Nanoligands for affinity chromatography and methods of use therefore

Abstract

A graphene substrate covalently bound to a ligand of Formula A. The ligand provides bis-chelation of a metal ion. The ligand has a porphyrin ring or a nitrilotriacetic acid moiety.

Description

NANOLIGANDS FOR AFFINITY CHROMATOGRAPHY AND METHODS OF USE THEREFORE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non-provisional of, U.S. Patent Application 63 / 709,104 (filed October 18, 2024), the entirety of which is incorporated herein by reference. STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number FA9550-16-1-0279 awarded by the Air Force Office of Scientific Research and grant number 1746198 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Immobilized metal affinity chromatography (IMAC) stationary phases are used in the biotechnology sector in different ways: batch, microplate, sensor chip, and column formats. Surface exposed amino acids in native proteins such as Glu, Asp, Tyr, Cys, His, Arg, Lys, and Met can naturally coordinate metals thanks to electron donating groups in their side chains. However, recombinant proteins with polyhistidine affinity extensions (His-tags) of different length (e.g. His6 and His10) can be engineered at the N- or C-terminus for stronger metal coordination, and therefore easier and more specific protein isolation. Most proteins can be genetically modified for efficient binding to metals provided that the His-tags are sterically accessible. His6-tags are commonly used and assure effectiveness in protein binding along with adequate metal binding strength. His-tags may be cleaved using a protease enzyme, especially when biological activity is expected to be negatively affected by their presence. Existing chelating resins allow the separation of His-tagged proteins from mixtures that have either lower or no affinity for the same resins. Purification strategies under native or denaturing conditions are modifiedby applying different metal ions (including Ni2+, Cu2+, Zn2+, Ag+, Fe3+, Ga3+, Zr3+, and Co2+). For instance, Ni2+has low epitope-tag affinity and a high nonspecific affinity, in contrast with Co2+which has high epitope-tag affinity and low nonspecific affinity.

[0004] Prior research in the field of graphene oxide (GO)-based resins focused on a GO-streptavidin complex that used bio-recognition as a first step towards affinity purification. Other work has been published on covalent tethering of proteins to GO. For example, it is possible to covalently attach proteins to GO via diimide-activated amidation. Additionally, surface assembly of GO nanosheets on SiO2 particles has been used for the selective isolation of hemoglobin, paving the way towards reverse phase chromatography. A hybrid mesoporous material based on GO / TiO2 / SiO2was proposed as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides. Other investigations have shown that carbon 2D- nanomaterials can be used for chromatographic applications including HPLC beyond protein isolation purposes. For instance, graphene and GO sheets supported on silica has been shown to provide versatile high-performance adsorbents for solid-phase purification. A gold nanoparticle decorated GO / SiO2 composite stationary phase for HPLC was demonstrated. Superparamagnetic GO-based dispersive-solid phase extraction for preconcentration and determination of tamsulosin hydrochloride in human plasma. GO-based resins are characterized by an aspect ratio typical of nanosheets. This geometrical property does not favor its use in column format, though it does not completely prevent it in small-columns. Suitable materials chemistry modification may result in the possibility of using GO-based resin in large columns.

[0005] The IMAC technology, developed over 40 years ago, has long been envisioned as the most effective low-cost solution for addressing all of these needs, but high hopes were dimmed by significant drawbacks for which no practical remedies are available: mainly metal ion leaching, size-variant protein capacity, and denaturation of some proteins. Contrary to common belief, the need to cleave the epitope-tag in pharmaceutical applications has not been the reason for the limited success of IMACresins. While GO-based resin in conjunction with new metal coordination ligands hold great promise in solving these issues they are still incompatible with large columns. Accordingly, a need still exists for alternative GO-based resins.

[0006] The discussion above is merely provided for general background informationand is not intended to be used as an aid in determining the scope of the claimed subject matter. SUMMARY

[0007] This disclosure provides a graphene substrate covalently bound to a ligandhaving a structure of:where n is an to are C1-C10 alkanes or a linker of Formula D, G is a trivalent moiety, and L is selected from:wherein R is, and m and oand 3; or a salt thereof.

[0008] This brief description of the invention is intended only to provide a briefoverview of subject matter disclosed herein according to one or more illustrative embodiments and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplified form that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] So that the manner in which the features of the invention can be understood, adetailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompassesother equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0010] FIG.1 is a schematic diagram of a method for carboxylating GO to CGO.

[0011] FIG.2 is a schematic diagram of a method to functionalize GO or CGO (xGO) with a ligand. The ligand may chelate a nickel salt.

[0012] FIG.3A is an example of a synthetic scheme to produce one ligand for use with the disclosed resins.

[0013] FIG.3B, FIG.3C and FIG.3D is an example of a synthetic scheme to produce another ligand for use with the disclosed resins.

[0014] FIG.4 depicts the bis-chelation of a histidine residue by the ligands of the disclosed resin. DETAILED DESCRIPTION OF THE INVENTION

[0015] This disclosure provides ligands for use with any kind of polysaccharide- based resins and also with GO-based resins. The ligands increase the coordination strength of metal ions and minimize metal loss. Additionally, this disclosure provides a method that is more naturally compatible with the features of GO-based resins.

[0016] In one embodiment, the ligands have a structure of Formula A.Formula A wherein n is an integer from 0 to 4, S and S are independently selected C1-C10 alkanes or a linker of Formula D, G is a trivalent moiety, a L is a ligand. In one embodiment, S1and S2are identical. In one such embodiment, S1and S2are each a C3 alkane (e.g. propyl). In another such embodiment, S1and S2are each a C4 alkane. G may be, for example, a triazine, such as a 1, 3, 5 triazine.

[0017] In one embodiment, L is a ligand of Formula B.Formula B

[0018] In another embodiment, L is a ligand of Formula C.Cwherein R is Formula C-1 Formula C-1 wherein m2 and 3. In one embodiment, o is 1 (i.e. CH3). In one embodiment, m is 2. In some embodiments, the ligand of Formula B or Formula C chelates a metal, such as Ni2+.

[0019] In one embodiment, S1 and S2 are independently selected C1-C10 alkanes ora linker of Formula D. Formula D

[0020] Carboxylated GO (CGO) was synthesized from GO to increase the amount ofcarboxylic groups on the surface (FIG.1) using a published procedure (Journal ofMaterials Chemistry B 1, 5003-5013, 2013). In one embodiment, CGO increases thecarboxylic acid group by at least 10 mol / mol % relative to the corresponding GO. Chloroacetic acid reacts with exposed hydroxides in the GO to yield the ether, and a finalwash with dilute HCl neutralizes the solution. The resulting CGO was characterized by asignificant increase in the amount of carboxylic acid groups. The surface modifications were indicated by the significantly increased aqueous solubility of the material. TEM and EDX analysis of the product show the particle size is reduced and only C, O, and some Na remain, with sulfur no longer present. The degree of conjugation of CGO somewhatincreases due to water elimination catalyzed by the treatment with base, as indicated by the darker color of the dry material with respect to GO. CGO appears to be very suitable as a 2-dimensional resin for IMAC supports.

[0021] In one embodiment, GO or CGO (xGO) in suspension is used to synthesize IMAC resins where the target molecules are immobilized by means of coordinative linkages (see United States patent 9,822,151, the content of which is hereby incorporated by reference). For example, a first-generation IMAC resin was synthesized from 5 m size GO platelets with about 20% oxygen content, produced by a modified Hummers’ method (Electrophoresis 32, 870-876, 2011 and Chemical Society Reviews 39, 228-240, 2010). The chemical modification used two to three steps, as shown in FIG.2. First, the carboxylic acid groups on the GO were activated by forming the mixed anhydride with N-hydroxysuccinimide (NHS) and N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide (EDC). Second, the carboxyl groups were coupled to an amine-terminated ligand by displacement at basic pH to yield the functionalized GO-Ligand. In those embodiments wherein the ligand already comprises a metal ion (e.g. the porphyrin of FIG.3D), the resin synthesis is complete and a GO-NiLand is formed. In those embodiments wherein the ligand does not comprise a metal ion, a third step is executed wherein the GO-Ligand was complexed with Ni2+ions to obtain GO-NiLigand. These steps were carried out in situ without purification, using only mild centrifugation steps to remove unreacted reagents. The resulting GO-NiLigand resin was decorated with Ni2+-Ligand sites available for His-tagged protein binding. This disclosure provides two new GO / CGO ligands. In some embodiments, carboxylates that do not bear ligands are further modified with polyethylene glycol (PEG) moieties to further modulate the surface chemistry of the resin. Methods for attaching PEG moieties to GO and CGO resins are known in the art.

[0022] FIG.3A depicts the synthesis and structure of bis nitrilotriacetic acid (NTA) ligand L1, which provides a ligand of Formula B. Compound 1 coupled with trivalent compound 2 in the presence of N,N-diisopropylenethylamine (DIEA) in acetonitrile (ACN) to yield compound 3. Compound 3 was coupled to compound 4 by exposure toDIEA in acetonitrile. The protecting groups (e.g. t-butyl alkoxides) were removed by treating with trifluoroacetic acid (TFA) in methanol (MeOH) to yield compound 5 (ligand L1).

[0023] FIG.3B to FIG.3D depicts the synthesis and structure of porphyrin (Por) ligand L2of Formula C. In FIG.3B compound 5 is coupled with compound 1 in the presence of N-methyl-2-pyrrolidone (NMP) to yield compound 7. In FIG.3C, compound 7 is exposed to a polyethylene glycol (PEG) to yield compound 8. In FIG.3D, compound 8 was exposed to a nickel salt (e.g. nickel (II), acetylacetonate, Ni(acac)2, nickel sulfate, etc.) followed by removal of the protecting groups with trifluoroacetic acid (TFA) to yield compound 9 (NiLigand-L2).

[0024] These ligands feature a free amine-terminated tether which is coupled to a carboxylic acid group on the GO or CGO through dehydration with, for example, N- hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in basic carbonate buffer (pH 10). The number of both the stochastic metal binding sites and the engineered bis-chelates can be evaluated by assays determining the % metal ion in the IMAC resins. The bis-chelates (FIG.4) have nM andsubnanomolar ( 10-29 M) binding constants, respectively. At the same time, they havechemical and thermal stability compared to commercial polysaccharide-based resins. Referring to FIG.4, advantageously, they provide double-point binding to a histidine residue of a protein that is exposed to the disclosed resins.

[0025] In one embodiment, graphite with a narrow size distribution (3 m) was used as a precursor to attain a reproducible and consistent synthesis of GO and highly hydrophilic CGO. AFM and TEM measurements provided an assessment of size distribution, and the resulting materials were used to synthesize resins. The high density of metal ion binding ligands resulted in a surface with a distribution of multi-point binding sites. The adjacent bis-metal ion complexes of ligand L1and ligand L2shows reversible nanomolar and subnanomolar (vide supra) affinity binding constants and permits the role of multi-point attachment in purification efficiency. Moreover, thesquare planar metalloporphyrins do not demetallate, allowing for the cooperative binding of His-tagged proteins with no subsequent metal leaching.

[0026] The high capacity of all disclosed nanoresins can be utilized in TFF devices. An IMAC nanoresin may be mixed with clarified serum or lysate and a protein of interest is bound to the nanoresin. The unbound proteins and cell material pass through the tangential flow membrane in the form of permeate, while the bound protein-resin bioconjugate remain as retentate. By changing the buffer conditions, the target protein is released and removed as permeate. Tangential flow is used as a final concentration and buffer exchange step in many pharmaceutical practices. Accordingly, the same equipment can be applied to the affinity purification step.

[0027] Tangential flow filtration (TFF) is typically used for the separation and purification of biomolecules on the base of their sizes. It is used in immunology, protein chemistry, molecular biology, biochemistry and microbiology. TFF can be used to fractionate large from small biomolecules, harvest cell suspensions, and clarify fermentation broths and cell lysates. GO-based resins can be used in combination with TFF to achieve separation of proteins, enzyme and antobodies not merely on the basis of size, but also on the basis of immunoaffinity and metal affinity functionalities on the nanomaterials.

[0028] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A graphene derivative comprising a graphene substrate covalently bound to a ligand having a structure of: where nselected C1-C10 alkanes or a linker of Formula D, G is a trivalent moiety, and L is selected from:wherein R is ,m and o are integers independently selected from 1, 2 and 3; wherein Formula D is or a salt2. The graphene derivative as recited in claim 1, wherein the graphene derivativeconsists essentially of the graphene substrate and the ligand.

3. The graphene derivative as recited in claim 1, wherein the graphene substrate is agraphene oxide (GO).

4. The graphene derivative as recited in claim 1, wherein the graphene substrate is acarboxylated graphene oxide (CGO).

5. The graphene derivative as recited in claim 1, wherein the graphene derivativecomprises polyethylene glycol (PEG) moieties covalently bonded directly to the graphene derivative.

6. The graphene derivative as recited in claim 5, wherein the graphene derivativeconsists essentially of the graphene substrate, the polyethylene glycol (PEG) moieties and the ligand.

7. The graphene derivative as recited in claim 1, wherein n is 2 and G is a triazine.

8. The graphene derivative as recited in claim 1, wherein G is a 1, 3, 5 triazine.

9. The graphene derivative as recited in claim 1, wherein L is:.

10. The graphene derivative as recited in claim 1, wherein L is: ,11. The graphene as n is 4, G is 1, 3, 5 triazine, S1and S2are butyl, and L is .

12. The graphene derivative as recited in claim 1, wherein n is 4, G is 1, 3, 5 triazine,S1and S2are:is wherein R is Formula C-1: Formula C-1,wherein m is 2 and o is 1.

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