Total synthesis of the collagen glycosylated cross-link β-d-galactopyranosyl-O-pyridinoline and of its unnatural epimer β-d-galactopyranosyl-O-epipyridinoline

Article abstract of DOI:10.1016/j.tetasy.2007.07.006

A short parallel synthesis of β-d-galactopyranosyl-O-pyridinoline (Gal-PYD), a collagen glycoconjugated cross-link, and of Gal-epiPYD, a (5S)-epimer, useful as internal standard in the analytical evaluation of the natural isomer in human tissue, is report

Full text of DOI:10.1016/j.tetasy.2007.07.006

Tetrahedron: Asymmetry 18 (2007) 1750–1757
Total synthesis of the collagen glycosylated cross-link b-D-
galactopyranosyl-O-pyridinoline and of its unnatural epimer b-D-
galactopyranosyl-O-epipyridinoline
Pietro Allevi,* Raffaele Colombo, Elios Giannini and Mario Anastasia
`
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita degli Studi di Milano, via Saldini 50, I-20133 Milano, Italy
Received 4 July 2007; accepted 10 July 2007
Available online 7 August 2007
Abstract—A short parallel synthesis of b-D-galactopyranosyl-O-pyridinoline (Gal-PYD), a collagen glycoconjugated cross-link, and of
Gal-epiPYD, a (5S)-epimer, useful as internal standard in the analytical evaluation of the natural isomer in human tissue, is reported.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
access to all known reducible and non-reducible collagen
cross-links.7 Moreover, we also report the parallel synthesis
of Gal-epiPYD 4, with an unnatural (5S)-hydroxylysine
side chain, confident that it could be differentiated by
HPLC from its natural isomer and consequently could be
used as internal standard in the analytical measurement
of Gal-PYD 2 in bone or in other biological media.
Glucosyl-galactosyl pyridinoline (Glc-Gal-PYD) 1 and
galactosyl pyridinoline (Gal-PYD) 2 are two glycoconju-
gated cross-links of collagen (Fig. 1), the first formed by
the glycosylation of pyridinoline (PYD) 3 mainly present
in synovial tissue, the second formed from PYD mainly
present in bone.1 Recently,2 we reported the first chemical
synthesis of Glc-Gal-PYD 1, which is currently under
extensive investigation as a possible marker of the varia-
tions in joint-tissue remodelling and of the progression of
articular cartilage destruction in osteoarthritis.1,3–5 In fact,
according to Gineyts et al.,3 Glc-Gal-PYD 1 is strongly
associated with the presence of osteoarthritis at the tibio-
femoral and patellofemoral joints in men, and its urinary
levels are considered more specific markers of joint pro-
gressive destruction in various diseases, including rheuma-
toid arthritis.3–5
Herein, we report the different possibilities of synthesising
diastereomerically pure Gal-PYD 2 and Gal-epiPYD 4,
together with their differentiation by HPLC. The final short
protocol proposed starts from a diastereomeric mixture
of tert-butyl (2S,5R)- and tert-butyl (2S,5S)-6-azido-2-ben-
zyloxycarbonylamino-5-hydroxyhexanoate 5 and 6, two
protected synthons of natural (5R)-hydroxylysine and of
its unnatural (5S)-epimer, respectively.8
2. Results and discussion
Gal-PYD 2 has been detected, after alkaline hydrolysis of
different tissues6 but, until now, it has not been available
by synthesis. Its potential utility in the diagnosis and ther-
apy management of metabolism of bone or osteoporosis
has also not been checked.
On the basis of our previous work,2 acquired during the
synthesis of Glc-Gal-PYD 1, the synthesis of Gal-PYD 2
and Gal-epiPYD 4, required the solution of two crucial
synthetic problems as the stereoselective b-glycosylation
of the hydroxylysine side chain and the subsequent assem-
bling of its 1,4,5-trisubstituted 3-hydroxypyridinium ring.
With our previous results in mind,2,9 we first prepared a
series of glycosylated congeners 7, starting from the known
azide 5,8 and then, through the reaction sequence depicted
in Scheme 1, Gal-PYD 2. With a parallel sequence of
reactions, starting from one of the glycosylated azides 11
Intrigued by the possibility of exploring the potential bio-
logical relevance of Gal-PYD 2, we report herein its syn-
thesis, as point of efforts directed towards providing
*
Corresponding author. Fax: +39 02503 16040; e-mail: pietro.allevi@
unimi.it
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.07.006

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
1751
+
+
+
NH3
NH3
CO2- NH3
CO2
NH3
CO2- NH3
+
+
CO2- NH3
+
-O
O
-O
-O
O
-
-
-
CO2
HO
HO
CO2
N+
OH
O
N+
N+
OH
O
HO
HO
5
5
HO
O
OH
OH
+
+
+
NH3
NH3
NH3
-O2C
-O2C
O
-O2C
HO
Glc-Gal-PYD 1
Gal-PYD 2
3
OH
HO
+
NH3
+
CO2- NH3
N3
R1
R2
-O
-
CO2
N+
OH
O
HO
HO
NHCbz
ButO2C
O
5: R1 = OH; R2 = H
6: R1 = H; R2 = OH
OH
+
NH3
-O2C
Gal-epiPYD 4
Figure 1. Pyridinoline and glycosylated pyridinolines.
NHBoc
tBuO2C
t
NHBoc
CO2 Bu
NHBoc
CO2 Bu
-O
O
t
O
9
OBn
O
OBn
O
N3
NH2
BnO
BnO
BnO
BnO
i
OBn
O
Br
N+
BnO
BnO
O
O
Gal-PYD 2
ii
OR1
tBuO2C
R2
OR1
tBuO2C
R2
N
N
OBn
tBuO2C
Bn
Cbz
Cbz
Cbz
N
7
8
10
NHBoc
tBuO2C
O
t
NHBoc
CO2 Bu
NHBoc
CO2 Bu
-O
t
9
OBn
O
OBn
O
N3
NH2
BnO
BnO
BnO
BnO
i
OBn
O
Br
N+
BnO
BnO
O
O
Gal-epiPYD 4
OR1
R2
OBn
Bn
ii
O
N
N
tBuO2C
tBuO2C
OBn
tBuO2C
Bn
Cbz
Cbz
Cbz
N
11
12
13
c: R1 = Bn; R2 = H
d: R1 = Bn; R2 = Bn
a: R1 = H; R2 = H
b: R1 = Ac; R2 = H
Scheme 1. Synthesis of protected galactopyridinolines. Reagents and conditions: (i) SnCl2, PhSH, Et3N, THF, rt, 2 h, 83–86%; (ii) Na2CO3, MeCN, rt,
6 h, then Na2CO3, O2, MeOH, rt, 7 d, 51–61%.
derived2 from the epimeric azide 6, we prepared Gal-5epi-
PYD 4 (Scheme 1).
of thiophenol and triethylamine in THF.10 The obtained
glycosylated amine 8a was then reacted with an excess of
bromoketone 9 (1:2.5), in CH3CN containing Na2CO3, to
promote the initial dialkylation of the amino group of 8a
and to start the sequence of ‘one-pot reactions’ leading to
the 4,5-disubstituted 3-hydroxypyridinium compound 10
As shown in Scheme 1, azide 7a was reduced to the corre-
sponding amine 8a by reaction with a tin(II) complex
formed by treatment of SnCl2 with appropriate amounts

1752
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
(inner condensation of the initially formed amino diketone,
followed by air oxidation of the cyclic dienol formed).11
Moreover, the reaction afforded poor results and only trace
amounts of a fluorescent compound, having the molecular
mass of 10, could be detected in the reaction mixture (iso-
lated by TLC). We considered that this result could be due
to the concurrent interaction of the free 2-hydroxyl group
with bromoketone 9. In fact, we obtained similar results
starting from the glycosylated hydroxylysine 8b, obtained
by acetylation of 7a to 7b and successive reduction of the
azide group, since the 2-acetate undergoes hydrolysis under
the basic conditions of the reaction. At this point we
decided to protect the 2-hydroxy group as a benzyl deriva-
tive and to start the synthesis with the compound 8c. This
protection should not prolong the synthesis since, at the
end, a contemporaneous regeneration of all groups protect-
ing the galactose portion of the molecule could still be pos-
sible. Moreover, when we tried to obtain compound 8c by
selective benzylation of the 2-hydroxy group of 7a and sub-
sequent chemical reduction, we observed, under different
reaction conditions, that a concurrent benzylation of the
free amidic hydrogen, with the formation of the dibenzy-
lated azide 7d (TLC and MS evidences) was always opera-
tive. Thus we decided to start the synthesis from the
completely benzylated amine 8d. This was obtained by benz-
ylation of 7a, with benzyl bromide and sodium hydride in
the presence of tetrabutylammonium iodide, and successive
chemical reduction of the azide group. The glycosylated
amine 8d was then reacted with an excess of bromoketone
derivative 9 (1:2.5 molar ratio) in CH3CN containing
Na2CO3 to prepare, in a ‘one-pot reaction’, glycoconjugate
10. The initial dialkylation showed the expected course and
was complete after 6–8 h. At this point, according to our
protocol,2,11 the CH3CN was replaced by MeOH and the
resulting mixture vigorously shaken under a slight pressure
of oxygen (1.3 atm) at room temperature for 70 h. Stan-
dard work-up and chromatography afforded the glycocon-
jugate compound 10 in satisfactory yields (61%), as a glass
that showed the expected physico-chemical properties, ele-
mental analysis and molecular ion, together with appropri-
ate NMR evidences, which was consistent with the
presence of the pyridinium ring, three carboxylic groups
and an intact b-bonded galactosidic ring. Treatment of gly-
coconjugate 10 with aqueous trifluoracetic acid (Scheme 2)
regenerated all the carboxylic groups and the Boc protected
amino groups, affording benzylated compound 14, which
in turn, via catalytic reduction, afforded the desired Gal-
PYD 2 (Scheme 2).
A parallel sequence of reactions, starting from the epimeric
azide 11a, afforded the epimeric compounds 12 and 13
(Scheme 1). Hydrolysis of compound 13 (Scheme 2) affor-
ded amino acid 15 and finally the desired Gal-epiPYD 4.
Thus, we reached our synthetic goal but however we did
not consider it completely satisfactory due to the laborious
preparation8,9 of the starting compounds 7a and 11a,
which reduces the relative simplicity of the total syntheses
of Gal-PYD 2 and Gal-epiPYD 4. In fact, the preparation
of glycoconjugated azides 7a and 11a (starting compounds
of the syntheses) also suffers from the difficulty of the prep-
aration of the starting material, that is, the diastereomeri-
cally pure (5R)- and (5S)-masked hydroxylysines 5 and 6,
which at best are obtainable in diastereomeric pure form
after a laborious three step separation8,12,13 of their diaste-
reomeric mixture.
Thus, we considered the possibility of simplifying the prep-
aration of glycoconjugated azides 7a and 11a, avoiding the
initial separation of the masked hydroxylysines 5 and 6.
+
NHBoc
NH3
t
-
CO2
+
CO2 Bu
NHBoc
CO2 Bu
NH3
-O
-O
t
-
CO2
N+
N+
OBn
O
OBn
O
BnO
BnO
BnO
BnO
i
ii
Gal-PYD 2
O
O
OBn
tBuO2C
Bn
Cbz
OBn
Bn
Cbz
N
N
-O2C
14
10
+
NHBoc
NH3
t
-
CO2 Bu
CO2
+
-
NHBoc
CO2 Bu
NH3
CO2
-O
-O
O
t
N+
N+
OBn
O
OBn
O
BnO
BnO
BnO
BnO
i
ii
Gal-epiPYD 4
O
OBn
Bn
OBn
Bn
N
N
Cbz
tBuO2C
Cbz
-O2C
15
13
Scheme 2. Deprotection to obtain the galactopyridinolines. Reagents and conditions: (i) TFA, rt, 1 h; (ii) H2, PdCl2, MeOH–H2O–AcOH, rt, 12 h, 88–
89% over two steps.

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
1753
OBn
O
OBn
O
N3
BnO
BnO
BnO
BnO
NH
OCCCl3
O
OBn
O
N3
BnO
BnO
N3
OH
tBuO2C
OAc
16
silica gel
column
NHCbz
O
HO
OR1
tBuO2C
17: R1 = Ac
7a+11a: R1 = H
7a
N3
i
NHCbz
NHCbz
tBuO2C
OBn
O
BnO
BnO
5+6
ii
O
OH
NHCbz
tBuO2C
11a
t
˚
Scheme 3. Shortened parallel preparation of the starting materials. Reagents and conditions: (i) BuMe2SiSO3CF3, molecular sieves 3 A, Et2O, rt, 1 h,
51%; (ii) Cs2CO3, MeOH, rt, 6 h, 39% (7a) and 41% (11a).
With the glycosylated azides (5R)-7a and (5S)-11a in hand,
we found that it was possible to separate them by simple
rapid column chromatography. This allowed their prepara-
tion to be shortened, which could start from a diastereo-
meric mixture of the hydroxyazides, 5 and 6, thus
avoiding their initial laborious separation. Moreover, we
were able to additionally shorten the synthesis of glycosyl-
ated azides (5R)-7a and (5S)-11a using as a galactosyl
donor, in the glycosidation of 5 and 6, the tribenzyl-2-
acetylgalactosyl-1-trichloracetimidate 16 in place of the
corresponding 2-chloroacetate used previously,9 done
before experiencing the lability of the 2-acetate to basic
medium (Scheme 3). Acetate 16 can be easily prepared in
6 steps from commercial galactose14 while the preparation
of the tribenzyl-2-chloroacetylgalactosyl-1-trichloracetami-
date requires 8 steps from the same parent sugar.9
4. Experimental
4.1. General methods
Nuclear magnetic resonance spectra were recorded at
298 K on Bruker AM-500 spectrometer operating at
500.13 MHz for 1H and 125.76 MHz for 13C. Chemical
shifts are reported in parts for million (ppm, d units) and
are referenced to residual CHCl3 (dH = 7.26 ppm) and to
CDCl3 (dC = 77.0 ppm) for solutions in CDCl3 or to inter-
nal CH3OD (dH = 3.34 ppm and dC = 49.5 ppm) for solu-
tions in D2O. 1H NMR data are tabulated in the
following order: number of protons, multiplicity (s, singlet;
d, doublet; br s, broad singlet; m, multiplet), coupling con-
stant(s) in Hertz, assignment of proton(s). The H and 13C
1
resonances were assigned by 1H decoupling, H–1H COSY
1
and H–13C correlation experiments. The nomenclature of
1
Thus a convenient and relatively short parallel synthesis
has been achieved of the unreported glycoconjugated
Gal-PYD 2 and Gal-epiPYD 4, preparing the starting
azides according to the sequence of reaction reported in
Scheme 3 and pursuing the synthesis according to the reac-
tion sequence reported in Schemes 1 and 2.
the single positions is given as follows (Fig. 2).
+
NH3
4chain
2
-
+
-
1
CO2
NH3
-O
2
3
1
4
CO2
3
5
5chain
1
Gal
N+
With the galactosylated pyridinolines in the hand, we also
found the analytical HPLC conditions (see Section 4)
required to separate the native and the unnatural isomer
which then is a suitable inner standard for the analysis of
Gal-PYD 2.
OH
HO
4
HO
6
6
O
5
O
Hyl
2
4
1
3
OH
3
+
NH3
2
-O2C
Figure 2. Carbon numeration used in this work.
3. Conclusion
In conclusion a short, simple synthesis of Gal-PYD 2 and
Gal-epiPYD 4 both of interest for studies in the biological
relevance of GalPYD 2 levels in bone and in other biolog-
ical tissues has been achieved.
Optical rotations were taken on a Perkin–Elmer 241 polar-
imeter and [a]D values are given in 10ꢀ1 deg cm2 gꢀ1. Mass
spectra were obtained using
a Finnigan LCQdeca
(ThermoQuest) ion trap mass spectrometer fitted with an
electrospray source (ESI). The spectra were collected in
continuous flow mode by connecting the infusion pump
directly to the ESI source. Solutions of compounds were
infused at a flow rate of 0.5 mL/min. The spray voltage
was set at 5.0 kV in the positive and at 4.5 kV in the nega-
tive ion mode with a capillary temperature of 220 ꢁC. Full-
scan mass spectra were recorded by scanning a m/z range
of 100–2000. All reactions were monitored by thin-layer
The results of the present work have also allowed us to sim-
plify and shorten the preparation of galactosyl hydroxyly-
sine and galactosyl epihydroxylysine, two compounds of
biological interest. The shortening of the preparation of
azides 7a and 11a represents a formal shortening of our
previously reported synthesis of these glycosylated amino
acids.9

1754
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
chromatography (TLC) carried out on 0.25 mm E. Merck
Silica Gel plates (60 F254) using UV light, 50% sulfuric acid,
anisaldehyde/H2SO4/EtOH solution or 0.2% ninhydrin in
ethanol and heat as developing agent. E. Merck 230–
400 mesh silica gel was used for flash column chromato-
graphy.15 Work-up refers to washing with water, drying
over Na2SO4 and evaporation of the solvent.
3.64 (2H, m, HGal-6), 3.59 (1H, dd, J = 11.4, 4.9, HGal-5),
3.54 (1H, dd, J = 10.1, 2.7, HGal-3), 3.32 (1H, dd,
J = 13.0, 4.0, HHyl-6a), 3.20 (1H, dd, J = 13.0, 6.5, HHyl
-
6b), 2.06 (3H, s, CH3CO), 1.92 and 1.81 (2 · 1H, 2m,
HHyl-3), 1.56 (2H, m, HHyl-4), 1.42 [3H, s, C(CH3)3]; 13C
NMR (CDCl3) d: 171.4 (COOtBu), 169.6 (CH3COO),
155.9 (NCOOR), 139–127 (aromatics), 100.7 (CGal-1),
82.1 [C(CH3)3], 80.4 (CGal-3), 76.6 (CHyl-5), 74.5
(OCH2Ph), 73.5 (OCH2Ph), 73.5 (CGal-5), 72.6 (CGal-4),
72.0 (OCH2Ph), 71.3 (CGal-2), 68.5 (CGal-6), 66.7
(OCH2Ph), 54.7 (CHyl-6), 53.8 (CHyl-2), 28.2 (CHyl-4),
28.2 (CHyl-3) 27.9 [C(CH3)3], 21.0 (CH3CO); ESI-MS (po-
sitive) m/z: 875.4 (M+Na+). Anal. Calcd for C47H58-
N2O11: C, 68.26; H, 7.07; N, 3.39. Found: C, 68.39; H,
7.20; N, 3.31.
4.2. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonylamino-
5-(3,4,6-tri-O-benzyl-2-O-acetyl-b-D-galactopyranosyl)hex-
anoate 7b
The tert-butyl (2S,5R)-6-azido-2-benzyloxycarbonylamino-
5-(3,4,6-tri-O-benzyl-2-hydroxy-b-D-galactopyranosyl)hex-
anoate 7a (330 mg; 0.41 mmol) was dissolved in pyridine
(1 mL) and treated with Ac2O (0.9 mL) at room tempera-
ture for 3 h. The addition of methanol followed by dilution
with ethyl acetate and washing with a solution of citric acid
afforded, after the usual work-up, the 2-acetylated com-
pound 7b (270 mg; 78%): an oil, showing in TLC
Rf = 0.30 (eluting with hexane/AcOEt; 70:30; v/v);
4.4. tert-Butyl (2S,5R)-6-azido-2-[benzyl(benzyloxycarb-
ony)amino]-5-(2,3,4,6-tetra-O-benzyl-b-D-galactopyranos-
yl)hexanoate 7d
In a dried round bottom flask, a solution of galactosyl
azide 7a (338 mg; 0.417 mmol) in anhydrous THF (4 mL)
was prepared and cooled to 0 ꢁC. NaH (64 mg; 60% in min-
eral oil; 1.73 mmol) was added and the reaction was stirred
at the same temperature for 10 min. Benzyl bromide
(205 lL; 1.73 mmol) and tetrabutylammonium iodide
(127 mg; 0.345 mmol) were then added and the reaction
allowed to warm to room temperature and left under
stirring overnight.
20
1
½aꢁD ¼ þ9:3 (c 1, CH2Cl2); H NMR (CDCl3) d: 7.1–7.2
(aromatic protons), 5.36 (2H, overlapping, d, J 7.5, NH
and dd, J = 10.1, 7.9, HGal-2), 5.2–4.4 (8H, benzylic pro-
tons), 4.43 (HGal-1, overlapped with benzylic protons),
4.24 (1H, m, HHyl-2), 3.94 (1H, d, J = 2.8, HGal-4), 3.67
(1H, m, HHyl-5), 3.63 (2H, m, HGal-6), 3,58 (1H, dd,
J = 6.6, <1, HGal-5), 3.50 (1H, dd, J = 10.1, 2.8, HGal-3),
3.40 (2H, m, HHyl-6), 2.02 (3H, s, CH3CO), 1.88 (1H, m,
HHyl-3a), 1.71 (0.5H, m, HHyl-3b), 1.62 (2.5H, m, HHyl-4
and HHyl-3b), 1.48 [3H, s, C(CH3)3]; 13C NMR (CDCl3)
d: 171.1 (COOtBu), 169.3 (CH3COO), 155.7 (NCOOR),
139–127 (Aromatics), 101.6 (CGal-1), 82.4 [C(CH3)3], 80.3
(CGal-3), 78.3 (CHyl-5), 74.4 (OCH2Ph), 73.7 (CGal-5),
73.6 (OCH2Ph), 72.4 (CGal-4), 72.0 (OCH2Ph), 71.4
(CGal-2), 68.7 (CGal-6), 66.9 (OCH2Ph), 54.5 (CHyl-6),
54.1 (CHyl-2), 28.3 (CHyl-3), 27.9 (CHyl-4) 27.9 [C(CH3)3],
20.9 (CH3CO); ESI-MS (positive) m/z: 875.4 (M+Na+).
Anal. Calcd for C47H56N4O11: C, 66.18; H, 6.62; N, 6.57.
Found: C, 66.29; H, 6.48; N, 6.61.
The reaction was quenched by adding an NaH2PO4 aque-
ous solution (1.2 M, 5 mL), diluted with AcOEt (20 mL)
and worked-up. The flash chromatography purification of
the crude material, (eluting with hexane/AcOEt; 80:20;
v/v), gave the title compound 7d (289 mg; 70% yield), in pure
20
form, as an amorphous solid: ½aꢁD ¼ ꢀ2:4 (c 1, CH2Cl2);
1H NMR (CDCl3, T = 323 K) d: 7.1–7.2 (30H, aromatic
protons), 5.23–4.48 (12H, benzylic protons), 4.32 (HGal-1,
overlapping with benzylic protons), 4.16 (1H, b, HHyl-2),
3.89 (1H, dd, J 3.1, 3.0, HGal-4), 3.80 (1H, dd, J = 9.7,
7.6, HGal-2), 3.64 (2H, m, HGal-6), 3.57 (1H, m, HHyl-5),
3.51 (1H, dd, J 6.4, 3.0, HGal-5), 3.48 (1H, dd, J = 9.7,
3.0, HGal-3), 3.3 and 3.2 (2 · 1H, 2m, HHyl-6), 2.1 and 1.8
(2 · 1H, 2m, 2 conformers in the ratio 6:4, HHyl-3), 1.5
(2H, 2m, 2 conformers in the ratio 6:4, HHyl-4), 1.35 [3H,
s, C(CH3)3]; 13C NMR (CDCl3, T = 323 K) d: 169.9
(COOR), 156.7 (NCOOR), 139–127 (aromatics), 103.4
(CGal-1), 82.4 (CGal-3), 81.6 [C(CH3)3], 79.6 (CGal-2), 78.1
(CHyl-5), 75.2 (OCH2Ph), 74.5 (OCH2Ph), 73.9 (CGal-4),
73.6 (CGal-5), 73.6 (OCH2Ph), 73.1 (OCH2Ph), 69.0
(CGal-6), 67.5 (OCH2Ph), 60.9 (CHyl-2), 54.5 (CHyl-6),
4.3. tert-Butyl (2S,5R)-6-amino-2-benzyloxycarbonylamino-
5-(3,4,6-tri-O-benzyl-2-O-acetyl-b-D-galactopyranosyl)hex-
anoate 8b
Anhydrous SnCl2 (79 mg; 0.42 mmol) was dissolved under
stirring into anhydrous THF (1.0 mL) at 25 ꢁC, after which
PhSH was added (174 lL; 1.69 mmol) followed by Et3N
(174 lL; 1.27 mmol). After 20 min, the azide 7b (240 mg;
0.28 mmol), dissolved in anhydrous THF (1.0 mL), was
added and stirring continued for 2 h. At this time, the sol-
vent was evaporated under reduced pressure and the resi-
due was recovered with dichloromethane and washed
with aqueous NaOH (1 M ). After the usual work-up, the
residue (250 mg) was chromatographed (eluting with
50.5 (NCH2Ph), 29.4 (CHyl-4), 27.9 [C(CH3)3], 26.1 (CHyl
-
3). ESI-MS (positive) m/z: 1008.3 (M+NH4þ), 10013.5
(M+Na+). Anal. Calcd for C59H66N4O10: C, 71.49; H,
6.71; N, 5.65. Found: C, 71.35; H, 6.59; N, 5.51.
CH2Cl2/MeOH; 100:4; v/v) to afford the desired amine
20
8d (200 mg; 86% yield); ½aꢁD ¼ þ5:2 (c 1, CH2Cl2); 1H
4.5. tert-Butyl (2S,5R)-6-amino-2-[benzyl(benzyloxycarb-
ony)amino]-5-(2,3,4,6-tetra-O-benzyl-b-D-galactopyranos-
yl)hexanoate 8d
NMR (CDCl3) d: 7.1–7.2 (aromatic protons), 5.36 (2H,
overlapping, d, J 8.3, NH, dd, J = 10.1, 7.9, HGal-2), 5.1–
4.4 (8H, benzylic protons), 4.49 (HGal-1, overlapped with
benzylic protons), 4.28 (1H, ddd, J = 12.8, 7.8, 4.5, HHyl
-
To a solution of anhydrous SnCl2 (73 mg; 0.385 mmol) in
2), 3.98 (1H, d, J = 2.7, HGal-4), 3.75 (1H, m, HHyl-5),
anhydrous THF (3 mL), PhSH (150 lL; 1.36 mmol) and

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
1755
Et3N (150 lL; 1.08 mmol) were added and left under stir-
ring for 5 min. A solution of the azide 7d (227 mg;
0.229 mmol in 1 mL of dry THF) was then added. The
reaction was stirred at room temperature for 2 h, after
which the solvent was evaporated and the crude residue
purified by flash chromatography. By-products were eluted
using CH2Cl2/MeOH (98:2; v/v) and the glycosylated
amine 8d using CH2Cl2/MeOH (92:8; v/v) (168 mg; 76%
(OCH2Ph), 75.1 (OCH2Ph), 74.6 (OCH2Ph), 73.5 (OCH2Ph),
73.4 (CGal-4), 73.3 (CGal-5), 72.9 (OCH2Ph), 68.6
(CGal-6), 67.6 (OCH2Ph), 32.9 (C5Ch-2), 28.4 and 28.3
[3 · C(CH3)3], 28.3 (C4Ch-1), 28.0, 28.0 and 27.8
[3 · C(CH3)3], 26.3 (C5Ch-1), 25.5 and 25.1 (CHyl-3 and
4). ESI-MS (positive) m/z:1565.6 (20%; M+Na), 1543.6
(100%, M+H+). Anal. Calcd for C89H114N4O19: C, 69.24;
H, 7.44; N, 3.63. Found: C, 69.19; H, 7.35; N, 3.57.
yield). The compound, obtained as a glassy material,
20
showed: ½aꢁD ¼ ꢀ0:7 (c 1, CH2Cl2); 1H NMR (CDCl3,
4.7. Preparation of b-D-galactopyranosyl-O-pyridinoline
Gal-PYD 2
323 K) d: 7.2–7.1 (30H, aromatic protons), 5.2–4.2
(12H, benzylic protons), 4.31 (HGal-1, overlapping with
benzylic protons), 4.05 (1H, m, HHyl-2), 3.86 (1H, br s,
HGal-4), 3.77 (1-H, m, HGal-2), 3.66 (1H, m, HGal-6a), 3.6
(m, HHyl-5, partially overlapped), 3.57–3.52 (3H, overlap-
ping HGal-5, HGal-6b and HGal-3), 2.8 (2H, m, HHyl-6),
2.1 and 1.8 (2 · 1H, 2m, HHyl-3), 1.5 (2H, m, HHyl-4),
1.34 [3H, s, C(CH3)3]; 13C NMR (CDCl3, T = 323 K) d:
169.8 (COOR), 156.5 (NCOOR), 139–127 (aromatics),
Compound 10 (110 mg; 0.071 mmol) was dissolved in
CF3COOH/H2O (2 mL; 95:5, v/v) and the resulting solu-
tion was stirred at room temperature for 1 h. The solvent
was then evaporated under reduced pressure and the resi-
due triturated with a 1:1 mixture of diisopropyl ether/
hexane, affording the partially protected glycosylated
pyridinoline 14 as its tris-trifluoracetate salt as a powder.
1
103.4 (CGal-1), 82.4 (CGal-3), 81.5 [C(CH3)3], 80.2 (CGal
-
The H NMR spectrum was recorded in order to verify
5), 79.3 (CGal-2), 75.1 (OCH2Ph), 74.7 (OCH2Ph), 73.8
(CGal-4), 73.5 (OCH2Ph) 73.5 (CGal-5), 73.18 (OCH2Ph),
68.6 (CGal-6), 67.5 (OCH2Ph), 60.9 (CHyl-2), 50.6
(NCH2Ph), 44.8 (CHyl-6), 30.4 (CHyl-4), 27.9 [C(CH3)3],
25.6 (CHyl-3). ESI-MS (positive) m/z: 965.5 (M+H+). Anal.
Calcd for C59H68N2O10: C, 73.42; H, 7.10; N, 2.90. Found:
C, 72.85; H, 7.25; N, 3.07.
the complete cleavage of the Boc groups and of the tert-
butyl esters. The obtained compound was dissolved in
55 mL of a MeOH/H2O/AcOH mixture (8:2:1; v/v/v).
PdCl2 (50 mg) was added and the reaction mixture was sha-
ken overnight, at room temperature, under an atmospheric
pressure of hydrogen. The solution was then filtrated, after
which MeOH was evaporated under reduced pressure and
the remaining solution diluted with water and loaded on a
strong acidic resin column (2 mL; Dowexꢂ 50WX8-200).
The resin was washed with water and finally with a 1 M
NH3 solution in H2O/MeOH (2:1; v/v) to recover the prod-
uct. After evaporation of MeOH the solution was freeze-
dried and the residue was dissolved in water and freeze-
dried twice to afford the title compound 2 (37 mg; 88%
yield over two steps) as a fluffy material which showed:
½aꢁD ¼ ꢀ4:4 (c 0.5, H2O); H NMR (D2O) d: 7.70 (2H, br
s, pyridinium protons), 4.58 (1H, m, HHyl-6a), 4.38 (1H,
d, J = 7.7, HGal-1), 4.32 (1H, m, HHyl-6b), 4.26 (1H, m,
HHyl-5), 4.02 (1H, dd, J = 6.6, 4.6, H4Ch-2), 3.86–3.75
(3H, overlapping, HHyl-2, H5Ch-3 and HGal-4), 3.59 (1H,
dd, J = 9.8, 3.5, HGal-3), 3.54–3.41 (4H, overlapping,
HGal-2, HGal-5, HGal-6a and HGal-6b), 3.32-3.24 (2H, AB
system, H4Ch-1a and H4Ch-1b), 2.96 (1H, m, H5Ch-1a),
4.6. Completely protected b-D-galactopyranosyl-O-pyridin-
oline 10
To a solution of glycosylated amine 8d (168 mg; 0.174
mmol) in CH3CN (15 mL) containing Na2CO3 (368 mg;
3.48 mmol), the protected bromoketone
6 (165 mg;
0.435 mmol) was added and the mixture was stirred under
nitrogen for 6 h. At this time, the disappearance of both the
starting glycosylated amine 8d and of the initially formed
monoalkylated product was observed (TLC; CH2Cl2/
MeOH; 100:5; v/v; Rf = 0.3 and 0.7, respectively). The sol-
vent was then evaporated under reduced pressure after
which the crude residue was recovered with MeOH
(30 mL) and vigorously shaken, under a slight pressure of
oxygen (1.3 atm), at room temperature for 7 days. After
this period of time, the mixture was diluted with CH2Cl2
(50 mL) and filtered on a pad of Celite. Evaporation of
the solvent afforded a crude residue, which was purified
by flash chromatography on silica. Elution with CH2Cl2/
MeOH (100:4; v/v) afforded the protected glycosylated
pyridinoline 10 (136 mg; 61% yield) as a resinous material:
20
1
2.72 (1H, m, H5Ch-1b), 2.21–2.08 (3H, overlapping, H5Ch
-
2a, H5Ch-2b and HHyl-3a), 2.00 (1H, m, HHyl-3b), 1.81
(1H, m, HHyl-4a), 1.69 (1H, m, HHyl-4b); 13C NMR
(D2O, T = 318 K) d: 172.4, 172.2, 171.7 (3 · COOH),
163.3, 140.6, 137.4, 130.9, 128.2 (pyridinium ring), 101.0
(CGal-1), 75.8 (CHyl-5), 72.8 (CGal-5), 70.9 (CGal-3), 69.1
(CGal-2), 66.6 (CGal-4), 61.7 (CHyl-6), 58.9 (CGal-6), 52.7
(CHyl-2), 52.6 (C4Ch-2), 52.5 (C5Ch-3), 29.3 (C5Ch-2), 26.5
(C4Ch-1), 25.9 (CHyl-4), 24.3 (CHyl-3), 24.1 (C5Ch-1). Anal.
Calcd for C24H38N4O13: C, 48.81; H, 6.49; N, 9.49. Found:
C, 48.89; H, 6.55; N, 9.56.
20
1
½aꢁD ¼ ꢀ0:7 (c 1, CH2Cl2); H NMR (CDCl3, T = 318 K)
d: 7.5–7.0 (30H, aromatic protons), 5.20–4.44 (12H, benz-
ylic protons), 4.2 (overlapping, HGal-1, HHyl-2 and
H4Ch-2), 4.07 (1H, b, H5Ch-3), 3.83 (1H, br s, HGal-4),
3.69 (1H, m, HGal-2), 3.47–3.37 (5H, overlapping, HHyl-5,
HGal-5, HGal-6 HGal-3), 3.25 (1H, dd, J 11.4, 11.2, H4Ch
-
1a), 2.92 (1H, dd, J = 12.6, <1, H4Ch-1b), 2.55 (2H, m,
H5Ch-1), 2.1–1.5 (overlapping, HHyl-3, H5Ch-2, HHyl-4),
1.48, 1.47, 1.45, 1.41 and 1.33 [5 · 3H, 5s, 5 · C(CH3)3];
13C NMR (CDCl3, T = 318 K) d: 171.3, 169.6, 169.6
(COOR), 156.5, 155.1, 143.3, 136.4, 130.5 (pyridinium
ring), 103.8 (CGal-1), 82.2 (CGal-3), 82.0, 81.7, 81.0
[3 · C(CH3)3], 79.2 (CGal-2), 79.1, 77.8 [2 · C(CH3)3], 75.2
4.8. tert-Butyl (2S,5S)-6-azido-2-[benzyl(benzyloxycarb-
ony)amino]-5-(2,3,4,6-tetra-O-benzyl-b-D-galactopyran-
osyl)hexanoate 11d
Starting with the hydroxy azide 11a (316 mg; 0.389 mmol)
and following the procedure described above for com-
pound 7d, the protected azide 11d was prepared (290 mg;

1756
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
20
1
75% yield); ½aꢁD ¼ þ7:0 (c 1, CH2Cl2); H NMR (CDCl3,
T = 323 K) d: 7.1–7.2 (30H, aromatic protons), 5.23
(12H, benzylic protons), 4.42 (HGal-1, overlapping with
benzylic protons), 3.89 (1H, d, J = 1.8, HGal-4), 3.80 (1H,
(OCH2Ph), 73.9 (CGal-4), 73.6 (CGal-5), 73.5 (OCH2Ph),
72.9 (OCH2Ph), 68.6 (CGal-5), 67.6 (OCH2Ph), 32.9
(C5Ch-2), 28.4 and 28.3 [2 · C(CH3)3], 28.3 (C4Ch-1), 28.0,
28.0 and 27.8 [3 · C(CH3)3], 26.3 (C5Ch-1) 25.5 and 25.1
(CHyl-3 and C5Ch-4); ESI-MS (positive) m/z:1565.6 (20%;
M+Na), 1543.6 (100%, M+H+). Anal. Calcd for
C89H114N4O19: C, 69.24; H, 7.44; N, 3.63. Found: C,
69.39; H, 7.31; N, 3.77.
dd, J = 9.7, 7.7, HGal-2), 3.7–3.5 (5H, overlapping, HGal
-
6, HHyl-5, HGal-5 and HGal-3), 3.2 (2H, m, HHyl-6), 2.1–
1.4 (4H, m, HHyl-3 and HHyl-4), 1.35 [3H, s, C(CH3)3];
13C NMR (CDCl3, T = 323 K) d: 170.1 (COOR), 156.6
(NCOOR), 138–127 (aromatics), 103.0 (CGal-1), 82.6
(CGal-3), 81.7 (CHyl-5), 81.4 [C(CH3)3], 79.5 (CGal-2), 75.4
(OCH2Ph), 74.7 (OCH2Ph), 73.9 (CGal-4), 73.5 (OCH2Ph),
73.3 (CGal-5), 72.9 (OCH2Ph), 68.8 (CGal-6), 67.4
(OCH2Ph), 60.9 (CHyl-2), 54.3 (CHyl-6), 51.0 (NCH2Ph),
29.9 (CHyl-4), 27.9 [C(CH3)3], 25.8 (CHyl-3). ESI-MS (posi-
tive) m/z: 1008.3 (M+NH4þ), 10013.5 (M+Na+). Anal.
Calcd for C59H66N4O10: C, 71.49; H, 6.71; N, 5.65. Found:
C, 71.55; H, 6.63; N, 5.51.
4.11. b-D-Galactopyranosyl-O-epipyridinoline 4
Starting with the completely protected compound 13
(90 mg, 0.058 mmol) and following the procedure described
above for compound 2, title compound 4 was prepared
20
(30 mg, 88% yield over two steps): ½aꢁD ¼ þ3:8 (c 0.5,
1
H2O); H NMR (D2O) d: 7.81 (1H, br s, pyridinium pro-
ton), 7.77 (1H, br s, pyridinium proton), 4.58 (1H, m,
HHyl-6a), 4.38–4.34 (2H, overlapping, HHyl-5 and HHyl
-
4.9. tert-Butyl (2S,5S)-6-amino-2-[benzyl(benzyloxycarb-
ony)amino]-5-(2,3,4,6-tetra-O-benzyl-b-D-galactopyranos-
yl)hexanoate 12
6b), 4.14 (1H, d, J = 7.7, HGal-1), 4.04 (1H, dd, J = 6.6,
4.9, H4Ch-2), 3.89-3.71 (5H, overlapping, HHyl-2, H5Ch-3,
HGal-4, HGal-6a and HGal-6b), 3.60 (1H, m, HGal-5), 3.54
(1H, dd, J = 10.1, 3.1, HGal-3), 3.45 (1H, dd, J = 10.1,
7.7, HGal-2), 3.32 (1H, dd, J = 14.1, 6.6, H4Ch-1a), 3.26
(1H, dd, J = 14.1, 4.9, H4Ch-1b), 2.95 (1H, ddd, J = 14.3,
12.2, 5.2, H5Ch-1a), 2.73 (1H, ddd, J = 14.3, 11.9, 5.2,
H5Ch-1b), 2.20-2.02 (4H, overlapping, H5Ch-2a, H5Ch-2b,
HHyl-3a and HHyl-3b), 1.75 (1H, m, HHyl-4a), 1.63 (1H,
m, HHyl-4b); 13C NMR (D2O, T = 318 K) d: 172.3, 171.9,
171.2 (3 · COOH), 163.3, 140.7, 137.3, 130.0, 127.4 (pyrid-
inium ring), 100.4 (CGal-1), 75.0 (CHyl-5), 73.0 (CGal-5),
70.6 (CGal-3), 68.6 (CGal-2), 66.4 (CGal-4), 60.7 (CHyl-6),
58.9 (CGal-6), 52.2, 52.1, 52.1 (C4Ch-2, C5Ch-3, CHyl-2),
28.9 (C5Ch-2), 26.1 (C4Ch-1), 25.5 (CHyl-4), 23.8, 23.6
(C5Ch-1, CHyl-3). Anal. Calcd for C24H38N4O13: C, 48.81;
H, 6.49; N, 9.49. Found: C, 48.73; H, 6.42; N, 9.43.
Starting with azide 11d (330 mg; 0.332 mmol) and follow-
ing the procedure described above for compound 8d, title
compound 12 was prepared (268 mg; 83% yield):
20
1
½aꢁD ¼ þ4:8 (c 1, CH2Cl2); H NMR (CDCl3, T = 318 K)
d: 7.1–7.2 (30H, aromatic protons), 5.2–4.4 (12H, benzylic
protons), 4.60 (HHyl-2, overlapped with benzylic protons),
4.43 (HGal-1, overlapped with benzylic protons), 3.93
(1H, br s, HGal-4), 3.80 (1H, dd, J 9.7, 7.8, HGal-2), 3,66
(1H, dd, J = 8.5, 7.9, HGal-6a) 3.6–3.5 (3H, overlapped,
HGal-6b, HGal-5 and HGal-3), 3.45 (1H, b, HHyl-5), 3.2
(2H, m, HHyl-6), 2.1–1.4 (4H, m, HHyl-3 and HHyl-4),
1.33 [3H, s, C(CH3)3]; 13C NMR (CDCl3) d: 170.1
(COOR), 156.7 (NCOOR), 138–127 (aromatics), 103.3
(CGal-10), 82.8 (CGal-30), 81.7 (CHyl-5), 81.3 [C(CH3)3],
79.7 (CGal-20), 75.2 (OCH2Ph), 74.7 (OCH2Ph), 73.9
(CGal-40), 73.5 (OCH2Ph), 73.3 (CGal-50), 73.2 (OCH2Ph),
4.12. 3 tert-Butyl (2S,5R)- and (2S,5S)-6-azido-2-benzyl-
oxycarbonylamino-5-(3,4,6-tri-O-benzyl-2-hydroxy-b-D-
galactopyranosyl)hexanoate 7a and 11a
68.9 (CGal-60), 67.5 (OCH2Ph), 61.0 (CHyl-2), 54.3 (CHyl
-
6), 51.0 (NCH2Ph), 30.0 (CHyl-4), 27. [C(CH3)3], 25.8
(CHyl-3). ESI-MS (positive) m/z: 965.5 (M+H+). Anal.
Calcd for C59H68N2O10: C, 73.42; H, 7.10; N, 2.90. Found:
C, 73.74; H, 7.02; N, 2.77.
A mixture of tert-butyl (2S,5R)- and (2S,5S)-6-azido-2-
benzyloxycarbonylamino-5-hydroxyhexanoate
5 and 6
(2.6 g; 6.9 mmol), O-(3,4,6-tri-O-benzyl-2-O-acetyl-a-D-
galactopyranosyl) trichloroacetimidate 16 (5.4 g; 8.5 mmol)
˚
4.10. Completely protected b-D-galactopyranosyl-O-epipyri-
dinoline 13
and powdered molecular sieves (3 A, 0.8 g) in anhydrous
diethyl ether (40 mL) was stirred for 15 min at 25 ꢁC. After
this time, tert-butyldimethylsilyl trifluoromethanesulfonate
(250 lL; 1.08 mmol) was added and stirring was continued
for 1 h under argon. The powdered molecular sieves were
then filtered off and washed with AcOEt and the organic
solution was worked-up. The residue was purified by flash
chromatography (eluting with hexane/AcOEt; 75:25; v/v)
to give an inseparable mixture of the crude diastereomeric
title compounds 7b and 11b (2.98 g; 51% yield): an oil;
Starting with amine 12 (251 mg, 0.260 mmol) and following
the procedure described above for compound 10, title com-
20
pound 13 was prepared (205 mg, 51% yield): ½aꢁD ¼ þ12:2
(c 1, CH2Cl2); 1H NMR (CDCl3 T = 323 K) d: 7.5–7.0
(30H, aromatic protons), 5.2–4.5 (12H, benzylic protons),
4.2 (3H, overlapping, HGal-1, H4Ch-2, H5Ch-3), 3.90 (1H,
br s, HGal-4), 3.74 (1H, dd, J 8.5, 8.5, HGal-2), 3.6–3.4
(5H, overlapping, HHyl-5, HGal-5, HGal-6, HGal-3), 3.25
and 2.82 (2 · 1H, 2m, H4Ch-1), 2.50 (m, H5Ch-1), 2.1–1.5
(overlapping, HHyl-4, H5Ch-2, HHyl-3), 1.47 [s, 27H,
3 · C(CH3)3], 1.41 and 1.33 [2s, 2 · 9H, 2 · C(CH3)3]; 13C
NMR (CDCl3) d: 171.2, 169.8, 1698 (COOR), 156.1,
1
Rf = 0.30 (hexane/AcOEt; 70:30; v/v). The H NMR of
the obtained mixture showed the presence of the previously
obtained isomer 7b and that of its (5S)-epimer 11b in a 1:1
ratio.
155.5, 143.8, 136.5, 130.1 (pyridinium ring), 102.5 (CGal
1), 82.6 (CGal-3), 82.0, 81.7, 81.0 [3 · C(CH3)3], 79.2
(CGal-2), 79.1, 77.8 [2 · C(CH3)3], 75.2 (OCH2Ph), 74.6
-
The mixture of stereomers (2.00 g; 2.30 mmol) was then
dissolved in methanol (40 mL), after which Cs2CO3 (2 g;
6.1 mmol) was added and the mixture was stirred for 6 h

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 1750–1757
1757
`
2. Allevi, P.; Cribiu, R.; Giannini, E.; Anastasia, M. Bioconju-
at room temperature. The solution was concentrated and
the Cs2CO3 filtered. The solvent was then evaporated to
afford a crude residue, which was chromatographed
(eluting with hexane/AcOEt; 75:25; v/v) to afford first the
amine 7a (713 mg; 39% yield) and then the (5S)-epimer
11a (750 mg; 41% yield).
gate Chem. 2005, 16, 1045–1048.
3. Jordan, K. M.; Syddall, E. E.; Garnero, P.; Gineyts, E.;
Dennison, E. M.; Sayer, A. A.; Delmas, P. D.; Cooper, C.;
Arden, N. K. Ann. Rheum. Dis. 2006, 65, 871–877, and
references cited therein.
4. Garnero, P.; Delmas, P. D. Curr. Opin. Rheumatol. 2003, 15,
641–646.
5. Garnero, P.; Gineyts, E.; Christgau, S.; Finck, B.; Delmas, P.
D. Arthritis Rheum. 2002, 46, 21–30.
6. Robins, S. P. Biochem. J. 1983, 215, 167–173.
4.13. Comparison of HPLC behavior of Gal-Pyd 2 and of
Gal-epiPyd 4
`
7. For our studies on reducible cross-links see: Cribiu, R.;
A sample of each compound was dissolved in water and
analyzed using the best found chromatographic conditions
to separate compound 2 from its epimer 4: the HPLC col-
umn was a LiChrocartꢂ 4-125, LiChrosphere RP-18
(5 lm); the mobile phase was a solution of water/CH3CN
(90:10; v/v) containing eptafluorobutyrric acid (0.02 M);
the flow rate was 1 mL/min and the detection was per-
formed by fluorescence (kex = 297 nm; kemiss = 380 nm).
The galactosyl-O-pyridinoline 2 was eluted after 24.8 min
while galactosyl-O-epi-pyridinoline 3 was eluted after
27.5 min showing a satisfactory peak resolution (Rs > 1).
Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry 2005, 16,
3059–3069; For those on not reducible cross links see: Allevi,
P.; Cribiu, R.; Giannini, E.; Anastasia, M. J. Org. Chem.
`
2007, 3478–3483, and references cited therein.
8. Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry 2000, 11,
3151–3160.
9. (a) Allevi, P.; Anastasia, M.; Paroni, P.; Ragusa, A. Tetra-
hedron: Asymmetry 2004, 15, 3139–3148; (b) Allevi, P.;
Anastasia, M.; Paroni, P.; Ragusa, A. Bioorg. Med. Chem.
Lett. 2004, 14, 3319–3321.
`
10. Bartra, M.; Romea, P.; Urpı, F.; Vilarrasa, J. Tetrahedron
1990, 46, 587–594.
11. For the rationalization of the mechanism of the reaction see:
Allevi, P.; Longo, A.; Anastasia, M. Chem. Commun. 1999,
559–560.
Acknowledgement
12. Allevi, P.; Anastasia, M. Patent No. WO 02/055499 A1.
13. van den Nieuwendijk, A. M. C. H.; Kriek, N. M. A. J.;
Brussee, J.; van Boom, J. H.; van der Gen, A. Eur. J. Org.
Chem. 2000, 3683–3691.
This work was financially supported by Italian MiUR
`
(Ministero dell’Universita e della Ricerca).
14. Lay, L.; Nicotra, F.; Panza, L.; Russo, G. Helv. Chim. Acta
1994, 77, 509–514.
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