Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase

Article abstract of DOI:10.1002/1521-3765(20010917)7:18<3911::AID-CHEM3911>3.0.CO;2-1

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for

Full text of DOI:10.1002/1521-3765(20010917)7:18<3911::AID-CHEM3911>3.0.CO;2-1

FULL PAPER
Convergent Strategies for the Attachment of Fluorescing Reporter Groups to
Peptide Nucleic Acids in Solution and on Solid Phase
Oliver Seitz* and Olaf Köhler[a]
Abstract: The site-selective conjugation
of peptide nucleic acids (PNA) with
fluorescent reporter groups is essential
for the construction of hybridisation
probes that can report the presence of
a particular DNA sequence. This paper
describes convergent methods for the
solution- and solid-phase synthesis of
multiply labelled PNA oligomers. The
solid-phase synthesis of protected PNA
enabled the selective attachment of
fluorescent labels at the C-terminal end
(3' in DNA) which demonstrated that
further manipulations on protected
PNA fragments are feasible. For the
conjugation to internal sites, a method is
introduced that allows for the on-resin
assembly of modified monomers there-
by omitting the need to synthesise an
entire monomer in solution. Further-
more, it is shown that the application of
a highly orthogonal protecting group
strategy in combination with chemose-
lective conjugation reactions provides
access to a rapid and automatable solid-
phase synthesis of dual labelled PNA
probes. Real-time measurements of nu-
cleic acid hybridisation were possible by
taking advantage of the fluorescence
resonance energy transfer (FRET) be-
tween suitably appended fluorophoric
groups. Analogously to DNA-based mo-
lecular beacons, the dual labelled PNA
probes were only weakly fluorescing in
the single-stranded state. Hybridisation
to a complementary oligonucleotide,
however, induced a structural reorgan-
isation and conferred a vivid fluores-
cence enhancement.
Keywords: DNA recognition ´ do-
nor± acceptor systems
´
fluores-
cence spectroscopy ´ peptide nucleic
acids ´ solid-phase synthesis
NH2
Introduction
NH2
N
N
NH
O
A large portion of current approaches towards the diagnosis
and therapy of diseases is gene-oriented by its very nature.
The successful completion of the human genome project will
likely intensify the research efforts aiming at the identifica-
tion, detection and remedy of disease-related genes or gene
sequences. Synthetic compounds that specifically recognise
and bind to a specific DNA or RNA sequence of interest are
therefore of great value.[1±3] A particularly successful DNA
binding agent is a recently developed class of DNA analogues,
the peptide nucleic acids (PNA) (Figure 1).[4±9] The complete
replacement of the ribose phosphate backbone with an
artificial pseudopeptide backbone resulted in the remarkably
improved binding to complementary nucleic acid sequences
occurring with both high affinity and high selectivity. Despite
its name, PNA is not an acid rendering the hybridisation event
N
O
O
N
O
O
O
O
N
N
O
N
O
N
N
NH
NH2
N
N
P
NH
NH2
O-
O
NH
O
O
N
O
O
P
O-
O
O
DNA
PNA
Figure 1. DNA and its analogue PNA.
largely independent of the salt concentration.[10] A partic-
ularly attractive feature is that PNAs are not subject to
nuclease or protease mediated degradation and are thus
highly stable even in living cells. These properties have been
advantageously employed for example in pre-gel hybrid-
isations as alternative to Southern hybridisation,[11] for the
identification of point mutations by PCR clamping[12, 13] and
fluorescently labelled probes,[14, 15] in the fabrication and
usage of PNA probe arrays,[16] and in the isolation,[17] block-
ing[18±21] and restriction[22±24] of genes or mRNAs.
[a] Dr. O. Seitz, O. Köhler
MPI for Molecular Physiology, Department of Chemical Biology
and Institut für Organische Chemie, Universität Dortmund
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Fax : (‡49) 231 1332499
For applications as diagnostical probes PNAs as well as
oligonucleotides have to be equipped with reporter groups.
E-mail: oliver.seitz@mpi-dortmund.mpg.de
Chem. Eur. J. 2001, 7, No. 18
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3911

O. Seitz and O. Köhler
FULL PAPER
However, the highly versatile labelling techniques that
involve the use of enzymes such as terminal transferase are
not applicable since PNA is not a substrate of any of the
known DNA-modifying enzymes. The functionalisation and
labelling of PNA, therefore, has to be accomplished by
chemical means. Most commonly, fluorophores were append-
ed to the N-terminal amino group of PNA oligomers which
corresponds to the 5'-end of oligonucleotides.[25±27] The con-
jugation to the C-terminal carboxyl group (3') is difficult since
access is blocked during the solid-phase synthesis. An alter-
native would be the use of internal conjugation sites that
could be provided by modified nucleobases or backbones.
However, this usually requires the often cumbersome syn-
thesis of modified PNA monomers. It is the aim of the work
presented in here to extend the repertoire of the existing
methodology available for PNA labelling. It will be shown
that the attachment of fluorophores to the C-terminus[28] and
to internal conjugation sites[15] is possible by employing
convergent strategies and both solution- and solid-phase
labelling of PNAwill be discussed. Focus of these studies is the
rapid synthesis of dual labelled PNA probes[29, 30] which may
prove suitable for the sequence specific DNA-detection in
homogeneous solution.
Figure 2. Unhybridised PNA forms intra- or intermolecular associates of
unknown structure. The representation of the dual labelled PNA-probe 1 is
intended to illustrate that appropriately appended fluorescence donor and
fluorescence quencher groups could be located in close proximity. The
DNA-based molecular beacons 2 contain target-unrelated arm sequences
and are designed to form a stem-and-loop structure. In both 1 and 2,
fluorescence is quenched due to collisional quenching and fluorescence
resonance energy transfer (FRET). When the probe sequence anneals to
the target sequence T, a structural reorganisation increases the donor±
quencher distance within the duplexes 1 ´ T and 2 ´ T and fluorescence can
occur.
Design principles: It was observed that thermal denaturation
of single-stranded PNA in contrast to DNA showed a phase
transition along with a considerable hyperchromicity, which
indicated that base stacking might be a favourable process
even in unhybridised PNA.[31] We reckoned that a suitably
appended fluorescence donor could be located in close
proximity to the fluorescence quencher due to a possible
inter- or intramolecular association of PNA single strands
(Figure 2). As a result collisional quenching and fluorescence
resonance energy transfer (FRET)[32, 33] would diminish the
fluorescence of the single-strand 1. The hybridisation to a
complementary nucleic acid (!1 ´ T), however, would induce
a structural reorganisation that would lead to an increase of
the donor± quencher distance. Thus, in the duplex 1 ´ T,
fluorescence would occur serving as a means to detect the
complementary nucleic acid in homogeneous solution. This
process is similar to the fluorescence dequenching that is
observed for structured DNA-based molecular beacons (2 !
2 ´ T).[34, 35] Structured molecular beacons based on PNA[36]
and PNA/DNA hybrids[37] have also been reported. It is
important to note, however, that in the PNA-based approach
outlined in here target-unrelated arm sequences, which serve
to maintain the structural integrity of the probe, would not be
required.
The above-mentioned approach involves the attachment of
two reporter groups, a fluorescence donor and a fluorescence
quencher. Due to the unknown structure of single-stranded
PNA it was unclear how the markers have to be arranged in
order to maximise fluorescence quenching of the unhybrid-
ised PNA 1. Hence, a flexible conjugation strategy was
desired. An approach, that combines terminal and internal
labelling allows for the optimisation of the donor± quencher
distance without simultaneously changing the length of the
PNA oligomer. For terminal labelling a reporter group can be
appended to either the N-terminus or the C-terminus. Both
conjugation sites were envisioned to be useful for the
approach outlined in Figure 2. The N-terminal amino group
is readily accessible during the solid-phase synthesis of PNA
and therefore most labelling techniques were focussed on this
particular site.[25±27] However, within the framework of a
research project aiming at the development of PNA ligation
probes, we were also in need for C-terminally modified PNA
conjugates. Unfortunately, during solid-phase synthesis the
growing PNA oligomer is anchored through this carboxyl
group. The liberation of the PNA from the solid phase by
routine procedures usually furnishes unprotected oligomers.
Since selective modification of the C-terminus are difficult to
achieve in the presence of the unprotected exocyclic amino
groups of the nucleobases,[38, 39] it was planned to employ
protected PNA oligomers. Surprisingly, when we became
aware of this synthetic need there had been no method
reported for the solid-phase synthesis of protected peptide
nucleic acids suitable for further modifications at the C-ter-
minal carboxyl group.[40]
For the attachment of reporter groups to internal positions
conjugation sites are required which can be provided by either
the nucleobase or the aminoethylglycine backbone. In PNA,
the introduction of modified nucleobases or surrogates there-
of can be accomplished by applying the well-established
methodology of amide bond forming reactions rather than the
complex glycosylation reactions that would be necessary for
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Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
installing the corresponding modification in DNA. A con-
vergent strategy can therefore readily be implemented by
employing modified nucleobases. The ease of altering PNA at
the nucleobase level had been exploited in several stud-
ies.[41±44] However, in all these studies, the non-standard
nucleobases were incorporated by using preformed mono-
meric building blocks. A strategy that would omit the need to
synthesise an entire monomer building block in solution
would facilitate the rapid synthesis of modified PNA oligo-
mers.[45, 46] As the central building block in this approach an
orthogonally protected backbone module such as the Boc/
Fmoc-protected aminoethylglycine 3b[47] would be incorpo-
rated during PNA assembly. After removal of the Fmoc group
the non-standard nucleobase would be coupled, thereby
furnishing the modified monomer on the solid phase.
ester[54] yielded the Boc-protected HYCRON conjugate 5a.
An analogous reaction scheme provided the conjugates 5b
and 5c containing Boc/Fmoc-protected aminoethylglycine 3b
and the N-Boc-protected glycine 3c, respectively. Unfortu-
nately, in all attempts in which a Boc/Z-protected guanosine
building block was subjected to the nucleophilic esterification,
a complex mixture was obtained with a major product that
contained two HYCRON tethers. However, a concomitant
allylation of other nucleophilic structures can be avoided by
activating the carboxylic group in presence of the anchor
alcohol or by constructing the Boc-GZ monomer on the solid
phase (see below).
The
Boc/Fmoc-protected
aminoethylglycine
3b[47]
(Scheme 2) was obtained in a one-pot synthesis starting from
the N-Boc-aminoethylglycine ethyl ester 7,[48] an intermediate
Fmoc
Results and Discussion
H
a
N
COOEt
N
COOH
Boc-HN
Boc-HN
7
3b
Synthesis of building blocks: Currently, two different PNA-
building blocks are commercially available, Boc/Z- and Fmoc/
Bhoc-protected monomers. The former combine the TFA-
removable Boc group for temporary protection with a HF (or
TFMSA)-cleavable Z-protecting group for permanent pro-
tection of the exocyclic amino groups of the nucleobases.[48, 49]
The latter offer a real orthogonal protecting scheme com-
prised of the base-labile Fmoc group for temporary protection
and the acid-labile benzhydryloxycarbonyl group for perma-
nent blockage.[50] Recently, Dts/Z protection was demonstrat-
ed to allow for the solid-phase synthesis of fully protected
PNA amides.[51] In developing a strategy for the solid-phase
synthesis of protected PNA bearing a C-terminal carboxyl
group, a linker of general utility was desirable that could
enable the use of both types of commercially available PNA
building blocks. The allylic HYCRON linker was chosen since
the allylic linkage provides a high degree of orthogonality in
combination with the Boc- as well as the Fmoc strategy.[52] For
the synthesis of preformed starting monomer± HYCRON
conjugates such as the cytosine conjugate 6a a nucleophilic
esterification was employed by using the Boc-protected
starting monomers 3a and the allylic bromide 4 (Scheme 1).[53]
The subsequent reductive removal of the phenacyl (Pac)
Scheme 2. a) i) 0.4m NaOH/EtOH (5:8); ii) AcOH; iii) Fmoc-OSu, 91%.
in the synthesis of the conventional Boc/Z-protected PNA
monomers. Saponification of the ethyl ester was followed by a
careful adjustment to pH 8 and subsequent addition of Fmoc-
OSu. Crystallisation furnished the backbone building block
3b in high purity and high yield.
In order to link fluorescent reporter groups to nucleobases
additional conjugation sites have to be introduced. Scheme 3
shows the synthesis of the adenine modification 11. The
NH(CH2)6NH2
N
Cl
N
NH(CH2)6NH-Fmoc
N
N
N
a
b
N
N
N
N
N
N
N
8
9
CO2tBu
CO2tBu
CO2R
10: R = tBu
11: R = H
c
Scheme 3. a) H2N(CH2)6NH2, nBuOH, 38%; b) Fmoc-Cl, NMM, CH2Cl2,
72%; c) TFA, 95%.
N-Boc protected N6-alkylamino tether was chosen because it
directs the attachment of fluorophores to the major groove of
a DNA duplex without detriment to the Watson ± Crick base
pairing.[55] The synthesis was achieved starting from 6-chloro-
purine, which was first carboxymethylated to obtain 8.[56]
After separation from the N7-isomer, 8 was treated with
hexamethylenediamine. Subsequent reaction of 9 with Fmoc-
Cl blocked the primary amino group and treatment with TFA
cleaved the tBu ester to yield 11.
O
+
Boc-X-OH
Br
COOPac
4
Br-HYCRON-OPac
a
4
3a, b, c
O
Boc-X-O
COOR
4
Z-HN
N
5a-c: R = Pac
b
6a-c: R = H
O
N
A rapid synthesis of multiply labelled PNA oligomers is
provided by a strategy in which the attachment of the
fluorescent reporter groups is performed on the solid phase.
When a particular nucleobase modification is intended to be
repeatedly used it might be preferable to employ preformed
monomer modifications. Scheme 4 describes the synthesis of
O
Fmoc
N
CO
N
CO
HN
HN
CO
X =
HN
(3a→) 6a
(3b→) 6b
(3c→) 6c
Scheme 1. a) sat. aq. NaHCO3, Bu4NBr, CH2Cl2 (5a, 83%; 5b, 92%; 5c,
76%); b) Zn, AcOH (6a, 79%; 6b, 94%; 6c, 89%).
Chem. Eur. J. 2001, 7, No. 18
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3913

O. Seitz and O. Köhler
FULL PAPER
H
O
COOH
H
Ac-O
O
N
N
Cl
N
HN
Boc
HN
R
3
n
20 Ac-HYCRON
n
N
N
N
N
a
c
N
N
N
a
N
N
N
N
O
COOH
O
N
H
Ac-O
O
12
R = H: 13a,b (n=3, 1)
R = Boc: 14a,b (n=3, 1)
15a: n=3
15b: n=1
b
3
21
b, c
O
H
H
N
d
N
CO2All
CO2tBu
O
Fmoc-HN
Fmoc-HN
N
H
Boc-X-O
O
3
16
17
22a (X=CZ)
22b (X=Gly)
H
N
d, e
HN
N
Boc
n
N
23: Boc-Phe-GZ-GZ-CZ-OH
24: Ac-CZ-CZ-GZ-Gly-OH
R = All: 18a,b (n=3, 1)
R = H: 19a,b ( n=3, 1)
e
f
N
15a,b
+ 17
N
O
N
COOR
Fmoc-HN
Z-HN
N
Scheme 4. a) 13a: H2N(CH2)4NH2, nBuOH, 42%; 13b: H2N(CH2)2NH2,
n-BuOH; b) Boc2O, DMAP, Pyr (14a, 51%; 14b, 44% based on 12);
c) i) O3, CH2Cl2, MeOH; ii) NaClO2, NaH2PO4, 2-methyl-2-butene,
tBuOH (15a, 58%; 15b, 71%); d) i) TFA; ii) SOCl2, All-OH, 84%;
e) HOBt, DCC, CH2Cl2, DMF (18a, 76%; 18b, 84%); f) MeNHPh,
[Pd(PPh3)4], THF (19a, 81%; 19b, 71%). (DCC ˆ N,N'-dicyclohexylcar-
bodiimide).
O
N
N
HN
O
N
Z-HN
N
O
O
N
CO
N
CO
HN
HN
CZ
GZ
Scheme 5. a) HBTU, HOBt, NMM, CH2Cl2, aminomethyl-polystyrene;
b) 1n NaOH/dioxane (1:3); c) Boc-X-OH, DIC, DMAP, d) i) TFA/m-
cresol (95:5); ii) Boc-BZ-COOH, HBTU, iPr2NEt, Pyr; iii) Ac2O/Pyr;
iv) repeat i) ± iii); e) [Pd(PPh3)4], morpholine, DMSO, DMF (23, 36%, 24,
35% based on aminomethyl-polystyrene). (DIC ˆ N,N'-diisopropylcarbo-
diimide, HBTU ˆ O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium-
hexafluorophosphate, HOBt ˆ 1-hydroxy-benzotriazol, NMM ˆ N-meth-
ylmorpholine).
Fmoc/Boc-protected adenine monomers suitable for a syn-
thetic strategy in which all reactions including the labelling
reactions can be performed on the solid phase. As starting
material 6-chloropurine was subjected to a regioselective
allylation[57, 58] followed by treatment with tetramethylenedi-
amine or ethylenediamine. Then the primary amino groups of
13a and 13b were protected by reaction with Boc2O. The
conversion of 14a and 14b to the carboxymethylene deriva-
tives proceeded smoothly by ozonolysis and oxidative workup
providing the Boc-protected N6-aminoalkyl-N9-carboxymeth-
yladenine 15a and 15b. The protected aminoethylglycine unit
17 suitable for the coupling with 15a and 15b was synthesised
from the known backbone 16.[59] Treatment of 16 with TFA
liberated the carboxyl group, which in a Brenner-type
esterification was converted to the allyl ester 17.[60] This
seemingly tedious scheme avoids the d-lactam formation
which can be observed when N-deprotected aminoethylgly-
cine allyl esters are employed. The couplings of the carboxy-
methylated nucleobases 15a and 15b with the backbone 17
were accomplished by activation with DCC[61] and HOBt.[62]
Finally, Pd0-catalysed allyl transfer to N-methylaniline fur-
nished the Fmoc/Boc-protected adenine modifications 19a
and 19b.
starting monomers. The subsequent solid-phase synthesis was
performed according to the Boc strategy using the loaded
resins 22a and 22b. In this and all other syntheses acetylation
was employed as a means to cap eventually unreacted amino
groups. The final detachment was accomplished by a Pd0-
catalysed allyl transfer to the nucleophile morpholine. Puri-
fication by silica gel chromatography or size-exclusion
chromatography afforded the protected PNA-oligomers 23
and 24 in 36 and 35% overall yield, respectively. It should be
mentioned that the protected PNA oligomers were poorly
soluble, which might also be responsible for the broad peaks
observed in HPLC analysis. MALDI-TOF analysis, however,
clearly confirmed the identity of the protected PNAs 23
and 24.
The determination of the relatively low overall yields
reached in the synthesis of 23 and 24 was based on the initial
loading of resin 21 with reactive acetyl groups. It thus seemed
that the esterification using polymer-bound alcohols did not
reach completion. In order to test this hypothesis it was
planned to draw on amide bond forming reactions rather than
esterification reactions, thereby employing an amino func-
tionalised resin and preformed conjugates of the starting
monomer and the HYCRON linker. Hence, HYCRON-
conjugates 6a and 6c were coupled with the solid support
(Scheme 6). Application of the solid-phase synthesis protocol
as described above was followed by the Pd0-catalysed
cleavage of the allylic linkage. The subsequent chromato-
Solid-phase synthesis: For the synthesis of the model tetra-
mers 23 and 24 the loading was commenced by coupling the
acetylated HYCRON handle 20[53] to aminomethylated poly-
styrene resin (Scheme 5). For the determination of the loading
yield an NMR assay was developed. A few beads of resin 21
were treated with a mixture of deuterated sodium methoxide
in CDCl3 that contained toluene as an internal standard. The
acetate formed was quantified by NMR which revealed a
quantitative loading reaction. Saponification of the HY-
CRON-resin 21 liberated the allylic hydroxyl group which
was subjected to a esterification using N-Boc protected
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Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
apparent when a part of the resulting resin 27 was subjected to
the Pd0-catalysed allyl cleavage. The Boc/Z-protected guano-
sine monomer was released in a yield of 61% based on the
initial loading of resin 26 with Fmoc groups, which exceeds the
yield of 52% that can be accomplished in the solution-phase
synthesis.[63] For elaboration to the oligomer 29a resin 27 was
extended using the described Boc/Z methodology. The final
Pd0-catalysed cleavage and subsequent HPLC purification
afforded the protected 7-mer 29a in an overall yield of 36%.
However, the coupling of the carboxymethyl guanine to the
polymer-bound aminoethylglycine did not reach completion
and the corresponding acetylated derrivative 29b, which was
easier to purify, was isolated in 39% yield.
6a,c
a
O
Boc-X-O
CO-HN
4
(6a→) 22a
(6c→) 22c
b-d
(22a→) 25
Z-GZ-CZ-OH
Boc-Gly-G
(22c→) 24 Ac-CZ-CZ-GZ-Gly-OH
Scheme 6. a) aminomethyl polystyrene, HBTU, iPr2NEt, HOBt, DMF;
b) Ac2O, Pyr; c) Boc/Z-PNA solid-phase synthesis (see Scheme 5);
d) [Pd(PPh3)4], morpholine, DMSO, DMF (24, 59%; 25, 67%).
The on-resin synthesis of PNA monomers was advanta-
geously applied for the assembly of PNA-conjugate 32, which
provides an conjugation site at an internal position. Starting
from resin 26 the linear solid-phase synthesis proceeded as
described. Introduction of the aminoethylglycine 3b yielded
the orthogonally protected PNA-backbone resin 30. After
removal of the Fmoc group the adenine modification 11 was
coupled by using HATU[64] activation and prolonged coupling
times. The photometric determination of the Fmoc loading
revealed a quantitative yield for the incorporation of 11. The
subsequent chain elongation was followed by the Pd0-cata-
lysed cleavage in presence of N-methylaniline as an allyl
scavenger. As the analysis of 29a and 29b had suggested it
seemed possible that the on-resin synthesis of the starting
guanosine derivative did not reach completion. MALDI-TOF
analysis showed two major peaks (Figure 3). One was
assigned to the desired product and the other to a product
from incomplete coupling of the carboxymethyl guanine gZ-
OH. A compound indicative for a failed introduction of the
adenine modification 11 was not detectable by MALDI-TOF
MS. This nicely confirmed the Fmoc-loading analysis which
had indicated a quantitative yield for this on-resin coupling.
The final HPLC purification furnished the PNA heptamer 32
graphic purification afforded the protected PNA-oligomers 24
and 25 in 59% and 67% overall yield, respectively. This high
overall yield was based on the initial loading of the
polystyrene resin with reactive amino groups. It is, thus,
noticable that the attachment of the starting monomers
through preformed linker conjugates allowed a highly effi-
cient solid-phase synthesis.
It has to be noted that for a general applicability of the
above-mentioned strategy a set of at least four different
starting monomer± HYCRON conjugates would be required.
In addition, the synthesis of conjugates that contain acidic
structures such as the guanine imide system has to be
performed by acylating esterification, which for the HY-
CRON anchor is less efficient than nucleophilic esterification.
An alternative loading procedure employs the Boc/Fmoc-
protected aminoethylglycine conjugate 6b as a common
precursor (Scheme 7). Attachment of 6b onto the solid
support furnished the precursor resin 26. Treatment with
DMF/morpholine liberated the secondary amino group,
which subsequently was coupled to carboxymethyl guanine
gZ-OH. The success of this on-resin synthesis[45,46] became
O
O
N
HN
N
HN
Z-HN
N
Z-HN
N
O
N
N
Fmoc
N
O
O
gZ-OH
COOH
a, b
N
HYCRON-NH
27
HYCRON-NH
Boc-NH
Boc-NH
6b
c, d
Boc-GZ-HYCRON-PS
Boc-Aeg(Fmoc)-HYCRON-PS
O
26
(CH2)6NH-Fmoc
N
HN
Ac-GZ-CZ-CZ-A-CZ-GZ-GZ-OH
32
N
Z-HN
N
g
O
e, g
COOH
N
28
Boc-NH
NH(CH2)6NH-Fmoc
e, g
N
N
Ac-GZ-CZ-CZ-AZ-CZ-GZ-Aeg(R)-OH
Fmoc
29a: R=gZ
29b: R=Ac
27
N
N
O
CO-CZGZGZ-HYCRON-NH
N
c, f
CO-CZGZGZ-HYCRON-NH
e
N
Boc-NH
Boc-NH
31
30
Scheme 7. a) aminomethyl polystyrene, HBTU, iPr2NEt, HOBt, DMF; b) Ac2O, Pyr; c) DMF/morpholine; d) HATU, iPr2NEt, DMF; e) Boc/Z-PNA solid-
phase synthesis (see Scheme 5); f) 11, HATU, iPr2NEt, DMF; g) [Pd(PPh3)4], morpholine (MeNHPh for 32), DMSO, DMF (28, 61%; 29a, 36%; 29b, 39%;
32, 50%).(Aeg ˆ aminoethylglycine, HATU ˆ O-(1-oxy-7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyl-uronium hexafluorophosphate, PS ˆ polystyrene).
Chem. Eur. J. 2001, 7, No. 18
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3915

O. Seitz and O. Köhler
FULL PAPER
obtained in high yield. However, the acid sensitivity of the
fluoresceinyl group precluded a complete deprotection of
34a. Hence, a coupling to dansylethylenediamine 33b was
used for C-terminal labelling of 25. The successive treatment
of 34b with TFA and trifluoromethanesulfonic acid in TFA
removed the Boc and Z groups. HPLC purification furnished
the C-terminally dansylated PNA 35 in good yield.
In addition to a selective modification of the C-terminus the
protected PNA conjugate 32 enables the attachment of
reporter groups to the Fmoc-protected primary alkylamino
group. First, 32 was coupled to the dansylethylenediamine
33b as described. The subsequent treatment with DMF/
morpholine removed the Fmoc group which was followed by
the acidolytic removal of the Z-protecting groups. A second
reporter group was attached to 36 by using the liberated
primary alkylamino group. The reaction with the fluorescence
quenching 4-(4'-dimethylaminophenylazo)benzene sulfonic
acid chloride (dabsyl chloride) proceeded in high selectivity
forming the dual labelled PNA conjugate 37 in high purity.
The examples presented in Scheme 8 demonstrate that
protected PNAs indeed serve as suitable substrates for the
attachment of reporter groups to the C-terminal carboxyl
group. However, it should be mentioned that although the
conjugation reactions proceeded smoothly, the sometimes
unpredictable solubility properties of protected PNA oligo-
mers can render work-up and purification procedures cum-
bersome.
Figure 3. MALDI-TOF-MS of a) crude 32 as obtained after resin cleavage
and of b) purified 32 that was mixed with the b-chain of bovine insulin as
internal reference.
protected by six Z groups and one Fmoc group in an overall
yield of 50% (based on the Fmoc loading of 26). It can be
concluded that the introduction of the adenine modification
11 proceeded smoothly whereas it proved difficult to achieve
a quantitative on-resin synthesis of the guanosine monomer
(Scheme 7).
C-terminal and internal PNA labelling in solution: Fully
protected PNA oligomers such as 23 ± 25, 29a and 32 allow for
selective modifications at the C-terminal carboxyl group. In
order to assess the feasibility of coupling reactions which
employ oligomeric PNA as acylating agents, the tetramer 25
was used as a model compound, activated with EDC[65] in
presence of HOAt and coupled to the fluorescein ± ethylene-
diamine conjugate 33a[66] (Scheme 8). After size exclusion
chromatography the C-terminally labelled PNA 34a was
PNA labelling on the solid phase: In the work described
above the labelling steps have been performed in solution by
using protected PNA oligomers that were obtained by solid-
phase synthesis. The conjugation reactions and the subsequent
work-up involved can be time-consuming, particularly when
more than one reporter group has to be attached. A strategy,
in which all reactions including the labelling steps can be
performed on the solid phase, is thus desirable. For the on-
resin labelling it seemed advantageous to combine internal
labelling with N-terminal labelling since access to the
C-terminus can be hindered on solid phases. The internal
conjugation site would be provided by the N6-aminoalkyl
modified adenine which enabled the efficient conjugation of
36 with the dabsyl moiety (Scheme 8). In the examples
presented above Boc/Z-protected PNA monomers were used.
Many fluorescent labels, however, are subject to degradation
under the conditions required for the removal of the
permanent Z-protecting groups. A solution to this problem
is offered by the combination of a highly orthogonal blocking
group strategy with chemoselective conjugation reactions.
Hence, the use of Fmoc- and Bhoc-protecting groups in
combination with the HYCRON linkage was fashioned. It
enables the application of much milder cleavage conditions in
that the removal of all protecting groups would proceed on
the solid phase being followed by an on-resin labelling
procedure. Finally, Pd0-catalysed cleavage from the HY-
CRON resin would provide particularly mild cleavage
conditions that leave labile fluorophores unaffected.
a
c, b
H2N(CH2)2NH-Dye
33a : Dye= FIT
33b : Dye= Dans
Boc-Gly-GZ-GZ-CZ-X-Dye
(CH2)6NH2
Ac-GCCACGG-X-Dans
34a : Dye= FIT; 34b : Dye= Dans
36
X= HN(CH2)2NH
b
b, d
H-Gly-G-G-C-X-Dans
(CH ) NH-
Dabs
2
6
35
Ac-GCCACGG-X-Dans 37
O
R
O
OH
Me2N
O
S
Dans
COOH
O
O
S
Me2N
N
N
HN
O
S
FIT
Dabs (Dabs-Cl, R = Cl)
Scheme 8. a) 25, EDC, HOAt, DMF, CH2Cl2 (34a, 64%); b) m-cresol,
Me2S, TFA, TFMSA (1:1:10:1) (35, 60% based on 25; 36, 66% based on
32); c) i) 32, EDC, HOAt, DMF, CH2Cl2; ii) DMF/morpholine (1:1);
d) 4 equiv Dabs-Cl, sat. aq. NaHCO3, DMF, 50%. (Dabs ˆ 4-(4'-dimethyl-
aminophenylazo)benzene sulfonyl, Dans ˆ 5-dimethylaminonaphthaline-
1-sulfonyl, EDC ˆ Et2(CH2)3N ˆ C ˆ NEt ´ HCl, HOAt ˆ 1-hydroxy-7-aza-
benzotriazol).
First, the solid-phase synthesis of 42 was performed, in
order to examine whether the incorporation of the adenine
modification 19a, which contains an unprotected N6-amino
3916
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Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
group, into a Fmoc-based solid-phase synthesis would allow
for an efficient solid-phase assembly (Scheme 9). Fmoc/Bhoc-
protected monomers as illustrated in the inset were coupled
using the HYCRON-loaded resin 38. All coupling reactions
succeeded through HATU activation.[64] For the introduction
of the internal conjugation site, the Fmoc/Boc-protected N6-
modified adenine building block 19a was incorporated. After
completion of the chain elongation all protecting groups
including the Bhoc and Boc groups were removed on the solid
phase by treating 40a with a TFA/ethanedithiol/water mix-
ture. The fully unprotected PNA 41a remained bound to the
solid support, thus, allowing for a simple and efficient removal
of scavengers and cleaved by-products. Finally, the Pd0-
catalysed cleavage accomplished the liberation of the PNA-
conjugate 42. After HPLC purification an overall yield of
30% (based on 39) was reached, demonstrating that the use of
the N6-unprotected derivative 19a enables the effective
synthesis of PNA conjugates with an internal conjugation site.
This result was promising for the fashioning of an on-resin
labelling protocol. The solid-phase synthesis of the dual
labelled PNA-oligomers 44 and 45 is shown in Scheme 9.
The chain assembly proceeded as described for 42. After its
completion the resin 40b was prepared for the chemoselective
conjugation reactions by which the reporter groups should be
appended. The treatment of the fully protected PNA-resin
40b with TFA/ethanedithiol/water mixture removed all
protecting groups including the S-Trityl, N-Boc and Bhoc
groups. The unprotected PNA-oligomer 41b, again, remained
attached to the solid phase. After neutralisation the PNA
resin 41b was reacted with the 4-(4'-dimethylaminophenyl-
azo)benzoic acid hydroxysuccinimide ester (DABCYL-SE).
This reaction led to acylation of both the primary amino group
of the N6-modified adenine and the cysteine thiol group. The
latter was cleaved by subsequent treatment with DMF/
piperidine. Since unprotected thiol groups are prone to form
disulfides, a preventive dithiothreitol reduction was per-
formed. The liberated thiol group was selectively alkylated
by a reaction with 5-(2'-iodoacetamidoethyl)aminonaphtha-
lene sulfonic acid (IAEDANS) furnishing resin 43. The final
Pd0-catalysed detachment proceeded in presence of morpho-
line yielding the dual labelled PNA-conjugate 44. It should be
emphasised that although the chemical yield was modest the
additional hydrophobicity as conferred by the attachment of
the two labels greatly facilitated the purification procedure.
For example, HPLC analysis of a material obtained by simple
C18-SepPak extraction showed two peaks only (Figure 4).
The minor peak resulted from incomplete coupling to the
modified adenine as judged by MALDI-TOF analysis. The
subsequent RP-HPLC purification furnished 44 and the
analogously prepared positional isomer (41c ! 45) in high
purity. Interestingly, even after repeated HPLC purification,
the MALDI-TOF-MS spectra consistently showed a second
peak with a m/z ratio that was 133 units lower than the
molecular mass. This peak, however, seemed to be associated
with a photochemically induced fragmentation by the MAL-
DI-laser since ESI-MS analysis revealed the presence of a
single peak with the expected molecular mass.
Hybridisation experiments: Both conjugates the dual labelled
PNA-probes 44 and 45 were used in hybridisation experi-
ments targeting the oligonucleotides 46 and 47. Figure 5 shows
the fluorescence spectra of 44 and 45. It became apparent that
the single stranded PNA-conjugates indeed exhibited a
quenched EDANS fluorescence for reasons outlined in
Scheme 9. a) i) HBTU, HOBt, iPr2NEt, DMF, amino-Tentagel; ii) Ac2O, Pyr (1:10); b) i) TFA; ii) iPr2NEt/DMF (1:9); c) Fmoc-T, HBTU, HOBt, iPr2NEt,
DMF; d) iterative cycles of: i) piperidine/DMF (1:4); ii) Fmoc-BBhoc-OH, HATU, iPr2NEt, Pyr, DMF; iii) Ac2O, Pyr, DMF; e) i) piperidine/DMF (1:4);
ii) Ac2O, Pyr, DMF; f) TFA/ethanedithiol/H2O (95:2.5:2.5); g) 10 equiv DABCYL-SE, DMF/Pyr/NMM (7:1:1); h) piperidine/DMF (1:4); i) 10 equiv
dithiothreitol, DMF/H2O/NMM (9:3:1); j) 10 equiv IAEDANS, DMF/H2O/NMM (9:3:1); k) [Pd(PPh3)4], morpholine, DMSO, DMF, 10% (based on 39).
Chem. Eur. J. 2001, 7, No. 18
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3917

O. Seitz and O. Köhler
FULL PAPER
Figure 2. Addition of the mismatched oligonucleotide 47 led
only to a minor change of the fluorescence spectrum.
However, when the complementary oligonucleotide 46 was
added at 258C the fluorescence spectra changed dramatically.
The emission of PNA-probes 44 and 45 was enhanced by a
factor of 4.6 and 3, respectively. The fluorescence enhance-
ment was even more pronounced when the hybridisation was
allowed to occur at 208C. At this temperature, 108C below the
TM, presumably 100% of the PNA probes are in the
hybridised state leading to a fluorescence intensification by
factors of 6.4 and 4.3, respectively. A spectral shift that could
be indicative for a hydrophobicity-induced increase of the
fluorescence was not detectable. It is thus likely that the
mechanism by which the fluorescence increased followed the
dequenching process that is observed for equally labelled
molecular beacons.
Analogous to molecular beacons, probes 44 and 45 began to
fluoresce almost immediately after addition of the nucleic
acid target strand. Figure 6 shows that the fluorescence of
Figure 4. Analytical HPLC trace at l ˆ 250 nm of 44 as a) crude material
after SepPak C18 extraction and b) after purification (conditions as
specified in the experimental part). The c) MALDI-TOF-MS of purified
44 consistently showed a second peak, which presumably originates from
fragmentation, with a m/z ratio that was 133 units lower than the molecular
mass. The d) ESI-MS trace, however, revealed the presence of a single peak
with the expected molecular mass.
Figure 6. Fluorescent emission of probe 44 after addition of 1 equiv 47
(0 min), 3 equiv 47 (21 min), 1 equiv 46 (40 min) and 3 equiv 46 (51 min).
Measurement conditions: 100 mm NaCl, 10 mm NaH2PO4, 0.1 mm EDTA,
pH 7, 298 K, 1 mm in 45, excitation at 335 nm.
PNA-probe 44 reached a plateau five minutes thereafter. The
half-maximal enhancement was obtained in less than one
minute indicating that hybridisation of the dual labelled
probes is a rapid process. Noteworthy the preparatory
denaturation or renaturation procedures were not required.
The dual labelled PNA 45 spans an 11-base segment
between the two labels. If the hybridisation-induced increase
of the fluorescence intensity were a general, sequence-
independent phenomenon, the PNA-probe 48, which accom-
modates a 10-mer mixed-base segment between the termi-
nally appended dansyl and DABCYL labels, should exhibit a
similar fluorescence enhancement. Indeed, addition of the
oligonucleotide 49, a synthetic fragment of the pig-H-Ras
gene, led to a fluorescence intensification by a factor of 3
(Figure 7). The PNA-probes 45 and 48 differ in sequence and
length. However, the hybridisation of both PNA probes was
accompanied by the same fluorescence increase. This result
suggests that the fluorescence behaviour of the dual labelled
Figure 5. Fluorescence spectra (arbitrary units, calibration based on the
fluorescence of single stranded 44) of the a) PNA-probe 44 and b) PNA-
probe 45. The inset shows the oligomers used (Cys* ˆ Cys(EDANS), A* ˆ
A(N6(CH2)4NH-DABCYL)) Measurement conditions: 100 mm NaCl,
10 mm NaH2PO4, 0.1 mm EDTA, pH 7, 298 K, 1 mm in 44 and 45, 4 mm in
46 or 47 when added, excitation at 335 nm. The uppermost curves were
measured at 1.5 mm probe concentration and 293 K.
3918
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Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
temperatures of the 44 ´ 46 and the 45 ´ 46 duplex compared
well with the TM of 30.48C for the unmodified DNA ´ DNA
duplex 46 ´ 52. This data supports the notion that PNA
through its favorable base pairing properties is able to
tolerate modifications that if installed in DNA might result
in a greatly limited affinity to nucleic acid targets.
Conclusion
The convergent strategies elaborated in these studies provid-
ed access to multiply labelled PNA conjugates, which are
difficult to synthesise by alternative means. Both solution- and
solid-phase methods were used. The first solid-phase synthesis
of protected PNA enabled a selective functionalisation of the
C-terminal carboxyl group as demonstrated by the synthesis
of C-terminally dansylated PNA such as conjugates 35 and 37.
For adding new functionality to PNA, non-standard nucleo-
bases such as the modified adenine 11 were introduced. In
contrast to previous studies, a strategy was fashioned that
allowed for the introduction of the modified nucleobase to
occur on the solid phase, thereby omitting the need to
synthesise an entire monomer in solution. It has to be
emphasised that an analogous modification of oligodeoxy-
ribonucleotides would require a glycosylation reaction to be
performed on the solid support, which by current techniques
is virtually impossible. In this approach, the key building
block was the Boc/Fmoc-protected aminoethylglycine 3b
which was used in combination with the allylic HYCRON
linker. Both Boc- and Fmoc-protecting groups were removed
while the PNA oligomer remained bound to the polymeric
resin. This strategy proved particularly successful for the on-
resin labelling of PNA. The alliance of orthogonal protecting
group techniques with chemoselective conjugation reactions
gave rapid access to dual labelled PNA probes such as 44 and
45. It was demonstrated that the single-stranded PNA probes
were only weakly fluorescing but experienced a substantial
fluorescence enhancement when bound to complementary
DNA. Applications such as real-time PCR monitoring and
real-time RNA detection in living cells could be feasible and
benefit from the increased biostability of the PNA-based
hybridisation probes.
Figure 7. Fluorescence spectra (arbitrary units) of the terminally labelled
PNA-probe 48 as single strand and in complex with DNA 49 (Lys* ˆ
Cys(Dans), Lys# ˆ Lys(DABCYL)). Measurement conditions: 100 mm
NaCl, 10 mm NaH2PO4, pH 7, 298 K, 1 mm in 48, 1 mm in 49 when added,
excitation at 330 nm.
PNA-oligomers 44, 45 and 48 is a generalisable phenomenon
that is independent of the sequence context.
In order to determine the DNA binding affinity of the
internally modified PNA conjugates 44 and 45, the temper-
ature-dependent UV absorbance was analysed. This revealed
a melting temperature TM of 30.58C for the 44 ´ 46 duplex and
a TM of 28.58C for the 45 ´ 46 duplex (Table 1). A comparison
with the TM of 37.08C determined for the hybridisation of
unmodified PNA 50 indicated that the introduction of the
fluorescent labels in 44 and 45 led to a destabilisation of the
duplex with complementary DNA. If the modifications would
block parts of the duplex formation, the TM values of the 44 ´
46 and the 45 ´ 46 duplex would correspond to that of
unmodified PNAs spanning 14 and 11 base pairs, respectively.
The PNA-oligomer 51 spans a 13-base segment when
hybridised with DNA 46. However, the TM of 27.08C for the
51 ´ 46 duplex is lower than the TM of 28.58C obtained for the
45 ´ 46 duplex, which suggests that the modification distorts
duplex geometry but not to the extent that base pairing of the
nucleobases located C-terminal of the modification site would
be completely prevented.
It is well known that the introduction of fluorescent labels
affects the duplex stability particularly when attached to
internal nucleobases.[67] However, the PNA backbone confers
a substantial stabilisation which compensates for the pertur-
bation introduced by the labelling. Indeed, the melting
Experimental Section
General methods: Boc/Z-protected PNA monomers were prepared
according to literature procedures.[48] Fmoc/Bhoc-protected PNA-mono-
mers were purchased from Applied Biosystems. Oligonucleotides were
custom-made by MWG-Biotech (Ebersberg, Germany). Reactions were
carried out at room temperature if no other specifications are given. Solid-
phase synthesis was performed manually using 5 mL polyethylene syringe
reactors which are equipped with a fritted disc. All column chromatog-
raphy was performed on SDS 60 ACC silica gel and TLC on E. Merck silica
gel 60 F254 glass/plastic/aluminium coated plates. The photometric deter-
mination of the Fmoc loadings was performed following a literature
method.[53] 1H- and 13C NMR spectra were recorded on Bruker AC250,
AM400, or DRX-500 spectrometers. The signals of the residual protonated
solvent (CDCl3 or [D6]DMSO) were used as reference signals. Coupling
constants J are reported in Hz. Mass spectra were measured on a Finnigan
MAT MS70 spectrometer for FAB-MS, on a PerSeptive Biosystems
Voyager for MALDI-TOF-MS and on a Finnigan-MAT LCQ for ESI-MS.
Table 1. Duplex stabilities.
Duplex (with 5'-(ATA)5A-3', 46)
Melting temperature[b]
Ac-Cys(EDANS)-(TTA)4TTA*T-GlyC, 44[a]
30.58C
Ac-Cys(EDANS)-(TTA)3TTA*TTAT-GlyC, 45[a] 28.58C
Ac-(TTA)5T-GlyC, 50
Ac-(TTA)4T-GlyC, 51
5'-(TTA)5T-3', 52
37.08C
27.08C
30.48C
[a] A* ˆ A(N6(CH2)4NH-DABCYL). [b] Determined as denaturation
curves at 1.25 mm probe concentration in a buffered solution (100mm
NaCl, 10mm NaH2PO4, 0.1mm EDTA, pH 7).
Chem. Eur. J. 2001, 7, No. 18
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0947-6539/01/0718-3919 $ 17.50+.50/0
3919

O. Seitz and O. Köhler
FULL PAPER
Fluorescence spectroscopy was performed with a Perkin ± Elmer LS-50B.
Analytical HPLC was performed with a Merck ± Hitachi system, for
preparative HPLC Rainin SD-300 pumps were used. A Nucleosil C18-HD
column (5m, 250 Â 4 mm, Macherey&Nagel) was used for analytical HPLC
and a Nucleosil C18-HD column (5m, 250 Â 10 mm) for preparative HPLC.
Gradients of solvent A (H2O, 1 % CH3CN, 0.1% TFA) and B (CH3CN, 1%
H2O, 0.1% TFA) were used as specified.
124.73, 119.81, 79.18, 70.59, 70.52, 70.45, 70.40, 70.34, 70.30, 69.59, 69.56,
67.64, 67.45, 66.32, 65.94, 65.03, 64.98, 49.89, 49.51, 48.88, 48.44, 47.10, 38.89,
34.62, 28.28; C46H58N2O13 (846.96).
(E)-17-[N-(2-(tert-Butyloxycarbonyl-amino)ethyl)-N-[N4-(benzyloxycar-
bonyl)cytosine-1-ylacetyl]glycinyloxy]-4,7,10,13-tetraoxa-15-heptadeceno-
ic acid, Boc-CZ-HYCRON-OH (6a): Zinc (0.1 g, activated by treatment
with 1n HCl and subsequent washing with H2O and glacial acetic acid) was
added to a solution of 5a (51 mg, 56.8 mmol) in glacial acetic acid (2 mL).
After 1.5 h of vigirous stirring zinc was removed by filtration over Hyflo.
The filtrate was concentrated in vacuo. The residue was repeatedly
coevaporated with toluene and purified by chromatography (CHCl3/
MeOH/AcOH). Coevaporation with toluene and subsequent drying in
vacuo yielded a colorless oil (35 mg, 79%). Rf (CHCl3/MeOH/AcOH
N-[2-(tert-Butyloxycarbonyl-amino)ethyl]-N-fluorenylmethyloxycarbo-
nylglycine, Boc-Aeg(Fmoc)-OH (3b): H2O (3 mL) and 1m NaOH (2 mL)
were added to N-[2-(tert-butyloxycarbonyl-amino)ethyl]-glycine ethyl ester
(116 mg, 0.50 mmol) in ethanol (8 mL). After 1 h of stirring the pH was
adjusted to pH 8 by addition of acetic acid. Subsequently, Fmoc-OSu
(138 mg, 0.41 mmol) was added. After 14 h of stirring the pH was adjusted
to pH 8 by addition of 1m NaOH. Again Fmoc-OSu (30 mg, 0.09 mmol)
was added. The pH adjustment and Fmoc-OSu addition was repeated until
all starting material was converted. Then, acetic acid was added for
neutralisation and the volatiles were removed by evaporation in vacuo. The
residue was dissolved in CHCl3 (20 mL). The organic phase was washed
with 0.5m KHSO4 (10 mL). The aqueous phase was extracted with CHCl3
(5 mL) before the combined organic layers were washed with brine (5 mL).
The organic layer was dried over MgSO4, and solvents were evaporated in
vacuo. The residue was dissolved in ethylacetate (20 mL) and n-hexane was
added until a precipate formed. Filtration and repeated washing with n-
hexane furnished an off-white powder (114 mg, 91%). 1H,13C NMR and
FAB-MS analysis confirmed the identity of 3b.[47]
1
85:15:1) ˆ 0.50; H NMR (two rotamers, 250 MHz, CDCl3): d ˆ 7.65 ± 7.52
(m, 1H, C6), 7.31 ± 7.09 (m, 6H, Zar, C6), 5.82 ± 5.72 (m, 2H, HYCRON-
H14,15), 5.53 ± 5.51 (m, 0.7H, Aeg-H6), 5.16 (s, 2H, Z-CH2), 4.99 ± 4.98 (m,
0.3H, Aeg-H6'), 4.68 (s, 1.4H, CCH ), 4.61 (d, J ˆ 3.3, 0.6H, HYCRON-H17'),
2
4.55 (d, J ˆ 4.3, 1.4H, HYCRON-H17), 4.50 (s, 0.6H, CCH '), 4.28 (s, 0.4H,
2
Aeg-H2'), 4.00 ± 3.95 (m, 3.6H, Aeg-H2, HYCRON-H14), 3.70 (m, 2H,
HYCRON-H3), 3.56 ± 3.45 (m, 14H, HYCRON, Aeg-H4'), 3.29 ± 3.19 (m,
2H, Aeg-H5), 2.54 (t, J ˆ 5.8, 2H, HYCRON-H2), 1.36 (s, 9H, tBu);
13C NMR (two rotamers, 62.5 MHz, CDCl3): d ˆ 169.28, 167.19, 163.40,
156.16, 155.33, 152.58, 150.11, 135.20, 132.34, 131.56, 129.01, 128.61, 128.47,
128.20, 125.74, 125.27, 95.23, 79.83, 70.56, 70.35, 69.72, 69.59, 67.72, 66.61,
‡
65.78, 65.19, 50.76, 49.58, 49.15, 48.85, 38.70, 35.13, 28.39; HR-MS (FAB ,
‡
glycerine/H2O): m/z: 778.344 [M‡H] , calcd for C36H51N5O14: 778.351.
(E)-17-[N-(2-(tert-Butyloxycarbonyl-amino)ethyl)-N-[N4-(benzyloxycar-
bonyl)cytosine-1-ylacetyl]glycinyloxy]-4,7,10,13-tetraoxa-15-heptadeceno-
ic acid phenacyl ester, Boc-CZ-HYCRON-OPac (5a): Br-HYCRON-OPac
4 (149 mg, 0.32 mmol), tetrabutylammonium bromide (63 mg, 0.19 mmol)
and saturated aqueous NaHCO3 solution (4 mL) were added to Boc-CZ-
OH 3a (95 mg, 0.19 mmol) in CH2Cl2 (4 mL). The two-phase system was
vigorously stirred for 14 h. The organic layer was separated and the aqeous
phase extracted twice with CH2Cl2. The combined organic layers were
dried over MgSO4, and solvents were evaporated in vacuo. Chromatog-
raphy (CHCl3/MeOH) yielded a slightly yellowish oil (140 mg, 83%). Rf
(CHCl3/MeOH 95:5) ˆ 0.19; 1H NMR (two rotamers, 250 MHz, CDCl3):
d ˆ 7.87 (dd, Jo,m ˆ 8.6, Jo,p ˆ 1.3, 2H, o-Pac), 7.62 ± 7.49 (m, m-Pac, p-Pac,
4H, C6), 7.47 ± 7.45 (m, 5H, Zar), 7.43 ± 7.35 (m, 1H, C6), 5.85 ± 5.75 (m, 2H,
HYCRON-H14,15), 5.64 ± 5.58 (m, 0.7H, Aeg-H6), 5.34 (s, 2H, Pac-CH2),
(E)-17-[N-[2-(tert-Butyloxycarbonyl-amino)ethyl]-N-fluorenylmethyloxy-
carbonyl-glycinyloxy]-4,7,10,13-tetraoxa-15-heptadecenoic acid, Boc-
Aeg(Fmoc)-HYCRON-OH (6b): A suspension comprised of 6a (1.24 g,
1.49 mmol), activated zinc (1 g) and glacial acetic acid (15 mL) was
vigirously stirred for 1.5 h. The work-up was performed as described for 6a.
Chromatography (CHCl3/MeOH/AcOH) yielded a yellowish oil (1.0 g,
1
94%). Rf (CHCl3/MeOH/AcOH 85:15.1) ˆ 0.58; H NMR (two rotamers,
250 MHz, CDCl3): d ˆ 7.71 ± 7.67 (m, 2H, Fmocar), 7.52 ± 7.44 (m, 2H,
Fmocar), 7.36 ± 7.22 (m, 4H, Fmocar), 5.79 ± 5.73 (m, 2H, HYCRON-H14,15),
5.03 ± 5.01 (m, 0.5H, Aeg-H6), 4.72 ± 4.70 (m, 0.5H, Aeg-H6'), 4.55 (d, J ˆ
4.2, 1H, HYCRON-H17), 4.50 (d, J ˆ 4.6, 1H, HYCRON-H17), 4.46 (d, J ˆ
5.8, 1H, Fmoc-CH2), 4.36 (d, J ˆ 6.5, 1H, Fmoc-CH2'), 4.19 (t, J ˆ 5.8, 0.5H,
Fmoc-H9), 4.12 (t, J ˆ 6.2, 0.5H, Fmoc-H9), 3.97 ± 3.87 (m, 4H, Aeg-H2,
HYCRON-H14), 3.69 (t, J ˆ 6.2, 2H, HYCRON-H3), 3.67 ± 3.50 (m, 12H,
HYCRON), 3.38 ± 3.35 (m, 1H), 3.21 ± 3.16 (m, 2H), 2.93 ± 2.91 (m, 1H)
2.54 (t, J ˆ 6.1, 2H, HYCRON-H2), 1.33 (s, 9H, tBu); 13C NMR (two
rotamers, 100.6 MHz, CDCl3): d ˆ 174.79, 169.80, 169.56, 156.24, 156.01,
143.84, 143.77, 141.30, 141.26, 131.69, 131.50, 127.73, 127.21, 127.06, 125.99,
124.89, 124.83, 119.93, 79.43, 70.68, 70.55, 70.46, 70.37, 70.29, 69.60, 67.79,
67.57, 66.47, 65.12, 49.98, 49.56, 48.90, 48.52, 47.18, 38.93, 34.86, 28.37; HR-
5.19 (s, 2H, Z-CH2), 4.99 ± 4.97 (m, 0.3H, Aeg-H6'), 4.73 (s, 1.4H, CCH ), 4.64
2
(d, J ˆ 4.9, 0.6H, HYCRON-H17'), 4.59 (d, J ˆ 4.6, 1.4H, HYCRON-H17),
2
2
4.53 (s, 0.6H, CCH '), 4.30 (s, 0.4H, Aeg-H '), 4.05 ± 3.98 (m, 3.6H, Aeg-H ,
HYCRON-H14), 3.81 (t, J ˆ 6.6, 2H, HYCRON-H3), 3.63 ± 3.48 (m, 14H,
HYCRON, Aeg-H4), 3.35 ± 3.15 (m, 2H, Aeg-H5), 2.77 (t, J ˆ 6.5, 2H,
HYCRON-H2), 1.40 (s, 9H, tBu); 13C NMR (two rotamers, 62.5 MHz,
CDCl3): d ˆ 192.09, 175.87, 171.01, 169.33, 167.72, 167.22, 163.06, 162.62,
156.08, 155.48, 152.42, 149.85, 135.11, 134.15, 133.91, 132.56, 131.80, 128.87,
128.64, 128.55, 128.19, 127.76, 125.63, 125.15, 95.10, 79.81, 77.26, 70.68, 70.56,
70.44, 70.40, 69.77, 69.68, 67.80, 66.42, 66.06, 65.83, 65.28, 50.79, 49.39, 49.09,
48.91, 38.69, 36.49, 34.72, 31.44, 21.67, 28.39, 27.86, 27.39, 20.99; C44H57N5O15
(895.95).
2
‡
‡
MS (FAB , glycerine/H2O): m/z: 715.349 [M‡H] , calcd for C37H50N2O12
:
715.344.
(N6-(1-Aminohex-6-yl)adenine-9-yl)acetic acid tert-butyl ester (9): 1,6-
Diaminohexane (2.15 g, 18.50 mmol) was added to the 6-chloropurine 8
(0.78 g, 2.90 mmol) in n-butanol (15 mL). After 1 h reflux the solvent was
removed in vacuo. The residue was purified by chromatography (CHCl3/
MeOH 8:2 ! CHCl3/MeOH/Me2NEt 7:3:0.01) yielding a yellowish oil
(380 mg, 38%). Rf (CHCl3/MeOH/EtNMe2 7:3:0.1) ˆ 0.53; 1H NMR
(250 MHz, CDCl3): d ˆ 8.36 (s, 1H), 7.79 (s, 1H), 5.97 (t, J ˆ 5.5, 1H),
4.84 (s, 2H, CH2COOtBu), 3.65 ± 3.48 (m, 2H), 3.15 ± 2.99 (m, 2H), 2.70 ±
2.49 (m, 2H), 1.70 ± 1.60 (m, 2H), 1.52 ± 1.39 (m, 15H); 13C NMR
(62.5 MHz, CDCl3): d ˆ 166.38 (CO), 155.07 (A6), 153.37 (A2), 149.18
(A4), 140.11 (A8), 119.11 (A5), 83.42 (CMe3), 46.92, 44.76 (CH2COOtBu),
32.73, 29.67, 28.00 (tBu), 26.66, 26.53.
(E)-17-[N-[2-(tert-Butyloxycarbonyl-amino)ethyl]-N-fluorenylmethyloxy-
carbonyl-glycinyloxy]-4,7,10,13-tetraoxa-15-heptadecenoic acid phenacyl
ester, Boc-Aeg(Fmoc)-HYCRON-OPac (5b): A two-phase system con-
sisting of Boc-Aeg(Fmoc) (1.01 g, 2.29 mmol), Br-HYCRON-Pac 4 (1.15 g,
2.44 mmol), Bu4NBr (0.72 g, 2.22 mmol), CH2Cl2 (20 mL) and saturated
NaHCO3 (20 mL) was treated as described for 5a. Purification was
achieved by chromatography (n-hexane/EtOAc), which yielded of
a
colorless oil (1.75 g, 92%). Rf (n-hexane/EtOAc 1:2) ˆ 0.47; 1H NMR
(two rotamers, 250 MHz, CDCl3): d ˆ 7.89 (d, J ˆ 7.3, 2H, o-Pac), 7.74 (2 Â
d, J ˆ 7.3, 2H), 7.62 ± 7.25 (m, 9H, Pacar, Fmocar), 5.84 ± 5.71 (m, 2H,
HYCRON-H14,15), 5.34 (s, 2H, Pac-CH2), 5.09 ± 5,06 (m, 0.5H, Aeg-H6),
4.80 ± 4.77 (m, 0.5H, Aeg-H6'), 4.61 (d, J ˆ 4.5, 1H, HYCRON-H17), 4.56 (d,
J ˆ 4.7, 1H, HYCRON-H17), 4.50 (d, J ˆ 6.0, 1H, Fmoc-CH2), 4.41 (d, J ˆ
6.5, 1H, Fmoc-CH2'), 4.24 (t, J ˆ 6.0, 0.5H, Fmoc-H9), 4.17 (t, J ˆ 6.4, 0.5H,
Fmoc-H9), 3.99 ± 3.91 (m, 4H, Aeg-H2, HYCRON-H14), 3.81 (t, J ˆ 6.6, 2H,
HYCRON-H3), 3.64 ± 3.53 (m, 12H, HYCRON), 3.44 ± 3.41 (m, 1H),
3.27 ± 3.24 (m, 2H), 2.99 ± 2.94 (m, 1H), 2.78 (t, J ˆ 6.5, 2H, HYCRON-H2),
1.38 (s, 9H, tBu); 13C NMR (two rotamers, 100.6 MHz, CDCl3): d ˆ 191.94,
170.85, 169.47, 156.17, 156.04, 155.83, 143.76, 143.67, 141.19, 141.15, 134.05,
133.77, 131.75, 131.56, 128.74, 127.63, 127.11, 126.96, 125.73, 125.50, 124.78,
(N-6-(1-(Fluorenylmethoxycarbonyl-amino)hex-6-yl)adenine-9-yl)acetic
acid tert-butyl ester (10): Fmoc-Cl (148 mg, 0.57 mmol) and NMM (66 mL,
0.60 mmol) were added to amine 9 (190 mg, 0.54 mmol) in CH2Cl2 (5 mL).
After 4 h CH2Cl2 (60 mL) and an aqueous pH 4 buffer (70 mL) comprised
of 0.5m KHSO4 and sat. NaHCO3 was added. The resulting two-phase
system was stirred. The organic layer was separated and the aqueous phase
was extracted with CH2Cl2. The combined organic phases were washed
with brine and dried over MgSO4. Chromatography (EtOAc) yielded a
white amorphous solid (223 mg, 72%). Rf (EtOAc) ˆ 0.34; 1H NMR
(250 MHz, CDCl3): d ˆ 8.37 (s, 1H), 7.80 (s, 1H), 7.74 (d, J ˆ 7.4, 2H, Fmoc-
H4,5), 7.58 (d, J ˆ 7.3, 2H, Fmoc-H1,8), 7.39 (t, J ˆ 7.3, 2H, Fmoc-H3,6), 7.30 (t,
3920
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0718-3920 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
J ˆ 7.3, 2H, Fmoc-H2,7), 6.00 ± 5.85 (m, 1H), 5.09 ± 5.00 (m, 1H), 4.84 (s, 2H,
CH2COOtBu), 4.40 (d, J ˆ 7.0, 2H, Fmoc-CH2), 4.25 (d, 1H, Fmoc-H9),
3.70 ± 3.62 (m, 2H), 3.20 ± 3.13 (m, 2H), 1.73 ± 1.64 (m, 2H), 1.53 ± 1.40 (m,
15H); 13C NMR (62.5 MHz, CDCl3): d ˆ 166.16 (CO), 156.33 (A6), 154.69
(A2), 153.08 (Fmoc-CO), 149.06 (A4), 143.86 (Fmoc-C4a,b), 141.13 (Fmoc-
C8a,b), 140.06 (A8), 127.48 (Fmoc-C3,6), 126.86 (Fmoc-C2,7), 124.90 (Fmoc-
C1,8), 119.78 (Fmoc-C4,5), 118.89 (A5), 83.33 (CMe3), 66.29, 47.13, 44.60
(CH2COOtBu), 40.75, 29.68, 29.45, 27.83 (tBu), 26.31, 26.20; C32H38N6O4
(570.68).
A8), 6.46 (brs, 1H, NH), 6.05 ± 5.89 (m, 1H, All-H2), 5.26 ± 5.09 (m, Jcis
ˆ
10.2, Jtrans ˆ 16.8, 3H, NH, All-H3), 4.73 (d, J ˆ 6.6, 2H, All-H1), 3.74 (brs,
6
N6-CH2CH2
1H, AN -CH CH ), 3.39 ± 3.34 (m, 2H, A
), 1.34 (s, 9H, Boc); 13C NMR
(62 MHz, CDCl3): d ˆ 156.25, 154.91, 152.88, 139.76, 131.84, 119.93, 118.80,
2
2
79.30, 45.64, 40.75, 28.30; C15H22N6O2 (318.38).
(N6-(1-tert-Butyloxycarbonyl-aminobut-4-yl)adenine-9-yl)acetic
acid
(15a): The ozonolysis of 14a (0.59 g, 1.70 mmol) was performed as
described by Thomson and co-workers.[59] For work-up the reaction
mixture was concentrated to 1ꢁ3 of the original volume. The precipitate
was collected yielding a yellowish powder (346 mg, 58%). Rf (CHCl3/
(N6-(1-(Fluorenylmethoxycarbonyl-amino)hex-6-yl)adenine-9-yl)acetic
acid (11): TFA (5 mL) was added to the tert-butyl ester 10 (115 mg,
0.20 mmol). After 5 min stirring the solvent was removed in vacuo. The
residual TFAwas removed by repeated coevaporation with toluene. During
the chromatography (CHCl3/MeOH/AcOH) precipitation occured. Elu-
tion with DMF and subsequent removal of the solvent in vacuo yielded a
yellowish amorphous solid (98 mg, 95%). It was not possible to remove
DMF entirely. Since analytical RP-HPLC revealed that no by-products but
DMF were present, the material was used for further reactions. Rf (CHCl3/
MeOH/AcOH 85:15:1) ˆ 0.4; tR ˆ 27 min (grad: 0 min (0%B) ! 40 min
(80%B), 1 mLmin 1, 508C); 1H NMR (250 MHz, [D6]DMSO): d ˆ 8.16 (s,
1H), 8.05 (s, 1H), 7.87 (d, J ˆ 7.5, 2H, Fmoc-H4,5), 7.73 ± 7.65 (m, J ˆ 7.3, 3H,
NH, Fmoc-H1,8), 7.39 (t, J ˆ 7.3, 2H, Fmoc-H3,6), 7.32 ± 7.25 (m, 3H), 4.81 (s,
2H, CH2COOtBu), 4.27 (d, J ˆ 6.9, 2H, Fmoc-CH2), 4.19 (d, J ˆ 6.7, 1H,
Fmoc-H9), 3.44, 3.35 (2 Â brs, incl. H2O), 2.97 ± 2.92 (m, 2H), 1.73, 1.57 ±
1.54 (m, 2H), 1.39 ± 1.21 (m, 6H); 13C NMR (100.6 MHz, [D6]DMSO): d ˆ
170.21 (CO), 156.21 (A6), 154.56 (Fmoc-CO), 152.45 (A2), 149.0 (A4),
144.03 (Fmoc-C4a,b), 141.41 (A8), 140.82 (Fmoc-C8a,b), 127.69 (Fmoc-C3,6),
127.13 (Fmoc-C2,7), 125.24 (Fmoc-C1,8), 120.19 (Fmoc-C4,5), 118.73 (A5),
65.23, 46.87, 45.01, 40.26, 29.46, 29.24, 26.26, 26.14; C28H30N6O4 (514.58).
1
MeOH/AcOH 85:15:1) ˆ 0.17; H NMR (250 MHz, [D6]DMSO): d ˆ 8.17
(brs, 1H, A2), 8.08 (s, 1H, A8), 7.76 (brs, 1H, NH), 6.81 ± 6.77 (m, 1H, NH),
9
6
6
4.95 (s, 2H, AN -CH ), 3.59 ± 3.22 (m, 2H, AN -Bu-1), 2.92 (ddd, 2H, AN -Bu-4
,
2
6
6
J1 ꢀ J2 ꢀ J3 ꢀ 6.3), 1.59 ± 1.32 (m, 13H, AN -Bu-2, AN -Bu-3, Boc); 13C NMR (62.5
MHz, [D6]DMSO): d ˆ 169.39 (COOH), 155.61, 154.55 (Boc-CO, A6), 152.51
9
(A2), 148.97 (A4), 141.08 (A8), 118.56 (A5), 77.35 (Boc), 43.98 (AN -CH ),
2
6
6
6
6
39.72 (AN -Bu-4, AN -Bu-1), 28.29 (Boc), 27.04, 26.60 (AN -Bu-3, AN -Bu-2); anal.
calcd for C16H24N6O4: C 52.7, H 6.6, N 23.0; found: C 52.5, H 6.5, N 22.1.
(N6-(1-(tert-Butyloxycarbonyl-amino)eth-2-yl)adenine-9-yl)acetic
acid
(15b): The ozonolysis of 14b (710 mg, 2.23 mmol) and the subsequent
work-up was performed as described above yielding a colorless powder
1
(529 mg, 71%). H NMR (250 MHz, [D6]DMSO): d ˆ 8.18 (brs, 1H, A2),
8.10 (s, 1H, A8), 7.74 (brs, 1H, NH), 6.94 ± 6.90 (m, 1H, NH), 4.95 (s, 2H,
N9-CH2
N6-CH2CH2
N6-CH2CH2
A
), 3.50 ± 3.35 (m, 2H, A
), 3.16 (m, 2H, A
), 1.35 (s, 9H,
Boc); 13C NMR (100.6 MHz, [D6]DMSO): d ˆ 169.40 (COOH), 155.72,
154.49 (Boc-CO, A6), 152.48 (A2), 149.04 (A4), 141.30 (A8), 118.73 (A5),
9
N6-CH2CH2
‡
77.66 (Boc), 44.00 (AN -CH ), 39.90 (A
), 28.26 (Boc); HR-MS (FAB ,
2
‡
3-NBA): m/z: 337.166 [M‡H] , calcd for C14H20N6O4: 337.162.
N-(2-(Fluorenylmethoxycarbonyl-amino)ethyl)-glycine allyl ester (17): A
solution of Fmoc-aminoethylglycine tert-butyl ester 16 (1.54 g, 3.66 mmol)
in TFA (10 mL) was stirred for 30 min. The TFA was removed in vacuo.
The residue was coevaporated with toluene (3 Â ). The residue was
supended in allyl alcohol (10 mL). At 08C thionylchloride (0.90 mL) was
added in portions. The mixture was stirred at 1008C for 1 h. After cooling to
room temperature diethylether (25 mL) was added. The precipitate was
collected by filtration. Repeated washings with diethyl ether yielded a
colorless powder (1.25 g, 84%). The material was used without further
purification.
1-(N6-(1-Aminobut-4-yl)adenine-9-yl)prop-2-ene (13a): 1,4-Diaminobu-
tane (3.62 g, 41.1 mmol) was added to a solution of 12 (1.0 g, 5.14 mmol)
in n-butanol (20 mL). After 1 h stirring at 808C the solvent was removed in
vacuo. Chromatography (CHCl3/MeOH/H2O/EtNMe2 60:30:5:1) and sub-
sequent drying in vacuo furnished a yellowish oil (0.53 g, 42%). Apart from
1H NMR analysis this material was used without further characterisation.
Rf (CHCl3/MeOH 3:2) ˆ 0.27; 1H NMR (400 MHz, [D6]DMSO): d ˆ 8.20
(brs, 1H, A2), 8.09 (s, 1H, A8), 7.79 (brs, 1H, NH), 6.09 ± 6.00 (m, 1H, All-
H2), 5.16 (dd, J1 ˆ 10.3, J2 ˆ 1.0, 1H, All-H3cis), 5.02 (d, J ˆ 17.3, 1H, All-
6
6
H3trans), 4.77 (d, J ˆ 5.3, 2H, All-H1), 3.80 ± 3.50 (m, NH2, AN -Bu-1, AN -Bu-4
,
N-[2-(Fluorenylmethoxycarbonyl-amino)ethyl]-N-[N6-(1-(tert-butyloxy-
carbonyl-amino)but-4-yl)adenine-9-yl)acetyl]glycine allyl ester, Fmoc-
6
6
H2O), 1.64 (m, 4H, AN -Bu-2, AN -Bu-3).
1-(N6-(1-Aminoeth-2-yl)adenine-9-yl)prop-2-ene (13b): The reaction of
purine 12 (1.00 g, 5.12 mmol) and ethylenediamine (3.09 g, 51.40 mmol)
was performed as described above. Chromatography (CHCl3/MeOH/
Et2NMe/H2O 6:4:1:1) yielded the title compound (1.9 g) which was used
without further purification.
A(CH ) NH-Boc-OAll (18a): HOBt (41 mg, 0.23 mmol) and DCC (48 mg,
0.23 mmol) were added to a solution of the amine 17 (97 mg, 0.23 mmol)
and the acid 15a (84 mg, 0.23 mmol) in a mixture of DMF (1 mL) and
CH2Cl2 (1 mL). After 20 h stirring the solvent was removed in vacuo. The
residue was suspended in CHCl3 (10 mL) and filtrated. The filtrate was
washed twice with sat. NaHCO3. The organic layer was washed with 0.1m
KHSO4 and with brine. The combined organic layers were dried with
MgSO4 before the solvent was removed in vacuo. Chromatography
2
4
1-(N6-(1-(tert-Butyloxycarbonyl-amino)but-4-yl)adenine-9-yl)prop-2-ene
(14a): Boc2O (137 mg, 0.63 mmol) was added to amine 13a (136 mg,
0.53 mmol) in dry pyridine (3 mL). TLC control after 16 h showed the
presence of starting material. Thus, DMAP (20 mg, 0.16 mmol) and Boc2O
(65 mg, 0.3 mmol) were added. After 4 h the solvent was removed in vacuo.
The residue was dissolved in CH2Cl2 (10 mL) and washed twice with 1m
NaH2PO4 buffer (pH 5.6). The combined aqueous phases were extracted
with CH2Cl2 before the combined organic layers were dried over MgSO4.
The subsequent chromatography (EtOAc) yielded an amorphous solid
(94 mg, 51%). Rf (CHCl3/MeOH 9:1) ˆ 0.27; 1H NMR (400 MHz, CDCl3):
d ˆ 8.39 (brs, 1H, A2), 7.75 (brs, 1H, A8), 6.09 ± 5.99 (m, 1H, All-H2), 5.79
(brs, 1H, NH), 5.30 (d, J ˆ 10.0, 1H, All-H3cis), 5.19 (d, J ˆ 17.0, 1H, All-
H3trans), 4.80 (d, J ˆ 5.8, 2H, All-H1), 3.74 (brs, 1H, NH), 3.68 (brs, 3H,
(EtOAc ! EtOAc/EtOH 8:1) yielded
a colorless amorphous solid
(127 mg, 76%). Rf (EtOAc/EtOH 9:1) ˆ 0.4; 1H NMR (two rotamers,
400 MHz, [D6]DMSO): d ˆ 8.12 (brs, 1H), 7.87 (d, J ˆ 7.5, 2H, Fmoc-H4,5),
7.78 (brs, 0.7H), 7.69 ± 7.64 (m, 2.3H), 7.52 ± 7.49 (m, 0.7H), 7.40 (t, J ˆ 7.5,
2H, Fmoc-H3,6), 7.32 ± 7.27 (m, 2.3H), 6.81 (brs, 1H), 5.98 ± 5.81 (m, 2H,
All-H2), 5.40 ± 5.16 (m, 3.4H, All-H3, ACH ), 5.04 (s, 0.6H, A ), 4.68 (d,
CH2'
2
J ˆ 5.5, 0.6H, All-H1'), 4.55 (d, J ˆ 5.3, 1.4H, All-H1), 4.48 (s, 0.6H, Aeg-
H2'), 4.35 (d, J ˆ 6.7, 1.4H, FmocCH ), 4.29 ± 4.19 (m, 2.6H, Fmoc , Fmoc-
CH2'
2
H9), 4.11 (s, 1.4H, Aeg-H2), 3.58 ± 3.54 (m, 1.4H), 3.46 ± 3.34 (m, incl. H2O),
3.14 ± 3.10 (m, 0.6H), 2.94 ± 2.90 (m, 2H), 1.58 ± 1.53 (m, 2H), 1.41 ± 1.33 (m,
11H); 13C NMR (two rotamers, 100.6 MHz, [D6]DMSO): d ˆ 172.09, 169,
25, 168.69, 167.14, 156.46, 156.18, 155.62, 154.43, 152.31, 149.05, 143.88,
141.41, 140.78, 132.26, 127.64, 127.07, 125.11, 120.14, 118.34, 117.88, 77.34,
65.55, 64.91, 47.92, 47.15, 46.76, 43.78, 28.28, 27.04, 26.57, 21.07; C38H46N8O7
(726.83).
6
6
AN -Bu-1), 3.21 ± 3.18 (m, 2H, AN -Bu-4), 1.76 ± 1.70 (m, 2H), 1.64 ± 1.57 (m,
2H), 1.43 (s, 9H, Boc); 13C NMR (125.7 MHz, CDCl3): d ˆ 156.03, 154.66,
153.26, 148.76, 139.32, 131.70, 118.91, 78.83, 45.67, 39.97, 29.57, 28.35, 26.84,
21.21; C17H26N6O2 (346.43).
1-(N6-(1-(tert-Butyloxycarbonyl-amino)eth-2-yl)adenine-9-yl)prop-2-ene
(14b): Boc2O (2.31 g, 10.56 mmol) and DMAP (0.21 g, 1.76 mmol) were
added to the raw compound 13b (1.9 g) in dry pyridine (42 mL). After 19 h
a further portion of Boc2O (1.01 g, 4.58 mmol) and DMAP (0.21 g,
1.76 mmol) were added. After 4 h aqueous work-up was performed as
described for 14a. Chromatography (EtOAc ! EtOAc/EtOH 3:1) yielded
a yellowish amorphous solid (0.71 g, 44% based on 12). Rf (CHCl3/MeOH
N-[2-(Fluorenylmethoxycarbonyl-amino)ethyl]-N-[N6-(1-(tert-butyloxy-
carbonyl-amino)eth-2-yl)adenine-9-yl)acetyl]glycine allyl ester, Fmoc-
A(CH ) NH-Boc-OAll (18b): The reaction including amine 17 (174 mg,
0.42 mmol) and acid 15b (140 mg, 0.42 mmol) was performed as described
above. Chromatography (EtOAc ! EtOAc/EtOH 8:1) yielded a colorless
amorphous solid (245 mg, 84%). The product was used without further
characterisation. Rf (EtOAc/EtOH 9:1) ˆ 0.45.
2
2
1
9:1) ˆ 0.50; H NMR (250 MHz, CDCl3): d ˆ 8.31 (s, 1H, A2), 7.73 (s, 1H,
Chem. Eur. J. 2001, 7, No. 18
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0718-3921 $ 17.50+.50/0
3921

O. Seitz and O. Köhler
FULL PAPER
N-[2-(Fluorenylmethoxycarbonyl-amino)ethyl]-N-[N6-(1-(tert-butyloxy-
dioxane (3 Â 3 mL), 1,4-dioxane/H2O (1:1, 3 Â 3 mL), H2O (3 Â 3 mL) and
dry DMF (8 Â 3 mL). To the resin was added a 3 ± 5-fold excess (based on
Ac-loading of resin 21) of a solution of the symmetrical anhydride of the
starting monomers. After 18 h the resin was washed with DMF (6 Â 3 mL)
and CH2Cl2 (4 Â 3 mL).
Boc Cleavage: TFA/m-Cresol (95:5, 1.5 mL) was added to the resin. After
4 min and washing with CH2Cl2 this procedure was repeated once. Finally
the resin was washed with CH2Cl2 (6 Â 2 mL) and DMF (6 Â 2 mL).
Coupling: 2.5 equiv of the building block (final concentration ꢀ0.1m) were
added to the resin, which was preactivated for 2 min using 4 equiv HOBt,
2.5 equiv HBTU in a solution of 0.5m DIPEA in pyridine/DMF (1:4). After
3 h the resin was washed with DMF (5 Â 2 mL).
Capping: Ac2O/Pyr (1:50, 2 mL) was added to the resin. After 3 min the
resin was washed with DMF and the procedure was repeated once. Finally
the resin was washed with DMF (6 Â 2 mL) and CH2Cl2 (6 Â 2 mL).
carbonyl-amino)but-4-yl)adenine-9-yl)acetyl]glycine, Fmoc-A(CH ) NH-Boc
-
2
4
OH (19a): N-Methylaniline (0.56 mL) was added to a solution of allyl
ester 18a (205 mg, 0.28 mmol) in THF (6 mL). The solution was degassed
by freeze-thaw-pump cycles. After addition of [Pd(PPh3)4] the mixture was
stirred for 14 h under exclusion of light. The solvent was removed in vacuo.
To the residue was added methanol (5 mL). The resulting precipitate was
removed by filtration. The filtrate was concentrated to dryness in vacuo
before CHCl3 (20 mL) was added. The solution was washed twice with 0.3m
KHSO4. The aqueous phase was extracted twice with CHCl3. The
combined organic layers were washed with brine and dried over MgSO4
before the solvent was removed in vacuo. Chromatography (CHCl3/
MeOH/AcOH) and subsequent lyophilisation from benzene yielded an off-
white powder (155 mg, 81%). Rf (CHCl3/MeOH/AcOH 85:15:1) ˆ 0.10;
1H NMR (500 MHz, 1H,13C-COSY, HMQC, [D6]DMSO): d ˆ 8.14 ± 8.12
(m, 1H, A2), 7.94 (s, 1H, A8), 7.87 (d, J ˆ 7.5, 2H, Fmoc-H4,5), 7.76 (brs, 1H,
NH), 7.66 ± 7.62 (m, 2H, Fmoc-H1,8), 7.53 (t, J ˆ 6.8, 0.6H, NH), 7.43 ± 7.40
Cleavage: A degassed solution of [Pd(PPh3)4] in morpholine/DMF/DMSO
(1:5:5) was added to the resin. After 16 h the resin was washed repeatedly
with DMF. The filtrate was evaporated in vacuo and the residue repeatedly
coevaporated with DMF until dryness.
(m, J ˆ 7.4, 2.4H, NH, Fmoc-H3,6), 7.25 (t, J ˆ 7.3, 2H, NH, Fmoc-H2,7), 6.94
9
N9-CH2'
(brs, 1H, NH), 5.19 (s, 1.2H, AN -CH ), 5.01 (s, 0.8H, A
), 4.36 ± 4.17 (m,
2
3.8H, Fmoc-CH2, Fmoc-H9, Aeg-H2'), 3.98 (s, 1.2H, Aeg-H2), 3.57 ± 3.52
6
(m, 1.2H, Aeg-H4), 3.50 ± 3.39 (m, 2H, AN -Bu-1), 3.37 ± 3.30 (m, 2H, Aeg-
Boc-Phe-GZ-GZ-CZ-OH (23): 31 mg (25 mmol) resin 21 were used. Gra-
dient silica gel chromatography (CHCl3/MeOH/AcOH 85:15:1 ! CHCl3/
MeOH/AcOH/H2O 4:2:1:0.35) furnished a white powder (13.5 mg, 36%).
Rf (CHCl3/MeOH/AcOH/H2O 4:2:1:0.2) ˆ 0.26; MS (MALDI-TOF, DHB,
6
H4', Aeg-H5), 3.16 ± 3.10 (m, 0.8H, Aeg-H5'), 2.94 ± 2.91 (m, 2H, AN -Bu-4),
6
6
1.60 ± 1.52 (m, 2H, AN -Bu-3) 1.44 ± 1.30 (s, 11H, AN -Bu-2, Boc); 13C NMR (two
rotamers, 125.6 MHz, 1H,13C-COSY, HMQC, [D6]DMSO): d ˆ 170.93,
9
170.47 (Aeg-C1), 167.47, 167.00 (AN -CH CO), 156.53, 156.16 (Fmoc-CO),
2
‡
pos): m/z: 1502.11 [M(monoisotop)‡H] , calcd for C70H76N20O19: 1501.72.
155.65 (Boc-CO), 154.57 (A6), 152.31 (A2), 149.29 (A4), 143.96 (Fmoc-
C8a,b), 141.45 (A8), 140.88 (Fmoc-C4a,b), 127.84 (Fmoc-C3,6), 127.08 (Fmoc-
C2,7), 125.18 (Fmoc-C1,8), 120.09 (Fmoc-C4,5), 118.63 (A5), 77.41 (Boc),
65.64, 65.58 (Fmoc-CH2), 49.24, 47.76, 47.02 (Aeg-C2, Aeg-C4), 46.78
Ac-CZ-CZ-GZ-Gly-OH (24): 37 mg (30 mmol) resin 21 were used. LH20
chromatography (CHCl3/MeOH 1:1) and subsequent n-hexane-mediated
precipitation from CH2Cl2/MeOH (1:1) and drying in vacuo furnished a
white powder (13.6 mg (35%). Rf (CHCl3/MeOH/AcOH/H2O 4:2:1:0.5) ˆ
0.5; tR ˆ 31 min (0 ± 40 min: 10 ± 80% B in A, 1 mLmin 1, 508C); MS
9
6
6
(Fmoc-C9), 43.81, 43.49 (AN -CH ), 39.92 (AN -Bu-4), 39.63 (AN -Bu-1), 38.92,
2
6
6
38.12 (Aeg-C5), 27.66 (Boc), 26.39 (AN -Bu-3), 24.52 (AN -Bu-2); HR-MS (FAB,
‡
‡
(MALDI-TOF, DHB, pos): m/z: 1314.4 [M(average)‡H] , calcd for
3-NBA/DMSO, pos): m/z: 687.318 [M‡H] , calcd for C35H42N8O7: 687.325.
C59H64N18O18: 1314.3.
N-[2-(Fluorenylmethoxycarbonyl-amino)ethyl]-N-[N6-(1-(tert-butyloxy-
carbonyl-amino)eth-2-yl)adenine-9-yl)acetyl]glycine, Fmoc-A(CH ) NH-Boc
-
Solid-phase synthesis by loading preformed starting monomer-HYCRON
conjugates: Boc-Gly-GZ-GZ-CZ-OH (25): A solution of conjugate 6a
(80.0 mg, 103 mmol), HOBt (23.6 mg, 150 mmol), DIPEA (35.2 mL,
206 mmol) and HBTU (39 mg, 103 mmol) in DMF (1 mL) was added to
aminomethylpolystyrene (62.4 mg, 1.1 mmolg 1). After 15 h the resin was
washed with CH2Cl2 and capped by a 10 min treatment with Ac2O/Pyr
(1:50, 2 mL). Washing with DMF and CH2Cl2 and drying afforded resin 22a
(140 mg). The subsequent synthesis proceeded as decribed using resin 22a
(70 mg). After cleavage the crude material was purified by LH20
chromatography (CHCl3/MeOH 1:1) and gradient silica gel chromatog-
2
2
OH (19b): The reaction including allyl ester 18b (245 mg, 0.35 mmol)
was performed as described above. Two-fold chromatography (CHCl3/
MeOH/AcOH) and subsequent lyophilisation from benzene yielded an off-
white powder (165 mg, 71%). However, even after repeated lyophilisation
it was not possible to remove DMF entirely. Rf (CHCl3/MeOH/AcOH
85:20:2) ˆ 0.50; 1H NMR (two rotamers, 400 MHz, 1H,13C-COSY, HMQC,
[D6]DMSO): d ˆ 8.12 (brs, 1H, A2), 7.97 (s, 1H, A8), 7.87 (d, J ˆ 7.5, 2H,
Fmoc-H4,5), 7.69 ± 7.64 (m, 3H, NH, Fmoc-H1,8), 7.50 (t, J ˆ 6.8, 0.6H, NH),
7.38 (t, J ˆ 7.4, 2H, Fmoc-H3,6), 7.32 ± 7.28 (m, J ˆ 7.3, 2.4H, NH, Fmoc-H2,7),
9
N9-CH2'
6.91 (brs, 1H, NH), 5.20 (s, 1.2H, AN -CH ), 5.02 (s, 0.8H, A
), 4.36 ± 4.14
raphy
(CHCl3/MeOH/AcOH
85:20:2 ! CHCl3/MeOH/AcOH/H2O
2
(m, 3.8H, Fmoc-CH2, Fmoc-H9, Aeg-H2'), 3.98 (s, 1.2H, Aeg-H2), 3.53 ±
4:2:1:0.35). Drying in vacuo furnished a white powder (32.2 mg, 67%). Rf
(CHCl3/MeOH/AcOH/H2O 4:2:1:0.5) ˆ 0.42; tR ˆ 20 min (0 ± 40 min: 25 ±
6
3.51 (m, 3.2H, AN -CH CH , Aeg-H ), 3.33 ± 3.30 (m, 1.6H, Aeg-H , Aeg-H ),
4
4'
5'
2
2
75% B in A, 1 mLmin 1, 508C); MS (FAB , 3-NBA/DMSO): m/z: 1411.82
‡
6
3.16 ± 3.13 (m, 3.2H, AN -CH CH , Aeg-H ), 1.35 (s, 9H, Boc); 13C NMR (two
5
2
2
‡
rotamers, 100.6 MHz, 1H,13C-COSY, HMQC, [D6]DMSO): d ˆ 171.00,
[M(monoisotop)‡H] , calcd for C63H70N20O19: 1411.52.
9
170.46 (Aeg-C1), 167.32, 166.85 (AN -CH CO), 156.42, 156.14 (Fmoc-CO),
Ac-CZ-CZ-GZ-Gly-OH (24):
A solution of conjugate 6c (110.0 mg,
2
155.70 (Boc-CO), 154.47 (A6), 152.31 (A2), 149.20 (A4), 143.87 (Fmoc-
C8a,b), 141.64 (A8), 140.75 (Fmoc-C4a,b), 127.63 (Fmoc-C3,6), 127.08 (Fmoc-
C2,7), 125.11 (Fmoc-C1,8), 120.14 (Fmoc-C4,5), 118.52 (A5), 77.62 (Boc), 65.54
240 mmol), HOBt (53.0 mg, 350 mmol), NMM (52.0 mL, 480 mmol) and
HBTU (39 mg, 232 mmol) in CH2Cl2/DMF (2:1, 1.5 mL) was added to
aminomethylpolystyrene (90.0 mg, 1.1 mmol per g). After 15 h the resin
was washed with CH2Cl2 and capped by a 5 min treatment with Ac2O/Pyr
(1:3, 2 mL). Washing with DMF and CH2Cl2 and drying afforded resin 22c
(170 mg). The subsequent synthesis proceeded as decribed using 56 mg of
resin 22c. After cleavage the crude material was purified by LH20
chromatography (CHCl3/MeOH 1:1) and silica gel chromatography
(CHCl3/MeOH/AcOH/H2O 7:2.5:1:0.5). Drying in vacuo furnished a white
powder (25.6 mg, 59%). Analytical data: see above.
(Fmoc-CH2), 50.00, 47.34 (Aeg-C2), 47.01, 46.96 (Aeg-C4), 46.73 (Fmoc-C9),
N6-CH2CH2
N6-CH2CH2
9
43.65, 43.47 (AN -CH ), 39.66 (A
, A
), 38.70, 37.89 (Aeg-C5),
2
‡
‡
28.24 (Boc); HR-MS (FAB , 3-NBA/DMSO): m/z: 659.301 [M‡H] , calcd
for C33H38N8O7: 659.294.
Solid-phase synthesis by esterification of the starting monomer with the
anchor alcohol: A solution of acid 20 (1.14 g, 3.41 mmol), HOBt (0.92 g,
5.11 mmol), NMM (0.75 mL, 6.82 mmol) and HBTU (1.29 g, 3.41 mmol) in
CH2Cl2 (25 mL) was added to aminomethylpolystyrene (1.24 g, 1.1 mmol
per g). After 15 h the resin was washed with CH2Cl2 and capped by a 15 min
treatment with 16 mL Ac2O/Pyr (1:3). Washing with CH2Cl2 and drying
afforded polymer 21 (1.74 g). NMR-in-situ-cleavage assay: Minute
amounts of sodium were dissolved in [D4]MeOH (0.5 mL). This solution
was added to CDCl3 (3 mL). To the resin 21 (62.4 mg) was added toluene
(4.15 mL) and the freshly prepared solution of sodium methylate in CDCl3
(2 mL). After 30 min 1H NMR revealed a toluene/Ac-ratio of 1.28/1
translating into a loading of 0.8 mmol per g.
Solid-phase synthesis by on-resin synthesis of the starting monomer
Boc-GZ-HYCRON-PS (27):
A solution of conjugate 6b (0.98 g,
1.37 mmol), HOBt (0.32 g, 2.06 mmol), NMM (0.3 mL, 2.74 mmol) and
HBTU (0.52 g, 1.37 mmol) in DMF (7 mL) was added to aminomethylpo-
lystyrene (0.62 g, 1.1 mmol per g). After 15 h the resin was washed with
DMF and capped by a 10 min treatment with Ac2O/Pyr (1:6, 7 mL).
Washing with DMF and CH2Cl2 and drying afforded resin 26, displaying a
Fmoc-loading of 0.6 mmol per g. To resin 26 (96 mg, 60 mmol) was added
DMF/morpholine (1:1, 3 mL). After 45 min the resin was washed with
DMF (8 Â 3 mL). DIPEA (61 mL, 360 mmol), pyridine (100 mL) and HATU
(64 mg, 170 mmol) were added subsequently to a solution of the N9-
carboxymethylene-N2-(benzyloxycarbonyl)-guanine (61.8 mg, 180 mmol)
Loading: Resin 21 was suspended in a solution of 1n NaOH (3 mL) in 1,4-
dioxane (1:3). After 15 min the resin was washed with 1,4-dioxane (4 Â
3 mL). This procedure was repeated once. The resin was washed with 1,4-
3922
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0718-3922 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
in DMF (1.3 mL). After 5 min of preactivation this solution was added to
the resin and shaken for 16 h. Washing with DMF and dry CH2Cl2 and
drying over P4O10 yielded resin 27 (88 mg). To 19.2 mg of resin 27 was
added a degassed solution of [Pd(PPh3)4] in morpholine/DMF/CH2Cl2
(1:5:3, 10 mL). After 16 h the resin was washed repeatedly with DMF.
The filtrate was evaporated in vacuo and the residue repeatedly coevapo-
rated with DMF until dryness. LH20 chromatography was followed by
gradient silica gel chromatography (CHCl3/MeOH/AcOH 85:15:1 !
CHCl3/MeOH/AcOH/H2O 70:30:3:10). Suspended silica gel was removed
by repeated filtration of a solution of the residue in CH3CN/EtOH (1:1).
Subsequent evaporation of the filtrate yielded building block 28 (4.3 mg,
61%) which proved identical to an authentic reference (purchased from
Applied Biosystems).
TFA/Me2S/m-cresol-mixture (2:10:1:1, 1 mL). After 3 h the solution was
concentrated in vacuo before addition of cold diethyl ether (10 mL). The
precipitate was purified by HPLC. Lyophilisation yielded a fluffy powder
(0.2 mg, 66%). tR ˆ 18.5 min (0 ± 30 min: 0 ± 25% B in A, 30 ± 40 min: 25 ±
80% B in A, 1 mLmin 1, 508C); MS (MALDI-TOF, DHB, pos): m/z: 2337
‡
[M(average)‡H] , calcd for C96H125N47O23S: 2338.
Ac-G-C-C-A(CH ) NH-Dabs-C-G-G-NHCH2CH2NH-DANS (37): A 0.1m NaH-
CO3 solution (40 mL) and a 30 mm dabsyl chloride solution (10 mL) in DMF
were added to oligomer 36 (0.2 mg, ꢀ0.08 mmol). After 16 h the mixture
was passed through an water-equilibrated RP18-SepPak cartridge and
washed with water. Elution with CH3CN/H2O (6:4) and drying in vacuo
yielded a red solid (0.1 mg, ꢀ50%). tR ˆ 20 min (0 ± 30 min: 0 ± 25% B in
A, 30 ± 40 min: 25 ± 80% B in A, 1 mLmin 1, 508C); UV/Vis: lmax ˆ 269,
462 nm; C110H138N50O25S2 (2624.75).
2
6
Ac-GZ-CZ-CZ-AZ-CZ-GZ-GZ-OH (29a): Starting from resin 27 (22 mg) the
linear chain assembly was performed as described above. The crude
material obtained after the Pd0-catalysed cleavage was purified by gradient
silica gel chromatography (CHCl3/MeOH/AcOH 80:20:2 ! CHCl3/
MeOH/AcOH/H2O 4:2:1:0.5) yielding to fraction A (35 mg) and fraction B
(26 mg), which both contained product 29a. Preparative HPLC of
fraction A (4.5 mg) yielded Ac-GZ-CZ-CZ-AZ-CZ-GZ-Aeg(Ac)-OH 29b
(2.0 mg, 39%) and product 29a (0.4 mg, 7%). Preparative HPLC
fraction B (4.1 mg) yielded additional product 29a (2.0 mg, 29%, overall:
36%). tR ˆ 31 min (0 ± 40 min: 10 ± 80% B in A, 1 mLmin 1, 508C); MS
Ac-TTATTATTATTA(CH ) NH TTAT-Gly-OH (42): The Boc-Gly-HY-
CRON conjugate 6c was coupled as described above to Tentagel
(500 mg, 0.29 mmol per g) to yield resin 38 (535 mg). CH2Cl2/TFA (1:1,
2 mL) was added to the resulting resin (135 mg). After 50 min the resin was
washed with CH2Cl2 and with DMF. This resin was treated with a solution
of Fmoc-T (56 mg, 0.11 mmol) in DMF (1 mL), to which HOBt (19 mg,
0.11 mol), NMM (24 mL, 0.22 mmol) and HBTU (19 mg, 0.11 mmol) were
added. After 3 h the resin was washed with DMF (5 Â ) and was subjected
to capping as described. Final washing with CH2Cl2 and subsequent drying
2
4
2
‡
(MALDI-TOF, DHB, pos): m/z: 2902 [M(average)‡H] , calcd for
1
in vacuo furnished resin 39 (148 mg), which contained 0.19 mmol Fmocg
C
132H137N43O36: 2903.
resin. Resin 39 (33 mg, 5 mmol) was used for the synthesis of 41.
Ac-GZ-CZ-CZ-A(CH ) NH-Fmoc-CZ-GZ-GZ-OH (32): Starting from resin 26
(20 mg) the guanosine base was coupled as decribed for 27. The linear
chain assembly was performed until incorporation of 3. The resin was dried
yielding 30 (41 mg). To resin 30 (16.4 mg) was added DMF/morpholine
(1:1, 2 mL). After 60 min the resin was washed with DMF. The adenine
modification 11 (9.0 mg, 19.2 mmol) was preactivated for 5 min by dissolv-
ing in DMF (200 mL) and adding NMM (4.4 mL) and HATU (6.1 mg,
16.0 mmol) before the resulting mixture was added to the resin. After 13 h
the resin was washed with DMF and CH2Cl2 and dried. The Fmoc group
was removed from 0.3 mg of the 16.0 mg of resin 31 obtained and the
deprotection quantified. The Fmoc loading was 0.4 mmol per g. The chain
assembly was completed and the Pd0-catalysed cleavage was performed as
described. C18-SepPak-filtration and preparative HPLC was followed by
drying in vacuo to afford a yellowish powder (7.3 mg, 50%). tR ˆ 15.5 min
(0 ± 30 min: 50 ± 90% B in A, 30 ± 35 min: 90%B in A, 1 mLmin 1, 508C);
2
6
Fmoc removal: DMF/piperidine (4:1), 1 Â 1 ± 2 min, 1 Â 8 min, 7 washings
with DMF.
Couplings: The resin was suspended in a solution of 20 mmol Fmoc-
protected building block in 0.266m NMM in DMF (2% pyridine, 150 mL)
which was preactivated by addition of HATU (7.6 mg, 20 mmol). After 1.5 h
the resin was washed 5 Â with DMF.
Capping: Ac2O/pyr (1:50, 1.5 mL), 10 min, 7 washings with DMF. The final
Fmoc cleavage was monitored to reveal a loading with 2.9 mmol Fmoc. The
N-terminus was acetylated by a subsequent capping step.
Removal of Bhoc- and Boc-protecting groups: The dried resin (46 mg) was
suspended in TFA/ethanedithiol/H2O (95:2.5:2.5, 1.5 mL) and shaken for
15 min. After filtration the resin was washed with CH2Cl2 (3 Â ), the TFA
mixture (1.5 mL) was added again and the resin was shaken for further
40 min. The procedure was repeated for another 5 min until the CH2Cl2
filtrates proved colorless. The resin was washed with CH2Cl2 (5 Â ),
pyridine (5 Â ) and DMF (5 Â ).
‡
MS (MALDI-TOF, DHB, pos): m/z: 3091 [M(average)‡H] , calcd for
C
145H154N44O36: 3090.
Boc-Gly-GZ-GZ-CZ-NHCH2CH2NH-FITC (34a): A 50 mm HOBt solution
(100 mL, 5 mmol) in DMF and a 50 mm EDC solution (50 mL, 2.5 mmol) in
CH2Cl2 was added to a solution of protected PNA 25 (1.9 mg, 1.35 mmol) in
DMF (500 mL). After addition of the amine 33a (1.3 mg, 2.84 mmol) the
solution was stirred for 40 h. Sephadex-LH20 chromatography (CHCl3/
MeOH 1:1) furnished a bright yellow solid (1.6 mg, 64%) after drying in
Cleavage: The Pd0-catalysed cleavage was performed as described for 23.
After 16 h the resin was filtered and washed excessively with DMF. The
combined filtrates were concentrated to dryness in vacuo. Traces of DMSO
were removed by repeated coevaporation of DMF.
Purification: A solution (7 mL) of 0.1% TFA in H2O was added to the
residue. The precipitate was removed by filtration. The volatiles were
removed from the filtrate in vacuo and to the residue was added 5%
MeOH in H2O (2 mL). After removal of the precipitate by filtration, the
filtrate was passed through a H2O equilibrated Kromafix C18 cartridge.
Elution with H2O (5 mL) and subsequent evaporation yielded the residue
(10.8 mg). RP-HPLC purification (Nucleosil 5-100, C18 HD, 250 Â 10 mm,
3.5 mLmin 1) of 7.7 mg and subsequent lyophilisation yielded a white fluffy
powder (4.5 mg, 27% based on the TFA salt of 42). tR ˆ 16 min (0 ± 40 min:
0% ! 40% B in A, 1 mLmin 1, 508C); MS (MALDI-TOF, DHB, pos):
‡
vacuo. MS (MALDI-TOF, DHB, pos): m/z: 1843.5 [M(average)‡H] ,
calcd for C88H87N23O23S: 1843.8.
H-Gly-G-G-C-NHCH2CH2NH-DANS (35): Analogously to 34a protected
PNA 25 (2.0 mg, 1.41 mmol) was subjected to the coupling with dansyl-
amine 33b. Sephadex-LH20 chromatography (CHCl3/MeOH 1:1) furnish-
ed a yellow powder (3.3 mg), which was dissolved in TFA (4 mL). After
15 min the TFA was removed by evaporation in vacuo. To the residue was
added dimethylsulfide (300 mL), m-cresol (300 mL), TFA (3 mL) and
trifluoromethanesulfonic acid (300 mL). After 2.5 h precipitation was
induced by the addition of cold diethyl ether (40 mL). The precipitate
was purified by size-exclusion chromatography (Biogel P2, 0.05m
NH4HCO3) and preparative HPLC. Lyophilisation yielded a yellow solid
(1.0 mg, 60% based on 25). tR ˆ 18 min (0 ± 40 min: 0 ± 50% B in A,
‡
m/z: 4494 [M(average)‡H] , calcd for C184H235N81O57: 4494.
Ac-Cys(EDANS)-TTATTATTATTATTA(CH ) NH-DABCYLT-Gly-OH
(44):
2
4
Starting from resin 39 (19.5 mg, 2.9 mmol) the chain assembly (building
blocks: Fmoc-T, Fmoc-ABhoc, Fmoc-Cys(Trt) and 19a) and the removal of
the Trt-, Boc- and Bhoc-groups was performed as decribed for 42. Before
attachment of the reporter groups the resin 40b was washed with pyridine
(4 Â ). To the resin was added DABCYL-SE (2.5 mg, 6.8 mmol) in a DMF/
pyr/NMM solution (7:1:1, 90 mL). After 15 min a further DABCYL-SE
(1.4 mg, 3.8 mmol) was added. The suspension was shaken for 4 h. The resin
was washed with DMF (5 Â ) and treated with DMF/piperidine (4:1). After
5 min the resin was washed with DMF (5 Â ). The resin was supended in a
solution of dithiothreitol (10 mmol) in a mixture of DMF/H2O (3:1, 250 mL)
and NMM (20 mL). After 15 min the resin was washed with degassed DMF/
H2O (3:1) and a solution of IAEDANS (4.0 mg, 9.2 mmol) in DMF/H2O
1 mLmin 1, 508C); (FAB , NBA): m/z: 1206.53 [M(monoisotop)‡Na] ,
‡
‡
calcd for C48H61N23O12S Na: 1206.45.
Ac-G-C-C-A(CH ) NH -C-G-G-NHCH2CH2NH-DANS (36): A 30 mm DMF/
HOAt solution (25 mL) and the amine 33b were added to PNA-oligomer 32
(0.4 mg, 0.13 mmol). Subsequently, a 15 mm solution (20 mL) of EDC in
CH2Cl2 was added. After 25 h of stirring HPLC purification afforded 1 mg
material, which was dissolved in DMF/morpholine (1:1, 1 mL). The
resulting solution was stirred for 50 min before DMF (3 mL) was added.
To the residue obtained after evaporation in vacuo was added a TFMSA/
2
6
2
Chem. Eur. J. 2001, 7, No. 18
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0718-3923 $ 17.50+.50/0
3923

O. Seitz and O. Köhler
FULL PAPER
(3:1, 200 mL) containing 5% NMM. After 4 h the resin was washed with
DMF/H2O (3:1, 5 Â ) and with DMF (5 Â ).
[3] E. Uhlmann, A. Peymann, Chem. Rev. 1990, 90, 543 ± 584.
[4] P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254,
1497 ± 1500.
Cleavage: see 23.
[5] P. E. Nielsen, G. Haaima, Chem. Soc. Rev. 1997, 73 ± 78.
[6] B. Hyrup, P. E. Nielsen, Bioorg. Med. Chem. 1996, 4, 5 ± 23.
[7] P. E. Nielsen, Pure Appl. Chem. 1998, 70, 105 ± 110.
[8] E. Uhlmann, A. Peyman, G. Breipohl, D. W. Will, Angew. Chem. 1998,
110, 2954 ± 2983; Angew. Chem. Int. Ed. 1998, 37, 2797 ± 2823.
[9] K. N. Ganesh, P. E. Nielsen, Curr. Org. Chem. 2000, 4, 931 ± 943.
[10] S. Tomac, M. Sarkar, T. Ratilainen, P. Wittung, P. E. Nielsen, B.
Norden, A. Graslund, J. Am. Chem. Soc. 1996, 118, 5544 ± 5552.
[11] H. Perry-OꢂKeefe, X. W. Yao, J. M. Coull, M. Fuchs, M. Egholm, Proc.
Natl. Acad. Sci. USA 1996, 93, 14670 ± 14675.
[12] H. érum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, C.
Stanley, Nucleic Acids Res. 1993, 21, 5332 ± 5336.
[13] C. Thiede, E. Bayerdorffer, R. Blasczyk, B. Wittig, A. Neubauer,
Nucleic Acids Res. 1996, 24, 983 ± 984.
[14] E. M. Kyger, M. D. Krevolin, M. J. Powell, Anal. Biochem. 1998, 260,
142 ± 148.
[15] O. Seitz, F. Bergmann, D. Heindl, Angew. Chem. 1999, 111, 2340 ±
2343; Angew. Chem. Int. Ed. 1999, 38, 2203 ± 2206.
[16] J. Weiler, H. Gausepohl, N. Hauser, O. N. Jensen, J. D. Hoheisel,
Nucleic Acids Res. 1997, 25, 2792 ± 2799.
[17] L. C. Boffa, E. M. Carpaneto, V. G. Allfrey, Proc. Natl. Acad. Sci. USA
1995, 92, 1901 ± 1905.
[18] L. Good, P. E. Nielsen, Nat. Biotechnol. 1998, 16, 355 ± 358.
[19] P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Nucleic Acids Res.
1993, 21, 197 ± 200.
[20] L. Good, P. E. Nielsen, Proc. Natl. Acad. Sci. USA 1998, 95, 2073 ±
2076.
[21] S. E. Hamilton, C. G. Simmons, I. S. Kathiriya, D. R. Corey, Chem.
Biol. 1999, 6, 343 ± 351.
[22] V. Demidov, M. D. Frank-Kamenetskii, M. Egholm, O. Buchardt, P. E.
Nielsen, Nucleic Acids Res. 1993, 21, 2103 ± 2107.
[23] A. G. Veselkov, V. V. Demidov, M. D. Frank-Kamenetskii, P. E.
Nielsen, Nature 1996, 379, 214.
[24] M. Footer, M. Egholm, S. Kron, J. M. Coull, P. Matsudaira, Biochem-
istry 1996, 35, 10673 ± 10679.
Purification: The combined DMF filtrates obtained after the cleavage were
concentrated in vacuo. Repeated coevaporation with DMF yielded a solid
residue. H2O was added and the precipitate removed by filtration. The
concentrated filtrate was applied on a C18-SepPak cartridge, which was
ªactivatedº before by passing through MeOH followed by water equili-
bration. First H2O (5 mL) was eluted, then CH3CN/H2O (4:1, 5 mL). The
red-colored eluent obtained after addition of CH3CN/H2O (3:1, 5 mL) was
collected and lyophylised. Two-fold semipreparative HPLC (Nucleosil
5-100, C18 HD, 250 Â 10 mm, 0 ± 30 min: 10% ! 40%B in A,
3.5 mLmin 1) yielded 25.2 OD260. tR ˆ 22 min (0 ± 40 min: 10% ! 40% B
in A, 1 mLmin 1, 508C); ESI-MS (nanospray, pos): m/z: 5153.0, calcd for
C216H267N87O63S2: 5154.0; UV/Vis (H2O): lmax ˆ 265, 485 nm.
Ac-Cys(EDANS)-TTATTATTATTA(CH ) NH-DABCYLTTAT-Gly-OH
(45):
2
4
Starting from resin 39 (19.5 mg, 2.9 mmol) the synthesis was performed as
described for 44. A yield of 16.3 OD260 was obtained. tR ˆ 23 min (0 ±
40 min: 10% ! 40% B in A, 1 mLmin 1, 508C); ESI-MS (nanospray,
pos): m/z: 5153.0, calcd for C216H267N87O63S2: 5154.0; UV/Vis (H2O): lmax
265, 485 nm.
ˆ
Ac-Lys(Dans)-ACCTACAGCC-Lys(DABCYL)-NH2 (48): Fmoc-Lys-
(DABCYL) (purchased from Molecular Probes, Leiden, Netherlands)
was coupled to the Tentagel-resin loaded with the Rink-linker. The
subsequent solid-phase synthesis was performed in 3 mmol scale with a
Multisyntech Syro synthesizer and with the methods as described for 42. At
the N-terminal end Fmoc-Lys(Boc)-OH was coupled, the Fmoc group was
removed and the liberated amino group subsequently acetylated. Cleavage
from the solid support was performed using TFA/ethanedithiol/thioanisol/
H2O (92.5:2.5:2.5:2.5). The purification of the PNA ± DABCYL conjugate
was performed by solid-phase extraction followed by HPLC (see above)
which furnished 10.8 OD260. To the purified PNA-DABCYL conjugate (2.1
OD260, 65 nmol) in CH3CN/H2O (4:6, 48 mL) DIPEA (4.2 mL) was added.
Within 30 min a solution of 5-dimethylaminonaphthaline-1-sulfonyl chlor-
ide in 40% CH3CN/H2O (0.16m) was added in four portions (1 mL). The
mixture was vortexed for 1 h. The purification proceeded by chromatog-
raphy on a C18-SepPak cartridge as described for 44. The fraction eluting
with CH3CN/H2O (4:6) was collected. HPLC analysis showed a single peak.
Lyophilisation yielded 1.4 OD260. tR ˆ 14 min (C18-PPN (Macherey&Na-
Â
[25] P. Wittung, J. Kajanus, K. Edwards, G. Haaima, P. Nielsen, B. Norden,
B. G. Malmström, FEBS Lett. 1995, 375, 317 ± 320.
[26] L. D. Mayfield, D. R. Corey, Bioorg. Med. Chem. Lett. 1999, 9, 1419 ±
1422.
[27] L. D. Mayfield, D. R. Corey, Anal. Biochem. 1999, 268, 401 ± 404.
[28] O. Seitz, Tetrahedron Lett. 1999, 40, 4161 ± 4164.
[29] O. Seitz, Angew. Chem. 2000, 112, 3389 ± 3392; Angew. Chem. Int. Ed.
2000, 39, 3249 ± 3252.
[30] X. H. Liu, S. Balasubramanian, Tetrahedron Lett. 2000, 41, 6153 ±
6156.
[31] K. L. Dueholm, P. E. Nielsen, New J. Chem. 1997, 21, 19 ± 31.
[32] T. Förster, Ann. Phys. 1948, 2, 55 ± 75.
[33] L. Stryer, Annu. Rev. Biochem. 1978, 47, 819 ± 846.
[34] S. Tyagi, F. R. Kramer, Nat. Biotechnol. 1996, 14, 303 ± 308.
[35] J. W. J. Li, X. H. Fang, S. M. Schuster, W. H. Tan, Angew. Chem. 2000,
112, 1091 ± 1094; Angew. Chem. Int. Ed. 2000, 39, 1049 ± 1052.
[36] B. Armitage, D. Ly, T. Koch, H. Frydenlund, H. érum, G. B. Schuster,
Biochemistry 1998, 37, 9417 ± 9425.
[37] E. Ortiz, G. Estrada, P. M. Lizardi, Mol. Cell. Probes 1998, 12, 219 ±
226.
[38] S. S. Gosh, G. F. Musso, Nucleic Acids Res. 1987, 15, 5353 ± 5372.
[39] M. N. Erout, A. Troesch, C. Pichot, P. Cros, Bioconjugate Chem. 1996,
7, 568 ± 575.
gel), 250 Â 4 mm, 0 ± 2 min: 10% B in A, 2 ± 40 min: 10% ! 50% B in A,
1
1 mLmin
,
508C); MS (MALDI-TOF, DHB, pos): m/z: 3440.5
‡
[M(average)‡H] , calcd for C146H184N66O34S: 3440.6.
Hybridisation experiments: Stock solutions of the PNA probes (50
1
1
pmolmL in CH3CN/H2O (1:1)) and the DNA-targets (400 pmolmL in
H2O) were prepared. The PNA probe solution (20 mL) was added into a
quartz cuvette and diluted with aq. buffer (100 mm NaCl, 10 mm NaH2PO4,
0.1 mm EDTA, pH 7, 980 mL). The fluorescence spectrum (excitation at
335 nm) was recorded at 298 K sample temperature. DNA target was
added (10 mL) and the fluorescence spectrum was measured after 10 min.
In an alternative experiment (uppermost curves in Figure 5) the PNA
probe solution (30 mL) was diluted with the buffer (970 mL) and fluo-
rescence spectra (excitation at 335 nm) were measured at 293 K sample
temperature before and after addition of DNA target (10 mL).
Hybridisation kinetics: To the aq. buffer (100 mm NaCl, 10 mm NaH2PO4,
0.1 mm EDTA, pH 7, 980 mL) was added an aliquot of the stock solution of
1
PNA-probe 44 (50 pmolmL in CH3CN/H2O (1:1), 20 mL). Oligonucleo-
tide 47 was added (400 pmolmL 1 in H2O, 2.5 mL) and the emission (298 K,
excitation at 335 nm) at 485 nm was monitored. After 21 min further 47
(400 pmolmL 1 in H2O, 7.5 mL) was added, after 40 min 46 (400 pmolmL 1 in
H2O, 2.5 mL) was added and after 51 min further 46 (400 pmolmL 1 in H2O,
7.5 mL).
[40] For analogous modifications of protected 3'-alkylamino-oligonucleo-
tides: D. L. McMinn, M. M. Greenberg, J. Am. Chem. Soc. 1998, 120,
3289 ± 3294.
[41] B. Armitage, D. Ly, T. Koch, H. Frydenlund, H. érum, H. G. Batz,
G. B. Schuster, Proc. Natl. Acad. Sci. USA 1997, 94, 12320 ± 12325.
[42] B. P. Gangamani, V. A. Kumar, K. N. Ganesh, Chem. Commun. 1997,
7, 1913 ± 1914.
Acknowledgement
This work was supported by the DFG and the Fonds der chemischen
Industrie. O.S. gratefully acknowledges a Liebig- and DFG fellowship.
[43] H. Challa, M. L. Styers, S. A. Woski, Org. Lett. 1999, 1, 1639 ± 1641.
[44] H. Challa, S. A. Woski, Tetrahedron Lett. 1999, 40, 8333 ± 8336.
[1] A. DeMesmaeker, R. Haner, P. Martin, H. E. Moser, Acc. Chem. Res.
1995, 28, 366 ± 374.
Â
[45] G. Aldrian-Herrada, A. Rabie, R. Wintersteiger, J. Brugidou, J. Pept.
[2] J. F. Milligan, M. D. Matteucci, J. C. Martin, J. Med. Chem. 1993, 36,
Sci. 1998, 4, 266 ± 281.
1923 ± 1937.
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0947-6539/01/0718-3924 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 18

Peptide Nucleic Acids
3911±3925
[46] L. S. Richter, R. N. Zuckermann, Bioorg. Med. Chem. Lett. 1995, 5,
1159 ± 1162.
[47] D. Muller, I. Zeltser, G. Bitan, C. Gilon, J. Org. Chem. 1997, 62, 411 ±
416.
[56] M. Rasmussen, J. M. Hope, Aust. J. Chem. 1982, 35, 525 ± 534.
[57] C. Dalby, C. Bleasdale, W. Clegg, M. R. J. Elsegood, B. T. Golding,
R. J. Griffin, Angew. Chem. 1993, 105, 1822 ± 1823; Angew. Chem. Int.
Ed. Engl. 1993, 32, 1696 ± 1697.
 Â
[48] K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen,
T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J.
Org. Chem. 1994, 59, 5767 ± 5773.
[49] T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson,
H. érum, Int. J. Pept. Protein Res. 1997, 49, 80 ± 88.
[50] J. M. Coull, M. Egholm, R. P. Hodge, M. Ismail, S. B. Rajur
(Perseptive Biosystems, Inc.), WO 9640685.
[51] M. Planas, E. Bardaji, K. J. Jensen, G. Barany, J. Org. Chem. 1999, 64,
7281 ± 7289.
[52] O. Seitz, H. Kunz, Angew. Chem. 1995, 107, 901 ± 904; Angew. Chem.
Int. Ed. Engl. 1995, 34, 803 ± 805.
[58] J. Thibon, L. Latxague, G. Deleris, J. Org. Chem. 1997, 62, 4635 ± 4642.
[59] S. A. Thomson, J. A. Josey, R. Cadilla, M. D. Gaul, C. F. Hassman,
M. J. Luzzio, A. J. Pipe, K. L. Reed, D. J. Ricca, R. W. Wiethe, S. A.
Noble, Tetrahedron 1995, 51, 6179 ± 6194.
[60] M. Brenner, H. R. Müller, W. Pfister, Helv. Chim. Acta 1950, 33, 568.
[61] J. C. Sheehan, G. P. Hess, J. Am. Chem. Soc. 1955, 77, 1067.
[62] W. König, R. Geiger, Chem. Ber. 1970, 103, 788.
[63] O. Seitz, unpublished results.
[64] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397 ± 4398.
[65] J. C. Sheehan, P. A. Cruickshank, G. L. Boshart, J. Org. Chem. 1961,
26, 2525.
[53] O. Seitz, H. Kunz, J. Org. Chem. 1997, 62, 813 ± 826.
[54] J. P. Tam, S. B. H. Kent, T. W. Wong, R. B. Merrifield, Synthesis 1979,
955.
[66] K. Brinkmann, H. Linnertz, E. Amler, E. Lanz, P. Herman, W.
Schoner, Eur. J. Biochem. 1997, 249, 301 ± 308.
[67] C. Wojczewski, K. Stolze, J. W. Engels, Synlett 1999, 10, 1667 ± 1678.
[55] C. R. Allerson, S. L. Chen, G. L. Verdine, J. Am. Chem. Soc. 1997, 119,
7423 ± 7433.
Received: December 14, 2000 [F2939]
Chem. Eur. J. 2001, 7, No. 18
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0947-6539/01/0718-3925 $ 17.50+.50/0
3925