Diffusion-ordered Spectroscopy Research Articles

New Tools to Probe the Protein Surface: Ultrasmall Gold Nanoparticles Carry Amino Acid Binders.

A strategy toward epitope-selective functionalized nanoparticles is introduced in the following: ultrasmall gold nanoparticles (diameter of the metallic core about 2 nm) were functionalized with molecular tweezers that selectively attach lysine and arginine residues on protein surfaces. Between 11 and 30 tweezer molecules were covalently attached to the surface of each nanoparticle by copper-catalyzed azide alkyne cycloaddition (CuAAC), giving multiavid agents to target proteins. The nanoparticles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and 1H NMR spectroscopy (diffusion-ordered spectroscopy, DOSY, and surface composition). The interaction of these nanoparticles with the model proteins hPin1 (WW domain; hPin1-WW) and Survivin was probed by NMR titration and by isothermal titration calorimetry (ITC). The binding to the WW domain of hPin1 occurred with a KD of 41 ± 2 μM, as shown by ITC. The nanoparticle-conjugated tweezers targeted cationic amino acids on the surface of hPin1-WW in the following order: N-terminus (G) ≈ R17 > R14 ≈ R21 > K13 > R36 > K6, as shown by NMR spectroscopy. Nanoparticle recognition of the larger protein Survivin was even more efficient and occurred with a KD of 8 ± 1 μM, as shown by ITC. We conclude that ultrasmall nanoparticles can act as versatile carriers for artificial protein ligands and strengthen their interaction with the complementary patches on the protein surface.

A Light-Harvesting/Charge-Separation Model with Energy Gradient Made of Assemblies of meta-Pyridyl Zinc Porphyrins.

Self-assembly of porphyrins is a fascinating topic, not only for mimicking chlorophyll assemblies in photosynthetic organisms, but also for the potential of creating molecular-level devices. Herein, zinc porphyrin derivatives bearing a meta-pyridyl group at the meso position were prepared and their assemblies studied in chloroform. Among the porphyrins studied, one with a carbamoylpyridyl moiety gave a distinct 1 H NMR spectrum in CDCl3 , which allowed the supramolecular structure in solution to be probed in detail. Ring-current-induced chemical-shift changes in the 1 H NMR spectrum, together with vapor-pressure osmometry and diffusion-ordered NMR spectroscopy, among other evidence, suggested that the porphyrin molecules form a trimer with a triangular cone structure. Incorporation of a directly linked porphyrin-ferrocene dyad with the same assembling properties in the assemblies led to a rare example of a light-harvesting/charge-separation system in which an energy gradient is incorporated and reductive quenching occurs.

The Influence of the Strength of Drug-Polymer Interactions on the Dissolution of Amorphous Solid Dispersions.

In an earlier report, ionic interactions between ketoconazole (KTZ), a weakly basic drug, and poly(acrylic acid) (PAA), an anionic polymer, resulted in a dramatic decrease in molecular mobility as well as reduced crystallization propensity of amorphous solid dispersion (ASD) in the solid state. On the other hand, weaker dipole-dipole interactions between KTZ and polyvinylpyrrolidone (PVP) resulted in ASDs with higher crystallization propensity (Mistry Mol Pharm., 2015, 12 (9), 3339-3350). In this work, we investigated the behavior of the ketoconazole (KTZ) solid dispersions in aqueous media. In vitro dissolution tests showed that the PAA ASD maintained the level of supersaturation for a longer duration than the PVP ASD at low polymer contents (4-20% w/w polymer). Additionally, the PAA ASDs were more resistant to drug crystallization in aqueous medium when measured with synchrotron X-ray diffractometry. Two-dimensional 1H nuclear Overhauser effect spectroscopy (NOESY) NMR cross peaks between ketoconazole and PAA confirmed the existence of drug-polymer interactions in D2O. The interaction was accompanied by a reduced drug diffusivity as monitored by 2D diffusion ordered spectroscopy (DOSY) NMR and enthalpy-driven when characterized by isothermal titration calorimetry (ITC). On the other hand, drug-polymer interactions were not detected between ketoconazole and PVP in aqueous solution, with NOESY, DOSY, or ITC. The results suggest that interactions that stabilize ASDs in the solid state can also be relevant and important in sustaining supersaturation in solution.

Controlling the Surface Functionalization of Ultrasmall Gold Nanoparticles by Sequence‐Defined Macromolecules

Ultrasmall gold nanoparticles (diameter about 2 nm) were surface‐functionalized with cysteine‐carrying precision macromolecules. These consisted of sequence‐defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1H NMR spectroscopy, 1H NMR diffusion‐ordered spectroscopy (DOSY), small‐angle X‐ray scattering (SAXS), and high‐resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule‐conjugated gold nanoparticles.

Self-Assembly of Benzimidazole-Derived Tris-NHC Ligands and AgI -Ions to Hexanuclear Organometallic Cages and Their Unusual Transmetalation Chemistry.

Multi-ligand self-assembly to attain the AgI -N-heterocyclic carbene (NHC)-built hexanuclear organometallic cages of composition [Ag6 (3 a,b)4 ](PF6 )6 from the reaction of benzimidazole-derived tris(azolium) salts [H3 -3 a,b](PF6 )3 with Ag2 O was achieved. The molecular structures of the cages were established by X-ray diffraction studies along with NMR and MS analyses. The existence of a single assembly in solution was supported by diffusion-ordered spectroscopy (DOSY) 1 H NMR spectra. Further, transmetalation reactions of these self-assembled complexes, [Ag6 (3 a,b)4 ](PF6 )6 , with CuI /AuI -ions provided various coinage metal-NHC complexes having diverse molecular compositions, which included the first example of a hexanuclear CuI -dodecacarbene complex, [Cu6 (3 b)4 ](PF6 )6 .

Synthesis of Terpyridine End-Modified Polystyrenes through ATRP for Facile Construction of Metallo-Supramolecular P3HT-b-PS Diblock Copolymers.

Complementary complexation between 2,2′:6′,2″-terpyridine (tpy) and 6,6″-dianthracenyl-substituted tpy in the presence of Zn(II) ions provided an efficient strategy for construction of metallo-supramolecular diblock copolymers. To synthesize well-defined tpy-modified polystyrenes (PSs), an Fe(II) bis(tpy) complex bearing α-bromoester as a metallo-initiator was applied to atom transfer radical polymerization (ATRP) to avoid poisoning the Cu(I) catalyst. Subsequently, a series of tpy-functionalized PSs was obtained after the decomplexation of <tpy-Fe(II)-tpy> junction by tetrakis(triethylammonium) ethylenediaminetetraacetate (TEA-EDTA) under mild conditions. The metallo-supramolecular poly(3-hexylthiophene) (P3HT)-block-PS diblock copolymers were prepared by simply mixing the corresponding terminally tpy-modified homopolymers with Zn(II) ions, and further characterized by 1H NMR and diffusion ordered spectroscopy (DOSY) experiments. The approach using metallo-initiators for ATRP offers an opportunity to construct tpy-functionalized polymers with controllable molecular weights and low polydispersities. Through the spontaneous heteroleptic complexation, a variety of metallo-supramolecular diblock copolymers with tunable block ratios can be easily constructed.

Hemisynthesis of coumarin derivatives from Ammodaucus leucotrichus oil extract: organobase-catalyzed reaction, analytical study by diffusion-ordered spectroscopy NMR and differential scanning calorimetry

This paper presents an in-depth chemical and analytical study of a natural substance extracted from Ammodaucus leucotrichus Coss. & Dur and its derivatives after hemisynthesis. The analysis was performed using Diffusion-Ordered Spectroscopy (NMR DOSY) and Differential Scanning Calorimetry (DSC) as general methods. The results show an interesting chemical reactivity towards coumarin-derived bisnucleophiles (4-hydroxycoumarin and triacetic acid lactone), and products obtained by hemisynthesis of pyrano[4,3-b]pyrane derivatives following Knoevenagel condensation and Michael's addition on this natural substance with the use of 4-pyrolidinopyridine organobase as catalyst.

Analysis of LDL and HDL size and number by nuclear magnetic resonance in a healthy working population: The LipoLab Study.

Atherosclerosis is the underlying process in cardiovascular disease (CVD), the first cause of death in developed countries. We aimed to identify people with no known CVD and normal values of LDL-C and HDL-C, but with alterations in the number and size of lipoprotein particles (as measured by nuclear magnetic resonance [NMR]) and to analyse their sociodemographic, clinical and biochemical characteristics. Cross-sectional study in occupational risks prevention centre in Castellón (Spain) in 2017 and 2018, in consecutively recruited adults (18-65years) with no known CVD. Sociodemographic, clinical and biochemical variables were collected. Lipid profiles were analysed (Liposcale test), along with the concentration, size and number of the main types of lipoprotein particles, determined by 2D diffusion-ordered NMR spectroscopy. Using contingency tables, we analysed the characteristics of people with normal LDL and HDL cholesterol but abnormal levels of LDL and HDL particles. The magnitude of association between explanatory variables and abnormal levels of each kind of lipoprotein was assessed with multivariable logistic regression models. Of the 400 total participants (31.3% women; age 46.4±4.3years), 169 had normal LDL and HDL cholesterol. Abnormal lipoprotein particle values depended on the subtype: prevalence of abnormal LDL levels ranged from 8.3% to 36.7%; and of HDL, from 28.4% to 42.6%. High systolic blood pressure and total cholesterol were significantly associated with abnormal LDL levels. Male sex and high systolic blood pressure were associated with abnormalities in HDL. An extended lipids profile, obtained by NMR, enables the identification of people with normal HDL-C and LDL-C levels who present abnormal levels of LDL-P and/or HDL-P. Higher total cholesterol, systolic blood pressure, BMI and male sex were significantly associated with these abnormal values.

2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems.

Many bioactive substances face the problem of limited bioavailability, mainly due to low aqueous solubility and poor metabolic stability. Their complexation with drug delivery systems offers a more optimum pharmacological profile. Some of these drug delivery systems that have promising potential form complexes with bioactive compounds such as cyclodextrins and calixarenes. The monitoring of the success and the type of the complexation are of great importance and two-dimensional diffusion-ordered NMR spectroscopy (2D DOSY) is a valuable tool for the studying of these complexes and described as "NMR chromatography." Herein we report the procedure for the complexation of the natural product quercetin in 2-hydroxypropyl-β-cyclodextrin and the anticancer drug temozolomide in p-sulfonatocalix[4]arene and the determination of the complexation with 2D DOSY spectroscopy.

Stability Evaluation and Degradation Studies of DAC® Hyaluronic-Polylactide Based Hydrogel by DOSY NMR Spectroscopy.

The stability and the degradation of polymers in physiological conditions are very important issues in biomedical applications. The copolymer of hyaluronic acid and poly-D,L-lactic acid (made available in a product called DAC®) produces a hydrogel which retains the hydrophobic character of the poly-D,L-lactide sidechains and the hydrophilic character of a hyaluronic acid backbone. This hydrogel is a suitable device for the coating of orthopedic implants with structured surfaces. In fact, this gel creates a temporary barrier to bacterial adhesion by inhibiting colonization, thus preventing the formation of the biofilm and the onset of an infection. Reabsorbed in about 72 h after the implant, this hydrogel does not hinder bone growth processes. In the need to assess stability and degradation of both the hyaluronan backbone and of the polylactic chains along time and temperature, we identified NMR spectroscopy as a privileged technique for the characterization of the released species, and we applied diffusion-ordered NMR spectroscopy (DOSY-NMR) for the investigation of molecular weight dispersion. Our diffusion studies of DAC® in physiological conditions provided a full understanding of the product degradation by overcoming the limitations observed in applying classical chromatography approaches by gel permeation UV.

Controlling the Chain Folding for the Synthesis of Single-Chain Polymer Nanoparticles Using Thermoresponsive Polymers

Synthetic polymer single-chain nanoparticles (SCNPs) are an emerging new class of nanomaterials that possess similar folded structures as natural proteins. However, most SCNPs reported so far are p...

Interactions of Cationic Diruthenium Trithiolato Complexes with Phospholipid Membranes Studied by NMR Spectroscopy.

To apprehend the possible mechanisms involved in the cellular uptake and the membrane interactions of cytotoxic dinuclear p-cymene trithiolato ruthenium(II) complexes, the interactions of the complexes [(η6-p-MeC6H4Pri)2Ru2(R1)2(R2)]+ (R1 = R2 = SC6H4-m-Pri:1; R1 = SC6H4-p-OMe, R2 = SC6H4-p-OH:2; R1 = SCH2C6H4-p-OMe, R2 = SC6H4-p-OH:3) with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles were studied using nuclear magnetic resonance (NMR) spectroscopy. 1H NMR, nuclear Overhauser effect (NOE), diffusion ordered spectroscopy (DOSY), and T1 and T2 relaxation data provided information on interactions between the complexes and the model membranes and on the submolecular localization of the complexes at the membrane interface. The results suggest that (a) the interaction takes place without new covalent adduct formation, (b) the cationic diruthenium complexes interact with DOPC head groups most likely involving electrostatic interactions while remaining structurally unchanged, (c) the changes indicating interactions are more pronounced for the most lipophilic complex 1, and (d) the diruthenium complexes remain at the exterior vesicle surface and are unlikely inserted between the phospholipid chains. The complexes also interact with micellar/free DHPC and seem to induce micellization or aggregation in solutions below critical micelle concentration (CMC). Our study suggests high affinity of the Ru complexes for the membrane surface that likely plays a key role in cellular uptake and possibly also in redistribution in mitochondria.

Dimerization of 2-Ethylhexylphosphonic Acid Mono-2-ethylhexyl Ester (HEH[EHP]) as Determined by NMR Spectrometry

ABSTRACT The aggregation state of extractant molecules is an important consideration in characterizing or modeling solvent extraction systems used in hydrometallurgy. For example, previously reported efforts to measure organic-phase dimerization constants have required the presence of an additional phase, either aqueous phase or gas phase. NMR spectroscopy can be used to probe organic-phase inter-molecular interactions without requiring the presence of an additional phase, making it possible to study the simplest organic system, that contain only extractant and diluent. In this report, the dimerization of 2-ethylhexylphosphonic acid mono-2-ethylhexl ester (HEH[EHP]) in n-dodecane and in toluene was investigated by two different NMR-based methods: chemical shifts and Diffusion Ordered SpectroscopY (DOSY). The chemical-shift analysis requires monitoring the chemical shift of the acidic proton as the concentration of HEH[EHP] changes. DOSY is a 2D NMR technique used to probe the size of molecules. The size of diffusing species was related back to the average aggregate molecular weight via a calibration curve. Because the DOSY method had not been used in this manner before, a validation of the method using the interaction constant between HDEHP and CMPO was performed. After this validation, DOSY was applied to HEH[EHP] dimerization. DOSY results demonstrated that the chemical shift of the acidic proton was the peak most affected by the dimerization state of the HEH[EHP]. All other peaks, including that of 31P, were affected more significantly by the changing dielectric constant of the solution. The dimerization constants determined were significantly lower than those reported in prior literature, possibly due to the effect of water in the literature values.

Thermodynamic Control over Molecular Aggregate Assembly Enables Tunable Excitonic Properties across the Visible and Near-Infrared.

Specific molecular arrangements within H-/J-aggregates of cyanine dyes enable extraordinary photophysical properties, including long-range exciton delocalization, extreme blue/red shifts, and excitonic superradiance. Despite extensive literature on cyanine aggregates, design principles that drive the self-assembly to a preferred H- or J-aggregated state are unknown. We tune the thermodynamics of self-assembly via independent control of the solvent/nonsolvent ratio, ionic strength, or dye concentration, obtaining a broad range of conditions that predictably stabilize the monomer (H-/J-aggregate). Diffusion-ordered spectroscopy, cryo-electron microscopy, and atomic force microscopy together reveal a dynamic equilibrium between monomers, H-aggregated dimers, and extended J-aggregated 2D monolayers. We construct a model that predicts the equilibrium composition for a range of standard Gibbs free energies, providing a vast aggregation space which we access using the aforementioned solvation factors. We demonstrate the universality of this approach among several sheet-forming cyanine dyes with tunable absorptions spanning visible, near, and shortwave infrared wavelengths.

Di(hydroperoxy)cycloalkane Adducts of Triarylphosphine Oxides: A Comprehensive Study Including Solid-State Structures and Association in Solution.

Four new di(hydroperoxy)cycloalkane adducts (Ahn adducts) of p-Tol3PO (1) and o-Tol3PO (2), namely, p-Tol3PO·(HOO)2C(CH2)5 (3), o-Tol3PO·(HOO)2C(CH2)5 (4), p-Tol3PO·(HOO)2C(CH2)6 (5), and o-Tol3PO·(HOO)2C(CH2)6 (6), have been synthesized and fully characterized. Their single crystal X-ray structures have been determined and analyzed. The 31P NMR data are in accordance with hydrogen bonding of the di(hydroperoxy)alkanes to the P═O groups of the phosphine oxides. Due to their high solubility in organic solvents, natural abundance 17O NMR spectra of 1-6 could be recorded, providing the signals for the P═O groups and additionally the two different oxygen nuclei in the O-OH groups in the adducts 3-6. The association and mobility of 3-6 were explored by 1H DOSY (diffusion ordered spectroscopy) NMR, which indicated persistent hydrogen bonding of the adducts in solution. Competition experiments with phosphine oxides allowed ranking of the affinities of the di(hydroperoxy)cycloalkanes for the different phosphine oxide carriers. On the basis of variable temperature 31P NMR investigations, the Gibbs energies of activation ΔG‡ for the adduct dissociation processes of 3-6 at different temperatures, as well as the enthalpy ΔH‡ and entropy ΔS‡ of activation, have been determined. IR spectroscopy of 3-6 corroborated the hydrogen bonding, and in the Raman spectra, the ν(O-O) stretching bands have been identified, confirming the presence of peroxy groups in the solid materials. The high solubilities in selected organic solvents have been quantified.

Aliphatic polycarbonates derived from epoxides and CO2: A comparative study of poly(cyclohexene carbonate) and poly(limonene carbonate)

The ring-opening copolymerization (ROCOP) of epoxides and CO2 provides an alternative approach towards polycarbonates and due to their aliphatic nature, they represent an interesting alternative to bisphenol-A based polycarbonates. A Lewis acidic β-diiminate (BDI)CF3-Zn-(SiMe3)2 complex 1 is used in the ROCOP of CO2 with cyclohexene oxide (CHO) and limonene oxide (LO), respectively. The knowledge gained from polymerizations monitored via in situ IR spectroscopy was used to upscale the reactions to a 1 L reactor. The two products poly(cyclohexene carbonate) (PCHC) and poly(limonene carbonate) (PLC) were then characterized via thermal analysis, a multiaxial pressure test, and dynamic mechanical analysis and compared with commercial polymers. While PCHC and PLC were both thermally stable at 150 °C for 20 min and only minor decomposition occurred at 180 °C, PLC is prone to cross-linking at elevated temperatures. This could be prevented by hydrogenation of the double bond or by the addition of an antioxidant. In the mechanical performance, the aliphatic polymers ranged between the highly impact resistant Durabio® and the brittle PMMA but broke without a splintering of the material. Overall, this study enabled a classification of CO2-based polycarbonates, especially of the novel PLC. Additionally, complex 1 was active in the terpolymerization of CHO, LO, and CO2. The formation of an actual terpolymer was confirmed via aliquot gel-permeation chromatography and diffusion-ordered NMR spectroscopy. High-pressure NMR techniques reveal an interesting kinetic feature. CHO gets copolymerized with CO2 exclusively, and LO incorporation only starts when CHO is fully consumed.

PBI‐type Polymers and Acidic Proton Conducting Ionic Liquids – Conductivity and Molecular Interactions

AbstractProton conducting ionic liquids (PILs) are discussed as new electrolytes for the use as non‐aqueous electrolytes at operation temperatures above 100 °C. During fuel cell operation the presence of significant amounts of residual water is unavoidable. The highly Brønsted‐acidic PIL 2‐Sulfoethylmethylammonum triflate [2‐Sema][TfO] is able to perform fast proton exchange processes with H2O, resulting from 1H‐NMR and pulsed field gradient (PFG)/diffusion ordered spectroscopy (DOSY) self‐diffusion measurements. Proton conduction takes place by a vehicle mechanism via PIL cations or H3O+, but also by a cooperative mechanism involving both species. Thus, highly Brønsted‐acidic PILs are promising candidates for the use as non‐aqueous electrolytes. To use [2‐Sema][TfO] as electrolyte in a proton electrolyte fuel cell (PEFC) it has to be immobilized in a host polymer. There is a (slow) uptake of the PIL by polybenzimidazole (PBI) up to a weight increase of ∼130%, due to a swelling process. A protonation of the basic imidazole moieties takes place. NMR analysis was applied to elucidate the molecular interactions between PBI, PIL, and residual water. Proton exchange, respectively an interaction between the polar groups and water can be observed in spectra, indicating a network of H‐bonds in doped PBI. Therefore, highly acidic PILs are promising candidates for the use as non‐aqueous electrolytes.

Exploring IDP-Ligand Interactions: tau K18 as A Test Case.

Over the past decade intrinsically disordered proteins (IDPs) have emerged as a biologically important class of proteins, many of which are of therapeutic relevance. Here, we investigated the interactions between a model IDP system, tau K18, and nine literature compounds that have been reported as having an effect on tau in order to identify a robust IDP–ligand system for the optimization of a range of biophysical methods. We used NMR, surface plasmon resonance (SPR) and microscale thermophoresis (MST) methods to investigate the binding of these compounds to tau K18; only one showed unambiguous interaction with tau K18. Several near neighbors of this compound were synthesized and their interactions with tau K18 characterized using additional NMR methods, including 1D ligand-observed NMR, diffusion-ordered spectroscopy (DOSY) and 19F NMR. This study demonstrates that it is possible to detect and characterize IDP–ligand interactions using biophysical methods. However, care must be taken to account for possible artefacts, particularly the impact of compound solubility and where the protein has to be immobilized.

Elucidating the Molecular Interactions of Encapsulated Doxorubicin within a Nonionic, Thermoresponsive Polyester Coacervate.

Thermoresponsive polymers that display a lower critical solution temperature (LCST) are attractive drug delivery systems (DDSs) due to their potential to encapsulate and release therapeutics in a sustained manner as a function of temperature input. To attain the full potential of such DDSs, methods that illustrate the details of drug-polymer interactions are necessary. Here, we synthesized a nonionic, coacervate-forming, thermoresponsive polyester to encapsulate doxorubicin (Dox) and used solution state NMR spectroscopy and fluorescence microscopy techniques to probe the interactions between the polymer and Dox at the molecular level. The incomplete dehydration provides a matrix for encapsulation of sensitive therapeutics and preserving their activity, while the low hysteresis property of the polyester provides rapid transition from soluble to coacervate phase. Saturation transfer difference (STD) NMR revealed the Dox-polymer interactions within the coacervates. 1H-1H nuclear Overhauser effect spectroscopy (NOESY) cross-peak differences of Dox confirmed the Dox-polymer interactions. Diffusion-ordered spectroscopy (DOSY) revealed the slower diffusion rate of Dox in the presence of polyester coacervates. These studies illustrate how the state of the polyester (below and above LCST) affects the polyester-Dox interactions and offers details of the specific functional groups involved in these interactions. Our results provide a framework for future investigations aimed at characterizing fundamental interactions in polymer-based DDSs.

Kinetic Stabilities and Exchange Dynamics of Water-Soluble Bis-Formamide Caviplexes Studied Using Diffusion-Ordered NMR Spectroscopy (DOSY).

A deep cavitand binds long-chain trans,trans- and trans,cis-bis-formamide isomers in water solution giving a pair of caviplexes in a ca. 60:40 ratio. Both caviplexes display in/out guest exchange dynamics that are slow on the 1 H NMR chemical shift timescale, but fast on the EXSY timescale. We apply diffusion-ordered NMR spectroscopy (DOSY) to characterize the caviplexes. On the diffusion timescale, the guest in/out exchange processes feature intermediate dynamics allowing the assessment of their kinetic stabilities. We found that the trans,cis-bis-formamide isomers form kinetically more stable caviplexes than the trans,trans-counterparts. We also show that the kinetic stabilities of the bis-formamide caviplexes relate well with their relative thermodynamic stabilities. Fortunately, the tuning of the DOSY parameters allowed the observation of the exchange dynamics as slow processes on the experiment timescale.