Inhibition of vibrational energy flow within an aromatic scaffold via heavy atom effect.

The regulation of intramolecular vibrational energy redistribution (IVR) to influence energy flow within molecular scaffolds provides a way to steer fundamental processes of chemistry, such as chemical reactivity in proteins and design of molecular diodes. Using two-dimensional infrared (2D IR) spectroscopy, changes in the intensity of vibrational cross-peaks are often used to evaluate different energy transfer pathways present in small molecules. Previous 2D IR studies of para-azidobenzonitrile (PAB) demonstrated that several possible energy pathways from the N3 to the cyano-vibrational reporters were modulated by Fermi resonance, followed by energy relaxation into the solvent [Schmitz et al., J. Phys. Chem. A 123, 10571 (2019)]. In this work, the mechanisms of IVR were hindered via the introduction of a heavy atom, selenium, into the molecular scaffold. This effectively eliminated the energy transfer pathway and resulted in the dissipation of the energy into the bath and direct dipole-dipole coupling between the two vibrational reporters. Several structural variations of the aforementioned molecular scaffold were employed to assess how each interrupted the energy transfer pathways, and the evolution of 2D IR cross-peaks was measured to assess the changes in the energy flow. By eliminating the energy transfer pathways through isolation of specific vibrational transitions, through-space vibrational coupling between an azido (N3) and a selenocyanato (SeCN) probe is facilitated and observed for the first time. Thus, the rectification of this molecular circuitry is accomplished through the inhibition of energy flow using heavy atoms to suppress the anharmonic coupling and, instead, favor a vibrational coupling pathway.

Unraveling Complex Local Protein Environments with 4-Cyano-l-phenylalanine.

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography. It was then genetically incorporated individually at three unique sites (74, 133, and 149) in sfGFP to probe these local protein environments. The incorporation of the UAA site-specifically in sfGFP utilized an engineered, orthogonal tRNA synthetase in E. coli using the Amber codon suppression protocol, and the resulting UAA-containing sfGFP constructs were then explored with this approach. This methodology was effectively utilized to further probe the local environments of two surface sites (sites 133 and 149) that we previously explored with room temperature IR spectroscopy and X-ray crystallography and a new interior site (site 74) featuring a complex local environment around the nitrile group of pCNPhe. Site 133 was found to be solvent-exposed, while site 149 was partially buried. Site 74 was found to consist of three distinct local environments around the nitrile group including nonspecific van der Waals interactions, hydrogen-bonding to a structural water, and hydrogen-bonding to a histidine side chain.

Design and synthesis of a bis-macrocyclic host and guests as building blocks for small molecular knots.

The thread-link-cut (TLC) approach has previously shown promise as a novel method to synthesize molecular knots. The modular second-generation approach to small trefoil knots described herein involves electrostatic interactions between an electron-rich bis-macrocyclic host compound and electron-deficient guests in the threading step. The bis-macrocyclic host was synthesized in eight steps and 6.6% overall yield. Ammonium and pyridinium guests were synthesized in 4-5 steps. The TLC knot-forming sequence was carried out and produced a product with the expected molecular weight, but, unfortunately, further characterization did not produce conclusive results regarding the topology of the product.

Extending the vibrational lifetime of azides with heavy atoms.

The development of novel vibrational reporters (VRs), aka infrared (IR) probes, to study local environments and dynamic processes in biomolecules and materials continues to be an important area of research. Azides are important VRs because of their small size and large transition dipole strengths, however, their relatively short vibrational lifetimes (<2 ps) have limited their full potential. Herein we report that the vibrational lifetimes of azides can be increased by attaching them to heavy atoms and by using heavy 15N isotopes. Three group 14 atom triphenyl azides (Ph3CN3, Ph3SiN3, Ph3SnN3), and their triple-15N isotopomers, were synthesized in good yields. Tributyltin azide and its heavy isotopomer (Bu3Sn15N3) were also prepared to probe the effect of molecular scaffolding. The extinction coefficients for the natural abundance azides were determined, ranging from 900 to 1500 M-1 cm-1. The vibrational lifetimes of all azides were measured by pump-probe IR spectroscopy and each showed a major component with a short-to-moderate vibrational lifetime and a minor component with a much longer vibrational lifetime. Based on these results, the lifetime, aka the observation window, of an azide reporter can be extended from ∼2 ps to as long as ∼300 ps by a combination of isotopic labeling and heavy atom effect. 2D IR measurements of these compounds further confirmed the ability to observe these azide transitions at much longer timescales showing their utility to capture dynamic processes from tens to hundreds of picoseconds.

2D-IR studies of cyanamides (NCN) as spectroscopic reporters of dynamics in biomolecules: Uncovering the origin of mysterious peaks.

Cyanamides (NCN) have been shown to have a larger transition dipole strength than cyano-probes. In addition, they have similar structural characteristics and vibrational lifetimes to the azido-group, suggesting their utility as infrared (IR) spectroscopic reporters for structural dynamics in biomolecules. To access the efficacy of NCN as an IR probe to capture the changes in the local environment, several model systems were evaluated via 2D IR spectroscopy. Previous work by Cho [G. Lee, D. Kossowska, J. Lim, S. Kim, H. Han, K. Kwak, and M. Cho, J. Phys. Chem. B 122(14), 4035-4044 (2018)] showed that phenylalanine analogues containing NCN show strong anharmonic coupling that can complicate the interpretation of structural dynamics. However, when NCN is embedded in 5-membered ring scaffolds, as in N-cyanomaleimide and N-cyanosuccinimide, a unique band structure is observed in the 2D IR spectrum that is not predicted by simple anharmonic frequency calculations. Further investigation indicated that electron delocalization plays a role in the origins of the band structure. In particular, the origin of the lower frequency transitions is likely a result of direct interaction with the solvent.

Tuning Molecular Vibrational Energy Flow within an Aromatic Scaffold via Anharmonic Coupling.

From guiding chemical reactivity in synthesis or protein folding to the design of energy diodes, intramolecular vibrational energy redistribution harnesses the power to influence the underlying fundamental principles of chemistry. To evaluate the ability to steer these processes, the mechanism and time scales of intramolecular vibrational energy redistribution through aromatic molecular scaffolds have been assessed by utilizing two-dimensional infrared (2D IR) spectroscopy. 2D IR cross peaks reveal energy relaxation through an aromatic scaffold from the azido- to the cyano-vibrational reporters in para-azidobenzonitrile (PAB) and para-(azidomethyl)benzonitrile (PAMB) prior to energy relaxation into the solvent. The rates of energy transfer are modulated by Fermi resonances, which are apparent by the coupling cross peaks identified within the 2D IR spectrum. Theoretical vibrational mode analysis allowed the determination of the origins of the energy flow, the transfer pathway, and a direct comparison of the associated transfer rates, which were in good agreement with the experimental results. Large variations in energy-transfer rates, approximately 1.9 ps for PAB and 23 ps for PAMB, illustrate the importance of strong anharmonic coupling, i.e., Fermi resonance, on the transfer pathways. In particular, vibrational energy rectification is altered by Fermi resonances of the cyano- and azido-modes allowing control of the propensity for energy flow.

Paired Spectroscopic and Crystallographic Studies of Proteases.

The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X-ray crystallographic studies. The active site serines of the proteases were reacted with 4-cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water-soluble inhibitor model, potassium 4-cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H2O/DMSO and the frequency-temperature line-slope (FTLS) method in H2O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin-CBS and trypsin-CBS in aqueous buffer were both measured to be -3.5×10-2 cm-1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active-site nitriles in both proteases are mostly solvated. The X-ray crystal structures of the subtilisin-CBS and trypsin-CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin-CBS and in one conformation in the trypsin-CBS. The crystallographic data support the FTLS data that the active-site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X-ray crystallography provides a powerful approach to studying protein structure.

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.

Synthesis and Evaluation of the Sensitivity and Vibrational Lifetimes of Thiocyanate and Selenocyanate Infrared Reporters.

Two novel 2'-deoxyadenosine (dA) analogues, Si2-dA-SCN and Si2-dA-SeCN, and two novel phenylalanine (Phe) analogues, Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN, have been synthesized and the thiocyanate (SCN) and selenocyanate (SeCN) functional groups evaluated as vibrational reporters. The syntheses of Si2-dA-SCN and Si2-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, and the syntheses of Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively. The SCN and SeCN stretch vibrational modes were shown to be sensitive to the local environment by frequency shifts and full-width half-maximum (fwhm) changes in response to tetrahydrofuran (THF) and THF/water solvent mixtures. The vibrational lifetimes of the Si2-dA-SeCN (237±12 ps) and Boc-Me-PheCH2SeCN (295±31 ps) in THF solution were determined by ultrafast infrared pump-probe spectroscopy to be 1.5 to 3 times longer than those for Si2-dA-SCN (140±6 ps) and Boc-Me-PheCH2SCN (102±4 ps). The longer lifetimes for the SeCN analogues were attributed to the better insulating effects of the heavier selenium atom compared to the sulfur atom. The solvent sensitivity and longer vibrational lifetimes compared to other vibrational reporters suggest that SCN and SeCN vibrational reporters are well suited to studying several dynamic processes including protein and nucleic acid hydration and conformational changes, however stability issues may require post-synthetic modification methods to incorporate these reporters into biomacromolecules.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.

An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.

Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.

Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

Profiles in chemistry: a historical perspective on the national organic symposium.

This perspective delineates the history of the National Organic Chemistry Symposium (NOS) and, in doing so, traces the development of organic chemistry over the past 88 years. The NOS is the premier event sponsored by the ACS Division of Organic Chemistry (ORGN) and has been held in odd-numbered years since 1925, with the exceptions of 1943 and 1945. During the 42 symposia, 332 chemists have given 549 plenary lectures. The role the NOS played in the launch of The Journal of Organic Chemistry and Organic Reactions and the initiation of the Roger Adams Award are discussed. Representative examples highlighting the chemistry presented in each era are described, and the evolution of the field is examined by assigning each NOS talk to one of seven subdisciplines and analyzing how the number of talks in each subdiscipline has changed over time. Comparisons of the demographics of speakers, attendees, and ORGN members are made, and superlatives are noted. Personal interest stories of the speakers are discussed, along with the relationships among them, especially their academic lineage. Logistical aspects of the NOS and their historical trends are reviewed. Finally, the human side of science is examined, where over the past century, the NOS has been intertwined with some of the most heated debates in organic chemistry. Conflicts and controversies involving free radicals, reaction mechanisms, and nonclassical carbocations are discussed.

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 -N(3) asymmetric stretch vibration with 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 while others remained complex. The origins of the observed profiles are discussed. 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.

A direct comparison of azide and nitrile vibrational probes.

The synthesis of 2'-azido-5-cyano-2'-deoxyuridine, N(3)CNdU (1), from trityl-protected 2'-amino-2'-deoxyuridine was accomplished in four steps with a 12.5% overall yield. The IR absorption positions and profiles of the azide and nitrile group of N(3)CNdU were investigated in 14 different solvents and water/DMSO solvent mixtures. The azide probe was superior to the nitrile probe in terms of its extinction coefficient, which is 2-4 times larger. However, the nitrile IR absorbance profile is generally less complicated by accidental Fermi resonance. The IR frequencies of both probes undergo a substantial red shift upon going from water to aprotic solvents such as THF or DMSO. DFT calculations supported the hypothesis that the molecular origin of the higher observed frequency in water is primarily due to hydrogen bonds between the probes and water molecules.

2D IR photon echo of azido-probes for biomolecular dynamics.

The vibrations in the azido-, N(3), asymmetric stretching region of 2'-azido-2'-deoxyuridine (N(3)dU) are examined by two-dimensional infrared spectroscopy. In water and tetrahydrofuran (THF), the spectra display a single sharp diagonal peak that shows solvent sensitivity. The frequency-frequency correlation time in water is 1.5 ps, consistent with H-bond making and breaking dynamics. The 2D IR spectrum is reproduced for N(3)dU in water based on a model correlation function and known linear response functions. Its large extinction coefficient, vibrational frequency outside the protein and nucleic acid IR absorption, and sensitivity to water dynamics render -N(3) a very useful probe for 2D IR and other nonlinear IR studies: its signal is ca. 100 times that of nitriles.

(15)N NMR studies of a nitrile-modified nucleoside.

Nitrile-modified molecules have proven to be excellent probes of local environments in biomolecules via both vibrational and fluorescence spectroscopy. The utility of the nitrile group as a spectroscopic probe has been expanded here to (15)N NMR spectroscopy by selective (15)N incorporation. The (15)N NMR chemical shift (δ((15)N)) of the (15)N-labeled 5-cyano-2'-deoxyuridine (C(15)NdU, 1a) was found to change from 153.47 to 143.80 ppm in going from THF-d(8) to D(2)O. A 0.81 ppm downfield shift was measured upon formation of a hydrogen-bond-mediated heterodimer between 2,6-diheptanamidopyridine and a silyl ether analogue of 1a in chloroform, and the small intrinsic temperature dependence of δ((15)N) of C(15)NdU was measured as a 0.38 ppm downfield shift from 298 to 338 K. The experiments were complemented with density functional theory calculations exploring the effect of solvation on the (15)N NMR chemical shift.

A sensitive multispectroscopic probe for nucleic acids.

Azides have recently been used as vibrational probes of proteins, but their incorporation into nucleic acids has been limited to photo-cross-linking or click chemistry applications. The utility of 2'-azido-2'-deoxyuridine (N(3)-dU, 1) as an IR and (15)N NMR spectroscopic probe of the sugar phosphate backbone region of nucleic acids was investigated by measuring the effects of solvent, heterodimer formation, and temperature on peak frequencies and IR bandwidth. The azide IR asymmetric stretching band (nu(N(3))) of N(3)-dU was sensitive to its environment, undergoing a blue shift of 13.5 cm(-1) when changing the solvent from THF to water. The solvent effects on (15)N chemical shifts (delta((15)N)) of each of the nitrogen atoms in the azido group was studied, and the terminal nitrogen atom was the most sensitive to solvent, shifting downfield by 3.8 ppm when changing the solvent from THF-d(8) to D(2)O. Formation of a base-pair-like heterodimer between 3 (a silyl ether analogue of 1) and 2,6-diheptanamidopyridine (4) in chloroform resulted in minimal changes in the IR and (15)N NMR spectral frequency and chemical shift, respectively, as expected given the location of the azido moiety. The intrinsic temperature dependence of nu(N(3)) and delta((15)N) were found to be minimal over the temperature range studied especially compared to the solvent dependence of these spectral observables. The analysis of the experimental studies was complemented by density functional theory (DFT) calculations on model systems.

A vibrational probe for local nucleic acid environments: 5-cyano-2'-deoxyuridine.

Nitriles have been shown to be effective vibrational probes of local environments in proteins but have yet to be fully utilized for the study of nucleic acids. The potential utility of 5-cyano-2'-deoxyuridine ( 1) as a probe of local nucleic acid environment was investigated by measuring the dependence of the IR nitrile stretching frequency (nu CN), line shape, and absorbance on solvent and temperature. The nu CN was found to be sensitive to solvent with an observed blue shift of 9.2 cm (-1) in going from THF to water. The dependence of the nitrile IR absorbance band was further investigated in water-THF mixtures. Global line shape analysis, difference FTIR spectroscopy, and singular value decomposition (SVD) were used to show the presence of three distinct local environments around the nitrile group of 1 in these mixtures. A modest blue shift in nu CN was observed upon a hydrogen-bond-mediated heterodimer formation between 2 (a silyl ether analogue of 1) and 2,6-diheptanamido-pyridine ( 3a) in chloroform. The intrinsic temperature dependence of the nu CN was found to be minimal and linear over the temperature range studied. The experimental studies were complemented by density functional theory (DFT) calculations on the dependence of the nitrile stretching frequency on solute-solvent interactions and upon heterodimer formation with model systems.