SYNTHESIS OF SHIGELLA FLEXNERI SPECIFIC OLIGOSACCHARIDES

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

Carbohydrates displayed at the surface of cells and pathogens are of great therapeutic potential. On the one hand, the human glycome is being scrutinized in detail, on the other hand increasing knowledge on microbial carbohydrates and carbohydrate binding proteins offers new openings for therapeutic and prophylactic interventions. Among a large diversity of applications, carbohydrates are actively investigated as vaccine components. In this context, synthetic carbohydrates represent an attractive alternative to carbohydrate antigens of biological origin. The licensing of QuimiHib®, over a decade ago, demonstrated feasibility. Other vaccine candidates derived from synthetic oligosaccharides are under development whether targeting infectious diseases or non-transmittable diseases such as cancer.

Owing to an increasing interest in well-defined carbohydrates, progress in synthetic methodologies to complex oligosaccharides evolve rapidly. Reports on the use of scaffolds compatible with customized modifications opening the way to a diversity of targets have emerged, especially related to the synthesis of highly diverse complex N-gl yeans. Chemo- enzymatic strategies, mostly relying on the use of glycosyltransferases at the latest stages of the synthesis, are highly attractive. These two approaches were successfully combined to deliver a small library of N-glycans (Science (2013) 341 (6144), 379-83). Similarly, glycorandomization/glycodiversification find wide applications. Yet, drawbacks in the use of glycosyltransferases include enzyme availability, added to cost and availability of the sugar- nucleotide donors.

An alternative strategy consists in the use of "engineered transglycosidase/low cost donor" systems adapted to the customization of non-natural acceptors for the chemo- enzymatic synthesis of carbohydrates and glycoconjugates (Cell. Mol. Life Sci. (2016) 73, 2661-79). This strategy involves mono- and disaccharide acceptors, demonstrating feasibility for simple non-natural acceptors (J. Am. Client. Soc. (2009) 131 , 7379-89, Ghent, Commiin. (2015) 51 , 2581-4). However, this method provides an access to a limited number of targets only, due to its partially divergent character. Accordingly, it is an object of the present invention to provide versatile core precursors, able to yield a great number of oligosaccharides in a highly efficient divergent maimer.

Another aim of the present invention is to provide a way to a large variety of selected targets in the context of vaccine development against shigellosis. Most of the known 15 5. flexneri serotypes are pathogenic for human and a multivalent vaccine providing broad serotype coverage is required {Clin, Infect. Dis. (2014) 59, 933). Additional 5". flexneri O- antigen diversity has been described {Biochemistry (Moscow) (2015) 80, 901-14).

Inventors have for the first time demonstrated that enzymes are being able to perform the in vitro a-D-glucosylation of the ABCD and DABC tetrasaccharides, corresponding to the backbone repeating unit, and frame-shift thereof, respectively, of S. flexneri O-antigens {Biochemistry (Moscow) (2015) 80, 901-14). Surprisingly, branching sucrases, and more generally glucansucrases, can in particular glucosylate the non-natural ABCIA _C CCBA _C D-AII tetrasaccharide acceptor, to access S. flexneri specifically ct-D-glucosylated pentasaccharides.

Most of the known S. flexneri O-antigens are defined by a repeating unit encompassing a common (ABCD) _n tetrasaccharide backbone. This feature offers major opportunities for the development of a broad serotype coverage vaccine against S. flexneri by use of synthetic carbohydrate haptens.

In contrast to other strategies whereby the design of a n-valent polysaccharide-based vaccine requires the independent preparation of n monovalent polysaccharide antigens and their conversion into immunogens (see for example Prevnar®, Synflorix®, and other licensed polysaccharide-protein conjugate vaccines), synthesis opens the way to a divergent strategy to the target carbohydrate haptens built on a single versatile core precursor inspired from the tetrasaccharide backbone repeat of the O-antigens of interest.

Thus, in one aspect, the present invention relates to a compound of following formula

(¾) :

( _T D) _x AB _z C( _T D) _r R (lo)

wherein:

x and y are 0 or 1 , providing x + y = 1 ;

A is 2)-a-L-Rha/ -( 1→ ;

B is 2)-a-L-Rha/?-(l→ ;

C is 3)-a-L-Rha/ ^? -(l→ ;

D is 3)-a-D-Glc/?N-(l→ ; Z is ClAc, BrAc, Ac or 0;

T is a protecting group capable of anchimeric assistance and being orthogonal to Z, when Z is ClAc, BrAc or Ac, or is azide ( 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 C1CH ₂ -C(0)-;

BrAc is BrCH ₂ -C(0)-.

In one aspect, the present invention relates to a compound of following formula (¾):

( _T D) _x ABzC( _T D) _y -R (Io)

wherein:

x and y are 0 or 1 , providing x + y = 1 ;

A is 2}-a-L-Rha/ -( 1 - ;

B is 2)-a-L-Rhap-(l→ ;

C is 3)-a-L-Rha/?-(l→ ;

D is 3)-a -D-GlcpN-( 1→ or 3)-β-0-01 Ν-( 1→ ;

Z is ClAc, BrAc, Ac or 0;

T is a protecting group capable of anchimeric assistance and being preferentially orthogonal to Z, in particular when Z is Ac, or is azide (N 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 C1C¾-C(0)-;

BrAc is BrCH ₂ -C(0)-.

In a particular embodiment:

when x = 0 and y = 1, D is 3)-a-D-Glc/>N-(l→ or 3)-P-r GlcpN-( l→ ;

when x = 1 and y = 0, D is 3)-p-D-Glc/?N-( 1— > ; Regarding formulae (¾) and (I), D is in particular ;

In a particular embodiment, TD means that T is in position 2D.

In a particular embodiment, means that Z is in position 2c-

Orthogonality and orthogonal protecting groups in carbohydrate chemistry are well known from the skilled in the art, and are in particular described in Agoston et al. (Tetrahedron: Asymmetry 27 (2016) 707-728).

R is in particular chosen from allyl (All), wra-methoxyphenyl (PMP), pentenyl (Pent), phenyl (Ph), triisopropylsilyl (TIPS) or /er/-butyldiphenylsilyl (TBDPS) group. R is in particular on the O in position lc, when x = 1, or on the O in position ID, when y = i-

When R is Ph, the atom in position lc, when x = 1, and in position ID, when y = 1, is in fact a S.

T is in particular chosen from tricMoroacetyl (CBAc), 2,2,2-trichloroethoxycarbonyl

(Troc) or allyloxycarbonyl (Alloc).

In another aspect, the present invention relates to a compound of following formula

(I) :

(ci3AcD) _x AB _z C(ci3AcD) _y -All (I)

wherein:

x and y are 0 or 1 , providing x + y = 1 ;

A is 2)-a-L-Rha/?-(l→ ;

B is 2)-(x-L-Rha/?-(l→ ;

C is 3)-a-L-Rha/?-(l-> ;

D is 3)-a-D-Glc/ N-(l -> ;

Z is ClAc, BrAc, Ac or 0 ;

All is allyl ;

CBAc is C1 ₃ C-C(0)- ;

ClAc is C1CH ₂ -C(0)- ;

BrAc is BrCH ₂ -C(0)-.

In another aspect, the present invention relates to a compound of following formula

(I):

(ci3AcD) _x AB _z C(ci3AcD) _y -All (I)

wherein:

x and y are 0 or 1, providing x + y = 1 ;

A is 2)-a-L-Rha -(l- ;

C is 3)-<x-i.-Rhap-(l→ ;

D is 3)-a-D-GlcpN-(l→ or 3)-p-D-Glc N-(l→ ;

Z is ClAc, BrAc, Ac or 0 ;

All is allyl ;

CBAc is C1 ₃ C-C(0)- ;

ClAc is C1CH ₂ -C(0)- ; BrAc is BrCH ₂ -C(0)-.

In a particular embodiment:

when x = 0 and y = 1, D is 3)-a-D-GlcpN-(l→ or 3)-p-D-GlqpN-(l -> ;

when x = 1 and y = 0, D is 3)-p-D-Glc N-( l→ ;

Compounds of formula (I) offer compatibility with chain elongation into O-antigen fragments, which may require a protecting group able to ensure anchimenc assistance at position 2 of the reducing residue.

The orthogonal protecting group in position 2c enables the 2c-0-acetylation pattern of importance for some serotypes.

Lastly, orthogonal protection at the anomeric position of the reducing residue enables to avoid α/β mixtures while facilitating subsequent chemical modification and in particular chain elongation into O-antigen fragments.

In a particular embodiment, the present invention relates to a compound of formula (I), wherein:

C13 Ac is in position 2D,

Z is in position 2c, and/or

All is in position lc, when x = 1 , or in position I D, when y = 1.

In a particular embodiment, Z is ClAc, corresponding to a compound of formula (CBA _C D) _X ABC!A _C C(CBA _C D) _V -A11.

In a particular embodiment, Z is BrAc, corresponding to a compound of formula

(ci3AcD) _x ABBrAcC(ci3AcD) _r All.

In a particular embodiment, Z is Ac, corresponding to a compound of formula (ci3AcD) _x AB _AC C(ci3A _C B) AlL

In a particular embodiment, Z is 0, corresponding to a compound of formula

(c)3AcD) _x ABC(ci3AcD) Alh

In a particular embodiment, the present invention relates to a compound of formula (I), wherein x is 0 and y is 1 , corresponding to the following formula (la):

AB _z Cci3AcD-AU (la).

In a particular embodiment, said compound is of formula ABCIA _C CCBA _C D-AU, ABBrAcCcuAcD-All, AB., ..CcBAcD-All or

The compound of formula (I) is in particular of the following formula:

more particularly with Z = H or Z = ClAc as in the following:

In a particular embodiment, the present invention relates to a compound of formula (I), wherein x is 1 and y is 0, corresponding to the following formula (lb):

CBAcDAB/C-All (lb).

In a particular embodiment, said compound is of formula CBA _C DABCIA _C C-AH, CBAcDABBrAcC-All, _C i3 _Ac DAB _Ac C-Ali or CBA _C DABC-AH.

The compound of formula (I) is in particular of the following formula:

more particularly with Z = H or Z = ClAc as in the following:

In another aspect, the present invention relates to a process of preparation of a compound of formula (¾) as defined above, comprising the following steps:

(i) a step of contacting a protected ABzC-triosyl donor with a protected jD-R acceptor to yield a protected ABzCjD-R compound ;

(ii) one or more steps of deprotection of the protected compound obtained in step (i) to give a compound of formula ABzCjD-R.

In another aspect, the present invention relates to a process of preparation of a compound of formula (I) as defined above, comprising the following steps:

(i) a step of contacting a protected ABzC-triosyl donor with a protected CBA _C D- M acceptor to yield a protected ABZCCU _AC D-AII compound ;

(ii) one or more steps of deprotection of the protected compound obtained in step (i) to give a compound of formula ABZCCDA _C D- H.

In a particular embodiment, the ABzC-triosyl donor is of formula AB _/ C-Z', wherein Z' is PTFA or TCA, PTFA representing N-phenyltrifiuoroacetimidyl and TCA representing trichloroacetimidyl, Z' being more particularly TCA.

In a particular embodiment, the protected ABzC-triosyl donor is of one of the following formulae:

TCA

, and notably

wherein :

TES is triethylsilyl ;

Lev is levulinyl ;

Nap is 2-naphytlmcthyl ;

PMB is /jfzra-rnethoxybenzy! .

By levulinyl (or levuiinoyl) is meant the group CH3-CO-CH2-CH2-CO-.

In the whole description, a wavy bond such as indicates that the corresponding substifuent is in axial and/or in equatorial position.

Thus, a compound containing such a wavy bond exist as a mixture of the alpha and beta anomers, or only as the alpha or beta anomer.

In a particular embodiment, the protected ABzC-triosyl donor is of one of the above- mentioned formulae, wherein at least one of the TES groups or each TES group is independently replaced by a group chosen from TBS (tert-butyldimethylsilyl), TIPS (triisopropylsilyl), PMB, Nap or Lev.

In a particular embodiment, the protected ABzC-triosyl donor is of one of the above- mentioned formulae, wherein at least one of the BDA groups or each BDA (butane 2,3- diacetal) group is independently replaced by a group chosen from the 1 ,2-diacetaI family, and in particular by CDA (cyclohexane- 1 ,2-diacetal). The CDA group is for example described in Chem. Rev. (2001 ) 101 , 53-80.

In a particular embodiment, the protected ABzC-triosyl donor is of one of the above- mentioned formulae, wherein at least one of the Nap groups or each Nap group is independently replaced by a group chosen from TBS, TIPS or PMB.

In a particular embodiment, the protected G3 _.\C D-A 11 acceptor is of following formula: CI ₃ In. a particular embodiment, the protected ABZCCBA _C D-AH compound is of one of the following formulae:

In a particular embodiment, the protected ABZCCBA _C D-AII compound is of one of the above-mentioned formulae, wherein at least one of the TES groups or each TES group is independently replaced by a group chosen from TBS, TIPS, PMB, Nap or Lev.

In a particular embodiment, the protected ABZCCBA _C D-AII compound is of one of the above-mentioned formulae, wherein at least one of the vicinal Nap/TES pairs or each vicinal Nap/TES pair is independently replaced by a .group chosen from BDA or CDA.

By Nap TES pair, is in particular meant a Nap that is vicinal to a Nap or TES group, as following:

In a particular embodiment, the protected ABZCCBA _C D-AII compound is of one of the above-mentioned formulae, wherein at least one of the Nap groups or each Nap group is independently replaced by a group chosen from TBS, TIPS or PMB.

In another aspect, the present invention relates to a process of preparation of a compound of formula (¾) as defined above, comprising the following steps:

(i) a step of contacting a protected ABzC-R acceptor with a protected i D donor to yield a protected xDAB/C-R compound ;

(ii) one or more steps of deprotection of the protected compound obtained in step

(i) to give a compound of formula rDAB/C-R.

In another aspect, the present invention relates to a process of preparation of a compound of formula (I) as defined above, comprising the following steps:

(i) a step of contacting a protected AB/C-A11 acceptor with a protected CBA _C D donor to yield a protected CBA _C DAB/C-AH compound ; (ii) one or more steps of deprotection of the protected compound obtained to give a compound of formula CBA _C DABZC-AII

In particular, the AB C-A11 acceptor is of one of the following formulae:

wherein:

TES is triethylsilyl ;

Lev is levulinyl ;

Nap is 2-naphty!methyl ;

PMB is /?ara-methoxybenzyl.

In a particular embodiment, the ABzC-A!l acceptor compound is of one of the above- mentioned fonnulae, wherein at least one of the TES groups or each TES group is independently replaced by a group chosen from TBS, TIPS, PMB or Nap.

In a particular embodiment, the protected ABZCCBA _C D- U compound is of one of the above-mentioned formulae, wherein at least one of the Nap groups or each Nap group is independently replaced by a group chosen from TBS, TIPS or PMB.

In a particular embodiment, the protected ABzC-All acceptor is of one of the above- mentioned formulae, wherein at least one of the vicinal Nap/TES pairs or each vicinal Nap/TES pair is independently replaced by a group chosen from BDA or CD A.

In particular, the CBA _C D donor is of formula CBAJD-Z', wherein Z' is PTFA or TCA, PTFA representing Λ'-ph en yl tri fl uoroaceti m i dyl and TCA representing trichloroacetimidyl.

In particular, the protected CBA _C D donor is of one of the following formulae or the corresponding oxazolines:

I ₃ Ph-Τ O

TESO ^' OTCA TESO OPTFA

I ₃

and notably:

By "corresponding oxazoline" is meant a group as following:

The 1 ,2-oxazoline may result from intramolecular cyclisation and loss of leaving group at position 1.

In a particular embodiment, the CBA _C D donor compound is of one of the above- mentioned formulae, wherein the TES group is independently replaced by a group chosen

When the ABzC-AU acceptor is of formula (a) and the CDA _C D donor bears a TES protecting group in position 3D, there is in particular one step (ii) of dcprotection only. In the other cases, there may be two steps of deprotection.

In a particular embodiment, the protected C A _C DAB/C-AH compound is of one of the following formulae:

In a particular embodiment, the protected CBA _C DABZC-AII compound is of one of the above-mentioned formulae, wherein at least one of the TES groups or each TES group is independently replaced by a group chosen from TBS, TIPS, PMB or Nap.

In a particular embodiment, the protected CBA _C DABZC-AU compound is of one of the above-mentioned formulae, wherein at least one of the vicinal Nap/TES pairs or each vicinal Nap/TES pair is independently replaced by a group chosen from BDA or CD A.

In a particular embodiment, the protected CBA _C DABZC-AII compound is of one of the above-mentioned formulae, wherein at least one of the TBS groups or each TBS group is replaced by a group chosen from PMB, Nap, Lev, TIPS or Lev.

In a particular embodiment, the protected CBA _C DAB/C-AII compound is of one of the above-mentioned formulae, wherein the 4,6-0-benzylidene acetal is replaced by an 4,6-0- isopropylidene acetal.

In another aspect, the present invention relates to a compound as defined by the following formulae, wherein Z is ClAc, Br Ac or Ac : notably , and

I ₃ In a particular embodiment, the compound is of one of the above-mentioned formulae, wherein at least one of the vicinal Nap/TES pairs or each vicinal Nap/TES pair is independently replaced by a group chosen from BDA or CDA.

In another aspect, the present invention relates to a method of preparation of a saccharide comprising the following steps:

(i) a step of a-D-glucosylation of a compound of formula (¾) as defined above 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 a a-D-glucosylated ( _t D) _X ABKC(TD) _V -R compound;

(ii) optionally, and in particular when y is 1 , a step of acetylation of the 3A position and/or the 6p position to obtained a 3A- and/or δο-0-acetylated compound;

(iii) optionally, a step of cleavage of the ClAc or BrAc protecting group bome by C to obtain a 2c-hydroxylated compound;

(iv) optionally, a step of (a) conversion of the ClAc or BrAc protecting group bome by C into an acetyl group (Ac), (b) conversion of the C13Ac masking group bome by D into an acetyl group (Ac), (c) conversion of the allyl protecting group bome by D into a propyl (Pr) moiety followed if needed by the de-O-acylation of the 2c position to obtained either a 2(-acylated or 2c-hydroxylated compound;

(v) optionally, a step of chain elongation at one or both of the ends of the compound resulting from the enzymatic a-D-glucosylation of the compound of formula (1) as defined above.

In another aspect, the present invention relates to a method of preparation of a saccharide comprising the following steps:

(i) a step of a-D-glucosylation of a compound of formula (I) as defined above 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 a α-D-glucosylated (cBAcD) _x AB C(c;3AcD)y-All compound;

(ii) optionally, and in particular when y is 1, a step of acetylation of the 3A position and/or the 6D position to obtained a 3A- and/or 6o-O-acetylated compound;

(iii) optionally, a step of cleavage of the ClAc or BrAc protecting group bome by C to obtain a 2c-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 CBAc masking group borne by D into an acetyl group (Ac), (c) conversion of the ally! protecting group borne by D into a propyl (Pr) moiety followed if needed by the de-O-acylation of the 2c position to obtained either a 2c-acylated or 2c-hydroxylated compound;

(v) optionally, a step of chain elongation at one or both of the ends of the compound resulting from the enzymatic oc-D-glucosylation of the compound of formula (I) as defined above.

For example, step (ii) is a regioselective O-acylation at a primary hydroxyl group as well as at the equatorial hydroxyl group of 1 ,2-cis diol systems, which has been for instance described for polyols including carbohydrates by means of a borinic-acid catalyzed regioselective step (J. Am. Chem. Soc. (2011) 133, 3724-7). Alternatively, methods selective for the O-acylation of primary hydroxyl groups have been described including enzymatic O- acylation (Tetrahedron: Asymm. (2000) 11, 3647-51). Such transformations could also be in particular envisioned using an a-D-glucosylated AB _Ac C _A cD-Pr substrate (wherein Pr = propyl, Ac ^~ acetyl), as discussed below, or an a-D-glucosylated ABCA _C D-ΡΓ substrate.

Step (iii) may be achieved using thiourea {Carbohydr. Res. (2012) 356, 1 15-31).

About step (iv), the transformation of an α-D-glucosylated (ci3AcD) _x BzC(ci3AcD) _y - ll compound into the corresponding ( _Ac D) _x ABAcC( _A cI3y)- r (Pr = propyl, Ac = acetyl) may be achieved by Pd/ ^' C- or Pd(OH) ₂ -mediated hydrogenation {Chem. Asian J. (2017) 12, 419-39), while the subsequent de-O-acetylation at position 2c is easily achievable by conventional transesterification, for example by use of a methanol ic solution of sodium methoxide {Chem. Eur. J. (2016) 22, 10892 - 91 1).

About step (v), and, for example, taking advantage of the cw-vicinal diol in residue A (not applicable in the case enzymatic a-D-glucosylation would occur at position 3A as in the S. flexneri 3a O-antigen), the 2 _A and 3A hydroxyl groups may be masked as an isopropylidene acetal either directly or post selective masking of the primary hydroxyl groups (J. Org. Chem. (2015) 80, 1 1237-57). Following the unmasking of the primary alcohols if needed, the remaining hydroxyl groups may then be masked with permanent protecting groups, preferentially benzyl ethers (J. Org. Chem. (2015) 80, 1 1237-57). Removal of the isopropylidene group under acid hydrolysis conditions, followed by selective masking of the 3 _A -OH by use of a protecting group orthogonal to all those in place (PMB or a silyl ether such as TES for example) may provide a pentasaccharide acceptor compatible with chain elongation at the 2 _A -OH. Alternatively, levulinylation of the hydroxyl group at position 2 _A may provide a folly orthogonally protected pentasaccharide building block, which may be converted into a donor by selective removal of the ally! ether followed by activation of the obtained hemiacetal into an imidate donor (anomeric OPTFA or OTCA substitution) (J, Org. Chem. (2015) 80, 1 1237-57).

Herein, a chemo-enzymatic strategy to customized glycobricks suitable for the efficient synthesis of fragments of a diversity of S. flexneri O-antigens is disclosed. These glycobricks could be assembled into homo-oligomers, therefore providing a novel route to S. flexneri type-specific haptens, in particular through step (v) of chain elongation.

Homo-oligomers correspond to fragments encompassing n repeating units of the O- antigen from a selected S. flexneri serotype. Such a 15mer hapten, corresponding to a three repeating unit fragment of the O-antigen, was for example identified for 5". flexneri 2a (J. Immunol (2009) 182, 2241-7, Bioconjugate Chem. (2016) 27, 883-92). Besides, available data suggest that at least a 15mer, most probably a 20mer, corresponding to three and four repeating unit fragments of the O-antigen would act as a suitable S. flexneri 3 a hapten.

Glycoside hydrolases EC 3.2.1. and EC 2.4.1 are a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycoside hydrolases, based on sequence similarity, has led to the definition of > 100 different families. This classification is well known for the skilled in the art and is in particular available on the CAZy (http://www.cazy.org) web site.

By enzymes of the G.H13 family is meant enzymes of glycoside hydrolase family 13 active on sucrose, that is a family of glycoside hydrolases, in particular amylosucrases (subfamily), which are also included in the « glucansucrase » family. This classification is well known for the skilled in the art and is in particular available on the CAZy (GH13.4 http://www.cazy.org/GH13_4.html) web site.

By enzymes of the GH13 family is meant in particular a wild type glycoside hydrolase, more particularly a transglucosylase, even more particularly an amylosucrase (EC 2.4.1.4) or a sucrose hydrolase (EC 3.2.1.-), as described in the patent application EP 2 100 966, even more particularly an amylosucrase from Neisseria polysaccharea, preferably selected from the group consisting of 1G5A, 1ZS2, 1 MVY, 1MW0, 1 S46, UGI, 1MW2, 1MW3, 1MW1 and UG9 proteins as found in the Protein Data Base (PDB, https://www.rcsb.org/) and as described in the patent application EP 2 100 966, or a mutant of the protein, as described in the patent application EP 2 100 966. By enzymes of the GH70 family is meant transglucosylases produced by lactic acid bacteria from, e.g., Streptococcus, Leuconostoc, Weisella or Lactobacillus genera. This classification is well known for the skilled in the art and is in particular available on the CAZy (http://www.cazy.org GH70.html) web site. In particular the enzymes of the GH70 family are branching sucrases and glucansucrases (EC 2.4.1). An enzyme of the GH70 family that can be used in the framework of this invention is an altemansucrase from Leuconostoc citreum, more particularly of strain NRRL B-1355.

In particular, said enzyme is selected from the group consisting of the BRS-B, BRS-B- Dl, BRS-B-D2, BRS-C, BRS-A, BRS-D, BRS-E, GBD-CD2, GBD-CD2 W2135V, GBD- CD2 W2135C-F2136I, GBD-CD2 W2135S-F2136L, 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 enzymes.

In particular, said enzyme is selected from the group consisting of the BRS-B, BRS-B- Dl , BRS-B-D2, BRS-C, BRS-A, BRS-D, BRS-E, GBD-CD2, GBD-CD2 W2135V, GBD- CD2 W2135C-F2136I, GBD-CD2 W2135S-F2136L, GBD-CD2 W2135I-F2136C, GBD-CD2 W2135C, 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 F2163G L2166I, GBD-CD2 A2162E F2163L, GBD-CD2 F2163L, GBD-CD2 F2163I-D2164E-L21661 enzymes.

These enzymes are described in the art, as mentioned below in the examples.

In particular, said enzyme is a mutant of the BRS-B-D2 enzyme (SEQ ID NO : 4), as defined in the following table:

in particular, said enzyme is BRS-B.

In particular, said enzyme is BRS-B-D2.

In particular, said enzyme is GBD-CD2 F2163G.

In particular, said enzyme is GBD-CD2 W21351-F2136C or GBD-CD2 W2135L- F2136L or GBD-CD2 W2 I35S-F2136L.

In particular, said saccharide is a fragment of O-antigens from S. flexneri, in particular of serotype la, lb, 2a, 2b, 3a, X, 4a, 4b, 5a, 5b, 7a or 7b.

In particular, said saccharide comprises the following fragment:

-[((El→2) _a - (El→4), (M) _{b A} cD) _x (L) _c A (El→3) _d B (E l --->4) _{e (Ac)} ,C ((El→2) _a . (El→4) _a (M) _b whcrcin:

L is El- 3 or Ac in position 3A

M is El— >6 or Ac in position 6D;

a and b is 0 or 1, providing a + b = 0 or 1 ;

a' is:

- 0 or 1 when a is 1 ;

- 0 when a is 0 ;

c, d and e are 0 or 1 ;

a, b, c, d and e being in particular such as a + b + c + d + e = l or 2 ;

z is at each occurrence independently 0 or 1, z being in particular at all occurrences 0 or 1 ;

E represents a residue a-D-Glcp- ;

n is an integer superior or equal to 1, in particular comprised between 1 and 10.

In particular, said saccharide comprises the following fragment:

-[((El→2) _a . (El→4) _a (M) _{b Ac} D) _x (L) _c (El→4) - A (El→3) _d (El >4) _d - B (El→4) _{e {Ac)z} C i(El→2) _a - (El→4) _a ( ) _{b Ac} D) _y ] _n - wherein:

L is El ->3 or Ac in position 3A;

M is El -»6 or Ac in position 6D;

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, c being in particular at all occurrences 0 or 1 , providing c + c' = 0 or 1 ;

when L is E l ->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, b being in particular at all occurrences 0 or 1 , providing a + b = 0 or 1 ;

when M is E 1— >6, b is 0 or 1 , providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = 1 , 2 or 3.

z is at each occurrence independently 0 or 1 , z being in particular at all occurrences 0 or 1 ;

E represents a residue a-D-Glcp- ;

n is an integer superior or equal to 1 , in particular comprised between 1 and 10.

In the above and below paragraphs, L and/or M are in particular Ac at at least one occurrence, more particularly at all occurrences.

In. the above and below paragraphs, L and/or M are in particular El - ->3 and El— >6 respectively, at all occurrences.

In particular, said saccharide comprises the following fragment:

-[((El→2) _a - (El→4) _a (M) _{b Ac} D) _x (L) _e A (El→3) _d B (El→4) _{e (Α} € ((E l - 2) _fl - (El→4) _a (M) _b

_Ac D) _y ] _n -Ail

wherein:

L is El— >3 or Ac in position 3A ;

M is El - >6 or Ac in position 6 _D ;

a and b is 0 or 1 , providing a + b = 0 or 1 ;

a' is:

- 0 or 1 when a is 1 ; - 0 when a is 0 ;

c, d and e are 0 or 1 ;

a, b, c, d and e being in particular such as a + b + c + d + e = l or 2 ;

z is at each occurrence independently 0 or 1 , z being in particular at all occurrences 0 or 1 ; E represents a residue a-D-Glcp- ;

n is an integer superior or equal to 1, in particular comprised between 1 and 10.

In particular, said saccharide comprises the following fragment:

-[«E l→2) _a - (El→4) _a (M) _{b Ac} D) _x (L) _c (El→4) _c . A (El→3) _d (El→4) _cl - B (E 1→4) _{e (Ac)/} C

((El ->2) _a . (El→4) _a (M) _{b Ac} D) _y ] _n ^All

wherein:

L is E ! ^■ -- ^■ 3 or Ac in position 3 A;

M is El-»6 or Ac in position 6D;

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, c being in particular at all occurrences 0 or 1, providing c + c' = 0 or 1 ;

when L is El->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, b being in particular at all occurrences 0 or 1, providing a + b = 0 or 1 ;

when M is El— >6, b is 0 or 1, providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = 1, 2 or 3.

In particular, said saccharide comprises the following fragment:

-[(<E1→2),. (El→4) _a (M) _{b Ac} D) _x (L) _c A (El→3) _d B (El→4) _{e (Ac)z} C ((El ->2) _a - (El→4). (M) _b

wherein:

L is El~>3 or Ac in position 3A ;

M is El— >6 or Ac in position 6D ;

a and b is 0 or 1 , providing a + b = 0 or 1 ; a' is:

- 0 or 1 when a is 1 ;

- 0 when a is 0 ;

c, d and e are 0 or 1 ;

a, b, c, d and e being in particular such as a + b + c + d + e = 1 or 2 ;

z is at each occurrence independently 0 or 1, z being in particular at all occurrences 0 or 1 ; E represents a residue α-D-Glcp- ;

n is an integer superior or equal to 1, in particular comprised between 1 and 10 ;

Pr is propyl.

In particular, said saccharide comprises the following fragment:

-[((El→2) _a - (El→4) _a (M) _{b Ac} D) _x (L) _c (El→4) _c . A (El→3) _d (El→4) _d . B (El→4) _{e (Ac)z} C

((El→2) _a . (E l→4) _a (M) _{b Ac} D) _y ] _n -Pr

wherein:

L is El->3 or Ac in position 3 _A ;

M. is El→6 or Ac in position 6D;

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, c being in particular at all occurrences 0 or 1, providing c + c' = 0 or 1 ;

when L is El— >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, b being in particular at all occurrences 0 or 1 , providing a + b = 0 or 1 ;

when M is El— >6, b is 0 or 1, providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = 1, 2 or 3;

Pr is propyl.

In particular, said saccharide comprises the following fragment:

-[((El→2) ₀ - (El→4) _a (El→6) _{b Ac} D) _x (El→3) _c A (El→3) _d B (El→4) _{c (Ac)z} C ((El→2) _a - (El→4) _a (El--»6) _{b Ac} D) _y ] _n - wherein:

a and b is 0 or 1 , providing a + b = 0 or 1 ;

a' is:

- 0 or 1 when a is 1 ;

- 0 when a is 0;

c, d and e are 0 or 1 ;

a, b, c, d and e being in particular such as a + b + c + d + e = 1 or 2 ;

z is at each occurrence independently 0 or 1 , z being in particular at all occurrences 0 or 1 ;

E represents a residue cx-D-Glc/?- ;

n is an integer superior or equal to 1 , in particular comprised between 1 and 10.

In particular, said saccharide comprises the following fragment:

-[((E 1→2) _a - (E 1→4) _a (El→6) _{b Ac} D) _x (E 1→3) _c (El -→4) _c - A (El- 3) _d (E1→¾ B (El→4) _e

(Ac ₎₂ C ((E l→2) _a - (El→4) _a (El→6) _b D) _y ]„- wherein:

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 ;

c is 0 or 1 , providing c + c' = 0 or 1 ;

b is 0 or 1 , providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = l, 2 or 3.

In particular, said saccharide comprises the following fragment:

-[((E 1→2).v (E 1→4) _a (Ac 1→6) _{b Ac} D) _x (Ac 1→3) _c (El→4) _c - A (El→3) _d (El→4) _d . B (El→4) _e

_(Ac) zC ((El→2) _a . (El→4) _a (El→6) _b D)v] _n - wherein:

a is 0 or 1 ,

a' is:

- 0 or I when a is 1 ;

- 0 when a is 0 ;

e is 0 or 1 ; c' is 0 or 1 ;

(1 and d' are 0 or 1, providing d + d' = 0 or 1 ;

c is at each occurrence independently 0 or 1 , c being in particular at all occurrences 0 or 1, providing c + c' = 0 or 1 ;

b is at each occurrence independently 0 or 1 , b being in particular at all occurrences 0 or 1 , providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = 1, 2 or 3.

In particular, said saccharide comprises the following fragment:

-[((El→2) _a - (El→4), (El→6 _Ac D)x (Ac 1→3) _c (El→4) _c . A (El→3) _d (El ->4) _d . B (El ->4) _{c {Ac)z} C ((El→2) _a . (El→4) _a (El→6) _b D) _y ] _n - whcrcin:

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 ;

c is at each occurrence independently 0 or 1 , c being in particular at all occurrences 0 or 1, providing c + c' = 0 or 1 ;

b is 0 or 1, providing a + b = 0 or 1 ;

a, b, c, c', d, d' and e being in particular such as a + b + c + c' + d + d' + e = 1, 2 or 3.

In particular, said saccharide comprises the following fragment:

-f((E 1→2)a- (E 1→4) _a (Ac 1→6) _{b Ac} D) _x (E 1→3) _c (E 1→4) _c - A (El→3) _d (E 1→4) _d - B (E 1→4) _e , _Ac)z C ((El→2) _a . (El→4) _a (Ε\→6 D) _y ] _n - wherein:

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 ; , c is 0 or 1 , providing c + c' = 0 or 1 ;

b is at each occurrence independently 0 or 1 , b being in particular at all occurrences 0 or 1 , providing a + b = 0 or 1 ;

a, b, c, c', d, d ^* and e being in particular such as a + b + c + c' + d + d' + e = 1, 2 or 3..

By "z is at each occurrence independently 0 or 1" is meant that for each occurrence of the repeating unit repeated n times, z can be independently 0 or 1. In other terms, for n superior or equal to 2, position 2c can be noii-O-acetylated, rally O-acetylated or partially acetylated within said saccharide.

By "when L is Ac, c is at each occurrence independently 0 or 1" is meant that for each occurrence of the repeating unit repeated ri times, c can be independently 0 or 1 , when L is Ac, In other terms, for n superior or equal to 2 _» position 3A can be non-O-acetylated, fully O- acetylated or partially acetylated within said saccharide.

By "when M is Ac, b is at each occurrence independently 0 or 1" is meant that for each occurrence of the repeating unit repeated n times, b can be independently 0 or 1, when M is Ac. In. other terms, for n superior or equal to 2, position 6D can be non-O-acetylated, fully O-acetylated or partially acetylated within said saccharide.

More particularly, said saccharide comprises the following fragment:

-[AB _(At)2 C(E l→6) _A cDj _n - In a particular embodiment, the sucrose-active enzyme is selected from the group consisting of the BRS-B, BRS-B-D2, BRS-C, BRS-A, BRS-D, BRS-E, GBD-CD2, GBD- CD2 W2135V, GBD-CD2 W2135C-F2136I, GBD-CD2 W2135S-F2136L, GBD-CD2 W2135I-F2136C, GBD-CD2 W2135C, 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 F2163G L2166I, GBD-CD2 A2162E F2163L, GBD- CD2 F2163L, GBD-CD2 F21631-D2164E-L2166I enzymes, and the BRS-B-D2 M6, BRS-B- D2 M21 , BRS-B-D2 M23, BRS-B-D2 M28, BRS-B-D2 M30, BRS-B-D2 M31 , BRS-B-D2 M34, BRS-B-D2 M35, BRS-B-D2 M40 and BRS-B-D2 M41 enzymes, the enzyme being more particularly BRS-B.

More particularly, said saccharide comprises the following fragment:

-[(El→3)AB ₍ Ac) _Z C _A cD]n-

In a particular embodiment, the sucrose-active enzyme is selected from the group consisting of the BRS-B, BRS-B-D2, BRS-C, BRS-A, BRS-D, BRS-E, GBD-CD2, GBD- CD2 W21 35V, GBD-CD2 W2135C-F2136I, GBD-CD2 W2135S-F2136L, GBD-CD2 W2135I-F2136C, GBD-CD2 W2135N-F2136Y, GBD-CD2 W2135N, GBD-CD2 W21351- 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-L21661 enzymes, and the BRS-B-D2 M14, BRS-B-D2 M l 8, BRS-B-D2 M21 , BRS-B-D2 M23, BRS-B-D2 M28, BRS-B-D2 M30, BRS-B-D2 M34, BRS-B-D2 M35, BRS-B-D2 M40 and BRS-B-D2 M41 enzymes, the enzyme being more particularly BRS-B, GBD-CD2 W2135G or GBD-CD2 F2163I-D2164E-L2166I.

More particularly, said saccharide comprises the following fragment:

-[(El→4)AB ₍ Ac) _Z C _A cD]n-

In a particular embodiment, the sucrose-active enzyme is selected from the group consisting of the BRS-B, BRS-B-D2, BRS-C, BRS-A, BRS-D, BRS-E, GBD-CD2, GBD- CD2 W2135V, GBD-CD2 W2135C-F2136I, GBD-CD2 W2135S-F2136L, 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-F21361, GBD-CD2 W2135C-F2136N, GBD-CD2 W2135G, GBD-CD2 W2135F, GBD-CD2 F2163G, GBD-CD2 L21661, GBD-CD2 F2163H, GBD-CD2 F2163G L2166I, GBD-CD2 A2162E F2163L, GBD-CD2 F2163L, GBD-CD2 F2163I-D2164E-L2166I enzymes, and the BRS-B-D2 Ml 4, BRS-B-D2 M l 8, BRS-B-D2 M21 , BRS-B-D2 M23, BRS-B-D2 M28, BRS-B-D2 M30, BRS-B-D2 M34, BRS-B-D2 M35, BRS-B-D2 M40 and BRS-B-D2 M41 enzymes, the enzyme being more particularly BRS-B, GBD-CD2 W2135G or GBD-CD2 F2163I-D2164E-L2166I.

More particularly, said saccharide comprises the following fragment:

-[A(El→4)B _(A c) _Z C _A cD] _n - In a particular embodiment, the sucrose-active enzyme is selected from the group consisting of the GBD-CD2 W2135S-F2136L, GBD-CD2 W2135I-F2136C, GBD-CD2 W2135L-F2136L enzymes, and the BRS-B-D2 M14, BRS-B-D2 M18, BRS-B-D2 M21, BRS-B-D2 M23, BRS-B-D2 M28, BRS-B-D2 M30, BRS-B-D2 M35, BRS-B-D2 M40 and BRS-B-D2 M41 enzymes.

I another aspect, the present invention relates to a compound of one of the following formulae:

((E 1→2) _a .(E 1→4) _a (E 1→6) _bC i3AcD) _x (E 1→3) _C (E 1→4) _C A(E 1→3) _cf (E 1 ->4) _d -B(E 1→4) _e

CI _AC C((E 1 ->2) _a .(E 1→4) _a (E 1→6 _b ) _C i3A _C D) _y » All; (t E I >2) _A .(E 1→4) _a (E 1→6)bci3AcD) _x (E 1→3) _C (E 1→4) _C -A(E 1→3) _d (E 1→4) _D -B(E 1 - 4) _C C((E 1→2) _A - (E 1→4)„(E 1→6) _B cD _Ac D All;

((E 1 >2),-(E 1 >4) _U (E 1→6) _bC i3AcD) _x (E 1→3) _C (E 1→4) _C A(E 1 ~>3) _d (E 1→4) _d -B(E 1→4) _E

_Ac C((El-→2) ₃ .(El→4) _a (El→6) _b cBAcD) _y -AIl;

((E 1→2) _A .(E 1→4) _a (E 1→6) _bC .A )x(E 1→3) _C (E 1→4 -A(E 1 ->3) _d (E 1→4) _D -B(E i >4) _e

_Ac C((El→2) _a .(El→4MEl→6) _{b c} ,3AcD) _y ;

((E 1→2) _a E 1→4) _a (E 1→6) _b ci3AcD) _x (E 1→3) _C (E 1→4) _C A(E 1→3)„(E 1 >4) _d -B(E 1→4) _E

ciAcC((E 1 >2).·(Ε 1 >4) _a (E 1→6) _{b C} u _Ac D) _y ;

((E 1→2) _a .(E 1→4) _a (E 1→6)bCB A _C D) _X (E 1 ->3) (E 1→4) _C . A(E 1→3) _d (E 1→4) _D -B(E 1→4),C((E 1→2) _a -

(El→4) _a (El→6) _{b C} ,3AcD) _y ;

((El→4).(E 1→6) _bC 3A D) _x (E 1→3) _S (E i ~>4) _C A(E I ->3) _d (E 1→4) _D .B(E 1→4) _E

CIA _C C((E 1→4) _a (E 1→6 _b ) _c ,3AcD) _r All;

((E 1→4) _a (E 1→6) _b ci3 A _C D) _X (E1→3) _C (E 1→4) _C -A(E 1→3) _d (E 1→4) _D -B(E 1→4) _E C((E 1→4) _A (E 1 ->6) _b

CBAcD)y-All;

((E 1→4) _a (E 1→6)κ;; _3Α ,Ι ),(Ε 1→3) _C (E 1→4) _c A(E 1→3) _d (E 1→4) _D -B(E 1 ~»4). _Ac C((El→4) _a (El→6) _b

((El→4) _a (E 1 >6) _{bC i} AcD)x(E 1→3).(E 1 ->4) _c A(E 1→3) _d (E 1→4),·Β(Ε 1→4) _{E Ac} C((E 1 - 4) _a (E 1 · >6)

((E 1→4) _a (E 1→6) _bC i3 A _C D) _X (E 1→3) _C (E 1→4) _C A(E 1→3) _d (E 1 - 4) _D -B (E 1→4%

CIA _C C((E 1→4) _a (E 1→6)„ ci3AcD) _y ;

((E 1→4) _a (E 1→6)„ c,3AcD) _x (E 1→3) _C (E 1→4) _C -A(E 1→3) _D (E 1→4) _D .B(E 1→4) _e C((E 1→4) _a (E 1→6) _b

((E 1→4) _a (E ! >6 , C!3A _C D) _x (E 1→3) _c A(E 1→3) _d B(E 1→4) _c CIA.C((E 1→4) _a (El→6) _{B C} IM. D) _y -All;

((E 1→4) _a (E 1→6) _b ci3 AcD) _x (E 1→3) _C A(E 1→3) _D B(E I→4) _e C((E 1→4) _a (E 1→6) _{b C} B _Ac D) _y - All;

((E 1→4) _a (E 1→6) _B ci3AcD) _x (E l→3) _c A(E 1→3) _d B(E 1 ->4) _{e AC} C((E 1→4) _a (E 1 ->6) _{b a3} AcD) _r All; ((E 1→4) _a (E 1→6) _b ci3AcD) _x (E 1→3) _c A(E 1→3) _d B(E 1→4) _{e Ac} C((E 1→4) _a (E l→6) _{B a3 Ac} D) _y ;

((E 1→4) _a (E 1→6) _B cBAcD) _x (E 1→3) _c A(E 1→3) _d B(E 1→4 _C IA _C C((E 1→4) _a (El→6) _b ci3A _C D) _y ;

((E 1→4) _a (E 1 ~>6) _B ci3AcD) _x (E 1 -->3) _c A(E 1→3) _d B(E 1 >4) _c C((E 1→4) _a (E 1→6) _B COA _C D),;

ABA _C C(E1 - 6) CI3A _C D-A11;

AB _Ac C(El→6) ci3AcD;

ABciAeC(El→6) _a3Ac D; ABC(El→6) cBAcD

(El→3)ABci _Ac C _c ,3AcD-All;

(El→3)ABCci _3Ac D-All;

(E1→3)ABACC _c , ₃ A _O D-A11;

(E1→3)AB _A CQ:BACD;

(E1→3).ABCIACCCI3ACD;

(El→3)ABCCI3ACD;

(E1 -->4)ABCIACC C',3ACD-A11;

(El ->4)ABCC,3A _C D-A11;

(E 1 ->4)ABACC I3ACD-A11;

(E 1 ->4)ABACCCI3ACD;

(E1→4)ABCIACCCI3ACD;

A(E1→4)B _CI ACC _c ,3ACD-A11;

A(E 1 ->4)BCCI3ACD-A11;

A(E1→4)BACC _C I3ACD-A11;

A(E1->4)B _AC C _C1 3ACD;

A(E 1 - 4)BCIACCCI3ACD;

In another aspect, the invention concerns an enzyme selected from the group comprising the enzymes BRS-D-2 M6, BRS-D-2 M14, BRS-D-2 M18, BRS-D-2 M21 , BRS-

D-2 M23, BRS-D-2 M28, BRS-D-2 M30, BRS-D-2 M31 , BRS-D-2 M34, BRS-D-2 M35, BRS-D-2 M40, BRS-D-2 M41 as defined above, and their variants.

Synthesis

Compounds of formula I may be obtained thanks to a [3+1] strategy as shown below. For instance, an ABQA _C C rhamnotriosyl donor encompassing protecting groups orthogonal to an allyl ether (All), a N-trichloroacetyl (Cl ₃ Ac) and chloroacetyl moiety (CiAc), was reacted with a D acceptor. A two-step deprotection process gave the ABCIA _C CCUA _C D-AU acceptor. Protection and deprotection techniques are for instance described by P. G. M. Wuts and T. W. Greene (Greene's Protective Groups in Organic Synthesis, Fourth Edition; Wiley- Interscience, 2006; or Greene's Protective Groups in Organic Synthesis, fifth Edition; Wiley- Interscience, 2014),

Advantageously, rhamnoses A, B and C are in particular built from a single precursor. A suitable synthetic pathway may be the following:

Definitions

The following terms and expressions contained herein are defined as follows:

As used herein, a range of values in the form "x-y" or "x to y", or "x through y", include integers x, y, and the integers there between. For example, the phrases "1-6", or "1 to 6" or "1 through 6" are intended to include the integers 1 , 2, 3, 4, 5, and 6. Preferred embodiments include each individual integer in the range, as well as any subcombination of integers. For example, preferred integers for "1-6" can include 1 , 2, 3, 4, 5, 6, 1-2, 1-3, 1-4, 1- 5, 2-3, 2-4, 2-5, 2-6, etc.

As used herein, the term (E) means that the saccharide following the term (E) bears a residue a-D-Glc -. For example, A(E)B means that B bears a residue ct-D-Glc -. Furthermore, (E l— 6) AcD means that the residue A _C D bears a residue ot-D-Glc/?- linked at its primary hydroxyl group (position 6D-OH).

As used herein, the term "donor" more particularly refers to a mono-, oligo- or polysaccharide bearing a leaving group at the anomeric position.

As used herein, the term "acceptor" more particularly refers to a mono-, oligo- or polysaccharide having at least a free hydroxyl group, in general other than the anomeric hydroxyl, preferably at least the free hydroxyl group corresponding to the elongation site of the growing chain.

A variant is derived from an enzyme of the GH13 or GII70 family, such as amylosucrases in the case of GH13 family, and branching sucrases and glucansucrases in the case of GH70 family, by the introduction of mutations (deletion(s), insertion(s) and/or substitutions(s)) at specific positions in the sequence of said enzyme, while retaining the ability of said enzyme to catalyze ct-D-glucosylation.

In particular, the amino acid sequence of said variant has at least 50% identity, or by order of increasing preference at least 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity, with the amino acid sequence of the corresponding enzyme of the Gill 3 or GH70 family.

The percent amino acid sequence identity is defined as the percent of amino acid residues in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity. The Percent identity is then determined according to the following formula: Percent identity = 100 x [1- (C/ ^' R)], wherein C is the number of differences between the Reference Sequence and the Compared sequence over the entire length of the Reference sequence, wherein (i) each amino acid in the Reference Sequence that does not have a corresponding aligned amino acid in the Compared Sequence, (ii) each gap in the Reference Sequence, and (iii) each aligned amino acid in the Reference Sequence that is different from an amino acid in the Compared Sequence constitutes a difference; and R is the number amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as an amino acid.

Unless otherwise specified, the percent of identity between two protein sequences which are mentioned herein is calculated from the BLAST results performed either at the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or at the GRYC (http://gryc.inra.fr/) websites using the BlastP program with the default BLOSUM62 parameters as described in Altschul el al. (1997).

By "ABC-triosyl" is meant an ABC rhamnotriosyl, i.e. an ABC triose group.

FIGURES

Figure 1 A presents the HSQC spectrum corresponding to pentasaccharide 1 . Figure IB 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 ani C

AHyl 4-0-(2-naphtylmethyI)-a-L-rhamnopyranoside (1) 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 ramol) 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 NaHC0 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 ₃ N (4 mL), solvents were evaporated under reduced pressure. The residue was dissolved in DCM (600 mL) and washed with H?0 (3x300 mL) and brine (200 mL). The organic layer was dried by passing through phase separator filter and concentrated to dryness.

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 ₂ 0 (3 x 300 mL) and brine (150 mL). The organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated to dryness.

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 it 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 (3 x 400 mL) to give a brown solid. Filtration over a pad of silica eluting with a 4:1 mixture of cilex/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 (1 1.6 g). The total yield of diol 1 is 81 % over 4 steps.

Ή NMR (400 MHz, CDC1 ₃ ) δ 7.87 - 7.78 (m, 411, H _ArNap ), 7.52 - 7.44 (m, 3H, H _Ar Nap)» 5.89 (dddd, I II, J - 17.2, 10.4, 6.0, 5.2 Hz, CH=C¾), 5.28 (dq _app , I II, J = 17.2, 1.5 Hz,

CH=Ci¾), 5.19 (dq _ap p, 1H, J - 10.4, 1.5 Hz, CH=G%), 4.95 - 4.86 (m, 2H, H _ArNap ), 4.81 (d,

J= 1 .4 Hz, HI, H-l), 4.17 (ddt, I H, J= 12.9, 5.1, 1.5 Hz, 1 H, HAH), 4.02 - 3.93 (m, 3H, H _A ii,

H-2, H-3), 3.79 (dq, 1H, J = 9.2, 6.3 Hz, H-5), 3.41 (t _app , 1H, J = 9.2 Hz, H-4), 2.45 (brs, 2H,

OH), 1.38 (d, 3H, J= 6.3 Hz, H-6).

i ³ C NMR (100 MHz, CDC1 ₃ ) δ 133.8 (CH=C¾), 128.4 (CivAr), 128.0 (C _1VAr ), 127.7 (C _IVAr ),

126.7-125.8 (7C, C _Ar ), 1 17.4 (CH=€H ₂ ) _» 98.5 (C- l 'J _C . _H = 170.1 Hz), 75.1 (CH _2Nap ), 71.6,

71.3 (2C, C-2, C-3), 68.0 (C _A „), 67.3 (C-5), 18.1 (C-6).

HRMS (ES ): mlz 362.1985 (calcd for C,6H ₂₂ 0 ₅ Na [M+NH ₄ ]' mlz 362.1967); mlz 367.1576 (calcd for C ₄ 3H ₅ ,C10 ₁₂ Na [M+Naf mlz 367.1521).

Synthesis of the Aamnopyranosyl donors (5 and 5a) used as precursor to residues A and B

[Ir] = [IrH ₂ {THF) ₂ (PPh ₂ Me) ₂ ]PF ₆

The TES derivative

AIM 4-0-(2-naphthylmethyl)-3-i?-triethylsilyI-a-L-rharnnopyranos ide (2)

To a solution of allyl 4-O-(2-naphthylmethyl)-a-L-rharnnopyranoside (diol 1) (5.0 g, 14.5 mmol) in anhydrous acetonitrile (MeCN, 200 mL) stirred at 0°C were successively added dropwise chlorotriethylsilane (TESC1 99%, 2.9 mL, 17.4 mmol, 1.2 equiv) and NJV- 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 N- treated silica gel (toluene/ethyl acetate 95:5) to give compound 2 as a yellow oil (4.9 g,

74%).

Allyl 2-0-levHlnyl-4-0-(2-naphtliylmetliyl)-3-£l-trietliylslyl-e- L-rhamnopyraiiosMe (3) Route 1: To a solution of allyl 4-O-(2-naphthylmethyl)-3-( -tricthylsilyl-a-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 ₃ N-treated silica gel (cyclohexane/EtOAc 9: 1 to 5:5) to give the fully protected 3 as a yellow oil (4.5 g, 74%).

Route 2: To a solution of allyl 4-O-(2-naphthylmethyl)-a-i.-rhamnopyranoside (diol 1) (20.0 g, 58.1 mmol) in anhydrous MeCN (800 mL) stirred at 0°C were successively added dropwise TESC1 (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.

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 ₃ N-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%).

2-0-LevuIInyl-4-0-(2-naplitliyimethyi)-3-0-trIetIiylsIlyl-a p-L-rliamiiopyranose (4) To a solution of allyl 2-0-levulinyl-4-O-(2-naphthylmethyl)-3-0-triethylsilyl-a-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(I)hexafluorophosphate (76.0 mg, 90 μπιοΐ, 0.02 equiv). After stirring the reaction mixture for 2 hours at room temperature, TLC (cyclohexane/EtOAe 7:3) showed complete conversion of the starting material (Rf = 0.53) into a less polar product (Rf = 0.56). Λ-Iodosuccinimide (MS 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 I¾N-treated silica gel (cyclohexane EtOAc 8:2 to 6:4) to give hemiacetal 4 as a yellow oil (2.1 g, 89%, α/β 8:2). 2-0-Levulinyl-4-0-(2-naphthylmethyl)-3-0-tri^

trichloroacetimidate (5)

Route 1: To a solution of 2-0-levulinyl-4-0-(2-naphthyImethyl)-3-0-triethyisilyl-L- rhamnopyranose (hemiacetal 4) (2.00 g, 3.87 mmol) in anhydrous 1 ,2-dichlorocthane (DCE, 30 mL) stirred at room temperature were successively added trichloroacetonitrile (C1 ₃ CCN 98%, 1.16 mL, 11.6 mmol, 3.0 equiv) and l,8-diazabicyclo[5A0]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). Volatiies 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%, α/β 95:5).

Route 2: To a solution of ally! 2-0-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-a-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- (mcthyldiphenylphosphine)iridiura(I)hexafluorophosphate ([lr], 456 mg, 540 μιτιοΐ, 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 (cyclohexanc'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).

To a solution of the crude material (13.9 g) in anhydrous DCE (210 mL) stirred at room temperature were successively added CI ₃ CCN (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 ₃ N-treated silica gel (cyclohexane/EtOAc 9: 1 to 5:5) to give donor 5 as a whitish crystalline solid (15.04 g, 84%, α/β 95:5).

The BDA derivative

L-rhamno

monohydr

[irH ₂ (THF) ₂ (PPh ₂ Me) ₂ ]PF ₆

Allyl 3,4-0-(2 ',3 '-dimethoxybutaii-2 ^s ,3 '-diyI)-a-L-rhamnopyranoside (2a)

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 NaHC0 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 tri fluoride etherate (11.7 mL, 95 mrnol, 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 ₃ 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 2-0-leviilinyl-3,4-0-(2',3'-dimethoxybutaii-2' _» 3'-iiyl>a-L- rhamnopyranoside (3a)

To a solution alcohol 2a (27.0 g, 84.8 mrnol) in anhydrous dichlorornethane (650 mL) stirred at rt were successively added DCC (59.5 g, 0.29 mol, 3.4 equiv.), DMAP (8.29 g, 67.9 mrnol, 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 NaHC0 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).

2-0-Levulinyl-3,4-0-(2%3'-diraethoxybutan-2' '-diyl)-a-L-rhamnopyranosyl

trichloroacetimidate (5a)

To a solution of rhamnoside 3a (37.5 g, 90.0 mrnol) 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 mrnol, 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 (NTS, 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 NaHCOs (300 mL) and then brine (300 mL). The organic layer was dried over anhyd. sodium sulfate, filtered and concentrated under vacuum.

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 N-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

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).

Route A

Route B 88%

Synthesis of the glucosamine D donor.

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.

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

11 84% 12

[ir] = [!r(COD) ₂ (PPh ₂ Me) ₂ ]PF ₆

A synthesis of donor 12a, which encompasses a 4,6- -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.

ΤΒδθ-^- ^ OPTFA

TBSOTf _Ph --Y-o-A 1 [Ir], THF NHC(0)CCI ₃

2,6-lutidine 2. aq I ₂ , THF, NaHC0 ₃ 12a

Acceptor TBSO^-^-OAII „ _{+ 2: 1}

10 DCM. -78 NHC(0)CCI ₃ 3. PTFA-Ci, K ₂ C0 ₃

92% 11a Acetone

[Ir] = [irH ₂ {THF) ₂ (PPh ₂ Me) ₂ ]PF ₆ ^65%

Ailyl 4,6-0-benzylidene-3-0-/e^butyldimethylsilyl-2-deoxy-2-trichl oroacetamido-P-D- glucopyranoside (11a)

To a solution of allyl 4,6-0-benzylidene-2-deoxy-2-trichloroacetamido-P-D-glucopyra noside

(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

trifluoromethanesulfonate (2.54 mL, 1 1.05 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 %).

4,6-0-Benzylidene-3-0-fcrz butyldim

glucopyranosyl N-(phenyl)trifluoroacctimidate (12a)

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- c clooctadienebis(methyldiphenylphosphine)iridium(I)hexafluoro phosphate (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? (200 mL) and then brine (200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum.

To a solution of crude hemiacetal (4.42 mmol) in anhydrous acetone (36 mL), stirred at room temperature, were successively added A ^r -(phenyl)trifluoroacetimidyl chloride (1.80 mL, 6.63 mmol, 1.5 equiv) and potassium carbonate (1.53 g, 1 1.05 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 ABoxeC-Z'

Selected examples

Allyl 2-i?-chIoroacetyl-4-0-(2-napht\lraethyl)-a-L-rhamnopyranosid e (13)

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 (2 x 40 mL). The organic phase was washed with saturated aqueous NaHC0 ₃ (2 x 25 mL) and brine (25 mL). The aqueous phase was extracted with DCM (2 x 25 mL). The combined organic phases were dried over Na ₂ S0 ₄ , filtered, evaporated and finally co-evaporated with toluene to yield the crude alcohol 13 as a 92:8 mixture of regioisomers. Allyl 2-0-levulinyl-4-0-(2-naphtylmethyl)-3-(7-triethylsilyl-a-L-r hamnopyranosyI- (l→3)-2-0^hloroacetyl-4-0-(2-naphtylmcthvI)-a-L-rharnnopyr anoside (14)

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. ter/-Butyldimethylsilyl trifluoromethanesulfonate (TBSOTf, 100 μΐ-, 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 ₃ N 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%). Ally! 2-0-levulinyl-4-0-(2-naphtyImcthyl)-3-(9-tricthylsilyI-a-L-r hamnopyranosyI-

(l→2)-4-0-(2-naph<ylmetliyl)-3-0-triethyIsIlyI-a-L-r hamnopyranosyl-(l→3)-2-0- chloroacetyl-4-0-(2-naphtylmethyl)-a-i.-rhamnopyranoside (16)

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 μί, 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 ₃ N 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 ABQA _C C trisaccharide 16 (1.36 g, 85%).

HRMS (ESI+): m/z 1336.6191 (calcd for C73H99CI4NO16S.2 [M+N¾f) found m/z 1336.6171.

2-0-LevuHnyM- -(2-naphtylmethyI)-3-0-triethylsilylHi-L-rhamnopyranosyl-(l� ��2)-4- 0-(2-naphtyImethyI)-3-0-triethylsily!-a^^

0-(2-naphtyimethyl)-a/p-L-rhamnopyranose (17)

To a solution of the fully protected ABQA _C C (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 ₃ N- 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^oH sC NO^Sia [M+NII ₄ ] ⁺ ) found m/z 1296.5922. 2-0-Levulinyl-4-0-(2-naphtyImethyl)-3-0-^

0-(2-naphtylmethyl)-3-0-triethy!silyl-a-L-rhamnopyranosyl-(l -→3)-2-0-chloroacety (9-(2-naphtylmethyI)-a/ -L-rhamnopvTanosyl trichloroacetimidate (li)

To a solution of the AB _C IA _C C triose 17 (700 mg, 550 μπιοΐ) in anhydrous DCE (5.0 rriL) stirred at room temperature were successively added CI3CCN (165 pL, 3.0 equiv) and DBU (40 μΐ,, 0,5 equiv). After stirring the reaction mixture for 45 min at room temperature, the same amounts of CCI3CN 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 EtsN-treated silica gel (toluene/EtOAc 8:2 containing 3%o I¾N) to give donor IS (830 mg, 84%).

FIRMS (ESI+): m/z 1439.4974 (calcd for C72¾ ₅ Cl ₄ N ₂ 0 ₁₆ Si ₂ [M+N¾ ) found m/z 1439.4912.

2-0-Levulinyl-4-0-(2-naphtylmethyI)-3-0-triethylsilyl-o-L -rhaninopyranosyl-(l *2)-4- 0-(2-naphtylmethyl) -( riethylsilyl-a-L-rt

0-(2-naphtyimetliyl)-a/p-L-rliaiiinopyraiiosyl Λ-phenyitrifluoroacetimidate (19)

To a solution of the ABCIA _C C triose 17 (1.56 g, 1.22 mmol) in acetone (24.4 iiiL) stirred at room temperature were successively added K ₂ C0 ₃ (337 ml, 2.0 equiv) and N- phenyltrifluoroacetimidyl chloride (PTFACl, 290 pL, 1.5 equiv). After stirring the reaction mixture overnight at room temperature, a TLC (cyclofaexane/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%). ABciAcCcBAcD- ll may also be obtained through the alternative route B as defined below, whereby the protecting group differs from that shown above and are: R ¹ = Nap, R ³ , R ⁴ = R ⁶ , R ⁴ = BDA, R ² = CIAc, R ⁵ = Lev, R ⁸ = All, R ⁹ = C13Ac).

[Ir] = [lrH ₂ {THF) ₂ (PPh ₂ ye) ₂ ]PF ₅

Selected examples

Allyl 2-(?-levulinyl-3,4-0-(2' '-diniethoxybutan-2'^'-diyl)-a-L-rharanopyranosyl- (l-^3)-2-0-chloroacetyl-4-0-(2-naphtylmethyl)-a-L-rhamnopyra noside (14a)

To a solution of cmde allyl 2-0-chloroacetyl-4-0-(2-methylnaphthyl)- -L-rhamnopyranoside

(5, 6.91 mmol, 1.2 equiv.) in anhyd. diethyl ether (60 mL), stirred at it, 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 %).

Aliyl 3,4-0-(2V^'-dirnethoxybutan-2' '-diyl)-a-L-rhamnopyranosyl-(l'^3)-2-0- chIoroacetyI-4-0-(2-naphtyImethyl)-a-L-rhamnopyranoside (15a)

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 μί, 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 NaHCOi (15 mL) was added and the aqueous layer was extracted twice with dicMoromethane (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 di saccharide 15a as a yellow oil (0.63 g, 72 %).

AIM 3,4-0-(2 ',3 '-dimethoxybutan-2 ',3 '-diyl)-2-0-levulinyl-a-L-rhamnopyranosyl- (1 -»2)-3,4-0-(2' '-dImethoxybutan-2 ',3 '-diyl)-a-L-rhamnopyranosyi-(l ->3)-2-0- chloroacetyl-4-0-(2-naphtylmethyl)-a-L-rhamnopyranoside (16a)

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 μΙ. _-5 44 μιιιοΐ, 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 ABCIA _C CCUA _C D-AII

ABc'.AcCcDAeD-All (ABC'D', 21) was obtained following the generic route B (the first steps of route B up to the triosyl donor IS being in particular described above) as defined below. The activation glycosylation and a final two-step orthogonal deprotcction 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 ¹ = R ⁴ = Nap, R ³ = R ⁶ = TES, R ² = C Ac, R ⁵ = Lev, R ⁸ = All, R ⁹ = C13Ac).

ABciAcCc AeD-All may also be obtained through the alternative route A as defined in the overview scheme below. ABCIA _C CCI3A _C D-A11 may also be obtained through the alternative route B as defined below, whereby the protecting group differ from that shown above and are: R ¹ = PMB, R ³ = R ⁴ = Nap, R ⁶ = TES, R ² = ClAc, R ⁵ = Lev, R ⁸ = All, R ⁹ = CBAc).

Selected examples

Ally! 2-0-levuiinyl-4-0-(2-naphtylinethyI)-3-i?-triethylsilyl-a-L- rhamnopyranosyI-

(l→2)-4-0-(2-naplitylitietliyl)-3-0-triethy!silyI-a-L-r hameopyranosyl-(l→3)-2-0- chloroacetyM-0-(2-naphtylmethyl)-a-L-rhamnopyranosyKl→3)- ,6- ?-benzylidene-2- deoxy-2-N-trichloroacetyl- ^D-glucopyranoside (20)

A solution of the triosyl trichloroacetimidate 18 (924 mg, 649 μηιοΐ) 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 μΐ., 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. I¾N 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 ABCIA _C CCBA _C D tetrasaccharide (20, 932 mg, 84%).

HRMS (ESI+): m/z 1729.6128 (calcd for C ₈₈ Hn3Cl ₄ N ₂ 0 ₂ ,Si ₂ [M+NH ₄ ] ') found m/z 1729.6161. Allyl a-L-rhamnopyranosyI-(i→2)-a-L-rhainnopyranosyl-(l→3)-2-i ?-chloroacetyI-a-L- rhamnopyranosyl-(l→3)-2-deoxy-2-A ^, -trichloroacetyl- ^, --)-glucopyranoside (21)

To a solution of the alcohol (222 mg, 137 μηιοΐ) - issued from the delevulinylation of the fully protected 20 - in toluene stirred at 0 °C was added TFA to reach a TF A/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 ₂ 0/MeCN ()→50%), then by RP-HPLC (MeCN/¾0 20.5%) to give the target AB _C ) _A cCc!3 _A cD-All 21 following freeze-drying (70 mg, 36%). RP-HPLC (C I 8 ^® RP fusion

(4.6x250 mm, 4.0 μηι, 80 A, CH ₃ CN in H ₂ 0 (30% for 4 mill, then 30→4Q% over 7 min, at 1.0 mL.min ⁵ ), 40 °C, λ: 220 nm) = 7.6 min.

^! H NMR (800 MHz, D ₂ 0), δ 5.84 (m, 1H, -CH=), 5.25 (m, IH, J _trans = 17.3 Hz, =CH ₂ ), 5.19 (m, 1 H, J _cis = 10.5 Hz, =C¾), 5.13 (dd, 1 H, J _2>3 = 3.0 Hz, J ₂ ,i = 1.9 Hz, H-2 _C ), 5.1 1 (d, IH, J _!>2 = 1.3 Hz, H-1 _B ), 4.86 (d, IH, J _1>2 = 1.6 Hz, H-l _c ), 4.86 (d, IH, J _i)2 = 1.5 Hz, II- 1 _A ), 4.66 (d, 1H, J _1>2 = 8.0 Hz, H-l _D ), 4.28 (m, 1 H, HAH), 4.27 (d, 1H, J _gem =15.4 Hz, H _OA ), 4.23 (d, I H, J _GEM =15.4 Hz, HCIA _C ), 4.12 (m, I H, H _A1I ), 4.05 (m, IH, H-5 _C ), 3.99 (dd, IH, ½ = 33 Hz, J _2;1 = 1.7 Hz, H-2 _A ), 3.93 (dd, I H, J ₃ , ₄ = 9.7 Hz, J ₃ , ₂ = 3.1 Hz, H-3 _C ), 3.89 (dd, 1H, J _2>3 = 4.7 Hz, J _2, i = 1 .7 Hz, H-2 _B ), 3.88 (d, 1H, ½ = 2.1 Hz, H-6b _D ), 3,86 (m, 1H, H-2 _D ), 3.72 (m, 2H, H-3 _D) H-3 _A ), 3.71 (d, 1H, J _6>5 = 3.3 Hz, H-6a _D ), 3.69 (m, 1H, H-5 _B ), 3.63 (dd, 1 H, J ₃ , ₄ = 9.9 Hz, J _3,2 = 3.5 Hz, H-3 _B ), 3.62 (m, 1H, H-5 _A ), 3.54 (dd, 1H, J _4,5 = 9.9 Hz, J _4>3 = 9.9 Hz, H-4 _D ), 3.52 (dd, 1H, J _4>5 = 9.8 Hz, J ₄ , ₃ = 9.8 Hz, H-4 _C ), 3.42 (m, 1H, H-5 _D ), 3.38 (dd, 1H, J _4>5 = 9.8 Hz, J ₄₍₃ = 9,8 Hz, H-4 _B ), 3.37 (dd, 1H, J _4>5 = 9.8 Hz, ½ = 9.8 Hz, H-4 _A ), 1.25 (d, 3H, J ₆₎₅ = 6.2 Hz, H- 6B), 1.20 (d, 3H, ¾ ₅ = 6.4 Hz, H-6 _A ), 1.1 (d, 3H, J ₆ , ₅ = 6.4 Hz, H-6 _C ).

¹³ C NMR (800 MHz, D ₂ 0), δ 168.6 (CO _aAc ), 164.7 (CO _NHC (0)cc: ₃ ), 133.0 (CH= _A „), 1 18.7 (=CH _2A11 ), 102,3 (C-1 _A ), 100.5 (C- 1 _H ), 99.1 (C-1 _D ), 98.5 (C-l _c ), 91.6 (CC1 ₃ ), 81.7 (C-3 _D ), 78.0 (C-2 _B ), 76.1 (C-5 _D ), 74.5 (C-3 _C ), 73.4 (C-2 _C ), 72,2 (C-4 _C ), 72.0 (2C, C-4 _B , C-4 _A ), 70.8 (- CH ₂ - _Aii ), 70.0 (2C, C-3 _A , C-2 _A ), 69.9 (C~3 _B ), 69.5 (C-5 _B ), 69.2 (C-5 _C ), 69.1 (C-5 _A ), 68.5 (C- 4 _D ), 60.7 (C-6 _D ), 57.1 (C-2 _D ), 40.7 (-CH ₂ - _aA c), 16.7 (C-6 _B ), 16,6 (C-6 _A ), 16.2 (C-6 _C ).

HRMS (ESI+): m/z 895.1840 (calcd for C ₃ :H ₄₇ Cl ₄ Oi9NH4 [M+NH ₄ ] ⁺ ) found m/z 895.1860.

Allyl a-L-rhamnopyranosyI-(l-→2)-a-L-rhamnopyranosyI-(l→3)-2-( -chloroacetyl-a-L- rhamnopyranosyI-(l→3)-2-deoxy-2-A-tr!chIoroacet l- -i>-glucopyranoside (21a)

To a solution of the lightly protected tetrasaccharide ABQA _C CCBACD-AH (21, 200 mg, 0.23 mmol) in anhydrous methanol (31 rnL) was added MeONa (25% w/w in MeOH, 126 pL, 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 if 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 ABCci _3A cD-All 21a as a white powder (76 mg, 42%). RP- HPLC (CI 8 RP fusion (4.6x250 mm, 4.0 μιη, 80 A, CH ₃ CN in H ₂ 0 (30% for 4 min, then

30→40% over 7 min, at 1.0 mL.min ^"1 ), 40 °C, λ: 220 nm) = 3.8 min.

HRMS (ESI+): m/z 819.2124 (calcd for C30H ₄ 6CI3 O18NH4 [M+NH ₄ ] ') found m/z 819.2745; m/z 824.1618 (calcd for C ₃ oH4 ₆ Cl ₃ NQ _{! 8} Na [M+Na] ') found m/z 824.2239.

Synthesis of an alternative B donor (25)

Toluene, 60°C, overnight overnight

22 23

AHyl 3,4-di-0-(2-naphtylmethyl)-a-L-rhamnopyranoside (22)

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 it, 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.

Ή NMR (CDCI3) «5 7.81 (m, 8H, HA _T N. _p ), 7.50 (m, 6H, H _ArNap ), 5.93 (m, 1 11, J _mils = 17.1 Hz,

J _gem = 1.5 Hz, CH=CH ₂ ), 5.31 (m, 1 H, J _cb = 10.4 Hz, CH=C¾), 5.22 (m, 1 H, CH=Ci¾), 5.10 (d, 3H, J = 1 1 .2 Hz, CH _2Na p), 4.91 (m, 2H, CH _2Nap , H- l ), 4.21 (m, 1H, HAH), 4.16 (m, 1 H, H- 2), 4.02 (m, 2H, H _A! i, H-3), 3.85 (m, 1H, H-5), 3.60 (pt, 1 H, J _3A = J4.5 = 9.3 Hz, H-4), 2.58 (bs, 1H, Jj.oH = 9.6 Hz, OH), 1.40 (d, 3H, J _5i6 = 6.3 Hz, H-6).

HEMS (ESf ): m/z 502.2608 (calcd for C31H36O5N [M+N¾] ⁺ m/z 502.2593)

Allyl 2-0-levulinyI-3,4-di-(9-(2-naphtylinethyl)-a-L-rhamnopyranos ide (23)

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. 2-0-LevuIinyl-3,4-di-i?-(2-naphtylmeth l)-a-L-rhamnopyranose (24)

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 trig, 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/ ^' EtOAe 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. 2-0-Levulinyl-3,4-di-0-(2-naphty lrnethyl)-a-L-rhamnopyranosyl trichloroacetimidate

(25)

To a solution of hemiacetal 24 (from 22, 9.0 mmol) in anhydrous DCE (50 mL) stirred at room temperature were successively added CI3CC (2.7 mL, 3.0 equiv) and DBU (670 μί, 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 EtsN-treated silica gel (toluene/EtOAc 95:5 to 8:2 containing 1¾N 5%o) to give donor 25 as a brown oil (5.05 g, 88% over three steps). Ή NMR (CDCI3) δ 8.67 (s, 1H, H), 7.81 (m, 8H, H^ _p ), 7.49 (m, 6H, H _ArNap ), 6.22 (d, 1 H, H-l), 5.56 (dd, 1H, J _u = 2.1 Hz, J ₂ = 3,3 Hz, H-2), 5.14 (d, III, J = 1 1.1 Hz, CH _2Nap ), 4.92 (d, 1H, J= 1 1.4 Hz, CH _2Nap ), 4.87 (d, 1 H, CH _2Nap ), 4.77 (d, 1H, CH _2Nap ), 4.10 (dd, 1 H, H-3), 4.00 (m, 1H, H-5), 3.62 (pt, 1 H, J ₄ = J ₄ = 9.5 Hz, H-4), 2.78 (m, 4H, CH _2Le v), 2.18 (s, 3H, CHj v), 1.40 (d, 3H, J _5i6 = 6.2 Hz, H-6)

¹³ C NMR (CDCI3) δ 206.0 (CO _Lev ), 171.9 (C0 _2Lcv ), 160.1 (NHCO), 135.6433.2 (6C, Qv), 128.2-126.1 (14C, C _ArNap ) ₅ 95.2 (C- l , ^! J _C . = 179.5 Hz), 79.4 (C-4), 77.1 (C-3), 75.7 (C _Nap ), 72.0 (C _Nap ), 70.8 (C-5), 67.9 (C-2), 38.0 (CH _2Lev ), 29.8 (CH _3Le v), 28.1 (CH _2L ev), 18.1 (C-6). HRMS (ES ): mlz 703.1744 (calcd for C ₃₅ H ₃₈ C1 ₃ N0 ₇ [M+ NH ₄ ] ⁺ miz 703.1749)

Synthesis of an alternative C acceptor (28)

1 ) AIIOH, AcCl

70 °C, 2.5h then 1 ) PMBCI, NaH

40°C overnight DMF, RT, overnight

2) Me ₂ C(OMe) ₂ . PTSA 2) 80% aq. AcOH

acetone, RT, 3h 60 °C, 3 d

26 27

0 °C, 10 min 28 Ally! 4-0- 7flra-methoxybenzyI-a-L-rhamnopyranoside (27)

Grade allyl 2,3-O-isopropylidene-a-L-rhamnopyranoside (26, from L-rhamnose, 1 10 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 portioewise to this suspension. The mixture was stirred for 2 h at rt, then /wra-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 ₂ 0 (3 x 120 mL) and brine (120 mL). The organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated to dryness to give the fully protected intermediate. The crade 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 (2 x 100 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).

Ή NMR (CDCI3) δ 730 (m, 2H, Η _ΑΓΡΜ Β), 6,91 (m, 2H, Η _ΑΓΡΜ Β), 5.89 (m, 1H, C7/=€H ₂ ), 5.29 (m, 1 H, J _TRANS =17,2 Hz, J _gem = 1.6 Hz, CH=CI¾), 5.20 (m, 1H, J _trans = 10.4 Hz, CH=Ci¾) _» 4.81 (d, 1H, Ji,2 = L I Hz, H-l), 4.69 (m, 2H, CH _2P MB), 4.17 (m, 1H, H _A!! ), 3.97 (m, 3H, H-All, H- 2, H-3), 3.82 (s, 3H, CH _3P MB), 3.75 (m, 111, H-5), 3.35 (pt, 1 H, J _? , ₄ = J ₄ .5 = 9.2 Hz, H-4), 2.45

(bs, 2H, OH), 136 (d, 3H, J ₅ , ₆ = 6.3 Hz, H-6).

Allyl 2-<?-chloroacetyI-4-0- >arfl-methoxybenzyl-a-L-rhamnopyranoside (28)

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 (2 x 25 mL). The organic phase was washed with saturated aqueous NaHC(¾ (2 x 25 mL) and brine (25 mL). The aqueous phase was extracted with DCM (2 x 12.5 mL). The combined organic phases were dried over Na ₂ S0 , filtered, evaporated and finally co-evaporated with toluene to yield alcohol 28 as a mixture of regioisomers. The crade material is used as such in the next step.

Synthesis of COA _C DABCL _AC C-AII

c _DAc D Bci _Ac C-All is obtained following route B' as defined below.

In particular, it can be obtained from donor 12 or its 4,6-0-benzylidene analog and the ABC trioside acceptor 29 following conventional delevulinylation at position 2 _A of the fully protected precursor 16. A synthesis is highlighted in the scheme below whereby R ¹ = R ⁴ = Nap, R ³ = R ⁶ = TES, R ² = ClAc, R ⁵ = Lev, R ⁷ = TBS, R ^{8 «} All, R ⁹ = C13Ac).

Allyl 4,6-0-benzyIidene-3-0-/ r^butyldimethylsilyl-2-deoxy-2-trichioroacetamido- -D- glucopyranosyl-(i ^→ 2)-4-0-(2-naphtylmethyl)-a-L-rIiamiiopyranosyI-(l ^→ 2)-4-0-(2- naphtylmethyl)-a-L-rliamHopyranosy!-(l ^→ 3)-2-0-cliioroacetyl-4-0-(2-napIiiylmetliyl)- a-L-rhamnopyranoside (32) and allyl 4,6-0-beHzyIidene-2-deoxy-2-trkli!oroacetaiiiido- P-D-glucopyranosyl-(l— *2)-4-0-(2-naph^lmethyl)-a-L-rhamnopyranosyi-(l— »2)-4-0-(2- naphtyIraethyl)-a-L-rhamnopyranosyl-(l ^→ 3)-2-0-chloroacetyW-0^2-naphtylmethyl)- a-L-rhamnopyranoside (31)

To a solution of crade tetrasacchande 30 (1.48 mmol) in THF/AcOH (4: 1, 74 mL) was slowly added 1 M 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. NaHC0 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 elutioii diol 32 (820 mg, 37%) and triol 31 (334 mg, 16%).

Ally! 2^eoxy-2-trichIoroacetamido- -D-gIucopyranosyl-(l— ^» 2)-a-L-rhamnopyranosyl-(l -→2)-a-L-rhanmopyranosyl-(l- ^→ 3)-2-0-chIoroacet l-a-L-rhamnopvTanoside (33)

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 CI 8 column (¾0/MeCN 0→ 40%) to give the lightly protected tetrasaccharide D 'ABC '-All (33) as a white powder (74 mg, 35%). RP-HPLC (CI 8 ^® P fusion (4.6x250 mm, 4.0 μτη, 80 A, C¾CN in H ₂ 0 (30% for 4 min, then 30→40% over 7 min, at 1.0 mL.mm '), 40 °C, λ: 220 ran) = 10.2 min.

HUMS (ESI+): m/z 900.1394 (calcd for CsiituCLjNGtgNa [M+ af) found m/z 900.1246.

Route B

5 Example 2: Enzymatic a-D-glucosvlation of a compound of formula (I) with branching siicrases

Enzymes

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

Osidlc

Enzyme linkage on

Source organism References

accronym natural

acceptor

Leuconostoc US2016136199 (Al ),

BRS-B citreum NRRL-B WO2016146764 (Al )

742 Vuillemin et al. (JBC, 2016)

US2016136199 (A1 ),

Leuconostoc fallax

BRS-C erf ,3 WO2016146764 (Al )

KCTC 3537

Vuillemin et al (JBC, 2016)

Leuconostoc

BRS-E mesenteroides

F I-MG

Leuconostoc US2017152489 (Al),

BRS-A citreum NRRL-B US2017101484 (Al ), ES2398227 (T3)

1299 Vuillemin et al. (JBC, 2016)

US2017152489 (Al ),

Lactobacillus

BRS-D US2017101484 (Al), ES2398227 (T3) kunkei EFB6

a-1,2 Vuillemin et al. (JBC, 2016)

From truncated

US2017152489 (Al),

DSM-E

US2017101484 (Al ), ES2398227 (T3)

GBD-CD2 Leuconostoc

Fabre et al. (J. Bact, 2004), Brison et al. citreum NRRL-B

(JBC, 2012)

1299

GBD-CD2 Yarmick Malbert. Flavonoid

GBD-CD2

W2135V* glucodiversifi cation with engineered

mutants

GBD-CD2 sucrose-active enzymes. Biotechnology. W2135C- INSA Toulouse

F2136I* (PhD, 2014)

GBD-CD2

W2135S-

F2136L*

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.

The sequence of said enzymes is as follows:

SEQ ID NO : 1 (BRS-E)

SEQ ID NO : 2 (BRS-A)

SEQ ID NO : 3 (BRS-B-D1)

SEQ ID NO : 4 (BRS-B-D2)

SEQ ID NO : 5 (BRS-C)

SEQ ID NO : 6 (BRS-D) SEQ ID NO : 7 (GBD-CD2).

BRS-E (L. MAHHHHHHVTSLY AGSAAAPFTMQQ NATLQVSPT NDNSVA NTTS Mesenteroides TANVA VDITTD TRDISSAN VN LITNQYKENSNGSWSYYD NGQIV G KFRI-MG) LQTINGNIQYFDSTTGEQVKGQTLTIDGVIYSFD DSGNGTKTEVASLPTTGS

YATKDGSNWQYEDQQQQ PIKGLYTDKG NLRYFNETDG TQVKGTVVSV DNNTYYFDKDSGNGQLVPSVTGGQYGTIQLNNQTVWVYRNANGEIVKGLQ NINGNIQYFDPNTGEQLKGKVATVNGVTYY FEASDGNLVG TVSDGLVTVN GQIQ YFDP ATGEQAKNKQ I VNNVTYYFDDNGYGQYLFTNAILSTTPDAY SAHTQAYNTDQSSFTNVVDGFLTADSWYRPKEV1ADATGS AWQTSSENDY RPIITVWWPNKNVEVNYLKLMQDNDLLSTQ TQFTIFSDQY TLNEAAQAAQ NEIEKRIYREKSTDWLKDLLFEA1 IGDTPSF VKQQFIWNKD SEYQGAGGGN LWSLQGGYLK YVNDSETSWS DSTSRKHDYY EYLLGNDIDN SNPQVIAENI NWLYYLMNFGSLTGNDDTANFDGVRMDAVIYMKGEASTKVYQFLHKSDE L TKNEKIANEH ISIVEDGTDh 1 1 NNSAL1V SKWNENIASS LAKIASGKDT SLEALVKTDE KTSVSRIMNS SVESDVNPNY SMIRSHDRGS QDEVINASKV ANNDQSIALD NINLNQLENG LKLYYEDQAS PTKNYNYYNI PASYALLLSN KDTVPRLYYSDMYQDYDQNPDTPQQYMSKP TIYYSAIDAL LKARIKYVAG GQS AVEKVGDNKDQEVLTSVRYGKNVMTATDTGTVESRTEGMGVIVSN NTKLKLSTTDQIVLHMGAAHANQAYQALMLT DEGIQLYNDNAPVVWTD HNGDLVFNGNDINGQKNTSIKGYFNPQVAGYLAVWVPVGATDTQDARTK ASTNATTDGKVFHSNAALDSNVIYEGFSNFQPIARNHNDFSNVKIAENVDLF KKWGi rSFELAPQYRSADVSDLVGSTFVDVVTKNGYGLSDRYDLGFVTPTK YGSDSDLRNAISSLHAQGIQAMADFVGNQIYALNDGQEVVTAQRSDMFNN TLNNAFGTELYVVNSIGGGKYQAKYGGNYLEEIASLYPDLFTNQDGTKIDIN TKIKQWSAKYMNGTNVLGRGMGYVLKDWNTATYFKLDGEHTVLPAALTL SG LK V ENG VT YYYKNNERQTGTQT VDD VT YFFDPKTG AMKKDYFDFT AD NKVYYYGKGGRADPAFLYKV VSAWSHPQFE K

BRS-A, 1878 MRQKETITRKKLYKSGKSWVAAATAFAVMGVSAVTTVSADTQTPVGTTQS amino acids QQDLTGQRGQDKPTTKEVIDKKEPVPQVSAQNAGDLSADAKTTKADDKQD (L, citreum TQPTNAQLPDQGNKQTNSNSDKGVKESTTAPVKTTDVPSKSVTPETNTSING NRRL B- GQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDN

1299) AYYFDKNSGNGRTFTKISNGSYSEKDG WQYVDSHDKQPVKGLYDVEGN

LQYFDLSTGNQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQT

TKDKDGNETSYWAYLDNQGNAIKGLNDVNGEIQYFDEHTGEQLKGHTATL

DGTTYYFEGNKGNLVSVVNTAPTGQYKINGDNVYYLDNNNEAIKGLYGIN

GNLNYFDLATGIQLKGQAKNIDGIGYYFDKDTGNGSYQYTLMAPSNKNDY

TQHNVVNNLSESNFKNLVDGFLTAETWYRPAQILSHGTDWVASTDKDFRPL

ITVWWPNKDIQVNYLRLMQNEGVLNQSAVYDLNTDQLLLNEAAQQAQIGI

EKKISQTGNTDWLNNVLFTTHDGQPSFIKQQYLWNSDSEYHTGPFQGGY

LKYQNSDLTPNVNSKYRNADNSLDFLLANDVDNSNPIVQAEDLNWLYYLL

NFGSITTQGKENNSNFDSIRIDAVDFVSNDLIQRTYDYLRAAYGVDKNDKEA

NAHLSLVEAGLDAGTTTIHQDALIESDIREAMKKSLTNGPGSNISLSNLIQDK

EGDKLIADRANNSTENVAIPNYSIIHAHDKDIQDKVGAAITDATGADWTNFT

PEQLQKGLSLYYEDQRKIEKKYNQYNIPSAYALLLTNKDTVPRVYYGDMY

QDDGQYMQKQSLYFDTITALMEARKQFVAGGQTINVDDNGVLTSVRFGKG

AMTANDIGTNETRTQGIGVVIANDPSLKLSKDSKVTLHMGAAHRNQNYRA

LLLTTDNGIDSYSSSKNAPVIKTDDNGDLVFSNQDINDQLNTKVHGFLNSEV

SGYLSAWVPLDATEQQDARTLPSEKSVNDGKVLHSNAALDSNLIYEAFSNF

QPMPTNRNEYTNVVIADKADTFKSWGITSFEMAPQYRSSQDKTFLDSTIDN

GYAFTDRYDLGFEKPTKYGNDEDLRQAIKQLHSSGMQVMADWANQIYNL

PGKEVASTNRVDW GNNLS TPFGTQMYVVNTVGGGKYQNKYGGEFLDKL

KAAYPDIFRSKNYEYDVKNYGGNGTGSVYYTVDSKTRAELDTDTKIKEWS A YMNGT VLGLGMGYVLKDWQTGQYFNVSNQNMKFLLPSDLISNDITV

QLGVPVTD IIFDPASAY MYSNLPEDMQVMDYQDD KSTPSIKPLSSYN

NKQVQVTRQYTDS GVSWNL1TFAGGDLQGQ LWVDSRALTMTPFKTMN

QISFISYANRNDGLFLNAPYQVKGYQLAGMSNQYKGQQVTIAGVANVSGK

DWSLISFNGTQYWIDSQALNTNFTHDMNQKVFVNTTSNLDGLFLNAPYRQP

GY LAGLA NYNNQTVTVSQQYFDDQGTVWSQVVLGGQTVWVDNHALA

QMQVRDTNQQLYVNSNGRNDGLFLNAPYRGQGSQLIGMTADYNGQHVQV

T QGQDAYGAQWRLITL NQQVWVDSRALSTTIMQAMNDDMYVNSSQRT

DGLWLNAPYTMSGA WAGDTRSANGRYVHIS AYSNEVGNTYYLTNLNG

QSTWID RAFTATFDQVVALNATIVARQRPDGMF TAPYGEAGAQFVDYV

TNYNQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRSFSPVVT VVDY

QA IVPRTTRDGVFSGAPYGEVNAKLV MATAYQNQVVHATGEYTNASGI

TWSQFALSGQED LWIDKRALQA

BRS-B MEM ETITRKKLYKSG SWVAAATAFAVMGVSAVTTVSADTQTPVGTTQS

QQDLTGQTGQD PTT EVIDK EPVPQVSAQNVGDLSADA TP ADD QD

TQPTNAQLPDQGNKQTNSNSD GVKESTTAPV TTDVPSKSVAPETNTSIN

GGQYVE DGQFVYIDQSG QVSGLQN1EGHTQYFDPKTGYQT GELKNIDD

NAYYFD NSGNGRTFT ISNGSYSEKDGMWQYVDSHDK.QPVKGLYDVEG

NLQYFDLSTGNQAKHQIRSVDGVTYYFDADSGNATAF AVTNGRYAEQTT

KD DGNETSYWAYLDNQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATV

DGTTYYFEGN GNLVSVVNTAPTGQYKiNGDNVYYLDNNNEAI GLYGIN

GNLNYFDLATGIQL GQA NIDGIGYYFDQNNGNGEYRYSLTGPVV DVY

SQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSHGTDWVASTD DFRP

LITVVVWPNKDIQVNYLKLMQQIGILDNSVVFDTN DQLVLNKGAESAQIGI

EKKVSETGNTDWLNELLFAPNGNQPSF1 QQYLWNVDSEYPGGWFQGGYL

AYQNSDLTPYANTNPDYRTH GLEFLLANDVDNSNPVVQAEQLNWLYYL

MNFGQITANDSNANFDSMRiDAISFVDPQIA KAYDLLDKMYGLTDNEAV

ANQHISrVEAPKGETPITVEKQSALVESNWRDRMKQSLS NATLD LDPDPA

INSLEKLVADDLVNRSQSSD DSSTIPNYSIVHAHDKDIQDTViHIMKIVNNN

PNISMSDFTMQQLQNGL AFYEDQHQSVK Y QYNIPSAYALLLT DTVP

RVFYGDMYQDYGDDLDGGQYMATKSIYYNAIEQMMKARLKYVAGGQIM

AVT I NDGIN DGTNKSGEVLTSVRf G DIMDAQGQGTAESRNQGIGV1V

SNSSGLELKNSDS1TLHMGIAHKNQAYRALMLTND GIVNYDQDNNAPIAW

TNDHGDL1FTNQMINGQSDTAVKGYLNPEVAGYLAVWVPVGANDNQDAR

TVTTNQK TDG VLHTNAALDSKLMYEGFSNFQ MPTRGNQYANVVIT N

IDLF SWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGFKTPT YGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNIN

GDTKLVVDPRYGTQMTVVNSVGGGDYQAKYGGEYLDTIS LYPGLLLDSN

GQ IDLSTKIKEWSAKYLNGS IPQVGMGYVLKDWNNGQYFHILD EGQY

SLPTQLVSNDPETQIGESVNYKYFIGNSDATYN YH LPNTVSLINSQEGQI

KTQQSGVTSDYEGQQVQVTRQYTDSKGVSWNLITFAGGDLQGQKLWVDS

RALTMTPF TMNQISFISYANRNDGLFLNAPYQVKGYQLAGMSNQYKGQQ

VT1AGVANVSGKDWSLISFNGTQYWIDSQALNTNFTHDMNQ VFVNTTSNL

DGLFLNAPYRQPGYKLAGLAKNYNNQTVTVSQQYFDDQGTVWSEVVLGG

QTVWVDNHALAQMQVSDTSQQLYV SNGRNDGLFLNAPYRGQGSQLIGM

TADYNGQHVQVT QGQDAYGAQWRLlTLNNQQVWVDSRALSniVQAMN

DDMYV SNQRTDGLWLN'APYTMSGAKWAGDTRSANGRYVHIS AYS EV

GNTYYLTNLNGQSTW1DKRAFTATFDQWALNATIVARQRPDGMFKTAPY

GEAGAQFVDYVTNY QQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRS

FSPVVTKVVDYQAKIVPRTTRDGVFSGAPYGEV A LVNMATAYQNQVV

HATGEYTNASGITWSQFALSGQED LWIDKRALQA

BRS-B-D1 (L. DTQTPVGTTQSQQDLTGQTGQDKPTTKEVIDK EPVPQVSAQNVGDLSADA citreum KTPKADD QDTQPTNAQLPDQGNKQTNSNSD GVKESTTAPV TTDVPSK NRRL B-742) SVAPETNTSINGGQYVE DGQFVYIDQSGKQVSGLQNIEGHTQYFDP TGY

QTKGELK IDDNAYYFDKNSGNGRTFT ISNGSYSE DGMWQYVDSHD Q PVKGLYDVEGNLQYFDLSTGNQAKHQIRSVDGVTYYFDADSGI* CAV TNGRYAEQTTKDKDGNETSYWAYLDNQGNAIKGLNDVNGEIQYFDEHT

GEQLKGHTATVDGTTYYFEGNKGNLVSWNTAPTGQY I GDNVYYLDN EAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFDQNNGNGEYRY

SLTGPVVKDVYSQHNAVN LSA NFKNLVDGFLTAETWYRPAQILSHGTD

WVASTDKDFRPL1TVWWPN DIQVNYL LMQQIGILDNSVVFDTNNDQLV

LNKGAESAQIGIE KVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSE

YPGGWFQGGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVV

QAEQLNWLYYLMNFGQITANDSNANFDSMRIDAISFVDPQIAKKAYDLLDK

MYGLTDNEAVANQHISIVEAP GETPITVEKQSALVESNWRDRMKQSLSKN

ATLDKLDPDPAINSLEKLVADDLV RSQSSDKDSSTIPNYSrVHAHDKDIQD

TVIHIMKIV N PNISMSDFTMQQLQNGL AFYEDQHQSVKKYNQYNIPSA

YALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMATKSIYYNAIEQMMKA

RL YVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGKDIMDAQ

GQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND GIVNYDQDN APIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYL

AVWVPVGANDNQDARTVTTNQ NTDG VLHTNAALDS LMYEGFSNFQK

MPTRGNQYANVVITKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQ

NGYGLSDRYDLGFKTPT YGTDQDLRKAIERLHQAGMSVMADFVANQIYG

LHADKEVVSAQHVNINGDT LVVDPRYGTQMTVVNSVGGGDYQAKYGG

EYLDTIS LYPGLLLDSNGQKIDLSTK1KEWSAKYLNGSNIPQVGMGYVLKD

WNNGQYFH 1 LD EGQYS LPTQL

BRS-B-D2 (L. MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDP TGYQT GEL ID citreum DNAYYFDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPV GLYDVE NRRL B-742) GNLQYFDLSTGNQA HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQT

TKD DGNETSYWAYLDNQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATV

DGTTYYFEGN GNLVSVVNTAPTGQYKINGDNVYYLDNKNEAI GLYGIN

GNLNYFDLATGIQLKGQAKNIDGIGYYFDQNNGNGEYRYSLTGPVV DVY

SQHNAVNNLSANNFKNLVDGFLTAETWYRPAQILSHGTDWVASTDKDFRP

LITVWWPNKDIQVNYL LMQQIGILDNSVVFDTNNDQLVLNKGAESAQIG1

EKKVSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGWFQGGYL

AYQNSDLTPYANTNPDYRTH GLEFLLANDVDNSNPVVQAEQLNWLYYL

MNFGQITANDSNANFDSMRIDAISFVDPQIA AYDLLD MYGLTD EAVA

NQHISIVEAPKGETPITVEKQSALVESNWRDRM QSLS NATLD LDPDPA!

NSLE LVADDLVNRSQSSD DSSTIPNYSIVHAHD DIQDTVIHIM IVN NP

NISMSDFTMQQLQNGL AFYEDQHQSVKKYNQYNIPSAYALLLTN DTVP

RVFYGDMYQDYGDDLDGGQYMAT SIYYNAIEQMMKARL YVAGGQIM

AVT I NDGIN DGTN SGEVLTSVRFGKDIMDAQGQGTAESRNQGIGVIV

SNSSGLEL NSDSITLHMGIAH NQAYRALMLTNDKGIVNYDQDNNAPIAW

TNDHGDLIFTNQMINGQSDTAV GYLNPEVAGYLAVWVPVGANDNQDAR

TVTTNQKNTDG VLHTNAALDS LMYEGFSNFQ MPTRGNQYANVVITKN

IDLFKSWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGFKTPT

KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNIN

GDT LVVDPRYGTQMTVVNSVGGGDYQAKYGGEYLDTIS LYPGLLLDSN

GQKIDLSTKI EWSA YLNGSNIPQVGMGYVL DWNNGQYFHILD EGQY

SLPTQLKGGRADPAFLY VVHHHHHH

BRS-C M QQESITRK LYKAGKSWVVAATLFAATLFAAMGAAGATTVASADVQ

(GH Leucono DTVVVTADKNTTDKDKEPI TAGANVVD GVAQTTDTNTTD TIEVG S stoc fallax K VDMSATDKKVTETV SVDTSATDK TTEAVKPVDTNATDKKATEAVKPV CTC3537 1) DTNATDKKTTEAVKPVDTNTTD VTEAIKPVNTNADD TAEPV TISAT

DTVKTIANKQKGATEEQAVITEGHYEAQGDGFVYIT DG QLTGLQNINGN

TQYFDPATGQQL GDIKAVAGTVYYFDKNSGNARVYQKVADGTYSENNE

HWQYIS VDNKPVEGLYNVQGNLQYFDMSTGNQVKNDIRSVDGVTYYFD DSGNGSAFNALSAGEYVE KETDAQGNQNSYWTYSGLDGNPVKGLYDIN

GSLQYFDE NGAQL GGTATV GVTYYFEQD GNLISVV SVESGQY ID NDNVYYID QGNTL GLYAINGQLNYFDMSTGVQLKGASE.NANGVGYYF

DKDKGNGQYQYSLITSTLANAFSKHNAANDYTQSSFTHTVDGFLTADTWY

RPTEIL NGTTWVASTSQDLRPMITVWVVPN NVQLNYL LMQTEGLLDSG

QVYDLNSDQALL QAAQTVQVNIEKRITKAGNSDWLNDLLYNSHGirrPSF

V QQAIW ADSEYHGGWFQGGYLAYRNSDLTPYANSSYRHYTGMEFLLA DVD SNPlVQAEDLNWLYYLMNFGTETGNDPQA FDSrRIDAISFVD QV

A KAYELLHDMYGLSASDAVA HVSiVEASADQTPVITENHDALlESYW

RDTM NSLS DASiDSSAGSLSAMINDGNVDRANDSTTESSIFPNYTlVHAH

D DIQDAVSNVMKIVNNDPSISLDGFTMEQLE GLSAFYADQRSAV QYN

QYNIPSAYAVMLTNKDTVPRTFYGDMYQDDGQYMANKSLYYDAIDTMMK

ARL YVSGGQTMSVT INNANSQKSGEVLTSVRFGKGVMDATDAGSAESR

TQGIGVVVSNSSGLQLNDNDKIVLHMGAAHKNQEYRALMLTTNDGIKSFN

NDEAPINYTDDNGDLIFDGHNIDGQENTAIRGYLNPQVAGYLAVWVPTGA

DDQDARTQPSNEKSTDGKVLHTNAALDSELIYEGFSNFQPMPTT DEYTOV

M1AKNIDLF SWGITNFELAPQYRSSDGKN1 DRF1DSLVQNGYGLSDRY

DLGFETPTKYGTDQDLRTAI TLHQAGMTVMADYVANQIYGLNTSQEVVD

AQRVNSD NAVEVRYGQHLNVVNSIGGGEYQ LYGG YLEILN LYPDLL

VDENGN IDIDT IKQWSAKYLNGSNVTGLGMGYVLKDWSNGQYFNISNT

DGKVMLPEQLVKHMPAVEIGTQTNYTAYISSTIRRDGLYNNMPWGVTATG

QDGNEIKWERQGSTSDYNHQKVQVNRQYVD QGVVWNLIN

FDDKDLWVDSNALVTVNFTSQKPT HFVQFGMRQGKYDGFYLSAPYKQTE

S WVASTRTHQGQLLEVVGQYTTGSGSRKVTWYLVGLDG QVWVDSRAV

GTNFSHKTNiNLLINSATRNDGMYLNAPYGQKGYKRETSSRFYNE LVTVS

QQYYDNKGVIWNL1TLNG KLWVDSRAFATVID VNQSLYINSRNDGMY

LNAPYRAQGA RYASTKTYTGQRVQVTLQRKDTHGVTWYLT VDS QLW

VDSHAFAPTFTRNVSLNVKVNSSKRNDGIYLNAPYGN A RIASTKAYNG

KRVKAS EYKDA GVTWYLVNLNNKQVWIDKRAF

Brs-D (Lb MNINSNER VRF MY SGKQWIVAGLTTAVISIAVYGGSSIANGGIEAKAD kunkei EFB6) AQ AATSSIVNTNNSTNSSNANS1ASLPQNGTYSTNDNGQTW YVSQ DI

QGLY DNNDQLRYFNEYDGTQAKGDIVNVNNDNYYFDKDSGQGHKIDSY

TGGSYSESKVN QDGWIYKSSDNNDVKGVATVDGNIQYFDQNTGLQLKGG

SAQIGGVDYYFDPNKGNLVGKVDQVVNSNDYSDNKLLDSN NVVKGLVV

NNGQLQFFDTSNGNQAKNKQVIANGITYYFDTNGNGQYLFTNTGKSAVDD

rrQRNAANSVNPSDY NVVDGFFTADTWYRP QILDNGlTWRNSNSNELR

PMITAWWPN DVQVNYL LMQNNGLLDKSNSYS1QSDQQTLNQAAQKAQ

VN1E KISQTGNTDWLNDLLFKG GDNPSFV QQYIWSSDSESPWQGDAWF

QGGYLKYGNSVMTPNmSNYRDSNNLFDFLLA DVDNSNPAVQAEDLNW

LYYLTNFGTITANDS ANFDSIRIDAVDFISNDIIQRSYDYLRQ FNL QSDA

NADSHISLVEGGVDAGTTSYSNDGLVEAPFRLDAYPLLHKQDGDVFKNLID

EEDSGIDISNHNGETNTNNTIGGITLSGG PNYSlVHAHDKDVQEKVGQAIiD

TTGI DWTDFTPSQLAQGLETFYNDQRQTVK YNDYNVPSAYAIMLTNKG

TVPRIYYGDMYQDDGQFMQ SLYYDDiANLMTAR YVSGGQSMVDNN

GILTS VRFG GANTVSDSGTEDTRNQGIGLIVGSAPKKVLNDGDTVVLHMG

AAHKNQKYRALMLTTENGIQNYNSDDNAPVAETDDNGDLVFSN DINGQA

NTAI QVANPEVNGYLAAWVPVGASDDQDSRTAPSTSQNNDG VLHENDA

LDSNLIFEGFSNFQPTPTOHDEYANVVIAKNASLFKDWGVTSFEMAPQYRSS

QDHTFVDSTIDNGYAFSDRYDLGFGTPTKYGTDEDLRNAI SLHDNGMQV

MADVVY QLYNLPGQEVVSATRAGVTGNTNALPFGTQLYVVNTIGGGDY

QK YGGAFLNELQEQYPSLF SQKY YYYKNYA GAGPGYLTVNDAER

SDIPYNQPITEWSA YM GTNILGRGMGYVL DW TGDYFKLSGSDSTLPS

SLTY SGWVENPDSTWSYYEKNNIDKLTGSQVINEERVFFD NGIQVKGGW

V NSNGTYSYYD SGNILTGDQLIDGEHFFFDNNGVQV G WI NSDGS

KSYYDSHLG LI TDK VSSNARKKKSKEELLYENAL VLRKDK RLD N TKANIR YNKSLKKYRKA LLAIT RVANAR AI IAK VLSl RKNI

NNE RYY AL EYYVAE SYLKITGNYN KYYYEFD LTP VKWKNIYS YKSRHFT RV I GTLVRVKSIVRSG VARINIGNGHFITSS DFI MF K

GBD-CD2 AQAGHYITKNGNDWQYDTNGELAKGLRQDSNG LRYFDLTTGIQA GQFV (L. citreum TIGQETYYFSKDHGDAQLLPMVTEGHYGTITLKQGQDTKTAWVYRDQNNT NRRL B-12 9 IL GLQNINGTLQFFDPYTGEQLKGGVAKYDDKLFYFESGKGNLVSTVAGD (tronque YQDGHYISQDGQTRYADKQNQLVKGLVTVNGALQYFDNATGNQIKNQQVI depuis DSR- VDGKTYYFDDKGNGEYLFTNTLDMSTNAFSTKNVAFNHDSSSFDHTVDGF E) LTADTWYRP SILANGTTWRDSTD DMRPLITVWWPNKNVQV YLNFM

ANGLLTTAAQYTLHSDQYDLNQAAQDVQVAIERRIASEHGTDWLQKLLFES

QNN PSFV OQFIWN DSEYHGGGDAWFQGGYL YGNNPLTPTTNSDYR

QPGNAFDFLLANDVDNSNPVVQAENLNWLHYLMNFGTITAGQDDANFDSI

R1DAVDFIH DTIQRTYDYLRDAYQVQQSEA ANQHISLVEAGLDAGTSTIH

NDALIESNLREAATLSLTNEPG N PLTNMLQDVDGGTLITDHTQNSTENQ

ATPNYSIIHAHDKGVQE VGAAITDATGADWTNFTDEQLKAGLELFYKD

QRATNKKYNSYNIPSIYALMLTNKDTVPRMYYGDMYQDDGQYMANKSIY

YDALVSLMTARKSYVSGGQTMSVDNHGLL SVRFG DAMTANDLGTSAT

RTEGLGVIIGNDPKLQLNDSDKVTLDMGAAHKNQ YRAVILTTRDGLATFN

SDQAPTAWTNDQGTLTFSNQEINGQDNTQIRGVANPQVSGYLAVWVPVG

ASD QDARTAA 111 b HDG VLMS AALDS LIYEGFS FQP ATTHDELT

NVVIAKNADVFN GITSFEMAPQYRSSGDHTFLDSTIDNGYAFTDRYDLG

FNTPTKYGTDGDLRATIQALHHANMQVMADVVDNQVYNLPGKEVVSATR

AGVYGNDDATGFGTQLYVTNSVGGGQYQEKYAGQYLEALKAKYPDLFEG

KAYDYWY NYANDGSNPYYTLSHGDRESIPADVAIKQWSAKYMNGTNVL

GNGMGYVL DWHNGQYF LDGD STLPQI GEL LEG PIPNPLLGLDSTR

TGHHHHHH

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

Mutant Sequence

MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGEL NIDDNAYY FDKNSGNGRTFT ISNGSYSEKDGMWQYVDSHDKQPV GLYDVEGNLQYFDLSTG

NQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLD

NQGNAIKGLNDV GEIQYFDEHTGEQL GHTATVDGTTYYFEGNKGNLVSVVNTAP

TGQY INGDNVYYLDN NEAIKGLYGINGNL YFDLATGIQL GQA NIDGIGYYFD

QNNGNGEYRYSLTGPVV DVYSQH AVN LSANNF NLVDGFLTAETWYRPAQII.

SHGTDWVASTD DFRPLITVWWPN DIQVNYLKLMQQIGILDNSVVFDTN DQLVL

N GAESAQIGIE VSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGAFQ

GGYLAYQNSDLTPYANTNPDYRTHNGI VLLANDVDNSNPVVQAEQLNVVLYYEMN

FGQITANDSNANFDSMRIDGLAFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISrVE

AP GETPITVEKQSALVESNWIDRMLQSLS NATLDKLDPDPAINSLE LVADDLVN

RSQSSDKDSSTIPNYSIVHAHDLLLVDTVIHIM IVN NPNISMSDFTMQQLQNGLKA

FYEDQHQSV YNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA

T SlYYNAIEQMM ARL YVAGGQIMAVT I NDGINKD iTN SGEVLTSVRFG DI

MDAQGQGTAESRNQGIGVIVSNSSGLEL NSDS1TLHMGIAHKNQAYRALMLTNDK

GIVNYDQD NAPIAWTNDHGDI.IFTNQMINGQSDTAVKGYLNPEVAGYEAVWVPV

GANDNQDARTVTTNQK TDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANV

VIT NIDLF SWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGF TPT

YGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDT LVV

DPRYGTQMTW SVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQ IDLSTKIKEW

SA YLNGSNIPQVGMGYVL DWN GQYFHILDKEGQYSLPTQLKGGRADPAFLY

M6 VVHHHHHH MASQYVE DGQFVYIDQSG QVSGLQNIEGHTQYFDP TGYQTKGEL .NIDD AYY FD NSGNGRTFT ISNGSYSE DGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTG QAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTT D DGNETSYWAYLD NQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATVDGITYYFEGNKGNLVSVVNTAP TGQY INGD VYYLDN NEAIKGLYGINGNLNYFDLATGIQL GQAK IDGIGYYFD Q NGNGEYRYSLTGPVV DVYSQHNAVNNLSANNF NLVDGFLTAETWYRPAQIL SHGTDWVASTD DFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVL N GAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGMM QGGYLAYQNSDLTPYANTNPDYRTFINGV FMLANDVDNSNPVVQAEQLNWLYYL MNFGQ1TANDSNANFDSMRIDGMWLVDPQIA KAYDLLD MYGLTDNEAVANQHI SIVEAP GETP1TVE QSALVES WVDRMLQSLSK.NATLD LDPDPAINSLE LVAD DLV RSQSSD DSSTIP YSIVHAHDLNLVDTVIHIM IVNNNPNISMSDFTivlQQEQ GL AFYEDQHQSVKKYNQY IPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQ Y AT SIYYNAIEQMMKARLKYVAGGQI AVT I NDGI KDGTNKSGEVLTSVRF GKDIMDAQGQGTAESRNQGIGVIVSNSSGLEL NSDSITLHMGIAH NQAYRALMLT NDKGIVNYDQDN APIAWTNDHGDLIFTNQMINGQSDTAV GYLNPEVAGYLAVW VPVGANDNQDARTVTTNQKNTDGKVLHTNAALDS LMYEGFSNFQ MPTRGNQY ANVVITKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGF TPTKYGTDQDLR A1E.RLHQAGMSVMADFVANQIYGLHAD EVVSAQHVNINGDT KLVVDPRYGTQMTVV SVGGGDYQA YGGEYLDTIS LYPGLLLDSNGQKIDLSTK IKEWSA YLNGSNIPQVGMGYVL DWNNGQYFHILD EGQYSLPTQLKGGRADPAF

M14 .VVHHHHHH

MASQYVE DGQFVYIDQSGKQVSGLQNIEGHTQYFDP TGYQTKGEL NIDDNAYY

FDKNSGNGRTFT ISNGSYSE DGMWQYVDSHDKQPVKGLYDVEGNLQYFDLSTG

NQA HQIRSVDGVTYYFDADSGNATAF AVTNGRYAEQTTKD DGNETSYWAYLD

NQGNAIKGLNDV GEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVV TAP

TGQY INGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQL GQA NIDGIGYYFD

Q NGNGEYRYSLTGPVV DVYSQHNAVNNLSANNFKNLVDGFLTAETWYRPAQIL

SHGTDWVASTDKDFRPLITVWWPN DIQVNYLKLMQQIGILDNSVVFDTNNDQLVL

N GAESAQIGIE VSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQ

GGYLAYQNSDLTPYANTNPDYRTHNGIRYLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAPDWVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIV

EAP GETPITVE QSALVESNWIDRMLQSLSKNATLDKLDPDPAINSLEKLVADDLV

NRSQSSD. .DSSTIPNYSIVHAHDINVLDTV1HIMK1VNNNPNISMSDFTMQQLQNGL

AFYEDQHQSVK YNQYNIPSAYALLLTN DTVPRVFYGDMYQDYGDDLDGGQYM

AT SIYYNAIEQMMKARL YVAGGQIMAVTKI NDGINKDGTN SGEVLTSVRFG

DIMDAQGQGTAESR QGIGVIVSNSSGLELKNSDSITLHMGIAFi QAYRAL LTND

KGIVNYDQDNNAPL WTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVP

VGANDNQDARTVTTNQKNTDG VLHT AALDS LMYEGFSNFQKMPTRGNQYAN

VVITKNIDLFKSWGITDFELAPQYRSSDG DITDRFLDS1VQ GYGLSDRYDLGFKTPT

KYGTDQDLR AIERLHQAGMSVMADFVANQ1YGLHADKEVVSAQHVNINGDTKLV

VDPRYGTQMTW SVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQ IDLST I E

WSAKYLNGSNIPQVGMGYVLKDWN GQYFHILDKEGQYSLPTQLKGGRADPAFLY

M18 KWHHHHHH

MASQYVEKDGQF iSG QVSGLQNIEGHTQYFDPKTGYQT GEL IDDIs

FDKNSGNGRTFT ISNGSYSE DGMWQYVDSHD QPV GLYDVEGNLQYFDLSTG

NQAKHQIRSVDGVTYYFDADSGNATAF AVTNGRYAEQTTKDKDGNETSYWAYLD

NQGNAI GLNDWGEIQYFDEHTGEQL GHTATVDGTTYYFEGNKGNLVSVVNTAP

TGQY INGD VYYLDNNNEAI GLYGINGNLNYFDLATGIQL GQA NIDGIGYYFD

Q NGNGEYRYSLTGPVV E ⁾ VYSQFINAVNNLSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTD DFRPLFrVWWP KDIQV YLKLMQQIGILDNSVVFDTNNDQLVL

N GAESAQIG1E KVSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGLFQ

GGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAIDFVDPQIA AYDLLDKMYGLTDNEAVANQHISIVE

M21 ADKGETPITVE QSALVESNWWLRMKQSLSKNATLDKLDPDPAINSLEKLVADDLV NRSQSSD DSSTIP YSIVHAHDLDIQETVVHIM IVN NPNISWTDFTMQQLQNGLK

AFYEDQHQSV YNQYNIPSAYALLLTN DTVPRVFYGDMYQDYGDDLDGGQYM

AT SIYYNAIEQMMKARL YVAGGQ1MAVTKIKNDGINKDGTN SGEVLTSVRFG

D1MDAQGQGTAESRNQGIGVIVSNSSGLELKNSDS1TLHMGLAH NQAYRALMLTND

KGIVNYDQDNNAPIAWTNDHGDLIFTNQMI GQSDTAV GYL PEVAGYLAVWVP

VGANDNQDARTVTTNQKNTDG VLHTNAALDSKLMYEGFSNFQKMPTRGNQYAN

VVITKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGF TPT YGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHV INGDTKLV

VDPRYGTQMTVV SVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQKIDLSTKI E

WSAKYLNGSNIPQVGMGYVL DWN GQYFHILDKEGQYSLPTQLKGGRADPAFLY

KVVHHHHIIH

MASQYVEKDGQFVYIDQSG QVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY

FDKNSGNGRTFTKISNGSYSE DGMWQYVDSHD QPVKGLYDVEGNLQYFDLSTG

NQA HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLD

NQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVVNTAP

TGQY INGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQL GQA NIDGIGYYFD

Q NGNGEYRYSLTGPVVKDVYSQHNAV LSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTDKDFRPLITVWWPN DIQVNYLKLMQQIGILDNSVVFDTNNDQLVL

N GAESAQIGIEK VSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGPFQ

GGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAIMFVDPQIA AYDLLDKMYGLTDNEAVANQHiSIVE

AD GETPITVE QSALVESNWLLRM QSLSKNATLDKLDPDPAINSLEKLVADDLVN

RSQSSDKDSSTIPNYSIVHAHD DILETVTHIM IVNN PNISDTDFTMQQLQNGL A

FYEDQHQSV YNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYMA

T SIYYNAIEQMM ARLKYVAGGQIMAVT IKNDGINKDGTN SGEVLTSVRFGKDI

MDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAH NQAYRALMLTNDK

GIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVPV

GANDNQDARTVTTNQ NTDG VLHTNAALDS LMYEGFSNFQKMPTRGNQYANV

V1T NIDLF SWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGFKTPT

YGTDQDLRKAIERLHQAGMSV ADFVANQIYGLIIADKEVVSAQHV INGDTKLVV

DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTIS LYPGLLLDSNGQ IDLST I EW

SA YLNGSN1PQVGMGYVL DWNNGQYFHILD EGQYSLPTQL GGRADPAFLYK

M23 VVHHHHHH

MASQYVEKDGQFVYIDQSG QVSGLQN1EGHTQYFDPKTGYQT GELKNIDDNAYY FDKNSGNGRTFTKISNGSYSE DGMWQYVDSHD QPV GLYDVEGNLQYFDLSTG NQAKHQIRSVDGVTYYFDADSGNATAF AVTNGRYAEQTTKDKDGNETSYWAYLD NQGNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGN GNLVSVVNTAP

TGQY INGDNVYYLDNNNEAIKGLYGI GNLNYFDLATGIQLKGQA NIDGIGYYFD

QN GNGEYRYSLTGPVV DVYSQHNAV NLSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTDKDFRPLITVWWPN DIQV YLKLMQQIGILDNSVVFDTNNDQLVL

N GAESAQIGIEK VSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGMF

QGGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLM

NFGQITANDSNANFDSMRIDAIMFVDPQIA AYDLLDKMYGLTDNEAVANQHISIV

EDSKGETPITVE QSALVESNWWLRMKQSLSKNATLDKLDPDPAINSLE LVADDLV

NRSQSSDKDSSTIPNYSIVHAHDLDILETVVHIM IVNN PNISWTDFTMQQLQNGLK

AFYEDQHQSV YNQYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYM

AT SIYY AIEQMMKARL YVAGGQIMAVT IK DGIN DGTN SGEVLTSVRFG

DIMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAH NQAYRALMLTND

KGIVNYDQDN APIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVP

VGANDNQDARTVTTNQKNTDG VLHTNAALDS LMYEGFSNFQKMPTRGNQYAN

WIT NIDLFKSWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGFKTPT YGTDQDLRKAIERLHQAGMSVMADFVANQiYGLHAD EVVSAQHVNI GDT LV

VDPRYGTQMTVV SVGGGDYQA YGGEYLD ^' HSKLYPGLLLDSNGQ IDLSTKI E

WSAKYLNGSNIPQVGMGYVLKDW GQYFHILDKEGQYSLPTQLKGGRADPAFLY

M28 VVHHHHHH MASQYVE DGQFVYIDQSG QVSGLQNIEGHTQYFDPKTGYQT GELKNIDDNAYY

FD NSGNG TFTKISNGSYSE DGMWQYVDSHD QPVKGLYDVEGNLQYFDLSTG

NQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKD DGNETSYWAYLD QGNAI GLNDVNGEIQYFDEHTGEQLKGHTATVDGITYYFEGNKGNLVSVV TAP

TGQY rNGDNVYYLDNN EAIKGLYGI G L YFDLATGIQL GQAKNIDGIGYYFD

QNNGNGEYRYSLTGPVV DVYSQH AVNNLSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTD DFRPLITVWWPN DIQVNYL LMQQIGILDNSVVFDTNNDQLVL

N GAESAQIGlEKKVSETGNTDWLNELLFAPNGNQPSFl QQYLWiWDSEYPGGLFQ

GGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAlSWVDPQIAK AYDLLDKMYGLTDNEAVANQHISr E

ALKGETPiTVE QSALVESNWMQRM QSLS NATLD LDPDPAINSLE LVADDLV

NRSQSSDKDSSTIPNYSIVHAHDVDrVETVTHMKIVNNNPNISMTDFTMQQLQNGL

AFYEDQHQSV KY QYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYM

AT SrrYNAlEQMMKARLKYVAGGQIMAVTKIKNDGIN DGTN SGEVLTSVRFGK

DIMDAQGQGTAESRNQGIGVIVSNSSGLEL NSDSITLHMGIAH NQAYRALMLTND

KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWVP

VGANDNQDARTVTTNQKNTDG VLHTNAALDSKLMYEGFSNFQKMPTRGNQYAN

VV1T NIDLFKSWGITDFELAPQYRSSDG DITDRFLDSWQNGYGLSDRYDLGFKTPT YGTDQDLR AIERLHQAGMSVMADFVANQIYGLHAD EVVSAQHVNINGDTKLV

VDPRYGTQMTVV SVGGGDYQAKYGGEYLDTIS LYPGLLLDS GQ IDLST IKE

WSA YLNGSNIPQVGMGYVL DWWJGQYFHILD EGQYSLPTQL GGRADPAFLY

M30 KVVHHHHHH

MASQYYEKDGQFVYIDQSG QVSGLQN1EGHTQYFDP TGYQTKGELKN1DDNAYY

FD NSGNGRTFT IS GSYSEKDG WQYVDSHDKQPVKGLYDVEGNLQYFDLSTG

NQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTT DKDGNETSYWAYLD

NQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGNKGNLVSVV TAP

TGQYKINGDNVYYLDNNNEAI GLYGINGNLNYFDLATGIQLKGQA NIDGIGYYFD

Q G GEYRYSLTGPVVKDVYSQHNAV LSA F NLVDGFLTAET RPAQ1L

SHGTDWVASTDKDFRPLITVWWPN DIQVNYL LMQQIGILDNSVVFDTNNDQLVL

NKGAESAQIGIEKKVSETGNTDWLNELLFAPNGNQPSFI QQYLWNVDSEYPGGLFQ

GGYLAYQNSDLTPYANTNPDYRTFINGLEFLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAISWVDPQIAK AYDLLDKMYGLTDNEAVANQHISIVE

AL GETPITVE QSALVESNWMERM QSLS NATLD LDPDPAI SLE LVADDLV

NRSQSSDKDSSTIPNYSIVHAHDIDIILTVWH K1VNNNPNISVTDFTMQQLQNGLKA

FYEDQHQSVK YNQY IPSAYALLLT DTVPRVFYGDMYQDYGDDLDGGQYMA

T SIYYNAIEQMMKARL YVAGGQIMAVT I DGIN DGTNKSGEVLTSVRFG D1

MDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAH NQAYRALMLTND

GIVNYDQDNNAPIAWTNDHGDLIFTNQMI GQSDTAV GYLNPEVAGYLAVWVPV

GANDNQDARTVTTNQ NTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYANV

V1T NIDLF SWG1TDFELAPQYRSSDG D1TDRFLDSIVQNGYGLSDRYDLGF TPTK

YGTDQDLR AIERLHQAGMSVMADFVA QIYGLHAD EVVSAQHV INGDTKLVV

DPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQ IDLSTKIKEW

SA YLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQL GGRADPAFLY

M31 VVHHHHHH

MASQYVEKDGQFVYIDQSG QVSGLQNIEGHTQYFDPKTGYQT GEL NIDDNAYY

FDK SGNGRTFI ^' ISNGSYSE DGMWQYVDSHDKQPVKGLYDVEG LQYFDLSTG

NQA HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKD DGNETSYWAYLD

NQGNAIKGLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGN GNLVSVVNTAP

TGQYKINGDNVYYLDNNNEAI GLYGINGNLNYFDLATGIQL GQA NIDGIGYYFD

Q NGNGEYRYSLTGPVVKDVYSQHNAVN LSANNFKNLVDGFLTAETWYRPAQIL

SHGTDWVASTD DFRPLITVWWPN DIQVNYLKLMQQIGE.DNSVVFDTNNDQLVL

NKGAESAQIGIE VSETGNTDWL ELLFAPNGNQPSFIKQQYLWNVDSEYPGGLFQ

GGYLAYQNSDLTPYANTNPDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLMN

FGQITANDSNANFDSMRIDAISWVDPQlA AYDLLDKMYGLTDNEAVANQHISrVE

M34 ALKGETPITVEKQSALVESNWMARMKQSLSKNATLDKLDPDPAI SLEKLVADDLV NRSQSSDKDSSTIPNYSIVHAHDVDILETVVHIM IV NNPNISPTDFTMQQLQNGLK

AFYEDQHQSV Y QYNIPSAYALLLTNKDTVPRVFYGDMYQDYGDDLDGGQYM

ATKSIYYNAIEQMMKARLKYVAGGQIMAVTKIKNDGINKDGTNKSGEVLTSVRFGK

DIMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHKNQAYRALMLTND

KGIVNYDQDNNAPIAWTNDHGDLIFTNQMINGQSDTAV GYLNPEVAGYLAVWVP

VGANDNQDARTVTTNQKNTDGKVLHTNAALDSKLMYEGFSNFQKMPTRGNQYAN

WITKNIDLFKSWGITDFELAPQYRSSDG DITDRFLDSIVQNGYGLSDRYDLGFKTPT

KYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTKLV

VDPRYGTQMTVVNSVGGGDYQA YGGEYLDTISKLYPGLLLDSNGQ IDLSTKIKE

WSAKYLNGSNIPQVGMGYVLKDW NGQYFHILD EGQYSLPTQL GGRADPAFLY VVHHHHHH

MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGEL IDDNAYY FDKNSGNGRTFT ISNGSYSEKDGMWQYVDSHDKQPV GLYDVEGNLQYFDLSTG NQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTT DKDGNETSYWAYLD NQGNAIKGLNDVNGEIQYFDEHTGEQL GHTATVDGTTYYFEGNKGNLVSVVNTAP

TGQY INGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQAKNIDGIGYYFD

QN GNGEYRYSLTGPVV DVYSQHNAVNNLSA NFKNLVDGFLTAETWYRPAQIL

SHGTDWVASTD DFRPLITVWWPNKDIQVNYLKLMQQIGILDNSVVFDTNNDQLVL

NKGAESAQIGIEK VSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGAFQ

GGYLAYQNSDLTPYANTNPDYRTH GLEFMLANDVDNSNPVVQAEQL WLYYLM

NFGQITANDSNANFDSMRIDAISFVDPQIAK AYDLLDKMYGLTDNEAVANQHISIV

EAPKGETPITVEKQSALVESNWRDRM QSLSKNATLDKLDPDPAINSLEKLVADDLV

NRSQSSDKDSSTIPNYSIVHAHDADVQVTVIGTM IVNN PNISHTDFTMQQLQNGL

KAFYEDQHQSV YNQYNIPSAYALLLTN DTVPRVFYGDMYQDYGDDLDGGQY

MAT SIYYNAIEQMM ARLKYVAGGQIMAVTKI NDGI DGTNKSGEVLTSVRFG

KDIMDAQGQGTAESRNQG1GVIVSNSSGLEL NSDSITLHMG1AHKNQAYRALMLTN

DKGIVNYDQDNNAP1AWTNDHGDL1FTNQM1NGQSDTAVKGYLNPEVAGYLAVWV

PVGANDNQDARTVTTNQ NTDG VLHTNAALDS LMYEGFSNFQKMPTRGNQYA

NVVIT NIDLF SWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKT

PTKYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHAD EVVSAQHVNINGDT

LVVDPRYGTQMTVVNSVGGGDYQA YGGEYLDTISKLYPGLLLDSNGQKIDLSTKI

KEWSA YLNGSNIPQVGMGYVLKDWNNGQYFHILD EGQYSLPTQL GGRADPAF

M35 LYKVVHHHHHH

MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDP TGYQT GELKNIDDNAYY

FDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPV GLYDVEGNLQYFDLSTG

NQA HQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKD DGNETSYWAYLD

NQGNAI GLNDVNGEIQYFDEHTGEQLKGHTATVDGTTYYFEGN GNLVSVV TAP

TGQYKINGDNVYYLDNN EAIKGLYGINGNL YFDLATGIQLKGQA NirXiIGYYFD

QNNGNGEYRYSLTGPVVKDVYSQH AVN LSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTDKDFRPLITVWWPN DIQVNYLKLMQQIGILDNSVVFDTNNDQLVL

NKGAESAQIGIEK VSETGNTDWLNELLFAPNGNQPSFIKQQYLW VDSEYPGGDFQ

GGYLAYQNSDLTPYANTNPDYRTHNGLEFLMANDVDNSNPVVQAEQLNWLYYLM

NFGQITANDSNANFDSMRIDAISFVDPQIAKKAYDLLDKMYGLTDNEAVANQHISIV

EAPKGETPITVE QSALVESNWRDRMKQSLSKNATLD LDPDPAI SLEKLVADDLV

NRSQSSD DSSTIPNYSIVHAHDVDITITVVSLM IV NNPNISSTDFTMQQLQNGLKA

FYEDQHQSVK YNQYNIPSAYALLLTN DTVPRVFYGDMYQDYGDDLDGGQYMA

T SIYYNAIEQMMKARL YVAGGQIMAVTKIKNDGIN DGTN SGEVLTSVRFGKDI

MDAQGQGTAESRNQGIGVTVSNSSGLELKNSDSITLHMGIAH NQAYRALMLTNDK

GIVNYDQDNNAPIAWTNDHGDLIFTNQM1 GQSDTAV GYLNPEVAGYLAVWVPV

GANDNQDARTVTTNQ NTDG VLHTNAALDS LMYEGFSNFQKMPTRGNQYA V

VITKNIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGF TPT

YGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEWSAQHVNINGDTKLW

DPRYGTQ TVV SVGGGDYQAKYGGEYLDTIS LYPGLLLDSNGQKIDLSTKi EW

SA YLNGSNIPQVGMGYVL DW NGQYFHILDKEGQYSLPTQLKGGRADPAFLY

M40 WHHHHHH MASQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGELKNIDDNAYY

FDKNSGNGRTFTKISNGSYSE DGMWQYVDSHD QPV GLYDVEGNLQYFDLSTG

NQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYAEQTTKDKDGNETSYWAYLD

NQGNAI GLNDVNGEIQYFDEHTGEQL GHTATVDGTTYYFEGN GNLVSVVNTAP

TGQYKINGDNVYYLDNNNEAIKGLYGINGNLNYFDLATGIQLKGQA NIDG1GYYFD

QNNGNGEYRYSLTGPVV DVYSQHNAVNNLSANNF NLVDGFLTAETWYRPAQIL

SHGTDWVASTD DFRPLITVWWPN DIQVNYLKLMQQIGILDNSVVFDTONDQLVL

N GAESAQIGIE KVSETGNTDWLNELLFAPNGNQPSFIKQQYLWNVDSEYPGGLM

QGGYLAYQNSDLTPYANT PDYRTHNGLEFLLANDVDNSNPVVQAEQLNWLYYLM

NFGQITANDSNANFDSMRJDAISFVDPQ1AKKAYDLLD MYGLTDNEAVANQHISIV

EAP GETPITVEKQSALVESNWRDRM QSLS NATLD LDPDPAINSLE LVADDLV

NRSQS SD D S STIPNYS I VHAHDLDLEDT VVSLMKIVNNNPNISMTDFTMQQLQNGL

KAFYEDQHQSVKKYNQY IPSAYALLLT KDTVPRVFYGDMYQDYGDDLDGGQY

MAT SIYYNAIEQMM ARL YVAGGQIMAVTKJKNDGIN DGTN SGEVLTSVRFG

KDIMDAQGQGTAESRNQGIGVIVSNSSGLELKNSDSITLHMGIAHK.NQAYRALMLT N

DKGIVNYDQDNNAP1AWTNDHGDLIFTNQMINGQSDTAVKGYLNPEVAGYLAVWV

PVGANDNQDARTVTTNQKNTDG VLHTNAALDS LMYEGFSNFQKMPTRGNQYA

NVVIT NIDLFKSWGITDFELAPQYRSSDGKDITDRFLDSIVQNGYGLSDRYDLGFKT

PTKYGTDQDLRKAIERLHQAGMSVMADFVANQIYGLHADKEVVSAQHVNINGDTK

LVVDPRYGTQMTVVNSVGGGDYQAKYGGEYLDTISKLYPGLLLDSNGQ IDLSTKI

KEWSA YLNGSNIPQVGMGYVLKDWNNGQYFHILDKEGQYSLPTQLKGGRADPAF

M41 LYKVVHHHHHH

Isolation of brsE gene

The brsE gene was identified in Leiiconostoc mesenteroides KFM-MG genome (NCBI Reference Sequence : CP000574) by performing a nucleotide BLAST against a GH70 a- transglucosyiase encoding gene database. The protein sequence of BRS-E is deposited under the GenBank accession number AHF 19404.1.

Recombinant expression of BRS-E in E. coli

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 pENT /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 μg ml-1 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. For enzyme production, E. coli BL21 * DE3 cells were freshly transformed by 55 brsE. Twenty milliliters of LB medium, supplemented with ampicillin (100 μg niL- 1 ), were inoculated with 100 μΐ, of transformation mix and incubated overnight at 37 °C under agitation (200 rpm). Then, Erlenmeyer flasks culture containing a modified ZYM5052 medium with 100 μg mL ampicillin, 0.1% lactose, 0% glucose and 1 % glycerol were inoculated with the starter culture at an OD600nm 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 OD600nm of 80 and disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (1 1 ,000 g, 30 min, 8°C) of the crude cell extract.

Enzyme production

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 Δ2, brsC, brsD, brsE and pBAD49/brsA, respectively. Twenty milliliters of LB medium, supplemented with ampicillin ( 100 μg mL ^"1 ), were inoculated with 100 μL· of transformation mix and incubated overnight at 37 °C under agitation (200 rpm).

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-A2, 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 ^"1 ) and inoculated with the corresponding starter culture at an ODeoonm 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 _600nm of 80 for BRS-A, BRS-B-A2, BRS-C, BRS-D, BRS-E, and an OD _600nm of 30 for GBD-CD2 and mutants. Cells were disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (1 1 ,000 g, 30 min, 8 °C) of the crude cell extract.

Enzyme purification by affinity chromatography Recombinant enzymes are produced in fusion with a 6xHis 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 _600nni 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 μηι cartridge, lysates were applied at 10 °C onto a 1 ml HisTrap HP ^® column that had been equilibrated with the binding buffer, using an A TAXpress 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, CA, 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 Superosel2 resin.

Enzymatic activity assay

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 tetrasaccharide using branching sucrases

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.mL ^"1 of enzyme, 50 mM to 1 M sucrose, and 10 mM to 100 mM tetrasaccharide of formula (la), in particular ABC; _AC CC _! 3 _AC .D-A11 (also referred as ABC'D'). Reactions were incubated in glass tubes for 8 to 24 h.

Specifically, the pentasaccharides were produced in the following conditions:

Pentasaccharide 1 (PI) was produced in particular by BRS-B-.A2 in a 500 uL scale reaction using 200 pL of ABC'D' acceptor preparation at 110 g.L ^"1 , 206 μΐ, of sucrose at 830 g.L ^"! _, 50 μΐ, of sodium acetate buffer 500 mM at pH 5.1, 9.62 μΐ, of purified enzyme BRS-B-A2 at 52 U / mL and H ₂ 0 to 500 μ

Pentasaccharides 2 (P2) and 2' (2') were produced in particular by GBD-CD2 F2163G in a 2 mL scale reaction using 800 pL of ABC'D' acceptor preparation at 110 g.L ^"1 , 823 pL of sucrose at 830 g.L ^"1 _, 200 μL· of sodium acetate buffer 500 mM at pH 5.1, 130.7 μΐ. of purified enzyme GBD-CD2 F2163G at 15.3 U / mL and H ₂ 0 to 2 mL.

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 μΐ. of ABC'D' acceptor preparation at 110 g.L ^"1 , 823 μL· of sucrose at 830 g.L/ ¹ 200 μL· of sodium acetate buffer 500 mM at pH 5.1, 130.7 L of purified enzyme at 15.3 U / mL and H ₂ 0 to 2 mL.

Methods for separation, detection and purification of the compounds of Interest:

The presence of residual acceptor ABC'D' and glucosylated products (pentasaccharides PI and P2, P2' and P3) was determined by HPLC-MS (High performance Liquid Chromatography coupled with Mass Spectrometry) using a C18RP Fusion (4 μηι, 80 A, 250 x 4.6 mm) analytical column placed in an oven at 40 °C and eluting with a 20-minute iLO/acetonitrile gradient from 70:30 to 60:40 at a flow of 1 mL.min ^"1 . Reaction media were diluted 10 times in H ₂ 0/acetonitrile (70:30, v/v) + 0.08% trifiuoroacetic acid (TFA) before injection of 20 μΐ. 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 - 1 50) 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.

Pentasaccharides PI , 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 ₂ 0 for NMR analyses. NMR analyses for pentasaccharides structure elucidation:

The samples were dissolved in DCl-containing D ₂ 0 at pH 4.9 For NMR studies, the samples were lyophilized three times and dissolved in 180 μΐ. of 99.9% DCl-containing D ₂ 0.

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).

^! H and °C I D NMR spectra were accumulated at 30 °C, 65536 data points were acquired with 32 and 2048 scans respectively for proton and carbon experiments.

' H- ¹³ C HSQC (Heteronuclear Single Quantum Coherence spectroscopy), HMBC (I leteronuclear 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

The structure of Pentasaccharide PI was determined by MR spectroscopy (950MHz), revealing an a- 1 ,6 glucosylation of D', characteristic of 5. flexneri serotype 4a.

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

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

D