NMR Spectroscopic Monitoring Research Articles

Developing MR reporter genes: promises and pitfalls

MR reporter genes have the potential to monitor transgene expression non-invasively in real time at high resolution. These genes can be applied to interrogate the efficacy of gene therapy, to assess cellular differentiation, cell trafficking, and specific metabolic activity, and also assess changes in the microenvironment. Efforts toward the development of MR reporter genes have been made for at least a decade, but, despite these efforts, the field is still in its early developmental stage. This reflects the fact that there are potential pitfalls, caused by the low sensitivity of detection, the need for substrates with their associated undesirable pharmacokinetics, and/or the difficult and, in some cases, delayed interpretation of signal changes. Nevertheless, significant progress has been made during the last few years. Whereas enzyme-based reporters were initially applied to NMR spectroscopic monitoring of changes in phosphor and fluorine metabolism, MRI-based approaches are now emerging that rely on: (1) enzyme-based cleavage of functional groups that block water (proton) exchange or protein binding of MR contrast agents; (2) expression of surface receptors that enable binding of specific MR contrast agents; (3) expression of para- and anti-ferromagnetic (metallo)proteins involved with iron metabolism, such as tyrosinase, transferrin receptor, and ferritin. After an introduction to the basic principles of designing promoters, expression vectors, and cloning of transgenes, a fresh look is provided on the use of reporter genes for optical (including bioluminescent) and nuclear imaging, with which MR reporter genes compete. Although progress in the use of MR reporter genes has been slow, newer strategies that use metalloproteins or alternative contrast mechanisms, with no need for substrates, promise rapid growth potential for this field.

Synthesis and Anion‐Selective Complexation of Homobenzylic Tripodal Thiourea Derivatives

AbstractCryptand‐ and tripod‐type thiourea derivatives 4b and 5a–d, which have binding functionalities at the homobenzylic positions, were synthesized as possible neutral receptors toward anions with an expectation that the three binding sites work cooperatively to bind an anion selectively. 1H NMR spectroscopic monitoring of the titration of cryptand 4b with CH3CO2–, Cl–, and F– in CDCl2CDCl2 at 100 °C showed that the binding constants were considerably smaller than those of tripodal thiourea 5a, presumably owing to the presence of strong intramolecular hydrogen bonding in 4b. Complexation constants of tripodal receptors 5a–d with H2PO4–, CH3CO2–, Cl–, and Br– anions were evaluated by 1H NMR and/or UV/Vis spectroscopic analysis of the titration in DMSO. Though tripodal receptors 5a,b undergo complexation with phosphate anion in a 1:1 stoichiometry, their association constants were not as large as simple reference compound 14 probably because of the steric hindrance around the binding sites and the large entropy cost for cooperative binding. Receptor 5c exhibits complexation in a 1:2 stoichiometry with H2PO4– and CH3CO2–, whereas it forms 1:1 complexes with chloride and bromide anions because of the subtle balance between the steric hindrance and the binding ability. However, by increasing the acidity of the thiourea functionality, receptor 5d exhibited remarkably enhanced binding ability and selectivity toward H2PO4– compared to those of reference compound 15 presumably through cooperative complexation of the three binding sites to the guest anion. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Living Isospecific Styrene Polymerization by Chiral Benzyl Titanium Complexes That Contain a Tetradentate [OSSO]-Type Bis(phenolato) Ligand

Dibenzyl complexes [Ti(OC6H2-R1-6-R2-4)2{S(CH2)2S}(CH2Ph)2] (2a−c; R1 = tBu, R2 = Me, a; R1, R2 = tBu, b; R1 = H, R2 = Me, c) with a tetradentate dianionic [OSSO]-type ligand were synthesized from the corresponding titanium dichloro complexes [Ti(OC6H2-R1-6-R2-4)2{S(CH2)2S}Cl2] and benzylmagnesium chloride in toluene. NMR spectroscopic monitoring of the reaction of the dibenzyl complex 2b with B(C6F5)3 in bromobenzene indicates the formation of a thermally sensitive benzyl cation [Ti(OC6H2-tBu2-4,6)2{S(CH2)2S}(CH2Ph)]+. 1H NMR spectra at −30 °C show two C1-symmetric diastereomers in a 55:45 ratio, suggesting slow epimerization on the NMR time scale at this temperature. The benzyl complex 2c, which does not bear bulky ortho-substituents, was found to be fluxional and unstable in solution. Related benzyl complexes of the 1,5-dithiapentanediyl-linked ligand [Ti(OC6H2-tBu-6-R-4)2{S(CH2)3S}(CH2Ph)2] (4a,b; R = Me, a; R = tBu, b) were synthesized in an analogous manner and were also shown to be highly fluxional...

The modelling and kinetic investigation of the lipase-catalysed acetylation of stereoisomeric prostaglandins

The lipase-catalysed acetylation of the hydroxyl groups of five stereoisomeric prostaglandins of type F was investigated by means of molecular dynamics simulations and the results compared with experimental observations. An NMR spectroscopic monitoring was performed to estimate reaction velocities and the regioselectivity. A molecular modelling protocol that could qualitatively differentiate between the OH groups of prostaglandins being either accessible or unaccessible to the Candida antarctica lipase B (CALB) catalysed acetylation was developed. The protocol developed analysed the protein structure deformation, the content of essential hydrogen bonds and the function-based subset energy of tetrahedral intermediates along the molecular dynamics simulations trajectory. The tetrahedral intermediates displaying a deformation RMS value lower than 3.0 Å, an essential hydrogen bond content over 50% and a subset energy less than −95 kJ/mol were classified active. In total, the accessibility of 16 out of 17 different prostaglandin OH groups was correctly predicted.

Zirconium‐Mediated Conversion of Amides to Nitriles: A Surprising Additive Effect.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Methodology for applied 4 MHz RF hyperthermia concomitant with 31P NMR spectroscopic monitoring of murine tumours

It has been generally found that solid tumours in vivo are more susceptible to destruction by heat than normal tissues. Hyperthermia has, thus, been employed in the treatment of cancer either applied alone or in combination with other modalities such as chemotherapy and radiotherapy. However, the critical mechanism(s) by which heat sensitizes and kills cells in the solid tumour remains poorly defined. Magnetic resonance spectroscopic monitoring of tumour metabolism during application of hyperthermia may provide important insight into the response to hyperthermic challenge. The implementation of dual antenna-coil methodology that provides for NMR spectroscopic monitoring (31P at 121 MHz) concomitant with applied 4 MHz RF hyperthermia in murine tumours is described herein, in some detail. This technology, which does not require advanced (and expensive) magnetic resonance imaging systems, should be readily adaptable by other laboratories with an interest in murine tumour models.

Another Aspect of the Reaction Behavior of Cyclopentadienones: 1,5‐Sigmatropic Rearrangement of the 1,4‐Addition Products of Amines

AbstractTreatment of 2,5‐bis(methoxycarbonyl)‐3,4‐diphenylcyclopentadienone (1) with aniline (2a) gave the stereoisomeric 2‐anilino‐2,5‐bis(methoxycarbonyl)‐3,4‐diphenylcyclopentenones (cis‐ and trans‐4a). 1H NMR spectroscopic monitoring of the reaction indicates that both compounds are derived from the 1,5‐sigmatropic rearrangement of the 1,4‐addition products cis‐ and trans‐3a. The structure of 3a was established by the X‐ray analysis of the product after methylation (3a‐Enol‐Me) with diazomethane. The sequential pericyclic reaction behavior of 1 with amines is discussed on the basis of X‐ray crystal structures and the optimized structure of the transition state at the B3LYP/6−31G(d) level of theory. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Preparation of 5‐Diaminomethylenebarbiturates by Barbituric Acid Addition to Carbodiimides.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Preparation of 5-diaminomethylenebarbiturates by barbituric acid addition to carbodiimides

Through NMR spectroscopic monitoring of barbituric acid addition to carbodiimide, a general synthetic procedure for the preparation of 5-diaminomethylenebarbiturates (DABA) was developed. This procedure is very simple and applicable to the preparation of large quantities of DABA derivatives. Through an X-ray structural study of one of the DABA derivatives, it was established that these compounds have an ylide-type structure with strong charge separation within the molecule.

Stereoselective oxidation of linalool with tert-butyl hydroperoxide, catalyzed by a vanadium(V) complex with a chiral terpenoid ligand

Stereoselective epoxidation of (−)-( R)-linalool by tert-butylhydroperoxide (TBHP) catalyzed by a vanadium(V) complex formed by interaction of [VO(acac) 2] or [VO(O n-Bu) 3] with a new chiral terpenoid ligand (L) is reported. Moderate diastereomeric excess (de 56%) was attained in toluene at 20 °C. 51 V , 13 C and 17 O NMR spectroscopic monitoring of the studied catalytic system showed that a new complex 1 forms quantitatively upon interaction of [VO(O n-Bu) 3] with L in CH 2Cl 2. Compound 1 is a mononuclear complex of vanadium(V), which incorporates the tridentate chiral ligand L, as well as VO moieties. It has been shown that TBHP binds to 1 giving a 1·TBHP adduct with the association constant K=1.36±0.11 l mol −1 at room temperature and enthalpy Δ r H 298 0=−6.5±2.0 kJ mol −1 and entropy Δ r S 298 0=−20±6 J mol −1 K −1. These thermodynamic parameters are typical for outer sphere complexes (OSCs). Adduct 1·TBHP is suggested to be a precursor of the active intermediate of the stereoselective epoxidation process.

Polymerization of Ethylene Catalyzed by Iron Complex Bearing 2,6-Bis(imine)pyridyl Ligand: 1H and 2H NMR Monitoring of Ferrous Species Formed via Catalyst Activation with AlMe3, MAO, AlMe3/B(C6F5)3 and AlMe3/CPh3(C6F5)4

1H and 2H NMR spectroscopic monitoring of ferrous species formed via interaction of 2,6-bis[1-(2,6-dimethylphenylimino)ethyl]pyridineiron(II) chloride (1) with AlMe3, MAO, AlMe3/B(C6F5)3 and AlMe3/CPh3 (C6F5)4 is reported. At interaction of 1 with MAO in toluene solution, the new stable heterodinuclear neutral complexes with proposed structures LFe(II)(Cl)(μ-Me)2AlMe2 and LFe(II)(Me)(μ-Me)2AlMe2 are formed (L is initial tridentate ligand). Complex LFe(II)(Cl)(μ-Me)2AlMe2 predominates at low Al/Fe ratios (less than 50), while LFe(II)(Me)(μ-Me)2AlMe2 at high Al/Fe ratios (more than 500). Complex assigned to LFe(II)(Me)(μ-Me)2AlMe2 can be prepared via interaction of 1 with AlMe3. Activation of LFe(II)(Me)(μ-Me)2AlMe2 by B(C6F5)3 and CPh3B(C6F5)4 gives rise to formation of new complexes with proposed structures [LFe(μ-Me)2AlMe2]+[MeB(C6F5)3]– and [LFe(μ-Me)2AlMe2]+ [B(C6F5)4]–. Unexpectedly, the activity at ethylene polymerization was even higher for 1/AlMe3 than for 1/MAO catalytic system. The co-catalytic activity of MAO towards 1 dramatically decreased with the diminishing of AlMe3 content in the composition of MAO. Activity of the catalyst 1/AlMe3 and the molecular structure of polyethylene produced do not change noticeably at the addition of B(C6F5)3 to 1/AlMe3.These data allow to suggest, that active species of 1/AlMe3 and 1/MAO systems are neutral methylated ferrous complexes but not cationic intermediates. Probably, complex LFe(II)(Me)(μ-Me)2 AlMe2 is the closest precursor of these active species.

1H NMR and EPR spectroscopic monitoring of the reactive intermediates of (Salen)Mn III catalyzed olefin epoxidation

Using 1H NMR and EPR spectroscopy, manganese species formed in the catalytic systems 1+iodosobenzene (PhIO) and 1+meta-chloroperoxybenzoic acid ( m-CPBA), where 1 is ( R, R)-(−)- N, N′-bis(3,5-di- tert-butylsalicylidene)-1,2-cyclohexanediamino-manganese(III) chloride ([(Salen)Mn III]) were studied. Three types of manganese complexes were characterized in the catalytic system 1+PhIO ( 4– 6). Complex 4 is very unstable and reacts with styrene at −20°C to afford styrene oxide. It exhibits three signals of tBu groups at 1.68, 1.64 and 1.42 ppm. This pattern closely resembles that for a model complex [(Salen)Mn V≡N]. Based on these data, 4 was identified as d 2 low-spin oxomanganese(V) complex [(Salen)Mn V=O] +. Complexes 5 and 6 are relatively stable at −20°C and poorly reactive towards styrene at this temperature. They display 1H NMR spectra characteristic for antiferromagnetically coupled μ-oxo-dinuclear Mn IV species and are identified as dinuclear complexes [(Salen)LMn IV- O-Mn IV(Salen)L′] with L, L′=Cl − and PhIO. It was found by EPR that the acylperoxo complex (Salen)Mn III(OOCOAr) ( 7) was formed at the first stage of the interaction of 1 with m-CPBA in CH 2Cl 2. Complex 7 is unstable and converts into manganese(IV) oxo complex [(Salen)Mn IV(O)] ( 8). The evaluated first order rate constant of this conversion is 0.25±0.08 min −1 at −70°C. Complex 7 reacts with styrene with the rate constant 1.1±0.4 M −1 min −1 at −70°C to give epoxide and restore 1. Complex 8 is inert towards styrene at low temperature. The effect of donor ligand N-methylmorpholine- N-oxide (NMO) on the epoxidation of styrene by the system 1+ m-CPBA was studied. Addition of NMO (2–5 equiv.) to the solution of 1 in CH 2Cl 2 before interaction with m-CPBA was found to dramatically increase the rate of undesirable transformation of the reactive acylperoxo complex 7-NMO into relatively inert oxo complex- 8-NMO. However, in the presence of styrene, such undesirable conversion is entirely suppressed by very rapid reaction of 8-NMO with styrene to afford styrene oxide and restore 1-NMO.

Carbonylation-decarbonylation reactions of the carbomethoxymethylcobalt complexes CH 3O 2CCHRCo(CO) 3L (R [sbnd] H, CH 2CO 2CH 3; L [sbnd] CO, PPh 3) and their acyl derivatives

The primary and secondary alkyl- and acylcobalt carbonyl complexes CH 3O 2CCHRCo(CO) 3L and CH 3O 2CCHRC(O)Co(CO) 3L (R H or CH 2CO 2CH 3; L CO, PPh 3) ( 1–8) have been prepared and studied by a variety of IR and 1H, 13C 1H, 31P 1H and 17O 1H NMR spectroscopic methods. Unlike their primary analogues, all the secondary complexes readily undergo a reversible carbonylation-decarbonylation reaction. The new alkyl complex trans-CH 3O 2CCH 2Co(CO) 3PPh 3 ( 3) was characterized in solution, and exhibited remarkably different properties from the well-known homologue trans-CH 3CH 2O 2CCH 2Co(CO) 3PPh 3, i.e. it readily decomposes to [Co(CO) 3PPh 3] 2, suggesting homolytic dissociation of the carbon-cobalt bond, and its IR spectrum exhibits an organic ν(CO) absorbance at an unusually low frequency, pointing to an oxygen-cobalt interaction. The corresponding acyl complex trans-CH 3O 2CCH 2C(O)Co(CO) 3PPh 3 ( 4) was isolated as a stable solid, but smoothly decarbonylated in solution to 3. Multinuclear NMR spectroscopic monitoring of this reaction suggested the intermediate formation of cis-CH 3O 2CCH 2Co(CO) 3PPh 3, providing the first direct evidence for alkyl migration in cobalt carbonyl complexes.

Covalent reversible binding of alkoxides or thiolates to colchicinoids

1 H NMR spectroscopic monitoring in 28:1 (CD3)2SO–CD3OD at room temp, shows that colchicine 1a reacts extensively with equimolar NaOCD3 giving reversibly the σ-adduct 2a, via CD3O– addition at C-10, as proved by similar experiments with [8,11-2H2]colchicine 1b, in which 2b was obtained. Under similar conditions, dithiocolchicine 1d also reacts extensively with equimolar NaSMe via MeS– addition at C-10 to give adduct 4. In contrast, isocolchicine 8 and equimolar NaOCD3 give only very little σ-adduct (of alternative structure 9 or 10) whereas in the case of thiocolchicine 1c and equimolar NaSEt only broadening of ring-C 1H NMR signals, most markedly of 11-H, was observed, indicating rapid exchange at C-10. It is concluded that, among a few analogies, the behaviour of colchicinoids toward bases differs sharply from that of troponoids.