NMR (400 MHz, CDCl3): 8.17-7.13 (m, 15H, H-Ar), 5.99 (m, 1H,

OCH2CH=CH2), 5.63 (t, 1H, J4,3=J4,5 9.8 Hz, H-4A), 5.58 (d, 1H, J2,3 3.6 Hz, H-2A), 5.37 (s, 1H, CHPh), 5.36 (d, 1H, Jvic 17.1 Hz, trans OCH2CH=CHH), 5.27 (d, 1H, Jvic 10.3 Hz, cis OCH2CH=CHH), 5.04 (bs, 1H, H-1A), 4.43 (dd, 1H, J3,4 9.7 Hz, J3,2 3.6 Hz, H-3A), 4.36 (d, 1H, J2,1 7.9 Hz, H-1B), 4.28-4.22 (m, 2H, H-6aB, OCHHCH=CH2), 4.14-3.99 (m, 3H, H-4B, H-5A, OCHHCH=CH2), 3.89 (d, 1H, Jgem 12.3 Hz, H-6bB), 3.39-3.26 (m, 3H, H-2B, H-3B, H-5B), 1.32 (d, 3H, J6,5 6.2 Hz, H-6A); 13C NMR (100 MHz, CDCl3):  166.1, 165.7 (2 COPh), 137.2 (Cipso), 133.5126.3 (OCH2CH=CH2, C-Ar), 118.1 (OCH2CH=CH2), 102.5, 101.2, 96.6 (C-1A, C-1B, CHPh), 76.2, 74.1, 73.1, 72.2, 71.4, 68.6, 68.5, 66.5, 66.3, 64.1 (C-2A, C-2B, C-3A, C-3B, C-4A, C-4B, C-5A, C-5B, C-6B, OCH2CH=CH2), 17.6 (C-6A). MALDI TOF-MS for C36H37N3O11 (m/z): Mr (calcd) 687.24, Mr (found) 710.39 (M+Na)+. Anal calcd: C. 62.87, H 5.42, N, 6.11; found C 62.66, H 5.49, N 6.03.

Allyl [2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-naphthalenylmethyl)--D-galactopyranosyl](1→3)-(2-azido-4,6-O-benzylidene-2-deoxy--D-galactopyranosyl)-(1→3)-2,4-di-O-benzoyl--

16

L-rhamnopyranoside

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(14)  A mixture of 13 (31.6 mg, 46.0 mol) and 11 (46.4 mg, 81.0 mol)

was coevaporated three times with dry toluene (1 mL). The residue was dried, mixed with freshly activated AW-300 4Å molecular sieves and then suspended under argon atmosphere in CH2Cl2 (1.5 mL). The mixture was stirred at -35°C for 10 min. A 0.42 M TMSOTf solution in CH2Cl2 (9.7 L, 4.1 mol) was then added. The mixture was stirred for 2.5 h at -35°C and then quenched by adding one drop of Et3N, filtered over a Celite pad and concentrated. Flash chromatography (3:1 to 1:1 v/v petroleum ether-ethyl acetate) afforded 14(32.7 mg, 65%) as a yellowish oil. []D +93.0 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.15-7.21 (m, 27H, H-Ar), 5.98 (m, 1H, OCH2CH=CH2), 5.63 (t, 1H, J4,3=J4,5 9.7 Hz, H-4A), 5.56 (d, 1H, J2,3 1.7 Hz, H-2A), 5.42 (s, 1H, CHPh), 5.38 (d, 1H, Jvic 16.5 Hz, trans OCH2CH=CHH), 5.37 (s, 1H, CHPh), 5.28 (d, 1H, Jvic 10.0 Hz, cis OCH2CH=CHH), 5.10 (d, 1H, J1,2 3.3 Hz, H-1C), 5.05 (bs, 1H, H-1A), 4.86 (d, 1H, Jgem 12.0 Hz, OCHH-naphthyl), 4.74 (d, 1H, Jgem 12.0 Hz, OCHH-naphthyl), 4.44 (dd, 1H, J3,4 9.7 Hz, J3,2 3.6 Hz, H-3A), 4.40 (d, 1H, J1,2 8.1 Hz, H-1B), 4.30 (d, 1H, Jgem 12.3 Hz, H-6aC), 4.25 (dd, 1H, Jgem 12.8 Hz, Jvic 5.4 Hz, OCHHCH=CH2), 4.15-3.98 (m, 6H, H-3C, H-4B, H-4C, H-5A, H-6bC, OCHHCH=CH2), 3.94 (d, 1H, Jgem 12.2 Hz, H-6aB), 3.87 (dd, 1H, J2,3 10.7 Hz, J2,1 3.3 Hz, H-2C), 3.82 (d, 1H, Jgem 12.2 Hz, H-6bB), 3.67 (dd, 1H, J2,3 10.1 Hz, J2,1 8.1 Hz, H-2B), 3.56 (bs, 1H, H5C), 3.33 (dd, 1H, J3,2 10.1 Hz, J3,4 3.2 Hz, H-3B), 3.32 (bs, 1H, H-5B), 1.31 (d, 3H, J6,5 6.2 Hz, H6A);

13

C NMR (100 MHz, CDCl3):  166.1, 165.4 (2 COPh), 137.2, 137.1, 135.1 (Cipso), 133.3-

125.5 (OCH2CH=CH2, C-Ar), 117.8 (OCH2CH=CH2), 102.3, 100.6, 100.5, 96.3, 94.6 (C-1A, C-1B, C-1C, 2 CHPh), 75.7, 73.9, 73.8, 73.1, 73.0, 72.0, 71.6, 70.1, 68.8, 68.5, 68.4, 66.2, 66.0, 63.0, 61.3, 58.8 (C-2A, C-2B, C-2C, C-3A, C-3B, C-3C, C-4A, C-4B, C-4C, C-5A, C-5B, C-5C, C-6B, C-6C, OCH2CH=CH2, OCH2-naphthyl), 17.4 (C-6A). MALDI TOF-MS for C60H58N6O15 (m/z): Mr (calcd) 1102.40, Mr (found) 1125.18 (M+Na)+. Anal calcd: C. 65.33, H 5.30, N, 7.62; found C 65.20, H 5.40, N 7.56.

17

Allyl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(2-azido-4,6-O-benzylidene-2-deoxy-3-O-methoxycarbonyl--D-galactopyranosyl)-

(1→3)-(2-azido-4,6-O-benzylidene-2-deoxy--D-galactopyranosyl)-(1→3)-2,4-di-O-benzoyl-L-rhamnopyranoside

(15)  A mixture of 13 (76.8 mg, 0.112 mmol) and 6 (92.8 mg, 0.187

mmol) was coevaporated three times with dry toluene (2 mL). The residue was dried, mixed with freshly activated AW-300 4Å molecular sieves and then suspended under argon atmosphere in CH2Cl2 (3.5 mL). The mixture was stirred at -35°C for 10 min. A 0.42 M TMSOTf solution in CH2Cl2 (23.3 L, 9.7 mol) was then added. The mixture was stirred for 2.5 h at -35°C and then quenched by adding one drop of Et3N, filtered over a Celite pad and concentrated. Flash chromatography (2:1 to 1:1 v/v petroleum ether-ethyl acetate) afforded 15(78.0 mg, 68%) as a yellowish oil. []D +141.0 (c 1.0, CH2Cl2); 1H NMR (400 MHz, CDCl3): 8.13-7.22 (m, 20H, HAr), 5.99 (m, 1H, OCH2CH=CH2), 5.64 (t, 1H, J4,3=J4,5 9.8 Hz, H-4A), 5.56 (dd, 1H, J2,3 3.5 Hz, J2,1 1.7 Hz, H-2A), 5.50 (s, 1H, CHPh), 5.42 (s, 1H, CHPh), 5.37 (dd, 1H, Jvic 17.2 Hz, Jgem 1.6 Hz, trans OCH2CH=CHH), 5.28 (dd, 1H, Jvic 10.3 Hz, Jgem 1.6 Hz, cis OCH2CH=CHH), 5.17 (d, 1H, J1,2 3.5 Hz, H-1C), 5.07-5.03 (m, 2H, H-1A, H-3C), 4.48-4.43 (m, 3H, H-1B, H-3A, H-6aB), 4.32-4.22 (m, 2H, H-6aC, OCHHCH=CH2), 4.16-3.94 (m, 5H, H-4B, H-4C, H-5A, H-6bB, OCHHCH=CH2), 3.87-3.83 (m, 2H, H-2C, H-6bC), 3.78 (s, 3H, OCH3), 3.74 (bs, 1H, H-5C), 3.69 (dd, 1H, J2,3 10.5 Hz, J2,1 7.9 Hz, H-2B), 3.35 (dd, 1H, J3,2 10.5 Hz, J3,4 3.4 Hz, H-3B), 3.32 (bs, 1H, H-5B), 1.31 (d, 3H, J6,5 6.3 Hz, H-6A);

13

C NMR (100 MHz, CDCl3):  166.1, 165.4 (2 COPh), 154.6 (CO2CH3),

137.2, 135.5, 133.1 (Cipso, OCH2CH=CH2), 133.0-126.1 (C-Ar), 118.1 (OCH2CH=CH2), 102.7, 100.8, 100.6, 96.6, 94.6 (C-1A, C-1B, C-1C, 2 CHPh), 75.8, 74.2, 73.4, 73.0, 72.3, 72.0, 70.2, 68.8, 68.7, 68.6, 66.5, 66.3, 62.9, 61.5, 56.4, 55.2 (C-2A, C-2B, C-2C, C-3A, C-3B, C-3C, C-4A, C-4B, C-4C, C-5A, C-5B, C-5C, C-6B, C-6C, OCH2CH=CH2, COCH3), 17.6 (C-6A). MALDI TOF-MS for C51H52N6O17 (m/z): Mr (calcd) 1020.34, Mr (found) 1043.09 (M+Na)+. Anal calcd: C. 60.00, H 5.13, N, 8.23; found C 59.85, H 5.22, N 8.18.

18

Allyl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(2-azido-4,6-O-benzylidene-2-deoxy--D-galactopyranosyl)-(1→3)-(2-azido-4,6-O-

benzylidene-2-deoxy--D-galactopyranosyl)-(1→3)-2,4-di-O-benzoyl--L-rhamnopyranoside (16)  From 14A solution of 14(53 mg, 48.0 mol) in CH2Cl2 (2.0 mL) was treated with water (230 L) and then with DDQ (16.4 mg, 72.0 mol). The mixture was vigorously stirred at rt for 5 h and then a second aliquot of DDQ (5.5 mg, 24.0 mol) was added. The mixture was stirred at rt for an additional hour and then diluted with CH2Cl2 (100 mL) and washed with saturated aqueous NaHCO3 (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (3:1 to 3:2 v/v petroleum ether-ethyl acetate) to afford 16(31.4 mg, 68%) as a white waxy solid.  From 15: Pyridine (2.3 mL) and acetic acid (32 L) were added to a mixture of 15(107 mg, 0.105 mmol) and LiI (42.2 mg, 0.315 mmol). The mixture was heated to reflux (120°C) and stirred for 5 h. It was then diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by flash chromatography (3:1 to 3:2 v/v petroleum ether-ethyl acetate) to afford 16(85.0 mg, 84%) as a white waxy solid. []D +100.5 (c 1.0, CH2Cl2); 1H NMR (500 MHz, CDCl3): 8.15-7.21 (m, 20H, H-Ar), 5.99 (m, 1H, OCH2CH=CH2), 5.62 (t, 1H, J4,3=J4,5 9.5 Hz, H-4A), 5.56 (dd, 1H, J2,3 3.5 Hz, J2,1 1.0 Hz, H-2A), 5.52 (s, 1H, CHPh), 5.43 (s, 1H, CHPh), 5.40 (dd, 1H, Jvic 17.5 Hz, Jgem 1.5 Hz, trans OCH2CH=CHH), 5.28 (dd, 1H, Jvic 10.5 Hz, Jgem 1.5 Hz, cis OCH2CH=CHH), 5.09 (d, 1H, J1,2 3.0 Hz, H-1C), 5.06 (d, 1H, J1,2 1.0 Hz, H-1A), 4.46 (dd, 1H, J3,4 10.0 Hz, J3,2 3.5 Hz, H-3A), 4.43 (d, 1H, J1,2 8.0 Hz, H-1B), 4.31 (d, 1H, Jgem 12.0 Hz, H-6aC), 4.24 (dd, 1H, Jgem 12.5 Hz, Jvic 5.5 Hz, OCHHCH=CH2), 4.20 (d, 1H, J4,3 3.5 Hz, H-4C), 4.15 (d, 1H, J4,3 3.0 Hz, H-4B), 4.12-3.93 (m, 6H, H-2C, H-5A, H-6aB, H-6bB, H-6bC, OCHHCH=CH2), 3.70 (bs, 1H, H-5C), 3.67 (dd, 1H, J2,3 10.0 Hz, J2,1 8.0 Hz, H-2B), 3.51 (dd, 1H, J3,2 11.0 Hz, J3,4 3.5 Hz, H-3C), 3.33 (dd, 1H, J3,2 10.5 Hz, J3,4 3.0 Hz, H-3B), 3.31 (bs, 1H, H-5B), 1.31 (d, 3H, J6,5 6.5 Hz, H-6A);

13

C NMR (125 MHz, CDCl3): 

166.1, 165.4 (2 COPh), 137.3, 137.2 (Cipso), 133.5-126.1 (OCH2CH=CH2, C-Ar), 118.0 19

(OCH2CH=CH2), 102.6, 101.2, 100.8, 96.6, 95.1 (C-1A, C-1B, C-1C, 2 CHPh), 75.9, 75.3, 74.5, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

73.4, 72.3, 70.4, 69.0, 68.7, 68.6, 66.6, 66.5, 66.4, 63.1, 61.6, 59.8 (C-2A, C-2B, C-2C, C-3A, C-3B, C-3C, C-4A, C-4B, C-4C, C-5A, C-5B, C-5C, C-6B, C-6C, OCH2CH=CH2), 17.6 (C-6A). MALDI TOFMS for C49H50N6O15 (m/z): Mr (calcd) 962.33, Mr (found) 985.20 (M+Na)+. Anal calcd: C. 61.12, H 5.23, N, 8.73; found C 61.20, H 5.32, N 8.62.

Allyl

(2,3,4,6-tetra-O-benzoyl--D-glucopyranosyl)-(1→3)-(2-azido-4,6-O-benzylidene-2-

deoxy--D-galactopyranosyl)-(1→3)-(2-azido-4,6-O-benzylidene-2-deoxy--Dgalactopyranosyl)-(1→3)-2,4-di-O-benzoyl--L-rhamnopyranoside (18)  A mixture of 16 (21.3 mg, 22.2 mol) and 17 (32.5 mg, 45.9 mol) was coevaporated three times with dry toluene (1 mL). The residue was dried, mixed with freshly activated AW-300 4Å molecular sieves and then suspended under argon atmosphere in CH2Cl2 (1.0 mL). The mixture was stirred at 0°C for 10 min. A 0.21 M TMSOTf solution in CH2Cl2 (12.0 L, 2.5 mol) was then added. The mixture was stirred for 2 h at 0°C and then quenched by adding one drop of Et3N, filtered over a Celite pad and concentrated. Flash chromatography (3:1 to 3:2 v/v petroleum ether-ethyl acetate) afforded 18(22.4 mg, 67%) as a yellowish waxy solid. []D +91 (c 0.8, CH2Cl2); 1H NMR (500 MHz, CDCl3):

8.17-7.14 (m, 40H, H-Ar), 6.00 (m, 1H, OCH2CH=CH2), 5.84 (t, 1H, J3,4=J3,2 9.5 Hz, H-3D), 5.68-5.62 (m, 2H, H-4A, H-4D), 5.58 (dd, 1H, J2,3 3.0 Hz, J2,1 2.0 Hz, H-2A), 5.56 (dd, 1H, J2,3 10.0 Hz, J2,1 8.0 Hz, H-2D), 5.39 (s, 1H, CHPh), 5.38 (d, 1H, Jvic 17.0 Hz, trans OCH2CH=CHH), 5.35 (s, 1H, CHPh), 5.28 (d, 1H, Jvic 10.5 Hz, cis OCH2CH=CHH), 5.05 (d, 1H, J1,2 2.0 Hz, H-1A), 5.02 (d, 1H, J1,2 3.5 Hz, H-1C), 4.99 (d, 1H, J1,2 8.0 Hz, H-1D), 4.65 (dd, 1H, Jgem 12.0 Hz, J6a,5 3.0 Hz, H6aD), 4.49-4.43 (m, 2H, H-3A, H-6bD), 4.40 (d, 1H, J1,2 8.0 Hz, H-1B), 4.34 (d, 1H, J4,3 3.0 Hz, H4C), 4.29-4.24 (m, 2H, H-5D, OCHHCH=CH2), 4.12-4.05 (m, 4H, H-4B, H-5A, H-6aB, OCHHCH=CH2), 4.01 (dd, 1H, J2,3 11.0 Hz, J2,1 3.5 Hz, H-2C), 3.90 (d, 1H, Jgem 11.0 Hz, H-6bB), 3.84 (d, 1H, Jgem 12.0 Hz, H-6aC), 3.69-3.64 (m, 2H, H-2B, H-3C), 3.57 (d, 1H, Jgem 12.0 Hz, H-

20

6bC), 3.47 (bs, 1H, H-5C), 3.35 (dd, 1H, J3,2 10.5 Hz, J3,4 3.5 Hz, H-3B), 3.29 (bs, 1H, H-5B), 1.33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(d, 3H, J6,5 6.0 Hz, H-6A); 13C NMR (125 MHz, CDCl3):  166.1, 165.9, 165.7, 165.5, 165.1, 164.9 (6 COPh), 137.5, 137.3 (Cipso), 133.5-126.1 (OCH2CH=CH2, C-Ar), 118.1 (OCH2CH=CH2), 102.7, 102.3, 100.8, 100.5, 96.6, 94.7 (C-1A, C-1B, C-1C, C-1D, 2 CHPh), 76.1, 75.8, 75.6, 74.3, 73.1, 73.0, 72.3, 72.2, 71.6, 70.2, 69.3, 68.7, 68.6, 68.4, 66.5, 66.2, 63.5, 62.3, 61.6, 57.7 (C-2A, C-2B, C-2C, C2D, C-3A, C-3B, C-3C, C-3D, C-4A, C-4B, C-4C, C-4D, C-5A, C-5B, C-5C, C-5D, C-6B, C-6C, C-6D, OCH2CH=CH2), 17.6 (C-6A). MALDI TOF-MS for C83H76N6O24 (m/z): Mr (calcd) 1540.49, Mr (found) 1563.25 (M+Na)+. Anal calcd: C. 64.67, H 4.97, N, 5.45; found C 64.49, H 5.05, N 5.40.

Allyl (2,3,4,6-tetra-O-benzoyl--D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-O-benzylidene-2deoxy--D-galactopyranosyl)-(1→3)-(2-acetamido-4,6-O-benzylidene-2-deoxy--Dgalactopyranosyl)-(1→3)-2,4-di-O-benzoyl--L-rhamnopyranoside (19)  A solution of 18 (11.0 mg, 7.3 mol) in THF (330 L) was treated with Ac2O (220 L) and AcOH (110 L). Zn/Cu alloy (33 mg) was then added and the mixture was vigorously stirred at rt for 5 h, after that a second aliquot of Zn/Cu alloy (33 mg) was added. After overnight stirring at rt, the mixture was diluted with ethyl acetate (10 mL), filtered over a Celite pad and concentrated. The residue was purified by flash chromatography (99:1 to 97:3 v/v CH2Cl2-MeOH) to afford 19 (5.6 mg, 50%). []D +74 (c 0.4, CH2Cl2);

1

H NMR (600 MHz, CDCl3): 8.16-7.08 (m, 40H, H-Ar), 5.97 (m, 1H,

OCH2CH=CH2), 5.81 (t, 1H, J3,4=J3,2 9.5 Hz, H-3D), 5.65-5.61 (m, 2H, H-2A, H-4D), 5.54 (t, 1H, J4,3=J4,5 9.7 Hz, H-4A), 5.43-5.35 (m, 4H, H-2D, 2 NH, trans OCH2CH=CHH), 5.27 (d, 1H, Jvic 10.4 Hz, cis OCH2CH=CHH), 5.24 (s, 2H, 2 CHPh), 5.04-4.99 (m, 4H, H-1A, H-1B, H-1C, H-1D), 4.704.68 (m, 2H, H-6aD, H-6bD), 4.44-4.33 (m, 2H, H-2C, H-3A), 4.25-4.16 (m, 3H, H-3B, H-4B, OCHHCH=CH2), 4.10-4.06 (m, 4H, H-4C, H-5A, H-5D, OCHHCH=CH2), 3.97 (d, 1H, Jgem 12.2 Hz, H-6aC), 3.85 (d, 1H, Jgem 12.2 Hz, H-6bC), 3.78 (dd, 1H, J3,2 11.1 Hz, J3,4 3.8 Hz, H-3C), 3.67-3.64 (m, 2H, H-6aB, H-6bB), 3.47 (m, 1H, H-2B), 3.36 (bs, 1H, H-5C), 3.31 (bs, 1H, H-5B), 1.30 (d, 3H,

21

J6,5 6.0 Hz, H-6A), 1.24 (s, 3H, COCH3), 0.88 (s, 3H, COCH3); 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

C NMR (100 MHz, CDCl3): 

13

170.1, 169.9 (2 NCOCH3), 166.0, 165.9, 165.8, 165.6, 165.1, 164.8 (6 COPh), 137.6, 137.4 (Cipso), 133.5-126.2 (OCH2CH=CH2, C-Ar), 118.1 (OCH2CH=CH2), 101.3, 101.1, 100.7, 99.6, 96.6, 94.1 (C-1A, C-1B, C-1C, C-1D, 2 CHPh), 75.5, 75.4, 75.1, 73.4, 72.8, 72.3, 72.2, 71.8, 71.2, 70.5, 69.1, 68.8, 68.6, 68.5, 66.2, 65.9, 63.2, 62.2 (C-2A, C-2D, C-3A, C-3B, C-3C, C-3D, C-4A, C-4B, C-4C, C4D, C-5A, C-5B, C-5C, C-5D, C-6B, C-6C, C-6D, OCH2CH=CH2), 52.9, 47.3 (C-2B, C-2C), 22.6, 21.8 (2 COCH3), 17.6 (C-6A). MALDI TOF-MS for C87H84N2O26 (m/z): Mr (calcd) 1572.53, Mr (found) 1595.35 (M+Na)+. Anal calcd: C. 66.40, H 5.38, N, 1.78; found C 66.24, H 5.46, N 1.81.

Allyl

(2,3,4,6-tetra-O-benzoyl--D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-

deoxy--D-galactopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy--Dgalactopyranosyl)-(1→3)-2,4-di-O-benzoyl--L-rhamnopyranoside (21)  A solution of 19 (10.0 mg, 6.5 mol) in 9:1 v/v AcOH-H2O (600 L) was stirred at 50°C for 14 h, then cooled to rt and concentrated. The residue was dissolved in Ac2O (300 L) and treated with pyridine (300 L). The solution was stirred at rt overnight, then diluted with CHCl3 (20 mL) and washed with 0.1 M HCl (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give a residue, that was purified by preparative thin-layer chromatography (PLC) (5:1 v/v ethyl acetatetoluene). Pure 21 (7.0 mg, 70%) was obtained as a white waxy solid. []D +60 (c 0.5, CH2Cl2); 1H NMR (600 MHz, CDCl3): 8.14-7.24 (m, 30H, H-Ar), 6.50 (d, 1H, JH,NH 9.5 Hz, NH), 5.95 (m, 1H, OCH2CH=CH2), 5.86-5.83 (m, 2H, H-3D, NH), 5.62 (t, 1H, J4,3=J4,5 9.7 Hz, H-4D), 5.54 (dd, 1H, J2,3 3.5 Hz, J2,1 1.9 Hz, H-2A), 5.52 (t, 1H, J4,3=J4,5 9.8 Hz, H-4A), 5.38-5.33 (m, 4H, H-2D, H4B, OCH2CH=CH2), 5.28 (d, 1H, J1,2 10.4 Hz, H-1B), 5.04 (d, 1H, J4,3 3.3 Hz, H-4C), 4.99 (bs, 1H, H-1A), 4.83 (d, 1H, J1,2 7.8 Hz, H-1D), 4.63 (d, 1H, J1,2 3.5 Hz, H-1C), 4.53-4.52 (m, 2H, H-6aD, H6bD), 4.37-4.32 (m, 3H, H-2C, H-3A, H-3B), 4.23 (dd, 1H, Jgem 12.8 Hz, Jvic 5.3 Hz, OCHHCH=CH2), 4.08-3.93 (m, 6H, H-5A, H-5D, H-6aB, H-6bB, H-6aC, OCHHCH=CH2), 3.83-3.75

22

(m, 2H, H-5B, H-5C), 3.72 (dd, 1H, Jgem 11.4 Hz, J6b,5 4.6 Hz, H-6bC), 3.53 (dd, 1H, J3,2 11.1 Hz, J3,4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3.0 Hz, H-3C), 2.95 (m, 1H, H-2B), 2.03 (s, 3H, OCOCH3), 2.02 (s, 3H, OCOCH3), 1.89 (s, 3H, OCOCH3), 1.88 (s, 3H, OCOCH3), 1.46 (s, 3H, NCOCH3), 1.26 (d, 3H, J6,5 6.3 Hz, H-6A), 0.95 (s, 3H, NCOCH3);

13

C NMR (125 MHz, CDCl3):  170.6, 170.3, 170.1, 169.5 (2 NCOCH3, 4

OCOCH3), 165.9, 165.8, 165.7, 165.6, 165.0, 164.7 (6 COPh), 133.6-128.3 (OCH2CH=CH2, C-Ar), 118.1 (OCH2CH=CH2), 101.6, 100.8, 99.9, 96.5 (C-1A, C-1B, C-1C, C-1D), 76.2, 73.5, 72.6, 72.4, 72.0, 71.5, 69.6, 69.3, 68.7, 68.4, 67.9, 67.8, 66.2, 63.1, 62.6, 61.0 (C-2A, C-2D, C-3A, C-3B, C-3C, C-3D, C-4A, C-4B, C-4C, C-4D, C-5A, C-5B, C-5C, C-5D, C-6B, C-6C, C-6D, OCH2CH=CH2), 55.6, 48.2 (C-2B, C-2C), 22.6, 22.2 (2 NCOCH3), 20.8, 20.7, 20.6, 20.5 (4 OCOCH3), 17.6 (C-6A). MALDI TOF-MS for C81H84N2O30 (m/z): Mr (calcd) 1564.51, Mr (found) 1587.39 (M+Na)+. Anal calcd: C. 62.14, H 5.41, N, 1.79; found C 61.88, H 5.65, N 1.74.

Allyl

-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy--D-galactopyranosyl-(1→3)-2-

acetamido-2-deoxy--D-galactopyranosyl-(1→3)--L-rhamnopyranoside (2)  A solution of 21 (6.9 mg, 4.4 mol) in CH3OH (600 L) was treated with a 0.47 M methanolic solution of CH3ONa (49 L, 23.0 mol). After 6 h stirring at rt, the solution was diluted with H2O (3 mL), neutralized with Amberlist-15 (H+ form) and centrifuged at 15°C (4000 rpm, 5 min). The supernatant was freeze-dried. Pure 2 (3.1 mg, 91%) was obtained as a white fluffy solid. []D +51 (c 0.2, H2O); 1H NMR (400 MHz, D2O): 5.97 (m, 1H, OCH2CH=CH2), 5.36 (d, 1H, Jvic 17.0 Hz, trans OCH2CH=CHH), 5.28 (d, 1H, Jvic 10.5 Hz, cis OCH2CH=CHH), 5.07 (d, 1H, J1,2 4.0 Hz, H1C), 4.83 (d, J1,2 1.8 Hz, 1H, H-1A), 4.70 (d, 1H, J1,2 9.0 Hz, H-1B), 4.51 (d, 1H, J1,2 8.0 Hz, H-1D), 4.39 (dd, 1H, J2,3 11.0 Hz, J1,2 4.0 Hz, H-2C), 4.24-4.20 (m, 2H, H-4C, OCHHCH=CH2), 4.17 (dd, 1H, J2,3 3.0 Hz, J2,1 1.8 Hz, H-2A), 4.12 (d, 1H, J4,3 2.0 Hz, H-4B), 4.11-4.05 (m, 2H, H-2B, OCHHCH=CH2), 3.88-3.71 (m, 11H, H-3A, H-3B, H-3C, H-5A, H-5C, H-6aB, H-6aC, H-6aD, H-6bB, H-6bC, H-6bD), 3.64 (dd, 1H, J5,6a 8.0 Hz Hz, J5,6b 3.0 Hz, H-5B), 3.51 (t, 1H, J4,5=J4,3 9.5 Hz, H-

23

4A), 3.50-3.42 (m, 3H, H-3D, H-4D, H-5D), 3.28 (t, 1H, J2,3=J2,1 8.0 Hz, H-2D), 2.06 (s, 3H, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

NCOCH3), 2.02 (s, 3H, NCOCH3), 1.28 (d, 3H, J6,5 6.5 Hz, H-6A); 13C NMR (100 MHz, CDCl3):  175.5, 175.3 (2 NCOCH3), 133.8 (OCH2CH=CH2), 119.3 (OCH2CH=CH2), 105.0 (C-1D), 103.5 (C1B), 99.2 (C-1A), 94.3 (C-1C), 80.7 (C-3A), 78.0 (C-3C), 76.4 (C-4D), 76.2 (C-3D), 75.6 (C-5B), 75.3 (C-3B), 73.6 (C-2D), 71.8 (C-5C), 71.6 (C-4A), 70.7 (C-2A), 70.1 (C-5D), 69.7 (C-5A), 69.2 (C-4C), 68.9 (OCH2CH=CH2), 64.2 (C-4B), 61.7 (C-6C), 61.4 (C-6B), 61.1 (C-6D), 51.6 (C-2B), 48.7 (C-2C), 23.0, 22.7 (2 NCOCH3), 17.2 (C-6A). MALDI TOF-MS for C31H52N2O20 (m/z): Mr (calcd) 772.31, Mr (found) 795.18 (M+Na)+. Anal calcd: C. 48.18, H 6.78, N, 3.63; found C 47.91, H 6.96, N 3.52.

Acknowledgments This research was supported by the Ministero dell‟Istruzione dell‟Università e della Ricerca (MIUR), project PRIN 2010-2011 “Metodologie chimiche innovative per biomateriali intelligenti”. A.M. and A.S. acknowledge COST Action BM1003 “Microbial cell surface determinants of virulence as targets for new therapeutics in Cystic Fibrosis.” This work has been partially supported to A.M. by Italian Cystic Research Foundation, grant FFC 11#2010 with the contribution of Pastificio Giovanni Rana s.p.a.

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6. Astronomo, R.D.; Burton, D.R. Nat. Rev. Drug Disc. 2010, 9, 308–324. 7. Silipo, A.; De Castro, C.; Lanzetta, R.; Parrilli, M.; Molinaro, A. In Prokaryotic Cell Wall Compounds. Structure and Biochemistry; König, H., Claus, H., Varma, A., Eds.; Springer: Heidelberg, Germany, 2010; pp 133–154. 8. Cryz Jr, S.J.; Sadoff, J.C.; Cross, A.S.; Furer, E. Antibiot. Chemother. 1989, 42, 177–183. 9. Döring, G.; Pier, G.B. Vaccine 2008, 26, 1011–1024. 10. Faurè, R.; Shiao, T.C.; Lagnoux, D.; Giguère, D.; Roy, R. Org. Biomol. Chem. 2007, 5, 2704– 2707. 11. Komarova, B.S.; Tsvetkov, Y.E.; Pier, G.B.; Nifantiev, N.E. J. Org. Chem. 2008, 73, 8411– 8421. 12. Blaukopf, M.; Müller, B.; Hofinger, A.; Kosma, P. Eur. J. Org. Chem. 2012, 119–131. 13. De Soyza, A.; Silipo, A.; Lanzetta, R.; Govan, R.J.; Molinaro, A. Innate Immun. 2008, 14, 127– 144. 14. Ortega, X.; Silipo, A.; Saldias, M.S.; Bates, C.C.; Molinaro, A.; Valvano, M.A. J. Biol. Chem. 2009, 284, 21738-21751. 15. Knirel, Y. In Bacterial Lipopolysaccharides: Structure, Chemical Synthesis, Biogenesis and Interaction with Host Cells; Knirel, Y., Valvano, M.A., Eds.; Springer: Heidelberg, Germany, 2011; pp 41–115. 16. Ieranò, T.; Silipo, A.; Sturiale, L.; Garozzo, D.; Brookes, H.; Khan, C.M.A.; Bryant, C.; Gould, F.F.; Corris, P.A.; Lanzetta, R.; Parrilli, M.; De Soyza, A.; Molinaro, A. Glycobiology 2008, 18, 871–881. 17. Ieranò, T.; Silipo, A.; Sturiale, L.; Garozzo, D.; Bryant, C.; Lanzetta, R.; Parrilli, M.; Aldridge, C.; Gould, F.K.; Corris, P.A.; Khan, C.M.A.; De Soyza, A.; Molinaro, A. Glycobiology 2009, 19, 1214–1223. 18. Bedini, E.; Cirillo, L.; Parrilli, M. Carbohydr. Res. 2012, 349, 24–32. 25

19. Bongat, A.F.G.; Demchenko, A.V. Carbohydr. Res. 2007, 342, 374–406. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

20. Kalikanda, J.; Li, Z. J. Org. Chem. 2011, 76, 5207–5218. 21. Kalikanda, J.; Li, Z. Carbohydr. Res. 2011, 346, 2380–2383. 22. Ingle, A.B.; Chao, C.-S.; Hung, W.-C.; Mong, K.-K.T. Org. Lett. 2013, 15, 5290–5293. 23. Yang, L.; Ye, S. Carbohydr. Res. 2010, 345, 1713–1721. 24. Geng, Y.; Ye, X.-S. Synlett 2010, 2506–2512. 25. Manabe, S.; Ishii, K.; Ito, Y. J. Am. Chem. Soc. 2006, 128, 10666–10667. 26. Plattner, C.; Höfener, M.; Sewald, N. Org. Lett. 2011, 13, 545–547. and references cited therein. 27. Emmadi, M.; Kulkarni, S.S. J. Org. Chem. 2011, 76, 4703–4709 and references cited therein. 28. Tseng, P.-H.; Jiaang, W.-T.; Chang, M.-Y.; Chen, S.-T. Chem. Eur. J. 2001, 7, 585–590. 29. Adinolfi, M.; Barone, G.; Guariniello, L.; Iadonisi, A. Tetrahedron Lett. 2000, 41, 9305–9308. 30. Aspinall, G. O.; Crane, A. M.; Gamman, D.W.; Ibrahim, I. H.; Khare, N. K.; Chatterjee, D.; Rivoire, B.; Brennan, P. J. Carbohydr. Res. 1991, 216, 337–355. 31. Mong, K.-K.T.; Yen, Y.-F.; Hung, W.-C.; Lai, Y.-H.; Chen, J.-H. Eur. J. Org. Chem. 2012, 3009-3017. 32. Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem. 1985, 1537–1545. 33. Wang, L.-X.; Li, C.; Wang, Q.-C.; Hui, Y.-Z. Tetrahedron Lett. 1993, 34, 7763–7766. 34. Yang, Y.; Li, Y.; Yu, B. J. Am. Chem. Soc. 2009, 131, 12076–12077. 35. Cirillo, L.; Bedini, E.; Molinaro, A.; Parrilli, M. Tetrahedron Lett. 2010, 51, 1117–1120. 36. Bedini, E.; Cirillo, L.; Marchetti, R.; Basso, S.; Tufano, D.; Molinaro, A.; Parrilli, M. Synlett 2014, 25, 365-370. 37. Crich, D.; Vinogradova, O. J. Org. Chem. 2006, 71, 8473–8480. 38. Cid, M.B.; Alfonso, F.; Martin-Lomas, M. Chem. Eur. J. 2005, 11, 928-938. 39. Adinolfi, M.; Iadonisi, A.; Pastore, A. Tetrahedron Lett. 2009, 50, 7051-7054. 40. Adinolfi, M.; Iadonisi, A.; Ravidà, A.; Schiattarella, M. Tetrahedron Lett. 2004, 45, 4485-4488. 41. Svarovsky, S.A.; Szekely, Z.; Barchi Jr., J.J. Tetrahedron-Asimmetry 2005, 16, 587-598. 26

42. Kakita, K.; Tsuda, T.; Suzuki, N.; Nakamura, S.; Nambu, H.; Hashimoto, S. Tetrahedron 2012, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

25, 5005-5017. 43. Jamois, F.; Le Goffic, F.; Yvin, J.C.; Plusquellec, D.; Ferrières, V. Open Glycosci. 2008, 1, 1924.

27

Table headings and Figure captions 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 1: Structure of the saccharide epitopes exposed on the external membrane of the three most prevalent clinical Bcc species

Figure 2: Synthetic oligosaccharides of the outer core from Bcc species

Figure 3: HSQC-DEPT and 1H NMR spectra of target tetrasaccharide 2 (400 MHz, 298K, D2O, acetone as internal standard). All the C-H correlations are assigned.

Scheme 1: Synthesis of monosaccharide building blocks. Reagents and conditions: (a) see ref. 28; (b) methyl chloroformate, TMEDA, CH2Cl2, 0°C, 99%; (c) NAPBr, NaH, DMF, rt, 86% (d) NBS, 4:1 v/v THF-H2O, 0°C, 83% (/=2.5:1) for 5, 89% (/=2.5:1) for 10; (e) Cl3CCN, DBU, CH2Cl2, rt, 82%; (f) Cl3CCN, K2CO3, CH2Cl2, rt, 84% for 6, 68% for 11; (g) see ref. 30; (h) see ref. 43. NAP = 2-naphthylmethyl

Scheme 2: Synthesis of target tetrasaccharide. Reagents and conditions: (a) see Table 1; (b) LiI, AcOH, pyridine, reflux, 98% for 13, 84% for 16; (c) DDQ, 4:1 v/v CH2Cl2-MeOH, rt, 91% for 13 68% for 16; (d) 17, TMSOTf, CH2Cl2, 0°C, 67%; (e) Zn/Cu, 3:2:1 v/v/v THF-Ac2O-AcOH, rt, 50%; (f) 9:1 v/v AcOH-H2O, 50°C; (g) Ac2O, py, rt, 70% over two steps from 19; (h) Na, CH3OH, rt, 91%.

Table 1: Glycosylation reactions with GalNAc glycosyl donors

28

B. cenocepacia 14,15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3)-D-GalNAc-(1

3)--D-GalNAc-(1

4)--L-Rha-(1

n

3)--D-QuiNAc -(1

7)-L,D-Hep INNER

CORE

B. multivorans 16 -D-Glc-(1

3)--D-GalNAc-(1

3)--D-GalNAc-(1

3)--L-Rha -(1

INNER

3)-L,D-Hep CORE

B. vietnamiensis 17 -D-Gal-(1

3)--D-GalNAc-(1

3)--D-GalNAc -(1

3)-L,D-Hep INNER

CORE

Figure 1

29

LIPID A

LIPID A

LIPID A

OH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

HO OH OH O

O

HO OH

O

HO

O

AcHN

OH

O

1

O AcHN O

HO OH OH O

O HO HO

HO OH

OH

O HO O

O AcHN

AcHN

2

Figure 2

30

O OH

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 3

31

Ph

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Ph

O O

3,4,6-tri-O-acetyl-D-galactal

O O

a

d

O

O

RO

RO N3

b c

N3 SePh

3: R=OH

4: R=COOCH3

e f

9: R=NAP

f OAll

X

5: R=COOCH3, X=OH 10: R=NAP, X=OH 6: R=COOCH3, X=-OC(NH)CCl3 6: R=COOCH3, X=-OC(NH)CCl3 11: R=NAP, X=-OC(NH)CCl3

g L-rhamnose

O BzO HO OBz

7 OBz

h D-glucose

O BzO BzO

17

BzO OC(NH)CCl3

Scheme 1

32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

O O

O O

7 + 6/6/11

Ph

OAll

Ph

O O

BzO O

a

+

O

RO

OBz

RO

N3

OBz

N3

O

8: R=COOCH3

b

BzO O

12: R=NAP 13: R=H

c Ph

8: R=COOCH3 OAll

12: R=NAP

a

OAll

Ph

O O O

O O

O BzO O

RO N3

O OBz

O N3

15: R=COOCH3 14: R=NAP 16: R=H

b c Ph

d

Ph

O O

OAll

OBz O

O

BzO BzO

O O

O BzO O

O X

BzO

O OBz

O X

18: X=N3

e

19: X=NHAc f

R'O OR'

OAll

OR O

O RO RO

R'O OR'

OR

O RO O

O AcHN

AcHN

g h

O OR

O

20: R=Bz, R'=H 21: R=Bz, R'=Ac 2: R=R'=H

Scheme 2 .

33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

acceptora

promoterb

entry

donora

1

6

7

TMSOTf (0.25 eq.)

2

6

7

BF3.OEt2 (0.25 eq.)

3:2 v/v CH2Cl2-n-hexane

7

TMSOTf (0.25 eq.)

3 4

6 6

solvent

T

2:1:1 v/v/v -70°C to CH3CN-EtCN-CH2Cl2 -50°C

product

yieldc (/)d

8

no reactione

-85°C to -75°C

8

19% (only )

3:2 v/v CH2Cl2-n-hexane

-85°C to -75°C

8

58% (only )

7

TMSOTf (0.50 eq.)

EtCN

-80°C to -70°C

8

36% (70:30)

EtCN

-80°C to -70°C

8

no reactione

5

6

7

TMSOTf (0.10 eq.)

6

6

7

TMSOTf (0.10 eq.)

CH3CN

-40°C

8

70% (45:55)

7

11f

7f

TMSOTf (0.10 eq.)

CH3CN

-40°C

12

80% (48:52)

13f

TMSOTf (0.05 eq.)

-35°C

14

tracese

CH2Cl2

-35°C

14

65% (only )

8

11f

1:1 v/v CH2Cl2-DME

9

11f

13f

TMSOTf (0.05 eq.)

10

11f

13f

TMSOTf (0.10 eq.)

CH2Cl2

-75°C

14

52% (only )

11

6f

f

TMSOTf (0.05 eq.)

CH2Cl2

-35°C

15

68% (only )

13

a

Donor/acceptor molar ratio = 1.5 unless otherwise indicated Promoter equivalents calculated with respect to the donor Isolated yield d Anomeric ratio measured by isolation of the two anomers e Determined by analysis of the crude reaction mixture by NMR spectroscopy f Donor/acceptor molar ratio = 1.7 b c

Table 1

34

Figure(s)

Figure(s)

Figure(s)

Figure(s)

Figure(s)

Table(s)

Highlights



Outer core fragment of Burkholderia cepacia pv. multivorans lipooligosaccharide



Synthetic tetrasaccharide composed of rhamnose, glucose and N-acetylgalactosamine



Allyl aglycone on the target for future conjugation with immunogenic protein



- or -glycosylation with the same 2-azido-2-deoxy-galactosyl donor