Unusual carbohydrate transfer reactions of cyclodextrin transglucosylase

Unusual carbohydrate transfer reactions of cyclodextrin transglucosylase

Tlic cyclodestxin transglucosylase from has been shown by 1Gench a kansfer of glucose wits from t,lrc alpha Schardinger de&in t,o a number of carbohy...

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cyclodestxin transglucosylase from has been shown by 1Gench a kansfer of glucose wits from t,lrc alpha Schardinger de&in t,o a number of carbohydrates. It was demonstrated t’hat’ coupling was retarded or in some cases completely inhibited by changes in colifiguration or structure at any position other than the iiumber one of the glucopyranosc acceptor. l’azur c’t al. (2) lat,er used similar methods to prepare a series of methyl glycosides of malt,o-oligosaccharides. The R. m,ucexzns enzyme cffrctjcd transfer of glucose units from the cyclohexaamylose t’o t’he a- and fi-methyl glucosides, yielding methyl maltoand methyl side, methyl maltotriosidc, maltot&ao&e as tdle products of the rcdist,I.it,lit,ioii react ions. The currtwt, study was initiated to rcesamiw the stereo rec~uiremcnts for accept01 act)ivity of the cyclod&rin transglucosylase from 13. maccraus. The advisability of this rc-csnminatjion was suggested by the demollat,ratioll that, there was some latitude in the strwt ural rec~uirrmetitjs of the acceptor molrculc ill carbohydrat,e trallsfcr rractjions. l3acilZrrs ~maccrans cl al. (1) to effect,

l’cat et ~11.(:3) have drmonstrat~cd this witjh the 1) enzyme from potatoes. This t,ransglucosylase which catalyzes the syntheses of a series of malto-ohgosaccharides was shown to be able to effect t,raiisfcrs t,o monosaccharides which differ from glucose. Coupling reactions were tried with t,hc: CX- and P-methyl-wxylopyranosides, with (Y- methyl-2-deosy-n-glucopyranoside, w%h methyl-l)-mannopyl,alloside, and with t8hc methyl glycosides of :Z-o-mctl~yl-l~-glll~opyranoside and (i-o-methyl-I)-glricopyralloside. The sensitiw t,hin layer chromatographic techniques developed for t#hemalt’ooligosaccharides (1) allowed these rcact,iotl mixtures to be scanned on a micro basis.

rthmc precipit:ttion to increase ttle yield of 1Ike a-dextrin. The cuupling reactions were conducted as dcscribed by Pazur 12), cscept, that 0.623, of his qu:mtitics werr enlploycd. Twenty-five mg. of the nrctlryl glycoside and 25 mg. of the cyctohexamylose were dissolved in 0.10 ml. of witcr. A 0.22.ml. aliquot of the cgctodcxtrin tr:tnsgtyc*osyt:~se solution was added to wch reaction tube. Samptcs were removed for :malysis after 20 xntl 68 tIr. incubation at room temper:Lt~ure. I~Lictl reaci,ion mixture vas cxnlllint=d by the ttlin layer chromatogrnphic prcjcedures drveloped in this tnt~mxfory (4). Ttle samples were :tlso examined by a modific:k tion of the thin layer analyses of (:ee (8). Silica gut (i was used as the adsorbent in these tests. TIIP sotvrnt system was t,olnrric-c,thrr-ett13not (10:50: 10, v/v). A larger amount of mztcrial was prepared from r~-rnettrgt-2-deos~-~~-~l~~(~~~t)~r:~~~osi(l~?. This was frxtionatcd by c~~ttulosc ~llroln:ttoRrupll~ (2). A portion of t.he trioside wts trydrolyzed in 1.0 .‘1 HCl and analyzed for 2~(leox?l)~~tucot~,yl.:lnose by thin layer ctlrorrlatogr:lt~t~~.


The results of experiments whcrcin cyclohexaamylose and cyclodcxt’rin t,ransglucosylase were incubated for 68 hr. with a variety of glycosides are shown in Fig. 1. In each case t’he dark spot at the top represents the remainder of tllc unreacted glycoside, and the material at the origin is t)lle cyclohexaamylose. In the absence of a11 acceptor, the cyclohexaamylose plus enzyme mixture (sample 2) showed only the spot) at blic origin. The intermediatr spots are the ~1~1 products of the transfer reactious. Sample 1 shows the a-methyl maltoside, maltotrioside, and maltot,etraoside in descclrdilg order. The same series appear ill sample 5 in which t,he a-methyl maltoside was tlrn acceptSor. The JI’~ values are the same as those obtained from a reaction mistlnv which had been fractionated according to the procedure of I’azur (2). The products iu samples I and 3 were more readily drtc&Mt~

t,han thr others. This had t)etn expected. Ttw results of I~IwKI~ (1) indicated that a compo~md with the structure and tollliguration of glucose would tw the most active accfptor. The readily detect,ahlc plodu%s formed u-itti t)oth LY-and 8-inctllyl-u-?cylopy~airosidt: WC st~ow~~i11samples :3 and 1. The I<,, vales (Jf the iiWrmcdiatc3 spotmu are slightly smallcl thatl t,twsc of the cu-mct,hyl maltodestl%w ill samples 1 and 3. Smaller I?/ \-alucs were also otktiiwd 011 tlir silica gel plates using t.hc t,otlicilc~-cttlrl.-ctialiol (10:X:40, v, ‘v) solvent, mistwc. 7’11~mistluxl which contained the mctIhyl I)-nlalurop.vl.a1’osidc, x(J. (i, showed only a few failit ilrt,ermrdiate spots, which could he sw11 otlty OII very careful csamillation. They uwo Iiot visit+ at, all OILthe silica gel plates. It) \vas Ilot possible to oht’ain targci~ yields of ttwso products l)ecaust iiicuhat,ion twyolid 68 1~. wusc~l the fragmentation of the ryclotic~xanniyto~c ill ttic atwncc of an acceptor. Samples T aild !) show t hc intermediatjc spots for the products formed whc~r t’hc rnct1ry1



‘,-dt~or;y-~~-~l~~cc~py~a~~~~s~~ was a part of ttw lllolccl~lr.


misturr which coiitailiod glycosidt of I:-o-mct,tlyl-lb glrlcopyranosc did Ilot’ yktd any detcctahl(~ i~kwniediatc products uilder the colitlit~ioiis of tlic esperimelks. \$‘tiilr t hc mos;t active acccpt,or motccrilcs arc those ha\-illg t,tle iitmwiific~d ~liicosc~ kkructure, ttitw clsperimcnts have dcmonst8ratcd that, ttw cyclodcxtrill transglucosylasc from 13. ~vznwi~auscan effect tjrallsfws to ccttaiil deli\-at i\-cs t)tiat arc stiwf~ural modifications of glucose. In two cases it has trcii shown t,tiat stluctrual modificat~iot~s al, posithi Six Of the glucose a(!(!epkw d0 ll(Jf, prevcwt coiipling. The sylopyl.allositlea, which lack the six hytllospm~~tllyl gror~p, and, also ilicttlyl-pi-ci-nictlryl-I)-~tlicopyl.alloside cstiihit roiiptilig. t11t

I. 2.


pymuosidc a1~1 (i-o-rnetllyl-I)-gllicopyi.alloside ww riwtl as acceptors. ‘lY~c KJ \-aliies ii1 t trrsc cuscs arc tiigtirr than those in saniplc 1. IN fact t.tw II), value of t Ire disacctraridc cwlitaining ttic a -methyl 2 - dcosy I) - gtll(~op?-~anosidc is jrlst twlow the spot of ttlc~ 1llllYwtcd acceptol~. A\ lai~gw amouilt of the oligosac~ctial~id(~ colltaillillg the deoxy compomld was pwpaw1 and fract iollatctl o11a cellulose colmnll CL). ‘1%~ triosittc \vas hydrolyzed in dihkc acid. C‘til~omato~t~aptiic ailatysrs pro~~~l t,trat

3 1. 5. Ii.

i. x.