Three-step Cascade Research Articles

Multicomponent Synthesis of Fused Benzimidazolopiperazines

We present a novel protocol for the efficient synthesis of fused benzimidazolo piperazines starting from a four-component Ugi-Smiles reaction and a subsequent three-step cascade involving an acid-catalyzed cyclization, an intramolecular reductive cyclization, and an oxidation.

Synthesis of 3-substituted 8,9-didehydroazepino[4,5-b]indolines via ring expansion reaction of pyrroloindolines

An unexpected ring expansion reaction was observed to give the 3-substituted 8,9-didehydroazepinoindolines when 3-substituted pyrroloindolines reacted with electron-deficient acetylenes in the presence of catalytic amount of CuI. The ring expansion reaction proceeded via a three-step one-pot cascade pathway of aza-Michael-addition/ring-opening/iminium-cyclization.

Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.

Technical Note: Unsupervised C‐arm pose tracking with radiographic fiducial

C-arm fluoroscopy reconstruction, such as that used in prostate brachytherapy, requires that the relative poses of the individual C-arm fluoroscopy images must be known prior to reconstruction. Radiographic fiducials can provide excellent C-arm pose tracking, but they need to be segmented in the image. The authors report an automated and unsupervised method that does not require prior segmentation of the fiducial. The authors compute the individual C-arm poses relative to a stationary radiographic fiducial of known geometry. The authors register a filtered 2D fluoroscopy image of the fiducial to its 3D model by using image intensity alone without prior segmentation. To enhance the C-arm images, the authors investigated a three-step cascade filter and a line enhancement filter. The authors tested the method on a composite fiducial containing beads, straight lines, and ellipses. Ground-truth C-arm pose was provided by a clinically proven method. Using 111 clinical C-arm images and +/- 10 degrees and +/- 10 mm random perturbation around the ground-truth pose, a total of 2775 cases were evaluated. The average rotation and translation errors were 0.62 degrees (STD = 0.31 degrees) and 0.72 mm (STD = 0.55 mm) for the three-step filter and 0.67 degrees (STD = 0.40 degrees) and 0.87 mm (STD = 0.27 mm) using the line enhancement filter. The C-arm pose tracking method was sufficiently accurate and robust on human patient data for subsequent 3D implant reconstruction.

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

High osmolarity glycerol (HOG) pathway in Candida albicans

Signal transduction pathways mediated by mitogen-activated protein kinases (MAPKs) play crucial roles in eukaryotie cells.In C.albicans,HOG-MAPK pathway is mainly involved in the regulating of morphogenesis,virulence and cellular response to external stress.The pathway is composed of two upstream osmosensing branches,i.e.,SLNl and SHO1 branches,and a downstream three-step MAP kinase cascade system.A deep insisht into the roles of HOG-MAPK pathway-associated signal transduction proteins and their regulation will offer new strategies for the prevention and treatment of candidiasis. Key words: Candida; albicans; Signal transduction

Spectroscopy of rubidium Rydberg states with three diode lasers

This paper presents a new method of purely optical spectroscopy of rubidium Rydberg states in a room-temperature gas cell. The atoms are excited into the Rydberg state from the ground state with a three-step diode laser cascade setup. The excitation of Rydberg states is monitored by observing the absorption of the laser performing the first excitation step on the strong rubidium D2 line. Different polarization schemes and possible excitation geometries are discussed.

Fine Tuning Reactivity: Synthesis and Isolation of 1,2,3,12b-Tetrahydroimidazo[1,2-f]phenanthridines

A facile route for the synthesis and isolation of 1,2,3,12b-tetrahydroimidazo[1,2-f]phenanthridines (TIPs) has been developed. The heterocycle is a reactive intermediate in the three-step cascade synthesis of 2,3-dihydro-1H-imidazo[1,2-f]phenanthridinium cations (DIPs), a biologically active DNA intercalating framework; however, the intermediate has previously only been characterized in situ. Derivatization of the structure at the imidazo-N position controls the reactivity of the intermediate with respect to electronic potential and pK(a) allowing isolation of a selection of TIP structures. Correlations between these parameters and reaction outcome have been made, and other influences such as steric and solvent effects have also been investigated.

A network of interactions among seminal proteins underlies the long-term postmating response in Drosophila

Despite the importance of seminal proteins in fertility and their capacity to alter mated females' physiology, the molecular pathways and networks through which they act have not been well characterized. Drosophila seminal fluid includes proteins that fall into biochemical classes conserved from insects to mammals, making it an excellent model with which to address this question. Drosophila seminal fluid also contains a "sex peptide" (SP, Acp70A) that plays a major role in regulating egg production and mating behavior in females for several days after mating. This long-term postmating response (LTR) initially requires the association of SP with sperm. The LTR also requires members of the conserved seminal protein classes (two lectins, a protease, and a cysteine-rich secretory protein). Here, we show that these seminal proteins function interdependently, regulating a three-step cascade (first, at the level of seminal protein transfer to the female; second, at the level of stability; and third, at the level of localization within females), leading to the normal localization of SP to sperm-storage organs. This localization is, in turn, necessary for successful induction of the LTR. The requirements for manifestation of the LTR in Drosophila establish the paradigm that multiple seminal proteins can exert their actions through a multistep, multicomponent network of interactions.

Chemoselective Synthesis of Substituted Imines, Secondary Amines, and β‐Amino Carbonyl Compounds from Nitroaromatics through Cascade Reactions on Gold Catalysts

Substituted imines, alpha,beta-unsaturated imines, substituted secondary amines, and beta-amino carbonyl compounds have been synthesized by means of new cascade reactions with mono- or bifunctional gold-based solid catalysts under mild reaction conditions. The related synthetic route involves the hydrogenation of a nitroaromatic compound in the presence of a second reactant such as an aldehyde, alpha,beta-unsaturated carbonyl compound, or alkyne, which circumvents an ex situ reduction process for producing the aromatic amine. The process is shown to be highly selective towards other competing groups, such as double bonds, carbonyls, halogens, nitriles, or cinnamates, and thereby allows the synthesis of different substituted nitrogenated compounds. For the preparation of imines, substituted anilines are formed and condensed in situ with aldehydes to provide the final product through two tandem reactions. High chemoselectivity is observed, for instance, when double bonds or halides are present within the reactants. In addition, we show that the Au/TiO2 system is also able to catalyze the chemoselective hydrogenation of imines, so that secondary amines can be prepared directly through a three-step cascade reaction by starting from nitroaromatic compounds and aldehydes. On the other hand, Au/TiO2 can also be used as a bifunctional catalyst to obtain substituted beta-amino carbonyl compounds from nitroaromatics and alpha,beta-unsaturated carbonyl compounds. Whereas gold sites promote the in situ formation of anilines, the intrinsic acidity of Ti species on the support surface accelerates the subsequent Michael addition. Finally, two gold-catalyzed reactions, that is, the hydrogenation of nitro groups and a hydroamination, have been coupled to synthesize additional substituted imines from nitroaromatic compounds and alkynes.

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by beta-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation.

Total Synthesis of the Akuammiline Alkaloid (±)-Vincorine

The first total synthesis of the akuammiline alkaloid (+/-)-vincorine (6) has been accomplished in about 1% overall yield in 31 steps. A concise assembly of the core 1,2-disubstituted 1,2,3,4-tetrahydro-4a,9a-iminoethanocarbazole (1), a distinctive feature of akuammiline and strychnos alkaloids, was developed via a three-step one-pot cascade reaction consisting of copper-catalyzed intramolecular cyclopropanation, ring-opening, and ring closure. The construction of the last seven-membered E-ring in a rigid two-ring moiety (31, 45 to 47) through Heck coupling, Michael addition, pi-allyl/Heck or pi-allyl/Stille coupling failed, leading us to seek an alternative method. After successful addition of an acetate side chain on C15 of the cyclohexenyl ring (D-ring) in Boc-protected 35b by a Johnson-Claisen rearrangement and multistep modification of the functionality in the rearrangement product 33a, the E-ring formation was then realized for providing pentacyclic lactam 32 through intramolecular condensation of the acid group on the D-ring and the amine group on the C-ring with Mukaiyama's reagent. An E-ethylidenyl group on the E-ring was stereoselectively added to afford lactam 56a through a two-step reaction of 32 consisting of aldol addition with acetaldehyde and cis-elimination of the resulting hydroxyl group. Final elaboration of 56a, including opening of the seven-membered E-ring, selective reduction of the alpha,beta-unsaturated ester, and reclosure of the seven-membered E-ring completed the total synthesis of 6.

Crystallographic Evidence for Cyclopropane Ring Formation in Isotwistenes Obtained by Thermal Cascade Reactions of Cyclopentadienone with Acyclic Conjugated Dienes

The molecular structure of the double Diels-Alder (DDA) adduct derived from three-step thermal cascade reactions of 2,5-bis(methoxycarbonyl)-3,4-diphenylcyclopentadienone with ethyl sorbate is elucidated by single crystal X-ray analysis. The structural features and the reaction mechanism of the adduct are discussed on the basis of density functional theory (DFT) calculation results.

Asymmetric synthesis of trans-disubstituted cyclopropanes using phosphine oxides and phosphine boranes

The stereocontrolled synthesis of trans-disubstituted cyclopropylketones has been achieved from beta-alkyl, gamma-benzoyl phosphine oxides via a three-step cascade reaction incorporating an acyl transfer, phosphinoyl transfer and cyclisation to form the cyclopropane. Using Evans' chiral oxazolidinone auxiliary and by masking the phosphine oxide moiety as a phosphine borane we have extended the method to the synthesis of enantiomerically-enriched trans-disubstituted cyclopropyl ketones.

Total Synthesis of (−)-Ardeemin

Total synthesis of potent anti-MDR indole alkaloids (-)-ardeemin and its N-acyl analogues has been accomplished from L-tryptophan with about 2% overall yield in 20 steps. The key step depended on the newly developed three-step one-pot cascade reaction of 7 with diazoester 8 via intermolecular cyclopropanation, ring opening, and ring closure to assemble the chiral 3-substituted hexahydropyrrolo[2,3-b]indole 4a.

Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

Capecitabine: an overview of the side effects and their management

Xeloda (capecitabine), a thymidine phosphorylase activated fluoropyrimidine carbamate, is currently the only universally approved orally administered 5-fluorouracil (5-FU) prodrug. It belongs to a newer generation of orally administered fluoropyrimidines. It has been developed because of the clinical need for efficient, tolerable and convenient agents, which do not require continuous infusion. Capecitabine is not a cytotoxic drug in itself, but via a three-step enzymatic cascade, it is converted to 5-FU mainly within human cancer cells. While the drug compares favorably with 5-FU in patients with advanced or metastatic colorectal cancer and pretreated breast cancer, it also has an improved toxicity profile, mainly of gastrointestinal and dermatologic effects with a significantly lower incidence of grade 3/4 myelotoxicity compared with infusional 5-FU-based chemotherapy. Capecitabine's selective activation within the tumor allows for less systemic toxicity events. A gradient of fluoropyrimidine toxicity is observed: high in the US and low in East Asia. In addition, there is a discrepancy in tolerance of dose among patients treated in the US vs. Europe. Although patients can take the drug orally in the convenience of their own home, the key to successful management of capecitabine is the clinician's awareness of its severe, but low in incidence, adverse effects, and the patients' education, emphasizing compliance with the treatment plan, prevention and timely recognition of its toxicities.

A Three-step Proteolytic Cascade Mediates the Activation of the Peptidoglycan-induced Toll Pathway in an Insect

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway.

3D space-varying coefficient models with application to diffusion tensor imaging

The present methodological development and the primary application field originate from diffusion tensor imaging (DTI), a powerful nuclear magnetic resonance technique which enables the quantification of microscopical tissue properties. The current analysis framework of separate voxelwise regressions is reformulated as a 3D space-varying coefficient model (SVCM) for the entire set of diffusion tensor images recorded on a 3D voxel grid. The SVCM unifies the three-step cascade of standard data processing (voxelwise regression, smoothing, interpolation) into one framework based on B-spline basis functions. Thereby strength is borrowed from spatially correlated voxels to gain a regularization effect right at the estimation stage. Two SVCM variants are conceptualized: a full tensor product approach and a sequential approximation, rendering the SVCM numerically and computationally feasible even for the huge dimension of the joint model in a realistic setup. A simulation study shows that both approaches outperform the standard method of voxelwise regression with subsequent regularization. Application of the fast sequential method to real DTI data demonstrates the inherent ability to increase the grid resolution by evaluating the incorporated basis functions at intermediate points. The resulting continuous regularized tensor field may serve as basis for multiple applications, yet, ameloriation of local adaptivity is desirable.

Unmasking of aminoanthroquinone moiety through a ring opening in the presence of copper salts and a subsequent cross-coupling/recyclization cascade

In the presence of copper(I) salts, 3-bromo- or 3-iodoisoxazoles undergo isoxazole ring opening to give keto amines that can undergo further one-pot cascade cross-coupling/recyclization transformation into an extended flat polyaromatic ring system that can provide an interesting new platform for the design of DNA intercalators. If necessary, the three-step reaction cascade can be interrupted at a desired intermediate step through a judicious choice of reaction temperature and catalyst.