Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc₄Xyl₃Gal₂ (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K_m for individual acceptors increased in the order [1-³H]XLLG < XLLG-SR < [1-³H]XLLGol < XLLG-FITC < XLLG-ANTS < XLLG-AA, while the turnover numbers characterized by k_cat decreased in the order XLLG-FITC > XLLG-SR > XLLG-ANTS > [1-³H]XLLGol > [1-³H]XLLG > XLLG-AA. Catalytic efficiency (expressed as k_cat/K_m) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-³H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O177

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the ¹H and ¹³C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Radioactive contamination of the marine environment: Uptake and distribution of3H inDunaliella bioculata

The marine flagellateDunaliella bioculata, which is easily cultivated under laboratory conditions, is a suitable organism for assessing the importance of the radioactive contamination by³H bound to organic molecules. We have studied the uptake of the following tritiated precursors: thymidine-methyl-³H, adenine-2-³H, uridine-5-³H, l-leucine-4-³H, glycine-2-³H, l-arginine-3.4-³H, 1-aspartic acid-2. 3-³H, 1-phenylalanine-2.3-³H, D-glucose-2-³H and D-glucose-6-³H. Under the experimental conditions (2000 lux; incubation time 30 min), all tritiated molecules are taken up byD. bioculata. Their intracellular concentration may reach that of the external medium. However, leucine and adenine accumulate in the algae: their respective concentrations are 10 and 100 times higher than in the culture medium. The molecular distribution of³H has been studied by various biochemical techniques and by sieve chromatography on sepharose 4B. It has been found that more l-leucine-4-³H is incorporated into acid and acetone soluble substances than into proteins. Adenine-2-³H is mainly incorporated into macromolecules of biological significance (RNA, DNA). CsCl gradient centrifugation has shown that the total DNA ofDunaliella is constituted by a major (ϖ=1.707 g/cm³) and by a minor (ϖ=1.693 g/cm³) component. Fellow of the Commission of the European Communities     

Binding of all-trans-retinoic acid to MLTC-1 proteins

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 mean ± SD) fmoles/8 × 10⁴ cells at 37 °C. About 90% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.9 μM and 0.65 μM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 μM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 μM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [³H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [³H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells. (Mol Cell Biochem 276: 55–60, 2005)This paper is dedicated to the memory of Prof. E. Quagliariello.     

Heterogeneous set of cell wall teichoic acids in strains of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> VKM B-760 and VKM B-764

Cell walls of Bacillus subtilis VKM B-760 and VKM B-764 are characterized by heterogeneous composition of teichoic acids. Polymer I with structure -6)-β-D-Galp-(1→1)-sn-Gro-(3-P-, polymer II with structure -6)-α-D-Glcp-(1→1)-sn-Gro-(3-P-, and a small amount of unsubstituted 1,3-poly(glycerol phosphate) were detected in strain VKM B-760. Strain VKM B-764 contains an analogous set of teichoic acids, but a characteristic feature of polymer II is the presence of disubstituted glycerol residue with α-glucopyranose localization in the integral chain at C-1 hydroxyl and β-glucopyranose as a side branch at C-2 hydroxyl (polymer III): -6)-α-D-Glcp-(1→1)-[β-D-Glcp-(1→2)]-sn-Gro-(3-P-. The structures of polymer I in bacilli and polymer III in Gram-positive bacteria are described for the first time. Teichoic acids were studied by chemical methods and on the basis of combined analysis of one-dimensional ¹H-, ¹³C-, and ³¹P-NMR spectra, homonuclear two-dimensional ¹H/¹H COSY, TOCSY, and ROESY, and heteronuclear two-dimensional ¹H/¹³C gHSQC- and HMQC-TOCSY experiments. Simultaneous presence of several different structure teichoic acids in the bacillus cell walls as well as chemotaxonomical perspectives of the application of these polymers as species-specific markers for members of the Bacillus genus is discussed.     

Deuterium incorporation experiments from (3R)- and (3S)-[3-2H]leucine into characteristic isoprenoidal lipid-core of halophilic archaea suggests the involvement of isovaleryl-CoA dehydrogenase

The stereochemical reaction course for the two C-3 hydrogens of leucine to produce a characteristic isoprenoidal lipid in halophilic archaea was observed using incubation experiments with whole cell Halobacterium salinarum. Deuterium-labeled (3R)- and (3S)-[3-²H]leucine were freshly prepared as substrates from 2,3-epoxy-4-methyl-1-pentanol. Incorporation of deuterium from (3S)-[3-²H]leucine and loss of deuterium from (3R)-[3-²H]leucine in the lipid-core of H. salinarum was observed. Taken together with the results of our previous report, involving the incubation of chiral-labeled [5-²H]leucine, these results strongly suggested an involvement of isovaleryl-CoA dehydrogenase in leucine conversion to isoprenoid lipid in halophilic archaea. The stereochemical course of the reaction (anti-elimination) might have been the same as that previously reported for mammalian enzyme reactions. Thus, these results suggested that branched amino acids were metabolized to mevalonate in archaea in a manner similar to other organisms.     

Transport, degradation and cell wall-integration of XXFGol, a growth-regulating nonasaccharide of xyloglucan, in pea stems

When [glucitol-³H]XXFGol (a NaB³H₄-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were conducted to discover whether such ³H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to be postulated. After 24 h, ³H from [glucitol-³H]XXFGol and [glucitol-³H]XXXGol showed U-shaped distributions, with most ³H at the base and apex of the stem. Radioactivity from [fucosyl-³H]XXFG and [xylosyl-³H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10⁻⁷ M [glucitol-³H]XXFGol was supplied, about 14 fmol ·  seedling^–1 of apparently intact [³H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including free [³H]glucitol) and some wall-bound ³H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation). We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are maintained at low steady-state concentrations by both hydrolysis and transglycosylation. Received: 1 April 1997 / Accepted: 28 May 1997     

Radiometric determination of cytidine 5′-triphosphate in biological materials

A simple and sensitive radioenzymic method is described for the direct determination of cytidine 5′-triphosphate (CTP) in the range of 1 to 9 nmol in perchloric acid extracts of tissue. The method involves a specific conversion of CTP and [1,2-¹⁴C]phosphorylethanolamine to labeled CDP-ethanolamine using purified CTP:phosphorylethanolamine cytidylyltransferase (EC 2.7.7.14). The [¹⁴C]CDP-ethanolamine formed is isolated on a Dowex 1-formate column taking advantage of the selectivity of borate complexing. A spectrophotometric method for determining CTP in standard solutions is also described.     

Synthesis and characterization of glucose-grafted biodegradable amphiphilic glycopolymers P(AGE-glucose)-b-PLA

Novel glucose-grafted biodegradable amphiphilic glycopolymers P(AGE-glucose)-b-PLA were synthesized through a facile and efficient way. First, the block copolymer intermediates PAGE-b-PLA bearing double bonds in the side chains were synthesized by ring-opening polymerization of LA using PAGE as macroinitiator and Sn(Oct)₂ as catalyst; Then, 2-mercaptoethyl-β-glucoside (MEGlu) was conjugated to the side chains of PAGE-b-PLA via free-radical coupling reaction to give the glycopolymer P(AGE-glucose)-b-PLA. The micellization behavior of the glycopolymers P(AGE-glucose)-b-PLA in aqueous media was investigated by fluorescence (FL), ¹H nuclear magnetic resonance spectroscopy (¹H NMR), dynamic light scattering (DLS), and transmission electron microscope (TEM). The results showed that these glycopolymers P(AGE-glucose)-b-PLA formed spherical micelles with diameters about 200 nm.     

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg²⁺ and Mn²⁺. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.     

The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

When pig liver microsomal preparations were incubated with GDP-[¹⁴C]mannose, 10–40% of the ¹⁴C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [¹⁴C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [¹⁴C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [¹⁴C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [³H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[¹⁴C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.     

Xyloglucan−pectin linkages are formed intra-protoplasmically,contribute to wall-assembly,and remain stable in the cell wall

We tested two hypotheses for the mechanism by which xyloglucan–pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using α-(1→5)-l-[³H]arabino- or β-(1→4)-d-[³H]galacto-oligosaccharides as model acceptor substrates. The ³H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [³H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [³H]Xyloglucan–pectin complexes were detectable <4 min after [³H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan–pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [³H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [³H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [³H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan–pectin bonds are stable and the pectic moiety aids wall-assembly.     

Kaempferol triosides from Silphium perfoliatum

Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, ¹H, ¹³C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1′→6)-O-β- -galactopyranoside and kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1→ 6)-O-β- (2-O-E-caffeoylgalactopyranoside).     

Triterpene saponins from Salsola imbricata

Continuing our investigations on medicinal plants of the Egyptian desert, two new triterpene glycoside derivatives, along with three known compounds have been isolated from the roots of Salsola imbricata, a shrub widely growing in Egypt. Their structures have been established as 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-akebonic acid 28-O-β-d-glucopyranoside and 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-29-hydroxyoleanolic acid 28-O-β-d-glucopyranoside on the basis of spectroscopic methods including 1D- (¹H, ¹³C) and 2D-NMR (DQF-COSY, HSQC, HMBC) experiments as well as mass spectrometry analysis.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

Synthesis of mixed N,N′-(Ar,Ar′-diaryl)iminoisoindolines for applications in palladacycle formations

The synthesis of N,N′-(Ar,Ar′-diaryl)iminoisoindolines containing different aryl groups bound to the two nitrogen atoms is described. The iminoisoindolines were obtained by a three component, one-pot reaction of phthalaldehyde with 1 equivalent p-NO₂-aniline and 1 equivalent p-R-aniline, where R = H, Me, MeO or ⁱPr, resulting in formation of non-symmetrically substituted (mixed) iminoisoindolines, 1-p-nitrophenylimino-2-p-R-phenylisoindoline (R = H (1), Me (2), MeO (3), and ⁱPr (4)), as analytically pure precipitates requiring no further purification. Only one isomer precipitates from solution wherein the nitro group resides exclusively at the imine position while the more electron donating substituent ends up on the isoindoline ring position. Further reaction with Pd(OAc)₂ in dichloromethane at room temperature results in formation of six-membered [C,N] dinuclear cyclopalladated complexes with the general formula [(Ar,Ar′-diaryliminoisoindoline)Pd{μ-OAc}]₂.