Preparation of spin-labeled 2-amino-dA, dA, dC and 5-methyl-dC phosphoramidites for the automatic synthesis of EPR active oligonucleotides

Article abstract of DOI:10.1055/s-2001-12355

2,2,6,6-Tetramethyl-1-piperidinyloxy free radical (TEMPO) labeled phosphoramidites of 2-amino-dA, dA, dC and 5-methyl-dC were synthesized and used for the automatic synthesis of mono-labeled oligodeoxynucleotides (ODNs), which proved active to EPR. It is now possibile to insert a paramagnetic probe into nucleic acids in a site- and type-specific manner. The combination of this synthetic approach with EPR spectroscopy can be exploited for performing studies on dynamics and local structural modifications in nucleic acids.

Full text of DOI:10.1055/s-2001-12355

PAPER
565
Preparation of Spin-Labeled 2-Amino-dA, dA, dC and 5-Methyl-dC
Phosphoramidites for the Automatic Synthesis of EPR Active Oligonucleotides
Cesare Giordano,*a Federica Fratini,b Donato Attanasio,c Luciano Cellaib
a Centro di Studio per la Chimica del Farmaco, CNR, Dipartimento di Studi Farmaceutici, Università "La Sapienza", P.le A Moro 5, 00185,
Rome, Italy
Fax +39(6)491491; E-mail: cesare.giordano@uniroma1.it
b Istituto di Strutturistica Chimica "G. Giacomello", CNR, P. O. Box 10, 10016 Monterotondo Stazione, Rome, Italy
c Istituto per la Chimica dei Materiali, CNR, P.O. Box 10, 00016 Monterotondo Stazione, Rome, Italy
Received 3 August 2000; revised 11 December 2000
bring about only minor structural perturbations into du-
Abstract: 2,2,6,6-Tetramethyl-1-piperidinyloxy free radical
plex structures, since the label protrudes into the major
(TEMPO) labeled phosphoramidites of 2-amino-dA, dA, dC and 5-
methyl-dC were synthesized and used for the automatic synthesis of
mono-labeled oligodeoxynucleotides (ODNs), which proved active
groove of the double helix.9 In addition to spin-labeled dA
and dC, we planned to synthesize also the analogues
to EPR. It is now possibile to insert a paramagnetic probe into nu- 2-amino-dA and 5-methyl-dC since both are known to sta-
cleic acids in a site- and type-specific manner. The combination of
bilize duplexes relative to dA and dC. An enhanced bind-
this synthetic approach with EPR spectroscopy can be exploited for
ing can thus be achieved by using the modified bases and
performing studies on dynamics and local structural modifications
this can turn out to be particularly useful with short ODNs
in nucleic acids.
duplexes or if the spin-label is positioned close to the
ends.
Key words: DNA, nucleosides, ODNs, nitroxides, EPR
Introduction
The insertion of a spin-label like TEMPO into nucleic ac-
ids allows the application of EPR spectroscopy to the
study of dynamics and local structure modifications.1
C5-Spin-labeled dU analogues were prepared for antiviral
activity evaluation2 and for incorporation into ODNs by
enzymatic3 or automatic synthesis.4 Furthermore, spin-la-
beling was obtained, on a micro scale, by treating haloge-
nated adenylic coenzymes with a large excess of 4-amino-
TEMPO (Tempamine).5 Spin-labels were attached to dif-
ferent positions of nucleosides through tethers varying in
length and flexibility.6
Here we report the synthesis of 6-TEMPO-labeled 2-ami-
no-dA (1a) and dA (1b), 4-TEMPO-labeled dC (2a) and
5-methyl-dC (2b) phosphoramidites (Figure 1), which
carry a nitroxide spin-label directly linked to the exocyclic
amino group. They were prepared with the aim of increas-
ing the possibility of inserting a spin-label into nucleic ac-
ids by automatic synthesis, in a site- and type- specific
manner. In fact the availability of labeled dA and dC al-
lows one to insert the probe at any base pair along the se-
quence. The two pyrimidine derivatives have already been
prepared, but by a different procedure and with lower
yields.7 We decided to attach TEMPO directly to the exo-
cyclic amino group of the above mentioned nucleic bases
for the following reasons: i) the presence of linkers con-
necting the reporter group to the nucleoside would greatly
influence EPR spectra in dependence of the length and the
rigidity of the linker;8 ii) the present derivatives are able
to give Watson Crick pairings as normal bases do; and
iii) the introduction of the label at those positions should
Figure 1 TEMPO-labeled 2-amino-dA, dA, dC and 5-methyl-dC
phosphoramidites
Preparation of the Spin-Labeled Phosphoramidites
The synthesis of 1a,b is outlined in Scheme 1. At first,
N2,3’,5’-triisobutyryl-2’-deoxyguanosine
(Triibu-dG)10
(3a) and 3’,5’-diisobutyryl-2’-deoxyinosine (Diibu-dI)11
(3b) were converted to the O6-sulfonylated derivatives
4a,b by reaction with 2,4,6-triisopropylbenzenesulfonyl
chloride (TPS-Cl) in the presence of an organic base ac-
cording to a known procedure.11 Sulfonylation of 3a gave
4a as the only product in 90% yield; on the contrary, the
reaction with 3b produced a mixture of N1- and O6-sulfo-
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Reagents and conditions: i) TPS-Cl/TEA/DMAP/CH2Cl2, r.t., 18 h, 4a: 90%, 4b: 50%; ii) Tempamine/CH2Cl2, reflux, 18 h, 54 69%; iii) 6a,b:
NH4OH/MeOH, r.t., 4 h, 90 95%; 10a,b: TBAF/THF, r.t., 18 h, 95%; iv) DMTr-Cl/pyridine, r.t., 3 h, 69 77%; v) NCCH2CH2OP(Cl)Ni-Pr2,
i-Pr2NEt/CH2Cl2, r.t., 30 min, 81 91%
Scheme 1
Reagents and conditions: See Scheme 1
Scheme 2
nylated isomers, in a ratio of nearly 1:1, which were easily Nucleophilic displacement with tempamine, by using the
separated by flash chromatography.11
same procedure used for the purine derivatives, gave spin-
labeled 9a,b in 69% and 65% yields, respectively. Quan-
titative removal of the 3’,5’-tetraisopropyldisiloxanyl
group was achieved by a slight excess of fluoride ion in
THF to generate 10a,b in good yields. 5’-O Dimethoxytri-
tylation and 3’-O activation afforded 11a,b and the phos-
phoramidites 2a,b, respectively.
The spin-labeled analogues were prepared by nucleo-
philic substitution of the respective sulfonylated com-
pounds with two equivalents of tempamine in boiling
CH2Cl2 overnight and gave 5a,b in 54% and 68% yields,
respectively. Deprotection of the sugar hydroxyl groups
took place smoothly on treatment with methanolic ammo-
nia at room temperature and gave 6a,b in nearly quantita-
tive yield. The regioselective protection of the 5’-position
with 4,4’-dimethoxytrityl cloride (DMTr-Cl) afforded
7a,b which were activated at the 3’-position by 2-cyanoet-
hyl-N,N-diisopropylchlorophosphoramidite to give the
desired phosphoramidites 1a,b.
Preparation of the ODNs and EPR measurements
0.1 M solutions of the TEMPO-labeled phosphoramidites
1a,b and 2a,b in anhydrous MeCN were used for the prep-
aration of four mono-labeled ODNs different in length
and composition. The position of insertion of the label
from the 5’-terminal along the sequence was at +11 for the
40-mer 5’-d(ACT TAC AGC CAT GCA CTC CGC AAC
GGC GAA TAG CCA TCC C)-3’ (A), at +7 for the 20-
mer 5’-d(ACA CTC ATC ACA CAT AAC GT)-3’ (B), at
+10 for the 20-mer 5’-d(ACA CTC GGG CAA GCG
ACC GA)-3’ (C) and at +15 for the 30-mer 5’-d(ACA
TAC AGC CTA GCT CTC CGA CAC GGC GAA)-3’
(D). The analogous non-labeled ODNs A0, B0, C0 and D0
were also synthesised as controls. The ODNs were all
The synthesis of 2a,b (Scheme 2) started from the sulfo-
nylated derivatives 8a,b, obtained from 2’-deoxy-3’,5’-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine
3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)thymi-
dine,12 respectively, and TPS-Cl by the method used for
4a,b. This reaction occurred regioselectively and cleanly
at the O4-position to give the sulfonate esters 8a,b in near-
ly quantitative yield and no O4-N3 isomerization was ob-
served on standing.
and
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Preparation of Spin-Labeled 2-Amino-dA, dA, dC and 5-Methyl-dC Phosphoramidites
567
checked by PAGE staining with ethidium bromide (Fig-
ure 2).
Figure 2 Comparison by PAGE (20%) of couples of spin-labeled
(left spot) and control (right spot) 40-mers (A, A0), 20-mers (B, B0),
20-mers (C, C0) and 30-mers (D, D0) stained with ethidium bromide
Figure 3 shows the EPR spectra of all four A, B, C and D
in comparison with the spectra of the respective labeled
monomer phosphoramidites 1a,b and 2a,b. As expected,
the EPR spectra of the ODNs are considerably broadened.
Figure 3 Comparison of the EPR spectra of 2-amino-dA (A), dA
(B), dC (C) and 5-methyl-dC (D) TEMPO-labeled phosphoramidites
(left side) and of mono-labeled-ODNs (right side) 40-mer (A), 20-
Extremely marked and characteristic changes are present mer (B), 20-mer (C) and 30-mer (D)
in the high-field line, which is somewhat broadened rela-
tive to the other two and its lineheight is decreased. All
dard) spectra were recorded on a Varian XL-300 spectrometer. Be-
four labeled ODNs spectra display essentially the same
line shapes, which are characteristic of a mobile nitroxide
label.
fore recording the NMR spectra, samples containing a nitroxide
group were treated with 1.5 equiv of phenylhydrazine as reducing
agent.13 Optical rotations were determined with a Schmidt-Haensch
1604 polarimeter. Fast atom bombardment (FAB) mass spectra
were recorded on a VG 70-70 EQ-MF instrument equipped with a
standard FAB source (8 keV, Xe atoms, glycerol/1-thioglicerol so-
lution). The mass spectra of 1a,b and 2a,b were obtained in electro-
spray (ES) conditions by a ZABSpec oa-TOF (Micromass Ltd.
Manchester UK) instrument. Silica gel for column chromatography
and TLC silica gel plates were from Merck AG (Darmstadt, Germa-
ny). The EPR spectra were performed with a Varian E109 spec-
trometer, X band, modulation amplitude of 0.5 gauss, time constant
300 ms, field scan time 120 s, 20 scans, microwave power 1 mV.
The sample holder was a glass capillary tube and the measurements
were performed on 0.1 mM solutions in MeCN for the monomers
and in water for the ODNs, at 20 °C. Elemental analyses were per-
formed by Servizio Microanalisi of CNR, Area della Ricerca di Ro-
ma, and were within ± 0.4% of the theoretical values.
Conclusion
In conclusion, we have synthesised four spin-labeled
phosphoramidites useful for the preparation of mono- and
poly-labeled ODNs by automatic synthesis. These probes
can be inserted in a site- and type-specific manner along
the sequence in any double-strand nucleic acids. They of-
fer a greater possibility of using EPR spectroscopy, which
is a technique extremely effective not only for studies on
nucleic acid dynamics and structural modifications, but
also for studying the interactions of nucleic acids with
other nucleic acids, or with nucleic acids binding proteins,
or with small molecules, which can themselves carry a
spin probe.
Sulfonylated Nucleosides 8a,b; General Procedure
A solution of the protected nucleoside (1.0 mmol) in anhyd CH2Cl2
(5.0 mL) was treated overnight at r.t. with TPS-Cl (606 mg,
2.0 mmol) in the presence of Et3N (404 mg, 4.0 mmol) and DMAP
(12 mg, 0.1 mmol). The mixture was evaporated and the oily residue
purified by flash chromatography on silica gel (CH2Cl2/EtOAc
80:20). Products were recovered as white foams and used without
further purification.
Melting points (Büchi oil apparatus) are uncorrected. UV and IR
spectra were obtained with a Shimadzu UV-2101PC and a Perkin-
1
Elmer 521 recording spectrophotometers, respectively. H NMR
(TMS as internal standard) and 31P NMR (H3PO4 as external stan-
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4-O-[(2,4,6-Triisopropylphenyl)sulfonyl]-3’,5’-O-(1,1,3,3-tet-
raisopropyldisiloxane-1,3-diyl)-2’-deoxyuridine (8a)
2.80 (m, 3 H, Me2CHCO and CHpiperidine), 4.00 4.20 (m, 3 H, H-4’,
H-5’and H-5”), 4.17 (br s, 1 H, H-3’), 5.86 (br s, 1 H, H-1’), 7.51
and 7.62 (2 s, 2 H, H-2 and H-8).
From
3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-de-
oxyuridine12 (638 mg, 1.35 mmol); yield: 780 mg (78%); Rf 0.70
FAB-MS: Calcd mass for C27H40N6O6: m/z = 545, found 546 (M +
(CHCl3/MeOH, 98:2); [ ]D20 +47.8 (c = 0.5, MeCN).
H)+.
UV (MeCN): max = 288 (9800), 236 (10700) nm.
N4-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-3’,5’-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-2’-deoxycytidine (9a)
From 8a (684 mg, 0.9 mmol). Chromatographed with CH2Cl2/i-
PrOH (98:2); yield: 405 mg (69%); Rf 0.27 (CHCl3/MeOH, 98:2);
[ ]D20 +51.5 (c = 1, EtOH).
1
IR (CHCl3): = 2949, 1667, 1625, 1462, 1383, 1115 cm .
1H NMR (CDCl3): = 0.71 1.26 [m, 42 H, (CH3)2C], 1.53 1.76
(m, 2 H, H-2’ and H-2”), 2.00 2.37 (m, 4 H, Me2CHSi), 2.46 2.95
(m, 3 H, Me2CHPh), 3.41 4.13 (m, 4 H, H-3’, H-4’, H-5’ and H-
5”), 5.78 (d, 1 H, J = 8.0 Hz, H-5), 6.15 (d, 1 H, J = 6.0 Hz, H-1’),
7.05 (s, 2 H, Ar), 7.85 (d, 1 H, J = 8.0 Hz, H-6).
UV (EtOH): max = 273 (10900), 243 (9600) nm.
1
IR (CHCl3): = 3431, 2947, 1641, 1494, 1325, 1116 cm .
FAB-MS: Calcd mass for C36H60N2O8SSi2: m/z = 737, found 738
1H NMR (CDCl3): = 0.74 1.04 [m, 24 H, (CH3)2C], 1.11 (s, 12 H,
CH3piperidine), 1.28 1.60 (m, 4 H, CH2piperidine), 1.62 2.05 (m, 2 H, H-
2’ and H-2”), 2.09 2.52 (m, 5 H, Me2CHSi and CHpiperidine), 3.25
4.25 (m, 4 H, H-3’, H-4’, H-5’ and H-5”), 5.80 (d, 1 H, J = 8.0 Hz,
H-5), 6.18 (br s, 1 H, H-1’), 7.90 (d, 1 H, J = 8.0 Hz, H-6).
(M + H)+.
4-O-[(2,4,6-Triisopropylphenyl)sulfonyl]-3’,5’-O-(1,1,3,3-tet-
raisopropyldisiloxane-1,3-diyl)thymidine (8b)
From
3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)thy-
midine12 (670 mg, 1.38 mmol); yield: 717 mg (69%); Rf 0.83
FAB-MS: Calcd mass for C30H54N4O6Si2: m/z = 623, found 624 (M
(CHCl3/MeOH, 98:2); [ ]D20 +46.6 (c = 0.5, MeCN).
+ H)+.
UV (MeCN): max = 289 (6700), 233 (11000) nm.
5-Methyl-N4-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-3’,5’-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-deoxycytidine
(9b)
From 8b (753 mg, 1.0 mmol). Chromatographed with CH2Cl2/
MeOH (98:2); yield: 421 mg (65%); Rf 0.50 (CHCl3/MeOH, 95:5);
[ ]D20 +37.6 (c = 1, EtOH)
1
IR (CHCl3): = 3417, 2948, 1668, 1533, 1462, 1385, 1115 cm .
1H NMR (CDCl3): = 0.78 1.43 [m, 42 H, (CH3)2C], 1.62 1.98
(m, 2 H, H-2’ and H-2”), 1.82 (s, 3 H, C5-CH3), 2.10 2.56 (m, 4 H,
Me2CHSi), 2.60 3.02 (m, 3 H, Me2CHPh), 3.53 3.82 (m, 2 H, H-
5’ and H-5”), 3.90 4.06 (m, 1 H, H-4’), 4.10 4.37 (m, 1 H, H-3’),
5.67 (d, 1 H, J = 6.0 Hz, H-1’), 7.01 (s, 2 H, Ar), 7.69 (s, 1 H, H-6).
UV (EtOH): max = 275 (10100), 247 (9700) nm.
1
FAB-MS: Calcd mass for C37H62N2O8SSi2: m/z = 751, found 752
IR (CHCl3): = 3441, 2946, 1657, 1497, 1333, 1117 cm .
(M + H)+.
1H NMR (CDCl3): = 0.79 1.10 [m, 24 H, (CH3)2C], 1.16 (s, 12 H,
CH3piperidine), 1.20 1.46 (m, 4 H, CH2piperidine), 1.60 1.92 (m, 2 H, H-
2’ and H-2”), 1.72 (s, 3 H, C5-CH3), 2.07 2.34 (m, 5 H, Me2CHSi
and CHpiperidine), 3.40 3.62 (m, 2 H, H-5’ and H-5”), 3.70 3.86 (m,
1 H, H-4’), 4.15 4.36 (m, 1 H, H-3’), 5.64 (br s, 1 H, H-1’), 7.60 (s,
1 H, H-6).
Spin-Labeled Nucleosides 5a,b and 9a,b; General Procedure
A solution of the appropriate sulfonylated nucleoside 4 and 8
(1 mmol) and tempamine (342 mg, 2.0 mmol) in CH2Cl2 (5 mL) was
refluxed overnight. The mixture was diluted with CH2Cl2 (30 mL)
and washed with sat. aq NaHCO3 solution (2 î 20 mL) and brine
(20 mL). The organic layer was dried (Na2SO4), evaporated at re-
duced pressure and the residue purified by silica gel flash chroma-
tography. All products were obtained as pale pink foams.
FAB-MS: Calcd mass for C31H56N4O6Si2: m/z = 637, found 638 (M
+ H)+.
Deprotection of 3’,5’-OH; General Procedures
3’,5’-Di-O-isobutyryl-2-isobutyramido-N6-(1-oxyl-2,2,6,6-tet-
ramethyl-4-piperidinyl)-2’-deoxyadenosine (5a)
Procedure A (for 6a,b): The protected nucleoside 5a,b (1.0 mmol)
was treated with a mixture of MeOH (15 mL) and aq 25% NH4OH
(5 mL) for 4 h at r.t. After evaporation to dryness, the residue was
purified by silica gel flash chromatography.
From 4a11 (800 mg, 1.1 mmol). Chromatographed with EtOAc/hex-
ane (70:30); yield: 366 mg (54%); Rf 0.22 (EtOAc/hexane, 1:1);
20
[ ]Hg 5.2 (c = 2, EtOH).
Procedure B (for 10a,b): To a solution of the protected nucleoside
9a,b (1.0 mmol) in THF (2 mL) was added TBAF (523 mg,
2.0 mmol) and the mixture was kept overnight at r.t. After removal
of the solvent at reduced pressure, the residue was dissolved in H2O
(20 mL) and washed with Et2O (2 î 15 mL). The aqueous phase
was evaporated to dryness and the product purified by silica gel
chromatography.
UV (MeOH): max = 272 (22900), 232 (34900) nm.
1
IR (CHCl3): = 3421, 2986, 1735, 1620, 1461, 1387, 1154 cm .
1H NMR (CDCl3): = 0.90 1.12 [m, 18 H, (CH3)2C], 1.20 (s, 12 H,
CH3piperidine), 1.35 1.66 (m, 4 H, CH2piperidine), 1.70 1.94 (m, 2 H, H-
2’ and H-2”), 2.11 2.48 (m, 3 H, Me2CHCO), 2.51 2.81 (m, 1 H,
CHpiperidine), 4.05 4.31 (m, 3 H, H-4’, H-5’ and H-5”), 4.92 5.11
(m, 1 H, H-3’), 5.91 (t, 1 H, J = 6.0 Hz, H-1’), 7.30 (s, 1 H, H-8).
2-Isobutyramido-N6-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-
2’-deoxyadenosine (6a)
FAB-MS: Calcd mass for C31H47N7O7: m/z = 630, found 631 (M +
From 5a (320 mg, 0.5 mmol). Chromatographed with EtOAc/i-
PrOH (98:2); pale pink foam; yield: 220 mg (90%); Rf 0.35
H)+.
3’,5’-Di-O-isobutyryl-N6-(1-oxyl-2,2,6,6-tetramethyl-4-piperid-
inyl)-2’-deoxyadenosine (5b)
20
(EtOAc); [ ]D 3.5 (c = 1.5, EtOH).
UV (EtOH): max = 277 (17900), 232 (25500) nm.
IR (CHCl3): = 3419, 2990, 1711, 1621, 1460, 1384, 1104 cm-1.
From 4b11 (784 mg, 1.2 mmol). Chromatographed with CHCl3/
EtOAc (80:20); yield: 431 mg (68%); Rf 0.20 (EtOAc/hexane,
20
70:30); [ ]D 17.3 (c = 1, EtOH).
1H NMR (DMSO-d6): = 0.90 and 0.94 [2 s, 6 H, (CH3)2C], 1.23 (s,
12 H, CH3piperidine), 1.18 1.64 (m, 4 H, CH2piperidine), 1.88 2.18 (m,
2 H, H-2’ and H-2”), 2.22 2.40 (m, 1 H, Me2CHCO), 2.65 2.88
(m, 1 H, CHpiperidine), 3.06 3.20 (m, 3 H, H-4’, H-5’ and H-5”),
3.99 4.28 (m, 1 H, H-3’), 5.82 (t, 1 H, J = 6.0 Hz, H-1’), 7.70 (s, 1
H, H-8).
UV (EtOH): max = 267 (21000), 213 (22100) nm.
IR (CHCl3): = 3418, 2990, 1735, 1618, 1470, 1366, 1152 cm-1.
1H NMR (CDCl3): = 1.00 1.38 [m, 24 H, (CH3)2C and
CH3piperidine], 1.45 2.20 (m, 6 H, CH2piperidine, H-2’ and H-2”), 2.22
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Preparation of Spin-Labeled 2-Amino-dA, dA, dC and 5-Methyl-dC Phosphoramidites
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FAB-MS: Calcd mass for C23H35N7O5: m/z = 490, found 491 (M +
IR (CHCl3): = 3394, 2936, 1726, 1619, 1507, 1462, 1378, 1177
cm .
1
H)+.
N6-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-2’-deoxyadenos-
ine (6b)
1H NMR (CDCl3): = 1.08 and 1.12 [2 s, 6 H, (CH3)2C], 1.26 (s, 12
H, CH3piperidine), 1.20 1.60 (m, 4 H, CH2piperidine), 1.70 2.06 (m, 2 H,
H-2’ and H-2”), 2.18 2.60 (m, 2 H, Me2CHCO and CHpiperidine),
3.10 3.28 (m, 3 H, H-4’, H-5’ and H-5”), 3.55 (s, 6 H, CH3O),
3.90 4.15 (m, 1 H, H-3’), 6.04 (br s, 1 H, H-1’), 6.31 6.60 and
6.75 7.18 (2 m, 13 H, Ar), 7.43 (s, 1 H, H-8).
From 5b (370 mg, 0.7 mmol). Chromatographed with EtOAc/i-
PrOH (98:2); pale pink foam; yield: 255 mg (90%); Rf 0.20
20
(EtOAc); [ ]D 16.4 (c = 1, EtOH).
UV (EtOH): max = 266 (15200), 213 (14200) nm.
FAB-MS: Calcd mass for C44H53N7O7: m/z = 792, found 793 (M +
1
IR (CHCl3): = 3304, 2993, 1728, 1618, 1479, 1372, 1136 cm .
H)+.
1H NMR (DMSO-d6): = 1.08 (s, 12 H, CH3), 1.49 1.90 (m, 6 H,
CH2piperidine, H-2’ and H-2”), 2.00 2.40 (m, 1 H, CHpiperidine), 3.31
3.47 (m, 2 H, H-5’ and H-5”), 3.70 (br s, 1 H, H-4’), 4.19 4.28 (m,
1 H, H-3’), 6.01 (t, 1 H, J = 6.0 Hz, H-1’), 7.75 and 7.88 (2 s, 2 H,
H-2 and H-8).
FAB-MS: Calcd mass for C19H28N6O4: m/z = 405, found 405 (M)+.
N4-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-2’-deoxycytidine
(10a)
5’-O-(4,4’-Dimethoxytrityl)-N6-(1-oxyl-2,2,6,6-tetramethyl-4-
piperidinyl)-2'-deoxyadenosine (7b)
From 6b (162 mg, 0.4 mmol). Chromatographed with EtOAc/hex-
ane/TEA (70:29:1); pale pink foam; yield: 218 mg (77%); Rf 0.36
20
(EtOAc/MeOH, 95:5); [ ]D 5.1 (c = 1, EtOH).
UV (EtOH): max = 268 (19600), 235 (22400) nm.
IR (CHCl3): = 3316, 2991, 1729, 1618, 1507, 1474, 1373, 1177
1
cm .
From 9a (371 mg, 0.6 mmol). Chromatographed with EtOAc/i-
PrOH (80:20); light pink oil; yield: 217 mg (95%); Rf 0.27 (EtOAc/
i-PrOH, 70:30); [ ]D20 +52.0 (c = 1, EtOH).
1H NMR (CDCl3): = 1.24 (s, 12 H, CH3), 1.60 2.00 (m, 6 H,
CH2piperidine, H-2’ and H-2”), 2.10 2.45 (m, 1 H, CHpiperidine), 3.18
3.39 (m, 2 H, H-5’ and H-5”), 3.50 (s, 6 H, CH3O), 3.68 (br s, 1 H,
H-4’), 4.15 4.24 (m, 1 H, H-3’), 6.04 (t, 1 H, J = 6.0 Hz, H-1’),
6.24 6.55 and 6.64 7.20 (2 m, 13 H, Ar), 7.47 and 7.85 (2 s, 2 H,
H-2 and H-8).
FAB-MS: Calcd mass for C40H46N6O6: m/z = 706, found 706 (M)+.
5’-O-(4,4’-Dimethoxytrityl)-N4-(1-oxyl-2,2,6,6-tetramethyl-4-
piperidinyl)-2'-deoxycytidine (11a)
UV (EtOH): max = 273 (11100), 242 (9500) nm.
1
IR (CHCl3): = 3409, 2935, 1641, 1499, 1333, 1096 cm .
1H NMR (DMSO-d6): = 1.08 (s, 12 H, CH3), 1.58 2.28 (m, 6 H,
CH2piperidine, H-2’ and H-2”), 2.30 2.48 (m, 1 H, CHpiperidine), 3.30
3.46 (m, 2 H, H-5’ and H-5”), 3.50 3.71 (m, 1 H, H-4’), 3.95 4.11
(m, 1 H, H-3’), 5.74 (d, 1 H, J = 8.0 Hz, H-5), 6.15 (t, 1 H, J = 6.0
Hz, H-1’), 7.84 (d, 1 H, J = 8.0 Hz, H-6).
From 10a (225 mg, 0.6 mmol). Chromatographed with EtOAc/i-
PrOH/TEA (95:4:1); pale pink oil; yield: 290 mg (72%); Rf 0.27
(EtOAc/MeOH, 80:20); [ ]D20 +48.8 (c = 1, EtOH).
FAB-MS: Calcd mass for C18H28N4O5: m/z = 380, found 381 (M +
H)+.
5-Methyl-N4-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-2’-
deoxycytidine (10b)
From 9b (410 mg, 0.6 mmol). Chromatographed with EtOAc/i-
PrOH (80:20); amorphous pink solid; yield: 225 mg (95%);
mp 238 240 °C; Rf 0.17 (EtOAc/i-PrOH, 80:20); [ ]D20 +15.3 (c =
0.5, EtOH)
UV (EtOH): max = 277 (13700), 235 (25300) nm.
1
IR (CHCl3): = 3429, 2991, 1642, 1507, 1252, 1118 cm .
1H NMR (CDCl3): = 1.10 (s, 12 H, CH3), 1.57 2.15 (m, 6 H,
CH2piperidine, H-2’ and H-2”), 2.33 2.47 (m, 1 H, CHpiperidine), 3.15
3.33 (m, 2 H, H-5’ and H-5”), 3.55 (s, 6 H, CH3O), 3.67 3.93 (m,
1 H, H-4’), 4.03 4.36 (m, 1 H, H-3’), 5.10 (d, 1 H, J = 8.0 Hz, H-
5), 5.95 (t, 1 H, J = 6.0 Hz, H-1’), 6.30 6.58 and 6.67 7.10 (2 m,
13 H, Ar), 7.95 (br s, 1 H, H-6).
UV (EtOH): max = 280 (6600), 246 (6300) nm.
1
IR (CHCl3): = 3413, 2931, 1663, 1620, 1555, 1353, 1108 cm .
1H NMR (DMSO-d6): = 1.05 (s, 12 H, CH3), 1.15 1.80 (m, 6 H,
CH2piperidine, H-2’ and H-2”), 1.85 (s, 3 H, C5-CH3), 1.98 2.25 (m, 1
H, CHpiperidine), 3.00 3.28 (m, 3 H, H-4’, H-5’ and H-5”), 4.11 4.29
(m, 1 H, H-3’), 6.12 (br s, 1 H, H-1’), 7.63 (s, 1 H, H-6).
FAB-MS: Calcd mass for C39H46N4O7: m/z = 683, found 684 (M +
H)+.
5’-O-(4,4’-Dimethoxytrityl)-5-methyl-N4-(1-oxyl-2,2,6,6-tet-
ramethyl-4-piperidinyl)-2'-deoxycytidine (11b)
From 10b (158 mg, 0.4 mmol). Chromatographed with EtOAc/i-
PrOH/TEA (95:4:1); amorphous pink solid; yield: 209 mg (75%);
mp 172 175 °C; Rf 0.58 (EtOAc/i-PrOH, 70:30); [ ]D20 +16.5 (c =
0.5, EtOH).
FAB-MS: Calcd mass for C19H30N4O5: m/z = 395, found 395 (M)+.
5’-O-Dimethoxytritylation; General Procedure
The required derivative (1.0 mmol), previously coevaporated with
anhyd pyridine and dried under high vacuum overnight, was dis-
solved in anhyd pyridine (12 mL) and then DMTr-Cl (678 mg,
2.0 mmol) was added at r.t. After 3 h, the reaction was quenched
with MeOH (0.1 mL) and the mixture concentrated to small vol-
ume. The residue was diluted with CH2Cl2 (25 mL) and washed with
aq sat. NaHCO3 solution (2 î 20 mL) and brine (20 mL). The or-
ganic extract was dried (Na2SO4), filtered, evaporated and finally
coevaporated with toluene to remove the excess of pyridine. The
products were isolated by silica gel chromatography.
5’-O-(4,4’-Dimethoxytrityl)-2-isobutyramido-N6-(1-oxyl-
2,2,6,6-tetramethyl-4-piperidinyl)-2'-deoxyadenosine (7a)
From 6a (200 mg, 0.47 mmol). Chromatographed with EtOAc/hex-
ane/TEA (70:29:1); pale pink foam; yield: 223 mg (69%); Rf 0.53
(EtOAc); [ ]D20 +11.6 (c = 2, EtOH).
UV (EtOH): max = 276 (14300), 234 (31300) nm.
IR (CHCl3): = 3443, 2934, 1660, 1625, 1550, 1338, 1250, 1116
1
cm .
1H NMR (CDCl3): = 1.09 (s, 12 H, CH3), 1.33 (s, 3 H, C5-CH3),
1.55 2.29 (m, 6 H, CH2piperidine, H-2’ and H-2”), 2.33 2.55 (m, 1 H,
CHpiperidine), 3.09 3.29 (m, 2 H, H-5’ and H-5”), 3.58 (s, 6 H,
CH3O), 3.88 4.05 (m, 1 H, H-4'), 4.24 4.54 (m, 1 H, H-3'), 6.30 (br
s, 1 H, H-1’), 6.61 6.89 and 7.25 7.44 (2 m, 13 H, Ar), 7.59 (s, 1
H, H-6).
FAB-MA: Calcd mass for C40H48N4O7: m/z = 697, found 698 (M +
H)+.
Phosphoramidites 1a,b and 2a,b; General Procedure
UV (EtOH): max = 276 (19300), 232 (41100) nm.
Compounds 7a,b and 11a,b were dried under high vacuum over-
night after coevaporation with anhyd pyridine. To a stirred solution
Synthesis 2001, No. 4, 565–572 ISSN 0039-7881 © Thieme Stuttgart · New York

570
C. Giordano et al.
PAPER
of the appropriate nucleoside (1.0 mmol) and i-Pr2NEt (0.7 mL,
4.0 mmol) in anhyd CH2Cl2 (12 mL) was added 2-cyanoethyl-diiso-
propylchlorophosphoramidite (473 mg, 2.0 mmol) by a syringe
over 2 min under N2. The mixture was left for 30 min at r.t., filtered
to remove the precipitated amine hydrochloride and diluted with
CH2Cl2 (30 mL). The solution was washed with ice-cooled brine
(2 î 15 mL) and the organic phase was dried (Na2SO4), filtered,
concentrated to small volume at low pressure and added dropwise
to stirred hexane cooled to 78 °C. Products 1a and 2a separated as
an oil and were lyophilised from benzene; 1b and 2b separated as a
solid and were recovered by filtration and dried. All compounds
were as mixture of diastereoisomers.
IR (CHCl3): = 3431, 2963, 1642, 1502, 1462, 1326, 1252, 1178
cm .
1
1H NMR (CDCl3): = 1.09 1.18 [m, 12 H, (CH3)2C], 1.25 (s, 12 H,
CH3piperidine), 1.74 2.20 (m, 6 H, CH2piperidine, H-2’ and H-2”), 2.42 (t,
2 H, J = 6.0 Hz, CH2CN), 2.33 2.47 (m, 1 H, CHpiperidine), 3.28
3.43 (m, 2 H, Me2CHN), 3.45 3.65 (m, 3 H, H-4’, H-5’ and H-5”),
3.78 (s, 6 H, CH3O), 4.12 (br s, 2 H, CH2OP), 4.62 (br s, 1 H, H-3’),
5.35 (br s, 1 H, H-5), 6.25 6.40 (m, 1 H, H-1’), 6.76 6.90 and
7.16 7.46 (2 m, 13 H, Ar), 7.80 (d, 1 H, J = 8.0 Hz, H-6).
13C NMR (CDCl3): = 40.87 (C-2’), 55.24 (C-5’), 74.52 (C-3’),
86.03 (C-1’), 86.70 (C-4’), 95.73 (C-5), 144.66 (C-6), 150.49 (C-2),
158.64 (C-4).
5’-O-(4,4’-Dimethoxytrityl)-2-isobutyramido-N6-(1-oxyl-
2,2,6,6-tetramethyl-4-piperidinyl)-2’-deoxyadenosine-3’(2-cya-
noethyl-N,N-diisopropyl)phosphoramidite (1a)
31P NMR (CDCl3): = 150.34, 150.29.
HRMS: m/z Calcd for C48H63N6O8P 882.444, found 882.435 (M+),
From 7a (120 mg, 0.15 mmol); yield: 122 mg (81%); Rf 0.54, 0.65
918.432 (+ 2H2O).
20
(EtOAc/hexane, 70:30); [ ]D 7.5 (c = 1, MeCN)
5’-O-(4,4’-Dimethoxytrityl)-5-methyl-N4-(1-oxyl-2,2,6,6-tet-
ramethyl-4-piperidinyl)-2'-deoxycytidine-3'-(2-cyanoethyl-
N,N-diisopropyl) phosphoramidite (2b)
From 11b (241 mg, 0.34 mmol); yield: 282 mg (91%); mp 126
130 °C; Rf 0.76, 0.83 (EtOAc/i-PrOH, 95:5); [ ]D20 +17.2 (c = 1,
MeCN).
UV (MeCN): max = 276 (18300), 233 (39900) nm.
IR (CHCl3): = 3395, 2963, 1710, 1619, 1508, 1462, 1383, 1178
1
cm .
1H NMR (CDCl3): = 1.05 1.28 [m, 18 H, (CH3)2C], 1.42 (s, 12 H,
CH3piperidine), 1.35 1.54 (m, 4 H, CH2piperidine), 1.85 2.20 (m, 2 H, H-
2’ and H-2”), 2.38 2.50 (m, 3 H, Me2CHCO and CHpiperidine), 2.61
(br s, 2 H, CH2CN), 3.23 3.50 (m, 2 H, Me2CHN), 3.52 3.90 (m,
2 H, H-5’ and H-5”), 3.78 (s, 6 H, CH3O), 4.11 4.38 (m, 3 H, H-4’
and CH2OP), 4.68 4.80 (m, 1 H, H-3’), 6.37 (t, 1 H, J = 6.0 Hz, H-
1’), 6.72 7.00 and 7.15 7.38 (2 m, 13 H, Ar), 7.85 (2 s, 1 H, H-8).
13C NMR (CDCl3): = 39.76 (C-2’), 63.57 (C-5’), 76.40 (C-3’),
84.20 (C-1’), 86.43 (C-4’), 126.90 (C-5), 137.52 (C-8), 144.55 (C-
4), 158.54 (C-6), 160.95 (C-2).
UV (MeCN): max = 277 (10100), 235 (24200) nm.
IR (CHCl3): = 3443, 2992, 1663, 1501, 1464, 1337, 1245, 1177
1
cm .
1H NMR (CDCl3): = 1.13 1.29 [m, 24 H, (CH3)2C and
CH3piperidine], 1.28 (s, 3 H, C5-CH3), 1.41 1.56 (br s, 4 H,
CH2piperidine), 2.01 (br s, 2 H, H-2’ and H-2”), 2.38 (t, 2 H, J = 6.0 Hz,
CH2CN), 3.20 3.38 (m, 3 H, Me2CHN and CHpiperidine), 3.40 3.65
(m, 3 H, H-4’, H-5’ and H-5”), 3.76 (s, 6 H, CH3O), 4.13 (br s, 2 H,
CH2OP), 4.60 (br s, 1 H, H-3’), 6.35 6.44 (m, 1 H, H-1’), 6.79
6.84 and 7.23 7.35 (2 m, 13 H, Ar), 7.68 (s, 1 H, H-6).
13C NMR (CDCl3): = 40.72 (C-2’), 55.27 (C-5’), 74.28 (C-3’),
85.87 (C-1’), 86.18 (C-4’), 110.56 (C-5), 144.47 (C-6), 151.27 (C-
2), 158.72 (C-4).
31P NMR (CDCl3): = 151.30, 149.48.
HRMS: m/z Calcd for C53H70N9O8P 991.508, found 991.532 (M+),
1027.532 (+ 2H2O).
5’-O-(4,4’-Dimethoxytrityl)-N6-(1-oxyl-2,2,6,6-tetramethyl-4-
piperidinyl)-2'-deoxyadenosine-3'-(2-cyanoethyl-N,N-diisopro-
pyl) phosphoramidite (1b)
31P NMR (CDCl3): = 150.50, 150.07.
HRMS: m/z Calcd for C49H65N6O8P 896.460, found 896.459 (M+),
From 7b (180 mg, 0.25 mmol); yield: 193 mg (84%); mp 82 84 °C;
20
Rf 0.63, 0.70 (EtOAc); [ ]D 37.3 (c = 1.5, MeCN).
932.498 (+ 2H2O)
UV (MeCN): max = 267 (25600), 237 (26300) nm.
Synthesis and Nucleoside Composition Analysis of ODNs
1
IR (CHCl3): = 3319, 2958, 1615, 1508, 1470, 1375, 1178 cm .
The automatic synthesis of the ODNs was performed at 1 µmol
scale on an ABI (Applied Biosystems, Inc., Forster City, CA) 392/
8 synthesiser, leaving the terminal 5’-O-DMTr-group on. The la-
beled phosphoramidites were used for the synthesis of the ODNs
without having to change any parameter in the standard DNA syn-
thesis protocol of the machine, in particular the coupling time was
kept at 25 s and average coupling yields in the range 96 98% were
obtained. Having used Ac-protected dC for the synthesis, the ODNs
were cleaved from the solid support by a mixture of ammonia and
methylamine (AMA, Beckmann) in 10 min at r.t., then the solution
was heated for 10 min at 55 °C in order to deprotect the exocyclic
amino groups. After concentration, the samples were loaded onto an
HPLC "Pure DNA" Dynamax column module (C4), 300 Å, 5µ,
21.4 î 50 mm eluted at a flow-rate of 6 mL/min. First the failure
sequences were removed with a linear gradient of MeCN 3 18% in
triethylammonium acetate buffer 0.1 M, pH 7.0, in 15 min. After-
wards the terminal DMTr was removed on column from the full-
length ODN by 0.5% TFA. Then the DMTr-free full-length ODN
was eluted by a second linear gradient 3 40% MeCN in the same
buffer as above, in 20 min. The pooled fractions were lyophilised
and checked by PAGE on 20% polyacrylamide, 7 M urea gels (1.5
mm) containing 50 mM Tris-borate buffer pH 8.0, 0.1 mM EDTA.
The four labeled (A, B, C, D) and the corresponding unlabeled (A0,
B0, C0, D0) ODNs, each 0.5 O.D., were digested in 100 L of 10 mM
1H NMR (CDCl3): = 1.04 1.29 [m, 12 H, (CH3)2C], 1.35 (s, 12 H,
CH3piperidine), 1.53 1.89 (m, 4 H, CH2piperidine), 1.95 2.14 (m, 2 H, H-
2’ and H-2”), 2.38 2.50 (m, 3 H, Me2CHCO and CHpiperidine), 2.60
(t, 2 H, J = 6.0 Hz, CH2CN), 3.25 3.49 (m, 2 H, Me2CHN), 3.52
3.90 (m, 2 H, H-5’ and H-5”), 3.75 (s, 6 H, CH3O), 3.93 4.20 (m,
1 H, H-4’), 4.26 (br s, 2 H, CH2OP), 4.73 (br s, 1 H, H-3’), 6.45 (br
s, 1 H, H-1’), 6.70 6.95 and 7.10 7.40 (2 m, 13 H, Ar), 7.93 and
7.95 (2 s, 2 H, H-2 and H-8).
13C NMR (CDCl3): = 39.26 (C-2’), 63.48 (C-5’), 76.35 (C-3’),
84.01 (C-1’), 85.32 (C-4’), 126.52 (C-5), 138.20 (C-8), 144.56 (C-
4), 151.25 (C-2), 158.51 (C-6).
31P NMR (CDCl3): = 149.97, 148.70.
HRMS: m/z Calcd for C49H63N8O7P 906.455, found 906.434 (M+),
942.372 (+ 2H2O).
5’-O-(4,4’-Dimethoxytrityl)-N4-(1-oxyl-2,2,6,6-tetramethyl-4-
piperidinyl)-2'-deoxycytidine-3'-(2-cyanoethyl-N,N-diisopro-
pyl) phosphoramidite (2a)
From 11a (277 mg, 0.4 mmol); yield: 297 mg (83%); Rf 0.63, 0.75
(EtOAc/i-PrOH, 95:5); [ ]D20 +38.4 (c = 2, MeCN).
UV (EtOH): max = 275 (9400), 235 (19400) nm.
Synthesis 2001, No. 4, 565–572 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Preparation of Spin-Labeled 2-Amino-dA, dA, dC and 5-Methyl-dC Phosphoramidites
571
Figure 4 HPLC of enzymatic digestion of the labeled ODNs A (A10C16G7T6+6a), B (A7C7GT4+6b), C (A6C6G6T+10a) and D
(A9C11G6T3+10b). The peaks at 5.1 5.2 min correspond to dC, those at ca. 9.1 to dG, those at ca. 9.8 to T, those at 10.6 10.7 to dA. The peaks
corresponding to the labeled nucleosides are marked with an asterisk
potassium phosphate buffer, pH 7.0, with a mixture of phosphodi-
esterase from Crotalus adamanteus, 30 mU (Sigma) and alkaline
phosphatase from bovine intestine, 10 U (Sigma), for 6 h at 37 °C,
then filtered through a Millex-HV filter unit, 25 mm, 0.45 m, and
analysed by HPLC on a Supelco-C18 column, 250 î 4.6 mm,
5 m, eluting at 1 mL/min, with a linear gradient of MeCN, 3-40%
in 15 min, in triethylammonium acetate buffer, 0.1 M, pH 7.0, mon-
itoring at 255 nm. When the digestion mixtures of the labeled ODNs
were analysed, a second linear-gradient step was added, bringing
MeCN to 60% in 10 min. One tetramer of composition 5'-d(ACGT)-
3' was also synthesised and analysed in the same way, as reference
standard mixture. The relative heights of the peaks in the chromato-
grams, corresponding to the four deoxynucleosides dC, dG, T, dA,
which elute in the order, were used for the evaluation of the compo-
sition of the eight ODNs above. The peaks of the labeled nucleo-
sides 6a, 6b, 10a, 10b were matched with the corresponding
standards. The digestion mixtures of the labeled ODNs proved to
contain also the labeled nucleosides (Figure 4). Nucleoside 6b re-
sulted only half than expected, although the amount of enzymes and
the digestion time were double as usual.14 The accordance of the
theoretical composition of each ODN with the composition evaluat-
ed in this way resulted as follows for dC, dG, T and dA, and for the
labeled nucleosides, in the order of elution, arbitrarily assigning a
value of 1.00 to dC, the one eluting first: (A0) 1.00, 1.02, 0.92, 0.96;
(A) 1.00, 1.06, 0.83, 0.72, 0.73 (6a); (B0) 1.00, 0.95, 0.90, 0.90; (B)
1.00, 0.76, 0.95, 0.80, 0.49 (6b); (C0) 1.00, 1.00, 1.11, 0.89; (C)
1.00, 1.00, 1.00, 1.00, 0.87 (10a); (D0) 1.00, 1.00, 1.00, 0.91; (D)
1.00, 0.90, 0.90, 0.71, 1.10 (10b).
References
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Article Identifier:
1437-210X,E;2001,0,04,0565,0572,ftx,en;T01200SS.pdf
Synthesis 2001, No. 4, 565–572 ISSN 0039-7881 © Thieme Stuttgart · New York