IDEI PCE 168/2017

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The information to be found on the project page is the following:

  1. Project full title; Reservoirs and magnetisation transfer for hyperpolarised nuclear spins.
  2. The acronym; It’s not necessary
  3. Project summary
  4. The results obtained at each stage of the project
  5. Final results of the project

The information conveyed by magnetic resonance relies on the amount of the magnetisation used and on the way this collective magnetic state is preserved during the experiment. Recently, significant progress was made in summoning larger amounts of polarised nuclear spins using sparse unpaired electrons, via the procedure known as Dynamic Nuclear Polarisation (DNP). This procedure is a revolution for NMR, a spectroscopy that was previously notorious for its lack of sensitivity, as it can enhance the signal-to-noise by factors up to 10’000-fold. Though powerful, the new applications of this very recent technique remain challenging to implement, especially due to issues related to the lifetimes of hyperpolarised magnetisation. Conveying enhanced magnetisation to the desired molecular location, the intended interaction site within a cell, or the intended imaging site within the body is critically dependent on its characteristic lifetime. Improvements in magnetisation lifetimes can be designed even in a standard NMR context, optimizing molecular probes and excitation techniques before implementation in a DNP context. The NMR methods proposed in this project are intended to decisively contribute, at this point, to the transformation of enhanced magnetic resonance into a more versatile technique. New ways of preserving magnetisation are put forward. Optimal spin eigenstates to preserve magnetisation in molecules with several coupled magnetic nuclei will be theoretically evaluated and experimentally tested. Long-lived magnetisation can travel long distances almost unaltered – that is, preserving its original amplitude, and, therefore, detection sensitivity. This can lead to applications ranging from studies of the mechanism and dynamics of biomolecular interactions, and up to magnetic-resonance angiography of endogenous molecular probes in humans.

  1. General objectives and expected results to be achieved

Characterization of molecules, polarization offset transfer, using J couplings; creating a strategy adapted to support magnetization in the chosen molecules; control of polarization transfer in proposed molecules; publication and dissemination of the results.

  1. The coordinating institution, possibly with the institution logo and contact details; It’s not necessary
  2. Consortium component; It’s not necessary
  3. Contracting Authority (project financier); UEFISCDI
  4. Project duration;12.07.2017 – 31.12.2019
  5. The activities and responsibilities of each participant (the implementation plan);

The projected team consists of four members with diverse competences and at various stages of advancement in their professional career. One of the team members, recruited as a post-doctoral researcher will bring expertise in DNP substrates and state-of-the-art theory and experiments. This researcher will use their know-how to also perform DNP-enhanced experiments on local molecules in EU laboratories. A key researcher is Anamaria Hanganu, well-anchored scientist in the NMR environment at the University of Bucharest. The person will be in charge of the spectroscopic characterization of molecules and keeping the project close to the environment: identifying molecular probes for the production of which the expertise of local synthesis chemists can have a major impact and valorizing the outputs of the project for other groups: using enhanced lifetimes for slow diffusion and slow exchange experiments. Master students will be trained within and contribute to the project. NB: a PhD student may join the project, after the first year, once administrative procedures linked to doctoral guidance for the PI are finalized. The student will have a scholarship from distinct funds.

4. The results obtained at each stage of the project :

Stage 1

In this project stage the following were achieved:

  1. A strategy has been developed to support magnetization in molecules containing an aromatic ring adjacent to nitrogen nuclei.
  2. Models have been established for polarization transfer study for purchased substances: pyruvate, lactate, Ala-Gly, glutamine, glutamic acid, aspartic acid, asparagine. For the same type of experiment, a number of compounds were also purchased: glycine 1-13C, glucose 6-13C and asparagine 4-13

Broad description of the experiments performed and results presentation:

Long-lived states (LLS) of nuclear spin order were used for the first time to probe interactions between molecules and diamagnetic metal ions. LLS are currently employed in two contexts: (i) hyperpolarized NMR experiments using dissolution Dynamic Nuclear Polarization (d-DNP) or parahydrogen for sensitivity enhancement: in these experiments, LLS are used to maintain hyperpolarized magnetization and can subsequently detect changes in the chemical environment of the polarized molecular probe; (ii) NMR experiments that start from thermal-equilibrium magnetization to encode LLS. In this context, the LLS information can be complementary to data obtained from standard longitudinal relaxation rates.

Oxadiazole derivatives are electron transporting and luminescent molecules that have recognized potential for the fabrication of organic light-emitting diodes (OLEDs). Such interactions were initially described in oxadiazole derivatives bearing substituents that feature donor groups able to coordinate metal ions. It was recently observed that the two nitrogen atoms of the oxadiazole ring itself feature interactions with silver that can lead to the formation of coordination polymers even without the aid of any other donor group. It is therefore worth investigating the interactions between the two adjacent nitrogens of the heterocycle and metal ions.

NMR experiments were recorded in a field B0 = 11.75 T using a Bruker Avance spectrometer operating at the 1H Larmor frequency m0 = 500.13 MHz. The temperature was T = 25 0C. NMR spectra were acquired using TopSpin and spectral intensities extracted using Topspin, Mestrec, and Matlab functions. The relaxation curves were fitted using the dedicated Matlab function and errors were calculated from a Monte-Carlo analysis performed using 100 points for each fit.

Natural-abundance nitrogen-15 spectra were recorded to probe the effect of the nonsymmetrical substituents on the 15N shifts. Direct 15N detection was first tested on a standard formamide sample. Proton reference spectra and LLS experiments were recorded

with 64 transients and a recovery delay of 20 s, proton T1 experiments were recorded using 16 transients and a recovery delay of 25 s, and nitrogen detection was performed with 8192 transients and a 15 s recovery delay.

NMR 1H and 15N spectra recorded for the studied oxadiazole molecule is shown in Figure 1:

Figure 1. Oxodiazole: 2-(4-metoxifenil)-5-(p-tolil)-1,3,4-oxadiazol.

Figure 1. Oxodiazole: 2-(4-metoxifenil)-5-(p-tolil)-1,3,4-oxadiazol.

Structural characterization of oxodiazole was performed by recording the 1D and 2D-NMR spectra (Figures 2A-2G).

1H-NMR (500.13 MHz, MeOD-d4, δ ppm, J Hz): 8.02 (d, 2H, H-S, H-S’, 8.5 Hz), 7.96 (d, 2H, H-R, H-R’, 8.0 Hz), 7.37 (d, 2H, H-K, H-K’, 8.0 Hz), 7.09 (d, 2H, H-I, H-I’, 8.5 Hz), 3.87 (s, 3H, A3), 2.41 (s, 3H, H-B3).

13C-NMR (125.77 MHz, MeOD-d4, δ ppm): 165.7 (CQ4), 165.6 (CQ3), 164.0 (CQ1), 143.8 (CQ6), 130.8 (C-K, C-K’), 129.67 (C-S, C-S’), 127.6 (C-R, C-R’), 121.7 (CQ5), 116.7 (CQ2), 115.6 (C-I, C-I’), 55.9 (C-A3), 21.7 (C-B3).

Figure 2A. 1D 1H NMR spectra of oxodiazole (blank)

Figure 2A. 1D 1H NMR spectra of oxodiazole (blank)

Figure 2B. 1D 13C NMR spectra of oxodiazole (blank)

Figure 2B. 1D 13C NMR spectra of oxodiazole (blank)

Figure 2C. 1D Apt-NMR spectra of oxodiazole (blank)

Figure 2C. 1D Apt-NMR spectra of oxodiazole (blank)

Figure 2E. 2D HMQC-NMR spectra of oxodiazole (blank)

Figure 2E. 2D HMQC-NMR spectra of oxodiazole (blank)

Figure 2F. 2D HMB-NMR spectra of oxodiazole (blank)

Figure 2F. 2D HMB-NMR spectra of oxodiazole (blank)

Figure 2G. 2D HMBC- NMR spectra of oxodiazole (blank)

Figure 2G. 2D HMBC- NMR spectra of oxodiazole (blank)

A strategy was developed to study the interactions between organic compound (oxodiazole) and metal ions (silver Ag (I)) using LLS method. A stock solution of CF3SO3Ag was prepared. Portions of this solution were added to the NMR tube over the oxodiazole solution (Figure 3).

Figure 3. 1D 1H NMR spectra of: A – control sample of oxadiazol (blank); B–0.25:1[CF3SO3Ag]:[ oxadiazol]; C–0.5:1 [CF3SO3Ag]:[ oxadiazol]; D–0.75:1 [CF3SO3Ag]:[ oxadiazol].

Figure 3. 1D 1H NMR spectra of: A – control sample of oxadiazol (blank); B–0.25:1[CF3SO3Ag]:[ oxadiazol]; C–0.5:1 [CF3SO3Ag]:[ oxadiazol]; D–0.75:1 [CF3SO3Ag]:[ oxadiazol].

LLS with relaxation time constants (‘lifetimes’) up to TLLS = 1/RLLS ~ 10 s were detected on aromatic protons in oxadiazole and were used to probe molecular interactions with Ag(I) ions. The pulse sequence used for LLS excitation and detection is shown in Fig. 4, alongside a standard inversion-recovery experiment designed to measure the spin-lattice relaxation rate constant.

Figure 4: (A) NMR experiment used to probe LLS. (B) LLS relaxation profile obtained for free oxadiazole dissolved in methanol at B0 = 11.75 T, spins (K, R). (C) NMR inversion -recovery experiment. (D) Inversion-recovery curve detected on spin R in the free oxadiazole derivative.

Figure 4: (A) NMR experiment used to probe LLS. (B) LLS relaxation profile obtained for free oxadiazole dissolved in methanol at B0 = 11.75 T, spins (K, R). (C) NMR inversion -recovery experiment. (D) Inversion-recovery curve detected on spin R in the free oxadiazole derivative.

Table 1 lists the longitudinal relaxation constants of oxadiazole derivatives.

Table 1. Values of R1 and RLLS for free compound 1 and mixtures of compound 1 with increasing concentrations of Ag(I) salt (from 0.25 molar equivalents to 0.75 molar equivalents).

R1 (s-1)RLLS (s-1)Detected on signals of spin R, spin I (in bold) and, respectively, detected on signals of spin K, spin S (in italics)
Eq. de Ag(I)00.250.50.7500.250.50.75
Spin R0.22±0.010.27±0.010.30±0.010.31±0.010.096±0.0050.139±0.0050.152±0.0040.166±0.005
Spin K0.29±0.010.36±0.010.40±0.010.41±0.01(0.094±0.005)(0.138±0.005)(0.153±0.005)(0.162±0.006)
Spin I0.33±0.010.40±0.010.43±0.020.45±0.010.122±0.0040.172±0.0080.179±0.0080.204±0.012
Spin S0.22±0.010.27±0.010.30±0.010.32±0.01(0.110±0.010)(0.150±0.020)(0.171±0.008)(0.180±0.016)

The longitudinal relaxation time constants T1=1/R1, on the order of five seconds, are significantly shorter than the detected LLS lifetimes. Sample degassing extends all relaxation rate constants by a factor of 2. The R1 rates measured for oxadiazole in presence of silver ions increase by cu 0.05-0.07 s-1 (ca. 20%) upon addition of 0.25 molar equivalents of silver salt and remain almost the same at higher Ag(I) concentrations. As far as LLS are concerned, the addition of silver(I) to the oxadiazole solution leads to a increase by 70% in LLS relaxation rates, RLLS, of the (I, S) pair and by 65% for the (S,K) pair. Therefore, variations in RLLS = 1 / TLLS upon silver addition offer significantly improved contrast compared to variations in R1 = 1 / T1, making LLS a sensitive probe for mapping the interaction (Fig. 5 and Fig.6).

Figure 5. LLS relaxation rate constants of the aromatic protons of free oxadiazole (blue), on a 1:0.25 molar mixture of oxadiazole and silver salt (green), on a 1:0.50 molar mixture of oxadiazole and silver salt (yellow) and on a 1:0.75 molar mixture of oxadiazole and silver salt (red) at B0 = 11.75 T.

Figure 5. LLS relaxation rate constants of the aromatic protons of free oxadiazole (blue), on a 1:0.25 molar mixture of oxadiazole and silver salt (green), on a 1:0.50 molar mixture of oxadiazole and silver salt (yellow) and on a 1:0.75 molar mixture of oxadiazole and silver salt (red) at B0 = 11.75 T.

Figure 6. T1 relaxation rate constants of the aromatic protons of free oxadiazole (blue), on a 1:0.25 molar mixture of oxadiazole and silver(I) salt (green), on a 1:0.50 molar mixture of oxadiazole and silver(I) salt (yellow) and on a 1:0.75 molar mixture of oxadiazole and silver(I) salt (red) at B0 = 11.75.

Figure 6. T1 relaxation rate constants of the aromatic protons of free oxadiazole (blue), on a 1:0.25 molar mixture of oxadiazole and silver(I) salt (green), on a 1:0.50 molar mixture of oxadiazole and silver(I) salt (yellow) and on a 1:0.75 molar mixture of oxadiazole and silver(I) salt (red) at B0 = 11.75.

The interaction between an oxadiazole derivative and silver(I) ions was sensed using proton LLS. The relaxation rate constants of LLS excited on hydrogen nuclei situated in the immediate vicinity of donor atoms were seen to be more sensitive probes than standard NMR longitudinal relaxation for the interaction.

Hyperpolarization of the detected nuclear spins can offer additional insights on this type of interactions at low sample concentrations.

The experimental results obtained at this stage were disseminated by publishing an article in Journal of Magnetic Resonance (FI = 2.432):

Cristina Stavarache, Anamaria Hanganu, Anca Paun, Codruta Paraschivescu, Mihaela Matache, Paul R. Vasos, “Long-lived states detect interactions between small molecules and diamagnetic metal ions”, Journal of Magnetic Resonance, 2017, 284, 15-19, http://dx.doi.org/10.1016/j.jmr.2017.09.001.

Also, the preliminary results obtained were disseminated through oral presentations within the following scientific manifestations by the members of the research team:

  • Paul Vasos, “Enhanced NMR to follow enzymatic reactions in living cells”, Oral presentation, 20th Romanian International Conference on Chemistry and Chemical Engineering, 6-9 September 2017, Poiana Brasov, Romania.
  • Aude Sadet, CRUK Cambridge Institute, Workshop, 6-9 December 2017, Molecular probes useful for in vivo MRI experiments
  1. Description / presentation of various events within the project (exp: conferences, seminars, partner meetings, etc.);
  • Paul Vasos, “Enhanced NMR to follow enzymatic reactions in living cells”, Oral presentation, 20th Romanian International Conference on Chemistry and Chemical Engineering, 6-9 September 2017, Poiana Brasov, Romania.
  • Aude Sadet, CRUK Cambridge Institute, Workshop, 6-9 December 2017, Molecular probes useful for in vivo MRI experiments
  • Aude Sadet, Glasgow, United Kingdom 25 – 27 June 2018,Artificial Water Channels Faraday Discussion
  1. Links to various other pages of interest to the visitor. Ex: legislative pages in the project field of activity, similar or complementary projects, webpages of the partner institutions in the project, etc;
  2. Contact details of the project director; Vasos Paul

e-mail: paul.vasos@icub.unibuc.ro

Phone: 021.3159249

  1. Final results of the project. It’s not necessary

Project code: PN-III-P4-ID-PCE-2016-0887

  • Cristina Stavarache, Anamaria Hanganu, Anca Paun, Codruta Paraschivescu, Mihaela Matache, Paul R. Vasos, “Long-lived states detect interactions between small molecules and diamagnetic metal ions”, Journal of Magnetic Resonance, 2017, 284, 15-19, http://dx.doi.org/10.1016/j.jmr.2017.09.001.

https://www.ncbi.nlm.nih.gov/pubmed/28938134

  • Viorel Vasile Nastasa, Cristina Stavarache, Anamaria Hanganu, Adina Coroaba, Alina Nicolescu, Calin Deleanu, Aude Sadet, Paul Vasos, “Hyperpolarised NMR to Follow Water Proton Transport through Membrane Channels via Exchange with Biomolecules”, Faraday Discussions, 2018, DOI: 10.1039/C8FD00021B.

Stage 2

New methods for structural characterisation of biomolecules

Transfer of hyperpolarisation using J couplings

Summary of the results obtained:

1) Demonstration of polarization transfer from hyperpolarized protons of water to molecules; discussing the transfer rate in the context of the interposition of a lipid membrane in which channels are located along the transferred protons (article published in Faraday Discussions, 2018, 209, 67-82 2, DOI: 10.1039 / c8fd00021b);

2) Demonstration of the effects of the J coupling between Ala-Gly protons on the polarization transfer and the interactions of Long Lived Coherences (LLC’s) with water magnetization – (results contributed to the articles: Sadet et al., JACS 2019, Sadet et al., Sci. Rep. 2019);

3) Hierarchy of magnetization lifetimes that can be achieved using Long-lived states (LLS) methods for different biomolecules with potential use as biomarkers for in vivo NMR – article in preparation;

4) Study of the magnetization transfer between the reduced form and the oxidized form of glutathione: ongoing experiments to propose new methods for monitoring oxidant stress by monitoring the conversion between the reduced form and the oxidized form via magnetic resonance.

Organization of the project and scientific communication:  The results were communicated in an international scientific conference organized by the UK Royal Society, the Faraday Discussions Meeting, Glasgow, June 2018, communication by Dr A. Sadet and published in Faraday Discussions (article located in the maximum area of ​​influence in the field of physical chemistry, after the influence score quoted in June 2018 by uefiscdi.ro);  the project team was supplemented by the arrival of a MSc student who ensures the advance of the theoretical and calculation part of the project (Florin Teleanu); a collaboration was initiated and an experimental measurement campaign was performed using hyperpolarization (dissolution – Dynamic Nuclear Polarisation) at the Normal School of Paris, Laboratoire des Biomolecules and the University of Vienna); the results are used in the course held annually at the Doctoral School of the Faculty of Chemistry, the University of Bucharest and the results of the project contribute to the realization of a contracted book chapter (in preparation) for the book published by the Royal Society of Chemistry: “Long-Lived Nuclear Spin Order”.

The experimental results obtained at this stage were disseminated by publishing an article in Faraday Discussions (FI = 3,427) “Hyperpolarized NMR to Follow Water Proton Transport through Membrane Channels via Exchange with Biomolecules”, Faraday Discussions, 2018, 209, 67-82, DOI : 10.1039 / C8FD00021B.

Stage 3

Development of a 2D method for hyperpolarized NMR.

Completion of scientific activities, publication and dissemination

NMR spectroscopy based on protons can be used to study peptide and protein folding and conformational stability in membranes.

Proton−proton correlation spectroscopy (COSY) is the most straightforward route for acquiring atomic-resolution structural information for peptides with masses of less than 10 kDa whenever isotope labelling is not necessary or feasible.

We describe an experiment that allows high-resolution characterization of peptide conformations in less than 1 min. The very short duration of the described experiments is obtained using signal enhancement by D-DNP. For the proof of concept, we chose the dipeptide alanine-glycine (AlaGly) dissolved in hyperpolarized HDO as a model system to study peptide-liposome interactions by hyperpolarized COSY (Figure 1).

Figure 1 (adapted from our paper Sadet et al., JACS 2019). Proton spectrum of AlaGly obtained without hyperpolarization)

To obtain hyperpolarized COSY data, we followed a previously reported experimental strategy and dissolved the AlaGly peptide in a hyperpolarized mixture of 4% H2O in D2O, with enhancements of the HDO protons, εHDO, in the range 100-200. The peptide signal intensities in the first point of DDNP-enhanced 2D COSY, stemming from HDO are enhanced by a factor ε = IHyperpolarized/Iconventional ≈ 405. In the hyperpolarized COSY spectrum (Figure 2), in addition to the Gly-HN/Gly-Hα and Ala-HN/Ala-Hα cross-peaks originating from magnetization relayed via intraresidue J couplings to Hα protons, we note the appearance of several cross-peaks that benefit from enhanced signal intensities. Because of the high sensitivity of the experiment, the Ala-HN/Ala-Hβ correlation stemming from transfer via 4J couplings is observed and interresidue correlations of a similar long-range nature between Gly-HN and Ala-Hα protons can be inferred, despite overlap at these sites.

Figure 2 (adapted from our paper Sadet et al., JACS 2019)

(A) Hyperpolarized 2D COSY proton correlations in the amide region of AlaGly and signal assignments. The hyperpolarized COSY spectrum recorded in 1 min (in red) is overlaid with a conventional COSY spectrum obtained without hyperpolarization in the same time (in blue). Both spectra were processed in magnitude mode. Signal assignments are based on a conventional COSY spectrum covering both the amide and aliphatic regions; (B) Overlay of projections of proton correlation spectra from hyperpolarized (red) and conventional (blue, magnified by a factor 400) COSY spectra.

Conventional COSY in thermal equilibrium (i.e., without hyperpolarization) revealed only magnetization transfer from Ala-HN to Ala-Hα and from Gly-HN to Gly-Hα1 and Gly-Hα2.

The signal enhancements obtained for different positions on the 2D map as well as for newly detected signals are listed in Table 1, column II.

Longitudinal relaxation of magnetization during sample transfer between the polarizer and the detection system is crucial for the obtained enhancement. The key advantage of hyperpolarized HDO is that its relaxation time constant is relatively long, T1(HDO) ≈ 8 s, allowing magnetization to be observed for up to tens of seconds after hyperpolarization. By comparison, direct polarization of 1H nuclei in AlaGly followed by dissolution, transfer, and injection is expected to result in lower signal enhancements, even for the slowest-relaxing methyl protons in Ala, with typical T1(1H) values of ∼2 s in high magnetic fields and even shorter during the transfer step in low fields. Therefore, one can obtain multidimensional hyperpolarized spectra within the time window imposed by HDO relaxation rather than being limited by the much faster relaxation of the target peptide. The obtained enhancements (Table 1) allowed us to elevate the signal amplitudes of formerly invisible correlations above the detection threshold.

Proton correlations were adequately detected in the hyperpolarized 2D map. Three points need to be considered in comparison with a conventional COSY experiment:

(i) A small spectral window was used in the indirect dimension in order to preserve resolution. Even with reduced spectral windows, resolution in the indirect dimension of the hyperpolarized experiment remains an issue because of the limited evolution time, t1max. To improve the resolution under these conditions, “SOFT-COSY” experiments may be useful.

(ii) The method is hindered by very intense water resonances, which impose the challenge of avoiding radiation damping effects that would distort the acquired spectra; furthermore, the

HDO hyperpolarization reservoir has to be maintained. Hence, the direct dimension of the spectra in Figure 2 is reduced, and selective excitation of the peptide resonances was chosen, as proposed in earlier work.

(iii) Finally, the experiment does not require a conventional recovery delay (typically τrecovery = 4 s), as the proton polarization is replenished by chemical and magnetic exchange with the solvent.

We studied the interaction of AlaGly with liposomes (Figure 3). The liposomes consisted of large unilamellar vesicles (LUVs).

Figure 3 (adapted from our paper Sadet et al., JACS 2019)

Changes in hyperpolarized COSY maps due to interactions with liposomes. (A, B) Hyperpolarized COSY and projections thereof (integrals over the indirect dimension) for AlaGly without liposomes (shown in red) and in the presence of liposomes (shown in blue; the scale is multiplied by a factor of 4 in the projection). All of the COSY spectra were processed in magnitude mode. (C) Representation of free AlaGly and a conformation interacting with the liposome. (D) Snapshot from an MD trajectory showing the peptide forming a hydrogen bond (yellow line) between the Ala residue and dioleoylphosphocholine (DOPC) on the bilayer surface.

We observed a pronounced difference between spectra recorded in the presence and absence of liposomes. Relative to free AlaGly, an overall reduction of signal intensities of the amide region was immediately detected in the liposome containing sample. The signal losses in spectra recorded in the presence of liposomes, relative to free AlaGly, can be traced back to two factors: (i) hindered exchange between HDO and HN sites and (ii) accelerated relaxation of peptide protons in the presence of LUVs via slowed dynamics upon binding. While signal losses via factor (ii) trigger a mostly uniform scaling of spectral intensities in a small peptide, losses due to effect (i) lead to more pronounced decreases in signal amplitudes for sites that are shielded from the solvent. We observed a site-specific effect, i.e., a reduction of the intensity of Ala-NH3-based cross-signals compared with those based on Gly-HN (Table 1, column III). The signal enhancement of the Ala-HN/Ala-Hα correlation was reduced 14-fold, while the Gly-HN/Gly-Hα correlation was reduced by only a factor of 6. Hence, the signal changes are compatible with effect (i), and the impact of liposome binding on the AlaGly signal enhancement indicates stronger interactions of the Ala residue with the liposome surface.

The transfer of hyperpolarization from HDO to the detected amides can be based on (i) chemical proton exchange, (ii) direct magnetic exchange, i.e., NOEs, from the solvent to the HN protons, and (iii) exchange-relayed NOEs. 1D waterselective and 2D NOESY experiments showed that the transfer of polarization is based on a combination of both NOEs from the solvent and chemical exchange (Figure 4).

Figure 4 (adapted from the supporting material of our paper Sadet et al., JACS 2019)

a) Pulse sequence employed for the Selective Water-NOESY;b) 1D selective NOE experiments starting from water magnetization. Overall magnetization transfer towards HN and aliphatic sites reaches a maximum towards transfer times TM approximatively 0.5 s c) 2D 1H-1H NOESY of AlaGly for 300 ms mixing time. Positive signals are shown in green and negative signals in red.

Recent advances in observation techniques showed that even small peptides have defined structural propensities. These preferences are more marked in the presence of interaction partners. Amphiphilic peptides of a few amino acids bind to lipid vesicles through exothermic reactions with binding free energies, ΔG, that depend on the peptide sequence and vary between −10 and −80 kJ/mol.35 Energy gaps on this order of magnitude are typical for strongly associated partners. The increase in spectral dispersion observed upon liposome addition (Figure 3B) indicates a spread in peptide conformations. This spread occurs over ca. 0.5 ppm, i.e., 400 Hz under our experimental conditions. The major conformations that appear upon liposome introduction feature signals with similar intensities. This implies that the energy gaps that separate these conformations at the temperature of the study, T = 300 K, are on the order of ΔG ≈ −RT = −2.5 kJ/mol, where R = 8.3 J K−1 mol−1 is the universal gas constant.  To validate the interaction model obtained from the experiments, we employed molecular dynamics (MD) simulations. The peptide interacting with the lipid bilayer was sampled for 200 ns using a CHARMM based simulation platform. The peptide, initially assumed to be in a random conformation, was seen to spend a significant time in close contact with the bilayer surface. Most of the hydrogen bonds (∼92%,) occurred when the peptide was oriented with the Ala residue toward the bilayer surface (Figure 3D), forming hydrogen bonds between the Ala N-terminus and phosphatidylcholine anions. This corroborates our deduction that the positively charged peptide N-terminus forms the primary interaction site with the membrane surface. The binding energies calculated from MD span the range −0.98 to −12.89 kJ/mol, reasonably fitting with the experimentally inferred value mentioned above.

To conclude, structural information can be obtained within 1 min by 2D proton correlation spectroscopy using hyperpolarized HDO protons. The residue-dependent enhancement factors yield information about binding sites and can facilitate the assignment of magnetic resonance signals to atoms within molecular and intermolecular frameworks. The detection of biomolecular interactions without any recourse to isotopic enrichment makes possible fast and straightforward applications such as drug screening for binding to defined biomolecular sites via hyperpolarized correlation spectroscopy.

The experimental results obtained at this stage were disseminated by publishing an article in Journal of the American Chemical Society (FI = 14.695).

Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions”, Journal of the American Chemical Society, 2019, 141, 12448-12452, DOI: 10.1021/jacs.9b03651.

Also, the results obtained were disseminated in an oral presentation in a scientific event by the members of the research team:

Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Water-Exchanging Hydrogens’ Positions in Biomolecules Detected via Long-Lived Coherences and Hyperpolarized 2D COSY”, 60th Experimental Nuclear Magnetic Resonance Conference, Oral presentation, April 7 – 12, 2019, Asilomar Conference Center in Pacific Grove, California, USA.

  1. Tanaka, Radu, Doria, Vasos, Medical Physics, 2019, 46 (10), 726-734, doi: 10.1002/mp.13741
  2. Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions”, Journal of the American Chemical Society, 2019, 141, 12448-12452, DOI: 10.1021/jacs.9b03651.
  3. Aude Sadet, Cristina Stavarache, Florin Teleanu, Paul R. Vasos, “Water hydrogen uptake in biomolecules detected via nuclear magnetic phosphorescence”, Scientific Report, 2019, 9, 17118, https://doi.org/10.1038/s41598-019-53558-8.

The results obtained were disseminated through presentations in the following scientific manifestations by the members of the research team:

  • Paul Vasos, “Enhanced NMR to follow enzymatic reactions in living cells”, invited oral presentation, 20th Romanian International Conference on Chemistry and Chemical Engineering, 6-9 September 2017, Poiana Brasov, Romania.
  • Aude Sadet, Cancer Research Institute, University of Cambridge, 6-9 December 2017, Molecular probes useful for in vivo MRI experiments “.
  • Aude Sadet, “Hyperpolarised NMR to Follow Water Proton Transport through Membrane Channels via Exchange with Biomolecules”, UK Royal Society, oral presentation, Faraday Discussions Meeting, Glasgow, June 2018.
  • Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Water-Exchanging Hydrogens’ Positions in Biomolecules Detected via Long-Lived Coherences and Hyperpolarized 2D COSY”, 60th Experimental Nuclear Magnetic Resonance Conference, Oral presentation, April 7 – 12, 2019, Asilomar Conference Center in Pacific Grove, California, USA.

5. Final results of the project

General conclusions

Between August 2017 – November 2019, 6 ISI articles were published. Two book chapters are in preparation. A patent has been filed at OSIM. Of the articles already published, 2 articles are located in the maximum area of ​​influence according to UEFICSDI classification in the field of physical chemistry.

  1. Cristina Stavarache, Anamaria Hanganu, Anca Paun, Codruta Paraschivescu, Mihaela Matache, Paul R. Vasos, “Long-lived states detect interactions between small molecules and diamagnetic metal ions”, Journal of Magnetic Resonance, 2017, 284, 15-19, http://dx.doi.org/10.1016/j.jmr.2017.09.001.
  2. Viorel Vasile Nastasa, Cristina Stavarache, Anamaria Hanganu, Adina Coroaba, Alina Nicolescu, Calin Deleanu, Aude Sadet, Paul Vasos, “Hyperpolarised NMR to Follow Water Proton Transport through Membrane Channels via Exchange with Biomolecules”, Faraday Discussions, 2018, 209, 67-82, DOI: 10.1039/C8FD00021B
  3. Manda, Hinescu, Neagoe, Ferreira, Boscenco, Vasos, Basaga, Cuadrado, Emerging Therapeutic Targets in Oncologic Photodynamic Therapy, Current Pharmaceutical Design, 24, 44, 5268-5295, 2018, doi: 10.2174/1381612825666190122163832
  4. Asavei, Bobeica, Nastasa, Manda, Naftanaila, Bratu, Mischianu, Cernaianu, Ghenuche, Savu, Stutman, Tanaka, Radu, Doria, Vasos, Medical Physics, 2019, 46 (10), 726-734, doi: 10.1002/mp.13741
  5. Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions”, Journal of the American Chemical Society, 2019, 141, 12448-12452, DOI: 10.1021/jacs.9b03651.
  6. Aude Sadet, Cristina Stavarache, Florin Teleanu, Paul R. Vasos, “Water hydrogen uptake in biomolecules detected via nuclear magnetic phosphorescence”, Scientific Report, 2019, 9, 17118, https://doi.org/10.1038/s41598-019-53558-8.

The results obtained were disseminated through presentations in the following scientific manifestations by the members of the research team:

  • Paul Vasos, “Enhanced NMR to follow enzymatic reactions in living cells”, invited oral presentation, 20th Romanian International Conference on Chemistry and Chemical Engineering, 6-9 September 2017, Poiana Brasov, Romania.
  • Aude Sadet, Cancer Research Institute, University of Cambridge, 6-9 December 2017, Molecular probes useful for in vivo MRI experiments “.
  • Aude Sadet, “Hyperpolarised NMR to Follow Water Proton Transport through Membrane Channels via Exchange with Biomolecules”, UK Royal Society, oral presentation, Faraday Discussions Meeting, Glasgow, June 2018.
  • Aude Sadet, Cristina Stavarache, Mihaela Bacalum, Mihai Radu, Geoffrey Bodenhausen, Dennis Kurzbach, Paul R. Vasos, „Water-Exchanging Hydrogens’ Positions in Biomolecules Detected via Long-Lived Coherences and Hyperpolarized 2D COSY”, 60th Experimental Nuclear Magnetic Resonance Conference, Oral presentation, April 7 – 12, 2019, Asilomar Conference Center in Pacific Grove, California, USA.

 

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