**2. Resolution of different hydrogen-bond states by 240GHz ESR in bulk organic solvents**

It has been well established that the g-tensor and hyperfine components of nitroxide radicals are very sensitive to the local environment, to polarity and proticity in particular. In general, changing the local environment of the nitroxide moiety from water (polar) to hydrocarbon (non-polar) causes an increase in the *g-*tensor components and a concomitant decrease in the values of the components of the hyperfine tensor. This effect is most pronounced for the tensor components gxx and Azz. However, the separation of the polarity and proticity effects could complicate the analysis of the ESR spectra. Proticity refers to the propensity to donate hydrogen bonds; whereas aprotic refers to solvents which cannot donate a hydrogen bond. At relatively low frequencies, up to 95GHz, the separation of hydrogen-bonded vs. non hydrogen-bonded states of the nitroxide often relied upon different g versus A plots, discovered for these two states17. However, as shown in a recent study using TEMPO, if the correlations are indeed different for TEMPO in protic and aprotic solvents, the difference is rather small18.

On the other hand, superior g-factor resolution of HF ESR allows for observation of two resolved spectral components corresponding to two (Smirnova et al.19 at 130GHz) or possibly more (Bordignon et al.20 at 95, 275 and 360GHz ) states of hydrogen-bonding. Two hydrogen bonding states coexisting in frozen deuterated alcohols were previously demonstrated for perdeuterated TEMPONE by X-band ESR21.

Tables 1 and 2 show the values of *Azz* and *aiso* hyperfine splitting and the gxx component of the g-tensor for several nitroxide radicals.

As one sees from Fig.1, whereas at X-band one sees a continuous increase in the 14N hyperfine splitting with change of the local environment from non-polar/aprotic to polar/protic, the 240 GHz ESR shows three distinct values of the gxx parameter. Although the presence/ratio of these components strongly depends on the polarity-proticity of the solvent, there is little variation in the gxx measured for each such component. For all four spin labels studied three distinct components could be detected: (1) "non-polar", as in toluene, DBPh or the minor component in alcohols, (2) "polar", the major component for ethanol and major or minor component in TFE and water/glycerol , depending on the nitroxide used, and (3) "very polar" component observable in TFE and water/glycerol. Although components 2 and 3 cannot be separated at 240GHz as two distinct peaks, their presence is quite obvious (compare Figs.1A-D for different nitroxides). We assign these components to different


\*\* 2Azz value at 77K cannot be reliably determined due to the presence of a singlet-like background.

168 Nitroxides – Theory, Experiment and Applications

**organic solvents** 

rather small18.

in this behavior and a new look at the vast body of experimental data accumulated with PC spin labels in the last 30 years. In particular, we revisit so-called "polarity profiles" determined from the *g*-factor values and hyperfine splittings of PC spin labels in frozen phospholipid membranes with or without cholesterol and show that these values are affected by a number of factors in the membrane composition, chain packing in the lipid phase and folding properties of the *sn-2* spin labeled chain PC labels rather than reflect

**2. Resolution of different hydrogen-bond states by 240GHz ESR in bulk** 

It has been well established that the g-tensor and hyperfine components of nitroxide radicals are very sensitive to the local environment, to polarity and proticity in particular. In general, changing the local environment of the nitroxide moiety from water (polar) to hydrocarbon (non-polar) causes an increase in the *g-*tensor components and a concomitant decrease in the values of the components of the hyperfine tensor. This effect is most pronounced for the tensor components gxx and Azz. However, the separation of the polarity and proticity effects could complicate the analysis of the ESR spectra. Proticity refers to the propensity to donate hydrogen bonds; whereas aprotic refers to solvents which cannot donate a hydrogen bond. At relatively low frequencies, up to 95GHz, the separation of hydrogen-bonded vs. non hydrogen-bonded states of the nitroxide often relied upon different g versus A plots, discovered for these two states17. However, as shown in a recent study using TEMPO, if the correlations are indeed different for TEMPO in protic and aprotic solvents, the difference is

On the other hand, superior g-factor resolution of HF ESR allows for observation of two resolved spectral components corresponding to two (Smirnova et al.19 at 130GHz) or possibly more (Bordignon et al.20 at 95, 275 and 360GHz ) states of hydrogen-bonding. Two hydrogen bonding states coexisting in frozen deuterated alcohols were previously

Tables 1 and 2 show the values of *Azz* and *aiso* hyperfine splitting and the gxx component of

As one sees from Fig.1, whereas at X-band one sees a continuous increase in the 14N hyperfine splitting with change of the local environment from non-polar/aprotic to polar/protic, the 240 GHz ESR shows three distinct values of the gxx parameter. Although the presence/ratio of these components strongly depends on the polarity-proticity of the solvent, there is little variation in the gxx measured for each such component. For all four spin labels studied three distinct components could be detected: (1) "non-polar", as in toluene, DBPh or the minor component in alcohols, (2) "polar", the major component for ethanol and major or minor component in TFE and water/glycerol , depending on the nitroxide used, and (3) "very polar" component observable in TFE and water/glycerol. Although components 2 and 3 cannot be separated at 240GHz as two distinct peaks, their presence is quite obvious (compare Figs.1A-D for different nitroxides). We assign these components to different

gradients of polarity or water content present in the membrane16.

demonstrated for perdeuterated TEMPONE by X-band ESR21.

the g-tensor for several nitroxide radicals.

**Table 1.** Values of isotropic hyperfine splitting constant *aiso* and 2Azz determined at X-band at 295 and 77K respectively for several solvents. These Azz values were also used to obtain the best fits for the corresponding 240 GHz rigid limit spectra.


**Table 2.** gxx component of the g-tensor determined by 240GHz ESR at 80-85K in several glass forming solvents. A common value of 2.00233 was assigned as gzz for all spin labels and gxx value was accurately determined relative to this gzz value from the corresponding spectral splitting29, 52. If two components are present in the spectrum, the component with higher fraction is marked bold. "Sh" denotes the presence of a high/low field component, which manifests itself not as a distinct peak but as a shoulder on the main component.

hydrogen-bonding states of nitroxide radicals and speculate that state 2 corresponds to a single hydrogen bond, while state 3 is double-bonded. Existence of multiple hydrogen bonding to a nitroxide has been predicted theoretically22-24 and later suggested as an explanation for complex ESR lineshapes observed in spin labeled proteins20. Interestingly, the gxx value of the non-hydrogen bonded component for all four spin labels studied shows little dependence on the polarity of the frozen glass-forming solvent (Table 3). This contrasts with some theoretical predictions for the g-factor25, as well as some room temperature measurements for *giso* pointing to a higher g-factor for lower dielectric constants ε17, 18 .

(A)

(B)

**Figure 1.** 240 GHz ESR spectra of 4-oxo-TEMPO (TEMPONE) (from 16) (A), 4-hydroxy-TEMPO (TEMPOL) (B), TEMPO (C) and 2,2,5,5-Tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid free radical (D) in a series of glass-forming solvents at 80-85K. Black dotted lines in A and D show two-component rigid simulation of the ethanol spectra. In (A), g values 2.009450, 2.008830 and 2.008500 are noted "1", "2" and "3" respectively.


**Table 3.** Hyperfine splitting parameter 2Azz in DMPC and DPPC membranes with and without cholesterol determined by X-band ESR at 77K. To record the ESR spectra the samples in 1.2 mm ID capillaries after long exposure at 190C were quickly submerged into liquid nitrogen.
