(R)-2,4-Dihydroxybutyramide seco-Pseudonucleosides: New Versatile Homochiral Synthons for Synthesis of Modified Oligonucleotides

Article abstract of DOI:10.1021/ol0269289

(Matrix Presented) Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-α-hydroxy-γ-butyrolactone and (R)-(-)-pantolactone by aminolysis, side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an oligonucleotide chain during solid-phase assembly.

Full text of DOI:10.1021/ol0269289

ORGANIC
LETTERS
2002
Vol. 4, No. 26
4607-4610
(R)-2,4-Dihydroxybutyramide
seco-Pseudonucleosides: New Versatile
Homochiral Synthons for Synthesis of
Modified Oligonucleotides
,†
Natalia N. Dioubankova,†,‡ Andrey D. Malakhov,† Dmitry A. Stetsenko,*
Vladimir A. Korshun,‡ and Michael J. Gait†
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK, and
Shemyakin-OVchinnikoV Institute of Bioorganic Chemistry, Russian Academy of
Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia
ds@mrc-lmb.cam.ac.uk
Received September 18, 2002
ABSTRACT
Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-r-hydroxy-γ-butyrolactone and (R)-(−)-pantolactone by aminolysis,
side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were
used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain
reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an
oligonucleotide chain during solid-phase assembly.
Synthetic nonisotopically labeled oligonucleotide probes have
found widespread application in molecular biology and
various nucleic acids-based assays in medicine.1 The most
expeditious way to obtain these materials is machine-assisted
solid-phase synthesis. Despite significant progress made over
the last two decades in the area of oligonucleotide techno-
logy,2 there is still a need to develop new modified reagents
for oligonucleotide labeling during solid-phase assembly,
which may allow introduction of various reporter groups into
any predetermined position of a nucleic acid chain.
groups and nonradioactive labels into an oligonucleotide
chain during solid-phase synthesis.4-11 Frequently, the
precursors were shaped to be spatial surrogates of nucleo-
(3) Bannwarth, W.; Dorn, A.; Iaiza, P.; Pannekouke, X. HelV. Chim. Acta
1994, 77, 182.
(4) (a) Nelson, P. S.; Kent, M.; Muthini, S. Nucleic Acids Res. 1992,
23, 6253. (b) Nelson, P. S.; Kent, M. A. US Patents 5,451,463, 1995, and
5,942,610, 1999.
(5) (a) Daniher, A. T.; Bashkin, J. K. Chem. Commun. 1998, 1077. (b)
Putnam, W. C.; Bashkin, J. K. Chem. Commun. 2000, 767.
(6) (a) De Vos, M. J.; Elsen, A. Van; Bollen, A. Nucleosides Nucleotides
1994, 13, 2245. (b) Neunier, P. Bioorg. Med. Chem. Lett. 1996, 6, 147.
(7) (a) Fino, J. R.; Mattingly, P. G.; Ray, K. A. Bioconjug. Chem. 1996,
7, 274. (b) Korshun, V. A.; Balakin, K. V.; Proskurina, T. S.; Mikhalev, I.
I.; Malakhov, A. D.; Berlin, Y. A. Nucleosides Nucleotides 1999, 18, 2661.
(8) (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990, 112,
453. (b) Yamana, K.; Takei, M.; Nakano, H. Tetrahedron Lett. 1997, 38,
6051.
A number of nonnucleoside reagents have been suggested
to allow site-specific incorporation of various functional
† MRC Laboratory of Molecular Biology.
‡ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry.
(1) (a) Goodchild, J. Bioconjug. Chem. 1990, 1, 165. (b) Beaucage, S.
L.; Iyer, R. P. Tetrahedron 1993, 49, 6123.
(9) (a) Lin, K.-Y.; Matteucci, M. Nucleic Acids Res. 1991, 19, 3111. (b)
Korshun, V. A.; Pestov, N. B.; Nozhevnikova, E. V.; Prokhorenko, I. A.;
Gontarev, S. V.; Berlin, Y. A. Synth. Commun. 1996, 26, 2531.
(2) Applied Antisense Technology; Stein, C. A., Krieg, A. M., Eds.;
Wiley-Liss: New York, 1998.
10.1021/ol0269289 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/27/2002

sides, e.g. maintain a three-carbon atom spacing between the
phosphate groups3 essential in the case of internal incorpora-
tion to minimize duplex destabilization. Among the reagents
containing a 1,3-propanediol backbone are racemic deriva-
tives of 2-(4-aminobutyl)-1,3-propanediol,4 serinol (2-amino-
1,3-propanediol),5 4-amino-1,3-butanediol,6 and some 1-sub-
stituted 1,3-propanediols,7 as well as O2-alkylated glycerols.8
However, most of the compounds, while simple and readily
obtainable, suffer from several disadvantages. First, â-cy-
anoethyl phosphoramidites derived from primary alcohols
are generally less stable than those from secondary alcohols.
Second, use of racemic reagents leads to a mixture of
diastereomeric oligonucleotides, which may complicate their
isolation and purification. Few attempts to obviate the second
problem by using achiral synthons based on N-substituted
diethanolamine9 gave even less close nucleoside mimics.
Most successful approaches so far made use of natural
materials, trans-4-hydroxy-L-prolinol10 and L-threoninol.11
Several other homochiral skeletons were suggested recently,
including 2-hydroxymethyl-3-hydroxypyrrolidine12 and aba-
sic 2′-deoxynucleoside,13 but these compounds proved to be
much more expensive to synthesize.
group, if present, was masked by reaction with 9-fluorenyl-
methyl succinimidyl carbonate (3a,b) or methyl trifluoro-
acetate (3e). The primary hydroxy group of intermediates
4a-e was selectively 4,4′-dimethoxytritylated,14 and the
resulting alcohols 5a-e were purified by column chroma-
tography and converted into phosphoramidites15 (6a-c) and
solid-supported reagents16 (7a-e), respectively, by known
procedures. A reporter group (biotin or fluorescein) may be
introduced at this stage by transient N-protecting group
cleavage by aqueous Na2CO3 (5e) or piperidine (5a) treat-
ment and subsequent reaction with O,O′-diisobutyryl-5(6)-
carboxyfluorescein pentafluorophenyl ester17 or D-(+)-biotin
pentafluorophenyl ester10b to afford labeled precursors 5f and
5g, correspondingly. After column purification, these com-
pounds were used to produce two additional solid supports,
7f,g. The loading range of these polymeric reagents was
found to be 16-50 µmol/g. Attachment yields in the case
of pantolactone-derived supports 7b,d were lower, and the
reaction took longer to proceed, which indicates a steric
hindrance introduced by two extra methyl groups. All the
phosphoramidites are white solids that can be stored at -20
°C for at least several months without any loss of reactivity.
We describe here a novel family of reagents for nucleic
acid labeling and modification based on (R)-2,4-dihydroxy-
butyramide seco-pseudonucleosides, which are easily obtain-
able, chirally pure, and suitable for incorporation on both
3′- and 5′-ends, internal and multiple substitutions within
an oligonucleotide chain. Advantages of our approach
compared to, e.g., threoninol functionalization,11 are that it
offers a wider range of a functional group incorporation and,
furthermore, is stereochemically simpler, since only one
chiral center is involved.
Scheme 1. Synthesis of (R)-2,4-Dihydroxybutyramide
seco-Pseudonucleoside Reagents
We decided to use readily available homochiral precursors,
(R)-(+)-R-hydroxy-γ-butyrolactone (1, R ) H) and (R)-
(-)-pantolactone (2, R ) Me) as starting materials since they
possess the required three-carbon chain in a configuration
analogous to natural ribose, with a chiral carbon atom with
a secondary hydroxy group representing 3′-OH and a
carbonyl group replacing the ribose C-2′. In addition,
introduction of geminal methyl groups into acyclic backbone
in pantolactone series offers restraint of its flexibility and
adds favorably to the solubility of these derivatives.
Lactones 1 and 2 were treated with an excess (1.1-5.0
equiv) of one of the primary amines (ethylenediamine,
1-pyrenemethylamine, or 4,7,10-trioxa-1,13-tridecanedi-
amine) at 55 °C for 24-48 h. Then the side-chain amino
(10) (a) Reed, M. W.; Adams, A. D.; Nelson, J. S.; Meyer, R. B.
Bioconjug. Chem. 1991, 2, 217. (b) He´bert, N.; Davis, P. W.; DeBaets, E.
L.; Acevedo, O. L. Tetrahedron Lett. 1994, 35, 9509. (c) Reed, M. W.;
Lukhtanov, E. A.; Gorn, V. V.; Lucas, D. D.; Zhou, J. H.; Pai, S. B.; Cheng,
Y.; Meyer, R. B., Jr. J. Med. Chem. 1995, 38, 4587. (d) Prokhorenko, I.
A.; Korshun, V. A.; Petrov, A. V.; Gontarev, S. V.; Berlin, Y. A. Bioorg.
Med. Chem. Lett. 1995, 5, 2081.
(11) (a) Reynolds, M. A.; Beck, T. A.; Hogrefe, R. I.; McCaffrey, A.;
Arnold, L. J., Jr.; Vaghefi, M. M. Bioconjugate Chem. 1992, 3, 366. (b)
Fukui, K.; Iwane, K.; Shimidzu, T.; Tanaka, K. Tetrahedron Lett. 1996,
37, 4983. (c) Asanuma, H.; Tarakada, T.; Yoshida, T.; Tamaru, D.; Liang,
X.; Komiyama, M. Angew. Chem., Int. Ed. 2001, 40, 2671.
(12) Ceulemans, G.; Van Aerschot, A.; Rozenski, J.; Herdewijn, P.
Tetrahedron 1997, 53, 14957.
(R)-2,4-Dihydroxybutyramide reagents 6a-c and 7a-g
were evaluated in machine-assisted oligodeoxyribonucleotide
synthesis by standard 2-cyanoethyl phosphoramidite chem-
(14) Smith, M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H. G. J.
Am. Chem. Soc. 1962, 84, 430.
(15) (a) Barone, A. D.; Tang, J.-Y.; Caruthers, M. H. Nucleic Acids Res.
1984, 12, 4051. (b) Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nuclic
Acids Res. 1984, 12, 4539.
(16) Damha, M. J.; Giannaris, P. A.; Zabarylo, S. V. Nucleic Acids Res.
1990, 18, 3813.
(13) Smith, T. H.; Kent, M. A.; Muthini, S.; Boone, S. J.; Nelson, P. S.
Nucleosides Nucleotides 1996, 15, 1581.
(17) Haralambidis, J.; Angus, K.; Pownall, S.; Duncan, L.; Chai, M.;
Tregear, G. W. Nucleic Acids Res. 1990, 18, 501.
4608
Org. Lett., Vol. 4, No. 26, 2002

Table 1. Side-Chain Substituents of
(R)-2,4-Dihydroxybutyramide seco-Pseudonucleoside Reagents
(Scheme 1)
no.
R
R1 - R3
a
b
c
d
e
H
-CH2CH2NHX, R1 (X ) H), R2 ) R3 (X ) Fmoc)
Me -CH2CH2NHX, R1 (X ) H), R2 ) R3 (X ) Fmoc)
H
R1 ) R2 ) R3 ) -CH2(1-pyrenyl)
Me R1 ) R2 ) R3 ) -CH2(1-pyrenyl)
H
-(CH2)2(CH2OCH2)3(CH2)2NHX, R1 (X ) H),
R2 ) R3 (X ) trifluoroacetyl)
R3 ) -(CH2)2(CH2OCH2)3(CH2)2NH(biotin)
R3 ) -(CH2)2NHCO-5(6)-(fluorescein diisobutyrate)
f
g
H
H
istry.18 Use of modified seco-pseudonucleoside phosphora-
midites 6a-c allows the introduction of substituted 2,4-
dihydroxybutyramide unit(s) most preferably on the 5′- or
3′-end of an oligonucleotide chain, while multiple incorpora-
tion and/or introduction at the internal position(s) are also
possible. The 3′ case requires a special solid-phase linker,
e.g., a support generating a 3′-phosphate group19 or one of
7a-g to ensure subsequent liberation of an oligonucleotide
during ammonia deprotection. The polymeric reagents 7a-g
can also be used independently to append the modification
to the 3′-terminus. The average coupling efficiency of the
modified phosphoramidites 6a-c at a 0.15 M concentration
in dry MeCN and 10-15 min reaction time was found to be
greater than 97%. Several model oligonucleotides were thus
assembled, cleaved from their respective solid supports,
deprotected by concentrated aqueous ammonia treatment at
55 °C overnight, and analyzed by reverse-phase HPLC and
MALDI-TOF MS.20 Examples of typical RP-HPLC traces
are shown in Figure 1. Sequences and properties of the
oligodeoxyribonucleotides incorporating various (R)-2,4-
dihydroxybutyramide modifications are summarized in Table
Figure 1. Typical RP-HPLC traces of crude (R)-2,4-dihydroxy-
butyramide seco-pseudonucleoside-containing oligodeoxyribonucle-
otides: (a) 2-aminoethyl V and (b) 1-pyrenemethyl XIV (Table
2). For HPLC conditions, see Supporting Information.
2. Yields obtained were good, and all the molecular masses
correspond to the expected values within the accuracy of
the measurement.
Next, we studied the influence of substituted (R)-2,4-
dihydroxybutyramide modification(s) on thermal stability of
the duplexes formed by modified oligodeoxyribonucleotides
with complementary DNA (Table 3). It is evident that many
of the modifications analyzed, whether 3′- or 5′-terminal,
caused very little disturbance of the corresponding duplex
(∆Tm from -0.4 to +0.1 °C), while pyrene seco-pseudo-
nucleoside was in fact quite stabilizing in terminal positions
(from +2.4 to +4.0°), which is in good agreement with
Table 2. Properties of Oligodeoxyribonucleotides Bearing (R)-2,4-Dihydroxybutyramide seco-Pseudonucleoside Units
no.a
sequence, 5′ to 3′, (modification)b
R
MALDI-TOF, calcd/foundc
RP-HPLC retention time, minc
purity, %c,d
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
CTCCCAGGCTCAAAT(6a )pe
CTCCCAGGCTCAAAT(7a )
CTCCCAGGCTCAAAT(6b)pe
(6b)CTCCCAGGCTCAAAT
CTCCCAGGCTCAAAT(6b)(6b)pe
CTCCCAGGCTCAAAT(7b)
CTCCCAGGCTCAAAT(6c)pe
(6c)CTCCCAGGCTCAAAT
CTCCCAGGC(6c)CAAAT
C(6c)CCCAGGCTCAAAT
C(6c)CCCAGGC(6c)CAAAT
CTCCCAGGCTCAAAT(7c)
ATTTGAGCCTGGGAG(7d )
CTCCCAGGCTCAAAT(7e)
CTCCCAGGCTCAAAT(7f)
CTCCCAGGCTCAAAT(7g)
H
H
4801.1/4796.8
4718.8/4718.7
4826.8/4824.8
4749.2/4750.7
5078.9/5077.1
4749.2/4747.9
4969.8/4970.1
4892.3/4892.2
4588.1/4586.7
4588.1/4589.5
4677.0/4676.7
4892.3/4890.5
5071.4/5070.4
4881.3/4884.2
5097.6/5105.1
5076.9/5076.8, 5079.2
15.2
12.8
12.7
12.5
13.2
12.5
22.2
21.0
19.4
20.3
23.6
24.9
25.3
13.2
17.1
13.9, 14.9
89.0
82.1
84.5
83.9
82.3
81.4
81.3
86.1
85.1
84.6
79.7
89.1
84.4
83.7
81.8
24.8, 57.7
Me
Me
Me
Me
H
H
H
H
H
H
Me
H
H
H
a Roman numerals indicate the number of an oligonucleotides, starting with an unmodified 15-mer (II) and its complementary template (I) (Table 3).
b Numbers in brackets indicate modification from a phosphoramidite (6) or a solid support (7) (Table 1). c Values separated by a comma refer to 5(6)-
isomers of carboxyfluorescein. d Integrated from HPLC traces. e 3′-Phosphate.
Org. Lett., Vol. 4, No. 26, 2002
4609

effect is not additive, since substitution at both positions led
to only -3.4° per modified residue, a marginal further
decrease from a single pyrene seco-pseudonucleoside in the
middle. Switching from a butyrolactone to a pantolactone
unit in terminal positions seems to have little effect on Tm
either. It should also be noted that the presence of a
3′-phosphate group led to a decrease in Tm by ca. 1.6°.
Hybridization of pyrene-modified oligodeoxyribonucleotides
XI-XIV to the complementary RNA follows the same trend
both in 3′-terminal and internal positions, with slightly higher
and more additive destabilization in the latter case (data not
shown).
In conclusion, we describe here a new family of oligo-
nucleotide modification reagents comprising (R)-2,4-dihy-
droxybutyramide seco-pseudonucleosides, which are homo-
chiral and readily accessible from (R)-(+)-R-hydroxy-γ-
butyrolactone and (R)-(-)-pantolactone. These may be either
phosphoramidites for 3′-, internal, and 5′-terminal modifica-
tion, or solid-supported linkers for 3′-modification of oligo-
nucleotides during solid-phase synthesis. By choice of side-
chain functional groups, one can incorporate various functional
(short- or long-chain amine) or reporter groups (pyrene,
biotin, fluorescein) into an oligonucleotide chain. In addition,
the 3′- and/or 5′-terminal modifications do not decrease the
ability of a modified oligonucleotide to form a duplex with
a complementary DNA sequence.
Table 3. DNA Duplex Stability of Various
(R)-2,4-Dihydroxybutyramide seco-Pseudonucleoside-Modified
Oligodeoxyribonucleotides
no.a
modificationb
R
position Tm, °C ∆Tm/modc, °C
II-I
IV-I
V-I
VI-I
IX-I
XI-I
XII-I
XIII-I
XIV-I
XVI-I
XVII-I
none
7a
57.6
H
3′
3′
5′
3′
inte
inte
inte
3′
3′
3′
57.3
56.2
57.7
60.0
51.4
55.0
50.7
61.6
57.7
57.2
-0.3
-1.5
+0.1
+2.4
-6.3
-2.7
-3.4
+4.0
+0.1
-0.4
6bd
Me
Me
H
H
H
H
H
H
H
6b
6cd
6c
6c
6c
7c
7e
7f
a Roman numerals indicate the number of an oligonucleotide (II-IX)
in a duplex with a complementary DNA template GAGGGTCCGAGTTTA
(I). b Coming from phosphoramidite 6 or solid support 7 (Table 1). c ∆Tm
per one modification. d 3′-Phosphate. e Internal position (Table 2).
previous findings.21 Whereas pyrene is relatively well toler-
ated when substituting one of the internal thymidines close
to the 5′-end (-2.7°), it is noticeably more destabilizing
(-6.3°) when placed in the midst of the sequence in place
of another thymidine, which is in line with our previous
results on pyrene 2′-carbamate modification.22 However, this
Acknowledgment. We thank Prof. Vladimir A. Efimov
for his encouraging interest and Donna Williams for advice
on oligonucleotide synthesis. N.N.D. and V.A.K. acknow-
ledge the support of RFBR Grants 00-15-97947, 00-03-
32701, and 02-03-32376.
(18) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J.-Y.
Methods Enzymol. 1987, 154, 287.
(19) Koizumi, M.; Koga, R.; Hotoda, H.; Momota, K.; Ohmine, T.;
Furukawa, H.; Agatsuma, T.; Nishigaki, T.; Abe, K.; Kosaka, T.; Tsutsumi,
S.; Sone, J.; Kaneko, M.; Kimura, S.; Shimada, K. Bioorg. Med. Chem.
1997, 5, 2235.
(20) Brown, T.; Brown, D. J. S. In Oligonucleotides and Analogues: A
Practical Approach; Eckstein, F., Ed.; Oxford University Press: Oxford,
1991, p 1.
(21) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.;
Paris, P. L.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 1996, 118,
8182.
Supporting Information Available: Experimental de-
tails, compound characterization, HPLC traces, MALDI-TOF
spectra, and melting curves for some of the oligonucleotides.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(22) Korshun, V. A.; Stetsenko, D. A.; Gait, M. J. J. Chem. Soc., Perkin
Trans. 1 2002, 1092.
OL0269289
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Org. Lett., Vol. 4, No. 26, 2002