Many children suffering from autism spectrum disorder (ASD) experience gastrointestinal (GI) conditions. Enterocloster bolteae has been regularly detected in the stool of individuals suffering from GI symptoms and autism. Literature has suggested that E. bolteae strains WAL 16351 and WAL 14578 produce an immunogenic capsular polysaccharide (CPS) comprised of disaccharide repeating units: α-D-Man-(1→4)-β-Rha-(1→3) that could be used for the development of an immunotherapeutic vaccine. Ambiguity in the configuration of rhamnose led to the synthesis of tri- and disaccharide analogues containing D-rhamnose and L-rhamnose, respectively. ROESY-NMR spectra showed that CH3-6 of rhamnose and H-2 of mannose in the L-Rha containing disaccharide gave correlation. No such correlation was seen between the CH3-6 of rhamnose and the H-2 of mannose in the D-Rha containing trisaccharide. Molecular dynamics studies on hexasaccharide containing L-Rha or D-Rha confirmed that these structures adopt conformations resulting in different distances between the C6-rhamnose and the H-2 mannose of the preceding residue. We also demonstrate that assignment of the absolute configuration of the rhamnosyl residue in the β-Rhap-(1→3)-D-Man linkage can be determined using the 13C chemical shift of C-2 in of D-Mannose. While β-D-Rha will lead to an upfield shift of C-2 due to γ-gauche interaction between H-1 Rha and H-2 Man, β-L-Rha will not. Our results provide insights to distinguish between D- and L-rhamnose in the α-D-Manp-(1→4)-β-Rhap-(1→3) repeating motif.

NMR, conformation, synthesis, structure, D-mannose, capsular polysaccharide, molecular dynamics, L-rhamnose, vaccine, D-rhamnose

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@@H](C)O[C@@H]([*])[C@@H](O)[C@H]2O)[C@H]1O

308.283 g/mol (C₁₂H₂₀R₂O₉, R = next and previous repeats, not counted in mol weight)

Many children suffering from autism spectrum disorder (ASD) experience gastrointestinal (GI) conditions. Enterocloster bolteae has been regularly detected in the stool of individuals suffering from GI symptoms and autism. Literature has suggested that E. bolteae strains WAL 16351 and WAL 14578 produce an immunogenic capsular polysaccharide (CPS) comprised of disaccharide repeating units: α-D-Man-(1→4)-β-Rha-(1→3) that could be used for the development of an immunotherapeutic vaccine. Ambiguity in the configuration of rhamnose led to the synthesis of tri- and disaccharide analogues containing D-rhamnose and L-rhamnose, respectively. ROESY-NMR spectra showed that CH3-6 of rhamnose and H-2 of mannose in the L-Rha containing disaccharide gave correlation. No such correlation was seen between the CH3-6 of rhamnose and the H-2 of mannose in the D-Rha containing trisaccharide. Molecular dynamics studies on hexasaccharide containing L-Rha or D-Rha confirmed that these structures adopt conformations resulting in different distances between the C6-rhamnose and the H-2 mannose of the preceding residue. We also demonstrate that assignment of the absolute configuration of the rhamnosyl residue in the β-Rhap-(1→3)-D-Man linkage can be determined using the 13C chemical shift of C-2 in of D-Mannose. While β-D-Rha will lead to an upfield shift of C-2 due to γ-gauche interaction between H-1 Rha and H-2 Man, β-L-Rha will not. Our results provide insights to distinguish between D- and L-rhamnose in the α-D-Manp-(1→4)-β-Rhap-(1→3) repeating motif.

NMR, conformation, synthesis, structure, D-mannose, capsular polysaccharide, molecular dynamics, L-rhamnose, vaccine, D-rhamnose

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,3,4	aDManp	104.1	73.1	76.1	69.4	76.4	63.8
1,3	bDRhap	99.9	74.3	73.1	81.9	73.6	20.5
1	aDManp	103.5	69.9	80.4	68.0	75.1	63.7
	Me


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,3,4	aDManp	5.29	4.05	3.80	3.67	3.80	3.80-3.87
1,3	bDRhap	4.77	4.01	3.77	3.58	3.51	1.37
1	aDManp	4.81	4.12	3.96	3.76	3.64	3.77-3.90
	Me


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,3,4	aDManp	104.1/5.29	73.1/4.05	76.1/3.80	69.4/3.67	76.4/3.80	63.8/3.80-3.87
1,3	bDRhap	99.9/4.77	74.3/4.01	73.1/3.77	81.9/3.58	73.6/3.51	20.5/1.37
1	aDManp	103.5/4.81	69.9/4.12	80.4/3.96	68.0/3.76	75.1/3.64	63.7/3.77-3.90
	Me

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@H](O)[C@@H]2O)[C@@H]1O

502.466 g/mol (C₁₉H₃₄O₁₅)

Many children suffering from autism spectrum disorder (ASD) experience gastrointestinal (GI) conditions. Enterocloster bolteae has been regularly detected in the stool of individuals suffering from GI symptoms and autism. Literature has suggested that E. bolteae strains WAL 16351 and WAL 14578 produce an immunogenic capsular polysaccharide (CPS) comprised of disaccharide repeating units: α-D-Man-(1→4)-β-Rha-(1→3) that could be used for the development of an immunotherapeutic vaccine. Ambiguity in the configuration of rhamnose led to the synthesis of tri- and disaccharide analogues containing D-rhamnose and L-rhamnose, respectively. ROESY-NMR spectra showed that CH3-6 of rhamnose and H-2 of mannose in the L-Rha containing disaccharide gave correlation. No such correlation was seen between the CH3-6 of rhamnose and the H-2 of mannose in the D-Rha containing trisaccharide. Molecular dynamics studies on hexasaccharide containing L-Rha or D-Rha confirmed that these structures adopt conformations resulting in different distances between the C6-rhamnose and the H-2 mannose of the preceding residue. We also demonstrate that assignment of the absolute configuration of the rhamnosyl residue in the β-Rhap-(1→3)-D-Man linkage can be determined using the 13C chemical shift of C-2 in of D-Mannose. While β-D-Rha will lead to an upfield shift of C-2 due to γ-gauche interaction between H-1 Rha and H-2 Man, β-L-Rha will not. Our results provide insights to distinguish between D- and L-rhamnose in the α-D-Manp-(1→4)-β-Rhap-(1→3) repeating motif.

NMR, conformation, synthesis, structure, D-mannose, capsular polysaccharide, molecular dynamics, L-rhamnose, vaccine, D-rhamnose

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4	aDManp	104.3	73.2	73.3	69.4	76.0	63.5
	bLRhap	90.6	74.5	72.5	84.4	71.5	19.5


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4	aDManp	5.00	4.03	3.85	3.74	3.97	3.80-3.88
	bLRhap	5.45	4.22	3.94	3.63	4.30	1.28


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4	aDManp	104.3/5.00	73.2/4.03	73.3/3.85	69.4/3.74	76.0/3.97	63.5/3.80-3.88
	bLRhap	90.6/5.45	74.5/4.22	72.5/3.94	84.4/3.63	71.5/4.30	19.5/1.28

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[C@@H]1O[C@H](O)[C@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O

326.298 g/mol (C₁₂H₂₂O₁₀)