Chemical Shift Analysis Research Articles

1H, 13C, and 15N resonance assignments of the amyloidogenic peptide SEM2(49-107) by NMR spectroscopy.

It has been shown that human seminal fluid is a major factor in enhancing HIV activity. The SEM2(49-107) peptide is a product of cleavage after ejaculation by internal prostheses of the semenogelin 2 protein, expressed in seminal vesicles. It is established that the peptide SEM2(49-107) forms amyloid fibrils, which increase probability of contracting HIV infection. In this nuclear magnetic resonance (NMR) study, we present almost complete (86%) resonance assignments for the 1H 15N and 13C atoms of the backbone and side-chain of the SEM2(49-107) peptide (BioMagResBank accession number 52356). The secondary structure of SEM2(49-107) peptide was estimated by using two approaches, secondary chemical shifts analysis (CSI) and TALOS-N prediction. Analysis of the secondary structure of the SEM2(49-107) peptide using both methods revealed that the peptide contains helical segments at the C-terminus. Also in this work, we used phase-sensitive 2D HSQC 1H- 15N experiments measuring longitudinal T1 and transverse T2 NMR relaxation times to report predicted secondary structure and backbone dynamics of the SEM2(49-107) peptide. This resonance assignment will form the basis of future NMR research, contributing to a better understanding of the peptide structure and internal dynamics of the molecule.

Nonlinear optical properties of La3+-Doped tellurite-zinc glasses: Impact of chemical bonding and physical properties via Raman and XPS

Understanding the connection between the local environments of lanthanum ions in tellurite glasses and their nonlinear optical (NLO) properties is essential for various technological applications. Rare earth ions can serve as probes for the local environments of the host material if the structure-property correlations are well understood. In this study, we investigated glasses with compositions 70TeO2 - (30-x)ZnO - (x)La2O3 (mol%) (x = 0, 5, 7, 9) fabricated by the melt-quenching technique. The capabilities of combined Raman and X-ray photoemission (XPS) spectroscopy were used to identify these glasses and define spectral deconvolution for distinguishing them in the formation of bridging oxygens (BO) and non-bridging oxygens (NBO). NLO properties were evaluated using open-aperture (OA) and closed-aperture (CA) Z-scan measurements with femtosecond (fs) laser pulses (∼220 fs, 750 Hz) at wavelengths ranging from 600 to 1500 nm. Raman spectra revealed a shift to higher frequencies from 731 to 746 cm−1, with increasing of La3+ ions, indicating the conversion of TeO4 polyhedra to TeO3 structural units, affecting the Te-O- Zn2+ or Te=O bonds and resulting in a variation of the polarizability. Besides the conventional analysis of oxidation state and chemical shifts of the core-level peaks, XPS spectroscopy included a careful analysis of the type oxygen bridges and metal-oxygen bond length to distinguish otherwise similar spectral contributions of different TZL glasses. Glasses with a high fraction of Zn2⁺ ions bonded with oxygen form NBO and modify the local structure of the glass network, while La³⁺ ions coordinate with oxygen atoms or interact with the lone pairs of tellurium atoms. By analyzing the correlation in different concentrations of La3+ ions, the Te-O bonds and NBO/(BO + NBO) ratio were clearly demonstrated. These findings suggest charge compensation between the Zn2⁺ and La³⁺ ions, for maintaining structural stability within the glass network. This study also reveals an enhancement of NLO properties as a function of La3+ ions concentration in TZL glasses under fs laser excitation due to resonant nonlinearity. The third-order optical nonlinearities showed an increase in the two-photon absorption coefficient (β) from 0.26 × 10−2 cm GW−1 (TZL5) to 0.62 × 10−2 cm GW−1 (TZL9), along with an increase in the nonlinear refractive index (n2) from 0.32 × 10−18 m2 W−1 to 0.65 × 10−18 m2 W−1. This enhancement is attributed to the increased number of NBOs and the high polarizability of La3+ ions. For all-optical switching (AOS) applications, two figures of merit were applied using the n2, α0, and β coefficients. The results suggest that the TZL glasses studied are potential candidates for AOS devices. Thus, this work not only elucidates the factors underlying the NLO properties but also highlights the promising potential of these glasses for use in AOS devices.

Exploring the dynamics and interactions of the N-myc transactivation domain through solution nuclear magnetic resonance spectroscopy.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Long-range conformational changes in the nucleotide-bound states of the DEAD-box helicase Vasa

DEAD-box helicases use ATP to unwind short double-stranded RNA (dsRNA). The helicase core consists of two discrete domains, termed RecA_N and RecA_C. The nucleotide binding site is harbored in RecA_N, while both RecA_N and RecA_C are involved in RNA recognition and ATP hydrolysis. In the absence of nucleotides or RNA, RecA_N and RecA_C do not interact (“open” form of the enzyme). In the presence of both RNA and ATP the two domains come together (“closed” form), building the composite RNA binding site and stimulating ATP hydrolysis. Because of the different roles and thermodynamic properties of the ADP-bound and ATP-bound states in the catalytic cycle, the conformations of DEAD-box helicases in complex with ATP and ADP are assumed to be different. However, the available crystal structures do not recapitulate these supposed differences and show identical conformations of DEAD-box helicases independent of the identity of the bound nucleotide. Here, we use NMR to demonstrate that the conformations of the ATP- and ADP-bound forms of the DEAD-box helicase Vasa are indeed different, contrary to the results from x-ray crystallography. These differences do not relate to the populations of the open and closed forms, but are intrinsic to the RecA_N domain. NMR chemical shift analysis reveals the regions of RecA_N where the average conformations of Vasa-ADP and Vasa-ATP are most different and indicates that these differences may contribute to modulating the affinity of the two nucleotide-bound complexes for RNA substrates.

1H, 13C and 15N resonance assignments of a shark variable new antigen receptor against hyaluronan synthase.

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1H,15N,13C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.

Manual and automatic assignment of two different Aβ40 amyloid fibril polymorphs using MAS solid-state NMR spectroscopy

Amyloid fibrils from Alzheimer’s amyloid-beta peptides (Aβ) are found to be polymorphic. So far, 14 Aβ40 fibril structures have been determined. The mechanism of why one particular protein sequence adopts so many different three-dimensional structures is yet not understood. In this work, we describe the assignment of the NMR chemical shifts of two Alzheimer’s disease fibril polymorphs, P1 and P2, which are formed by the amyloid-beta peptide Aβ40. The assignment is based on 13C-detected 3D NCACX and NCOCX experiments MAS solid-state NMR experiments. The fibril samples are prepared using an extensive seeding protocol in the absence and presence of the small heat shock protein αB-crystallin. In addition to manual assignments, we obtain chemical shift assignments using the automation software ARTINA. We present an analysis of the secondary chemical shifts and a discussion on the differences between the manual and automated assignment strategies.

The role of chelating agent in the self-assembly of amphoteric surfactants

Hypothesis: Limited research has been conducted on the influence of chelating agents on the self-assembly process in surfactant solutions. The traditional approach assumes the chelating agent only interferes as a salting-out ion, therefore promoting surfactant separation. However, the opposite behavior has been observed for iminodipropionate based surfactants, in which the presence of chelating agents of the aminopolycarboxylate type increases solubility of nonionic ethoxylated surfactants in mixed micellar systems. Specific interaction between chelating agents-surfactants can be an important parameter in the self-assembly processes.Experiments: Physicochemical properties of solutions containing amphoteric surfactant and tetrasodium glutamatediacetate have been investigated. Macroscopic properties, such as viscosity and cloud point, were evaluated in the presence of a non-water-soluble alkyl ethoxylated surfactant. Interactions between amphoteric surfactant and chelating agent were monitored by NMR spectroscopy, including 13C chemical shift and lineshape analysis as well as 1H diffusometry.Findings: The study reveals that there is an interaction between the head group of the surfactant and the chelating agent forming oligomeric surfactant analogues with larger hydrophilic moieties, which results in smaller, more spherical micelles. The combined interactions provide possibilities for tuning the aggregation behavior of systems containing surfactants and chelating agents, and with that, the macroscopic properties of the system.

Origin of Reactivity Trends of an Elusive Metathesis Intermediate from NMR Chemical Shift Analysis of Surrogate Analogues.

Olefin metathesis has become an efficient tool in synthetic organic chemistry to build carbon-carbon bonds, thanks to the development of Grubbs- and Schrock-type catalysts. Olefin coordination, a key and often rate-determining elementary step for d0 Schrock-type catalysts, has been rarely explored due to the lack of accessible relevant molecular analogues. Herein, we present a fully characterized surrogate of this key olefin-coordination intermediate, namely, a cationic d0 tungsten oxo-methylidene complex bearing two N-heterocyclic carbene ligands─[WO(CH2)Cl(IMes)2](OTf) (1) (IMes = 1,3-dimesitylimidazole-2-ylidene, OTf-triflate counteranion), resulting in a trigonal bipyramidal (TBP) geometry, along with its neutral octahedral analogue [WO(CH2)Cl2(IMes)2] (2)─and an isostructural oxo-methylidyne derivative [WO(CH)Cl(IMes)2] (3). The analysis of their solid-state 13C and 183W MAS NMR signatures, along with computed 17O NMR parameters, helps to correlate their electronic structures with NMR patterns and evidences the importance of the competition among the three equatorial ligands in the TBP complexes. Anchored on experimentally obtained NMR parameters for 1, computational analysis of a series of olefin coordination intermediates highlights the interplay between σ- and π-donating ligands in modulating their stability and further paralleling their reactivity. NMR spectroscopy descriptors reveal the origin for the advantage of the dissymmetry in σ-donating abilities of ancillary ligands in Schrock-type catalysts: weak σ-donors avoid the orbital-competition with the oxo ligand upon formation of a TBP olefin-coordination intermediate, while stronger σ-donors compromise M≡O triple bonding and thus render olefin coordination step energy demanding.

Computationally Assisted Analysis of NMR Chemical Shifts as a Tool in Conformational Analysis.

A key to understanding the properties of functional molecules is to determine their conformation in solution. A conformational analysis procedure that relies on quantum mechanical calculations and the widely used DP4+ probability was evaluated to decipher the structural information encoded in NMR chemical shifts. The results underscore the potential utility of using NMR chemical shifts in advancing conformational analysis studies of complex molecules in solution.

Including the Ensemble of Unstructured Conformations in the Analysis of Protein's Native State by High-Pressure NMR Spectroscopy.

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1H-15N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1H and 15N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts.

Analyse des Nativen Zustands von Proteinen durch Hochdruck NMR‐Spektroskopie unter Berücksichtigung des Ensembles Unstrukturierter Konformationen

AbstractDie Analyse druckabhängiger Änderungen der chemischen Verschiebung von Proteinen erlaubt es, Aussagen über strukturelle Fluktuationen zu machen, die diese bereits bei Umgebungsdruck aufweisen. Das Problem, allgemeine Effekte, die mit einer Erhöhung des hydrostatischen Drucks einhergehen, von strukturell bedingten Effekten auf die chemische Verschiebung zu trennen, wurde bisher durch das Einbeziehen der Eigenschaften unstrukturierter Modellpeptide angegangen. In dieser Arbeit wurden chemische Verschiebungen in 2D 1H‐15N‐HSQC Spektren von chemisch als auch druckdenaturierten Zuständen des Kälteschockproteins B aus Bacillus Subtilis (BsCspB) zugeordnet und ihre Abhängigkeit von Änderungen des hydrostatischen Drucks ausgewertet. Die durch hydrostatischen Druck denaturierte Polypeptidkette wurde verwendet, um allgemeine Effekte von strukturbedingten Effekten auf 1H und 15N chemische Verschiebungen zu trennen. Daraus resultierende Interpretationen der druckabhängigen Änderungen der chemischen Verschiebungen werden im strukturellen Kontext diskutiert. Es wurde festgestellt, dass das Ensemble unstrukturierter Konformationen von BsCspB anders auf eine Erhöhung des Drucks reagiert, als es unstrukturierte Modellpeptide tun. Daraus kann geschlussfolgert werden, dass der Ansatz, mit dem man allgemeine Effekte, die durch eine Erhöhung des Drucks hervorgerufen werden, abschätzt, die strukturellen Rückschlüsse ändert, die anhand einer Analyse von chemischen Verschiebungen getroffen werden.

NMR chemical shift assignment of Drosophila odorant binding protein 44a in complex with 8(Z)-eicosenoic acid

The odorant binding protein, OBP44a is one of the most abundant proteins expressed in the brain of the developing fruit fly Drosophila melanogaster. Its cellular function has not yet been determined. The OBP family of proteins is well established to recognize hydrophobic molecules. In this study, NMR is employed to structurally characterize OBP44a. NMR chemical shift perturbation measurements confirm that OBP44a binds to fatty acids. Complete assignments of the backbone chemical shifts and secondary chemical shift analysis demonstrate that the apo state of OBP44a is comprised of six α-helices. Upon binding 8(Z)-eicosenoic acid (8(Z)-C20:1), the OBP44a C-terminal region undergoes a conformational change, from unstructured to α-helical. In addition to C-terminal restructuring upon ligand binding, some hydrophobic residues show dramatic chemical shift changes. Surprisingly, several charged residues are also strongly affected by lipid binding. Some of these residues could represent key structural features that OBP44a relies on to perform its cellular function. The NMR chemical shift assignment is the first step towards characterizing the structure of OBP44a and how specific residues might play a role in lipid binding and release. This information will be important in deciphering the biological function of OBP44a during fly brain development.

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Dimeric eremophilane-type sesquiterpenoids from the peeled stems of Syringa pinnatifolia

A continued phytochemical investigation guided by 1H NMR and LC-MS data on the ethanol extract from the peeled stems of Syringa pinnatifolia Hemsl. led to the isolation of 16 undescribed dimeric eremophilane sesquiterpenoids, namely syringenes R–Z (1–9) and A1–G1 (10–16). These structures were elucidated by extensive analysis of spectroscopic data, including HRESIMS, NMR, quantum-mechanics-based computational analysis of NMR chemical shifts, and single-crystal X-ray diffraction analyses, and a concise rule for determination of relative configuration of angular methyl was proposed. The results of the cardioprotective assay demonstrated that 3 exhibits a protective effect against hypoxia-induced injuries in H9c2 cells. This effect was observed at a concentration of 10 μM, with a protective rate of 28.43 ± 11.80%.

Proline-Tyrosine Ring Interactions in Black Widow Dragline Silk Revealed by Solid-State Nuclear Magnetic Resonance and Molecular Dynamics Simulations.

Selective one-dimensional 13C-13C spin-diffusion solid-state nuclear magnetic resonance (SSNMR) provides evidence for CH/π ring packing interactions between Pro and Tyr residues in 13C-enriched Latrodectus hesperus dragline silk. The secondary structure of Pro-containing motifs in dragline spider silks consistently points to an elastin-like type II β-turn conformation based on 13C chemical shift analysis. 13C-13C spin diffusion measurements as a function of mixing times allow for the measurement of spatial proximity between the Pro and Tyr rings to be ∼0.5-1 nm, supporting strong Pro-Tyr ring interactions that likely occur through a CH/π mechanism. These results are supported by molecular dynamics (MD) simulations and analysis and reveals new insights into the secondary structure and Pro-Tyr ring stacking interactions for one of nature's toughest biomaterials.

Dissociation Kinetics and Antimicrobial Activity of Ofloxacin Antibiotic in Artificial Tears Via 1H-NMR, Raman, and UV-Vis Spectroscopic Analysis.

Introduction: The hydrogen-bonded networks play a significant role in influencing several physicochemical properties of ofloxacin in artificial tears (ATs), including density, pH, viscosity, and self-diffusion coefficients. The activities of the ofloxacin antibiotic with Ats mixtures are not solely determined by their concentration but are also influenced by the strength of the hydrogen bonding network which highlight the importance of considering factors such as excessive tear production and dry eye conditions when formulating appropriate dosages of ofloxacin antibiotics for eye drops. Objectives: Investigating the physicochemical properties of ofloxacin-ATs mixtures, which serve as a model for understanding the impact of hydrogen bonding on the antimicrobial activity of ofloxacin antibiotic eye drops. Determine the antimicrobial activities of the ofloxacin-Ats mixture with different concentration of ofloxacin. Methods: The ofloxacin-ATs mixtures were analyzed using 1H-NMR, Raman, and UV-Vis spectroscopies, with variation of ofloxacin concentration to study its dissociation kinetics in ATs, mimicking its behavior in human eye tears. The investigation includes comprehensive analysis of 1H-NMR spectral data, self-diffusion coefficients, Raman spectroscopy, UV-Vis spectroscopy, liquid viscosity, and acidity, providing a comprehensive assessment of the physicochemical properties. Results: Analysis of NMR chemical shifts, linewidths, and self-diffusion coefficient curves reveals distinct patterns, with peaks or minima observed around 0.6 ofloxacin mole fraction dissociated in ATs, indicating a strong correlation with the hydrogen bonding network. Additionally, the pH data exhibits a similar trend to viscosity, suggesting an influence of the hydrogen bonding network on protonic ion concentrations. Antibacterial activity of the ofloxacin-ATs mixtures is evaluated through growth rate analysis against Salmonella typhimurium, considering varying concentrations with mole fractions of 0.1, 0.4, 0.6, 0.8, and 0.9. Conclusions: The antibiotic-ATs mixture with a mole fraction of 0.6 ofloxacin exhibited lower activity compared to mixtures with mole fractions of 0.1 and 0.4, despite its lower concentration. The activities of the mixtures are not solely dependent on concentration but are also influenced by the strength of the hydrogen bonding network. These findings emphasize the importance of considering tear over-secretion and dry eye problems when designing appropriate doses of ofloxacin antibiotics for eye drop formulations.

Exploring the structure of type V deep eutectic solvents by xenon NMR spectroscopy.

Hydrophobic non-ionic (type V) deep eutectic solvents (DESs) have recently emerged as a new class of sustainable materials that have shown unique properties in several applications. In this study, type V DESs thymol : camphor, menthol : thymol and eutectic mixtures (EMs) based on menthol : carboxylic acids with variable chain length, are experimentally investigated using xenon NMR spectroscopy, with the aim to clarify the peculiar nanostructure of these materials. The results, obtained from the analysis of the 129Xe chemical shifts and of the longitudinal relaxation times, reveal a correlation between the deviation from ideality of the DESs and their structure free volume. Furthermore, the effect of varying the composition of DESs and EMs on the liquid structure is also studied.

Global minimum and a heap of low-lying isomers with planar tetracoordinate carbon in the CAl3MgH2- system.

A global minimum and a heap of low-lying isomers with planar tetracoordinate carbon (ptC) are identified in the CAl3MgH2- system by computational quantum chemical investigations. The nature of the chemical bonding in the global minimum ptC isomer is examined using the conceptual quantum chemical tools. The atoms in molecule (AIM) analysis reveals that the global minimum isomer possesses a ptC geometry. Additionally, the adaptive natural density partitioning (AdNDP), electron localization function (ELF), and nucleus-independent chemical shifts (NICS) analysis corroborate the presence of delocalization in the ptC isomer. The delocalization of electron density in the global minimum ptC isomer contributes to attaining structural stability. The results also suggest that the bridging hydrogen plays a crucial role in stabilizing the ptC system. Furthermore, the ab initio molecular dynamics study supports the structural stability of the ptC isomer.

Quantifying Intermolecular Interactions in Asymmetric Peptide Organocatalysis as a Key toward Understanding Selectivity.

The kinetic resolution of trans-cyclohexane-1,2-diol with a lipophilic oligopeptide catalyst shows extraordinary selectivities. To improve our understanding of the factors governing selectivity, we quantified the Gibbs free energies of interactions of the peptide with both enantiomers of trans-cyclohexane-1,2-diol using nuclear magnetic resonance (NMR) spectroscopy. For this, we use advanced methods such as transverse relaxation (R2), diffusion measurements, saturation transfer difference (STD), and chemical shift (δ) analysis of peptide-diol mixtures upon varying their composition (NMR titrations). The methods employed give comparable and consistent results. The molecular recognition by the catalyst is approximately 3 kJ mol-1 in favor of the preferentially acetylated (R,R)-enantiomer in the temperature range studied. Interestingly, the difference of 3 kJ mol-1 is also confirmed by results from reaction monitoring of the acylation step under catalytic conditions, indicating that this finding is true regardless of whether the investigation is performed on the acetylated species or on the free catalyst. To arrive at these conclusions, the self-association of both the catalyst and the substrate in toluene was found to play an important role and thus needs to be taken into account in reaction screening.

1H, 31P NMR, Raman and FTIR spectroscopies for investigating phosphoric acid dissociation to understand phosphate ion kinetics in body fluids

The study investigates the formation and transportation of ionic charge carriers in phosphoric acid-water system. This investigation encompasses an analysis of 1H and 31P NMR chemical shifts, self-diffusion coefficients, spin-lattice relaxation rates, spin-spin relaxation rates, activation energies, dissociation constants, electrical conductivity, and Raman shifts, along with FTIR spectra across various water concentrations. Significantly, the maxima observed in these curves at around 0.8 water molar fraction predominantly from the unique molecular arrangement between phosphoric acid and water molecules, influenced by a hydrogen bonding network. These findings yield valuable insights into phosphate ion kinetics within body fluids, covering essential aspects like hydrogen bonding networks, ionization processes, and the energy kinetics of phosphoric dissociation. A customized semiempirical model is applied to calculate dissociated species (water, phosphoric acid, and hydronium ion) at different water contents within a wide range of water mole fraction. Furthermore, this investigation extends to the dissociation of phosphoric acid in DMEM cell culture media, offering a more precise model for phosphate ionic kinetics within body fluids, especially at nominal phosphate concentrations of approximately 1:700μL.