2 Ethoxyethanol Synthesis Essay

Description

2-Ethoxyethanol is a common solvent. It, like other glycol ethers, is used in the semiconductor industry. It is also used in surface coatings such as lacquers and paints. It is used in varnish removers, printing inks, duplicating fluids, wood stains, and epoxies.

Substance details

Substance name: 2-Ethoxyethanol

CASR number: 110-80-5

Molecular formula: C4H10O2

Synonyms: Ethylene glycol monoethyl ether; Glycol ethyl ether; cellosolve; Glycol monoethyl ether; Ethoxyethanol; ethylene glycol ethyl ether; glycol ether ee; Ethyl cellosolve; Ethyl Glycol

Physical properties

2-Ethoxyethanol is a colourless liquid, organic solvent with a sweet odour. It dissolves readily in both water and organic solvents (acetone, benzene, carbon tetrachloride, etc)

Melting Point: -90°C

Boiling Point: 135°C

Vapour Density: 3.1

Specific Gravity: 0.93

Flash point: 40°C (combustible liquid)

Chemical properties

Lower explosive limit: 1.8%

Upper explosive limit: 14%

Reacts with strong oxidizers.

Further information

The National Pollutant Inventory (NPI) holds data for all sources of 2-Ethoxyethanol emissions in Australia.

Description

2-Ethoxyethanol can effect you when breathed in or by passing through your skin. Short-term exposures may irritate the eyes, nose, and throat. Very high levels may cause you to feel dizzy, lightheaded and to pass out. Long-term effects from exposure to 2-Ethoxyethanol are possible kidney damage, damaged blood cells, damaged testes in males, and decreased fertility in males. 2-Ethoxyethanol has been shown to be a teratogen in animal studies, and is a possible human teratogen. A teratogen is a substance that harms a foetus.

Entering the body

2-Ethoxyethanol will enter the body if we breathe in contaminated air or drink contaminated water. It can also pass through the skin.

Exposure

Workers in the industries that use or produce 2-Ethoxyethanol are at risk of exposure. Consumers can be exposed to 2-Ethoxyethanol by exposure to air from production and processing facilities using 2-Ethoxyethanol. Consumers may also be exposed to 2-Ethoxyethanol when using consumer products containing 2-Ethoxyethanol, especially if there is not good ventilation. Household hard surface cleaners, paints, varnishes, lacquers, inks and paint removers are some of the consumer products that may release 2-Ethoxyethanol.

Health guidelines

According to Worksafe Australia, it is allowable for workers to be exposed to 5 parts per million 2-Ethoxyethanol over an eight hour workshift. Worksafe Australia has determined that 2-Ethoxyethanol may cause birth defects and may be teratogenic (harm a foetus).

Description

2-Ethoxyethanol has slight short-term and slight long-term toxicity to aquatic life. Insufficient data are available to predict the effects of 2-Ethoxyethanol on plants, birds or land animals.

Entering the environment

Industrial emissions of 2-Ethoxyethanol can produce elevated, but still low level concentrations in the atmosphere around the source. 2-Ethoxyethanol is readily washed out of the air, this in turn means that about 95% of the 2-Ethoxyethanol emitted to the environment will eventually end up in water. Once in the water it will take between a few weeks to half a year for it to be completely degraded out of the water. Because 2-Ethoxyethanol is used in many consumer products, short-term indoor concentrations may be elevated above the levels considered safe for workers.

Where it ends up

2-Ethoxyethanol is rapidly degraded by chemical and biological processes. 2-Ethoxyethanol is not expected to bio-accumulate.

Environmental guidelines

There are no specific Australian environmental guidelines for 2-Ethoxyethanol.

Industry sources

The primary stationary sources of 2-Ethoxyethanol are the industries that manufacture it or use it in production. Some of the industries that use it in production are the paint, varnish, wood stain, and lacquer industries. It is used in printing and in the semiconductor industry.

Diffuse sources, and industry sources included in diffuse emissions data

Diffuse emissions to air are from commercial and household painting, staining and use of varnish and lacquers. Some inks will also give off low levels of 2-Ethoxyethanol.

Natural sources

2-Ethoxyethanol does not occur naturally in the environment.

Transport sources

There are no known sources of mobile emissions of 2-Ethoxyethanol.

Consumer products

Household hard surface cleaners, ink markers, various paint and paint-related products, paint thinners, polishing preparations.

Sources used in preparing this information

  • Australian and New Zealand Environment and Conservation Council (ANZECC) (1992), Australian Water Quality Guidelines for Fresh and Marine Waters.
  • ChemFinder WebServer Project (1995), 2-Ethoxyethanol (accessed, March, 1999)
  • Cornell University, Planning Design and Construction, MSDS, Ethylene Glycol MonoEthyl Ether, Tech (accessed, March, 1999)
  • Environmental Defense Fund (1998), Ethylene Glycol Monoethyl Ether: The Chemical Scorecard: (accessed, March, 1999)
  • Environmental Health Center, a division of the National Safety Council, Environment Writer – Chemical Backgrounders Glycol Ethers Chemical Backgrounder (March, 1999) (accessed, March, 1999)
  • Meagher, D (1991), The Macmillan Dictionary of The Australian Environment, Macmillan Education Australia Pty Ltd.
  • National Environment Protection Council (1998), National Environment Protection Measure for the National Pollutant Inventory. (accessed, March, 1999)
  • National Health and Medical Research Council (NHMRC) and Agriculture and Resource Management Council of Australia and New Zealand (ARMCANZ) (1996), Australian Drinking Water Guidelines.
  • New Jersey Department of Health and Senior Services (1995), Hazardous Substance Fact Sheet, 2-ETHANOXYETHANOL, PO Box 368, Trenton, NJ.
  • New Jersey Department of Health, Right to Know Program (1986), TRIFacts, 2-Ethoxyethanol (accessed, March, 1999)
  • NTP Chemical Repository, Radian Corporation, 2-Ethoxyethanol (AUGUST 29, 1991) (accessed, March, 1999)
  • Richardson, M (1992), Dictionary of Substances and their Effects, Royal Society of Chemistry, Clays Ltd, England.
  • Sittig, M (1991), Handbook of Toxic and Hazardous Chemicals and Carcinogens, 3rd edition, Noyes Publications, USA.
  • Technical Advisory Panel (1999), Final Report to the National Environment Protection Council.
  • US Department of Health and Human Services (1990), NIOSH Pocket Guide to Chemical Hazards, Publication No. 90-117.
  • The Environmental Chemicals Data and Information Network (ECDIN) Ethanol, 2-ethoxy (accessed, April, 1999)
  • U. S. Environmental Protection Agency (EPA), The Integrated Risk Information System (IRIS) database ( 1 March, 1997 ) 2-Ethoxyethanol (accessed, March, 1999)
  • Worksafe Australia (1996), Exposure Standard 2-Ethoxyethanol (accessed, March, 1999)
  • Worksafe Australia (1996), Hazardous Substance 2-Ethoxyethanol (accessed, March, 1999)

1. Introduction

Pseudomonas aeruginosa is an opportunist Gram-negative human pathogen responsible for a variety of nosocomical infections and life-threatening diseases in immunocompromised and debilitated patients [1,2]. P. aeruginosa infections are notoriously difficult to eradicate, which has been attributed to the predilection of P. aeruginosa cells to form antibiotic-resistant biofilms, and high levels of intrinsic antibiotic resistance [1,2,3]. Indeed, multi-drug resistance P. aeruginosa nosocomical infections are increasingly being detected across the globe [4]. Thus the exploration of new strategies for tackling infections caused by this notorious pathogen is urgently warranted [1,2,5]. The ability of P. aeruginosa to cause disease is dependent upon the production of agents called ‘virulence factors’ that actively cause damage to host tissues [1,6,7,8]. The targeting of virulence factors (for example, inhibition of their production) has been identified as a potential new therapeutic approach to treating P. aeruginosa infections; in principle, this would attenuate the pathogenicity of the bacterium, increasing the likelihood that the host immune system can clear the infection before too much tissue damage is caused [1,7,9,10,11]. One of the many virulence factors produced by P. aeruginosa is pyocyanin (Figure 1) [12]. There is a large body of evidence that this low molecular weight redox-active phenazine dye is important to the pathogenesis of P. aeruginosa infections [1,5,12,13,14]. Unsurprisingly therefore, the inhibition of pyocyanin production has been identified as a potential antivirulence strategy against this organism [1,5,14,15]. Indeed, there has been much interest in the discovery of compounds with the ability to inhibit pyocyanin biosynthesis in recent years [1,16,17,18,19,20].

Figure 1. BHL and OdDHL are two natural AHL autoinducers used by P. aeruginosa in quorum sensing. Pyocyanin is a virulence factor produced by P. aeruginosa. Compound 1, an abiotic OdDHL-mimic, is capable of strongly inhibiting the production of pyocyanin in cultures of wild type P. aeruginosa [1].

Figure 1. BHL and OdDHL are two natural AHL autoinducers used by P. aeruginosa in quorum sensing. Pyocyanin is a virulence factor produced by P. aeruginosa. Compound 1, an abiotic OdDHL-mimic, is capable of strongly inhibiting the production of pyocyanin in cultures of wild type P. aeruginosa [1].

Pyocyanin production in P. aeruginosa is regulated by an intercellular signaling process known as quorum sensing [21,22]. Many species of bacteria use quorum sensing systems, which allows for concerted interactions between the cells comprising a population [9]. This communication process is mediated by small diffusible signaling molecules termed autoinducers [9,20,23]. In the majority of Gram-negative species, N-acylated-L-homoserines (AHLs) serve as the autoinducers [20,23]. These are produced by LuxI-type synthase enzymes and bind to cyctoplasmic LuxR-type receptors to initiate the expression of genes associated with bacterial group processes [1,20,23,24,25]. In general, each bacterial species responds specifically to its own unique AHL(s), and uses different LuxI-type synthases and LuxR-type receptors [23,26]. Two AHL-based quorum sensing systems are present in P. aeruginosa. One employs N-butanoyl-L-homoserine lactone (BHL, Figure 1) as the signaling molecule (generated by RhlI and detected by RhlR) and the other uses N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL, Figure 1, generated by LasI with LasR as the cognate receptor). There is a third quorum sensing system in P. aeruginosa which employs a chemically distinct autoinducer (termed the Pseudomonas quinolone signal, PQS). The PQS system is interlinked with the two AHL-based systems, forming an intricate hierarchical quorum sensing network, with the las system generally regarded as standing at the apex [1,23,27,28]. The production of pyocyanin is regulated by RhlR and transcription of the rhlR gene itself is regulated by LasR [1,21]. Thus, inhibitors of LasR would be expected to attenuate the biosynthesis of pyocyanin [1,20,23,29,30,31]. The structure of OdDHL, the natural LasR agonist, has often been used as a template to guide the design and synthesis of abiotic LasR antagonists [1,11,16,20,23]. We recently reported the synthesis of OdDHL analogues containing non-native head groups in place of the natural homoserine lactone moiety [1]. These compounds were evaluated for their ability to inhibit the production of pyocyanin in cultures of wild type P. aeruginosa, with 1 (Figure 1) found to be the most potent (note that compound 1 was not screened in any LasR-based reporter systems).

Given that 1 is closely related in structure to OdDHL (which is known to interact with the LasR receptor) and the fact that pyocyanin production is generally considered to be regulated by LasR-based quorum sensing, it was postulated that 1 reduces the level of pyocyanin production by disrupting OdDHL-dependent activation of LasR [1]. Experimental evidence suggested that 1 is capable of binding to LasR and it was inferred that 1 might be an antagonist of the LasR receptor and an inhibitor of LasR-based quorum sensing in P. aeruginosa. This could have important implications; quorum sensing is known to regulate many additional facets of P. aeruginosa pathogenicity [15,32,33,34] and there is tremendous interest in finding small molecules that can disrupt AHL-mediated signaling in this organism [9,20,23]. However, our previous studies did not provide any direct evidence for an interaction between compound 1 and the LasR receptor, or indeed any other molecular targets. We were therefore interested in examining the molecular basis for the activity of 1 in more detail. Such information should assist in the design of next-generation agents with improved potency. Towards this end, we envisaged the design and synthesis of an affinity-based (“pull down”) chemical probe incorporating 1. This could potentially be employed in affinity-based (“pull down”) proteomic assays in order to directly detect the biological target(s) of 1 and thus better delineate the mechanism by which it reduces the level of pycocyanin production in P. aeruginosa [35,36,37,38,39,40,41].

2. Results and Discussion

2.1. Probe Design

Typically, affinity-based probes are composed of the biologically active molecule of interest tethered via a chemical linker to an insoluble support [38]. Usually, the probe is then incubated with the cell lysate of the relevant organism [42]. The small molecule’s macromolecular targets can then be extracted by virtue of specific binding; washing steps are used to remove non-binding proteins, and the remaining high affinity binders can be eluted from the support, separated using polyacrylaminde gel electrophoresis and identified using various mass spectrometry techniques [35,36,37,42]. Biotin is often used as an equivalent of an insoluble support (“tag”) in affinity probes, since immobilization on streptavidin beads (either before or after incubation with the biological system) is possible by virtue of the strong non-covalent biotin-streptavidin interaction [35,37,42]. Indeed, biotinylated probes have been widely used for the identification of many small molecule biological targets [38]. An advantage of biotinylated probes over solid-phase supports in that they are often cell permeable. Thus in addition to carrying out experiments using cell lysates, it is also possible for such probes to be incubated with live cells and interact with target protein(s) in their native environment inside a living cell or organism [37]. After cell lysis the probe can be pulled out of solution with streptavidin resin, which will also pull out any bound protein(s) [37]. Based on these considerations, we targeted the synthesis of 2 (Figure 2), a biotinylated affinity probe that could potentially be used for detecting the molecular targets of the active compound 1.

Figure 2. The target biotinylated affinity probe.

Figure 2. The target biotinylated affinity probe.

It was decided to use a polyethylene glycol (PEG)-based chain as the linker. PEG chains are commonly employed in this regard; they are usually long enough to mitigate undesired steric interactions steric hindrance between the support and small molecule-biomolecule interactions [38] and flexible enough to allow the target molecule to adopt multiple orientations in three-dimensions (and so access a favorable macromolecular binding pose). Furthermore, PEG linkers are also hydrophilic, increasing the solubility of molecules in aqueous solution [38]. A crucial consideration when preparing an affinity-based probe is where on the molecule of interest the linker should be introduced, as it is important that its’ biological activity is not affected significantly [38]. Our previous studies [1] indicated that further substitution of the aromatic ring portion would not be appropriate, as the nature of the aromatic head group was found to have a profound effect upon the ability of OdDHL-mimics of the type of 1 to inhibit pyocyanin production (with strongest inhibition associated with the meta-methoxy aromatic ring of 1). There exists a subtle interplay between the structural and electronic properties of the aromatic ring group governing compound activity, meaning that the impact of further substitution could not be reliably predicted. Furthermore, evidence suggests that the natural 3-oxo-dodecanoyl tail group of OdDHL is important for the inhibition of pyocyanin production by compounds which mimic the structure of AHLs. We therefore decided to retain the dicarbonyl unit and the nine-carbon alkyl chain, and attach the linker at the end of the alkyl chain.

2.2. Probe Synthesis

The synthesis of compound 2 (Scheme 1) began with the reaction of 2-(2-(2-chloroethoxy)ethoxy)-ethanol and sodium azide, which furnished azide 3 in quantitative yield. The desired nine-carbon alkyl chain was installed by reaction deprotonation of the hydroxyl group and treatment with 10-bromodecanoic acid.

Scheme 1. The synthesis of biotin-tagged affinity probe 2. rt = room temperature.

Scheme 1. The synthesis of biotin-tagged affinity probe 2. rt = room temperature.

The resulting acid 4 was converted to the corresponding acid chloride and reacted with Meldrum’s acid to generate adduct 5. Subsequent treatment of this crude material with methanol yielded β-ketoester 6. Acetal protection to form 7 was followed by reduction of the azide group to yield 8. Subsequent HATU-mediated coupling with D-biotin proceeded smoothly to generate the protected ester 9. Hydrolysis using lithium hydroxide provided the carboxylate 10. HATU-mediated coupling of 10 with m-anisidine furnished the protected precursor 11 in reasonable yield. Finally, acid-catalyzed deprotection provided the desired compound 2.

Typical affinity-based pull down assays involve the use of one-dimensional (1D) gel electrophoresis to separate proteins binding to the bioactive molecule under investigation. However, this method can suffer from a lack of sensitivity. We have previously proposed a strategy to address this issue based around combining biotin-mediated affinity capture from cell lysates with 2D difference gel electrophoresis (DIGE) [42]. This method of electrophoresis should allow a greater sensitivity compared to 1D techniques and thus facilitate the identification of weak binding-protein targets [42]. Conceivably, such an approach could be used with the affinity probe 2 in order to elucidate molecular targets for the anti-pyocyanin compound 1.

0 Thoughts to “2 Ethoxyethanol Synthesis Essay

Leave a comment

L'indirizzo email non verrà pubblicato. I campi obbligatori sono contrassegnati *