Synthetic access to spacer-linked 3,6-diamino-2,3,6-trideoxy-α-d-glucopyranosides-potential aminoglycoside mimics for the inhibition of the HIV-1 TAR-RNA/Tat-peptide complex

Article abstract of DOI:10.1016/j.carres.2007.05.015

The synthesis of spacer-linked neoaminoglycoside 5 is described. Key steps of the synthesis are the introduction of nitrogen functionalities at C-3 and C-6 and the olefin cross metathesis of allyl glycoside 16. Although it is known that Grubbs catalysts t

Full text of DOI:10.1016/j.carres.2007.05.015

Carbohydrate Research 342 (2007) 1704–1714
Synthetic access to spacer-linked
,6-diamino-2,3,6-trideoxy-a-D-glucopyranosides—potential
aminoglycoside mimics for the inhibition of the
HIV-1 TAR-RNA/Tat-peptide complex
3
Thomas J o¨ ge, Martin Jesberger, Patrick Br o¨ ker and Andreas Kirschning*
Institut f u¨ r Organische Chemie Leibniz, Universit a¨ t Hannover and Zentrum f u¨ r Biomolekulare Wirkstoffe (BMWZ),
Schneiderberg 1B, D-30167 Hannover, Germany
Received 9 March 2007; received in revised form 1 May 2007; accepted 11 May 2007
Available online 18 May 2007
Abstract—The synthesis of spacer-linked neoaminoglycoside 5 is described. Key steps of the synthesis are the introduction of nitro-
gen functionalities at C-3 and C-6 and the olefin cross metathesis of allyl glycoside 16. Although it is known that Grubbs catalysts
tolerate nitrogen functionalities, difficulties were encountered in the cross metathesis reaction. Factors that govern this dimerization
are the steric and electronic demands of the catalyst and the substrate. Preliminary biological evaluation of homodimer 5, by study-
ing the inhibition of HIV-1 TAR-RNA/Tat-peptide complex using a method based on fluorescence titration, revealed an inhibitory
effect of 5.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Olefin metathesis; Aminoglycosides; Antibiotics; Azidosugars
1
. Introduction
efforts of different research groups were dedicated to
the synthesis of aminoglycoside antibiotics and deriva-
tives as well as mimetics that are able to selectively bind
to RNA. In this context, a wide range of modified
Small molecule natural products and natural product-
like analogues have aided in understanding the role
and function of many biomacromolecules critical to
the progression and maintenance of the cell cycle. The
identification of the direct target of such small molecules
gives investigators a tool to control a specific aspect of
4
aminoglycosides, in which substitution of one or more
5
carbohydrates was systematically varied, have been
prepared. Furthermore, orthogonally protected sugar
diamino acids have been exploited as building blocks
in the synthesis of linear and branched aminoglycoside
1
the cell cycle. Aminoglycoside antibiotics like neomycin
6
B (1), streptomycin (2), and analogues derived from
them are highly potent antibiotics. By interacting with
the 16S subunit of the bacterial ribosome they interfere
with the fidelity of translation and translocation, which
ultimately disrupts protein biosynthesis of bacteria and
derivatives. Some of these efforts also demonstrated
that synthetic derivatives of natural aminoglycosides
can exert improved antibacterial performance while tar-
geting resistance-causing enzymes at the same time
7
(Fig. 1).
2
causes miscoding. The structural basis of the recogni-
tion process has been determined. Recently, many
Recently, we described the first preparation of novel
macrocyclic 1,4-butanediol-linked aminodeoxyglyco-
3
8
sides 3 and 4. The coupling of the aminodeoxyhexose
units was achieved by olefin metathesis of appropriately
allylated aminosugar precursors and finally, in the case
of macrocycle 4, by ring closing metathesis (RCM).
*
Corresponding author. E-mail: andreas.kirschning@oci.uni-hannover.
de
0
008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.05.015

T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1705
HN
Indeed, aminoglycoside antibiotics like 1 and 2 often
contain more than one nitrogen functionality per hexose
or cyclitol unit. All mimetics that we have prepared so
far, including 3 and 4, bear only one amino group per
hexose unit. En route to new amino-rich macrocycles
like 4, we therefore first investigated synthetic routes
to access spacer-linked glycomimetics like 5 that contain
a larger density of functionalities with nitrogen. The
synthetic challenge was to deal with more than one N-
functionality while employing an olefin cross metathesis
as the key step to combine the subunits.
NH2
HN
H N
2
HO
OH
OH
HO
O
NH2
H2N(HNC)HN
O
O
HO
O
H N
OHC
2
O
O
H N
OH
2
OH
Me
2
H N
2
O
OH
H N
OH
O
2
HO
HO
HO
1
O
O
HO
NHMe
HO
O
O
NH2
O
O
Me
HO
O
Me
O
H N
O
2
H2N
Me
2
3
2. Results and discussion
O
Me
NH2
O
The syntheses of neoaminoglycoside 5 commenced with
the three step transformation of D-ulose 6 (available
Me
O
O
HO
H N
O
NH2
O
2
O
10
from D-mannose ) to the 3-azido-3-deoxy-glycoside 7
H N
O
2
O
1
1
5
2
(Scheme 1). Another three steps (removal of benzyl-
idene protection, 6-O-tosylation and introduction of azi-
do group) led to 3,6-bisazido glycoside 8. At this point,
we introduced the allyl moiety by first transforming the
methyl glycoside into the glycosyl chloride, followed by
hydrolysis and glycosidation in the presence of allyl
4
Figure 1. Structures of neomycin (1), streptomycin (2), linear and
macrocyclic neoaminoglycosides 3 and 4 and target neoaminoglycoside
5.
1
2
Dot blot binding experiments of 4 with TAR-RNA in
the presence of the Tat protein and multi-dimensional
heteronuclear NMR spectroscopic studies on the com-
plex of HIV-2 TAR-RNA revealed that the novel
macrocyclic aminoglycoside analogue simultaneously
contacts the crucial residues of the bulge required for
Tat binding and the A35 of the dynamic hexanucleotide
alcohol, which furnished allyl glycoside 9. Transfor-
mation of the methyl glycoside into the free sugar by
employing different Brønsted and Lewis acids, as well
as direct conversion of the glycosyl chloride into the
desired product via glycosidation, resulted in poor yields.
Staudinger reaction employing the conditions first
described by Letsinger and co-workers yielded the cor-
responding diamine, which was directly transformed into
bis(trifluoroacetamide) 11 in good yield for both steps.
Alternatively, we also prepared the 4-O-benzyl protected
derivative 12 via bisazido glycoside 10.
1
3
9
loop. This novel mode of binding, together with the
easy accessibility of new aminoglycoside antibiotic
mimetics, revealed that glycomimetics of type 3 and 4
are biologically and pharmaceutically interesting syn-
thetic targets. From these structural studies we also con-
cluded that additional amino groups in 4 should lead to
an increased number of contact points with the RNA,
hence leading to even tighter interaction.
An alternate synthetic approach, which is depicted in
Scheme 2, started from tri-O-acetyl glucal 13. It is well
established that glycals can be converted into 3-azido-
3-deoxy hexoses in aqueous acetic acid using sodium
1. p-TsOH (cat.), MeOH
2. p-TsCl, pyr.
N3
O
ref. 11
1%
O
Ph
O
Ph
O
O
O
3. NaN , DMF
O
3
HO
N3
N3
7
5
3%
O
OMe
OMe
OMe
6
7
8
N3
NHTFA
O
1
2
. BCl , CH Cl , -78°C
3 2 2
. Ag CO , MeCN, H O
2
3
2
O
1. PPh3, pyr., NH3
. (TFA) O, Et N (81%)
RO
N3
3
. allyl alcohol, AcCl (cat.) (56%)
RO
TFAHN
2
2
3
O
O
1
2
3
. NaH, BnBr, TBAI, DMF
. BCl , CH Cl , -78°C
R= H
R= Bn 10
9
R= H 11
R= Bn 12
3
2
2
. Ag CO , MeCN, H O
2
3
2
4
. allyl alcohol, AcCl (cat.) (82%)
Scheme 1. Preparation of allyl glycosides 9–12.

1706
T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1
2
3
4
. Amberlyst A-26, MeOH (99%)
. Me NHCl, Et N, TsCl, CH Cl (53%)
3
3
2
2
. Ac O, pyr. (80%)
2
. NaN , DMF, (31%)
3
1
. ref. 14 (52%)
OAc
O
OAc
O
5. LiAlH , Et O then H O, 10% NaOH
4 2 2
2. Dowex 50 X,
6. TFA O, Et N (27% for 3 steps)
2
3
allyl alcohol (77%)
AcO
N3
AcO
AcO
O
1
3
14a
(
and β-arabino 14b, α-ribo 14c, β-ribo 14d)
14a:14b:14c:14d= 80 :1 : 9 :10)
NHTFA
O
HO
TFAHN
O
1
1
Scheme 2. Alternate preparation of allyl glycoside 11 from glycal 13.
1
4
azide as the nitrogen source. The mixture of stereoiso-
mers was directly transformed into the corresponding
allyl glycosides 14, which could be separated by column
chromatography. From here, the synthesis followed a
similar route as described in Scheme 1. Complete
de-O-acylation allowed us to introduce the tosyl group
at C-6. Exchange by azide gave the bisazido compound,
which is a 4-O-acylated analogue of glycoside 9. Trans-
formation into bisamide 11 was carried out under reduc-
tive conditions, which turned out to be less efficient
compared to the Staudinger reaction. We ascribe this
observation to severe difficulties when removing inor-
ganic materials derived from lithium aluminium hydride
after the reduction step.
ization of the intermediate chloro 4-O-benzyl-2,3,6-tri-
deoxy-3,6-trifluoroacetamido-a/b-arabino-hexopyranoside
with 1,4-butanediol in the presence of silver triflate only
yielded about 30% of the desired homodimer, which was
part of a complex reaction mixture. A similar result was
obtained for the corresponding acetimidate.
Preliminary biological evaluation of homodimer 5 was
conducted by studying the inhibition of HIV-1 TAR-
RNA/Tat-peptide complex in the presence of homodi-
mer 5. We established a method based on fluorescence
1
6,17
titration
by using the arginine-rich motif of the
Tat-peptide composed of the nine amino acids, 49–58,
which was labelled with rhodamine green. A solution
of this peptide in the presence of 5 (60 lmol) was titrated
with unlabelled TAR-RNA. The results collected from
the fluorescence-titration measurements are listed in
Figure 2. By increasing the amount of homodimer 5
with respect to the Tat-peptide, a strong signal reduction
caused by homodimer 5 was encountered, which clearly
reveals a pronounced inhibitory effect of 5 on the Tat/
TAR-RNA complex. This preliminary result, along with
With allyl glycosides 11 and 12 in hand, we
approached the dimerization step. Earlier work has
demonstrated that a conventional approach, which
involves glycosylation of a spacer diol with suitable gly-
cosyl donors, very inefficiently (low yield, mixture of
8
e
anomers) leads to spacer-linked disaccharides.
Although many ruthenium-based complexes for pro-
moting olefin metathesis are available, only the first gen-
eration Grubbs catalyst 15 is suited to efficiently
promote this transformation. The more reactive precat-
alysts (Grubbs II or Hoveyda-type complexes) tend to
catalyze double bond migration yielding the correspond-
9
our previous work, is an accordance with Tor’s ratio-
nale, as to why compounds should contain flexible link-
ers and rigid domains when interacting with RNAs in
1
8
general. For example, when two moderate or good
RNA binders are covalently linked, the ribozyme inhib-
itory activity of the derivate surpasses that of any natu-
8
,15
ing enol ethers instead.
1
9
To our surprise, allyl glycoside 11, as well as the 4-O-
benzyl derivative, did not furnish metathesis products
under varying conditions (12, CH Cl /MeCN, 45 ꢁC,
starting material; 12, benzene, 50 ꢁC, starting material;
2, CH Cl , 50 ꢁC, decomp.; 11, benzene, 80 ꢁC, de-
comp.) (Scheme 3). Only after O-silylation of the hydr-
oxyl group at C4 did the resulting allyl glycoside 16
undergo cross metathesis under conventional reaction
conditions. The product was subjected to hydrogenation
conditions to afford 1,4-butanediol linked homodimer
ral aminoglycoside antibiotic. In principle, if two
binding sites are in close proximity, a dimeric derivative
can bind simultaneously to the two sites, resulting in
stronger binding affinity.
2
2
1
In conclusion, we describe the first preparation of
aminoglycoside mimetic 5, which contains two 2,3,6-tri-
deoxy-3,6-diamino glycoside units. Unlike analogues
with only one amino group per unit such as homodimer
2
2
1
7,20
18,
homodimer 5 showed an inhibitory effect on the
Tat/TAR-RNA complex. Detailed studies are underway
in our laboratories to structurally study this inhibitory
1
7. The target aminoglycoside mimetic 5 was collected
in excellent yield after removal of all protecting groups.
At this point, it should be mentioned that direct dimer-
9
effect and to create macrocyclic derivatives of 5 for fur-
ther in vitro testing.

T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1707
PCy3
Cl
Ru
15
Cl
Ph
NHTFA
O
PCy3
(different conditions)
RO
TFAHN
O
R= H 11
R= Bn 12
NHTFA
O
NHTFA
1
. 15, CH Cl
2 2
O
2. Pd(OH) , H
2
2
TBSO
TFAHN
RO
TFAHN
58%
O
O
1
7
2
TBSOTf,
R=H
R= TBS 16
11
1. TBAF, THF
94%
2
,6-lutidine
0%
2. NaOH, MeOH
7
NH2
O
HO
H N
2
O
5
2
Scheme 3. Olefin metathesis reactions.
3
2
3
1
G
Pyranosid-Dimer 81-Inhibition (60 µM)
U
G
400
C
G
fluorescence
Signal [V]
C
A
G
G
2
7
390
C
A
6
2
U38
C 39
G
U
3
80
70
C
2
3U
A U
G C
A U
C G
C G
G C
G C
3
49
58
RKKRRQRRR
360
Tat-petide
3
50
40
5
' 3'
3
TAR-RNA
0
100
200
300
400
500
600
Ratio 5 / Tat-peptide
NH2
OH
OH
O
HO
O
OH
1
8
NH2
no signal in fluorescence titration
assay (up to 7.1 mM 18).
Figure 2. Structures of TAR-RNA and Tat-peptide; effect of dimer 5 on Tat/TAR-complex, determined by fluorescence titration (200 nM TAR-
RNA, 100 nM Tat-peptide; maximum concentration of 5 = 60 lM); structure of homodimer 18.
3. Experimental
(Bruker) with 200 MHz (50 MHz), Avance 400/DPX
Bruker) with 400 MHz (100 MHz) and Avance 500/
(
3
.1. General methods
DRX (Bruker), respectively, using tetramethylsilane as
the internal standard. Unless otherwise noted, CDCl3
is the solvent for all NMR experiments. Multiplicities are
described using the following abbreviations: s = singlet,
1
13
1
13
H NMR, C NMR and H, C–COSY as well as
NOESY spectra were measured on Avance 200/DPX

1708
T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
0
0
d = doublet, t = triplet, q = quartet, m = multiplet,
3.2. 1 ,4 -O-Di-(3,6-diamino-2,3,6-trideoxy-a-D-arabino-
hexopyranoside)-1,4-butane (5)
1
3
br = broad. Chemical shift values of C NMR spectra
are reported as values in parts per million relative to
residual CHCl (77 ppm) or CD OD (49 ppm) as inter-
0
0
To a solution of 1 ,4 -O-Di-(4-tert-butyldimethylsilyl-
2,3,6-trideoxy-3,6-trifluoroacetamido-a-D-arabino-hexo-
pyranoside)-1,4-butane (17) (40 mg, 40 lmol) in THF
(1 mL) was added TBAF (37 mg, 118 lmol). The reac-
tion mixture was stirred at room temperature for 2 h,
preadsorbed on silica gel and purified by column chro-
matography over silica gel using petroleum ether–
EtOAc (v/v = 4/1). The product was dissolved in MeOH
(1 mL) and aq NaOH (2 mL, 1 M) was added. The reac-
tion mixture was stirred at room temperature for 3 h.
The solvent was removed in vacuo and the crude prod-
uct was purified by column chromatography on reversed
3
3
nal standards. The multiplicities refer to the resonances
in the off-resonance spectra and were elucidated using
the distortionless enhancement by polarization transfer
(
DEPT) spectral editing technique, with secondary
pulses at 90ꢁ and 135ꢁ. Multiplicities are reported using
the following abbreviations: s = singlet (due to quater-
nary carbon), d = doublet (methine), q = quartet
(
methyl), t = triplet (methylene). Mass spectra were
recorded on a type LCT-spectrometer (Micromass)
and on a type VG autospec (Micromass). Ion mass
(
m/z) signals are reported as values in atomic mass units
followed, in parentheses, by the peak intensities relative
to the base peak (100%). Optical rotations [a] were col-
lected on a Polarimeter 341 (Perkin Elmer) at a wave-
phase silica gel using H O–MeOH (v/v = 1/0 to 0/1) as
2
eluent. Title compound 5 was obtained as a colourless
2
D
2
1
powder (28 mg, 94%): ½aꢁ +18.5 (c 1.0, H O);
H
2
ꢀ1
2
ꢀ1
length of 589 nm and are given in 10 deg cm g
.
NMR (400 MHz, MeOD, MeOD = 3.31 ppm): d 1.45–
1.55 (m, 1H, 2-H), 1.62–1.71 (m, 2H, OCH CH –H),
Combustion analyses were performed at the Institut f u¨ r
Organische Chemie, Leibniz Universit a¨ t Hannover.
The fluorescence titration was measured with a FP-
2
2
1.96–2.03 (m, 1H, 2-H), 2.60 (dd, J = 13.8, 7.5 Hz, 1H,
4-H), 2.93–3.15 (m, 3H, 3-H, 6-H), 3.36–3.49 (m, 2H,
5-H, OCH CH –H), 3.68–3.74 (m, 1H, OCH CH –H),
6
500 spectral photometer of JASCO. All solvents used
2
2
2
2
1
3
were of reagent grade and were further dried. Reactions
were monitored by thin-layer chromatography (TLC) on
4.95 (d, J = 2.6 Hz, 1H, 1-H); C NMR (100 MHz,
MeOD, MeOD = 49.0 ppm): d 24.2 (t, C–OCH CH ),
2
2
2
54
silica gel 60 F (E. Merck, Darmstadt) and spots were
detected either by UV-absorption or by charring with
H SO /4-methoxybenzaldehyde in MeOH. Preparative
27.7 (t, C-2), 38.4 (t, C-6), 44.0 (d, C-3), 68.1 (t, C–
OCH CH ), 74.1 (d, C-5), 75.5 (d, C-4), 98.2 (d, C-1);
2
2
+
HRMS m/z calcd for C H N O
6
[M+Na] :
2
4
16 34
4
column chromatography was performed on silica gel 60
E. Merck, Darmstadt). D-Glucal 13 is commercially
available. D-Ulose 6 was prepared according to Ref.
401.2376. Found: 401.2376.
(
3.3. Methyl 3-azido-4,6-O-benzylidene-2,3-dideoxy-a-D-
arabino-hexopyranoside (7)
10. The synthesis of homodimer 18 is described in Ref.
0. The trans-activation-response (TAR) fragment, used
2
for the fluorescence-titration assay, was obtained as a
To a solution of methyl 4,6-O-benzylidene-2-deoxy-a-D-
erythro-hexopyranoside-3-ulose (6) (5.7 g, 22 mmol) in
dry MeOH (200 mL) was added sodium borohydride
(1.65 mg, 43.7 mmol) at 0 ꢁC. The suspension was stir-
0
non labelled oligonucleotide (31 nucleotides: 5 -GGC
0
CAG AUC UGA GCC UGG GAG CUC GGC C-3 )
from Purimex (Grebenstein, Germany). The extinction
coefficient was 192,300 L/(mol cm) at 260 nm. The fluo-
rescence-labelled Tat-peptide (sequence: RhoG-DOA-
RKKR RQRRR-COOH; rhodamine green (RhoG) as
fluorophore, attached via a linker with the peptide) was
obtained as hydrochloride from NMI-Peptides (Reutlin-
gen, Germany) (95% HPLC purity; MW = 1840 g/mol;
extinction coefficient 74,000 L/(mol cm) at 530 nm).
The fluorophore RhoG showed an excitation wave-
length at 504 nm and an emission wavelength at 530 nm.
red at room temperature for 2 h. H O (50 mL) and
2
CH Cl were added, the phases were separated and the
2
2
organic phase was washed with satd aq NaHCO , dried
3
with MgSO4 and concentrated in vacuo. The crude
product was dissolved in CH Cl (30 mL) and added
2
2
dropwise to a solution of trifluoromethanesulfonic
anhydride (5.0 mL, 29.5 mmol) in dry pyridine
(14 mL). The reaction mixture was stirred at room tem-
perature for 45 min and then poured into ice cold satd
aq NaHCO . The aqueous phase was extracted with
3
CH Cl , the organic phase was dried with MgSO and
2
2
4
H2N
O
NH2
concentrated in vacuo. The red residue was dissolved
in dry DMF (100 mL). Sodium azide (5.5 g, 84.7 mmol)
was added and the reaction mixture was stirred for 1.5 h
at room temperature. The reaction was quenched with
H O. The aqueous layer was extracted with Et O, the
CO2
2
2
O
O
combined organic phases were dried (MgSO ) and con-
4
NH
centrated in vacuo. The residue was purified by column
chromatography over silica gel using petroleum ether–
HN

T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1709
EtOAc (v/v = 4/1) as eluent. Title compound 7 was
obtained as a colourless syrup (4.5 g, 71%): H NMR
dried with MgSO , concentrated in vacuo and purified
4
1
by column chromatography over silica gel using petro-
leum ether–EtOAc (v/v = 6/1) as eluent. Title com-
pound 8 was obtained as a colourless syrup (104 mg,
(
400 MHz, CDCl3, TMS = 0 ppm):
d 1.72 (ddd,
J = 13.5, 12.0, 3.5 Hz, 1H, 2-H ), 2.17 (ddd, J = 13.5,
ax
2
D
2
1
5
.0, 1.0 Hz, 1H, 2-H ), 3.35 (s, 3H, OCH –H), 3.56 (t,
67%): ½aꢁ
+139.4 (c 1.0, CHCl3);
H
NMR
eq
3
J = 9.4 Hz, 1H, 4-H), 3.76 (t, J = 10.2 Hz, 1H, 6-Hax),
.88 (ddd, J = 10.2, 9.4, 4.5 Hz, 1H, 5-H), 4.06 (ddd,
J = 12.0, 9.4, 5.0 Hz, 1H, 3-H), 4.30 (dd, J = 10.2,
.5 Hz, 1H, 6-H ), 4.78 (d, J = 3.5 Hz, 1H, 1-H), 5.63
(400 MHz, CDCl3, TMS = 0 ppm):
d
1.73 (td,
3
J = 12.7, 3.5 Hz, 1H, 2-H), 2.18 (ddd, J = 13.2, 4.9,
1.1 Hz, 1H, 2-H), 3.03 (s, 1H, OH–H), 3.38 (s, 3H,
OMe–H), 3.39 (t, J = 9.7 Hz, 1H, 4-H), 3.48 (dd,
J = 13.1, 5.5 Hz, 1H, 6-H), 3.56 (dd, J = 13.1, 2.6 Hz,
1H, 5-H), 3.73–3.81 (m, 2H, 3-H, 6-H), 4.82 (d,
4
eq
1
3
(
s, 1H, PhCH–H), 7.31–7.54 (m, 5H, Ph–H);
C
NMR (100 MHz, CDCl , CDCl = 77 ppm): d 35.51
3
3
1
3
(
t, 2-C), 54.80 (q, OCH –C), 56.37 (d, 3-C), 63.15 (d,
J = 3.0 Hz, 1H, 1-H); C NMR (100 MHz, CDCl3,
3
5
1
-C), 69.06 (t, 6-C), 82.08 (d, 4-C), 98.14 (d, 1-C),
CDCl = 77.2 ppm): d 34.5 (t, C-2), 51.3 (t, C-6), 54.8
3
01.54 (d, PhCH–C), 125.97 (d, Ph–C), 128.24 (d, Ph–
(q, C–OMe), 60.3 (d, C-3), 70.8 (d, C-5), 70.9 (d, C-4),
1
13
ꢀ
C), 128.96 (s, Ph–C), 137.08 (s, Ph–C). H and
NMR spectral data matched those reported before.
C
97.5 (d, C-1); HRMS m/z calcd for C H N O [M] :
7
12
6
3
8
d
227.0893. Found: 227.0897.
3.4. Methyl 3,6-azido-2,3,6-trideoxy-a-D-arabino-hexo-
pyranoside (8)
3
.5. Allyl 3,6-azido-2,3,6-trideoxy-a-D-arabino-hexo-
pyranoside (9)
3
.4.1. Methyl 3-azido-2,3-dideoxy-a-D-arabino-hexopyr-
A solution of methyl 3,6-azido-2,3,6-trideoxy-a-D-ara-
bino-hexopyranoside (8) (200 mg, 0.88 mmol) in CH Cl
anoside. To a solution of methyl 3-azido-4,6-O-benzyl-
idene-2,3-dideoxy-a-D-arabino-hexopyranoside (7) (300
mg, 1.0 mmol) in MeOH (20 mL) was added p-toluene-
sulfonic acid (20 mg, 0.1 mmol). The reaction was stir-
2
2
3
(
(
2 mL) was cooled to ꢀ78 ꢁC and treated with BCl
1.1 mL, 1.1 mmol, 1 M in hexanes). The reaction mix-
ture was stirred for 1 h while slowly warming to room
red at room temperature for 5 h. Et N (1 mL) was
3
temperature. It was cooled to ꢀ78 ꢁC again and treated
added, the reaction mixture was concentrated in vacuo
and the residue was purified by column chromatography
over silica gel using petroleum ether–EtOAc (v/v = 4/1)
with H O. The aqueous phase was extracted with
2
CH Cl (3·) and the combined organic phases were con-
2
2
centrated in vacuo. The crude product was dissolved in
as eluent. Title compound was obtained as a yellow oil
acetonitrile (4.5 mL), treated with H O (1 mL) and
1
2
(
163 mg, 80%): H NMR (400 MHz, CDCl , TMS =
3
Ag CO (728 mg, 2.6 mmol) and stirred at room temper-
2
3
0
ppm): d 1.64 (td, J = 12.7, 3.4 Hz, 1H, 2-H), 2.11
ature for 24 h. The reaction mixture was diluted with
Et O and Na SO was added. After filtration, the solu-
(
dd, J = 12.7, 4.4 Hz, 1H, 2-H), 3.32 (s, 3H, MeO–H),
2
2
4
3
1
.50 (t, J = 9.0 Hz, 1H, 4-H), 3.57 (dt, J = 9.6, 3.4 Hz,
tion was concentrated in vacuo and allyl alcohol
4.8 mL) was added, followed by the addition of acetyl
chloride (150 lL). The reaction mixture was stirred at
H, 5-H), 3.74–3.86 (m, 3H, 3-H, 6-H), 4.77 (d,
(
1
3
J = 3.1 Hz, 1H, 1-H); C NMR (100 MHz, CDCl3,
CDCl = 77.2 ppm): d 34.8 (t, C-2), 54.7 (q, C–OMe),
3
5
0 ꢁC for 16 h, treated with Et N and purified by col-
3
6
0.1 (d, C-3), 61.9 (t, C-6), 70.4 (d, C-4), 71.5 (d, C-5),
umn chromatography on silica gel using petroleum
1
13
9
7.7 (d, C-1). H and C NMR spectral data matched
ether–EtOAc (v/v = 6/1) as eluent. Title compound 9
2
1
those reported before.
22
D
was obtained as a colourless oil (40 mg, 56%): ½aꢁ
1
+
95.2 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ,
3
3
3
.4.2. Methyl 3,6-azido-2,3,6-trideoxy-a-D-arabino-hexo-
CDCl = 7.26 ppm):
d
1.76 (ddd, J = 13.0, 12.5,
3
pyranoside (8). To a solution of methyl 3-azido-2,3-
dideoxy-a-D-arabino-hexopyranoside described above
3.5 Hz, 1H, 2-H), 2.21 (ddd, J = 13.0, 5.0, 1.0 Hz, 1H,
2-H), 3.42 (t, J = 9.5 Hz, 1H, 4-H), 3.48 (dd, J = 13.1,
5.6 Hz, 1H, 6-H), 3.55 (dd, J = 13.1, 2.6 Hz, 1H, 6-H),
3.78–3.86 (m, 2H, 5-H, 3-H), 3.99 (ddt, J = 12.9, 6.1,
1.3 Hz, 1H, CH @CH–CH –H), 4.18 (ddd, J = 12.9,
(
138 mg, 0.68 mmol) in dry pyridine (2 mL) was added
p-toluenesulfonyl chloride (142 mg, 0.75 mmol) and the
reaction mixture was stirred at room temperature for
2
2
1
6 h. The solvent was removed in vacuo, EtOAc was
5.2, 1.5 Hz, 1H, CH @CH–CH –H), 4.98 (d,
2
2
added and washed successively with 1 N HCl and satd
aq NaHCO . The organic phase was dried (Na SO )
J = 3.0 Hz, 1H, 1-H), 5.23 (dq, J = 10.2, 1.2 Hz, 1H,
CH @CH–CH –H), 5.32 (dd, J = 17.2, 1.5 Hz, 1H,
3
2
4
2
2
and concentrated in vacuo. The crude product was dis-
solved in DMF (2 mL) and sodium azide (221 mg,
CH @CH–CH –H), 5.86–5.96 (m, 1H, CH @CH–
2
2
2
1
3
CH –H);
C NMR (100 MHz, CDCl , CDCl =
2
3 3
3
.4 mmol) was added. The reaction mixture was stirred
77.2 ppm): d 29.7 (t, C-2), 34.6 (t, C-6), 51.5 (d, C-3),
60.4 (d, C-5), 68.1 (t, C–CH @CH–CH ), 71.1 (d, C-
at 50 ꢁC for 20 h. H O was added and the aqueous phase
was extracted (3·) with EtOAc. The organic phase was
2
2
2
4), 95.6 (d, C-1), 117.7 (t, C–CH @CH–CH ), 133.5
2
2

1710
T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
(
d, C–CH @CH–CH ); HRMS m/z calcd for
filtered through silica gel and directly used for the next
step. A suspension of the crude material (68 g) was
refluxed in allyl alcohol (300 mL) for 30 min, filtered
off and washed with abs. allyl alcohol several times.
2
2
ꢀ
C H N O [M] : 253.1049. Found: 253.1057.
9
14
6
3
3
arabino-hexopyranoside (11)
.6. Allyl 2,3,6-trideoxy-3,6-trifluoroacetamido-a-D-
The crude product was dissolved in allyl alcohol
ꢂ
(
680 mL) and treated with the Dowex 50X8 at 90 ꢁC
for 2 h. The reaction mixture was filtered and the ion ex-
change resin was washed several times with CH Cl . The
A solution of allyl 3,6-azido-2,3,6-trideoxy-a-D-arabino-
hexopyranoside (9) (46 mg, 0.18 mmol) in dry pyridine
2
2
combined organic phases were concentrated in vacuo
and the mixture of title compounds was collected
(
1 mL) was treated with PPh (190 mg, 0.72 mmol) and
3
stirred at room temperature for 1 h. NH3 (105 lL,
.45 mmol; 28% in H O) was added and the reaction
(46.1 g, 40%).
1
2
mixture was allowed to stand at room temperature for
6 h. The solvents were removed in vacuo, the crude
1
3
.7.2. Allyl 3-azido-2,3-dideoxy-6-O-tosyl-a-D-arabino-
product was dried by azeotropic distillation with toluene
and the dry crude product was dissolved in CH Cl
hexopyranoside. 3-Azido-4,6-di-O-acetyl-2,3-dideoxy-a-
D-arabino-hexopyranoside described above (1 g, 3.19
mmol) was dissolved in MeOH (15 mL) and treated with
Amberlyst IRA 900 (5 g) and was shaken overnight at
rt. After filtration and washing of the resin with MeOH
2
2
(
1 mL). Et N (2.9 mmol, 0.4 mL) and trifluoroacetic
3
anhydride (1.45 mmol, 0.2 mL) were added and stirred
at room temperature for 3 h. MeOH was added, the
reaction mixture was preadsorbed on silica and purified
by column chromatography on silica gel using petro-
leum ether–EtOAc (v/v = 2/1) as eluent. Title com-
pound 11 was obtained as colourless oil (20 mg, 81%):
ꢂ
(25 mL), the combined organic phases were concentrated
in vacuo to yield the crude product (0.73 g, 3.16 mmol),
which was pure enough to be used for the next step.
The crude product (65 mg, 0.28 mmol) was dissolved in
CH Cl (3 mL) and treated with trimethylammonium
chloride (38.5 mg, 0.28 mmol) and Et N (97 lL,
1
H NMR (400 MHz, MeOD, MeOD = 3.31 ppm): d
1
.77 (td, J = 12.8, 3.5 Hz, 1H, 2-H), 1.98 (d,
J = 4.8 Hz, 1H, 2-H), 3.23–3.28 (m, 1H, 4-H), 3.39
dd, J = 13.9, 8.5 Hz, 1H, 6-H), 3.68–3.75 (m, 2H, 6-
2
2
3
0
.70 mmol). The solution was cooled to ꢀ10 ꢁC and p-
toluenesulfonyl chloride (65 mg, 0.34 mmol) in CH Cl
2
(
H, 5-H), 3.91 (ddt, J = 12.9, 5.9, 1.3 Hz, 1H,
CH @CH–CH –H), 4.08 (ddd, J = 12.9, 5.3, 1.4 Hz,
2
(3 mL) was slowly added. After 30 min, the reaction
2
2
was terminated by the addition of satd aq NaCl and ex-
tracted with CH Cl (4 · 5 mL). The combined organic
phases were dried (MgSO ) and purified by column chro-
1
H, CH @CH–CH –H), 4.21 (ddd, J = 12.3, 10.0,
2
2
4
.5 Hz, 1H, 4-H), 4.88 (d, J = 2.9 Hz, 1H, 1-H), 5.13
2
2
(
dd, J = 10.4, 1.6 Hz, 1H, CH @CH–CH –H), 5.24
4
2
2
matography over silica gel using petroleum ether–EtOAc
(
dd, J = 17.3, 1.7 Hz, 1H, CH @CH–CH –H), 5.85
2
2
(v/v = 3/1) as eluent. The title compound was obtained as
(
ddt, J = 17.0, 10.9, 5.5 Hz, 1H, CH @CH–CH –H);
C NMR (100 MHz, MeOD, MeOD = 49.0 ppm): d
2
2
22
1
3
a colourless oil (57 mg, 53%): ½aꢁ +64.9 (c 0.81, CHCl3);
D
1
H NMR (400 MHz, CDCl , CDCl = 7.26 ppm): d 1.67
3
6.0 (t, C-2), 41.1 (t, C-6), 50.4 (d, C-3), 68.7 (t, C–
3
3
(ddd, J = 12.8, 12.5, 3.4 Hz, 1H, 2b-H), 2.14 (dd,
CH @CH–CH ), 71.6 (d, C-4), 72.3 (d, C-5), 96.8 (d,
2
2
J = 12.8, 5.0 Hz, 1H, 2a-H), 2.45 (s, 3H, CH Ph–H),
2
C-1), 117.4 (t, C–CH @CH–CH ), 117.5 (q,
3
2
2
.64 (s, 1H, OH–H), 3.50 (dd, J = 9.7, 9.6 Hz, 1H, 4-
J = 287.0 Hz, C–CF ), 117.6 (q, J = 286.6 Hz, C–CF ),
3
3
H), 3.75 (ddd, J = 9.7, 3.9, 2.0 Hz, 1H, 5-H), 3.83 (ddd,
J = 12.5, 9.6, 5.0 Hz, 1H, 3-H), 3.90 (ddt, J = 12.9, 5.4,
1
35.4 (d, C–CH @CH–CH ), 159.0 (q, J = 36.8 Hz, C–
2
2
CF CO), 159.2 (q, J = 36.9 Hz, C–CF CO); HRMS
m/z calcd for C H F N O [M+Na+MeCN] :
3
3
+
1
.4 Hz, 1H, CH @CH–CH H O–H), 4.07 (ddt,
2
a
b
1
3
16
6
2
5
J = 12.9, 5.4, 1.4 Hz, 1H, CH @CH–CH H O–H), 4.18
4
58.1135. Found: 459.1127.
2
a
b
(
3
dd, J = 11.1, 2.0 Hz, 1H, 6b-H), 4.41 (dd, J = 11.1,
.9 Hz, 1H, 6a-H), 4.89 (d, J = 3.4 Hz, 1H, 1-H), 5.19
(dq, J = 10.4, 1.6 Hz, 1H, HtransH C@CH –CH O–H),
3.7. Allyl 2,3,6-trideoxy-3,6-trifluoroacetamido-a-D-
arabino-hexopyranoside (11)
cis
2
2
5.26 (dq, J = 16.8, 1.6 Hz, 1H, HtransH C@CH –
cis
2
CH O–H), 5.84 (ddt, J = 16.8, 10.4, 5.8 Hz, 1H,
2
3
.7.1. Allyl 3-azido-4,6-di-O-acetyl-2,3-dideoxy-a-D-ara-
bino-hexopyranoside (14a). To a suspension of glycal
13) (100 g, 0.37 mol) in H O (430 mL) were added gla-
CH
7.81 (d, J = 8.3 Hz, 2H, Ts–H); C NMR (100 MHz,
CDCl , CDCl = 77.2 ppm): d 21.7 (q, C–CH Ph), 34.6
(t, C-2), 68.0, 68.7 (t, C-6, C–CH @CH–CH O), 59.8,
69.9, 70.0, (d, C-3, C-4, C-5), 95.8 (d, C-1), 117.6 (t, C–
2
@CH–CH
2
O–H), 7.38 (d, J = 8.3 Hz, 2H, Ts–H),
1
3
(
3
3
3
2
cial acetic acid (95 mL) and sodium azide (47.7 g,
.74 mol). The mixture was stirred at 80 ꢁC for 5 h and
2
2
0
then cooled to room temperature. Satd aq NaHCO3
was added and the aqueous phase was extracted with
EtOAc (3·). The combined organic phases were dried
CH
2
@CH–CH
@CH–CH
2
O), 128.0, 129.9 (d, C–Ts), 133.5 (d, C–
O), 132.7, 145.1 (s, C–Ts). Elemental
CH
2
2
Anal. Calcd for C16H N O S: C, 50.12; H, 5.52; N,
21 3 6
(
MgSO ) and concentrated in vacuo. The mixture was
4
10.96. Found: C, 50.22; H, 5.39; N, 11.01.

T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1711
3
.7.3. Allyl 4-O-acetyl-3-azido-2,3-dideoxy-6-O-tosyl-a-
D-arabino-hexopyranoside. Allyl 3-azido-2,3-dideoxy-
-O-tosyl-a-D-arabino-hexopyranoside described above
17 mg, 44.3 lmol) was dissolved in a mixture of pyri-
4-H), 5.00 (d, J = 3.3 Hz, 1H, 1-H), 5.24 (dq, J = 10.3,
1.4 Hz, 1H, HtransH C@CH–CH O–H), 5.33 (dq,
cis
2
6
(
J = 17.1, 1.4 Hz, 1H, HtransH C@CH–CH O–H), 5.92
cis
2
(ddt, J = 17.1, 10.3, 5.6 Hz, 1H, CH @CH–CH O–H);
2
2
1
3
dine–Ac O (v/v = 2/1, 3 mL) and stirred for 16 h. The
C NMR (100 MHz, CDCl , CDCl = 77.2 ppm): d
3 3
2
reaction was terminated by the addition of satd aq
20.8 (q, C–COCH ), 34.9 (t, C-2), 51.4 (t, C-6), 68.2 (t,
3
NaHCO and extracted with CH Cl (3 · 5 mL). The
C–CH @CH–CH O), 57.2, 69.7, 71.6 (d, C-3, C-4, C-
3
2
2
2
2
combined organic phases were dried (MgSO ) and puri-
5), 95.3 (d, C-1), 117.9 (t, C–CH @CH–CH O), 133.4
4
2
2
fied by column chromatography over silica gel using
petroleum ether–EtOAc (v/v = 4/1) as eluent. The title
compound was obtained as a colourless oil (15 mg,
(d, C–CH @CH–CH O), 169.9 (s, C–COCH ); LRMS
2
2
3
+
m/z calcd for C H N O [M+Na] : 319.1. Found:
1
1
16
6
4
319.1.
22
1
8
0%): ½aꢁ +68.3 (c 0.73, CHCl3); H NMR (400
D
MHz, CDCl3, CDCl = 7.26 ppm):
d
1.69 (ddd,
3.7.5. Allyl 2,3,6-trideoxy-3,6-trifluoroacetamido-a-D-
arabino-hexopyranoside. Allyl 4-O-acetyl-3,6-diazido-
3
J = 13.2, 12.6, 3.3 Hz, 1H, 2b-H), 2.15 (ddd, J = 13.2,
.8, 0.8 Hz, 1H, 2a-H), 2.45 (s, 3H, CH Ph–H), 3.89
4
2,3,6-trideoxy-a-D-arabino-hexopyranoside
described
3
(
ddd, J = 12.6, 9.7, 4.8 Hz, 1H, 3-H), 3.91 (ddt,
above (50 mg, 169 lmol) was dissolved in Et O (7 mL)
2
J = 12.8, 5.9, 1.4 Hz, 1H, CH @CH–CH H O–H),
and treated with LiAlH (51.2 mg, 1.35 mmol) at 0 ꢁC.
2
a
b
4
3
.95 (ddd, J = 10.0, 5.6, 3.2 Hz, 1H, 5-H), 4.03 (dd,
J = 10.8, 5.6 Hz, 1H, 6b-H), 4.08 (dd, J = 10.8, 3.2 Hz,
H, 6a-H), 4.10 (ddt, J = 12.8, 5.5, 1.4 Hz, 1H,
CH @CH–CH H O–H), 4.75 (dd, J = 10.0, 9.7 Hz,
The reaction was stirred at 0 ꢁC for 2 h and was then ter-
minated by the addition of H O (0.17 mL), NaOH (10%,
2
1
3 mL) and H O (3 mL). The reaction was filtered
2
TM
through Celite and the organic phase was concentrated
2
a
b
1
H, 4-H), 4.89 (d, J = 3.3 Hz, 1H, 1-H), 5.21 (dq,
and dried in vacuo. The resulting crude product was di-
J = 10.4, 1.4 Hz, 1H, HtransH C@CH –CH O–H),
rectly used for the next step by dissolving it in Et N
cis
2
2
3
5
.28 (dq, J = 17.1, 1.4 Hz, 1H, HtransH C@CH –
(2 mL) and cooling to 0 ꢁC. Trifluoroacetic acid
(0.5 mL) was slowly added and the reaction was stirred
at 0 ꢁC for 1 h. The reaction was stopped by addition of
satd aq NaHCO and extracted with CH Cl (3 · 5 mL).
cis
2
CH O–H), 5.87 (ddt, J = 17.1, 10.4, 5.5 Hz, 1H,
CH @CH–CH O–H), 7.34 (d, J = 8.2 Hz, 2H, Ts–H),
2
2
2
1
3
7
.78 (d, J = 8.2 Hz, 2H, Ts–H); C NMR (100 MHz,
3
2
2
CDCl , CDCl = 77.2 ppm): d 20.7 (q, C–CH CO),
The combined organic phases were washed with satd aq
3
3
3
2
1.7 (q, C–CH Ph–), 34.7 (t, C-2), 68.1, 68.3 (t, C-6,
NaCl, dried (MgSO ) and purified by column chroma-
3
4
C–CH @CH–CH O), 57.5, 68.0, 70.4 (d, C-3, C-4, C-
tography over silica gel using petroleum ether–EtOAc
2
2
5
), 128.1, 129.8 (d, C–Tos), 133.3 (d, C–CH @CH–
(2/1) as eluent. Title compound 11 is obtained as a col-
ourless oil (18 mg, 27%): H and C NMR spectral data
matched those reported above.
2
1
13
CH O), 132.6, 144.9 (s, C–Ts), 169.8 (s, C–COCH );
LRMS m/z calcd for C H N O S [M+Na] : 448.1.
2
3
+
1
8
23
3
7
Found: 448.1.
3.8. Allyl 4-O-benzyl-2,3,6-trideoxy-3,6-trifluoroacet-
3
.7.4. Allyl 4-O-acetyl-3,6-diazido-2,3,6-trideoxy-a-D-
amido-a-D-arabino-hexopyranoside (12)
arabino-hexopyranoside. Allyl 4-O-acetyl-3-azido-2,3-
dideoxy-6-O-tosyl-a-D-arabino-hexopyranoside described
above (14 mg, 32.9 lmol) was dissolved in DMF
3.8.1. Methyl 3,6-azido-4-O-benzyl-2,3,6-trideoxy-a-D-
arabino-hexopyranoside. To a solution of methyl 3,6-
(
3 mL), treated with NaN (10 mg, 150 lmol) and stir-
azido-2,3,6-trideoxy-a-D-arabino-hexopyranoside
(8)
3
red at 80 ꢁC for 4 h. The reaction was terminated by
(100 mg, 0.44 mmol) in DMF (3 mL) were added succes-
sively NaH (24.5 mg, 0.61 mmol), BnBr (105 lL,
0.88 mmol) and tetra-n-butylammonium iodide (1 mg).
The reaction mixture was stirred at room temperature
addition of satd aq NaCl and extracted with CH Cl2
2
(
(
2 · 5 mL). The combined organic phases were dried
MgSO ) and purified by column chromatography over
4
silica gel using petroleum ether–EtOAc (v/v = 10/1) as
eluent. The title compound was obtained as a colourless
for 24 h and was then treated with excess of H O. The
2
aqueous phase was extracted with CH Cl (3 · 10 mL),
2
2
2
2
1
oil (3 mg, 31%): ½aꢁ +101.1 (c 1.0, CHCl ); H NMR
preadsorbed on silica gel and purified by column chro-
matography over silica gel using petroleum ether–
EtOAc (v/v = 6/1) as eluent. The title compound was
D
3
(
400 MHz, CDCl , CDCl = 7.26 ppm): d 1.77 (ddd,
3 3
J = 13.1, 12.5, 3.3 Hz, 1H, 2b-H), 2.13 (s, 3H,
2
D
2
CH CO–H), 2.21 (ddd, J = 13.1, 4.5, 1.3 Hz, 1H, 2a-
obtained as a colourless oil (134 mg, 95%): ½aꢁ
3
1
H), 3.22 (dd, J = 13.3, 2.6 Hz, 1H, 6b-H), 3.33 (dd,
J = 13.3, 7.1 Hz, 1H, 6a-H), 3.89 (ddd, J = 9.8, 7.1,
+187.3 (c 1.0, MeOH); H NMR (400 MHz, CDCl ,
3
CDCl = 7.26 ppm): d 1.71 (td, J = 12.8, 3.4 Hz, 1H,
3
2
3
.6 Hz, 1H, 5-H), 3.93 (ddd, J = 12.5, 9.7, 4.5 Hz, 1H,
2-H), 2.16 (ddd, J = 13.6, 4.9, 0.3 Hz, 1H, 2-H), 3.31
(d, J = 9.6 Hz, 1H, 4-H), 3.34 (s, 3H, OMe–H), 3.38
(dd, J = 13.0, 5.1 Hz, 1H, 6-H), 3.49 (dd, J = 13.0,
2.4 Hz, 1H, 5-H), 3.76 (ddd, J = 9.7, 5.3, 2.4 Hz, 1H,
-H), 4.01 (ddt, J = 12.9, 5.6, 1.4 Hz, 1H, CH @CH–
2
CH H O–H), 4.20 (ddt, J = 12.9, 5.6, 1.4 Hz, 1H,
CH @CH–CH H O–H), 4.81 (dd, J = 9.8, 9.7 Hz, 1H,
a
b
2
a
b

1712
T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
6
-H), 3.90 (ddd, J = 12.4, 9.3, 2.1 Hz, 1H, 3-H), 4.59 (d,
3.73–4.06 (m, 3H, 3-H, 5-H, CH @CH–CH –H), 4.15
2
2
J = 10.9 Hz, 1H, Bn–H), 4.79 (d, J = 3.1 Hz, 1H, 1-H),
(ddt, J = 13.0, 5.0, 1.4 Hz, 1H, CH @CH–CH –H),
2
2
1
3
4
.91 (d, J = 10.9 Hz, 1H, Bn–H); C NMR (100 MHz,
4.59 (d, J = 10.8 Hz, 1H, Bn–H), 4.91 (d, J = 10.8 Hz,
1H, Bn–H), 4.98 (d, J = 3.0 Hz, 1H, 1-H), 5.25 (dd,
J = 3.3, 1.6 Hz, 1H, CH @CH–CH –H), 5.34 (dd,
CDCl , CDCl = 77.2 ppm): d 35.3 (t, C-2), 51.3 (t, C-
3
3
6
(
1
), 54.8 (q, C–OMe), 60.1 (d, C-3), 70.5 (d, C-5), 75.0
t, C–Bn), 78.4 (d, C-4), 97.4 (d, C-1), 128.1 (d, C–Ph),
28.2 (d, C–Ph), 128.6 (d, C–Ph), 137.4 (s, C–Ph);
2
2
J = 3.3, 1.6 Hz, 1H, CH @CH–CH –H), 5.78–6.04 (m,
2
2
1H, CH @CH–CH –H), 7.29–7.50 (m, 5H, Ph–H);
2
2
+
+
HRMS m/z calcd for C H N O
[M+Na] :
HRMS m/z calcd for C H N O
3
[M+Na] :
1
4
18
6
3
16 20
6
3
41.1338. Found: 341.1342.
367.1495. Found: 367.1488.
3
.8.2. 3,6-Azido-4-O-benzyl-2,3,6-trideoxy-D-arabino-hexo-
3.8.4. Allyl 4-O-benzyl-2,3,6-trideoxy-3,6-trifluoroacetam-
ido-a-D-arabino-hexopyranoside (12). To a solution of
allyl 3,6-azido-4-O-benzyl-2,3,6-trideoxy-a-D-arabino-
hexopyranoside 10 (218 mg, 0.36 mmol) in dry pyridine
pyranose. To a solution of methyl 3,6-azido-4-O-benz-
yl-2,3,6-trideoxy-a-D-arabino-hexopyranoside described
above (568 mg, 1.78 mmol) in CH Cl (15 mL) was added
2
2
BCl (2 mL, 2.0 mmol) at ꢀ78 ꢁC. The solution was al-
(4 mL) was added PPh (665 mg, 2.5 mmol). The reac-
tion mixture was stirred at room temperature for 1 h,
3
3
lowed to warm to room temperature while being stirred
for 1 h, cooled to ꢀ78 ꢁC, treated with a phosphate buffer
solution and stirred for additional 30 min at room tem-
was treated with NH (410 lL, 5.1 mmol) and was al-
3
lowed to stand for 16 h. The solvent was removed in va-
cuo, CH Cl (20 mL), Et N (1.4 mL, 10.1 mmol) and
perature. The aqueous phase was extracted with Et O
2
2
2
3
(
3 · 20 mL) and concentrated in vacuo. The crude prod-
trifluoroacetic anhydride (705 lL, 5.1 mmol) were added
and the reaction mixture was stirred at room tempera-
ture for 3 h. MeOH was added, the reaction mixture
was preadsorbed on silica gel and purified by column
chromatography over silica gel using petroleum ether–
EtOAc (v/v = 4/1) as eluent. Title compound 12 was ob-
tained as colourless crystals (194 mg, 63%): mp 237 ꢁC;
uct was dissolved in MeCN (22 mL), and H O (5 mL) and
Ag CO were added. The mixture was stirred at room
temperature for 16 h. Na SO and Et O were added
and the mixture was filtered through a pad of Celite .
The crude product was preadsorbed on silica gel and
purified by column chromatography over silica gel using
petroleum ether–EtOAc (v/v = 6/1) as eluent. The title
compound was obtained as a colourless oil (485 mg,
9
2
2
3
2
4
2
TM
2
2
1
½aꢁ +52.8 (c 1.0, MeOH); H NMR (400 MHz, ace-
D
tone-d , acetone = 2.05 ppm): d 1.95–2.10 (m, 1H, 2-
6
1
0%): H NMR (400 MHz, CDCl , CDCl = 7.26 ppm):
H), 3.51 (t, J = 9.8 Hz, 1H, 4-H), 3.55–3.70 (m, 2H, 6-
H), 3.87 (ddd, J = 9.8, 7.3, 2.8 Hz, 1H, 5-H), 4.91
(ddt, J = 12.9, 5.6, 1.4 Hz, 1H, CH @CH–CH –H),
3
3
d 1.70 (ddd, J = 13.0, 12.6, 3.4 Hz, 1H, 2-H), 2.19 (ddd,
J = 13.3, 4.9, 1.0 Hz, 1H, 2-H), 3.34 (d, J = 9.5 Hz, 1H,
2
2
4
6
-H), 3.40 (d, 1H, 6-H), 3.52 (dd, J = 13.1, 2.5 Hz, 1H,
-H), 3.97 (dd, J = 9.4, 4.8 Hz, 1H, 3-H), 4.03 (ddd,
4.10 (ddt, J = 12.9, 5.6, 1.4 Hz, 1H, CH @CH–CH –
2
2
H), 4.45 (m, 1H, 3-H), 4.66 (d, J = 4.8 Hz, 2H, Bn–
H), 4.91 (d, J = 2.4 Hz, 1H, 1-H), 5.11 (ddd, J = 10.4,
3.3, 1.6 Hz, 1H, CH @CH–CH –H), 5.23 (ddd,
J = 9.6, 4.9, 2.6 Hz, 1H, 5-H), 4.60 (d, J = 10.8 Hz, 1H,
Bn–H), 4.91 (d, J = 10-8 Hz, 1H, Bn–H), 5.36 (d,
J = 2.9 Hz, 1H, 1-H). C NMR (100 MHz, CDCl3,
2
2
1
3
J = 17.0, 3.3, 1.6 Hz, 1H, CH @CH–CH –H), 5.85
2
2
CDCl = 77.2 ppm): d 35.4 (t, C-2), 51.3 (t, C-6), 59.5
(ddt, J = 17.0, 10.4, 5.6 Hz, 1H, CH @CH–CH –H),
8.45, 8.55 (m, 2H, NHTfa–H); C NMR (100 MHz,
3
2
2
13
(
9
(
d, C-3), 70.5 (d, C-5), 75.0 (t, C–Bn), 78.5 (d, C-4),
1.1 (d, C-1), 128.2 (d, C–Ph), 128.3 (d, C–Ph), 128.6
d, C–Ph), 137.3 (s, C–Ph); LRMS m/z calcd for
acetone-d , acetone = 29.8 ppm): d 36.1 (t, C-2), 41.5
6
(t, C-6), 49.5 (d, C-3), 68.3 (t, C–CH @CH–CH ), 70.7
2
2
+
C H N O [M+Na] : 327.3 Found: 327.3.
(d, C-5), 74.8 (t, C–Bn), 78.7 (d, C-4), 96.2 (d, C-1),
1
3
16
6
3
1
17.2 (t, C–CH @CH–CH ), 117.5 (q, J = 287.0 Hz,
2
2
3
.8.3. Allyl 3,6-azido-4-O-benzyl-2,3,6-trideoxy-a-D-ara-
bino-hexopyranoside (10). 3,6-Azido-4-O-benzyl-2,3,6-
trideoxy-D-arabino-hexopyranose described above
200 mg, 0.66 mmol) was added to a solution of acetyl
C–CF ), 128.6, 128.9, 129.1, 132.8 (C–Ph), 139.0 (d,
3
C–CH @CH–CH ), 159.0 (q, J = 36.8 Hz, C–CF CO);
2
2
3
+
HRMS m/z calcd for C H F N O [M+Na+MeCN] :
2
0
22
6
2
5
(
483.1355. Found: 483.1368.
chloride (150 lL) in allyl alcohol (5 mL). The reaction
was stirred for 18 h at room temperature and was
0
0
3.9. 1 ,4 -O-Di-(4-tert-butyldimethylsilyl-2,3,6-trideoxy-
3,6-trifluoroacetamido-a-D-arabino-hexopyranoside)-1,4-
butane (17)
stopped with Et N (200 lL). The crude product was
3
preadsorbed on silica gel and purified by column chro-
matography over silica gel using petroleum ether–
EtOAc (v/v = 6/1) as eluent. Title compound 10 was ob-
3.9.1.
Allyl
4-tert-butyldimethylsilyl-2,3,6-trideoxy-
tained as a mixture of a- and b-anomers (4:1) (218 mg,
3,6-trifluoroacetamido-a-D-arabino-hexopyranoside (16).
To a solution of allyl 2,3,6-trideoxy-3,6-trifluoroacet-
amido-a-D-arabino-hexopyranoside (11) (55 mg, 0.14
mmol) in CH Cl (3 mL) were added 2,6-lutidine
1
9
6%): H NMR (200 MHz, CDCl , CDCl = 7.26 ppm):
3
3
d 1.72 (td, J = 13.0, 3.3 Hz, 1H, 2-H), 2.19 (dd, J = 13.0,
.0 Hz, 1H, 2-H), 3.25–3.55 (m, 3H, 6-H, 6-H, 4-H),
5
2
2

T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
1713
2
D
2
1
(
49 lL, 0.42 mmol) and TBSOTf (64 lL, 0.28 mmol)
(25 mg, 58%): ½aꢁ +87.0 (c 1.0, MeOH); H NMR
and the reaction mixture was stirred at room tempera-
ture for 16 h. Additional 2,6-lutidine (49 lL, 0.42 mmol)
and TBSOTf (64 lL, 0.28 mmol) were added and the
(400 MHz, MeOD, MeOD = 3.31 ppm): d 0.06 (s, 3H,
CH Si–H), 0.13 (s, 3H, CH Si–H), 0.90 (s, 9H,
(CH ) CSi–H), 1.59–1.71 (m, 2H, OCH CH –H), 1.84
3
3
3
3
2
2
reaction mixture was stirred for another 2 h. H O was
(td, J = 12.6, 3.3 Hz, 1H, 2-H), 1.92 (dd, J = 12.6,
5.1 Hz, 1H, 2-H), 3.22 (dd, J = 13.1, 9.9 Hz, 1H, 4-H),
3.37–3.44 (m, 1H, OCH CH –H), 3.51 (t, J = 9.3 Hz,
2
added and the reaction was extracted with CH Cl
2
2
(
3 · 10 mL). The combined organic phases were pread-
2
2
sorbed on silica gel and purified by column chromatog-
raphy over silica gel using petroleum ether–EtOAc
1H, 6-H), 3.58–3.64 (m, 1H, OCH CH –H), 3.73 (td,
2 2
J = 9.5, 2.3 Hz, 1H, 5-H), 3.86 (dd, J = 13.2, 2.1 Hz,
1H, 6-H), 4.25 (ddd, J = 12.1, 9.4, 4.9 Hz, 1H, 3-H),
(
v/v = 6/1) as eluent. Title compound 16 was obtained
1
13
as a colourless oil (50 mg, 70%): H NMR (400 MHz,
4.88 (d, J = 2.9 Hz, 1H, 1-H); C NMR (100 MHz,
MeOD, MeOD = 3.31 ppm): d 0.06 (s, 3H, CH Si–H),
MeOD, MeOD = 49.0 ppm): d ꢀ4.1 (q, C–CH Si),
3
3
0
1
.14 (s, 3H, CH Si–H), 0.90 (s, 9H, (CH ) CSi–H),
.85 (td, J = 13.0, 3.5 Hz, 1H, 2-H), 1.92 (ddd,
ꢀ3.6 (q, C–CH Si), 18.8 (q, C–(CH ) CSi), 26.3 (s, C–
3
3 3
3
3 3
(CH ) CSi), 27.4 (t, C–OCH CH ), 36.4 (t, C-2), 42.6
3 3 2 2
J = 13.0, 5.2, 1.0 Hz, 1H, 2-H), 3.22 (dd, J = 13.2,
0 Hz, 1H, 4-H), 3.51 (t, J = 9.3 Hz, 1H, 6-H), 3.73
td, J = 9.6, 2.2 Hz, 1H, 5-H), 3.86 (dd, J = 13.2,
.1 Hz, 1H, 6-H), 3.93 (ddt, J = 12.8, 6.0, 1.3 Hz, 1H,
CH @CH–CH –H), 4.10 (ddd, J = 12.8, 5.3, 1.4 Hz,
(t, C-6), 51.0 (d, C-3), 68.0 (t, C–OCH CH ), 72.2 (d,
2 2
C-4), 73.0 (d, C-5), 96.1 (d, C-1), 117.4 (q,
1
(
2
J = 286.9 Hz, C–CF ), 117.6 (q, J = 286.7 Hz, C–CF ),
3
3
158.7 (q, J = 36.7 Hz, C–CF CO), 159.1 (q, J =
3
36.9 Hz, C–CF CO); HRMS m/z calcd for C H F -
2
2
3
36 58 12
ꢀ
1
H, CH @CH–CH –H), 4.27 (ddd, J = 12.0, 9.4,
N O Si [M] : 989.3422. Found: 989.3439.
4 10 2
2
2
5
.1 Hz, 1H, 3-H), 4.88 (d, J = 2.9 Hz, 1H, 1-H), 5.17
(
(
5
dd, J = 10.3, 1.3 Hz, 1H, CH @CH–CH –H), 5.27
2
2
3
3
.10. Fluorescence titration
.10.1. TAR (trans-activation-response) fragment. For
dd, J = 17.2, 1.6 Hz, 1H, CH @CH–CH –H), 5.86–
2
2
1
3
.98 (m, 1H, CH @CH–CH –H);
C NMR (100
2
2
MHz, MeOD, MeOD = 49.0 ppm): d ꢀ4.1 (q, C–
the fluorescence titration an unlabelled 31-nucleotides
containing TAR fragment (sequence: 5 -GGC CAG
AUC UGA GCC UGG GAG CUC GGC C-3 ) was
used. The TAR fragment showed an extinction coeffi-
cient of 192300 L/(mol cm) at 260 nm.
CH Si), ꢀ3.6 (q, C–CH Si), 18.8 (q, C–(CH ) CSi),
3
3
3 3
0
2
(
7
6.3 (s, C–(CH ) CSi), 36.3 (t, C-2), 42.6 (t, C-6), 50.8
3 3
0
d, C-3), 68.7 (t, C–CH @CH–CH ), 72.4 (d, C-4),
2
2
3.0 (d, C-5), 96.3 (d, C-1), 117.4 (q, J = 287.2 Hz, C–
CF ), 117.6 (q, J = 286.6 Hz, C–CF ), 117.6 (t, C–
3
3
CH @CH–CH ), 135.3 (d, C–CH @CH–CH ), 158.5
2
2
2
2
(
q, J = 36.6 Hz, C–CF CO), 159.0 (q, J = 36.9 Hz,
3.10.2. F-Tat (transactivator of transcription)-pep-
tide. For the fluorescence titration the Tat-peptide
marked with a chromophore, namely rhodamine green,
which was bound via a DOA-linker to the Tat-peptide,
was used (sequence: RhoG-DOA-RKKR RQRRR-
COOH). The peptide was purified by HPLC to 95% pur-
ity. It has a molecular weight of 1840 g/mol and an
extinction coefficient of 74,000 L/(mol cm) at 530 nm.
Rhodamine Green (RhoG) is excited at 504 nm and
emitted at 530 nm.
3
C–CF CO); HRMS m/z calcd for C H F N O Si
3
19 29
6
2
5
ꢀ
[
M] : 508.1828. Found: 508.1835.
0 0
1 ,4 -O-Di-(4-tert-butyldimethylsilyl-2,3,6-tride-
3
.9.2.
oxy-3,6-trifluoroacetamido-a-D-arabino-hexopyranoside)-1,
-butane (17). To a solution of allyl 4-tert-butyldimeth-
4
ylsilyl-2,3,6-trideoxy-3,6-trifluoroacetamido-a-D-arabino-
hexopyranoside (16) (44 mg, 86 lmol) in CH Cl (2 mL)
was added Grubbs precatalyst 15 (3 mg, 3.7 lmol). The
2
2
reaction mixture was stirred at room temperature for
The titration experiments were carried out in cuvettes,
which had been pretreated with dimethyldichloro silane
prior to use. A solution consisting of TAR-RNA
(200 nM) and Tat-peptide (100 nM) was added to the
cuvette. Under these conditions about 90% of the Tat/
TAR-complex had formed. Defined concentrations (up
to 60 lM) of 5 were titrated to the solution and the fluo-
rescence signal was measured.
1
3
6 h and a second portion of Ru-complex 15 (3 mg,
.7 lmol) was added. The reaction mixture was stirred
at 40 ꢁC for 8 h. A third portion of 15 (3 mg, 3.7 lmol)
was added and the reaction mixture was stirred at 40 ꢁC
for 48 h. The reaction mixture was filtered through a
TM
pad of Celite and the solvent was removed in vacuo.
The crude product was dissolved in a solvent mixture
consisting of EtOAc–CH Cl –MeOH (1 mL, v/v/v =
2
2
1
6/8/1) and PtO (10 mg) was added. The reaction mix-
2
ture was stirred under an atmosphere of H at room
Acknowledgements
2
temperature for 16 h, was preadsorbed on silica gel
and purified by column chromatography over silica gel
using petroleum ether–EtOAc (v/v = 6/1) as eluent.
Title compound 17 was obtained as a colourless oil
This work was supported by the Deutsche Forschungs-
gemeinschaft (SFB 416, project B2) and by the Fonds
der Chemischen Industrie. We thank J. Alves (Medical

1714
T. J o¨ ge et al. / Carbohydrate Research 342 (2007) 1704–1714
University Hannover, MHH) for helpful support with
respect to fluorescence titration.
6. Sicherl, F.; Wittmann, V. Angew. Chem. 2005, 117, 2133–
136; Angew. Chem., Int. Ed. 2005, 44, 2096–2099.
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Science 1994, 264, 375–382; (c) Shaw, K. J.; Rather, P. N.;
Hare, R. S.; Miller, G. H. Microbiol. Rev. 1993, 57, 138–
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