A new synthetic route to 4-methylumbelliferyl-β-D-glucopyranosiduronic acid (MUG)

Article abstract of DOI:10.1055/s-2007-967972

A synthetic route to prepare 4-methylumbelliferyl-β-D- glucopyranosiduronic acid (MUG) from 4-methylumbelliferyl-β-D- glucopyranoside (MUGluc) was developed. The primary hydroxyl group in MUGluc was protected by tritylation followed by acetylation of secondary hydroxyls. The triphenylmethyl group was selectively removed by treatment with iodine-methanol in benzene and the free hydroxyl was transformed into the carboxylic acid by phase-transfer oxidation with sodium hypochlorite and TEMPO as catalyst. Finally, the acetate groups were removed by reaction with barium methoxide in methanol to afford the MUG with an overall yield of 37% from the MUGluc. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-967972

LETTER
649
A New Synthetic Route to 4-Methylumbelliferyl-b-D-glucopyranosiduronic
Acid (MUG)
cid el A. López-López, Alexander Balbuzano-Deus, Juan C. Rodríguez-Domínguez, Miriam Mesa Hernández,a
a
a
a,b
4
M
-Methylumbelliferyl-
i
-
D
-glu
g
copyranosid
u
uronic
A
a
a
b
Anais Fernández Villalobo, Yulianela Ibarra Reyes, Gilbert Kirsch*
a
Departamento de Química, Centro de Química Farmacéutica, Calle 200 y 21, Atabey, Playa, 11600 Ciudad de la Habana, Cuba
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique (LIMBP), Institut Jean Barriol, Université Paul Verlaine Metz,
b
1
Boulevard Arago, 57070 Metz, France
Fax +33(3)87315801; E-mail: kirsch@univ-metz.fr
Received 17 November 2006
methylumbelliferyl-b-D-glucopyranoside
However, there are no reports in the literature related with
the first of both approaches, probably because of the in-
(MUGluc).
Abstract: A synthetic route to prepare 4-methylumbelliferyl-b-D-
glucopyranosiduronic acid (MUG) from 4-methylumbelliferyl-b-D-
glucopyranoside (MUGluc) was developed. The primary hydroxyl
group in MUGluc was protected by tritylation followed by acetyla- stability of typical glucopyranuronic acid derivatives such
tion of secondary hydroxyls. The triphenylmethyl group was selec- as bromides or iodides. In effect, it is known that during
tively removed by treatment with iodine-methanol in benzene and
the free hydroxyl was transformed into the carboxylic acid by
phase-transfer oxidation with sodium hypochlorite and TEMPO as
catalyst. Finally, the acetate groups were removed by reaction with
barium methoxide in methanol to afford the MUG with an overall
yield of 37% from the MUGluc.
the preparation of the umbelliferyl-b-D-glucopyrano-
siduronic acid (a MUG analogue) by reaction between
methyl
2,3,4-tri-O-acetyl-a-D-glucopyranosyluronate
bromide or iodide and umbelliferone, low yields (6–10%)
of the conjugate are obtained.9
–11
Key words: synthesis, MUG, 4-methylumbelliferyl-b-D-gluco- The only reported procedure in order to synthesize the
pyranoside, 4-methylumbelliferyl-b-D-glucopyranosiduronic acid
MUG carries out the oxidation of primary hydroxyl group
of the easily available MUGluc by oxygen gas in aqueous
12
alkaline media using platinum as catalyst. However, this
approach has some drawbacks: high purity oxygen must
be used to avoid catalyst poisoning, large charge of plati-
num catalyst is necessary (approximately 50% in weight
related to MUGluc), ion-exchange column chromatogra-
phy must be used to isolate the desired product and
finally, large volumes of water have to be eliminated by
freeze-drying and relatively low yields of MUG are
commonly obtained (18–20%).
Substrates based on 4-methylumbelliferone (4-MU) have
been extensively used for the detection of enzymes in
1
,2
microbiological diagnostics. The facility of hydrolysis
of these kind of compounds by a specific enzyme makes
possible its detection by the fluorescence expressed by
3
–5
the 4-MU in the media. The 4-methylumbelliferyl-b-D-
glucopyranosiduronic acid (MUG, Figure 1) is a commer-
cially available fluorogenic substrate (Biosynth, Fluka)
widely used for quantitatively identifying and differenti-
ating the presence of the Coliform group and specially
Escherichia coli in water, food and other products for
In order to overcome the previously cited drawbacks, we
developed an alternative way for obtaining MUG
(
Scheme 1).
6
–8
human consumption.
In the literature there is a report about an unsuccessful
attempt of using this approach to synthesize 4-methyl-
umbelliferyl a-L-idopyranosiduronic acid.13 In that case,
the work was interrupted after detecting the acyl migra-
tion and low yields during the elimination of the trityl
group by treatment with hydrogen bromide in acetic acid.
HOOC
O
HO
HO
O
O
O
OH
MUG
In the present work, the primary hydroxyl group of
MUGluc was selectively protected by reaction with trityl
chloride in pyridine, followed by acetylation of the sec-
ondary hydroxyl groups with acetic anhydride to obtain
compound 1 with good yield (73%). Although the hydro-
gen bromide in acetic acid is widely used in carbohydrate
chemistry to remove selectively the trityl protecting
group, this technique was not used because very short
reaction times are required (below 1 min). As a conse-
quence, the process is difficult to control and relatively
low yields are obtained due the sensibility of the glyco-
sidic linkage in acidic media. An attempt of effecting the
deprotection using anhydrous cupric sulfate in refluxing
Figure 1 Chemical structure of 4-methylumbelliferyl-b-D-gluco-
pyranosiduronic acid (MUG)
From a synthetic point of view, there are two possible
approaches of preparing MUG: (a) direct glycosylation of
-MU or its salts with glucopyranuronic acid derivatives
4
and (b) oxidation of primary hydroxyl group of the 4-
SYNLETT 2007, No. 4, pp 0649–0651
Advanced online publication: 21.02.2007
DOI: 10.1055/s-2007-967972; Art ID: G34106ST
Georg Thieme Verlag Stuttgart · New York
0
1.
0
3.
2
0
0
7
©

6
50
M. A. López-López et al.
LETTER
OH
OCPh3
O
O
i,ii
HO
HO
AcO
AcO
O
O
O
73%
O
O
O
OH
OAc
MUGluc
1
92% iii
OH
HOOC
O
iv
O
AcO
AcO
AcO
O
O
O
68%
O
O
O
AcO
OAc
OAc
3
2
85%
v
HOOC
O
HO
HO
O
O
O
OH
4
MUG
Scheme 1 Synthetic route to prepare MUG. Reagents and conditions: (i) Ph CCl, pyridine, 90 °C, 5 h; (ii) Ac O, r.t., 24 h; (iii) I , MeOH,
3
2
2
benzene, 65 °C, 24 h; (iv) NaOCl, TEMPO, NaBr, NaHCO , Bu NBr, CH Cl –H O, 0 °C, 3 h; (v) Ba(OMe) , MeOH, 0–5 °C, 24 h.
3
4
2
2
2
2
benzene was made, according to the literature report.14 known that the hydrolysis of acetylated coumarin glyco-
However; no reaction was detected by TLC after 24 hours, sides requires some attention, because a slight excess of
18
a longer time than that reported (5 h). Moreover, the detri- base leads to the irreversible lactone opening. If the al-
1
5
tylation activity of several silica gels is well documented kali-metal salt of the glucuronide is desired, potassium or
since in the original work the reaction mixture was puri- sodium hydroxide (or preferably the corresponding car-
fied by column chromatography over silica gel. It is pos- bonates) in aqueous alcohols may be used. If the free glu-
sible that the reaction observed in that case was promoted curonic acid is required, it may be useful to carry out the
by this acidic absorbent and not by the cupric sulfate.
deacetylation step by the Zemplen procedure (sodium
methoxide–methanol), followed by treatment with an ion-
exchange resin.19
As a consequence, the detritylation step was performed
1
6
using an iodine–methanol reagent. This method has the
advantage of being selective for the trityl group whereas Considering that the acetylated glycoside 3 has an acid
the glycosidic linkage is stable to the reaction conditions. group, it was decided to carry out the deacetylation with
Detritylation probably occurs due to the in situ generation barium methoxide in methanol in order to simplify the
of acid traces (hydroiodic acid) produced by the oxidation final work-up for obtaining the MUG. It was necessary to
of methanol by iodine. Thus, compound 2 was obtained add barium methoxide, not only to remove acetate groups
with an excellent yield (92%) and good purity after re- but also to neutralize the acid function. Altough the
moval of the triphenylmethanol (a by-product) with di- formed barium salt of 3 is slightly soluble in methanol,
isopropyl ether, 24 hours at 65 °C were required for the the reaction was complete, as was demonstrated by TLC
completion of the reaction and no acyl migration was analysis.
detected.
The barium salt of the deacetylated product was also in-
Oxidation of the primary hydroxyl group in compound 2 soluble in methanol, but when the reaction mixture was
to the corresponding acid was achieved by using a catalyt- treated with the calculated amount of sulfuric acid to re-
ic amount of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEM- move barium all the obtained product become soluble in
PO), 5% aqueous sodium hypochlorite as oxidant and a methanol and it was possible to quantitatively separate the
basic medium generated by sodium hydrogencarbonate.17 barium sulfate to obtain MUG (4) in good yield (85%) and
A biphasic system (dichloromethane–water), in the pres- high purity, by removing the solvent under vacuum and
ence of tetrabutylammonium bromide (Bu NBr) as phase- crystallization from diethyl ether. It may be noted that
4
transfer catalyst, was used to run the reaction because of during the process no lactone opening was observed.
the insolubility of 2 in water. Under these conditions,
In conclusion, the developed synthetic route is very useful
to prepare MUG. Despite of some synthetic steps more
than the reported procedure, the overall yield obtained
from MUGluc was 37%, it means almost twice the yield
reported (18–20%) when this fluorogenic substrate was
synthesized by oxidation with oxygen gas in the presence
compound 3 was readily obtained (2–3 h) with an accept-
able yield (68%) and good purity after acidification of the
aqueous phase to pH 3, simultaneous extraction with
dichloromethane and final solvent evaporation under re-
duced pressure.
20
The last step of this synthetic approach was the removal of platinum as catalyst.
of acetate groups from secondary hydroxyls of 3. It is well
Synlett 2007, No. 4, 649–651 © Thieme Stuttgart · New York

LETTER
4-Methylumbelliferyl-b-D-glucopyranosiduronic Acid
651
13C NMR (CDCl
6
1
): d = 18.61, 20.31, 20.59, 20.60, 62.02,
8.40, 71.15, 72.78, 73.91, 86.89, 98.52, 104.40, 113.18,
13.74, 115.48, 125.72, 127.07 (3 C), 127.81 (6 C), 128.60
References and Notes
3
(
1) Manafi, M.; Kneifel, W.; Bascomb, S. Microbiol. Rev. 1991,
5, 335.
5
(6 C), 143.40 (3 C), 152.02, 154.83, 159.42, 160.76, 168.96,
(
(
(
2) Dealler, S. F. Rev. Med. Microbiol. 1993, 4, 198.
3) Shadix, L. C.; Rice, E. W. Can. J. Microbiol. 1991, 37, 908.
4) Berg, J. D.; Fiksdal, L. Appl. Environ. Microbiol. 1988, 54,
1
6
4
69.28, 170.20. Anal. Calcd for C H O (722.73): C,
8.14; H, 5.30. Found: C, 68.20; H, 5.17.
-Methylumbelliferyl-2,3,4-tri-O-acetyl-b-D-gluco-
41 38 12
2118.
pyranoside (2)
A solution of iodine (0.3 g, 1.18 mmol) in MeOH was added
to a solution of compound 1 (1.8 g, 2.55 mmol) in benzene
(
5) Brenner, K. P.; Rankin, C. C.; Roybal, Y. R.; Stelma, G. N.;
Scarpino, P. V.; Dufour, A. P. Appl. Environ. Microbiol.
1
993, 59, 3534.
6) Feng, P. C. S.; Hartman, P. A. Appl. Environ. Microbiol.
982, 43, 1320.
7) Kilian, M.; Bulow, P. Acta Pathol. Microbiol. Scand., Sect.
B 1976, 84, 245.
(18 mL) and the mixture was stirred at 65 °C for 24 h. The
(
(
(
(
solvents were evaporated under vacuum and the residue was
dissolved in EtOAc (60 mL), washed with 10% aq Na S O
1
2
2
3
(
30 mL), followed by H O (3 × 10 mL), and dried over
2
anhyd Na SO . The organic extract was evaporated to
2
4
8) Rippey, S. R.; Chandler, L. C.; Watkins, W. D. J. Food Prot.
dryness under reduced pressure and the residue was stirred
h with diisopropyl ether (30 mL). The resulting solid was
1987, 50, 685.
2
9) Walsh, J. S.; Patanella, J. E.; Halm, K. A.; Facchine, K. L.
filtered off, washed with diisopropyl ether (3 × 5 mL) and
Drug Metab. Dispos. 1995, 23, 869.
vacuum-dried at 40 °C to afford 2. Yield: 1.10 g (92%); mp
(
(
(
(
(
10) Bollemback, J.; Long, J. W.; Bejamin, D. G.; Lindquist, J. A.
J. Am. Chem. Soc. 1955, 77, 3310.
11) Berrang, B.; Twine, C. E.; Hennessee, G. L.; Carroll, F. I.
Synth. Commun. 1975, 5, 231.
1
1
76–178 °C. H NMR (CDCl ): d = 2.04 (6 H, s), 2.07 (3 H,
3
s) 2.37 (3 H, s), 3.72 (3 H, m), 5.21 (4 H, m), 6.15 (1 H, s),
13
6.70–7.56 (3 H, m). C NMR (CDCl ): d = 18.56, 20.52 (3
3
C), 60.91, 68.21, 71.04, 72.42, 74.65, 98.23, 103.95, 113.03,
12) Courtin-Duchateau, M. C.; Veyrières, A. Carbohydr. Res.
1
1
5
4
13.61, 115.41, 125.75, 152.20, 154.70, 159.17, 160.81,
69.24, 170.06 (2 C). Anal. Calcd for C H O (480.42): C,
5.00; H, 5.04. Found: C, 55.33; H, 5.20.
-Methylumbelliferyl-2,3,4-tri-O-acetyl-b-D-gluco-
1
978, 65, 23.
13) Baggett, N.; Samra, A. K.; Smithson, A. Carbohydr. Res.
983, 124, 63.
2
2
24 12
1
14) Randazzo, G.; Capasso, R.; Cicala, M. R.; Evidente, A.
Carbohydr. Res. 1980, 85, 298.
pyranosiduronic Acid (3)
A sat. solution of aq NaHCO (15 mL) and a 5% solution of
3
(
(
15) Lehrfeld, J. J. Org. Chem. 1967, 32, 2544.
16) Wahlstrom, J. L.; Ronald, R. C. J. Org. Chem. 1998, 63,
NaOCl (19 mL) were added to a solution of 2 (1.25 g, 2.7
mmol), NaBr (0.06 g, 0.583 mmol), TBAB (0.06 g, 0.186
mmol), and TEMPO (0.06 g, 0.384 mmol) in a 1:1 mixture
of CH Cl –H O (20 mL) at 0 °C. After 2–3 h the aqueous
6021.
(
(
17) Yeung, B. K. S.; Hill, D. C.; Janicka, M.; Petillo, P. A. Org.
Lett. 2000, 2, 1279.
18) Brown, R. T.; Scheinmann, F.; Stachulski, A. V. J. Chem.
Res., Synop. 1997, 370.
2
2
2
phase was separated and washed with CH Cl (2 × 10 mL).
2
2
The, CH Cl (20 mL) was added; the mixture was cooled to
2
2
0–5 °C and adjusted to pH 3 with 10% HCl. The organic
(
(
19) Stachulski, A. V.; Jenkins, G. N. Nat. Prod. Rep. 1998, 173.
20) General Methods
phase was separated, washed with H O (10 mL) and dried
2
over anhyd Na SO . The solvent was evaporated to dryness
2
4
TLC was performed on precoated plates of silica gel GF-254
under reduced pressure and the resulting solid was vacuum-
dried to afford 3. Yield 0.87 g (68%); mp 218–221 °C. H
(Merck); EtOAc–CHCl –AcOH (8:6:1) and n-BuOH–H O–
1
3
2
AcOH (5:1:1) were used as mobile phases. The
chromatograms were visualized in a Camag UV/Vis lamp
l = 254 and 360 nm). Compounds were also detected by
spraying the plates with 5% H SO –EtOH reagent followed
NMR (CDCl + MeOD ): d = 2.00 (6 H, s), 2.02 (3 H, s),
3
4
2.36 (3 H, s), 4.22 (1 H, m), 5.21–5.36 (4 H, m), 6.14 (1 H,
(
13
s), 6.87–7.55 (3 H, m). C NMR (CDCl + MeOD ): d =
3
4
2
4
18.46, 20.37 (3 C), 68.86, 70.78, 71.92, 72.13, 98.11,
104.22, 112.80, 113.90, 115.50, 125.73, 152.70, 154.49,
159.09, 161.30, 167.94, 169.35, 169.69, 170.17. Anal. Calcd
by heating. Melting points(mp) were determined using a
1
Büchi capillary apparatus and are uncorrected. H NMR and
13
C NMR spectra were registered on a Bruker AC 250F
for C H O (494.40): C, 53.45; H, 4.49. Found: C, 53.77;
2
2
22 13
spectrometer for 250 MHz and 62.5 MHz, respectively,
H, 5.03.
-Methylumbelliferyl-b-D-glucopyranosiduronic Acid
MUG, 4).
using CDCl , CDCl + MeOD or DMSO-d as solvents and
3
3
4
6
4
(
TMS as internal standard. Optical rotation of MUG was
measured at 20 °C with an ADP220 Bellenghan + Stanley
Ltd. apparatus. Elemental analysis was done in a
Thermofinnigan FlashEA 1112 equipment.
A suspension of 3 (2.0 g, 4.18 mmol) in MeOH (30 mL) was
stirred at 0–5 °C with a solution of Ba(OMe) (0.503 g, 2.52
mmol) in MeOH (10 mL). After 24 h, concentrated H SO
2
2
4
4-Methylumbelliferyl-2,3,4-tri-O-acetyl-6-O-trityl-b-D-
(
0.14 mL, 2.52 mmol) was added, the precipitated BaSO4
glucopyranoside (1)
Trityl chloride (4.25 g, 15.25 mmol) was added to a stirred
suspension of 4-methylumbelliferyl-b-D-glucopyranoside
was filtered off, the filtrate was evaporated to dryness under
reduced pressure and the residue was crystallized with Et O
2
(
20 mL). The resultant solid was separated by filtration,
(5.0 g, 14.78 mmol) in pyridine (11 mL). The mixture was
washed with Et O (3 × 5 mL) and vacuum-dried to afford 4.
2
heated at 90–100 °C for 5 h. After cooling to 0–5 °C, Ac O
2
Yield 1.25 g (85%); mp 140–143 °C; [a] –116 (c 0.25,
D
(7.5 mL, 79.34 mmol) was added and the mixture was left to
12
1
H O); {lit. mp 139–145 °C; [a] –114 (c 0.25, H O)}. H
2
D
2
stand overnight at r.t., poured into cold H O (225 mL) and
stirred for 2 h. The resultant solid was filtered off, washed
2
NMR (DMSO-d ): d = 2.40 (3 H, s), 3.23 (1 H, m), 3.56 (1
6
H, m), 4.98–5.19 (2 H, m), 5.38 (1 H, m), 6.22 (1 H, s), 6.99–
with cold H O (3 × 50 mL), vacuum-dried at 40 °C and
13
2
7
7
1
.75 (3 H, m). C NMR (DMSO-d ): d = 17.93, 71.82,
6
recrystallized from EtOH to afford 1. Yield 7.60 g (73%);
2.96, 73.63, 76.43, 100.00, 103.29, 111.46, 113.43, 113.89,
26.14, 153.13, 154.22, 159.92, 160.23, 172.22.
1
mp 212–215 °C. H NMR (CDCl ): d = 1.78 (3 H, s), 2.03 (3
3
H, s), 2.08 (3 H, s) 2.40 (3 H, s), 3.25 (2 H, m), 3.76 (1 H,
m), 5.09–5.43 (4 H, m), 6.21 (1 H, s), 6.99–7.60 (18 H, m).
Synlett 2007, No. 4, 649–651 © Thieme Stuttgart · New York