ß-amino acids reduce ternary complex stability and alter the translation elongation mechanism.

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-ß2 isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by approximately one order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D-α-Phe. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include consideration of the efficiency and stability of ternary complex formation.

Mapping the influence of ligand electronics on the spectroscopic and 1O2 sensitization characteristics of Pd(II) biladiene complexes bearing phenyl-alkynyl groups at the 2- and 18-positions.

Photodynamic therapy (PDT) is a promising treatment for certain cancers that proceeds via sensitization of ground state 3O2 to generate reactive 1O2. Classic macrocyclic tetrapyrrole ligand scaffolds, such as porphyrins and phthalocyanines, have been studied in detail for their 1O2 photosensitization capabilities. Despite their compelling photophysics, these systems have been limited in PDT applications because of adverse biological side effects. Conversely, the development of non-traditional oligotetrapyrrole ligands metalated with palladium (Pd[DMBil1]) have established new candidates for PDT that display excellent biocompatibility. Herein, the synthesis, electrochemical, and photophysical characterization of a new family of 2,18-bis(phenylalkynyl)-substituted PdII 10,10-dimethyl-5,15-bis(pentafluorophenyl)-biladiene (Pd[DMBil2-R]) complexes is presented. These second generation biladienes feature extended conjugation relative to previously characterized PdII biladiene scaffolds (Pd[DMBil1]). We show that these new derivatives can be prepared in good yield and, that the electronic nature of the phenylalkynyl appendages dramatically influence the PdII biladiene photophysics. Extending the conjugation of the Pd[DMBil1] core through installation of phenylacetylene resulted in a ∼75 nm red-shift of the biladiene absorption spectrum into the phototherapeutic window (600-900 nm), while maintaining the PdII biladiene's steady-state spectroscopic 1O2 sensitization characteristics. Varying the electronics of the phenylalkyne groups via installation of electron donating or withdrawing groups dramatically influences the steady-state spectroscopic and photophysical properties of the resulting Pd[DMBil2-R] family of complexes. The most electron rich variants (Pd[DMBil2-N(CH3)2]) can absorb light as far red as ∼700 nm but suffer from significantly reduced ability to sensitize formation of 1O2. By contrast, Pd[DMBil2-R] derivatives bearing electron withdrawing functionalities (Pd[DMBil2-CN] and Pd[DMBil2-CF3]) display 1O2 quantum yields above 90%. The collection of results we report suggest that excited state charge transfer from more electron-rich phenyl-alkyne appendages to the electron deficient biladiene core circumvents triplet sensitization. The spectral and redox properties, as well as the triplet sensitization efficiency of each Pd[DMBil2-R] derivative is considered in relation to the Hammett value (σp) for each biladiene's R-group. More broadly, the results reported in this study clearly demonstrate that biladiene redox properties, spectral properties, and photophysics can be perturbed greatly by relatively minor alterations to biladiene structure.

Photoredox-Nickel Dual-Catalyzed C-Alkylation of Secondary Nitroalkanes: Access to Sterically Hindered α-Tertiary Amines.

The preparation of tertiary nitroalkanes via the nickel-catalyzed alkylation of secondary nitroalkanes using aliphatic iodides is reported. Previously, catalytic access to this important class of nitroalkanes via alkylation has not been possible due to the inability of catalysts to overcome the steric demands of the products. However, we have now found that the use of a nickel catalyst in combination with a photoredox catalyst and light leads to much more active alkylation catalysts. These can now access tertiary nitroalkanes. The conditions are scalable as well as air and moisture tolerant. Importantly, reduction of the tertiary nitroalkane products allows rapid access to α-tertiary amines.

Elucidation of complex triplet excited state dynamics in Pd(II) biladiene tetrapyrroles.

Pd(II) biladienes have been developed over the last five years as non-aromatic oligotetrapyrrole complexes that support a rich triplet photochemistry. In this work, we have undertaken the first detailed photophysical interrogation of three homologous Pd(II) biladienes bearing different combinations of methyl- and phenyl-substituents on the frameworks' sp3-hybridized meso-carbon (i.e., the 10-position of the biladiene framework). These experiments have revealed unexpected excited-state dynamics that are dependent on the wavelength of light used to excite the biladiene. More specifically, transient absorption spectroscopy revealed that higher-energy excitation (λexc ∼ 350-500 nm) led to an additional lifetime (i.e., an extra photophysical process) compared to experiments carried out following excitation into the lowest-energy excited states (λexc = 550 nm). Each Pd(II) biladiene complex displayed an intersystem crossing lifetime on the order of tens of ps and a triplet lifetime of ∼20 µs, regardless of the excitation wavelength. However, when higher-energy light is used to excite the complexes, a new lifetime on the order of hundreds of ps is observed. The origin of the 'extra' lifetime observed upon higher energy excitation was revealed using density functional theory (DFT) and time-dependent DFT (TDDFT). These efforts demonstrated that excitation into higher-energy metal-mixed-charge-transfer excited states with high spin-orbit coupling to higher energy metal-mixed-charge-transfer triplet states leads to the additional excitation deactivation pathway. The results of this work demonstrate that Pd(II) biladienes support a unique triplet photochemistry that may be exploited for development of new photochemical schemes and applications.

Electrochemically Mediated Oxidation of Sensitive Propargylic Benzylic Alcohols.

The electrochemical oxidation of sensitive propargylic benzylic alcohols having varying substituents is reported. We describe the preparation and characterization of N-hydroxytetrafluorophthalimide (TFNHPI) and pseudo-high-throughput development of a green electrochemical oxidation protocol for sensitive propargylic benzylic alcohols that employs TFNHPI as a stable electrochemical mediator. The electrochemical oxidation of propargylic benzylic alcohols was leveraged to develop short synthetic pathways for preparing gram quantities of resveratrol natural products such as pauciflorols.

Synthesis, Electrochemistry, and Photophysics of Pd(II) Biladiene Complexes Bearing Varied Substituents at the sp3-Hybridized 10-Position.

A set of Pd(II) biladiene complexes bearing different combinations of methyl- and phenyl-substituents on the sp3-hybridized meso-carbon (the 10-position of the biladiene framework) was prepared and studied. In addition to a previously described Pd(II) biladiene complex bearing geminal dimethyl substituents a the 10-position (Pd[DMBil]), homologous Pd(II) biladienes bearing geminal methyl and phenyl substituents (Pd[MPBil1]) and geminal diphenyl groups(Pd[DPBil1]) were prepared and structurally characterized. Detailed electrochemical as well as steady-state and time-resolved spectroscopic experiments were undertaken to evaluate the influence of the substituents on the biladiene's tetrahedral meso-carbon. Although all three biladiene homologues are isostructural, Pd[MPBil1] and Pd[DPBil1] display more intense absorption profiles that shift slightly toward lower energies as geminal methyl groups are replaced by phenyl rings. All three biladiene homologues support a triplet photochemistry, and replacement of the geminal dimethyl substituents of Pd[DMBil1] (ΦΔ = 54%) with phenyl groups improves the ability of Pd[MPBil1] (ΦΔ = 76%) and Pd[DPBil1] (ΦΔ = 66%) to sensitize 1O2. Analysis of the excited-state dynamics of the Pd(II) biladienes by transient absorption spectroscopy shows that each complex supports a long-lived triplet excited-state (i.e., τ > 15 µs for each homologue) but that the ISC quantum yields (ΦT) varied as a function of biladiene substitution. The observed trend in ISC efficiency matches that for singlet oxygen sensitization quantum yields (ΦΔ) across the biladiene series considered in this work. The results of this study provide new insights to guide future development of biladiene based agents for PDT and other photochemical applications.

Synthesis, Spectroscopic, and 1O2 Sensitization Characteristics of Extended Pd(II) 10,10-Dimethylbiladiene Complexes Bearing Alkynyl-Aryl Appendages.

Photodynamic therapy (PDT), which involves the photoinduced sensitization of singlet oxygen, is an attractive treatment for certain types of cancer. The development of new photochemotherapeutic agents remains an important area of research. Macrocyclic tetrapyrrole compounds including porphyrins, phthalocyanines, chlorins, and bacteriochlorins have been pursued as sensitizers of singlet oxygen for PDT applications but historically are difficult to prepare/purify and can also suffer from high nonspecific dark toxicity, poor solubility in biological media, and/or slow clearance from biological tissues. In response to these shortcomings, we have developed a series of novel linear tetrapyrrole architectures complexed to late transition metals as potential PDT agents. We find that these dimethylbiladiene (DMBil1) tetrapyrrole complexes can efficiently photosensitize generation of 1O2 oxygen upon irradiation with visible light. To extend the absorption profile of the DMBil1 platform, alkynyl-aryl groups have been conjugated to the periphery of the tetrapyrrole using Sonogashira methods. Derivatives of this type containing ancillary phenyl (DMBil-PE), naphthyl (DMBil-NE), and anthracenyl (DMBil-AE) groups have been prepared and characterized. In addition to structurally characterizing Pd[DMBil-NE] and Pd[DMBil-AE], we find that extension of the tetrapyrrole conjugation successfully red-shifts the absorption of the DMBil-Ar family of biladienes further into the phototherapeutic window (i.e., 600-900 nm). Photochemical sensitization studies demonstrate that our series of new palladium biladiene complexes (Pd[DMBil-Ar]) can sensitize the formation of 1O2 with quantum yields in the range ΦΔ = 0.59-0.73 upon irradiation with light of λ ≥ 650 nm. The improved absorption properties of the Pd[DMBil-Ar] complexes in the phototherapeutic window, together with their high 1O2 quantum yields, highlight the promise of these compounds as potential agents for PDT.

Synthesis, Redox, and Spectroscopic Properties of Pd(II) 10,10-Dimethylisocorrole Complexes Prepared via Bromination of Dimethylbiladiene Oligotetrapyrroles.

Two brominated 10,10-dimethylisocorrole (10-DMIC) derivatives containing Pd(II) centers have been prepared and characterized. These compounds were prepared via bromination of 10,10-dimethylbiladiene-based oligotetrapyrroles. Bromination of free base 10,10-dimethylbiladiene (DMBil1) followed by metalation with Pd(OAc)2, as well as bromination of the corresponding Pd(II) dimethylbiladiene complex (Pd[DMBil1]) provide routes to Pd(II) hexabromo-10,10-dimethyl-5,15-bis(pentafluorophenyl)-isocorrole (Pd[10-DMIC-Br6]) and Pd(II) octabromo-10,10-dimethyl-5,15-bis(pentafluorophenyl)-isocorrole (Pd[10-DMIC-Br8]). The solid-state structures of the two brominated isocorrole complexes are presented, as is that for a new decabrominated dimethylbiladiene derivative (DMBil-Br10). The electronic and spectroscopic properties of the brominated biladiene and isocorrole derivatives were probed using a combination of voltammetric methods and steady-state UV-vis absorption and emission experiments. Data obtained from these experiments allow the properties of the brominated biladiene and isocorrole derivatives to be compared to previously studied biladiene derivatives (i.e., DMBil1 and Pd[DMBil1]). CV and DPV experiments demonstrate that Pd[10-DMIC-Br6] and Pd[10-DMIC-Br8] support well-behaved multielectron redox chemistry, similar to that which has been observed for other nonaromatic tetrapyrroles containing sp3-hybridized meso-carbons. Spectroscopic experiments reveal that bromination of the dimethylbiladiene core shifts this system's UV-vis absorption profile to lower energy and that the dimethylisocorrole complexes support panchromatic absorption profiles that extend across the UV-vis and into the near-IR region. Photosensitization experiments demonstrate that unlike previously studied Pd(II) biladiene constructs, DMBil-Br10, Pd[10-DMIC-Br6], and Pd[10-DMIC-Br8] support limited triplet excited state chemistry with O2, indicating that the novel nonaromatic tetrapyrrole derivatives described in this work may be best suited for applications other than singlet oxygen sensitization.

The Distinct Conformational Landscapes of 4S-Substituted Prolines That Promote an endo Ring Pucker.

4-Substitution on proline directly impacts protein main chain conformational preferences. The structural effects of N-acyl substitution and of 4-substitution were examined by NMR spectroscopy and X-ray crystallography on minimal molecules with a proline 4S-nitrobenzoate. The effects of N-acyl substitution on conformation were attenuated in the 4S-nitrobenzoate context, due to the minimal role of the n→π* interaction in stabilizing extended conformations. By X-ray crystallography, an extended conformation was observed for most molecules. The formyl derivative adopted a δ conformation that is observed at the i+2 position of ß-turns. Computational analysis indicated that the structures observed crystallographically represent the inherent conformational preferences of 4S-substituted prolines with electron-withdrawing 4-position substituents. The divergent conformational preferences of 4R- and 4S-substituted prolines suggest their wider structure-specific application in molecular design. In particular, the proline endo ring pucker favored by 4S-substituted prolines uniquely promotes the δ conformation [(ϕ, ψ) ≈(-80°, 0°)] found in ß-turns. In contrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with a close n→π* interaction in the crystalline state, despite the endo ring pucker, suggesting its special capabilities in promoting compact conformations in ϕ due to its strongly electron-donating character.

pH-Driven Mechanistic Switching from Electron Transfer to Energy Transfer between [Ru(bpy)3]2+ and Ferrocene Derivatives.

The metal-to-ligand charge transfer excited states of [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) may be deactivated via energy transfer or electron transfer with ferrocene derivatives in aqueous conditions. Stern-Volmer quenching analysis revealed that the rate constant for [Ru(bpy)3]2+ excited-state quenching depends on solution pH when a ferrocenyl-amidinium derivative (Fc-am) containing a proton-responsive functionality tethered to the ferrocene center was present. By contrast, the rate constant with which the [Ru(bpy)3]2+ excited state is quenched by an analogous ferrocene derivative (ferrocenyl-trimethylammonium, Fc-mam) that lacks a protonic group does not depend on pH. These results show that the presence (or absence) of a readily transferrable proton modulates quenching rate constants in bimolecular events involving [Ru(bpy)3]2+ and ferrocene. More surprisingly, transient absorption spectroscopy reveals that the mechanism by which the [Ru(bpy)3]2+ excited state is quenched by Fc-am appears to be modulated by solution proton availability, switching from energy transfer at low pH when Fc-am is protonated, to electron transfer at high pH when Fc-am is deprotonated. The mechanistic switching that is observed for this system cannot be aptly explained using a simple driving force dependence argument, suggesting that more subtle factors dictate the pathway by which the [Ru(bpy)3]2+ excited state is deactivated by ferrocene in aqueous solutions.