Rigid crosslinking of the CD3 complex leads to superior T cell stimulation.

Functionally bivalent non-covalent Fab dimers (Bi-Fabs) specific for the TCR/CD3 complex promote CD3 signaling on T cells. While comparing functional responses to stimulation with Bi-Fab, F(ab')2 or mAb specific for the same CD3 epitope, we observed fratricide requiring anti-CD3 bridging of adjacent T cells. Surprisingly, anti-CD3 Bi-Fab ranked first in fratricide potency, followed by anti-CD3 F(ab')2 and anti-CD3 mAb. Low resolution structural studies revealed anti-CD3 Bi-Fabs and F(ab')2 adopt similar global shapes with CD3-binding sites oriented outward. However, under molecular dynamic simulations, anti-CD3 Bi-Fabs crosslinked CD3 more rigidly than F(ab')2. Furthermore, molecular modelling of Bi-Fab and F(ab')2 binding to CD3 predicted crosslinking of T cell antigen receptors located in opposing plasma membrane domains, a feature fitting with T cell fratricide observed. Thus, increasing rigidity of Fab-CD3 crosslinking between opposing effector-target pairs may result in stronger T cell effector function. These findings could guide improving clinical performance of bi-specific anti-CD3 drugs.

Novel phosphorylation-dependent regulation in an unstructured protein.

This work explores how phosphorylation of an unstructured protein region in inhibitor-2 (I2) regulates protein phosphatase-1 (PP1) enzyme activity using molecular dynamics (MD). Free I2 is largely unstructured; however, when bound to PP1, three segments adopt a stable structure. In particular, an I2 helix (i-helix) blocks the PP1 active site and inhibits phosphatase activity. I2 phosphorylation in the PP1-I2 complex activates phosphatase activity without I2 dissociation. The I2 Thr74 regulatory phosphorylation site is in an unstructured domain in PP1-I2. PP1-I2 MD demonstrated that I2 phosphorylation promotes early steps of PP1-I2 activation in explicit solvent models. Moreover, phosphorylation-dependent activation occurred in PP1-I2 complexes derived from I2 orthologs with diverse sequences from human, yeast, worm, and protozoa. This system allowed exploration of features of the 73-residue unstructured human I2 domain critical for phosphorylation-dependent activation. These studies revealed that components of I2 unstructured domain are strategically positioned for phosphorylation responsiveness including a transient α-helix. There was no evidence that electrostatic interactions of I2 phosphothreonine74 influenced PP1-I2 activation. Instead, phosphorylation altered the conformation of residues around Thr74. Phosphorylation uncurled the distance between I2 residues Glu71 to Tyr76 to promote PP1-I2 activation, whereas reduced distances reduced activation. This I2 residue Glu71 to Tyr76 distance distribution, independently from Thr74 phosphorylation, controls I2 i-helix displacement from the PP1 active site leading to PP1-I2 activation.

Analysis of protein phosphatase-1 and aurora protein kinase suppressors reveals new aspects of regulatory protein function in Saccharomyces cerevisiae.

Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates.

How phosphorylation activates the protein phosphatase-1 • inhibitor-2 complex.

Phosphorylation regulates activity of many proteins; however, atomic level details are known for very few examples. Inhibitor-2 (I2) squelches the ubiquitous protein phosphatase-1 (PP1) enzyme activity by blocking access to the metal-containing active site. I2 Thr74 phosphorylation results in PP1 activation without I2 dissociation from the PP1-I2 complex. The dynamic disordered structure of the 73-residue segment of I2 containing Thr74, prevented visualization by X-ray crystallography of PP1-I2. In this work, I generated structures of this segment using simulated annealing to NMR restraints, fused them to the crystallographic PP1-I2 coordinates, and used molecular dynamics to study the impact of Thr74 phosphorylation on structural alterations leading to PP1 activation. Frequencies of I2 Tyr149 displacement from the PP1 active site, rotation of the phenolic Tyr149 side chain to prevent its reinsertion, and repositioning the I2 inhibitory helix to expose the PP1 active site to solvent and substrates significantly increased upon I2 Thr74 phosphorylation. After these steps, a second metal bound to produce PP1-Mn(2)-I2, which held the phosphorylated form of I2 to its active site less tightly than it held dephosphorylated I2. I2 Thr74 lies on the edge of variable dynamic communities of residues where it forms various allosteric pathways that induce motions at the PP1 active site 20Å away. These molecular dynamics simulations show how an unstructured region of I2 can harness enhanced rapid movements around phosphorylated Thr74 to pry I2 residues away from the PP1 active site in early steps of PP1-I2 activation.

Function of protein phosphatase-1, Glc7, in Saccharomyces cerevisiae.

Budding yeast, Saccharomyces cerevisiae, and its close relatives are unique among eukaryotes in having a single gene, GLC7, encoding protein phosphatase-1 (PP1). This enzyme with a highly conserved amino acid sequence controls many processes in all eukaryotic cells. Therefore, the study of Glc7 function offers a unique opportunity to gain a comprehensive understanding of this critical regulatory enzyme. This review summarizes our current knowledge of how Glc7 function modulates processes in the cytoplasm and nucleus. Additionally, global Glc7 regulation is described.

Synthesis and antiviral evaluation of 6-(alkyl-heteroaryl)furo[2,3-d]pyrimidin-2(3H)-one nucleosides and analogues with ethynyl, ethenyl, and ethyl spacers at C6 of the furopyrimidine core.

Sonogashira coupling strategies were employed to synthesize new furo[2,3-d]pyrimidin-2(3H)-one (FuPyrm) 2'-deoxynucleoside analogues. Partial or complete reduction of ethyne-linked compounds afforded ethenyl- and ethyl-linked derivatives. Levels of inhibition of varicella-zoster virus (VZV), human cytomegalovirus (HCMV), a broad range of other DNA and RNA viruses, and several cancer cell lines were evaluated in cell cultures. The anti-VZV potency decreased with increasing rigidity of the side chain at C6 of the FuPyrm ring in the order dec-1-yn-1-yl < dec-1-en-1-yl < decan-1-yl. In contrast, compounds with a rigid ethynyl spacer between C6 of the FuPyrm ring and a 4-alkylphenyl moiety were more potent inhibitors of VZV than the corresponding derivatives with an ethyl spacer. Replacement of the phenyl moiety in 6-(4-alkylphenyl) derivatives with a pyridine ring (in either regioisomeric orientation) gave analogues with increased solubility in methanol but reduced anti-VZV potency, and replacement with a pyrimidine ring reduced the anti-VZV activity even further. The pyridine-ring-containing analogues were approximately 20-fold more potent inhibitors of VZV than acyclovir but were approximately 6-fold less potent than BVDU and approximately 60-fold weaker than the most active 6-(4-pentylphenyl)-substituted prototype.

Addition of difluorocarbene to 3',4'-unsaturated nucleosides: synthesis of 2'-deoxy analogues with a 2-oxabicyclo[3.1.0]hexane framework.

Treatment of protected 2'-deoxy-3',4'-unsaturated nucleosides derived from adenosine and uridine with difluorocarbene [generated from bis(trifluoromethyl)mercury and sodium iodide] gave fused-ring 2,2-difluorocyclopropane compounds. Stereoselective alpha-face addition to the dihydrofuran ring resulted from hindrance by the protected beta-anomeric nucleobases. A protected uracil compound was converted smoothly into the cytosine derivative via a 4-(1,2,4-triazol-1-yl) intermediate. Removal of the protecting groups gave new difluorocyclopropane-fused nucleoside analogues. The solid-state conformation of the nearly planar furanosyl ring in the uracil compound had a shallow 2E pucker, and a more pronounced 1E conformation was present in the furanosyl ring of the cytosine derivative.

N-oxides of adenosine-type nucleosides undergo pyrimidine ring opening and closure to give 5-amino-4-(1,2,4-oxadiazol-3-yl)imidazole derivatives.

Treatment of acylated adenosine N-oxides with carboxylic anhydrides and thiophenol resulted in pyrimidine ring opening followed by exocyclic ring closure. Ammonolysis gave 5-amino-4-(5-substituted-1,2,4-oxadiazol-3-yl)-1-(beta-d-ribofuranosyl)imidazole derivatives, whereas iodine in methanol selectively unmasked the 5-amino group. Related flexible nucleoside analogues can be prepared from adenine-type precursors.

Structure and synthesis of 6-(substituted-imidazol-1-yl)purines: versatile substrates for regiospecific alkylation and glycosylation at N9.

X-ray crystal structures of several 6-(azolyl)purine base and nucleoside derivatives show essentially coplanar conformations of the purine and appended 6-(azolyl) rings. However, the planes of the purine and imidazole rings are twisted approximately 57 degrees in a 2-chloro-6-(4,5-diphenylimidazol-1-yl)purine nucleoside, and a twist angle of approximately 61 degrees was measured between the planes of the purine and pyrrole rings in the structure of a 6-(2,5-dimethylpyrrol-1-yl)purine nucleoside derivative. Shielding "above" N7 of the purine ring by a proximal C-H on the 6-azolyl moiety is apparent with the coplanar compounds, but this effect is diminished in those without coplanarity. Syntheses of 6-(azolyl)purines from both base and nucleoside starting materials are described. Treatment of 2,6-dichloropurine with imidazole gave 2-chloro-6-(imidazol-1-yl)purine. Modified Appel reactions at C6 of trityl-protected hypoxanthine and guanine derivatives followed by detritylation gave 6-(imidazol-1-yl)- and 2-amino-6-(imidazol-1-yl)purines. Imidazole was introduced at C6 of 2',3',5'-tri-O-acetylinosine by a modified Appel reaction, and solvolysis of the glycosyl linkage gave 6-(imidazol-1-yl)purine. Guanosine triacetate was transformed into the protected 2,6-dichloropurine nucleoside, which was subjected to S(N)Ar displacement with imidazoles at C6 followed by glycosyl solvolysis to provide 2-chloro-6-(substituted-imidazol-1-yl)purines. Potential applications of these purine derivatives are outlined.

Sylviside, a diterpene glucoside derivative from Gnaphalium sylvaticum.

A new diterpene glucoside (1), named sylviside, was isolated from the aerial parts of Gnaphalium sylvaticum. Its structure was elucidated as 2beta,15alpha,20alpha-trihydroxy-19,20-dicarboxy-ent-kaur-16-ene 2beta-O-(2'-angelate)-beta-D-glucopyranoside, on the basis of spectroscopic analysis ((1)H NMR, (13)C NMR, HMQC, HMBC, NOESY), and was confirmed by X-ray crystallographic analysis. Sylviside (1) displayed weak cytotoxicity against HeLa WT (human epitheloid cervical carcinoma) cells and was also evaluated for its effects on reversing multidrug resistance in HeLa cells overexpressing MDR1.

Double-stranded metal-organic networks for one-dimensional mixed valence coordination polymers.

The design of new types of metal-organic networks and the search for unusual crystal architecture represents an important task for modern inorganic and materials chemistry research. A group of new monosubstituted phenylcyanoximes, containing F, Cl, and Br atoms at the 2, 3, or 4 positions, were synthesized using the high yield nitrosation reaction with CH3-ONO and were spectroscopically (1H NMR, 13C NMR, UV-visible, IR, mass spectrometry) and structurally characterized. Results of X-ray analysis revealed nonplanar trans-anti geometry for 2-chlorophenyl(oximino)acetonitrile, H(2Cl-PhCO); a nonplanar anti configuration for 4-chlorophenyl(oximino)acetonitrile, H(4Cl-PhCO); and planar cis-syn geometry for 3-fluorophenyl(oximino)acetonitrile, H(3F-PhCO). All arylcyanoximes undergo deprotonation in solutions with the formation of colored anions exhibiting pronounced negative solvatochromism in a series of polar protic and aprotic solvents. Nine thallium(I) cyanoximates were obtained using the reaction between hot (approximately 95 degrees C) aqueous solutions of Tl2CO3 and solid powdery monohalogenated arylcyanoximes HL. Crystal structures of two Tl(I) cyanoximates [Tl(2Cl-PhCO) and Tl(4Br-PhCO)] contained centrosymmetric dimeric units (TlL)2 that are connected to a coordination polymer by means of an oxygen atom of the oxime group of the neighboring molecule. Cyanoxime anions act as bridging ligands in both structures where the polymeric motif consists of double-stranded Tl-O chains interconnected with the formation of zigzagging Tl2O2 planar rhombes. Thallium atoms form infinite linear arrays with close intermetallic separations. The nearest Tl(I)...Tl(I) distances are 3.838 and 4.058 angstroms in the Tl(2Cl-PhCO) and Tl(4Br-PhCO) structures, respectively, close to that in metallic thallium (3.456 angstroms). Monosubstituted phenyl groups are well aligned in pi-stacking columns that are perpendicular to the array of Tl(I) atoms and stabilize formed structures. Coordination polyhedrons of thallium(I) in these complexes represent distorted trigonal pyramids with stereoactive lone pair.

Asymmetric halo-Mannich-type reaction provides access to pyrrolidines and beta-proline derivatives.

[reaction: see text] A new halo-Mannich-type reaction is reported using cyclopropyl carbonyl-derived enolates and sulfonyl-protected imines. Chiral oxazolidinones auxiliaries were found to be effective for completely controlling the stereochemistry of the products. Variations in the oxazolidinone, protecting group, and imine components show this to be a quite general reaction. The initial iodo-Mannich products were found to be readily cyclized in the presence of triethylamine to afford the resulting protected pyrrolidines, which could be readily deprotected under standard conditions.

Asymmetric glycolate aldol reactions using cinchonium phase-transfer catalysts.

Cinchona phase-transfer catalysts (PTC) were developed for glycolate aldol reactions to give differentially protected 1,2-diol products. Silyl enol ether 9 reacted to generate benzhydryl-protected products. O-Allyl trifluorobenzyl cinchonium hydrofluoride CN-4 (20 mol %) catalyzed the addition of 9 to benzaldehyde to give 8 as a single syn-product in 76% yield and 80% ee. Recrystallization enriched the product to 95% ee, and a Baeyer-Villiger reaction transformed the product into useful ester intermediates. [reaction: see text]

Functionalization of alpha,beta-unsaturated esters and ketones: a facile and highly stereoselective one-pot approach to N-protected alpha,beta-dehydroamino acid derivatives.

A new, facile, and highly stereoselective protocol toward alpha,beta-dehydroamino acid derivatives has been developed. The one-pot synthesis was very convenient to perform by using the aminohalogenation reaction of alpha,beta-unsaturated esters and ketones followed by treatment with specific bases. Only two [2.2.2] bicyclic organic bases were found to be effective for this transformation. Good yields (58-68%) and excellent Z-selectivity were obtained for 12 examples. [reaction: see text]

Ionic liquid media resulted in the first asymmetric aminohalogenation reaction of alkenes.

[reaction: see text] The first asymmetric aminohalogenation of functionalized alkenes has been established. The ionic liquid [bmim][BF4] was found to be the only effective media for success as normal organic solvents failed to give any product for this reaction. The reaction is also very convenient to perform by simply mixing the three reactants, cinnamates, N,N-dichloro-p-toluenesulfonamide, and catalyst, together with 4 A molecular sieves at room temperature in [bmim][BF4] in any convenient vial of appropriate size without special protection from inert gases. Good chemical yields (60-72%) and diastereoselectivities (up to 75% de) have been obtained with a good scope of substrates. The resulting individual diastereomers have been cleanly separated via column chromatography. The absolute stereochemistry of the reaction was unambiguously determined by X-ray structural analysis.

Synthesis and properties of gem-(difluorocyclopropyl)amine derivatives of bicyclo[n.1.0]alkanes.

[reaction: see text] Generation of difluorocarbene(carbenoid) in the presence of enamines derived from cyclic ketones results in overall insertion of CF2 to produce bicyclic difluorocyclopropylamines. These adducts are very weakly basic, and their thermal stabilities vary markedly with their structures.

New asymmetric halo aldol reaction provides a novel approach to biologically important chiral cyclothers and cycloamines.

[reaction: see text] A new asymmetric halo aldol reaction has been developed by reacting cyclopropyl carbonyl derived enolates with aldehydes. The absolute structure was unambiguously confirmed by X-ray structural analysis. Eight examples were reported with good yields and up to complete control of diastereomeric excesses. These halo aldol products have been readily cyclized in the presence of weak bases to produce chiral 2,3-disubstituted tetrahydrofuran derivatives in good yield without any observed epimerization.

Borylation of aryldiazonium ions with N-heterocyclic carbene-palladium catalysts formed without added base.

[reaction: see text] A highly efficient catalytic borylation process with aryldiazonium ions was developed using a carbene-palladium catalyst formed in situ to give arylpinacolatoborane products. An X-ray structure for the N-heterocyclic carbene-palladium complex, used as the catalyst formed from bis(2,6-diisopropylphenyl)-4,5-dihydroimidazolium chloride, was obtained without added base.

Selective synthesis of the para-quinone region of geldanamycin.

[structure: see text] The quinone portion of the ansamycin geldanamycin was made with complete selectivity from the 1,4-dihydroquinone generated from a 1,4-bis-methoxymethyl (MOM) ether intermediate. Palladium catalysis with air gave the desired product in 98% isolated yield. The structure was established using NMR, UV, and X-ray analysis with comparisons to geldanamycin, ortho-quino-geldanamycin and a model compound.

A catalytic reaction of alkynes via multiple-site functionalization.

The first multiple-site activation of alkynes with amine/halogen functionalities has been established. The reaction was performed by treating alkyne with N,N-dichlorobenzenesulfonamide at 80 degrees C in the presence of palladium acetate catalyst. A new mechanism was proposed which involves the novel formation of beta-halovinyl palladium and pi-allylpalladium species. Excellent regio- and stereoselectivities were achieved with the absolute structure determined by X-ray structural analysis.