Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

Ou e e ence: CAR 5203 P-au ho que y- 7 AUTHOR QUERY FORM Jou nal: CAR A icle Numbe : 5203 Please e-mail o ax you esponses and any co ec ions o: E-mail: co ec ions.essd@else ie .sps.co.in Fax: +31 2048 52799 Dea Au ho , Any que ies o ema ks ha ha e a isen du ing he p ocessing o you manusc ip a e lis ed below and highligh ed by flags in he p oo . Please check you p oo ca e ully and ma k all co ec ions a he app op ia e place in he p oo (e.g., by using on-sc een anno a ion in he PDF file) o compile hem in a sepa a e lis . Fo co ec ion o e ision o any a wo k, please consul h p://www.else ie .com/a wo kins uc ions. A icles in Special Issues: Please ensu e ha he wo ds ‘ his issue’ a e added (in he lis and ex ) o any e e ences o o he a icles in his Special Issue. Unci ed e e ences: Re e ences ha occu in he e e ence lis bu no in he ex – please posi ion each e e ence in he ex o dele e i om he lis . 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Ch ysina, Ma ia-Despoina Cha a gi, Ky a-Melinda Alexacou, László Somsák * O OR' R'O R'O OR' H NH N O H N O R O OR' R'O R'O OR' H NH N OO R R=Ph,(R'O) 4 - β - D -Glc p , (R'O) 4 -β- D -Gal p , (R'O) 3 -β- D -Xyl p R' = Ac, Bz, H The dep o ec ed biu e de i a i es showed mode a e inhibi o y e ec agains abbi muscle glycogen phospho ylase band human sali a y a -amylase. CAR 5203 No. o Pages 1, Model 5G 12 No embe 2009 ARTICLE IN PRESS 1 UNCORRECTED PROOF Syn hesis o new glycosyl biu e and u ea de i a i es as po en ial glycoenzyme inhibi o s Nó a Fel öldi a , Ma ie a Tó h a , E angelia D. Ch ysina b , Ma ia-Despoina Cha a gi b , Ky a-Melinda Alexacou b , László Somsák a,* a Depa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, H-4010 Deb ecen, Hunga y b Ins i u e o O ganic and Pha maceu ical Chemis y, The Na ional Hellenic Resea ch Founda ion, 48, Vas. Cons an inou A e. 116 35 A hens, G eece a icle in o A icle his o y: Recei ed 14 July 2009 Recei ed in e ised o m 11 Oc obe 2009 Accep ed 20 Oc obe 2009 A ailable online xxxx Keywo ds: Glycosyl u ea Glycosyl biu e 20 Inhibi o a -Amylase Glycogen phospho ylase abs ac O-Pe ace yla ed 1-(b-D-glucopy anosyl)-5-phenylbiu e was p epa ed in he eac ion o O-pe ace yla ed b-D-glucopy anosylisocyana e and phenylu ea. The eac ion o O-pe ace yla ed N-b-D-glucopy anosylu- ea wi h phenylisocyana e u nished he co esponding 1-(b-D-glucopy anosyl)-3,5-diphenyl- as well as 3-(b-D-glucopy anosyl)-1,5-diphenyl biu e s besides 1-(b-D-glucopy anosyl)-3-phenylu ea. O-Pe ace - yla ed 1-(b-D-glucopy anosyl)-5-(b-D-glycopy anosyl)biu e s we e ob ained in one-po eac ions o O-pe ace yla ed b-D-glucopy anosylamine wi h OCNCOCl ollowed by a second glycopy anosylamine o b-D-gluco,b-D-galac o and b-D-xylo configu a ions. O-Acyl p o ec ed 1-(b-D-glucopy anosyl)-3-(b-D-glyco- py anosylca bonyl)u eas we e ob ained om he eac ion o b-D-glucopy anosylisocyana e wi h C-(gly- copy anosyl) o mamides o b-D-gluco and b-D-galac o configu a ions. The O-acyl p o ec ing g oups we e emo ed unde acid- o base-ca alyzed anses e ifica ion condi ions, excep o he N-acylu ea de i a i es whe e he clea age o he N-acyl g oups was as e han dep o ec ion. Some o he new compounds exhibi ed mode a e inhibi ion agains abbi muscle glycogen phospho ylase band human sali a y a -amylase. Ó2009 Else ie L d. All igh s ese ed. 1. In oduc ion Ca bohyd a e p ocessing enzymes (glycoenzymes) ca alyze he assembly and deg ada ion o i al oligo- and polysaccha ides. Disco e y o inhibi o s o glycoenzymes and e ealing hei s uc- u e–ac i i y ela ionship (SAR) is a p incipal end in he de elop- men o ca bohyd a e-based d ugs. 1 Du ing ou p e ious esea ch se e al inhibi o s o glycosidase 2–5 and glycogen phospho ylase 6,7 (GP) enzymes we e syn hesized and cha ac e ized. The fi s nano- mola glucose-based inhibi o o abbi muscle GPb(RMGPb) was 50 iden ified among N-acyl-N 0 -b- D -glucopy anosyl u eas B( o selec ed examples see Table 1, en ies 5–7). The s ong binding o he 2-naph hyl de i a i e (en y 7) was a ibu ed o i s ex ensi e in e ac ions upon binding wi h he esidues lining he so-called b-pocke o he ca aly ic channel o he enzyme. 8 GP’s b-pocke is loca ed nex o he ca aly ic si e o he enzyme in he di ec ion o he b-anome ic subs i uen o bound D -glucose de i a- i es su ounded by bo h pola and apola amino acid side chains. 9 In he na i e RMGPb, his si e is occupied by wa e molecules he posi ions o which gi e insigh s o he design o new glucose 60 analogues wi h subs i uen s ha would op imize he ne wo k o in e ac ions wi h he esidues in close icini y. In o de o ack down he na u e o in e ac ions in he b-pocke , and he ole o he linke be ween he suga and he a oma ic pa o he molecule some N-a yl-N 0 -b- D -glucopy anosyl u eas A( o selec ed examples see Table 1, en ies 1–3) ha e been in es iga ed so a . De i a i es Aexhibi ed weake binding o RMGPbin compa ison o B. To s udy he e ec o a longe linke o simila composi ion, syn hesis o biu e de i a i es Cwas en isaged. As he se ies o compounds B in es iga ed so a con ained mainly apola esidues 7 (R = e.g., 70 me hyl, cyclohexyl, (subs i u ed)phenyl and naph hyl), an e o o exploi pola in e ac ions in he b-pocke by subs i u ing suga ings o R in bo h Band Cwas also planned. O OH HO HO OH H NH NR O A O OH HO HO OH H NH N O O R B O OH HO HO OH H NH N O O H NR C 0008-6215/$ - see on ma e Ó2009 Else ie L d. All igh s ese ed. doi:10.1016/j.ca es.2009.10.016 *Co esponding au ho . Tel.: +36 52512900x22348; ax: +36 52453836. E-mail add ess: [email p o ec ed] (L. Somsák). Ca bohyd a e Resea ch xxx (2009) xxx–xxx Con en s lis s a ailable a ScienceDi ec Ca bohyd a e Resea ch jou nal homepage: www.else ie .com/loca e/ca es CAR 5203 No. o Pages 7, Model 5G 12 No embe 2009 ARTICLE IN PRESS Please ci e his a icle in p ess as: Fel öldi, N.; e al. Ca bohyd . Res. (2009), doi:10.1016/j.ca es.2009.10.016 UNCORRECTED PROOF 2. Resul s and discussion The syn hesis o he p o ec ed 1-b- D -glucopy anosyl-5-phenyl biu e 4was achie ed by a eac ion o phenylu ea wi h b- D -gluco- 80 py anosylisocyana e 3 10 gene a ed in si u om glucosylamine 2 11 ob ained by ca aly ic educ ion o glucosylazide 1 12 (Scheme 1). Compound 4was isola ed by c ys alliza ion om MeOH, and dep o- ec ion was e ec ed by acid-ca alyzed anses e ifica ion o gi e 5. We ha e also a emp ed o p oduce 4in a somewha sho e way om b- D -glucosyl u ea 6 13 and PhNCO. In efluxing E OAc no eac ion occu ed be ween 6and 1.5 equi o PhNCO. Using he same a io o he eagen s in boiling oluene allowed isola ion o 1-b- D -glucosyl-3-phenyl u ea 7 10 in 41% yield. Pe o ming he eac ion in nea , boiling PhNCO ga e compound 7as well as biu e 90 de i a i es 8and 9in 4%, 24% and 31% isola ed yields, espec i ely. This unexpec ed esul could be explained by he p esence o wa e in he eac ion mix u e. When 6was eac ed wi h 1 equi o PhNCO in efluxing oluene in he p esence o 1 equi o H 2 O, he o ma ion o 7could be obse ed. U ea 7was also o med when 6and 1 equi o PhNH 2 we e eac ed in boiling oluene. Finally, hea ing o 7in nea PhNCO ga e biu e 9. All a emp s o a oid he o ma ion o hese p oduc s by ca e ully d ying he sol en s and eac an s as well as he use o molecula sie es in he eac ion mix u es ailed. S uc u al elucida ion o he new biu e s 8and 9 100 was s aigh o wa d by MS and NMR measu emen s as ollows om he selec ed cha ac e is ic da a shown in Scheme 1. Coupling o a glycosylu ea and a glycosylisocyana e could gi e 1,5-bis-glycosyl biu e de i a i es. Howe e , he use o comme - cial OCNCOCl as a bielec ophilic eagen 14,15 ,and glycosylamines as nucleophiles o e ed a simple and sho e syn he ic pa hway owa ds such a ge compounds. Thus, eac ion o glucopy anosyl- amine 2 11 wi h 0.5 equi o OCNCOCl ga e cleanly he expec ed 1,5-bis-glucosyl biu e 12 (Scheme 2). Asymme ic de i a i es could also be ob ained in wo-s ep, one-po eac ions om 2by 110 he addi ion o 1 equi o OCNCOCl ollowed by a second glycosyl- amine 10 16 o 11 17,18 o gi e 14 o 16, espec i ely. Deace yla ion was pe o med by he Zemplén p o ocol o esul in high yields o biu e s 13,15 and 17. To ob ain N-acyl-N 0 -b- D -glucopy anosyl u eas wi h a suga pa in he acyl g oup he eac ion o isocyana e 3 10 wi h O-pe ace yla - ed anhyd o-aldonamide 18 19 was in es iga ed fi s (Scheme 3). When 3and 18 we e eac ed in efluxing E OAc in equimola amoun s he con e sion o 18 was 25%, and he expec ed acylu ea 20 could be isola ed in 32% yield. Raising he empe a u e o he 120 boiling poin o oluene ga e a 56% con e sion o 18 and 50% iso- la ed yield o 20. A sa is ac o y esul was achie ed by applying 3in a wo old excess o a ull con e sion o 18, and he yield o 20 inc eased o 89%. F om a eac ion o 3and O-pe benzoyla ed anhyd o-aldonamide 19 20 (mola a io 2:1) in boiling oluene 23 was ob ained in a 98% yield. In each o he abo e eac ions bis-glu- copy anosyl u ea 21 was also isola ed in a ious amoun s which could be due o he p esence o aces o wa e in he mix u es. 21 A emp ed dep o ec ion o acyl u eas 20 and 23 was success ul un- de nei he basic no acidic anses e ifica ion condi ions because 130 clea age o he N-acyl moie y was as e han emo al o he O- acyl-p o ec ing g oups. Bis-glucopy anosyl u ea 21 was dep o ec- ed unde Zemplén condi ions o gi e 22 22 in sa is ac o y yield. The dep o ec ed compounds we e es ed o hei po ency o in- hibi abbi muscle glycogen phospho ylase bac i i y acco ding o he p o ocol desc ibed ea lie , 23,24 and he esul s a e summa ized in Table 1. Compounds wi h wo suga moie ies a ached o he e minal ni ogens o ei he u ea 22 (en y 4) o biu e s 13,15 and 17 (en ies 9–11) showed e y low inhibi ion o he enzyme ac i i y. Compa ison wi h he inhibi ion shown by he phenylbi- 140 u e de i a i e 5(en y 8) his may e eal ha he highly pola su- ga esidues opposi e o he b- D -glucopy anosyl pa o he compounds a e un a ou able o he binding. Among biu e s he b- D -xylopy anosyl de i a i e 17 p o ed he mos e ficien , and his may be in acco d wi h he less pola cha ac e o his esidue wi h h ee OH g oups compa ed o ou ones in he b- D -gluco- and galac opy anosyl pa s o 13 and 15, espec i ely. A compa ison o he phenyl subs i u ed de i a i es (en ies 1, 5and 8) allows o conclude ha he acyl u ea linke is supe io o he u ea and he biu e ype ones. 150 Compounds 13 and 22 we e also es ed agains human sali a y a -amylase acco ding o he me hod epo ed ea lie , 3 and exhib- i ed inhibi ion in he low millimola ange (IC 50 10.7 and 8.3 mM, espec i ely). In conclusion, syn hesis o 1-(b- D -glucopy anosyl) biu e s wi h an a oma ic and se e al b- D -glycopy anosyl esidues in he 5-posi- ion allowed o ex end s uc u e–ac i i y ela ionships o glucose analogue inhibi o s o glycogen phospho ylase. In oduc ion o he highly pola suga moie ies esul ed in weak binding. The leng h o he linke composed o NHCO elemen s be ween he b- 160 D -glucopy anosyl and he a oma ic pa s o he inhibi o s p o ed o be op imal in he acyl u ea se ies. Table 1 Inhibi ion o abbi muscle glycogen phospho ylase b(RMGPb) by selec ed glucose de i a i es and he new compounds O OH HO HO OH H NH NR O A O OH HO HO OH H NH N O R O B En y Inhibi ion ( l M) R En y Inhibi ion (K i ( l M)) 1K i 18 7 5 4.6 8 2IC 50 350 7 6 15.2 6,7 3K i 5.2 7 7 0.35 6,7 4IC 50 1209 ± 8 K ia 725 ± 5 O OH HO HO OH 22 O OH HO HO OH H NH N O H N O R C Inhibi ion IC 50 ( l M) K ia ( l M) 8 5 41.6 ± 4 20.8 ± 2 9 O OH HO HO OH 13 37% a 1 mM 10 O OH HO HO OH 15 32% a 1 mM 11 O OH HO HO 17 987 ± 127 592 ± 76 a K i alues we e calcula ed o compa ison pu poses by he Cheng–P uso equa ion: 25 K i =IC 50 /(1 + [S]/K m ). 2N. Fel öldi e al. / Ca bohyd a e Resea ch xxx (2009) xxx–xxx CAR 5203 No. o Pages 7, Model 5G 12 No embe 2009 ARTICLE IN PRESS Please ci e his a icle in p ess as: Fel öldi, N.; e al. Ca bohyd . Res. (2009), doi:10.1016/j.ca es.2009.10.016 UNCORRECTED PROOF 3. Expe imen al 3.1. Gene al me hods Mel ing poin s we e measu ed in open capilla y ubes o on a Kofle ho -s age and a e unco ec ed. Op ical o a ions we e de e - mined wi h a Pe kin–Elme 241 pola ime e a . NMR spec a we e eco ded wi h B uke 360 (360/90 MHz o 1 H/ 13 C) o B uke 400 (400/100 MHz o 1 H/ 13 C) o A ance DRX 500 (500/125 MHz o 1 H/ 13 C) spec ome e s. Chemical shi s a e e e enced o in e - 170 nal TMS ( 1 H), o o he esidual sol en signals ( 13 C). 1 HNMR assignmen s we e es ablished on he basis o g adien enhanced DQF-COSY spec a. 26 P o on chemical shi s and scala coupling cons an s we e ex ac ed om he esolu ion enhanced 1D p o on spec a. COSY spec a we e eco ded wi h 512 2 k da a poin s, spec al wid hs 4000 Hz, numbe o ansien s 4 and ecycle delay o 1.8 s. Mic oanalyses we e pe o med on a Ca lo-E ba analyse Type 1106. ESIMS we e eco ded wi h a B uke mic OTOF-Q ins u- men . TLC was pe o med on DC-Alu olle Kieselgel 60 F 254 (Me ck), and he pla es we e isualized unde UV ligh and by gen le hea - 180 ing. Fo column ch oma og aphy Kieselgel 60 (Me ck, pa icle size 0.063–0.200 mm) was used. Flasks we e flame-d ied be o e pe - o ming he eac ions. O ganic solu ions we e d ied o e anhy- d ous MgSO 4 ,and concen a ed unde diminished p essu e a 40–50 °C (wa e ba h). 3.2. 1-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)-5-phenyl biu e (4) 2,3,4,6-Te a-O-ace yl-b- D -glucopy anosylisocyana e (3) p e- pa ed in si u by Ichikawa’s me hod 10 om glucosylamine 2 11 (0.6 g, 1.73 mmol) was dissol ed in oluene (12 mL) and hen ea- 190 ed wi h phenyl u ea (0.47 g, 3.46 mmol). The mix u e was e- fluxed and moni o ed by TLC (2:1 E OAc–hexane). When he eac ion was comple e, he sol en was e apo a ed, and he esi- due was c ys allized om MeOH o gi e 0.37 g (42%) o 4.Mp: 207–209 °C; [ a ] D 23 (c1.68, ace one); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.42 (s, 1H, NH), 7.44–7.12 (m, 6H, A , NH), 5.33 ( , 1H, 3 J 3,4 = 10.0 Hz, H-3), 5.23 ( , 1H, H-1), 5.10 ( , 1H, 3 J 4,5 = 9.5 Hz, H-4), 4.98 ( , 1H, 3 J 2,3 = 9.2 Hz, H-2), 4.33 (dd, 1H, 2 J 6,6 0 = 12.6 Hz, H-6), 4.13 (dd, 1H, 3 J 5,6 0 = 2.1 Hz, H-6 0 ), 3.87 (ddd, 1H, 3 J 5,6 = 5.0 Hz, H-5), 2.44 (s, 1H, NH), 2.10, 2.08, 2.05 (s, 12H, 200 4OCOCH 3 ). 13 C NMR (CDCl 3 , 90 MHz) d(ppm) 170.7, 170.4, 170.0, 169.5 (CO), 155.0, 152.3 (NHCONH), 136.8, 129.4, 129.1, 124.4, 120.5 (A ), 78.9 (C-1), 73.3, 72.9, 70.0, 68.0 (C-2–C-5), 61.7 (C-6), 20.7, 20.6, 20.56, 20.5 (CH 3 ). ESIMS: [M+Na] + calcd 532.46, O OAc H NH N O O OAc NH N O O OAc H NN O Ph Ph Ph OH NPh OH NPh O OAc AcO AcO OAc N3O OR RO RO OR H NH N O O OAc NH2O OAc NCO H NNH2 O H N O Ph 608.56 10.24 (s); 6.44 (d, 3 J = 8.8 Hz) 5.11 (pseudo , 3 J = 9.6 Hz) 155.8; 152.1 608.56 10.73 (s); 6.32 (d, 3 J = 10.0 Hz) 151.8; 151.1 489.14 7.69 (s); 6.24 (d, 3 J = 9.2 Hz) 5.28 (pseudo , 3 J = 9.2 Hz) 154.6 [M+Na]+ NH (δ, ppm) H-1 (δ, ppm) C=O (δ, ppm) 8.75 (s) ++ 4321 R = Ac (42%) 5 R = H (91%) 76 (4%) 8 (24%) 9 (31%) abc e d O OAc AcO AcO OAc Scheme 1. Reagen s and condi ions: (a) H 2 , Raney-Ni, E OAc, ; (b) (Cl 3 CO) 2 CO, NaHCO 3 ,CH 2 Cl 2 ,H 2 O, ; (c) PhNHCONH 2 , oluene, eflux; (d) AcCl, CHCl 3 –MeOH, ; (e) PPh 3 , E OAc, NH 3 ,CO 2 , ; and ( ) nea PhNCO, eflux. O OAc AcO AcO OAc NH 2 O R R R RH NH N O H N O O R R R 3 R 1 R 2 NH 2 1 . a o 12 b o 14 and 16 2. c o 14 and 16 2 10-11 12-17 OR 3 R R 2 R 1 R R R1 R 2 R 3 Yield 12 OAc CH2OAc H OAc 91% d 13 OH CH2OH H OH 96% 10, 14 OAc CH2OAc OAc H 83% d 15 OH CH2OH OH H 98% 11, 16 OAc H H OAc 86% d 17 OH H H OH 96% Scheme 2. Reagen s and condi ions: (a) OCNCOCl, E 3 N, d y THF, N 2 a m, ; (b) OCNCOCl, d y THF, N 2 a m, 26 °C; (c) 10 o 11,E 3 N, d y THF, N 2 a m, 0–25 °C; and (d) ca . NaOMe, abs MeOH, . N. Fel öldi e al. / Ca bohyd a e Resea ch xxx (2009) xxx–xxx 3 CAR 5203 No. o Pages 7, Model 5G 12 No embe 2009 ARTICLE IN PRESS Please ci e his a icle in p ess as: Fel öldi, N.; e al. Ca bohyd . Res. (2009), doi:10.1016/j.ca es.2009.10.016 UNCORRECTED PROOF ound: 532.16. Anal. Calcd o C 22 H 27 N 3 O 11 (509.47): C, 51.87; H, 5.22; N, 7.51. Found: C, 51.97; H, 5.24; N, 7.50. 3.3. 1-(b- D -Glucopy anosyl)-5-phenyl biu e (5) Biu e 4(250 mg, 0.49 mmol) was dissol ed in a mix u e o MeOH and CHCl 3 (1:1). A ca aly ic amoun o AcCl was added and he mix u e was s i ed a . The eac ion was moni o ed by 210 TLC (1:1 CHCl 3 –MeOH). When he eac ion was comple e, i was neu alized wi h solid NaHCO 3 ,and hen fil e ed and he sol en was e apo a ed. The esidue was pu ified by column ch oma og a- phy (9:1 CHCl 3 –MeOH) o gi e 162 mg (97%) o 5as a whi e pow- de . Mp: 191–193 °C; [ a ] D +4 (c0.68, MeOH);. 1 H NMR (DMSO- d 6 +D 2 O, 360 MHz) d(ppm) 7.41 (d, 2H, A ), 7.29 ( , 2H, A ), 7.04 ( , 1H, A ), 4.68 (d, 1H, 3 J 1,2 = 8.9 Hz, H-1), 3.63 (dd, 1H, 3 J 5,6 0 = 1.6 Hz, H-6 0 ), 3.42 (dd, 1H, 2 J 6,6 0 = 12.1 Hz, H-6), 3.19–3.14 (m, 1H, 3 J 5,6 = 5.8 Hz, H-5), 3.22, 4.26, 4.25 ( , 1H, 3 J 2,3 = 3 J 3,4 = 3 J 4,5 = 8.9 Hz, H-2,3,4). 13 C NMR (DMSO-d 6 , 90 MHz) d(ppm) 154.6, 152.2 220 (NHCONH), 138.2, 128.9, 123.1, 119.0 (A ), 80.3 (C-1), 78.5, 77.3, 72.9, 69.8 (C-2–C-5), 60.8 (C-6). Anal. Calcd o C 14 H 19 N 3 O 7 (341.32): C, 49.27; H, 5.61; N, 12.31. Found: C, 49.35; H, 5.66; N, 12.30. 3.4. Reac ion o 2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl u ea 13 (6) wi h phenylisocyana e U ea 6(100 mg, 0.26 mmol) was efluxed in nea phenylisocya- na e(1 mL). The eac ion was moni o ed by TLC (2:1 E OAc–hex- ane). When he eac ion was comple e, he excess amoun o phenylisocyana e was emo ed by dilu ing he mix u e wi h hex- 230 ane. The o med p ecipi a e was fil e ed, dissol ed in CH 2 Cl 2 and washed wi h sa d aq NaHCO 3 . The o ganic laye was sepa a ed, d ied and he sol en was e apo a ed. The esidue was sepa a ed by column ch oma og aphy (50:1 CH 2 Cl 2 –ace one) o gi e, in he o de o elu ion, compounds 8,9and 7 10 (4%). 3.4.1. 3-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)-1,5- diphenyl biu e (8) Yield: 36 mg (24%), colou less sy up. R = 0.89 (25:1 CHCl 3 –ace- one) [ a ] D +0.2 (c1.71, DMSO). 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.73 (s, 1H, N(CONHC 6 H 5 ) 2 ), 8.75 (s, 1H, N(CONHC 6 H 5 ) 2 ), 7.51– 240 7.09 (m, 10H, A ), 6.32 (d, 1H, 3 J 1,2 = 10.0 Hz, H-1), 5.66 ( , 1H, 3 J 3,4 = 9.5 Hz, H-3), 5.39 ( , 1H, 3 J 4,5 = 9.5 Hz, H-4), 5.16 ( , 1H, 3 J 2,3 = 10.0 Hz, H-2), 4.52 (dd, 1H, 2 J 6,6 0 = 12.6 Hz, H-6), 4.18 (dd, 1H, 3 J 5,6 0 = 2.1 Hz, H-6 0 ), 4.05 (ddd, 1H, 3 J 5,6 = 3.7 Hz, H-5), 2.13, 2.06, 2.04, 2.00 (s, 12H, 4OCOCH 3 ). 13 C NMR (CDCl 3 + DMSO-d 6 , 90 MHz) d(ppm) 168.6, 167.9, 167.8, 167.7 (CO), 151.8, 151.1 (NCON), 138.3–116.0 (A ), 79.0 (C-1), 73.1, 71.4, 66.3, 66.1 (C-2– C-5), 60.1 (C-6), 19.2, 19.1, 19.0, 18.9 (CH 3 ). ESIMS: [M+Na] + calcd o C 28 H 31 N 3 O 11 (585.57): 608.56, ound: 608.19. 3.4.2. 1-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)-3,5- 250 diphenyl biu e (9) Yield: 46 mg (31%), whi e powde . R = 0.80 (25:1 CHCl 3 –ace- one). Mp: 226–229 °C; [ a ] D 4(c0.64, DMSO). 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.24 (s, 1H, NHCON(C 6 H 5 )CONHC 6 H 5 ), 7.56– 7.06 (m, 10H, A ), 6.44 ( , 1H, 3 J H-1,NH = 8.8 Hz, NHCON(C 6 H 5 )CON- HC 6 H 5 ), 5.28 ( , 1H, 3 J 3,4 = 9.6 Hz, H-3), 5.11 ( , 1H, 3 J 1,2 = 9.6 Hz, H- 1), 5.02 ( , 1H, 3 J 4,5 = 9.6 Hz, H-4), 4.78 ( , 1H, 3 J 2,3 = 9.6 Hz, H-2), 4.31 (dd, 1H, 2 J 6,6 0 = 12.3 Hz, H-6), 4.10 (dd, 1H, 3 J 5,6 0 = 1.8 Hz, H- 6 0 ), 3.81 (ddd, 1H, 3 J 5,6 = 4.4 Hz, H-5), 2.09, 2.024, 2.019, 1.98 (s, 12H, 4OCOCH 3 ). 13 C NMR (CDCl 3 , 90 MHz) d(ppm) 170.6, 260 170.1, 169.8, 169.4 (CO), 155.8, 152.1 (NCON), 137.4, 135.6, 130.3, 129.9, 129.4, 128.9, 124.1, 120.1 (A ), 79.8 (C-1), 73.5, 72.5, 69.8, 67.9 (C-2–C-5), 61.5 (C-6), 20.7, 20.5 (CH 3 ). ESIMS: [M+Na] + calcd o C 28 H 31 N 3 O 11 (585.57): 608.56, ound: 608.18. 3.5. 1,5-Bis-(2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl)biu e (12) Glucosylamine 2 11 (400 mg, 1.15 mmol) was dissol ed in d y THF (5 mL), hen E 3 N (80 l L, 0.58 mmol) and OCNCOCl (46 l L, 0.58 mmol) we e added. The mix u e was s i ed a unde ni o- gen a mosphe e. A e he eac ion was comple e (TLC, 10:1 E OAc–hexane) he mix u e was dilu ed wi h wa e (5 mL), and 270 washed wi h E OAc (3 5 mL). The o ganic phase was d ied and he sol en was e apo a ed unde educed p essu e o yield 402 mg (91%) colou less sy up. R = 0.83 (10:1 E OAc–hexane); [ a ] D 19 (c0.97, CHCl 3 ); 1 H NMR (DMSO-d 6 ): d(ppm) 9.11 (s, 1H, NH), 8.02 (d, 2H, J=9.5 Hz, 2 NH), 5.42, 5.34, 4.92, 4.86 (4 pseudo , 8H, J=9.5, 9.6 Hz in each, 2 H-1, 2 H-2, 2 H-3, 2H-4), 4.16–3.94 (m, 6H, 2 H-5, 2 H-6, 2 H-6 0 ), 2.00, 1.99, 1.98, 1.95 (4s, 24H, 8 CY 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.4 (COCH 3 ), 154.3 (2 NHCO), 78.7 (C-1), 73.1, 72.8, 70.0, 67.9 (C-2 o C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. O OAc AcO AcO OAc NCO 3 O OAc AcO AcO OAc CONH 2 O OBz BzO BzO OBz CONH 2 O OAc AcO AcO OAc H N O O OAc AcO AcO OAc H N O 18 19 20 R = Ac (89%) PhCH 3 , Δ PhCH 3 , Δ O OR RO RO OR H NH N O 21 R = Ac 22 R = H (79%) + + NaOMe, MeOH, O RO OR OR RO O BzO OBz OBz BzO H N O H N O O AcO OAc AcO AcO 23 R=Ac,R'= Bz (98%) Scheme 3. 4N. Fel öldi e al. / Ca bohyd a e Resea ch xxx (2009) xxx–xxx CAR 5203 No. o Pages 7, Model 5G 12 No embe 2009 ARTICLE IN PRESS Please ci e his a icle in p ess as: Fel öldi, N.; e al. Ca bohyd . Res. (2009), doi:10.1016/j.ca es.2009.10.016 UNCORRECTED PROOF 280 Calcd o C 30 H 41 N 3 O 20 (763.65): C, 47.18; H, 5.41; N, 5.50. Found: C, 47.23; H, 5.50; N, 5.58. 3.6. Gene al p ocedu e I o he syn hesis o 1-(2,3,4,6- e a-O- ace yl-b- D -glucopy anosyl)-5-(pe -O-ace yl-b- D - glycopy anosyl)biu e s 14 and 16 Glucosylamine2 11 (100 mg,0.29 mmol)wasdissol edind yTHF (2 mL), and some eshly hea ed molecula sie es we e added. The mix u e was cooled o 20 °C, OCNCOCl (23 l L, 0.29 mmol) was added, and s i ed a 26 °C unde ni ogen a mosphe e o a day. Then a solu ion o 2,3,4,6- e a-O-ace yl-b- D -galac opy anosyl- 290 amine 16 (10, 100 mg, 0.29 mmol) o 2,3,5- i-O-ace yl-b- D -xylopy - anosylamine 17,18 (11, 80 mg, 0.29 mmol) in d y THF (2 mL) and E 3 N (40 l L, 0.29 mmol) we e added, and he mix u e was allowed o wa m up o . When he eac ion was comple e (TLC, 10:1 E OAc– hexane) he insoluble ma e ials we e fil e ed o wi h suc ion, and hesol en was emo edunde educedp essu e.Thec udep oduc was pu ified by column ch oma og aphy (7:1 E OAc–hexane). 3.6.1. 1-(2,3,4,6-Te a-O-ace yl-b- D -galac opy anosyl)-5- (2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl)biu e (14) P epa ed acco ding o Gene al p ocedu e I (Sec ion 3.6) om 300 glucosylamine 2(100 mg, 0.29 mmol) and galac osylamine 10 (100 mg, 0.29 mmol). Yield: 183 mg (83%) colou less sy up. R = 0.58 (10:1 E OAc–hexane); [ a ] D 15 (c0.98, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 7.83 (b s, 1H, NH), 7.60–7.41 (m, 2H, 2 NH), 5.37 (pseudo d, 1H, J=3.0 Hz, H-4-Gal), 5.34 (pseudo , J=9.6 Hz H-3-Glc), 5.23 (pseudo , 1H, J=9.3, 9.4 Hz, H-1-Glc), 5.20–5.15 (m, 2H, H-1-Gal, H-3-Gal), 5.09 (pseudo , 1H, J=9.3 Hz, H-2-Gal), 5.07–4.98 (m, 2H, H-2-Glc, H-4-Glc), 4.18 (dd, 1H, J=4.5, 12.4 Hz, H-6a-Glc), 4.13–4.09 (m, 1H, H-5-Gal), 4.09–4.00 (m, 3H, H-6a- Gal, H-6b-Gal, H-6b-Glc), 3.94 (m, 1H, H-5-Glc), 2.21, 2.11, 2.01, 310 1.99, 1.98, 1.96 (6b s, 24H, 8 CH 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.4 (COCH 3 ), 154.3 (2 NHCO), 79.1, 78.9 (C-1-Glc, C-1-Gal), 73.2, 72.8, 71.9, 71.0, 70.1, 68.0, 67.8, 67.2 (C-2- Glc o C-5-Glc, C-2-Gal o C-5-Gal), 61.6, 61.0 (C-6-Glc, C-6-Gal), 20.6, 20.5 (CH 3 ). Anal. Calcd o C 30 H 41 N 3 O 20 (763.65): C, 47.18; H, 5.41; N, 5.50. Found: C, 47.29; H, 5.54; N, 5.61. 3.6.2. 1-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)-5-(2,3,5- i- O-ace yl-b- D -xylopy anosyl)biu e (16) P epa ed acco ding o Gene al p ocedu e I (Sec ion 3.6) om glucosylamine 2(100 mg, 0.29 mmol) and xylosylamine 11 320 (80 mg, 0.29 mmol). Yield: 171 mg (86%) colou less sy up. R = 0.58 (10:1 E OAc–hexane); [ a ] D 28 (c0.58, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 7.75 (b s, 1H, NH), 7.49 (b s, 2H, 2 NH), 5.35 (pseudo , 1H, J=9.4 Hz, H-3-Glc), 5.29 (pseudo , 1H, J=9.1 Hz, H-3-Xyl), 5.23 (pseudo , 1H, J=9.4 Hz, H-1-Glc), 5.13 (pseudo , 1H, J=9.1 Hz, H-1-Xyl), 5.06–4.88 (m, 4 H, H-4-Glc, H- 2-Glc, H-2-Xyl, H-4-Xyl), 4.17 (dd, J=4.8, 12.4 Hz, H-6a-Glc), 4.04 (dd, 1H, J=2.0, 12.4 Hz, H-6b-Glc), 3.98 (dd, J=5.4, 11.5 Hz, H- 5a-Xyl), 3.93–3.88 (m, 1 H, H-5-Glc), 3.51–3.44 (ddd, 1H, J=1.3 Hz, 11.5 Hz, H-5b-Xyl), 2.18, 2.01, 2.00, 1.99, 1.98, 1.96 330 (5b s, 21 H, 7 CY 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.8, 169.4 (COCH 3 ), 154.4 (2 NHCO), 79.0, 78.7 (C-1- Glc, C-1-Xyl), 73.1, 72.8, 71.8, 70.0, 69.9, 68.6, 68.0 (C-2-Glc o C- 5-Glc, C-2-Xyl o C-4-Xyl), 63.7, 61.5 (C-6-Glc, C-5-Xyl), 20.5, 20.4 (CH 3 ). Anal. Calcd o C 27 H 37 N 3 O 18 (691.59): C, 46.89; H, 5.39; N, 6.08. Found: C, 46.99; H, 5.31; N, 6.15. 3.7. Gene al p ocedu e II o he emo al o O-acyl p o ec ing g oups An O-pe ace yla ed compound (100 mg) was dissol ed in d y MeOH (1 mL), and a solu ion o NaOMe (1 M in MeOH) was added 340 o he solu ion in a ca aly ic amoun . The eac ion mix u e was kep a . When he eac ion was comple e (TLC, 7:3 CHCl 3 –MeOH) he solu ion was neu alized wi h a ca ion exchange esin Ambe - lys 15 (H + o m). Fil a ion and emo al o he sol en esul ed in he co esponding deace yla ed suga de i a i es. 3.7.1. 1,5-Bis-(b- D -glucopy anosyl)biu e (13) P epa ed acco ding o Gene al p ocedu e II (Sec ion 3.7) om biu e 12 (100 mg, 0.13 mmol). Yield: 54 mg (96%) colou less sy - up. R = 0.45 (1:3 CHCl 3 –me hanol); [ a ] D 4(c0.51, MeOH); 1 H NMR (D 2 O): d(ppm) 4.91 (d, 2H, J=9.3 Hz, 2 H-1), 3.86 (dd, 350 2H, J=1.6, 12.3 Hz, 2 H-6a), 3.70 (dd, 2H, J=5.3, 12.3 Hz, 2H-6b), 3.52, 3.43, 3.40 (3 pseudo , 6H, J=9.0, 9.3 Hz in each 2H-2, 2 H-3, 2 H-4), 3.51–3.47 (m, 2H, 2 H-5); 13 CNMR (Me 2 SO-d 6 ): d(ppm) 154.2 (NHCO), 80.3, 78.4, 77.3, 72.8, 69.7 (C-1-C-5), 60.8 (C-6). Anal. Calcd o C 14 H 25 N 3 O 12 (427.36): C, 39.35; H, 5.90; N, 9.83. Found: C, 39.46; H, 5.99; N, 9.90. 3.7.2. 1-(b- D -Galac opy anosyl)-5-(b- D -glucopy anosyl)biu e (15) P epa ed acco ding o Gene al p ocedu e II (Sec ion 3.7) om biu e 14 (100 mg, 0.13 mmol). Yield: 53 mg (98%) colou less sy - 360 up. R = 0.35 (1:3 CHCl 3 –MeOH); [ a ] D 7(c0.54, H 2 O); 1 HNMR (DMSO-d 6 ): d(ppm) 4.65–4.56 (m, 2H), 3.69–3.57 (m, 2H), 3.47– 3.00 (m, 9H), 2.95 ( , 1H); 13 C NMR (D 2 O): d(ppm) 159.6 (2 NHCO), 81.5 (C-1-Glc, C-1-Gal), 77.9, 77.2, 72.6, 70.0 (C-2- Glc o C-5-Glc, C-2-Gal o C-5-Gal), 61.3 (C-6-Glc, C-6-Gal). Anal. Calcd o C 14 H 25 N 3 O 12 (427.36): C, 39.35; H, 5.90; N, 9.83. Found: C, 39.44; H, 5.98; N, 9.89. 3.7.3. 1-(b- D -Glucopy anosyl)-5-(b- D -xylopy anosyl)biu e (17) P epa ed acco ding o Gene al p ocedu e II (Sec ion 3.7) om biu e 16 (100 mg, 0.14 mmol). Yield: 55 mg (96%) colou less sy - 370 up. R = 0.63 (1:3 CHCl 3 –MeOH); [ a ] D 12 (c0.52, H 2 O); 1 HNMR (D 2 O): d(ppm) 4.86 (d, 1 H, J=9.3 Hz), 3.81 (dd, 1H, J=2.1, 12.6 Hz), 3.65 (dd, 1H, J=5.1 Hz, 12.6 Hz), 3.51–3.25 (m, 10 H); 13 C NMR (D 2 O): d(ppm) 156.3 (2 NHCO), 81.6, 80.9 (C-1-Glc, C-1-Xyl), 78.1, 77.1, 72.6, 72.4, 69.9, 69.7 (C-2-Glc o C-5-Glc, C- 2-Xyl o C-4-Xyl), 67.3, 61.2 (C-6-Glc, C-5-Xyl). Anal. Calcd o C 13 H 23 N 3 O 11 (397.34): C, 39.30; H, 5.83; N, 10.58. Found: C, 39.39; H, 5.90; N, 10.65. 3.8. 1-(2,3,4,6-Te a-O-ace yl-b- D -galac opy anosylca bonyl)-3- (2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl)u ea (20) 380 C-(2,3,4,6-Te a-O-ace yl-b- D -galac opy anosyl) o mamide 19 (18, 100 mg, 0.27 mmol) was dissol ed in d y oluene (3 mL). Then some molecula sie es and c ys alline isocyana e 3 10 (202 mg, 0.54 mmol) we e added. The eac ion was s i ed a eflux empe - a u e. A e one day he eac ion mix u e was wo ked up: he molecula sie es we e fil e ed o wi h suc ion and he solu ion was concen a ed unde educed p essu e. The esidue was pu ified by column ch oma og aphy (100:1 CHCl 3 –MeOH). Two p oduc s we e isola ed: 20 (177 mg, 89%) and 21 21 (52 mg). Cha - ac e iza ion o 20: colou less sy up; R = 0.55 (5:1 E OAc–hexane); 390 [ a ] D +18 (c0.84, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 8.66 (b s, 1H, NH), 8.64 (d, 1H, J=9.3 Hz, NH), 5.41 (pseudo d, J=2.2 Hz, H-4- Gal), 5.35 (pseudo , J=9.6 Hz, H-3-Glc), 5.28 (pseudo J=9.3 Hz, H-1-Glc), 5.22–5.15 (m, 2H, H-2-Gal, H-3-Gal), 5.04, 5.03 (2 pseudo , J=9.3, 9.6 Hz in bo h, H-4-Glc, H-2-Glc), 4.24–4.02 (m, 6 H, H-6a- Glc, H-6b-Glc, H-1-Gal, H-5-Gal, H-6a-Gal, H-6b-Gal), 3.91 (ddd, 1H, J=2.3, 4.8, 9.9 Hz, H-5-Glc), 2.12, 2.03, 2.02, 2.01, 1.99, 1.98, 1.96, 1.93 (8b s, 24H,8 CH 3 ). 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 170.1, 169.9, 169.7, 169.4 (COCH 3 ), 168.0, 152.1 (2 CONH), 78.8, 76.7 (C-1-Glc, C-1-Gal), 74.9, 73.5, 73.0, 70.6, 69.7, 68.2, 67.0, 400 65.9 (C-2-Glc o C-5-Glac, C-2-Gal o C-5-Gal), 61.7, 61.4 (C-6-Glc, N. Fel öldi e al. / Ca bohyd a e Resea ch xxx (2009) xxx–xxx 5 CAR 5203 No. o Pages 7, Model 5G 12 No embe 2009 ARTICLE IN PRESS Please ci e his a icle in p ess as: Fel öldi, N.; e al. Ca bohyd . Res. (2009), doi:10.1016/j.ca es.2009.10.016 UNCORRECTED PROOF C-6-Gal), 20.7, 20.6, 20.5 (CH 3 ). Anal. Calcd o C 30 H 40 N 2 O 20 (748.64): C, 48.13; H, 5.39; N, 3.74. Found: C, 48.20; H, 5.43; N, 3.79. 3.9. 1,3-Bis-(b- D -glucopy anosyl)u ea (22) P epa ed acco ding o Gene al p ocedu e II (Sec ion 3.7) om u ea 21 (190 mg, 0.26 mmol). Yield 80 mg (79%) amo phous solid. Li . 27 Mp: 207 °C (dec.); R = 0.45 (1:3 CHCl 3 -me hanol); [ a ] D +23 (c 0.59, DMSO), li . 27 [ a ] D 32.8 (c2, wa e ); 1 H NMR (D 2 O): d(ppm) 4.86 (d, 1H, J=9.3 Hz, H-1), 3.86 (dd, J=1.5, 12.3 Hz, H-6a), 3.69 410 (dd, 1H, J=5.2, 12.3 Hz, H-6b), 3.53, 3.38, 3.37 (3 pseudo , 3H, J=9.2, 9.7 Hz in each, H-2, H-3 ,H-4), 3.51–3.48 (m, 1H, H-5). 13 C NMR (D 2 O): d(ppm) 159.6 (CO), 81.5 (C-1), 77.9, 77.2, 72.6, 70.0 (C-2 o C-5), 61.3 (C-6). 3.10. 1-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)-3-(2,3,4,6- e a-O-benzoyl-b- D -glucopy anosylca bonyl)-u ea (23) C-(2,3,4,6-Te a-O-benzoyl-b- D -glucopy anosyl) o mamide 20 (19, 54 mg, 0.086 mmol) was dissol ed in d y oluene (1 mL), and some molecula sie es we e added ollowed by c ys alline isocya- na e 3 10 (64 mg, 0.172 mmol) mmol). The eac ion mix u e was 420 hea ed o eflux empe a u e. When he eac ion was comple e (TLC, 5:1 E OAc–hexane) he molecula sie es we e fil e ed o wi h suc ion and he esidue was concen a ed unde educed p essu e. The c ude p oduc was pu ified by column ch oma og a- phy (1:1 E OAc–hexane). Two p oduc s we e isola ed: 23 (84 mg, 98%) and 21 21 (23 mg). Cha ac e iza ion o 23: colou less sy up; R = 0.53 (1:1 E OAc–hexane); [ a ] D 6(c0.61, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 9.01 (b s, 1H, NH), 8.56 (d, 1H, J=9.2 Hz, NH), 8.05, 7.94, 7.90, 7.78 (4d, 8H, A ), 7.63–7.32 (m, 12H, A ), 6.02 (pseudo , 1H, J=9.4 Hz, H-3-GlcBz), 5.77 (pseudo , 1H, 430 J=10.1 Hz, H-4-GlcBz), 5.75 (pseudo , 1H, J=9.8 Hz, H-2-GlcBz), 5.38 (pseudo , 1H, J=9.6 Hz, H-3-GlcAc), 5.34 (pseudo , 1H, J=9.4 Hz, H-1-GlcAc), 5.00, 4.99 (2 pseudo , 2H, J=9.4, 9.8 Hz in bo h, H-4-GlcAc, H-2-GlcAc), 4.65 (dd, 1H, J=2.3, 12.3 Hz, H-6a- GlcBz), 4.57 (dd, 1H, J=4.6, 12.3 Hz, H-6b-GlcBz), 4.54 (pseudo , 1H, J=9.9 Hz, H-1-GlcBz), 4.43–4.39 (m, 1H, H-5-GlcBz), 4.14 (dd, 1H, J=4.8, 12.3 Hz, H-6a-GlcAc), 4.01 (dd, 1H, J=1.7, 12.3 Hz, H-6b-GlcAc), 3.94 (ddd, 1H, J=1.9, 4.4, 9.9 Hz), 2.19, 1.96, 1.94 (3b s, 12H, 4CH 3 ). 13 C NMR (CDCl 3 ): d(ppm) 170.5, 170.0, 169.5, 169.3, 166.0, 165.5, 165.2, 165.1 (COCH 3 ), 168.2, 440 152.6 (2 CONH), 133.6, 133.3, 133.2, 129.8, 129.7, 129.6, 128.2, 128.3 (CH–A ), 129.1, 128.7 (C–A ), 78.4, 76.6 (2 C-1), 76.1, 73.1, 72.8, 69.8, 69.6, 68.8, 68.1(2 C-2 o C-5), 62.8, 61.6 (2 C- 6), 20.5, 20.4 (CH 3 ). Anal. Calcd o C 50 H 48 N 2 O 20 (996.92): C, 60.24; H, 4.85; N, 2.81. Found: C, 60.19; H, 4.90; N, 2.89. Acknowledgemen s This wo k was suppo ed by he Hunga ian Scien ific Resea ch Fund(OTKA 45927). This wo k was suppo ed by he EU Ma ie Cu - ie Ea ly S age T aining (EST) Con ac No. MEST-CT-020575 a Ma ie Cu ie Hos Fellowships o he T ans e o Knowledge (ToK) Con- 450 ac No. MTKD-CT-2006–042776. The au ho s hank P o esso K. E. Kö é and M . M. He czeg o ad ice on and help wi h 2D NMR spec a, and D . Gy. Gyémán o pe o ming he a -amylase assay. Re e ences 1. Wong, C. H. Ca bohyd a e-based D ug Disco e y; Wiley-VCH: Weinheim, 2003. 2. Kiss, L.; Somsák, L. Ca bohyd . Res. 1996,291, 43–52. 3. Gyémán , G.; Kand a, L.; Nagy, V.; Somsák, L. Biochem. Biophys. Res. Commun. 2003,312, 334–339. 4. Elek, R.; Kiss, L.; P aly, J. P.; Somsák, L. Ca bohyd . Res. 2005,340, 1397–1402. 4605. Kand a, L.; Remenyik, J.; Ba a, G.; Somsák, L.; Gyémán , G.; Pa k, K. H. Ca bohyd . Res. 2005,340, 1311–1317. 6. 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