Recognition of the True and False Resonance Raman Optical Activity

Abstract Resonance Raman optical activity (RROA) possesses all aspects of a sensitive tool for molecular detection, but its measurement remains challenging. We demonstrate that reliable recording of RROA of chiral colorful compounds is possible, but only after considering the effect of the electronic circular dichroism (ECD) on the ROA spectra induced by the dissolved chiral compound. We show RROA for a number of model vitamin B12 derivatives that are chemically similar but exhibit distinctively different spectroscopic behavior. The ECD/ROA effect is proportional to the concentration and dependent on the optical pathlength of the light propagating through the sample. It can severely alter relative band intensities and signs in the natural RROA spectra. The spectra analyses are supported by computational modeling based on density functional theory. Neglecting the ECD effect during ROA measurement can lead to misinterpretation of the recorded spectra and erroneous conclusions about the molecular structure.


MD simulations
(CN)13-epi-Cbl(e-lactone) and (CN)Cbl initial geometries have been built in Avogadro software. [1] The molecular dynamics (MD) conformational search tool implemented in Gabedit software [2] was recruited to generate 10 lowestenergy conformers of the studied molecular systems. Amber99 force field [3] was employed. Simulated annealing procedure (T=1000 K) included heating, equilibration and production runs which lasted 1, 1, and 10 ps, respectively. The time step was 1 fs. Velocity Verlet algorithm was employed in trajectory calculations. No constraints were used. At the end of the molecular dynamics calculations the selected lowest energy conformers were optimized using quasi Newton Raphson procedure (10 000 steps). The optimization convergence criteria are the default ones as defined in Gabedit software.

Quantum chemical calculations
All quantum chemical calculations were performed by of Gaussian G16.C01 software, [4] and analyzed by means of GaussView 6, [5] VMD 1.9.4, [6] GaussSum 3 software, [7] and a set of home-made programs and scripts. A set of conformers of (CN)13-epi-Cbl(e-lactone) and (CN)Cbl obtained from the MD simulations was optimized at the CAM-B3LYP/GD3/6-31G(d) theory level. The MDF10 pseudopotential and basis set were used for the Co atom. The solvent (water) was modeled using the CPCM model. [8] Electronic absorption energies and intensities (oscillator and rotatory strengths) were calculated using TD-DFT, for the first 100 electronic states, at the same level of theory as for the geometries. Similarly, vibrational frequencies and pre-resonance Raman and ROA intensities were calculated employing the same level for the lowest-energy conformers of (CN)13-epi-Cbl(e-lactone) and (CN)Cbl. The excitation wavelength used in the polarizability calculation was close to the first electronic transitions, to mimic the experimental conditions. In particular, although the incident laser wavelength is 532 nm, theoretical electronic transition energies are blue-shifted compared to the experiment, and the excitation wavelength was adapted accordingly, the value of 430 nm provided the best results.

(CN)13-epi-Cbl(e-lactone)
and Δε values of the solute, and theoretical frequencies, DOC factors and Raman intensities (solute or solvent), 0.2 cm pathlength L, 0.1 cm pathlength L', and experimental concentrations. The frequencies, and Raman intensities (430 nm excitation) of the solute were calculated as described above.

Electronic Absorption (UV-Vis) and Electronic Circular Dichroism (ECD) measurements
UV-Vis and ECD spectra of vitamin B12 and its derivatives were recorded in the 230-650 nm spectral range at room temperature in distilled water or DMSO. The solutions in various concentrations were measured in the 10, 2 and 1 mm quartz cells. All spectra were recorded in a single scan on Jasco J−1500 spectropolarimeter with 100 nm min −1 scanning speed, step size of 0.2 nm, 1 nm bandwidth, and a response time of 1 s. They were background-corrected using solvents recorded under the same conditions. UV-Vis/ECD spectra of studied samples were also measured after Raman/ROA experiments to control sample stability. Because of the limited amount of solution (~140 μL), in this case they were registered in quartz optical cell with a path length of 1 mm and accumulation of 3-5 scans (Figure 1, S11 and S13).

Raman and Raman Optical Activity (ROA) measurements
Raman and ROA spectra of vitamin B12 and its analogs dissolved in distilled water as well as DMSO were obtained on a ChiralRAMAN-2X TM spectrometer (BioTools Inc.) at 7 cm -1 resolution within 250-2500 cm -1 employing the excitation wavelength of 532 nm. Before starting the measurements, all solutions were passed through the Millex®  Table S5.
We emphasis that Raman/ROA spectra were registered with long (all samples listed in Table S5) and short path lengths. The measurements using the short path length included aqueous solutions of (CN)13-epi-Cbl(elactone) at 0.1 and 0.8 mg/mL concentrations (Figure 5 and S22). To prevent degradation, for most of the samples, many Raman/ROA measurements of freshly prepared solutions were averaged (Table S5). Minor baseline corrections of both Raman and ROA was also applied (Figure 3, 6 and S24, S25). Raw spectra are provided below (Figures S13-S22). At least three independent experiments for each sample were conducted, but the obtained ROA spectra were quite reproducible ( Figure S24).

Organic synthesis
The procedure for the synthesis of (CN)13-epi-Cbl was repeated with the following changes: (CN)Cbl (0.221 mmol, 300 mg) was suspended in trifluoroacetic acid (5.8 mL) and the mixture was stirred at room temperature for 2 h. Subsequently the acid was removed under reduced pressure and the residue was dried in vacuo for 20 min. The crude product was suspended in diethyl ether, mixed thoroughly, filtered and washed with another portion of diethyl ether. The red solid was redissolved in water and purified via reversed-phase column chromatography using LiChroprep RP-18 (40-63 mm) silica with redistilled water and HPLC-grade CH3CN as eluents (gradually form 5% to 15% CH3CN/H2O, v/v). The first intense red band was collected and concentrated in vacuo. The pure (CN)13-epi-Cbl was isolated as a red solid (36 mg, 12% yield). 1