Glycoconjugates and their use as potential vaccines against infection by Shigella flexneri

Abstract

The present invention relates to a conjugate comprising an oligo- or polysaccharide selected from the group consisting of: (X).sub.x-{BCDA}.sub.n-(Y).sub.y (X).sub.x-{CDAB}.sub.n-(Y).sub.y (X).sub.x-{DABC}.sub.n-(Y).sub.y (X).sub.x-{ABCD}n-(Y).sub.y wherein A, B, C, D, X and Y, x, y and n are as defined in claim 1, said oligo- or polysaccharide being bound to a carrier.

Claims

1. A vaccine composition comprising a physiologically acceptable vehicle and a conjugate comprising an oligo- or polysaccharide selected from the group consisting of:
(X).sub.x-{BCDA}.sub.n-(Y).sub.y
(X).sub.x-{CDAB}.sub.n-(Y).sub.y
(X).sub.x-{DABC}.sub.n-(Y).sub.y
(X).sub.x-{ABCD}.sub.n-(Y).sub.y wherein: n is an integer comprised between 2 and 10, A is a beta-d-Galacturonic acid (1,3) residue, B is a N-acetyl-beta-d-Galactosamine (1,2) residue, C is independently, at each occurrence, an alpha-I-Rhamnose (1,2) residue, wherein at most one of its 3c or 4c positions is OAc (i.e OC(O)CH.sub.3), provided that there is at least one occurrence of C wherein the 3c position is not OAc, and provided that when C is a non reducing end residue, its 2c position may be acetylated or not, D is an alpha-I-Rhamnose (1,4) residue, x and y are independently selected among 0 and 1, X and Y are independently selected among A, B, C, D, AB, BC, CD, DA, ABC, BCD, CDA, DAB, provided that the ratio of 3C-OAc/4C-OAc is greater than 1, said oligo- or polysaccharide being bound to a carrier.

2. The vaccine composition according to claim 1, wherein the oligo- or polysaccharide is liable to be bound by an anti-SF6 and/or anti-SF6a antibody.

3. The vaccine composition according to claim 1, wherein said oligo- or polysaccharide is selected from the group consisting of:
{BCDP}.sub.n,
{CDAB}.sub.n,
{DABC}.sub.n, and
{ABCD}.sub.n wherein A, B, C, and D are as defined in claim 1 and n is an integer comprised between 2 and 10.

4. The vaccine composition according to claim 1, wherein n is comprised between 2 and 6.

5. The vaccine composition according to claim 1, wherein the carrier is selected among a protein or a peptide comprising at least one T-helper epitope, or a derivative thereof.

6. The vaccine composition according to claim 5, wherein the carrier is a Shigella protein able to induce a protective immune response against several Shigella serotypes.

7. The vaccine composition according to claim 1, wherein the carrier is tetanus toxoid or a fragment thereof.

8. The vaccine composition according to claim 1, wherein the carrier is a liposome.

9. The vaccine composition according to claim 1, wherein the oligo- or polysaccharide is bound to the carrier via a spacer which does not contain any carbohydrate residue.

10. The vaccine composition according to claim 1, wherein the oligo- or polysaccharide to carrier ratio is comprised between 1:1 and 30:1.

11. The vaccine composition according to claim 1, wherein the oligo- or polysaccharide is {CDAB}.sub.n, wherein A, B, C, and D are as defined in claim 1 and n is an integer comprised between 2 and 10.

12. The vaccine composition of claim 1, further comprising an immunogen which affords protection against another pathogen, such as for example, S. flexneri serotype 1 b, 2a and 3a, members of other Shigella species such as S. sonnei and S. dysenteriae type 1, or pathogens responsible for diarrhoeal disease in humans.

13. The vaccine composition according to claim 1, which is formulated for parenteral, oral, intranasal, intradermal, subcutaneous or transcutaneous administration.

14. A vaccine composition comprising a physiologically acceptable vehicle and a conjugate comprising an oligo- or polysaccharide (Ia) selected from the group consisting of:
(X).sub.x-{BCDA}.sub.n-(Y).sub.y-OQ
(X).sub.x-{CDAB}.sub.n-(Y).sub.y-OQ
(X).sub.x-{DABC}.sub.n-(Y).sub.y-OQ
(X).sub.x-{ABCD}.sub.n-(Y).sub.y-OQ Wherein: A, B, C, D, X, Y, x, y and n are as defined in claim 1, provided that the ratio of 3C-OAc/4C-OAc is greater than 1, O is the C.sub.1 oxygen atom of the reducing end residue of the oligo- or polysaccharide, Q is H, or a group LZ, L is a divalent C.sub.1-C.sub.12 alkyl or alkenyl chain optionally interrupted by one or more heteroatoms, notably selected from an oxygen atom, a sulphur atom or a nitrogen atom, said nitogen and sulphur atoms being optionally oxidized, and the nitrogen atom being optionally involved in an acetamide bond, and Z is a terminal reactive function, optionally protected, able to form a covalent bond with a carrier and/or a solid support.

15. The vaccine composition of claim 14, wherein Z is Hal, biotin, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, azido, alkoxy, epoxyde, acetal, C(O)H, SR.sub.1, NH.sub.2 or NHC(O)CH.sub.2Hal, R.sub.1 being H, C(O)CH.sub.3 or SR.sub.2, and R.sub.2 being a C.sub.1-C.sub.6 alkyl, a C.sub.6-C.sub.10 aryl, or a 5 to 7 membered heteroaryl, such as pyridyl, or any group allowing to convert SSR.sub.2 into SH.

16. The vaccine composition according to claim 14, which is immobilized on a solid support.

17. A vaccine composition comprising a physiologically acceptable vehicle and a conjugate comprising an oligo or polysaccharide (Ib) selected from the group consisting of:
(X).sub.x-{BCDA}.sub.n-(Y).sub.y-W
(X).sub.x-{CDAB}.sub.n-(Y).sub.y-W
(X).sub.x-{DABC}.sub.n-(Y).sub.y-W
(X).sub.x-{ABCD}.sub.n-(Y).sub.y-W Wherein: A is a beta-d-Galacturonic (1,3) acid residue, B is a N-protected-beta-d-Galactosamine (1,2) residue, wherein said N-protecting group is an acetyl group, or a precursor thereof such as a N-trichloroacetyl group, C is an alpha-I-Rhamnose (1,2) residue, wherein at most one of 3c or 4c is OAc, D is an alpha-I-Rhamnose (1,4) residue, x and y are independently selected among 0 and 1, n is an integer comprised between 2 and 10, X and Y are independently selected among A, B, C, D, AB, BC, CD, DA, ABC, BCD, CDA, DAB, provided that the ratio of 3C-OAc/4C-OAc is greater than 1, wherein each OH and/or CO.sub.2H group of said residues are optionally protected by a protecting group, W is OR.sup.i, SR.sup.ii or Hal, wherein said O, S and Hal are the C.sub.1 heteroatom of the reducing end residue of the oligo- or polysaccharide chain, S being optionally oxidized, R.sup.i is H, a hydroxyl protecting group, an anomeric hydroxyl activating group, or a LZ group, L and Z being as defined above for formula (Ia), R.sup.ii is C.sub.1-C.sub.12 alkyl, C.sub.6-C.sub.10 aryl, an imidate leading preferably to S-thiazolinyl (STaz) or S-benzoxazolyl (SBox).

18. An oligosaccharide which is selected from the group consisting of: Benzyl (4-O-benzyl-3-O-para-methoxybenzyl--I-rhamnopyranosyl)-(1.fwdarw.2)-(3,4-di-O-benzyl--I-rhamnopyranosyl)-(1.fwdarw.4)-(allyl 2,3-di-O-benzyl--d-galactopyranosid)uronate, Allyl (4-O-benzyl-3-O-para-methoxybenzyl--I-rhamnopyranosyl)-(1.fwdarw.2)-(3,4-di-O-benzyl--I-rhamnopyranosyl)-(1.fwdarw.4)-(benzyl 2,3-di-O-benzyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--d-galactopyranoside, 3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-/-d-galactopyranosyl N-phenyltrifluoroacetimidate, Allyl (2,3-di-O-benzyl--d-galactopyranosyl)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--d-galactopyranoside, Allyl (benzyl 2,3-di-O-benzyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--d-galactopyranoside, Allyl (benzyl 2,3-di-O-benzyl-4-O-levulinoyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--d-galactopyranoside, (Benzyl 2,3-di-O-benzyl-4-O-levulinoyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-d-galactopyranose, Benzyl 2,3-di-O-benzyl-4-O-levulinoyl--d-galactopyranosyluronate-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-d-galactopyranosyl N-phenyltrifluoroacetimidate, Benzyl 2,3-di-O-benzyl-4-O-levulinoyl--d-galactopyranosyluronate-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-d-galactopyranosyl trichloroacetimidate, (3,4-Di-O-benzyl-2-O-levulinoyl--I-rhamnopyranosyl)-(1.fwdarw.4)-(benzyl 2,3-di-O-benzyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-d-galactopyranosyl N-phenyltrifluoroacetimidate, (4-O-Benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--I-rhamnopyranosyl)-(1.fwdarw.2)-(3,4-di-O-benzyl--I-rhamnopyranosyl)-(1.fwdarw.4)-(benzyl 2,3-di-O-benzyl--d-galactopyranosyluronate)-(1.fwdarw.3)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--d-galactopyranosyl N-phenyltrifluoroacetimidate.

Description

EXAMPLES

(1) I. Synthesis of Oligo- and/or Polysaccharides

(2) General Methods:

(3) Nuclear magnetic resonance (NMR) spectra were recorded at 303 K on a Bruker Avance spectrometer at 400 MHz (.sup.1H) and 100 MHz (.sup.3C) equipped with a BBO probe. Signals are reported as m (multiplet), s (singlet), d (doublet), t (triplet), dd (doublet of doublet), q (quadruplet), qt (quintuplet), sex (sextuplet), dt (doublet of triplet), dq (doublet of quadruplet), ddd (doublet of doublet of doublet), m (multiplet). The signals can also be described as broad (prefix b), pseudo (prefix p), overlapped (suffix .sub.o) or partially overlapped (suffix .sub.po) The coupling constants are reported in hertz (Hz). The chemical shifts are reported in ppm () relative to residual solvent peak. 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 are marked with an asterisk. Sugar residues are lettered according to the lettering of the repeating unit of the SF6 OAg (A, B, C and D) and identified by a subscript in the listing of signal assignments. For oligosaccharides larger than one repeating unit, the A, B, C, D lettering is turned into A, B, C, if and A, B, C, D, starting with A, B, C, D from the reducing end.

(4) HRMS spectra were recorded on a WATERS QTOF Micromass instrument in the positive-ion electrospray ionisation (ESI.sup.+) mode. Solutions were prepared using 1:1 MeCN/H.sub.2O containing 0.1% formic acid.

(5) Analytical RP-HPLC analyses of the final compounds were run on an Agilent 1200 Series instrument equipped with a UV detector (=215 nm) and a Symmetry 3.5 m C.sub.18 100 2.1100 mm analytical column eluting with a 0-35% linear gradient of MeCN in 0.01 N aq. TFA over 20 min at a flow rate of 0.3 mL.Math.min.sup.1).

(6) A. Linear Synthetic Strategy

(7) Linear synthesis stands for a process whereby the chain elongation required to obtain the oligo- or polysaccharide target involves the predetermined ordered glycosylation of an acceptor with one residue at a time in alternance with the selective unmasking of the site of subsequent glycosylation within the newly introduced residue.

(8) Example of a Linear Synthesis: Access to Tetrasaccharides BCDA-Pr (XXX) and B.sub.AcCDA-Pr (XXXI)

(9) ##STR00044## ##STR00045##

(10) The galacturonate acceptor 154 (A) is first glycosylated with the rhamnosyl trichloroacetimidate donor 92 (D) to give the fully protected disaccharide 164. The site of elongation is selectively deprotected, turning this disaccharide into an acceptor (165). The newly synthesized 165 is reacted, under selected imidate-based glycosylation conditions, with a second rhamnosyl donor 119 that is a precursor to residue C. Interestingly, its 3-OH is orthogonally protected in the form of a para-methoxybenzyl ether. Hydrazinolysis of the single levulinoyl ester of the resulting trisaccharide allowed the selective unmasking of the 2.sub.C-OH, thus turning the glycosylation product 167 into acceptor 169. Glycosylation of trisaccharide 169 with the galactosaminyl residue 24 (B), in this case a thioglycoside donor, provided the -linked glycosylation product 183 as required. The fully protected tetrasaccharide 183 was submitted to a two step process involving first the oxidative cleavage of the PMB ether, and then the acetylation of the thus unmasked 3.sub.C-OH to give the 3-OAc analogue 187. Treatment of tetrasaccharides 183 and 187 with Pd/C in a hydrogen atmosphere led to propyl glycosides XXX and XXXI, issued from the hydrogenolysis of the benzyl ester, benzyl and when needed para-methoxybenzyl ethers in concomitance with the trichloroacetamide to acetamide and allyl to propyl reduction. Tetrasaccharide XXX and its 3.sub.C-OAc analogue XXXI are synthetic frame-shifted mimics of the biological repeating unit of SF6, SF6a and/or E. coli O147 O-antigens.

Benzyl (3,4-di-O-benzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranoside)uronate (164)

(11) A suspension of acceptor 154.sup.[27] (300 mg, 595 mol), trichloroacetimidate 92.sup.[28] (419 mg, 713 mol) and freshly activated 4 AW 300 MS (450 mg) in anhyd. toluene (5.9 mL) was stirred for 1 h at rt under an Ar atmosphere. The reaction mixture was cooled to 10 C., and TMSOTf (5 L, 30 mol) was added. The reaction mixture was stirred at that temperature for 30 min. The reaction was quenched by adding Et.sub.3N. The resulting mixture was filtered and the filtrate was concentrated. The residue was purified by flash chromatography (Tol/Acetone 97:3 to 96:4) to give disaccharide 164 (519 mg, 94%) as a white oil.

(12) .sup.1H NMR (CDCl.sub.3), 7.41-7.23 (m, 25H, H.sub.Ar), 5.99 (m, 1H, CH.sub.All), 5.55 (dd, 1H, J.sub.1,2=2.1 Hz, J.sub.2,3=3.3 Hz, H-2.sub.D), 5.36 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.28 (d, 1H, J=12.3 Hz, H.sub.CO2Bn) 5.24-5.20 (m, 2H, H-1.sub.D, CH.sub.2All), 5.12 (d, 1H, H.sub.CO2Bn), 4.94 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.91 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.79 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.77 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.74 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.64 (d, 1H, J=11.1 Hz, H.sub.Bn), 4.61 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.50 (m, 1H, H.sub.All), 4.45 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.43-4.39 (m, 2H, H-4.sub.A, H-1.sub.A), 4.17 (m, 1H, H.sub.All), 4.05 (bs, 1H, H-5.sub.A), 3.88 (dd, 1H, J.sub.3,4=9.4 Hz, H-3.sub.D), 3.80-3.71 (m, 2H, H-2.sub.A, H-5.sub.D), 3.56 (dd, 1H, J.sub.2,3=9.7 Hz, J.sub.3,4=2.9 Hz, H-3.sub.A), 3.37 (pt, 1H, J.sub.4,5=9.4 Hz, H-4.sub.D), 2.72-2.62 (m, 4H, CH.sub.2Lev), 2.16 (s, 3H, CH.sub.3Lev), 1.30 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D).

(13) .sup.13C NMR (CDCl.sub.3) partial, 206.4 (CO.sub.Lev), 171.6 (CO.sub.2Lev), 167.4 (C-6.sub.A), 102.6 (C-1.sub.A, .sup.1J.sub.CH=161.3 Hz), 98.9 (C-1.sub.D, .sup.1J.sub.CH=172.7 Hz), 67.4 (C.sub.CO2Bn), 38.1 (COCH.sub.2Lev), 29.9 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

(14) HRMS (ESI.sup.+): m/z 951.3846 (calcd for C.sub.55H.sub.60O.sub.13Na [M+Na].sup.+: m/z 951.3932).

Benzyl (3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (165)

(15) To a solution of disaccharide 164 (1.25 g, 1.35 mmol) in dry pyridine (8.3 mL) stirred at 0 C. under an Ar atmosphere was slowly added acetic acid (5.6 mL) followed by hydrazine monohydrate (326 L, 6.73 mmol). The reaction mixture was stirred at rt for 1.5 h. Volatiles were evaporated and co-evaporated twice with toluene. The residue was taken up in DCM and washed with water. The aq. layer was re-extracted twice with DCM, and the combined organic phases were washed with brine, passed through a phase separator filter and concentrated. The residue was purified by flash chromatography (Chex/EtOAc 8:2 to 7:3) to give alcohol 165 (1.05 g, 90%) as a light yellow oil.

(16) .sup.1H NMR (CDCl.sub.3), 7.42-7.27 (m, 25H, H.sub.Ar), 6.01 (m, 1H, CH.sub.All), 5.39 (d.sub.o, 1H, J.sub.1,2=1.7 Hz, H-1.sub.D), 5.38 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.32 (d, 1H, J=12.2 Hz, H.sub.CO2Bn), 5.25 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2All), 5.14 (d, 1H, H.sub.CO2Bn), 4.96 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.90 (d, 1H, J=11.3 Hz, H.sub.Bn), 4.80 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.76 (d.sub.po, 1H, J=10.9 Hz, H.sub.Bn), 4.75 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.67-4.64 (m, 3H, H.sub.Bn), 4.54 (m, 1H, H.sub.All), 4.49 (dd, 1H, H-4.sub.A), 4.43 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.23-4.16 (m, 2H, H-2.sub.D H.sub.All), 4.08 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 3.89 (dd, 1H, J.sub.2,3=3.2 Hz, J.sub.3,4=9.0 Hz, H-3.sub.D), 3.80-3.71 (m, 2H, H-2.sub.A, H-5.sub.D), 3.58 (dd, 1H, J.sub.2,3=9.8 Hz, J.sub.3,4=3.0 Hz, H-3.sub.A), 3.47 (pt, 1H, J.sub.4,5==9.3 Hz, H-4.sub.D), 2.43 (bs, 1H, OH), 1.32 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D).

(17) .sup.13C NMR (CDCl.sub.3) partial, 167.3 (C-6.sub.A), 102.7 (C-1.sub.A, .sup.1J.sub.CH=159.4 Hz), 100.4 (C-1.sub.D, .sup.1J.sub.CH=172.7 Hz), 67.3 (C.sub.CO2Bn)

(18) HRMS (ESI.sup.+): m/z 853.3478 (calcd for C.sub.50H.sub.54O.sub.11Na [M+Na].sup.+: m/z 853.3564).

Benzyl (4-O-benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (167)

(19) A mixture of acceptor 165 (300 mg, 361 mol), trichloroacetimidate 119 (267 mg, 433 mol) and freshly activated 4 powdered MS (750 mg) in anhyd. toluene (10.8 mL) was stirred for 1 h at rt under an Ar atmosphere. The reaction mixture was cooled to 10 C., and TMSOTf (3.3 L, 18 mol) was added. The reaction mixture was stirred at that temperature for 20 min. The reaction was quenched by adding Et.sub.3N. The resulting suspension was filtered and concentrated. The residue was purified by flash chromatography (Tol/EtOAc 95:5 to 85:15) to give trisaccharide 167 (426 mg, 91%) as a light yellow oil.

(20) .sup.1H NMR (CDCl.sub.3), 7.43-7.17 (m, 32H, H.sub.Ar), 6.86 (d, 2H, J=8.5 Hz, H.sub.ArPMB), 5.99 (m, 1H, CH.sub.All), 5.52 (dd, 1H, J.sub.1,2=1.9 Hz, J.sub.2,3=3.1 Hz, H-2.sub.C), 5.36 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.33 (bs.sub.o, 1H, H-1.sub.D), 5.27 (d, 1H, J=12.2 Hz, H.sub.CO2Bn), 5.22 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2All), 5.12 (d, 1H, H.sub.CO2Bn), 4.94 (d.sub.po, 1H, J=11.1 Hz, H.sub.Bn), 4.92-4.88 (m.sub.o, 3H, H-1.sub.C, 2H.sub.B), 4.80 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.75 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.70-4.57 (m, 6H, H.sub.Bn), 4.50 (m.sub.po, 1H, H.sub.All), 4.48 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.39 (m.sub.o, 1H, H-4.sub.A), 4.38 (d.sub.o, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.17 (m, 1H, H.sub.All), 4.11 (pt, 1H, H-2.sub.D), 4.02 (d, 1H, J.sub.4,5=1.0 Hz, H-5.sub.A), 3.94 (dd, 1H, J.sub.3,4=9.3 Hz, H-3.sub.C), 3.86 (dd.sub.po, 1H, J.sub.2,3=2.9 Hz, J.sub.3,4=9.4 Hz, H-3.sub.D), 3.82 (dq, 1H, H-5.sub.C), 3.78 (s, 3H, CH.sub.3PMB), 3.75-3.64 (m, 2H, H-2.sub.A, H-5.sub.D), 3.51 (dd, 1H, J.sub.2,3=9.8 Hz, J.sub.3,4=2.8 Hz, H-3.sub.A), 3.42 (pt, 1H, J.sub.4,5=9.5 Hz, H-4.sub.D), 3.37 (pt, 1H, J.sub.4,5=9.5 Hz, H-4.sub.C), 2.77-2.66 (m, 4H, CH.sub.2Lev), 2.18 (s, 3H, CH.sub.3Lev), 1.28 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(21) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.7 (CO.sub.2Lev), 167.3 (C-6.sub.A), 159.2 (C.sub.IVPMB), 113.7 (2C, C.sub.ArPMB), 102.7 (C-1.sub.A, .sup.1J.sub.CH=157.6 Hz), 100.0 (C-1.sub.D, .sup.1J.sub.CH=178.6 Hz), 99.2 (C-1.sub.C, .sup.1J.sub.CH=171.6 Hz), 67.3 (C.sub.CO2Bn), 55.1 (CH.sub.3PMB), 38.2 (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.3 (CO.sub.2CH.sub.2Lev).

(22) HRMS (ESI.sup.+): m/z 1307.5554 (calcd for C.sub.76H.sub.84O.sub.18Na [M+Na].sup.+: m/z 1307.5555).

Benzyl (4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (169)

(23) Acetic acid (11.3 mL) followed by hydrazine monohydrate (492 L, 10.1 mmol) were slowly added to a solution of the fully protected 167 (2.60 g, 2.0 mmol) in dry pyridine (16.8 mL) stirred at 0 C. under an Ar atmosphere. The reaction mixture was stirred at rt for 30 min. Volatiles were evaporated and co-evaporated twice with toluene. The residue was taken up in DCM and washed with water. The aq. layer was re-extracted twice with DCM, and the combined organic phases were washed with brine, passed through a phase separator filter, and concentrated. The residue was purified by flash chromatography (Tol/EtOAc 9:1 to 7:3) to give alcohol 169 (2.23 g, 93%) as a white foam.

(24) .sup.1H NMR (CDCl.sub.3), 7.45-7.16 (m, 32H, H.sub.Ar), 6.89 (d, 2H, J=8.6 Hz, H.sub.ArPMB), 6.00 (m, 1H, CH.sub.All), 5.37 (m, 1H, J.sub.trans=17.3 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.35 (bs.sub.o, 1H, H-1.sub.D), 5.28 (d, 1H, J=12.1 Hz, H.sub.CO2Bn), 5.23 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2All), 5.13 (d, 1H, H.sub.CO2Bn), 5.02 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.C), 4.94 (d.sub.po, 1H, J=10.9 Hz, H.sub.Bn), 4.90 (d.sub.po, 1H, J=11.2 Hz, H.sub.Bn), 4.89 (d.sub.po, 1H, J=10.9 Hz, H.sub.Bn), 4.81 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.75 (d, 1H, 1=10.9 Hz, H.sub.Bn), 4.70-4.59 (m, 7H, H.sub.Bn), 4.51 (m, 1H, H.sub.All), 4.40 (m.sub.o, 1H, H-4.sub.A), 4.39 (d.sub.o, 1H, J.sub.1,2=7.7 Hz, H-1.sub.A), 4.17 (m.sub.po, 1H, H.sub.All), 4.15 (m.sub.o, 1H, H-2.sub.D), 4.12 (m, 1H, H-2.sub.C), 4.03 (d, 1H, J.sub.4,5=0.9 Hz, H-5.sub.A), 3.91-3.86 (m, 2H, H-3.sub.C, H-3.sub.D), 3.84 (dq.sub.po, 1H, H-5.sub.C), 3.79 (s, 3H, CH.sub.3PMB), 3.76-3.66 (m, 2H, H-2.sub.A, H-5.sub.D), 3.52 (dd, 1H, J.sub.2,3=9.8 Hz, J.sub.3,4=2.8 Hz, H-3.sub.A), 3.45 (pt.sub.po, 1H, J.sub.4,5=9.5 Hz, H-4.sub.C), 3.41 (pt, 1H, J.sub.3,4=J.sub.4,5=9.6 Hz, H-4.sub.D), 1.30 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.21 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(25) .sup.13C NMR (CDCl.sub.3) partial, 167.3 (C-6.sub.A), 159.2 (C.sub.IVPMB), 114.0 (2C, C.sub.ArPMB), 102.7 (C-1.sub.A, .sup.1J.sub.CH=161.1 Hz), 100.8 (C-1.sub.D, .sup.1J.sub.CH=169.0 Hz), 100.2 (C-1.sub.C, .sup.1J.sub.CH=171.9 Hz), 67.3 (C.sub.CO2Bn), 55.2 (CH.sub.3PMB).

(26) HRMS (ESI.sup.+): m/z 1210.5117 (calcd for C.sub.71H.sub.78O.sub.16Na [M+Na].sup.+: m/z 1210.5222), m/z 613.2374 (calcd for C.sub.71H.sub.79O.sub.16K [M+H+K].sup.2+: m/z 613.2502).

Benzyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (183)

(27) A mixture of acceptor 169 (300 mg, 253 mol), thioglycoside donor 24 (260 mg, 379 mol), and freshly activated 4 powdered MS (750 mg) in anhyd. DCM (2.9 mL) was stirred for 1 h at rt under an Ar atmosphere. The reaction mixture was cooled to 78 C., then NIS (114 mg, 505 mol) and TMSOTf (4.6 L, 25 mol) were added. The reaction mixture was stirred for 30 min allowing the bath to reach 60 C. A TLC control (Tol/EtOAc 9:1) indicated the absence of acceptor 169 (rf=0.18) and the presence of a new less polar product (rf=0.38). The reaction was quenched by addition of Et.sub.3N. The resulting suspension was filtered and concentrated. The residue was purified by flash chromatography (Tol/EtOAc 98:2 to 92:8) to give tetrasaccharide 183 (356 mg, 80%) as a white foam.

(28) .sup.1H NMR (CDCl.sub.3), 7.41-7.05 (m, 47H, H.sub.Ar), 6.91 (d, 1H, J.sub.NH,2=7.5 Hz, NH), 6.83 (d, 2H, J=8.6 Hz, H.sub.ArPMB), 5.97 (m, 1H, CH.sub.All), 5.35 (m, 1H, J.sub.trans=17.3 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.26 (bs, 1H, H-1.sub.D), 5.25 (d.sub.po, 1H, H.sub.CO2Bn), 5.21 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2ll), 5.10 (d, 1H, J=12.2 Hz, H.sub.CO2Bn) 4.98-4.92 (m, 4H, H-1.sub.B, H-1.sub.C, 2H.sub.Bn), 4.90 (d.sub.po, 1H, J=10.9 Hz, H.sub.Bn), 4.84 (d.sub.po, 1H, J=11.1 Hz, H.sub.Bn), 4.76 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.72 (d.sub.po, 1H, J=10.5 Hz, H.sub.Bn), 4.71-4.67 (m, 2H, H.sub.Bn), 4.65-4.55 (m, 3H, H.sub.Bn), 4.58-4.45 (m, 6H, 5H.sub.Bn, H.sub.All), 4.36 (d.sub.o, 1H, J.sub.1,2=7.7 Hz, H-1.sub.A), 4.34 (bd.sub.o, 1H, H-4.sub.A), 4.27 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.22 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.18-4.10 (m, 2H, H-2.sub.B, H.sub.All), 4.08 (pt, 1H, H-2.sub.C), 4.03-3.96 (m, 4H, H-5.sub.A, H-3.sub.B, H-4.sub.B, H-2.sub.D), 3.87 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.6 Hz, H-3.sub.C), 3.81 (dd.sub.po, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.5 Hz, H-3.sub.D), 3.79 (dq.sub.po, 1H, H-5.sub.C), 3.71 (s, 3H, CH.sub.3PMB), 3.70-3.59 (m, 3H, H-2.sub.A, H-6a.sub.B, H-5.sub.D), 3.51-3.42 (m, 3H, H-3.sub.A, H-5.sub.B, H-4.sub.C), 3.34 (pt.sub.po, 1H, J.sub.4,5=9.2 Hz, H-4.sub.D), 3.31 (m.sub.o, 1H, H-6b.sub.B), 1.28 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.18 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(29) .sup.13C NMR (CDCl.sub.3) partial, 167.3 (C-6.sub.A), 161.6 (NHCO), 159.4 (C.sub.IVPMB), 113.9 (2C, C.sub.ArPMB), 102.7 (C-1.sub.A, .sup.1J.sub.CH=162.0 Hz), 101.3 (C-1.sub.C, .sup.1J.sub.CH=173.5 Hz), 100.7 (C-1.sub.B, .sup.1J.sub.CH=162.4 Hz), 100.4 (C-1.sub.D, .sup.1J.sub.CH=175.4 Hz), 92.9 (CCl.sub.3), 67.3 (C.sub.C02Bn), 55.2 (CH.sub.3PMB).

(30) HRMS (ESI.sup.+): m/z 1784.6198 (calcd for C.sub.100H.sub.106Cl.sub.3NO.sub.21Na [M+Na].sup.+: m/z 1784.6221).

Benzyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(3-O-acetyl-4-O-benzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (187)

(31) Water (1.6 mL) and CAN (870 mg, 1.59 mmol) were added to a solution of tetrasaccharide 183 (700 mg, 397 mol) in MeCN (15.9 mL). The reaction mixture was stirred at rt for 30 min. The reaction was quenched with satd. aq. NaHCO.sub.3. The reaction mixture was diluted with water and DCM and the aq. phase was extracted three times with DCM. The combined extracts were washed with brine, passed through a phase separator filter, and concentrated. The resulting crude oil was dissolved in pyridine (20 mL), and excess acetic anhydride (3.75 mL) and DMAP (48 mg, 397 mol) were added to the solution kept under stirring at rt. Volatiles were evaporated and co-evaporated three times with toluene. The residue was purified by flash chromatography (Tol/EtOAc 95:5 to 9:1) to afford the monoacetylated tetrasaccharide 187 (582 mg, 87% over two steps) as a white foam.

(32) .sup.1H NMR (CDCl.sub.3), 7.41-7.04 (m, 45H, H.sub.Ar), 7.02 (d, 1H, J.sub.NH,2=7.0 Hz, NH), 5.98 (m, 1H, CH.sub.All), 5.36 (dd.sub.po, 1H, J.sub.2,3=3.1 Hz, H-3.sub.C), 5.35 (m, 1H, J.sub.gem=1.6 Hz, CH.sub.2All), 5.32 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.D), 5.26 (d, 1H, J=12.2 Hz H.sub.CO2Bn), 5.22 (m, 1H, J.sub.cis 10.5 Hz, CH.sub.2All), 5.09 (d, 1H, H.sub.CO2Bn), 5.03 (d.sub.po, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 5.01 (d.sub.po, 1H, J.sub.1,2=2.0 Hz, H-1.sub.C), 4.89 (d.sub.po, 1H, J 10.8 Hz, H.sub.Bn), 4.88 (d, 2H, J=11.1 Hz, 2H.sub.Bn), 4.77 (d, 1H, J=11.8 Hz, H.sub.Bn), 4.70 (d, 2H, J=11.2 Hz, H.sub.Bn), 4.66 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.62-4.52 (m.sub.po, 7H, H.sub.Bn), 4.50 (m.sub.po, 1H, H.sub.All), 4.40-4.35 (m, 4H, H-1.sub.A, H-4.sub.A, H-3.sub.B, H-2.sub.C), 4.26 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.20 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.16 (m, 1H, H.sub.All), 4.05 (d, 1H, J.sub.3,4=2.4 Hz, H-4.sub.B), 4.01 (d, 1H, J.sub.4,5=0.9 Hz, H-5.sub.A), 4.00 (m, 1H, H-2.sub.D), 3.92-3.82 (m, 2H, H-2.sub.B, H-5.sub.C), 3.80 (dd, 1H, J.sub.2,3=2.9 Hz, J.sub.3,4=9.5 Hz, H-3.sub.D), 3.70-3.58 (m, 3H, H-2.sub.A, H-6a.sub.B, H-5.sub.D), 3.56-3.47 (m, 4H, H-3.sub.A, H-5.sub.B, H-4.sub.C, H-4.sub.D), 3.35 (dd, 1H, J.sub.5,6b=4.5 Hz, J.sub.6a,6b=8.3 Hz, H-6b.sub.B), 2.17 (s, 3H, H.sub.Ac), 1.29 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.14 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(33) .sup.13C NMR (CDCl.sub.3) partial, 170.7 (CO.sub.Ac) 167.2 (C-6.sub.A), 161.5 (NHCO), 102.6 (C-1.sub.A, .sup.1J.sub.CH=156.6 Hz), 101.3 (C-1.sub.C, .sup.1J.sub.CH=176.3 Hz), 100.0 (C-1.sub.D, .sup.1J.sub.CH=171.1 Hz), 99.6 (C-1.sub.B, .sup.1J.sub.CH=163.3 Hz), 92.9 (CCl.sub.3), 67.3 (C.sub.CO2Bn), 21.3 (CH.sub.3Ac).

(34) HRMS (ESI.sup.+): m/z 1684.5983 (caled for C.sub.94H.sub.101Cl.sub.3NO.sub.21 [M+H].sup.+: m/z 1684.5931), m/z 1706.5928 (calcd for C.sub.94H.sub.100Cl.sub.3NO.sub.21Na [M+Na].sup.+: m/z 1706.5751).

Propyl 2-acetamido-2-deoxy--D-galactopyranosyl-(12)--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosiduronic acid (XXX)

(35) To a stirred solution of tetrasaccharide 183 (254 mg, 140 mol) in THF/H.sub.2O (4:1, 10.2 mL), was added 10% Pd/C (200 mg). The suspension was stirred under a hydrogen atmosphere for 2 days. After this time, MS analysis indicated a single molecular weight corresponding to that of the target tetrasaccharide. The reaction mixture was filtered. Evaporation of the volatiles, freeze-drying furnished and purification of the crude material by preparative RP-HPLC (Kromasil 5 m C18 100 10250 mm semi-preparative column, 0-20% linear gradient of CH.sub.3CN in 0.08% aq. TFA over 20 min at a flow rate of 5.5 mL.Math.min.sup.1) gave tetrasaccharide XXX (61.8 mg, 59%) as a white solid following repeated freeze-drying.

(36) .sup.1H NMR (D.sub.2O), 5.24 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.D), 5.08 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.C), 4.55 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.38 (d, 1H, J.sub.1,2=8.0 Hz, H-1.sub.A), 4.35 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.27 (dd, 1H, J.sub.3,4=2.8 Hz, H-4.sub.A), 4.05 (dd, 1H, 1.sub.2,3=3.0 Hz, H-2.sub.C), 4.02 (dd, 1H, J.sub.2,3=3.1 Hz, H-2.sub.D), 3.87-3.74 (m, 6H, H-4.sub.B, OCH.sub.2Pr, H-2.sub.B, H-3.sub.A, H-3.sub.D, H-3.sub.C), 3.74-3.63 (m, 3H, H-6a.sub.B, H-6b.sub.B, H-3.sub.B), 3.63-3.51 (m, 4H, H-5.sub.B, H-5.sub.C, OCH.sub.2Pr, H-5.sub.D), 3.50 (dd, 1H, J.sub.2,3=9.9 Hz, H-2.sub.A), 3.34 (pt, 1H, J.sub.3,4=J.sub.4,5=9.8 Hz, H-4.sub.D), 3.27 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.C), 1.97 (s, 3H, CH.sub.3NHAc), 1.57 (sex, 2H, J=7.4 Hz, CH.sub.2Pr), 1.20-1.15 (m, 6H, H-6.sub.C, H-6.sub.D), 0.84 (t, 3H, CH.sub.3Pr)

(37) .sup.13C NMR (D.sub.2O), 175.1 (NHCO), 171.6 (C-6.sub.A), 103.1 (C-1.sub.B, .sup.1J.sub.CH=163.2 Hz), 102.3 (C-1.sub.A, .sup.1J.sub.CH=161.7 Hz), 101.0 (C-1.sub.C, J.sub.CH=175.8 Hz), 99.9 (C-1.sub.D, .sup.1J.sub.CH=174.7 Hz), 78.6 (C-2.sub.C), 78.5 (C-2.sub.D), 76.7 (C-4.sub.A), 75.0 (C-5.sub.B), 72.9 (C-3.sub.A), 72.8 (C-5.sub.A), 72.3 (2C, C-4.sub.C, OCH.sub.2Pr), 71.9 (C-4.sub.D), 70.8 (C-3.sub.B), 70.2 (C-2.sub.A), 69.7 (2C, C-3.sub.D, C-3.sub.C), 69.2 (C-5.sub.D), 69.1 (C-5.sub.C), 67.7 (C-4.sub.B), 60.9 (C-6.sub.B), 52.8 (C-2.sub.B), 22.0 (2C, CH.sub.2Pr, CH.sub.3NHAc), 16.6 (2C, C-6.sub.D, C-6.sub.C), 9.6 (CH.sub.3Pr).

(38) HRMS (ESI.sup.+): m/z 754.2722 (calcd for C.sub.29H.sub.49NO.sub.20Na [M+Na].sup.+: m/z 754.2745). RP-HPLC (215 nm): R.sub.t=8.5 min.

Propyl 2-acetamido-2-deoxy--D-galactopyranosyl-(12)-(3-O-acetyl--L-rhamnopyranosyl)-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosiduronic acid (XXXI)

(39) To a stirred solution of tetrasaccharide 187 (187 mg, 110 mol) in THF/H.sub.2O (4:1, 8.4 mL), was added 10% Pd/C (150 mg). The suspension was stirred under a hydrogen atmosphere for 2 days. After this time, MS analysis indicated a molecular weight corresponding to that of the target tetrasaccharide. The reaction mixture was filtered. Evaporation of the volatiles, freeze-drying and purification by preparative RP-HPLC (Kromasil 5 m C18 100 10250 mm semi-preparative column, 0-20% linear gradient of CH.sub.3CN in 0.08% aq. TFA over 20 min at a flow rate of 5.5 mL.Math.min.sup.1) gave tetrasaccharide XXXI (45.3 mg, 53%) as a white solid following repeated freeze-drying.

(40) .sup.1H NMR (D.sub.2O), 5.23 (d, 1H, J.sub.1,2=1.3 Hz, H-1.sub.D), 5.03 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.C), 4.92 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=10.0 Hz, H-3.sub.C), 4.33 (d, 1H, J.sub.1,2=8.0 Hz, H-1.sub.A), 4.31 (bs.sub.o, 1H, H-5.sub.A), 4.30 (d.sub.po, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 4.22 (dd, 1H, J.sub.3,4=2.8 Hz, J.sub.4,5=1.1 Hz, H-4.sub.A), 4.12 (pt, 1H, H-2.sub.C), 3.99 (dd, 1H, J.sub.2,3=3.1 Hz, H-2.sub.D), 3.83-3.70 (m, 5H, H-4.sub.B, OCH.sub.2Pr, H-2.sub.B, H-3.sub.A, H-3.sub.D), 3.70-3.57 (m, 4H, H-5.sub.C, H-6a.sub.B, H-6b.sub.B, H-3.sub.B), 3.52-3.46 (m, 3H, H-5.sub.B, OCH.sub.2Pr, H-5.sub.D), 3.45 (dd.sub.po, 1H, J.sub.2,3=10.0 Hz, H-2.sub.A), 3.34 (pt.sub.po, 1H, J=9.3 Hz, J=9.8 Hz, H-4.sub.C), 3.35 (pt.sub.po, 1H, J=9.6 Hz, J=9.8 Hz, H-4.sub.D), 2.09 (s, 3H, CH.sub.3Ac), 1.97 (s, 3H, CH.sub.3NHAc), 1.56 (sex, 2H, J=7.4 Hz, CH.sub.2Pr), 1.16 (d, 3H, J.sub.5,6=6.3 Hz, H-6.sub.D), 1.14 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 0.80 (t, 3H, CH.sub.3Pr) .sup.13C NMR (D.sub.2O), 174.5 (NHCO), 173.5 (CO.sub.Ac), 171.5 (C-6.sub.A), 103.2 (C-1.sub.B, .sup.1J.sub.CH=163.2 Hz), 102.3 (C-1.sub.A, .sup.1J.sub.CH=161.8 Hz), 101.0 (C-1.sub.C, .sup.1J.sub.CH=174.7 Hz), 99.8 (C-1.sub.D, .sup.1J.sub.CH=173.6 Hz), 78.8 (C-2.sub.D), 76.7 (C-4.sub.A), 76.4 (C-2.sub.C), 74.8 (C-5.sub.B), 72.8 (C-3.sub.A), 72.7 (C-5.sub.A), 72.6 (C-3.sub.C), 72.2 (OCH.sub.2Pr), 71.8 (C-4.sub.D), 70.2 (C-3.sub.B), 70.1 (C-2.sub.A), 70.0 (C-4.sub.C), 69.6 (C-3.sub.D), 69.2 (C-5.sub.D), 69.1 (C-5.sub.C), 67.6 (C-4.sub.B), 60.8 (C-6.sub.B), 52.4 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 22.1 (CH.sub.2Pr), 20.5 (CH.sub.3Ac), 16.6, 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.6 (CH.sub.3Pr)

(41) HRMS (ESI.sup.+): m/z 796.2866 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 796.2852).

(42) HPLC (215 nm): R.sub.t=9.0 min.

(43) Characterization of Additional Propyl Tetrasaccharides Mimicking Frame-Shifted Repeating Units of SF6, SF6a and/or E. coli O147 OAgs, Obtained According to a Linear Synthetic Strategy.

Propyl -D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)--L-rhamnopyranosyl-(12)--L-rhamnopyranoside (XIX)

(44) .sup.1H NMR (D.sub.2O), 5.01 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.C), 4.76 (d, 1H, J.sub.1,2=1.2 Hz, H-1.sub.D), 4.55 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.36 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.23 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.12 (dd, 1H, J.sub.3,4=3.4 Hz, H-4.sub.A), 4.09 (d, 1H, J.sub.3,4=3.1 Hz, H-4.sub.B), 4.02 (dd, 1H, J.sub.2,3=2.8 Hz, H-2.sub.C), 3.89 (dd, 1H, J.sub.2,3=10.9 Hz, H-2.sub.B), 3.79 (dd, 1H, J.sub.2,3=3.4 Hz, H-2.sub.D), 3.77-3.69 (m, 3H, H-3.sub.B, H-3.sub.C, H-3.sub.D), 3.66 (dd, 1H, J.sub.5,6a=7.8 Hz, J.sub.6a,6b=11.9 Hz, H-6a.sub.B), 3.64-3.53 (m, 5H, H-6b.sub.B, H-5.sub.C, H-3.sub.A, H-5.sub.B, H-5.sub.D), 3.51 (dt.sub.po, 1H, J=7.0 Hz, J=9.8 Hz, OCH.sub.2Pr), 3.41 (dd.sub.po, 1H, J.sub.2,3=9.9 Hz, H-2.sub.A), 3.36 (dt.sub.po, 1H, J=6.3 Hz, OCH.sub.2Pr), 3.31 (pt.sub.po, 1H, J.sub.3,4=J.sub.4,5=9.6 Hz, H-4.sub.D), 3.22 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.C), 1.89 (s, 3H, CH.sub.3NHAc), 1.47 (psex, 2H, CH.sub.2Pr), 1.15 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.12 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 0.78 (t, 3H, J=7.4 Hz, CH.sub.3Pr)

(45) .sup.13C NMR (D.sub.2O), 174.9 (NHCO), 172.0 (C-6.sub.A), 104.6 (C-1.sub.A, .sup.1J.sub.CH=162.3 Hz), 102.7 (C-1.sub.B, .sup.1J.sub.CH=163.4 Hz), 101.1 (C-1.sub.C, .sup.1J.sub.CH=173.5 Hz), 98.1 (C-1.sub.D, .sup.1J.sub.CH=171.8 Hz), 79.9 (C-3.sub.B), 78.9 (C-2.sub.D), 78.4 (C-2.sub.C), 74.6 (C-5.sub.B), 73.7 (C-5.sub.A), 72.1 (C-4.sub.C), 72.0 (C-4.sub.D), 71.9 (C-3.sub.A), 69.9 (C-3.sub.D), 69.8 (C-2.sub.A), 69.7 (OCH.sub.2Pr), 69.6 (C-4.sub.A), 69.4 (C-3.sub.C), 69.1 (C-5.sub.C), 68.6 (C-5.sub.D), 67.6 (C-4.sub.B), 60.9 (C-6.sub.B), 51.5 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.8 (CH.sub.2Pr), 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.7 (CH.sub.3Pr).

(46) HRMS (ESI.sup.+): m/z 754.2789 (calcd for C.sub.29H.sub.49NO.sub.20Na [M+Na].sup.+: m/z 754.2745).

(47) HPLC (215 nm): R.sub.t=12.5 min.

Propyl -D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-3-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranoside (XII)

(48) .sup.1H NMR (D.sub.2O), 5.03 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.C), 4.93 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=10.0 Hz, H-3.sub.C), 4.80 (d, 1H, J.sub.1,2=1.1 Hz, H-1.sub.D), 4.40 (d.sub.po, 1H, J.sub.1,2=7.7 Hz, H-1.sub.A), 4.39 (d.sub.po, 1H, J.sub.1,2=8.2 Hz, H-1.sub.B), 4.23 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.15 (pt, 1H, H-2.sub.C), 4.13 (dd, 1H, J.sub.3,4=3.4 Hz, H-4.sub.A), 4.11 (d, 1H, J.sub.3,4=2.9 Hz, H-4.sub.B), 3.87 (dd.sub.po, 1H, J.sub.2,3=10.9 Hz, H-2.sub.B), 3.83 (dd, 1H, J.sub.2,3=3.3 Hz, H-2.sub.D), 3.79 (dd, 1H, H-3.sub.B), 3.75-3.68 (m, 2H, H-3.sub.D, H-5.sub.C), 3.68-3.56 (m, 4H, H-6a.sub.B, H-6b.sub.B, H-3.sub.A, H-5.sub.D), 3.56-3.47 (m, 2H, H-5.sub.B, OCH.sub.2Pr), 3.46-3.38 (m, 3H, H-2.sub.A, H-4.sub.D, H-4.sub.C), 3.37 (dt.sub.po, 1H, J=6.4 Hz, J=9.9 Hz, OCH.sub.2Pr), 2.07 (s, 3H, CH.sub.3Ac), 1.95 (s, 3H, CH.sub.3NHAc), 1.48 (psex, 2H, CH.sub.2Pr), 1.17 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D*), 1.16 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C*), 0.79 (t, 3H, J=7.4 Hz, CH.sub.3Pr)

(49) .sup.13C NMR (D.sub.2O), 174.7 (NHCO), 173.6 (CO.sub.Ac), 172.2 (C-6.sub.A), 104.6 (C-1.sub.A, .sup.1J.sub.CH=162.1 Hz), 102.7 (C-1.sub.B, .sup.1J.sub.CH=163.9 Hz), 101.1 (C-1.sub.C, .sup.1J.sub.CH=176.5 Hz), 98.0 (C-1.sub.D, .sup.1J.sub.CH=171.9 Hz), 79.4 (C-2.sub.D), 79.1 (C-3.sub.B), 76.3 (C-2.sub.C), 74.5 (C-5.sub.B), 73.9 (C-5.sub.A), 72.6 (C-3.sub.C), 72.0, 71.9 (2C, C-3.sub.A, C-4.sub.D), 69.9-69.8 (3C, C-4.sub.C, C-2.sub.A, C-3.sub.D), 69.7 (OCH.sub.2Pr), 69.5 (C-4.sub.A), 69.2 (C-5.sub.C), 68.6 (C-5.sub.D), 67.4 (C-4.sub.B), 60.8 (C-6.sub.B), 51.4 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.8 (CH.sub.2Pr), 20.5 (CH.sub.3Ac), 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.7 (CH.sub.3Pr).

(50) HRMS (ESI.sup.+): m/z 796.2831 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 796.2852).

(51) HPLC (215 nm): R.sub.t=13.5 min

Propyl -D-galactopyranosyl uronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-4-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranoside (XII)

(52) .sup.1H NMR (D.sub.2O), 5.06 (d, 1H, 1.sub.1,2=1.6 Hz, H-1.sub.C), 4.80 (d, 1H, J.sub.1,2=1.1 Hz, H-1.sub.D), 4.73 (pt, 1H, J.sub.4,5=9.8 Hz, H-4.sub.C), 4.63 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.39 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.20 (d, 1H, 1.sub.4,5=1.1 Hz, H-5.sub.A), 4.16-4.13 (m, 3H, H-2.sub.C, H-4.sub.A, H-4.sub.B), 4.00 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.9 Hz, H-3.sub.C), 3.94 (dd, 1H, J.sub.2,3=10.9 Hz, H-2.sub.B), 3.86 (dd, 1H, 1.sub.2,3=3.3 Hz, H-2.sub.D), 3.84-3.74 (m, 3H, H-3.sub.B, H-3.sub.D, H-5.sub.C), 3.72 (dd.sub.po, 1H, J.sub.5,6a=7.7 Hz, J.sub.6a,6b=12.0 Hz, H-6a.sub.B), 3.67 (dd.sub.po, 1H, 1.sub.5,6b=4.7 Hz, H-6b.sub.B), 3.64-3.56 (m, 3H, H-3.sub.A, H-5.sub.D, H-5.sub.B), 3.55 (pt.sub.po, 1H, J=6.9 Hz, J=9.8 Hz, OCH.sub.2Pr), 3.46 (dd, 1H, J.sub.2,3=10.0 Hz, H-2.sub.A), 3.40 (dt, 1H, J=6.3 Hz, OCH.sub.2Pr), 3.36 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.D), 2.06 (s, 3H, CH.sub.3Ac), 1.93 (s, 3H, CH.sub.3NHAc), 1.51 (psex, 2H, CH.sub.2Pr), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.06 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 0.82 (t, 3H, J=7.4 Hz, CH.sub.3Pr).

(53) .sup.13C NMR (D.sub.2O), 175.0 (NHCO), 173.7 (CO.sub.Ac), 172.6 (C-6.sub.A), 104.3 (C-1.sub.A, .sup.1J.sub.CH=162.6 Hz), 102.4 (C-1.sub.B, .sup.1J.sub.CH=164.1 Hz), 101.1 (C-1.sub.C, .sup.1J.sub.CH=175.0 Hz), 98.1 (C-1.sub.D, .sup.1J.sub.CH=171.4 Hz), 79.9 (C-3.sub.B), 79.1 (C-2.sub.D), 77.6 (C-2.sub.C), 74.8 (C-5.sub.B), 74.1 (2C, C-5.sub.A, C-4.sub.C), 72.2 (C-4.sub.D), 72.1 (C-3.sub.A), 70.0 (2C, C-2.sub.A, C-3.sub.D), 69.8 (OCH.sub.2Pr), 69.7 (C-4.sub.A), 68.6 (C-5.sub.D), 68.1 (C-3.sub.C), 67.7 (C-4.sub.B), 67.1 (C-5.sub.C), 61.0 (C-6.sub.B), 51.6 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 20.5 (CH.sub.3Ac), 16.6, 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.8 (CH.sub.3Pr).

(54) HRMS (ESI.sup.+): m/z 796.2927 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 786.2852).

(55) HPLC (215 nm): R.sub.t=17.3 min.

Propyl -L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)--L-rhamnopyranoside (XVI)

(56) .sup.1H NMR (D.sub.2O), 5.09 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.D), 4.90 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.C), 4.63 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.44 (d, 1H, J.sub.1,2=7.7 Hz, H-1.sub.A), 4.33 (d, 1H, J.sub.4,5=1.1 Hz, H-5.sub.A), 4.29 (dd, 1H, 1.sub.3,4=3.1 Hz, H-4.sub.A), 4.15 (d, 1H, J.sub.3,4=3.1 Hz, H-4.sub.B), 4.01 (dd, 1H, J.sub.2,3=3.4 Hz, H-2.sub.D), 3.96 (dd.sub.po, 1H, J.sub.2,3=10.8 Hz, H-2.sub.B), 3.94 (dd.sub.o, 1H, H-2.sub.C), 3.83-3.73 (m, 3H, H-3.sub.A, H-3.sub.B, H-3.sub.C), 3.73-3.67 (m, 3H, H-6a.sub.B, H-6b.sub.B, H-3.sub.D), 3.64-3.49 (m, 5H, H-5.sub.C, H-5.sub.B, OCH.sub.2Pr, H-5.sub.D, H-2.sub.A), 3.42 (dt, 1H, J=6.2 Hz, J=9.8 Hz, OCH.sub.2Pr), 3.33 (pt, 1H, J.sub.3,4=J.sub.4,5=9.8 Hz, H-4.sub.D), 3.27 (pt, 1H, J.sub.3,4=J.sub.4,5=9.6 Hz, H-4.sub.C), 1.96 (s, 3H, CH.sub.3NHAc), 1.53 (sex, 2H, CH.sub.2Pr), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 1.17 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 0.83 (t, 3H, J=7.5 Hz, CH.sub.3Pr).

(57) .sup.13C NMR (D.sub.2O), 175.0 (NHCO), 171.6 (C-6.sub.A), 104.5 (C-1.sub.A, .sup.1J.sub.CH=163.1 Hz), 102.7 (C-1.sub.B, .sup.1J.sub.CH=163.5 Hz), 101.5 (C-1.sub.D, .sup.1J.sub.CH=174.2 Hz), 98.5 (C-1.sub.C, .sup.1J.sub.CH=174.6 Hz), 80.3 (C-3.sub.B), 78.5 (C-2.sub.C), 76.3 (C-4.sub.A), 74.7 (C-5.sub.B), 72.9 (C-5.sub.A), 72.7 (C-3.sub.A), 70.0 (C-4.sub.C), 71.7 (C-4.sub.D), 72.3 (C-2.sub.D), 70.1 (C-3.sub.C), 69.6 (C-3.sub.D), 69.8 (C-2.sub.A), 69.7 (OCH.sub.2Pr), 69.2 (C-5.sub.D), 68.6 (C-5.sub.C), 67.7 (C-4.sub.B), 60.9 (C-6.sub.B), 51.6 (C-2.sub.B), 22.4 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 16.6, 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.8 (CH.sub.3Pr)

(58) HRMS (ESI.sup.+): m/z 754.2788 (calcd for C.sub.29H.sub.49NO.sub.20Na [M+Na].sup.+: m/z 754.2745).

(59) HPLC (215 nm): R.sub.t=10.5 min.

Propyl -L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-3-O-acetyl--L-rhamnopyranoside (XV)

(60) .sup.1H NMR (D.sub.2O), 5.03 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.D), 4.93 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4 10.0 Hz, H-3.sub.C), 4.84 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.C), 4.42 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.38 (d, 1H, J.sub.1,2=8.2 Hz, H-1.sub.B), 4.28 (d, 1H, J.sub.4,5=1.1 Hz, H-5.sub.A), 4.23 (dd, 1H, J.sub.3,4=2.8 Hz, H-4.sub.A), 4.10 (d, 1H, J.sub.3,4=2.9 Hz, H-4.sub.B), 4.00 (dd, 1H, H-2.sub.C), 3.96 (dd, 1H, J-.sub.2,3=3.3 Hz, H-2.sub.D), 3.88 (dd, 1H, J.sub.2,3=10.8 Hz, H-2.sub.B), 3.78 (dd, 1H, H-3.sub.B), 3.73 (dd, 1H, J.sub.2,3=10.0 Hz, H-3.sub.A), 3.69-3.59 (m, 4H, H-6a.sub.B, H-6b.sub.B, H-3.sub.D, OCH.sub.2Pr), 3.58-3.43 (m, 4H, H-5.sub.C, H-5.sub.B, H-5.sub.D, H-2.sub.A), 3.43-3.35 (m, 2H, OCH.sub.2Pr, H-4.sub.C), 3.27 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.D), 2.06 (s, 3H, CH.sub.3Ac), 1.94 (s, 3H, CH.sub.3NHAc), 1.48 (sex, 2H, CH.sub.2Pr), 1.17 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 1.11 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 0.78 (t, 3H, J=7.5 Hz, CH.sub.3Pr).

(61) .sup.13C NMR (D.sub.2O), 174.5 (NHCO), 173.4 (CO.sub.Ac), 171.6 (C-6.sub.A), 104.3 (C-1.sub.A, .sup.1J.sub.CH=163.6 Hz), 102.6 (C-1.sub.B, .sup.1J.sub.CH=165.7 Hz), 101.4 (C-1.sub.D, .sup.1J.sub.CH=174.6 Hz), 98.5 (C-1.sub.C, .sup.1J.sub.CH=175.3 Hz), 79.8 (C-3.sub.B), 76.4 (C-2.sub.C), 76.3 (C-4.sub.A), 74.4 (C-5.sub.B), 72.8 (2C, C-3.sub.A, C-3.sub.C), 72.6 (C-5.sub.A), 71.7 (C-4.sub.D), 70.2 (2C, C-4.sub.C, C-2.sub.D), 69.8 (C-2.sub.A), 69.7 (C-3.sub.D), 69.6 (OCH.sub.2Pr), 69.1 (C-5.sub.D), 68.6 (C-5.sub.C), 67.5 (C-4.sub.B), 60.8 (C-6.sub.B), 51.4 (C-2.sub.B), 22.5 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 20.5 (CH.sub.3Ac), 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.7 (CH.sub.3Pr).

(62) HRMS (ESI.sup.+): m/z 796.5854 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 796.2852).

(63) HPLC (215 nm): R.sub.t=12.7 min.

Propyl -L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-4-O-acetyl--L-rhamnopyranoside (XV)

(64) .sup.1H NMR (D.sub.2O), 5.09 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.D), 4.90 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.C), 4.73 (pt, 1H, J.sub.3,4=J.sub.3,4=9.8 Hz, H-4.sub.C), 4.66 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.44 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.30 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.28 (dd, 1H, J.sub.3,4=2.9 Hz, H-4.sub.A), 4.16 (d, 1H, J.sub.3,4=3.1 Hz, H-4.sub.B), 4.04-4.00 (m, 2H, H-2.sub.C, H-2.sub.D), 4.00-3.94 (m, 2H, H-3.sub.C, H-2.sub.B), 3.83-3.67 (m, 6H, H-3.sub.B, H-3.sub.A, H-6a.sub.B, H-6b.sub.B, H-3.sub.D, H-5.sub.C), 3.62-3.50 (m, 4H, OCH.sub.2Pr, H-5.sub.B, H-5.sub.D, H-2.sub.A), 3.43 (dt, 1H, J=6.4 Hz, J=9.7 Hz, OCH.sub.2Pr), 3.32 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.D), 2.07 (s, 3H, CH.sub.3Ac), 1.95 (s, 3H, CH.sub.3NHAc), 1.53 (sex, 2H, CH.sub.2Pr), 1.16 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.09 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 0.83 (t, 3H, J=7.3 Hz, CH.sub.3Pr).

(65) .sup.13C NMR (D.sub.2O), 175.1 (NHCO), 173.7 (CO.sub.Ac), 171.8 (C-6.sub.A), 104.5 (C-1.sub.A, .sup.1J.sub.CH=162.7 Hz), 102.3 (C-1.sub.B, .sup.1J.sub.CH=164.9 Hz), 101.4 (C-1.sub.D, .sup.1J.sub.CH=173.9 Hz), 98.7 (C-1.sub.C, .sup.1J.sub.CH=174.4 Hz), 80.3 (C-3.sub.B), 77.6 (C-2.sub.C), 76.2 (C-4.sub.A), 74.8 (C-5.sub.B), 74.3 (C-4.sub.C), 72.6 (C-5.sub.A), 72.8 (C-3.sub.A), 71.8 (C-4.sub.D), 70.2 (C-2.sub.D), 69.9 (C-3.sub.D), 69.8 (2C, OCH.sub.2Pr, C-2.sub.A), 69.2 (C-5.sub.D), 68.4 (C-3.sub.C), 67.8 (C-4.sub.B), 66.5 (C-5.sub.C), 61.0 (C-6.sub.B), 51.6 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 22.0 (CH.sub.2Pr), 20.5 (CH.sub.3Ac), 16.6, 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.8 (CH.sub.3Pr).

(66) HRMS (ESI.sup.+): m/z 796.2884 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 798.2852).

(67) HPLC (215 nm): =16.4 min.

Propyl -L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranoside (XXXV)

(68) .sup.1H NMR (D.sub.2O), 5.20 (d, 1H, J.sub.1,2=1.3 Hz, H-1.sub.D), 4.80 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.C), 4.36 (d, 2H, J.sub.1,2=8.0 Hz, H-1.sub.A, H-1.sub.B), 4.36 (d, 1H, J.sub.4,5=1.1 Hz, H-5.sub.A), 4.20 (dd, 1H, J.sub.3,4=2.8 Hz, H-4.sub.A), 4.08 (dd, 1H, J.sub.2,3=3.0 Hz, H-4.sub.B), 3.93 (dd, 1H, H-2.sub.D), 3.90 (dd, 1H, J.sub.2,3=3.4 Hz, H-2.sub.C), 3.86 (dd, 1H, J.sub.1,2=8.6 Hz, J.sub.2,3=10.8 Hz, H-2.sub.B), 3.72 (dd.sub.po, 1H, J.sub.2,3=3.3 Hz, H-3.sub.D), 3.71-3.63 (m, 5H, OCH.sub.2Pr, H-3.sub.A, H-3.sub.B, H-6a.sub.B, H-6b.sub.B), 3.61 (dd.sub.po, 1H, H-3.sub.C), 3.57-3.50 (m, 2H, H-5.sub.C, H-5.sub.B), 3.46 (dd.sub.po, 1H, J.sub.2,3=9.8 Hz, H-2.sub.A), 3.45-3.38 (m, 2H, OCH.sub.2Pr, H-5.sub.D), 3.29 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.D), 3.28 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.C), 1.86 (s, 3H, CH.sub.3NHAc), 1.40 (sex, 2H, CH.sub.2Pr), 1.10 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 1.09 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 0.72 (t, 3H, J=7.3 Hz, CH.sub.3Pr)

(69) .sup.13C NMR (D.sub.2O), 174.7 (NHCO), 171.5 (C-6.sub.A), 104.5 (C-1.sub.A, .sup.1J.sub.CH=164.1 Hz), 102.0 (C-1.sub.C, .sup.1J.sub.CH=172.0 Hz), 101.1 (C-1.sub.B, .sup.1J.sub.CH=161.8 Hz), 99.9 (C-1.sub.D, .sup.1H.sub.CH=175.7 Hz), 80.7 (C-3.sub.B), 78.2 (C-2.sub.D), 76.3 (C-4.sub.A), 74.6 (C-5.sub.B), 72.7 (C-3.sub.A), 72.5 (C-5.sub.A), 72.1 (OCH.sub.2Pr), 71.9, 71.7 (2C, C-4.sub.C, C-4.sub.D), 69.9 (2C, C-2.sub.C, C-3.sub.C), 69.7, 69.6 (2C, C-2.sub.A, C-3.sub.D), 69.2 (C-5.sub.D), 68.9 (C-5.sub.C), 67.7 (C-4.sub.B), 60.8 (C-6.sub.B), 51.2 (C-2.sub.B), 22.1 (CH.sub.3NHAc), 22.0 (CH.sub.2Pr), 16.6, 16.5 (2C, C-6.sub.D, C-6.sub.C), 9.5 (CH.sub.3Pr)

(70) HRMS (ESI.sup.+): m/z 754.2769 (calcd for C.sub.29H.sub.49NO.sub.20Na [M+Na].sup.+: m/z 754.2745).

(71) HPLC (215 nm): R.sub.t=8.9 min.

Propyl 3-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranoside (XXXVI)

(72) .sup.1H NMR (D.sub.2O), 5.32 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.D), 4.90 (dd.sub.po, 1H, J.sub.2,3=3.3 Hz, J.sub.3,4=9.9 Hz, H-3.sub.C), 4.90 (d.sub.o, 1H, H-1.sub.C), 4.44 (d, 1H, J.sub.1,2=8.6 Hz, H-1.sub.B), 4.43 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.34 (d, 1H, J.sub.4,5=1.1 Hz, H-5.sub.A), 4.28 (dd, 1H, J.sub.3,4=2.8 Hz, H-4.sub.A), 4.16 (d, 1H, J.sub.3,4=3.0 Hz, H-4.sub.B), 4.12 (dd, 1H, J.sub.1,2=1.9 Hz, H-2.sub.C), 4.02 (dd, 1H, J.sub.2,3=3.2 Hz, H-2.sub.D), 3.94 (dd, 1H, J.sub.2,3=10.8 Hz, H-2.sub.B), 3.80 (dd, 1H, H-3.sub.D), 3.79-3.68 (m, 5H, H-3.sub.A, H-3.sub.B, OCH.sub.2Pr, H-6a.sub.B, H-6b.sub.B), 3.61 (m, 1H, H-5.sub.B), 3.56 (pt.sub.o, 1H, J.sub.3,4=J.sub.4,5=9.9 Hz, H-4.sub.C), 3.58-3.51 (m, 3H, H-5.sub.C, H-5.sub.D, H-2.sub.A), 3.49 (dt, 1H, J=10.2 Hz, OCH.sub.2Pr), 3.41 (pt, 1H, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.D), 2.09 (s, 3H, CH.sub.3Ac), 1.94 (s, 3H, CH.sub.3NHAc), 1.48 (sex, 2H, CH.sub.2Pr), 1.22 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 1.19 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 0.79 (t, 3H, CH.sub.3Pr).

(73) .sup.13C NMR (D.sub.2O), 174.8 (NHCO), 173.6 (CO.sub.Ac), 171.6 (C-6.sub.A), 104.6 (C-1.sub.A, .sup.1J.sub.CH=163.6 Hz), 101.7 (C-1.sub.C, .sup.1J.sub.CH=172.6 Hz), 101.2 (C-1.sub.B, .sup.1J.sub.CH=163.6 Hz), 99.9 (C-1.sub.D, .sup.1J.sub.CH=173.5 Hz), 80.7 (C-3.sub.B), 78.7 (C-2.sub.D), 76.4 (C-4.sub.A), 74.7 (C-5.sub.B), 73.5 (C-3.sub.C), 72.9 (C-5.sub.A), 72.7 (C-3.sub.A), 72.3 (OCH.sub.2Pr), 71.8 (C-4.sub.D), 69.8 (C-4.sub.C), 69.7 (2C, C-3.sub.D, C-2.sub.A*), 69.3 (C-5.sub.C*), 69.0 (C-5.sub.D), 68.0 (C-2.sub.C), 67.8 (C-4.sub.B), 60.9 (C-6.sub.B), 51.3 (C-2.sub.B), 22.2 (CH.sub.3NHAc), 22.1 (CH.sub.2Pr), 20.4 (CH.sub.3Ac), 16.7, 16.6 (2C, C-6.sub.D, C-6.sub.C), 9.6 (CH.sub.3Pr)

(74) HRMS (ESI.sup.+): m/z 796.2878 (calcd for C.sub.31H.sub.51NO.sub.21Na [M+Na].sup.+: m/z 796.2852).

(75) HPLC (215 nm): R.sub.t=9.5 min.

(76) Characterization of Additional Propyl Pentasaccharides Mimicking Frame-Shifted Repeating Units of SF6, SF6a and/or E. coli O147 O-Ags, Obtained According to a Linear Synthetic Strategy.

Propyl 3-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-3-O-acetyl--L-rhamnopyranoside (XIX)

(77) .sup.1H NMR (D.sub.2O), 5.31 (d, 1H, J.sub.1,2=1.2 Hz, H-1.sub.D), 4.96 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.0 Hz, H-3.sub.C), 4.88 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.8 Hz, H-3.sub.C), 4.88-4.87 (m, 2H, H-1.sub.C, H-1.sub.C), 4.76 (d, 1H, J.sub.1,2=7.9 Hz, H-1.sub.A), 4.42 (d, 1H, J.sub.1,2=8.2 Hz, H-1.sub.B), 4.33 (d.sub.po, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.26 (dd.sub.o, 1H, H-4.sub.A), 4.14 (bd, 1H, J.sub.3,4=2.6 Hz, H-4.sub.B), 4.11 (dd, 1H, J.sub.1,2=1.9 Hz, H-2.sub.C), 4.03 (pt, 1H, H-2.sub.C), 4.01 (dd, 1H, J.sub.2,3=3.3 Hz, H-2.sub.D), 3.92 (bdd, 1H, J.sub.2,3=10.6 Hz, H-2.sub.B), 3.82 (dd, 1H, H-3.sub.B), 3.80-3.64 (m, 6H, H-3.sub.D, H-3.sub.A, H-6a.sub.B, H-6b.sub.B, H-5.sub.C, H-5.sub.C), 3.62-3.47 (m, 5H, H-5.sub.B, H-5.sub.D, OCH.sub.2Pr, H-2.sub.A, H-4.sub.C), 3.47-3.33 (m, 3H, OCH.sub.2Pr, H-4.sub.D, H-4.sub.C), 2.10 (s, 3H, CH.sub.3Ac-3C), 2.07 (s, 3H, CH.sub.3Ac-3C), 1.98 (s, 3H, CH.sub.3NHAc), 1.52 (psex, 2H, J=7.0 Hz, CH.sub.2Pr), 1.23-1.15 (m, 9H, H-6.sub.D, H-6.sub.C, H-6.sub.C), 0.83 (t, 3H, J=7.4 Hz, CH.sub.3Pr).

(78) .sup.13C NMR (D.sub.2O), 174.5 (NHCO), 173.6 (CO.sub.Ac-3C), 173.4 (CO.sub.Ac-3C), 171.6 (C-6.sub.A), 104.3 (C-1.sub.A), 102.6 (C-1.sub.B), 101.7 (C-1.sub.C), 99.9 (C-1.sub.D), 98.5 (C-1.sub.C), 79.8 (C-3.sub.B), 78.7 (C-2.sub.D), 76.4 (C-4.sub.A), 76.2 (C-2.sub.C), 74.5 (C-5.sub.B), 73.5 (C-3.sub.C), 72.9 (2C, C-3.sub.C, C-5.sub.A), 72.6 (C-3.sub.A), 71.8 (C-4.sub.D), 70.9 (C-4.sub.C), 69.8 (C-4.sub.C), 69.7-68.7 (6C, C-3.sub.D, OCH.sub.2Pr, C-2.sub.A, C-5.sub.C, C-5.sub.D, C-5.sub.C), 68.0 (C-2.sub.C), 67.5 (C-4.sub.B), 60.8 (C-6.sub.B), 51.5 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 20.5, 20.4 (2C, CH.sub.3Ac), 16.6-16.4 (3C, C-6.sub.D, C-6.sub.C, C-6.sub.C), 9.8 (CH.sub.3Pr).

Propyl 2-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-3-O-acetyl--L-rhamnopyranoside (XIX)

(79) .sup.1H NMR (D.sub.2O), 5.28 (d, 1H, J.sub.1,2=1.2 Hz, H-1.sub.D), 5.12 (dd, 1H, J.sub.1,2=1.7 Hz, J.sub.2,3=3,4 Hz, H-2.sub.C), 4.96 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=9.0 Hz, H-3.sub.C), 4.92 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.C), 4.88 (d.sub.o, 1H, H-1.sub.C), 4.75 (d, 1H, J.sub.1,2=7.9 Hz, H-1.sub.A), 4.42 (d, 1H, J.sub.1,2=8.2 Hz, H-1.sub.B), 4.33 (d.sub.po, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.26 (dd.sub.o, 1H, H-4.sub.A), 4.14 (bd, 1H, J.sub.3,4=2.6 Hz, H-4.sub.B), 4.03 (pt, 1H, H-2.sub.C), 4.01 (dd, 1H, J.sub.2,3=3.3 Hz, H-2.sub.D), 3.92 (bdd, 1H, J.sub.2,3=10.6 Hz, H-2.sub.B), 3.87 (dd, 1H, J.sub.3,4=9.8 Hz, H-3.sub.C), 3.82 (dd, 1H, H-3.sub.B), 3.80-3.64 (m, 6H, H-3.sub.D, H-3.sub.A, H-6a.sub.B, H-6b.sub.B, H-5.sub.C, H-5.sub.C), 3.62-3.47 (m, 4H, H-5.sub.B, H-5.sub.D, OCH.sub.2Pr, H-2.sub.A), 3.47-3.33 (m, 4H, OCH.sub.2Pr, H-4.sub.D, H-4.sub.C, H-4.sub.C), 2.10 (s, 3H, CH.sub.3Ac-3C), 2.06 (s, 3H, CH.sub.3Ac-2C), 1.98 (s, 3H, CH.sub.3NHAc), 1.52 (psex, 2H, J=7.0 Hz, CH.sub.2Pr), 1.23-1.15 (m, 9H, H-6.sub.D, H-6.sub.C, H-6.sub.C), 0.83 (t, 3H, J=7.4 Hz, CH.sub.3Pr).

(80) .sup.13C NMR (D.sub.2O), 174.5 (NHCO), 173.4 (CO.sub.Ac-3C), 173.1 (CO.sub.Ac-2C), 171.6 (C-6.sub.A), 104.3 (C-1.sub.A), 102.6 (C-1.sub.B), 99.9 (C-1.sub.D), 99.1 (C-1.sub.C), 98.5 (C-1.sub.C), 79.8 (C-3.sub.B), 78.8 (C-2.sub.D), 76.4 (C-4.sub.A), 76.2 (C-2.sub.C), 74.5 (C-5.sub.B), 72.9 (2C, C-3.sub.C, C-5.sub.A), 72.6 (C-3.sub.A), 72.3 (C-2.sub.C), 72.2 (C-4.sub.C), 71.8 (C-4.sub.D), 70.9 (C-4.sub.C), 69.7-68.7 (6C, C-3.sub.D, OCH.sub.2Pr, C-2.sub.A, C-5.sub.C, C-5.sub.D, C-5.sub.C), 68.5 (C-3.sub.C), 67.5 (C-4.sub.B), 60.8 (C-6.sub.B), 51.5 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 20.5, 20.3 (2C, CH.sub.3Ac), 16.6-16.4 (3C, C-6.sub.D, C-6.sub.C, C-6.sub.C), 9.8 (CH.sub.3Pr).

(81) HRMS (ESI.sup.+): m/z 984.3530 (calcd for C.sub.36H.sub.63NO.sub.26Na [M+Na].sup.+: m/z 984.3530).

(82) HPLC (215 nm): R.sub.t=13.2 min.

Propyl 4-O-acetyl--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)-3-O-acetyl--L-rhamnopyranoside (XIX)

(83) .sup.1H NMR (D.sub.2O), 5.24 (d, 1H, 1.sub.1,2=1.3 Hz, H-1.sub.D), 4.93 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.3,4=10.0 Hz, H-3.sub.C), 4.86 (d.sub.o, 1H, J.sub.1,2=1.7 Hz, H-1.sub.C), 4.85 (d.sub.o, 1H, J.sub.1,2=1.7 Hz, H-1.sub.C), 4.75 (t, 1H, 1.sub.3,4=J.sub.4,5=9.9 Hz, H-4.sub.C), 4.41 (d, 1H, J.sub.1,2=7.8 Hz, H-1.sub.A), 4.38 (d, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 4.21 (bs, 2H, H-4.sub.A, H-5.sub.A), 4.11 (d, 1H, J.sub.3,4=3.0 Hz, H-4.sub.B), 4.01 (dd, 1H, J.sub.2,3=2.7 Hz, H-2.sub.C), 3.97 (dd.sub.po, 1H, J.sub.2,3=3.5 Hz, H-2.sub.C), 3.96 (dd.sub.po, 1H, H-2.sub.D), 3.88 (dd.sub.po, 1H, J.sub.2,3=11.0 Hz, H-2.sub.B), 3.85 (dd.sub.po, 1H, J.sub.2,3=3.4 Hz, J.sub.3,4=9.9 Hz, H-3.sub.C), 3.79-3.69 (m, 4H, H-3.sub.A, H-3.sub.B, H-3.sub.D, H-5.sub.C), 3.68-3.62 (m, 3H, H-5.sub.C, H-6a.sub.B, H-6b.sub.B), 3.56-3.47 (m, 4H, H-5.sub.D, OCH.sub.2Pr, H-2.sub.A, H-5.sub.B), 3.40 (1, J.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.C), 3.39 (dt.sub.po, 1H, J=6.3 Hz, J=9.8 Hz, OCH.sub.2Pr), 3.41 (pt, 1H, J.sub.3,4=J.sub.4,5=9.8 Hz, H-4.sub.D), 2.06 (s, 3H, CH.sub.3Ac), 2.03 (s, 3H, CH.sub.3Ac), 1.94 (s, 3H, CH.sub.3NHAc), 1.49 (sex, 2H, J=7.1 Hz, CH.sub.2Pr), 1.17 (d, 3H, J.sub.5,6=6.3 Hz, H-6.sub.C), 1.19 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.03 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 0.79 (t, 3H, J=7.4 Hz, CH.sub.3Pr),

(84) .sup.13C NMR (D.sub.2O), 174.5 (NHCO), 173.7, 173.4 (2C, CO.sub.AC), 171.6 (C-6.sub.A), 104.3 (C-1.sub.A), 102.5 (C-1.sub.B,), 102.4 (C-1.sub.C), 99.7 (C-1.sub.D), 99.3 (C-1.sub.C), 79.8 (C-3.sub.B), 78.6 (C-2.sub.D), 76.2 (2C, C-2.sub.C, C-4.sub.A), 74.6 (C-5.sub.B), 73.8 (C-4.sub.C), 73.2 (C-5.sub.A), 73.7 (2C, C-3.sub.C, C-3.sub.A), 71.8 (C-4.sub.D), 69.9-69.2 (5C, C, C-2.sub.C, C-4.sub.C, C-2.sub.A, C-3.sub.D, OCH.sub.2Pr), 68.4 (C-5.sub.D), 68.1 (C-5.sub.C), 67.5 (C-3.sub.C), 66.8 (2C, C-4.sub.B, C-5.sub.C), 60.7 (C-6.sub.B), 51.2 (C-2.sub.B), 22.3 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 20.5, 20.4 (2C, CH.sub.3Ac), 16.5, 16.4 (3C, C-6.sub.D, C-6.sub.C, C-6.sub.C), 9.7 (CH.sub.3Pr).

Propyl -L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyluronic acid-(13)-2-acetamido-2-deoxy--D-galactopyranosyl-(12)--L-rhamnopyranoside (XX)

(85) .sup.1H NMR (D.sub.2O), 5.28 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.D), 4.90 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.C), 4.88 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.C), 4.63 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 4.45 (d, 1H, J.sub.1,2=7.9 Hz, H-1.sub.A), 4.34 (d, 1H, J.sub.4,5=1.1 Hz, H-5.sub.A), 4.28 (dd, 1H, J.sub.3,4=2.9 Hz, H-4.sub.A), 4.16 (d, 1H, J.sub.3,4=3.2 Hz, H-4.sub.B), 4.01 (dd, 1H, J.sub.2,3=3.2 Hz, H-2.sub.D), 4.00-3.93 (m, 3H, H-2.sub.B, H-2.sub.C, H-2.sub.C), 3.83-3.67 (m, 7H, H-3.sub.A, H-3.sub.B, H-3.sub.C, H-3.sub.C, H-6a.sub.B, H-6b.sub.B, H-3.sub.D), 3.66-3.49 (m, 6H, H-5.sub.C, H-5.sub.C, H-5.sub.B, OCH.sub.2Pr, H-5.sub.D, H-2.sub.A), 3.43 (dt, 1H, J=6.3 Hz, J=9.8 Hz, OCH.sub.2Pr), 3.37 (pt, 1H, J.sub.3,4=J.sub.4,5=9.8 Hz, H-4.sub.D), 3.36 (pt, 1H, 1.sub.3,4=J.sub.4,5=9.7 Hz, H-4.sub.C), 3.27 (pt, 1H, 1.sub.3,4=J.sub.4,5=9.6 Hz, H-4.sub.C), 1.96 (s, 3H, CH.sub.3NHAc), 1.53 (psex, 2H, CH.sub.2Pr), 1.21-1.16 (m, 9H, H-6.sub.C, H-6.sub.C, H-6.sub.D), 0.83 (t, 3H, J=7.5 Hz, CH.sub.3Pr)

(86) .sup.13C NMR (D.sub.2O), 175.0 (NHCO), 171.6 (C-6.sub.A), 104.5 (C-1.sub.A, .sup.1J.sub.CH=163.1 Hz), 102.7 (C-1.sub.B, .sup.1J.sub.CH=163.1 Hz), 102.1 (C-1.sub.C, .sup.1J.sub.CH=171.3 Hz), 100.0 (C-1.sub.D, .sup.1J.sub.CH=175.3 Hz), 98.5 (C-1.sub.C, .sup.1J.sub.CH=174.0 Hz), 80.4 (C-3.sub.B), 78.5 (C-2.sub.C), 78.3 (C-2.sub.D), 76.3 (C-4.sub.A), 74.7 (C-5.sub.B), 72.9 (C-5.sub.A), 72.6 (C-3.sub.A), 72.3 (C-4.sub.C), 72.1, 71.9 (2C, C-4.sub.D, C-4.sub.C), 70.1 (C-2.sub.C), 70.0 (2C, C-3.sub.C, C-3.sub.C), 69.8 (C-2.sub.A), 69.7 (C-3.sub.D), 69.6 (OCH.sub.2Pr), 69.3 (C-5.sub.D), 69.0 (C-5.sub.C), 68.6 (C-5.sub.C), 67.7 (C-4.sub.B), 60.9 (C-6.sub.B), 51.6 (C-2.sub.B), 22.4 (CH.sub.3NHAc), 21.9 (CH.sub.2Pr), 16.7-16.5 (3C, C-6.sub.D, C-6.sub.C, C-6.sub.C), 9.8 (CH.sub.3Pr).

(87) HRMS (ESI.sup.+): m/z 900.3400 (calcd for C.sub.35H.sub.59NO.sub.24Na [M+Na].sup.+: m/z 900.3325).

(88) HPLC (215 nm): R.sub.t=10.7 min.

(89) B. Convergent Synthetic Strategy

(90) Convergent synthesis stands for a process whereby chain elongation engaged to obtain the oligo- or polysaccharide target involves pre-defined building blocks comprisingat least in partmore than one residue. Building blocks of interest include acceptors (bearing the reducing endchain residue), donors (bearing the non reducing endchain residue), or donors acting as potential acceptors (designed to allow selective unmasking of the hydroxyl group involved in the next glycosylation step), which are often used repeatedly in the synthesis of targets larger than 1 repeating unit, preferentially more than 2 repeating unit. Selected building blocks are combined in a predetermined order. Convergent strategies are advantageously used on one hand to reach oligo- and polysaccharide targets comprising more than one repeating unit and on the other hand to avoid difficult glycosylations at an advanced stage.

(91) The need for highly convergent routes to synthetic oligo- and polysaccharides mimicking large fragments of the OAg of SF6, SF6a and/or E. coli O147 (up to 10 repeating units), led us to design a set of building blocks (see Table I), the combination of which provides an accessin theoryto the required targets. Advantageously, the selected building blocks take into account the diversity of both endchain residues and chain length within the OAg fragments of interest. In the search for efficiency, they were designed according to a disconnection at the B-C linkage.

Synthesis of Selected Building Blocks Shown in Table I from Key Intermediates

(92) One major achievement is that all selected building blocks derive from common precursors designed on purpose. In particular, disaccharide (HO).sub.2-AB (241) and rhamnosyl donors protected at position 3 in the form of a para-methoxybenzyl ether (43, 119, 166) were identified as important intermediates.

(93) Disaccharide AB as a template: as seen in scheme 6, upon appropriate tuning encompassing the controlled oxidation/benzylation of its free primary hydroxyl group, the key diol 241 is converted to the HO-AB acceptor 242. On the one hand, masking the 4.sub.A-OH of the later followed by appropriate anomeric deallylation/activation of the fully protected intermediate 244, provided the useful AB-TCA and AB-PTFA donors (246, 247), via hemiacetal 245. On the other hand, acceptor 242 was efficiently glycosylated at position 4.sub.A, to give trisacharide 277, which was converted to the corresponding DAB-PTFA donor (283). Selective delevulinoylation of the fully protected 277, and subsequent glycosylation at the 2.sub.D-OH gave tetrasaccharide 279, which was advantageously converted to the HO-CDAB acceptor (280) and to the CDAB-PTFA donor (284), following appropriate protecting group manipulation.

(94) ##STR00046## ##STR00047##

(95) Rhamnosyl donors C as major intermediates: as seen in scheme 7, when reacted with the known allyl 3,4-di-O-benzyl--L-rhamnoside acceptor (D),.sup.[29] the trichloroacetimidate donor 43 gave the fully protected rhamnobioside 47, which was easily turned into the corresponding acceptor 48, upon selective unmasking of OH-2.sub.C under transesterification conditions. Alternatively, when an appropriate HO-DA acceptor is used instead of the above mentioned D rhamnoside, for coupling to the C-TCA (119) or C-PTFA (166) donors, the fully protected CDA trisaccharide is isolated in yields above 90%, and next may be easily converted to the required HO-CDA acceptor (169), upon controlled hydrazinolysis of the levulinoyl ester at position 2.sub.C in a buffered medium. In a similar fashion, changing the above mentioned HO-DA acceptor for a suitable HO-DAB acceptor in the glycosylation reaction, allowed chain elongation at the later to give the fully protected CDAB tetrasaccharide (279), which was advantageously converted either to the HO-CDAB acceptor (280) or to the CDAB-PTFA donor (284), as already mentioned.

(96) ##STR00048## ##STR00049##

Synthetic Protocols and Characterization of Key Intermediates and all Novel Building Blocks Listed in Table 1

Phenyl 3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido-1-thio--D-galactopyranoside (24)

(97) A solution of thioglycoside 21.sup.[30] (5.00 g, 9.21 mmol) in anhyd. MeOH (100 mL) was treated by MeONa (0.5 Min MeOH, 311 L, 1.44 mmol). The solution was stirred for 2 h at rt under an Ar atmosphere. The reaction was quenched with Dowex-H.sup.+ resin. The resin was filtered and the filtrate was evaporated. Benzyl bromide (6.6 mL, 55.3 mmol, 6.0 equiv.) and NaH (60% in oil, 2.21 g, 55.3 mmol, 6.0 equiv.) were successively added to a solution of the crude triol in anhyd. DMF (74 mL) cooled to 10 C. The mixture was stirred under an Ar atmosphere keeping the temperature below 0 C. After 1.5 h, the reaction was quenched with the minimum amount of MeOH. The reaction mixture was diluted with EtOAc, and washed with water and brine. The organic layer was dried over anhyd. Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by flash chromatography (Tol/EtOAc 98:2 to 95:5) to give the target tri-O-benzyl derivative (24, 4.90 g, 77%), as a white solid.

(98) .sup.1H NMR (CDCl.sub.3), 7.56-7.22 (m, 2H, H.sub.Ar), 7.40-7.20 (m, 18H, H.sub.Ar), 6.84 (d, 1H, J.sub.NH,2=7.5 Hz, NH), 5.30 (d, 1H, J.sub.1,2=10.2 Hz, H-1), 4.90 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.68 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.58 (d, 1H, H.sub.Bn), 4.54 (d, 1H, H.sub.Bn), 4.53 (d, 1H, J 11.8 Hz, H.sub.Bn), 4.48 (d, 1H, H.sub.Bn), 4.28 (dd, 1H, J.sub.2,3=10.5 Hz, J.sub.3,4=2.7 Hz, H-3), 4.09 (d, 1H, H-4), 3.94 (pdt, 1H, H-2), 3.78-3.68 (m, 3H, H-5, H-6a, H-6b).

(99) .sup.13C NMR (CDCl.sub.3), 161.7 (NHCO), 138.4, 137.8, 137.3 (3C, C.sub.IVAr), 132.8 (2C, C.sub.Ar), 132.2 (C.sub.IVAr), 130.0-127.6 (18C, C.sub.Ar), 92.5 (CCl.sub.3), 84.3 (C-1), 78.2 (C-3), 77.6 (C-5), 74.5, 73.6 (2C, C.sub.Bn), 72.4 (C-4), 72.3 (CBn), 68.4 (C-6), 53.7 (C-2).

(100) HRMS (ESI.sup.+): m/z 708.1128 (calcd for C.sub.35H.sub.34Cl.sub.3NO.sub.5SNa [M+Na].sup.+: m/z 708.1121).

3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-/-D-galactopyranosyl N-phenyltrifluoroacetimidate (181)

(101) The hemiacetal (1.85 g, 3.11 mmol), obtained from the thioglycoside 24, was dissolved in acetone (31 mL). N-(phenyl)trifluoroacetimidoyl chloride (1.29 g, 6.22 mmol) and Cs.sub.2CO.sub.3 (1.12 g, 3.42 mmol, 1.1 equiv.) were added to the solution. The mixture was stirred for 4 h at rt. The reaction mixture was filtered and concentrated. The residue was purified by flash chromatography (Chex/EtOAc 85:15 to 7:3+1% Et.sub.3N) to give N-phenyltrifluoroacetimidate 181 (2.10 g, 88%) as a light yellow oil.

(102) .sup.1H NMR (CDCl.sub.3), 7.61-7.09 (m, 18H, H.sub.Ar), 6.77 (d, 2H, J=7.6 Hz, H.sub.Ar), 6.48 (d.sub.o, 1H, J.sub.NH,2=7.7 Hz, NH), 6.47 (bs.sub.o, 1H, H-1), 4.97 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.76 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.73 (m, 1H, H-2), 4.64 (d, 1H, H.sub.Bn), 4.57-4.48 (m, 3H, H.sub.Bn), 4.20 (bs, 1H, H-4), 4.07 (pt, 1H, H-5), 3.88 (dd, 1H, J.sub.2,3=11.0 Hz, J.sub.3,4=1.4 Hz, H-3), 3.72 (pt, 1H, J.sub.5,6a=7.8 Hz, H-6a), 3.62 (dd, 1H, J.sub.5,6b=5.7 Hz, J.sub.6a,6b=9.1 Hz, H-6b).

(103) .sup.13C NMR (CDCl.sub.3), 161.8 (NHCO), 143.1, 138.0, 137.7, 137.0 (4C, C.sub.IVAr), 129.4-119.3 (20C, C.sub.Ar), 94.3 (C-1, .sup.1J.sub.CH=162.9 Hz), 92.3 (CCl.sub.3), 75.5 (C-3), 74.8, 73.7 (2C, C.sub.B), 72.5 (C-5), 71.6 (C-4), 71.3 (C.sub.Bn), 68.1 (C-6), 50.6 (C-2).

(104) Allyl 4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranoside (9)..sup.[26] Bu.sub.2SnO (37.2 g, 149.5 mmol, 1.1 equiv.) was added to a solution of the known allyl 4-O-benzyl--L-rhamnopyranoside.sup.[31] (40.0 g, 135.8 mmol) in anhyd. toluene (1.1 L). The mixture was stirred for 5 h at reflux using a Dean-Stark apparatus and subsequently concentrated under reduced pressure to give a 0.4 M solution. After cooling to rt, dry CsF (21.05 g, 138.6 mmol, 1.0 equiv.), dry TBAI (65.25 g, 175.6 mmol, 1.3 equiv.) and PMBCl (20.3 mL, 149.5 mmol, 1.1 equiv.) were successively added. The reaction mixture was stirred for 5 h at reflux, at which time monitoring by TLC (Chex/EtOAc 6:4) showed the conversion of the starting material (rf=0.22) into a major less polar product (rf=0.51). The temperature was lowered to 0 C., salts were removed by filtration (toluene wash) and volatiles were evaporated under reduced pressure. The residue was purified by flash chromatography (Chex/EtOAc 9:1 to 7:3) to give acceptor 9 (48.1 g, 81%) as an orange-yellow oil. Analytical data were as deseribed..sup.[26]

2-O-Benzoyl-4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl trichloroacetimidate (43)

(105) The hemiacetal (7.89 g, 16.49 mmol) obtained from the known allyl glycoside 9,.sup.[26] benzoylated at position 2, was dissolved in DCE (25 mL). Trichloroacetonitrile (6.6 mL, 66.0 mmol) and DBU (616 L, 4.12 mmol) were added, and the solution was stirred under an Ar atmosphere at rt for 30 min. The mixture was directly purified by flash chromatography (Chex/EtOAc 8:2+1% Et.sub.3N) to give donor 43 (9.71 g, 95%) as a yellow oil.

(106) .sup.1H NMR (CDCl.sub.3), 8.70 (s, 1H, NH), 8.13-6.81 (m, 14H, CH.sub.Ar), 6.33 (d, 1H, J.sub.1,2=1.9 Hz, H-1), 5.73 (dd, 1H, H-2), 4.95 (d, 1H, J=11.1 Hz, H.sub.Bn), 4.76 (d, 1H, J=11.1 Hz, H.sub.PMB), 4.68 (d, 1H, H.sub.Bn), 4.57 (d, 1H, H.sub.PMB), 4.11 (dd, 1H, J.sub.2,3=3.3 Hz, J.sub.3,4=9.6 Hz, H-3), 4.03 (dq, 1H, J.sub.4,5=9.6 Hz, H-5), 3.80 (s, 3H, CH.sub.3PMB), 3.64 (pt, 1H, H-4), 1.41 (d, 3H, J.sub.5,6=6.0 Hz, H-6).

(107) .sup.13C NMR (CDCl.sub.3), 165.8 (CO), 160.2 (CNH), 159.4 (C.sub.IVPMB), 138.2 (C.sub.IVBn), 133.3 (C.sub.Bz), 130.0 (C.sub.IVPMB), 129.9, 129.7, 128.5, 128.4, 128.3, 127.8, (12C, 11C.sub.Ar, C.sub.IVBz), 113.8 (2C, C.sub.ArPMB), 95.4 (C-1), 90.9 (CCl.sub.3), 79.3 (C-4), 77.0 (C-3), 75.5 (C.sub.Bn), 71.4 (C.sub.PMB), 70.8 (C-5), 68.2 (C-2), 55.2 (CH.sub.3PMB), 18.2 (C-6).

Allyl (2-O-benzoyl-4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-3,4-di-O-benzyl--L-rhamnopyranoside (47)

(108) A mixture of the known allyl 2,4-di-O-benzyl--L-rhamnoside acceptor.sup.[28] (3.00 g, 7.81 mmol), donor 43 (5.84 g, 9.37 mmol), and powdered MS 4 (6.0 g) in anhyd. toluene (78 mL) was stirred at rt under an Ar atmosphere for 1 h. The suspension was cooled to 40 C., and TMSOTf (71 L, 0.39 mmol) was added. The reaction mixture was stirred for 20 min at this temperature and Et.sub.3N was added to quench the reaction. The suspension was filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (Chex/EtOAc 9:1) to give disaccharide 47 (6.30 g, 95%) as a yellow oil.

(109) .sup.1H NMR (CDCl.sub.3), 8.13-8.08 (m, 2H, H.sub.Ar), 7.62 (m, 1H, H.sub.Ar), 7.53-7.47 (m, 2H, H.sub.Ar), 7.37-7.15 (m, 17H, H.sub.Ar), 6.83-6.78 (m, 2H, H.sub.ArPMB), 5.89 (m, 1H, CH.sub.All), 5.77 (dd, 1H, H-2.sub.C), 5.27 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.20 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2All), 5.14 (d, 1H, J.sub.1,2=1.8 Hz, H-1.sub.C), 4.93 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.92 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.80 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.D), 4.78 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.71 (bs, 2H, H.sub.Bn), 4.67 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.63 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.54 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.15 (m, 1H, H.sub.All), 4.08 (dd, 1H, J.sub.2,3=3.2 Hz, J.sub.3,4=9.3 Hz, H-3.sub.C), 4.05 (dd, 1H, J.sub.2,3=2.9 Hz, H-2.sub.D), 3.99-3.86 (m, 3H, H.sub.All, H-3.sub.D, H-5.sub.C), 3.76 (s, 3H, CH.sub.3PMB), 3.73 (dq, 1H, J.sub.4,5=9.2 Hz, J.sub.5,6=6.2 Hz, H-5.sub.D), 3.54 (pt, 1H, J.sub.4,5=9.4 Hz, H-4.sub.C), 3.48 (pt, 1H, J.sub.3,4=9.4 Hz, H-4.sub.D), 1.36 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C), 1.32 (d, 3H, H-6.sub.D).

(110) .sup.13C NMR (CDCl.sub.3) partial, 165.5 (CO.sub.Bz), 159.2 (C.sub.IVPMB), 113.7 (2C, C.sub.arPMB), 99.4 (C-1.sub.C, .sup.1J.sub.CH=171.0 Hz), 97.9 (C-1.sub.D, .sup.1J.sub.CH=170.0 Hz), 55.2 (CH.sub.3PMB).

(111) HRMS (ESI.sup.+): m/z 867.3601 (calcd for C.sub.51H.sub.56O.sub.11Na [M+Na].sup.+: m/z 867.3720).

Allyl (4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-3,4-di-O-benzyl--L-rhamnopyranoside (48)

(112) A solution of disaccharide 47 (6.27 g, 7.42 mmol) in anhyd. MeOH (74 mL) was treated with 0.5 M methanolic MeONa (7.4 mL, 3.7 mmol) at 60 C. The mixture was stirred at this temperature under an Ar atmosphere for 3 h, then at rt for 16 h. The reaction mixture was neutralized by addition of Dowex-H.sup.+ resin. The suspension was filtered and volatiles were evaporated. The residue was purified by flash chromatography (Tol/EtOAc 8:2) to give acceptor 48 (5.2 g, 94%) as a white foam.

(113) .sup.1H NMR (CDCl.sub.3), 7.39-7.27 (m, 17H, H.sub.Ar), 6.91-6.87 (m, 2H, H.sub.ArPMB), 5.88 (m, 1H, CH.sub.All), 5.27 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.19 (m, 1H, J.sub.cis=10.4 Hz, CH.sub.2All), 5.09 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.C), 4.89 (d, 2H, J=11.0 Hz, H.sub.Bn), 4.78 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.D), 4.71 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.67 (bs.sub.o, 2H, H.sub.Bn), 4.66 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.65 (d.sub.po, 1H, J=11.3 Hz, H.sub.Bn), 4.63 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.17-4.10 (m, 2H, H.sub.All, H-2.sub.C), 4.05 (dd, 1H, J.sub.2,3=2.9 Hz, H-2.sub.D), 3.98-3.86 (m, 3H, H-3.sub.C, H.sub.All, H-3.sub.D), 3.82 (dq, 1H, J.sub.4,5=9.6 Hz, J.sub.5,6=6.2 Hz, H-5.sub.C), 3.80 (s, 3H, CH.sub.3PMB), 3.71 (dq, 1H, J.sub.4,5=9.4 Hz, J.sub.5,6=6.2 Hz, H-5.sub.D), 3.46 (pt.sub.po, 1H, J.sub.3,4=9.3 Hz, H-4.sub.C), 3.41 (pt, 1H, J.sub.3,4=9.7 Hz, H-4.sub.D), 2.46 (d, 1H, J.sub.OH,2=1.8 Hz, OH-2.sub.C), 1.32 (d, 3H, H-6.sub.D), 1.31 (d, 3H, H-6.sub.C).

(114) .sup.13C NMR (CDCl.sub.3) partial, 159.4 (C.sub.IVPMB), 114.0 (2C, C.sub.arPMB), 100.7 (C-1.sub.C, .sup.1J.sub.CH=171.7 Hz), 98.0 (C-1.sub.D, .sup.1J.sub.CH=170.1 Hz), 55.2 (CH.sub.3PMB).

(115) HRMS (ESI.sup.+): m/z 763.3458 (calcd for C.sub.44H.sub.52O.sub.10Na [M+Na].sup.+: m/z 763.3449).

4-O-Benzyl-2-O-levulinoyl-3-O-para-methoxybenzyl-/-L-rhamnopyranosyl trichloroacetimidate (119)

(116) The hemiacetal (10.0 g, 21.16 mmol) obtained from the known allyl glycoside 9,.sup.[26] levulinoylated at position 2. was dissolved in anhyd. DCE (42 mL) and stirred under an Ar atmosphere. Trichloroacetonitrile (10.6 mL, 105.8 mmol) and DBU (0.95 mL, 6.3 mmol) were added at rt. The reaction mixture was purified as such by flash chromatography (Chex/EtOAc 7:3 to 1:1+1% Et.sub.3N) to give a 7.5:1 / mixture of trichloroacetimidate 119 (12.66 g, 97%) as a light yellow oil (a/3 ratio 7.5:1).

(117) .sup.1H NMR (CDCl.sub.3), 8.67 (s, 1H, NH), 7.41-7.26 (m, 7H, H.sub.Ar), 6.89-6.84 (m, 2H, 8.7 Hz, H.sub.ArPMB), 6.19 (d, 1H, J.sub.1,2=1.9 Hz, H-1), 5.47 (dd, 1H, J.sub.2,3=3.3 Hz, H-2), 4.94 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.66 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.65 (d, 1H, H.sub.Bn), 4.51 (d, 1H, H.sub.Bn), 3.98 (dd, 1H, J.sub.3,4=9.6 Hz, H-3), 3.94 (dq, 1H, J.sub.4,5=9.4 Hz, H-5), 3.82 (s, 3H, CH.sub.3PMB), 3.50 (pt, 1H, H-4), 2.84-2.72 (m, 4H, CH.sub.2Lev), 2.22 (s, 3H, CH.sub.3LeV), 1.36 (d, 3H, J.sub.5,6=6.2 Hz, H-6).

(118) .sup.13C NMR (CDCl.sub.3), 206.3 (CO.sub.Lev), 171.9 (CO.sub.2Lev), 160.1 (CNH), 159.4 (C.sub.IVArPMB), 138.1 (C.sub.IVar), 130.0-127.9 (8C, 7C.sub.Ar, C.sub.IVArPMB),), 113.8 (2C, C.sub.ArPMB), 95.1 (C-1), 90.8 (CCl.sub.3), 79.2 (C-4), 76.6 (C-3), 75.6 (C.sub.Bn), 71.5 (C.sub.PMB), 70.7 (C-5), 67.8 (C-2), 55.3 (CH.sub.3PMB), 38.0 (COCH.sub.2Lev), 29.9 (CH.sub.3Lev), 28.1 (CO.sub.2CH.sub.2Lev), 18.0 (C-6).

4-O-Benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl-/-L-rhamnopyranosyl N-phenyltrifluoroacetimidate (166)

(119) The hemiacetal (5.0 g, 10.58 mmol) obtained from the known allyl glycoside 9,.sup.[26] levulinoylated at position 2, was dissolved in acetone (36 mL). N-(phenyl)trifluoroacetimidoyl chloride (4.39 g, 21.2 mmol) and Cs.sub.2CO.sub.3 (3.79 g, 11.6 mmol) were added to the solution. The mixture was stirred for 4 h at rt. The reaction mixture was filtered and concentrated. The residue was purified by flash chromatography (Chex/EtOAc 9:1 to 7:3+1% Et.sub.3N) to give a 5:1 / mixture of N-phenyltrifluoroacetimidate 166 (6.70 g, 98%) as a light yellow oil.

(120) .sup.1H NMR (CDCl.sub.3), 7.42-6.81 (m, 14H, H.sub.Ar), 6.17 (bs, 1H, H-1), 5.46 (bs, 1H, H-2), 4.94 (d, 1H, J=10.6 Hz, H.sub.Bn), 4.70-4.62 (m, 2H, H.sub.Bn), 4.53 (d, 1H, J=10.6 Hz, H.sub.Bn), 3.98 (dd, 1H, J.sub.2,3=3.3 Hz, J.sub.3,4=9.6 Hz, H-3), 3.90 (dq, 1H, J.sub.4,5=9.1 Hz, J.sub.5,6=6.1 Hz, H-5), 3.83 (s, 3H, CH.sub.3PMB), 3.49 (pt, 1H, H-4), 2.82-2.69 (m, 4H, CH.sub.2Lev), 2.21 (s, 3H, CH.sub.3Lev) 1.32 (d, 3H, H-6).

(121) .sup.13C NMR (CDCl.sub.3, partial), 206.2 (CO.sub.Lev), 171.8 (CO.sub.2Lev), 159.4 (C.sub.IVArPMB), 143.3, 138.1 (2C, C.sub.IVAr), 130.0-119.4 (13C, 12C.sub.Ar, C.sub.IVArPMB), 113.8 (2C, C.sub.ArPMB), 93.9 (C-1), 79.2 (C-4), 76.9 (C-3), 75.6 (C.sub.Bn), 71.7 (C.sub.PMB), 70.4 (C-5), 67.8 (C-2), 55.3 (CH.sub.3PMB), 38.0 (COCH.sub.2Lev), 29.9 (CH.sub.3Lev), 28.0 (CO.sub.2CH.sub.2Lev), 18.0 (C-6).

Allyl (benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (242)

(122) TEMPO (171 mg, 1.10 mmol) and BAIB (4.41 g, 13.69 mmol) were added to a solution of diol 241 (4.86 g, 5.48 mmol) in DCM/H.sub.2O (2:1, 99 mL) vigorously stirred at rt. After 1.5 h, the reaction was quenched by addition of a 10% aq. NaHSO.sub.3 solution. The biphasic mixture was diluted with DCM, and the layers were separated. The aq. phase was acidified with a 10% aq. HCl solution and re-extracted twice with DCM. The combined organic extracts were washed with brine, dried by passing through a phase separator filter and concentrated. Benzyl bromide (2.4 mL, 21.9 mmol, 4.0 equiv.) and KHCO.sub.3 (2.12 g, 21.9 mmol, 4.0 equiv.) were added to the crude intermediate in dry DMF (82 mL) and the reaction mixture was stirred under an Ar atmosphere for 16 h. The reaction mixture was diluted with water and EtOAc. The layers were separated and the organic phase was washed with brine, dried over anhyd. Na.sub.2SO.sub.4, filtered and evaporated. The residue was purified by flash chromatography (Tol/EtOAc 9:1 to 85:15) to give the benzyl uronate 242 (4.25 g, 78%) as a white solid.

(123) .sup.1H NMR (CDCl.sub.3), 7.44-7.24 (m, 25H, H.sub.Ar), 6.86 (d, 1H, J.sub.NH,2=6.7 Hz, NH), 5.88 (m, 1H, CH.sub.All), 5.27 (m, 1H, J.sub.trans=17.3 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.26 (bs, 2H, H.sub.CO2Bn), 5.18 (m, 1H, J.sub.cis=10.4 Hz, CH.sub.2All), 5.05 (d, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 5.01 (d, 1H, J=11.8 Hz, H.sub.Bn), 4.79 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.74 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.73 (bs.sub.o, 2H, H.sub.Bn), 4.71 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.68 (dd.sub.po, 1H, J.sub.2,3=11.1 Hz, J.sub.3,4=3.1 Hz, H-3.sub.B), 4.52 (d.sub.po, 1H, J=11.9 Hz, H.sub.Bn), 4.49 (d.sub.po, 1H, J.sub.1,2=7.7 Hz, H-1.sub.A), 4.46 (d, 1H, J=11.9 Hz, H.sub.Bn), 4.39-4.33 (m, 2H, H.sub.All, H-4.sub.A), 4.25 (d, 1H, H-4.sub.B), 4.12-4.05 (m, 2H, H.sub.All, H-5.sub.A), 3.76-3.67 (m, 2H, H-2.sub.B, H-5.sub.B), 3.67-3.61 (m, 2H, H-2.sub.A, H-6a.sub.B), 3.53 (dd.sub.po, 1H, J.sub.5,6b 5.9 Hz, J.sub.6a,6b=10.9 Hz, H-6b.sub.B), 3.50 (dd.sub.po, 1H, J.sub.2,3=9.2 Hz, J.sub.3,4=3.4 Hz, H-3.sub.A), 2.32 (bs, 1H, OH-4.sub.A).

(124) .sup.13C NMR (CDCl.sub.3) partial, 167.3 (C-6.sub.A), 162.1 (NHCO), 103.7 (C-1.sub.A), 97.5 (C-1.sub.B), 92.2 (CCl.sub.3), 67.1 (C.sub.CO2Bn).

(125) HRMS (ESI.sup.+): m/z 1012.2621 (calcd for C.sub.52H.sub.54Cl.sub.3NO.sub.12Na [M+Na].sup.+: m/z 1012.2609).

(Benzyl 2,3-di-O-benzyl-4-O-levulinoyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--n-galactopyranosyl trichloroacetimidate (246)

(126) Levulinic acid (1.03 mL, 10.09 mmol), DCC (1.87 g, 9.08 mmol) and DMAP (123 mg, 1.01 mmol,) were added to a solution of uronate 242 (5.00 g, 5.04 mmol) in anhyd. DCM (50 mL). The solution was stirred under an Ar atmosphere for 16 h. The reaction mixture was filtered through a pad of Celite, diluted with DCM and washed successively with a 10% aq. HCl solution, a satd. aq. NaHCO.sub.3 solution and brine, dried by passing through a phase separator filter and concentrated. The residue was purified by flash chromatography (Tol/EtOAc 9:1 to 85:15) to give the fully protected disaccharide 244 (4.99 g, 91%) as a light yellow solid.

(127) 1,5-Cyclooctadienebis(methyldiphenylphosphine)-iridium hexafluorophosphate (194 mg, 230 mol) was dissolved in anhyd. THF (60 mL). Hydrogen was bubbled through the solution for 15 min, and the resulting yellow solution was concentrated to dryness. The residue was taken up in anhyd. THF (92 mL), and a solution of the fully protected disaccharide 244 (4.99 g, 4.58 mmol) in anhyd. THF (92 mL) was added. The mixture was stirred for 2 h at rt under an Ar atmosphere. A solution of iodine (2.33 g, 9.16 mmol) in THF/H.sub.2O (4:1, 110 mL,) was added, and stirring went on for 2 h at rt. The reaction was quenched by the addition of a freshly prepared solution of 10% aq. sodium bisulfite. The mixture was concentrated to volume under reduced pressure and the aq. phase was extracted three times with DCM. The combined organic layers were washed with brine, dried by passing through a phase separator filter and evaporated to dryness. The resulting oil was purified by flash chromatography (Tol/EtOAc 7:3) to give the corresponding hemiacetal 245 (4.80 g, qqtive) in a 6.7:1 / ratio as a light yellow foam.

(128) Trichloroacetonitrile (193 L, 1.92 mmol) and DBU (17 L, 0.12 mmol) were successively added to a solution of the resulting hemiacetal (245, 403 mg, 380 mol) in anhyd. DCM (7.7 mL). The mixture was stirred for 30 min at rt under an Ar atmosphere. Following concentration to volume (reduced pressure, rt) the reaction mixture was directly purified by flash chromatography (Chex/EtOAc 7:3+1% Et.sub.3N) to give trichloroacetimidate 246 (412 mg, 91%) in a 12:1 / ratio as a white foam. The isomer had:

(129) .sup.1H NMR (CDCl.sub.3), 8.74 (s, 1H, NH), 7.44-7.22 (m, 25H, H.sub.Ar), 6.75 (d, 1H, J.sub.NH,2=8.7 Hz, NH), 6.55 (d, 1H, J.sub.1,2=3.6 Hz, H-1.sub.B), 5.85 (bdd, 1H, H-4.sub.A), 5.20 (d, 1H, J=11.8 Hz, H.sub.CO2Bn), 5.16 (d, 1H, H.sub.CO2Bn), 5.12 (d, 1H, J=11.8 Hz, H.sub.Bn), 4.96 (ddd, 1H, J.sub.2,3=11.1 Hz, H-2.sub.B), 4.90 (d, 1H, J=12.2 Hz, H.sub.Bn), 4.75-4.68 (m, 4H, 3H.sub.Bn, H-1.sub.A), 4.60 (bd, 1H, H-4.sub.B), 4.50-4.46 (m, 2H, H.sub.Bn), 4.42 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.27 (d, 1H, J.sub.4,5=1.2 Hz, H-5.sub.A), 4.22 (t, 1H, J.sub.5,6a=J.sub.5,6b=6.4 Hz, H-5.sub.B), 4.15 (dd, 1H, J.sub.2,3=11.0 Hz, J.sub.3,4=2.7 Hz, H-3.sub.B), 3.65-3.60 (m, 4H, H-6a.sub.B, H-6b.sub.B, H-2.sub.A, H-3.sub.A), 2.57-2.37 (m, 4H, CH.sub.2Lev), 1.99 (s, 3H, CH.sub.3Lev),

(130) .sup.13C NMR (CDCl.sub.3) partial, 205.7 (CO.sub.Lev), 171.2 (CO.sub.2Lev), 165.9 (C-6.sub.A), 161.9 (NHCO), 160.2 (CNH), 104.5 (C-1.sub.A), 95.3 (C-1.sub.B), 92.3 (CCl.sub.3C(O)NH), 91.0 (CCl.sub.3OC(NH)), 67.7 (C.sub.CO2Bn), 37.8 (CH.sub.2Lev), 29.5 (CH.sub.3Lev), 27.9 (CH.sub.2Lev).

Benzyl 2,3-di-O-benzyl-4-O-levulinoyl--D-galactopyranosyluronate-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-D-galactopyranosyl N-phenyltrifluoroacetimidate (247)

(131) Hemiacetal 245 obtained from acceptor 242 as described above (5.67 g, 5.40 mmol) was dissolved in acetone (59 mL). N-(phenyl)trifluoroacetimidoyl chloride (2.24 g, 10.8 mmol) and Cs.sub.2CO.sub.3 (1.94 g, 5.94 mmol) were added and the mixture was stirred for 1.5 h at rt. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by flash chromatography (Chex/EtOAc 8:2 to 7:3+1% Et.sub.3N) to give donor 247 (5.38 g, 89%) in a 13:1 / ratio as a white foam.

(132) .sup.1H NMR (CDCl.sub.3), 7.43-7.22 (m, 27H, H.sub.Ar), 7.10 (m, 1H, H.sub.Ar), 6.76 (d, 2H, J=7.6 Hz, H.sub.Ar), 6.72 (d, 1H, J.sub.NH,2=8.2 Hz, NH-2.sub.B), 6.52 (bs, 1H, H-1.sub.B), 5.87 (dd, 1H, J.sub.3,4=2.8 Hz, J.sub.4,5=1.2 Hz, H-4.sub.A), 5.23 (d, 1H, J=11.8 Hz, H.sub.CO2Bn), 5.18 (d, 1H, H.sub.CO2Bn), 5.07 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.89 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.84 (m, 1H, H-2.sub.B), 4.75 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.73 (d.sub.po, 1H, J=12.6 Hz, H.sub.Bn), 4.71 (bs.sub.o, 1H, H.sub.Bn), 4.69 (d, 1H, J.sub.1,2=6.0 Hz, H-1.sub.A), 4.55 (bd, 1H, H-4.sub.B), 4.52 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.50 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.46 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.29 (d, 1H, H-5.sub.A), 4.18-4.12 (m, 2H, H-5.sub.B, H-3.sub.B), 3.68-3.59 (m, 3H, H-6a.sub.B, H-2.sub.A, H-3.sub.A), 3.54 (dd, 1H, J.sub.5,6b=6.3 Hz, J.sub.6a,6b=9.8 Hz, H-6b.sub.B), 2.61-2.39 (m, 4H, CH.sub.2Lev), 2.00 (s, 3H, CH.sub.3Lev).

(133) .sup.13C NMR (CDCl.sub.3), 205.7 (CO.sub.Lev), 171.3 (CO.sub.2Lev), 165.9 (C-6.sub.A), 162.0 (NHCO), 143.1, 138.7, 138.1, 138.0, 137.3, 135.0 (6C, C.sub.IVAr), 129.0-119.2 (30C, C.sub.Ar), 104.4 (C-1.sub.A), 94.2 (C-1.sub.B), 92.3 (CCl.sub.3), 78.6 (C-2.sub.A), 77.3, 77.2 (2C, C-3.sub.B, C-3.sub.A), 75.8 (C-4.sub.B), 75.0, 74.8, 73.4 (3C, C.sub.Bn), 72.5 (C-5.sub.A), 72.4 (C-5.sub.B), 72.2 (C.sub.Bn), 68.9 (C-6.sub.B), 67.7 (C.sub.CO2Bn), 67.5 (C-4.sub.A), 50.7 (C-2.sub.B), 37.8 (CH.sub.2Lev), 29.5 (CH.sub.3Lev), 27.9 (CH.sub.2Lev).

Allyl (3,4-di-O-benzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl-P-D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (277)

(134) A mixture of acceptor 242 (7.26 g, 7.32 mmol), known 3,4-di-O-benzyl-2-O-levulinoyl--L-rhamnopyranosyl trichloroacetimidate.sup.[28] (5.59 g, 9.52 mmol), and powdered MS 4 (10.9 g) in anhyd. toluene (146 mL) was stirred at rt under an Ar atmosphere for 1 h. The reaction mixture was cooled to 10 C. and TMSOTf (66 L, 370 mol) was added. After 20 min, the reaction was quenched with Et.sub.3N. Solids were filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (Tol/EtOAc 9:1 to 8:2) to give trisaccharide 277 (9.19 g, 89%) as a white foam.

(135) .sup.1H NMR (CDCl.sub.3), 7.43-7.10 (m, 35H, H.sub.Ar), 6.90 (d, 1H, J.sub.NH,2=6.7 Hz, NH), 5.89 (m, 1H, CH.sub.All), 5.46 (dd, 1H, J.sub.1,2=1.9 Hz, J.sub.3,4=3.0 Hz, H-2.sub.D), 5.28 (m.sub.o, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.27 (d.sub.o, 1H, H.sub.CO2Bn), 5.19 (d.sub.o, 1H, H-1.sub.D), 5.18 (m.sub.o, 1H, J.sub.cis=10.4 Hz, CH.sub.2All), 5.15 (d, 1H, J=12.2 Hz, H.sub.CO2Bn), 5.04 (d, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 4.96 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.84 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.82 (s, 2H, H.sub.Bn), 4.76 (s, 2H, H.sub.Bn), 4.67 (dd.sub.po, 1H, J.sub.2,3=10.9 Hz, J.sub.3,4=3.3 Hz, H-3.sub.B), 4.66 (d.sub.po, 1H, J=10.9 Hz, H.sub.Bn), 4.58 (d, 1H, J=11.1 Hz, H.sub.B), 4.53 (d, 1H, J.sub.1,2=7.6 Hz, H-1.sub.A), 4.49-4.44 (m, 2H, H.sub.Bn, H-4.sub.A), 4.42 (d, 1H, J=12.0 Hz, H.sub.Bn), 4.37 (m.sub.po, 1H, H.sub.All), 4.33 (d.sub.po, 1H, J 10.7 Hz, H.sub.Bn), 4.24 (bd, 1H, H-4.sub.B), 4.13-4.07 (m, 2H, H.sub.All, H-5.sub.A), 3.95 (d, 1H, J 10.8 Hz, H.sub.Bn), 3.77 (dq.sub.po, 1H, J.sub.4,5=9.8 Hz, H-5.sub.D), 3.77-3.67 (m, 4H, H-3.sub.D, H-2.sub.B, H-2.sub.A, H-5.sub.B), 3.60 (dd, 1H, J.sub.5,6a=6.6 Hz, J.sub.6a,6b 10.0 Hz, H-6a.sub.B), 3.52 (dd, 1H, J.sub.2,3=9.7 Hz, J.sub.3,4=2.8 Hz, H-3.sub.A), 3.38-3.32 (m, 2H, H-6b.sub.B, H-4.sub.D), 2.71-2.61 (m, 4H, CH.sub.2Lev), 2.16 (s, 3H, CH.sub.3Lev), 1.33 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D).

(136) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.5 (CO.sub.2Lev), 167.0 (C-6.sub.A), 162.2 (NHCO), 103.9 (C-1.sub.A, .sup.1J.sub.CH=163.6 Hz), 98.9 (C-1.sub.D, .sup.1J.sub.CH=170.6 Hz), 97.5 (C-1.sub.B, J.sub.CH=163.2 Hz), 92.3 (CCl.sub.3), 67.4 (C.sub.CO2Bn), 57.0 (C-2.sub.B), 38.1 (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

(137) HRMS (ESI.sup.+): m/z 1436.4559 (calcd for C.sub.77H.sub.82Cl.sub.3NO.sub.18Na [M+Na].sup.+: m/z 1436.4495).

(3,4-Di-O-benzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-D-galactopyranosyl N-phenyltrifluoroacetimidate (283)

(138) 1,5-Cyclooctadienebis(methyldiphenylphosphine)-iridium hexafluorophosphate (91 mg, 0.11 mmol) was dissolved in anhyd. THF (43 mL) and hydrogen was bubbled through the solution for 15 min (H-cube, full H.sub.2 mode). The resulting yellow solution was evaporated to dryness. The residue was taken up in anhyd. THF (43 mL) and poured to a solution of allyl glycoside 277 (3.05 g, 2.15 mmol) in anhyd. THF (43 mL). The mixture was stirred under Ar at rt for 2 h. A solution of iodine (1.09 g, 4.31 mmol) in THF/H.sub.2O (4:1, 52 mL) was added, and the mixture was stirred for 1.5 h at rt. The reaction was quenched with 10% aq. sodium bisulfite. The mixture was concentrated to volume and the aq. phase was extracted twice with EtOAc. The organic layers were pooled, washed with brine, dried over anhyd. Na.sub.2SO.sub.4, filtered and concentrated to dryness. The residue was purified by flash chromatography (Tol/EtOAc 7:3) to give the corresponding hemiacetal 281 (2.84 g, 96%) as a light yellow foam. (/, 7.5:1).

(139) This hemiacetal (2.74 g, 2.0 mmol) was dissolved in acetone (20 mL). N-(phenyl)trifluoroacetimidoyl chloride (827 mg, 3.98 mmol) followed by Cs.sub.2CO.sub.3 (714 mg, 2.19 mmol) were added to the solution. The mixture was stirred for 2 h at rt. More Cs.sub.2CO.sub.3 (649 mg, 2.0 mmol) was added. After 1 h, the reaction mixture was filtered and concentrated. The residue was purified by flash chromatography (Chex/EtOAc 65:35 to 6:4+1% Et.sub.3N) to give a 7:1 / mixture of N-phenyltrifluoroacetimidate 283 (3.00 g, 97%) as a light yellow foam.

(140) .sup.1H NMR (CDCl.sub.3), 7.63-7.05 (m, 38H, H.sub.Ar), 6.74 (d, 2H, J=7.5 Hz, H.sub.Ar), 6.68 (d, 1H, J.sub.NH,2=8.3 Hz, NH), 6.50 (bs, 1H, H-1.sub.B), 5.38 (dd, 1H, J.sub.1,2=2.0 Hz, J.sub.2,3=3.1 Hz, H-2.sub.D), 5.26 (d, 1H, J=12.0 Hz, H.sub.CO2Bn), 5.10 (d.sub.o, 1H, H-1.sub.D), 5.11 (d.sub.po, 1H, H.sub.CO2Bn), 4.98 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.91 (d, 1H, J=12.2 Hz, H.sub.Bn), 4.86-4.71 (m, 5H, H-2.sub.B, 4H.sub.Bn), 4.64 (d, 1H, J.sub.1,2=7.5 Hz, H-1.sub.A), 4.55 (d, 1H, J=11.1 Hz, H.sub.Bn), 4.53 (d, 1H, J=11.6 Hz, H.sub.Bn), 4.48 (d.sub.po, 1H, J.sub.3,4=3.2 Hz, H-4.sub.B), 4.47 (d.sub.o, 1H, J=11.7 Hz, H.sub.Bn), 4.45 (bd.sub.o, 1H, H-4.sub.A), 4.41 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.23 (d, 1H, J=10.6 Hz, H.sub.Bn), 4.16 (d, 1H, 1.sub.4,5=0.8 Hz, H-5.sub.A), 4.12-4.05 (m, 2H, H-5.sub.B, H-3.sub.B), 3.81 (d, 1H, J=10.7 Hz, H.sub.Bn), 3.77 (dd.sub.po, 1H, J.sub.2,3=9.5 Hz, H-2.sub.A), 3.73 (dq.sub.po, 1H, 1.sub.4,5=9.6 Hz, H-5.sub.D), 3.63 (dd, 1H, J.sub.2,3=3.2 Hz, J.sub.3,4.fwdarw.9.4 Hz, H-3.sub.D), 3.59 (dd.sub.po, 1H, 1.sub.3,4=2.7 Hz, H-3.sub.A), 3.56 (dd.sub.po, 1H, J.sub.5,6a=6.7 Hz, J.sub.6a,6b=10.3 Hz, H-6a.sub.B), 3.36 (dd, 1H, J.sub.5,6b=5.2 Hz, H-6b.sub.B), 3.31 (pt, 1H, H-4.sub.D), 2.71-2.59 (m, 4H, CH.sub.2Lev), 2.15 (s, 3H, CH.sub.3Lev), 1.30 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D).

(141) .sup.13C NMR (CDCl.sub.3), 206.1 (CO.sub.Lev), 171.4 (CO.sub.2Lev), 166.8 (C-6.sub.A), 161.9 (NHCO), 104.9 (C-1.sub.A), 99.0 (C-1.sub.D), 94.0 (C-1.sub.B), 92.3 (CCl.sub.3), 67.6 (C.sub.CO2Bn), 38.1 (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

Allyl (4-O-benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (XX)

(142) To a solution of the fully protected trisaccharide 277 (8.84 g, 6.24 mmol) in anhyd. pyridine (52 mL) stirred at 0 C. under an Ar atmosphere was added dropwise AcOH (35 mL) followed by hydrazine monohydrate (1.52 mL, 31.2 mmol). The reaction mixture was stirred for 1 h allowing the cooling bath to reach rt. Following addition of DCM and water, the two layers were separated and the aq. one was re-extracted twice with DCM. The combined organic extracts were washed with brine, dried by passing through a phase separator filter and volatiles were evaporated. The residue was purified by flash chromatography (Chex/EtOAc 75:25 to 7:3) to give the corresponding acceptor (7.56 g, 92%) as a white foam.

(143) A mixture of this acceptor (7.03 g, 5.34 mmol), trichloroacetimidate donor 119 (3.96 g, 6.42 mmol), and powdered MS 4 (10.6 g) in anhyd. toluene (134 mL) was stirred at rt under an Ar atmosphere for 1 h. The reaction mixture was cooled to 10 C. and TMSOTf (48 L, 267 mol) was added. After 30 min, the reaction was quenched with Et.sub.3N. Solids were filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (Tol/EtOAc 95:5 to 8:2) to give tetrasaccharide 279 (8.66 g, 91%) as a white foam.

(144) .sup.1H NMR (CDCl.sub.3), 7.45-7.11 (m, 42H, H.sub.Ar), 6.89-6.84 (m, 3H, 2H.sub.ArPMB, NH), 5.88 (m, 1H, CH.sub.All), 5.49 (dd, 1H, J.sub.1,2=1.8 Hz, H-2.sub.C), 5.31 (d, 1H, J.sub.1,2=1.6 Hz, 14-1.sub.D), 5.26 (d.sub.po, 1H, J=12.1 Hz, H.sub.CO2Bn), 5.25 (m.sub.po, 1H, J.sub.trans 17.2 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.17 (m, 1H, J.sub.cis=10.4 Hz, CH.sub.2All), 5.13 (d, 1H, H.sub.CO2Bn), 5.03 (d, 1H, J.sub.1,2=8.5 Hz, H-1.sub.B), 4.97 (d.sub.po, 1H, J=11.6 Hz, H.sub.Bn), 4.96 (bs.sub.o, 1H, H-1.sub.C), 4.90 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.82 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.79 (d.sub.po, 1H, J=12.4 Hz, H.sub.Bn), 4.77 (bs.sub.o, 2H, H.sub.Bn), 4.68-4.61 (m, 6H, 5H.sub.Bn, H-3.sub.B), 4.58 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.49 (d, 1H, J.sub.1,2=7.6 Hz, H-1.sub.A), 4.47 (d, 1H, J=11.9 Hz, H.sub.Bn), 4.43 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.40 (bd, 1H, H-4.sub.A), 4.36 (m, 1H, H.sub.All), 4.25 (d, 1H, J.sub.3,4=3.0 Hz, H-4.sub.B), 4.22 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.14 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.12-4.04 (m, 3H, H.sub.All, H-2.sub.D), 4.04 (d, 1H, J.sub.4,5=0.6 Hz, H-5.sub.A), 3.87 (dd, 1H, J.sub.2,3=3.2 Hz, J.sub.3,4=9.1 Hz, H-3.sub.C), 3.83 (dq, 1H, 1.sub.4,5=9.4 Hz, J.sub.5,6=6.2 Hz, H-5.sub.C), 3.78 (s, 3H, CH.sub.3PMB), 3.73-3.63 (m, 5H, H-5.sub.D, H-3.sub.D, H-2.sub.B, H-2.sub.A, H-5.sub.B), 3.61 (dd, 1H, J.sub.5,6a=6.6 Hz, J.sub.6a,6b=10.0 Hz, H-6a.sub.B), 3.47 (dd.sub.po, 1H, J.sub.2,3=9.7 Hz, J.sub.3,4=2.7 Hz, H-3.sub.A), 3.45 (pt.sub.po, 1H, H-4.sub.C), 3.39 (dd, 1H, J.sub.5,6b=5.5 Hz, H-6b.sub.B), 3.37 (pt, 1H, H-4.sub.D), 2.79-2.66 (m, 4H, CH.sub.2Lev), 2.19 (s, 3H, CH.sub.3Lev), 1.32 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.22 (d, 3H, H-6.sub.C).

(145) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.7 (CO.sub.2Lev), 167.0 (C-6.sub.A), 162.1 (NHCO), 159.4 (C.sub.IVArPMP), 113.9 (2C, C.sub.ArPMB), 103.9 (C-1.sub.A), 100.7 (C-1.sub.C), 100.2 (C-1.sub.D), 97.5 (C-1.sub.B), 92.3 (CCl.sub.3), 67.4 (C.sub.CO2Bn), 55.2 (CH.sub.3PMB), 38.2 (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.3 (CO.sub.2CH.sub.2Lev).

(146) HRMS (ESI.sup.+): m/z 1792.6085 (calcd for C.sub.98H.sub.106Cl.sub.3NO.sub.23Na [M+Na].sup.+: m/z 1792.6119).

(4-O-Benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-/-D-galactopyranosyl N-phenyltrifluoroacetimidate (284)

(147) Cyclooctadienebis(methyldiphenylphosphine)-iridium hexafluorophosphate (97 mg, 0.11 mmol) was dissolved in anhyd. THF (46 mL) and hydrogen was bubbled through the solution for 15 min (H-cube, full H.sub.2 mode). The resulting yellow solution was evaporated to dryness. The residue was taken up in anhyd. THF (46 mL) and poured to a solution of the allyl glycoside 279 (4.05 g, 2.29 mmol) in anhyd. THF (46 mL). The mixture was stirred under Ar at rt for 3.5 h. A solution of iodine (1.16 g, 4.57 mmol) in THF/H.sub.2O (4:1, 55 mL) was added, and the mixture was stirred for 1.5 h at rt. The reaction was quenched with 10% aq. sodium bisulfite. The mixture was concentrated to volume and the aq. phase was extracted twice times with EtOAc. The organic layers were pooled, washed with brine, dried over anhyd. Na.sub.2SO.sub.4, filtered and concentrated to dryness. The residue was purified by flash chromatography (Tol/EtOAc 75:25 to 6:4) to give the coresponding hemiacetal (3.85 g, 97%) as a light yellow solid (/, 5:1).

(148) This hemiacetal (3.73 g, 2.15 mmol) was dissolved in acetone (43 mL). N-(phenyl)trifluoroacetimidoyl chloride (893 mg, 4.30 mmol) followed by Cs.sub.2CO.sub.3 (1.40 g, 4.30 mmol) were added to the solution. The mixture was stirred for 2 h at rt. The reaction mixture was filtered and concentrated. The residue was purified by flash chromatography (Chex/EtOAc 75:25 to 6:4+1% Et.sub.3N) to give a 5:1 / mixture of N-phenyltrifluoroacetimidate 284 (3.66 g, 89%) as a white foam.

(149) .sup.1H NMR (CDCl.sub.3), 7.46-7.05 (m, 45H, H.sub.Ar), 6.87-6.82 (m, 2H, H.sub.ArPMB), 6.77-6.72 (m, 2H, H.sub.Ar), 6.67 (d, 1H, J.sub.NH,2=8.3 Hz, NH), 6.50 (bs, 1H, H-1.sub.B), 5.47 (dd, 1H, J.sub.1,2=1.9 Hz, J.sub.2,3=3.1 Hz, H-2.sub.C), 5.27 (s.sub.o, 1H, H-1.sub.D), 5.26 (d, 1H, J=12.1 Hz, H.sub.CO2Bn), 5.11 (d, 1H, H.sub.CO2BN), 5.03 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.94-4.85 (m, 3H, 2H.sub.Bn, H-2.sub.B), 4.84-4.72 (m, 4H, 3H.sub.Bn, H-1.sub.C), 4.65 (2d.sub.o, 2H, J=11.5 Hz, H.sub.Bn), 4.63-4.55 (m, 4H, 3H.sub.Bn, H-1.sub.A), 4.51 (bd, 1H, J.sub.3,4=2.4 Hz, H-4.sub.B), 4.50-4.42 (m, 4H, H.sub.Bn), 4.41 (hd.sub.po, 1H, J.sub.3,4=3.6 Hz, H-4.sub.A), 4.13-4.06 (m, 4H, H.sub.Bn, H-5.sub.B, H-5.sub.A, H-3.sub.B), 4.04 (d, 1H, J=11.6 Hz, H.sub.Bn), 3.94-3.89 (m, 2H, H-2.sub.D, H-3.sub.C), 3.81 (dq, 1H, J.sub.4,5=9.4 Hz, J.sub.5,6=6.3 Hz, H-5.sub.C), 3.77 (s, 3H, CH.sub.3PMB), 3.73 (dd, 1H, J.sub.1,2=7.5 Hz, J.sub.2,3=10.0 Hz, H-2.sub.A), 3.67 (dq, 1H, J.sub.4,5=9.4 Hz, J.sub.5,6=6.2 Hz, H-5.sub.D), 3.62-3.52 (m, 3H, H-3.sub.D, H-6a.sub.B, H-3.sub.A), 3.43-3.33 (m, 3H, H-4.sub.C, H-4.sub.D, H-6b.sub.B), 2.77-2.67 (m, 4H, CH.sub.2Lev), 2.19 (s, 3H, CH.sub.3Lev), 1.29 (d, 3H, H-6.sub.D), 1.20 (d, 3H, H-6.sub.C).

(150) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.6 (CO.sub.2Lev) 166.8 (C-6.sub.A), 162.0 (NHCO), 159.2 (C.sub.IVArPMB), 143.1 (CNPh), 113.7 (2C, C.sub.ArPMB), 105.0 (C-1.sub.A), 100.1 (C-1.sub.D), 99.2 (C-1.sub.C), 94.3 (C-1.sub.B), 92.3 (CCl.sub.3), 67.5 (C.sub.CO2Bn), 55.2 (CH.sub.3PMB), 38.1, (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

Allyl (4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosylurortate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (280)

(151) AcOH (20 mL), followed by hydrazine monohydrate (443 L, 9.12 mmol), was added dropwise to a solution of 279 (3.23 g, 1.82 mmol) in anhyd. pyridine (30 mL) stirred at 0 C. under an Ar atmosphere. The reaction mixture was stirred for 1 h allowing the cooling bath to reach it Following addition of DCM and water, the two layers were separated and the aq. one was re-extracted twice with DCM. The combined organic extracts were washed with brine, dried by filtration through a phase separator filter and volatiles were evaporated. The residue was purified by flash chromatography (Chex/EtOAc 75:25 to 65:35) to give alcohol 280 (2.87 g, 94%) as a white foam.

(152) .sup.1H NMR (CDCl.sub.3), 7.42-7.10 (m, 42H, H.sub.Ar), 6.91-6.87 (m, 2H, J=8.6 Hz, H.sub.ArPMB), 6.86 (d, 1H, J.sub.NH,2=6.8 Hz, NH), 5.88 (m, 1H, CH.sub.All), 5.31 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.D), 5.26 (d.sub.po, 1H, J=12.1 Hz, H.sub.CO2Bn), 5.25 (m.sub.po, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.17 (m, 1H, J.sub.cis=10.4 Hz, CH.sub.2All), 5.13 (d, 1H, H.sub.CO2Bn), 5.03 (d, 1H, J.sub.1,2=8.5 Hz, H-1.sub.B), 4.97 (d.sub.po, 1H, J=11.6 Hz, H.sub.Bn), 4.96 (bs.sub.o, 1H, H-1.sub.C), 4.90 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.82 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.79 (d.sub.po, 1H, J=12.4 Hz, H.sub.Bn), 4.77 (bs.sub.o, 2H, H.sub.Bn), 4.68-4.61 (m, 6H, 5H.sub.Bn, H-3.sub.B), 4.58 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.49 (d, 1H, 42=7.6 Hz, H-1.sub.A), 4.47 (d, 1H, J=11.9 Hz, H.sub.Bn), 4.43 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.40 (bd, 1H, H-4.sub.A), 4.36 (m, 1H, H.sub.All), 4.25 (d, 1H, J.sub.3,4=3.0 Hz, H-4.sub.B), 4.22 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.14 (d, 1H, J=11.4 Hz, H.sub.Bn), 4.12-4.04 (m, 3H, H.sub.All, H-2.sub.C, H-2.sub.D), 4.04 (d, 1H, J.sub.4,5=0.6 Hz, H-5.sub.A), 3.87 (dd, 1H, J=.sub.2,3=3.2 Hz, J.sub.3,4=9.1 Hz, H-3.sub.C), 3.83 (dq, 1H, J.sub.4,5=9.4 Hz, H-5.sub.C), 3.78 (s, 3H, CH.sub.3PMB), 3.73-3.63 (m, 5H, H-5.sub.D, H-3.sub.D, H-2.sub.B, H-2.sub.A, H-5.sub.B), 3.61 (dd, 1H, J.sub.5,6a=6.6 Hz, J.sub.6a,6b=10.0 Hz, H-6.sub.B), 3.47 (dd.sub.po, 1H, J.sub.2,3=9.7 Hz, J.sub.3,4=2.7 Hz, H-3.sub.A), 3.45 (pt.sub.po, 1H, J.sub.3,4=9.3 Hz, J.sub.4,5 9.4 Hz, H-4.sub.C), 3.39 (dd, 1H, J.sub.5,6b=5.5 Hz, H-6b.sub.B), 3.37 (pt, 1H, H-4.sub.D), 2.39 (bs, 1H, OH-2.sub.C), 1.32 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.22 (d, 1H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(153) .sup.13C NMR (CDCl.sub.3) partial, 167.0 (C-6.sub.A), 162.1 (NHCO), 159.4 (C.sub.IVArPMB), 113.9 (2C, C.sub.ArPMB), 103.9 (C-1.sub.A), 100.7 (C-1.sub.C), 100.2 (C-1.sub.D), 97.5 (C-1.sub.B), 92.3 (CCl.sub.3), 67.4 (C.sub.CO2Bn), 55.2 (CH.sub.3PMB). HRMS (ESI.sup.+): m/z 1694.5691 (calcd for C.sub.93H.sub.100Cl.sub.3NO.sub.21Na [M+Na].sup.+: m/z 1694.5751).

(154) Convergent Synthesis of Fully Protected Precursors to SF6, SF6a, and/or E. coli O147 O-Antigen Fragments Using the Building Blocks Shown in Table 1.

(155) Selected Examples:

(156) Protected pentasaccharide ABCDA (248). Taking advantage of the design of proper building blocks, the fully protected 248 is obtained in one single step in high yield, upon glycosylation of acceptor 169 with donor (Scheme 8).

(157) ##STR00050##

Benzyl (benzyl 2,3-di-O-benzyl-4-O-levulinoyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(allyl 2,3-di-O-benzyl--D-galactopyranosid)uronate (248)

(158) A mixture of trisaccharide acceptor 169 (2.15 g, 1.81 mmol), disaccharide donor 247 (2.65 g, 2.17 mmol) and freshly activated 4 powdered MS (5.4 g) in anhyd. DCM (36.2 mL) was stirred for 1 h at rt under an Ar atmosphere, then at 78 C. TMSOTf (16.4 L, 91 mol) was added and the reaction mixture was stirred at that temperature for 20 min. The reaction was quenched with Et.sub.3N. The suspension was filtered and the filtrate was concentrated. The residue was purified by flash chromatography (Tol/EtOAc 95:5 to 85:5) to give pentasaccharide 248 (3.47 g, 86%) as a white foam.

(159) .sup.1H NMR (CDCl.sub.3), 7.47-7.43 (m, 2H, H.sub.Ar), 7.41-7.02 (m, 53H, H.sub.Ar), 7.16 (m, 1H, H.sub.Ar), 7.08 (m, 1H, H.sub.Ar), 7.03 (d, 1H, J.sub.NH,2=6.8 Hz, NH), 6.82 (d, 2H, J=8.7 Hz, H.sub.ArPMB), 5.98 (m, 1H, CH.sub.All), 5.82 (bd, 1H, H-4.sub.A), 5.35 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CH.sub.2All), 5.27 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.D), 5.27-5.19 (m, 4H, H-1.sub.B, CH.sub.2All, 2H.sub.CO2Bn), 5.18 (d.sub.po, 1H, J=11.9 Hz, H.sub.CO2Bn), 5.10 (d.sub.po, 1H, J=12.1 Hz, H.sub.CO2Bn), 5.08 (d, 1H, J=11.4 Hz, H.sub.Bn), 5.00 (d, 1H, J.sub.1,2=1.2 Hz, H-1.sub.C), 4.92 (d, 1H, J=10.9 Hz, H.sub.Bn), 4.91 (d, 1H, J 10.9 Hz, H.sub.Bn), 4.87 (d, 1H, J=11.7 Hz, H.sub.Bn), 4.82 (d, 1H, J=11.1 Hz, H.sub.Bn), 4.80-4.69 (m, 6H, 5H.sub.Bn, H-3.sub.B), 4.66-4.59 (m, 4H, H-1.sub.A, 3H.sub.Bn), 4.57-4.46 (m, 6H, 5H.sub.Bn, H.sub.All), 4.38-4.33 (m, 3H, H-1.sub.A, H-4.sub.A, H-4.sub.B), 4.28 (d, 1H, J=11.8 Hz, H.sub.Bn), 4.22 (d, 1H, J.sub.4,5=1.0 Hz, H-5.sub.A), 4.19 (d.sub.po, 1H, J=11.7 Hz, H.sub.Bn), 4.15 (m.sub.o, 1H, H.sub.All), 4.08 (pt, 1H, H-2.sub.C), 4.01 (pt, 1H, H-2.sub.D), 3.99 (s, 1H, H-5.sub.A), 3.86 (dd.sub.po, 1H, J.sub.2,3=2.9 Hz, H-3.sub.C), 3.85-3.78 (m, 2H, H-2.sub.B, H-3.sub.D), 3.78 (dq.sub.pc, 1H, J.sub.4,5=9.5 Hz, H-5.sub.C), 3.71-3.61 (m, 6H, H-5.sub.D, CH.sub.3PMB, H-2.sub.A, H-5.sub.B), 3.59-3.52 (m, 3H, H-6a.sub.B, H-2.sub.A, H-3.sub.A), 3.49 (dd, 1H, J.sub.2,3=9.8 Hz, J.sub.3,4=2.8 Hz, H-3.sub.A), 3.43 (pt, 1H, J.sub.3,4=9.5 Hz, H-4.sub.C), 3.38 (dd, 1H, J.sub.5,6b=5.2 Hz, J.sub.6a,6b=9.0 Hz, H-6b.sub.B), 3.32 (pt, 1H, J.sub.3,4=J.sub.4,5=9.4 Hz, H-4.sub.D), 2.59-2.41 (m, 4H, CH.sub.2Lev), 2.00 (s, 3H, CH.sub.3Lev), 1.28 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(160) .sup.13C NMR (CDCl.sub.3), 205.9 (CO.sub.Lev), 171.3 (CO.sub.2Lev), 167.3, 166.2 (2C, C-6.sub.A, C-6.sub.A), 161.7 (NHCO), 113.7 (2C, C.sub.ArPMB), 103.6 (C-1.sub.A, .sup.1J.sub.CH=163.2 Hz), 102.6 (C-1.sub.A, .sup.1J.sub.CH=155.6 Hz), 101.2 (C-1.sub.D, .sup.1J.sub.CH=176.2 Hz), 100.2 (C-1.sub.C, .sup.1J.sub.CH=173.5 Hz), 98.9 (C-1.sub.B, .sup.1J.sub.CH=162.4 Hz), 92.6 (CCl.sub.3), 67.5, 67.3 (2C, C.sub.CO2Bn), 55.1 (CH.sub.3PMB), 37.9 (COCH.sub.2Lev), 29.5 (CH.sub.3Lev), 28.0 (CO.sub.2CH.sub.2Lev).

(161) HRMS (ESI.sup.+): m/z 2238.8015 (calcd for C.sub.125H.sub.132Cl.sub.3NO.sub.29Na [M+Na].sup.+: m/z 2238.7849).

(162) Protected heptasaccharide DABCDAB (294). Taking advantage of the design of proper building blocks, the fully protected 294 is obtained in one single step in high yield, upon glycosylation of the tetrasaccharide acceptor 280 with the trisaccharide donor 283 (Scheme 9).

(163) ##STR00051##

Allyl (3,4-di-O-benzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (294)

(164) A mixture of tetrasaccharide acceptor 280 (0.50 g, 0.30 mmol), trisaccharide donor 283 (0.65 g, 0.42 mmol), and powdered 4 MS (1.25 g) in anhyd. DCM (7.5 mL) was stirred at rt under an Ar atmosphere for 1 h. The suspension was cooled to 90 C., and TMSOTf (2.7 L, 15 mol) was added. The reaction mixture was stirred for 30 min allowing the cooling bath to reach 78 C. and Et.sub.3N was added to quench the reaction. The suspension was filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (Tol/EtOAc 88:12 to 8:2) to give heptasaccharide 294 (0.81 g, 89%) as a white foam.

(165) .sup.1H NMR (CDCl.sub.3), 7.41-6.95 (m, 78H, 77H.sub.Ar, NH-2.sub.B), 6.82 (d, 1H, J.sub.NH,2=6.7 Hz, NH-2.sub.B), 6.81-6.78 (m, 2H, H.sub.ArPMB), 5.87 (m, 1H, CH.sub.All), 5.42 (dd, 1H, J.sub.1,2=1.9 Hz, J.sub.2,3.sup.=3.0 Hz, H-2.sub.D), 5.29-5.20 (m, 5H, H-1.sub.D, CH.sub.2All, 2H.sub.CO2Bn, H-1.sub.B), 5.16 (m, 1H, J.sub.cis=10.4 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.14 (d, 1H, J.sub.1,2=1.7 Hz, H-1.sub.D), 5.11 (d, 1H, J=12.2 Hz, H.sub.CO2Bn), 5.10 (d, 1H, J=12.1 Hz, H.sub.C02Bn), 5.02 (d.sub.po, 1H, J.sub.1,2=8.2 Hz, H-1.sub.B), 4.01 (d.sub.po, 1H, J=11.6 Hz, H.sub.Bn), 4.96 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.C), 4.93 (2d.sub.o, 2H, H.sub.Bn), 4.88 (d, 1H, J=11.8 Hz, H.sub.Bn), 4.81 (d, 1H, J=11.1 Hz, H.sub.Bn), 4.79 (d, 1H, J=12.1 Hz, H.sub.Bn), 4.78-4.68 (m, 7H, H-3.sub.B, 6H.sub.Bn), 4.66-4.51 (m, 10H, 8H.sub.Bn, H-1.sub.A, H-3.sub.B), 4.49-4.39 (m, 5H, 3H.sub.Bn, H-1.sub.A, H-4.sub.A), 4.37-4.32 (m, 2H, H.sub.All, H-4.sub.A), 4.31 (bd, 1H, J.sub.3,4=3.0 Hz, H-4.sub.B), 4.26 (d, 1H, J=10.8 Hz, H.sub.Bn), 4.21-4.16 (m, 3H, H-4.sub.B, 2H.sub.Bn), 4.11-4.04 (m, 5H, H-2.sub.C, H-5.sub.A, H.sub.All, 2H.sub.Bn), 3.98 (bs, 1H, H-5.sub.A), 3.93 (pt, 1H, H-2.sub.D), 3.88-3.80 (m, 3H, H-3.sub.C, H-2.sub.B, H.sub.Bn), 3.79-3.56 (m, 14H, H-5.sub.C, H-5.sub.D, H-2.sub.A, H-3.sub.D, H-3.sub.D, CH.sub.3PMB, H-2.sub.B, H-6a.sub.B, H-2.sub.A, H-5.sub.B, H-5.sub.D, H-5.sub.B), 3.49 (dd, 1H, J.sub.2,3=9.7 Hz, J.sub.3,4=2.8 Hz, H-3.sub.A), 3.49-3.40 (m, 2H, H-4.sub.C, H-3.sub.A), 3.40-3.34 (m, 2H, H-6b.sub.B, H-6a.sub.B), 3.33-3.26 (m, 3H, H-4.sub.D, H-6b.sub.B, H-4.sub.D), 2.70-2.60 (m, 4H, CH.sub.2Lev), 2.14 (s, 3H, CH.sub.3Lev), 1.30 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.29 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C).

(166) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.4 (CO.sub.2Lev) 166.9 (2C, C-6.sub.A, C-6.sub.A) 162.1 (NHCO-2.sub.B), 161.7 (NHCO-2.sub.B), 159.2 (C.sub.IVPMB), 130.7 (C.sub.IVPMB), 113.7 (2C, C.sub.ArPMB), 104.0 (C-1.sub.A), 103.8 (C-1.sub.A), 101.0 (C-1.sub.C), 100.4 (C-1.sub.D), 99.0 (C-1.sub.D), 98.8 (C-1.sub.B), 97.5 (C-1.sub.B), 92.7, 92.2 (2C, CCl.sub.3), 67.4, 67.3 (2C, C.sub.CO2Bn), 55.1 (CH.sub.3PMB), 38.1 (COCH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

(167) HRMS (ESI.sup.+): m/z 1536.4961 (calcd for C.sub.167H.sub.176Cl.sub.6N.sub.2O.sub.38Na.sub.2 [M+2Na].sup.2+: m/z 1536.4913).

(168) Protected Octasaccharide (CDAB).sub.2 (300) and Dodecasaccharide (CDAB).sub.3 (302): Example of an Iterative Process.

(169) As illustrated in the following, the building blocks listed in Table 1 are advantageously involved in an iterative process for the synthesis of structures larger than two repeating units. At least three glycosylation steps are required to reach the corresponding oligo- or polysaccharides. In this regard, tetrasaccharide CDAB-PTFA (284) was identified as crucial in the elongation process. It was designed to act as a donor and potential acceptor, and for that reason its 2.sub.C-OH is masked with an orthogonal protecting group, namely a levulinoyl ester (Lev). Interestingly, the key tetrasaccharide 284 can thus be used repeatedly according to an iterative glycosylation/delevulinoylation process to reach the desired oligo- or polysaccharide length. Indeed, when performed at 90 C. under controlled conditions, the condensation of the HO-CDAB acceptor (280) and CDAB-PTFA donor (284) gave the fully protected (CDAB).sub.2 octasaccharide (300) in high yield. Hydrazynolysis of the levulinoyl ester located at position 2 of the non reducing end residue gave the octasaccharide acceptor HO-(CDAB).sub.2 (301), which was set to react with the same CDAB-PTFA donor (284), again at 90 C., to give the fully protected dodecasaccharide (CDAB).sub.3 in high yield (302). Please note that the reaction temperature is critical to ensure the required glycosylation. Dodecasaccharide 302 is thus obtained in 3 high yielding steps by appropriate combination of two out of the eight newly available building blocks (Scheme 10).

(170) ##STR00052##

Allyl (4-O-benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (300)

(171) A mixture of tetrasaccharide acceptor 280 (1.40 g, 0.84 mmol), tetrasaccharide donor 284 (2.23 g, 1.17 mmol), and powdered 4 MS (3.5 g) in anhyd. DCM (29 mL) was stirred at rt under an Ar atmosphere for 1 h. The suspension was cooled to 90 C., and TMSOTf (7.6 L, 42 mol) was added. The reaction mixture was stirred for 30 min allowing the cooling bath to reach 78 C. and Et.sub.3N was added to quench the reaction. The suspension was filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (SiO.sub.2 20-40 m, Tol/EtOAc 92:8 to 8:2) to give octasaccharide 300 (2.28 g, 81%) as a white foam.

(172) .sup.1H NMR (CDCl.sub.3), 7.41-6.95 (m, 85H, 84H.sub.Ar, NH-2.sub.B), 6.87-6.74 (m, 5H, 4H.sub.ArPMB, NH-2.sub.B), 5.87 (m, 1H, CH.sub.All), 5.46 (dd, 1H, J.sub.1,2=1.9 Hz, J.sub.2,3=3.1 Hz, H-2.sub.C), 5.28-5.20 (m, 6H, H-1.sub.D, H-1.sub.D, CH.sub.2All, 2H.sub.CO2Bn, H-1.sub.B), 5.16 (m, 1H, J.sub.cis=10.4 Hz, J.sub.gem=1.5 Hz, CH.sub.2All), 5.16-5.08 (m, 2H, H.sub.CO2Bn), 5.04 (d, 1H, J=12.2 Hz, H.sub.Bn), 5.03 (d, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 4.96 (d, 1H, J.sub.1,2=1.2 Hz, H-1.sub.C), 4.95-4.86 (m, 4H, H.sub.Bn), 4.81 (d.sub.po, 1H, J=10.5 Hz, H.sub.Bn), 4.79-4.71 (m, 7H, H-1.sub.C, 6H.sub.Bn), 4.69 (dd.sub.po, 1H, J.sub.2,3=10.9 Hz, J.sub.3,4=10.9 Hz, H-3.sub.B), 4.67-4.56 (m, 10H, H-3.sub.B, 9H.sub.Bn), 4.54 (d.sub.po, 1H, J.sub.1,2=7.5 Hz, H-1.sub.A), 4.53 (d, 1H, J=11.0 Hz, H.sub.Bn), 4.49-4.39 (m, 5H, 4H.sub.Bn, H-1.sub.A), 4.38-4.30 (m, 4H, H-4.sub.A, H-4.sub.A, H.sub.All, H-4.sub.B), 4.21-4.16 (m, 3H, 2H.sub.Bn, H-4.sub.B), 4.21-4.04 (m, 6H, H-2.sub.C, H.sub.All, 4H.sub.Bn), 4.04 (bs, 1H, H-5.sub.A), 3.98 (bs, 1H, H-5.sub.A), 3.96-3.93 (m, 2H, H-2.sub.D, H-2.sub.D), 3.91 (ddpo, 1H, J.sub.2,3=3.2 Hz, J.sub.3,4=9.4 Hz, H-3.sub.C), 3.86-3.74 (m, 7H, H-3.sub.C, H-2.sub.B, H-5.sub.C, H-5.sub.C, CH.sub.3PMB), 3.70-3.56 (m, 13H, H-5.sub.D, H-2.sub.A, H-3.sub.D, H-3.sub.D, CH.sub.3PMB, H-2.sub.B, H-6a.sub.B, H-2.sub.A, H-5.sub.B, H-5.sub.D, H-5.sub.B), 3.46-3.25 (m, 9H, H-3.sub.A, H-3.sub.A, H-4.sub.C, H-6b.sub.B, H-6a.sub.B, H-4.sub.D, H-6b.sub.B, H-4.sub.D), 2.76-2.66 (m, 4H, CH.sub.2Lev), 2.18 (s, 3H, CH.sub.3Lev), 1.29 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.28 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D), 1.20 (2d.sub.o, 6H, J.sub.5,6=6.2 Hz, H-6.sub.C, H-6.sub.C).

(173) .sup.13C NMR (CDCl.sub.3) partial, 206.1 (CO.sub.Lev), 171.6 (CO.sub.2Lev), 166.9 (2C, C-6.sub.A, C-60, 162.1 (NHCO-2.sub.B), 161.7 (NHCO-2.sub.B), 159.2 (2C, C.sub.IVPMB), 130.6, 130.3 (2C, C.sub.IVPMB), 113.7 (4C, C.sub.ArPMB), 103.9 (C-1.sub.A), 103.8 (C-1.sub.A), 101.0 (C-1.sub.C), 100.4 (C-1.sub.D), 100.1 (C-1.sub.D), 99.2 (C-1.sub.C), 98.8 (C-1.sub.B), 97.5 (C-1.sub.B), 92.7, 92.2 (2C, CCl.sub.3), 67.3 (2C, C.sub.CO2Bn), 55.2, 55.1 (2C, C.sub.H3PMB), 38.1 (COCH.sub.2LEV), 29.8 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

(174) HRMS (ESI.sup.+): m/z 1714.5948 (calcd for C.sub.188H.sub.200Cl.sub.6N.sub.2O.sub.43Na.sub.2 [M+2Na].sup.2+: m/z 1714.5725).

Allyl (4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-meth oxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (301)

(175) To a solution of the fully protected 300 (1.31 g, 0.39 mmol) in anhyd. pyridine (9.6 mL) stirred at 0 C. under an Ar atmosphere was added dropwise AcOH (6.4 mL) followed by hydrazine monohydrate (94 L, 1.93 mmol). The reaction mixture was stirred for 1.5 h allowing the cooling bath to reach rt. Following addition of DCM and water, the two layers were separated and the aq. one was re-extracted twice with DCM. The combined organic extracts were washed with brine, dried by passing through a phase separator filter and volatiles were evaporated. The residue was purified by flash chromatography (Tol/EtOAc 9:1 to 75:25) to give alcohol 301 (1.05 mg, 82%) as a white foam.

(176) .sup.1H NMR (CDCl.sub.3), 7.41-6.94 (m, 85H, 84H.sub.Ar, NH-2.sub.B), 6.90-6.86 (m, 2H, H.sub.ArPMB), 6.84 (d, 1H, J.sub.NH,2=6.7 Hz, NH-2.sub.B), 6.82-6.78 (m, 2H, H.sub.ArPMB), 5.87 (m, 1H, CH.sub.All), 5.29-5.20 (m, 6H, H-1.sub.D, H-1.sub.D, CH.sub.2All, 2H.sub.CO2Bn, H-1.sub.B), 5.16 (m, 1H, J.sub.cis 10.4 Hz, J.sub.gem, =1.5 Hz, CH.sub.2All), 5.16-5.09 (m, 2H, H.sub.CO2Bn), 5.04 (d, 1H, J=11.4 Hz, H.sub.Bn), 5.03 (d, 1H, J.sub.1,2=8.3 Hz, H-1.sub.B), 4.96 (d, 1H, J.sub.1,2=1.4 Hz, H-1.sub.C), 4.95-4.86 (m, 5H, H-1.sub.C, H.sub.Bn), 4.83-4.68 (m, 8H, 7H.sub.Bn, H-3.sub.B), 4.68-4.51 (m, 13H, 11H.sub.Bn, H-3.sub.B, H-1.sub.A), 4.49-4.40 (m, 4H, 3H.sub.Bn, H-1.sub.A), 4.39-4.31 (m, 4H, H-4.sub.A, H-4.sub.A, H.sub.All, H-4.sub.B), 4.22-4.16 (m, 3H, 2H.sub.Bn, H-4.sub.B), 5.14 (d, 1H, J=11.2 Hz, H.sub.Bn), 4.12-4.04 (m, 7H, H-2.sub.C, H.sub.All, 3H.sub.Bn, H-2.sub.C H-5.sub.A), 4.00 (pt.sub.po, 1H, H-2.sub.D), 3.99 (s.sub.o, 1H, H-5.sub.A), 3.94 (pt, 1H, H-2.sub.D), 3.88-3.74 (m, 8H, H-3.sub.C, H-3.sub.C, H-2.sub.B, H-5.sub.C, H-5.sub.C, CH.sub.3PMB), 3.72-3.56 (m, 13H, H-5.sub.D, H-2.sub.A, H-3.sub.D, H-3.sub.D, CH.sub.3PMB, H-2.sub.B, H-6a.sub.B, H-2.sub.A, H-5.sub.B, H-5.sub.D, H-5.sub.B), 3.48-3.26 (m, 9H, H-3.sub.A, H-4.sub.C, H-3.sub.A, H-4.sub.C, H-6b.sub.B, H-6a.sub.B, H-4.sub.D, H-6b.sub.B, H-4.sub.D), 2.37 (bs, 1H, OH-2.sub.C), 1.30 (d.sub.po, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D*), 1.29 (d.sub.po, 3H, J.sub.5,6=6.2 Hz, H-6.sub.D*), 1.21 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C*), 1.20 (d, 3H, J.sub.5,6=6.2 Hz, H-6.sub.C*).

(177) .sup.13C NMR (CDCl.sub.3) partial, 166.9 (2C, C-6.sub.A, C-6.sub.A), 162.1 (NHCO-2.sub.B), 161.7 (NHCO-2.sub.B), 159.4, 159.2 (2C, C.sub.IVPMB), 130.7, 130.2 (2C, C.sub.IVPMB), 113.9, 113.7 (4C, C.sub.ArPMB), 104.0 (C-1.sub.A), 103.8 (C-1.sub.A), 101.0 (C-1.sub.C), 100.7 (C-1.sub.C), 100.4 (C-1.sub.D), 100.3 (C-1.sub.D), 98.8 (C-1.sub.B), 97.4 (C-1.sub.B), 92.7, 92.2 (2C, CCl.sub.3), 67.3 (2C, C.sub.CO2Bn), 55.2, 55.1 (2C, CH.sub.3PMB).

(178) HRMS (ESI.sup.+): m/z 1665.5668 (calcd for C.sub.183H.sub.194Cl.sub.6N.sub.2O.sub.41Na.sub.2 [M+2Na].sup.2+: m/z 1665.5542).

Allyl (4-O-benzyl-3-O-para-methoxybenzyl-2-O-levulinoyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-(4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(12)-(4-O-benzyl-3-O-para-methoxybenzyl--L-rhamnopyranosyl)-(12)-(3,4-di-O-benzyl--L-rhamnopyranosyl)-(14)-(benzyl 2,3-di-O-benzyl--D-galactopyranosyluronate)-(13)-4,6-di-O-benzyl-2-deoxy-2-trichloroacetamido--D-galactopyranoside (302)

(179) A mixture of octasaccharide acceptor 301 (153 g, 46 mol), tetrasaccharide donor 284 (121 mg, 64 mol), and powdered 4 MS (375 mg) in anhyd. DCM (1.6 mL) was stirred at rt under an Ar atmosphere for 1 h. The suspension was cooled to 90 C., and TMSOTf (0.4 L, 2 mol) was added. The reaction mixture was stirred for 30 min allowing the cooling bath to reach 78 C. and Et.sub.3N was added to quench the reaction since a TLC control (Tol/acetone 9:1) showed the presence of a new major compound (rf=0.24) and that no acceptor (rf=0.19) nor donor (rf=0.41) remained. The suspension was filtered over and the filtrate was concentrated to dryness. The residue was purified by flash chromatography (SiO.sub.2 20-40 m, Tol/EtOAc 92:8 to 8:2) to give dodecasaccharide 302 (156 mg, 68%) as a white foam.

(180) .sup.1H NMR (CDCl.sub.3), 7.42-6.94 (m, 129H, 126H.sub.Ar, NH-2.sub.B, NH-2.sub.B, NH-2.sub.B), 6.87-6.77 (m, 6H, H.sub.ArPMB), 5.87 (m, 1H, CH.sub.All), 5.48 (dd, 1H, J.sub.1,2=1.7 Hz, J.sub.2,3=3.1 Hz, H-2.sub.C), 5.29-5.15 (m, 10H, H-1.sub.D, H-1.sub.D, H-1.sub.D, CH.sub.2All, 3H.sub.CO2Bn, H-1.sub.B, CH.sub.2All), 5.13-5.07 (m, 3H, H.sub.Co2Bn), 5.05 (d, 1H, J=11.2 Hz, H.sub.Bn), 5.03 (d, 1H, J.sub.1,2=8.4 Hz, H-1.sub.B), 5.00 (d, 1H, J=11.3 Hz, H.sub.Bn), 4.98-4.88 (m, 7H, H-1.sub.C, H-1.sub.C, 5H.sub.Bn), 4.87-4.67 (m, 14H, H-1.sub.C, H-3.sub.B, H-3.sub.B, 11H.sub.Bn), 4.68-4.39 (m, 25H, H-3.sub.B, H-1.sub.A, H-1.sub.A, H-1.sub.A, 21H.sub.Bn), 4.38-4.31 (m, 5H, H-4.sub.A, H-4.sub.A-, H-4.sub.A, H.sub.All, H-4.sub.B*), 4.28 (d, 1H, J.sub.3,4=3.2 Hz, H-4.sub.B*), 4.22-4.16 (m, 4H, 3H.sub.Bn, H-4.sub.B), 4.16-3.98 (m, 13H, H-2.sub.C, H.sub.All, H-2.sub.C H-5.sub.A, H-5.sub.A, H-5.sub.A, 7H.sub.Bn), 3.97-3.90 (m, 3H, H-2.sub.D, 2.sub.D, H-3.sub.C), 3.89 (pt, 1H, H-2.sub.D), 3.87-3.74 (m, 10H, H-3.sub.C, H-3.sub.C, H-2.sub.B, H-2.sub.B, H-5.sub.C, H-5.sub.C, H-5.sub.C, CH.sub.3PMB), 3.71-3.55 (m, 20H, H-5.sub.D, H-5.sub.D, H-5.sub.D, H-2.sub.B, H-5.sub.B, H-5.sub.B, H-5.sub.B, H-2.sub.A, H-2.sub.A, H-2.sub.A, H-3.sub.D, H-3.sub.D, H-3.sub.D, 2CH.sub.3PMB, H-6a.sub.B), 3.47-3.24 (m, 14H, H-3.sub.A, H-3.sub.A, H-4.sub.C, H-3.sub.A, H-4.sub.C, H-4.sub.C, H-6b.sub.B, H-6a.sub.B, H-6a.sub.B, H-4.sub.D, H-6b.sub.B, H-6b.sub.D, H-4.sub.D), 2.77-2.67 (m, 4H, CH.sub.2Lev), 2.12 (s, 3H, CH.sub.3Lev), 1.32-1.29 (m, 9H, H-6.sub.D, H-6.sub.D, H-6.sub.D), 1.23-1.19 (m, 9H, H-6.sub.C, H-6.sub.C, H-6.sub.C).

(181) .sup.13C NMR (CDCl.sub.3) partial, 206.2 (CO.sub.LEV), 171.7 (CO.sub.2Lev), 166.9 (3C, C-6.sub.A, C-6.sub.A, C-6.sub.A), 162.1 (NHCO-2.sub.B), 161.7 (2C, NHCO-2.sub.B, NHCO-2.sub.B), 159.2, 159.1 (3C, C.sub.IVPMB), 130.7, 130.2 (3C, C.sub.IVPMB), 113.9, 113.7 (6C, C.sub.ArPMB), 103.9-103.8 (3C, C-1.sub.A, C-1.sub.A, C-1.sub.A), 101.0 (2C, C-1.sub.C, C-1.sub.C), 100.4, 100.2 (3C, C-1.sub.D, C-1.sub.D, C-1.sub.D), 99.2 (C-1.sub.C), 98.8 (C-1.sub.B-, C-1.sub.B), 97.4 (C-1.sub.B), 92.6, 92.2 (3C, CCl.sub.3), 67.4-67.3 (3C, C.sub.C02Bn), 55.2, 55.1 (3C, CH.sub.3PMB), 38.2 (COCH.sub.2Lev), 29.9 (CH.sub.3Lev), 28.2 (CO.sub.2CH.sub.2Lev).

(182) II. Antigenicity of the Oligo- and/or Polysaccharides of the Invention

(183) A. Materials and Methods

(184) 1) Production and Characterization of mAbs Specific for S. flexneri Serotype 6 LPS

(185) Mice were immunized with heat-killed bacteria prepared with the strain Shigella flexneri serotype 6 (SF6) named Sc544. Briefly, mice were immunized first intraperitoneally (i.p.) with 10.sup.8 heat-killed bacteria mixed with incomplete Freund adjuvant, then twice i.p. with 10.sup.8 heat-killed bacteria without adjuvant at one month interval. Immunogenicity was measured by ELISA (as described for the identification of the appropriate concentration of IgG to use for IC50 measurement) against SF6 LPS purified from the strain SF6 Sc544 according to Westphal O and Jann K (Methods Carbohydr. Chem. 5 83 (1965)). Mice displaying an anti-LPS SF6 antibody response were then submitted to an intravenous (i.v.) injection boost 3 days prior to be sacrificed for spleen recovery to perform cell fusion for the obtention of hybridoma, as described in Phalipon A. et al (J. Immunol. 176 1686 (2006).

(186) Hybridoma were selected from the supernatant of the cell culture issued from the fusion by ELISA against purified SF6 LPS. Positive hybridoma were used for the production of a large amount of the corresponding mAb, that was purified by G-protein chromatography. Thus, the hybridoma A22-4 secreting the monoclonal antibody mIgG A22-4 was deposited at the Collection Nationale de Culture de Microorganismes (CNCM, Paris, France) according to the Budapest Treaty under number I-4813, on Oct. 28, 2013.

(187) 2) ELISA for IC50 Measurements

(188) The binding of the available mIgG A22-4 to the synthetic oligosaccharides was measured as previously described (Phalipon A. et al (J. Immunol. 176 1686 (2006)). Briefly, the mIgG concentration to be used was defined in the first step. To do so, a standard curve was established for mIgG A22-4. The mIgG was incubated at different concentrations, overnight at 4 C., on microtiter plates coated with SF6 LPS purified from the strain SF6 Sc544 at a concentration of 2.5 m/mL in a carbonate buffer (pH 9.6), then with PBS-BSA 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 1/5,000 (Sigma-Aldrich) for 1 h at 37 C. After washing with PBS-Tween 20 (0.05%), the substrate was added (12 mg of p-nitrophenyl phosphate in 1.2 mL of 1 M Tris-HCl buffer (pH 8.8) and 10.8 mL of 5 M NaCl). Once the color developed, the plate was read at 405 nm Dynatech MR400 microplate reader). A standard curve OD=f([Ab]) was fitted to the quadratic equation Y=aX2+bX+c, where Y is the OD and X is the Ab concentration. A correlation factor (r.sup.2) of 0.99 was routinely obtained. The mIgG A22-4 concentration to be used in the second step for measuring the IC50, which is defined as the concentration of oligosaccharides required to inhibit 50% of mIgG binding to LPS, was chosen as follows. It corresponds to the minimal concentration of mAb which gives the maximal OD on the standard curve.

(189) Then for IC50 measurement, the mIgG A22-4, used at the concentration of 0.025 g/ml, was incubated overnight at 4 C. with different concentrations of the oligosaccharides to be tested in PBS-BSA 1%. The maximum concentration tested was 2.5 mM for all oligosaccharides. Then, measurement of unbound mIgG was performed as described above using microtiter plates coated with purified SF6 LPS. The mAb concentration was deduced from the standard curve. A 100% unbound mIgG concentration was defined for the mAb incubated in the same conditions but without any oligosaccharide. Finally, a curve corresponding to % unbound mIgG=f([oligosaccharide]) allows to calculate the IC50 for each oligosaccharide tested.

(190) B. Results

(191) The binding of mIgG A22-4 to the synthetic and oligo- or polysaccharides was evaluated in inhibition ELISA (Table B)

(192) TABLE-US-00002 Synthetic Oligosaccharides tested as propyl glycoside IC.sub.50 (mM).sup.# CDAB 0.016 .sub.AcCDAB 0.036 .sub.AcCDAB.sub.AcC 0.5 CDABCDA 0.015 CDABCDAB 0.004 BCDAB 0.025 BCDABC 0.5 BCDABCDA 0.018 DABCDAB 0.053 ABCDAB 0.019 .sup.# corresponds to values inferior to but close to the mentionned value.

REFERENCES

(193) [1] K. Hygge Blakeman, A. Weintraub, G. Widmalm, Eur. J. Biochem. 1998, 251, 534-537. [2] M. M. Levine, Vaccine 2006, 24, 3865-3873. [3] S. K. Niyogi, J. Microbiol. 2005, 43, 133-143. [4] L. von Seidlein, D. R. Kim, M. Ali, H. Lee, X. Wang, V. D. Thiem, G. Canh do, W. Chaicumpa, M. D. Agtini, A. Hossain, Z. A. Bhutta, C. Mason, O. Sethabutr, K. Talukder, G. B. Nair, J. L. Deen, K. Kotloff, J. Clemens, PLoS Med. 2006, 3, e353. [5] a) M. M. Levine, K. L. Kotloff, E. M. Barry, M. F. Pasetti, M. B. Sztein, Nat. Rev. Microbiol. 2007, 5, 540-553; b) M. N. Kweon, Curr. Opin. Infect. Dis. 2008, 21, 313-318. [6] K. L. Kotloff, J. P. Winickoff, B. Ivanoff, J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, M. M. Levine, Bull. World Health Organ. 1999, 77, 651-666. [7] a) C. Ferreccio, V. Prado, A. Ojeda, M. Cayyazo, P. Abrego, L. Guers, M. M. Levine, Am. J. Epidemiol. 1991, 134, 614-627; b) 3. H. Passwell, S. Ashkenzi, Y. Banet-Levi, R. Ramon-Saraf, N. Farzam, L. Lerner-Geva, H. Even-Nir, B. Yerushalmi, C. Y. Chu, J. Shiloach, J. B. Robbins, R. Schneerson, I. S. S. Grp, Vaccine 2010, 28, 2231-2235. [8] F. R. Noriega, F. M. Liao, D. R. Maneval, S. Ren, S. B. Formal, M. M. Levine, Infect. Immun. 1999, 67, 782-788. [9] a) D. A. Simmons, E. Romanowska, J. Med. Microbiol. 1987, 23, 289-302; b) A. V. Perepelov, M. E. Shekht, B. Liu, S. D. Shevelev, V. A. Ledov, S. N. Senchenkova, V. L. Lvov, A. S. Shashkov, L. Feng, P. G. Aparin, L. Wang, Y. A. Knirel, FEMS Immunol. Med. Microbiol. 2012, DOI: 10.1111/j.1574-1695X.2012.01000.x. [10] H. L. DuPont, R. B. Hornick, M. J. Snyder, J. P. Libonati, S. B. Formal, E. J. Gangarosa, J. Infect. Dis. 1972, 125, 12-16. [11] a) A. Phalipon, M. Tanguy, C. Grandjean, C. Guerreiro, F. Belot, D. Cohen, P. J. Sansonetti, L. A. Mulard, J. Immunol. 2009, 182, 2241-2247; b) M. Wacker, C. Waechter, in WO 2011/062625 A1, Vol. WO 2011/062625 A1 (Ed.: Glycovaxyn), CH, 2011, pp. 1-156. [12] R. W. Kaminski, E. V. Oaks, Expert Rev. Vaccines 2009, 8, 1693-1704. [13] a) P. Costantino, R. Rappuoli, F. Berti, Expert Opin Drug Discov 2011, 6, 1045-1066; b) E. Jessouroun, I. A. Freitas Brasileiro Da Silveira, C. Chagas Bastos, C. E. Frasch, C. H. Lee, in US 2007/0110762 A1, Vol. US 2007/0110762 A1, USA, 2007, pp. 1-18; c) P. J. Kniskem, S. Marburg, in Development and clinical uses of Haemophilus influenzae b conjugate vaccines (Eds.: R. W. Ellis, D. M. Granoff), Marcel Dekker, New York, 1994, pp. 37-69. [14] G. T. Hermanson, Bioconjugate techniques, 2nd Edition, Academic Press Inc., San Diego, 2008. [15] A. Fattom, R. Schneerson, D. C. Watson, W. W. Karakawa, D. Fitzgerald, I. Pastan, X. Li, J. Shiloach, D. A. Bryla, J. B. Robbins, Infect Immun 1993, 61, 1023-1032. [16] a) G. S. Bixler, Jr., R. Eby, K. M. Demiody, R. M. Woods, R. C. Seid, S. Pillai, Adv Exp Med Biol 1989, 251, 175-180; b) P. Costantino, S. Viti, A. Podda, M. A. Velmonte, L. Nencioni, R. Rappuoli, Vaccine 1992, 10, 691-698. [17] J. Kim, F. J. Michon, in WO 2005/000346 A1, Vol. WO 2005/000346 A1, USA, 2005. [18] J. H. Passwell, S. Ashkenazi, E. Harley, D. Miron, R. Ramon, N. Farzam, L. Lerner-Geva, Y. Levi, C. Chu, J. Shiloach, J. B. Robbins, R. Schneerson, Pediatr Infect Dis J 2003, 22, 701-706. [19] P. R. Paradiso, D. A. Hagerman, D. V. Madore, H. Keyserling, J. King, K. S. Reisinger, M. M. Blatter, E. Rothstein, H. H. Bernstein, J. Hackell, Pediatrics 1993, 92, 827-832. [20] D. Vahnori, A. Pessi, E. Bianchi, G. Corradin, J Immunol 1992, 149, 717-721. [21] J. Alexander, J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H. M. Serra, R. T. Kubo, A. Sette, et al., Immunity 1994, 1, 751-761. [22] S. Kaeothip, P. Pornsuriyasak, N. P. Rath, A. V. Demchenko, Org Lett 2009, 11, 799-802. [23] A. F. Bongat, M. N. Kamat, A. V. Demehenko, J Org Chem 2007, 72, 1480-1483. [24] a) P. J. Kocienski, Protecting groups, 3rd ed., Thieme, Stuttgart, 2004; b) P. G. M. Wuts, T. W. Greene, Greene's protective groups in organic synthesis, 4th ed., Wiley-Interscience, Hoboken, N.J.; Chichester, 2007. [25] A. V. Demchenko, Handbook of chemical glycosylation: advances in stereoselectivity and therapeutic relevance, Wiley-VCH, Weinheim; Chichester, 2008. [26] M. K. Gurjar, A. S. Mainkar, Tetrahedron 1992, 48, 6729-6738. [27] S. Kramer, B. Nolting, A. J. Ott, C. Vogel, J. Carbohydr. Chem. 2000, 19, 891-921. [28] J. Boutet, L. A. Mulard, Eur. J. Org. Chem. 2008, 5526-5542. [29] P. Westerduin, P. E. de Haan, M. J. Dees, J. H. van Boom, Carbohydrate Research 1988, 180, 195-205. [30] F. Belot, J. C. Jacquinet, Carbohydr. Res. 1996, 290, 79-86. [31] B. M. Pinto, D. G. Morissette, D. R. Bundle, J. Chem. Soc. Perkin Trans. 1 1987, 9-14.