C-glycoside phosphoramidite building block for versatile functionalization of oligodeoxyribonucleotides

Article abstract of DOI:10.1039/a703059b

A chiral non-nucleosidic phosphoramidite building block containing a thioester function in its structure, 4, is synthesized in six steps starting from '2-deoxy-D-ribose'. It is used in the machine-assisted oligonucleotide synthesis in a conventional manner. Upon completion of the oligonucleotide chain assembly the thioester bond is cleaved with various nucleophiles (hydroxide ion, propane-1,3-diamine, cystamine, histamine), resulting in oligonucleotide conjugates with a tether group in the middle of the oligonucleotide chain.

Full text of DOI:10.1039/a703059b

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C-Glycoside phosphoramidite building block for versatile
functionalization of oligodeoxyribonucleotides
Jari Hovinen*,† and Harri Salo
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
A chiral non-nucleosidic phosphoramidite building block containing a thioester function in its structure,
4, is synthesized in six steps starting from ‘2-deoxy-D-ribose’. It is used in the machine-assisted
oligonucleotide synthesis in a conventional manner. Upon completion of the oligonucleotide chain
assembly the thioester bond is cleaved with various nucleophiles (hydroxide ion, propane-1,3-diamine,
cystamine, histamine), resulting in oligonucleotide conjugates with a tether group in the middle of the
oligonucleotide chain.
groups. For the introduction of a functional group in the 3Ј- or
Introduction
5Ј-end of an oligonucleotide the method is straightforward.
However, for the introduction of reporter groups in the
middle of the oligonucleotide chain a multistep synthesis of
a phosphoramidite building block derived from 3Ј-deoxy-
psicothymidine 3 is needed.19,20 After completion of the
oligonucleotide-chain assembly the laevulinyl group (Lv) is
removed selectively with hydrazinium acetate, and the phos-
phoramidite 2 is coupled to the free hydroxy group.18 Here is
presented a simple synthesis of a non-nucleosidic phosphor-
amidite building block, 4, starting from ‘2-deoxy--ribose’ (2-
deoxy--erythro-pentose). This C-glycoside derivative enables
attachment of various functional groups in the middle of the
oligonucleotide chain by use of our tethering strategy. The use
of compound 4 results in oligonucleotide conjugates where the
structure of the entire oligomer is minimally modified. The
introduction of an abasic site in the middle of the oligonucleo-
tide chain destabilizes the duplex formed upon hybridization
with a complementary oligonucleotide to the same extent as a
spot mutation.21
Synthetic oligonucleotides covalently linked to various ligands
have been increasingly used as research tools in molecular
biology.1 They have been applied to genetic analysis, and to
elucidate the mechanisms of gene function.2–6 Oligonucleotides
carrying reporter groups have had widespread use in automated
sequencing,7–9 hybridization affinity chromatography10,11 and
fluorescence microscopy.12,13 Oligonucleotide–biotin conjugates
are widely used as hybridization probes.6 Antisense oligo-
nucleotides covalently linked to intercalators, chain-cleaving or
alkylating agents have been shown to be efficient as gene-
expression regulators. The sequence-specific artificial nucleases,
when targeted against messenger RNA (mRNA), may find
applications even as chemotherapeutics.
We have recently developed a versatile strategy for oligo-
nucleotide tethering: an ester14–17 or thioester18 function is
attached to the oligonucleotide during chain assembly, and
upon completion of the oligonucleotide synthesis the desired
functional group is introduced by treating the oligomer, when
still anchored to the solid support, with an appropriate nucleo-
phile (Scheme 1). Finally, the oligonucleotide is allowed to react
Results and discussion
CN
O
O
O
P
Synthesis of the phosphoramidite
H
N
DMTrO
P
O
Preparation of the phosphoramidite building block, 4, is out-
lined in Scheme 2. Treatment of ‘2-deoxy--ribose’22 with an
equimolar amount of 4,4Ј-dimethoxytrityl chloride (DMTrCl)
yielded 2-deoxy-5-O-(4,4Ј-dimethoxytrityl)--ribose 5 in mod-
erate yield (51%). When compound 5 was allowed to react with
(ethoxycarbonylmethylene)triphenylphosphophorane in dry
tetrahydrofuran (THF) at reflux, compound 6 was formed. It
was cyclized without isolation to the epimeric furanoses 7a,b by
treatment with ethanol containing traces of sodium ethoxide.
The epimers were separated on a silica gel column and were
S
NPri2
Ph
S
O
1
2
1. Oligonucleotide synthesis using substrates 1 - 4
2. Deprotection with nucleophiles (e.g., 0.1 M aq.
alkane-α,ω-diamine, hydroxide ion, cystamine,
histamine followed by conventional ammoniolysis)
1
1
characterized based on the following observations: H, H 2D
nuclear Overhauser enhancement (NOESY) spectrum of the
faster migrating isomer had distinct cross-peaks between H-2b
and H-3 (the latter proton is undoubtedly on the β-face of the
C-glycoside ring; for numbering, see Scheme 2) as well as
between H-2b and H-3. By contrast, there was no cross-peak
between H-1 and H-2a. Thus, H-2b and the anomeric proton
are on the same face of the ring, and the compound is the
α-anomer, 7a. The opposite effect was observed in the case of
the slower migrating epimer: there was a cross-peak between
H-3 and H-2b, H-2a and H-2, but not between H-2b and H-1.
Hence this compound can be judged as the β-anomer, 7b. The
Nu
oligo
O
DMTrO
Thy
DMTrO
O
O
OLv
COSCH2Ph
O
P
O
P
3
4
NC
NC
NPri2
O
NPri2
O
Scheme 1
1
in solution with electrophiles, such as succinimido, isothio-
cyanato, maleimido or chlorosulfonyl derivatives of conjugate
α:β ratio according to H NMR analysis‡ of the unpurified
‡ Determination of the α:β ratio is based on the integral ratio of the
well separated 3-protons of isomers 7a,b.
† Present address: Wallac Oy., P.O. Box 10, FIN-20101, Turku, Finland.
J. Chem. Soc., Perkin Trans. 1, 1997
3017

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DMTrO
O
(i)
O
O
OH
OH
DNA
O
O
DMTr-T
CPG
HO
synthesis
OH
OH
COSCH2Ph
5
O
P
O
CN
(ii)
O
(ii), (iii)
11
DMTrO
HO
CO2Et
OH
O
(i), (ii)
CHCO2Et
OH
OH
O
6
O
8
O
(iii)
C-X
(i)
O
P
O
O–
2b
CO2Et
5
DMTrO
O
DMTrO
O
H
H
O
1
6
3
4
+
H
CO2Et
2
OH
H
12 X = NH[CH2]3NH2
OH
(iv)
2a
13 X = NH[CH2]2SS[CH2]2NH2
7a
7b
N
14 X = NH[CH2]2
N
H
15 X = OH
Scheme 3 Reagents: (i) aq. amine or NaOH; (ii) aq. ammonia
DMTrO
(v)
DMTrO
O
O
nucleotide tethering a model sequence dT6XT7 (X = abasic site)
was prepared (Scheme 3). The phosphoramidite 4 was used in
CO2–Py+
COSCH2Ph
the 7th coupling step in conventional manner (0.1 mol dmϪ3
4
OH
OH
9
in acetonitrile, coupling time 25 s). No difference between the
coupling efficiency of compound 4 and commercial nucleosidic
phosphoramidite building blocks was detected. When the
oligonucleotide-chain assembly was completed the solid-
support-bound material was treated with various nucleophiles
as shown in Table 1. We have already shown that thioesters
react smoothly with nitrogen nucleophiles under mild con-
ditions.16 Indeed, also in the present case, treatment of the pro-
tected oligonucleotide 11 with dil. aq. amines gave the desired
oligonucleotide conjugates 12–14, according to HPLC analysis,
in over 90% yield (peaks of capped sequences are not included).
When 0.1 mol dmϪ3 NaOH was used as the cleavage agent, the
corresponding acid, 15, was obtained. The deprotection pro-
cedure was completed by conventional ammonolysis to verify
that all of the oligonucleotide was released from the solid
support.
10
(vi)
DMTrO
O
COSCH2Ph
O
P
4
NCCH2CH2O
NPri2
Scheme 2 Reagents and work-up conditions: (i) DMTrCl in pyridine;
(ii) Ph3P᎐CHCO2Et in THF; (iii) NaOEt (trace) in EtOH; (iv) 1 mol
᎐
dmϪ3 NaOH–1,4-dioxane (1:4 v/v); then Dowex-50 (pyridinium form);
(v) DCC and toluene-α-thiol in dichloromethane; (vi) 2-cyanoethyl
N,N,NЈ,NЈ-tetraisopropylphosphorodiamidite and 1H-tetrazole in
acetonitrile
Characterization of the oligonucleotides prepared
reaction mixture was 1:2.4. This is quite surprising, since it
is known that Wittig-mercurio-23 and -iodocyclizations24 of
2-deoxy-5-O-trityl--ribofuranoses afford a 1:1 mixture of
epimers. Furthermore, when ‘2-deoxy--ribose’ was converted
into compound 8 as described by Rokach et al.25 followed by
dimethoxytritylation, products 7a and 7b were obtained in an
equimolar ratio.
The α-epimer 7a was converted into the phosphoramidite
building block, 4, as follows: saponification of the ester func-
tion gave the corresponding acid, 9, dicyclohexylcarbodiimide
(DCC)-assisted thioesterification of which with toluene-α-thiol
gave rise to thioester 10 (75% overall yield). An excess of the
thiol was used in order to suppress the reaction of the
unprotected 3-hydroxy group. Compound 10 was finally phos-
phitylated using 2-cyanoethyl N,N,NЈ,NЈ-tetraisopropyl-
phosphorodiamidite in the presence of 1H-tetrazole to give,
after silica gel column chromatography, thioester 4 as the final
product.
The oligonucleotides prepared were isolated on ion-exchange
high-performance liquid chromatography (HPLC), further
purified on a reversed-phase (RP) column and desalted by gel
filtration.14 An ion-exchange HPLC profile of the oligonucleo-
tide tethered to cystamine, compound 13 (crude reaction
mixture), is shown in Fig. 1 as an illustrative example. The
structures of the oligonucleotide conjugates were verified on
matrix-isolated laser-desorption time-of-flight (MALDI-TOF)
mass spectrometry.26 In all the cases the observed relative
molecular masses were in agreement with the expected values
(Table 1).
Experimental
General
Reagents for oligonucleotide synthesis were purchased from
Cruachem. 2-Cyanoethyl N,N,NЈ,NЈ-tetraisopropylphosphoro-
diamidite was prepared according to a published procedure.27
Cystamine dihydrochloride was converted into the free-base
form 28 which was used without purification. NMR spectra were
recorded on a JEOL LA-400 spectrometer operating at 399.8
Synthesis of the oligonucleotide conjugates
In order to demonstrate the suitability of thioester 4 for oligo-
3018
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 1 Deprotection procedures for compound 11 to obtain tethered oligonucleotides 12–15, their ion-exchange HPLC retention timesa and
relative molecular masses obtained by MALDI-TOF mass spectrometryb
Product
Deprotection procedure
tR/minc
M(Found)
M(Calc.)
12
14
13
15
(i) 0.1  aq. propane-1,3-diamined for 6 h, (ii) ammonolysise
(i) 0.2  aq. histamine for 6 h,d (ii) ammonolysise
(i) 0.1  aq. cystamine for 6 h,d (ii) ammonolysise
(i) 0.1  aq. NaOH for 4 h,d (ii) ammonolysise
13.3
13.5
13.2
17.3
4184.3
4221.2
4267.3
4128.7
4186.8
4223.9
4265.0
4130.7
a For conditions, see Experimental section. b The molecular ion of dT14 (4196.8 Da) was used as an external standard. c tR of dT14 was 15.0 min.
d At room temperature. e Conc. aq. ammonia, 7 h, at 55 ЊC.
ations with dry pyridine and was dissolved in the same solvent
(500 cm3). DMTrCl (12.6 g, 37.3 mmol) was added and the
mixture was stirred overnight at room temperature. The reac-
tion was quenched with ice–water, and all volatile material
was removed in vacuo. The residue was dissolved in dichloro-
methane and washed with saturated aq. NaHCO3. The organic
phase was dried over MgSO4 and concentrated. Purification on
silica gel (eluent CH2Cl2–MeOH–Et3N 97:3:0.1; v/v/v) yielded
the title compound as a foam (8.3 g, 51%); δH([2H6]DMSO)
7.44–7.20 (9 H, m), 6.88 (4 H, d, J 6.9), 6.21 and 6.17 (tot. 1 H,
J 2 × 4.9, exch. with D2O), 5.37 (1 H, m), 5.00 and 4.96 (tot. 1
H, 2 d, J 5.0 and 5.4, exch. with D2O), 4.08 and 3.96 (tot. 1 H, 2
m), 3.89 and 3.77 (tot. 1 H, m), 3.76 (6 H, s), 3.03 (2 H, m), 2.27
(1 H, m) and 1.83 (1 H, m).
Ethyl [2-deoxy-5-O-(4,4Ј-dimethoxytrityl)-á- and -â-D-erythro-
pentofuranosyl]acetate 7a,b
Method A. Compound 5 (10 g, 22.9 mmol) was dissolved
in dry THF (100 cm3). (Ethoxycarbonylmethylene)triphenyl-
phosphorane (Fluka; 8.88 g, 25.2 mmol) was added, and the
mixture was heated at reflux for 6 h before being allowed to
cool, and all volatile material was removed in vacuo. The residue
was dissolved in ethanol (100 cm3) containing traces of sodium
ethoxide and kept 1 h at ambient temperature. The solvent was
removed, the residue was dissolved in dichloromethane, and the
solution was washed with brine and dried (MgSO4). The residue
was applied onto a silica gel column and eluted with a mixture
of hexane and ethyl acetate (3:2, v/v) in order to remove
triphenylphosphine oxide. Fractions containing a mixture
Fig. 1 Ion-exchange HPLC profile of 13 (crude reaction mixture) at
of the epimers 7a,b were pooled and the rechromatographed
260 nm. The large peak at tR ~ 3 min is the result of non-nucleosidic
(eluent CH2Cl2–MeOH–Et3N, 98:2:0.1, v/v/v) to give the pure
material. For gradient limits, see the Experimental section.
anomers. Compound 7a (2.32 g, 20%) (Found: C, 71.4; H, 6.8.
and 161.9 MHz for 1H and 31P, respectively. SiMe4 was used as
C30H34O7 requires C, 71.1; H, 6.8%); δH(CDCl3) 7.42–7.19 (9
H, m, ArH), 6.82 (4 H, d, J 6.8, ArH), 4.50 (1 H, m, H-1), 4.31
an internal (1H) and H3PO4 as an external (31P) reference.
(1 H, m, H-3), 4.15 (2 H, q, J 7.2, OCOCH2), 4.05 (1 H, m,
H-5), 3.79 (6 H, s, 2 × OCH3), 3.24 (1 H, dd, J 4.6 and 9.6,
Ha-5), 3.07 (1 H, dd, J 6.2 and 9.6, Hb-5), 2.73 (1 H, dd, J 6.4
and 15.9, Ha-6), 2.65 (1 H, dd, J 5.9 and 15.9, Hb-6), 2.48 (2 H,
m, Ha-2 and 3-OH), 1.79 (1 H, m, Hb-2) and 1.26 (3 H, t, J 7.2,
CH3). Isomer 7b (5.67 g, 49%) (Found: C, 71.3; H, 6.5%);
δH(CDCl3) 7.69–7.19 (9 H, m, ArH), 6.82 (4 H, d, J 6.8, ArH),
4.55 (1 H, m, H-1), 4.33 (1 H, m, H-3), 4.15 (2 H, q, J 7.2,
OCOCH2), 3.93 (1 H, m, H-4), 3.79 (6 H, s, 2 × OCH3), 3.23 (1
H, dd, J 4.4 and 9.6, Ha-5), 3.07 (1 H, dd, J 6.0 and 9.6, Hb-5),
2.66 (1 H, dd, J 7.2 and 15.4, Ha-6), 2.51 (1 H, dd, J 6.0 and
15.4, Hb-6), 2.06 (1 H, ddd, J 2.4, 5.7 and 13.1, Ha-2), 1.84 (2 H,
m, Hb-2 and 3-OH) and 1.25 (3 H, t, J 7.2, CH3).
Method B. ‘2Ј-Deoxy--ribose’ (5.0 g, 37.3 mmol) was treated
with (ethoxycarbonylmethyl)triphenylphosphorane (1.5 mol
equiv.) as described above for compound 5. After base-
catalyzed cyclization, all volatile material was removed in vacuo.
The residue was coevaporated with dry pyridine and dissolved
in the same solvent (50 cm3). DMTr-Cl (12.6 g, 37.3 mmol) and
a catalytic amount of 4-(dimethylamino)pyridine (DMAP)
were added and the mixture was stirred overnight at ambient
temperature. Work-up and purification was performed as
described above in Method A. The isolated yields of isomers 7a
and 7b were both 35%.
J-Values are given in Hz. When reported, characterization of
1
1
NMR signals is based on H, H 2D homonuclear chemical-
shift correlation spectroscopy (COSY) and NOESY experi-
ments. MALDI-TOF mass spectra of oligonucleotides were
recorded on a Finnigan Lasermat spectrometer (negative detec-
tion mode) using the procedure described by Pieles et al.26
Elemental analyses were performed on a Perkin-Elmer 2400
Series II instrument.
HPLC Techniques
HPLC analyses were carried out as described previously in
detail.14 Chromatographic conditions in the present work were:
Ion exchange (SynChropak AX-300, 4.6 × 250 mm, 6 µm), flow
rate 1 cm3 minϪ1, buffer A = 0.05 mol dmϪ3 KH2PO4 in 50%
(v/v) aq. formamide, pH 5.6; Buffer B = A ϩ 0.6 mol dmϪ3
(NH4)2SO4, from 10 to 60% B in 30 min. Reversed phase
(Nucleosil 300-5C18, 4.0 × 250 mm, 5 µm), flow rate 1 cm3
minϪ1, Buffer A = 0.05 mol dmϪ3 NH4OAc, Buffer B = A ϩ
50% (v/v) MeCN, from 0 to 40% B in 40 min. Gel filtration
(TSKgel G2000SW, 7.5 × 300 mm), flow rate 1 cm3 minϪ1
water.
,
2-Deoxy-5-O-(4,4Ј-dimethoxytrityl)-D-erythro-pentofuranose 5
‘2-Deoxy--ribose’ (5.0 g, 37.3 mmol) was dried by coevapor-
J. Chem. Soc., Perkin Trans. 1, 1997
3019

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S-Benzyl [2-deoxy-5-O-(4,4Ј-dimethoxytrityl)-á-D-erythro-
pentofuranosyl]thioacetate 10
References
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Compound 7a (2.0 g, 3.95 mmol) was dissolved in a mixture of
1,4-dioxane and 0.1 mol dm3 NaOH (20 cm3; 1:1 v/v) and the
solution was stirred for 1 h at room temperature. Solvent was
evaporated off, the residue was dissolved in a mixture of
pyridine–water–methanol (2:1:1, v/v/v) and the solution was
passed through a Dowex-50 resin (pyridinium form) using the
same solvent system as the eluent. All volatile material was
removed in vacuo. After coevaporation with pyridine the resi-
due was dissolved in dichloromethane (20 cm3). Toluene-α-thiol
(0.93 cm3, 7.90 mmol) and DCC (0.82 g, 3.95 mmol; predis-
solved in 5 cm3 of CH2Cl2) were added and the mixture was
stirred for 4 h at ambient temperature. The dicyclohexylurea
formed was filtered off, and the filtrate was washed with brine
and dried (MgSO4). Purification on a silica gel column (eluent
CH2Cl2–MeOH 97:3, v/v) yielded the title compound as an oil
(1.73 g, 75%) (Found: C, 72.1; H, 6.43; S, 5.75. C35H36O6S
requires C, 71.9; H, 6.21; S, 5.48%); δH(CDCl3) 7.42–7.19 (14 H,
m, ArH), 6.82 (4 H, d, J 6.8, ArH), 4.58 (1 H, m, H-1), 4.30 (1
H, m, H-3), 4.12 (2 H, AB, J 13.9, SCH2Ph), 3.83 (1 H, m, H-4),
3.76 (6 H, s, 2 × OCH3), 3.18 (1 H, dd, J 4.2 and 9.8, Ha-5), 3.10
(1 H, dd, J 5.4 and 9.8, Hb-5), 2.93 (1 H, dd, J 7.1 and 14.9,
Ha-6), 2.76 (1 H, dd, J 5.6 and 14.9, Hb-6), 2.02 (1 H, ddd, J 2.4,
5.6 and 7.8, Ha-2), 1.54 (1 H, m, Hb-2) and 1.74 (1 H, br, 3-OH).
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Synthesis of the phosphoramidite 4
Predried alcohol 10 (1.75 g, 3.0 mmol) and 2-cyanoethyl
N,N,NЈ,NЈ-tetraisopropylphosphorodiamidite (1.39 g, 4.60
mmol) were dissolved in dry acetonitrile (1.5 cm3). 1H-
Tetrazole (3.0 mmol, 6.70 cm3; 0.45 mol dmϪ3 in acetonitrile)
was added, and the mixture was stirred for 30 min at room
temperature before being poured into 5% aq. NaHCO3 (150
cm3) and extracted with dichloromethane (2 × 75 cm3). The
extracts were combined, dried (MgSO4), and concentrated.
Purification on a silica gel column (eluent hexane–ethyl acetate–
triethylamine, 5:4:1, v/v/v) yielded compound 4 as an oil (1.8 g,
80%), which could be stored at Ϫ20 ЊC; δH(CDCl3) 7.45–7.20
(14 H, m, ArH), 6.82 (4 H, 2 × d, J 6.8, ArH), 4.69 (1 H, m,
H-1), 4.46 (1 H, m, H-3), 4.20 (1 H, m, H-4), 3.78 and 3.77 (tot.
6 H, 2 × s, OCH3), 3.71 (3 H, m, OCH2CH2CN and CHMe2),
3.31 (3 H, m, Ha-6a and H2-5), 2.85 (1 H, m, Hb-6), 2.53 and
2.40 (tot. 2 H, CH2CN), 2.40 (1 H, m, Ha-2), 1.92 and 1.82 (tot.
1 H, 2 × m, Hb-2) and 1.15 (12 H, Pri); δP(CDCl3) 148.31 (0.5 P)
and 148.22 (0.5 P).
Preparation of the oligonucleotides
The oligonucleotides were assembled on an Applied Bio-
systems 392 DNA Synthesizer in 0.2 µmol scale using
phosphoramidite chemistry and recommended protocols
(DMTr-Off synthesis). The phosphoramidite 4 was used in the
7th coupling step in a conventional manner (0.1 mol dm3 solu-
tion in dry acetonitrile, coupling time 25 s). When the oligo-
nucleotide-chain assembly was completed the fully protected
oligonucleotide was treated with aqueous nucleophiles and
ammoniolysed (for reaction times and concentrations, consult
Table 1).
Acknowledgements
Financial support from the Academy of Finland is gratefully
acknowledged.
Paper 7/03059B
Received 6th May 1997
Accepted 24th June 1997
3020
J. Chem. Soc., Perkin Trans. 1, 1997