The first synthesis of glucosylgalactosyl hydroxylysine (Glu-Gal-Hyl) an important biological indicator of collagen turnover

Article abstract of DOI:10.1016/j.bmcl.2004.03.068

This paper reports the first chemical synthesis of α-D- glucopyranosyl-(1→2)-β-D-galactopyranosyl-O-hydroxylysine, a glycoside of hydroxylysine important as indicator of skin and bone collagen turnover, starting with commercial compounds.

Full text of DOI:10.1016/j.bmcl.2004.03.068

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3319–3321
The first synthesis of glucosylgalactosyl hydroxylysine
(Glu-Gal-Hyl) an important biological indicator
of collagen turnover
Pietro Allevi,a,* Mario Anastasia,b Rita Paronia and Andrea Ragusac
a
ꢀ
Dipartimento di Medicina, Chirurgia e Odontoiatria, Universitꢀa di Milano, via A. Di Rudinı 8, I-20142 Milano, Italy
bDipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universitꢀa di Milano, via Saldini 50, I-20133 Milano, Italy
cDepartment of Chemistry, University of Southampton, Southampton SO171BJ, UK
Received 23 January 2004; revised 12 March 2004; accepted 19March 2004
Abstract—This paper reports the first chemical synthesis of a-
glycoside of hydroxylysine important as indicator of skin and bone collagen turnover, starting with commercial compounds.
D
-glucopyranosyl-(1 fi 2)-b-D-galactopyranosyl-O-hydroxylysine, a
Ó 2004 Elsevier Ltd. All rights reserved.
The glucosylgalactosyl hydroxylysine 1 (Scheme 1) is a
component of collagen, isolated from hydrolysates of
various soluble collagens from marine sponges and from
vertebrates.1–5 The human glycoside, formed by a post-
translational oxidation collagen lysine and successive
glycosylation, is released during collagen breakdown
and excreted in urine.2;6
relatively free of contaminants.2;5;14 Therefore, the
accessibility of the glycoside 1 by synthesis represents an
important target for the correct standardization of the
analytical techniques normally used in clinical determi-
nations of this biochemical marker.
On the other hand the availability of a simple protocol
for the preparation of the glycoside 1 and of its cong-
eners is of self-consistent interest due to the difficulty of
assembling a glycosidic a-amino alcohol moiety in an
intact amino acid, which in addition is prone to an easy
lactamization. Herein we report the first chemical syn-
thesis and complete characterization of the glycoside 115
(Scheme 1). The synthesis starts from commercial amino
acids and sugars and mimes the natural assembling of 1,
which is known to involve first the b-galactosylation of
hydroxylysine residue and then the a-glucosylation, at
position 2, of the formed galactoside. The first key step
of the synthesis is the b-O-galactosylation of the
hydroxyazide 2 (Scheme 1), a masked hydroxylysine.
This reaction was efficiently performed, after various
unsatisfactory attempts, using as galactosyl donor the
differently protected galactose derivative 3 and the tri-
chloroacetimidate protocol of Schmidt16 (Scheme 1).
The synthesis does not use an expensive protected nat-
Actually, glycoside 1 has attracted considerable atten-
tion both as component of collagen useful in research
aimed at understanding how collagen induces rheuma-
toid arthritis7 (an inflammatory disease characterized by
cartilage degradation and bone erosion), and as indica-
tor, together with the shorter glycoside (5R)-5-O-(b-
galactopyranosyl)-5-hydroxy- -lysine, of total collagen
D-
L
turnover.8 Therefore, between some synthetic methods
affording mono or diglycosylated hydroxylysine build-
ing blocks, useful in solid-phase synthesis of glycopep-
tides from collagen,9 various analytical methods for the
quantitative detection of the glycoside 1 or of galactosyl
hydroxylysine in biological samples have been re-
ported.10–13 However, no synthesis of the free glucos-
ylgalactosyl hydroxylysine 1 has, until now, been
reported and the reference standards of this compound
are obtained from different natural sources, in forms
ural
amino acidic hydroxyazide 2, prepared in pure form
(Scheme 2), from
-lysine17a and from other more
L-hydroxylysine but starts from the protected
L
Keywords: Glucosylgalactosyl hydroxylysine; Collagen turnover
readily available amino acids (L
-glutamic17b acid and
markers; Glycosylated -d-hydroxylysine.
L
glycine17c), according to our recent work on the syn-
thesis of pyridinolines, other important biological
* Corresponding author. Tel.: +39-250316047; fax: +39-250316040;
e-mail: pietro.allevi@unimi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.068

3320
P. Allevi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3319–3321
N3
OBn
O
OBn
O
BnO
BnO
BnO
BnO
HO
H
i
ii
tBuO2C
N3
+
O
NH
CCl3
O
ClCH2CO2
NHCbz
NHCBz
ClCH2CO2
t
CO2 Bu
3
2
4
OBn
O
BnO
BnO
N3
OBn
O
O
CCl3
NH
N3
BnO
OBn
O
BnO
BnO
iii
O
iv
NHCBz
O
BnO
BnO
BnO
O
HO
O
OBn
t
NHCBz
CO2 Bu
CO2 Bu
t
6
5
OBn
BnO
NH3
H2N
OH
O
+
OH
HO
HO
HO
O
v
O
O
-
HO
HO
2 CF3CO2
O
O
NH2
O
+
OH
O
NH3
CO2H
t
HO
OH
CO2 Bu
1
7
OH
HO
OH
HO
Scheme 1. Reagents and conditions: (i) t-BuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25 °C, 1 h, 52%; (ii) Zn(OAc)2Æ2H2O, MeOH, reflux, 9h, 93%;
(iii) t-BuMe2SiSO3CF3, molecular sieves 3A, Et2O, 25 °C, 1 h, 45%; (iv) H2, Pd/C, THF–EtOH, 1 atm, 25 °C, 90%; (v) CF3CO2H–H2O (95:5), 25 °C,
1 h, 92%.
2, in diethyl ether at 25 °C, affording the b-O-galactoside
HO
H
H
HO
4 in 52% yield.21 In the crude product of the reaction,
the obtained b-O-galactoside 4 is not accompanied by
any detectable quantity of the corresponding a-anomer
or ortho-ester. On the contrary, the a-isomer is present,
as a more polar companion (TLC and ESI-MS evi-
dences) when the reaction is catalyzed by boron triflu-
oride. As expected, the regeneration of the 2-hydroxy
group of 4, affording compound 5, was performed in
quantitative yields, by refluxing the chloroacetate 4 with
Zn(OAc)2Æ2H2O in methanol. These very mild reaction
conditions, we found useful for dichloroacetate hydro-
lysis in the synthesis of the glycoside etoposide,22 allows
the preservation of all the remaining functions of the
derivative 4. The successive a-glucosylation of 5 was
i
tBuO2C
N3
HO2C
N3
NHCbz
NHCbz
2
Scheme 2. Reagents and conditions: (i) O-tert-butyl-N,N0-
dicyclohexylisourea, CH2Cl2, rt, 5 days, 89%.
markers of collagen turnover.18 More importantly, the
use of the hydroxyazide 2, in place of hydroxylysine,
avoided the protection, and the obligatory deprotection,
of the e-amino group of this amino acid, and various
troubles due to its possible lactamization.19 The prepa-
ration of the tert-butyl ester 2 from the parent acid was
performed using the procedure reported by Liebe and
Kunz,20 which was very efficient.
performed using the O-(2,3,4,6-tetra-O-benzyl-b-D-
glucopyranosyl)trichloroacetimidate and tert-butyldi-
methylsilyl triflate, which in this case should favour23 the
formation of the a-anomer 6 which, in fact, was
obtained in 45% yield after chromatographic purifica-
tion.24 At this point, the strategic choice of the protec-
tive groups, has permitted the simultaneous cleavage of
the benzyl and benzyloxycarbonyl groups and the gen-
eration of the e-amino group of the hydroxylysine
aglycone by catalytic hydrogenation.
As glycosyl donor was chosen the galactosyl trichloro-
acetimidate 3 (Scheme 3), with the 2-hydroxy group
protected as chloroacetate. This protective group was
selected both for its capacity to favour, as neighbouring
group participation, the formation of the desired b-
anomeric linkage and for its cleavability, under nearly
neutral conditions, which could open the way to extend
the obtained galactose derivative 4 in the 2-direction.
Under the best conditions found, tert-butyldimethylsilyl
triflate promoted the galactosylation of the hydroxyazide
The formed glucosylgalactosyl hydroxylysine tert-butyl
ester 7 was finally treated with trifluoroacetic acid to
OBn
O
OBn
O
OBn
O
OBn
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
iii
i
ii
OH
OH
NH
CCl3
OCOCH2Cl
OH
O
ClCH2CO2
ClCH2CO2
ClCH2CO2
3
Scheme 3. Reagents and conditions: (i) ClCH2COCl, Et2O–Py, )10 to 25 °C, 2 h, 98%; (ii) 25% aqueous NH3, THF, 1 h, 85%; Cl3CCN, DBU,
CH2Cl2, )30 °C, 4 h, 79%.

P. Allevi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3319–3321
3321
12. Leigh, S. D.; Julia Ju, H.-S.; Lundgard, R.; Daniloff, G.
Y.; Liu, V. J. Immunol. Methods 1998, 220, 169–178.
13. Casetta, B.; Romanello, M.; Moro, L. Rapid Commun.
Mass Spectrosc. 2000, 14, 2238–2241, and references cited
therein.
14. Tenni, R.; Rimoldi, D.; Zanaboni, G.; Cetta, G.; Castel-
lani, A. A. Ital. J. Biochem. 1984, 33, 117–127.
15. Selected data for (2S,5R)-2,6-diamino-5-[a-D-glucopyr-
afford the target glycoconjugate 1 as ditrifluoroacetate
salt.
Our work makes available in a relatively easy way the
commercially unavailable hydroxylysine glycoconjugate
1, and facilitates its use in biological researches on col-
lagen. More generally, we feel that present results
appear to be very attractive for the assembling, via our
previously reported protocols,17;18 of other more com-
plex glycoside of cross-linked hydroxylysine (glycosyl-
ated pyridinolines) present in collagen.25
anosyl-(1 fi 2)-b-
D-galactopyranosyloxy]hexanoic acid 1
difluoroacetate (C18H36N2O13 2CF3COOÀ): a syrup; ½aŠ
D
+21.9( c 1, MeOH); 1H NMR (D2O): 5.34 (1H, d, J ¼ 3:6,
100-H), 4.62 (1H, d, J ¼ 7:5, 10-H), 4.11 (1H, m, 5-H), 4.08
(1H, dd, J ¼ 6:5, 6.5, 2-H), 3.66 (1H, dd, J ¼ 9:5, 7.5,
20-H), 3.50 (1H, dd, J ¼ 9:9, 3.6, 200-H); 13C NMR (D2O):
172.4 (C-1), 173.7 (q, JCF ¼ 34:3, CF3COOÀ), 117.3 (q,
JCF ¼ 289:9, CF3COOÀ), 102.5 (C-10), 98.5 (C-100), 72.5
(C-5), 53.4 (C-2), 43.6 (C-6). This and all other new
compounds gave correct C, H, and N elemental analyses
that were within 0.3% of the theoretical values.
Acknowledgements
This work was supported financially by Italian MIUR
ꢀ
(Ministero dell’Istruzione, dell’Universita e della Ric-
erca).
16. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212–235.
17. (a) Allevi, P.; Galligani, M.; Anastasia, M. Tetrahedron:
Asymmetry 2002, 13, 1901–1910; (b) Allevi, P.; Anastasia,
M. Tetrahedron: Asymmetry 2000, 11, 3151–3160;
(c) Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry
2003, 14, 2005–2012.
18. For our previous work on pyridinoline synthesis, see also:
Anastasia, L.; Anastasia, M.; Allevi, P. J. Chem. Soc.,
Perkin Trans. 1 2001, 2404–2408; Allevi, P.; Longo, A.;
Anastasia, M. J. Chem. Soc., Perkin Trans. 1 1999, 2867–
2868; Allevi, P.; Longo, A.; Anastasia, M. J. Chem. Soc.,
Chem. Commun. 1999, 559–560.
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19. Unpublished results from our laboratory.
20. (a) Liebe, B.; Kunz, H. Angew. Chem. 1997, 36, 618–621;
(b) Liebe, B.; Kunz, H. Helv. Chim. Acta 1997, 80, 1473–
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1993, 4, 1205–1220.
21. Selected data for (2S,5R)-6-azido-2-benzyloxycarbonyl-
amino-5-(3,4,6-tri-O-benzyl-2-O-chloroacetyl-b-D-galacto-
5. Garza, H.; Bennett, N., Jr.; Rodriguez, G. P. J. Chroma-
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pyranosyloxy)hexanoate tert-butyl ester 4: an oil; ½aŠD +7.9
1
(c 1, CHCl3); H NMR (CDCl3): 5.34 (1H, dd, J ¼ 10:0,
0
7.9, 20-H), 4.43 (1H, d, J ¼ 7:9 , 1-H), 4.01 and 3.92
(2 · 1H, 2 · d, J ¼ 14:7, ClCHHCO2), 3.63 (1H, m, 5-H),
1.44 (9H, s, C(CH3)3); 13C NMR (CDCl3): 171.0 (C-1),
165.9(ClCH 2CO2), 155.8 (NHCO), 101.5 (C-10), 54.4
(C-6), 54.1 (C-2).
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D
2)-(3,4,6-tri-O-benzyl-b-D-galactopyranosyloxy)]hexanoate
tert-butyl ester 6: an oil; ½aŠ +25.6 (c 1, CHCl3); 1H NMR
D
(CDCl3): 5.62 (1H, d, J ¼ 3:5, 100-H), 4.63 (1H, d, J ¼ 7:7,
10-H), 4.21 (1H, dd, J ¼ 10:0, 7.7, 20-H), 3.88 (1H, m, 5-H),
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