Category Archives: CIDNP

Nuclear spin-hyperpolarization generated in a flavoprotein under illumination: experimental field-dependence and theoretical level crossing analysis

Ding, Yonghong, Alexey S. Kiryutin, Alexandra V. Yurkovskaya, Denis V. Sosnovsky, Renad Z. Sagdeev, Saskia Bannister, Tilman Kottke, et al. “Nuclear Spin-Hyperpolarization Generated in a Flavoprotein under Illumination: Experimental Field-Dependence and Theoretical Level Crossing Analysis.” Scientific Reports 9, no. 1 (December 2019): 18436.

https://doi.org/10.1038/s41598-019-54671-4

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The effect can be observed in various natural photosynthetic reaction center proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, and in a flavin-binding light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin. In the latter system, a functionally instrumental cysteine has been mutated to interrupt the natural cysteineinvolving photochemistry allowing for an electron transfer from a more distant tryptophan to the excited flavin mononucleotide chromophore. We explored the solid-state photo-CIDNP effect and its mechanisms in phototropin-LOV1-C57S from the green alga Chlamydomonas reinhardtii by using fieldcycling solution NMR. We observed the 13C and, to our knowledge, for the first time, 15N photo-CIDNP signals from phototropin-LOV1-C57S. Additionally, the 1H photo-CIDNP signals of residual water in the deuterated buffer of the protein were detected. The relative strengths of the photo-CIDNP effect from the three types of nuclei, 1H, 13C and 15N were measured in dependence of the magnetic field, showing their maximum polarizations at different magnetic fields. Theoretical level crossing analysis demonstrates that anisotropic mechanisms play the dominant role at high magnetic fields.

Photochemically induced dynamic nuclear polarization NMR on photosystem II: donor cofactor observed in entire plant #CIDNP

Janssen, Geertje J., Pavlo Bielytskyi, Denis G. Artiukhin, Johannes Neugebauer, Huub J. M. de Groot, Jörg Matysik, and A. Alia. “Photochemically Induced Dynamic Nuclear Polarization NMR on Photosystem II: Donor Cofactor Observed in Entire Plant.” Scientific Reports 8, no. 1 (December 2018): 17853.

https://doi.org/10.1038/s41598-018-36074-z.

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect allows for increase of signal and sensitivity in magic-angle spinning (MAS) NMR experiments. The effect occurs in photosynthetic reaction centers (RC) proteins upon illumination and induction of cyclic electron transfer. Here we show that the strength of the effect allows for observation of the cofactors forming the spin-correlated radical pair (SCRP) in isolated proteins, in natural photosynthetic membranes as well as in entire plants. To this end, we measured entire selectively 13C isotope enriched duckweed plants (Spirodela oligorrhiza) directly in the MAS rotor. Comparison of 13C photo-CIDNP MAS NMR spectra of photosystem II (PS2) obtained from different levels of RC isolation, from entire plant to isolated RC complex, demonstrates the intactness of the photochemical machinery upon isolation. The SCRP in PS2 is structurally and functionally very similar in duckweed and spinach (Spinacia oleracea). The analysis of the photo-CIDNP MAS NMR spectra reveals a monomeric Chl a donor. There is an experimental evidence for matrix involvement, most likely due to the axial donor histidine, in the formation of the SCRP. Data do not suggest a chemical modification of C-131 carbonyl position of the donor cofactor.

New insights into the nature of short-lived paramagnetic intermediates of ketoprofen. Photo-CIDNP study

Babenko, Simon V., Polina S. Kuznetsova, Nikolay E. Polyakov, Aleksandr I. Kruppa, and Tatyana V. Leshina. “New Insights into the Nature of Short-Lived Paramagnetic Intermediates of Ketoprofen. Photo-CIDNP Study.” Journal of Photochemistry and Photobiology A: Chemistry 392 (April 2020): 112383.

https://doi.org/10.1016/j.jphotochem.2020.112383

The short-lived paramagnetic particles formed during the UV irradiation of nonsteroidal antiinflammatory drug – ketoprofen (KP) have been investigated using chemically induced dynamic nuclear polarization (CIDNP). This study answers the questions about the nature of paramagnetic particles which can be responsible for KP phototoxic effects. Phototoxic side effects of NSAIDs, according modern point of view, are associated with the action of free radicals, however, there is insufficient information regarding the nature of the radical species. In contrast, most ketoprofen photodegradation schemes include carbanion as a precursor of products. CIDNP effects analysis has shown that all the major products of KP photodegradation can form via radical pairs (RPs) involving benzyl (2’), ketyl (3’) and CO2H● or CO2-● free radicals and solvated electron. Radical ways of KP photodegradation include: decarboxylation via RP with benzyl radical formation (I) in nonaqueous solution and both, (I) and photoreduction with formation of ketyl radicals in the presence of water. Moreover, it was found that the photoinduced radical decarboxylation of KP represents a reversible process.

13C → 1H transfer of light-induced hyperpolarization allows for selective detection of protons in frozen photosynthetic reaction center

Bielytskyi, Pavlo, Daniel Gräsing, Kaustubh R. Mote, Karthick Babu Sai Sankar Gupta, Shimon Vega, P.K. Madhu, A. Alia, and Jörg Matysik. “13C → 1H Transfer of Light-Induced Hyperpolarization Allows for Selective Detection of Protons in Frozen Photosynthetic Reaction Center.” Journal of Magnetic Resonance 293 (August 2018): 82–91.

https://doi.org/10.1016/j.jmr.2018.06.003

In the present study, we exploit the light-induced hyperpolarization occurring on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photoCIDNP) effect to boost the NMR signal intensity of selected protons via inverse crosspolarization. Such hyperpolarization transfer is implemented into 1H-detected twodimensional 13C-1H correlation magic-angle-spinning (MAS) NMR experiment to study protons in frozen photosynthetic reaction centers (RCs). As a first trial, the performance of such an experiment is tested on selectively 13C labeled RCs from the purple bacteria of Rhodobacter sphaeroides. We observed response from the protons belonging to the photochemically active cofactors in their native protein environment. Such an approach is a potential heteronuclear spin-torch experiment which could be complementary to the classical heteronuclear correlation (HETCOR) experiments for mapping proton chemical shifts of photosynthetic cofactors and to understand the role of the proton pool around the electron donors in the electron transfer process occurring during photosynthesis.

Assignment of NMR resonances of protons covalently bound to photochemically active cofactors in photosynthetic reaction centers by 13C–1H photo-CIDNP MAS-J-HMQC experiment

Bielytskyi, Pavlo, Daniel Gräsing, Stefan Zahn, Kaustubh R. Mote, A. Alia, P.K. Madhu, and Jörg Matysik. “Assignment of NMR Resonances of Protons Covalently Bound to Photochemically Active Cofactors in Photosynthetic Reaction Centers by 13C–1H Photo-CIDNP MAS-J-HMQC Experiment.” Journal of Magnetic Resonance 298 (January 2019): 64–76.

https://doi.org/10.1016/j.jmr.2018.11.013

Modified versions of through-bond heteronuclear correlation (HETCOR) experiments are presented to take advantage of the light-induced hyperpolarization that occurs on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. Such 13C–1H photoCIDNP MAS-J-HMQC and photo-CIDNP MAS-J-HSQC experiments are applied to acquire the 2D 13C–1H correlation spectra of selectively 13C-labeled photochemically active cofactors in the frozen quinoneblocked photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (R.) sphaeroides wildtype (WT). Resulting spectra contain no correlation peaks arising from the protein backbone, which greatly simplifies the assignment of aliphatic region. Based on the photo-CIDNP MAS-J-HMQC NMR experiment, we obtained assignment of selective 1H NMR resonances of the cofactors involved in the electron transfer process in the RC and compared them with values theoretically predicted by density functional theory (DFT) calculation as well as with the chemical shifts obtained from monomeric cofactors in the solution. We also compared proton chemical shifts obtained by photo-CIDNP MAS-J-HMQC experiment under continuous illumination with the ones obtained in dark by classical crosspolarization (CP) HETCOR. We expect that the proposed approach will become a method of choice for obtaining 1H chemical shift maps of the active cofactors in photosynthetic RCs and will aid the interpretation of heteronuclear spin-torch experiments.

13C → 1H transfer of light-induced hyperpolarization allows for selective detection of protons in frozen photosynthetic reaction center #DNPNMR

Bielytskyi, Pavlo, Daniel Gräsing, Kaustubh R. Mote, Karthick Babu Sai Sankar Gupta, Shimon Vega, P. K. Madhu, A. Alia, and Jörg Matysik. “13C → 1H Transfer of Light-Induced Hyperpolarization Allows for Selective Detection of Protons in Frozen Photosynthetic Reaction Center.” Journal of Magnetic Resonance 293 (August 1, 2018): 82–91.

https://doi.org/10.1016/j.jmr.2018.06.003

In the present study, we exploit the light-induced hyperpolarization occurring on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect to boost the NMR signal intensity of selected protons via inverse cross-polarization. Such hyperpolarization transfer is implemented into 1H-detected two-dimensional 13C–1H correlation magic-angle-spinning (MAS) NMR experiment to study protons in frozen photosynthetic reaction centers (RCs). As a first trial, the performance of such an experiment is tested on selectively 13C labeled RCs from the purple bacteria of Rhodobacter sphaeroides. We observed response from the protons belonging to the photochemically active cofactors in their native protein environment. Such an approach is a potential heteronuclear spin-torch experiment which could be complementary to the classical heteronuclear correlation (HETCOR) experiments for mapping proton chemical shifts of photosynthetic cofactors and to understand the role of the proton pool around the electron donors in the electron transfer process occurring during photosynthesis.

Effect of heavy atoms on photochemically induced dynamic nuclear polarization in liquids

Okuno, Y. and S. Cavagnero, Effect of heavy atoms on photochemically induced dynamic nuclear polarization in liquids. Journal of Magnetic Resonance, 2018. 286: p. 172-187.

http://www.sciencedirect.com/science/article/pii/S1090780717302938

Given its short hyperpolarization time (∼10−6 s) and mostly non-perturbative nature, photo-chemically induced dynamic nuclear polarization (photo-CIDNP) is a powerful tool for sensitivity enhancement in nuclear magnetic resonance. In this study, we explore the extent of 1H-detected 13C nuclear hyperpolarization that can be gained via photo-CIDNP in the presence of small-molecule additives containing a heavy atom. The underlying rationale for this methodology is the well-known external-heavy-atom (EHA) effect, which leads to significant enhancements in the intersystem-crossing rate of selected photosensitizer dyes from photoexcited singlet to triplet. We exploited the EHA effect upon addition of moderate amounts of halogen-atom-containing cosolutes. The resulting increase in the transient triplet-state population of the photo-CIDNP sensitizer fluorescein resulted in a significant increase in the nuclear hyperpolarization achievable via photo-CIDNP in liquids. We also explored the internal-heavy-atom (IHA) effect, which is mediated by halogen atoms covalently incorporated into the photosensitizer dye. Widely different outcomes were achieved in the case of EHA and IHA, with EHA being largely preferable in terms of net hyperpolarization.

Liquid-State 13C Polarization of 30% through Photo-Induced Non-Persistent Radicals #DNPNMR

Capozzi, A., et al., Liquid-State 13C Polarization of 30% through Photo-Induced Non-Persistent Radicals. The Journal of Physical Chemistry C, 2018.

https://doi.org/10.1021/acs.jpcc.8b01482

Hyperpolarization via dissolution Dynamic Nuclear Polarization (dDNP) is crucial to significantly increase the magnetic resonance imaging (MRI) sensitivity, opening up for in vivo real-time MRI using in particular 13C-labelled substrates. The range of applications is however limited by the relatively fast decay of the nuclear spin polarization together with the constraint of having to polarize the spins near the MRI magnet. As recently demonstrated, the employment of UV-induced non-persistent radicals represents an elegant solution to tackle these drawbacks. Nevertheless, since its introduction, the spread of the technique has been prevented by the relatively low achievable polarization, slow buildup time and time-consuming sample preparation. In the present work, thanks to a thorough investigation of the radical generation step, we provide a robust protocol to enhance the efficiency and performance of the UV-radical technique. Under optimal conditions, it was possible to produce up to 60 mM radical in less than 5 min, and reach maximum DNP enhancement with a buildup time constant of approx. 25 min, at 6.7 T and 1 K, resulting in 30% 13C liquid-state polarization.

Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives #DNPNMR

Liu, G., et al., Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives. Phys. Chem. Chem. Phys., 2017. 19(47): p. 31823-31829.

https://www.ncbi.nlm.nih.gov/pubmed/29171613

We report on radical polarization and optically-driven liquid DNP using nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to transient nitroxide radical polarization that is one order of magnitude larger than that at the Boltzmann equilibrium. The life time of the radical polarization increases with the size of the fullerene derivative and is correlated with the electronic spin-lattice relaxation time T1e. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.

Photo-induced radical polarization and liquid-state dynamic nuclear polarization with fullerene nitroxide derivatives

DNP without microwave radiation? A very interesting approach.

Liu, G., et al., Photo-induced radical polarization and liquid-state dynamic nuclear polarization with fullerene nitroxide derivatives. Phys. Chem. Chem. Phys., 2017.

http://dx.doi.org/10.1039/C7CP06073D

We report on radical polarization and optically-driven liquid DNP with nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to a transient nitroxide radical polarization that is one order of magnitude larger than at Boltzmann equilibrium. Life time of radical polarization increases with size of the fullerene derivative and correlates with the electronic spin lattice relaxation time T1e. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.

Have a question?

If you have questions about our instrumentation or how we can help you, please contact us.