Hydroxylysine containing glycoconjugates: An efficient synthesis of natural galactosylhydroxylysine (Gal-Hyl) and glucosylgalactosylhydroxylysine (Glu-Gal-Hyl) and of their (5S)-epimers

Article abstract of DOI:10.1016/j.tetasy.2004.08.006

The paper reports the first chemical synthesis of α-d-glucopyranosyl- (1→2)-β-d-galactopyranosyl-5-O-hydroxylysine, a biological marker of bone resorption and of its unnatural (5S)-epimer, starting from commercial sugars and amino acids. Moreover, the synthetic protocol set-up has resulted in a new procedure for the synthesis of the β-d-galactopyranosyl-5-O- hydroxylysine and its unnatural (5S)-epimer.

Full text of DOI:10.1016/j.tetasy.2004.08.006

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3139–3148
Hydroxylysine containing glycoconjugates: an efficient synthesis
of natural galactosylhydroxylysine (Gal-Hyl) and
glucosylgalactosylhydroxylysine (Glu-Gal-Hyl) and of their
(5S)-epimers
Pietro Allevi,a,* Rita Paroni,a Andrea Ragusab,ꢀ and Mario Anastasiab
a
`
`
Dipartimento di Medicina, Chirurgia e Odontoiatria, Universita di Milano, via A. Di Rudinı 8, I-20142 Milano, Italy
`
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di Milano, via Saldini 50, I-20133 Milano, Italy
b
Received 13 July 2004; accepted 3 August 2004
Available online 16 September 2004
Abstract—The paper reports the first chemical synthesis of a-D-glucopyranosyl-(1!2)-b-D-galactopyranosyl-5-O-hydroxylysine, a
biological marker of bone resorption and of its unnatural (5S)-epimer, starting from commercial sugars and amino acids. Moreover,
the synthetic protocol set-up has resulted in a new procedure for the synthesis of the b-D-galactopyranosyl-5-O-hydroxylysine and its
unnatural (5S)-epimer.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
HO
HO
NH2
OH
O
HO
HO
NH2
OH
O
Collagen is a family of structurally related proteins
formed of a helix of three alpha chains, each of which
has its own amino acid composition, genetic mechanism
and tissue distribution. Some lysine (Lys) residues, spe-
cifically located in the collagen fibrils of bone and skin,
are susceptible of post-translational hydroxylation and
subsequent glycosylation by either b-D-galactopyranosyl
or a-D-glucopyranosyl-(1!2)-b-D-galactopyranosyl moi-
eties.1,2 In the human body, during collagen breakdown,
the glycosylated hydroxylysine residues are released into
the serum as b-D-galactopyranosyl-5-O-hydroxylysine
O
O
O
OH
OH
O
+
NH3
HO
+
-O2C
NH3
-O2C
OH
HO
2
1
HO
HO
HO
NH2
NH2
OH
O
OH
O
O
O
HO
HO
[(5R)-GaHL]
1
and a-D-glucopyranosyl-(1!2)-b-
O
OH
OH
D-galactopyranosyl-O-hydroxylysine [(5R)-GgaHL] 2
(Fig. 1). Then they are not recycled or significantly
metabolized3 but are quantitatively excreted into urine.4
O
+
+
NH3
NH3
-O2C
-O2C
OH
3
4
HO
Thus, glycosides 1 and 2 have attracted considerable
attention as biochemical markers of total collagen turn-
Figure 1.
over,5 correlated to diseases of bone resorption,6 useful
for a better understanding of various pathologies and
for clinical diagnosis. As a consequence, pure (5R)-
GaHL 1 and (5R)-GGaHL 2 are essential for studies
*
Corresponding author. Tel.: +39 0250316047; fax: +39
0250316040; e-mail: pietro.allevi@unimi.it
ꢀ A. Ragusa is currently a Ph.D. student in the Department of
Chemistry, University of Southampton, Southampton, UK.
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.08.006

3140
P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
of collagen biochemistry and for the development of
analytical methods to monitor the metabolic status of
bone collagen. In fact, even if a large number of analyt-
ical methods for the quantitative detection of the glyco-
sides 1 and 2 in biological samples has been reported,7
only recently a racemic8 and a homochiral9 chemical
syntheses of the (5R)-GaHL 1 have been described,
while no synthesis of the (5R)-GGaHL 2 is to date avail-
able.10,11 Thus the reference standards for physico-
chemical analyses are generally obtained from different
natural sources,12 in forms sometimes not completely
free of contaminants. Moreover, the synthesis of the gly-
cosides (5R)-GaHL 1 and of the (5R)-GGaHL 2 also
represents an attractive synthetic target due to the inter-
esting structural features of these glycoconjugated
hydroxylysines. For this we enclosed the synthesis of
glycosides (5R)-GaHL 1 and (5R)-GGaHL 2 in our pro-
grammes on the synthesis and the utilization of collagen
metabolites.13 In addition, we planned the synthesis of
the glycosides (5S)-GaHL 3 and (5S)-GGaHL 4, two
glycosides of unnatural (5S)-hydroxylysine, epimers of
the glycosides 1 and 2, respectively. In fact, we consid-
ered that these unnatural epimers could be suitable
internal standards for the detection of the natural
metabolites 1 and 2, in almost all the analytical methods
reported, and in particular the more sophisticated liquid
chromatography–tandem mass spectrometry (LC/MS/
MS).7a
masked (5R)-5-hydroxy-L-lysine (5R)-5 (Scheme 1), pre-
pared by esterification, according to Kunz and co-work-
ers14 of the corresponding acid,13d and a galactosyl
donor, conveniently activated, at the anomeric hydr-
oxyl, to be used in a Schimdt15 stereocontrolled protocol
of b-glycosylation.
Our synthetic scheme prompted us to use as galactosyl
acceptor the masked hydroxylysine (5R)-5, in place of
other hydroxylysine esters, in order to avoid the protec-
tion, and the obligatory deprotection, of the e-amino
group of the amino acid and various troubles due to
possible lactamizations. On the other hand, we decided
to construct, as glycosyl donor, the galactosyl derivative
a-6 (Scheme 2), orthogonally protected as the chloroac-
etate at the 2-hydroxyl group. In fact, we considered
that the trichloroacetimidate a-6, having a participating
protecting group at the 2 position, could favour the for-
mation of the desired b-anomeric bond. In addition, the
chloroacetate group, after the glycosylation, could be
selectively removed in the presence of the glycosidic
bond, thus allowing to extend the saccharidic chain in
the 2-direction.
With this in mind, we prepared the required glycosyl
donor a-6 from the easily available16 3,4,6-tri-O-ben-
zyl-D-galactopyranose in three steps (Scheme 2). In fact,
the preparation of a-6 requires first the chloroacetyl-
ation of both the hydroxyl groups of the benzylated
galactose, by reaction with chloroacetyl chloride, afford
the 1,2-O-dichloroacetyl derivative as an 1:1 anomeric
mixture (1H NMR), then, a controlled treatment of
the O-dichloroacetates mixture of with ammonia in
acetonitrile17 to allow the selective regeneration of the
anomeric hydroxyl, thus affording the 3,4,6-tri-O-benz-
yl-2-O-chloroacetylgalactopyranose (as a chromato-
graphically inseparable mixture of a and b epimers;
2. Results and discussion
In planning our synthetic work for the preparation of
the (5R)-GaHL 1 and of the (5R)-GGaHL 2 (Fig. 1),
we decided to establish a common protocol, which could
permit to approach the synthesis of both the (5R)-GaHL
1 and of the (5R)-GGaHL 2 and possibly of their (5S)-
epimers 3 and 4. With this in mind, we programmed to
start the synthesis with the formation of a b-galactoside
bond between a suitable O-glycosyl acceptor as the
1
8:2; H NMR) and finally, the activation of the glycos-
idic carbon by reaction with CCl3CN, in the presence
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The first
5R-GaHL 1
N3
PO
PO
PO
PO
PO
PO
NHZ
N3
N3
OP
O
OP
O
OP
O
HO
H
+
O
O
RO
RO
HO
O
NH
NHZ
tBuO2C
NHZ
CCl3
tBuO2C
tBuO2C
(5R)-5
5R-GGaHL 2
Scheme 1. P = protecting group; R = acyl group removable with mild condition; Z = benzyloxycarbonyl group.
OBn
O
OBn
O
OBn
O
OBn
O
OBn
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
i
iii
ii
OCOCH2Cl
O
+
O
NH
CCl3
HO
ClCH2CO2
ClCH2CO2
ClCH2CO2
ClCH2CO2
OH
OH
O
NH
CCl3
α−6
β−6
Scheme 2. Reagents and conditions: (i) ClCH2COCl, Et2O/Py, ꢀ10 to 25ꢁC, 2h, 98%; (ii) NH3, CH3CN, 25ꢁC, 85%; (iii) Cl3CCN, DBU, CH2Cl2,
ꢀ30ꢁC, 4h, 79%.

P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
3141
two reactions occur in nearly quantitative yields, while
the formation of the desired trichloroacetimidate a-6
was obtained, in the best case, in 79% yields. In all cases,
the a-6 anomer was accompanied by its b-anomer b-6,
which was isolated and completely characterized.
(5R)-GaHL 1 but also for reaching the synthesis of the
more complex disaccharidic glycoside (5R)-GGaHL 2
(Scheme 3).
In fact, catalytic hydrogenation of the glycoside 8 causes
the simultaneous generation of the amino group from
the azide and the removal of the benzyl and benzyloxy-
carbonyl groups with formation of an amino tert-butyl
ester, which, by simple treatment with aqueous trifluor-
acetic acid, affords the desired 5R-GaHL 1 as its
trifluoracetate.
With the stating material in the hands, we performed the
galactosylation of the masked hydroxylysine (5R)-5
(Scheme 3) using as galactosyl donor the a-anomer a-6
and tert-butyldimethylsilyl triflate as catalyst.15 In these
conditions, we obtained the b-O-galactoside 7, which, in
the crude product of the reaction, was not accompanied
by any detectable quantity of the corresponding a-ano-
mer or ortho-ester. On the contrary, the a-isomer was
present, as a more polar companion (TLC and
ESI-MS evidences) when the reaction was catalyzed by
boron trifluoride etherate.
Obtainment of the disaccharide 2 required the formation
of a a-glucosidic bond at the 2 position of the key inter-
mediate 8. This glucosylation has been affected, in the
best conditions, coupling the acceptor 8 with O-
(2,3,4,6-tetra-O-benzyl-b-D-glucopyranosyl)trichloroacet-
imidate 919 in diethyl ether at 25ꢁC and in the presence
of tert-butyldimethylsilyl triflate. In absence of a partic-
ipant group at the 2 position of glycosyl donor, these
conditions, as expected on the base of Wegmann and
Schmidt work20, allow to obtain with the higher-diaster-
eoselectivity and the more convenient yield (45% after
chromatographic purification), the protected diglycosi-
dic compound 10. Moreover also under these condi-
tions, set-up by Wegmann and Schimdt20 for the use
of the glucosyl donor 9, the compound 10 was accompa-
nied by the undesired b-anomer (18% yield).
In a final preparation we used, as galactosyl donor, also
the purified b-trichloroacetimidate b-6 and obtained a
super imposable result, thus supporting the conclusion
that in the Schmidt glycosylation the geometry of the tri-
chloroacetimidate is irrelevant.15
The successive selective regeneration of the 2-hydroxy
group of 7 was performed by refluxing in methanol the
chloroacetate 7 in the presence of Zn(OAc)22H2O.
These very mild reaction conditions, were already found
by us to be useful for the hydrolysis of a dichloroacetate
in the synthesis of the glycoside etoposide.18 The selec-
tive removal of the chloroacetate group generates the
galactoside 8 having a free hydroxy group at position
2 of the galactose portion. This key intermediate 8 re-
sulted useful not only for pursuing the synthesis of the
The correct a-stereochemistry at glucosidic anomeric
carbon was confirmed by the observed chemical shift
of the anomeric proton (d = 5.62ppm) and from its cou-
pling constant (J = 3.5Hz) with the adjacent H-2 proton
in the glucopyranosidic ring.
N3
+
BnO
BnO
BnO
BnO
BnO
BnO
HO
HO
N3
NH3
N3
OBn
O
OBn
O
OBn
O
OH
O
i
HO
H
ii
iii
+
O
O
O
-
2CF3COO
HO
HO
ClCH2CO2
ClCH2CO2
O
NH
NHZ
+
NHZ
NH3
NHZ
tBuO2C
tBuO2C
HO2C
CCl3
tBuO2C
1
(5R)-5
α−6
8
7
OBn
O
BnO
BnO
NH
CCl3
iv
O
OBn
9
+
HO
HO
BnO
NH2
HO
N3
NH3
OH
O
OBn
O
OH
O
O
O
O
BnO
BnO
HO
-
v
vi
O
OH
O
OBn
O
OH
2CF3COO
O
O
O
+
NH2
NHZ
NH3
HO
HO
tBuO2C
tBuO2C
HO2C
OH
OBn
OH
HO
BnO
HO
10
2
11
Scheme 3. Reagents and conditions: (i) tBuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25ꢁC, 1h, 52%; (ii) Zn(OAc)2 2H2O, MeOH, reflux, 9h, 93%;
(iii) a. H2, Pd/C, THF–EtOH, 1atm, 25ꢁC, 90%; b. CF3CO2H–H2O (95:5), 25ꢁC, 1h, 92%; (iv) tBuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25ꢁC,
15min, 45%; (v) H2, Pd/C, THF–EtOH, 1atm, 25ꢁC, 9h, 90%; (vi) CF3CO2H–H2O (95:5), 25ꢁC, 1h, 92%.

3142
P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
N3
+
BnO
BnO
BnO
BnO
BnO
BnO
HO
HO
N3
NH3
N3
OBn
O
OBn
O
OBn
O
OH
O
i
H
ii
iii
+
O
O
O
HO
-
2CF3COO
HO
HO
ClCH2CO2
ClCH2CO2
O
NH
NHZ
+
NHZ
NH3
NHZ
tBuO2C
tBuO2C
HO2C
CCl3
tBuO2C
3
(5S)-5
α−6
13
12
OBn
O
BnO
BnO
NH
CCl3
iv
O
OBn
9
+
HO
HO
BnO
NH2
HO
N3
NH3
OH
O
OBn
O
OH
O
O
O
O
BnO
BnO
HO
-
v
vi
O
OH
O
OBn
O
OH
2CF3COO
O
O
O
+
NH2
NHZ
NH3
HO
HO
tBuO2C
tBuO2C
HO2C
OH
OBn
OH
HO
BnO
HO
14
4
15
Scheme 4. Reagents and conditions: (i) tBuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25ꢁC, 1h, 50%; (ii) Zn(OAc)2 2H2O, MeOH, reflux, 9h, 96%;
(iii) a. H2, Pd/C, THF–EtOH, 1atm, 25ꢁC, 93%; b. CF3CO2H–H2O (95:5), 25ꢁC, 1h, 95%; (iv) tBuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25ꢁC,
15min, 42%; (v) H2, Pd/C, THF–EtOH, 1atm, 25ꢁC, 9h, 92%; (vi) CF3CO2H–H2O (95:5), 25ꢁC, 1h, 95%.
Catalytic hydrogenation of the glycoside 10 cleaved the
benzyl and benzyloxycarbonyl groups and generated the
e-amino group of the hydroxylysine aglycone, thus
affording the tert-butyl esters 11, which, by treatment
with trifluoracetic acid, afforded the corresponding acid
5R-GGaHL 2, isolated as ditrifluoracetate.
and 2 deriving from collagen breakdown and required
in various biological studies. In addition, our
work makes available the two glycoconjugate (5S)-
hydroxylysines 3 and 4, which appear very useful as
internal standards in biological analyses directed to the
quantification of glycosides (5R)-GaHL 1 and (5R)-
GGaHL 2.
A parallel series of reactions starting with the galactose
trichloroacetimidate a-6 and the epimeric hydroxyazide
(5S)-5 allows to prepare the galactoside (5S)-GaHL 3
and the diglycoside (5S)-GGaHL 4 as trifluoroacetates
(Scheme 4).
More importantly, we feel that the present results are
very attractive for assembling other more complex
glycoside of cross-linked hydroxylysine (glycosylated
pyridinolines) present in collagen,21 adopting our previ-
ously reported protocols for the contraction of the 3-
hydroxypyridine nucleus.13 Work in this direction is
ongoing in our laboratory and will be published in the
due course.
All reactions occur in a similar fashion and the condi-
tions already set-up for the glycosylation of the (5R)-
hydroxyazide (5R)-5 and for its galactoside 8, were
found suitable for the preparation of the unnatural iso-
mers 3 and 4.
4. Experimental
In particular the formation of the disaccharidic glyco-
side 14 appears to be independent from the stereochem-
istry of aglycone and, under the same conditions used
for the (5R)-series, the compound 14 was obtained in
42% yield, accompanied by the b-anomer in 16% yield.
Also in this case, the appropriate a-stereochemistry of
the formed O-glucosidic bond was derived from the
appropriate chemical shift (d = 5.50ppm) and from its
correct coupling constant (J = 3.5Hz) with the adjacent
H-2 proton.
4.1. General methods
Nuclear magnetic resonance spectra were recorded at
303K on Bruker AM-500 spectrometer operating at
1
500.13MHz for H and 125.76MHz for 13C. Chemical
shifts are reported in parts for million (ppm, d units)
and are referenced to residual CHCl3 (dH = 7.24ppm)
and to CDCl3 (dC = 77.0ppm) for solutions in CDCl3
or to internal CH3OD (dH = 3.30ppm and
dC = 49.0ppm) for solutions in D2O. 1H NMR data
are tabulated in the following order: number of protons,
multiplicity (s, singlet; d, doublet; bs, broad singlet; m,
multiplet), coupling constant(s) in hertz, assignment of
proton(s). The 1H and 13C resonances were assigned
3. Conclusions
In conclusion, our work makes available in a relatively
easy way the two glycoconjugate (5R)-hydroxylysines 1
1
1
1
by H decoupling, H–1H COSY and H–13C correla-

P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
3143
tion experiments. Optical rotations were taken on a
Perkin–Elmer 241 polarimeter and [a]D values are given
epimer (5R)-5. After flash chromatography (eluting
with hexane/AcOEt; 70:30; v:v,) the tert-butyl (2S,5S)-
6-azido-2-benzyloxycarbonylamino-5-hydroxyhexanoate
in 10ꢀ1 degcm2 gꢀ1
.
(5S)-5 (817mg; 87% yield): an oil; Rf 0.39 (silica gel,
27
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 com-
pounds were infused at a flow rate of 5mL/min. The
spray voltage was set at 5.0kV in the positive and at
4.5kV in the negative ion mode with a capillary temper-
ature of 220ꢁC. Full-scan mass spectra were recorded by
scanning a m/z range of 100–2000. All reactions were
monitored by thin-layer chromatography (TLC) carried
out on 0.25mm E. Merck silica gel plates (60 F254) using
UV light, 50% sulfuric acid or 0.2% ninhydrin in ethanol
and heat as developing agent. E. Merck 230–400mesh
silica gel was used for flash column chromatography.22
Usual work-up refers to washing the organic layer with
water, drying over Na2SO4 and evaporating the solvent
under reduced pressure.
hexane/AcOEt; 60:40; v:v); ½aꢁ ¼ þ0:6 (c 1, CHCl3);
D
1H NMR (CDCl3): d 7.34–7.27 (5H, aromatics-H),
5.39 (1H, d, J = 7.5, NH), 5.08 (2H, s, OCH2Ph), 4.27
(1H, m, 2-H), 3.76 (1H, m, 5-H), 3.32 (1H, dd,
J = 12.4, 3.2, 6-Ha), 3.22 (1H, dd, J = 12.4, 7.1, 6-Hb),
2.30 (1H, d, J = 4.2, OH), 1.89 (1H, m, 3-Ha), 1.79
(1H, m, 3-Hb), 1.52 (2H, m, 4-H2), 1.44 [9H, s,
C(CH3)3]; ESI-MS (positive) m/z: 379.0 (37%; M+H+),
401.0 (100%; M+Na+), 402.0 (20%), 778.9 (46%;
2M+Na+), 779.9 (19%). Anal. Calcd for C18H26N4O5:
C, 57.13; H, 6.93; N, 14.81. Found: C, 57.2; H, 6.9; N,
14.7.
4.4. Preparation of the O-(3,4,6-tri-O-benzyl-2-O-chlo-
roacetyl-a-D-galactopyranosyl)trichloroacetimidate a-6
4.4.1. Preparation of the 3,4,6-tri-O-benzyl-1,2-O-dichlo-
roacetyl-D-galactopyranose. A solution of chloroacetyl
chloride (3.7mL; 46.4mmol) in diethyl ether (20mL)
was slowly added to a solution of 3,4,6-tri-O-benzylgal-
actopyranose16 (7.00g; 15.6mmol) and pyridine
(6.27mL; 77.6mmol) in diethyl ether (80mL), maintain-
ing the solution temperature at ꢀ10ꢁC. After warming
to 25ꢁC and a 2h stirring, the reaction mixture was di-
luted with ethyl acetate (150mL) and washed with a cold
aqueous solution of citric acid (200mL; 10%) and then
with brine (3 · 100mL). Usual work-up afforded the
crude 3,4,6-tri-O-benzyl-1,2-O-dichloroacetyl-D-galacto-
pyranose (9.20g; 98% yield), as an oil sufficiently pure
for direct use in the next reaction: Rf 0.29 and 0.23 (a
and b epimers) (silica gel, hexane/AcOEt; 80:20; v:v);
1H NMR showed what it was a mixture of a and b epi-
4.2. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-hydroxyhexanoate (5R)-5
A
mixture of dicyclohexylcarbodiimide (3.7g;
17.9mmol), tert-butyl alcohol (2.1mL; 22.0mmol) and
copper(I) chloride (19mg) was stirred at room tempera-
ture for 5days in order to obtain the O-tert-butyl-N,N0-
dicyclohexylisourea. Then the mixture was diluted with
CH2Cl2 (10mL) and a solution of (2S,5R)-6-azido-2-
benzyloxycarbonylamino-5-hydroxyhexanoic
acid13d
(700mg; 2.17mmol) in CH2Cl2 (20mL) was added.
The obtained mixture was then stirred at room temper-
ature for 5days, diluted with hexane (30mL) and filtered
on a short column of silica gel. The column was first
eluted with hexane (400mL) and then with diethyl ether
(150mL). Elution with diethyl ether afforded a partially
purified product, which was then subjected to rapid
chromatography (eluting with hexane/AcOEt; 70:30;
v:v) to afford pure tert-butyl (2S,5R)-6-azido-2-benzyl-
oxycarbonylamino-5-hydroxyhexanoate (5R)-5 (731
1
mers in a 1:1 ratio. H NMR (CDCl3) for a-epimer: d
6.39 (1H, d, J = 3.5, 1-H), 5.56 (1H, d, J = 10.5, 3.5, 2-
H), 4.06 (1H, dd, J = 2.8, <1, 4-H), 3.93 (1H, dd,
J = 10.5, 2.8, 3-H); for b-epimer: d 5.63 (1H, d,
J = 8.4, 1-H), 5.50 (1H, d, J = 9.8, 8.4, 2-H), 4.02 (1H,
dd, J = 3.0, <1, 4-H), 3.60 (1H, dd, J = 9.8, 3.0, 3-H);
ESI-MS (positive) m/z: 625.1 (100%), 626.2 (34%),
627.1 (64%), 628.1 (22%), 629.1 (11%), 641.2 (11%),
643.2 (7%). Anal. Calcd for C31H32Cl2O8: C, 61.70; H,
5.34. Found: C, 61.5; H, 5.4.
mg; 89% yield): an oil; Rf 0.39 (silica gel, hexane/AcOEt;
26
D
60:40; v:v); ½aꢁ ¼ þ10:9 (c 1, CHCl3); 1H NMR
(CDCl3): d 7.35–7.23 (5H, aromatics-H), 5.48 (1H, d,
J = 7.7, NH), 5.08 (2H, s, OCH2Ph), 4.29 (1H, m, 2-
H), 3.77 (1H, m, 5-H), 3.29 (1H, dd, J = 12.4, 3.6, 6-
Ha), 3.22 (1H, dd, J = 12.4, 6.8, 6-Hb), 2.72 (1H, d,
J = 5.3, OH), 1.98 (1H, m, 3-Ha), 1.68 (1H, m, 3-Hb),
1.52 (2H, m, 4-H2), 1.44 [9H, s, C(CH3)3]; ESI-MS (pos-
itive) m/z: 378.8 (39%; M+H+), 401.0 (100%; M+Na+),
402.0 (22%), 778.8 (48%; 2M+Na+), 779.9 (21%). Anal.
Calcd for C18H26N4O5: C, 57.13; H, 6.93; N, 14.81.
Found: C, 57.1; H, 6.8; N, 14.7.
4.4.2. Preparation of the 3,4,6-tri-O-benzyl-2-O-chloro-
acetyl-D-galactopyranose. The obtained 1,2-O-dichlo-
roacetyl derivative (7.70g; 12.8mmol) dissolved in
CH3CN (200mL), was added to a solution of ammonia
in acetonitrile (70mL; prepared by bubbling ammonia
gas through the solvent at 25ꢁC for 10min). The mixture
was stirring at 25ꢁC until the starting material has dis-
appeared (TLC, hexane/AcOEt; 80:20; v:v) and then
concentrated at temperature <40ꢁC. After dilution with
AcOEt (80mL) and usual work-up, the residue was puri-
fied by flash chromatography (eluting with hexane/
AcOEt; 70:30; v:v) to afford the 3,4,6-tri-O-benzyl-2-
O-chloroacetyl-D-galactopyranose as a mixture (5.72g;
85% yield) of a and b epimers in a 80:20 ratio (1H
4.3. tert-Butyl (2S,5S)-6-azido-2-benzyloxycarbonyl-
amino-5-hydroxyhexanoate (5S)-5
The ester was prepared starting with (2S,5S)-6-azido-
2-benzyloxycarbonylamino-5-hydroxyhexanoic acid13d
(800mg; 2.48mmol) in CH2Cl2 (23mL) and using the
procedure described above for the preparation of the
NMR): an oil; Rf 0.32 (silica gel, hexane/AcOEt;
26
D
70:30; v:v); ½aꢁ ¼ þ43:3 (c 1, CHCl3); 1H NMR

3144
P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
(CDCl3) for a-epimer: d 7.36–7.25 (15H, aromatics),
5.47 (1H, d, J = 3.5, 1-H); 5.35 (1H, dd, J = 10.5, 3.5,
2-H), 4.90 (1H, d, J = 11.4, benzylic), 4.69 (1H, d,
J = 12.2, benzylic), 4.62 (1H, d, J = 12.2, benzylic),
4.53 (1H, d, J = 11.4, benzylic), 4.46 (1H, d, J = 11.9,
benzylic), 4.40 (1H, d, J = 11.9, benzylic), 4.14 (1H,
ddd, J = 6.6, 6.1, <1, 5-H), 4.04 (1H, d, J = 15.4,
ClCHHCO2), 3.98 (1H, dd, J = 10.5, 2.8, 3-H), 3.97
(1H, d, J = 15.4, ClCHHCO2), 3.94 (1H, dd, J = 2.8,
<1, 4-H), 3.57 (1H, dd, J = 9.5, 6.6, 6-Ha), 3.44 (1H,
dd, J = 9.5, 6.1, 6-Hb); ESI-MS (positive) m/z: 549.2
(100%; M+Na+), 550.2 (33%), 551.2 (39%), 565.3
(11%; M+K+), 566.3 (3%), 567.3 (4%). Anal. Calcd
for C29H31ClO7: C, 66.09; H, 5.93. Found: C, 65.8; H,
5.8.
4.5. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-b-D-
galactopyranosyloxy)hexanoate 7
A mixture of tert-butyl (2S,5R)-6-azido-2-benzyloxycar-
bonylamino-5-hydroxyhexanoate
(5R)-5
(1.09g;
2.88mmol), O-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-a-
D-galactopyranosyl)trichloroacetimidate a-6 (3.3g;
4.92mmol) and powdered molecular sieves (3A,
500mg) in anhydrous diethyl ether (120mL) was stirred
for 15min at 25ꢁC. At this time tert-butyldimethylsilyl
trifluoromethanesulfonate (330lL; 1.44mmol) was
added and stirring was continued for 1h under argon.
At this time, the powdered molecular sieves were filtered
off and washed with AcOEt. The organic layers were
washed with a saturated aqueous solution of NaHCO3
(100mL) and worked-up to afford a residue, which
was purified by flash chromatography (eluting with hex-
ane/AcOEt; 75:25; v:v) to give the title compound 7
4.4.3. Preparation of the title compound a-6. 1,8-Diaza-
bicyclo[5.4.0]undec-7-ene (DBU; 248lL; 0.0017mmol)
was added to a solution of the 3,4,6-tri-O-benzyl-2-O-
chloroacetyl-D-galactopyranose (4.38g; 8.3mmol) and
trichloroacetonitrile (8.3mL; 83.0mmol) in CH2Cl2
(80mL) at ꢀ30ꢁC. After a stirring of 4h at ꢀ30ꢁC,
the solvent was removed under reduced pressure and
the residue was purified by flash column chromatogra-
phy (eluting with hexane/AcOEt/Et3N; 85:15:1; v:v:v)
to afford the O-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-a-
D-galactopyranosyl)trichloroacetimidate a-6 (4.41g;
(1.33g; 52% yield): an oil; Rf 0.20 (silica gel, CH2Cl2/
26
diisopropyl ether; 100:5; v:v); ½aꢁ ¼ þ7:9 (c 1, CHCl3);
D
1H NMR (CDCl3): d 7.33–7.23 (20H, aromatics), 5.34
(1H, dd, J = 10.0, 7.9, 20-H), 5.33 (1H, d, J = 7.7,
NH), 5.09 (1H, d, J = 12.1, part A of AB system, Cbz
OCHHPh), 5.06 (1H, d, J = 12.1, part B of AB system,
Cbz OCHHPh), 4.90 (1H, d, J = 11.6, benzylic), 4.63
(1H, d, J = 12.1, benzylic), 4.56 (1H, d, J = 11.6, benzy-
lic), 4.44 (1H, d, J = 12.1, benzylic), 4.43 (1H, d,
J = 11.7, part A of AB system, benzylic), 4.43 (1H, d,
J = 7.9, 10-H), 4.41 (1H, d, J = 11.7, part B of AB sys-
tem, benzylic), 4.18 (1H, m, 2-H), 4.01 (1H, d,
J = 14.7, ClCHHCO2), 3.92 (1H, dd, J = 2.6, <1,
40-H), 3.92 (1H, d, J = 14.7, ClCHHCO2), 3.63 (1H,
m, 5-H), 3.59–3.54 (3H, overlapping, 50-H, 60-Ha, 60-
Hb), 3.50 (1H, dd, J = 10.0, 2.6, 30-H), 3.37 (1H, dd,
J = 13.0, 4.6, 6-Ha), 3.33 (1H, dd, J = 13.0, 5.9, 6-Hb),
1.82 (1H, m, 3-Ha), 1.61–1.51 (3H, overlapping, 3-Hb,
4-Ha, 4-Hb), 1.44 [9H, s, C(CH3)3]; 13C NMR (CDCl3):
d 171.0 (C-1), 165.9 (ClCH2CO2), 155.8 (NHCO), 101.5
(C-10), 82.5 [C(CH3)3], 80.1 (C-30), 78.3 (C-5), 74.5,
73.6, 72.1 (3 · OCH2Ph), 73.7 (C-50), 73.1 (C-20), 72.4
(C-40), 68.6 (C-60), 67.0 (NHCO2CH2Ph), 54.4 (C-6),
54.1 (C-2), 40.7 (ClCH2CO2), 28.4 (C-3), 27.9
[C(CH3)3], 27.7 (C-4); ESI-MS (positive) m/z: 909.4
79% yield) as an oil; Rf 0.56 (silica gel, hexane/AcOEt;
26
D
70:30; v:v); ½aꢁ ¼ þ66:7 (c 1, CHCl3); 1H NMR
(CDCl3): d 8.51 (1H, s, @NH), 7.35–7.25 (15H, aromat-
ics), 6.54 (1H, d, J = 3.5, 1-H); 5.57 (1H, dd, J = 10.3,
3.5, 2-H), 4.96 (1H, d, J = 11.3, benzylic), 4.72 (1H, d,
J = 12.2, benzylic), 4.61 (1H, d, J = 12.2, benzylic),
4.60 (1H, d, J = 11.3, benzylic), 4.47 (1H, d, J = 11.7,
benzylic), 4.41 (1H, d, J = 11.7, benzylic), 4.17 (1H,
ddd, J = 8.4, 5.6, <1, 5-H), 4.11 (1H, dd, J = 2.8, <1,
4-H), 4.06 (1H, dd, J = 10.3, 2.8, 3-H), 3.90 (2H, s,
ClCH2CO2), 3.65 (1H, dd, J = 9.1, 8.4, 6-Ha), 3.57
(1H, dd, J = 9.1, 5.6, 6-Hb); ESI-MS (positive) m/z:
692.4 (76%), 693.4 (21%), 694.2 (100%), 695.2 (31%),
696.1 (49%), 697.1 (15%), 698.0 (12%). Anal. Calcd for
C31H31Cl4NO7: C, 55.46; H, 4.65; N, 2.09. Found: C,
55.5; H, 4.5; N, 2.1.
(100%;
35ClꢀM+Na+),
910.4
(50%),
911.4
Further elution affords the O-(3,4,6-tri-O-benzyl-2-O-
chloroacetyl-b-D-galactopyranosyl)trichloroacetimidate
b-6 (0.610g; 11% yield) as an oil; Rf 0.39 (silica gel, hex-
(43%; 37ClꢀM+Na+), 925.3 (23%; 35ClꢀM+K+),
926.4 (12%), 927.4 (11%; 37ClꢀM+K+). Anal. Calcd
for C47H55ClN4O11: C, 63.61; H, 6.25; N, 6.31. Found:
C, 63.5; H, 6.3; N, 6.3.
26
D
1
ane/AcOEt; 70:30; v:v); ½aꢁ ¼ þ20:3 (c 1, CHCl3); H
NMR (CDCl3): d 8.59 (1H, s, @NH), 7.36–7.25 (15H,
aromatics), 5.71 (1H, d, J = 8.1, 1-H), 5.64 (1H, dd,
J = 9.8, 8.1, 2-H), 4.92 (1H, d, J = 11.6, benzylic), 4.67
(1H, d, J = 12.3, benzylic), 4.60 (1H, d, J = 11.6, benz-
ylic), 4.48 (1H, d, J = 12.3, benzylic), 4.46 (1H, d,
J = 11.6, benzylic), 4.42 (1H, d, J = 11.6, benzylic),
4.03 (1H, dd, J = 2.6, <1, 4-H), 3.89 (1H, d, J = 14.9,
ClCHHCO2), 3.84 (1H, d, J = 14.9, ClCHHCO2), 3.77
(1H, ddd, J = 7.5, 5.5, <1, 5-H), 3.68–3.61 (3H, overlap-
ping, 3-H, 6-H2); ESI-MS (positive) m/z: 692.4 (76%),
693.4 (21%), 694.2 (100%), 695.2 (31%), 696.1 (49%),
697.1 (15%), 698.0 (12%). Anal. Calcd for
C31H31Cl4NO7: C, 55.46; H, 4.65; N, 2.09. Found: C,
55.4; H, 4.6; N, 2.2.
4.6. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-b-D-galacto-
pyranosyloxy)hexanoate 7 from the b-trichloroacet-
imidate b-6
Starting with the tert-butyl (2S,5R)-6-azido-2-benzyl-
oxycarbonylamino-5-hydroxyhexanoate (5R)-5 (165
mg; 0.44mmol) and the O-(3,4,6-tri-O-benzyl-2-O-chlo-
roacetyl-b-D-galactopyranosyl)trichloroacetimidate b-6
(500mg; 0.75mmol) and following the glycosylation
procedure described above, the title compound 7
(190mg; 49% yield) was obtained, identical in all
respects with that described above.

P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
3145
4.7. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-b-D-galactopyranosyl-
oxy)hexanoate 8
5-H), 3.88 (1H, dd, J = 2.7, <1, 40-H), 3.73–3.71 (2H,
AB system, 60-Ha, 60-Hb), 3.66 (1H, ddd, J = 7.4, 4.7,
<1, 50-H), 3.61 (1H, dd, J = 10.0, 2.7, 30-H), 3.51 (1H,
dd, J = 10.0, 7.7, 20-H), 3.17 (1H, dd, J = 13.1, 1.4, 6-
Ha), 2.99 (1H, dd, J = 13.1, 9.5, 6-Hb), 2.20 (1H, m,
3-Ha), 2.00 (1H, m, 3-Hb), 1.78 (1H, m, 4-Ha), 1.72
(1H, m, 4-Hb); 13C NMR (D2O): d 172.6 (C-1), 163.7
(q, JCF = 36.2, CF3COOꢀ), 117.3 (q, JCF = 290.9,
CF3COOꢀ), 103.4 (C-10), 76.7 (C-5), 76.4 (C-50), 73.7
(C-30), 72.1 (C-20), 69.8 (C-40), 62.3 (C-60), 53.7 (C-2),
44.0 (C-6), 29.1 (C-4), 26.5 (C-3); ESI-MS (positive) m/
z: 325.2 (100%; M+H+), 326.2 (13%), 347.2 (27%;
M+Na+), 363.3 (5%; M+K+). Anal. Calcd for
C12H24N2O8Æ2CF3COOH: C, 34.79; H, 4.74; N, 5.07.
Found: C, 34.7; H, 4.6; N, 5.1.
A mixture of chloroacetylated galactoside 7 (1.2g;
1.35mmol) and Zn(CH3COO)2 2H2O (648mg;
2.95mmol) in MeOH (30mL) was refluxed for 9h. Then
the solvent was evaporated and the residue was dis-
solved in AcOEt (50mL) and worked-up. Flash chroma-
tography (eluting with hexane/AcOEt; 75:25; v:v)
afforded the pure title compound 8 (1.02g; 93% yield):
an oil; Rf 0.35 (silica gel, hexane/AcOEt; 70:30; v:v);
26
D
1
½aꢁ ¼ þ0:7 (c 1, CHCl3); H NMR (CDCl3): d 7.37–
7.26 (20H, aromatics), 5.76 (1H, d, J = 7.7, NH), 5.13–
5.09 (2H, AB system, benzylic), 4.90 (1H, d, J = 11.6,
benzylic), 4.69 (2H, s, benzylic), 4.60 (1H, d, J = 11.6,
benzylic), 4.47 (1H, d, J = 11.7, benzylic), 4.43 (1H, d,
J = 11.7, benzylic), 4.31 (1H, d, J = 7.7, 10-H), 4.25
(1H, ddd, J = 7.7, 6.3, 6.3, 2-H), 3.93 (1H, ddd,
J = 9.8, 7.7, 2.3, 20-H), 3.85 (1H, dd, J = 2.5, <1, 40-
H), 3.74 (1H, m, 5-H), 3.59–3.54 (3H, overlapping, 50-
H, 60-Ha, 60-Hb), 3.41 (2H, d, J = 4.7, 6-H2), 3.37
(1H, dd, J = 9.8, 2.5, 30-H), 3.19 (1H, d, J = 2.3, 20-
OH), 1.95 (1H, m, 3-Ha), 1.85 (1H, m, 3-Hb), 1.68
(1H, m, 4-Ha), 1.63 (1H, m, 4-Hb), 1.45 [9H, s,
C(CH3)3]; 13C NMR (CDCl3): d 171.2 (C-1), 155.8
(NHCO), 103.8 (C-10), 82.1 [C(CH3)3], 81.9 (C-30),
78.1 (C-5), 74.3, 73.3, 72.6 (3 · OCH2Ph), 73.6
(C-50), 73.0 (C-40), 71.3 (C-20), 68.7 (C-60), 66.8
(NHCO2CH2Ph), 54.6 (C-6), 54.0 (C-2), 27.8
[C(CH3)3], 27.7 (C-4), 27.5 (C-3); ESI-MS (positive)
m/z: 833.4 (100%; M+Na+), 834.4 (51%), 835.0
(15%), 849.5 (29%; M+K+), 850.5 (15%). Anal.
Calcd for C45H54N4O10: C, 66.65; H, 6.71; N, 6.91.
Found: C, 66.5; H, 6.6; N, 6.8.
4.9. tert-Butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-[(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-
(1fi2)-(3,4,6-tri-O-benzyl-b-D-galactopyranosyloxy)]hex-
anoate 10
A mixture of galactoside 8 (1.61g; 1.98mmol), O-
(2,3,4,6-tetra-O-benzyl-b-D-glucopyranosyl)trichloroacet-
imidate 919,20 (2.66g; 3.96mmol) and powdered molecu-
lar sieves (3A; 500mg) in anhydrous diethyl ether
(120mL) was stirred for 15min at room temperature.
Then tert-butyldimethylsilyl triflate (227lL; 0.99mmol)
was added and stirring was continued, at room temper-
ature, for 1h under argon. The powdered molecular
sieves were filtered off and washed with AcOEt. The or-
ganic phase was washed with a saturated NaHCO3
aqueous solution (100mL) and worked-up to afford a
residue, which was purified by flash chromatography
(eluting with hexane/AcOEt; 80:20; v:v) to give the title
compound 10 (1.19g; 45% yield) as an oil; Rf 0.28 (silica
29
D
gel, hexane/AcOEt; 80:20; v:v); ½aꢁ ¼ þ25:6 (c 1,
1
4.8. (2S,5R)-2,6-Diamino-5-(b-D-galactopyranosyl-
oxy)hexanoic acid 1
CHCl3); H NMR (CDCl3): d 7.38–7.06 (20H, aromat-
ics), 5.62 (1H, d, J = 3.5, 100-H), 5.20 (1H, d, J = 7.9,
NH), 5.08–4.45 (16H, benzylic) 4.63 (1H, d, J = 7.7,
10-H), 4.33 (1H, ddd, J = 10.0, 2.2, 2.2, 500-H), 4.21
(1H, dd, J = 10.0, 7.7, 20-H), 4.14 (1H, m, 2-H), 4.01
(1H, dd, J = 9.5, 9.5, 300-H), 3.99 (1H, dd, J = 3.0, <1,
40-H), 3.88 (1H, m, 5-H), 3.69 (1H, dd, J = 10.0, 9.5,
400-H), 3.63 (1H, dd, J = 9.5, 3.5, 200-H), 3.64–3.61 (3H,
overlapping, 50-H, 60-Ha, 60-Hb), 3.62 (1H, dd,
J = 10.0, 3.0, 30-H), 3.54 (1H, dd, J = 12.8, 4.0, 6-Ha),
3.45–3.39 (2H, AB system, 600-Ha, 600-Hb), 3.35 (1H,
dd, J = 12.8, 3.9, 6-Hb), 1.75–1.78 (2H, overlapping,
3-Ha, 4-Ha), 1.61–1.54 (2H, overlapping, 3-Hb, 4-Hb),
1.44 [9H, s, C(CH3)3]; ESI-MS (positive) m/z: 1355.6
(100%; M+Na+), 1356.6 (82%), 1357.6 (40%), 1358.5
(11%), 1371.5 (31%; M+K+), 1372.5 (24%), 1373.5
(13%), 1374.4 (4%). Anal. Calcd for C79H88N4O15: C,
71.15; H, 6.65; N, 4.20. Found: C, 71.0; H, 6.7; N, 4.2.
The tert-butyl (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-b-D-galactopyranosyloxy)-
hexanoic acid 8 (200mg; 0.25mmol) in tetrahydrofuran
(8.6mL) and ethanol (28.6mL) was hydrogenated in the
presence of palladium (generated from 30mg of PdCl3),
at room temperature under 1atm of hydrogen. After 6h,
the reaction was monitored on TLC (silica gel, MeOH/
25% aqueous NH3; 10:2; v:v), which indicated the pres-
ence of a single spot at Rf 0.21. Then the catalyst was fil-
tered off on a pad of Celite and washed with methanol
(15mL). Evaporation of the solvent mixture under re-
duced pressure (below 40ꢁC) afforded a residue, which
was dissolved into TFA–H2O (1.5mL; 95:5; v:v) and
the resulting solution was stirred at room temperature
for 1h. The crude product obtained by evaporation of
the solvent was dissolved in distilled water (3mL) and
washed with toluene (2 · 4mL). The aqueous solution
was co-evaporated with toluene under reduced pressure
(below 40ꢁC) to afford the pure glucosylgalactosyl
hydroxylysine 1 as ditrifluoroacetate (125mg; 92%
4.10. tert-Butyl (2S,5R)-2,6-diamino-5-[a-D-glucopyra-
nosyl-(1fi2)-b-D-galactopyranosyloxy]hexanoate 11
The protected compound 10 (300mg; 0.22mmol) in
tetrahydrofuran (39mL) and ethanol (114mL) was
hydrogenated in the presence of palladium (10% on acti-
vated carbon, 300mg) at room temperature under 1atm
of hydrogen. After 9h, the reaction was monitored on
yield); a syrup; Rf 0.21 (silica gel, MeOH/25% aqueous
26
D
1
NH3; 10:2; v:v); ½aꢁ ¼ ꢀ0:9 (c 1, MeOH) [lit.9 ꢀ1.4 (c
0.18, MeOH)]; H NMR (D2O): d 4.42 (1H, d, J = 7.7,
10-H), 4.10 (1H, dd, J = 6.3, 6.3, 2-H), 4.04 (1H, m,

3146
P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
TLC (silica gel, MeOH/25% aqueous NH3; 10:1.5; v:v),
which indicated the presence of a single spot at Rf 0.27.
Then the catalyst was filtered off on a pad of Celite
and thoroughly rinsed with a methanolic solution of
distilled water (20mL; 5:1; v:v). Evaporation of the com-
bined filtrates under reduced pressure (below 40ꢁC)
afforded a residue, which was dissolved in distilled water
(2mL) and washed with toluene (2 · 4mL). The aqueous
solution was co-evaporated with toluene under
reduced pressure (below 40ꢁC) to give the title com-
pound 11, (110mg; 90% yield) as a syrup; Rf 0.27
C18H34N2O13Æ2CF3COOH: C, 36.98; H, 5.08; N, 3.92.
Found: C, 37.0; H, 5.2; N, 3.9.
4.12. tert-Butyl (2S,5S)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-b-D-
galactopyranosyloxy)hexanoate 12
Starting with the tert-butyl (2S,5S)-6-azido-2-benzyloxy-
carbonylamino-5-hydroxyhexanoate (5S)-5 (900mg;
2.38mmol) and the O-(3,4,6-tri-O-benzyl-2-O-chloroace-
tyl-a-D-galactopyranosyl)trichloroacetimidate a-6 (2.72
g; 4.05mmol) and following the glycosylation procedure
described above for the epimeric ester (5R)-5, the title
compound 12 (1.06g; 50% yield) was obtained, after
flash chromatography (eluting with hexane/AcOEt;
75:25; v:v): an oil Rf 0.20 (silica gel, CH2Cl2/diisopropyl
(silica gel, MeOH/25% aqueous NH3; 10:1.5; v:v);
27
½aꢁ ¼ þ25:9 (c 1, MeOH); 1H NMR (D2O): d 5.35
D
(1H, d, J = 3.6, 100-H), 4.63 (1H, d, J = 7.5, 10-H), 4.12
(1H, m, 5-H), 4.03 (1H, ddd, J = 9.5, 1.6 and 1.6,
500-H), 4.02 (1H, dd, J = 6.4, 6.4, 2-H), 3.90 (1H, dd,
J = 3.0, <1, 40-H), 3.80 (1H, dd, J = 12.2, 1.6, 600-Ha),
3.75–3.62 (6H, overlapping, 30-H, 50-H, 60-H2, 300-H,
600-Hb), 3.68 (1H, dd, J = 9.5, 7.5, 20-H), 3.50 (1H, dd,
J = 9.9, 3.6, 200-H), 3.39 (1H, dd, J = 9.5, 9.5, 400-H),
3.19 (1H, dd, J = 13.3, 1.9, 6-Ha), 3.01 (1H, dd,
J = 13.3 and 9.9, 6-Hb), 2.10 (1H, m, 3-Ha), 2.00 (1H,
m, 3-Ha), 1.85 (1H, m, 4-Ha), 1.69 (1H, m, 4-Hb),
1.48 [9H, s, C(CH3)3]; 13C NMR (D2O): d 170.1 (C-1),
102.4 (C-10), 98.6 (C-100), 86.9 [C (CH3)3], 76.1, 75.8
(C-20, C-300), 75.1 (C-5), 73.8 (C-30), 72.6 (C-500), 72.5
(C-50), 72.3 (C-200), 70.4 (C-400), 69.9 (C-40), 62.1 (C-60),
61.4 (C-600), 54.0 (C-2), 43.6 (C-6), 28.7 (C-4),
28.1 [C(CH3)3], 26.3 (C-3); ESI-MS (positive) m/z:
543.1 (100%; M+H+), 544.1 (23%), 565.1 (23%;
M+Na+), 566.2 (6%). Anal. Calcd for C22H42N2O13:
C, 48.70; H, 7.80; N, 5.16. Found: C, 48.8; H, 7.8; N,
5.1.
27
D
1
ether; 100:5; v:v); ½aꢁ ¼ þ1:7 (c 1, CHCl3); H NMR
(CDCl3): d 7.35–7.23 (20H, aromatics), 5.34 (1H, dd,
J = 9.8, 7.7, 20-H), 5.31 (1H, d, J = 7.2, NH), 5.06 (2H,
s, Cbz OCH2Ph), 4.89 (1H, d, J = 11.6, benzylic), 4.65
(1H, d, J = 12.1, benzylic), 4.54 (1H, d, J = 11.6, benz-
ylic), 4.47 (1H, d, J = 7.7, 10-H), 4.46 (1H, d, J = 11.7,
benzylic), 4.44 (1H, d, J = 12.1, benzylic), 4.38 (1H, d,
J = 11.7, benzylic), 4.22 (1H, m, 2-H), 3.99 (1H, d,
J = 14.7, ClCHHCO2), 3.95 (1H, dd, J = 2.8, <1, 40-H),
3.90 (1H, d, J = 14.7, ClCHHCO2), 3.71 (1H, m, 5-H),
3.63 (1H, ddd, J = 7.7, 7.7, <1, 50-H), 3.59–3.55 (2H,
overlapping, 60-Ha, 60-Hb), 3.52 (1H, dd, J = 9.8, 2.8,
30-H), 3.27 (1H, dd, J = 12.6, 3.5, 6-Ha), 3.13 (1H, dd,
J = 12.6, 6.3, 6-Hb), 1.87 (1H, m, 3-Ha), 1.75 (1H, m,
3-Hb), 1.52–1.48 (2H, overlapping, 4-Ha, 4-Hb), 1.37
[9H, s, C(CH3)3]; 13C NMR (CDCl3): d 171.4 (C-1),
166.1 (ClCH2CO2), 155.9 (NHCO), 100.2 (C-10), 82.1
[C(CH3)3], 80.1 (C-30), 76.8 (C-5), 74.5, 73.5, 72.0
(3 · OCH2Ph), 73.5 (C-50), 73.0 (C-20), 72.6 (C-40), 68.3
(C-60), 66.7 (NHCO2CH2Ph), 54.6 (C-6), 53.7 (C-2),
40.8 (ClCH2CO2), 28.3 (C-4), 28.2 (C-3), 28.0
[C(CH3)3]; ESI-MS (positive) m/z: 909.4 (100%;
35ClꢀM+Na+), 910.4 (51%), 911.3 (43%; 37ClꢀM+Na+),
925.4 (25%; 35ClꢀM+K+), 926.4 (12%), 927.4 (10%;
37ClꢀM+K+). Anal. Calcd for C47H55ClN4O11: C,
63.61; H, 6.25; N, 6.31. Found: C, 63.6; H, 6.2; N, 6.2.
4.11. (2S,5R)-2,6-Diamino-5-[a-D-glucopyranosyl-(1fi2)-
b-D-galactopyranosyloxy]hexanoic acid 2
The tert-butyl ester 11 (300mg; 0.55mmol) was dis-
solved into TFA–H2O (3.0mL; 95:5; v:v) and the result-
ing solution was stirred at room temperature for 1h.
Evaporation of the solvent afforded the pure title com-
pound 2 as ditrifluoroacetate (363mg; 92% yield); a syr-
up; Rf 0.22 (silica gel, MeOH/25% aqueous NH3; 10:3;
26
v:v); ½aꢁ ¼ þ21:9 (c 1, MeOH); 1H NMR (D2O): d
4.13. tert-Butyl (2S,5S)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-b-D-galactopyranosyl-
oxy)hexanoate 13
D
5.33 (1H, d, J = 3.6, 100-H), 4.61 (1H, d, J = 7.5, 10-H),
4.10 (1H, m, 5-H), 4.07 (1H, dd, J = 6.5, 6.5, 2-H),
4.02 (1H, ddd, J = 9.7, 1.5 and 1.5, 500-H), 3.89 (1H,
dd, J = 2.3, <1, 40-H), 3.80 (1H, dd, J = 11.3, <1,
600-Ha), 3.74–3.66 (6H, overlapping, 30-H, 50-H, 60-H2,
300-H, 600-Hb), 3.65 (1H, dd, J = 9.5, 7.5, 20-H), 3.49
(1H, dd, J = 9.9, 3.6, 200-H), 3.38 (1H, dd, J = 9.7, 9.7,
400-H), 3.18 (1H, dd, J = 12.9, <1, 6-Ha), 3.00 (1H,
dd, J = 12.9 and 10.5, 6-Hb), 2.14 (1H, m, 3-Ha),
2.00 (1H, m, 3-Hb), 1.87 (1H, m, 4-Ha), 1.71 (1H,
m, 4-Hb); 13C NMR (D2O): d 172.4 (C-1), 163.7 (q,
JCF = 34.3, CF3COOꢀ), 117.3 (q, JCF = 289.9,
CF3COOꢀ), 102.5 (C-10), 98.5 (C-100), 76.1, 75.8 (C-20,
C-300), 75.2 (C-5), 73.8 (C-30), 72.6 (C-500), 72.4 (C-200),
72.2 (C-50), 70.4 (C-400), 69.9 (C-40), 62.0 (C-60), 61.3
(C-600), 53.4 (C-2), 43.6 (C-6), 28.8 (C-4), 26.2 (C-3);
ESI-MS (positive) m/z: 487.0 (100%; M+H+), 488.1
(19%), 489.1 (4%), 509.2 (38%; M+Na+), 525.1 (5%;
M+K+), 973.0 (7%; 2M+H+). Anal. Calcd for
Starting with chloroacetylated galactoside 12 (900mg;
1.01mmol) and following the procedure described above
for the selective hydrolysis of the epimer 7, the title com-
pound 13 (748mg; 91% yield) was obtained after flash
chromatography (eluting with hexane/AcOEt; 75:25;
v:v): an oil; Rf 0.26 (silica gel, hexane/AcOEt; 70:30;
30
1
v:v); ½aꢁ ¼ ꢀ1:9 (c 1, CHCl3); H NMR (CDCl3): d
D
7.34–7.23 (20H, aromatics), 5.35 (1H, d, J = 8.1, NH),
5.06 (2H, s, benzylic), 4.87 (1H, d, J = 11.6, benzylic),
4.72–4.66 (2H, AB system, benzylic), 4.57 (1H, d,
J = 11.6, benzylic), 4.44 (1H, d, J = 11.9, benzylic),
4.37 (1H, d, J = 11.9, benzylic), 4.30 (1H, d, J = 7.7,
10-H), 4.23 (1H, m, 2-H), 3.91 (1H, ddd, J = 9.8, 7.7,
2.3, 20-H), 3.89 (1H, dd, J = 2.6, <1, 40-H), 3.78 (1H,
m, 5-H), 3.62–3.54 (3H, overlapping, 50-H, 60-Ha, 60-
Hb), 3.41 (1H, dd, J = 9.8, 2.8, 30-H), 3.37 (1H, dd,

P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
3147
J = 12.8, 3.7, 6-Ha), 3.26 (1H, dd, J = 12.8, 6.3, 6-Hb),
2.57 (1H, d, J = 2.3, 20-OH), 1.89 (1H, m, 3-Ha), 1.80
(1H, m, 3-Hb), 1.59 (2H, m, 4-H2), 1.38 [9H, s,
C(CH3)3]; 13C NMR (CDCl3): d 171.3 (C-1), 155.9
(NHCO), 102.8 (C-10), 81.9 (C-30), 81.8 [C(CH3)3],
77.4 (C-5), 74.4, 73.3, 72.5 (3 · OCH2Ph), 73.5 (C-50),
73.0 (C-40), 71.3 (C-20), 68.5 (C-60), 66.7
(NHCO2CH2Ph), 54.3 (C-6), 53.8 (C-2), 28.3 (C-4),
28.1 (C-3), 27.8 [C(CH3)3]; ESI-MS (positive) m/z:
833.4 (100%; M+Na+), 834.4 (51%), 835.0 (15%), 849.5
(25%; M+K+), 850.5 (13%). Anal. Calcd for
C45H54N4O10: C, 66.65; H, 6.71; N, 6.91. Found: C,
66.6; H, 6.7; N, 6.8.
overlapping, 50-H, 60-Ha, 60-Hb), 3.58 (1H, dd,
J = 9.8, 2.5, 30-H), 3.41–3.37 (2H, AB system, 600-Ha,
600-Hb), 3.20 (1H, dd, J = 12.6, 5.1, 6-Ha), 3.00 (1H,
dd, J = 12.6, 6.1, 6-Hb), 1.83 (1H, m, 3-Ha), 1.68 (1H,
m, 3-Hb), 1.58 (1H, m, 4-Ha), 1.43 (1H, m, 4-Hb),
1.37 [9H, s, C(CH3)3]; ESI-MS (positive) m/z: 1355.6
(100%; M+Na+), 1356.6 (80%), 1357.6 (39%), 1358.5
(13%), 1371.5, (31%; M+K+), 1372.5 (26%), 1373.5
(13%), 1374.4 (4%). Anal. Calcd for C79H88N4O15: C,
71.15; H, 6.65; N, 4.20. Found: C, 71.0; H, 6.6; N, 4.1.
4.16. tert-Butyl (2S,5S)-2,6-diamino-5-[a-D-glucopyrano-
syl-(1fi2)-b-D-galactopyranosyloxy]hexanoate 15
4.14. (2S,5S)-2,6-Diamino-5-(b-D-galactopyranosyl-
oxy)hexanoic acid 3
Starting with tert-butyl (2S,5S)-6-azido-2-benzyloxycar-
bonylamino-5-[(2,3,4,6-tetra-O-benzyl-a-D-glucopyranos-
yl)-(1!2)-(3,4,6-tri-O-benzyl-b-D-galactopyranosyloxy)]-
hexanoate 14 (300mg; 0.22mmol) and following the
procedure described above for the hydrogenolysis of
the epimer 10, the title compound 15 was obtained
Starting with the tert-butyl (2S,5S)-6-azido-2-benzyl-
oxycarbonylamino-5-(3,4,6-tri-O-benzyl-b-D-galactopyr-
anosyloxy)hexanoate 13 (250mg; 0.31mmol) and
following the same procedure above reported for the
(2S,5R)-isomer 8, the title compound 3 as ditrifluoro-
acetate was obtained (153mg; 90% yield) as a syrup;
(112mg; 92% yield) as a syrup; Rf 0.27 (silica gel,
27
MeOH/25% aqueous NH3; 10:1.5; v:v); ½aꢁ ¼ þ41:1(c
D
1, MeOH); 1H NMR (D2O): d 5.21 (1H, d, J = 3.2,
100-H), 4.62 (1H, d, J = 7.9, 10-H), 4.18 (1H, m, 5-H),
4.08 (1H, dd, J = 6.0, 6.0, 2-H), 4.00 (1H, ddd,
J = 9.8, 1.6 and 1.6, 500-H), 3.92 (1H, dd, J = 2.5, <1,
40-H), 3.80 (1H, dd, J = 11.5, 1.5, 600-Ha), 3.76–3.64
(6H, overlapping, 30-H, 50-H, 60-H2, 300-H, 600-Hb), 3.61
(1H, dd, J = 9.7, 7.5, 20-H), 3.58 (1H, dd, J = 10.5,
3.2, 200-H), 3.43 (1H, dd, J = 9.8, 9.8, 400-H), 3.25 (1H,
dd, J = 12.6, 1.9, 6-Ha), 3.16 (1H, dd, J = 12.6 and
8.0, 6-Hb), 2.09 (2H, ddd, J = 7.2, 7.2, 7.2, 3-H2), 1.86
(1H, m, 4-Ha), 1.75 (1H, m, 4-Hb), 1.50 [9H, s,
Rf 0.22 (silica gel, MeOH/25% aqueous NH3; 10:2;
27
v:v); ½aꢁ ¼ þ21:3 (c 1, MeOH); 1H NMR (D2O): d
D
4.46 (1H, d, J = 7.7, 10-H), 4.12 (1H, dd, J = 6.3, 6.3,
2-H), 4.10 (1H, m, 5-H), 3.88 (1H, dd, J = 2.8, <1, 40-
H), 3.73–3.70 (2H, AB system, 60-Ha, 60-Hb), 3.66
(1H, ddd, J = 6.5, 5.7, <1, 50-H), 3.62 (1H, dd,
J = 10.2, 2.8, 30-H), 3.53 (1H, dd, J = 10.2, 7.7, 20-H),
3.20 (1H, dd, J = 13.3, 2.5, 6-Ha), 3.12 (1H, dd,
J = 13.3, 8.3, 6-Hb), 2.09 (2H, m, 3-H2), 1.85 (1H, m,
4-Ha), 1.79 (1H, m, 4-Hb); 13C NMR (D2O): d 172.7
(C-1), 163.7 (q, JCF = 36.2, CF3COOꢀ), 117.3 (q,
JCF = 290.9, CF3COOꢀ), 103.7 (C-10), 77.5 (C-5), 76.3
(C-50), 73.7 (C-30), 72.0 (C-20), 69.6 (C-40), 62.0 (C-60),
53.5 (C-2), 43.5 (C-6), 28.4 (C-4), 26.2 (C-3); ESI-MS
(positive) m/z: 325.2 (100%; M+H+), 326.2 (13%),
347.2, (30%; M+Na+), 363.3 (4%; M+K+). Anal. Calcd
for C12H24N2O8Æ2CF3COOH: C, 34.79; H, 4.74; N, 5.07.
Found: C, 34.8; H, 4.7; N, 5.1.
13
C(CH3)3]; C NMR (D2O): 169.8 (C-1), 103.0 (C-10),
100.2 (C-100), 86.8 [C(CH3)3], 79.2 (C-20), 76.2 (C-300),
76.1 (C-5), 73.9 (C-30), 73.0 (C-500), 72.5 (C-50), 72.4
(C-200), 70.3 (C-400), 69.9 (C-40), 62.0 (C-60), 61.2 (C-
600), 54.0 (C-2), 43.2 (C-6), 28.5 (C-4), 28.1 [C(CH3)3],
26.3 (C-3); ESI-MS (positive) m/z: 543.1 (100%;
M+H+), 544.1 (25%), 565.2 (22%; M+Na+), 566.2
(6%). Anal. Calcd for C22H42N2O13: C, 48.70; H, 7.80;
N, 5.16. Found: C, 48.6; H, 7.7; N, 5.2.
4.15. tert-Butyl (2S,5S)-6-azido-2-benzyloxycarbonyl-
amino-5-[(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-
(1fi2)-(3,4,6-tri-O-benzyl-b-D-galactopyranosyloxy)]hex-
anoate 14
4.17. (2S,5S)-2,6-Diamino-5-[a-D-glucopyranosyl-(1fi2)-
b-D-galactopyranosyloxy]hexanoic acid 4
Starting with tert-butyl (2S,5S)-2,6-diamino-5-[a-D-glu-
copyranosyl-(1!2)-b-D-galactopyranosyloxy]hexanoate
15 (300mg; 0.55mmol) and following the procedure de-
scribed above for the hydrolysis of the analogue 11, the
title compound 4 was obtained as ditrifluoroacetate
Starting with tert-butyl (2S,5S)-6-azido-2-benzyloxycar-
bonylamino-5-(3,4,6-tri-O-benzyl-a-D-galactopyranosyl-
oxy)hexanoate 13 (1.81g; 2.23mmol), and following the
procedure described above for the glycosylation of the
epimer 8, the title compound 14 was obtained (1.25g;
42% yield) as a pure oil after flash chromatography
(355mg; 90% yield); a syrup; Rf 0.22 (silica gel,
27
MeOH/25% aqueous NH3; 10:3; v:v); ½aꢁ ¼ þ33:4 (c
D
1
(eluting with hexane/AcOEt; 80:20; v:v); Rf 0.28 (silica
1, MeOH); H NMR (D2O): d 5.20 (1H, d, J = 3.6, 100-
H), 4.59 (1H, d, J = 7.2, 10-H), 4.14 (1H, m, 5-H), 3.98
(1H, ddd, J = 9.8, 4.8, 2.9, 500-H), 3.90–3.87 (2H, over-
lapping, 2-H, 40-H), 3.77–3.65 (5H, overlapping, 50-H,
60-H2, 600-H2), 3.69 (1H, dd, J = 10.0, 3.5, 30-H), 3.66
(1H, dd, J = 10.5, 10.0, 300-H), 3.59 (1H, dd, J = 10.0,
7.2, 20-H), 3.55 (1H, dd, J = 10.5, 3.6, 200-H), 3.41 (1H,
dd, J = 10.0, 9.8, 400-H), 3.22 (1H, dd, J = 13.6, 2.9, 6-
Ha), 3.14 (1H, dd, J = 13.6, 7.2, 6-Hb), 2.02 (2H, ddd,
J = 7.2, 7.2, 7.2, 3-H2), 1.83 (1H, m, 4-Ha), 1.74 (1H,
32
D
gel, hexane/AcOEt; 80:20; v:v); ½aꢁ ¼ þ31:7 (c 1,
1
CHCl3); H NMR (CDCl3): d 7.37–7.05 (20H, aromat-
ics), 5.50 (1H, d, J = 3.5, 100-H), 5.28 (1H, d, J = 8.4,
NH), 5.08–4.35 (16H, benzylic) 4.55 (1H, d, J = 7.7,
10-H), 4.30 (1H, ddd, J = 10.2, 2.3, 2.3, 500-H), 4.21
(1H, m, 2-H), 4.10 (1H, dd, J = 9.8, 7.7, 20-H), 4.01
(1H, dd, J = 9.5, 9.5, 300-H), 3.96 (1H, dd, J = 2.5, <1,
40-H), 3.79 (1H, m, 5-H), 3.67 (1H, dd, J = 10.2, 9.5,
400-H), 3.61 (1H, dd, J = 9.5, 3.5, 200-H), 3.64–3.53 (3H,

3148
P. Allevi et al. / Tetrahedron: Asymmetry 15 (2004) 3139–3148
m, 4-Hb); 13C NMR (D2O): d 174.1 (C-1), 163.7
(q, JCF = 34.3, CF3COOꢀ), 117.0 (q, JCF = 289.9,
CF3COOꢀ), 102.7 (C-10), 99.9 (C-100), 78.7 (C-20), 76.0
(C-5 and C-300), 73.6 (C-30), 72.7 (C-500), 72.3 (C-50),
72.1 (C-200), 70.0 (C-400), 69.7 (C-40), 61.7 (C-60), 60.9
(C-600), 54.4 (C-2), 42.8 (C-6), 28.2 (C-4), 26.1 (C-3);
ESI-MS (positive) m/z: 487.0 (100%; M+H+), 488.1
(20%), 489.1 (3%), 509.2 (36%; M+Na+), 525.1 (4%;
M+K+), 973.0 (6%; 2M+H+). Anal. Calcd for
C18H34N2O13Æ2CF3COOH: C, 36.98; H, 5.08; N, 3.92.
Found: C, 37.1; H, 4.9; N, 3.9.
1622; (j) Rodriguez, G. P.; Claus-Walker, J. J. Chromatogr
1984, 308, 65–73.
8. Manor, C.; Wei, J.; Mac Farlane, G. D. Eur. Pat. Appl.
1998, EP-827963.
9. Adamczyk, M.; Reddy, R. E.; Rege, S. D. Synth.
Commun. 2000, 30, 3281–3290.
10. For a preliminary report on the synthesis of the 5R-
GGaHL 2 see: Allevi, P.; Anastasia, M.; Paroni, R.;
Ragusa, A. Bioorg. Med. Chem. Lett. 2004, 14, 3319–
3321.
11. Some protected mono and diglycosylated hydroxylysine
building blocks have previously been prepared and used in
solid-phase synthesis of collagen glycopeptides or to
prepare immunogens: (a) Holm, B.; Broddefalk, J.;
Flodell, S.; Wellner, E.; Kihlberg, J. Tetrahedron 2000,
56, 1579–1586; (b) Malkar, N. B.; Lauer-Fields, J. L.;
Fields, G. B. Tetrahedron Lett. 2000, 41, 1137–1140; (c)
Broddefalk, J.; Forsgreen, M.; Sethson, I.; Kihlberg, J. J.
Org. Chem. 1999, 64, 8948–8953; (d) Adamczyk, M.;
Reddy, R. E. Tetrahedron: Asymmetry 1999, 10, 3157–
3165; (e) Broddefalk, J.; Backlund, J.; Almqvist, F.;
Johansson, M.; Holmdahl, R.; Kihlberg, J. J. Am. Chem.
Soc. 1998, 120, 7676–7683; (f) Koeners, H. J.; Schatten-
kerk, C.; Verhoeven, J. J.; van Boom, J. H. Tetrahedron
1981, 37, 1763–1771.
12. (a) Garza, H.; Bennett, N., Jr.; Rodriguez, G. P. J.
Chromatogr. A 1996, 732, 385–389; (b) Napoli, R. M.;
Middleditch, B. S.; Cintron, N. M.; Chen, Y. M. Chro-
matographia 1989, 28, 497–501; (c) Tenni, R.; Rimoldi, D.;
Zanaboni, G.; Cetta, G.; Castellani, A. A. Ital. J. Biochem.
1984, 33, 117–127; (d) Smith, D. F. Anal. Biochem. 1976,
71, 106–113; (e) Markiw, R. T. Biochem. Med. 1974, 9,
233–236.
13. (a) Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry
2003, 14, 2005–2112; (b) Allevi, P.; Galligani, M.; Anas-
tasia, M. Tetrahedron: Asymmetry 2002, 13, 1901–1910;
(c) Anastasia, L.; Anastasia, M.; Allevi, P. J. Chem. Soc.,
Perkin Trans. 1 2001, 2404–2408; (d) Allevi, P.; Anastasia,
M. Tetrahedron: Asymmetry 2000, 11, 3151–3160; (e)
Allevi, P.; Longo, A.; Anastasia, M. J. Chem. Soc., Perkin
Trans. 1 1999, 2867–2868; (f) Allevi, P.; Longo, A.;
Anastasia, M. J. Chem. Soc., Chem. Commun. 1999, 559–
560.
References
1. For a comprehensive review on biology of bones see: (a)
Bilezikian, J. P., Raisz, L. G., Rodan, G. A., Eds.;
Academic: San Diego, 2001; For a review on this topic see:
(b) Moro, L.; Romanello, M. Cells Mater. 1997, 7, 329–
332.
2. (a) Spiro, R. G.; Fukushi, S. J. Biol. Chem. 1969, 244,
2049–2058; (b) Spiro, R. G. J. Biol. Chem. 1969, 244, 602–
612; (c) Cunningham, L. W.; Ford, J. D. J. Biol. Chem.
1968, 243, 2390–2398.
3. Moro, L.; Noris-Suarez, K.; Michalsky, M.; Romanello,
M.; de Bernard, B. Biochim. Biophys. Acta 1993, 1156,
288–290.
4. Cunningham, L. W.; Ford, J. D.; Segrest, J. P. J. Biol.
Chem. 1967, 242, 2570–2571.
5. For a review on this topic see: (a) Szulc, P.; Seeman, E.;
Delmas, P. D. Osteoporosis Int. 2000, 11, 281–294; (b)
Bettica, P.; Taylor, A. K.; Talbot, J.; Moro, L.; Talmini,
R.; Baylink, D. J. J. Clin. Endocrinol. Metab. 1996, 81,
542–546; (c) Rauch, F.; Schnabel, D.; Seibel, M. J.;
Remer, T.; Stabrey, A.; Michalk, D.; Schonau, E. J. Clin.
Endocrinol. Metab. 1995, 80, 1295–1300; (d) Krane, S. M.;
Kantrowitz, F. G.; Byrne, M.; Pinnell, S. R.; Singer, F. R.
J. Clin. Invest. 1977, 59, 819–827; (e) Segrest, J. P.;
Cunningham, L. W. J. Clin. Invest. 1970, 49, 1497–1509.
6. (a) Leigh, S. D.; Julia Ju, H.-S.; Lundgard, R.; Daniloff,
G. Y.; Liu, V. J. Immunol. Methods 1998, 220, 169–178;
(b) Michalsky, M.; Stepan, J. J.; Wilczek, H.; Formank-
ova, J.; Moro, L. Clin. Chim. Acta 1995, 234, 101–108; (c)
LoCascia, V.; Braga, V.; Bertoldo, F.; Bettica, P.; Pasini,
A. F.; Stefani, L.; Moro, L. Clin. Endocrinol. 1994, 41, 47–
51.
7. (a) Casetta, B.; Romanello, M.; Moro, L. Rapid Commun.
Mass Spectrom. 2000, 14, 2238–2241; (b) Al-Dehaimi, A.
W.; Blumsohn, A.; Eastell, R. Clin. Chem. 1999, 45, 676–
681; (c) Bank, R. A.; Beekman, B.; Tenni, R.; TeKoppele,
J. M. J. Chromatogr. B 1997, 703, 267–272; (d) Alfano,
M.; Casari, E.; Stenico, A.; Murone, M.; Grazioli, V. Clin.
Chem. 1994, 40, 2113–2114; (e) Yoshihara, K.; Mochi-
dome, N.; Shida, Y.; Hayakawa, Y.; Nagata, M. Biol.
Pharm. Bull. 1993, 16, 604–607; (f) Grazioli, V.; Casari, E.;
Murone, M.; Bonini, P. A. J. Chromatogr. B 1993, 615,
59–66; (g) Grazioli, V.; Casari, E.; Murone, M. Chroma-
tographia 1993, 36, 144–146; (h) Moro, L.; Battista, C.;
Modricky, C.; Rovis, L.; de Bernard, B. J. Chromatogr.
1989, 490, 285–292; (i) Moro, L.; Modricky, C.; Stagni,
N.; Vittur, F.; de Bernard, B. Analyst 1984, 109, 1621–
14. (a) Liebe, B.; Kunz, H. Angew. Chem. 1997, 36, 618–621;
(b) Liebe, B.; Kunz, H. Helv. Chim. Acta 1997, 80, 1473–
1482; (c) Schultz, M.; Kunz, H. Tetrahedron: Asymmetry
1993, 4, 1205–1220.
15. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212–235.
16. Asai, N.; Fusetani, N.; Matsunaga, S. J. Nat. Prod. 2001,
64, 1210–1215.
17. Fiandor, J.; Garcia-Lopez, M. T.; DeLasHeras, F. G.;
´
Mendez-Castrillon, P. P. Synthesis 1985, 1121–1123.
18. Allevi, P.; Anastasia, M.; Ciuffreda, P.; Bigatti, E.;
Macdonald, P. J. Org. Chem. 1993, 58, 4175–4178.
19. Schmidt, R. R.; Michel, J. Tetrahedron Lett. 1984, 25,
821–824.
20. Wegmann, B.; Schmidt, R. R. J. Carbohydr. Chem. 1987,
6, 357–375.
21. Gineyts, E.; Garnero, P.; Delmas, P. D. Rheumatology
2001, 40, 315–323.
22. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923–2925.