Base-Pairing Systems Related to TNA: α-Threofuranosyl Oligonucleotides Containing Phosphoramidate Linkages

Article abstract of DOI:10.1021/ol020015x

(Matrix Presented) (3′NH)- and (2′NH)-TNA, two isomeric phosphoramidate analogues of TNA (α-threofuranosyl-(3′→2′) oligonucleotides), are shown to be efficient Watson-Crick base-pairing systems and to undergo intersystem cross-pairing with TNA, RNA, and D

Full text of DOI:10.1021/ol020015x

ORGANIC
LETTERS
2002
Vol. 4, No. 8
1279-1282
Base-Pairing Systems Related to TNA:
r-Threofuranosyl Oligonucleotides
Containing Phosphoramidate Linkages1
Xiaolin Wu,† Sreenivasulu Guntha,† Mathias Ferencic,†,‡
,†,‡,
Ramanarayanan Krishnamurthy,*,†,§ and Albert Eschenmoser*
The Skaggs Institute for Chemical Biology at the Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and Laboratory of
Organic Chemistry, Swiss Federal Institute of Technology, Hoenggerberg, HCI-H309,
CH-8093 Zu¨rich, Switzerland
eschenmoser@org.chem.ethz.ch
Received January 16, 2002
ABSTRACT
(3′NH)- and (2′NH)-TNA, two isomeric phosphoramidate analogues of TNA (r-threofuranosyl-(3′f2′) oligonucleotides), are shown to be efficient
Watson−Crick base-pairing systems and to undergo intersystem cross-pairing with TNA, RNA, and DNA.
In systematic studies toward a chemical etiology of nucleic
acid structure, we discovered that R-threofuranosyl-(3′f2′)
oligonucleotides (TNA) represent an efficient Watson-Crick
base-pairing system, able to undergo informational intersys-
tem cross-pairing with both RNA and DNA.2 This finding
deserves our special interest since, among the potentially
natural nucleic acid alternatives3 investigated so far, TNA
is structurally the simplest. More specifically, in view of its
four-carbon-only carbohydrate building block, TNA may be
considered to represent a simpler type of oligomer structure
than RNA.2,4 Since, in an etiological context, structural
complexity of a molecule is to be assessed by criteria that
define a structure’s generational complexity, we previously
argued that any experimental analysis of this aspect of the
TNA structure should not be confined to TNA itself but
include the backbones of a whole family of hypothetical
oligomer structures that could derive from (C2 + C2 f C4)
assembly processes involving nitrogenous starting materials
and intermediates.2 From such a point of view, the TNA
backbone might represent just the oxygenous phosphodiester-
type representative in a library of related nitrogenous
oligomer systems, some of which might have the ability to
communicate with RNA by intersystem cross-pairing. It is
from this perspective that we synthesized two new members
of the family, containing in place of the phosphodiester
groups the isomeric phosphoramidate linkages depicted in
the formulas given in the abstract. Both systems were found
to indeed show base-pairing properties that are quite similar
to those of TNA.
† Scripps Research Institute.
‡ Swiss Federal Institute of Technology.
§ Fax: ++1-858-784-9573. E-mail: rkrishna@scripps.edu.
Fax: ++41-1-632-1043.
Modes of formation, synthesis, and properties of phos-
phoramidate analogues of oligonucleotides in the DNA and
RNA series have been widely studied with regard to their
potential as antisense reagents5-7 and have also been the
subject of experiments8,9 and considerations8,10 in an etiologi-
cal context.
(1) Chemistry of R-Aminonitriles. 35. Part 34: Wagner, T.; et al. HelV.
Chim. Acta 2002, 85, 399.
(2) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy,
R.; Eschenmoser, A. Science 2000, 290, 1347.
(3) Eschenmoser, A. Science 1999, 284, 2118.
(4) Orgel, L. E. Science 2000, 290, 1306. Herdewijn, P. Angew. Chem.,
Int. Ed. 2001, 40, 2249.
10.1021/ol020015x CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/16/2002

The nucleoside derivatives required for our study, namely,
the R-L-3′-deoxy-3′-N-(4′′-methoxytrityl)amino threofura-
nosyl nucleosides 8 and 9 and the isomeric R-L-2′-deoxy-
2′-N-(4′′-methoxy trityl)amino threofuranosyl nucleosides 16
and 22 were prepared according to Schemes 1, 2, and 3.11
The synthesis of the nucleosides 8 and 9 starts with 1,2,3-
tri-O-acetyl erythrose 112 (Scheme 1) and proceeds by
mesyloxy nucleosides 4 and 5, and substitution of the
mesyloxy group by azide ions leads from the erythro into
the threo series through inversion of configuration. The latter
is evidenced by an X-ray analysis of the azido alcohol 6 in
the thymine series.14 Catalytic hydrogenation of the azide
group, followed by protection of the amino group by mono-
4-methoxytrityl chloride (MMT),15 affords the target nucleo-
side 8 and 9.
Scheme 1a
Scheme 2a
a Numbers in parentheses denote molar equivalents referring to
starting compound or ratios. Numbers before these parentheses
denote concentrations in M. (a) (i) 0.3 M (1.1 molar equiv) ABz or
T, 0.6 (2.5) BSA, CH3CN, 60˚C, 1 h, then 0.8 (3.0) SnCl4 60 ˚C,
20 min; (ii) for B ) ABz, 1 M NaOH in THF/MeOH/H2O 5:4:1, 4
˚C, 10 min, 68%; for B ) T, 2 M NH3 (MeOH) in THF, rt 14 h,
65%; (b) (i) 0.3-0.9 (2.0) DMT-Cl, Py, rt, 14 h, then 0.2-0.6 (1.3)
Ms-Cl, 4 °C f rt, 16 h; (ii) 0.4 (11.7) TFA, CH2Cl2, MeOH (3:
96:1), rt, 30 min., 49% for ABz, 74% for T; (c) 2.1 (7.0) NaN3,
DMF/H2O (4:1), 100 ˚C, 4 h, 82% for ABz, 78% for T; (d) (i) H2,
10% Pd/C, MeOH, rt, 1 h; (ii) 0.3 (1.5) MMT-Cl, Py, rt, 2 h, 92%
for ABz, 96% for T; (e) 0.3 (1.2) chloro(2-cyanoethoxy)(diisopro-
pylethylamino)phosphine, 0.7 (2.5) NEt(i-Pr)2, CH2Cl2, rt ,1 to 3h,
87% for ABz, 90% for T.
a (a) 2.1 M (8.0 molar equiv) NaN3, 0.3 (1.2) BzOH, HMPA,
150 °C, 2 H, 82%; (b) 10% Pd-C, H2, MeOH, rt, 2 h, 97%; (c)
1.4 (1.2) MMT-Cl, Py, rt, 1.5 h, 91%; (d) 0.3 (1.5) chloro(2-
cyanoethoxy)(diisopropylethylamino)phosphine, 0.5 (4.0) NEt(i-
Pr)2, CH2Cl2, rt, 92%.
The synthesis of the isomeric nucleosides 16 (Scheme 2)
and 22 (Scheme 3) starts from the thymine-derived anhydro-
nucleoside 13, the preparation of which from R-L-threofura-
nosyl thymine 12 was reported earlier.2 Proton-catalyzed ring
opening in 13 by reaction with sodium azide16 at elevated
temperature, catalytic reduction of the azide group of 14,
and protection of the amino group of 15 to give the thymine
nucleoside 16 are all reactions that proceeded in high yield.
The corresponding adenine-containing nucleoside in this
series (Scheme 3) is obtained by the (TfA)-transnucleosi-
dation17 18 f 19. The reaction is performed under modified
Vorbru¨ggen nucleosidation conditions13 and proceeds in a
satisfactory yield of 74%, provided that the 2′-amino group
is protected with the trifluoroacetyl group.17 Base-catalyzed
nucleosidation under Vorbru¨ggen-Hilbert-Johnson condi-
tions13 followed by selective base-catalyzed hydrolysis of
the O-acetyl groups to 2 and 3. Monotritylation with 4,4′-
dimethoxytrityl chloride (DMT-Cl) in pyridine in both series
occurs selectively at the 2′-hydroxyl (for B ) thymine, 84%
2′-O-DMT, 3% 3′-O-DMT, 6% 2′,3′-bis-O-DMT; for B )
N6-benzoyladenine, 43% 2′-O-DMT, 33% of a mixture of
2′- and 3′-O-DMT and 6% of 2′,3′-bis-O-DMT; percentages
refer to isolated material). Mesylation of the free 3′-hydroxyls
followed by acid-catalyzed detritylation affords the 3′-
(11) All compounds were fully characterized by H and 13C NMR and
1
(5) Letsinger, R. L.; Mungall, W. S. J. Org. Chem. 1970, 35, 3800.
Gryaznov, S. M.; Letsinger, R. L. Nucleic Acids Res. 1992, 20, 3403.
(6) Gryaznov, S.; Chen, J. K. J. Am. Chem. Soc. 1994, 116, 3143. Chen,
J. K.; Schultz, R. G.; Lloyd, D. H.; Gryaznov, S. M. Nucleic Acids Res.
1995, 23, 2661.
(7) Matray, T. J.; Gryaznov, S. M. Nucleic Acids Res. 1999, 27, 3976.
(8) Lohrmann, R.; Orgel. L. E. Nature 1976, 261, 342. Lohrmann, R.;
Orgel. L. E. J Mol. EVol. 1976, 7, 253. Lohrmann, R.; Orgel. L. E. J Mol.
EVol. 1977, 9, 323. Zielinski, W. S.; Orgel, L. E. Nature 1987, 327, 3467.
Zielinski, W. S.; Orgel, L. E. Nucleic Acids Res. 1987, 15, 1699.
(9) Kiedrowsky, G. v.; Wlotzka, B.; Helbing, J.; Matzen, M.; Gordon,
S. Angew. Chem., Int. Ed. Engl. 1991, 30, 423. Sievers, D.; Kiedrowsky,
G. v. Chem. Eur. J. 1998, 4, 629.
mass spectral data after purification by column chromatography on silica
gel. A full experimental account will be published in HelV. Chim. Acta.
(12) Kline, P. C., Serianni, S. J. Org. Chem. 1992, 57, 1772.
(13) Vorbru¨ggen, H.; Bennua, B. Chem. Ber. 1981, 114, 1279. Bo¨hringer,
M.; Roth, H.-J.; Hu¨nziker, J.; Go¨bel, M.; Krishnan, R.; Giger, A.; Schweizer,
B.; Schreiber, J.; Leumann, C.; Eschenmoser, A. HelV. Chim. Acta 1992,
75, 1416.
(14) The X-ray analysis was carried by Raj K. Chadha, TSRI. Crystal-
lographic data for the structure have been deposited with the Cambridge
Crystallographic Data Center as deposition No. CCDC 172143. Copies of
the data can be obtained, free of charge, on application to the CCDC, 12
Union Road, Cambridge CB12 1EZ, UK (fax +44 (1233) 336 0333; e-mail
deposit@ccdc.cam.ac.uk).
(10) Gryaznov, S. M.; Lloyd, D. H.; Chen, J.-K.; Schultz, R. G.;
DeDionisio, L. A.; Ratmeyer, L.; Wilson, W. D. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 5798.
(15) Bannwarth, W. HelV. Chim. Acta 1988, 71, 1517.
(16) Verheyden, J. P. H.; Wagner, D.; Moffatt, J. G. J. Org. Chem. 1971,
36, 250.
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Org. Lett., Vol. 4, No. 8, 2002

minimal purity of 95% and shown to have the expected
molecular mass by MALDI-TOF MS.
Scheme 3a
An exploratory base-pairing study was carried out in the
(3′NH)-TNA series on the self-complementary sequence
(
3′NHA3′NHT)8, a sequence that was chosen because TNA
strands with alternating purine and pyrimidine bases had been
observed to form the most stable duplexes among isomeric
sequences.2 Remarkably, (3′NHA3′NHT)8 was found to form a
duplex that turned out to be both thermally and thermody-
namically more stable than the corresponding TNA duplex
(Figure 1).
a (a) (i) 2.4 M (5.0 molar equiv) TFAA, DMF, rt, 14 h; (ii) 3.3
(3.2) Ac2O, Py, rt, 3 h, 94%.; (b) 0.1 (3.0) ABz, 0.4 (9.0) BSA, 0.1
(3.0) SnCl4, CH3CN, 80 °C, 12 h, 74%; (c) 2 M NH3 in CH3OH,
rt, 3 days, quant.; (d) 0.1 (2.5) MMT-Cl, Py, rt, 1.5 h, 73%; (e) (i)
0.5 (2.5) BzCl, Py, rt, 2 h; (ii) 1 M NaOH, THF/MeOH/H2O 5:4:
1, 20 min, 0 ˚C, 90%; (f) 0.2 (1.5) chloro(2-cyanoethoxy)(diiso-
propylethylamino)phosphine, 0.5 (4.0) NEt(i-Pr)2, CH2Cl2, rt, 88%.
removal of all three protecting groups after the trans-
nucleosidation step and stepwise reprotection of the two
amino groups of 20 gave the desired adenine nucleoside 22
in excellent yields.
The conversion of the target nucleosides 8, 9, 16, and 22
to the corresponding phosphoroamidites 10, 11, 17, and 23s
the activated derivatives for solid support oligomer synthesiss
was carried out under standard conditions.18
The assembly on a DNA synthesizer19 of oligonucleotide
(3′NH)- and (2′NH)-TNA strands containing exclusively
phosphoramidate linkages was accomplished using a modi-
fied version of the phosphoroamidite method described by
Gryaznov.7 Nucleoside derivatives 8, 9, 16, and 22 were
attached to CPG solid support via a succinate linker.20 4,5-
Dicyanoimidzaole (pKa 5.2)21 was used as coupling activator
instead of the more common 5-ethylthio-1-H-tetrazole (pKa
3.8)22 in order to cope with the relative lability of the MMT
protecting group toward protons. In performing the auto-
mated synthesis, unreacted amino groups were capped with
0.5 M isobutyric anhydride in 2,6-lutidine/THF (1:1), and
the detachment of the oligos from the solid support with
concomitant deprotection of the phosphate and nucleobase
protecting groups was achieved by treatment with 1.0 M
methylamine in H2O/EtOH (1:1) at rt during 2 h. Oligos were
purified by HPLC (SAX 1000-8 (Macherey-Nagel)) up to a
Figure 1. (A) UV-spectroscopic melting curve (heating) and (B)
temperature-dependent CD curves of the duplex formed by the self-
complementary (3′NH)-TNA sequence (3′NHA3′NHT)8 (temperature
ranges are 20-95 °C). Tm values and thermodynamic data were
determined in 1.0 M NaCl, 10 mM NaH2PO4, 0.1 mM Na2EDTA,
pH 7.0. All Tm curves were reversible (no hysteresis). Tm values
are taken from the maxima of the first-derivative curve (software
(17) Imazawa, M.; Eckstein, F. J. Org. Chem. 1979, 44, 2039. Shimizu,
B.; Miyaki, M. Tetrahedron Lett. 1968, 7, 855.
(18) For instance, see: Reck, F.; Wippo, H.; Wagner, T.; Krishnamurthy,
R.; Eschenmoser, A. HelV. Chim. Acta 2001, 84, 1778.
(19) Oligonucleotides were synthesized on an Expedite 8909 Nucleic
Acid Synthesis system (Perseptive Biosystems). For details, see experimental
part of ref 18.
(20) Pon, R. T.; Yu, S.; Sanghvi, Y. S. Bioconjugate Chem. 1999, 10,
1051. Wu, X. Ph. D Dissertation (No. 13567), ETH-Zu¨rich, Zu¨rich, 2000.
(21) Vargeese, C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.; Kropp,
E.; Peterson, K.; Pieken, W. Nucleic Acids Res. 1998, 26, 1046.
(22) Terent’ev, A. P.; Vinogradova, E. V.; Chetverikov, V. P.; Dash-
kevich, S. N. The Chemistry of Heterocyclic compounds; Interscience: New
York, 1970; Vol. 6, p 146.
-1
Kaleidagraph). Thermodynamic data are derived from plots of Tm
with versus ln (c) (experimental error estimated in ∆H values (5%).
Tm values and thermodynamic data for the TNA duplex are taken
from ref 2.
The main part of our study focused on the pairing
properties of the non-selfcomplementary but antiparallel-
complementary sequences 3′-(A4T3ATAT2AT2A)-2′ and 3′-
Org. Lett., Vol. 4, No. 8, 2002
1281

(TA2TA2TATA3T4)-2′ prepared before in the TNA as well
as in the RNA and DNA series.2 Table 1 summarizes the Tm
however, of interest to juxtapose these differences with the
pairing properties of Gryaznov’s amidate-DNA- and -RNA-
type oligomers: there, the (3′NH)-DNA6 and the (3′NH)-
RNA7 were found to pair strongly with the natural parent
systems, whereas the (5′NH)-isomer of DNA was reported
not to pair with DNA and RNA at all.10
Table 1
The similarity of CD curves in Figure 2 of the sequence-
Figure 2. Comparison of CD spectra of duplexes formed by the
(2′NH)-TNA sequence A4T3ATAT2AT2A with its complementary
sequence TA2TA2TATA3T4 in the DNA, RNA, TNA,and (2′NH)-
and (3′NH)-TNA series (c ≈ 5 + 5 µM, T ≈ 0 °C). For conditions,
see the caption of Figure 1.
identical hexadecamer duplexes formed by the (2′NH)-TNA
sequence A4T3ATAT2AT2A with its complementary se-
quence TA2TA2TATA3T4 from the RNA, DNA, TNA, and
(3′-NH)- and (2′NH)-TNA series further characterize the
structural kinship between the TNA family and the natural
systems (see also Figure 1).
The (3′NH)-phosphoramidate-TNA sequence TA2TA2-
TATA3T4 has been shown to be stable toward hydrolytic
cleavage in slightly basic medium (1.0 M NaCl, 0.25 M
MgCl2, 0.1 M HEPES buffer, 37 °C, pH 8, 25 days) quite
similar to TNA itself.2 Not unexpectedly,5 such is not the
case under acidic conditions; the half-life of the hexadecamer
(2′NH)-phosphoramidate-TNA sequence A4T3ATAT2AT2A
in 80% aqueous acetic acid (pH ≈ 0.3) at rt is about 1 h.
We consider the experimental demonstration of the base-
pairing capacity of the (2′NH)- and (3′NH)-analogues of
TNA as a step in a systematic experimental mapping of the
base-pairing properties and the potential for self-assembly
of the members of a family of threose-related informational
oligomer systems that, in principle, can all be derived from
a narrow set of C2- and C1-starting materials.2
a (3′NH)-TNA ) 3′-3′NHA43′NHT33′NHA3′NHT3′NHA3′NHT23′NHA3′NHT23′NHA-
2′; (2′NH)-TNA is defined in an analogous manner. The color of the
acronyms relates to oligonucleotide sequence indicated by the same color.
The labels 3′ and 2′ denote strand orientation in TNA sequences; for RNA
and DNA sequences, the corresponding labels are 5′ and 3′. Base sequences
are written in the 3′f2′ or 5′f3′ direction. Tm values in the shaded diagonal
refer to intrasystem pairing. For conditions, see caption of Figure 1. Tm
values refer to a concentration of ca. 5 + 5 µM. An asterisk indicates that
Tm values were taken from ref 2.
values and the thermodynamic data of duplexes formed by
intrasystem as well as intersystem cross-pairing of these two
sequences in all possible combinations involving the back-
bones of TNA, RNA, DNA, (3′NH)-TNA, and (2′NH)-TNA.
The data in the shaded rectangles refer to intrasystem pairing
and reveal the (3′NH)-TNA duplex to be the thermally (but
not thermodynamically) most stable duplex among all, while
the (2′-NH)-isomer is the thermally (as well as thermody-
namically) weakest. Intersystem cross-pairings of both
(3′NH)- and (2′NH)-TNA are stronger with RNA than with
DNA; in fact, the combinations (3′NH)-TNA/RNA and
(2′NH)-TNA/RNA turn out to be comparable in strength to
that of the natural DNA/RNA combination. Rather remark-
ably, (2′NH)-TNA forms a distinctly stronger cross-pairing
duplex with TNA than its (3′-NH)-isomer. A rationale for
these subtle behavioral differences is not in sight. It is,
Acknowledgment. This work was supported by the
Skaggs Research Foundation. M.F. thanks The Austrian
Science Fund (FWF) for an Erwin Schro¨dinger Scholarship
(No. J-1825 CHE) and S.G. thanks the NASA/NSCORT
program for fellowship support. X.W. is a Skaggs Postdoc-
toral Fellow.
OL020015X
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Org. Lett., Vol. 4, No. 8, 2002