Monofunctional Reagents Research Articles

Synthesis and blend behavior of high performance homo- and segmented thermoplastic polyimides

New synthetic methodology was developed as part of an effort to increase the processibility of high Tg polyimide homo− and copolymers, suitable as matrix resins and structural adhesives. Molecular weight and end group control together with solution imidization techniques were successfully employed to convert a variety of poly(amic acid) intermediates to fully cyclized polyimides. The solution imidization was conducted in N-methylpyrrolidone (NMP) with o-dichlorobenzene used as the azeotroping agent at 165–190°C. This technique has produced products which are more soluble than polyimides prepared previously by bulk thermal cyclization of poly(amic acids) at temperature of 300°C. They are also more stable than “chemically” imidized materials. In addition, incorporation of the monofunctional reagent phthalic anhydride provides nonreactive phthalimide end groups and controlled molecular weight. This latter feature significantly further improved the melt and solution processibility of the resulting polyimides. In this study thermoplastic, fully cyclized polyimides of 10 000, 20 000, and 30 000 Mn were prepared which displayed glass transition temperatures ranging from 260–353°C, with the highest Tg observed with phthalimide capped polyimide systems derived from 6F-dianhydride and p-phenylene diamine. Tough, transparent films were prepared from polymers of 20 000 and 30 000 g/mol by casting from NMP solution or by compression molding at 50–70°C above the glass transition temperature. For purposes of molecular weight assessment, t-butyl phthalic anhydride was used as the end blocker. This permitted 400 M-Hertz proton NMR to be used for assessing the concentration of end groups. Comparison of the 18 aliphatic protons at the end of the chain allowed Mn values to be determined, which agreed well with theory. A series of poly(arylene ether ketone)/aromatic polyimide blends were investigated to determine the influence of structural variation and composition on miscibility. As an extension to the PEEKTM/UltemTM blend system, which has been reported to be miscible over all proportions, this study examined how structural variations in both the poly(arylene ether ether ketone) and the polyimide portions affect miscibility. In particular, replacement of the hydroquinone fraction in PEEKTM with bisphenol A or sulfonyl diphenol produced an amorphous polymer which was no longer miscible with UltemTM. Polyimide structures modified by employing 3,3′,4,4′-benzophenonetetracarboxylic dianhydride (BTDA) and 4,4′-[1,4-phenylene-bis-(1-methyl ethylidene)] bisaniline (Bis P) diamine to obtain higher glass transition temperatures were also investigated. This system afforded homogeneous blends with PEEKTM when the (Bis P) diamine was utilized in the synthesis of the polyimide. Furthermore, up to 50 mole percent of hexafluoro-bis-dianhydride (6FDA) could be substituted for BTDA without loss of miscibility. However, when the more polar 3,3′-diaminodiphenylsulfone diamine was employed, immiscible blends resulted. An additional important variant has been to incorporate polyimide siloxane segmented copolymers into the PEEKTM blend system. The polyimide segment can be designed to be miscible whereas the siloxane portion is homogeneously dispersed into a second phase which, in fact, enriches the surface behavior quite dramatically in siloxane content. The latter could be of some importance in allowing for atomic oxygen resistance and possibly improved flame resistance behavior.

Photochemical and photobiological properties of 4,8-dimethyl-5′-acetylpsoralen

The photochemical and photobiological properties of 4,8-dimethyl-5′-acetylpsoralen (AcPso), proposed for the photochemotherapy of some skin diseases, were investigated. The photoreaction of AcPso with DNA is weaker in the presence of air than in a nitrogen atmosphere, in terms of total photobinding and DNA cross-linking; when UVA irradiation is performed in air, AcPso behaves as a monofunctional reagent. The quenching effect of oxygen is related to the high capacity of AcPso to produce singlet oxygen. Furthermore, it is demonstrated that AcPso photoadducts are better producers of singlet oxygen than free AcPso in solution. Using DNA sequencing methodology, two modes of DNA photosensitization by AcPso are shown, these lead to the formation of photoadducts mainly at T residues (and at C to a lesser extent) and to photo-oxidized G residues probably via singlet oxygen. Chemical or enzymatic cleavage were used as probes in these experiments. A rapid assay for the detection of the photodynamic effect of a photosensitizer on DNA, involving oxygen, is also described. Finally, the cytotoxicity and genotoxicity of AcPso on E. coli WP2 cells appear to be related to its ability to form photoadducts, in particular cross-links, rather than to its capacity to produce singlet oxygen.

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179-188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257-261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.

ChemInform Abstract: The Reactions of Phosphazenes with Difunctional and Polyfunctional Nucleophilic Reagents

Abstract After a brief introduction of the reaction patterns of N3P3Cl6 and N4P4Cl8 with monofunctional nucleophilic reagents, the reactions of these two chlorocyclophosphazenes with difunctional, trifunctional and tetrafunctional nucleophilic reagents are surveyed. These reagents contain aliphatic and aromatic hydroxyl and primary and secondary amino groups. Spiro, ansa, dangling and bridging units have been observed as well as a combination of these. The stability of the various ring systems is discussed. Crystal structures and 1H, 13C and 31P n.m.r. spectroscopic studies are reported.

Polysulfone Modified Thermoplastic Polyimide Segmented Copolymer Matrix Resin Systems

Several methods have been investigated which improve the processability of polyimide homo- or copolymer systems. In particular, the utilization of a solution imidiza tion technique in converting the poly(amic acid) intermediate to the fully cyclized polyimide has been successfully exploited. The solution imidization was conducted in N- methylpyrollidone (NMP) and an azeotroping agent, such as cyclohexylpyrollidone (CHP), at approximately 160°C for 24 hours. This technique has been shown to yield more soluble products than the corresponding bulk thermal imidization which is con ducted at higher temperatures of ∼300°C. Another important method of improving melt as well as solution processability involves the incorporation of a monofunctional reagent to obtain nonreactive end groups and controlled molecular weights. Structural modifica tion by copolymerization with more flexible oligomers such as poly(arylene ether)s may also enhance processability and possibly provide improved impact strength. In this paper, the effects of incorporating varying concentrations of poly(arylene ether) sulfone and ketone oligomers into the polyimide backbone are reported. The overall copolymer molecular weight was consistently controlled to —25,000 g/mole, with the use of small molar quantities of the monofunctional reagent phthalic anhydride. Very tough, trans parent films were obtained in all cases by compression molding copolymers at 340°C. Thermal analysis indicated that microphase separation is achievable in some cases as judged by the presence of two glass transition temperatures.

Novel procedures for derivatization of ribosomes for crystallographic studies

Undecagold and tetrairidium clusters have been used for the preparation of heavy-metal derivatives of ribosomal particles, necessary for the evaluation of phases in the x-ray structure determination of these large particles. To obtain specific binding, monofunctional reagents of the clusters were prepared and were covalently bound to free sulfhydryl groups on the surface of the ribosome. In addition, a mutant of Bacillus stearothermophilus which lacks one ribosomal protein (BL11) was grown. The heavy-atom clusters were covalently bound to isolated protein BL11, and the modified protein was consequently reconstituted into the mutated ribosomal subunits. Crystallographic data have been collected from crystals of native particles, from the mutated ones, and from the iridium- and gold-derivatized subunits. All these crystal forms are isomorphous within the experimental error.

Low Dielectric, Hydrophobic Polyimide Homopolymers and Poly(Siloxane Imide) Segmented Copolymers for Electronic Applications

AbstractPolyimides generally possess excellent thermal and mechanical properties, making them attractive candidates for high performance applications. To be useful for microelectronic applications, however, these materials must also be good insulators, as well as be readily processable.The incorporation of flexible polysiloxane segments into the polyimide backbone structure has been shown to yield soluble, processable copolyimides with good thermal and mechanical properties. In addition, the siloxane component imparts a number of other significant benefits for electronic applications. These include reduced water sorption, surface modification, good thermal and ultraviolet stability, and resistance to degradation in oxygen plasma environments. For polar polyimide systems, siloxane incorporation will also reduce the dielectric constant. The use of other less polar, more hydrophobic monomers will consistently yield soluble systems with lower dielectric constants as well.In this work, a series of high molecular weight, soluble polyimide homopolymers and segmented polysiloxane-polyimide copolymers were synthesized by a solution technique. The solution procedure, conducted at lower temperatures (˜170°C) than the classical bulk thermal imidization (300°C), has been shown to yield polyimides of enhanced solubility. In order to further enhance processability, molecular weight was controlled through the incorporation of monofunctional reagents such as phthalic anhydride and maleic anhydride, yielding nonreactive or potentially reactive endgroups, respectively. A series of maleic anhydride terminated imide oligomers with varying molecular weights were synthesized based upon the hexafluoropropane linked dianhydride and bisaniline diamine. In their oligomeric state, they exhibited enhanced solubility compared with their linear high molecular weight analogue. As these monomers were relatively nonpolar and hydrophobic, they afforded polyimides of low dielectric constant and a low level of water sorption. After thermally crosslinking the endgroups, the advantages of insoluble network systems could be realized. Particular advantages for electronic applications include thermal and dimensional stability over a wide temperature range, good mechanical properties, and chemical resistance. Structure-property characterization, including water sorption, dielectric constants, solubility behavior and thermal/mechanical properties will be reported.

Pyrrolocoumarin derivatives: DNA-binding properties

The spectroscopic and DNA-binding properties of a number of pyrrolocoumarin derivatives, including linear tricyclic, angular tricyclic, linear tetracyclic and angular tetracyclic compounds were investigated. The compounds we examined form non-covalent complexes with duplex DNA, probably of the intercalation type. The binding constants are comparable with the constants found for the furocoumarin analogues. Although for some of the compounds the photoreactivity with DNA is comparable with that of 8-MOP, pyrrolocoumarins behave as monofunctional reagents. This fact is explained in terms of an increased delocalization of the 4′,5′ double bond in the pyrrole moiety. Denaturation—renaturation experiments and HPLC analysis of the photoadducts confirm that pyrrolocoumarins are essentially monofunctional DNA-photobinding agents.

Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents

To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating.

Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides.

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein.

On-column preparation of chemically bonded stationary phase with maximum surface coverage and high reproducibility, and its application to packed microcapillary columns

A method for the on-column preparation of chemically bonded stationary phase with monofunctional reagents was studied in conventional stainless-steel columns and in glass capillary columns for packed microcapillary chromatography. Simple reactions of the silica surface with octadecyldimethyl-N,N-diethylaminosilane in packed columns resulted in a surface coverage of more than 3.0 μmol/m 2, comparable to the stationary phase of maximum coverage prepared in batch. The extent of surface coverage and the amount of residual silanols were carefully examined by using positively charged amines and neutral compounds. Although the results indicate a slight difference between the C 18 phases in stainless-steel columns and in packed microcapillary columns, excellent reproducibility was found for the on-column bonding reaction, as expected for the reaction using monofunctional reagents.

Interaction of specific platelet membrane proteins with collagen: evidence from chemical cross-linking.

Two recently developed membrane-impermeant cross-linkers, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3), have been used to examine the interaction of human platelets with collagen. Reaction of human platelets with either of the two cross-linking reagents at micromolar concentrations completely inhibited platelet aggregation in response to collagen but not in response to thrombin. Platelet adhesion to collagen was, however, not affected by these reagents. Inhibition of collagen-induced platelet aggregation by DTSSP or BS3 appears to be due to cross-linking and not simply to the chemical modification of membrane proteins, since the homologous monofunctional reagent sulfosuccinimidyl propionate had no effect on platelet aggregation. Inhibition of platelet aggregation by BS3 was accompanied by a decrease in the intensity of glycoprotein bands IIb, IIIa, and IV when analyzed on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. In order to determine if collagen is directly interacting with a specific platelet membrane glycoprotein, 3H-labeled platelets were allowed to adhere to collagen and then cross-linked with various concentrations of DTSSP. Proteins which remain associated with collagen after lysis and washing were analyzed on NaDodSO4 gels. At concentrations of 16-50 microM DTSSP, glycoproteins IIb and IIIa appeared to be specifically cross-linked to collagen. These results suggest that the glycoprotein IIb-IIIa complex, which has previously been implicated as the fibrinogen receptor in activated platelets, may also be directly involved in collagen-induced platelet aggregation.

Dense particles and slow sedimenting particles produced by ultraviolet irradiation of poliovirus.

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.

The cross-linking of tubulin with imidoesters.

Tubulin was reacted with a monofunctional imidoester, ethyl acetimidate, and three bifunctional imidoesters ranging in extension from 5 to 10 A. Extensive cross-linking was found to occur with the three bifunctional reagents resulting in the formation of dimers, trimers, tetramers, pentamers, and hexamers. In addition, a similar cross-linking patterns was observed with the monofunctional reagent. This type of cross-linking is rarely seen when proteins are reacted with imidoesters. Given that the cross-linking span of ethyl acetimidate is only 3 A it is reasonable to infer that there are nucleophilic groups in close proximity within the tubulin dimer. Amidinated tubulin is still capable of assembling into microtubules.

Cross-linking studies of the protein topography of rat liver microsomes

A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0°C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an M I of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with M r of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercapto-ethanol or dithiothreitol were not successful.

Nucleosides and Nucleotides. Part 17. A simple preparation of protected deoxynucleoside‐3'‐phosphates

AbstractA simple method for the preparation of fully protected 2′‐deoxynucleoside‐3′‐phosphates is described. The main step, phosphorylation of partially protected deoxynucleosides, is performed with the monofunctional reagent p‐chlorophenyl (2‐cyanoethyl) phosphochloridate3) (2) in dioxane. This reagent is quickly prepared from readily accessible p‐chlorophenyl phosphodichloridate by reaction with 3‐hydroxypropionitrile and triethylamine in dioxane or ether. The crude reagent is used directly for phosphorylation. After chromatography, protected deoxynucleoside‐3′‐phosphates were isolated in yields of 80–87%. The method described here, which has been optimized in detail, represents a simple alternative to published methods.

3-Amino- and 3-oxy-derivatives of psoralen: preparation and interactions with DNA.

3-Amino- and 3-oxy-derivatives of psoralen, thus having electron-donating groups at the 3-position of the alpha-pyronic ring, were prepared and studied as regards their capacities of interaction and photoreaction with native DNA in vitro. Very interesting properties were shown especially by the 3-amino-derivatives, namely 3-amino-4'-methylpsoralen and 3-diethylaminoacetylamido-4'-methylpsoralen, having a very high photobinding capacity with DNA and behaving as pure, or fairly pure, monofunctional reagents. These properties appear to be quite suitable for the therapeutic treatment of psoriasis and other skin diseases characterized by an increased reproducing activity of epidermal cells.

ChemInform Abstract: NEW MONOFUNCTIONAL REAGENTS FOR DNA AS POSSIBLE AGENTS FOR THE PHOTOCHEMOTHERAPY OF PSORIASIS: DERIVATIVES OF 4,5′‐DIMETHYLANGELICIN

AbstractDas Dimethylangelicin (I) führt zum Chlormethylderivat (II), aus dem man die Verbindungen (III) erhält.

Topology of membrane sulfhydryl groups in the human erythrocyte Demonstration of a non-reactive population in intrinsic proteins

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, N- ethylmaleimide . About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations. These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled N- ethylmaleimide or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with N-[ethyl-2- 3H] ethylmaleimide . The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive. After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards N- ethylmaleimide . It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.

New monofunctional reagents for DNA as possible agents for the photochemotherapy of psoriasis: derivatives of 4,5'-dimethylangelicin.

With the aim of obtaining new agents for the photochemotherapy of psoriasis, we have prepared monofunctional reagents for DNA by starting from 4,5'-dimethylangelicin (2), an angular furocoumarin, and introducing in a 4'-(hydroxymethyl) (3), 4'-(methoxymethyl) (4), or 4'-(aminomethyl) group (5), in way analogous to what other authors have done previously on trioxsalen, a DNA bifunctional reagent. These new compounds form complexes with DNA in the ground state and by successive irradiation (UV-A) undergo monofunctional photoaddition to the macromolecule. Photobinding to DNA was highest for 3 and gradually lower for 4 and 5, respectively. These compounds do not form interstrand photocross-linkages in DNA and do not show any skin phototoxicity. Fluorimetric studies show that their 4',5' double bond is involved in the photoaddition to DNA. Their photobiological activity evaluated on Ehrlich ascites tumor cells and on T2 phages was strictly connected with their photobinding to DNA. The effect of the introduction of hydroxymethyl and methoxymethyl groups in angular 2 is somewhat similar to that previously described for trioxsalen: the introduction of an aminomethyl group in 2 markedly increases the affinity in the dark for DNA but under UV-A irradiation strongly inhibits photobinding to the macromolecule. By contrast, in the analogous derivative of trioxsalen both the affinity for DNA in the dark and the photobinding to DNA increased.