High affinity, sequence specific DNA binding by synthetic tripyrrole-peptide conjugates

Article abstract of DOI:10.1002/chem.200500010

Linking the basic region of a bZIP transcription factor to a distamycin-like tripyrrole peptide by means of a nitrogen-containing tether produces a hybrid capable of high-affinity recognition of specific, designated DNA sequences. The importance of the nitrogen in the tether is shown by the considerable reduction in affinity (more than 10-fold) caused by its replacement with an ether linkage. Attachment of an aminopropyl chain on the pyrrole adjacent to the pyrrole bearing the ni trogen-containing tether increases affinity approximately one order of magnitude. These results confirm that a suitable location of protonated amine groups on designed DNA-binding peptides provides for higher affinities, most probably because of the generation of salt bridged contacts with the phosphodiester backbone.

Full text of DOI:10.1002/chem.200500010

FULL PAPER
High Affinity, Sequence Specific DNA Binding
by Synthetic Tripyrrole–Peptide Conjugates
Juan B. Blanco,[a] Olalla Vꢀzquez,[a] Josꢁ Martꢂnez-Costas,[b] Luis Castedo,[a] and
Josꢁ L. MascareÇas*[a]
Abstract: Linking the basic region of a
bZIP transcription factor to a distamy-
cin-like tripyrrole peptide by means of
a nitrogen-containing tether produces a
hybrid capable of high-affinity recogni-
tion of specific, designated DNA se-
quences. The importance of the nitro-
gen in the tether is shown by the con-
siderable reduction in affinity (more
than 10-fold) caused by its replacement
with an ether linkage. Attachment of
an aminopropyl chain on the pyrrole
adjacent to the pyrrole bearing the ni-
trogen-containing tether increases af-
finity approximately one order of mag-
nitude. These results confirm that a
suitable location of protonated amine
groups on designed DNA-binding pep-
tides provides for higher affinities,
most probably because of the genera-
tion of salt bridged contacts with the
phosphodiester backbone.
Keywords:
distamycin
conjugation
DNA recognition
·
·
·
peptides · pyrroles
Introduction
One of the simplest structural families of transcription
factor proteins is the bZIP family, which bind dsDNA as
leucine zipper-mediated homo- or heterodimers, and insert
two N-terminal peptidic chains called basic regions (BR)
into adjacent DNA major grooves.[3] Interestingly, the basic
regions are largely unstructured in the absence of DNA, but
they fold into an a-helix upon specific DNA binding.[4] Al-
though the leucine zipper region does not contact the DNA
directly, DNA binding is strongly dependent on such dimeri-
zation, as prevention of this process through specific muta-
tions precludes DNA recognition.[5] It has been shown that
the leucine zipper unit can be replaced by other noncovalent
or by covalent artificial dimerizing units without significant-
ly compromising the recognition capabilities of the system.[6]
However, monovalent bZIP BRs exhibit very poor DNA-
binding affinities unless the important DNA-contacted resi-
dues are appropriately grafted into a preorganized a-helix.[7]
We have recently demonstrated that appropriate tethering
of a monomeric bZIP BR domain to a distamycin-like tri-
pyrrole that binds with moderate-to-good affinity in the
minor groove adjacent to the BR target site, provides for
specific binding of the peptide to its cognate major groove
site, binding that most probably occurs according to the hy-
pothetical model depicted in the Figure 1.[8] Most important,
the resulting tripyrrole–peptide conjugate exhibits higher af-
finity for its target hybrid site than either the BR monomer
or the tripyrrole. In particular we have found that the deriv-
ative 1, which has a peptidic region of only 22 natural amino
acids, is capable of binding relatively long specific DNA
The proper functioning of cells involves accurate control of
DNA transcription by the concerted and regulated action of
transcription factors. These proteins interact, with high affin-
ity and specificity, with short DNA sequences that are usual-
ly located upstream of the promoter of the gene.[1] The
design of molecules that are smaller than natural transcrip-
tion factors but have similar DNA-binding properties is an
important goal of current research at the chemistry/biology
interface, not only because of their potential utility for eluci-
dation of the molecular basis of DNA recognition by natural
proteins, but also as potential chemotherapeutic agents in
the postgenomic age.[2]
[a] J. B. Blanco, O. Vꢀzquez, Prof. Dr. L. Castedo,
Prof. Dr. J. L. MascareÇas
Departamento de Quꢁmica Orgꢀnica
y Unidad Asociada al CSIC, Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
Fax : (+34)981-595-012
E-mail: qojoselm@usc.es
[b] Dr. J. Martꢁnez-Costas
Departamento de Bioquꢁmica y Biologꢁa Molecular
Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author: Synthesis of 3, 6,
10, 12, and 15 and characterization data, as well as gel shift titration
data for the binding of 11 to T/CREhs at 48C.
Chem. Eur. J. 2005, 11, 4171 – 4178
DOI: 10.1002/chem.200500010
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4171

sites with low nanomolar affinity (Kd ~3 nm) at 48C. It
should be noted, however, that part of this affinity is lost
when the interaction assay is carried out at 238C, a fact that
somewhat restricts the potential utility of the system as a se-
quence-specific DNA probe.
result suggests that such amino group, which should be
mostly protonated at neutral pH, is critical for a successful
DNA binding by this type of conjugates, most probably be-
cause it is engaged in a salt-bridged type of contact with the
phosphodiester oxygens.
This finding supported the
hypothesis that the presence of
amino groups in appropriate
positions of
a DNA-binding
ligand strengthens the interac-
tion, and suggested that the
DNA affinity of tripyrrole–pep-
tide hybrids such as 1 might be
further increased by facilitating
more interactions of this kind.
Indeed in this work we also
show that attaching a propyla-
mine chain to the nitrogen
Figure 1. Structure of the peptide–tripyrrole hybrids, and qualitative working model for the hypothetical inter- atom of the middle pyrrole in
action of 1 with the specific DNA target, showing a possible position for the secondary NH group of the
tether (Caution: the drawing doesnꢃt necessarily reflect the real structure of the complex).
hybrid 1 leads to a conjugate 11
capable of binding the designat-
ed composite DNA sequence
with a Kd in the low nanomolar
Significantly, we have found that the linking tether is not
a mere connector that keeps the DNA-binding modules at
an appropriate distance, but apparently it plays a more
active role in the recognition process. Hence, replacement
of the pentylpropylamine fragment of tether with a propyl-
triglycine unit markedly reduces affinity for DNA, even
though both tethers span very similar distances.[8b] This
result raised the question of whether such a decrease in af-
finity is due to geometric restrictions imposed by the rigidity
of the peptidyl tether or to the absence of the secondary ni-
trogen atom present in the linker of 1, or perhaps to both
factors. Herein we demonstrate that replacing the secondary
amino group present in the linker of hybrid 1 by an oxygen
atom results in a considerably drop in DNA affinity. This
range even at room temperature (238C).
Results and Discussion
Synthesis of hybrid 2, which contains an ether type of link-
age: In order to dissect the relevance of the secondary
amine group of the tether in the hybrid 1 we prepared the
analogue 2 which bears an oxygen atom in place of the NH
group. The synthesis of 2 required assembly of aminotripyr-
role 8, which was accomplished both by linking monopyrrole
4 first to the tether and then to the N-aminodipyrrole 7[9]
(route A in Scheme 1), and also by installing the tether di-
rectly on the N-terminal ring of tripyrrole 9 (route B).[8b]
Although route A is slightly more convergent, route B is
shorter, and is more versatile in that it allows the tripyrrole
to be endowed with a variety of different tethers by just
changing the alkylating species in the final step. With inter-
mediate 8 in hand, we assembled compound 2 as shown in
Scheme 2, by coupling 8 with a peptide consisting essentially
of amino acids 226–248 of the GCN4 BR (R245 of this BR
was replaced by glutamic acid for attachment of the
tether).[10] The coupling reaction was carried out while the
peptide was still attached to the solid support and fully pro-
tected except at the key glutamic acid; following isolation of
2, the overall yield of the process of peptide assembly and
coupling to 8 was approximately 36%.
Abstract in Spanish: Conectando la region bꢀsica de un
factor de transcripciꢁn de tipo bZIP con un tripirrol similar
a la Distamicina, a travꢂs de una cadena que contiene un
grupo amino, se obtiene un hꢃbrido capaz de reconocer se-
cuencias especꢃficas de ADN con gran afinidad. Si se reem-
plaza el grupo amino de la cadena por un ꢂter se observa
una importante reducciꢁn en la afinidad (mꢀs de 10 veces),
lo que demuestra la importancia del grupo NH presente en
dicha cadena. Por otra parte, la introducciꢁn de un grupo
aminopropilo en el pirrol contiguo al que porta la cadena de
conexiꢁn al pꢂptido conlleva un aumento de afinidad de
aproximadamente un orden de magnitud. Estos resultados
confirman que una localizaciꢁn adecuada de grupos amino
protonados en pꢂptidos diseÇados para interaccionar con
ADN proporciona mejores afinidades, seguramente debido a
la generaciꢁn de puentes salinos con los fosfodiꢂsteres del
ADN.
DNA binding properties of 2 in comparison with 1: Since
specific binding of bZIP BRs to DNA is concomitant with
the formation of an a-helix, circular dichroism (CD) is a
particularly useful technique to detect such interactions, pro-
vided that the basic region is not already highly helical in
the absence of DNA. Clearly, evidence for an increase in
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Tripyrrole–Peptide Conjugates
FULL PAPER
This band is accompanied by increased positive ellipticity at
330 nm due to the tripyrrole moiety binding in the minor
groove (Figure 2a). Addition of compound 2 to the same
oligonucleotide caused parallel spectral changes to those
induced by 1, and the specificity of the interaction was
supported by the failure to induce spectral changes at
222 nm when the oligonucleotide used was T/CREhsm,
which has a single mutation in the peptide-binding site, or
CREhs, which lacks the A-rich sequence responsible for the
minor-groove binding of the tripyrrole moiety. The intensity
of the band at 222 nm in the complex 2-T/CREhs is slightly
lower than that observed in the complex with 1 but still sig-
nificant, suggesting that the basic region of 2 also folds into
an a-helix upon binding to the cognate composite DNA se-
quence.
Although this result could suggest a relatively high affini-
ty binding, electrophoretic mobility shift titrations in poly-
acrylamide gels by using 32P-end-labelled T/CREhs showed
that compound 2 was only weakly bound: even at a concen-
tration of 200 nm it failed to saturate T/CREhs (Figure 2c,
lanes 1–8), whereas near-saturation was achieved by just a
20 nm concentration of compound 1 (Figure 2b). Using as
DNA probe 32P-T/CREhsm (Figure 2c, lanes 9–14) we ob-
serve the formation of a slightly slower migrating band
which probably arises from a complex in which the peptide
moiety is not folded inside the groove but electrostatically
bound to the phosphate surface.
Scheme 1. Synthesis of the tripyrrole unit 8: A) a) 1) NaH, THF, 238C;
2) acrylonitrile; b) Et3N, Boc2O, THF, H2, Pd/C; c) Ph3P, I2, imidazole;
d) 4, K2CO3, acetone, reflux; e) TFA, CH2Cl2; f) Boc-Gly-OSu, Et3N,
CH2Cl2; g) NaOH, EtOH/H2O; h) 7, DECP, Et3N, DMAP, THF; i) 1) H2,
Pd/C; 2) AcCl, Et3N; j) TFA, CH2Cl2; B) a) DECP, Et3N, DMAP, THF, 7;
b) 1) H2, Pd/C; 2) AcCl, Et3N; c) 1) 3, K2CO3, acetone, reflux; 2) TFA,
CH2Cl2; d) 1) Boc-Gly-OSu, DIEA, DMF; 2) TFA, CH2Cl2.
That 1, in which the tether contains an NH group which is
most probably protonated at the working pH, has a greater
affinity for T/CREhs than 2, in
which the NH is replaced by an
ether linkage, seems likely to
be due to the NH group of 1
forming a salt bridge with phos-
phodiester oxygens in the sec-
tion of oligonucleotide back-
bone straddled by the peptide–
tether–tripyrrole conjugate (see
Figure 1). This conclusion is
consistent with the notion that
hydrogen bonds between DNA-
binding proteins and DNA
backbone phosphates, which ac-
Scheme 2. Coupling step for the synthesis of 2.
count for about half of the
direct hydrogen bonds observed
in protein–DNA complexes,
helicity by CD does not necessarily correlate with high affin-
ity binding, as the concentrations required for the CD ex-
periments are in the micromolar range; however, tight, spe-
cific binding does require the induction of an a-helical struc-
ture, a characteristic that can be readily detected by obser-
vation of the change in the negative ellipticity at 222 nm in
the CD spectrum.[11] As previously reported, addition of a
20 base-pair duplex oligonucleotide (T/CREhs) containing
the designated target hybrid sequence (P5’-GTCATAAAAT-
3’) to 1 at 48C, produces a significant variation of the CD
signal at 222 nm, which is consistent with specific binding.
contribute in a great measure to their association con-
stants.[12] In the case of our hybrid systems this type of inter-
action could even be more relevant for the stability of the
complex owing to the special location of the amine group in
the tether connecting the major and minor groove binding
counterparts.
Design, synthesis and DNA-binding properties of a peptide–
tripyrrole derivative containing a propylamino chain in the
middle pyrrole: The affinity of hybrid 1 for its specific
target dsDNA decreases over 10-fold when the interaction
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J. L. MascareÇas et al.
of compound 11, in which an
aminopropyl chain has been in-
stalled on the middle pyrrole
of 1.
Compound 11 was synthe-
sized by linking the aminopro-
pylated monopyrrole 12 to the
terminal pyrrole 13 on one side
and to the tether-bearing pyr-
role 15 on the other, and then
coupling the resulting tripyrrole
peptide to the same GCN4 BR
segment as in 2 (Scheme 3). An
important aspect of the strategy
was the use of “orthogonal”
protecting groups for distinct
terminal amines present in each
of the N-pyrrole chains, so that
it is possible to selectively
unmask the amino group pres-
ent in the tether of the N-termi-
nal pyrrole. After TFA treat-
ment to remove protecting
groups and cleave the BR
moiety from the resin, com-
pound 11 was obtained in ap-
proximately 17% overall yield.
As shown in Figure 4a, the
CD difference spectrum of the
peptide conjugate 11 in the
Figure 2. DNA-binding properties of 2. a) CD difference spectra of hybrid 1 in the presence of T/CREhs
and of 2 in the presence or absence of ds-oligonucleotides: in the absence of DNA (&), in the presence T/
(
),
*
hs
hs
CREhs ( ), in the presence CRE
( ), in the presence of T/CRE m ( ); CD spectra were obtained at 48C as
~
~
&
described in the Experimental Section, and were slightly smoothed to facilitate viewing. The difference spectra
are the spectra of ligand + DNA mixture minus that of the DNA. b) Autoradiogram showing the binding of
hybrid 1 to 32P-labelled T/CREhs, lanes 1–10: 0, 1, 2, 5, 10, 20, 40, 60, 80, 100 nm. c) Autoradiograms showing
the binding of hybrid 2 to 32P-labelled DNAs, lanes 1–8, T/CREhs: 0, 20, 40, 60, 80, 100, 150, 200 nm; lanes 9–
14: T/CREhsm: 200, 150, 100, 80, 40, 0 nm. d) Sequences of duplex oligonucleotides used. The BR subsite
(CREhs) is underlined and the tripyrrole binding site (T) is in italics.
assay is carried out at room
temperature instead of at 48C.
This is a major hurdle if one
wants to develop this DNA rec-
ognition approach, based on si-
multaneous minor and major
groove binding, into a viable
strategy to compete with the
DNA-binding ability of natural-
ly occurring transcription fac-
tors. Therefore it would be very
convenient and highly desirable
Figure 3.
to obtain derivatives that bind
with low nanomolar dissocia-
tion constants at higher temper-
atures. The results described above, which support the hy-
pothesis that the good affinity of 1 was in part due to its
tether containing an amino group that interacted with the
oligonucleotide backbone, suggested that affinity might be
further increased by facilitating more ligand–backbone inter-
actions of this kind. In view of the literature on related oli-
gopyrroles,[13] and after examining a simple qualitative work-
ing model of the conjugate-oligonucleotide complex
(Figure 3), we decided to investigate the binding properties
presence of the target DNA (T/CREhs) at 208C revealed a
slightly higher helicity than that observed for its relative 1
(Figure 4a).[14] A similar pattern of relative intensities is ex-
hibited by the band around 330 nm that reflects binding by
the tripyrrole moiety. As could be expected, the CD spectra
acquired in the presence of a duplex oligomer having a mu-
tated peptide-binding half site (T/CREhsm) or lacking an AT
rich sequence (CREhs) show a significantly weaker a-helical
folding transition.
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Tripyrrole–Peptide Conjugates
FULL PAPER
peptides are able to address se-
quences containing relatively
high number of bases, so they
could target particular sites in
the genome with higher specif-
icity. These molecules, in addi-
tion to providing basic informa-
tion about the factors that
govern sequence-specific pro-
tein–DNA interactions, might
find important biomedical ap-
plications as designed genome
interference agents.
Building on previous work in
which monomeric bZIP BRs
had been found capable of low-
temperature specific DNA rec-
ognition when linked by
a
tether to tripyrroles capable of
minor-groove binding to flank-
ing sequences, in this work we
investigated the contributions
to binding affinity made by the
tether and by an appropriate
tripyrrole side chain. High-af-
finity binding was found to re-
quire the presence in the tether
of a suitably placed secondary
Scheme 3. Synthesis of the peptide–tripyrrole hybrid 11. a) 1) H2, Pd/C; 2) DECP, Et3N, DMAP, THF, 12;
b) 1) H2, Pd/C; 2) DECP, Et3N, DMAP, THF, 15; c) 1) H2, Pd/C; 2) AcCl, DIEA; d) piperidine/DMF;
e) 1) HATU, DIEA, DMF, 10; 2) TFA, scavengers.
Gratifyingly, mobility shift titrations using 32P-end-label-
led T/CREhs showed that at 238C half-saturation with 11 oc-
curred at a concentration of only about 5 nm, as against
more than 50 nm for compound 1 at this temperature (Fig-
ure 4b and c).[15] Like compound 2, compound 11 also
formed a less mobile complex with T/CREhsm, which we
again attribute to non-specific binding without insertion of
the BR moiety into the major groove; in this case half-satu-
ration was achieved at a concentration of about 40 nm, near
one order of magnitude greater than for the oligonucleotide
with the full BR target sequence (Figure 4c, lanes 8–14).
The affinity of 11 for CREhs, which lacks the tripyrrole-bind-
ing AAAAT motif, was negligible (Figure 4d). These elec-
trophoresis results combined with the CD spectra confirm
that the new hybrid peptide–tripyrrole derivative 11 is able
to bind at room temperature (~238C) to designated dsDNA
sequences with low nanomolar dissociation constants, being
enormously selective with respect to sequences that lack an
AT rich region and almost 10-fold more selective with
regard to mismatch DNA sites containing this type of re-
gions.
amino group, probably because it forms a salt bridge with
phosphates in the segment of DNA backbone straddled by
the tether. This conclusion led us to design a new peptide–
tripyrrole hybrid deliberately equipped with an aminopropyl
residue in one of the pyrroles, anticipating that it might ex-
hibit higher DNA affinities owing to the generation of simi-
lar kind of electrostatic contacts. Addition of the tripyrrole
moiety to an aminopropyl side chain capable of similar in-
teractions resulted in compound 11 which achieves low
nanomolar affinity for the composite consensus half site T/
CREhs even at room temperature (approximately 10-fold
improvement with respect to the analogue lacking this side
chain). As far as we know, compound 11 is the smallest arti-
ficially designed transcription-factor-based peptide deriva-
tive to have proved capable of recognizing such long DNA
sequences (9 or 10 bp) with such high affinity. Work to fur-
ther improve the design process so that in addition to high
affinity we can also attain even better specificities, as well as
to obtain more structural and thermodynamic data to fur-
ther characterize these DNA–peptide complexes, is under
way.
Conclusion
Experimental Section
There is a great deal of interest in obtaining artificial
mimics of transcription factors that being smaller in size are
yet capable of reproducing their DNA-binding specificity
and affinity. It is desirable that these artificial DNA-binding
General procedures and protocols: All dry solvents were freshly distilled
under argon from the appropriate drying agent before use. THF and
Et2O were distilled from sodium/benzophenone. CH2Cl2 was distilled
from CaH2. All reactions were conducted in dry solvents under argon at-
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J. L. MascareÇas et al.
standard protected, except the Fmoc-Glu(OAll)-OH introduced at posi-
tion 245.
CD measurements were made in a 2 mm cell. Samples contained 10 mm
phosphate buffer (pH 7.5), 100 mm NaCl, 5 mm peptide and 5 mm ds-oligo
when present. The peptide–DNA mixtures were incubated for 15 min
before registering. The spectra are the average of 5 scans and were slight-
ly smoothed using the “smooth” macro implemented in the program Ka-
leidagraph (v 3.5 by Synergy Software). Spectra of the peptides in the
presence of DNA were calculated as the difference between the spectra
of the peptide–DNA mixture and the spectrum of free DNA.
For gel mobility shift assays, binding reactions were performed over
30 min by using ~45 pM labeled dsDNAs (unless otherwise stated) in a
binding mixture (20 mL) containing 18 mm Tris (pH 7.5), 90 mm KCl,
1.8 mm MgCl2, 1.8 mm EDTA, 9% glycerol, 0.3 mgmLꢀ1 BSA and 2.2%
NP-40. Products were resolved by PAGE by using a 10% nondenaturing
acrylamide gel and 0.5X TBE buffer, and analyzed by autoradiography
(TBE 0.5X buffer is 44 mm boric acid, 44 mm Tris base and 0.1 mm
EDTA pH 8).
Synthesis of the tripyrrole derivative 8
{3-[5-(4-Acetylamino-2-{5-[5-(3-dimethylaminopropyl]carbamoyl)-1-
methyl-1H-pyrrol-3-yl-carbamoyl]-1-methyl-1H-pyrrol-3-yl-carbamoyl}-
pyrrol-1-yl)-pentyloxy]-propyl}-carbamic acid tert-butyl ester: K2CO3
(330 mg) and iodide 3 (299 mg, 0.9 mmol) were added to a solution of tri-
pyrrole 9 (100 mg, 0.2 mmol) in dry acetone (6 mL). The reaction mixture
was refluxed for 8 h and the resulting suspension was filtered through
Celite. The filtrate was concentrated and the residue purified by flash
chromatography (neutral aluminium oxide, 5% MeOH/CH2Cl2) to afford
the expected product as a pale-yellow solid. 1H NMR (CD3OD): d =
1.25–1.45 (brs, 13H), 1.52–1.58 (m, 2H), 1.62–1.70 (m, 4H), 2.00–2.05 (m,
5H), 3.04 (brs, 10H), 3.22–3.38 (m, 6H), 3.83–3.86 (2 s, 6H), 6.92–6.98
(m, 3H), 7.19–7.22 (m, 3H); 13C NMR (CD3OD): d = 23.1 (CH3), 23.4
(CH2), 24.2 (CH2), 24.4 (CH2), 28.8 (CH3), 30.1 (CH2), 31.1 (CH2), 36.9
(CH3), 38.9 (CH2), 49.9 (CH3), 51.5 (CH3), 63.1 (CH2), 65.3 (CH2), 69.4
(CH2), 71.4 (CH2), 79.8 (C), 104.0 (CH), 106.6 (CH), 114.4 (CH), 120.8
(CH), 123.2 (C), 123.4 (C), 124.0 (C), 124.5 (C), 124.6 (C), 125.3 (C),
158.4 (C), 160.5(C), 161.2 (C), 164.3 (C), 170.3 (C); MS (FAB+): m/z:
740 (36) [M+H]+, 618 (14); HRMS: m/z: calcd for C37H58N9O7: 740.4459,
found 740.4459.
Figure 4. DNA-binding properties of 11. a) CD difference spectra of
hybrid 1 in the presence of T/CREhs
(
) and of peptide 11 in the pres-
*
({3-[5-(4-Acetylamino-2-{5-[5-{3-(dimethylaminopropylcarbamoyl)-1-
methyl-1H-pyrrol-3-yl-carbamoyl]-1-methyl-1H-pyrrol-3-yl-carbamoyl}-
pyrrol-1-yl)-pentyloxy]-propylcarbamoyl}-methyl)-carbamic acid (8): The
above-mentioned tripyrrole derivative (150 mg, 0.2 mmol) was dissolved
in CH2Cl2 (3 mL) and cooled to 08C. TFA (3 mL) was added dropwise
and the resulting orange solution was stirred at 08C for 1 h and at room
temperature for another 2 h. The solvents were removed under reduced
pressure and the residual TFA was removed by codistillation with
CH2Cl2. The resulting residue was dissolved in DMF (5 mL) and to this
solution was added DIEA (0.2 mL, 0.98 mmol) and Boc-Gly-OSu (54 mg,
0.20 mmol). The resulting solution was stirred for 1 h at room tempera-
ture, the solvent evaporated and the residue purified by flash chromatog-
raphy (neutral aluminium oxide, 5% MeOH/CH2Cl2) and submitted to
the standard protocol for removal of the Boc protecting group (TFA/
CH2Cl2). The residue resulting from removal of the solvents consisted of
the desired tripyrrole 8 (43 mg, 31%). 1H NMR (CD3OD): d = 1.15 (m,
4H), 1.39 (m, 2H), 1.57 (m, 4H), 1.83 (m, 2H), 1.93 (s, 3H), 2.74 (s, 6H),
3.01 (t, J=7.3 Hz, 2H), 3.12 (t, J=6.9 Hz, 2H), 3.20–3.30 (m, 4H), 3.50
(s, 2H), 3.72–3.74 (2 s, 6H), 4.16 (t, J=6.3 Hz, 2H), 6.67 (s, 1H), 6.72 (s,
2H), 6.79 (s, 2H), 7.01 (s, 1H); 13C NMR (CD3OD): d = 22.9 (CH3),
24.3 (CH2), 26.4 (CH2), 30.3 (CH2), 30.4 (CH2), 32.7 (CH2), 36.7 (CH2),
36.8 (CH3), 36.9 (CH3), 38.0 (CH2), 41.5 (CH2), 43.4 (CH3), 49.6 (CH2),
56.6 (CH2), 69.1 (CH2), 71.7 (CH2), 106.2 (CH), 106.6 (CH), 119.6 (CH),
120.9 (CH), 123.2 (C), 123.9 (C), 124.0 (C), 124.5 (C), 161.4 (C), 161.5
(C), 164.9 (C), 167.2 (C), 170.4 (C); MS (FAB+): m/z: 697 (100)
[M+H]+; HRMS: m/z: calcd for C34H53N10O6 697.4150, found 697.4151.
ence or absence of ds-oligonucleotides: in the absence of DNA (&), in
hs
the presence T/CREhs
(
), in the presence CRE ( ), in the presence of
~
*
hs
&
T/CRE m ( ); CD spectra were obtained at 208C as described in the Ex-
perimental Section. The contribution of the DNA has been subtracted.
b) Autoradiogram showing the binding of hybrid 1 to 32P-labelled T/
CREhs at 238C: lanes: 5, 10, 15, 20, 30, 40, 50 nm. c) Autoradiograms
showing the binding of hybrid 11 to 32P-labelled DNAs at 238C: lanes 1–
7: T/CREhs: 0, 5, 10, 15, 20, 30, 40 nm; lanes 8–14: T/CREhsm: 5, 10, 15,
20, 30, 40, 50 nm. d) Autoradiogram showing the binding of hybrid 11 to
32P-labelled CREhs at 238C: 0, 100, 200, 300, 400, 500, 600, 700 nm.
mosphere unless otherwise stated. Thin-layer chromatography (TLC) was
performed on silica gel plates and components were visualized by obser-
vation under UV light, or by treating the plates with a p-anisaldehyde,
phosphomolybdic or ninhidrin reagent followed by heating. Dryings were
performed with anhydrous Na2SO4 and concentrations were carried out
1
in a rotary evaporator. H and 13C NMR spectra were recorded in CDCl3,
at 250 MHz and 62.9 MHz, respectively, and in some cases at 300 or
500 MHz (75.4 or 125.7 for 13C NMR). Carbon types were determined
from DEPT and 13C NMR experiments.
Peptide synthesis was performed using standard Fmoc solid phase synthe-
sis method on a Rink-MBHA amide resin (~0.46 mmolgꢀ1), using mix-
tures of HBTU/HOBt as coupling agents, DIEA as base and DMF as sol-
vent. The cleavage/deprotection step was performed by treatment of the
resin-bound peptide with the following mixture: 830 mL TFA, 25 mL
EDT, 50 mg PhOH, 50 mL tioanisole and 50 mL H2O (300 mL of this mix-
ture for each 10 mg of resin). The amino acids used in the synthesis were
Synthesis of the tripyrrole derivative 16
(3-{2-[5-(3-Aminopropylcarbamoyl)-1-methyl-1H-pyrrol-3-yl-carbamoyl]-
4-nitropyrrol-1-yl}-propyl)- carbamic acid tert-butyl ester (14): A solution
of nitropyrrole 13 (615 mg, 2.42 mmol) in MeOH (30 mL) was hydrogen-
4176
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 4171 – 4178

Tripyrrole–Peptide Conjugates
FULL PAPER
ated over 10% palladium on charcoal (400 mg) at room temperature for
1.5 h (balloon pressure). The catalyst was removed by filtration through
Celite, and the filtrate concentrated under reduced pressure. The residue
was dissolved in DMF (8 mL) and added over another solution of nitroa-
cid 12 (584 mg, 1.86 mmol), DECP (0.366 mL, 2.42 mmol), Et3N
(1.23 mL, 9.3 mmol) and DMAP (10 mg) in THF (25 mL) cooled at 08C.
The resulting mixture was allowed to stir at room temperature for 8 h
and concentrated under reduced pressure. The product was purified by
flash column chromatography in neutral aluminium oxide (5% MeOH/
CH2Cl2) to afford 14 (870 mg, 90%). 1H NMR (CDCl3): d = 1.41 (s,
9H), 1.75 (q, J=7.2 Hz, 2H), 1.96 (q, J=6.7 Hz, 2H), 2.25 (s, 6H), 2.39
(m, 2H), 3.04 (t, J=6.6 Hz, 2H), 3.30 (m, 2H), 3.84 (s, 3H), 4.44 (t, J=
6.8 Hz, 2H), 6.77 (s, 1H), 7.19 (s, 1H), 7.38 (s, 1H), 7.90 (s, 1H);
13C NMR (CDCl3): d = 28.2 (CH2), 28.8 (CH3), 32.6 (CH2), 36.8 (CH3),
38.4 (CH2), 38.7 (CH2), 45.6 (CH3), 48.6 (CH2), 58.4 (CH2), 80.1 (C),
105.8 (CH), 109.1 (CH), 120.4 (CH), 122.9 (C), 124.7 (C), 127.2 (CH),
128.0 (C), 136.4 (C), 158.4 (C), 159.4 (C), 164.0 (C); MS (FAB+): m/z:
520 (100) [M+H]+, 420 (5); HRMS: m/z: calcd for C24H38N7O6 520.2884,
found 520.2904.
tion was stirred at room temperature for 30 min. Solvents were removed
and the residue was purified by HPLC (gradient 5 ! 95% B, tR
=
30 min; A: TFA/H2O 0.1%, B: TFA/CH3CN 0.1%) to afford the desired
product 16 (165 mg, 69%). 1H NMR (CD3OD): d = 1.22–1.35 (m, 4H),
1.43–1.44 (2 s, 18H), 1.55–1.60 (m, 2H), 1.70–1.89 (m, 4H), 1.94 (t, J=
6.6 Hz, 2H), 2.08 (s, 3H), 3.09 (s, 6H), 3.15–3.41 (m, 14H), 3.66 (s, 2H),
3.89 (s, 3H), 4.38 (t, J=6.41 Hz, 2H), 6.90 (s, 1H), 6.93 (s, 1H), 6.98 (s,
1H), 7.19 (s, 1H), 7.20 (s, 1H), 7.26 (s, 1H); MS (FAB+): m/z: 939 (9)
[M+H]+, 839 (3), 739 (10); HRMS: m/z: calcd for C46H75N12O9:
939.5780, found 939.5771.
Synthesis of the peptide–tripyrrole hybrids: The following is the general
procedure for the coupling of the amino tripyrrole derivatives with the
solid-phase bound peptide, exemplified for the coupling of tripyrrole 8 to
the resin-linked peptide 10 to give hybrid 2. Resin-bound peptide 10
(25 mg, Eppendorf tube) was suspended in DMF (1 mL) and mixture was
shaken for 1 h to ensure a good resin swelling. The DMF was removed a
solution of HATU in DMF (2,6 mg in 170 mL) and DIEA (28 mL, 0.5m in
DMF) was added. The resulting mixture was shaken for 5 min, and a so-
lution of the tripyrrole 8 (10 mg in 70 mL of DMF) and 28 mL of DIEA
(0.5m in DMF) was added. The reaction mixture was shaken for 2 h, and
the resin washed with DMF (3ꢄ0.6 mL, for 5 min), and Et2O (2ꢄ
0.5 mL). Cleavage/deprotection of the bound peptide under standard
conditions afforded a major product that was purified by RP-HPLC (gra-
dient 10 ! 35%, tR =24.32 min). MALDI-MS analysis confirmed the for-
mation of the desired hybrid 2 (~36% yield, considering also the peptide
synthesis): m/z: calcd for C143H244N53O39: 3327.9, found 3327.4 [M+H]+;
1: [~26% yield]; MALDI-TOF: calcd for C143H245N54O38: 3326.9, found
3326.6 [M+H]+; RP-HPLC: tR =23.38 min; 11: [~17% yield]; MALDI-
TOF: calcd for C145H250N55O38: 3369.9, found 3370.0 [M+H]+; RP-HPLC:
tR =21.0 min.
[5-(4-Acetylamino-2-{1-(3-tert-butoxycarbonylaminopropyl)-5-[5-(3-dime-
thylamino-propylcarbamoyl)-1-methyl-1H-pyrrol-3-yl-carbamoyl]-1H-
pyrrol-3-yl-carbamoyl}-pyrrol-1-yl)-pentyl]-[3-(2-aminoacetylamino)-
propyl]-carbamic acid tert-butyl ester (16): A solution of the dipyrrole 14
(300 mg, 0.58 mmol) in MeOH (30 mL) was hydrogenated over 10% pal-
ladium on charcoal (200 mg) at room temperature for 1.5 h (balloon pres-
sure). The catalyst was removed by filtration through Celite, and the fil-
trate concentrated under reduced pressure. The residue was dissolved in
DMF (8 mL) and added over another solution previously prepared of 15
(510 mg, 0.75 mmol), DECP (0.123 mL, 0.81 mmol), Et3N (0.402 mL,
2.9 mmol) and DMAP (10 mg) in THF (20 mL) cooled at 08C. The re-
sulting mixture was allowed to stir at room temperature for 8 h and con-
centrated under reduced pressure. The product was purified by flash
column chromatography in neutral aluminium oxide (5% MeOH/
CH2Cl2) to afford the expected nitrotripyrrole (439 mg, 66%). 1H NMR
(CD3OD): d = 1.17–1.22 (m, 2H), 1.33–1.36 (2 s, 18H), 1.36–1.40 (m,
2H), 1.48–1.63 (m, 2H), 1.70–1.75 (m, 2H), 1.83–1.94 (m, 4H), 2.84 (s,
6H), 2.96 (t, J=6.6 Hz, 2H), 3.06–3.12 (m, 8H), 3.33 (t, J=6.3 Hz, 2H),
3.68 (s, 2H), 3.81 (s, 3H), 4.12 (t, J=6.7 Hz, 1H), 4.26–4.38 (m, 6H), 6.83
(s, 1H), 6.92 (s, 1H), 7.13 (s, 1H), 7.21–7.34 (m, 6H), 7.54 (s, 1H), 7.57
(s, 1H), 7.69 (s, 1H), 7.72 (s, 1H), 7.84 (s, 1H); 13C NMR (CD3OD): 24.6
(CH2), 26.6 (CH2), 28.8 (CH3), 32.2 (CH2), 33.0 (CH2), 36.5 (CH2), 37.0
(CH3), 38.7 (CH2), 43.5 (CH3), 45.1 (CH2), 47.2 (CH2), 48.2 (CH2), 50.8
(CH2), 56.6 (CH2), 68.2 (CH2), 80.0 (C), 81.0 (C), 106.7 (CH), 109.2
(CH), 119.8 (CH), 121.0 (CH), 123.3 (C), 123.8 (C), 124.3 (C), 126.2
(CH), 127.3 (C), 128.0 (CH), 128.2 (CH), 128.9 (CH), 136.4 (C), 142.6
(C), 145.3 (C), 158.5 (C), 159.1 (C), 159.6 (C), 161.2 (C), 165.0 (C), 172.3
(C); HRMS (ESI-TOF): m/z: calcd for C59H81N12O12 1149.6097, found
1149.609.
Acknowledgements
This work was supported by the E.R.D.F. and the Spanish Ministry of
Education and Science (SAF2004–01044) and the Xunta de Galicia (PGI-
DIT02BTF20901PR). J.B.B. thanks the Spanish Ministry of Science and
Education and the University of Santiago for his predoctoral fellowship.
We are very grateful to Prof. J. Benavente for allowing us to use radioac-
tivity facilities.
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Received: January 4, 2005
Published online: April 28, 2005
4178
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Chem. Eur. J. 2005, 11, 4171 – 4178