The first chemical synthesis of UDP[6-3H]-α-D- galactofuranose

Article abstract of DOI:10.1002/ejoc.200500056

Galactofuranose metabolism is a good target for the development of novel chemotherapeutic agents for the treatment of some microbial infections. This is a valid objective because galactofuranose is absent in mammals. Two enzymes are involved in the biosynthesis of molecules containing galactofuranose: a mutase, which catalyzes the interconversion of UDP-Galp and UDP-Galf, and D-galactofuranosyltransferases. The mechanism of action of the mutase and its inhibition is currently being investigated, whereas studies on the galactofuranosyltransferases have been hampered by the lack of a labeled galactofuranose nucleotide. In the present work we describe the chemical synthesis of UDP-α-D-[6-³H]Galf and we prove its effectiveness for incorporation of radioactive galactofuranose into a natural acceptor. This is the first report on the chemical synthesis of a labeled donor of galactofuranose with the potential for studying the galactofuranosyltransferases independently from the UDP-Galp mutase. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500056

FULL PAPER
The First Chemical Synthesis of UDP[6-3H]-α-
-galactofuranose
D
Karina Mariño,[a] Carla Marino,[a] Carlos Lima,[a] Luciana Baldoni,[a] and
Rosa M. de Lederkremer*[a]
Keywords: Carbohydrates / Galactofuranosyltransferase / Isotopic labeling / Nucleotides / UDP-Galf
Galactofuranose metabolism is a good target for the develop-
ment of novel chemotherapeutic agents for the treatment of
some microbial infections. This is a valid objective because
galactofuranose is absent in mammals. Two enzymes are in-
volved in the biosynthesis of molecules containing galactofu-
ranose: a mutase, which catalyzes the interconversion of
lack of a labeled galactofuranose nucleotide. In the present
work we describe the chemical synthesis of UDP-α- -[6-
D
3H]Galf and we prove its effectiveness for incorporation of
radioactive galactofuranose into a natural acceptor. This is
the first report on the chemical synthesis of a labeled donor
of galactofuranose with the potential for studying the galac-
tofuranosyltransferases independently from the UDP-Galp
mutase.
UDP-Galp and UDP-Galf, and
D-galactofuranosyltransfer-
ases. The mechanism of action of the mutase and its inhibi-
tion is currently being investigated, whereas studies on the
galactofuranosyltransferases have been hampered by the
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
trisaccharides resulting from the enzymatic reaction have
been characterized by fast atom bombardment mass spec-
trometry. The sugar donor used for the reaction was UDP-
[14C]Galp, which must be converted to UDP-[14C]Galf be-
fore transference.
The availability of labeled substrates is important for the
detection of galactofuranose intermediates and the enzymes
involved in Galf metabolism. We have previously described
the synthesis of methyl β-d-[6-3H]galactofuranoside, which
is useful for the detection of β-d-galactofuranosidase.[14]
A drawback of the preparation of UDP-Galf from la-
beled UDP-Galp by using the mutase in vitro is the low
yield of UDP-Galf, as only 7% of the furanose nucleotide
is present at equilibrium.[15] A microtiter assay for the mu-
tase is based on the release of tritiated formaldehyde by
periodate oxidation of the UDP-[6-3H]Galf formed from
UDP-[6-3H]Galp.[16]
The synthesis of nonlabeled UDP-Galf[17,18] from α-d-
Galf-1-phosphate (1), which was synthesized for the first
time in our laboratory, has been described.[19] In the present
report we describe a chemical synthesis of α-d-[6-3H]Galf-
1-phosphate (1*) and UDP-[6-3H]Galf (3*) from the pre-
cursor 5.[14] The radioactive donor substrate 3* should be
useful for discrimination of inhibitors of both the UDP-
α-d-Galp mutase and the galactofuranosyl transferases in
different species. The α-d-galactofuranose 1,2-cyclic phos-
phate (4), a product usually formed by decomposition of
UDP-Galf,[20] was also prepared from the monophosphate
1 for the identification of the reaction products. Radioactive
galactose from the UDP-[6-3H]Galf (3*) was incorporated
into the peptidophosphogalactomannan by a membrane
preparation of Penicillium fellutanum.
Introduction
The metabolic pathways involved in the incorporation of
galactofuranose into microbial glycoconjugates are attract-
ive targets for the development of chemotherapeutic agents,
since the furanose galactose is absent in mammalian cells.
β-d-Galactofuranosyl residues are constituents of patho-
genic microorganisms such as the bacteria Mycobacterium
tuberculosis and M. leprae,[1,2] trypanosomatids like Trypa-
nosoma cruzi and Leishmania,[3–5] and fungi like Paracocci-
dioides brasiliensis.[6] In the latter species, the rare α-d-galac-
tofuranosyl linkage has also been detected, depending on
the phase of the fungus.
At least two enzymes are necessary for the construction
of the galactofuranosyl linkage: a mutase, UDP-galactopyr-
anosylmutase (EC 5.4.99.9),[7,8] which converts UDP-α-d-
Galp into UDP-α-d-Galf, and a UDP-galactofuranosyl
transferase, which is responsible for the incorporation of
the sugar into the glycan. The mechanism of action of the
mutase is currently under investigation by several
groups.[9–11] However, few studies have been reported for
the transferase.[12,13] It has been only described in Mycobac-
terium tuberculosis, using synthetic glycosides of Galf disac-
charides as acceptor substrates.[12] The transferase is a bi-
functional enzyme that catalyzes the formation of β(1,5)
and β(1,6) linkages (gene product of Rv 3808c).[12] The Galf
[a] CIHIDECAR (CONICET), Departamento de Química Orgá-
nica, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires
Pabellón II, Ciudad Universitaria, 1428 Buenos Aires, Ar-
gentina
Fax: +54-11-45763352
E-mail: lederk@qo.fcen.uba.ar
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200500056
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First Chemical Synthesis of UDP[6-3H]-α-d-galactofuranose
FULL PAPER
with the nonlabeled compounds. The structures for the tar-
get, UDP-Galf, and for intermediate and degradation prod-
ucts were confirmed by the chemical shifts for the diagnos-
tic signals in the NMR spectra (Table 1), which were in
agreement with those reported in the literature.[17,19,20,28]
Standard acetolysis of 6 caused partial isomerization to the
pyranosic forms, according to the 13C NMR spectrum of
the nonlabeled product. Ring expansion was avoided by
previous benzoylation of O-6 and mild acetolysis in CH2Cl2
solution to afford 1-O-acetyl-2,3,5,6-tetra-O-benzoyl-d-gal-
actofuranose (7).[22] Compound 7* was used for the prepa-
ration of α-d-[6-3H]Galf-phosphate (1*) following the pro-
cedure previously described in our laboratory,[19] with slight
modifications for the nonradioactive analog. Thus, treat-
ment of 7* with bromotrimethylsilane and condensation
with dibenzyl phosphate in the presence of triethylamine
Results and Discussion
The synthesis of UDP-Galf (3) relies on the coupling of
α-d-Galf-1-P (1) with an activated derivative of 5Ј-UMP (2)
(Scheme 1). The tritium-labeled Galp analog, namely UDP-
[6-3H]Galp, was synthesized from the cold nucleotide by
treatment with galactose oxidase followed by reduction
with NaB3H4.[21] The introduction of a tritium label directly
in UDP-Galf (3), or in 1, was not possible, first because
there is no enzyme available for oxidation of C-6 in Galf,
and secondly due to the extreme lability of UDP-Galf. In a
previous study, we have developed a strategy for the specific
tritium labeling of the 6-position of methyl β-d-galactofur-
anoside,[14] and now we extended it to the synthesis of
UDP-[6-3H]Galf (3*) starting from compound
(Scheme 2). All the reactions were performed in parallel
5
Scheme 1. i) 1,1Ј-carbonyldiimidazole, DMF; ii) DMF.
Scheme 2. i) NaB3H4, MeOH; ii) BzCl, Py; iii) 2.5% H2SO4/Ac2O in CH2Cl2 (1:20); iv) Si(CH3)3Br, CH2Cl2; v) (BnO)2PO2H, Et3N,
toluene; vi) H2 (35 psi), Pd/C, EtOAc, NEt3; vii) NEt3/H2O/MeOH (1:2:5).
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K. Mariño, C. Marino, C. Lima, L. Baldoni, R. M. de Lederkremer
Table 1. Chemical shifts for the diagnostic signals of compounds 1, 3, 4, 8α, 8β, and 10.
FULL PAPER
Compounds
1H NMR
(JH,P
(4.9)
13C NMR
31P NMR
Ref.
H-1
H-2
C-1
(JC,P
C-2
(JC,P)
(J1,2
)
)
(J2,3
)
(JH,P
)
)
α-d-Galf 1-P (1)[a]
UDP-Galf (3)[a] Galf
Ribf
5.42
5.45
5.85
5.93
6.34
6.13
5.80
4.00[c]
(m)
4.03
96.9
(5.3)
98.0
(5.5)
88.6
77.9
(7.6)
77.0
(8.1)
+2.5
[19]
[17]
(4.5)
(4.4)
(4.5)
(4.5)
(4.5)
(Ͻ0.5)
(8.9)
–10.1, –12.0
(5.1)
(8.5)
(2.1)
(2.1)
(6.9)
α-d-Galf 1,2-P (4)[a]
8α[b]
4.75
5.73
5.53
4.32
102.6
(4.1)
97.6
(4.4)
103.3
(5.5)
98.7
85.7
+16.9
–3.7
[20]
[19]
[19]
[28]
(14.0)
(5.8)
(4.9)
76.7
(6.0)
82.1
8β[b]
(Ͻ0.5)
α-d-Galp 1,2-P (10)
79.0
(2.7)
+10.4
(4.7)
(19.1)
(8.8)
[a] In D2O. [b] In CDCl3. [c] Center of a complex multiplet.
led to the anomeric mixture 8*, which was separated by
column chromatography. The fractions were counted,
pooled, and analyzed by TLC and fluorography (Figure 1,
A). The first radioactive fraction eluted from the column
contained a mixture of 8β*, 2,3,5,6-tetra-O-benzoyl-d-
[6-3H]galactofuranose (9*), and traces of 8α* (Figure 1,
part A, lane 1). The second radioactive fraction (Figure 1A,
lane 2) contained 8α* (Rf = 0.16). The double spots shown
are due to partial transesterification during the previous
acetolysis step, as proved by NMR experiments carried out
on the compounds isolated from the corresponding nonlab-
eled synthesis, which showed the presence of more than one
acetate group. This preparation was also analyzed for com-
1
parison (Figure 1, B). We again confirmed by 13C and H
NMR spectroscopy (Table 1) that the β-anomer (8β) spon-
taneously decomposes to the stable tetrabenzoate 9 (Rf =
0.24).[19] This observation has been contested,[18] probably
based on the fact that the β-phosphate 8β and its decompo-
sition product, 9, could not be distinguished (Figure 1 part
B, lane 3). Compound 8α* was obtained in 42% yield from
7* and could be stored at –20 °C for several months. The
yield could be improved by further acetylation of 9*, to
obtain 7*.
Conditions for the removal of the protecting groups in
8α* were optimized for minimum decomposition. Thus, de-
benzylation of 8α* was performed in a shorter time than
previously described,[19] and a pressure of 35 psi was used,
followed by debenzoylation under mild conditions. α-d-[6-
3H]Galf 1-phosphate, as the triethylamonium salt (1*),
showed the same retention time (Rt = 9.75, Condition 1, Lane 1: fractions 15–35, corresponding to 2,3,5,6-tetra-O-benzoyl-
Figure 2, A) as the nonradioactive compound when ana-
lyzed by HPAEC-PAD. The peak of galactose at 1.5 min
could be due to partial decomposition of the radioactive
Figure 1. A) Thin layer chromatography and fluorography of frac-
tions obtained from column chromatography purification of 8α*.
d-[6-3H]galactofuranose (9*), dibenzyl 2,3,5,6-tetra-O-benzoyl-β-d-
[6-3H]galactofuranosylphosphate (8β*), and dibenzyl 2,3,5,6-tetra-
O-benzoyl-α-d-[6-3H]galactofuranosylphosphate (8α*). Lane 2:
fractions 36–43, corresponding to dibenzyl 2,3,5,6-tetra-O-benzoyl-
α-d-[6-3H]galactofuranosylphosphate
(8α*).
B)
Column
sample 1* under the basic conditions of the analysis.
For the coupling of 1* with UMP, activation with 1,1Ј-
carbonyldiimidazole, as described previously for the nonla-
beled nucleotide,[17] gave better yields (26% for nonlabeled
3 and 13% for the radioactive nucleotide 3*) than morphol-
chromatography of unlabeled 8α. Lane 3: 2,3,5,6-tetra-O-benzoyl-
α,β-d-galactofuranose (9) and dibenzyl 2,3,5,6-tetra-O-benzoyl-β-
d-galactofuranosylphosphate (8β). Lane 4: dibenzyl 2,3,5,6-tetra-
O-benzoyl-α-d-galactofuranosylphosphate (8α). TLC was per-
formed on silica gel 60 plates with solvent A and detected as de-
idate activation[23] (Scheme 1). Thus, the triethylammonium scribed in the Experimental Section.
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First Chemical Synthesis of UDP[6-3H]-α-d-galactofuranose
FULL PAPER
cific activity of 3* (50 μCiμmol–1) was calculated by using
the absorbance of UDP-Galp uridine at 260 nm as internal
standard to determine the molar concentration, and scintil-
lation counting to determine the radioactivity of the sample
separated. Fractions B and C (Figure 3), when analyzed by
high pressure anion exchange chromatography with pulse
amperometric detection (HPAEC-PAD), showed a peak co-
incident with galactose and another peak at 5.45 min (Con-
dition 1, Figure 2, B). The latter was identified as the α-d-
galactofuranose 1,2-cyclic phosphate 4 as described below.
Figure 3. Sephadex G-10 chromatography of UDP-[6-3H]Galf and
byproducts of the coupling reaction. The reaction was performed
as described in the Experimental Section and chromatographed on
a column of Sephadex G-10. Fractions of 1 mL were collected and
analyzed by UV at 260 nm (♦) and liquid scintillation counting (ο).
Figure 2. HPAEC-PAD analysis of isomeric galactose phosphates.
A) Preparation of α-d-[6-3H]Galf 1-P (1*). B) Included fraction B
(Figure 3) from the Sephadex purification of UDP-[6-3H]Galf. C)
Authentic samples of α-d-Galp 1,2-P (10), α-d-Galf 1,2-P (4), α-d-
Galp 1-P (11), α-d-Galf 1-P (1), and d-Gal 2-P (12). The separation
was performed on a CarboPac PA-10 column under Condition 1,
as described in the Experimental Section.
salt of nucleotide 2a was converted into the activated UMP-
imidazolide 2b. For the characterization of UDP-[6-3H]Galf
(3*), the nonradioactive analog 3 was prepared under the
same reaction conditions for the coupling and was charac-
terized by NMR spectroscopy as described previously.[17]
Attempts to analyze the reaction product by TLC using se-
veral eluent systems were unsuccessful due to the instability
of UDP-[6-3H]Galf (3*). Gel chromatography (Sephadex
G-10) of the reaction mixture showed the profile depicted
in Figure 3. Three fractions were obtained: the excluded
volume (A) with 12% of the radioactivity, and two included
peaks slightly resolved at 70 mL (B, 45%) and 77 mL (C,
43%). The fractions were counted, pooled, and analyzed by
HPLC as described in the Experimental Section. Fraction
A from a radioactive and nonlabeled preparation was ana-
lyzed by reversed-phase C-18 HPLC using UDP-Galp as
internal standard (Rt = 4.6 min, Figure 4). A radioactive
Figure 4. HPLC purification of UDP-[6-3H]Galf. The excluded
fraction from Sephadex G-10, was chromatographed on an RP-
18 column, as described in the Experimental Section. An internal
standard of UDP-Galp was used. (᭿) Liquid scintillation counting;
(––) UV detection at 260 nm.
The degradation of 3 to 4 and 5Ј-UMP (Scheme 1), or
peak with Rt = 5.2 min was coincident with UDP-Galf, as glycosidic bond cleavage to afford d-galactose and 5Ј-UDP,
detected by UV spectroscopy (Figure 4). UMP (Rt are the main problems when handling the furanose nucleo-
6.6 min, Figure 4) was also present in fraction A. The spe- tide 3.[17,20] The α-d-Galf 1,2-cyclic phosphate 4* could be
=
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K. Mariño, C. Marino, C. Lima, L. Baldoni, R. M. de Lederkremer
FULL PAPER
formed during the coupling step or by decomposition of active α-d-Galf 1,2-P (4*) in the presence of UDP-glucose
the nucleotide in the course of its purification. With the aim with membranes or with a lysate of mycelium did not show
of confirming the identity of 4*, an authentic sample was incorporation of radioactivity.
prepared starting from compound 1. α-d-Galf 1-P (1) was
In order to confirm the incorporation of Galf, the ex-
treated with dicyclohexylcarbodiimide, as described for d- cluded radioactive fraction from the column was hy-
fructose 1-phosphate.[24] TLC examination showed total drolyzed with 20 mm trifluoroacetic acid under conditions
conversion into a compound with a higher Rf value. No that lead to hydrolysis of galactofuranosyl linkages. All the
cyclization was obtained upon treatment of 1 with carbon- radioactivity eluted as an included peak (Figure 5) which
yldiimidazole under the reaction conditions used for the corresponded to galactose when analyzed by HPAEC-PAD.
coupling of 1 with UMP, thus indicating that cyclic com- Galactose eluted at 12.7 min (Condition 2, data not shown).
pound 4 is formed from nucleotide 3. We also proved that
In conclusion, the present synthesis of a labeled donor
the peak at 5.45 min observed during HPAEC-PAD analy- substrate of galactofuranose provides a tool for studies on
sis of fraction B (Figure 2, B) corresponds to the formation galactofuranosyltransferases, which are valuable targets for
of 4 as a decomposition product from 3. The NMR spectro- the inhibition of the biosynthesis of glycoconjugates essen-
scopic data of 4 (Table 1) are in agreement with the signals tial to the growth of microbial pathogens.
assigned by Köplin et al. in the spectrum of crude UDP-
Galf obtained enzymatically.[20] The chromatographic reso-
Experimental Section
lution of α-d-Galp 1,2-P (10), α-d-Galf 1,2-P (4), α-d-Galp
1-P (11), α-d-Galf 1-P (1), and d-Gal 2-P (12) by HPAEC-
PAD is shown in Figure 2, part C; their retention times are
3.05, 5.45, 7.93, 9.75, and 13.35 min, respectively. The fu-
ranosic derivatives eluted later than the pyranosic analogs,
as previously observed for other compounds.[25] Compound
12 can be prepared from either of the cyclic phosphates by
mild acid hydrolysis, as described in the Experimental Sec-
tion. We proved that the synthesized UDP-[6-3H]Galf (3*)
is a good donor of [6-3H]Galf by using a membrane prepa-
ration of Penicillium fellutanum and added peptidophos-
phogalactomannan (pPGM)[26] obtained from a three day
culture, as described in the Experimental Section. To avoid
decomposition of the furanosic nucleotide, which is ex-
tremely labile,[17,18,20] we used the radioactive fraction A
(Figure 3) containing the donor UDP-[6-3H]Galf obtained
from the Sephadex separation without further purification.
The incubated reaction mixture was dialyzed for removal of
the donor substrate and the labeled pPGM was separated
on a BioGel P6 column (Figure 5). Incubations with radio-
General: Thin layer chromatography (TLC) was performed on
0.2 mm silica gel 60 F254 aluminum-supported plates (Merck), and
developed using the following solvent systems: (A) toluene/ethyl
acetate (9:1, v/v); (B) 1-propanol/concd. aqueous NH3/water (7:1:2,
v/v). The radioactivity in the fractions was determined with a
Rack-beta Wallac liquid scintillation counter using a scintillation
cocktail (Optiphase “Hisafe” 3, LKB). Radiolabeled compounds
were detected by fluorography. The TLC plates were sprayed with
EN3HANCE (New England Nuclear) and were exposed to Kodak
X-Omat A-R5 films at –70 °C. The nonradioactive standards were
detected by exposure to UV light or by spraying with 5% (v/v)
sulfuric acid in ethanol and charring. Column chromatography was
performed on silica gel 60 (230–400 mesh). The H, 13C, and 31P
NMR spectra were recorded on a Bruker AM 500 spectrometer.
The chemical shift reference for 31P was that of external phosphoric
acid (85%) in D2O set at 0.0 ppm.
1
Protein concentrations were obtained by the Lowry method
adapted for membrane proteins.[27] Solutions were concentrated by
rotary evaporation with a bath temperature below 40 °C.
α-d-Galp 1-P (sodium salt) was purchased from Sigma and con-
verted into the triethylammonium salt by passage through Dowex
AG 50W-X12 (triethylammonium form). All other reagents, unless
otherwise stated, were purchased from Sigma–Aldrich.
HPLC Analysis of UDP-Galf: Nucleoside mono- and diphosphates
and nucleotide sugars were analyzed by reversed-phase HPLC with
a Phenomenex SphereClone 5 μm ODS(2) column (250×4.60 mm,
RP-18) and 36 mm KH2PO4 as eluent (flow rate = 0.6 mLmin–1).
The eluted fractions were analyzed by UV or liquid scintillation
counting.
HPAEC-PAD Analysis: Analysis by HPAEC-PAD was performed
with a Dionex DX 300 HPLC system equipped with a CarboPac
PA-10 anion-exchange analytical column (4×250 mm), and a
guard column PA-10 (4×50 mm). The following conditions were
used:
Condition 1: 2 mm NaOH and 150 mm sodium acetate, isocratically
at a flow rate of 1 mLmin–1.
Condition 2: 20 mm NaOH, isocratically, at
a flow rate of
1 mLmin–1.
Figure 5. BioGel P-6 chromatography of radiolabeled pPGM. (᭿),
and its mild acid hydrolysis product, (᭡). Samples were obtained
as indicated in the Experimental Section and chromatographed on
a column of BioGel P-6. Fractions of 1 mL were collected and
aliquots were analyzed by liquid scintillation counting.
The pulsed sensitivity in both cases was set at 30 nA and the pulsed
potentials were as follows: E1 = +0.05 V, E2 = +0.60 V, and E3 =
–0.60 V.
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First Chemical Synthesis of UDP[6-3H]-α-d-galactofuranose
1-O-Acetyl-2,3,5,6-tetra-O-benzoyl-
-[6-3H]galactofuranose
FULL PAPER
D
(7*):
nucleotide[17] was essentially followed. Thus, 1,1Ј-carbonyldiimid-
azole (1.3 mg, 9 μmol) was added to a solution of uridine 5Ј-mono-
phosphate (2 mg, 4 μmol) in DMF (1 mL). The solution was kept
under argon for 3 h at room temperature and then evaporated to
obtain the imidazolide 2b as described previously.[17] A mixture of
α-d-[6-3H]galactofuranosyl bis(triethylammonium) phosphate salt
(1*, 10.5 μCi) and the imidazolide 2b, previously dried by co-evapo-
ration with pyridine, was redissolved in DMF (1 mL) and kept un-
der argon at room temperature for 19 h. The reaction was stopped
by adding cold water (8 mL) and rapidly extracted with chloroform
(2×3 mL). The aqueous solution was coevaporated several times
with water in order to eliminate DMF, concentrated by lyophiliza-
tion, and purified by size-exclusion chromatography (1.4 μCi, Se-
phadex G-10 column, 1.8×80 cm; eluent: water, flow: 1 mLmin–1,
Compound 5 (3 mg, 6 μmol), prepared from 1,2,3,5,6-penta-O-ben-
zoyl-α,β-d-galactofuranose as described previously,[14] was reduced
in methanol (0.3 mL) with 10 mCi of NaB3H4 in 0.1 m KOH and,
after 3 h at room temperature, solid NaBH4 (3 mg, 80 μmol) was
added. The mixture was left overnight at 4 °C and the excess of
reagent was destroyed with acetone (100 μL). The solution was
stirred for 15 min and deionized by elution through a column of
Bio-Rad AG 50W-X12 resin (H+ form) and an Amberlite MB-3A
(mixed form) column to eliminate the boric acid. Compound 6* (Rf
= 0.52, solvent A) was benzoylated in the 6-position with benzoyl
chloride (0.4 mL) in pyridine (0.5 mL) during 4 h. The reaction was
quenched by addition of cold water and stirred for 30 min. After
extraction with CH2Cl2, the organic phase was washed with
NaHCO3 and water. The organic extracts were dried over anhy- Figure 3).
drous MgSO4, filtered, and taken to dryness (42 μCi).The acetolysis
The yield of compound 3 obtained from nonlabeled compound 1
(9 mg) was 26%.
α- -Galactofuranosyl 1,2-Cyclic Phosphate (4) and D-Galactose 2-
step was performed in dry CH2Cl2 (1 mL) with Ac2O/H2SO4 (40:1,
52 μL) for 16 h at 30 °C. The reaction was stopped by dilution with
CH2Cl2 (10 mL) and addition of cold water. After stirring for
30 min, the organic phase was washed and treated as above. Crude
compound 7* (35 μCi, Rf = 0.46, solvent A) was obtained by evap-
oration of the solvent.
D
Phosphate (12): Triethylamine (28 μL, 0.2 mmol), dicyclohexylcar-
bodiimide (140.3 mg, 0.6 mmol), and water (0.1 mL) were added to
a solution of compound 1 (31.4 mg, 0.078 mmol, Rf = 0.1, solvent
B) in pyridine (2 mL). This solution was stirred at 30 °C for 20 h.
TLC examination showed total conversion of the starting material
into a compound with a higher Rf (Rf = 0.4, solvent B). Water
(8 mL) was added and cyclohexylurea was filtered off. The aqueous
filtrate was extracted three times with diethyl ether, and the com-
bined ether extracts were washed once with water. The aqueous
solution was lyophilized to obtain 4 (26.8 mg, 86%). 1H NMR
The yield of compound 7 obtained from nonlabeled compound 5
(25 mg) was 84%.
Dibenzyl
2,3,5,6-Tetra-O-benzoyl-α-D
-[6-3H]galactofuranosyl-
phosphate (8α*): Compound 7* (35 μCi) was dissolved in dry
CH2Cl2 (1 mL) and bromotrimethylsilane (0.2 mL, 1.5 mmol) was
added, with external cooling (0 °C). After 24 h of stirring at room
temperature, the solution was concentrated in vacuo, and co-evapo-
rated several times with toluene to remove the excess of reagent.
The resulting bromide was dissolved in anhydrous toluene (1.2 mL)
and treated with dibenzylphosphate (20 mg, 70 μmol) and triethyl-
amine (15 μL, 105 μmol). After 4 h of stirring, the solution was
filtered, and the filtrate was concentrated in vacuo at room tem-
perature and chromatographed on a silica gel column (4×1.5 cm)
with toluene/EtOAc (95:5) as eluent. Fractions (1 mL) were col-
lected and aliquots were taken to estimate the radioactivity by li-
quid scintillation counting. Fractions containing the compound of
Rf = 0.16 (solvent A, Figure 1) were evaporated to dryness to afford
14 μCi. A mixture of compounds 8β and the tetrabenzoate 9 (Rf =
0.24) was obtained from fractions 15–35 (20 μCi).
3
3
(500 MHz, D2O): δ = 5.93 (dd, J1,2 = 4.5, JH,P = 14.0 Hz, 1 H,
3
3
H1), 4.75 (dd, J2,3 = 2.1, JH,P = 6.9 Hz, 1 H, H2) ppm. The
spectroscopic data were in agreement with those previously re-
ported.[20]
The same procedure was followed to prepare α-d-galactopyranosyl
1,2-cyclic phosphate (10, Rf = 0.29, 90%), whose structure was con-
firmed by spectroscopic data, which were found to be coincident
with those described in the literature.[28]
Compound 4 (15 mg, 0.044 mmol) was treated with 20 mm TFA
(100 μL) at 100 °C during 15 min. TLC examination showed total
conversion of the starting material into a product showing a lower
Rf value (Rf = 0.13, solvent B). Triethylamine was added, and after
evaporation of the reagents, d-galactose 2-phosphate (12, 75%,
8.5 mg, 0.033 mmol) was obtained as an anomeric mixture. 1H
The yield of compound 8α obtained from nonlabeled compound 7
(20 mg) was 49%.
3
NMR (500 MHz, D2O): δ = 5.32 (d, J1,2 = 3.8 Hz, 1 H, H1 α
3
anomer), 4.58 (d, J1,2 = 7.6 Hz, 1 H, H1 β anomer) ppm. 13C
α-D
-[6-3H]Galactofuranosyl Bis(triethylammonium)phosphate Salt
NMR (125 MHz, D2O): β anomer: δ = 97.01 (d, JC,P = 5.9 Hz,
C1), 76.22 (d, JC,P = 5.1 Hz, C5), 73.90 (d, JC,P = 2.5 Hz, C3),
72.58 (d, JC,P = 5.1, C2), 69.41 (C4), 61.8 (C6) ppm; α anomer: δ
= 92.38 (d, JC,P = 3.4 Hz, C1), 71.20 (C5), 69.97 (C4), 69.72 (d,
JC,P = 3.4 Hz, C2), 69.61 (C3), 62.00 (C6) ppm.
(1*): A solution of 8α* (14 μCi) in EtOAc (1 mL) containing trieth-
ylamine (20 μL, 144 μmol) and 10% Pd/C (1 mg) was hydrogenated
at 35 psi during 4 h. The mixture was filtered through Celite and,
after evaporation of the solvent, the resulting syrup was dried un-
der vacuum and debenzoylated with 2 mL of 0.07 m NaOMe in 1:1
CH2Cl2/MeOH, under argon. After 30 min, the solution was di-
luted with MeOH (5 mL) and the solvents evaporated. Water was
added and the solution was washed with toluene (1 mL) to elimin-
ate methyl benzoate. Both fractions were analyzed by liquid scintil-
lation counting, and no radioactivity was found in the toluene frac-
tion. The aqueous solution was lyophilized, and compound 1*
(10.5 μCi) was identified by comparison with an authentic cold
sample[19] by HPAEC-PAD (Condition 1, Figure 2A).
The same product was obtained from α-d-galactopyranosyl 1,2-
cyclic phosphate (10).
Preparation of Cell-Free Extracts and Membranes of P. fellutanum:
Cultures of Penicillium fellutanum[26] were obtained using 0.5% ga-
lactose as carbon source.[29] The cultures were filtered after 3 d of
growing and the mycelial pads were finely ground in a mortar pre-
viously cooled to –10 °C. For one standard pad (52 g wet weight),
Al2O3 (20 g) was added and the mass was ground vigorously for 2–
4 min at 4 °C, whereupon 12 mL of 0.1 m sodium phosphate buffer
(pH 6.8, Buffer A) was added and the grinding continued for a
further 3 min. A total of 25 mL buffer (approx. 1 mL per gram of
wet weight of mycelium) was used in the grinding and in washing
out the mortar.
The yield of compound 1 obtained from nonlabeled compound 8α
(10 mg) was 88%.
Uridine 5Ј-(α-
monium) Salt (3*): The procedure described for the nonradioactive
D
-[6-3H]Galactofuranosyl Diphosphate) Di(triethylam-
Eur. J. Org. Chem. 2005, 2958–2964
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2963

K. Mariño, C. Marino, C. Lima, L. Baldoni, R. M. de Lederkremer
FULL PAPER
The homogenate was centrifuged at 8000 g for 30 min at 4 °C to
remove Al2O3 and cell debris. The supernatant containing
0.5 mgmL–1 of protein (pH 6.5–6.8) was centrifuged at 200000 g
for 1 h at 4 °C to sediment the membranes, which were resuspended
in buffer A and again centrifuged. The final suspension of mem-
branes was obtained at a protein concentration of 3 mgmL–1. This
membrane suspension can be stored at –70 °C for at least 6 months
without significant loss of activity.
Pict 06-3036 and ANPCYT, BID 1201/OC-AR PICT 06-05133)
and UBACyT. RML and CM are research members of CONICET,
and KM was supported by a fellowship from CONICET.
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Abbreviations: UDP-Galp: Uridine 5Ј-(α-d-galactopyranosyl di-
phosphate); UDP-Galf: Uridine 5Ј-(α-d-galactofuranosyl diphos-
phate); α-d-Galp 1-P: α-d-galactopyranosyl bis(triethylammonium)
phosphate salt; α-d-Galf 1-P: α-d-galactofuranosyl bis(triethylam-
monium) phosphate salt; d-Gal 2-P: galactose 2-phosphate; α-d-
Galf 1,2-P: α-d-galactofuranose 1,2-cyclic phosphate; α-d-Galp
1,2-P: α-d-galactopyranose 1,2-cyclic phosphate; pPGM: peptido-
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Received: January 24, 2005
Acknowledgments
This work was supported by the Agencia Nacional de Promoción
Científica y Tecnológica, Argentina (ANPCYT, BID 802/OC-AR
2964
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 2958–2964