Resonance Raman spectroscopy of chemically modified retinals: Assigning the carbon-methyl vibrations in the resonance Raman spectrum of rhodopsin

To assign the observed vibrationsl modes in the resonance Raman spectrum of the retinylidene chromophore of rhodopsin, we have studied chemically modified retinals. The series of analogs investigated are the n-butyl retinals substituted at C₉ and C₁₃. The results obtained for the 11-cis isomer have clearly assigned the CCH₃ vibrational frequencies observed in the spectrum of the retinylidene chromophore. The data show that the C₍₉₎CH₃ stretching vibration can be assigned to the vibrational mode observed in the 1017 cm^?1 region, and the vibration detected at 997 cm^?1 can be assigned to the C₍₁₃CH₃ vibration. The C₍₅₎CH₃ stretching mode does not contribute to the vibrations observed in this region. The splitting in the C_(n)CH₃ (n = 9, 13) vibration is characteristic of the 11-cis conformation. The results on the modified retinals do not support the hypothesis that the splitting arises from equilibrium mixtures of 11-cis, 12-s-cis and 11-cis, 12-s-trans in solution. Thus, this splitting cannot be used to determine whether the chromophore in rhodopsin is in a 12-s-cis or 12-s-trans conformation. However, our results demonstrate that there are other vibrational modes in the spectra which are sensitive to this conformational equilibrium and we use the presence of a strong ~ 1271 cm^?1 mode in bovine and squid rhodopsin spectra as an indication that the chromophore in these pigments is 11-cis, 12-s-trans.     

Conformation of cyclic analogs of enkephalin. III. Effect of varying ring size

A comparative study has been made using molecular mechanics of the ring entity of the active enkephalin analogs, Tyr-cyclo(-N^ω-D -XXX-Gly-Phe-Leu-), where XXX is variously A₂pr, A₂bu, and Orn. Several conformations are favored for all three, and the lower-energy models are compatible with a Gly³-Phe⁴ bend in the active form of enkephalin. Some difficulties in assuming standard geometries in conformational surveys are illustrated.     

Stereochemistry of protected ornithine side chains in gramicidin S derivatives and their resistance to N-methylation

Summary Derivatives of gramicidin S (GS) and its mono- and di-d-cyclohexylalanined-Cha) analogs possessing various protecting groups on Orn side chains were prepared.¹H NMR spectra of the unsymmetrically protected analogs [Orn(X)², Orn(X^′)^2′,d-Cha⁴]GS were similar to the composites of the spectra of the symmetrical derivatives [Orn(X)^2,2′,d-Cha^4,4′]GS and [Orn(X^′)^2,2′]Gs, revealing the proximity of the protecting groups of N^δH of Orn residues at the 2 and 2^′ positions to the side chains ofd-Phe (ord-Cha) residues at the 4 and 4^′ positions, respectively. The results indicated the presence of H-bonds between the N^°H of Orn and the carbonyl ofd-Phe residues in the i→i+2 sense and not in i→i-3, which was also supported by the ROESY analysis. The substantially strong H-bonds can explain the observed resistance of the urethane NH of the Orn side chains in the GS derivatives to the N-methylation with CH₃I−Ag₂O in DMF.     

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C¹X₃C²X_nC³X₃C⁴), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C¹–C⁴ and C²–C³. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.     

The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

Configurational assignments of C-nitromethyl-C-hydroxy branched-chain carbohydrate derivatives by use of a europium shift-reagent in 1 H-N.M.R. spectroscopy

Configurational assignments for the tertiary alcoholic centers of four branched-chain 3-C-nitromethylglycopyranosides, namely, methyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (1), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-glucopyranoside (4), benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-C-nitromethyl-α-D-allopyranoside (5), and methyl 4,6-O-benzylidene-3-C-nitromethyl-2-O-p-tolylsulfonyl-α-D-glucopyranoside (8), were made on the basis of the downfield chemical shifts of their identifiable protons per molar equivalent of added Eu(fod)₃, as compared with those of model compounds, of known configuration, having a close structural relationship. In some cases, the assignments were corroborated by the position of the acetyl resonances in the unshifted 60-MHz p.m.r. spectra of the corresponding O-acetyl derivatives.     

Proton magnetic resonance of the triple helix of poly(prolylprolylglycyl)n with defined degree of polymerization

Proton magnetic resonance spectra at 220 MHz were obtained for deuterium oxide and aqueous solutions of the polytripeptides (Pro-Pro-Gly)_n, which were synthesized as collagen models by the modified solid phase method. At higher temperatures, the signals of the proline C_a-protons for the peptides with n ≦ 5 and for those with n = 10 and 15 demonstrate the presence of cis and trans isomers with respect to the Gly-Pro or Pro-Pro C-N bonds. Glycine C_a-protons give typical AB type patterns. At lower temperatures, as the peptides with n = 5, 10 and 15 form triple helices, all of the resonance peaks become broad, but the whole form of the spectrum is quite similar to that of poly(l-proline) form II. The glycine C_a-proton resonances become barely detectable and the upfield peak of the two proline C_a-proton resonances fade away. At the same time, a new glycine NH resonance appears at a field slightly higher than that of a random coil. It seems to suggest that the formation of triple helices accompanies the conversion of cis proline peptide bonds into all trans bonds, and that the glycine residue environment completely changes in the helix.     

Initial reaction dynamics of proteorhodopsin observed by femtosecond infrared and visible spectroscopy

We present a comparative study using femtosecond pump/probe spectroscopy in the visible and infrared of the early photodynamics of solubilized proteorhodopsin (green absorbing variant) in D₂O with deprotonated (pD 9.2) and protonated (pD 6.4) primary proton acceptor Asp-97. The vis-pump/vis-probe experiments show a kinetic isotope effect that is more pronounced for alkaline conditions, thus decreasing the previously reported pH-dependence of the primary reaction of proteorhodopsin in H₂O. This points to a pH dependent H-bonding network in the binding pocket of proteorhodopsin, that directly influences the primary photo-induced dynamics. The vis-pump/IR-probe experiments were carried out in two different spectral regions and allowed to monitor the retinal CC (1500 cm⁻¹-1580 cm⁻¹) and CN stretching vibration as well as the amide I mode of the protein (1590 cm⁻¹-1680 cm⁻¹). Like the FTIR spectra of the K intermediate (PR_K-PR difference spectra) in this spectral range, the kinetic parameters and also the quantum efficiency of photo-intermediate formation are found to be virtually independent of the pD value.     

Synthesis of 1,2,4-tri-O-acetyl-5-deoxy-5-C-[(R and S)-methoxy-phosphinyl]-3-O-methyl-α- and -β- d-xylopyranose,and their structural analysis by 400-MHz,proton nuclear magnetic resonance spectroscopy

5-Deoxy-5-iodo-1,2-O-isopropylidene-3-O-methyl-α- d-xylofuranose, prepared quantitatively from its 5-Op-tolylsulfonyl precursor, readily gave the 5-C-(diethoxy-phosphinyl) derivative. Treatment of this compound with sodium dihydrobis(2-methoxyethoxy)aluminate, followed by hydrogen peroxide, mineral acid, and hydrogen peroxide, yielded 5-deoxy-5-C-(hydroxyphosphinyl)-3-O-methyl-α,β- d-xylopyranoses in 65% overall yield. The structures of these sugar analogs were effectively established on the basis of the mass and 400-MHz, ¹H-n.m.r. spectra of the four title compounds, derived by treatment with diazomethane and then acetic anhydride in pyridine. 5-C-[(S)-(1-Acetoxyethenyl)phosphino]-1,2,4-tri-O-acetyl-5-deoxy-3-O-methyl-β- d-xylopyranose was also isolated and characterized.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

13C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and trans$\text{N-}\text{acetyl}\text{-}\text{dl}\text{-}\text{proline}$ in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4′-C-aminoalkyl-2′-O-methyluridine adopts an C2′-endo sugar puckering in dsRNA. The 4′-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.     

The Discovery of Intramolecular Stereoelectronic Forces That Drive The Sugar Conformation in Nucleosides and Nucleotides

Abstract

This report summarizes our results⁸ on how the determination of the thermodynamics of the two-state North (N, C2′-exo-C3′-endo) ? South (S,C2′-endo-C3′-exo) pseudorotational equilibrium in aqueous solution (pD 0.6 - 12.0) basing on vicinal ³J_HH extracted from ¹H-NMR spectra measured at 500 MHz from 278K to 358K yields an experimental energy inventory of the unique stereoelectronic forces that dictate the conformation of the sugar moiety in β-D-ribonucleosides (rNs), β-D-nucleotides, in the mirror-image β-D- versus β-L-2′-deoxynucleosides (dNs) as well as in α-D- or L- versus β-D- or L-2′-dNs. Our work shows for the first time that the free-energies of the inherent internal flexibilities of β-D- versus β-L-2′-dNs and α-D- versus α-L-2′-dNs are identical, whereas the aglycone promoted tunability of the constituent sugar conformation is grossly affected in the α-nucleosides compared to the β-counterparts.     

Synthetic and spectroscopic studies of 2-C-methyl-erythritol and 2-C-methyl-threito

The synthesis of 2-C-methyl-d,l-threitol and two isopropylidene derivatives of 2-C-methyl-d-erythritol is described. ¹H and ¹³C NMR spectra of these and several related compounds are discussed.     

The coordination chemistry of bidentate tellurium ligands. I. Complexes of palladium,platinum, and mercury with large chelate rings

Complexes formed from the reaction of palladium(II) and platinum(II) halides with (p-EtO·C₆H₄)Te(CH₂)_nTe(C₆H₄OEt-p) (Lⁿ, n = 6, 7, 8, 9, 10) are reported together with data for some mercury(II) complexes, [HgLⁿCl₂] which are used for comparative purposes. The compounds [MLⁿX₂] (M  Pd, Pt; n = 7, 8 (Pt only), 9, 10; X  Cl, Br) have molecular weights in molten naphthalene which fluctuate about the monomer value. [ML⁶X₂] (M  Pd, Pt; X  Cl, Br) are totally insoluble and are believed to be polymeric. The palladium(II) complexes have trans dichloro groups whereas the platinum compounds show cis dichloro groups in the solid state.¹³C NMR spectra are valuable to confirm the coordination of the ligand; the methylene resonance of the TeCH₂ group undergoes a 19–20 ppm downfield shift on coordination. ¹²⁵Te NMR spectra of the Pd(II) and Pt(II) complexes show two broad resonances the chemical shifts of which imply the presence of cis and trans isomers in CDCl₃ solution. A more detailed variable temperature high field study of [PtL⁸Cl₂] (¹²⁵Te and ¹⁹⁵Pt NMR) reveals a complex solution chemistry involving at least two cis and two trans species. The temperature range over which the solution is stable (−10 to 70 °C) is insufficient to allow a totally unambiguous interpretation but a model based on monomer ⇍ dimer equilibria provides a self consistent interpretation.     

Molecular mobilities of soluble components in the aqueous phase of chromaffin granules

NMR relaxation times have been used to characterize molecular motion and intermolecular complexes in the aqueous phase of bovine chromaffin granules. Partially relaxed ¹³C and proton spectra have been obtained at 3 and 25°C. T₁ measurements of five protonated carbons on epinephrine (C₂, C₅, C₆ CHOH and NCH₃) give a correlation time of 0.15 (10^?9) s at 25°C for the catechol ring and methine carbon, while the effective correlation time for the NCH₃ group is somewhat shorter due to its internal degree of rotational freedom. Resonances of protonated carbons on the soluble protein chromogranin give very similar corerlation times: 0.20 (10^?9) s for the peptide α-carbon and 0.2 (10^?9) s for the methylene sidechain carbons of glutamic acid. The correlation time (τ_R) of ATP was not measured direrctly using ¹³C T₁ data due to the weakness of its spectrum, but its reorinetation appears to be substantially slower than that of epinephrine or chromogranin. This conclusion is based on three observations: (1) the qualitative temperature dependence of T₁ for H₂ and H₈ on the adenine ring places τ_R for ATP to the right of the T₁ minimum, or τ_R ? 1.0 (10^?9) s; (2) ¹³C resonances of ATP have anomalously low amplitudes compared with epinphrine resonances, a fact that is readily explained only if ATP undergoes substantially slower reorientation; and (3) a comparision of the T₁ data on H₈ on chromaffin granules and in a dilute aqueous solution, where ρ_R for ATP cam be measured directly, indicates that τ_R ～ 1.0 (10^?9 s at 25°C in the granules. The relaxation data are consistent with the concept of a storage complex based on electrostatic interaction between a polyion (chromogranin) and its counterious (ATP and epinephrine), in which ATP cross-links cationic sidechains of the protein.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

NMR and amber analysis of the neamine pharmacophore for the design of novel aminoglycoside antibiotics

The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D₂O at 25 °C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ∼80% and ∼20%, respectively, were detected at pD 7.4 with kinetic constants k_on(P→S) = 1.9771 s⁻¹ and k_off(S→P) = 1.1319 s⁻¹. A tertiary, T-state, neamine species populated at ∼3% was also detected by NMR at pD 7.4. The pKa values determined by NMR titration experiments are pKa1 6.44 ± 0.13 for N3 of ring-II, pKa2 7.23 ± 0.09 for N2′ of ring-I, pKa3 7.77 ± 0.19 for N1 of ring-II, and pKa4 8.08 ± 0.15 for N6′ of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the ⁴C₁ chair conformation between pD 3.3 and pD = 7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the ⁶rH₁ half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.     

Multinuclear magnetic resonance studies of the hydrolyzed and aquated species of the antitumor complexes cis-Pt(cyclobutylamine)2X2 and cis-Pt(NH3)(cyclobutylamine)X2 and antitumor testing of mixed-amine Pt(II) compounds

The aquated and hydrolyzed species formed from the complexes cis-Pt(cba)₂I₂ and cis-Pt(NH₃)(cba)I₂ (cba = cyclobutylamine) were studied by multinuclear (¹⁹⁵Pt, ¹⁵N and ¹H) magnetic resonance spectroscopy. The iodo ligands were removed with AgNO₃. In acidic medium, the aqueous product consists of the diaqua and the aqua-nitrato cations, although some monohydroxo-bridged dimers are formed after several hours, especially for the mixed-ligand compound. In basic medium, the main species are the dihydroxo compounds. At neutral pD, several species exist in solution, especially with the mixed-amine system, which contained also a small quantity of the symmetric cis-Pt(cba)₂ complexes. Difficulties were encountered because of the insolubility of several oligomeric species, contrary to the cis-Pt(NH₃)₂ system, probably due to the greater lipophilicity of cba compared to NH₃. Monohydroxo-bridged dimers are formed in large quantities and the stereochemistry of the mixed-amine species was determined by ¹⁵N NMR spectroscopy. For the latter system, the cyclic dihydroxo-bridged dimers are the predominant species at neutral pD after a few hours. After an extended period of time, most of the oligomers precipitate, leaving the more soluble monohydroxo-bridged dimers as the major species in solution. The preliminary antitumor testing results on several dichloro mixed-ligand compounds are listed. The results on further testing on the most active compound cis-Pt(NH₃)(cba)Cl₂ are also included.     

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4–10) by nmr spectroscopy

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle¹⁰]-neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the ¹³C chemical shifts of the C_α (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C_α shifts show that the main helical region for SPOMe lies between ⁴P and ⁹G. The C-terminus segment L? M? NH₂ is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides. © 1993 John Wiley & Sons, Inc.