L-Valine assisted distinction between the stereo-isomers of D-hexoses by positive ion ESI tandem mass spectrometry

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Research ArticleReceived: 5 August 2009 Accepted: 13 April 2010 Published online in Wiley Interscience: 12 May 2010

(www.interscience.com) DOI 10.1002/jms.1752

L-Valine assisted distinction betweenthe stereo-isomers of D-hexoses by positive ion ESItandem mass spectrometryGang Li,a Zhongbin Huang,a Chuan Fu,a Pengxiang Xu,a Yan Liua

and Yu-fen Zhaoa,b,c∗

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry(ESI-ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 aminoacids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigatedby tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave richfragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could becomplexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexosecomplexes coordinated by potassium ion, the MS2 spectra of the ion at m/z 453 yielded unambiguous differentiation. And thefragmentation ions are sensitive to the stereochemical differences at the carbon-4 of hexoses in the complexes, as proved bythe MS2. Copyright c© 2010 John Wiley & Sons, Ltd.

Keywords: tandem mass spectrometry; electrospray ionization; complex; hexose; amino acid

Introduction

Carbohydrates play an essential role in a wide range of biologicalrecognition systems. In particular, they are specifically recognizedby proteins, such as monoclonal anti-saccharide antibodies,enzymes, saccharide transporters, and lectin. And their interactionsmay stimulate biological responses, such as antigen–antibody[1]

and host–parasite[2,3] interactions, cell differentiation,[4] etc.Therefore, many studies have been conducted to determinethe mechanism of action of these ubiquitous molecules whichare involved in molecular targeting.[5] Since both carbohydratesand proteins are very complex molecules, a study of theinteractions between amino acids and monosaccharides may bea practical initial step to understand the characteristic mechanismof protein–carbohydrate interactions.

Although nuclear magnetic resonance (NMR) spectroscopyhas been the key tool for the study of molecular interactions,usually it requires a substantial amount of highly purifiedmaterial. Furthermore, the results are often difficult to interpretfor the carbohydrate complexes. On the other hand, currently,electrospray ionization mass spectrometry (ESI-MS) can offer theprecise mass spectrum for the complexes with high sensitivity forthe study of noncovalent complexes,[6] which provides importantinformation on the molecular recognition processes. We employedtandem mass spectrometry (MSn) for the investigation on theinteractions between amino acids and monosaccharides.

The complexes between carbohydrates and other compoundshave been studied by ESI-MS for the structural elucidation ofcarbohydrates[7] as well as for the distinction among monosac-charide stereo-isomers such as hexoses, hexosamines, andN-acetylhexosamines.[8] For example, Zhu and Sato have investi-gated fragmentation differences between ammonium-cationizedmonosaccharides using tandem mass spectrometry.[3,8] Leary and

coworkers have analyzed monosaccharides chemically conjugatedwith metal complexes such as [Co(DAP)2Cl2]Cl, and performed thestructural elucidation of hexose with diastereomeric Zn(dien)2Cl2derivatives[9] and Ni(NH2(CH2)3NH2)3Cl2 derivatives.[10,11] The dis-sociation ions specific to stereochemical differences at C2 andC4 in hexose complexes were observed in the MS2 and MS3

spectra.[9] Salpin and Tortajada have investigated stereo-isomersof metal–hexose complexes such as [Pb(monosaccharide)m −H]+ ions.[12] All these studies showed that the ESI-MSn of di-astereomeric monosaccharide complexes exhibited characteristicdifferences.

Glucose (Glc), galactose (Gal), mannose (Man), and fructose (Fru)(Scheme 1) as the most common hexoses present in nature[13] havebeen chosen for our studies owing to their biological relevance.Fru is a ketohexose, and the other three are aldohexoses whichdiffer in the configuration of their hydroxyl groups. For MS/MSstudies presented here, three rare hexoses (aldohexoses), allose(All), gulose (Gul), talose (Tal) (Scheme 1), were also investigatedto compare them with the four common hexoses. Proteins

∗ Correspondence to: Yu-fen Zhao, Department of Chemistry and The KeyLaboratory for Chemical Biology of Fujian Province, College of Chemistryand Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China.E-mail: [email protected]

a Department of Chemistry and The Key Laboratory for Chemical Biology of FujianProvince, College of Chemistry and Chemical Engineering, Xiamen University,Xiamen 361005, P. R. China

b Department of Pharmaceutical Science, Medical College, Xiamen University,Xiamen 361005, P. R. China

c The Key Laboratory for Bioorganic Phosphorous and Chemical Biology, Ministryof Education, Department of Chemistry, Tsinghua University, Beijing 100084,P. R. China

J. Mass. Spectrom. 2010, 45, 643–650 Copyright c© 2010 John Wiley & Sons, Ltd.

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Scheme 1. Chemical structures of the D-hexose isomers used in this study.

that interact with carbohydrates noncovalently occur widely innature. Prominent examples are carbohydrate-specific enzymesand anticarbohydrate antibodies. In recent years, the lectins, aclass of agglutinin, have come into the forefront of biologicalresearch. Lectins bind mono- and oligosaccharides reversibly andwith high specificity, but they are devoid of catalytic activity andare not the products of an immune response. They interact withsaccharides by a network of hydrogen bonds and hydrophobicinteractions; coordination with metal ions may also play a role.Not all amino acids are equally abundant in the lectins. It wasreported that many lectins are composed of rich valine (Val); forexample, Val accounts for 14.7% of the total residues for Geodialectin I.[14 – 16]

For the studies of noncovalent complexes of hexoses (Hex) andamino acids (AA), the binary mixtures were easily prepared bydissolving one molar equivalent of hexose and amino acid in amethanol/water mixed solvent. The mixture was then ionized byESI and examined by ion trap mass spectrometry. It was found thathexoses can complex with the amino acid in the molecular ratioof 1 : 1 (cationized by proton or Na+) or 2 : 1 (cationized by K+ onlyfor Val). MS2 spectra of AA–Hex complexes could provide morestructural information than simple mass spectra, especially for Val.The fragmentation patterns of Val–Hex complexes cationized bythe proton and Na+ were simple and showed no significantdifference between each other. However, the complexes ofHex and 2 equivalents of Val cationized by K+ showed uniquefragmentation patterns and could be used to differentiate sevenhexose diastereomers.

Experimental

Chemicals

All carbohydrate standards (All, Gal, Glc, Gul, Man, Tal, Fru), aminoacid standards (alanine (Ala), arginine (Arg), asparagine (Asn),aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid(Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu),lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine(Val)) used in this study were purchased from J&K ChemicalLtd (Shanghai, China). Their purities reached 98–99%. Methanol(for HPLC) was purchased from Merck. Water of the required

purity for preparing monosaccharide standard stocks was obtainedusing a Milli-Q water purification system (Millipore, USA). Othercompounds in this study were used as purchased HPLC or analysisgrade, such as sodium chloride, potassium chloride, and aceticacid.

Mass spectrometry

ESI-MSn spectra were recorded using an ion trap mass spec-trometer equipped with an ESI source (Esquire 3000 plus, Bruker,German). In the positive ion mode, the entrance to the capillarywas −4 kV relative to the needle and −500 V relative to the end-cap. The spray was stabilized with nitrogen sheath gas at 8 psi anddrying gas heated at 250 ◦C, and a flow rate of 4 l/min was used toevaporate the solvent in the spray chamber. In MS2 experiments,a mass range of m/z 50–600 was scanned. The width of isolationwas set to 2.0 and the fragmentation amplitude was set from 0.40to 0.80 V in the ‘smart fragmentation’ mode. All experiments wererepeated at least twice on different days.

Hexoses (50 µM) were dissolved in a methanol/water (1 : 1, v/v)solvent mixture supplemented with one type of amino acid (50 µM).The concentration of sodium ions was 10 µM. The concentration ofpotassium ions was 20 µM. All samples were injected for analysisby ESI-MS using direct infusion with a syringe pump at a flow rateof 4 µl/min after filtration with a 0.45 µm filter membrane (Millex,Millipore, USA).

Results and Discussion

Complexation of hexoses with amino acids

In this work, the ESI-MS of hexose complexes with amino acids werestudied. Each of the hexoses was dissolved in a methanol/water(1 : 1, v/v) solvent containing one type of amino acid and thenexamined by ESI-MS. Among all 20 amino acids, Ala, Cys, Gly, Ile,Leu, Met, Phe, Pro, Ser, Thr, Tyr, and Val could generate adducts withhexoses. These amino acid–hexose complexes could be ionizedby proton, sodium ion, or potassium ion (Scheme 2). Figure 1shows a typical full ESI-MS spectrum for the Val and Gal mixturein a methanol/water solution of K+. Table 1 shows the m/z andrelative abundance of the AA–Hex complex ions in the ESI massspectra for 12 amino acids. To get more information of the adduct

www.interscience.wiley.com/journal/jms Copyright c© 2010 John Wiley & Sons, Ltd. J. Mass. Spectrom. 2010, 45, 643–650

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L-Valine assisted distinction between the stereoisomers of D-hexoses

Scheme 2. Complexation of the hexoses with amino acids.

Figure 1. The spectrum of Gal and Val complexes in a methanol/water solution of KCl.

Table 1. The relative intensitiesa of the AA–Hex complex ions in theMS spectra for the 12 amino acids

m/z (relative intensity %)Aminoacid [AA + Hex + H]+ [AA + Hex + Na]+ [2AA + Hex + K]+

Ala 270 (6–10) 292 (2–10)

Cys 302 (5–12)

Gly 256 (3–7) 278 (11–19)

Ile 312 (2–7) 334 (4–8)

Leu 312 (6–9) 334 (3–13)

Met 330 (5–11) 352 (2–4)

Phe 346 (4–10)

Pro 296 (9–15) 318 (9–17)

Ser 286 (2–4) 308 (3–9)

Thr 322 (3–5)

Tyr 362 (2–5) 384 (10–15)

Val 298 (8–14) 320 (26–45) 453 (10–20)

a The relative intensities were obtained from the individual spectrumof hexoses.[AA + Hex + H]+, [AA + Hex + Na]+ , and [2AA + Hex + K]+ wererespectively generated in methanol/water (1 : 1, v/v), methanol/water(1 : 1, v/v) solution of NaCl (10 µM), and methanol/water (1 : 1, v/v)solution of KCl (20 µM).

ions, MS2 spectra were investigated. Some of the adducts couldlose one neutral amino acid or hexose. Protonated complexeslose neutral hexose: for example, the Gly–Gal complex (Fig. 2a).But there could be two results for the complexes ionized by thesodium ion: for example, the Val–All complex (Fig. 2b) loses Val,and the Met–Glc complex (Fig. 2c) loses Met or Glc. Some complexions could give rich fragmentation patterns in the MS2 spectra,such as for Ile, Phe (Fig. 2d), and Tyr.

However, there were no adduct ions detected for the other eightamino acids, Arg, Asn, Asp, His, Gln, Glu, Lys, and Trp, which havestrong polar side chains. It was assumed that the polar functionalgroup is not a favorable structure for adduct formation. On theother hand, aliphatic or hydrophobic side chains could enhancethe stability of the adducts. Furthermore, these eight amino acidshave high proton affinities (except for Asp and Glu)[17] and sodiumion affinities.[18]

We repeated all experiments with different concentrations ofNa+ and K+. For the formation of adducts, 10 µM of Na+ and 20 µM

of K+ were appropriate. Lower concentrations would weaken thepeaks ionized by the metal ions. We changed the pH and decreasedthe declustering potential, but found that these conditions did notdetermine the generation of the Hex–AA complex. The variation ofpH affects the intensities of the protonated species. Low pH gaverise to stronger peaks of [AA + H]+ at the cost of weakening the

J. Mass. Spectrom. 2010, 45, 643–650 Copyright c© 2010 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jms

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Figure 2. Positive ion ESI-MS2 spectra of hexose complex for Gly (a), Val (b), Met (c) and Phe (d).

peaks of [AA + Hex + H]+ . High pH increases the intensities of thespecies ionized by the metal ion, but no new adduct was detected.With pH >9 or <5, it was difficult to get any stable spectrum. Themixture of methanol and water (1 : 1, v/v) with a pH of about 6.9(measured by a pH meter) gave the best signal-to-noise ratio formost samples. Although the declustering potential was decreasedto 60 V, Arg, Asn, Asp, His, Gln, Glu, Lys, and Trp did not complexwith any hexose, and only Val could generate the 2 : 1 adductswith hexoses ionized by the potassium ion.

Because of the inefficient protonation of free carbohydrates,[19]

the [AA + Hex + H]+ ion should be formed by the interactionbetween the protonated amino acid and the free hexose. The highproton affinities of amino acids mean that the protonated aminoacids have strong intramolecular hydrogen bonds,[20] which canhinder the intermolecular interaction, and explains why aminoacids with high proton affinities do not generate the protonatedcomplexes with hexoses.

In sodium-ionized amino acids, bonding mainly results fromnoncovalent electrostatic interactions, which are fundamentallydifferent from the hydrogen bonds in protonated amino acids.[18]

The sodium ion affinities of amino acids range from 161 to225 kJ/mol,[18] as the functional side chains provide extra ligandsto the sodium ion, whereas the sodium affinities for hexosesrange from 174 to 179 kJ/mol.[21] It was found that amino acidswith sodium affinities exceeding 201 kJ/mol could not generateadducts with hexoses, such as Arg, Asn, Asp, His, Gln, Glu, Lys, andTrp. It seems that the sodium ion could be seized tightly by theamino acid and cannot be complexed by hexose if the differencebetween their sodium affinities becomes more than 20 kJ/mol.

The heterodimers of the amino acids have been studied byWesdemiotis.[22] In fact, only 30 heterodimers gave acceptablesignals. But the heterodimers mentioned in this paper weregenerated by pairs of amino acids without great differencebetween their sodium ion affinities (<10 kJ/mol). We think thatthe proton and metal ion affinities could be the main factorsresponsible for the formation of AA–Hex complexes.

Valine

In the case of Val, there was a very interesting phenomenon: stable2 : 1 adducts were observed in its mixed solutions with Hex. InFig. 3, the MS2 spectra generated from [2Val + Hex + K]+ at m/z453 shows a series of ions. If M+

1 , M+2 , and M+

3 represented [2Val +Hex + K]+ (m/z 453), [Val + Hex + K]+ (m/z 336), and [Val + Hex+ H2O + K]+ (from hydration of the complex by residual waterinside the ion trap, m/z 354), the ions at m/z at 435 and 393 may beconsidered as [M1 − H2O]+ and [M1 − C2H4O2]+; the ions at m/z306, 276, and 216 as [M2 − CH2O]+ , [M2 − C2H4O2]+, and [M2 −C4H8O4]+; and ions at m/z 294 and 234 as [M3 − C2H4O2]+ and[M3 − C4H8O4]+. Although these formulas did not represent thereal dissociation pathways, there were many peaks obviously dueto the breaking down of the carbohydrate ring skeleton.[23]

It is interesting to note that four common hexose isomers (Gal,Glc, Man, Fru) gave some similar daughter ions, but with differentrelative ratios: the ratios of m/z 276 to m/z 336 are 0.2 for Gal, 1.6 forGlc, 1.1 for Man, and only 0.03 for Fru. The significant change of thepeak ratio might provide a potential marker for the differentiationamong these four isomers. In addition, the complexes of Fru andGal also showed the m/z 306 peak but not for Glc or Man. Of these


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Figure 3. Positive ion ESI-MS2 spectra of Gal (a), Glc (b), Man (c), and Fru (d) generated from precursor ion [2Val + Hex + K]+ at m/z 453. Fragmentationamplitude was 0.6 V.

Figure 4. Positive ion ESI-MS2 spectra of All (a), Gul (b), and Tal (c) generated from precursor ion [2Val + Hex + K]+ at m/z 453. Fragmentation amplitudewas 0.6 V.


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Table 2. The main fragment ions in the MS2 spectra of the [2Val + Hex + K]+ ions (m/z 453)

Relative abundance (%)

Peaks (m/z) Fragment released from m/z 453 Gal Glc Man Fru All Gul Tal

435 H2O 3.0 4.9 3.8

393 C2H4O2 26.8 37.1

363 C3H6O3 2.4

354 Val + H2O 3.9 8.1 6.4 8.0 3.7 5.3

336 Val 100 62.2 88.9 100 31.1 100 100333 C4H8O4 2.6 15.3

306 Val, CH2O 2.7 18.1 2.6

294 Val + H2O, C2H4O2 5.2 5.3 6.8

276 Val, C2H4O2 15.6 100 100 3.0 100 8.6 9.3

234 Val + H2O, C4H8O4 7 10.4 4.8

216 Val, C4H8O4 3

four isomers, Glc was the only that had the product ion at m/z 393.Fru also had its special product ion at m/z 363.

Based on the above data, through the assistance of the Valadduct, it seems that the four common Hex isomers mightbe distinguished from each other. How about the rare hexoseisomers All, Gul, and Tal? It was found that the MS2 spectraproduced from the precursor ion at m/z 453 also showed somedifferences at the fragmentation voltage above 0.6 V (Fig. 4). Forexample, m/z 393 appeared (Fig. 4a) for All but not for Gul (Fig. 4b)and Tal (Fig. 4c). Instead, these spectra showed intense m/z 336peaks.

Most of the fragmentation ions of All showed similar MS2

spectrum as Glc, except for the additional peaks at m/z 333 andm/z 306; so All could be distinguished from other isomers by theadducts with Val. MS2 spectra of Gul and Tal were similar to thatof Gal, except that the ion at m/z 306 was missing for Gul, and Talshowed neither m/z 306 nor m/z 435 peaks (a clean spectrum).Therefore, the potassium ion adducts could be used as a toolto distinguish the hexose isomers with the assistance of valine.Table 2 summarizes the results of MS2 experiments for the sevenhexose complexes with Val.

It is worth noting that in the MS2 spectra of [2Val + Hex +K]+ (m/z 453), Glc, Man, and All with equatorial C4 hydroxylsgave abundant daughter ions at m/z 276, whereas the other threealdohexoses, Gal, Gul, and Tal, with an axial C4 hydroxyl group,did not yield strong m/z 276 peak and had the strongest peaksat m/z 336. The reproducibility of these results was confirmedby altering the fragmentation amplitude from 0.4 to 0.8 V. Therelative abundance ratios of ions at m/z 276 to 336 at allfragmentation amplitudes are shown in Fig. 5. The abundantproduct ion m/z 276 is a positive indication of the presence ofan equatorial C4 hydroxyl group. In other words, the equatorialor axial C4 hydroxyl is an ON/OFF switch for the ions at m/z276.

How was the ion at m/z 276 generated? The [2Val + Hex + K]+

complexes easily lose one molecule of Val to yield the m/z 336peak for all the stereoisomers of the hexose. The ion at m/z 276would be gained if the ion at m/z 336 lost C2H4O2 from hexose bya cross-ring cleavage. The ions at m/z 336 in the MS2 spectra wereisolated to examine MS3 spectra. The m/z 276 peak was detectedin all seven spectra. The MS3 spectrum of [2Val + Glc + K]+ (Fig. 6a)showed an abundant ion at m/z 276. The MS3 spectrum of [2Val+ Gal + K]+ generated the ion at m/z 306 by the loss of CH2O as

Figure 5. The relative abundance ratios of m/z 276 to 336 in the MS2

spectra at different fragmentation amplitudes for six aldohexoses.

well as the ion at m/z 276 (Fig. 6b). The MS3 spectra for the Man,Gul, and Tal were similar to Fig. 6a and those of All and Fru weresimilar to Fig. 6b.

The m/z 336 ion dissociates through a cross-ring cleavageto generate an ion at m/z 276 by elimination of C2H4O2. Inprevious studies,[24] the loss of C1–C2 (0,2A cleavage according tothe Domon and Costello nomenclature) has been systematicallyobserved. However, the stereochemistry on the C4 hydroxyl groupplays an important role for the loss of C2H4O2. Two mechanismsare proposed in Scheme 3, one corresponding to 0,2A cleavage andone involving the loss of C5–C6 (0,4A cleavage). The latter wouldbe affected by the stereochemistry of the C4 if the hydroxylson C4 and C6 were involved together in the interaction like inScheme 4. Consequently, the experiment was repeated with alabeled monosaccharide, 1-13C-D-glucose. In this case, the 0,4Acleavage would correspond to the loss of 60 Da, while the 0,2Acleavage would be characterized by the elimination of 61 Da. Inthe result, both they were observed, as shown in Fig. 7. For thehexoses with an axial C4 hydroxyl, the extraction of a molecule ofC2H4O2 could be suppressed by the Val. Table 3 gives the summaryof the information derived from the daughter peaks of [2Val + Hex+ K]+ ions.


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Figure 6. Positive ion ESI-MS3 spectra generated from the precursor ion [Val + Hex + K]+ at m/z 336 for Glc (a) and Gal (b). Fragmentation amplitude was0.4 V.

Scheme 3. Proposed mechanisms of generation of the m/z 276 ions from [Val + Hex + K]+ complexes.

Conclusions

The investigation of amino acid–hexose binary mixtures yieldedimportant results. Twelve of the 20 standard amino acidscould generate adducts with hexoses, and some of the adductions produced characteristic fragmentation patterns in the MS2

process. The generation of amino acid–hexose complexes showeda close relationship with the proton affinity and the sodium ionaffinity. The MS2 spectra of AA–Hex complexes cationized by theproton were very simple. Because the proton affinities of aminoacids were larger than those of hexoses, an AA–Hex complexionized by proton was readily split into a protonated amino acid

Scheme 4. The possible interaction between Val and hexoses with the axial C4 hydroxyl group.


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Figure 7. Positive ion ESI-MS3 spectra generated from the precursor ion[2Val + Hex + K]+ at m/z 454 for 1-13C-D-glucose. The fragmentationamplitude was 0.4 V.

Table 3. The differences between the MS2 spectraa of the [2Val +Hex + K]+ ions (m/z 453)

Characteristic peaks (m/z)Axial C4 Ratios of

Nob Hexose 435 393 363 333 306 294 hydroxyl 276/336

3a Gal + + � 0.2

3b Glc + + + + 1.6

3c Man + 1.1

3d Fru + + ∼0c

4a All + + + + 3.2

4b Gul + � 0.1

4c Tal � 0.1

a Fragmentation amplitude was 0.6 V.b The no. is according to Figs 3 and 4.c Fru is a ketohexose and has different fragmentation pathway, so theabundance of ion at m/z 276 cannot be the indictor of axial C4 hydroxylgroup for it.

and a neutral hexose. The MS2 spectra of AA–Hex complexescationized by the sodium ion were similar to each other for thesame amino acid. This fact suggested that the sodium was bindingto the amino acid in an AA–Hex complex. If sodium was bindingto the hexose, the fragmentation patterns could show somedifferences for seven hexoses because of the different bindingways of hexoses to Na+.[21]

Among the 20 amino acids, Val is a unique amino acid thatforms 2 : 1 adducts with hexoses which are readily ionized by thepotassium ion, [2Val + Hex + K]+. We think that the potassium ionaffinities and the molecular sizes of amino acids are the reasonsfor this phenomenon. Val has a smaller molar volume than Ileand Leu[25] which have the similar potassium ion affinities (about128 kJ/mol) as Val.[26] Although there is more space around K+(radius = 1.36 Å) for ligands than Na+ (radius = 1.02 Å),[27] Val hasmore possibilities to form 2 : 1 complexes than Ile and Leu.

In the tandem mass spectra of [2Val + Hex + K]+ for theseven hexose isomers, the dissociation products of their precursor

ions provide enough information to distinguish them from eachother. In the complex ions, these hexose skeletons were brokenby the release of CnH2nOn (n = 1–4). Here, it should be pointedout that the main fragmentation patterns of the complex camefrom the cross-ring cleavage of the hexose. This phenomenonreflects that there are so strong interactions between the hexoseand the valine that the nonconvalent bonding cannot be easilybroken. The MS2 spectra of [Hex + K]+ (m/z 219) showed nosignificant difference among all seven complexes (data not shown)and no sensitivity to the configuration of hexoses. Therefore, thestereoselectivity of valine to hexoses should involve the interactionbetween the valine and the hexose in [2Val + Hex + K]+. The effectof the stereochemistry on the C4 hydroxyl group implies that thenoncovalent interactions in the complexes arise in different ways.That will be the next goal of our study.

Acknowledgements

The authors would like to acknowledge the support from theKey Foundation of Science and Technology Project of China 2006(DFA430430) and National Natural Science Foundation of China(No. 20572061).

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L-Valine assisted distinction between the stereo-isomers of D-hexoses by positive ion ESI tandem mass spectrometry