Fmoc-protected iminosugar modified asparagine derivatives as building blocks for glycomimetics-containing peptides

Article abstract of DOI:10.1016/j.bmc.2007.04.007

CSF114(Glc) is the first synthetic Multiple Sclerosis Antigenic Probe able to identify autoantibodies in a statistically significant number of Multiple Sclerosis patients. The β-turn conformation of this glucopeptide is fundamental for a correct presentat

Full text of DOI:10.1016/j.bmc.2007.04.007

Bioorganic & Medicinal Chemistry 15 (2007) 3965–3973
Fmoc-protected iminosugar modified asparagine derivatives
as building blocks for glycomimetics-containing peptides
a,b
a,b
b,c
a,b
Francesca Nuti, Ilaria Paolini, Francesca Cardona, Mario Chelli,
a,d
b,c
b,c
Francesco Lolli, Alberto Brandi, Andrea Goti,
a,e
a,b,
*
Paolo Rovero and Anna M. Papini
a
Laboratory of Peptide & Protein Chemistry & Biology, Polo Scientifico e Tecnologico, University of Florence,
I-50019 Sesto Fiorentino (FI), Italy
b
Department of Organic Chemistry ‘‘Ugo Schiff’’, Polo Scientifico e Tecnologico, University of Florence and CNR-ICCOM,
Via della Lastruccia 13, I-50019 Sesto Fiorentino (FI), Italy
c
Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente attivi, Polo Scientifico e Tecnologico,
University of Florence, I-50019 Sesto Fiorentino (FI), Italy
d
Department of Neurological Sciences, University of Florence, Viale Morgagni 85, I-50134 Firenze, Italy
Department of Pharmaceutical Sciences, Polo Scientifico e Tecnologico, University of Florence, Via Ugo Schiff 6,
I-50019 Sesto Fiorentino (FI), Italy
e
Received 15 November 2006; revised 30 March 2007; accepted 5 April 2007
Available online 10 April 2007
Abstract—CSF114(Glc) is the first synthetic Multiple Sclerosis Antigenic Probe able to identify autoantibodies in a statistically sig-
nificant number of Multiple Sclerosis patients. The b-turn conformation of this glucopeptide is fundamental for a correct presen-
tation of the epitope Asn(Glc). To verify the influence of sugar mimics in antibody recognition in Multiple Sclerosis, we synthesized
Fmoc-protected Asn derivatives containing alkaloid-type sugar mimics. The corresponding glycomimetics-containing peptide deriv-
atives of the CSF114-type sequence were tested in competitive and solid-phase non-competitive ELISA on Multiple Sclerosis
patients’ sera.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
Multiple Sclerosis (MS) using different glycomimetic
derivatives of CSF114 peptide sequence. CSF114(Glc)
is a structure-based designed glucosylated peptide, char-
acterized by a b-turn, able to identify autoantibodies
in a statistically significant number of MS patients com-
The importance of carbohydrate recognition in biologi-
cal events is well established on many experimental find-
ings. How post-translational protein modifications, in
particular glycosylation, can have a role in the origin
of autoimmune responses is still not characterized but
almost all of the key molecules involved in innate and
adaptive immune responses are glycoproteins. More-
over, in the last years, a number of autoimmune diseases
2
3
pared to healthy blood donors and other autoimmune
4
diseases. We demonstrated that the presence of a b-D-
glucopyranosyl moiety on an Asn residue at position 7
of CSF114(Glc) is fundamental for auto-Ab recogni-
tion. In fact, no Abs could be identified by the corre-
sponding unglycosylated peptide sequence. Moreover,
the specific autoantibody recognition is most likely dri-
ven by direct interactions of the antibody binding site
with the Asn-linked sugar moiety and not with the
CSF114 peptide sequence. These data let us to assess
that in MS, autoantibody recognition is strictly corre-
1
have been associated with glycosylation defects.
Our interest was to further investigate the role of glyco-
syl moiety in autoantibody (auto-Ab) recognition in
3
lated with specific glycosylated epitopes.
Keywords: Iminosugars; Glycopeptides; Multiple Sclerosis; Solid-phase
peptide synthesis.
*
Corresponding author. Tel.: +39 055 4573561; fax: +39 055
573584; e-mail: annamaria.papini@unifi.it
To extend auto-Ab recognition to glycosylated epitopes
in MS and to verify the influence of sugar mimics, we
4
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.007

3966
F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
synthesized glycomimetics-containing peptide deriva-
tives of the CSF114-type sequence containing alkaloid-
type sugar mimics having pyrrolidine and piperidine
OH
AcO
N
HO
OH
AcO
i, ii
OAc
HO
or
OH
AcO
OAc
5
OH
or
OAc
structures, well known as glycosidase inhibitors.
N
H
N
N
H
Fmoc
2a
Fmoc
The activity of polyhydroxylated alkaloids found in
plants and microorganisms is related to their ability of
mimicking the pyranosyl or furanosyl structures of
monosaccharides. These sugar mimics, in which the oxy-
gen ring has been replaced by a nitrogen, are one of the
most interesting discoveries in the field of natural prod-
ucts in recent years. Naturally occurring sugar mimics
containing nitrogen are classified into several structural
classes: polyhydroxylated piperidines and pyrrolidines,
and polyhydroxylated alkaloids containing bicyclic skel-
etons that can be divided into fused compounds, such as
pyrrolizidines and indolizidines (bearing a bridgehead
nitrogen atom) and bridged bicyclic compounds such
as nortropanes (possessing a secondary amine group).
In these bicyclic alkaloids, the configuration of the stere-
ogenic carbons bearing the hydroxyl groups relates to
1
a
1b (DNJ)
2b
AcO
AcO
OAc
AcO
OAc
OAc
iii
or
N
N
H
H
3
a (R-H)
3b (R'-H)
R or R'
R or R'
iv
O
v
O
O
O
NHFmoc
NHFmoc
OtBu
OH
5a or 5b
4
a or 4b
6
that of the corresponding carbohydrates.
Scheme 1. Reagents and conditions: (i) Fmoc-OSu, dry Py, N
Ac O, Py, N
iv) Fmoc-L-Asp-Ot-Bu, HATU, NMM, DMF (4a 37%; 4b 89%); (v)
TFA/DCM (1:1) (5a 98%; 5b 90%).
2
; (ii)
2
2
(2a 70%; 2b 84%); (iii) Pip 20%, THF (3a 64%; 3b 80%);
(
Iminosugars can be regarded as potential therapeutic
agents and as tools for understanding biological recog-
nition processes, because of the formation of specific
7
,8
bonds to the active sites of glycosidases. Since the
mode of action of glycosidases involves the cleavage of
glycosidic bonds between sugar molecules, individual
glycosidases show specificity for certain sugar molecules
and for a specific anomeric configuration of the
2. Chemistry
We undertook the synthesis of new asparagine Fmoc-
protected building blocks bearing orthogonally pro-
tected polyhydroxylated iminosugars on the side chain:
9
,10
sugar.
(
S)-a-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-c-
These enzymes are involved in the biosynthesis of the
oligosaccharide portions of glycoproteins and glycolip-
ids, which play a crucial role in mammalian cellular
structures and functions. For instance, the oligosaccha-
ride chains regulate the correct functioning of glycopro-
teins by stabilizing them and ensuring their correct
conformation. In particular, 1-deoxynojirimycin [(2S-
hydroxymethyl)-3R,4R,5S-piperidinetriol or 1,5-dide-
oxy-1,5-imino-D-glucitol, DNJ] has demonstrated
interesting anti-diabetic, anti-cancer, and anti-HIV
properties, and showed to possess potent inhibitory
oxo-[3S,4S-bis(acetyloxy)-1-pyrrolidine]butanoic acid
[Fmoc-L-Asn(DHPyrAc2)-OH, 5a], (S)-a-[[(9H-fluoren-
9-ylmethoxy)carbonyl]amino]-c-oxo-[2R-[(acetyloxy)
methyl]-3R,4R,5S-tris(acetyloxy)-1-piperidine]butanoic
acid [Fmoc-L-Asn(DNJAc4)-OH, 5b], (S)-a-[[(9H-fluo-
ren-9-ylmethoxy)carbonyl]amino]-c-oxo-[2R-[2-deoxy-
1,3,4,6-tetra-O-acetyl-2R-D-glucopyranosyl]-3R,4R-bis
(1,1-dimethylethoxy)-1-pyrrolidine]butanoic acid 1-(pen-
tafluorophenyl) ester [Fmoc-L-Asn(DHPyrt-Bu2-2-deoxy
GlcAc4)-OPfp, 9a], and (S)-a-[[(9H-fluoren-9-ylmeth-
oxy)carbonyl]amino]-c-oxo-[2S-[2-deoxy-1,3,4-tri-O-
acetyl-2S-L-rhamnopyranosyl]-3S,4S-bis(1,1-dimethyl-
ethoxy)-1-pyrrolidine]butanoic acid 1-(pentafluorophe-
nyl) ester [Fmoc-L-Asn(DHPyrt-Bu2-2-deoxyRhaAc3)-
OPfp, 9b] (Fig. 1).
1
1–13
activity of glycosidase enzymes.
The iminosugar
N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor
of a-glucosidase I, a cellular enzyme removing terminal
glucose residues from nascent oligosaccharide.
1
4,15
In addition to their ability to inhibit processing of exo-
glycosidases, lysosomal glycosidases, and the intestinal
disaccharidases involved in carbohydrate digestion, imi-
nosugars appear to have additional activities, including
immunomodulatory properties and inhibition of glyco-
lipid synthesis, which continue to expand their range
Since the iminosugars employed are not commercially
available, it was necessary to produce polyhydroxylated
nitrogen heterocycles, that is, 3,4-dihydroxypyrrolidine
(DHPyr, 1a), deoxynojirimycin (DNJ, 1b), 2-deoxy-
2-[(2R,3R,4R)-3,4-dimethylethoxy-2-pyrrolidinyl]-3,4,6-
tri-O-acetyl-D-glucopyranose (6a), and 2-deoxy-2-
[(2S,3S,4S)-3,4-dimethylethoxy-2-pyrrolidinyl]-3,4-di-
1
7
18
1
6
of potential uses.
1
9,20
O-acetyl-L-rhamnopyranose (6b).
(3S,4S)-1-benzylpyrrolidine over Pd(OH) /C gave 3,4-
Hydrogenolysis of
We were especially interested in preparing building
blocks containing N-linked iminosugars and providing
a general high yielding method to covalently bind them
to the Asp side chain. The building blocks were pro-
tected for solid-phase peptide synthesis (SPPS), follow-
ing the Fmoc/t-Bu strategy (Scheme 1).
2
2
1
dihydroxypyrrolidine 1a. Deoxynojirimycin 1b, an
iminosugar with the same number of hydroxyl functions
and configuration of glucose, was prepared following
the method of Matos et al. monitoring the deprotec-
tion of the hydroxyl functions by electrospray ionization
1
8

F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
3967
OH
O
OH
N
OH
HO
OH
H
HO
HO
OH
HO
HO
HO
OH
OH
HO
HO
OH
O
CH3
OH
N
H
OH
N
N
O
NH
HO
H
OH
OH
O
O
O
O
O
O
O
O
O
O
N
N
H
N
H
N
N
H
H
H
Figure 1. N-Linked derivatives of Asn: DHPyr, DNJ, DHPyr-2-deoxyGlc, DHPyr-2-deoxyRha, and Glc.
mass spectrometry (ESI-MS). Two pseudoimino-C-
OAc
N
AcO
OAc
disaccharides containing a dihydroxypyrrolidine linked
to deoxyglucose 6a or deoxyrhamnose 6b were obtained
by an intermolecular 1,3-dipolar cycloaddition between
an enantiopure pyrroline N-oxide and the appropriate
AcO
OAc
OAc
N
1
9,20
O
1
,2-glycal that produces a tricyclic isoxazolidine.
O
O
O
NHFmoc
NHFmoc
The target molecules were obtained by isoxazolidine
ring-opening and sequential steps of protection, Fmoc-
deprotection, and coupling with Fmoc-L-Asp-OH
protected as tert-butyl or pentafluorophenyl ester. For
temporary secondary amine protection, the iminosugars
OH
OH
5
a
5b
Ot
Bu
OtBu
t
BuO
t
BuO
AcO
H
OAc
1
a and b and 6a and b were treated with N-(9H-fluore-
OAc
AcO
AcO
nylmethoxycarbonyloxy)succinimide (Fmoc-OSu) in
pyridine. Then, O-acetylation of hydroxyl functions
was achieved in situ by addition of acetic anhydride,
O
CH3
N
H
OAc
N
O
OAc
H
O
O
O
O
2
2
as reported by Meldal and Bock. After purification
by FCC, the fully protected iminosugars 2a and b and
NHFmoc
NHFmoc
7
a and b were obtained in good yields. Treatment of
the Fmoc-protected iminosugars with a solution of
0% piperidine in THF gave the corresponding Fmoc-
OPfp
OPfp
9
a
9b
2
Figure 2. Building blocks 5a and b and 9a and b.
deprotected compounds 3a and b and 8a and b.
Coupling between the amino acid and the iminosugar
moieties was performed using in situ coupling reagents,
or pre-activation of the a-carboxyl group. In the present
work, both methods were employed in good yield.
Fmoc-L-Asp-Ot-Bu was coupled with monocyclic imi-
nosugars 3a and b using HATU as coupling reagent
and NMM in DMF to obtain 4a and b, while Fmoc-
L-Asp(Cl)-OPfp was coupled with bicyclic iminosugars
OtBu
OtBu
H
t
BuO
t
BuO
HO
OH
OH
HO
O
CH3
or
N
H
O
N
H
H
OH
HO
OH
6
a
8
dry THF and NMM. Fmoc-L-Asp(Cl)-OPfp was ob-
a and b to obtain 9a and b (Fig. 2 and Scheme 2) using
6b
i, ii
2
3
tained as described by Meldal et al. After deprotection
of a-carboxyl group of asparagine derivatives 4a and b
with TFA, compounds 5a and b were obtained (Fig. 2).
OtBu
H
t
BuO
OtBu
AcO
OAc
CH3
t
BuO
AcO
OAc
O
OAc
N
O
or
AcO
Fmoc
N
H
OAc
The strategy involving pre-activated Fmoc-Asp(Cl)-
OPfp was chosen to not interfere with hydroxyl protec-
tion of the imino-C-disaccharides. The building
blocks 5a and b and 9a and b, containing the sugar mim-
ics, have been introduced in the CSF114 sequence,
Fmoc
7a (Fmoc-R'')
7b (Fmoc-R''')
iii
R'' or R'''
7
obtaining glycopeptides [Asn (DHPyr)]CSF114 (10),
[
Glc)]CSF114 (12).
7
Asn (DNJ)]CSF114 (11), and [Asn (DHPyr-2-deoxy-
7
O
O
iv
R''-H or R'''-H
NHFmoc
8
a
8b
OPfp
a or 9b
The synthesis of the peptide containing the amino sugar
9
9
b was unsuccessful possibly because of problems re-
lated to the steric hindrance of the rhamnose-containing
imino-C-disaccharide. All the glycopeptides were
synthesized following the Fmoc/t-Bu strategy and the
Scheme 2. Reagents and conditions: (i) Fmoc-OSu, dry Py, N
2
; (ii)
Ac O, Py, N (7a 52%; 7b 45%); (iii) Pip 20%, THF (8a 68%; 8b 73%);
2 2
(iv) Fmoc-L-Asp(Cl)-OPfp, NMM, dry THF (9a 41%; 9b 44%).

3968
F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
Table 1. Chemical data for the synthesized CSF114-type glycopeptides 10–12
ꢀ1
2+
a
Compound
Peptide
Gradient at 3 mL min for
semi-preparative HPLC
ESI-MS [M+2H]
Found (Calcd)
:
HPLC
(t , min)
R
7
[Asn (DHPyr)]CSF114
10
11
12
25–50% B in 30 min
25–40% B in 30 min
20–60% B in 30 min
1265.9 (2529.3)
1296.1 (2590.3)
1338.2 (2676.4)
13.67
10.23
10.25
7
[Asn (DNJ)]CSF114
7
[Asn (DHPyr-2-deoxyGlc)]CSF114
a
ꢀ1
Analytical HPLC gradient at 1 mL min : 20–60% B in 15 min.
3. Immunoassays of CSF114-type glycopeptides 10–12
The autoantibody titer in MS patients’ sera by CSF114-
type glycopeptides was evaluated by competitive and
3
solid-phase non-competitive ELISA.
The inhibition curves (Fig. 3) showed that the glycopep-
tides 11 and 12 display inhibitory activity only at higher
concentration, while the glycopeptide 10 showed no
activity at all. CSF114(Glc) is the glycopeptide with
the lowest IC50 value. None of the CSF114-type glycom-
imetics-containing peptides was able to inhibit anti-
CSF114(Glc) autoantibodies in MS patients.
In solid-phase non-competitive ELISA (Fig. 4), only
CSF114(Glc) detected increased IgG antibodies in MS
patients’ sera compared to healthy blood donors.
Figure 3. Inhibition test of antibodies binding to CSF114(Glc) with
the glycopeptides 10–12. The results are expressed as % of a
representative MS positive serum (ordinates axis). The concentrations
of the peptides are plotted on the x-axis.
In conclusion, we have described an efficient method to
synthesize new asparagine derivatives orthogonally pro-
tected for Fmoc/t-Bu SPPS, bearing alkaloid-type sugar
mimics containing pyrrolidine and piperidine structures.
The building blocks were successfully introduced in the
standard synthetic protocol described in the general pro-
cedure. The glycopeptides 10–12 were synthesized by
introducing Fmoc-L-Asn(DHPyrAc2)-OH (5a), Fmoc-
L-Asn(DNJAc4)-OH (5b), and Fmoc-L-Asn(DHPyrt-
Bu2-2-deoxyGlcAc4)-OPfp (9a) during the SPPS at po-
sition 7, as described in the general procedure. Peptide
cleavage from the resin and deprotection of the amino
acid side chains were carried out as described in the gen-
eral procedure. After lyophilization, deprotection of the
hydroxyl functions of the sugar linked to the peptide
was accomplished by a methanolic solution of MeONa.
0
type I b-turn structure CSF114. The CSF114-type gly-
copeptides were tested in MS patients’ sera both by
competitive and solid-phase non-competitive ELISA.
Biological data obtained with the new alkaloid-type su-
gar mimics containing peptides supported by our previ-
3
ous results confirmed that Asn(Glc) is up to now the
unique minimal and fundamental epitope recognizing
auto-Abs in a relapsing-remitting form of MS.
The crude products were purified and analyzed by RP-
HPLC. Characterization of the products was performed
with ThermoFinnigan LCQ Advantage LC-ESI-MS
4. Experimental
4.1. General
(
Table 1).
THF was distilled over sodium/benzophenone and
DCM over CaH . Flash column chromatographies
2
2
4
(
FCC) were performed according to Still et al. on
SiO (Merck, Silica Gel 60, 40–63 lm). Thin layer chro-
2
matographies (TLC) were carried out on SiO (Merck,
Silica Gel 60 F plastic plates) and spots located with:
UV light (254 and 366 nm), methanolic ninhydrin, Flu-
2
ꢁ
ram
2(3H),1 (3 H)-isobenzofuran]-3,3 -dione) in acetone,
and ethanolic p-anisaldehyde (EtOH/p-anisaldehyde/
(Fluka; fluorescamine, 4-phenyl-spyro[furan-
0 0 0
AcOH/H SO , 90:2:1:3). Elemental analyses were per-
2
4
formed on a Perkin-Elmer 240 C Elemental Analyzer.
1
13
H and C NMR spectra were recorded at 200 and
0 MHz, respectively, on a Varian spectrometer. Glyco-
5
Figure 4. Abs titers of MS patients’ sera and of blood donors’ sera to
CSF114(Glc) and to the glycopeptides 10–12.
peptides were analyzed by analytical RP-HPLC (Waters
Alliance, 2695 separation module equipped with a 2996

F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
3969
diode array detector) using a Jupiter C18 (5 lm,
2H, Pyr 2-H and 5-H), 3.53 (pdd, J = 12.4 Hz, 2H, Pyr
0
ꢀ
1
0
13
2
50 · 4.6 mm) column (Phenomenex) at 1 mL min
.
The solvent system used was A (0.1% TFA in H O)
2 -H and 5 -H), 2.08 (s, 3H, Ac), 2.07 (s, 3H, Ac).
C
NMR (CDCl ) d 169.5 (COCH ), 169.0 (COCH ),
2
3
3
3
and B (0.1% TFA in CH CN). Glycopeptides were puri-
3
154.6 (CONH), 143.7, 141.2, 127.7, 127.0, 125.0 and
120.0 (Fmoc Carom), 74.9 (Pyr CH), 74.0 (Pyr CH),
67.4 (Fmoc CH ), 50.2 (Pyr CH ), 49.9 (Pyr CH ),
fied by preparative RP-HPLC (model 600, Waters) on a
Jupiter C18 column (10 lm, 250 · 10 mm) at
2
2
2
ꢀ
1
mL min using the same solvent systems reported
4
47.2 (Fmoc CH), 20.8 (COCH ). ESI-MS (m/z)
3
+
[M+H] : found 410.2. Anal. Calcd for C H NO : C,
above. Characterization of the products was performed
with the LCQ Advantage liquid chromatography elec-
trospray ionization mass spectrometer (ThermoFinni-
gan). Glycopeptides were lyophilized with an Edwards
Modulyo apparatus.
2
3
23
6
67.47; H, 5.66; N, 3.42. Found: C, 67.58; H, 5.78; N,
3.12.
4.2.2. N-Fmoc-2R-[(acetyloxy)methyl]-3R,4R,5S-piperid-
inetriol triacetate (2b). 1-Deoxynojirimycin (1b) (270 mg,
1.65 mmol) yielded 2b as a white solid (766 mg, 84%). Rf
4
1
.1.1. 3S,4S-Pyrrolidinediol (1a). To a solution of 3S,4S-
-benzylpyrrolidinediol (1 g, 5.2 mmol) in MeOH
1
[AcOEt/hexane, 1:1; UV, vanilline] = 0.4. H NMR
(CDCl ) d 7.74 (d, J = 7 Hz, 2H, Fmoc 4-H and 5-H),
(
15 mL) was added Pd(OH) /C (1 mmol). The mixture
2
3
was stirred for 2 days at room temperature under H2
1 atm), filtered through Celite, washed with MeOH,
and evaporated to dryness. The product 1a was dis-
7.54 (d, J = 7 Hz, 2H, Fmoc 1-H and 8-H), 7.36–7.27
(m, 4H, Fmoc 2-H, 7-H, 3-H, and 6-H), 4.93 (pt, 1H,
DNJ 4-H), 4.84–4.79 (m, 2H, Fmoc CH –O), 4.43–
2
(
solved in water and lyophilized (498 mg, 93%). Rf
NMR
4.33 (m, 6H, DNJ CH OAc, 2-H, 3-H, 5-H and Fmoc
2
1
[
DCM/MeOH, 10:1; ninhydrin] = 0.1.
H
9-H), 3.55–3.50 (m, 2H, DNJ 6-H), 2.11, 2.09, 2.07
1
3
(
DMSO-d ) d 3.76 (pdd, 2H, 3-H and 4-H), 3.4 (br s,
and 2.05 (4 s, 12H, 4· Ac). C NMR (CDCl ) d
6
3
2
H), 2.47 (dd, J = 4.2, 11.4 Hz, 2H, 2-H and 5-H).
H, 2· OH), 2.9 (dd, J = 4.4, 11.6 Hz, 2H, 2-H, and 5-
165.5–169.6 (COCH , CONH), 155.7 (urethane CO),
3
1
3
C
143.7, 141.2, 127.7, 127.0, 125.0 and 120.0 (Fmoc
Carom), 67.2–67.6 (C-3, C-5 and C-4), 67.1 (Fmoc
CH –O), 60.1 (2-CH –OAc), 53.2 (C-2), 47.0 (C-6),
NMR (DMSO-d ) d 77.3 (CHOH), 52.9 (CH ). ESI-
6
2
+
MS (m/z) [M+H] : found 104.1. Anal. Calcd for
2
2
+
C H NO : C, 46.59; H, 8.80; N, 13.58. Found: C,
4
39.6 (Fmoc C-9), 17.4 (CH ). ESI-MS (m/z) [M+H] :
3
9
2
4
6.78; H, 8.78; N, 13.42.
found 554.2. Anal. Calcd for C H NO : C, 62.92;
31
2
9
10
H, 5.64; N, 2.53. Found: C, 62.84; H, 5.56; N, 2.73.
4.1.2. 2R-(Hydroxymethyl)-3R,4R,5S-piperidinetriol (1b).
To a solution of 2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-
4.2.3. N-Fmoc-2-deoxy-2R-[3R,4R-bis(1,1-dimethyleth-
oxy)-2R-pyrrolidinyl]-1,3,4,6-tetra-O-acetyl-D-glucopyra-
nose (7a). Compound 6a (930 mg, 2.46 mmol) yielded 7a
D-glucitol (1 g, 1.73 mmol) in MeOH (10 mL) was added
Pd(OH) /C (0.35 mmol). The mixture was stirred at
2
room temperature under H (1 atm) for 3 weeks. The
2
as a solid (976 mg, 52%). R [AcOEt/hexane, 1:3; UV, p-
f
1
deprotection reaction was controlled by ESI-MS until
the main signal was [M+H] = 164.9. Then the mixture
anisaldehyde] = 0.32. H NMR (CDCl ) d 7.74 (d,
3
+
J = 7 Hz, 2H, Fmoc 4-H and 5-H), 7.54 (d, J = 7 Hz,
2H, Fmoc 1-H and 8-H), 7.36–7.27 (m, 4H, Fmoc 2-
was filtered through Celite, washed with MeOH, and
the solvent evaporated to dryness. The product 1b was
recrystallized from EtOH/H O giving a pale yellow solid
2
0
H, 7-H, 3-H, and 6-H), 5.69–5.63 (m, 2H, 5 -H ),
2
0
0
4.99–4.94 (m, 1H, 1-H ), 4.38–3.82 (m, 11H, 3 -H, 4 -
a
0
H, and 2 -H, deoxyGlc 3-H, 4-H, 5-H and 6-H , Fmoc
(
literature.
245 mg, 86%). Spectra are in accordance with the
1
2
8
CH O and 9-H), 3.13–3.09 (m, 1H, deoxyGlc 2-H),
2
2
.06, 2.05, 2.03 and 2.00 (4 s, 12H, 4· Ac), 1.26–1.12
13
4
.2. Fmoc-protection of amino group: general procedure
(m, 18H, 2· t-Bu). C NMR (CDCl ) d 170.6–169.1
3
(
125.0, and 120.0 (Fmoc Carom), 92.3 (C-1), 80.7 (C-4),
COCH ), 156.0 (CONH), 143.7, 141.2, 127.7, 127.0,
3
Fmoc-OSu (1.1 equiv) was added to the various imi-
nosugars (1 equiv) dissolved in dry pyridine under nitro-
gen. The mixture was stirred at room temperature
0
0
76.4 (C-3 ), 74.0 (C-4 ), 72.1 (3-C), 71.9 and 69.3
(CMe ), 67.4 (Fmoc CH ), 62.9 (C-6), 62.1 (C-5), 53.6
3
2
0
overnight. To the pyridine solution was added Ac O
2
(Pyr C-5), 47.0 (Fmoc CH and C-2), 43.8 (C-2 ), 34.0
(CH O), 29.2 and 28.3 [C(CH ) ], 21.1 and 20.6
(
8 equiv), and the reaction mixture was stirred for 16 h
2
3 3
+
(COCH ). ESI-MS (m/z) [M+Na] : found 790.4. Anal.
at room temperature under nitrogen in the dark. The
solvent was removed by co-evaporation with toluene.
The crude products were purified by FCC to provide
the protected iminosugars 2a and b and 7a and b.
3
Calcd for C H NO : C, 64.13; H, 6.96; N, 1.82.
4
Found: C, 64.43; H, 6.78; N, 1.89.
1
53
13
4.2.4. N-Fmoc-2-deoxy-2S-[3S,4S-bis(1,1-dimethylethoxy)-
4
4
.2.1. N-Fmoc-3S,4S-pyrrolidinediol diacetate (2a). 3S,
S-Pyrrolidinediol (1a) (480 mg, 0.46 mmol) yielded 2a
as a white solid (132 mg, 70%). R [AcOEt/hexane, 1:2;
2S-pyrrolidinyl]-1,3,4-tri-O-acetyl-L-rhamnopyranose (7b).
Compound 6b (1.22 g, 3.37 mmol) yielded 7b as a solid
(1.07 g, 45%). R [AcOEt/hexane, 1:3; UV, p-anisalde-
f
f
1
1
UV] = 0.2. H NMR (CDCl ) d 7.75 (d, J = 7.4 Hz,
hyde] = 0.48. H NMR (CDCl ) d 7.73 (d, J = 7.4 Hz,
3
3
2
H, Fmoc 4-H and 5-H), 7.60 (d, J = 7.4 Hz, 2H, Fmoc
-H and 8-H), 7.34–7.27 (m, 4H, Fmoc 2-H, 7-H, 3-H,
2H, Fmoc 4-H and 5-H), 7.54 (d, J = 8 Hz, 2H, Fmoc
1-H and 8-H), 7.36–7.27 (m, 4H, Fmoc 2-H, 7-H, 3-H
1
and 6-H), 5.16 (d, J = 3.6 Hz, 2H, CH –O), 4.23 (dd,
J = 1.4, 7.6 Hz, 2H, Pyr 3-H and 4-H), 4.24 (pt,
J = 6.6 Hz, 1H, Fmoc 9-H), 4.23 (dd, J = 4.4, 12.8 Hz,
0
and 6-H), 5.73–5.60 (m, 2H, 5 -H ), 4.76–4.62 (m, 3H,
2
2
0
0
0
1-H , 3 -H and 4 -H), 4.45–3.76 (m, 7H, 2 -H, deoxyRha
a
3-H, 4-H and 5-H, Fmoc CH O and 9-H), 3.14–3.10 (m,
2

3970
F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
1
1
H, deoxyRha 2-H), 2.05, 2.04 and 2.01 (3 s, 9H, 3· Ac),
4.3.4. 2-Deoxy-2S-[3S,4S-bis(1,1-dimethylethoxy)-2S-pyr-
rolidinyl]-1,3,4-tri-O-acetyl-L-rhamnopyranose (8b).
Compound 7b (840 mg, 1.18 mmol) yielded 8b as an
oil (422 mg, 73%). Rf [DCM/MeOH, 10:1; vanil-
1
3
.27–1.12 (m, 21H, CH and 2· t-Bu). C NMR (CDCl )
3
3
d 170.3–169.8 (COCH ), 157.0 (CONH), 143.7, 141.2,
3
1
8
7
4
2
27.7, 127.0, 125.0, and 120.0 (Fmoc Carom), 92.1 (C-1),
0
0
1
0
1.0 (C-4), 76.2 (C-3 ), 74.9 (C-4 ), 72.1 (C-3), 70.1 and
0
lin] = 0.31. H NMR (CDCl ) d 5.62–5.52 (m, 2H, 5 -
3
0
0.0 (CMe ), 67.4 (Fmoc CH ), 61.1 (C-5), 52.0 (C-5 ),
3
H ), 5.02–4.98 (m, 1H, 1-H ), 4.68–4.56 (m, 2H, 3 -H
2
2
a
0
0
0
7.0 (Fmoc CH and C-2), 45.9 (C-2 ), 35.0 (CH O),
2
and 4 -H), 4.23–3.91 (m, 2H, 2 -H and 3-H), 3.76–3.74
(m, 1H, 4-H), 3.47–3.39 (m, 1H, 5-H), 2.75 (pdt,
J = 11.0, 2.2 Hz, 1H, 2-H), 2.06, 2.04 and 2.00 (3· s,
9.3 and 28.4 [C(CH ) ], 21.2 and 20.8 (COCH ), 17.6
3
3
3
+
(CH ). ESI-MS (m/z) [M+Na] : found 732.3. Anal. Calcd
3
for C H NO : C, 65.99; H, 7.24; N, 1.97. Found: C,
6
1
3
9H, 3· Ac), 1.25–1.09 (m, 21H, CH and 2· t-Bu).
C
3
9
51
11
3
5.69; H, 7.14; N, 2.05.
NMR (CDCl ) d 170.5–170.2 (COCH ), 92.4 (C-1),
3 3
0
0
8
and 68.8 (CMe ), 63.5 (C-5), 54.5 (C-5 ), 49.4 (C-2),
0.5 (C-4), 76.6 (C-3 ), 75.9 (C-4 ), 73.9 (C-3), 72.5
0
4
.3. Deprotection of amino group: general procedure
3
0
4
(
4.7 (C-2 ), 28.6 and 28.2 [C(CH ) ], 22.6 and 20.8
3
COCH ), 17.8 (CH ). ESI-MS (m/z) [M+Na] : found
3 3
3
+
Fmoc-iminosugars 2a and b and 7a and b were treated
with 20% piperidine in THF at room temperature for
510.33. Anal. Calcd for C H NO : C, 59.12; H, 8.48;
24 41 9
N, 2.87. Found: C, 59.31; H, 8.34; N, 2.81.
3
0 min. The solvent was removed and the residues were
purified from THF/hexane to provide compounds 3a
and b and 8a and b.
4
.4. Coupling reaction: general procedure I
A solution of 3a or b (1 equiv) in DMF was added to a
solution of Fmoc-L-Asp-Ot-Bu (1 equiv), HATU
(1 equiv), and NMM (1 equiv) in DMF. The reaction
mixture was stirred for 2 h at room temperature. Evap-
oration of the solvent yielded the crude products, which
were purified with FCC to obtain 4a or b.
4
2
.3.1. 3S,4S-Pyrrolidinediol diacetate (3a). Compound
a (1 g, 2.44 mmol) yielded 3a as an oil (292 mg, 64%).
1
Rf [AcOEt/hexane, 1:2; UV, ninhydrin] = 0.15.
NMR (CDCl ) d 5.06 (d, J = 3.6 Hz, 2H, 3-H and 4-
H
3
H), 3.68 (dd, J = 9.6, 13.6 Hz, 2H, 2-H and 5-H), 3.56
pdd, J = 16.2 Hz, 2H, 2-H and 5-H), 2.13 (s, 3H, Ac),
(
2
1
3
.08 (s, 3H, Ac). C NMR (CDCl ) d 169.8 (COCH ),
3 3
4
.4.1. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
7
5.2 (CHOAc), 50.4 (CH ), 20.8 (COCH ). ESI-MS
2
3
+
m/z) [M+H] : found 188.1. Anal. Calcd for
c-oxo-[3S,4S-bis(acetyloxy)-1-pyrrolidine]butanoic acid
tert-butyl ester (4a). Compound 3a (290 mg, 1.53 mmol)
yielded 4a as a white solid (418 mg, 47%). R [AcOEt/
(
C H NO : C, 51.33; H, 7.00; N, 7.48. Found: C,
8
13
4
f
5
1.08; H, 6.90; N, 7.54.
1
hexane, 1:2; UV, ninhydrin] = 0.23. H NMR (CDCl )
3
d 7.75 (d, J = 6.8 Hz, 2H, Fmoc 4-H and 5-H), 7.60
4.3.2. 2R-[(Acetyloxy)methyl]-3R,4R,5S-piperidinetriol
triacetate (3b). Compound 2b (760 mg, 1.37 mmol)
(
4
d, J = 7 Hz, 2H, Fmoc 1-H and 8-H), 7.34–7.25 (m,
H, Fmoc 2-H, 7-H, 3-H, and 6-H), 6.2 (d, J = 6.2 Hz,
yielded 3b as a white solid (363 mg, 80%). R [isopropa-
f
1H, Asn NH), 5.17–5.10 (m, 2H, Pyr 3-H and 4-H),
4.6 (m, 1H, Asn a-H), 4.24 (pt, J = 6.7 Hz, 1H, Fmoc
9-H), 3.86–3.75 (m, 4H, Pyr 2-H and 5-H ), 3.04 (dd,
1
nol/AcOEt/H O, 6:1:3; UV, ninhydrin] = 0.67. H NMR
2
(
J = 8.6 Hz, 2H, 3-H and 5-H), 4.05 (br s, 2H, CH OAc),
CDCl ) d 5.08 (pd, J = 9.6 Hz, 1H, 4-H), 4.86 (pt,
3
2
2
2
J = 17.1, 4.4 Hz, 1H, Asn b-H), 2.68 (dd, J = 17.2, 4.4
3
.77–3.74 (m, 1H, 2-H), 3.43 (pdt, J = 9, 12.6 Hz, 2H, 6-
0
Hz, 1H, Asn b -H), 2.07 (s, 3H, Ac), 2.03 (s, 3H, Ac),
1
3
1
3
H ), 2.05 (s, 12H, 4· Ac). C NMR (CDCl ) d 169.8
2
3
1.45 (s, 9H, t-Bu). C NMR (CDCl ) d 170.0 (COOt-
3
(
(
CO), 75.1 (C-3), 51.2 (C-2), 20.8 (COCH ). ESI-MS
3
Bu), 169.2–169.6 (COCH ), 156.3 (CONH), 143.7,
141.2, 127.7, 127.0, 125.0, and 120.0 (Fmoc Carom),
81.9 (CMe ), 75.0–73.4 (Pyr CH), 67.1 (Fmoc CH ),
3
+
m/z) [M+H] : found 332.1. Anal. Calcd for
C H NO : C, 50.75; H, 6.39; N, 4.23. Found: C,
1
4
21
8
3
2
5
0.82; H, 6.43; N, 4.42.
5
3
1.0 (Asn C-a), 50.5–49.7 (Pyr CH ), 47.1 (Fmoc CH),
2
5.6 (CH O), 28.1 [C(CH ) ], 20.8 (COCH ). ESI-MS
2
3 3
3
+
4
.3.3. 2-Deoxy-2R-[3R,4R-bis(1,1-dimethylethoxy)-2R-
(m/z) [M+H] : found 581.3. Anal. Calcd for
C H N O : C, 64.13; H, 6.25; N, 4.82. Found: C,
64.76; H, 6.65; N, 4.67.
pyrrolidinyl]-1,3,4,6-tetra-O-acetyl-D-glucopyranose (8a).
Compound 7a (750 g, 0.98 mmol) yielded 8a as an oil
3
1
36
2
9
(
363 mg, 68%). R [DCM/MeOH, 10:1; vanillin] = 0.46.
f
1
0
H NMR (CDCl ) d 5.65–5.60 (m, 2H, 5 -H ), 5.09
4.4.2. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
c-oxo-[2R-[(acetyloxy)methyl]-3R,4R,5S-tris(acetyloxy)-
1-piperidine]butanoic acid tert-butyl ester (4b). Com-
pound 3b (360 mg, 1.09 mmol) yielded 4b as a white
3
2
0
0
(
(
pd, 1H, 1-H ), 4.94–4.86 (m, 2H, 3 -H and 4 -H), 4.70
a
0
d, J = 8.9 Hz, 1H, 1-H ), 4.26–3.94 (m, 3H, 2 -H, 2-H
b
and 3-H), 3.76–3.74 (m, 1H, 4-H), 3.49–3.38 (m, 3H,
5
-H and 6-H ), 2.05, 2.03, 2.00 and 1.99 (4 s, 12H, 4·
solid (702 mg, 89%). Rf [AcOEt/hexane, 1:1; UV,
2
1
3
1
Ac), 1.27–1.09 (m, 18H, 2· t-Bu). C NMR (CDCl )
vanillin] = 0.29. H NMR (CDCl ) d 7.74 (d, J = 7 Hz,
3
3
d 170.8–170.4 (COCH ), 92.6 (C-1), 80.4 (C-4), 76.4
2H, Fmoc 4-H and 5-H), 7.54 (d, J = 7 Hz, 2H, Fmoc
1-H and 8-H), 7.36–7.20 (m, 4H, Fmoc 2-H, 7-H, 3-H,
and 6-H), 6.07 (br d, J = 8 Hz, 1H, Asn NH), 4.71–
3
0
0
(
6
C-3 ), 74.6 (C-4 ), 72.5 (C-3), 70.9 and 70.3 (CMe ),
0
3
5.1 (C-6), 62.6 (C-5), 54.5 (C-5 ), 46.4 (C-2), 44.5
0
(
ESI-MS (m/z) [M+Na] : found 569. Anal. Calcd for
C-2 ), 29.6 and 28.4 [C(CH ) ], 22.6 and 20.7 (COCH ).
4.65 (m, 2H, Fmoc OCH ), 4.57–4.34 (m, 2H, Asn a-
3
3
3
2
+
H and Fmoc 9-H), 4.25–4.06 (m, 4H, DNJ 3-H, 5-H,
and CH OAc), 3.01 (dd, J = 16.4, 4.4 Hz, 1H, Asn b-
C H NO : C, 57.23; H, 7.94; N, 2.57. Found: C,
2
6
43
11
2
0
5
7.45; H, 7.76; N, 2.61.
H), 2.85 (dd, J = 16.8, 4 Hz, 1H, Asn b -H), 2.79–2.70

F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
3971
(
m, 2H, DNJ 6-H ), 2.04 (br s, 12H, 4· Ac), 1.48 (s, 9H,
Fmoc 9-H), 3.75–3.60 (m, 1H, deoxyRha 4-H), 3.54–
3.43 (m, 2H, Asn b-H ), 3.47–3.20 (m, 1H, deoxyRha
2
1
3
t-Bu). C NMR (CDCl ) d 177.2 (C-c), 169.6 (COOt-
3
2
Bu), 156.3 (urethane CO), 143.7, 141.2, 127.7, 127.0,
1
5-H), 2.08, 2.01, and 1.98 (3· s, 9H, 3· Ac), 1.24–1.15
1
3
25.0, and 120.0 (Fmoc Carom), 82.6 (CMe ), 76.2 (C-
3
(m, 21H, CH and 2· t-Bu). C NMR (CDCl ) d
3
3
4
), 67.1 (Fmoc CH –O), 66.9–66.4 (C-3 and C-5), 64.0
2
170.5–169.7 (COCH ), 168.3 (COOPfp), 160.0 (CONH),
143.7, 141.2, 127.7, 127.0, 125.0, and 120.0 (Fmoc Carom),
3
(
CH OAc), 54.4 (Asn C-a), 47.2 (Fmoc C-9), 44.7 (C-
2
0
0
2
MS (m/z) [M+Na] : found 747.3. Anal. Calcd for
), 42.5 (C-6), 35.9 (Asn C-b), 14.04 [C(CH ) ]. ESI-
94.6 (C-1), 80.1 (C-4), 76.5 (C-3 ), 75.5 (C-4 ), 74.3
(C-3), 73.7 and 72.2 (CMe ), 68.6 (Fmoc CH ), 63.5
3
3
+
3
2
0
C H N O : C, 61.32; H, 6.12; N, 3.87. Found: C,
3
(C-5), 55.2 (Asn C-a), 52.4 (C-5 ), 49.0 (Fmoc CH),
46.8 (C-2), 43.6 (C-2 ), 36.3 (CH O), 29.4 and 28.3
7
44
2
13
0
6
1.44; H, 6.36; N, 3.68.
2
[
MS (m/z) [M+Na] : found 991.4. Anal. Calcd for
C(CH ) ], 22.2 and 20.6 (COCH ), 17.6 (CH ). ESI-
3 3 3 3
+
4
.5. Coupling reaction: general procedure II
C H F N O : C, 59.39; H, 5.59; F, 9.59; N, 2.83.
14
4
9
55
5
2
Fmoc-L-Asp(Cl)-OPfp (1 equiv) dissolved in dry THF
was added to a solution containing the appropriate imi-
nosugar 8a or b (1 equiv) and NMM (1 equiv) in dry
THF at 0 ꢂC and stirred at room temperature for
Found: C, 59.51; H, 5.80; F, 9.51; N, 2.89.
4.6. tert-Butyl-deprotection of Fmoc-protected amino
acids: general procedure
3
0 min. Then, filtration of the precipitate and concentra-
tion under reduced pressure afforded an oil that was
purified from THF/hexane to afford compound 9a or b.
The pure t-Bu-protected monomers 4a or b were dis-
solved in a mixture of TFA and DCM (1:1). The result-
ing mixture was stirred for 3 h at room temperature. The
solvents were evaporated off. After dissolution in water
and lyophilization, Fmoc-protected amino acids 5a and
b were obtained.
4
.5.1. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
c-oxo-[2R-[2-deoxy-1,3,4,6-tetra-O-acetyl-2R-D-glucopyr-
anosyl]-3R,4R-bis(1,1-dimethylethoxy)-1-pyrrolidine]buta-
noic acid 1-(pentafluorophenyl) ester (9a). Compound 8a
(
600 mg, 1.10 mmol) yielded 9a (473 mg, 41%). Rf
4.6.1. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
c-oxo-[3S,4S-bis(acetyloxy)-1-pyrrolidine]butanoic acid
(5a). tert-Butyl ester 4a (460 mg, 0.79 mmol) yielded 5a
1
[
AcOEt/hexane, 1:1; vanillin] = 0.52. H NMR (CDCl )
3
d 7.75 (d, J = 6.6 Hz, 2H, Fmoc 4-H and 5-H), 7.58 (d,
J = 6.4 Hz, 2H, Fmoc 1-H and 8-H), 7.41–7.30 (m, 4H,
Fmoc 2-H, 7-H, 3-H, and 6-H), 6.22 (d, J = 8.8 Hz, 1H,
a-NH), 5.67–5.54 (m, 2H, 2-H and 5 -H), 5.14 (d,
J = 2.6 Hz, 1H, 1-H ), 4.95–4.84 (m, 2H, 3 -H and 4 -
a
H), 4.71 (d, J = 8.8 Hz, 1H, 1-H ), 4.68–4.62 (m, 1H,
Asn a-H), 4.39–4.34 (m, 1H, Fmoc 2 -H), 4.27–4.16
(
deoxyGlc 2-H and 3-H), 3.79–3.67 (m, 1H, deoxyGlc
4
6
Ac), 1.20–1.09 (m, 18H, 2· t-Bu). C NMR (CDCl )
d
as a white solid (408 mg, 98%). R [AcOEt/hexane 2:1;
f
1
UV] = 0.08. H NMR (CDCl ) d 7.75 (d, J = 6.8 Hz,
3
0
2H, Fmoc 4-H and 5-H), 7.60 (d, J = 7 Hz, 2H, Fmoc
1-H and 8-H), 7.34–7.27 (m, 4H, Fmoc 2-H, 7-H, 3-H,
and 6-H), 6.2 (d, J = 6.2 Hz, 1H, Asn NH), 5.18 (m,
2H, Pyr 3-H and 4-H), 4.23–4.19 (m, 4H, Asn a-H,
Fmoc OCH and 9-H), 3.80–3.75 (m, 4H, Pyr 2-H
2
0
0
b
0
m, 3H, Fmoc CH O and 9-H), 4.13–4.03 (m, 2H,
2
2
and 5-H ), 3.04 (dd, J = 16.8, 4.4 Hz, 1H, Asn b-H),
2
0
-H), 3.53–3.21 (m, 5H, Asn b-H, deoxyGlc 5-H and
2.7 (dd, J = 16.8 4.4 Hz, 1H, Asn b -H), 2.07 (s, 3H,
OAc), 2.04 (s, 3H, OAc). C NMR (CDCl ) d 172.6
0
13
-H and 6 -H), 2.08, 2.06, 2.02 and 1.98 (4 s, 12H, 4·
3
1
3
(COOH), 169.5–169.7 (COCH ), 155.9 (CONH), 143.7,
141.2, 127.7, 127.0, 125.0, and 120.0 (Fmoc Carom),
3
3
170.8–170.0 (COCH ), 168.3 (COOPfp), 156.0
3
(
(
(
CONH), 143.7, 141.2, 127.7, 127.0, 125.0 and 120.0
0
74.6–73.3 (Pyr CH), 67.2 (Fmoc CH ), 50.9 (Asn C-a),
2
Fmoc C
0
), 92.5 (C-1), 80.3 (C-4), 76.4 (C-3 ), 74.7
C-4 ), 72.3 (C-3), 70.9 and 70.3 (CMe ), 67.7 (Fmoc
50.3–50.0 (Pyr CH ), 47.0 (Fmoc CH), 36.7 (CH O),
arom
2
2
+
20.8 (COCH ). ESI-MS (m/z) [M+Na] : found 547.2.
3
3
CH ), 65.1 (C-6), 62.6 (C-5), 54.6 (Asn C-a), 52.6 (C-
Anal. Calcd for C H N O : C, 61.83; H, 5.38; N,
27 28
5.34. Found: C, 61.46; H, 54.97; N, 5.29.
2
2
9
0
0
5
), 48.9 (Fmoc CH), 46.6 (C-2), 42.9 (C-2 ), 35.6
CH O), 29.6 and 28.5 [C(CH ) ], 21.8 and 20.6
(
(
2
3 3
+
COCH ). ESI-MS (m/z) [M+Na] : found 1049.4. Anal.
4.6.2. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
c-oxo-[2R-[(acetyloxy)methyl]-3R,4R,5S-tris(acetyloxy)-
1-piperidine]butanoic acid (5b). tert-Butyl ester 4b
3
Calcd for C H F N O : C, 58.39; H, 5.48; F, 9.06; N,
5
1
57
5
2
16
2
.67. Found: C, 58.19; H, 5.51; F, 8.98; N, 2.73.
(80 mg, 0.11 mmol) yielded 5b as a white solid (66 mg,
90%). Rf [AcOEt/Esano, 2:1; UV] = 0.1. H NMR
1
4
.5.2. (S)-a-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
c-oxo-[2S-[2-deoxy-1,3,4-tri-O-acetyl-2S-L-rhamnopyrano-
syl]-3S,4S-bis(1,1-dimethylethoxy)-1-pyrrolidine]butanoic
acid 1-(pentafluorophenyl) ester (9b). Compound 8b
(CDCl ) d 7.74 (d, J = 7 Hz, 2H, Fmoc 4-H and 5-H),
3
7.54 (d, J = 7 Hz, 2H, Fmoc 1-H and 8-H), 7.36–7.28
(m, 4H, Fmoc 2-H, 7-H, 3-H, and 6-H), 6.07 (d,
J = 8 Hz, 1H, Asn NH), 4.71–4.66 (m, 2H, Fmoc
(
750 mg, 1.53 mmol) yielded 9b (671 mg, 44%). Rf
AcOEt/hexane, 1:1; vanillin] = 0.60. H NMR (CDCl )
1
[
OCH ), 4.57–4.34 (m, 2H, Asn a-H, Fmoc 9-H), 4.25–
3
2
d 7.75 (d, J = 6.6 Hz, 2H, Fmoc 4-H and 5-H), 7.58 (d,
J = 6.4 Hz, 2H, Fmoc 1-H and 8-H), 7.41–7.30 (m, 4H,
Fmoc 2-H, 7-H, 3-H and 6-H), 5.96 (d, J = 8 Hz, 1H, a-
4.06 (m, 4H, DNJ 3–H, 5–H and CH OAc), 3.01 (dd,
J = 16.4, 4.4 Hz, 1H, Asn b-H), 2.85 (dd, J = 16.8,
2
0
4 Hz, 1H, Asn b -H), 2.79–2.76 (m, 1H, DNJ 6-H),
0
13
NH), 5.63–5.53 (m, 2H, 5-H, and 5 -H), 5.05 (d,
0
2.04 (br s, 12H, 4· OAc). C NMR (CDCl ) d 176.0
3
0
J = 2.2 Hz, 1H, 1-H ), 4.69–4.57 (m, 3H, 3 -H, 4 -H,
a
(C-c), 173.0 (COOH), 156.3 (urethane CO), 143.7,
141.2, 127.7, 127.6, 125.0, and 120.0 (Fmoc Carom),
0
and Asn a-H), 4.40–4.34 (m, 3H, Fmoc 2 -H and
CH O), 4.23–4.18 (m, 3H, deoxyRha 2-H, 3-H, and
74.6 (C-4), 67.1 (Fmoc CH –O), 66.9–66.4 (C-3 and
2
2

3972
F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
C-5), 64.0 (CH OAc), 54.4 (Asn C-a), 47.2 (Fmoc C-9),
4.8. Immunological assays: general procedure
2
4
[
4.7 (C-2), 42.5 (C-6), 35.9 (Asn CH O). ESI-MS (m/z)
2
+
M+Na] : found 691.2. Anal. Calcd for C H N O :
Antibody titers were determined in solid-phase ELISA
3
3
36
2
13
2
5
C, 59.28; H, 5.43; N, 4.19. Found: C, 59.04; H, 4.98;
N, 4.10.
(SP-ELISA). 96-Well activated Polystyrene ELISA
plates (Limbro Titertek, ICN Biomedicals, Inc., Aurora,
Ohio, USA) were coated with 1 lg/100 lL/well of pep-
tides or glycopeptides in pure carbonate buffer 0.05 M
4.7. General procedure for the solid-phase peptide
synthesis (SPPS): automated synthesis
(
pH 9.6) and incubated at 4 ꢂC overnight. After five
washes with saline containing 0.05% Tween 20, nonspe-
cific binding sites were blocked by fetal calf serum
Glycopeptides were synthesized on an automatic batch
synthesizer (APEX 396, Advanced ChemTech) equipped
with a 40-well reaction block, using a Wang resin pre-
loaded with the C-terminal amino acid of the sequence,
following the Fmoc/t-Bu SPPS strategy. Fmoc-amino
acids and resin were purchased from Novabiochem
AG (Laufelfingen, Switzerland). Fmoc deprotections
were performed in 30 min with 20% piperidine in
DMF. Coupling reactions (repeated twice) were per-
formed for 45 min by using a 0.5 M solution of the
Fmoc-protected amino acids and HOBt in DMF
(
FCS), 10% in saline Tween (100 lL/well) at room tem-
perature for 60 min. Sera diluted from 1:100 were ap-
plied at 4 ꢂC for 16 h in saline Tween 10% FCS. After
five washes, we added 100 lL/well of alkaline phospha-
tase conjugated anti-human IgM or IgG Fab2-specific
affinity-purified antibodies (Sigma, St. Louis, Missouri,
USA) diluted 1:500 in saline Tween/FCS. After an over-
night incubation and five washes, 100 lL of substrate
solution consisting of 2 mg/mL p-nitrophenylphosphate
(Sigma, St. Louis, Missouri, USA) in 10% diethanol-
amine buffer was applied. After 30 min, the reaction
was blocked with 50 lL of 1 M NaOH and the absor-
bance read in a multichannel ELISA reader (SUNRISE,
TECAN, Austria) at 405 nm. ELISA plates, coating
conditions, reagent dilutions, buffers, and incubation
times were tested in preliminary experiments. Each ser-
um was individually titrated to check for parallellism
of antibody absorbances in dilutions. Within-assays
and between-assays coefficients of variation were below
(
(
2.5 equiv), a 0.5 M solution of TBTU in DMF
2.5 equiv), and 4 M NMM in DMF (5 equiv). Peptide
cleavage from the resin and deprotection of the amino
acid side chains were carried out in 3 h with TFA/thio-
anisole/EDT/phenol/H O (82.5:5:2.5:5:5) (vol:vol:vol:-
2
vol:vol). The resin was filtered off, and the solution
was concentrated. The crude products were precipitated
with cold Et O, centrifuged, and lyophilized. Deprotec-
2
tion of the sugar moiety was performed by adding 0.1 M
MeONa to a solution of the crude material in dry
MeOH to pH 12. After 2 h of stirring, the reaction
was quenched with dry CO to neutrality, the solvent
2
was evaporated to dryness, and the residue was
lyophilized.
1
0%. The antibody levels revealed by SP-ELISA are ex-
pressed as absorbance value at a dilution of 1:100 as a
ratio of positive controls in the same experiment. Posi-
tive samples were analyzed twice to evaluate the differ-
ences between the two determinations. The reference
values were set as the mean + 2SD of the control groups.
Within- and between-assays coefficients of variation
were below 10%.
4.7.1. Parallel synthesis of H-Thr-Pro-Arg-Val-Glu-Arg-
Asn(DHPyr)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-
7
Trp-Met-Val-Lys-OH [Asn (DHPyr)]CSF114 (10); H-
Antibody affinity and antibody affinity heterogeneity
were measured by competitive ELISA following the
methods previously published. In preliminary titration
Thr-Pro-Arg-Val-Glu-Arg-Asn(DNJ)-Gly-His-Ser-Val-Phe-
Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-OH [Asn (DNJ)]-
7
26
CSF114 (11); H-Thr-Pro-Arg-Val-Glu-Arg-Asn(DHPyr-
2
curves the semi-saturation dilution was calculated
(absorbance 0.7). At this dilution, antibody was pre-
incubated with increasing antigen concentration (6 h at
25 ꢂC). Unblocked antibodies were revealed by ELISA,
and the absorbance was graphically represented in rela-
tion to the antigen concentration. Data are expressed
as % of absorbance of positive serum in reception to
peptide concentration.
-deoxyGlc)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-
7
Trp-Met-Val-Lys-OH [Asn (DHPyr-2-deoxyGlc)]CSF114
12). Glycopeptides 10–12 were synthesized using Fmoc-
(
L-Lys(Boc)-Wang resin (0.57 mmol/g, 100 mg). The
introduction of sugar moieties was performed by using
a 2-fold excess (0.114 mmol) of the building blocks
Fmoc-L-Asn(DHPyrAc2)-OH (5a) or Fmoc-L-Asn(DN-
JAc4)-OH (5b) dissolved in DMF, HATU (2 equiv), and
NMM (3.5 equiv) for 1.5 h. Coupling with Fmoc-L-
Asn(DHPyrt-Bu2-2-deoxyGlcAc4)-OPfp (9a) was per-
formed by using 2-fold excess (0.114 mmol) of the
building block, HOBt (2 equiv), and NMM (3.5 equiv)
dissolved in DMF for 1.5 h. All glycopeptides were
cleaved and side chains deprotected at room tempera-
ture, and then deacetylated as described in the general
procedure. Glycopeptides 10–12 were purified by semi-
preparative HPLC. Fractions containing homogeneous
material as monitored by HPLC were combined and
lyophilized. Characterization of the products was per-
formed using analytical HPLC and ESI-MS spectrome-
try. The analytical data are reported in Table 1.
Acknowledgments
We thank the Fondazione Ente Cassa di Risparmio di
Firenze (Italy) for the financial support to the Labora-
tory of Peptide and Protein Chemistry and Biology of
the University of Florence. This work was funded in
part by PRIN 2005 (Ministero dell’Universit a` e della
Ricerca, prot. 2005032959).
References and notes
1. Doyle, H. A.; Mamula, M. J. Trends Immunol. 2001, 22, 443.

F. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 3965–3973
3973
2
. Carotenuto, A.; D’Ursi, A. M.; Mulinacci, B.; Paolini, I.;
Lolli, F.; Papini, A. M.; Novellino, E.; Rovero, P. J. Med.
Chem. 2006, 49, 5072.
1993, 268, 570; (d) Hancock, S. M.; Vaughan, M. D.;
Withers, S. G. Curr. Opin. Chem. Biol. 2006, 5, 509; (e)
Aharoni, A.; Thieme, K.; Chiu, C. P.; Buchini, S.;
Lairson, L. L.; Chen, H.; Strynadka, N. C.; Wakarchuk,
W. W.; Withers, S. G. Nat. Methods 2006, 8, 609; (f)
Mullegger, J.; Chen, H. M.; Warren, R. A.; Withers, S.
G. Angew. Chem. Int. Ed. Engl. 2006, 45, 2585; (g)
Gloster, T. M.; Meloncelli, P.; Stick, R. V.; Zechel, D.;
Vasella, A.; Davies, G. J. J. Am. Chem. Soc. 2007, 129,
2345.
3
. (a) Mazzucco, S.; Mata, S.; Vergelli, M.; Fioresi, R.;
Nardi, E.; Mazzanti, B.; Chelli, M.; Lolli, F.; Ginanneschi,
M.; Pinto, F.; Massacesi, L.; Papini, A. M. Bioorg. Med.
Chem. Lett. 1999, 9, 167; (b) Lolli, F.; Mulinacci, B.;
Carotenuto, A.; Bonetti, B.; Sabatino, G.; Mazzanti, B.;
D’Ursi, A. M.; Novellino, E.; Pazzagli, M.; Lovato, L.;
Alcaro, M. C.; Peroni, E.; Pozo-Carrero, M. C.; Nuti, F.;
Battistini, L.; Borsellino, G.; Chelli, M.; Rovero, P.;
Papini, A. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
11. (a) Watson, A. A.; Fleet, G. W. J.; Asano, N.; Molyneux,
R. J.; Nash, R. J. Phytochemistry 2001, 56, 265; (b)
Vasella, A.; Davies, G. J.; Bohm, M. Curr. Opin. Chem.
Biol. 2002, 6, 619; (c) Kaper, T.; van Heusden, H. H.; van
Loo, B.; Vasella, A.; van der Oost, J.; de Vos, W. M.
Biochemistry 2002, 41, 4147; (d) Heightman, T. D.;
Vasella, A. T. Angew. Chem. Int. Ed. 1999, 38, 750; (e)
Zechel, D. L.; Withers, S. G. Curr. Opin. Chem. Biol. 2001,
5, 643; (f) Yip, V. L. Y.; Withers, S. G. Org. Biomol.
Chem. 2004, 2, 2707.
1
0273; (c) Papini, A. M. Nat. Med. (NY) 2005, 11, 13.
4
. Lolli, F.; Mazzanti, B.; Pazzagli, M.; Peroni, E.; Alcaro,
M. C.; Lanzillo, R.; Brescia Morra, V.; Santoro, L.;
Gasperini, C.; Galgani, S.; D’Elios, M. M.; Zipoli, V.;
Sotgiu, S.; Pugliatti, M.; Rovero, P.; Chelli, M.; Papini, A.
M. J. Neuroimmunol. 2005, 167, 131.
5
. For recent reviews on alkaloid glycosidase inhibitors, see:
(
a) Asano, N. Glycobiology 2003, 13, 93R; (b) Asano, N.
Curr. Top. Med. Chem. 2003, 3, 471; (c) Lillelund, V. H.;
Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102,
12. Lasky, L. A. Science 1992, 258, 964.
13. Dewek, R. A. Chem. Rev. 1996, 96, 683.
14. Block, T. M.; Lu, X.; Platt, F. M.; Foster, G. R.; Gerlich,
W. H.; Blumberg, B. S.; Dwek, R. A. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 2235.
5
15; (d) Watson, A. A.; Fleet, G. W. J.; Asano, N.;
Molyneux, R. J.; Nash, R. J. Phytochemistry 2001, 56,
65; (e) Elbein, A. D.; Molyneux, R. J. In Comprehensive
2
Natural Products Chemistry; Barton, D., Nakanishi, K.,
Meth-Cohn, O., Eds.; Elsevier, 1999; Vol. 3, p 179; (f)
Simmonds, M. S. J.; Kite G. C.; Porter, E. A. In
Iminosugars as Glycosidase Inhibitors; Stutz, A. Ed.;
Wiley-VCH: Weinheim, Germany, 1999; p 8; (g) Ossor,
A.; Elbein, A. D. In Carbohydrates in Chemistry and
Biology; Ernst, B.; Hart, G. W.; Sinay, P. Eds.; Wiley-
VCH: Weinheim, Germany, 2000; Vol. 3, Part II, p 513.
. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J.
Tetrahedron: Asymmetry 2000, 11, 1645.
15. Datema, S.; Olofsson, P.; Romero, P. A. Pharmacol. Ther.
1987, 33, 221.
16. Jacob, G. S. Curr. Opin. Struct. Biol. 1995, 5, 605.
17. Nagel, U.; Kinzel, E.; Andrade, J.; Prescher, G. Chem.
Ber. 1986, 119, 3326.
18. Matos, C. R. R.; Lopes, R. S. C.; Lopes, C. C. Synthesis
1999, 4, 571.
19. Cardona, F.; Valenza, S.; Picasso, S.; Goti, A.; Brandi, A.
J. Org. Chem. 1998, 63, 7311.
6
7
20. Cardona, F.; Valenza, S.; Goti, A.; Brandi, A. Eur. J. Org.
Chem. 1999, 6, 1319.
. (a) Walter, S.; Fassbender, K.; Gulbins, E.; Liu, Y.;
Rieschel, M.; Herten, M.; Bersch, T.; Engelhardt, B.
J. Neuroimmunol. 2002, 132, 1; (b) Liu, J.; Shikman, A. R.;
Lotz, M. K.; Wong, C.-H. Chem. Biol. 2001, 8, 701.
. Gross, P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W.
Clin. Cancer Res. 1995, 1, 935.
21. Caldwell, C. G.; Chen, P.; He, J.; Parmee, E. R.; Leiting,
B.; Marsilio, F.; Patel, R. A.; Wu, J. K.; Eiermann, G. J.;
Petrov, A.; He, H.; Lyons, K. A.; Thornberry, N. A.;
Weber, A. E. Bioorg. Med. Chem. Lett. 2004, 14, 1265.
22. Meldal, M.; Bock, K. Tetrahedron Lett. 1990, 31,
6987.
23. Christiansen-Brams, I.; Meldal, M.; Bock, K. J. Chem.
Soc., Perkin Trans. 1993, 1461.
24. Still, W. C.; Khan, M.; Mitra, A. J. Org. Chem. 1985, 50,
2394.
8
9
. For reviews on inhibition of glycosidases, see: (a) Elbein,
A. D. Annu. Rev. Biochem. 1987, 56, 497; (b) Sinnott, M.
L. Chem. Rev. 1990, 90, 1171; (c) Legler, G. Adv.
Carbohydr. Chem. Biochem. 1990, 48, 319; (d) Franck,
R. W. Bioorg. Chem. 1992, 20, 77.
1
0. For reviews on bioactivity of glycosidase inhibitors, see
25. Loomans, E. E.; Gribnau, T. C.; Bloemers, H. P.;
Schielen, W. J. J. Immunol. Methods 1998, 221, 119.
26. Rath, S.; Stanley, C. M.; Steward, M. W. J. Immunol.
Methods 1988, 106, 245.
(a) Winchester, B.; Fleet, G. J. Glycobiol. 1992, 2, 199;
b) Vogel, P. Chim. Oggi 1992, 10, 9; (c) Karlsson, G. B.;
(
Butters, T. D.; Dwek, R. A.; Platt, F. M. J. Biol. Chem.