RESUMO
Delivery of macromolecular cargos such as siRNA to the cytosol after endocytosis remains a critical challenge. Numerous approaches including viruses, lipid nanoparticles, polymeric constructs, and various peptide-based approaches have yet to yield a general solution to this delivery issue. In this manuscript, we describe our efforts to design novel endosomolytic peptides that could be used to facilitate the release of cargos from a late endosomal compartment. These amphiphilic peptides, based on a chimeric influenza hemagglutinin peptide/cell-penetrating peptide (CPP) template, utilize a pH-triggering mechanism in which the peptides are protonated after acidification of the endosome, and thereby adopt an alpha-helical conformation. The helical forms of the peptides are lytically active, while the non-protonated forms are much less or non-lytically active at physiological pH. Starting from an initial lead peptide (INF7-Tat), we systematically modified the sequence of the chimeric peptides to obtain peptides with greatly enhanced lytic activity that maintain good pH selectivity in a red blood cell hemolysis assay.
Assuntos
Motivos de Aminoácidos , Peptídeos Penetradores de Células , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Concentração de Íons de Hidrogênio , Sequência de Aminoácidos , Peptídeos Penetradores de Células/química , Dicroísmo Circular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemólise , Humanos , Proteólise , Análise EspectralRESUMO
An efficient multicomponent orthogonal protocol was developed for post-synthetic oligonucleotide modification using commercially available 2'- O-methyl ester and 2'- O-propargyl nucleoside scaffolds. Amidation of methyl esters with primary amines was achieved in the presence of 2'-propargyl groups which were utilized for subsequent copper catalyzed cycloaddition with GalNAc-azide. The methodology was applied to generate siRNA composed of multiple amide and triazole conjugates. Computational methods were used to illustrate the impact of substitution at the 2'-position. This a powerful post-oligomerization technique for rapidly introducing diversity to oligonucleotide design.
Assuntos
Acetilgalactosamina/química , Amidas/química , Azidas/química , Cobre/química , Reação de Cicloadição/métodos , Oligonucleotídeos/química , RNA Interferente Pequeno/química , Acetilgalactosamina/síntese química , Amidas/síntese química , Azidas/síntese química , Catálise , Química Click/métodos , Esterificação , Células HeLa , Humanos , Modelos Moleculares , Oligonucleotídeos/síntese química , RNA Interferente Pequeno/síntese química , Triazóis/síntese química , Triazóis/químicaRESUMO
Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Relação Estrutura-Atividade , Alcinos/química , Azidas/química , Catálise , Cromatografia Líquida de Alta Pressão/métodos , Química Click , Cobre/química , Reação de Cicloadição , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Técnicas de Síntese em Fase SólidaRESUMO
The synthesis, computer modeling, and biological activity of an octawalled molecular umbrella short interfacing RNA (siRNA) conjugate is described. This molecular umbrella-siRNA conjugate exhibited mRNA knockdown activity in vitro in the absence of a transfection reagent. Evaluation of this molecular umbrella conjugate in vivo, using the rat eye via intravitreal injection, resulted in sequence specific mRNA knockdown in the retina with no obvious signs of toxicity, as judged by ophthalmic examination.
Assuntos
Portadores de Fármacos , Olho , RNA Interferente Pequeno/administração & dosagem , Vias de Administração de Medicamentos , Células HEK293 , Humanos , Simulação de Dinâmica MolecularRESUMO
Herpesvirus infections are ubiquitous, with over 95% of the adult population infected by at least one strain. While most of these infections resolve without treatment in healthy individuals, they can cause significant morbidity and mortality in immunocompromised, stem cell, or organ transplant patients. Current nucleoside standards of care provide meaningful benefit but are limited due to poor tolerability, resistance, and generally narrow spectrum of activity. Herpesviruses share a conserved DNA polymerase, the inhibition of which is validated as an effective strategy to disrupt viral replication. By utilizing a non-nucleoside inhibitor of the viral DNA polymerase, we sought to develop agents covering multiple herpesviruses (e.g., CMV, VZV, HSV1/2, EBV, and HHV6). Herein is described the invention of an oxazolidinone class of broad-spectrum non-nucleoside herpes antiviral inhibitors. A lead compound (42) with potent biochemical and broad-spectrum cellular activity was found to be efficacious in murine models against both HSV-1 and CMV infection.
RESUMO
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endossomos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , Autoantígenos/genética , Autoantígenos/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Eficiência , Endossomos/metabolismo , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/metabolismo , Interferência de RNA/efeitos dos fármacos , Interferência de RNA/fisiologia , Estabilidade de RNA/genética , RNA Interferente Pequeno/farmacologia , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.
Assuntos
Mastócitos , Medicamentos Sintéticos , Animais , Degranulação Celular , Chumbo , Mastócitos/metabolismo , Peptídeos/química , Peptídeos/toxicidade , Ratos , Medicamentos Sintéticos/metabolismoRESUMO
Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer's disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.
Assuntos
Doença de Alzheimer , Acetilcolinesterase , Doença de Alzheimer/diagnóstico por imagem , Animais , Humanos , Macaca mulatta , Tomografia por Emissão de Pósitrons/métodos , Receptores MuscarínicosRESUMO
Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/síntese química , Animais , Degranulação Celular/efeitos dos fármacos , Cistina/química , Desenho de Fármacos , Temperatura Alta , Mastócitos/efeitos dos fármacos , Modelos Moleculares , Medição da Dor/efeitos dos fármacos , Ratos , Venenos de Aranha/farmacologiaRESUMO
Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.
Assuntos
Reagentes de Ligações Cruzadas/química , RNA Interferente Pequeno/química , Amidas/síntese química , Aminas/química , Ácidos Carboxílicos/química , Métodos , SoluçõesRESUMO
Development of a practical synthesis of MK-7009, a 20-membered [corrected] macrocycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization. Ring closure via macrolactamization was found to give the highest yields under relatively high reaction concentrations. Optimization of the ring formation step and the synthesis of key intermediates en route to MK-7009 are reported.
Assuntos
Técnicas de Química Sintética/métodos , Indóis/química , Indóis/síntese química , Lactamas/química , Compostos Macrocíclicos/química , Catálise , Ciclização , Ciclopropanos , Hidrogenação , Isoindóis , Lactamas Macrocíclicas , Leucina/análogos & derivados , Paládio/química , Prolina/análogos & derivados , SulfonamidasRESUMO
All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Herpesviridae/efeitos dos fármacos , Herpesviridae/enzimologia , Antivirais/farmacologia , Sítios de Ligação , DNA Polimerase Dirigida por DNA/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Exodesoxirribonucleases , Nucleotídeos , Quinolinas/farmacologia , Proteínas Virais , Replicação ViralRESUMO
By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.
RESUMO
The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Piridinas/farmacologia , Receptor Muscarínico M4/agonistas , Animais , Células CHO , Radioisótopos de Carbono/química , Radioisótopos de Carbono/farmacologia , Cricetulus , Humanos , Macaca mulatta , Agonistas Muscarínicos/química , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Tomografia por Emissão de Pósitrons , Piridinas/química , Receptor Muscarínico M4/metabolismoRESUMO
Increasing amounts of biological data are accumulating in the pharmaceutical industry and academic institutions. However, data does not equal actionable information, and guidelines for appropriate data capture, harmonization, integration, mining, and visualization need to be established to fully harness its potential. Here, we describe ongoing efforts at Merck & Co. to structure data in the area of chemogenomics. We are integrating complementary data from both internal and external data sources into one chemogenomics database (Chemical Genetic Interaction Enterprise; CHEMGENIE). Here, we demonstrate how this well-curated database facilitates compound set design, tool compound selection, target deconvolution in phenotypic screening, and predictive model building.
Assuntos
Bases de Dados Factuais , Descoberta de Drogas , Genômica , Modelos Teóricos , FenótipoRESUMO
DESIGN: The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. METHODS: Human CD4+ T cells were treated ex vivo with Phorbol 12-myristate 13-acetate, prostatin or ingenol-3-angelate. At 3 h and 24 h post-treatment, cells were harvested and RNA-Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT-PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. RESULTS: Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. CONCLUSION: Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation.
Assuntos
HIV-1/fisiologia , Proteína Quinase C/metabolismo , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Biomarcadores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Diterpenos/química , Diterpenos/farmacologia , Agonismo de Drogas , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 3 de Resposta de Crescimento Precoce/genética , Expressão Gênica , Infecções por HIV/virologia , Humanos , Células Jurkat , Masculino , Forbóis/farmacologia , Análise de Sequência de RNARESUMO
There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.
Assuntos
Membrana Celular/química , Peptídeos/administração & dosagem , Peptídeos/química , Engenharia de Proteínas/métodos , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , MicroRNAs/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Solubilidade , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
[reaction: see text] Trifluoropropanoic acid reacts with 1 equiv of POCl3 in DMF to generate the trifluoromethyl enamine (7). At this stage, two reaction manifolds are available. The expected reaction with additional POCl3 generates the 2-trifluoromethyl vinamidinium salt (3c). However, thermally driven loss of fluoride generates an iminium ion, which sets the stage for a [2 + 2] cycloaddition to ultimately generate the dimethylaminomethylene vinamidinium salt (1).
Assuntos
Dimetilaminas/síntese química , Compostos Heterocíclicos/síntese química , Vimblastina/síntese química , Química Farmacêutica , Fluorocarbonos/química , Iminas/química , Vimblastina/análogos & derivados , Compostos de Vinila/químicaRESUMO
Reaction of Cp(PMe(3))IrPh(OH) (1) with nitriles is undetectably slow in benzene solution at room temperature. However, in the presence of Cp(PMe(3))IrPh(OTf) (2) (OTf = O(3)SCF(3)), the reaction is strongly catalyzed, leading to iridium(III) carboxamides Cp(PMe(3))IrPh[NHC(O)R] (6a-d) [R = C(6)H(4)CH(3) (6a), C(6)H(5) (6b), C(6)H(4)CF(3) (6c), CH(3) (6d)]. We propose that these transformations occur by initial displacement of the trifluoromethanesulfonate ("triflate") anion of 2 by a molecule of nitrile, leading to a nitrile-substituted iridium cation, [Cp(PMe(3))IrPh(NCR)](+) (10). Following this, the nucleophilic hydroxide group of 1 attacks the (activated) nitrile molecule bound in 10, leading (after proton transfer) to the iridium carboxamide complex. In the case of nitriles possessing hydrogens alpha to the cyano group, competitive loss of one of these protons is observed, leading to iridium C-bound cyanoenolates such as Cp(PMe(3))(Ph)Ir(CH(2)CN) (7). Protonolysis of carboxamides 6a-d with HCl yields Cp(PMe(3))IrPh(Cl) (9) and the free amides. A pronounced solvent effect is observed when the reaction between 1 and nitriles catalyzed by 2 is carried out in THF solution. The basic hydroxide ligand of 1 induces an overall dehydration/cyclization reaction of the coordinated aromatic nitrile. For example, the reaction of 1 with p-trifluorotolunitrile and a catalytic amount of 2 leads to the formation of 6c, water, [Ph(PMe(3))Ir[C(5)Me(4)CH(2)C(C(6)H(4)CF(3))N]] (12), and [Ph(PMe(3))Ir(C(5)Me(4)CH(2)C(C(6)H(4)CF(3))NH)]OTf (13). A mechanism to explain the formation of both 12 and 13 and the role each compound plays in the formation of the iridium carboxamides is proposed.
RESUMO
Ruthenium complexes employing axially chiral ligands were found to be effective asymmetric hydrogenation catalysts for the reduction of alpha,beta-unsaturated ene acid 1-E to give 2, a prostaglandin D2 (PGD2) receptor antagonist. With [(S-BINAP)Ru(p-cymene)Cl2]2 (3, S-BINAP = (S)-(+)-2,2'-bis(diphenylphospino)-1,1'-binapthyl), it was discovered that low hydrogen pressures (<30 psi) were essential to achieve high enantioselectivities (92% ee). A detailed mechanistic study was undertaken to elucidate this pressure dependence. It was determined that compound 1-E is in a ruthenium-catalyzed equilibrium with endocylic isomer 1-Endo and in photochemical equilibrium with Z isomer 1-Z. Each isomer could be hydrogenated to give 2, albeit with different rates and enantioselectivities. Hydrogenation of 1-Endo with 3 was found to give 2 in high enantiomeric excess, regardless of pressure and at a rate substantially faster than that of hydrogenation of 1-E and 1-Z. In contrast, isomers 1-E and 1-Z exhibited pressure-dependent enantioselectivities, with higher enantiomeric excesses obtained at lower pressures. A rationale for this pressure dependence is described. Deuterium labeling studies with 1-Endo and tiglic acid were used to elucidate the mechanism of hydride insertion and product release from ruthenium. Under neutral conditions, protonolysis was the major pathway for metal-carbon cleavage, while under basic conditions, hydrogenolysis of the metal-carbon bond was predominant.