Response to Comment on "Following Molecular Mobility during Chemical Reactions: No Evidence for Active Propulsion" and "Molecular Diffusivity of Click Reaction Components: The Diffusion Enhancement Question".

In their Comment (DOI: 10.1021/jacs.2c02965) on two related publications by our groups (J. Am. Chem. Soc. 2021, 143, 20884-20890; DOI: 10.1021/jacs.1c09455) and another (J. Am. Chem. Soc. 2022, 144, 1380-1388; DOI: 10.1021/jacs.1c11754), Huang and Granick discuss the diffusion NMR measurements of molecules during a copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. Here we respond to these comments and maintain that no diffusion enhancement was observed for any species during the reaction. We show that the relaxation agent does not interfere with the CuAAC reaction kinetics nor the diffusion of the molecules involved. Similarly, the gradient pulse length and diffusion time do not affect the diffusion coefficients. Peak overlap was completely removed in our study with the use of hydrazine as the reducing agent. The steady-state assumption does not hold for these diffusion measurements that take several minutes, which is the reason monotonic gradient orders are not suitable. Finally, we discuss the other reactions where similar changes in diffusion have been claimed. Our conclusions are fully supported by the results represented in our original JACS Article and the corresponding Supporting Information.

1H NMR Studies of Intramolecular OH/OH Hydrogen Bonds via Titratable Isotope Shifts.

Methanol titrations of partially deuterated 1,4- and 1,3-diols dissolved in nonpolar solvents such as CD2Cl2 and benzene-d6 have provided 1H NMR measurements of OH/OD isotope shifts, diagnostic for intact intramolecular hydrogen bonds, under conditions of increasing protic solvent concentration. 1,4- and 1,3-diols with conformationally favored intramolecular OH/OH hydrogen bonds can be titrated to constant isotope shift values, albeit with variable sign, in the presence of excess methanol equivalents, providing evidence for intact intramolecular hydrogen bonds under these conditions. Conversely, the isotope shift in a 1,3-diol with a conformationally labile intramolecular hydrogen bond titrated to zero when in the presence of excess equivalents methanol, consistent with intramolecular hydrogen bond rupture under these conditions. Additionally, the titration behavior of hydroxyl chemical shifts in diols and protected derivatives has revealed significant OH/OD isotope shifts in the absence of chemical shift differences (δOHin = δOHout) that are necessary for an equilibrium isotope effect, lending evidence for an intrinsic contribution to the isotope effect. OH/OD isotope shift titration thus provides a means for understanding the origins of these isotope effects and for probing the intact or nonintact nature of intramolecular OH/OH hydrogen bonds in response to intermolecular hydrogen bonds provided by a protic solvent.

A flow electrochemistry-enabled synthesis of 2-substituted N-(methyl-d)piperidines.

A synthesis of N-monodeuteriomethyl-2-substituted piperidines is described. An efficient and readily scalable anodic methoxylation of N-formylpiperidine in an undivided microfluidic electrolysis cell delivers methoxylated piperidine 3, which is a precursor to a N-formyliminium ion and enables C-nucleophiles to be introduced at the 2-position. The isotopically labelled N-deuteriomethyl group is installed using the Eschweiler-Clarke reaction with formic acid-d2 and unlabelled formaldehyde. Monodeuterated N-methyl groups in these molecular systems possess small isotropic proton chemical shift differences important in the investigation of molecules that are able to support long-lived nuclear spin states in solution nuclear magnetic resonance.

Following Molecular Mobility during Chemical Reactions: No Evidence for Active Propulsion.

The reported changes in self-diffusion of small molecules during reactions have been attributed to "boosted mobility". We demonstrate the critical role of changing concentrations of paramagnetic ions on nuclear magnetic resonance (NMR) signal intensities, which led to erroneous measurements of diffusion coefficients. We present simple methods to overcome this problem. The use of shuffled gradient amplitudes allows accurate diffusion NMR measurements, even with time-dependent relaxation rates caused by changing concentrations of paramagnetic ions. The addition of a paramagnetic relaxation agent allows accurate determination of both diffusion coefficients and reaction kinetics during a single experiment. We analyze a copper-catalyzed azide-alkyne cycloaddition "click" reaction, for which boosted mobility has been claimed. With our methods, we accurately measure the diffusive behavior of the solvent, starting materials, and product and find no global increase in diffusion coefficients during the reaction. We overcome NMR signal overlap using an alternative reducing agent to improve the accuracy of the diffusion measurements. The alkyne reactant diffuses slower as the reaction proceeds due to binding to the copper catalyst during the catalytic cycle. The formation of this intermediate was confirmed by complementary NMR techniques and density functional theory calculations. Our work calls into question recent claims that molecules actively propel or swim during reactions and establishes that time-resolved diffusion NMR measurements can provide valuable insight into reaction mechanisms.

Origins of Small Proton Chemical Shift Differences in Monodeuterated Methyl Groups.

We have recently shown that the small proton chemical shift difference in 2-methyl-1-(methyl-d)piperidine supports a long-lived nuclear spin state. To identify additional candidate molecules with CH2D groups exhibiting accessible long-lived states, and to investigate the factors governing the magnitude of the shift differences, we report a computational and experimental investigation of methyl rotational equilibria and proton chemical shifts in a variety of 2-substituted 1-(methyl-d)piperidines. The polarity and size of the 2-substituent affect the 1,2-stereoisomeric relationship, and consequently, the strength of the rotational asymmetry within the CH2D group. Nonpolar and large 2-substituents prefer the equatorial position, and relatively large shift differences (i.e., > 13 ppb) are observed. Polar and small substituents, however, increasingly prefer the axial position, and medium to small shift differences (i.e., 0 to 9 ppb) are observed. In addition, the diastereotopic CH2D proton chemical shift difference for tricarbonyl(1-chloro-2-deuteriomethylbenzene) chromium(0) was computed, showing that reasonable predictions of these small shift differences can be extended to more complex, organometallic species.

Z-Selective olefin metathesis on peptides: investigation of side-chain influence, preorganization, and guidelines in substrate selection.

Olefin metathesis has emerged as a promising strategy for modulating the stability and activity of biologically relevant compounds; however, the ability to control olefin geometry in the product remains a challenge. Recent advances in the design of cyclometalated ruthenium catalysts has led to new strategies for achieving such control with high fidelity and Z selectivity, but the scope and limitations of these catalysts on substrates bearing multiple functionalities, including peptides, remained unexplored. Herein, we report an assessment of various factors that contribute to both productive and nonproductive Z-selective metathesis on peptides. The influence of sterics, side-chain identity, and preorganization through peptide secondary structure are explored by homodimerization, cross metathesis, and ring-closing metathesis. Our results indicate that the amino acid side chain and identity of the olefin profoundly influence the activity of cyclometalated ruthenium catalysts in Z-selective metathesis. The criteria set forth for achieving high conversion and Z selectivity are highlighted by cross metathesis and ring-closing metathesis on diverse peptide substrates. The principles outlined in this report are important not only for expanding the scope of Z-selective olefin metathesis to peptides but also for applying stereoselective olefin metathesis in general synthetic endeavors.

Carboxylate-assisted C(sp³)-H activation in olefin metathesis-relevant ruthenium complexes.

The mechanism of C-H activation at metathesis-relevant ruthenium(II) benzylidene complexes was studied both experimentally and computationally. Synthesis of a ruthenium dicarboxylate at a low temperature allowed for direct observation of the C-H activation step, independent of the initial anionic ligand-exchange reactions. A first-order reaction supports an intramolecular concerted metalation-deprotonation mechanism with ΔG(‡)(298K) = 22.2 ± 0.1 kcal·mol(-1) for the parent N-adamantyl-N'-mesityl complex. An experimentally determined ΔS(‡) = -5.2 ± 2.6 eu supports a highly ordered transition state for carboxylate-assisted C(sp(3))-H activation. Experimental results, including measurement of a large primary kinetic isotope effect (k(H)/k(D) = 8.1 ± 1.7), agree closely with a computed six-membered carboxylate-assisted C-H activation mechanism where the deprotonating carboxylate adopts a pseudo-apical geometry, displacing the aryl ether chelate. The rate of cyclometalation was found to be influenced by both the electronics of the assisting carboxylate and the ruthenium ligand environment.

The Cambridge Anti-myopia Study: variables associated with myopia progression.

PURPOSE: To identify variables associated with myopia progression and to identify any interaction between accommodative function, myopia progression, age, and treatment effect in the Cambridge Anti-Myopia Study. METHODS: Contact lenses were used to improve static accommodation by altering ocular spherical aberration, and vision training was performed to improve dynamic accommodation. One hundred forty-two subjects, aged 14-21 years, were recruited who had a minimum of -0.75D of myopia. Subjects were assigned to contact lens treatment only, vision training only, contact lens treatment and vision training, or control group. Spherical aberration, lag of accommodation, accommodative convergence/accommodation (AC/A) ratio, accommodative facility, ocular biometry, and refractive error were measured at regular intervals throughout the 2-year trial. RESULTS: Ninety-five subjects completed the 24-month trial period. There was no significant difference in myopia progression between the four treatment groups at 24 months. Age, lag of accommodation, and AC/A ratio were significantly associated with myopia progression. There was a significant treatment effect at 12 months in the contact lens treatment group in younger subjects, based on a median split, aged under 16.9 years (p = 0.005). This treatment effect was not maintained over the second year of the trial. Younger subjects experienced a greater reduction in lag of accommodation with the treatment contact lens at 3 months (p = 0.03), compared to older contact lens treatment and control groups. There was no interaction between AC/A ratio and contact lens treatment effect. CONCLUSIONS: Age, lag of accommodation, and AC/A ratio were significantly associated with myopia progression. Although there was no significant treatment effect at 24 months, an interaction between age and contact lens treatment suggests younger subjects may be more amenable, at least in the short term, to alteration of the visual system using optical treatments.

A randomised clinical trial to assess the effect of a dual treatment on myopia progression: the Cambridge Anti-Myopia Study.

PURPOSE: To evaluate the effect of a dual treatment modality for myopia, by improving accommodative functions, on myopia progression. METHODS: A double blind randomised control trial was conducted on 96 subjects. The treatment modality for the trial employed custom designed contact lenses which control spherical aberration in an attempt to optimise static accommodation responses during near-work, and a vision-training programme to improve accommodation dynamics. Myopia progression was assessed over a 2 year period using cycloplegic autorefraction and biometry. RESULTS: The mean progression was found to be -0.33 Dioptres (D) over the 2 years of the study. There was no interaction between contact lens treatment and vision training treatment at 24 months (p = 0.72). There was no significant treatment effect of either Vision Training or Contact Lens Spherical Aberration control on myopia progression. CONCLUSIONS: This study is unable to demonstrate that the progression of myopia can be reduced over a 2 year period by either of the two treatments aimed at improving accommodative function. Neither treatment group (contact lens or vision training) progressed at a slower rate over the 2 years of the study than did the appropriate control group.

Enthalpy/entropy contributions to conformational KIEs: theoretical predictions and comparison with experiment.

Previous theoretical studies of Mislow's doubly-bridged biphenyl ketone 1 and dihydrodimethylphenanthrene 2 have determined significant entropic contributions to their normal (1) and inverse (2) conformational kinetic isotope effects (CKIEs). To broaden our investigation, we have used density functional methods to characterize the potential energy surfaces and vibrational frequencies for ground and transition structures of additional systems with measured CKIEs, including [2.2]-metaparacyclophane-d (3), 1,1'-binaphthyl (4), 2,2'-dibromo-[1,1'-biphenyl]-4,4'-dicarboxylic acid (5), and the 2-(N,N,N-trimethyl)-2'-(N,N-dimethyl)-diaminobiphenyl cation (6). We have also computed CKIEs in a number of systems whose experimental CKIEs are unknown. These include analogs of 1 in which the C=O groups have been replaced with CH2 (7), O (8), and S (9) atoms and ring-expanded variants of 2 containing CH2 (10), O (11), S (12), or C=O (13) groups. Vibrational entropy contributes to the CKIEs in all of these systems with the exception of cyclophane 3, whose isotope effect is predicted to be purely enthalpic in origin and whose Bigeleisen-Mayer ZPE term is equivalent to DDH‡. There is variable correspondence between these terms in the other molecules studied, thus identifying additional examples of systems in which the Bigeleisen-Mayer formalism does not correlate with DH/DS dissections.

Breaking the Link Between Negative Emotion and Unhealthy Eating: the Role of Emotion Regulation.

Stressful experiences frequently lead to increased consumption of unhealthy foods, high in sugar and fat yet low in nutrients. Can emotion regulation help break this link? In a laboratory experiment (N = 200), participants were encouraged to ruminate on a current, distressing personal problem, followed by instruction to use a specific emotion regulation strategy for managing feelings around that problem (challenge appraisal, relaxation/distraction, imagined social support, no-instruction control). Participants then spent 15 min on an anagram task in which 80% of items were unsolvable-a frustrating situation offering a second, implicit opportunity to use the regulation strategy. During the anagram task they had free access to a snack basket containing various options. Analyses revealed significant differences among regulation conditions in consumption of candy versus healthy snack options; challenge appraisal led to the healthiest snack choices, imagined social support to the least healthy snack choices. Supplementary Information: The online version contains supplementary material available at 10.1007/s42761-023-00190-5.

1H and 13C NMR assignments for the cyanine dyes SYBR Safe and thiazole orange.

Analysis of (1)H and (13)C NMR and mass spectral data for the fluorescent nucleic acid stain SYBR Safe indicates that it contains a cyanine-based cationic core structure identical to thiazole orange. The difference between these two compounds is the type of N-substitution on the quinolinium ring system (SYBR Safe, n-Pr; thiazole orange, Me). The (1)H and (13)C NMR resonances for both compounds were assigned on the basis of one- and two-dimensional (COSY, ROESY, HSQC, and HMBC) experiments. The preferred conformation of these compounds was computed by ab initio methods and found to be consistent with the NMR data.

Dimethyl 4,5-di-chloro-phthalate.

While endeavoring to synthesize new chlorinated ligands for ruthenium-based metathesis catalysts, the title compound dimethyl 4,5-di-chloro-phthalate, C10H8Cl2O4, was prepared from commercially available 4,5-di-chloro-phthalic acid in ∼77% yield. The title mol-ecule, which also finds utility as a precursor mol-ecule for the synthesis of drugs used in the treatment of Alzheimer's disease, shows one carbonyl-containing methyl ester moiety lying nearly co-planar with the chlorine-derivatized aromatic ring while the second methyl ester shows a significant deviation of 101.05 (12)° from the least-squares plane of the aromatic ring. Within the crystal, structural integrity is maintained by the concerted effects of electrostatic inter-actions involving the electron-deficient carbonyl carbon atom and the electron-rich aromatic ring along the a-axis direction and C-H⋯O hydrogen bonds between neighboring mol-ecules parallel to b.

Theoretical and NMR studies of deuterium isotopic perturbation of hydrogen bonding in symmetrical dihydroxy compounds.

Deuterium equilibrium isotope effects (EIEs) for a cage diol and 2,6-dihydroxyacylaromatics and complexes thereof containing intra- or intermolecular hydrogen bonds have been calculated using harmonic and anharmonic vibrational frequencies using Gaussian '03 and the HF, B3LYP, and MP2 levels of theory. The predicted isotope effects have been compared with experimental NMR data, and the origins of the isotope effects have been characterized in terms of zero-point vibrational energy differences and enthalpic and entropic contributions to the free energy difference between labeled species. Reliable free energy predictions based upon harmonic frequencies were found for systems whose isotope effects are governed by bond stretching effects and for systems whose isotope effects are determined by low-frequency vibrational modes. In contrast, thermochemical predictions based upon anharmonic frequencies were found to be far less consistent. Vibrational entropy is predicted to play an important role in modulating and, in some cases, governing isotopic site preferences in hydroxyl-derived intra- and intermolecular hydrogen bonds.

Conformations of N-heterocyclic carbene ligands in ruthenium complexes relevant to olefin metathesis.

The structure of ruthenium-based olefin metathesis catalyst 3 and model pi-complex 5 in solution and in the solid state are reported. The N-tolyl ligands, due to their lower symmetry than the traditional N-mesityl substituents, complicate this analysis, but ultimately provide explanation for the enhanced reactivity of 3 relative to standard catalyst 2. The tilt of the N-tolyl ring provides additional space near the ruthenium center, which is consistent with the enhanced reactivity of 3 toward sterically demanding substrates. Due to this tilt, the more sterically accessible face bears the two methyl substituents of the N-aryl rings. These experimental studies are supported by computational studies of these complexes by DFT. The experimental data provides a means to validate the accuracy of the B3LYP and M06 functionals. B3LYP provides geometries that match X-ray crystal structural data more closely, though it leads to slightly less (approximately 0.5 kcal mol-1) accuracy than M06 most likely because it underestimates attractive noncovalent interactions.

The effect of altering spherical aberration on the static accommodative response.

PURPOSE: To investigate the effect of altering the spherical aberration (SA) of the eye on the static accommodative response. METHODS: Participants were fitted with nominally afocal contact lenses with controlled amounts of SA of either -0.2, -0.1, 0.0, +0.1 or +0.2 microm for a 5-mm pupil. Measurements of SA and root mean square (RMS) total aberration for the eye plus lens for each participant were determined with a Complete Ophthalmic Analysis System aberrometer. Accommodation was stimulated either by placing targets at different dioptric distances from the eye, or by using a fixed distance target and placing negative-powered lenses in front of the eye. Accommodation responses were determined with a Shin-Nippon autorefractor. RESULTS: For both stimuli situations, the slope of the accommodation stimulus-response function was lowest for the lenses with +0.2 microm SA, and increased as the amount of SA was reduced. There was a significant negative correlation between SA and slope. Lag of accommodation at 33 cm correlated well with added SA, but did not correlate with total RMS error. There was no significant difference between the responses at 30 min after lens wear started and the responses after 1 h. CONCLUSIONS: Adding negative SA to the eye generally improves the slope of the accommodation stimulus-response curve and decreases lag of accommodation, and positive added SA depresses the slope of the stimulus-response curve and increases lag. The effect seems to be specific to SA, as there was no relationship between lag and RMS error. Altering SA may be a viable way of changing accommodative functions in clinical situations.

Increasing negative spherical aberration with soft contact lenses improves high and low contrast visual acuity in young adults.

PURPOSE: To evaluate the effects of a customised manipulation of spherical aberration (SA) on the high and low contrast visual acuities and contrast sensitivity of young adult myopes. METHODS: A placebo-controlled double masked trial of customised spherical aberration controlling soft contact lenses was undertaken in myopes aged 15-23. Participants wore customised soft contact lenses with either (i) zero spherical aberration or (ii) negative spherical aberration that resulted in a net SA (eye plus lens) of -0.1 micron. High and low contrast log MAR visual acuities and Pelli Robson contrast sensitivity were assessed in 196 eyes of 98 subjects after a period of 12 months wearing the lenses. RESULTS: Both high and low contrast acuities were significantly better in the group wearing the contact lenses with negative spherical aberration (high contrast log MAR, p = 0.043; low contrast log MAR, p = 0.043) which was not due to differences in residual astigmatism or pupil size between the two groups. Pelli Robson contrast sensitivity was not significantly different in the two groups. CONCLUSIONS: Manipulation of spherical aberration, taking account of the participants' baseline level of aberration, can cause statistically significant improvements in high and low contrast distance visual acuity although these improvements are too small in magnitude to be of clinical significance.

The Tripeptide RER Mimics Secreted Amyloid Precursor Protein-Alpha in Upregulating LTP.

Secreted amyloid precursor protein-alpha (sAPPα), generated by enzymatic processing of the APP, possesses a range of neurotrophic and neuroprotective properties and plays a critical role in the molecular mechanisms of memory and learning. One of the key active regions of sAPPα is the central APP domain (E2) that contains within it the tripeptide sequence, RER. This sequence is exposed on the surface of a coiled coil substructure of E2. RER has by itself displayed memory-enhancing properties, and can protect newly formed engrams from interference in a manner similar to that displayed by sAPPα itself. In order to determine whether RER mimics other properties of sAPPα, we investigated the electrophysiological effects of the N-terminal protected acetylated RER (Ac-RER) and an isoform containing a chiral switch in the first amino acid from an l- to a d-orientation (Ac-rER), on synaptic plasticity. We found that, like sAPPα, exogenous perfusion with nanomolar concentrations of Ac-RER or Ac-rER enhanced the induction and stability of long-term potentiation (LTP) in area CA1 of rat and mouse hippocampal slices, in a protein synthesis- and trafficking-dependent manner. This effect did not occur with a control Ac-AAA or Ac-IFR tripeptide, nor with a full-length sAPPα protein where RER was substituted with AAA. Ac-rER also protected LTP against amyloid-beta (Aß25 - 35)-induced LTP impairment. Our findings provide further evidence that the RER-containing region of sAPPα is functionally significant and by itself can produce effects similar to those displayed by full length sAPPα, suggesting that this tripeptide, like sAPPα, may have therapeutic potential.

Vicinal deuterium perturbations on hydrogen NMR chemical shifts in cyclohexanes.

The substitution of a deuterium for a hydrogen is known to perturb the NMR chemical shift of a neighboring hydrogen atom. The magnitude of such a perturbation may depend on the specifics of bonding and stereochemical relationships within a molecule. For deuterium-labeled cyclohexanes held in a chair conformation at -80 degrees C or lower, all four possible perturbations of H by D as H-C-C-H is changed to D-C-C-H have been determined experimentally, and the variations seen, ranging from 6.9 to 10.4 ppb, have been calculated from theory and computational methods. The predominant physical origins of the NMR chemical shift perturbations in deuterium-labeled cyclohexanes have been identified and quantified. The trends defined by the Delta delta perturbation values obtained through spectroscopic experiments and by theory agree satisfactorily. They do not match the variations typically observed in vicinal J(H-H) coupling constants as a function of dihedral angles.