Cholera is a serious enteric disease, which re-mains a persistent problem worldwide [1–4].The most recent of the eight pandemics occurred
in Peru and India/Bangladesh, in 1991 and 1992, re-spectively. The cholera problem is augmented by thefact that strains of Vibrio cholerae have become resistantto many antimicrobial agents. Systematic prevention ofcholera by immunization has not yet been achievedbecause of a lack of an efficient protective vaccine. Thiswork was carried out within studies of antigenic deter-minants of lipopolysaccharides (LPS) of Vibrio choleraeO:1. Previously, the monomeric models were studied byelectron ionization mass spectrometry [5]. Here, westudy synthetic mono- to hexasaccharides (Figure 1)that mimic the fragments of the O-antigen of Ogawaand Inaba O-PS by electrospray mass spectrometry [6]in positive mode. The structure of O-specific polysac-charide (O-PS or O-antigen) of Vibrio cholerae O:1 con-sists of a relatively short chain of 4-amino-4,6-dideoxy-�-D-mannopyranose (perosamine), the amino group ofwhich is acylated with 3-deoxy-L-glycero-tetronic acid(DGT). The O-PS of the Ogawa strain differs from that
Published online April 5, 2006Address reprint requests to Dr. V. Kovácik, Institute of Chemistry, Slovak
Academy of Sciences, Dúbravska cesta 9, 84538 Bratislava, Slovakia. E-mail:[email protected]
of Inaba in that the upstream, terminal 4-acylated4-amino-4,6-dideoxy-�-D-mannopyranose residue inthe O-PS of the Ogawa strain carries a methyl group atO-2.
Experimental
The Ogawa and Inaba oligosaccharide models 1-6Ogand 1-6In (Figure 1), were synthesized as describedpreviously [7–11] The purity of the substances wasverified by MALDI-TOF MS and 13C NMR spectros-copy. Molecular mass was determined on MALDI TOFIV (Shimadzu, Kratos Analytical, Manchester, UK)instrument. 2,5-Dihydroxy benzoic acid was used as amatrix. Ion acceleration voltage was 5 kV. Sampleswere irradiated by 337 nm photons from the nitrogenlaser. Typically, 100 shots were summed into a singlemass spectrum. 13C NMR spectra were measured onBruker Avance-DPX FT (300 MHz) instrument indeuteriochloroform.
Positive ion ESI MS1–5 were measured on Esquire3000 ion-trap mass spectrometer (Bruker Daltonik, Bre-men, Germany) equipped with an electrospray ioniza-tion source. One mg of the sample was dissolved in 1 mlof acetonitrile/water 1:1. Sample solutions were intro-duced into the ion source at the flow rate of 3 �l/min
via a metal capillary held at high voltage (3.5 kV). Other
r Inc. Received January 10, 2006Revised February 8, 2006
Accepted February 9, 2006
750 KOVACIK ET AL. J Am Soc Mass Spectrom 2006, 17, 749–756
instrument conditions were as follows: drying gas tem-perature, 250 °C; drying gas flow, 5 �l/min; nebulizerpressure, 14 psi. Nitrogen was used as both nebulizinggas and drying gas. The nozzle-skimmer potential and
Figure 1. Compounds investigated.
Table 1. Schematic reperesentation of the (Z)-anti conformer ofangles (deg) of 1In conformers and sodium complexes calculatedconformers calculated using different basis sets
aE � �1 013.097183 a.u.; bE � �1 013.436271 a.u.; cE � �1175.285486 a.u.;
octopole potential were modified and optimized beforeeach experiment. For CID ESI MS measurement, a valueof 0.95 was applied to all substances by way of frag-mentation amplitude. Two or three measurements wererun with every sample, if necessary.
The quantum chemical calculations were carried outusing the Jaguar program [12] on QS8-2800 C computer[13]. The optimizations of the geometry were performedusing the B3LYP density functional method [14] (DFT)with the standard 6-31G* basis set. The geometries for aset of low-energy candidate structures for the monosac-charide (1In) complexes with the sodium cation werefully optimized with no constraints on geometricalparameters. The final energies were obtained using thesingle point calculations at the B3LYP/6-311 �� G**levels from the 6-31G* optimized geometry. Mass Fron-tier software [15] 4.0 (MF) was used to assist in theelucidation the fragmentation [16].
Results and Discussion
Conformational Study
The conformation of C(4) amide 3-deoxy-L-glycero-tet-ronic acid substituent NHDGT-C(4) influences stabilityof 1In conformations and formation of Na� complexes.Five torsional angles describe the conformation of theNHDGT-C(4) group (Table 1), namely �1 � [C(3)–C(4)–
In molecule and the labeling of the atoms. Selected torsionalLYP/6-31G* level and the relative energy �E (kcal mol�1) of
751J Am Soc Mass Spectrom 2006, 17, 749–756 ESI IT OF SYNTHETIC ANALOGS OF SACCHARIDE
N–C(8)], �2 � [C(4)–N–C(8)–C(9)], �3 � [N–C(8)–C(9)–C(10)], �4 � [C(8)–C(9)–C(10)–C(11)], and �5 � [C(9)–C(10)–C(11)–O (11)]. The torsional angle around C(4)–Nbond (�1) may adopt either the antiperiplanar or syn-periplanar arrangement relative to the C(4)–N andN–C(8) bonds. The four possible conformers are (Z)-anti, (Z)-syn, (E)-anti, and (E)-syn.The initial structuresof the (Z)-anti, (Z)-syn, (E)-anti, and (E)-syn conformersfor 1In were built using the preferred 4C1 ring confor-mation and with conformations around three C–Cbonds (�3–�5) in the antiperiplanar orientation. Thesefour structures of 1In were optimized using fully re-laxed calculations at the B3LYP/6-31G* level, and thenthe energy was calculated at the B3LYP/6-311 �� G**level. The (Z)-anti conformer (�1 � 112.5° and �2 ��168.2°) is 2.05, 7.55, and 12.81 kcal/mol more stablethan the (Z)-syn (�62.9°, 161.1°), (E)-anti (97.5°, 25.4°),and (E)-syn (�70.7°, �8.9°) conformers, respectively.Orientation of the hydroxyl groups in all conformers is
Figure 2. The B3LYP/6-31G* calculated structures of three pre-ferred complexes for (Z)- anti conformer.
Table 2. Schematic reperesentation of seven starting locations (Nin the 1In complexes calculated at B3LYP/6-31G* level and the Bdifferent basis sets
anti-clockwise with the HO(3) proton pointing to theO(2) oxygen and forming a hydrogen bond. The re-maining part of the NHDGT-C(4) group adopts a sim-ilar conformation in all four conformers exhibitingO(9)-H-O(11) hydrogen bond. This is documented bythe interval of values for torsional angles �3 (�95°–�102°), �4 (�174°–�178°), and �5 (�72°–�73°), respec-tively. The calculated preference of the (Z)-anti con-former is in conformity with the experimental data on�-d-glucopyranosides [17]. Therefore, the optimizedstructures of the (Z)-anti conformer was selected togenerate the initial structures of Na� complexes posi-tioning the sodium cation at seven different locationsdepicted in Figure 2. These structures were optimizedin fully relaxed calculations.
The optimization at the B3LYP/6-31G* level led toseven (Z)-anti distinct minima. The values of the tor-sional angles �1–�5 together with the relative energies ofthe seven resulting complexes (Na1–Na7) are given inTable 1. The relative binding energies calculated at theB3LYP/6-311 �� G**//6-31G* level are reported inTable 2. The complexation of 1In with the sodium cationchanges the conformation of the 1In. The most sensitiveto this complexation is the orientation around theC(8)–C(9) bond characterized by the �3 torsional angle.This is reflected in a large interval (from �130° to 64°)of the �3 values. It can be seen from the relative energyvalues in Table 2 that Na3, Na4, and Na6 complexes arethe preferred ones. These three optimized complexstructures are shown in Figure 2. To evaluate an influ-ence of a conformation of the NHDGT-C(4) group onstability of complexes, we have calculated also the threepreferred location of the sodium cation for the second
Na7) for the sodium complexes of 1In. Position of the sodiumrelative binding energies �Eb (kcal.mol�1) calculated using
752 KOVACIK ET AL. J Am Soc Mass Spectrom 2006, 17, 749–756
lowest energy conformers (Z)-syn. The results areshown in Tables 1 and 2. In Na3 and Na4 complexes,one of the oxygens coordinating the sodium cation isthe carbonyl oxygen O(8) of the NDGT group. Thelowest energy complex Na3 exhibits interactions of thesodium cation with two oxygen atoms, O(8) and O(9),whereas complexes Na4 and Na6 show interactions ofsodium atom with three oxygens of 1In molecule. Na�
� O distances from 2.15 to 2.37 Å are characteristic forthe sodium complexes of 1In. It is interesting to notethat the nitrogen atom is not involved in the coordina-tion of the sodium cation, although one of startingpositions (Na3) was from the “N-location”. In two Na2and Na3 complexes, the same oxygen atoms are in-volved in coordination of sodium. However the struc-ture of the resulting complex is different. The bindingenergy of these complexes decreases with an enlarge-ment of basis set, e.g., for the complex Na3 decreasesfrom 67.2 kcal/mol at the 6-31G* level to 57.8 kcal/molat the 6-311 �� G** level. We have observed the similartrend during our previous work [18].
ESI-IT MS Study of Mass SpectrometricFragmentation Rules of Basic StructuralUnits of LPS
Fragmentation of monomeric substances 1In and 1Oghas been elucidated from the ESI IT multistage massspectra. The aim of this work was to obtain tandemmass spectrometric fragmentation rules of basic struc-tural units of the LPS. Examples of ion trap mass spectraMS1–3 obtained from 1In are shown in Figure 3. Thetandem MS2 spectrum of the 1Og monomer is presentedin Table 3, which summarizes the ion trap MS data of allOgawa compounds investigated. The Mass Frontiersoftware has assisted in the formulation of the fragmen-tation mechanisms shown in the Scheme 1. In the firstroute, a molecule of water is eliminated from thesodium-cationized molecule. The [M � Na � H2O]�
ions are denoted as a species. In the following steps,elimination of a molecule of ethene, carbon monoxide,water, or hydroxypropanal gives rise to the c ,d, and especies, as proven by MS 2 and MS3 measurements. Themarked eliminations are formulated as products of
0
2
4
5x10
Intens.
14 16 18 20 22
200 210
2
0
1000
2000
3000
4000
5000
14 16 18 20 22
148
182210
2
0
200
400
600
140 160 180 200 220
f1e1
e1
g1
OH
NH O
OH
O
O
OH
OH
Na+
Figure 3. ESI IT multistage m
decay of the adducts coordinated on the carbonyl
oxygen of the NDGT group. The less energetic isomer,the adduct with the Na cation in coordination to hemi-acetal oxygen atom is supposed. In the less favoredroute, the elimination of molecule of methanol from [M� Na]� adducts takes place. The presence of a methoxygroup at the C(2) position in 1Og reflects the shift of m/zvalues of the respective ions by 14 u. The fragmentationof hemi-acetal coordinated adducts involves elimina-tion of a molecule of �-hydroxy-�-butyrolactone (f andg fragments) giving rise to sodium ions of the 4-aminomannopyranoside type. We are not able to provide astraightforward explanation for the ion at m/z 148 in theMS3 spectrum of 1In (Figure 3). Fragmentation of di- tohexa-oligomers was elucidated in the same manner(Figures 4 and 5, Tables 3 and 4) as that applied to themonomer. In the spectra of the dimer, the cleavage ofthese ions results in formation of the monomer, havingthe same structure as the Inaba monomer, with thehydroxyl group at the C(2) position. Similar shorteningof the oligosaccharide chain is observed also for thehigher oligomers. In the spectrum of the trimer, theseions represent two species, the dimeric and monomericInaba type ions. In the spectrum of the tetramer, twosimilar ions, the trimeric and dimeric, are again present(Figure 4). On the other hand, the pentamer spectrum(Figure 5) shows three intense species: tetra-, tri- anddimer. And finally in the spectrum ESI IT of thehexamer, four shorter species containing Inaba units arepresent. The intensity of monomer 1In fragment isnegligibly. A comparison of the ESI MSn spectra of theoligomers of Ogawa and Inaba types confirms that theleaving residue in the first step of the chain-shorteningpathway is the upstreaming mannose unit with the C(2)hydroxyl or methoxyl C-2 group of the oligomer. For-mally the chain shortening pathways correspond tothose resulting in Y ions from oligosaccharides de-scribed by Domon and Costello [19] and others. Thefragmentation of hemi-acetal coordinated adducts in-volves the elimination of molecule of �-hydroxy-�-butyrolactone (f and g fragments) in the MS2 spectra,giving rise to sodium adducts of the 4-amino mannopy-ranoside type. In the MS3 and higher multistage spectra,the elimination of other molecules of �-hydroxy-�-butyrolactone (minus 102 mu) or other species also
302
318
+MS
24 26 28 30 32 m/
256 270
284
302
24 26 28 30 32 m/
256284
240 260 280 300 320 m/z
[M+Na]+1In
[M+K]+
a1
b1c1
1In
a1c1
+MS2(302)
+MS3(302->284)
pectra of Inaba monomer 1In.
28
28
d1
d1
from product ions was observed. Example routes in
*The peak abundances under 5% are not included
753J Am Soc Mass Spectrom 2006, 17, 749–756 ESI IT OF SYNTHETIC ANALOGS OF SACCHARIDE
Table 3. The ion abundances in ESI IT tandem mass spectra of[M�Na]� ions of LPS models, serotype Ogawa 1Og�6Og
Scheme 1. The basic fragmentation mechanism pathways of the 1In unit.
754 KOVACIK ET AL. J Am Soc Mass Spectrom 2006, 17, 749–756
Figure 4. ESI IT multistage mass spectra of Inaba tetramer 4In.
ss sp
755J Am Soc Mass Spectrom 2006, 17, 749–756 ESI IT OF SYNTHETIC ANALOGS OF SACCHARIDE
Figure 4 show formation of da4 and fa4 species equiv-alent to e and g ions in MS 2 in the MS3 spectrum of a4
ions, as well as of cd4, dd4, and fd4 ions in the MS3
spectrum of d4. Similarly, the af4, cf4, df4, and ff4 speciesare visible in the MS3 fragmentation of f4 ions. In thespectrum of the Ogawa pentamer 5Og (Figure 5), thesecondary ions da5 and fa5 ions are visible at the MS3
fragmentation of a5 species, and ac5, cc5, dc5, ec5, and fc5
are present in the fragmentation of c5 product ions.Similarly, the ad5, cd5, dd5, ed5, and fd5 fragments areproduced in the fragmentation of d5 ions, as well as theaf5, cf5, df5, ef5, and ff5 species arising from f5 ions. Themechanism of the formation of the ions under discus-sion is similar to that, described in Scheme 1.
Conclusions
The oligomeric models (mono- through hexasaccharide)of the O-antigens of Vibrio cholerae O:1 have been studiedby electrospray ion trap multistage (MSn, n � 1–3) massspectrometry. The ESI IT technique has merits in that itprovides information on the length of the repeating unit ofthe natural O-chain and his biological heterogeneity ofO-antigens from Vibrio cholerae LPS. In the first route, amolecule of water is eliminated from the sodium cation-ized molecular ion. In the following steps, elimination of amolecule of ethene, carbon monoxide, water, or 1-hy-droxypropanal take place, as proven by MS 2 and MS3
Figure 5. ESI IT multistage ma
measurements. The fragmentation of hemi-acetal coordi-
nated adducts involves elimination of a molecule of �-hy-droxy-�-butyrolactone. The predominant pathway offragmentation of the oligomers is the elimination shorten-ing the length of the oligosaccharide. In this way, conver-sion of the Ogawa to Inaba fragments takes place underthe conditions of measurement. Comparing various stagesof multistage ESI IT mass spectra shows that the tandemMS2 spectra (Tables 3 and 4) are most informative for thestructure elucidation of compounds of this type. Thehigher order ion trap spectra were found to be an excellenttool for the formulation of the fragmentation pathways.
AcknowledgmentsThis investigation was supported by VEGA grant no. 2/3159/23of the Slovak Academy of Sciences and Ministry of Education ofSlovakia.
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