influence of coordination number and ligand size on the dissociation mechanisms of transition...

Influence of coordination number and ligand size on thedissociation mechanisms of transition

metal-monosaccharide complexes

Glenn Smith, Azita Kaffashan, Julie A. Leary*

Department of Chemistry, University of California, Berkeley, CA 94720, USA

Received 31 July 1998; accepted 19 October 1998

Abstract

Several features of metal-carbohydrate complexes, in the form of deprotonated metal/N-glycoside ions, were varied in orderto determine which types of complexes would best enable mass spectrometric differentiation of the stereochemical features ofthe coordinated monosaccharides. Metal complexes were generated by using transition metals such as nickel, copper, and zinc.Additionally, the size and coordination number of ligands in the complex, as well as the number of these ligands coordinatedto the metal, were varied. By using a quadrupole ion trap mass spectrometer, multistage mass spectrometric experiments wereperformed on the electrospray-generated metalN-glycoside complexes. Several tricoordinate NiN-glycoside systems werecapable of differentiating the stereochemistry about the C-2 center in the monosaccharide ring, whereas the tricoordinate Cu/ensystem allowed for the differentiation of both the C-2 and C-4 stereocenters. No stereochemical differentiation was possiblefrom four- or five-coordinate species with the exception of the four-coordinate Zn/diencomplexes. Changes in the metal centeror size of theN-glycoside ligand generated the greatest changes in the product ion spectra of the tricoordinate complexes. Suchalterations in four- or five-coordinate complexes often did not result in greatly differing product ion spectra. Whereas theproduct ion spectra of most metal/N-glycoside complexes could be easily categorized according to structural features of theprecursor ion, exceptional species such as the four coordinate [Zn(dien/monosaccharide)–H]1 precursor ion, also capable ofdifferentiating the stereochemical features about C-2 and C-4, can give unexpected stereochemical information. (Int J MassSpectrom 182/183 (1999) 299–310) © 1999 Elsevier Science B.V.

Keywords:Transition metal; Monosaccharide; Electrospray ionization; Quadrupole ion trap; Stereochemistry.

1. Introduction

As the diverse composition and roles of carbohy-drates in biological systems become increasinglyapparent [1], so does the need for a rapid, sensitive

methodology that would allow for the identification ofthe sequence and structural features of these importantbiomolecules. Tandem mass spectrometry (MS/MS)is attractive because it can be used to analyze smallquantities of heterogeneous samples [2] such as thoseoften encountered with biological samples. Numerousexamples of the successful use of MS/MS can be citedwhere information has been obtained about the num-ber of monomer units [3], substitution patterns (e.g.

* Corresponding author. E-mail: [email protected] is with great admiration and respect that we dedicate this

research to the memory of Professor Ben S. Freiser.

1387-3806/99/$20.00 © 1999 Elsevier Science B.V. All rights reservedPII S1387-3806(98)14244-6

International Journal of Mass Spectrometry 182/183 (1999) 299–310

occurrence of amine,N-acetylamine, or carboxylicacid functionalities) [3–5], linkage position or branch-ing points [6–17] in a carbohydrate. However, struc-tural characterization is not complete without distin-guishing stereochemical features of the carbohydrateincluding those arising from the presence of differingdiastereomeric monosaccharides (i.e. glucose, man-nose, etc.). Mass spectrometric techniques may seemill suited for an application requiring differentiation ofstereochemical features of isomeric molecules, never-theless, a large number of examples displaying thepotential sensitivity of mass spectrometry towardstereochemical features of molecules are readilyavailable, as demonstrated by the publication of anentire text on the subject [18].

Complete differentiation of all eight unsubstituted,diastereomeric D-monosaccharides would require ananalytical technique that is capable of discriminatingconfigurations of three stereocenters in a monosaccha-ride ring. A more pragmatic goal might involve thedifferentiation of the most biologically relevant un-substituted monosaccharides: glucose, mannose, andgalactose. Such a task still requires discerning theconfigurations of two stereocenters (Table 1). Previ-ous work in our laboratory has utilized various metal-ligand systems (metalN-glycoside complexes) as aroute to generating cationized carbohydrates [19–21].It was often observed that the dissociation mecha-nisms of these metal-carbohydrate cations were re-flective of specific stereochemical features of thecoordinated carbohydrate. However, the use of somemetal systems allowed for differentiation of no morethan a single stereocenter [19,20].

Investigations into the formation of metal-carbo-hydrate complexes by different groups have beenongoing [22–25], with the complexes often applied tochromatographic and electrophoretic separation ofcarbohydrates. The ability of metal cations to act asseparation agents in carbohydrate chromatographyhas been suggested to result from the presence ofdiffering metal complexation sites on diastereomericmonosaccharides. These differing complexation sitesare probably generated by variations in the numberand position of axial and equatorial substituents on themonosaccharide. For example, the largest stabilityconstants observed with cuprammonium complexesof carbohydrates occurred for those monosaccharidespossessing anaxial, equatorial, axial sequence ofthree oxygen atoms [26]. It may be generally con-cluded that the interactions between the metal andcarbohydrate within a metal-carbohydrate complexwill differ for a given metal depending on the struc-ture of the complexation environment presented bythe carbohydrate. This concept is clearly illustrated bythe crystal structure of the distrontium complex ofdi-b-D-fructopyranose 29,1:2,19-dianhydride, wheretwo structurally distinct coordination sites on a singlesugar molecule yielded bidentate and tetradentatesites that were coordinated to different Sr21 cations[27]. The structure of metal-carbohydrate complexescan also vary greatly with the metal cation. Suchstructural differences are immediately apparent fromthe observation that the metal-carbohydrate stoichi-ometry in a methyl furanoside complex is 1:1 for theCa21 complex and 1:2 for the Pb21 complex [28].

The goal of this work is to identify metal-carbo-hydrate complexes that may be used for the completemass spectrometric differentiation of the diastereo-meric monosaccharides listed in Table 1. An analysisof the behavior of the different complex stabilities andcompositions reported for the condensed-phase metal-carbohydrate complexes allows for the formulation ofa strategy to be used for identifying such complexes.First, changes in theN-glycoside ligands can be madethat result in complexation sites that emphasize thedependence of the dissociation pathways upon themonosaccharide stereochemistry. Features of theN-glycoside ligands that may potentially be varied are

Table 1Ring substituent configuration ofD-aldohexoses

Monosaccharide C-2–OH C-4–OH

glucose equatorial equatorialmannose axial equatorialgalactose equatorial axialtalose axial axial

300 G. Smith et al./International Journal of Mass Spectrometry 182/183 (1999) 299–310

the size of the amine used to generate theN-glycosideligand and the coordination number of the resultingligand. Additionally, the number of ligands coordi-nated to the metal may be varied. Second, an attemptcan be made to further “tune” the complex structureby taking advantage of a metal’s preference for aspecific coordination geometry. Thus, in the researchpresented herein, an assay has been undertaken thatprobes the effects of varying the metal center andligand characteristics with an emphasis toward under-standing how these changes influence the dissociationpathways of diastereomeric metalN-glycoside com-plexes. The various complexes were generated viaelectrospray ionization (ESI) and their product ionspectra obtained by using a quadrupole ion trap. Suchinstrumentation and its ability to perform multistageMS experiments (MSn) have been shown to be usefulin the stereochemical differentiation of a series of Zncomplexes [21].

2. Experimental

2.1. Instrumentation

All experiments were carried out on a FinniganLCQ quadrupole ion trap mass spectrometer (Finni-gan MAT, San Jose, CA) equipped with an electro-spray ionization source. The pressure inside the vac-uum chamber was 1.93 1023 Torr or less during allacquisitions. Samples were infused into the instru-ment at 4mL/min. Ionization was performed at either4 kV for the Ni complexes or 5 kV for the Zn and Cucomplexes. All spectra were acquired at a capillarytemperature of 150 °C and all ion guide voltages weretuned so as to maximize the abundance of the relevantprecursor ion current. A total of 30 scans wereaveraged to produce each spectrum. Precursor ionswere isolated over a mass range of 1.5–1.7 Da prior toexcitation. rf voltages between 0.45 and 1.00 V( p2p),depending on the precursor ion, were applied toendcaps of the ion trap for 30 ms during the excitationperiod. The amplitude of the rf voltage was chosen tooptimize any stereochemically differentiating featuresof the product ion spectra. All subsequent acquisitions

were performed at this preset rf voltage. Additionally,all acquisition parameters were identical for a givenset of diastereomeric complexes. A criterion of 3%relative abundance has been utilized to determine thepresence or absence of a product ion for all datacollected on diastereomeric differentiation. Ions oflower relative abundance are not reproducible, or areincluded in the noise level background.

2.2. Synthesis

Transition metal-amine complexes that were usedas reagents to generate the metalN-glycoside com-plexes were synthesized by using one of three amineligands: 1,3-diaminopropane (dap), ethylenediamine(en), and diethylenetriamine (dien). All metal aminereagents were synthesized using previously publishedprocedures. The preparations and purifications aredescribed briefly below

2.2.1. Ni (dap)3 z 2Cl [29], Ni (en)3 z 2Cl [30], Zn(dap)3 z 2Cl [31], Zn (en)3 z 2Cl [31]

Three molar equivalents of the appropriate aminewere added slowly to a methanolic solution of eitherNiCl2 z 6H2O or ZnCl2. Solids precipitated soon afterstirring. Each solid was purified by recrystallizationfrom an isopropanol/methanol mixture.

2.2.2. Cu (dap)2 z 2Cl [29], Cu (en)2 z 2Cl [32], Ni(dien)2 z 2Cl [33] andZn (dien)2 z 2Cl [34]

Two molar equivalents of the appropriate aminewere added slowly to a methanolic solution of CuCl2 z

H2O. Solids precipitated soon after stirring. Eachsolid was purified by recrystallization from an isopro-panol/methanol mixture.

2.2.3. Cu (dien)z 2Cl [35]One molar equivalent of the diethylenetriamine

was added slowly to a methanolic solution of CuCl2 z

H2O. A solid precipitated soon after stirring. The solidwas purified by recrystallization from an isopropanol/methanol mixture.

301G. Smith et al./International Journal of Mass Spectrometry 182/183 (1999) 299–310

2.2.4. Metal/N-glycoside complexesLabeled and unlabeled metal-N-glycoside com-

plexes were synthesized by using a microscaled ad-aptation of the procedure previously published byYano et al. [36–39]. Briefly, 4 mg of one of fourdiastereomeric D-aldohexoses (Table 1) and threemolar equivalents of a Ni, Zn, or Cu amine complexwere dissolved in 200mL of methanol, yielding finalconcentrations of 0.04 M and; 0.12 M, respectively.Samples were refluxed at 80 °C for 20 min unlessotherwise noted. All reaction mixtures were kept at; 5 °C until used. No purification was performedprior to analysis.

2.3. ESI-MS analysis

Solutions for ESI-MS were prepared by dilutingthe reaction mixture (; 0.04 M) 100-fold by using 9:1MeOH/H2O. Final concentrations of all solutionswere; 400 mM.

2.4. Chemicals and materials

D-Glucose (A. C. S. reagent grade), D-mannose(99%), 1,3-diaminopropane (99%), ethylenediamine(99%), and diethylenetriamine (99%) were obtainedfrom Aldrich Chemical Co. (Milwaukee, WI). D-Galactose (99%), D-talose, and HPLC grade waterand methanol were obtained from Sigma ChemicalCo. (St. Louis, MO). D-mannose-6-2H2 (98% D) andD-mannose-3-13C (99% 13C) were obtained fromOmicron Biochemical Co. (South Bend, IN). Zincdichloride, nickel dichloride hexahydrate, andA. C. S. grade methanol were purchased from FisherChemical Co. (Fairborn, NJ). Copper dichloride dihy-drate was purchased from Mallinckrodt, Inc. (Paris,KN). All materials were used as received withoutfurther purification.

3. Results and discussion

The original reports of the synthesis of these typesof metal N-glycoside complexes typically discussedonly one product complex that had been isolated.

Fortunately, in the performance of the desired studieson the various metalN-glycoside complexes of inter-est, it was not necessary to specifically synthesize andisolate only the desired complex prior to the prepara-tion of any analyte solution. The low resolution massspectra of a given reaction mixture always showed thepresence of several types of metalN-glycoside com-plexes. The appropriate precursor ions were merelyisolated in the ion trap prior to any MSn experiments.

Epimerization of the reactant monosaccharideswas also a concern for all reactions. It has been shownthat D-glucose can undergo rapid epimerization in thepresence of metal cations and amine bases, evenunder mild reflux conditions [37]. However, suchtransformations of the monosaccharides were ob-served with transition metal cations only when usingmethylated amines (i.e.N, N9-dimethylethylenedia-mine). None of the amine ligands used in this studywere methylated. Epimerization of monosaccharidesin the presence of the transition metal cations and theamine ligands used in this study has not been ob-served previously.

3.1. Tricoordinate complexes

Scheme 1 displays the various types of tricoordi-nate, deprotonated species that were investigated inthis study. All complexes consisted of a single triden-tate N-glycoside ligand coordinated to a doublycharged metal ion. The ligands possess a deprotona-tion site, yielding the singly charged species ofinterest. The tridentateN-glycoside ligands were gen-erated by using eitheren or dap. Metal N-glycosidecomplexes of all four monosaccharides (Table 1) were


generated for all tricoordinate metal/enand metal/dapsystems.

Ions consistent with the monoisotopic, deproto-nated tricoordinate species were present in the low-resolution mass spectra of all reaction mixtures. Thepresence of isobaric interferences was not obvious inmost low-resolution mass spectra as suggested by thetypically excellent agreement between the theoreticaland experimental isotope patterns of the complexes.However, for the Cu/en/talose and all Cu/dapreactionmixtures, the low-resolution mass spectra clearlyindicated the presence of isobaric interferences. Insuch cases, the deprotonated species was alternatelygenerated via a corresponding HCl adduct present inall reaction mixtures. Collision induced dissociation(CID) of these adducts resulted primarily in the loss ofHCl, yielding the desired deprotonated species:

[M 1 Cl]1 3CID

M 1 HCl

The product ion spectra of the deprotonated species ofsuch complexes were thus generated in MS3 experi-ments.

It was of great concern that this alternate method ofproducing the deprotonated species would result inthe formation of a different precursor ion than wouldhave otherwise been produced in-source in the ab-sence of the isobaric interferences. To alleviate suchconcerns, product ion spectra of the in-source gener-ated deprotonated species (MS2 experiments) werecompared to the product ion spectra of similar com-plexes generated via CID of the HCl adduct (MS3

experiments) in reaction mixtures where such isobaricinterferences were not detected. The product ionspectra obtained in the MS2 and MS3 experimentswere identical to each for all reaction mixtures wheresuch comparisons could be made. Therefore, it wasassumed that when isobaric interferences were likelyto be present along with the deprotonated species,relevant product ion spectra could be obtained byusing the precursor ion generated via the HCl adduct.

Although the product ion spectra varied somewhatwith different metal centers and ligand sizes, similartypes of neutral losses were observed from all trico-ordinate species. Most product ions resulted from

cross-ring cleavages in the monosaccharide moiety(neutral loss of CnH2nOn, wheren ranges from 1 to 4)[8,9,40–42] and/or losses of one or more watermolecules. Similar product ion spectra have also beenreported for the tricoordinate Ni/dapprecursor ions byusing either a sector instrument fast-atom bombard-ment (FAB-generated complexes) [19] or a triplequadrupole mass spectrometer (ESI-generated com-plexes) [20].

The dissociation of the tricoordinate Ni/dap pre-cursor ions resulted in the formation of a large varietyof product ions as represented by the product ionspectrum of the glucose complex in Fig. 1(A). Onenotable product ion, them/z 245 ion (combinedneutral loss of CH2O and H2O) was useful forstereochemical characterization. Them/z 245 production was fairly intense for both complexes possessingequatorial C-2 hydroxyl substituents as indicated inFig. 1(A) for the glucose complex. Conversely, com-plexes possessing axial C-2 substituents were charac-terized by a low intensity (, 5% of the base peak) ofthis same product ion.

A number of product ions were also detectedfollowing the dissociation of some Ni/en complexes

Fig. 1. Product ion spectra (MS2) of tricoordinate deprotonated NiN-glycoside precursor ions: (A) [Ni (dap/glucose)–H]1 precursorion (m/z 293); (B) [Ni (en/mannose)–H]1 precursor ion (m/z279). M represents the precursor ion in each spectrum.


as represented by the product ion spectrum of [Ni(en/mannose)–H]1 in Fig. 1(B). Unlike the Ni/dapcomplexes, the intensity of the product ion resultingfrom the combined losses of CH2O and H2O (m/z231) was notsensitive to the stereochemical featuresof the complexed monosaccharides. Instead, it wasobserved that, at the collision energies utilized, prod-uct ions at a mass-to-charge ratio smaller thanm/z219 (e.g.m/z 189)were observed only for complexespossessing C-2 axial substituents. Such lower massproduct ions were entirely absent from the product ionspectra of those complexes possessing equatorial C-2substituents.

Both tricoordinate Ni complexes can be used todifferentiate the orientation of the C-2 hydroxyl groupof a monosaccharide. However, such structural sensi-tivity manifests itself differently depending on theligand size (i.e.en- versusdap-basedN-glycosidicligands). One feature shared by the dissociation pro-cesses of all tricoordinate Ni complexes is the inabil-ity to differentiate the configurations of the C-4substituent of any of the monosaccharides. The abilityto differentiate the substituent orientations about bothstereocenters (C-2 and C-4) is necessary to distinguishall four diastereomeric monosaccharides.

A representative product ion spectrum of a trico-ordinate Zn/dap complex is shown in Fig. 2(A). Aswas observed with the Ni/dap system, a number ofproduct ions were present. Unfortunately, the generalfeatures of the product ion spectra of the four diaste-reomeric complexes were not significantly varied toallow for any stereochemical differentiation. Mostnotably, the intensity of them/z 251 product ion(combined losses of CH2O and H2O) was not sensi-tive to the stereochemical features of the C-2 substitu-ent as it was in the comparable Ni/dap complexes.

The formation of product ions from the tricoordi-nate Zn precursor ions was also dependent upon thesize of the coordinated ligand. Fewer product ionswere generated from the dissociation of the smallerZn/en complexes as shown by a representative prod-uct ion spectrum in Fig. 2(B). Product ions at amass-to-charge ratio less than 225 were very weak(, 1% of the base peak) for all four Zn/encomplexes.The product ion spectra of the tricoordinate Zn/en

precursor ions also did not include any apparentfeatures allowing for stereochemical differentiation.

The product ion spectra of all tricoordinate Cu/dapcomplexes were very simple, possessing a singleprominent product ion atm/z 178 (C4H8O4). This isdemonstrated by the product ion spectrum of the [Cu(dap/mannose)–H]1 precursor ion [Fig. 3(A)]. It isclear that such uncomplicated product ion spectra donot possess features that would allow for any stereo-chemical differentiation.

The product ion spectra of the tricoordinate Cu/encomplexes present a stark contrast to the uninforma-tive spectra obtained with the Cu/dap tricoordinatespecies. The product ion spectrum of the [Cu (en/mannose)–H]1 precursor ion [Fig. 3(B)] is an exam-ple of the increased diversity of the dissociations thatoccur from this smaller CuN-glycoside complex.Inspection of the spectra obtained under identicalexperimental conditions from all four diastereomericCu/encomplexes revealed unique product ion spectrafor each of these precursor ions. Table 2 summarizesthe various product ions that were generated from thedifferent tricoordinate Cu/en species.

It is clear that the types of neutral losses obtainedfrom the dissociation of deprotonated, tricoordinate

Fig. 2. Product ion spectra (MS2) of tricoordinate deprotonated ZnN-glycoside precursor ions: (A) [Zn (dap/mannose)–H]1 precursorion (m/z 299); (B) [Zn (en/mannose)–H]1 precursor ion (m/z285). M represents the precursor ion in each spectrum.


metal/N-glycoside complexes vary when comparingthe dap-based N-glycoside ligands to the smalleren-basedN-glycoside ligands. Additionally, changingthe metal center also affected the ability to achievestereochemical differentiation from the product ionspectra of tricoordinate complexes. The orientation ofonly the C-2 substituent was discernible from thedissociation mechanisms of the Ni/en and Ni/dapcomplexes, whereas a full differentiation of the fourdiastereomeric monosaccharides was achieved withthe Cu/en complexes. Contrasting these results, theproduct ion spectra of all tricoordinate Zn complexes

were devoid of features indicative of a clear stereo-chemical differentiation.

3.2. Four-coordinate complexes

All deprotonated, four-coordinate complexes con-sisted of a single tridentateN-glycoside ligand coor-dinated to a doubly charged metal ion as shown inScheme 2. Two classes ofN-glycoside ligands wereutilized: dien-based ligands having a 1:1 amine/monosaccharide molar ratio, anden- or dap-basedligands having a 1:2 molar ratio. The ligands possessa deprotonation site, yielding the singly chargedspecies of interest. The four-coordinate complexescould be generated from all reaction mixtures byusing the four monosaccharides in Table 1, except forthe Ni/dien reaction mixtures. Low-resolution massspectra of the Ni/dien reaction mixtures often did notshow any NiN-glycoside ions sufficiently intense forMS2 experiments, even after extended reflux times

Fig. 3. Product ion spectra (MS3) of tricoordinate deprotonated CuN-glycoside precursor ions: (A) [Cu (dap/mannose)–H]1 precursorion (m/z 298); (B) [Cu (en/mannose)–H]1 precursor ion (m/z284). M represents the precursor ion in each spectrum. Thedeprotonated precursor ions were generated via a HCl adduct (seetext).

Table 2Product ions from [Cu (en/monosaccharide–H]1 precursor ions (m/z 298) (“X” represents product ions that were observed at relativeintensities greater than 3% of the base peak)

Product ions

m/z 266 m/z 253 m/z 248 m/z 224 m/z 206 m/z 194 m/z 188 m/z 176 m/z 164

glucose . . . . . . . . . X X . . . X . . . Xgalactose X X . . . X X . . . X . . . Xmannose X . . . X X X X X X Xtalose X . . . X . . . . . . X . . . . . . X


(; 12 h). Evidence of isobaric interferences with thefour-coordinate species was not found in the low-resolution mass spectra as suggested by the typicallyexcellent agreement between the theoretical and ex-perimental isotope patterns of the complexes. There-fore, all product ion spectra were generated directlyfrom the deprotonated precursor ions produced in thesource.

Product ion spectra of representative four-coordi-nate CuN-glycoside complexes possessing differentamine/monosaccharide molar ratios and ligand sizesare shown in Fig. 4. The product ion spectra offour-coordinate Ni and Zn complexes (data notshown) were qualitatively similar to those in Fig. 4 inthat identical neutral losses were observed with thedifferent metal centers. Also evident from Fig. 4, theproduct ion spectra of different classes of four-

coordinate precursor ions were remarkably similar toeach other; i.e. changes in the ligand size or amine/monosaccharide molar ratio had little effect on theneutral losses observed. A notable exception to thesesimilarities occurred for the previously studied Zn/dien complexes [21], as discussed below.

Two features occurring consistently in the production spectra of most four-coordinate complexes are theproduct ions resulting from neutral losses of 90 Da(C3H6O3) and 120 Da (C4H8O4). These 90 and 120Da losses are most likely similar to those mentionedabove, which involve cross-ring cleavages in themonosaccharide ring. Other types of cross-ring cleav-ages were relatively weak or absent. Losses of one ortwo water molecules were often observed, especiallyfrom the Zn and Cu complexes. Concurrent losses ofCnH2nOn and additional water molecules (e.g. CH2Oand H2O), having been shown above to be useful forstereochemical differentiation of some tricoordinatecomplexes, were absent.

Large mass neutral losses (180, 210, and 240 Da)were present only in the product ion spectra ofcomplexes containing ligands with a 1:2 amine/monosaccharide molar ratio. Such neutral losses arelikely to result from a loss of 90 or 120 Da from onemonosaccharide ring and an additional loss of 90 or120 Da from the second monosaccharide ring. Cleav-age of theN-glycoside bond, resulting in the loss of aneutral anhydromonosaccharide (162 Da) was alsoobserved only for complexes possessing this class ofligands.

Unique product ions, characteristic of the config-uration of a stereocenter, were not observed as shownby the similarity amongst the product ion spectra ofmost of the diastereomeric, four-coordinate com-plexes. Therefore, stereochemical differentiation wasnot possible amongst most four-coordinate com-plexes. Unlike the tricoordinate ions, changes in themetal center and ligand size typically did not signif-icantly alter the product ion spectra of the four-coordinate species.

One notable exception amongst the four-coordi-nate complexes is the Zn/dien species. As has beenpreviously reported in detail [21], characteristic prod-uct ion spectra of the four diastereomeric precursor

Fig. 4. Product ion spectra (MS2) of four-coordinate deprotonatedCu N-glycoside precursor ions: (A)dap-based ligand possessing1:2 amine/monosaccharide molar ratio (m/z 460); (B) en-basedligand possessing 1:2 amine/monosaccharide molar ratio (m/z446); (C)dien-based ligand possessing 1:1 amine/monosaccharideratio (m/z 327). M represents the precursor ion in each spectrum.Product ions marked with “*” result from cleavages in bothmonosaccharide rings.


ions can be obtained from the four-coordinate [Zn(dien/monosaccharide)–H]1 complexes, as summa-rized in Table 3.

3.3. Five-coordinate complexes

The two types of deprotonated, five-coordinatecomplexes studied are shown in Scheme 3. One groupof complexes consisted of a single pentadentatedien-basedN-glycoside ligand (1:2 amine/monosaccharidemolar ratio) coordinated to a doubly charged metalion. The second group of complexes consisted of adoubly charged metal ion coordinated by two ligands:a tridentateen-basedN-glycoside ligand and a biden-tate amine ligand. All complexes possess a deproto-nation site, yielding the singly charged species ofinterest.

Examples of single-ligand, five-coordinate com-plexes were limited only to the species obtained fromthe Cu/dien reaction mixtures. Complexes of this sortwere generated for all four diastereomeric monosac-charides (Table 1). Although other types of ZnN-glycoside complexes were produced from the Zn/dienreaction mixtures (i.e. [Zn (dien/monosaccharide)–

H]1), the five coordinate species were not sufficientlyintense to allow for MSn experiments. The problemsencountered with generating any NiN-glycoside com-plexes from the Ni/dien reaction mixtures have beendiscussed above. The deprotonated, single-ligand CuN-glycoside precursor ions were those generated in-source and not via a HCl adduct.

A representative product ion spectrum of a single-ligand, five-coordinate complex is shown in Fig. 5.Such a spectrum is very reminiscent of the product ionspectra of the four-coordinate complexes possessingligands with a 1:2 amine/monosaccharide molar ratio[Fig. 4(A) and (B)]. Cross-ring cleavages within asingle monosaccharide ring resulting in neutral lossesof 90 Da (C3H6O3) or 120 Da (C4H8O4) are the mostprominent dissociation pathways. Concurrent lossesfrom the second monosaccharide ring (product ionsmarked with a “*”) were also present. As was the casefor most four-coordinate complexes, characteristicproduct ions were not obtained from the four diaste-reomeric single-ligand, five-coordinate systems.

Difficulties were also encountered when attempt-ing to make many of the two-ligand, five-coordinateprecursor ions. The low-resolution mass spectra indi-cated that such species were not produced for many ofthe metal/dap reaction mixtures. However, the two-ligand complexes could be generated from mostNi/en, Cu/en, and Zn/en reaction mixtures; only the[Cu (en)(en/glucose)–H]1 precursor ion could not beformed. All deprotonated, two-ligand, five-coordinate

Table 3Product ions from [Zn (dien/monosaccharide)–H]1 precursorions (m/z 328) (“X” represents product ions that were observedat relative intensities greater than 3% of the base peak)

Product ions

m/z310

m/z298

m/z292

m/z280

m/z268

m/z238

m/z226

m/z208

glucose . . . X . . . X X . . . X Xgalactose X X . . . X X X X Xmannose X X . . . X X X . . . Xtalose X X X . . . X X . . . X

Fig. 5. Product ion spectrum (MS2) of five-coordinate [Cu (dien/mannose2)–H]1 precursor ion (m/z 489).M represents the precur-sor ion in each spectrum. Product ions marked with “*” result fromcleavages in both monosaccharide rings.


precursor ions utilized in these experiments wereobtained directly from those generated in-source andnot via CID of the corresponding HCl adduct.

Fig. 6(A) and (B) show representative product ionspectra of the [Zn (en)(en/mannose)–H]1 and [Cu(en)(en/mannose)–H]1 precursor ions, respectively.Similar spectra were obtained from the correspondingprecursor ions from all other Zn/enand Cu/enreactionmixtures. Prominent product ions resulting from theloss of 60 and 120 Da were always present. Twoassignments exist for each of these neutral losses. The60 Da loss may be assigned to a neutral C2H4O2

molecule following the typical cross-ring cleavages inthe monosaccharide ring. However, because the bi-dentateen ligand also has a molecular weight of 60Da, the loss of the amine ligand cannot be ruled out.Similarly, the 120 Da losses may result from either across-ring cleavage in the monosaccharide ring (lossof C4H8O4) or a concurrent loss of theen ligand andC2H4O2.

Isotopic labeling experiments were performed toidentify the molecular formula of the 60 and 120 Daneutral losses.N-glycoside complexes of Cu and Znwere synthesized by using either mannose-6-D2 ormannose-3-13C. Such labeled compounds were cho-sen because it has been shown that the C2H4O2 neutralloss may result from losses of either C-3 and C-4, orC-5 and C-6 [42]. Losses of other combinations ofcarbon centers (i.e. C-4 and C-5) or carbon centerspossessing substituents coordinated directly to themetal center (i.e. C-1 or C-2) are likely to involvelarge barriers and have not been previously observedin the product ion spectra of metalN-glycosidecomplexes [20,21,42]. These labeled complexes canthus be used to distinguish neutral losses consisting ofen or C2H4O2. A neutral loss of 60 Da was observedin the product ion spectra of all the labeled [metal(en)(en/mannose)–H]1 complexes (metal5 Cu, Zn).Such results conclusively show that the 60 Da lossfrom the two-ligand, five-coordinate complexes wasbecause of the loss of only the bidentateen ligandfrom the precursor ion.

The loss of 120 Da was also easily assigned.Neutral losses of 122 Da were present in the production spectra of the mannose-6-D2 complexes, indicat-ing that C-6 and its labeled atoms are lost in thisdissociation pathway. However, neutral losses of only120 Da were observed from the mannose-3-13C com-plexes, which showed that C-3 was never lost fromthe precursor ion following this dissociation. It hasbeen previously demonstrated that the loss of C4H8O4

from Zn N-glycoside complexes resulted from thelosses of C-3, C-4, C-5, and C-6. Because C-3 isretained by the product ion, the 120 Da loss can onlybe due to a concurrent loss of the bidentateen ligandand C2H4O2.

The product ion spectra of the five-coordinatecomplexes generated from the Ni/enreaction mixturesalso displayed prominent losses of 60 and 120 Da, asrepresented by the [Ni (en)(en/mannose)–H]1 com-plex in Fig. 6(C). Several additional product ions (m/z249 andm/z 189) were also present. As was ob-served with the Zn and Cu complexes, no stereo-chemically characteristic product ions were generatedfrom any of the four diastereomeric precursor ions.

Fig. 6. Product ion spectrum (MS2) of two-ligand, five-coordinateprecursor ions: (A) [Zn (en)(en/mannose)–H]1 precursor ion (m/z345); (B) [Cu (en)(en/mannose)–H]1 precursor ion (m/z 344); (C)[Ni (en)(en/mannose)–H]1 precursor ion (m/z 339). M representsthe precursor ions in each spectrum.


Isotopically labeled Ni/enreaction mixtures were alsoprepared by using mannose-6-D2 and mannose-3-13C.Again, the losses of 60 and 120 Da could be assignedto the loss of only theen ligand, and a concurrent lossof en and C2H4O2, respectively. The labeling experi-ments that were performed did not allow for theunequivocal assignment of the other neutral lossesfrom the Ni complex. Experiments that will allow fora conclusive assignment are currently in the planningstages for all metal-coordinated complexes. However,the loss of 150 Da (m/z 189) is likely to be becauseof a successive loss of C3H6O3 (90 Da) following across-ring cleavage anden ligand (60 Da). Cross-ringcleavages in the monosaccharide ring leading to theformation of the 150 Da neutral loss, C5H10O5, havenot been observed from theseN-glycoside complexes.The loss of 90 Da may represent either the loss ofC3H6O3 or simultaneous losses of CH2O and theenligand.

Although a complete survey of all relevant five-coordinate species could not be performed, somegeneral trends in the dissociation pathways could stillbe discerned. First, the types of neutral losses gener-ated were largely dependent upon the number ofligands coordinated to the metal. The single-ligand,five-coordinate species generated product ion spectrathat were very similar to those of the four-coordinatespecies. The product ion spectra of the two-ligand,five-coordinate precursor ions varied for differentmetal complexes. Both the Cu and Zn complexesgenerated fewer product ions than the Ni complexes.However, the dissociation pathways of all such com-plexes were dominated by mechanisms involving lossof the bidentate amine ligand coordinated to the metal.Although the product ion spectra of both types offive-coordinate precursor ions were quite different, nei-ther type of complex was capable of generating productions that were characteristic of any of the stereochem-ical features of the coordinated monosaccharide.

4. Conclusions

Several features of deprotonated metal/N-glyco-side complexes were varied in order to determine

which types of complexes would best enable stereo-chemical differentiation of the coordinated monosac-charides. When possible, different coordination num-bers, metal centers, and types of ligands were utilized.This work has demonstrated that the monosaccharidesare typically best differentiated by MS/MS of thetricoordinate complexes, with the exception of thefour coordinate Zn/dien species. The tricoordinateCu(en) N-glycoside complexes displayed characteris-tic product ion spectra for each of the four diastereo-meric monosaccharides. Therefore, these complexesallowed for the differentiation of the stereochemicalfeatures about the C-2 and C-4 centers of themonosaccharides. Differentiation of the stereochemi-cal features about a single carbon center, C-2, wasachieved by the tricoordinate NiN-glycoside systems.However, the information obtained from a singlestereocenter is insufficient to differentiate the fourdiastereomeric monosaccharides in Table 1. Changesin the metal center or size of theN-glycoside ligandgenerated the greatest changes in the product ionspectra of the tricoordinate complexes. Such alter-ations in four- or five-coordinate complexes typicallydid not result in greatly differing product ion spectra.

Although the product ion spectra of most metal/N-glycoside complexes could be easily categorized ac-cording to structural features of the precursor ion,exceptional species such as the four coordinate [Zn(dien/monosaccharide)–H]1 precursor ion can giveunexpected stereochemical information. It is presentlyunclear whether factors such as complex geometry,electronic configuration or sterics, or a combination ofthe three, dictate the dissociation pathways of themetal N-glycoside complexes, thus allowing for thedifferentiation of diastereomeric monosaccharides.Further detailed studies of the effects of these factorswill be necessary to answer such questions.

Acknowledgement

The authors acknowledge NIH grant no. GM47356for financial support.


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influence of coordination number and ligand size on the dissociation mechanisms of transition...

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