PROTECTED TETRASACCHARIDES, THEIR PROCESS OF PREPARATION AND THEIR USE AS TRANSGLUCOSYLASE ACCEPTOR SUBSTRATES IN THE CHEMO-ENZYMATIC SYNTHESIS OF SHIGELLA FLEXNERI SPECIFIC OLIGOSACCHARIDES

Abstract

The present invention provides protected tetrasaccharides, their process of preparation and their use in the synthesis of oligosaccharides, in particular fragments of O-antigens from Shigella flexneri, for example of serotype 1a, 1b, 2a, 2b, 3a, X, 4a, 4b, 5a, 5b, 7a or 7b.

Claims

1-22. (canceled)

23. A compound of following formula (I.sub.0):
(.sub.TD).sub.xAB.sub.ZC(.sub.TD).sub.y-R(I.sub.0) wherein: x and y are 0 or 1, providing x+y=1; A is 2)--l-Rhap-(1.fwdarw.; B is 2)--l-Rhap-(1.fwdarw.; C is 3)--l-Rhap-(1.fwdarw.; D is 3)--d-GlcpN-(1 or 3)--d-GlcpN-(1.fwdarw.; Z is ClAc, BrAc, Ac or ; T is a protecting group capable of anchimeric assistance, or is azide (N.sub.3); R is a protecting group compatible with chain elongation into O-antigen fragments, and being orthogonal to T and Z, when Z is ClAc, BrAc or Ac; ClAc is ClCH.sub.2C(O); BrAc is BrCH.sub.2C(O).

24. A compound according to claim 23, of following formula (I):
(.sub.Cl3AcD).sub.xAB.sub.ZC(.sub.Cl3AcD).sub.y-All(I) wherein: x and y are 0 or 1, providing x+y=1; A is 2)--l-Rhap-(1.fwdarw.; B is 2)--l-Rhap-(1.fwdarw.; C is 3)--l-Rhap-(1.fwdarw.; D is 3)--d-GlcpN-(1.fwdarw. or 3)--d-GlcpN-(1.fwdarw.; Z is ClAc, BrAc, Ac or ; All is allyl; Cl3Ac is Cl.sub.3CC(O); ClAc is ClCH.sub.2C(O); BrAc is BrCH.sub.2C(O).

25. The compound according to claim 23, wherein: Cl3Ac is in position 2.sub.D, Z is in position 2.sub.C, and/or All is in position 1.sub.C, when x=1, or in position 1.sub.D, when y=1.

26. The compound according to claim 23, wherein x is 0 and y is 1, corresponding to the following formula (Ia):
AB.sub.ZC.sub.Cl3AcD-All(Ia).

27. The compound according to claim 23, of one the following formulae: ##STR00038##

28. The compound according to claim 23, wherein x is 1 and y is 0, corresponding to the following formula (Ib):
.sub.Cl3AcDAB.sub.ZC-All(Ib).

29. The compound according to claim 23, of the following formula: ##STR00039##

30. A process of preparation of a compound according to claim 23, comprising the following steps: (i) a step of contacting a protected AB.sub.ZC-triosyl donor with a protected TD-R acceptor to yield a protected AB.sub.ZC.sub.TD-R compound; and (ii) one or more steps of deprotection of the protected compound obtained in step (i) to give a compound of formula AB.sub.ZC.sub.TD-R or (i) a step of contacting a protected AB.sub.ZC-R acceptor with a protected TD donor to yield a protected .sub.TDAB.sub.ZC-R compound; and (ii) one or more steps of deprotection of the protected compound obtained in step (i) to give a compound of formula .sub.TDAB.sub.ZC-R.

31. The process according to claim 30, wherein the AB.sub.ZC-triosyl donor is of formula AB.sub.ZC-Z, wherein Z is PTFA or TCA, PTFA representing N-phenyltrifluoroacetimidyl and TCA representing trichloroacetimidyl.

32. The process according to claim 30, wherein the protected AB.sub.ZC-triosyl donor is of one of the following formulae: ##STR00040## wherein: TES is triethylsilyl Lev is levulinyl; Nap is 2-naphytlmethyl; PMB is para-methoxybenzyl.

33. The process according to claim 30, wherein the protected .sub.Cl3AcD-All acceptor is of the following formula: ##STR00041##

34. The process according to claim 30, wherein the protected AB.sub.ZC.sub.Cl3AcD-All compound is of one of the following formulae: ##STR00042##

35. The process according to claim 30, wherein the AB.sub.ZC-All acceptor is of one of the following formulae: ##STR00043## wherein: TES is triethylsilyl PMB is para-methoxybenzyl.

36. The process according to claim 30, wherein the .sub.Cl3AcD donor is of formula .sub.Cl3AcD-Z, wherein Z is PTFA or TCA, PTFA representing N-phenyltrifluoroacetimidyl and TCA representing trichloroacetimidyl.

37. The process according to claim 30, wherein the protected .sub.Cl3AcD donor is of one of the following formulae or the corresponding oxazolines: ##STR00044##

38. A method of preparation of a saccharide comprising the following steps: (i) a step of -d-glucosylation of a compound of formula (I) according to claim 1 by a sucrose-active enzyme selected from the group consisting of the enzymes of the GH13 family, the enzymes of the GH70 family, their variants, peptide fragments of said enzymes or variants, and modified enzymes/peptides derived from said enzymes, variants or fragments, to obtained an -d-glucosylated (.sub.Cl3AcD).sub.xAB.sub.ZC(.sub.Cl3AcD).sub.y-All compound; (ii) optionally, a step of acetylation of the 3.sub.A position and/or the 6.sub.D position to obtained a 3.sub.A- and/or 6.sub.D-acetylated compound; (iii) optionally, a step of cleavage of the ClAc or BrAc protecting group borne by C to obtain a 2.sub.C-hydroxylated compound; (iv) optionally, a step of (a) conversion of the ClAc or BrAc protecting group borne by C into an acetyl group (Ac), (b) conversion of the Cl3Ac masking group borne by D into an acetyl group (Ac), (c) conversion of the allyl protecting group borne by D into a propyl (Pr) moiety followed if needed by the de-O-acylation of the 2.sub.C position to obtained either a 2.sub.C-acylated or 2.sub.C-hydroxylated compound; (v) optionally, a step of chain elongation at one or both of the ends of the compound resulting from the enzymatic -d-glucosylation of the compound of formula (I) as defined above.

39. The method according to claim 38, wherein said saccharide is a fragment of O-antigens from S. flexneri.

40. The method according to claim 38, wherein said saccharide comprises one of the following fragments:
-[((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.x(L).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.e(Ac)zC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.y].sub.n-
-[((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.x(L).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.e(Ac)zC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.y].sub.n-All
-[((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.x(L).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.e(Ac)zC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(M).sub.bAcD).sub.y].sub.n-Pr wherein: L is E1.fwdarw.3 or Ac in position 3.sub.A; M is E1.fwdarw.6 or Ac in position 6.sub.D; a is 0 or 1, a is: 0 or 1 when a is 1; 0 when a is 0; e is 0 or 1 c is 0 or 1; d and d are 0 or 1, providing d+d=0 or 1; when L is Ac, c is at each occurrence independently 0 or 1, providing c+c=0 or 1; when L is E1.fwdarw.3, c is 0 or 1, providing c+c=0 or 1; when M is Ac, b is at each occurrence independently 0 or 1, providing a+b=0 or 1; when M is E1.fwdarw.6, b is 0 or 1, providing a+b=0 or 1

41. The method according to claim 38, wherein said saccharide comprises the following fragment:
-[AB.sub.(Ac)zC(E1.fwdarw.6).sub.AcD].sub.n-, or
-[(E1.fwdarw.3)AB.sub.(Ac)zC.sub.AcD].sub.n-, or
-[(E1.fwdarw.4)AB.sub.(Ac)zC.sub.AcD].sub.n-, or
-[A(E1.fwdarw.4)B.sub.(Ac)zC.sub.AcD].sub.n-.

42. A compound of one of the following formulae:
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6.sub.b).sub.Cl3AcD).sub.y-All;
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eAcC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eAcC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eC((E1.fwdarw.2).sub.a(E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6.sub.b).sub.Cl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.4).sub.a(E1.fwdarw..sup.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.c(E1.fwdarw.4).sub.cA(E1.fwdarw.3).sub.d(E1.fwdarw.4).sub.dB(E1.fwdarw.4).sub.eC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.e C((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.eAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y-All;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.eAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.eClAcC((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.x(E1.fwdarw.3).sub.cA(E1.fwdarw.3).sub.d B(E1.fwdarw.4).sub.e C((E1.fwdarw.4).sub.a(E1.fwdarw.6).sub.bCl3AcD).sub.y;
AB.sub.ClAcC(E1.fwdarw.6).sub.Cl3AcD-All;
ABC(E1.fwdarw.6).sub.Cl3AcD-All;
AB.sub.ClAcC(E1.fwdarw.6).sub.Cl3AcD;
AB.sub.AcC(E1.fwdarw.6).sub.Cl3AcD-All;
AB.sub.AcC(E1.fwdarw.6).sub.Cl3AcD;
ABC(E1.fwdarw.6).sub.Cl3AcD;
(E1.fwdarw.3)AB.sub.ClAcC.sub.Cl3AcD-All;
(E1.fwdarw.3)ABC.sub.Cl3AcD-All;
(E1.fwdarw.3)AB.sub.AcC.sub.Cl3AcD-All;
(E1.fwdarw.3)AB.sub.AcC.sub.Cl3AcD;
(E1.fwdarw.3)AB.sub.ClAcC.sub.Cl3AcD;
(E1.fwdarw.3)ABC.sub.Cl3AcD;
(E1.fwdarw.4)AB.sub.ClAcC.sub.Cl3AcD-All;
(E1.fwdarw.4)ABC.sub.Cl3AcD-All;
(E1.fwdarw.4)AB.sub.AcC.sub.Cl3AcD-All;
(E1.fwdarw.4)AB.sub.AcC.sub.Cl3AcD;
(E1.fwdarw.4)AB.sub.ClAcC.sub.Cl3AcD;
(E1.fwdarw.4)ABC.sub.Cl3AcD;
A(E1.fwdarw.4)B.sub.ClAcC.sub.Cl3AcD-All;
A(E1.fwdarw.4)BC.sub.Cl3AcD-All;
A(E1.fwdarw.4)B.sub.AcC.sub.Cl3AcD-All;
A(E1.fwdarw.4)B.sub.AcC.sub.Cl3AcD;
A(E1.fwdarw.4)B.sub.ClAcC.sub.Cl3AcD;
A(E1.fwdarw.4)BC.sub.Cl3AcD; or of one of the following formulae, wherein Z is ClAc, BrAc or Ac: ##STR00045## ##STR00046##

Description

FIGURES

[0188] FIG. 1A presents the HSQC spectrum corresponding to pentasaccharide 1.

[0189] FIG. 1B presents the HMBC spectrum corresponding to pentasaccharide 1.

EXAMPLES

Example 1: Synthesis of Compound of Formula (I)

Synthesis of a common precursor (1) to residues A, B and C

Allyl 4-O-(2-naphtylmethyl)--L-rhamnopyranoside (1)

[0190] ##STR00022##

[0191] Acetyl chloride (50 mL, 0.70 mol, 2.5 equiv.) was added dropwise to allyl alcohol (610 mL) at 0 C., the solution was stirred for 25 min, then L-rhamnose monohydrate (50 g, 277 mmol) was added. The mixture was heated for 2.5 h at 70 C. then for 15 h at 40 C. Follow up by TLC (DCM/MeOH 8:2) indicated the total conversion of the starting hemiacetal (Rf 0.2) into a less polar product (Rf 0.7). The bath temperature was cooled to 0 C. and the solution was neutralized by addition of NaHCO.sub.3 (102.5 g). The suspension was filtered over a pad of Celite and solvents were evaporated and co-evaporated three times with toluene. The brown oily residue was dissolved in anhydrous acetone (300 mL), then 2,2-dimethoxypropane (100 mL, 0.81 mol, 3.0 equiv.) and PTSA (3.04 g, 16 mmol, 0.05 equiv.) were successively added. The mixture was stirred for 3 h at rt. Follow up by TLC (DCM/MeOH 9:1) indicated the total conversion of the intermediate allyl glycoside (Rf 0.3) into a less polar product (Rf 0.6). The solution was neutralized by adding Et.sub.3N (4 mL), solvents were evaporated under reduced pressure. The residue was dissolved in DCM (600 mL) and washed with H.sub.2O (3300 mL) and brine (200 mL). The organic layer was dried by passing through phase separator filter and concentrated to dryness.

[0192] The residue was dissolved in DMF (800 mL) under Ar, the bath temperature was cooled to 5 C., and NaH (60% oil dispersion, 29.1 g, 0.73 mol, 2.4 equiv.) was added portionwise to this suspension. The mixture was stirred for 2 h at rt, then 2-bromomethylnaphthalene (73.5 g, 0.33 mol, 1.2 equiv.) was added portionwise at 5 C. and the reaction mixture was stirred at rt for 2 h. Follow up by TLC (cyclohexane/EtOAc 7:3) indicated the total conversion of the intermediate alcohol (Rf 0.3) into a less polar product (Rf 0.67). The reaction was quenched at 0 C. by addition of MeOH (50 mL). Solvents were eliminated under reduced pressure and volatiles were co-evaporated with toluene. The residue was taken up in EtOAc (400 mL) and washed with H.sub.2O (3300 mL) and brine (150 mL). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness.

[0193] The residue was dissolved in 80% aq. AcOH (500 mL) and the solution was stirred for 6 h at 80 C. then over the weekend at rt and heating was continued for 5 h at 80 C. Follow up by TLC (cyclohexane/EtOAc 5:5) indicated the total conversion of the intermediate acetal (Rf 1.0) into a more polar product (Rf 0.2). Solvents were removed under vacuum and traces of AcOH were eliminated by co-evaporation with toluene (3400 mL) to give a brown solid. Filtration over a pad of silica eluting with a 4:1 mixture of cHex/EtOAc then 1:1 mixture of cyclohexane/EtOAc then recrystallization in hot cyclohexane afforded the expected diol (65.5 g, 69%) as a pale brown solid. Mother liquors were further purified by flash column chromatography (cyclohexane/EtOAc 100:0 to 50:50) to give an additional amount of expected diol (11.6 g). The total yield of diol 1 is 81% over 4 steps.

[0194] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.87-7.78 (m, 4H, H.sub.ArNap), 7.52-7.44 (m, 3H, H, H.sub.ArNap), 5.89 (dddd, 1H, J=17.2, 10.4, 6.0, 5.2 Hz, CHCH.sub.2), 5.28 (dq.sub.app, 1H, J=17.2, 1.5 Hz, CHCH), 5.19 (dq.sub.app, 1H, J=10.4, 1.5 Hz, CHCH.sub.2), 4.95-4.86 (m, 2H, H.sub.ArNap), 4.81 (d, J=1.4 Hz, 1H, H-1), 4.17 (ddt, 1H, J=12.9, 5.1, 1.5 Hz, 1H, H.sub.All), 4.02-3.93 (m, 3H, H.sub.All, H-2, H-3), 3.79 (dq, 1H, J=9.2, 6.3 Hz, H-5), 3.41 (t.sub.app, 1H, J=9.2 Hz, H-4), 2.45 (brs, 2H, OH), 1.38 (d, 3H, J=6.3 Hz, H-6).

[0195] .sup.13C NMR (100 MHz, CDCl.sub.3) 133.8 (CHCH.sub.2), 128.4 (C.sub.IVAr), 128.0 (C.sub.IVAr), 127.7 (C.sub.IVAr), 126.7-125.8 (7C, C.sub.Ar), 117.4 (CHCH.sub.2), 98.5 (C-1 .sup.1J.sub.C,H=170.1 Hz), 75.1 (CH.sub.2Nap), 71.6, 71.3 (2C, C-2, C-3), 68.0 (C.sub.All), 67.3 (C-5), 18.1 (C-6).

[0196] HRMS (ESI): m/z 362.1985 (calcd for C.sub.16H.sub.22O.sub.5Na [M+NH.sub.4].sup.+ m/z 362.1967); m/z 367.1576 (calcd for C.sub.43H.sub.51ClO.sub.12Na [M+Na].sup.+ m/z 367.1521).

Synthesis of the Rhamnopyranosyl Donors (5 and 5a) Used as Precursor to Residues A and B

[0197] ##STR00023##

[0198] The TES Derivative

Allyl 4-O-(2-naphthylmethyl)-3-O-triethylsilyl--L-rhamnopyranoside (2)

[0199] To a solution of allyl 4-O-(2-naphthylmethyl)--L-rhamnopyranoside (diol 1) (5.0 g, 14.5 mmol) in anhydrous acetonitrile (MeCN, 200 mL) stirred at 0 C. were successively added dropwise chlorotriethylsilane (TESCl 99%, 2.9 mL, 17.4 mmol, 1.2 equiv) and N,N-diisopropylethylamine (DIPEA 99.5%, 3.8 mL, 21.8 mmol, 1.5 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (cyclohexane/EtOAc 8:2) showed complete consumption of the starting material (Rf=0.13) and the presence of a main product (Rf=0.70). Methanol (1.2 mL, 29.0 mmol, 0.5 equiv) was added, the mixture was stirred for 15 min, and volatiles were evaporated under reduced pressure. Toluene was added and a precipitate appeared. The suspension was filtered and the filtrate was concentrated to dryness under vacuum. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (toluene/ethyl acetate 95:5) to give compound 2 as a yellow oil (4.9 g, 74%).

Allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl--L-rhamnopyranoside (3)

[0200] Route 1: To a solution of allyl 4-O-(2-naphthylmethyl)-3-O-triethylsilyl--L-rhamnopyranoside (alcohol 2) (5.0 g, 10.9 mmol) in anhydrous dichloromethane (DCM, 65 mL) stirred at room temperature were successively added 1,3-dicyclohexylcarbodiimide (DCC 99%, 7.65 g, 37.1 mmol, 3.4 equiv), 4-dimethylaminopyridine (DMAP, 1.07 g, 8.73 mmol, 0.8 equiv) and levulinic acid (LevOH 98%, 4.69 mL, 45.8 mmol, 4.2 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete consumption of the starting material (Rf=0.60) and the presence of a more polar compound (Rf=0.53). The reaction mixture was concentrated under reduced pressure. The crude material was taken in EtOAc (50 mL) and the resulting suspension was filtered on a pad of Celite. Water (30 mL) was added to the filtrate and the organic layer was washed successively with 10% aqueous copper (II) sulfate (30 mL), water (30 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by eluting from a column of Et.sub.3N-treated silica gel (cyclohexane/EtOAc 9:1 to 5:5) to give the fully protected 3 as a yellow oil (4.5 g, 74%).

[0201] Route 2: To a solution of allyl 4-O-(2-naphthylmethyl)--L-rhamnopyranoside (diol 1) (20.0 g, 58.1 mmol) in anhydrous MeCN (800 mL) stirred at 0 C. were successively added dropwise TESCl (11.70 mL, 69.7 mmol, 1.2 equiv) and DIPEA (15.2 mL, 87.1 mmol, 1.5 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (cyclohexane/EtOAc 8:2) showed complete consumption of the starting material (Rf=0.13) and the presence of a main product (Rf=0.70). Methanol (1.2 mL, 29.0 mmol, 0.5 equiv) was added, the mixture was stirred for 15 min, and volatiles were evaporated under reduced pressure. Toluene was added and a precipitate appeared. The suspension was filtered and the filtrate was concentrated to dryness under vacuum.

[0202] To a solution of the crude alcohol 2 in anhydrous DCM (200 mL) stirred at room temperature were successively added DCC (40.74 g, 197.4 mmol, 3.4 equiv), DMAP (5.7 g, 46.5 mmol, 0.8 equiv) and levulinic acid (23.8 mL, 232.3 mmol, 4.0 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete consumption of the starting material (Rf=0.60) and the presence of a more polar product (Rf=0.53). The reaction mixture was concentrated under reduced pressure. The crude material was taken in EtOAc (50 mL) and the resulting suspension was filtered on a pad of Celite. Water (30 mL) was added to the filtrate and the organic layer was washed successively with 10% aqueous copper (II) sulfate (30 mL), water (30 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by eluting from a column of Et.sub.3N-treated silica gel (cyclohexane/ethyl acetate 9:1 to 5:5) to give the fully protected 3 as a yellow oil with (30.7 g, 95%).

2-O-Levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-/-L-rhamnopyranose (4)

[0203] To a solution of allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl--L-rhamnopyranoside (allyl glycoside 3) (2.5 g, 4.49 mmol) in anhydrous THF (60 mL) stirred at room temperature was added hydrogen-activated 1,5-cyclooctadienebis-(methyldiphenylphosphine)iridium(1)hexafluorophosphate (76.0 mg, 90 mol, 0.02 equiv). After stirring the reaction mixture for 2 hours at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete conversion of the starting material (Rf=0.53) into a less polar product (Rf=0.56). N-Iodosuccinimide (NIS 95%, 1.21 g, 5.39 mmol, 1.2 equiv) in 1:5 water/THF (30 mL) and then distilled water (40 mL) were added to the mixture stirred at 0 C. After stirring the reaction mixture for 2 h at this temperature, TLC (cyclohexane/EtOAc 7:3) showed complete conversion of the intermediate (Rf=0.56) into a more polar product (Rf=0.26). 10% aqueous sodium metabisulfite (50 mL) was added. THF was evaporated under reduced pressure and DCM (50 mL) was added. The aqueous layer was extracted twice with DCM (30 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate (30 mL) and then brine (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (cyclohexane/EtOAc 8:2 to 6:4) to give hemiacetal 4 as a yellow oil (2.1 g, 89%, / 8:2).

2-O-Levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsllyl-/-L-rhamnopyranosyl trichloroacetimidate (5)

[0204] Route 1: To a solution of 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-L-rhamnopyranose (hemiacetal 4) (2.00 g, 3.87 mmol) in anhydrous 1,2-dichloroethane (DCE, 30 mL) stirred at room temperature were successively added trichloroacetonitrile (Cl.sub.3CCN 98%, 1.16 mL, 11.6 mmol, 3.0 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU 98%, 289 L, 1.94 mmol, 0.5 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (cyclohexane/ethyl acetate 7:3) showed complete consumption of the starting material (Rf=0.26) into a less polar product (Rf=0.53). Volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of triethylamine-treated silica gel (cyclohexane/ethyl acetate 9:1 to 5:5) to give donor 5 as a whitish crystalline solid (2.3 g, 90%, / 95:5).

[0205] Route 2: To a solution of allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl--L-rhamnopyranoside (allyl glycoside 3) (15.0 g, 26.9 mmol) in anhydrous THF (300 mL) stirred at room temperature was added hydrogen-activated 1,5-cyclooctadienebis-(methyldiphenylphosphine)iridium(I)hexafluorophosphate ([Ir], 456 mg, 540 mol, 0.02 EtOAc 7:3) showed complete conversion of the starting material (Rf=0.53) into a less polar product (Rf=0.56). NIS (7.27 g, 32.3 mmol, 1.2 equiv) in 1:5 water/THF (150 mL) and then distilled water (250 mL) were added to the mixture stirred at 0 C. After stirring the reaction mixture for 2 h at this temperature, TLC (cyclohexane/EtOAc 7:3) showed complete conversion of the intermediate (Rf=0.56) into a more polar product (Rf=0.26). 10% aqueous sodium metabisulfite (100 mL) was added. THF was evaporated under reduced pressure and DCM (500 mL) was added. The aqueous layer was extracted twice with DCM (300 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate (200 mL) and then brine (200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum (m: 14.3 g).

[0206] To a solution of the crude material (13.9 g) in anhydrous DCE (210 mL) stirred at room temperature were successively added Cl.sub.3CCN (8.1 mL, 80.8 mmol, 3.0 equiv) and DBU (2.01 mL, 13.5 mmol, 0.5 equiv). After stirring the reaction mixture for 2 h 50 at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete consumption of the starting material (Rf=0.26) into a less polar product (Rf=0.53). Volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (cyclohexane/EtOAc 9:1 to 5:5) to give donor 5 as a whitish crystalline solid (15.04 g, 84%, a/3 95:5).

The BDA Derivative

[0207] ##STR00024##

Allyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)--L-rhamnopyranoside (2a)

[0208] Acetyl chloride (34 mL, 475 mmol, 2.5 equiv) was added dropwise to allyl alcohol (420 mL) at 0 C. The solution was stirred for 25 min and L-rhamnose monohydrate (34.3 g, 190 mmol) was added. The mixture was heated for 2.5 h at 70 C. then for 15 h at 40 C. Follow up by TLC (DCM/MeOH 8:2) indicated the total conversion of L-rhamnose (Rf=0.2) into a less polar product (Rf 0.7). The bath temperature was cooled to 0 C. and the solution was neutralized by addition of solid NaHCO.sub.3 (102.5 g). The suspension was filtered off a pad of Celite and solvents were evaporated and co-evaporated three times with toluene. To a solution of crude allyl rhamnoside (190 mmol) in anhydrous methanol (1.0 L) stirred at room temperature were successively added butan-2,3-dione (18.3 mL, 0.21 mol, 1.1 equiv), trimethyl orthoformate (83 mL, 0.76 mol, 4.0 equiv) and boron trifluoride etherate (11.7 mL, 95 mmol, 0.5 equiv). After stirring the reaction mixture for 1.5 h under reflux, a TLC follow up (DCM/MeOH 95:5) showed complete consumption of the starting material (Rf=0.3) and the presence of a main product (Rf=0.5). Et.sub.3N was slowly added to the reaction mixture at 0 C. until neutralization and volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (cyclohexane/EtOAc 8:2 to 6:4) to give compound 2a as a brown oil (57.8 g, 96%, 2 isomers 9:1).

Allyl 2-O-levulinyl-3,4-O-(2,3-dimethoxybutan-2,3-diyl)--L-rhamnopyranoside (3a)

[0209] To a solution alcohol 2a (27.0 g, 84.8 mmol) in anhydrous dichloromethane (650 mL) stirred at rt were successively added DCC (59.5 g, 0.29 mol, 3.4 equiv.), DMAP (8.29 g, 67.9 mmol, 0.8 equiv.) and levulinic acid (36.5 mL, 0.36 mol, 4.2 equiv.). After stirring the reaction mixture at rt for 2 h, a TLC follow up (DCM/MeOH 98:2) showed complete consumption of the starting material (Rf=0.25) and the presence of two less polar compounds (Rf=0.6, 0.65). The reaction mixture was concentrated under reduced pressure. Saturated aqueous NaHCO.sub.3 (300 mL) was added to the reaction mixture. The aqueous layer was extracted once with DCM (300 mL) and the combine organic phases were washed twice with brine (150 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of silica gel (DCM/MeOH 1:0 to 9:1) to give the fully protected 3a as a brown oil (37.5 g, 95%, 2 isomers 9:1).

2-O-Levulinyl-3,4-O-(2,3-dimethoxybutan-2,3-diyl)--L-rhamnopyranosyl trichloroacetimidate (5a)

[0210] To a solution of rhamnoside 3a (37.5 g, 90.0 mmol) in anhyd. tetrahydrofuran (THF, 500 mL) stirred at rt was added hydrogen-activated 1,5-cyclooctadienebis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (1.5 g, 1.80 mmol, 0.02 equiv.). After stirring the reaction mixture at room temperature for 2 h, TLC (cyclohexane/EtOAc 5:5) showed complete conversion of the starting material (Rf=0.8, 0.85) into two closely eluting products (Rf=0.8, 0.9). N-iodosuccinimide (NIS, 24.3 g, 108 mmol, 1.2 equiv.) in water/THF (1:5, 250 mL) and then additional water (370 mL) were added to the mixture stirred at 0 C. After stirring the reaction mixture for 2 h at this temperature, TLC (cyclohexane/EtOAc 5:5) showed complete conversion of the intermediate into two more polar products (Rf=0.45, 0.5). Saturated aqueous sodium metabisulfite (500 mL) and then ethyl acetate (500 mL) were added. The aq. layer was extracted twice with ethyl acetate (300 mL) and the combined organic phases were washed with saturated aqueous NaHCO.sub.3 (300 mL) and then brine (300 mL). The organic layer was dried over anhyd. sodium sulfate, filtered and concentrated under vacuum.

[0211] To a solution of crude hemiacetal 4a (90.0 mmol) in anhydrous DCE (500 mL) stirred at rt were successively added trichloroacetonitrile (27.1 mL, 0.27 mol, 3.0 equiv.) and DCC (6.7 mL, 45.0 mmol, 0.5 equiv.). After stirring the reaction mixture for 2.5 h at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete consumption of the starting material (Rf=0.45, 0.5) into two less polar product (Rf=0.75, 0.5). Volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (cyclohexane/EtOAc 8:2 to 6:4) to give donor 5a as a yellow to brownish oil (38.6 g, 82%, a only, 2 isomers 9:1).

Synthesis of the Glucosamine D Acceptor

[0212] The known glucosamine D acceptor was obtained following the procedure below, through route A or the improved route B (Carbohydrate Chemistry: Proven Synthetic Methods, Eds P. Murphy and C. Vogel, 2017, vol. 4, chap. 39, in press).

##STR00025##

Synthesis of the Glucosamine D Donor.

[0213] The known glucosamine D donor 12 was obtained as published (Tetrahedron Lett. (2008) 49, 5339-42). Alternatively, the analogue of donor 12, equipped with a 4,6-O-benzylidene acetal instead of a 4,6-O-isopropylidene acetal could be obtained from the D acceptor 9 according to the procedure described for the conversion of alcohol 10 into donor 12.

[0214] It is noteworthy that alcohol 10 can also serve as a suitable acceptor in the synthesis of compounds of the formula (Ia).

##STR00026##

[0215] A synthesis of donor 12a, which encompasses a 4,6-O-benzylidene acetal and a TBS ether in place of the 4,6-O-isopropylidene acetal and the Lev group, respectively, is exemplified in the following.

##STR00027##

Allyl 4,6-O-benzyildene-3-O-tert-butyldimethysiyl-2-deoxy-2-trichloroacetamido--D-glucopyranoside (11a)

[0216] To a solution of allyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido--D-glucopyranoside (10) (2.0 g, 4.42 mmol) in anhydrous dichloromethane (40 mL), stirred at 78 C., were successively added 2,6-lutidine (2.06 mL, 17.7 mmol, 4.0 equiv) and tert-butyldimethylsilyl trifluoromethanesulfonate (2.54 mL, 11.05 mmol, 2.5 equiv). After stirring the reaction mixture overnight at room temperature, a TLC follow up (toluene/ethyl acetate 8:2) showed complete consumption of the starting material (Rf=0.25) and the presence of a less polar product (Rf=0.7). Distilled water (50 mL) and ethyl acetate (200 mL) were added. The aqueous layer was extracted with ethyl acetate (100 mL) and the combined organic phases were washed with water (50 mL), 10% aq. citric acid (50 mL) and then water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 95:5 to 9:1) to give the fully protected 11a as a white powder (2.32 g, 92%).

4,6-O-Benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-trichloroacetamido-/-D-glucopyranosyl N-(phenyl)trifluoroacetimidate (12a)

[0217] To a solution of glucosaminide 11a (2.27 g, 4.0 mmol) in anhydrous THF (200 mL) stirred at room temperature was added hydrogen-activated 1,5-cyclooctadienebis(methyldiphenylphosphine)iridium(I)hexafluorophosphate (170 mg, 0.20 mmol, 0.05 equiv). After stirring the reaction mixture for 4 hours at room temperature, a TLC follow up (toluene/ethyl acetate 9:1) showed complete conversion of the starting material (Rf=0.6) into a less polar product (Rf=0.6). N-Iodosuccinimide (1.35 g, 6.0 mmol, 1.5 equiv) in 1:5 water/THF (56 mL) was then added to the mixture stirred at room temperature. After stirring for 2 h at this temperature, a TLC follow up (toluene/EtOAc 9:1) showed complete conversion of the intermediate into a more polar product (Rf=0.2). Saturated aqueous sodium metabisulfite (200 mL) and then ethyl acetate (500 mL) were added. The aqueous layer was extracted twice with ethyl acetate (250 mL) and the combined organic phases were washed with saturated aqueous NaHCO.sub.3 (200 mL) and then brine (200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum.

[0218] To a solution of crude hemiacetal (4.42 mmol) in anhydrous acetone (36 mL), stirred at room temperature, were successively added N-(phenyl)trifluoroacetimidyl chloride (1.80 mL, 6.63 mmol, 1.5 equiv) and potassium carbonate (1.53 g, 11.05 mmol, 2.5 equiv). After stirring the reaction mixture for 3 hours at room temperature, TLC (toluene/EtOAc 8:2) showed complete consumption of the starting material (Rf=0.2) and the presence of a mixture of less polar products (Rf=0.75 and 0.8). The mixture was filtered off a pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (toluene/EtOAc 95:5 to 9:1) to give a 2:1 mixture of the N-(phenyl)trifluoroacetimidate donor 12a and oxazoline 12b as a brown oil (2.57 g, 65% over two steps).

Synthesis of the AB.SUB.ClAc.C-Z

Selected Examples

[0219] ##STR00028## ##STR00029##

Allyl 2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (13)

[0220] Diol 1 (2.0 g, 6.0 mmol) was solubilized in anhydrous MeCN (5.0 mL). To the solution was added trimethylchloroorthoacetate (2.35 mL, 3.0 equiv) and PTSA (90 mg, 0.08 equiv). The solution was stirred at room temperature for 1 hour (reaction followed by TLC Toluene/EtOAc 7:3). To the reaction medium cooled to 0 C. was added a 90% aqueous TFA (3.0 mL) and the reaction mixture was stirred at room temperature for 15 min. Water (20 mL) was added. The product was extracted with DCM (240 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (225 mL) and brine (25 mL). The aqueous phase was extracted with DCM (225 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered, evaporated and finally co-evaporated with toluene to yield the crude alcohol 13 as a 92:8 mixture of regioisomers.

Allyl 2-O-levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (14)

[0221] A solution of trichloroacetimidate 5 (5.84 g, 8.83 mmol) and crude acceptor 13 (4.09 g, 1.1 equiv) in toluene (88 mL) containing 4A-MS (1.25 g) was stirred at room temperature for 15 min, then at 60 C. for 15 min. tert-Butyldimethylsilyl trifluoromethanesulfonate (TBSOTf, 100 L, 0.05 equiv) was added to reaction mixture stirred at 60 C. and the bath was left to reach 40 C. After stirring for 1 h, Et.sub.3N was added to the suspension at 35 C., the mixture was filtered through a pad of Celite, and the filtrate was concentrated to dryness. Rapid filtration of the residue over silica gel and crystallization in EtOAc/pentane 5:1 (400 mL) gave the fully protected 14 (3.83 g, 72%).

Allyl 2-O-levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl--L-rhamnopyranosyl-(12)-4-O-(2-naphtylmethyl)-3-O-triethylsilyl--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (16)

[0222] A solution of trichloroacetimidate 5 (1.04 g, 1.24 equiv) and alcohol 15 (1.0 g, 1.22 mmol) in toluene (30 mL) containing 4A-MS (1.38 g) was stirred at room temperature for 15 min, then at 20 C. for 15 min. TMSOTf (11 L, 0.05 equiv) was added to reaction mixture stirred at 20 C. and the bath was left to reach 10 C. After stirring for 1 h at this temperature, stirring is pursued for 1 h while the bath slowly reached room temperature. Et.sub.3N was added to the suspension, the mixture was filtered through a pad of Celite, and the filtrate was concentrated to dryness. Column chromatography gave the fully protected AB.sub.ClAcC trisaccharide 16 (1.36 g, 85%).

[0223] HRMS (ESI+): m/z 1336.6191 (calcd for C.sub.73H.sub.99Cl.sub.4NO.sub.16Si.sub.2 [M+NH.sub.4].sup.+) found m/z 1336.6171.

2-O-Levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl--L-rhamnopyranosyl-(12)-4-O-(2-naphtylmethyl)-3-O-triethylsilyl--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)-/-L-rhamnopyranose (17)

[0224] To a solution of the fully protected ABC.sub.ClAcC (1.04 g, 7.6 mmol) in anhydrous THF (35 mL) stirred at room temperature was added hydrogen-activated [Ir] (13.0 mg, 0.02 equiv). After stirring the reaction mixture for 45 min at room temperature, TLC (toluene/EtOAc 9:1) showed complete conversion of the starting material into a less polar product. The reaction mixture was cooled to 0 C., NIS (205 mg, 1.2 equiv) in 1:5 water/THF (17.5 mL) and then distilled water (25 mL) were added to the mixture stirred at 0 C. After stirring the reaction mixture for 1.5 h at this temperature, TLC (toluene/ethyl acetate 9:1) showed complete conversion of the intermediate into a more polar product. 10% aqueous sodium metabisulfite (50 mL) was added. THF was evaporated under reduced pressure and DCM (100 mL) was added. The aqueous layer was extracted twice with DCM (50 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate (50 mL) and then brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (toluene/ethyl acetate 85:15 to 0:100) to give hemiacetal 17 (825 mg, 85%). HRMS (ESI+): m/z 1296.5878 (calcd for C.sub.70H.sub.95Cl.sub.4NO.sub.16Si.sub.2 [M+NH.sub.4].sup.+) found m/z 1296.5922.

[0225] To a solution of the AB.sub.ClAcC triose 17 (700 mg, 550 mol) in anhydrous DCE (5.0 mL) stirred at room temperature were successively added Cl.sub.3CCN (165 L, 3.0 equiv) and DBU (40 L, 0.5 equiv). After stirring the reaction mixture for 45 min at room temperature, the same amounts of CCl.sub.3CN and DBU were added and the reaction was stirred for 1 h more at room temperature. Volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (toluene/EtOAc 8:2 containing 3% Et.sub.3N) to give donor 18 (830 mg, 84%). HRMS (ESI+): m/z 1439.4974 (calcd for C.sub.72H.sub.95Cl.sub.4N.sub.2O.sub.16Si.sub.2 [M+NH.sub.4].sup.+) found m/z 1439.4912.

[0226] To a solution of the AB.sub.ClAcC triose 17 (1.56 g, 1.22 mmol) in acetone (24.4 mL) stirred at room temperature were successively added K.sub.2CO.sub.3 (337 mL, 2.0 equiv) and N-phenyltrifluoroacetimidyl chloride (PTFACl, 290 L, 1.5 equiv). After stirring the reaction mixture overnight at room temperature, a TLC (cyclohexane/EtOAc 2:8) indicated that that the starting 17 had been converted to a less polar product. The suspension was filtered over a pad of Celite and volatiles were evaporated under reduced pressure. The residue was purified by column chromatography to give donor 19 (1.56 g, 88%).

[0227] AB.sub.ClAcC.sub.Cl3AcD-All may also be obtained through the alternative route B as defined below, whereby the protecting group differs from that shown above and are: R.sup.1=Nap, R.sup.3, R.sup.4=R.sup.6, R.sup.4=BDA, R.sup.2=ClAc, R.sup.5=Lev, R.sup.8=All, R.sup.9=Cl3Ac).

##STR00030##

Selected Examples

Allyl 2-O-levulinyl-3,4-O-(2,3-dimethoxybutan-2,3-diyl)-act-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (14a)

[0228] To a solution of crude allyl 2-O-chloroacetyl-4-O-(2-methylnaphthyl)--L-rhamnopyranoside (5, 6.91 mmol, 1.2 equiv.) in anhyd. diethyl ether (60 mL), stirred at rt, were successively added donor 5a (3.0 g, 5.76 mmol) and activated 4A molecular sieves (3.0 g). After stirring for 15 min at rt, the reaction mixture was cooled down to 20 C. and stirred for an additional 15 min at this temperature. Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.18 mL, 1.15 mmol, 0.2 equiv.) was then slowly added. After stirring the reaction mixture for 1 h at 20 C., TLC (toluene/ethyl acetate 7:3) showed complete consumption of the donor 5a (Rf=0.4) and the presence of a less polar product (Rf=0.6). Triethylamine was then added until neutralization. The reaction mixture was stirred for 15 min at 20 C. and then filtered off a pad of Celite. The filtrate was concentrated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (cyclohexane/ethyl acetate 97:3 to 7:3) to give disaccharide 14a as a yellow oil (3.1 g, 69%).

Allyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (15a)

[0229] To a solution of disaccharide 14a (1.0 g, 1.28 mmol) in pyridine/acetic acid (3:2, 13 mL), stirred at room temperature, was added hydrazine monohydrate (125 L, 2.57 mmol, 2.0 equiv.). After stirring the reaction mixture at room temperature for 1 h, a TLC follow up (toluene/ethyl acetate 7:3) showed complete consumption of the starting material (Rf=0.6) and the presence of a closely eluting product (Rf=0.6). Saturated aqueous NaHCO.sub.3 (15 mL) was added and the aqueous layer was extracted twice with dichloromethane (15 mL) and the combine organic phases were washed once with brine (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 9:1 to 7:3) to give disaccharide 15a as a yellow oil (0.63 g, 72%).

Allyl 3,4-O-(2,3-dimethoxybutan-2,3-diyl)-2-O-levulinyl--L-rhamnopyranosyl-(12)-3,4-O-(2,3-dimethoxybutan-2,3-diyl)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (16a)

[0230] To a solution of alcohol 15a (300 mg, 0.44 mmol) in anhyd. diethyl ether (4.0 mL), stirred at rt, were successively added donor 5 (252 mg, 0.48 mmol, 1.1 equiv.) and activated 4A MS (0.3 g). After stirring the reaction mixture for 15 min at rt, the reaction mixture was cooled down to 20 C. and stirred for an additional 15 min at this temperature. Trimethylsilyl trifluoromethanesulfonate (6.8 L, 44 mol, 0.1 equiv.) was then slowly added. After stirring the reaction mixture for 2 h at 20 C., TLC (toluene/diethyl ether/ethyl acetate 5:4:1) showed complete consumption of disaccharide 7 (Rf=0.65) and the presence of a less polar product (Rf=0.7). Triethylamine was then added until neutralization. The reaction mixture was stirred for 15 minutes at 20 C. and then filtered off a pad of Celite. The filtrate was concentrated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 9:1 to 7:3) to give trisaccharide 16a as a yellow to brown oil (370 mg, 81%).

Synthesis of AB.SUB.ClAc.C.SUB.Cl3Ac.D-All

[0231] AB.sub.ClAcC.sub.Cl3AcD-All (ABCD, 21) was obtained following the generic route B (the first steps of route B up to the triosyl donor 18 being in particular described above) as defined below. The activation glycosylation and a final two-step orthogonal deprotection of route B are in particular performed with a rhamnotriosyl donor and a monosaccharide D acceptor 9 as shown in the more detailed scheme, which just follows. In the exemplified synthesis (R.sup.1=R.sup.4=Nap, R.sup.3=R.sup.6=TES, R.sup.2=ClAc, R.sup.5=Lev, R.sup.8=All, R.sup.9=Cl3Ac).

##STR00031##

[0232] AB.sub.ClAcC.sub.Cl3AcD-All may also be obtained through the alternative route A as defined in the overview scheme below.

[0233] AB.sub.ClAcC.sub.Cl3AcD-All may also be obtained through the alternative route B as defined below, whereby the protecting group differ from that shown above and are: R.sup.1=PMB, R.sup.3=R.sup.4=Nap, R.sup.6=TES, R.sup.2=ClAc, R.sup.5=Lev, R.sup.8=All, R.sup.9=Cl3Ac).

Selected Examples

[0234] A solution of the triosyl trichloroacetimidate 18 (924 mg, 649 mol) and the D acceptor 9 (440 mg, 1.5 equiv) in toluene/DCM (3:1, 37 mL) containing 4A-MS (1.91 g) was stirred at room temperature for 15 min, then at 15 C. for 15 min. TBSOTf (22 L, 0.15 equiv) was added to reaction mixture stirred at 15 C. and the bath was left to reach 0 C. in 1.3 h. A follow up by TLC (toluene/EtOAc 9:1) indicated consumption of the donor. Et.sub.3N was added to the suspension, the mixture was filtered through a pad of Celite, and the filtrate was concentrated to dryness. Column chromatography gave the fully protected AB.sub.ClAcC.sub.Cl3AcD tetrasaccharide (20, 932 mg, 84%).

[0235] HRMS (ESI+): m/z 1729.6128 (calcd for C.sub.88H.sub.113Cl.sub.4N.sub.2O.sub.21Si.sub.2 [M+NH.sub.4].sup.+) found m/z 1729.6161.

Allyl -L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl--L-rhamnopyranosyl-(13)-2-deoxy-2-N-trichloroacetyl--D-glucopyranoside (21)

[0236] To a solution of the alcohol (222 mg, 137 mol)issued from the delevulinylation of the fully protected 20in toluene stirred at 0 C. was added TFA to reach a TFA/toluene ratio of 9:1. The reaction mixture was stirred overnight, at which point a follow up by TLC indicated total consumption of the starting tetrasaccharide and the presence of a major more polar product. Volatiles were coevaporated twice with toluene, then once with acetonitrile and finally with MeOH. The crude material was purified by reverse phase flash chromatography (H.sub.2O/MeCN 0.fwdarw.50%), then by RP-HPLC (MeCN/H.sub.2O 20.5%) to give the target AB.sub.ClAcC.sub.Cl3AcD-All 21 following freeze-drying (70 mg, 36%). RP-HPLC (C18 RP fusion (4.6250 mm, 4.0 m, 80 , CH.sub.3CN in H.sub.2O (30% for 4 min, then 30-40% over 7 min, at 1.0 mL.Math.min.sup.1), 40 C., : 220 nm)=7.6 min.

[0237] .sup.1H NMR (800 MHz, D.sub.2O), 5.84 (m, 1H, CH), 5.25 (m, 1H, J.sub.trans=17.3 Hz, =CH.sub.2), 5.19 (m, 1H, J.sub.cis=10.5 Hz, CH.sub.2), 5.13 (dd, 1H, J.sub.2,3=3.0 Hz, J.sub.2,1=1.9 Hz, H-2c), 5.11 (d, 1H, J.sub.1,2=1.3 Hz, H-1.sub.B), 4.86 (d, 1H, J.sub.1,2=1.6 Hz, H-1.sub.C), 4.86 (d, 1H, J.sub.1,2=1.5 Hz, H-1.sub.A), 4.66 (d, 1H, J.sub.1,2=8.0 Hz, H-1.sub.D), 4.28 (m, 1H, H.sub.All), 4.27 (d, 1H, J.sub.gem=15.4 Hz, H.sub.ClAc), 4.23 (d, 1H, J.sub.gem=15.4 Hz, H.sub.ClAc), 4.12 (m, 1H, H.sub.All), 4.05 (m, 1H, H-5.sub.C), 3.99 (dd, 1H, J.sub.2,3=3.3 Hz, J.sub.2,1=1.7 Hz, H-2.sub.A), 3.93 (dd, 1H, J.sub.3,4=9.7 Hz, J.sub.3,2=3.1 Hz, H-3.sub.C), 3.89 (dd, 1H, J.sub.2,1=4.7 Hz, J.sub.2,1=1.7 Hz, H-2.sub.B), 3.88 (d, 1H, J.sub.6,5=2.1 Hz, H-6b.sub.D), 3.86 (m, 1H, H-2.sub.D), 3.72 (m, 2H, H-3.sub.D, H-3.sub.A), 3.71 (d, 1H, J.sub.6,5=3.3 Hz, H-6a.sub.D), 3.69 (m, 1H, H-5s), 3.63 (dd, 1H, J.sub.3,4=9.9 Hz, J.sub.3,2=3.5 Hz, H-3.sub.B), 3.62 (m, 1H, H-5.sub.A), 3.54 (dd, 1H, J.sub.4,5=9.9 Hz, J.sub.4,3=9.9 Hz, H-4.sub.D), 3.52 (dd, 1H, J.sub.4,5=9.8 Hz, J.sub.4,3=9.8 Hz, H-4.sub.C), 3.42 (m, 1H, H-5.sub.D), 3.38 (dd, 1H, J.sub.4,5=9.8 Hz, J.sub.4,3=9.8 Hz, H-4.sub.B), 3.37 (dd, 1H, J.sub.4,5=9.8 Hz, J.sub.4,3=9.8 Hz, H-4.sub.A), 1.25 (d, 3H, J.sub.6s, =6.2 Hz, H-6.sub.B), 1.20 (d, 3H, J.sub.6,5=6.4 Hz, H-6.sub.A), 1.19 (d, 3H, J.sub.6,5=6.4 Hz, H-6.sub.C).

[0238] .sup.13C NMR (800 MHz, D.sub.2O), 168.6 (CO.sub.ClAc), 164.7 (CO.sub.NHC(O)CCl.sub.3), 133.0 (CH.sub.All), 118.7 (CH.sub.2All), 102.3 (C-1.sub.A), 100.5 (C-1.sub.B), 99.1 (C-1.sub.D), 98.5 (C-1.sub.C), 91.6 (CCl.sub.3), 81.7 (C-3.sub.D), 78.0 (C-2.sub.B), 76.1 (C-5.sub.D), 74.5 (C-3.sub.C), 73.4 (C-2.sub.C), 72.2 (C-4.sub.C), 72.0 (2C, C-4.sub.B, C-4.sub.A), 70.8 (CH.sub.2.sub.All), 70.0 (2C, C-3.sub.A, C-2.sub.A), 69.9 (C-3.sub.B), 69.5 (C-5.sub.B), 69.2 (C-5.sub.C), 69.1 (C-5.sub.A), 68.5 (C-4.sub.D), 60.7 (C-6.sub.D), 57.1 (C-2.sub.D), 40.7 (CH.sub.2-.sub.ClAc), 16.7 (C-6.sub.B), 16.6 (C-6.sub.A), 16.2 (C-6.sub.C).

[0239] HRMS (ESI+): m/z 895.1840 (calcd for C.sub.31H.sub.47Cl.sub.4NO.sub.19NH.sub.4 [M+NH.sub.4].sup.+) found m/z 895.1860.

Allyl -L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl--L-rhamnopyranosyl-(13)-2-deoxy-2-N-trichloroacetyl--D-glucopyranoside (21a)

[0240] To a solution of the lightly protected tetrasaccharide AB.sub.ClAcC.sub.Cl3AcD-All (21, 200 mg, 0.23 mmol) in anhydrous methanol (31 mL) was added MeONa (25% w/w in MeOH, 126 L, 0.55 mmol, 2.4 equiv.). After stirring the reaction mixture for 3 h, a TLC follow up (cyclohexane/ethyl acetate 8:2) showed complete consumption of the starting material (Rf=0.15). Dowex H.sup.+ was then added until neutralization and filtered off, volatiles were evaporated under reduced pressure. The residue was purified by reverse phase chromatography eluting from a C18 column (water/MeCN 1:0 to 6:4) to give, after lyophilization, tetrasaccharide ABC.sub.Cl3AcD-All 21a as a white powder (76 mg, 42%). RP-HPLC (C18 RP fusion (4.6250 mm, 4.0 m, 80 , CH.sub.3CN in H.sub.2O (30% for 4 min, then 30.fwdarw.40% over 7 min, at 1.0 mL.Math.min.sup.1), 40 C., : 220 nm)=3.8 min.

[0241] HRMS (ESI+): m/z 819.2124 (calcd for C.sub.30H.sub.46Cl.sub.3NO.sub.18NH.sub.4 [M+NH.sub.4].sup.+) found m/z 819.2745; m/z 824.1618 (calcd for C.sub.30H.sub.46Cl.sub.3NO.sub.18Na [M+Na].sup.+) found m/z 824.2239.

Synthesis of an Alternative B Donor (25)

[0242] ##STR00032##

Allyl 3,4-di-O-(2-naphtylmethyl)--L-rhamnopyranoside (22)

[0243] Dibutyltin oxide (4.0 g, 1.1 equiv) was added to a solution of diol 1 (5.0 g, 15.0 mmol) in anhydrous toluene (100 mL). The mixture was stirred for 2 h at reflux using a Dean-Stark apparatus. After cooling to rt, dry CsF (2.2 g, 1.0 equiv), dry tetrabutylammoniun iodide (6.97 g, 1.3 equiv) and 2-naphtylmethyl bromide (3.54 g, 1.1 equiv.) were successively added. After heated at 60 C. overnight, a TLC control (toluene/EtOAc 8:2) showed the total consumption of starting diol 1. After cooling to 0 C., salts were removed by filtration over a pad of Celite and solvents were evaporated under reduced pressure. The crude was purified by flash chromatography to give alcohol 22 (5.02 g, 69%) as a brown oil.

[0244] .sup.1H NMR (CDCl.sub.3) 7.81 (m, 8H, H.sub.ArNap), 7.50 (m, 6H, H.sub.ArNap), 5.93 (m, 1H, J.sub.trans=17.1 Hz, J.sub.gem=1.5 Hz, CHCH.sub.2), 5.31 (m, 1H, J.sub.cis=10.4 Hz, CHCH.sub.2), 5.22 (m, 1H, CHCH), 5.10 (d, 3H, J=11.2 Hz, CH.sub.2Nap), 4.91 (m, 2H, CH.sub.2Nap, H-1), 4.21 (m, 1H, H.sub.All), 4.16 (m, 1H, H-2), 4.02 (m, 2H, H.sub.All, H-3), 3.85 (m, 1H, H-5), 3.60 (pt, 1H, J.sub.3,4=J.sub.4,5=9.3 Hz, H-4), 2.58 (bs, 1H, J.sub.2,OH=9.6 Hz, OH), 1.40 (d, 3H, J.sub.5,6=6.3 Hz, H-6).

[0245] HRMS (ESI.sup.+): m/z 502.2608 (calcd for C.sub.31H.sub.36O.sub.5N [M+NH.sub.4].sup.+ m/z 502.2593)

Allyl 2-O-levulinyl-3,4-di-O-(2-naphtylmethyl)--L-rhamnopyranoside (23)

[0246] To a solution of alcohol 22 (5.02 g, 9.0 mmol) in anhydrous DCM (42 mL) stirred at room temperature were successively added DCC (3.16 g, 1.7 equiv), DMAP (440 mg, 0.4 equiv) and levulinic acid (1.94 mL, 4.2 equiv). After stirring the reaction mixture overnight at room temperature, TLC (toluene/EtOAc 7:3) showed complete consumption of the starting material. The reaction mixture was filtered over a pad of Celite, and volatiles were evaporated under reduced pressure. The crude material was taken in ethyl acetate (50 mL) and the organic layer was washed thrice with 10% aqueous copper (II) sulfate (30 mL), water (30 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude material was used as such in the next step.

2-O-Levulinyl-3,4-di-O-(2-naphtylmethyl)--L-rhamnopyranose (24)

[0247] To a solution of the fully protected 23 (from 22, 9.0 mmol) in anhydrous THF (50 mL) stirred at room temperature was added hydrogen-activated [Ir] (152 mg, 0.02 equiv) in anhydrous THF (10 mL). After stirring the reaction mixture for 2 hours at room temperature, another 0.02 equiv of hydrogen-activated [Ir] vas added and the reaction mixture was stirred overnight at room temperature. TLC (cyclohexane/EtOAc 6:4) showed that the starting allyl rhamnoside had been consumed. Iodine (2.74 g, 1.2 equiv) in water/THF (1:5, 72 mL). After stirring the reaction mixture for 1 h at this temperature, TLC (cyclohexane/EtOAc 6:4) showed complete conversion of the intermediate into a more polar product. 10% aqueous sodium metabisulfite was added. THF was evaporated under reduced pressure and DCM (50 mL) was added. The aqueous layer was extracted twice with DCM (30 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate and then brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude material was used as such in the next step.

2-O-Levulinyl-3,4-di-O-(2-naphtylmethyl)--L-rhamnopyranosyl trichloroacetimidate (25)

[0248] To a solution of hemiacetal 24 (from 22, 9.0 mmol) in anhydrous DCE (50 mL) stirred at room temperature were successively added Cl.sub.3CCN (2.7 mL, 3.0 equiv) and DBU (670 L, 0.5 equiv). After stirring the reaction mixture for 2 h at room temperature, TLC (toluene/EtOAc7:3) showed complete consumption of the starting material. Volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of Et.sub.3N-treated silica gel (toluene/EtOAc 95:5 to 8:2 containing Et.sub.3N 5%) to give donor 25 as a brown oil (5.05 g, 88% over three steps).

[0249] .sup.1H NMR (CDCl.sub.3) 8.67 (s, 1H, NH), 7.81 (m, 8H, H.sub.ArNap), 7.49 (m, 6H, H.sub.ArNap), 6.22 (d, 1H, H-1), 5.56 (dd, 1H, J.sub.1,2=2.1 Hz, J.sub.2,3=3.3 Hz, H-2), 5.14 (d, 1H, J=11.1 Hz, CH.sub.2Nap), 4.92 (d, 1H, J=11.4 Hz, CH.sub.2Nap), 4.87 (d, 1H, CH.sub.2Nap), 4.77 (d, 1H, CH.sub.2Nap), 4.10 (dd, 1H, H-3), 4.00 (m, 1H, H-5), 3.62 (pt, 1H, J.sub.3,4=J.sub.4,5=9.5 Hz, H-4), 2.78 (m, 4H, CH.sub.2Lev), 2.18 (s, 3H, CH.sub.3Lev), 1.40 (d, 3H, J.sub.5,6=6.2 Hz, H-6)

[0250] .sup.13C NMR (CDCl.sub.3) 206.0 (CO.sub.Lev), 171.9 (CO.sub.2Lev), 160.1 (NHCO), 135.6-133.2 (6C, C.sub.IV), 128.2-126.1 (14C, C.sub.ArNap), 95.2 (C-1, .sup.1J.sub.C-H=179.5 Hz), 79.4 (C-4), 77.1 (C-3), 75.7 (C.sub.Nap), 72.0 (C.sub.Nap), 70.8 (C-5), 67.9 (C-2), 38.0 (CH.sub.2Lev), 29.8 (CH.sub.3Lev), 28.1 (CH.sub.2Lev), 18.1 (C-6).

[0251] HRMS (ESI.sup.+): m/z 703.1744 (calcd for C.sub.35H.sub.38Cl.sub.3NO.sub.7 [M+NH.sub.4].sup.+ m/z 703.1749)

Synthesis of an Alternative C Acceptor (28)

[0252] ##STR00033##

Allyl 4-O-para-methoxybenzyl--L-rhamnopyranoside (27)

[0253] Crude allyl 2,3-O-isopropylidene--L-rhamnopyranoside (26, from L-rhamnose, 110 mmol) was dissolved in DMF (320 mL) under Argon, the bath temperature was cooled to 5 C. and NaH (60% oil dispersion, 10.6 g, 2.4 equiv.) was added portionwise to this suspension. The mixture was stirred for 2 h at rt, then para-methoxybenzyl chloride (17.9 mL, 1.2 equiv.) was added dropwise at 5 C. and the reaction mixture was stirred at rt overnight. Follow up by TLC (toluene/EtOAc 8:2) indicated the total conversion of the intermediate alcohol into a less polar product. The reaction was quenched at 0 C. by addition of MeOH (20 mL). Solvents were eliminated under reduced pressure and volatiles were co-evaporated with toluene. The residue was taken up in EtOAc (200 mL) and washed with H.sub.2O (3120 mL) and brine (120 mL). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness to give the fully protected intermediate.

[0254] The crude intermediate was dissolved in 80% aq. AcOH (200 mL) and the solution was stirred for 3 d at 60 C. Follow up by TLC (toluene/EtOAc 7:3) indicated the total conversion of the intermediate acetal into a more polar product. Solvents were removed under vacuum and traces of AcOH were eliminated by co-evaporation with cyclohexane (2100 mL) to give a brown solid. Crystallization from cyclohexane and column chromatography of the mother liquor eluting with toluene/EtOAc 8:2 to 6:4) gave diol 28 (27.9 g, 78% over four steps), m.p.=72 C. (cyclohexane).

[0255] .sup.1H NMR (CDCl.sub.3) 7.30 (m, 2H, H.sub.ArPMB), 6.91 (m, 2H, H.sub.ArPMB), 5.89 (m, 1H, CHCH.sub.2), 5.29 (m, 1H, J.sub.trans=17.2 Hz, J.sub.gem=1.6 Hz, CHCH.sub.2), 5.20 (m, 1H, J.sub.trans=10.4 Hz, CHCH.sub.2), 4.81 (d, 1H, J.sub.1,2=1.1 Hz, H-1), 4.69 (m, 2H, CH.sub.2PMB), 4.17 (m, 1H, H.sub.All), 3.97 (m, 3H, H-All, H-2, H-3), 3.82 (s, 3H, CH.sub.3PMB), 3.75 (m, 1H, H-5), 3.35 (pt, 1H, J.sub.3,4=J.sub.4,5=9.2 Hz, H-4), 2.45 (bs, 2H, OH), 1.36 (d, 3H, J.sub.5,6=6.3 Hz, H-6).

Allyl 2-O-chloroacetyl-4-O-para-methoxybenzyl--L-rhamnopyranoside (28)

[0256] Diol 27 (1.0 g, 3.0 mmol) was solubilized in anhydrous acetonitrile (MeCN, 5 mL). To the solution was added trimethylchloroorthoacetate (1.39 mL, 3.0 equiv) and APTS (59 mg, 0.1 equiv). The solution was stirred at room temperature for 1 hour (reaction followed by TLC toluene/EtOAc 7:3). To the reaction medium cooled to 0 C. was added a 90% aqueous TFA (2.0 mL) and the reaction mixture was stirred at room temperature for 10 min. Water was added until the mixture became completely cloudy. The product was extracted with DCM (225 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (225 mL) and brine (25 mL). The aqueous phase was extracted with DCM (212.5 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered, evaporated and finally co-evaporated with toluene to yield alcohol 28 as a mixture of regioisomers. The crude material is used as such in the next step.

Synthesis of .sub.Cl3AcDAB.sub.ClAc-All

[0257] .sub.Cl3AcDAB.sub.ClAcC-All is obtained following route B as defined below.

[0258] In particular, it can be obtained from donor 12 or its 4,6-O-benzylidene analog and the ABC trioside acceptor 29 following conventional delevulinylation at position 2.sub.A of the fully protected precursor 16. A synthesis is highlighted in the scheme below whereby R.sup.1=R.sup.4=Nap, R.sup.3=R.sup.6=TES, R.sup.2=ClAc, R.sup.5=Lev, R.sup.7=TBS, R.sup.8=All, R.sup.9=Cl3Ac).

##STR00034## ##STR00035##

Allyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-trichloroacetamido--D-glucopyranosyl-(12)-4-O-(2-naphtylmethyl)--L-rhamnopyranosyl-(12)-4-O-(2-naphtylmethyl)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (32) and allyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido--D-glucopyranosyl-(12)-4-O-(2-naphtylmethyl)--L-rhamnopyranosyl-(12)-4-O-(2-naphtylmethyl)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl-4-O-(2-naphtylmethyl)--L-rhamnopyranoside (31)

[0259] To a solution of crude tetrasaccharide 30 (1.48 mmol) in THF/AcOH (4:1, 74 mL) was slowly added 1M TBAF in THF (14.8 mL, 14.8 mmol, 10.0 equiv.). After stirring the reaction mixture overnight at rt, were added TBAF (1M solution in THF, 14.8 mL, 14.8 mmol, 10.0 equiv.) and AcOH (14.8 mL). After stirring the reaction mixture for 2 days at rt, a TLC follow up (cyclohexane/ethyl acetate 7:3) showed the presence of a complex mixture of products (Rf=0.05, 0.25, 0.35, 0.45 and 0.55). Distilled water (20 mL) and toluene (50 mL) were added. The organic layer was washed with satd aq. NaHCO.sub.3 (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated and co-evaporated with toluene under vacuum. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 95:5 to 4:6) to give by order of elution diol 32 (820 mg, 37%) and triol 31 (334 mg, 16%).

Allyl 2-deoxy-2-trichloroacetamido--D-glucopyranosyl-(12)--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(13)-2-O-chloroacetyl--L-rhamnopyranoside (33)

[0260] A solution of tetrasaccharide 31 (334 mg, 0.24 mmol) in trifluoroacetic acid/1,1,1,3,3,3-hexafluoro-2-propanol (9:1, 3.0 mL) is stirred at rt for 2 h. Toluene (10 mL) was added and volatiles were evaporated and co-evaporated with toluene (five times) under reduced pressure. The residue was dissolved in water (10 mL) and dichloromethane (5 mL). The organic phase was washed with water (10 mL) and the aq. phases were freeze-dried and lyophilized. The residue was purified by reverse phase chromatography eluting from a C18 column (H.sub.2O/MeCN 0.fwdarw.40%) to give the lightly protected tetrasaccharide DABC-All (33) as a white powder (74 mg, 35%). RP-HPLC (C18 RP fusion (4.6250 mm, 4.0 m, 80 , CH.sub.3CN in H.sub.2O (30% for 4 min, then 30-40% over 7 min, at 1.0 mL.Math.min.sup.1), 40 C., : 220 nm)=10.2 min.

[0261] HRMS (ESI+): m/z 900.1394 (calcd for C.sub.31H.sub.47Cl.sub.4NO.sub.19Na [M+Na].sup.+) found m/z 900.1246.

Example 2: Enzymatic -D-Glucosylation of a Compound of Formula (I) with Branching Sucrases

Enzymes

[0262] Table 1 presents some enzymes that were used in the context of the present invention.

TABLE-US-00002 Osidic linkage on Enzyme natural Source accronym acceptor organism References BRS-B -1,3 Leuconostoc US2016136199 (A1), citreum NRRL-B WO2016146764 (A1) 742 Vuillemin et al. (JBC, 2016) BRS-C Leuconostoc fallax US2016136199 (A1), KCTC 3537 WO2016146764 (A1) Vuillemin et al. (JBC, 2016) BRS-E Leuconostoc mesenteroides KFRI-MG BRS-A -1,2 Leuconostoc US2017152489 (A1), citreum NRRL-B US2017101484 (A1), 1299 ES2398227 (T3) Vuillemin et al. (JBC, 2016) BRS-D Lactobacillus US2017152489 (A1), kunkei EFB6 US2017101484 (A1), ES2398227 (T3) Vuillemin et al. (JBC, 2016) GBD-CD2 From truncated US2017152489 (A1), DSR-E US2017101484 (A1), Leuconostoc ES2398227 (T3) citreum NRRL-B Fabre et al. (J. Bact, 2004), 1299 Brison et al. (JBC, 2012) GBD-CD2 GBD-CD2 Yannick Malbert. W2135V* mutants Flavonoid GBD-CD2 glucodiversification W2135C- with engineered F2136I* sucrose-active enzymes. GBD-CD2 Biotechnology. W2135S- INSA Toulouse F2136L* (PhD, 2014) GBD-CD2 W2135I- F2136C* GBD-CD2 W2135N- F2136Y* GBD-CD2 W2135N* GBD-CD2 W2135I- F2136Y* GBD-CD2 W2135L* GBD-CD2 W2135C* GBD-CD2 W2135N- F2136H* GBD-CD2 W2135L- F2136L* GBD-CD2 W2135F- F2136I* GBD-CD2 W2135C- F2136N* GBD-CD2 W2135G* GBD-CD2 W2135F* GBD-CD2 F2163G* GBD-CD2 L2166I* GBD-CD2 F2163H* GBD-CD2 F2163G L2166I* GBD-CD2 A2162E F2163L* GBD-CD2 F2163L* GBD-CD2 F2163I- D2164E- L2166I* *The mutations are given relatively to the sequence of the GBD-CD2 wild type.

[0263] The sequence of said enzymes is as follows: [0264] SEQ ID NO: 1 (BRS-E) [0265] SEQ ID NO: 2 (BRS-A) [0266] SEQ ID NO: 3 (BRS-B-D1) [0267] SEQ ID NO: 4 (BRS-B-D2) [0268] SEQ ID NO: 5 (BRS-C) [0269] SEQ ID NO: 6 (BRS-D) [0270] SEQ ID NO: 7 (GBD-CD2).

TABLE-US-00003 BRS-E(L. MAHHHHHHVTSLYKKAGSAAAPFTMQQNNATLQVSPTKNDNSVAKNTTSTANV Mesenteroides AKVDITTDNTRDISSANNVNNLITNQYKENSNGSWSYYDNNGQIVKGLQTING KFRI-MG) NIQYFDSTTGEQVKGQTLTIDGVIYSFDKDSGNGTKTEVASLPTTGSYATKDG SNWQYEDQQQQPIKGLYTDKGNLRYFNETDGTQVKGTVVSVDNNTYYFDK DSGNGQLVPSVTGGQYGTIQLNNQTVWVYRNANGEIVKGLQNINGNIQYFDPN TGEQLKGKVATVNGVTYYFEASDGNLVGTVSDGLVTVNGQIQYFDPATGEQ AKNKQVIVNNVTYYFDDNGYGQYLFTNAILSTTPDAYSAHTQAYNTDQSSFT NVVDGFLTADSWYRPKEVIADATGSAWQTSSENDYRPIITVWWPNKNVEVNY LKLMQDNDLLSTQTQFTIFSDQYTLNEAAQAAQNEIEKRIYREKSTDWLKD LLFEAHGDTPSFVKQQFIWNKDSEYQGAGGGNLWSLQGGYLKYVNDSETS WSDSTSRKHDYYEYLLGNDIDNSNPQVIAENINWLYYLMNEGSLTGNDDT ANFDGVRMDAVIYMKGEASTKVYQFLHKSDELTKNEKIANEHISIVEDGTD ETTKNNSALIVSKWNENIASSLAKIASGKDTSLEALVKTDEKTSVSRIMNS SVESDVNPNYSMIRSHDRGSQDEVINASKVANNDQSIALDNINLNQLENG LKLYYEDQASPTKNYNYYNIPASYALLLSNKDTVPRLYYSDMYQDYDQNPD TPQQYMSKPTIYYSAIDALLKARIKYVAGGQSMAVEKVGDNKDQEVLTSVR YGKNVMTATDTGTVESRTEGMGVIVSNNTKLKLSTTDQIVLHMGAAHANQAYQ ALMLTNDEGIQLYNDNAPVVWTDHNGDLVFNGNDINGQKNTSIKGYFNPQVAG YLAVWVPVGATDTQDARTKASTNATTDGKVFHSNAALDSNVIYEGFSNFQPIA RNHNDFSNVKIAENVDLFKKWGITSFELAPQYRSADVSDLVGSTFVDVVTKNG YGLSDRYDLGFVTPTKYGSDSDLRNAISSLHAQGIQAMADFVGNQIYALNDGQ EVVTAQRSDMFNNTLNNAFGTELYVVNSIGGGKYQAKYGGNYLEEIASLYPDL FTNQDGTKIDINTKIKQWSAKYMNGTNVLGRGMGYVLKDWNTATYFKLDGEHT VLPAALTLSGLKVENGVTYYYKNNERQTGTQTVDDVTYFFDPKTGAMKKDYFD FTADNKVYYYGKGGRADPAFLYKVVSAWSHPQFEK BRS-A,1878 MRQKETITRKKLYKSGKSWVAAATAFAVMGVSAVTTVSADTQTPVGTTQSQQD aminoacids LTGQRGQDKPTTKEVIDKKEPVPQVSAQNAGDLSADAKTTKADDKQDTQPTNA (L.citreum QLPDQGNKQTNSNSDKGVKESTTAPVKTTDVPSKSVTPETNTSINGGQYVEKD NRRLB- GQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYYFDKNSG 1299) NGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGNQAK HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATLDGTTYYFEGNKGNLVSVVNTA PTGQYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGI GYYFDKDTGNGSYQYTLMAPSNKNDYTQHNVVNNLSESNEKNLVDGFLTAETW YRPAQILSHGTDWVASTDKDFRPLITVWWPNKDIQVNYLRLMQNEGVLNQSAV YDLNTDQLLLNEAAQQAQIGIEKKISQTGNTDWLNNVLETTHDGQPSFIKQQY LWNSDSEYHTGPFQGGYLKYQNSDLTPNVNSKYRNADNSLDELLANDVDNSNP IVQAEDLNWLYYLLNFGSITTQGKENNSNFDSIRIDAVDFVSNDLIQRTYDYL RAAYGVDKNDKEANAHLSLVEAGLDAGTTTIHQDALIESDIREAMKKSLTNGP GSNISLSNLIQDKEGDKLIADRANNSTENVAIPNYSIIHAHDKDIQDKVGAAI TDATGADWTNFTPEQLQKGLSLYYEDQRKIEKKYNQYNIPSAYALLLTNKDTV PRVYYGDMYQDDGQYMQKQSLYFDTITALMEARKQFVAGGQTINVDDNGVLTS VRFGKGAMTANDIGTNETRTQGIGVVIANDPSLKLSKDSKVTLHMGAAHRNQN YRALLLTTDNGIDSYSSSKNAPVIKTDDNGDLVFSNQDINDQLNTKVHGFLNS EVSGYLSAWVPLDATEQQDARTLPSEKSVNDGKVLHSNAALDSNLIYEAFSNF QPMPTNRNEYTNVVIADKADTFKSWGITSFEMAPQYRSSQDKTFLDSTIDNGY AFTDRYDLGFEKPTKYGNDEDLRQAIKQLHSSGMQVMADVVANQIYNLPGKEV ASTNRVDWNGNNLSTPFGTQMYVVNTVGGGKYQNKYGGEFLDKLKAAYPDIFR SKNYEYDVKNYGGNGTGSVYYTVDSKTRAELDTDTKIKEWSAKYMNGTNVLGL GMGYVLKDWQTGQYFNVSNQNMKFLLPSDLISNDITVQLGVPVTDKKIIFDPA SAYNMYSNLPEDMQVMDYQDDKKSTPSIKPLSSYNNKQVQVTRQYTDSKGVSW NLITFAGGDLQGQKLWVDSRALTMTPFKTMNQISFISYANRNDGLFLNAPYQV KGYQLAGMSNQYKGQQVTIAGVANVSGKDWSLISFNGTQYWIDSQALNTNFTH DMNQKVFVNTTSNLDGLFLNAPYRQPGYKLAGLAKNYNNQTVTVSQQYFDDQG TVWSQVVLGGQTVWVDNHALAQMQVRDTNQQLYVNSNGRNDGLFLNAPYRGQG SQLIGMTADYNGQHVQVTKQGQDAYGAQWRLITLNNQQVWVDSRALSTTIMQA MNDDMYVNSSQRTDGLWLNAPYTMSGAKWAGDTRSANGRYVHISKAYSNEVGN TYYLTNLNGQSTWIDKRAFTATFDQVVALNATIVARQRPDGMFKTAPYGEAGA QFVDYVTNYNQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRSFSPVVTKVV DYQAKIVPRTTRDGVFSGAPYGEVNAKLVNMATAYQNQVVHATGEYTNASGIT WSQFALSGQEDKLWIDKRALQA BRS-B MEMKETITRKKLYKSGKSWVAAATAFAVMGVSAVTTVSADTQTPVGTTQSQQD LTGQTGQDKPTTKEVIDKKEPVPQVSAQNVGDLSADAKTPKADDKQDTQPTNA QLPDQGNKQTNSNSDKGVKESTTAPVKTTDVPSKSVAPETNTSINGGQYVEKD GQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYYFDKNSG NGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGNQAK HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTA PTGQYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGI GYYFDQNNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETW YRPAQILSHGTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVV FDTNNDQLVLNKGAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQY LWNVDSEYPGGWFQGGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSN PVVQAEQLNWLYYLMNFGQITANDSNANFDSMRIDAISFVDPQIAKKAYDLLD KMYGLTDNEAVANQHISIVEAPKGETPITVEKQSALVESNWRDRMKQSLSKNA TLDKLDPDPAINSLEKLVADDLVNRSQSSDKDSSTIPNYSIVHAHDKDIQDTV IHIMKIVNNNPNISMSDFTMQQLQNGLKAFYEDQHQSVKKYNQYNIPSAYALL LTNKDTVPRVFYGDMYQDYGDDLDGGQYMATKSIYYNAIEQMMKARLKYVAGG QIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKDIMDAQGQGTAESRNQGIGVI VSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTNDKGIVNYDQDNNAPIAWT NDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPVGANDNQDARTVTTN QKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVITKNIDLFKSWG ITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPTKYGTDQDL RKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVVDPRYG TQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEWSA KYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLVSNDPETQIGES VNYKYFIGNSDATYNMYHNLPNTVSLINSQEGQIKTQQSGVTSDYEGQQVQVT RQYTDSKGVSWNLITFAGGDLQGQKLWVDSRALTMTPFKTMNQISFISYANRN DGLFLNAPYQVKGYQLAGMSNQYKGQQVTIAGVANVSGKDWSLISFNGTQYWI DSQALNTNFTHDMNQKVFVNTTSNLDGLFLNAPYRQPGYKLAGLAKNYNNQTV TVSQQYFDDQGTVWSEVVLGGQTVWVDNHALAQMQVSDTSQQLYVNSNGRNDG LFLNAPYRGQGSQLIGMTADYNGQHVQVTKQGQDAYGAQWRLITLNNQQVWVD SRALSTTIVQAMNDDMYVNSNQRTDGLWLNAPYTMSGAKWAGDTRSANGRYVH ISKAYSNEVGNTYYLTNLNGQSTWIDKRAFTATFDQVVALNATIVARQRPDGM FKTAPYGEAGAQFVDYVTNYNQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQ RSFSPVVTKVVDYQAKIVPRTTRDGVFSGAPYGEVNAKLVNMATAYQNQVVHA TGEYTNASGITWSQFALSGQEDKLWIDKRALQA BRS-B-D1(L. DTQTPVGTTQSQQDLTGQTGQDKPTTKEVIDKKEPVPQVSAQNVGDLSADAKT citreum PKADDKQDTQPTNAQLPDQGNKQTNSNSDKGVKESTTAPVKTTDVPSKSVAPE NRRLB-742) TNTSINGGQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELK NIDDNAYYFDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEG NLQYFDLSTGNQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDK DGNETSYWAYLDNQGNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYF EGNKGNLVSVVNTAPTGQYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLAT GIQLKGQAKNIDGIGYYFDQNNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANN FKNLVDGFLTAETWYRPAQILSHGTDWVASTDKDFRPLITVWWPNKDIQVNYL KLMQQIGILDNSVVFDTNNDQLVLNKGAESAQIGIEKKVSETGNTDWLNELLF APNGNQPSFIKQQYLWNVDSEYPGGWFQGGYLAYQNSDLTPYANTNPDYRTHN GLEFLLANDVDNSNPVVQAEQLNWLYYLMNFGQITANDSNANFDSMRIDAISF VDPQLAKKAYDLLDKMYGLTDNEAVANQHISIVEAPKGETPITVEKQSALVES NWRDRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVNRSQSSDKDSSTIPNY SIVHAHDKDIQDTVIHIMKIVNNNPNISMSDFTMQQLQNGLKAFYEDQHQSVK KYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMATKSIYYNAI EQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKDIMDAQG QGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTNDKGI VNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYAN VVITKNIDLFKSWGITDFELAPQYRSSDGKDITDRELDSIVQNGYGLSDRYDL GFKTPTKYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVN INGDTKLVVDPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNG QKIDLSTKIKEWSAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPT QL BRS-B-D2(L. MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDN citreum AYYFDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYF NRRLB-742) DLSTGNQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNET SYWAYLDNQGNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKG NLVSVVNTAPTGQYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLK GQAKNIDGIGYYFDQNNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLV DGFLTAETWYRPAQILSHGTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQ IGILDNSVVFDTNNDQLVLNKGAESAQIGIEKKVSETGNTDWLNELLFAPNGN QPSFIKQQYLWNVDSEYPGGWFQGGYLAYQNSDLTPYANTNPDYRTHNGLEFL LANDVDNSNPVVQAEQLNWLYYLMNFGQITANDSNANFDSMRIDAISFVDPQI AKKAYDLLDKMYGLTDNEAVANQHISIVEAPKGETPITVEKQSALVESNWRDR MKQSLSKNATLDKLDPDPAINSLEKLVADDLVNRSQSSDKDSSTIPNYSIVHA HDKDIQDTVIHIMKIVNNNPNISMSDFTMQQLQNGLKAFYEDQHQSVKKYNQY NIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMATKSIYYNAIEQMMK ARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKDIMDAQGQGTAE SRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTNDKGIVNYDQ DNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPVGANDN QDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVITK NIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGEKTP TKYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDT KLVVDPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDL STKIKEWSAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGG RADPAFLYKVVHHHHHH BRS-C MKQQESITRKKLYKAGKSWVVAATLFAATLFAAMGAAGATTVASADVQKDTVV (GH_Leucono VTADKNTTDKDKEPIKTAGANVVDKGVAQTTDTNTTDKKTIEVGKSVDMSATD stoc_fallax_K KKVTETVKSVDTSATDKKTTEAVKPVDTNATDKKATEAVKPVDTNATDKKTTE CTC3537_1) AVKPVDTNTTDKKVTEAIKPVNTNADDKTAEPVKTISATKDTVKTIANKQKGA TEEQAVITEGHYEAQGDGFVYITKDGKQLTGLQNINGNTQYFDPATGQQLKGD IKAVAGTVYYFDKNSGNARVYQKVADGTYSENNEHWQYISKVDNKPVEGLYNV QGNLQYFDMSTGNQVKNDIRSVDGVTYYFDKDSGNGSAFNALSAGEYVEKKET DAQGNQNSYWTYSGLDGNPVKGLYDINGSLQYFDEKNGAQLKGGTATVNGVTY YFEQDKGNLISVVNSVESGQYKIDNDNVYYIDNQGNTLKGLYAINGQLNYFDM STGVQLKGASENANGVGYYFDKDKGNGQYQYSLITSTLANAFSKHNAANDYTQ SSFTHTVDGFLTADTWYRPTEILKNGTTWVASTSQDLRPMITVWWPNKNVQLN YLKLMQTEGLLDSGQVYDLNSDQALLNQAAQTVQVNIEKRITKAGNSDWLNDL LYNSHGETPSFVKQQAIWNADSEYHGGWFQGGYLAYRNSDLTPYANSSYRHYT GMEFLLANDVDNSNPIVQAEDLNWLYYLMNFGTETGNDPQANFDSIRIDAISF VDKQVAKKAYELLHDMYGLSASDAVANKHVSIVEASADQTPVTTENHDALIES YWRDTMKNSLSKDASIDSSAGSLSAMINDGNVDRANDSTTESSIFPNYTIVHA HDKDIQDAVSNVMKIVNNDPSISLDGFTMEQLEKGLSAFYADQRSAVKQYNQY NIPSAYAVMLTNKDTVPRTFYGDMYQDDGQYMANKSLYYDAIDTMMKARLKYV SGGQTMSVTKINNANSQKSGEVLTSVRFGKGVMDATDAGSAESRTQGIGVVVS NSSGLQLNDNDKIVLHMGAAHKNQEYRALMLTTNDGIKSFNNDEAPINYTDDN GDLIFDGHNIDGQENTAIRGYLNPQVAGYLAVWVPTGAKDDQDARTQPSNEKS TDGKVLHTNAALDSELIYEGFSNFQPMPTTKDEYTNVMIAKNIDLFKSWGITN FELAPQYRSSDGKNINDRFIDSLVQNGYGLSDRYDLGFETPTKYGTDQDLRTA IKTLHQAGMTVMADYVANQIYGLNTSQEVVDAQRVNSDNNAVEVRYGQHLNVV NSIGGGEYQNLYGGKYLEILNKLYPDLLVDENGNKIDIDTKIKQWSAKYLNGS NVTGLGMGYVLKDWSNGQYFNISNTDGKVMLPEQLVKHMPAVEIGTQTNYTAY ISSTIRRDGLYNNMPWGVTATGQDGNEIKWERQGSTSDYNHQKVQVNRQYVDK QGVVWNLINFDDKDLWVDSNALVTVNFTSQKPTKHFVQFGMRQGKYDGFYLSA PYKQTESKWVASTRTHQGQLLEVVGQYTTGSGSRKVTWYLVGLDGKQVWVDSR AVGTNFSHKTNINLLINSATRNDGMYLNAPYGQKGYKRETSSRFYNEKLVTVS QQYYDNKGVIWNLITLNGKKLWVDSRAFATVIDKKVNQSLYINSRNDGMYLNA PYRAQGAKRYASTKTYTGQRVQVTLQRKDTHGVTWYLTKVDSKQLWVDSHAFA PTFTRNVSLNVKVNSSKRNDGIYLNAPYGNKKAKRIASTKAYNGKRVKASKEY KDAKGVTWYLVNLNNKQVWIDKRAF Brs-D(Lb MNINSNERKVRFKMYKSGKQWIVAGLTTAVISIAVYGGSSIANGGIEAKADAQ kunkeiEFB6) NAATSSIVNTNNSTNSSNANSIASLPQNGTYSTNDNGQTWKYVSQNKDIQGLY KDNNDQLRYFNEYDGTQAKGDIVNVNNDNYYFDKDSGQGHKIDSYTGGSYSES KVNNQDGWIYKSSDNNDVKGVATVDGNIQYFDQNTGLQLKGGSAQIGGVDYYF DPNKGNLVGKVDQVVNSNDYSDNKLLDSNKNVVKGLVVNNGQLQFFDTSNGNQ AKNKQVIANGITYYFDTNGNGQYLFTNTGKSAVDDFTQRNAANSVNPSDYKNV VDGFFTADTWYRPKQILDNGTTWRNSNSNELRPMITAWWPNKDVQVNYLKLMQ NNGLLDKSNSYSIQSDQQTLNQAAQKAQVNIEKKISQTGNTDWLNDLLFKGNG DNPSFVKQQYIWSSDSESPWQGDAWFQGGYLKYGNSVMTPNTNSNYRDSNNLF DFLLANDVDNSNPAVQAEDLNWLYYLTNFGTITANDSNANFDSIRIDAVDFIS NDIIQRSYDYLRQKFNLMQSDANADSHISLVEGGVDAGTTSYSNDGLVEAPFR LDAYPLLHKQDGDVFKNLIDEEDSGIDISNHNGETNTNNTIGGITLSGGKPNY SIVHAHDKDVQEKVGQAIIDTTGIKDWTDFTPSQLAQGLETFYNDQRQTVKKY NDYNVPSAYAIMLTNKGTVPRIYYGDMYQDDGQFMQKKSLYYDDIANLMTARK KYVSGGQSMVDNNGILTSVRFGKGANTVSDSGTEDTRNQGIGLIVGSAPKKVL NDGDTVVLHMGAAHKNQKYRALMLTTENGIQNYNSDDNAPVAETDDNGDLVFS NKDINGQANTAIKQVANPEVNGYLAAWVPVGASDDQDSRTAPSTSQNNDGNVL HENDALDSNLIFEGFSNFQPTPTNHDEYANVVIAKNASLFKDWGVTSFEMAPQ YRSSQDHTFVDSTIDNGYAFSDRYDLGFGTPTKYGTDEDLRNAIKSLHDNGMQ VMADVVYNQLYNLPGQEVVSATRAGVTGNTNALPFGTQLYVVNTIGGGDYQKK YGGAFLNELQEQYPSLFKSQKYKYYYKNYANNGAGPGYLTVNDAERSDIPYNQ PITEWSAKYMNGTNILGRGMGYVLKDWNTGDYFKLSGSDSTLPSSLTYKSGWV ENPDSTWSYYEKNNIDKLTGSQVINEERVFFDNNGIQVKGGWVKNSNGTYSYY DKNSGNILTGDQLIDGEHFFFDNNGVQVKGKWIKNSDGSKSYYDSHLGKLIKT DKKVSSNARKKKSKEELLYENALKVLRKDKKRLDKNKTKANIRKYNKSLKKYR KAKKKLLAITKNRVANARKAIKIAKKVLSKRKNINNEKRYYKALKEYYVAEKS YLKITGNYNKKYYYEFDKLTPKVKVVKNIYSYKSRHFTKKNRVKKIKKGTLVR VKSIVRSGKVARINIGNGHFITSSKDFIKMFK GBD-CD2 AQAGHYITKNGNDWQYDTNGELAKGLRQDSNGKLRYFDLTTGIQAKGQFVTIG (L.citreum QETYYFSKDHDAQLLPMVTEGHYGTITLKQGQDTKTAWVYRDQNNTILKGLQN NRRLB-1299 INGTLQFFDPYTGEQLKGGVAKYDDKLFYFESGKGNLVSTVAGDYQDGHYISQ tronqu DGQTRYADKQNQLVKGLVTVNGALQYFDNATGNQIKNQQVIVDGKTYYFDDKG depuisDSR- NGEYLFTNTLDMSTNAFSTKNVAFNHDSSSFDHTVDGFLTADTWYRPKSILAN E) GTTWRDSTDKDMRPLITVWWPNKNVQVNYLNFMKANGLLTTAAQYTLHSDQYD LNQAAQDVQVAIERRIASEHGTDWLQKLLFESQNNNPSFVKQQFIWNKDSEYH GGGDAWFQGGYLKYGNNPLTPTTNSDYRQPGNAFDFLLANDVDNSNPVVQAEN LNWLHYLMNFGTITAGQDDANFDSIRIDAVDFIHNDTIQRTYDYLRDAYQVQQ SEAKANQHISLVEAGLDAGTSTIHNDALIESNLREAATLSLTNEPGKNKPLTN MLQDVDGGTLITDHTQNSTENQATPNYSIIHAHDKGVQEKVGAAITDATGADW TNFTDEQLKAGLELFYKDQRATNKKYNSYNIPSIYALMLTNKDTVPRMYYGDM YQDDGQYMANKSIYYDALVSLMTARKSYVSGGQTMSVDNHGLLKSVRFGKDAM TANDLGTSATRTEGLGVIIGNDPKLQLNDSDKVTLDMGAAHKNQKYRAVILTT DGLATFNSDQAPTAWTNDQGTLTFSNQEINGQDNTQIRGVANPQVSGYLAVWV PVGASDNQDARTAATTTENHDGKVLHSNAALDSNLIYEGFSNFQPKATTHDEL TNVVIAKNADVFNNWGITSFEMAPQYRSSGDHTFLDSTIDNGYAFTDRYDLGF NTPTKYGTDGDLRATIQALHHANMQVMADVVDNQVYNLPGKEVVSATRAGVYG NDDATGFGTQLYVTNSVGGGQYQEKYAGQYLEALKAKYPDLFEGKAYDYWYKN YANDGSNPYYTLSHGDRESIPADVAIKQWSAKYMNGTNVLGNGMGYVLKDWHN GQYFKLDGDKSTLPQIKGELKLEGKPIPNPLLGLDSTRTGHHHHHH

[0271] Table 2 presents some mutants of BRS-B-D2 that were used in the context of the present invention.

TABLE-US-00004 Mutant Sequence M6 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGAFQG GYLAYQNSDLTPYANTNPDYRTHNGIKVLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDGLAFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWIDRMLQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDLLLVDTVIHIMKIVNNNPNISMSDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M14 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGMMQG GYLAYQNSDLTPYANTNPDYRTHNGVKFMLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDGMWLVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWVDRMLQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDLNLVDTVIHLMKIVNNNPNISMSDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M18 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQG GYLAYQNSDLTPYANTNPDYRTHNGIRYLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAPDWVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWIDRMLQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDINVLDTVIHIMKIVNNNPNISMSDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M21 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAIDFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA DKGETPITVEKQSALVESNWWLRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDLDIQETVVHIMKIVNNNPNISWTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M23 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGPFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAIMFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA DKGETPITVEKQSALVESNWLLRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDKDILETVTHIMKIVNNNPNISDTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVREGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGESNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M28 ASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYYF DKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGNQ AKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQG NAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTGQ YKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQN NGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSHG TDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNKG AESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGMFQGG YLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFGQ ITANDSNANFDSMRIDAIMFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEDS KGETPITVEKQSALVESNWWLRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVNR SQSSDKDSSTIPNYSIVHAHDLDILETVVHIMKIVNNNPNISWTDFTMQQLQNGLK AFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMAT KSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKDI MDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTNDK GIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPVG ANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVIT KNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPTK YGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVVD PRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEWS AKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKVV HHHHHH M30 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISWVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA LKGETPITVEKQSALVESNWMQRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDVDIVETVTHIMKIVNNNPNISMTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M31 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISWVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA LKGETPITVEKQSALVESNWMERMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDIDIILTVWHIMKIVNNNPNISVTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M34 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISWVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA LKGETPITVEKQSALVESNWMARMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDVDILETVVHIMKIVNNNPNISPTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVREGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGESNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M35 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGAFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFMLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWRDRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDADVQVTVIGTMKIVNNNPNISHTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M40 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGDFQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLMANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWRDRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDVDITITVVSLMKIVNNNPNISSTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH M41 MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTGN QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLDNQ GNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAPTG QYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQ NNGNGEYRYSLTGPVVKDVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSH GTDWVASTDKDFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVLNK GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLMQG GYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMNFG QITANDSNANFDSMRIDAISFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIVEA PKGETPITVEKQSALVESNWRDRMKQSLSKNATLDKLDPDPAINSLEKLVADDLVN RSQSSDKDSSTIPNYSIVHAHDLDLEDTVVSLMKIVNNNPNISMTDFTMQQLQNGL KAFYEDQHQSVKKYNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA TKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKD IMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV GANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANVVI TKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKTPT KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLVV DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKIKEW SAKYLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAFLYKV VHHHHHH
Isolation of brsE Gene

[0272] The brsE gene was identified in Leuconostoc mesenteroides KFRI-MG genome (NCBI Reference Sequence: CP000574) by performing a nucleotide BLAST against a GH70 -transglucosylase encoding gene database. The protein sequence of BRS-E is deposited under the GenBank accession number AHF19404.1.

Recombinant Expression of BRS-E in E. coli

[0273] A synthetic brsE gene was designed in order to optimize its expression in E. coli (Biomatik, Cambridge, ON, Canada), and cloned in pET28b vector. The gene was then amplified by PCR from pET28b/BrsE plasmid DNA template using the forward primer 5-atgggctacaaggccgg-3 and the reverse primer 5-accataataatacaccttattatcggc-3. The PCR product was then inserted into the pENTR/D-TOPO vector (Life Technologies). From a positive entry clone, LR recombination (Gateway LR Clonase II enzyme mix, Life technologies) was performed with pET-55-DEST destination vector (Merck Millipore). Expression clones were selected on LB agar plates supplemented with 100 g ml1 of ampicillin. Plasmids were then extracted using the GenElute HP Plasmid Miniprep kit (Sigma-Aldrich), verified by restriction analyses and sequenced (GATC Biotech). E. coli TOP 10 competent cells (Life Technologies) were used for all cloning experiments.

[0274] For enzyme production, E. coli BL21* DE3 cells were freshly transformed by 55/brsE. Twenty milliliters of LB medium, supplemented with ampicillin (100 g mL-1), were inoculated with 100 L of transformation mix and incubated overnight at 37 C. under agitation (200 rpm). Then, Erlenmeyer flasks culture containing a modified ZYM5052 medium with 100 g mL ampicillin, 0.1% lactose, 0% glucose and 1% glycerol were inoculated with the starter culture at an OD600 nm of 0.05. Cultures were incubated at 21 C. under agitation (150 rpm). After 26-hour incubation, cells were harvested by centrifugation, dispersed in 50 mM sodium acetate buffer (pH 5.75) at a final OD600 nm of 80 and disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (11,000 g, 30 min, 8 C.) of the crude cell extract.

Enzyme Production

[0275] Cloning of branching sucrase genes in inducible vectors (pET53, pET55 or pBAD49, Life technologies) for heterologous expression in E. coli cells was previously described (Vuillemin et al., J Biol Chem. 2016; 291(14):7687-702). E. coli BL21* DE3 and E. coli BL21 AI cells were freshly transformed by pET53-55/brsB A2, brsC, brsD, brsE and pBAD49/brsA, respectively. Twenty milliliters of LB medium, supplemented with ampicillin (100 g mL.sup.1), were inoculated with 100 L of transformation mix and incubated overnight at 37 C. under agitation (200 rpm).

[0276] Enzyme production were performed in Erlenmeyer flasks with modified ZYM5052 medium that contains i) 0% lactose, 0% glucose, 0.5% glycerol and 0.01% L-arabinose for BRS-A production, ii) 0.1% lactose, 0% glucose and 1% glycerol for BRS-B-2, BRS-C, BRS-D, BRS-E production or iii) 0.75% lactose, 0.05% glucose and 1.5% glycerol for GBD-CD2 (wild type and mutants) production. All culture media were supplemented with ampicillin (100 g mL.sup.1) and inoculated with the corresponding starter culture at an OD.sub.600nm of 0.05. Cultures were incubated at 21 C. or 23 C. under agitation (150 rpm). After 26-hour incubation, cells were harvested by centrifugation, dispersed in 50 mM sodium acetate buffer (pH 5.75) at a final OD.sub.600nm of 80 for BRS-A, BRS-B-2, BRS-C, BRS-D, BRS-E, and an OD.sub.600nm of 30 for GBD-CD2 and mutants. Cells were disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (11,000 g, 30 min, 8 C.) of the crude cell extract.

Enzyme Purification by Affinity Chromatography

[0277] Recombinant enzymes are produced in fusion with a 6His tag allowing purification by affinity chromatography. For that purpose, cells were centrifuged and resuspended in binding buffer (20 mM phosphate sodium buffer, pH 7.4, 500 mM NaCl, 20 mM imidazole, 2.5% (v/v) glycerol) at a final OD.sub.600nm of 200 for BRS-B productions, and 30 for GBD-CD2 and mutants productions. After disruption by sonication, centrifugation (18,000 g, 30 min, 4 C.) and filtration through a 0.22 m cartridge, lysates were applied at 10 C. onto a 1 ml HisTrap HP column that had been equilibrated with the binding buffer, using an AKTAXpress system (GE Healthcare). The proteins were eluted by imidazole gradient from 10 to 500 mM, over 25 minutes. Eluate fractions of 3 mL were desalted onto 10-DG column (Biorad, Hercules, Calif., USA), with 50 mM sodium acetate buffer at pH 5.75 with 100 mM NaCl, or purified for a second round by gel-filtration on a Superose12 resin.

Enzymatic Activity Assay

[0278] One unit of branching sucrase (wild-type and mutants) is defined as the amount of enzyme which catalyzes the production of one micromole of fructose per min, at 30 C., in 50 mM sodium acetate buffer at pH 5.1 or pH 5.75 depending on the enzyme, and from 292 mM sucrose. The enzyme activities were determined by measuring the amount of reducing sugars using the dinitrosalycilic acid (DNS) method (G. L. Miller, Anal. Chem. 1959, 31, 426-428).

Glucosylation of Tetrasaccharlde Using Branching Sucrases

[0279] Transglucosylation assays were performed at a temperature between 20 to 37 C. in 50 mM sodium acetate buffer, pH 5.0 to 6.0, supplemented with 0.05 to 5 U.Math.mL.sup.1 of enzyme, 50 mM to 1 M sucrose, and 10 mM to 100 mM tetrasaccharide of formula (Ia), in particular AB.sub.ClAcC.sub.Cl3AcD-All (also referred as ABCD). Reactions were incubated in glass tubes for 8 to 24 h.

[0280] Specifically, the pentasaccharides were produced in the following conditions:

[0281] Pentasaccharide 1 (P1) was produced in particular by BRS-B-2 in a 500 L scale reaction using 200 L of ABCD acceptor preparation at 110 g.Math.L.sup.1, 206 L of sucrose at 830 g.Math.L.sup.1, 50 L of sodium acetate buffer 500 mM at pH 5.1, 9.62 L of purified enzyme BRS-B-2 at 52 U/mL and H.sub.2O to 500 L.

[0282] Pentasaccharides 2 (P2) and 2 (2) were produced in particular by GBD-CD2 F2163G in a 2 mL scale reaction using 800 L of ABCD acceptor preparation at 110 g.Math.L.sup.1, 823 L of sucrose at 830 g.Math.L.sup.1, 200 L of sodium acetate buffer 500 mM at pH 5.1, 130.7 L of purified enzyme GBD-CD2 F2163G at 15.3 U/mL and H.sub.2O to 2 mL.

[0283] Pentasaccharide 3 (P3) was produced in particular by GBD-CD2 W2135S-F2136L or GBD-CD2 W2135I-F2136C in a 2 mL scale reaction using 800 L of ABCD acceptor preparation at 110 g.Math.L.sup.1, 823 L of sucrose at 830 g.Math.L.sup.1 200 L of sodium acetate buffer 500 mM at pH 5.1, 130.7 L of purified enzyme at 15.3 U/mL and H.sub.2O to 2 mL.

Methods for Separation, Detection and Purification of the Compounds of Interest:

[0284] The presence of residual acceptor ABCD and glucosylated products (pentasaccharides P1 and P2, P2 and P3) was determined by HPLC-MS (High performance Liquid Chromatography coupled with Mass Spectrometry) using a C18RP Fusion (4 m, 80 , 2504.6 mm) analytical column placed in an oven at 40 C. and eluting with a 20-minute H.sub.2O/acetonitrile gradient from 70:30 to 60:40 at a flow of 1 mL.Math.min.sup.1. Reaction media were diluted 10 times in H.sub.2O/acetonitrile (70:30, v/v)+0.08% trifluoroacetic acid (TFA) before injection of 20 L samples. UV Detection was carried out at 220 nm wavelength. The mass of the different compounds was determined by mass spectrometry with a 0.5 s full scan (m/z 200-1950) both in positive and negative modes. Needle was set on 3.5 kV, cone on 60 V, and the probe temperature was maintained at 450 C.

[0285] Pentasaccharides P1, P2, P2 and P3 were purified by automatic fractionation on an Agilent 1260 Infinity HPLC, during 20 min of separation (same method as above). Several rounds of purification were performed if necessary. Products detected by UV-RI peaks were collected, and reanalyzed by HPLC. Fractions containing single peak products were pooled, concentrated to dryness using a SpeedVac before exchange in D.sub.2O for NMR analyses.

NMR Analyses for Pentasaccharides Structure Elucidation:

[0286] The samples were dissolved in DCl-containing D.sub.2O at pH 4.9 For NMR studies, the samples were lyophilized three times and dissolved in 180 L of 99.9% DCl-containing D.sub.2O.

[0287] All NMR spectra were recorded on a Bruker Avance spectrometer operating at a proton frequency of 950 MHz and at a carbon frequency of 238 MHz with a 5-mm gradient indirect cryoprobe. All spectra were processed and analyzed with Topspin software (Bruker).

[0288] .sup.1H and .sup.13C 1D NMR spectra were accumulated at 30 C., 65536 data points were acquired with 32 and 2048 scans respectively for proton and carbon experiments.

[0289] .sup.1H-.sup.13C HSQC (Heteronuclear Single Quantum Coherence spectroscopy), HMBC (Heteronuclear single quantum coherence spectroscopy) and Double Quantum Filtered COrrelation SpectroscopY (QDF COSY) experiments were performed at 30 C. Homo and heteronuclear spectra were recorded under the following experimental conditions: 512 increments of 2048 complex points are acquired with an accumulation of 16 scans. Spectral widths were 16025 Hz for proton dimension and 44267 Hz for carbon dimension.

Results

[0290] The structure of Pentasaccharide P1 was determined by NMR spectroscopy (950 MHz), revealing an -1,6 glucosylation of D, characteristic of S. flexneri serotype 4a.

[0291] The structure of Pentasaccharide P2 was determined by NMR spectroscopy (950 MHz), revealing an -1,3 glucosylation of A, characteristic of S. flexneri serotype 3a.

[0292] The structure of Pentasaccharides P2 and P3 were also determined by NMR spectroscopy (950 MHz), revealing respectively an -1,4 glucosylation of residue A and an -1,4 glucosylation of residue B.

##STR00036## ##STR00037##

PROTECTED TETRASACCHARIDES, THEIR PROCESS OF PREPARATION AND THEIR USE AS TRANSGLUCOSYLASE ACCEPTOR SUBSTRATES IN THE CHEMO-ENZYMATIC SYNTHESIS OF SHIGELLA FLEXNERI SPECIFIC OLIGOSACCHARIDES

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C12P19/28

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C07H15/10

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C12P19/14

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International classification

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C07H15/10

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C12P19/28

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