Titanium-carbon functionalities on an oxo surface defined by a calix [4] arene moiety and its redox chemistry

Lithiation of [p-Bu^t-calix[4]-(OMe)₂(OH)₂] (1), followed by reaction with TiCl₃(thf)₃ or TiCl₄(thf)₂, led to the corresponding titanium-calix[4]arene complexes [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl] (2) and [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl₂] (3), respectively. Reaction of 1 with TiCl₄(thf)₂ results in demethylation of the calix[4]arene and the obtention of [p-Bu^t-calix[4]-(OMe)₂(O)₃]TiCl] (4), whose hydrolysis led to [p-Bu^t-calix[4]-(OMe)(OH)₃] (6). The preparation of 6 can be carried out as a one-pot synthesis. Both 2 and 4 undergo alkylation reactions using conventional procedures, thus forming surprisingly stable organometallic species, namely [p-Bu^t-calix[4]-(OMe)₂(O)₂Ti(R)] (R = Me (7); CH₂Ph (8), p-MeC₆H₄ (9) and [p-Bu^t-calix[4]-(OMe)(O)₃Ti(R)] (R = Me (10); CH₂Ph (11); p-MeC₆H₄ (12)). Complexes 7 and 9 undergo a thermal oxidative conversion into 10 and 12, occurring with the demethylation of one of the methoxy groups. A solid state structural property of 9 and 12 has been revealed by X-ray analysis showing a self-assembly of the monomeric units into a columnar polymer, where the p-tolyl substituent at the metal functions as a guest group for an adjacent titanium-calixarene. Reductive alkylation of 3 with Mg(CH₂Ph)₂ gave 8 instead of forming the corresponding dialkyl derivative. Two synthetic routes have been devised for the synthesis of the Ti(III)-Ti(III) dimer [p-Bu^t-calix[4]-(OMe)(O)₃Ti]₂] (13): the reduction of 4 and the reaction of TiCl₃(thf)₃ with the lithiated form of 6. A very strong antiferromagnetic coupling is responsible for the peculiar magnetic behavior of 13. The proposed structures have been supported by the X-ray analyses of 4, 9, 12 and 13.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo[Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)2, and cyclo(Glu-Pro)2, were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)-Pro]2 and cyclo(Glu-Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.     

Atomic mapping of the sugar interactions in one-site and two-site mutants of cyanovirin-N by NMR spectroscopy

The details of the interaction between two mutants of Cyanovirin-N (CV-N), an HIV inactivating protein, and di- and trimannosides, substructures of Man-9, were investigated by STD NMR spectroscopy. One mutant, CV-N (mutDB), contains only one carbohydrate-binding site on domain A, whereas in CV-N (mutDA), the specificity of domain A for trimannose was changed while the site in domain B was kept intact, allowing for a dissection of the overall binding. Results of the STD NMR experiments revealed close contact between the protein binding site on domain A and H2, H3, and H4 of the nonreducing terminal mannose unit for Manalpha(1-2)Manalpha OMe, Manalpha(1-2)Manalpha(1-3)Manalpha OMe, and Manalpha(1-2)Manalpha(1-6)Manalpha OMe. The Manalpha(1-2)Manalpha(1-2)Manalpha OMe trisaccharide interacted with CV-N with the highest affinity. Further dissection of the interaction was achieved by NMR experiments with synthetic 2'-, 3'-, 4'-, and 6'-deoxy analogues of the disaccharide Manalpha(1-2)Manalpha OMe. STD and (1)H- (15)N HSQC NMR spectroscopy revealed that the 2'- and 6'-deoxy dimannosides were recognized by CV-N, whereas no binding was detected for the 3'- and 4'-deoxy sugars. These results demonstrate that the 3'- and 4'-hydroxyl groups on the terminal residue are engaged in key polar interactions with the protein and are required for high-affinity binding.     

Separation of D-forms and L-forms of DL-aspartic acid derivatives by the enzyme subtilisin DY

The ability of the enzyme subtilisin DY for the synthesis of derivatives of DL-aspartic acid which are differently N and C-terminal protected and semiproducts of the peptide synthesis was investigated. The enzyme reaction was characterized by high yields and a comparatively short reaction time. Two of the substrates, Z-D,L-Asp-(OMe)₂ and PhAc-D,L-Asp-(OMe)₂, were hydrolyzed for about 15 min; the reaction time for Boc-D,L-Asp-(OMe)₂ was 2.5 h. The values for the MICHAELIS constants obtained for Z-D,L-Asp-(OMe)₂ (K_m = 0.576 mM) and PhAc-D,L-Asp-(OMe)₂ (K_m = 0.300 mM) showed a high affinity of the enzyme to the substrates. For Boc-D,L-Asp-(OMe)₂ the affinity of the enzyme is considerable lower (K_m = 14.07 mM). The results of these investigations can be effectively used for the separation of N-protected derivatives of D,L-aspartic acid and with a high probability also for other amino and racemic forms.     

Synthesis and selected properties of oligonucleotidyl-(Pm----O)-amino acids

N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was synthesized as a diastereomeric mixture of approx. (1:1) by reacting d(TpT) with 2,4,6-triisopropylbenzenesulfonyl chloride and N-Cbz-Ser-OMe. The phosphoesteric bond of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was found to be stable in acid (1 N HCl, 1 h, 37 degrees C), but labile in alkaline solution (0.1-1 N NaOH, 1 h, 37 degrees C). The products of alkaline hydrolysis were determined to be d(TpT) and the amino acid derivatives. Furthermore, the phosphotriester N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was more labile than the diesteric analogue N-Cbz-Ser (OMe)-pdT. The internucleotide phosphotriester linkage of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was also found to be resistant to enzymatic digestion with spleen and snake venom phosphodiesterases.     

Living ring-opening polymerization of a carbohydrate-derived lactone for the synthesis of protein-resistant biomaterials

An efficient living ring-opening polymerization (ROP) of a permethoxylated epsilon-caprolactone [(OMe)CL] catalyzed by yttrium(III) isopropoxide was developed for the synthesis of degradable protein-resistant polymers [P(OMe)CL]. The lactone monomer was efficiently prepared from a reduced sugar, D-dulcitol. Kinetic studies of the ROP revealed a linear dependence of ln[M]0/[M] on polymerization time as well as a linear correlation between the number-averaged molecular weight (M(n)) and monomer conversion; both support it is a living polymerization. A series of block copolymers of our permethoxylated lactone with epsilon-caprolactone [P(OMe)CL-b-PCL] were synthesized and fully characterized. In thermal analyses only single T(g)s were observed in all the block copolymers, suggesting that P(OMe)CL and PCL blocks are fully miscible. Finally, surface plasmon resonance (SPR) sensograms demonstrated that both P(OMe)CL and the P(OMe)CL-b-PCL block copolymers exhibit excellent resistance to fibrinogen and lysozyme.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis, characterization and cytotoxic activity of substituted benzyl iminoether Pt(II) complexes of the type cis- and trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-R}2] (R=Me, OMe, F). X-ray structure of trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-F}2

New substituted benzyl iminoether derivatives of the type cis- and trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-R}(2)] (R=Me (1a, 2a), OMe (3a, 4a), F (5a, 6a)) have been synthesized and characterized by elemental analyses, FT-IR spectroscopy and NMR techniques. The iminoether ligands are in the E configuration, which is stable in solution and in the solid state, as confirmed by the (1)H NMR data. Complex trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-F}(2)] (6a) was also characterized by an X-ray diffraction study. Complexes 1a-6a have been tested against a panel of human tumor cell lines in order to evaluate their cytotoxic activity. cis-Isomers were significant more potent than the corresponding trans-isomers against all tumor cell lines tested; moreover, complexes 1a and 5a showed IC(50) values from about 2-fold to 6-fold lower than those exhibited by cisplatin, used as reference platinum anticancer drug.     

Structural studies with weak oxytocin antagonists

Summary [Aib³,Thr⁵]OT, [Aib³,Thr(OMe)⁵]OT, [Aib³,Orn⁸]OT, [Thr(OMe)⁵,Orn⁸]OT and [Phe²,Thr(OMe)⁵,Orn⁸]OT were synthesized by solid-phase techniques. From the biological properties of these peptides, it seems that the simultaneous replacement of positions 3 and 5 of oxytocin with Aib and Thr(OMe) results in an analogue devoid of antagonistic activity in comparison with the singly substituted compounds. Simultaneous Orn⁸ substitution does the same in the case of the Aib³ analogue and even leads to agonistic activity in the case of the Thr(OMe)⁵ analogue. Replacement of Tyr² by Phe², e.g. [Phe²,Thr(OMe)⁵,Orn⁸]OT, again favors the appearance of minor antagonistic potency.     

Supported organometallic complexes. Part 27: novel sol-gel processed rhodium(I) complexes: synthesis, characterization, and catalytic reactions in interphases

A novel T-silyl functionalized cationic (COD)(dppp)rhodium(I) complex was sol-gel processed with various amounts of the co-condensing agents MeSi(OMe)₂(CH₂)₆(OMe)₂SiMe and MeSi(OMe)₂(CH₂)₃(C₆H₄)(CH₂)₃(OMe)₂SiMe to give novel stationary phases for ‘Chemistry in Interphases’. The polysiloxane matrices and the integrity of the rhodium(I) complex centers were investigated by means of multinuclear solid-state NMR (¹³C, ²⁹Si, ³¹P) and EXAFS spectroscopies. Dynamic NMR measurements show an increasing mobility of the matrix and the reactive centers with a higher amount of the co-condensing component. The accessibility of the anchored rhodium(I) centers was scrutinized by the metal catalyzed hydrogenation of 1-hexene. All applied xerogels show remarkable activities and selectivities. An enhancement of the activities is achieved when polar solvents are used. SEM micrographs reveal the morphology of the hybrid materials and energy dispersive X-ray spectroscopy (EDX) suggests that the distribution of the elements is in satisfying agreement with the applied composition.     

Resolution of DL-2-aminosuberic acid via protease-catalyzed ester hydrolysis

Summary Papain-catalyzed regioselective cleavage of-methyl ester in Z-DL-Asu(OMe)-OMe leads to Z-L-Asu(OMe)-OH and Z-D-Asu(OMe)-OMe. Subsequent saponifications yield Z-L-Asu-OH and Z-D-Asu-OH. The enzymatic-ester hydrolysis was also achieved by subtilisin BPN in organic solvent with low water content.Abbreviations Asu 2-aminosuberic acid - Z benzyloxycarbonyl - OMe methyl ester - DCHA dicyclohexylamine     

O-acylation of hydroxyproline residues: effect on peptide-bond isomerization and collagen stability

In collagen, strands of the sequence XaaYaaGly form a triple-helical structure. The Yaa residue is often (2S,4R)-4-hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O-acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cgamma atom: AcHyp[C(O)CF3]OMe congruent with Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe congruent with Achyp[C(O)CH3]OMe > or = AcHypOMe congruent with AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)(10), which is O-acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)(10) > {ProHyp[C(O)CH3]Gly}(10) > (ProProGly)(10). Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group.     

An access to various sulfation patterns in dermatan sulfate: chemical syntheses of sulfoforms of trisaccharide methyl glycosides

The syntheses are reported for the first time of alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc4SO(3)-(1-->4)-alpha-L-IdopA2SO(3)-(1-->OMe), its disulfated analogue alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-L-IdopA2SO(3)-(1-->OMe), and of beta-D-GalpNAc4SO(3)-(1-->4)-alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc4SO(3)-(1-->OMe), which represent structural fragments of dermatan sulfate, unavailable directly by chemical or enzymatic degradation of the glycosaminoglycan polymer. These molecules were readily obtained from a pair of key disaccharide intermediates, in which the relative difference of stability of the D-GalNAc 4-hydroxy protecting groups (acetate or pivalate) toward saponification conditions allowed access to various sulfoforms from a common precursor. For the preparation of these blocks, the 4-O-pivaloyl-D-galacto moiety was readily obtained through a one-pot stereospecific intramolecular nucleophilic displacement on an easily available 3-O-pivaloyl-D-gluco precursor, and the L-IdoA moiety through selective radical oxidation at C-6 of a L-ido 4,6-diol derivative with oxoammonium salts.     

Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins

We report the deduced amino acid sequences of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.     

Synthesis, immobilization and catalytic activity of some silylated cyclopentadienyl rhodium(I) complexes

The mixture of isomers of silylated cyclopentadiene derivative C₅H₅CH₂CH₂Si(OMe)₃ (1) has been used for the syntheses of the mononuclear Rh(I) complexes [η⁵-C₅H₄(CH₂)₂Si(OMe)₃]Rh(CO)₂ (3). [η⁵-C₅H₄(CH₂)₂Si(OMe)₃]Rh(COD) (4) and [η⁵-C₅H₄(CH₂)₂Si(OMe)₃]Rh(CO)(PPh₃) (5). Upon entrapment of 3–5 in silica sol-gel matrices, air stable, leach-proof and recyclable catalysts 6–8 resulted. Their catalytic activities in some hydrogenation processes were compared with those of the non-immobilized complexes 3–5, as well as with those of homogeneous and heterogenized non-silylated analogs, 9–14.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.