Bis-quaternary Ammonium Research Articles

Synthesis and characterization of photo-curable bis-quaternary ammonium dimethacrylate with antimicrobial activity for dental restoration materials

In this study, new quaternary ammonium bis-phenol A glycerolate dimethacrylate (QABGMA) was synthesized and proposed as a possible antimicrobial dental monomer. Such monomer has the potential to copolymerize with other methacrylate monomers and impart antimicrobial activity in dental resins and other systems. QABGMA monomer was synthesized and incorporated (at 0–15wt.%) into a 1/1 (wt./wt.) mixture of bisphenol A glycerolate dimethacrylate (BisGMA)/triethylene glycol dimethacrylate (TEGDMA). The mixture was cured by visible light irradiation and degree of conversion (DC) was measured by Fourier Transform Infrared Spectrophotometer (FT-IR). Weight increase and solubility of the cured specimens were measured by immersion in water and ethanol/water solution (75/25, v/v) at 37±1°C for 30days. The antimicrobial activity of QABGMA monomer was investigated against Gram-positive bacteria (Streptococcus mutans, Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and a fungus Candida albicans. Finally, cytotoxicity and viability of resin on L929 fibroblast cells were quantified. The incorporated difunctional QABGMA reduced degree of conversion slightly and increased water absorption of the cured polymer. The dental materials obtained from the QABGMA polymerization system showed good antimicrobial activity against tested microorganisms. Also, resin with ⩾10wt.% QABGMA showed significantly reduced viability.

A new class of bifunctional chiral phase transfer catalysts for highly enantioselective asymmetric epoxidation of α,β-unsaturated ketones at ambient temperature

A new type of bis-quaternary ammonium bromide as chiral multifunctional phase transfer catalysts derived from readily available inexpensive cinchona alkaloids has been developed and evaluated for the enantioselective asymmetric epoxidation of various chalcones in the presence of lower concentrations of various oxidants, bases, solvents and ambient temperature conditions. Under optimized reaction conditions, highest chemical yields of up to 98% along with the excellent enantioselectivities of about 99% were obtained by using the cinchona based chiral multifunctional phase transfer catalysts.

Synthesis, Characterization, Crystal Structure, and Properties of a Mercury(Ii) Complex Containing Dmit Ligand and N,N’-Ethylidenedipyridium Cation

A new mercury(II) complex containing dmit ligand and biquaternary ammonium cation, DPyE[Hg(dmit)2] (1) (DPyE2+ = N,N’-ethylidenedipyridinium, dmit = 1,3-dithiol-2-thione-4,5-dithiolate), was synthesized and fully characterized by elemental analysis, IR, 1H-NMR, UV–Vis, TG, single crystal X-ray diffraction, and powder X-ray diffraction. Moreover, the electrochemical properties and conductivities of complex 1 were studied by cyclic voltammetry and four-probe AC technique, respectively. Complex 1 crystallizes in the monoclinic space group P 21/n with a = 8.1815(2) Å, b = 12.8701(5) Å, c = 23.2681(7) Å, β = 90.922(3)°, Z = 4, V = 2449.75(14) Å3, Dc = 2.114 g·cm–3. The crystal structure shows that the dihedral angle of two dmit ligands of [Hg(dmit)2]2– anion is closed to vertical, which results in the formation of a distorted HgS4 tetrahedral geometry. The S···S short contacts assemble [Hg(dmit)2]2– anions into 1D, 2D, and 3D supramolecular network. The DPyE2+ cations are located in the caves, providing additional stabilization to crystal packing via C–H···S hydrogen bonds to [Hg(dmit)2]2– anions. The crystal packing of 1 is characterized by face-to-face π-π contacts between DPyE2+ cations and [Hg(dmit)2]2– anions, which result in two orthogonally oriented and mixed stacking columns. These stacking columns interweave into a 2D network, which is further connected to a 3D network via interplanar S···S contacts. In addition, the temperature-dependent conductivity in the range of 50–300 K reveals semi-conducting properties of complex 1, and a room temperature conductivity of 1.53 × 10–3 S·cm–1 is observed.

The synergistic inhibition effect between imidazoline-based dissymmetric bis-quaternary ammonium salts and thiourea on Q235 steel in CO2 corrosion process

The inhibition effect of imidazoline-based dissymmetric bis-quaternary ammonium salt (DBA) and Thiourea (TU) on Q235 steel in CO2-saturated simulated oilfield-produced water was studied by means of the weight-loss method, polarization and electrochemical impedance spectroscopy (EIS). The polarization measurements reveal that the compound inhibitors are anodic-type inhibitors that mainly inhibit the anodic process. EIS results show that the changes in the impedance parameters are due to the formation of a protective layer on the Q235 steel surface by the adsorption of inhibitor molecules. The results of energy dispersive X-ray spectroscopy and scanning electron microscopy confirm the existence of the protective layer. The results also show that adsorption model obeys to the Flory–Huggins adsorption isotherm. The synergism parameter suggests that the synergistic effect is obvious at concentrations of 100 and 200 mg L−1, and there is a cooperative mechanism between the DBA and TU.

Effect of siloxane spacer length on organosilicon bi-quaternary ammonium amphiphiles

A series of organosilicon Bola-form bi-quaternary ammonium amphiphiles, [OH5C3(C2H5)2N+-(CH2)3-Si(CH2)3-O-(Si(CH3)2O)n-Si(CH2)3⋯(CH2)3-N+(C2H5)2C3H5O]Cl2− (SinN2Cl2, n=0, 4, 6, 8), with the same headgroups and different length of hydrophobic linkage has been synthesized. The critical micelle concentration (CMC) of each amphiphiles was determined by equilibrium surface tension. With the increased length of hydrophobic siloxane spacer, the CMC values follow the order of Si8N2Cl2<Si6N2Cl2<Si4N2Cl2<Si0N2Cl2. At the same time, thermodynamic parameters (ΔGm0, ΔHm0, ΔSm0) of micellization were calculated by Gibbs equation under the mass action model. It indicates that the micellization of the amphiphiles in aqueous solution is entropy-driven. Especially, the surface area (Amin) of Si4N2Cl2 is the largest attributing to the interaction of free-rotated siloxane spacer and its internal oxygen atom, which make the molecular stretch free. The antimicrobial property of Si4N2Cl2 is more effective than others below the concentration of CMC, while the antimicrobial property of Si8N2Cl2 is more effective above the concentration of CMC, which indicated that both the adsorbability in formation of micellar and the hydrophobicity arising from the different length of siloxane spacer are important to inhibit microbe. Moreover, their wetting ability has been characterized by contact angles on various material surfaces. It shows that the higher weight of lipophilic siloxane spacer leads to lower contact angels.

Crystalline Inclusion Compounds: Thiourea and Bis-quaternary Ammonium Salts

Two new inclusion compounds, namely 3(CH3)3N+(CH2)3N+(CH3)3·6Br–·8(NH2)2CS·H2O (1) and (CH3)3N+(CH2)4N+(CH3)3·2Br–·2(NH2)2CS (2), have been prepared and characterized by X-ray crystallography. In the crystal structure of 1, uni-directional double channels with peanut-shaped cross-section running paralleling to the a axis are generated by N–H···S and N–H···Br hydrogen bonds between thiourea molecules and bromide anions, and trimethylenebis(trimethylammonium) dications are arranged separately in each hemisphere of the double channel. In 2, tetramers comprising pairs of thiourea molecules and bromide anions by N–H···S and N–H···Br hydrogen bonds are joined with the methyl groups of tetramethylenebis(trimethylammonium) dications to generate similar quasi-rectangle channels. The present study shows that new anionic host lattices can be constructed from thiourea molecules and bromide anions as building blocks, which readily adopt different topologies for the accommodation of bis-quaternary ammonium dications of various sizes. Hydrogen bonding constitutes a principal driving force that dominates the crystal packing of the two compounds, in which weak hydrogen bonds are of particular significance in the construction. In the crystal structure of 1, uni-directional double channels with peanut-shaped cross-section running paralleling to the a axis are generated by N–H···S and N–H···Br hydrogen bonds between thiourea molecules and bromide anions, and trimethylenebis(trimethylammonium) dications are arranged separately in each hemisphere of the double channel. In 2, tetramers comprising pairs of thiourea molecules and bromide anions by N–H···S and N–H···Br hydrogen bonds are joined with the methyl groups of tetramethylenebis(trimethylammonium) dications to generate similar quasi-rectangle channels.

Variable Electrospray Ionization and Matrix-Assisted Laser Desorption/ Ionization Mass Spectra of the Bisquaternary Ammonium Salt Ethonium

Determination of a single “fingerprint” mass spectrum of a biologically active compound, required for compound identification in mixtures and biological materials in such applied tasks as ecological monitoring and biological imaging, may become a nontrivial problem for labile compounds whose mass spectra depend strongly on the applied experimental conditions. In the present communication, qualitatively different mass spectral patterns obtained for the bisquaternary ammonium salt ethonium Cat2+•2Cl– under varied conditions of electrospray ionization and matrixassisted laser desorption/ionization (MALDI) are described and systematized. It is shown that qualitative changes in the electrospray mass spectra of ethonium occur upon a cone voltage increase from 10 V to 100 V, which are caused by the subsequent appearance and destruction of several primary ions: the intact dication Cat2+, the dication-counterion cluster Cat2+•Cl– and[Cat ‑ H]+ and [Cat ‑ CH3]+ ions. Novel experimental evidence of survival of the gas-phase dication Cat2+ with the shortest possible distance of ca 4 Co between the quaternary nitrogen atoms provided by two CH2 groups and Cat2+ fragmentation with preservation of the doubly charged state of the fragments are revealed under soft electrospray conditions. The interpretation of the MALDI mass spectrum is made taking into account the formation of a salt of the bisquaternary ammonium base and 2,5-dihydroxybenzoic acid, which results in the absence of any chlorine-containing ions in the mass spectra and a fragment ion distribution that is different from that observed under electrospray ionization conditions. The main mass spectral features revealed for ethonium may aid in the identification of other types of bisquaternary ammonium compounds.

Quaternary ammonium derivatives of natural terpenoids. Synthesis and properties

In this review, we summarized and analyzed the literature data (from 1963 to March, 2014) on the synthesis and properties of quaternary ammonium derivatives of citronellal, menthol, camphor, lambertian acid, dehydroabietic acid, levopimaric acid, isosteviol, steviol, betulinic acid, dihydrobetulinic acid, platanic acid, oleanolic acid, ursolic acid, and polyprenols. Functionalization of terpenoids with ammnoium groups offers them new properties, such as cholinotropic, antiviral, and antimicrobial activities, or transforms them into chiral ionic liquids having a practical potential for application as chiral phasetransfer catalysts. The synthesis of ammonium derivatives of terpenoids possessing properties of cationic lipids or dimeric bis-quaternary ammonium surfactants was proved to have a promising outlook. A specific attention was paid to the method developed for transformation of any hydrophobic terpenoid or steroid into an amphiphilic water-soluble compound self-organized in the aqueous medium into vesicles (liposomes) forming cell-penetrating complexes with DNA.

Design, Synthesis, and X‐ray Structural Analyses of Diamantane Diammonium Salts: Guests for Cucurbit[n]uril (CB[n]) Hosts

AbstractNew bisprimary and bisquaternary diamantane‐1,6‐ and ‐4,9‐diammonium/diaminium salts were synthesized, and characterized by NMR spectroscopy and X‐ray crystallography. The impetus for these syntheses were previously reported X‐ray crystallographic investigations of adamantane mono‐ and bisquaternary ammonium ions [3,5‐diMeAda‐1‐NH3 or Ada‐1,3‐di(NMe3)] complexed with cucurbit[n]uril (n = 7, 8). The crystal structures were analyzed to ascertain possible structural hypotheses for high binding affinity guests bound within various diameter pumpkin‐shaped hosts. Although Diam‐4,9‐di(NMe3I) 5 could be readily prepared from the bisprimary precursor, corresponding Diam‐1,6‐di(NMe3I) 14 could not be obtained even under strong reaction conditions. Stereochemical analysis of this situation suggests very severe steric non‐bonding cis‐1,3‐diaxial type H···H interactions between the “axial”‐type NMe3 group and neighboring “axial” proton neighbors. These same interactions are found in Ada‐2,6‐di(NMe3I) analogues, but they are alleviated in this smaller polycyclic skeleton through tilting the axial C(methylene)–NMe3 bond away from its axial neighbors. However, similar structural relief for Diam‐1,6‐di(NMe3I) is not possible, because the C(methine)–NMe3 bond therein is ligated to the diamondoid's rigid skeleton.

Probing of the combined effect of bisquaternary ammonium antimicrobial agents and acetylsalicylic acid on model phospholipid membranes: differential scanning calorimetry and mass spectrometry studies

A model molecular biosystem of hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers that mimics cell biomembranes is used to probe combined membranotropic effects of drugs by instrumental techniques of molecular biophysics. Differential scanning calorimetry reveals that doping of the DPPC model membrane with individual bisquaternary ammonium compounds (BQAC) decamethoxinum, ethonium, thionium and acetylsalicylic acid (ASA) leads to lowering of the membrane melting temperature (Tm) pointing to membrane fluidization. Combined application of the basic BQAC and acidic ASA causes an opposite effect on Tm (increase), corresponding to the membrane densification. Thus, modulation of the membranotropic effects upon combined use of the drugs studied can be revealed at the level of model membranes. Formation of noncovalent supramolecular complexes of the individual BQACs and ASA with DPPC molecules, which may be involved in the mechanism of the drug-membrane interaction at the molecular level, is demonstrated by electrospray ionization (ESI) mass spectrometry. In the ternary (DPPC + ASA + BQAC) model systems, the stable complexes of the BQAC dication with the ASA anion, which may be responsible for modulation of the membranotropic effects of the drugs, were recorded by ESI mass spectrometry. The proposed approach can be further developed for preliminary evaluation of the combined effects of the drugs at the level of model lipid membranes prior to tests on living organisms.

Ultrasonic assisted dimeric cinchona based chiral phase transfer catalysts for highly enanatioselective synthesis of epoxidation of α,β-unsaturated ketones

New types of bis-quaternary ammonium bromide as chiral multisite phase transfer catalysts derived from cinchona alkaloids have been developed and evaluated for the enantioselective epoxidation of chalcones in the presence of lower concentrations of various oxidants, bases and ultrasonic irradiation conditions.

Wettability of compound organobentonites with ultra-low swelling modified by a series of bis-quaternary ammonium salts

A series of compound organobentonites were prepared by using three kinds of bis-quaternary ammonium salts (BQAS), hexamethyl ethyl diammonium methylsulfate (EDAS), hexamethyl hexyl diammonium methylsulfate (HDAS) and hexamethyl decyl diammonium methylsulfate (DDAS), with different alkyl chain lengths. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to characterize the structure of organobentonites. The results suggested that these organic cations lie flatly as a monolayer between the siloxane layers, the swelling of organobentonites was obviously decreased and the morphology of bentonite was largely changed after modification. In addition, the wettability of organobentonites for deionized water and n-heptane were measured by Washburn's pressure method simultaneous with height method. The simplified lipophilic-to-hydrophilic ratio (SLHR) and relative wettability index (RWI) were introduced to evaluate the relative lipophilic and hydrophilic properties of different organobentonites. The hydrophilic increased in an order of E–B, H–B and D–B for the unitary organobentonites; ED–B, EH–B and HD–B for the binary organobentonites; and 2.0CEC, 1.5CEC, 1.0CEC and 0.5CEC for the ternary organobentonites. It should be noted that, from the presence of ultra-low swelling and wettability of compound organobentonites, we can see a synergistic effect, the compounding of different chain-length of BQAS had, on the wettability of organobentonites.

Model mass spectrometric study of competitive interactions of antimicrobial bisquaternary ammonium drugs and aspirin with membrane phospholipids

Aim. The aim of the study is to reveal molecular mechanisms of possible activity modulation of antimicrobial bisquaternary ammonium compounds (BQAC) and aspirin (ASP) through noncovalent competitive complexation under their combined introduction into the model systems with membrane phospholipids. Methods. Binary and triple systems containing either decamethoxinum or ethonium, or thionium and aspirin, as well as dipalmitoylphosphatidylcholine (DPPC) have been investigated by electrospray ionization mass spectrometry. Results. Basing on the analysis of associates recorded in the mass spectra, the types of nonocovalent complexes formed in the systems studied were determined and the supposed role of the complexation in the BQAC and ASP activity modulation was discussed. The formation of associates of BQAC dications with ASP anion is considered as one of the possible ways of deactivation of ionic forms of the medications. The formation of stable complexes of BQAC with DPPC and ASP with DPPC in binary systems as well as the complexes distribution in triple-components systems BQAC:ASP:DPPC point to the existence of competition between drugs of these two types for the binding to DPPC. Conclusions. The results obtained point to the competitive complexation in the model molecular systems containing the BQAC, aspirin and membrane phospholipids. The observed phenomenon testifies to the possibility of modulating the activity of bisquaternary antimicrobial agents and aspirin under their combined usage, due to the competition between the drugs for binding to the target membrane phospholipid molecules and also due to the formation of stable noncovalent complexes between BQAC and ASP.

Orientations and Proximities of the Extracellular Ends of Transmembrane Helices S0 and S4 in Open and Closed BK Potassium Channels

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.

Inhibition Performance of Novel Dissymmetric Bisquaternary Ammonium Salt with an Imidazoline Ring and an Ester Group

AbstractIn this paper, the inhibition performance of a novel dissymmetric bis‐quaternary ammonium salt with an imidazoline ring and an ester group (DBAS) for Q235 steel in 2 % NaCl solution saturated with CO2 was investigated using weight loss, the polarization curve, electrochemical impedance spectroscopy methods and the quantum chemistry calculation. The results show that DBAS, which is good for the environment and have many activated centers and low ELUMO, has a high inhibition performance for Q235 steel in 2 % NaCl solution saturated with CO2. The inhibition efficiency of the studied corrosion inhibitor increased with increasing inhibitor concentrations. The inhibitor is a mixed‐type inhibitor which inhibits both anodic and cathodic reactions.

Bis-quaternary ammonium cation-based organic ionic plastic crystals: plastic crystal behaviour and ionic liquid properties above melting points

A new family of organic ionic plastic crystals (OIPCs) based on bis-quaternary ammonium cations was synthesized and characterized. The synthesized bis-quaternary ammonium salts with bis(trifluoromethanesulfonyl) imide (TFSI−) anions exhibit plastic crystal properties as determined by differential scanning calorimetry (DSC). The high ionic conductivity of the compounds in the solid-state indicated that this new family of OIPCs can be promising solid-state electrolytes for electrochemical devices. Cyclic voltammetry characterization of OIPCs in the liquid-state revealed their wide electrochemical windows and highly reversible lithium metal electrodeposition properties.

Biological Evaluation of Quaternary bis Ammonium Salt and Cetylpyridinum Bromide Against S. epidermidis biofilm

Quaternary ammonium compounds are broad-spectrum bacteriocides widely used as antiseptics, disinfection and preservation agents. The aim of this study was to examine the activity of two quaternary ammonium salts, cetylpyridinum bromide and a newly synthesized quaternary bis ammonium salt, against S. epidermidis biofilm. The average values of killing efficiency for cetylpyridinum bromide ranged from 26.6% to 64.1% for all tested concentrations (0.125 to 8.0 microg x mL(-1)) and for quaternary bis ammonium salt the percentage of killing efficiency ranged from 59.7% to 88.4% for tested concentrations (from 2.0 to 128.0 microg x mL(-1)). Both tested compounds significantly affect staphylococcal biofilms, but any of used concentrations caused a total eradication of bacterial biofilm.

A dissymmetric bis-quaternary ammonium salt gemini surfactant as effective inhibitor for Q235 steel in hydrochloric acid

The inhibition effect and adsorption behavior of a novel dissymmetric bisquaternary ammonium salt (DBAL) for Q235 steel in 1M hydrochloric acid medium were investigated using weight loss method, polarization and electrochemical impedance spectroscopy (EIS). The result of weight loss method indicates that the inhibition efficiency increased with DBAL concentration and temperature in the studied range. The inhibition efficiency is above 90% at a concentration of 3.28×10−4M in the temperature range of 298–328K. The polarization measurements reveal that DBAL is a mix-type inhibitor which mainly inhibits cathodic process. EIS results show that changes in the impedance parameters (Rct and Cd) were due to the formation of a protective layer on the Q235 steel surface by the adsorption of inhibitor molecules. Adsorption follows the Langmuir isotherm via chemical adsorption on the Q235 steel surface. Thermodynamic and kinetic parameters were evaluated from the effect of temperature on corrosion and inhibition processes to discuss the adsorption mechanism.

Synthesis of a Rosin Gemini Surfactant and Its Properties

A novel rosin Gemini surfactant, bisquaternary ammonium salt (BQA) of rosinate, was synthesized in high yield and purity. First, rosin reacted with ethanol to produce ethyl rosinate. Then the ethyl rosinate was modified with fumaric acid. Also, triethylamine reacted with epoxy chloropropane to synthesize epoxy quaternary ammonium salt (QAS). Finally, the epoxy QAS was reacted with the modified rosinate to prepare the Gemini surfactant, BQA of rosinate, at the following conditions: the intermediate and the modified rosinate mole ratio of 3:1, reaction temperature of 82.5°C, and reaction time of 8.5 h. Gravimetric analysis showed that its BQA content was 90.9% (w/w) of the product. The chemical structure of the BQA was conformed by high-performance liquid chromatogram, Fourier transform infrared spectroscopy, and nuclear magnetic resonance analysis. As a BQA Gemini surfactant, it has good surface activities and antifungal activity to most of the tested wood decay or mold fungi. Besides these great properties, its raw material is renewable. Herein, the BQA of rosinate will be a great Gemini surfactant alternative in related industries.

Novel class of bisquaternary ammonium salts in inhibition of mild steel corrosion in HCl and H2SO4

A series of new bisquaternary ammonium inhibitor molecules p-xylylenebis[trialkylammonium chloride] were prepared in excellent yields by reacting α,α′-dichloro-p-xylene with a group of tertiary amines such as N,N-diallyl-N′-formyl-1,12-diaminododecane, N,N-diallyl-, N,N-diethyldodecylamine and N-dodecylpyrrolidine. Inhibition efficiencies (IEs) for different concentrations of bisquaternary salts for the inhibition of corrosion of mild steel in 1, 4 and 7·7M HCl as well as 0·5M H2SO4 exposed for 6 h at 60°C were determined gravimetrically. While in the presence of 400 ppm all the inhibitor molecules exhibited excellent %IEs of ⩾99 in 1M HCl, p-xylylenebis[N,N-diallyl, N-dodecylammonium chloride] provided very good %IE of 97 and 94 respectively in 4 and 7·7M HCl. The inhibitors imparted good to very good IE in arresting mild steel corrosion in 0·5M H2SO4; p-xylylenebis[N,N-diallyl, N-dodecylammonium chloride] performed the best, giving %IE of 94 at 400 ppm. The interactions of the π electrons in the diallyl moiety with the low lying vacant d orbitals of iron help the inhibitor molecules to undergo adsorption and interfere with the anodic dissolution process. Comparable results for the IEs were obtained by the electrochemical method using Tafel plots of the synthesised compounds. The adsorption of these compounds on the mild steel surface was found to obey Frumkin and Temkin adsorption isotherms in 1M HCl and 0·5M H2SO4 respectively.