Covalently Bound Azido Groups Are Very Specific Water Sensors, Even in Hydrogen-Bonding Environments

Abstract

Covalently bound azido groups are found in many commercially available biomolecular precursors and substrates, and the NNN asymmetric stretching band of these groups is a strong infrared absorber that appears in a spectral region clear of other signals. In order to evaluate comprehensively the solvatochromism of the asymmetric azido NNN stretching band for site-specific use in biomolecular contexts, infrared spectra of the model compounds 5-azido,1-pentanoic acid and 3-(p-azidophenyl),1-propanoic acid were acquired in a large variety of nonpolar, polar, and hydrogen-bond-donating solvents, as well as mixed aqueous-organic solvents. Spectra in pure solvents indicated that the aliphatic NNN stretching frequency maximum does not depend on solvent polarity, while the aromatic NNN frequency displays a weak but nonzero sensitivity to polarity. In both cases, the NNN frequency exhibits a blue-shift in H-bond-donating solvents, but the frequency in water is higher than in any other H-bond-donating solvent including solvents that are stronger H-bond donors. In nonfluorinated H-bond donor solvents, the frequency blue shift scales with the density of H-bond donors. This sensitivity to the presence of water was further explored in several mixed solvent environments, with the conclusion that this vibrational mode is a highly specific sensor of hydration, even in environments containing other H-bond donors like amides and alcohols, due to the very high local density of H-bond donors in water. The relatively uncomplicated (compared to nitriles, for example), water-specific response of this vibrational mode should lead to its adoption as a site-specific probe of hydration in many different possible systems in which the presence and role of molecular water is of primary interest.

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

“…The increased tolerance to incorporation of these probes at surface-exposed positions, along with their sensitivity to solvation, suggests that they are likely most useful as probes of surface hydration, as suggested previously based on solvent studies. [32,34] From this perspective, the uniquely large blueshift observed upon thermally induced folding of (CN)Phe186 is particularly interesting, as it suggests that solvation at the proteinwater interface is particularly strong, which is consistent with other studies indicating that proteins are hydrated by a layer of high-density water molecules. [51,52] This is also consistent with (CN)Phe186 being the only case where modification stabilizes the folded state of the protein.…”

“…[21][22][23][24][25][26][27][28] The CN and N 3 probes have the advantage of being strong chromophores; however, unlike CÀD labels, CN and N 3 substituents are extrinsic probes that may introduce interactions that are not present in the native protein, and sensitivity to these artificial interactions may desensitize them to their native protein environment. Indeed, previous studies have indicated that both probes are predominantly sensitive to hydrogen bonding, [5,[29][30][31][32][33][34] interactions introduced by the probes themselves. Moreover, as extrinsic probes they might perturb the protein, a possibility exacerbated by the size of the N 3 group and by the H-bonding propensity of both CN and N 3 groups.…”

“…Based on solvent studies such redshifts are expected to result from reduced H bonding, [33,34] suggesting that the N 3 probes are not fully solvent exposed in the unfolded state. As the CÀD and CN absorptions at the same positions in the unfolded protein are virtually identical to those of the free amino acid, the protected environment must be unique to the azide probes.…”

A new fluorescence method to analyze water traces in gasoline based on the breakup of diphenylquinoxaline-6-amine–Zn–bis-(2,4,6-trichlorophenyl) oxalate

“…Among these methods, the spectroscopic methods especially fluorescence spectroscopy have been considered as a powerful alternative approach for the quantification of water content (Srivastava et al 2011;Wolfshorndl et al 2012;Luzinova et al 2012;Samadi-Maybodi et al 2012;Ooyama et al 2011). Various fluorescent dyes such as merocynanine dyes (Kumoi et al 1972), flavone derivatives (Cho et al 2005), luminescent nanospheres (Gao et al 2009), rhodamine 6G (Choi and Tse 1999), 10-allyl-acridine orange (Yang et al 2001), naphthalimide and its derivatives (Niu et al 2007) were introduced as fluorescence dye sensors.…”

4‐Cyano‐α‐methyl‐l‐phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane Interactions

“…Thedata described in this work, corroborated by recent applications and advances in our understanding of its chromophoric properties [65 -68],p oint to ac lear superiority of Phe(p-CN)o ver Phe(p-NO 2 )a savibrational sensor.I nt his context, however, it is worth noting that recent publications have highlighted the overall, even more positive,p roperties of the azido-benzene IR label, including that occurring in Phe(p-N 3 )[ 69] [70]. As tudy on trichogin GA IV containing this latter residue is currently planned in our laboratory.…”