Patent Application
20200181212
This application incorporates in its entirety the Sequence Listing entitled “FAM_N_PRV_SEQ_LISTING_2015_04_03_ST25.txt” (106,014 bytes), which was created on Apr. 3, 2015, and filed electronically herewith.
This invention relates to chemical and mechanical methods to modify natural and hybrid physiologically active peptides such as peptide toxins related to, or inspired from, the toxins found in venomous spiders, snails, mollusks and other animals.
Typically high heat and pressure, such as the conditions produced by autoclaves and used for sterilization, are used to neutralize and inactivate biological samples like fungi, bacteria and viruses. Often proteins are denatured or even destroyed by such a process. Usually when organisms are exposed to high temperatures and pressures, they fail to thrive or even survive because their proteins are denatured and consequently the organisms become inactive and die. The only biological process that follows is decay. Acidic conditions alone can sometimes produce a similar result. Expose most active peptides, like toxic proteins, to low pH or acid conditions and the peptide denatures and no longer functions like the native peptide or protein. Autoclaves are often used by medical offices to treat instruments, devices to make them safe and sterile for reuse and increasingly they are used to treat biologically contaminated waste to turn it into safe neutral harmless waste for disposal. Here we report the artificially induced conversion of certain toxic peptides to create both different forms of those peptides and new and useful derivatives of the original peptides that are both useful by themselves and useful as new compounds and new stable intermediates that useful to make other important compounds.
This invention has two parts. In Part 1 we describe a process of using artificially induced chemical and mechanical methods modify a peptide, including a toxic peptide comprising the following steps, optionally in the letter order: a) mix said peptide with water to make an aqueous solution or aqueous emulsion of said peptide in a liquid or semi-liquid form, wherein the aqueous solution or aqueous emulsion is comprised of at least 10% water; b) measure the pH of said peptide in the aqueous solution or aqueous emulsion; c) adjust the pH of said solution or emulsion to less than pH 7.0. The pH may be between about 1.0 and about 6.5, between about 2.0 and about 6.0, between about 2.5 and about 5.5, between about 3.0 and about 5.0, between about 3.0 and about 4.0, about 3.2, 3.4, 3.5, 3.6, or 3.8.
The process wherein after said pH adjustment the peptide is dried to a dry powder or granular form. The pH adjustment can be made using a strong or weak acid. Strong acid examples are any of the following acids—chloric acid (HClO3), hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid (HI), phosphoric acid (H3PO4), sulfuric acid (H2SO4). Perchloric acid (HClO4), and Nitric acid (HNO3). Weak acid examples are acetic acid and/or oxalic acid. During the pH adjustment, the aqueous solution or aqueous emulsion is exposed to a dry heat i.e. a temperature increase without steam or pressure or heat, pressure and steam. Heat and heat and pressure conditions described in the specification can also be used with any of the procedures including the dry powder procedures described herein.
The process of removing any one or more covalently bound 2H+O or molecules from a peptide while said peptide is in an aqueous solution or emulsion by the reduction of the pH of the solution or emulsion to less than 7.0. The peptides that work especially well with the process are the peptides described in the specification or in the sequence listing and particularly SEQ ID NO:119 and SEQ ID NO:121.
In addition to the process we describe insecticidal compositions of the peptides and formulations suitable for application to the locus of an insect to be treated with the peptide. In addition to the process and compositions we describe toxic peptides per se, with any one or more covalently bound 2H+O or molecules removed pH of the peptide in aqueous solution or emulsion is reduced to less than 7.0.
We describe a process of modifying a peptide, comprising the following steps: a) prepare said peptide as a pure Form 1 peptide, or peptide acid or composition containing less than about 10% water; b) place said Form 1 peptide in a controllable chamber or heating platform; c) heat said peptide to a desired temperature, with or without pressure, with or without steam; d) maintain the heated peptide at the desired temperature, pressure and steam until the desired amount of Form 1 peptide, called peptide acid, Converts to Form 2 peptide, called peptide lactone. The controllable chamber can maintain temperatures from 0 to 500° C. and pressures from atmospheric to 500 psi. The peptide can be heated to about the following temperatures; heated to at least about 10° C. but to no more than a maximum temperature selected from about 200° C., 300° C., or at most 400° C.
We describe a process where the peptide is: heated to at least from a temperature selected from about any of the following temperatures, temperature ranges or combinations of ranges of temperatures: 10° C. to 20° C.; 20° C. to 30° C.; 30° C. to 40° C.; 40° C. to 50° C.; 50° C. to 60° C.; 60° C. to 70° C.; 70° C. to 80° C.; 80° C. to 90° C.; 90° C. to 100° C.; 100° C. to 110° C., 110° C. to 120° C., 120° C. to 130° C., 130° C. to 140° C., 140° C. to 150° C., 150° C. to 160° C., 160° C. to 170° C., 170° C. to 180° C., 180° C. to 190° C., 190° C.-200° C., 200° C. to 210° C., 210° C. to 220° C., 220° C. to 230° C., 230° C. to 240° C., 240° C. to 250° C., 250° C. to 260° C., 260° C. to 270° C., 270° C. to 280° C., 280° C. to 290° C., 290° C. to 300° C., 300° C. to 400° C. and 400° C. to 500° C.
We describe a process where the peptide, or peptide acid is exposed to any of the following pressures or ranges of pressures: a) from about 10 psi to about 40 psi; b) from about 15 psi to about 35 psi; c) from about 18 psi to about 25 psi; d) about 21 psi. The chosen temperature and pressure range from the following periods depending on the temperature and pressure chosen: a) from about 5 minutes to about 40 minutes; b) from about 10 minutes to about 30 minutes; c) from about 15 minutes to about 25 minutes; d) about 21 minutes.
The following conditions may be used, the peptide should be maintained at the following temperatures and pressures and times: a) between from about 100° C. to about 140° C.; at a pressure of from about 10 psi to about 40 psi; for from about 5 minutes to about 40 minutes; b) between from about 110° C. to about 130° C.; at a pressure of from about 15 psi to about 35 psi; for from about 10 minutes to about 30 minutes; c) between from about 115° C. to about 125° C.; at a pressure of from about 18 psi to about 25 psi; for from about 15 minutes to about 25 minutes; d) of about 121° C., at a pressure of about 21 psi, for about 20 minutes. In cases the pressure is no greater than atmospheric pressure and the temperature is selected from the temperatures of at least 50° C. to 60° C. or greater. In some cases the following temperatures, temperature ranges or combinations of ranges of temperatures are used: 50° C. to 60° C.; 60° C. to 70° C.; 70° C. to 80° C.; 80° C. to 90° C.; 90° C. to 100° C.; 100° C. to 110° C., 110° C. to 120° C., 120° C. to 130° C., 130° C. to 140° C., 140° C. to 150° C., 150° C. to 160° C., 160° C. to 170° C., 170° C. to 180° C., 180° C. to 190° C., 190° C.-200° C., 200° C. to 210° C., 210° C. to 220° C., 220° C. to 230° C., 230° C. to 240° C., 240° C. to 250° C., 250° C. to 260° C., 260° C. to 270° C., 270° C. to 280° C., 280° C. to 290° C., 290° C. to 300° C., 300° C. to 400° C. and 400° C. to 500° C.
The process may use the following temperatures and times, where the peptide is a) heated and maintained at a temperature of more than about 100° C. for at least about 1 hr.; b) heated and maintained at a temperature of between about from 80° C. to about 120° C. for at least about 2 hr.; c) heated and maintained at a temperature of between about from 50° C. to about 80° C. for at least about 3 hr. Alternatively the peptide may be a) heated and maintained at a temperature of more than about 180° C., and a pressure of at least about 5 psi for at least about 5 minutes; b) heated and maintained at a temperature of more than about 100° C., and a pressure of at least about 10 psi for at least about 10 minutes; c) heated and maintained at a temperature of between about from 80° C. to about 120° C., and a pressure of at least about 10 psi, for at least about 30 minutes; or d) heated and maintained at a temperature of between about from 50° C. to about 80° C. for at least about 1 hr.
The peptide may be converted using the following conditions: a) heated and maintained at a temperature of between about 200° C. to about 300° C., and a pressure of between about 5 to about 10 psi for between about 5 to about 10 minutes; b) heated and maintained at a temperature of between about 150° C., and about 200° C., and a pressure of between about 10 to about 30 psi for between about 5 to about 30 minutes; c) heated and maintained at a temperature of between about from 80° C. to and about 150° C., and a pressure of between about 10 to about 20 psi for between about 20 to about 60 minutes; or d) heated and maintained at a temperature of between about from 50° C. to about 80° C. and a pressure of between about 10 to about 40 psi for between about 30 to about 60 minutes.
Alternative conditions are where the peptide is a) heated and maintained at a temperature of between about 110° C., and about 130° C., and a pressure of between about 10 to about 20 psi for between about 10 to about 20 minutes; orb) heated and maintained at a temperature of about 121° C., and a pressure about 21 psi for about 20 minutes.
In general we describe a process of removing any one or more covalently bound 2H+O, or H2O or molecules from a peptide by the heating of said peptide under any of the conditions, temperatures and pressures as described herein. A process of removing any one or more covalently bound 2H+O, or H2O or molecules from any peptide in the sequence listing by the heating of said peptide under any of the conditions, temperatures and pressures as described herein. We describe any peptide in the sequence listing after Conversion. We describe the peptides produced from any of the procedures described in the specification or claims. We describe insecticidal composition of the peptides produced by any of the processes of claims in a formulation suitable for application to the locus of an insect to be treated with the peptide. We describe a toxic peptide, and call it a peptide lactone when any one or more covalently bound 2H+O or molecules removed when the peptide is heated to any of the conditions, temperatures and pressures as described herein. We describe a toxic peptide described in any or produced by any of the procedures here where one or more covalently bound 2H+O or H2O molecules removed, and then it is called a peptide lactone, herein and in Part 2.
Especially suitable conditions for conversion are to heat the peptide and maintain it at a temperature of about 121° C., and a pressure about 21 psi for about 20 minutes.
In Part 2 of this application we describe how the peptide lactone can be converted into a peptide hydrazide and the peptide hydrazide converted into a peptide hydrazone. We describe the process of making, and the peptide hydrazide product made by the process of converting an insect predator peptide from the peptide lactone form to the peptide hydrazide form comprising mixing an insect predator peptide lactone with hydrazine and purifying to obtain the peptide hydrazide. We describe how a peptide lactone is prepared in water, hydrazine monohydrate is added and the mixture is stirred to form the peptide hydrazide which is optionally frozen, thawed and purified to obtain purified peptide hydrazide. If desired the insect predator peptide can vary in size from about 20 amino acids to about 50 amino acids and has 2, 3 or 4 cystine bonds, or alternatively it has 3 or 4 cystine bonds or 2 or 3 cystine bonds. The peptide lactone can be prepared from any peptide in the sequence listing and any peptide in the sequence listing or any peptide with more than 80% homology to any peptide in the sequence listing, or any sequence having more than 85%, 90%, 95% or 99% homology and 3 or 4 cystine bonds.
We have demonstrated how to use these methods with the peptide named the Hybrid +2 peptide wherein either method a or method b can be used, comprising: method a; a) start with a solution of 100 mg of purified Form 2 peptide, the Hybrid +2 peptide lactone, in 1 mL of water, b) treat the 1 mL of 100 mg peptide lactone with 100 uL of hydrazine monohydrate and stir at room temperature to form the peptide hydrazide, optionally for 2 hours, c) purify the solution of peptide hydrazide on a prep HPLC (eluted with a gradient of acetonitrile/water/trifluoroacetic acid), d) select appropriate fractions of peptide hydrazide, e) combine appropriate fractions of peptide hydrazide and concentrating under vacuum to reduce the volume, f) freeze the reduced volume of peptide hydrazide, at below zero temperature, optionally at −80° C., g) freeze-dry the Hybrid +2 peptide hydrazide, optionally on a lyopholizer, to obtain Hybrid +2 peptide hydrazide (I); or method b, wherein method b comprises: a) stir a solution of 25 mL of Super Liquid Concentrate, which is a mixture of Form 1, the peptide acid and Form 2, optionally at about 50° C. to 90° C., optionally at 75° C., b) let the solution cool, c) treat solution with hydrazine monohydrate, optionally 2 mL, and stir, optionally at room temperature for 2 hours; d) purify portions on a prep HPLC, optionally eluted with a gradient of (acetonitrile/water/trifluoroacetic acid) e) combine and concentrate fractions, reduce volume, optionally under vacuum, f) freeze remaining liquid, optionally freeze at −80° C. and lyopholize to produce Hybrid +2 peptide hydrazide.
We also show how to use the peptide hydrazide and react it with a carbonyl to make a useful peptide hydrazone. This is done by converting an insect predator peptide from the peptide hydrazide to the peptide hydrazone comprising, a) mix a solution of hydrazide in water and add hexanal in ethanol, stir, b) treat with a stock solution made of hexanal, acetic acid and ethanol, stir, c) add a stock solution made from hexanal, acetic acid and ethanol, d) mix, let stand and then optionally heat to produce the hydrazone. We used this process to make Hydrazone (II). This was done by a) mixing a solution hydrazide (I) in water with hexanal in ethanol, stir, b) add some stock solution of claim 16, d) mix and let stand then optionally heat to produce Hydrazone (II).
The hydrazone is both a key stable intermediate and can also be a final product. The product being a pegylated peptides or PEG peptide. The hydrazone can be other things as well but we believe that it is most useful when it is pegylated. We also show an alkylated hydrazone. The pegylated peptide actually takes the form of a hydrazone. See Example 9 and Hydrazone (III) and Example 11 and (IX). Compounds like this have never existed before and the chemistry to make them has never been taught before. These peptide hydrazones are novel, the pegylated peptide hydrazones like Hydrazone (IX) are novel in two aspects. First, the unsaturated carbonyl linkage shown in Examples 10(b) and 11(b) have never been used before to link a PEG with a peptide. Second, starting this reaction with the aldehyde or ketone on the “pegylation side” that is where the aldehyde or ketone is bound to PEG and then reacting that with the peptide hydrazide has never been shown before. Usually the aldehyde or ketone is put on the peptide and then the peptide ketone or peptide aldehyde is reacted or combined with the PEG. Using an unsaturated carbonyl in this reaction makes the bond more stable and harder to break because the imine nitrogen is less basic. So a comparison can be made of the carbonyl in Example 9 where PEG is joined to the peptide with a saturated carbonyl with Example 11 where PEG is joined to the peptide with an unsaturated carbonyl. The unsaturated carbonyl linkage of Example 11 is especially important because it forms a stronger bond making a more durable linkage between the peptide and PEG. This stronger bond is the result of the unsaturated carbonyl making the imine nitrogen less basic and not as readily protonated which is the first step in hydrolysis of the hydrazone linkage. These types of bonds have never been used before to link peptides and PEG or alkyl groups.
Pegylated peptides are well known but this method of making them, from a peglated hydrazone made from a peptide lactone that is converted to a hydrazide is novel and unknown until now. The pegylated toxic insecticidal peptides are extremely important because when these insecticides are delivered to the insect via ingestion of plants, oral bioavailability is critically important. In a way this is very similar to how important oral bioavailability is to for a drug taken by a human when taken by mouth. In both situations the factor that controls how well the medicine “works” is its oral bioavailability. Pegylation of proteins increases the size and molecular weight of molecules. Pegylation decreases cellular protein clearance by reducing elimination through the retiduloendothelial system or by specific cell-protein interactions. In addition, pegylation forms a protective ‘shell’ around the protein. This shell and its associated waters of hydration shield the protein from immunogenic recognition and increase resistance to degradation by proteolytic enzymes, such as trypsin, chymotrypsin and Streptomyces griseus protease. See, Pegylation A Novel Process of Modifying Pharmacokinetics. J. Milton Harris, Nancy E. Martin and Marlene Modi, in Clin Pharmacology 2001; 40(7): 539-551 at 543. Pegylation increases bioavailability by giving the peptide a greater half-life. For example, pegylation reduced the degradation of asparaginase by trypsin: after a 50 minute incubation period, there was 5, 25 and 98% residual activity of native asparaginase, PEG-asparaginase and branched-PEG-asparaginase, respectively. Id.
We show how to convert an insect predator peptide from the peptide lactone to the peptide hydrazide and finally to the peptide hydrazone, which is a pegylated peptide. We give an example of the Peptide Hydrazide (I) mixing with an Aldehyde (IV) to make Peptide Hydrazone (III), a pegylated protein. The process involves, acidifying complex glycols with a strong or weak acid, adding hydrazide and mixing well to make peptide hydrazone. The peptide hydrazone can be a pegylated peptide depending on the carbonyl used to make the hydrazone. We show how to make the peptide Hydrazone (III) by a) adding 1 drop of acetic acid to a stock solution of the mixture of compounds referred to as O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV) in ethanol, b) use the stock solution of O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV) (MW-2,000) treated with acetic acid from step a and add it to a solution of hydrazide (I) in water, c) mix and allow to stand at room temperature, d) add the remainder of the stock solution of O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV)(MW-2,000) in portions and allow the mixture to stand overnight after mixing to produce Peptide Hydrazone (III). We show how an insect predator peptide hydrazide can be converted to the peptide hydrazone comprising, adding an acrylic ketone to a hydrazide to make a hydrazone. The latter process is demonstrated with the process for making the peptide Hydrazone (VI) comprising, adding acrylic ketone (V) in ethanol to a solution of hydrazide (I) in water and mixing. We also make the peptide Hydrazone (IX) comprising adding PEG4 Ketone (VIII) to a solution of hydrazide (I) in water, and mixing to make Hydrazone (IX). The peptide hydrazone is thus shown to be a key intermediate needed to make the pegylated peptides according to our process.
We describe a process of preparing a peptide and or the peptide produced by the process and or an insecticidal composition produced by the process described as removing any one or more covalently bound 2H+O, or H2O or molecules from a peptide; including any toxic peptide with any one or more covalently bound 2H+O or molecules removed under any of the conditions, temperatures, pressures and pH or acidic conditions, either alone or in combination as described herein or found in the specification or claims including any of the peptides, hydrazides or hydrazones produced from any of the procedures described in the specification and claims or use of any of these peptides as insecticidal compositions of the peptides produced by any of the processes described in the specification and claims and then used in a formulation suitable for application to the locus of an insect.
The definitions should be read and understood in view of the application as a whole, its descriptions, examples and claims.
AI means active ingredient.
Autoclave means a device, with a pressure vessel that can be closed or locked and that allows for the addition of steam and or heated water, typically allowing for the removal of dry air with steam, sometimes with vacuum pumps, optionally allowing for steam pulsing or cycling in order to produce higher temperatures either with dry heat and/or with high pressure and optionally steam, if desired. It usually powered from an attached electric cord, a power cord, that carries current from a wall outlet to the device to power the heat and pressure made by the device, but it can refer to a simple pressure vessel that could be heated on a stove top.
Carbonyl means an aldehyde or ketone.
Chamber means an enclosed vessel or space.
Centigrade is a unit of temperature, usually as degree, it may be abbreviated C as in 40 C or ° C. as in 40° C.
Convert and Conversion means the transformation of a peptide from what is described as Form 1 to Form 2, using the methods described herein of heat, heat and steam and/or pressure or acid conditions either alone or in combination with other factors. Conversion is more fully described and exemplified herein.
DI means deionized water.
Form 1 or Form 1 peptide, refers to the form of a peptide, form suggesting the way it is folded or presents its active sites and its number or degree of internal bonding, and specifically Form I or Form 1 means a peptide as it exists when it is first formed and without the loss of 2H plus 0 or 18 daltons from its molecular weight. Form 1 is also known as the acid form of the peptide sometimes called here the peptide acid.
Form 2 or Form 2 peptide, refers to the form of a peptide, form suggesting the way it is folded or presents its active sites and its number or degree of internal bonding, and specifically Form II or Form 2 means a peptide that began as Form 1 peptide but was transformed through the application of any one of a combination of treatments described herein such as: heat, temperatures, pressure, steam, acid, low pH conditions resulting in the loss of a 18 daltons equivalent to a water molecule, when measured before and after it Converts from Form 1 to Form 2. When a peptide begins in one form and then looses 2H plus 0 or 18 daltons from its molecular weight it then exists as a Form 2 peptide. Form 2 is also known as the lactone form of the peptide or peptide lactone. See the first paragraph in Part 2 for the definition of lactone, as it is used in this document.
Formulation means a mixture of ingredients usually including the active ingredient, here typically a toxic peptide with other ingredients to increase the solubility, stability, spreadability, effectiveness, safety or other desired properties usually associated with storing or delivering the active ingredient.
Insect and Insect to be treated means an insect that a person having knowledge of the insect would like the insect controlled in some fashion such as limiting its food consumption, limiting its growth or shortening its life because it is perceived to consume or destroy food or materials or by its nature and presence it is undesirable.
Locus of an insect means the place where an insect normally lives, eats, sleeps or travels to or from.
Physiologically active peptide means a toxic peptide that is biologically active.
Pressure vessel means an enclosed container capable of holding a high pressure, with dry or wet pressured device that can, with the addition of water, produce heated steam and high temperatures. A pressure vessel needs to receive power from an external source, such as from a stove top heating ring, or as part of a autoclaved device.
Strong acid means an acid that ionizes completely in a solution of water. It has a low pH, usually between 1 and 3. Examples include: hydrochloric acid—HCl, hydrobromic acid—HBr, hydroiodic acid—HI, sulfuric acid—H2SO4, phosphoric acid (H3PO4), perchloric acid HClO4, nitric acid HNO3 and chloric acid HClO3.
Toxic peptide means a peptide, natural, artificial or synthetic, composed of amino acids, natural or artificial that produces harmful effect on insects when they are exposed to the peptides. Toxic peptides includes venomous peptides which are peptides from or related to venomous creatures like spiders, snakes, molluscs and snails. Toxic peptides includes the peptides identified and described in U.S. Pat. Nos. 8,217,003 and 8,501,684.
Water about 10% or a least about 10% or 10% or more or less means any formulation or mixture than has at least about 10% of its total weight or amount, available as water, that is water molecules not covalently bound as part of a larger molecule and capable of ionization of the H2O molecules, that is capable of maintaining a pH.
Weak acid means an acid that does not dissociate completely when in a water solution. They usually have a pH between 3 and 6. Examples include: acetic acid and oxalic acid. Weak acids exist in equilibrium between molecules that are ionized and those that are not.
General Descriptions and Procedures
Described herein are various treatments including heat alone, heat in combination with heated water, steam, heat and pressure and/or independently acid treatments that can modify a peptide . . .
These peptides undergo what is essentially a dehydration by rearrangement process. We call this transformation “Conversion.” Conversion happens when a normally toxic peptide is transformed into a much more active and more toxic peptide using elevated temperature, or heat, with or without steam and pressure, or acid, or heat with acid, or acid with heat plus steam and/or pressure or various combinations of temperature, heat, heat with pressure, heat with steam and pressure, acidity or low pH, acid or low pH with heat, acid or low pH with heat and pressure, acid or low pH with heat, steam and pressure. Conversion can be made to occur relatively quickly when heat is applied or if the peptides are in water, when low pH is applied to an aqueous solution of peptides. A temperature increase, that is heat, with or without an increase in pressure; with or without steam; or a decrease in pH, that is by applying an acid or acidic conditions to liquid formulation; or a combination of both temperature and acid results in a modification of certain toxin peptides that are described herein. Further observations, measurements and analysis of various embodiments related to this discovery are disclosed and claimed.
In some embodiments, peptides, toxic to insects, are treated with the following conditions: heat alone or heat in combination with steam and pressure, such as in a typical autoclave, operating at about 100° C. to 150° C. If steam and pressure are used with a pressure of about 100 kPa or 15 psi. for anywhere from 3, 5, 10, 20, 30, 40, 50, 60, 70, 75, 80 or 90 minutes depending on the variables of temperature, pressure and acidity then Conversion will result in a relatively short period of time. Suitable conditions for conversion are to heat the peptide and maintain it at a temperature of about 121° C., and a pressure about 21 psi for about 20 minutes. Some of the procedures described herein, in some embodiments, are similar to standard procedures used when autoclaving biological samples for reuse or safe disposal.
If lower temperatures and pressures than those described above are used, then Conversion takes longer than the times suggested above. The process can be used on dry powder or crystal forms of peptides or the peptides can be put into solution and then Converted. When peptides are put into aqueous solutions then pH becomes an important factor to monitor, adjust and control. In general, lower the pH solutions convert faster than higher pH solutions and Conversion just about stops above pH 7.0.
Typical autoclave operating conditions suitable for the methods described herein are: steam heated to about 120° C. to 135° C. for about 15 minutes, or about 10 to 20 minutes, at a pressure of about 100 kPa or 15 psi, or about 10 to 20 psi, will be enough to make the Conversion in a reasonable period of time. One skilled in the art will be able to change and vary the conditions to monitor and control the rates of Conversion, by using measurements and assays as described herein.
The method of modifying a peptide requires some heat over and above room temperature. Heat by itself or heat in the presence of steam and or heat in the presence of pressure can be used. The time it takes to convert depends on how much heat, and or steam and pressure and if relevant the acidity of the solution the peptides are in. Heat plus time is sufficient to make the make the changes or Conversion identified herein. How much time is required depends on how much heat is used and whether or not steam and pressure are used with the heat. Similarly, how much heat is required depends on how much time the peptides are heated and whether or not steam and pressure is used.
A few examples of possible heat options, with and without, steam; as well as various pressures that can be used to modify peptides are disclosed. One skilled in the art would be able to use these teachings and examples to determine many other possible temperatures, pressures, pH conditions and combinations thereof.
Examples of temperatures, times and pressures, with and without steam.
With steam: a) 110° C., 30 psi, 20 min.; b) 120° C., 15 psi, 15 min.; c) 130° C., 30 psi, 3 min., 8 min., 10 min. to 15 min. depending on container and whether covered or not.
Without steam (dry normal pressure): a) 120° C., 0 psi, 12 hrs; b) 130° C., 0 psi, 6 hrs.; c) 140° C., 0 psi, 3 hrs.; d) 150° C., 0 psi, 2.5 hrs.; e) 160° C., 0 psi, 2 hrs.; f) 170° C., 0 psi, 1 hr.
It should be noted and understood that even moderate increases in temperature can effectuate the desired changes in the peptide, provided enough time is given for the reaction to proceed. For example, room temperature is typically in the range of about 20 to 25° C. When the temperature of the preparations is raised to as little as 40° C., the reaction can take place in a number of hours or days; however, the reaction at 40° C., with no steam and no pressure will proceed very slowly and could take as long as 2 years to complete. The reaction at 100° C., with no steam and no pressure could take as long as 6 months to complete. But if the reaction is run at 120° C., 15 psi, Conversion could be completed in 15 minutes.
The examples of heat, time, steam, and pressure, provided above can be used with wet or dry preparations. Dry preparation activity is important because in the commercial preparations of the peptide toxin, a dry preparation is easy to measure, transport, sell and use. The method of exposing dry powder to steam heat is especially preferred because the steam heat can also be used to disable and deactivate most living materials such as yeast hybrids that may be undesirable left over contaminates from the manufacture of the toxic peptides.
Another independent factor, in addition to heat, steam and pressure that can be used to modify peptides is pH or acidity. Low pH, i.e. below 7, or acidity, can be used when the peptides are in solution and either at room temperature or in combination with the time, temperatures, pressure and steam factors discussed above.
Acidity and Acid conditions is believed to be an important factor that can influence the rate of Conversion. First it should be appreciated that the processes described above can take place when the peptides are in a dry form without water, but they can also be converted to their more active form when mixed with water, or when hydrated with sufficient water to form a measurable pH. Low pH or acid conditions, 7.0 or less has been found to be an independent factor that can be used to increase the rate and speed of Conversion. The optimal pH appears to be between about 1.5 and about 6, preferably between about 2 and about 5, more preferably between about 3 and about 4, more preferably about 3.5 but any acid conditions, 7.0 or lower, will increase the rate of reaction when the peptides are in solution. This is essentially an equilibrium reaction driven by pH. At a pH above 7.0 the reaction will be slow, the higher the pH the slower until it becomes so slow as to be essentially ineffective, when using aqueous reaction conditions. There will be some conversion at a pH slightly above pH 7.0 to about 7.5. At higher pH conditions the Conversion will be so slow as to effective and is generally considered to be of little commercial value.
In one embodiment the peptides are mixed with water, put in solution at a pH of 6.0 or less and Converted under steam and pressure at a temperature of between about 120° C. to about 150° C. for a rapid Conversion in less than about 10 minutes.
The Reaction. Without wishing to be bound by theory, and the procedures described do not require it, but to further advance the disclosure of the discovery, and to improve the teaching herein, we think the following reactions may take place during Conversion. When certain peptides are processed according the heat, pressure, steam, and acid regimes described herein they appear to lose the equivalent of a water molecule and so we sometimes call the process on of dehydration.
For purposes of illustration, we provide data for 2 sequences, SEQ ID NO:119 (also called Hybrid +2) and SEQ ID NO:121, both are provided in the examples and the sequence listing. These are two toxic peptides that differ only their N-terminal amino acids. SEQ ID NO:119 has an N-terminal GS. SEQ ID NO:121 does not have an N-terminal GS. SEQ ID NO:121 has 39 amino acids and they are the same 39 C-terminal amino acids found in SEQ ID NO:119. These toxic peptides are useful to demonstrate and explain Conversion.
We begin by explaining what Conversion is not. Conversion is not when a peptide with an N-terminal having an amino acid like glutamine, or Q, as in SEQ ID NO:121, spontaneously forms a cyclic compound like pyroglutamic acid. For example the N-terminal glutamic acid of SEQ ID NO:121 can form pyroglutamic acid. Here we call the spontaneous cyclization of either an N-terminal or internal amino acid having a free NH3 group, the “NH3 reaction.” The NH3 reaction is not Conversion and it is not comparable to Conversion. We call Conversion the “2H+O reaction” or “H2O reaction” or “dehydration reaction,” and it is completely different than the NH3 reaction. Both can occur with the same peptide as we prove in Example 5. The existence of two forms of a single peptide and the controlled ability to change one form into the other or at least the form having 2H+O into a form not having it is demonstrated with these two peptides and is characterized and explained below in the examples.
Optimal peptides for Conversion.
We believe many peptides are suitable for Conversion, including those described in detail below. Toxic insect peptides or insect predator peptides have 2, 3 or 4 cystine bonds, which means they have 4, 6, or 8 cysteines. They are peptides of greater than about 10 amino acid residues and less than about 300 amino acid residues. More preferably they range in amino acid or aa size from about 20 aa to about 50 amino acids. They range in molecular weight from about 550 Da to about 350,000 Da. They show surprising stability when exposed to high heat and low pH. Toxic insect peptides have some type of insecticidal activity. Typically they show activity when injected into insects but most do not have significant activity when applied to an insect topically. The insecticidal activity of toxic insect peptides is measured in a variety of ways. Common methods of measurement are widely known to those skilled in the art. Such methods include, but are not limited to determination of median response doses (e.g., LD50, PD50, LC50, ED50) by fitting of dose-response plots based on scoring various parameters such as: paralysis, mortality, failure to gain weight, etc. Measurements can be made for cohorts of insects exposed to various doses of the insecticidal formulation in question. Analysis of the data can be made by creating curves defined by probit analysis and/or the Hill Equation, etc. In such cases, doses would be administered by hypodermic injection, by hyperbaric infusion, by presentation of the insecticidal formulation as part of a sample of food or bait, etc.
Toxic insect peptides are defined here as all peptides shown to be insecticidal upon delivery to insects either by hypodermic injection, hyperbaric infusion, or upon per os delivery to an insect (i.e., by ingestion as part of a sample of food presented to the insect). This class of peptides thus comprises, but is not limited to, many peptides produced naturally as components of the venoms of spiders, mites, scorpions, snakes, snails, etc. This class also comprises, but is not limited to, various peptides produced by plants (e.g., various lectins, ribosome inactivating proteins, and cystine proteases), and various peptides produced by entomopathogenic microbes (e.g. the Cryl/delta endotoxin family of proteins produced by various Bacillus species.)
The following documents are incorporated by reference in the US in their entirely, in other jurisdictions where allowed and they are of common knowledge given their publication. In addition they are incorporated by reference and known specifically for their sequence listings to the extent they describe peptide sequences. See the following:
U.S. Pat. No. 7,354,993 B2, issued Apr. 8, 2008 specifically the peptide sequences listed in the sequence listing, and those numbered 1-39, and those named U-ACTX polypeptides, toxins that can form 2-4 intrachain disulphide bridges, and variants thereof, and the peptides appearing on columns 4-9 of the specification and in
Described and incorporated by reference to the peptides identified herein are homologous variants of sequences mentioned, have homology to such sequences or referred to herein which are also identified and claimed as suitable for Conversion according to the processes described herein including but not limited to all homologous sequences including homologous sequences having at least any of the following percent identities to any of the sequences disclosed her or to any sequence incorporated by reference: 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater identity to any and all sequences identified in the patents noted above, and to any other sequence identified herein, including each and every sequence in the sequence listing of this application. When the term homologous or homology is used herein with a number such as 30% or greater then what is meant is percent identity or percent similarity between the two peptides. When homologous or homology is used without a numeric percent then it refers to two peptide sequences that are closely related in the evolutionary or developmental aspect in that they share common physical and functional aspects like topical toxicity and similar size within 100% greater length or 50% shorter length or peptide.
Described and incorporated by reference to the peptides identified herein that are derived from any source mentioned in the US and EP patent documents referred to above, including but not limited to the following: Toxins isolated from plants and insects, especially toxins from spiders, scorpions and plants that prey on or defend themselves from insects, such as, funnel web spiders and especially Australian funnel web spiders, including toxins found in, isolated from or derived from the genus Atrax or Hadronyche, including the genus species, Hadronyche versuta, or the Blue Mountain funnel web spider, Atrax robustus, Atrax formidabilis, Atrax infensus including toxins known as “atracotoxins,” “co-atracotoxins,” “kappa” atracotoxins, “omega” atracotoxins also known as w-atracotoxin, U-ACTX polypetides, U-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, or mutants or variants, especially peptides of any of these types and especially those less than about 200 amino acids but greater than about 10 amino acids, and especially peptides less than about 150 amino acids but greater than about 20 amino acids, especially peptides less than about 100 amino acids but greater than about 25 amino acids, especially peptides less than about 65 amino acids but greater than about 25 amino acids, especially peptides less than about 55 amino acids but greater than about 25 amino acids, especially peptides of about 37 or 39 or about 36 to 42 amino acids, especially peptides with less than about 55 amino acids but greater than about 25 amino acids, especially peptides with less than about 45 amino acids but greater than about 35 amino acids, especially peptides with less than about 115 amino acids but greater than about 75 amino acids, especially peptides with less than about 105 amino acids but greater than about 85 amino acids, especially peptides with less than about 100 amino acids but greater than about 90 amino acids, including peptide toxins of any of the lengths mentioned here that can form 2, 3 and or 4 or more intrachain disulphide bridges, including toxins that disrupt calcium channel currents, including toxins that disrupt potassium channel currents, especially insect calcium channels or hybrids thereof, especially toxins or variants thereof of any of these types, and any combination of any of the types of toxins described herein that have topical insecticidal activity, can be Converted by the processes described herein.
It should be understood that the same or other peptides can be conjugated to the peptides described herein. The conversion from Form 1 to Form is an internal conversion, the N and C terminal peptides are not affected and thus the N and C terminal amino acids can have covalent binding partners, be they long or short. We describe in detail binding partners that at up to 1000 amino acids in size, in addition to 900, 800, 700, 600, 500, 400, 300, 200, 100, 50 or fewer amino acids peptide conjugates are described.
Venomous peptides from the Australian Funnel Web Spider, genus Atrax and Hadronyche are particularly suitable and work well when treated by the methods, procedures or processes described by this invention. These spider peptides, like many other toxic peptides, including especially toxic scorpion and toxic plant peptides, become topically active or toxic when treated by the processes described by this invention. Examples of suitable peptides tested and with data are provided herein. In addition to the organisms mentioned above, the following species may also carry toxins suitable for Conversion by the process of this invention. The following species are named: Agelenopsis aperta, Androctonus australis Hector, Antrax formidabillis, Antrax infensus, Atrax robustus, Bacillus thuringiensis, Bothus martensii Karsch, Bothus occitanus tunetanus, Buthacus arenicola, Buthotus judaicus, Buthus occitanus mardochei, Centruroides noxius, Centruroides suffusus, Hadronyche infensa, Hadronyche versuta, Hadronyche versutus, Hololena curta, Hottentotta judaica, Leiurus quinquestriatus, Leiurus quinquestriatus hebraeus, Leiurus quinquestriatus, Oldenlandia affinis, Scorpio maurus palmatus, Tityus serrulatus, Tityus zulianu. Any peptidic toxins from any of the genus listed above could be considered for Conversion according to the process in this invention.
The Examples in this specification are not intended to, and should not be used to limit the invention, they are provided only to illustrate the invention.
As noted above, many peptides are suitable candidates for conversion. The sequences noted above, below and in the sequence listing are especially suitable peptides that can be converted. Some of these peptides have been converted according to the procedures described herein as is described in the examples below.
SEQ ID NO:60 is named “ω-ACTX-Hv1a” it has disulfide bridges at positions: 4-18, 11-22 and 17-36. The molecular weight is 4096.
SEQ ID NO:117 is named “ω-ACTX-Hv1a+2” it has disulfide bridges at positions: 6-20, 13-24 and 19-38. The molecular weight is 4199.
SEQ ID NO:118 is named “ω-ACTX-Hv1c” it has disulfide bridges at positions: 5-19, 12-24, 15-16, 18-34. The molecular weight is 3912.15.
SEQ ID NO:119 is named “rU-ACTX-Hv1a (“Hybrid”)+2” it has disulfide bridges at positions: 5-20, 12-25, 19-39. The molecular weight is 4570.51.
The examples below are intended to illustrate and provide further written description and support to this disclosure. They are not intended to limit the disclosure or the claims.
SEQ ID NO:119 is GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA.
SEQ ID NO:119 has 41 amino acids. When properly folded, it has 3 disulfide bonds. It has the elemental composition of C185H276N56O68S6. SEQ ID NO:119 may be called the “+2 hybrid,” “Hybrid +2,” or the “plus 2 hybrid.”
SEQ ID NO:121 is QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA. SEQ ID NO:121 has 39 amino acids and they are the same as the 39 “C” terminal amino acids in SEQ ID NO:119. SEQ ID NO:121 may be called, “native” or “native hybrid” OR “native hybrid peptide.”
The N-terminal amino acid of SEQ ID NO:119 is “G,” glycine or Gly. The 2 N-terminal amino acids in SEQ ID NO:119 are “GS” these amino acids are not part of the N-terminal of SEQ ID NO:121. The N-terminal of SEQ ID NO:121 is “Q” or glutamine.
It is possible for a sequence ending in glutamine, Q or Gln, like SEQ ID NO:121 to spontaneously undergo cyclization, from glutamine to pyroglutamic acid. The reaction can be quick, can be spontaneous and does not require the addition of heat or acid. This amino group, sometimes N-terminal cyclization, is known to occur in peptides and it results in the peptide losing 17 mass units, atomic units or daltons, correlating with the 17 daltons of NH3 lost upon the cyclization. We refer to this reaction as the “NH3 reaction,” it is not what we call Conversion. We explain this reaction in more detail in Example 5, below.
We think a very different reaction occurs when heat is applied to a toxic peptide like SEQ ID NO:121, and we refer to this reaction as Conversion or the “2H+0 reaction.” Conversion results in a modificatio in the peptide which is an altogether different reaction, with the peptide having different properties as compared to what happens to a peptide that experiences the NH3 reaction.
When a toxic peptide is exposed to any of the Conversion conditions we describe herein, i.e. heat, pressure, steam, acid conditions for aqueous solutions, then we believe the “2H+O reaction” results.
The Conversion, or 2H+O reaction, results in a compound having one less water molecule than before the reaction starts. Below, we provide data showing that the peptide that results from Conversion results in a peptide with one less H2O, or 18 dalton and is essentially dehydrated but is also much more robust and toxic a peptide, than the peptide was before it was Converted. That is the form of the peptide changes, such that the original form, herein called form 1 or Peak 1, has 18 more daltons as compared to the Converted Form 2 peptide.
Mass spec. evidence that shows the 2H+O reaction is not the same as the NH3 reaction, rather, the 2H+O reaction results in the loss of 18 Daltons correlating with 18 Daltons of H2O, rather than 17 Daltons of NH3. We show the daltons of H2O lost in the 2H+O reaction in Example 1; and the 17 Daltons of NH3 lost is provided in Example 5.
Mass Spectrograph Peak 1/Form 1 and Peak 2/Form 2. In
When the two mass values from
The peptides and their forms, indicated by Peaks 1 and 2, were isolated and compared. The examples below provide comparisons of the activity of the original form, called any of the following: Peak 1, Form 1, native, acid form, peptide acid, original, preConverted, unconverted, or not Converted form of the peptide. Form 1 is the form or acid form that heated or acidified in order to turn it into Form 2 or the lactone form or peptide lactone as lactone is defined in Part 2. In some of these examples the heat treatment is an autoclave treatment, at about 121° C. for 20 minutes at 21 psi., or if the peptide is in liquid form it means lowering the pH to under 7.0 in order to Convert the peptide to what is called any of the following: Peak 2, Form 2, the lactone form, the peptide lactone, (as lactone is defined in Part 2.) the dehydrated form of the peptide, or the Converted form of the peptide.
Diet Incorporation Study. The graph in
Four days after the insects were fed, the difference in the percent mortality between the traditional 618 (before Conversion) and the autoclaved 618 (after Conversion) becomes pronounced. The autoclave treatment lead to a quicker speedier death following the caterpillars eating treated food. The number of insects dying that were treated with Form 2, is about 95%, compared to the 618 dry powder Form 1, native peptide or peptide not Converted, Form 1, is about 22%. Autoclaving the normal peptide did not deactivate the hybrid protein as expected.
Methods: Insects: SCR are purchased from Crop Characteristics (Farmington, Minn.). Insects were received as “ready to hatch” on filter paper. The insects were hatched at room temperature (26 C) and left in the plastic bag they were shipped in. The insects were hatched after 1-2 days and were used the day of hatch for the assay.
Media: SCR larval diet was purchased from Bioserve (Product # F9800B, Frenchtown, N.J.). To make 100 mL of diet, 100 mL of DI water is boiled with 1.44 g of the provided agar. Solution is boiled until the agar is fully dissolved. Then 13.28 g of diet and 460 ul of KOH are added and media is mixed on warm stir plate until homogeneous. Media is then aliquoted into 20 mL portions and cooled to 65 C in a water bath.
Treatments: The 618 treatments were prepared using the calculation of 25% AI. A 10 ppt solution was made (10 mg/mL) by mixing 260 mg of powder with 6.5 mL of water. The solution is mixed thoroughly and sonicated if necessary to dissolve all the powder completely. 200 mg of 618 powder was put in a glass jar with a screw on top. The powder was then autoclaved on the 20 minute Dry cycle with the cap loosened. After the autoclave cycle, the powder had absorbed some liquid. 5 ml of water was then added to the powder and mixed well to dissolve. 5 mL of either water or treatment is then added to the 20 mL of 65 C food and mixed well and 1 mL of DI is then transferred to each well of the bug condos (Bioserve Product # BAW128) using a repeat pipetter and allowed to cool.
Insects are then applied once media has cooled and set (20 min), one per well, using a paint brush to transfer SCR. Wells are then sealed with perforated lids (Bioserve Product # BACV16) and left on the light cart in the insect lab.
Bioassay comparison. Results of a bioassay comparison are shown in
The results of the Bioassay comparisons as lethal dose 50 are provided in Table 1, below.
Stability pH Study. Example 4 shows the effect of heat and/or acid on conversion trends for both Form 1 and Form 2. This was both a stability and a pH study. It compares pre Conversion or Form 1, to post Conversion or Form 2 peptides. The study used the peptide of SEQ ID NO:119 and shows that, in addition to heat, a decrease in pH, that is the lowering of the pH of a solution of peptide, with acid or any means to lower the pH to make it 7.0 or below, will result in increased Conversion of the peptide from Form 1 to Form 2.
The Stability pH Study results are shown in
Observation: Slight decrease in peak 2 height in both pH solutions.
Observation. Slight decrease in peak 2 height in both pH solutions
Observation. Larger decrease in peak 2 height in higher pH solution
Observation. Decrease in peak 1 height in lower pH solution. Decrease in peak 2 in higher pH solution.
Observation. Slight decrease in peak 2 height in higher pH solutions
Observation. Decrease in peak 1 height in pH 3.9 solution. Decrease in peak 2 height in pH 7.5 solution. Loss of peak 2 in pH 9.4 solution.
The results of the study undertaken in Example 4 demonstrates the rate of conversion from Peak 1 (i.e., the pre Conversion peptide Form 1) to Peak 2 (i.e., the Converted peptide Form 2), after they are taken up in aqueous solution and adjusted to different pH or acidity levels. Here, the results demonstrate that conversion from Form 1 to Form 2 requires the application of heat and/or acidity to influence conversion rations in a sample of Super Liquid Concentrate (i.e., a solution comprising a mixture of Form 1, the peptide acid, and Form 2, the peptide lactone). Thus, in addition to heat, the conversion ratios observed in Tables 2-7 illustrate that conversion from the peptide form (i.e., Form 1 or Peak 1) to the peptide lactone form (i.e., Form 2 or Peak 2) requires a pH of 7.0 or less (e.g., a pH of 5.0, 4.5, 4.0, 3.5, 3.0. 2.5, 2.0 or less), with the most drastic conversion occurring at pH values ranging between 3 and 4.
Additionally, Example 4 shows that increasing the temperature and/or the length of time at a higher temperature will support a conversion trend that favors Form 2. Accordingly, this phenomenon can be observed by examining the ratio of Form 1, which is seen to shift from 1.8 to 1.2 to 1.05, as temperature, and time-at-temperature increases; this conversion trend clearly shows that conversion of Form 1 and temperature are inversely proportional. Indeed, as shown in Table 5, when the temperature is increased to 40° C., the ratio of Form 1 to Form 2 at pH 3.9 flips, resulting in a ratio of 0.6. In summary, Example 4 demonstrates that treating a mixture of the two forms to a higher temperature, and a lower pH, would yield a greater rate of conversion.
Non Converting isoforms. We have shown that SEQ ID NO:119 can form an isoform with the loss of 18 Daltons in M.W. at higher temperature. In Example 2 we showed Conversion does not affect insecticidal potency when the original form of SEQ ID NO:119, Form 1, as a powder, was autoclaved to make it Convert to Form 2 and then it was tested by adding to the diet of the Southern Corn Rootworm, larva set. However, this transformation in SEQ ID NO:119, a hybrid peptide, has not been noticed in a peptide like SEQ ID NO:121, a native peptide. In contrast to SEQ ID NO:119, in SEQ ID NO:121 there is a an N-terminal Gln, which may cyclized itself to N-Pyr with loss of a NH3, i.e. loss of 17 daltons in M.W. These two chemical modifications, loss of H2O and loss of NH3, are difficult to differentiate because the loss of M.W. in these two processes is so close. We used analytical HPLC and sensitive TOF LC/MS methods to evaluate whether both of these chemical modifications can happen to a sequence like SEQ ID NO:121, which we also call the native hybrid peptide. The data below shows the Conversion can be induced in a native hybrid peptide when it is subjected to appropriate conditions as we describe herein for this process.
Materials and Methods. SEQ ID NO:121 was made from Hybrid-ACTX-Hvla K. lactis strain, pLB12D-YCT-24-1. Agilent HPLC system with Onyx 100 monolithic C18 HPLC column was used to analyze the SEQ ID NO:121 peptide production and isoform formation.
The LC-MS system is located at Launch MI Lab in SMIC, and consists of a Waters/Micromass quadrupole time-of-flight (Q-Tof Premier) mass spectrometer on-line with a Waters NanoAcquity UPLC system. Sample was diluted 1:50 in 0.1% formic acid in water.
Method A.
Five μL of sample was injected onto a Waters BEH130 C-18 Symmetry column (0.3 mm ID×15 cm) at a flow rate of 5 uL/min. Reverse-phase separation was achieved using a linear gradient from 0.1% mobile phase B (water with 0.1% formic acid) to 40% mobile phase B (100% acetonitrile with 0.1% formic acid) over 25 minutes, 85% B at 25.5 minutes, 85% B at 27.5 minutes, and 0.1% B at 28 minutes.
Method B.
Ten to 30 uL μL of sample was injected onto a Waters C-18 X-Bridge Column (4.6 mm ID×50 mm) at a flow rate of 1 mL/min. Reverse-phase separation was achieved over 15 minutes using a linear gradient of 99% mobile phase A (water with 0.1% formic acid) to 95% mobile phase B (100% acetonitrile with 0.1% formic acid) over 6 minutes, 95% B at 11 minutes, and 1% B at 11.2 minutes for a total run time of 18 minutes.
Column effluent was sampled by the mass spectrometer via an electrospray ionization source. Waters Masslynx 4.1 software was used for instrument control and MS and MS/MS data acquisition. Within Masslynx the MaxEnt 3 algorithm was used for deconvolution of multiply charged ions to a calculated monoisotopic M+H mass value.
Method C.
The LC-MS system consisted of a Waters/Micromass ZQ spectrometer with an electrospray ionization source. The sample was injected onto a Zorbax SB-C18 column (2.1×30 mm) at a flow rate of 1 mL/min. Reverse-phase separation was achieved over 3.1 minutes using a linear gradient of 96% mobile phase A (water with 0.1% formic acid) to 98% mobile phase B (100% acetonitrile with 0.07% formic acid) using a diode array detector (210 to 300 nm).
Results and Discussion.
Production of SEQ ID NO:121, aka native hybrid peptide, production strain, pLB24-YCT-24-1, was cultured in Defined Medium with 2% sorbitol as carbon source at 23.5 C for 6 days. The OD600 reached 30 at the time when the condition medium was collected after removal of cells. 300 μL of the conditioned medium was injected into Agilent HPLC analytic system and a yield of native hybrid peptide was determined as 164 mg/L.
Agilent HPLC evaluation of native hybrid isoforms. The collected native hybrid conditioned medium was treated at 4 C, room temperature (˜23 C) and 50 C for 24 hours before analysis by Agilent analytic HPLC with loading of 300 μL of each. The HPLC chromatographs of native hybrid peptide samples treated at different temperature are shown in
Peak 1, indicating Form 1, was the most abundant isoform initially, but Peak 1/Form 1 can transformed into isoforms Peak 2 and Peak 3 with time and higher temperature. We demonstrate that a 50° C. treatment for 24 hr. will almost make Peak 1 disappear (to only 5.6%). Conversely, Peak 2 and Peak 3 isoforms increase with temperature and increase faster with higher temperature.
One isoform detected by TOF MS was the one with M.W. of 4417.6826, which represents the “native” native hybrid peptide, i.e. unmodified native hybrid, it is labeled as Peak 1 in
A second isoform detected had a M.W of 4399.6455. This isoform has 18 dalton loss in M.W. from the “native” isoform, indicating loss of a water molecule. This isoform, with a loss of H2O, is not labeled in
A third isoform detected had a M.W. of 4400.6660. This isoform had 17 dalton loss in M.W. from the “native” isoform and likely a loss of NH3. This isoform with a loss of NH3 is labeled as Peak 2 in
A fourth isoform is the combination of loss of both a H2O and a NH3 molecule, resulting in an isoform with M.W. of 4382.6313. The isoform with a loss of both H2O and NH3 is labeled as Peak 3 in
These results show there are at least two chemical modifications possible in the native hybrid peptide molecule, both N-terminal glutamine cyclization to pyroglutamic acid, and a dehydration reaction. From the TOF MS based peak intensity chromatograph, the isoform with only a H2O loss was barely detectable. This is consistent with the HPLC evaluation in which only 3 peaks have been detected. Loss of a H2O molecule can make the peptide more hydrophobic and further loss of a NH3 can make the peptide even more hydrophobic. We can predict that loss of H2O will shift the native hybrid peak to a later retention time in HPLC chromatograph. Loss of both H2O and NH3 will further shift the peak to an even later retention time.
In Part 1 we describe how it is possible to artificially manipulate a toxic peptide with mechanical or chemical means such as temperature, pressure, strong and/or weak acids, in order to transform a peptide from its native state or what we call Form 1 into the useful state we call Form 2. The Form 2 composition may be referred to herein as the “carbonyl”, “activated carbonyl”, “lactone”, “lactone like”, and/or “lactone like form.” In this document we usually refer to this Form 2 composition simply as a lactone or peptide lactone. The structure of these compounds has no dictionary definition in this document, here they are defined by the characteristics we describe here. Here a “lactone” has the properties we attribute to the Form 2 compound. We use the word “lactone” and peptide “lactone” because these compounds react like a lactone. We describe how to make them, how to identify them, how to isolate them and how to use them. We provide data to show these peptide lactones are more biologically active than the native peptides and that they are very useful and versatile. They are stable intermediates that can be used to make other valuable compounds. In Part 2 we show how the peptide lactone can be made into two different and stable active compounds, useful as stable intermediates to make a variety of other compounds.
In Part 2 we describe peptide hydrazides, peptide hydrazones and we teach how to make and use them. A hydrazide or peptide hydrazide results from the reaction of the Part I peptide lactone with hydrazine. The other stable intermediate compound we describe we call a hydrazone or peptide hydrazone. A peptide hydrazone results from the reaction of a peptide hydrazide with a carbonyl compound. What is especially useful about peptide hydrazones is that they can be covalently bonded with other useful moieties such as alkyl chains and or pegylated products and then used for a variety of purposes, some of which we describe here. The ability to create an alkylated protein, in the manner we describe, is very useful. The ability to easily produce a pegylated protein, in the manner we describe is, perhaps, even more useful. Pegylated proteins have been used to reduce the immunogenicity of proteins, to decrease the metabolism of proteins and to increase the bioavailability of proteins. We believe our techniques, disclosed here for the first time, can be used to create pegylated proteins with exceptional value. These techniques can be used to make alkylated and pegylated proteins, and other types of proteins, more easily, quicker and at a lower cost than previously possible. One protein enhanced by pegylation is insulin.
We are able to demonstrate that the peptide lactone, the peptide hydrazide, and the peptide hydrazone, these can be either “peptide intermediates,” novel, chemically stable, chemically useful compounds used to react with other compounds, like PEG4Ketone (VIII) in Example 11. The peptide lactone and peptide hydrazide provide a single discrete site on these peptides or peptide acids where functional groups are added. The peptide and toxic peptide products and intermediates provide a single discrete chemical handle with unique chemistry synthetic or biological molecules more useful and functional. For example, this chemistry allows one to mono functionalize with a pegylation chain at a single site of the polypeptide. Another example is that it could allow one to mono attach molecules at one discrete site on the peptide or peptide acid such as a periodated digested glycosylated peptide or other carbohydrate. These peptide intermediates can be used to produce a wide range of products. We show that these toxic peptide intermediates are useful and provide more reaction options than the typical toxic peptide. We understand that pegylated toxic petides are even more active than unpegylated peptides.
PEGylation or pegylation is the linking of a peptide to polyethylene glycol and/or polypropropylene glycol or (PEG). Once linked to a peptide, each PEG subunit becomes tightly associated with two or three water molecules, which have the dual function of rendering the peptide more soluble in water and making its molecular structure larger. In a first generation protein pegylation the PEG attaches to one or more of several potential sites on the protein, such as to lysine and N-terminal amines. A problem with this approach is that a population of modified peptides can contain a mixture of molecules with PEG attached to different lysines, as well as molecules with different numbers of linked PEGs. This variability in modification diminishes the purity of the finished product and impedes reproducibility.
There have basically been two other more modern approaches used to add PEG to proteins in a more controlled manner; either A) alter the PEG to make it more reactive or B) alter the protein to provide special sites for PEG attachment.
A PEG method type A), alter the PEG, is described in U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979, incorporated herein by reference, specifically as to its descriptions of polymers suitable for pegylation. This patent describes modifying the polymer at one end either by the alteration of the terminal group or by the addition of a coupling group having activity to the peptide and reacting the activated polymer with the peptide. This method was used to pegylate insulin and other hormones. See U.S. Pat. No. 4,179,337.
A PEG method of type B), modify the peptide rather than the PEG, is to add a cysteine where desired to generate site-specific PEGylation at places chosen to minimize interference with the peptide's biological function, while decreasing the peptide's immunogenicity. PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, and PEG orthopyridyl disulfide are thiol reactive PEGs that have been created to PEGylate free cysteine residues. This approach has been used in a number of ways including making monoPEGylated human growth hormone analog. See Peptide PEGylation: The Next Generation, by Baosheng Liu, Pharmaceutical Technology, Volume 35.
The process described herein is a new and different method compared to anything used before and it allows for specific attachment of the PEG to a specific site on the protein. The novel method we describe provides for PEG attachment to the peptide using a PEG carbonyl reaction to a peptide hydrazide and is described in detail below. It can be used with any linear or branch polymer having a molecular weight of between about 500 to about 20,000 daltons selected from the group consisting of polyethylene glycol and polypropropylene glycol. The polymer may be unsubstituted or substituted by alkoxy or alkyl groups where the substituting groups possessing less than 5 carbon atoms. The benefits of being able to make a PEG toxic insect peptide are substantial and described above in the Summary of Invention.
General Chemical Reactions and Molecules Derived Therefrom
I. The Peptide Hydrazide.
The peptide hydrazide is made from the peptide lactone (see Part 1) and hydrazine to form a peptide hydrazide. The peptide hydrazide is essentially made in a three step procedure. The peptide lactone is mixed with hydrazine monohydrate. The mixture is stirred to solution to form the peptide hydrazide, and the peptide hydrazide is purified.
One of ordinary skill in the art would be able to produce many versions of this procedure, for example, the mixture of peptide lactone and hydrazine should be stirred well to form a solution. The peptide hydrazine formed in this solution can then be purified by a variety of methods, such as by prepative HPLC.
We teach both mixing the aqueous solution of the peptide lactone and the solution of hydrazine added as hydrazine monohydrate and then stirring well at room temperature. The peptide hydrazide can then be purified. We used HPLC for purification, other options, known to one skilled in the art are available. Procedures of this type are well known to the ordinary chemist and the procedures outlined herein may be varied considerable by one skilled in the art. Other options for collection and purification could be used. In the examples below both relatively pure and impure samples of lactone were used as the starting material and both of these resulted in high purity Peptide Hydrazide (I) and are described in Example 6. Other procedures could be used. In Example 6a and 6(b) the peptide lactone and hydrazide are mixed together and stirred. Purification steps can vary widely and various options are available and known to one skilled in the art.
II. The Peptide Hydrazone.
The peptide hydrazone and peptide hydrazide are important intermediates. Different types of peptide hydrazones can be made depending on what functional groups are desired for the peptide. Here we show various examples of different peptide hydrazones. Examples of hydrazones are shown in Examples 8-11. One skilled in the art will understand these are but representative and illustrative not limiting examples, other reagents and conditions could be used.
These examples use the peptide hydrazide with one or another type of carbonyl to create novel peptide hydrazones like the examples of Formula (II), (III), (VI) and (IX). Some examples of reactive carbonyl compounds producing novel pegylated proteins are provided.
Hexanal is added to a hydrazide to produce Hydrazone (II).
The reactions discussed above are shown in the structures below with details provided in the descriptions below and supporting data can be found in
The Examples and experiments performed for Part 2 are as follows:
Example 6, shows the peptide hydrazide, referred to as Peptide Hydrazide (I) or Hydrazide (I), can be made from the peptide lactone. Mass Spec. data is provided in
Example 7, provides data showing the peptide hydrazide is quicker acting when the normal acid form of the peptide is made into its hydrazide form. The toxic peptide used for both compounds began with Hybrid +2. After Hybrid +2 is converted to the hydrazide the two compounds (peptide acid form and peptide hydrazide form) are different compounds but they are very similar and have the same peptide backbone. The net difference essentially is that one peptide had hydrazine was added to create the Hydrazide (I) of Hybrid +2. These two samples were then tested on flies. One of the samples, either the normal acid form of the peptide or the hydrizide form of the peptide, i.e. the Hydrazide (I), were exposed to one of two groups of flies. One group of flies was exposed to the toxic peptide in hydrazide form, i.e. Hydrazide (I), the other group of flies was exposed to the toxic peptide in its native acid form. The data provided below in Example 7 shows the hydrazide kills insects faster than the native acid form of the same peptide.
Example 8, shows how hexanal can be used to make the hydrazone form of a peptide. Example 8 starts with the hydrazide (I), hexanal is added and the result is a hydrazone, referred to here as Formula (II) or Hydrazone (II). Mass spec. data is provided in
Example 9, provides for the preparation of a different hydrazone than Example 8. In Example 9 the compound “O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV) (MW˜2,000)” is used to make a peptide hydrazone. Mass Spec. data is provided in
Example 10, shows another way to make a hydrazone. Here it is a hydrazone made from a hydrazide and an acrylic ketone. It is the preparation of Hydrazone (VI) from Hydrazide (I) using Acrylic Ketone (V). Mass Spec. data is provided in
Example 11, describes the preparation of Hydrazone (IX) using a PEG4 Ketone (VIII). This example starts with Example 11(a) where 3-acetylacrylic acid and a carbodiimide are used to make PEG4 Ketone (VIII). Then, in Example 11(b), the PEG4 Ketone (VIII) and Hydrazide I are used to make Hydrazone (IX). Mass Spec. data is provided in
Representative formula to describe the peptide in its native acid form, the peptide lactone described in Part 1 and the peptide hydrazide of Part 2 is shown. Other hydrazides and hydrazones are described in Examples 8-11.
This example shows two methods to prepare peptide hydrazide (I).
Example 6(a).
A solution of 100 mg of purified Form 2 peptide, the peptide lactone, in 1 mL of water was treated with 100 uL of hydrazine monohydrate and stirred at room temperature for 2 hours. The material was purified in portions on a prep HPLC (eluted with a gradient of acetonitrile/water/trifluoroacetic acid). Appropriate fractions were combined and concentrated under vacuum to a reduced volume. The liquid was frozen in a freezer at −80° C. and then freeze-dried on a lyopholizer to yield 36.94 mg of peptide hydrazide (I) as a white solid.
Example 6(b).
A solution (25 mL) of Super Liquid Concentrate (mixture of Form 1 and Form 2 peptide, aka peptide lactone, at 14 mg/mL) was stirred overnight at 75° C. After cooling, HPLC showed mostly Form 2 peptide, the peptide lactone. The solution was treated with 2 mL of hydrazine monohydrate and stirred at room temperature for 2 hours. The material was purified in portions on a prep HPLC (eluted with a gradient of acetonitrile/water/trifluoroacetic acid). Appropriate fractions were combined and concentrated under vacuum to a reduced volume. The liquid was frozen in a freezer at −80° C. and then freeze-dried on a lyopholizer to yield 252.2 mg of peptide hydrazide (I) as a white solid. Hydrazide (I) LCMS by method B ESI/MS 4578.00 (M+H), retention time 3.6-4.1 minutes. See
In Example 7 we compare a toxic peptide in its typical acid form, Form 1 or the peptide form, with the same toxic peptide after it is converted to the peptide hydrazide, or Peptide Hydrazide (I) as it is labeled in the formula provided here. The following samples are prepared for injection:
100 ng/uL solution of Hydrazide (I) in water. This solution was diluted with water to make 50 ng/uL and 5 ng/uL solutions
100 ng/uL solution of Hybrid +2 in water. This solution was diluted with water to make 50 ng/uL and 5 ng/uL solutions.
Next, we performed the experiments according to the following steps:
(1) Prepare injected fly containers with proper labels, and punch air hole in the container lids with an 18-gauge needle. Choose flies. Use the flies on day 1 and day 2 after fully hatch-out and day 1 is the day of fully hatch-out. Turn on the CO2 gas line and immobilize the flies in the box by input of the CO2 gas with a needle. After flies are immobilized, transfer flies to a CO2 plate and keep them in sleep. The flies are massed and those with mass of 12-18 mg are used for the injection bioassay.
(2) Perform the injection. Load 100-200 μl solution into a 1 ml syringe with a 30-gauge needle and mount the loaded syringe into the microapplicator. Remove the air bubble from the syringe by turning the pushing pole of the microapplicator. Then set injection volume to 0.5 μl in the microapplicator.
(3) Inject the houseflies with 0.5 μl of the prepared solutions above from the dorsal thorax by turning the pushing pole. Inject 10 flies for each prepared solution above. Put the injected flies into the prepared cups with a lid with air holes. Add 2 Whatman #4, 4.2 cm filter paper discs. Add 1 mL of sterile nanopure water.
(4) Keep all the injected flies at room temperature. At 3 hour, 5 hour, 21 hour and 24 hour post-injection, score the injected flies. If there is more than 20% mortality in the negative control, redo the fly injection as described above. At all four scoring time points, the water and anesthesia controls had 0% mortality.
At the 5 hour time point, the 100 ng/uL concentration of hydrazide had 80% mortality while Hybrid +2 at the same concentration achieved 10% mortality.
A solution of 5 mg (0.00109 mmol) of hydrazide (I) in 100 uL of water was treated with 0.16 uL (0.00133 mmol) of hexanal in 10 uL of absolute ethanol. The mixture was stirred for 1 hour. Made a stock solution of 5 mg of hexanal and 2.86 uL of acetic acid in 490 uL of absolute ethanol. Reaction was treated with 10.9 uL of the stock solution and after mixing let stand for 2 hours. The mixture was heated at −60° C. for 1 hour. LCMS by method B ESI/MS 4661.60 (M+H, hydrazone), retention time 6.8-7.1 minutes. See
A stock solution of O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (MW˜-2,000) (IV)(10.9 mg) in 100 uL of absolute ethanol was treated with 1 drop of acetic acid. Note: O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (MW˜2′000) is a mixture of compounds with a distribution around a MW of 2000 and not a single compound. A solution of 5 mg (0.00109 mmol) of hydrazide (I) in 100 uL of water was treated with 22 uL (0.0012 mmol) of the stock solution of O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV) (MW˜2,000). After mixing, the mixture was allowed to stand at room temperature. The remainder of the stock solution of O-[2-(6-Oxocaproylamino)ethyl]-O′-methylpolyethylene glycol (IV)(MW˜2,000) was added in portions and the mixture allowed to stand overnight after mixing. LCMS by method B ESI/MS retention time 7.2-7.6 minutes. See
A mixture of 0.5 g (4.38 mmol) of 3-Acetylacrylic acid, 0.924 g (4.82 mmol) of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 0.651 g (4.82 mmol) of 1-Hydroxybenzotriazole hydrate (HOBt) in 4 mL of dichloromethane and 4 mL of tetrahydrofuran was stirred under nitrogen at room temperature for 10 minutes. The reaction was cooled in an ice bath and treated with a solution of 0.443 g (4.38 mmol) of hexylamine in 8 mL of dichloromethane. The reaction was stirred cold for 1 hour and overnight at room temperature. The reaction was diluted with dichloromethane and the organics were washed with a saturated sodium bicarbonate solution followed by a wash with water. The organic layer was dried over magnesium sulfate, filtered and concentrated under vacuum to yield a yellow solid. The solid was taken up in dichloromethane and purified on a column of silica gel (eluting with 50% ethyl acetate/hexanes). The appropriate fractions were combined and concentrated under vacuum to yield 537.06 mg of acrylic ketone (V) as a white solid. LCMS by method C ESI/MS 198.1 (M+H), 196.2 (M−H). See
A solution of 5 mg (0.00109 mmol) of hydrazide (I) in 150 uL of water was treated in portions with 0.96 mg (0.0048 mmol) of acrylic ketone (V) in 48 uL of absolute ethanol. The mixture was stirred for ½ hour after each addition and then overnight. LCMS by method B ESI/MS 198.24 (M+H, acrylic ketone); 4760.60 (M+H, hydrazone), retention time 5.1-5.8 minutes. See
A mixture of 137.6 mg (1.21 mmol) of 3-Acetylacrylic acid, 254.3 mg (1.327 mmol) of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 179.25 mg (1.327 mmol) of 1-Hydroxybenzotriazole hydrate (HOBt) in 1 mL of dichloromethane and 1 mL of tetrahydrofuran was stirred under nitrogen at room temperature for 10 minutes. The reaction was cooled in an ice bath and treated with a solution of 250 mg (1.21 mmol) of m-PEG4-amine (VII) in 2 mL of dichloromethane. The reaction was stirred cold for 1 hour and overnight at room temperature. The reaction was diluted with dichloromethane and the organics were washed with a saturated sodium bicarbonate solution followed by a wash with water. The organic layer was dried over magnesium sulfate, filtered and concentrated under vacuum to yield 302.29 mg of PEG4 Ketone (VIII) as an oil. LCMS by method C ESI/MS 304.1 (M+H), 302.1 (M−H). See
A solution of 5 mg (0.00109 mmol) of hydrazide (I) in 150 uL of water was treated in portions with 2.0 mg (0.0066 mmol) of PEG4 Ketone (VIII). The mixture was stirred for ½ hour after each addition. LCMS by method B ESI/MS 304.28 (M+H, PEG4 ketone); 4867.70 (M+H, hydrazone) retention time 4.7-5.1 minutes. See
The examples are intended to illustrate and not limit the claims and the claimed invention. One ordinarily skilled in the art would be expected to be able to make numerous variations and different version of what is shown here.
The present application is a continuation of U.S. Ser. No. 15/301,030, filed Sep. 30, 2016, which is a 35 U.S.C. § 371 to Patent Cooperation Treaty Application No. PCT/US2015/024344, filed Apr. 3, 2015, which claims the benefit of U.S. Patent Application No. 61/975,147, filed Apr. 4, 2014, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61975147 | Apr 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15301030 | Sep 2016 | US |
| Child | 16713975 | US |
