Synthesis of cyclic Py-im polyamide libraries

Article abstract of DOI:10.1021/jo302053v

Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC50 values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.

Full text of DOI:10.1021/jo302053v

Article
pubs.acs.org/joc
Synthesis of Cyclic Py-Im Polyamide Libraries
Benjamin C. Li, David C. Montgomery, James W. Puckett, and Peter B. Dervan*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
S
* Supporting Information
ABSTRACT: Cyclic Py-Im polyamides containing two GABA
turn units exhibit enhanced DNA binding affinity, but
extensive studies of their biological properties have been
hindered due to synthetic inaccessibility. A facile modular
approach toward cyclic polyamides has been developed via
microwave-assisted solid-phase synthesis of hairpin amino acid
oligomer intermediates followed by macrocyclization. A
focused library of cyclic polyamides 1−7 targeted to the
androgen response element (ARE) and the estrogen response
element (ERE) were synthesized in 12−17% overall yield. The Fmoc protection strategy also allows for selective modifications
on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of
cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides.
Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines.
The IC50 values of compounds 1−7 and 9−11 were determined, revealing remarkably varying levels of cytotoxicity.
cyclic polyamides by intramolecular coupling of a cysteine and a
chloroacetyl residue, but the modification of the optimal three-
carbon GABA turn into a sulfur-containing four-atom linker
compromises its DNA binding affinity and may alter its biological
properties.23
INTRODUCTION
■
The selective modulation of eukaryotic gene expression by small
molecules may have important implications in the field of
chemical biology and human medicine. Pyrrole-imidazole
polyamides are a class of synthetic ligands that can be
programmed to bind the minor groove of specific DNA
sequences.1 Antiparallel, side-by-side N-methylpyrrole (Py)
and N-methylimidazole (Im) carboxamide (Im/Py) pairs
distinguish G·C from C·G base pairs, N-methyl-3-hydroxypyr-
role (Hp)/Py shows specificity for T·A over A·T, whereas Py/Py
pairs are specific for both T·A and A·T.2−5 By linking two strands
of these heterocyclic oligomers via a γ-amino butyric acid
(GABA) turn unit, hairpin Py-Im polyamides can be
programmed to bind a large library of DNA sequences with
affinities comparable to natural DNA binding proteins.6−8
Hairpin polyamides have been shown to localize to the nuclei
of living cells and regulate endogenous gene expression by
disrupting protein/DNA interfaces.9−17 Cyclic polyamides
containing a second GABA turn unit exhibit further enhanced
DNA binding properties.18−21 We have recently demonstrated
their gene regulatory effects on AR-activated gene expression in
prostate cancer models.22
This discovery has opened a new area of research toward
transcriptional regulation with small molecules, but the relative
synthetic inaccessibility of cyclic polyamides has remained a
bottleneck for examining libraries of structural variants that
would modulate affinity, cell uptake, and biological activity.
Initial solid-phase methods were low yielding and required
substantial premodifications of the PAM resin.18−20 While the
solution-phase synthesis of cyclic polyamides remains useful in
large-scale target-oriented synthesis, it has limited practicality
toward libraries for screening biological activities.22 The recent
report by Morinaga et al. offers a modular approach to achieve
We report here a solid-phase polyamide synthesis of a key
hairpin amino acid oligomer intermediate, which followed by
intramolecular cyclization affords cyclic polyamides 1−8 in good
yields. The polyamides were synthesized stepwise on 2-
chlorotrityl resin. The modular approach led to rapid access of
a focused library of cyclic polyamides 1−7 (Scheme 1) with
various core sequences and turn unit modifications. The
utilization of Fmoc chemistry allowed for differentially protected
turn units, which were modified selectively to complement
existing cellular imaging and cell uptake enhancement
technologies.24 We examined the DNA binding properties and
cytotoxicity profiles of compounds 1−11 and the cellular
localization of cyclic polyamides 12−14 by fluorescence
microscopy.
RESULTS AND DISCUSSION
■
Microwave-Assisted Solid-Phase Synthesis. Due to
previously observed decomposition of the conjugated C-terminal
free carboxylic acid in polyamide intermediates, 2-chlorotrityl
chloride (2-Cl-Trt-Cl) resin was chosen for its mild synthesis and
cleavage conditions. Polyamide synthesis on this resin has been
previously reported by Aldrich-Wright and co-workers, but a
resin-bound β-alanine linker was used in both instances, and a
Special Issue: Robert Ireland Memorial Issue
Received: September 24, 2012
Published: October 29, 2012
© 2012 American Chemical Society
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solution. In light of the recent improvements in both efficiency
and yield, the couplings were performed under microwave-
assisted conditions using the desired PyBOP-activated mono-
mers.27 Initial syntheses performed at 60 °C led to premature
cleavage of intermediates off the 2-Cl-Trt resin, and 50 °C
couplings were therefore preferable. The challenging Im to Py
coupling required an FmocPyImOH dimer, which was obtained
via an optimized procedure by Weltzer and Wemmer.28 The
deprotection and coupling conditions are detailed in Table 1.
Scheme 1. Microwave-Assisted Synthesis of Cyclic
Polyamides 1−7
a
Table 1. Standard Fmoc Deprotection and Microwave-
Assisted Coupling Times for Solid-Phase Polyamide Synthesis
b
coupling times (min)
a
resin-bound nucleophile deprotection times
Py
20
Im GABA/β-Ala
Py
3 × 10 min
3 × 10 min
2 × 5 min
20
30
20
20
30
20
c
Im
IR
20
GABA/β-Ala
a
b
All deprotections were performed in 50% piperidine in DMF. All
coupling reactions were conducted under microwave-assisted con-
ditions at 50 °C with a 0.3 M solution of the activated monomers (3
equiv of monomer acid, 3 equiv of PyBOP, 8 equiv of DIEA, DMF).
c
FmocPyOH coupling onto resin with N-terminal Im was incomplete
even at 60 °C for up to 1 h. Synthesis of polyamide sequences that
require this linkage should use the FmocPyImOH dimer instead,
demonstrated later in the synthesis of 8.
This two-step deprotection−coupling procedure was repeated
until the desired polyamide sequence was achieved. To build the
small library of polyamides in a modular fashion, the resin was
split into different batches at corresponding steps for further
derivatization. Upon completion, the N-terminal Fmoc-
protected polyamide oligomer was cleaved from the resin with
30% hexafluoroisopropanol (HFIP) in dichloromethane
(DCM), concentrated in vacuo, and the resulting residue was
subjected to a 20% piperidine solution to remove the Fmoc
group. Direct cleavage of the free amine polyamide oligomer was
attempted, but found to be ineffective due to poor solubility of
the zwitterion intermediate in the cleavage solution. After
purification by high-performance liquid chromatography
(HPLC), the desired polyamide intermediates 18−24 were
obtained in 31−40% yields (Table 2).
DPPA-Mediated Macrocyclization. The polyamide mac-
rocyclization step was achieved by a DPPA-mediated ring-closing
reaction between the N-terminal amino group and the C-
terminal carboxylic acid. This method was first employed by Cho
et al. in the synthesis of cyclic polyamides.18,29,30 In order to
obtain a general workup procedure applicable to polyamides of
various lipophilicities, diisopropylethylamine (DIEA) was used
as the base in place of sodium bicarbonate (NaHCO3) in the
original conditions.31 Deprotection of the turn units with
trifluoromethanesulfonic acid (TFMSA) or trifluoroacetic acid
(TFA), followed by HPLC purification, afforded polyamides 1−
7 in 37−43% yields over two steps.
a
All PyBOP-mediated coupling conditions were performed under
microwave-assisted conditions (see Table 1). Reagents and conditions:
(i) 50% piperidine, DMF; (ii) FmocPyOH, PyBOP, DIEA, DMF; (iii)
50% piperidine, DMF; (iv) FmocPyOH, PyBOP, DIEA, DMF; (v)
50% piperidine, DMF; (vi) FmocImOH, PyBOP, DIEA, DMF; (vii)
50% piperidine, DMF; (viii) Z-β-Dab(Fmoc)-OH (for 1−6) or Boc-β-
Dab(Fmoc)-OH (for 7), PyBOP, DIEA, DMF; (ix) 50% piperidine,
DMF; (x) FmocPyOH, PyBOP, DIEA, DMF; (xi) 50% piperidine,
DMF; (xii) FmocPyOH, PyBOP, DIEA, DMF; (xiii) 50% piperidine,
DMF; (xiv) FmocPyOH (for 1−3) or FmocImOH (for 4−7),
PyBOP, DIEA, DMF; (xv) 50% piperidine, DMF; (xvi) FmocImOH,
PyBOP, DIEA, DMF; (xvii) 50% piperidine, DMF; (xviii) Z-β-
Dab(Fmoc)-OH (for 1 and 4 and 7) or Fmoc-GABA-OH (for 2 and
5) or Boc-β-Dab(Fmoc)-OH (for 3 and 6), PyBOP, DIEA, DMF;
(xix) 30% HFIP, DCM; (xx) 20% piperidine, DMF; (xxi) DPPA,
DIEA, DMF; (xxii) 10% TFMSA, TFA (for 1, 2, 4, and 5) or TFA (for
3, 6, and 7).
Selective Derivatization of Cyclic Polyamide Turn
Units. By taking advantage of the Fmoc protection scheme,
the two GABA β-amino groups in 3 were differentially protected.
This is further highlighted in 6 and 7, which share the same
asymmetric polyamide core targeted to the 5′-WGGWCW-3′
sequence, and allowed for the selective conjugation of a benzoic
acid moiety on a single turn unit in polyamides 9−11 that has
been recently developed to enhance the cellular localization
properties of hairpin polyamides (Scheme 2).24 Monosubsti-
new loading procedure was therefore needed.25,26 2-Cl-Trt-Cl
resin was first loaded with the Fmoc-protected Py monomer in
N,N-dimethylformamide (DMF) and capped with methanol.
Resin substitution levels were determined by the Fmoc test and
confirmed by weighing the dry mass of the loaded resin. Fmoc
deprotection was achieved using a 50% piperidine in DMF
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Table 2. Summary Table of MALDI-TOF Data and Synthetic Yields for Cyclic Polyamides 1−8 and Intermediates 18−25
a
Scheme 2. Preparation of Cyclic Polyamides 9−11
Supporting Information). Cyclic polyamides 12−14 with a
fluorescein dye were synthesized in a similar fashion and imaged
in living cells via fluorescence microscopy (Scheme 3).
Furthermore, both the solubility and the pharmacokinetic
profiles of cyclic polyamides have been shown to be highly
dependent on subtle structural modifications, and this method
allows for the modular synthesis of these structural variants in an
efficient manner.31−33
Synthesis of Cyclic and Hairpin Polyamides with C-
Terminal Imidazole Units. Previously established solid-phase
polyamide synthesis methods have been generally limited to
sequences beginning with a pyrrole monomer.34,35 Solid-phase
synthesis of polyamides starting with imidazole units on the
commonly used Kaiser oxime resin have been low yielding,
mainly attributed to the sensitivity of the oxime−imidazole
linkage that leads to premature cleavage of resin-bound
intermediates. The addition of an aliphatic linker (e.g., Boc-β-
Ala-PAM resin) circumvents this issue, but previous studies on
hairpin polyamides with C-terminal β-alanine motifs have shown
reduced cellular uptake properties and thus diminished gene
regulatory effects of these products.9 Using the microwave-
assisted conditions reported above, cyclic polyamide 8 targeted
to the 5′-WCGWGW-3′ sequence found in E-Box binding sites
has been synthesized in 13% yield overall.
Initial attempts starting with FmocImOH-loaded resin
resulted in undesired cleavage during subsequent steps, and so
syntheses began with loading of the FmocPyImOH dimer onto
2-chlorotrityl resin. After deprotection−coupling of the corre-
sponding monomer units, followed by resin cleavage and Fmoc
removal, polyamide intermediate 25 was isolated by HPLC
purification in 34% yield. DPPA-mediated cyclization of 25,
followed by Cbz deprotection afforded cyclic polyamide 8 in 39%
yield over two steps (Scheme 4). Hairpin polyamide 17 was
a
Reagents and conditions: (i) BzOH, PyBOP, DIEA, DMF; (ii) 10%
TFMSA, TFA.
tuted benzyl carbamate (Cbz) polyamides 3, 6, and 7 were
chosen as targets based on unpublished results indicating that
Cbz-functionalized polyamides are biologically active (see
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a
a
Scheme 3. Preparation of Cyclic Polyamides 12−14
Scheme 4. Preparation of Cyclic Polyamide 8
a
Reagents and conditions: (i) FITC, DIEA, DMF; (ii) 10% TFMSA,
TFA.
synthesized in a similar stepwise manner to afford the C-terminal
acid intermediate, which was then coupled to a 3,3′-diamino-N-
methyldipropylamine linker, followed by isophthalic acid
conjugation, Boc deprotection, and isolated in 24% yield over
16 steps (see Scheme S1). This is a step forward which allows for
the synthesis of both cyclic and non-β-alanine-linked hairpin
polyamides with sequences beginning with an imidazole unit,
further expanding the scope of targetable DNA sequences and
inhibition of transcription-factor-mediated gene expression by
Py-Im polyamides.
Thermal Stabilization of DNA Duplexes by Poly-
amides. Py-Im polyamide−DNA binding affinities and
specificities have historically been measured by quantitative
DNase I footprinting assays.36 As previously reported, however,
cyclic polyamides have exceptionally high DNA binding affinities
that exceed the detection limit of this experiment (i.e., Ka ≥ 2 ×
1010 M−1).22,37,38 The DNA binding affinities of cyclic
polyamides 1−14 have been rank ordered by magnitude of
DNA thermal stabilization (ΔTm) and compared to the
corresponding hairpin polyamides 14−16. Spectroscopic
measurements were performed on 12-mer DNA duplexes with
sequences 5′-CGATGTTCAAGC-3′, 5′-CGATGGTCAAGC-
3′, and 5′-CGATCGTGAAGC-3′, each containing a match
binding site for the corresponding polyamides.
a
All PyBOP-mediated coupling conditions were performed under
microwave-assisted conditions (see Table 1). Reagents and conditions:
(i) 50% piperidine, DMF; (ii) FmocPyOH, PyBOP, DIEA, DMF; (iii)
50% piperidine, DMF; (iv) FmocPyOH, PyBOP, DIEA, DMF; (v)
50% piperidine, DMF; (vi) Z-β-Dab(Fmoc)-OH, PyBOP, DIEA,
DMF; (vii) 50% piperidine, DMF; (viii) FmocImOH, PyBOP, DIEA,
DMF; (ix) 50% piperidine, DMF; (x) Fmoc-β-Ala-OH, PyBOP, DIEA,
DMF; (xi) 50% piperidine, DMF; (xii) FmocPyImOH, PyBOP, DIEA,
DMF; (xiii) 50% piperidine, DMF; (xiv) Z-β-Dab(Fmoc)-OH,
PyBOP, DIEA, DMF; (xv) 30% HFIP, DCM; (xvi) 20% piperidine,
DMF; (xvii) DPPA, DIEA, DMF; (xviii) 10% TFMSA, TFA.
duplexes. Mono-unsubstituted cyclic polyamide 2 provides less
DNA stabilization compared to 1 (ΔTm = 20.4 °C), which is
likely due to the loss of a positive charge and thus the loss of
favorable electrostatic interactions with the negatively charged
DNA backbone. Perhaps more surprising is the high ΔTm values
retained by monoprotected cyclic polyamides 3 (ΔTm = 27.3 °C)
and 9 (ΔTm = 28.0 °C), each containing a lone free amino group
and net +1 charge. As shown in Figure 1, the benzoyl (Bz) group
in 9 projects straight down the minor groove, avoiding
unfavorable steric interactions with the groove wall, and may
offer insight into the high degree of DNA stabilization by 3 and 9
comparable to the bisamino cycle 1. Fluorescein-conjugated 12
(ΔTm = 21.8 °C) has a DNA binding affinity lower than 3 and 9,
perhaps due to increased steric clashes from the larger
substitution group and unfavorable electrostatic interactions
from the negatively charged fluorescein group, but still binds
DNA at a similar level to 15 (Table 3).
Consistent with the findings of Chenoweth et al., the ΔTm
value for bisamino cyclic polyamide 1 (ΔTm = 26.1 °C) was
calculated to be significantly higher than that of hairpin
polyamide 15 (ΔTm = 22.0 °C).22 So while 15 has an established
binding affinity to the match androgen response element (ARE)
half-site (5′-WGWWCW-3′) in the subnanomolar range, cyclic
polyamide 1 provides even greater stabilization to such DNA
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Table 3. Tm Values for the Polyamide Library
Figure 1. Molecular model of benzoyl-substituted cyclic polyamide turn
along the DNA minor groove, based on published crystal structure by
Chenoweth et al. (PDB ID: 3OMJ).
The magnitude of stabilization provided by cyclic polyamides
4−7, 10−11, and 13−14 targeted to estrogen response element
(ERE) half-sites (5′-WGGWCW-3′) follows a similar pattern to
the aforementioned ARE targeting series. Mono-unsubstituted
cycle 5 (ΔTm = 18.6 °C) stabilizes the duplex at a comparable
level to hairpin polyamide 16 (ΔTm = 16.7 °C), whereas
bisamino compound 4 (ΔTm = 23.2 °C) has a higher ΔTm value.
The mono-Cbz cycles 6 (ΔTm = 23.6 °C) and 7 (ΔTm = 24.3
°C), mono-Bz-substituted 10 (ΔTm = 26.0 °C) and 11 (ΔTm =
25.1 °C), and monofluorescein conjugates 13 (ΔTm = 22.2 °C)
and 14 (ΔTm = 21.0 °C) each bind DNA similar to 4.
Cyclic polyamide 8 (ΔTm = 13.4 °C) binds the match 5′-
WCGWGW-3′ sequence at an elevated level compared to
hairpin 17 (ΔTm = 6.6 °C), which may prove important toward
targeting oncogenic transcription factors such as c-Myc that act
through binding canonical E-Box (5′-CACGTG-3′) sequences.
Sulforhodamine B Cytotoxicity Assay for Compounds
1−11. The cytotoxicity of compounds 1−11 was assessed in
A549 human lung carcinoma and T47D human breast cancer cell
lines (Tables 4 and 5). The cyclic polyamides targeted to the 5′-
WGWWCW-3′ sequence generally exhibit a higher level of
cytotoxicity than the 5′-WGGWCW-3′ series, which is
consistent with the trends observed in the DNA thermal
denaturation analysis and confocal microscopy studies. Detailed
inspection of the IC50 values within the series of compounds and
across the cell lines, however, offers some unanticipated insights
into the different biological properties of these minor structural
variants.
Bisamino cycle 1, which has previously been shown to be
biologically active, did not display any significant level of
cytotoxicity in either cell line (IC50 > 30 μM). Mono-
unsubstituted compound 2 (IC50 = 4.9 μM) and mono-Bz 9
(IC50 = 1.0 μM) were comparably cytotoxic to hairpin polyamide
15 (IC50 = 3.1 μM) in A549 cells but an order of magnitude more
μM) again shares a comparable level of cytotoxicity with the
reference hairpin 16 (IC50 = 1.1 μM), whereas mono-Bz
compounds 10 (IC50 = 13.3 μM) and 11 (IC50 = 7.5 μM) are
both an order of magnitude less cytotoxic.
Perhaps most interestingly, mono-Cbz compound 7 did not
exhibit observable levels of cytotoxicity (IC50 > 30 μM) in either
A549 or T47D cells. Considering that 7 is a regioisomer of 6,
where the Cbz group is simply swapped onto the other turn, and
that 7 only differs from 3 by a single −CH to −N substitution, it
is rather surprising that 7 is more than 15- to 65-fold less
cytotoxic than 6 and at least 180- to 1200-fold less cytotoxic than
3 in the two examined cell lines. Given the comparable DNA
cytotoxic (IC50 = 74 and 79 nM, respectively) than 15 (IC50
=
710 nM) in T47D cells. The mono-Cbz cycle 3 was consistently
the most cytotoxic compound in both A549 (IC50 = 160 nM) and
T47D (IC50 = 25 nM) cell lines.
For the 5′-WGGWCW-3′ targeting polyamides, the only
compound that exhibited an IC50 value lower than 30 μM in
A549 cells was the mono-Cbz compound 6 (IC50 = 1.9 μM). In
T47D cells, consistent with the 5′-WGWWCW-3′ series,
bisamino cycle 4 (IC50 > 30 μM) was found to be not
significantly cytotoxic and 6 (IC50 = 460 nM) was the most
cytotoxic compound. Mono-unsubstituted cycle 5 (IC50 = 0.82
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ization of cyclic polyamides, fluorescein conjugates 12−14 were
synthesized and visualized in living cells via confocal microscopy
(Figures 2 and 3). The selective conjugation of a single
fluorescein molecule not only helped retain a free amino group
for solubility purposes but also allowed for the qualitative
comparison of the two fluorescein conjugates 13 and 14 that
share the same asymmetric polyamide core.
Table 4. SRB Cytotoxicity Data on Compounds 1−3, 9, and
15, in A549 and T47D Cells, 72 Hour Incubation
In each of the cases examined, cyclic polyamides 12−14
appear to permeate through the cellular membrane and localize
in the cell nucleus, which was confirmed by the colocalization
with Hoechst 33258 DNA stain. For ease of qualitatively
assessing compound uptake, all A549 images were taken at a 660
fluorescence gain level, and all T47D images were taken at 600
fluorescence gain. Comparing Figures 2 and 3, the fluorescence
levels of compounds 12−14 in T47D cells are all significantly
higher than in A549, which is only further amplified by this
difference in gain levels.
Compound 12 matched to the 5′-WGWWCW-3′ sequence
exhibits the highest level of nuclear localization in both cell lines.
Among the two 5′-WGGWCW-3′ targeting cycles, polyamide 14
qualitatively appears to have a relatively higher fluorescence
signal in the cell nuclei in both A549 and T47D cells. This may
help explain the cytotoxicity data reported above, where
compounds 6 and 11 with Cbz and Bz substitutions on the
same side as the fluorescein in 14 consistently display larger
biological effects than 7 and 10 that are more structurally similar
to cyclic polyamide 13.
Table 5. SRB Cytotoxicity Data on Compounds 4−7, 10, 11,
and 16, in A549 and T47D Cells, 72 Hour Incubation
CONCLUSION
■
We have described a modular solid-phase synthesis method,
which, when combined with an established DPPA-mediated
macrocyclization step, afforded cyclic polyamides in a high-
yielding and time-efficient manner. Using this method, we have
overcome previous limitations and synthesized both cyclic and
hairpin polyamides that start with an imidazole unit. The binding
affinities of all synthesized cycles have been assessed by DNA
thermal denaturation assays and compare favorably to hairpin
polyamides that bind their match DNA sequences at
subnanomolar concentrations. Furthermore, the protection
strategy of our method allows for selective modification of the
GABA turn units, which we have used to rapidly generate a
focused library of compounds. The cytotoxicity and uptake
analysis of the cyclic polyamides revealed unexpected properties
that further highlight the need for an efficient method to
synthesize structural variants of cyclic polyamides for future
studies.
EXPERIMENTAL SECTION
■
General Experimental Methods. 2-Chlorotrityl chloride (2-Cl-
Trt-Cl) resin was purchased from Bachem. FmocPyOH and
FmocImOH monomers were purchased from Wako. PyBOP was
purchased from NovaBioChem. Boc-β-Dab(Fmoc)-OH was purchased
from Peptides International. All DNA oligomers were purchased HPLC
purified from Integrated DNA Technologies. Cell culture medium was
purchased from Gibco. Fetal bovine serum was purchased from Omega
Scientific. Microwave-assisted coupling reactions were conducted on a
Biotage Initiatior Eight synthesizer. Polyamide concentrations were
measured in 20% MeCN in 0.1% (v/v) aqueous TFA using an
approximated extinction coefficient of 69 200 M−1 cm−1 at λmax near 310
nm, unless otherwise specified.32,37
stabilization properties between 6 and 7, and their common core
sequence, we would not have predicted this vast discrepancy in
cytotoxicity.
This study has demonstrated the large and somewhat
unpredictable effects in biological activity induced by small
structural variations of cyclic polyamides. On the basis of our
preliminary work, the aggregation and pharmacokinetic proper-
ties of polyamides also vary greatly depending on structural
modifications.31−33 All this combines to highlight the importance
of a fast and reliable method to generate focused libraries of cyclic
polyamides for future research.
Monomer Loading onto 2-Cl-Trt Resin. Prior to manual
microwave-assisted synthesis, 2-Cl-Trt-Cl resin (1.0 g, 1.59 mmol/g)
was first loaded by mixing with 576 mg (1.59 mmol, 1 equiv) of
FmocPyOH monomer, followed by addition of 6 mL of dimethylfor-
Confocal Microscopy of Cyclic Polyamide−Fluorescein
Conjugates 12−14. To directly examine the cellular local-
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Figure 2. Confocal microscopy of cyclic polyamide−fluorescein conjugates 12 (top), 13 (middle), and 14 (bottom) in A549 cells. In order to confirm
nuclear localization, the fluorescence panel (left) was compared with Hoechst 33258 staining (middle) and overlay (right).
mamide (DMF) and 1.38 mL of diisopropylethylamine (DIEA) (7.59
mmol, 5 equiv). The suspended mixture was stirred for 18 h, then
capped by addition of 1 mL of methanol (MeOH) and stirred for 1 h.
The orange-colored, loaded resin was then collected on a fritted peptide
synthesis vessel, washed with DMF (2×), MeOH (2×), DMF (2×),
MeOH (2×), and diethylether (Et2O). [Owing to the sensitivity of the
2-Cl-Trt resin toward hydrolysis/methanolysis, this final Et2O wash was
found to be essential and all loaded resin was sealed and stored at −20
°C.] The loading efficiency was quantitated via the Fmoc test and
confirmed by measuring the mass of the dried resin. Typical monomer
loading was calculated to be 0.4−0.8 mmol/g.
Microwave-Assisted Solid-Phase Synthesis (18−24). All solid-
phase polyamide coupling reactions were performed manually on a
Biotage Initiator Eight microwave synthesizer on a 200−500 mg scale of
loaded resin. Prior to each monomer coupling reaction, the N-terminal
Fmoc group was first removed in a piperidine solution. The Fmoc
deprotections were performed in a fritted peptide synthesis vessel at
room temperature, and the specific conditions for each N-terminal
monomer are as follows:
N-Fmoc-GABA/β-Alanine: (a) swell resin in DCM; (b) wash with
DMF; (c) add 50% piperidine in DMF; (d) shake suspension for 5 min;
(e) wash with DMF; (f) repeat steps a−e once.
Following Fmoc removal, the resin was deswelled in MeOH, washed
with Et2O, dried in vacuo, and transferred to a microwave synthesis
vessel as a dry powder. The corresponding monomer acid (3 equiv) was
activated with PyBOP (3 equiv) and DIEA (6 equiv) in DMF (0.3 M
concentration of monomer), and added to the resin. The coupling
reactions were then setup in the microwave reactor at 50 °C for the time
durations described in Table 1. After the listed microwave-assisted
coupling times, the reaction mixture was filtered into a peptide synthesis
vessel, and the collected resin was washed with DMF (3×), MeOH
(3×), Et2O, and dried in vacuo. To ensure completion of each
deprotection and coupling step, analytical HPLC spectra were taken by
cleaving a small resin sample in 30% hexafluoroisopropanol (HFIP) in
DCM.
The polyamide core was synthesized on 2-Cl-Trt resin in an iterative
manner by repeating the deprotection−coupling procedures described
above using the corresponding monomeric units. Upon completion of
the sequence, 100−200 mg of the resin was suspended in 1 mL of 30%
HFIP in DCM and stirred for 1 h to yield the crude N-terminal Fmoc-
protected polyamide intermediate. The reaction mixture was then run
through a cotton filter to remove the resin, and the filtrate was
concentrated in vacuo. The residual oil was resuspended in 5 mL of a 1:1
N-Fmoc-Pyrrole/Imidazole: (a) swell resin in DCM; (b) wash with
DMF; (c) add 50% piperidine in DMF; (d) shake suspension for 10
min; (e) wash with DMF; (f) repeat steps a−e twice.
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Figure 3. Confocal microscopy of cyclic polyamide−fluorescein conjugates 12 (top), 13 (middle), and 14 (bottom) in T47D cells. In order to confirm
nuclear localization, the fluorescence panel (left) was compared with Hoechst 33258 staining (middle) and overlay (right).
MeOH/DCM mixture and reconcentrated in vacuo to give an off-
white/beige solid. To remove the N-terminal Fmoc group, the solid was
redissolved in 800 μL of DMF, followed by addition of 200 μL of
piperidine, and the solution was stirred for 30 min. Upon confirmation
of complete deprotection by analytical HPLC, the solution was added to
4 mL of 30% MeCN in 0.1% aqueous TFA. The precipitated 9-
methylenefluorene side product was then removed by centrifugation
and washed twice with 2 mL of 30% MeCN in 0.1% aqueous TFA. The
combined aqueous solution was purified by reverse-phase HPLC and
lyophilized to dryness to yield precyclic polyamide intermediates 18−
24. All dried samples of 18−24 were stored at −20 °C prior to DPPA-
mediated macrocyclization.
Synthetic yields and MALDI-TOF characterization data for 18−24
are summarized below:
(18): 12.6 μmol recovered (38.0 μmol theoretical, 33% yield).
MALDI-TOF [M + H]+ calcd for C70H77N22O15+ = 1465.4, observed =
1465.9.
(21): 15.1 μmol recovered (49 μmol theoretical, 31% yield). MALDI-
TOF [M + H]+ calcd for C69H76N23O15+ = 1466.5, observed = 1466.9.
(22): 17.2 μmol recovered (51 μmol theoretical, 34% yield). MALDI-
TOF [M + H]+ calcd for C61H69N22O13+ = 1317.5, observed = 1317.2.
(23): 16.2 μmol recovered (51 μmol theoretical, 32% yield). MALDI-
+
TOF [M + Na]+ calcd for C66H77N23NaO15 = 1454.5, observed =
1455.0.
(24): 8.2 μmol recovered (25 μmol theoretical, 33% yield). MALDI-
+
TOF [M + Na]+ calcd for C66H78N23NaO15 = 1454.5, observed =
1454.9.
DPPA-Mediated Macrocyclization (1−7). The macrocyclization
reactions were run on a 2−16 μmol scale. Intermediates 18−24 were
first dissolved in DMF (0.25 mM) in a round-bottom flask equipped
with a magnetic stir bar, followed by addition of DIEA (200 equiv), and
purged with argon for 15 min. Diphenylphosporyl azide (DPPA) (50
equiv) was then added to the reaction mixture in a dropwise manner,
while rapidly stirring. Upon full addition of the DPPA, the solution was
allowed to react and stirred at room temperature for 16−20 h. After
confirmation of reaction completion by analytical HPLC, the reaction
mixture was concentrated in vacuo and the resulting oil residue was
dissolved in 3 mL of MeCN and transferred to a 15 mL Falcon tube. The
MeCN was then removed with air flow, and 3 mL of 0.1% aqueous TFA
was added to the remaining oil layer to yield an off-white suspension,
which was isolated via centrifugation and lyophilized to dryness.
(19): 15.2 μmol recovered (38.0 μmol theoretical, 40% yield).
MALDI-TOF [M + H]+ calcd for C62H70N21O13+ = 1316.5, observed =
1316.9.
(20): 12.8 μmol recovered (38 μmol theoretical, 34% yield). MALDI-
+
TOF [M + Na]+ calcd for C67H79N22NaO15 = 1453.6, observed =
1453.9.
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For reactions starting with 18, 19, 21, and 22, the lyophilized residue
was submitted to 1 mL of 10% trifluoromethanesulfonic acid (TFMSA)
in TFA, stirred for 5 min, frozen in LN2, and thawed by layering 1 mL of
DMF. For reactions starting with 20, 23, and 24, the lyophilized residue
was submitted to 1 mL of neat TFA, stirred for 15 min, frozen in LN2,
and thawed by layering 1 mL of DMF. All of the thawed solutions were
then diluted with 6 mL of 0.1% aqueous TFA, purified by reverse-phase
HPLC, and lyophilized to dryness to yield cyclic polyamides 1−7.
Synthetic yields and MALDI-TOF characterization data for 1−7 are
summarized below:
first loaded with FmocPyImOH dimer (96 mg, 0.20 mmol), which was
obtained from published procedures.28 Experimental details were
analogous to the monomer loading protocol reported above. The
obtained Fmoc-Py-Im-(2-Cl-Trt) resin (265 mg, 0.59 mmol/g) was
subjected to the previously described microwave-assisted solid-phase
synthesis conditions to build the corresponding polyamide sequence. A
quarter of the resin (0.15 μmol theoretical) was then cleaved and
purified by reverse-phase HPLC to yield precyclic polyamide
intermediate 25 as an off-white powder (13.3 μmol, 34% yield). The
isolated 25 (2.0 μmol) was subjected to DPPA-mediated macro-
cyclization conditions analogous to that for compounds 1−7, with the
Cbz groups removed with 10% TFMSA in TFA, and purified by reverse-
(1): 4.5 μmol recovered (12.9 μmol theoretical, 35% yield). MALDI-
TOF [M + H]+ calcd for C54H63N22O10+ = 1179.5, observed = 1179.9.
(2): 0.84 μmol recovered (2.0 μmol theoretical, 42% yield). MALDI-
TOF [M + H]+ calcd for C54H62N21O10+ = 1164.5, observed = 1164.6.
(3): 3.2 μmol recovered (6.7 μmol theoretical, 47% yield). MALDI-
TOF [M + H]+ calcd for C62H69N22O12+ = 1313.6, observed = 1314.0.
(4): 3.1 μmol recovered (8.0 μmol theoretical, 39% yield). MALDI-
TOF [M + H]+ calcd for C53H62N23O10+ = 1180.5, observed = 1180.9.
(5): 0.96 μmol recovered (2.0 μmol theoretical, 48% yield). MALDI-
TOF [M + H]+ calcd for C53H61N22O10+ = 1165.5, observed = 1165.5.
(6): 3.7 μmol recovered (10.0 μmol theoretical, 37% yield). MALDI-
TOF [M + H]+ calcd for C61H68N23O12+ = 1314.5, observed = 1314.5.
(7): 2.2 μmol recovered (5.7 μmol theoretical, 38% yield). MALDI-
TOF [M + H]+ calcd for C61H68N23O12+ = 1314.5, observed = 1314.8.
Selective Conjugation of Benzoic Acid Derivatives (9−11). A
solution of benzoic acid (3.0 mg, 0.025 mmol, 25 equiv) and PyBOP (13
mg, 0.025 mmol, 25 equiv) in DMF (0.5 mL) and DIEA (44 μL, 0.25
mmol, 250 equiv) was stirred at room temperature for 10 min. The
activated solution was then added to 3 (1.0 μmol) and stirred for 3 h.
After confirmation of complete reaction by analytical HPLC, 12 mL of
cold Et2O was added to the reaction mixture and cooled at −20 °C for 16
h. The precipitate was then isolated by centrifugation and allowed to air-
dry. The resulting residue was submitted to 1 mL of 10%
trifluoromethanesulfonic acid (TFMSA) in TFA, stirred for 5 min,
frozen in LN2, and thawed by layering 1 mL of DMF. The thawed
solution was then diluted with 6 mL of 0.1% aqueous TFA, purified by
reverse-phase HPLC, and lyophilized to dryness to yield cyclic
polyamide 9 (684 nmol, 68% yield). Using the same procedure
described above, starting with 6 (1.60 μmol) and 7 (750 nmol), yielded
monobenzoyl-substituted cyclic polyamides 10 (1.05 μmol, 67% yield)
and 11 (367 nmol, 49% yield), respectively.
phase HPLC to afford cyclic polyamide 8 (773 nmol, 39% yield).
+
(25): MALDI-TOF [M − CO2 + H]+ calcd for C65H675N22O13
=
1371.6, observed = 1371.7.
+
(8): MALDI-TOF [M + H]+ calcd for C61H67N22O11 = 1129.5,
observed = 1130.0.
Detailed experimental procedures and characterization data for 17 are
provided in the Supporting Information.
Thermal Denaturation Analysis. Melting temperature analysis
was performed on a Varian Cary 100 spectrophotometer equipped with
a thermocontrolled cell holder possessing a cell path length of 1 cm. A
degassed aqueous solution of 10 mM sodium cacodylate, 10 mM KCl,
10 mM MgCl2, and 5 mM CaCl2 at pH 7.0 was used as analysis buffer.
DNA duplexes and polyamides were mixed in 1:1 stoichiometry to a
final concentration of 2 μM for each experiment. Prior to analysis,
samples were heated to 95 °C and cooled to a starting temperature of 25
°C with a heating rate of 5 °C/min for each ramp. Denaturation profiles
were recorded at λ = 260 nm from 25 to 95 °C with a heating rate of 0.5
°C/min. The reported melting temperatures were defined as the
maximum of the first derivative of the denaturation profile.
Cell Culture. Cell lines were cultured at 37 °C under 5% CO2 using
standard cell culture and sterile techniques. Cell medium was
supplemented with 10% fetal bovine serum. Ham’s F-12K (Kaighn’s)
medium was used for A549 cells, and RPMI 1640 was used for T47D
cells.
Confocal Microscopy. For each experiment, cells were plated in
200 μL of the proper medium onto glass-bottom cell culture plates at a
density of 1 × 105 (A549) or 1.5 × 105 cells/mL (T47D). Cells were
grown for 24 h, and media were replaced with fresh media containing
polyamide to give a final DMSO concentration of 0.1%. Next, cells were
incubated for 16 h, followed by removal of media, washing, and addition
of fresh media. Hoechst 33258 was added 2 h prior to imaging. Imaging
was performed at the Caltech Beckman Imaging Center using a Zeiss
LSM 510 Meta NLO two-photon inverted laser scanning microscope
equipped with a 40× oil-immersion objective lens. Polyamide−
fluorescein conjugates 12−14 were imaged in multi-track mode using
488 nm laser excitation at 15% output with a pinhole of 375 μm and a
standard fluorescein filter set. Hoechst was imaged using 800 nm two-
photon excitation with an HFT KP680 dichroic and a 390 to 465 nm
band-pass filter with a fully open pinhole. All images were analyzed using
Zeiss LSM Zen software.
Sulforhodamine B Cytotoxicity. For cytotoxicity assays, cell lines
were plated in 96-well cell culture plates in 100 μL of media at a density
of 1 × 104 (A549) or 5 × 104 cells/mL (T47D). IC50 values were
determined using the sulforhodamine B (SRB) colorimetric assay as
previously described.39 Cells were grown for 24 h, before polyamides in
100 μL of media were added in serial dilution, in quadruplicate for each
concentration. After incubation for 72 h, cell medium was replaced with
100 μL of fresh media, and cells were allowed to recover for an additional
24 h. Cells were then fixed by adding 100 μL of 10% trichloroacetic acid
directly to each well and stored at 4 °C for 1 h, before being washed,
dried, stained with 100 μL of 0.057% SRB solution per well for 30 min,
and washed and dried again as described. After solubilizing the bound
dye with 200 μL of 10 mM Tris (pH 10.5) per well, absorbance at 490
nm was measured on a PerkinElmer Victor microplate reader. The data
are charted as a percentage of untreated controls, corrected for
background absorbance. IC50 is defined as the concentration that
inhibits 50% of control cell growth. These values were determined by
nonlinear least-squares regression fit to Y = A + (B − A)/(1 + 10∧((log
+
(9): MALDI-TOF [M + H]+ calcd for C61H67N22O11 = 1283.5,
observed = 1284.1.
+
(10): MALDI-TOF [M + H]+ calcd for C60H66N23O11 = 1284.5,
observed = 1284.5.
+
(11): MALDI-TOF [M + H]+ calcd for C60H66N23O11 = 1284.5,
observed = 1284.9.
Cyclic Polyamide−Fluorescein Conjugates (12−14). A solution
of fluorescein isothiocyanate (FITC) (2.7 mg, 7.0 μmol, 25 equiv) in
DMF (0.2 mL) and DIEA (12 μL, 0.07 mmol, 250 equiv) was added to 3
(0.28 μmol) and stirred for 2 h. After confirmation of complete reaction
by analytical HPLC, 12 mL of cold Et2O was added to the reaction
mixture and cooled at −20 °C for 16 h. The precipitate was then isolated
by centrifugation and allowed to air-dry. The resulting residue was
submitted to 1 mL of 10% trifluoromethanesulfonic acid (TFMSA) in
TFA, stirred for 5 min, frozen in LN2, and thawed by layering 1 mL of
DMF. The thawed solution was then diluted with 6 mL of 0.1% aqueous
TFA, purified by reverse-phase HPLC, and lyophilized to dryness to
yield cyclic polyamide 12 (65 nmol, 23% yield). Using the same
procedure described above, starting with 6 (0.40 μmol) and 7 (0.40
μmol), yielded cyclic polyamide−fluorescein-conjugate 13 (345 nmol,
86% yield) and 14 (118 nmol, 29% yield), respectively.
(12): ESI-MS [M + H]+ calcd for C75H74N23O15S+ = 1568.6,
observed = 1568.3.
(13): ESI-MS [M + H]+ calcd for C74H73N24O15S+ = 1569.5,
observed = 1569.2.
(14): ESI-MS [M + H]+ calcd for C74H73N24O15S+ = 1569.5,
observed = 1569.3.
Cyclic and Hairpin Polyamides Targeted to 5′-WCGWGW-3′
Sequence (8 and 17). 2-Cl-Trt-Cl resin (200 mg, 1.59 mmol/g) was
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Article
EC50 − X) × H)), where A = max, B = min, and H = Hill slope. All
calculations were performed using Prism 4 (GraphPad) software. Stated
IC50 values represent the mean and standard deviation of three
independent biological replicates.
(16) Matsuda, H.; Fukuda, N.; Ueno, T.; Tahira, Y.; Ayame, H.; Zhang,
W.; Bando, T.; Sugiyama, H.; Saito, S.; Matsumoto, K.; Mugishima, H.;
Serie, H. J. Am. Soc. Nephrol. 2006, 17, 422−432.
(17) Raskatov, J. A.; Meier, J. L.; Puckett, J. W.; Yang, F.;
Ramakrishnan, P.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 2012,
109, 1023−1028.
ASSOCIATED CONTENT
■
(18) Cho, J.; Parks, M. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 10389−10392.
S
* Supporting Information
Experimental procedures and synthetic scheme for 17, HPLC
spectra for 1−8 and 17−26, detailed confocal microscopy data
for 12−14, and cytotoxicity data for compound S1. This material
is available free of charge via the Internet at http://pubs.acs.org.
(19) Herman, D. M.; Turner, J. M.; Baird, E. E.; Dervan, P. B. J. Am.
Chem. Soc. 1999, 121, 1121−1129.
(20) Melander, C.; Herman, D. M.; Dervan, P. B. Chem.Eur. J. 2000,
6, 4487−4497.
(21) Zhang, Q.; Dwyer, T. J.; Tsui, V.; Case, D. A.; Cho, J.; Dervan, P.
B.; Wemmer, D. E. J. Am. Chem. Soc. 2004, 126, 7958−7966.
(22) Chenoweth, D. M.; Harki, D. A.; Phillips, J. W.; Dose, C.; Dervan,
P. B. J. Am. Chem. Soc. 2009, 131, 7182−7188.
AUTHOR INFORMATION
■
Corresponding Author
*Address correspondence to dervan@caltech.edu.
(23) Morinaga, H.; Bando, T.; Takagaki, T.; Yamamoto, M.; Hashiya,
K.; Sugiyama, H. J. Am. Chem. Soc. 2011, 133, 18924−18930.
(24) Meier, J. L.; Montgomery, D. C.; Dervan, P. B. Nucleic Acids Res.
2012, 40, 2345−2356.
Notes
The authors declare no competing financial interest.
(25) Taleb, R. I.; Jaramillo, D.; Wheate, N. J.; Aldrich-Wright, J. R.
Chem.Eur. J. 2007, 13, 3177−3186.
ACKNOWLEDGMENTS
■
(26) van Holst, M.; Le Pevelen, D.; Aldrich-Wright, J. R. Eur. J. Inorg.
Chem. 2008, 29, 4608−4615.
This work was supported by the National Institutes of Health
(GM27681). B.C.L. is grateful to the California Tobacco-Related
Disease Research Program (18DT-0015) for a dissertation
award. D.C.M. is thankful to the National Institutes of Health for
a predoctoral research training grant (5T32GM007616). The
authors would like to thank Dr. Amanda E. Hargrove for helpful
discussions, and Dr. Jordan L. Meier for assistance with
molecular modeling.
(27) Puckett, J. W.; Green, J. T.; Dervan, P. B. Org. Lett. 2012, 14,
2774−2777.
(28) Weltzer, M.; Wemmer, D. E. Org. Lett. 2010, 12, 3488−3490.
(29) Brady, S. F.; Varga, S. L.; Freidinger, R. M.; Schwenk, D. A.;
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(31) Raskatov, J. A.; Hargrove, A. E.; So, A. Y.; Dervan, P. B. J. Am.
Chem. Soc. 2012, 134, 7995−7999.
DEDICATION
■
(32) Hargrove, A. E.; Raskatov, J. A.; Meier, J. L.; Montgomery, D. C.;
Dervan, P. B. J. Med. Chem. 2012, 55, 5425−5432.
(33) Synold, T. W.; Xi, B.; Wu, J.; Yen, Y.; Li, B. C.; Yang, F.; Phillips, J.
W.; Nickols, N. G.; Dervan, P. B. Cancer Chemother. Pharmacol. 2012,
70, 617−625.
Dedicated to Robert E. Ireland.
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