New regioselective derivatives of sucrose with amino acid and acrylic groups

Article abstract of DOI:10.1016/j.carres.2005.11.033

We report here a range of new sucrose derivatives obtained from '3-ketosucrose' in aqueous medium with few reaction steps. As an intermediate, 3-amino-3-deoxy-α-d-allopyranosyl β-d-fructofuranoside (1) was obtained via the classical route of reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized. Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids. Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccharides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.

Full text of DOI:10.1016/j.carres.2005.11.033

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 322–331
New regioselective derivatives of sucrose with amino acid
and acrylic groups
Jan Anders,a Rachel Buczys,b Elmar Lampe,a Martin Walter,b
Emile Yaacouba and Klaus Buchholza,*
aChair for Carbohydrate Technology, Technical University, Langer Kamp 5, D 38106 Braunschweig, Germany
bInnoSweet GmbH, Langer Kamp 5, D 38106 Braunschweig, Germany
Received 28 February 2005; received in revised form 10 November 2005; accepted 24 November 2005
Available online 27 December 2005
Abstract—We report here a range of new sucrose derivatives obtained from Ô3-ketosucroseÕ in aqueous medium with few reaction
steps. As an intermediate, 3-amino-3-deoxy-a-D-allopyranosyl b-D-fructofuranoside (1) was obtained via the classical route of
reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized
by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized.
Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids.
Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccha-
rides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Sucrose derivatives; Amino derivatives of sucrose; 3-Ketosucrose; Regioselective substitution; Polymer building blocks; Allosucrose
aminoacyl conjugates
1. Introduction
fructofuranoside (Ô3-ketosucroseÕ).1 We have already
described the conditions of fermentation where sufficient
enzymatic activity is produced, and optimized condi-
tions for oxidation, in order to achieve good yields
and low byproduct concentration levels for the 3-keto
derivatives of sucrose, isomaltulose, leucrose, lactose,
and a sugar alditol, a-D-glucopyranosyl-(1!6)-manni-
tol.2 The biotransformation has been optimized and
scaled up to give 70% yield of 3-ketosucrose, and even
a 90% yield with 3-keto-isomaltulose.3
Subsequent chemical steps provide access to a range
of products, including a-D-allopyranosyl b-D-fructofur-
anoside (allosucrose) and allose,4 the corresponding
cyanohydrin,5 oximes, and 3-amino-3-deoxy-a-D-allo-
pyranosyl b-D-fructofuranoside (1).6 N-Acylation
yielded the dodecanamide, which exhibits surfactant
properties.6 All of these routes proceed with high regi-
oselectivity, and mostly also with high stereoselectivity
and yield, in aqueous solution without protecting
groups. Furthermore, Grignard reactions gave C–C
Sucrose chemistry has elicited much interest since
sucrose (a-D-glucopyranosyl b-D-fructofuranoside) is
available on a large scale with high purity at low cost.
However, the functionality of sucrose, with eight nearly
equivalent hydroxyl groups, makes selective synthesis of
derivatives laborious and difficult. Furthermore, the pre-
ferred route for potential technical applications are reac-
tions in water, avoiding protecting groups, which
require highly selective catalysis. Enzymes offer the
advantage of high regio- and stereoselectivity. Agrobac-
terium tumefaciens elaborates a dehydrogenase, which
oxidizes sucrose and other disaccharides at the 3-
position yielding a-D-ribo-hex-3-ulopyranosyl-b-D-
*
Corresponding author. Tel.: +49 531 391 7260; fax: +49 531 391
7263; e-mail: k.buchholz@tu-bs.de
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.11.033

J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
323
linked derivatives with alkyl as well as allyl substitu-
ents.6 The reductive amination could be improved to
give substantially higher yields of 70% of 1, using hydra-
zine, fresh Raney nickel catalyst, and providing control
of the pH, as is described here.
Further reactions leading to polymer building-blocks
of different structures, and the corresponding polymers,
as well as amino acid conjugates, are described in this
paper. Polymers having saccharide building blocks of
different structural architecture, where the saccharide
units are integrated into the polymer main chain,7–9 or
appended as side chains, bound via amino functional
groups,11,12 or by C–C-bonds13 have found major
interest.
derivative with a spacer in order to distance the acrylic
group from the rigid sucrose moiety, giving more flexi-
bility and less steric hindrance for subsequent polymer-
ization steps, to be described subsequently. Compound 1
is reacted in an equimolar amount of 2-isocyanato
methyl methacrylate at 0–7 ꢁC. The product was ex-
tracted with diethyl ether and freeze dried to yield over
90% of 3-deoxy-3-N0-methacryloyloxyethylureido-a-D-
allopyranosyl bD-fructofuranoside (MAE-sucrose) (3),
an acrylic acid derivative with a urea spacer.
(1) 3-Amino-3-deoxy-a-D-allopyranosyl b-D-fructofuran-
oside
Glycoconjugates have become a field of great interest
due to their manifold biological functions. A recent
example is 1,5-anhydro-D-fructose, used as a synthon
for various approaches.14 Conjugates of carbohydrates
and amino acids occur naturally in glycopeptides, glyco-
proteins, and in minor amounts in bacterial lipopolysac-
charides. All of these substances are of major biological
importance in various immunological processes.15,16
Conjugates of carbohydrates also play a major role in
a range of important antibiotics. Chemical synthesis of
model compounds such as asparagine derivatives linked
to oligosaccharides, generally require the use of protect-
ing groups in the saccharide and the amino acid moiety
in order to achieve selective coupling, and many block-
ing and deblocking steps are required for such synthe-
ses.17 Sucrose, despite its obvious advantages, has
found much less attention as a building block, because
of the difficulties of sucrose chemistry already men-
tioned. Here we present the synthesis of some aminoacyl
derivatives of 3-amino-3-deoxy-a-D-allopyranosyl b-D-
fructofuranoside, which requires protecting groups only
for the amino acid moiety.
OH
OH
O
OH
HO
O
HO
O
OH
H2N
HO
(2) 3-Deoxy-3-N-methacrylamido-a-D-allopyranosyl b-D-
fructofuranoside (MA-sucrose)
OH
OH
O
OH
HO
N
O
HO
O
OH
H
HO
O
(3) 3-Deoxy-3-N0-methacryloyloxyethylureido-a-D-allo-
pyranosyl b-D-fructofuranoside (MAE-sucrose)
2. Results and discussion
OH
2.1. Polymer building blocks and polymers
OH
O
OH
HO
N
Polyvinylsaccharides have interesting potential in such
fields as cosmetics, pharmaceuticals, and as polymeric
surfactants.10–13 Most vinylsaccharides have been syn-
thesized from reducing mono- and di-saccharides via
reductive amination, a simple technique that proceeds
in water with no need for protecting groups.10–12 Under
such conditions, sucrose derivatives of regioselective
substitution pattern are accessible only via 3-ketosu-
crose and by subsequent reductive amination to 3-ami-
no-3-deoxy-a-D-allopyranosyl b-D-fructofuranoside (1),
followed by reaction with methacrylic anhydride to give
the methacrylamide derivative: 3-deoxy-3-N-methacry-
lamido-a-D-allopyranosyl b-D-fructofuranoside (MA-
sucrose) (2).6 It seemed advisable to design an additional
O
HO
O
OH
H
H
HO
O
N
O
O
Polymerization to give homopolymers was performed
in oxygen-free water under a nitrogen atmosphere with a

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J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
radical initiator (Na2S2O8–Na2S2O5, usually 1%). Reac-
tions with MA-sucrose gave, at a monomer concentra-
tions in the range of 0.244 mol/L, 40–60 ꢁC after 6–
48 h, dialysis and freeze drying, a 38–50% yield of poly-
mer 4. The molar mass was in the range of 61,000
(60 ꢁC) to 350,000 (g/mol) (40 ꢁC) (for details and fur-
ther examples, see Experimental). 1H NMR spectra
show that the signals associated with the double bond
of the methacrylic group at 5.78 and 5.50 ppm had dis-
appeared. Polymerization of MAE-sucrose (3) gave, at
60 ꢁC, under otherwise similar conditions, a 58–88%
yield of polymer 5, with a molar mass in the range of
490,000–1,900,000 (g/mol). Thus the introduction of a
spacer between the sucrose moiety and the acrylic substi-
tuent had a dramatic effect on the molar mass, which
was higher by an order of magnitude, and the yield,
which was increased significantly. It may be assumed
that steric hindrance restricts the growth rate of MA-su-
crose, affecting both yield and chain length, as has been
observed with similar systems.10
Copolymerization of MA-sucrose both with acrylam-
ide and acrylonitrile was successfully performed, with
significantly higher yields as compared to the
homopolymerization.18
2.2. Amino acid conjugates of sucrose
Here we present the synthesis of some aminoacyl
derivatives of 3-amino-3-deoxy-a-D-allopyranosyl b-D-
fructofuranoside, which requires protecting groups only
for the amino acid moiety. The resulting amide linkage
is similar to the unconventional binding of saccharides
and amino acids in bacterial lipopolysaccharides,19,20
whereas glycoproteins have a N- or O-glycosylic linkage
connecting the two moieties.
The sucrose–amino acid conjugates are new com-
pounds, which might be useful in different aspects of bio-
chemical or medical research. Coupling of amino acids to
the 3-position in the sucrose molecule via 3-amino-
allosucrose is rather unconventional. The synthesis does
not require protecting groups in the sucrose derivative,
although it is not trivial, because of its limited range of
stability,6 undergoing decomposition both at elevated
pH and hydrolysis of the glycosidic bond at acidic pH.
In many glycoproteins, the carbohydrate residue is
linked to the protein backbone through an aspartoyl–
glycosylamine bond. Since aspartic acid often occurs
in natural glycoconjugates, 1 was coupled with the pro-
tected amino acid benzyl-N-benzyloxycarbonyl-L-aspar-
tate (Boc-Asp-OBzl) by treatment with N,N0-
dicyclohexylcarbodiimide in pyridine–water according
to the method of Kiyozumi et al.21 After removing the
side product (N,N0-dicyclohexylurea) and the residual
amino acid, compound 6 was separated by column chro-
matography on silica gel (Fig. 1). Identification was per-
formed by fast-atom bombardment MS and 13C NMR
spectroscopy. Selective deprotection of the C-terminus
was possible by hydrogenolysis of 6 with Pd/charcoal
catalyst. Complete deprotection of 7 was accomplished
by cleavage of the benzyloxycarbonyl group with tri-
fluoroacetic acid (TFA, 90%) to yield the amino acid
conjugate 3-(4-L-aspartylamido)-3-deoxy-a-D-allopyran-
osyl b-D-fructofuranoside 8 (Fig. 1). Purification was in
all cases possible by means of column chromatography.
As well as the aspartic acid derivative, some other amino
acids were used for the condensation reaction with 1.
Products of these reactions are shown in Figure 2.
By acidic hydrolysis it was possible to cleave the
glycosidic linkage of 8. Fructose and 3-(4-L-aspartyl-
amido)-3-deoxy-a-D-allopyranose (13) were separated by
ion-exchange chromatography. This result also shows
that the corresponding monosaccharide derivatives are
readily accessible by this route.
(4) Poly-[3-deoxy-3-N-methacrylamido-a-D-allopyran-
osyl b-D-fructofuranoside] (poly-MA-sucrose)
OH
OH
O
HO
O
HO
OH
OH
O
H
O
N
H
OH
(
)
n
H
(5) Poly-[3-deoxy-3-N0-methacryloyloxyethylureido-a-D-
allopyranosyl
sucrose)
b-D-fructofuranoside]
(poly-MAE-
OH
OH
O
OH
HO
O
HO
O
OH
H
N
H
HO
O
N
O
O
H
(
)
n
Another convenient protecting group used is fluoren-
yl-9-methoxycarbonyl-group (Fmoc), which gives
H

J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
325
OH
OH
OH
O
HO
O
OH
O
HO
OH
OH
HO
Boc
DCC, pyridine/H2O
OBzl
Asp
O
O
HN
OH
OH
HO
O
H2N
OH
O
OH
CH
CH
6
Boc
HN
COOBzl
H2/Pd
OH
OH
OH
OH
O
O
HO
HO
HN
O
O
OH
OH
OH
OH
HO
HO
O
O
CF3COOH
HN
OH
OH
O
O
CH
H2NC CH COOH
CH
HN CH COOH
8
7
Boc
Figure 1. Synthesis of 3-(N-tert-butoxycarbonyl-L-aspartic acid-4-amido-1-benzyl ester)-3-deoxy-a-D-allopyranosyl b-D-fructofuranoside (6), 3-(N-
tert-butoxycarbonyl-L-aspartic acid 4-amido)-3-deoxy-a-D-allopyranosyl b-D-fructofuranoside (7), and 3-(L-aspartic acid 4-amido)-3-deoxy-a-D-
allopyranosyl b-D-fructofuranoside (8).
OH
OH
O
HO
O
O
O
O
O
CH
NH Boc
R =
R =
R =
R =
HO
OH
OH
O
9
HN
R
CH3
OH
CH
NH
Boc
10
CH(CH3)2
CH2
CH
NH Boc
NH
11
12
(CH2)4
Z
CH
NH Boc
C6H5
Figure 2. Synthesis of further aminoacyl derivatives of 1: 3-(N-tert-butoxycarbonyl-L-alanylamido) (9), 3-(N-tert-butoxylcarbonyl-L-leucylamido)
(10), 3-(Na-tert-butoxylcarbonyl-Ne-Z-L-lysyl amido) (11), and 3-(N-tert-butoxylcarbonyl-L-phenylalanylamido) (12).

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J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
quantitative yields. It is stable during the coupling pro-
cedure and is easily removed under mild conditions by,
for instance, morpholine at a pH of ꢀ8. Activation of
the carboxyl group was performed with ethyl-2-eth-
oxy-1,2-dihydro-1-quinolinecarboxylate (EEDQ) in the
following examples, with yields around 60%. The prod-
ucts were characterized as mentioned before, except that
electrospray ionization was used for MS. Fmoc-trypto-
phan was thus coupled to 1, using nearly equimolar
quantities, in a solvent mixture of ethanol, 2-propanol,
and acetone, with activation by EEDQ. Extraction,
freeze drying, and purification with silica gel gave a
60% yield of the 3-Deoxy-3-(N-Fmoc-L-tryptophanyl-
amido)-a-D-allopyranosyl b-D-fructofuranoside (14).
A dipeptide conjugate was synthesized with glycine,
and subsequent phenylalanine coupling to the aminosu-
crose. Fmoc-glycine was coupled with 1 in a water–etha-
nol–2-propanol mixture with activation by EEDC. After
purification with silica gel and freeze drying, a 64% yield
of 3-deoxy-3-(N-Fmoc-L-glycylamido)-a-D-allopyrano-
syl b-D-fructofuranoside was obtained. Deprotection
was straightforward with diethylamine, and extraction
with ether to give 95% of 3-deoxy-3-(L-glycylamido)-a-
D-allopyranosyl b-D-fructofuranoside (15). To this gly-
cine derivative Fmoc-L-phenylalanine was coupled with
activation by EEDC in ethanol. Purification by silica
gel gave 50% of the dipeptide conjugate of allosucrose,
3-deoxy-3-(N-Fmoc-L-phenylalanyl-L-glycylamido)-a-D-
allopyranosyl b-D-fructofuranoside (16).
(15) 3-Deoxy-3-(L-glycylamido)-a-D-allopyranosyl b-D-
fructofuranoside
(16) 3-Deoxy-3-(N-Fmoc-L-phenylalanyl-L-glycylamido)-
a-D-allopyranosyl b-D-fructofuranoside
The synthesis of new amino acid and dipeptide conju-
gates of sucrose via 3-amino-allosucrose thus with an
unconventional regio-substitution pattern is demon-
strated. The amino acids introduced may be neutral,
or acidic, or basic, no protecting groups for the saccha-
ride are required, and good yields can be obtained.
(14)
3-Deoxy-3-(N-Fmoc-L-tryptophanylamido)-a-D-
3. Experimental
3.1. General
allopyranosyl b-D-fructofuranoside
TLC was conducted on aluminum sheets, precoated
with 0.2 mm layers of Silica Gel 60F-254 (E. Merck,
Darmstadt, Germany); the components were located
either by exposure to UV light or by spraying with A
(0.2% naphthoresorcin in ethanol–20% H2SO4 (1:1)),
or B (MeOH–concd H2SO4 (1:1)) followed by heating
at 120 ꢁC. Column chromatography was performed on
silica gel (230–400 mesh, E. Merck) with eluent C
(EtOAc–EtOH–H2O (5:3:1)) or D (MeOH–H2O–NH3
(25%) (5:1:0.5)). Other eluents are mentioned in individ-
ual sections. Preparation of 1 is described in Ref. 6.
Analysis of molar mass Mw and d.p. was accom-
plished by size-exclusion chromatography (SEC) with
a multi-angle laser-light detector (MALLS) system.
Two systems were applied for SEC: one from Shimadsu

J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
327
with a differential refractometer detector RID-6A, a
MALLS Dawn DSP detector (Laser photometer, Wyatt
Technology Co. Santa Barbara, CA, USA) and a col-
umn of Polymer Standard Service (PSS Suprema 1000,
10 lm); second a system from Merck–Hitachi with a
Waters Associates Differential Refractometer R 401, a
column from Tosohaas Bioseparation Specialists (TSK-
gel GMPWXL, 7.8 mm ID · 300 mm, 13 mm), together
with the same MALLS system as already described. As
solvent, twice distilled water with 0.05% NaN3 (Aldrich)
was used.
NMR spectra were recorded with a Bruker AM-400
instrument; 1H NMR spectra at 400 MHz and 13C
NMR spectra at 100 MHz, if not stated otherwise.
Several 13C NMR spectra were recorded with a Bruker
AM-300 instrument at 75.5 MHz, as noted. Mass spec-
tra were mostly recorded with a Finnigan MAT 8430
instrument. Electrospray-MS (MS(ES)) was performed
with a Fisons VG Trio 2000 instrument. Microanalyses
were performed by Analytisches Labor des Institutes
ture of acetone–ether. The product 2 was filtered and
washed with ice-cold ether to yield 1.6 g of 2 as white
crystals (69%). H NMR (400 MHz, D2O) d 5.78 (d,
1
J 0.9 Hz, 1H, C@CAHa), 5.51 (d, J 0.9 Hz, 1H,
C@CAHb), 5.45 (d, J1 ,2 4.0 Hz, 1H, H-10), 4.73 (t,
0
0
J2 ,1 =J2 ,3 4.0 Hz, 1H, 20-H), 4.27 (d, J3,4 8.7 Hz, 1H,
3-H), 4.06 (t, J3,4 = J4,3 8.6 Hz, 1H, 4-H), 4.01–3.84
(m, 8H, 5-H, 6-H2, 30-H, 40-H, 50-H, 60-H2), 3.64 (s,
2H, 1-H2), 1.98 (s, 3H, CH3). 13C NMR (100 MHz,
D2O) d 174.2 (NHCO), 140.8 (HC@CACH3), 122.0
(HC@CACH3), 104.6 (C-2), 92.4 (C-10), 82.5 (C-5),
76.7 (C-3), 74.4 (C-4), 69.1 (C-50), 65.9, 65.8 (C-20, C-
40), 63.1 (C-1), 62.8 (C-6), 60.9 (C-60), 53.5 (C-30), 18.7
(CH3).
0
0
0
0
3.4. 3-Deoxy-3-N0-methacryloyloxyethylureido-a-D-allo-
pyranosyl b-D-fructofuranoside (3)
2-Isocyanato methyl methacrylate (0.83 mL, 5.9 mmol)
was added dropwise to a cooled (0 ꢁC) solution of 2 g
(5.9 mmol) 1 in water. The mixture was stirred for 1 h
at 0 ꢁC and 16 h at 7 ꢁC. After 1 h at room temperature,
the mixture was extracted three times with 10 mL of
ether. The product 3 was isolated by freeze drying, yield
of 2.92 g (99%). IR (cmꢁ1): 3500–3020 (OH), 2934, 2894
(C–H), 1713 (C@O) (ester), 1642 (C@O) (urea), 1561
(N–H), 1176, 1121, 1052, 992 (C–OH); 1H NMR
(400 MHz, D2O) d 6.09 (d, J 0.9 Hz, 1H, C@C–Ha),
fur Pharmazeutische Chemie, Technische Universita¨t,
¨
Braunschweig.
L-Tryptophan, L-phenylalanine, Fmoc-Cl and EEDQ
were purchased from Merck, Fmoc-L-glycine and Fmoc-
L-phenylalanine from Novabiochem (all with purity
>99%).
3.2. 3-Amino-3-deoxy-a-D-allopyranosyl b-D-fructo-
furanoside (1)
0
0
5.66 (d, J 0.9 Hz, 1H, C@C–Hb), 5.32 (d, J1 ,2 3.5 Hz,
1H, H-10), 4.72 (m, 1H, 20-H under D2O), 4.21 (d, J3,4
8.6 Hz, 1H, 3-H), 4.17 (t, J 4.9 Hz, 2H, NCH2CH2O),
4.05 (t, J3,4 = J4,3 8.6 Hz, 1H, 4-H), 3.87–3.48 (m,
10H, 1-H2, 5-H, 6-H2, 30-H, 40-H, 50-H, 60-H2), 3.41 (t,
J 4.9 Hz, 2H, NCH2CH2O), 1.87 (s, 3H, CH3). 13C
NMR (100 MHz, D2O) d 172.3 (COO), 163.5 (NHCO),
138.4 (HC@C–CH3), 129.5 (HC@C–CH3), 106.5 (C-2),
94.7 (C-10), 84.1 (C-5), 79.1 (C-3), 76.3 (C-4), 71.2 (C-
50), 68.0, 67.7 (C-20, C-40), 67.0 (NCH2CH2O), 64.6
(C-1), 64.1 (C-6), 62.7 (C-60), 55.7 (C-30), 41.4
(NCH2CH2O), 18.7 (CH3). Anal. Calcd for C19H32O13:
C, 45.97; H, 6.45; N, 5.65. Found: C, 45.00; H, 6.78;
N, 5.45.
The preparation was modified to give improved yields.
A sample (90.0 mL, 1.86 mol) of hydrazine hydrate
was dissolved in water (1.5 L) and the pH adjusted to
6 with concd HCl. A solution (120.0 g, 0.35 mol) of 3-
ketosucrose in 600 mL water was added over 5 h. The
mixture was stirred at room temperature while the pH
was held between 6.0 and 6.5 by adding hydrazine
hydrate. After 19 h, 100 g Raney alloy was added and
the suspension was added to a high-pressure apparatus.
Hydrogenation was carried out at 50 ꢁC and 80 bar H2
pressure for 24 h. The alloy particles were filtered off
and the filtrate was freeze dried. The crude material
was passed through an ion-exchange column and the
product isolated by chromatography (ion-exchanger
Amberlite CG 120II in the NHþ4 -form, 0.5% aq NH3
eluent) to yield 95.2 g (80%) of 1. Analytical data were
given in Ref. 6.
3.5. Poly-(3-deoxy-3-N-methacrylamido-a-D-allopyranos-
yl b-D-fructofuranoside) (poly-MA-sucrose) (4)
Typical procedure for polymerization of 2 under nitro-
gen: A sample (0.5 g, 1.22 mmol) of 2 and 1 mol %
Na2S2O8 was dissolved in 0.5 mL water in a glass tube.
Oxygen was removed by the freeze–thaw method and
the mixture heated for 48 h to 60 ꢁC. Then the tube
was opened, cooled, and the solution was added to
10 mL water and dialyzed against water. The product
4 was isolated by freeze drying in a yield of 40%. Other
reaction conditions assayed and results are collected in
Table 1.
3.3. 3-Deoxy-3-N-methacrylamido-a-D-allopyranosyl b-
D-fructofuranoside (MA-sucrose) (2) (compare Ref. 6)
Methacrylic anhydride (1.1 mL, 6.4 mmol) was added
dropwise to a cooled (ꢁ10 ꢁC) suspension of 2.0 g
(5.8 mmol) 1 in 10 mL MeOH. The mixture was stirred
for 1 h at ꢁ10 ꢁC, 3 h at ꢁ5 ꢁC and 15 h at 4 ꢁC. It was
warmed to room temperature and poured into a 4:1 mix-

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J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
Table 1. Reaction conditions assayed for polymerization of 2
Sample
Radical initiator (mol %)
Reaction temperature (ꢁC)
Reaction time (h)
Yield [g (%)]
Molecular weight Mw (g/mol)
1
2
3
4
1a
1b
2b
2b
60
40
40
40
48
24
48
6
0.20 (40)
0.25 (50)
0.23 (46)
0.19 (38)
61,400
194,000
349,000
96,400
a Na2S2O8.
b Na2S2O8/Na2S2O5.
[a]D +20.3 (c 1.3, H2O); IR (cmꢁ1): 3500–3020 (OH),
2927 (C–H), 1643 (C@O) (urea), 1526 (N–H), 1128,
3.7. 3-(N-tert-Butoxycarbonyl-L-aspartic acid-4-amido-
1-benzyl ester)-3-deoxy-a-D-allopyranosyl b-D-fructo-
furanoside (6)
1
1112, 1050, 990 (C–OH); H NMR (400 MHz, D2O) d
5.43 (H-10), 4.73 (20-H), 4.60–3.30 (1-H, 3-H, 4-H, 5-
H, 6-H, 30-H, 40-H, 50-H, 60-H), 2.70–1.60 (CHCH3),
1.60–0.70 (CH3). 13C NMR (100 MHz, D2O) d 184.0
(NHCO), 106.6 (C-2), 92.5 (C-10), 84.5 (C-5), 78.2 (C-
3), 75.5 (C-4), 68.3 (C-50), 64.7 (C-20, C-40), 62.7 (C-1),
62.8 (C-6), 58.1 (C-60), 54.7 (CHCH3), 48.3 (C-30), 25–
17 (CH2CHCH3, CH2CHCH3).
To a solution of 1 (0.59 mmol, 201 mg) in 4:1 pyridine–
water (13 mL) N-tert-butoxycarbonyl-L-aspartic acid
1-benzyl ester (0.59 mmol, 193.5 mg) was added. The
resulting solution was cooled to 0 ꢁC. N,N0-Dicylclo-
hexylcarbodiimide (1.0 mmol dissolved in 0.5 mL
pyridine–water) was added dropwise with stirring. The
mixture was warmed to room temperature and stirred
for 17 h. Glacial acetic acid (one drop) was added and
stirring was continued for 15 min. The mixture was fil-
tered to remove the N,N0-dicyclohexylurea. Pyridine
was coevaporated with several portions of toluene.
The aqueous residue was extracted with Et2O. The
water phase was lyophilized and the crude product
was applied for hydrogenation without further
purification.
3.6. Poly-(3-deoxy-3-N0-methacryloyloxyethylureido-
a-D-allopyranosyl b-D-fructofuranoside) (poly-MAE-
sucrose) (5)
Typical procedure for polymerization of 3 under nitro-
gen: A sample (0.5 g, 1 mmol) of 3 and 3 mol %
Na2S2O8 was dissolved in 0.5 mL water in a glass tube.
Oxygen was removed by the freeze–thaw method and
the mixture heated for 48 h to 60 ꢁC. Then the tube
was opened, cooled, and the solution was added to
10 mL water and dialyzed against water. The product
5 was isolated by freeze drying in a yield of 88%. Other
reaction conditions assayed and results are collected in
Table 2.
3.8. 3-(N-tert-Butoxycarbonyl-L-aspartic acid 4-amido)-
3-deoxy-a-D-allopyranosyl b-D-fructofuranoside (7)
A suspension of 6 (0.31 mmol, 200 mg) in 50% aq
MeOH (22 mL) was hydrogenated in the presence of
10% palladium–charcoal (50 mg) at atmospheric pres-
sure for 4 h at room temperature. The catalyst was
filtered off and washed with aq MeOH and the filtrates
were evaporated. Compound 7 was purified by chroma-
tography on silica gel. Elution with C gave the product
(yield 44%). 13C NMR (75 MHz, D2O) d 179.1 (COO),
175.1 (NHCOR), 158.2 (NHCOOR), 104.7 (C-2), 93.0
(C-10), 82.6 (C-5), 82.0 (C(CH3)3), 77.3 (C-3), 75.0 (C-
4), 69.5 (C-50), 65.9, 65.8, (C-20, C-40), 63.2, 62.3 (C-1,
C-6), 60.9 (C-60), 54.1, 53.5 (C-30, C-2asp), 40.3 (C-
[a]D +31.8 (c 1.5, H2O); IR (cmꢁ1): 3500–3020 (OH),
2926 (C–H), 1720 (C@O) (ester), 1651 (C@O) (urea),
1
1520 (N–H), 1163, 1115, 1048, 971 (C–OH); H NMR
(400 MHz, D2O) d 5.40 (H-10), 4.73 (20-H), 4.60–3.30
(1-H, 3-H, 4-H, 5-H, 6-H, 30-H, 40-H, 50-H, 60-H,
NCH2CH2O, NCH2CH2O), 2.70–1.60 (CHCH3), 1.60–
0.70 (CH3). 13C NMR (100 MHz, D2O) d 182.3
(COO), 163.4 (CONH), 106.6 (C-2), 94.7 (C-10), 84.3
(C-5), 79.2 (C-3), 76.5 (C-4), 71.6 (C-50), 68.2, 67.9 (C-
20, C-40), 66.1 (NCH2CH2O), 64.7 (C-1), 64.3 (C-6),
62.9 (C-60), 55.9 (C-30), 47.4 (CHCH3), 41.0
(NCH2CH2O), 21.3 (CH2CHCH3), 19.4 (CH2CHCH3).
3
asp), 28.5 (3 · CH3). FABMS (glycerol) m/z 555
[MꢁH]ꢁ, 393 [MꢁfructosylꢁH]ꢁ.
Table 2. Reaction conditions assayed for polymerization of 3
Sample
Radical initiator (mol %)
Reaction temperature (ꢁC)
Reaction time (h)
Yield [g (%)]
Molecular weight Mw (g/mol)
1
2
3
4
0.5
3.0
3.0
3.0
60
60
60
60
24
24
48
6
0.29 (58)
0.38 (76)
0.44 (88)
0.19 (38)
494,000
1,115,000
1,880,000
162,000

J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
329
3.9. 3-(L-Aspartic acid 4-amido)-3-deoxy-a-D-allo-
pyranosyl b-D-fructofuranoside (8)
10), 82.4 (C-5), 81.0 (C(CH3)3), 77.4 (C-3), 74.3 (C-4),
69.2, 65.7, 65.4 (C-20, C-40, C-50), 66.5, 62.4 (C-1,
C-6), 60.5 (C-60), 56.0 (C-30), 53.0 (C-2lys), 40.6, 40.5
(C-3lys, C-4lys, C-5lys, C-6lys, CH2Ph), 28.1 (C(CH3)3).
FABMS (glycerol) m/z 726 [M+Na]+.
Compound 7 (0.2 mmol, 112 mg) was dissolved in
cooled 90% trifluoroacetic acid (0.8 mL) and kept for
15 min at 4 ꢁC. Cold dried Et2O (60 mL) was added
and the precipitated product was filtered off and washed
with Et2O. Purification by chromatography on silica gel
and elution with D gave the product (yield 77%). 13C
NMR (75 MHz, D2O, acetone-d6) d 174.7 (COO),
174.1 (NHCOR), 104.8 (C-2), 93.2 (C-10), 82.7 (C-5),
77.3 (C-3), 74.9 (C-4), 69.6, 66.2, 65.9 (C-20, C-40, C-
50), 63.2, 62.3 (C-1, C-6), 61.1 (C-60), 53.5, 52.5 (C-30,
C-2asp), 37.3 (C-3asp). FABMS (glycerol) m/z 457
[M+H]+, 295 [Mꢁfructosyl+H]+.
3.14. 3-(N-tert-Butoxycarbonyl-L-phenylalanylamido)-3-
deoxy-a-D-allogyranosyl b-D-fructofuranoside (12)
Chromatography with eluent C gave the product (yield
33%). 13C NMR (75 MHz, D2O, acetone-d6) d 176.3
(NHCOR), 158.0 (NHCOOR), 137.0 (quaternary aro-
matic carbon CH2Ph), 128.9, 128.5, 128.1 (five tertiary
aromatic carbons CH2Ph), 104.5 (C-2), 92.5 (C-10),
82.4 (C-5), 81.0 (C(CH3)3), 77.4 (C-3), 74.3 (C-4), 69.2,
65.7, 65.4 (C-20, C-40, C-50), 66.5, 62.4 (C-1, C-6), 60.5
(C-60), 56.0 (C-30), 53.0 (C-2phe), 40.5 (C-3phe), 28.1
(C(CH3)3). FABMS (glycerol) m/z 611 [M+Na]+, 589
[M+H]+, 427 [Mꢁfructosyl+H]+.
3.10. Synthesis of the aminoacyl derivatives 9, 10, 11,
and 12
Compound 1 (0.59 mmol, 201 mg) was coupled with the
protected amino acid as described for the preparation of
6. After lyophilization the product was purified by col-
umn chromatography on silica gel. The following prod-
ucts were thus obtained.
3.15. 3-(L-Aspartic acid 4-amido)-3-deoxy-a-D-allo-
pyranose (13)
A solution of 8 in 0.5 N HCI (5 mL) was heated at 60 ꢁC
for 10 min. After cooling to 20 ꢁC, the solution was neu-
tralized with NaOH. Compound 13 was separated from
the reaction mixture by ion-exchange chromatography
(separation conditions: column size 4 · 250 mm; pack-
ing: cation exchange resin, Ca2+; eluent: H2O).
3.11. 3-(N-tert-Butoxycarbonyl-L-alanylamido)-3-deoxy-
a-D-allopyranosyl b-D-fructofuranoside (9)
Chromatography with eluent C gave the product (yield
40%). 13C NMR (75 MHz, (CD3)2SO)
d
170.8
(NHCOR), 154.9 (NHCOOR), 104.2 (C-2), 91.6 (C-
10), 82.7 (C-5), 78.2 (C(CH3)3), 76.4 (C-3), 73.7 (C-4),
69.0, 65.5, 65.0 (C-20, C-40, C-50), 61.8 (C-1, C-6), 60.1
(C-60), 55.3 (C-30), 49.5 (C-2ala), 28.1 (C(CH3)3), 17.9
(C-3ala).
3.16. Fmoc-L-tryptophan22
L-Tryptophan (100 mg, 0.49 mmol) in 50 mL of acetone/
10% Na2CO3 solution was cooled with ice, and then
fluorenyl-9-methoxycarbonyl chloride (Fmoc) (135 mg,
0.52 mmol), in 2 mL of acetone was added. The solution
was stirred for 90 min at 0 ꢁC and then 4 h at room
temperature, and then poured into 100 mL of water
and extracted three times with Et2O. The pH of the
solution was adjusted to 2 under cooling with ice, and
the solution kept for 15 h at 5 ꢁC. The white precipitate
was then filtered off and washed three times with water
to yield the product (190 mg, 91%). Mr 426.47 g/mol.
1H and 13C NMR spectra data are in accordance with
the literature.19 ES+MS m/z 875 [2M+Na]+, 465
[M+K]+, 449 [M+Na]+, 427 [M+H]+.
3.12. 3-(N-tert-Butoxycarbonyl-L-leucylamido)-3-deoxy-
a-D-allopyranosyl b-D-fructofuranoside (10)
Chromatography with eluent C gave the product (yield
30%). 13C NMR (75 MHz, D2O, acetone-d6) d 178.0
(NHCOR), 158.3 (NHCOOR), 105.0 (C-2), 93.0 (C-
10), 82.6 (C-5), 82.2 (C(CH3)3), 77.5 (C-3), 74.6
(C-4), 69.4, 66.1, 65.7 (C-20, C-40, C-50), 62.9, 62.4
(C-1, C-6), 60.8 (C-60), 55.3 (C-30), 53.4 (C-2leu), 40.8
(C-3leu), 28.5 (C(CH3)3), 25.1 (C(CH3)2), 23.1
(C(CH3)2). FABMS (glycerol) m/z 555 [M+H]+, 393
[Mꢁfructosyl+H]+.
3.17. 3-Deoxy-3-(N-Fmoc-L-tryptophanylamido)-a-D-
allopyranosyl b-D-fructofuranoside (14)
3.13. 3-(Na-tert-Butoxycarbonyl-Ne-Z-L-lysylamido)-3-
deoxy-a-D-allopyranosyl b-D-fructofuranoside (11)
Fmoc-L-tryptophan (1.37 g, 3.2 mmol) was dissolved in
a mixture of 30 mL EtOH, 30 mL 2-propanol, and
10 mL acetone, and then a solution of 1 (1 g, 2.9 mmol)
in water (40 mL) and ethanol (40 mL) was added, and
then solution of EEDQ (0.87 g, 3.5 mmol) in a mixture
of 2-propanol (20 mL), EtOH (10 mL), and 10 mL
13C NMR (75 MHz, D2O, acetone-d6)
d
176.3
(NHCOR), 158.0, 157.9 (NHCOOR), 137.0 (quaternary
aromatic carbon CH2Ph), 128.9, 128.1, 128.0 (five ter-
tiary aromatic carbons CH2Ph), 104.5 (C-2), 92.5 (C-

330
J. Anders et al. / Carbohydrate Research 341 (2006) 322–331
acetone was added slowly. After 24 h of stirring, addi-
tional EEDQ (0.45 g, 1.8 mmol) in the same solvent
mixture was added, and stirring was continued for
48 h, followed by the addition of water (100 mL). The
alcohol was evaporated and the aqueous solution then
extracted with 30 mL petroleum ether and three times
with Et2O (30 mL). The water solution was concentrated
followed by freeze drying. The product was purified by
chromatography on silica gel and elution consecutively
with 100 mL petroleum ether, a 1:3 mixture of petro-
leum ether and Et2O (100 mL), Et2O (100 mL) and
2-propanol (200 mL), monitored by TLC (Rf in 2-propa-
nol 0.67). The yield of the freeze-dried product was 60%.
IR (cmꢁ1): 3350 (OH), 2926 (C–H), 1700 (C@O) (ester),
1671 (C@O) (amide), 1526 (N–H), 1478, 1450 (CH2),
8.7 Hz, 1H, 4-H), 3.90 (m, 2H, 5-H, 50-H), 3.83–3.62
(m, 10H, 1-H2, 6-H2, 20H, 40-H, 60-H2, NH2CH2–)
3.50 (t, J 5.5 Hz, 1H, 30-H). 13C NMR (100 MHz,
D2O) d 176.0 (CONH), 104.9 (C-2), 92.2 (C-10), 82.3
(C-5), 77.1 (C-3), 74.7 (C-4), 68.9 (C-50), 65.9, 65.6 (C-
20, C-40), 63.0 (C-6), 62.7 (C-1), 60.9 (C-60), 57.4
(NH2CH2–), 53.4 (C-30). ES+MS m/z 399 [M+H]+,
365, 237, 219, 181.
3.19. 3-Deoxy-3-(N-Fmoc-L-phenylalanyl-L-glycylam-
ido)-a-D-allopyranosyl b-D-fructofuranoside (16)
Compound 15 (0.16 g, 0.4 mmol) and Fmoc-L-phenylal-
anine (0.155 g, 0.4 mmol) were dissolved in EtOH
(50 mL) and then EEDQ (0.1 g, 0.4 mmol) in EtOH
(20 mL) was added while stirring. The solution was stir-
red for a further 45 h at room temperature, and then
water (250 mL) was added and the EtOH removed in va-
cuo. The product was purified by chromatography on
silica gel with petroleum ether (100 mL), 100 mL of 1:3
EtOH–petroleum ether, and finally 2-propanol. The
yield was 0.153 g (0.2 mmol, 50%), Mr = 767.79 g/mol.
IR (cmꢁ1): 3350 (OH), 3065, 2929 (C–H), 1703 (C@O)
(ester), 1603, (C@O) (amide), 1535 (N–H), 1498, 1477,
1
1249, 1105, 1050 (C–OH); H NMR (300 MHz, D2O,
acetone-d6) d 7.74 (d, J 7.5 Hz, 2H, Aryl-HFmoc), 7.65
(d, J 7.6 Hz, 1H, 30trp-H), 7.52 (d, J 7.40 Hz, 2H, Aryl-
H
Fmoc), 7.31 (m, 3H, 2 · Aryl-HFmoc, 60trp-H), 7.22 (m,
2H, 2 · Aryl-HFmoc), 7.04 (s, 1H, 10trp-H), 6.99 (m, 2H,
40trp-H, 5t0rp-H), 5.38 (d, J 3.2 Hz, 1H, 10-H), 4.57 (t, J
4.3 Hz, 1H, Aryl-CH–CH2–O), 4.23 (d, J3,4 8.2 Hz,
1H, 3-H), 4.11 (m, 4H, Aryl-CH–CH2–O, 2trp-H, 4-H,
5H), 3.98 (m, 1H, 50-H), 3.80 (m, 3H, 5-H, 20-H,
50-H), 3.70 (m, 6H, 6-H2, 40-H, 50-H, 60-H2), 3.58 (m,
3H, 1-H2, 30-H), 3.29 (m, 2H, 3trp-H). ES+MS m/z 788
[M+K]+, 772 [M+Na]+, 750 [M+H]+ 612, 588, 570,
181, 179.
1
1451, 1413 (CH2), 1258, 1106, 1050 (C–OH); H NMR
(300 MHz, pyridine-d5) d 7.82 (d, J 7.4 Hz, 2H, Aryl-
H
Fmoc), 7.65 (t, J 7.5 Hz, 2H, Aryl-HFmoc), 7.41–7.29
(m, 9H, 5 · Ph–Hphe, 4 · Aryl-HFmoc), 5.38 (d, J
3.2 Hz, 1H, 10-H), 4.61–4.18 (m, 10H, CH2NH2, Aryl-
CH–CH2–O, Aryl-CH–CH2–O, 3-H, 5-H, 20-H, 40-H,
50-H), 4.08 (d, J3,4 7.1 Hz, 1H, 3-H), 3.96 (m, 1H,
3.18. 3-Deoxy-3-(L-glycylamido)-a-D-allopyranosyl b-D-
fructofuranoside (15)
2
phe-H), 3.85 (m, 1H, 30-H), 3.70 (m, 2H, 3phe-H2). 13C
To a solution of 1 (1.5 g, 4.4 mmol) in water (70 mL)
and EtOH was added a solution of Fmoc-glycine
(1.45 g, 4.9 mmol) in a 3:2:1 mixture of EtOH–2-propa-
nol–water and then a solution of EEDQ (1.31 g,
5.3 mmol) in 2-propanol (50 mL) was added slowly with
stirring. After 20 h of reaction at room temperature
water (200 mL) was added, the alcohol is removed in va-
cuo and the product isolated by freeze drying. For puri-
fication it was suspended in petroleum ether,
ultrasonicated for 30 min, followed by filtration, and
purification by chromatography on silica gel in 2-propa-
nol. Freeze drying yielded 1.74 g (64%) of 3-deoxy-3-
(Fmoc-L-glycylamino)-a-D-allopyranosyl b-D-fructofur-
anoside (Rf in 2-propanol 0.60), Mr = 620.61 g/mol.
For deprotection 0.45 g (0.73 mmol) of the product
was treated with Et2NH (2 mL), stirred for 60 min at
room temperature, and then water (150 mL) was added.
The solution was then extracted three times with petro-
leum ether and two times with Et2O. The aqueous solu-
tion was freeze dried and the product purified by
chromatography on silica gel with 2-propanol. The yield
was 0.275 g (0.7 mmol, 95%), Mr = 398.37 g/mol. 1H
NMR (75 MHz, pyridine-d5)
d
172.6, 171.4
(2 · NHCOR), 157.2 (NHCOOR), 144.6, 141.6 (four
aromatic quaternary carbons Fmoc), 138.5 (aromatic
quaternary carbon phe), 130.0, 129.8, 129.7 (five tertiary
aromatic carbons phe), 128.9, 128.0, 126.7, 121.7 (eight
tertiary aromatic carbons Fmoc) 105.3 (C-2), 93.3 (C-
10), 84.4 (C-5), 79.9 (C-3), 75.4 (C-4), 71.3 (C-50), 67.2
(Aryl-CH–CH2–O), 67.0 (C-2phe), 54.1 (C-30), 46.8
(NHCOCH2–), 38.9 (C-3phe). ES+MS m/z 790
[M+Na]+, 768 [M+H]+, 740, 696, 630, 612, 553, 522.
Acknowledgements
Suggestions by Dr. J. Seibel on the manuscript and
support by the Federal Government/Fachagentur fur
¨
Nachwachsende Rohstoffe e.V. (Grant No. 22001498)
is gratefully acknowledged.
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