Influence of the type of junction in DNA-3'-peptide nucleic acid (PNA) chimeras on their binding affinity to DNA and RNA

Article abstract of DOI:10.1002/(SICI)1522-2675(19991215)82:12<2151::AID-HLCA2151>3.0.CO;2-0

The automated on-line synthesis of DNA-3'-PNA (PNA = Polyamide Nucleic Acids) chimeras 1-3 is described, in which the 3'-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X = S), a N-(2-

Full text of DOI:10.1002/(SICI)1522-2675(19991215)82:12<2151::AID-HLCA2151>3.0.CO;2-0

Helvetica Chimica Acta ± Vol. 82 (1999)
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Influence of the Type of Junction in DNA-3'-Peptide Nucleic Acid
(PNA) Chimeras on Their Binding Affinity to DNA and RNA
1
by Beate Greiner, Gerhard Breipohl, and Eugen Uhlmann* )
Hoechst Marion Roussel Deutschland GmbH, Chemical Research G 838, D-65926 Frankfurt a.M.
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday
The automated on-line synthesis of DNA-3'-PNA (PNA ˆ Polyamide Nucleic Acids) chimeras 1 ± 3 is
described, in which the 3'-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA
either via a N-(2-mercaptoethyl)- (X ˆ S), a N-(2-hydroxyethyl)- (X ˆ O), or a N-(2-aminoethyl)- (X ˆ NH)
N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3'-PNA chimera 4 without a nucleobase at the
linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their Tm
values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to
the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable
duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on
duplex stabilization was determined to be in the order O > S ꢀ NH, rendering the phosphodiester bridge the
most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base
mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest
that the base at the linking unit contributes significantly to duplex stabilization.
Introduction. ± Peptide or Polyamide Nucleic Acids (PNAs) are nucleic acid
mimetics, in which the entire sugar-phosphate backbone is replaced by non-ionic N-(2-
aminoethyl)glycine units [1]. They bind to complementary DNA or RNA targets with
higher affinity than natural oligonucleotides following the Watson-Crick rules of base
pairing [2], and they are resistant to enzymatic degradation [3]. These properties make
them attractive candidates for the use as antisense therapeutics and diagnostics [4].
Recently, we have shown that some limitations of PNAs, such as low cellular uptake,
ambiguous orientation on hybridization (parallel or antiparallel), the inability to
activate RNase H, and the propensity to self-aggregation, can be overcome by using
DNA-PNA chimeras [5][6]. In these chimeras (see 1), the 3'-part of the oligodeoxy-
nucleotide was linked to a terminal (hydroxyethyl)glycine unit (X ˆ O) of the PNA via
a phosphodiester linkage [5][7]. For PNA-5'-DNA chimeras with the 5'-part of the
DNA linked to the carboxy terminus of the PNA, a significant influence of the type of
linkage at the DNA/PNA junction has been reported previously [8]. Here, we describe
the synthesis and binding properties of novel DNA-3'-PNA chimeras containing all
four nucleobases, which are linked by a N-(2-mercaptoethyl)glycine (2; X ˆ S) or an
N-(2-aminoethyl)-glycine (3; X ˆ NH) [9] unit, respectively. To aid the optimal future
design of DNA-3'-PNA chimeras, we directly compare the binding affinity of all three
differently linked DNA-3'-PNA chimeras 1 ± 3 in the duplex with complementary
1
)
Fax: ‡ 49-69-305 5595; e-mail: eugen.uhlmann@hmrag.com

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DNA and RNA. To evaluate the contribution of the linking moiety in these chimeras
to their binding affinity, we have also prepared and investigated the DNA-3'-PNA
chimera 4 with an abasic N-(2-hydroxyethyl)glycine unit at the DNA/PNA junction
(R ˆ H, X ˆ O).
Results and Discussion. ± Synthesis of the Monomeric Linker Units. For the
incorporation of the abasic linker unit (X ˆ O, R ˆ H) at the DNA/PNA junction of the
chimeras, we synthesized first the orthogonally protected monomeric linker unit 8
(Scheme 1). Reductive amination of 2-aminoethanol with glyoxylic acid in H2O with H2
as reducing agent and Pd/C as catalyst [10] resulted in N-(2-hydroxyethyl)glycine (6) in
83% yield. The amino function of 6 was then protected by reaction with (9H-fluoren-9-
yl) methyl N-succinimidyl carbonate in dioxane to give the [(9H-fluoren-9-yl)methoxy]-
carbonyl (Fmoc) derivative 7 in 77% yield. Finally, the abasic linker unit 8 was
obtained in 70% yield by reaction of the OH group of 7 with (4-methoxyphenyl)di-
phenylmethyl (Mmt) chloride in pyridine.
The monomeric linker unit 11 (Scheme 2), which is based on N-(2-mercapto-
ethyl)glycine, was prepared from 2-mercaptoethylamine hydrochloride by reaction with
bis(4-methoxyphenyl)phenylmethyl (Dmt) chloride and successive N-alkylation with
ethyl 2-bromoacetate and NEt3 in DMF to give N-[2-(Dmt-thio)ethyl]glycine ethyl
ester (10). Intermediate 10 was coupled without further purification with thymine-3-
Scheme 1

Helvetica Chimica Acta ± Vol. 82 (1999)
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Scheme 2
acetic acid using O-{[(2-cyanoethoxycarbonyl)methylidene]amino}-1,1,3,3-tetramethyl-
uronium tetrafluoroborate (TOTU) and then saponified with 2n NaOH in dioxane/
H2O 2 :1 (v/v). After purification of the crude product by silica-gel chromatography
with 1% NEt3 in CH2Cl2/MeOH, the (mercaptoethyl)glycine-based monomer 11 was
obtained in 32% overall yield.
Solid-Phase Synthesis of DNA-3'-PNA Chimeras 15 ± 18. Mmt-protected monomers
12 based on N-(2-aminoethyl)glycine and a universal 6-aminohexanol-derived CPG
solid support 14 [10] were used for the synthesis of the PNA part of the DNA-3'-PNA
(Scheme 3) [5][10][11]. After synthesis of the PNA part of the chimeras, the novel
linker units 8 or 11, respectively, were coupled at the junction to allow on-line synthesis
of the DNA part of the chimeras by standard phosphoramidite chemistry [12], which,
after cleavage from the support and deprotection, yielded the chimeras 15 and 17,
respectively. The DNA-3'-PNA chimera 16 was synthesized using the N-(2-hydroxy-
ethyl)glycine-derived monomer 13 [13] as linker unit, while the phosphoramidate-
linked chimera 18 was synthesized with the standard PNA monomer 12 and subsequent
coupling of a protected nucleoside phosphoramidite to the terminal amino group of the
PNA [9]. All DNA-3'-PNA chimeras were synthesized at 2-mmol scale. Deprotection of
the Mmt or Dmt group was carried out with 3% trichloroacetic acid (TCA) in CH2Cl2
including one intermediate washing step with MeCN and subsequent neutralization of
the support with EtN(i-Pr)2 (DIPEA; or Huenigꢀs base). For efficient coupling of the
monomers, a pre-activation step with either O-(1H-benzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate (HBTU), O-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU), or (7-aza-1H-benztriazol-1-yloxy)-
tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) in the presence of
DIPEA was performed, whereby the required pre-activation time strongly depended

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Helvetica Chimica Acta ± Vol. 82 (1999)
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on the type of coupling reagent. While HATU and PyAOP gave sufficient coupling
efficiencies (90 ± 96%) using short pre-activation times (10 ± 60 s), HBTU required
longer pre-activation (15 min) to obtain similar results. Unreacted amino functions
were capped with the standard DNA-capping mixture consisting of Ac2O/N-
methylimidazole in THF to prevent the growth of failure sequences due to incomplete
coupling reactions.
The orthogonally protected monomeric PNA building blocks with base-labile
protecting groups at the heterocyclic bases and an acid-labile Mmt group for temporary
protection of the terminal amino function, or a Dmt group for the OH and SH functions,
respectively, are fully compatible with standard phosphoramidite DNA synthesis.
Consequently, the protected chimeras could be cleaved from the solid support (2 h at
508) and simultaneously deprotected by treatment with concentrated aqueous NH3 (16 h
at 508). The crude products were purified by denaturing preparative polyacrylamide gel
electrophoresis (PAGE) and desalted via C-18 columns. All DNA-3'-PNA chimeras
were characterized by ion-exchange HPLC using a Gen Pack Fax column (Millipore-
Waters) and by negative-ion electrospray mass spectroscopy (see Exper. Part).
Binding Affinity of the DNA-3'-PNAChimeras. To study the influence of the type of
linking unit at the DNA/PNA junction on the binding affinity of the chimeras, the UV
melting curves of the corresponding duplexes with DNA and RNA were measured at
260 nm under physiological salt conditions (140 mm KCl, 10 mm NaH2PO4, 0.1 mm Na-
EDTA, pH 7.4) and the Tm values were calculated. All three DNA-3'-PNA chimeras
16 ± 18, which have a nucleobase at the DNA/PNA junction, form more stable duplexes
with complementary DNA 20 or RNA 21 than the natural oligonucleotide 19 (Table 1).
The duplexes with complementary DNA were stabilized by ‡ 1.7 to ‡ 4.3 K, whereas
the stabilization for complementary RNA was even larger (DTm ‡ 4.7 to ‡ 6 K). The
influence of X in the linking unit on duplex stabilization was in the order O > S ꢀ NH,
rendering the phosphodiester bridge the most favored linkage at the DNA/PNA
junction. If the chimera was linked with just N-(2-hydroxyethyl)glycine without a
nucleobase at the junction, such as in oligomer 15, then the duplex with complementary
DNA was destabilized by 7.1 K relative to the natural duplex, and by as much as
11.4 K relative to the DNA-PNA chimera 16 with a thymine base at the junction. This
result implies that the nucleobase at the PNA unit of the junction actually forms a base
pair with the complementary base of the second strand.

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Table 1. Tma) Values of Duplexes of DNA-3'-PNA Chimeras 15 ± 18 with Complementary DNA or RNA
Tm [8C]
X
R
DNAb)
RNAc)
15
16
17
18
19d)
O
O
S
NH
±
H
42.8
54.2
52.1
51.6
49.9
43.3
54.0
52.7
53.0
48.0
R1
R1
R1
±
a) 140 mm KCl, 10 mm NaH2PO4, 0.1 mm Na-EDTA, pH 7.4
b) Complementary DNA:
c) Complementary RNA:
3'- T G T AG T A C C AG C -5' (20)
3'-UGUAGUA C C AG C -5' (21)
d) Unmodified oligonucleotide: 5'-A C A T C A T GG T C G-3' (19)
To further analyze the contribution of the nucleobase-containing linker unit, we
also measured the Tm values of the different chimeras 15 ± 18 (Table 2) against
complementary DNA having one base mismatch opposite to the base at the linking unit
(mm-DNA; 22) and against an oligonucleotide lacking the nucleobase at the junction
(ab-DNA; 23). Interestingly, introduction of a base mismatch destabilized the duplex
to a similar extent (DTm 7.7 to 11.5 K) as the removal of the nucleobase of the
linking PNA unit (DTm 11.4 K). Incorporation of an abasic unit in the comple-
mentary strand (ab-DNA; 23) directly opposite to the linker unit confirmed the
contribution of the nucleobase to binding: the Tm values of the duplexes with a base
mismatch were between 40.6 to 45.48, while the Tm values of the duplexes with an abasic
unit were between 45.1 to 47.38. Remarkably, the DNA ´ DNA duplex 23 ´ 24 with two
abasic sites opposite to each other was significantly less stable (DTm 14.5 K) than the
DNA ´ DNA-PNA duplex 15 ´ 23, in which the abasic site of the linker unit is opposite to
the abasic site in the complementary DNA.
In conclusion, all investigated DNA-3'-PNA chimeras with a nucleobase at the
junction formed more stable duplexes with complementary DNA and RNA than the
corresponding unmodified DNA. The destabilizing effect of base mismatches or abasic
Table 2. Influence of Base-Mismatches or Abasic Sites on the Tm Values
Tm [8C]
X
R
DNAa)
mm-DNAb)
ab-DNAc)
15
16
17
O
O
S
NH
±
H
R1
R1
R1
±
42.8
54.2
52.1
51.6
49.9
34.0
45.6
45.4
40.6
43.9
±
46.0
47.3
45.5
45.1
31.5
31.5
18
19d)
24e)
±
±
±
a) Complementary DNA:
b) Complementary mismatch-DNA: 3'- T G T AG T T C C AG C -5' (22)
c) Complementary abasic-DNA:
3'- T G T AG T D C C AG C -5' (23)
d) Unmodified sense oligonucleotide: 5'-A C A T C A T GG T C G-3' (19)
e) Abasic sense oligonucleotide:
5'-A C A T C ADGG T C G-3' (24)
D: abasic linker derived from phosphoric acid mono-[2-(hydroxymethyl)tetrahydrofuran-3-yl] ester
3'- T G T AG T A C C AG C -5' (20)

Helvetica Chimica Acta ± Vol. 82 (1999)
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sites opposite to the nucleobase at the DNA/PNA junction suggests that this base very
likely contributes to duplex stabilization through a Watson-Crick base pair. Further-
more, the phosphoramidite linkage (X ˆ NH) may be an attractive alternative to the
phosphodiester linkage (X ˆ O) in DNA-3'-PNA chimeras, especially when hybridized
to complementary RNA, since no additional building block for its incorporation is
required.
We thank H. Wenzel, S. Hein, and L. Hornung for expert technical assistance, and Dr. A. Schäfer for
measurement of mass spectra. The work was funded in part by the Deutsche Forschungsgemeinschaft and
Bundesministerium für Forschung und Technologie (BMBF; project No. 0311143).
Experimental Part
1. General. The peptide coupling reagents HATU/1-hydroxy-7-aza-1H-benzotriazole (HOAt), and PyAOP
were purchased from Perseptives Biosystems, HBTU, (9H-fluoren-9-yl)methyl N-succinimidyl carbonate
(Fmoc-ONSu) and aminopropyl CPG from Fluka (Neu-Ulm, Germany). Standard nucleoside phosphorami-
dites and the reagent for introduction of the abasic linker (dSpacer phosphoramiditeꢁ) were purchased from
Eurogentec (Seraing, Belgium). TLC was carried out on Merck DC Kieselgel 60 F-254 glass plates. HPLC
Analysis of PNAs: on a Beckman System Gold HPLC system, with a NaCl gradient (buffer A: 10 mm NaH2PO4,
100 mm NaCl in MeCN/H2O 1:4 (v/v), pH 6.8; buffer B: 10 mm NaH2PO4, 1.5m NaCl in MeCN/H2O 1:4 (v/v); 0
to 30% B in 30 min). Tm Values [8C Æ 0.3] were measured under approximately physiological salt conditions
(140 mm KCl, 10 mm NaH2PO4, 0.1 mm Na-EDTA, pH 7.4) in the cooling phase from 908 to 108 with a temp.
1
ramp of 0.38/min at 260 nm on a Varian Carey 1 Bio UV/VIS Spectrometer at 1 mm oligomer concentration.
1H-NMR Spectra: at 270 MHz in the solvents indicated; chemical shifts (d) in ppm downfield relative to the
internal standard. MS: either fast atom bombardement (FAB), electrospray (ES), or direct chemical ionization
(DCI). NBA: 3-Nitrobenzyl alcohol.
2. Monomeric Building Units. N-(2-Hydroxyethyl)glycine (6). Glyoxylic acid monohydrate (46 g,
500 mmol) was dissolved in H2O (1 l), and 2-aminoethanol (30.2 ml, 500 mmol) was added under stirring and
cooling. The mixture was treated with 10% Pd/C catalyst (10 g) and hydrogenated (10 bar) in the autoclave at
r.t. Then, the catalyst was filtered off, and the filtrate was concentrated in vacuo. The residue was co-evaporated
twice with a small amount of toluene. The crude product was triturated using 250 ml of hot MeOH, filtered off,
washed with MeOH, and dried: 49.56 g (83%). M.p. 178 ± 1808(dec.). TLC (silica gel; BuOH/AcOH/H2O/
AcOEt 1:1:1:1 (v/v/v/v)): Rf 0.36. 1H-NMR (D2O): 3.77 (2 H, (m, HOCH2); 3.68 (s, CH2COOH); 3.22
‡
(m,CH2NH). DCI-MS: 120 ([M ‡ H] ).
N-[(9H-Fluoren-9-yl)methoxycarbonyl]-N-(2-hydroxyethyl)glycine (7). NaHCO3 (10.08 g, 120 mmol) was
dissolved in H2O (150 ml) and 6 (7.15 g, 60 mmol) was added under stirring. After a clear soln. was obtained,
Fmoc-ONSu (20.22 g, 60 mmol) in dioxane (300 ml) was added dropwise. After the final addition, stirring was
continued for 3 h at r.t. The soln. was filtered, and the filtrate was concentrated in vacuo. The residue was solved
in H2O (100 ml), and the pH was adjusted to 2 with an KHSO4 soln. Then, the mixture was extracted with
AcOEt (3 Â 150 ml), the combined org. layers were washed with H2O (4 Â 50 ml), dried (Na2SO4), filtered, and
evaporated in vacuo. Precipitation was achieved with (i-Pr)2O. The precipitate was filtered off, recrystallized
from AcOEt/(i-Pr)2O, filtered, and dried: 15.8 g (77%) of 7. M.p. 114 ± 1168 (dec.). TLC (silica gel; BuOH/
AcOH/H2O 3 : 1 : 1 (v/v/v)): Rf 0.62. 1H-NMR ((D6)DMSO): 12.71 (br. s, 1 H); 7.24 ± 7.95 (m, 8 H); 4.63 (br. s,
‡
1 H); 4.19 ± 4.39 (m, 3 H); 3.92 ± 4.11 (m, 2 H); 3.11 ± 3.56 (m, 4 H). ES-MS (pos. mode): 342 ([M ‡ H] ).
N-[(9H-Fluoren-9-yl)methoxycarbonyl]-N-{2-[(4-methoxyphenyl)diphenylmethoxy]ethyl}glycine
(8).
Compound 7 (14.74 g, 43 mmol) was dissolved in pyridine (160 ml), (4-methoxyphenyl)diphenylmethyl
chloride (13.31 g, 43 mmol) was added, and the mixture was stirred for 2 h at r.t. After evaporation in vacuo, the
residue was taken up in AcOEt (300 ml) and extracted with sat. aq. NaHCO3 (3 Â 30 ml) and H2O (3 Â 30 ml).
The org. layer was dried (Na2SO4), filtered, evaporated, and precipitated with (i-Pr)2O. The product was filtered
and dried. 19.95 g (70%) of 8. TLC (silica gel; AcOEt/MeOH/Et3N 60 : 40 : 1 (v/v/v)): Rf 0.45. 1H-NMR
((D6)DMSO): 6.80 ± 7.95 (m, 22 H); 4.09 ± 4.31 (m, 3 H); 3.80, 3.91 (2s, 2 H); 3.76 (s,3 H); 2.92 ± 3.56 (m, 4 H).
‡
FAB-MS (MeOH/NBA): 658.3 ([M ‡ 2Na] ).
N-(2-{[Bis(4-methoxyphenyl)phenylmethyl]thio}ethyl)-N-[(thymin-1-yl)acetyl]glycine (11). To a soln. of
Dmt-Cl (10.17 g, 30 mmol) in AcOH (100 ml), 2-mercaptoethylamine hydrochloride (4.43 g, 39 mmol)

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Helvetica Chimica Acta ± Vol. 82 (1999)
dissolved in H2O (70 ml) was added. The soln. was stirred for 2 h at r.t. and then evaporated in vacuo to a volume
of 50 ml. After addition of H2O (200 ml), the pH was adjusted to 10 using 2n NaOH. The mixture was extracted
with AcOEt (2 Â 100 ml), and the combined org. phases were washed with sat. NaCl soln., dried, filtered, and
concentrated in vacuo (14.75 g). Part of the resulting crude product (12.56 g) was dissolved in dry DMF
(100 ml), treated with Et3N (4.6 ml, 33 mmol) and ethyl bromoacetate (3.66 ml, 33 mmol) and stirred for 3 h.
The soln. was concentrated in vacuo, and the residue was taken up in AcOEt (150 ml), washed with H2O (4 Â
40 ml). Then, the org. layer was dried (Na2SO4), filtered, and evaporated in vacuo (12.92 g). Part of the
resulting crude product (10; 5.95 g) was dissolved in dry DMF (50 ml) and (thymin-2-yl)acetic acid (2.36 g,
12.8 mmol), Et3N (4.45 ml, 32 mmol), and TOTU (4.2 g, 12.8 mmol) were added to the soln. The mixture was
stirred for 2 h at r.t., then evaporated in vacuo. The residue was taken up in AcOEt (150 ml) and washed with
sat. NaHCO3 soln. (3 Â 10 ml) and H2O (2 Â 10 ml). The org. layer was dried (Na2SO4), filtered, and
concentrated in vacuo. The resulting residue was dissolved in a mixture of dioxane (80 ml) and H2O (40 ml),
and the ester was hydrolyzed by adding 2n NaOH soln. in portions (13 ml). The soln. was concentrated in vacuo
to a volume of 50 ml and extracted with AcOEt (4 Â 50 ml). The pH of the aq. layer was adjusted to 5 with 2n
HCl, and the soln. was then extracted with AcOEt (4 Â 50 ml). The combined org. layers were washed twice
with H2O (30 ml), dried (Na2SO4), filtered, and evaporated in vacuo. The crude product was purified by silica-
gel chromatography using a step gradient of 0 ± 5% MeOH in CH2Cl2 (1% Et3N in all eluents). The fractions
containing the product were pooled and dried in vacuo: 2.38 g (32% from Dmt-Cl) of 11. TLC (silica gel;
‡
CH2Cl2/MeOH/Et3N 100 : 10 : 1 (v/v/v)): Rf 0.38. FAB-MS (NBA ‡ LiCl): 610.2 ([M ‡ Li] ), 616.2 ([M ‡ 2Li
‡
H] ). 1H-NMR ((D6)DMSO): 11.4 (s, H C(3) of thymine); 6.91 ± 7.38 (m, 14 H, Dmt, H C(6) of thymine);
4.16, 4.39 (2s, NCOCH2); 3.69 (2s, 2 MeO); 3.56, 3.61 (2s, NCH2COOH); 2.98 (m, CH2N); 2.31 (m, CH2S); 1.76
(s, Me of thymine).
2. Solid-Phase Synthesis of the DNA-3'-PNA Chimeras. The Mmt/acyl-protected monomer 12 and Dmt/
acyl-protected monomer 13 were prepared as described in [13]. The Mmt-protected 6-aminohexan-1-ol/
succinylamidopropyl CPG [10] with a loading 36 mmol/g was used as solid-support. Synthesis was performed on
a modified Eppendorf Biotronik Ecosyn D-300 DNA synthesizer or on an ABI 394 DNA synthesizer at 2-mmol
scale. The DNA part was synthesized according to standard procedures [12]. The following synthesis conditions
were used for the synthesis of the PNA part including the N-(2-mercaptoethyl)glycine, N-(2-hydroxyethyl)-
glycine, or N-(2-aminoethyl)glycine derived linkers: 1) washing step with MeCN; 2) deprotection of the Mmt
group: 3% TCA in CH2Cl2; 110 s total treatment time interrupted by one wash with MeCN for 20 s; 3) washing
step with DMF/MeCN 1 :1 (v/v); 4) syringe wash; 5) neutralization: washing with DMF/MeCN 1:1 (v/v) and
EtN(i-Pr)2; 6) coupling of monomers: Monomers (0.2 to 0.3m solutions in DMF); etN(i-Pr)2 (0.2 to 0.3m in
DMF); coupling reagent (0.2 ± 0.3m in DMF); reagents were pre-mixed, pre-activated and delivered onto the
solid support. Due to the use of different coupling reagents, we had to adjust the pre-activation and reaction
time; 7) capping. Reagent A: 10% Ac2O/10% lutidine in THF; reagent B: 16% N-methyl-1H-imidazole in THF
(the DNA capping reagents A and B were mixed just before use).
After synthesis was complete, the chimeras were cleaved from the support (2.5 h at 508) and deprotected
(6 h at 508) with concentrated aq. NH3 soln. The crude product was analyzed by HPLC using a Gen Pack Fax
column (Millipore-Waters), eluting with a NaCl gradient (buffer A: 10 mm NaH2PO4, 100 mm NaCl in MeCN/
H2O 1:4 (v/v) pH 6.8; buffer B: 10 mm NaH2PO4, 1.5m NaCl in MeCN/H2O 1:4 (v/v); 0 ± 30% B in 30 min).
Purification was achieved by prep. PAGE (15% polyacrylamide) and desalted via a C-18 column. The purified
PNA/DNA chimeras were further analyzed by negative-ion ES-MS.
3. Synthesis of 5'-ACA TCA oeg(X ˆ O, R ˆ H)gg tcg-hex-OH (15). As described in Exper. 2, with HATU/
HOAt (0.3m) as coupling reagent with a pre-activation time of 1 min, and a total coupling time of 45 min. Yield
of crude product was 101 OD. Purification of 42 OD of the crude product resulted in 3.2 OD purified product.
Characterization by negative-ion ES-MS: 3473.75 Æ 0.13 (M; calc. for C124H162N55O54P6: 3473.86).
4. Synthesis of 5'-ACA TCA oeg-t(X ˆ O, R ˆ R1)gg tcg-hex-OH (16). As described in Exper. 2, with
HBTU (0.2m) as coupling reagent with permanent pre-activation (DNA synthesizer ABI 394), and a total
coupling time of 15 min. Yield of crude product was 26 OD. Purification of the crude product (26 OD) resulted
in 13.6 OD purified product. Characterization by negative-ion ES-MS: 3596.65 Æ 0.77 (M; calc. for
C
129H167N57O56P6: 3597.94).
5. Synthesis of 5'-ACATCA seg-t(X ˆ S, R ˆ R1)gg tcg-hex-OH (17). As described in Exper. 2, with HBTU
(0.25m) as coupling reagent with a pre-activation time of 15 min, and a total coupling time of 15 min. Yield of
crude product was 60 OD. Purification of 44 OD of the crude product resulted in 9.5 OD purified product.
Characterization by negative-ion ES-MS: 3613.51 Æ 0.13 (M; calc. for C129H167N57O55P6S: 3614.00).

Helvetica Chimica Acta ± Vol. 82 (1999)
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6. Synthesis of 5'ACA TCA aeg-t(X ˆ NH, R ˆ R1)gg tcg-hex-OH (18). As described in Exper. 2, with
PyAOP (0.3m) as coupling reagent with a pre-activation time of 10 s, and a total coupling time of 15 min. Yield
of crude product was 52 OD. Purification of 51 OD of the crude product resulted in 1.9 OD of purified product.
Characterization by negative-ion ES-MS: 3596.49 Æ 0.09 (M; calc. for C129H168N58O55P6: 3596.98).
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Received August 30, 1999