Patent Application
20130184436
Peptides are short polymers of amino acids containing peptide bonds, which link the carboxyl group of one amino acid to the amino group of a second amino acid. Peptides are distinguished from proteins on the basis of size, typically containing fewer than 100 monomer units (amino acid units). Peptides are involved in many processes in living organisms. Therefore, as small molecule drugs, antigens, hormones, ligands, vaccines, antibiotics, toxins, etc., peptides are playing more and more important roles in modern biology research. To meet the need of large quantities of peptides, the chemical synthesis of peptides has become an immeasurably valuable tool in the field of scientific research.
Early syntheses of peptides were performed in solution, requiring significant bench work. Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield (J. Am. Chem. Soc. 85 (14): 2149-2154, 1963), resulted in a paradigm shift within the peptide synthesis community. This is now the commonly accepted method for creating peptides in the lab in a synthetic manner. SPPS can be performed either manually or automatically. Manual synthesis often suffers from low efficiency, whereas automatic synthesis requires automatic synthesizers, which are typically very expensive.
Thus, there is a need for a method of high-efficiency peptide synthesis which is simpler, more flexible, and less expensive than using existing automatic synthesizers.
The invention is directed to an apparatus for semi-automated parallel synthesis of multiple macromolecules, wherein the apparatus comprises at least two liquid reservoirs each in liquid communication with at least one multi-channel liquid dispenser having an array of n nozzles, an array of n reaction containers containing solid substrates, wherein each nozzle is positioned above or adjacent a different reaction container, and at least one base chamber connected to the array of reaction containers for receiving waste from the n reaction containers, wherein n is an integer from two to about 100.
A method for semi-automated parallel solid phase peptide synthesis comprises adding amino acid reagents and non-amino acid reagents to a plurality of reaction containers, each containing a substrate for the synthesis, and removing non-amino reagents from the reaction containers, wherein the amino acid reagents are added manually and the non-amino acid reagents are added and removed using an automated apparatus.
In a method for solid-phase peptide synthesis which method comprises steps of providing an activated polymer resin to a reaction container; performing a first washing step comprising adding a washing liquid to the reaction container to swell the polymer resin and removing the washing liquid to yield a swelled, activated polymer resin; performing a deprotecting step comprising adding a deprotecting liquid to the reaction container, mixing the swelled, activated polymer resin with the deprotecting liquid, and removing the deprotecting liquid from the reaction container; performing a second washing step three times, wherein the second washing step comprises adding a washing liquid to the reaction container and removing the washing liquid from the reaction container; performing a coupling step comprising adding a protected amino acid in a liquid to the reaction container and allowing the amino acid to react; repeating the second washing step; repeating the deprotecting, second washing, coupling, and second washing steps, wherein each coupling step comprises adding a new amino acid to couple to the previously added amino acid; removing the resin from the reaction container; and cleaving the peptide from the resin;
the improvement comprising performing the first and second washing steps and the deprotecting step using an automated apparatus and performing the adding steps manually.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
In the drawings:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference.
The invention relates to a semi-automated method for the parallel synthesis of multiple peptides or other macromolecules and a semi-automated, high through-put peptide synthesizer apparatus.
As described in more detail below, the semi-automated method for parallel peptide synthesis according to the invention is based on conventional solid phase peptide synthesis (SPPS) techniques. As in conventional SPPS, the method involves first preparing an activated polymer resin which will act as the solid phase (solid support) on which the peptide synthesis will occur. Subsequently, amino acids are added sequentially to form the desired peptide in stepwise fashion. The first amino acid reacts directly with the activated polymer resin to form a linkage; subsequent amino acids couple to the previously added amino acid(s) to form a growing peptide. After each addition step, washing and deprotecting liquids are added and removed to take away excess (unreacted) amino acid and deprotect the appropriate functional groups, ensuring that the desired peptide bonds will be formed with each subsequently added amino acid. Rather than being a completely manual or a completely automated process, as are known in the art, the method according to the invention involves both manual and automated steps: the addition and removal of washing and deprotecting liquids are performed in an automated manner, whereas the additions of amino acids are performed manually.
The apparatus for peptide synthesis according to the invention provides for simple, efficient, low-cost, high through-put, semi-automated peptide synthesis, and may be used for building sequence defined peptide chains. It may also be utilized for performing other solid phase reactions known in the art or to be developed. It may be understood that “solid phase reactions” are those that are performed on a solid phase support, such as a polymer resin, typically for the step-by-step growth of a molecule. In addition to peptides, solid phase synthesis may also be used for synthesizing other macromolecules, such as DNA, RNA, and modified oligonucleotides, and in combinatorial chemistry. Accordingly, while the apparatus is described below with respect to the synthesis of peptides, it should be understood that its use is not limited thereto.
The apparatus comprises an array of reaction containers (which are designed to hold the solid supports or substrates for the peptide synthesis (activated polymer resins) and the growing peptides), at least one multi-channel liquid dispenser having an array of nozzles, each positioned above a separate reaction container in the array, two or more liquid reservoirs, each reservoir in liquid communication with the liquid dispenser(s), and at least one base chamber connected to the reaction containers for removing liquid therefrom. In one embodiment, the addition and removal of liquids is controlled by programmable electromagnetic valves. The number of reaction containers in the system may be as large as desired, and is only limited by the practicality of building, maintaining, and controlling the apparatus. Accordingly, the use of a multi-channel liquid dispenser allows for the synthesis of multiple (different) peptides simultaneously in parallel with simple manipulation, requiring minimal bench labor. This represents a significant improvement relative to the conventional manual synthesis which yields only a single peptide at one time.
An apparatus according to one embodiment of the invention is shown schematically in
For example, when used for peptide synthesis, liquid A may be a washing liquid, such as dichloromethane (DCM) or dimethylformamide (DMF). Other washing liquids which are known in the art or to be developed would also be appropriate for use as liquid A. Liquid B may be a deprotecting liquid, for example. Appropriate deprotecting liquids for peptide synthesis are well known in the art and need not be described further; the deprotecting liquid used for a particular synthesis may be determined on a case-by-case basis depending on the protection desired.
Each reservoir is connected to and in liquid communication with a multi-channel liquid dispenser 29, 31. As shown in
The multi-channel liquid dispenser(s) 29, 31 each comprises an array of n nozzles 21, with each nozzle positioned above or adjacent to a reaction container 23 in an array of n reaction containers. It is within the scope of the invention for the number of nozzles and reaction containers (n) to be as few as two to as many as about 100. Preferably, the number of reaction containers is about eight to about sixteen. The liquids in the reservoirs 1, 3 may be transferred automatically and in tandem to the multi-channel liquid dispensers 29, 31 and then to the reaction containers 23 such as by using a pump 5, 7 or a compressed gas such as air, nitrogen, argon, etc. (not shown) and controlled by electromagnetic valves 13, 15. Thus, by utilizing the apparatus, a liquid A or B may be simultaneously added to n reaction containers in an automated fashion.
The reaction containers are designed to contain solid substrates (supports), such as polymer resins, for the solid phase synthesis. The reaction containers have at least one opening for receiving and removing reactants and liquids. Preferably, each reaction container has different openings for receiving and removing reactants and liquids, e.g., at least one opening for receiving and at least one opening for removing. Liquids common to the reactions performed in the reaction containers, such as washing and deprotecting liquids, are introduced to the openings via the multi-channel liquid dispenser, whereas reagents, such as amino acids, are added manually via the same or different openings in the reaction containers. Preferably, reagents and liquids are removed automatically from the reaction containers via the base chamber, as described below. The reaction containers may be open at the top for easy addition or polymer resin and amino acids, but preferably have a top that maybe covered or closed to prevent contamination.
The n reaction containers 23 are also connected to one or more base chamber(s) 25 for receiving waste from the reaction containers. The removal of reaction mixtures or wash liquids from the reaction containers 23 through the base chamber(s) may be controlled by a vacuum pump 33, or by two vacuum pumps 27 and 33, as shown in
In one embodiment, as illustrated in
The number of reaction containers in the array of reaction containers and the number of nozzles in the array of nozzles is preferably the same, and may be about 2 to about 100, more preferably about eight to about sixteen. The apparatus in
A preferred reaction container is shown schematically in
It is within the scope of the invention to utilize a control (computer) interface to monitor and/or control the functions of the valves, pumps, and pressure regulators. The control interface may be programmed to realize automated or semi-automated parallel simultaneous synthesis of multiple peptides. In contrast with prior art apparatuses for peptide synthesis, the apparatus according to the invention is low cost and offers increased flexibility. The addition and removal of liquids are controlled by pumps and programmable electromagnetic valves, thus reducing the cost of the apparatus (commercial automated machines typically cost tens of thousands of dollars) and increasing the efficiency of the peptide synthesis relative to manual synthesis. Another advantage of the apparatus is the flexibility in scale, providing the ability to synthesize peptides on a small or large scale as desired.
The semi-automated method for parallel peptide synthesis according to the invention is based on conventional solid phase peptide synthesis techniques. However, rather than being a completely manual or a completely automated process, as are known in the art, the method involves both manual and automated steps. Use of the apparatus described above described above provides for an efficient, simple, semi-automated synthetic process.
The first step of the method involves providing activated polymer resin(s) to reaction container(s). The number of reaction containers may be two or more, depending on the number of peptides which are to be synthesized in parallel. Methods for preparing activated polymer resins and appropriate resins for SPPS are well known in the art and need not be described. The resins are provided manually to the reaction container(s) which are open at the top or have an easily removable cover for access. The amount of resin may be determined based on the desired scale of the reaction.
In a second step, washing liquid (solvent or solution), such as dimethylformamide (DMF), is added to the reaction container(s) to swell the resin; the DMF is then removed. This step, as well as all subsequent washing steps, is an automated step. Specifically, DMF is present in one reservoir of the apparatus of the invention and is added to the reaction container(s) via the multi-channel liquid dispenser. DMF is then removed from the reaction containers via the base chamber. The washing liquid is not limited to DMF and other washing liquids known in the art or to be developed, such as dichloromethane (DCM), would also be appropriate. The introduction and removal of the washing liquid to and from the reaction containers is controlled by valves and pumps, preferably using a computer or control interface.
The third step of the method involves adding a deprotecting liquid to the reaction container(s) containing the swelled activated resin(s). This step is automated and is performed using the apparatus of the invention by adding the deprotecting liquid from the second reservoir to the reaction containers via the multi-channel liquid dispenser. The reaction of resin with deprotecting liquid is allowed to proceed for at least about five minutes with mixing, such as with a magnetic stirring apparatus present in the reaction containers. The length of mixing will be understood by one skilled in the art to depend on the particular reaction to be carried out and the nature and volume of the reactants. The deprotecting liquid is then removed from the reaction containers via the base chamber using pumps and valves controlled by a computer interface. Appropriate deprotecting liquids are well known in the art and need not be described further.
Subsequently, a washing liquid, such as DMF, is added to the reaction container(s) to wash the resin and remove excess deprotecting liquid. The washing liquid is then removed. This automated step, as previously described, is preferably performed three times. The washing liquid is preferably the same liquid as used to swell the resin in a previous step.
In a fifth step, a first amino acid, preferably dissolved in a solvent such as DMF, is added manually to each reaction container, such as by syringe, pipette, etc. As well known in the art, the amino acid is protected to avoid the formation of random polymer. The amino acids added to each reaction container may be the same or different, depending on the peptides which are desired. Since the particular amino acid added may be different for each reaction container, this is a manual or non-automated step. In this first coupling step, the amino acid will react with the activated polymer resin to form a linkage. The reaction is allowed to proceed with mixing until complete and is preferably monitored to determine when it has been completed.
Subsequently, an automated washing step as previously described is performed (for example, three times) to remove excess unreacted amino acid from the reaction container(s).
The polymer resin is now coupled to a single amino acid. In order to form a dipeptide, the deprotecting, washing, addition/coupling, and washing steps are repeated so that a further newly added amino acid couples to the amino acid attached to the polymer resin. Subsequently, in order to increase the length of the dipeptide to produce the desired peptide, the deprotecting, washing, addition/coupling, and washing steps are repeated as many times as necessary. In each coupling step, a further newly added amino acid couples to the previously formed intermediated peptide attached to the polymer resin and forms a new peptide bond.
Finally, the resin is removed from the reaction container as previously described for cleavage of the peptide and the crude peptide is purified, such as with HPLC. The liquids remaining in the reaction containers are removed, such as via the base chamber of the apparatus.
The method steps described herein are well known in the art for the synthesis of peptides via SPPS. However, known methods for peptide synthesis using this protocol are either all performed manually or all using an automated device, and are both slow and time consuming or extremely expensive. The method according to the invention includes both manual and automated steps, thus providing a low cost method for preparing peptides.
The method for semi-automated parallel solid phase peptide synthesis according to the invention may also be understood to be a method which involves adding amino acid reagents and non-amino acid reagents to a reaction container containing a substrate for the synthesis and removing non-amino acid reagents from the reaction container. In the method, the amino acid reagents are added to the reaction container manually and the non-amino acid reagents are added to and removed from the reaction container using an automated apparatus, such as that described above according to an embodiment of the present apparatus invention.
As explained previously, the method according to the invention has been described in detail with respect to the synthesis of peptides. However, the invention is not limited to peptides, and may also be appropriate for the synthesis of other macromolecules such as DNA, RNA, and modified oligonucleotides, which are synthesized from nucleoside building blocks rather than from amino acids.
Various embodiments of the invention have now been described. It is to be noted, however, that this description of these specific embodiments is merely illustrative of the principles underlying the inventive concept. It is therefore contemplated that various modifications of the disclosed embodiments will, without departing from the spirit and scope of the invention, be apparent to persons skilled in the art.
The following specific example of the invention is further illustrative of the nature of the invention, it needs to be understood that the invention is not limited thereto.
The synthesis of twelve different peptides was performed using an apparatus according to the invention as shown in
Specifically, peptides with the sequences RAYSPSA, ERDYSPS, CNYYSNS, KIIPFNR, ERTYSPS, NETSYRH, DNYYSNS, ERAYSPS, AMTYPKE, ERTASPS, CKETTMK and ERDYSPS were synthesized simultaneously following the standard solid phase peptide synthesis (SPPS) procedure using the apparatus of the current invention as shown in
Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201220019160.7 | Jan 2012 | CN | national |
