Solid-phase synthesis of oligomers carrying several chromophore units linked by phosphodiester backbones

Article abstract of DOI:10.1016/j.bmcl.2008.02.074

A method for the preparation of oligomers by linking chromophore units is described. Specifically, the synthesis of chromophore units having a protected-hydroxyl group and a phosphoramidite function is described, along with a method to link several units

Full text of DOI:10.1016/j.bmcl.2008.02.074

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2306–2310
Solid-phase synthesis of oligomers carrying several
chromophore units linked by phosphodiester backbones
a,b,e
´
Anna Avino,
Isabel Navarro,d,e Josep Farrera-Sinfreu,c Miriam Royo,b,c
˜
d,g
Juan Aymamı, Antonio Delgado,f,h Amadeu Llebaria,f
´
Fernando Albericioa,b,i and Ramon Eritjaa,b,e,
*
aInstitute for Research in Biomedicine (IRB Barcelona), Josep Samitier 1-5, E-08028 Barcelona, Spain
bNetworking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Josep Samitier 1-5, E-08028 Barcelona, Spain
cCombinatorial Chemistry Unit, Barcelona Scientific Park (PCB), Josep Samitier 1-5, E-08028 Barcelona, Spain
dCrystax Pharmaceuticals S.L., Josep Samitier 1-5, E-08028 Barcelona, Spain
eInstitute for Advanced Chemistry of Catalonia (IQAC), Spanish Research Council (CSIC), Jordi Girona 18-26,
E-08034 Barcelona, Spain
fResearch Unit on BioActive Molecules (RUBAM), Spanish Research Council (CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain
g
`
Dpt. d’Enginyeria Qu´ımica, Universitat Politecnica de Catalunya (UPC), Diagonal 647, E-08028 Barcelona, Spain
h
`
Unitat de Qu´ımica Farmaceutica (Unitat Associada al CSIC),
Facultat de Farmacia, Universitat de Barcelona (UB), Avgda Joan XXIII, s/n, E-08028 Barcelona, Spain
`
Department of Organic Chemistry, University of Barcelona (UB), Martı i Franque´s 1, E-08028 Barcelona, Spain
i
´
Received 30 January 2008; revised 20 February 2008; accepted 29 February 2008
Available online 5 March 2008
Abstract—A method for the preparation of oligomers by linking chromophore units is described. Specifically, the synthesis of chro-
mophore units having a protected-hydroxyl group and a phosphoramidite function is described, along with a method to link several
units using solid-phase phosphite-triester protocols.
Ó 2008 Elsevier Ltd. All rights reserved.
DNA-intercalating drugs are planar molecules formed
by several fused aromatic rings that form stacks between
DNA base pairs, thus reducing the opening and unwind-
ing of the double helix. Each intercalating drug binds
strongly to particular base pairs due to several interac-
tions, ranging from van der Waals interactions to the
formation of hydrogen bonds with adjacent nucleo-
bases. Intercalating drugs have varying selectivity, which
may be improved by linking several intercalating units.
Various authors have described the synthesis of bis- or
tris-intercalating drugs with promising activity and
selectivity.1,2 However, there is no general methods for
the rapid synthesis of a small library of oligomers carry-
ing DNA-intercalating compounds.
The linkage of one single DNA-intercalating drug to
peptides and oligonucleotides has been reported.3,4 Oli-
gonucleotides carrying one DNA-intercalating drug
have better hybridization properties, nuclease resistance
and cellular uptake. Intercalating agents have also been
introduced in peptide nucleic acids (PNA)5 and pep-
tides.6 Recently Kool et al. described the preparation
and fluorescent properties of oligomeric fluorophores
based on C-glycoside backbones.7,8
In these two papers we describe the synthesis of oligo-
meric compounds containing several chromophore units
using solid-phase protocols. This paper reports the syn-
thesis of oligomers with phosphodiester linkages and the
accompanying paper reports the synthesis of oligomers
linked by amide bonds.
We chose the L-threoninol and (R)-3-amino-1,2-propan-
diol backbones for the oligomers with phosphodiester
links for several reasons. They have the adequate length
to hold the chromophore units between DNA base
Keywords: Chromophore; Fluorescence; Oligonucleotides; Solid-phase
synthesis; Phosphite-triester; DNA-intercalating drugs.
*
Corresponding author. Tel.: +34 93 4039942; fax: +34 93 2045904;
e-mail: recgma@cid.csic.es
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.074

´
A. Avin˜o et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2306–2310
2307
O
pairs. Both compounds can be obtained in enantiomeri-
O
cally pure form from commercial sources and they have
two hydroxyl groups (one primary and the other second-
ary) and one amino group. The chromophore can be at-
tached at the amino group position. Then, the primary
hydroxyl group can be protected by the 4,40-dimethoxy-
trityl (DMT) group. Finally the secondary alcohol will
be used to prepare the phosphoramidite or the hemisuc-
cinate derivatives needed for the assembly of oligomers
by solid-phase methods (Scheme 1).
NH2
HN
R
HN
R
HO
OH
R-COOH, 1
HO
OH
DMTO
OH
DMT-Cl
3a-i
2a = Qut
2b = Act
2c = Qgt
2d = Agt
2e = Pht
2f = Obt
2g = Fmt
2h = Nct
2i = Git
O
HN
R
N(isopropyl)2
DMTO
O
P
O
CN
O
4a, b, e, f, g, i
The threoninol molecule has been used to introduce sev-
eral units of Methyl Red moieties in oligonucleotides9
and the 3-amino-1,2-propandiol backbone has been
used to introduce a phenanthridinium unit into oligonu-
cleotides.10 Recently aminopropyl nucleic acids with a
HN
R
O
DMTO
O
NH
CPG
O
5a-i
1c
1a
1b
3-amino-1,2-propanediol
described.11
backbone
have
been
N
N
N
N
H
N
H
Threoninol derivatives of general formula 2a–d can be
prepared according to the process summarized in
Scheme 2. As monomeric units we chose several hetero-
cyclic derivatives carrying a carboxylic acid function:
10H-indolo[3,2-d]quinoline (1a), acridine (1b), phenyl-
quinoline (1e), 3-oxo-3H-benzo[f]chromene (1f), and
fluorenylmethyl (1g), neocriptolepine (1h), and ben-
zo[de]-1,3-diketoisoquinoline (1i). In order to increase
the distance between the intercalating drug and the thr-
eoninol linker molecule, and to give more flexibility to
the units a glycine unit was added in compounds 1a
and 1b by reaction of the corresponding carboxyl
derivative with glycine methyl ester and subsequent
hydrolysis of the methyl ester intermediate to yield
derivatives 1c–d.
COOH
N
CONHCH2COOH
COOH
1f
1d
1e
O
O
N
COOH
CONHCH2COOH
COOH
1i
1h
1g
N
N
O
O
N
COOH
COOH
COOH
Scheme 2. Synthesis of threoninol derivatives.
i). The DMT derivatives (3a–i) were reacted with
chloro-N,N-diisopropylamino-O-2-cyanoethoxy phos-
phine to yield phosphoramidites 4a, b, e, f, g, and i or
loaded to controlled pore glass (CPG) solid supports
using the succinyl linkage (5a–i).
L-Threoninol was reacted with carboxyl derivatives (1a–
i) using diisopropylcarbodiimide and 1-hydroxybenzo-
triazole, yielding compounds 2a–i. The threoninol deriv-
atives (2a–i) were reacted with dimethoxytrityl (DMT)
chloride in pyridine to yield the DMT derivatives (3a–
The synthesis of the 3-amino-1,2-propandiol derivatives
is shown in Scheme 3. The R isomer of 3-amino-1,2-
propandiol was reacted with the chloro acridine deriva-
tive (6a) or the chloro indoloquinoline derivative (6b),
yielding the corresponding diol derivatives 7a–b. In
addition, anhydride 6c was reacted with 3-amino-1,2-
propandiol to yield diol 7c. The resulting diols (7a–c)
were reacted with DMT chloride in pyridine to yield
DMT derivatives 8a–c, followed by the reaction with
chloro-N,N-diisopropylamino-O-2-cyanoethoxy phos-
phine to yield phosphoramidites 9a and c.
NH2
NH
OH
NH
HO
OH
HO
R
N
R
In order to synthesize oligomers using solid-phase pro-
tocols, DMT derivatives of the chromophores described
above were coupled to CPG supports using the succinyl
linker as described elsewhere.12 For this purpose, the
DMT-derivatives described above were reacted with
succinic anhydride, followed by coupling of the resulting
hemisuccinates with amino functionalized CPG yielding
glass beads loaded with the appropriate chromophores.
O
P
DMTO
CN
O
NH
NH
O
OH
O
P
O
H
O
O-
n
The phosphoramidites described above were assembled
into strands of up to 6 units (Table 1) using the appro-
Scheme 1. General preparation of the monomers.

´
A. Avin˜o et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2306–2310
2308
and 3) and the last letter indicates the backbone (t for
threoninol and a for 3-amino-1,2-propandiol).
R
R
H2N
HO
6a-b
HN
HN
Standard phosphoramidite chemistry was used.13 This
consists of cycles of four chemical reactions: (1) removal
of the DMT group with 3% trichloroacetic acid in
dichloromethane; (2) phosphoramidite coupling using
5-fold excess of phosphoramidite and 20-fold excess of
tetrazole; (3) capping with acetic anhydride and N-meth-
ylimidazole; and (4) oxidation of phosphite to phos-
phate with 0.01 M iodine in tetrahydrofurane/pyridine/
water. Coupling yields were between 80% and 95%.
For the synthesis of the dimer containing a phosphoro-
thioate linkage (1-Act-ps-Cra-3) the oxidizing solution
was replaced by a solution of 3H-1,2-benzodithiol-3-
one 1,1-dioxide14 in acetonitrile.
HO
OH
DMTO
OH
OH
8a-b
6a
7a = Aca
7b = Cra
N
R
HN
Cl
N
N(isopropyl)2
DMTO
6b
O
P
9a
O
CN
N
Cl
O
H2N
HO
N
O
R-O
After the assembly of the sequences, supports were trea-
ted with conc. aqueous ammonia to yield the desired
unprotected oligomers. Good yields and purities were
obtained during the synthesis of oligomers with L-thr-
eoninol backbone as reported in Figure 1.
OH
OH
7c, R = H, Pia
8c, R = DMT
O
O
6c
O
O
Some oligomers decomposed in hot ammonia solutions,
for example, oligomers carrying the chromene ring (1f,
Obt) and those with the propan-1,2-diol backbone car-
rying acridine (7a, Aca) and benzo[d,e]-1,3-diketoiso-
quinoline (7c, Pia). Alternatively oligomers with the
propan-1,2-diol backbone were deprotected either with
(1) a 0.5 M 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
solution in pyridine, or (2) NH3 in methanol at room
temperature for 2–4 h. In both cases yields were very
low. Best results were obtained using an acid labile lin-
ker. In this strategy 4-trityloxybutanoic acid was reacted
with polystyrene loaded with Rink-amide linker.15 The
N
O
DMTO
N(isopropyl)2
O
P
O
CN
9c
Scheme 3. Synthesis of 3-amino-1,2-propandiol derivatives.
priate solid supports (1–4 lmol scale). We used a three-
letter code for each monomer unit. The first two letters
of the code define the heterocyclic moiety (see Schemes 2
Table 1. Oligomers prepared in this study
Compound
MS (expected)
MS (found)
HPLC, retention time (min)
1-Act-p-Qut-3a
721.7
760.7
1133.0
2171.8
693.6
709.6
708.7
734.7
708.7
747.7
1151.0
684
766.5 [M+2Na+]
761.3
1139.8
12.7
14.4
1-Qut-p-Qut-3a
1-Act-p-Qut-p-Qut-3a
1-Act-(p-Act)5-3a
1-Act-p-Cra-3a
15.3
11.6
2172.3
694.6
13.0
15.0, 15.5 (2 isomers)
1-Act-ps-Cra-3a
708.4
707.8
1-Pht-p-Act-3a
14.5
19.1
14.9
16.8
12.3
18.8
17.9
14.4
15.3
11.3
11.3
16.8
11.6
5.8
1-Pht-p-Pht-3a
733.7
707.6
746.6
1-Act-p-Pht-3a
1-Pht-p-Qut-3a
1-Act-p-Qut-p-Agt-3a
1-Fmt-p-Fmt-3a
1-Qut-p-Fmt-3a
1152.2
707 [M+Na+]
780.5 [M+Na++K+]
733
722
1-Qut-p-Cra-2a
732.7
1172.0
1154.9
1093.9
1146.0
769.5
763.6
881.6
911
1-Qut-p-Qut-p-Qut-3a
1-Act-p-Act-p-Act-3a
1-Act-p-Qut-p-Act-3a
1-Act-p-Qut-p-Nct-3a
1-Pia-p-Pia-p-bab
1-Aca-p-Aca-p-bab
1-Obt-p-Obt-p-bab
1-Git-p-Git-p-bab
1-Git-p-Obt-p-bab
1238 [M+3Na+]
1077.1 [M+Na+]
1093.9
1146
792.4
764.6
880.1
934.3 [M+Na+]
919 [M+Na+]
13.8
12.7
14.1
896
p, phosphate; ps, phosphorothioate; ba, (CH2)3CONH2.
a Deprotection using ammonia.
b Deprotection using trifluoroacetic acid.

´
A. Avin˜o et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2306–2310
2309
tion). Optimal excitation wavelengths ranged from
247 to 433 nm and the corresponding emission wave-
lengths ranged from 395 to 499 nm. The phenyl-
quinoline derivatives had the lowest emission
wavelengths.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
In summary, we report the synthesis of several fused
heterocyclic derivatives functionalized with threoninol
and propan-1,2-diol that are useful for the rapid syn-
thesis of oligomers of more than 6 units linked
through phosphodiester or phosphorothioate bonds.
The resulting oligomers are water soluble. The binding
properties of these molecules are under investigation.
Preliminary results show that the presence of the neg-
atively charged phosphodiester backbone does not
compromise the binding to DNA. Finally, the deriva-
tives described in this work can also be used to intro-
duce fluorescent and intercalating agents into synthetic
DNA and RNA.3,4
0
5
10
Tr(min)
15
20
Figure 1. HPLC chromatogram of hexamer 1-Act-(p-Act)5-3 after
ammonia deprotection.
resulting support was used for the assembly of the poly-
mers using phosphoramidite derivatives (4f, 9a, 9c).
After the assembly of the sequences, supports were trea-
ted with 0.5 M DBU solution, washed in acetonitrile
and treated with a solution containing 95% trifluoroace-
tic acid in water. Oligomers with the propan-1,2-diol
backbone (7a and 7c) and the chromene ring (1f) were
obtained in excellent yields under these conditions.
Acknowledgments
This study was supported by the OTRI (PTR1995-0976-
OP), Crystax Pharmaceuticals S.L., the Spanish Minis-
try of Education (BFU2004-02048, CTQ2005-00315,
BFU2007-63287), the Generalitat de Catalunya (2005/
SGR/00693).
Drugs that bind nucleic acids may show preference for
certain sequences. One of the methods to analyze the se-
quence specificity of DNA-binding drugs is the competi-
tion dialysis.16 In this method different nucleic acid
structures are dialyzed against a common ligand solu-
tion. Figure 2 shows the competitive dialysis experiment
for the monomer Act and the trimer Act-p-Act-p-Act
with oligonucleotide sequences representing alternated
or consecutive A-T or G-C duplex sequences. While
the monomer has little variation on the affinity for each
model duplex, the trimer has a clear affinity for contig-
uous A-T sequence.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.02.074.
References and notes
1. Arya, D. P.; Willis, B. J. Am. Chem. Soc. 2003, 125,
12398.
2. Fechter, E.; Olenyuk, B.; Dervan, P. B. Angew. Chem. Int.
Ed. 2004, 43, 3591.
The excitation and emission wavelengths of the new
compounds were measured (see Supporting informa-
´ `
3. Asseline, U.; Thuong, N. T.; Helene, C. New J. Chem.
1997, 212, 5.
4. Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res.
2002, 30, 4918.
5. Shiraishi, T.; Nielsen, P. E. Nucleic Acids Res. 2004, 32,
4893.
6. Carlson, C. B.; Beal, P. A. Bioorg. Med. Chem. Lett. 2000,
10, 1979.
7. Cuppoletti, A.; Cho, Y.; Park, J. S.; Stra¨ssler, C.; Kool, E.
T. Bioconjug. Chem. 2005, 16, 528.
8. Wilson, J. N.; Gao, J.; Kool, E. T. Tetrahedron 2007, 63,
3427.
AAAATTTT
ATATATAT
Act-p-Act-p-Act
Act
CCCCGGGG
CGCGCGCG
9. Asanuma, H.; Shirasuka, K.; Takarada, T.; Kash-
ida, ; Komiyama, M. J. Am. Chem. Soc. 2003,
125, 2217.
10. Huber, R.; Amann, N.; Wagenknecht, H. A. J. Org.
Chem. 2004, 69, 744.
0
1
2
3
4
5
6
11. Zhou, D.; Froeyen, M.; Rozenski, J.; Van Aerschot, A.;
Herdewijn, P. Chem. Biodivers 2007, 4, 740.
12. Pon, R. T. In Methods in Molecular Biology Vol. 20:
Protocols for Oligonucleotides and Analogs; Agrawal, S.,
relative fluorescence (arbritary units)
Figure 2. Competitive dialysis of monomer Act and trimer 1-Act-p-
Act-p-Act-3 (see experimental conditions in Supplementary material).

´
A. Avin˜o et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2306–2310
2310
Ed.; Humana Press Inc.: Totowa, NJ, USA, 1993; pp 465–
496.
14. Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan, J.; Beacauge,
S. J. Org. Chem. 1990, 55, 4693.
13. Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds,
D. R.; Fischer, E. F.; McBride, L. J.; Matteucci, M.;
Stabinsky, Z.; Tang, J. Y. Methods Enzymol. 1987, 154, 287.
15. Rink, H. Tetrahedron Lett. 1987, 28, 3787.
16. Ren, J.; Chaires, J. B. Biochemistry 1999, 38,
16067.