One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of (1)H-(1)H and (1)H-(13)C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo.

NMR, core, O-antigen, Kdo, Salmonella, MS

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
5,3,3,3,2,2,2	Ac	174.6	23.2
5,3,3,3,2,2	aDGlcpN	94.4	54.4	72.4	71.2	73.2	61.8
5,3,3,3,2	aDGlcp	91.7	74.3	72.5	70.2	73.0	61.4
5,3,3,3	aDGalp	96.4	71.4	69.1	70.0	71.1	61.8
5,3,3,6	aDGalp	99.6	69.3	70.7	70.2	71.9	?
5,3,3	aDGlcp	101.8	71.3	77.9	72.3	72.1	67.8
5,3,7	a?Hepp	101.3	70.7	81.0	66.3	73.6	?	?
5,3	a?Hepp	103.3	70.7	81.0	66.3	73.6	?	70.4
5	a?Hepp	101.0	71.1	80.4	66.4	?	?	?
	aXKdop	176.5	97.3	35.1	67.0	76.1	?	?	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
5,3,3,3,2,2,2	Ac	-	2.09
5,3,3,3,2,2	aDGlcpN	5.11	3.99	3.84	3.50	3.92	3.77
5,3,3,3,2	aDGlcp	5.60	3.74	3.91	3.51	3.72	3.77-3.88
5,3,3,3	aDGalp	5.66	3.95	4.00	4.06	4.41	3.75
5,3,3,6	aDGalp	4.99	3.84	3.84	4.03	?	?
5,3,3	aDGlcp	5.24	3.66	4.04	3.40	4.13	3.80-3.90
5,3,7	a?Hepp	4.93	4.00	3.86	3.86	3.63	?	?
5,3	a?Hepp	5.14	4.30	4.00	4.03	3.75	?	3.75
5	a?Hepp	5.12	4.11	4.02	?	?	?	?
	aXKdop	-	-	1.85-2.17	4.07	4.15	?	?	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
5,3,3,3,2,2,2	Ac		23.2/2.09
5,3,3,3,2,2	aDGlcpN	94.4/5.11	54.4/3.99	72.4/3.84	71.2/3.50	73.2/3.92	61.8/3.77
5,3,3,3,2	aDGlcp	91.7/5.60	74.3/3.74	72.5/3.91	70.2/3.51	73.0/3.72	61.4/3.77-3.88
5,3,3,3	aDGalp	96.4/5.66	71.4/3.95	69.1/4.00	70.0/4.06	71.1/4.41	61.8/3.75
5,3,3,6	aDGalp	99.6/4.99	69.3/3.84	70.7/3.84	70.2/4.03	71.9/?	?/?
5,3,3	aDGlcp	101.8/5.24	71.3/3.66	77.9/4.04	72.3/3.40	72.1/4.13	67.8/3.80-3.90
5,3,7	a?Hepp	101.3/4.93	70.7/4.00	81.0/3.86	66.3/3.86	73.6/3.63	?/?	?/?
5,3	a?Hepp	103.3/5.14	70.7/4.30	81.0/4.00	66.3/4.03	73.6/3.75	?/?	70.4/3.75
5	a?Hepp	101.0/5.12	71.1/4.11	80.4/4.02	66.4/?	?/?	?/?	?/?
	aXKdop			35.1/1.85-2.17	67.0/4.07	76.1/4.15	?/?	?/?	?/?

The spectrum also has 10 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = a?Hepp
[1*][1*]([1*]O[C@@H]1[C@H](O)C[C@](O)(C(=O)O)O[C@@H]1[C@H](O)CO)O[C@H]1O[C@H](CO[C@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H](O)[C@H](O[C@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)[C@H]1O

1105.949 g/mol (C₄₀H₆₇R₃NO₃₄, R = residue superclass(es), not counted in mol weight)

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of (1)H-(1)H and (1)H-(13)C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo.

NMR, core, O-antigen, Kdo, Salmonella, MS

There are 2 chemically distinct structures. Please, select:
Ac(1-2)aDGlcpN(1-2)aDGlcp(1-2)aDGalp(1-3)[aDGalp(1-6)]aDGlcp(1-3)[P-?),a?Hepp(1-7)]a?Hepp(1-3)[xXEt?N(1-P-P-4)]a?Hepp(1-5)?XKdop Ac(1-2)aDGlcpN(1-2)aDGlcp(1-2)aDGalp(1-3)[aDGalp(1-6)]aDGlcp(1-3)[P-?),a?Hepp(1-7)]a?Hepp(1-3)[xXEt?N(1-P-P-4)]a?Hepp(1-5)xXKdoa

SVGMWSMILES3D molIonize

 

R1 = a?Hepp
[1*][1*](O[C@H]1O[C@H](CO[C@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H](O)[C@H](O[C@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2NC(C)=O)[C@H]1O)(OP(=O)(O)O)[1*](O[C@@H]1[C@H](O)CC(O)(C(=O)O)O[C@@H]1[C@H](O)CO)OP(=O)(O)OP(=O)(O)OCCN

1422.969 g/mol (C₄₂H₇₇R₃N₂O₄₅P₃, R = residue superclass(es), not counted in mol weight)