2,6-Diaminopurine in TNA: Effect on Duplex Stabilities and on the Efficiency of Template-Controlled Ligations

Article abstract of DOI:10.1021/ol020016p

(Matrix Presented) Replacement of adenine by 2,6-diaminopurine - two nucleobases to be considered equivalent from an etiological point of view - strongly enhances the stability of TNA/TNA, TNA/RNA, or TNA/DNA duplexes and efficiently accelerates template-

Full text of DOI:10.1021/ol020016p

ORGANIC
LETTERS
2002
Vol. 4, No. 8
1283-1286
2,6-Diaminopurine in TNA: Effect on
Duplex Stabilities and on the Efficiency
of Template-Controlled Ligations1
Xiaolin Wu,† Guillermo Delgado,† 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, Ho¨nggerberg, HCI-H309,
CH-8093 Zu¨rich, Switzerland
eschenmoser@org.chem.ethz.ch
Received January 16, 2002
ABSTRACT
Replacement of adenine by 2,6-diaminopurinestwo nucleobases to be considered equivalent from an etiological point of viewsstrongly
enhances the stability of TNA/TNA, TNA/RNA, or TNA/DNA duplexes and efficiently accelerates template-directed ligation of TNA ligands.
The constitutional simplicity and remarkable base-pairing
properties of TNA (R-threofuranosyl-(3′f2′)-oligonucle-
otides) raise the question of a possible role of threose-derived
informational oligomers in the origin of RNA.2-4 Such a
proposition calls for a comprehensive experimental inves-
tigation of the chemical properties of TNA and related
oligomer systems containing threose-derived backbones.2,3
With this in mind, we have extended our investigations on
the base-pairing properties of TNA to sequences that contain
2,6-diaminopurine as a nucleobase in place of adenine and
have tested the propensity of TNA and RNA sequences to
act as templates for the ligation of TNA ligands. Here we
report that replacement of adenine by 2,6-diaminopurine in
TNA5snot unexpectedlysstrongly enhances the stability of
TNA/TNA, TNA/RNA, and TNA/DNA duplexes and ef-
ficiently accelerates template-directed ligation reactions.
2,6-Diaminopurine’s capacity to stand for adenine in
Watson-Crick base pairs, and to enhance duplex stability
by virtue of its three (instead of two) hydrogen bonds to
uracil or thymine, has been demonstrated in a variety of
6-9
studies for a number of oligonucleotide systems.
N9-R-L-Threofuranosyl nucleoside of 2,6-diaminopurine in
its N2,N6-protected form (4), whose structure is supported
by an X-ray analysis,10 was prepared from the L-threofura-
nose derivative (1) and 2,6-diaminopurine-N2,N6-dibenzoate11
(2), closely following the procedures described earlier2 for
(5) From an etiological point of view, 2,6-diaminopurine is not to be
considered an unnaturally complex nucleobase; it can assemble from the
same type of elementary building blocks as guanine can.
(6) Howard, F. B.; Frazier, J.; Miles, H. T. J. Biol. Chem. 1966, 241,
4293. Rackwitz, H. R.; Scheit, K. H. Eur. J. Biochem. 1977, 72, 191.
Gaffney, B. L.; Marky, L. A.; Jones, R. A. Tetrahedron 1984, 40, 3. Cheong,
C.; Tinoco, I. Jr.; Chollet, A. Nucleic Acids Res. 1988, 16, 5115.
(7) Seela, F.; Becher, G. Nucleic Acids Res. 2001, 29, 2069 and literature
cited therein.
† Scripps Research Institute.
‡ Swiss Federal Institute of Technology.
§ Fax: ++1-858-784-9573. E-mail: rkrishna@scripps.edu.
Fax: ++41-1-632-1043.
(1) Chemistry of R-Aminonitriles. 36. Part 35: ref 3.
(2) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy,
R.; Eschenmoser, A. Science 2000, 290, 1347.
(3) Wu, X.; S. Guntha, Ferencic, M.; Krishnamurthy, R.; Eschenmoser,
A. Org. Lett. 2002, 4, 1279.
(4) Orgel, L. E.; Science, 2000, 290, 1306. Herdewijn, P. Angew. Chem.,
Int. Ed. 2001, 40, 2249.
(8) Matray, T. J.; Gamsey, S.; Pongracz, K.; Gryaznov, S. M. Nucleosides
Nucleotides 2000, 19, 1553. Matray, T. J.; Gryaznov, S. M. Nucleic Acids
Res. 1999, 27, 3976. Boudou, V.; Kerremans. L.; De Bouvere, B.; Lescrinier,
E.; Schepers, G.; Busson, R.; Van Aerschot, A.; Heredewijn, P. Nucleic
Acids Res. 1999, 27, 1450.
(9) Krishnamurthy, R.; Pitsch, S.; Minton, M.; Miculka, C.; Windhab,
N.; Eschenmoser, A. Angew. Chem., Int. Ed. Engl. 1996, 35, 1537.
10.1021/ol020016p CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/16/2002

Scheme 1a
a Numbers in parentheses denote molar equivalents referring to
starting compound or ratios. Numbers before these parentheses
denote concentrations in M. (a) 0.16 M (1.2 molar equiv) of 2,
0.43 (3.1) BSA, 0.18 (1.3) TMSOTf, CH3CN, 65 °C, 3 h; 59%;
(b) 2 M NaOH, THF/MeOH/H2O (5:4:1), 0 °C, 15 min; 87%; (c)
0.16 M (1.3) DMTCl, CH2Cl2/DMF (1:1), 0.7 (5.9) 2,6-lutidine,
0.18 (1.5) AgOTf, rt, 4 h, then (0.2) DMTCl, (0.2) AgOTf, rt, 20
h; 3′-O-DMT 40%, 2′-O-DMT 20%, 2′,3′-bis-O-DMT 6%; (d) 0.98
(5.7) chloro(cyanoethoxy)(diisopropylamino)phosphine, 0.53 (3.1)
N(i-Pr)2Et, CH2Cl2, rt, 2 h; 94%. X-ray structure analysis of 4:
torsion angles: O-C(2′)-C(3′)-O, 77.7°; O-C(1′)-N(9)-C(4),
166.8°.
other TNA nucleosides (Scheme 1). TNA sequences contain-
ing 2,6-diaminopurine () D) required for our studies were
synthesized on solid support according to the previously
reported procedure2 using intermediate 6.12
The duplex t(A12)/t(T12), exemplifying the purine/pyrimi-
dine sequence motif that is known to be associated with weak
pairing in TNA,13 raises its Tm value from 14 to 45 °C by
(A f D) replacement (standard conditions, see Figure 1).
A similar enhancement of duplex stability is observed in
TNA/TNA, TNA/RNA, and TNA/DNA duplexes composed
of the hexadecamer sequence A4T3ATAT2AT2A and its
antiparallel complement (the RNA sequence containing A
Figure 1. (A) UV-spectroscopic Tm curves (heating) of the
duplexes t(T12)‚t(A12) and t(T12)‚t(D12) (no hysteresis of cooling
curves). Tm values are derived from the maxima of the first-
derivative curve (software Kaleidagraph). Thermodynamic data are
derived from plots of Tm-1 with versus ln (c);25 experimental error
estimated in ∆H values (5%. (B) Temperature-dependent CD
curves of the duplex formed between t-(3′-DTTDTTDTDTTTD-
DDD-2′) and its complementary sequence (c ≈ 5 + 5 µM,
temperature range 30-100 °C). All measurements were made in
1.0 M NaCl, 10 mM NaH2PO4, 0.1 mM Na2EDTA, pH 7.0.
(10) Carried out by Raj K. Chadha, TSRI. Crystallographic data for the
structure has been deposited with the Cambridge Crystallographic Data
Center as deposition No. CCDC 175169. 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).
(11) Thomas, H. J.; Johnson, J. A.; Fitzgibbon, W. E.; Clayton, S. J.;
Baker, B. R.; Synthetic Proceedures in Nucleic acids Chemistry; Zorbach,
W. W., Tipson, R. S., Eds. Interscience Publishers: New York, 1968; Vol.
1, p 249.
and U, the DNA sequence A and T) (Table 1). The duplex
containing D in both of its TNA strands is more stable than
the duplexes from all other combinations (Table 1). Those
data of Table 1 which refer to differences induced by (A f
D) replacement correspond to an average enhancement of
duplex stability per single (A f D) replacement of about
-0.7 kcal/mol DDG (25 °C).14
Our experiments on the effect of (A f D) replacements
on the efficiency of template-directed ligations15,16 of TNA
ligands focused on the same hexadecamer duplex which we
used for the stability studies, taking 3′(or 5′)-TA2TA2-
TATA3T4-2′(or 3′) as the template sequence and using a
water-soluble carbodiimide as the activating agent17 (Table
1
(12) All compounds were characterized by H and 13C NMR and mass
spectral data after purification by column chromatography on silica gel. A
full experimental account will be published in HelV. Chim. Acta. Oligo-
nucleotides were synthesized on an Expedite 8909 Nucleic Acid Synthesizer
(Perseptive Biosystems), purified by HPLC (ion exchange column SAX
1000-8 (Macherey-Nagel)) up to a minimal purity of 95% and shown to
have the expected molecular mass by MALDI-TOF MS (Supporting
Information). Ligand sequences containing a 2′-phosphate group were made
using a DMTO-ethylsulfonyl derivatized-CPG solid support (see Bolli, M.;
Micura, R.; Pitsch, S.; Eschenmoser, A. HelV. Chim. Acta 1997, 80, 1901;
formula in footnote 5). Ligand sequences containing 3′-amino threofuranosyl
adenine as the end group were synthesized using the required building block
described in ref 3. RNA oligonucleotides were purchased from Xeragon
Inc. and DNA(A,T) oligonucleotides from Operon Technologies Inc.
1284
Org. Lett., Vol. 4, No. 8, 2002

ligations involving other artificial base-pairing systems have
been recently described.20
Table 1. Tm Values (c ≈ 5 + 5 µM) and Thermodynamic
Data of the Duplexes Formed by Cross-Pairing between the
Hexadecamer Base Sequences Indicated Bearing a TNA, RNA,
or DNA Backbonea
The formation of a phosphodiester bond between the
unmodified TNA ligands t(A4T3AT)-2′-phosphate and t(AT2-
AT2A)-3′-OH (see Table 2 and Figure 2 in the Supporting
Information) driven by EDC17 under the control of the
unmodified TNA(A,T)-hexadecamer sequence mentioned
above as a template is, under the conditions chosen (see Table
2), a slow and rather unproductive reaction.21 However, the
same process with ligands containing the nucleobase D
instead of A produces the corresponding ligation product in
essentially quantitative yields within hours. The same pattern
holds for ligations involving the TNA template containing
D instead of A. Furthermore, a similar acceleration due to
(A f D) replacement is observed when ligations leading to
a phosphoramidate bond are compared. Phosphate-to-phos-
phoramidate ligation of the D-containing TNA ligands is so
fast that side reactions do not discernibly interfere, and the
ligation products are obtained in essentially quantitative yield
(HPLC) within minutes. Whereas (A f D) replacement in
the TNA ligands strongly accelerates ligation in either
phosphodiester or phosphoramidite formation, such is not
observed when A is replaced by D in the TNA template
sequence; both templates give rise to comparable ligation
efficiencies. Remarkably, an RNA template sequence (con-
taining A and U) is as efficient in bringing about ligation of
the TNA ligands as the TNA templates themselves. However,
DNA as a template fails to induce phosphate-to-phosphodi-
ester ligations to any discernible extent; yet it brings about
an essentially quantitative phosphate-to-phosphoramidate
ligation when the TNA ligands contain D in place of A.
Figure 3 summarizes experiments carried out to assess
sequence fidelity. The medium-fast phosphate-to-phosphodi-
ester ligation of the D-containing TNA ligands was chosen
for comparing combinations that involve a single mismatch
in the 9-mer ligand and/or a single mismatch (in two
a The color of the acronyms relates to oligonucleotide sequence of the
same color in the duplex. The labels 3′ and 2′ indicate strand orientation in
TNA sequences; for RNA and DNA sequences, the corresponding labels
are 5′ and 3′. Sequences are written in the 3′ f 2′ or 5′ f 3′ Ddirection.
T
m values in the shaded diagonal refer to intrasystem pairing; all others to
intersystem-crosspairing. For conditions, see caption of Figure 1.
2). Ligation rate enhancements by (A f D) replacement have
been demonstrated for the natural oligonucleotide series.18
We also addressed questions referring to the sequence fidelity
to be expected in TNA ligation, as well as to the degree by
which in the TNA series phosphate-to-phosphoramidate
ligations are more efficient19 than phosphate-to-phosphodi-
ester ligations. Examples of intersystem-template-controlled
Table 2. TNA(A,T)-, TNA(D,T)-, RNA-(A,U)-, and DNA(A,T)-Templated Ligations of TNA Ligands: Effects of (A f D)
Replacement in Templates and TNA Ligands and of (OH f NH2) End-Group Replacement in the Ligand-Nucleophile on the Rate of
Ligationa
ligands
templates
TNA(A,T)
TNA(D,T)
RNA(A,U)
DNA(A,T)
2-
TNA-2′-OPO3
TNA HO -3′-H2N-
(A,T)
(D,T)
HO-
HO-
4 h
19%
57%
14%
51%
30%
53%
4 h
1 h
4 h
4 h
>95%
5 h
>95%
4 h
>95%
(A,T)
(D,T)
H2N-
H2N-
10 min
30 min
10 min
13 min
59%
52%
95%
80%
43%
>95%
94%
33 min
20 min
50 min
45 min
>95%
60%
4 h
5 h
37%
>95%
>95%
>95%
>95%
a Percentages refer to the yield of ligation product formed after the reaction times indicated as monitored by ion-exchange HPLC (see Figure 2 in the
Supporting Information). Ligations were carried out in 0.1 M aqueous HEPES buffer with 0.1 mM template, 0.2 mM ligand (each), 0.2 M EDC, pH 7.5, rt
All ligation products have been identified by MALDI-TOF mass spectroscopy and most of them, in addition by co-injection with authentic material. Test
experiments involving the (A,T)-TNA-3′-NH2 and (A,T)-TNA-2′-phosphate ligands in the absence of any template did not show any discernible ligation.
Note that the nucleoside unit that bears the 3′-amino end group has A instead of D.
Org. Lett., Vol. 4, No. 8, 2002
1285

discernible extent either.23 The requisites (sequence lengths,
nucleobases, type of activation and conditions) for a tran-
scription of TNA into RNA sequences remain to be explored.
Among the observed TNA ligations, relative efficiencies
qualitatively parallel the stability of complexes between
ligands, or between ligation products and templates. Thus,
the remarkably different response of the three templates
toward the single mismatch in the nonamer ligand reflects
itself in the Tm values of the corresponding ternary ligation
complexes (see caption of Figure 3). Expectedly, there are
exceptions, such as the observations according to which (A
f D) replacement in the TNA ligands accelerates ligations
whereas (A f D) replacement in the TNA template does
not (Table 2)24 or that DNA, as compared to RNA, seems
unproportionately inefficient as template relative to its cross-
pairing capability with TNA. A decisive factor for the
efficiency of intersystem template-controlled ligations must
be the constellational positioning of the two ligands’ reaction
districts by the template.16 Within the studies presented here,
this factor seems to express itself most prominently in the
observed asymmetry between the efficiency of RNA-
templated TNA ligation and that of TNA-templated RNA
ligations.
Figure 3. Effect of mismatches (M) in TNA 9-mer and 7-mer
ligands on the efficiency of phosphate-to-phosphodiester ligation
of TNA ligands (containing D and T) mediated by templates of
the TNA and RNA series. Tm values of ligation triplexes (phos-
phodiester series). L-9 + L-7 + template (5 + 5 + 5 µM): TNA-
(A,T), 22 °C; TNA(D,T), 37 °C. RNA(A,U): 32 °C. L-9(M) +
L-7 + template: TNA(A,T), <0 °C; TNA(D,T), <0 °C; RNA-
(A,U), 13 °C. For conditions of ligations, see caption of Table 2.
Acknowledgment. This work was supported by the
Skaggs Research Foundation. X.W. is a Skaggs Postdoctoral
Fellow. G.D. thanks the Skaggs Research foundation and la
Universidad Nacional Auto´noma de Me´xico for fellowship
support.
Supporting Information Available: X-ray structure of
4, MALDI-TOF mass data of sequences, HPLC traces and
relative rate curves of selected ligations, and Tm values of
ligand-template, product-template, and ligand(mismatch)-
template complexes. This material is available free of charge
via the Internet at http://pubs.acs.org.
versions) in the 7-mer ligand. Expectedly, a single mismatch
in the 9-mer ligand turns out to be less harmful to the ligation
than one in the 7-mer ligand, the relative rates depending,
however, on the template. Offering both monomismatched
ligands concomitantly to any of the three templates does not
lead to ligation at all. This amounts to a remarkably high
sensitivity of the ligation to the presence of mismatches in
the TNA series.22
Under the conditions where the RNA template (containing
A and U) can direct the phosphate-to-phosphodiester ligation
of the TNA ligands even somewhat more efficiently than
the two TNA templates themselves (Figure 2 in the Sup-
porting Information), the latter do not induce the phosphate-
to-phosphodiester ligation of the RNA ligands r(AAAAU-
UUAU) and r-5′-phosphate-(AUUAUUA). To keep this
result in perspective, it must be pointed out that under
otherwise identical conditions the RNA template was found
not to induce ligation of the two RNA ligands to any
OL020016P
(17) N-(3-Dimethyl(aminopropyl)-N′-ethylcarbodiimide hydrochloride ()
EDC); see, e.g., Shabarova, Z. A.; Dolinnaya, N. G.; Drusta, V. L.;
Melnikova, N. P.; Purmal, A. A. Nucleic Acids Res. 1981, 9, 5747.
(18) Hartel, C.; Go¨bel, M. W. HelV Chim. Acta 2000, 83, 2541. Kozlov,
I.; Orgel, L. E. HelV. Chim. Acta 1999, 82, 1799. See also the ligation
experiments in the p-RNA series: Bolli, M.; Micura, R.; Eschenmoser, A.
Chem. Biol. 1997, 4, 309.
(19) Dolinnaya, N. G.; Sokolava, N. I., Grayznova, O. I.; Shabarova, Z.
A. Nucleic Acids Res. 1988, 9, 3721. Lohrmann, R.; Orgel, L. E. Nature
1976, 261, 342. Zielinski, W. S.; Orgel, L. E. Nature 1987, 327, 346.
Kiedrowsky, G. v.; Wlotzka, B.; Helbing, J.; Matzen, M.; Jordan, S. Angew.
Chem., Int. Ed. Engl. 1991, 30, 423.
(20) Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E. Nucl. Acids Res. 1997,
25, 4797 and literature cited therein. Kozlov, I. A.; De Bouevere, B.;
Aerschot, A. v.; Herdewijn, P.; Orgel, L. E. J. Am. Chem. Soc. 1999, 121,
5856. Kozlov, I. A.; Zelinski, M.; Allart, B.; Kerremans, L.; Aerschot, A.
v.; Busson, R.; Herdewijn, P.; Orgel, L. E. Chem. Eur. J. 2000, 6, 151.
Koppitz, M.; Nielsen, P. E.; Orgel, L. E. J. Am. Chem. Soc. 1998, 120,
4563.
(13) Model considerations point to poor interstrand base stacking in the
(weakly base pairing) homopurine-homopyrimidine duplexes of TNA as
opposed to the (much more stable) duplexes in which purines and
pyrimidines are alternating (compare the low hyperchromicity in t(A12)/
t(T12) and its D-analogue in Figure 1, as compared to a hyperchromicity of
35-40% for TNA duplexes (AT)8, (AU)8, or (TA)8).
(14) Stability rise increments are expected to depend on sequences and
are not expected to remain the same with increasing the number of (A f
D) replacements. See: Sa´gi, J.; Szakanyi, E.; Vorl´ıckova´, M.; Kypr, J. J.
Biomol. Struct. Dyn. 1996, 13, 1035.
(21) Slow ligation rates allow side reactions (due to large excess of the
carbodiimide activation agent) to hamper product formation.
(22) For recent studies on the fidelity of template-controlled ligations
see, for examle, Kenneth, J.; Ellington, A. D. Chem. Biol. 1997, 4, 595.
Bolli, M.; Micura, R.; Pitsch, S.; Eschenmoser, A. HelV. Chim. Acta 1997,
80, 1901. Mattes, A.; Seitz, O. Chem. Commun. 2001, 2050.
(23) For the inefficiency of chemical ligation of RNA ligands on DNA
templates, see the following: Dolinnaya, N. G.; Sokolova, N. I.; Ashir-
bekova, D. T.; Shabarova, Z. A. Nucleic Acids Res. 1991, 19, 3067.
(24) Self-pairing of template strands are expected to contribute to this
phenomenon. Tm values (c ≈ 5 µM, 1 M NaCl) of template strands: TNA-
(A,T), <5 °C; TNA(D,T), 30 °C.
(15) Naylor, R.; Gilham, P. T. Biochemistry 1966, 5, 2722.
(16) Dolinnaya, N. G.; Shabarova, Z. A. Russ. Chem. Bull. 1996, 45,
1787 (review).
(25) Marky, L. A.; Breslauer, R. J. Biopolymers 1987, 26, 1601.
1286
Org. Lett., Vol. 4, No. 8, 2002