A simple and efficient method to label l-fucose

Article abstract of DOI:10.1016/j.tetlet.2006.07.047

This letter reports a new labeling method of fucoidan and more precisely of its monomer, l-fucose. We studied the coupling processes of new aryliodide precursors to l-fucose in order to prepare the next step, that is, the fucoidan radiolabeling. The use o

Full text of DOI:10.1016/j.tetlet.2006.07.047

Tetrahedron Letters 47 (2006) 6869–6873
A simple and efficient method to label L-fucose
a
b
a
a
`
Emmanuelle Jestin, Karine Bultel-Riviere, Alain Faivre-Chauvet, Jacques Barbet,
Anthony Loussouarnb and Jean-Franc¸ois Gestina,b,
*
aDe´partement de Recherche en Cance´rologie, INSERM U601, Universite´ de Nantes, 9 quai Moncousu,
44093 Nantes Cedex, France
bCHELATEC, 9 quai Moncousu, 44093 Nantes Cedex, France
Received 28 April 2006; revised 11 July 2006; accepted 13 July 2006
Available online 8 August 2006
Abstract—This letter reports a new labeling method of fucoidan and more precisely of its monomer, L-fucose. We studied the cou-
pling processes of new aryliodide precursors to L-fucose in order to prepare the next step, that is, the fucoidan radiolabeling. The use
of precursors containing hydrazide and hydroxylamines is an alternative procedure avoiding the use of toxic borohydride or organic
borane used for reductive amination. These coupling reactions are efficient and stereoselective. They provide stable products.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Fucoidans, polysaccharides containing substantial per-
centages of L-fucose and sulfate ester groups are ex-
tracted from Phaeophycophyta (or brown algae) such
as Fucus vesiculosus or Ascophyllum nodosum.1–4 They
have potent biological activities: anticoagulant/ anti-
thrombotic,5,6 anti-inflammatory,7 antiviral,8 anti-poi-
son,9 and anti-angiogenic.10 The aim of this work was
to develop a labeling technique for fucose containing
polysaccharides using a simple chemical reaction in
order to simplify their detection for biological applica-
tions. It is essential to label macromolecules without
altering their biological properties. According to the lit-
erature, labeling polysaccharides with fluorescence or
UV-detectable11–15 groups is the most commonly used,
but these techniques may not be appropriate for
in vivo studies because of their low sensitivity.
out the use of a costly scintillation cocktail. The second
advantage is the relative shortness of their half-lives
(max 60 days for 125I in comparison to 12.35 years for
3H and 5730 years for 14C).
A previous paper17 described the radioiodination of a
macromolecule, scleroglucan. The reaction consisted
first in a hydroxyl group activation as a p-tosylate that
was displaced by iodide in the second step. This method
was useful and did not alter the polysaccharide. In our
case, unfortunately, this method could not be employed
because of a lack of reactive hydroxyl groups on the
macromolecule.
The purpose of this work was thus to develop original
precursors for fucoidan radioiodination on the reducing
end. In order to achieve this goal, we designed several
molecules containing an aromatic ring carrying two
active functions: one used for iodination while the other
forms a covalent link with the macromolecule. It should
be then possible to radiolabel16,18 the precursor prior to
coupling it to the desired molecule in two successive
reactions. The advantage of this method is to decrease
the risk of degradation of the molecule by avoiding
reductive conditions used during usual radiolabeling
methods. This method could be then adapted to other
non reduced polysaccharides.
The introduction of radioactive atoms associated to the
macromolecule could solve this problem. The usefulness
of radioactively labeled compounds in general stems
from the ease of detecting and measuring extremely
small amounts. Carbon-14 and tritium are extensively
used as radiolabels. The isotopes of iodine16 have two
3
major advantages over 14C and H. All available iodine
isotopes are c emitters that can be detected directly with-
Keywords: Carbohydrate; Fucoidan; L-fucose; Sugar labeling;
Iodination.
According to the literature,18 three coupling functions
can be used (amine, hydrazide, and hydroxylamine).
Amines19,20 condense with aldehydes to form a Schiff
*
Corresponding author. Tel.: +33 240 084 719; fax: +33 240 356
697; e-mail: jfgestin@nantes.inserm.fr
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.07.047

6870
E. Jestin et al. / Tetrahedron Letters 47 (2006) 6869–6873
base linkage. This reaction is reversible and requires a
second step, the imine reduction, to stabilize the linkage.
Usually toxic borohydride or organic boranes are used
for that purpose. Furthermore, the ring opening of the
reducing end residue, which occurs during reductive
amination, may have a detrimental effect on the biolog-
ical activity or immunogenicity of the carbohydrate.21
O
O
NH2
N
O
OH
O
b
a
I
I
I
4
5
6
c
c'
In contrast to reductive amination, hydrazides,22–25 and
hydroxylamines26–29 condense with aldehydes to form
stable osazones and oximes, respectively.
O
N
O N
I
I
This letter describes the syntheses of two types of
prosthetic groups (Scheme 1): 3-iodophenylhydrazide
3, O-(3-iodobenzyl)-hydroxylamine 6, N-methyl-O-(3-iod-
obenzyl)-hydroxylamine 9, and N-isopropyl-O-(3-iod-
obenzyl)-hydroxylamine 10.
7
8
d
d
H
O
NHCH3
O
N
To define appropriate conditions for the coupling reac-
tion toward carbohydrate, a preliminary chemistry work
was performed. This study permitted to define the cou-
pling reaction conditions and their stereoselectivity.
For that purpose, we substituted the polysaccharide
with its L-fucose monomer during chemical reactions.
This allowed us to work with non radioactive iodinated
precursors in order to characterize the reaction products
using conventional methods. Of course, the final goal is
to substitute stable iodine by radioactive iodine.
I
I
9
10
Scheme 3. Syntheses of O-(3-iodobenzyl)-hydroxylamines 6, 9, and 10.
Reagents and conditions: (a) N-hydroxyphthalimide (2 equiv), PPh3
(2 equiv), diethylazodicarboxylate (2 equiv), anhydrous CHCl3, rt,
15 h, 71%; (b) NH2–NH2ÆH2O (10 equiv), EtOH abs, reflux, 3 h, 80%;
(c) HCHO (1.5 equiv), anhydrous CHCl3, reflux, 1 h, 95%; (c0)
acetone, reflux, 1 h, 90%, and (d) NaCNBH3 (3 equiv), EtOH, HCl
(12 N) until pH 3, rt, 30 min, quantitative.
3-Iodophenylhydrazide 3 (Scheme 2) was prepared30 in a
two-step reaction with 95% global yield. This efficient
reaction started with 3-iodobenzoic acid 1. The starting
material was activated as N-hydroxysuccinimidyl ester
using TSTU (tetrafluoroborate N,N,N0,N0-tetramethyl-
O-(N-succinimidyl)-uronium) according to the Al-Jam-
maz method31 to give compound 2 in a reproducible
yield. Hydrazide 3 was then obtained quantitatively by
reacting with hydrazine monohydrate.
In the following synthesis (Scheme 3), we used a pros-
thetic group containing O-substituted-hydroxylamine
function. It was also interesting to study the N-substitu-
tion effect regarding glycosylation reaction. O-Substi-
tuted hydroxylamines 6, 9, and 10 were obtained32
according to a Mitsunobu method33,34 starting with
3-iodobenzylalcohol 4. This method permitted the
introduction of a protected amine using a phthalimido
protective group. O-(3-Iodobenzyl)-N-hydroxyphthali-
mide 5 was obtained in 71% yield. Amine deprotection
was performed with hydrazine monohydrate to give
O-(3-iodobenzyl)-hydroxylamine 6 with 80% yield.
Aldehyde and ketone carbonyls were reacted with the
highly nucleophilic aminooxy group to form oximes 7
and 8. NaCNBH3 reduction of these oximes was
performed at pH 3 and gave quantitatively N-substi-
tuted hydroxylamines 9 and 10.
H
O
N NH2
O NHR1
I
I
3
6 : R1 = H
9 : R1 = CH3
10 : R1 = CH(CH3)2
Once the synthesis of these four precursors was
performed, we studied the glycosylation reactions with
L-fucose.35 The reactions (Scheme 4) proceeded in polar
organic solvent (MeOH/CH3COOH glacial (85/15)) at
room temperature. The iodinated precursors were con-
densed on the anomeric position. Hydrazides and
hydroxylamines condensed with the aldehyde to form,
respectively, osazone or oxime. These acyclic intermedi-
ates were more or less stable and also existed in cyclic
forms. The reactions were complete within 20 h and pro-
vided glycosylated compounds in good and reproducible
yields (Table 1).
Scheme 1. Precursors used for the iodination of carbohydrates.
O
O N
H
N NH2
O
O
COOH
a
b
O
I
I
I
3
2
1
Scheme 2. Synthesis of 3-iodophenylhydrazide 3. Reagents and
conditions: (a) TSTU (2.5 equiv), NEt3 (2 equiv), anhydrous CH3CN,
rt, 95% and (b) NH2–NH2ÆH2O (5 equiv), NEt3 (1 equiv), anhydrous
CH3CN, rt, 100%.
Regarding each glycosylation reaction, the resulting
products consisted of a mixture of two cyclic forms

hydroxylamine 6 on L-fucose resulted in a stable oxime,
E. Jestin et al. / Tetrahedron Letters 47 (2006) 6869–6873
6871
#
H
H
H
H2N N
O
N
NH
OH
OH
NH N
OH
O
O
OH
O
O
6-exo-trig
OH
OH
OH
OH
OH
+
OH
OH
I
I
I
osazone
3
11
(β majority)
R1
R1
R1
#
OH
OH
OH
OH
O
HN
O
O
N
O
N O
+
6-exo-trig
OH
OH
OH
OH
OH
OH
OH
I
I
I
oxime
12 : R1 = H
13 : R1 = CH3
14 : R1 = CH(CH3)2
6 : R1 = H
9 : R1 = CH3
10 : R1 = CH(CH3)2
(β majority)
R1
R2
#
N
R1
HO
O
R1
N
5-exo-trig
OH
OH
OH
O
R2
HN
OH
R2
OH
OH
HO
OH
OH
OH
3: R1 = H ; R2 = NH-CO-Ar
6: R1 = H ; R2 = O-CH2-Ar
9: R1 = CH3 ; R2 = O-CH2-Ar
(α−furanose minority)
10: R1 = CH(CH3)2 ; R2 = O-CH2-Ar
Scheme 4. Glycosylation of iodinated precursors.
Table 1. Yields and stereoselectivity of glycosylations on L-fucose
which may explain the equilibrium observed between the
cyclic and acyclic forms. N-Substitution of the hydroxyl-
amines seems to destabilize the oxy-iminium intermedi-
ate inducing ring closure.
Product
Precursor
Yield
(%)
% Cyclic
form
% (b-Pyranose/
a-furanose)
11
12
13
14
3
6
96
90
98
85
100
50
100
100
95/5
95/5
92.5/7.5
95/5
9
10
We have studied the feasibility of L-fucose glycosylation
prior to working with the polysaccharide structure,
fucoidan. We have demonstrated that these reactions
proceeded chemoselectively on the aldehyde without
activation of the anomeric center. Glycosylations were
obtained in good and reproducible yields giving prod-
ucts stable in a fridge for several months. Addition of
the precursors gave essentially the final cyclic forms,
with b-pyranose prevailing, with the exception of O-(3-
iodobenzyl)-hydroxylamine 6, which gave 50% of acyclic
form. We observed that addition of N-methyl and
N-isopropyl substituted O-(3-iodobenzyl)-hydroxy-
lamine induced cyclisation.
(b-pyranose and a-furanose) and also of the acyclic form
for hydroxylamine 12. These compounds were not iso-
lated but characterized by NMR spectroscopy. It is
3
noteworthy that the analysis of the JH1ꢀH2 coupling
constant values36 indicated that the reactions proceeded
with high stereoselectivity. We noticed a large excess of
b-pyranose forms, that is, the cis-position of the precur-
sor on C1 toward CH3-5.37 The presence of the a-fura-
nose form was ascertained on the basis of the typical
deshielded chemical shift values36 as well as the coupling
constants observed by NMR spectroscopy.
In conclusion, all precursors have shown good proper-
ties and could be potential candidates for polysaccharide
radiolabeling. Regarding radiolabeling applications, the
essential factor would be the resulting stability of the
radiolabeled polysaccharides in vivo. In fact, this study
was important to optimize the coupling reaction be-
tween precursors and polysaccharides prior to substitut-
ing cold iodine by radioactive iodine. We defined
conditions smooth enough to be used for polysaccharide
radiolabeling without denaturation.
Iodinated precursors gave the b-pyranose form in
majority, except for O-(3-iodobenzyl)-hydroxylamine 6
where acyclic forms were detected in equal proportions.
The b-pyranose major formation can be rationalized
(Scheme 4) by the bulky OH-2 group which induces a
preferential 6-exo-trig ring closure with OH-5 attacking
the Si oxy-iminium face. The a-furanose forms arise
from 5-exo-trig ring closure with OH-4. Addition of

6872
E. Jestin et al. / Tetrahedron Letters 47 (2006) 6869–6873
29. Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffmann,
Acknowledgements
M.; Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
12305–12310.
30. Synthesis of 3: 3-Iodobenzoic acid
´
This work was supported by the ‘Region des Pays de la
Loire’. Special thanks to Chelatec for their advices and
experimental supports.
1 (1 equiv, 2 g,
8.06 mmol) was dissolved in 5 ml of anhydrous CH3CN
under inert atmosphere. NEt3 (2 equiv, 2.25 ml,
16.12 mmol), and 2.5 equiv of TSTU (6.1 g, 20.16 mmol)
were added in the round-bottom flask at room tempera-
ture. The reaction mixture was stirred at room tempera-
ture. A brown colour appeared immediately. After 15 min,
the reaction was finished. The mixture was concentrated in
vacuo. The product was purified by column chromatog-
raphy in 95% yield (2.65 g, 7.68 mmol). Eluent: CHCl3/
References and notes
1. Percival, E.; McDowell, R. H. Chemistry and Enzymology
of Marine Algal Polysaccharides; Academic Press: Lon-
don, 1967; 157–164.
2. Chevolot, L.; Mulloy, B.; Ratiskol, J.; Foucault, A.;
Colliec-Jouault, S. Carbohydr. Res. 2001, 330, 529–535.
3. Marais, M. F.; Joseleau, J. P. Carbohydr. Res. 2001, 336,
155–159.
4. Patankar, M. S.; Oehninger, S.; Barnett, T.; Williams, R.
L.; Clark, G. F. J. Biol. Chem. 1993, 268, 21770–21776.
5. Mauray, S.; Sternberg, C.; Theveniaux, J.; Millet, J.;
AcOEt (95/5); Rf: 0.9; [M+H+]: 346; H NMR (CDCl3):
1
3
2.9 (s, 4H, 2 · CH2); 7.27 (t, 1H, H5, J = 7.9 Hz); 8 (d,
1H, H4, 3J = 7.9 Hz); 8.1 (d, 1H, H6, 3J = 7.9 Hz); 8.47 (s,
1H, H2). 13C NMR (CDCl3): 27.7 (2 · CH2); 96 (C3); 129
(C6); 131.7 (C5); 132.5 (C1); 141.2 (C2); 145.8 (C4); 162.6
(C@O); 170.8 (2 · C@O).
O-(N-Succinimidyl)-3-iodobenzoate ester 2 (1 equiv, 0.5 g,
1.45 mmol) was dissolved with 5 ml of anhydrous CH3CN
under inert atmosphere. Monohydrate hydrazine (5 equiv,
203 ll, 7.25 mmol) and 1 equiv of NEt3 (202 ll, 1.45
mmol) were dissolved in 5 ml of anhydrous CH3CN and
were added dropwise to the reaction mixture. A white
suspension appeared after few minutes. The mixture was
filtered on celite. The filtrate was concentrated. The final
product was purified by flash chromatography on silica gel
in a quantitative yield (0.38 g, 1.45 mmol). Eluent: CHCl3/
MeOH (9/1); Rf: 0.4; [M+H+]: 263; 1H NMR (DMSO-d6):
´
`
Sinquin, C.; Tapon-Bretaudiere, J.; Fisher, A. M. Thromb.
Haemost. 1995, 74, 1280–1285.
6. Chevolot, L.; Foucault, A.; Chaubet, F.; Kervarec, N.;
Sinquin, C.; Fisher, A. M.; Boisson-Vidal, C. Carbohydr.
Res. 1999, 319, 154–165.
7. Blondin, C.; Chaubet, F.; Nardella, A.; Sinquin, C.;
Jozefonvicz, J. Biomaterials 1996, 17, 597–603.
8. Baba, M.; Schols, D.; Pauwels, R.; Nakashima, H.; De
Clercq, E. J. Acquir. Immune Defic. Syndr. 1990, 3, 493–
499.
9. Angulo, Y.; Lomonto, B. Biochem. Pharmacol. 2003, 66,
1993–2000.
10. Hahnenberger, R.; Jakobson, A. M. Glycoconjugate J.
1991, 8, 350–353.
11. Roger, O.; Colliec-Jouault, S.; Ratiskol, J.; Sinquin, C.;
Guezennec, J.; Fisher, A. M.; Chevolot, L. Carbohydr.
Polym. 2002, 50, 273–278.
12. Ramsay, S. L.; Freeman, C.; Grace, P. B.; Redmond, J.
W.; MacLeod, J. K. Carbohydr. Res. 2001, 333, 59–71.
13. O’Shea, M. G.; Samuel, M. S.; Konik, C. M.; Morell, M.
K. Carbohydr. Res. 1998, 307, 1–12.
14. Rothenberg, B. E.; Hayes, B. K.; Toomre, D.; Manzi, A.
E.; Varki, A. Proc. Natl. Acad. Sci. 1993, 90, 11939–
11943.
15. Araki, Y.; Andoh, A.; Fujiyama, Y.; Hata, K.; Makino,
J.; Okuno, T.; Nakanura, F.; Bamba, T. J. Chromatogr. B
2001, 753, 209–215.
3
4.56 (s, 2H, NH2); 7.29 (t, 1H, H5, J = 7.9 Hz); 7.86 (d,
3
3
1H, H4, J = 8.3 Hz); 7.91 (d, 1H, H6, J = 7.9 Hz); 8.18
(s, 1H, H2); 9.9 (s, 1H, NH). 13C NMR (DMSO-d6): 94.7
(C3); 127.4 (C6); 131.4 (C5); 136.3 (C1); 137.3 (C2); 141.6
(C4); 167.9 (C@O).
31. Al-Jammaz, I.; Al-Otaibi, B.; Amarey, J. K. Appl. Radiat.
Isot. 2002, 57, 743–747.
32. Synthesis of 6: 3-iodobenzyl alcohol
4
(0.27 ml,
2.14 mmol) was dissolved into 30 ml of anhydrous CHCl3
under nitrogen atmosphere. PPh3 (1.12 g, 4.3 mmol) and
N-hydroxyphthalimide (0.7 g, 4.3 mmol) were added to
the reaction mixture. The reaction was stirred at room
temperature to dissolve all components. Diethylazodi-
carboxylate (0.67 ml, 4.3 mmol) was added. The reaction
mixture was stirred for 15 h at room temperature. The
solvent was evaporated under reduced pressure. The
product was purified by column chromatography in 71%
yield (0.57 g, 1.52 mmol). Eluent: CHCl3 (100); Rf: 0.6;
16. Seevers, R. H.; Counsell, R. E. Chem. Rev. 1982, 82, 575–
590.
17. Boeykens, S. P.; Vasquez, C.; Temprano, N.; Rosen, M.
Carbohydr. Polym. 2004, 55, 129–137.
18. Wilbur, D. S. Bioconjugate Chem. 1992, 3, 433–470.
19. Dahl, L. B.; Laurent, T. C.; Smedsrod, B. Anal. Biochem.
1988, 175, 397–407.
[M+H+]: 380; H NMR (CDCl3): 5.06 (s, 2H, CH2); 7.05
1
(t, 1H, H5, 3J = 7.8 Hz); 7.45 (d, 1H, H4, 3J = 7.7 Hz), 7.7
(m, 5H, H6=H2 ;3 ;4 ;5 ); 7.8 (s, 1H, H2). 13C NMR (CDCl3):
0
0
0
0
0
0
0
0
78.8 (CH2); 94 (C3); 123.6 ðC2 =C5 Þ; 128.86 ðC3 =C4 Þ;
0
0
130.3 ðC1 =C6 =C6Þ; 134.5 (C5); 135.9 (C1); 138.3 (C2/C4);
160.3 (C@O); 163.4 (C@O).
O-(3-Iodobenzyl)-N-hydroxyphthalimide 5 (0.57 g, 1.51
mmol) was dissolved in 50 ml of absolute ethanol.
Hydrazine monohydrate (10 equiv, 727 ll, 15.1 mmol)
was added. The reaction mixture was stirred and heated
at reflux for 3 h. The mixture was cooled: a white
precipitate appeared. The solid was filtered and rinsed
with ethanol. The filtrate was concentrated. The product
was obtained after purification by column chromatogra-
phy in 80% yield (0.3 g, 1.21 mmol). Eluent: CHCl3/
20. Keck, K. Immunochem. 1972, 9, 359–360.
21. Prasad A. V.; Richards J. C.; Method of producing syn-
thetic N-linked glycoconjugates. 1997, WO9702277.
22. Buckingham, J. Q. Rev. Chem. Soc. 1969, 23, 37–56.
23. Randerath, K. Anal. Biochem. 1981, 115, 391–397.
24. Flinn, N. S.; Quibell, M.; Monk, T. P.; Ramjee, M. K.;
Urch, C. J. Bioconjugate Chem. 2005, 16, 722–728.
25. Heindel, N. D.; Van Dort, M.; Cahn, M.; Schneider, R.;
Burns, H. D. J. Heterocycl. Chem. 1985, 22, 209–210.
26. Peluso, S.; Ufret, M.; O’Reilly, M. K.; Imperiali, B. Chem.
Biol. 2002, 9, 1323–1328.
1
MeOH (90/10); Rf: 0.3; [M+H+]: 250; H NMR (CDCl3):
4.61 (s, 2H, CH2); 5.43 (s, 2H, NH2); 7.09 (t, 1H, H5,
3J = 7.7 Hz); 7.31 (d, 1H, H4, 3J = 7.7 Hz), 7.64 (d, 1H,
27. Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54,
12269–12278.
3
H6, J = 8 Hz); 7.72 (s, 1H, H2). 13C NMR (CDCl3): 75.3
(CH2); 97.5(C3); 126.2 (C6); 130.3 (C5); 136.2 (C2); 136.3
(C4); 142.5 (C1).
28. Lees, A.; Sen, G.; Lopez Acosta, A. Vaccine 2006, 24, 716–
729.

E. Jestin et al. / Tetrahedron Letters 47 (2006) 6869–6873
6873
Synthesis of 9: Paraformaldehyde (54 mg, 1.8 mmol) was
dissolved in 10 ml of anhydrous CHCl3 and was heated at
30 ꢁC for 5 min for depolymerisation. O-(3-Iodobenzyl)-
hydroxylamine 6 (0.3 g, 1.2 mmol) was added. The reac-
tion mixture was heated at reflux for 1 h and then cooled.
The solvent was evaporated. Product was obtained after
purification by column chromatography in 95% yield
(0.3 g, 1.14 mmol). Eluent: CHCl3/MeOH (90/10); Rf: 0.3;
Synthesis of 11: L-fucose (12.5 mg, 0.076 mmol);
3-iodophenylhydrazide 3 (40 mg, 0.15 mmol). Eluent of
chromatography: CHCl3/MeOH (80/20). Rf = 0.4. Yield:
96% (29.9 mg, 0.073 mmol). [MꢀH+] = 407. 1H NMR
(DMSO-d6): 1.12 (d, CH3 a form, 3J = 6.4 Hz); 1.14 (d,
3
0
0
0
0
CH3 b form, J = 6.4 Hz); 3.4 (m; 8H, H2 =H3 =H4 =H5
b
0
form + H2/H3/H4/H5
a
form); 3.81 (d, 1H, H1 b,
J = 8.4 Hz); 4.39 (d, 1H, H1 a, 3J = 5.2 Hz); 5 (s, 3H,
OH); 5.9 (s, 1H, NH); 7.28 (t, 1H, H5, 3J = 8 Hz); 7.87 (d,
3
0
1
[M+H+]: 262; H NMR (CDCl3): 5.06 (s, 2H, CH2); 6.49
(d, 1H, CH2@N, 3J = 8.2 Hz); 7.1 (d, 1H, CH2@N,
3J = 7.9 Hz); 7.1 (t, 1H, H5, 3J = 7.6 Hz); 7.33 (d, 1H,
1H, H4, J = 7.6 Hz); 7.91 (d, 1H, H6, J = 8 Hz); 8.2 (s,
3
3
1H, H2); 10.2 (s, 1H, NH). 13C NMR (DMSO-d6): 18.6
3
3
0
0
0
H4, J = 7.6 Hz), 7.65 (d, 1H, H6, J = 7.6 Hz ); 7.73 (s,
1H, H2). 13C NMR (CDCl3): 75.6 (CH2); 93 (C3); 128.4
(C6); 130 (C5); 135.4 (C2); 135.9 (C4); 136.4 (CH2@N); 141
(C1). Oxime 7 (0.3 g, 1.14 mmol) was dissolved in 5 ml of
absolute ethanol. Sodium cyanoborohydride (0.2 g,
3.4 mmol) was added. The pH was adjusted to 3 with
HCl 12 N. The reaction was stirred for 30 min at room
temperature under nitrogen atmosphere. The solvent was
evaporated. A NaOH aqueous solution was used to obtain
a pH 8. The aqueous layer was extracted with CH2Cl2.
The organic layer was dried over Na2SO4, filtered and
evaporated. Product 9 was obtained quantitatively (0.3 g,
1.14 mmol). [M+H+]: 264; 1H NMR (CDCl3): 2.73 (s, 3H,
CH3); 4.64 (s, 2H, CH2); 5.12 (s, 1H, NH); 7.08 (t, 1H, H5,
3J = 7.6 Hz); 7.32 (d, 1H, H4, 3J = 7.3 Hz), 7.63 (d, 1H,
(CH3); 55.9 ð C5 ðaÞÞ; 71.2 ðC2 Þ;71.9 ðC3 ðaÞÞ; 72.2
0
0
0
0
ðC5 ðbÞ þ C3 ðbÞÞ; 73.6 ðCH2 þ C4 Þ; 87 ðC1 ðaÞÞ; 91
0
ð C1 ðbÞÞ; 95.2 (C3); 126.7 (C6); 130.5 (C5); 134.9 (C2);
135.5 (C4); 139.7 (C1); 163.9 (C@O).
Synthesis of 12: L-Fucose (9.8 mg, 0.06 mmol); O-(3-
iodobenzyl)-hydroxylamine 6 (30 mg, 0.12 mmol). Eluent
of chromatography: CHCl3/MeOH(90/10). Rf = 0.2.
Yield: 90% (21.4 mg, 0.054 mmol). [MꢀH+] = 394. 1H
NMR (DMSO-d6): 1.05 (d, 3H, CH3); 3.1–3.6 (m,
0
0
0
0
0
8H, H2 =H3 cyclics þ H3 =H4 =H5 acyclics þ 3OH); 3.83
3
0
0
(quint, 1H, H5 cyclic, J = 6.4 Hz); 4.1 (d, 1H, H4 cyclic,
3
3
0
J = 6.4 Hz); 4.3 (d, 1H, H1 b, J = 7.63 Hz); 4.55 (d, 1H,
H1 a, 3J = 5.17 Hz); 4.6 (d, 1H, H2 acyclic, 3J = 7 Hz);
0
0
4.96 (s, 2H, CH2); 7.17 (t, 1H, H5, 3J = 7.6 Hz); 7.35
(d, 1H, H4, 3J = 7.6 Hz); 7.49 (d, 1H, H1 acyclic,
0
3
H6, J = 8 Hz); 7.73 (s, 1H, H2). 13C NMR (CDCl3): 33.9
3J = 7.3 Hz); 7.65 (d, 1H, H6, 3J = 7.6 Hz); 7.69 (s, 1H,
H2). 13C NMR (DMSO-d6): 20 (CH3); 64.9 ðC5 Þ; 68 ðC2 Þ;
0
0
(CH3); 73.3 (CH2); 94.5 (C3); 127.2 (C6); 130.3 (C5); 135.9
(C2); 136.3 (C4); 141.5 (C1).
0
0
0
72.2/72.7 ðC3 =CH2Þ; 73.6 ðC1 cyclic=C4 Þ; 94.7 (C3); 127.3
Synthesis of 10: O-(3-Iodobenzyl)-hydroxylamine 6 (0.1 g,
0.4 mmol) was dissolved in acetone. The mixture was
heated at reflux for 1 h and was then cooled. The solvent
was evaporated. The product was purified by column
chromatography in 90% yield (104 mg, 0.36 mmol). Elu-
ent: CHCl3/MeOH (90/10); Rf: 0.3; [M+H+]: 290; 1H
NMR (CDCl3): 1.88 (s, 3H, CH3); 1.9 (s, 3H, CH3); 5 (s,
(C6); 130.5 (C5); 136.3 (C2/C4); 140.6 (C1); 153.4
0
ðC1 acyclicÞ.
Synthesis of 13: L-Fucose (9.9 mg, 0.06 mmol); N-methyl-
O-(3-iodobenzyl)-hydroxylamine 9 (32 mg, 0.12 mmol).
Eluent of chromatography: CHCl3/MeOH(90/10).
Rf = 0.3. Yield: 98% (24.4 mg, 0.059 mmol). [MꢀH+] =
408. 1H NMR ((CD3)2CO): 1.17 (d, CH3 a form,
3J = 6.4 Hz); 1.18 (d, CH3 b form, 3J = 6.4 Hz); 2.6 (s,
3
2H, CH2); 7.08 (t, 1H, Hc, J = 8 Hz); 7.32 (d, 1H, Hb,
3J = 7.6 Hz), 7.62 (d, 1H, Hd, 3J = 7.3 Hz); 7.7 (s, 1H,
Ha). 13C NMR (CDCl3): 15.8 (CH3); 21.9 (CH3); 74.19
(CH2); 94.3 (C3); 126.9 (C6); 130 (C5); 136.7 (C2/C4); 140.8
(C1); 155.7 (N@CH2).
3H, CH3–N); 3.6 (m, 8H, H2 =H3 =H4 = H5 b formþ
0
0
0
0
H2 = H30=H4 =H5 a form); 3.97 (d, 1H, H1 ðbÞ, 3J =
0
0
0
0
3
0
8.9 Hz); 4.38 (d, H1 ðaÞ, J = 5.2 Hz); 4.68 (s, 2H, CH2);
7.13 (t, 1H, H5, 3J = 7.9 Hz); 7.41 (d, 1H, H4,
3J = 7.6 Hz); 7.63 (d, 1H, H6,3J = 7.9 Hz); 7.74 (s, 1H,
H2). 13C NMR ((CD3)2CO): 16.9 (CH3); 39 (CH3–N); 68.9
Oxime 8 (104 mg, 0.36 mmol) was dissolved in 5 ml of
absolute ethanol. Sodium cyanoborohydride (67.8 mg,
1.08 mmol) was added. The pH was adjusted to 3 with
12 N HCl. The reaction was stirred for 30 min at room
temperature under nitrogen atmosphere. The solvent was
evaporated. A 6 N NaOH aqueous solution was used to
obtain a pH 8. The aqueous layer was extracted with
CH2Cl2. The organic layer was dried over Na2SO4, filtered
and evaporated. Product 10 was obtained quantitatively
0
0
0
0
ðC5 ðaÞÞ; 72.5 ðC2 Þ; 72.9 ðC5 ðbÞÞ; 74.7 ðC3 ðaÞÞ; 74.9
0
0
0
0
ðC3 ðbÞÞ; 76 (CH2); 88.2 ðC4 Þ; 94.4 ð C1 ðaÞÞ; 94.5 ðC1 ðbÞÞ;
101.3 (C3); 129 (C6); 131.1 (C5); 137.5 (C2); 138.5 (C4);
141.7 (C1).
Synthesis of 14: L-Fucose (8.5 mg, 0.052 mmol);
N-isopropyl-O-(3-iodobenzyl)-hydroxylamine 10 (30 mg,
0.1 mmol). Eluent of chromatography: CHCl3/MeOH (90/
10). Rf = 0.4. Yield: 85% (19.2 mg, 0.044 mmol).
[MꢀH+] = 436. 1H NMR (CD3OD): 1.05 (d, 6H, CH3–
1
(104.7 mg, 0.36 mmol). [M+H+]: 292; H NMR (CDCl3):
1.07 (d, 6H, 2 · CH3, 3J = 6.1 Hz); 3.17 (m, 1H, CH,
3J = 6.4 Hz); 4.65 (s, 2H, CH2); 5.37 (s, 1H, NH); 7.08 (t,
CH, J = 6.1 Hz); 1.18 (d, CH3 a form, 3J = 6.4 Hz); 1.21
3
3
3
3
1H, H5, J = 7.6 Hz); 7.32 (d, 1H, H4, J = 7.6 Hz), 7.63
(d, 1H, H6, 3J = 7.9 Hz); 7.72 (s, 1H, H2). 13C NMR
(CDCl3): 21.7 (2 · CH3); 53.3 (CH); 76.9 (CH2); 95.9 (C3);
128.9 (C6); 131.6 (C5); 138.3 (C2); 138.7 (C4); 142.1 (C1).
33. Mitsunobu, O. Synthesis 1981, 1–28.
(d, CH3 b form, J = 6.4 Hz); 2.97 (m, 1H, CH); 3.5 (m,
0
0
0
0
0
0
0
0
8H, H2 =H3 =H4 =H5 b form þ H2 =H3 =H4 =H5 a form);
4.3 (d, 1H, H1 (b), J = 8.9 Hz); 4.5 (d, H1 ðaÞ, 3J =
4.8 Hz); 4.7 (s, 2H, CH2); 6.96 (t, 1H, H5, 3J = 7.6 Hz);
7.18 (d, 1H, H4, 3J = 7.7 Hz); 7.7 (d, 1H, H6, 3J = 7.8 Hz);
7.8 (s, 1H, H2). 13C NMR (CD3OD): 17.6 (CH3); 19
3
0
0
34. Baalam, B.; Hamon, A.; Maudet, M. Tetrahedron Lett.
1998, 39, 7865–7868.
0
0
0
(2 · CH3); 47 (CH); 64.1 ðC5 ðaÞÞ; 66.4 ðC2 Þ; 67.1 ðC5 ðbÞÞ;
0
0
0
35. General method for the coupling to L-fucose: L-Fucose
(1 equiv) and 2 equiv of aryliodide precursors were
dissolved in a mixture of solvent MeOH/CH3COOH
glacial (85/15). The reaction mixture was stirred for 20 h
at room temperature. The solvent was evaporated. The
product was purified by chromatography.
71.1 ðC3 ðaÞÞ; 74.1 ðC3 ðbÞÞ; 75 ðC4 Þ; 75.1 (CH2); 82.1
0
ðC1 Þ; 97.5 (C3); 126.2 (C6); 130.3 (C5); 135.9 (C2); 136
(C4); 143 (C1).
36. Kett, W. C.; Batley, M.; Redmond, J. W. Carbohydr. Res.
1997, 299, 129–141.
37. IUPAC-IUBMB Carbohydr. Res. 1997, 297, 1–92.