Capillary electrophoresis analysis of glucooligosaccharide regioisomers

Gilles Joucla1

Thérèse Brando2

Magali Remaud-Simeon1

Pierre Monsan1

Germain Puzo2

1Département de GénieBiochimique et Alimentaire,Centre de Bioingénierie,Gilbert Durand,Toulouse, France

2Institut de Pharmacologie et deBiologie Structurale du CNRS,Toulouse, France

Capillary electrophoresis analysis of gluco-oligosaccharide regioisomers

Complex gluco-oligosaccharide mixtures of two regioisomer series were successfullyseparated by CE. The gluco-oligosaccharide series were synthesized, employing adextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive gluco-pyranosyl transfers from sucrose to the acceptor glucose or maltose. The glucosyltransfer to both acceptors, occurring through the formation of a1?6 linkages, differedfor the two series only in the glucosidic bond to the reducing end namely a1?6 ora1?4 bond for glucose or maltose acceptor, respectively. Thus, the combination ofthe two series results in mixed pairs of gluco-oligosaccharide regioisomers with differ-ent degrees of polymerization (DP). These regioisomer series were first derivatized byreductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS). Under acidic con-ditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosacchar-ides of each series were separated enabling unambiguous size determination by cou-pling CE to electrospray-mass spectrometry. However, neither these acidic conditionsnor alkaline buffer systems could be adapted for the separation of the gluco-oligosac-charide regioisomers arising from the two combined series. By contrast, increasedresolution was observed in an alkaline borate buffer, using differential complexation ofthe regioisomers with the borate anions. Such conditions were also successfullyapplied to the separation of glucodisaccharide regioisomers composed of a1?2,a1?3, a1?4, and a1?6 linkages commonly synthesized by glucansucrase enzymes.

Keywords: Capillary electrophoresis / Glucansucrase / Gluco-oligosaccharides / Mass spec-trometry / Regioisomers DOI 10.1002/elps.200305737

1 Introduction

Oligosaccharides have been traditionally used in food,animal feed, pharmaceutical and cosmetic industries assweeteners, stabilizers or bulking agents [1]. Over thepast 15 years, a new field of application based on the pre-biotic properties of some of such nondigestible moleculeshas emerged [2]. Indeed, oligosaccharides as prebioticsretained attention for their ability to resist the attack ofdigestive enzymes and to enhance the growth of “health-promoting” bacteria (mainly Bifidobacteria and Lactoba-cilli) in the intestinal tract. This concept greatly stimulatedthe emergence of a prebiotics industry, which has grownrapidly to provide oligomers such as fructo-oligosacchar-ides, lactulose, galacto-oligosaccharides, xylo-oligosac-charides, soybean oligosaccharides or isomalto-oligo-saccharides, almost exclusively obtained via biological

processes or plant extraction. Today, the research effortin this field is focused on the production of new oligosac-charide structures, the so-called “second generation ofprebiotics”, with new physical-chemical properties andpossible different and more specific bioactivities [3].

In this context, glucansucrases from lactic acid bacteriaare attractive tools. These transglycosidases are classi-fied from sequence similarities in the family 70 of glyco-side hydrolases [4–7]. Their primary physiological func-tion is the synthesis of glucan from sucrose, a simpleand widely available agro-resource. Various types of glu-cosidic bonds can be formed by these enzymes: a1?2,a1?3, a1?4, or a1?6 bonds depending on their regio-specificity [8–10]. In addition, when acceptors are addedto the reaction mixture, the enzymatic activity can beredirected from polymer synthesis toward oligosacchar-ide synthesis [11–14]. The glucose residue is first trans-ferred to the acceptor (usually a carbohydrate) and thereaction product may in turn play the role of an acceptor.In that case, successive glucopyranosyl transfers to theacceptor molecule lead to the formation of oligosacchar-ide series. Thus, using glucansucrases of distinctive spe-cificities in the presence of different acceptors givesaccess to a large variety of oligosaccharide structures,

Correspondence: Dr. Germain Puzo, Institut de Pharmacologieet de Biologie Structurale du CNRS, UMR 5089, 205 route deNarbonne, F-31077, Toulouse Cedex 4, FranceE-mail: [email protected]: 133-5-6117-5994

Abbreviations: APTS, 9-aminopyrene-1,4,6-trisulfonate; DP,degree of polymerization

Electrophoresis 2004, 25, 861–869 861

CE

and

CE

C

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

862 G. Joucla et al. Electrophoresis 2004, 25, 861–869

as e.g., (i) leucrose (5-a-D-glucopyranosyl-D-fructopyra-nose, isomer of sucrose) [15], (ii) dextrans of controlledmolecular weight [16], (iii) a-glucosylated cellobiose [17],and (iv) gluco-oligosaccharides with a1?2 glucosidicbonds produced on the industrial scale (50 t/year) [18, 19].

To identify the various oligosaccharide structures that canbe obtained by acceptor reaction catalyzed by glucansu-crases of various specificities, the prerequisite is to deter-mine the composition of the acceptor reaction mixture.The analytical method used must be fast, of high resolu-tion to separate structures differing only by slight struc-tural changes, sensitive to avoid the use of large amountsof sample, and preferably quantitative. To date, the mostwidely employed method for oligosaccharide separationwas high-performance liquid chromatography (HPLC)[20, 21], which allows quantitative analysis when coupledto refractive index (RI) detection. However, HPLC-RI suf-fers from poor sensitivity and lack of resolution in theseparation of regioisomer oligosaccharides. High-perfor-mance anion exchange chromatography is a good alter-native. Amperometric detection is highly sensitive forunderivatized carbohydrates [22] but separation requiresthe use of high pH, which impedes coupling to MS andnecessitates desalting. Moreover, amperometric detec-tion differs according the oligosaccharide size hinderingtheir quantitative analysis.

Compared to these methods, capillary electrophoresis(CE) is a powerful and highly sensitive technique for theanalysis of neutral and charged carbohydrates. Generally,CE is coupled with laser-induced fluorescence (LIF). Thisapproach requires oligosaccharide derivatization whichcan be performed with fluorescent probes such as 8-ami-nonaphthalene-1,3,6-trisulfonate (ANTS) or 9-aminopyr-ene-1,4,6-trisulfonate (APTS). This method proved to beefficient for the separation of fluorescent mono- and oli-gosaccharide derivatives, allowing their detection up tothe femtomole level [23–25]. Chen et al. [25, 26] per-formed LIF-CE to resolve APTS-derivatized monosac-charides with electrolytes at neutral pH conditions, in buf-fers such as 120 mM 3-(N-morpholino)-propanesulfonicacid (MOPS, pH 7.0) or 50 mM sodium phosphate(pH 7.4). APTS-sugar separation was shown to be gener-ally enhanced by using borate buffer which forms com-plexes with sugars. Indeed, borate alters both hydrody-namic volumes and charge-to-mass ratios due to the ad-ditional charge contribution. This method was applied tothe separation of several disaccharide derivatives: gen-tiobiose Glcp-(b1?6)-Glc-APTS, maltose Glcp-(a1?4)-Glc-APTS, lactose Galp-(b1?4)-Glc-APTS, cellobioseGlcp-(b1?4)-Glc-APTS, and melibiose Galp-(a1?6)-Glc-APTS with 175 mM borate at pH 10.2. Separationsof maltotetraose-APTS Glcp-(a1?4)-Glcp-(a1?4)-Glcp-(a1?4)-Glc-APTS and a corresponding regioisomer Glcp-

(a1?6)-Glcp-(a1?4)-Glcp-(a1?4)-Glc-APTS were alsodescribed [25, 26].

Here, we report the use of LIF-CE and CE coupled to MSfor separation and mass determination of gluco-oligosac-charide regioisomers with a degree of polymerization (DP)ranging from 2 to 9, synthesized with glucansucrase fromLeuconostoc mesenteroides NRRL B-512F [27]. We alsoinvestigated the ability of CE to resolve several glucosedisaccharide regioisomers in order to validate separationof compounds presenting the various linkage types gen-erated by the glucansucrase family.

2 Materials and methods

2.1 Chemicals

Disaccharide glucose isomers were purchased fromSigma-Aldrich (St. Louis, MO, USA) and isomaltose fromSeikagaku America (East Falmouth, MA, USA). Oligo-saccharide series were synthesized by enzymatic reactionusing L. mesenteroides NRRL B-512F dextransucrase, withglucose or maltose as acceptor sugars. The oligosacchar-ides generated were either individually purified by prepara-tive HPLC to use them as standards or directly derivatizedas a crude mixture after removal of polymer by ethanolprecipitation (ratio 1:1). APTS was obtained from Fluka(St. Quentin Fallavier, France). Sodium cyanoborohydride(NaBH3CN), 1 M in tetrahydrofuran (THF), was purchasedfrom Sigma-Aldrich.

2.2 Oligosaccharide synthesis byL. mesenteroides NRRL B-512Fdextransucrase

L. mesenteroides NRRL B-512F, provided by the NCAURstock culture collection in Peoria, (IL, USA), was the dex-transucrase source. Cells were grown at 277C on stand-ard medium as previously described [28]. Expression ofdextransucrase genes was obtained with sucrose supple-mentation. Dextransucrase, an extracellular enzyme, wasseparated from cells by centrifugation in order to obtaincrude enzymatic preparation containing enzymes andglucan, the polymer produced during the culture growth.The crude enzymatic preparation was then purified byliquid-liquid extraction with polyethylene glycol (PEG).The protocol, adapted from Paul et al. [29], allows purifi-cation from other contaminant activities like levansu-crase, which also uses sucrose as substrate. Purifieddextransucrase was the enzyme source for the synthesisof oligosaccharides. Acceptor reactions were performedat 307C in acetic acid buffer (20 mM, pH 5.2) supplement-ed with sucrose (100 g?L21), glucose (100 g?L21) or mal-tose (50 g.L21) as acceptor molecule, CaCl2 (0.05 g?L21)


Electrophoresis 2004, 25, 861–869 CE analysis of gluco-oligosaccharide regioisomers 863

and 1 U?mL21 of dextransucrase. Oligosaccharide syn-thesis was carried out for 1.5 h and completion of sucroseconsumption was monitored with a C18 HPLC column (asdescribed below). Polymer synthesis is redirected to oli-gosaccharide synthesis when an acceptor is added. Typi-cally, the reaction gives 70% and 15% of oligosacchar-ides with maltose or glucose as acceptor, respectively.Ethanol was added in a volume ratio of 1/1 v/v to precipi-tate the coproduced polymer (dextran), and the mixtureswere centrifuged at 10 000 rpm for 30 min. The superna-tants, separated from dextran pellets, were then evapo-rated to eliminate ethanol. These oligosaccharide mix-tures were directly derivatized with APTS or were purifiedon preparative HPLC for single standard purposes.

2.3 Oligosaccharide purification by preparativeHPLC

A sample of the oligosaccharide synthesis mixture withoutpolymer was submitted to purification in order to obtainpure standards, ranging from a degree of glucose polymer-ization of 2 (DP2) to 5 (DP5). Separations of oligosacchar-ides in each series were carried out by HPLC on a C18column (10 mm, 250620 mm; Bischoff Chromatography,Leonberg, Germany) coupled to refractive index detec-tion (I.C.S.) [20, 21]. Supernatant volumes of 1 mL wereinjected, then eluted with deionized water at a flow rate of5 mL?min21. The purification quality was monitored on aC18 HPLC column (5 mm, 25064 mm; Bishoff chromatog-raphy) with a flow rate of 0.5 mL?min21 of deionized water.

2.4 Derivatization of oligosaccharides withAPTS

The derivatization protocol first described by Jackson [30]and adapted by Chen et al. [25] was modified as follows.Dried oligosaccharide standards (DP2, DP3, DP4, andDP5, 5 nmol of each), and mixtures of synthesized oligo-saccharides purified from polymer (6 nmol) were sub-mitted to derivatization by adding 0.3 mL of 0.2 M APTS(60 nmol) in 15% acetic acid and 0.3 mL of 1 M NaBH3CNin tetrahydrofuran (THF). After incubation at 557C for90 min, the mixture was suitably diluted with deionizedwater before injection in CE or CE-MS.

2.5 Capillary electrophoresis

All experiments were carried out on a P/ACE 5000 capil-lary electrophoresis system (Beckman Coulter, Palo Alto,CA, USA), equipped with an argon-ion laser of 4 mW.Detection was performed with a laser induction fluores-cence detector module (LIF; excitation at 488 nm and

emission at 520 nm). Separations were monitored on anuncoated fused-silica capillary (47 cm650 mm ID) at aconstant temperature of 257C. Samples were injected byapplying 0.5 psi (3.45 kPa) pressure for 5 s. Separationswere performed with acetic acid (1% v/v), triethylamine(15 mM), pH 3.7, or lithium borate (20 mM), pH 9.15, at220 kV or 120 kV, respectively.

2.6 CE-ESI-MS experiments

Analyses were performed on a CE system P/ACE MDQ(Beckman Coulter) with a 75 mm680 cm fused-silicacapillary. The outlet was integrated into the ESI needle,directly coupled to an ion-trap MS system (LCQ DUO;Thermofinnigan, Les Ulis, France). Separations were car-ried out with an electrolyte composed of acid acetic (1%v/v) and triethylamine (15 mM), pH 3.7, and under anapplied voltage of 220 kV. Migration was monitored bythe total ion current (TIC). During analysis, the tempera-ture was maintained constant (257C) along the capillaryand the outlet end of the capillary was at a spray voltageof 4 kV. The sheath liquid, consisting of water:isopropanol(20:80), was infused to the ESI needle through a syringepump at a flow rate of 3 mL?min21 using nitrogen asnebulizing gas. For measurements, negative mode wasused and all data were collected on Xcalibur Software.MS calibration was performed with Ultramark 1621(Lancaster Synthesis, Pelham, NA, USA).

3 Results and discussion

3.1 APTS gluco-oligosaccharide derivatives:separation and characterization by CE andCE-MS

Recently, Nigou et al. [31] achieved the separation ofAPTS-disaccharide regioisomers consisting of two man-nopyranosyl units differing by the interglycosidic linkagetype: a1?2, a1?3, a1?4 or a1?6 bonds. The separa-tion was performed with acetic acid (1% v/v), triethyla-mine (30 mM), pH 3.7, as electrolyte. These electrophoret-ic conditions were applied to two series of gluco-oligosac-charide mixtures synthesized with glucose or maltose asacceptor, using glucansucrase from L. mesenteroidesNRRL B-512F. This enzyme mainly synthesizes oligosac-charides with a1?6 glucosidic linkages.

The first mixture, synthesized with maltose Glcp-(a1?4)-Glc as acceptor, contained gluco-oligosaccharides aris-ing from the maltose addition of a1?6 linked glucopyra-nosyl units (Fig. 1A). The enzyme catalyses the transfer ofglucosyl from sucrose onto the nonreducing end of themaltose moiety, to yield Glcp-(a1?6)-Glcp-(a1?4)-Glc.



Figure 1. Series of gluco-oligosaccharides synthesized from sucrose with dextransucrase purified from L. mesenteroidesNRRL B-512F. (A) Synthesized with maltose as acceptor, (B) synthesized with glucose as acceptor.

This trisaccharide is glucosylated on turn and so forth, tosynthesize a series of linear gluco-oligosaccharides withan increasing DP. This crude mixture, purified from poly-mer, was derivatized with APTS and analyzed by CE inuncoated fused-silica capillaries using electrolytes atacidic pH, in order to suppress ionization of the surfacesilanol groups leading to an electroosmotic flow valuenear zero. Thus, the three negative charges derived fromthe sulfonate groups provide the electrophoretic mobilityallowing the APTS gluco-oligosaccharide derivatives toreach the detector located at the anode. As the moleculeshave the same global negative charge, migration velocitydepends on size and hydrodynamic volume.

The electropherogram in Fig. 2 illustrates the separation ofgluco-oligosaccharide APTS derivatives. The first peak cor-responds to the Glc-APTS derivative. Glucose is producedby the low hydrolysis rate of sucrose by glucansucrase dur-ing the acceptor reaction with maltose. The nine followingpeaks observed were tentatively assigned to gluco-oligo-saccharide APTS derivatives differing by the DP. The firstAPTS-gluco-oligosaccharide derivative was attributed toDP2, Glcp-(a1?4)-Glc-APTS (maltose derivative, remain-ing acceptor), followed by DP3, Glcp-(a 1?6)-Glcp-(a1?4)-Glc-APTS, DP4, Glcp-(a1?6)-Glcp-(a1?6)-Glcp-

(a1?4)-Glc-APTS, DP5, [Glcp-(a1?6)-Glcp]2-(a1?4)-Glc-APTS, DP6, [Glcp-(a1?6)-Glcp]2-Glcp-(a1?4)-Glc-APTS,DP7, [Glcp-(a1?6)-Glcp]3-(a1?4)-Glc-APTS, DP8, [Glcp-(a1?6)-Glcp]3-Glcp-(a1?4)-Glc-APTS, and finally DP9[Glcp-(a1?6)-Glcp]4-(a1?4)-Glc-APTS. Mainly due to theenzyme reaction specificity [27], we observed a decreasein abundance of increasing size glycoforms. Indeed, underthese conditions, up to DP10 gluco-oligosaccharide APTSderivatives can be found in small amounts. All these APTS-gluco-oligosaccharides produced by the dextransucraseusing maltose as acceptor were successfully separated inless than 15 min.

In order to support these findings, the gluco-oligosac-charide mixture was analyzed by CE coupled to ESI-MS.A sheath liquid (water/isopropanol 20/8 v/v) was coaxiallydelivered to the capillary to improve spray stability and toallow completion of the CE electric field. The capillary wasempirically located at the same level of the ESI needle,providing ESI current stability. According to Fig. 3A, theTIC electropherogram shows that the CE separation ofAPTS-gluco-oligosaccharides was not drastically alteredby the CE-MS coupling. Each separated gluco-oligo-saccharide APTS derivative was analyzed by ESI-MS inthe negative-ion mode. The negative ESI-mass spectra



Figure 2. Separation of gluco-oligosaccharide APTSderivatives. Gluco-oligosaccharides were synthesizedby L. mesenteroides NRRL B-512F dextransucrase withmaltose as acceptor (see Fig. 1A). CE Conditions: fluores-cence detection; fused-silica capillary, 50 mm (ID)647 cm;buffer, 1% v/v acetic acid, 15 mM triethylamine, pH 3.7;outlet, anode. Peak identification: (Glc) glucose APTSderivative, (DP2), (DP3), (DP4), (DP5), (DP6), (DP7), (DP8),(DP9), and (DP10), gluco-oligosaccharide APTS deriva-tives with a DP of 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.The gluco-oligosaccharides are composed of a-1,6 linkedglucose units with a maltose unit at the reducing end (seeFig. 1A).

(Fig. 3B) are dominated by peaks assigned to singly andmultiply charged deprotonated molecular ions [M-zH]z-,illustrated by three representative APTS-gluco-oligosac-charide spectra. Peaks at m/z 782.1, m/z 390.6, andm/z 260.2 assigned to [M-H]2, [M-2H]2- and [M-3H]3-

respectively, typify DP2 APTS derivative; likewise peaksat m/z 1106.1 [M-H]2, m/z 552.7 [M-2H]2-, m/z 368.2[M-3H]3- typify DP4 APTS derivative; and finally m/z714.8 [M-2H]2-, m/z 476.4 [M-3H]32 typify DP6 APTSderivative. In conclusion, it can be proposed that thegluco-oligosaccharides synthesized with acceptor mal-tose by L. mesenteroides NRRL B-512F dextransucraseis composed of a mixture of glycoforms with a degree ofpolymerization from DP2 to DP10, corresponding to thelast detected APTS derivative. In addition, among theseglycoforms DP2 and DP4 are the most abundant molec-ular species.

Figure 3. CE-MS analysis of gluco-oligosaccharide APTSderivatives. Gluco-oligosaccharides were synthesized byL. mesenteroides NRRL B-512F dextransucrase with mal-tose as acceptor (see Fig. 1A). (A) TIC profile. Conditionsand peak identification were the same as in Fig. 2, exceptfused-silica capillary, 75 mm (ID)680 cm; (B) Negative ESI-mass spectra of DP2, DP4 and DP6 APTS derivatives.

The second gluco-oligosaccharide mixture was synthe-sized by the same acceptor reaction process asdescribed above. The use of glucose as acceptor insteadof maltose leads to a gluco-oligosaccharide series thatonly contains a1?6 linked glucopyranosyl units (Fig. 1B).Likewise, the crude mixture was derivatized and theAPTS-gluco-oligosaccharides analyzed by CE. The elec-tropherogram (Fig. 4) is similar to the previous one show-ing the presence of DP2, Glcp-(a1?6)-Glc-APTS, fol-lowed by DP3, Glcp-(a1?6)-Glcp-(a1?6)-Glc-APTS,DP4, [Glcp-(a1?6)]3-Glc-APTS, DP5, [Glcp-(a1?6)]4-Glc-APTS, DP6, [Glcp-(a1?6)]5-Glc-APTS, DP7, [Glcp-(a1?6)]6-Glc-APTS, DP8, [Glcp-(a1?6)]7-Glc-APTS andfinally DP9, [Glcp-(a1?6)]8-Glc-APTS. The observedAPTS-gluco-oligosaccharide migration times are similarto those of the corresponding DP gluco-oligosaccharidesobtained with maltose as acceptor. However, the electro-pherogram profile differs drastically showing a decreasein abundance of glycoforms DP2 to DP9.

The APTS derivatives of gluco-oligosaccharides rangingup to DP9 are separated and characterized in less than15 min, whereas such an analysis requires more time



Figure 4. Separation of gluco-oligosaccharide APTSderivatives. Gluco-oligosaccharides were synthesizedby L. mesenteroides NRRL B-512F dextransucrase withglucose as acceptor (see Fig. 1B). CE conditions andpeak identification were the same as in Fig. 2. The gluco-oligosaccharide APTS derivatives are solely composedof a-1,6 linked glucose units (see Fig. 1B).

with classical analysis by reverse-phase HPLC. In addi-tion, the advantage of the reductive amination with APTSis the absence of reducing ends avoiding large and unre-solved doublets in the analysis of native gluco-oligosac-charides reflecting the a/b anomeric equilibrium, as pre-viously described for reverse phase HPLC analysis [17].Therefore, we investigated the capacity of CE to separategluco-oligosaccharide regioisomers resulting from thesimultaneous use of both maltose and glucose acceptorsleading to a mixture of gluco-oligosaccharides which dif-fer by their disaccharide residue located at the reducingend assigned to maltose and isomaltose (Fig. 1). This mix-ture was derivatized and the APTS-gluco-oligosacchar-ides were analyzed by CE using the conditions describedabove. The CE electropherogram (Fig. 5) shows pairs ofpoorly resolved peaks assigned to DP2, DP6, DP7, DP8,and DP9 regioisomers. By contrast, DP3, DP4, and DP5regioisomers appear as one peak. Despite our effort usingCE with running electrolyte at acidic pH we were unsuc-cessful in resolving gluco-oligosaccharide regioisomersover a large DP range.

Figure 5. Separation of a mixture of APTS derivativesof two regioisomer gluco-oligosaccharide series. Gluco-oligosaccharides were synthesized by L. mesenteroidesNRRL B-512F dextransucrase with glucose or maltoseas acceptor (see Fig. 1). CE conditions and peak identifi-cation were the same as in Fig. 2. Each peak is composedof two unresolved APTS-gluco-oligosaccharide regioi-somers as represented in Fig. 1.

3.2 CE separation of gluco-oligosaccharideregioisomers

Chen et al. [25] reported the separation of two APTS-tetrasaccharide regioisomers, [Glcp-(a1?4)]3-Glc-APTSand Glcp-(a1?6)-[Glcp-(a1?4)]2-Glc-APTS, under alka-line conditions. The electroosmotic flow is predominantcompared to the electrophoretic mobility, allowing themigration of the negatively charged oligosaccharideAPTS derivatives toward the cathode. The authors sug-gested that the separation of the APTS-tetrasaccharideregioisomers was induced by differences in their hydrody-namic volumes. Another approach to allow discriminationof closely related structures is to perform separation inborate buffer, successfully employed for the separationof various APTS derivatives of regioisomers of di- and oli-gosaccharides [25, 26]. Borate forms complexes with oli-gosaccharides via the vicinal hydroxyl groups, and com-plex stability differs between cis-vicinal or trans-vicinaldihydroxyl orientations in the sugar moiety. Indeed,borate complexation introduces charges and thus modi-



fies both the charge-to-mass ratios and the hydrody-namic volumes of the APTS sugars, according to theirstructures.

First attempts were performed with sodium borate bufferswhich proved to be poorly reproducible and of low reso-lution. Further separations were performed with lithiumborate buffer. Lithium co-ion mobility is 20% lower thansodium, causing an increase of the plate numbersdescribed by Kibler and Bächmann [32]. Resolution effi-ciency was studied with the DP2 regioisomers, maltoseGlcp-(a1?4)-Glc and isomaltose Glcp-(a1?6)-Glc APTSderivatives, using different lithium borate concentrations(Table 1). The migration time increased by increasing buf-fer concentrations, resulting in enhanced resolution. Thisprolonged separation analysis, which is presumably dueto the lower electroosmotic flow, can be adapted forseparation of gluco-oligosaccharide regoisomers withvarious ranges of DP. Lithium borate buffer (20 mM,pH 9.15) allows separation of maltose and isomaltosewith a resolution coefficient of R = 1.26. Such a lithiumborate concentration was suitable for the separation ofAPTS-gluco-oligosaccharide regioisomers up to DP8(Fig. 6), and corresponds to a good compromise betweenresolution and analysis time. From gluco-oligosaccharideDP2 to DP8, for each APTS-gluco-oligosaccharide typi-fied by a given DP, two peaks are successfully resolved.It should be noticed that resolution decreases withincreasing DP. Assignment of DP2 to DP4 gluco-oligosac-charide regioisomers was performed by spiking the crudemixture with APTS derivatives from single gluco-oligosac-charide purified by preparative HPLC. Using these stan-dards, it was unambiguously established that gluco-oli-gosaccharides containing a maltose unit at the reducingend were detected later than the gluco-oligosaccharideregioisomers only composed of a1?6 bonds (glucose asacceptor). This result can be explained by the fact thatAPTS-gluco-oligosaccharides with a maltose moiety atthe reducing end form more stable complexes withborate. Borate complexation seems to play a crucial rolefor the separation of the gluco-oligosaccharide regioi-

Table 1. Influence of the borate concentration on theresolution of maltose and isomaltose APTS deri-vatives (DP2)

Lithiumborate (mM)

pH Resolution Maltose migrationtime (min)

12.5 – 0.46 5.7820 9.15 1.26 9.0325 9.22 1.50 9.1550 9.73 2.96 14.60

CE conditions: fused-silica capillary, 50 mm ID647 cm;buffer, lithium tetraborate

Figure 6. Separation of a mixture of gluco-oligosacchar-ide APTS derivatives, same as in Fig. 5. CE conditionsfused-silica capillary, 50 mm ID647 cm; buffer, 20 mM

lithium tetraborate, pH 9.15; outlet, cathode. Peak identi-fication as in Fig. 2. M and G labeling corresponds to thegluco-oligosaccharides obtained from acceptor reactionwith maltose or glucose, respectively (see Fig. 1).

somers described in the present work, as classical alka-line conditions did not permit separation of the regioi-somers in a wide DP range.

As previously indicated, during the acceptor reaction withacceptor maltose, glucose is released because of the lowhydrolysis rate of sucrose. So, it can be assumed that glu-cose can also be recognized as acceptor to producegluco-oligosaccharides. In order to investigate this pro-spect, analysis conditions similar to those describedabove were applied to the gluco-oligosaccharide seriessynthesized with acceptor maltose. However, no APTS-gluco-oligosaccharides solely composed of a1?6 lin-kages were detected, only APTS-gluco-oligosaccharideswith maltose units at the reducing end, suggesting thatglucose is not released in sufficient concentrations toplay the role of acceptor (data not shown).

As mentioned above, the conditions were set to resolveAPTS-gluco-oligosaccharide regioisomers. Such a reso-lution led us to investigate identification of regioisomerssimply on the base of their migration times. As the migra-tion times vary with minor differences in experimentalconditions, we focus our effort on finding a suitable inter-nal standard (IS) as a reference to express the migration ofevery compound. The obvious reference standard would



have been glucose as this sugar is present in all reactionmixtures involving glucansucrases. However, APTS-glu-cose migrates far from other APTS-gluco-oligosacchar-ides, resulting in a low reliability standard. In the presentwork we suggest maltose be taken as a migration refer-ence. The inset in Fig. 6 gives the migration of regioisomerAPTS derivatives from DP2 to DP4 expressed in “maltosemigration units”, corresponding to the ratio betweenmigration times of APTS-gluco-oligosaccharides andAPTS-maltose. These values, obtained in 20 mM lithiumborate (pH 9.15), were calculated from eight electropher-ograms collected over two years. The migration timesvaried over a wide range: e.g., 7.08–9.19 min, in the caseof APTS-glucose (data not shown). However, using mal-tose migration units, each APTS-gluco-oligosaccharideregioisomer up to DP4 was unambiguously assigned.Accordingly, this migration calculation will help in theidentification of gluco-oligosaccharides in glucansucrasereaction mixtures at the regioisomer level.

3.3 Application to the separation ofglucodisaccharide regioisomers

In the present work, we synthesized gluco-oligosacchar-ides with dextransucrase from L. mesenteroides NRRL B-512F, a glucansucrase that produces mainly a1?6 link-ages. However, some other glucansucrases, producedby other orgamisms, synthesize gluco-oligosaccharidescomposed of a-linked glucose residues, via a1?2,a1?3 or a1?4 glucosidic linkages, depending on the en-zyme [8, 10]. Then the ability to resolve disaccharidescontaining these typical bonds by CE was investigated.Four reducing disaccharides were derivatized and mixed:kojibiose (a1?2 linked), nigerose (a1?3 linked), maltose(a1?4 linked), and isomaltose (a1?6 linked). In order toidentify a suitable migration standard, two additionalAPTS derivatives of glucodisaccharides were added:laminaribiose (b1?3 linked), cellobiose (b1?4 linked),and finally glucose. As the borate buffer system was pre-viously found to be efficient for the separation of regioi-somers, we applied these conditions (20 mM lithumborate, pH 9.15) to the mixture of the four a-linked andthe two b-linked APTS-glucodisaccharides. Figure 7depicts the resolution of all the a-linked glucodisacchar-ide regioisomers. The first eluted compound is the a1?3linked, followed by the a1?6 then the a1?2 and the lasteluted compound is the a1?4 linked APTS-glucodisac-charide. The two b-linked disaccharides composed ofglucose units are also resolved among the a-linked disac-charide group. Since laminaribiose and cellobiose areeluted close to the a-linked disaccharides, they mightnot be suitable as standards. As suggested above, wetook into account APTS-maltose migration time as areference to determine the migration of a-linked glucodi-

Figure 7. Separation of glucose disaccharide regioi-somers and glucose APTS derivatives. Conditions as inFig. 6. Peak identification: APTS derivatives of (1) niger-ose (a1?3); (2) isomaltose (a1?6); (3) kojibiose (a1?2);(4) maltose (a1?4); (L) laminaribiose (b1?3); (C) cello-biose (b1?4), and (Glc) glucose.

saccharides as “maltose migration units” calculated fromseven electropherograms (Table 2). Migration times ofAPTS derivatives did not provide any help in identificationof the compounds because of the standard variation.Based on migration time, it appeared that nigerose can-not be discriminated from isomaltose, the isomaltosemigration time also overlapped the kojibiose migrationtime, and the same was true for kojibiose and maltose.However, when calculations were based on maltosemigration time, all the a-linked APTS-glucodisaccharide

Table 2. Migration of a-linked APTS-disaccharides com-pared to their “maltose migration unit”, corre-sponding to the ratio between APTS-disacchar-ide migration time and the APTS-maltose migra-tion time

APTS-disaccharide

Migration time(min)

Maltose migrationunit

Nigerose 5.68 6 0.28 0.916 6 0.007Isomaltose 5.84 6 0.15 0.961 6 0.007Kojibiose 6.01 6 0.14 0.989 6 0.003Maltose 6.08 6 0.13 1.000Glucose 7.51 6 0.44 1.203 6 0.013

CE conditions: fused-silica capillary, 50 mm ID647 cm;buffer, 20 mM lithium tetraborate



regioisomers were identified. Therefore, the “maltosemigration unit” calculation is a complementary method forthe identification of APTS derivatives of the glucodisac-charide regioisomers. As mentioned above, this identifica-tion method can be adapted to APTS derivatives of variousoligosaccharides like the gluco-oligosaccharide seriesdescribed in the present work, in order to assign DPs andhelp in the designation of regioisomers without anyrequirement for spiking the mixture with pure standards.

4 Concluding remarks

Gluco-oligosaccharides synthesized via an acceptor re-action by L. mesenteroides NRRL B-512F dextransucrasewere separated by CE in acidic buffer after derivatizationwith APTS. However, such conditions did not permit res-olution of regioisomers from two series of gluco-oligosac-charides synthesized with two different acceptors. Cur-rent saline alkaline buffers were not suitable either for theseparation of such regioisomers with identical charge-to-mass ratios. This report demonstrates that the resolutionof APTS-gluco-oligosaccharide regioisomers over a wideDP range was more appropriately performed with boratebuffer systems, based on differentiation of borate/regioi-somer complexes. Their migrations were calculated inmigration units of maltose, the standard chosen in orderto sort out the regioisomers. Moreover, coupling to ESI-MS was employed for the determination of the DP ofgluco-oligosaccharides. In approaches to identify com-pounds with prebiotic properties, these combined meth-ods look promising for profiling mixtures of gluco-oligo-saccharides synthesized by glucansucrases.

We gratefully acknowledge Georgy Couram and AgnèsLendrat for their helpful work. We also appreciate technicalassistance from Pierre Escalier for dextransucrase purifi-cation. We thank Beckman (France) and Thermofinnigan(France) for their supply of a CE-MS system. This work is apart of the “Pôle Aliment Santé Midi-Pyrénées” programand was financially supported by the “Conseil RégionalMidi-Pyrénées” (AO DAER-Recherche 99.008161, DAER-Recherche 01.002720, DAER-Recherche 01.009056 andCPER SDV3.2).

Received July 17, 2003

5 References

[1] Monsan, P., Paul, F., FEMS Microbiol. Rev. 1995, 16, 187–192.

[2] Gibson, G. R., Roberfroid, M. B., J. Nutr. 1995, 125, 1401–1412.

[3] Tannock, W. G., Probiotics and Prebiotics: Where are WeGoing?, Caister Academic Press, Wymondham, UK 2002.

[4] Coutinho, P. M., Henrissat, B., http://afmb.cnrs-mrs.fr/CAZY/, 1999.

[5] Monchois, V., Remaud-Simeon, M., Russell, R. R., Monsan,P., Willemot, R. M., Appl. Microbiol. Biotechnol. 1997, 48,465–472.

[6] Arguello-Morales, M. A., Remaud-Simeon, M., Pizzut, S.,Sarcabal, P., Willemot, R., Monsan, P., FEMS Microbiol.Lett. 2000, 182, 81–85.

[7] Bozonnet, S., Dols-Laffargue, M., Fabre, E., Pizzut, S.,Remaud-Simeon, M., Monsan, P., Willemot, R. M., J. Bacter-iol. 2002, 184, 5753–5761.

[8] Jeanes, A., Haynes, W., Williams, C., Rankin, J., Melvin, E.,Austin, M., Clusray, J., Fisher, B., Tsuchiya, H., Rist, C., J.Am. Chem. Soc. 1954, 76, 5041–5052.

[9] Kralj, S., van Geel-Schutten, G. H., Rahaoui, H., Leer, R. J.,Faber, E. J., van der Maarel, M. J. E. C., Dijkhuizen, L., Appl.Envir. Microbiol. 2002, 68, 4283–4291.

[10] Monchois, V., Willemot, R. M., Monsan, P., FEMS Microbiol.Rev. 1999, 23, 131–151.

[11] Koepsell, H. J., Tsuchiya, H. M., Hellman, N. N., Kasenko, A.,Hoffmen, C. A., Sharpe, E. S., Jackson, R. W., J. Biol. Chem.1953, 200, 793–801.

[12] Remaud-Simeon, M., Willemot, R. M., Sarcabal, P., Potockide Montalk, G., Monsan, P., J. Mol. Catal. B: Enzym. 2000,10, 117–128.

[13] Robyt, J. F., Walseth, T. F., Carbohydr. Res. 1978, 61, 433–445.

[14] Côté, G. L., Robyt, J. F., Carbohydr. Res. 1982, 111, 127–142.

[15] Demuth, B., Jordening, H. J., Buchholz, K., Biotechnol.Bioeng. 1999, 62, 583–592.

[16] Paul, F., Oriol, E., Auriol, D., Monsan, P., Carbohydr. Res.1986, 149, 433–441.

[17] Arguello-Morales, M. A., Remaud-Simeon, M., Willemot, R.M., Vignon, M. R., Monsan, P., Carbohydr. Res. 2001, 331,403–411.

[18] Dols-Lafargue, M., Willemot, R. M., Monsan, P. F., Remaud-Simeon, M., Biotechnol. Bioeng. 2001, 74, 498–504.

[19] Dols-Lafargue, M., R. M.,Monsan, P. F., Remaud-Simeon,M., Biotechnol. Bioeng. 2001, 75, 276–284.

[20] Remaud, M., Paul, F., Monsan, P., Heyraud, A., Rinaudo, M.,J. Carbohydr. Chem. 1991, 10, 861–876.

[21] Remaud, M., Paul, F., Monsan, P., Lopez-Munguia, A., Vig-non, M. R., J. Carbohydr. Chem. 1992, 11, 359–378.

[22] Rocklin, R. D., Pohl, C. A., J. Liq. Chromatogr. 1983, 6,1577–1590.

[23] Chiesa, C., O’Neill, R. A., Electrophoresis 1994, 15, 1132–1140.

[24] Oefner, P. J., Chiesa, C., Glycobiology 1994, 4, 397–412.[25] Chen, F. T., Evangelista, R. A., Anal. Biochem. 1995, 230,

273–280.[26] Evangelista, R. A., Liu, M. S., Chen, F. T. A., Anal. Chem.

1995, 67, 2239–2245.[27] Robyt, J. F., Eklund, S. H., Carbohydr. Res. 1983, 121, 279–

286.[28] Dols, M., Remaud, S., Willemot, R. M., Vignon, M. R., Mon-

san, P. F., Carbohydr. Res. 1997, 305, 549–559.[29] Paul, F., Monsan, P., Auriol, D., Patent 2545501, France

1984.[30] Jackson, P., Biochem. J. 1990, 270, 705–713.[31] Nigou, J., Vercellone, A., Puzo, G., J. Mol. Biol. 2000, 299,

1373–1382.[32] Kibler, M., Bachmann, K., J. Chromatogr. A 1999, 836, 325–

331.


Documents

Capillary electrophoresis analysis of glucooligosaccharide regioisomers