List of Figures

Teaching Chem 145 with her was one of the highlights of my time at Caltech. My interactions with Doug and his research group were among the highlights of my graduate school experience at Caltech.

Nomenclature and Symbology

Introduction to Molecular Recognition of DNA

Background and Signifi cance
Nucleic Acid Structure
Molecular Recognition of DNA
DNA Recognition by Minor-Groove Binders
Scope of this work

The monocationic natural product distamycin also binds AT regions of the DNA minor groove in a 1:1 (PDB: 2DND) and 2:1 (PDB: 378D) ligand:DNA stoichiometry. The minor groove edge of a G•C base pair contains a steric hydrogen bond that donates the "bump" in the form of the exocyclic amine of guanine.

Solution-Phase Synthesis of Pyrrole–Imidazole Polyamides

Introduction
Results and Discussion
Conclusion
Spectra and Supplemental Information

The resulting solid was washed with copious amounts of anhydrous Et 2 O and dried in vacuo to yield dimer 12 as a tan solid (3.3 g, 99%). The resulting solid was washed with copious amounts of anhydrous Et 2 O and dried in vacuo to yield trimer 4 as a brown-orange solid (3.45 g, 99%). The resulting solid was washed with copious amounts of anhydrous Et 2 O and dried in vacuo to yield tetramer 14 as a light brown solid (886 mg, 83%).

The resulting solid was washed with copious amounts of anhydrous Et 2 O and dried in vacuo to give tetramer 3 as a brown solid (728 mg, 93%). The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give dimer 5 as a white solid (432 mg, 99%). The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give ImPyPyPy-(R)β-CbzHNγ-Im-CO2Et 16 as a tan solid (2.2 g, 97%).

The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to yield ImPyPyPy-(R)β-CbzHNy-ImPyPyPy-CO2Me2 as a tan solid (2.09 g, 96%). The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to yield ImPyPyPy-(R)β-CbzHNy-ImPyPyPy-CO2H22 as a tan solid (442 mg, 89%). The protecting groups are cleaved at different rates, and early reaction samples reveal a mono-protected compound.] The reaction was then filtered through a Sep-Pak cartridge (5 g C-18 sorbent) and the Sep-Pak was washed. with DMF (4 ml), aqueous MeCN (50%, 20 ml), MeCN (250 ml) and MeOH (250 ml).

The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give ImPyPyPy-(R)β-H2Ny-ImPyPyPy-(+)-IPA1 as a light tan solid (18.9 mg, 81%) .

Cyclic Pyrrole–Imidazole Polyamides Targeted to the

Androgen Response Element

Introduction

Modulation of the expression of eukaryotic gene networks by small molecules is a challenge at the frontier of chemical biology. Pyrrole-imidazole polyamides are a class of cell-permeable small molecules that bind to the minor groove of DNA in a sequence-specific manner.1,2 Side-by-side stacked N-methylpyrrole (Py) and N-methylimidazole (Im )carboxamides (Im/Py pairs) distinguish G•C from C•G base pairs, while Py/Py pairs specify for both T•A and A•T.3 Py-Im hairpin polyamides are programmed for a broad repertoire of DNA sequences with similar affinities to endogenous transcription factors.4 They are cell permeable and affect gene transcription by disrupting protein-DNA interfaces. (AR),8 and AP-19 have been described in recent years, providing a new approach to gene control by small molecules. In parallel with our gene regulation studies, considerable effort has been devoted to maximizing the biological potency of hairpin Py-Im polyamides through structural modifications.

In particular, we have recently demonstrated that hairpin polyamides bearing the (R)-β-amino-γ turn, such as polyamide 4, have favorable binding to DNA and are useful in studies of gene regulation (Figure 3.1). 5g A Considerable effort exists in our laboratory to regulate aberrant AR-activated gene expression in prostate cancer.8 To further optimize lead oligomer 4 , it would seem reasonable to cap the hairpin with a linker identical, yielding a cyclic 1 structure, would further improve DNA affinity (Figure 3.1). Ball-and-stick representation legend: black and white circles represent N-methylimidazole and N-methylpyrrole units, respectively, the semicircle with the - sign represents the terminal isophthalic acid substituent and the white half-diamond with the + sign represents the triamine linker unit . In addition, we examined the DNA binding properties of these compounds by thermally melting duplex DNA and performed preliminary studies of their ADMET properties in vitro.

Cyclic Py-Im polyamides 1–3 have been shown to regulate endogenous gene expression in cell culture experiments.

Results and Discussion

With all atoms in place for the target cyclic polyamide 1, compound 9 was elaborated to

Conclusion

As shown in Table 3.1, polyamides 1-5 produced an increase in the duplex-DNA melting temperature relative to the individual DNA duplex, confirming polyamide-DNA binding. The resulting solid, which contained a small amount of residual H 2 O, was frozen and lyophilized to dryness and then suspended in excess anhydrous Et 2 O and filtered, and the filter cake washed with copious amounts of anhydrous Et 2 O. The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give BocHN-(R)β-CbzHNy-ImPyPyPy-CO2Me9 as a tan solid (518 mg, 96%).

The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give HCl•H2N-(R)β-CbzHNγ-ImPyPyPy-CO2Me 10 as a brown solid (114 mg, 99%). The resulting solid was washed with copious amounts of anhydrous Et2O and dried in vacuo to give BocHN-(R)β-CbzHNγ-ImPyPyPy-(R)β-CbzHNγ-ImPyPyPy-CO2Me 12 as a tan solid (44 mg, 94% ). . The resulting solid, containing a small amount of residual H2O, was frozen and lyophilized to dryness and then suspended in excess anhydrous Et2O, triturated and filtered, and the filter cake was washed with copious amounts of anhydrous Et2O.

Note: The solution becomes cloudy as the macrocyclization proceeds.] The reaction mixture was concentrated to a volume of 11 mL and added to a solution of H2O (30 mL) and aqueous HCl (1 N, 9.2 mL) at 0 ºC. The solid was suspended in excess anhydrous Et2O, triturated, filtered, and the filter cake was washed with large amounts of anhydrous Et2O.

Oligomerization Route to Polyamide Macrocycles

Introduction
Conclusion

Furthermore, preliminary studies of the in vitro ADMET properties of 1 revealed excellent metabolic stability.11,12. The oligomerization strategy for the synthesis of 1 and related polyamides is reported here. The pentafluorophenyl ester 4 was prepared in one step from the previously reported carboxylic acid of 4.11 Acidic deprotection of the γ-amino functionality of 4 followed by drying in vacuo affords intermediate 5 which is the substrate for homodimerization/. This result is apparently due to the inherent geometric constraints of 2 , preventing side-by-side antiparallel alignment of the PyPyPyIm strands, a motif well accommodated by the minor groove of DNA.

In summary, we have demonstrated that macrocyclic polyamides can be synthesized by Figure 4.3 (Top) Reversed phase HPLC analysis (2. hour time point) of the oligomerization reaction showing 1z, 2z, 3z and acyclic intermediates. In addition, we show that certain cyclic polyamide geometries are completely devoid of the ability to bind dsDNA, a result that can be used in the design of highly specific molecules for targeting non-B-form DNA structures or other higher-order nucleic acid motifs. The propagated error in ΔTm measurements is the square root of the sum of the square of the standard deviations for the Tm values.

The yield of 1z is calculated from the mass of the purified and isolated material (0.5 mg). The reported melting temperatures were defined as the maximum of the first derivative of the denaturation profile.

Allosteric Modulation of DNA by Small Molecules

Introduction
Conclusion
Notes and References
Spectra, Data Statistics, and Supplemental Information

We observe significant structural allosteric perturbations of the DNA helix induced by binding of GABA (γ-aminobutyric acid) spin-linked polyamides in the minor groove. In the DNA complex, the aromatic amino acids are bound with an N to C orientation of each ImImPyPy strand in the cycle adjacent to the 5' to 3' direction of the DNA. Binding of polyamide widens the minor groove up to 4 Å while compressing the major groove.

The polyamide bends the DNA strand >18° toward the major groove, as shown in Figure 5.3, and shortens the overall length of the helix by ~1 Å (Figure 5.2). It is possible that there is an intrinsic preference for conformation A, which alleviates the β-methylene interaction with the minor groove floor. The hydration pattern around the turn is highly conserved at both ends of the structure and the water-mediated hydrogen bonds are within ~2.7–2.9 Å from ammonium to water to the lone adenine pair (Figure 5.5a).

Structural basis for the twist preference for AT versus GC is demonstrated by the β-methylene conformational preference, which points to the DNA minor groove floor within van der Waals contact distance of the adenine base. Schematic diagram of native DNA crystal structure showing the sugar conformation at each position and the electron density map of the DNA asymmetric unit at the 1.0 σ level.

Structural Elucidation of a β-amino-γ-linked Cyclic

Polyamide-DNA Complex and RNA Binding Studies

Introduction
Overall Structure
Overall structure of DNA-polyamide complex
Turn conformation
Allosteric Perturbations
Solvation
Conclusion
Notes and References
Supplemental Information
Programmable Oligomers Targeting 5’-GGGG-3’ in the

We observe significant allosteric structural perturbations of the DNA helix induced upon binding of substituted GABA (γ-aminobutyric acid) twist-linked polyamides in the DNA minor groove. Geometry of the alpha-amino turn that interacts through water-mediated hydrogen bonds with the adenine and guanine base pairs in the floor of the DNA minor groove. Isolated view of one half of the polyamide (divided along a plane through the long axis of the polyamide and the DNA helical axis) showing hydrogen bond distances made to the DNA minor groove floor.

Hydrogen bonding interactions of the DNA-polyamide complex with electron density contoured at the 1.0 σ level. Comparison of minor groove width for DNA in the absence of polyamide (yellow curve and structure) and in the presence of bound polyamide (blue curve and structure). Comparison of the width of the major groove for DNA in the absence of polyamide (yellow curve and structure) and in the presence of bound polyamide (blue curve and structure).

Additionally, 6 waters hydrate the polyamide ammonium turns (3 at each turn) with 4 of the 6 anchoring the polyamide to the floor of the DNA minor groove by bridging hydrogen bonds. First, thiamine (T) is replaced by uracil (U) which presents the addition of a 5'-methyl group to the major groove of the helix.

Minor Groove of DNA and NF-κB Binding Inhibition

Introduction