Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity.

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a gamma-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a beta-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in gamma-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.

In this Phase I trial, patients' peripheral blood dendritic cells were pulsed with peptides eluted from the surface of autologous glioma cells. Three biweekly intradermal vaccinations of peptide-pulsed dendritic cells were administered to seven patients with glioblastoma multiforme and two patients with anaplastic astrocytoma. Dendritic cell vaccination elicited systemic cytotoxicity in four of seven tested patients. Robust intratumoral cytotoxic and memory T-cell infiltration was detected in two of four patients who underwent reoperation after vaccination. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed dendritic cell vaccine for patients with malignant glioma.

Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.

Total serum glycosylated proteins in detection and monitoring of gestational diabetes.

The goal of this study was to determine whether serum glycosylated protein levels (i.e., fructosamine) can reliably screen for gestational diabetes and whether these levels are valid markers of short-term glycemic control in the third trimester of pregnancy. Ninety-seven pregnant women at 26-28 wk gestation were evaluated over 9 mo. HbA1c and serum glycosylated protein (serum fructosamine) were determined at the baseline venipuncture of the 100-g oral glucose tolerance test performed to detect gestational diabetes. Of the 97 women studied, 13 tested positive for gestational diabetes (National Diabetes Data Group criteria). There were significant differences in the fasting and 1-, 2-, and 3-h glucose values between nondiabetic and diabetic patients (P less than 0.005 at each time point). No difference was noted in the baseline serum glycosylated protein level (2.02 +/- 0.08 vs. 1.98 +/- 0.02 mM, NS) or HbA1c level (4.42 +/- 0.2 vs. 4.6 +/- 0.3%, NS) between gestational and nondiabetic patients. Diabetic patients were followed at 2-wk intervals, with serum glycosylated protein analysis, HbA1c, fasting glucose, and mean glucose determined by outpatient monitoring. Serum glycosylated protein correlated significantly to fasting blood glucose (r = 0.81, P less than 0.001) and mean outpatient glucose (r = 0.62, P less than 0.001) at the 2-wk follow-up visits. No correlation was found between HbA1c and fasting blood glucose (r = 0.11, NS) or mean outpatient glucose (r = -0.12, NS) during the follow-up period. The serum glycosylated protein level (serum fructosamine) is not a useful screening test for gestational diabetes. However, this assay shows potential as an objective marker of short-term control in evaluating the maternal glycemic state.

Tissue distribution of the cytofectin component of a plasmid-DNA/cationic lipid complex following intravenous administration in mice.

Allovectin-7 is a gene therapy agent that consists of plasmid DNA (pDNA) encoding the human HLA-B7 class I and beta2-microglobulin genes (VCL-1005), complexed with the cationic lipid DMRIE Br and DOPE. A tritiated version of the cytofectin component, DMRIE Br, was synthesized by regiospecific isotope incorporation to a very high specific activity. The 3H-labeled DMRIE/DOPE mixture was complexed with VCL-1005 to produce a radiolabeled version of Allovectin-7. The VCL-1005/3H-DMRIE/DOPE complex was administered intravenously to mice, and the tissue distribution of radioactivity was analyzed 24 hr later. Excretion of radioisotope was monitored for 96 hr post dosing. At 24 hr post administration, a tissue distribution for the radioisotope of liver >> spleen > lung >> heart > brain approximately muscle approximately blood was observed. During the 96-hr period post dose, very little administered radioactivity (<17%) was excreted and the majority of the isotope (83%) remained in the animal. This is the first report on the biodistribution of the cytofectin component of a pDNA-cationic lipid complex for which the distribution of the plasmid component has also been reported.

A new cationic liposome DNA complex enhances the efficiency of arterial gene transfer in vivo.

An important goal of gene therapy for cardiovascular diseases and cancer is the development of effective vectors for catheter-based gene delivery. Although adenoviral vectors have proven effective for this purpose in animal models, the ability to achieve comparable gene transfer with nonviral vectors would provide potentially desirable safety and toxicity features for clinical studies. In this report, we describe the use of a new cationic DNA-liposome complex using an improved expression vector and lipid, N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine (GAP-DL-RIE/DOPE) to optimize catheter-mediated gene transfer in porcine arteries. The efficiency of this vector was compared to DNA alone, DNA with a previously described cationic liposome complex, (+/-)-N-(2-hydroxyethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE/DOPE), and a replication-defective adenoviral vector in a porcine artery gene transfer model. When used in optimal ratios, GAP-DL-RIE/DOPE liposomes provided a 15-fold higher level of gene expression in arteries compared to DNA alone or DMRIE/DOPE. Gene expression was observed in intimal and medial cells. However, when compared to adenoviral vectors (10(10) pfu/ml), gene expression following GAP-DLRIE/DOPE transfection was approximately 20-fold lower. Following intravenous injection of GAP-DLRIE/DOPE in mice, biochemical, hematological, and histopathological abnormalities were not observed. Significant improvements in the efficacy of arterial gene expression can be achieved by optimization of transfection condition with DNA-liposome complexes in vivo that may prove useful for arterial gene delivery in cardiovascular diseases and cancer.

Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy.

Among the potential nonviral vectors for human gene therapy are DNA-liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA-liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA-liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.

Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.

Improved cationic lipid formulations for in vivo gene therapy.

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

Structural requirements for cationic lipid mediated phosphorothioate oligonucleotides delivery to cells in culture.

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Increased levels of surgical adhesions in TGFbeta1 heterozygous mice.

Adhesion formation and fibrosis represent a major complication of surgical intervention. Reducing the morbidity associated with adhesions requires an understanding of the mechanisms underlying their formation. Since increased levels of transforming growth factor-beta1 (TGFbeta1) have been associated with inflammation and adhesion production, we investigated the requirement of TGFbeta1 in peritoneal adhesion formation utilizing mice carrying a targeted disruption of the TGFbeta1 allele. Mice that were either wild-type (+/+), containing two normal alleles of TGFbeta1, or heterozygous (+/-) for the TGFbeta1 null allele received injections of magnesium silicate (talc), and the extent of abdominal adhesions was determined utilizing a standard grading score. Wild-type (+/+) animals had at least twofold more TGFbeta1 protein in peritoneal fluids at 2 h posttrauma compared to heterozygous (+/-) mice (727 vs. 243 pg TGFbeta1/mg protein by enzyme-linked immunosorbent assay (ELISA) in +/+ and +/- mice, respectively), and had significantly less scar and adhesion formation (p < .05) at 7 days posttrauma (1.8 +/- 0.8 vs. 3.4 +/- 1.4, graded from 0 to 5, in +/+ and +/- mice, respectively). These results demonstrate that haploid insufficiency in TGFbeta1 levels can lead to inappropriate matrix and adhesion production during inflammation, and together with previous studies suggest that any perturbation of normal TGFbeta1 levels can modulate the injury response that regulates the extent of adhesion formation.

Monoterpene cyclases. Stereoelectronic requirements for substrate binding and ionization.

Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.

Monoterpene cyclases: physicochemical features required for pyrophosphate binding determined from inhibition by structural analogs.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate:(+)-alpha-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage (Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.

Terpene cyclase catalysis in organic solvent/minimal water media: demonstration and optimization of (+)-alpha-pinene cyclase activity.

A partially purified and lyophilized preparation of (+)-alpha-pinene cyclase from sage (Salvia officinalis) was shown to convert geranyl pyrophosphate to the monoterpene olefins alpha-pinene, camphene, limonene, and myrcene, in hexane with the addition of 0.1 to 10% water. This constitutes the first report of catalysis by a terpene cyclase in "non-aqueous" media. The relative proportions of olefinic products, metal ion requirement, pH optimum, temperature response, and time course of the enzymatic conversion were determined. The cyclase was shown to be stabilized with respect to temperature and time by the use of hydrocarbon solvent, and, in all other characteristics, to exhibit properties closely resembling those observed in aqueous media.

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction.

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.

Direct demonstration of the isomerization component of the monoterpene cyclase reaction using a cyclopropylcarbinyl pyrophosphate substrate analog.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene cyclases has precluded direct observation of the topologically required isomerization step leading from geranyl pyrophosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl pyrophosphate, which ultimately cyclizes to the various monoterpene skeletons. By using a partially purified monoterpene cyclase preparation and 2,3-cyclopropylgeranyl pyrophosphate, a substrate analog designed to uncouple the reaction sequence, the production of the corresponding tertiary homoallylic pyrophosphate isomer was demonstrated. This provides direct evidence for the usually cryptic isomerase component of the overall catalytic cycle. A number of other related products generated by reaction of cyclase with the analog were also identified, the structures and proportions of which were consistent with the intermediacy in catalysis of a cyclopropylcarbinyl cation X pyrophosphate anion pair. Kinetic parameters for the analog were compared with those of the natural substrate geranyl pyrophosphate. The results presented confirm mechanistic similarities in the enzymatic ionization and subsequent transformation of allylic pyrophosphate and cyclopropylcarbinyl pyrophosphate intermediates of isoprenoid metabolism.

Coherent oscillations in membrane potential synchronize impulse bursts in central olfactory neurons of the crayfish.

Lateral protocerebral interneurons (LPIs) in the central olfactory pathway of the freshwater crayfish Procambarus clarkii reside within the lateral protocerebrum and receive direct input from projection neurons of the olfactory midbrain. The LPIs exhibit periodic (0.5 Hz) changes in membrane potential that are imposed on them synaptically. Acute surgical experiments indicate that the synaptic activity originates from a group of oscillatory neurons lying within the lateral protocerebrum. Simultaneous intracellular recordings from many LPI pairs indicate that this periodic synaptic input is synchronous and coherent among the population of approximately 200 LPIs on each side of the brain. In many LPIs, specific odors applied to antennules in isolated head preparations generate long-lasting excitatory postsynaptic potentials and impulse bursts. The impulse bursts are generated only near the peaks of the ongoing depolarizations, approximately 1 s after stimulus application, and so the periodic baseline activity is instrumental in timing burst generation. Simultaneous recordings from pairs of LPIs show that, when impulse bursts occur in both cells after an odorant stimulus, they are synchronized by the common periodic depolarizations. We conclude that the common, periodic activity in LPIs can synchronize impulse bursts in subsets of these neurons, possibly generating powerful long-lasting postsynaptic effects in downstream target neurons.

An immunological role for the CD8 beta-chain.

Mature T cells can be functionally divided into two categories distinguished by surface expression of either CD4 or CD8, which in turn corresponds to restriction by and binding to class II or class I major histocompatibility complex proteins, respectively. CD8 can be expressed as a homodimer of the alpha-chain, or as a heterodimer of alpha- and beta-chains on human and mouse T cells, although most peripheral T cells seem to express CD8 alpha beta heterodimers exclusively (reviewed in ref. 9). Functional characterization of CD8 has focused primarily on the effect of the alpha-chain, which enhances or reconstitutes T-cell responses in homodimeric form and may play a specific role in thymic selection. In contrast, no role has been ascribed to CD8 beta or alpha beta heterodimers specifically. Here we show that CD8 alpha beta transfectants produce more interleukin-2 than CD8 alpha transfectants in response to specific stimuli. Increased interleukin-2 is also observed in cells expressing hybrid CD8 beta-alpha molecules (extracellular CD8 beta plus CD8 alpha transmembrane and cytoplasmic regions) on their surface. These results indicate that external portions of CD8 beta could be critical and that they may act independently of CD8 alpha in mediating their augmentation effect.

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.