One-pot stereoselective synthesis of β-N-aryl-glycosides by N-glycosylation of aromatic amines: application to the synthesis of tumor-associated carbohydrate antigen building blocks

Article abstract of DOI:10.1016/j.tet.2007.02.092

We studied the stereoselective synthesis of several β-N-aryl-glycosides by glycosylation of aromatic primary amines using unprotected carbohydrates in aqueous solution. This was the first report showing an efficient method for the synthesis with one step of β-N-glycosyl-para-amino-phenyl alanine building blocks for the tumor-associated carbohydrate antigen (TACA) glycopeptides synthesis. Analysis of products by ¹H and ¹³C NMR indicated that the Amadori rearrangement had not occurred after formation of the stereoselective β-N-glycoside bond (natural N-glycoprotein linkage). The study of the chemical and enzymatic stability in aqueous media of β-N-aryl-glycosides synthesized was also investigated. For the first time we have shown that the N-glycosidic bond was relatively stable at pH near to 7 and more stable than the O-glycosidic bond to enzymatic hydrolysis. This higher enzymatic and chemical stability of the N-glycosidic bond is essential to envisage further development of stable TACA building blocks.

Full text of DOI:10.1016/j.tet.2007.02.092

Tetrahedron 63 (2007) 4178–4183
One-pot stereoselective synthesis of b-N-aryl-glycosides by
N-glycosylation of aromatic amines: application to the synthesis
of tumor-associated carbohydrate antigen building blocks
*
Nicolas Bridiau, Moulay Benmansour, Marie Dominique Legoy and Thierry Maugard
´
Laboratoire de Biotechnologie et de Chimie Bio-organique, CNRS FRE 2766, Universite de La Rochelle,
´
Avenue Michel Crepeau, 17042 La Rochelle, France
Received 29 January 2007; accepted 21 February 2007
Available online 24 February 2007
Abstract—We studied the stereoselective synthesis of several b-N-aryl-glycosides by glycosylation of aromatic primary amines using un-
protected carbohydrates in aqueous solution. This was the first report showing an efficient method for the synthesis with one step of b-N-gly-
cosyl-para-amino-phenyl alanine building blocks for the tumor-associated carbohydrate antigen (TACA) glycopeptides synthesis. Analysis of
products by 1H and 13C NMR indicated that the Amadori rearrangement had not occurred after formation of the stereoselective b-N-glycoside
bond (natural N-glycoprotein linkage). The study of the chemical and enzymatic stability in aqueous media of b-N-aryl-glycosides synthe-
sized was also investigated. For the first time we have shown that the N-glycosidic bond was relatively stable at pH near to 7 and more stable
than the O-glycosidic bond to enzymatic hydrolysis. This higher enzymatic and chemical stability of the N-glycosidic bond is essential to
envisage further development of stable TACA building blocks.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
a (aNeuNAc-2,3-bGal-1,3-[aFuc-1,4-]bGlcNAc-) antigens,
are b-N-glycosidically linked to an asparagine.12
It is well established that oncological transformations are
accompanied by the change of cell surface glycosylation
patterns.1,2 The abnormal glycans expressed on tumor cells
are known as tumor-associated carbohydrate antigens (TA-
CAs). Since the early 1970s, many TACAs have been iden-
tified2–6 and become useful molecular templates and targets
in the design and development of therapeutic cancer vac-
cines.4–10
Our laboratory has actively been engaged in the chemo-en-
zymatic synthesis of molecules with biological activities us-
ing green conditions and particularly in the synthesis of
complex oligosaccharides and glycoconjugates by hydro-
lases.13–18 As a result of our advances in this area, we began
a program to explore the synthesis of N-linked glycosyl
amino acid building blocks for TACA glycopeptides elabo-
ration.
Traditionally, cancer vaccines are created by coupling TA-
CAs to a carrier protein, such as keyhole limpet hemocyanin
(KLH). Resultant glycoconjugates can be employed to im-
munize cancer patients to fight tumors that bear the same
antigens. This method has witnessed success with some
TACAs, and a few cancer vaccines thus developed are now
in clinical trials.11
2. Results and discussion
2.1. Synthesis of b-N-aryl-glycosides
As a first step toward this goal, we chose to study the N-gly-
cosylation of aniline. Few synthetic methods have been
reported for the N-glycosylation of aromatic compounds
starting from aniline derivatives and sugars.19–25 Usually
the reaction could be accomplished by heating at reflux in
protic solvents such as MeOH. The N-aryl-glycosylamines
were obtained in variable yields depending on the reactivity
of the aniline derivatives. Moreover, the reaction time re-
quired was very long (about 6 days) and the stereochemistry
at the anomeric carbon depended on the sugar and on the
steric features of the amine and the sugar. In some cases,
Generally, TACAs such as TN (aGalNAc-), T (Thomsen-
Friedenreich, bGal-1,3-aGalNAc-), sialyl-TN (aNeuNAc-
2,6-aGalNAc-), and sialyl-T (aNeuNAc-2,3-bGal-1,3-
aGalNAc- and bGal-1,3-[aNeuNAc-2,6-]aGalNAc-) are
a-O-glycosidically linked to a serine or a threonine in the
peptide backbone. Others, such as sialyl Lewis x (aNeu-
NAc-2,3-bGal-1,4-[aFuc-1,3-]bGlcNAc-) and sialyl Lewis
* Corresponding author. Fax: +33 546458247; e-mail: tmaugard@univ-lr.fr
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.02.092

N. Bridiau et al. / Tetrahedron 63 (2007) 4178–4183
4179
a mixture of epimers a/b was obtained. Optimization of the
N-glycosylation reaction in terms of yield, reaction time,
and diastereoselection represented a significant challenge.
carried out at 40 ꢀC for 5 h with the same concentrations
of D-glucose 1a and aniline 2a, we observed that the opti-
mum pH of the N-glycosylation was close to 6.5 (55%)
and that the reaction was slightly favored in alkaline condi-
tions (Fig. 1). When the effect of the temperature was studied
with the same concentrations, we observed that the reaction
was favored with temperatures lower than 50 ꢀC (Fig. 2). In
fact, in alkaline conditions and at higher temperatures, sev-
eral others products were obtained, probably resulting of the
Amadori rearrangement and characterized by the formation
of brown pigments.
In this context, we chose to study the N-glycosylation reac-
tion in aqueous media as a continuation of our research
devoted to the development of green organic chemistry. It
is well accepted that the reaction between an amine such as
aniline 2a and aldoses such as D-glucose 1a in water, at high
temperature and in slightly acid conditions first proceeds
through the corresponding N-glycoside, 3a, which is, how-
ever, not stable and can be rearranged to produce the so-
called Amadori product, 4a (Scheme 1), as the first relatively
stable glycoconjugate.26
60
40
20
0
Therefore, in a preliminary experiment, we decided to carry
out the condensation of aniline 2a (250 mM) and an unpro-
tected D-glucose 1a (250 mM) in aqueous phosphate buffer
pH 8 and at 40 ꢀC. By HPLC analysis, the decrease of 2a
concentration (12% after 5 h) was proportional with the syn-
thesis of only one product. Products formed were purified by
semi-preparative HPLC. Analysis of products by 1H and 13
C
0
2
4
6
pH
8
10
12
NMR like the mass spectrometry analysis indicated that the
Amadori rearrangement had not occurred after formation of
the desired product 3a. In the H NMR spectra of 3a, the
1
Figure 1. Effect of pH on the N-glycosylation yield. Reaction was carried
out at 40 ꢀC in aqueous buffers pH 2–5 (citrate phosphate), pH 6–8 (phos-
phate), and pH 9–11 (100 mM, glycine NaOH).
anomeric hydrogen is a doublet with a coupling constant
of 8.8 Hz. Such coupling constant is in the range for those
resulting from an axial–axial configuration of H1 and H2
and therefore, for these sugar, a b-N-glycosidic linkage.
55
50
45
40
35
The preference for formation of the b isomer can be rational-
ized in terms of the competition between substitution at the
sterically least hindered position (b) versus substitution at
a position which incurs stabilization from the anomeric ef-
fect (a). The substitution is driven predominantly by sterics
and the reaction is stereoselective for the b configuration.
Although these reaction conditions permit the desired prod-
uct (b-3a) to be obtained, they are not particularly efficient,
consequently other pHs and temperatures were therefore ex-
amined. We investigated the effect of pH and temperature on
the N-glycosylation reaction yield. When the reaction was
15
25
35
45
55
65
75
85
T °C
Figure 2. Effect of temperature on the N-glycosylation yield. Reaction was
carried out in aqueous buffer pH 6.5 (phosphate).
OH
H
OH
H2N
OH
N
O
HO
HO
HO
OH
+
OH
HO
OH
OH
2a
1a
H2O
OH
OH
OH
H
H
N
OH
N
OH
O
OH
OH
N
HO
HO
HO
HO
HO
HO
OH
OH
O
H
N
O
HO
HO
N
H
HO
HO
OH
OH
OH
3a
4a
Scheme 1. Formation of N-glucosyl-aniline 3a and Amadori product 4a from D-glucose 1a and aniline 2a in aqueous alkaline conditions.

4180
N. Bridiau et al. / Tetrahedron 63 (2007) 4178–4183
Using a temperature of 40 ꢀC and a pH of 6.5, we investi-
gated the preparation in aqueous media of several N-aryl-
glycosides, 3b–3m using D-glucose (1a), D-galactose (1b),
lactose (1c), N-acetyl-D-glucosamine (1d), N-acetyl-D-ga-
lactosamine (1e), and N-acetyl-D-lactosamine (1f). In all re-
actions tested under equimolar conditions, acceptable yields
from 30% to 55% were obtained with complete b-selectivity
(Table 1). These yields can be increased using a higher sugar
concentration (3j and 3k footnote b, 3m).
100
80
60
40
20
0
pH 8
pH 6,5
pH 4,5
pH 2
The product 3k can be considered as an analog of TN antigen
and as a precursor of T (Thomsen-Friedenreich), sialyl-TN,
and sialyl-T antigen synthesis. The products 3j and 3l can
be considered as precursors of sialyl Lewis x and sialyl
Lewis a antigens.
0
1
2
3
Time (h)
4
5
The product 2d is an intermediary of the synthesis of thiazo-
loquinazolinones with a glycogen synthase kinase-3 inhibi-
tory activity.27
Figure 3. Influence of the pH on the time course of N-glucosyl-aniline (3a)
decomposition. Reactions were carried out at 40 ꢀC in aqueous buffer pH 2
(B) to 4.5 (-) (citrate phosphate), pH 6.5 (,) to 8 (C) (phosphate).
2.2. Chemical hydrolysis of b-N-aryl-glycosides
To investigate the kinetics of the chemical hydrolysis of syn-
thesized b-N-aryl-glycosides, 3a was maintained at 40 ꢀC
under different pH conditions and the hydrolysis was moni-
tored by HPLC over time. As shown in Figure 3, 3a was hy-
drolyzed relatively slowly under alkaline conditions. After
5 h at pH 8, b-N-glycoside 3a was still the major constituent
with some glucose and aniline liberated. The stability of 3a
at pH 8 is well reflected in the long half-life, t1/2, of about
26 h and in the poor initial rate of hydrolysis. In contrast, hy-
drolysis of 3a was found to be faster under neutral and
slightly acidic conditions. The low pH of 2 induced a sponta-
neous hydrolysis of 3a, which did not allow the measure-
ment of the half-life and the initial rate of hydrolysis.
Between these extreme pH values, the rate of decomposition
increased with acidity; for example, the t1/2 of 3a was about
2 h at pH 6.5 and about 8 min at pH 4.5. A similar comport-
ment was observed with b-N-galactoside 3b (Table 2).
The hydrolytic instability of the b-N-glycosidic bond in b-N-
glucosyl-aniline (3a) and b-N-galactosyl-aniline (3b) under
acidic and neutral pH conditions is well in line with earlier
observations on the stability of N-glycosides of amino acids
in aqueous solutions.28,29 In contrast to b-N-glucosyl-aniline
(3a) and b-N-galactosyl-aniline (3b), we observed that prod-
ucts 3j and 3k were more stable under pH near to the neutral-
ity (Table 2).
Table 1. N-Glycosylation of aromatic compounds
H2N
OH
OH
R2
R1O
O
R1O
HO
H
N
O
aqueous
HO
R2
R
OH
R
Na2HPO4/NaH2PO4
(100 mM, pH 6.5)
40 °C
Sugars
R
R1
Amines
R2
Product (yield %)a
1a
1b
1c
1d
1a
1b
1a
1b
1c
1d
1e
1f
OH
OH
OH
NHAc
OH
OH
OH
OH
H equatorial
H axial
Gal (b-1)
H equatorial
H equatorial
H axial
H equatorial
H axial
Gal (b-1)
H equatorial
H axial
2a
2a
2a
2a
2b
2b
2c
2c
2c
2c
2c
2c
H
H
H
H
3a (55)
3b (51)
3c (52)
3d (37)
3e (40)
3f (38)
3g (54)
3h (34)
mCH2OH
mCH2OH
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
OH
3i (40)
NHAc
NHAc
NHAc
3j (41), 3j (74)b
3k (41), 3k (75)b
3l (30)
Gal (b-1)
O
N
N
1c
OH
Gal (b-1)
2d
3m (80)b
a
Yields were determined by HPLC analysis after 5 h of reaction.
Reaction was carried out with 4 equiv of sugar.
b

N. Bridiau et al. / Tetrahedron 63 (2007) 4178–4183
Table 2. Stability of b-N-aryl-glycosides in aqueous media
4181
OH
R1O
HO
H
N
OH
O
R1O
HO
H2N
H2O
O
R2
R2
+
R
40 °C
R
OH
Product
R
R1
R2
pH
vi (103 mmol minꢁ1
)
t1/2
3a
3a
3a
3a
3b
3b
3b
3b
3j
3j
3j
3k
3k
3k
OH
OH
OH
OH
OH
OH
OH
OH
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
H equatorial
H equatorial
H equatorial
H equatorial
H axial
H axial
H axial
H axial
H equatorial
H equatorial
H equatorial
H axial
H axial
H axial
H
H
H
H
H
H
H
H
8
0.28
2.53
29.5
nd
1.22
10.90
nd
w26.5 h
w2.25 h
w8.5 min
Less of 3 min
w4.45 h
w24 min
w2 min
Less of 1 min
w8 days
w3 days
w22 h
6.5
4.5
2
8
6.5
4.5
2
nd
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
pCH2–CH–(COOH)NH2
8
0.031
0.08
0.27
0.089
0.486
1.763
7.35a
6.5
8
w2 days
w13 h
w3 h
7.35a
6.5
a
pH physiologique, t1/2: half-life, vi: initial rate of chemical hydrolysis, nd: not determined.
2.3. Enzymatic hydrolysis of b-N-aryl-glycosides
3. Conclusion
With an aim of knowing if a O-glycosidic bond is more stable
than a N-glycosidic bond to enzymatic hydrolysis, we
compared the hydrolysis rates of b-O-phenyl-glucopyrano-
side and b-O-phenyl-galactopyranoside with the hydrolysis
rate of b-N-phenyl-glucopyranoside 3a and b-N-phenyl-
galactopyranoside 3a by using a commercial enzymatic
preparation of glucosidase (Novarom G) and galactosidase
(Lactozym). Table 3 clearly demonstrates that a N-glyco-
sidic bond is very stable in the presence of glycosidase in
comparison to a similar compound with a O-glycosidic
bond.
A short (one step) and efficient method was developed in
aqueous media for b-N-selective glycosylation of aromatic
primary amines such as aniline from unprotected sugar. The
Amadori rearrangement had not occurred after formation of
the stereoselective b-N-glycoside bond. This method allows
the synthesis of various stable b-N-aryl-glycosides, which
can be considered as analogs or precursors of TACAs.
The study of the chemical and enzymatic stability in aque-
ous media was also investigated. We have shown that the
N-glycosidic bond was relatively stable in pH near to 7
and more stable than the O-glycosidic bond to enzymatic
hydrolysis.
This higher chemical and enzymatic stability is essential
to envisage further development of TACA building blocks,
especially by chemo-enzymatic synthesis. This strategy is
currently in progress in the laboratory for the synthesis of
TACA building blocks from products 3j, 3k, and 3l. These
TACA building blocks could be incorporated into target
glycopeptides using solid-phase peptide synthesis on the
Wang resin.
4. Experimental
4.1. Biological and chemical materials
All chemicals were purchased from Sigma Co. (USA) except
compound 2d, which was previously synthesized in the
Table 3. Comparison of stability of b-N-aryl-glycosides and b-O-aryl-glycosides to enzymatic hydrolysis
OH
R O
1
OH
O
R
R1O
HO
HR
O
glycosyl hydrolase
aqueous
+
HO
OH
OH
OH
Na2HPO4/NaH2PO4
(100 mM, pH 6.5)
40 °C
R=NH or O
Product
pH
6.5
Enzyme
vi (mmol minꢁ1 gꢁ1
)
t1/2
b-N-Phenyl-glucopyranoside 3a
b-O-Phenyl-glucopyranoside
b-N-Phenyl-galactopyranoside 3b
b-O-Phenyl-galactopyranoside
b-Glucosidase (Novarom G)
b-Galactosidase (Lactozym)
14
323
0
w2 h
w1 h 10 min
w26 min
Less of 1 min
18
Reactions were carried out at 40 ꢀC with 15 g Lꢁ1 of Novarom G or Lactozym in aqueous buffer pH 6.5. t1/2: half-life, vi: initial rate of enzymatic hydrolysis.

4182
N. Bridiau et al. / Tetrahedron 63 (2007) 4178–4183
laboratory.27 Deionized water was obtained via a Milli-Q
system (Millipore, France).
Nitrogen was used as the drying gas at 15 L/min and
350 ꢀC at a nebulizer pressure of 4 bars. The scan range
was 50–1000 m/z using five averages and 13,000 m/z per
second resolution. The capillary voltage was ꢁ4000 V for
negative ion detection. Processing was done off-line using
HP Chemstation.
Lactozym 3000 L HP-G is a soluble commercial preparation
of b-galactosidase from Kluyveromyces lactis manufactured
by Novo Nordisk A/S (Bagsvaerd, Denmark), Batch
DKN08649 with an activity of 3530 U mlꢁ1. One unit is de-
fined as the amount of enzyme which releases 1 mmol glu-
cose per minute under the following standard conditions:
4.75% (w/w) lactose, milk buffer system pH 6.5, 37 ꢀC,
reaction time 30 min.
4.5. Libraries characterization of b-N-aryl-glycosides
4.5.1. Compound 3a. m/z (LR-ESI+) C12H17NO5H (M+H+),
found: 255.9, calcd: 256.2809. H NMR (400 MHz, D2O):
1
d 7.10 (dd, 2H, H-3, H-5), 6.72–6.69 (m, 3H, H-2, H-4, H-
6), 4.58 (d, 1H, J¼8.8 Hz, H-10), 3.69 (dd, 1H, H-40), 3.52
(dd, 1H, H-50), 3.42–3.35 (m, 2H, H-20, H-30), 3.25 (dd,
2H, H-60). 13C NMR (D2O): d 146.39 (C-1), 130.38 (C-3,
C-5), 120.38 (C-4), 115.16 (C-2, C-6), 85.62 (C-10), 77.63
(C-50), 77.24 (C-30), 73.44 (C-20), 70.50 (C-40), 61.53 (C-60).
Novarom G is a dry commercial preparation of b-1-4-gluco-
sidase derived from Aspergillus niger manufactured by
Novo Nordisk A/S (Bagsvaerd, Denmark) with an activity
of 80 BGDU gꢁ1. One unit (BGDU) is defined as the amount
of enzyme, which releases 1 mmol of p-nitrophenol from
p-nitrophenyl-b-D-glucopyranoside (pNPG) per minute at
23 ꢀC and pH 3.5.
4.5.2. Compound 3b. m/z (LR-ESI+) C12H17NO5H (M+H+),
found: 255.9, calcd: 256.2809. H NMR (400 MHz, D2O):
1
4.2. Synthesis of N-aryl-glycosides
d 7.10 (dd, 2H, H-3, H-5), 6.72–6.68 (m, 3H, H-2, H-4,
H-6), 4.53 (d, 1H, J¼8.4 Hz, H-10), 3.81 (dd, 1H, H-40),
3.62 (dd, 1H, H-50), 3.56 (m, 1H, H-30), 3.53 (dd, 2H, H-
60), 3.49 (dd, 2H, H-20). 13C NMR (D2O): d 146.48 (C-1),
130.37 (C-3, C-5), 120.28 (C-4), 115.12 (C-2, C-6), 86.06
(C-10), 76.27 (C-50), 74.50 (C-30), 71.08 (C-20), 69.61
(C-40), 61.65 (C-60).
In standard conditions, aromatic amine (250 mM) was sus-
pended in phosphate buffer (100 mM, pH 6.5). Sugar
(250 mM) was added to the suspension. The mixture was
stirred at 40 ꢀC. The reactions were carried out until the for-
mation of a steady state obtained around 5 h. These condi-
tions were used except when otherwise stated in the text.
4.5.3. Compound 3c. m/z (LR-ESI+) C18H27NO10H
(M+H+), found: 417.9, calcd: 418.4245. 1H NMR
(400 MHz, D2O): d 7.13 (dd, 2H, H-3, H-5), 6.78–6.66 (m,
3H, H-2, H-4, H-6), 4.61 (d, 1H, J¼9.2 Hz, H-10), 4.27 (dd,
1H, J¼8 Hz, H-100), 3.76–3.28 (m, 12H, H-20, H-30, H-40,
H-50, H-60, H-200, H-300, H-400, H-500, H-600). 13C NMR (D2O):
d 146.35 (C-1), 130.40 (C-3, C-5), 120.42 (C-4), 115.18 (C-2,
C-6), 103.70 (C-100), 85.48 (C-10), 79.24 (C-50), 76.22 (C-500),
76.13 (C-30), 76.08 (C-300), 73.31 (C-20), 73.14 (C-200), 71.76
(C-400), 69.33 (C-40), 61.83 (C-60), 60.89 (C-600).
4.3. Chemical and enzymatic stability of N-aryl-glyco-
sides
Solution of N-aryl-glycosides in water (5 mM) was main-
tained at 40 ꢀC under different pH conditions with or without
glycosylhydrolases (15 g Lꢁ1). The hydrolysis was moni-
tored by HPLC over time.
4.4. HPLC and structural analyses
Quantitative and structural analyses of reactants and products
were conducted using an LC/MS-ES system from Agilent
(1100 LC/MSD Trap mass spectrometer VL and differential
refractometer, Waters, model 410), with a Prontosyl C18 re-
versed-phase column (250ꢂ4 mm, 5 mm) eluted with aceto-
nitrile/water (15/85, v/v) at room temperature and at a flow
rate of 0.2 ml/min. Quantification was carried out at 280 nm.
4.5.4. Compound 3d. m/z (LR-ESI+) C14H20N2O5Na
(M+Na+), found: 318.9, calcd: 319.3087. 1H NMR
(400 MHz, D2O): d 7.09 (dd, 2H, H-3, H-5), 6.72 (dd, 1H,
H-4), 6.65 (d, 2H, H-2, H-6), 4.62 (d, 1H, J¼8.4 Hz, H-10),
3.69 (dd, 2H, H-60), 3.55 (dd, 1H, H-40), 3.45 (dd, 1H,
H-50), 3.37 (m, 1H, H-30), 3.31 (dd, 1H, H-20), 1.81 (s, 3H,
13
H-80). C NMR (D2O): d 175.73 (C-70), 146.12 (C-1),
130.39 (C-3, C-5), 120.56 (C-4), 115.20 (C-2, C-6), 85.24
(C-10), 77.27 (C-50), 75.41 (C-30), 70.74 (C-40), 61.51 (C-
60), 55.87 (C-20), 22.81 (C-80).
1
Products formed were characterized by H and 13C NMR
(DEPT) after purification via preparative HPLC, using
a Prontosyl C18 (250ꢂ20 mm, 10 mm) reversed-phase col-
umn, eluted with acetonitrile/water (15/85, v/v) at room tem-
perature and at a flow rate of 5 ml/min. Lyophilization of the
solvent gave white crystalline products.
4.5.5. Compound 3e. m/z (LR-ESI+) C13H19NO6Na
(M+Na+), found: 307.9, calcd: 308.2893. 1H NMR
(500 MHz, D2O): d 7.19 (dd, 1H, H-5), 6.78–6.72 (m, 3H,
H-2, H-4, H-6), 4.67 (d, 1H, J¼8.9 Hz, H-10), 4.47 (s, 1H,
H-7), 3.78 (d, 1H, H-40), 3.61 (m, 1H, H-50), 3.51–3.45 (m,
2H, H-20, H-30), 3.35 (dd, 2H, H-60). DEPT 13C NMR
(D2O): d 129.81 (C-5), 118.38 (C-6), 113.64 (C-2), 113
(C-4), 84.76 (C-10), 76.81 (C-50), 76.44 (C-30), 72.64 (C-
20), 69.71 (C-40), 63.86 (C-7), 60.76 (C-60).
1H and 13C NMR (DEPT, Distortionless Enhancement by
Polarization Transfer) were recorded on a JEOL-JNM
LA400 spectrometer (400 MHz) (Laboratoire Commun
d’Analyse, Universite de La Rochelle, France), with tetra-
methylsilane as an internal reference. Samples were studied
as solutions in D2O.
ꢀ
4.5.6. Compound 3f. m/z (LR-ESI+) C13H19NO6Na
(M+Na+), found: 308.27, calcd: 308.2893. 1H NMR
(500 MHz, D2O): d 7.20 (dd, 1H, H-5), 6.79–6.75 (m, 3H,
Low-resolution mass spectral analyses were obtained by
electrospray in the positive and negative detection modes.

N. Bridiau et al. / Tetrahedron 63 (2007) 4178–4183
4183
H-2, H-4, H-6), 4.64 (d, 1H, J¼8.6 Hz, H-10), 4.49 (s, 1H, H-
7), 3.91 (dd, 1H, H-40), 3.71 (m, 1H, H-50), 3.68–3.58 (m,
3H, H-20, H-30, H-60). DEPT 13C NMR (D2O): d 129.79
(C-5), 118.28 (C-6), 113.61 (C-2), 112.95 (C-4), 85.18 (C-
10), 75.47 (C-50), 73.67 (C-30), 70.25 (C-20), 68.80 (C-40),
63.87 (C-7), 60.87 (C-60).
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4.5.7. Compound 3g. m/z (LR-ESI+) C15H22N2O7H
(M+H+), found: 342.9, calcd: 343.3597.
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4.5.9. Compound 3i. m/z (LR-ESI+) C21H32N2O12H
(M+H+), found: 504.9, calcd: 505.5033.
4.5.10. Compound 3j. m/z (LR-ESI+) C17H25N3O7H
(M+H+), found: 405.9, calcd: 406.3945. 1H NMR
(400 MHz, D2O): d 7.02 (d, 2H, H-5, H-9), 6.69 (d, 2H, H-
6, H-8), 4.57 (d, 1H, J¼8.4 Hz, H-10), 3.80–3.31 (m, 7H,
H-2, H-20, H-30, H-40, H-50, H-60), 2.97 (2dd, 2H, H-3),
13
1.87 (s, 3H, H-80). C NMR (D2O): d 175.85 (C-70),
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174.88 (C-1), 145.53 (C-7), 131.25 (C-5, C-9), 126.67 (C-
4), 115.70 (C-6, C-8), 85.19 (C-10), 77.32 (C-50), 75.44 (C-
30), 70.77 (C-40), 61.52 (C-60), 56.91 (C-2), 55.86 (C-20),
36.31 (C-3), 22.88 (C-80).
4.5.11. Compound 3k. m/z (LR-ESI+) C17H25N3O7H
(M+H+), found: 405.9, calcd: 406.3945. 1H NMR
(400 MHz, D2O): d 6.97 (d, 2H, H-5, H-9), 6.69 (d, 2H, H-
6, H-8), 4.46 (d, 1H, J¼8.3 Hz, H-10), 3.96–3.46 (m, 7H,
H-2, H-20, H-30, H-40, H-50, H-60), 2.91 (2dd, 2H, H-3),
13
1.81 (s, 3H, H-80). C NMR (D2O): d 186.97 (C-70),
176.08 (C-1), 145.61 (C-7), 131.23 (C-5, C-9), 115.19 (C-
6, C-8), 126.30 (C-4), 85.61 (C-10), 76.35 (C-50), 72.48 (C-
30), 70.23 (C-40), 61.67 (C-60), 56.94 (C-2), 54.47 (C-20),
36.38 (C-3), 22.91 (C-80).
4.5.12. Compound 3l. m/z (LR-ESI+) C23H35N3O12Na
(M+Na+), found: 567.9, calcd: 568.5381.
4.5.13. Compound 3m. m/z (LR-ESI+) C23H35N3O12Na
(M+Na+), found: 514.0, calcd: 514.5138.
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27. Testard, A.; Picot, L.; Lozach, O.; Blairvacq, M.; Meijer, L.;
Acknowledgements
ꢀ
Murillo, L.; Piot, J. M.; Thiery, V.; Besson, T. J. Enzyme
Financial support from Region Poitou Charente and from
ꢀ
Universite de La Rochelle is acknowledged.
Inhib. Med. Chem. 2005, 20, 557–568.
28. Von Euler, H.; Brunius, E. Chem. Ber. 1926, 59B, 1581–
1585.
References and notes
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