WO2005084773A1 - Crystallisation button and use method thereof - Google Patents

Abstract

The invention relates to a crystallisation button which has been specially designed for the crystallisation of small molecules, but which can also be used for the crystallisation of macromolecules. The inventive device can be used with any type of solvent, regardless of whether it is polar or apolar, and can be employed using techniques involving direct mixing, evaporation, a change in temperature or a combination of same. In addition, the device enables the crystallisation phenomenon and the phenomena of double refraction to be viewed using crossed polarisers and the solution to be accessed easily in order to collect the crystals.

Description

TITLE

GLASS BUTTON AND USE PROCEDURE

OBJECT OF THE INVENTION The object of the present invention is a crystallization button, specially designed for the crystallization of small molecules, but which can also be used for crystallization of macromolecules. The device can be used with any type of solvent, whether polar or non-polar and can be used by direct mixing, evaporation, or temperature change or combination techniques.

It also allows the visualization of the crystallization phenomenon and birefringence phenomena using cross polarizers and easy access to the solution to collect the crystals.

STATE OF THE TECHNIQUE

The crystallization of biological, inorganic and synthetic compounds is an important requirement for many lines of research in the chemical, pharmaceutical, biomedical and biotechnological industry. This crystallization is usually made from its supersaturated solutions. To achieve supersaturation, different mechanisms such as evaporation, temperature variation, solubility change, pressure change, etc. are used. Through these mechanisms, the supersaturation of the solution is increased until the precipitation of the solid phase occurs. However, the important thing to reduce the nucleation density of crystals and their size and quality is the speed at which the supersaturation is increased (JM García-Ruiz, Counterdiffusion methods for protein crystallisation. Methods in Enzimology 368 (2003) 130-154). This speed cannot be controlled with the existing crystallization devices on the market. Currently the crystallization of monocrystals of biological macromolecules is carried out in commercially available devices for this purpose, including: 1) Limbro and Limbro type boxes, which are plates of plastic material (polystyrene or polycarbonate) that have between 24, 96 or more wells of several milliliters of volume that is closed by means of a lid of the same plastic material. A solution of the compound to be crystallized or a reagent can be introduced into each well and each well can be individually covered by a glass cover (A. Ducruix and R. Giege, in "Crystallisation of Nucleic Acids and Proteins: A Practical Approach" ( A. Ducruix and R. Giegé, eds), p. 121, IRL Press at Oxford University, 1999)

2) Nextal type boxes that have 24 wells of several milliliters of volume that are closed by means of thread. The whole set is made of plastic.

3) Crystallization devices known as Granada Crystallization Box (patents ES-2 172 363 and ES-2 164 032 and JM García-Ruiz, LA González- Ramírez, JA Gavira and F. Otálora. Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques Acta Crystallographica D58 (2002) 1638-1642.

4) Dialysis buttons (A. McPherson, "Crystallisation of Biological Macromolecules" Cold Spring Harbor Laboratory Press, 1999).

The technical problem that the present invention intends to solve is that there are currently no specific commercial devices for the crystallization of small molecule compounds.

EXPLANATION OF THE INVENTION

The object of the present invention is a crystallization device (button) formed by the following elements: a) male body of cylindrical, prismatic or pyramidal geometry made of a chemically inert material to any solvent that can be used in crystallization of proteins with one end closed that has a flat surface of optical quality glass welded to the body of inert material and another open end provided with a threaded cap with a seal ("O-ring") b) female body made of cylindrical, prismatic or pyramidal geometry manufactured in a chemically inert material to any solvent that can be used in crystallization of proteins with an open end provided with a threaded cap that engages with the corresponding threaded end of the male body and another closed end that presents, welded to the body of inert material, a flat surface of optical quality glass on which at least d The valves or holes that allow the inside of the button to communicate with the atmosphere when the threaded male and female body form a tight assembly.

The male and female bodies are made of chemically inert materials to all types of solvents, both polar protic and water, methanol or acetic acid, or aprotic dipolars such as nitrobenzene or acetonitrile, or aprotic apolar such as hexane or benzene. In particular, the male and female bodies are made of Teflon or the male body can also be made of glass.

It is also an object of the present invention a method of using said crystallization button comprising the following steps: a) dissolution of the compound to crystallize in an appropriate solvent forming an approximately saturated solution. b) Placing a drop of the solution prepared in the previous stage on the flat surface of optical quality glass of the male body of the button. c) Close the male body with the female body of the button by threading. d) Opening of at least one of the valves or orifices located in the female body of the button to allow evaporation of the solution. e) Monitoring of the crystalline nucleation process by observing the microscope button. f) Close the valves or open holes in the previous stages when the crystalline nucleation begins to be observed. g) Once equilibrium has been reached and crystalline growth has stopped, button opening and monocrystals are collected.

When the temperature change technique is used, the method of using the crystallization button comprises the following steps: a) dissolution of the compound to be crystallized in an appropriate solvent forming an approximately saturated solution at room temperature T ₀ . b) Placing a drop of the solution prepared in the previous stage on the flat surface of optical quality glass of the male body of the button. c) Close the male body with the female body of the button by threading. d) closing of the valves located in the female body of the button to prevent evaporation of the solution. e) Placing the device at a temperature Ti lower than the environment for a time sufficient for the solution to reach said temperature Ti f) Monitoring the crystalline nucleation process by observing the microscope button. g) Once equilibrium has been reached and crystalline growth has stopped, button opening and monocrystals are collected.

In all cases, the solvents used can be of any type, both protic polar like water, methanol or acetic acid, or aprotic dipolars such as nitrobenzene or acetonitrile, or aprotic apolar such as hexane or benzene.

BRIEF DESCRIPTION OF THE FIGURE Figure 1: Representation of a crystallization button in which:

(1) corresponds to flat glass surfaces of optical quality in both the male body and the female body.

(2) corresponds to the valves or evaporation holes.

(3) corresponds to the male and female bodies made of a chemically inert material.

(4) corresponds to the seal placed on the male body.

(5) corresponds to the threads in both the male and female bodies.

DETAILED DESCRIPTION AND MODE OF EMBODIMENT OF THE INVENTION

The object of the present invention is a crystallization device (button) which allows the degree of supersaturation of a small volume solution to be controlled by active control of its temperature and evaporation thereof. The button is designed for the crystallization of small molecule compounds but which can also be used for crystallization of macromolecules. The concept is based on the following properties: 1. This device allows the use of any type of solvent, whether polar or apolar. 2. This device allows to use the direct mixing technique 3. This device allows to use the evaporation technique 4. This device allows to use the temperature change technique 5. This device allows to combine the three previous techniques 6. This device allows visualization of the crystallization phenomenon and birefringence phenomena using cross polarizers. 7. This device allows easy access to the solution to collect the crystals

The device (button) consists of two male and female bodies that are screwed together (see Figure 1). Each body has a flat surface of optical quality glass (1) that is welded to the body itself (3) made of Teflon or a material that is chemically inert to acidic or basic solvents, polar or non-polar, alcohols, ketones, dimethylsulfoxide, and in general any solvent that can be used in protein crystallization. The welding or joining between the Teflon piece and the optical quality glass should be such that the tightness of the body is ensured. The tightness of the two bodies once screwed is secured with a seal (4) (O-ring) placed in the male body. The female body has at least two valves or orifices (2) placed in the Teflon part that allow the interior of the device to communicate with the atmosphere in such a way that evaporation of the inner solvent can occur if desired. The male body can also be only made of glass as long as it can be attached to the female body ensuring the tightness of the device and has based on an optical quality surface to allow microscopic observation.

Examples of using the crystallization buttons are indicated below:

Example of use 1 for a small molecule inorganic compound: 1. A saturated aqueous solution of calcium sulfate is prepared at a temperature of 50 ° C by any of the usual procedures. 2. Take a few microliters of the previous solution with a micropipette, pipette or syringe or any suitable device for it. Between 10 nanoliters and 100 microliters of the saturated solution is poured into the male of the device. 3. The device is closed tightly with the female. 4. The device is transferred to the microscope or binocular magnifying glass at room temperature and it is observed that there has been no precipitation. 5. The valves located in the female are opened so that the saturated solution evaporates and becomes oversaturated. 6. When the microscope or binocular magnifying glass is observed that crystallization begins to occur, the valves are closed.

Example of use 2 for a small molecule organic compound: 1. An approximately saturated solution of ecteinascidin-743 is prepared at room temperature by dissolving 15 mg of ecteinascidin-743 in 1 milliliter of a mixture of isopropanol / water at a volume ratio of 1/19. 2. Take a few microliters of the previous solution with a micropipette, pipette or syringe or any suitable device for it. Between 10 nanoliters and 100 microliters of the saturated solution is poured into the male of the device. 3. The device is closed tightly with the female. 4. The device is transferred to the microscope or binocular magnifying glass at room temperature and it is observed that there has been no precipitation. 5. The valves located in the female are opened so that the saturated solution evaporates and becomes oversaturated. 6. The device is moved to a refrigerator or refrigerator at a temperature of 4 ° C. 7. Periodically observe if the solution has precipitated. 8. When the microscope or binocular magnifying glass is observed or when the crystallization begins to occur, the valves close.

Example of use 3 for a macromolecular compound: 1. An approximately saturated solution of chicken egg lysozyme is prepared at room temperature by dissolving 25 mg of lysozyme in 350 microliters of a sodium acetate buffer solution (pH = 4.7) at which add 350 microliters of a 10% weight / volume NaCI solution. 2. Take a few microliters of the previous solution with a micropipette, pipette or syringe or any suitable device for it. Between 10 nanoliters and 100 microliters of the saturated solution is poured into the male of the device. 3. The device is closed tightly with the female. 4. The device is transferred to the microscope or binocular magnifying glass at room temperature and it is observed that there has been no precipitation. 5. The valves located in the female are opened so that the saturated solution evaporates and becomes oversaturated. When the microscope or binocular magnifying glass is observed that crystallization begins to occur, the valves close.

Claims

1.- Crystallization button formed by the following elements: a) male body of cylindrical, prismatic or pyramidal geometry made of a chemically inert material to any solvent that can be used in protein crystallization with a closed end that has a flat glass surface of optical quality welded to the body of inert material and another open end provided with a threaded end with a seal ("O-ring"). b) female body of cylindrical, prismatic or pyramidal geometry made of a chemically inert material to any solvent that can be used in crystallization of proteins with an open end provided with a threaded cap that engages with the corresponding threaded end of the male body and another end closed that presents, welded to the body of inert material, a flat surface of optical quality glass on which at least two valves or holes are located that allow to communicate the inside of the button with the atmosphere when the threaded male and female body form a set watertight. 2. Crystallization button according to claim 1, characterized in that the male and female bodies are made of chemically inert materials to all types of protic polar solvents such as water, methanol or acetic acid, or aprotic dipolars such as nitrobenzene or acetonitrile, or aprotic apolar such as hexane or benzene. 3. Crystallization button according to claim 2, characterized in that the male and female bodies are made of Teflon. 4. Crystallization button according to claim 2, characterized in that the male body is made of glass. 5. Method of using a crystallization button according to claims 1-4 characterized in that it comprises the following steps: a) dissolution of the compound to be crystallized in an appropriate solvent forming an approximately saturated solution. b) placing a drop of the solution prepared in the previous stage on the flat surface of optical quality glass of the male body of the button. c) closing the male body with the female body of the button by threading. d) opening of at least one of the valves or holes located in the female body of the button to allow evaporation of the solution. e) monitoring of the crystalline nucleation process by observing the microscope button. f) closing of the valves or openings in the previous stages when the crystalline nucleation begins to be observed. g) once equilibrium has been reached and crystalline growth has been stopped, button opening and monocrystals collected. 6. Method of use according to claim 5, characterized in that the solvents used can be of any type, both protic polar like water, methanol or acetic acid, or aprotic dipolars such as nitrobenzene or acetonitrile, or aprotic apolar such as hexane or benzene. 7. Method of using a crystallization button according to claims 1-4 characterized in that it comprises the following steps: a) dissolution of the compound to be crystallized in an appropriate solvent forming an approximately saturated solution at room temperature T ₀ . b) Placing a drop of the solution prepared in the previous stage on the flat surface of optical quality glass of the male body of the button. c) closing the male body with the female body of the button by threading. d) closing of the valves or holes located in the female body of the button to prevent evaporation of the solution. e) placing the device at a temperature Ti lower than the environment for a time sufficient for the solution to reach said temperature Ti f) monitoring the crystalline nucleation process by observing the microscope button. g) once equilibrium has been reached and crystalline growth has been stopped, button opening and monocrystals collected. 8. Method of use according to claim 7, characterized in that the solvents used are both protic polar water, methanol or acetic acid, or aprotic dipolars such as nitrobenzene or acetonitrile, or aprotic apolar such as hexane or benzene.