Equivalent Reaction Conditions Research Articles

Precursor prioritization for p-cymene production through synergistic integration of biology and chemistry

The strategy of synergistic application of biological and chemical catalysis is an important approach for efficiently converting renewable biomass into chemicals and fuels. In particular, the method of determining the appropriate intermediate between the two catalytic methods is critical. In this work, we demonstrate p-cymene production through the integration of biosynthesis and heterogenous catalysis and show how a preferred biologically derived precursor could be determined. On the biological side, we performed the limonene and 1,8-cineole production through the mevalonate pathway. Titers of 0.605 g/L and a 1.052 g/L were achieved, respectively. This difference is in agreement with the toxicity of these compounds toward the producing microorganisms, which has implications for subsequent development of the microbial platform. On the heterogeneous catalysis side, we performed the reaction with both biological precursors to allow for direct comparison. Using hydrogenation/dehydrogenation metals on supports with acid sites, both limonene and 1,8-cineole were converted to p-cymene with similar yields under equivalent reaction conditions. Thus, we could determine that the most promising strategy would be to target 1,8-cineole, the higher titer and lower toxicity bio-derived precursor with subsequent catalytic conversion to p-cymene. We further optimized the biological production of 1,8-cineole via fed-batch fermentation and reached the titer of 4.37 g/L which is the highest known 1,8-cineole titer from microbial production. This work provides a valuable paradigm for early stage considerations to determine the best route for the high-efficiency production of a target biobased molecule using an integration of biology and chemistry.

Physical mixing of a catalyst and a hydrophobic polymer promotes CO hydrogenation through dehydration.

In many reactions restricted by water, selective removal of water from the reaction system is critical and usually requires a membrane reactor. We found that a simple physical mixture of hydrophobic poly(divinylbenzene) with cobalt-manganese carbide could modulate a local environment of catalysts for rapidly shipping water product in syngas conversion. We were able to shift the water-sorption equilibrium on the catalyst surface, leading to a greater proportion of free surface that in turn raised the rate of syngas conversion by nearly a factor of 2. The carbon monoxide conversion reached 63.5%, and 71.4% of the hydrocarbon products were light olefins at 250°C, outperforming poly(divinylbenzene)-free catalyst under equivalent reaction conditions. The physically mixed CoMn carbide/poly(divinylbenzene) catalyst was durable in the continuous test for 120 hours.

Insight into Temperature Dependency and Design of Experiments towards Process Development for Cysteine‐Based Antibody‐Drug Conjugates

AbstractIn the synthetic organic community, antibody‐drug conjugates (ADCs) have become a popular topic of interest. The field is expanding beyond cytotoxic drug synthesis, and into other bioconjugation applications to have a greater impact on drug discovery and development. Bioconjugation chemistry is a complex domain for synthetic chemists, and there are limited published reports that can serve as guidance to overcome technical gaps between small molecule synthesis and large molecule bioconjugation to make ADCs. Here, we report a thorough evaluation of the temperature dependency for the tris‐(2‐carboxyethyl)‐phosphine (TCEP) reduction of antibody interchain disulfide bonds, which is a crucial step for creating cysteine‐conjugated ADCs. This investigation was conducted using three different antibodies with different isoelectric points, and under equivalent reaction conditions. Additionally, an initial stability assessment of the resulting ADCs was performed and revealed appropriate material storage conditions. The experimental data described herein can be a useful guide for synthetic organic chemists designing future ADC preparation processes.

Mechanistic origin of the diverging selectivity patterns in catalyzed ethane and ethene oxychlorination

In the context of vinyl chloride monomer (VCM) production, an oxychlorination catalyst that allows direct VCM formation from gas-derived ethane instead of expensive oil-derived ethene is intensively sought after. A wide range of stable ethane oxychlorination catalysts for this purpose have recently been reported, yet they mainly yield ethene, while VCM remains a minor by-product. Strikingly, the same catalysts are active in ethene oxychlorination, resulting in selective VCM formation under equivalent reaction conditions. This work reveals the origin of these diverging selectivity patterns by combining quantitative catalytic tests, temporal analysis of products (TAP), and density functional theory (DFT) on iron phosphate. Ethane oxychlorination is found to proceed sequentially through ethyl chloride (EtCl) dehydrochlorination to ethene, while ethene oxychlorination directly yields VCM without formation of the intermediate dichloroethane (EDC) on iron phosphate. Furthermore, by co-feeding ethane in ethene oxychlorination, we demonstrate that the alkane suppresses the formation of VCM in ethene oxychlorination. The reason for this VCM inhibition is found in the hydrocarbon competition for a combination of the active, free and chlorinated iron centers. As ethane activation exhibits half of the barrier of ethene activation, the presence of ethane leads to active site depletion, hindering VCM formation. These observations are extended by ethane co-feeding tests in ethene oxychlorination over a wide range of known oxychlorination catalysts (EuOCl, LaOCl, CeO2, and CuCl2-KCl-LaCl3/γ-Al2O3), and corresponding DFT calculations, indicating that the described phenomenon is material independent. The gathered molecular-level understanding explains the major hurdle of using ethane as feedstock for vinyl chloride production.

Microwave-assisted rapid synthesis of poly(butylene succinate): principal effect of microwave irradiation of accelerating the polycondensation reaction

To determine the origin of the reaction rate acceleration in the synthesis of poly(butylene succinate) (PBS) upon microwave irradiation, we herein investigated the effect of the microwave-assisted activation of the reactants, viz. the monomers 1,4-butanediol (BD) and succinic acid (SA), and of the water produced as a by-product during the reaction. The rate of removal of the water by-product from the reaction solution was precisely measured under equivalent reaction conditions (i.e., time and temperature) using both microwave and conventional heating methods. Microwave heating selectively activated and removed the water from the reaction solution more rapidly and to a greater extent than oil bath heating. Measurement of the temperature-dependent dielectric properties of the reactants revealed that the dielectric loss factor (eʹʹ) of BD increased with increasing temperature, peaking at 80 °C. In contrast, the eʹʹ of the mixture of BD and SA was significantly lower than that of BD alone from 70–140 °C. These results indicate that the principal effect of microwave irradiation in the synthesis of PBS was the accelerated removal of the water by-product. The origin of the reaction rate acceleration in the microwave-assisted synthesis of poly(butylene succinate) (PBS) was investigated by measuring the rate of removal of the water by-product from the reaction solution under equivalent reaction conditions using both microwave and conventional heating methods. Microwave heating selectively activated and removed the water from the reaction solution more rapidly and to a greater extent than oil bath heating. These results indicate that the principal effect of microwave irradiation in the synthesis of PBS was the accelerated removal of the water by-product.

From Rate Measurements to Mechanistic Data for Condensed Matter Reactions: A Case Study Using the Crystallization of [Zn(OH2)6][ZnCl4

The kinetics of crystallization of the R = 3 hydrate of zinc chloride, [Zn(OH2)6][ZnCl4], is measured by time-resolved synchrotron x-ray diffraction, time-resolved neutron diffraction, and by differential scanning calorimetry. It is shown that analysis of the rate data using the classic Kolmogorov, Johnson, Mehl, Avrami (KJMA) kinetic model affords radically different rate constants for equivalent reaction conditions. Reintroducing the amount of sample measured by each method into the kinetic model, using our recently developed modified-KJMA model (M-KJMA), it is shown that each of these diverse rate measurement techniques can give the intrinsic, material specific rate constant, the velocity of the phase boundary, vpb. These data are then compared to the velocity of the crystallization front directly measured optically. The time-resolved diffraction methods uniquely monitor the loss of the liquid reactant and formation of the crystalline product demonstrating that the crystallization of this hydrate phase proceeds through no intermediate phases. The temperature dependent vpb data are then well fit to transition zone theory to extract activation parameters. These demonstrate that the rate-limiting component to this crystallization reaction is the ordering of the waters (or protons) of hydration into restricted positions of the crystalline lattice resulting in large negative entropy of activation.

The effect of thermal pre-treatments on the nucleation of uranium hydride

Uranium hydriding experiments are reported in which samples experienced a single thermal cycle within the range of 35°C to 75°C, before undergoing reaction with hydrogen at a pressure of 110mbar. Under equivalent reaction conditions, elevated nucleation kinetics were observed for samples that had been thermally cycled prior to hydrogen exposure, as compared to samples that had not been cycled. These observations have been rationalised in terms of two alternative hypotheses based on i) the anisotropic thermal expansion of uranium leading to disruption of the surface uranium oxide overlayer during cooling of the experimental samples or ii) differing levels of water present on the oxide overlayer following pre-treatment.

Well-defined coinage metal transfer agents for the synthesis of NHC-based nickel, rhodium and palladium macrocycles.

With a view to use as carbene transfer agents, well-defined silver(i) and copper(i) complexes of a macrocyclic NHC-based pincer ligand, bearing a central lutidine donor and a dodecamethylene spacer [CNC-(CH2)12, 1], have been prepared. Although the silver adduct is characterised by X-ray diffraction as a dinuclear species anti-[Ag(μ-1)]2(2+), variable temperature measurements indicate dynamic structural interchange in solution involving fragmentation into mononuclear [Ag(1)](+) on the NMR time scale. In contrast, a mononuclear structure is evident in both solution and the solid-state for the analogous copper adduct partnered with the weakly coordinating [BAr(F)4](-) counter anion. A related copper derivative, bearing instead the more coordinating cuprous bromide dianion [Cu2Br4](2-), is notable for the adoption of an interesting tetranuclear assembly in the solid-state, featuring two cuprophilic interactions and two bridging NHC donors, but is not retained on dissolution. Coinage metal precursors [M(1)]n[BAr(F)4]n (M = Ag, n = 2; M = Cu, n = 1) both act as carbene transfer agents to afford palladium, rhodium and nickel complexes of 1 and the effectiveness of these precursors has been evaluated under equivalent reaction conditions.

Analogies and differences between calcium-based and europium-based graphite intercalation compounds

An original method using lithium-based liquid alloys has been developed, allowing studies on bulk graphite intercalation compounds with calcium and europium. We showed that binary and ternary compounds belonging to graphite-lithium-calcium and graphite-lithium-europium systems are synthesized in both cases for equivalent reaction conditions but amazingly with many different structural and physical properties. Concerning CaC6 and EuC6, even if their 2D unit cells are hexal, their c-axis stacking sequences lead to different symmetries. Regarding kinetical data, formation mechanisms of these graphite intercalation compounds appear comparable however different, with a first common step before differenciation in the intercalation mechanism. Obviously, their physical properties are strongly different due to the nature of the intercalated metallic element. So, the different ternary compounds from these systems also show very considerable differences concerning their electronic properties: complex magnetic ordering for Eu-based ternary GIC and superconducting behaviour for Ca-based ternary GIC. However, common points are highlighted.

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

Controlled selectivity for palladium catalysts using self-assembled monolayers

The selective reaction of one part of a bifunctional molecule is a fundamental challenge in heterogeneous catalysis and for many processes including the conversion of biomass-derived intermediates. Selective hydrogenation of unsaturated epoxides to saturated epoxides is particularly difficult given the reactivity of the strained epoxide ring, and traditional platinum group catalysts show low selectivities. We describe the preparation of highly selective Pd catalysts involving the deposition of n-alkanethiol self-assembled monolayer (SAM) coatings. These coatings improve the selectivity of 1-epoxybutane formation from 1-epoxy-3-butene on palladium catalysts from 11 to 94% at equivalent reaction conditions and conversions. Although sulphur species are generally considered to be indiscriminate catalyst poisons, the reaction rate to the desired product on a catalyst coated with a thiol was 40% of the rate on an uncoated catalyst. Interestingly the activity decreased for less-ordered SAMs with shorter chains. The behaviour of SAM-coated catalysts was compared with catalysts where surface sites were modified by carbon monoxide, hydrocarbons or sulphur atoms. The results suggest that the SAMs restrict sulphur coverage to enhance selectivity without significantly poisoning the activity of the desired reaction.

Surface nano-structuring of reverse osmosis membranes via atmospheric pressure plasma-induced graft polymerization for reduction of mineral scaling propensity

Surface nano-structuring of polyamide desalination membrane with a hydrophilic poly(methacrylic acid) was shown to reduce the membrane mineral scaling propensity as demonstrated with calcium sulfate dihydrate (gypsum). A two-step approach was employed, whereby the active polyamide (PA) layer of a thin-film composite (TFC) synthesized membrane was activated with impinging atmospheric plasma, followed by a solution free-radical graft polymerization (FRGP) of a water soluble methacrylic acid (MAA) monomer, at 60 °C and initial monomer concentration of 5–20% (v/v), onto the surface of the PA-TFC membrane. The approach of creating a layer of end-grafted poly(methacrylic acid) (PMAA) surface chains was first developed and evaluated using a surrogate polyamide membrane layer interfacially polymerized onto a thin poly(ethyleneimine) (PEI) film coated onto a silicon wafer. The resulting PMAA–PA-PEI–Si surrogate membrane surface was hydrophilic with a water contact angle range of 10–17°. Structuring of the PA-TFC membrane at equivalent FRGP reaction conditions resulted in membranes of higher permeability (by a factor of 1.3–2.26) relative to a commercial RO membrane of a similar surface roughness (∼70 nm) and salt rejection. Flux tests of membrane mineral scaling demonstrated that membrane mineral scaling propensity can be measurably reduced, relative to commercial membrane of the same salt rejection, while yielding equivalent or higher water permeability. The onset time for gypsum scaling for the optimal membrane surface (prepared at 10% (v/v) initial MAA concentration) was retarded by a factor of 2–5 relative to the commercial RO membrane. Current work is ongoing to further optimize the surface structure in order to increasing scaling resistance and assess the impact of surface structuring on nucleation of mineral salt crystals.

Methylene-functionalized saponite: A new type of organoclay with CH 2 groups substituting for bridging oxygen centers in the tetrahedral sheet

The synthesis of saponite in the presence of bis(triethoxysilyl)methane (BTESM) as an organosilicon reagent results in the replacement of up to 33.3% of the oxygen atoms in the tetrahedral sheet by bridging methylene groups. The methylene-functionalized saponites represent a new form of covalently bonded organoclay that truly is isomorphic with purely inorganic saponite made under equivalent reaction conditions from sodium silicate as the silicon source. The isoelectronic and isolobal relationship between methylene and bridging oxygen centers is essential for methylene saponite formation. Bridging ethylene groups are not incorporated into the Kagome net of the basal surfaces due to a mismatch in bridging group size. The textural properties of the methylene saponites are similar to those for purely inorganic magnesium saponite made under equivalent synthetic conditions in the absence of BTESM. Layer stacking disorders afford large surface areas (∼ 550 to 650 m 2/g), making the methylene saponites attractive candidates for use as adsorbents and functional fillers for polymer composites.

Preparation and characterization of Ce-Zr and Ce-Mn based oxides for n-hexane combustion: Application to catalytic membrane reactors

Abstract Ce-Mn and Ce-Zr catalytic samples with different Ce/metal molar ratio have been prepared by coprecipitation followed by calcination at moderate temperatures (350–450 °C) and characterized by XRD, XPS, BET, TPR and SEM–EDX techniques. The preparation procedure of analogous catalytic membranes over γ-Al2O3 ceramic supports has been also studied in order to control loading, distribution and composition of the catalytic material inside the membrane thickness. To evaluate their catalytic performance, the combustion of n-hexane from air-diluted streams has been carried out in both a conventional fixed bed reactor and a flow-through catalytic membrane reactor operating in Knudsen diffusion regime. Surface area alone cannot account for the reaction performance achieved; however, the redox properties and oxygen mobility of the mixed oxides have been revealed as key parameters controlling the catalytic activity. Although Ce-Mn powdered catalysts appear more active in n-hexane combustion than their Ce-Zr counterparts; a strong deactivation phenomenon, more severe for Mn rich samples, is observed with time on stream in contrast with Ce-Mn based membranes showing catalytic stability under equivalent reaction conditions.

Experimental Evaluation of a Three-Phase Downflow Capillary Reactor

One reason for the few commercial successes of intensified catalytic multiphase reactors might be that many of these developments inherently incorporate a fixed catalyst, which may not suit an industry that is based on principles of batch manufacture and multiproduct plants. This study evaluates an intensified reactor that encompasses the opportunities demonstrated from structured flows and thin channels, together with a mobile, slurry catalyst, namely, a multicapillary reactor with gas/liquid/suspended catalyst flow. Using the single capillary arrangement, the reaction of resorcinol hydrogenation was shown to be first order in hydrogen and to be operating under kinetic control. The increase of the gas-to-liquid flow ratio showed a positive effect on the observed reaction rate. Comparison of the rate of resorcinol hydrogenation in the capillary reactor with an autoclave under equivalent reaction conditions demonstrated that the rate in the single capillary reactor (SCR) was enhanced by a factor of 7.

Differences between human and rabbit coagulation factor X—implications for in vivo models of thrombosis

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX–glutathione S-transferase (RafX–GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA·HCl], S2765 [ Z- d-Arg-Gly-Arg-pNA·HCl] and Spectrozyme Xa [MeO-CO- d-CHG-Gly-Arg-pNA·AcOH]). Michaelis constants ( K m) for the rabbit protein were 187, 72 and 69 μM, respectively, and for the human analog, 255, 63 and 135 μM, respectively. Comparing the extent of substrate turnover ( V max) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.

Synthesis and characterization of four new europium group XIV chalcogenides: K(2)EuTSe(5) and KEuTS(4) (T = Si, Ge).

Four europium group XIV chalcogenides have been synthesized using the reactive flux method: K(2)EuTSe(5) (I, II) and KEuTS(4) (III, IV) where T = Si, Ge. K(2)EuSiSe(5), I, crystallizes in the monoclinic space group P2(1)/c with cell parameters a = 11.669(3) A, b = 9.844(2) A, c = 8.917(2) A, beta = 91.583(5) degrees, and Z = 4. K(2)EuGeSe(5), II, crystallizes in the monoclinic space group P2(1)/c with cell parameters a = 11.8056(3) A, b = 9.9630(1) A, c = 8.9456(1) A, beta = 91.195(1) degrees, and Z = 4. Both K(2)EuSiSe(5) and K(2)EuGeSe(5) are semiconductors with optical band-gaps of approximately 2.00 and 1.84 eV, respectively. Raman spectroscopy shows vibrations from the (TSe(5))(4-) (T = Si, Ge) unit. KEuSiS(4), III, crystallizes in the monoclinic space group P2(1) with cell parameters a = 6.426(4) A, b = 6.582(5) A, c = 8.566(7) A, beta = 107.83(6) degrees, and Z = 2. KEuGeS(4), IV, crystallizes in the monoclinic space group P2(1) with cell parameters a = 6.510(2) A, b = 6.649(2) A, c = 8.603(3) A, beta = 107.80(2) degrees, and Z = 2. Band-gap analysis shows that both compounds are semiconductors with optical band-gaps of 1.72 and 1.71 eV, respectively. The Raman spectrum of KEuGeS(4) shows the vibrations of the (GeS(4))(4-) unit. Fluorescence spectroscopy confirms the presence of Eu(III) in III and IV instead of Eu(II) as in I and II. These four crystalline products were formed under equivalent stoichiometric reaction conditions. The fact that two different products are observed can be used to understand the relationship between the oxidative and reductive potentials within these flux reactions.

Novel Bifunctional Catalysts of Cobalt Phthalocyanine Bonded to Organic-Functionalized Basic Calcined Mg–Al Hydrotalcite for Autoxidation of Mercaptans

Two novel bifunctional catalysts, cobalt(II) 2,9,16,23-tetraaminophthalocyanine CoPc(NH2)4 bound to basic calcined Mg–Al hydrotalcite (Mg/Al=3:1) modified with organic functional groups by complexation and by covalent linkage, respectively, have been found to exhibit higher catalytic activity and stability in 1-octanethiol autoxidation than the corresponding supported CoPc(NH2)4 catalyst under equivalent reaction conditions.

Development of a catalytic combustor for a heavy-duty utility gas turbine

Catalytic combustion is an attractive technology for gas turbine applications where ultra-low emission levels are required. Recent tests of a catalytic reactor in a full scale combustor have demonstrated emissions of 3.3 ppm NO x , 2.0 ppm CO, and 0.0 ppm UHC. The catalyst system is designed to only convert about half of the natural gas fuel within the catalyst itself, thus limiting the catalyst temperature to a level that is viable for long-term use. The remainder of the combustion occurs downstream from the catalyst to generate the required inlet temperature to the turbine. Catalyst development is typically done using subscale prototypes in a reactor system designed to simulate the conditions of the full scale application. The validity of such an approach is best determined experimentally by comparing catalyst performance at the two size scales under equivalent reaction conditions. Such a comparison has recently been achieved for catalysts differing in volume by two orders of magnitude. The performance of the full scale catalyst was similar to that of the subscale unit in both emission levels and internal temperatures. This comparison lends credibility to the use of subscale reactors in developing catalytic combustors for gas turbines.

Substrate Selectivities and Lipid Modulation of Plant Phospholipase Dα, -β, and -γ

Three classes of phospholipase D (PLD), designated PLDα, -β, and -γ, have been cloned from plants, but their substrate selectivities have not been established. Using active PLDs expressed from their cDNAs inEscherichia coli,we compared the hydrolytic activities of these three PLDs toward various phospholipids and the influence of substrate composition on their substrate selectivities. When single-class phospholipid vesicles of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2),N-acylphosphatidylethanolamine (NAPE), and cardiolipin (CL) were examined, PLDα hydrolyzed PC, PE, and PG but PLDβ and -γ showed no activity toward any of these lipids. When PIP2was included in mixed vesicles with the phospholipids above, PLDα showed the same PC-, PE-, and PG-hydrolyzing ability, whereas PLDβ and -γ were able to hydrolyze both PE and PS. When both PE and PIP2were included in substrate vesicles, PLDβ and PLDγ hydrolyzed PC, PG, and NAPE, showing that both PE and PIP2are required for PC, PG, and NAPE hydrolysis by PLDβ and -γ. The PE activation of PLDβ and -γ required lipid vesicles made of mostly PE, suggesting that PE may affect the substrate presentation rather than serve as a cofactor of these PLDs. Under equivalent reaction conditions, PLDβ displayed a similar preference for PC and NAPE, whereas PLDγ preferred NAPE to PC by nearly three times. None of the three PLDs used PI, CL, or PIP2as substrates. These results have identified PS- and NAPE-hydrolyzing PLDs and have indicated an important role for lipid composition in regulating the substrate selectivity of PLDβ and -γ.