Structural Properties of Macrodontain I,a Cysteine Protease from <Emphasis Type="Italic">Pseudananas macrodontes</Emphasis> (Morr.) Harms (Bromeliaceae)

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman’s degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M^?1 cm^?1. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α?+?β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.     

Synthesis and evaluation of macrocyclic transition state analogue inhibitors for alpha-chymotrypsin

Lactams 1 and 2 are readily formed from acyclic precursors in the presence of trypsin and chymotrypsin, respectively, identifying the macrocyclic ring system as a potential motif for constrained transition state analogue inhibitors of the serine peptidases. Ketone 3 was synthesized and shown to be a modest inhibitor of chymotrypsin (Ki = 220 microM), albeit 4-fold more potent than the acyclic hydroxy acid 25 (Ki = 1.5 mM as a mixture of epimers). A precursor (31) to the amino boronic acid 4 was also prepared; although this derivative was a potent inhibitor of chymotrypsin (Ki = 130 nM) by virtue of the boronic acid moiety, it showed no advantage over the des-amino analogue 32 (Ki = 120 nM), which is not capable of cyclizing.     

Diastereoisomeric singly bridged cyclophosphazene-macrocyclic compounds

31P NMR spectroscopy and added chiral shift reagent (CSR) or chiral solvating agent (CSA) have been used to show that unsymmetrically substituted singly bridged macrocyclic phosphazene compounds exist as 1:1 diastereoisomers of two racemic mixtures, in contrast to previous work (ref 2) on symmetrically substituted diastereoisomeric analogues, which exist as meso and racemic forms. The cis-ansa cyclotriphosphazatriene-macrocycle, 1, is meso and monosubstitution of the >P(O-macrocycle)Cl group with 2-naphthol gives a racemic product (7), in which the macrocyclic ring exists in a trans-ansa configuration. Reaction of 7 with the di-secondary amine, piperazine, gives an unsymmetrically disubstituted racemic compound (8) having a cis-ansa configuration of the macrocyclic ring. Reaction of 8 with a further quantity of 1 forms a singly bridged derivative (9) with the macrocyclic rings in cis-trans configurations, and further reaction of 9 with pyrrolidine gives compound 10 with the macrocyclic rings in cis-cis configurations. Both 9 and 10 have four stereogenic centers giving rise to diastereoisomeric compounds existing as mixtures of two racemates. The results are consistent with inversion of configuration at phosphorus at each step of the reaction of >P(OR)Cl groups with nucleophile Z (i.e., Z = naphthoxy, piperazino, pyrrolidino) to form >P(OR)Z derivatives.     

Synthesis of jasplakinolide analogues containing a novel omega-amino acid

The synthesis of the omega-amino acid 4 is described utilizing a two-dimensional synthesis strategy combined with an enzymatic differentiation of homotopic ester groups. The amino acid 4 features two non-bonded interactions that result in conformational constraints on a cyclic construct. This amino acid was incorporated into the four macrolactams 17, 22, 31, and 37. The ring in 17 and 22 is 18-membered, whereas 31 and 37 have a 19-membered ring. The pairs with the same ring size differ in a N-methyl group. For the larger macrolactams (31 and 37) conformational analysis showed that the macrocyclic rings are somewhat more rigid than in the natural lead, the depsipeptide jasplakinolide. Nevertheless, their conformations are comparable to the natural product. There are no intramolecular hydrogen bonds, neither is the cis-rotamer populated in the N-methyl compound 37. Due to the increased flexibility of the smaller macrolactams 17 and 22 and signal overlap, a distinct solution structure could not be obtained for these compounds. The amino acid 4 should be useful for restricting the conformation of other small peptides.     

Construction of an advanced tetracyclic intermediate for total synthesis of the marine alkaloid sarain A

In work directed toward a total synthesis of the marine alkaloid sarain A (1), the advanced intermediate 54, containing all the key elements and the seven stereogenic centers of sarain A, has been successfully synthesized from bicyclic lactam 4, previously prepared via an intramolecular stereospecific [3 + 2]-azomethine ylide dipolar cycloaddition. Intermediate lactam 4 could be efficiently converted to N-Boc derivative 12. Introduction of a two-carbon fragment into lactam 12 which eventually becomes the C-7',8' syn diol of the "eastern" ring was then achieved by C-acylation of the corresponding enolate with methoxyacetyl chloride followed by a highly stereoselective ketone reduction with Zn(BH4)2 to afford alcohol 16. Intermediate 16 has the incorrect C-7' relative stereochemistry for sarain A, but this problem was conveniently remedied by inverting the C-7' center via an intramolecular Ohfune-type cyclization of the silyl carbamate derived from Boc mesylate 27 to produce the key cyclic carbamate 28. It was then possible to convert acetal 28 to allylsilane 32 followed by cyclization to the alkaloid tricyclic core 33 via an allylsilane/N-sulfonyliminium ion cyclization. Formation of the "western" macrocyclic ring has been successfully addressed using functional group handles at C-3' and N-1' on the tricyclic core via a ring-closing olefin metathesis (RCM) strategy with the second-generation Grubbs ruthenium catalyst to produce intermediate macrolactam 47. A chelation-controlled addition of ethynylmagnesium bromide to advanced aldehyde 51 afforded a single diastereomeric adduct 53 which is tentatively assigned to have the correct C-7',8' syn-diol stereochemistry. This adduct could be rearranged to the conveniently protected amino carbonate 54 which is set up for construction of the remainder of the eastern ring of sarain A.     

Incorporation of a flexible, pyridine-functionalized Diaza-crown ether into discrete supramolecules via coordination-driven self-assembly

A flexible, pyridine-functionalized diaza-crown ether was self-assembled into discrete supramolecules of differing stoichiometries upon combination with various organoplatinum molecules. They are characterized by electrospray ionization mass spectrometry and (31)P[(1)H] and (1)H NMR. In one case, (1)H-(1)H NOE enhancements of a [1 + 1] assembled structure demonstrate the puckered shape of the macrocyclic ring. Despite its inherent flexibility, the dipyridyl-substituted 18-membered diaza-crown ligand prefers to self-assemble into closed systems when reacted with platinum-containing acceptors.     

Ion-exchange chromatographic assay of peptidases acting on the C-terminal hexapeptide sequence of substance P

A rapid and sensitive assay for peptidases acting on the C-terminal hexapeptide sequence of the neuropeptide substance P is described. The radiolabelled substrate, N alpha-[125I]desaminotyrosyl-substance P (6-11) is easily prepared by coupling commercially available radioiodinated Bolton-Hunter reagent with substance P (6-11). Peptidase activity is determined by quantitative separation of the degradation products from the intact substrate on small QAE-Sephadex columns. The assay has been used to measure degradation of the substrate by rat parotid and diencephalon slices. The peptidase activity in the latter system was inhibited by substance P and substance P fragments and was sensitive to metal chelators and thiol reagents.     

Metal ion recognition via 'selective detuning'. The interaction of selected transition and post-transition metal ions with a mono-N-benzylated O2N3-donor macrocycle and its xylyl-bridged ring analogue

The interaction of cobalt(II), nickel(II), copper(II), zinc(II), cadmium(II), silver(I) and lead(II) with symmetrical mono-N-benzylated and xylyl-linked macrocyclic ligands derived from the O2N3-macrocycle, 1,12,15-triaza-3,4:9,10-dibenzo-5,8-dioxacycloheptadecane, has been investigated. The log K values for the respective 1 : 1 complexes in 95% methanol (I= 0.1; Et4NClO4, 25 degrees C) of the mono-benzylated derivative have been determined potentiometrically and the results compared with the values obtained previously for the parent (non-benzylated) ring system as well as for related di- and tri-benzylated macrocyclic species. Mono-benzylation results in slightly enhanced stability for the 1 : 1 silver(I) complex while the values for the corresponding complexes of the other six ions are in each case decreased even though the highest stability is still maintained for the 1 : 1 copper(II) complex. The log K results are in accord with a previous proposal that N-benzylation of amine-containing macrocyclic rings of the present type will normally have only a minor (positive or negative) influence on the affinity towards silver(I) while the corresponding binding strengths towards the remaining six metal ions are significantly reduced-behaviour we term 'selective detuning'. Competitive seven-metal transport experiments across a bulk chloroform membrane have been performed using both ligand systems as ionophores. In parallel to the log K results, transport selectivity for copper(II) was exhibited by both systems, with similar transport efficiencies being evident when compared on a 'per macrocyclic cavity' basis.     

A convenient approach to heterocyclic building blocks: synthesis of novel ring systems containing a [5,6]Pyrano[2,3-c]pyrazol-4(1H)-one moiety

Starting from commercially available educts, a straightforward synthetic route to new heterocyclic building blocks is exemplified with the one- or two-step synthesis of tri-, tetra-, or pentacyclic ring systems. Representatives of the following novel ring systems are prepared from 3-methyl-1-phenyl-2-pyrazolin-5-one and the corresponding o-halo-arenecarbonyl chloride using calcium hydroxide in refluxing 1,4-dioxane: pyrimidino[4',5':5,6]pyrano[2,3-c]pyrazol-4(1H)-one, thieno[3',2':5,6]pyrano[2,3c]pyrazol- 4-(1H)-one, thieno[3',4':5,6]pyrano[2,3-c]pyrazol-4(1H)-one, thieno[3',2':4',5']thieno[2',3':5,6]-pyrano[2,3-c]pyrazol-4(1H)-one, [1,3]dioxolo[5',6'][1]benzothieno[2',3':5,6]pyrano-[2,3-c]- pyrazol-4(1H)-one, pyridazino[4',3':5,6]pyrano[2,3-c]pyrazol-4(1H)-one and pyrazolo-[4',3':5',6']pyrido[3',4':5,6]pyrano[2,3-c]pyrazol-4(1H)-one. While the latter two ring systems are directly obtained due to a spontaneous intramolecular substitution reaction, in the other reactions uncyclised 4-aroylpyrazol-5-ols are produced, which are cyclised into the target heterocycles in a subsequent synthetic step (i.e. treatment with NaH in DMF). Detailed NMR spectroscopic investigations ((1)H-, (13)C-, (15)N-) with the obtained compounds were undertaken to unambiguously prove the new structures.     

Controlled peptide solvation in portion-mixing libraries of FRET peptides: improved specificity determination for Dengue 2 virus NS2B-NS3 protease and human cathepsin S

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P2-P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.     

Cloning, Sequencing, and Identification Using Proteomic Tools of a Protease from Bromelia hieronymi Mez

Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3'RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M(-1) cm(-1)). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α?+?β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.     

Stable glucopyranosylpalladium complexes with cis-beta-hydrogen. A six-membered ring metallocycle with an oxygen donor ligand

Two stable glucopyranosylpalladium complexes, chloro[1,3-dimethyl-5-(3,4,6-tri-O-acetyl-2-deoxy-alpha-D -arabinohexopyranosyl)-2,4(1H,3H)-pyrimidinedionnato] (triphenylphosphine)-palladium and the corresponding triphenylarsine analog, were studied using fast atom bombardment mass spectrometry, 1H, 13C and 31P nuclear magnetic resonance, UV and IR spectroscopy to establish structures for these complexes. The data obtained indicate that the pyranosyl ring is in a chair conformation in which palladium (C2'), acetoxy (C3' C4') and acetoxymethyl (C5') are equatorial and 1,3-dimethyl-2,4(1H,3H) pyrimidinedion-5-yl (C1') is axial. The palladium(II) ion is encompassed in a six-membered ring metallocycle in which C2' of the glucopyranosyl ring and the oxygen of the C4 carbonyl of the pyrimidinedionyl group occupy adjacent ligand sites. The other two ligand sites on square planar palladium are occupied by triphenylphosphine (or triphenylarsine) cis to C2' and trans to carbonyl oxygen, and chloride trans to C2' and cis to oxygen. This stable metallocycle has three unusual features, a cis-beta-hydrogen, a six-membered Pd-containing ring and an oxygen donor ligand. Its surprising stability is due to conformational barriers to the proper alignment of Pd with pyranosyl ring substituents required for elimination reactions.     

Lifting of the degeneracy in semibullvalenes by remote and direct substituents: a quantitative study using variable-temperature carbon-13 NMR spectroscopy

A series of six 1,5-(ethylmethyl)semibullvalenes (1a <==> 1a', 2 <==> 2', 3 <==>3') and two 4(2)-substituted semibullvalenes (4 <==> 4'), each undergoing Cope equilibria between nondegenerate valence tautomers, was investigated by carbon-13 NMR spectroscopy at a range of temperatures in several different solvents. Gompper's treatment of substituent perturbation was extended, specifically accounting for the effects of the substituents on chemical shifts, to allow the determination of the thermodynamic parameters for these skewed equilibria. These new treatments were used to determine the population difference (f - f ') between the valence tautomers and the perturbation thermodynamic quantities DeltaH(P), DeltaS(P), and DeltaG(P). The slow-exchange limit was reached for the parent 1,5-(ethylmethyl)semibullvalenes 3a <==> 3a' from which it was established that the preferred valence tautomer is 3a with the ethyl group on the cyclopropane ring. Despite considerable effort, the slow-exchange limit could not be reached in any of our other remotely substituted semibullvalenes. Provided that the ethyl group always prefers the cyclopropyl position as in 3a, the 1-ethyl-5-methylsemibullvalenes 1a, 2, and 3 are more stable by DeltaH(P) = 0.7-1.7 kJ mol(-1) than their valence tautomers 1a', 2', and 3'. In the directly substituted semibullvalenes (4 left harpoon ovet right harpoon 4'), the preferred valence tautomers 4a and 4b have the bromine atom or the nitrile group on the vinyl position (C(4)) rather than on the cyclopropane ring (C(2)) and are more stable than 4a' and 4b' by DeltaH(P) = 4.8 and 7.0 kJ mol(-1), respectively.     

Tandem reactions of α-diazo ketones with macrocyclic olefins: diastereoselective synthesis of oxanorbornane fused macrocyclic lactones

Tandem cyclization-cycloaddition reactions of α-diazo ketones with macrocyclic olefins in the presence of rhodium(II) acetate catalyst led to the oxanorbornane fused macrocyclic di- or tetralactone ring systems in moderate yield. This forms the first example of 1,3-dipolar cycloaddition reactions with a macrocyclic olefin as a dipolarophile, affording a variety of new oxanorbornane fused macrocycles with diastereoselectivity.     

二十四元六氮大环双核铜(I)配合物的氧化去甲基作用: 单加氧酶的模拟

以5-溴-2-甲氧基-1, 3-苯二甲醛与二乙烯三胺通过[2+2]缩合,合成了一个新的六氮杂二十四元大环配体, 并在[Cu(CH3CN)4]ClO4存在下生成Cu(I)大环配合物, 然后在空气(或氧气)中氧化, 得到了新的大环双核Cu(II)配合物, 用多种方法对其进行了表征, 用1H NMR谱等方法鉴定了氧化产物。实验结果表明: 在Cu(I)配合物氧化过程中,能使配体环上的一个甲氧基发生断裂, 形成苯氧桥和水桥联的Cu(II)配合物。在木质素酶等单加氧酶的氧化过程中也伴随着氧化去甲基作用。本文首次用大环配合物对这一过程进行了模拟, 并测定了氧化反应中的吸氧量和吸氧速率常数。     

Electrochemical, catalytic and antimicrobial activity of N-functionalized tetraazamacrocyclic binuclear nickel(II) complexes

The five binuclear nickel(II) complexes have been synthesized by the Schiff base condensation of 1,8-[bis(3-formyl-2-hydroxy-5-methyl)benzyl]-l,4,8,11-tetraazacyclo-tetradecane (PC) with appropriate aliphatic diamines and nickel(II) perchlorate. All the five complexes were characterized by elemental and spectral analysis. The electronic spectra of the complexes show three d-d transition in the range of 550-1055 nm due to 3A2g→3T2g(F), 3A2g→3T1g(F) and 3A2g→3T1g(P). These spin allowed electronic transitions are characteristic of an octahedral Ni2+ center. Electrochemical studies of the complexes show two irreversible one electron reduction waves at cathodic region. The reduction potential of the complexes shifts towards anodically upon increasing the chain length of the macrocyclic ring. All the nickel(II) complexes show two irreversible one electron oxidation waves at anodic region. The oxidation potential of the complexes shift towards anodically upon increasing the chain length of the macrocyclic ring. The catalytic activities of the complexes were observed to be increase with increase the macrocyclic ring size. The observed rate constant values for the catalytic hydrolysis of 4-nitrophenyl phosphate are in the range of 5.85×10(-3) to 9.14×10(-3) min(-1). All the complexes were screened for antimicrobial activity.     

Polyhalogenoheterocyclic compounds: Part 51. [1] Macrocycles from 4-alkoxy-tetrafluoropyridine derivatives

4-Alkoxy-tetrafluoropyridine derivatives were used as building blocks for the synthesis of 14- and 16-membered macrocyclic ring systems comprising pyridine and either poly-ether or amine subunits; two of the macrocycles were characterised by X-ray crystallography.     

Controlled formation and topologies of thiophenolate-based macrocycles: rings, cylinders and bowls

The Schiff-base condensations of 1,3-diaminopropane with a protected thiophenol dialdehyde in the presence of Ni(2+), Pd(2+) or Zn(2+) can be controlled to yield either mononuclear acyclic, or 2 + 2 and 4 + 4 macrocyclic complexes by the choice of both metal cation and counteranion. The Ni(2+) complex of the 2 + 2 macrocycle contains two square-planar nickel ions and shows an arrangement similar to one observed previously: the mu-S atoms of the thiophenolate groups are pyramidal and lie on the same side of the plane defined by the four N atoms of the macrocycle to give a V-shaped molecule. By contrast, the Zn(2+) complex of the 2 + 2 macrocycle undergoes oligomerization to yield a bowl-shaped hexanuclear complex that includes a mu(3)-carbonate anion. Essential for this topology is the presence of three mu(3)-S-thiophenolato groups that link the three macrocyclic units to form a Zn(3)S(3) ring that seals the bottom part of the bowl. In this arrangement, one of the pyramidal mu(3)-S atoms in each dinuclear Zn(2+) complex is inverted relative to the arrangement observed for the dinickel complexes. Molecular modelling suggests that inversion about the mu-S atoms of the 2 + 2 macrocyclic complexes is readily accessible at room temperature and that the contrasting arrangements observed for the Ni(2+) and Zn(2+) complexes are those energetically most favourable for the respective metal ions. Rare 4 + 4 macrocyclic complexes are isolated as neutral dinuclear complexes for Ni(2+) and Pd(2+) and as a tetranuclear complex cation for Zn(2+). The topologies of these systems contrast significantly: those with two square-planar Ni(2+) or Pd(2+) ions form extended rings, while that with Zn(2+) forms a sulfur-lined cylinder which hosts acetonitrile molecules in the crystalline state. Reaction conditions can also be optimised to produce 2 + 1 acyclic ligands as their mononuclear Ni(2+) and Pd(2+) complexes, providing potentially useful building blocks for production of more complicated macrocyclic and supramolecular systems.     

'Upenamide: An unprecedented macrocyclic alkaloid from the Indonesian sponge Echinochalina sp

'Upenamide (1) represents a new class of macrocyclic marine alkaloid possessing both spirooxaquinolizidinone and hemiaminal ring systems. It was isolated from the Indonesian sponge Echinochalina sp. The gross structure of 1 was elucidated by spectroscopic methods and accurate mass measurements. A suggestion is made as to its biogenetic origin.     

Slippage of a Porphyrin Macrocycle over Threads of Varying Bulkiness: Implications for the Mechanism of Threading Polymers through a Macrocyclic Ring

Threading of a polymer through a macrocyclic ring may occur directly, that is, by finding the end of the polymer chain, or by a process in which the polymer chain first folds and then threads through the macrocyclic ring in a hairpin‐like conformation. We present kinetic and thermodynamic studies on the threading of a macrocyclic porphyrin receptor ( H₂1 ) onto molecular threads that are blocked on one side and are open on the other side. The open side is modified by groups that vary in ease of folding and in bulkiness. Additionally, the threads contain a viologen binding site for the macrocyclic receptor, which is located close to the blocking group. The rates of threading of H₂1 were measured under various conditions, by recording as a function of time the quenching of the fluorescence of the porphyrin, which occurs when receptor H₂1 reaches the viologen binding site. The kinetic data suggest that threading is impossible if the receptor encounters an open side that is sterically encumbered in a similar way as a folded polymer chain. This indicates that threading of polymers through macrocyclic compounds through a folded chain mechanism is unlikely.