SILICONE LIQUIDS AS TEMPERATURE REGULATING MEDIA FOR PROCESSES INVOLVING MICROWAVE FACILITATED TRANSFORMATIONS
FIELD OF THE INVENTION
The present invention relates to the use of low-viscous silicone liquids as temperature regulating (e.g. cooling) media in processes involving microwave facilitated transformations, in particular processes for microwave facilitated protein folding.
BACKGROUND OF THE INVENTION
Microwave facilitated transformations have been met with increasing interest within recent years. Microwave energy has traditionally been used for rapidly heating reaction mixtures, but has also recently been used to specifically induce transformations in proteins (see e.g. Bohr et al. in WO 96/30394).
Especially in the latter case where the "heating" effect of the microwave energy is unnecessary or even undesirable, it can be useful to apply microwave energy and simultaneously cool (or temperature control) the mixture comprising the compounds (e.g. proteins) intended to undergo a transformation. Also, it can be relevant to establish and maintain a temperature considerably higher than ambient temperature before application of microwave energy.
Previously, it has been necessary to use water cooling which then needs to be disengaged before application of microwave energy in that water otherwise would absorb the microwave energy intended for the sample (reaction mixture/protein solution). This necessitates the utilisation of cycles of independent microwave application and cooling (or heating).
Alternatively, it has been necessary to use air cooling which, however, is far less effective.
Thus, there is a desire for more suitable temperature regulating media for systems where microwave energy is to be applied.
DESCRIPTION OF THE INVENTION
In the work with microwave induced protein folding, the present inventor has found that low-viscous silicone liquids appear to be particularly interesting and useful as temperature regulating media in the instances where microwave energy is applied to mixtures comprising compound(s) (e.g. protein(s)) intended to undergo a transformation, where the transformation is intended to be induced by the application of energy to the collective twist modes of the compound rather than by the heating of the mixture.
Thus, the present invention provides the use of a low-viscous silicone liquid as a temperature regulating medium in a microwave facilitated process.
It should be understood that the "temperature regulating media" can also be used to keep the temperature at a predetermined high level. Preferably, the silicone liquid is used as a cooling medium.
The usefulness of the such silicone liquids is believed to be due to the "transparency" of the medium to electromagnetic radiation in the microwave frequency range, i.e. in the range of 0.1-100 GHz, in particular at the frequency of domesitic microwave ovens, i.e. at 2.45 GHz.
The term "silicone liquid" is intended to mean a compound/composition which is in the liquid form within the temperature range of from -10°C to 200°C and which essentially is consisting of oligomers or polymers comprising repeating units of the following formula:
~[-SiR1R2-O-]~
where R1and R2 independently are monovalent organic radicals. Particularly interesting silicone liquids are those where R1 and R2 independently are selected from C1-4-alkyl and phenyl, preferably methyl.
Typical oligomers and polymers are those of the following formula:
R3-O-[-SiR1R2-O-]n-R4
where R3 and R4 are as defined for R1 and R2 above and n is in the range of 2-100, preferably in the range of 2-50, in particular in the range of 2-25. Preferably R -R4 are all methyl.
The silicone liquids to be used within the present invention should preferably have a viscosity in the range of 0.5-10,000 cp, such as in the range of 1.0-1000 cp, preferably in the range of 1.0-500 cp, in particular in the range of 1.0-100 cp.
Suitable commercially available silicone fluids are such as those produced by Dow Corning, e.g. the Dow Coming 200® Fluid series. Examples hereof are Dow Corning 200® Fluid, 0.65 CST, Dow Corning 200® Fluid, 1.0 CST, Dow Corning 200® Fluid, 1.2 CST (1.2 cp), Dow Corning 200® Fluid, 5 CST, Dow Corning 200® Fluid, 20 CST, Dow Corning 200® Fluid, 350 CST, Dow Corning 200® Fluid, 500 CST, etc.
It is advantageous that the silicone liquid has a flash point of at least 40°C, such as at least 45°C, preferably at least 50°C, in particular at least 55°C. A relatively high flash point is desirable in order to avoid the risk of self-ignition.
The present invention also provides a process for facilitating the transformation of a compound (e.g. a protein) in a mixture by application of microwave energy, wherein the temperature of the mixture is regulated simultaneously by using a temperature regulating medium comprising a low-viscous silicone liquid. The cooling is advantageously effected by circulating the silicone oil through the microwave oven (or cavity) and a cooling bath. The cooling bath can then be set to the desired temperature. Thus, silicone oil is advantageously circulated through the cavity used for application of microwaves to the mixture and an external temperature regulated bulk, e.g. a water bath. The circulation may be effected subsequently or intermittently to the application of microwave to the mixture. In the main embodiment, the temperature is regulated simultaneously, thus, in these instances, the circulation is effected simultaneously to the application of microwaves to the sample.
Particularly interesting fields of utilisation of the present invention are for systems where a chemical transformation is to take place in a microwave radiation absorbing medium such as water, dimethylformamide, acetonitrile, DMSO, N-methylpyrrolidone, ethanol, methanol, acetone, tetrahydrofuran, dichloromethane, and chloroform. Examples of such
systems are industrial protein or enzyme manufacture, non-heat microwave facilitated organic reactions (transformation), interference with biological systems, kinetic evaluations of biological systems, microwave application to aqueous solutions in order to avoid protein fibriliation, etc.
In an alternative approach where the temperature effect of microwaves is desirable, the silicone oil is used as a cooling medium after application of microwaves to a sample (e.g. reaction mixture). In these instances, the silicon oil is utilised in order to rapidly cool the sample (e.g. reaction mixture) or the microwave oven (or cavity). Due to the transparency of the silicone oil (to microwaves), the silicone oil may be present in the microwave oven (or cavity) during application of the microwaves to the sample (e.g. reaction mixture). In this intriguing embodiment, the a sample holder (holder for a reaction container) may be cooled, e.g. in order to avoid side product formation in a reaction mixture. It should be noted that care should be taken when heating the reaction mixture above the flash point for the silicone oil.
Actually, the coolant can be used to cool all relevant microwave aided processes in which excessive heat is generated and/or in systems where heat is generated as a side effect of microwave induction.
It is known from the art that microwaves can enhance many chemical reactions often many orders of magnitudes (Tetrahedron letters 1999, 40, 9273-9276; J. Org. Chem. 1998, 4854-4856) and that different special devises have been produced in which such reactions can be performed. Examples of microwave rate enhanced reactions are: the Suzuki reaction, benzylation-, silylation-, nitrogroup reduction, Heck-, and Mitsunobo reactions. Special instruments can be produced in which microwaves can be applied directly to the reaction mixture while the instrument also can perform other manipulations such as pipetting of reagents, cooling etc. An example of such an instrument is the Smith Synthesiser from Personal Chemistry. The cooling liquid comprised in this patent is very suitable to cool devises used to carry out such chemical reactions. The reaction chambers are slots made in a reaction devise of glass, metal, polymer etc. that is placed on the instrument. In order to use the coolant optimally the reaction slots has to be in intimate contact with the coolant only separated by a thin wall of the solid material by which the devise is composed of. Optimally the devise is composed of a material with a high heat conductance so that heat evolved by the microwave irradiation in the reaction chamber of
the reaction mixture can be removed efficiently. Each reaction devise may be composed of one or several reaction slots, i.e. as a multiple reaction format (e.g. the ELISA format). It is important to note that the reaction scale is not restricted to high throughput small scale syntheses in that any reaction scale can be appropriate. In fact efficient cooling is often more important for large scale than for small scale.
In systems where microwaves are induced to species that interacts specifically with the electromagnetic radiation heat generation poses a special problem. In these systems heat is generated a "side product" and has to be removed before unspecific thermal effects are induced. Examples of such systems are disperse/colloidal systems where macroscopic species interacts with the microwaves. Also proteins interact by a non-thermal mechanism with microwaves (see Bohr et al. in WO 96/30394) and excessive heat is naturally deteriorating for such biological systems.
EXAMPLES
Example 1
The experiment was performed using a conventional microwave oven with a magnetron (2.24 GHz) operating at 600 W. Two samples each in a cylindrical glass container (one being distilled water (20 ml) and the other silicone oil (20 ml - Dow Corning 200 fluid, 1.2 cp)) were placed in the oven. After 10, 30, 60 and 90 seconds, the temperature of each sample was measured using a thermocoupled wire thermometer. The measurements are listed below in table I.
Table I
The increase in temperature for the water sample was as expected for application of 600 W in a conventional oven. The silicone oil, however, did not absorb any radiation but showed a minor temperature increase due to the heating of the surrounding glass.
Example 2
The silicone oil (Dow Corning 200 fluid, 1.2 cp) was used as a cooling liquid circulating in a sleeve (50 ml) around a sample container (10 ml). The cooling liquid was connected through plastic tubes with a water-bath regulated at 10 °C. The sample container was placed in a microwave oven (2.4 GHz - 10 W), and the temperature of the sample was measured with a thermocoupled wire thermometer. The temperature measurements are given in Table II below.
Table
*see Example 3.a.
The experiments only showed a marginal increase in temperature. These results demonstrate that silicone oils are effective as cooling liquid in typical microwave experiments where the thermal effects and the temperature can be controlled reasonably well.
Example 3
This example illustrates the applicability of a low-viscous silicone liquid as a temperature regulating medium for a microwave-facilitated protein refolding process.
a. Slow refolding of β-lactoglobulin
5 mg β-lactoglobulin protein was dissolved in a mixture of 3 ml 0.1 M KCI and 4 ml 4 M urea. The mixture was adjusted to approx. pH 2 with HCI.
The protein in this solution can be present in three states: a cold denatured state, a folded state and a hot denatured state depending on the temperature. Approx. 3 ml of the sample was injected into an inner tube of a sample-holder of a polarimeter apparatus. 365 nm wavelength light could pass through the inner tube and the polarisation of the light could be measured before and after passage of the protein matter of the inner tube. A liquid cooling system was established allowing a cooling medium to circulate through a temperature bath and through a sleeve outside of the inner tube. The cooling medium was a low-viscous silicone liquid (Dow Corning 200 fluid, 1.2 cp).
The experimental procedure started by measuring the polarisation of the cold denatured state at about 5°C after 3 hours of equilibration of the mixture. The temperature was subsequently stepped at increments of 2 K up to approx. 44°C. After each increment, the mixture was allowed to equilibrate the temperature for 5 min. This, however, did not equilibrate the folding state of the β-lactoglobulin sample as this equilibration is considered to be much slower. The polarisation was measure after each temperature equilibration. Due to the slow equilibration of the folding state of the β-lactoglobulin sample, heating and subsequent cooling revealed an effective hysteresis loop for the polarisation of the mixture.
b. Microwave facilitated folding/refolding of β-lactoglobulin
Microwave radiation was provided from a standard magnetron of a domestic microwave oven operating at 2.45 GHz with a total effect of 800 W. The microwave oven was remodelled so that the sample holder of the polarimeter could be inserted without disconnecting the cooling system. Several containers containing a total of 2 litres of water was placed in the oven in order to absorb some of the microwave effect released from the magnetron. The temperature of the mixture was measured before and after microwave irradiation. The microwave irradiation was applied for 5 sec. at 800 W causing the temperature of the mixture to increase by about 0.3 K. The power absorbed by the 3 ml
sample was estimated about 0.75 W. Immediately after microwave irradiation, the sample holder was reinserted in the polarimeter and the polarisation was re-measured.
The experiment from (a) was repeated starting with the denatured state of β-lactoglobulin in the KCI/urea mixture equilibrated at 4°C. First the temperature was incrementally increased to 48°C without the application of microwaves. Then the temperature was incrementally lowered to 4°C, however with application of microwaves at the 20°C stage. After re-equilibration at 4°C, the temperature was again incrementally increased to 48°C, however with application of microwaves at the 8°C stage. The polarisation was measured during the temperature cycles.
It appears from the example that the silicone fluid has turned out to be an efficient temperature regulating medium. In an alternative process, water was used as a temperature regulating medium, however, this required that the cooling medium (water) was removed from the sleeve (or the sleeve was separated from the inner tube) before application of microwave energy.