Modulating Accidental Fermi Resonance: What a Difference a Neutron Makes

Vibrational reporters have shown significant promise as sensitive probes of local environments in proteins and nucleic acids. The utility of two potential vibrational probes, the cyanate and azide groups in phenyl cyanate and 3-azidopyridine, respectively, has been hindered by accidental Fermi resonance. Anharmonic coupling, between the fundamental −OCN or −N₃ asymmetric stretch vibration and a near resonant combination band, results in an extremely broad and complex absorption profile for each of these probes. A total of eight phenyl cyanate and six 3-azidopyridine isotopomers were synthesized and studied. Isotopic editing effectively modulated the accidental Fermi resonance; the absorption profiles of several isotopomers were greatly simplified, whereas others remained complex. The origins of the observed profiles are discussed. The addition of a single neutron to the middle atom of the oscillator converted the absorption profile to essentially a single band, resulting from either the cyanate or azide asymmetric stretch vibration

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4‑Azidomethyl‑l‑Phenylalanine

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-l-phenylalanine (pN₃CH₂Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN₃CH₂Phe are compared to the vibrational reporters: 4-cyano-l-phenylalanine (pCNPhe) and 4-azido-l-phenylalanine (pN₃Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN₃CH₂Phe to different protein environments to be measured. The photostability of pN₃CH₂Phe was also measured relative to the photoreactive UAA, pN₃Phe

Expanding the Utility of 4‑Cyano‑l‑Phenylalanine As a Vibrational Reporter of Protein Environments

The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-l-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers (¹⁵N, ¹³C, ¹³C¹⁵N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-l-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-l-phenylalanine

Kinetic Isotope Effect Provides Insight into the Vibrational Relaxation Mechanism of Aromatic Molecules: Application to Cyano-phenylalanine

Varying the reduced mass of an oscillator via isotopic substitution provides a convenient means to alter its vibrational frequency and hence has found wide applications. Herein, we show that this method can also help delineate the vibrational relaxation mechanism, using four isotopomers of the unnatural amino acid <i>p</i>-cyano-phenylalanine (Phe-CN) as models. In water, the nitrile stretching frequencies of these isotopomers, Phe-¹²C¹⁴N (<b>1</b>), Phe-¹²C¹⁵N (<b>2</b>), Phe-¹³C¹⁴N (<b>3</b>), and Phe-¹³C¹⁵N (<b>4</b>), are found to be equally separated by ∼27 cm^–1, whereas their vibrational lifetimes are determined to be 4.0 ± 0.2 (<b>1</b>), 2.2 ± 0.1 (<b>2</b>), 3.4 ± 0.2 (<b>3</b>), and 7.9 ± 0.5 ps (<b>4</b>), respectively. We find that an empirical relationship that considers the effective reduced mass of CN can accurately account for the observed frequency gaps, while the vibrational lifetime distribution, which suggests an intramolecular relaxation mechanism, can be rationalized by the order-specific density of states near the CN stretching frequency

Two-Dimensional Infrared Study of Vibrational Coupling between Azide and Nitrile Reporters in a RNA Nucleoside

The vibrations in the azide, N₃, asymmetric stretching region and nitrile, CN, symmetric stretching region of 2′-azido-5-cyano-2′-deoxyuridine (N₃CNdU) are examined by two-dimensional infrared (2D IR) spectroscopy. At earlier waiting times, the 2D IR spectrum shows the presence of both vibrational transitions along the diagonal and off-diagonal cross peaks indicating vibrational coupling. The coupling strength is determined from the off-diagonal anharmonicity to be 66 cm^–1 for the intramolecular distance of ∼7.9 Å, based on a structural map generated for this model system. In addition, the frequency–frequency correlation decay is detected, monitoring the solvent dynamics around each individual probe position. Overall, these vibrational reporters can be utilized in tandem to simultaneously track global structural information and fast structural fluctuations