Phosphoramidites and solid supports based on N-substituted 2,4-dihydroxybutyramides: universal reagents for synthesis of modified oligonucleotides

Article abstract of DOI:10.1016/j.tet.2006.05.008

A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-α-hydroxy-γ-butyrolactone or (R)-(-)-pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues) into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.

Full text of DOI:10.1016/j.tet.2006.05.008

Tetrahedron 62 (2006) 6762–6773
Phosphoramidites and solid supports based on N-substituted
2,4-dihydroxybutyramides: universal reagents for synthesis of
modified oligonucleotides
Natalia N. Dioubankova,a Andrei D. Malakhov,a Dmitry A. Stetsenko,b,y
Michael J. Gaitb and Vladimir A. Korshuna,
*
aShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10,
117997 Moscow, Russian Federation
bMedical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Received 24 November 2005; revised 12 April 2006; accepted 4 May 2006
Available online 2 June 2006
Abstract—A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites
is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-a-hydroxy-g-butyrolactone or (R)-(ꢀ)-
pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After
protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide
synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues)
into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
A 1,3-diol system with one primary and one secondary
hydroxyl group mimicking the 50- and 30-OH groups of
nucleosides is an obvious structural element in the design
of non-nucleoside reagents suitable for machine-aided
assembly of modified oligonucleotides. We recently reported
that it can be obtained easily by acylation of amines with
a-hydroxy-g-butyrolactones.5 In the procedure, a suitable
compound with an aliphatic primary or even secondary
amino group can be converted into the corresponding
N-substituted 2,4-dihydroxybutyramide. The precursor is
then subjected to successive steps of side-chain protection
(if required), 4,40-dimethoxytritylation of the primary
hydroxyl group, and conversion into the phosphoramidite
or attachment to a solid support via the secondary hydroxyl
group. Herewe describe the synthesis of several new reagents
that allow the introduction of reactive (amine), catalytically
active (imidazole) and fluorescent (pyrene) residues into
oligonucleotides as well as the preparation and characterisa-
tion of various oligonucleotide conjugates.
Although modified oligonucleotides have diverse use in
chemistry, biology and medicine, the emergence of new areas
of their application is a current trend.1 Solid-phase phosphor-
amidite oligonucleotide synthesis, a highly efficient and reli-
able technique developed in the 1980s,2 is still a mainstay of
chemical DNA preparation. Numerous modifying phosphor-
amidite reagents for oligonucleotide synthesis also have been
developed3 and many of them are commercially available.
The potential of phosphoramidites for combinatorial chemis-
try has been recognised.4 Our aim was to develop reagents
applicable for the preparation of oligonucleotide conjugates
in combinatorial mode, for instance, for synthesis of artificial
ribonucleases that have an oligonucleotide part for comple-
mentary recognition and a variable catalytic part carrying
active functional groups, e.g., amine, imidazole, etc.
Keywords: Modified oligonucleotides; Phosphoramidites; 2,4-Dihydroxy-
butyramides; Imidazole; Pyrene.
Abbreviations: DIC, 1,3-diisopropylcarbodiimide; DMAP, 4-dimethylamino-
pyridine; Dmt, 4,40-dimethoxytrityl; Fmoc, 9-fluorenylmethoxycarbonyl;
FmocOSu, 9-fluorenylmethyl N-succinimidyl carbonate; LCAA-CPG,
long-chain alkylamino controlled pore glass; Py, pyridine; RP-HPLC,
reversed-phase HPLC; Tfa, trifluoroacetyl.
2. Results and discussion
2.1. Synthesis of phosphoramidites
* Corresponding author. Tel./fax: +7 495 3306738; e-mail: korshun@mail.
ibch.ru
Present address: School of Biomedical and Molecular Sciences, Univer-
y
a-Hydroxy-g-butyrolactones 1a,b are known to react
smoothly with amines.6 No racemisation at the a-carbon
sity of Surrey, Guildford GU2 7XH, UK.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.008

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6763
atom was reported. We found that in the case of a-hydroxy-g-
butyrolactones 1a and 1b a simple un-catalysed reaction with
primary amines at a slightly elevated temperature gives very
good yields of the corresponding 2,4-dihydroxybutyramides
after 24–48 h. Primary amines with a bulky substituent, e.g.,
pyrene or secondary amine, e.g., N,N0-dimethylethylenedi-
amine require much longer reaction times.
Imidazole residues of histidines are present in catalytic cen-
tres of DNA- and RNA-cleaving enzymes.11 Many syntheses
of imidazole–oligonucleotide conjugates as potential target-
addressed RNA cleavers are reported.12 It was found that the
Boc group is suitable for histidine NIm-protection during
phosphoramidite DNA synthesis, undergoing smooth cleav-
age upon standard ammonolytic deprotection conditions.13
This prompted us to investigate its use for imidazole protec-
tion in phosphoramidite reagents 10a,b.
a,u-Diamines served as precursors for aminolinker re-
agents. Lactones 1a and 1b were treated with an excess
(5.0 equiv) of ethylenediamine or N,N0-dimethylethylenedi-
amine at 55 ꢁC for 1–7 days to give mono-acylated products
2 (Scheme 1).
The synthesis of the imidazole reagents is outlined in
Scheme 2. The primary amino group of histamine was acyl-
ated with lactones 1a or 1b to give dihydroxybutyramide 7a
The remaining amino function was masked with Fmoc or
Tfa, common amino protecting groups compatible with oli-
gonucleotide synthesis,3 affording 3a–d. Compound 3a pre-
cipitated readily from the reaction mixture. By contrast, the
presence of two geminal 3-methyl groups in the pantolac-
tone-derived compound 3b or N-methylation in 3c,d allow
their purification by column chromatography. The hydroxyl
groups in compounds 3 were functionalised by known proce-
dures of nucleoside chemistry:7 the primary hydroxyl group
was 4,40-dimethoxytritylated8 selectively, and the resulting
compounds 4 were converted into phosphoramidites 5a–c9
and solid-supported reagents 6a–c.10 The loading of the sup-
ports was estimated to be in the range of 35–52 mmol/g. Phos-
phoramidites were found to decompose significantly onsilica
gel during column chromatography and thus were isolated by
precipitation into hexane. Reagents 5a–c are white solids that
can be stored at ꢀ20 ꢁC for at least several months without
any substantial loss of reactivity. Phosphoramidite 5b is
appreciably more stable upon storage compared to 5a, which
indicates a positive shielding effect of the two methyl groups.
The reagents allow introduction of one or several primary or
secondary amino groups into any position within an oligo-
nucleotide sequence.
OH
OH
R
R
R
R
a
b
H
N
H
N
π
1a,b
N
N
HO
HO
Boc
O
7a,b
O
NH
N
τ
8a,b1,b2
a R = H
b R = Me
a
R = H
b1 R = Me, τ-Boc
b2 R = Me, π-Boc
ODmt
ODmt
R
R
R
R
H
N
d
c
N
H
N
O
P
N
HO
O
Boc
Pri2N
O
NBoc
O
N
10a,b
9a,b1,b2
CN
a R = H
b R = Me
a
R = H
b1 R = Me, τ-Boc
b2 R = Me, π-Boc
Scheme 2. Synthesis of imidazole phosphoramidites. Reaction conditions
and yields: (a) histamine, 55 ꢁC, 83% (7a); (b) Boc2O, 76% (8a), 38%
and 17% (8b1 and 8b2 from 1b); (c) DmtCl, Py, 0 ꢁC to rt, 1–3 h, 86%
(9a), 55% and 37% (9b1 and 9b2 from 8b1); (d) (iPr2N)2PO(CH2)2CN,
diisopropylammonium tetrazolide, DCM, 80% (10a), 60% (10b).
OH
OH
ODmt
R
O
R
R
R
R
R
R1
N
c or d
R
R
R1
N
e
a or b
R1
N
OH
NHR1
HO
NR1R2
HO
NR1R2
HO
O
O
2a-c
O
3a-d
O
4a-d
1a,b
a R = R1 = H
a R = R1 = H, R2 = Fmoc
a R = H
b R = Me
b R = Me, R1 = H
c R = H, R1 = Me
b R = Me, R1 = H, R2 = Fmoc
c R = H, R1 = Me, R2 = Fmoc
d R = H, R1 = Me, R2 = Tfa
ODmt
ODmt
R
R
R
R
R1
N
R1
N
f or g
NR1R2
O
P
NR1R2
O
O
O
Pri2N
O
O
support
CN
5a-c
6a-c
a R = R1 = H, R2 = Fmoc
a R = R1 = H, R2 = Fmoc
b R = Me, R1 = H, R2 = Fmoc
c R = H, R1 = Me, R2 = Tfa
b R = Me, R1 = H, R2 = Fmoc
c R = H, R1 = Me, R2 = Fmoc
Scheme 1. Synthesis of amino-modifier reagents and solid supports. Reaction conditions and yields: (a) (CH2NH2)2, 55 ꢁC, overnight, quant. (2a–b);
(b) (CH2NHMe)2, 55 ꢁC, 7 days, quant. (2c); (c) FmocOSu, aq MeCN, 88% (3a), 75% (3b), 64% (3c); (d) CF3CO2Me, rt, 30 min; (e) DmtCl, Py, 0 ꢁC
to rt, 1–3 h, 81% (4a), 97% (4b), 89% (4c), 93% (4d from 2c); (f) (iPr2N)2PO(CH2)2CN, diisopropylammonium tetrazolide, DCM, rt, 2 h, 85% (5a),
67% (5b), 77% (5c); (g) succinylated LCAA-CPG, DIC, DMAP, Py, rt, loading (mmol/g): 35 (6a), 52 (6b), 44 (6c).

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N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
or 7b, respectively. These were treated with Boc2O to afford
imidazole-protected compounds 8. Only the t-Boc isomer
was isolated in the case of 8a, whilst 7b gave a mixture of
t- and p-isomers (8b1 and 8b2), easily separable by column
chromatography. Dimethoxytritylation of 8 followed by
phosphitylation yielded phosphoramidites 10. Only the
major isomer 8b1 was used in the reaction with DmtCl in
pyridine.
ODmt
OH
R
R
R
R
b
a
H
N
H
N
1a,b
HO
HO
O
O
12a,b
a R = H
b R = Me
11a,b
a R = H
b R = Me
c
Surprisingly, two separable products 9b1 and 9b2 were
again isolated after a usual workup and column chromato-
graphy. This indicates that partial t/p migration of the
Boc group takes place during dimethoxytritylation, possibly
catalysed by pyridine hydrochloride formed in the reaction.
p-Isomers (8b2 and 9b2) were found to be unstable: after
storage of the compounds as dried solids for one month at
+4 ꢁC, 8b2 gave ca. 1:1 mixture of 8b1 and 8b2, and 9b2
was completely converted into 9b1. The structures of com-
pounds 8b1, 8b2 and 9b1 were determined using Rapoport’s
NMR criteria.14,15 Moreover, a ROESY experiment for 9b1
(spectral width 10 ppmꢂ10 ppm, 16,000ꢂ512 of complex
points) showed similar cross-peaks of medium intensity
between Boc protons and both H-2 and H-5 of the imidazole
moiety.
d
ODmt
ODmt
R
R
R
R
H
N
H
N
O
O
O
P
O
Pri2N
O
O
support
CN
13a,b
14a,b
a R = H
b R = Me
a R = H
b R = Me
Scheme 3. Synthesis of pyrene phosphoramidites. Reaction conditions and
yields: (a) 1-pyrenemethylamine, 55 ꢁC, 100% (11a), 92% (11b); (b)
DmtCl, Py, 62% (12a), 83% (12b); (c) (iPr2N)2PO(CH2)2CN, diisopropyl-
ammonium tetrazolide, DCM, 70% (13a), 92% (13b); (d) succinylated
LCAA-CPG, DIC, DMAP, pyridine, rt, loading (mmol/g): 20 (14a), 16 (14b).
Interestingly, the less stable p-Boc isomers and their rear-
rangement to thermodynamically more stable t-Boc com-
pounds were detected in the ‘b’ series only. The influence
of the remote gem-dimethyl group in 7b on regioselectivity
of imidazole acylation in comparison with 7a seems to be
unlikely. The observed difference could be explained ratio-
nally by the following assumptions: (1) p-Boc isomer is
also formed from 7a, but converts rapidly into the stable
t-Boc compound; (2) the substantial difference in p/
t-migration rates between a and b series is caused by spatial
hindrance of the hydroxyl(s) that probably facilitates the
rearrangement.
2.2. Synthesis and characterisation of modified
oligonucleotides
All the phosphoramidites and supports obtained were tested
in machine-assisted solid-phase oligonucleotide synthesis.
The coupling time for the modified phosphoramidites 5a–
c, 10a,b and 13a,b was extended to 10 min. The coupling
yields were comparable to those for nucleoside phosphor-
amidites (determined by the released Dmt cation). The conju-
gates prepared were cleaved from their supports, deprotected
by concd aq ammonia treatment at 55 ꢁC overnight, analysed
by reversed-phase HPLC and MALDI-TOF MS and purified
by PAGE for thermal denaturation studies. Some loss of the
50-terminal modified unit(s) was observed when the Dmt
group was removed prior to ammonolysis, possibly resulting
from the attack of the 50-terminal hydroxyl on the neighbour-
ing phosphodiester bond. To obtain uniformly high yields of
the 50-modified oligonucleotides these were synthesised rou-
tinely in ‘Dmt On’ mode, and the Dmt protecting group was
cleaved after ammonia treatment. Sequences and some prop-
erties of the modified oligodeoxyribonucleotides are sum-
marised in Table 1. Examples of HPLC traces are shown
in Figure 1. Even in the crude mixtures after synthesis, the
content of the desired conjugates was usually more than
90% with almost no detectable amount of any side products.
Compound 9b1 was then phosphitylated to give 10b, a pair
of diastereomers arising from the phosphoramidite group,
as a single product. No migration of the Boc group was
observed during the reaction.
Of course, the exact position of the Boc group in the reagents
10a,b does not influence the structure of the resultant imida-
zole–oligonucleotide conjugates, since both t- and p-Boc
isomers should be deprotected in concd aq ammonia.
To the best of our knowledge, amongst pyrene non-nucleo-
side reagents described to date, only a few examples contain
a 1,3-diol backbone.16 Multistep syntheses of deoxyribosyl
pyrene C-nucleosides are complex, time consuming and
include separation of a mixture of a- and b-anomers.17
Scheme 3 depicts a straightforward synthesis of new pyrene
phosphoramidite reagents 13a,b and supports 14a,b from
1-pyrenemethylamine.
As expected, the incorporation of the hydrophobic pyrene
moiety into oligonucleotides increases the retention time of
conjugates on the HPLC column whilst other 2,4-dihydroxy-
butyramide substituents influence the chromatographic
mobility of the corresponding oligomers only negligibly.
The structures of 2,4-dihydroxybutyramide derivatives 2a,
3a,b, 7a, 8a,b1 and 11a,b were confirmed by 1H–13C
NMR correlations. The assignment of signals in the 13C
NMR spectra was performed using HMQC (cross-peaks
from proton-bound carbon atoms) and HMBC (cross-peaks
from interaction through two, three and four bonds) spectra.
Thermal stability experiments show, as expected,18 an increase
in Tm for duplexes with pyrene 2,4-dihydroxybutyramide in

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6765
Table 1. Properties of oligodeoxyribonucleotides bearing (R)-2,4-dihydroxybutyramide non-nucleoside units
a
#
Sequence, 50 to 30
Amidite or support
source of X
MALDI-TOF,
found/calcd
RP-HPLC retention
time, minb
ON01
ON02
ON03
ON04
ON05
ON06
ON07
ON08
ON09
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
ON18
ON19
ON20
ON21
ON22
ON23
ON24
ON25
ON26
CTCCCAGGCTCAAATXp
CTCCCAGGCTCAAATXp
XCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATX
CTCCCAGGCTCAAATXX
CTCCCAGGCTCAAATXp
XCTCCCAGGCTCAAATX
CTCCCAGGCTCAAATX
CTCCCAGGCTCAAATX
ATTTGAGCCTGGGAGX
ATTTGAGCCTGGGAGX
CTCCCAGGCTCAAATX
CTCCCAGGCTCAAATXp
XCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATXp
XCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATXXp
XXCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATXXp
XXCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATX
CTCCCAGGCTCAAATXp
XCTCCCAGGCTCAAAT
CTCCCAGGCTCAAATXXp
XXCTCCCAGGCTCAAAT
XCTCCCAGGCTCAAATXp
5a
5b
5b
6a
4801.1/4796.8
4824.8/4826.9
4750.8/4749.2
4718.7/4719.2
5078.8/5078.9
4970.1/4969.9
5341.7/5341.0
4890.5/4889.9
4920.6/4920.3
5041.9/5040.9
5070.4/5071.4
4747.9/4749.2
4851.8/4850.8
4773.5/4772.1
4888.9/4881.2
4804.7/4801.2
5124.6/5124.9
5044.2/5044.9
5182.8/5183.4
5102.5/5103.5
4750.3/4749.2
4847.3/4844.1
4748.0/4749.2
5081.7/5080.0
5001.7/5001.4
5079.0/5080.0
15.20
12.68
12.48
12.75
13.23
22.21
34.80
24.85
27.71
21.42
25.30
12.51
12.79
12.63
12.55
13.17
12.52
12.54
13.02
14.14
13.78
12.90
12.64
13.26
12.71
12.59
6b, 5b
13a
13b, 14b
14a
14b
14a
14b
6b
10a
10a
10b
10b
10a
10a
10b
10b
6c
5c
5c
5c
5c
5c
d
—
ON27
CUCCCAXGCUCAc
(5a+10a+dG)
3731.7/3729.1
3784.5/3780.0
3836.7/3833.2
d
—
ON28
CUCCCAXGCUCAc
(5b+10b+20OMe-rG)
3761.5/3762.5
3812.6/3814.1
3868.6/3873.3
a
p—30-phosphate.
HPLC conditions are given in Section 4.
20-O-Methylribonucleotides are in bold.
Broad unresolved peak.
b
c
d
a dangling position and a slight destabilisation for duplexes
with the 30-phosphate group (Table 2). Other terminal modi-
fications influence the Tm values insignificantly.
3. Conclusions
Our results demonstrate that various primary and secondary
aliphatic amines may be easily converted into 2,4-dihydroxy-
butyramide phosphoramidites and solid supports and used
for the preparation of modified oligonucleotides carrying
diverse functional groups, e.g., amine, imidazole or pyrene.
When a mixture of modifying phosphoramidites is used,
a mixture of conjugates is produced that shows the potential
of the reagents for application in combinatorial chemistry.
Next, we tested the suitability of 2,4-dihydroxybutyramide
reagents for the generation of combinatorial libraries of oli-
gonucleotides. We used a 1:1:1 (mol) mixture of 5a and
10a with dG phosphoramidite for synthesis of ON27 and
5b and 10b with 20-OMe-rG phosphoramidite in the same ra-
tio for synthesis of ON28. The sum of the concentrations of
phosphoramidites was 0.1 M in acetonitrile. Oligonucleo-
tides were synthesised in ‘Dmt On’ mode and purified by
RP-HPLC (for conditions see Section 4). The purified Dmt-
containing oligomers were treated with 80% acetic acid at
room temperature for 30 min, isolated by 1 M LiClO4–ace-
tone precipitation, and analysed by RP-HPLC and MALDI-
TOF MS. In both cases, HPLC showed only one broad
peak at ca. 15 min (data not shown). In the mass spectra, three
main signals corresponding to individual library members
were present (Fig. 2).
4. Experimental
4.1. General
500 MHz 1H, 125.7 MHz 13C and 202.4 MHz 31P NMR spec-
tra were recorded on a Bruker DRX-500 spectrometer and ref-
erenced to DMSO-d6 (2.50 ppm for H and 39.70 ppm for
1
1
13C), MeCN-d3 (1.96 ppm for H), MeOH-d3 (3.34 ppm for
1H and 49.00 ppm for 13C) and 85% aq H3PO4 (0.00 ppm
for31P). 1H–13Cgradient-selectedHMQCand HMBC spectra
were obtained by using 2048 (t2)ꢂ256 (t1) complex point
data sets, zero filled to 2048 (F2)ꢂ1024 (F1) points. The
spectral widths were 13 and 200 ppm for 1H and 13C dimen-
sions, respectively. HMBC spectra were measured with
50 ms delay for evolution of long-range couplings. Varian
The spectra indicate that the reactivity of phosphoramidites
derived from 2,4-dihydroxybutyramide is similar to 20-
deoxyribonucleoside phosphoramidites, and of those from
2,4-dihydroxy-3,3-dimethylbutyramide to 20-O-methyl-
ribonucleoside phosphoramidites.

6766
N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
a
a
15000
10000
3
5000
0
2
1
0
10
20
30
40
t, min
2000
3000
4000
5000
m/z
b
b
15000
10000
5000
0
3
2
1
0
10
20
t, min
30
40
2000
2500
3000
3500
m/z
4000
4500
5000
Figure 1. (a) Typical RP-HPLC traces of crude (R)-2,4-dihydroxybutyr-
amide-containing oligodeoxyribonucleotides: (1) 2-aminoethyl ON04,
(2) N,N0-dimethyl-2-aminoethyl ON21 and (3) 1-pyrenemethyl ON08
(Table 1). (b) Typical RP-HPLC traces of crude (R)-2,4-dihydroxy-3,3-
dimethylbutyramide-containing oligodeoxyribonucleotides: (1) 2-amino-
ethyl ON02, (2) 2-(imidazol-40-yl)ethyl ON15 and (3) 1-pyrenemethyl
ON09 (Table 1). For HPLC conditions, see Section 4.
Figure 2. MALDI-TOF mass spectra of combinatorial mixtures ON27 (a)
and ON28 (b).
were recorded on Micromass LCTreflection TOF mass spec-
trometer. IR spectra were recorded using Bruker Vector 22
spectrometer. Melting points were determined using a Boe-
tius heating table and are uncorrected. Analytical thin-layer
chromatography was performed on Kieselgel 60 F254 pre-
coated aluminium plates (Merck), spots were visualised un-
der UV light (254 nm). Silica gel column chromatography
was performed using Merck Kieselgel 60 0.040–0.063 mm.
Unity NMR spectrometer (600 MHz) was used for measur-
ing DQF-COSY and ROESY spectra. H NMR coupling
constants are reported in Hertz and refer to apparent multi-
plicities. ESI-TOF HRMS spectra in positive ion mode
1
Table 2. Stability of various (R)-2,4-dihydroxybutyramide-modified oligo-
deoxyribonucleotides as duplexes with an unmodified complementary DNA
strand
Reagents obtained from commercial suppliers were used
without further purification unless otherwise noted. Diiso-
propylammonium tetrazolide was prepared as described.19
DCM was used freshly distilled from CaH2. THF was freshly
#
Modifying
reagent
Position of
modification
Tm, ꢁC
DTm/mod, ꢁC
ON04
ON08
ON09
ON10
ON11
ON13
ON15
ON16
ON17
ON18
ON19
ON20
ON22
ON23
ON24
ON25
ON26
6a
30-
57.3
61.6
62.0
59.1
58.5
57.4
57.3
58.9
56.9
57.9
56.9
58.0
57.0
58.1
56.7
58.4
56.7
ꢀ0.3
+4.0
+4.4
+1.5
+0.9
ꢀ0.2
ꢀ0.3
+1.3
ꢀ0.4
+0.2
ꢀ0.4
+0.2
ꢀ0.6
+0.5
ꢀ0.5
+0.4
ꢀ0.5
˚
14a
14b
14a
14b
10a
10b
10b
10a
10a
10b
10b
5c
30-
distilled from powdered LiAlH4 and stored over 4 A molec-
ular sievesunder argon. Other solvents were used as received.
30-
30-
30-
Oligonucleotide synthesis was carried out on a ABI 380B
DNA/RNA synthesiser either on 0.2 or 1 mmol scale using
20-deoxynucleoside phosphoramidites from Cruachem
(Scotland). The molecular mass of each oligonucleotide
was checked by MALDI-TOF MS on a Perseptive Biosys-
tems Voyager DE workstation in positive ion mode using a
mixture (1:1 v/v) of 2,6-dihydroxyacetophenone (40 mg/
mL in MeOH) and aq diammonium hydrogen citrate
(80 mg/mL) as a matrix premixed just before loading the
samples onto a plate. Thermal denaturation experiments
were performed on a Perkin–Elmer Lambda 40 UV/vis
30-
30-
50-
30-
50-
30-
50-
30-
5c
5c
5c
5c
50-
30-
50-
50- and 30-

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6767
Spectrometer with PTP 6 (Peltier Temperature Programmer)
in a buffer containing 100 mM NaCl, 10 mM Na-phosphate,
0.1 mM EDTA, pH 7.0.
as a white crystalline powder. An additional amount of prod-
uct (0.90 g) was obtained by evaporation of combined fil-
trate, dissolving the residue in EtOAc (100 mL), washing
with water (2ꢂ70 mL), drying (Na2SO4), evaporation and
i
4.1.1. (2R)-N-(2-Aminoethyl)-2,4-dihydroxybutyramide
(2a).
crystallisation from PrOH. Total yield 3.40 g (88%), mp
A
solution of (R)-(+)-a-hydroxy-g-butyrolactone
133.5–134.0 ꢁC (iPrOH). Rf 0.13 (CHCl3–MeOH 9:1 v/v).
ESI-TOF HRMS: m/z¼385.1759 [M+H]+, calcd for
[C21H24N2O5+H]+ 385.1758. IR (KBr): 3321 cmꢀ1, n (OH,
NH); 2944 cmꢀ1, n (sp3-CH); 1694 cmꢀ1, n (CaO);
1536 cmꢀ1, n (NH); 1451 cmꢀ1, n (sp3-CH). 1H NMR
(DMSO-d6) d 7.88 (m, 2H, ArH (H-4, H-5)), 7.77 (br s, 1H,
CHCONH), 7.68 (m, 2H, ArH (H-1, H-8)), 7.40 (m, 2H,
ArH (H-3, H-6)), 7.33 (m, 2H, ArH (H-2, H-7)), 7.28 (br s,
1H, OCONH), 5.40 (d, 1H, J¼5.4 Hz, CHOH), 4.40 (t, 1H,
J¼5.1 Hz, CH2OH) (both exchangeable with D2O), 4.30
(m, 2H, CH2OCO), 4.21 (t, 1H, J¼6.9 Hz, fluorene H-9),
3.94 (m, 1H, CHO) (in the presence of D2O gives dd,
J1¼3.7 Hz, J2¼8.4 Hz), 3.49 (m, 2H, CH2OH), 3.16 (m,
2H), 3.07 (m, 2H) (NCH2CH2), 1.83 (m, 1H), 1.55 (m, 1H)
(CH2CH2CH). 13C NMR (DMSO-d6) d 174.91 (HNCaO),
156.64 (OCaO), 144.35 (2C), 141.18 (2C), 128.05 (2C),
127.51 (2C), 125.57 (2C), 120.55 (2C) (Ar, Fmoc), 69.04
(CHOH), 65.81 (CH2OCO), 57.91 (CH2OH), 47.18 (fluorene
C-9),w40z NCH2), 38.66 (NCH2), 38.04 (CCH2C).
(1.02 g, 10 mmol) in ethylenediamine (3.5 mL, 50 mmol) was
kept at 55 ꢁC overnight, evaporated, co-evaporated with toluene
(30 mL) and N,N-diisopropylethylamine (1.8 mL, 10 mmol).
The residue was triturated in Et2O under argon and dried
in vacuo to yield amide 2a (1.63 g, 100%) as a white solid. ESI-
TOF HRMS: m/z¼163.1074 [M+H]+, calcd for [C6H14N2O3+
1
H]+ 163.1077. H NMR (DMSO-d6) d 7.81 (br s, 0.19H),
7.67 (br s, 0.81H) (CONH, exchangeable with D2O), 3.93
(dd, 1H, J1¼3.6 Hz, J2¼8.6 Hz, CHO), 3.49 (m, 2H, CH2O),
3.40–3.00 (br, 2H,z NH2, exchangeable with D2O), 3.07 (appar-
ent q, 2H, J¼6.3 Hz, CH2NH), 2.57 (t, 2H, J¼6.3 Hz,
CH2NH2), 1.83 (m, 1H), 1.55 (m, 1H) (CH2CH2CH). 13C
NMR (DMSO-d6) d 174.63 (CaO), 69.07 (CHOH), 57.92
(CH2OH), 41.97, 41.65 (CH2CH2), 38.07 (CCH2C).
4.1.2. (2R)-N-(2-Aminoethyl)-2,4-dihydroxy-3,3-di-
methylbutyramide (2b). Compound 2b was prepared in
a similar manner as 2a from (R)-(ꢀ)-pantolactone (3.25 g,
25 mmol) and ethylenediamine (9 mL, 125 mmol). After co-
evaporation with toluene (100 mL) and N,N-diisopropylethyl-
amine (4.4 mL, 25 mmol), trituration in Et2O and drying,
the desired amide 2b (4.76 g, 100%) was obtained as a white
solid. ESI-TOF HRMS: m/z¼191.1388 [M+H]+, calcd for
[C8H18N2O3+H]+ 191.1390. IR (KBr): 3342 cmꢀ1, n (OH,
NH); 2963 cmꢀ1, n (sp3-CH); 1654 cmꢀ1, n (CaO);
1540 cmꢀ1, n (NH). 1H NMR (DMSO-d6) d 7.81 (br s,
0.08H), 7.67 (br s, 0.92H) (CONH, exchangeable with D2O),
3.71 (s, 1H, CHO), 3.29 (d, 1H), 3.18 (d, 1H) (2J¼10.4 Hz,
CH2O), 3.20–2.80 (br, 2H, NH2, exchangeable with D2O),
3.07 (m, 2H, JHCCH¼JHNCH¼6.4 Hz, 2J¼12.8 Hz, CH2NH),
4.1.5. (2R)-N-[2-(9-Fluorenylmethoxycarbonylamino)-
ethyl)-2,4-dihydroxy-3,3-dimethylbutyramide (3b).
Compound 3b was prepared by treatment of amine 2b
(1.90 g, 10 mmol) in 50% aq MeCN (20 mL) by a solution
of FmocOSu (3.38 g, 10 mmol) in MeCN (20 mL) at room
temperature for 48 h. The mixture was filtered, evaporated,
the residue was dissolved in EtOAc (200 mL), washed with
water (150 mL), 5% NaHCO3 (2ꢂ150 mL), then dried
(Na2SO4), evaporated, co-evaporated with toluene
(3ꢂ30 mL) and chromatographed on silica gel column using
stepwise gradient elution with 0.5/1/2/3/5/8%
MeOH in CHCl3 (v/v). Fractions containing product were
combined, evaporated and the residue was dried in vacuo to
afford pure 3b as a white solid (3.10 g, 75%), mp 64.0–
65.0 ꢁC. Rf 0.17 (CHCl3–MeOH 9:1 v/v). ESI-TOF HRMS:
m/z¼435.1909 [M+Na]+, calcd for [C23H28N2O5+Na]+
2
2.57 (m, 2H, JHCCH¼6.4 Hz, J¼12.6 Hz, CH2NH2), 0.81
(s, 3H), 0.80 (s, 3H) (C(CH3)2). 13C NMR (DMSO-d6)
d 173.84 (CaO), 75.20 (CHOH), 66.18 (CH2OH), 42.12,
41.53 (CH2CH2), 38.39 (CCMe2C), 21.42, 20.81 (CH3).
4.1.3. (2R)-N-Methyl-N-(2-methylaminoethyl)-2,4-di-
hydroxybutyramide (2c). A solution of (R)-(+)-a-hydroxy-
g-butyrolactone (1.02 g, 10 mmol) in N,N0-dimethylethyl-
enediamine (5.2 mL, 50 mmol) was kept at 55 ꢁC for 7 days,
then evaporated, co-evaporated with N,N-diisopropylethyl-
amine (1.8 mL, 10 mmol) and then toluene (3ꢂ30 mL) to
give diol 2c as a yellowish viscous oil (1.90 g, quant.).
ESI-TOF HRMS: m/z¼191.1381 [M+H]+, calcd for
[C8H18N2O3+H]+ 191.1390. The compound was used with-
out further purification in the next steps.
435.1890. IR (KBr): 3329 cmꢀ1, n (OH, NH); 2957 cmꢀ1
,
n (sp3-CH); 1697 cmꢀ1, n (CaO); 1537 cmꢀ1, n (NH);
1450 cmꢀ1, n (sp3-CH bending). 1H NMR (DMSO-d6)
d 7.88 (d, 2H, J¼7.3 Hz, ArH (H-4, H-5)), 7.74 (br s, 1H,
CHCONH, exchangeable with D2O), 7.67 (d, 2H,
J¼7.5 Hz, ArH (H-1, H-8)), 7.41 (m, 2H, ArH (H-3, H-6)),
7.33 (m, 2H, ArH (H-2, H-7)), 7.25 (br s, 1H, OCONH,
exchangeable with D2O), 5.33 (d, 1H, J¼5.5 Hz, CHOH),
4.44 (t, 1H, J¼5.5 Hz, CH2OH) (both exchangeable with
D2O), 4.29 (m, 2H, CH2OCO), 4.20 (m, 1H, fluorene H-9),
3.71 (d, 1H, J¼5.5 Hz, CHO), 3.29 (m, 1H,z CHHOH),
3.25–3.03 (m, 5H, NCH2CH2, CHHOH), 0.80 (s, 3H), 0.78
(s, 3H) (C(CH3)2). 13C NMR (DMSO-d6) d 173.66
(HNCaO), 156.63 (OCaO), 144.35 (2C), 141.18 (2C),
128.05 (2C), 127.50 (2C), 125.56 (2C), 120.55 (2C) (Ar,
Fmoc), 75.63 (CHOH), 68.48 (CH2OCO), 65.80 (CH2OH),
47.17 (fluorene C-9), w40 (2C,z NCH2CH2), 38.53
(CCMe2C), 21.40, 20.79 (CH3).
4.1.4. (2R)-N-[2-(9-Fluorenylmethoxycarbonylamino)-
ethyl]-2,4-dihydroxybutyramide (3a). To a stirred solution
of crude amine 2a (1.63 g, 10 mmol) in 50% aq MeCN
(20 mL), a solution of FmocOSu (3.38 g, 10 mmol) in
MeCN (20 mL) was added in one portion and stirring con-
tinued overnight at ambient temperature. The precipitate
formed was filtered off, washed with MeCN (10 mL), Et2O
(30 mL) and dried in vacuo to yield diol 3a (2.50 g, 65%)
4.1.6. (2R)-N-Methyl-N-(2-[methyl(9-fluorenylmethoxy-
carbonyl)amino]ethyl)-2,4-dihydroxybutyramide (3c).
The crude amine 2b (1.14 g, 6 mmol) in 50% aq MeCN
z
Calculated value; the signal of water or a solvent is also present in the
region.

6768
N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
(15 mL) was treated with a solution of FmocOSu (2.03 g,
6 mmol) in MeCN (10 mL) for 48 h. The workup was similar
to the above procedure. After column chromatography in
0.5/1/1.5/2/2.5% MeOH in CHCl3 (v/v) the desired
compound 3c was isolated as a white foam (1.58 g, 64%),
Rf 0.36 (CHCl3–MeOH 9:1 v/v). ESI-TOF HRMS: m/z¼
435.1878 [M+Na]+, calcd for [C23H28N2O5+Na]+
m/z¼737.3205 [M+Na]+, calcd for [C44H46N2O7+Na]+
1
737.3197. H NMR (DMSO-d6) d 7.88 (d, 2H, J¼7.3 Hz,
ArH (fluorene H-4, H-5)), 7.71 (br s, 1H, CHCONH), 7.66
(d, 2H, J¼7.3 Hz, ArH (fluorene H-1, H-8), 7.43–7.16 (m,
14H, ArH (fluorene H-2, H-3, H-6, H-7, Dmt), OCONH),
6.86 (d, 4H, J¼8.9 Hz, ArH (Dmt)), 5.36 (d, 1H, J¼
5.3 Hz, OH, exchangeable with D2O), 4.28 (m, 2H,
CH2OCO), 4.21 (m, 1H, fluorene H-9), 3.80 (d, 1H, J¼
5.3 Hz, CHO), 3.72 (s, 6H, OCH3), 3.17–2.98 (m, 5H,
NCH2CH2, CHHODmt), 2.75 (d, 1H, 2J¼8.3 Hz,
CHHODmt), 0.92 (s, 3H), 0.78 (s, 3H) (C(CH3)2). 13C
NMR (DMSO-d6) d 172.73 (HNCaO), 157.97, 157.95,
157.93, 156.22 (OCaO), 149.66, 145.49, 143.94, 140.78,
136.21, 136.16, 136.11, 129.87, 129.83, 128.96, 128.94,
128.24, 127.88, 127.69, 127.65, 127.33, 127.09, 126.50,
125.36, 125.15, 123.94, 121.42, 120.16, 113.03, 84.95,
75.27, 68.36, 65.40, 55.05, 55.03, 55.00 (OMe), 46.78,
38.60, 38.12, 22.21, 21.08, 20.51.
1
435.1890. H NMR (MeCN-d3) d 7.85 (d, 2H, J¼7.3 Hz,
ArH, H-4, H-5), 7.79 (d, 0.57H, J¼7.5 Hz, ArH), 7.65 (d,
1.43H, J¼7.3 Hz, ArH) (H-1, H-8), 7.43 (m, 2H, ArH, H-3,
H-6), 7.36 (m, 2H, ArH, H-2, H-7), 4.59–4.25 (m, 4H,
CHO, CHCH2OCO), 3.85–2.33 (m, 12H, CH2OH,
N(CH3)CH2CH2NCH3), 1.89–1.26 (m, 2H, CH2CH2CH).
13C NMR (MeCN-d3) d 172.24 (MeNCaO), 155.80
(OCaO), 144.16 (2C), 141.09 (2C), 128.01 (2C), 127.35
(2C), 125.46 (2C), 120.47 (2C) (Ar, Fmoc), 68.88
(CHOH), 68.42 (CH2OCO), 65.74 (CH2OH), 47.10 (fluorene
C-9), 39.55, 39.16 (NCH2CH2), 38.44 (CCH2C), 35.10,
34.52 (CH3).
4.1.9. (2R)-O4-(4,40-Dimethoxytrityl)-N-methyl-N-
(2-[methyl-N0-(9-fluorenylmethoxycarbonyl)amino]-
ethyl)-2,4-dihydroxybutyramide (4c). Compound 4c was
prepared from 3c (1.48 g, 3.6 mmol) and DmtCl (1.37 g,
3.95 mmol). The product was purified by column chromato-
graphy in CHCl3–toluene/1:4/1:1/2:1/CHCl3+1%
Py (v/v/v). Yield 2.28 g (89%), white foam, Rf 0.27
(CHCl3–EtOAc 1:1+1% Et3N v/v/v). ESI-TOF HRMS:
m/z¼737.3202 [M+Na]+, calcd for [C44H46N2O7+Na]+
737.3197. 1H NMR (DMSO-d6) d 7.87 (m, 2H, ArH (fluo-
rene H-4, H-5)), 7.58 (m, 2H, ArH (fluorene H-1, H-8)),
7.42–7.12 (m, 13H, ArH (fluorene H-2, H-3, H-6, H-7,
Dmt)), 6.84 (m, 4H, ArH (Dmt)), 4.79 (m, 1H, OH, ex-
changeable with D2O), 4.63–4.20 (m, 4H, CHO,
CHCH2OCO), 3.72 (s, 6H, OCH3), 3.55–2.55 (m, 12H,z
N(CH3)CH2CH2NCH3, CH2ODmt), 1.83–1.43 (m, 2H,
CH2CH2CH). 13C NMR (DMSO-d6) d 173.00, 172.93
(MeNCaO), 158.03, 157.68 (OCaO), 143.97, 143.90,
143.85, 142.63, 140.81, 139.47, 137.48, 136.06, 136.00,
129.65, 128.96, 128.94, 128.24, 127.80, 127.70, 127.65,
127.33, 127.12, 126.61, 125.35, 124.95, 121.41, 120.11,
120.06, 120.04, 113.15, 113.11, 109.76, 85.46, 65.06,
64.98, 59.87, 55.06, 55.05, 55.02 (OMe), 34.45, 21.08.
4.1.7. (2R)-O4-(4,40-Dimethoxytrityl)-N-[2-(9-fluorenyl-
methoxycarbonylamino)ethyl]-2,4-dihydroxybutyr-
amide (4a). Diol 3a (2.50 g, 6.5 mmol) was co-evaporated
with pyridine (3ꢂ20 mL), dissolved in dry pyridine
(50 mL), cooled in an ice bath, and DmtCl (2.40 g,
7.1 mmol) was added in three portions within 1 h. After dis-
appearance of the starting material, the excess of DmtCl was
quenched with MeOH (1 mL), and after 10 min the mixture
was diluted with CHCl3 (300 mL), washed with 2.5%
NaHCO3 (3ꢂ200 mL), water (2ꢂ200 mL), 20% NaCl
(200 mL), then dried (Na2SO4), evaporated, co-evaporated
with toluene (3ꢂ30 mL) and the residue was chromato-
graphed on silica gel column using stepwise gradient
CHCl3–toluene 1:2/1:1/2:1/CHCl3+1% Py (v/v/v).
Fractions containing the product were combined, evaporated
and the residue was dried in vacuo to afford 4a as a white
foam (3.6 g, 81%), Rf 0.16 (CHCl3–EtOAc 1:1+1% Et3N
v/v/v). ESI-TOF HRMS: m/z¼709.2869 [M+Na]+, calcd
for [C42H42N2O7+Na]+ 709.2884. 1H NMR (DMSO-d6)
d 7.88 (d, 2H, J¼7.5 Hz, ArH (fluorene H-4, H-5)), 7.78
(br s, 1H CHCONH), 7.67 (d, J¼7.3 Hz, ArH, fluorene, H-
1, H-8), 7.41–7.12 (m, 14H, ArH (Dmt+fluorene H-2, H-3,
H-6, H-7), OCONH), 6.86 (d, 4H, J¼8.9 Hz, ArH (Dmt)),
5.37 (d, 1H, J¼5.3 Hz, CHOH, exchangeable with D2O),
4.29 (d, 2H, J¼6.7 Hz, CH2OCO), 4.18 (t, 1H, J¼6.7 Hz,
fluorene H-9), 3.96 (m, 1H, CHO) (in the presence of D2O
gives dd J1¼3.5 Hz, J2¼8.4 Hz), 3.72 (s, 6H, CH3), 3.16–
2.98 (m, 6H, NCH2CH2, CH2ODmt), 1.98 (m, 1H), 1.70
(m, 1H) (CH2CH2CH). 13C NMR (DMSO-d6) d 174.17,
174.11 (HNCaO), 158.02, 158.00, 156.20 (OCaO),
149.69, 149.66, 149.64, 145.31, 143.95, 142.63, 140.79,
139.47, 137.48, 136.16, 136.11, 129.67, 128.97, 128.95,
128.25, 127.79, 127.73, 127.65, 127.34, 127.31, 127.11,
126.57, 125.36, 125.17, 123.94, 123.92, 121.42, 121.40,
120.15, 120.07, 120.05, 113.15, 109.76, 85.35, 68.65,
59.77, 55.05 (OMe), 46.79, 34.74, 21.08.
4.1.10. (2R)-O4-(4,40-Dimethoxytrityl)-N-methyl-N-(2-
[N0-methyl-N0-trifluoroacetylamino]ethyl)-2,4-dihydroxy-
butyramide (4d). The crude diol 2c (0.75 g, 4 mmol) was
co-evaporated with dry MeOH (20 mL), dissolved in the
mixture of MeOH (10 mL), Et3N (0.6 mL, 4 mmol) and
CF3CO2Me (2.0 mL 10 mmol). The mixture was stirred at
ambient temperature for 30 min and evaporated to give crude
3d. This was co-evaporated with pyridine and tritylated with
DmtCl (2.02 g, 6.0 mmol) as above. The product 4d was
chromatographed on silica gel eluting with CHCl3–toluene
1:2/1:1/CHCl3+0.25% Et3N (v/v/v). Yield 1.44 g
(93%) as a white foam, Rf 0.23 (CHCl3–EtOAc 1:1+1%
Et3N v/v/v). ESI-TOF HRMS: m/z¼611.2331 [M+Na]+,
calcd for [C31H35F3N2O6+Na]+ 611.2339. 1H NMR
(DMSO-d6) d 7.38–7.21 (m, 9H, ArH (Dmt)), 6.87 (d, 4H,
J¼8.7 Hz, ArH (Dmt)), 5.07 (d, 0.04H, J¼7.8 Hz), 4.91 (d,
0.16H, J¼7.8 Hz), 4.75 (d, 0.19H, J¼7.8 Hz), 4.59 (d,
0.61H, J¼8.0 Hz) (OH, exchangeable with D2O), 4.42 (m,
1H, CHO), 3.73 (s, 6H, OCH3), 3.80–3.26 (m, 4H,z CH2O,
CH2NTfa), 3.15–2.78 (m, 8H, N(CH3)CH2CH2N(CH3)Tfa),
4.1.8. (2R)-O4-(4,40-Dimethoxytrityl)-N-[2-(9-fluorenyl-
methoxycarbonylamino)ethyl]-2,4-dihydroxy-3,3-di-
methylbutyramide (4b). Diol 3b (2.10 g, 5 mmol) was
tritylated in a similar manner with DmtCl (1.90 g,
5.5 mmol). Yield 3.50 g (97%), white foam, Rf 0.22
(CHCl3–EtOAc 1:1+1% Et3N v/v/v). ESI-TOF HRMS:

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6769
1.86–1.50 (m, 2H, CH2CH2CH). 13C NMR (DMSO-d6)
d 173.90, 173.58, 172.96 (MeNCaO), 158.05, 156.13,
155.85, 155.57, 145.24, 145.18, 136.03, 129.62, 128.93,
128.24, 127.81, 127.77, 127.72, 127.66, 126.62, 125.35,
117.49, 115.18, 113.17, 113.13, 85.46, 65.13, 64.98, 59.86,
55.05 (OMe), 45.94, 45.76, 43.76, 40.19, 40.02, 39.86,
35.39, 35.03, 34.85, 34.82, 34.80, 34.53, 34.44, 34.36, 33.46.
157.51 (OCaO), 146.53, 145.17, 142.08, 137.23, 131.21,
131.13, 129.19, 129.08, 128.61, 128.05, 127.55, 126.05,
120.91, 119.41, 113.78, 86.54, 78.96, 69.24, 68.85, 66.99,
66.87, 59.79, 59.64, 59.06, 55.81 (OMe), 55.24, 48.06,
47.27, 45.91, 44.18, 44.04, 43.94, 41.48, 40.06, 39.78,
25.03, 24.83, 23.05, 22.83, 22.24, 21.41, 20.87, 20.19.
4.1.13. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-methyl-N-(2-
[methyl(trifluoroacetyl)amino]ethyl)-2,4-dihydroxy-
butyramide (5c). Compound 4d (1.18 g, 2.0 mmol) was
phosphitylated with diisopropylammonium tetrazolide
(510 mg, 3.0 mmol) and bis(N,N-diisopropylamino)-2-cyano-
ethoxyphosphine (1.0 mL, 3 mmol) under argon for 2 h. The
conversion of the starting material was monitored by TLC
(CHCl3–EtOAc 1:1+1% Et3N v/v/v). The compound was
precipitated from DCM (30 mL) into cold hexane. Yield
1.18 g (77%), white foam, Rf 0.29, 0.39 (CHCl3–EtOAc
1:1+1% Et3N v/v/v). ESI-TOF HRMS: m/z¼811.3442
[M+Na]+, calcd for [C40H52F3N4O7P+Na]+ 811.3418. 31P
NMR (MeCN-d3) d 150.42 (0.19P), 150.03 (0.36P), 149.86
4.1.11. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-[2-(9-fluorenyl-
methoxycarbonylamino)ethyl]-2,4-dihydroxybutyr-
amide (5a). Compound 4a (1.38 g, 2 mmol) was co-
evaporated with dry DCM (2ꢂ20 mL), dissolved in dry
DCM, diisopropylammonium tetrazolide (0.51 g, 3 mmol)
and bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
(1 mL, 3 mmol) were added, and the mixture was stirred un-
der argon for 2 h. After conversion of the starting compound
is complete (monitoring by TLC, CHCl3–EtOAc 1:1+1%
Et3N v/v/v) the mixture was diluted with EtOAc and washed
with 5% NaHCO3 (2ꢂ100 mL) and 20% NaCl (100 mL).
The organic layer was dried over Na2SO4, evaporated to dry-
ness, and the residue was dissolved in 30 mL DCM and pre-
cipitated into cold (ꢀ10 ꢁC) hexane (500 mL), the solid was
filtered off, co-evaporated with dry DCM (3ꢂ20 mL) and
dried in vacuo to afford phosphoramidite 5a (1.51 g, 85%)
as a white foam, Rf 0.34, 0.43 (CHCl3–EtOAc 1:1+1%
Et3N v/v/v). ESI-TOF HRMS: m/z¼909.4066 [M+Na]+,
calcd for [C51H59N4O8P+Na]+ 909.3963. 31R NMR
(MeCN-d3) d 151.13, 148.73 (diastereomers, w1:2). 1O
NMR (MeCN-d3) d 7.85–7.19 (m, 18H, ArH (fluorene,
Dmt), CCONH), 6.94 (m, 1H, OCONH), 6.84 (m, 4H, ArH
(Dmt)), 4.35–4.01 (m, 4H, CHCH2OCO, CHO), 3.76 (m,
6H, OCH3), 3.65–2.48 (m, 12H, NCH2CH2, RPSO2SO2,
NCH, CH2ODmt), 2.18–2.00 (m, 2H, CH2CH2CH), 1.30–
0.96 (m, 12H, CH3 (iPr)). 13C NMR (MeCN-d3) d 173.57,
173.27, 173.16 (HNCaO), 159.49, 157.49 (OCaO),
146.37, 145.14, 142.08, 137.31, 130.86, 128.96, 128.67,
128.63, 128.06, 127.60, 126.34, 126.10, 120.91, 113.91,
86.88, 86.77, 72.09, 66.98, 60.55, 60.26, 59.74, 59.59,
59.12, 58.07, 55.81 (OMe), 55.24, 48.09, 47.19, 45.96,
43.99, 43.89, 41.49, 39.73, 35.29, 34.84, 24.87, 23.12,
23.05, 21.91, 20.86, 20.51, 19.99.
1
(0.11P), 149.30 (0.34P). H NMR (MeCN-d3) d 7.49–7.20
(m, 9H, ArH (Dmt)), 6.89 (m, 4H, ArH (Dmt)), 4.88–4.64
(m, 0.44H), 4.20–4.02 (m, 0.56H) (CHO), 3.79 (m, 6H,
OCH3), 3.71–2.48 (m, 18H, RPSO2SO2, NCH, CH2ODmt,
CH3NCH2CH2NCH3), 2.14–1.81 (m, 2H,z CH2CH2CH),
1.29–1.03 (m, 12H, CH3 (iPr)). 13C NMR (MeCN-d3)
d 172.59 (MeNCaO), 159.57, 146.41, 146.11, 137.23,
130.85, 128.87, 128.72, 127.68, 119.49, 113.95, 87.04,
86.88, 69.15, 60.88, 60.49, 59.61, 59.45, 59.28, 59.12,
55.83, 55.24 (OMe), 47.18, 46.72, 45.97, 45.22, 43.95,
43.85, 35.83, 35.68, 34.95, 24.90, 24.80, 24.74, 23.13,
23.06, 20.88.
4.1.14. (2R)-N-[2-(Imidazol-4-yl)ethyl]-2,4-dihydroxy-
butyramide (7a). A solution of (R)-(+)-a-hydroxy-g-butyro-
lactone (1.02 g, 10 mmol) and histamine (1.17 g, 10 mmol)
in EtOH (10 mL) was kept at 55 ꢁC for 24 h. The solid
formed was filtered off, washed with EtOH (5 mL), Et2O
(2ꢂ5 mL) and dried in vacuo to afford 7a (1.77 g, 83%) as
white crystals, mp 149.0–149.5 ꢁC (EtOH). ESI-TOF
HRMS: m/z¼214.1187 [M+H]+, calcd for [C9H15N3O3+
H]+ 214.1186. IR (KBr): 3392 cmꢀ1, n (OH); 3164 cmꢀ1
,
4.1.12. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-[2-(9-fluorenyl-
methoxycarbonylamino)ethyl]-2,4-dihydroxy-3,3-di-
methylbutyramide (5b). Compound 4b (1.87 g, 2.6 mmol)
was phosphitylated as above with diisopropylammonium
tetrazolide (0.67 g, 3.9 mmol) and bis(N,N-diisopropyl-
amino)-2-cyanoethoxyphosphine (1.27 mL, 3.9 mmol) to
afford 5b (1.60 g, 67%) as a white foam. ESI-TOF HRMS:
m/z¼937.4284 [M+Na]+, calcd for [C53H63N4O8P+Na]+
937.4276. 31P NMR (MeCN-d3) d 152.38, 149.04 (diastereo-
mers,w1:2). 1H NMR (MeCN-d3) d 7.84 (d, 2H, J¼7.4 Hz,
ArH (fluorene H-4, H-5)), 7.66 (m, 2H, ArH (fluorene H-1,
H-8)), 7.48–7.22 (m, 15H, ArH (fluorene H-2, H-3, H-6, H-
7, Dmt), CHCONH, OCONH), 6.86 (m, 4H, ArH (Dmt)),
4.37–4.31 (m, 2H, CH2OCO), 4.23 (m, 1H, fluorene H-9),
4.03–3.92 (m, 1H, CHO), 3.77 (c, 6H, OCH3), 3.73–2.70
(m, 10H, NCH2CH2, RPSO2, NCH, CH2ODmt), 2.46 (t,
0.8O, J¼6.0 Hz), 2.29 (t, 1.2O, J¼6.0 Hz) (CH2CN, dia-
stereomers), 1.28–0.95 (m, 18H, CH2C(CH3)2CH, CH3
(iPr)). 13C NMR (MeCN-d3) d 171.71 (HNCaO), 159.46,
n (NH); 2904 cmꢀ1, n (sp3-CH); 1644 cmꢀ1, n (CaO);
1533 cmꢀ1, n (NH). 1H NMR (CD3OD) d 7.61 (m, 1H, imi-
dazole H-2), 6.89 (br s, 1H, imidazole H-5), 4.14 (dd, 1H,
J1¼3.7 Hz, J2¼8.6 Hz, CHO), 3.71 (m, 2H, CH2O), 3.51
(t, 2H, J¼7.0 Hz, NCH2), 2.82 (t, 2H, J¼7.0 Hz, NCH2CH2),
2.01 (m, 1H), 1.74 (m, 1H) (CH2CH2CH). 13C NMR
(CD3OD) d 177.21 (CO), 136.13 (3C, imidazole C-2, C-4,
C-5), 70.56 (CHOH), 59.57 (CH2OH), 39.88, 38.23
(NCH2CH2), 27.80 (CH2CH2CH).
4.1.15. (2R)-N-{2-[1-(tert-Butoxycarbonyl)imidazol-4-
yl]ethyl}-2,4-dihydroxybutyramide (8a). To a stirred solu-
tion of diol 7a (0.745 g, 3.5 mmol) in 50% aq dioxan
(50 mL), Boc2O (0.839 g, 3.8 mmol) was added at room tem-
perature and stirring was continued for 5 h. After completion
of the reaction (monitored by TLC, CHCl3–MeOH 4:1 v/v),
the solvent was evaporated, and the residue was chromato-
graphed on a silica gel column using stepwise gradient
elution with 0/2/4/6/8% MeOH in CHCl3. Yield
0.83 g (76%), white crystals, mp 92.0–93.0 ꢁC. Rf 0.37

6770
N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
(CHCl3–MeOH 5:1 v/v). ESI-TOF HRMS: m/z¼314.1715
[M+H]+, calcd for [C14H23N3O5+H]+ 314.1710. IR (KBr):
3428 cmꢀ1, n (OH); 3316 cmꢀ1, 3103 cmꢀ1, n (NH);
amide (9a). Diol 8a (0.78 g, 2.5 mmol) was tritylated with
DmtCl (0.93 g, 2.75 mmol) according to the procedure for
4a. Theproductwaspurifiedbychromatographyusingastep-
wise gradient CHCl3–toluene 1:2/1:1/2:1/CHCl3+
0.25% Py (v/v/v), then 10%/50% EtOAc in CHCl3+
0.25% Py (v/v/v). Yield 1.31 g (86%), white foam, Rf 0.46
(CHCl3–MeOH 20:1+1% Et3N v/v/v). ESI-TOF HRMS:
m/z¼638.2766 [M+Na]+, calcd for [C35H41N3O7+Na]+
638.2837. 1H NMR (DMSO-d6) d 8.10 (br s, 1H, imidazole
H-2), 7.80 (m, 1H, CONH (exchangeable with D2O)),
7.37–7.22 (m, 10H, ArH (Dmt), imidazole H-5), 6.87 (d,
4H, J¼8.9 Hz, ArH (Dmt)), 5.40 (d, 1H, J¼5.7 Hz, CHOH,
exchangeable with D2O), 3.98 (m, 1H, CHO), 3.72 (s, 6H,
OCH3), 3.30 (m, 2Hz), 3.04 (m, 2H) (CH2N, CH2O), 2.58
(t, 2H, J¼7.0 Hz, NCH2CH2), 1.96 (m, 3H), 1.69 (m, 3H)
(CH2CH2CH), 1.55 (s, 9H, CH3 (Boc)). 13C NMR (DMSO-
d6) d 173.76 (HNCaO), 158.01, 146.80, 145.29, 141.03,
136.74, 136.10, 136.07, 129.66, 128.94, 128.24, 127.78,
127.72, 126.56, 125.36, 113.42, 113.14, 85.35, 85.08,
68.60, 59.73, 55.05 (OMe), 37.60, 34.77, 27.81, 27.49,
27.48, 27.45, 27.43, 27.41 (CMe3), 21.08.
2938 cmꢀ1, n (sp3-CH); 1653 cmꢀ1, n (CaO); 1536 cmꢀ1
,
1
n (NH). H NMR (DMSO-d6) d 8.10 (br s, 1H, imidazole
H-2), 7.79 (m, 1H, CONH, exchangeable with D2O), 7.28
(br s, 1H, imidazole H-5), 5.41 (d, 1H, J¼5.5 Hz, CHOH),
4.39 (t, 1H, J¼5.3 Hz, CH2OH) (both exchangeable with
D2O), 3.91 (m, 1H, CHO), 3.48 (m, 2H, CH2O), 3.33 (m,
2H,z NCH2), 2.63 (t, 2H, J¼7.0 Hz, NCH2CH2), 1.81 (m,
1H, CH2CHHCH), 1.52 (m, 10H, CH3 (Boc), CH2CHHCH).
13C NMR (DMSO-d6) d 174.53 (HNCaOC), 147.19
(NCOO), 141.45 (imidazole C-4), 137.14 (imidazole C-2),
113.86 (imidazole C-4), 85.48 (C(CH3)3), 69.01 (CHOH),
57.91 (CH2OH), 37.97 (2C, NCH2CH2), 28.22
(CH2CH2CH), 27.85 (3C, CH3).
4.1.16. (2R)-N-{2-[1-(tert-Butoxycarbonyl)imidazol-4-
yl]ethyl}-2,4-dihydroxy-3,3-dimethylbutyramide (8b1)
and (2R)-N-{2-[1-(tert-butoxycarbonyl)imidazol-5-
yl]ethyl}-2,4-dihydroxy-3,3-dimethylbutyramide (8b2).
A solution of (R)-(ꢀ)-pantolactone (4.3 g, 33 mmol) and his-
tamine (3.3 g, 30 mmol) in abs EtOH (20 mL) was kept at
55 ꢁC for 72 h, then evaporated, co-evaporated with toluene
to afford (2R)-N-[2-(imidazol-4-yl)ethyl]-2,4-dihydroxy-
3,3-dimethylbutyramide 7b as a yellow oil. The product
was used without further purification. ESI-TOF HRMS:
m/z¼242.1501 [M+H]+, calcd for [C11H19N3O3+H]+
242.1499. The crude 7b was treated with Boc2O (7.20 g,
33 mmol) in 50% aq dioxane (50 mL) as above to afford
two isomers, 8b1 (3.88 g, 38%) and 8b2 (1.75 g, 17%) as
white solids, Rf 0.54 and 0.48, correspondingly (CHCl3–
MeOH 4:1 v/v). ‘Fast moving’ product 8b1: ESI-TOF
HRMS: m/z¼342.2019 [M+H]+, calcd for [C16H27N3O5+
H]+ 342.2023. 1H NMR (DMSO-d6) d 8.10 (br s, 1H, imida-
zole H-2), 7.75 (m, 1H, CONH, exchangeable with D2O),
7.29 (br s, 1H, imidazole H-5), 5.31 (d, 1H, J¼5.5 Hz,
CHOH), 4.43 (t, 1H, J¼5.4 Hz, CH2OH) (both exchangeable
with D2O), 3.69 (d, 1H, J¼5.5 Hz, CHO), 3.44–3.14 (m, 4H,
CH2O (2J¼10.3 Hz, JHCOH¼5.4 Hz), NCH2), 2.64 (m, 2H,
NCH2CH2), 1.55 (s, 9H, CH3 (Boc)), 0.77 (s, 3H), 0.75 (s,
3H) (C(CH3)2). 13C NMR (DMSO-d6) d 173.30 (HNCaOC),
147.19 (NCOO), 141.47 (imidazole C-4), 137.13 (imidazole
C-2), 113.94 (imidazole C-4), 85.43 (C(CH3)3), 75.58
(CHOH), 68.52 (CH2OH), w40,z 37.97 (NCH2CH2), 28.23
(CH2CCH), 27.85 (3C, CH3), 21.29, 20.75 (C(CH3)2).
‘Slow moving’ product 8b2: ESI-TOF HRMS: m/z¼
342.2013 [M+H]+, calcd for [C16H27N3O5+H]+ 342.2023.
The compound purified by column chromatography was
kept at 4 ꢁC for one month before recording its NMR spec-
trum. NMR (as well as TLC) showed considerable p/t
4.1.18. (2R)-O4-(4,40-Dimethoxytrityl)-N-{2-[1-(tert-
butoxycarbonyl)imidazol-4-yl]ethyl}-2,4-dihydroxy-3,3-
dimethylbutyramide (9b1) and (2R)-O4-(4,40-dimethoxy-
trityl)-N-{2-[1-(tert-butyloxycarbonyl)imidazol-5-
yl]ethyl}-2,4-dihydroxy-3,3-dimethylbutyramide (9b2).
The ‘fast moving’ product 8b1 (3.7 g, 11 mmol) was trity-
lated with DmtCl (4.15 g, 12 mmol) as described above
and isolated from silica gel column eluted with CHCl3–
toluene 1:2/1:1/2:1/CHCl3+0.25% Py (v/v/v), then
2.5%/5% EtOAc in CHCl3+0.25% Py (v/v/v). Fractions
containing products were combined, evaporated and the resi-
dues were dried in vacuo to afford 9b1 (3.95 g, 55%) and 9b2
(2.60 g, 37%) as white foams, Rf 0.53 and 0.47, correspond-
ingly (CHCl3–MeOH 20:1+1% Et3N v/v/v). ‘Fast moving’
product 9b1: ESI-TOF HRMS: m/z¼666.3163 [M+Na]+,
1
calcd for [C37H45N3O7+Na]+ 666.3150. H NMR (MeCN-
d3) d 7.96 (m, 1H, imidazole H-2), 7.48–7.15 (m, 10H,
ArH (Dmt), imidazole H-5), 6.94 (br s, 1H, CONH (ex-
changeable with D2O)), 6.86 (d, 4H, J¼8.8 Hz, ArH
(Dmt)), 3.94 (d, 1H, J¼5.7 Hz, OH (exchangeable with
D2O)), 3.78 (s, 6H, OCH3), 3.67 (d, 1H, J¼5.7 Hz, CHO),
3.37 (m, 2H, CH2N), 3.07 (d, 1H, 2J¼8.7 Hz), 2.85 (d, 1H,
2J¼8.7 Hz) (CH2O), 2.61 (m, 2H, NCH2CH2), 1.60 (s, 9H,
CH3 (Boc)), 0.96 (s, 3H), 0.88 (s, 3H) (C(CH3)2). 13C
NMR (MeCN-d3) d 172.96 (HNCaO), 158.47, 145.98,
145.46, 141.37, 136.89, 136.58, 136.39, 129.90, 128.78,
128.55, 127.83, 127.72, 126.91, 125.40, 113.88, 113.69,
86.55, 86.38, 68.85, 59.37, 55.03 (OMe), 38.43, 34.87,
28.01, 27.87, 27.63, 27.57, 27.39, 27.31 (CMe3), 21.29,
21.18, 19.95. Compound 9b2 was stored at 4 ꢁC for one
month before recording its NMR spectrum. NMR and TLC
showed complete conversion 9b2/9b1 during storage.
1
Boc migration (conversion >50%). H NMR (DMSO-d6)
of ‘slow moving’ product 8b2 (deduced from NMR spectrum
of the mixture of 8b1 and 8b2) d 8.08 (br s, 1H, imidazole H-
2), 7.76 (m, 1H, CONH, exchangeable with D2O), 6.79 (br s,
1H, imidazole H-4), 5.31 (d, 1H, J¼5.5 Hz, CHOH), 4.44 (t,
1H, J¼5.4 Hz, CH2OH) (both exchangeable with D2O), 3.70
(d, 1H, J¼5.5 Hz, CHO), 3.45–3.13 (m, 4H,z CH2O, NCH2),
2.91 (m, 2H, NCH2CH2), 1.57 (s, 9H, CH3 (Boc)), 0.77
(s, 3H), 0.76 (s, 3H) (C(CH3)2).
4.1.19. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-{2-[1-(tert-butoxy-
carbonyl)imidazol-4-yl]ethyl}-2,4-dihydroxybutyramide
(10a). Compound 9a (1.23 g, 2.0 mmol) was phosphitylated
with bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
(0.95 mL, 3 mmol) and diisopropylammonium tetrazolide
(510 mg, 3.0 mmol) to afford phosphoramidite 10a (1.29 g,
80%) as a white foam, Rf 0.37, 0.26 (CHCl3–EtOAc
4.1.17. (2R)-O4-(4,40-Dimethoxytrityl)-N-{2-[1-(tert-
butoxycarbonyl)imidazol-4-yl]ethyl}-2,4-dihydroxybutyr-

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6771
1:1+1% Et3N v/v/v). ESI-TOF HRMS: m/z¼838.3908
[M+Na]+, calcd for [C44H58N5O8P+Na]+ 838.3915. 31R
NMR (MeCN-d3) d 150.61, 148.64 (diastereomers,w9:10).
1H NMR (MeCN-d3) d 8.01 (br s, 1H, imidazole H-2),
7.46–7.17 (m, 10H, ArH (Dmt), imidazole H-5), 7.07 (m,
0.59H), 7.01 (m, 0.41H) (CONH, diastereomers), 6.87 (m,
4H, ArH (Dmt)), 4.31 (m, 0.41H), 4.24 (m, 0.59H) (CHO,
diastereomers), 3.78 (s, 6H, OCH3), 3.67–2.51 (m, 12H,
CH2O, NCH2CH2, RPSO2SO2, NCH), 2.18–2.00 (m, 2H,
CH2CH2CH), 1.61 (s, 9H, CH3 (Boc)), 1.27–0.98 (m, 12H,
CH3 (iPr)). 13C NMR (MeCN-d3) d 170.23 (HNCaO),
158.87, 147.16, 145.97, 141.67, 137.12, 136.19, 130.27,
129.71, 128.47, 127.98, 127.36, 126.87, 119.99, 113.88,
113.11, 85.44, 77.77, 68.32, 59.12, 58.84, 55.14 (OMe),
44.88, 42.52, 42.35, 38.05, 37.86, 27.54 (CMe3), 24.78,
24.17, 23.06, 22.24, 22.08, 19.97.
125.62, 125.56, 125.11, 124.44 (2C), 123.69 (pyrene),
69.19 (CHOH), 57.94 (CH2OH), w40z (NCH2), 38.22
(CCH2C).
4.1.22. (2R)-N-(Pyren-1-ylmethyl)-2,4-dihydroxy-3,3-
dimethylbutyramide (11b). A solution of (R)-(ꢀ)-panto-
lactone (3.25 g, 25 mmol) and 1-pyrenemethylamine
(4.6 g, 20 mmol) in acetone (50 mL) was kept at 55 ꢁC
for 7 days, then evaporated and chromatographed on silica
gel column using stepwise gradient CHCl3–toluene 1:1/
2:1/CHCl3 (v/v), then 0.5%/1%/2% MeOH in
CHCl3 (v/v). Fractions containing the product were com-
bined, evaporated and the residue was dried in vacuo
to afford pure 11b (6.64 g, 92%) as a yellow foam, Rf
0.24 (CHCl3–MeOH 4:1 (v/v)). ESI-TOF HRMS:
m/z¼384.1577 [M+Na]+, calcd for [C23H23NO3+Na]+
384.1570. IR (KBr): 3396 cmꢀ1, n (OH); 3041 cmꢀ1
,
4.1.20. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-{2-[1-(tert-
butoxycarbonyl)imidazol-4-yl]ethyl}-2,4-dihydroxy-3,3-
dimethylbutyramide (10b). Compound 9b1 (3.22 g,
5.0 mmol) was phosphitylated with bis(N,N-diisopropyl-
amino)-2-cyanoethoxyphosphine (2.38 mL, 7.5 mmol) and
diisopropylammonium tetrazolide (1.28 g, 7.5 mmol) to
give compound 10b as a white foam (2.36 g, 60%), Rf 0.38,
0.30 (CHCl3–EtOAc 1:1+1% Et3N v/v/v). ESI-TOF
HRMS: m/z¼866.4238 [M+Na]+, calcd for [C46H62N5
O8P+Na]+ 866.4228. 31R NMR (MeCN-d3) d 152.29,
149.11 (diastereomers,w9:10). 1H NMR (MeCN-d3) d 7.97
(br s, 1H, imidazole H-2), 7.49–7.18 (m, 11H, ArH (Dmt),
imidazole H-5, CONH), 6.87 (m, 4H, ArH (Dmt)), 3.97 (d,
0.55H, JHCOP¼12.9 Hz), 3.93 (d, 0.45O, JHCOP¼9.9 Hz)
(CHO, diastereomers), 3.79 (m, 6H, OCH3), 3.75–2.51 (m,
12H, CH2O, NCH2CH2, RPSO2SO2, NCH), 1.59 (s, 9H,
CH3 (Boc)), 1.27–0.94 (m, 18H, C(CH3)2, CH3 (iPr)). 13C
NMR (MeCN-d3) d 169.16 (HNCaO), 157.99, 146.76,
145.27, 140.91, 136.72, 135.88, 129.94, 129.81, 127.93,
127.80, 127.67, 126.54, 118.79, 113.58, 112.98, 85.08,
77.31, 67.91, 58.62, 58.23, 55.03 (OMe), 44.58, 42.82,
42.55, 37.76, 37.53, 27.43 (CMe3), 24.27, 24.08, 22.66,
22.16, 21.96, 21.10, 20.67, 19.81, 19.42.
n (sp2-CH); 1649 cmꢀ1, n (CaO); 1529 cmꢀ1, n (NH);
1041 cmꢀ1, n (C–O); 841 cmꢀ1, n (Ar-H). 1H NMR
(DMSO-d6) d 8.49 (d, 1H, J¼8.1 Hz, ArH (H-10)), 8.38
(m, 1H, NH, exchangeable with D2O), 8.32–8.06 (m, 8H,
ArH), 5.43 (d, 1H, J¼4.1 Hz, CHOH, exchangeable with
2
D2O), 5.04 (m, 2H, JHNCH¼5.1 Hz, J¼12.2 Hz, NCH2),
4.45 (m, 1H, CH2OH, exchangeable with D2O), 3.85 (d,
J¼4.1 Hz, CHO), 3.30 (m, 1Hz), 3.20 (m, 1H) (CH2O, in
the presence of D2O gives two d, 2J¼9.2 Hz), 0.82 (s, 3H),
0.79 (s, 3H) (C(CH3)2). 13C NMR (DMSO-d6) d 173.38
(CO), 133.67, 131.24, 130.78, 130.47, 128.47, 127.83
(2C), 127.40, 126.84, 126.63, 125.60 (2C), 125.04, 124.45
(2C), 123.93 (pyrene), 75.71 (CHOH), 68.46 (CH2OH),
w40z (NCH2), 37.21 (C(CH3)2), 21.51, 20.83 (CH3).
4.1.23. (2R)-O4-(4,40-Dimethoxytrityl)-N-(pyren-1-yl-
methyl)-2,4-dihydroxybutyramide (12a). Diol 11a
(1.29 g, 3.9 mmol) was tritylated with DmtCl (1.40 g,
4.1 mmol) as described for 4a. The product was purified
by chromatography in CHCl3–toluene 1:21:1/2:1/
CHCl3+0.25% Py (v/v/v), then 10%/50% EtOAc in
CHCl3+0.25% Py (v/v/v). Yield 1.33 g (62%), white foam,
Rf 0.28 (CHCl3–EtOAc 1:1+1% Et3N v/v/v). ESI-TOF
HRMS: m/z¼658.2572 [M+Na]+, calcd for [C42H37NO5+
Na]+ 658.2564. 1H NMR (DMSO-d6) d 8.42–7.99 (m,
10H, NH, ArH (pyrene)), 7.36–7.13 (m, 9H, ArH (Dmt)),
6.81 (m, 4H, ArH (Dmt)), 5.50 (d, 1H, J¼4.1 Hz, OH,
exchangeable with D2O), 5.01 (m, 2H, JHNCH¼5.1 Hz,
2J¼12.2 Hz, NCH2), 4.12 (m, 1H, CHO) (in the presence
of D2O gives dd with J1¼3.1 Hz and J2¼6.1 Hz), 3.69 (s,
6H, CH3), 3.07 (m, 2H, CH2O), 2.04 (m, 1H), 1.82 (m,
1H) (CH2CH2CH). 13C NMR (DMSO-d6) d 173.80 (CO),
157.98, 145.33, 137.39, 136.13, 136.07, 133.11, 130.85,
130.35, 130.05, 129.65, 128.94, 128.24, 128.03, 127.78,
127.69, 127.49, 127.43, 127.01, 126.60, 126.55, 126.25,
125.35, 125.24, 125.17, 124.68, 124.06, 124.01, 123.29,
113.10, 85.36, 68.73 (CHOH), 59.68, 55.01 (OMe), 34.88
(CCH2C).
4.1.21. (2R)-N-(Pyren-1-ylmethyl)-2,4-dihydroxybutyr-
amide (11a). A solution of (R)-(+)-a-hydroxy-g-butyro-
lactone (1.02 g, 10 mmol) and 1-pyrenemethylamine (2 g,
9 mmol) in ethanol (5 mL) was kept at 55 ꢁC for one
week, evaporated and co-evaporated with toluene. The resi-
due was triturated in EtOAc–toluene and dried in vacuo
to yield amide 11a (2.62 g, 88%) as white crystals, mp
162.0–164.0 ꢁC (EtOAc–toluene). ESI-TOF HRMS:
m/z¼356.1260 [M+Na]+, calcd for [C21H19NO3+Na]+
356.1257. IR (KBr): 3330 cmꢀ1, n (OH); 3273 cmꢀ1
,
n (NH); 3040 cmꢀ1, n (sp2-CH); 1642 cmꢀ1, n (CaO);
1537 cmꢀ1, n (NH); 1044 cmꢀ1, n (C–O); 839 cmꢀ1
,
n (ArH). 1H NMR (DMSO-d6) d 8.42 (m, 2H, ArH
(H-10), NH), 8.33–8.04 (m, 8H, ArH), 5.52 (d, 1H, J¼
5.1 Hz, CHOH, exchangeable with D2O), 5.05 (d, 2H,
J¼5.1 Hz, NCH2), 4.43 (m, 1H, CH2OH, exchangeable
with D2O), 4.07 (m, 1H, CHO) (in the presence of D2O
gives dd with J1¼3.1 Hz and J2¼7.1 Hz), 3.53 (m, 2H,
CH2O), 1.92 (m, 1H), 1.64 (m, 1H) (CH2CH2CH). 13C
NMR (DMSO-d6) d 174.68 (CO), 133.61, 131.25, 130.76,
130.44, 128.41, 127.90, 127.83, 127.39, 126.90, 126.65,
4.1.24. (2R)-O4-(4,40-Dimethoxytrityl)-N-(pyren-1-yl-
methyl)-2,4-dihydroxy-3,3-dimethylbutyramide (12b).
Diol 11b (6.48 g, 18 mmol) was tritylated with DmtCl
(6.80 g, 20 mmol) as above. The compound was chromato-
graphed on a silica gel column eluted with 2.5%/5%/
7.5%/10%/12.5%/15% EtOAc in toluene+0.25%
Et3N (v/v/v). Yield 10.0 g (83%), yellow foam, Rf 0.35

6772
N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
(CHCl3+1% Et3N v/v/v). ESI-TOF HRMS: m/z¼686.2872
127.90, 127.75, 127.70, 127.65, 127.59, 127.40, 127.16,
127.08, 127.04, 126.51, 126.31, 126.24, 125.32, 125.25,
125.21, 125.17, 124.68, 124.57, 124.09, 123.98, 123.95,
123.49, 123.25, 118.76, 118.57, 112.96, 85.13, 85.11,
68.01, 67.66, 58.68, 58.23, 58.19, 55.00 (OMe), 54.96,
44.58, 44.53, 42.69, 42.65, 42.59, 42.55, 24.35, 24.28,
24.23, 24.13, 24.04, 23.98, 22.65, 22.30, 22.22, 21.12,
20.70, 19.84, 19.79, 19.64, 19.42, 19.37.
1
[M+Na]+, calcd for [C44H41NO5+Na]+ 686.2877. H NMR
(DMSO-d6) d 8.46–8.02 (m, 10H, NH, ArH (pyrene)),
7.40–7.16 (m, 9H, ArH (Dmt)), 6.83 (d, 4H, J¼7.1 Hz,
ArH (Dmt)), 5.46 (d, 1H, J¼4.1 Hz, OH, exchangeable
with D2O), 4.99 (m, 2H, NCH2), 3.92 (m, 1H, CHO),
3.70 (s, 6H, OCH3), 3.01 (d, 1H, 2J¼7.1 Hz), 2.78 (d,
1H, 2J¼7.1 Hz) (CH2O), 0.93 (s, 3H), 0.82 (s, 3H)
(C(CH3)2). 13C NMR (DMSO-d6) d 172.47 (CO), 157.93,
145.50, 136.19, 136.12, 133.25, 130.84, 130.36, 130.04,
129.85, 128.94, 128.24, 128.02, 127.85, 127.69, 127.42,
126.29, 126.89, 126.48, 126.23, 125.35, 125.21, 125.15,
124.63, 124.05, 124.01, 123.46, 113.01, 84.95, 75.31, 68.42
(CHOH), 55.01 (OMe), 38.91 (CCMe2C), 22.30, 20.60
(CC(CH3)2C).
4.1.27. Solid supports (6a–c) and (14a,b). The solid sup-
ports were prepared by a modification of the published
method.10 A solution of succinic anhydride (300 mg,
3 mmol) and DMAP (80 mg, 0.66 mmol) in 10 mL of dry
˚
pyridine was added to 300 mg of LCAA-CPG-500 A, and
the mixture was left at room temperature for 24 h with
occasional swirling. After filtration, successive washes
with 10 mL portions of pyridine, CH2Cl2, and Et2O, and
drying, the succinylated LCAA-CPG was suspended in
4 mL of DMF–pyridine (1:1 v/v); compound 4a–c or 12a,b
(0.25 mmol), 1,3-diisopropylcarbodiimide (DIC) (0.28 mL,
1.8 mmol) and DMAP (20 mg) were added; and the slurry
was left for 48 h at room temperature. To block the remaining
carboxylic acid groups, methanol (0.25 mL) (supports 6a–c)
or a solution of pentachlorophenol (100 mg) in pyridine
(1 mL) (supports 14a,b) was added, and the mixture was
kept at room temperature for the next 12 h. Then, the support
was filtered and for 14a,b only, re-suspended in 5% v/v solu-
tion of piperidine in pyridine (3 mL), reacted for 10 min and
filtered again. All the supports were washed successively
with 10 mL portions of CHCl3, MeOH, MeCN and Et2O,
and dried in vacuo. Loadings were determined by treatment
of a portion of the support (5 mg) with 3% w/v CCl3CO2H
in 1,2-dichloroethane (1 mL) and measuring the absorbance
4.1.25. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-(pyren-1-yl-
methyl)-2,4-dihydroxybutyramide (13a). Compound
12a (0.95 g, 1.5 mmol) was phosphitylated with bis(N,N-
diisopropylamino)-2-cyanoethoxyphosphine
(0.73 mL,
2.25 mmol) and diisopropylammonium tetrazolide (0.38 g,
2.25 mmol) to afford 13a (0.945 g, 70%) as a white foam,
Rf 0.54, 0.62 (CHCl3–EtOAc 1:1+1% Et3N v/v/v). ESI-
TOF HRMS: m/z¼858.3640 [M+Na]+, calcd for
[C51H54N3O6P+Na]+ 858.3642. 31R NMR (MeCN-d3) d
150.71, 149.03 (diastereoisomers,w1:1). 1H NMR (MeCN-
d3) d 8.29–7.90 (m, 9H, ArH (pyrene)), 7.42–7.12 (m, 10H,
NH, ArH (Dmt)), 6.81 (m, 4H, ArH (Dmt)), 5.10–5.04 (m,
2H, NCH2), 4.44 (m, 0.5H), 4.37 (m, 0.5O) (CHO, diastereo-
mers), 3.70 (s, 6H, CH3), 3.57–2.83 (m, 6O, RPSO2, NCH,
CH2O), 2.46 (t, 1O, J¼6.0 Hz), 2.29 (t, 1O, J¼6.0 Hz)
(CH2CN, diastereomers), 2.18 (m, 2H, CH2CH2CH), 1.98–
0.72 (m, 12O, CH3 (iPr)). 13C NMR (MeCN-d3) d 172.25,
172.03 (CO), 132.17, 131.79, 131.68, 130.84, 129.49,
128.88, 128.80, 128.68, 128.34, 128.19, 128.11, 128.04,
127.92, 127.57, 127.18, 126.23, 125.70, 125.56, 125.47,
125.33, 124.05, 123.98, 113.83, 86.78, 72.52, 72.40, 71.87,
60.17, 60.07, 59.98, 59.76, 59.54, 59.38, 59.10, 58.94,
55.75, 55.24 (OMe), 46.66, 45.91, 43.89, 43.79, 41.75,
41.61, 34.81, 34.38, 32.24, 24.61, 24.43, 23.28, 23.11,
23.04, 21.91, 20.85, 20.53, 19.62, 14.31.
of Dmt+ at 504 nm (3¼76 mL cmꢀ1 mmolꢀ1 10
)
and were
found to be (in mmol/g): 35 (6a), 52 (6b), 44 (6c), 20 (14a)
and 16 (14b).
4.1.28. Synthesis of oligodeoxynucleotides. Oligonucleo-
tide synthesis was carried out on a DNA/RNA synthesiser
using standard manufacturer’s protocols. Modified phos-
phoramidites 5a–c, 10a,b and 13a,b were used in solid-phase
oligodeoxyribonucleotide synthesis with their coupling time
increased to 10 min. After assembly, the CPG-bound oligo-
nucleotides were treated with concd ammonium hydroxide
at 55 ꢁC overnight, evaporated and precipitated from 1 M
LiClO4 (0.4 mL) by dilution with acetone (1.6 mL). Oligo-
nucleotides were isolated by electrophoresis in 20% denatur-
ing (7 M urea) polyacrylamide gel in Tris–borate buffer, pH
8.3, and desalted using NAP-10 column and standard purifi-
cation procedure.
4.1.26. (2R)-O4-(4,40-Dimethoxytrityl)-O2-(N,N-diisopro-
pylamino-2-cyanoethoxyphosphinyl)-N-(pyren-1-yl-
methyl)-2,4-dihydroxy-3,3-dimethylbutyramide (13b).
The precursor 12b (2.65 g, 4.0 mmol) was phosphitylated
with bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
(2.0 mL, 6 mmol) and diisopropylammonium tetrazolide
(1.02 g, 6 mmol) to give phosphoramidite 13b as a white
foam (3.16 g, 92%), Rf 0.30, 0.39 (hexane–EtOAc 1:1+1%
Et3N v/v/v). ESI-TOF HRMS: m/z¼886.3949 [M+Na]+,
calcd for [C53H58N3O6P+Na]+ 886.3955. 31R NMR
RP-HPLC of oligonucleotides was carried out using a Phe-
nomenex RP-C18 column (3.90ꢂ300 mm) and dual-wave-
length (215 and 254 nm) UV detection using gradient of
acetonitrile in 0.1 M aq triethylammonium acetate (0–5%,
5 min, 5–15%, 10 min, 15–40%, 30 min, 40–80%, 10 min,
80–0%, 10 min).
1
(MeCN-d3) d 152.49, 149.36 (diastereomers, w9:10). O
NMR (MeCN-d3) d 8.32–7.90 (m, 9H, ArH (pyrene)),
7.45–7.18 (m, 9H, ArH (Dmt)), 6.94 (m, 1H, NH), 6.79–
6.68 (m, 4H, ArH (Dmt)), 5.02 (m, 2H, NCH2), 4.04 (d,
0.55H, JHCOP¼12.6 Hz), 3.99 (d, 0.45O, JHCOP¼9.9 Hz)
(CHO, diastereomers), 3.70 (m, 6H, OCH3), 3.57–2.75 (m,
6O, RPSO2, NCH, CH2O), 2.47 (t, 1.1O, J¼6.0 Hz),
2.33 (t, 0.9O, J¼6.0 Hz) (CH2CN, diastereomers), 1.26–
0.70 (m, 18O, CH2C(CH3)2CH, CH3 (iPr)). 13C NMR
(DMSO-d6) d 169.29 (CO), 129.79, 128.21, 128.16,
Acknowledgements
We thank Professor Vladimir A. Efimov and Igor A. Pro-
khorenko for their encouraging interest, Donna Williams

N. N. Dioubankova et al. / Tetrahedron 62 (2006) 6762–6773
6773
11. (a) Schneider, F. Angew. Chem., Int. Ed. Engl. 1978, 17, 583–
592; (b) Perreault, D. M.; Anslyn, E. V. Angew. Chem., Int. Ed.
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for advice on oligonucleotide synthesis, and Irina A. Stepa-
nova for help in manuscript preparation. This work was
partially supported by the Russian Foundation for Basic
Research, Grant Nos. 03-03-32196 and 06-04-81019. We
are grateful to the Shemyakin–Ovchinnikov Institute NMR
Spectrometry Facility (Registry No. 98-03-08) for NMR
spectra.
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