Synthesis of an aryl 2-deoxy-β-glycosyl tetrasaccharide to probe retaining endo-glycosidase mechanism

Article abstract of DOI:10.1016/j.tetasy.2009.03.008

The synthesis of the 1,3-1,4-β-glucanase substrate analogue 4-nitrophenyl O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-2-desoxi-β-d-glucopyranoside 2 is reported. Starting from the main tetrasaccharide obtained by enzym

Full text of DOI:10.1016/j.tetasy.2009.03.008

Tetrahedron: Asymmetry 20 (2009) 851–854
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of an aryl 2-deoxy-b-glycosyl tetrasaccharide to probe retaining
endo-glycosidase mechanism
*
Mireia Abel, Antoni Segade, Antoni Planas
Laboratory of Biochemistry, Department of Bioengineering, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of the 1,3–1,4-b-glucanase substrate analogue 4-nitrophenyl O-b-D-glucopyranosyl-(1?4)-
O-b-D-glucopyranosyl-(1?4)-O-b-D-glucopyranosyl-(1?3)-2-desoxi-b-D-glucopyranoside 2 is reported.
Starting from the main tetrasaccharide obtained by enzymatic depolymerization of barley b-glucan,
the synthetic scheme involves preparation of the corresponding 3-O-substituted glycal which was con-
Received 4 February 2009
Accepted 10 March 2009
Available online 22 April 2009
verted into a 2-deoxy-a-glycosyl iodide as a glycosyl donor. The key glycosylation step was successfully
achieved by nucleophilic substitution of the iodide donor with 4-nitrophenolate with high b-selectivity.
Dedicated to Professor George Fleet on
occasion of his 65th birthday
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
compared to the canonical mechanism of retaining glycosidases,
in particular the high efficiency shown by the enzyme for hydra-
Deoxy glycosides have been traditionally used in mechanistic
and kinetic studies of glycosidases as analogues of their natural
substrates to probe hydrogen bonding interactions in the active
site of the enzyme and their role in specificity and catalytic effi-
ciency. For retaining b-glycosidases the 2-OH of the sugar unit in
subsite ꢀ1 has a major contribution to transition state stabiliza-
tion. Kinetic studies for a number of glycosidases1–6 have shown
that the substrate 2-OH group contributes 5–10 kcalꢁmolꢀ1 to tran-
sition state stabilization whereas other hydroxyl groups may only
contribute 1–2 kcalꢁmolꢀ1. Even though 2-deoxy glycosides are
more reactive than the parent 2-hydroxy compounds in terms of
spontaneous hydrolysis,7,8 they are often poor substrates or inhib-
itors of retaining b-glycosidases. The hydrogen bond interactions
between the 2-OH group of the substrate and active site residues
of the enzyme are optimized upon ring distortion leading to the
transition state, which transiently increases the acidity of the hy-
droxyl group resulting in stabilization of the oxocarbenium-like
tion of glycals in which the 1,2-double bond of the sugar unit in
subsite ꢀ1 is hydrated to the 2-deoxy glycoside, prompted us to
evaluate the importance of the 2-OH group of the substrates in
the hydrolytic mechanism of the enzyme.
Herein we report the synthesis of the 2-deoxy analogue 2 of the
aryl tetrasaccharide substrate 1 (Fig. 1). Compound 1 is a good sub-
strate of B. licheniformis 1,3–1,4-b-glucanase, which catalyzes the
hydrolysis of the glycosidic bond between the 3-O-substituted glu-
cosyl unit and the aglycon with release of 4-nitrophenol.15,16 The
2-deoxy analogue 2 may follow the same enzyme-catalyzed hydro-
lysis and comparative kinetic analysis will allow evaluation of the
role of the 2-OH in the enzymatic mechanism.
OH
O
OH
O
OH
O
OH
O
HO
O
HO
HO
HO
O
O
O
HO
OH
OH
OH
X
transition state in the enzymatic mechanism of the enzyme.9,10
A
NO2
1
X = OH
number of X-ray structures of enzyme–ligand complexes and cova-
lent glycosyl–enzyme intermediates show strong H-bond interac-
tions of the 2-OH group with the catalytic nucleophile, the short
distance between oxygen atoms (ca. 2.5 Å) close to a low barrier
hydrogen bond (LBHB).4,5,10–13
2
X = H
Figure 1. Aryl b-glycoside substrates for 1,3–1,4-b-glucanases.
We have been extensively working with Bacillus 1,3–1,4-b-glu-
canases that belong to family 16 of glycoside hydrolases.14 They
are endo-glycosidases that hydrolaze mixed-linked b1?3 and
b1?4 glucans such as barley b-glucan and lichenan, with a strict
cleavage specificity for b1?4 linkages in 3-O-substituted glucopyr-
anosyl units. Due to a number of mechanistic differences as
2. Results and discussion
The main challenge in the synthesis of 2 is the construction of
the aryl 2-deoxy-b-glucopyranosidic linkage. The stereoselective
preparation of 2-deoxy-b-glycosides is difficult due to the lack of
substituents at C-2 that provide anquimeric assistance to direct
b-glycosylation.17–19 The most common methods involve the use
of a heteroatom substituent at C2 of the glycosyl donor followed
by its reductive removal after glycosylation.20 Other methods
* Corresponding author. Tel.: +34 932672000; fax: +34 932056266.
E-mail address: antoni.planas@iqs.es (A. Planas).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.03.008

852
M. Abel et al. / Tetrahedron: Asymmetry 20 (2009) 851–854
include the use of
a
-glycosyl phosphates,21 displacement of
a
-gly-
glycoside 9 was obtained with a b:
a selectivity higher than 20:1
cosyl halides,22 palladium-catalyzed glycosylation reactions,23 and
utilization of alkoxy-substituted anomeric radicals.24 However
most of these methods fail or provide low stereoselectivity for
the glycosylation of phenols to obtain aryl 2-deoxy-b-glycosides.
Only few methods25–28 show b-selectivity. They involve the prepa-
ration of rather complex glycosyl donors, usually 2-deoxy-2-phen-
ylthio- or 2-deoxy-2-phenylseleno-glycosyl donors, and need
further reactions for removal of the temporary group at C-2. In
(the
a
-anomer was detected by 1H NMR in enriched fractions dur-
ing the purification), along with the expected E2-elimination prod-
uct (glycal 5) and the tetrasaccharide 7, generated by the reaction
of 8 with remains of TMSAcO which were not efficiently eliminated
after the glycosyl iodide synthesis. After careful chromatographic
purification, the 4-nitrophenyl 2-deoxy-b-glycoside 9 was isolated
with an overall 30% yield starting from 7. Deprotection of the O-
acetyl groups using sodium methoxide in methanol (88% yield)
gave the desired analogue 2.
selecting a direct b-glycosylation method, a-glycosyl halides have
shown to readily undergo substitution with aryl alkoxy anions to
Since the 1H NMR data of compound 6 showed that the anomer-
provide aryl 2-deoxy-b-glycosides.29
ic hydroxyl group at C-1 was mainly in the a-configuration, a one-
The synthesis (Fig. 2) started from readily available per-O-acet-
ylated tetrasaccharide 3 (obtained by enzymatic hydrolysis of
step glycosylation of 6 by the method reported by Roush and Li35
based on a Mitsunobu reaction was also attempted. Treatment of
the 2-deoxy tetrasaccharide 6 with 4-nitrophenol in the presence
of triphenylphosphine and diethylazodicarboxylate (DEAD) in
CH2Cl2/toluene afforded the per-O-acetylated 4-nitrophenyl-2-
barley b-glucan)15 which was transformed to the
a-glycosyl bro-
mide 4 with HBr in acetic acid and then reduced to the glycal 5.
Reduction of the bromide 4 was studied by different methods be-
cause the presence of a good leaving group (the 3-O-cellotriosyl
group) in the allylic position of the formed glycal double bond
leads to rearrangements and bond cleavages that lower the yield
of glycal formation when compared with the reduction of other
glycosyl bromides. Reduction with Zn/acetic acid in the presence
of Cu2+ rendered the glycal 5, but best yields after optimization
through an experimental design approach were 36%. Other meth-
ods attempted were reduction with Cr2+ in basic media,30 reduc-
tion with Zn/silver/graphite31, and electrochemical reduction in
aprotic solvents,32 but the yields did not surpass 40%. The best re-
sults were obtained by using Mn and bis(cyclopentadienyl)tita-
nium(III) chloride (Cp2TiCl2) in THF,33 with an overall yield of
50% from 4. The glycal 5 was converted in the 2-deoxy-tetrasac-
charide 6 through the mercuration–demercuration procedure of
Bettelli et al.34 First, addition of mercurium acetate to the double
bond of 5 gave an organomercuric intermediate at C-2 which
was directly reduced by addition of NaBH4. Again, allylic reactions
seemed to be responsible for the low yield obtained in this step (ca.
50%). Acetylation of the reducing end of 6 quantitatively yielded
the per-O-acetylated 2-deoxy-tetrasaccharide 7, which was ready
for b-glycosylation.
deoxy saccharide 9 (a + b) in 48% overall yield. Despite of the good
yield, stereoselectivity was poor, with a b:a ratio of 2.2 to 1.
3. Conclusion
In conclusion, the target 4-nitrophenyl 2-deoxy-b-glycoside 2
was obtained in seven steps from per-O-acetylated 3-O-b-cellotri-
osyl-D-glucopyranose with an overall yield of 6.1%. The synthetic
sequence involves preparation of the glycal 5 under mild condi-
tions since it its prone to rearrangements (3-O-substituted glycal).
Mercuration–demercuration yielded the 2-deoxy saccharide which
was converted into the 2-deoxy-
sylation step was successfully achieved by nucleophilic substitu-
tion of the -glycosyl iodide 8 with 4-nitrophenolate with high
a-glycosyl iodide 8. The key glyco-
a
b-selectivity. Comparative kinetics of the 2-deoxy analogue 2 and
substrate 1 to evaluate the contribution of the 2OH group of the
substrate in the enzymatic hydrolysis by 1,3–1,4-b-glucanases will
be reported elsewhere.
4. Experimental
4.1. General
The a-glycosyl iodide 8 used as glycosyl donor was obtained by
treatment of 7 with iodotrimethyl silane in anhydrous CH2Cl2 at
0 °C,29 and subsequently used without purification in the glycosyl-
ation step with sodium 4-nitrophenoxide. The desired aryl 2-deoxy
All materials were purchased from commercial sources, except
tetrasaccharide 3, which was obtained following the procedure de-
OAc
O
OAc
OAc
O
OAc
O
OAc
i
O
AcO
AcO
AcO
AcO
O
AcO
O
O
AcO
OAc
O
O
AcO
OAc
OAc
OAc
OAc
Br
3
4
ii
OAc
O
OAc
O
OAc
O
iv
iii
AcO
AcO
O
AcO
OAc
OH
O
O
7
6
5
v
OAc
O
OR
O
OR
O
OR
O
OR
O
RO
O
AcO
vi
RO
RO
RO
O
O
O
O
RO
OR
OR
OR
I
8
NO2
9
2
R = AcO
R = H
vii
Figure 2. (i) HBr/HAcO 33%, rt, 30 min (99%); (ii) Cp2TiCl2, Mn powder, THF, rt, N2 atm, 13 h (50% from 3); (iii) (a) Hg(AcO)2, THF/H2O (4:1), 0 °C, 45 min, (b) NaBH4, H2O/THF
(4:1), 0 °C, 1 min (47%); (iv) Ac2O/Py (1:1), rt, 7 h (99%); (v) TMSI, CH2Cl2, 0 °C, N2 atm, 45 min; (vi) 4-nitrophenol, NaHMDS, 15-crown-5, THF, rt, 45 min (30% from 7); (vii)
20 mM NaOMe in MeOH, rt, 13.5 h (88%).

M. Abel et al. / Tetrahedron: Asymmetry 20 (2009) 851–854
853
scribed in the literature.15 Mn powder was dried under vacuum at
60 °C for 6 h before being used. All anhydrous solvents were dis-
tilled prior to use: THF was distilled from LiAlH4; CH2Cl2 was dis-
tilled from CaH2; toluene was distilled from P2O5; and MeOH
was distilled from magnesium/iodine. TLC was performed on
Macherey-Nagel Polygram SIL G/UV254 (0.20 mm) plates, and
detection was achieved by observation under UV light at 254 and
360 nm, and by spraying with 50% H2SO4 and heating. Flash chro-
91.6 (C1I), [76.2, 76.1, 74.8, 72.8, 72.7, 72.6, 72.6, 72.5, 72.0, 71.7,
71.7, 71.5, 69.4, 68.3, 67.7] (C2II–IV, C3I–IV, C4I–IV, C5I–IV), [62.6,
62.1, 61.8, 61.4] (C6I–IV), 35.3 (C2I), 20.8–20.4 (CH3CO).
4.1.3. Acetyl O-(2,3,4,6-tetra-O-acetyl)-b-
(1?4)-O-(2,3,6-tri-O-acetyl)-b- -glucopyranosyl-(1?4)-O-
(2,3,6-tri-O-acetyl)-b- -glucopyranosyl-(1?3)-4,6-di-O-acetyl-
2-deoxy-b- -glucopyranoside 7
D-glucopyranosyl–
D
D
D
matography was carried out on silica gel (SDS, 35–70
spectra were recorded on a Varian-Gemini 300 instrument using
tetramethylsilane as internal standard. Yields are not optimized.
l
m). NMR
At first, 2.07 g (1.79 mmol) of 6 was dissolved in 48 mL of
Ac2O/pyridine (1:1) and the mixture was stirred at rt, protected
from light and moisture, for 7 h. The mixture was poured into
300 mL of ice water and the solid was filtered and redissolved
in 200 mL of CHCl3. The filtrate was extracted with
3 ꢂ 100 mL of CHCl3. These organic layers were combined, and
were washed with 3 ꢂ 100 mL of saturated aqueous NaHCO3,
dried over MgSO4, and distilled off. The obtained white solid
was dried under vacuum to yield 2.13 g (1.78 mmol, 99%) of 7
4.1.1. O-(2,3,4,6-Tetra-O-acetyl-b-
(2,3,6-tri-O-acetyl-b- -glucopyranosyl)-(1?4)-(2,3,6-tri-O-
acetyl-b- -glucopyranosyl)-(1?3)-4,6-di-O-acetyl-1,5-anhydro-
2-deoxy- -arabino-hex-1-enitol 5
D-glucopyranosyl-(1?4)-O-
D
D
D
At first, 5.48 g (4.37 mmol) of tetrasaccharide 3 was suspended
in 45 mL of 33% HBr in HAcO. The system was protected with a
CaCl2 tube and was stirred vigorously for 30 min. The resulting
solution was poured onto 300 mL of ice/water and the precipitated
solid was filtered and redissolved in 150 mL of CHCl3. The filtrate
was extracted with 3 ꢂ 150 mL of CHCl3. The organic layers were
combined, washed with 4 ꢂ 150 mL of saturated aqueous NaHCO3,
and dried over MgSO4. The solvent was distilled under reduced
pressure and the glycosyl bromide was dried under vacuum. Com-
pound 4 was obtained as a white solid and was used in the next
synthetic step without further purification.
as a 4:1 mixture of b:
a
anomers. 1H NMR (CDCl3): dH = 6.23
(dd, J1eq,2ax = 3.0 Hz, J(1eq,2eq) = 1.0 Hz, 0.2H; H-1eqI), 5.73 (dd,
J1ax,2ax = 9.5 Hz, J1ax,2eq = 2.0 Hz, 0.8H; H-1axI), 5.16–3.56 (m,
26H; H-1II–IV H-2II–IV H-3I–IV H-4I–IV, H-5I–IV H-6I–IV), 2.15–
, , , ,
1.97 (m, 40H; CH3CO, H-2eqI), 1.90-1.70 (m, 1H; H-2axI); 13C
NMR (CDCl3): dC = 170.7-169.0 (CH3CO), [100.8, 100.5, 98.9,
91.2] (C-1I–IV), [77.4, 76.2, 76.1, 75.8, 72.9, 72.8, 72.7, 72.6,
72.4, 72.0, 71.7, 71.6, 71.5, 68.4, 67.7] (C-2II–IV, C-3I–IV, C-4I–IV
,
C-5I–IV), [62.3, 62.1, 61.7, 61.5] (C-6I–IV), 34.5 (C-2I), 21.0-20.5
(CH3CO).
Next, 2.36 g (9.48 mmol) of Cp2TiCl2 and 1.00 g (18.18 mmol) of
Mn powder were added to the glycosyl bromide 4. The reaction
flask was sealed under an N2 atmosphere. Then, 35 mL of anhy-
drous THF was added with a syringe and the solution was stirred
for 13 h. The reaction mixture was filtered over Celite and collected
over 30 g of silica gel. The solvent was distilled off and the silica
was dried under vacuum and charged in a chromatographic col-
umn, which was eluted with cyclohexane–ethyl acetate 2:1?3:4
to yield 2.49 g (2.19 mmol, 50%) of glycal 5.
4.1.4. 4-Nitrophenyl O-(2,3,4,6-tetra-O-acetyl)-b-
anosyl–(1?4)-O-(2,3,6-tri-O-acetyl)-b- -glucopyranosyl-(1?4)-
O-(2,3,6-tri-O-acetyl)-b- -glucopyranosyl-(1?3)-4,6-di-O-
acetyl-2-deoxy-b- -glucopyranoside 9
At first, 2.13 mg (1.78 mmol) of tetrasaccharide 7 was added to
15 mL of freshly distilled CH2Cl2 and the resulting solution was
cooled to 0 °C. Two hundred and seventy microliters (1.98 mmol)
of iodotrimethylsilane were added and the mixture was stirred at
0 °C for 45 min. After evaporation of the solvent under reduced pres-
sure, the residue was dissolved in 10 mL of anhydrous toluene and
the solvent was evaporated again. The orange oil thus obtained
D-glucopyr-
D
D
D
1H NMR (CDCl3): dH = 6.45 (dd, J1,2 = 6.5, J1,3 = 1.0, 1H; H-1I),
[5.23–4.80, 4.59–3.54] (m, 24H; H-2I–IV, H-3I–IV, H-4I–IV, H-5I–IV
,
H-6I–IV), 4.65 (d, J1,2 = 8.0, 1H; H-1II), 4.47-4.48 (2d, J1,2 = 8.0, 2H;
H-1III, IV), 2.15–1.97 (12s, 36H; CH3CO); 13C NMR (CDCl3):
dC = 170.3–169.0 (CH3CO), 144.7 (C-1I), [99.4, 99.2, 98.1, 97.4] (C-
2I, C-1II–IV), [76.3, 76.0, 73.2, 72.3, 72.2, 71.9, 71.8, 71.4, 71.3,
71.1, 71.0, 70.4, 69.8, 67.7, 67.1] (C-2 II–IV, C-3 I–IV, C-4 I–IV, C-5 I–IV),
[62.2, 62.0, 61.4, 61.0] (C-6 I–IV), 20.5–20.2 (CH3CO).
(the
THF and added, under a nitrogen atmosphere, to a 15 mL anhydrous
THF solution of 401 mg (2.88 mmol) 4-nitrophenol, 430
a-glycosyl iodide 8) was redissolved in 15 mL of anhydrous
lL
(2.17 mmol) of 15-crown-5, and 2.20 mL sodium hexamethyldisi-
lazide (1 M in THF). After stirring for 45 min, the mixture was diluted
with 100 mL of CHCl3 and washed with 4 ꢂ 100 mL of aqueous 1 N
NaOH and 100 mL of saturated aqueous NaCl. The organic layer
was dried over MgSO4, the solvent was evaporated, and the residue
was purified by flash chromatography (CHCl3–ethyl acetate 3:2), to
yield 390 mg (0.31 mmol) of 9. Other fractions containing 9 were
purified again by flash chromatography, to yield an additional
291 mg (0.23 mmol) of 9, resulting in a total yield of 30% from 7.
1H NMR (CDCl3): dH = 8.20–7.08 (2d, J = 9.5 Hz, 4H; C6H4NO2),
5.30 (dd, J1,2ax = 8.5 Hz, J1,2eq = 2.5 Hz, 1H; H-1I), 5.19–3.54 (m,
26H; H-1II–IV, H-2II–IV, H-3I–IV, H-4I–IV, H-5I–IV, H-6aI–IV, H-6bI–IV),
2.45 (ddd, Jgem = 13.0 Hz, J2eq,3 = 4.5 Hz, J1,2eq = 2.0, 1H; H-2eqI),
2.15-1.98 (m, 37H; CH3CO, H-2axI); 13C NMR (CDCl3): dC = 170.5-
169.1 (CH3CO), 161.3 (C-1), 142.7 (C-4), 125.7 (C-3, C-5), 116.2
4.1.2. O-(2,3,4,6-Tetra-O-acetyl)-b-
(2,3,6-tri-O-acetyl)-b- -glucopyranosyl-(1?4)-O-(2,3,6-tri-O-
acetyl)-b- -glucopyranosyl-(1?3)-4,6-di-O-acetyl-2-deoxy-b-
glucopyranose 6
D-glucopyranosyl-(1?4)-O-
D
D
D-
A solution of 1.83 g (5.74 mmol) of Hg(AcO)2 in 15 mL of water
at 0 °C was added to a solution of 2.16 g (1.90 mmol) of the glycal 5
in 60 mL of THF at 0 °C. The resulting yellow suspension was stir-
red at 0 °C for 45 min, then it was diluted with 225 mL of water at
0 °C and 430 mL (11.4 mmol) of NaBH4 was added to the mixture.
It was stirred for 1 min observing the appearance of a gray solid,
and it was neutralized adding crushed CO2. The reaction mixture
was extracted with 4 ꢂ 100 mL of AcOEt and the combined organic
layers were washed with 4 ꢂ 100 mL of saturated aqueous NaCl,
dried over MgSO4, and the solvent was distilled under vacuum.
The crude reaction mixture was purified by flash chromatography
(C-2, C-6), [100.8, 100.5, 99.0, 96.0] (C-1I–IV), 76.2-67.6 (C-2II–IV
,
C-3I–IV, C-4I–IV, C-5I–IV), 62.7-61.4 (C-6I–IV), 34.7 (C-2I), 20.8-20.4
(CH3CO).
(cyclohexane–ethyl acetate 3:2?1:3) and 1.03 g (891 lmol, 47%)
4.1.5. 4-Nitrophenyl O-b-
glucopyranosyl-(1?4)-O-b-
-glucopyranoside 2
At first, 403 mg (0.32 mmol) of 9 was suspended in 12 mL of
freshly distilled MeOH, and then 2.2 mL of a 0.165 M solution of
D
-glucopyranosyl-(1?4)-O-b-
D-
of 6 was obtained as a white solid. 1H NMR (CDCl3): dH = 5.41 (s,
D-glucopyranosyl-(1?3)-2-deoxy-b-
1H; H-1I), 5.15–3.52 (m, 26H; H-1II–IV, H-2II–IV, H-3I–IV, H-4I–IV, H-
D
5
I–IV, H-6I–IV), 2.15–1.97 (m, 38H; CH3CO, H-2Ia, H-2Ib); 13C NMR
(CDCl3): dC = 170.9–169.1 (CH3CO), [100.8, 100.5, 99.3] (C1II–IV),

854
M. Abel et al. / Tetrahedron: Asymmetry 20 (2009) 851–854
10. Vocadlo, D.; Davies, G. J. Curr. Opin. Chem. Biol. 2008, 12, 539–555.
11. Burmeister, W. P.; Cottaz, S.; Driguez, H.; Iori, R.; Palmieri, S.; Henrissat, B.
Structure 1997, 5, 663–675.
12. Sidhu, G.; Withers, S. G.; Nguyen, N. T.; McIntosh, L. P.; Ziser, L.; Brayer, G. D.
Biochemistry 1999, 38, 5346–5354.
13. Juers, D. H.; Heightman, T. D.; Vasella, A.; McCarter, J. D.; MacKenzie, L.;
Withers, S. G.; Matthews, B. W. Biochemistry 2001, 40, 14781–14791.
14. Planas, A. Biochim. Biophys. Acta 2000, 1543, 361–382; Viladot, J. LL.; Canals, F.;
Batllori, X.; Planas, A. Biochem. J. 2001, 355, 79–86; Faijes, M.; Pérez, X.; Pérez,
O.; Planas, A. Biochemistry 2003, 42, 13304–13318; Gaiser, O. J.; Piotukh, K.;
Ponnuswamy, M. N.; Planas, A.; Borriss, R.; Heinemann, U. J. Mol. Biol. 2006,
357, 1211–1225; Biarnés, X.; Nieto, J.; Planas, A.; Rovira, C. J. Biol. Chem. 2006,
281, 1432–1441.
15. Malet, C.; Vallés, J.; Bou, J.; Planas, A. J. Biotechnol. 1996, 48, 209–219.
16. Malet, C.; Planas, A. Biochemistry 1997, 36, 13838–13848; Planas, A.; Abel,
M.; Millet, O.; Palasí, J.; Pallarés, C.; Viladot, J. Ll. Carbohydr. Res. 1998, 310,
53–64.
NaOMe in MeOH was added. After stirring with exclusion of light
and moisture for 13.5 h, the precipitated solid was filtered and
dried under vacuum to yield 215 mg (0.28 mmol, 88%) of 2. 1H
NMR (DMSO-d6): dH = 8.22–7.23 (2d, J = 9.5 Hz, 4H; C6H4NO2),
5.49 (dd, J1,2ax = 9.0 Hz, J1,2eq = 1.0 Hz, 1H; H-1I), [4.39, 4.33, 4.24]
(3d, J1,2 = 8.0 Hz, 3H; H-1II–IV), 3.85–2.96 (m, 23H; H-2II–IV, H-3I–IV
,
H-4I–IV
, , ,
H-5I–IV H-6aI–IV H-6bI–IV), 2.45 (ddd, Jgem = 11.0 Hz,
J2eq,3 = 4.5 Hz, J1,2eq = 1.0 Hz, 1H; H-2eqI), 1.67 (ddd, Jgem = 11.0 Hz,
J1,2ax = J2ax,3 = 9.5 Hz, 1H; H-2axI); 13C NMR (DMSO-d6): dC = 161.7
(CAr-10), 141.7 (CAr-40), 125.7 (CAr-30, CAr-50), 116.5 (CAr-20, CAr-60),
[103.2, 102.8, 100.9, 96.2] (C-1I–IV), [80.6, 80.4, 78.4, 77.1, 76.8,
II–IV
76.5, 74.9, 74.8, 74.8, 74.8, 73.2, 73.0, 72.7, 70.0, 68.6] (C-2
,
C-3 I–IV, C-4 I–IV, C-5 I–IV), [61.0, 60.5, 60.4, 60.4] (C-6 I–IV), 36.1
(C-2I).
17. Thiem, J.; Klaffke, W.. In Topics in Current Chemistry; Springer: Berlin,
Heidelberg, 1990; Vol. 154, pp 285–332.
18. Kirschning, A.; Bechtold, A. F. W.; Rohr, J. Top. Curr. Chem. 1997, 188, 1–84.
19. Marzabadi, C. H.; Franck, R. W. Tetrahedron 2001, 56, 8385–8417.
20. Beaver, M. G.; Billings, S. B.; Woerpel, K. A. Eur. J. Org. Chem. 2008, 771–781.
21. Pongdee, R.; Wu, B.; Sulikowski, G. A. Org. Lett. 2001, 3, 3523–3525.
22. Binkley, R. W.; Koholic, D. J. J. Org. Chem. 1989, 54, 3577–3581.
23. Zhou, M.; O’Doherty, G. A. J. Org. Chem. 2007, 72, 2485–2493.
24. Crich, D.; Ritchie, T. J. J. Chem. Soc., Perkin Trans. 1 1990, 945–954.
25. Ramesh, S.; Kaila, N.; Grewal, G.; Franck, R. W. J. Org. Chem. 1990, 55, 5–7.
26. Gervay, J.; Danishefsky, S. J. J. Org. Chem. 1991, 56, 5448–5451.
27. Roush, W. R.; Lin, X.-F. J. Am. Chem. Soc. 1995, 117, 2236–2250.
28. Franck, R. W.; Marzadabi, C. H. J. Org. Chem. 1998, 51, 3950–3954.
29. Lam, S. N.; Gervay-Hague, J. Org. Lett. 2003, 5, 4219–4222.
30. Pollon, J. H.; Llewellyn, G.; Williams, J. M. Synthesis 1989, 758–759.
31. Czuk, R.; Fürstner, A.; Glänzer, B. I.; Weidmann, H. J. Chem. Soc., Chem. Commun.
1986, 1149–1150.
Acknowledgment
This work was supported by Grant BIO2007-67904-C02-02
from the Ministry of Science and Innovation, Spain.
References
1. Roeser, K.-R.; Legler, G. Biochim. Biophys. Acta 1981, 657, 321–333.
2. Mega, T.; Matsushima, Y. J. Biochem. (Tokio) 1983, 94, 1637–1647.
3. Rivera-Sagrero, A.; Cañada, F. J.; Nieto, O.; Jiménez-Barbero, J.; Martín-Lomas,
M. Eur. J. Biochem. 1992, 209, 415–422.
4. White, A.; Tull, D.; Johns, K.; Withers, S. G.; Rose, D. R. Nat. Struct. Biol. 1996, 3,
149–154.
5. Notenboom, V.; Birsan, C.; Nitz, M.; Rose, D. R.; Warren, R. A. J.; Withers, S. G.
Nat. Struct. Biol. 1998, 5, 812–818.
6. Zechel, D. L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11–18.
7. Lee, Y. C. Biochem. Biophys. Res. Commun. 1969, 35, 161–167.
8. Lehmann, J.; Schröter, E. Carbohydr. Res. 1972, 23, 359–368.
9. Vasella, A.; Davies, G. J.; Böhm, M. Curr. Opin. Struct. Biol. 2002, 6, 619–629.
32. Maran, F.; Vianello, E. Electrochim. Acta 1989, 34, 587–589.
33. Hansen, T.; Krintel, S. L.; Daasbjerg, K.; Skrydstrup, T. Tetrahedron Lett. 1999, 40,
6087–6090.
34. Bettelli, E.; Cherubini, P.; D’Andrea, P.; Passacantilli, P.; Piancatelli, G.
Tetrahedron 1998, 54, 6011–6018.
35. Roush, W. R.; Li, X. F. J. Am. Chem. Soc. 1995, 117, 2236–2250.