METHODS OF MAKING AND USING PLATFORMS FOR PEPTIDE SYNTHESIS AND COMPOSITIONS THEREOF

Abstract

Methods are disclosed of making and using platforms for peptide synthesis and compositions thereof, such peptide-anchored resins or beads for use in solid-phase peptide synthesis. The platform includes a plurality of platform particles, which particles are insoluble carrier material particles (microparticles/nanoparticles) having a plurality of different linkers coupled to them. The plurality of linkers includes, in various combinations and combinations, (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; (c) 2-Hydroxy-5-dibenzosuberone; (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene). In some embodiments, the insoluble carrier material particles have a plurality of linkers that are each a different type from one another. Such platforms can be used in solid-phase peptide synthesis processes.

Claims

1. A method of making a platform for a solid-state peptide synthesis process, wherein the method comprises: (i) selecting an insoluble carrier material, wherein the insoluble carrier material is in the form of a plurality of particles; (ii) coupling a plurality of different linkers to each of the plurality of particles of the insoluble carrier material to form a plurality of platform particles of the platform, wherein the plurality of different linkers comprises: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; (c) 2-Hydroxy-5-dibenzosuberone; (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

2. The method of claim 1, wherein the insoluble carrier material is a resin material.

3. The method of claim 1, wherein the particles are microparticles.

4. The method of claim 1, wherein the particles are nanoparticles.

5. The method of claim 4, wherein the nanoparticles have an average diameter of less than 100 nm.

6. The method of claim 1, wherein the platform is a universal platform for a solid-state peptide synthesis process.

7. A method of making a platform for a solid-state peptide synthesis process, wherein the method comprises: (i) selecting an insoluble carrier material, wherein the insoluble carrier material is in the form of a plurality of particles; (ii) coupling a plurality of different linkers to each of the plurality of particles of the insoluble carrier material to form a plurality of platform particles of the platform, wherein the plurality of different linkers comprises at least three of the linkers selected from the group consisting of: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; (c) 2-Hydroxy-5-dibenzosuberone; (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

8. The method of claim 7, wherein the plurality of different linkers comprises: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; and (c) 2-Hydroxy-5-dibenzosuberone.

9. The method of claim 8, wherein the plurality of different linkers further comprises one or more of the linkers selected from the group consisting of: (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

10. The method of claim 7, wherein the insoluble carrier material is a resin material.

11. The method of claim 7, wherein the particles are microparticles.

12. The method of claim 7, wherein the particles are nanoparticles.

13. The method of claim 12, wherein the nanoparticles have an average diameter of less than 100 nm.

14. The method of claim 1, wherein the platform is a universal platform for a solid-state peptide synthesis process.

15. A platform composition capable of being used in a solid-state peptide synthesis process to synthesize peptides, wherein the platform composition comprises: (i) an insoluble carrier material, wherein the insoluble carrier material is in the form of a plurality of particles; (ii) a plurality of different linkers, wherein each of the plurality of particles of the insoluble carrier material is coupled to at least one of each of the different linkers in the plurality of linkers, and wherein the plurality of different linkers comprises: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; (c) 2-Hydroxy-5-dibenzosuberone; (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

16. The platform composition of claim 15, wherein the insoluble carrier material is a resin material.

17. The platform composition of claim 15 wherein the particles are microparticles.

18. The platform composition of claim 15, wherein the particles are nanoparticles.

19. The platform composition of claim 18, wherein the nanoparticles have an average diameter of less than 100 nm.

20. The platform composition of claim 15, wherein the platform composition is capable of being used as a universal platform in a solid-state peptide synthesis process to produce peptides.

21. A platform composition capable of being used in a solid-state peptide synthesis process to synthesize peptides, wherein the platform composition comprises: (i) an insoluble carrier material, wherein the insoluble carrier material is in the form of a plurality of particles; (ii) a plurality of different linkers, wherein each of the plurality of particles of the insoluble carrier material is coupled to at least one of each of the different linkers in the plurality of linkers, and wherein the plurality of different linkers comprises at least three of the linkers selected from the group consisting of: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; (c) 2-Hydroxy-5-dibenzosuberone; (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

22. The platform composition of claim 21, wherein the plurality of different linkers comprises: (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); (b) 4-Formyl-3-methoxy-phenoxyacetic acid; and (c) 2-Hydroxy-5-dibenzosuberone;

23. The platform composition of claim 22, wherein the plurality of different linkers further comprises one or more of the linkers selected from the group consisting of: (d) 4-Hydroxymethylbenzoic acid (HMBA); (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); (f) 4-(Fmoc-hydrazino)-benzoic acid; (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

24. The platform composition of claim 21, wherein the insoluble carrier material is a resin material.

25. The platform composition of claim 21, wherein the particles are microparticles.

26. The platform composition of claim 21, wherein the particles are nanoparticles.

27. The platform composition of claim 26, wherein the nanoparticles have an average diameter of less than 100 nm.

28. The platform composition of claim 21, wherein the platform is a universal platform for a solid-state peptide synthesis process.

29. A method comprising: (a) loading a platform composition of any one of claims 15-28 into a reaction vessel of a solid-phase peptide synthesis synthesizer; and (b) utilizing the solid-phase peptide synthesis synthesizer to synthesize a first peptide using the platform composition as the platform for peptide synthesis.

30. The method of claim 29 further comprising: (c) after the step of synthesizing the first peptide, utilizing the solid-phase peptide synthesis synthesizer to synthesize a second peptide, wherein (i) the solid-state peptide synthesis synthesizer uses the platform composition as the platform for peptide synthesis and (ii) the second peptide is a different peptide from the first peptide.

31. The method of claim 29, wherein the solid-phase peptide synthesis synthesizer is a continuous flow peptide synthesizer.

32. The method of claim 31 further comprising measuring the flow in the continuous flow peptide synthesizer to quantitatively measure the synthesis of the first peptide.

Description

DESCRIPTION OF DRAWINGS

[0058] FIG. 1 is a schematic of a flow peptide synthesis known in the prior art.

[0059] FIG. 2 is a schematic of an automated fast-flow peptide synthesizer known in the prior art.

[0060] FIG. 3 is an overview of a representative SPPS peptide synthesis process.

[0061] FIG. 4A is an illustration of a particle of the platform.

[0062] FIG. 4B is an illustration of a platform having a plurality of particles shown in FIG. 4A.

[0063] FIG. 4C is an illustration of an alternative particle of the platform.

[0064] FIG. 5A is a photograph of a continuous flow SPPS setup in which embodiments of the platform of the present invention can be utilized.

[0065] FIG. 5B is a schematic of the continuous flow SPPS setup shown in FIG. 4A.

[0066] FIG. 6 is a flow time diagram for an embodiment of the present invention.

DETAILED DESCRIPTION

[0067] The present invention relates to methods of making and using platforms for peptide synthesis and compositions thereof, such peptide-anchored resins or beads for use in solid-phase peptide synthesis. The resins and beads are universal platforms in that these can be used in varieties of peptide synthesis, and do not need to be replaced in the system even when a different peptide synthesis is to be performed.

Platform

[0068] A universal platform (in resin and bead form) has been discovered that is a universal platform for peptide synthesis. The platform is a carrier material made of particles, with each particle having a plurality of distinct types of linkers (otherwise referred to anchors). Generally, the particles are microparticle (or nanoparticle) in size and are generally of the same size (i.e., generally these are monosized with the majority within 20% of the average size of the particles)

[0069] The linkers are protective groups that are bifunctional molecules that anchor the growing peptide to the insoluble carrier particle. Due to this plurality of distinct types of linkers, the platform is universal in that it can be used for various SPPS processes without having to be replaced between the transition from the production of a first peptide to the production of a second (and distinct) peptide.

[0070] The present invention utilizes insoluble carrier material suitable for SPPS, which generally can be a resin material. While polystyrene-based resins are typically utilized in SPPS processes, other polymer resins can be utilized due to the coupling of the plurality of linkers.

[0071] In embodiments of the present invention, the plurality of linkers includes: [0072] (a) Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker); [0073] (b) 4-Formyl-3-methoxy-phenoxyacetic acid; [0074] (c) 2-Hydroxy-5-dibenzosuberone; [0075] (d) 4-Hydroxymethylbenzoic acid (HMBA); [0076] (e) 4-Hydroxymethyl-phenoxyacetic acid (HMP linker); [0077] (f) 4-(Fmoc-hydrazino)-benzoic acid; [0078] (g) 4(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butyric acid; and [0079] (h) Fmoc-Suberol (5-Fmoc-amino-2-carboxymethoxy-10,11-dihydro-5H-dibenzo[a,d] cycloheptene).

[0080] FIG. 4A illustrates a platform particle 400 that includes an insoluble carrier material particle 401 having a plurality of linkers (a)-(h) (which linkers (a)-(h) are designated as L.sub.a-L.sub.h, respectively). FIG. 4B is an illustration of a platform 402 that includes a plurality of the platform particles 400. In such embodiment, each of the linkers (a)-(h) (L.sub.a-L.sub.h) of platform particle 400 is coupled to each of the insoluble carrier material particles 401. For instance, the C-terminal Fmoc amino acid may be coupled to the linker yielding the so-called handle, which can be purified before loading the polymer. Regardless of the bulkiness of the amino acid, high loads are obtained by coupling these handles.

[0081] In other embodiments, a subset of three of more of linkers (a)-(h) is coupled to the insoluble carrier. For example, by coupling linkers (a)-(c) to the insoluble carrier, what would result is a viable platform for the synthesis of all naturally occurring peptides and the synthesis of about 20% of non-naturally occurring peptides. The term universal platform as used in this paragraph and elsewhere herein is not intended to mean that the platform is a viable platform for all SPPS processes; rather, it means that the platform is capable of being utilized in a variety of different SPPS processes to yield the majority of naturally occurring peptides.

[0082] In other embodiments, the subset of linkers includes linkers (a)-(c) plus one or more of linkers (d)-(h). And, in still further embodiments, the subset of linkers includes one or two of linkers (a)-(v) plus one or more of linkers (d)-(h).

[0083] The number of each of linkers (a)-(f) attached to an individual particle is generally more than one, i.e., the individual particles have two or more of linker (a), two or more of linker (b), etc.

[0084] In some embodiments, such as shown in the platform particle 403 illustrated in FIG. 4C, the individual particles have a one-to-one correspondence in that there is generally one of linker (a), one of linker (b), etc. coupled to the individual particle. In such instance, the individual particles can be nanoparticles, including nanoparticles having an average diameter of less than 100 nm.

SPPS Method Utilizing The Platform

[0085] The platform of the present invention can then be utilized in an SPPS setup, such as the continuous flow SPPS setup shown in FIGS. 5A-5B. For example, the platform as described above can be used for a first SPPS process in which the synthesis employs the Fmoc-2,4-dimethoxy-4-(carboxymethyloxy)-benzhydrylamine (Rink amide linker) linker for the first amino group. Once this has occurred, the washing and deactivation step then precludes the other linkers from being involved in subsequent steps occurring in the process. Moreover, during this process, to prevent byproducts of deprotection and cleavage from reacting with the peptide and forming undesired impurities, scavengers can be added.

[0086] Once this first SPPS process is completed, the platform can be used for a second SPPS process in the setup without having to replace the platform. For example, the platform as described above can be used for a second SPPS process in which the synthesis employs the 2-Hydroxy-5-dibenzosuberone linker for the first amino group.

[0087] FIG. 6 is a flow time diagram for a system or an SPPS process, which system uses three pumps (pumps one through three) and the representative SPPS process has pumping profiles 601-603, respectively, that can be performed with the platform. As reflected in FIG. 6, (a) pump one is pumping the activating agent, solvent (DMF), and deprotection reagent, at 50 mi/mm; (b) pump two is pumping amino acid, solvent (DMF) at 50 ml/min; and (c) pump three is pumping DIEA at 5 ml/min. By way of example, the activating agent can be such as activators in DMF, such as 0.38 M actuators in DMF; the deprotection reagent can be 4000 piperidine+1% FA in DMF, and the amino acids can be 0.4 M amino acids in DMF.

[0088] TABLE I (below) reflects the various materials and approximate times that are utilized during the first SPPS process for the profile shown in FIG. 6.

TABLE-US-00001 TABLE I Step Description Time Pump Material Flow Rate 1 Prewash 0 s to 12 s None 0 ml/min 2 Prewash 12 s to 60 s 1 Solvent 50 ml/min 3 Coupling Loop - 60 s to 64.2 s 1 Activating Agent 50 ml/min Prime 2 Amino Acid 50 ml/min 4 Coupling Loop - 64.2 s to 67.2 s 1 Activating Agent 50 ml/min Coupling 2 Amino Acid 50 ml/min 3 DIEA 5 ml/min 5 Coupling Loop - 67.2 s to 71.4 s 1 Solvent 50 ml/min Wash 2 Solvent 50 ml/min 3 DIEA 5 ml/min 6 Coupling Loop - 71.4 s to 90.2 s 1 Solvent 50 ml/min Wash 2 Solvent 50 ml/min 7 Coupling Loop - 90.2 s to 96.5 s 1 Deprotect 50 ml/min Deprotection 2 Solvent 50 ml/min 8 Coupling Loop - 96.5 s to 115.7 s 1 Solvent 50 ml/min Wash 2 Solvent 50 ml/min 9 Post Coupling 115.7 s to 125 s None 0 ml/min Loop

[0089] Without replacing the platform, a second SPPS can be performed. TABLE I (below) reflects the various materials and approximate times that can be utilized during the second SPPS process.

TABLE-US-00002 TABLE II Descrip- Step tion Time Pump Material Flow Rate 1 Wash 0 s to 90 s 1 DMI 2.5 ml/min 2 DMI 2.5 ml/min 2 Wash 90 s to 120 s 1 DCM 2.5 ml/min 2 DCM 2.5 ml/min 3 Depro- 120 s to 234 s 1 DCM 2.5 ml/min tection 2 Detritylation 2.5 ml/min stock 4 Wash 234 s to 252 s 1 DCM 2.5 ml/min 2 DCM 2.5 ml/min 5 Neutral- 252 s to 264 s 1 DCM 2.5 ml/min ization 2 Neutralization 2.5 ml/min stock 6 Wash 264 s to 288 s 1 DCM 2.5 ml/min 2 DCM 2.5 ml/min 7 Wash 288 s to 318 s 1 DMI 2.5 ml/min 2 DMI 2.5 ml/min 8 Coupling 318 s to 323 s 1 Coupling base 2 ml/min stock 2 Monomer stock 2 ml/min 9 Wash 323 s to 330 s 1 DMI 2 ml/min 2 DMI 2 ml/min 10 Wash 330 s to 750 s 1 DMI 0.1 ml/min 2 DMI 0.1 ml/min

[0090] Another advantage of the platform particles is that these can be quantitatively measured by flow, particularly for those particles that have a one-to-one correspondence of linker to particle (such as platform particle 403 illustrated in FIG. 4C).

[0091] This present invention provides a flow chemistry advantage that will reduce the reagent consumption, have inherent automation potential, be suitable for real time/in-line monitoring, be more repeatable, have improved heat and mass transfer (mixing and heating), and be utilized at extreme reaction conditions (as there is more variation in the insoluble carrier material).

[0092] Additional advantages include that the platform has the added versatility in view of its being a universal platform, the ability to use the platform in scalable processes, and the ability to expand the peptides that can be synthesized with the same platform.

[0093] While embodiments of the invention have been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. The embodiments described and the examples provided herein are exemplary only and are not intended to be limiting. Many variations and modifications of the invention disclosed herein are possible and are within the scope of the invention. The scope of protection is not limited by the description set out above, but is only limited by the claims which follow, that scope including all equivalents of the subject matter of the claims.

[0094] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated herein by reference in their entirety, to the extent that they provide exemplary, procedural, or other details supplementary to those set forth herein.

[0095] Amounts and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly recited limits of 1 to approximately 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as less than approximately 4.5, which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[0097] Following long-standing patent law convention, the terms a and an mean one or more when used in this application, including the claims.

[0098] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0099] As used herein, the term about and substantially when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of the following: in some embodiments 20%, in some embodiments 10%, in some embodiments 5%, in some embodiments 1%, in some embodiments 0.5%, and in some embodiments 0.1%, each with respect to or from the specified amount, as such variations are appropriate to perform the disclosed method.

[0100] As used herein, the term and/or when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase A, B, C, and/or D includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.