Frequency Changes in Terminal Alkynes Provide Strong, Sensitive, and Solvatochromic Raman Probes of Biochemical Environments

Abstract

The C�C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C�C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C�C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor−acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed p-ethynylphenylalanine into the interior of the Escherichia coli fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C�C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

“…Further, when we calculated the peak wavenumber and FWHMs with Lorentzian fitting, we observed red-shifting of the peak wavenumber in 90% DMSO. This can be explained in terms of the solvent effect, considering that Raman tags such as nitriles and alkynes are sensitive to the local environment (vibrational solvatochromism). − Also, the peak broadening of 9CN-DEP at low DMSO concentration is consistent with the reported tendency that the nitrile peak width is broader in water than in organic solvent . On the other hand, the peak width of 9CN-DER tended to be narrower due to aggregate formation at low DMSO concentration (Figure S3 and Table S1).…”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…While such strongly absorbing alkynes with long vibrational lifetimes are useful IR probes for nonlinear vibrational spectroscopy, they have not been widely used in biological studies. 244…”

“…In addition, Cho and co-workers , showed that addition of a heavy atom, such as silicon (Si) or selenium (Se), between the probe alkyne group and the parent molecule can significantly enhance the transition dipole moment and the vibrational lifetime of the corresponding CC stretching vibration. While such strongly absorbing alkynes with long vibrational lifetimes are useful IR probes for nonlinear vibrational spectroscopy, they have not been widely used in biological studies …”

Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences

“…(2) Wei and co-workers 137 demonstrated a hydrogen− deuterium exchange (HDX) strategy to sense local chemical and environment changes in cells (e.g., acidity) using terminal alkyne-tagged Raman probes, which is based on the idea that deuteration of the alkynyl hydrogen can cause a large red shift in its Raman peak (>100 cm −1 in their cases); in addition, they reported a strategy to generate alkyne Raman probes via photoinduced cyclopropenone caging, allowing them to perform multicolor live-cell SRS imaging and tracking; 150 (3) Numata and co-workers 151 demonstrated that terminal alkynebased Raman probes that mimic choline and octadecanoic acid can be used to track and quantify such metabolite's uptake and the corresponding metabolic pathways within intact Arabidopsis thaliana (A. thaliana) roots and tobacco Bright Yellow-2 (BY-2) culture cells; (4) Liu and co-workers 152 developed a urinary biomarker, which consists of two alkyne-containing moieties with one behaving as an internal standard and one acting as a disease responsive unit, for quantitatively monitoring cancer development in real time via surface enhanced Raman spectroscopy; and (5) Londergan and coworkers 153 demonstrated that the Raman signals arising from both aliphatic and aromatic terminal alkynes show strong solvatochroism and, hence, can be used to report local electronic interactions in proteins (i.e., via a genetically incorporable alkyne-containing nonnatural amino acid, such as p-ethynylphenylalanine). Furthermore, various strategies have been developed to increase the Raman scattering cross section of alkynes used in biological studies or expand their utilities.…”

“…For example, they have been used to image and interrogate a wide variety of biological molecules, such as proteins, nucleic acids, glycans, and small metabolites, in live cells via stimulated Raman scattering (SRS) microscopy. − Since this topic has been extensively reviewed, − ,,− below we only briefly discuss several recent studies in this area, highlighting the increased utility of the CC stretching vibration in biological science. For example, (1) Min and co-workers developed a new set of ultrabright and compact Raman-active nanoparticles (Rdots) that are composed of polyyne-based dyes (or other alkyne-containing molecules) and further demonstrated that, when used in conjunction with SRS microscopy, such Rdots can lead to a detection limit down to pM scale in solution and even allow single-particle imaging; (2) Wei and co-workers demonstrated a hydrogen–deuterium exchange (HDX) strategy to sense local chemical and environment changes in cells (e.g., acidity) using terminal alkyne-tagged Raman probes, which is based on the idea that deuteration of the alkynyl hydrogen can cause a large red shift in its Raman peak (>100 cm –1 in their cases); in addition, they reported a strategy to generate alkyne Raman probes via photoinduced cyclopropenone caging, allowing them to perform multicolor live-cell SRS imaging and tracking; (3) Numata and co-workers demonstrated that terminal alkyne-based Raman probes that mimic choline and octadecanoic acid can be used to track and quantify such metabolite’s uptake and the corresponding metabolic pathways within intact Arabidopsis thaliana ( A. thaliana ) roots and tobacco Bright Yellow-2 (BY-2) culture cells; (4) Liu and co-workers developed a urinary biomarker, which consists of two alkyne-containing moieties with one behaving as an internal standard and one acting as a disease responsive unit, for quantitatively monitoring cancer development in real time via surface enhanced Raman spectroscopy; and (5) Londergan and co-workers demonstrated that the Raman signals arising from both aliphatic and aromatic terminal alkynes show strong solvatochroism and, hence, can be used to report local electronic interactions in proteins (i.e., via a genetically incorporable alkyne-containing nonnatural amino acid, such as p -ethynylphenylalanine).…”