Solid-phase supported mimic of GDP-l-galactose

Article abstract of DOI:10.1016/j.tetasy.2009.03.005

A C-glycoside mimetic of l-galactose 1-diphosphate, a potential ligand of GDP-l-galactose hydrolase, an enzyme involved in the biosynthesis of vitamin C, has been designed, stereoselectively synthesised by C-allylation of tribenzylated l-fucose, by period

Full text of DOI:10.1016/j.tetasy.2009.03.005

Tetrahedron: Asymmetry 20 (2009) 744–745
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Tetrahedron: Asymmetry
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Solid-phase supported mimic of GDP-L-galactose
*
Barbara La Ferla, Laura Russo, Cristina Airoldi, Francesco Nicotra
Department of Biotechnology and Bioscience, University of Milano Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A C-glycoside mimetic of L-galactose 1-diphosphate, a potential ligand of GDP-L-galactose hydrolase, an
Received 26 February 2009
Accepted 4 March 2009
Available online 30 March 2009
enzyme involved in the biosynthesis of vitamin C, has been designed, stereoselectively synthesised by
C-allylation of tribenzylated -fucose, by periodate-osmium tetroxide degradation of the double bond,
L
by condensation of the obtained aldehydes with benzylacetate and by deprotection. The obtained
mimetic was linked to a sepharose resin.
This paper is dedicated to Professor George
Fleet, on the occasion of his 65th birthday
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
b
a
D-mannose 1-P
GTP
D-mannose 6-P
D-fructose 6-P
The world production of vitamin C (
80000 tons/year, and is obtained from
L
-ascorbic acid) is around
GMP
e
c
D-glucose exploiting a syn-
Ppi
d
thetic procedure developed by Reichstein in 1933 with few subse-
quent improvements.1 In light of the economical relevance of this
compound considerable efforts are being made to develop biotech-
nological procedures, which would allow vitamin C to be produced
by fermentation, and therefore the enzymes involved in its biosyn-
thesis are relevant targets.
The biosynthesis of
been extensively studied. The process starts from
is converted into -fructose 6-phosphate, which in turn undergoes
L-galactose 1-P
f
GDP-L-galactose
GDP-D-mannose
NADH NAD+
g
h
L-ascorbic acid
L-galactonolactone
L-galactose
a, mannose 6-P isomerase
e, hydrolase
L
-ascorbic acid in vegetal organisms has
b, phosphomannomutase
f, L-galactose-1-P phosphatase
g, L-galactose dehydrogenase
h, galactonolactoneo xidase
D-glucose, which
c, mannose1-P guanilyl transferase
d, GDP-mannose-3,5-epimerase
D
the transformations reported in Scheme 1.
The enzymes involved in this pathway have been isolated and
cloned, but one of them, the phosphohydrolase, which converts
Scheme 1.
GDP-L-galactose into L-galactose 1-phosphate, was unknown until
2. Results and discussion
very recently, and is now the subject of great interest.2
In this context, we planned the synthesis of stable mimics of
GDP-galactose or L-galactose 1-phosphate with the aim of not only
facilitating the purification of the enzyme by affinity chromatogra-
phy, but also to find an artificial ligand that could eventually act as
an inhibitor or as a chaperone.3
The synthesis of compound 1 was performed starting from
commercially available 2,3,4-tri-O-benzyl -fucose, which, after
acetylation, was submitted to a Sakurai reaction with allyltri-
L
methylsilane (BF3ꢀOEt2, MeCN, 98% yield) to stereoselectively
afford 2-(tri-O-benzyl-
a
-L
-fucopyranosyl)-1-propene 24 (no traces
L
-Galactose is a rare sugar, which in not commercially available,
therefore we decided to take advantage on the structural similarity
with -fucose, and designed the -fuco derivative 1 as mimic of the
-galactose 1-diphosphate moiety of GDP- -Gal (Fig. 1).
L
L
2
L
L
2
Mg
Mg
The C-glycosidic nature of compound 1 guarantees its stability,
O
O
the two oxygen atoms, properly positioned to chelate Mg2+ favour
the interaction with the active site of the enzyme, and the carbox-
ylic function makes the compound suitable for ligation to a resin.
O
O
O
O
HO
G
P
P
O
O
OH
HO
O
OH
O
OH
OH
HO
OH
HO
1
GDP-L-galactose
* Corresponding author.
E-mail address: francesco.nicotra@unimib.it (F. Nicotra).
Figure 1.
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.03.005

B. La Ferla et al. / Tetrahedron: Asymmetry 20 (2009) 744–745
745
of the b anomer were detected). This procedure improves that
described in the literature,5 in which compound 2 was obtained
by the acetylation of -fucose, a Sakurai reaction, saponification
L
of the acetates and finally benzylation (Scheme 2).
O
NaIO4, OsO4
tBuOH, acetone,
H2O
O
O
OBn
OBn
OBn
3
BnO
OBn
2
68% yield
BnO
MeCO2Bn
LDA,THF
88% yield
Figure 2. Second derivative of IR absorption spectra of ligand, resin before coupling
reaction and resin after coupling reaction. Spectra were collected in ATR-FTIR
O
O
OH
OH
OH
OH
(Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy) at
a
OBn
H2, Pd(OH)2
AcOEt, MeOH
93% yield
resolution of 2 cmꢂ1
.
O
O
OBn
OH
5
OBn
HO
BnO
3. Conclusion
4
Scheme 2.
A mimic of
L-galactose 1-diphosphate, a potential ligand of the
enzyme that converts GDP-
L
-galactose into -galactose 1-phos-
L
phate was designed, synthesised stereoselectively and supported
on a sepharose resin as an interesting tool to isolate and purify
the enzyme, and to study the structural requirements for good
inhibitors/chaperones.
2-(Tri-O-benzyl-
a-L-fucopyranosyl)-1-propene 2 was submit-
ted to periodate-osmium tetroxide cleavage of the double bond
and the aldehyde obtained, 2-(tri-O-benzyl-a-L-fucopyranosyl)-1-
acetaldehyde 36 (68% yield) was condensed with benzyl acetate
(LDA, THF, 88% yield) in order to generate benzyl 4-(tri-O-benzyl-
a-L
-fucopyranosyl)-3-hydroxybutanoate 47 as a mixture of epi-
References
mers (1.1/0.9 ratio). This mixture was not separated since both
stereoisomers were expected to be ligands of the target enzyme.
Catalytic hydrogenation (H2, Pd(OH)2, AcOH–MeOH, 96% yield)
1. Reichstein, T. U.S. Patent 2,265,121, 1941 and 2,301,811, 1942.
2. Linster, C. L.; Adler, L. N.; Webb, K.; Christensen, K. C.; Brenner, C.; Clarke, S.
G. J. Biol. Chem. 2008, 283, 18483.
3. For
a review on the role of glycomimetics as chaperons for refolding of
afforded the completely deprotected 4-(
a-L-fucopyranosyl)-3-
hydroxybutanoic acid 5,8 the
L-galactose 1-diphosphate mimetic
carbohydrate processing enzyme see: Fan, J.-Q. Iminosugars as Active-Site-
Specific Chaperones for the Treatment of Lysosomal Storage Disorders in
Iminosugars. Compain, P., Martin, O. R., Eds.; John Wiley & Sons Ltd: Chichester,
UK, 2007, pp 225–247.
and potential ligand of GDP-galactose phosphohydrolase.
Compound 5 was finally supported on an EAH Sepharose resin
(–NH2 functionalised, see Scheme 3) by activation of the carboxylic
acid with 1-ethyl-3-dimethylaminopropyl-carbodiimide (EDC) and
N-hydroxysuccinimide, performing the reaction in water.
4. Selected analytical data of 3-(tri-O-benzyl-a-L
-fucopyranosyl)-1-propene 2: 1H
NMR (400 MHz, CDCl3) d ppm 7.41–7.21 (m, 5H, HAr), 5.87–5.71 (m, 1H, H(20)),
5.16–4.98 (m, 2H, H830a),(30b)), 4.81–4.52 (m, 6H, 6CHPh), 4.16–4.04 (m, 1H,
H(1)), 4.04–3.94 (m, 1H, H(5)), 3.85–3.74 (m, 3H, H(2),(3),(4)), 2.50–2.27 (m, 2H,
H(10a),(10b)), 1.31 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) d 139.0,
138.8, 138.6, 135.6, 129.0–127.8, 116.9, 77.12, 76.82, 76.03, 73.32, 73.23, 73.23,
70.41, 68.93, 15.43; ½a D20
¼ ꢂ16:6 (c 1.5, CHCl3).
ꢁ
O
5. Uchiyama, T.; Woltering, T. J.; Wong, W.; Lin, C.-C.; Kaijmoto, T.; Takebayashi,
M.; Weitz-Schmidt, G.; Asakura, T.; Noda, M.; Wong, C.-H. Bioorg. Med. Chem.
1996, 4, 1149.
OH
OH
6. Selected analytical data of 2-(tri-O-benzyl-a-L H
-fucopyranosyl)-1-acetaldehyde 3: 1
O
NMR (400 MHz, CDCl3) d ppm 9.69 (m, 1H, CHO), 7.41–7.21 (m, 5H, HAr), 4.77
(d, J = 11.92 Hz, 2H, 2CHPh), 4.72–4.59 (m, 4H, 3CHPh, H(1)), 4.50 (d,
J = 11.71 Hz, 1H, CHPh), 3.96–3.89 (m, 1H, H(5)), 3.86 (dd, J = 6.5, 4.2 Hz, 1H,
H(2)), 3.79–3.77 (m, 1H, H(4)), 3.75 (dd, J = 6.5, 2.9 Hz, 1H, H(3)), 2.68–2.58 (m,
2H, H(10a), H(10b), 1.29 (d, J = 6.7 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) d
201.2, 138.7, 138.6, 138.1, 129.0–127.8, 77.24, 76.35, 75.74, 73.52, 73.462,
OH
OH
HO
OH
H
N
5
O
H2N
EDC, NHS,
H2O
73.280, 69.54, 67.04, 43.10, 15.55; ½a D20
¼ ꢂ26:1 (c 1.8, CHCl3).
L
ꢁ
7. Selected analytical data for benzyl 4-(tri-O-benzyl-a- -fucopyranosyl)-3-
hydroxybutanoate 4: (* isomer signals) 1H NMR (400 MHz, CDCl3) d ppm 7.53–
7.11 (m, HAr), 5.16 (br s, CH2Ph), 4.82–4.45 (m, 6CHPh), 4.37–4.29 (m, H(20)),
OH
H
N
O
OH
O
4.29–4.15 (m, H(20 ),(1),(1*)), 4.09–3.99 (m, H(5*), 3.97–3.85 (m, H(5)), 3.83–
*
N
H
3.70 (m, H(2),(3),(4),(2*),(3*),(4*)), 3.65–3.59 (m, OH), 3.14–3.06 (m, OH*), 2.63–
2.43 (m, H(10a),(10b),(10a*),(10b*)), 1.97–1.80 (m, H(30a),(30a*)), 1.69–1.54 (m,
H(30b),(30b*)), 1.36–1.26 (m, CH3, CH3*); 13C NMR (100 MHz, CDCl3) d 172.6,
172.0, 138.9, 138.8, 138.7, 138.6, 138.3, 138.3, 136.0, 135.9, 129.0–127.8, 77.15,
77.11, 76.88, 76.84, 75.97, 75.94, 75.69, 75.67, 75.64, 75.61, 73.48, 73.40, 73.36,
73.31, 73.28, 73.22, 69.45, 69.15, 68.42, 66.67, 66.60, 65.70, 42.03, 41.95, 15.51,
15.47.
O
OH
6
OH
HO
Scheme 3.
8. Selected analytical data for 4-(
L
-fucopyranosyl)-3-hydroxybutanoic acid 5: 1H NMR
*
(400 MHz, CD3OD)
d
ppm 4.23–4.05 (m, H(1), (1*),(20),(20 ), 3.95–3.77 (m,
H(2),(5),(2*),(5*), 3.74–3.54 (m, H(3),(4),(3*),(4*)), 2.57 (dd, J = 15.4, 4.3 Hz,
H(10a)), 2.52–2.45 (m, H(10a*),(10b*)), 2.39 (dd, J = 15.4, 8.6 Hz, H(10b)), 2.00–
The functionalised resin 6 obtained was submitted to the col-
orimetric TNBS test9 which showed the absence of residual free
amino groups, therefore the loading was 0.11 mol of compound
5 per ml of resin. IR analysis confirmed the presence of the li-
gand (Fig. 2).
1.76 (m, H(30a),(30b),(30a*)), 1.73–1.63 (m, H(30b*)), 1.25 (m, CH3, CH3 ); 13C NMR
*
(100 MHz, CD3OD) d 178.6, 178.5, 76.05, 75.79, 75.77, 75.64, 75.05, 75.00, 72.53,
72.33, 72.18, 71.51, 70.60, 68.93, 46.55, 45.11, 35.99, 35.60, 19.49, 19.23.
9. Honocock, W. S.; Battersby, J. Anal. Biochem. 1976, 71, 261.