Richard C. D. Brown

Tetrazine–trans-cyclooctene ligation for the rapid construction of 18F labeled probes

A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 3,6-diaryl-s-tetrazines and an (18)F-labeled trans-cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Significance Channelrhodopsin-2 is a dimeric membrane protein functioning as a light-gated ion channel, which has triggered numerous optogenetic applications. We present the first NMR study, to our knowledge, by which structural details of the retinal cofactor could be resolved. This study was only possible by enhancing the detection sensitivity 60-fold through dynamic nuclear polarization (DNP), a highly promising hybrid method linking EPR with solid-state NMR spectroscopy. Our data show that ground-state channelrhodopsin-2 contains the retinal cofactor in its all-trans configuration with a slightly perturbed polyene chain. Three different photointermediates could be trapped and analyzed. Our study shows that DNP-enhanced solid-state NMR is a key method for bridging the gap between X-ray–based structure analysis and functional studies toward a highly resolved molecular picture. Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15N-labeled channelrhodopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P1500 K-like state, the slowly decaying late intermediate P4480, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.

A nuclear singlet lifetime of more than one hour in room-temperature solution

Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) are supremely important techniques with numerous applications in almost all branches of science. However, until recently, NMR methodology was limited by the time constant T1 for the decay of nuclear spin magnetization through contact with the thermal molecular environment. Long-lived states, which are correlated quantum states of multiple nuclei, have decay time constants that may exceed T1 by large factors. Here we demonstrate a nuclear long-lived state comprising two 13C nuclei with a lifetime exceeding one hour in room-temperature solution, which is around 50 times longer than T1. This behavior is well-predicted by a combination of quantum theory, molecular dynamics, and quantum chemistry. Such ultra-long-lived states are expected to be useful for the transport and application of nuclear hyperpolarization, which leads to NMR and MRI signals enhanced by up to five orders of magnitude.

Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization

Substances containing rapidly rotating methyl groups may exhibit long-lived states (LLSs) in solution, with relaxation times substantially longer than the conventional spin-lattice relaxation time T1. The states become long-lived through rapid internal rotation of the CH3 group, which imposes an approximate symmetry on the fluctuating nuclear spin interactions. In the case of very low CH3 rotational barriers, a hyperpolarized LLS is populated by thermal equilibration at liquid helium temperature. Following dissolution, cross-relaxation of the hyperpolarized LLS, induced by heteronuclear dipolar couplings, generates strongly enhanced antiphase NMR signals. This mechanism explains the NMR signal enhancements observed for (13)C-γ-picoline (Icker, M.; Berger, S. J. Magn. Reson. 2012, 219, 1-3).

Recent developments in solid-phase organic synthesis

This review covers the literature published during the period from October 1996 to October 1997, with an emphasis placed upon solid-phase small molecule synthesis, linkers and cleavage strategies.

TEMPO‐Mediated Electrooxidation of Primary and Secondary Alcohols in a Microfluidic Electrolytic Cell

A general procedure for the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated electrooxidation of primary and secondary alcohols modified for application in a microfluidic electrolytic cell is described. The electrocatalytic system utilises a buffered aqueous tert-butanol reaction medium, which operates effectively without the requirement for additional electrolyte, providing a mild protocol for the oxidation of alcohols to aldehydes and ketones at ambient temperature on a laboratory scale. Optimisation of the process is discussed along with the oxidation of 15 representative alcohols.

Long-lived nuclear singlet order in near-equivalent 13c spin pairs

Molecules that support (13)C singlet states with lifetimes of over 10 min in solution have been designed and synthesized. The (13)C(2) spin pairs in the asymmetric alkyne derivatives are close to magnetic equivalence, so the (13)C long-lived singlet states are stable in high magnetic field and do not require maintenance by a radiofrequency spin-locking field. We suggest a model of singlet relaxation by fluctuating chemical shift anisotropy tensors combined with leakage associated with slightly broken magnetic equivalence. Theoretical estimates of singlet relaxation rates are compared with experimental values. Relaxation due to antisymmetric shielding tensor components is significant.

Hyperpolarized singlet NMR on a small animal imaging system.

Nuclear spin hyperpolarization makes a significant advance toward overcoming the sensitivity limitations of in vivo magnetic resonance imaging, particularly in the case of low‐gamma nuclei. The sensitivity may be improved further by storing the hyperpolarization in slowly relaxing singlet populations of spin‐1/2 pairs. Here, we report hyperpolarized 13C spin order transferred into and retrieved from singlet spin order using a small animal magnetic resonance imaging scanner. For spins in sites with very similar chemical shifts, singlet spin order is sustained in high magnetic field without requiring strong radiofrequency irradiation. The demonstration of robust singlet‐to‐magnetization conversion, and vice versa, on a small animal scanner, is promising for future in vivo and clinical deployments. Magn Reson Med, 2012.

Solid-phase synthesis of cyclic sulfonamides employing a ring-closing metathesis–cleavage strategy

Abstract Ring-closing olefin metathesis using the Grubbs catalyst was applied to the cyclisative release of resin-bound sulfonamides. Flexible linkers apparently facilitated the cyclisation–cleavage, allowing a number of novel cyclic sulfonamides to be prepared in good yields using catalytic amounts of the Grubbs catalyst.

Oxidative cyclization reactions of trienes and dienynes: total synthesis of membrarollin.

Trienes and dienynes containing one electron-deficient double bond were shown to undergo regio- and stereoselective oxidative cyclization in the presence of permanganate ion to afford 2,5-bis-hydroxyalkyltetrahydrofurans (THF diols). The THF diols produced retained either alkene or alkyne functionalities, which provided convenient handles for the metal oxo-mediated introduction of an adjacent THF ring with overall control of relative and absolute stereochemistry. Adjacent bis-THFs possessing threo-cis-threo-trans-erythro, threo-cis-threo-trans-threo, threo-cis-threo-cis-erythro, threo-cis-erythro-cis-threo, or threo-cis-erythro-trans-threo relationships were synthesized by appropriate selection of alkene geometry and methodology for the closure of the second ring. The threo-cis-threo-cis-erythro stereochemical arrangement is embodied within the bis-THF core units of a number of Annonaceous acetogenins including membrarollin, while trilobacin has a threo-cis-erythro-trans-threo configured core. As an application of the selective oxidative cyclization approach, a total synthesis of membrarollin was completed in 17 linear steps from dodecyne. The C21,C22 double epimer of membrarollin was also synthesized in 15 linear steps and without recourse to the use of hydroxyl group protection.