Solid-supported synthesis and click conjugation of 4′- C -alkyne functionalized oligodeoxyribonucleotides

Article abstract of DOI:10.1021/bc100268w

4′-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine 3′-(2- cyanoethyl-N,N-diisopropyl)phosphoramidites (1, 2) were synthesized, and one or two such monomers were incorporated into a 15-mer oligodeoxyribonucleotide. After chain assembly, azido-functionalized ligands, including appropriate derivatives of 1,4-phenylenedimethaneamine, mannose, paromamine, and neomycin, were conjugated to the alkynyl groups by the click chemistry on a solid support. The influence of the 4′-modifications on the melting temperature with DNA and 2′-O-methyl RNA targets was studied. Oligonucleotides containing one to four mannose ligands in the central part of the chain (up to two 4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine units) form equally stable duplexes with complementary 2′-OMe RNA as the corresponding unmodified DNA sequence. At high salt content, the mannose conjugation is even stabilizing. On using a DNA target, a modest destabilization occurs. All the amino group bearing conjugates stabilized the duplexes, the DNA?DNA duplexes more than the DNA?2′-O-methyl RNA duplexes.

Full text of DOI:10.1021/bc100268w

1890
Bioconjugate Chem. 2010, 21, 1890–1901
Solid-Supported Synthesis and Click Conjugation of 4′-C-Alkyne
Functionalized Oligodeoxyribonucleotides
Anu Kiviniemi,* Pasi Virta, and Harri Lo¨nnberg
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received June 11, 2010; Revised Manuscript Received
August 23, 2010
4′-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine
3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites (1, 2) were synthesized, and one or two such monomers were
incorporated into a 15-mer oligodeoxyribonucleotide. After chain assembly, azido-functionalized ligands, including
appropriate derivatives of 1,4-phenylenedimethaneamine, mannose, paromamine, and neomycin, were conjugated
to the alkynyl groups by the click chemistry on a solid support. The influence of the 4′-modifications on the
melting temperature with DNA and 2′-O-methyl RNA targets was studied. Oligonucleotides containing one to
four mannose ligands in the central part of the chain (up to two 4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine
units) form equally stable duplexes with complementary 2′-OMe RNA as the corresponding unmodified DNA
sequence. At high salt content, the mannose conjugation is even stabilizing. On using a DNA target, a modest
destabilization occurs. All the amino group bearing conjugates stabilized the duplexes, the DNA ·DNA duplexes
more than the DNA ·2′-O-methyl RNA duplexes.
INTRODUCTION
study, the corresponding alkyne-derivatized nucleosides, viz.,
4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-C-
[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine monomers
(1, 2), have been synthesized and incorporated into oligonucle-
otides by phosphoramidite chemistry. The 4-pentynyl group was
chosen as the alkyne spacer to provide the 4′-C-modified
nucleoside with sufficient flexibility to ensure efficient click
reaction with azides (48) and proper alignment of the conjugate
groups upon duplex formation. A related strategy has previously
been applied to solid-supported derivatization of oligonucle-
otides containing ω-alkynylated nucleobases (48, 49). Monomers
1 and 2 allowed the attachment of two or one conjugative group
per modified residue, respectively (Figure 1). The ligands used
for the conjugation included azido derivatives of mannose (3),
p-aminomethylbenzene (4), and aminoglycosides paromamine
(5) and neomycin (6). The effects of these conjugate groups on
the melting temperature of the duplexes with cDNA and 2′-O-
methyl RNA sequences were determined to assess the contribu-
tions of electrostatic interactions and sterical factors to the
efficiency of hybrization.
Conjugation to peptides, carbohydrates, or small molecule
compounds has been extensively studied as a possible way to
improve the delivery and targeting of siRNA and antisense
oligonucleotides (1-15), and the synthesis of such conjugates
has been recently reviewed (16-21). Both carbohydrate-carbohy-
drate (22, 23) and carbohydrate-protein interactions (24-26)
are known to play a central role in cellular recognition. These
interactions are multipodal; simultaneous interaction with several
monosaccharide ligands is required for high-affinity binding
(27-30). Accordingly, the most attention has been paid to
preparation of oligonucleotide conjugates bearing a multiarmed
glycocluster at either terminus of the chain (31-36). As far as
glycotargeting of siRNA is concerned, another approach appears,
however, possible, viz., decoration of the sense strand with
sugars. A prerequisite for this approach is that the sugar ligands
do not markedly destabilize the duplex. In other words, the sense
strand is aimed at serving as a scaffold to which the lectin-
binding sugar ligands are tethered at appropriate distances
throughout the molecule. Since the carrier is simultaneously able
to hybridize with the antisense strand, the latter becomes
enriched at the cell surface and, hence, internalizated by
receptor-mediated endocytosis.
Several examples of oligonucleotide glycoconjugates bearing
the sugars at intrachain positions have been published. These
cannot, however, be exploited as sense strands in siRNA, since
the sugar ligands are attached either to base moieties (37-41)
or to non-nucleosidic units incorporated in the sugar-phosphate
backbone (42-44). Both of these conjugations markedly
destabilize the duplex. In fact, the only example of conjugation
that does not markedly affect the duplex stability is offered by
4′-C-tethered glycoconjugates obtained by subjecting a 4′-C-
azidomethyl incorporating oligonucleotide to Cu(I) catalyzed
1,3-dipolar cycloaddition, the so-called click reaction (45, 46),
with an alkyne-derivatized sugar (47). The latter approach,
however, suffers from the limitation that the 4′-C-azidomethyl
unit has to be introduced into the chain by H-phosphonate
coupling, since the intramolecular Staudinger reaction prevents
preparation of the phosphoramidite building block. In the present
EXPERIMENTAL PROCEDURES
General Remarks. The NMR spectra were recorded at 500
MHz. The mass spectra were recorded using EI or ESI
ionization. The RP HPLC analyses of the oligonucleotide
conjugates were performed using a Thermo ODS Hypersil C18
(250 × 4.6 mm, 5 µm) analytical column, gradient elution from
0% to 50% MeCN in 0.1 mol L-1 triethylammonium acetate
(aq) buffer over 60 min, flow rate 1 mL min-1, and detection at
260 nm.
4′-C-Aminomethyl-3′-O-(4,4′-dimethoxytrityl)thymidine (8).
4′-C-Azidomethyl-3′-O-(4,4′-dimethoxytrityl)thymidine (2.14 g,
3.6 mmol) (47) was dissolved in THF (24 mL). Triphenylphos-
phine (1.50 g, 5.7 mmol), water (64 µL), and a drop of 33% aq
NH3 (∼20 µL) was added, and the reaction was stirred
overnight. The reaction mixture was evaporated to dryness and
purified by silica gel chromatography (10-20% MeOH in DCM
containing 0.1% triethylamine). The yield was 1.54 g (75%) as
a white solid foam.1H NMR (500 MHz, CD3CN) δppm 7.50-7.47
10.1021/bc100268w  2010 American Chemical Society
Published on Web 09/09/2010

4′-C-Alkyne Functionalized Oligodeoxyribonucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1891
Figure 1. (a) Solid-supported conjugation of oligodeoxyribonucleotides by the click reaction. (b) Azido-functionalized ligands used in the click
reaction.
(m, 2H), 7.40-7.32 (m, 6H), 7.27 (m, 1H), 7.09 (br d, 1H, J )
1.0 Hz), 6.93-6.88 (m, 4H), 6.12 (dd, 1H, J ) 8.0 Hz, 4.0
Hz), 4.34 (dd, 1H, J ) 7.5 Hz, 7.5 Hz), 3.79 (s, 3H), 3.78 (s,
3H), 3.60 (d, 1H, J ) 12.0 Hz), 3.40 (d, 1H, J ) 12.0 Hz),
3.34 (d, 1H, J ) 13.5 Hz), 3.09 (d, 1H, J ) 14.0 Hz), 1.98 (m,
1H), 1.78 (br d, 3H, J ) 1.0 Hz), 1.32 (ddd, 1H, J ) 14.5 Hz,
8.3 Hz, 4.3 Hz). 13C NMR (125 MHz, CD3CN) δppm 163.7,
159.0, 159.0, 150.6, 145.6, 136.4, 136.1, 136.0, 130.5, 130.5,
128.2, 128.0, 127.1, 113.2, 113.2, 110.4, 87.4, 87.2, 83.6, 73.7,
64.6, 55.0, 43.5, 38.2, 11.5. ESI-MS: [M + Na]+ C32H35N3NaO7
requires 596.2367, found 596.2376.
4′-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine (9). Com-
pound 8 (1.4 g, 2.4 mmol) was dissolved in MeOH (40 mL)
and 4-pentynal (0.4 g, 4.9 mmol), NaBH3CN (0.46 g, 7.3 mmol),
and acetic acid (140 µL) were added on an ice bath. The reaction
was allowed to proceed overnight at room temperature. To
remove the 4,4′-dimethoxytrityl group, 80% aqueous acetic acid
was added, and after 2 h, the reaction mixture was evaporated
to dryness. The residue was purified by silica gel chromatog-
raphy (5-20% MeOH in DCM) yielding product 9 (0.65 g,
66%) as a powder. 1H NMR (500 MHz, CD3CN) δppm 7.59 (d,
1H, J ) 1.0 Hz), 6.28 (dd, 1H, J ) 7.8 Hz, 6.3 Hz), 4.41 (dd,
1H, J ) 5.0 Hz, 3.0 Hz), 3.61 (d, 1H, J ) 11.5 Hz), 3.52 (d,
1H, J ) 11.5 Hz), 2.92 (d, 1H, J ) 14.5 Hz), 2.86 (d, 1H, J )
14.5 Hz), 2.78-2.71 (m, 2H), 2.68-2.61 (m, 2H), 2.32-2.20
(m, 6H), 2.19 (t, 2H, J ) 2.8 Hz), 1.86 (br d, 3H, J ) 1.0 Hz),
1.76-1.66 (m, 4H). 13C NMR (125 MHz, CD3CN) δppm 163.9,
150.6, 136.3, 109.9, 87.3, 84.9, 84.0, 74.1, 69.0, 65.8, 56.7, 53.9,
40.2, 25.4, 15.7, 11.6. HRMS (EI): M+ C21H29N3O5 requires
403.2107, found 403.2113.
chloride (0.35 g, 1.0 mmol) was added and the mixture was
stirred overnight at room temperature. The volatiles were
removed under reduced pressure, and the residue was purified
by silica gel chromatography (50-90% ethyl acetate in petro-
leum ether + 0.1% triethylamine). The yield was 0.29 g (42%)
as a white solid foam. 1H NMR (500 MHz, CDCl3) δppm
7.40-7.37 (m, 2H), 7.33-7.23 (m, 8H), 6.87-6.82 (m, 4H),
6.46 (dd, 1H, J ) 9.0 Hz, 5.5 Hz), 4.69 (dd, 1H, J ) 4.0 Hz,
4.0 Hz), 3.79 (s, 6H), 3.12 (d, 1H, J ) 9.5 Hz), 3.03 (d, 1H, J
) 14.5 Hz), 3.01 (d, 1H, J ) 9.5 Hz), 2.91-2.83 (m, 2H), 2.80
(d, 1H, J ) 14.0 Hz), 2.58-2.50 (m, 2H), 2.38 (m, 1H),
2.25-2.10 (m, 5H), 1.95 (t, 2H, J ) 2.8 Hz), 1.77-1.65 (m,
4H), 1.54 (d, 3H, J ) 1.0 Hz). 13C NMR (125 MHz, CDCl3)
δppm 163.7, 158.8, 150.3, 144.1, 135.5, 135.2, 135.0, 130.1,
128.1, 128.1, 127.3, 113.3, 110.9, 87.2, 86.1, 84.9, 83.6, 76.1,
69.0, 67.2, 58.0, 55.3, 53.9, 41.4, 25.1, 16.4, 12.0. HRMS (EI):
M+ C42H47N3O7 requires 705.3414, found 705.3403.
3′-O-[(2-Cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-
4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]-5′-O-(4,4′-dimethox-
ytrityl)thymidine (1). Compound 12 (0.25 g, 0.35 mmol) was
dried over P2O5 in a vacuum desiccator and dissolved in dry
DCM (5 mL). Triethylamine (248 µL, 1.8 mmol) and 2-cya-
noethyl N,N-diisopropylphosphoramidochloridite (94.5 µL, 0.4
mmol) were added to the reaction solution under nitrogen. After
1 h, the reaction was completed and the mixture was eluted
through a short, dried silica gel column (50% ethyl acetate in
hexane, 0.1% triethylamine). The faster-eluting diastereomer I
was partly obtained as a pure compound, while diastereomer II
remained contaminated by diastereomer I (see Supporting
Information). Yield was 0.29 g (91%) as colorless oil. Diaste-
reomer I: 1H NMR (500 MHz, CD3CN) δppm 9.42 (s, 1H), 7.68
(br d, 1H, J ) 1.0 Hz), 7.53-7.49 (m, 2H), 7.41-7.32 (m,
6H), 7.28 (m, 1H), 6.93-6.89 (m, 4H), 6.24 (dd, 1H, J ) 6.5
4′-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]-5′-O-(4,4′-dimethox-
ytrityl)thymidine (12). To a solution of compound 9 (0.39 g,
1.0 mmol) in 20 mL of dry pyridine, 4,4′-dimethoxytrityl

1892 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kiviniemi et al.
1
Hz, 5.5 Hz), 5.01 (ddd, 1H, J ) 10.5 Hz, 7.0 Hz, 7.0 Hz), 3.80
(s, 6H), 3.74-3.58 (m, 5H), 3.13 (d, 1H, J ) 11.0 Hz),
2.68-2.46 (m, 8H), 2.32 (m, 2H), 2.10 (t, 2H, J ) 2.8 Hz),
2.07-1.94 (m, 4H), 1.52-1.40 (m, 4H), 1.39 (br d, 3H, J )
1.0 Hz), 1.21 (d, 6H, J ) 6.5 Hz), 1.20 (d, 6H, J ) 6.5 Hz);
13C NMR (125 MHz, CD3CN) δppm 163.9, 158.8, 150.5, 145.1,
136.0, 135.8, 135.7, 130.3, 130.3, 128.2, 127.9, 127.0, 118.3,
113.1, 113.1, 109.9, 88.7, 88.7, 86.6, 84.5, 83.0, 72.5, 72.4, 68.7,
64.7, 58.6, 58.5, 55.8, 55.0, 54.2, 43.1, 43.0, 38.6, 38.5, 26.5,
24.0, 24.0, 24.0, 23.9, 20.1, 20.1, 15.7, 11.2; 31P NMR (200
MHz, CDCl3) δppm 148.7. Diastereomer II: 1H NMR (500 MHz,
CD3CN) δppm 9.45 (s, 1H), 7.67 (s, 1H), 7.53-7.47 (m, 2H),
7.41-7.31 (m, 6H), 7.27 (m, 1H), 6.94-6.87 (m, 4H), 6.28
(dd, 1H, J ) 6.0 Hz, 6.0 Hz), 4.95 (ddd, 1H, J ) 9.5 Hz, 6.8
Hz, 6.8 Hz), 3.87 (m, 1H), 3.83-3.71 (m, 7H), 3.68-3.55 (m,
3H), 3.12 (d, 1H, J ) 10.5 Hz), 2.72-2.42 (m, 8H), 2.30 (m,
2H), 2.09 (t, 2H, J ) 2.8 Hz), 2.06-1.93 (m, 4H), 1.51-1.38
(m, 7H), 1.18 (d, 6H, J ) 6.5 Hz), 1.10 (d, 6H, J ) 6.5 Hz);
13C NMR (125 MHz, CD3CN) δppm 163.9, 158.8, 150.5, 145.1,
136.1, 135.7, 135.6, 130.3, 130.3, 128.2, 127.9, 127.0, 118.6,
113.2, 113.1, 110.1, 88.4, 88.4, 86.7, 84.4, 82.9, 73.7, 73.6, 68.7,
64.9, 58.2, 58.0, 55.8, 55.0, 54.2, 43.1, 43.1, 38.8, 26.5, 24.3,
24.2, 23.8, 23.8, 20.2, 20.1, 15.7, 11.2; 31P NMR (200 MHz,
CD3CN) δppm 148.9. ESI-MS: [M+H]+ C51H65N5O8P requires
906.4565, found 906.4552.
was 0.27 g (40%) as a white solid foam. H NMR (500 MHz,
CD3CN) δppm 7.49-7.45 (m, 2H), 7.37-7.31 (m, 7H), 7.26 (m,
1H), 6.92-6.87 (m, 4H), 6.29 (dd, 1H, J ) 7.5 Hz, 6.5 Hz),
4.63 (dd, 1H, J ) 5.5 Hz, 3.5 Hz), 3.79 (s, 6H), 3.22 (d, 1H, J
) 10.0 Hz), 3.10 (d, 1H, J ) 10.0 Hz), 2.85 (d, 1H, J ) 14.0
Hz), 2.69 (d, 1H, J ) 14.5 Hz), 2.56 (m, 1H), 2.45 (m, 1H),
2.30-2.20 (m, 5H), 2.16-2.10 (m, 3H), 1.62 (m, 2H), 1.54 (s,
3H); 13C NMR (125 MHz, CD3CN) δppm 163.8, 158.8, 150.6,
144.9, 135.8, 135.7, 135.5, 130.2, 130.2, 128.1, 128.0, 127.0,
113.2, 110.1, 86.8, 86.7, 84.2, 84.1, 74.4, 68.9, 66.5, 59.9, 57.8,
55.0, 42.9, 40.4, 26.0, 15.6, 11.4. ESI-MS: [M+Na]+
C38H43N3NaO7 requires 676.2993, found 676.2997.
3′-O-[(2-Cyanoethoxy)-(N,N-diisopropylamino)phosphinyl)]-
4′-C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]-5′-O-(4,4′-
dimethoxytrityl)thymidine (2). Compound 13 (0.27 g, 0.4
mmol) was dried over P2O5 in a vacuum desiccator and
dissolved in dry DCM (5 mL). Triethylamine (293 µL, 2.1
mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochlo-
ridite (111 µL, 0.5 mmol) were added to the reaction solution
under nitrogen. After 1 h, the reaction was completed and the
mixture was eluted through a short, dried silica gel column (50%
ethyl acetate in hexane, 0.1% triethylamine). The faster eluting
diastereomer I was partly obtained as a pure compound, while
diastereomer II remained contaminated by diastereomer I (see
Supporting Information). Yield was 0.31 g (87%) as a white
solid foam. Diastereomer I: 1H NMR (500 MHz, CD3CN) δppm
8.89 (s, 1H), 7.54-7.49 (m, 3H), 7.40-7.32 (m, 6H), 7.28 (m,
1H), 6.92-6.88 (m, 4H), 6.24 (dd, 1H, J ) 7.0 Hz, 5.0 Hz),
4.98 (ddd, 1H, J ) 10.5 Hz, 7.3 Hz, 7.3 Hz), 3.80 (s, 6H),
3.75-3.59 (m, 4H), 3.42 (d, 1H, J ) 10.5 Hz), 3.22 (d, 1H, J
) 10.5 Hz), 2.62-2.47 (m, 6H), 2.41-2.29 (m, 2H), 2.20 (s,
3H), 2.09-2.03 (m, 3H), 1.53 (m, 2H), 1.44 (br d, 3H, J ) 1.0
Hz), 1.21 (d, 6H, J ) 7.5 Hz), 1.20 (d, 6H, J ) 7.0 Hz); 13C
NMR (125 MHz, CD3CN) δppm 163.6, 158.8, 150.3, 145.1,
135.8, 135.8, 135.7, 130.3, 128.2, 127.9, 127.0, 118.6, 113.1,
110.0, 88.9, 88.8, 86.5, 84.4, 83.2, 72.7, 72.5, 68.6, 64.3, 58.7,
58.5, 58.0, 55.0, 43.4, 42.9, 38.5, 26.5, 24.0, 23.9, 20.1, 20.1,
15.6, 11.3; 31P NMR (200 MHz, CD3CN) δppm 148.6. Diaste-
4′-C-[C-(4-Pentyn-1-yl)aminomethyl]thymidine (10). Com-
pound 8 (0.92 g, 1.6 mmol) was dissolved in MeOH (20 mL)
and 4-pentynal (0.18 g, 2.2 mmol), NaBH3CN (0.30 g, 4.8
mmol) and acetic acid (92 µL) were added on an ice bath. The
reaction was allowed to proceed overnight at room temperature.
To remove the 4,4′-dimethoxytrityl group, 80% aqueous acetic
acid was added, and after 2 h, the reaction mixture was
evaporated to dryness. The residue was purified by silica gel
chromatography (5-20% MeOH in DCM), giving compound
1
10 in 94% yield (0.51 g) as a white powder. H NMR (500
MHz, MeOD/CD3Cl (1:1)) δppm 7.61 (s, 1H), 6.38 (dd, 1H, J
) 8.3 Hz, 6.3 Hz), 4.53 (dd, 1H, J ) 4.0 Hz, 4.0 Hz), 3.65 (s,
2H), 3.37 (m, 1H), 3.23-3.13 (m, 3H), 2.48 (m, 1H), 2.38-2.31
(m, 3H), 2.20 (br s, 1H), 1.96 (m, 2H), 1.91 (s, 3H); 13C NMR
(125 MHz, MeOD/CD3Cl (1:1)) δppm 164.7, 151.2, 136.5, 111.4,
86.1, 85.7, 81.5, 74.1, 70.7, 65.1, 49.7, 47.8, 39.4, 24.1, 15.6,
12.0. ESI-MS: [M+H]+ C16H24N3O5 requires 338.1711, found
338.1725.
1
reomer II: H NMR (500 MHz, CD3CN) δppm 8.94 (s, 1H),
7.52-7.48 (m, 3H), 7.38-7.31 (m, 6H), 7.27 (m, 1H),
6.91-6.87 (m, 4H), 6.27 (dd, 1H, J ) 7.0 Hz, 6.0 Hz), 4.90
(ddd, 1H, J ) 9.5 Hz, 7.0 Hz, 6.0 Hz), 3.87 (m, 1H), 3.79 (s,
6H), 3.77-3.56 (m, 3H), 3.43 (d, 1H, J ) 10.5 Hz), 3.21 (d,
1H, J ) 10.0 Hz), 2.69 (t, 2H, J ) 6.0 Hz), 2.62-2.47 (m,
4H), 2.42-2.26 (m, 2H), 2.20 (s, 3H), 2.09-2.02 (m, 3H), 1.52
(m, 2H), 1.45 (br d, 3H, J ) 1.0 Hz), 1.19 (d, 6H, J ) 6.5 Hz),
1.12 (d, 6H, J ) 6.5 Hz); 13C NMR (125 MHz, CD3CN) δppm
163.5, 158.8, 150.3, 145.1, 135.8, 135.8, 135.7, 130.3, 128.2,
127.9, 127.0, 118.6, 113.1, 110.2, 88.5, 88.4, 86.6, 84.4, 83.1,
74.0, 73.9, 68.6, 64.5, 58.8, 58.2, 58.0, 54.9, 43.4, 43.0, 38.6,
26.5, 24.2, 24.1, 23.8, 23.8, 20.2, 20.1, 15.6, 11.2; 31P NMR
(200 MHz, CD3CN) δppm 148.9. ESI-MS: [MH]+ C47H61N5O8P
requires 854.4252, found 854.4247.
4′-C-[N-Methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine (11).
Compound 10 (0.45 g, 1.3 mmol) was dissolved in MeOH (10
mL), and paraformaldehyde (44 mg), NaBH3CN (92 mg, 1.5
mmol), and acetic acid (76 µL) were added on an ice bath. The
mixture was stirred at room temperature over the weekend, and
paraformaldehyde (22 mg) and NaBH3CN (50 mg, 0.8 mmol)
were added to complete the reaction. The reaction was
evaporated to dryness and purified by silica gel chromatography
1
(10-20% MeOH in DCM). The yield was 0.44 g (93%). H
NMR (500 MHz, CD3CN) δppm 7.48 (s, 1H), 6.40 (dd, 1H, J )
8.5 Hz, 6.0 Hz), 4.45 (dd, 1H, J ) 4.5 Hz, 4.5 Hz), 3.66 (d,
1H, J ) 11.5 Hz), 3.61 (d, 1H, J ) 12.0 Hz), 3.41 (d, 1H, J )
14.0 Hz), 3.31 (m, 1H), 3.17 (m, 2H), 2.86 (s, 3H), 2.44-2.28
(m, 5H), 1.95-1.87 (m, 5H); 13C NMR (125 MHz, CD3CN)
δppm 163.7, 150.6, 136.1, 110.5, 85.9, 85.6, 82.5, 74.3, 70.1,
65.3, 59.0, 58.1, 43.6, 38.8, 23.2, 15.2, 11.6. ESI-MS: [M+H]+
C17H26N3O5 requires 352.1867, found 352.1849.
N-[4-(azidomethyl)benzyl]-2,2,2-trifluoroacetamide (4). N-[4-
(Azidomethyl)benzyl]-(4-methoxytrityl)amine (15) was first
synthesized from N-(4-methoxytrityl)-1,4-phenylenedimetha-
namine (14). Compound 14 (1.00 g, 2.4 mmol) was dissolved
in DCM (4 mL), and an aqueous solution of K2CO3 (3.7 mmol)/
CuSO4 (15 µmol) (7 mL) and MeOH (30 mL) were added.
Triflyl azide (4.9 mmol, theoretical amount calculated from
trifluoromethanesulfonyl anhydride used in the preparation) was
prepared as previously reported (50) and added to the reaction
mixture. The reaction was stirred overnight at room temperature
and evaporated to dryness. The residue was dissolved in ethyl
acetate and extracted with water. The organic fraction was dried
with Na2SO4, evaporated, and purified by silica gel chroma-
tography (10-20% ethyl acetate in petroleum ether, 0.1%
4′-C-[N-Methyl-N-(4-pentyn-1-yl)aminomethyl]-5′-O-(4,4′-
dimethoxytrityl)thymidine (13). To a solution of compound
11 (0.36 g, 1.0 mmol) in 15 mL of dry pyridine, 4,4′-
dimethoxytrityl chloride (0.42 g, 1.2 mmol) was added, and the
mixture was stirred overnight at room temperature. The reaction
mixture was evaporated and purified by silica gel chromatog-
raphy (5% MeOH in DCM + 0.1% triethylamine). The yield

4′-C-Alkyne Functionalized Oligodeoxyribonucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1893
triethylamine). Compound 15 was obtained in 96% yield (1.03
g) yield. 1H NMR (500 MHz, CDCl3) δppm 7.57-7.52 (m, 4H),
7.47-7.40 (m, 4H), 7.32-7.24 (m, 6H), 7.22-7.17 (m, 2H),
6.85-6.81 (m, 2H), 4.32 (s, 2H), 3.79 (s, 3H), 3.34 (s, 2H),
1.84 (br s, 1H); 13C NMR (125 MHz, CDCl3) δppm 158.0, 146.2,
141.4, 138.1, 129.8, 128.5, 128.4, 128.3, 127.9, 126.3, 113.2,
70.5, 55.2, 54.6, 47.6. To the solution of compound 15 (0.95 g,
2.2 mmol) in 10 mL of DCM, trifluoroacetic acid (0.34 mL,
4.4 mmol) and MeOH were added. After 1 h stirring at room
temperature, the reaction mixture was evaporated. Without
further purification, the detritylated product was subjected to
trifluoroacetylation with methyl trifluoroacetate (1.60 g, 12.5
mmol) in a mixture of triethylamine (1.6 mL) and MeOH (9
mL). After 2.5 h, the mixture was evaporated and purified by
silica gel chromatography (5-50% ethyl acetate in petroleum
ether). The yield was 0.50 g (88%) as a yellowish solid foam.
1H NMR (500 MHz, CDCl3) δppm 7.35-7.30 (m, 4H), 6.65 (br
s, 1H), 4.54 (d, 2H, J ) 6.0 Hz), 4.35 (s, 2H); 13C NMR (125
MHz, CDCl3) δppm 157.2, 136.0, 135.7, 128.9, 128.5, 115.8,
54.3, 43.6. ESI-MS: [M+Na]+ C10H9F3N4NaO requires 281.0621,
found 281.0602.
for 3 h and evaporated to dryness. The residue was dissolved
in ethyl acetate and extracted with saturated aq NaHCO3. The
organic fraction was dried with Na2SO4, evaporated, and purified
by silica gel chromatography (30% ethyl acetate in DCM +
0.1% triethylamine). The product (19, 1.96 g) was isolated as
white foam in 98% yield.1H NMR (500 MHz, CD3CN) δppm
7.72 (d, 1H, J ) 9.0 Hz), 7.56 (d, 1H, J ) 9.0 Hz), 7.52-7.48
(m, 2H), 7.39-7.30 (m, 7H), 7.27 (m, 1H), 6.95-6.90 (m, 4H),
5.42 (dd, 1H, J ) 10.0 Hz, 9.5 Hz), 5.36 (d, 1H, J ) 3.5 Hz),
5.31 (dd, 1H, J ) 10.0 Hz, 9.5 Hz), 5.08 (dd, 1H, J ) 10.0 Hz,
10.0 Hz), 5.06 (dd, 1H, J ) 10.5 Hz, 10.0 Hz), 4.43 (ddd, 1H,
J ) 10.0 Hz, 9.5 Hz, 3.8 Hz), 4.29 (m, 1H), 4.20 (m, 1H), 4.07
(dd, 1H, J ) 10.0 Hz, 10.0 Hz), 3.87 (m, 1H), 3.81 (s, 3H),
3.81 (s, 3H), 3.47 (dd, 1H, J ) 10.5 Hz, 1.5 Hz), 2.83 (dd, 1H,
J ) 10.5 Hz, 2.5 Hz), 2.10 (ddd, 1H, J ) 13.0 Hz, 4.5 Hz, 4.5
Hz), 2.02-1.94 (m, 7H), 1.92 (s, 3H), 1.71 (s, 3H); 13C NMR
(125 MHz, CD3CN) δppm 170.3, 170.2, 169.4, 168.7, 158.7,
158.7, 144.9, 136.1, 135.6, 130.1, 130.0, 128.1, 127.9, 126.9,
113.1, 113.0, 96.1, 85.6, 75.1, 74.6, 72.9, 70.9, 69.4, 67.4, 60.2,
55.0, 52.0, 48.6, 48.1, 31.1, 20.0, 19.8, 19.8, 19.7 (peaks
referring to Tfa are not listed). ESI-MS: [M+Na]+
C47H48F9N3NaO16 requires 1104.2783, found 1104.2695.
1,3,2′-Tri-N-trifluoroacetylparomamine (17). Paromamine
trihydrochloride (16) was obtained by hydrolysis of paromo-
mycin sulfate under acidic conditions (1 mol L-1 HCl aq in
MeOH, reflux overnight), as described previously (51). To the
suspension of compound 16 (2.96 g, 6.8 mmol) in a mixture of
MeOH (60 mL) and triethylamine (14.4 mL), methyl trifluo-
roacetate (8.76 g, 68.4 mmol) was added. After a few minutes,
the solution cleared for a while, but soon the product began to
precipitate. The reaction was stirred for 1 h at room temperature,
and the precipitated product was filtered and washed with
MeOH. Compound 17 was received in 76% yield (3.20 g) as a
white powder. 1H NMR (500 MHz, D2O/acetone-d6) δppm 5.38
(d, 1H, J ) 3.5 Hz), 4.02 (m, 1H), 3.90 (dd, 1H, J ) 10.5 Hz,
3.5 Hz), 3.84 (m, 1H), 3.73-3.65 (m, 4H), 3.58 (dd, 1H, J )
9.5 Hz, 9.0 Hz), 3.55-3.40 (m, 3H), 1.93 (m, 1H), 1.70 (ddd,
1H, J ) 12.5 Hz, each); 13C NMR (125 MHz, D2O/acetone-d6)
δppm 97.6, 79.4, 76.9, 73.6, 73.0, 70.5, 70.0, 60.6, 54.7, 50.1,
49.3, 31.9 (peaks referring to Tfa are not listed). ESI-MS:
[M+Na]+ C18H22F9N3NaO10 requires 634.1054, found 634.1052.
5,6,3′,4′-Tetra-O-acetyl-1,3,2′-tri-N-trifluoroacetylparom-
amine (20). A solution of 3% dichloroacetic acid in DCM (30
mL) was added to compound 19 (1.90 g, 1.8 mmol) and MeOH
was added. The reaction was stirred at room temperature for
4 h and evaporated to dryness. The residue was dissolved in
ethyl acetate and extracted with saturated aq NaHCO3. The
organic fraction was dried with Na2SO4, evaporated, and purified
by silica gel chromatography (70% ethyl acetate in petroleum
1
ether). The product (20, 1.27 g) was isolated in 92% yield. H
NMR (500 MHz, CD3CN) δppm 7.84 (d, 1H, J ) 9.0 Hz), 7.58
(d, 1H, J ) 8.5 Hz), 7.30 (d, 1H, J ) 9.0 Hz), 5.31 (dd, 1H, J
) 9.5 Hz, 9.5 Hz), 5.27 (d, 1H, J ) 4.0 Hz), 5.14 (dd, 1H, J )
10.5 Hz, 9.5 Hz), 5.07 (dd, 1H, J ) 10.0 Hz, 9.5 Hz), 5.03 (dd,
1H, J ) 10.5 Hz, 10.5 Hz), 4.38-4.17 (m, 3H), 4.09 (dd, 1H,
J ) 10.0 Hz, 9.5 Hz), 3.81 (ddd, 1H, J ) 10.0 Hz, 4.0 Hz, 2.0
Hz), 3.66 (ddd, 1H, J ) 12.5 Hz, 6.5 Hz, 2.0 Hz), 3.53 (ddd,
1H, J ) 12.5 Hz, 6.0 Hz, 4.0 Hz), 2.96 (t, 1H, J ) 6.3 Hz),
2.13 (ddd, 1H, J ) 13.5 Hz, 4.5 Hz, 4.5 Hz), 2.04-1.96 (m,
4H), 1.96 (s, 3H), 1.95 (s, 6H); 13C NMR (125 MHz, CD3CN)
δppm 170.5, 170.2, 169.8, 169.4, 96.1, 75.5, 74.5, 72.9, 70.5,
70.3, 67.6, 59.7, 52.1, 48.7, 48.0, 31.2, 19.9, 19.8, 19.8, 19.7
(peaks referring to Tfa are not listed). ESI-MS: [M+Na]+
C26H30F9N3NaO14 requires 802.1476, found 802.1474.
6′-O-(4,4′-Dimethoxytrityl)-1,3,2′-tri-N-trifluoroacetylpa-
romamine (18). Compound 17 (1.96 g, 3.2 mmol) was dried
over P2O5 in a vacuum and dissolved in dry pyridine (70 mL).
4,4′-Dimethoxytrityl chloride (1.09 g, 3.2 mmol) was added and
the mixture was stirred overnight at room temperature. After
evaporation, the residue was partitioned between ethyl acetate
and saturated aq NaHCO3. The organic fraction was dried with
Na2SO4, evaporated, and purified by silica gel chromatography
(10% MeOH in DCM + 0.1% triethylamine) yielding product
6′-Azido-6′-deoxy-5,6,3′,4′-tetra-O-acetyl-1,3,2′-tri-N-tri-
fluoroacetylparomamine (21). Compound 20 (0.73 g, 0.9
mmol) was dried over P2O5 in a vacuum and dissolved in dry
DCM (6 mL). 10% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene,
1.4 mL, 0.9 mmol) solution in MeCN and trifluoromethane-
sulfonyl anhydride (0.19 mL, 1.1 mmol) were added on an ice
bath. After 30 min, the ice bath was removed, and the reaction
was stirred at room temperature for 1.5 h. The reaction mixture
was partitioned between ethyl acetate and water. The organic
phase was separated, dried with Na2SO4, and evaporated to
dryness. The residue was purified by silica gel chromatography
(50% ethyl acetate in hexane) yielding triflate product (0.46 g,
53%), which was directly subjected to azidation step. To a
solution of triflate product (0.45 g, 0.5 mmol) in dry DMF (3
mL), sodium azide (0.16 g, 2.5 mmol) was added and the
mixture was stirred overnight at room temperature. The reaction
mixture was partitioned between diethyl ether and water. The
organic phase was separated, dried with Na2SO4, and evaporated
to dryness. The residue was purified by silica gel chromatog-
raphy (50% ethyl acetate in petroleum ether) yielding product
1
18 (2.00 g, 68%) as a purplish foam. H NMR (500 MHz,
CD3CN) δppm 8.07 (br d, 1H, J ) 7.0 Hz), 7.54 (br d, 2H, J )
8.5 Hz), 7.52-7.49 (m, 2H), 7.39-7.32 (m, 6H), 7.26 (m, 1H),
6.92-6.88 (m, 4H), 5.30 (d, 1H, J ) 3.0 Hz), 4.05-3.92 (m,
2H), 3.89-3.75 (m, 10H), 3.70-3.61 (m, 3H), 3.53 (dd, 1H, J
) 9.5 Hz, 9.0 Hz), 3.49 (m, 1H), 3.42-3.34 (m, 2H), 3.27 (dd,
1H, J ) 10.0 Hz, 3.5 Hz), 3.23 (dd, 1H, J ) 10.0 Hz, 2.5 Hz),
2.01 (ddd, 1H, J ) 13.0 Hz, 4.5 Hz, 4.5 Hz), 1.71 (ddd, 1H, J
) 13.0 Hz, each); 13C NMR (125 MHz, CD3CN) δppm 158.6,
145.3, 136.2, 136.2, 130.2, 128.2, 127.8, 126.7, 113.0, 98.4,
85.6, 80.1, 76.5, 73.7, 72.0, 71.5, 70.3, 62.0, 54.9, 54.8, 50.1,
49.6, 31.5 (peaks referring to Tfa are not listed). ESI-MS:
[M+Na]+ C39H40F9N3NaO12 requires 936.2360, found 936.2349.
6′-O-(4,4′-Dimethoxytrityl)-5,6,3′,4′-tetra-O-acetyl-1,3,2′-
tri-N-trifluoroacetylparomamine (19). Compound 18 (1.68 g,
1.8 mmol) was dissolved in dry pyridine and acetic anhydride
(1.4 mL, 14.8 mmol), and a catalytic amount of 4-dimethy-
laminopyridine was added. The mixture was heated at 60 °C
1
21 (0.39 g, 98%). H NMR (500 MHz, CD3CN) δppm 7.83 (d,
1H. J ) 9.0 Hz), 7.58 (d, 1H, J ) 8.0 Hz), 7.31 (d, 1H, J ) 8.5

1894 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kiviniemi et al.
Hz), 5.32 (dd, 1H, J ) 10.0 Hz, 9.5 Hz), 5.28 (d, 1H, J ) 4.0
Hz), 5.15-5.07 (m, 2H), 5.04 (dd, 1H, J ) 10.5 Hz, 10.5 Hz),
4.40-4.27 (m, 2H), 4.22 (m, 1H), 4.06 (dd, 1H, J ) 10.0 Hz,
9.5 Hz), 3.92 (m, 1H), 3.56 (dd, 1H, J ) 13.5 Hz, 2.5 Hz),
3.36 (dd, 1H, J ) 13.5 Hz, 4.3 Hz), 2.13 (ddd, 1H, J ) 13.0
Hz, 4.8 Hz, 4.8 Hz), 2.04 (ddd, 1H, J ) 13.0 Hz each), 1.99 (s,
3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H); 13C NMR (125
MHz, CD3CN) δppm 170.4, 170.2, 169.4, 96.0, 75.8, 74.5, 72.9,
70.1, 69.3, 68.1, 52.0, 50.1, 48.7, 48.0, 31.1, 19.9, 19.8, 19.7,
19.7 (peaks referring to Tfa are not listed). ESI-MS: [M+Na]+
C26H29F9N6NaO13 requires 827.1541, found 827.1496.
mL, 37.0 mmol) and a catalytic amount of 4-dimethylaminopy-
ridine were added, and the reaction was stirred overnight at room
temperature. After evaporation, the residue was partitioned
between ethyl acetate and saturated aq NaHCO3. The organic
phase was separated, dried with Na2SO4, and evaporated to
dryness. The residue was dissolved in 3% dichloroacetic acid
in DCM (40 mL) and MeOH was added. After stirring the
reaction 3 h at room temperature, the mixture was evaporated
and extracted with ethyl acetate and saturated aq NaHCO3. The
organic phase was dried with Na2SO4, evaporated to dryness,
and purified by silica gel chromatography (5-10% MeOH in
DCM) to yield 3.90 g (87%) of the product 24 as a white foam.
1H NMR (500 MHz, CDCl3 + drop of CD3OD) δppm 5.84 (d,
1H, J ) 2.0 Hz), 5.17 (d, 1H, J ) 2.5 Hz), 5.14 (dd, 1H, J )
9.5 Hz, 9.5 Hz), 4.97 (dd, 1H, J ) 3.0 Hz, 3.0 Hz), 4.91 (m,
1H), 4.89-4.81 (m, 2H), 4.80 (d, 1H, J ) 1.5 Hz), 4.76 (dd,
1H, J ) 5.8 Hz, 2.3 Hz), 4.37-4.24 (m, 2H), 4.17-4.05 (m,
4H), 3.99 (m, 1H), 3.95 (dd, 1H, J ) 9.5 Hz, 8.5 Hz), 3.78 (dd,
1H, J ) 13.0 Hz, 2.5 Hz), 3.74-3.64 (m, 3H), 3.63-3.53 (m,
2H), 3.32 (dd, 1H, J ) 14.3 Hz, 5.8 Hz), 3.23 (dd, 1H, J )
14.0 Hz, 9.5 Hz), 2.18 (s, 3H), 2.15 (s, 3H), 2.10 (s, 3H),
2.08-2.02 (m, 7H), 1.99 (s, 3H), 1.81 (ddd, 1H, J ) 13.0 Hz
each); 13C NMR (125 MHz, CDCl3 + drop of CD3OD) δppm
171.0, 170.8, 170.5, 170.3, 168.7, 168.5, 107.4, 97.4, 95.0, 82.8,
80.9, 75.1, 75.0, 74.9, 73.3, 72.3, 69.9, 69.5, 68.2, 66.8, 66.2,
59.0, 51.7, 48.6, 48.1, 47.4, 40.1, 39.9, 31.3, 20.5, 20.2, 20.2,
20.2 (peaks referring to Tfa are not listed).
6′-Azido-6′-deoxy-1,3,2′-tri-N-trifluoroacetylparomamine (5).
To a solution of compound 21 (0.40 g, 0.5 mmol) in dry MeOH
(12 mL), sodium methoxide (160 µL of 0.6 mol L-1 solution
in MeOH) was added. After 1 h, the mixture was neutralized
with a strong cation-exchange resin (Dowex 50WX8-200, H+-
form) and filtered. The solution was evaporated, yielding pure
product 5 (0.28 g, 88%) as a solid powder. 1H NMR (500 MHz,
CD3CN + drop of CD3OD) δppm 8.54 (d, 1H. J ) 9.0 Hz),
8.15 (d, 1H, J ) 9.0 Hz), 8.01 (d, 1H, J ) 8.0 Hz), 5.21 (d,
1H, J ) 3.5 Hz), 4.02 (m, 1H), 3.95 (m, 1H), 3.84 (m, 1H),
3.69 (ddd, 1H, J ) 10.0 Hz, 3.0 Hz, 3.0 Hz), 3.63-3.56 (m,
2H), 3.54 (dd, 1H, J ) 13.5 Hz, 2.5 Hz), 3.50 (dd, 1H, J ) 9.5
Hz, 9.0 Hz), 3.47-3.41 (m, 2H), 3.34 (dd, 1H, J ) 10.0 Hz,
9.5 Hz), 2.00 (ddd, 1H, J ) 13.0 Hz, 4.3 Hz, 4.3 Hz), 1.68
(ddd, 1H, J ) 13.0 Hz each); 13C NMR (125 MHz, CD3CN +
drop of CD3OD) δppm 98.6, 81.2, 76.2, 73.7, 72.0, 71.0, 70.3,
54.7, 50.5, 50.1, 49.5, 31.6 (peaks referring to Tfa are not listed).
ESI-MS: [M+Na]+ C18H21F9N6NaO9 requires 659.1119, found
659.1122.
Compound 24 (1.07 g, 0.7 mmol) was dried over P2O5 in
vacuum and dissolved in dry DCM (5 mL). 10% DBU (1.1
mL, 0.7 mmol) in MeCN and trifluoromethanesulfonyl anhy-
dride (0.15 mL, 0.9 mmol) were added on an ice bath. After 30
min, the ice bath was removed and the reaction was stirred at
room temperature for 2 h. The reaction mixture was partitioned
between ethyl acetate and water. The organic phase was
separated, dried with Na2SO4, and evaporated to dryness. The
residue was purified by silica gel chromatography (50-80%
ethyl acetate in hexane) yielding the triflate product (0.59 g,
50%) as reddish foam. The crude product was subjected as such
to azidation. Accordingly, to a solution of the triflate product
(0.59 g, 0.4 mmol) in dry DMF (3.5 mL), sodium azide (0.13
g, 2.0 mmol) was added, and the mixture was stirred overnight
at room temperature. The reaction mixture was partitioned
between diethyl ether and water. The organic phase was
collected, dried with Na2SO4, and evaporated to dryness. The
residue was purified by silica gel chromatography (50-70%
5′′-O-(4,4′-Dimethoxytrityl)-1,3,2′,6′,2′′′,6′′′-hexa-N-trifluo-
roacetylneomycin (23). Triethylamine (3.6 mL, 26 mmol) and
methyl trifluoroacetate (2.6 mL, 26 mmol) were added to a
mixture of neomycin trisulfate (22, 2.0 g, 2.2 mmol) and MeOH
(10 mL). The mixture was stirred overnight at room temperature
and evaporated to dryness. Saturated NaHCO3 was added, and
the crude product was extracted with ethyl acetate. The organic
layers were combined, dried over Na2SO4, filtered, and evapo-
rated to dryness. The residue was further dried by coevaporation
with dry pyridine and dissolved in dry pyridine, and then 4,4′-
dimetoxytrityl chloride (0.80 g, 2.4 mmol) was added to the
mixture. After overnight reaction, saturated NaHCO3 was added
to the mixture and the product was extracted with ethyl acetate.
The organic layers were combined, dried over anhydrous
Na2SO4, filtered, and evaporated to dryness. The residue was
purified by silica gel chromatography (10% MeOH, 1% tri-
ethylamine in DCM) to yield 2.35 g (73%) of the product (23)
as white foam.1H NMR (500 MHz, CD3OD) δppm 7.46 (m, 2H),
7.36-7.30 (m, 6H), 7.21 (m, 1H), 6.90 (m, 4H), 5.78 (d, 1H, J
) 3.3 Hz), 5.16 (d, 1H, J ) 4.6 Hz), 5.09 (d, 1H, J ) 1.5 Hz),
4.29 (dd, 1H, J ) 4.8 Hz, both), 4.21-4.20 (m, 2H), 4.14 (m,
1H), 4.11 (dd, 1H, J ) 6.7 Hz, both), 4.01 (m, 1H), 3.97 (m,
1H), 3.92 (m, 1H), 3.87 (dd, 1H, J ) 9.9 and 8.8 Hz), 3.80 (s,
6H), 3.75-3.63 (m, 5H), 3.60 (dd, 1H, J ) 13.5 and 7.4 Hz),
3.56-3.51 (m, 2H), 3.35-3.31 (m, 3H), 3.19 (dd, 1H, J ) 10.5
and 3.1 Hz), 2.00 (ddd, 1H, J ) 12.9 Hz, 4.2 and 4.2 Hz), 1.77
(ddd, 1H, J ) 12.7 Hz, each); 13C NMR (125 MHz, CD3OD)
δppm 157.93, 157.85, 145.0, 136.0, 135.6, 130.2, 129.9, 128.0,
127.4, 126.3, 112.7, 109.0, 97.6, 96.3, 87.4, 86.3, 81.9, 76.9,
75.6, 74.8, 73.0, 72.1, 71.5, 70.3, 69.8, 69.2, 67.1, 62.5, 54.3,
53.5, 51.3, 49.6, 49.1, 40.4, 39.6, 31.5 (peaks referring to Tfa
are not listed). ESI-MS: [M+Na]+ C56H58F18N6NaO21 requires
1515.3260, found 1515.3239.
1
ethyl acetate in hexane) yielding 25 (0.38 g, 69%). H NMR
(500 MHz, CD3CN) δppm 7.87 (d, 1H, J ) 9.5 Hz), 7.59 (br t,
1H), 7.54 (d, 1H, J ) 8.5 Hz), 7.50-7.42 (m, 2H), 7.23 (d,
1H, J ) 9.5 Hz), 5.86 (d, 1H, J ) 3.5 Hz), 5.24-5.17 (m, 2H),
4.99 (d, 1H, J ) 3.0 Hz), 4.98-4.84 (m, 5H), 4.44-4.34 (m,
2H), 4.28 (m, 1H), 4.22 (m, 1H), 4.13-4.04 (m, 3H), 4.01 (ddd,
1H, J ) 10.5 Hz, 3.5 Hz, 3.5 Hz), 3.89-3.81 (m, 2H), 3.69
(dd, 1H, J ) 13.5 Hz, 3.0 Hz), 3.62 (ddd, 1H, J ) 14.5 Hz, 5.0
Hz, 3.0 Hz), 3.56-3.41 (m, 4H), 2.12 (s, 3H), 2.10 (s, 3H),
2.08-2.04 (m, 4H), 2.03 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H),
1.96 (m, 1H); 13C NMR (125 MHz, CD3CN) δppm 170.4, 170.3,
170.2, 170.0, 169.2, 168.7, 107.8, 96.9, 95.5, 82.7, 79.5, 76.5,
74.8, 74.7, 74.6, 71.3, 69.9, 68.4, 68.1, 67.6, 65.5, 51.7, 50.5,
48.8, 48.5, 47.6, 39.5, 38.9, 30.8, 20.1, 20.1, 19.9, 19.8, 19.8,
19.7 (peaks referring to Tfa are not listed). ESI-MS: [M+Na]+
C47H51F18N9NaO24 requires 1490.2652, found 1490.2649.
5′′-Azido-5′′-deoxy-1,3,2′,6′,2′′′,6′′′-hexa-N-trifluoroacetyl-
neomycin (6). To a solution of compound 25 (0.43 g, 0.3 mmol)
in dry MeOH (7 mL), sodium methoxide (200 µL of 0.6 mol
L-1 solution in MeOH) was added. After 6 h, the mixture was
neutralized with a strong cation-exchange resin (Dowex
50WX8-200, H+-form) and filtered. The solution was evapo-
5′′-Azido-5′′-deoxy-6,3′,4′,2′′,3′′′,4′′′-hexa-O-acetyl-
1,3,2′,6′,2′′′,6′′′-hexa-N-trifluoroacetylneomycin (25). Com-
pound 23 (4.60 g, 3.1 mmol) was dried over P2O5 in vacuum
and dissolved in dry pyridine (10 mL). Acetic anhydride (3.5

4′-C-Alkyne Functionalized Oligodeoxyribonucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1895
Scheme 1a
a Reagents: (i) PPh3, H2O, NH3 (aq), THF; (ii) 4-pentynal (1 or 2 equiv), NaBH3CN, AcOH, MeOH; (iii) paraformaldehyde, NaBH3CN, AcOH,
MeOH; (iv) DMTrCl, pyridine; (v) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, Et3N, DCM.
rated yielding pure product 6 (0.34 g, 95%) as a solid powder.
1H NMR (500 MHz, CD3OD) δppm 9.35 (d, 1H, J ) 9.0 Hz),
9.27 (t, 1H, J ) 5.5 Hz), 9.23 (d, 1H, J ) 8.0 Hz), 8.82 (t, 1H,
J ) 5.5 Hz), 8.73 (d, 1H, J ) 9.5 Hz), 8.33 (d, 1H, J ) 9.5
Hz), 5.73 (d, 1H, J ) 3.0 Hz), 5.12-5.08 (m, 2H), 4.23-4.08
(m, 5H), 4.05 (ddd, 1H, J ) 10.0 Hz, 10.0 Hz, 4.0 Hz),
3.97-3.87 (m, 3H), 3.80-3.51 (m, 11H), 3.42 (dd, 1H, J )
13.3 Hz, 3.3 Hz), 3.23 (dd, 1H, J ) 9.5 Hz, 9.0 Hz), 1.99 (ddd,
1H, J ) 13.0 Hz, 4.3 Hz, 4.3 Hz), 1.73 (ddd, 1H, J ) 12.5 Hz
each); 13C NMR (125 MHz, CD3OD) δppm 110.3, 97.6, 96.5,
87.6, 79.9, 76.2, 74.6, 73.6, 72.0, 71.7, 70.3, 70.2, 69.3, 67.7,
53.7, 51.3, 51.2, 49.8, 49.0, 40.4, 40.0, 31.5 (peaks referring to
Tfa are not listed). ESI-MS: [M+Na]+ C35H39F18N9NaO18
requires 1238.2018, found 1238.2027.
Synthesis of Oligodeoxyribonucleotides. The modified oli-
godeoxyribonucleotides were synthesized on an Applied Bio-
systems 392 DNA synthesizer in a 1.0 µmol scale using
commercial 500 Å CPG-Q-adenosine support (Q ) hydro-
quinone-O,O′-diacetate). For phosphoramidites 1 and 2 (0.13
mol L-1 solution in dry MeCN), a prolonged coupling time of
10 min was used. Otherwise, standard DNA protocol was
applied. The oligonucleotides were released from the support
with concentrated ammonia (33% aqueous NH3) at 55 °C
overnight. For the click reactions, the support-bound material
26-29 were treated for 5-8 min with 10% DBU in MeCN (1
mL, dried over molecular sieves), after which the support was
sequentially washed with MeCN, DMF, MeOH, water, and
MeOH.
solution was added. The mixture was shaken at room temper-
ature for 5 h. The support was collected by filtration and washed
first with water, DMF, and MeOH. To remove the copper salt
and TBTA ligand as quantitatively as possible, the support was
then treated for 10 min with aq EDTA solution (0.1 mol L-1),
washed with water, and then for another 10 min with aqueous
solution of neomycin trisulfate (0.1 mol L-1). Finally, the
support was washed with water, DMF, DCM, MeOH, and
MeCN and subjected to standard ammonolytic release. After
evaporation, the oligonucleotide conjugate was purified by RP
HPLC.
Melting Temperature Studies. The melting curves (absor-
bance versus temperature) were measured at 260 nm on a
Perkin-Elmer Lambda 35 UV-vis spectrometer equipped with
a multiple cell holder and a Peltier temperature-controller. The
temperature was changed at a rate of 0.5 °C/min (from 20 to
90 °C). The measurements were performed in 10 mmol L-1
potassium phosphate buffer (pH 7) containing 0.1 mol L-1 or
1.0 mol L-1 NaCl. The oligonucleotide conjugates and their
cDNA or 2′-O-methyl RNA targets were used at a concentration
of 2 µmol L-1. Tm values were determined as the maximum of
the first derivate of the melting curve.
RESULTS AND DISCUSSION
Synthesis of 4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thy-
midine and 4′-C-[N-methyl-N-(4-pentyn-1-yl)aminometh-
yl]thymidine 3′-(2-Cyanoethyl-N,N-diisopropylphosphor-
amidites (1, 2). 4′-C-Azidomethyl-3′-O-(4,4′-dimethoxytrityl)-
thymidine (7) was synthesized in good yield (75%) from 4′-C-
hydroxymethylthymidine as described previously (47). Because
the azido group can be easily converted to amino group by
Staudinger reaction, it allows further functionalization. Accord-
ingly, 4′-C-aminomethyl-3′-O-(4,4′-dimethoxytrityl)thymidine
(8) was obtained by the reaction of compound 7 with triph-
enylphosphine and water (Scheme 1). Compound 8 was then
subjected to reductive alkylation in the presence of NaBH3CN
under acidic conditions. On using 2 equiv of 4-pentynal (52),
4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine (9) was ob-
Click Conjugation. Azide-functionalized ligands, 3-6, were
conjugated to the alkynyl groups of oligonucleotides 26-29.
The solutions of ligands (50 mmol L-1) in MeOH were added
onto the support-bound oligonucleotides as follows: 15 equiv
for 27, 30 equiv for 26 and 29, and 40 equiv for 28. After that,
CuSO4 and tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA-
ligand) were added as a 25 mmol L-1 solution in a 12:3:1
mixture of water, DMSO, and 2-butanol. The amounts added
were as follows: 7.5 equiv for 27, 15 equiv for 26 and 29, and
20 equiv for 28. Finally, sodium ascorbate (15, 30, and 40 equiv
for 27, 26, 29, and 28, respectively) as 0.1 mol L-1 aqueous

1896 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kiviniemi et al.
Scheme 2a
a Reagents: (i) TfN3, K2CO3, CuSO4, H2O, MeOH, DCM; (ii) DCA, DCM, MeOH; (iii) TfaOMe, Et3N, MeOH; (iv) DMTrCl, pyridine; (v)
Ac2O, DMAP, pyridine; (vi) Tf2O, 10% DBU in MeCN, DCM; (vii) NaN3, DMF; (viii) NaOMe, MeOH.
tained. 4′-C-[N-Methyl-N-(4-pentyn-1-yl)aminomethyl]thymi-
dine (11) was, in turn, prepared by two consecutive alkylation
steps. Reaction with 4-pentynal gave 4′-C-[N-(4-pentyn-1-
yl)aminomethyl]thymidine (10), which was then N-methylated
with paraformaldehyde. After the reductive alkylations, the 3′-
O-4,4′-dimethoxytrityl group was removed with 80% acetic acid.
Compounds 9 and 11 were then converted to 5′-O-(4,4′-
dimethoxytrityl) ethers (12, 13) and phosphitylated to phos-
phoramidites 1 and 2.
Synthesis of Ligands 3-6. The azido-functionalized ligands
(4-6) used in the click conjugation were synthesized as outlined
in Scheme 2. The azidation of previously synthesized (47) N-(4-
methoxytrityl)-1,4-phenylenedimethanamine (14) was carried
out by the Cu2+-catalyzed diazo transfer described in the
literature (50). The acid-labile trityl protection of the product
(15) was changed to a base-labile trifluoroacetyl protection,
removable by the conventional ammonolysis used for the release
and deprotection of support-bound oligonucleotides.
azidation. Since the azidation gave poor yields using mesyl
esters as a starting material, conversion of the 6′-hydroxyl group
to a triflate was attempted. The conventional treatment with
triflic anhydride in the presence of pyridine, however, yielded
a 6′-pyridinium salt instead of the desired product. Replacement
of pyridine by less nucleophilic DBU then provided the desired
triflate, which may be azidated to product 21 in 52% overall
yield. This was finally deacetylated by methoxide ion-catalyzed
transesterification in methanol to obtain product 5, applicable
to postsynthetic conjugation of the alkynylated oligonucleotides.
5′′-Azido-1,3,2′,6′,2′′′,6′′′-hexa-N-trifluoroacetylneomycin (6)
was synthesized starting from neomycin trisulfate (22) according
to the same protocol as described for the paromamine derivative
5 above. The mannose-derived ligand, methyl 2,3,4-tri-O-acetyl-
6-azido-6-deoxy-R-D-mannopyranoside (3), was prepared as
previously reported (53).
Oligonucleotide Synthesis and Click Conjugation. 4′-C-
[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-C-[N-
methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine monomers 1
and 2 were incorporated into 15-mer oligodeoxyribonucleotides
26-29 using standard phosphoramidite coupling chemistry
except for a prolonged 10 min coupling time. The coupling
yields were 97%. Interestingly, we noticed that these modified
oligonucleotides did not withstand solid-supported click con-
Paromamine trihydrochloride (16) was obtained by acidic
hydrolysis of commercially available paromomycin sulfate (51).
After trifluoroacetylation of the amino groups, the primary 6′-
hydroxyl group was protected as a 4,4′-dimethoxytrityl ether
(18) and the remaining hydroxyl functions were acetylated. The
6′-position was then detritylated, sulfonated, and subjected to

4′-C-Alkyne Functionalized Oligodeoxyribonucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1897
Scheme 3. Synthesis of the Conjugated Oligodeoxyribonucleotides Containing One 4′-C-Modified Monomer
Table 1. Melting Experiments of the Oligonucleotide Conjugates and Their Complementary DNA or 2′-O-Methyl-RNA Sequencesa
compl. DNA Tm/°C and (∆ Tm/°C)
compl. 2′-O-methyl RNA Tm/°C and (∆ Tm/°C)
0.1 mol L-1 NaCl
1.0 mol L-1 NaCl
0.1 mol L-1 NaCl
1.0 mol L-1 NaCl
5′-CAT CTG GTT CTA CGA
52.8
61.9
54.1
65.0
5′-CAT CTG GTBCTA CGA (26)
5′-CAT CBG GTT CBA CGA (28)
5′-CAT CTG GTD CTA CGA (27)
5′-CAT CDG GTT CDA CGA (29)
5′-CAT CTG GTE CTA CGA (30)
5′- CAT CEG GTT CEA CGA (36)
5′-CAT CTG GTF CTA CGA (31)
5′-CAT CTG GTH CTA CGA (32)
5′-CAT CHG GTT CHA CGA (37)
5′- CAT CIG GTT CIA CGA (38)
5′- CAT CTG GTJ CTA CGA (33)
5′-CAT CTG GTK CTA CGA (34)
5′- CAT CKG GTT CKA CGA (39)
5′-CAT CTG GTL CTA CGA (35)
54.5 (+1.7)
52.5 (-0.3)
54.6 (+1.8)
53.5 (+0.7)
53.8 (+1.0)
51.3 (-1.5)
54.6 (+1.8)
57.9 (+5.1)
62.0 (+9.2)
61.0 (+8.2)
57.9 (+5.1)
57.8 (+5.0)
58.2 (+5.4)
56.1 (+3.3)
61.3 (-0.6)
59.7 (-2.2)
62.5 (+0.6)
61.6 (-0.3)
62.1 (+0.2)
59.6 (-2.3)
62.2 (+0.3)
63.7 (+1.8)
66.6 (+4.7)
66.5 (+4.6)
62.2 (+0.3)
63.5 (+1.6)
62.8 (+0.9)
62.1 (+0.2)
54.7 (+0.6)
53.0 (-1.1)
55.7 (+1.6)
52.4 (-1.7)
54.3 (+0.2)
53.8 (-0.3)
54.6 (+0.5)
54.2 (+0.1)
56.7 (+2.6)
57.3 (+3.2)
53.2 (-0.9)
54.1 ((0.0)
55.1 (+1.0)
52.5 (-1.6)
65.1 (+0.1)
65.4 (+0.4)
66.4 (+1.4)
64.7 (-0.3)
65.5 (+0.5)
66.1 (+1.1)
65.3 (+0.3)
64.3 (-0.7)
66.5 (+1.5)
67.1 (+2.1)
64.8(-0.2)
65.1 (+0.1)
65.2 (+0.2)
64.2 (-0.8)
a In 10 mmol L-1 potassium phosphate buffer (pH 7) containing 0.1 mol L-1 or 1.0 mol L-1 NaCl. The oligonucleotide conjugates and their cDNA
or 2′-O-methyl RNA targets were used a concentration of 2 µmol L-1
.
jugation as long as their phosphate groups were 2-cyanoethyl
protected, but a chain cleavage took place leaving the
modified monomer attached to the 5′-terminus of the support-
bound oligonucleotide. By contrast, the 4-pentyn-1-yl func-
tionalized oligonucleotides that had not been subjected to
the conditions of click reaction could be isolated in intact
form by conventional ammonolysis. To carry out the click
reaction on a solid support, the 2-cyanoethyl groups had to
be removed prior to the click reaction, which was achieved
by treatment with 10% DBU in MeCN (5-8 min). Since the

1898 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kiviniemi et al.
succinyl linker is not fully compatible with the DBU
treatment, but undergoes a premature cleavage via succin-
imide formation (54), all the conjugate syntheses were carried
out on a Q-linker loaded CPG support (55). After exposure
of the internucleosidic linkages as phosphodiesters, the azido-
functionalized ligands 3-6 were attached by the click
reaction (CuSO4/TBTA/sodium ascorbate, 5 h, r.t) and the
conjugates obtained were released from the support and
deprotected by ammonolysis (Scheme 3). The crude products
(30-39; see Table 1) were purified by RP-HPLC, and their
identity was verified by ESI-MS. Figure 2 shows representa-
tive examples of the HPLC traces of the crude oligonucle-
otides 35, 36, and 39 and the homogeneous products after
purification. In each case, the desired conjugate was clearly
a main product.
In our previous study on oligonucleotides containing the 4′-
C-azidomethylthymidine monomer, we were not able to achieve
homogeneous products by the solid-supported click conjugation
in case the ligand contained amino groups in their structure,
but capillary electrophoresis revealed the presence of two peaks
with the same molecular mass. When the reactions were
performed in solution, this problem was not encountered. With
4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-C-
[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine monomers
(1, 2) used in the present study, capillary electrophoretic, HPLC,
and ESI-MS data all indicated the presence of only one pure
conjugate for each oligonucleotide. Accordingly, all conjugations
could now be conveniently made on a solid support without
complications.
well as the corresponding unmodified 15-mer oligodeoxyribo-
nucleotide. At high salt concentration, the hybridization was
even slightly more efficient compared to the unmodified DNA
sequence.
All conjugates bearing amino groups (32-35, 37-39) mark-
edly stabilized the duplex with DNA target. With the p-
aminomethylbenzene conjugates, the DNA-hybrids incorporat-
ing one or two H (32, 37) or I (38) monomers were stabilized
by 4-5 °C/modification. Toward 2′-O-methyl RNA, the stabi-
lization was less prominent. The stabilization was more marked
at low ionic strength, but it still existed at high ionic strength.
Undoubtedly, the major part of the stabilization results from
reduced electrostatic repulsion between negatively charged
sugar-phosphate backbones and positively charged amino
groups present at neutral pH. This influence plays a more
important role at low ionic strength.
When one paromamine derived monomer, J (33) or K (34)
was incorporated into the strand, the DNA-hybrid was stabilized
by 5 °C, but the second monomer (39) did not result in any
extra stabilization. With 2′-O-Me RNA target, two monomers
K were needed to achieve even a modest increase in Tm (∆Tm
) +1.0 °C). Compared to the paromamine monomers, the
neomycin group (monomer L in 35) was less stabilizing than
the paromamine group (monomer K in 34) toward DNA, and
toward 2′-O-Me-RNA, the effect is even destabilizing. Similarly
to the p-aminomethylphenyl ligands, the duplex stabilizing effect
of the aminoglycosides ligands is more marked at low ionic
strength.
The hybridization properties of the 4′-modified oligonucle-
otides are encouraging. Evidently the 4′-position, facing the
minor groove upon hybridization, can accommodate even rather
large molecule clusters without appreciable destabilization of
the duplex. The 4′-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thy-
midine monomer (1) allows attachment of two ligands per
residue, which may well be useful when applying the glyco-
targeting concept. A short segment of the duplex may be
decorated with several sugar units. The results received with
the mannose conjugated oligonucleotide, 36, lend some support
to the feasibility of sugar-coated oligonucleotides as carriers of
RNA-type oligonucleotides. On the other hand, 4′-C-[N-methyl-
N-(4-pentyn-1-yl)aminomethyl]thymidine monomer (2) can be
preferable for attachment of bulky ligands such as neomycin
derivatives to oligonucleotides. Among the amino group-
bearing ligands, the most remarkable hybridization enhance-
ment was obtained with p-aminomethylphenyl conjugates (32,
37, 38). Sterically more demanding aminoglycoside ligands
(33-35, 39) stabilized the DNA · DNA duplex less, but still
notably. All amino group-bearing conjugates preferred DNA
over 2′-OMe RNA. Compared to the results of the present
paper, the previously observed duplex stabilizations achieved
with some conjugates of the 4′-C-azidomethylthymidine
incorporating oligonucleotides are modest (47). This implies
that the alkyl chain of the 4′-C-(4-pentyn-1-ylaminometh-
yl)thymidine monomers (1, 2) allows a more flexible align-
ment of the triazole ring and conjugate groups upon
hybridization. The tertiary amino group, which probably is
protonated under physiological conditions, further stabilizes
the duplex formation by diminishing the electrostatic repul-
sion between the complementary chains, as evidenced by the
fact that the melting temperatures observed for the oligo-
nucleotides containing 4′-C-(alkynylaminomethyl)thymidine
units (26-29) are higher than those observed for their 4′-
C-azidomethylthymidine counterparts.
Melting Temperature Studies. Melting temperatures (Tm)
for the duplexes of oligonucleotides 26-29 and their conjugates
30-39 with the cDNA and 2′-O-methyl RNA sequences were
measured at pH 7 at the ionic strength of 0.1 and 1.0 mol L-1
(Table 1).
When one 4′-C-modified monomer, B (26) or D (27), was
incorporated into the DNA strand, the stability of the duplex
increased with both DNA and 2′-O-Me RNA. When two such
monomers were incorporated in the sequence (28, 29), a slight
destabilization took place.
With mannose-conjugated oligonucleotides containing a
single modified monomer, either E (30) or F (31), the Tm of
the DNA-hybrid was again increased, but the presence of two
modified units (36) is destabilizing. By contrast, on using a 2′-
O-methyl RNA target, the conjugate containing two E mono-
mers and, hence, four mannose residues hybridized almost as
CONCLUSION
4′-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4′-
C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine 3′-phos-

4′-C-Alkyne Functionalized Oligodeoxyribonucleotides
Bioconjugate Chem., Vol. 21, No. 10, 2010 1899
Figure 2. RP HPLC traces of the crude (A) and purified (B) oligonucleotide conjugates 35, 36, and 39.
phoramidites (1, 2) have been synthesized as a continuation of
the previously reported preparation of 4′-C-azidomethylthymi-
dine 3′-hydrogenphosphonate. Together, these building blocks
enable the exploitation of the click conjugation on both ways,
i.e., to have azido function present on either the oligonucleotide
or ligand side. Nucleoside 1 bearing two 4-pentyn-1-yl groups
is designed for high-density functionalization of modified
oligonucleotides, whereas nucleoside 2 allows one-armed
conjugations. Azido-derivatized ligands may be efficiently
conjugated to the alkynyl groups of these monomers by the click
chemistry on a solid support. However, the 2-cyanoethyl
protections must be removed from the phosphates prior to the
click conjugation, since the 4′-modifications used here under
the click reaction conditions cause cleavage of the 5′-linked
phosphate triester bond. Melting temperature studies reveal that
oligonucleotides containing one to four mannose ligands in the
central part of the chain (i.e., one or two modified nucleoside
residue) form equally stable duplexes with complementary 2′-
OMe RNA as the corresponding unmodified DNA sequence.
At high salt content, the mannose conjugation is even stabilizing.
The results with the space-demanding aminoglycoside ligands
further demonstrate the utility of 4′-modified nucleosides 1 and
2 in glycoconjugations. All amino group-bearing conjugates
stabilized the DNA ·DNA duplex significantly and the DNA ·2′-
O-methyl RNA duplex notably.
ACKNOWLEDGMENT
Financial support from the Academy of Finland is gratefully
acknowledged.
Supporting Information Available: NMR spectral data for
1, 2, 4-6, 12, and 13. ESI-MS and RP HPLC data for
oligonucleotides 26-39. In addition, examples of melting
temperature curves for 26-39 are presented. This material is
available free of charge via the Internet at http://pubs.acs.org.
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