A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis

Article abstract of DOI:10.1080/15257770701795938

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701795938

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A Novel Thymidine Phosphoramidite
Synthon for Incorporation of
Internucleoside Phosphate Linkers
During Automated Oligodeoxynucleotide
Synthesis
David Tabatadze a , Paul Zamecnik a , Ivan Yanachkov b , George
Wright b , Katherine Pierson a , Surong Zhang c , Alexei Bogdanov, Jr.
c & Valeri Metelev c d
a Cancer Center, Massachusetts General Hospital , Charlestown,
Massachusettes, USA
b GL Synthesis, Inc. , Worcester, Massachusetts, USA
c Department of Radiology , University of Massachusetts Medical
School , Worcester, Massachusetts, USA
d Department of Chemistry , Moscow State University , Moscow,
Russian Federation
Published online: 01 Feb 2008.
To cite this article: David Tabatadze , Paul Zamecnik , Ivan Yanachkov , George Wright ,
Katherine Pierson , Surong Zhang , Alexei Bogdanov, Jr. & Valeri Metelev (2008) A Novel Thymidine
Phosphoramidite Synthon for Incorporation of Internucleoside Phosphate Linkers During Automated
Oligodeoxynucleotide Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 27:2, 157-172, DOI:
10.1080/15257770701795938
To link to this article: http://dx.doi.org/10.1080/15257770701795938
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Nucleosides, Nucleotides, and Nucleic Acids, 27:157–172, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701795938
A NOVEL THYMIDINE PHOSPHORAMIDITE SYNTHON FOR
INCORPORATION OF INTERNUCLEOSIDE PHOSPHATE LINKERS
DURING AUTOMATED OLIGODEOXYNUCLEOTIDE SYNTHESIS
David Tabatadze,1 Paul Zamecnik,1 Ivan Yanachkov,2 George Wright,2
Katherine Pierson,1 Surong Zhang,3 Alexei Bogdanov, Jr.,3 and
Valeri Metelev3,4
1Cancer Center, Massachusetts General Hospital, Charlestown, Massachusettes, USA
2GL Synthesis, Inc., Worcester, Massachusetts, USA
3Department of Radiology, University of Massachusetts Medical School, Worcester,
Massachusetts, USA
4Department of Chemistry, Moscow State University, Moscow, Russian Federation
A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorpo-
2
ration of primary amino groups, attached through a triethylene glycol linker to the internucleoside
phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized
amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-
Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared
fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides
containing base-attached aminolinkers. We anticipate that the developed synthetic approach will
be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter
molecules.
Keywords Thymidine phosphoramidite synthon; sequence-specific attachment; single
ligand; multiple ligands; reporter molecules
INTRODUCTION
The use of synthetic phosphodiester oligonucleotides as therapeutic
agents faces two major challenges: 1) rapid degradation of the compounds
Received 9 April 2007; accepted 2 August 2007.
This work was supported in part by grants from the G. Harold and Leila V. Mathers Foundation and
National Institutes of Health Grant 5 R01 AI060872–02. The work of S. Z., V. M., and A. B. was supported
in part by National Institutes of Health Exploratory Grant R21 CA116144. The authors thank Mrs. Karen
Pierson for technical assistance.
Address correspondence to Paul Zamecnik, Cancer Center, Massachusetts General Hospital, 149
Thirteenth St, Charlestown, MA 02129. E-mail: paul zamecnik@hms.harvard.edu
157

158
D. Tabatadze et al.
in vivo and 2) low uptake and inefficient transport through plasma
membranes. To overcome these problems, various approaches have been
tested in recent decades. One successful strategy has been synthesis of
chemically modified oligonucleotides, particularly chemical modification
of the backbones of oligonucleotides, insofar as this approach provides
preservation of Watson-Crick hybridization properties of compounds. Syn-
thetic oligonucleotides with phosphorothioate,[1] methylphosphonate,[2]
boranophosphate,[3] benzylphosphonate,[4] modifications, locked nucleic
acid,[5,6] peptide nucleic acid,[7] morpholino derivatives,[8–9] and oligonu-
cleotides with modified terminal groups[10–13] have been widely tested as
novel therapeutic agents[14–16] and diagnostic tools.[17]
For some applications, native or modified synthetic oligonucleotides
have to be linked to nonnucleotide molecules or surfaces. The nonnu-
cleotide molecules could be fluorescent dyes or quenchers, metal chelators,
ligands for various proteins, enzymes, receptors, transporters, or other
biologically active molecules, hydrophobic residues, or even other oligonu-
cleotides. The resultant conjugates may be useful as hybridization probes in
DNA sequencing and microarray technology, as diagnostic and therapeutic
agents, electron and fluorescent microscopy probes, and have roles in
crystallography, affinity chromatography, and in cell biology research.[18–22]
Because oligonucleotides, with the exception of phosphorothioates, lack
appropriately reactive functional groups for conjugate synthesis, introduc-
tion of such groups is necessary. One of the most convenient and widely
used groups for conjugation purposes is the aliphatic amino group. It can
be coupled selectively and under mild conditions with ligands (reporters)
bearing carboxylic groups or their activated derivatives, sulfonyl chlorides,
isocyanates and isothiocyanates, aldehydes and alkylating residues, or other
electrophilic functionalities. The amino group is usually attached to the
oligonucleotide by a linker (spacer). The nature and the length of the
linkers are important for the synthesis and the function of the conjugates.
Short linkers can create spatial restrictions, which lower the reactivity of
the amino group and interfere with the function of the ligand and the
oligonucleotide. Hydrophobic linkers, even when of sufficient length, tend
to collapse in aqueous environments, assuming globular conformations and
again creating spatial crowding. The site of the spacer attachment is another
important factor determining the properties of the conjugates. Tradition-
ally, the spacer is attached at the 5ꢁ-end of the oligonucleotide upon the
completion of the automated synthesis.[23–26] The attachment to the 3ꢁ-end
on nonstandard supports also has been explored.[27] The drawbacks of
both methods are that they do not allow position-specific modifications.
If multiple reporters are to be attached, they cluster at the end of the
oligonucleotide with no convenient strategy to vary their relative positions.
When the reporter has to be placed within a certain sequence, or when

A Novel Thymidine Phosphoramidite Synthon
159
multiple reporters are to be placed within a desired distance, the intrastrand
modification is necessary. The standard approach is to attach the linker
to C-5 of the pyrimidine bases or C-8 of the purines. Deoxyribonucleoside
phosphoramidites of this type, with trifluoroacetyl-protected amino groups,
are commercially available. It is obvious that the design of the spacer and the
place of its attachment in those synthons are driven more by consideration
of synthetic convenience than by functionality. In the case of purines, an
aliphatic spacer is attached to C-8 through an amino group, and in the case
of pyrimidines, a carbamoylvinyl group introduced at C-5 is in conjugation
with the base. Both alternatives could potentially result in interfering with
normal base pairing. The attachment of the linker to the internucleoside
phosphates enables sequence-specific and multi-site labeling, minimally
perturbing the Watson-Crick base pairing and, importantly, providing the
stabilization of the oligonucleotides toward nucleases.[28,29] Two major
types of internucleoside modifications have been described: 1) using a
spacer linked to the sulphur of phosphorothioates: 2) phosphoramidate
nitrogen-linked spacers.[24] The first type of linker is prepared by alkylation
of phosphorothioates, and the second by oxidation of H-phosphonates in
the presence of 1,ω-diamines. The drawbacks of these strategies include
incompatibility with the commonly used phosphoramidite-based automated
oligonucleotide synthesis, and the possibility that they may require addi-
tional post-synthetic modifications.
The attachment of a linker to the oxygen of the internucleoside
phosphates has been explored by using a sequence-specific attachment
of 2-aminoethyl groups.[30] However, the short two-carbon atom spacer is
not optimal for further attachment of reporters or ligands to the amino
group. Increasing the length of the spacer results in its destabilization,
because of the intramolecular attack at the terminal amino group of
carbon atom adjacent to the phosphate through a favorable five or six
atom cyclic transition state, resulting in the spacer’s scission. In fact, the
4-trifluoroacetamidobutyl group has been developed as an alternative to the
cyanoethyl group for phosphate protection in oligonucleotide synthesis.[31]
Therefore, we hypothesized that by increasing the length of the spacer to 9
atoms an unfavorable 10 atom cyclic transition state would be created.[32]
We decided to test ω-aminoalkyl derivatives that are sufficiently stable under
alkaline deblocking conditions for position- specific attachment of ligands
or reporter groups, to the internucleoside phosphates.
In this paper we report the synthesis of a thymidine phosphoramidite
monomer, which was used for incorporating aliphatic primary amino groups
through a triethylene glycol spacer to the internucleoside phosphate at
desired locations. Various fluorescent or quenching reporter groups were
covalently attached to the amino groups of the synthesized oligonucleotides
in high yields.

160
D. Tabatadze et al.
MATERIALS AND METHODS
2-(2-(2-Aminoethoxy)ethoxy)ethanol, 3. A mixture of 2-(2-(2-chloro-
ethoxy)ethoxy)ethanol, 1 (50.0 g, 0.297 mol) and sodium azide (19.28 g,
0.297 mol) in 450 ml N,N-dimethylformamide was stirred overnight at
90◦C. The mixture was cooled, diluted with 500 ml tetrahydrofuran, and,
after stirring for 1 hour, it was filtered. The solid salts were washed with
tetrahydrofuran. The combined filtrate and washings, which contain the
azide 2, were stirred and treated with triphenylphosphine (83.76 g, 0.319
mol), added in two portions, and stirred continuously for 48 hours while
protected from contact with air. Water (8.1 ml) was added, and stirring
was continued for another 48 hours. The mixture was evaporated on a
membrane pump vacuum, and then on an oil pump vacuum, at a bath
temperature of 45◦C. The residue was stirred with 850 ml of water and
filtered. The solid was washed with water, and the combined filtrate and
washings were concentrated on a membrane pump vacuum at a bath
temperature of 70◦C. The resulting yellowish oil was distilled under vacuum
(0.2 mm Hg) to give 3 as a colorless oil: 39.3 g, 88% yield. 1H NMR (CDCl3):
δ 3.54–3.72 (m, 8H, CH2-O), 3.51 (t, 2H, CH2-OH), 2.82 (t, 2H, CH2-NH2),
2.30 (bs, 3H, NH2+OH); 13C NMR (Me2SO-d6): δ 73.82, 73.24, 70.63,
70.49, 61.06, 42.14; ESI+ MS: [M+H]+ 150.3, calculated for C5H16NO3
150.2.
Trifluoro-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)acetamide, 4. Methyl
trifluoroacetate (3.20 g, 2.52 ml, 25 mmol) was added drop wise through
a septum with stirring and gentle cooling to 3 (2.98 g, 20 mmol), and the
mixture was sealed and left at room temperature for 24 hours. The volatiles
were evaporated under membrane pump vacuum and bath temperature
of 65◦C to give 4 as a slightly yellowish oil which crystallized when stored
1
in the refrigerator: 4.83 g, 98% yield. H NMR (Me2SO-d6): δ 9.41 (t, 1H,
NH), 4.57 (bs, 1H, OH), 3.55–3.28 (m, 12H, CH2); 13C NMR (Me2SO-d6):
δ 156.60 (q, J 36.1 Hz, CO), 115.96 (q, J 116 Hz, CF3), 73.15, 70.50, 70.46,
68.67, 61.08, 39.99.
5ꢁ-O-DMTr-thymidine-3ꢁ-O-(2-(2-(2-trifluoroacetamidoethoxy)ethoxy)et-
hyl)-N,N-diisopropylphosphoramidite, 6. Bis-(diisopropylamino)chloroph-
osphine (1.334 g, 5 mmol) was weighed in an argon-filled glove bag and
loaded in a flame-dried, then cooled under argon vial equipped with a
stirring bar. The vial was sealed with a Teflon-lined septum, and 20 ml
of dry benzene were added through the septum under argon, followed
by dry diisopropylamine (0.607 g, 0.848 ml, 6 mmol). The clear solution
was cooled to 10◦C, and a solution of 4 (1.23 g, 5 mmol) in 2.6 ml of dry
benzene was added drop wise with vigorous stirring and cooling during
ca. 3 minutes. The mixture was stirred under argon for 1 hour at room
temperature. At that time a 31P NMR spectrum of the reaction mixture
showed complete consumption of bis-(diisopropylamino)chlorophosphine.

A Novel Thymidine Phosphoramidite Synthon
161
The reaction mixture was filtered under positive pressure of argon through
a glass fiber filter, and the solid was washed with dry benzene. The combined
filtrate and washings were evaporated under membrane vacuum to yield 5
as oil. The oil was dissolved in 10 ml of dry N,N-dimethylformamide, and
the solution was added through a septum to 5ꢁ-O-DMTr-thymidine (3.00 g,
5.5 mmol) in an argon-filled, flame dried flask, followed by 3 ml of a
0.45 M tetrazole solution in dry acetonitrile. This mixture was stirred for
1 hour under argon at room temperature. Triethylamine (0.5 ml)
was added, and the reaction mixture was evaporated under vacuum.
The resulting oil was dissolved in 50 ml dichloromethane and loaded
onto a silica gel column (30×5 cm), equilibrated with hexane:ethyl
acetate:triethylamine, 66:33:2, and eluted with a gradient of hexane:ethyl
acetate:triethylamine, 66:33:2, to ethyl acetate:triethylamine, 100:2 (4 l),
at a flow rate of 50 ml/min. The fractions containing 6 were pooled and
evaporated to give 2.40 g of solid. This material was 95% pure by HPLC and
contained 1.7 mol% of bis-(DMTr-T)-N,N-diisopropylphosphoroamidite.
Because this impurity can cause branching during oligonucleotide synthesis,
the product was repurified on a silica gel column which was eluted first
with an isocratic mixture of hexane:ethyl acetate:triethylamine, 59:39:2,
(2 l), and then with a gradient of hexane:ethyl acetate:triethylamine,
59:39:2, -hexane:ethyl acetate:triethylamine, 39:59:2, (4 l). The fractions
containing pure 6 were pooled and evaporated under vacuum. The residue
was dissolved in dry benzene and lyophilized to give 1.84 g (40% yield) of
6 as a colorless powder. HPLC: two diastereomers in ratio 2:1; purity, 96%
1
and no bis-(DMTdT)-phosphoramidite contamination. H NMR (C6D6): δ
10.3 (bs, 1H, NH-1), 7.8–7.5 (m, 4H, H-6+Ar-H+NH), 7.48–7.37 (m, 4H,
Ar-H), 7.25–7.15 (m, 2H, Ar-H), 7.13–7.04 (m, 1H, Ar-H), 6.83–6.75 (m,
4H, Ar-H), 6.65–6.53 (m, 1H, H-1ꢁ), 4.86–4-76 (m, 1H, H-4ꢁ), 4.33 and 4.18
(2 m in ratio 2:1, 1H, H-3ꢁ), 3.83–3.20 (m, 16H, CH2O, CH2NH, H-5ꢁ5ꢁꢁ,
NCHMe2), 3.37 (s, 6H, CH3O), 2.64–2.26 (m, 2H, H-2ꢁ2ꢁꢁ), 1.56 (s, 3H,
5-Me), 1.18–1.02 (12H, CHMe2); 13C NMR∗ (C6D6): δ 164.82b, 164.75a;
159.73a, 159.71b; 157.64b (q; 2JC-F = 36.1 Hz), 157.60a (q; 2JC-F = 36.1 Hz);
151.52b, 151.48a; 145.65a, 145.55b; 136.37; 136.32b, 136.00a; 131.01; 129.07;
1
1
128.92; 128.63; 127.72; 117.13b (q; JC-F = 288.0 Hz), 117.18a (q; JC-F
=
288.0 Hz); 114.04; 111.57; 110.22; 87.67; 86.33a (d; JC-P = 4.0 Hz), 86.01b
3
(d; JC-P = 6.3 Hz); 85.72; 74.00a (d; JC-P = 15.5 Hz), 73.89b (d; JC-P
=
3
2
2
17.2 Hz); 71.86b (d; JC-P = 6.9 Hz), 71.79a (d; JC-P = 7.4 Hz); 71.11b,
3
3
71.05a; 70.76b, 70.71a; 64.08a, 63.87b; 63.46b (d; JC-P = 16.1 Hz), 63.33a
2
(d; JC-P = 16.6 Hz); 55.25; 43.76 (2JC-P = 12.6 Hz); 40.90b (d; JC-P = 3.4
2
3
∗Most of the 13C resonances are split due to the presence of two diastereomers in ratio 2:1. In those
cases the signal associated with the more abundant diastereomer is indicated with the superscript “a,”
and the signal ue to the less abundant diastereomer—with the superscript “b.”

162
D. Tabatadze et al.
3
Hz), 40.60a (d; JC-P = 4.6 Hz); 40.35b, 40.27a; 25.06; 24.97; 12.25; 31P
NMR (1H dec., C6D6): δ 149.69, 149.51 (s, in ratio 1:2); 19F NMR (1H dec.,
C6D6): δ −75.86, −75.92 (s, in ratio 1:2); ESI+ MS: [M+Et3NH]+ 1020.23,
calculated for C51H74F3N5O11P 1020.51; ESI-MS: [M-H]− 917.50, calculated
for C45H57F3N4O11P 917.37.
Synthesis of oligonucleotides was performed using phenoxyacetyl
protected dA, 4-isopropyl-phenoxyacetyl protected dG, acetyl protected dC
and dT phosphoramidites (all from Glen Research, Sterling VA) on a 394
DNA/RNA synthesizer (Applied Biosystems) by using a standard protocol
for phosphoramidite synthesis. Synthesis was performed on 1 µM dT-CPG
columns (Glen Research). Coupling duration for the incorporation of
the novel dT phosphoramidite monomer 6 at the targeted location of
the oligonucleotide sequence was increased up to 3 minutes. A 50 mM
solution of monomer in anhydrous acetonitrile was used. Coupling
efficiency according to the DMTr monitor reading was 99%. Deprotection
of oligonucleotides was carried out for 5 hours at room temperature using
concentrated ammonium hydroxide. Four oligonucleotides containing
either one (I-III) or two (IV) internucleoside aminolinkers were synthesized
(Table 1).
INITIAL PURIFICATION OF OLIGONUCLEOTIDES
The ion-pair reverse-phase HPLC method was used for the isolation and
purification of modified oligonucleotides on a C18 column (Microsorb-MV
100-5, 250 × 4.6 mm, Varian, USA). For the linear eluting gradient the
following buffers were used: A, 2% acetonitrile in 0.1 M triethylammonium
acetate (TEAA), pH 7 (Glen Research); B, 50% acetonitrile in 0.1 M TEAA,
pH 7. The column was eluted at 1 ml/min at room temperature. To isolate
trityl-on oligonucleotides the following gradient was used: 90% A-10% B for
3 minutes, then to 100% B in 22 minutes. Detritylation was performed by
treating with 80% acetic acid for 30 minutes followed by ethanol/sodium ac-
etate precipitation. The purity of obtained oligonucleotides was determined
by reverse phase HPLC using a gradient of 90% A-10% B for 3 minutes, then
to 50% A-50% B in 47 minutes.
Conjugation of Oligonucleotides Bearing Aminolinkers with
Dabcyl and QSY21 NHS Esters
15 nmol of oligonucleotide bearing an aminolinker were dissolved
in 40 µl of 0.1 M NaHCO3. Three 10 µl portions of a saturated so-
lution of dabcyl NHS ester (AnaSpec, USA) in N,N-dimethylformamide
were added to the oligonucleotide solution at 20-minute intervals, and
the inhomogeneous mixture was left overnight at room temperature in the
dark. Aqueous 2 M LiClO4 (0.1 ml) and 0.7 ml of acetone were added, the

A Novel Thymidine Phosphoramidite Synthon
163
TABLE 1 Synthesized oligonucleotides bearing one or two amino reporter groups at
various positions and the corresponding dye-conjugated products
Molecular massb
No.
Oligonucleotidesa
Calculated
Observed
I
II
III
IV
CGGAAAGT∗CCCTCATAGCT
5904.0
5964.1
5964.1
6110.3
NA
5904.2
5964.4
5965.2
6111.6
TGGAAAGCTTTT∗TACAGTT
TGGAAAGCTTTCTAT∗AGTT
TGGAAAGCTTTT∗TAT∗AGTT
V
CGGAAAGˆTCCCTCATAGCT
VI
CGGAAAGTCy3.5CCCTCATAGCT
CGGAAAGTQSY21CCCTCATAGCT
CGGAAAGT800CWCCCTCATAGCT
CGGAAAGTCy7CCCTCATAGCT
CGGAAAGTdabcylCCCTCATAGCT (fraction 1)
CGGAAAGTdabcylCCCTCATAGCT (fraction 2)
TGGAAAGCTTTT800CWTACAGTT
TGGAAAGCTTTTdabcylTACAGTT
TGGAAAGCTTTTCy7TACAGTT
TGGAAAGCTTTCTATdabcylAGTT
TGGAAAGCTTTCTAT800CWAGTT
TGGAAAGCTTTTdabcylTATdabcylAGTT
CGGAAAG800CWTCCCTCATAGCT
AGCTATGAG800CWGGACTTTCCG
NA
NA
6888.9
NA
VII
VIII
IX
X1
X2
6888.0
6155.8
6155.8
6949.0
6215.3
NA
6215.3
6949.0
6612.8
6870.9
6942.9
6155.6
6155.4
6950.0
6216.0
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
6216.4
6950.0
6613.2
6870.8
6942.0
aT∗ is T-O-P (=O)-OCH2CH2OCH2CH2OCH2CH2NH2; TCy3.5 is T-O-P(=O)-OCH2-
CH2OCH2CH2OCH2CH2NH-Cy3.5; TQSY21 is T-O-P(=O)-OCH2CH2OCH2CH2OCH2-
CH2NH-QSY21; T800CW T-O-P(=O)-OCH2CH2OCH2CH2OCH2CH2NH-800CW; Tdabcyl
-
is T-O-P(=O)-OCH2CH2OCH2CH2OCH2CH2NH-dabcyl; TCy7 T-O-P(=O)-OCH2CH2-
OCH2CH2OCH2CH2NH-Cy7; Gˆ is -NH-(CH2)6-NH2 at the 8 position of guanine; and
G800CW is –NH-(CH2)6-NH-800CW at the 8 position of guanine.
bBy MS.
NA, not analyzed.
mixture was vortexed, and precipitates were collected after centrifugation
at 10000 rpm for 7 minutes. The oligonucleotides were then re-precipitated
as above followed by Micro Bio-Spin 6 column (Bio-Rad Laboratories)
purification according to the manufacturer’s recommendations. After spin-
chromatography, oligonucleotides were purified by reverse phase HPLC
using the conditions described above. Purified oligonucleotides X, XII, XIV
and XVI (Table 1) were concentrated in a centrifugal vacuum concentra-
tor (SpeedVac, Savant) to 50–100 µl, and were precipitated by standard
ethanol/sodium acetate treatment.
Conjugation of Oligonucleotides Bearing an Aminolinker with
Cy3.5, Cy7, or 800CW NHS Esters
10 nmol of aminolinker-bearing oligonucleotide were dissolved in
30 µl of 0.1 M NaHCO3. Two 7.5 µl portions of dye solutions (0.5 mg in
30 µl dimethylsulfoxide; Cy3.5, Cy7 NHS esters were from Amersham-GE

164
D. Tabatadze et al.
Healthcare (USA), and heptamethine cyanine IRDye 800CW NHS ester was
from Li-COR (USA) were added over a 1 hour interval, and the homoge-
neous reaction mixture was left overnight at room temperature in the dark.
The oligonucleotides were precipitated in a mixture of 0.1 ml water, 16 µl
3M sodium acetate (pH 5.5), and 0.7 ml ethanol. The mixture was left at
−80◦C for 3 hours, and centrifuged at 10000 rpm for 8.5 minutes. The la-
beled oligonucleotides VI, VIII, IX, XI, XIII, XV, XVII, and XVIII (Table 1)
were purified by spin-chromatography, then by reverse phase HPLC, and
finally by ethanol/sodium acetate precipitation, as described above.
Spectral measurements were performed using Cary 50 Bio UV-visible
spectrophotometer. Melting temperatures were determined with a step-wise
Peltier temperature ramping bath in 2-degree mode in a temperature-
controlled cuvette holder. Electrospray mass spectrometry (MS) was per-
formed with a LTQ Linear Ion trap mass spectrometer (Thermo, Inc) with
use of a Nanomate (Advion, Inc.) robot. Spectra were obtained in the
negative ion mode with 1.8 kV spray voltage and 0.8 PSI back pressure
(UMMS Proteomic and Mass Spectrometry Core Facility, University of
Massachusetts Medical School, Worcester, MA, USA). Sample solutions were
adjusted to 75% methanol before MS analysis. The full mass spectra were
acquired for 30 sec from m/z 400–2000 using 2 ms scans and 10 ms
maximum injection time.
RESULTS AND DISCUSSION
Synthesis of Synthon 6
In order to introduce one or more amino groups attached by an
appropriate linker to the internucleoside phosphate group in a position
specific manner, using a standard phosphoramidite chemistry with no
post-synthetic modifications, we designed a phosphoramidite synthon—5ꢁ-
O-DMTr-thymidine-3ꢁ-O-(2-(2-(2-trifluoroacetamidoethoxy)ethoxy)ethyl)-
N,N-diisopropylphosphoramidite, (6, see Figure 1). The 9 atom, triethylene
glycol linker was expected to have minimal tendency to undergo cyclization
by a nucleophilic attack of the terminal amino group on the carbon
atom adjacent to the phosphate group. In addition, the polyethylene
glycol spacers are hydrophilic in nature, conformationally flexible in
both organic and aqueous environments, and are biologically inert.[33,34]
The trifluoroacetyl protection of the amino group was chosen due to its
stability under the automated oligonucleotide synthesis conditions, and
easy removal during the basic deprotection of the oligonucleotide.
The synthesis of amidite 6 is outlined in Figure 1. First, commercially
available 2-(2-(2-chloroethoxy)ethoxy)ethanol, 1, was converted, via the
azide 2, to 2-(2-(2-aminoethoxy)ethoxy)ethanol, 3, in a one pot synthesis in
88% yield. The amino group of 3 was protected as the trifluoroacetamide by

A Novel Thymidine Phosphoramidite Synthon
165
FIGURE 1 A scheme of synthon 6 synthesis.
reaction with excess of methyl trifluoroacetate to give 4 in 98% yield. Next,
the diamidite 5 was prepared by reaction of 4 with bis(diisopropylamino)-
chlorophosphine and diisopropylamine, and, without isolation, was con-
verted to the targeted amidite 6 by reaction with a slight excess of 5ꢁ-O-
DMTr-dT in the presence of tetrazole. Synthon 6 was isolated in 52% yield
(from 4) and 95% purity by silica gel chromatography. This material was
repurified to remove a small amount (1.7 mol%) of (DMTr-T)2PN(i-Pr)2,
which could cause branching during oligonucleotide synthesis, to give, after
lyophilization from benzene, 6 in 40% final yield.
Stability and Coupling Efficiency of Synthon 6
After storing synthon 6 for six months at −20◦C, no apparent decrease
in coupling efficiency was detected. Usually the compound was dissolved
in anhydrous acetonitrile under argon and was kept on the synthesizer for
two weeks at room temperature. The coupling yield of the monomer for
the 3 minutes coupling duration was 98–99% after 7–8 days, and for the
same coupling conditions coupling yield decreased to 90% after additional
10–12 days. Coupling yields were assessed using a DMTr detector reading
on the synthesizer. Based on this finding we conclude that dry monomer
stored at −20◦C preserves its intactness over six months and is stable in dry
acetonitrile for at least 1 week.
OLIGONUCLEOTIDE SYNTHESIS
Initially we attempted to synthesize an oligonucleotide by combining
standard phosphoramidite monomers (benzoyl protected dA and dC, and

166
D. Tabatadze et al.
iso-butyryl protected dG) with our novel thymidine phosphoramidite syn-
thon, 6. The average coupling yield for 6 was 96–97% under standard
conditions. When coupling time was increased up to 3 minutes, the
coupling efficiency increased to 99%. The HPLC analysis after standard
deprotection showed that major portion of the final product was cleaved
into several small fragments (data not shown). Since phosphotriesters are
less stable in basic environment than phosphodiesters[35,36] we decided
to use “ultramild” phosphoramidite monomers (phenoxyacetyl protected
dA, 4-isopropyl-phenoxyacetyl protected dG and acetyl protected dC from
Glen Research) instead of standard ones. The use of “ultramild” monomers
enabled deprotection under milder conditions (5 hours of ammonium
hydroxide treatment at room temperature instead of 14–16 hours at 55◦C),
which resulted in a great improvement of the yield of the final product.
We demonstrated the wide applicability of internucleotide amino linkers
by synthesizing four 19-mer oligonucleotides containing one or two amino
linker groups at different positions (see Table 1). Also, for comparison
purposes, few 19-mer oligonucleotides (as example V) were synthesized
using a commercial G-phosphoramidite monomer with an amino group
attached through an aminohexyl linker to C-8 of the base. Ion-paired
reverse phase HPLC on a C18 column was used for the post-deprotection
purification of the modified oligonucleotides. Their purity and identity were
determined by analytical reverse phase HPLC and ESI mass spectrometry
(see Table 1).
Watson-Crick base paring properties of the modified oligonucleotides
were evaluated by comparison of melting temperatures of a duplex formed
between the amino-functionalized oligonucleotide CGG AAA GT∗C CCT
CAT AGC T (I) and its complementary unmodified oligonucleotide 3ꢁ GCC
TTT CAG GGA GTA TCG A 5ꢁ (duplex 1) and a control, unmodified
duplex with the same sequence (duplex 2). The melting temperatures were
almost equal, i.e. 65 and 66◦C for duplexes 1 and 2, respectively. Therefore,
the presence of aminolinker phosphotriester group in the middle of the
oligonucleotide sequence did not appear to interfere with Watson-Crick
hybridization properties of the oligonucleotides.
Conjugation of the Amino-Modified Oligonucleotides with
Fluorescent Dyes
Amino-functionalized oligonucleotides were conjugated with three dif-
ferent red and far-red fluorochromes (Cy3.5, Cy7, and 800CW) as well as
with two quenchers (dabcyl and QSY21). The conjugates were purified
by ion-pair reverse phase HPLC, and characterized by HPLC analysis,
mass spectrometry and UV/VIS spectroscopy (for details, see Materials
and Methods and Table 1). The results of mass spectrometric analysis
of the products correlated well with their calculated molecular masses

A Novel Thymidine Phosphoramidite Synthon
167
FIGURE 2 Reverse phase HPLC analysis of reaction of amino-linker modified oligonucleotide I with
dabcyl NHS ester leading to CGGAAAGTdabcylCCCTCATAGCT (X). Panel A, steep gradient. Panel B,
shallow gradient. In the case of the shallow gradient the product is separated into two fractions, X1 and
X2, due to the presence of two diasteriomers, identical by mass spectrometry (Table 1).
(Table 1). HPLC analysis showed that covalent attachment of dyes to
the aminolinker phosphotriester groups afforded high yields of target
products (80–90%). For example, Figures 2A and 2B show HPLC profiles
of CGGAAAGTdabcylCCCTCATAGCT (X) under conditions of steep and
shallow gradients of acetonitrile, respectively. In the case of the shallow
gradient the product X showed separation into two peaks, probably due
to two different diastereomeric forms of the product as a result of cre-
ation of additional chiral center at the triester phosphorus atom. Those
two peaks (X1 and X2) were collected separately, and their mass spectra
were compared. The mass-to-charge values for both isomers were equal
and in good agreement with the calculated masses of expected product
(Table 1).
High yields of dye conjugation to the oligonucleotides at the inter-
nucleotide phosphotriester were observed for all compounds synthesized.
The major factors explaining the high yields presumably are the steric
accessibility and hydrophilic nature of the amino linker moiety. Covalent
attachment of a dye to an aminolinker group at the internucleotide triester

168
D. Tabatadze et al.
FIGURE 3 Reverse phase HPLC analysis of reaction mixtures of coupling of oligonucleotides
with heptamethine cyanine IRDye 800CW. Panel A, CGGAAAGˆTCCCTCATAGCT (V). Panel B,
CGGAAAGT∗CCCTCATAGCT (I).
provided significantly higher yield than the attachment via an aminolinker
of the heterocyclic base. Figure 3 shows the HPLC analysis of the reac-
tion mixtures resulting from labeling of CGGAAAGˆTCCCTCATAGCT (V)
where the amino group is attached via hexylamino linker to C-8 of G, and
CGGAAAGT∗CCCTCATAGCT (I) where the amino group is attached to
the phosphotriester, with IRDye 800CW NHS ester. Both reactions were
performed at the same time and under identical conditions. Yield of VIII
FIGURE 4 Reverse phase HPLC analysis of the reaction mixture of coupling of
TGGAAAGCTTTT∗TAT∗AGTT (IV) with dabcyl NHS ester. The presence of residual IV, the
mono-adduct and the target product (TGGAAAGCTTTTdabcylTATdabcylAGTT, XVI) are shown.

A Novel Thymidine Phosphoramidite Synthon
169
where dye is attached to the aminolinker at the phosphotriester (panel A,
∼90%) was approximately 2.5 times higher than the yield of XVII where
linking was performed via the aminolinker on the nucleotide base (panel B,
∼35%). The same trend was observed in other experiments with NHS ester
of heptamethine cyanine IRDye 800CW dye (the yield of XVIII was about
35%, however, thecompounds XI and XV were obtained with the 85% yield)
as well as with dabcyl NHS ester and the corresponding oligonucleotides
(data not shown).
A di-amino-functionalized oligonucleotide was labeled successfully to
give the doubly labeled oligonucleotide in 50% yield. Figure 4 shows
FIGURE 5 UV/VIS spectra of dye-labeled oligonucleotides. Panel A, CGGAAAGT800CW
CCCTCATAGCT (VIII, dotted line) and TGGAAAGCTTTT800CWTACAGTT (XI, solid line). Panel B,
TGGAAAGCTTTCTATdabcylAGTT (XIV, solid line) and TGGAAAGCTTTTdabcylTATdabcylAGTT (XVI,
dotted line).

170
D. Tabatadze et al.
HPLC analysis of the reaction mixture of TGGAAAGCTTTT∗TAT∗AGTT
(IV) and dabcyl NHS ester (for coupling conditions see Materials and
Methods). The chromatogram shows three major peaks that corre-
spond to the starting material, the mono-adduct, and the target prod-
uct TGGAAAGCTTTTdabcylTATdabcylAGTT (XVI), respectively. Further evi-
dence for labeling of oligonucleotides was found by comparison of
UV/VIS spectra of a mono and di-labelled oligonucleotides. In Figure
5A are shown the UV/VIS spectra of CGGAAAGT800CWCCCTCATAGCT
(VIII) and TGGAAAGCTTTT800CWTACAGTT (XI), in which the absor-
bance maximum at 780 nm indicates the presence of covalently
attached 800CW dye. In Figure 5B are the UV/VIS spectra of
dabcyl-labelled TGGAAAGCTTTCTATdabcylAGTT (XIV) and TGGAAA-
GCTTTTdabcylTATdabcylAGTT (XVI), both of which show absorbance max-
ima at 470 nm, indicating the presence of dabcyl groups. As expected,
the double-labeled oligonucleotide XVI has a twice higher 470/260 nm
absorbance ratio than oligonucleotide XIV (Figure 5B).
We performed a limited stability study of one of the novel oligonu-
cleotide conjugates, VIII, which contained 800CW dye at the eighth nucle-
oside from the 5ꢁ-end (see Table 1). When dissolved in deionized water and
stored at −20◦C for approximately four months, HPLC analysis showed no
signs of degradation (Figure 6).
In conclusion, we have synthesized and successfully used a novel phos-
phoramidite synthon that enables facile incorporation of primary amino
groups attached via a triethylene glycol linker to the internucleoside phos-
phates during the automatic oligonucleotide synthesis. The novel amino-
linker bearing oligonucleotides have preserved Watson-Crick base-pairing
properties, are stable under deprotection and conjugation conditions,
and are superior in their labeling yields to oligonucleotides containing
FIGURE 6 Reverse phase HPLC analysis of CGGAAAGT800CWCCCTCATAGCT (VIII) dissolved in
deionized water and stored at −20◦C for approximately 4 months.

A Novel Thymidine Phosphoramidite Synthon
171
base-attached aminolinkers. The novel synthon and ligand-conjugation
method can be used for high yield, sequence-specific attachment of single
or multiple ligands or reporter molecules to synthetic oligonucleotides.
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