A simple strategy for the synthesis of the glycosylated collagen biomarkers α-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-O-pyridinoline (Glc-Gal-PYD), α-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-5-O-hydroxylysine (Glc-Gal-Hyl) and of their unnatural ep

Article abstract of DOI:10.1016/j.tetasy.2007.10.037

A short synthetic strategy for the preparation of the α-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-O-pyridinoline (Glc-Gal-PYD), of the α-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-5-O-hydroxylysine (Glc-Gal-Hyl) and of their respective epimers, Glc-Gal

Full text of DOI:10.1016/j.tetasy.2007.10.037

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2689–2694
A simple strategy for the synthesis of the glycosylated collagen
biomarkers a-D-glucopyranosyl-(1!2)-b-D-galactopyranosyl-O-
pyridinoline (Glc-Gal-PYD), a-D-glucopyranosyl-(1!2)-b-D-
galactopyranosyl-5-O-hydroxylysine (Glc-Gal-Hyl) and of their
unnatural epimers Glc-Gal-epiPYD and Glc-Gal-epiHyl
Pietro Allevi,* Eti A. Femia, 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 1 October 2007; accepted 26 October 2007
Abstract—A short synthetic strategy for the preparation of the a-D-glucopyranosyl-(1!2)-b-D-galactopyranosyl-O-pyridinoline (Glc-
Gal-PYD), of the a-D-glucopyranosyl-(1!2)-b-D-galactopyranosyl-5-O-hydroxylysine (Glc-Gal-Hyl) and of their respective epimers,
Glc-Gal-epiPYD and Glc-Gal-epiHyl, useful for the analytical evaluation of collagen biomarkers in human sinovium and bone, is
reported.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
for the quantitative evaluation of Glc-Gal-PYD 1 and of
other glycosylated natural pyridinolines in biological
media.
In some recent interesting work, Gineyts et al.1 have
demonstrated that a-D-glucopyranosyl-(1!2)-b-D-galacto-
pyranosyl-O-pyridinoline 1 (Glc-Gal-PYD 1; Fig. 1), a
glycoconjugate cross-link of collagen, is a biomarker
strongly associated with the presence of osteoarthritis at
the tibiofemoral and patellofemoral joints in men. More-
over urinary levels of Glc-Gal-PYD 1 are considered to
be more specific markers of joint destruction progression
in various diseases, including rheumatoid arthritis.2–4
Herein we report the first synthesis of Glc-Gal-epiPYD 2
based on a relatively short procedure, which allows a par-
allel preparation of the natural epimer 1, in a way which is
shorter than that previously available. This synthetic strat-
egy is also applied to the preparation of Glc-Gal-Hyl 3 and
its unnatural epimer Glc-Gal-epiHyl 4.
Considering the biological relevance of Glc-Gal-PYD 1, in
previous work we realized the first synthesis of this bio-
marker, which is now available5 for studies. Afterwards
we started the synthesis of its epimer, the a-D-glucopyran-
osyl-(1!2)-b-D-galactopyranosyl-O-epipyridinoline 2 (Glc-
Gal-epiPYD 2), having an unnatural (5S)-hydroxylysine
side chain, considering that this unnatural glycosylated
pyridinoline was of wide interest as an internal standard,
equally fluorescent, to set up suitable analytical methods
2. Results and discussion
We have obtained the synthesis of Glc-Gal-epiPYD 2
according to a relatively short protocol (Scheme 1) which
noticeably modifies the procedure already set up for the
synthesis of the natural Glc-Gal-PYD 1 since it avoids
the laborious initial separation of the diastereoisomeric
masked hydroxylysines 5a and 5b required here.5
This was possible since we recently observed6 that the
epimeric galactosylated (5S)-hydroxylysine 6a (Scheme 1)
was easily separable by simple column chromatography
from its known natural (5R)-epimer 6b, thus offering the
*
Corresponding author. Tel.: +39 02503 16047; 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.2007.10.037

2690
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 2689–2694
+
+
NH3
NH3
-
-
+
-
+
-
CO2
CO2
NH3
NH3
-O
O
-O
O
CO2
CO2
N+
N+
OH
O
OH
O
HO
HO
HO
HO
O
O
O
O
+
OH
+
OH
NH3
NH3
-O2C
-O2C
HO
HO
OH
OH
HO
HO
Glc-Gal-PYD 1
Glc-Gal-epi PYD 2
NH2
NH2
OH
OH
O
HO
HO
O
O
O
HO
HO
O
O
O
O
+
OH
+
OH
NH3
NH3
-O2C
-O2C
HO
HO
OH
OH
HO
HO
Glc-Gal-epi Hyl 4
Glc-Gal-Hyl 3
Figure 1. Glycosylated pyridinolines and hydroxylysines.
BnO
OBn
OBn
N3
N3
BnO
O
BnO
OBn
N3
O
NH
CCl3
BnO
O
O
O
BnO
BnO
OBn
5
HO
ref. 6
O
BnO
O
OBn
O
HO
i
NHCbz
tBuO2C
NHCbz
NHCbz
tBuO2C
tBuO2C
6
5
OBn
7
BnO
ii
BnO
BnO
NH2
BnO
BnO
OBn
O
NH2
OBn
O
(5R)
O
(5S)
O
O
OBn
+
O
OBn
O
O
NHCbz
NHCbz
BnO
BnO
tBuO2C
tBuO2C
a: (5
S)
b: (5
R
)
OBn
OBn
8b
BnO
8a
BnO
t
˚
Scheme 1. Shortened parallel syntheses of glycosylated protected hydroxylysines. Reagents and conditions: (i) BuMe2SiSO3CF3, molecular sieves 3 A,
Et2O, rt, 1 h, 77%; (ii) (a) SnCl2, PhSH, Et3N, THF, rt, 1 h, (b) column chromatography: 8a (41%), 8b (39%).
possibility to start with these separated azides and perform
an abbreviated synthesis of the glycoconjugate pyridinoline
1 and its epimer 2. Moreover, we decided to postpone the
separation of the epimeric intermediates to the level of
the isomeric diglycosylated amines 8a and 8b, confident
that the introduction of an additional saccharidic molecule
could preserve or increase the differences of chromato-
graphic behaviour between the (R)- and (S)-isomers. In
effect, on reacting the mixture of epimeric (5S)- and (5R)-
hydroazides 6a and 6b with a glucosyl donor (Scheme 2),
we obtained first a chromatographically inseparable mix-
ture of azides 7a and 7b and then after chemical reduction
with SnCl2–PhSH,7 the mixture of amines 8a and 8b were
easily separated by simple chromatography. At this
point we reacted amine 8a with the known bromoketone
9 in acetonitrile containing Na2CO3, according to our
protocol.6
After exchange of the solvent (MeOH instead of MeCN),
we obtained the protected Glc-Gal-epiPYD 10 in satisfac-
tory yield (61%) and with the expected physico-chemical
properties. In particular, the presence, in compound 10,
of the three amino acidic functionalities was supported
by the 13C resonances of the carboxylic groups, while that
of the 3,4-hydroxypyridinium ring was supported by the
13C resonances and the corrected UV absorption (kmax
340.0 and 258.5 nm, in EtOH).5
The glycoconjugate compound 10 was then hydrolyzed by
treatment with aqueous trifluoroacetic acid to afford tricarb-

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 2689–2694
2691
+
NHBoc
NH3
t
-
+
-
CO2 Bu
CO2
NHBoc
NH3
-O
O
-O
t
CO2 Bu
CO2
O
N+
N+
t
OBn
O
OBn
O
Br
CO2 Bu
BnO
BnO
BnO
BnO
O
2
O
iii
NHCbz
ii
9
NHBoc
Glc-Gal-epiPYD
8a
O
O
i
2
OBn
O
OBn
NHCbz
tBuO2C
10
HO2C
BnO
BnO
OBn
BnO
OBn
BnO
11
Scheme 2. Synthesis of glucogalactoepipyridinoline. Reagents and conditions: (i) Na2CO3, MeCN, rt, 8 h, then Na2CO3, O2, MeOH, rt, 72 h, 61%; (ii)
TFA, rt, 1 h, 93%; (iii) H2, PdCl2, MeOH–H2O–AcOH, rt, 16 h, 73%.
oxylic acid 11, which was finally debenzylated by hydro-
genolysis, in aqueous acidic medium under catalysis by
PdCl2, to afford the desired epimeric Glc-Gal-epiPYD 2.
3. Conclusion
In conclusion, in this work we report the first synthesis of
the unnatural Glc-Gal-epiPYD 2 together with a procedure
which offers a simplified access to the preparation of the
natural epimer Glc-GalPYD 1 and of the glycosylated
hydroxylysines 3 and 4. All of these glycoconjugated
cross-links are now easily obtained for biochemical studies
and can allow us to set up robust analytical HPLC
methods.
Glc-Gal-epiPYD 2 obtained showed physico-chemical
1
properties (mass spectrum, H NMR, etc.) in agreement
with the assigned structure. These were very close to those
of the natural Glc-Gal-PYD 1 obtained5 from amine 8b.
Due to these nearly superimposable physical properties of
Glc-Gal-PYD 1 and Glc-Gal-epiPYD 2, only after an
extensive study of the HPLC behaviour of these epimers,
under various conditions of analysis, were we able to find
that they could be satisfactorily separated. This qualifies
Glc-Gal-epiPYD 2 as a useful internal standard for HPLC
analyses of Glc-Gal-PYD 1 in biological media.
4. Experimental
4.1. General methods
Nuclear magnetic resonance spectra were recorded at
303 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 per million (ppm, d units) and
are referenced to residual CHCl3 (dH = 7.24 ppm) and to
CDCl3 (dC = 77.0 ppm) for solutions in CDCl3 or to inter-
nal CH3OD (dH = 3.30 ppm and dC = 49.0 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
Interestingly, our results also allow to obtain in a new sim-
pler way the recently reported8,9 Glc-Gal-hydroxylysines 3
and 4 (Scheme 3), two glycoconjugated amino acids, of
wide interest in many biological studies. This additional
possibility was demonstrated subjecting epimers 8a and
8b to two successive deblocking reactions (Scheme 3) and
obtaining the corresponding glycosylated hydroxylysines
3 and 4.
1
constant(s) in Hz, assignment of proton(s). The H and
1
13C resonances were assigned by H decoupling, 1H–1H
1
COSY and H–13C correlation experiments. The carbon
HO
HO
NH2
(5S)
OH
O
numeration used in the NMR description is that given in
Figure 2.
O
i
O
OH
8a
ii
O
+
NH3
HO
N3
-OOC
+
NH3
HO
OH
4chain
2
4
HO
HO
HO
-
+
-
1
CO2
NH3
-O
2
1
NHCbz
3
CO2
tBuO2C
OH
4
3
NH2
(5R)
5
Gal
1
5chain
O
5
N+
OH
O
HO
4
HO
Hyl
i
ii
6
6
8b
O
O
5
O
O
OH
+
2
4
O 1
NH3
HO
-OOC
3
1OH
O
5
+
NH3
2
6
-O2C
HO
OH
3
3
4
HO
OH
Glc
HO
Scheme 3. Preparation of glycosylated hydroxylysines. Reagents and
conditions: (i) H2, Pd/C, THF–H2O, rt, 9 h, 81%; (ii) TFA, rt, 1 h, 82%.
Figure 2. Carbon numeration used in this work.

2692
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 2689–2694
Optical rotations were taken on a Perkin–Elmer 241 polar-
imeter and [a]D values are given in 10ꢀ1 deg cm2 gꢀ1
THF (37 mL). After 10 min, the epimeric mixture of azides
7 (4.56 g, 3.42 mmol) was added. The analyte mixture was
stirred at room temperature for 1 h. After this time, the sol-
vent was evaporated and the crude residue was purified by
flash chromatography. Elution with hexane–AcOEt (30:70,
v/v) afforded first some by-products, then, with hexane–
AcOEt–MeOH (30:70:2, v/v), 5S-isomer 8a (1.83 g;
Y = 41%), a glass: ½aꢁD ¼ þ40:5 (c 1, CH2Cl2); H NMR
(CDCl3): d 7.3–7.0 (aromatics), 5.51 (1H, d, J 3.5 Hz,
HGlc-1), 5.36 (1H, d, J 8.4 Hz, N–H), 4.60 (1H, d, J
.
HPLC analyses were carried out on a ODS-2 column
(Waters Spherosorb, 150 mm, 4.6 mm ID, 5 lm); elution
was performed with 0.02 M heptafluorobutanoic acid
(HFBA) in a water/MeCN solution containing variable ra-
tio of these solvents and the detection was carried out by
fluorescence (kex = 297 nm; kemiss = 380 nm).
20
1
Mass spectra were carried out using a Finnigan LCQdeca
(ThermoQuest) ion trap mass spectrometer equipped 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 10 lL/min. The spray voltage
was set at 5.0 kV, operating in the positive ionization
mode with capillary temperature of 220 ꢁC. Full-scan mass
spectra were recorded by scanning a m/z range of 100–
2000.
7.7 Hz, HGal-1), 4.31 (1H, ddd, J 9.5, 3.0, 2.5 Hz, HGlc-
5), 4.19 (1H, m, HHyl-2), 4.10 (1H, dd, J 9.9, 7.7 Hz,
HGal-2), 4.03 (1H, dd, J 9.5, 9.5 Hz, HGlc-3), 3.96 (1H,
dd, J 3.5, <1 Hz, HGal-4), 3.67 (1H, dd, J 9.5, 9.5 Hz,
HGlc-4), 3.63–3.55 (3H, overlapping, HGal-5, HGal-6a,
HGal-6b), 3.60 (1H, dd, J 9.5, 3.5 Hz, HGlc-2), 3.56 (1H,
dd, J 9.9, 3.2 Hz, HGal-3), 3.38 (2H, br s, HGlc-6a and
HGlc-6a), 2.58 (1H, dd, J 12.9, 6.6 Hz, HHyl-6a), 2.52
(1H, dd, J 12.9, 4.5 Hz, HHyl-6b), 1.81 (1H, m, HHyl-3a),
1.73 (1H, m, HHyl-3b), 1.51 (1H, m, HHyl-4a), 1.45 (1H,
m, HHyl-4b), 1.37 [3H, s, C(CH3)3]; 13C NMR (CDCl3): d
171.6 (CHyl-1), 155.9 (NHCO2Bn), 138.8–127.0 (aromat-
ics), 102.7 (CGal-1), 96.7 (CGlc-1), 82.4 (CGlc-3), 81.7
(CMe3), 81.5 (CGal-3), 79.4 (CGlc-2), 78.8 (CHyl-5), 78.3
(CGlc-4), 73.4 (CGal-4), 73.3 (CGal-2), 73.1 (CGal-5), 69.9
(CGlc-5), 68.5 (CGal-6), 68.1 (CGlc-6), 54.2 (CHyl-2), 45.4
(CHyl-6), 28.4 (CHyl-4), 28.4 (CHyl-3), 27.9 [C(CH3)3]; ESI-
MS (positive) m/z: 1307.5 (100%), 1308.5 (66%),1309.6
(33%), 1310.7 (9%; M+H+). Anal. Calcd for
C79H90N2O15: C, 72.57; H, 6.94; N, 2.14. Found: C,
72.73; H, 6.89; N, 2.19.
All reactions were monitored by thin-layer chromatogra-
phy (TLC) carried out on 0.25 mm E. Merck silica gel
plates (60 F254) using UV light, 50% sulfuric acid, anisalde-
hyde–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 chromatography.10
Work-up refers to washing with water, drying with Na2SO4
and evaporation of the solvent.
4.2. Preparation of tert-butyl (2S,5S)- and (2S,5R)-6-amino-
2-benzyloxycarbonylamino-5-[(2,3,4,6-tetra-O-benzyl-a-D-
glucopyranosyl)-(1!2)-(3,4,6-tri-O-benzyl-b-D-galacto-
pyranosyl)]hexanoates 8a and 8b
Elution with hexane–AcOEt–MeOH (30:70:5, v/v) afforded
(5R)-isomer 8b (1.74 g; Y = 39%). This compound showed
the correct elemental analysis and the physico-chemical
properties already described.6
4.2.1. Preparation of the epimeric mixture of (2S,5S)- and
(2S,5R)-6-azido-2-benzyloxycarbonylamino-5-[(2,3,4,6-tetra-
O-benzyl-a-D-glucopyranosyl)-(1!2)-(3,4,6-tri-O-benzyl-b-
4.3. Preparation of completely protected a-D-glucopyran-
osyl-(1!2)-b-D-galactopyranosyl-O-5-epipyridinoline 10
D-galactopyranosyloxy)]hexanoate 7.
A
mixture of
epimeric galactosides 66 (3.67 g; 4.52 mmol), O-(2,3,4,6-
tetra-O-benzyl-a-D-glucopyranosyl) trichloroacetimidate8
(13.2 g; 19.3 mmol) and powdered molecular sieves (3 A,
The protected bromoketone 9 (654 mg, 1.72 mmol) was
added to a solution of amine 8a (900 mg, 0.69 mmol) in
CH3CN (45 mL) containing Na2CO3 (1.35 g, 12.7 mmol),
and the mixture was stirred at room temperature under
nitrogen for 8 h. At this time was observed the disappear-
ance of the starting glycosylated amine 8a and of the ini-
tially formed monoalkylated product (TLC:CH2Cl2–
MeOH, 100:5, v/v; Rf = 0.43 and 0.79, respectively). The
solvent was then evaporated under reduced pressure and
the crude residue was dissolved in MeOH (45 mL) and sha-
ken under a slight pressure of oxygen (1.3 atm) and at
room temperature for 72 h. After this time, the mixture
was diluted with dichloromethane (50 mL) and filtered on
a pad of Celite. After evaporation of the solvent we
obtained a crude residue which was chromatographed on
silica gel and eluted with CH2Cl2–MeOH (100:4, v/v) to
˚
2 g), in anhydrous diethyl ether (150 mL), was stirred
for 15 min at room temperature. Then tert-butyldimethyl-
silyl triflate (208 lL; 0.91 mmol) was added and stirring
was continued for 1 h, at room temperature, under argon
atmosphere. At this time, the powdered molecular sieves
were filtered off and washed with AcOEt. The organic
phase was worked up to afford a residue, which was
purified by flash chromatography (eluting with hexane–
AcOEt, 80:20 v/v) to give the mixture of epimers 7
(4.56 g; Y = 76%) as an oil. The product showed the
correct elemental analysis and 1H NMR in agreement
with the presence of both known (5S) and (5R)-epimeric
compounds.8
4.2.2. Preparation of tert-butyl (2S,5S)- and of (2S,5R)-6-
amino-2-benzyloxycarbonylamino-5-[(2,3,4,6-tetra-O-benzyl-
a-D-glucopyranosyl)-(1!2)-(3,4,6-tri-O-benzyl-a-D-galacto-
pyranosyl)]hexanoates 8a and 8b. PhSH (2.25 mL, 21.9
mmol) and Et3N (2.25 mL, 16.1 mmol) were added to a
stirred solution of SnCl2 (1.00 g, 5.29 mmol) in anhydrous
afford the protected glycosylated pyridinoline 10 (790 mg;
20
Y = 61%) as a glass: ½aꢁD ¼ þ34:9 (c 1, CH2Cl2); UV kmax
-
(EtOH)/nm (e/dm3 molꢀ1 cmꢀ1), 258.5 (5200), 340 (5950);
1H NMR (CDCl3): d 7.32–7.04 (aromatics), 5.48 (d, J
7.7 Hz, N–H), 5.33 (1H, d, J 3.5 Hz, HGlc-1), 4.39 (1H,
d, J 7.7 Hz, HGal-1), 4.31 (1H, ddd, J 9.5, 3.0, 2.5 Hz,

P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 2689–2694
2693
HGlc-5), 4.14 (1H, m, HHyl-2), 4.07 (1H, dd, J 9.6, 7.7 Hz,
HGal-2), 4.06 (1H, dd, J 9.5, 9.5 Hz, HGlc-3), 3.81 (1H, m,
HHyl-5), 3.70 (1H, dd, J 9.5, 9.5 Hz, HGlc-4), 3.63 (1H,
dd, J 9.5, 3.5 Hz, HGlc-3), 3.56 (1H, dd, J 9.6, 3.5 Hz,
HGal-3), 3.46 (1H, dd, J 3.5, <1 Hz, HGal-4), 3.30 (2H, m,
HGlc-6a and HGlc-6b), 1.46, 1.45, 1.38 [27H, 3 · s,
3 · C(CH3)3], 1.44 [18H, s, 2 · C(CH3)3]; 13C NMR
(CDCl3): d 171.2, 171.1, 171.0 (3 · COOtBu), 168.7,
155.6, 143.2 (3 · pyridinium ring carbons), 156.0
(NHCOO), 102.3 (CGal-1), 97.1 (CGlc-1), 56.6, 53.7, 53.6
(3 · aminoacid a-carbons), 28.4, 28.3, 27.93, 27.91, 27.8
[5 · C(CH3)3]; ESI-MS (positive) m/z: 1885.7 (80%),
1886.6 (100%), 1887.6 (60%), 1888.5 (10%; M+H+). Anal.
Calcd for C109H136N4O24: C, 69.41; H, 7.27; N, 2.97.
Found: C, 69.52; H, 7.15; N, 3.06.
4.5. Comparison of HPLC behaviour of Glu-Gal-Pyd 1 and
of Glu-Gal-epiPyd 2
A sample of each compound was dissolved in water and ana-
lyzed using the best chromatographic conditions to separate
compound 1 from its epimer 2: the HPLC column has been
reported previously in a general method, the mobile phase
was a solution of water–CH3CN (93:7, v/v) containing hep-
tafluorobutyric acid (0.02 M), the flow rate was 1 mL/min
and the detection was achieved by fluorescence. Glucosyl-
galactosyl-O-epipyridinoline 2 was eluted after 45.5 min
while glucosyl-galactosyl-O-pyridinoline 1 was eluted after
48.4 min showing a satisfactory peak resolution (Rs > 1).
4.6. Preparation of (2S,5S)-2,6-diamino-5-[a-D-glucopyran-
osyl-(1!2)-b-D-galactopyranosyloxy]hexanoic acid 4
4.4. Preparation of the a-D-glucopyranosyl-(1!2)-b-D-
galactopyranosyl-O-epipyridinoline 2, by successive
regeneration of protecting functionalities
Amine 8a (200 mg; 0.153 mmol) dissolved in tetrahydrofu-
ran (25 mL) and ethanol (75 mL) was hydrogenated in the
presence of palladium (200 mg, 10% on activated carbon)
at room temperature under 1 atm of hydrogen. After 9 h,
the catalyst was filtered on a pad of Celite and thoroughly
rinsed with a solution of MeOH–distilled water (10 mL;
5:1, v/v). Evaporation of the combined filtrates at low tem-
perature (<30 ꢁC) and under reduced pressure afforded a
residue, which was dissolved in distilled water (2 mL) and
washed with toluene (2 · 4 mL). The aqueous solution
was co-evaporated with toluene under reduced pressure.
The residue was dissolved in wet trifluoroacetic acid
(1 mL, TFA–H2O, 95:5, v/v) and the resulting solution
was stirred at room temperature for 1 h. Evaporation of
the solvent afforded the pure title compound 4 as a ditrifluo-
roacetate (88 mg; Y = 81%), having the physico-chemical
properties identical with those already observed by us.8
4.4.1. Preparation of the partially protected Glc-Gal-
epiPYD 11. The completely protected Glc-Gal-epiPYD
10 (600 mg, 0.318 mmol) was dissolved in CF3COOH–
H2O (12 mL, 95:5, v/v) and stirred at room temperature
for 1 h. Evaporation of the solvent under reduced pressure
afforded a crude residue formed by the partially protected
Glc-Gal-epiPYD 11 as tri-trifluoroacetate salt (550 mg;
1
Y = 93%): a glass. The H NMR spectrum was recorded
to verify the complete cleavage of Boc groups and of the
tert-butyl esters.
4.4.2. Preparation of the a-D-glucopyranosyl-(1!2)-b-D-
galactopyranosyl-O-epipyridinoline 2. The partially pro-
tected Glc-Gal-epiPYD 11 (780 mg, 0.419 mmol) was dis-
solved in 390 mL of a mixture of MeOH–water–glacial
acetic acid (8:2:1, v/v/v) and hydrogenated in the presence
of PdCl2 (150 mg) at room temperature and atmospheric
pressure. When the hydrogen absorption was completed
(16 h), the catalyst was separated by filtration over a pad
of Celite. The solvent was eliminated under reduced
pressure to afford a residue, which was recovered by
water and then passed through a Dowex (50WX8-200)
column (20 mL) and eluted with a 1 M solution of NH3
in H2O–MeOH (1:1, v/v). The solution was freeze-dried to
4.7. Preparation of (2S,5R)-2,6-diamino-5-[a-D-glucopyran-
osyl-(1!2)-b-D-galactopyranosyloxy]hexanoic acid 3
Starting with amine 8b (200 mg; 0.153 mmol) and following
the procedure described above for the deprotection of the
epimeric amine 8a, the title compound 3 was obtained as
a ditrifluoroacetate (89 mg; Y = 82%) identical in all re-
spects with those already reported by us.8
Acknowledgement
obtain Glc-Gal-epiPYD
2
(225 mg; Y = 73%), an
20
amorphous material, which showed ½aꢁD ¼ þ17:4 (c 0.5,
H2O); 1H NMR (D2O) d: 8.22, 8.17 (2H, 2 · br s, pyridini-
um ring protons), 5.19 (1H, d, J 3.5 Hz, HGlc-1), 4.50 (1H,
d, J 7.5 Hz, HGal-1), 4.20 (1H, m, HHyl-5), 4.16 (1H, dd, J
6.0 and 6.0 Hz, H5chain-3), 4.02 (1H, dd, J 6.5 and 6.5 Hz,
HHyl-2), 3.84 (1H, dd, J 3.5 and <1 Hz, HGal-4), 3.39
(1H, dd, J 9.5 and 7.5 Hz, HGal-2), 3.54 (1H, dd, J 9.5
and 3.5 Hz, HGlc-2), 3.40 (1H, dd, J 9.5 and 9.5 Hz,
This work was financially supported by Italian MiUR
`
(Ministero dell’Universita e della Ricerca).
References
1. 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. Reum. Dis. 2006, 65, 871–877, and
references cited therein.
2. Gineyts, E.; Garnero, P.; Delmas, P. Rheumatology 2001, 40,
315–323.
3. Garnero, P.; Delmas, P. D. Curr. Opin. Rheumatol. 2003, 15,
641–646.
4. Garnero, P.; Gineyts, E.; Christgau, S.; Finck, B.; Delmas, P.
D. Arthritis Rheum. 2002, 46, 21–30.
HGlc-4), 3.00 (1H, m, H5chain-1a), 2.89 (1H, m, H5chain
-
1b); 13C NMR dC (CD3OD): 172.5, 172.4, 171.7
(3 · CO2H), 155.5 (C-3), 141.5, 141.0 (C-4 and C-5),
132.4, 130.8 (C-2 and C-6), 102.1 (CGal-1), 98.2 (CGlc-1),
78.2 (CHyl-5), 77.4 (CGal-2). ESI-MS (positive) m/z: 754.6
(25%), 753.2 (100%; M+H+). Anal. Calcd for
C30H48N4O18: C, 47.87; H, 6.43; N, 7.44. Found: C,
47.96; H, 6.28; N, 7.51.

2694
P. Allevi et al. / Tetrahedron: Asymmetry 18 (2007) 2689–2694
`
5. Allevi, P.; Cribiu, R.; Giannini, E.; Anastasia, M. Bioconju-
8. Allevi, P.; Anastasia, M.; Paroni, P.; Ragusa, A. Tetrahedron:
Asymmetry 2004, 15, 3139–3148.
gate Chem. 2005, 16, 1045–1048.
6. Allevi, P.; Colombo, R.; Giannini, E.; Anastasia, M. Tetra-
hedron: Asymmetry 2007, 18, 1750–1757.
9. Allevi, P.; Anastasia, M.; Paroni, P.; Ragusa, A. Bioorg. Med.
Chem. Lett. 2004, 14, 3319–3321.
`
7. Bartra, M.; Romea, P.; Urpı, F.; Vilarrasa, J. Tetrahedron
10. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923–2925.
1990, 46, 587–594.