The Preparation of DNA Duplexes Containing Internucleotide Phosphorothioate Groups in Various Positions of the Recognition Site for the EcoRII Restriction Endonuclease

Twenty four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as R _p and S _p diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the R _p and S _p diastereomers of 5–7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template.     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

6′-Methyl-5′-homoaristeromycin: A structural variation of the anti-orthopox virus candidate 5′-homoaristeromycin

The synthesis of 6′-methyl-5′-homoaristeromycin is described from a known 6′-ethyl ester. Antiviral analysis showed the (S)-6′ stereoisomer retained the vaccinia activity of the parent 5′-homoaristeromycin (1) while the (R)-6′ isomer was less active. Both were weaker than 1 towards cowpox. The diastereomers were equally active versus Epstein Barr virus while (S)-6′ was three times more active toward vesicular stomatitis virus than (R)-6′. The diastereomers were inactive towards numerous other viruses. The CC₅₀ for both diastereomers was >300 μM.     

Enzymic reduction of [6]-gingerol,a major pungent principle of ginger,in the cell-free preparation of rat liver

A reductive metabolism of S-(+)-[6]-gingerol [1-(4'-hydroxy-3'-methoxyphenyl)-5-hydroxydecan-3-one], the major pungent principle of ginger, was investigated in vitro with phenobarbital-induced rat liver 10,000 x g supernatant containing the NADPH-generating system. The ethyl acetate-extractable products were isolated and two metabolites were identified as diastereomers of [6]-gingerdiol by gas chromatrography/mass spectrometry. The ratio of two isomers formed in the above reaction was about 1:5, suggesting the stereospecific reduction of S-(+)-[6]-gingerol by carbonyl reductase activity present in the postmitochondrial supernatant fraction of rat liver. The enzymic reduction of S-(+)-[6]-gingerol thus introduces the second asymmetric carbon center in the molecule with concomitant production of S,S- and R,S-isomers of [6]-gingerdiol in different proportions. This stereospecific reduction of [6]-gingerol may be relevant to the clinical use of the compound.     

Synthesis of backbone-modified DNA analogues for biological applications

The phosphite triester method was adapted for automated synthesis of phosphate (backbone)-modified analogues of DNA. The use of phosphoramidite reagents to achieve substitution of the nonequivalent (diastereotopic) oxygens of an internucleotide phosphate linkage is nonselective, and leads to nonequivalent (diastereomeric) strands of DNA with R _p and S _p absolute configurations at the chiral, modified phosphorus position. Indications of the scope and limitations of the use of reversed-phase HPLC to separate these diastereomers have been obtained through studies of backbone-modified DNA analogues having phosphorothioate (P-S), phosphotriester (P-OR), and alkanephosphonate (P-R) linkages. Incorporation of these modifications in the self-complementary octanucleotide d(GGAATTCC) led to separable diastereomers, which form nonequivalent R _p · R _p and S _p · S _p duplexes. A combination of chemical and nuclear Overhauser effect NMR spectroscopic methods was developed to assign unambiguously, for the first time, the absolute configurations at phosphorus in these prototypal cases. The effects of backbone ethylation on DNA structure and dynamics were evaluated by NMR methods. Modified duplexes were used to probe for proposed phosphate contacts for EcoRI endonuclease, and to define, in concert with base-modified analogues, a recognition site for monoclonal anti-native DNA autoantibody.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H₂O₂/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site.     

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R² = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.     

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain. We have analyzed the epimeric purity of 4-methylsulfinylbutylglucosinolate by NMR methods using a chiral lanthanide shift reagent. The absolute configuration of the sulfoxide group has been established by comparing the ¹H NMR spectra of the two sulfoximine diastereomers of natural 4-methylsulfinylbutylglucosinolate. According to our data, 4-methylsulfinylbutylglucosinolate isolated from broccoli and A. thaliana is a pure epimer and its sulfoxide group has the R_S configuration. The product of the A. thaliana flavin monooxygenase has these same properties demonstrating that the enzyme is stereospecific and supporting its involvement in glucosinolate side chain formation.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Biosynthesis of flavan-3-ols and other secondary plant products from (2S)phenylalanine

(2S)-Phenyl[2-¹⁴C,3R-³H₁]alanine and (2S)-phenyl[2-¹⁴C,3S-³H₁]alanine have been employed as substrates to study procyanidin and flavan-3-ol biosynthesis. Parallel studies with the cyanogenic glucosides prunasin and sambunigrin, Winterstein's acid [(3R)-3-dimethylaminophenylpropionic acid] and tropic acid show these to be derived by stereospecific processes from (2S)-phenylalanine. New proposals for procyanidin biosynthesis are briefly commented upon.     

Structure of the active metal-adenosine 5′-triphosphate chelate complex used by light-triggered chloroplast ATPase

By employing phosphorothioate analogs of ATP in the presence of Mg²⁺ and Mn²⁺ as substrates in ATP hydrolysis, catalyzed by light and dithiothreitol-activated chloroplast ATPase, the structure of the reactive metal-nucleotide complex has been determined. Mg(S_P)-ATPαS and Mn(S_P)-ATPαS, in contrast to the corresponding R_P-isomers, are substrates in ATP hydrolysis. No metal-dependent change of specificity was observed. Mg(S_P)-ATPβS, having the Δ configuration, and Mn(S_P)-ATPβS and Mn(R_P)-ATPβS, consisting of a mixture of Δ and Λ configurations, were better substrates than Mg(R_P)-ATPβS, the isomer with almost exclusive Λ chelate structure. The same results were obtained when the competitive effect of the analogs on hydrolysis of ATP was studied. The competitive effect of the diastereomers on tight binding of ATP by membrane-associated CF₁, was investigated in the presence of Mg²⁺ and Cd²⁺. Mg(S_P)-ATPβS and Cd(R_P)-ATPβS, which both exhibit Δ structure, were more effective than Mg(R_P)-ATPβS and Cd(S_P)-ATPβS, showing the Λ configuration. No metal-dependent change of the preferred S_P-ATPαS specificity was detected. These results permit the conclusion that the actual substrate used by chloroplast ATPase is the β,γ-Δ-bidentate nucleotide chelate. Moreover, a stereospecific direct ionic interaction between the protein and α-phosphate is likely.     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of ɛ-hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.     

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S_p)-adenosine cyclic 3′:5′-monophosphorothioate ((S_p)-cAMPS)) and the other a diastereoisomeric antagonist ((R_p)-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91–244)·C-subunit, by using fluorescence spectroscopy and amide H/²H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91–244), and the effects of cAMP, (S_p)-cAMPS, and (R_p)-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R_p)-cAMPS on amide exchange of the RIα(91–244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91–244). Surprisingly, (R_p)-cAMPS not only increased the affinity of RIα(91–244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R_p)-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R_p)-cAMPS-induced increase in affinity of RIα(91–244) toward the C-subunit indicates that (R_p)-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.Cyclic AMP-dependent protein kinase (PKA)¹ plays a crucial role in a plethora of cellular functions. All isoforms of PKA are composed of two catalytic (C) subunits and homodimeric regulatory (R) subunits (1–3). As the name implies, cAMP is a major PKA regulator (4). Much progress has been made in the last decade in delineating the molecular basis of action of cAMP. An important tactic in this endeavor has been through the comparison of the effects of cAMP with those of two phosphorothioate cAMP analogs: (S_p)-cAMPS (a cAMP mimic) and (R_p)-cAMPS (an antagonist and a diastereoisomer of (S_p)-cAMPS). Although the importance of geometry of the sulfur substitution is critical in determining the pharmacological properties of the two phosphorothioate cAMP analogs, the molecular basis for this behavior is not fully understood. To date, these comparisons have only been made using either wild-type or truncated mutants of the type Iα regulatory subunit (RIα) that are free in solution, not complexed to the C-subunit. X-ray spectroscopic examination of ligand-bound RIα(92–379) complexes reveals few differences between ligand-bound complexes, but the (R_p)-cAMPS complex is structurally “looser” with higher thermal factors than complexes formed with either cAMP or (S_p)-cAMPS (5). This is consistent with the observation that both cAMP and (S_p)-cAMPS, but not (R_p)-cAMPS, raise the urea concentration required for wild-type RIα unfolding (6). Further insight into the structural basis of cAMP action stems from NMR spectroscopic comparison of the effects of (R_p)-cAMPS, cAMP, and (S_p)-cAMPS on chemical shifts and ¹⁵N relaxation of the RIα(119–244) mutant (7). In addition to producing fewer significant chemical shift changes than either cAMP or (S_p)-cAMPS, (R_p)-cAMPS binding is associated with enhanced millisecond to microsecond time scale backbone motions of a β-turn (β2,3 loop) and around the phosphate-binding cassette (PBC) (7).Further insight into the molecular basis of actions of cAMP and its analogs should come from the analysis of ligand-bound R·C complexes. Unfortunately, the large size of even the heterodimeric R·C complex (∼95 kDa) and the difficulty of preparing (R_p)-cAMPS·R·C-subunit crystals currently preclude the use of both NMR spectroscopy and x-ray crystallography. Consequently, we took two alternative lower resolution approaches to this issue. One approach involves the use of site-directed labeling combined with fluorescence spectroscopy to examine both the effects of cAMP and its analogs on R-C subunit binding kinetics and on the conformational dynamics of RIα(91–244). RIα(91–244) includes the “A” cyclic nucleotide binding (CNB) domain, the pseudosubstrate, and linker domains and represents the minimal segments necessary for high affinity C-subunit binding (Fig. 1) (8). The other approach involves an examination of the effects of cAMP and its analogs on solvent exposure/conformational flexibility of RIα(91–244)·C-subunit complex using H/²H amide exchange measured with a combination of mass spectrometry (ESI-Q-TOF) and proteolytic fragmentation. In the first approach, fluorescein maleimide (FM) was conjugated to three cysteine substitution mutants with the substitution sites located near or within the pseudosubstrate sequence, the linker domain, or αC (R92C, T104C, and R239C, respectively) of RIα(91–244) (Fig. 1). The time-resolved fluorescence anisotropy results suggest that cAMP and (S_p)-cAMPS reduce the interaction of the RIα linker domain and αC with the two peripheral R-C interaction sites on the C-subunit (so-called Sites 2 and 3) without affecting the interaction of the pseudosubstrate sequence with the active site cleft (so-called Site 1). Because of limitations of the amide H/²H exchange experiments, only the effects of (R_p)-cAMPS on H/²H amide exchange in RIα(91–244)·C-subunit complex could be investigated. The results showed that (R_p)-cAMPS induces a relatively widespread increase in amide exchange, indicating limited unfolding and/or enhanced conformational flexibility that is propagated almost globally through the C-subunit and, at least, part of RIα. These conformational changes were accompanied by a 5-fold increase in the affinity of RIα(91–244) toward C-subunit, suggesting that, at least, some of the (R_p)-cAMPS effects are mediated by an increase in internal entropy. Finally, the (R_p)-cAMPS-induced increase in R-C affinity indicates that (R_p)-cAMPS is better described as an inverse agonist because the basal activity of the PKA holoenzyme should be decreased by (R_p)-cAMPS.Open in a separate windowFig. 1.Overview of PKA structure and cAMP analogs. A, domain organization of RIα showing the domain boundaries of RIα(91–244) where the pseudosubstrate in green is connected to CNB-A domain in blue by a linker segment. B, structure of RIα(91–244) in the C-subunit-bound conformation (Protein Data Bank code 1U7E (23)) showing the pseudosubstrate in green, linker in yellow, and helical subdomain comprising helices αN, αA, αB, and αC in blue and β-subdomain in tan. The PBC is in red. C, structure of the C·RIα(91–244) holoenzyme showing the C-subunit in tan and RIα(91–244) in blue. Sites for introduction of cysteines by site-directed mutagenesis are represented by red circles. The cAMP binding site (PBC) is in red. D, structure of cAMP showing the 2′-OH group and 3′–5′ phosphodiester bond. The exocyclic oxygens upon replacement with sulfur atoms to generate the (S_p)-cAMPS and (R_p)-cAMPS diastereomers are highlighted.     

Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH₃ debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC₅₀ values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.