Legionaminic acid and 4-epi-legionaminic acid are 5,7-diacetamido nonulosonic acids and are assumed to play a crucial role in the virulence of Legionella pneumophila, the causative agent of Legionnaires' disease. Moreover, they are ideal target motifs for the development of vaccines and pathogen detection. Herein, we present a versatile de novo synthesis of legionaminic acid and 4-epi-legionaminic acid. Starting from simple d-serine, the C9-backbone is built up by two CC-bond formation reactions. First, the protected d-serine motif is elongated utilizing a highly stereoselective nitroaldol reaction to give a C6-precursor of desired d-rhamno configuration. Second, an indium-mediated allylation is employed to further elongate the carbon backbone and introduce a masked alpha-keto acid function.

nonulosonic acid, legionaminic acid, 4-epi-Legionaminic acid, Indium-mediated allylation, Nitroaldol reaction

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]([C@@H](C)O)[C@@H]1O[C@@](O[C@@H]2[C@@H](C(=O)O)O[C@@H](O[C@H]3[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]3O[C@@H]3[C@@H](C(=O)O)O[C@@H](O[C@@H]4[C@@H](CO)OC(O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](OS(=O)(=O)O)[C@H]2O)(C(=O)O)C[C@H](O)[C@H]1NC=O

1076.892 g/mol (C₃₆H₅₆N₂O₃₃S)

VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by beta-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyl-legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N-acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.

glycosylation, N-acetylneuraminic acid, N-linked, glycoprotein, virus, S-layer glycan, archaea, Haloferax volcanii

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
4,4,2,4,5	Fo	166.0
4,4,2,4,7	Ac	175.6	24.2
4,4,2,4	aXLegp	?	101.4	42.0	70.1	52.5	73.7	55.7	68.4	20.4
4,4,2,2	S
4,4,2	bDGlcpA	101.0	81.5	75.4	75.1	77.5	175.8
4,4	aDManp	99.7	79.7	71.4	68.1	75.0	62.6
4	bDGlcpA	104.1	75.0	77.8	78.3	77.1	176.0
	bDGlcp1N	80.9	73.3	76.7	79.8	78.2	61.6


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,4,2,4,5	Fo	8.111
4,4,2,4,7	Ac	-	2.115
4,4,2,4	aXLegp	-	-	1.758-2.799	3.641	3.709	3.897	3.826	4.040	1.169
4,4,2,2	S
4,4,2	bDGlcpA	4.710	4.132	3.764	4.187	3.930	-
4,4	aDManp	5.491	4.164	3.825	3.823	3.661	3.79
4	bDGlcpA	4.548	3.427	3.696	3.825	3.93	-
	bDGlcp1N	4.986	3.439	3.711	3.649	3.666	3.827-3.934


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,4,2,4,5	Fo	166.0/8.111
4,4,2,4,7	Ac		24.2/2.115
4,4,2,4	aXLegp			42.0/1.758-2.799	70.1/3.641	52.5/3.709	73.7/3.897	55.7/3.826	68.4/4.040	20.4/1.169
4,4,2,2	S
4,4,2	bDGlcpA	101.0/4.710	81.5/4.132	75.4/3.764	75.1/4.187	77.5/3.930	
4,4	aDManp	99.7/5.491	79.7/4.164	71.4/3.825	68.1/3.823	75.0/3.661	62.6/3.79
4	bDGlcpA	104.1/4.548	75.0/3.427	77.8/3.696	78.3/3.825	77.1/3.93	
	bDGlcp1N	80.9/4.986	73.3/3.439	76.7/3.711	79.8/3.649	78.2/3.666	61.6/3.827-3.934

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]([C@@H](C)O)[C@@H]1O[C@@](O[C@@H]2[C@@H](C(=O)O)O[C@@H](O[C@H]3[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]3O[C@@H]3[C@@H](C(=O)O)O[C@@H](O[C@@H]4[C@@H](CO)O[C@@H](N)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](OS(=O)(=O)O)[C@H]2O)(C(=O)O)C[C@H](O)[C@H]1NC=O

1075.908 g/mol (C₃₆H₅₇N₃O₃₂S)