Synthesis of β-Pyridyl α-Amino Acids: Conformationally Sensitive Charge Transfer-Based Fluorophores

Unnatural α-amino acids with charge transfer-based poly aromatic side chains have been designed as conformationally sensitive fluorophores. These were prepared using a hetero-Diels–Alder reaction and a Knoevenagel–Stobbe process to generate a biaryl pyridyl unit, followed by iron-catalyzed bromination and a Suzuki–Miyaura cross-coupling reaction to complete the triaryl system. A photophysical study led to the discovery of a p-methoxy analogue which exhibited viscosity-sensitive fluorescence in which emission could be controlled between twisted and planar conformations.


General Experimental
All reagents and starting materials were obtained from commercial sources and used as received.
Dimethyl (2S)-2-(tritylamino)butandioate (6) was prepared as previously described. 1Dry solvents were purified using a PureSolv 500 MD solvent purification system.All reactions were performed open to air unless otherwise mentioned.All reactions performed at elevated temperatures were heated using an oil bath.Brine refers to a saturated aqueous solution of sodium chloride.Flash column chromatography was carried out using Merck Millipore matrix silicagel 60 (40-63 µM).Merck aluminium-backed plates pre-coated with silica gel 60 (UV254) were used for thin-layer chromatography and visualized with a UV lamp. 1 H NMR and 13 C NMR spectra were recorded on a Bruker DPX 400 or 500 spectrometer, with chemical shift values in ppm relative to tetramethylsilane (δH 0.00 and δC 0.00), or for 1 H NMR, relative to residual chloroform (δH 7.26) or methanol (δH 3.31) as standard.For 13 C NMR the chemical shifts are reported relative to the central resonance of CDCl3 (δC 77.2) or CD3OD (δC 49.0) as standard.Carbon assignments are based on two-dimensional HMBC and DEPT experiments.Mass spectra were obtained either using a JEOL JMS-700 spectrometer for EI and CI, and Bruker Microtof-q or Agilent 6125B for ESI.Infrared spectra were obtained neat using a Shimadzu IR Prestige-21 spectrometer or Shimadzu 8400S spectrometer; wavenumbers are indicated in cm −1 .Melting points were determined on either a Reichert platform melting point apparatus or Stuart Scientific melting point apparatus.Optical rotations were determined as solutions irradiating with the sodium D line (λ = 589 nm) using an Autopol V polarimeter.[α]D values are given in units 10 −1 deg cm 2 g −1 .Absorption and emission data were recorded on one of two instruments: 1. UV-Vis spectra were recorded on a Pekin Elmer Lamda 25 instrument.Fluorescence spectra were recorded on a Shimadzu RF-5301PC spectrofluorophotometer. Emission data were measured using excitation and emission bandpass filters of 3 nm.
2. Both UV-Vis spectra and fluorescence spectra were recorded on a Horiba Duetta Fluorescence and Absorbance spectrometer.Absorbance spectra were recorded with an integration time of 0.05 s, and a band pass of 5 nm.Fluorescence spectra were recorded with excitation and emission band pass of 5 nm, an integration time of 0.1 s, and with detector accumulations set to 1. Respective standard samples were recorded with the same parameters.Quantum yields were determined using L-tryptophan (Φ = 0.14 in water) as the standard reference. 2e integrated fluorescence intensity of each compound was determined from the emission spectra given.Measurements were performed at five different concentrations.Concentrations were chosen to ensure the absorption value was below 0.1 to avoid re-absorption effects.Integrated fluorescence intensity was plotted as a function of the measured absorbance and a linear fit was calculated.The resultant gradient was then used to calculate the quantum yield, using the equation below: Subscript ST signifies the quantities associated with the quantum yield standard.Subscript X signifies the quantities associated with the novel compound.GradX is the determined gradient associated with the novel compound.GradST is the determined gradient associated with quantum yield standard.η is the refractive index of the solvent used in the fluorescence measurements.η = 1.333 for water, 1.361 for ethanol and 1.331 for methanol.

Experimental Procedures and Spectroscopic Data for all Compounds
Methyl (2S)-

Computational Details
All DFT calculations were performed in Gaussian 16 6 with the ωB97X-D functional 7 and the def2-TZVP basis set. 8The methanol solvent was included using the default PCM solvent model.The structure of amino acid 5a was fully optimized (no imaginary frequencies), and regular population analysis was performed.Visualizations of the HOMO and LUMO were generated using Avogadro software. 9timized structure and total energy of 5a:

5.
Photophysical Data for a-Amino Acids Spectra were recorded at 5 M in MeOH using an excitation and emission bandpass of 3 or 5 nm.
Absorption and Emission Spectra for 5a.