POLYSACCHARIDE AND METHODS
20210330799 · 2021-10-28
Inventors
- John McGiven (Surrey, GB)
- Laurence Howells (Surrey, GB)
- LUCY DUNCOMBE (Surrey, GB)
- David Bundle (Edmonton, CA)
- SATADRU SEKHAR MANDAL (Edmonton, CA)
- Susmita Sarkar (Edmonton, CA)
Cpc classification
A61K9/19
HUMAN NECESSITIES
A61K47/61
HUMAN NECESSITIES
C07H15/18
CHEMISTRY; METALLURGY
G01N2400/00
PHYSICS
G01N33/5308
PHYSICS
International classification
A61K47/61
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
C07H15/18
CHEMISTRY; METALLURGY
G01N33/53
PHYSICS
Abstract
There is provided a molecule comprising a chain of seven or more contiguous units of 4,6-dideoxy-4-acylamido-α-pyranose each pair of units joined by a C.sub.1-C.sub.2 or a C.sub.1-C.sub.3 link, the chain having a terminal end and a reducing end, wherein the pyranose ring in the unit of the chain most distal from the reducing end is linked to a cap structure. The cap structure is not a 4,6-dideoxy-4-acylamido-α-pyranose. There are also provided vaccine compositions comprising the molecule and methods of vaccinating an animal against infection by a Brucella organism, including methods of distinguishing between a vaccinated and an infected animal. There are further provided novel methods of detecting the presence in a sample of an anti-Brucella antibody.
Claims
1. A diagnostic conjugate comprising a trisaccharide consisting of three units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising only C.sub.1-C.sub.2 links and/or comprising a disaccharide consisting of two units of 4,6-dideoxy-4-acylamido-α-pyranose joined by a C.sub.1-C.sub.2 link, and/or a monosaccharide consisting of one unit of 4,6-dideoxy-4-acylamido-α-pyranose wherein the trisaccharide and/or disaccharide and/or monosaccharide is linked to a non-saccharide carrier via the reducing end.
2. The diagnostic conjugate according to claim 1, wherein any 4,6-dideoxy-4-acylamido-α-pyranose may be 4,6-dideoxy-4-formamido-α-pyranose, for example, 4,6-dideoxy-4-formamido-α-D-mannopyranose.
3. The diagnostic conjugate according to claim 2, wherein the trisaccharide is Structure XII and/or the disaccharide is Structure XI and/or the monosaccharide is Structure II.
4. The diagnostic conjugate according to claim 1 further comprising a monosaccharide consisting of one unit of 4,6-dideoxy-4-acylamido-α-pyranose linked to a non-saccharide carrier via the C.sub.1 carbon and/or a tetrasaccharide consisting of four units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising a central C.sub.1-C.sub.3 link and two C.sub.1-C.sub.2 links, the tetrasaccharide linked to a non-saccharide carrier via the reducing end.
5. The diagnostic conjugate according to claim 1, for use in a method for vaccinating an animal against infection by a Brucella organism, comprising administering to the animal a protective amount of a molecule comprising a chain of seven or more contiguous units of 4,6-dideoxy-4-acylamido-α-pyranose, adjacent units being joined by a C.sub.1-C.sub.2 or a C.sub.1-C.sub.3 link, the chain having a terminal end and a reducing end, wherein the pyranose ring in the unit of the chain most distal from the reducing end is linked to a cap structure.
6. The diagnostic conjugate according to claim 5, wherein the method further comprises obtaining a biological sample from the animal and contacting it with the diagnostic conjugate and/or composition.
7. The diagnostic conjugate according to claim 5, wherein the method further comprises contacting the sample with a universal antigen comprising at least 6 consecutive units of 4,6-dideoxy-4-acylamido-α-pyranose comprising C.sub.1-C.sub.2 links between most or all pairs of units and optionally comprising at least one C.sub.1-C.sub.3 link between a pair of units.
8. The diagnostic conjugate for use according to claim 7, wherein the universal antigen has or comprises the structure VIII, IX or XIX.
9. A method of detecting the presence in a sample of an anti-Brucella antibody comprising contacting the sample with an antigen comprising a trisaccharide consisting of three units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising only C.sub.1-C.sub.2 links and/or with an antigen comprising a disaccharide consisting of two units of 4,6-dideoxy-4-acylamido-α-pyranose joined by a C.sub.1-C.sub.2 link, wherein the trisaccharide and/or disaccharide is linked to a non-saccharide carrier via the reducing end.
10. The method of detecting the presence in a sample of an anti-Brucella antibody according to claim 9 comprising contacting the sample with an antigen comprising a monosaccharide consisting of one unit of 4,6-dideoxy-4-acylamido-α-pyranose linked to a non-saccharide carrier via the C.sub.1 carbon.
11. The method according to claim 9, further comprising contacting the sample with an antigen comprising a tetrasaccharide consisting of four units of 4,6-dideoxy-4-acylamido-α-pyranose and comprising a central C.sub.1-C.sub.3 link and two C.sub.1-C.sub.2 links, the tetrasaccharide linked to a non-saccharide carrier via the reducing end.
12. The method according to claim 9, wherein any 4,6-dideoxy-4-acylamido-α-pyranose may be 4,6-dideoxy-4-formamido-α-pyranose, for example, 4,6-dideoxy-4-formamido-a-D-mannopyranose.
13. The method according to claim 9, wherein the trisaccharide is Structure XII and/or the disaccharide is Structure XI.
14. The method according to claim 10, wherein the monosaccharide is Structure II.
15. The method according to claim 11, wherein the tetrasaccharide is Structure VI.
16. The method according to claim 9 for detecting an animal infected with a Brucella organism, wherein the method detects an infected animal from within a population of animals known to comprise individuals which have been vaccinated with a molecule comprising a chain of seven or more contiguous units of 4,6-dideoxy-4-acylamido-α-pyranose, adjacent units being joined by a C.sub.1-C.sub.2 or a C.sub.1-C.sub.3 link, the chain having a terminal end and a reducing end, wherein the pyranose ring in the unit of the chain most distal from the reducing end is linked to a cap structure, the method comprising contacting a biological sample obtained from the animal with a diagnostic conjugate, wherein detection of antibody binding to the diagnostic conjugate indicates that the sample was obtained from an animal infected with a Brucella organism.
17. The method according to claim 16 wherein the diagnostic conjugate comprises a trisaccharide consisting of three units of 4,6-dideoxy-4-acylamido-α-pyranose and C.sub.1-C.sub.2 links, or a disaccharide consisting of two units of 4,6-dideoxy-4-acylamido-α-pyranose joined by a C.sub.1-C.sub.2 link, or a monosaccharide consisting of one unit of 4,6-dideoxy-4-acylamido-α-pyranose, the trisaccharide or disaccharide or monosaccharide linked to a non-saccharide carrier.
18. The method according to claim 17 wherein the diagnostic conjugate has or comprises Structure XII and/or Structure XI and/or II and/or Structure III and/or Structure VI.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0111] Embodiments of the invention will now be described, by way of example only, with reference to
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EXAMPLES
Example 1: Initial Work to Develop a Possible Vaccine Candidate
[0128] The work disclosed in WO2014/170681 and in (Ganesh et al (2014) Journal of the American Chemical Society 136:16260-16269) and (McGiven et al (2015) Journal of Clinical Microbiology 53:1204-1210) suggested that it may be possible to develop a vaccine formed by chains of 4,6-dideoxy-4-formamido-α-D-mannopyranose units which are exclusively C.sub.1-C.sub.2 linked. This is because the shorter oligosaccharides described in those publications (such as di- or tetra-saccharides), that contain a single C.sub.1-C.sub.3 link and a limited number of C.sub.1-C.sub.2 links, were observed to be less likely to bind to antibodies induced by polysaccharides that are exclusively C.sub.1-C.sub.2 linked. It was suggested that vaccination with an exclusively C.sub.1-C.sub.2 linked polysaccharide would then be capable of discrimination from an animal infected with an organism having an OPS where C.sub.1-C.sub.3 links are present.
[0129] Therefore, initial experiments were carried out in which mice were immunised with an exclusively C.sub.1-C.sub.2 linked hexasaccharide, conjugated to tetanus toxoid, via a disuccinimidyl glutarate (DSG) linker (Structure I). Structure I is referred to as “TT-dsg-1,2hexa”.
##STR00008##
[0130] It was expected that these constructs would only raise antibodies against A and C/Y epitopes, but not against M epitopes, because of the lack of a C.sub.1-C.sub.3 link.
[0131] After immunising mice with TT-sq-1,2hexa and TT-dsg-1,2hexa, sera from the animals was tested against BSA-conjugated 1,2 hexasaccharide (Structure IX) and, as expected, showed a good response. The sera was also tested against the native bacterial antigens of lipopolysaccharides (LPS) from Brucella abortus, Brucella melitensis and Yersinia enterocolitica O:9 and, again, good responses were observed.
[0132] Sera were then tested with various synthetic oligosaccharide conjugate antigens as previously described, shown as Structures II, III, IV, V, VI, VII and IX in Table 2 below (in which, in the third column, “S” indicates a 4,6-dideoxy-4-formamido-α-D-mannopyranose unit, S2S indicates neighbouring units linked by C.sub.1-C.sub.2 and S3S indicates neighbouring units linked by C.sub.1-C.sub.3).
[0133] Surprisingly, it was found that the immunised sera were recognising the antigens including a C.sub.1-C.sub.3 glycosidic linkage, although there was no C.sub.1-C.sub.3 linkage present in the immunising antigen (
[0134] The unexpected antibody response to even the monosaccharide antigen (Structure II) led the inventors to conclude that development of the originally proposed DIVA vaccine would fail, since any oligosaccharide, irrespective of the presence or absence of C.sub.1-C.sub.3 links, would induce such a response as a minimum.
TABLE-US-00002 TABLE 2 synthetic oligosaccharide BSA conjugates Pattern Structure of sugars/ number linkages Structure Mono- saccharide II S
Methods Used for Example 1
[0135] Animal: Female CD1 mice (Charles River, Canada) of 6-8 weeks old were used to study the immune response. All the procedures and experiments involving animals were carried out using a protocol approved by the Animal Care Committee, Faculty of Bioscience, University of Alberta. The protocol was approved as per the Canadian Council on Animal Care (CCAC) guidelines.
[0136] Antigen: All synthetic oligosaccharide antigens were produced as described previously (Ganesh et al (2014) Journal of the American Chemical Society 136:16260-16269) or in the Appendix below. For animal experiments, a hexasaccharide of six units of perosamine all linked via 1,2 glycosidic bonds were conjugated to Tetanus toxoid (TT) using dsg-linker (disuccinimidyl glutarate), to form the molecule having Structure I above (also referred to as “TT-dsg-1,2hexa”). The hexasaccharide was synthesised with a reducing end amine terminated linker (Ganesh et al (2014) Journal of the American Chemical Society 136:16260-16269). A mixture of hexasaccharide and DSG (15 eq.) in DMF and 0.1 M PBS buffer (4:1, 0.5 mL) was stirred at room temperature for 6 h. The reaction mixture was concentrated under vacuum and the residue was washed with EtOAc 10 times to remove the excess DSG. The resultant solid was dried under vacuum for 1 h to obtain DSG activated oligosaccharide. Activated hexasaccharide (0.518 μmol) was added to the solution of tetanus toxoid (0.025 μmol) in 0.5 M borate buffer pH 9 and stirred slowly at 21° C. for 3 days. Then the reaction mixture was washed with PBS buffer, filtered through a millipore filtration tube (10,000 MWCO, 4×10 mL) and the resulting tetanus toxoid-conjugate was stored in PBS buffer. The MALDI-TOF mass spectrometry analysis indicated the conjugate had an average of 11.7 hexasaccharides per tetanus toxoid.
[0137] For screening the immune response via ELISA, the same hexasaccharide was conjugated to a different carrier protein, namely, bovine serum albumin (BSA), using squarate chemistry (Ganesh et al (2014) Journal of the American Chemical Society 136:16260-16269) as described previously (e.g., WO2014/170681), to form Structure IX. Additionally, immune responses were also screened using different synthetic oligosaccharides (Structures II-VI and Structure VIII in Table 1). Different native sLPS from Brucella abortus, Brucella melitensis and Yersinia enterocolitica were also used.
[0138] Vaccine formulation: Alum was prepared freshly at the very beginning of the immunization by following a published protocol (Lipinski et al (2012) Vaccine 30:6263-6269). Briefly, the solutions of 0.2 molar KAl(SO.sub.4).sub.2.12H.sub.2O and 1.0 molar NaHCO.sub.3 were prepared separately and filter sterilised. Then 10 mL of the second solution (bicarbonate solution) was added quickly to a 20 mL of the first solution with vigorous shaking. To avoid any material loss due to effervescence, the mixing step was carried out in a 200 mL beaker. The resulting alum precipitate was washed with PBS (which had previously been filtered and sterilised by autoclave) and spun down at 4000 g for 7 min. This washing cycle was continued till the pH of the supernatant was identical with PBS (pH 7.3). Finally, the alum was suspended in PBS at 50 mg/mL concentration, thimerosal (0.01% w/v) was added and the mixture stored at 4° C.
[0139] Alum was mixed with the TT-conjugate in 5:1 weight ratio and the mixture was allowed to rock overnight before administering on animals.
[0140] Immunization: Animals were immunised thrice at an interval of 21 days. A total volume of 250 μl comprising 12 μg TT-dsg-1,2hexa (equivalent to 1 μg of 1,2 hexasaccharide) was injected on each mouse of which 150 μl was injected inter peritoneally and the rest 100 μl was injected subcutaneously. Pre bleed were collected before the immunisation started. The animals were euthanized at the 10.sup.th day after final injection and final bleed was collected.
[0141] Serum processing: After collection, murine blood was incubated at 37° C. for one hour then spun at 1500 g for 10 min. Clear serum form the top was collected and stored at −20° C. until use.
[0142] Immunoassays: Antibody levels in the murine sera were studied using enzyme linked immuno-sorbent assay (ELISA). A published protocol (Bundle et al (2014) Bioconjugate Chemistry 25:685-697) was followed with little modification. Briefly, polystyrene microtiter plates were incubated with the coating antigen (1 μg/mL, 100 μL/well) at 4° C. overnight, then washed (5×) with PBST (0.05% Tween-20 in phosphate buffer saline, PBS). Then murine sera were added to the coated well at a serial I10 fold dilutions (100 μL/well). The starting dilution for the sera was 1:100. After incubation at room temperature for 2 h, the plates were washed (5×) with PBST. Then the plate was incubated with 100 μL/well of 1:5000 diluted goat anti-mouse IgG antibody, tagged with HRPO (KPL, 1.0 mg/mL stock) for 30 min at room temperature, then washed (5×) with PBST. A peroxidase substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB) with H.sub.2O.sub.2, was added. After 15 min the reaction was quenched by addition of phosphoric acid (1M, 100 μL/well). The plates were read at 450 nm and the data were processed using Origin software. 0.1% BSA in PBST was used to dilute all sera. End point dilution (xo) was recorded as the serum dilution giving an absorbance 0.2 above background and serum titer was calculated as reciprocal of xo. All the data were processed using Origin 9 and GraphPad Prism softwares.
Example 2: Investigation of Possible Epitope at Non-Reducing End of 4,6-dideoxy-4-formamido-α-D-mannopyranose Chain
[0143] Since the inventors were observing binding of antibodies raised against Structure I to even a monosaccharide antigen (Structure II), it was suspected that the terminal sugar provided an epitope for antibody binding (referred to herein as a “terminal epitope”). To investigate whether the binding potential of a single perosamine (4,6-dideoxy-4-formamido-α-D-mannopyranose) was dependent upon the specific structural features possessed only by the terminal perosamine (for example, the hydroxyls located on both C2 and C3), various synthetic oligosaccharides comprising a “cap” structure at the non-reducing end were prepared as shown below in Table 3. Some oligosaccharides were linked to BSA using squarate chemistry and some via DSG, as above.
TABLE-US-00003 TABLE 3 further synthetic oligosaccharide BSA conjugates, providing “cap” structures on the terminal perosamine Pattern Structure of sugars/ number linkages Structure Mannose- linked mono- saccharide XIII S
[0144] Sera from field infected cattle were tested with a selection of the above structures (
[0145] The “positive” data points in
[0146] There was a similar picture when the trisaccharides were evaluated although the contrast between the modified (Structure XIV) and non-modified (Structure V) antigens was not so extreme (a 4-8 fold reduction in titre). Presumably, the less extreme contrast reflects the increased capability of the trisaccharide within Structure XIV to act as a linear antigen. This pattern is also observed with the 1-2 hexasaccharide (Structure IX) and modified 1-2 pentasaccharide (Structure XVIII).
[0147] On the basis of this evidence, the inventors concluded that there was a significant subset of anti-OPS antibodies whose antigen binding to short oligosaccharides was dramatically affected by the presence or absence of a terminal 4,6-dideoxy-4-formamido-α-D-mannopyranose unit.
[0148] Similar experiments were carried out using the same serum from B. abortus infected cattle, using various oligosaccharide antigens modified by replacement of the C.sub.2—OH (hydroxyl) group on the terminal perosamine by an —OMe group. The overall results are summarised in Table 4 below.
[0149] Table 5 below shows more serological data from the application of the synthetic antigens to sera from cattle infected with B. abortus (n=20), “infected” samples, and sera from uninfected cattle (n=20) “non-infected” samples. The mannose-linked monosaccharide (Structure XIII) and mannose-linked trisaccharide (Structure XIV) antigens have poor diagnostic properties (low AUC [Area Under the dose response Curve] values), as they ineffectually differentiate between the “infected” and “non-infected” samples. Structure XIII is especially poor, set against the remarkable and completely unexpected diagnostic attributes of the non-modified (i.e., un-capped) monosaccharide (Structure II).
TABLE-US-00004 TABLE 4 Results from 6 serum samples from B. abortus infected cattle Antigen Strong pos Weak pos Negative monosaccharide squarate 6 0 0 (Structure II) 1-3 disaccharide squarate 6 0 0 (Structure III) t1-2 trisaccharide squarate 6 0 0 (Structure IV) t1-2 trisaccharide dsg 6 0 0 (Structure X) t1-3 trisaccharide 6 0 0 squarate (Structure V) Exclusively 1-2 hexasaccharide 6 0 0 squarate (Structure IX) mannose-linked exclusively 5 1 0 1-2 pentasaccharide squarate (Structure XVIII) mannose-linked t1-3 2 2 2 trisaccharide squarate (Structure XIV) OMe-modified t1-2 2 2 2 trisaccharide dsg (Structure XVII) OMe-modified 1-3 1 2 3 disaccharide dsg (Structure XV) mannose-linked 0 1 5 monosaccharide squarate (very weak) (Structure XIII)
[0150] On the basis of the results shown in Table 5, the inventors concluded that even the inclusion of a single OMe group to the C2 of the terminal monosaccharide was sufficient to abrogate much of the antibody response. This supported the concept that the terminal 4,6-dideoxy-4-formamido-α-D-mannopyranose was a specific structure distinct, in terms of antibody recognition, from a linear polymer of 4,6-dideoxy-4-formamido-α-D-mannopyranose units.
TABLE-US-00005 TABLE 5 Diagnostic performance attributes (YI.sub.max with DSn, DSp and AUC) for samples from animals culture Positive for B. abortus vs random field non-infected samples Antigen YImax DSn DSp AUC Mannose-linked monosaccharide 26.16 75 51.16 0.5558 (Structure XIII) Mannose-linked trisaccharide 53.02 60 93.02 0.7733 (Structure XIV) Mannose-linked pentasaccharide 78.72 95 83.72 0.9605 (Structure XVIII) Monosaccharide (Structure II) 83.02 90 93.02 0.9663 Hexasaccharide (Structure IX) 95 95 100 0.9942 Pentasaccharide (Structure VII) 100 100 100 1.00 Nonasaccharide (Structure XIX 100 100 100 1.00 below) Disaccharide (Structure III) 100 100 100 1.00 Tetrasaccharide (Structure VI) 100 100 100 1.00 (DSn = Diagnostic Sensitivity (%); DSP = Diagnostic Specificity (%); AUC = Area Under the (ROC) Curve; ROC = Receiver Operator Characteristic; YI = Youden Index (DSn + DSp − 100); YImax = the maximum YI value that can be achieved with variation of the +/− cut-off.)
[0151] Therefore, the inventors proposed that the response to the modified oligosaccharides from serum from infected animals might be similar to the response to the non-modified oligosaccharides from serum from animals immunised with antigens that possessed no tip epitope (i.e., no terminal 4,6-dideoxy-4-formamido-α-D-mannopyranose). In the first case, only the anti-linear antibodies would bind and the response would be low (very low with the short oligosaccharides). In the second case, there would be no anti-tip antibodies to bind and, therefore, the only response observed would be due to anti-linear antibodies. The response of these antibodies against the short oligosaccharides would also be low.
Methods Used for Example 2
[0152] Antigen: Oligosaccharides of perosamine were conjugated to Tetanus toxoid (TT) using dsg-linker (disuccinimidyl glutarate) or using squarate chemistry, as described above.
[0153] Bovine serology studies: Antibody levels in bovine sera were studied using enzyme linked immuno-sorbent assay (ELISA) as described previously (McGiven et al (2015) Journal of Clinical Microbiology 53:1204-1210).
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Example 3: Oxidation of OPS Terminal End Sugar to Disrupt Terminal Epitope
[0154] The inventors adapted the disclosure of Stefanetti et al. (Stefanetti et al (2014) Vaccine 32:6122-6129) to disrupt the structure of the terminal sugar in Brucella OPS. These workers subjected the OPS of Salmonella Typhimurium to mild oxidation with sodium metaperiodate. This opens the rhamnose ring to generate aldehydes, which can then be conjugated to the amines on CRM197 (genetically detoxified Diphtheria toxin) via reductive amination. The rhamnose sugar in that method is an internal sugar, rather than a terminal sugar. Therefore, the oxidation is possible because the polymer is linked via C.sub.1 and C.sub.4, so that the cis vicinal hydroxyl groups on C.sub.2 and C.sub.3 are available for oxidation. In the case of Brucella, the perosamines have a D-rhamnose framework (like D-mannose, but lacking OH on C.sub.6) but, because each non-terminal rhamnose in Brucella OPS is linked to its terminal end neighbour through either C.sub.2 or C.sub.3, the only rhamnose with cis vicinal hydroxyl groups on C.sub.2 and C.sub.3 is the terminal one.
[0155] Therefore, use of a similar method on the Brucella OPS can be used to generate a terminal structure shown below (Structure XX).
Methods Used for Example 3
[0156] Brucella OPS (from B. abortus S99 and B. suis biovar 2 [strain Thomsen]) was purified by hot-phenol extraction (Westphal et al (1952) Uber die Extraction von Bakterien mit Phenol/Wasser. Z. Naturforsch. 7:148-155) followed by mild acid hydrolysis and size exclusion chromatography (Meikle et al (1989) Infect Immun 57:2820-2828). The OPS for TT conjugation was oxidised at 2 mg/ml conc in 10 mM sodium metaperiodate (SMP) & 50 mM sodium acetate buffer (pH 5.5) for 1 hr in the dark. This was sufficient to oxidise the vicinal diol hydroxyl groups on the 2.sup.nd and 3.sup.rd carbons of the terminal sugar. Residual SMP was removed by desalting using a PD-10 column (Sephadex-G25 column) according to the manufacturer's instructions (GE Healthcare). A suitable volume of elution buffer allowed the OPS to flow through.
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Example 4: Vaccination with Capped Hexasaccharide
[0157] In view of the apparent importance of the tip epitope in antibody generation, a heptasaccharide linked to TT via the non-reducing terminal end of the sugar chain was prepared (TT-dsg-1,2-hepta.sub.(non-red)) (Structure XXI). This conjugation method disrupts the tip epitope, as no terminal 4,6-dideoxy-4-formamido-α-D-mannopyranose is available. The structure is Structure XXI below.
[0158] Therefore, this is a hexasaccharide capped with a structure which is not 4,6-dideoxy-4-formamido-D-mannopyranose.
[0159] Mice were immunised using this conjugate and sera were evaluated by iELISA against various antigens.
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Methods Used for Example 4
[0160] Animals; Vaccine formulation; Immunization; Serum processing; Immunoassays: All as described above for Example 1.
[0161] Antigen: The 4,6-dideoxy-4-formamido-α-D-mannopyranose hexasaccharide was prepared according to methods described previously (Eis & Ganem (1988) Carbohydrate Research 176:316-323).
[0162] For screening the immune response via ELISA, the same heptasaccharide was conjugated to bovine serum albumin (BSA), as described previously (e.g., WO2014/170681). The resulting heptasaccharide (in fact, a “capped” hexasaccharide) has the following structure:
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[0163] The further synthesis and conjugation methods used to prepare the TT-dsg-1,2-hepta.sub.(non-red) (Structure XXI) and BSA-dsg-1,2-hepta.sub.(non-red) (Structure XXII) can be found in the Appendix below.
[0164] Additionally, immune responses were also screened using different synthetic oligosaccharides (Structures III, VI and IX), as well as different native bacterial cell wall antigens from Brucella abortus and Yersinia enterocolitica O:9.
Example 5: Vaccination with Tip-Conjugated Polysaccharide
[0165] The inventors next attempted vaccination using a much longer polysaccharide, conjugated to the protein carrier via the non-reducing tip end, in a further attempt to obtain a vaccine which would be useable within a DIVA testing system. The object was to obtain a vaccine molecule which would generate antibodies which will not bind to the proposed diagnostic conjugate antigens (di- and tetra-saccharides, Structures III and VI respectively). As described in WO2014/170681, these antigens are already useful to distinguish between animals infected with Brucella and animals which are uninfected or infected with Yersinia enterocolitica O:9 (or strains of Brucella which have an OPS lacking α1,3 glycosidic linkages).
[0166] Mice were immunised, as outlined below, with OPS from B. abortus S99 (which has approximately 2% α1,3 linkages) and OPS from B. suis by 2 strain Thomsen (a strain with exclusively α1,2 linked polysaccharide), both conjugated to TT via the terminal sugar. Therefore, the B. abortus S99-derived structure was Structure XXIII below, in which conjugation to TT is achieved via C.sub.3, or a related structure in which conjugation to TT is achieved via C.sub.2, or via both C.sub.2 and C.sub.3.
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[0167] The B. suis by 2-derived structure was Structure XXIV, below. Again, in Structure XXIV conjugation to TT shown as achieved via C.sub.3, but the B. suis by 2-derived structure may be a related structure in which conjugation to TT is achieved via C.sub.2, or via both C.sub.2 and C.sub.3.
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[0168] Final bleed sera from the animals were tested then against the bacterial antigens of lipopolysaccharides (LPS) from B. abortus S99 and Brucella melitensis strain 16M (about 20% α1,3 linkages), whole cell antigens from B. abortus S99, B. melitensis 16M and B. suis biovar 2, as well as against tetanus toxoid. The results are shown in
[0169] The trisaccharide (Structure XII) was included in the analysis so that, together with the exclusively 1,2 linked hexasaccharide (Structure IX), an evaluation could be made of how the length of the exclusively 1,2 linked oligosaccharide impacts upon the binding of antibodies induced by the glycoconjugate immunogens having Structures XXIII and XXIV. When the sera was tested at a 1/100 dilution the there was no reaction against the trisaccharide (Structure XII). The results showed at least a ten-fold difference in average titre between the hexasaccharide (Structure IX) and the trisaccharide (Structure XIII). The magnitude of this difference was greater than expected, in view of the fact that the exclusively 1,2 linked trisaccharide antigen is considered to be an anti-C/Y antibody epitope (Table 1) and that these antibodies were considered likely to be those responsible for the observed cross reactions between A-dominant (e.g. B. abortus S99) and M-dominant (e.g. B. melitensis 16M) serotypes of Brucella antigen.
[0170] In order to demonstrate that the antigens are capable of detecting antibodies induced by infection with Brucella, these trisaccharide (Structure XIII) and hexasaccharide antigens (Structure IX) were evaluated against sera from naturally and experimentally infected animals. The ELISA results for both antigens when tested against sera from 12 naturally B. abortus infected cattle and 4 non-Brucella infected cattle are shown in
[0171] The results from the immunisations in mice with Structures XXIII and XXIV suggest that anti-tip epitope rather than anti-linear epitope antibodies are the primary types that bind to the short, exclusively 1,2-linked antigens. Prior to this evaluation, the absence of binding of antibodies induced by these structures to the shorter oligosaccharides containing the 1,3 link was thought to be primarily because the 1,3 link prevented antibodies against linear sequences of 1,2 links from binding; the tip epitope was thought to play an important but lesser role. The data now generated with the exclusively 1,2 linked trisaccharide shows that the tip epitope plays a more prominent role in serodiagnosis than previously thought.
[0172] Two of the exclusively 1,2 linked antigens (Structures VIII and XII, hexasaccharide and trisaccharide, respectively) were also tested against sera taken from four cattle experimentally infected with Brucella abortus strain 544 (an A-dominant strain); samples were taken on weeks 3, 7, 16, 24 and 53 weeks post infection. The average titres from these samples are shown in
[0173] For the same reasons, an exclusively 1,2 linked disaccharide (Structure XI) antigen is also an effective DIVA diagnostic. It is evident that it would not bind antibodies induced by a molecule comprising a cap structure, as described herein, for example Structures XXIII and XXIV. This is supported by the diagnostic data shown for the monosaccharide (Structure II) in
[0174] By way of further demonstration of its utility as a DIVA antigen, the exclusively 1,2 linked trisaccharide antigen (Structure XII) was used for the detection of anti-Brucella OPS antibodies in sera from 17 pigs infected with B. suis biovar 2 and in sera from 12 pigs that were not infected with Brucella. These samples were also tested with an equal mix (by mass of the conjugate diagnostic antigen) of the specific M-antigen tetrasaccharide (Structure VI) and the exclusively 1,2 linked trisaccharide conjugate (Structure XII). These results are presented by scatter plot in
[0175] From this, is can be seen that vaccination with an OPS conjugated to the carrier protein via the non-reducing terminal end raises antibodies capable of binding to the bacterial sLPS antigens, to whole Brucella cells, to an exclusively α1,2 hexasaccharide antigen (Structure IX) and also to a universal antigen (for example as described in WO2014/170681; Structure VIII). However, no binding is observed to the shorter antigens (Structures III, IV, V, VI and XII).
[0176] Comparing the results of this Example with the results of Example 4, for a DIVA vaccine to be provided the inventors concluded that a longer polymer of at least seven 4,6-dideoxy-4-formamido-α-D-mannopyranose units is required. The antibodies raised against such a polysaccharide, lacking the terminal tip epitope (disrupted as a result of conjugation to vaccine carrier protein through the non-reducing end of the polymer), are not detected by the short antigenic structures having Structures III, IV, V, VI and XII, so that these structures may be used as DIVA agents to distinguish between an animal which has been vaccinated using the modified polysaccharide and an animal which has been infected with Brucella.
Methods Used for Example 5
[0177] Preparation of Oxidised and TT-Conjugated OPS:
[0178] Brucella OPS was purified by hot-phenol extraction (Westphal et al (1952) Z. Naturforsch. 7:148-155) followed by mild acid hydrolysis and size exclusion chromatography (Meikle et al (1989) Infect Immun 57:2820-2828). The OPS for TT conjugation was oxidised at 2 mg/ml conc in 10 mM sodium metaperiodate (SMP) and 50 mM sodium acetate buffer (pH 5.5) for 1 hr in the dark. This was sufficient to oxidise the vicinal diol hydroxyl groups on the 2.sup.nd and 3.sup.rd carbons of the terminal sugar. Residual SMP was removed by desalting using a PD-10 column (Sephadex-G25 column) according to the manufacturer's instructions (GE Healthcare). A suitable volume of elution buffer allowed the OPS to flow through.
[0179] Oxidised OPS was then subjected to reductive amination. Oxidised OPS was incubated in PBS at final concentrations of 5 mg/ml OPS and 0.5 M ammonium chloride and 0.1 M sodium cyanoborohydride at 37° C. for 24 hours, before desalting into water with a Sephadex G-25 column and then freeze drying.
[0180] OPS was then incubated at 5 mg/ml with 5 mg/ml DSG in PBS for 45 mins on a rotary shaker before desalting with a Zeba 40 kDa column into fresh PBS. The OPS-DSG samples were then incubated with tetanus toxoid (TT) at final concentrations of approximately 2.5 and 0.5 mg/ml respectively. This was done for 2 hours at room temperature (in the dark) on a rotary shaker. Glycine was then added at a final concentration of 2 mg/ml and incubated for a further 15 mins. The samples were then subjected to fractionation by SEC-HPLC to separate the glycoconjugate from the unincorporated OPS. Binding of the glycoconjugates to anti-Brucella antibodies was confirmed by SDS-PAGE silver stain and Western blot.
[0181] Animals and immunisation: Three groups of 8 female CD1 mice were used, aged 7 weeks at the time of pre-bleed. A pre-bleed (100 μl) was taken from each mouse (from the tail vein) to prepare serum from which a baseline antibody titre against the native and proposed DIVA antigens was established. Antibody titre was evaluated by indirect ELISA assays.
[0182] Two days later the mice were immunised with 5 μg of the designated glycoconjugate antigen, suspended in physiological PBS without adjuvant. The dose was administered subcutaneously in a 100 μl volume. At 19 days post immunisation, a 100 μl blood sample were taken from each mouse via the tail vein. After another 2 days (21 days from the 1.sup.st immunisation) the mice were immunised a 2.sup.nd time with the same antigen, formulation, dose, volume and via the same route as for the 1.sup.st immunisation. After 33 days from the 1.sup.st immunisation, a 100 μl blood sample was taken from the mice via the tail vein. After another 2 days (35 days from the 1.sup.st immunisation) the mice were immunised for the 3.sup.rd time with the same antigen, formulation, dose, volume and via the same route as for the 1st immunisation. Two weeks after this (49 days from the first immunisation), the mice were euthanised, then dissected in order to extract blood from the chest cavity after cutting the aorta.
[0183] Immunoassays: The smooth LPS antigens B. abortus S99 and B. melitensis 16M were diluted 0.6 μg/ml and TT was diluted 2.5 μg/ml in carbonate buffer (Sigma). The whole cell antigens B. abortus S99, B. melitensis 16M and B. suis biovar 2 (Thomsen) were diluted 15.6 μg/ml in carbonate buffer (Sigma). Synthetic antigens (Structures III, VI, VII, VIII) were diluted 2.5 μg/ml in carbonate buffer (Sigma).
[0184] 100 μl per well of each antigen was added to Standard bind ELISA plates (Nunc). The plates were incubated overnight at 4-8° C. then washed four times with PBS-Tween, 200 μl per well and tapped dry on blotting paper.
[0185] Mouse sera were diluted in log dilutions at 1/100, 1/316.22, 1/1000, 1/3162.27, 1/10000, 1/31622.7, 1/100000, 1/316227, 1/1000000 and 1/3162270 in casein buffer and 100 μl per well was added to the antigen coated plates. Monoclonal antibody BM40 was diluted 5 μg/ml in casein buffer (Sigma) and added to the plates, 100 μl per well, as a control. A positive serum control, mouse sera from a mouse immunised with Hexasaccharide Structure I, and a negative serum control from a normal (non-immunised) mouse were also included, 100 μl per well, as controls.
[0186] The plates were incubated for 30 minutes at room temperature, on a rotator at 120 rpm, then washed four times with PBS-Tween, 200 μl per well and tapped dry on blotting paper. Anti-mouse immunoglobulins:HRP (Dako) was diluted 1 in 1000 in casein buffer (Sigma) and 100 μl/well was added to the plates. The plates were incubated for 60 minutes for the synthetic antigens and 30 minutes for sLPS and whole cell antigens at room temperature, on a rotator at 120 rpm, then washed four times with PBS-Tween, 200 μl per well and tapped dry on blotting paper. Substrate buffer (pH4.0) (Fluka) with 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma) and 3% hydrogen peroxide (Sigma) was added to the plates, 100 μl per well, and incubated at room temperature for 20 minutes. The reaction was slowed with 0.1M sodium azide, 100 μl per well, and the plates were read at 405 nm absorbance. Data was calculated as the blanked mean of duplicate wells as a percentage of the BM40 control wells tested with Disaccharide Structure III, as this was added to every test plate.
[0187] The optical densities (ODs) for each sample and dilution were blanked by subtracting the OD for control wells to which no sera had been added but were otherwise processed as described above. The quantitative data for the samples were then normalised by expressing the ODs as a percentage of the positive control. The end titres were calculated (using GraphPad Prism 6) as the dilution at which the signal (expressed as a percentage of the positive control) was equal to the positive/negative threshold. This threshold was calculated as the mean of the pre-bleed samples plus 1.96 times the standard deviation of the pre-bleed samples.
[0188] iELISAs on cattle and pig sera: To perform ELISA the oligosaccharide BSA conjugates (Structures II, VI, IX, XII) were immobilised onto the surface of standard polystyrene ELISA plates passively via overnight incubation in carbonate buffer at 4° C. at 2.5 μg/ml (1.25 μg each for mixed antigen coating), 100 μl/well. The plates were washed 4 times with 200 μl/well PBST (PBS containing 0.05% (v/v) Tween 20), tapped dry. Sera was diluted 1/50 in buffer (in duplicate) and 100 μl added per well. The plates were incubated for 30 mins at room temperature at 160 rpm, after which time they were washed and tapped dry as described above. For bovine sera, an HRP-conjugated mouse anti-bovine IgG1 conjugate was used. For porcine sera an HRP-conjugated recombinant protein A/G was used. The conjugates were diluted to working strength in buffer and the plates incubated, washed and tapped dry as for the serum incubation stage. The plates were then developed with ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and hydrogen peroxide substrate for 10-15 mins, stopped with 0.4 mM sodium azide and read at 405 nm wavelength. The optical density for the duplicates was averaged and the blank OD (buffer only instead of sera) was subtracted. This value was then expressed as a percentage of a common positive control serum sample from a B. abortus infected cow (for testing of cattle sera) or a positive control serum sample from a B. suis infected pig (for testing of porcine samples). In each case a negative control sample was always run in order to ensure the quality of the data.
[0189] The same ELISA method was used for testing using sLPS antigen. The sLPS was diluted to working strength and passive coated to standard polystyrene ELISA plates as described for the oligosaccharide conjugates. The rest of the procedure was conducted as described for the oligosaccharide conjugates.
[0190] Several populations of field sera were evaluated by iELISA using the antigens described above. The specific numbers of samples are described above. All samples classified as infected were from animals that had been confirmed as infected by bacteriological culture of B. abortus (cattle) or B. suis (pigs) from tissues derived from the animals themselves (most cattle samples and all swine samples) or the animals were serologically positive for brucellosis (using conventional serology) and were members of a herd that had been confirmed by bacteriological culture of B. abortus to be infected. The randomly collected samples from non-Brucella infected animals (cattle and pigs) have been collected from within Great Britain from 2007 onwards.
[0191] The oligosaccharide BSA conjugates (Structures II, VI, IX and XII), B. abortus sLPS and modified (i.e., capped) B. abortus OPS ELISAs were assessed against a panel of serum from cattle experimentally infected with either B. abortus or Y. enterocolitica O:9. Two groups of four Holstein/Fresian cross cattle were infected independently with either B. abortus strain 544 (109 colony forming units) via the ocular route, or Y. enterocolitica O:9 (1012 colony forming units) orally on 4 occasions on alternate days. The two animal groups were then kept apart to prevent cross infection. All cattle were confirmed free of both Yersinia and Brucella prior to experimental infection and microbiological investigations confirmed that subsequent infection had taken place. Serum from each animal was tested by at 3, 7, 16, 24, and 53 weeks post infection. All animal procedures were conducted in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986.
Example 6: Use of Capped OPS as a Diagnostic Antigen
[0192] A study was carried out in cattle to evaluate the potential of novel OPS based antigens to differentiate between antibodies induced by field strains of B. abortus or B. abortus S19 vaccine.
[0193] Two OPS based antigens were evaluated using standard iELISA methods. These were a standard preparation of smooth lipopolysaccharide (sLPS) from B. abortus S99 and purified OPS derived from B. abortus S99 which had been modified and conjugated to a carrier to assist attachment to the ELISA plate surface (cOPS). This modification and conjugation capped the OPS, i.e., disrupted the terminal epitope.
[0194] These antigens were evaluated against the following serum panel (there was no multiple sampling of animals: 20 samples taken 45 days after vaccination, 60 samples taken from herds confirmed by culture as infected with a field strains of B. abortus. Results are presented in
[0195] The sLPS antigen was the most effective at differentiating between the samples from the infected and non-infected animals. As might be expected, it was also the most susceptible to reaction with sera from vaccinated animals. The cOPS antigen also detected sera from the infected herds. The principle finding was that the cOPS antigen was less sensitive in detecting vaccine-induced antibodies whilst retaining sensitivity against field-induced antibodies due to true infection. This is demonstrated by the AUC values (for differentiation between the sera from infected herds and vaccinated animals) which were 0.8817 for the cOPS antigen and 0.6800 for the sLPS antigen. There was a highly significant difference between these two AUC values (P<0.01). This study indicates that the capped OPS antigen is a superior serological tool in areas where vaccination with B. abortus S19 is taking place.
Methods Used for Example 6
[0196] Preparation of antigens: Brucella sLPS, derived from B. abortus S99, was purified by hot-phenol extraction (Westphal et al (1952) Uber die Extraction von Bakterien mit Phenol/Wasser.Z.Naturforsch.7:148-155). The OPS was derived from this by mild acid hydrolysis and size exclusion chromatography (Meikle et al (1989) Infect Immun 57:2820-2828). The OPS was oxidised at 2 mg/ml conc in 10 mM sodium metaperiodate (SMP) and 50 mM sodium acetate buffer (pH 5.5) for 1 hr in the dark. This was sufficient to oxidise the vicinal diol hydroxyl groups on the 2.sup.nd and 3.sup.rd carbons of the terminal sugar. Residual SMP was removed by desalting using a PD-10 column (Sephadex-G25 column) according to the manufacturer's instructions (GE Healthcare). A suitable volume of elution buffer allowed the OPS to flow through.
[0197] Oxidised OPS was then subjected to reductive amination. Oxidised OPS was incubated in PBS at final concentrations of 5 mg/ml OPS and 0.5 M ammonium chloride and 0.1 M sodium cyanoborohydride at 37° C. for 24 hours, before desalting into water with a Sephadex G-25 column and then freeze drying. OPS was then activated by incubation at 5 mg/ml with 5 mg/ml DSG in PBS for 45 mins on a rotary shaker before desalting with a Zeba 40 kDa MWCO column into fresh PBS. Palmitic acid hydrazide (PAH) was dissolved to 10 mg/ml in DMSO and 1 part of this was added to 9 parts of OPS in PBS for a final dilution of 4.5 mg/ml OPS and 1 mg/ml PAH. The samples reacted for 2 hours at room temperature on a rotary shaker before excess PAH was removed by desalting into H.sub.2O with a Zeba 40 kDa MWCO column. The PAH conjugated OPS was then freeze dried.
[0198] Immunoassays: The sLPS and cOPS were diluted to 0.5 and 5 μg/ml respectively in carbonate buffer (pH 10). 100 μl per well of each antigen was added to standard bind ELISA plates. The plates were incubated overnight at 4-8° C. then washed 5 times with wash solution (0.0014% w/v di-sodium hydrogen orthophosphate and 0.1% Tween-20 in H.sub.2O) and tapped dry.
[0199] Cattle sera was diluted 1/50 in PBS containing 0.1% Tween-20 and 100 μl per well was added to the antigen coated plates. The plates were incubated for 1 hour at room temperature on a rotary shaker and then washed and tapped dry as described above. Protein A/G-HRP conjugate was diluted to 0.05 μg/ml in PBS containing 0.1% Tween 20 and 100 μl of this was added to every well. The plates were then incubated, washed and dried as above for the serum incubation. Substrate buffer was citric acid dibasic sodium phosphate at pH 5.5. One 10 mg tablet of OPD (o-phenylenediamine dihydrochloride) and 100 μl of 3% H.sub.2O.sub.2 was added per 25 mls of substrate buffer and 100 μl of this was added per well. Plates are developed for between 15-30 minutes and then optical densities (ODs) are read at 450 nm. The ODs for samples and controls are blanked by subtraction of the OD of a well to which buffer only was added (no sera). The blanked OD for each sample is expressed as a percentage of the blanked OD of a common positive control.
[0200] Vaccination studies: The protective efficacy of the vaccine formulation is tested in accordance with the OIE (World Organisation for Animal Health) requirements for the immunogenicity testing of B. abortus S19 and B. melitensis Rev1 vaccines (as described in the 2009 chapters on Bovine Brucellosis (chapter 2.4.3) and Caprine and Ovine Brucellosis (chapter 2.7.2) within the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The mice are immunised as described previously for Example 5, except that on day 49 they are challenged with a 100 μl dose, delivered intraperitoneally, containing 2×10.sup.5 CFU of B. abortus strain 544 (or B. melitensis strain 16M). Mice are killed 15 days later.
[0201] Reference lots of vaccines B. abortus S19 and B. melitensis Rev1 and a negative (PBS only) control are evaluated at the same time to demonstrate that the procedure has been conducted correctly and to provide reference points against which the protective efficacy of the novel vaccine will be assessed. The procedure for quantification of protective efficacy, by deriving the spleen weights and bacterial load, is described below.
[0202] Each spleen is excised aseptically, the fat is removed, and the spleen is weighed and homogenised. Alternatively, the spleens can be frozen and kept at −20° C. for from 24 hours to 7 weeks. Each spleen is homogenised aseptically with a glass grinder (or in adequate sterile bags in stomacher) in nine times its weight of PBS, pH 6.8 and three serial tenfold dilutions ( 1/10, 1/100 and 1/1000) of each homogenate made in the same diluent. 0.2 ml of each dilution is spread in quadruplicate in agar plates; two of the plates are incubated in a 10% CO.sub.2 atmosphere (allows the growth of both vaccine and challenge strains) and the other two plates are incubated in air (inhibits the growth of the B. abortus 544 CO.sub.2-dependent challenge strain), both at 37° C. for 5 days.
[0203] Colonies of Brucella are enumerated on the dilutions corresponding to plates showing fewer than 300 CPU. When no colony is seen in the plates corresponding to the 1/10 dilution, the spleen is considered to be infected with five bacteria. These numbers of Brucella per spleen are first recorded as X and expressed as Y, after the following transformation: Y=log (X/log X). Mean and standard deviation, which are the response of each group of six mice, are then calculated.
[0204] The conditions of the control experiment are satisfactory when: i) the response of unvaccinated mice (mean of Y) is at least of 4.5; ii) the response of mice vaccinated with the reference S19 vaccine is lower than 2.5; and iii) the standard deviation calculated on each lot of six mice is lower than 0.8.
Example 7: Intact Whole Cell Diagnostic Antigen (Rose Bengal Test) Comprising a Cap Structure
[0205] The process of eliminating the tip of the OPS can be performed when the OPS is also attached to other molecules. Through these attachments, the OPS may form a part of a larger entity including the whole bacterial cell from which it naturally extends.
[0206] The terminal perosamine in an OPS chain can be degraded by mild oxidation, thereby creating a cap structure at the distal end of the OPS chain, as described herein. This reaction, if maintained at the appropriate conditions, is very specific for the chemical groups that exist as part of the terminal perosamine of the OPS. Therefore, it is feasible that the degradation (capping) can be carried out on the OPS where this exists within a more complex combination of molecules and components without any significant or deleterious upon the non-OPS components. In consequence, it is possible to derive the diagnostic benefits from a capped OPS, as described in Example 6 above, even when the OPS is in an impure state.
[0207] This approach was evaluated using the diagnostic whole cell agglutination assay known as the Rose Bengal test (RBT). This test is commonly used as a screening assay for the serodiagnosis of brucellosis and is described as suitable for this purpose by the OIE (World Organisation for Animal Health). The diagnostic antigen consists of intact whole cells of B. abortus (stains S99 or S1119-3, both biovar 1 and A-dominant) that have been stained pink with rose bengal stain and then suspended in a pH 3.65 buffer (±0.05). This stain greatly assists the visualisation of the agglutination that occurs when the antigen is mixed with test sera that contains anti-Brucella antibodies. As with all the conventional diagnostic tests used for serodiagnosis of brucellosis caused by smooth Brucella strains (those from the species B. abortus, B. melitensis and B. suis), the principle diagnostic molecule within the antigen is the OPS, as this is the molecule against which most of the antibodies induced during infection are raised (Ducrotoy et al. (2016) Veterinary Immunology and Immunopathology 171:81-102).
[0208] To cap (i.e., de-tip) the OPS within the RBT antigen, which exists on the surface of the cells, the antigen was separated from the assay buffer by centrifugation and suspended in cold (4° C.) oxidation reagents (10 mM sodium metaperiodate in 0.1 M sodium acetate buffer pH 5.5). The cells were incubated in with these reagents in the dark at 4° C. until the mild oxidation reaction had been completed. This was verified by measuring the consumption of sodium metaperiodate over time and reaching a stage where replenishment of sodium metaperiodate back to 10 mM resulted in no further consumption. At this stage, it was considered that all the OPS on the surface of the cells had been capped (i.e., de-tipped). The cells were centrifuged to separate them from the oxidation buffer and resuspended in test buffer for serological evaluation.
[0209] The analytical sensitivity of the oxidised (capped) and non-oxidised (non-capped) antigens was compared using a dilution series (in negative bovine serum) of a known positive bovine sample from a B. abortus infected animal: neat, ½, ¼, ⅛, 1/16, 1/32, 1/64 and 1/128. The negative serum used for dilution was also tested. The results showed that the two antigens (before and after oxidation) performed the same, agglutination was observed down to a 1/64 dilution. The 1/128 dilution of the positive serum and the neat negative serum were negative with both antigens.
[0210] To evaluate the diagnostic sensitivity, the oxidised (capped) and non-oxidised (non-capped) antigens were applied to 17 sera from non-Brucella infected cattle and 17 sera from B. abortus infected cattle. The results showed that all samples from the Brucella infected cattle agglutinated with both antigens and all samples from the non-infected cattle did not agglutinate with either antigen. The positive and negative controls used in this evaluation also gave the correct results for both antigens.
[0211] This study shows that relatively crude antigens that contain OPS can be oxidised to completion under mild conditions and remain effective serodiagnostic antigens. It has already been shown that this reaction provides a cap structure to the OPS. In the findings from this study, the ability of the oxidised antigen to differentiate between samples from Brucella infected and non-infected animals was unchanged. This is in accordance with data presented in Example 6 where the diagnostic attributes of the oxidised (capped/detipped) OPS against these sample types was excellent. In this example, the capped OPS showed a reduced sensitivity to samples from animals vaccinated with B. abortus S19. The oxidised (i.e., capped) RBT antigen also exhibits this property and will, therefore, be superior than the non-oxidised antigen at differentiating between sera from animals infected with smooth Brucella strains and those vaccinated with smooth Brucella vaccines (such as B. abortus S19 and B. melitensis Rev1).
[0212] This method of capping (detipping) the OPS can also be applied to other Brucella OPS containing diagnostic antigens such as the sLPS used for ELISA (where the OPS is attached to core sugars which are in turn attached to the Lipid A), other antigens (whole cells, cell lysates or fractions) used in agglutination assays (such as the Serum Agglutination Assay, the Buffered Plate Agglutination Test, and the Complement Fixation Test).
Methods Used for Example 7
[0213] The RBT is performed was performed as described in the OIE manual (OIE 2016 Brucellosis Chapter 2.1.4. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. OIE, Paris). The test (and control) sera is added to the side of RBT test antigen, 30 μl of each, upon a smooth white surface. The two are then mixed together to produce an oval or circle approximately 2 cm in diameter. This mix is gently rocked at room temperature (18-26° C.) for 4 minutes. After this time the mix is observed and any visible agglutination is considered positive. All the samples that were tested, as described above, were done so using this method. Positive and negative control sera were run with every test.
[0214] To oxidise the RBT antigen a volume of working strength antigen was centrifuged at 3000 g in order to pellet the cells. The supernatant, the test buffer, was removed and the cells resuspended in an equal volume, as was removed, of cold (+4° C.) oxidation reagent (10 mM sodium metaperiodate in 0.1 M sodium acetate buffer pH 5.5). The cells were incubated for 30 mins in the dark and then centrifuged as before. The supernatant was removed and replaced with fresh oxidation reagent. This process was repeated four more times so the antigen has undergone 6 times 30 mins of oxidation in total with replenishment of oxidation reagent each hour. After the fifth hour the cells were centrifuged as describe above, the supernatant removed and replaced with the original volume of fresh test buffer. The replenishment process allowed the reaction to progress without limitation due to consumption of sodium metaperiodate and without deviating from the mild oxidation conditions that facilitate the specific reaction with the vicinal cis diols on the terminal perosamine of the OPS.
[0215] The sodium metaperiodate content within the extracted supernatant was measured by dispensing 100 μl of the oxidation reagent into separate wells of a 96 well ELISA plate. Then 100 μl of a 0.5 mg/ml concentration of ABTS (2,2-azinobis-3-ethylbenzthiazoline-6-sulfonic acid) in a pH 4.0 buffer was added to each test well. In the presence of sodium metaperiodate this drove a colour change that was proportional to the concentration of this oxidising agent. The colour change after 15 mins was measured using an ELISA plate reader set to measure optical density at 405 nm. The molarity of sodium metaperiodate in the test sample was calculated by reference to a standard curve which was established by using control wells containing a known concentration of sodium metaperiodate. The results of the oxidation reagent consumption are presented in
[0216] It is clear from this data that sodium metaperiodate is being consumed and that consumption slows and then effectively stops when cells that have already been subjected to sodium metaperiodate are introduced. The graph in the figure shows that, after five rounds of oxidation, no more significant reagent consumption is taking place. It was concluded from this that the antigen had been completely oxidised, all molecules capable of being oxidised by this mild process had been. After this oxidation process, the cells were centrifuged as described above, the supernatant removed, and resuspended in test buffer. These cells were then evaluated for diagnostic efficacy by application to the test sera described above. The oxidised RBT antigen was run in parallel with the original RBT antigen that had not been oxidised.
Example 8: Use of Exclusively 1,2 Linked Trisaccharide (Structure XII) and Disaccharide Antigens (Structure XI) as Serodiagnostic Antigens for Brucellosis
[0217] The properties of the exclusively 1,2 linked trisaccharide (Structure XII) and disaccharide antigens (Structure XI) as DIVA diagnostics has been described above in Example 5. The effectiveness of these antigens was shown by demonstration of their good diagnostic sensitivity for detection of B. abortus infection in cattle and B. suis (biovar 2) infection in pigs. The value of these antigens within the DIVA context is therefore well established.
[0218] A further study was carried out to assess the suitability and merits of these antigens for routine serology, in the presence or absence of vaccination. As the role of the tip epitope has been demonstrated, during the work described herein, to be an important aspect of the diagnostic sensitivity of these antigens, there was an expectation that the presence of an identical epitope on the OPS of Y. enterocolitica O:9 (an exclusively 1,2 linked 4,6-dideoxy-4-formamido-mannopyranosyl polymer) would lead to the generation of cross reactions and false positive results that could be excessive. This expectation was reinforced by the strong reactions exhibited against the exclusively 1,2 linked trisaccharide (Structure XII) antigen by the sera from pigs infected with B. suis biovar 2 (as the long repeating element of the OPS from this biovar is identical to that of Y. enterocolitica O:9).
[0219] To evaluate the extent of this cross reaction the sera from four cattle experimentally infected with Brucella abortus strain 544 and four cattle experimentally infected with Y. enterocolitica O:9 (using samples taken on weeks 3, 7, 16, 24 and 53 post infection) were evaluated. To measure the reactions relative to other diagnostic antigens serological the tests were performed using several different antigens and the results for these are presented as line graphs: B. abortus S99 sLPS (
TABLE-US-00006 TABLE 6 Number of samples from Y. enterocolitica O:9 infected cattle with serological results greater than the lowest serological result from the samples from the B. abortus 544 infected cattle. No. of positive Y. enterocolitica Antigen Tip present/absent O:9 samples B. abortus S99 sLPS Present 15 Exclusively 1, 2 linked Present 11 hexasaccharide BSA conjugate (Structure IX) Exclusively 1, 2 linked Present 7 trisaccharide BSA conjugate (Structure XII) Monosaccharide BSA conjugate Present 4 (Structure II)
[0220] The results show that when the diagnostic antigens possess the tip epitope, the number of Y. enterocolitica O:9 positive samples decreases, and so the specificity increases, as the length of the antigen becomes smaller: sLPS>hexasaccharide>trisaccharide>monosaccharide. This occurs even though the terminal perosamine of the B. abortus, B. melitensis, B. suis and the Y. enterocolticia O:9 OPS is exactly the same. The shorter the (exclusively 1,2 linked) oligosaccharide, the greater the boost to specificity; this could not have been predicted.
[0221] A detailed evaluation of the serological results shows that although the results for the samples from the Y. enterocolitica O:9 infected animals are initially high against the exclusively 1,2 linked trisaccharide (Structure XII), they then fall rapidly for all four animals. By week 16 the results for all four animals remain lower than the lowest result for the Brucella infected animals. The results for the Brucella infected animals do not begin to fall significantly until after week 16, even then two animals remain high. The serological profiles obtained with the B. abortus S99 sLPS antigen (the antigen recommended by the OIE for Brucella iELISA) show the results from the Y. enterocolitica O:9 infected animals falling immediately in 3 out of 4 cases (but not as dramatically as is the case for the exclusively 1,2 linked trisaccharide) and with one sample, from Brucella infected animal number 2, becoming relatively low at week 53. The results for Y. enterocolitica O:9 infected animal number 2 increase to week 7 and stay high until week 24.
[0222] The results from the exclusively 1,2 linked hexsaccharide (Structure IX) antigen show attributes of both the trisaccharide (Structure XII) and S99 sLPS antigens as befitting its intermediate length. Although the results for the samples from the Y. enterocolitica O:9 infected animals fall relatively quickly the result for animal 2 increases from week 2 to 7. The results for the monosaccharide show a good distinction between the infection types although some of the results for the samples from the Brucella infected animals are quite low, which reflects the more limited sensitivity of this antigen.
[0223] The exclusively 1,2 linked trisaccharide (Structure XII), the B. abortus S99 sLPS and a mix (50/50 by mass) of the exclusively 1,2 linked trisaccharide (Structure XII) and the tetrasaccharide (Structure VI) antigens were tested against 29 serum samples from cattle field infected with B. abortus, 20 serum samples from randomly selected non-Brucella infected cattle, and 31 samples from cattle that are false positive to conventional Brucella serodiagnostic assays. The data is presented in 3 scatter plots: B. abortus S99 sLPS against exclusively 1,2 linked trisaccharide (Structure XII) (
[0224] The scatter plots (
TABLE-US-00007 TABLE 7 Specificity against FPSR population when the test positive/negative cut-off is adapted to different antigen sensitivities (number of positive samples shown in brackets) Diagnostic Exclusively 1, 2 Sensitivity B. abortus S99 sLPS linked trisaccharide Mix 100.0% 0.0% (0) 12.9% (4) 9.7% (3) 96.6% 16.1% (5) 38.7% (12) 35.5% (11) 93.1% 58.1% (18) 45.2% (14) 38.7% (12)
[0225] The results and conclusions from the field sera and from the experimentally infected sera are in agreement. At the highest levels of diagnostic sensitivity, the specificity obtained with the exclusively 1,2 linked trisaccharide (Structure XII) is superior to that obtained with the natural sLPS antigen (the current standard antigen). Lowering the sensitivity requirement leads to a superior performance from the sLPS although the sensitivity compromise is unfavourable. The data from the experimental infections suggests that the specificity of the different antigens depends upon how close to the point of infection with a cross-reacting organism, such as Y. enterocolitica O:9, the sample is taken.
[0226] Therefore, not only are the exclusively 1,2 linked trisaccharide (Structure XII), disaccharide (Structure XI), and the monosaccharide (Structure II) unexpectedly highly sensitive diagnostic sensitivity antigens for brucellosis but they also have unexpectedly high diagnostic specificity.
[0227] In the present example, serology was performed on samples taken from infected animals where the infective Brucella biovar is an A-dominant strain. Such infections would be expected to give rise to a greater proportion of antibodies that react to sequences of exclusively 1,2 linked perosamines (4,6-dideoxy-4-formamido-mannopyranosyl), rather than antibodies against sequences containing 1,3 linked perosamines. The ability of the exclusively 1,2 linked trisaccharide (Structure XII) and the specific M-antigen tetrasaccharide (Structure VI) antigens to detect anti-OPS antibodies induced by such infections has been shown above or previously (WO2014/170681). The antigens may be useful when used on their own their own or, as shown above, work well when used together.
[0228] When infections with M-dominant strains occur, then the antibodies induced would likely shift towards a higher proportion of antibodies against sequences of perosamines containing 1,3 linkages. Under such circumstances, the specific M-antigen tetrasaccharide (Structure VI) would be a more sensitive diagnostic. The use of the two antigens in combination (Structures XII or XI in combination with Structure VI, for example applied as a mix) gives optimal sensitivity under both scenarios, namely, infection with A-dominant or M-dominant strains of B. abortus, B. melitensis and B. suis.
[0229] In sum, the work described herein provides an antigen combination which is a universal antigen that is sensitive, DIVA-compatible, more specific than native antigens such as the OIE-recommended antigen B. abortus S99 sLPS, and is cheaper to produce and use than a longer synthetic “universal” antigen.
Methods Used for Example 8
[0230] The serological methods and the samples used are the same as described for Example 5 with the addition of the false positive serological reactor samples (FPSRs). These sera were collected from within Great Britain between 1996 and 1999, more than 10 years since the declaration of its officially brucellosis-free status. These sera were all positive for at least one of four conventional serodiagnostic assays for bovine brucellosis, CFT, SAT, cELISA, or iELISA, that are approved by the OIE. Other than serology, there was no cultural or epidemiological evidence of the disease.
APPENDIX: POLYSACCHARIDE SYNTHESIS METHODS
Synthesis of Heptasaccharide
[0231] The synthesis uses three key build blocks a known protected methyl glycoside S12. Two glycosyl donors 11 and 13 (below) are used to extend the 1,2 linked oligosaccharide, donor 11 and a capping residue 13 bearing a tether to conjugate to protein. Compound 11 allows the chain extension one residue at a time and the temporary acetate protecting group at 0-2 allows for easy removal revealing the hydroxyl group for further chain extension. The preformed capping residue with attached tether 13 is prepared from the known methyl 2,3-O-isopropylidene-6-deoxy-α-D-mannopyranoside S5 and the protected tether 12 which is in turn prepared from commercially available benzyl (5-hydroxypentyl) carbamate in a two-step conversion to S13 and then 12 (Scheme 4S). A series of transformation allows for the reaction of S5 with 12 and then further reactions provide the thioglycoside 13.
[0232] The detailed construction of these intermediates proceeds as described below
Synthesis of thioglycoside donors 11
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (S11)
[0233] Analytical data for the title compound was essentially the same as previously described (Bundle et al (1988) Carbohydr Res 174:239-251).
1,2-di-O-acetyl-4-azido-4,6-dideoxy-α-D-mannopyranose (S12)
[0234] A solution of S11 (5 g, 17.05 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 50 mL) was stirred at 21° C. for 3 h, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (80 mL).
[0235] The mixture was then diluted with CH.sub.2Cl.sub.2 (˜100 mL) and washed with water (2×30 mL), sat. aq. NaHCO.sub.3 (35 mL), and brine (15 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound S12 (5.6 g, 91%) as a sticky liquid. Analytical data for S12: Rf=0.35 (ethyl acetate/hexane, 1/4, v/v); [α]D.sup.21=+30.71 (c=1.51, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.8, 168.3, 136.9, 128.5, 128.3, 128.1, 91.0, 75.7, 71.8, 69.3, 66.3, 63.5, 20.8 (×2), 18.5 ppm; HRMS (ESI): m/z calcd for C.sub.17H.sub.21N.sub.3O.sub.6Na [M+Na]+: 386.1323, found: 386.1322.
p-Tolyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (11)
[0236] To the stirred solution of S12 (0.78 g, 2.15 mmol) and p-toluenethiol (0.4 g, 3.22 mmol) in anhydrous CH.sub.2Cl.sub.2 (15 mL) at 0° C., BF.sub.3.Et.sub.2O (0.32 mL, 2.57 mmol) was added drop wise. When TLC showed the reaction was completed, the mixture was then diluted with CH.sub.2Cl.sub.2 (˜50 mL) and washed with water (2×10 mL), sat. aq. NaHCO.sub.3 (15 mL), and brine (10 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (Ethyl acetate-hexane gradient elution) to give 11 as a sticky liquid (0.854 g, 92.9%). Analytical data for 11: Rf=0.7 (Ethyl acetate/hexane, 1/3, v/v); [α]D.sup.21=+135.5 (c=2.25, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 170.0, 138.1, 137.0, 132.4, 132.3, 129.9, 129.8, 129.6, 128.5, 128.5, 128.4, 128.1, 86.4, 76.4, 71.7, 69.1, 68.2, 64.2, 21.1, 21.0, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.22H.sub.25N.sub.3O.sub.4SNa [M+Na]+: 450.1458, found: 450.1465.
Synthesis of Linker Bromoalkane 12
5-(N-benzyl((benzyloxy)carbonyl)amino)pentanol benzoate (S13)
[0237] Benzoyl chloride (0.88 mL, 7.59 mmol) was added dropwise to a stirred solution of benzyl (5-hydroxypentyl) carbamate (commercially available) (1.5 g, 6.32 mmol) in anhydrous CH.sub.2Cl.sub.2 (15 mL) containing Et.sub.3N (1.76 mL, 1.26 mmol) at 0° C. After 1 minute DMAP (1.7 g, 13.9 mmol) in anhydrous CH.sub.2Cl.sub.2 (10 mL) was added dropwise to the reaction mixture and stirred at rt overnight. The resulting mixture was diluted with CH.sub.2Cl.sub.2 (˜30 mL) and washed with aq. HCl (1M, 1×10 mL), water (60 mL), sat. aq. NaHCO.sub.3 (30 mL), and brine (30 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was quickly filtered off on silica gel (ethyl acetate-hexane gradient elution) to afford the almost pure compound as oil. This crude material was directly used for benzylation.
[0238] To the solution of benzoyl protected compound (0.9 g, 2.63 mmol) dissolved in anhydrous DMF (10 mL) was added NaH (0.12 g, 2.89 mmol) at 0° C. The mixture was stirred at 0° C. for 45 min, and then BnBr (0.37 mL, 3.16 mmol) were added. After stirring for another 12 h when TLC showed that the reaction was completed, it was quenched with H.sub.2O at 0° C., and the mixture was diluted with EtOAc. The aqueous layer was extracted with EtOAc (5×25 mL), and the organic phases were combined and dried over Na.sub.2SO.sub.4. The desired product S13 (1.093 g, 96.1%) was obtained upon flash column chromatography (ethyl acetate-hexane gradient elution) of the condensed product. Analytical data for S13: Rf=0.6 (ethyl acetate/hexane, 1/3.5, v/v .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 166.6, 156.7, 156.2, 137.9, 136.8, 132.8, 130.4, 129.5, 128.5, 128.4, 128.3, 127.8, 127.3, 127.2, 67.2, 64.8, 64.7, 50.5, 50.2, 47.0, 46.0, 28.4, 27.8, 27.4, 23.3 ppm; HRMS (ESI): m/z calcd for C.sub.27H.sub.29NO.sub.4Na [M+Na]+: 454.1989, found: 454.1986.
Benzyl N-benzyl(5-bromopentanyl)carbamate (12)
[0239] Sodium methoxide (˜0.8 mL, 0.5 M solution) was added to a solution of S13 (1.0 g, 2.32 mmol) in CH.sub.3OH (15 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and this crude material was directly used for bromination. To the solution of deprotected compound (0.96 g, 2.92 mmol) dissolved in anhydrous CH.sub.2Cl.sub.2 (15 mL) were added CBr.sub.4 (1.85 g, 5.55 mmol) and PPh.sub.3 (1.54 g, 5.86 mmol) at 0° C. The reaction was allowed to warmup to room temperature and stirring for another 3 h. When TLC showed the reaction was completed, it was quenched with H.sub.2O at 0° C., mixture was then diluted with CH.sub.2Cl.sub.2 (˜50 mL) and washed with water (2×10 mL), sat. aq. NaHCO.sub.3 (15 mL), and brine (15 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound 12 (1.085 g, 94.8%) as a liquid. Analytical data for 12: Rf=0.85 (ethyl acetate/hexane, 1/4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 156.7, 156.2, 137.8, 136.7, 128.5 (×2), 128.4, 128.0, 127.9, 127.4, 127.3, 127.2, 67.3, 67.2, 50.6, 50.3, 46.9, 46.0, 33.6, 33.4, 32.3 (×2), 27.2, 26.8, 25.3 ppm; HRMS (ESI): m/z calcd for C.sub.20H.sub.24NO.sub.2BrNa [M+Na]+: 412.0883, found: 412.0878.
Synthesis of p-tolyl Thioglycoside Donor 13
[0240] ##STR00032##
[0241] Scheme 4S. Conditions: a) CbzBnN(CH.sub.2).sub.5Br, NaH, DMF, 0° C. to rt, 18 h; b) TFA/H.sub.2O (9:1), CH.sub.2Cl.sub.2, rt, 10 min; c) BzCl, DMAP, Et.sub.3N, CH.sub.2Cl.sub.2, 0° C. to rt, 12 h; d) Ac.sub.2O, AcOH, H.sub.2SO.sub.4, rt, 4 h; e) BF.sub.3:Et.sub.2O, p-Toluenethiol, CH.sub.2Cl.sub.2, 0° C. to rt, 10 h.
Methyl 2,3-O-isopropylidene-6-deoxy-α-D-mannopyranoside (S5)
[0242] Analytical data for the title compound was essentially the same as previously described (Eis & Ganem (1988) Carbohydrate Research 176:316-323).
Methyl 4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl) 2,3-O-isopropylidene-6-deoxy-α-D-mannopyranoside (S14)
[0243] To the solution of S5 (2.0 g, 9.17 mmol) dissolved in anhydrous DMF (15 mL) was added NaH (0.4 g, 10.08 mmol) at 0° C. The mixture was stirred at 0° C. for 45 min, and then CbzBnN(CH.sub.2).sub.5Br (4.5 g, 11.01 mmol) were added. After stirring for another 12 h when TLC showed that the reaction was completed, it was quenched with H.sub.2O at 0° C., and the mixture was diluted with EtOAc. The aqueous layer was extracted with EtOAc (5×25 mL), and the organic phases were combined and dried over Na.sub.2SO.sub.4. The desired product S14 (3.26 g, 73.2%) along with eliminated alkene and small amount unreacted starting material S5 (0.16 g) were obtained upon flash column chromatography (ethyl acetate-hexane gradient elution) of the condensed product. Analytical data for S14: Rf=0.6 (ethyl acetate/hexane, 1/4, v/v); [α]D.sup.21=+20.48 (c=2.11, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 156.7, 156.1, 137.9, 136.9, 136.8, 128.5, 128.4, 127.9, 127.8, 127.3, 127.2, 109.0, 98.0, 82.0, 78.5, 75.9, 71.3, 67.1, 64.5, 54.7, 50.4, 50.1, 47.1, 46.1, 29.8, 28.0, 27.9, 27.5, 26.3, 23.4, 17.7 ppm; HRMS (ESI): m/z calcd for C.sub.30H.sub.41NO.sub.7Na [M+Na]+: 550.2775, found: 550.2785.
Methyl 4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-α-D-mannopyranoside S15)
[0244] A solution of S14 (1.0 g, 1.89 mmol) in TFA:H.sub.2O (9:1, 10 mL) was stirred at 21° C. for 30 min, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (50 mL). The mixture was then diluted with CH.sub.2Cl.sub.2 (˜50 mL) and washed with water (2×30 mL), sat. aq. NaHCO.sub.3 (25 mL), and brine (15 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound S15 (0.742 g, 80.3%) as oil. Analytical data for S15: Rf=0.4 (ethyl acetate/hexane, 1/1, v/v); [α]D.sup.21=+38.31 (c=1.27, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 156.7, 156.3, 137.8, 136.6, 129.6, 128.5, 128.4, 127.9, 127.8, 127.3, 127.2, 100.3, 81.7, 71.4, 71.3, 71.2, 67.2, 67.1, 54.8, 50.5, 50.3, 47.1, 46.1, 30.0, 29.8, 27.9, 27.2, 23.2, 17.9 ppm; HRMS (ESI): m/z calcd for C.sub.27H.sub.37NO.sub.7Na [M+Na]+: 510.2462, found: 510.2462.
Methyl 2,3-di-O-benzoyl-4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-α-D-mannopyranoside (S16)
[0245] Benzoyl chloride (0.23 mL, 1.97 mmol) was added dropwise to a stirred solution of S15 (0.4 g, 0.82 mmol) in anhydrous CH.sub.2Cl.sub.2 (10 mL) containing Et.sub.3N (0.46 mL, 3.28 mmol) at 0° C. After 2 minute DMAP (0.451 g, 3.69 mmol) in anhydrous CH.sub.2Cl.sub.2 (5 mL) was added dropwise to the reaction mixture and stirred at rt overnight. The resulting mixture was diluted with CH.sub.2Cl.sub.2 (˜20 mL) and washed with aq. HCl (1M, 2×5 mL), water (20 mL), sat. aq. NaHCO.sub.3 (10 mL), and brine (10 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound S16 (0.513 g, 90%) as oil. Analytical data for S16: Rf=0.7 (ethyl acetate/hexane, 1/3.5, v/v); [α]D.sup.21=−70.58 (c=1.71, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 165.5, 165.2, 156.7, 156.3, 137.9, 133.3, 133.0, 129.9, 129.8, 129.8, 129.6, 128.5, 128.4, 128.3, 127.9, 127.8, 127.1, 98.5, 79.5, 73.1, 72.9, 72.1, 71.1, 67.6, 67.1, 55.0, 50.4, 50.1, 47.0, 46.0, 29.9, 27.8, 27.4, 23.3, 18.0 ppm; HRMS (ESI): m/z calcd for C.sub.41H.sub.45NO.sub.9Na [M+Na]+: 718.2987, found: 718.298.
1-O-acetyl-2,3-di-O-benzoyl-4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-α-D-mannopyranose (S17)
[0246] A solution of S16 (0.5 g, 0.716 mmol) in acetic anhydride/acetic acid/sulfuric acid (50:20:0.5, 10 mL) was stirred at 21° C. for 3 h, and then poured into ice-cold 1M K.sub.2CO.sub.3 solution (50 mL). The mixture was then diluted with CH.sub.2Cl.sub.2 (˜20 mL) and washed with water (2×30 mL), sat. aq. NaHCO.sub.3 (15 mL), and brine (10 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound S17 (0.485 g, 92.2%) as a liquid. Analytical data for S17: Rf=0.55 (ethyl acetate/hexane, 1/4, v/v); [α]D.sup.21=−48.86 (c=1.51, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 168.6, 165.4, 165.2, 137.8, 136.7, 133.5, 133.2, 129.8, 129.6, 129.4, 129.0, 128.5, 128.5, 128.4, 128.2, 127.9, 127.8, 127.2, 127.1, 90.8, 79.1, 73.4, 71.8, 70.1, 69.9, 67.1, 50.4, 50.1, 46.9, 45.9, 29.9, 27.8, 27.4, 23.3, 21.0, 18.1, ppm; HRMS (ESI): m/z calcd for C.sub.42H.sub.45NO.sub.10Na [M+Na]+: 746.2936, found: 746.2931.
p-Tolyl 2,3-di-O-benzoyl-4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-1-thio-α-D-mannopyranoside (13)
[0247] To the stirred solution of S17 (1.2 g, 1.66 mmol) and p-toluenethiol (0.312 g, 2.48 mmol) in anhydrous CH.sub.2Cl.sub.2 (20 mL) at 0° C., BF.sub.3. Et.sub.2O (0.25 mL, 1.99 mmol) was added drop wise. When TLC showed the reaction was completed, the mixture was then diluted with CH.sub.2Cl.sub.2 (˜30 mL) and washed with water (2×10 mL), sat. aq. NaHCO.sub.3 (10 mL), and brine (20 mL). The organic phase was separated, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate-hexane gradient elution) to give 13 as a white solid (1.18 g, 90.7%). Analytical data for 13: Rf=0.65 (ethyl acetate/hexane, 1/4, v/v); [α]D.sup.21=−1.02 (c=0.9, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 165.4, 165.3, 156.7, 156.1, 138.1, 137.9, 136.9, 136.8, 133.4, 133.2, 132.7, 132.3, 130.0, 129.9 (×2), 129.8, 129.7 (×2), 129.6, 128.5 (×2), 128.4 (×2), 127.9, 127.8, 127.3 (×2), 127.2, 86.2, 79.7, 73.3, 73.1, 72.6, 72.4 (×2), 69.2, 67.1, 50.5, 50.2, 47.0, 46.0, 30.0, 27.9, 27.4, 23.3, 21.2, 18.0 ppm; HRMS (ESI): m/z calcd for C.sub.47H.sub.49NO.sub.8SNa [M+Na]+: 810.3071, found: 810.3069.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside
[0248] Analytical data for the title compound was essentially the same as previously described (Bundle et al (1988) Carbohydr Res 174:239-251), (Eis & Ganem (1988) Carbohydrate Research 176:316-323).
Assembly of Heptasaccharide
[0249] Glycosylation of the methyl glycoside by the activated thioglycoside 11 provides disaccharide 14. This is subjected to a transesterification reaction to remove the acetate ester revealing the hydroxyl group for a repeated sequence of glycosylation and transesterification. This is repeated a further 4 times leading in turn to trisaccharide 16 and 17, tetrasaccharides 18 and 19, pentasaccharides 21 and 22, and hexasaccharides 22 and 23. Then in a final chain extension reaction the capping residue with tether is attached by reacting 13 with 23 to yield the heptasaccharide 24 and after removal of benzoate ester the partial deprotected alcohol 25. Deprotection is achieved in a series of steps involving reduction of azido groups to amine followed by their N-formylation and then a hydrogenolysis step to remove benzyl ethers and amino protecting groups (Ganesh et al (2014) Journal of the American Chemical Society 136:16260-16269). Compound 8 is then conjugated to protein by selective activation of the tether amino group with bis-succinimide ester (DSG) or dibutyl squarate to give the activated intermediates S26 and S27. S26 was reacted with tetanus toxoid to provide the vaccine glyconconjugate 9 and S27 was reacted with BSA to provide the screening antigen 10.
Methyl 4-azido-2-O-acetyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (14)
[0250] The glycosyl acceptor compound S11 (1.42 g, 4.84 mmol), and glycosyl donor compound 11 (2.27 g, 5.33 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (20 mL), treated with freshly activated 4 Å molecular sieves (1.5 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (2.4 g, 9.71 mmol). After cooling to −10° C., TMSOTf (0.19 mL, 0.971 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (15 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) and water. After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×15), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give disaccharide 14 (2.66 g, 92.1%) as a sticky liquid. Analytical data for 14: Rf=0.5 (Ethyl acetate/Hexane 1:4, v/v); [α]D.sup.21=+36.24° (c=1.92, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.7, 137.6, 137.1, 128.5 (×2), 128.4, 128.0, 127.9, 127.8, 99.7, 99.4, 77.7, 75.4, 73.7, 72.0, 71.6, 67.6, 67.2, 66.9, 64.1, 63.8, 54.9, 20.9, 18.5 (×2) ppm; HRMS (ESI): m/z calcd for C.sub.29H.sub.36N.sub.6O.sub.8Na [M+Na]+: 619.2487, found: 619.2481.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (15)
[0251] Sodium methoxide (˜1.2 mL, 0.5 M solution) was added to a solution of 14 (2.6 g, 4.36 mmol) in CH.sub.3OH: THF [4:2] (20 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to deprotected disaccharide compound 15 (2.3 g, 95.4%) as white foam. Analytical data for 15: Rf=0.4 (Ethyl acetate/Hexane 1:4.5, v/v); [α]D.sup.21=+28.71 (c=1.56, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 137.5, 137.1, 128.6, 128.5, 128.3, 128.2 (×2), 128.0, 100.8, 99.9, 77.8, 77.6, 73.6, 72.1 (×2), 67.3, 67.2, 66.9, 64.3, 63.8, 54.9, 18.6, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.27H.sub.34N.sub.6O.sub.7Na [M+Na]+: 577.2381, found: 577.2381.
Methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (16)
[0252] The glycosyl acceptor compound 15 (2.25 g, 4.06 mmol), and glycosyl donor compound 11 (1.90 g, 4.46 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (25 mL), treated with freshly activated 4 Å molecular sieves (1.6 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (1.83 g, 8.11 mmol). After cooling to −10° C., TMSOTf (0.16 mL 0.893 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (15 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) [30 mL] and water (20 mL). After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×15), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give trisaccharide 16 (3.09 g, 88.9%) as a sticky liquid. Analytical data for 16: Rf=0.65 (Ethyl acetate/Hexane 1:5, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.7, 137.4, 137.3, 137.1, 128.5 (×2), 128.4, 128.1, 128.0 (×3), 100.3, 99.8, 99.1, 77.5, 76.8, 75.4, 73.5, 72.1, 72.0, 71.5, 67.8, 67.6, 67.1, 67.0, 64.4, 64.0, 63.8, 54.9, 21.0, 18.6 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.42H.sub.51N.sub.9O.sub.11Na [M+Na]+: 880.36, found: 880.3607.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (17)
[0253] Sodium methoxide (˜1.5 mL, 0.5 M solution) was added to a solution of 16 (3.0 g, 3.5 mmol) in CH.sub.3OH: THF [4:2] (20 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to afford the deprotected trisaccharide compound 17 (2.6 g, 91.2%) as white solid foam. Analytical data for 17: Rf=0.45 (Ethyl acetate/Hexane 1:5, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 137.3 (×2), 137.2, 128.6 (×3), 128.5, 128.3, 128.3, 128.2 (×2), 128.1 (×2), 128.0, 100.5, 100.4, 99.8, 77.6, 77.5, 76.8, 73.6, 73.3, 72.2, 72.1 (×2), 67.8, 67.3, 67.1, 67.0, 64.4, 64.2, 63.8, 54.9, 18.6 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.40H.sub.49N.sub.9O.sub.10Na [M+Na]+: 383.3495, found: 838.3501.
Methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (18)
[0254] The glycosyl acceptor compound 17 (2.05 g, 2.51 mmol), and glycosyl donor compound 11 (1.18 g, 2.76 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (20 mL), treated with freshly activated 4 Å molecular sieves (1.2 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (1.13 g, 5.02 mmol). After cooling to −10° C., TMSOTf (0.1 mL, 0.553 mmol) was added and the reaction was allowed to warmup to room temperature.
[0255] When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (10 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) and water. After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×10), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give tetrasaccharide 18 (2.49 g, 87.8%) as a syrup. Analytical data for 18: Rf=0.5 (Ethyl acetate/Hexane 1:4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.8, 137.4, 137.3, 137.1 (×2), 128.6 (×2), 128.5 (×2), 128.4, 128.3, 128.2, 128.1, 128.0 (×3), 100.3, 100.1, 99.7, 99.1, 77.4, 76.6, 75.4, 73.6, 73.4 (×2), 72.2, 72.1, 72.0, 71.5, 67.8, 67.6, 67.1, 66.9, 64.3, 64.2, 64.0, 63.8, 54.9, 21.0, 18.6 (×2), 18.5, 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.55H.sub.66N.sub.12O.sub.14Na [M+Na]+: 1141.4714, found: 1141.473.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (19)
[0256] Sodium methoxide (˜1.2 mL, 0.5 M solution) was added to a solution of 18 (2.2 g, 1.95 mmol) in CH.sub.3OH: THF [4:2] (15 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to afford the title compound 19 (1.86 g, 88.7%) as white solid. Analytical data for 19: Rf=0.4 (Ethyl acetate/Hexane 1:4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 137.3 (×2), 137.1, 128.6 (×2), 128.5, 128.4, 128.3 (×2), 128.2 (×3), 128.1, 128.0, 100.4, 100.3, 100.2, 99.7, 77.7, 77.4, 76.6, 73.6, 73.5, 73.2, 72.2, 72.1 (×3), 67.8, 67.3, 67.1, 66.9, 64.3, 64.2 (×2), 63.8, 54.9, 18.6 (×2), 18.5, 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.53H.sub.64N.sub.12O.sub.13Na [M+Na]+: 1099.4608, found: 1099.4625.
Methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (20)
[0257] The glycosyl acceptor compound 19 (1.63 g, 1.51 mmol), and glycosyl donor compound 11 (0.712 g, 1.66 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (15 mL), treated with freshly activated 4 Å molecular sieves (1 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (0.681 g, 3.03 mmol). After cooling to −10° C., TMSOTf (0.06 mL, 0.33 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (10 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) [15 mL] and water (15 mL). After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×10), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give pentasaccharide 20 (1.92 g, 91.9%) as a sticky liquid. Analytical data for 20: Rf=0.7 (Ethyl acetate/Hexane 1:4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.8, 137.4, 137.3, 137.2, 137.1, 128.6 (×3), 128.5 (×2), 128.4, 128.3 (×2), 128.2, 128.1 (×2), 128.0 (×3), 100.3, 100.2, 100.0, 99.7, 99.1, 77.4, 76.6, 76.5, 75.4, 73.7, 73.6, 73.4, 73.3, 72.2 (×2), 72.1, 72.0, 71.5, 67.8 (×2), 67.6, 67.1, 66.9, 64.3, 64.2 (×2), 64.1, 63.8, 54.9, 21.0, 18.6 (×2), 18.5 (×2), 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.68H.sub.81N.sub.15O.sub.17Na [M+Na]+: 1402.5827, found: 1402.5856.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (21)
[0258] Sodium methoxide (˜1.2 mL, 0.5 M solution) was added to a solution of 20 (1.8 g, 1.31 mmol) in CH.sub.3OH:THF [4:2] (15 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to afford the title compound 21 (1.57 g, 89.8%) as white foam. Analytical data for 21: Rf=0.55 (Ethyl acetate/Hexane 1:4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 137.3 (×2), 137.2, 129.0, 128.6 (×4), 128.4, 128.3 (×4), 128.2 (×2), 128.1, 128.0, 100.5, 100.3, 100.2 (×2), 99.7, 77.7, 77.4, 77.0, 76.6, 76.5, 73.7, 73.6, 73.4, 73.2, 72.2, 72.1 (×3), 67.8 (×2), 67.3, 67.1, 66.9, 64.4, 64.2, 63.8, 54.9, 18.6 (×2), 18.5 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.66H.sub.79N.sub.15O.sub.16Na [M+Na]+: 1360.5721, found: 1360.5749.
Methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (22)
[0259] The glycosyl acceptor compound 21 (1.45 g, 1.08 mmol), and glycosyl donor compound 11 (0.556 g, 1.3 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (15 mL), treated with freshly activated 4 Å molecular sieves (1 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (0.488 g, 2.16 mmol). After cooling to −10° C., TMSOTf (43 μL, 0.24 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (10 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) [10 mL] and water (15 mL). After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×10), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give hexasaccharide 22 (1.601 g, 90.1%) as a sticky liquid. Analytical data for 22: Rf=0.65 (Ethyl acetate/Hexane 1:4, v/v); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 169.8, 137.4, 137.3, 137.2, 137.1 (×3), 128.6 (×4), 128.5 (×2), 128.4, 128.3 (×2), 128.2, 128.1 (×2), 128.0 (×3), 100.3, 100.1 (×2), 100.0, 99.7, 99.1, 77.4, 76.7 (×2), 76.5, 75.4, 73.6 (×2), 73.5, 73.4, 73.3, 72.2, 72.1, 72.0, 71.5, 67.8 (×4), 67.6, 67.1, 66.9, 64.3 (×2), 64.2 (×2), 64.1, 63.8, 54.9, 21.0, 18.6 (×2), 18.5 (×3), 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.81H.sub.96N.sub.18O.sub.20Na [M+Na]+: 1663.694, found: 1663.6982.
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (23)
[0260] Sodium methoxide (˜1.0 mL, 0.5 M solution) was added to a solution of 22 (1.3 g, 0.792 mmol) in CH.sub.3OH: THF [4:2] (15 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to afford the title compound 23 (1.17 g, 92.3%) as oil. Analytical data for 23: Rf=0.5 (Ethyl acetate/Hexane 1:4, v/v .sup.13C NMR (126 MHz, CDCl.sub.3): δ: 137.3, 137.2 (×2), 128.7 (×3), 128.6 (×2), 128.4 (×3), 128.3 (×2), 128.2, 128.1 (×3), 100.5, 100.3, 100.2 (×2), 100.1, 99.8, 77.7, 77.5, 76.6 (×2), 73.7, 73.6, 73.5 (×2), 73.3, 72.2 (×2), 72.1, 67.9, 67.8, 67.4, 67.2, 67.0, 64.4, 64.2, 63.9, 54.9, 18.7, 18.6 (×2), 18.5 (×2), 18.3 ppm; HRMS (ESI): m/z calcd for C.sub.79H.sub.94N.sub.18O.sub.19Na [M+Na]+: 1621.6835, found: 1621.688.
Methyl 2,3-di-O-benzoyl-4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (24)
[0261] The glycosyl acceptor compound 23 (0.270 g, 0.169 mmol), and glycosyl donor compound 13 (0.146 g, 0.186 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (10 mL), treated with freshly activated 4 Å molecular sieves (0.3 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (0.076 g, 0.337 mmol). After cooling to −10° C., TMSOTf (6.4 μL, 0.037 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (5 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) [10 mL] and water (10 mL). After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×5), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate/Hexane gradient elution) to give heptasaccharide 24 (0.334 g, 87.4%) as a sticky liquid. Analytical data for 24: Rf=0.65 (Ethyl acetate/Hexane 1:4, v/v); [α]D.sup.21=−6.71° (c=1.23, CHCl.sub.3); .sup.13C NMR (126 MHz, CDCl.sub.3): δ: 165.3, 165.1, 156.6, 156.1, 137.9, 137.5, 137.4, 137.3, 137.2 (×2), 136.8, 133.3, 133.0 (×2), 129.9, 129.8, 129.6, 129.1, 128.7 (×2), 128.6 (×2), 128.5 (×2), 128.4 (×2), 128.3 (×2), 128.2, 128.1, 127.9, 127.8, 127.3, 125.3, 100.4 (×3), 100.2 (×2), 99.8, 99.1, 79.8, 77.5, 76.7, 76.6, 73.7, 73.6, 73.5, 73.3, 72.2 (×2), 72.1 (×2), 71.9, 70.9, 68.5, 68.1, 67.9, 67.8, 67.1, 67.0, 64.4, 64.2, 63.9, 54.9, 50.5, 50.2, 47.0, 46.1, 29.7, 29.4, 27.8, 27.4, 23.3, 18.7, 18.6 (×3), 18.5 (×2), 18.0 ppm; HRMS (ESI): m/z calcd for C.sub.119H.sub.135N.sub.19P.sub.27Na [M+Na]+: 2284.9667, found: 2284.9732.
Methyl 4-O-(5′-N-benzyl-5′-N-carboxybenzyl-pentanyl)-6-deoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (25)
[0262] Sodium methoxide (˜0.2 mL, 0.5 M solution) was added to a solution of 24 (0.26 g, 0.115 mmol) in CH.sub.3OH: THF [2:3] (10 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (Ethyl acetate-Hexane gradient elution) to afford the title compound 25 (0.215 g, 91.2%) as oil. Analytical data for 25: Rf=0.25 (Ethyl acetate/Hexane 1:3.3, v/v); [α]D.sup.21=+79.2 (c=2.21, CHCl.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ: 156.7, 156.3, 137.8, 137.4, 137.3, 137.2 (×2), 137.1 (×2), 136.7, 129.0, 128.6 (×2), 128.5, 128.4, 128.3 (×2), 128.2, 128.1, 128.0, 127.9, 127.8, 127.3, 100.8, 100.4, 100.3, 100.2, 100.1 (×2), 99.7, 81.6, 77.4, 76.5, 73.6, 73.6, 73.5, 73.5, 72.9, 72.2, 72.1, 72.1, 72.0, 71.7, 71.1, 68.2, 67.8, 67.7, 67.2, 66.9, 64.3, 64.2, 54.9, 50.5, 50.3, 47.1, 46.1, 29.7, 29.4, 27.9, 27.2, 23.3, 18.6 (×2), 18.5 (×4), 17.9 ppm; HRMS (ESI): m/z calcd for C.sub.105H.sub.131N.sub.20O.sub.25 [M+NH.sub.4]+: 2071.9589, found: 2071.9639.
Methyl 4-O-(5′-aminopentanyl)-6-deoxy-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-a-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (8)
[0263] To a stirred solution of 25 (0.11 g, 0.054 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and continued stirring for 16 h. After that, argon was bubbled for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The mass spectrometry analysis showed completion of reaction to corresponding amine compound and no products arising from incomplete reduction. HRMS (ESI): m/z calcd for C.sub.105H.sub.140N.sub.7O.sub.25 [M+H]+: 1898.9893, found: 1898.99.
[0264] This crude material was directly used for formylation Amine compound in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to 21° C. After that, solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford heptasaccharide. The high resolution mass spectrometry analysis showed completion of formylation reaction. HRMS (ESI): m/z calcd for C.sub.111H.sub.139N.sub.7O.sub.31Na [M+Na]+: 2088.9408, found: 2088.9405.
[0265] Formylated compound was dissolved in CH.sub.3OH/H.sub.2O (2:1, 10 mL), Pd(OH).sub.2 on carbon (20%, 0.060 g) was added. Then it was stirred under a pressure of hydrogen gas at 21° C. for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound 8 (0.0427 g, 61.2%, over 3 steps) as white foam. Analytical data for 8: [α]D.sup.21=+42.44 (c=1.02, H.sub.2O); .sup.13C NMR (126 MHz, CDCl.sub.3): δ: 168.8 (×2), 168.6, 165.9 (×4), 165.7, 103.2, 103.1 (×4), 102.7, 102.5, 101.5 (×2), 100.4, 100.3, 81.8, 78.2, 78.1 (×3), 78.0 (×2), 77.9, 73.5 (×2), 71.3, 70.8 (×2), 69.2, 68.7 (×2), 68.5, 68.4, 67.8, 57.9, 56.4, 55.9, 55.8 (×2), 52.9 (×2), 52.7 (×2), 40.4, 29.7, 27.5, 23.2, 17.9 (×2), 17.8 (×2), 17.7, 17.6 (×4) ppm; HRMS (ESI): m/z calcd for C.sub.54H.sub.92N.sub.7O.sub.29 [M+H]+: 1302.5934, found: 1302.5928.
Methyl 4-O-(5′-[N-succinimidyl]glutarylamidopentanyl)-6-deoxy-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (S26)
[0266] A mixture of heptasaccharide 8 (9 mg) and disuccinimidal glutarate (15 eq.) in DMF and 0.1 M PBS buffer (4:1, 1.5 mL) was stirred at rt for 6 h. The reaction mixture was concentrated under vacuum and the residue was washed with EtOAc 10 times to remove the excess disuccinimidal glutarate. The resultant solid was dried under vacuum for 1 h to obtain activated oligosaccharide S26 that was directly used for conjugation with BSA & tetanus toxoid. MALDI TOF MS (positive mode): calcd for C.sub.63H.sub.100N.sub.8O.sub.34Na [M+Na]+ m/z, 1535.6342; found, 1535.9996.
1-[(2′-Aminoethylamido)carbonylpentyl)-6-deoxy-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside] 2-butoxycyclobutene-3,4-dione (S27)
[0267] To a stirred solution of heptasaccharide 8 (0.006 g, 0.005 mmol) in water (0.5 mL) and EtOH (0.5 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 50 μL) was added and pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 1 h, mass spectrometry showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound S27 (0.005 g, 72.6%) as white foam. Analytical data for S27: .sup.13C NMR (126 MHz, CDCl.sub.3): δ: 190.3, 184.2, 183.8, 178.4, 178.0, 174.3, 168.8, 168.7, 168.6, 165.9, 165.7, 103.2, 103.2 (×4), 102.8, 102.6, 101.6, 100.4, 100.3, 81.9, 78.2, 78.1 (×2), 78.0 (×2), 77.9, 73.4, 71.3, 70.7, 69.8, 69.2, 69.1, 68.8, 68.6, 68.5, 68.4, 57.9, 56.4, 55.9, 55.8, 52.9 (×2), 52.7, 40.4, 32.4, 30.7, 30.5, 29.7, 27.5, 23.3, 23.2, 19.2, 19.0, 17.9, 17.8 (×2), 17.7 (×4), 17.6, 13.9 ppm; HRMS (ESI): m/z calcd for C.sub.62H.sub.99N.sub.7O.sub.32Na [M+Na]+: 1476.6335, found: 1476.6406.
[0268] Oligosaccharide Protein Conjugation:
[0269] Preparation of tetanus toxoid conjugate 9 (Structure XVI): Activated heptasaccharide S26 (0.8 mg, 0.518 μmol) was added to the solution of tetanus toxoid (4 mg, 0.026 μmol) in 0.5 M borate buffer pH 9 (1 mL) and stirred slowly at 21° C. for 3 days. Then the reaction mixture was washed with PBS buffer, filtered through millipore filtration tube (10,000 MWCO, 4×10 mL) and the resulting tetanus toxoid-conjugate 9 was stored in PBS buffer. The MALDI-TOF mass spectrometry analysis indicated the conjugate 9 had an average of 10.02 heptasaccharide per tetanus toxoid.
[0270] Preparation of BSA conjugate 10 (Structure XVII): BSA (10 mg) and activated heptasaccharide S27 (4.5 mg) were dissolved in 0.1 M PBS buffer pH 9 (1.2 mL) and stirred slowly at 21° C. for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through millipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 10 was obtained as a white foam (12.2 mg). The MALDI-TOF mass spectrometry analysis indicated the conjugate 10 had an average of 10.27 heptasaccharide per BSA.
Synthesis of exclusively 1,2-linked trisaccharide
Ethyl 4-azido-2,3-di-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1→2) 4-azido-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (S18)
[0271] Analytical data for the title compound was essentially the same as previously described (Bundle et al (1988) Carbohydr. Res. 174, 239-251).
5′-Methoxycarbonylpentyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (S19)
[0272] Analytical data for the title compound was essentially the same as previously described (Ganesh et al (2014) J. Amer. Chem. Soc. 136,16260-16269.
5′-Methoxycarbonylpentyl 4-azido-2,3-O-benzoyl-4,6-dideoxy-α-D-mannopyranosyl (1->2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1->2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (S20)
[0273] The glycosyl acceptor compound S19 (0.2 g, 0.491 mmol), and glycosyl donor compound S18 (0.414 g, 0.589 mmol), were combined, azeotroped twice with anhydrous toluene (5 mL), and placed under high vacuum for 2 h. The mixture was then dissolved in CH.sub.2Cl.sub.2 (15 mL), treated with freshly activated 4 Å molecular sieves (1 g), stirred under an Ar atmosphere at rt for 1 h. To the mixture was added NIS (0.221 g, 0.982 mmol). After cooling to −10° C., TMSOTf (19.5 μL, 0.108 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO.sub.3 (5 mL) and CH.sub.2Cl.sub.2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na.sub.2S.sub.2O.sub.3 (20%) and water. After extraction of the aqueous layer with CH.sub.2Cl.sub.2 (3×5), the combined organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuum, and purified by silica gel column chromatography (ethyl acetate-Hexane gradient elution) to give disaccharide S20 (0.418 g, 81.3%) as a sticky liquid. Analytical data for S20: Rf=0.7 (ethyl acetate/Hexane 1:4.5, v/v); [α]D.sup.21=−14.49° (c=1.79, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.02-8.05 (m, 2H, ArH), 7.95-7.97 (m, 2H, ArH), 7.64-7.68 (m, 1H, ArH), 7.50-7.57 (m, 3H, ArH), 7.33-7.41 (m, 8H, ArH), 7.22-7.26 (m, 3H, ArH), 7.13-7.17 (m, 1H, ArH), 5.71 (dd, J=3.3, 1.5 Hz, 1H, H-2.sub.C), 5.59 (dd, J=10.3, 3.3 Hz, 1H, H-3.sub.C), 5.06 (d, J=1.8 Hz, 1H, H-1.sub.B), 5.02 (d, J=1.8 Hz, 1H, H-1.sub.C), 4.76 (d, J=11.7 Hz, 1H, CHPh), 4.62-4.69 (m, 4H, 3 CHPh, H-1.sub.A), 3.95 (dd, J=2.2, 0.7 Hz, 1H, H-2.sub.B), 3.90 (dd, J=2.2, 0.7 Hz, 1H, H-2.sub.A), 3.76-3.81 (m, 2H, H-3.sub.B, H-5.sub.B), 3.74 (dd, J=9.9, 2.9 Hz, 1H, H-3.sub.A), 3.71 (s, 3H), 3.69 (t, J=9.9 Hz, 1H, H-4.sub.C), 3.55-3.65 (m, 3H, H-4.sub.B, H-5.sub.C, —O—CH.sub.2b), 3.43-3.49 (m, 1H, H-5.sub.A), 3.38 (dt, J=9.7, 6.4 Hz, 1H, —O—CH.sub.2a), 3.27 (t, J=9.9 Hz, 1H, H-4.sub.A), 2.33-2.39 (m, 2H, —CH.sub.2f), 1.64-1.72 (m, 2H, —CH.sub.2e), 1.56-1.64 (m, 2H, —CH.sub.2c), 1.35-1.42 (m, 2H, —CH.sub.2d), 1.38 (d, J=5.6 Hz, 3H, H-6.sub.C), 1.32 (d, J=5.9 Hz, 3H, H-6.sub.B), 1.29 (d, J=5.9 Hz, 3H, H-6.sub.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 165.2, 164.9, 137.5, 137.3, 133.4, 133.3, 129.8 (×2), 129.6, 129.3, 128.5 (×2), 128.4, 128.2, 128.1 (×2), 128.0, 100.3, 99.0, 98.8, 77.9, 73.9, 73.5, 72.3 (×2), 70.9, 69.5, 68.0, 67.5, 67.2, 64.5, 63.9, 63.5, 51.5, 34.0, 29.1, 25.7, 24.7, 18.6 (×2), 18.4 ppm; HRMS (ESI): m/z calcd for C.sub.53H.sub.61N.sub.9O.sub.14Na [M+Na]+: 1070.423, found: 1070.4248.
5′-Methoxycarbonylpentyl 4-azido-4,6-dideoxy-α-D-mannopyranosyl (1->2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl (1->2) 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (S21)
[0274] Sodium methoxide (˜0.3 mL, 0.5 M solution) was added to a solution of S20 (0.39 g, 0.372 mmol) in CH.sub.3OH: THF [4:2] (12 mL) until pH˜9 and the resulting mixture was stirred under argon for 6 h at 21° C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH.sub.3OH. The combined filtrate was concentrated in vacuo and purified by column chromatography on silica gel (ethyl acetate-Hexane gradient elution) to afford the title compound S21 (0.299 g, 95.6%) as white solid. Analytical data for S21: Rf=0.3 (ethyl acetate/Hexane 1:1.5, v/v); [α]D.sup.21=+84.18 (c=1.55, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.30-7.44 (m, 10H, ArH), 5.00 (d, J=1.8 Hz, 1H, H-1.sub.B), 4.90 (d, J=1.5 Hz, 1H, H-1.sub.C), 4.72 (d, J=11.4 Hz, 1H, CHPh), 4.61-4.67 (m, 4H, 3 CHPh, H-1.sub.A), 3.93-3.97 (m, 2H, H-2.sub.B, H-2.sub.C), 3.81-3.87 (m, 2H, H-2.sub.A, H-3.sub.A), 3.76 (dd, J=9.9, 2.9 Hz, 1H, H-3.sub.B), 3.73 (dd, J=10.0, 2.9 Hz, 1H, H-3.sub.C), 3.70 (s, 3H), 3.51-3.64 (m, 3H, H-5.sub.B, H-5.sub.C, —O—CH.sub.2b), 3.43-3.49 (m, 1H, H-5.sub.A), 3.40 (t, J=9.9 Hz, 1H, H-4.sub.C), 3.36 (dt, J=9.7, 6.4 Hz, 1H, —O—CH.sub.2a), 3.27 (t, J=9.9 Hz, 1H, H-4.sub.B), 3.40 (t, J=10.2 Hz, 1H, H-4.sub.A), 2.49 (d, J=6.9 Hz, 1 OH.sub.3C), 2.34 (t, J=7.4 Hz, 2H, —CH.sub.2f), 2.18 (d, J=3.9 Hz, 1 OH.sub.2C), 1.63-1.70 (m, 2H, —CH.sub.2e), 1.54-1.61 (m, 2H, —CH.sub.2c), 1.33-1.40 (m, 2H, —CH.sub.2d), 1.30 (d, J=6.2 Hz, 6H, H-6.sub.B, H-6.sub.C), 1.20 (d, J=6.2 Hz, 3H, H-6.sub.A); .sup.13C NMR (126 MHz, CDCl.sub.3): δ 174.0, 137.4 (×2), 128.6 (×2), 128.3, 128.2 (×2), 128.1, 100.7, 100.4, 98.7, 77.7, 77.2, 77.2, 73.8, 73.2, 72.3, 72.2, 70.2, 69.9, 67.8, 67.5, 67.4, 67.1, 65.8, 64.4, 64.2, 51.6, 33.9, 29.1, 25.7, 24.7, 18.6 (×2), 18.2 ppm; HRMS (ESI): m/z calcd for C.sub.39H.sub.53N.sub.9O.sub.12Na [M+Na]+: 862.3706, found: 862.3705.
5′-Methoxycarbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (4)
[0275] To a stirred solution of S21 (0.2 g, 0.239 mmol), in pyridine (5 mL) and water (2 mL) mixture, H.sub.2S was bubbled for 0.5 h at 40° C., and continued stirring for 16 h. After that, argon was bubbled for 10 min, solvents were removed in vacuo, and the residue was co-evaporated with toluene (3×10 mL) and dried. The mass spectrometry analysis showed completion of reaction to corresponding amine compound and no products arising from incomplete reduction.
[0276] This crude material was directly used for formylation Amine compound in CH.sub.3OH (5 mL) at −20° C. was added a freshly prepared formic anhydride (5 mL, ethereal solution) and stirred for 3 h, then slowly allowed to warm to 21° C. After that, solvents were evaporated and the residue was passed through column chromatography on silica gel (methanol-dichloromethane gradient elution) to afford trisaccharide. The high resolution mass spectrometry analysis showed completion of formylation reaction. HRMS (ESI): m/z calcd for C.sub.42H.sub.59N.sub.3O.sub.15Na [M+Na]+: 868.3838, found: 868.3837.
[0277] Formylated compound was dissolved in CH.sub.3OH/H.sub.2O (2:1, 15 mL), Pd(OH).sub.2 on carbon (20%, 0.090 g) was added. Then it was stirred under a pressure of hydrogen gas at 21° C. for 16 h. After filtration through celite pad and washed with CH.sub.3OH (3×10 mL), and solvents were removed in vacuo. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound 4 (0.094 g, 59.3%, over 3 steps) as white foam. Analytical data for 4: [α]D.sup.21=+31.58 (c=1.16, H.sub.2O); .sup.1H NMR (700 MHz, D.sub.2O): δ 8.20-8.24 (Z) and 8.03-8.06 07 (E) (m, 3H, NCHO), 5.16-5.22 (m, 1H, H-1.sub.B), 5.05-5.08 (m, 1H, H-1.sub.C), 4.89-4.93 (m, 1H, H-1.sub.A), 4.13-4.19 (m, 1H, H-2.sub.B), 4.06-4.13 (m, 2H, H-2.sub.C, H-3.sub.C), 3.92-4.03 (m, 6H, H-2.sub.A, H-3.sub.A, H-3.sub.B, H-4.sub.C, H-4.sub.B, H-4.sub.A), 3.87-3.92 (m, 2H, H-5.sub.A, H-5.sub.C), 3.80-3.84 (m, 1H, H-5.sub.B), 3.71-3.75 (m, 1H, —O—CH.sub.2b), 3.71 (s, 3H), 3.56 (dt, J=9.9, 5.9 Hz, 1H, —O—CH.sub.2a), 2.42 (t, J=7.4 Hz, 2H, —CH.sub.2f), 1.60-1.68 (m, 4H, —CH.sub.2e, —CH.sub.2c), 1.40 (dq, J=14.8, 7.3 Hz, 2H, —CH.sub.2d), 1.20-1.30 (m, 9H, 3×H-6); .sup.13C NMR (176 MHz, D.sub.2O): δ 178.4, 168.6 (×2), 165.7, 165.7 (×2), 102.9, 102.8, 101.5, 99.1, 78.5, 78.4, 78.2, 78.1, 78.0, 69.8, 69.1, 68.8, 68.7 (×2), 68.6, 68.5 (×2), 68.3 (×2), 67.9, 57.8, 52.9, 52.8, 52.7 (×2), 52.5, 34.4 (×2), 28.9, 25.7, 24.8, 17.8 (×2), 17.7 (×2), 17.6, 17.5 (×2) ppm. HRMS (ESI): m/z calcd for C.sub.28H.sub.47N.sub.3O.sub.15Na [M+Na]+: 688.2899, found: 688.2908.
(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside (S24)
[0278] A solution of 4 (0.06 g, 0.09 mmol) in freshly distilled 1,2-diaminoethane (3.0 mL) was stirred at 65° C. for 48 h. After that, excess reagent was removed in vacuo, and the residue was co-evaporated with CH.sub.3OH (3×10 mL) and dried. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound S24 (0.052 g, 83.15%) as white foam. Analytical data for S24: [α]D.sup.21=+37.05 (c=1.14, H.sub.2O); .sup.1H NMR (500 MHz, D.sub.2O): δ 8.24-8.33 (Z) and 8.05-8.12 (E) (m, 3H, NCHO), 5.23-5.26 (m, 1H, H-1.sub.B), 5.12 (s, 1H, H-1.sub.C), 4.93-4.97 (m, 1H, H-1.sub.A), 4.19-4.24 (m, 1H, H-2.sub.B), 4.10-4.18 (m, 2H, H-2.sub.C, H-3.sub.C), 3.96-4.08 (m, 6H, H-2.sub.A, H-3.sub.A, H-3.sub.B, H-4.sub.C, H-4.sub.B, H-4.sub.A), 3.91-3.96 (m, 2H, H-5.sub.A, H-5.sub.C), 3.84-3.89 (m, 1H, H-5.sub.B), 3.77 (dt, J=9.7, 6.8 Hz, 1H, —O—CH.sub.2b), 3.57-3.63 (m, 1H, —O—CH.sub.2a), 3.33 (t, J=6.2 Hz, 2H, —CH.sub.2g), 2.82 (t, J=6.2 Hz, 2H, —CH.sub.2h), 2.33 (t, J=7.4 Hz, 2H, —CH.sub.2f), 1.64-1.74 (m, 4H, —CH.sub.2e, —CH.sub.2c), 1.39-1.49 (m, 2H, —CH.sub.2d), 1.25-1.35 (m, 9H, 3×H-6); .sup.13C NMR (126 MHz, D.sub.2O): δ 178.3, 168.8 (×2), 165.8 (×2), 103.0, 102.9, 101.6, 99.3, 78.6, 78.3, 78.2, 78.1, 69.9, 69.2, 69.0, 68.9, 68.8 (×2), 68.6 (×2), 68.5, 68.4, 57.7, 53.0, 52.8 (×2), 52.7, 42.1, 42.1, 40.7, 36.7, 29.1, 26.0, 25.9, 17.9 (×2), 17.8 (×2), 17.7 (×2), 17.6 ppm; HRMS (ESI): m/z calcd for C.sub.29H.sub.51N.sub.5O.sub.14Na [M+Na]+: 716.3325, found: 716.333.
1-[(2′-Aminoethylamido)carbonylpentyl 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranosyl (1→2) 4,6-dideoxy-4-formamido-α-D-mannopyranoside] 2-butoxycyclobutene-3,4-dione (S25)
[0279] To a stirred solution of S24 (0.015 g, 0.022 mmol) in water (0.5 mL) and EtOH (0.5 mL), a solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (20% in ethanol, 70 μL) was added and pH was adjusted to 8 by careful addition of aq.NaHCO.sub.3 (1%) solution. After 1 h, mass spectrometry showed the reaction was complete; the reaction mixture was neutralized using CH.sub.3COOH (10%) and concentrated in vacuo. The residue was purified by reversed phase HPLC on C18 column in gradient water-acetonitrile and lyophilized, to give the title compound S25 (0.0133 g, 73.2%) as white foam. Analytical data for S25: .sup.1H NMR (700 MHz, D.sub.2O): δ 8.21-8.23 (Z) and 8.05 (E) (m, 3H, NCHO), 5.19 (s, 1H, H-1.sub.B), 5.07 (s, 1H, H-1.sub.C), 4.90-4.92 (m, 1H, H-1.sub.A), 4.68-4.75 (m, 2H, —CH.sub.2i), 4.14-4.19 (m, 1H, H-2.sub.B), 4.07-4.13 (m, 2H, H-2.sub.C, H-3.sub.C), 3.92-4.02 (m, 6H, H-2.sub.A, H-3.sub.A, H-3.sub.B, H-4.sub.C, H-4.sub.B, H-4.sub.A), 3.89 (m, 2H, H-5.sub.A, H-5.sub.C), 3.79-3.85 (m, 1H, H-5.sub.B), 3.73 (t, J=5.0 Hz, 1H, —CH.sub.2g), 3.65-3.71 (m, 1H, —O—CH.sub.2b), 3.62 (t, J=5.0 Hz, 1H, —CH.sub.2g), 3.51 (dd, J=9.6, 6.5 Hz, 1H, —O—CH.sub.2a), 3.40-3.45 (m, 2H, —CH.sub.2b), 2.19-2.27 (m, 2H, —CH.sub.2f), 1.77-1.84 (m, 2H, —CH.sub.2j), 1.51-1.64 (m, 4H, —CH.sub.2e, —CH.sub.2c), 1.46 (dt, J=15.5, 7.9 Hz, 2H, —CH.sub.2k), 1.30-1.36 (m, 2H, —CH.sub.2d), 1.20-1.30 (m, 9H, 3×H-6), 0.94-0.98 (m, 3H, —CH.sub.2l); .sup.13C NMR (176 MHz, D.sub.2O): δ 189.7, 184.1, 178.4, 177.8, 174.5, 168.6, 165.7, 165.7, 102.8, 101.5, 99.1, 98.9, 78.4, 78.1, 75.2, 75.1, 69.8, 69.1, 68.8, 68.7, 68.6, 68.4, 68.3 (×2), 57.8, 52.9, 52.7, 52.5, 45.0, 44.9, 40.2, 40.0, 36.6, 32.3, 29.1, 26.0, 25.9, 25.8, 25.7, 19.0, 18.9, 17.8 (×2), 17.7 (×2), 17.6, 17.5, 13.8 ppm; HRMS (ESI): m/z calcd for C.sub.37H.sub.59N.sub.5O.sub.17Na [M+Na]+: 868.3798, found: 868.3808.
[0280] Preparation of BSA conjugate 5: BSA (15 mg) and trisaccharide squarate S25 (3.8 mg, 6.77 μmol) were dissolved in 0.1 M PBS buffer pH 9 (600 μL) and stirred slowly at 21° C. for 3 days. Then the reaction mixture was diluted with Mili-Q water, filtered through millipore filtration tube (10,000 MWCO, 4×10 mL), lyophilized and the BSA-conjugate 5 was obtained as a white foam (17.6 mg). The MALDI-TOF mass spectrometry analysis indicated the conjugate 5 had an average of 16.2 disaccharides per BSA.