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

Article abstract of DOI:10.1016/j.carres.2009.10.016

O-Peracetylated 1-(β-d-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated β-d-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-β-d-glucopyranosylurea with phenylisocyanate furnished the corresponding 1

Full text of DOI:10.1016/j.carres.2009.10.016

Carbohydrate Research 345 (2010) 208–213
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of new glycosyl biuret and urea derivatives as potential
glycoenzyme inhibitors
Nóra Felföldi a, Marietta Tóth a, Evangelia D. Chrysina b, Maria-Despoina Charavgi b,
Kyra-Melinda Alexacou b, László Somsák a,
*
a Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary
b Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vas. Constantinou Ave. 116 35 Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2009
Received in revised form 11 October 2009
Accepted 20 October 2009
Available online 10 November 2009
O-Peracetylated 1-(b-
D
-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated
-glucopyranosylurea
-glucopyranosyl)-3,5-diphenyl- as well as
-glucopyranosyl)-3-phenylurea. O-Peracetylated
-glycopyranosyl)biurets were obtained in one-pot reactions of
-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine
-galacto and b- -xylo configurations. O-Acyl protected 1-(b- -glucopyranosyl)-3-
-glycopyranosylcarbonyl)ureas were obtained from the reaction of b- -glucopyranosylisocyanate
with C-(glycopyranosyl)formamides of b- -gluco and b- -galacto configurations. The O-acyl protecting
b-
with phenylisocyanate furnished the corresponding 1-(b-
3-(b- -glucopyranosyl)-1,5-diphenyl biurets besides 1-(b-
1-(b- -glucopyranosyl)-5-(b-
O-peracetylated b-
of b- -gluco, b-
(b-
D-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-b-D
D
D
D
D
D
D
Keywords:
D
D
D
D
Glycosyl urea
Glycosyl biuret
Inhibitor
D
D
D
D
groups were removed under acid- or base-catalyzed transesterification conditions, except for the
N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of
the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b
a
-Amylase
Glycogen phosphorylase
and human salivary
a-amylase.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
interactions with the residues in close vicinity. In order to track
down the nature of interactions in the b-pocket, and the role of
the linker between the sugar and the aromatic part of the molecule
Carbohydrate processing enzymes (glycoenzymes) catalyze the
assembly and degradation of vital oligo- and polysaccharides.
Discovery of inhibitors of glycoenzymes and revealing their struc-
ture–activity relationship (SAR) is a principal trend in the develop-
ment of carbohydrate-based drugs.1 During our previous research
several inhibitors of glycosidase2–5 and glycogen phosphorylase6,7
(GP) enzymes were synthesized and characterized. The first nano-
molar glucose-based inhibitor of rabbit muscle GPb (RMGPb) was
some N-aryl-N0-b-
D-glucopyranosyl ureas A (for selected examples
see Table 1, entries 1–3) have been investigated so far. Derivatives
A exhibited weaker binding to RMGPb in comparison to B. To study
the effect of a longer linker of similar composition, synthesis of
biuret derivatives C was envisaged. As the series of compounds B
investigated so far contained mainly apolar residues7 (R = e.g.,
methyl, cyclohexyl, (substituted)phenyl and naphthyl), an effort
to exploit polar interactions in the b-pocket by substituting sugar
rings for R in both B and C was also planned.
identified among N-acyl-N0-b-
D-glucopyranosyl ureas B (for
selected examples see Table 1, entries 5–7). The strong binding
of the 2-naphthyl derivative (entry 7) was attributed to its
extensive interactions upon binding with the residues lining the
so-called b-pocket of the catalytic channel of the enzyme.8 GP’s
b-pocket is located next to the catalytic site of the enzyme in the
OH
O
H
N
H
N
HO
HO
A
B
C
R
OH
direction of the b-anomeric substituent of bound
D-glucose deriva-
O
O
O
tives surrounded by both polar and apolar amino acid side chains.9
In the native RMGPb, this site is occupied by water molecules the
positions of which give insights for the design of new glucose
analogues with substituents that would optimize the network of
OH
O
H
N
H
N
HO
HO
R
OH
O
O
OH
O
H
N
H
N
H
N
HO
HO
R
* Corresponding author. Tel.: +36 52512900x22348; fax: +36 52453836.
E-mail address: somsak@tigris.unideb.hu (L. Somsák).
OH
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.10.016

N. Felföldi et al. / Carbohydrate Research 345 (2010) 208–213
209
Table 1
heating of 7 in neat PhNCO gave biuret 9. All attempts to avoid
the formation of these products by carefully drying the solvents
and reactants as well as the use of molecular sieves in the reaction
mixtures failed. Structural elucidation of the new biurets 8 and 9
was straightforward by MS and NMR measurements as follows
from the selected characteristic data shown in Scheme 1.
Inhibition of rabbit muscle glycogen phosphorylase b (RMGPb) by selected glucose
derivatives and the new compounds
OH
OH
H
H
H
H
O
O
HO
HO
HO
HO
N
N
N
N
R
R
OH
OH
Coupling of a glycosylurea and a glycosylisocyanate could give
1,5-bis-glycosyl biuret derivatives. However, the use of commer-
cial OCNCOCl as a bielectrophilic reagent14,15, and glycosylamines
as nucleophiles offered a simpler and shorter synthetic pathway
towards such target compounds. Thus, reaction of glucopyranosyl-
amine 211 with 0.5 equiv of OCNCOCl gave cleanly the expected
1,5-bis-glucosyl biuret 12 (Scheme 2). Asymmetric derivatives
could also be obtained in two-step, one-pot reactions from 2 by
the addition of 1 equiv of OCNCOCl followed by a second glycosyl-
amine 1016 or 1117,18 to give 14 or 16, respectively. Deacetylation
was performed by the Zemplén protocol to result in high yields
of biurets 13, 15 and 17.
O
O
O
A
B
Entry Inhibition
M)
R
Entry Inhibition (Ki
M))
(l
(l
1
Ki 187
5
6
7
4.68
2
IC50 3507
15.26,7
0.356,7
3
4
Ki 5.27
To obtain N-acyl-N0-b-
D-glucopyranosyl ureas with a sugar part
in the acyl group the reaction of isocyanate 310 with O-peracetylated
anhydro-aldonamide 1819 was investigated first (Scheme 3). When
3 and 18 were reacted in refluxing EtOAc in equimolar amounts
the conversion of 18 was 25%, and the expected acylurea 20 could
be isolated in 32% yield. Raising the temperature to the boiling point
of toluene gave a 56% conversion of 18 and 50% isolated yield for 20.
A satisfactory result was achieved by applying 3 in a twofold excess
for a full conversion of 18, and the yield of 20 increased to 89%. From
a reaction of 3 and O-perbenzoylated anhydro-aldonamide 1920
(molar ratio 2:1) in boiling toluene 23 was obtained in a 98% yield.
In each of the above reactions bis-glucopyranosyl urea 21 was
also isolated in various amounts which could be due to the presence
of traces of water in the mixtures.21 Attempted deprotection of acyl
ureas 20 and 23 was successful under neither basic nor acidic
transesterification conditions because cleavage of the N-acyl
moiety was faster than removal of the O-acyl-protecting groups.
Bis-glucopyranosyl urea 21 was deprotected under Zemplén condi-
tions to give 2222 in satisfactory yield.
OH
O
IC50 1209
8
HO
HO
Kia 725
5
OH 22
Inhibition
OH
O
H
H
H
HO
HO
IC50
(
lM)
Kia
(l
M)
N
N
N
R
OH
O
O
C
8
9
41.6
4
20.8
2
5
OH
O
HO
HO
37% at 1 mM
32% at 1 mM
OH
13
HO
HO
OH
O
10
11
The deprotected compounds were tested for their potency to in-
hibit rabbit muscle glycogen phosphorylase b activity according to
the protocol described earlier,23,24 and the results are summarized
in Table 1. Compounds with two sugar moieties attached to the
terminal nitrogens of either urea 22 (entry 4) or biurets 13, 15
and 17 (entries 9–11) showed very low inhibition of the enzyme
activity. Comparison with the inhibition shown by the phenylbi-
uret derivative 5 (entry 8) this may reveal that the highly polar su-
OH
15
17
O
HO
HO
987 127
592 76
OH
a
Ki values were calculated for comparison purposes by the Cheng–Prusoff
equation:25 Ki = IC50/(1 + [S]/Km).
gar residues opposite to the b-
compounds are unfavourable for the binding. Among biurets the
b- -xylopyranosyl derivative 17 proved the most efficient, and this
may be in accord with the less polar character of this residue with
three OH groups compared to four ones in the b- -gluco- and
galactopyranosyl parts of 13 and 15, respectively. A comparison
of the phenyl substituted derivatives (entries 1, 5 and 8) allows
to conclude that the acyl urea linker is superior to the urea and
the biuret type ones.
D-glucopyranosyl part of the
2. Results and discussion
D
The synthesis of the protected 1-b-
biuret 4 was achieved by a reaction of phenylurea with b-
D
-glucopyranosyl-5-phenyl
-gluco-
D
D
pyranosylisocyanate 310 generated in situ from glucosylamine 211
obtained by catalytic reduction of glucosylazide 112 (Scheme 1).
Compound 4 was isolated by crystallization from MeOH, and depro-
tection was effected by acid-catalyzed transesterification to give 5.
We have also attempted to produce 4 in a somewhat shorter
Compounds 13 and 22 were also tested against human salivary
way from b-D
-glucosyl urea 613 and PhNCO. In refluxing EtOAc
a
-amylase according to the method reported earlier,3 and exhib-
no reaction occurred between 6 and 1.5 equiv of PhNCO. Using
ited inhibition in the low millimolar range (IC50 10.7 and 8.3 mM,
the same ratio of the reagents in boiling toluene allowed isolation
respectively).
of 1-b-D
-glucosyl-3-phenyl urea 710 in 41% yield. Performing the
In conclusion, synthesis of 1-(b-
D-glucopyranosyl) biurets with
reaction in neat, boiling PhNCO gave compound 7 as well as biuret
derivatives 8 and 9 in 4%, 24% and 31% isolated yields, respectively.
This unexpected result could be explained by the presence of water
in the reaction mixture. When 6 was reacted with 1 equiv of
PhNCO in refluxing toluene in the presence of 1 equiv of H2O, the
formation of 7 could be observed. Urea 7 was also formed when
6 and 1 equiv of PhNH2 were reacted in boiling toluene. Finally,
an aromatic and several b- -glycopyranosyl residues in the 5-posi-
D
tion allowed to extend structure–activity relationships of glucose
analogue inhibitors of glycogen phosphorylase. Introduction of
the highly polar sugar moieties resulted in weak binding. The
length of the linker composed of NHCO elements between the b-
D-glucopyranosyl and the aromatic parts of the inhibitors proved
to be optimal in the acyl urea series.

210
N. Felföldi et al. / Carbohydrate Research 345 (2010) 208–213
OAc
O
OR
H
H
H
O
O
c
O
a
AcO
AcO
b
RO
RO
NH2
N3
NCO
N
N
N
Ph
OAc
OAc
OAc
OR
O
O
1
2
3
4 R = Ac (42%)
5 R = H (91%)
d
e
H
H
O
N Ph
O
N Ph
OAc
O
H
H
H
O
H
N
H
O
O
AcO
AcO
f
N
N
N
N
NH2
N
N
+
Ph
+
Ph
Ph
OAc
OAc
OAc
OAc
O
O
O
O
6
7 (4%)
8 (24%)
9 (31%)
[M+Na]+
489.14
7.69 (s);
608.56
608.56
NH (δ, ppm)
H-1 (δ, ppm)
C=O (δ, ppm)
10.73 (s);
8.75 (s)
6.32 (d, 3J = 10.0 Hz)
10.24 (s);
6.24 (d, 3J = 9.2 Hz)
6.44 (d, 3J = 8.8 Hz)
5.11 (pseudo t,
3J = 9.6 Hz)
5.28 (pseudo t,
3J = 9.2 Hz)
154.6
151.8; 151.1
155.8; 152.1
Scheme 1. Reagents and conditions: (a) H2, Raney-Ni, EtOAc, rt; (b) (Cl3CO)2CO, NaHCO3, CH2Cl2, H2O, rt; (c) PhNHCONH2, toluene, reflux; (d) AcCl, CHCl3–MeOH, rt; (e) PPh3,
EtOAc, NH3, CO2, rt; and (f) neat PhNCO, reflux.
mined with a Perkin–Elmer 241 polarimeter at rt. NMR spectra
were recorded with Bruker 360 (360/90 MHz for 1H/13C) or Bruker
400 (400/100 MHz for 1H/13C) or Avance DRX 500 (500/125 MHz
for 1H/13C) spectrometers. Chemical shifts are referenced to inter-
nal TMS (1H), or to the residual solvent signals (13C). 1H NMR
assignments were established on the basis of gradient enhanced
DQF-COSY spectra.26 Proton chemical shifts and scalar coupling
constants were extracted from the resolution enhanced 1D proton
spectra. COSY spectra were recorded with 512 ꢀ 2 k data points,
spectral widths 4000 Hz, number of transients 4 and recycle delay
of 1.8 s. Microanalyses were performed on a Carlo-Erba analyser
Type 1106. ESIMS were recorded with a Bruker micrOTOF-Q instru-
ment. TLC was performed on DC-Alurolle Kieselgel 60 F254 (Merck),
and the plates were visualized under UV light and by gentle heat-
ing. For column chromatography Kieselgel 60 (Merck, particle size
0.063–0.200 mm) was used. Flasks were flame-dried before per-
forming the reactions. Organic solutions were dried over anhy-
drous MgSO4, and concentrated under diminished pressure at
40–50 °C (water bath).
OAc
O
AcO
AcO
NH2
OAc
2
2. c to 14 and 16
1. a to 12
R2
R1
R3
O
b to 14 and 16
NH2
R2
R
R
10-11
R
R
H
O
H
H
R3
R
R
N
N
N
R1
O
R
R
O
O
12-17
R
R1
R2
H
R3
OAc 91%
Yield
12 OAc CH2OAc
d
3.2. 1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-5-phenyl
13 OH
CH2OH
H
OH
H
96%
83%
biuret (4)
10, 14 OAc CH2OAc OAc
2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylisocyanate (3) pre-
d
pared in situ by Ichikawa’s method10 from glucosylamine 211
(0.6 g, 1.73 mmol) was dissolved in toluene (12 mL) and then trea-
ted with phenyl urea (0.47 g, 3.46 mmol). The mixture was re-
fluxed and monitored by TLC (2:1 EtOAc–hexane). When the
reaction was complete, the solvent was evaporated, and the resi-
due was crystallized from MeOH to give 0.37 g (42%) of 4. Mp:
15 OH
11, 16 OAc
CH2OH
H
OH
H
H
98%
OAc 86%
d
17 OH
H
H
OH 96%
Scheme 2. Reagents and conditions: (a) OCNCOCl, Et3N, dry THF, N2 atm, rt; (b)
OCNCOCl, dry THF, N2 atm, ꢁ26 °C; (c) 10 or 11, Et3N, dry THF, N2 atm, 0–25 °C; and
(d) cat. NaOMe, abs MeOH, rt.
207–209 °C;
[
a
]
ꢁ23 (c 1.68, acetone); 1H NMR (CDCl3,
D
360 MHz) d (ppm) 9.42 (s, 1H, NH), 7.44–7.12 (m, 6H, Ar, NH),
3
5.33 (t, 1H, J3,4 = 10.0 Hz, H-3), 5.23 (t, 1H, H-1), 5.10 (t, 1H,
3J4,5 = 9.5 Hz, H-4), 4.98 (t, 1H, J2,3 = 9.2 Hz, H-2), 4.33 (dd, 1H,
3
2
3
J6,6 = 12.6 Hz, H-6), 4.13 (dd, 1H, J5,6 = 2.1 Hz, H-60), 3.87 (ddd,
0
0
3. Experimental
3
1H, J5,6 = 5.0 Hz, H-5), 2.44 (s, 1H, NH), 2.10, 2.08, 2.05 (s, 12H,
4 ꢀ OCOCH3). 13C NMR (CDCl3, 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 (Ar), 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 (CH3). ESIMS: [M+Na]+ calcd 532.46,
3.1. General methods
Melting points were measured in open capillary tubes or on a
Kofler hot-stage and are uncorrected. Optical rotations were deter-

N. Felföldi et al. / Carbohydrate Research 345 (2010) 208–213
211
OAc
H
AcO
H
O
AcO
AcO
N
N
O
AcO
AcO
OAc
O
OAc
AcO
OAc
O
O
CONH2
OAc
AcO
20 R = Ac (89%)
18
+
PhCH3, Δ
OAc
O
OR
OR
RO
H
H
N
O
RO
RO
RO
AcO
AcO
OR
NCO
N
O
OAc
OR
O
3
PhCH3, Δ
CONH2
21 R = Ac
22 R = H (79%)
NaOMe, MeOH, rt
OBz
O
BzO
BzO
+
OBz
19
OAc
BzO
H
O
H
N
AcO
AcO
N
O
OBz
OBz
BzO
OAc
O
O
23 R = Ac, R' = Bz (98%)
Scheme 3.
3
found: 532.16. Anal. Calcd for C22H27N3O11 (509.47): C, 51.87; H,
5.22; N, 7.51. Found: C, 51.97; H, 5.24; N, 7.50.
3J3,4 = 9.5 Hz, H-3), 5.39 (t, 1H, J4,5 = 9.5 Hz, H-4), 5.16 (t, 1H,
3
2
0
J2,3 = 10.0 Hz, H-2), 4.52 (dd, 1H, J6,6 = 12.6 Hz, H-6), 4.18 (dd,
3
3
1H, J5,6 = 2.1 Hz, H-60), 4.05 (ddd, 1H, J5,6 = 3.7 Hz, H-5), 2.13,
2.06, 2.04, 2.00 (s, 12H, 4ꢀOCOCH3). 13C NMR (CDCl3 + DMSO-d6,
90 MHz) d (ppm) 168.6, 167.9, 167.8, 167.7 (CO), 151.8, 151.1
(NCON), 138.3–116.0 (Ar), 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 (CH3). ESIMS: [M+Na]+ calcd
for C28H31N3O11 (585.57): 608.56, found: 608.19.
0
3.3. 1-(b-D-Glucopyranosyl)-5-phenyl biuret (5)
Biuret 4 (250 mg, 0.49 mmol) was dissolved in a mixture of
MeOH and CHCl3 (1:1). A catalytic amount of AcCl was added
and the mixture was stirred at rt. The reaction was monitored by
TLC (1:1 CHCl3–MeOH). When the reaction was complete, it was
neutralized with solid NaHCO3, and then filtered and the solvent
was evaporated. The residue was purified by column chromatogra-
phy (9:1 CHCl3–MeOH) to give 162 mg (97%) of 5 as a white
3.4.2. 1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-3,5-
diphenyl biuret (9)
Yield: 46 mg (31%), white powder. Rf = 0.80 (25:1 CHCl3–ace-
powder. Mp: 191–193 °C; [
a
]
D
+4 (c 0.68, MeOH);. 1H NMR
tone). Mp: 226–229 °C; [
a]
ꢁ4 (c 0.64, DMSO). 1H NMR (CDCl3,
D
(DMSO-d6 + D2O, 360 MHz) d (ppm) 7.41 (d, 2H, Ar), 7.29 (t, 2H,
360 MHz) d (ppm) 10.24 (s, 1H, NHCON(C6H5)CONHC6H5), 7.56–
3
3
Ar), 7.04 (t, 1H, Ar), 4.68 (d, 1H, J1,2 = 8.9 Hz, H-1), 3.63 (dd, 1H,
7.06 (m, 10H, Ar), 6.44 (t, 1H, JH-1,NH = 8.8 Hz, NHCON(C6H5)CON-
3
2
J5,6 = 1.6 Hz, H-60), 3.42 (dd, 1H, J6,6 = 12.1 Hz, H-6), 3.19–3.14
HC6H5), 5.28 (t, 1H, 3J3,4 = 9.6 Hz, H-3), 5.11 (t, 1H, 3J1,2 = 9.6 Hz, H-
0
0
3
3
3
3
(m, 1H, J5,6 = 5.8 Hz, H-5), 3.22, 4.26, 4.25 (t, 1H, J2,3
=
d
1), 5.02 (t, 1H, J4,5 = 9.6 Hz, H-4), 4.78 (t, 1H, J2,3 = 9.6 Hz, H-2),
3J3,4 = 3J4,5 = 8.9 Hz, H-2,3,4). 13C NMR (DMSO-d6, 90 MHz)
4.31 (dd, 1H, J6,6 = 12.3 Hz, H-6), 4.10 (dd, 1H, J5,6 = 1.8 Hz, H-
2
3
0
0
3
(ppm) 154.6, 152.2 (NHCONH), 138.2, 128.9, 123.1, 119.0 (Ar),
80.3 (C-1), 78.5, 77.3, 72.9, 69.8 (C-2–C-5), 60.8 (C-6). Anal. Calcd
for C14H19N3O7 (341.32): C, 49.27; H, 5.61; N, 12.31. Found: C,
49.35; H, 5.66; N, 12.30.
60), 3.81 (ddd, 1H, J5,6 = 4.4 Hz, H-5), 2.09, 2.024, 2.019, 1.98 (s,
12H, 4 ꢀ OCOCH3). 13C NMR (CDCl3, 90 MHz) d (ppm) 170.6,
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 (Ar), 79.8 (C-1), 73.5,
72.5, 69.8, 67.9 (C-2–C-5), 61.5 (C-6), 20.7, 20.5 (CH3). ESIMS:
[M+Na]+ calcd for C28H31N3O11 (585.57): 608.56, found: 608.18.
3.4. Reaction of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl
urea13 (6) with phenylisocyanate
3.5. 1,5-Bis-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)biuret (12)
Urea
6
(100 mg, 0.26 mmol) was refluxed in neat
phenylisocyanate(1 mL). The reaction was monitored by TLC (2:1
EtOAc–hexane). When the reaction was complete, the excess
amount of phenylisocyanate was removed by diluting the mixture
with hexane. The formed precipitate was filtered, dissolved in
CH2Cl2 and washed with satd aq NaHCO3. The organic layer was
separated, dried and the solvent was evaporated. The residue
was separated by column chromatography (50:1 CH2Cl2–acetone)
to give, in the order of elution, compounds 8, 9 and 710 (4%).
Glucosylamine 211 (400 mg, 1.15 mmol) was dissolved in dry
THF (5 mL), then Et3N (80 L, 0.58 mmol) and OCNCOCl (46 L,
l
l
0.58 mmol) were added. The mixture was stirred at rt under nitro-
gen atmosphere. After the reaction was complete (TLC, 10:1
EtOAc–hexane) the mixture was diluted with water (5 mL), and
washed with EtOAc (3 ꢀ 5 mL). The organic phase was dried and
the solvent was evaporated under reduced pressure to yield
402 mg (91%) colourless syrup. Rf = 0.83 (10:1 EtOAc–hexane);
[a
]
ꢁ19 (c 0.97, CHCl3); 1H NMR (DMSO-d6): d (ppm) 9.11 (s,
D
3.4.1. 3-(2,3,4,6-Tetra-O-acetyl-b-
diphenyl biuret (8)
D
-glucopyranosyl)-1,5-
1H, NH), 8.02 (d, 2H, J = 9.5 Hz, 2 ꢀ NH), 5.42, 5.34, 4.92, 4.86 (4
pseudo t, 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-60), 2.00,
1.99, 1.98, 1.95 (4s, 24H, 8 ꢀ CY3); 13C NMR (CDCl3): d (ppm)
170.6, 170.3, 169.9, 169.4 (COCH3), 154.3 (2 ꢀ NHCO), 78.7 (C-1),
73.1, 72.8, 70.0, 67.9 (C-2 to C-5), 61.5 (C-6), 20.6, 20.5 (CH3).
Yield: 36 mg (24%), colourless syrup. Rf = 0.89 (25:1 CHCl3–ace-
tone) [
a
]
D +0.2 (c 1.71, DMSO). 1H NMR (CDCl3, 360 MHz) d (ppm)
10.73 (s, 1H, N(CONHC6H5)2), 8.75 (s, 1H, N(CONHC6H5)2), 7.51–
7.09 (m, 10H, Ar), 6.32 (d, 1H, J1,2 = 10.0 Hz, H-1), 5.66 (t, 1H,
3

212
N. Felföldi et al. / Carbohydrate Research 345 (2010) 208–213
Anal. Calcd for C30H41N3O20 (763.65): C, 47.18; H, 5.41; N, 5.50.
Found: C, 47.23; H, 5.50; N, 5.58.
to the solution in a catalytic amount. The reaction mixture was
kept at rt. When the reaction was complete (TLC, 7:3 CHCl3–MeOH)
the solution was neutralized with a cation exchange resin Amber-
lyst 15 (H+ form). Filtration and removal of the solvent resulted in
the corresponding deacetylated sugar derivatives.
3.6. General procedure I for the synthesis of 1-(2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl)-5-(per-O-acetyl-b-D-
glycopyranosyl)biurets 14 and 16
3.7.1. 1,5-Bis-(b-D-glucopyranosyl)biuret (13)
Glucosylamine 211 (100 mg, 0.29 mmol) was dissolved in dry
Prepared according to General procedure II (Section 3.7) from
THF (2 mL), and some freshly heated molecular sieves were added.
biuret 12 (100 mg, 0.13 mmol). Yield: 54 mg (96%) colourless syr-
The mixture was cooled to ꢁ20 °C, OCNCOCl (23
was added, and stirred at ꢁ26 °C under nitrogen atmosphere for a
day. Then a solution of 2,3,4,6-tetra-O-acetyl-b- -galactopyrano-
sylamine16 (10, 100 mg, 0.29 mmol) or 2,3,5-tri-O-acetyl-b-
xylopyranosylamine17,18 (11, 80 mg, 0.29 mmol) in dry THF (2 mL)
and Et3N (40 L, 0.29 mmol) were added, and the mixture was al-
lL, 0.29 mmol)
up. Rf = 0.45 (1:3 CHCl3–methanol); [
a
]
D
ꢁ4 (c 0.51, MeOH); 1H
NMR (D2O): d (ppm) 4.91 (d, 2H, J = 9.3 Hz, 2 ꢀ H-1), 3.86 (dd,
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 t, 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); 13C NMR
(Me2SO-d6): 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 for C14H25N3O12 (427.36): C,
39.35; H, 5.90; N, 9.83. Found: C, 39.46; H, 5.99; N, 9.90.
D
D
-
l
lowed to warm up to rt. When the reaction was complete (TLC,
10:1 EtOAc–hexane) the insoluble materials were filtered off with
suction, and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography (7:1
EtOAc–hexane).
3.7.2. 1-(b-D-Galactopyranosyl)-5-(b-D-glucopyranosyl)biuret
(15)
Prepared according to General procedure II (Section 3.7) from
3.6.1. 1-(2,3,4,6-Tetra-O-acetyl-b-
D
-galactopyranosyl)-5-
biuret 14 (100 mg, 0.13 mmol). Yield: 53 mg (98%) colourless syr-
(2,3,4,6-tetra-O-acetyl-b- -glucopyranosyl)biuret (14)
D
up. Rf = 0.35 (1:3 CHCl3–MeOH); [
a]
ꢁ7 (c 0.54, H2O); 1H NMR
D
Prepared according to General procedure I (Section 3.6) from
glucosylamine 2 (100 mg, 0.29 mmol) and galactosylamine 10
(100 mg, 0.29 mmol). Yield: 183 mg (83%) colourless syrup.
(DMSO-d6): d (ppm) 4.65–4.56 (m, 2H), 3.69–3.57 (m, 2H), 3.47–
3.00 (m, 9H), 2.95 (t, 1H); 13C NMR (D2O): 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 to C-5-Glc, C-2-Gal to C-5-Gal), 61.3 (C-6-Glc, C-6-Gal). Anal.
Calcd for C14H25N3O12 (427.36): C, 39.35; H, 5.90; N, 9.83. Found:
C, 39.44; H, 5.98; N, 9.89.
Rf = 0.58 (10:1 EtOAc–hexane); [
a]
ꢁ15 (c 0.98, CHCl3); 1H NMR
D
(CD3CN): d (ppm) 7.83 (br 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 t, J = 9.6 Hz
H-3-Glc), 5.23 (pseudo t, 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 t, 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,
1.99, 1.98, 1.96 (6br s, 24H, 8 ꢀ CH3); 13C NMR (CDCl3): d (ppm)
170.6, 170.3, 169.9, 169.4 (COCH3), 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 to C-5-Glc, C-2-Gal to C-5-Gal), 61.6, 61.0 (C-6-Glc, C-6-Gal),
20.6, 20.5 (CH3). Anal. Calcd for C30H41N3O20 (763.65): C, 47.18; H,
5.41; N, 5.50. Found: C, 47.29; H, 5.54; N, 5.61.
3.7.3. 1-(b-D-Glucopyranosyl)-5-(b-D-xylopyranosyl)biuret (17)
Prepared according to General procedure II (Section 3.7) from
biuret 16 (100 mg, 0.14 mmol). Yield: 55 mg (96%) colourless syr-
up. Rf = 0.63 (1:3 CHCl3–MeOH); [
a]
ꢁ12 (c 0.52, H2O); 1H NMR
D
(D2O): 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);
13C NMR (D2O): 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 to C-5-Glc, C-
2-Xyl to C-4-Xyl), 67.3, 61.2 (C-6-Glc, C-5-Xyl). Anal. Calcd for
C13H23N3O11 (397.34): C, 39.30; H, 5.83; N, 10.58. Found: C,
39.39; H, 5.90; N, 10.65.
3.6.2. 1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-5-(2,3,5-tri-
O-acetyl-b- -xylopyranosyl)biuret (16)
D
3.8. 1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosylcarbonyl)-3-
Prepared according to General procedure I (Section 3.6) from
glucosylamine 2 (100 mg, 0.29 mmol) and xylosylamine 11 (80 mg,
0.29 mmol). Yield: 171 mg (86%) colourless syrup. Rf = 0.58 (10:1
(2,3,4,6-tetra-O-acetyl-b- -glucopyranosyl)urea (20)
D
C-(2,3,4,6-Tetra-O-acetyl-b-D
-galactopyranosyl)formamide19 (18,
EtOAc–hexane); [a D
]
ꢁ28 (c 0.58, CHCl3); 1H NMR (CD3CN): d (ppm)
100 mg, 0.27 mmol) was dissolved in dry toluene (3 mL). Then
some molecular sieves and crystalline isocyanate 310 (202 mg,
0.54 mmol) were added. The reaction was stirred at reflux temper-
ature. After one day the reaction mixture was worked up: the
molecular sieves were filtered off with suction and the solution
was concentrated under reduced pressure. The residue was
purified by column chromatography (100:1 CHCl3–MeOH). Two
products were isolated: 20 (177 mg, 89%) and 2121 (52 mg). Char-
acterization of 20: colourless syrup; Rf = 0.55 (5:1 EtOAc–hexane);
7.75 (br s, 1H, NH), 7.49 (br s, 2H, 2 ꢀ NH), 5.35 (pseudo t, 1H,
J = 9.4 Hz, H-3-Glc), 5.29 (pseudo t, 1H, J = 9.1 Hz, H-3-Xyl), 5.23
(pseudo t, 1H, J = 9.4 Hz, H-1-Glc), 5.13 (pseudo t, 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 (5br s, 21 H, 7 ꢀ CY3); 13C NMR (CDCl3): d
(ppm) 170.6, 170.3, 169.9, 169.8, 169.4 (COCH3), 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 to C-5-Glc, C-2-Xyl to C-4-Xyl), 63.7, 61.5 (C-6-Glc,
C-5-Xyl), 20.5, 20.4 (CH3). Anal. Calcd for C27H37N3O18 (691.59): C,
46.89; H, 5.39; N, 6.08. Found: C, 46.99; H, 5.31; N, 6.15.
[a]
+18 (c 0.84, CHCl3); 1H NMR (CD3CN): d (ppm) 8.66 (br s, 1H,
D
NH), 8.64 (d, 1H, J = 9.3 Hz, NH), 5.41 (pseudo d, J = 2.2 Hz, H-4-
Gal), 5.35 (pseudo t, J = 9.6 Hz, H-3-Glc), 5.28 (pseudo t 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
t, J = 9.3, 9.6 Hz in both, 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 (8br s, 24H,8 ꢀ CH3). 13C NMR (CDCl3): d (ppm) 170.6,
170.3, 170.1, 169.9, 169.7, 169.4 (COCH3), 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,
65.9 (C-2-Glc to C-5-Glc, C-2-Gal to C-5-Gal), 61.7, 61.4 (C-6-Glc,
3.7. General procedure II for the removal of O-acyl protecting
groups
An O-peracetylated compound (100 mg) was dissolved in dry
MeOH (1 mL), and a solution of NaOMe (1 M in MeOH) was added

N. Felföldi et al. / Carbohydrate Research 345 (2010) 208–213
213
C-6-Gal), 20.7, 20.6, 20.5 (CH3). Anal. Calcd for C30H40N2O20
(748.64): C, 48.13; H, 5.39; N, 3.74. Found: C, 48.20; H, 5.43; N, 3.79.
20.5, 20.4 (CH3). Anal. Calcd for C50H48N2O20 (996.92): C, 60.24;
H, 4.85; N, 2.81. Found: C, 60.19; H, 4.90; N, 2.89.
3.9. 1,3-Bis-(b-
D
-glucopyranosyl)urea (22)
Acknowledgements
Prepared according to General procedure II (Section 3.7) from
This work was supported by the Hungarian Scientific Research
Fund(OTKA 45927). This work was supported by the EU Marie Cur-
ie Early Stage Training (EST) Contract No. MEST-CT-020575 a Marie
Curie Host Fellowships for the Transfer of Knowledge (ToK) Con-
tract No. MTKD-CT-2006–042776. The authors thank Professor K.
E. Kövér and Mr. M. Herczeg for advice on and help with 2D
urea 21 (190 mg, 0.26 mmol). Yield 80 mg (79%) amorphous solid.
Lit.27 Mp: 207 °C (dec.); Rf = 0.45 (1:3 CHCl3-methanol); [
a]D +23 (c
0.59, DMSO), lit.27
[a
]
D ꢁ32.8 (c 2, water); 1H NMR (D2O): 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
(dd, 1H, J = 5.2, 12.3 Hz, H-6b), 3.53, 3.38, 3.37 (3 pseudo t, 3H,
J = 9.2, 9.7 Hz in each, H-2, H-3 ,H-4), 3.51–3.48 (m, 1H, H-5). 13C
NMR (D2O): d (ppm) 159.6 (CO), 81.5 (C-1), 77.9, 77.2, 72.6, 70.0
(C-2 to C-5), 61.3 (C-6).
NMR spectra, and Dr. Gy. Gyémánt for performing the
assay.
a-amylase
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-glucopyranosyl)formamide20
(19, 54 mg, 0.086 mmol) was dissolved in dry toluene (1 mL), and
some molecular sieves were added followed by crystalline isocya-
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8.05, 7.94, 7.90, 7.78 (4d, 8H, Ar), 7.63–7.32 (m, 12H, Ar), 6.02
(pseudo t, 1H, J = 9.4 Hz, H-3-GlcBz), 5.77 (pseudo t, 1H,
J = 10.1 Hz, H-4-GlcBz), 5.75 (pseudo t, 1H, J = 9.8 Hz, H-2-GlcBz),
5.38 (pseudo t, 1H, J = 9.6 Hz, H-3-GlcAc), 5.34 (pseudo t, 1H,
J = 9.4 Hz, H-1-GlcAc), 5.00, 4.99 (2 pseudo t, 2H, J = 9.4, 9.8 Hz in
both, 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 t,
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,
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152.6 (2 ꢀ CONH), 133.6, 133.3, 133.2, 129.8, 129.7, 129.6, 128.2,
128.3 (CH–Ar), 129.1, 128.7 (C–Ar), 78.4, 76.6 (2 ꢀ C-1), 76.1, 73.1,
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