PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability

Article abstract of DOI:10.1081/NCN-120025243

Three chimeric dimer synthons (oeg_t^NHT, oeg_up^NHT and oeg_uh^NHT) containing thymine (t), 5-(1-propynyl)-uracil (up) and 5-(1-hexyn-1-y1)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized

Full text of DOI:10.1081/NCN-120025243

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PNA-DNA Chimeras Containing 5-Alkynyl-pyrimidine
PNA Units. Synthesis, Binding Properties, and
Enzymatic Stability
Zoltán Bajor a , Gyula Sági a b , Zsuzsanna Tegyey a & Ferenc Kraicsovits a
a Chemical Research Center , Institute of Chemistry, Hungarian Academy of Sciences ,
Budapest, Hungary
b Chemical Research Center , Institute of Chemistry, Hungarian Academy of Sciences , 59–67
Pusztaszeri St., 1025 Budapest, Hungary
Published online: 31 Aug 2006.
To cite this article: Zoltán Bajor , Gyula Sági , Zsuzsanna Tegyey & Ferenc Kraicsovits (2003) PNA-DNA Chimeras Containing
5-Alkynyl-pyrimidine PNA Units. Synthesis, Binding Properties, and Enzymatic Stability, Nucleosides, Nucleotides and Nucleic
Acids, 22:10, 1963-1983, DOI: 10.1081/NCN-120025243
To link to this article: http://dx.doi.org/10.1081/NCN-120025243
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 10, pp. 1963–1983, 2003
PNA-DNA Chimeras Containing 5-Alkynyl-pyrimidine
PNA Units. Synthesis, Binding Properties,
and Enzymatic Stability
y
*
Zoltan Bajor, Gyula Sagi, Zsuzsanna Tegyey, and Ferenc Kraicsovits
´
´
Chemical Research Center, Institute of Chemistry, Hungarian Academy of
Sciences, Budapest, Hungary
ABSTRACT
Three chimeric dimer synthons (oeg tNHT, oeg upNHT and oeg uhNHT) contain-
ing thymine (t), 5-(l-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA
units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution
and incorporated into T20 oligonucleotide analogues, using standard P-amidite
chemistry. Insertion of dimer blocks led to destabilization of duplexes with
dA20 target. The smallest Tm drops were found for chimeras containing
oeg upNHT dimers. Incorporation of the chimeric synthons into the 30-end of
T20 brought about growing resistance to 30-exonucleolytic (SV PDE) cleavage
in the order of oeg tNHT < oeg upNHT < oeg uhNHT. Due to different endonucl-
ease activities of 30- and 50-exonucleases applied, placing of five consecutive
dimers at the 50-terminus resulted in a relatively smaller, but also side-chain
dependent, stabilization towards the hydrolysis by 50-exonuclease (BS PDE).
Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could
´
*Correspondence: Gyula Sagi, Chemical Research Center, Institute of Chemistry, Hungarian
Academy of Sciences, 59–67 Pusztaszeri St., 1025 Budapest, Hungary; Fax: þ36-1-438-4134;
E-mail: gsagi@chemres.hu.
yDeceased.
1963
DOI: 10.1081/NCN-120025243
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1964
Bajor et al.
hydrolyse the unnatural phosphodiester bond linking the 30-OH of thymidine
to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.
Key Words: 5-Alkynyl-uracils; PNA-DNA chimeras; T20-analogues; Melting
properties; Nuclease stability.
INTRODUCTION
Since their first synthesis by Nielsen et al.[1] peptide nucleic acids (PNA-s), con-
taining N-(2-aminoethyl)glycine instead of sugar-phosphate backbone of the natural
nucleic acids, have attracted great interest due to their higher duplex stability
and complete resistance to both nucleases and proteases. However, the more hydro-
phobic pure PNA-s are prone to self-aggregation[2] in addition, they are unable to
induce RNase H activity[3] which plays important role in the mechanism of action of
antisense oligos. Their celluar uptake was also found to be lower[4,5] than expected
earlier and they can form less stable, parallel duplexes, too.[2,6] Since these
disadvantageous properties are hurdles in the application of PNA-s, as potential
antisense or antigene drugs, some trials have been made to eliminate them.
According to the thorough and extended studies by Uhlmann et al.[2,6] PNA-
DNA chimeras consisting of PNA and DNA blocks retain the RNase H inducing
ability of natural DNA, in addition, they can form only antiparallel duplexes with
the complementary DNA or RNA strands. Binding affinity of chimeras, consisting
of separate homogenous PNA and DNA blocks, strongly depends on the
PNA=DNA ratio.[2,7] In general, the larger is the proportion of PNA units in a chi-
mera the higher is the Tm value of a chimera:DNA duplex relative to the native
DNA:DNA counterpart. The increase of thermal stability is even higher in cases
of chimera:RNA duplexes. However, beside the PNA proportion and the comple-
mentary strand, the degree of duplex stabilization depends on the type of chimera
as well.[8,9] In cases of pseudo-50-PNA-DNA-30 chimera:DNA duplexes a strong
structural perturbation occurs at the PNA-DNA junction which is likely due to
the rigid amide bond between the PNA carboxy group and the 50-amino group of
modified terminal nucleotide. Therefore insertion of a single PNA unit into a
DNA results in a significant drop of Tm especially when it is placed in the middle
of the sequence.[10,11] However, when PNA units are positioned at the 30- or 50-ter-
minus it leads to a relatively small decrease of Tm value.[11] Serum stability of
PNA-DNA chimeras has also been investigated. A model 13-mer chimera with
two PNA units at the 50- and 30-ends was found to be about 25 times more stable
in human serum than the corresponding DNA counterpart or the pseudo-50-PNA-
DNA-30 hybrids.[10] However, 50-DNA-PNA-pseudo-30 chimeras proved 50 times
more stable in fetal calf serum relative to the DNA analogue,[12] which can also be
explained by the major 30-exonuclease activity in the serum. Nevertheless there are
no data available about the stability of chimeras in the presence of pure endo- or
exonucleases. The cellular uptake and water solubility of PNA-s can also be
enhanced by their incorporation into PNA-DNA chimeras. Thus, the degree and

PNA-DNA Chimeras
1965
Figure 1. Dimer synthons incorporated into T20-analogue chimeras.
kinetics of uptake become similar to those observed for the pure DNA analogue.
Another alternative way to increase the cell-penetration of PNA-s is their conjuga-
tion with Trojan peptides[13] (transportan and penetratin) which led to considerable
increase of their in vitro[14] and in vivo[15] activity.
According to our and others’ earlier studies on the biophysical and biochem-
ical properties of base-modified DNA-s, the 5-alkynyl substitution of pyrimidine
bases results in a significant duplex stabilization[16,17] and a higher resistance to
enzymatic cleavage.[18] However, propynyl residues introduced into the 7-position
of 8-aza-7-deazapurines were found to be even more stabilizing than those at the
5-position of pyrimidine bases.[19] In addition, in the case of a heptanucleotide,
composed of exclusively 5-propynyl-pyrimidine deoxynucleotides, long-range coop-
erativity among the propynyl groups[20] and enhanced mismatch penalties[21] were
observed, compared to the dT and dC containing counterparts. In correlation with
the former beneficial biochemical properties, P-thio antisense oligonucleotides,
containing 5-(1-propynyl)-dU and 5-(1-hexyn-1-yl)-dU units, respectively in place
of thymidines, exhibited considerably higher antiviral[22] and antitumor[23] activity
in vitro.
To extend these studies to PNA-DNA chimeras and to investigate the biophysi-
cal and biochemical properties of chimeras with alternating PNA and DNA units
numerous T20 analogues, containing thymine, 5-(propyn-1-yl)-uracil or 5-(1-hexyn-
1-yl)-uracil PNA monomers with N-(2-hydroxyethyl)glycine backbone (oeg t,
oeg up, or oeg uh), using 50-amino-50-deoxythymidine (NHT) as PNA-DNA linker,
have been synthesized. Application of oeg tNHT, oeg upNHT and oeg uhNHT hybrid
dimer synthons (see Fig. 1) made possible the exclusive use of standard solid phase
P-amidite DNA synthesis protocol for the synthesis of chimeras without employing
any PNA coupling chemistry.
Hydrolysis of chimeras by exo- and endonucleases (SV PDE, BS PDE and
Nuclease Pl) and thermal stability of chimera:dA20 duplexes have been investigated.
Results are summarized in the present paper.

1966
Bajor et al.
RESULTS AND DISCUSSION
Synthesis
Starting from 5-iodo-uracil (1) first we prepared the 5-propynyl- and 5-(1-hexyn-
1-yl) uracils (2 and 3) via Pd-catalyzed coupling of 1 with the corresponding terminal
alkynes in dry DMF (see Sch. 1), by the analogy of the synthesis of 5-alkynyl-20-
deoxyuridines.[24] Carboxymethylation of 2 and 3 with bromoacetic acid in aq.
NaOH solution was carried out according to the procedure reported for similar alky-
lation of thymine[25] resulting in the required N1-carboxymethyl-5-substituted-uracils
(4, 5 and 6) with good yields. According to the comparative study of Greiner et al.[26]
to attain the highest duplex stability in a 50-DNA-PNA-pseudo-30 chimera, the phos-
phodiester bridge is the most favoured linkage at the DNA-PNA junction. Therefore
we coupled our base-acetic acid derivatives (4, 5 and 6) to N-(2-hydroxyethyl)glycine
tert-butyl ester with minor modification of the literature method.[27] Thus we isola-
ted the required N-(2-hydroxyethyl)-N-[(5-substituted-pyrimidin-1-yl)acetyl]glycine
tert-butyl esters (7, 8 and 9) with acceptable yields. Contrary to the strategy des-
cribed the coupled products were first dimethoxytritylated in dry pyridine to give the
corresponding DMT-protected intermediates. Due to the weak nucleophilicity of OH
group under these conditions we could not detect any unrequired lactone by-product
in the reaction mixture.
The crude DMT-protected esters were saponified with NaOH in aq. dioxane then
carefully acidified to pH ꢀ 5 to give the required free acids (10, 11 and 12), which were
isolated as triethylammonium salts in good overall yields (67–87%) after the necessary
silica gel chromatographic purifications. The coupling of acids with 50-amino-50-deox-
ythymidine[28] (13) in DMF, in the presence of NEt3 and TBTU, as activating agent,
proceeded smoothly within 2 h in each case resulting in the 30-free chimeric dimer syn-
thons: oeg tNHT, oeg upNHT and oeg uhNHT (14, 15 and 16) in DMT-protected
form. The 30-free dimers were then phosphitylated according to standard method
to give the corresponding 30-O-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidites
(17, 18 and 19), as internal building blocks for the solid-phase syntheses (see Sch. 2).
On the other hand, for the 30-terminal incorporation we prepared the corresponding
dimer-30-O-succinates (20, 21 and 22) which were then coupled to the solid support
˚
(LCAA-CPG, pore size 500 A) in the presence of EEDQ, as condensing agent.
On the basis of DMT assays the loading of CPG varied from 35 to 43 mmol=g.
According to the DMT-absorbance measurements coupling yields with the dimer-P-
amidites were found to be 84–88% using the standard coupling programme of
Synthesizer (total coupling time: 54 sec.) In order to improve the efficiency of these
couplings double coupling and 4-fold waiting times were applied resulting in some-
what higher coupling yields (91–94%). The overall yields thus obtained correlated
with the proportion of DMT-on final products in the crude mixtures, as determined
by HPLC. As it can be seen in Table 1, the synthesized 20-mer chimeras may be
arranged into three sets of trios according to the position of hybrid dimer units.
In the first set (24, 25 and 26) the chimeric blocks occupy a middle position, while
in the second one (27, 28 and 29) they are placed at the 30-end. Chimeras of the third
set (30 ,31 and 32) begin with a decamer of alternate PNA and DNA units at the 50-
terminus. T20 (23), T11 (33) and the 34 chimera served as reference compounds.

PNA-DNA Chimeras
1967
Scheme 1. (a) HCꢁC–R, (Ph3P)2PdCl2, CuI, Et3N, DMF, rt, 18 h; (b) BrCH2–COOH, 1 N
aq. NaOH, cc. aq. HCl; (c) (i) HO–(CH2)2–NH–CH2–COOt.Bu, TBTU, Et3N, DMF, rt, 3 h;
(ii) recrystallization from EtOAc; (d) (i) DMT-Cl, pyridine, rt, 18 h (ii) SiO2 chrom; (e)
(i) dioxane-water 4:1, 1 N aq. NaOH, pH ¼ 13, rt, 3 h; (ii) 1M aq. NaHSO4 to pH ꢀ 5.0;
(iii) SiO2 chrom; (f) TBTU, Et3N, DMF, rt, 2 h, DCFC.

1968
Bajor et al.
Scheme 2. (a) (i) (i.Pr2N)2–P–OCE, DIPAT, CH2Cl2, rt, 18 h; (ii) SiO2 chrom; (b) (i) Succinic
anhydride, DMAP, Et3N, CH2Cl2, rt, 18 h; (ii) SiO2 chrom.
Thermal Melting Studies
As shown in Table 1, placing of one oeg tNHT or oeg uhNHT dimer unit in the
middle of T20 (24 and 26) causes about ꢂ9ꢃC lowering of Tm value relative to that of
the reference T20:dA20 duplex. However, in the case of T9-oeg upNHT-T9 (25) the
Tm-drop is only ꢂ7.6ꢃC. The same substitutions at the 30-end (27, 28 and 29) have
less pronounced effect. The smallest drop of the Tm value (ꢂ1.8ꢃC) was found again
for the propynyl-substituted analogue (28). However, chimera:dA20 duplexes having
a decamer block of alternate PNA and DNA units at the 50-terminus (30, 31 and
32) showed significant drops (approx. 16–20ꢃC) in Tm values. Thermal stabilities
of these duplexes were close to that of T11:dA11 suggesting that chimeric parts of
these analogues are hardly involved in cooperative binding to the complementary
strand. According to Bergmann et al.[10] a phosphodiester ! phosphamide exchange

PNA-DNA Chimeras
Table 1. Melting points of the reference and chimera:dA20 duplexes.a
1969
Compd.
Sequence
Tm (ꢃC)
DTm (ꢃC)b
DTm (ꢃC)c
23
24
25
26
27
28
29
30
31
32
33
34
T20
57.4
48.3
49.8
48.5
54.7
55.6
55.0
36.7
41.6
37.9
38.6
11.3
0
T9-oeg tNHT-T9
T9-oeg upNHT-T9
T9-oeg uhNHT-T9
ꢂ9.1
ꢂ7.6
ꢂ8.9
ꢂ2.7
ꢂ1.8
ꢂ2.4
ꢂ20.7
ꢂ15.8
ꢂ19.5
ꢂ18.8
ꢂ46.1
0
þ1.5
þ0.2
0
þ0.9
þ0.3
0
T18-oeg tNH
T18-oeg upNH
T18-oeg uhNH
T
T
T
(oeg tNHT)5-T10
(oeg upNHT)5-T10
(oeg uhNHT)5-T10
T11
þ4.9
þ1.2
(T2-oeg tNHT)5
aMeasured in 0.5 M NaCl, 10 mM MgCl2, 20 mM Tris (pH 7.4) buffer.
bFor these DTm values the Tm of T20:dA20 duplex (57.4 ꢃC) is the reference.
cFor these DTm values Tm ꢂ s of the corresponding oeg tNHT containing chimera:dA20
duplexes are the references.
in the middle of a DNA sequence results in Tm drops of 1.0–2.4ꢃC, depending on the
modified nucleoside. The lower Tm value of our (oeg tNHT)5-T10:dA20 duplex
(36.7ꢃC) relative to that of T11:dA11 (38.6ꢃC) might also be explained by this small
destabilizing effect. (However, if the 5 oeg tNHT blocks are alternating with TT in
the full length of sequence (see compd. 34) it results in dramatic decrease of the
Tm (DTm ¼ ꢂ46.1ꢃC), consequently this arrangement may cause full destruction of
the duplex structure.) Similarly to the first and second set, the smallest Tm-drop
(ꢂ15.8ꢃC) was found again for the chimera having five oeg upNHT dimers (31).
Compared to 30, it means that, in spite of the alternating mixed backbone
structure, the propynyl group still has a duplex stabilization effect of about
DTm ¼ þ1.0ꢃC=dimer unit, relative to the methyl group. In the case of 25 and 28
similar Tm rises (DTm ¼ þ1.5 and þ0.9ꢃC, respectively) were observed which can
be attributed to the known increased p–p stacking interaction between the neigh-
bouring bases, caused by the conjugated triple bond.[17] A similar, but much smaller
effect (DTm ¼ þ0.2–0.3ꢃC=dimer unit in all the three sets) was observed for the hex-
ynyl group (compounds 26, 29, and 32). In these cases the duplex stabilizing effect of
triple bond is compensated by the hydrophobic destabilizing effect of long alkyl
chain, which protrudes into the major groove and probably disrupts the water struc-
ture around the phosphate moiety.[29,30]
Enzymatic Hydrolyses
The 30-modified analogues containing one dimer block at the 30-end (27, 28 and
29) were hydrolysed by snake venom phosphodiesterase (SV PDE) and were found
to be 33–53 times more resistant than T20, as shown by the corresponding 1=R values
(see Table 2). In accordance with our earlier results[18] the alkynyl analogues (28 and

1970
Bajor et al.
Table 2. SV PDE hydrolysis half-times of chimeras modified at the 30-end.a
Compd.
Sequence
t1=2 (min)
Relative rate (R)
1=R
23
27
28
29
T20
T18-oeg tNH
1.3
43
58
69
1
1
T
3.02 ꢄ 10ꢂ2
2.24 ꢄ 10ꢂ2
1.88 ꢄ 10ꢂ2
33.1
44.6
53.1
T18-oeg upNH
T18-oeg uhNH
T
T
aHydrolysis mixtures were incubated at 37ꢃC, in 5 mM MgCl2, 50 mM Tris (pH 8.8) buffer.
The enzyme (phosphodiesterase I. from Crotalus adamanteus venom, SIGMA) concentrations
were 4 ꢄ 10ꢂ3 and 4 ꢄ 10ꢂ4 unit=mL for chimeras and T20, respectively. The substrate concen-
tration was 4 A260 unit=mL in each experiment. Determination of t1=2 values is described in the
experimental.
29) proved more stable compard to the oeg tNHT-containing counterpart (27). It can
be seen that the longer is the 5-side chain of pyrimidine base in the PNA moiety the
more resistant is the chimera to the 30-exonucleolytic cleavage. The t1=2 values of pro-
pynyl and hexynyl analogues (28 and 29) were 1.3 and 1.6 times higher, respectively
compared to that of 27.
The resistance to SV PDE hydrolysis can also be enhanced significantly by incor-
poration of more consecutive dimer blocks. According to our recent findings,
reported in a preliminary form,[31] positioning of 1, 2 or 3 oeg tNHT blocks at the
30-end of T12 resulted in 50-, 96- and 145-fold increase of hydrolysis half-time,
respectively relative to the t1=2 of T12. In addition, RP HPLC profiles of the final
hydrolysis mixtures, after 1 day digestion, displayed different retention times (tR)
for the peaks of chimeric hydrolysis products. These tRvalues increased with increas-
ing number of dimer blocks incorporated, indicating longer and longer chimeric
fragments. It proves that even the double and triple chimeric blocks remained intact,
i.e., the enzyme could not hydrolyse the unnatural phosphodiester bond between the
30-OH of thymidine and the terminal OH of N-(2-hydroxyethyl)glycine PNA back-
bone. It is in good agreement with earlier results[32,33] connected with the enzymatic
stability of oligonucleotides containing different acyclic nucleosides.
The 50-modified analogues (30, 31 and 32) were hydrolysed by bovine spleen
phosphodiesterase (BS PDE). Compared to the SV PDE hydrolyses, in these cases
250 times higher enzyme concentrations (expressed in unit=mL) were necessary to
attain measurable hydrolysis rates. These chimeras, which contain five chimeric
dimer blocks at the 50-terminus, in spite of the much longer chimeric strands, were
found to be only 6–8 times more resistant than the reference T20 (see Table 3).
This resistance also increased with increasing length of 5-alkynyl substituent of the
PNA-uracil moiety. The propynyl analogue (31) and the hexynyl one (32) were 1.3
and 1.4 times more stable, respectively than the oeg tNHT containing counterpart (30).
Analogous incorporation of 1, 2 or 3 oeg tNHT units into the 50-terminus of T12
also led to increasing resistance to the hydrolysis by BS PDE. As indicated by the
HPLC profiles, similarly to those of the previous digestions by SV PDE, the longer
chimeric blocks also remained intact which refers to the complete resistance of this
phosphodiester bond to the 50-exonucleolytic cleavage, too.

PNA-DNA Chimeras
Table 3. BS PDE hydrolysis half-times of chimeras modified at the 50-end.a
1971
Compd.
Sequence
t1=2 (min)
Relative rate (R)
1=R
23
30
31
32
T20
7.7
45
59
64
1
1
(oeg tNHT)5-T10
(oeg upNHT)5-T10
(oeg uhNHT)5-T10
0.17
0.13
0.12
5.8
7.7
8.3
aHydrolysis mixtures were incubated at 37ꢃC, in 5 mM MgCl2, 50 mM Tris (pH 6.5) buffer.
The enzyme (phosphodiesterase II. from bovine spleen, SIGMA) concentrations were 1.0
and 0.1 unit=mL for chimeras and T20, respectively. The substrate concentration was 4
A
260 unit=mL in each case. Determination of t1=2 values is described in the experimental.
To investigate the stability of the longest chimeric blocks in the presence of an
endonuclease too, 50-T6-(oeg tNHT)3-30 and 50-(oeg tNHT)3-T6-30 were incubated
with Nuclease Pl. After 1 day digestion the peaks of HPLC profiles in both cases
were found to be identical with those obtained by the same analyses of the corre-
sponding SV PDE or BS PDE hydrolysis mixtures. It proves that the unnatural
phosphodiester bond is resistant not only to exonucleases but to endonuclease as
well.
The fact that the 50-modified analogues were found to be much less resistant to
the hydrolysis by 50-exonuclease (BS PDE) than the 30-modified derivatives to the
30-exonuclease (SV PDE) digestion, may be explained by the significant difference
between the endonuclease activity of the two enzymes.
CONCLUSIONS
In the present work a facile and simple synthetic route for the preparation of 3
chimeric PNA-DNA dimer synthons has been described. Longer coupling cycle and
larger reagent excess were necessary to attain acceptable (>90%) coupling yields
when dimer P-amidites were used for the solid-phase synthesis. Due to perturbation
of the duplex structure caused by the inhomogenous mixed backbone, incorporation
of chimeric blocks led to considerable drop of Tm values relative to that of the refer-
ence T20:dA20 duplex. The degree of Tm drops strongly depended on the number and
position of dimer units incorporated. The chimeric part of (oeg tNHT)5-T10 did not
form duplex at all, while the methyl ! propynyl exchange in the uracil PNA moiety
resulted in noticeable duplex stabilization, even if it was not enough to compensate
the destabilizing effect of the mixed backbone. For chimeras containing 5-hexynyl-
uracil PNA units, the duplex stabilizing effect of triple bond and destabilizing effect
of the long alkyl side-chain still gave a little positive DTm value (þ0.2ꢃC=mod).
Incorporation of dimer blocks increased the stability against exonucleases in each
case. While the unmodified T20 was rapidly digested by a 30-exonuclease (SV
PDE) PNA-DNA chimeras, having one chimeric dimer block at the 30-terminus
(27, 28 and 29), proved especially stable towards this exonucleolytic cleavage. We
have found that BS PDE had higher endonuclease activity compared to that of
SV PDE since, in spite of the incorporation of 5 dimer blocks, the t1=2 values of 50

1972
Bajor et al.
modified analogues (30, 31 and 32) increased only by 6–8 times relative to that of
T20. In cases of both enzymes the stability of chimeras depended on the length of
5-substituent of PNA base moiety. Within one set the hydrolysis rates decreased
in the order of oeg tNHT > oeg upNHT > oeg uhNHT, which can be attributed to
the increased inhibition of the substrate binding to the active site by the longer alky-
nyl side-chains. Neither exonucleases (SV PDE and BS PDE) nor an endonuclease
(Nuclease Pl) could hydrolyse the unnatural phosphodiester bond between the sec-
ondary OH of thymidine and the terminal OH of N-(2-hydroxyethyl)glycine PNA
moiety.
Our results call the attention to PNA-DNA chimeras having limited number of
PNA residues with 5-propynyl- or 5-hexynyl-uracil base at the 30- or 50-terminus, as
possible antisense candidates with suitable binding affinity to the complementary
targets and high resistance towards nucleolytic degradations.
EXPERIMENTAL SECTION
Materials and Methods
5-Iodo-uracil was purchased from Aldrich, 1,2-dibromo-propane, 1-hexyne,
Cul, EEDQ, TBTU and PdCl2 from Fluka. DMF and NEt3 were distilled from
˚
CaH2 and stored over molecular sieves (4 A). Precoated SiO2 plates (Kieselgel
60 HF254, 0.2 mm) were used for TLC and Kieselgel 60 (0.04–0.063 mm and 0.063–
0.2 mm) (Merck) for column chromatographic separations. Solvent systems used
were the following: A: CHCl3–MeOH 9:1, B: CHCl3–MeOH 2:1, C: CHCl3–MeOH
9:1 þ 1% TEA, D: CHCl3–MeOH 4:1 E: EtOAc-MeOH 9:1 þ 2% TEA. HPLC ana-
lyses and purification of the crude DMT-on oligonucleotides were performed with a
Jasco HPLC system using Hypersi ODS column (8 ꢄ 250 mm). The flow rate was
3 mL=min, the gradient was 5 ! 50% acetonitrile in 0.1M aq. NH4OAc solution.
Infrared spectra were recorded in KBr pellets with a Nicolet Magna 750 FT–IR spec-
trophotometer. 1H and 31P NMR spectra were recorded with a Varian XL-400 mul-
tinuclear instrument at 400 MHz. Chemical shifts are given in ppm relative to Me4Si
and H3PO4, as internal standards, respectively. ESI mass spectra were obtained with
a Perkin Elmer SCIEX, API 2000 tandem mass spectrometer equipped with electro-
spray ion source both in positive and negative ion mode. Samples were directly
injected into aq. acetonitrile. The flow rate was 0.2 mL=min. Elemental analyses were
carried out by the Environmental and Analytical Chemistry Department of the Che-
mical Research Center. Automated solid-phase oligonucleotide syntheses were run
on a MilliGen=Biosearch 8700 DNA Synthesizer using T- and dimer-loaded
˚
LCAA-CPG-s (pore size: 500 A), as solid supports. Tm points of duplexes with
dA20 target were measured with a Hewlett Packard 8452A UV-VIS spectrophot-
ometer (detection at 260 nm). Linear variations of temperature as a function of time
were regulated by a compatible microcomputer using Absorbe 3.0 software. In cases
of endonuclease (Nuclease P1 from Penicillium citrinum, SIGMA) digestions the
enzyme (c ¼ 0.02 unit=mL) and substrates (c ¼ 4 A260 unit=mL in each case) were
incubated at 37ꢃC, in 5 mM MgCl2, 50 mM Tris (pH 5.3) buffer. Half times of enzy-
matic hydrolyses (t1=2 values) were determined by IE HPLC analysis. Samples were

PNA-DNA Chimeras
1973
taken from the hydrolysis mixtures after given intervals (0, 5, 15, 30, 60 and
120 min.), freezed then applied to an analytical ion-exchange HPLC column. A260
absorbance values of the starting 20-mer were plotted against the time to give the
t1=2 value, belonging to the half-A260 value of the starting T20 analogue. The HPLC
analyses mentioned were performed with a Merck HPLC system (LaChrom L-7100
pump, Bischoff Lambda 1010 UV-VIS detector, detection at 260 nm), using MN
Nuleogen-DEAE 60-7 column (4 ꢄ 125 mm) equipped with a MN Nucleogen DEAE
60-7 guard column (4 ꢄ 30 mm). Elution was carried out using linear 0 ! 1.0 M KCl
gradient in aq. 20 mM NaOAc containing 20% acetonitrile programmed over a
200 min. period with a flow rate of 2 mL=min.
5-(1-Propynyl)-uracil (2). 5-Iodo-uracil (1) (9.52 g ¼ 40 mmol) was dissolved in
dry DMF (100 mL) and dry TEA (11 mL ¼ 80 mmol). The solution was purged with
argon then (Ph3P)2 PdCl2 (2.80 g ¼ 4 mmol) and CuI (1.52 g ¼ 8 mmol) were added.
After 5 min propyne (17 mL ¼ 0.3 mol), that was previously generated from 1,2-
dibromopropane,[34] was introduced with strirring at ambient temperature for 1 h.
The mixture was left to stand overnight then the solvent was evaporated in vacuo
to give yellow amorphous residue. It was suspended in 0.5 N aq. NaOH (150 mL),
the insoluble Pd salts were filtered and washed with further 0.5N aq. NaOH solution
(50 mL) and water (20 mL). The filtrate was treated with charcoal, filtered then acidi-
fied with cc. aq. HCl (7.0 mL) to pH ꢀ 5. The precipitate was filtered and washed
with 0.5 M aq. HCl. Since according to the TLC, it still contained Cu-salts and a
more polar impurity it was stirred with cc. aq. NH3 solution for 15 min then filtered.
Thus we isolated solid product that was pure by TLC. It was dried over P2O5 in
vacuo overnight to give 2.80 g ¼ 18.6 mmol of 5-(1-propynyl)-uracil (2), as a pale
grey powder. Yield: ꢀ47%. Rf(A): 0.33; IR n [cmꢂ1] ¼ 3214 m (NH), 2261 vw
(CꢁC), 1715 s (as C¼O), 1684 s (s C¼O), 1628 m (C¼C), 1434 m, 1234 m, 855 m;
1H NMR (DMSO-d6): d ¼ 2.00 (3H, s, CH3), 7.70 (1H, s, H6), 11.18 and 11.35
(2H, 2bs, 2NH); ESI MS m=z (%): 151.0 (42) [M þ H]þ, 157.0 (100) [M þ Li]þ,
C7H6N2O2 requires 150.13. Anal. calcd for C7H6N2O2; C, 56.00; H, 4.03; N,
18.66. Found: C, 55.78; H, 4.19; N, 18.47.
5-(1-Hexyn-1-yl)-uracil (3). Starting from the same amount (40.0 mmol) of
1 the alkynylation was carried out on similar way but in this case only 3
equiv ¼ 0.12 mol ¼ 13.5 mL of 1-hexyne was added. Due to the lower water solubility
of the product after removal of DMF the residue was stirred with 0.5 M aq. KOH
(88 mL ¼ 44 mmol) at 40ꢃC, for 1 h then filtered. Since the solid precipitate still
contained some main product it was further stirred with 150 mL of warm 1M aq.
KOH and the insoluble Pd and Cu salts were filtered. The filtrate was treated with
cc. aq. HCl (23.0 mL) which resulted in the precipitation of a pale yellow solid. After
1 h standing in the refrigerator the precipitate was filtered, washed with water
(15 mL) then dried in vacuo, over P2O5. Thus we obtained 4.15 g ¼ 21.6 mmol of
5-(1-hexyn-1-yl)-uracil (3). Yield: 57%, Rf(A): 0.40; IR n [cmꢂ1] ¼ 3200 m (NH),
2958 m (as CH2), 2933 m (s CH2), 2240 vw (CꢁC), 1730 s (as C¼O), 1686 s (s
C¼O), 1632 m (C¼C), 1429 m, 1215 m; 1H NMR (DMSO-d6): d ¼ 0.88(3H, t,
CH3), 1.30–1.65 (4H, m, (CH2)2), 2.38 (2H, t, CꢁC–CH2), 7.70 (1H, s, H6), 11.10
and 11.30 (2H, 2 bs, 2 NH); ESI MS m=z (%): 193.0 (90) [M þ H]þ, 199.0 (100)

1974
Bajor et al.
[M þ Li]þ, 215.2 (31) [M þ Na]þ, C10H12N2O2 requires 192.21. Anal. calcd for
C10H12N2O2:C, 62.48; H, 6.29; N, 14.57. Found: C, 62.31; H, 6.47; N, 14.40.
5-(1-Propynyl)-uracil-1-yl-acetic acid (4). Crude 2 (2.60 g ¼ 17.3 mmol) was
dissolved in 1 N aq. NaOH with stirring at 40ꢃC, cooled to room temperature then
the solution of bromoacetic acid (3.61 g ¼ 26 mmol) in water (20 mL) was added
dropwise. After 4 h stirring the mixture was carefully acidified with 1 N aq. HCl to
pH ꢀ 5. Some unreacted 2 was precipitated then removed by filtration. The pH of
the filtrate was then adjusted to ꢀ1.0 with cc. aq. HCl, which resulted in precipita-
tion of the required pure 5-(propyn-1-yl)-uracil-1-yl acetic acid (4). It was dried in
vacuo, over P2O5 for 2 days to give 2.11 g ¼ 10.14 mmol of 4, as a pale yellow pow-
der. Yield: 58.6%, Rf(B): 0.43–0.58 diffuse; IR n [cmꢂ1] ¼ 3170 m (NH), 3200–2800 m
(OH carboxylic), 2246 vw (CꢁC), 1738 s (as C¼O uracil), 1696 s (C¼O carboxylic),
1
1680 s (s C¼O uracil), 1632 m (C¼C), 1477 m, 1424 m, 1376 m, 1245 m, 1218 m; H
NMR (DMSO-d6): d ¼ 2.00 (3H, s, CH3), 4.40 (2H, s, CH2), 7.95 (1H, s, H6),
11.60 (1H, s, NH), 13.30 (1H, bs, COOH); ESI MS m=z (%): 209.2 (57) [M þ H]þ,
215.2 (100) [M þ Li]þ, 207.1 (100) [M ꢂ H]ꢂ, 415.3 (11) [2M ꢂ H]ꢂ,C9H8N2O4
requires 208.16. Anal. calcd for C9H8N2O4: C, 51.93; H, 3.87; N, 13.45. Found:
C, 51.74; H, 4.04; N, 13.31.
5-(1-Hexyn-1-yl)-uracil-1-yl-acetic acid (5). The procedure described for the
propynyl analogue had to be slightly modified due to the lower water solubility of
3. The crude 3 (1.92 g ¼ 10.0 mmol) was stirred with 0.5N aq. NaOH (70 mL) at
50ꢃC but it did not become quite homogeneous even after 1 h. The insoluble part
was filtered off then a solution of bromoacetic acid (2.08 g ¼ 15 mmol) in water
(10 mL) was added dropwise. After 24 h stirring the pH was set to ꢀ6.0 with 1 N
aq. HCl (10.0 mL) which led to the precipitation of some unreacted 3 along with a
little (<10%) main product. Further acidification of the filtrate with cc. aq. HCl
(1.7 mL) resulted in the precipitation of the required main product 5-(1-hexyn-1-
yl)-uracil-1-yl-acetic acid (5) that was filtered, washed with some cold water and
dried over P2O5, in vacuo. Thus we isolated 1.85 g ¼ 7.39 mmol of 5, as while solid.
Yield: 74%, Rf(A): 0.14; IR n [cmꢂ1] ¼ 3170 m (NH), 3200–2800 m (OH carboxylic),
2960 m (as CH2), 2925 m (s CH2), 2238 vw (CꢁC), 1736 s (as C¼O uracil), 1703 s
(C¼O carboxylic), 1680 s (s C¼O uracil), 1630 m (C¼C), 1467 m, 1201 m; 1H
NMR (DMSO-d6): d ¼ 0.90 (3H, t, CH3), 1.40 (4H, m, (CH2)2), 2.30 (2H, t,
CꢁC–CH2ꢂ), 4.42 (2H, s, CH2), 8.00 (1H, s, H6), 11.70 (1H, s, NH), 13.20 (1H,
bs, COOH); ESI MS m=z (%): 249.1 (100) [M ꢂ H]ꢂ, 499.3 (29) [2M ꢂ H]ꢂ, 749.2
(6) [3M ꢂ H]ꢂ, C12H14N2O4 requires 250.24. Anal. calcd for C12H14N2O4: C,
57.59; H, 5.64; N, 11.19. Found: C, 57.47; H, 5.78; N, 11.08.
Thymin-1-yl-acetic acid (6). This compound has been described in the litera-
1
ture[25] but only Rf and H NMR data were reported. IR n [cmꢂ1] ¼ 3185 m (NH),
3200–2800 m (OH carboxylic), 1740 s (as C¼O uracil), 1707 s (C¼O carboxylic),
1663 s (s C¼O uracil), 1633 s (C¼C); ESI MS m=z (%) 183.1 (100) [M ꢂ H]ꢂ,
367.0 (41) [2M ꢂ H]ꢂ, C7H8N2O4 requires 184.14. Anal. calcd for C7H8N2O4: C,
45.65; H, 4.38; N, 15.21. Found: C, 45.48; H, 4.55; N, 15.07.

PNA-DNA Chimeras
1975
General Procedure for the Coupling of N1-Carboxymethyl-5-substituted-uracils
with N-(2-Hydroxyethyl)glycine tert-butyl ester. N-(2-hydroxyethyl)glycine tert-butyl
ester[27] (0.96 g ¼ 5.5 mmol) was dissolved in DMF (10 mL) then any of N1-carboxy-
methyl-5-substituted-uracils (4, 5 or 6) (5.0 mmol), TBTU (1.76 g ¼ 5.5 mmol) and
TEA (1.54 mL ¼ 11 mmol) were added and the mixture was left to stir at ambient
temperature for 3 h. The solvent was removed in vacuo, the residue was dissolved
in CH2Cl2, evaporated to dryness again and the solid foam obtained was recrysta-
lized from EtOAc (25 mL). After 16 h standing at 4ꢃC the solid product was filtered,
washed with EtOAc and dried in vacuo to give the required coupled products, as
white solids.
N-(2-Hydroxyethyl)-N-(thymin-1-yl-acetyl)glycine tert-butyl ester (7). This
compound has been described and characterized in the literature.[27] We give only
some own, additional data: Yield: 76%, Rf(A): 0.39; mp.: 169–174ꢃC; IR n
[cmꢂ1] ¼ 3464 m (OH), 3163 w (NH), 2962 w (as CH2), 2933 w (s CH2), 1733 s
(C¼O ester), 1697 s (as C¼O uracil), 1670 s (s C¼O uracil), 1473 m, 1215 m,
1163 m; ESI MS m=z (%) 342.1 (78) [M þ H]þ, 348.1 (47) [M þ Li]þ, 359.2 (97)
[M þ NH4]þ, 364.3 (20) [M þ Na]þ, 380.2 (100) [M þ K]þ, C15H23N3O6 requires
341.44. Anal. calcd for C15H23N3O6: C, 52.76; H, 6.79; N, 12.31. Found: C, 52.68;
H, 6.87; N, 12.22.
N-(2-Hydroxyethyl)-N-[(5-propynyl-uracil-1-yl)acetyl]glycine tert-butyl ester (8).
Yield: 65%, Rf(A): 0.43; mp.: 189–192ꢃC; IR n [cmꢂ1] ¼ 3471 w (OH), 3170 w (NH),
2968 m (as CH2), 2935 m (s CH2), 1736 s (C¼O ester), 1700 s (as C¼O uracil), 1677 s
1
(s C¼O uracil), 1661 s (>N–C¼O), 1468 m, 1370 m, 1161 m; H NMR (DMSO-d6)
d ¼ 1.45–1.52 (9H, 3s, OCMe3), 2.03 (3H, s, CH3), 3.40–3.75 (5H, m, CH2–CH2–
OH), 4.05 and 4.28 (2H, 2s, glycine CH2, rotamers), 4.58 and 4.80 (2H, 2s, B–
CH2, rotamers), 7.82 and 7.85 (1H, 2s, H6, rotamers), 11.62 (1H, s, NH); ESI MS
m=z (%) 366.1 (54) [M þ H]þ, 372.3 (100) [M þ Li]þ, 383.2 (63) [M þ NH4]þ, 388.3
(25) [M þ Na]þ, 404.2 (13) [M þ K]þ, C17H23N3O6 requires 365.46. Anal. calcd for
C17H23N3O6: C, 55.87; H, 6.34; N, 11.50. Found: C, 55.72; H, 6.42; N, 11.39.
N-(2-Hydroxyethyl)-N-[(5-hexynyl-uracil-1-yl)acetyl]glycine tert-butyl ester (9).
Yield 58%, Rf(A): 0.50; mp.: 230–235ꢃC, IR n [cmꢂ1] ¼ 3480 w (OH), 3178 w (NH),
2970 m (as CH2), 2935 m (s CH2), 1738 s (C¼O ester), 1702 s (as C¼O uracil), 1680 s
(s C¼O uracil), 1664 s (>N–C¼O), 1466 m, 1375 m, 1160 m; 1H NMR (CDCl3)
d ¼ 0.90 (3H, t, CH3), 1.35–1.60 (13H, m, OCMe3 and –(CH2)2), 2.40 (2H, t,
CꢁC–CH2), 3.43–3.80 (5H, m, CH2–CH2–OH), 4.00 and 4.20 (2H, 2s, glycine, CH2,
rotamers), 4.48 and 4.75 (2H, 2s, B–CH2, rotamers), 7.30 and 7.38 (1H, 2s, H6,
rotamers), 9.20 (1H, bs, NH); ESI MS m=z (%) 408.1 (50) [M þ H]þ, 414.4 (100)
[M þ Li]þ, 425.2 (52) [M þ NH4]þ, 430.3 (27) [M þ Na]þ, 446.2 (17) [M þ K]þ
C20H29N3O6 requires 407.54. Anal. calcd for C20H29N3O6: C, 58.94; H, 7.17; N,
10.31. Found: C, 58.75; H, 7.30; N, 10.20.
N-(Thymin-1-yl-acetyl)-N-(2-dimethoxytrityloxy-ethyl)glycine triethylammonium
salt (10). (The reported synthesis[27] of this compound followed a different strategy)
Solution of 7 (1.70 g ¼ 5.0 mmol) in dry pyridine (10 mL) was cooled to 10ꢃC in water

1976
Bajor et al.
bath then DMT-Cl (1.86 g ¼ 5.5 mmol) was added in three portions within 1 h.
The mixture was stirred at ambient temperature for 3 h then it was poured into ice-
water (ꢀ150 mL). After separation of the supernatant aqueous phase the yellow syrup
was dissolved in CH2Cl2 (100 mL) and washed with water (2 ꢄ 20 mL). The organic
phase was dried with Na2SO4, filtered and evaporated to dryness. This crude
product was redissolved in CH2Cl2 (15 mL) and added dropwise to cold n.hexane
(300 mL). The off-white precipitate was filtered and dried in vacuo, over P2O5 to give
3.14 g ¼ 4.88 mmol of tritylated ester, as a white powder. Yield: 98%, Rf(A): 0.61.
This crude product (1.92 g ¼ 3.0 mmol) was dissolved in a mixture of dioxane
(13 mL) and water (3 mL) and the pH of solution was set to ꢀ13 with 1M aq. NaOH.
The mixture was stirred at 20ꢃC for 3h then cooled in ice-water bath and carefully
acidified to pH ꢀ 5.0 with 2N aq. NaHSO4. Then it was diluted with water (30 mL)
and extracted with CH2Cl2 (2 ꢄ 50 mL). The CH2Cl2 solution was evaporated, the
residue was dissolved in CHCl3–TEA (1%) (8.0 mL) and was applied to a silica gel
column (70 g of Kieselgel 60, 0.063–0.2 mm) that was eluted with CHCl3–MeOH
4:1 þ 2% TEA. The appropriate fractions were combined and evaporated to give
1.39 g ¼ 2.02 mmol of 10, as triethylammonium salt. Yield: 67,3%. Rf(C): 0.26; IR
n [cmꢂ1] ¼ 3210 w (NH), 3200–2800 w (OH carboxylic), 2939 m (as CH3 OMe),
2840 w (s CH3 OMe), 1716 s (as C¼O uracil), 1680 s (C¼O carboxylic), 1674 s (s
C¼O uracil), 1650 s (>N–C¼O), 1632 m (C¼C), 1608 m and 1509 m (aromatic),
1
1250 m, 117 m, 1033 m; H NMR (DMSO-d6):d ¼ 1.30 (9H, t, 3 Me of TEA), 1.75
and 1.90 (3H, CH3, rotamers), 3.00 (6H, q, 3CH2 of TEA), 3.25 and 3.38 (2H, 2t,
CH2–N, rotamers), 3.52–3.65 (2H, m, DMT–O–CH2), 3.80 (6H, s, 2 OMe of
DMT), 3.88 and 4.05 (2H, 2s, glycine CH2, rotamers), 4.58 and 4.85 (2H, 2s, B–
CH2, rotamers), 6.75–7.45 (14H, m, aromatic and H6), 11.30 (1H, bs, NH); ESI
MS m=z (%) 594.4 (100) [M þ Li]þ, 605.2 (31) [M þ NH4]þ, 586.3 [M ꢂ H]ꢂ,
C32H33N3O8 requires 587.68. Anal. calcd for C32H33N3O8: C, 65.40; H, 5.66; N,
7.15. Found: C, 65.51; H, 5.78; N, 7.21.
The alkynyl derivatives were prepared and purified on similar way resulting
in,the corresponding 5-propynyl- and 5-hexynyl-substituted PNA monomers (11
and 12).
N-[(5-Propynyl-uracil-1-yl)acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine triethyl-
ammonium salt (11). Yield: 81%, Rf(C): 0.27; IR n [cmꢂ1] ¼ 3220 w (NH), 3200–
2800 w (OH carboxylic), 2940 w (as CH3 OMe), 2852 w (s CH3 OMe), 1733 s (as
C¼O uracil), 1699 s (C¼O carboxylic), 1682 s (s C¼O uracil), 1651 s (>N–C¼O),
1
1608 m and 1509 m (aromatic), 1464 m, 1251 s, 1177 m, 1033 m; H NMR (CDCl3):
d ¼ 1.25 (9H, t, 3Me of TEA), 1.90 (3H, s, CꢁC–CH3), 3.03 (6H, q, 3 CH2 of
TEA), 3.27 and 3.40 (2H, 2s, H2C–N, rotamers), 3.54–3.68 (2H, m, DMT–O–
CH2), 3.82 (6H, s, 2 OMe of DMT), 3.90 and 4.07 (2H, 2s, glycine CH2, rotamers),
4.60 and 4.90 (2H, 2s, B–CH2, rotamers), 6.60–7.42 (14H, m, aromatic and H6),
11.38 (1H, bs, NH); ESI MS m=z 610.3 [M ꢂ H]ꢂ, C34H33N3O8 requires 611.70.
Anal. calcd for C34H33N3O8: C, 66.75; H, 5.44; N, 6.87. Found: C, 66.80; H, 5.62;
N, 6.95.
N-[(5-Hexynyl-uracil-1-yl)acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine triethyl-
ammonium salt (12). Yield: 87%, Rf(C): 0.30; IR n [cmꢂ1] ¼ 3200–2800 w

PNA-DNA Chimeras
1977
(OH carboxylic), 3180 w (NH), 2956 w (as CH3 OMe), 2933 w (s CH3 OMe), 1714 s
(as C¼O uracil), 1700 s (C¼O carboxylic), 1685 s (s C¼O uracil), 1654 s (>N–C¼O),
1
1608 s and 1510 s (aromatic), 1251 s, 1177 m, 1034 m; H NMR (CDCl3): d ¼ 0.85
(3H, t, CH3 of hexynyl), 1.10–1.65 (13H, m, 3 Me of TEA and 2 CH2 of hexynyl),
2.30 (2H, t, CꢁC–CH2), 3.04 (6H, q, 3, CH2 of TEA), 3.26 and 3.40 (2H, 2s,
H2C–N, rotamers), 3.55 and 3.68 (2H, 2s, DMT–O–CH2), 3.80 (6H, s, 2 OMe of
DMT), 3.92 and 4.10 (2H, 2s, glycine CH2, rotamers), 4.61 and 4.92 (2H, 2s, B–
CH2, rotamers), 6.50–7.50 (14H, m, aromatic and H6), 11.41 (1H, bs, NH); ESI
MS m=z 652.3 [M ꢂ H]ꢂ, C37H39N3O8 requires 653.78. Anal. calcd for
C37H39N3O8: C, 67.97; H, 6.01; N, 6.43. Found: C, 68.05; H, 6.12; N, 6.48.
General Method for the Coupling of PNA Monomers (10, 11 or 12) with 50-Amino-
50-deoxythymidine (13). The mixture of a PNA monomer (2.5 mmol), 13
(0.60 g ¼ 2.5 mmol) and TBTU (0.80 g ¼ 2.5 mmol) was dissolved in DMF (25 mL)
and TEA (0.70 mL ¼ 5.0 mmol) then stirred at room temperature for 2 h. The solvent
was removed then the residue was taken up with CH2Cl2 (50 mL) and washed with
brine (2 ꢄ 10 mL). The organic phase was dried with Na2SO4 and evaporated to
dryness. The residue was purified by dry column flash chromatography[35] (70 g of
Kieselgel 60 HF254, eluant: CHCl3–MeOH 1–10% þ 1% TEA). The pure fractions
were combined, evaporated and dried over P2O5 to give the following coupled
products:
N-[(Thymin-1-yl)acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxythymidin-
50-yl)amide (14). Yield: 59%, Rf(D): 0.68; IR n [cmꢂ1] ¼ 3180 w (NH), 1716 s
(as C¼O uracil), 1685 s (–NH–C¼O), 1672 s (s C¼O uracil), 1650 s (>N–C¼O),
1608 m and 1510 m (aromatic), 1252 m, 1178 w, 1033 w; 1H NMR (CDCl3):
d ¼ 1.70–1.95 (6H, 3s, 2 Me of T-s, rotamers), 2.55–2.72 (2H, m, H20ab), 3.15–3.60
(6H, m, H50ab and DMT–O–(CH2)2), 3.80 (6H, s, 2 OMe of DMT), 4.20–4.42
(3H, m, H40 and glycine CH2), 4.70–5.05 (3H, m, H30 and B–CH2), 5.33 (1H, d,
30-OH), 6.05 (1H, t, 50-NH), 6.22 (1H, dd, Hi0), 6.75–7.50 (15H, 2m, aromatic þ 2
H6), 11.28–11.35 (2H, bs, 2 NH); ESI MS m=z 817.3 [M þ Li]þ, C42H46N6O11
requires 810.91. Anan. calcd for C42H46N6O11: C, 62.20; H, 5.72; N, 10.36. Found:
C, 62.07; H, 5.85; N, 10.25.
N-[(5-Propynyl-uracil-1-yl)acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)amide (15). Yield: 82%, Rf(D): 0.70; IR n [cmꢂ1] ¼ 3200w (NH),
1713s (as C¼O uracil), 1686s (–NH–C¼O), 1670s (s C¼O uracil), 1656 s (>N–
1
C¼O), 1607 w and 1509m (aromatic), 1465m, 1250m, 1178w, 1033w; H NMR
(CDCl3): d ¼ 1.85 (3H, s, 5-CH3 of T), 2.03 (3H, s, CꢁCCH3), 2.48–2.70 (2H, m,
H20ab), 3.20–3.63 (6H, m, H50ab and DMT–O–(CH2)2), 3.82 (6H, s, 2 OMe of
DMT), 4.25–4.40 (3H, m, H40 and glycine CH2), 4.70–4.98 (3H, m, H30 and B–
CH2), 5.50–5.80 (2H, m, 30OH and 50-NH), 6.10 (1H, dd, H10), 6.80–7.50 (15H, 2 m,
aromatic and 2 H6), 11.30–11.38 (2H, bs, 2 NH); ESI MS m=z 841.3 [M þ Li]þ,
C44H46N6O11 requires 834.93. Anal. calcd for C44H46N6O11: C, 63.29; H, 5.55; N,
10.07. Found: C, 63.14; H, 5.71; N, 9.98.

1978
Bajor et al.
N-[(5-Hexnyl-uracil-1-yl)acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)amide (16). Yield: 77%, Rf(D): 0.77; IR n [cmꢂ1] ¼ 3180 w (NH),
1716 s (as C¼O uracil), 1687 s (–NH–C¼O), 1670 s (s C¼O uracil), 1653 s (>N–
1
C¼O), 1608 w and 1510 m (aromatic), 1465 m, 1252 m, 1178 w, 1034 w; H NMR
(CDCl3): d ¼ 0.90 (3H, t, CH3 of hexynyl), 1.35–1.70 (4H, m, (CH2)2 of hexynyl),
1.88 (3H, s, 5-CH3 of T), 2.39 (2H, t, CꢁC–CH2), 2.72–2.90 (2H, m, H20ab), 3.20–3.65
(6H, m, H50ab and DMT–O–(CH2)2), 3.80 (6H, s, 2 OMe of DMT), 4.20–4.50
(3H, m, H40 and glycine CH2), 4.70–4.90 (3H, m, H30 and B–CH2), 5.32 (1H, d, 30-
OH), 5.75 (1H, t, 50-NH), 6.15 (1H, dd, H10), 6.80–7.50 (15H, 2 m, aromatic and 2
H6), 11.32–11.41 (2H, bs, 2 NH); ESI MS m=z 883.3 [M þ Li]þ, C47H52N6O11 requires
877.01. Anal. calcd for C47H52N6O11: C, 64.36; H, 5.98; N, 9.58. Found: C, 64.21; H,
6.11; N, 9.47.
General Procedure for the Phosphitylation of Chimeric Dimers. The chimeric
dimer (14, 15 or 16) was dissolved in dry CH2Cl2 (20 mL) then diisopropylammo-
nium tetrazolide (DIPAT, 1.5 mmol) and bis-diisopropylamino-2-cyanoethyl-phos-
phite (0.87 mL ¼ 3.0 mmol) were added with stirring in Ar atmosphere. The
mixture was left to stir at ambient temperature overnight then it was diluted with
CH2Cl2 (30 mL) and washed with cold, 2% (w=v) aq. Na2CO3 (2 ꢄ 20 mL) then with
brine (20 mL). The CH2Cl2 solution was dried with Na2SO4, filtered and evaporated
to dryness. The residue was dissolved in EtOAc-TEA 19:1 (5 mL) and applied to a
silica gel column made of Kieselgel 60 (0.04–0.06 mm, 70 g) which was eluted with
linear gradient of EtOAc-TEA (5%)! EtOAc-MeOH (5%)–TEA (5%) (300–
300 mL). The appropriate pure fractions were combined, evaporated and dried in
vacuo, over P2O5 to give the required 30-O-(b-cyanoethyl-N,N-diisopropyl)-phos-
phoramidites (17, 18 and 19), as white solid foams.
N-(Thymin-1-yl-acetyl)-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxythymidin-
50-yl)amide-30-O-(N,N-diisopropylamino-2-cyanoethyl)phosphite (17). Yield: 84%,
1
Rf(E): 0.50; H NMR (CDCl3): d ¼ 1.05–1.20 (12H, 4s, 4Me), 1.81 (3H, s, 5-Me
of T), 1.87 (3H, s, 5-Me of t), 2.20–2.52 (2H, m, H20ab), 2.60 (2H, t, CH2–O),
3.17–3.30 (2H, m, HCMe2), 3.35–3.45 (2H, m, H50ab), 3.52–3.73 (6H, m, DMT–
O–(CH2)2 and NC–CH2), 3.78 (6H, s, OMe), 4.07 and 4.18 (2H, 2s, glycine CH2),
4.44 (1H, m, H40), 4.77 and 4.90 (2H, 2s, B–CH2), 5.12 (1H, m, H30), 5.85 (1H, t,
50-NH), 6.26 (1H, dd, H10), 6.75–7.48 (15H, m, aromatic and 2 H6), 11.20–11.35
(2H, bs, 2NH) 31P NMR (CDCl3): d ¼ 148.7 and 149.0 ppm. Anal. calcd for
C51H63N8O12P: C, 60.58; H, 6.28; N, 11.08; P, 3.06. Found: C, 60.34; H, 6.46; N,
11.02; P, 2.98.
N-[(5-Propynyl-uracil-1-yl)-acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)amide-30-O-(N,N-diisopropylamino-2-cyanoethyl)phosphite (18). Yield:
60%, Rf(E): 0.55; 1H NMR (CDCl3): d ¼ 1.02–1.16 (12H, 4s, 4Me), 1.79 (3H, s, 5-Me
of T), 1.98 (3H, s, CꢁC–Me), 2.21–2.54 (2H, m, H20ab), 2.62 (2H, t, CH2–O), 3.16–
3.28 (2H, m, HCMe2), 3.30–3.48 (2H, m, H50ab), 3.53–3.73 (6H, m, DMT–O–(CH2)2
and NC–CH2), 3.80 (6H, s, OMe), 4.10 and 4.21 (2H, 2s, glycine CH2), 4.47 (1H, m,
H40), 4.79 and 4.93 (2H, 2s, B–CH2), 5.15 (1H, m, H30), 5.90 (1H, t, 50-NH), 6.28

PNA-DNA Chimeras
1979
(1H, dd, H10), 6.72–7.53 (15H, m, aromatic and 2 H6), 11.20–11.33 (2H, bs, 2NH) 31P
NMR (CDCl3): d ¼ 149.4 ppm. Anal. calcd for C53H63N8O12P: C, 61.49; H, 6.13; N,
10.83; P, 2.99. Found: C, 61.37; H, 6.32; N, 10.70; P, 2.87.
N-[(5-Hexynyl-uracil-1-yl)-acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)amide-30-O-(N,N-diisopropylamino-2-cyanoethyl)phosphite (19). Yield:
1
59%, Rf(E): 0.59; H NMR (CDCl3): d ¼ 0.70 (3H, t, Me of hexynyl), 1.05–1.36
(16H, m, 4Me and 2CH2 of hexynyl), 1.81 (3H, s, 5-Me of T), 2.21–2.32 (1H, m,
H20a), 2.40 (2H, t, CꢁC–CH2), 2.43–2.53 (1H, m, H20b), 2.62 (2H, t, CH2–O), 3.14–
3.30 (2H, m, HCMe2), 3.33–3.48 (2H, m, H50ab), 3.52–3.73 (6H, m, DMT–O–(CH2)2
and NC–CH2), 3.80 (6H, s, OMe), 4.12 and 4.21 (2H, 2s, glycine CH2), 4.40 (1H, m,
H40), 4.80 and 4.94 (2H, 2s, B–CH2), 5.15 (1H, m, H30), 5.94 (1H, t, 50-NH), 6.28 (1H,
dd, H10), 6.70–7.55 (15H, m, aromatic and 2 H6), 11.22–11.36 (2H, bs, 2NH) 31P
NMR (CDCl3): d ¼ 148.9 and 148.6 ppm. Anal. calcd for C56H69N8O12P: C, 62.43; H,
6.46; N, 10.40; P, 2.87. Found: C, 62.30; H, 6.58; N, 10.27; P, 2.71.
General Procedure for the Succinylation of Chimeric Dimers. A 30-free dimer
(14, 15 or 16) (0.15 mmol) was dissolved in dry CH2Cl2 (4.0 mL) then TEA
(0.30 mmol ¼ 40 mL), 4-dimethylamino-pyridine (28 mg ¼ 0.23 mmol) and succinic
anhydride (45 mg ¼ 0.45 mmol) were added. The mixture was stirred at room tem-
perature overnight, then it was diluted with CH2Cl2 (30 mL), transferred to a separa-
tory funnel, washed with 10% (w=v) aq. NaH2PO4 (2 ꢄ 15 mL) and water (15 mL).
The organic phase was dried with Na2SO4, filtered and evaporated to dryness.
The crude product was purified by vacuum flash chromatography on a Kieselgel
60 HF254(30 g) column using CHCl3–MeOH (1–10%)-TEA (1%), as eluant. The pure
fractions were combined and evaporated then dried in vacuo, over P2O5. Thus we
isolated the corresponding dimer-30-O-succinate triethylammonium salts (20, 21
and 22), as white solids.
N-(Thymin-1-yl-acetyl)-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxythymidin-
50-yl)amide-30-O-succinate triethylammonium salt (20). Yield: 95%, Rf(D): 0.40; IR
n [cmꢂ1] ¼ 3180 w (NH), 3150–2800 w (OH carboxylic), 1737 s (C¼O ester), 1717 s
(as C¼O uracil), 1695 s (–NH–C¼O), 1675 s (C¼O carboxylic), 1656 s (>N–C¼O),
1
1608 w and 1510 m (aromatic), 1464 m, 1250 m, 117 w; H NMR (CDCl3): d ¼ 1.20
(9H, t, 3 Me of TEA), 1.78 (3H, s, 5-Me of T.), 1.92 (3H, s, 5-Me of t), 2.25–2.40
(2H, m, H20ab), 2.48–2.70 (4H, m, 2 CH2 of Su), 2.90 (6H, q, 3 CH2 of TEA),
3.38–3.47 (2H, m, H50ab), 3.55–3.72 (4H, m, DMT–O–(CH2)2), 3.80 (6H, s, 2
OMe), 4.10 and 4.19 (2H, 2s, glycine CH2), 4.50 (1H, m, H40), 4.80 and 4.90 (2H,
2s, B–CH2), 5.15 (1H, m, H30), 5.85 (1H, t, 50-NH), 6.05 (1H, t, H10), 6.80–7.40
(15H, 2m, 13 aromatic and 2 H6); ESI MS m=z 917.1 [M þ Li]þ, C46H50N6O14
requires 910.98. Anal. calcd for C46H50N6O14: C, 60.64; H, 5.53; N, 9.23. Found:
C, 60.71; H, 5.72; N, 9.30.
N-[(5-Propynyl-uracil-1-yl)-acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)amide-30-O-succinate triethylammonium salt (21). Yield: 77%,

1980
Bajor et al.
Rf(D): 0.44; IR n [cmꢂ1] ¼ 3150–2800 w (OH carboxylic), 3180 w (NH), 1739 s (C¼O
ester), 1715 s (as C¼O uracil), 1695 s (–NH–C¼O), 1670 s (C¼O carboxylic), 1660 s
(>N–C¼O), 1608 w and 1510 m (aromatic), 1463m, 1251 m, 117 w; 1H NMR (CDCl3):
d ¼ 1.25 (9H, t, 3 Me of TEA), 1.82 (3H, s, 5-CH3 of T), 2.03 (3H, s, CꢁC–CH3), 2.22–
2.42 (2H, m, H20ab), 2.46–2.72 (4H, m, (CH2)2 of Su), 2.97 (6H, q, 3 CH2 of
TEA), 3.35–3.45 (2H, m, H50ab), 3.50–3.75 (4H, m, DMT–O–(CH2)2), 3.79 (6H, s,
2OMe of DMT), 4.13 and 4.24 (2H, 2s, glycine CH2), 4.53 (1H, m, H40), 4.81 and 4.92
(2H, 2s, B–CH2), 5.19 (1H, m, H30), 5.90 (1H, t, 50-NH), 6.12 (1H, t, H10), 6.75–7.44
(15H, 2m, aromatic þ 2 H6); ESI MS m=z 941.2 [M þ Li]þ, C48H50N6O14 requires
935.00. Anal. calcd for C48H50N6O14: C, 61.66; H, 5.39; N, 8.99. Found: C, 61.78; H,
5.52; N, 9.10.
N-[(5-Hexynyl-uracil-1-yl)-acetyl]-N-(2-dimethoxytrityloxy-ethyl)glycine-(50-deoxy-
thymidin-50-yl)-amide-30-O-succinate triethylammonium salt (22). Yield: 86%, Rf(D):
0.50; IR n [cmꢂ1] ¼ 3180w (NH), 1741s (C¼O ester), 1715s (as C¼O) uracil),
1695s (–NH–C¼O), 1680s (C¼O carboxylic), 1667s (s C¼O uracil), 1658s (>N–
1
C¼O), 1607w and 1510m (aromatic), 1462m, 1251m, 1177w; H NMR (CDCl3):
d ¼ 0.83 (3H, t, CH3 of hexynyl), 1.28–1.62 (13H, m, 3 Me of TEA þ CH2)2 of hex-
ynyl), 1.83 (3H, s, 5–CH3 of T), 2.20–2.45 (4H, m, H20ab and CꢁC–CH2), 2.48–
2.75 (4H, m, (CH2)2 of Su), 3.00 (6H, q, 3 CH2 of TEA), 3.32–3.44 (2H, m, H50ab),
3.48–3.74 (4H, m, DMTO–(CH2)2), 3.80 (6H, s, 2 OMe), 4.15 and 4.25 (2H, 2s, glycine
CH2), 4.50 (1H, m, H40), 4.78 and 4.87 (2H, 2s, B–CH2), 5.17 (1H, m, H30), 5.95 (1H, t,
50–NH), 6.15 (1H, t, H10), 6.73–7.50 (15H, 2m, aromatic þ 2 H6); ESI MS m=z 983.2
[M þ Li]þ, C51H56N6O14 requires 977.08. Anal. calcd for C51H56N6O14: C, 62.69; H,
5.78; N, 8.60. Found: C, 62.77; H, 5.92; N, 8.72.
General Method for the Binding of 30-O-Succinates to LCAA-CPG. The mix-
ture of a dimer-30-succinate (20, 21 or 22) (0.50 mmol), LCAA-CPG (pore size:
˚
500 A) (0.25 g ꢀ 10 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
(124 mg ¼ 0.50 mmol) were suspended in dry CH3Cl2 (8.0 mL) and rotated slowly
for 2 days. The solid support was filtered, washed with CH2Cl2 (2 ꢄ 5 mL) and
CH3CN (5 mL), then it was resuspended in the mixture of CAP A (5 mL) and
CAP B (5 mL) reagents and rotated for additional 4 h in order to completely acety-
late the unreacted NH2 groups. The loaded CPG was filtered, washed with dry THF
(2 ꢄ 5 mL) and CH2Cl2 (5 mL) finally dried in dessicator over P2O5 and paraffin
shavings. The loadings, by DMT assays, were found to be 43, 38 and 35 mmol=g
for 20, 21 and 22, respectively.
ACKNOWLEDGMENTS
Financial supports of OTKA (Hungarian Scientific Research Fund, project no.:
T 026472) and NKFP MediChem (project no.: 1=047) are gratefully acknowledged.
´
The authors thank Mrs. Emma Belinszki and Mrs. Maria Baraczka for the valuable
technical assistance.

PNA-DNA Chimeras
1981
ABBREVIATIONS
SV PDE
BS PDE
TBTU
snake venom phosphodiesterase
bovine spleen phosphodiesterase
O-(benzotriazol-1-yl)-N,N,N1,N1-tetramethyluronium
tetrafluoroborate
DMT
4,40-dimethoxytriphenylmethyl
LCAA-CPG
EEDQ
DEAE
long-chain alkylamino controlled pore glass
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
diethylaminoethyl
DCFC
DIPAT
DMAP
dry column flash chromatography
diisopropylammonium tetrazolide
4-dimethylamino-pyridine
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Received March 3, 2003
Accepted July 18, 2003