Effects of sugar functional groups, hydrophobicity, and fluorination on carbohydrate-DNA stacking interactions in water

Article abstract of DOI:10.1021/jo402700y

Carbohydrate-aromatic interactions are highly relevant for many biological processes. Nevertheless, experimental data in aqueous solution relating structure and energetics for sugar-arene stacking interactions are very scarce. Here, we evaluate how structural variations in a monosaccharide including carboxyl, N-acetyl, fluorine, and methyl groups affect stacking interactions with aromatic DNA bases. We find small differences on stacking interaction among the natural carbohydrates examined. The presence of fluorine atoms within the pyranose ring slightly increases the interaction with the C-G DNA base pair. Carbohydrate hydrophobicity is the most determinant factor. However, gradual increase in hydrophobicity of the carbohydrate does not translate directly into a steady growth in stacking interaction. The energetics correlates better with the amount of apolar surface buried upon sugar stacking on top of the aromatic DNA base pair.

Full text of DOI:10.1021/jo402700y

Article
pubs.acs.org/joc
Effects of Sugar Functional Groups, Hydrophobicity, and Fluorination
on Carbohydrate−DNA Stacking Interactions in Water
Ricardo Lucas,† Pablo Penalver,† Irene Gomez-Pinto,‡ Empar Vengut-Climent,† Lewis Mtashobya,§
́
̃
Jonathan Cousin,§ Olivia S. Maldonado,† Violaine Perez,† Virginie Reynes,† Anna Avino,∥ Ramon Eritja,∥
́
́
̃
Carlos Gonzalez,‡ Bruno Linclau,§ and Juan C. Morales*,†
†Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC−Universidad de Sevilla, 49 Amer
́
́
ico Vespucio,
41092, Sevilla, Spain
‡Instituto de Química Física “Rocasolano”, CSIC, C. Serrano 119, 28006 Madrid, Spain
§Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom
∥Instituto de Química Avanzada de Cataluna (IQAC), CSIC, CIBER−BBN Networking Centre on Bioengineering, Biomaterials and
̃
Nanomedicine, Jordi Girona 18-26, E-08034 Barcelona, Spain
S
* Supporting Information
ABSTRACT: Carbohydrate−aromatic interactions are highly relevant for
many biological processes. Nevertheless, experimental data in aqueous
solution relating structure and energetics for sugar−arene stacking
interactions are very scarce. Here, we evaluate how structural variations in
a monosaccharide including carboxyl, N-acetyl, fluorine, and methyl groups
affect stacking interactions with aromatic DNA bases. We find small
differences on stacking interaction among the natural carbohydrates
examined. The presence of fluorine atoms within the pyranose ring slightly
increases the interaction with the C−G DNA base pair. Carbohydrate
hydrophobicity is the most determinant factor. However, gradual increase in
hydrophobicity of the carbohydrate does not translate directly into a steady
growth in stacking interaction. The energetics correlates better with the
amount of apolar surface buried upon sugar stacking on top of the aromatic DNA base pair.
A model based on carbohydrate−oligonucleotide conjugates
(COCs) has been employed by our group to study
carbohydrate−aromatic stacking interactions. We calculated
monosaccharide−phenyl interactions using a double dangling
motif at the 5′-end of a self-complementary CGCGCG
sequence.11 Energies of −0.15 to −0.4 kcal mol−1 were found
depending on the stereochemistry and number of hydroxyl
groups of the sugar. We have also observed mono- and
disaccharide stacking on top of DNA base pairs in COCs where
the sugar is directly linked to the 5′-end to DNA via an
ethylene glycol spacer.12 Extra stabilization of the DNA duplex
due to sugar stacking was observed on sequences with terminal
C−G and G−C base pairs (e.g., up to −0.25 kcal mol−1 for
glucose/DNA unit and −0.45 kcal mol−1 for cellobiose/DNA
unit). However, no stabilization was found on top of terminal
A−T or T−A base pairs most probably due to the enhanced
entropic cost of reducing the breathing of these more flexible
terminal pairs.
INTRODUCTION
■
Molecular interactions between biomolecules rule most of the
biological processes inside the cell. A detailed understanding of
these interactions provides information on these processes, and
also allows their exploitation in a variety of applications such as
in protein engineering or drug design. Stacking between
aromatics is among the most studied interactions, whereas full
comprehension of carbohydrate−aromatic stacking interactions
is still to be reached.1 This binding motif is commonly found in
carbohydrate−protein interactions such as in lectins2 and is also
found in aminoglycoside-RNA recognition.3
Approaches to study carbohydrate−aromatic stacking inter-
actions include molecular biology tools,4 NMR spectroscopy,5
IR spectroscopy,6 computational methods,7 and model systems
based in supramolecular architectures8 or in biomolecules.9
These studies have revealed that the hydrophobic effect and
CH−π interactions comprising dispersion and electrostatic
forces play a critical role. A recent work by Chen et al.10 has
used an enhanced aromatic sequon (sequence of amino acids
with an attached oligosaccharide) to study the relative
contributions of each of these forces. The authors have found
that the hydrophobic effect contributes significantly to
carbohydrate−aromatic stacking and is supplemented by
CH−π interactions with a dominating dispersive component.
In an effort to increase the energetics of carbohydrate−DNA
stacking, we synthesized COCs containing hydrophobic
versions of natural sugars (permethylated glucose or cellobiose)
Received: December 5, 2013
Published: February 19, 2014
© 2014 American Chemical Society
2419
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
Figure 1. Description of the oligonucleotide conjugates under study. (a) Schematic drawing of the dangling-ended DNA designed to study
carbohydrate−DNA interactions. (b) Enlarged view of the dangling-end area of a monosaccharide−oligonucleotide conjugate. (c) Carbohydrate−
−
oligonucleotide conjugates included in the study. R = −OCH2CH2−OPO2 −CGCGCG.
−
Figure 2. Top view of self-complementary DNA conjugates forming a double helix, permethylated glucose−CH2CH2−OPO2 −CGCGCG (left)
−
and permethylated glucose−CH2CH2−OPO2 −AGCGCT (right). The apolar carbohydrate stacks on top of a C−G base pair (left) and the apolar
carbohydrate stacks on top of an A−T base pair (right).
at the edge.13 In this case, the stability of the DNA double helix
increased significantly (e.g., up to −0.8 kcal mol−1 for glc(Me)/
DNA unit and −0.9 kcal mol−1 for cellob(Me)/DNA unit).
Additionally, these apolar carbohydrates were able also to
stabilize dsDNA with A−T or T−A terminal base-pairs. We
postulate that CH−π interactions together with the higher
hydrophobicity imparted by the methyl groups are responsible
for duplex stabilization.
In this work we decided to deepen our understanding
carbohydrate−DNA stacking interactions by studying three
different groups of carbohydrates in our dangling end COC
model (Figure 1). First, we studied a group of COCs
containing natural monosaccharides 5−8 (rhamnose, xylose,
N-acetylglucosamine, and glucuronic acid) to explore the
relevance of other functionalities on the pyranose. Second,
the influence on carbohydrate−DNA stacking of increasing
hydrophobicity in the pyranose unit was studied with a series of
COCs with partially hydrophobic glucose units 9−12. We have
observed in the NMR structures of permethylated glucose−
DNA conjugates (Figure 2) that one or two methyl groups
apparently do not participate contacting the DNA base pair
during stacking. The question arising was whether a gradual
increase in hydrophobicity of the sugar will gradually increase
stacking or whether the hydrophobic area contacting the
aromatic DNA bases will be more determinant. Finally, we
selected a group of COCs with fluorinated sugars 13−17 in
order to study the influence of this electron-withdrawing group
in the stacking interaction. At the same time, the reduced
hydrophilicity upon OH → F change could also have a role in
the carbohydrate−DNA interactions.
2420
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
corresponding trichloroacetimidate glycosyl donor 32.16 When
ethylene glycol was used as the glycosyl acceptor, very low
yields of glycosylated product were obtained. Then, 2-
benzyloxyethanol was reacted with 32 to give 33 (30% yield)
and subsequent hydrogenation resulted in alcohol 33 (46%
yield). Synthesis of the corresponding phosphoramidite
compound 35 was carried out as described above.
The synthesis of 6-O-acetyl-2,3,4-O-trimethyl glucose
phosphoramidite 41 started by differentiating the primary
hydroxyl group of 2-benzyloxyethyl β-D-glucopyranoside 3613
with a TBDPS group (Scheme 2). Further methylation of the
remaining OH groups, hydrogenation and phosphoramidite
synthesis yielded derivative 41. In the case of 2,3-O-diacetyl-
4,6-O-dimethyl glucose phosphoramidite 44 its preparation was
carried out from the reported 1,2,3-O-triacetyl-4,6-O-dimethyl
α,β-glucopyranoside 4217 by reaction of its corresponding α-
bromo derivative with ethylene glycol. Then, hydrogenation
and phosphoramidite preparation in similar conditions as
described above resulted in compound 44.
RESULTS AND DISCUSSION
■
Synthesis of Carbohydrate−Oligonucleotide Conju-
gates. The preparation of carbohydrate−oligonucleotide
conjugates was carried out using standard solid phase
automated oligonucleotide synthesis as reported previously.11,12
Different monosaccharides were attached via an ethylene glycol
spacer to the 5′-end of the self-complementary CGCGCG
sequence using the corresponding protected carbohydrate
phosphoramidite derivatives. Their synthesis was carried out
by glycosylation of the spacer followed by standard
phosphoramidite preparation (Scheme 1).
Scheme 1. Synthesis of Rhamnose, Xylose, 6-Deoxyglucose,
N-Acetylglucosamine and Glucuronate Methyl Ester
Phophoramidites 20, 23, 26, 31, and 35
a
Several mono- and difluorinated carbohydrate phosphor-
amidite derivatives were synthesized from the corresponding
acetylated α-bromide compounds (Scheme 3).18 Again classical
Koenigs−Knorr glycosylation was used and the alcohol
derivatives were obtained in moderate to good yields (44−
78%). The phosphoramidite derivatives of 3-fluoro-3-deoxy-
glucose 47, 4-fluoro-4-deoxy-glucose 50, 6-fluoro-6-deoxy-
glucose 53, 4-fluoro-4-deoxy-galactose 56, and 4,6-difluoro-
4,6-dideoxy-galactose 59 were synthesized using the same
reaction conditions used previously.
After standard solid phase automated oligonucleotide
synthesis, the resulting carbohydrate oligonucleotide conjugates
were cleaved from the resin, deprotected with ammonia, HPLC
purified, and characterized by MALDI-TOF mass spectrometry
(Supporting Information). In the case of glucuronic acid DNA
conjugate 8, preliminary treatment with Na2CO3 in MeOH/
H2O (2:1) for 24 h was carried out followed by normal
treatment with ammonia and HPLC purification.
Energetics of Carbohydrate−DNA Interactions. DNA
duplex stability was measured by UV-monitored thermal
denaturation experiments in a pH 7.0 phosphate buffer
containing 1 M NaCl. Thermodynamic parameters were
calculated from melting curve fitting and from van’t Hoff
plots (1/Tm versus ln[conjugate]).19 Thermodynamic param-
eters for the carbohydrate−oligonucleotide conjugates contain-
ing natural sugars at the 5′-end 1−8 are shown in Table 1. It is
important to clarify that the thermodynamic results obtained
are for each COC double helix that incorporates two
carbohydrate−DNA binding motifs within its structure. We
observed previously that the presence of glucose, galactose, and
fucose at the edge stabilized the DNA duplex by 3.1−3.5 °C
and by 0.3−0.6 kcal·mol−1.12 DNA stabilization by xylose and
N-acetylglucosamine is in the same range as for the previous
monosaccharides, whereas rhamnose and glucuronic acid are
less stabilizing, possibly due to the presence of an axial OH and
a negative charge in the pyranose, respectively.
a
Reagents and conditions: (a) ethylene glycol, Ag2CO3, CH2Cl2; (b)
2-cyanoethyl-N,N′-diisopropyamino-chlorophosphoramidite, DIPEA,
CH2Cl2; (c) 2-benzyloxyethanol, Ag2CO3, CH2Cl2; (d) MeNH2,
THF then pyridine/acetic anhydride, DMAP; (e) H2, Pd(OH)2,
EtOAc/MeOH (1:1); (f) ethylene glycol, BF3·OEt2, CH2Cl2.
In the case of the rhamnose, xylose, and 6-deoxyglucose,
their sugar phosphoramidites 20, 23, and 26 were synthesized
by attachment of ethylene glycol by Koenigs−Knorr glyco-
sylation with the corresponding acetobromosugars.11,14 The
promoter used was silver carbonate, and the isolated yields
were between 44 to 83% (Scheme 1). Reaction of the obtained
alcohols with 2-cyanoethyl-N,N′-diisopropylamino-chlorophos-
phoramidite in the presence of base resulted in the
carbohydrate phosphoramidite derivatives 20, 23, and 26 in
high yields (75−90%).
The N-acetylglucosamine phosphoramidite 31 was synthe-
sized by glycosylation of 2-benzyloxyethanol with acetobromo
N-phthalimide glucose 27.15 Next, phthalimide deprotection
with methylamine in THF and acetylation yielded the
corresponding N-acetamido derivative 29. Finally, hydro-
genation of the benzyl protecting group and phosphoramidite
incorporation produced GlcNAc derivative 31. Glucuronate
methyl ester phosphoramidite 35 was synthesized from the
In the series of hydrophobic glucose DNA conjugates 9−12,
all carbohydrates increased DNA stability with respect to the
natural glucose DNA conjugate 2 (Table 2). Whereas COC
double helix containing 6-deoxyglucose units showed a small
increase in Tm of 0.7 °C and 0.2 kcal·mol−1 with respect to
glucose, the other apolar sugar DNA conjugates containing 4,6-
dimethylated glucose, 2,3,4-trimethylated glucose, and 2,3,4,6-
2421
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
a
Scheme 2. Synthetic Route for the Preparation of Partially Methylated Glucose Phophoramidites 41 and 44
a
Reagents and conditions: (a) TBDPSCl, DIPEA, NEt3, DMF; (b) NaH, MeI, DMF; (c) TBAF 1.0 M, THF then pyridine/acetic anhydride,
DMAP; (d) H2, Pd(OH)2, EtOAc/MeOH (8:1); (e) 2-cyanoethyl-N,N′-diisopropyamino-chlorophosphoramidite, DIPEA, CH2Cl2; (f) HBr, AcOH,
CH2Cl2 then ethylene glycol, Ag2CO3, CH2Cl2.
tetramethylated glucose derivatives were more stable than 2 by
3.7−4.7 °C and by 0.7−0.9 kcal·mol−1.
Scheme 3. Synthetic Route for the Preparation of Fluoro
Monosaccharide Phophoramidites 47, 50, 53, 56, and 59
a
It is interesting to observe that the large increase in DNA
stability from the dimethylated glucose does not correlate with
the gradual increase in hydrophobicity in this series as can be
deduced from the log P values of the glucose derivatives (Table
3). In fact, it does not correlate either with the increase in
surface area of the apolar sugars. We estimated the actual
surface area buried as the result of the sugar stacking on top of
the C−G final DNA base pair for each of the glucose
derivatives. A good correlation between the buried surface and
the DNA stability is found since adding third and fourth methyl
groups to the glucose unit does not increase the stacking area in
the conjugate but slightly reduces it. In fact, a graph plotting
free energy of COCs (containing glucose, 2, and hydrophobic
sugars, 9−12) versus stacking area showed a good linear fit (the
coefficient of determination is R2 = 0.9193; see Figure S4,
Supporting Information). This seems to indicate that the free
energy of the conjugate is also directly related with the number
of water molecules displaced by the hydrophobic carbohydrates
from the area of contact with the DNA base pair and not with
the hydrophobicity of the carbohydrates. A similar result was
found by Kool et al. when studying π−π aromatic interactions
with nonpolar DNA bases (e.g., benzene, naphthalene, pyrene,
etc.) at the edge of dsDNA.20 Log P values were a poor
predictor of stacking interaction, whereas surface area of
a
Reagents and conditions: (a) ethylene glycol, Ag2CO3, CH2Cl2; (b)
2-cyanoethyl-N,N′-diisopropyamino-chlorophosphoramidite, DIPEA,
CH2Cl2.
Table 1. Melting Temperatures and Thermodynamic Parameters for Carbohydrate Oligonucleotide Conjugate Duplexes
Containing Natural Carbohydrates and the CGCGCG Sequence
d
dangling moiety in
abc
−ΔS° (van’t Hoff)
−ΔH° (van’t Hoff)
−ΔG°37 (van’t Hoff)
ΔG°37 (fits)
ΔΔG°37
,
,
5′-CGCGCG
Tm (°C)
(cal/k mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(none) 1
40.9 0.4
44.0 0.7
44.4 0.4
44.4 0.4
42.7 0.2
43.6 0.4
44.6 0.3
114
140
113
136
137
156
136
5
4
8
3
7
9
7
43.5
52.1
43.5
51.1
50.9
57.2
50.8
2
2
3
1
2
3
3
8.2 0.3
8.7 0.3
8.5 0.5
8.8 0.2
8.6 0.4
8.8 0.6
8.7 0.5
8.2 0.1
8.7 0.0
8.7 0.1
8.8 0.0
8.5 0.0
8.6 0.1
8.7 0.0
−
e
β-D-glucose-C2 2
−0.5
−0.3
−0.6
−0.4
−0.6
−0.5
e
β-D-galactose-C2 3
e
β-L-fucose-C2 4
α-L-rhamnose-C2 5
β-D-xylose-C2 6
N-acetyl-β-D-glucosamine-
C2 7
β-D-glucuronic acid-C2 8
42.4 0.7
141
2
52.2
1
8.5 0.1
8.5 0.0
−0.3
a
b
c
C2 stands for −CH2−CH2−. Buffer: 10 mM Na phosphate, 1 M NaCl, pH 7.0. Average value of three experiments measured at 5 μM conc.
d
e
ΔΔG°37 = ΔGvh − ΔG (CGCGCG control). Data from Lucas et al.12
2422
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
Table 2. Melting Temperatures and Thermodynamic Parameters for Carbohydrate Oligonucleotide Conjugate Duplexes
Containing Hydrophobic Sugars and the CGCGCG Sequence
d
dangling moiety in
abc
−ΔS° (van’t Hoff)
−ΔH° (van’t Hoff)
−ΔG°37 (van’t Hoff)
ΔG°37 (fits)
ΔΔG°37
,
,
5′-CGCGCG
(none) 1
β-D-glucose-C2 2
Tm (°C)
(cal/k mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
40.9 0.4
44.0 0.7
44.7 0.2
47.7 0.2
48.2 0.1
114
140
145
5
4
5
43.5
52.1
53.8
58.3
54.7
2
2
2
4
4
8.2 0.3
8.7 0.3
8.9 0.3
9.6 0.8
9.4 0.8
8.2 0.1
8.7 0.0
8.8 0.0
9.4 0.1
9.4 0.1
−
e
−0.5
−0.7
−1.4
−1.2
β-D-6-deoxyglucose-C2 9
β-D-4,6-dimethyl-glc-C2 10
157 11
146 11
β-D-2,3,4-trimethyl-glc-C2
11
β-D-2,3,4,6-tetramethyl-glc-
48.7 0.2
147 10
55.1
4
9.4 0.6
9.4 0.1
−1.2
f
C2 12
a
b
c
C2 stands for −CH2−CH2−. Buffer: 10 mM Na phosphate, 1 M NaCl, pH 7.0. Average value of three experiments measured at 5 μM conc.
d
e
f
ΔΔG°37 = ΔGvh − ΔG (CGCGCG control). Data from Lucas et al.12 Data from Lucas et al.13
Table 3. Molecular Weight and Partition Coefficient Data for Dangling Moieties Studied
a
b
c
dangling moiety in 5′-CGCGCG
MW
calc log P
surface area (Å2)
S-area folded
S-area unfolded
stacking area (Å2)
glucose
2
194.2
178.2
222.2
236.3
250.3
−2.01
−1.16
−1.29
−0.93
−0.57
186
179
207
227
243
2918
2905
2946
2954
2981
3093
3082
3147
3143
175
177
201
189
189
6-deoxyglucose
9
4,6-dimethyl-glucose
2,3,4-trimethyl-glucose
2,3,4,6-tetramethyl-glucose
10
11
12
3170
Log P values were calculated using the Crippen’s fragmentation21 in the ChemBioDraw Ultra 11.0 software. Half of the calculated surface area of
base as Connolly accessible area using Chem 3D Pro software. Estimated as buried surface by subtracting the unfolded from the folded
a
b
c
carbohydrate−DNA base pair.
Table 4. Melting Temperatures and Thermodynamic Parameters for Carbohydrate Oligonucleotide Conjugate Duplexes
Containing Fluorosugars and the CGCGCG Sequence
d
dangling moiety in
−ΔS° (van’t Hoff)
−ΔH° (van’t Hoff)
−ΔG°37 (van’t Hoff)
ΔG°37 (fits)
ΔΔG°37
abc
, ,
5′-CGCGCG
Tm (°C)
(cal/k mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(none) 1
40.9 0.4
44.0 0.7
44.5 0.1
44.7 1.3
44.3 0.0
44.4 0.4
44.6 0.9
114
140
141
141
150
113
5
4
2
6
5
8
43.5
52.1
52.4
52.5
55.3
43.6
51.2
50.4
2
2
1
2
2
3
5
3
8.2 0.3
8.7 0.3
8.8 0.1
8.9 0.4
8.9 0.3
8.5 0.5
8.6 1.0
8.6 0.6
8.2 0.1
8.7 0.0
8.8 0.0
8.9 0.1
8.8 0.0
8.7 0.1
8.6 0.0
8.6 0.1
−
β-D-glc-C2 2
−0.5
−0.6
−0.7
−0.7
−0.3
−0.4
−0.4
3-F-β-D-glc-C2 13
4-F-β-D-glc-C2 14
6-F-β-D-glc-C2 15
β-D-gal-C2 3
4-F-β-D-gal-C2 16
138 14
135
4,6-diF-β-D-gal-C2 17
45.0 0.5
b
9
a
c
C2 stands for −CH2−CH2−. Buffer: 10 mM Na phosphate, 1 M NaCl, pH 7.0. Average value of three experiments measured at 5 μM conc.
ΔΔG°37 = ΔGvh − ΔG (CGCGCG control).
d
overlap (buried surface by aromatic stacking) was a much
better predictor.
since the values are within the experimental error. This is a
similar situation to that found when comparing glucose and
galactose.
In the series of fluorinated sugars 13−17, DNA stability of
the carbohydrate oligonucleotide conjugates showed a minor
increase with respect to glucose and galactose derivatives 2 and
3 (0.3−0.7 °C in Tm and 0.1−0.2 kcal·mol−1 in ΔG, see Table
4). The incorporation of one or two electron-withdrawing
agents such as fluorine atoms in the pyranose could modify
CH−π interactions between the pyranose ring and the aromatic
DNA bases, but this effect seems to have little influence in the
carbohydrate−aromatic stacking interaction observed. These
results fit well with those obtained recently by Asensio et al.22
in a dynamic combinatorial approach using a disaccharide
model system. The authors observe a minor strengthening of
the carbohydrate/aromatic forces by incorporating electron-
withdrawing substituents at the anomeric position (−F or
−CH2CF3).
Finally, a couple of explicit comparisons between the three
groups of monosaccharides may yield some information on the
relevance of different modifications in the carbohydrate when
stacking with DNA bases. If we compare glucose, 6-
deoxyglucose and 6-fluoroglucose, we observe that replacement
of the 6-OH with a proton or a fluorine atom yields only a
small increase in stability, probably due to the small decrease in
hydrophilicity with this modification. In contrast, a large
difference in energetics is observed between 4,6-difluoroga-
lactose and 4,6-dimethyl-glucose (the latest 1.0 kcal·mol−1 in
ΔG more stable). Bearing in mind the different stereochemistry
of the carbohydrate, the increased hydrophobic surface in the
apolar carbohydrate has much more influence in the stacking
interaction than increasing the polarity of the −CH− protons
of the pyranose by the presence of the fluorine atoms.
Structural Features of Carbohydrate Oligonucleotide
Conjugates. As in previous NMR studies in related
systems,12,13 no distortion of the DNA double helix due to
If we consider the relevance of stereochemistry of
fluorosugars in the interaction with the DNA base pair, for
example by comparison of 4-fluoroglucose (4FGlc) and 4-
fluorogalactose (4FGal), we observe no difference in energetics
2423
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
solvent was removed, and the crude product was purified by flash
column chromatography (hexane:ethyl acetate mixtures) to afford the
corresponding glycosylated ethylene glycol.
the presence of the fluorinated carbohydrates is observed. Two
dimensional NMR spectra were recorded for conjugates 13 to
16. Complete resonance assignment was performed, and we
found significant chemical shift differences with the control
duplex only for some protons of the terminal base pair.
Observed NOEs between the sugar units and the terminal C−
G base pair were very similar to those observed for the
corresponding nonfluorinated natural sugars (see Table S2,
Supporting Information). A structural model of conjugate 13
was built on the basis of the observed experimental NOEs (see
Figure S5, Supporting Information). The carbohydrate stacks
on top of the terminal C−G base pairs mainly through its alpha
face. The fluorine atom is oriented toward the solvent and does
not participate in the interaction with the DNA, explaining the
little effect of the fluorine substitutions in the thermal stability
of these conjugates. Consequently, we conclude that the
structures of the fluorinated conjugates are very similar to those
reported previously, and no further modeling was carried out.
2-Hydroxyethyl 2,3,4-tri-O-acetyl-β-L-rhamnopyranose (19).
2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl bromide 18 (972 mg, 2.76
mmol) was reacted following the general glycosylation procedure. The
crude product was purified by flash column chromatography
(hexane:ethyl acetate from 2:1 to 1:2) to afford 19 (592 mg, 64%)
22
1
as glassy solid: [α]D −3.3 (c 1 in CHCl3); H NMR (300 MHz,
CDCl3) δ ppm 5.26−5.19 (m, 1H, H2), 5.02−4.96 (t, J = 9.8 Hz, 1H,
H3), 4.72 (s, 1H, H1), 3.90−3.83 (m, 1H, H4), 3.74−3.69 (m, 3H, H5,
CH2), 3.51−3.55 (m, 2H, CH2), 2.08, 1.99, 1.92 (3s, 9H, 3 ×
OCOCH3), 1.17−1.15 (d, J = 8 Hz, 3H, CH3); 13C NMR (75 MHz,
CDCl3) δ = 170.1, 170.0, 169.9 (3 × CO), 97.6 (C1), 70.9 (C3), 69.7
(C2), 69.6 (CH2), 69.10 (C5), 66.4 (C4), 61.3 (CH2), 20.8, 20.7, 20.6
(3 × OCH3), 17.3 (CH3); HRMS (FAB+) Calcd. for C14H22O9Na (M
+ Na) 357.1162, found 357.1152.
2-Hydroxyethyl 2,3,4-tri-O-acetyl-β-D-xylopyranose (22). 2,3,4-
Tri-O-acetyl-α-D-xylopyranosyl bromide 21 (850 mg, 2.50 mmol) was
reacted following the general glycosylation procedure. The crude
product was purified by flash column chromatography (hexane:ethyl
acetate from 2:1 to 1:2) to afford 22 (540 mg, 66%) as glassy solid:
CONCLUSION
■
22
1
[α]D −48.5 (c 1 in CHCl3); H NMR (300 MHz, CDCl3) δ ppm
5.18−5.12 (t, J = 9.0 Hz, 1H, H3), 4.97−4.88 (m, 2H, H2, H4), 4.50−
4.48 (d, J = 7.2 Hz, 1H, H1), 4.14−4.08 (dd, J = 5.4/5.1 Hz, 1H, H5),
3.84−3.79 (m, 4H, 2 × CH2), 3.39−3.32 (dd, J = 5.3 Hz, 1H, H5′),
2.03−2.00 (3s, 9H, 3 × OCOCH3); 13C NMR (75 MHz, CDCl3) δ =
170.4, 170.3, 169.9 (3 × CO), 100.5 (C1), 72.0 (C3), 71.8 (C2), 71.3
(CH2), 69.1 (C4), 62.5 (CH2), 61.9 (C5), 21.0. 20.9 (OCCH3);
HRMS (FAB+) Calcd. for C13H20O9Na (M + Na) 343.0999, found
343.1005.
Evaluation of stacking of natural monosaccharides on top of a
DNA base pair in water using our COC model system have
shown small differences due to changes in sugar stereo-
chemistry and to the presence of typical functional groups such
as N-acetyl or carboxylic acid in the pyranose ring. At the same
time, replacement of a hydroxyl group with a fluorine atom
should increase −CH− polarization and local hydrophobicity,
but none of these aspects showed large differences on stacking
of the carbohydrate derivatives. Finally, increasing the hydro-
phobicity of the sugars through methylation exhibited a
considerable enhancement in carbohydrate aromatic stacking.
Nevertheless, it is important to emphasize that energetics seem
to correlate better with the amount of surface contact area
between the carbohydrate and the aromatic DNA base pair
than with a net increase in sugar hydrophobicity. These results
appear to point out the relevance of the number of water
molecules displaced from the surface of contact between the
carbohydrate and the aromatics.
2-Hydroxyethyl 2,3,4-tri-O-acetyl-β-D-6-deoxyglucopyranose
(25). 2,3,4-Tri-O-acetyl-6-deoxy-α-D-glucosylbromide 24 (0.5 g, 1.50
mmol) was reacted following the general glycosylation procedure. The
crude product was purified by flash column chromatography
(hexane:ethyl acetate from 1:1 to 1:2) to afford 25 (196 mg, 44%)
22
1
as glassy solid: [α]D −23.8 (c 1 in CHCl3); H NMR (300 MHz,
CDCl3) δ ppm 5.11 (t, J = 9.0 Hz, 1H, H3), 4.92 (dd, J = 9.6 Hz, 1H,
H2), 4.77 (t, J = 9.5 Hz, 1H, H4), 4.46 (d, J = 8.1 Hz, 1H, H1), 3.72−
3.69 (m, 2H, CH2), 3.57−3.52 (m, 1H, H5), 2.40 (s, 1H, OH), 1.98,
1.97, 1.93 (3 × OCOCH3), 1.18 (d, 3H, J = 6.3 Hz, CH3) ; 13C NMR
(75 MHz, CDCl3) δ = 170.2, 169.6, 169.5 (CO), 100.9 (C1), 73.2
(C4), 72.7 (C3), 72.1 (CH2), 71.7 (C2), 70.0 (C5), 61.7 (CH2), 20.7,
20.6, 20.5 (OCCH3), 17.3 (CH3); HRMS (FAB+) Calcd. for
C14H22O9Na (M + Na) 357.1162, found 357.1168.
EXPERIMENTAL SECTION
■
2-Benzyloxyethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-
glucopyranose (28). Ag2CO3 (1.38 g, 5.01 mmol) was added to a
solution of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyrano-
syl bromide 2715 (1 g, 2.0 mmol) and 2-benzyloxyethanol (2.85 mL,
20.0 mmoL) in anhydrous CH2Cl2 (12 mL). The reaction was stirred
for 16 h and the mixture was then filtered over Celite and washed with
CH2Cl2. The solvent were removed and the crude purified by flash
column chromatography (hexane:ethyl acetate from 4:1 to 1:1) to
afford 28 (800 mg, 70%) as glassy solid: [α]D22 +19.7 (c 1 in CHCl3);
1H NMR (300 MHz, CDCl3) δ ppm 7.74−7.71 (m, 2H, Harom,
Phth), 7.63−7.59 (m, 2H, Harom, Phth), 7.17−7.00 (m, 5H, Ph), 5.74
(dd, J = 9.0/10.8 Hz, 1H, H3), 5.39 (d, J = 8.4 Hz, 1H, H1), 5.11 (dd, J
= 9.0/10.0 Hz, 1H, H4), 4.32−4.22 (m, 2H, H2, H6), 4.17 (s, 2H,
CH2Ph), 4.09 (dd, J = 2.4/12.3 Hz, 1H, H6′), 3.92−3.77 (m, 2H, CH2,
H5), 3.69−3.60 (m, 1H, CH2), 3.42−3.39 (m, 2H, CH2), 2.03, 1.95,
1.80 (3s, 9H, 3 × OCOCH3); 13C NMR (75 MHz, CDCl3) δ = 170.7,
170.2, 169.5 (3 × CO), 138.0, 134.2, 131.4, 128.4, 128.3, 127.8, 127.4,
127.3, 123.5, 98.3 (C1), 72.9 (CH2), 71.8 (C5), 70.7 (C3), 69.3 (C4),
69.0, 68.7 (2 × CH2), 62.0 (C6), 54.6 (C2), 20.8. 20.6, 20.5 (OCCH3);
HRMS (FAB+) Calcd. for C29H31NO11Na (M + Na) 592.1795, found
592.1786.
General Methods. All chemicals were obtained and used without
further purification, unless otherwise noted. All reactions were
monitored by TLC on precoated silica gel 60 plates F254 and
detected by heating with 5% sulfuric acid in ethanol or Mostain (500
mL of 10% H2SO4, 25 g of (NH4)6Mo7O24·4H2O, 1 g of Ce(SO4)2·
4H2O). Products were purified by flash chromatography with silica gel
60 (200−400 mesh). Low resolution mass spectra were obtained on an
ESI/ion trap mass spectrometer. High resolution mass spectra were
obtained on an ESI/quadrupole mass spectrometer. NMR spectra
were recorded on a 300, 400, or 500 MHz [300 or 400 MHz (1H), 75
or 100 (13C)] NMR spectrometers, at room temperature for solutions
in CDCl3, D2O, or CD3OD. Chemical shifts are referred to the solvent
signal. 2D experiments (COSY, TOCSY, ROESY, and HMQC) were
done when necessary to assign the carbohydrate derivatives and the
conjugates. Chemical shifts are in ppm. In all experiments the 1H
carrier frequency was kept at the water resonance. Data were
processed using manufacturer software, raw data were multiplied by
shifted exponential window function prior to Fourier transform, and
the baseline was corrected using polynomial fitting.
Synthetic Procedures. General Procedure for Glycosylation of
Ethylene Glycol. Ag2CO3 (1.7 g, 6.25 mmol) was added to a solution
of the acetyl protected glycosylbromide (2.5 mmol) and ethylenglycol
(1.3 mL, 25 mmoL) previously dried over molecular sieves, in
anhydrous CH2Cl2 (10 mL). The reaction was stirred for 24 h; the
mixture was then filtered over Celite and washed with CH2Cl2. The
2-Benzyloxyethyl 3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-
glucopyranose (29). MeNH2 (40% aq, 3 mL) was added to a
solution of 28 (390 mg, 0.68 mmol) in THF (6 mL). The reaction was
stirred for 16 h. The solvent was then removed and the reaction
2424
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
mixture was coevaporated with dry toluene. The crude was dissolved
in dry pyridine (10 mL) and acetic anhydride (3 mL) and a catalytic
amount of DMAP was added. The reaction mixture was stirred at
room temperature for 24 h. The solvent was removed in vacuo and the
crude product was purified by flash column chromatography
(hexane:ethyl acetate from 1:4 to 0:1) to afford 29 (200 mg, 62%
two steps) as a brown solid: [α]D −24.5 (c 1 in MeOH); H NMR
(300 MHz, CDCl3) δ ppm 7.30−7.19 (m, 5H, Ph), 5.76 (d, J = 8.7 Hz,
1H, NH), 5.16 (t, J = 9.3 Hz, 1H, H3), 4.99 (t, J = 9.3 Hz, 1H, H4),
4.69 (d, J = 7.2 Hz, 1H, H1), 4.46 (s, 2H, CH2Ph), 4.21−4.18 (m, 1H,
H6), 4.03−3.92 (m, 1H, H6′), 3.95−3.50 (m, 6H, 2 × CH2, H2, H5),
1.99, 1.95, 1.94, 1.78 (3s, 12H, 3 × OCOCH3, N-COCH3); 13C NMR
(75 MHz, CDCl3) δ = 170.8, 170.7, 170.3, 169.4 (4 × CO), 138.0,
128.5, 127.8, 127.6 (Carom), 100.9 (C1), 73.1 (CH2), 72.6 (C3), 71.8
(C5), 69.3 (CH2), 68.8 (C4), 68.6 (CH2), 62.1 (C6), 54.5 (C2), 23.2
(NCOCH3), 20.8. 20.7, 20.6 (OCCH3); HRMS (FAB+) Calcd. for
C23H31NO10Na (M + Na) 504.1846, found 504.1852.
2-Hydroxyethyl 3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-
glucopyranose (30). A solution of compound 29 (180 mg, 0.37
mmol) in ethyl acetate-MeOH (1:1, 6 mL) and Pd(OH)2 in catalytic
amount was stirred under an atmosphere of hydrogen for 48 h. The
mixture was filtered off over Celite and solvents were removed. The
crude product was purified by silica gel column chromathography
using as eluent (hexane:ethyl acetate, from 1:4 to 0:1) to give 30 (120
mg, 83%): [α]D22 −2.4 (c 1 in CHCl3); 1H NMR (400 MHz, CD3OD)
δ ppm 5.22 (t, J = 9.6 Hz, 1H, H3), 5.00 (t, J = 9.6 Hz, 1H, H4), 4.70
(d, J = 8.4 Hz, 1H, H1), 4.29 (d, J = 4.8/12.4 Hz, 1H, H6), 4.15 (d, J =
2.0/12.4 Hz, 1H, H6′), 3.91−3.80 (m, 3H, H2, H5, OCH2), 3.70−3.64
(m, 3H, OCH2, CH2OH), 2.08, 2.02, 1.99, 1.93 (4s, 12H, 3 ×
OCOCH3, N-COCH3); 13C NMR (75 MHz, CDCl3) δ = 172.2,
170.9, 170.4, 169.9 (4 × CO), 100.8 (C1), 72.8 (C3), 71.5 (C5), 70.9
(CH2O), 68.8 (C4), 61.9 (C6), 60.8 (CH2OH), 54.1 (C2), 21.4, 19.3,
19.2, 19.1; HRMS (FAB+) Calcd. for C16H25NO10Na (M + Na)
414.1376, found 414.1375.
(0. 476 mL, 4.64 mmoL) were added to a cooled solution of 2-
benzyloxyethyl β-D-glucopyranoside 3613 (1.325 g, 4.22 mmol) in
anhydrous DMF under argon atmosphere. Then tert-butyldiphenylsilyl
chloride (1.21 mL, 4.64 mmoL) was slowly added to the mixture and
the reaction was left to at room temperature until starting material had
disappeared in 1h. Ethyl acetate (300 mL) was then added to the
mixture and the organic layer was washed with brine (2 × 100 mL),
dried with anhydrous Na2SO4 and concentrated to dryness. The crude
product was purified by silica gel column chromatography using ethyl
22
1
1
acetate 100% as eluent to give 37 (1.3 g, 57%) as a white solid: H
NMR (400 MHz, CDCl3) δ ppm 7.63−7.57 (m, 5H, Ph), 7.32−7.25
(m, 5H, Ph), 7.23−7.14 (m, 5H, Ph), 4.52−4.35 (m, 2H, CH2), 4.20
(d, J = 7.6 Hz, 1H, H1) 3.84 (m, 2H, CH2), 3.76 (dd, J = 10.9, 5.3 Hz,
1H), 3.64−3.36 (m, 5H, CH2, H5, H6, H6′), 3.35−3.25 (m, 2H, H2,
H), 0.95 (s, 9H, 3 × CH3isopropyl); 13C NMR (101 MHz, CDCl3) δ =
137.7 (Cq-arom), 135.7 (2 × Carom), 135.6 (2 × Carom),133.3(Carom),
129.8(2 × Carom), 128.5 (2 × Carom), 127.9 (2 × Carom), 127.8 (2 ×
Carom), 127.7 (4 × Carom), 102.6 (C1), 76.4(CH2), 75.4, 73.4, 73.1,
71.4, 69.0, 68.3, 64.5 (C2−C5, 2 × CH2), 26.8 (3 × CH3 iPr),
19.3(CCH3 iPr); HRMS (FAB+) Calcd. for C31H40O7Na Si (M + Na)
575.2441, found 575.2451.
2-Benzyloxyethyl 2,3,4-tri-O-methyl-6-tert-butyldiphenylsilyl-β-D-
glucopyranose (38). Sodium hydride (1.25g, 60% conc., 30.8 mmol)
was slowly added to a cooled (0 °C) solution of 2-benzyloxyethyl 6-O-
tert-butyldiphenylsilyl-β-D-glucopyranose (37) (2.13 g, 3.86 mmol) in
anhydrous DMF (20 mL). After stirring at 0 °C for 10 min under
argon atmosphere, methyl iodide (3.6 mL, 58 mmol) was added and
the reaction was left to process overnight at room temperature. The
reaction was quenched with 2-propanol and treated with 50 mL sat.
NH4Cl. The aqueous layer was extracted with ethyl acetate (2 × 50
mL). The organic layer was then washed with brine and Na2S2O3,
dried with anhydrous Na2SO4 and concentrated to dryness. The crude
product was purified by silica gel column chromatography
(hexane:ethyl acetate, from 4:1 to 2:1) to give 38 (1.5 g, 67%) as a
1
white solid: H NMR (400 MHz, CDCl3) δ ppm 7.68−7.59 (m, 5H,
2-Benzyloxyethyl-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosy-
luronate) (33). BF3·OEt2 (50 mL, 0.67 mmol) was added to a
solution of trichloroacetimidate 3216 (800 mg, 1.67 mmol) and 2-
benzyloxyethanol (356 mL, 2.50 mmol) in anhydrous CH2Cl2 (8 mL).
After stirring at room temperature for 1 h under argon atmosphere,
triethylamine (0.3 mL) was added. Solvents were removed and the
crude product was purified by silica gel column chromatography
(toluene:acetone, from 10:1 to 6:1) to give the 33 (240 mg, 30%) as
Ph), 7.33−7.14 (m, 10H, Ph), 4.49 (s, 2H, CH2), 4.22 (d, J = 7.6 Hz,
1H, H1) 3.95 (m, 1H), 3.79 (d, J = 2.8 Hz, 2H, CH2), 3.68−3.59 (m,
3H), 3.55 (s, 3H, OCH3), 3.52 (s, 3H, OCH3), 3.47 (s, 3H, OCH3),
3.27 (t, 1H,), 3.09 (ddd, J = 8.9, 5.7, 2.8 Hz, 2H), 2.97 (t, 1H, H2),
0.96 (s, 9H, 3 × CH3 iPr); 13C NMR (101 MHz, CDCl3) δ =, 138.4
(Cq-arom), 135.9 (2 × Carom), 135.6 (2 × Carom),133.8(Carom),
133.3(Carom), 129.6(2 × Carom), 128.4 (2 × Carom), 127.8 (2 ×
Carom), 127.72 (2 × Carom), 127.69 (2 × Carom), 127.6 (3 × Carom),
103.4 (C1), 86.6, 83.8 (C2), 79.1, 75.6, 73.2, 69.4, 68.7, 62.8, 60.9,
60.5(2 × C), 26.8 (3 × CH3 iPr), 19.4(CCH3 iPr); HRMS (FAB+)
Calcd. for C34H46O7NaSi (M + Na) 617.2911, found 617.2899.
2-Benzyloxyethyl 2,3,4-tri-O-methyl-6-O-acetyl-β-D-glucopyra-
nose (39). TBAF (1 M in THF, 4.38 mL, 4.38 mmol) was slowly
added to a solution of 2-benzyloxyethyl 2,3,4-tri-O-methyl-6-O-tert-
butyldiphenylsilyl-β-D-glucopyranose (38) (1.3 g, 2.19 mmol) in
anhydrous THF (10 mL). The reaction was stirred for 90 min under
argon atmosphere at room temperature. 50 mL of CH2Cl2 were added
to the reaction mixture and the organic layer was then washed with
brine and NH4Cl, dried with anhydrous MgSO4 and concentrated to
dryness. The crude product was purified by silica gel column
chromatography using as eluent (hexane:ethyl acetate, from 1:2 to
1:4) to give 2-benzyloxyethyl 2,3,4-tri-O-methyl-β-D-glucopyranose
(650 mg, 1.83 mmol, 83%) as a white solid. The product was then
solved in anhydrous pyridine (10 mL) and acetic anhydride (1.38 mL,
14.6 mmol) and dimethylaminopyridine (catalytic amount) was added
to the reaction mixture. The reaction was left to process overnight at
room temperature under argon atmosphere. CH2Cl2 was then added
to the reaction mixture (50 mL) and the organic layer was washed with
brine and HCl (5% v/v), dried with anhydrous Na2SO4 and
concentrated to dryness. The crude product was purified by silica
gel column chromatography (hexane:ethyl acetate, from 1:1 to 1:3) to
give 39 (705 mg, 97%) as a white solid: 1H NMR (400 MHz, CDCl3)
δ ppm 7.35−7.13 (m, 5H, Ph), 4.48 (s, 2H, CH2), 4.26 (d, J = 4.8 Hz,
1H, H1), 4.22 (d, 1H), 4.11 (dd, 1H), 3.99−3.86 (m, 1H), 3.66 (dd,
1H), 3.62−3.56 (m, 2H), 3.54 (s, 3H, OCH3), 3.50 (s, 3H, OCH3),
22
glassy solid: [α]D −20.1 (c 1 in CHCl3); 1H NMR (300 MHz,
CDCl3) δ ppm 7.35−7.28 (m, 5H, Ph), 5.27−5.23 (m, 2H, H4, H3),
5.05 (t, J = 8.4 Hz, 1H, H2), 4.67(d, J = 7.8 Hz, 1H, H1), 4.58−4.50
(m, 2H, CH2Ph), 4.06−3.96 (m, 2H, CH2, H5), 3.81−3.75 (m, 4H,
CH3O, CH2), 3.66−3.63 (m, 2H, CH2), 2.06, 2.03, 199 (3s, 9H, 3 ×
OCOCH3); 13C NMR (75 MHz, CDCl3) δ = 170.0, 169.3, 169.2,
167.1 (4 × CO), 137.9 (Cq-arom), 128.3, 127.5, 127.4 (Carom), 101.9
(C1), 73.1 (CH2), 72.4 (C5), 71.9 (C3), 71.0 (C2), 70.3 (C4), 69.9,
69.3 (2 × CH2), 52.7 (CH3O), 20.5. 20.4 (OCCH3); HRMS (FAB+)
Calcd. for C13H20O9Na (M + Na) 343.0999, found 343.1005.
2-Hydroxyethyl-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosylur-
onate) (34). A solution of compound 33 (480 mg, 1.024 mmol) in
ethyl acetate-MeOH (1:1, 10 mL) and Pd(OH)2 in catalytic amount
was stirred under an atmosphere of hydrogen for 18 h. The mixture
was filtered off over Celite and solvents were removed. The crude
product was purified by silica gel column chromathography using as
eluent (hexane:ethyl acetate, 1:2) to give 34 (180 mg, 46%) as a syrup:
22
1
[α]D −19.0 (c 1 in CHCl3); H NMR (500 MHz, CDCl3) δ ppm
5.31−5.23 (m, 2H, H4, H3), 5.04 (t, J = 8.0 Hz, 1H, H2), 4.63 (d, J =
7.5 Hz, 1H, H1), 4.10 (d, J = 9.0 Hz, 1H, H5), 3.90−92 (m, 2H, CH2),
3.77 (s, 3H, CH3O) 3.76−3.73 (m, 2H, CH2), 2.07, 2.05, 2.04 (3s, 9H,
3 × OCOCH3); 13C NMR (125 MHz, CDCl3) δ = 170.1, 169.5,
169.4, 167.1 (4 × CO), 101.2 (C1), 72.9 (C5), 72.3 (C3), 71.9 (C2),
71.3 (C4), 69.1, 61.9 (2 × CH2), 53.0 (CH3O), 20.7, 20.6, 20.5
(OCCH3); HRMS (FAB+) Calcd. for C15H22O11Na (M + Na)
401.1060, found 401.1060.
2-Benzyloxyethyl 6-tert-butyldiphenylsilyl-β-D-glucopyranose
(37). Dimethylaminopyridine (catalytic amount) and triethylamine
2425
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
3.48 (s, 3H, OCH3), 3.28 (ddd, 1H, H5), 3.14−2.90 (m, 3H), 1.98 (s,
3H, OCOCH3); 13C NMR (101 MHz, CDCl3) δ = 170.7 (CO), 138.2
(Cq-arom), 128.3 (2 × Carom), 127.6 (2 × Carom), 127.55 (Carom), 103.5
(C1), 86.3(CH2), 83.6, 79.4, 73.1, 72.7, 69.25, 69.05, 63.3 (C2), 60. 8,
60.44, 60.37, 24.7, 20.8(OCOCH3); HRMS (FAB+) Calcd. for
C20H30O8Na (M + Na) 421.1838, found 421.1850.
1H NMR (300 MHz, CDCl3) δ ppm 5.03 (dt, JH,F= 15.0, J = 9.0 Hz,
1H, H3), 4.63 (dd, J= 7.8/9.6 Hz, 1H, H2), 4.30 (d, J= 7.8 Hz, 1H,
H1), 4.26−4.07 (m, 2H, H6, H4), 3.94−3.88 (ddd, J= 1.1/5.5/12.2 Hz,
1H, H6′), 3.58−3.47 (m, 4H, 2 × CH2), 3.41−3.38 (m, 1H, H5), 2.43
(br.s, 1H, OH), 1.80, 1.75, 1.73 (3 × OCOCH3); 13C NMR (75 MHz,
CDCl3) δ = 170.7, 170.1, 169.8 (CO), 101.3 (C1), 87.0 (d, JC,F = 184.0
2-Hydroxyethyl 2,3,4-tri-O-methyl-6-O-acetyl-β-D-glucopyranose
(40). Palladium hydroxyde (catalytic amount) was added to a solution
of 2-benzyloxyethyl 2,3,4-tri-O-methyl-6-O-acetyl-β-D-glucopyranose
(39) (705 mg, 1.77 mmol) in ethyl acetate (4 mL) and methanol
(0.5 mL). The reaction was stirred at room temperature under H2 (g)
atmosphere (4 psi) for 3 h. The mixture was then filtered over Celite
and washed with CH2Cl2 and methanol. The solvents were then
removed and the crude product was purified by flash column
chromatography (hexane:ethyl acetate, from 2:1 to 1:20) to afford 40
Hz, C4), 72.9 (OCH2), 72.6 (d, JC,F = 19.6 Hz, C3), 71.5 (d, JC,F
=
10.05 Hz, C5), 71.3 (d, JC,F = 5.47 Hz, C2), 62.2 (C6), 61.9 (CH2OH),
20.8 (OCCH3); 19F NMR (CDCl3, 323.6 MHz) −123.75 ppm;
HRMS (FAB+) Calcd. for C14H21O9FNa (M + Na) 375.1067, found
375.1073.
2-Hydroxyethyl 2,3,4-tri-O-acetyl-6-deoxy-6-fluoro-β-D-glucopyr-
anose (52). 2,3,4-Tri-O-acetyl-6-deoxy-6-fluoro-α-D-glucopyranosyl
bromide 5118e (742 mg, 2.00 mmol) was reacted following the
general glycosylation procedure. The crude product was purified by
flash column chromatography (hexane:ethyl acetate, from 1:2 to 1:3)
1
(518 mg, 95%) as a white solid: H NMR (400 MHz, CDCl3) δ ppm
22
4.32 (dd, 1H, H6′), 4.22 (d, J = 7.8 Hz, 1H, H1), 4.07 (dd, 1H, H6),
3.87 (ddd, 1H), 3.76 (ddd, 1H), 3.70−3.61 (m, 2H), 3.56 (s, 3H,
OCH3), 3.53 (s, 3H, OCH3), 3.45 (s, 3H, OCH3), 3.37 (ddd, 1H, H5),
3.12 (t, 1H, H3), 3.04−2.91 (m, 2H, H2, H4), 2.03 (s, 3H, OCOCH3);
13C NMR (101 MHz, CDCl3) δ = 170.8 (CO), 104.0 (C1), 86.4(C3),
83.6 (C2), 79.7 (C4), 73.7 (CH2), 72.8 (C5), 63.3 (C6), 62.4 (CH2),
60.9, 60.7, 60.6 (3 × OCH3), 20.7 (OCOCH3); HRMS (FAB+) Calcd.
for C13H24O8Na (M + Na) 331.1369, found 331.1367.
to afford 52 (310 mg, 44%) as glassy solid: [α]D −2.2 (c 1 in
CHCl3); 1H NMR (300 MHz, CDCl3) δ ppm 5.08 (t, J = 9.5 Hz, 1H,
H3), 4.90−4.79 (m, 2H, H4, H2), 4.44 (d, J= 8.0 Hz, 1H, H1), 4.40−
4.22 (dm, JH,F= 47.0 Hz, 2H, H6, H6′), 3.75−3.53 (m, 5H, 2 × CH2,
H5), 2.46 (br.s, 1H, OH), 1.90, 1.88, 1.84 (3 × OCOCH3); 13C NMR
(75 MHz, CDCl3) δ = 170.0, 169.4, 169.3 (CO), 100.9 (C1), 81.1 (d,
JC,F = 174.0 Hz, C6), 72.5 (d, JC,F = 19.3 Hz, C5), 72.4 (C3), 72.1
(OCH2), 71.1 (C2), 67.8 (d, JC,F = 6.82 Hz, C4), 61.5 (CH2OH), 20.5,
20.4 (OCCH3); 19F NMR (CDCl3, 323.6 MHz) −155.60 ppm;
HRMS (FAB+) Calcd. for C14H21O9FNa (M + Na) 375.1067, found
375.1071.
2-Hydroxyethyl 2,3-di-O-acetyl-4,6-di-O-methyl-β-D-glucopyra-
nose (43). HBr (2 mL, 33% in AcOH) were added dropwise to a
solution of 1,2,3-tri-O-acetyl-4,6-di-O-methyl-α/β-D-glucopyranose
4217 (1.0 g, 2.99 mmol) in anhydrous CH2Cl2 (5 mL). The reaction
was stirred at room temperature for 2 h. The mixture was then washed
with ice−water and NaHCO3, dried over Na2SO4 and concentrated.
2,3-Di-O-acetyl-4,6-di-O-methyl-β-D-glucopyranose bromide 42b (1.06
g) was obtained as a white solid and used without further purification
for next step. Ag2CO3 (2.06 g, 7.48 mmol) was added to a solution of
2,3-di-O-acetyl-4,6-di-O-methyl-β-D-glucopyranose bromide 42b (1.06
g, 2.99 mmol) and ethylene glycol (167 μL, 2.99 mmol), previously
dried over molecular sieves, in anhydrous CH2Cl2 (10 mL). The
reaction was then stirred at room temperature for 16 h under argon
atmosphere. The mixture was then filtered over Celite and washed
with CH2Cl2. The solvent was then removed and the crude purified by
flash column chromatography (hexane:ethyl acetate, from 1:2 to 1:20)
2-Hydroxyethyl 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-β-D-galacto-
pyranose (55). 2,3,6-Tri-O-acetyl-4-deoxy-4-fluoro-α-D-galactopyrano-
syl bromide 5418a,c (600 mg, 1.62 mmol) was reacted following the
general glycosylation procedure. The crude product was purified by
flash column chromatography (hexane:ethyl acetate, from 2:3 to 1:3)
to afford 55 (447 mg, 78%) as an oil: 1H NMR (400 MHz, CDCl3) δ
ppm 5.22 (dd, J = 8.0, 10.4 Hz,1H, H2), 4.94 (ddd, J = 2.4, 10.4, 27.6
Hz, 1H, H3), 4.81 (dd, J = 2.8, 50 Hz, 1H, H4), 4.51 (d, J = 7.9 Hz,
H1), 4.32−4.20 (m, 2H, H6, H6′), 3.89−3.75 (m, 5H, 2 × CH2, H5),
2.74 (s, 1H, OH), 2.04, 2.02, 2.00 (3s, 9H, 3 × OCOCH3); 13C NMR
(100 MHz, CDCl3) δ = 170.5, 170.3, 169.5 (CO), 101.4 (C1), 85.8 (d,
JC,F = 185.1 Hz, C4), 72.6 (CH2), 71.2 (d, JC,F = 17.6 Hz, C5), 70.9 (d,
JC,F = 18.1 Hz, C3), 68.7 (C2), 61.8 (CH2), 61.5 (d, JC,F = 5.6 Hz, C6),
20.7, 20.6 (OCCH3); 19F NMR (CDCl3, 323.6 MHz, internal
reference TFT) −216.1 ppm; HRMS (FAB+) Calcd. for C14H21O9FNa
(M + Na) 375.1067, found 375.1075.
1
to afford 43 (520 mg, 52%) as white solid: H NMR (400 MHz,
CDCl3) δ ppm 5.15 (t, 1H, H3), 4.90 (t, 1H, H2), 4.48 (d, J= 7.96 Hz,
1H, H1), 3.89−3.76 (m, 2H, CH2), 3.76−3.55 (m, 4H, CH2, H6, H6′),
3.49 (m, 1H, H5), 3.43−3.41 (m, 6H, 2 × OCH3), 3.40−3.35 (m, 1H,
H4), 2.89 (t, 1H, OH), 2.07−2.04 (m, 6H, 2 × OCOCH3); 13C NMR
(101 MHz, CDCl3) δ = 170.1, 169.8, (CO), 101.2 (s, C1), 77.6 (s, C4),
74.9 (s, C3), 74.4 (s, C5), 73.3 (s, CH2), 72.0 (s, C2), 71.0 (s, C6), 62.2
(CH2), 60.3, 59.3 (OCH3), 20.8, 20.7 (OCOCH3); HRMS (FAB+)
Calcd. for C14H24O9 (M + Na) 359.1318, found 359.1313.
2-Hydroxyethyl 2,3-di-O-acetyl-4,6-dideoxy-4,6-difluoro-β-D-gal-
actopyranose (58). 2,3-Di-O-acetyl-4,6-dideoxy-4,6-difluoro-α-D-gal-
actopyranosyl bromide 5718b (253 mg, 0.77 mmol) was reacted
following the general glycosylation procedure. The crude product was
purified by flash column chromatography (hexane:ethyl acetate, from
22
1:1 to 1:2) to afford 58 (158 mg, 66%): [α]D +5.8 (c 1 in CHCl3);
1H NMR (400 MHz, CDCl3) δ ppm 5.20 (t, J = 9.6 Hz, 1H, H2),
5.011−4.79 (m, 2H, H3, H4), 4.56 (dd, JH,F= 46.4, J= 6.4 Hz, 2H, H6,
H6′), 4.55 (d, J= 8.0 Hz, 1H, H1), 3.96−3.83 (m, 2H, H5, CH), 3.74−
3.66 (m, 3H, CH2), 2.72 (br.s, 1H, OH), 2.07, 2.03 (2 × OCOCH3);
13C NMR (100 MHz, CDCl3) δ = 170.3, 169.6 (CO), 101.2 (C1), 85.5
2-Hydroxyethyl 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-β-D-glucopyr-
anose (46). 2,4,6-Tri-O-acetyl-3-deoxy-3-fluoro-α-D-glucopyranosyl
bromide 4518d (1.05 g, 2.85 mmol) was reacted following the general
glycosylation procedure. The crude product was purified by flash
column chromatography (hexane:ethyl acetate, from 1:2 to 1:3) to
22
afford 46 (688 mg, 68%) as a glassy solid: [α]D −14.6 (c 1 in
(dd, J = 186.0, 5.0 Hz, C4), 80.4 (dd, J = 170.0, 6.0 Hz, C6), 72.9
(OCH2), 72.1 (CH2), 71.6 (dd, J = 24.0, 18.0 Hz, C5), 71.0 (d, J =
17.0 Hz, C3), 68.7 (C2), 61.6 (CH2OH), 20.7, 20.6 (OCH3); 19F
NMR (CDCl3, 323.6 MHz) −140.7, −155.1 ppm; HRMS (FAB+)
Calcd. for C12H18O7F2Na (M + Na) 335.0918, found 335.0917.
General Procedure for Synthesis of Carbohydrate Phosphor-
amidites. To a solution of 2-hydroxyethyl acetyl protected
carbohydrate (0.5 mmol) in dry CH2Cl2 (5 mL), DIEA (0.35 mL,
2.0 mmol) and 2-cyanoethyl-N,N′-diisopropylamino-chlorophosphor-
amidite (0.174 mL, 0.76 mmoL) were added at room temperature
under argon atmosphere. After 1 h the solvent was evaporated to
dryness. The product was purified by silica gel column chromathog-
raphy using a mixture of hexane, ethyl acetate and triethylamine.
2-(2,3,4-Tri-O-acetyl-β-L-rhamnopyranosyloxy)ethyl(2-cyano-
ethyl)(N,N-diisopropyl)phosphoramidite (20). 2-Hydroxyethyl 2,3,4-
tri-O-acetyl-β-L-rhamnopyranose 19 (0.12 g, 0.358 mmol) was reacted
CHCl3); 1H NMR (300 MHz, CDCl3) δ ppm 5.06−4.93 (m, 2H, H4,
H2), 4.37−4.31 (m, 2H, H1, H3), 4.06−4.04 (m, 2H, H6, H6′), 3.70−
3.52 (m, 5H, 2 × CH2, H5), 2.53 (s, 1H, OH), 1.98, 1.97, 1.95 (3 ×
OCOCH3); 13C NMR (75 MHz, CDCl3) δ = 170.9, 169.6, 169.5
(CO), 101.0 (d, JC,F = 11.1 Hz, C1), 91.8 (d, JC,F = 189.9 Hz, C3), 73.1
(CH2), 71.5 (d, JC,F = 18.7 Hz, C4), 71.2 (d, JC,F = 7.65 Hz, C5), 68.6
(d, JC,F = 18.6 Hz, C2), 62.1 (C6), 62.0 (CH2), 20.9, 20.8 (OCCH3);
19F NMR (CDCl3, 323.6 MHz) −119.6 ppm; HRMS (FAB+) Calcd.
for C14H21O9FNa (M + Na) 375.1067, found 375.1075.
2-Hydroxyethyl 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-β-D-glucopyr-
anose (49). 2,3,6-Tri-O-acetyl-4-deoxy-4-fluoro-α-D-glucopyranosyl
bromide 4818f (2.0 g, 5.00 mmol) was reacted following the general
glycosylation procedure. The crude product was purified by flash
column chromatography (hexane:ethyl acetate, from 1:2 to 1:4) to
22
afford 49 (1.06 g, 60%) as glassy solid: [α]D −12.5 (c 1 in CHCl3);
2426
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
following the general procedure. The crude product was purified by
flash column chromatography (hexane:ethyl acetate, 1:1 with 1%
NEt3) to obtain compound 20 as a syrup (165 mg, 86%): H NMR
2H, H3, H4), 4.97−4.90 (m, 2H, H2), 4.61 (dd, J = 7.8/9.6 Hz, 1H,
H1), 4.00−3.65 (m, 10H, 2 × OCH2, OCH2CH2CN, CH3O, H5),
3.60−3.48 (m, 2H, 2 × CHisopropyl), 2.59−2.55 (m, 2H, CH2CN), 2.04,
1
(300 MHz, CDCl3) δ 5.25−5.16 (m, 2H, H2, H3), 4.99 (t, J = 9.9 Hz,
1H, H4), 4.71 (dd, J = 3.7/1.6 Hz, 1H, H1), 3.94−3.48 (m, 9H, 2 ×
OCH2, OCH2CH2CN, 2 × CHisopropyl, H5), 2.63−2.57 (m, 2H,
CH2CN), 2.07, 1.98, 1.91 (3s, 3H, 3 × OCOCH3), 1.19−1.11 (m,
15H, CH3, 4 × CH3isopropyl); 13C NMR (75 MHz, CDCl3) δ = 170.1,
170.0, 169.9 (CO), 117.8 (CN), 97.7 (C1), 71.1, 69.7, 69.0, 67.8, 67.5,
63.3, 62.4, 62.2, 58.5, 43.2 (NCHisopropyl), 24.6, 24.5 (CH3isopropyl), 20.9,
20.8 (OCCH3), 20.6 (CH2CN), 17.4 (CH3); HRMS (FAB+) Calcd.
for C23H39N2O10PNa (M + Na) 557.2240, found 557.2222.
2-(2,3,4-Tri-O-acetyl-β-D-xylopyranosyloxy)ethyl(2-cyanoethyl)-
(N,N-diisopropyl)phosphoramidite (23). 2-Hydroxyethyl 2,3,4-tri-O-
acetyl-β-D-xylopyranose 22 (0.06 g, 0.197 mmol) was reacted
following the general procedure. The crude product was purified by
silica gel column chromathography (hexane:ethyl acetate, 1:1 with 1%
NEt3) to give compound 23 as a syrup (80 mg, 83%): 1H NMR (300
MHz, CDCl3) δ ppm 5.08 (t, J = 8.5 Hz, 1H, H3), 4.88−4.82 (m, 2H,
H2, H4), 4.51 (m, 1H, H1), 4.06 (dd, J = 5.3/11.8 Hz, 1H, H5), 3.89−
3.46 (m, 8H, 2 × OCH2, OCH2CH2CN, 2 × CHisopropyl), 3.34−3.26
(m, 1H, H5′), 2.61−2.54 (m, 2H, CH2CN), 1.99, 1.98, 1.97 (3s, 9H, 3
× OCOCH3), 1.11, 1.10 (2d, 12H, 4 × CH3isopropyl); 13C NMR (75
MHz, CDCl3) δ = 170.1, 169.9, 169.4 (3 × CO), 117.7 (CN), 100.7
(C1), 71.4, 70.6, 69.1, 68.8, 62.5, 62.3, 61.9, 58.5, 58.3, 43.1
(NCHisopropyl), 24.6, 24.5 (CH3isopropyl), 20.8, 20.6 (OCCH3), 20.5
(CH2CN); HRMS (FAB+) Calcd. for C22H37KN2O10P (M + K)
559.1823, found 559.1827.
2-(2,3,4-Tri-O-acetyl-6-deoxy-β-D-glucopyranosyloxy)ethyl(2-
cyanoethyl)(N,N-diisopropyl)phosphoramidite (26). 2-Hydroxyethyl
2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranoside 25 (0.140 g, 0.418
mmol) was reacted following the general procedure. The crude
product was purified by silica gel column chromathography using as
eluent (hexane:ethyl acetate, 1:1 with 1% NEt3) to give compound 26
as a syrup (200 mg, 90%): 1H NMR (500 MHz, CDCl3) δ 4.90 (t, J =
9.6 Hz, 1H, H3), 4.74−4.680 (m, 1H, H2), 4.57 (t, J = 9.6 Hz, 1H, H4),
4.36 (dd, JH,P= 14.5, J = 8.1 Hz, 1H, H1), 3.70−3.40 (m, 6H, 2 ×
OCH2, OCH2CH2CN), 3.39−3.29 (m, 3H, 2 × CHisopropyl, H5), 2.67−
2.62 (m, 2H, CH2CN), 2.05, 2.03, 1.99 (3s, 3H, 3 × OCOCH3), 1.23
(d, 3H, J = 6.0 Hz, CH3), 1.17 (t, 12H, 4CH3isopropyl); 13C NMR (75
MHz, CDCl3) δ = 170.2, 169.9, 169.6 (CO), 118.0 (CN), 100.8 (C1),
73.6 (C4), 73.1 (C3), 71.9 (C2), 70.2 (C5), 69.5, 62.8, 58.7 (3 × CH2),
43.3 (NCHisopropyl), 25.0, 24.9, 24.8, 24.7 (CH3isopropyl), 21.0, 20.9, 20.8
(OCCH3), 20.6 (CH2CN), 17.6 (CH3); HRMS (FAB+) Calcd. for
C23H39N2O10PNa (M + Na) 557.2240, found 557.2243.
2-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyrano-
syloxy)ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (31).
2-Hydroxyethyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyra-
noside 30 (0.20 g, 0.50 mmol) was reacted following the general
procedure. The crude product was purified by silica gel column
chromathography (hexane:ethyl acetate, 1:1 with 1% NEt3) to give
compound 31 as a syrup (265 mg, 90%): 1H NMR (300 MHz,
CDCl3) δ 5.92 (dd, J = 3.0/8.7 Hz, 1H, NH), 4.96 (t, J = 9.6 Hz, 1H,
H3), (td, J = 2.7/9.6 Hz, 1H, H4), 4.61 (t, J = 8.4 Hz, 1H, H1), 3.98
(dd, J = 4.8/12.3 Hz, 1H, H6), 3.84 (dd, J = 2.7/12.3 Hz, 1H, H6′),
3.72−3.40 (m, 9H, 2 × OCH2, OCH2CH2CN, H2, H5), 3.37−3.26 (m,
2H, 2 × CHisopropyl), 2.43−2.39 (m, 2H, CH2CN), 1.81, 1.73, 1.67 (3s,
12H, 3 × OCOCH3), 0.92−0.89 (m, 12H, 4 × CH3isopropyl); 13C NMR
(75 MHz, CDCl3) δ = 170.9, 170.6, 169.6 (CO), 118.4 (CN), 101.3
(C1), 73.0 (C3), 72.0 (C4), 69.6, 68.9 (CH2), 62.9 (CH2), 62.4 (C6),
58.5 (CH2), 54.6 (C2), 43.3 (NCHisopropyl), 24.9 (CH3isopropyl), 21.0,
20.9, 20.8 (OCCH3), 20.7 (CH2CN); HRMS (FAB+) Calcd. for
C25H42N3NaO11P (M + Na) 614.2455, found 614.2440.
1.99 (2s, 9H, 3 xOCOCH3), 1.15−1.09 (m, 12H, 4 × CH3isopropyl); 13
C
NMR (100 MHz, CDCl3) δ = 170.1, 169.4, 169.3, 167.2 (4 × CO),
117.8 (CN), 100.8 (C1), 72.5 (C5), 72.0 (C3), 71.1 (C2), 69.6 (CH2),
69.4 (C4), 62.4, 58.5, 52.8 (3 × CH2), 43.0 (NCHisopropyl), 24.6, 24.5
(CH3isopropyl), 20.7, 20.5, 20.4 (OCCH3), 20.3 (CH2CN); HRMS
(FAB+) Calcd. for C24H39N2O12PNa (M + Na) 601.2134, found
601.2138.
2-(2,3,4-Tri-O-methyl-6-O-acetyl-β-D-glucopyranosyloxy)ethyl(2-
cyanoethyl)(N,N-diisopropyl)phosphoramidite (41). 2-Hydroxyethyl
2,3,4-tri-O-methyl-6-O-acetyl-β-D-glucopyranose 40 (165 mg, 0.535
mmol) was reacted following the general procedure. The crude
product was purified by silica gel column chromatography
(hexane:ethyl acetate, from 3:2 to 1:2 with 1% NEt3) to give
1
compound 41 as a white foam (155 mg, 57%): H NMR (400 MHz,
CDCl3) δ ppm 4.30−4.20 (m, 2H, H6′, H1), 4.13 (dd, 1H, H6), 3.90
(m, 1H, CH2), 3.85−3.73 (m, 3H, CH iPr, CH2), 3.67 (dtt, 2H, CH2),
3.55 (s, 5H, OCH3+2H), 3.51 (s, 3H, OCH3), 3.44 (s, 3H, OCH3),
3.30 (ddd, 1H, H5), 3.10 (t, 1H, H3), 3.02 (t, 1H, H4), 2.92 (ddd, 1H,
H2), 2.58 (t, 2H, CH2), 2.02 (s, 3H, OCOCH3), 1.14−1.08 (m, 12H,
4 × CH3 isopropyl); 13C NMR (101 MHz, CDCl3) δ = 170.8 (CO),
117.7 (CN), 103.5 (C1), 86.3, 83.6, 79.4, 72.7, 69.6 (dd), 63.3, 62.4
(dd), 60.8, 60.6−60.2 (m, 2C), 58.4 (dd), 43.0 (dd, 2C), 24.7−24.4
(m, 4C), 20.8, 20.3; HRMS (FAB+) Calcd. for C22H42N2O9P (M + H)
509.2628, found 509.2653.
2-(2,3-Di-O-acetyl-4,6-di-O-methyl-β-D-glucopyranosyloxy)ethyl-
(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (44). 2-Hydrox-
yethyl 2,3-di-O-acetyl-4,6-di-O-methyl-β-D-glucopyranose 43 (0.150
g, 0.45 mmol) was reacted following the general procedure. The crude
product was purified by silica gel column chromathography
(hexane:ethyl acetate, 1:1 with 1% NEt3) to give compound 44 as a
white foam (111 mg, 53%): 1H NMR (400 MHz, CDCl3) δ ppm 5.14
(m, 1H, H3), 4.89 (ddd, 1H, H2), 4.55 (dd, 1H, H1), 3.98 (m, 2H,
CH2), 3.83 (m, 2H, CH2), 3.73 (m, 2H, CH2), 3.68−3.56 (m, 5H, H5,
H6/H6′, 2 × CHisopropyl), 3.48−3.40 (m, 7H, H4, 2 × OCH3), 2.66 (m,
2H, CH2), 2.06 (m, 6H, 2 × OCOCH3), 1.19 (m, 12H, 4 ×
CH3isopropyl); 13C NMR (101 MHz, CDCl3) δ = 170.2, 169.7 (CO),
117.8 (CN), 100.7 (C1), 75.1, 74.7, 71.9, 70.8, 69.3, 69.1, 62.5, 62.3,
60.3, 59.4, 58.6, 58.4, 43.1, 43.0, 24.7, 24.6, 24.5, 20.8, 20.4; HRMS
(FAB+) Calcd. for C23H41N2O10PNa (M + Na) 559.2392, found
559.2397.
2-(2,4,6-Tri-O-acetyl-3-deoxy-3-fluoro-β-D-glucopyranosyloxy)-
ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (47). 2-Hy-
droxyethyl 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-β-D-glucopyranose 46
(0.220 g, 0.62 mmol) was reacted following the general procedure.
The crude product was purified by silica gel column chromathography
(hexane:ethyl acetate, from 3:2 to 1:1 with 1% NEt3) to give
compound 47 as a syrup (260 mg, 76%): 1H NMR (300 MHz,
CDCl3) δ ppm 5.11−4.91 (m, 2H, H4, H2), 4.53−4.28 (m, 2H, H1,
H3), 4.12 (ddd, J= 2.0/4.7/12.4 Hz, 1H, H6), 3.99 (br. d, J= 12.3 Hz,
1H, H6′), 3.86−3.53 (m, 6H, 2 × OCH2, OCH2CH2CN) 3.52−3.40
(m, 3H, 2 × CHisopropyl, H5), 2.53−2.49 (m, 2H, CH2CN), 1.99, 1.97,
1.96 (s, 9H, 3 × OCOCH3), 1.05, 1.03 (2d, 12H, 4CH3isopropyl); 13C
NMR (75 MHz, CDCl3) δ = 170.9, 169.5, 169.4 (CO), 118.0 (CN),
101.0 (d, JC,F = 10.65 Hz, C1), 91.9 (d, JC,F = 189.1 Hz, C3), 71.4 (d,
JC,F = 18.5 Hz, C2), 71.1 (d, JC,F = 7.65 Hz, C5), 69.8 (CH2), 68.5 (d,
JC,F = 18.3 Hz, C4), 62.6 (CH2), 62.0 (C6), 58.6 (CH2), 43.3
(NCHisopropyl), 24.9, 24.8 (CH3 isopropyl), 21.1, 21.0, 20.9 (OCCH3),
20.6 (CH2CN). 19F NMR (CDCl3, 323.6 MHz, internal reference
TFT) −195.3 ppm; HRMS (FAB+) Calcd. for C23H38N2O10FNaP (M
+ Na) 575.2146, found 575.2158.
2-(Methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate-oxy)-
ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (35). 2-Hy-
droxyethyl methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate 34
(0.14 g, 0.37 mmol) was reacted following the general procedure.
The crude product was purified by silica gel column chromathography
(hexane:ethyl acetate, 1:1 with 1% NEt3) to give compound 35 as a
2-(2,3,6-Tri-O-acetyl-4-deoxy-4-fluoro-β-D-glucopyranosyloxy)-
ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (50). 2-Hy-
droxyethyl 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-β-D-glucopyranose 49
(0.365 g, 1.036 mmol) was reacted following the general procedure.
The crude product was purified by silica gel column chromathography
(hexane:ethyl acetate, from 3:2 to 1:1 with 1% NEt3) to give
compound 50 as a syrup (434 mg, 76%): 1H NMR (300 MHz,
1
syrup (160 mg, 75%): H NMR (300 MHz, CDCl3) δ 5.21−5.11 (m,
2427
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
CDCl3) δ ppm 5.03 (dt, JH,F= 15.0, J = 9.0 Hz, 1H, H3), 4.68 (dd, J=
7.8/9.6 Hz, 1H, H2), 4.40 (dd, J= 7.8/10.5 Hz, 1H, H1), 4.37−4.13
(m, 2H, H6, H4), 4.02−3.97 (dd, J= 5.5/12.2 Hz, 1H, H6′), 3.74−3.43
(m, 7H, 2 × OCH2, OCH2CH2CN, H5), 3.40−3.28 (m, 2H, 2 ×
CHisopropyl), 2.43−2.37 (m, 2H, CH2CN), 1.87, 1.83, 1.80 (3s, 9H, 3 ×
OCOCH3), 0.95, 0.93 (m, 12H, 4CH3isopropyl); 13C NMR (75 MHz,
CDCl3) δ = 170.4, 169.8, 169.3 (CO), 117.7 (CN), 101.6 (C1), 86.6
(CH2CN). 19F NMR (CDCl3, 323.6 MHz, internal reference TFT)
−217.1, −231.0 ppm; HRMS (FAB+) Calcd. for C21H35N2O8F2NaP
(M + Na) 535.1997, found 535.2005.
Synthesis of Carbohydrate−Oligonucleotide Conjugates.
Carbohydrate−oligonucleotide conjugates 6, 8, and 14 were
synthesized on a DNA automatic synthesizer by using standard β-
cyanoethylphosphoramidite chemistry. Conjugate 5, 7, 9, 10, 11, and
13−17 were prepared by Biomers following the same methodology.
Oligonucleotide conjugates were synthesized either on low-volume
200 nmols (LV200) or 1.0 μmol scale and using the DMT-off
procedure. Oligonucleotide supports were treated with 33% aqueous
ammonia for 16 h at 55 °C, and then the ammonia solutions were
evaporated to dryness and the conjugates were purified by reversed-
phase HPLC in a Waters Alliance separation module with a PDA
detector. HPLC conditions were as follows: Nucleosil 120 C18, 250 ×
8 mm, 10 μm column; flow rate: 3 mL/min. A 27 min linear gradient
0−30% B (solvent A: 5% CH3CN/95% 100 mM triethylammonium
acetate (TEAA; pH 6.5); solvent B: 70% CH3CN/30% 100 mM
TEAA (pH 6.5)).
Thermodynamic Measurements. Melting curves for the DNA
conjugates were measured in an UV/vis spectrophotometer at 280 nm
while the temperature was raised from 10 to 80 °C at a rate of 1.0 °C
min−1. Curve fits were excellent, with c2 values of 106 or better, and the
van’t Hoff linear fits were quite good (r2 = 0.98) for all
oligonucleotides. ΔH, ΔS, and ΔG errors were calculated as described
previously.19
NMR Spectroscopy. Samples of all the conjugates were purified
by HPLC, ion-exchanged with Dowex 50W resin and then suspended
in 500 μL of either D2O or H2O/D2O 9:1 in phosphate buffer, 100
mM NaCl, pH 7. NMR spectra were acquired in 600 or 800 MHz
NMR spectrometers. DQF-COSY, TOCSY and NOESY experiments
were recorded in D2O. The NOESY spectra were acquired with mixing
times of 150 and 300 ms, and the TOCSY spectra were recorded with
standard MLEV-17 spin-lock sequence, and 80-ms mixing time.
NOESY spectra in H2O were acquired with 100 ms mixing time. In 2D
experiments in H2O, water suppression was achieved by including a
WATERGATE23 module in the pulse sequence prior to acquisition.
Two-dimensional experiments in D2O were carried out at temper-
atures ranging from 5 to 25 °C, whereas spectra in H2O were recorded
at 5 °C to reduce the exchange with water. The spectral analysis
program Sparky24 was used for semiautomatic assignment of the
NOESY cross-peaks and quantitative evaluation of the NOE
intensities.
Conjugate Modeling and Buried Surface Area Calculations.
Computer models of conjugates 9−12 were built from the solution
structure of the permethylated conjugate 12 determined in a previous
study.13 The computer package Sybyl was used to perform the
adequate −CH3 to −OH substitutions. The coordinates were energy
minimized before surface area calculations. Models of the “open”
conjugates were built by manually changing the torsion angles of the
linking phosphate to locate the carbohydrate in a position where it is
completely exposed to the solvent.
(d, JC,F = 186.7 Hz, C4), 72.4 (d, JC,F = 19.5 Hz, C3), 71.2 (d, JC,F
=
11.1 Hz, C5), 70.9 (d, JC,F = 4.35 Hz, C2), 69.5, 62.3 (CH2), 61.9 (C6),
58.3 (CH2), 42.9 (NCHisopropyl), 24.5, 24.4 (CH3isopropyl), 20.6, 20.3
(OCCH3), 20.2 (CH2CN). 19F NMR (CDCl3, 323.6 MHz, internal
reference TFT) −199.9 ppm; HRMS (FAB+) Calcd. for
C23H38N2O10FNaP (M + Na) 575.2146, found 575.2164.
2-(2,3,4-Tri-O-acetyl-6-deoxy-6-fluoro-β-D-glucopyranosyloxy)-
ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (53). 2-Hy-
droxyethyl 2,3,4-tri-O-acetyl-6-deoxy-6-fluoro-β-D-glucopyranose 52
(0.150 g, 0.42 mmol) was reacted following the general procedure.
The crude product was purified by silica gel column chromathography
(hexane:ethyl acetate, from 3:2 to 1:1 with 1% NEt3) to give
compound 53 as a syrup (150 mg, 65%): 1H NMR (500 MHz,
CDCl3) δ ppm 5.08 (td, J = 3.0/9.5 Hz, 1H, H3), 5.05 (t, J = 9.5 Hz,
1H, H4), 5.00−4.97 (m, 1H, H2), 4.66 (dd, J= 8.0/12.0 Hz, 1H, H1),
4.40−4.22 (dm, JH,F= 47.0 Hz, 2H, H6, H6′), 4.00−3.69 (m, 7H, 2 ×
OCH2, OCH2CH2CN, H5), 3.64−3.57 (m, 2H, 2 × CHisopropyl), 2.67−
2.63 (m, 2H, CH2CN), 2.08, 2.06, 2.02 (3 × OCOCH3), 1.19 (t, 12H,
4CH3isopropyl); 13C NMR (125 MHz, CDCl3) δ = 170.3, 169.5, 169.3
(CO), 118.0 (CN), 100.9 (C1), 81.1 (d, JC,F = 174.0 Hz, C6), 72.7 (d,
JC,F = 4.3 Hz, C3), 72.5 (d, JC,F = 19.6 Hz, C5), 71.2 (C2), 69.4 (CH2),
68.1 (d, JC,F = 6.75 Hz, C4), 62.4, 58.5 (CH2), 43.3 (NCHisopropyl), 24.6,
24.5 (CH3isopropyl), 20.7, 20.6 (OCCH3), 20.4 (CH2CN). 19F NMR
(CDCl3, 323.6 MHz, internal reference TFT) −231.4 ppm; HRMS
(FAB+) Calcd. for C23H38N2O10FNaP (M + Na) 575.2146, found
575.2117.
2-(2,3,6-Tri-O-acetyl-4-deoxy-4-fluoro-β-D-galactopyranosyloxy)-
ethyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (56). 2-Hy-
droxyethyl 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-β-D-galactopyranose 55
(0.120 g, 0.34 mmol) was reacted following the general procedure.
The crude product was purified by silica gel column chromathography
(hexane:ethyl acetate, 1:1 with 1% NEt3) to give compound 56 as a
syrup (121 mg, 64%): 1H NMR (400 MHz, CDCl3) δ ppm 5.25−5.22
(m, 1H, H2), 5.01−4.90 (m, 1H, H3), 4.83 (d, J= 50.4 Hz, 1H, H4),
4.60 (dd, J= 8.0/11.8 Hz, 1H, H1), 4.37−4.32 (m, 1H, H5), 4.23−4.18
(dd, J= 6.8/11.3 Hz, 1H, H6), 3.96−3.70 (m, 7H, 2 × OCH2,
OCH2CH2CN, H6′), 3.62−3.54 (m, 2H, 2 × CHisopropyl), 2.66−2.62
(m, 2H, CH2CN), 2.1, 2.08, 2.06 (3s, 9H, 3 × OCOCH3), 1.17 (t, J =
6.6 Hz, 12H, 4 × CH3isopropyl); 13C NMR (100 MHz, CDCl3) δ =
170.4, 170.3, 169.3 (CO), 117.7 (CN), 100.9 (C1), 85.8 (dd, JC,P = 6.8
Hz, JC,F = 185.1 Hz, C4), 71.3 (dd, J = 4.7 Hz, J = 19.5 Hz), 70.8 (d, J =
18.0 Hz), 69.1 (dd, J = 25.2, 7.37 Hz), 68.6 (d, J = 4.8 Hz), 62.5 (dd, J
= 7.7, 16.6 Hz), 61.3, 58.4 (d, J = 19.0 Hz, CH2), 42.9 (dd, J = 4.2,
12.39 Hz, NCHisopropyl), 24.5, 24.4 (CH3isopropyl), 20.6, 20.3 (OCCH3),
20.2 (CH2CN). 19F NMR (CDCl3, 323.6 MHz, internal reference
TFT) −216.1 ppm; HRMS (FAB+) Calcd. for C23H38N2FO10NaP (M
+ Na) 575.2161, found 575.2146.
2-(2,3-Di-O-acetyl-4,6-dideoxy-4,6-difluoro-β-D-galacto-
pyranosyloxy)ethyl(2-cyanoethyl)(N,N-diisopropyl)phos-
phoramidite (59). 2-Hydroxyethyl 2,3-di-O-acetyl-4,6-dideoxy-4,6-
difluoro-β-D-galactopyranose 58 (0.120 g, 0.38 mmol) was reacted
following the general procedure. The crude product was purified by
silica gel column chromathography (hexane:ethyl acetate, 1:1 with 1%
NEt3) to give compound 59 as a syrup (125 mg, 62%): 1H NMR (400
MHz, CDCl3) δ ppm 5.23−5.21 (m, 1H, H2), 5.00−4.93 (m, 1H, H3),
4.86 (d, J= 50.4 Hz, 1H, H4), 4.62−4.52 (m, 3H, H6, H1, H6′), 3.97−
3.65 (m, 7H, H5, 2 × OCH2, OCH2CH2CN), 3.63−3.53 (m, 2H, 2 ×
CHisopropyl), 2.66−2.61 (m, 2H, CH2CN), 2.09−2.07 (m, 6H, 2 ×
OCOCH3), 1.20 (t, J = 6.6 Hz, 12H, 4CH3isopropyl); 13C NMR (100
MHz, CDCl3) δ = 170.3, 169.3 (CO), 117.9 (CN), 100.9 (C1), 85.7
(dd, J = 5.0 Hz, 186 Hz, C4), 71.3 (dd, J = 4.7, 19.5 Hz, C6), 71.6, 71.4,
71.3, 71.2, 69.3, 68.2, 62.5, 58.4 (d, J = 19.6 Hz, CH2), 42.9
(NCHisopropyl), 24.6, 24.5 (CH3isopropyl), 20.8, 20.7 (OCCH3), 20.3
The buried surface area is obtained as the difference between de
surface area of the “open” and the “folded” model of the
corresponding conjugate. Surface areas were calculated with the
program MOLMOL.25
ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of 1H NMR and 13C NMR spectra for compounds 19−
59. HPLC traces of carbohydrate oligonucleotide conjugates
(COCs) 5−17. Melting and van’t Hoff curves of the COCs.
NMR proton assignments, relevant NOEs, and chemical shift
changes for COCs 13−17. This material is available free of
charge via the Internet at http://pubs.acs.org.
2428
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429

The Journal of Organic Chemistry
Article
(16) Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb, H;
Keinan, E. J. Org. Chem. 1984, 49, 4988.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jcmorales@iiq.csic.es.
Notes
■
(17) Kovac, P.; Longauerova, Z. Chem. Zvesti 1972, 26, 179.
(18) (a) Koch, K.; Chambers, R. J. Carbohydr. Res. 1993, 241, 295.
(b) Withers, S. G.; Percival, M. D.; Street, I. P. Carbohydr. Res. 1989,
187, 43. (c) Xia, J.; Xue, J.; Locke, R. D.; Chandrasekaran, E. V.;
Srikrishnan, T.; Matta, K. L. J. Org. Chem. 2006, 71, 3696. (d) Csuk,
R.; Albert, S. Z. Naturforsch., B: J. Chem. Sci. 2011, 66b, 311.
(e) Nieschalk, J.; O’Hagan, D. J. Fluorine Chem. 1998, 91, 159.
(f) Berven, L. A.; Dolphin, D.; Withers, S. G. Can. J. Chem. 1990, 68,
1859.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support by Ministerio de Economia y Competitividad
(Grants CTQ2007-68014-C02-02, CTQ2009-13705,
CTQ2010-20541-C03-01, CTQ2012-35360), Generalitat de
Catalunya (2009/SGR/208), and Instituto de Salud Carlos III
(CIBER-BNN, CB06_01_0019) is gratefully acknowledged.
■
́
(19) (a) Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256.
(b) Ohmichi, T.; Nakano, S.; Miyoshi, D.; Sugimoto, N. J. Am. Chem.
Soc. 2002, 124, 10367.
(20) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X. F.; Sheils, C. J.;
Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213.
(21) Ghose, A. K.; Crippen, G. M. J. Chem. Inf. Comput. Sci. 1987, 27,
21.
(22) Santana, A. G.; Jimenez-Moreno, E.; Gomez, A. M.; Corzana, F.;
Gonzalez, C.; Jimenez-Oses, G.; Jimenez-Barbero, J.; Asensio, J. L. J.
Am. Chem. Soc. 2013, 135, 3347.
(23) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2, 661.
(24) Goddard, D. T., Kneller, D. G., SPARKY 3; University of
California: San Francisco.
(25) Koradi, R.; Billeter, M.; Wuthrich, K. J. Mol. Graphics 1996, 14,
29.
E.V.C. thanks Ministerio de Educacion
́
for a FPU fellowship.
REFERENCES
■
(1) (a) Asensio, J. L.; Arda, A.; Canada, F. J.; Jimenez-Barbero, J. Acc.
Chem. Res. 2013, 46, 946. (b) Nishio, M. Phys. Chem. Chem. Phys.
2011, 13, 13873.
(2) (a) Gabius, H.-J.; Siebert, H.-C.; Andre,
́
S.; Jimen
́
ez-Barbero, J.;
Rudiger, H. ChemBioChem 2004, 5, 740. (b) Lis, H.; Sharon, N. Chem.
̈
Rev. 1998, 98, 637. (c) Sharon, N.; Lis, H. Glycobiology 2004, 14, 53R.
(3) Vicens, Q.; Westhof, E. Biopolymers 2003, 70, 42.
(4) (a) Piotukh, K.; Serra, V.; Borriss, R.; Planas, A. Biochemistry
1999, 38, 16092. (b) Flint, J.; Nurizzo, D.; Harding, S. E.; Longman,
E.; Davies, G. J.; Gilbert, H. J.; Bolam, D. N. J. Mol. Biol. 2004, 337,
417.
(5) (a) Kogelberg, H.; Solis, D.; Jimenez-Barbero, J. Curr. Opin.
Struct. Biol. 2003, 13, 646. (b) Terraneo, G.; Potenza, D.; Canales, A.;
Jimenez-Barbero, J.; Baldridge, K. K.; Bernardi, A. J. Am. Chem. Soc.
2007, 129, 2890. (c) Ramirez-Gualito, K.; Alonso-Rios, R.; Quiroz-
Garcia, B.; Rojas-Aguilar, A.; Diaz, D.; Jimenez-Barbero, J.; Cuevas, G.
J. Am. Chem. Soc. 2009, 131, 18129.
(6) (a) Screen, J.; Stanca-Kaposta, E. C.; Gamblin, D. P.; Liu, B.;
Macleod, N. A.; Snoek, L. C.; Davis, B. G.; Simons, J. P. Angew. Chem.,
Int. Ed. Engl. 2007, 46, 3644. (b) Su, Z.; Cocinero, E. J.; Stanca-
Kaposta, E. C.; Davis, B. G.; Simons, J. P. Chem. Phys. Lett. 2009, 471,
17.
(7) (a) Tsuzuki, S.; Fujii, A. Phys. Chem. Chem. Phys. 2008, 10, 2584.
(b) Fernandez-Alonso, M. C.; Canada, F. J.; Jimenez-Barbero, J.;
Cuevas, G. J. Am. Chem. Soc. 2005, 127, 7379. (c) Sujatha, M. S.;
Sasidhar, Y. U.; Balaji, P. V. Protein Sci. 2004, 13, 2502. (d) Kozmon,
S.; Matuska, R.; Spiwok, V.; Koca, J. Phys. Chem. Chem. Phys. 2011, 13,
14215. (e) Kozmon, S.; Matuska, R.; Spiwok, V.; Koca, J. Chem.Eur.
J. 2011, 17, 5680.
(8) (a) Kobayashi, K.; Asakawa, Y.; Kato, Y.; Aoyama, Y. J. Am. Chem.
́
Soc. 1992, 114, 10307. (b) Morales, J. C.; Penades, S. Angew. Chem.,
Int. Ed. 1998, 37, 654. (c) Ke, C.; Destecroix, H.; Crump, M. P.; Davis,
A. P. Nat. Chem. 2012, 4, 718.
(9) (a) Laughrey, Z. R.; Kiehna, S. E.; Riemen, A. J.; Waters, M. L. J.
Am. Chem. Soc. 2008, 130, 14625. (b) Chavez, M. I.; Andreu, C.; Vidal,
P.; Aboitiz, N.; Freire, F.; Groves, P.; Asensio, J. L.; Asensio, G.;
Muraki, M.; Canada, F. J.; Jimenez-Barbero, J. Chem.Eur. J. 2005, 11,
7060.
(10) Chen, W.; Enck, S.; Price, J. L.; Powers, D. L.; Powers, E. T.;
Wong, C. H.; Dyson, H. J.; Kelly, J. W. J. Am. Chem. Soc. 2013, 135,
9877.
́
(11) Morales, J. C.; Reina, J. J.; Díaz, I.; Avino, A.; Nieto, P. M.;
̃
Eritja, R. Chem.Eur. J. 2008, 14, 7828.
(12) Lucas, R.; Gomez-Pinto, I.; Avinno,
Gonzalez, C.; Morales, J. C. J. Am. Chem. Soc. 2011, 133, 1909.
(13) Lucas, R.; Vengut-Climent, E.; Gomez-Pinto, I.; Avinno,
Eritja, R.; Gonzalez, C.; Morales, J. C. Chem. Commun. 2012, 48, 2991.
́
́
A.; Reina, J. J.; Eritja, R.;
́
́
́
A.;
́
(14) Sorg, B. L.; Hull, W. E.; Kliem, H. C.; Mier, W.; Wiessler, M.
Carbohydr. Res. 2005, 340, 181.
(15) (a) Kochetkov, N. K.; Byramova, N. E.; Tsvetkov, Y. E.;
Backinovskii, L. V. Tetrahedron 1985, 41, 3363. (b) Lemieux, R. U.;
Takwa, T.; Chung, B. Y. ACS Symp. SW 1976, 39, 90.
2429
dx.doi.org/10.1021/jo402700y | J. Org. Chem. 2014, 79, 2419−2429