Computationally and Experimentally Derived General Rules for

Anal. Chem. 2009, 81, 1108–1120

Computationally and Experimentally Derived General Rules for Fragmentation of Various Glycosyl Bonds in Sodium Adduct Oligosaccharides Hiroaki Suzuki,†,‡ Akihiko Kameyama,§ Kazuo Tachibana,‡ Hisashi Narimatsu,§ and Kazuhiko Fukui*,† Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-42 Aomi, Koto, Tokyo 135-0064, Japan, Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan, and Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Umezono, Tsukuba, Ibaraki 305-8568, Japan Mechanisms of fragmentation of glycosyl bond linkages in various saccharides were investigated by using computational calculations to find general rules of fragmentation of sodiated oligosaccharides in mass spectrometry. The calculations revealed that r-Glc, r-Gal, β-Man, r-Fuc, β-GlcNAc, and β-GalNAc linkages were cleaved more easily than β-Glc, β-Gal, and r-Man linkages because the transition states of the former were stabilized by the anomeric effect. The 1-6 linkage was more stable than the others, since saccharides with flexible 1-6 linkages were more stabilized in energy than the other linkages by the sodium cation. The sialyl linkage was the most labile of all the linkages investigated. Comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. The calculated stabilities ofglycosylbondswere:r-Man(Manr1-3Man,Manr1-4Man, Manr1-6Man) > β-Gal (Galβ1-4Gal) > r-GalNAc (GalNAcr1-4GalNAc) > β-Man (Manβ1-4GlcNAc) > r-Gal (Galr1-3Gal, Galr1-4Gal, Galr1-6Gal) > β-Man(Manβ1-4Man) > β-GalNAc (GalNAcβ1-4GalNAc) > r-Fuc (Fucr1-6GlcNAc) > r-Fuc (Fucr1-4GlcNAc) > β-GlcNAc(GlcNAcβ1-4GlcNAc)>r-Fuc(Fucr1-3GlcNAc) > r-NeuNAc (NeuNAcr2-3Gal, NeuNAcr2-6Gal); this result was close to the experimentally deduced trend. These theoretically and experimentally derived general rules for fragmentation should be useful for analyzing the experimentally obtained mass spectra of oligosaccharides. Post-translational protein modifications, such as phosphorylation and glycosylation, have been the targets of intense study in the field of proteomics because these modifications have widespread influences on protein function and structure. More than

half of the proteins in living organisms are glycosylated, and protein glycosylation is considered to add various functions to proteins. Glycoprotein oligosaccharides play essential roles in a variety of biochemical processes; they act as media for cell-cell recognition, aid in the processes of fertilization and inflammation, and add functionality to proteins by post-translational modification.1-3 Oligosaccharide structure directly affects carbohydrate function, and in glycomics the structural study of oligosaccharides has become an important part of efforts to gain further insights into glycosylation.4,5 As a sensitive and high-throughput method for the structural analysis of oligosaccharides, mass spectrometry has been applied widely.6-9 Tandem mass spectrometry, in particular, is regarded as a useful tool for structural characterization of oligosaccharides, since fragment ion spectra can be obtained via various fragmentation methods such as collision induced dissociation (CID)10 or postsource decay (PSD).11 Oligosaccharides can easily be ionized because they have a high affinity for alkali metal cations, which have a high content of negative oxygen atoms.12 The fragmentation of sodium ion adduct oligosaccharides ([M + Na]+) by lowenergy CID generated by matrix-assisted laser desorption/ ionization (MALDI) has been studied intensively.13-17 The (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

* To whom correspondence should be addressed. E-mail: [email protected]. † Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology. ‡ The University of Tokyo. § Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology.

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(14) (15)

Varki, A. Glycobiology 1993, 3, 97–130. Roseman, S. J. Biol. Chem. 2001, 276, 41527–41542. Dwek, R. A.; Butters, T. D. Chem. Rev. 2002, 102, 283–284. Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–2364. Dove, A. Nat. Biotechnol. 2001, 19, 913–917. Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349–450. Dell, A.; Morris, H. R. Science 2001, 291, 2351–2356. Zaia, J. Mass Spectrom. Rev. 2004, 23, 161–227. Park, Y. M.; Lebrilla, C. B. Mass Spectrom. Rev. 2005, 24, 232–264. Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321–369. Kaufmann, R.; Chaurand, P.; Kirsch, D.; Spengler, B. Rapid Commun. Mass Spectrom. 1996, 10, 1199–208. Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964–5970. Penn, S. G.; Cancilla, M. T.; Lebrilla, C. B. Anal. Chem. 1996, 68, 2331– 2339. Cancilla, M. T.; Penn, S. G.; Lebrilla, C. B. Anal. Chem. 1998, 70, 663– 672. Hanrahan, S.; Charlwood, J.; Tyldesley, R.; Langridge, J.; Bordoli, R.; Bateman, R.; Camilleri, P. Rapid Commun. Mass Spectrom. 2001, 15, 1141– 1151. 10.1021/ac802230a CCC: $40.75  2009 American Chemical Society Published on Web 12/31/2008

sodium cation forms a stable complex with the oligosaccharide molecule by forming a metal-oxygen coordination, thus avoiding the complications of generation of multiple charged ions and the rearrangement of fucose in an ion trap.18-20 Fragmentation from the sodium ion adduct oligosaccharide takes place mainly in glycosyl bonds at the nonreducing end side; this is called B/Ytype fragmentation.21 Much effort has been exerted to obtain a variety of structural data, including sequence and stereochemistry information such as anomeric configuration22,23 and linkage type,24-26 through the analysis of glycosyl bond fragmentations. The relative abundance of glycosyl bond cleavage depends on the stability and position of the glycosyl bond; thus the fragmentation patterns and their intensity profiles differ even in anomers or linkage isomers, making discrimination of various structural isomers possible. Differences in CID spectra should reflect the distribution of labile glycosyl bonds: the relative intensities of fragment ions derived from labile bond cleavage are greater, whereas those of fragment ions derived from stable bond cleavage are almost unobserved. For example, oligosaccharides containing a labile sialyl linkage bond27 afforded a prominent signal from the [M - NeuNAc + Na]+ ion. In contrast, oligosaccharides containing plural sites with similar stability afforded many signals with similar intensities in their CID spectra. Conversely, quantitative evaluation of the relative intensities of fragment ions will give us information on the relative stabilities of particular glycosyl bonds. Another factor in the differences in relative intensities of fragment ions is presumed to be the distribution of the sodium ion affinity domain in the saccharide molecule. This is based on the observation that all fragment ions derived from the [M + Na]+ ion were also sodium adducted. To acquire chemical insights into why each oligosaccharide affords a characteristic pattern, it is indispensable that we clarify the fragmentation mechanism. Clarification of the fragmentation mechanisms of a variety of oligosaccharides might also be useful in interpreting experimentally obtained fragmentation spectra and in assigning fragmentation spectra to fine structures. In searching for general rules of oligosaccharide fragmentation we focused on the sodium ion affinities of oligosaccharides and on glycosyl bond lability. As targets for our computational calculations we chose various modeled disaccharides whose linkages are commonly found in many classes of oligosaccharide. The linkages we investigated were R-Glc, β-Glc, R-Gal, β-Gal, R-Man, β-Man, R-Fuc, R-NeuNAc, (16) Kameyama, A.; Kikuchi, N.; Nakaya, S.; Ito, H.; Sato, T.; Shikanai, T.; Takahashi, Y.; Narimatsu, H. Glycobiology 2004, 14, 1069–1069. (17) Kameyama, A.; Kikuchi, N.; Nakaya, S.; Ito, H.; Sato, T.; Shikanai, T.; Takahashi, Y.; Takahashi, K.; Narimatsu, H. Anal. Chem. 2005, 77, 4719– 4725. (18) Brull, L. P.; Kovacik, V.; Thomas-Oates, J. E.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1998, 12, 1520–1532. (19) Franz, A. H.; Lebrilla, C. B. J. Am. Soc. Mass Spectrom. 2002, 13, 325– 337. (20) Harvey, D. J.; Mattu, T. S.; Wormald, M. R.; Royle, L.; Dwek, R. A.; Rudd, P. M. Anal. Chem. 2002, 74, 734–740. (21) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397–409. (22) Smith, G.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1996, 7, 953–957. (23) Gaucher, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1999, 10, 269–272. (24) Laine, R. A.; Pamidimukkala, K. M.; French, A. D.; Hall, R. W.; Abbas, S. A.; Jain, R. K.; Matta, K. L. J. Am. Chem. Soc. 1988, 110, 6931–6939. (25) Laine, R. A.; Yoon, E.; Mahier, T. J.; Abbas, S.; Delappe, B.; Jain, R.; Matta, K. Biol. Mass Spectrom. 1991, 20, 505–514. (26) Yoon, E. S.; Laine, R. A. Biol. Mass Spectrom. 1992, 21, 479–485. (27) Leavell, M. D.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2001, 12, 528–536.

R-GlcNAc, β-GlcNAc, R-GalNAc, and β-GalNAc. The fragmentation mechanism of each disaccharide was calculated at the HF/ 6-31G(d) level, and the chemical aspects of the lability of the glycosyl bonds are discussed. In this paper, we explore the general rules of fragmentation based on the vast amount of computational calculations and experimental data for mass spectra of the sodiated oligosaccharides. The general rules of fragmentation during CID can be useful in the development of novel tools for structural analysis in glycomics. EXPERIMENTAL SECTION Calculations. Theoretical calculations for disaccharides were performed to investigate the reaction mechanism. Quantum chemical calculations were performed with the Gaussian 03 program on a PC cluster at the CBRC. All calculations were performed by using the HF/6-31G(d) basis set. The initial geometries of the disaccharides were obtained from the Cambridge Structural Database (CSD version 5.25), and were fully optimized. A full geometric optimization was performed for the neutral disaccharide, a complex of each disaccharide and a sodium cation, and all of the optimized geometries were checked to have positive vibrational frequencies. In an attempt to verify the reaction pathway, the transition state was optimized by using quadratic synchronous guided transition-state optimization (QST3)28 at the HF/6-31G(d) level. The transition structures were checked against the frequencies with respect to the geometries having one imaginary frequency. All energy values were corrected with zeropoint vibrational energy from the frequency calculations. Experiments. Mass measurements were performed by using a MALDI quadrupole ion trap time-of-flight (TOF) mass spectrometer (AXIMA-QIT; Shimadzu). A nitrogen laser (wavelength 337 nm) was used for the laser desorption/ionization. A detailed configuration of the AXIMA-QIT is given elsewhere.29 All oligosaccharides were purchased from one of following distributors: Glyko (San Leandro, CA), Dextra Laboratories, (Reading, U.K.), and Sigma-Aldrich (St. Louis, MO). The structures of the oligosaccharides analyzed by AXIMA-QIT are shown in Figure 1. The samples were analyzed by placing 0.5 µL of analyte (approximately 2 µM) on the target plate followed by 0.5 µL of a 2,5-dihydroxybenzoic acid (2,5-DHB; Bruker-Daltonik) solution (10 mg/mL in 20% ethanol). The spot was allowed to dry and inserted into the mass spectrometer for analysis. All collision-induced dissociation (CID) spectra were obtained from sodium adduct ions. The collision energy was adjusted to reduce the intensity of the parent ion to less than 15% of the area of the base peak. RESULTS AND DISCUSSION Calculation of Binding Affinity of the Sodium Cation. We previously studied the sodium cation affinities of various oligosaccharides and found that stabilization by the sodium cation adduct may influence the lability of the glycosyl bond.30 It is theorized that metal ions interact with several oxygen atoms in oligosaccharides to stabilize the complex structure, and the increment in (28) Peng, C. Y.; Ayala, P. Y.; Schlegel, H. B.; Frisch, M. J. J. Comput. Chem. 1996, 17, 49–56. (29) Koy, C.; Mikkat, S.; Raptakis, E.; Sutton, C.; Resch, M.; Tanaka, K.; Glocker, M. O. Proteomics 2003, 3, 851–858. (30) Fukui, K.; Kameyama, A.; Mukai, Y.; Takahashi, K.; Ikeda, N.; Akiyama, Y.; Narimatsu, H. Carbohydr. Res. 2006, 341, 624–633.


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Figure 1. Structures of oligosaccharides analyzed by MALDI-CID MS.

the number of interactions with the sodium cation contributes to the stability of the saccharide molecule.31,32 To study fragmentation pathways and structure-reactivity relationships, we investigated the geometries of the stable complexes and affinities for the sodium cation. Figure 2 shows the stable complex structures of maltose and cellobiose optimized at the HF/6-31G(d) level. These complexes show a tridentate interaction of the sodium cation with the ring oxygen and two hydroxy oxygen atoms. The complexes of glucobioses that have 1-2 and 1-3 linkages also indicate a tridentate interaction between the sodium cation and the oxygen atoms, whereas glucobioses with 1-6 linkage, isomaltose and gentiobiose, showed tetradentate interaction (Supporting Information, Figure SI-1). Saccharides with 1-6 linkage

have three torsional axes in glycosyl bonds, such as C1-O, O-C6′, and C6′-C5′, and the presence of these torsional axes makes the 1-6 linkage more flexible than the other linkages. Because of this flexibility, isomaltose (GlcR1-6Glc) and gentiobiose (Glcβ1-6Glc) can form tetradentate interaction with the sodium cation, and they are more stable than the other glucobioses. The calculated energies of binding affinity of the sodium cation for the 26 disaccharides studied in this paper are listed in Table 1. Fragmentation of Glucobioses. In our previous study of three anomeric isomers of trehaloses (GlcR1-1RGlc, GlcR1-1βGlc, and Glcβ1-1βGlc), glycosyl bond cleavage was considered to take place by electron transfer from the ring oxygen and proton migration of the vicinal C-2 hydroxy group at the nonreducing end; the oxonium ion continuously forms 1,2-epoxy-D-glucopyranose (anGlc).33 Evidence for the hydroxy proton transfer was confirmed by H/D exchange experiments for three trehaloses. The relative abundance of B,Y-type fragment ions of GlcR1-1RGlc was the highest and that of Glcβ1-1βGlc was the lowest, indicating that R-Glc linkage was more labile than β-Glc linkage. The calculated activation energies of glycosyl bond fragmentation for the three linkages were in accordance with the trends of stabilities of glycosyl bonds derived from CID experiments, indicating that the fragmentation mechanism involving the attack

(31) Zhou, Z. R.; Ogden, S.; Leary, J. A. J. Org. Chem. 1990, 55, 5444–5446. (32) Cerda, B. A.; Wesdemiotis, C. Int. J. Mass Spectrom. 1999, 189, 189–204.

(33) Yamagaki, T.; Fukui, K.; Tachibana, K. Anal. Chem. 2006, 78, 1015–1022.

Figure 2. Stable complex structures with a sodium cation, calculated for maltose (a) and cellobiose (b). Distances (Å) from the sodium cation to the ring oxygen, the hydroxymethyl group, and the hydroxy group are shown.

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Table 1. Sodium Cation Binding Affinities (∆H298) of 26 Disaccharidesa binding affinity GlcR1-2Glc (kojibiose) Glcβ1-2Glc (sophorose) GlcR1-3Glc (nigerose) Glcβ1-3Glc (laminaribiose) GlcR1-4Glc (maltose) Glcβ1-4Glc (cellobiose) GlcR1-6Glc (isomaltose) Glcβ1-6Glc (gentiobiose) GalR1-3Gal GalR1-4Gal GalR1-6Gal Galβ1-4Gal ManR1-3Man ManR1-4Man ManR1-6Man Manβ1-4Man Manβ1-4GlcNAc FucR1-3GlcNAc FucR1-4GlcNAc FucR1-6GlcNAc NeuNAcR2-3Gal NeuNAcR2-6Gal GlcNAcR1-4GlcNAc GlcNAcβ1-4GlcNAc GalNAcR1-4GalNAc GalNAcβ1-4GalNAc a

-62.1 -64.3 -57.4 -58.4 -60.2 -63.9 -66.5 -75.4 -64.6 -64.1 -67.1 -55.0 -69.8 -62.9 -75.9 -61.1 -75.7 -56.8 -63.8 -66.6 -75.4 -77.8 -61.0 -61.3 -78.8 -76.9

Units are kcal/mol.

of the 2-OH group on C1 is feasible. We thus followed the fragmentation mechanism including the attack of the 2-OH group on C1 in the fragmentation analyses of glucobioses. Scheme 1 shows the fragmentation mechanism of the glycosyl bond cleavage for maltose. Fragmentation pathways of B,Y-type in glucobioses with various linkages (1-2, 1-3, 1-4, 1-6) were calculated at the HF/ 6-31G(d) level. The geometries of the stable complex with sodium cation, transition state, and product intermediate structures for gentiobiose are shown in Figure 3. In these three states, sodium cation interacts with C-1 (anomeric) oxygen, C-6 hydroxy oxygen, and two ring oxygen atoms. The coordination of metal ion to saccharide is loosely fixed through the reaction pathway in Figure 2. From the initial state to the transition state, the angle of O2-C2-C1 decreased from 111.0° (initial state) to 86.4° (transition state), indicating that O2 attacked C1 in the transition state. The distances of the ring oxygen from the sodium cation were 2.33 (initial state), 2.91 (transition state), and 2.39 (product intermediate) Å, respectively. It is intriguing that the distance from the ring oxygen to the sodium cation was increased in the transition state, whereas the distances remained shorter in the initial state and the product intermediate. This elongation of the distance indicates an electronic repulsion between the sodium cation and the ring oxygen, which has a positive charge relative to the oxygen in the initial state. Additionally, the bond lengths of Oring-C1 were 1.41 (initial), 1.29 (transition state), and 1.38 (product intermediate) Å, respectively, indicating that the chemical bond of Oring-C1 in the transition state is close to a double bond (CdO). Figure 4 shows the potential energy profiles of the glycosyl bond cleavage for maltose (R1-4), isomaltose (R1-6), and cellobiose (β1-4). The calculated activation energies of these

glucobioses increased in the order of maltose (R1-4) < isomaltose (R1-6) < cellobiose (β1-4). The difference in activated energy between the R1-4 and R1-6 linkages is attributable to the fact that the R1-6 linkage is stabilized more than the R1-4 linkage because of the formation of a tetradentate complex with the sodium cation. The experimental inclination of the stability of a variety of glycosyl bonds was studied by utilizing MALDIPSD and ESI-CID analyses.34,35 The order of the stabilities of these three glycosyl bonds was R1-4 < R1-6 < β1-4. Focusing on the energetic difference between the initial state and the product intermediate, the values for maltose and isomaltose were 12.2 and 14.3 kcal/mol. On the other hand, the value of the energetic difference for cellobiose was 72.8 kcal/mol, which was much higher than those of maltose and isomaltose. In the fragmentation of glucobioses the product intermediates of the fragmentation of the glycosyl bond consist of anGlc and Glc, regardless of linkage type. The energetic difference in the product intermediate is caused by the difference in energy between R-anGlc and β-anGlc. Figure 5a shows the structures and difference in total energy of R-anGlc and β-anGlc. The total energy of R-anGlc is lower than that of β-anGlc by 57.1 kcal/mol because of the anomeric effect. The anomeric effect is a stereoelectronic effect derived from an interaction between the lone pair of the ring oxygen and the antibonding molecular orbital of the axial C1-O bond as shown in Figure 5b.36 In the R-configuration, there is a hyperconjugation from the lone pair of the ring oxygen into the proximate C1-O antibonding molecular orbital, which has a conformation antiperiplanar to the lone pair. The anomeric effect is not expected in β-configuration, since the conformation between the lone pair and the C1-O bond is anticlinal. Thus the energy states of the product intermediate of glucobioses that have the β-configuration are always higher than those of glucobioses that have the R-configuration. According to the Hammond postulation, the transition state of an endothermic reaction resembles the product.37 In this sense, the stability and geometry of the product intermediate is important in discussing the stability of the transition state. In the R-linkage, the geometry of the transition state is similar to that of its product intermediate and is considered to be stabilized by the anomeric effect. On the other hand, the contribution of the anomeric effect is less expected in the β-linkage; consequently, the activation energy is higher than that of the R-linkage. Table 2 summarizes the total energies of the initial state, transition state, and product intermediate of glucobioses in accordance with the proposed fragmentation mechanism. The order of the lability of the glycosyl bonds is described as follows: R1-3 < R1-4 < R1-2 < R1-6 < β1-3 < β1-4 < β1-2 < β1-6. Glucobioses with the 1-6 linkage have the most stable glycosyl bonds among the R- and β-linkages, as discussed in the previous section. Comparison of activation energies within the same linkage type revealed that the order of activation energies was R-glycoside < β-glycoside. The contribution of the anomeric effect is considered to be a major factor in the lability of the glycosyl bonds in glucobioses. The lone pair of the ring oxygen has a conformation (34) Yamagaki, T.; Nakanishi, H. Proteomics 2001, 1, 329–339. (35) Kurimoto, A.; Daikoku, S.; Mutsuga, S.; Kanie, O. Anal. Chem. 2006, 78, 3461–3466. (36) Juaristi, E.; Cuevas, G. Tetrahedron 1992, 48, 5019–5087. (37) Hammond, G. J. Am. Chem. Soc. 1955, 77, 334–338.


1111

Scheme 1. Proposed Reaction Mechanism of Fragmentation of Glycosyl Bond of Maltose (r1-4 Linkage)

antiperiplanar to the C1-O bond in the R-glycoside, whereas the conformation of the lone pair and the C1-O bond is anticlinal in the β-glycoside. Generally, an antiperiplanar conformation is more favorable than an anticlinal conformation in the elimination reaction. From this perspective, the elimination of R-glycoside is considered to be more favorable than that of β-glycoside. Stabilization by the sodium cation is also an important factor. In the same linkage, the order of binding affinity of the sodium cation was R-linkage < β-linkage in the glucobioses. The binding affinities of the sodium cation for R-linked glucobioses increased in the order of R1-3 < R1-4 < R1-2 < R1-6, which is completely the same as the order of the activation energies. The same trend was

also observed in β-linked glucobioses. This result indicates that the binding affinity to the sodium cation was correlated with the activation energy, which will be discussed later. Fragmentation of Galactobioses. Galactose is an epimer of glucose at the 4 position. Fragmentations of galactobioses (GalGal) with R1-3, R1-4, β1-4, and R1-6 linkages were studied in

Figure 3. Optimized structures of the initial geometry of [gentiobiose + Na]+ (a), the transition state (b), and the product intermediate (c).

Figure 4. Potential energy profiles of the fragmentation pathways of maltose (a), isomaltose (b), and cellobiose (c). Units are kcal/ mol.

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Figure 5. Structures and relative energies of R-anGlc and β-anGlc (a), and the representation of the anomeric effect in R-configuration (b).

an attempt to verify the influence of stereochemical difference on the fragmentation of galactose and glucose. The binding affinities of the sodium cation to the galactobioses increased in the order of β1-4 < R1-4 < R1-3 < R1-6. In the galactobioses, β-linkage had less affinity to the sodium cation than R-linkage, a fact that is contrary to the trend in the glucobioses. Epimerization at the 4 position influences the structural features; therefore, the coordination of the sodium cation with the saccharide molecule may differ between the glucobioses and galactobioses. Such structural differences may cause an inversion of the affinity for the sodium cation. The fragmentation mechanism was calculated according to the reaction mechanism described in Scheme 1. Table 3 summarizes the energies calculated for the initial state, transition state, and product intermediate. The activation energies of the fragmentation are arranged in the order of R1-3 < R1-4 < R1-6 < β1-4. The trend in the activation energy is R-configuration < β-configuration, the same as the trend in glucobioses. We suspected that the relative configuration between 2-OH and the glycoside oxygen is a factor crucial to the fragmentation mechanism. In this sense, the relative configuration of the 2-OH group and the glycoside oxygen is the same in glucose and galactose; therefore, a trend by which the R-linkage is more fragile than the β-linkage is commonly observed in the fragmentation of glucoand galactobioses. The calculated activation energies of the fragmentation of GalR1-6 and GlcR1-6 were 59.3 and 64.5 kcal/mol, respectively. Note that R-Gal is a more labile linkage than R-Glc. In the experimental analyses of the fragmentation of the glycosyl bond, the fragmentations of Glc- and Gal-β-cyclodextrin with R1-6 linkages were examined by the MALDI-PSD method.34 Analyses of ion intensity revealed that the glycosyl bond of galactose was more labile than that of glucose. To compare theoretically derived stabilities of glycosyl bond in galactobioses to experimentally derived ones, MALDI-CID spectra were obtained. Supporting Information, Figure SI-2 shows the MALDI-CID spectra of galactobioses with R1-3, R1-4, and β1-4 linkages. To evaluate the relative intensity of glycosyl bond fragment ions (B,Y-type), the relative intensities of each peak in the CID spectra were calculated as a percentage to the area of the largest peak. The relative abundances of fragment ions

generated by B,Y-type glycosyl bond cleavage of R1-3, R1-4, and β1-4 linkages were described in the order of β1-4 (58%) < R1-4 (114%) < R1-3 (135%), indicating that the stabilities of three linkages are in the order of R1-3 < R1-4 < β1-4. Experimentally derived stabilities of glycosyl bonds were consistent with the calculated stabilities for these three linkages. Fragmentation of Mannobioses. Mannose is an epimer of glucose at the 2-position; this feature is characteristic of mannose but not glucose and galactose. Glucose and galactose have equatorial orientation at 2-OH, but mannose has axial orientation at 2-OH. In the fragmentation of mannobiose, the 2-OH group is supposed to attack C1 for glycosyl bond cleavage to take place. Table 4 summarizes the calculated energies for the fragmentation of mannobioses (Man-Man) with R1-3, R1-4, β1-4, and R1-6 linkages and Manβ1-4GlcNAc. The activation energies for the fragmentation increase in the order of Manβ1-4Man < Manβ14GlcNAc < ManR1-4Man < ManR1-3Man < ManR1-6Man. In a MALDI-PSD study of the fragmentation of R-Glc, R-Gal, and R-Man linkages, the order of stabilities increased as R-Gal < R-Glc < R-Man.34 This trend agreed with the order of our calculated activation energies for R-Glc, R-Gal, and R-Man. Examination of the calculated activation energies revealed that R1-6 was more stable than the other linkages, as was the case for gluco- and galactobiose. In the β-linkages, the activation energies of the glycosyl bond were in the order of Manβ1-4Man < Manβ1-4GlcNAc. The acetoamide group was more likely to be negatively charged than the hydroxy group; we surmised that the electronegativity of the glycoside oxygen was affected by the presence of the neighboring acetoamide group. We consider that the electronegative charge of the glycoside oxygen affects the stability of the glycosyl bond; the difference of activation energies between Manβ1-4Man and Manβ1-4GlcNAc is due to the presence of an acetoamide group at the GlcNAc moiety. The activation energies of β-linked mannobiose were notably lower than those of R-linked mannobiose; in contrast, the stability of the glycosyl bond in the gluco- and galactobioses was R-configuration < β-configuration. Figure 6 shows the structures and Newman projections of mannobioses. From the Newman projections, we characterized the stereochemical configuration of the 2-OH and glycoside oxygen in the R-configuration as trans (1,2-trans-glycoside) and as cis in β-configuration (1,2-cis-glycoside). In light of these stereochemical features, the lability of the β-linked mannose is due to the fact that the 2-OH group can easily attack the glycoside oxygen, since the 2-OH group is close to the glycoside oxygen. On the other hand, in the R-linked mannobioses, the 2-OH group is in a conformation antiperiplanar to the glycoside oxygen and distant from the glycoside oxygen; proton migration from 2-OH to the glycoside oxygen is unfavorable. Thus, the mannose moiety at the nonreducing end had to develop a distorted conformation to undergo fragmentation in the transition state (Supporting Information, Figure SI-3); this made the activation energy higher than those of the β-linked mannobioses. Fragmentation of r-Fuc and r-NeuNAc. Fucose, a deoxyhexose, is a biologically relevant monosaccharide that has been found in some tumor-associated blood-group glycosphingoAnalytical Chemistry, Vol. 81, No. 3, February 1, 2009

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Table 2. Activation Energies for Glucobioses Undergoing Glycosyl Bond Fragmentationa R-configuration linkage 1-2

1-3

1-4

1-6

a

total energy ZPVEb E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol)

β-configuration

initial

TS

product

initial

TS

product

-1452.409339 0.405660 -1452.003679

-1452.303063 0.400062 -1451.903001 63.2 -1452.284000 0.395138 -1451.888862 60.4 -1452.296461 0.399121 -1451.897340 60.9 -1452.296906 0.398598 -1451.898308 64.5

-1452.386599 0.402452 -1451.984147

-1452.409002 0.404311 -1452.004691

-1452.289566 0.400840 -1451.888726

-1452.371603 0.401159 -1451.970444

-1452.396724 0.403541 -1451.993183

-1452.377226 0.402198 -1451.975028

-1452.406758 0.404800 -1452.001958

-1452.379641 0.401299 -1451.978342

-1452.419977 0.404274 -1452.015703

-1452.245756 0.399106 -1451.846650 99.2 -1452.235813 0.398670 -1451.837143 97.9 -1452.245024 0.399695 -1451.845329 98.3 -1452.248541 0.398924 -1451.849617 104.2

-1452.390127 0.405034 -1451.985093 -1452.400362 0.405913 -1451.994449 -1452.405719 0.404666 -1452.001053

-1452.280018 0.400444 -1451.879574 -1452.287333 0.401353 -1451.885980 -1452.291881 0.401201 -1451.890680

Energy values are in Hartrees unless noted otherwise. b ZPVE, zero-point vibrational energy.

Table 3. Activation Energiesa of Galactobioses Undergoing Glycosyl Bond Fragmentation compound GalR1-3Gal

GalR1-4Gal

GalR1-6Gal

Galβ31-4Gal

a


initial

TS

product

-1452.407956 0.407029 -1452.000927

-1452.311047 0.398350 -1451.912697 55.4 -1452.301685 0.400262 -1451.901423 57.9 -1452.312111 0.397314 -1451.914797 59.3 -1452.255329 0.402239 -1451.853090 85.5

-1452.387217 0.403066 -1451.984151

-1452.399802 0.406157 -1451.993645 -1452.415281 0.405913 -1452.009368 -1452.396258 0.406840 -1451.989418

-1452.382902 0.40388 -1451.979022 -1452.388951 0.402581 -1451.986370 -1452.283169 0.403021 -1451.880148


lipids.38-40 In analyses of fucosyl oligosaccharides by mass spectrometry, the fucosyl linkage tends to be more labile than the other glycoside linkages.30 As a target of fucosyl disaccharide, Fuc-GlcNAc was chosen. The reaction mechanism of Fuc-GlcNAc can be followed in Scheme 1 and yields 1,2-epoxy-R-L-fucopyranose as a product. Sialic acids are a large family of 9-carbon carboxylated saccharides that are generally found in biosystems. One member of this family, N-acetylneuraminic acid (NeuNAc), is most commonly found at the terminal ends of glycoproteins and glycolipids.41 In general, the sialic acid residue is linked to the galactose residue in either an R2-3 or an R2-6 linkage by the enzymatic action of sialyltransferases. The sialyl linkage is known to be labile; therefore, there is inevitable cleavage of the sialic acid residue, which yields a Y-type product ion in the ionization process. The (38) Larkin, M.; Ahern, T. J.; Stoll, M. S.; Shaffer, M.; Sako, D.; Obrien, J.; Yuen, C. T.; Lawson, A. M.; Childs, R. A.; Barone, K. M.; Langersafer, P. R.; Hasegawa, A.; Kiso, M.; Larsen, G. R.; Feizi, T. J. Biol. Chem. 1992, 267, 13661–13668. (39) Erbe, D. V.; Watson, S. R.; Presta, L. G.; Wolitzky, B. A.; Foxall, C.; Brandley, B. K.; Lasky, L. A. J. Cell Biol. 1993, 120, 1227–1235. (40) Greenwell, P. Glycoconjugate. J. 1997, 14, 159–173. (41) Lasky, L. A. Science 1992, 258, 964–969.

1114


cleavage results in a loss of linkage information for sialic acid; a variety of methods to stabilize the sialic acid linkage have been studied.27,42-44 In the enzymatic reaction of sialidases, 2,3-dehydro-2-deoxyN-acetylneuraminicacid (NeuNAc2en) is a potent inhibitor of the reaction mechanism since this molecule is a transition state analogue of the reaction mechanism of sialidase.45 On consideration of the property of NeuNAc2en in the reaction mechanism of sialidase, the reaction mechanism which yields NeuNAc2en as a product intermediate was proposed as shown in Supporting Information, Scheme SI-1. This reaction takes place by the proton elimination at C2 and consequently generates NeuNAc2en and Gal as a product intermediate. The geometries of initial, transition (42) Powell, A. K.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 1027–1032. (43) Sekiya, S.; Wada, Y.; Tanaka, K. Anal. Chem. 2005, 77, 4962–4968. (44) Toyoda, M.; Ito, H.; Matsuno, Y. K.; Narimatsu, H.; Kameyama, A. Anal. Chem. 2008, 80, 5211–5218. (45) Vonitzstein, M.; Wu, W. Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron, J. M.; Penn, C. R. Nature 1993, 363, 418–423.

Table 4. Activation Energiesa of Mannobioses Undergoing Glycosyl Bond Fragmentation compound ManR1-3Man

ManR1-4Man

ManR1-6Man

Manβ1-4Man

Manβ1-4GlcNAc

a

total energy ZPVEb E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol)

initial

TS

product

-1452.399865 0.404933 -1451.994932

-1452.248500 0.399861 -1451.848639 91.8 -1452.25375 0.400739 -1451.853011 89.4 -1452.259449 0.399941 -1451.859508 94.4 -1452.323291 0.401851 -1451.976740 55.8 -1584.285683 0.457751 -1583.827932 63.5

-1452.284507 0.401409 -1451.883098

-1452.402494 0.406948 -1451.995546 -1452.414589 0.4047 -1452.009889 -1452.416553 0.406179 -1452.010374 -1584.389229 0.460054 -1583.929175

-1452.289553 0.401964 -1451.887589 -1452.294754 0.401504 -1451.893250 -1452.380221 0.403481 -1451.921440 -1584.356624 0.456452 -1583.900172


Figure 6. Mannobioses with R- and β-configurations, and their Newman projections in the direction of C-1 to C-2 at the nonreducing end. R represents the sugar residue at the reducing end.

state, and product intermediate structures were shown in Supporting Information, Figure SI-4. The calculated activation energy was 106.6 kcal/mol, which was not consistent with the fact that the sialyl linkage is a labile glycosyl bond. It is considered that C-2 proton is not reactive; consequently the elimination reaction required high energy. As another candidate of fragmentation mechanism of sialyl linkage, the reaction mechanism including the proton migration of carboxylic group was shown in Scheme 2. In this mechanism, fragmentation takes place by transfer of the lone pair of the ring oxygen. Then the proton of the carboxylic group, which is the most acidic proton in the molecule, migrates to the glycoside oxygen. The lability of sialyl glycoside is explained by the following calculation. Table 5 shows the energies calculated for the fragmentation of Fuc-GlcNAc and NeuNAc-Gal. Optimized structures for the initial state, transition state, and product intermediate of FucR13GlcNAc and NeuNAcR2-6Gal are shown in Supporting Information, Figures SI-5 and SI-6, respectively. The calculated activation energies are notably lower than those of the gluco-, galacto-, and mannobioses. The activation energies of Fuc-GlcNAc are lower than those of R-Glc, R-Gal, and β-Man by more than 10 kcal/mol, although the stereochemical orientation of 2-OH and glycoside oxygen is 1,2-cis-axial in these disaccharides. The difference

between the fucosyl disaccharides and the other disaccharides that have 1,2-cis-glycoside is the presence of a methyl group at the 6 position. In the transition state, the oxonium ion of fucose is stabilized by a proximate methyl group, which is electron rich; therefore, the transition state is energetically lowered. This stabilization effect is not expected in the disaccharides of R-Glc, R-Gal, and β-Man, since they have a hydroxy group instead of a methyl group at the 6 position. Thus the lability of the fucosyl linkage is due to the neighboring group effect of the methyl group in the fucose moiety. The order of the activation energies of the R2-3 and R2-6 linkages was R2-3 < R2-6, and the difference in activation energies was small. From the calculations, we considered that discrimination of the R2-3 and R2-6 linkages was difficult in the CID experiment using the precursor ion of [NeuNAc-Gal + Na]+. In the fragmentation analyses of sialyl saccharides in mass spectrometry, discrimination of these structural isomers is barely achieved. To overcome this problem, modification to sialyl saccharides by the attachment of transition metals27 or derivatization of hydroxy groups such as methylation has been reported.42,46,47 The energy of stabilization by the sodium cation was large in sialyl disaccharides (Table 1), but the activation energies for sialyl disaccharides were lower than those for the other disaccharides. A characteristic of the sialyl residue is the presence of a carboxyl group that has an acidic proton. The carboxyl group makes proton migration to the glycoside oxygen much easier; this is considered to be a major factor in lowering of the activation energy. The carboxyl group also contributes to stabilization of the transition state by delocalizing a negative charge at the carboxylic group. Fragmentation of r,β-GlcNAc and r,β-GalNAc. The structure of GlcNAcβ1-4GlcNAc that is linked to an asparagine residue in the β-configuration is a typical motif found in N-linked (46) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 4441– 4447. (47) Chen, P.; Werner-Zwanziger, U.; Wiesler, D.; Pagel, M.; Novotny, M. V. Anal. Chem. 1999, 71, 4969–4973.


1115

Scheme 2. Reaction Pathway of Glycosyl Bond Fragmentation in NeuNAcr2-3Gal

Table 5. Activation Energiesa of Fucosyl and Sialyl Saccharides Undergoing Glycosyl Bond Fragmentation compound FucR1-3GlcNAc

FucR1-4GlcNAc

FucR1-6GlcNAc

NeuNAcR2-3Gal

NeuNAcR2-6Gal

a

total energy ZPVEb E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol) total energy ZPVE E + ZPVE ∆Ea (kcal/mol)

initial

TS

product

-1509.511437 0.45394 -1509.057497

-1509.432452 0.445394 -1508.987058 44.2 -1509.435223 0.446356 -1508.988867 45.6 -1509.424686 0.445125 -1508.979561 49.6 -1924.819557 0.538364 -1924.281193 36.1 -1924.839113 0.540420 -1924.298693 36.4

-1509.480189 0.450869 -1509.029320

-1509.515950 0.454363 -1509.061587 -1509.512202 0.453625 -1509.058577 -1924.880122 0.541393 -1924.338729 -1924.899991 0.543212 -1924.356779

-1509.484215 0.451260 -1509.032955 -1509.476096 0.450963 -1509.025133 -1924.851454 0.539204 -1924.312250 -1924.851258 0.539146 -1924.312112


Figure 7. Stable complex structures of GlcNAcR1-4GlcNAc (a), and GlcNAcβ1-4GlcNAc (b) with a sodium cation. In (a), the distances from the sodium cation to the 6-OH at the nonreducing end, the ring oxygen, and the 3-OH at the reducing end are 2.34, 2.24, and 2.32 Å, respectively. In (b), the distances from the sodium cation to the carbonyl oxygen at the 2 position and the 6-OH at the reducing end are 2.14, 2.19 Å, respectively.

oligosaccharides. In this section, the mechanisms of fragmentation ofGlcNAcβ1-4GlcNAc,GlcNAcR1-4GlcNAc,GalNAcR1-4GalNAc, and GalNAcβ1-4GalNAc were studied. Figure 7 shows the stable complex structures of GlcNAcR1-4GlcNAc and GlcNAcβ14GlcNAc with a sodium cation. In the stable complex structure of GlcNAcR1-4GlcNAc, the saccharide molecule interacts with 6-OH, the ring oxygen at the nonreducing end, and the 3-OH of the glycosyl acceptor. Note that the 6-OH group at the nonreducing end is not flexible because of the interaction with the sodium cation, and the amide proton of the acetoamide group is in the vicinity of the glycoside oxygen. A possible fragmentation pathway that takes into account these structural features is shown in 1116


Scheme 3. This fragmentation takes place by (i) electron transfer of the lone pair on the ring oxygen and amide proton transfer; and (ii) successive formation of an aziridine group. In the complex structure of GlcNAcβ1-4GlcNAc in Figure 7b, the saccharide molecule forms a tridentate interaction with the sodium cation in the carbonyl oxygen at the 2 position and the 6-OH at the reducing end; this is notably a different geometry compared with that of GlcNAcR1-4GlcNAc. The fragmentation mechanism involving the 2-amide proton is unfavorable, since the amide proton is more distant from the glycoside oxygen than in the case of R-GlcNAc. In the fragmentation mechanism shown in Scheme 4 we therefore proposed attack of the C-1 carbon by the 6-OH group. In this mechanism, the GlcNAc residue at the nonreducing end has a boat conformation to undergo fragmentation. The proposed fragmentation takes place as follows: (i) electron transfer from the ring oxygen and proton migration of the C-2 hydroxy group at the nonreducing end; and (ii) continuous formation of 1,6-anhydro-D-GlcNAc by the oxonium ion. This mechanism is also applicable to GalNAcβ1-4GalNAc. In accordance with the proposed fragmentation mechanism, the calculated energies for the initial state, transition state, and the product intermediate are summarized in Table 6, and snapshots of the reaction pathways for GlcNAcR1-4GlcNAc and GlcNAcβ1-4GlcNAc are shown in Supporting Information, Figures SI-7 and SI-8, respectively. In the fragmentation of β-linked GlcNAc-GlcNAc and GalNAc-GalNAc, the energetic differences between the initial state and the product intermediate were -13.7 and -13.2 kcal/mol, indicating that the product intermediate was more stable than the initial geometry. It is interesting that the

Scheme 3. Reaction Pathway of Glycosyl Bond Fragmentation of GlcNAcr1-4GlcNAc

Scheme 4. Reaction Pathway of Glycosyl Bond Fragmentation of GlcNAcβ1-4GlcNAc

Table 6. Activation Energiesa of GlcNAc-GlcNAc and GalNAc-GalNAc Undergoing Glycosyl Bond Fragmentation compound GlcNAcR1-4GlcNAc

GlcNAcβ1-4GlcNAc

GalNAcR1-4GalNAc

GalNAcβ1-4GalNAc

a


initial

TS

product

-1716.341942 0.514634 -1715.827308

-1716.230468 0.509585 -1715.720883 66.8 -1716.244061 0.509291 -1715.734770 45.0 -1716.245479 0.508098 -1715.737381 66.0 -1716.260727 0.510169 -1715.750558 52.3

-1716.303603 0.510645 -1715.792958

-1716.322482 0.516074 -1715.806408 -1716.356908 0.514330 -1715.842578 -1716.349026 0.515044 -1715.833982

-1716.342130 0.513893 -1715.828237 -1716.304930 0.510577 -1715.794353 -1716.369180 0.514112 -1715.855068


product intermediate became more stable than the initial geometry although the product intermediate was a sterically hindered molecule. To understand this phenomenon, we focused on the energy of 1,6-anhydro-D-GlcNAc, which is a bis acetal molecule around O6, C1, and the ring oxygen. This acetal has a boat conformation (BO,3), and the stereochemical conformation between the lone pair of ring oxygen and the C1-O6 bond is antiperiplanar; in the same way, the stereochemical conformation between the lone pair of O6 and C1-Oring is also antiperiplanar. This acetal is stabilized by “doubly” anomeric effects, that is, a state of simultaneous occurrence of double anomeric effects from the lone pair of Oring to the antibonding

molecular orbital of C1-O6 and from the lone pair of O6 to the antibonding molecular orbital of C1-Oring. Comparison of the activation energies for R- and β-configurations revealed that the order of the activation energies were β < R in GlcNAcGlcNAc and GalNAc-GalNAc. We considered that the major factor causing the difference in the activation energies for the R- and β-linkages in GlcNAc-GlcNAc and GalNAc-GalNAc was the difference in the mechanism of fragmentation. The R-linked disaccharide yielded a strained aziridine structure (Scheme 3); on the other hand, the β-linked disaccharide yielded bis acetal, which consists of 5- and 6-membered rings (Scheme 4). Generally, a reaction that yields a strained 3-membered ring requires more Analytical Chemistry, Vol. 81, No. 3, February 1, 2009

1117

Figure 8. Scatterplot of activation energies and binding affinities of the sodium cation in 26 disaccharides. X and Y axes represent the affinity for the sodium cation and the activation energy, respectively. Linkage type is noted next to each mark. Blue, red, and green markers represent saccharides in groups (i), (ii), and (iii), respectively.

energy than one yielding a 5-membered ring; it is rational that fragmentation of the R-linked disaccharide requires more energy than that of the β-linked disaccharide. The energetic differences between the initial state and the product intermediate for R- and β-linked GlcNAc-GlcNAc were 22.6 and -13.2 kcal/mol, respectively. The product intermediate of β-GlcNAc was more stable than that of R-linked disaccharide. In fact, the geometries of the transition states for R- and β-GlcNAc were more similar to their product intermediates than those of the initial states (Supporting Information, Figures SI-7 and SI-8). The transition state of β-GlcNAc is surmised to be stabilized by “doubly” anomeric effects, whereas such stabilization is not expected in R-GlcNAc. This stabilization is a crucial point that accounts for discrimination of the activation energies of R- and β-GlcNAc. In the fragmentation of R- and β-GalNAc, the order of activation energies is β-GalNAc < R-GalNAc. The trend of fragmentations of R- and β-GalNAc can also be explained by the same discussions on R- and β-GlcNAc. Relationship between Activation Energy and Affinity for the Sodium Cation. We considered that the stability of the complex with the sodium cation influences the lability of the glycosyl bond. To investigate the structure-reactivity relationships of the sodiated disaccharides, the correlation between the affinities to the sodium cation and the calculated activation energies for each saccharide were plotted (Figure 8). In the scatter plot, we can divide the saccharides into three groups; (i) a group containing β-Glc, β-Gal, R-Man, β-GlcNAc, and β-GalNAc; (ii) a group containing R-Glc, R-Gal, β-Man, R-Fuc, R-GlcNAc, and R-GalNAc; and (iii) sialyl saccharides. The common feature of the saccharides in group (i) is that the type of glycosyl bond is classified as 1,2trans-glycoside. The β-Glc, β-Gal, and R-Man linkages in group (i) have higher activation energies than those in groups (ii) and (iii). On the other hand, the stabilities of the β-GlcNAc and β-GalNAc linkages are lower than the β-Glc, β-Gal, and R-Man linkages, since the mechanisms of fragmentation of these two 1118


linkages are different from those of β-Glc, β-Gal, and R-Man, as discussed earlier. It is intriguing that the affinity of the sodium cation was correlated with the calculated activation energies in the linkage isomers. The relationships between the calculated activation energy and the binding affinity of the sodium cation for R-Man, β-Glc, and R-Glc showed linear correlations, and their R2 values were 0.99, 0.93, and 0.91, respectively. In group (ii), the saccharide that had the high binding affinity had a higher activation energy. These results indicated that the stability of the glycosyl bonds of saccharides that had a high affinity for the sodium cation tended to be high. Notably, the 1-6 linkage was the most stable of all the linkages because this linkage is highly stabilized in energy by tetradentate interaction with the sodium cation. This characteristic may be a crucial factor in distinguishing between the 1-6 linkage and the other linkages. The major factor distinguishing group (i) from (ii) is the contribution of the anomeric effect. Saccharides in group (ii) that have 1,2-cis-glycoside are stabilized by the anomeric effect through the reaction pathways shown in Scheme 1; therefore, the activation energies are lower than those in group (i). Unlike in group (ii), in group (i) the anomeric effect is less likely to contribute to the fragmentation of β-Glc, β-Gal, and R-Man; consequently the activation energies in group (i) tend to be higher. However, the sialyl saccharides in group (iii) showed an exceptional trend in the relationship between binding affinity and activation energy. Although sialyl saccharides are stabilized by the sodium cation by 75-80 kcal/mol, the activation energies were the lowest among the saccharides investigated in this study. This exceptional behavior is due to the presence of a reactive acidic proton in the sialyl moiety. Proton migration can easily take place because of the high acidity of the proton, and the fragmentation reaction is less affected by stabilization by the sodium cation.

Table 7. Relative Intensities of Ions Generated from the Particular Glycosyl Bond Cleavages in CID Spectra of Oligosaccharides48

1

Numbers of glycans correspond to the numbers in Figure 1.

2

GlcNAcβ1-4GlcNAc.

Theoretically and Experimentally Deduced Inclinations of the Stability of Glycosyl Bonds. Extensive CID studies of the various oligosaccharides shown in Figure 1 have been performed to investigate the stabilities of the various glycosyl bonds under CID. As a parameter for comparison of the stability of glycosyl bonds during low-energy CID, we took the sum of the relative intensities of two types of fragment ion, B,C-series and Y,Z-series, generated by a single cleavage. In the oligosaccharides containing plural bonds of the same type of glycoside, the parameters for the corresponding glycoside were divided by the number of the corresponding glycosyl bond. The detailed procedure for the derivation of the relative intensities of glycosyl bond cleavages was shown in Supporting Information, Figure SI-9 and Table SI-1. Table 7 summarizes these values for the various types of glycoside normally found in glycolipids and glycoproteins. Values in Table 7 represent the stability of glycosyl bonds; the small value represents the glycosyl bond is stable, whereas the large value represents the glycosyl bond is labile. For the oligosaccharides containing both R-Fuc and R-NeuNAc, larger values were obtained from R-NeuNAc, which means that R-Fuc is more stable during CID (9 in Figure 1). The relationship of the calculated activation energies between R-Fuc and R-NeuNAc was consistent with the stabilities of these linkages during CID. Although R-Fuc is well-known to be labile under positive-ion CID, it is interesting that the intermediate values were obtained from R-Fuc in the N-linked oligosaccharides. This fact suggested that

a comparably labile glycoside exists in the N-linked oligosaccharides, that is, the GlcNAcβ1-4 bond in the chitobiose moiety of N-linked oligosaccharides (Table 7). The calculated activation energies of β-GlcNAc and R-Fuc reveal that the stability of β-GlcNAc is similar to that of R-Fuc. In saccharides with both β-GlcNAc and R-Fuc linkages, the fragmentations of the β-GlcNAc and R-Fuc linkages compete with each other in the CID process. We focused on the differences in stability of the anomeric linkages. In the cases of R- and β-Gal, whereas β-Gal afforded relatively small values the corresponding R-Gal afforded large values. On the other hand, the contrary trend was observed with the glycosides of Man, GalNAc, and GlcNAc: β-glycosides of these had large values, whereas R-glycosides had small values. These trends were in good accord with the theoretically deduced inclinations for these linkages. The stability of glycosides can be compared by using the values derived from relative ion intensities when the different types of glycosides exist in a single molecule. The variety of glycosides could be arranged in the order of stability under CID as follows: R-Man, β-Gal, R-GalNAc, R-Gal, β-Man, β-GalNAc, β-GlcNAc, and R-Fuc, R-Neu. In a previous study, we revealed that different trends in fragmentation were observed even for the same type of glycosides.48 β-GlcNAc residues at the branching position of the multi-antennary structure of N-linked oligosaccharides have different stabilities from each other. The (48) Kameyama, A.; Nakaya, S.; Ito, H.; Kikuchi, N.; Angata, T.; Nakamura, M.; Ishida, H. K.; Narimatsu, H. J. Proteome Res. 2006, 5, 808–814.


1119

trend in dissociation during low-energy CID as shown aboveswhich is generally applicablesmay be a major factor defining the intensity profiles of the CID spectra of oligosaccharides. The calculated activation energies of the variety of glycosides under CID are arranged as follows: R-Man (ManR1-3Man, ManR1-4Man, ManR1-6Man) > β-Gal (Galβ1-4Gal) > R-GalNAc (GalNAcR1-4GalNAc) > β-Man (Manβ1-4GlcNAc) > R-Gal (GalR1-3Gal, GalR1-4Gal, GalR1-6Gal) > β-Man (Manβ1-4Man) > β-GalNAc (GalNAcβ1-4GalNAc) > R-Fuc (FucR1-6GlcNAc) > R-Fuc (FucR1-4GlcNAc) > β-GlcNAc (GlcNAcβ1-4GlcNAc) > R-Fuc (FucR1-3GlcNAc) > R-NeuNAc (NeuNAcR2-3Gal, NeuNAcR2-6Gal). In the calculated trend, the order of stabilities for R-Fuc (FucR1-4GlcNAc) and β-GlcNAc was inverted compared with that of the experimentally deduced stabilities, and the difference in stabilities was 0.6 kcal/mol. This is because the calculated stabilities were derived from model disaccharides and not from the whole structures of the oligosaccharides investigated in the CID experiments. In the case of oligosaccharide 24 in Figure 1, which has R-Fuc, β-Gal, R-Man, β-Man, and β-GlcNAc linkages, the experimentally deduced trends in stability of these linkages are described in the order of R-Man (ManR1-3Man, ManR1-6Man) > β-Gal (Galβ1-4GlcNAc) > β-Man (Manβ1-4GlcNAc) > R-Fuc (FucR1-3GlcNAc) ≈ β-GlcNAc (GlcNAcβ1-4GlcNAc)sthe same order as that of the theoretically calculated stabilities of glycosyl bonds. The order of the calculated activation energies was mostly correlated with the stability of the glycosyl bonds in the CID experiments, indicating that the stabilities of various linkages in the CID process can be explained well by the theoretical calculations for disaccharides. This result indicates that the theoretical analyses of glycosyl bond fragmentations in the modeled disaccharides were sufficiently reliable to account for the experimental trends of fragmentation, even in the complicated glycans. CONCLUSION Fragmentation mechanisms were investigated for sodiated disaccharides with various glycosyl bonds to establish general rules for fragmentation. To elucidate the fragmentation mechanisms, we performed computational calculations of the proposed fragmentation mechanisms in consideration of complex structures with a sodium cation. In the fragmentation of disaccharides such as Glc-Glc, Gal-Gal, and Fuc-GlcNAc, where the 2-OH group at the nonreducing end residue is involved in the reaction mechanism, the R-linkage is more labile than the β-linkage. In contrast, this trend was inverted in the case of Man-Man. Focusing on the stereochemical orientation between the 2-OH group and the glycoside oxygen, R-Glc, R-Gal, R-Fuc, and β-Man were classified as 1,2-cis-glycosides, whereas β-Glc, β-Gal, R-Man were classified as 1,2-trans-glycosides. In the fragmentation of 1,2-cis-glycoside, the transition state and the product intermediate were stabilized by the anomeric effect, whereas this stabilization did not occur

1120


in 1,2-trans-glycoside. As a result, the activation energy for 1,2cis-glycoside was lower than that of 1,2-trans-glycoside. The sialyl linkage was the most labile among the various linkages studied in this paper. Binding affinities of the sodium cation for various saccharides were calculated in an attempt to determine the structure-activity relationships. Analysis of a scatterplot of activation energies and binding affinities revealed that the activation energy increased in accordance with the increment in binding affinity. For example, the 1-6 linkage was more stable than the other linkages in the same sequence of disaccharides because saccharides with flexible 1-6 linkages were stabilized to a greater degree by forming a tetradentate interaction with the sodium cation. The correlation between activation energy and binding affinity indicates that the stability of the glycosyl bond increased in proportion with the degree of stabilization by the sodium cation. However, sialyl disaccharides exhibited exceptional behavior: the activation energies for sialyl disaccharides were notably low, although these molecules were highly stabilized by the sodium cation. This phenomenon is attributable to the presence of a carboxyl group. Proton migration can easily take place because of the presence of the acidic proton, and the delocalization of the charge on the carboxyl group contributes to lower energy in the transition states. Various fragmentation mechanisms were calculated in this study, and the calculated activation energies were consistent with the experimentally deduced trend in the stability of the glycosyl bonds in CID. This result indicates that the theoretical analyses of fragmentation in the modeled disaccharides were appropriate strategies for analyzing the fragmentation trends of glycans with complicated structures. Further analyses of linkages not investigated in this paper may lead to more accurate interpretation of the fragmentation of glycans in mass spectrometry. The general rules described here should be useful in glycomics, in which analysis of oligosaccharides is normally performed by mass spectrometry using limited amounts of samples. ACKNOWLEDGMENT Part of this study was financially supported by the Global COE Program for Chemistry Innovation and the Japan Society for the Promotion of Science (JSPS, 18-11463). The experimental work was performed as a part of the R&D Project of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization (NEDO). SUPPORTING INFORMATION AVAILABLE Additional information was noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 11, 2008. AC802230A

October

21,

2008.

Accepted

Computationally and Experimentally Derived General Rules for

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