Oligonucleotides with 2′-O-carboxymethyl group: Synthesis and 2′-conjugation via amide bond formation on solid phase

Article abstract of DOI:10.1039/b409496d

An efficient method for synthesis of oligonucleotide 2′-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2′-O-carboxymethyl group were obtained by use of a novel uridine 3′-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2′-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2′-position of an oligonucleotide and the N-terminus of a peptide.

Full text of DOI:10.1039/b409496d

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A R T I C L E
Oligonucleotides with 2-O-carboxymethyl group: synthesis and
2-conjugation via amide bond formation on solid phase†
Anna Kachalova,a Eugeny Zubin,a Dmitry Stetsenko,*b Michael Gaitb and Tatiana Oretskayaa
a
Chemistry Department, M. V. Lomonossov Moscow State University, Leninskie Gory,
Moscow 119992, Russia. E-mail: oretskaya@genebee.msu.su; Fax: +7-095-9393181;
Tel: +7-095-9395411
MRC Laboratory of Molecular Biology, Hills Road, Cambridge UK CB2 2QH.
b
E-mail: ds@mrc-lmb.cam.ac.uk; Fax: +44-1223-402070; Tel: +44-1223-248011
Received 23rd June 2004, Accepted 30th July 2004
First published as an Advance Article on the web 3rd September 2004
An efficient method for synthesis of oligonucleotide 2-conjugates via amide bond formation on solid phase is
described. Protected oligonucleotides containing a 2-O-carboxymethyl group were obtained by use of a novel
uridine 3-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group
is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine
treatment. 2-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under
normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high
purity in good yield. The method is well suited in principle for the preparation of peptide–oligonucleotide conjugates
containing an amide linkage between the 2-position of an oligonucleotide and the N-terminus of a peptide.
peptides by amide bond formation on solid phase under peptide
Introduction
coupling conditions or in aqueous media by a water-soluble
There are many methodologies available for preparing natural
carbodiimide-mediated reaction.
oligonucleotides, as well as their analogues containing modified
We would like to report here the synthesis of the 2-O-
carboxymethylated oligonucleotides by use of a uridine 3-
phosphoramidite, where the carboxylic acid function carries an
allyl protecting group, which is readily removed under mild and
specific conditions of Pd(0)-catalysis. Subsequent conjugation
of the 2-carboxylic acid to a range of primary aliphatic and
phosphate groups, nucleobases and sugars.1 The advances in
solid-phase synthesis inspired various strategies for preparation
of oligonucleotide conjugates.2,3 Oligonucleotide conjugation
is predominantly carried out by use of a nucleophilic group
on an oligonucleotide to react with an electrophilic group on a
tag or a solid support. This strategy still predominates because
aromatic amines and peptides is achieved by peptide coupling
the common oligonucleotide deprotection is performed by base
reagent-promoted amide bond formation on a solid support. We
treatment e.g. ammonia or methylamine which are inherently
nucleophilic. Indeed, there are many methods of preparation of
demonstrate examples of the synthesis of 2-O-methyl oligoribo-
nucleotides containing a 2-O-carboxymethyl group and their
conjugation to a range of amines and peptides.
oligonucleotides modified with amino or thiol groups at a variety
of positions.1 On the other hand, there are many situations when
researchers wish to introduce an electrophilic group into oligo-
Results and discussion
nucleotides.4 In this vein, we developed a phosphoramidite incor-
porating a carboxylic acid function protected by a 2-chlorotrityl
group.5a The reagent is suitable for solid-phase synthesis of 5-
carboxylated oligonucleotides that may be conjugated to various
amines on solid supports after activation by a suitable peptide
coupling reagent.5 The monomer is now commercially available.5b
Similarly, 5-carboxy-modifier C10 is sold by Glen Research that
contains a preformed carboxylic acid N-hydroxysuccinimide
ester and can be used directly in solid-phase conjugation.6
In previous work in this field we7 and others8,9 described
syntheses of 2-O-7,8 or 2-S-carboxymethyloligonucleotides,9
where an alkyl ester was chosen as a carboxylic acid protecting
group.Aftercompletionof solid-phaseoligonucleotideassembly,
support-bound oligonucleotides containing methyl7,8b or ethyl8a
ester were hydrolysed by treating with aq. NaOH or treated
by an appropriate amine to afford either the carboxymethyl
group or the corresponding amides, respectively, after final
deprotection. As a result, these methods are frequently plagued
by low yields, long reaction times, the need for large excesses of
reactant(s), and the formation of by-products that are difficult
to separate.
The route to the 2-O-(allyloxycarbonyl)methyluridine build-
ing block 6 is illustrated in Scheme 1. The starting 3,5-O-
(tetraisopropyldisiloxan-1,3-diyl)uridine 1 was obtained from
uridine as reported previously12,13 and purified by column
chromatography on silica gel in CHCl3–EtOAc (9:1, then
2:1 v/v). Protection of the N3 of uracil is required, since the
subsequent alkylation of 3,5-O-protected uridine preferentially
occurs at the base moiety. To protect the imido function from
this side reaction, we chose to block the N3-position with an
ammonia-labile pivaloyloxymethyl (Pom) group. The group
also prevents undesirable side reactions during oligonucleotide
assembly. Furthermore, the greater lipophilicity of the Pom
derivative is helpful for chromatographic separation of the
target compound. Initially, Pom was reported as a protecting
group for the lactam function of a uracil moiety.14 This group
was introduced by the transient protection procedure, which
involved 2-O-trimethylsilylation followed by N3-protection.14b
On the other hand, Sekine15 showed earlier that various N3-
protected uridine derivatives may be obtained selectively in
the presence of an unprotected 2-OH group under conditions
of phase-transfer catalysis. These results led us to consider a
phase-transfer reaction to convert 1 into 2. Indeed, when 1 was
allowed to react with 10 equiv. of chloromethyl pivalate in a
biphasic system 0.2 M Na2CO3–CH2Cl2 (2:1 v/v) in the presence
of 0.2 equiv. of tetra-n-butylammonium hydrogen sulfate
(TBAHS) as a phase-transfer catalyst at room temperature
Recently, we reported our preliminary studies10,11 that show
that oligonucleotides containing a 2-carboxymethyl group
can be conjugated to amines, amino acid derivatives and small
† Electronic supplementary information (ESI) available: MALDI-TOF
spectra. See http://www.rsc.org/suppdata/ob/b4/b409496d/
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 9 3 – 2 7 9 7
2 7 9 3

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Table 1 Properties of 2-carboxy-modified 2-O-methyloligonucleotides and their conjugates
#
Oligonucleotide sequence, 5-3/conjugated molecule
MALDI-TOF MS calc./found (m/z)
Retention timea/min
Yieldb (%)
I
UUUUUU*UUUU
3183.9/3182.6
3942.6/3942.7
3311.6/3310.2
3264.6/3264.1
3256.5/3254.8
3276.5/3277.8
3367.7/3366.6
3481.8/3482.6
4141.8/4138.4
4155.9/4153.4
4088.8/4084.3
4054.8/4051.0
4235.9/4230.4
4201.9/4199.4
4243.0/4245.8
4208.9/4206.3
4889.7/4894.0
15.5
15.7
14.5
16.0
15.0
14.8
14.7
14.9
27.1
27.5
17.2
16.2
21.6
16.9
17.1
21.7
21.5
—
—
>90
>90
>90
75
85
80
65
85
>90
>90
>90
>90
>90
75
II
CUCCCAGGCU*CA
I.7
N,N,N-Tris(aminoethyl)amine (7)
Cyclohexylamine (8)
3-Amino-1,2-propanediol (9)
Histamine (10)
Spermine (11)
N,N,N,N-Tetrakis(3-aminopropyl)-1,4-butanediamine (12)
1-Aminopyrene (13)
I.8
I.9
I.10
I.11
I.12
II.13
II.14
II.15
II.16
II.17
II.18
II.19
II.20
II.21
1-Pyrenemethylamine (14)
H-Phe-NH2 15)
H-Leu-NH2 (16)
H-Phe-Phe-NH2 (17)
H-Leu-Phe-NH2 (18)
H-Gly-Leu-Met-NH2 (19)
H-Pro-Leu-Gly-NH2 (20)
H-Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2 (21)
80
a For conditions, see Experimental section. b Conversion of oligonucleotide peak to the conjugate peak, calculated from RP-HPLC traces.
for 48 h under vigorous stirring, N3-pivaloyloxymethyl-3,5-
O-(tetraisopropyldisiloxan-1,3-diyl)uridine 2 was isolated in
70% yield after column chromatography. General methods
of 2-O-alkylation of ribonucleosides have been reviewed
by Zatsepin et al.16 We previously adopted a convenient
procedure7 for 2-O-alkylation by use of a strong sterically
hindered organic base 2-tert-butylimino-2-diethylamino-1,3-
dimethylperhydro-1,3,2-diazaphosphorine (BEMP) described
in a number of publications.17,18 Later we replaced BEMP by
another phosphazene base P1-tert-butyltris(tetramethylene)
(BTPP), which is cheaper and even more basic, though
less sterically hindered than BEMP.19 Thus, we found that
alkylation of compound 2 with 2.5 equiv. of allyl chloroacetate
and 2.8 equiv. of BTPP gave 3 in 90% yield after 3–5 h.
Compound 3 was then desilylated smoothly with TBAF7 or,
better, triethylamine trihydrofluoride13 in THF and then
converted into the 5-O-dimethoxytrityl (DMTr) derivative
5 by the known procedure.20 Subsequent phosphitylation of
the 3-hydroxy group in an inert atmosphere using bis(N,N-
diisopropylamino)-2-cyanoethoxyphosphine in CH2Cl2 in the
presence of diisopropylammonium tetrazolide21 afforded the
phosphoramidite 6. This was used successfully in machine-
assisted solid-phase oligonucleotide synthesis. The average
coupling efficiency of 6 at a 0.2 M concentration in dry MeCN
and 30 min reaction time was found to be greater than 97%.
The modified phosphoramidite was utilised in the synthesis
of two 2-O-methyloligoribonucleotides I and II (Table 1).
Decamer I was synthesised as a model to select and optimise the
specific conditions of deblocking, purification and conjugation.
Oligonucleotide II is complementary to the HIV-1 TAR RNA
apical stem-loop, the binding site for the HIV-1 trans-activator
protein Tat.22
To remove the 2-allyl protecting group on the solid phase,
we used a mixture of tetrakis(triphenylphosphine)palladium(0),
triphenylphosphine and morpholine7 in dry CH2Cl2 for 50 min
(Scheme 2). During the procedure, other protecting groups
remain intact and the oligonucleotide is still linked to a polymer
support.23
Further conjugations of 2-O-carboxymethylated oligo-
nucleotides I and II were carried out on solid phase in organic
solvent (Scheme 2). The choice of amine was influenced by
the expected application for the conjugate e.g. introduction
of positive charge(s) or nucleophilic amino groups (N,N,N-
tris(aminoethyl)amine 7, histamine 10, spermine 11 and
N,N,N,N-tetrakis(3-aminopropyl)-1,4-butanediamine
12),
chemoselective ligation (3-amino-1,2-propanediol 9)22 or
fluorescent labeling (1-aminopyrene 13 and 1-pyrenemethyl-
amine 1413). In the case of spermine that contains both primary
and secondary amino groups, we expected selective reaction with
a primary amino group facilitated by the excess of amine.5a For
further conjugation experiments, we have chosen several amino
acid derivatives 15 and 16 and short peptides 17–20 and FMRF
amide-related peptide 21 demonstrated to be cardioactive neuro-
peptide.24 To prevent the formation of byproducts both amino
acids and peptides were utilised as C-terminal amides. To obtain
a high yield of structurally diverse types of conjugates (Table 1)
we used pre-activation of polymer-bound 2-O-carboxymethyl-
oligonucleotide with TBTU–HOBT (1:1) at 37 °C in dry DMF
Scheme 1 Preparation of 2-O-(allyloxycarbonyl)methyluridine 3-phosphoramidite (7). Abbreviations: TIPS - 1,1,3,3-tetraisopropyldisiloxan-
1,3-diyl, Pom - pivaloyloxymethyl, TBAHS - tetra-n-butylammonium hydrogen sulfate, BTPP - phosphazene base P1-tert-butyltris(tetramethylene),
DMTr - 4,4-dimethoxytrityl.
2 7 9 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 9 3 – 2 7 9 7

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Scheme 2 Solid-phase synthesis, selective deprotection and conjugation of 2-O-carboxymethylated oligonucleotides. Abbreviations: R, R1 =
protected oligonucleotide chain, R2 = amine or peptide residue, R3, R4 = unprotected oligonucleotide chain; TBTU - O-benzotriazol-1-yl-N,N,N,N-
tetramethyluronium tetrafluoroborate, HOBt - 1-hydroxybenzotriazole.
for 40 min, followed by addition of the amine, amino acid or
peptide and further incubation.25 Reaction times for the amines
differedsignificantlyfromthosefortheaminoacidsandpeptides.
Whilst all amines reacted within 3 h, amino acids and peptides
required overnight reaction. After completion of the reaction,
polymer-bound conjugates were cleaved from their solid
supports and deprotected by concentrated aqueous ammonia
treatment at 55 °C overnight. Reaction mixtures obtained were
analysed by reversed-phase HPLC and MALDI-TOF MS.
Only a single product was observed in the case of spermine
conjugate I.11. Examples of typical RP-HPLC traces are shown
in Fig. 1. Noteworthy, the two unprotected arginine residues in
peptide 21 did not interfere with its successful conjugation at
the N-terminus. We ascribe this to the presence of an excess of
HOBt that may protonate the guanidino group of arginine and
thus protect it from acylation.
or therapeutic evaluation. In addition, this conjugation method
may be a useful route for attachment of membrane-penetrating
peptides, particularly those containing multiple arginine
residues, to the 2-position of oligonucleotides and their
analogues for cellular uptake studies.
Experimental
General
Chemicals were obtained from commercial suppliers and used
without further purification unless otherwise noted. Chloro-
methyl pivalate, tetra-n-butylammonium hydrogen sulfate,
allyl chloroacetate, triethylamine trihydrofluoride, tetrakis(tri-
phenylphosphine)palladium(0), N,N,N-tris(aminoethyl)amine,
3-amino-1,2-propanediol, histamine, spermine, N,N,N,N-
tetrakis(3-aminopropyl)-1,4-butanediamine,
1-aminopyrene
and 1-pyrenemethylamine hydrochloride were purchased from
Aldrich. Phosphazene base P1-tert-butyltris(tetramethylene),
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine,
triphenylphosphine, morpholine, 1-hydroxybenzotriazole, O-
benzotriazol-1-yl-N,N,N,N-tetramethyluronium tetrafluoro-
borate (TBTU) and cyclohexylamine were from Fluka.
4,4-Dimethoxytrityl chloride (DMTrCl) was from Avocado.
Amino acids and peptides were bought from Bachem.
Diisopropylammonium tetrazolide was prepared from
diisopropylamine (BDH) and 1H-tetrazole solution in
acetonitrile (Glen Research).21 Dichloromethane (BDH) was
used after refluxing over and distillation from CaH2. DMF
(Fisher) was distilled in vacuo and used fresh. Other solvents:
benzene, chloroform, ethyl acetate, acetone, acetonitrile, THF,
hexane, absolute ethanol and methanol were used as received.
1H NMR spectra were recorded on a Bruker DRX 500 spectro-
meter (500.13 MHz for 1H and 202.4 MHz for 31P) and 2–5 mM
solutions. Chemical shifts (d, ppm) for 1H and 31P are referenced
to internal solvent resonances and reported relative to SiMe4 and
85% aq. H3PO4, respectively. 2D spectra involved use of adapted
COSY and HMQC techniques. Chemical shifts are accurate to
Fig. 1 Reversed-phase HPLC traces of crude oligonucleotide 2-con-
jugates: (1) oligonucleotide II; (2) conjugate II.20; (3) conjugate II.17;
(4) conjugate II.14. For HPLC conditions, see Experimental section.
In conclusion, we have described an efficient and reliable
method for preparation of 2-O-carboxymethyloligonucleo-
tides using protected uridine 3-phosphoramidite containing
a 2-carboxymethyl group protected as an allyl ester. After
deprotection under conditions of Pd(0) catalysis, such modified
oligonucleotides may be conjugated, while still attached to a
solid support, to a range of primary or secondary aliphatic
or aromatic amines or to the N-termini of short peptides.
The method described could prove useful in synthesis of small
molecule libraries of oligonucleotide conjugates for diagnostic
1
within 0.01 ppm for H. CSSI are accurate to within 0.25 Hz.
MALDI-TOF spectra were recorded on a Voyager DE system
(Applied Biosystems) or Reflex IV (Bruker) in positive ion mode
using either a 1:1 (v/v) mixture of 2,6-dihydroxyacetophenone
(2,6-DHAP) (40 mg cm−3 in MeOH) and aq. diammonium
hydrogen citrate (80 mg cm−3) for all oligonucleotides, and
2,5-dihydroxybenzoic acid (2,5-DHBA) (10 mg cm−3 in 50%
aq. MeOH) or 2,4,6-trihydroxyacetophenone (2,4,6-THAP)
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 9 3 – 2 7 9 7
2 7 9 5

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(10 mg cm−3 in 50% aq. MeOH) for low molecular weight
compounds as a matrix. TLC was carried out on Merck DC
Kieselgel 60 F254 aluminium sheets. Compounds were visualised
under short-wavelength UV and stained by trifluoroacetic acid
vapours for DMTr-containing species. Column chromatography
was carried out on Kieselgel 60 0.040–0.063 mm (Merck).
calc. m/z 495.56, found 497.04. 1H NMR (CDCl3): d 7.81 (d, 1H,
H-6, J5,6 = 7.5), 5.92 (AB system, 2H, NCH2OCOBut), 5.89 (ddt,
1H, CHCH2), 5.78 (d, 1H, H-1, J1,2 = 10), 5.76 (d, 1H, H-5,
J5,6 = 7.5), 5.53 (d, 1H,CH2-(Z)), 5.24 (d, 1H, CH2-(E)), 4.67
(br d, 2H, H-3, H-4), 4.61 (AB system, 2H, OCH2CO), 4.58,
4.43 (AB system, 2H, CO2CH2CH), 4.03, 4.0 (dd, 2H, H-3,
H-4), 3.88 (dd, 1H, H-2), 3.66 (dt, 1H, H-5), 3.58 (dt, 1H,
H-5), 3.0 (s, 1H, OH), 1.19 (s, 9H, But).
3,5-O-(Tetraisopropyldisiloxane-1,3-diyl)uridine (1). This
was prepared as described previously.13
2-O-Allyloxycarbonylmethyl-5-O-(4,4-dimethoxytrityl)-
N3-pivaloyloxymethyluridine (5). Compound 4 (2.3 g, 5 mmol)
was co-evaporated with dry pyridine (3 × 20 cm3), dissolved
in dry pyridine (50 cm3), cooled in an ice-bath, and DMTrCl
(2.05 g, 6.05 mmol) was added in one portion. The reaction
was monitored by TLC until the starting nucleoside disappears
completely. Then the excess of DMTrCl was quenched with
MeOH (2 cm3), and after 10 min the mixture was partially
evaporated, diluted with CHCl3 (100 cm3), washed with 5%
NaHCO3 (2 × 100 cm3), and brine (100 cm3), then dried
(Na2SO4), evaporated, co-evaporated with benzene (3 × 25 cm3)
and the residue was chromatographed on a silica gel column
(0→20→25→30→50→100% CHCl3 in benzene and further
1→2→5% MeOH in CHCl3 + 1% pyridine v/v/v). Yield
(3.4 g, 90%). Rf 0.4 (CHCl3–EtOH, 95:5 v/v). MALDI-TOF
N3-Pivaloyloxymethyl-3,5-O-(tetraisopropyldisiloxan-
1,3-diyl)uridine (2). To a two-phase solution of compound
1 (4.86 g, 10 mmol) in CH2Cl2 (200 cm3)–0.2 M aq. Na2CO3
(400 cm3) were added chloromethyl pivalate (14.3 cm3,
99.2 mmol) and tetra-n-butylammonium hydrogen sulfate
(0.74 g, 2.1 mmol). The resulting mixture was vigorously stirred
at ambient temperature for 48 h. Then the organic phase was
collected and washed with 5% NaHCO3 (2 × 200 cm3). The
organic layers were combined, dried (Na2SO4) and filtered.
The filtrate was evaporated, co-evaporated with benzene
(3 × 25 cm3) and the residue was chromatographed on silica
gel (stepwise gradient of 0→2→4→6→8→10% EtOAc in
benzene, v/v) to give compound 2 which was obtained as a white
foam (4.2 g, 70%). Rf 0.85 (CHCl3–EtOH, 9:1 v/v). MALDI-
TOF (2,5-DHBA): [M + Na]+ calc. m/z 623.85, found 624.05. 1H
NMR (CDCl3): d 7.81 (d, 1H, H-6, J5,6 = 7.5), 5.92 (AB system,
2H, NCH2OCOBut), 5.81 (s, 1H, H-1), 5.76 (d, 1H, H-5,
J5,6 = 7.5), 4.67 (br d, 2H, H-3, H-4), 4.22 (br s, 2H, H-5), 4.0
(d, 2H, H-2), 1.19 (s, 9H, But), 1.1–0.9 (m, 28H, Pri).
1
(2,4,6-THAP): [M + Na]+ calc. m/z 780.81, found 780.57. H
NMR (CDCl3): d 7.8 (d, 1H, H-6, J5,6 = 7.5), 7.38–7.17 (m, 10H,
Ar), 6.85 (d, 4H, o-Ar), 5.92 (AB system, 2H, NCH2OCOBut),
5.89 (m, 1H, CHCH2), 5.82 (d, 1H, H-1), 5.58 (d, 1H, H-5,
J5,6 = 7.5), 5.22 (d, 2H, CH2CHCH2), 4.69 (s, 2H, OCH2CO),
4.64 (d, 2H, CH2CHCH2), 4.39 (t, 1H, H-3), 4.32 (t, 1H,
H-2), 4.22 (br s, 1H, H-4), 3.8 (s, 6H, OCH3), 3,52, 3.43 (dd,
2H, H-5), 1.19 (s, 9H, But).
2-O-Allyloxycarbonylmethyl-N3-pivaloyloxymethyl-3,5-
O-(tetraisopropyldisiloxan-1,3-diyl)uridine (3). Compound 2
(4.2 g, 7 mmol) was dried by co-evaporation with dry MeCN
(3 × 20 cm3) and was then dissolved in dry MeCN–THF
(100 cm3, 1:1 v/v). Phosphazene base P1-tert-butyltris(tetra-
methylene) (6 cm3, 19.6 mmol) followed immediately by allyl
chloroacetate (2 cm3, 17.5 mmol) were added to the stirred
mixture at ambient temperature. TLC (CHCl3–EtOH, 97:3 v/v)
showed complete reaction after 3 h. The reaction mixture was
evaporated to dryness in vacuo. The yellow oil was dissolved
in CHCl3 (100 cm3) and washed with brine (1 × 100 cm3)
and water (2 × 100 cm3). The combined organic layers were
dried (Na2SO4), filtered, evaporated and co-evaporated with
benzene (3 × 25 cm3). The crude product was purified by
column chromatography on silica gel (stepwise gradient of
0→2→4→6→8% EtOAc in benzene, v/v). Compound 3 was
obtained as a pale yellow oil (4.4 g, 90%). Rf 0.58 (CHCl3–
EtOH, 97:3 v/v). MALDI-TOF (2,5-DHBA): [M + H]+ calc.
m/z 699.97, found 699.32. 1H NMR (CDCl3): d 7.81 (d, 1H, H-6,
J5,6 = 7.5), 5.92 (AB system, 2H, NCH2OCOBut), 5.89 (ddt, 1H,
CHCH2), 5.81 (s, 1H, H-1), 5.76 (d, 1H, H-5, J5,6 = 7.5), 5.53
(d, 1H, CH2-(Z)), 5.24 (d, 1H, CH2-(E)), 4.67 (br d, 2H,
H-3, H-4), 4.58, 4.43 (AB system, 2H, CO2CH2CH), 4.25
(A part of AB system, 2H, CO2CH2CH), 4.22 (br s, 2H, H-5),
4.0 (overlap, 2H, H-2, B part of AB system, CO2CH2CH),
1.19 (s, 9H, But), 1.1–0.9 (m, 28H, Pri).
2-O-Allyloxycarbonylmethyl-3-O-(N,N-diisopropylamino-
2-cyanoethoxyphosphinyl)-5-O-(4,4-dimethoxytrityl)-N3-
pivaloyloxymethyluridine (6). Compound 5 (3.4 g, 4.5 mmol)
was co-evaporated with dry CH2Cl2 (3 × 20 cm3), dissolved in
dry CH2Cl2, diisopropylammonium tetrazolide (1.1 g, 6.7 mmol)
and bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine
(2.3 cm3, 7.3 mmol) were added, and the reaction mixture
was stirred at 25 °C overnight. After TLC (CH2Cl2–Et3N,
98:2 v/v) showed the completion of the reaction, the
mixture was diluted with CH2Cl2 (50 cm3), washed with brine
(2 × 50 cm3), dried (Na2SO4), and evaporated to dryness. The
residue was dried in vacuo to afford 7 as a white foam (4.1 g,
95%). Rf 0.45 (CH2Cl2–Et3N, 98:2 v/v). MALDI-TOF (2,6-
DHAP–diammonium hydrogen citrate): [M + H]+ calc. m/z
960.06, found 960.13. 31P NMR (CD3CN): d 153.0, 153.5 (two
diastereomers).
Oligonucleotide synthesis
2-O-Methyloligoribonucleotides were assembled on an ABI
394B DNA Synthesizer by the phosphoramidite method
according to the manufacturer’s recommendations. Protected
2-O-methylribonucleoside phosphoramidites and S-ethylthio-
tetrazole were purchased from Glen Research (via Cambio).
Prepacked 0.4 lmol functionalised columns of controlled pore
glass (Glen Research) were used throughout. For couplings with
modified phosphoramidite 6, 0.2 M concentration in dry MeCN
was used, and the coupling time was increased to 30 min.
2-O-Allyloxycarbonylmethyl-N3-pivaloyloxymethyluridine
(4). To a solution of 3 (4.4 g, 6.3 mmol) in THF (15 cm3)
in a 30 cm3 screw-capped Teflon vial (Nalgene) was added
triethylamine trihydrofluoride (2.6 cm3, 15.7 mmol) and the
mixture was left for 1.5 h at room temperature, the completion
of deprotection was checked by TLC (CHCl3–EtOH, 9:1 v/v),
then diluted with EtOAc (50 cm3), washed with 5% NaHCO3
(2 × 50 cm3), water (50 cm3), 5% citric acid (2 × 50 cm3), and
brine (50 cm3), then dried (Na2SO4), evaporated to dryness
and co-evaporated with CHCl3 (3 × 25 cm3). The residue was
chromatographed on a silica gel column (0→1→2→3→4%
MeOH in CHCl3, v/v). Yield (2.3 g, 80%). Rf 0.15 (CHCl3–EtOH,
9:1 v/v). MALDI-TOF (2,5-DHBA): M+ calc. m/z 456.45, found
456.26, [M + Na]+ calc. m/z 479.44, found 479.11, [M + K]+
Deprotection of carboxylic acid function of oligonucleotides I, II
The 2-allyl protecting group was removed from the support-
bound modified oligonucleotides by treatment with a solution
of morpholine (0.03 cm3), tetrakis(triphenylphosphine)-
palladium(0) (5 mg) and triphenylphosphine (5 mg) in dry
CH2Cl2 (0.2 cm3) for 40 min at ambient temperature. The
supernatant was then decanted and the support was rinsed with
CH2Cl2 (2 × 0.15 cm3), EtOH (0.15 cm3), water (0.15 cm3) and
CH2Cl2 (0.15 cm3). The support was then dried on air.
2 7 9 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 9 3 – 2 7 9 7

View Online
85, 2409–2416; (b) further information on use of our reagent is
available from Link Technologies, Ltd. on www.linktech.co.uk/
Downloads/TIS-BC-02 v 1.3.pdf.
Coupling of amines, amino acids and peptides to 2-O-carboxy-
methyloligonucleotides I, II
6 R. I. Hogrefe and M. M. Vaghefi, US Pat., 6,320,041, 2001.
7 A. V. Kachalova, T. S. Zatsepin, E. A. Romanova, D. A. Stetsenko,
M. J. Gait and T. S. Oretskaya, Nucleosides, Nucleotides Nucleic
Acids, 2000, 19, 1693–1707.
8 (a) C. A. Buhr and M. D. Matteucci, Eur. Pat., EP 0942000A2, 1999;
(b) T. P. Prakash, A. M. Kawasaki, E. A. Lesnik, S. R. Owens and
M. Manoharan, Org. Lett., 2003, 5, 403–406.
9 H. Ozaki, S. Moriyama, K. Yokotsuka and H. Sawai, Tetrahedron
Lett., 2001, 42, 677–680.
10 A. V. Kachalova, E. M. Zubin, T. S. Zatsepin, Yu. V. Agapkina,
Yu. M. Ivanova, D. A. Stetsenko, M. J. Gait, T. S. Oretskaya, in
Innovation & Perspectives in Solid Phase Synthesis & Combinatorial
Libraries 2004, ed. R. Epton, Mayflower Worldwide, Kingswinford,
in press.
11 A. V. Kachalova, D. A. Stetsenko, M. J. Gait and T. S. Oretskaya,
Bioorg. Med. Chem. Lett., 2004, 14, 801–804.
12 B. Beijer, M. Grøtli, M. Douglas and B. Sproat, Nucleosides
Nucleotides, 1994, 13, 1905–1928.
13 V. A. Korshun, D. A. Stetsenko and M. J. Gait, J. Chem. Soc.,
Perkin Trans. 1, 2002, 1092–1104.
14 (a) M. Sekine and T. Hata, J. Am. Chem. Soc., 1986, 108, 4581–4586;
(b) M. Grøtli, R. Eritja and B. Sproat, Tetrahedron, 1997, 53,
11317–11346.
After the completion of the 2-carboxylic acid group
deprotection, a solution of TBTU (5 mg) and HOBt (2 mg)
in dry DMF (0.2 cm3) was added. The slurry was incubated at
37 °C for 40 min with occasional swirling, then an amine, amino
acid or peptide was added (100 equiv.). The reaction was carried
out at 37 °C for 3 h in the case of amines or overnight for amino
acid derivatives or peptides and shaking, then the supernatant
was discarded, and glass beads washed successively with DMF
(2 × 0.2 cm3), water (2 × 0.2 cm3) and EtOH (2 × 0.2 cm3).
Cleavage from the support and deprotection of phosphate and
nucleobase residues were performed using conc. aq. ammonia
overnight at 55 °C. Reaction mixtures were analysed by RP-
HPLC, conjugates were purified by RP-HPLC on a Gilson
HPLC system using a Beckman Ultrasphere ODS column
(4.6 × 250 mm) and dual wavelength detection (215 and 254 nm);
buffer A: 5% of MeCN (v/v) in 0.1 M triethylammonium
acetate, buffer B: MeCN; flow rate 1 cm3 min−1, gradient of B in
A: 0–5%, 5 min, 5–15%, 10 min, 15–40, 30 min, 40–80%, 10 min,
80–0%, 10 min. Combined fractions containing the conjugate
were evaporated, redissolved and precipitated by 4 M sodium
acetate solution (0.2 cm3) and ethanol (1.5 cm3) or 2 M LiClO4
(0.2 cm3) and acetone (1.5 cm3). Molecular masses of purified
conjugates were then checked by MALDI-TOF MS.
15 M. Sekine, J. Org. Chem., 1989, 54, 2321–2326.
16 T. S. Zatsepin, E. A. Romanova and T. S. Oretskaya, Russ. Chem.
Rev., 2002, 71, 513–534.
17 R. Schwesinger, Chimia, 1985, 39, 269–273.
18 M. Grøtli, M. Douglas, B. Beijer, R. G. Garsia, R. Eritja and
B. Sproat, J. Chem. Soc., Perkin Trans. 1, 1997, 2779–2788.
19 For further information on phosphazene bases see, Chem
Files (Fluka), 2003, 3, that can be downloaded from
http://www.sigmaaldrich.com/Brands/Fluka__Riedel_Home/
Literature/ChemFiles/Vol_3_No_1.html.
Acknowledgements
This work was supported by the Wellcome Trust CRIG
069419 and AstraZeneca UK, Ltd. The authors thank
Dr A. D. Malakhov and Mrs A. N. Muravieva for help with
HPLC purification.
20 M. Smith, D. H. Rammler, I. H. Goldberg and H. G. Khorana,
J. Am. Chem. Soc., 1962, 84, 430–440.
21 M. H. Caruthers, A. D. Barone, S. L. Beaucage, D. R. Dodds, E. F.
Fisher, L. J. McBride, M. Matteucci, Z. Stabinsky and J.-Y. Tang,
Methods Enzymol., 1987, 154, 287–313.
22 T. S. Zatsepin, D. A. Stetsenko, A. A. Arzumanov, E. A. Romanova,
M. J. Gait and T. S. Oretskaya, Bioconjugate Chem., 2002, 13,
822–830.
23 E. M. Zubin, E. A. Romanova, E. M. Volkov, V. N. Tashlitsky, G. A.
Korshunova, Z. A. Shabarova and T. S. Oretskaya, FEBS Lett.,
1999, 456, 59–62.
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