Substituent distribution in highly branched dextrins from methylated starches

Transcription

1 Crohydrte Reserch 327 (2000) Sustituent distriution in highly rnched dextrins from methylted strches Yuri E.M. vn der Burgt, Jck Bergsm, Ido P. Bleeker, Pul J.H.C. Mijlnd, Johnnis P. Kmerling, *, Johnnes F.G. Vliegenthrt Deprtment of Bio-Orgnic Chemistry, Bij oet Center, Utrecht Uni ersity, PO Box , NL-3508 TB Utrecht, The Netherlnds AVEBE Reserch nd De elopment, AVEBE-weg 1, NL-9607 PT Foxhol, The Netherlnds Received 2 Novemer 1999; ccepted 10 Ferury 2000 Astrct Grnulr potto strch nd mylopectin potto strch were methylted to molr sustitutions (MS) up to Extensive lph-mylse digestion gve mixtures of prtilly methylted oligomers. Precipittion of lrger frgments y methnol yielded minly -limit dextrins (84 99%). Methnol precipittes were extensively digested with etmylse yielding, -limit dextrins. The verge sustitution level of rnched glucose residues in the dextrins thus otined ws determined fter per deuteriomethyltion y using FAB mss spectrometry, nd compred with tht of the linerly linked glucose residues. The present work demonstrtes tht methyltion does not show ny preference for sustitution t either rnched or linerly linked glucose residues, tking into ccount the inherently lower mount of sustitution sites t rnched residues. The results corroorte erlier studies wherein it ws found tht sustituents in rnched regions re distriuted lmost rndomly. In ddition, the dt enle the determintion of the verge degree of rnching of prtilly methylted dextrins Elsevier Science Ltd. All rights reserved. Keywords: Methylted strches; Amylopectin potto strch; Brnching; Amylolysis; Sustituent distriution 1. Introduction Strch, the min energy reserve of higher plnts, consists of mylose nd mylopectin. Amylose is considered s n essentilly (1 4)-linked -D-glucn, wheres mylopectin contins up to 5% of rnched -D-glucose residues. The short mylopectin side chins re (1 6)-linked to longer chins [1] nd rrnged in doule helices, uilding up the orgnised crystlline frmework of the strch grnule [2,3]. Moleculr mss distriution, * Corresponding uthor. Tel.: /2184; fx: E-mil ddress: kme@oc.chem.uu.nl (J.P. Kmerling). mylose nd mylopectin content, nd the degree of rnching of these molecules depend on the otnicl source of the strch grnules [1,4,5]. Chemicl modifictions of strches provide strch products tht fulfil vrious demnds [6]. Detiled informtion on the distriution of sustituents cn contriute to the understnding of reltions etween moleculr structure nd functionl properties, thus opening wys to more-rtionl derivtistion processes. Previously, we reported tht in methylted strches, crystlline liner mylopectin side chins, which ply n importnt role in the retrogrdtion of geltinised strches, contin fewer sustituents thn morphous rnched prts [7,8]. The im of the present study is to /00/$ - see front mtter 2000 Elsevier Science Ltd. All rights reserved. PII: S (00)

2 424 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) exmine the sustitution pttern in rnched regions of methylted strches in more detil to determine whether preferences exist for sustitution sites t rnched glucose residues. 2. Results nd discussion Preprtion of methylted strches. Methylted potto strches (P) nd methylted mylopectin potto strches (A) were prepred y methyltion of strch grnules in n lkline queous suspension using dimethyl sulfte [8]. The molr sustitution (MS) vlues of the strch derivtives studied re listed in Tle 1. The MS vlues nd the monoscchride compositions of the intct grnules of P10, P20, P30, A10, A20 nd A30 were determined y using GLC nd hve een reported previously [7]. Preprtion of, -dextrins from methylted strches. Geltinised methylted strches P10 P30 nd A10 A30 were extensively digested with lph-mylse from Bcillus sutilis [9], yielding mixtures of (1 4)- -Dglucns of different sizes with vrying degrees of (1 6) rnching (DB). The so-clled limit dextrins, which re highly rnched nd hve degree of polymeristion (DP) 8 (s determined y high-performnce liquid chromtogrphy (HPLC) [7]), were seprted from the liner oligomers y precipittion with methnol. The MS vlues of the -limit dextrins were determined fter quntifying the mount of cross contmintion during methnol precipittion (Tle 1) [7]. The methnol precipittes thus otined (further referred to s -dextrin frctions) of P10 P30 nd A10 A30, minly contining -limit dextrins (84 99%) [7], were extensively digested with -mylse from Bcillus cereus, yielding, -limit dextrins nd smll oligomers [9,10]. For ech smple, the DP decresed during -mylolysis, s could e demonstrted y 1 H NMR spectroscopy [11,12]. The, -limit dextrins otined from P10 P30 nd A16 A30 were seprted from lierted mltose y using Bio-Gel P-2 chromtogrphy. Smll (1 4)-linked oligomers with or without single (1 6) rnching, originting from cross contmintion during the methnol precipittion [7] nd with DP up to 4, s determined y MALDI-TOF mss spectrometry, co-eluted with the mltose frction. Determintion of the sustitution level y using monoscchride nlysis shows tht the MS vlues of the, -limit dextrins re higher thn those of the corresponding -limit dextrins (Tle 1). This cn e rtionlised from the mode of ction of et-mylse, ecuse the inding of the mltosyl groups tht re susequently cleved will e stericlly hindered y the presence of methyl sustituents. Not only rnching points stop the digestion, ut lso sustituted glucose residues. Since predominntly non-sustituted mltose is relesed Tle 1 MS vlues of -limit dextrins nd, -limit dextrins from P10 P30 nd A10 A30 Smples Code MS MS ( -limit MS (, -limit MS MS c (grnule) dextrins) dextrins) (%) (, -limit dextrins ) Methylted potto strch P Methylted potto strch P Methylted potto strch P Methylted mylopectin A potto strch Methylted mylopectin potto strch A Methylted mylopectin potto strch A Molr sustitution (MS) is defined s mole of sustituents/mole of glucose residues. MS vries from 0 (ntive strch) to 3 (permethylted liner strch). All MS vlues re determined y using GLC in triplo (s 0.01). MS=[MS (, -limit dextrins) MS ( -limit dextrins)/ms ( -limit dextrins)] 100%. c Clculted MS of virtul, -limit dextrins using Eq. (1), with 0 MS terminl 0.15.

3 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) from the -limit dextrins, the, -limit dextrins will hve higher MS vlues. As cn e seen from Tle 1, the reltive increses in MS vlues ( MS) re lrger for the lower sustituted -limit dextrins. This oservtion is in greement with the steric hindrnce of etmylse lredy mentioned, which is expected to increse with the MS. The P-2 mltose frction contined smll mounts of methylted glucns, proly originting from the erlier mentioned cross contmintion. However, from these dt it cnnot e excluded tht et-mylse lso liertes mltose groups of low methyltion level. In conclusion, -mylolysis of -limit dextrins from P10 P30 nd A10 A30 results in reltive enrichment of prtilly methylted glucose residues in the generted, -limit dextrins s compred with the -limit dextrins. FAB mss spectrometric determintion of erge sustitution le els of constituting residues in per(deuterio)methylted, -limit dextrins. The -dextrin frctions (minly contining -limit dextrins) nd the, -limit dextrins otined from P10 P30 nd A10 A30 were perdeuteriomethylted with CD 3 I [13], then methnolysed with methnolic HCl [14,15]. This derivtistion procedure yielded 12 mixtures ll contining the three chemiclly different methyl glucosides (Scheme 1), nmely per(deuterio)methylted methyl glucosides (C ), (deuterio)methylted methyl glucosides hving HO-4 free (A nd D ), nd (deuterio)methylted methyl glucosides hving HO-4 nd HO-6 free (B ). Ech mixture of three chemiclly different methyl glucosides (Scheme 1) ws nlysed y FABMS. This resulted in three sets of sodiumctionised pseudomoleculr ions in the FAB spectrum. The first set hd the min pek t m/z 251 (B, with X=CD 3 ), the second t m/z 268 (A /D with X=CD 3 ), nd the third t m/z 285 (C, with X=CD 3 ). Ech set contins three pseudomoleculr ions hving zero, one, or two CH 3 groups. Within ech set, ll three compounds show the sme ionistion efficiency ecuse they re chemiclly identicl. Therefore, their FAB intensities represent the mount of ech compound present in one set. Note tht the intensities of the pseudomoleculr ions of the three different sets of methyl glucosides (B, A /D, nd C ) cnnot e compred with ech other, ecuse of their chemicl inequivlency. FAB spectr of mixtures derived from A10 A30 re shown in Fig. 1. The sodium-ctionised pseudomoleculr ion t m/z 251 nd its stellite ions t m/z 248 nd 245 (trce mounts) originte from residues linked t O-1, O-4, nd O-6 (B in Scheme 1, with X=H or CH 3 ). Their pek intensities correspond to the mounts of non-, mono-, nd disustituted residues, respectively. From these dt the verge sustitution level of glucose residues t rnching points (MS rnch ) cn e clculted. The sodium-ctionised pseudomoleculr ion t m/z 268 nd its stellite ions t m/z 265 nd 262 originte from residues linked t O-1 nd O-4, or the reducing end (A nd D in Scheme 1, respectively, with X=H orch 3 ). Using the rtios of the pek intensities, the verge sustitution level of glucose residues in liner (1 4)-linked chins (MS chin ) cn e determined. The results re summrised in Tle 2. In rnched glucose residues HO-6 is not ville for sustitution. Therefore, MS rnch is corrected (resulting in MS rnch,cor ) y ssuming tht HO-6 of rnched glucose residues is methylted in similr mounts s HO-6 of linerly linked glucose residues (tken from monoscchride nlysis of, limit dextrins). Interestingly, the corrected sustitution levels of rnched glucose residues (MS rnch,cor ) differ only slightly from those of linerly linked glucose residues (MS chin ) (see Tle 2). Therefore, no significnt preference for sustitution t either rnched or linerly linked glucose residues cn e concluded. In n erlier report we hve shown tht methyl sustituents re distriuted lmost rndomly in rnched regions of methylted strches ( -limit dextrins) [7]. The results shown here re in good greement with rndom distriution of methyl sustituents over rnched regions during methyltion. The sodium-ctionised pseudomoleculr ions t m/z 285 nd the stellite ions t m/z 282 nd 279 (trce mounts) originte from residues linked t O-1 only (C in Scheme 1, with X=H or CH 3 ). Since the mounts of mono- nd disustituted residues re too low for quntifiction, in ll, -limit dextrins the

4 426 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) Scheme 1. Methodology for the nlysis of methyltion t rnching points in -dextrin frctions nd in, -limit dextrins otined from P10 P30 nd A10 A30, nd for the determintion of the degree of rnching (DB). percentge of non-sustituted glucose residues t the non-reducing end must e higher thn 90%. Therefore, the verge sustitution level of terminl glucose residues is lower thn 0.15 (MS terminl 0.15). This implies tht lphnd et-mylse oth hve diminished ility for hydrolysis of the glycosidic linkges of scchrides in which the terminl non-reducing glucosyl group is methylted. Determintion of degree of rnching. The degree of rnching (DB) of hydrolystes of methylted strches cnnot e determined y 1D 1 H NMR spectroscopy ecuse of overlp of (prtilly) methylted 4)-Glc-(1 6)- residues with non-sustituted 4)-Glc-(1 residues, s is evident from Fig. 2. However, the DB my e determined y using GLC. For this purpose, ech mixture of methyl glucosides otined fter perdeuteriomethyltion nd methnolysis of, -limit dextrins from P10 P30 nd A10 A30 ws trimethylsilylted nd quntittively nlysed y GLC, using empiriclly determined molr response fctors [16,17] (Fig. 3). In this wy, the rtio for the residues B, C, nd A +D (Scheme 1, with X=H orch 3 ) ws determined. From this the

5 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) Fig H NMR spectrum of the methnol precipitte of A20 (only signls from nomeric protons re shown). Fig. 1. FAB spectr of sodium-ctionised methyl glucosides otined from A10, A20, nd A30 vi perdeuteriomethyltion nd methnolysis. The originl linkge of the methyl glucosides is indicted for A10, the corresponding structures re given in Scheme 1. DB could e derived (multiplied y 100% then gives the percentge of rnched glucose residues present in the originl polymer). The sme ws done for the -dextrin frctions from P10 P30 nd A10 A30. The verge DB vlues re given in Tle 3. From Tle 3 it is cler tht the DB vlues of ll -dextrin frctions increse fter -mylolysis. However, this increse shows no correltion with the MS. Using MS rnch nd MS chin from Tle 2, nd DB vlues from Tle 3, the MS vlues of, -limit dextrins (reconstructed virtul, -limit dextrins) were clculted ccording to Eq. (1). MS vlues thus otined re summrised in Tle 1. MS (, -limit dextrins ) =DB MS rnch +DB MS terminl +[100 (2 DB)] MS chin (1) The sustitution level of terminl glucose residues (MS terminl ) is estimted to e etween 0 nd 0.15 (see ove). As my e seen in Tle 2 Sustitution levels of residues originlly linked t O-1, O-4, nd O-6, nd of residues originlly linked t O-1 nd O-4 Smple MS rnch vlues MS contriution of 6- MS rnch,cor vlues MS chin vlues (residues MS d (%) (residues linked t O-1, O-sustituted residues (residues linked t O-1, linked t O-1 nd O-4) O-4, nd O-6) O-4, nd O-6) c P P P A A A A third deciml is included for clrity. However, the ccurcy of the mesurements justifies two decimls only. Positionl molr sustitution of 6-position (from monoscchride nlysis, counting one sustituent of 6-O-methyl-, 2,6-O-dimethyl-, 3,6-O-dimethyl-, nd 2,3,6-O-trimethyl methyl glucoside ech). c Clculted y dding the MS contriution of 6-O-sustituted residues to MS rnch. d MS chin =[MS chin MS rnch,cor /MS rnch,cor ] 100%.

6 428 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) Fig. 3. Gs chromtogrm of mixture of trimethylsilylted methyl glucosides (from, -limit dextrins of A10). The lelled peks re of interest. The first two peks correspond to per(deuterio)methylted methyl glucosides [originting from residues linked t O-1 only (C, / -pyrnose)], the next three with monosilylted (deuterio)methylted methyl glucosides [originting from residues linked t O-1 nd O-4 (A, / pyrnose/furnose)] nd the lst three with disilylted (deuterio)methylted methyl glucosides [originting from residues linked t O-1, O-4, nd O-6 (B, / -pyrnose/furnose)]. Tle 1, the mesured MS vlue of n, limit dextrin differs significntly from the one of reconstructed virtul, -limit dextrin. This is minly due to the inccurte vlue of MS terminl. To otin more specific vlue of MS terminl, detiled informtion is needed on which prtilly methylted mltose frgments re tolerted y et-mylse. 3. Mterils nd methods Preprtion of, -limit dextrins from methylted strches. Potto-strch grnules nd mylopectin potto-strch grnules were methylted in n lkline queous suspension y using Me 2 SO 4 (see Tle 1). The methylted strches otined, P10 P30 nd A10 A30 were geltinised nd extensively digested with lph-mylse from Bcillus sutilis [9] (BAN 240L from Novo). Oligomers with DP 8 precipitted y dding MeOH (5 vol equiv) [7]. The MeOH precipittes contined minly -limit dextrins nd were extensively digested with et-mylse from Bcillus cereus (Amno Phrmceuticl, Jpn) in shking wterth t 55 C nd ph 5.5, using 0.5 mss% of enzyme., -Limit dextrins were isolted fter seprtion from smller oligomers (DP 5) y using Bio-Gel P-2 column chromtogrphy. Anlyticl procedures. Monoscchride nlysis ws crried out y sujecting prtilly methylted oligomer or glucn smples to methnolysis (methnolic 1 M HCl, 18 h, 85 C). The resulting mixtures of methyl glucoside derivtives were trimethylsilylted (1:1:5 hexmethyldisilzne chlorotrimethylsilne pyridine), identified y GLC mss spectrometry, nd quntified y GLC using empiricl molr-response fctors [7]. GLC nlyses were performed on WCOT CP-SIL 5CB fused-silic cpillry column (25 m 0.32 mm) y using temperture progrm of C t 4 C/min nd mintining t 230 C for 5 min. GLC mss spectrometry of (O-methylted) glucose derivtives, mesured s trimethylsilylted methyl glucosides, ws crried out on n MD800/8060 system (Fisons instruments; electron energy, 70 ev), equipped with DB-1 fused-silic cpillry column (30 Tle 3 Degrees of rnching of -dextrin frctions nd, -limit dextrins from P10 P30 nd A10 A30 Smple DB ( -dextrin frctions) DB (, -limit dextrins) DB (%) P P P A A A The degree of rnching (DB) is defined s the percentge of glucose residues tht re rnched (clculted y dividing the mount of residues linked t O-1, O-4 nd O-6 y the totl mount of methyl glucosides in dextrin multiplied y 100%). DB=[DB (, -limit dextrins) DB ( -dextrin frctions)/db ( -dextrin frctions)] 100%.

7 Y.E.M. n der Burgt et l. / Crohydrte Reserch 327 (2000) m 0.32 mm, J&W Scientific), using temperture progrm of C t 4 C/min nd mintining t 230 C for 5 min. Prtilly methylted oligomers ( -limit dextrins otined fter -mylolysis of P10, P20, P30, A10, A20, nd A30 nd, -limit dextrins otined fter -mylolysis of -dextrin frctions) were permethylted with CD 3 I, s descried previously (Me 2 SO NOH) [13]. After methnolysis (methnolic 1 M HCl, 18 h, 85 C) the methyl glucosides were nlysed s such y FAB mss spectrometry; for gs-chromtogrphic purposes (determintion of DB) the methyl glucosides were trimethylsilylted s lredy descried efore injection. FAB mss spectrometry ws performed on Jeol JMS SX/SX 102A four-sector mss spectrometer, operted t 10 kv ccelerting voltge, equipped with Jeol MS-FAB 10 D FAB gun operted t 10 ma emission current, producing em of 6 kev xenon toms. The per(deuterio)methylted oligoscchride smples were mesured over mss rnge of m/z in mtrix of m-nitroenzyl lcohol sturted with NI, using the stndrd resolution of Prior to NMR nlysis, smples were exchnged once in D 2 O (99.9 tom% D, Isotec), then lyophilised nd dissolved in tom% D 2 O (Isotec). 1D 1 H NMR spectr were recorded on Bruker AC-300 spectrometer (Deprtment of Orgnic Chemistry, Utrecht University), equipped with 5 mm rod-nd proe, t proe temperture of 27 C. Chemicl shifts re expressed in ppm downfield from the signl for externl Me 4 Si, ut were ctully mesured y reference to externl cetone ( 2.225). The dt were collected in 16 K complex dt sets nd zerofilled to 32 K. After Fourier trnsformtion, using Gussin multipliction, the spectr were integrted with Bruker softwre. Acknowledgements This study ws supported y the PBTS Reserch Progrm with finncil id from the Ministry of Economic Affirs nd the Integrl Structure Pln for the Northern Netherlnds from the Dutch Development Compny. The uthors thnk Ms A. vn der Kerk-vn Hoof nd Mr C. Versluis for their contriutions to the FAB mss spectrometric results. References [1] Y. Tked, S. Tomook, S. Hizukuri, Crohydr. Res., 246 (1993) [2] A. Imerty, S. Pérez, Biopolymers, 27 (1988) [3] A. Imerty, H. Chnzy, S. Pérez, A. Buléon, V. Trn, Mcromolecules, 20 (1987) [4] S. Hizukuri, Crohydr. Res., 147 (1986) [5] D. Sievert, J. Holm, Strch/Stärke, 45 (1993) [6] O.B. Wurzurg (Ed.), Modified Strches: Properties nd Uses, CRC Press, Boc Rton, FL, [7] Y.E.M. vn der Burgt, J. Bergsm, I.P. Bleeker, P.J.H.C. Mijlnd, A. vn der Kerk-vn Hoof, J.P. Kmerling, J.F.G. Vliegenthrt, Crohydr. Res., 312 (1998) [8] Y.E.M. vn der Burgt, J. Bergsm, I.P. Bleeker, P.J.H.C. Mijlnd, A. vn der Kerk-vn Hoof, J.P. Kmerling, J.F.G. Vliegenthrt, Crohydr. Res., 320 (1999) [9] J.F. Royt, Strch, second ed., Acdemic Press, New York, 1984, pp [10] E. Bertoft, Crohydr. Res., 189 (1989) [11] M.J. Gidley, Crohydr. Res., 139 (1985) [12] J.D. Blke, M.L. Clrke, J. Littlemore, Crohydr. Res., 138 (1985) [13] I. Ciucnu, F. Kerek, Crohydr. Res., 131 (1984) [14] J.P. Kmerling, J.F.G. Vliegenthrt, in A.M. Lwson (Ed.), Clinicl Biochemistry Principles, Methods, Applictions, Vol. 1, Wlter de Gruyter, Berlin, 1989, pp [15] M.F. Chplin, Anl. Biochem., 123 (1982) [16] J.T. Scnlon, D.E. Willis, J. Chromtogr. Sci., 23 (1985) [17] A.D. Jorgensen, K.C. Picel, V.C. Stmoudis, Anl. Chem., 62 (1990)