Bis-histidyl hexacoordination in hemoglobins facilitates heme reduction kinetics

Hexacoordinate hemoglobins are a class of proteins that exhibit reversible bis-histidyl coordination of the heme iron while retaining the ability to bind exogenous ligands. One hypothesis for their physiological function is that they scavenge nitric oxide, a reaction that oxidizes the protein and requires reduction of the heme iron to continue. Reduction kinetics of hexacoordinate hemoglobins, including human neuroglobin and cytoglobin, and those from Synechocystis and rice, are compared to myoglobin, soybean leghemoglobin, and several relevant mutant proteins. In all cases, bis-histidyl coordination greatly increases the rate of reduction by sodium dithionite when compared to pentacoordinate hemoglobins. In myoglobin and leghemoglobin, reduction is limited by the rate constant for electron transfer, whereas in the hexacoordinate hemoglobins reduction is limited only by bimolecular binding of the reductant. These results can be explained by differences in the reorganization energy for reduction between hexacoordinate and pentacoordinate hemoglobins.     

The ferrous verdoheme-heme oxygenase complex is six-coordinate and low-spin

A biosynthetic and enzymatic method was developed for the preparation of 13C-labeled verdoheme, which permits the 13C NMR spectroscopic characterization of this elusive intermediate in the heme oxidation path catalyzed by the enzyme heme oxygenase. The 13C NMR data indicate that the ferrous verdoheme complex of Neisseria meningitides heme oxygenase is hexacoordinate and diamagnetic, with a proximal histidine and likely a distal hydroxide as axial ligands. The coordination number and spin state of the ferrous verdoheme-heme oxygenase complex is in stark contrast to the pentacoordinate and paramagnetic nature of the heme-heme oxygenase complex and heme centers in general.     

Determination of the 13C NMR chemical shifts in an a2u type iron(III) porphyrin cation radical

The 13C NMR chemical shifts of all the carbons in an a2u type iron(III) porphyrin radical cation, [Fe(TPP)Cl]+, have been determined for the first time by the titration method as well as by the chemical shift correlation; they are 2230, 1050, and -1910 ppm for the alpha-pyrrole, beta-pyrrole, and meso carbon atoms, respectively.     

Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using (13)C NMR and (1)H-(13)C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the (1)H chemical shift of the C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchanged upon binding. These changes in chemical shift are unexpected given the results of previous Raman studies which revealed that the C3=C2-C1=O HD enone fragment is polarized upon binding to MCAD such that the electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al. Biochemistry 2000, 39, 13982-13992). To investigate the apparent discrepancy between the NMR and Raman data for HD-CoA bound to MCAD, (13)C NMR spectra have been obtained for HD-CoA bound to enoyl-CoA hydratase, an enzyme system that has also previously been studied using Raman spectroscopy. Significantly, binding to enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by 4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement with the alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu, J.; Feng, Y.; Tonge, P. J. Biochemistry 2001, 40, 1725-33). The large increase in shielding experienced by the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring current field from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present when the enzyme is placed in an external magnetic field, also explains the differences in (13)C NMR chemical shifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used to rationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligands are bound to MCAD than when they are free in the protein solution.     

The hydroxide complex of Pseudomonas aeruginosa heme oxygenase as a model of the low-spin iron(III) hydroperoxide intermediate in heme catabolism: 13C NMR spectroscopic studies suggest the active participation of the heme in macrocycle hydroxylation

13C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe(III)-OH), where OH(-) has been used as a model of the OOH(-) ligand to gain insights regarding the elusive ferric hydroperoxide (Fe(III)-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe(III)-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S = (1)/(2) and S = (3)/(2) spin states, and the two less abundant populations exhibit pure, S = (3)/(2) and S = (1)/(2), (d(xy)())(1) electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH(-), is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH(-) in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.     

15N NMR chemical shifts for the identification of dipyrrolic structures

A variety of dipyrromethanes and dipyrromethenes have been prepared, and their 15N NMR chemical shifts have been measured by two-dimensional correlation to 1H NMR signals. The nitrogen atoms in five examples of dipyrromethanes consistently exhibit chemical shifts around -231 ppm, relative to nitromethane. Seven examples of hydrobromide salts of meso-unsubstituted dipyrromethenes consistently display 15N chemical shifts around -210 ppm, while their corresponding zinc(II) complexes exhibit chemical shifts around -170 ppm. The presence of electron-withdrawing substituents on one of the pyrrolic rings of dipyrromethenes affects the chemical shifts of both of the nitrogen nuclei in the molecule. Boron difluoride complexes of meso-unsubstituted dipyrromethenes display 15N chemical shifts around -190 ppm. Two examples of free-base dipyrromethenes bearing substituents at the meso-position exhibit 15N chemical shifts at approximately -156 ppm, and for the zinc complexes of these compounds at -162 ppm. One-bond nitrogen-hydrogen coupling constants, when measurable, were consistently in the range of -96 Hz. Since the measured 15N chemical shifts have such a high regularity correlated to structure, they can be used as diagnostic indications for identifying the structure of dipyrrolic compounds.     

Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.

Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.     

Carbon-13 NMR spectra of some 4-substituted phenacyl bromides

The ¹³C NMR signals for some 4- substituted phenacyl bromides were assigned. The experimental chemical shifts of the aromatic ring carbons are in close agreement with those calculated using substituent chemical shifts. Both the carbonyl and the α-methylene carbons exhibit upfield shifts compared with those of the corresponding 4-substituted acetophenones.     

Electronic structures of six-coordinate ferric porphyrin complexes with weak axial ligands: usefulness of 13C NMR chemical shifts

1H NMR, (13)C NMR, and EPR spectra of six-coordinate ferric porphyrin complexes [Fe(Por)L2]ClO4 with different porphyrin structures are presented, where porphyrins (Por) are planar 5,10,15,20-tetraphenylporphyrin (TPP), ruffled 5,10,15,20-tetraisopropylporphyrin (TiPrP), and saddled 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin (OETPP), and axial ligands (L) are weak oxygen ligands such as pyridine-N-oxide, substituted pyridine-N-oxide, DMSO, DMF, MeOH, THF, 2-MeTHF, and dioxane. These complexes exhibit the spin states ranging from an essentially pure high-spin (S = 5/2) to an essentially pure intermediate-spin (S = 3/2) state depending on the field strength of the axial ligands and the structure of the porphyrin rings. Reed and Guiset reported that the pyrrole-H chemical shift is a good probe to determine the spin state in the spin admixed S = 5/2,3/2 complexes (Reed, C. A.; Guiset, F. J. Am. Chem. Soc. 1996, 118, 3281-3282). In this paper, we report that the chemical shifts of the alpha- and beta-pyrrole carbons can also be good probes to determine the spin state because they have shown good correlation with those of the pyrrole-H or pyrrole-C(alpha). By putting the observed or assumed pyrrole-H or pyrrole-C(alpha) chemical shifts of the pure high-spin and pure intermediate-spin complexes into the correlation equations, we have estimated the carbon chemical shits of the corresponding complexes. The orbital interactions between iron(III) and porphyrin have been examined on the basis of these chemical shifts, from which we have found that both the d(xy)-a(2u) interaction in the ruffled Fe(T(i)PrP)L2+ and d(xy)-a(1u) interaction in the saddled Fe(OETPP)L2+ are quite weak in the high-spin and probably in the intermediate-spin complexes as well. Close inspection of the correlation lines has suggested that the electron configuration of an essentially pure intermediate-spin Fe(T(i)PrP)L2+ changes from (d(xy), d(yz))3(d(xy))1(d(z)2)1 to (d(xy))2(d(xz), d(yz))2(d(z)2)1 as the axial ligand (L) changes from DMF to MeOH, THF, 2-MeTHF, and then to dioxane. Although the DFT calculation has indicated that the highly saddled intermediate-spin Fe(OETPP)(THF)2+ should adopt (d(xy), d(yz))3(d(xy))1(d(z)2)1 rather than (d(xy))2(d(xz), d(yz))2(d(z)2)1 because of the strong d(xy)-a(1u) interaction (Cheng, R.-J.; Wang, Y.-K.; Chen, P.-Y.; Han, Y.-P.; Chang, C.-C. Chem. Commun. 2005, 1312-1314), our 13C NMR study again suggests that Fe(OETPP)(THF)2+ should be represented as (d(xy))2(d(xz), d(yz))2(d(z)2)1 because of the weak d(xy)-a(1u) interaction. The contribution of the S = 3/2 state in all types of the spin admixed S = 5/2,3/2 six-coordinate complexes has been determined on the basis of the (13)C NMR chemical shifts.     

Homo- and methano[60]fullerenes with chiral attached moieties--1H and 13C NMR chemical shift assignments and diastereotopicity effects

(1)H and (13)C NMR chemical shift predictions of homo- and methano[60]fullerenes containing chiral centers in attached fragment were made using the two-dimensional NMR technique (HH COSY, (1)H-(13)C HSQC and HMBC) and the quantum chemistry GIAO calculation method in the PBE/3ζ approach. The influence of a chiral substituent on the (13)C chemical shifts of diastereotopic fullerene carbons was estimated by comparing the calculated and experimental (13)C NMR spectra. The resonances of the fullerene carbons in α-, β- and δ-positions relative to the position of the substituent exhibit the greatest diastereotopic splitting.     

Synthesis and characterization of reduced heme and heme/copper carbonmonoxy species

Carbon monoxide readily binds to heme and copper proteins, acting as a competitive inhibitor of dioxygen. As such, CO serves as a probe of protein metal active sites. In our ongoing efforts to mimic the active site of cytochrome c oxidase, reactivity toward carbon monoxide offers a unique opportunity to gain insight into the binding and spectroscopic characteristics of synthetic model compounds. In this paper, we report the synthesis and characterization of CO-adducts of ((5/6)L)Fe(II), [((5/6)L)Fe(II)...Cu(I)](B(C(6)F(5))(4)), and [(TMPA)Cu(I)(CH(3)CN)](B(C(6)F(5))(4)), where TMPA = tris(2-pyridylmethyl)amine and (5/6)L = a tetraarylporphyrinate tethered in either the 5-position ((5)L) or 6-position ((6)L) to a TMPA copper binding moiety. Reaction of ((5/6)L)Fe(II) [in THF (293 K): UV-vis 424 (Soret), 543-544 nm; (1)H NMR delta(pyrrole) 52-59 ppm (4 peaks); (2)H NMR (from ((5)L-d(8))Fe(II)) delta(pyrrole) 53.3, 54.5, 55.8, 56.4 ppm] with CO in solution at RT yielded ((5/6)L)Fe(II)-CO [in THF (293 K): UV-vis 413-414 (Soret), 532-533 nm; IR nu(CO)(Fe) 1976-1978 cm(-1); (1)H NMR delta(pyrrole) 8.8 ppm; (2)H NMR (from ((5)L-d(8))Fe(II)-CO) delta(pyrrole) 8.9 ppm; (13)C NMR delta((CO)Fe) 206.8-207.1 ppm (2 peaks)]. Experiments repeated in acetonitrile, acetone, toluene, and dichloromethane showed similar spectroscopic data. Binding of CO resulted in a change from five-coordinate, high-spin Fe(II) to six-coordinate, low-spin Fe(II), as evidenced by the upfield shift of the pyrrole resonances to the diamagnetic region ((1)H and (2)H NMR spectra). Addition of CO to [((5/6)L)Fe(II)...Cu(I)](B(C(6)F(5))(4)) [in THF (293 K): UV-vis ((6)L only) 424 (Soret), 546 nm; (1)H NMR delta(pyrrole) 54-59 ppm (multiple peaks); (2)H NMR (from [((5)L-d(8))Fe(II).Cu(I)](B(C(6)F(5))(4))) delta(pyrrole) 53.4 ppm (br)] gave the bis-carbonyl adduct [((5/6)L)Fe(II)-CO...Cu(I)-CO](B(C(6)F(5))(4)) [in THF (293 K): UV-vis ((6)L only) 413 (Soret), 532 nm; IR nu(CO)(Fe) 1971-1973 cm(-1), nu(CO)(Cu) 2091-2093 cm(-1), approximately 2070(sh) cm(-1); (1)H NMR delta(pyrrole) 8.7-8.9 ppm; (2)H NMR (from [((5)L-d(8))Fe(II)-CO...Cu(I)-CO](B(C(6)F(5))(4))) delta(pyrrole) 8.9 ppm; (13)C NMR delta((CO)Fe) 206.8-208.1 ppm (2 peaks), delta((CO)Cu) 172.4 ((5)L), 178.2 ((6)L) ppm]. Experiments in acetonitrile, acetone, and toluene exhibited spectral features similar to those reported. The [((5/6)L)Fe(II)-CO.Cu(I)-CO](B(C(6)F(5))(4)) compounds yielded (CO)(Fe) spectra analogous to those seen for ((5/6)L)Fe(II)-CO and (CO)(Cu) spectra similar to those seen for [(TMPA)Cu(I)-CO](B(C(6)F(5))(4)) [in THF (293 K): IR nu(CO)(Cu) 2091 cm(-1), approximately 2070(sh) cm(-1); (13)C NMR delta((CO)Cu) 180.3 ppm]. Additional IR studies were performed in which the [((5)L)Fe(II)-CO...Cu(I)-CO](B(C(6)F(5))(4)) in solution was bubbled with argon in an attempt to generate the iron-only mono-carbonyl [((5)L)Fe(II)-CO.Cu(I)](B(C(6)F(5))(4)) species; in coordinating solvent or with axial base present, decreases in characteristic IR-band intensities revealed complete loss of CO from copper and variable loss of CO from the heme.     

Solution NMR determination of the seating(s) of meso-nitro-etioheme-1 in myoglobin: implications for steric constraints to meso position access in heme degradation by coupled oxidation

The highly stereoselective cleavage of hemin in myoglobin by coupled oxidation has been attributed to steric barriers that leave more space near the alpha- than the other meso-positions. The steric barriers near meso positions in myoglobin have been investigated by establishing the thermodynamics and dynamics of possible seatings in the pocket of horse myoglobin of a four-fold symmetric etioheme I modified with a bulky nitro group at a single meso position. The cyanomet complex of this reconstituted myoglobin exhibits three sets of (1)H NMR resonances that are linked dynamically and occur in approximate populations ratios of 0.82:0.10:0.08. Two dimensional (1)H NMR has been used to assign the hemin and heme pocket resonances in the major isomer in solution and to determine that the hemin is oriented with the nitro group at the canonical gamma-meso position of native hemin. The dominance of this isomer is attributed to the solvent exposure of this portion of the hemin which stabilizes the highly polar nitro group. Using a combination of magnetization transfer among methyl groups of the three isomers due to "hopping" of the hemin about its normal, the assigned resonances of an isoelectronic, bis-cyano complex of meso-nitro-etioheme I, and the known essentially constant rhombic perturbation of heme pocket sites on the hyperfine shifts of heme methyl (Kolczak, U.; Hauksson, J. B.; Davis, N. L.; Pande, U.; de Ropp, J. S.; Langry, K. C.; Smith, K. M.; LaMar, G. N. J. Am. Chem. Soc. 1999, 121, 835-843); the two minor isomers are shown to place their bulky nitro group at the canonical delta-meso (8%) and alpha-meso positions (10%). The comparable population of the isomers with nitro groups at the hydrophobic alpha- and delta-meso positions dictates that, while the static crystal structure finds more room near the alpha-meso position, the deformation at minimal energetic expense near the alpha- and delta-meso positions is comparable. These results argue that factors other than simple steric influences control the selectivity of the ring cleavage in myoglobin.     

Azide-inhibited bacterial heme oxygenases exhibit an S = 3/2 (dxz,dyz)3(dxy)1(dz2)1 spin state: mechanistic implications for heme oxidation

The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of (1)H and (13)C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S = (1)/(2), (d(xy))(2)(d(xz), d(yz))(3) electron configuration and planar heme and a second with a novel S = (3)/(2), (d(xz), d(yz))(3)(d(xy))(1)(d(z)(2))(1) spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the sigma-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH(-) (J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.     

Radical energies and the regiochemistry of addition to heme groups. Methylperoxy and nitrite radical additions to the heme of horseradish peroxidase

The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons and/or vinyl groups of the electrophilic or radical species generated in the catalytic oxidation of halides, pseudohalides, carboxylic acids, aryl and alkyl hydrazines, and other substrates. The determinants of the regiospecificity of these reactions, however, are unclear. We report here modification of the heme of HRP by autocatalytically generated, low-energy NO2* and CH3OO* radicals. The NO2* radical adds regioselectively to the 4- over the 2-vinyl group but does not add to the meso positions. Reaction of HRP with tert-BuOOH does not lead to heme modification; however, reaction with the F152M mutant, in which the heme vinyls are more sterically accessible, results in conversion of the heme 2-vinyl into a 1-hydroxy-2-(methylperoxy)ethyl group [-CH(OH)CH2OOCH3]. [18O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO* radical add to both the delta-meso carbon and the 2-vinyl group. The regiochemistry of these and the other known additions to the heme indicate that only high-energy radicals (e.g., CH3*) add to the meso carbon. Less energetic radicals, including NO2* and CH3OO*, add to heme vinyl groups if they are small enough but do not add to the meso carbons. Electrophilic species such as HOBr, HOCl, and HOSCN add to vinyl groups but do not react with the meso carbons. This meso- versus vinyl-reactivity paradigm, which appears to be general for autocatalytic additions to heme prosthetic groups, suggests that meso hydroxylation of the heme by heme oxygenase occurs by a controlled radical reaction rather than by electrophilic addition.     

Tryptophan chemical shift in peptides and proteins: a solid state carbon-13 nuclear magnetic resonance spectroscopic and quantum chemical investigation

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.     

1H and 13C NMR spectra of Strychnos alkaloids: Selected NMR updates

The PBE0/pcSseg-2//pcseg-2 calculations of ¹H and ¹³C NMR chemical shifts were performed for a classical series of 12 Strychnos alkaloids (except for the earlier studied parent strychnine), namely akuammicine, isostrychnine, rosibiline, tsilanine, spermostrychnine, diaboline, cyclostrychnine, henningsamide, strychnosilidine, strychnobrasiline, holstiine, and icajine. It was found that the calculated ¹H and ¹³C NMR chemical shifts show markedly good correlations with available experimental data, as characterized by a mean absolute error of 0.22 ppm for the range of 8 ppm for protons and 1.97 ppm for the range of 180 ppm for carbons. Complementarily, the present results provide essential NMR update and fill a gap in the NMR data of this distinguished group of vitally important natural products.     

The 1H and 13C NMR chemical shifts of Strychnos alkaloids revisited at the DFT level

The density functional theory calculation of ¹H and ¹³C NMR chemical shifts in a series of ten 10 classically known Strychnos alkaloids with a strychnine skeleton was performed at the PBE0/pcSseg-2//pcseg-2 level. It was found that calculated ¹H and ¹³C NMR chemical shifts provided a markedly good correlation with experiment characterized by a mean absolute error of 0.08 ppm in the range of 7 ppm for protons and 1.67 ppm in the range of 150 ppm for carbons, so that a mean absolute percentage error was as small as ~1% in both cases.     

Empirically predicted 13C NMR chemical shifts for 8-hydroxyflavone starting from 7,8,4'-trihydroxyflavone and from 7,8-dihydroxyflavone

8-Hydroxyflavone is not found in nature. While the (13)C chemical shifts of 8-hydroxyflavone have been reported previously, the observed (13)C chemical shifts were not assigned. A previously reported empirical predictive tool has been applied in reverse in order to deconvolute the (13)C chemical shifts for 8-hydroxyflavone from each of those of 7,8,4'-trihydroxyflavone and 7,8-dihydroxyflavone together with those of 7-hydroxyflavone, 4'-hydroxyflavone, and flavone. The two sets of calculated (13)C chemical shifts for 8-hydroxyflavone are in good agreement with each other in that the average absolute difference is 0.4 ppm. The previously reported but unassigned (13)C chemical shifts for 8-hydroxyflavone have been assigned by matching them with the averages of the two sets of calculated (13)C chemical shifts for 8-hydroxyflavone such that the minimum average absolute difference is 0.63 ppm. The assigned (13)C chemical shifts of 8-hydroxyflavone may be used, along with the (13)C chemical shifts of the remaining monohydroxyflavones, as part of a predictive tool to rapidly assess the (13)C NMR spectra of C8-hydroxylated flavonoids.     

13C-NMR-Spektren isomerer Diazaphenanthrene

The ¹³C chemical shifts of eleven isomeric diazaphenathrenes (1.5-? 1.10-, 2.7-, 4.5-? 4.7-, and 5.6-DAP) have been determined and iteratively assigned by means of comparison with suitable model compounds. The data obtained (132 points) were used to test the relationship between ¹³C chemical shifts and HMO charge densities. The best correlation with a standard deviation S(E) = 4.8 ppm was found for the chemical shifts, relative to phenanthrene, of tertiary carbons. The different slopes for correlations of tertiary and quarternary carbons (275–300 vs 540–550 ppm/electron) are most probably due to different ΔE values for both types of carbons.