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RO
OF
G aphical abs ac
pp xxx–xxxSyn 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, Ma ie a Tó h, E angelia D. 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
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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
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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.
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