Pyrrolidide formation as a side reaction during activation of carboxylic acids by phosphonium salt coupling reagents

Pyrrolidide derivatives are observed as unwanted by-products from slow reactions of activated carboxylates with nucleophilic amines, as mediated by phosphonium salt coupling reagents (PyAOP, PyBOP, PyBroP). This side reaction is attributed to the presence of small amounts (e.g., 0.5%, w/w) of pyrrolidine as a contaminant to commercial phosphonium salts, and does not occur when the reagents are crystallized before their use in coupling reactions.     

Pyrrolidide formation as a side reaction during activation of carboxylic acids by phosphonium salt coupling reagents

Summary Pyrrolidide derivatives are observed as unwanted by-products from slow reactions of activated carboxylates with nucleophilic amines, as mediated by phosphonium salt coupling reagents (PyAOP, PyBOP, PyBroP). This side reaction is attributed to the presence of small amounts (e.g., 0.5%, w/w) of pyrrolidine as a contaminant to commerical phosphonium salts, and does not occur when the reagents are crystallized before their use in coupling reactions.     

Structural dynamics of protein backbone φ angles: extended molecular dynamics simulations versus experimental 3 J scalar couplings

³ J scalar couplings report on the conformational averaging of backbone φ angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the experimental ³ J scalar couplings providing a slightly better reproduction of these experimental parameters than a high-resolution static structure.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

Inhibition of carbonic anhydrase isoforms I,II, IV,VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the carboxylates. This study proves that carboxylic acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA inhibitors.     

Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets

The p53 tetramer recognizes specifically a 20-bp DNA element. Here, we examined symmetries encoded in p53 response elements (p53REs). We analyzed base inversion correlations within the half-site, as well as in the full-site palindrome. We found that p53REs are not only direct repeats of half-sites; rather, two p53 half-sites couple to form a higher order 20bp palindrome. The palindrome couplings between the half-sites are stronger for the human than for the mouse genome. The full-site palindrome and half-site palindrome are controlled by insertions between the two half-sites. The most notable feature is that the full-site palindrome with coupling between quarter-sites one and four (H14 coupling) dominates the p53REs without insertions. The most frequently observed insertion in human p53REs of 3bp enhances the half-site palindrome. The statistical frequencies of the coupling between the half-sites in the human genome correlate with grouped experimental p53 affinities with p53REs. Examination of known p53REs indicates the H14 couplings are stronger for positive regulation than for negatively regulated p53REs, with repressors having the lowest H14 couplings. We propose that the palindromic sequence couplings may encode such potential preferred multiple binding modes of the p53 tetramer to DNA.     

Mapping the substrate scope of monoamine oxidase (MAO-N) as a synthetic tool for the enantioselective synthesis of chiral amines

A library of 132 racemic chiral amines (α-substituted methylbenzylamines, benzhydrylamines, 1,2,3,4-tetrahydronaphthylamines (THNs), indanylamines, allylic and homoallylic amines, propargyl amines) was screened against the most versatile monoamine oxidase (MAO-N) variants D5, D9 and D11. MAO-N D9 exhibited the highest activity for most substrates and was applied to the deracemisation of a comprehensive set of selected primary amines. In all cases, excellent enantioselectivity was achieved (e.e. >99%) with moderate to good yields (55–80%). Conditions for the deracemisation of primary amines using a MAO-N/borane system were further optimised using THN as a template addressing substrate load, nature of the enzyme preparation, buffer systems, borane sources, and organic co-solvents.     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Selective oxidation and N-coupling by purified laccase of Xylaria polymorpha MTCC-1100

The chemical route of oxidation of methyl group to its aldehyde is inconvenient because once a methyl group is attacked, it is likely to be oxidized to the carboxylic acid and it is very difficult to stop the reaction at the aldehyde stage. Fungal laccases can be used for such oxidation reaction and the reaction can be completed sharply within 1–2 h. Coupling of amines are another important reactions known for fungal laccases; coupling reactions generally take 3–7 h. We have used the purified laccase of molecular weight 63 kDa obtained from the fungal strain Xylaria polymorpha MTCC-1100 with activity of 1.95 IU/mL for selective oxidation of 2-fluorotoluene, 4-fluorotoluene, and 2-chlorotoluene to 2-fluorobenzaldehyde, 4-fluorobenzaldehyde, and 2-chlorobenzaldehyde, respectively, and syntheses of 3-(3, 4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines. In each oxidation reactions, ABTS was used as mediator molecule. All the syntheses are ecofriendly and were performed at room temperature.     

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Multinuclear magnetic resonance spectroscopy and crystal structures of iodo-bridged dinuclear Pt(II) complexes with amines

Complexes of the types cis-Pt(amine)₂I₂ were transformed into the iodo-bridged dimers, which were characterized mainly by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. For bulby amines, the dinuclear species were synthesized directly from K₂[PtI₄]. Compounds with several primary aliphatic and cyclic amines and two secondary amines were studied. In ¹⁹⁵Pt NMR, two signals were observed between −3899 and −4080 ppm in acetone. These species were assigned to the cis and trans dinuclear compounds I(amine)Pt(μ-I)₂PtI(amine). We suggest that the most shielded compound is the trans isomer. The difference between the two isomers is 12-13 ppm for the primary amine system and 26-27 ppm for the two secondary amines. There seems to be a slight dependence of the proton affinity in the gas phase of the amine (linear amines) with the δ(Pt) chemical shifts of the dinuclear Pt(II) compounds. The ²J(¹⁹⁵Pt-¹HN) coupling constants are slightly larger for the trans isomers (average 67 Hz, vs. 56 Hz). The ³J(¹⁹⁵Pt-¹H) coupling constants were detected only for the dimethylamine compounds, 46 Hz (trans) and 44 Hz (cis). In ¹³C NMR, the values of ²J(¹⁹⁵Pt-¹³C) and ³J(¹⁹⁵Pt-¹³C) were also found to be very slightly larger for the trans complexes (average 19 and 25 Hz vs. 15 and 18 Hz). The structures were confirmed by X-ray diffraction studies of the n-butylamine and diethylamine compounds. The two crystals were those of the trans dinuclear complexes.     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.     

Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and 3-methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones

The synthesis of 5-alkylamino- and 2,5-bis(alkylamino)-[1,4]-benzoquinones, showing structural similarity to natural mitomycins, was performed through coupling of 2-methoxy-3-methylhydroquinone with primary amines such as n-octylamine, geranylamine and cyclooctylamine using laccases from Myceliophthora thermophila (MtL) and Pycnoporus cinnabarinus SBUG-M 1044 (PcL). Product spectra of laccase reactions differ due to reaction systems pH values (pH 7.0 for MtL and pH 5.0 for PcL) applied to assure enzymes optimal catalytic efficiency. The MtL- and PcL-mediated formation of monoaminated products was achieved at equimolar reactant concentrations with amine coupling at the meta-position to benzoquinones methyl group. Increased formation of diaminated products occurred in PcL-mediated reactions and generally when the amine was supplied in excess. Diamination entailed elimination of the benzoquinone methoxy group (amination in para-position to the first amine substituent). Six products were synthesised and characterised by NMR and HR-MS analysis. The laccase-mediated amine coupling to 2-methoxy-3-methylhydroquinone confers two of the essential pharmaceutical active motifs from mitomycins: (i) a stable 1,4-benzoquinoic parent structure and (ii) a biological active alkylation function (NH).     

Protein backbone structure determination using only residual dipolar couplings from one ordering medium

Residual dipolar couplings provide significant structural information for proteins in the solution state, which makes them attractive for the rapid determination of protein folds. Unfortunately, dipolar couplings contain inherent structural ambiguities which make them difficult to use in the absence of additional information. In this paper, we describe an approach to the construction of protein backbone folds using experimental dipolar couplings based on a bounded tree search through a structural database. We filter out false positives via an overlap similarity measure that insists that protein fragments assigned to overlapping regions of the sequence must have self-consistent structures. This allows us to determine a backbone fold (including the correct C-C bond orientations) using only residual dipolar coupling data obtained from one ordering medium. We demonstrate the applicability of the method using experimental data for ubiquitin.     

Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of α-tocopherol and its model compound in solution

The electron nuclear double resonance (ENDOR) spectra of chromanoxyl radicals obtained by the PbO₂ oxidation of α-tocopherol and its model compound were observed in t-butylbenzene, and the proton hyperfine coupling constants were correctly determined. Each of the two β- and γ-methylene protons in the chromanoxyl ring shows an equivalent hyperfine splitting, suggesting that the heterocyclic ring attached to the aromatic ring are coplanar with the plane of the aromatic system. A comparison of the hyperfine couplings in α-tocopheroxyl radical and its model shows that the introduction of a long-isoprenoid-chain in the α-tocopherol in place of a methyl group in the model compound has very little effect on the unpaired spin distribution or molecular structure of the chromanoxyl skeleton. The results of McLachlan molecular orbital (MO) calculations were found to be in satisfactory agreement with the ‘experimental’ spin densities evaluated from the hyperfine coupling constants.     

Synthesis and evaluation of thymine-derived carboxamides against mitochondrial thymidine kinase (TK-2) and related enzymes

Based on the structure of our previously identified mitochondrial thymidine kinase (TK-2) inhibitors, three series of thymine-derived carboxamides have been synthesized and tested against TK-2 and related enzymes. The methodology employed has been a solution-phase parallel synthesis based on the coupling of three thymine-derived acids [4-(thymin-1-yl)butyric acid (I), [4-(thymin-1-yl)-butyrylamino]acetic acid (II) and 6-(thymin-1-yl)hexanoic acid (III)] with different commercially available primary amines that carry cyano and/or phenyl groups. The couplings were performed in good yields (from 60% to 90%), with the exception of those that incorporate the highly crowded triphenylmethylamine (e). From the new synthesized compounds, the N-trityl-6-(thymin-1-yl)hexanamide (IIIe) was the most active TK-2 inhibitor (IC(50)=19+/-2microM).     

Numerical method to analyze pH-profile from initial velocity data and semi-empirical procedure to identify catalytic residues of enzymes using pK and heat of ionization

A numerical computing method to estimate pK and heat of ionization directly from initial velocity data was developed for systematic analysis of rate parameters. Values of rate and thermodynamic parameters were determined together with standard deviations, without using Arrhenius and van't Hoff plots. Distributions of pK and heat of ionization of small-molecular derivatives of aliphatic carboxylates, imidazoles, thiols, and aliphatic amines as possible models for catalytic residues of enzymes were analyzed from a thermodynamic point of view. Each group was classified in a parallelogram without overlapping, except for a part of the imidazole and thiol groups. It was shown that values of pK and heat of ionization of carboxylates and histidine residues involved in the catalytic activity of enzymes determined from pH-profile experiments can be classified into similar, though somewhat extended, areas to those of the corresponding small-molecular derivatives. Identification of catalytic residues using values of pK and heat of ionization is proved to be a reliable method when the procedure is properly used.     

Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake

Chickpea and white lupin roots are able to exude large amounts of carboxylates, but the resulting concentrations in the rhizosphere vary widely. We grew chickpea in pots in eleven different Western Australian soils, all with low phosphorus concentrations. While final plant mass varied more than two-fold and phosphorus content almost five-fold, there were only minor changes in root morphological traits that potentially enhance phosphorus uptake (e.g., the proportion of plant mass allocated to roots, or the length of roots per unit root mass). In contrast, the concentration of carboxylates (mainly malonate, citrate and malate, extracted using a 0.2 mM CaCl₂ solution) varied ten-fold (averaging 2.3 mol g^–1 dry rhizosphere soil, approximately equivalent to a soil solution concentration of 23 mM). Plant phosphorus uptake was positively correlated with the concentration of carboxylates in the rhizosphere, and it was consistently higher in soils with a smaller capacity to sorb phosphorus. Phosphorus content was not correlated with bicarbonate-extractable phosphorus or any other single soil trait. These results suggest that exuded carboxylates increased the availability of phosphorus to the plant, however, the factors that affected root exudation rates are not known. When grown in the same six soils, three commonly used Western Australian chickpea cultivars had very similar rhizosphere carboxylate concentrations (extracted using a 0.2 mM CaCl₂ solution), suggesting that there is little genetic variation for this trait in chickpea. Variation in the concentration of carboxylates in the rhizosphere of white lupin did not parallel that of chickpea across the six soils. However, in both species the proportion of citrate decreased and that of malate increased at lower soil pH. We conclude that patterns of variation in root exudates need to be understood to optimise the use of this trait in enhancing crop phosphorus uptake.     

Synthesis of an O-alkynyl-chitosan and its chemoselective conjugation with a PEG-like amino-azide through click chemistry

N-Phthaloyl-chitosan O-prop-2-ynyl carbamate was prepared as a biopolymer amenable to undergo chemoselective conjugation by azide-alkyne coupling, while allowing upturn of chitosan's amines after dephthaloylation. N-phthaloylchitosan was prepared according to previously described methods and, due to its low solubility in current organic media, subsequent modifications were run in heterogeneous conditions. Activation of hydroxyls with carbonyl-1,1′-diimidazole and coupling to propargylamine yielded N-phthaloyl-chitosan O-prop-2-ynyl carbamate, then coupled to a model PEG-like azide by azide-alkyne coupling, giving the expected triazolyl conjugate. N-Dephthaloylation allowed recovery of the free amines, responsible for chitosan's bioadhesion and tissue-regeneration properties.The structures of all polymers were confirmed by Fourier-transformed infra-red (FT-IR) and X-ray photoelectron (XPS) spectroscopies, as well as by solid-state nuclear magnetic resonance (SSNMR). All chitosan derivatives were poorly soluble in both aqueous and organic media, which makes them suitable for topical applications or for removal of toxic substances from either the gastric intestinal tract or environmental sources.     

Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms I and II with (reduced) Schiff’s bases incorporating sulfonamide,carboxylate and carboxymethyl moieties

A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn²⁺-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The K_i values of the Schiff bases were in the range of 7.0–21,400 nM against hCA II and of 52–8600 nM against hCA I, respectively. The corresponding amines showed K_i values in the range of 8.6 nM–5.3 μM against hCA II, and of 18.7–251 nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications.