Patent Grant
11834468
The present invention provides protected tetrasaccharides, their process of preparation and their use in the synthesis of oligosaccharides, in particular fragments of O-antigens from Shigella flexneri, for example of serotype 1a, 1b, 2a, 2b, 3a, X, 4a, 4b, 5a, 5b, 7a or 7b.
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-glycans. 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. Chem. Soc. (2009) 131, 7379-89, Chem. Commun. (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 manner.
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 S. flexneri serotypes are pathogenic for human and a multivalent vaccine providing broad serotype coverage is required (Clin. Infect. Dis. (2014) 59, 933). Additional S. 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 α-
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 (I0):
(TD)xABZC(TD)y-R (I0)
wherein:
In one aspect, the present invention relates to a compound of following formula (I0):
(TD)xABZC(TD)y-R (I0)
wherein:
In a particular embodiment:
In a particular embodiment, TD means that T is in position 2D.
In a particular embodiment, ZC 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), para-methoxyphenyl (PMP), pentenyl (Pent), phenyl (Ph), triisopropylsilyl (TIPS) or tert-butyldiphenylsilyl (TBDPS) group.
R is in particular on the O in position 1C, when x=1, or on the O in position 1D, when y=1.
When R is Ph, the atom in position 1C, when x=1, and in position 1D, when y=1, is in fact a S.
T is in particular chosen from trichloroacetyl (Cl3Ac), 2,2,2-trichloroethoxycarbonyl (Troc) or allyloxycarbonyl (Alloc).
In another aspect, the present invention relates to a compound of following formula (I):
(Cl3AcD)xABZC(Cl3AcD)y-All (I)
wherein:
In another aspect, the present invention relates to a compound of following formula (I):
(Cl3AcD)xABZC(Cl3AcD)y-All (I)
wherein:
In a particular embodiment:
Compounds of formula (I) offer compatibility with chain elongation into O-antigen fragments, which may require a protecting group able to ensure anchimeric assistance at position 2 of the reducing residue.
The orthogonal protecting group in position 2C enables the 2C-O-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:
In a particular embodiment, Z is ClAc, corresponding to a compound of formula (Cl3AcD)xABClAcC(Cl3AcD)y-All.
In a particular embodiment, Z is BrAc, corresponding to a compound of formula (Cl3AcD)xABBrAcC(Cl3AcD)y-All.
In a particular embodiment, Z is Ac, corresponding to a compound of formula (Cl3AcD)xABAcC(Cl3AcD)y-All.
In a particular embodiment, Z is Ø, corresponding to a compound of formula (Cl3AcD)xABC(Cl3AcD)y-All.
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 (Ia):
ABZCCl3AcD-All (Ia).
In a particular embodiment, said compound is of formula ABCl3AcCCl3AcD-All, ABBrAcCCl3AcD-All, ABAcCCl3AcD-All or ABCCl3AcD-All.
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 (Ib):
Cl3AcDABZC-All (Ib).
In a particular embodiment, said compound is of formula Cl3AcDABClAcC-All, Cl3AcDABBrAcC-All, Cl3AcDABAcC-All or Cl3AcDABC-All.
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 (I0) as defined above, comprising the following steps:
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:
In a particular embodiment, the ABZC-triosyl donor is of formula ABZC—Z′, wherein Z′ is PTFA or TCA, PTFA representing N-phenyltrifluoroacetimidoyl and TCA representing trichloroacetimidoyl, Z′ being more particularly TCA.
In a particular embodiment, the protected ABZC-triosyl donor is of one of the following formulae:
and notably
wherein:
By levulinyl (or levulinoyl) is meant the group CH3—CO—CH2—CH2—CO—.
In the whole description, a wavy bond such as
indicates that the corresponding substituent 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-diacetal 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 Cl3AcD-All acceptor is of following formula:
In a particular embodiment, the protected ABZCCl3AcD-All compound is of one of the following formulae:
In a particular embodiment, the protected ABZCCl3AcD-All 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 ABZCCl3AcD-All 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 ABZCCl3AcD-All 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 (I0) as defined above, comprising the following steps:
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:
In particular, the ABZC-All acceptor is of one of the following formulae:
wherein:
In a particular embodiment, the ABZC-All acceptor 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 ABZCCl3AcD-All 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 CDA.
In particular, the Cl3AcD donor is of formula Cl3AcD-Z′, wherein Z′ is PTFA or TCA, PTFA representing N-phenyltrifluoroacetimidoyl and TCA representing trichloroacetimidoyl.
In particular, the protected Cl3AcD donor is of one of the following formulae or the corresponding oxazolines:
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 Cl3AcD donor compound is of one of the above-mentioned formulae, wherein the TES group is independently replaced by a group chosen from TIPS, PMB or Nap.
When the ABZC-All acceptor is of formula (a) and the Cl3AcD donor bears a TES protecting group in position 3D, there is in particular one step (ii) of deprotection only. In the other cases, there may be two steps of deprotection.
In a particular embodiment, the protected Cl3AcDABZC-All compound is of one of the following formulae:
In a particular embodiment, the protected Cl3AcDABZC-All 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 Cl3AcDABZC-All 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 a particular embodiment, the protected Cl3AcDABZC-All 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 Cl3AcDABZC-All compound is of one of the above-mentioned formulae, wherein the 4,6-O-benzylidene acetal is replaced by an 4,6-O-isopropylidene acetal.
In another aspect, the present invention relates to a compound as defined by the following formulae, wherein Z is ClAc, BrAc or Ac:
notably
and
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:
In another aspect, the present invention relates to a method of preparation of a saccharide comprising the following steps:
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 α-
Step (iii) may be achieved using thiourea (Carbohydr. Res. (2012) 356, 115-31).
About step (iv), the transformation of an α-
About step (v), and, for example, taking advantage of the cis-vicinal diol in residue A (not applicable in the case enzymatic α-
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 S. 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 3a 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 GH13 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, 1S46, 1JGI, 1MW2, 1MW3, IMW1 and 1JG9 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 alternansucrase 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-D1, 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-D1, 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-L2166I 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 W2135I-F2136C or GBD-CD2 W2135L-F2136L or GBD-CD2 W2135S-F2136L.
In particular, said saccharide is a fragment of O-antigens from S. flexneri, in particular of serotype 1a, 1b, 2a, 2b, 3a, X, 4a, 4b, 5a, 5b, 7a or 7b.
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)cA(E1→3)dB(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)c(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-
wherein:
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 E1→3 and E1→6 respectively, at all occurrences.
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)cA(E1→3)dB(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-All
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-All
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)cA(E1→3)dB(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-Pr
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(M)b AcD)x(L)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(M)b AcD)y]n-Pr
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(E1→6)b AcD)x(E1→3)cA(E1→3)dB(E1→4)c(Ac)zC((E1→2)a′(E1→4)a(E1→6)b AcD)y]n-
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(E1→6)b AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e(AC)zC((E1+2)a′(E1+4)e(E1→6)bD)y]n-
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(Ac1→6)b AcD)x(Ac1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(E1→6)bD)y]n-
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(E1→6)b AcD)x(Ac1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e(Ac)zC((E1→2)a′(E1→4)a(E1→6)bD)y]n-
wherein:
In particular, said saccharide comprises the following fragment:
-[((E1→2)a′(E1→4)a(Ac1→6)b AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e (Ac)zC((E1→2)a(E1→4)a(E1→6)bD)y]n-
wherein:
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 non-O-acetylated, fully 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 n 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(Ac)zC(E1→6)AcD]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 F2163I-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:
-[(E1→3)AB(Ac)zCAcD]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-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, 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 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:
-[(E1→4)AB(Ac)zCAcD]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-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, 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 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(E1→4)B(Ac)zCAcD]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.
In another aspect, the present invention relates to a compound of one of the following formulae:
((E1→2)a′(E1→4)a(E1→6)bCl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e ClAcC((E1→2)a(E1→4)a(E1→6b)Cl3AcD)y-All:
((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)eC((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e AcC((E1→2)a′(E1→4)e(E1→6)b Cl3AcD)y-All;
((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)x(E1→3)c(E1→4)cA(E1→3)d(E1→4)d′B(E1→4)e AcC((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)y;
((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)x(E1→3)(E1→4)c′A(E1→3)d(E1→4)dB(E1→4)e ClAcC((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)y;
((E1→2)a′(E1→4)a(E1→6)bCl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)eC((E1→2)a′(E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1→6)bCl3AcD)x(E1→3)e(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e ClAcC((E1→4)a(E1→6)bCl3AcD)y-All;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)eC((E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→4)a(E1→6)bCl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e AcC((E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→4)a(E1→6)bCl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)e AcC((E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1→6)bCl3AcD)(E1→3)x(E1→4)c′A(E1→3)d(E1→4)d′B(E1-4)e ClAcC((E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1+6)b Cl3AcD)x(E1→3)c(E1→4)c′A(E1→3)d(E1→4)d′B(E1→4)eC((E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)c′A(E1→3)dB(E1→4)e ClAcC((E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)cA(E1→3)dB(E1→4)e C((E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→4)a(E1→6)b Cl3AcD)(E1→3)cA(E1→3)dB(E1→4)e AcC((E1→4)a(E1→6)b Cl3AcD)y-All;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)cA(E1→3)dB(E1→4)e AcC((E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)cA(E1→3)dB(E1→4)e ClAcC((E1→4)a(E1→6)b Cl3AcD)y;
((E1→4)a(E1→6)b Cl3AcD)x(E1→3)cA(E1→3)dB(E1→4)e C((E1→4)a(E1→6)b Cl3AcD)y;
ABClAcC(E1→6)ClAcD-All;
ABC(E1→6)Cl3AcD-All;
ABAcC(E1→6)Cl3AcD-All;
ABAcC(E1→6)Cl3AcD;
ABClAcC(E1→6)Cl3AcD;
ABC(E1→6)Cl3AcD
(E1→3)ABCl3AcCCl3AcD-All;
(E1→3)ABCCl3AcD-All;
(E1→3)ABAcCCl3AcD-All;
(E1→3)ABAcCCl3AcD;
(E1→3)ABCl3AcCCl3AcD;
(E1→3)ABCCl3AcD;
(E1→4)ABClAcCCl3AcD-All;
(E1→4)ABCCl3AcD-All;
(E1→4)ABAcCCl3AcD-All;
(E1→4)ABAcCCl3AcD;
(E1→4)ABClAcCCl3AcD;
(E1→4)ABCCl3AcD;
A(E1→4)BCl3AcCCl3AcD-All;
A(E1→4)BCCl3AcD-All;
A(E1→4)BAcCCl3AcD-All;
A(E1→4)BAcCCl3AcD;
A(E1→4)BCl3AcCCl3AcD;
A(E1→4)BCCl3AcD.
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 ABClAcC rhamnotriosyl donor encompassing protecting groups orthogonal to an allyl ether (All), a N-trichloroacetyl (Cl3Ac) and chloroacetyl moiety (ClAc), was reacted with a D acceptor. A two-step deprotection process gave the ABClAcCCl3AcD-All 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:
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 α-
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 GH70 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 α-
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 GH13 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×[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 et al. (1997).
By “ABC-triosyl” is meant an ABC rhamnotriosyl, i.e. an ABC triose group.
Synthesis of a Common Precursor (1) to Residues A, B and C Allyl 4-O-(2-naphtylmethyl)-α-
Acetyl chloride (50 mL, 0.70 mol, 2.5 equiv.) was added dropwise to allyl alcohol (610 mL) at 0° C., the solution was stirred for 25 min, then L-rhamnose monohydrate (50 g, 277 mmol) was added. The mixture was heated for 2.5 h at 70° C. then for 15 h at 40° C. Follow up by TLC (DCM/MeOH 8:2) indicated the total conversion of the starting hemiacetal (Rf 0.2) into a less polar product (Rf 0.7). The bath temperature was cooled to 0° C. and the solution was neutralized by addition of NaHCO3 (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 Et3N (4 mL), solvents were evaporated under reduced pressure. The residue was dissolved in DCM (600 mL) and washed with H2O (3×300 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 H2O (3×300 mL) and brine (150 mL). The organic phase was dried over anhydrous Na2SO4, 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 rt and heating was continued for 5 h at 80° C. Follow up by TLC (cyclohexane/EtOAc 5:5) indicated the total conversion of the intermediate acetal (Rf 1.0) into a more polar product (Rf 0.2). Solvents were removed under vacuum and traces of AcOH were eliminated by co-evaporation with toluene (3×400 mL) to give a brown solid. Filtration over a pad of silica eluting with a 4:1 mixture of cHex/EtOAc then 1:1 mixture of cyclohexane/EtOAc then recrystallization in hot cyclohexane afforded the expected diol (65.5 g, 69%) as a pale brown solid. Mother liquors were further purified by flash column chromatography (cyclohexane/EtOAc 100:0 to 50:50) to give an additional amount of expected diol (11.6 g). The total yield of diol 1 is 81% over 4 steps.
1H NMR (400 MHz, CDCl3) δ 7.87-7.78 (m, 4H, HArNap), 7.52-7.44 (m, 3H, HArNap), 5.89 (dddd, 1H, J=17.2, 10.4, 6.0, 5.2 Hz, CH═CH2), 5.28 (dqapp, 11H, J=17.2, 1.5 Hz, CH═CH2), 5.19 (dqapp, 1 H, J=10.4, 1.5 Hz, CH═CH2), 4.95-4.86 (m, 2H, HArNap), 4.81 (d, J=1.4 Hz, 1H, H-1), 4.17 (ddt, 1H, J=12.9, 5.1, 1.5 Hz, 1H, HAll), 4.02-3.93 (m, 3H, HAll, H-2, H-3), 3.79 (dq, 1H, J=9.2, 6.3 Hz, H-5), 3.41 (tapp, 1H, J=9.2 Hz, H-4), 2.45 (brs, 2H, OH), 1.38 (d, 3H, J=6.3 Hz, H-6).
13C NMR (100 MHz, CDCl3) δ 133.8 (CH═CH2), 128.4 (CIVAr), 128.0 (CIVAr), 127.7 (CIVAr), 126.7-125.8 (7C, CAr), 117.4 (CH═CH2), 98.5 (C-1 1JC-H=170.1 Hz), 75.1 (CH2Nap), 71.6, 71.3 (2C, C-2, C-3), 68.0 (CAll), 67.3 (C-5), 18.1 (C-6).
HRMS (ESI+): m/z 362.1985 (calcd for C16H22O5Na [M+NH4]+ m/z 362.1967); m/z 367.1576 (calcd for C43H51ClO12Na [M+Na]+ m/z 367.1521).
Synthesis of the rhamnopyranosyl donors (5 and 5a) used as precursor to residues A and B
The TES Derivative
Allyl 4-O-(2-naphthylmethyl)-3-O-triethylsilyl-α-
To a solution of allyl 4-O-(2-naphthylmethyl)-α-
Allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-α-
Route 1: To a solution of allyl 4-O-(2-naphthylmethyl)-3-O-triethylsilyl-α-
Route 2: To a solution of allyl 4-O-(2-naphthylmethyl)-α-
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 Et3N-treated silica gel (cyclohexane/ethyl acetate 9:1 to 5:5) to give the fully protected 3 as a yellow oil with (30.7 g, 95%).
2-O-Levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-(a/p-
To a solution of allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-α-
After stirring the reaction mixture for 2 hours at room temperature, TLC (cyclohexane/EtOAc 7:3) showed complete conversion of the starting material (Rf=0.53) into a less polar product (Rf=0.56). N-Iodosuccinimide (NIS 95%, 1.21 g, 5.39 mmol, 1.2 equiv) in 1:5 water/THF (30 mL) and then distilled water (40 mL) were added to the mixture stirred at 0° C. After stirring the reaction mixture for 2 h at this temperature, TLC (cyclohexane/EtOAc 7:3) showed complete conversion of the intermediate (Rf=0.56) into a more polar product (Rf=0.26). 10% aqueous sodium metabisulfite (50 mL) was added. THF was evaporated under reduced pressure and DCM (50 mL) was added. The aqueous layer was extracted twice with DCM (30 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate (30 mL) and then brine (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of Et3N-treated silica gel (cyclohexane/EtOAc 8:2 to 6:4) to give hemiacetal 4 as a yellow oil (2.1 g, 89%, a/P 8:2).
2-O-Levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-a/P-
Route 1: To a solution of 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-
Route 2: To a solution of allyl 2-O-levulinyl-4-O-(2-naphthylmethyl)-3-O-triethylsilyl-α-
To a solution of the crude material (13.9 g) in anhydrous DCE (210 mL) stirred at room temperature were successively added Cl3CCN (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 Et3N-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
Allyl 3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-α-
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 NaHCO3 (102.5 g). The suspension was filtered off a pad of Celite® and solvents were evaporated and co-evaporated three times with toluene.
To a solution of crude allyl rhamnoside (190 mmol) in anhydrous methanol (1.0 L) stirred at room temperature were successively added butan-2,3-dione (18.3 mL, 0.21 mol, 1.1 equiv), trimethyl orthoformate (83 mL, 0.76 mol, 4.0 equiv) and boron trifluoride etherate (11.7 mL, 95 mmol, 0.5 equiv). After stirring the reaction mixture for 1.5 h under reflux, a TLC follow up (DCM/MeOH 95:5) showed complete consumption of the starting material (Rf=0.3) and the presence of a main product (Rf=0.5). Et3N was slowly added to the reaction mixture at 0° C. until neutralization and volatiles were evaporated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (cyclohexane/EtOAc 8:2 to 6:4) to give compound 2a as a brown oil (57.8 g, 96%, 2 isomers 9:1).
Allyl 2-O-levulinyl-3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-α-
To a solution alcohol 2a (27.0 g, 84.8 mmol) in anhydrous dichloromethane (650 mL) stirred at rt were successively added DCC (59.5 g, 0.29 mol, 3.4 equiv.), DMAP (8.29 g, 67.9 mmol, 0.8 equiv.) and levulinic acid (36.5 mL, 0.36 mol, 4.2 equiv.). After stirring the reaction mixture at rt for 2 h, a TLC follow up (DCM/MeOH 98:2) showed complete consumption of the starting material (Rf=0.25) and the presence of two less polar compounds (Rf=0.6, 0.65). The reaction mixture was concentrated under reduced pressure. Saturated aqueous NaHCO3 (300 mL) was added to the reaction mixture. The aqueous layer was extracted once with DCM (300 mL) and the combine organic phases were washed twice with brine (150 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of silica gel (DCM/MeOH 1:0 to 9:1) to give the fully protected 3a as a brown oil (37.5 g, 95%, 2 isomers 9:1).
2-O-Levulinyl-3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-α-
To a solution of rhamnoside 3a (37.5 g, 90.0 mmol) in anhyd. tetrahydrofuran (THF, 500 mL) stirred at rt was added hydrogen-activated 1,5-cyclooctadienebis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (1.5 g, 1.80 mmol, 0.02 equiv.). After stirring the reaction mixture at room temperature for 2 h, TLC (cyclohexane/EtOAc 5:5) showed complete conversion of the starting material (Rf=0.8, 0.85) into two closely eluting products (Rf=0.8, 0.9). N-iodosuccinimide (NIS, 24.3 g, 108 mmol, 1.2 equiv.) in water/THF (1:5, 250 mL) and then additional water (370 mL) were added to the mixture stirred at 0° C. After stirring the reaction mixture for 2 h at this temperature, TLC (cyclohexane/EtOAc 5:5) showed complete conversion of the intermediate into two more polar products (Rf=0.45, 0.5). Saturated aqueous sodium metabisulfite (500 mL) and then ethyl acetate (500 mL) were added. The aq. layer was extracted twice with ethyl acetate (300 mL) and the combined organic phases were washed with saturated aqueous NaHCO3 (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 Et3N-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).
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 (Ia).
A synthesis of donor 12a, which encompasses a 4,6-O-benzylidene acetal and a TBS ether in place of the 4,6-O-isopropylidene acetal and the Lev group, respectively, is exemplified in the following.
Allyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-trichloroacetamido-Q-
To a solution of allyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-
4,6-O-Benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-trichloroacetamido-α/β-
To a solution of glucosaminide 11a (2.27 g, 4.0 mmol) in anhydrous THF (200 mL) stirred at room temperature was added hydrogen-activated 1,5-cyclooctadienebis(methyldiphenylphosphine)iridium(I)hexafluorophosphate (170 mg, 0.20 mmol, 0.05 equiv). After stirring the reaction mixture for 4 hours at room temperature, a TLC follow up (toluene/ethyl acetate 9:1) showed complete conversion of the starting material (Rf=0.6) into a less polar product (Rf=0.6). N-Iodosuccinimide (1.35 g, 6.0 mmol, 1.5 equiv) in 1:5 water/THF (56 mL) was then added to the mixture stirred at room temperature. After stirring for 2 h at this temperature, a TLC follow up (toluene/EtOAc 9:1) showed complete conversion of the intermediate into a more polar product (Rf=0.2). Saturated aqueous sodium metabisulfite (200 mL) and then ethyl acetate (500 mL) were added. The aqueous layer was extracted twice with ethyl acetate (250 mL) and the combined organic phases were washed with saturated aqueous NaHCO3 (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 N-(phenyl)trifluoroacetimiodyl chloride (1.80 mL, 6.63 mmol, 1.5 equiv) and potassium carbonate (1.53 g, 11.05 mmol, 2.5 equiv). After stirring the reaction mixture for 3 hours at room temperature, TLC (toluene/EtOAc 8:2) showed complete consumption of the starting material (Rf=0.2) and the presence of a mixture of less polar products (Rf=0.75 and 0.8). The mixture was filtered off a pad of Celite® and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography eluting from a column of silica gel (toluene/EtOAc 95:5 to 9:1) to give a 2:1 mixture of the N-(phenyl)trifluoroacetimidate donor 12a and oxazoline 12b as a brown oil (2.57 g, 65% over two steps).
Synthesis of the ABClAcC-Z′
Selected Examples
Allyl 2-O-chloroacetyl-4-O-(2-naphtylmethyl)-α-
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 (2×40 mL). The organic phase was washed with saturated aqueous NaHCO3 (2×25 mL) and brine (25 mL). The aqueous phase was extracted with DCM (2×25 mL). The combined organic phases were dried over Na2SO4, filtered, evaporated and finally co-evaporated with toluene to yield the crude alcohol 13 as a 92:8 mixture of regioisomers.
Allyl 2-O-levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
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 4 Å-MS (1.25 g) was stirred at room temperature for 15 min, then at −60° C. for 15 min. tert-Butyldimethylsilyl trifluoromethanesulfonate (TBSOTf, 100 μL, 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, Et3N was added to the suspension at −35° C., the mixture was filtered through a pad of Celite®, and the filtrate was concentrated to dryness. Rapid filtration of the residue over silica gel and crystallization in EtOAc/pentane 5:1 (400 mL) gave the fully protected 14 (3.83 g, 72%).
Allyl 2-O-levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
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 4 Å-MS (1.38 g) was stirred at room temperature for 15 min, then at −20° C. for 15 min. TMSOTf (11 IL, 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. Et3N 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 ABClAcC trisaccharide 16 (1.36 g, 85%).
HRMS (ESI+): m/z 1336.6191 (calcd for C73H99Cl4NO16Si2 [M+NH4]+) found m/z 1336.6171.
2-O-Levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
To a solution of the fully protected ABClAcC (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 Et3N-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 C70H95Cl4NO16Si2 [M+NH4]+) found m/z 1296.5922.
2-O-Levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
To a solution of the ABClAcC triose 17 (700 mg, 550 μmol) in anhydrous DCE (5.0 mL) stirred at room temperature were successively added Cl3CCN (165 μL, 3.0 equiv) and DBU (40 μL, 0.5 equiv). After stirring the reaction mixture for 45 min at room temperature, the same amounts of CCl3CN 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 Et3N-treated silica gel (toluene/EtOAc 8:2 containing 3‰ Et3N) to give donor 18 (830 mg, 84%).
HRMS (ESI+): m/z 1439.4974 (calcd for C72H95Cl4N2O16Si2 [M+NH4]+) found m/z 1439.4912.
2-O-Levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
To a solution of the ABClAcC triose 17 (1.56 g, 1.22 mmol) in acetone (24.4 mL) stirred at room temperature were successively added K2CO3 (337 mL, 2.0 equiv) and N-phenyltrifluoroacetimidoyl chloride (PTFACl, 290 μL, 1.5 equiv). After stirring the reaction mixture overnight at room temperature, a TLC (cyclohexane/EtOAc 2:8) indicated that that the starting 17 had been converted to a less polar product. The suspension was filtered over a pad of Celite® and volatiles were evaporated under reduced pressure. The residue was purified by column chromatography to give donor 19 (1.56 g, 88%).
ABClAcCCl3AcD-All may also be obtained through the alternative route B as defined below, whereby the protecting group differs from that shown above and are: R1=Nap, R3, R4=R6, R4=BDA, R2=ClAc, R5=Lev, R8=All, R9=Cl3Ac).
Selected Examples
Allyl 2-O-levulinyl-3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-α-
To a solution of crude allyl 2-O-chloroacetyl-4-O-(2-methylnaphthyl)-α-
Allyl 3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-α-
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 μL, 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 NaHCO3 (15 mL) was added and the aqueous layer was extracted twice with dichloromethane (15 mL) and the combine organic phases were washed once with brine (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 9:1 to 7:3) to give disaccharide 15a as a yellow oil (0.63 g, 72%).
Allyl 3,4-O-(2′,3′-dimethoxybutan-2′,3′-diyl)-2-O-levulinyl-α-
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 4 Å 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 μL, 44 μmol, 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 ABClAcCCl3AcD-All
ABClAcCCl3AcD-All (ABC‘D’, 21) was obtained following the generic route B (the first steps of route B up to the triosyl donor 18 being in particular described above) as defined below. The activation glycosylation and a final two-step orthogonal deprotection of route B are in particular performed with a rhamnotriosyl donor and a monosaccharide D acceptor 9 as shown in the more detailed scheme, which just follows. In the exemplified synthesis (R1=R4=Nap, R3=R6=TES, R2=ClAc, R5=Lev, R8=All, R9=Cl3Ac).
ABClAcCCl3AcD-All may also be obtained through the alternative route A as defined in the overview scheme below.
ABClAcCCl3AcD-All may also be obtained through the alternative route B as defined below, whereby the protecting group differ from that shown above and are: R1=PMB, R3=R4=Nap, R6=TES, R2=ClAc, R5=Lev, R8=All, R9=Cl3Ac).
Selected Examples
Allyl 2-O-levulinyl-4-O-(2-naphtylmethyl)-3-O-triethylsilyl-α-
A solution of the triosyl trichloroacetimidate 18 (924 mg, 649 μmol) and the D acceptor 9 (440 mg, 1.5 equiv) in toluene/DCM (3:1, 37 mL) containing 4 Å-MS (1.91 g) was stirred at room temperature for 15 min, then at −15° C. for 15 min. TBSOTf (22 μL, 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. Et3N 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 ABClAcCCl3AcD tetrasaccharide (20, 932 mg, 84%).
HRMS (ESI+): m/z 1729.6128 (calcd for C88H13Cl4N2O21Si2 [M+NH4]+) found m/z 1729.6161.
Allyl a-
To a solution of the alcohol (222 mg, 137 μmol)—issued from the delevulinylation of the fully protected 20—in toluene stirred at 0° C. was added TFA to reach a TFA/toluene ratio of 9:1. The reaction mixture was stirred overnight, at which point a follow up by TLC indicated total consumption of the starting tetrasaccharide and the presence of a major more polar product. Volatiles were coevaporated twice with toluene, then once with acetonitrile and finally with MeOH. The crude material was purified by reverse phase flash chromatography (H2O/MeCN 0→50%), then by RP-HPLC (MeCN/H2O 20.5%) to give the target ABClAcCCl3AcD-All 21 following freeze-drying (70 mg, 36%). RP-HPLC (C18 ® RP fusion (4.6×250 mm, 4.0 μm, 80 Å, CH3CN in H2O (30% for 4 min, then 30→40% over 7 min, at 1.0 mL·min−1), 40° C., λ: 220 nm)=7.6 min.
1H NMR (800 MHz, D2O), δ 5.84 (m, 1H, —CH═), 5.25 (m, 1H, Jtrans=17.3 Hz, ═CH2), 5.19 (m, 1H, Jcis=10.5 Hz, ═CH2), 5.13 (dd, 1H, J2,3=3.0 Hz, J2,1=1.9 Hz, H-2C), 5.11 (d, 1H, J1,2=1.3 Hz, H-1B), 4.86 (d, 1H, J1,2=1.6 Hz, H-1C), 4.86 (d, 1H, J1,2=1.5 Hz, H-1A), 4.66 (d, 1H, J1,2=8.0 Hz, H-1D), 4.28 (m, 1H, HAll), 4.27 (d, 1H, Jgem=15.4 Hz, HClAc), 4.23 (d, 1H, Jgem15.4 Hz, HClAc), 4.12 (m, 1H, HAll), 4.05 (m, 1H, H-5C), 3.99 (dd, 1H, J2,3=3.3 Hz, J2,1=1.7 Hz, H-2A), 3.93 (dd, 1H, J3,4=9.7 Hz, J3,2=3.1 Hz, H-3C), 3.89 (dd, 1H, J2,3=4.7 Hz, J2,1=1.7 Hz, H-2B), 3.88 (d, 1H, J6,5=2.1 Hz, H-6bD), 3.86 (m, 1H, H-2D), 3.72 (m, 2H, H-3D, H-3A), 3.71 (d, 1H, J6,5=3.3 Hz, H-6aD), 3.69 (m, 1H, H-5B), 3.63 (dd, 1H, J3,4=9.9 Hz, J3,2=3.5 Hz, H-33), 3.62 (m, 1H, H-5A), 3.54 (dd, 1H, J4,5=9.9 Hz, J4,3=9.9 Hz, H-4D), 3.52 (dd, 1H, J4,5=9.8 Hz, J4,3=9.8 Hz, H-4C), 3.42 (m, 1H, H-5D), 3.38 (dd, 1H, J4,5=9.8 Hz, J4,3=9.8 Hz, H-4B), 3.37 (dd, 11H, J4,5=9.8 Hz, J4,3=9.8 Hz, H-4A), 1.25 (d, 3H, J6,5=6.2 Hz, H-6B), 1.20 (d, 3H, J6,5=6.4 Hz, H-6A), 1.19 (d, 3H, J6,5=6.4 Hz, H-6C).
13C NMR (800 MHz, D2O), δ 168.6 (COClAc), 164.7 (CONHC(O)CCl
HRMS (ESI+): m/z 895.1840 (calcd for C31H47Cl4NO19NH4 [M+NH4]+) found m/z 895.1860.
Allyl a-
To a solution of the lightly protected tetrasaccharide ABClAcCCl3AcD-All (21, 200 mg, 0.23 mmol) in anhydrous methanol (31 mL) was added MeONa (25% w/w in MeOH, 126 μL, 0.55 mmol, 2.4 equiv.). After stirring the reaction mixture for 3 h, a TLC follow up (cyclohexane/ethyl acetate 8:2) showed complete consumption of the starting material (Rf=0.15). Dowex H+ 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 ABCCl3AcD-All 21a as a white powder (76 mg, 42%). RP-HPLC (C18 ® RP fusion (4.6×250 mm, 4.0 μm, 80 Å, CH3CN in H2O (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 C30H46Cl3NO8NH4 [M+NH4]+) found m/z 819.2745; m/z 824.1618 (calcd for C30H46Cl3NO18Na [M+Na]+) found m/z 824.2239.
Synthesis of an Alternative B Donor (25)
Allyl 3,4-di-O-(2-naphtylmethyl)-α-
Dibutyltin oxide (4.0 g, 1.1 equiv) was added to a solution of diol 1 (5.0 g, 15.0 mmol) in anhydrous toluene (100 mL). The mixture was stirred for 2 h at reflux using a Dean-Stark apparatus. After cooling to rt, dry CsF (2.2 g, 1.0 equiv), dry tetrabutylammonium 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.
1H NMR (CDCl3) δ 7.81 (m, 8H, HArNap), 7.50 (m, 6H, HArNap), 5.93 (m, 1H, Jtrans=17.1 Hz, Jgem, =1.5 Hz, CH═CH2), 5.31 (m, 1H, Jcis=10.4 Hz, CH═CH2), 5.22 (m, 1H, CH═CH2), 5.10 (d, 3H, J=11.2 Hz, CH2Nap), 4.91 (m, 2H, CH2Nap, H-1), 4.21 (m, 11H, HAll), 4.16 (m, 11H, H-2), 4.02 (m, 2H, HAll, H-3), 3.85 (m, 1H, H-5), 3.60 (pt, 1H, J3,4=J4,5=9.3 Hz, H-4), 2.58 (bs, 1H, J2,OH=9.6 Hz, OH), 1.40 (d, 3H, J5,6=6.3 Hz, H-6).
HRMS (ESI+): m/z 502.2608 (calcd for C31H36O5N [M+NH4]+ m/z 502.2593)
Allyl 2-O-levulinyl-3,4-di-O-(2-naphtylmethyl)-α-
To a solution of alcohol 22 (5.02 g, 9.0 mmol) in anhydrous DCM (42 mL) stirred at room temperature were successively added DCC (3.16 g, 1.7 equiv), DMAP (440 mg, 0.4 equiv) and levulinic acid (1.94 mL, 4.2 equiv). After stirring the reaction mixture overnight at room temperature, TLC (toluene/EtOAc 7:3) showed complete consumption of the starting material. The reaction mixture was filtered over a pad of Celite®, and volatiles were evaporated under reduced pressure. The crude material was taken in ethyl acetate (50 mL) and the organic layer was washed thrice with 10% aqueous copper (II) sulfate (30 mL), water (30 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried by stirring over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude material was used as such in the next step.
2-O-Levulinyl-3,4-di-O-(2-naphtylmethyl)-α-
To a solution of the fully protected 23 (from 22, 9.0 mmol) in anhydrous THF (50 mL) stirred at room temperature was added hydrogen-activated [Ir] (152 mg, 0.02 equiv) in anhydrous THF (10 mL). After stirring the reaction mixture for 2 hours at room temperature, another 0.02 equiv of hydrogen-activated [Ir] vas added and the reaction mixture was stirred overnight at room temperature. TLC (cyclohexane/EtOAc 6:4) showed that the starting allyl rhamnoside had been consumed. Iodine (2.74 g, 1.2 equiv) in water/THF (1:5, 72 mL). After stirring the reaction mixture for 1 h at this temperature, TLC (cyclohexane/EtOAc 6:4) showed complete conversion of the intermediate into a more polar product. 10% aqueous sodium metabisulfite was added. THF was evaporated under reduced pressure and DCM (50 mL) was added. The aqueous layer was extracted twice with DCM (30 mL) and the combined organic phases were washed with saturated aqueous sodium bicarbonate and then brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude material was used as such in the next step.
2-O-Levulinyl-3,4-di-O-(2-naphtylmethyl)-α-
To a solution of hemiacetal 24 (from 22, 9.0 mmol) in anhydrous DCE (50 mL) stirred at room temperature were successively added Cl3CCN (2.7 mL, 3.0 equiv) and DBU (670 μL, 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 Et3N-treated silica gel (toluene/EtOAc 95:5 to 8:2 containing Et3N 5‰) to give donor 25 as a brown oil (5.05 g, 88% over three steps).
1H NMR (CDCl3) δ 8.67 (s, 1H, NH), 7.81 (m, 8H, HArNap), 7.49 (m, 6H, HArNap), 6.22 (d, 1H, H-1), 5.56 (dd, 1H, J1,2=2.1 Hz, J2,3=3.3 Hz, H-2), 5.14 (d, 1H, J=11.1 Hz, CH2Nap), 4.92 (d, 1H, J=11.4 Hz, CH2Nap), 4.87 (d, 1H, CH2Nap), 4.77 (d, 1H, CH2Nap), 4.10 (dd, 1H, H-3), 4.00 (m, 1H, H-5), 3.62 (pt, 1H, J3,4=J4,5=9.5 Hz, H-4), 2.78 (m, 4H, CH2Lev), 2.18 (s, 3H, CH3Lev), 1.40 (d, 3H, J5,6=6.2 Hz, H-6)
13C NMR (CDCl3) δ 206.0 (COLev), 171.9 (CO2Lev), 160.1 (NHCO), 135.6-133.2 (6C, CIV), 128.2-126.1 (14C, CArNap), 95.2 (C-1, 1JC-H=179.5 Hz), 79.4 (C-4), 77.1 (C-3), 75.7 (CNap), 72.0 (CNap), 70.8 (C-5), 67.9 (C-2), 38.0 (CH2Lev), 29.8 (CH3Lev), 28.1 (CH2Lev), 18.1 (C-6).
HRMS (ESI+): m/z 703.1744 (calcd for C35H38Cl3NO7 [M+NH4]+ m/z 703.1749)
Synthesis of an Alternative C Acceptor (28)
Allyl 4-O-para-methoxybenzyl-α-
Crude allyl 2,3-O-isopropylidene-α-
The crude intermediate was dissolved in 80% aq. AcOH (200 mL) and the solution was stirred for 3 d at 60° C. Follow up by TLC (toluene/EtOAc 7:3) indicated the total conversion of the intermediate acetal into a more polar product. Solvents were removed under vacuum and traces of AcOH were eliminated by co-evaporation with cyclohexane (2×100 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).
1H NMR (CDCl3) δ 7.30 (m, 2H, HArPMB), 6.91 (m, 2H, HArPMB), 5.89 (m, 1H, CH═CH2), 5.29 (m, 1H, Jtrans=17.2 Hz, Jgem=1.6 Hz, CH═CH2), 5.20 (m, 1H, Jtran=10.4 Hz, CH═CH2), 4.81 (d, 1H, J1,2=1.1 Hz, H-1), 4.69 (m, 2H, CH2PMB), 4.17 (m, 1H, HAll), 3.97 (m, 3H, H-All, H-2, H-3), 3.82 (s, 3H, CH3PMB), 3.75 (m, 1H, H-5), 3.35 (pt, 1H, J3,4=J4,5=9.2 Hz, H-4), 2.45 (bs, 2H, OH), 1.36 (d, 3H, J5,6=6.3 Hz, H-6).
Allyl 2-O-chloroacetyl-4-O-para-methoxybenzyl-α-
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 (2×25 mL). The organic phase was washed with saturated aqueous NaHCO3 (2×25 mL) and brine (25 mL). The aqueous phase was extracted with DCM (2×12.5 mL). The combined organic phases were dried over Na2SO4, filtered, evaporated and finally co-evaporated with toluene to yield alcohol 28 as a mixture of regioisomers. The crude material is used as such in the next step.
Synthesis of Cl3AcDABClAcC-All
Cl3AcDABClAcC-All is obtained following route B′ as defined below.
In particular, it can be obtained from donor 12 or its 4,6-O-benzylidene analog and the ABC trioside acceptor 29 following conventional delevulinylation at position 2A of the fully protected precursor 16. A synthesis is highlighted in the scheme below whereby R1=R4=Nap, R3=R6=TES, R2=ClAc, R5=Lev, R7=TBS, R8=All, R9=Cl3Ac).
Allyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-trichloroacetamido-β-
To a solution of crude tetrasaccharide 30 (1.48 mmol) in THF/AcOH (4:1, 74 mL) was slowly added IM 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. NaHCO3 (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated and co-evaporated with toluene under vacuum. The residue was purified by chromatography eluting from a column of silica gel (toluene/ethyl acetate 95:5 to 4:6) to give by order of elution diol 32 (820 mg, 37%) and triol 31 (334 mg, 16%).
Allyl 2-deoxy-2-trichloroacetamido-β-
A solution of tetrasaccharide 31 (334 mg, 0.24 mmol) in trifluoroacetic acid/1,1,1,3,3,3-hexafluoro-2-propanol (9:1, 3.0 mL) is stirred at rt for 2 h. Toluene (10 mL) was added and volatiles were evaporated and co-evaporated with toluene (five times) under reduced pressure. The residue was dissolved in water (10 mL) and dichloromethane (5 mL). The organic phase was washed with water (10 mL) and the aq. phases were freeze-dried and lyophilized. The residue was purified by reverse phase chromatography eluting from a C18 column (H2O/MeCN 0→40%) to give the lightly protected tetrasaccharide D′ABC′-All (33) as a white powder (74 mg, 35%). RP-HPLC (C18 ® RP fusion (4.6×250 mm, 4.0 μm, 80 Å, CH3CN in H2O (30% for 4 min, then 30→40% over 7 min, at 1.0 mL·min−1), 40° C., λ: 220 nm)=10.2 min.
HRMS (ESI+): m/z 900.1394 (calcd for C31H47Cl4NO19Na [M+Na]+) found m/z 900.1246.
Enzymes
Table 1 presents some enzymes that were used in the context of the present invention.
Leuconostoc
citreum NRRL-B
Leuconostoc fallax
Leuconostoc
mesenteroides
Leuconostoc
citreum NRRL-B
Lactobacillus
kunkei EFB6
Leuconostoc
citreum NRRL-B
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).
Mesenteroides
citreum
citreum
Table 2 presents some mutants of BRS-B-D2 that were used in the context of the present invention.
Isolation of brsE Gene
The brsE gene was identified in Leuconostoc mesenteroides KFRI-MG genome (NCBI Reference Sequence: CP000574) by performing a nucleotide BLAST against a GH70α-transglucosylase encoding gene database. The protein sequence of BRS-E is deposited under the GenBank accession number AHF19404.1.
Recombinant Expression of BRS-E in E. coli
A synthetic brsE gene was designed in order to optimize its expression in E. coli (Biomatik, Cambridge, ON, Canada), and cloned in pET28b vector.
The gene was then amplified by PCR from pET28b/BrsE plasmid DNA template using the forward primer 5′-atgggctacaaggccgg-3′ and the reverse primer 5′-accataataatacaccttattatcggc-3′. The PCR product was then inserted into the pENTR/D-TOPO vector (Life Technologies). From a positive entry clone, LR recombination (Gateway LR Clonase II enzyme mix, Life technologies) was performed with pET-55-DEST destination vector (Merck Millipore). Expression clones were selected on LB agar plates supplemented with 100 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 TOP10 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 mL-1), were inoculated with 100 μL 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 OD600 nm of 0.05. Cultures were incubated at 21° C. under agitation (150 rpm). After 26-hour incubation, cells were harvested by centrifugation, dispersed in 50 mM sodium acetate buffer (pH 5.75) at a final OD600 nm of 80 and disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (11,000 g, 30 min, 8° C.) of the crude cell extract.
Enzyme Production
Cloning of branching sucrase genes in inducible vectors (pET53, pET55 or pBAD49, Life technologies) for heterologous expression in E. co/i cells was previously described (Vuillemin et al., J Biol Chem. 2016; 291(14):7687-702). E. coli BL21*DE3 and E. coli BL21 AI cells were freshly transformed by pET53-55/brsB A2, brsC, brsD, brsE and pBAD49/brsA, respectively. Twenty milliliters of LB medium, supplemented with ampicillin (100 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-Δ2, 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 μg mL−1) and inoculated with the corresponding starter culture at an OD600 nm 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 OD600 nm of 80 for BRS-A, BRS-B-A2, BRS-C, BRS-D, BRS-E, and an OD600 nm of 30 for GBD-CD2 and mutants. Cells were disrupted by sonication. The recombinant enzymes were recovered in the soluble fraction after centrifugation (11,000 g, 30 min, 8° C.) of the crude cell extract.
Enzyme Purification by Affinity Chromatography
Recombinant enzymes are produced in fusion with a 6×His 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 OD600 nm 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 μm cartridge, lysates were applied at 10° C. onto a 1 ml HisTrap HP® column that had been equilibrated with the binding buffer, using an AKTAXpress system (GE Healthcare). The proteins were eluted by imidazole gradient from 10 to 500 mM, over 25 minutes. Eluate fractions of 3 mL were desalted onto 10-DG column (Biorad, Hercules, Calif., USA), with 50 mM sodium acetate buffer at pH 5.75 with 100 mM NaCl, or purified for a second round by gel-filtration on a Superose12 resin.
Enzymatic Activity Assay
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 (Ia), in particular ABClAcCCl3AcD-All (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 (P1) was produced in particular by BRS-B-Δ2 in a 500 μL scale reaction using 200 μL of ABC‘D’ acceptor preparation at 110 g·L−1, 206 μL of sucrose at 830 g·L−1, 50 μL of sodium acetate buffer 500 mM at pH 5.1, 9.62 μL of purified enzyme BRS-B-A2 at 52 U/mL and H2O to 500 μL.
Pentasaccharides 2 (P2) and 2′ (2′) were produced in particular by GBD-CD2 F2163G in a 2 mL scale reaction using 800 μL 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 μL of purified enzyme GBD-CD2 F2163G at 15.3 U/mL and H2O 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 μL 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 μL of purified enzyme at 15.3 U/mL and H2O 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 P1 and P2, P2′ and P3) was determined by HPLC-MS (High performance Liquid Chromatography coupled with Mass Spectrometry) using a C18RP Fusion (4 μm, 80 Å, 250×4.6 mm) analytical column placed in an oven at 40° C. and eluting with a 20-minute H2O/acetonitrile gradient from 70:30 to 60:40 at a flow of 1 mL·min1. Reaction media were diluted 10 times in H2O/acetonitrile (70:30, v/v)+0.08% trifluoroacetic acid (TFA) before injection of 20 μL samples. UV Detection was carried out at 220 nm wavelength. The mass of the different compounds was determined by mass spectrometry with a 0.5 s full scan (m/z 200-1950) both in positive and negative modes. Needle was set on 3.5 kV, cone on 60 V, and the probe temperature was maintained at 450° C.
Pentasaccharides P1, P2, P2′ and P3 were purified by automatic fractionation on an Agilent 1260 Infinity HPLC, during 20 min of separation (same method as above). Several rounds of purification were performed if necessary. Products detected by UV-RI peaks were collected, and reanalyzed by HPLC. Fractions containing single peak products were pooled, concentrated to dryness using a SpeedVac before exchange in D2O for NMR analyses.
NMR Analyses for Pentasaccharides Structure Elucidation:
The samples were dissolved in DCl-containing D2O at pH 4.9 For NMR studies, the samples were lyophilized three times and dissolved in 180 μL of 99.9% DCl-containing D2O.
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).
1H and 13C 1D NMR spectra were accumulated at 30° C., 65536 data points were acquired with 32 and 2048 scans respectively for proton and carbon experiments.
1H-13C HSQC (Heteronuclear Single Quantum Coherence spectroscopy), HMBC (Heteronuclear_single_quantum_coherence_spectroscopy) and Double Quantum Filtered COrrelation SpectroscopY (QDF COSY) experiments were performed at 30° C. Homo and heteronuclear spectra were recorded under the following experimental conditions: 512 increments of 2048 complex points are acquired with an accumulation of 16 scans. Spectral widths were 16025 Hz for proton dimension and 44267 Hz for carbon dimension.
Results
The structure of Pentasaccharide P1 was determined by NMR spectroscopy (950 MHz), revealing an α-1,6 glucosylation of D′, characteristic of S. flexneri serotype 4a.
The structure of Pentasaccharide P2 was determined by NMR spectroscopy (950 MHz), revealing an α-1,3 glucosylation of A, characteristic of S. flexneri serotype 3a.
The structure of Pentasaccharides P2′ and P3 were also determined by NMR spectroscopy (950 MHz), revealing respectively an α-1,4 glucosylation of residue A and an α-1,4 glucosylation of residue B.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/068013 | 7/3/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/007999 | 1/10/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20150050282 | Mulard | Feb 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 2008155487 | Dec 2008 | WO |
| Entry |
|---|
| Josephson et al., “Artificial carbohydrate antigens: the synthesis of the tetrasaccharide repeating unit of Shigella flexneri 0 antigen” Canadian Journal of Chemistry vol. 57 pp. 3073-3079 (Year: 1979). |
| Bundle et al., “Artificial Carbohydrate Antigens: The Synthesis of a Tetrasaccharide Hapten, A Shigella flexneri O-Antigen Repeating Unit” Carbohydrate Research vol. 80 pp. 75-85 DOI 10.1016/S0008-6215(00)85316-1 (Year: 1980). |
| Boutet et al., “Synthesis of Two Tetra- and Four Pentasaccharide Fragments of Shigella flexneri Serotypes 3a and X O-Antigens from a Common Tetrasaccharide Intermediate” Eur J Org Chem 2008 pp. 5526-5542 DOI 10.1002/ejoc.200800693 (Year: 2008). |
| Gauthier et al., “Non-stoichiometric O-acetylation of Shigella flexneri 2a O-specific polysaccharide: synthesis and antigenicity” Organic and Biomolecular Chemistry vol. 12 pp. 4218-4232 DOI: 10.1039/c3ob42586j (Year: 2014). |
| Hu et al., “Efficient Iterative Synthesis of O-Acetylated Tri- to Pentadecasaccharides Related to the Lipopolysaccharide of Shigella flexneri Type 3a through Di- and Trisaccharide Glycosyl Donors” Chemistry: an Asian Journal vol. 12 pp. 419-439 DOI : 10.1002/asia.201600819 (Year: 2017). |
| Agoston et al., “A new set of orthogonal protecting groups on a monosaccharide scaffold” Tetrahedron Letters vol. 56 pp. 5010-5012 (Year: 2015). |
| Wong et al., “Assembly of Oligosaccharide Libraries with a Designed Building Block and an Efficient Orthogonal Protection-Deprotection Strategy” J Am Chem Soc vol. 120 pp. 7137-7138 (Year: 1998). |
| Valdor et al., “A new set of orthogonal protecting groups on a monosaccharide scaffold” Progress in Biotechnology vol. 14, pp. 645-650 (Year: 1996). |
| Stephane Salamone, et al., “Investigation on the Synthesis of Shigella flexneri Specific Oligosaccharides Using Disaccharides as Potential Transglucosylase Acceptor Substrates,” Journal of Biological Chemistry, vol. 80, No. 22, pp. 11237-11257 (2015). |
| Julien Boutet, et al., “Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri 3a,” Chemistry—a European Journal, vol. 22, No. 31, pp. 10892-10911 (2016). |
| Number | Date | Country | |
|---|---|---|---|
| 20200165285 A1 | May 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62528277 | Jul 2017 | US |
