WO2015150775A1

WO2015150775A1 - Nucleant for macromolecule crystallisation

Info

Publication number: WO2015150775A1
Application number: PCT/GB2015/050978
Authority: WO
Inventors: Naomi Esther Chayen; Milo Sebastian Peter Shaffer; Hannah Siobhan LEESE; Lata GOVADA; Sahir KHURSHID; Robert Menzel
Original assignee: Imperial Innovations Limited
Priority date: 2014-03-31
Filing date: 2015-03-31
Publication date: 2015-10-08
Also published as: GB2559094A; GB201405739D0

Abstract

The present invention relates to a method of crystallising macromolecules, using a functionalised carbon material as a nucleant, wherein the functionalised carbon material comprises at least one chemical species covalently bound to the carbon material, wherein5 the chemical species comprises a polymeric component.

Description

NUCLEANT FOR MACROMOLECULE CRYSTALLISATION

TECHNICAL FIELD

The present invention relates to methods of crystallising macromolecules, such as proteins, using a functionalised carbon material as a nucleant.

BACKGROUND

Protein crystallisation is a key tool in drug discovery. Understanding the crystallisation of proteins is fundamental for the determination of new target protein structures. Nucleants have the potential to provide crystallographers with a tool to aid the crystallisation of new target proteins and also aid further understanding of the crystallisation mechanism of different proteins.

One way to obtain high quality crystals is to control nucleation. This is the first step that determines the entire crystallisation process. Once nucleated, crystal growth is optimal at metastable conditions, where crystals do not nucleate spontaneously but existing nuclei grow in a controlled manner. In recent years, there have been great advances in protein crystallisation at metastable conditions, utilising methods such as seeding and adding nucleants. The seeding method inserts crystal seeds into crystallisation trials however, this requires crystallites of the desired protein which are not always available. In the ongoing search for alternative heterogeneous materials, a variety of substances such as minerals, horse and human hair, thin films, charged surfaces, mesoporous materials such as bioglass and some carbon based materials (Gully, B.S., et al. Nanoscale 4, 5321-5324 (2012); Asanithi, P., et al. ACS Applied Materials & Interfaces 1, 1203-1210 (2009)) have been used as nucleants with varied success.

There is therefore still a need for improved nucleants to target protein crystallisation.

SUM MARY OF THE INVENTION

It has been found that polymer functionalised carbon materials are effective nucleants for the crystallisation of macromolecules.

Accordingly, in a first aspect, the present invention provides a method of crystallising a macromolecule comprising the step of adding a functionalised carbon material to a crystallisation sample comprising said macromolecule, wherein the functionalised carbon material comprises at least one chemical species covalently bound to the carbon material, wherein the chemical species comprises a polymeric component. The functionalised material acts as a nucleant for the crystallisation of the macromolecule. Thus the invention provides a method comprising adding the functionalised carbon material to induce the crystallisation of the macromolecule.

The versatility of carbon nanomaterial chemistry enables the tailoring of nucleants for certain proteins and aids the development of nucleants to specifically target protein crystallisation. Different chemical species, and in particular wherein the chemical species comprises a polymeric component, may be used to functionalise various carbon materials with differing surface geometries allowing the carbon material to be adapted to produce nucleants for different classes of macromolecules.

Moreover, by virtue of the functionalization, the carbon material can be rendered capable of being solubilised, for example, as a colloidal suspension, enabling dispensing of nucleant accurately as aliquots in the micro and nanoliter scale, which is beneficial for automation of crystallisation and for reducing the amount of nucleant and protein required to achieve crystallisation. Accordingly, the method may be automated. The polymeric component may comprise a homopolymer or a copolymer. The polymer may be linear or branched. The polymeric component may comprise a polyacrylate, polyvinyl, polyester, polyamide, polyurethane, polysaccharide or polyether polymer, or a copolymer thereof. Preferably, the polymeric component comprises polyethylene glycol. Preferably, the polymeric component has a number average molecular weight M_n of at least about 0.2 kDa, about 0.55 kDa, about 0.75 kDa, about 2 kDa, about 3 kDa, or about 4 kDa. The M_n may be up to about 6 kDa, about 7 kDa, about 8 kDa, about 10 kDa, about 12 kDa, about 13 kDa, or about 15 kDa. Preferably, the polymeric component has a M_n of from about 0.2 kDa to about 15 kDa, from about 2 kDa to about 10 kDa, from about 3 kDa to about 7 kDa, from about 4 kDa to about 6 kDa, or about 5 kDa.

The polymeric component may be directly or indirectly bound to the carbon material. The chemical species may comprise a linker which covalently links the polymeric component to the carbon material.

The carbon material may comprise a nanomaterial. The nanomaterial may comprise graphene, graphite nanoplatelets, graphene oxide, single wall nanotubes, multi-wall nanotubes, or graphene nanoribbons. The carbon material may comprise carbon black. Preferably, the carbon material comprises graphite nanoplatelets or graphene.

The method may be useful in a method for screening for crystals or for optimisation of the crystallisation conditions.

The macromolecule may be a biological macromolecule, for example, a protein. The method may comprise the further step of isolating the crystallised macromolecule from the crystallisation sample, for example by filtration.

The features described above in the first aspect of the invention may be present individually or in any combination thereof.

In a second aspect, the present invention provides the use of functionalised carbon nanomaterial as defined according to the first aspect of the invention in the crystallisation of a macromolecule.

In a third aspect, the present invention provides a nucleant for the crystallisation of a macromolecule comprising the functionalised carbon material as defined according to the first aspect of the invention.

In a fourth aspect, the present invention provides a heterogenous liquid nucleant comprising a functionalised carbon material as defined according to the first aspect of the invention and a solvent.

In a fifth aspect, the present invention provides a method of determining the structure of a macromolecule comprising the steps of:

(a) crystallising the macromolecule in the presence of a functionalised carbon material as defined according to the first aspect of the invention;

(b) analysing the crystal structure of the crystal produced in step (a).

In a sixth aspect, the present invention provides a kit of parts comprising functionalised carbon nanomaterial as defined according to the first aspect of the invention. In a seventh aspect, the present invention provides an automated method of crystallising a macromolecule comprising adding a functionalised carbon material as defined according to the first aspect of the invention to a crystallisation trial using an automated dispensing system. The crystallisation is in a screen or optimisation. Preferably, the functionalised carbon material is added as a suspension in a liquid.

In an eighth aspect, the present invention provides the use of an automated liquid dispensing system to dispense functionalised carbon material according to the first aspect of the invention.

In a ninth aspect, the present invention provides a crystal obtainable or obtained by the method of any one the first, fifth and seventh aspects of the invention.

All preferred features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will now be described by way of example only with reference to the accompanying figures, in which:

Figure 1 shows TGA under N₂ of mPEG 5 kDa functionalised carbon nanomaterials; Figure 2 shows the Raman spectra of carbon materials functionalised with mPEG 5 kDa;

Figure 3 shows the TEM of mPEG 5 kDa functionalised graphene;

Figure 4 shows the characterisation of the GNP grafted with varying mPEG chain length a) shows the TGA under N₂ and b) displays the relationship between grafting ratio and mPEG chain length, c) and d) summarises the Raman and D/G ratios;

Figure 5 shows the Raman spectra of as-received carbon black and functionalised mPEG 5 kDa carbon black;

Figure 6 shows the AFM of a few layers of graphene with height trace; Figure 7 shows the XPS of graphene functionalised mPEG 5 kDa; Figure 8 shows the TGA-MS of graphene functionalised mPEG 5 kDa.

DETAILED DESCRIPTION The present invention provides a method as described herein of crystallising a

macromolecule by adding a functionalised carbon material to a crystallisation sample to act as a nucleant to induce crystallisation of the macromolecule, wherein the

functionalised carbon material comprises at least one chemical species covalently bound to the carbon material, and the chemical species comprises a polymeric component.

Carbon materials according to the present invention include, but are not limited to, graphite, graphene, graphene oxide, carbon nanotubes or carbon black. The carbon material may also include multi-layered material comprising two or more layers of graphene, including graphite nanoplatelets (GNP), chemically modified graphene materials and materials made using graphene or another graphene material as a precursor. Carbon materials may be nanomaterials. Nanomaterials are materials with at least one external dimension in the size range from about 1 to 100 nm. Carbon nanomaterials are carbon materials with at least one external dimension in the size range from about 1 to 100 nm.

Graphene is a single-atom thick sheet of hexagonally arranged, sp²-bonded carbon atoms. Graphene may have a specific surface area of up to 2600 m²/g. Graphite nanoplatelets (GNP) are stacks of graphene sheets. They may have a thickness and/or lateral dimension less than 100 nm. They may be stacks with a total thickness of each graphite platelet of around about 2 nm and an average diameter of 1 to 2 μηι. GNPs have a high aspect ratio. Graphene nanoribbons may be single-atom-thick strips of hexagonally arranged, sp²-bonded carbon atoms and which have a longer lateral dimension which exceeds the shorter lateral dimension by at least an order of magnitude.

Graphene oxide (GO) is an oxidized form of graphene. Graphene oxide may be prepared by oxidation and exfoliation of graphite. Graphene oxide may comprise, for example, at least 20 atomic % oxygen.

Carbon nanotubes, according to the present invention, include, but are not limited to single-wall carbon nanotubes (SWNTs or SWCNTs), double-wall carbon nanotubes (DWNTs or DWCNTs), multi-wall carbon nanotubes (MWNTs or MWCNTs), small diameter carbon nanotubes, large wall carbon nanotubes, and combinations thereof. Carbon nanotubes may be any type of nanotube, that is, it may be any hollow tubular structure having at least one dimension measuring on the nanometer scale. For example, the nanotube may have a smallest inner diameter measuring between about 0.5 nm to about 50 nm, such as about 0.5 nm to about 20 nm, for example between about 0.7 nm to about 10 nm, e.g. between about 0.8 nm to about 2 nm. Small diameter multi-wall carbon nanotubes may be medium-sized carbon nanotubes with diameters of around 5 to 10 nm and lengths of several micrometers. Large diameter multi-wall carbon nanotubes may be large nanotubes with diameters of around about 100 nm and lengths of several tens of micrometers. The nanotube may be of any length. For example, the nanotube may have a length between about 5 nm to about 2 mm, for example, about 5 to about 500 μηι. The nanotube may have a length of 2 mm. The specific surface area of these small diameter multi-wall carbon nanotubes may be around 250 m²/g for 10 nm tubes, and 500 m²/g for 5 nm tubes.

Carbon black is a particulate amorphous carbon material with individual particles on the nanometer scale. Carbon black may also comprise agglomerates of particulate amorphous carbon material.

Functionalised carbon material may be prepared by the thermal grafting method or by the reduction method. In the thermal grafting method, the carbon material is activated under high temperature and then functionalised with a chemical species. In the reductive method, carbon materials are reduced and functionalised with a chemical species.

Functionalization or grafting may occur with free radical reactions, which may be divided into two general strategies, the "grafting to" or "grafting from" approach. In the "grafting to" approach, free radicals of the chemical species or pre-formed polymeric component thereof are generated in a supporting solvent and subsequently terminated on the carbon material surface, thereby forming a covalent bond thereto. The covalent bond may be directly to the carbon material or, indirectly, via a linker moiety covalently bound to the carbon material. In the "grafting from" approach, polymerisation of monomers or repeat units occurs from initiating species that have been first immobilised on the carbon material surface and on which the polymer is grown. The surface of the functionalised carbon material may be heterogeneously functionalised. Heterogeneous functionalization is the functionalization of a carbon surface irregularly over the hydrophobic carbon surface.

The functionalised carbon material may be porous. It may have a pore size distribution of from about 2-35nm, preferably from about 2-20 nm. It may have a specific surface area of at least about 10 m² g^"1 , preferably of at least about 210 m² g^"1 , as determined by BET measurements. In the context of this invention, the polymeric component represents the total polymeric material within the chemical species. This may be present as contiguous polymer or polymeric segments optionally interrupted by one or more linking moieties. The terms polymeric component and polymer may be used interchangeably. For the purpose of this invention, the term "polymer" is given the meaning of a molecular structure formed by a plurality (e.g. at least 5) monomers connected by covalent bonds. Preferably, the polymer may comprise at least about 10, about 30, about 50, or about 100 monomers. The polymer may comprise up to about 130, about 200, about 250, or about 350 monomers. The polymer may comprise from about 10 to about 350 monomers, about 30 to about 250 monomers, about 50 to about 200 monomers, or about 100 to about 130 monomers. The monomers may all be identical. In this case, the polymer is said to be a homopolymer. There may be more than one type of monomer present in the polymer. In this case, the polymer is said to be a copolymer which in the context of this invention, may have 2 types of monomer or more than two types of monomer. The monomers forming the copolymer may be arranged in any way. The monomers may be arranged randomly (random copolymer), alternating (alternating copolymer), or in blocks (block copolymer), or any combination thereof. Block polymers may have two or more repeating distinct blocks. The polymer may be a linear polymer comprising a single main polymer chain. The polymer may be a branched polymer comprising a main polymer chain with at least one polymeric side chain.

The polymeric component may comprise one or more polyacrylate, polyvinyl, polyester, polyamide, polypeptide, polyurethane, polysaccharide, polyether polymers, or copolymers thereof.

Polyacrylate polymers are derived from acrylate monomers. Polyacrylate polymers may comprise, for example, acrylic and methacrylic acid monomers, alkyl methacrylate monomers, amino alkyl methacrylate monomers, or any copolymers thereof. Polyacrylate polymers may include, for example, polyacrylic acid polymers, methacrylate polymers, alkyl methacrylate polymers, amino alkyl methacrylate polymers, or copolymers thereof. Such polyacrylate polymers may include, for example, polyacrylic acid, poly(2- (dimethylamino)ethylmethacrylate) (DMEAMA), polyacrylonitrile (AN), poly(2- (methylthio)ethyl methacrylate) (MTEMA), poly(dimethyl acryl amide) (DMAA), or polyethylene glycol methacrylate (PEGMA), or copolymers thereof. Vinyl polymers are derived from vinyl monomers. Vinyl polymers may include, for example, polyalkenes such as polyethylene, polybutadiene, polystyrene, polyvinyl acetate, polyvinyl alcohol, polyacrylonitrile or copolymers thereof. Such vinyl polymers may include, for example, poly(4-vinyl pyridine) (VP), or poly(N-methyl-4-vinyl pyridine) (MeVP).

Polyamide polymers are derived from monomers linked in the polymer chain by amide bonds. Polyamide polymers may include, for example, aliphatic polyamide polymers, aromatic polyamide polymers or copolymers thereof, e.g. polypeptides are derived from amino acid monomers. The amino acid monomers may comprise natural and/or unnatural amino acids.

Polyurethane polymers are derived from monomers linked in the polymer chain by carbamate bonds.

Polyester polymers are derived from monomers linked in the polymer chain by ester bonds, and may be derived, for example, from carboxylic acid and/or lactone monomers. Polyester polymers may include, for example, aliphatic polyesters or aromatic polyester polymers, or copolymers thereof. Such aliphatic polyesters may include, for example, polyglycolide, polylactic acid, polycaprolactone, polyhydroxyalkanoate,

polyhydroxybutyrate, polyethylene adipate, or copolymers thereof.

Polysaccharide polymers are derived from saccharide monomers. The monosaccharide monomers may be bound together by glycosidic bonds. Polysaccharides may include, for example, cellulose, cellulose derivatives, e.g. hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose.

Polyether polymers are derived from monomers linked in the polymer chain by ether bonds. Polyethers may include alkylene glycol monomers, e.g. C C₆ alkylene glycol, for example propylene glycol or ethylene glycol monomers, or copolymers thereof. Polyether polymers may include, for example, poly(alkylene glycol) polymers. Such poly(alkylene glycol) polymers may include, for example, poly(propylene glycol) or poly(ethylene glycol) polymers. Preferably, the polymeric component comprises polyethylene glycol. More preferably, the polymeric component comprises methoxy(polyethylene glycol) (mPEG).

The molecular weight of a polymeric component or chemical species is given as the number average molecular weight M_n. This may be determined by gel permeation chromatography, mass spectrometry or ¹ H NMR. The determination of M_n in this way may be used for "grafting to" reactions. For "grafting from" reactions, M_n is determined by determining the graft ratio (mass increase) and using the number of measured active sites to deduce M_n. In one embodiment, the polymeric component of the invention may have a number average molecular weight M_n of at least about 0.2 kDa, about 0.55 kDa, about 0.75 kDa, about 2 kDa, about 3 kDa, or about 4 kDa. The M_n may be up to about 6 kDa, about 7 kDa, about 8 kDa, about 10 kDa, about 12 kDa, about 13 kDa, or about 15 kDa. Preferably, the polymeric component has a M_n of from about 0.2 kDa to about 15 kDa, about 2 kDa to about 10 kDa, more preferably from about 4 kDa to about 6 kDa, about 3 kDa to about 7 kDa, or about 5 kDa.

Grafting ratio is the molar ratio of grafted chemical species against molar ratio of carbon within the raw carbon material, with both of these values taken from the weights calculated from thermogravimetric analysis (TGA). The derivative of percent weight with respect to temperature of the TGA is taken and smoothed with a Savitzky-Golay filter.

The resultant peak(s) allows the determination of an onset and ending temperatures of the grafted moiety degradation, whilst the plateau of the derivative gives the rate of degradation of the grafted species, which is presumed constant. The percentage weight loss between the two temperatures is calculated and the contribution of thermal degradation (calculated using the plateau rate multiplied by the difference in temperature) is subtracted. This weight loss is attributed to the grafting species minus the leaving group.

Functionalised carbon material wherein the polymeric component has a M_n of about 4 kDa to about 6 kDa (e.g. about 5 kDa) may have a grafting ratio of about 8 to 60%, more preferably 10 to 20%. Preferably, functionalised material wherein the polymeric component is PEG with a M_n of about 4 to about 6 kDa (e.g. about 5 kDa) may have a grafting ratio of about 8 to 60%, more preferably 10 to 20%. The polymeric component may be directly covalently bound to the carbon material. The polymeric component may be indirectly covalently bound to the carbon material, i.e. via another chemical moiety such as a linker. The linker may be any chemical moiety, for example, an optionally substituted aliphatic, heteroaliphatic, aryl, heteroaryl or

combination thereof.

The chemical species may be terminated distal to the carbon material by an end group formed by a simple termination of the polymeric component or an end group. The end group may include, for example, a hydrogen, hydroxyl, alkoxy group (such as a methoxy or other alkoxy group), an aliphatic group (such as a methyl or other alkyl group or an alkenyl or alkynyl group), a heteroaliphatic group, an aryl group, a heteroaryl group (such as an imidazole group), a carboxylic acid group, a cyano group, an amine, an amide, an acetyl group, an azide group, a maleimide group, an aldehyde group, a hydrazide group, a hydroxylamine group, an alkoxyamine, an epoxy, thiol or a phosphate group. These groups may be optionally substituted with one or more substituents selected from a halo group (such as F, CI, Br, I), oxo, or any of the groups as listed above. The end group may allow for the further functionalization of the chemical species by binding of a chemical moiety thereto.

Optional substituents referred throughout this disclosure may include, but are not limited to, substituents as mentioned above. For the purpose of this invention, the term "aliphatic" means straight-chain, branched or cyclic hydrocarbons which are completely saturated or which contain one or more units on unsaturation, but which are not aromatic, and include alkyl, alkenyl and alkynyl. An aliphatic group may have about 30 or fewer carbons in its backbone (e.g. CrC₃₀, C C₂o, C₁-C₁₂, Ci-C₆), wherein cyclic aliphatic is at least C₃ and alkene and alkyne are at least C₂. The term "heteroaliphatic" is an aliphatic as defined above wherein one or more carbon is replaced by a heteroatom. The term "aryl" refers to a C₆ to d₄ aromatic moiety

comprising one to three aromatic rings, which are optionally substituted. The term

"heteroaryl" refers to aromatic groups having 5 to 14 ring atoms and having one or more carbon atoms, from one to four heteroatoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen, or sulfur, and any

quartenized form of a basic nitrogen.

The versatility of carbon nanomaterial chemistry enables the functionalised carbon material to be tailored to have hydrophilic, hydrophobic, or amphiphilic properties. This allows tailoring of solubility, ability to disperse in solvents and nucleant efficiency.

In this invention, amphiphilic means possessing both hydrophilic and hydrophobic properties. The polymeric component may be hydrophilic, hydrophobic or amphiphilic. Accordingly, the chemical species may be hydrophilic, hydrophobic, or amphiphilic, and the

functionalised carbon material may be hydrophilic, hydrophobic, or amphiphilic. In some embodiments, the chemical species, functionalised carbon material or polymeric component may be amphiphilic. In another embodiment, the chemical species, functionalised carbon material or polymeric component may be hydrophilic. In another embodiment, the chemical species, functionalised carbon material or polymeric component may be hydrophobic.

It will be appreciated that solubility varies dependent on temperature, but in the context used herein, an entity is considered water soluble if at room temperature or on application of heat up to the boiling point of water, an amount which can be solubilised in liquid water. If heating is used to aid solubilisation, the entity should be able to remain in solution on cooling to room temperature. The solubility of a chemical species, functionalized carbon material or polymeric component is measured using methods known to a person skilled in the art. For example, the chemical species, functionalized carbon material or polymeric component is solubilized at room temperature, for example 21 °C, and then centrifuged, for example, at 2000g. The solubility of the resulting supernatant is measured using UV/vis absorbance according to methods known to a person skilled in the art.

In this invention, a hydrophilic polymeric component may be a water-soluble polymeric component having a solubility of at least 5 mg/ml in water at room temperature, e.g. where solubility is tested in isolation of the carbon material, for example, prior to grafting.

In this invention, dependent on the hydrophilic, hydrophobic or amphiphilic properties of the chemical species, the chemical species and/or the functionalised carbon material may be solubilized in water, an aqueous solution or in oil, for example paraffin oil or compositions comprising silicon. The functionalised carbon material in may be soluble in water or an aqueous solution at a concentration of least about 0.1 , about 10, about 20, about 25, about 30, about 100, about 200 μg/ml. The chemical species or polymeric component may be soluble in water or an aqueous solution at a concentration of up to 50 mg/ml. The functionalised carbon material may also be dispersed in water or an aqueous solution at a concentration of at least about 0.1 mg/ml, allowing dispersion at higher concentrations, e.g. about 5 mg/ml, to provide a stock solution, for example, for crystallisation trials. This allows for an accurate concentration of the carbon material to be dispensed, for example, into each drop in a crystallisation trial using the hanging drop method. A heterogenous liquid nucleant may comprise functionalised carbon material and a solvent. The solvent may comprise water, an aqueous solution, such as a buffer, or a hydrophobic solvent, such as an oil or composition comprising silicon. It will be understood that the term "macromolecule" includes any molecule over 1 kDa. Preferably, the macromolecule is a biological macromolecule. The macromolecule may be, for example a nucleic acid, or a polypeptide. More preferably, the polypeptide is a protein. Preferably, the polypeptide comprises at least 10, about 20 or about 50 amino acids. More preferably, the polypeptide comprises at least about 75, about 100, about 200, about 500, about 1000 amino acids.

The crystallisation sample may comprise a macromolecule in an aqueous solution.

Preferably, the crystallisation sample comprises a protein in an aqueous solution. The aqueous solution may be a buffer.

A chemical species may consist essentially of a polymeric component, optionally a linker and an end group. An exemplary chemical species is mPEG.

Determination of which supersaturation level is inadequate or insufficient for spontaneous nucleation is well known in the art of crystallisation. The level of supersaturation can be determined by setting-up screens covering a range of conditions around the conditions that yield crystals/microcrystals spontaneously.

It will be appreciated that the kits of the invention are suitable for all crystallisation methods, including the microbatch, vapour diffusion hanging drop, sitting drop, sandwich drop and the free interface diffusion crystallisation methods. These methods are conventional methods and are known to a person skilled in the art.

The kits of the invention may further comprise crystallisation plates or slides and/or filters or crystallisation chambers. Where the kit further comprises crystallisation plates, it is preferred if the plates are multi-well plates, which would be particularly suitable for a high throughput screening.

The kit may be one which is suitable for screening for the crystallisation of

macromolecules. In this embodiment, the kit may further include any one or more of a range of buffers (which may cover a range of pH values) and/or any one or more of a range of salts. Suitable buffers and salts are known in the art of crystallisation and include Na-HEPES pH 7.5, Tris hydrochloride pH 8.5 as buffers, and 0.2M ammonium sulphate and 0.2M sodium acetate trihydrate as salts. Advantageously, the kit may further comprise multi-well crystallisation plates and filters.

In the context used herein, "about" may refer to a variation of ± 10%.

Examples

A variety of functionalised carbon materials were prepared and are listed in Table 1. Samples 1 to 5 were functionalised with different repeat units using the thermal grafting method. The thermal grafting method is disclosed in WO 2010/001 123. Generally, the method comprises an optional pre-thermal oxidizing step (TO) of thermally oxidizing the MWNT in air at 670°C, for 6 cycles of 5 minutes to break up the entangled carbon nanotube (CNT) agglomerates. The carbon material is then heated and the resulting surface activated carbon material is incubated with a repeat unit.

Samples 6-10, 9, 10 were also prepared using the thermal grafting method, but without the pre-thermal oxidation step. Samples 15, 17 and 19 were prepared using a reduction methodology. This method uses Na/Naphthalene with either Ν,Ν-dimethylacetamide (DMAc) or THF. SWCNTs were provided by Hipco, MWCNTs were purchased from Arkema, natural flake graphite (Graphexel grade: 2369, Graphexel Ltd., UK) was obtained from the manufacturer, graphite nanoplatelets (GNPs) were purchased from XG Sciences and carbon blacks (Printex L8) were purchased from Degussa. A typical experiment for the preparation of functionalised graphene, nanotubes, and anionically charged carbon black involved heating the carbon nanomaterial to 400°C under vacuum (<10^"2 mbar) for 1 hr. The Na and naphthalene were used as received from Sigma Aldrich. The naphthalene was dried overnight in a vacuum oven with phosphorous pentoxide before transferring to a N₂ filled glove box. 1 mmol of sodium and 1 mmol of naphthalene were added to 10 ml of degassed THF and stirred for 1 day. A dark green colour was observed. 1 ml_ of the Na/naphthalene solution was added to dry nanocarbon and more degassed THF was added to obtain a charge ratio of 1 : 12 C:Na. The graphene/sodium naphthalide solution was sonicated for 15 minutes and stirred for 1 day before adding mPEG-Br to the mixture and stirred. The solution was removed from the glove box after 24 hours and quenched with dry 0₂. After bubbling dry 0₂ into the solution, the solution was stirred overnight under dry 0₂ for oxidation of any remaining charges on the functionalized nanocarbons. Ethanol (10 ml) was added slowly followed by water (20 ml) before extracting into hexane and washing several times with water. The mixture was filtered, washing thoroughly with hexane, THF, ethanol and water. After washing the sample with ethanol and THF again, the product was obtained after drying overnight under vacuum at 80°C.

Samples 13, 14, 16, 21 and 23 were not functionalised and were used as received.

Table 1 - Summary of the preparation of the functionalised carbon material samples 1 to 23

12 MWNT(B) VP (not thermally oxidized) grafted 4-vinyl pyridine

(VP) via the thermal grafting method

13 Graphite

14 GO

15 Graphene/graphite PEG (5 kDa) Reduction methodology

16 SWNT

17 SWNT PEG (5kDa) Reduction methodology

18 CB

19 CB PEG (5 kDa) Reduction methodology

20 MWNT(A) Acid oxidized

21 MWNT(A) Ung rafted

22 MWNT(A) Thermally oxidized

23 MWNT(B) Ung rafted

MWNT(A) is multi-wall carbon nanotubes (small diameter) and corresponds to medium- sized carbon nanotubes (commercial, Arkema) with diameters of around 10 nm and lengths of several micrometers, specific surface area of around 200 m²/g.

MWNT(B) is multi-wall carbon nanotubes (large diameter) and corresponds to large carbon nanotubes (In-House grown) with diameters of around 100 nm and lengths of several tens of micrometers, specific surface area of around 30 m²/g. The functionalised carbon materials in Table 1 were tested as nucleants in the crystallisation of catalase (10 mg/ml), lysozyme (40 mg/ml), trypsin (60 mg/ml), thaumatin (30 mg/ml) and ROAb 13 (a monoclonal antibody against CCR5) (10 mg/ml) over a period of 10 days of observation. Thaumatin crystallisation trials were performed in the metastable region (determined experimentally by systematically reducing the condition concentration) at a protein concentration of 20 mg ml^"1. The nineteen nucleants were individually dispersed into 0.25 M sodium potassium tartrate, 0.1 M bis-trispropane at pH 6.8. Lysozyme trials were conducted at 20 mg ml^"1 protein concentration. The nucleants were dispersed into 0.4 M sodium chloride, 0.1 M sodium acetate, pH 4.5.

Trypsin trials were conducted at 50 mg ml^"1. The nucleants were dispersed in 15% w/v PEG 8 kDa, 0.2 M ammonium sulphate, 0.1 M sodium cacodylate, pH 6.5. Table 2 Summary of protein concentrations and conditions

Protein Cone, mg ml^"1 Condition

Lysozyme 20 0.4 M sodium chloride, 0.1 M sodium acetate, pH 4.5. Thaumatin 20 0.25 M sodium potassium tartrate, 0.1 M bis- trispropane at pH 6.8.

Trypsin 50 16% w/v PEG 8 kDa, 0.2 M ammonium sulphate, 0.1

M sodium cacodylate, pH 6.5.

Catalase 10 0.1 M tri-ammonium citrate, pH 6.8 and 14-16 % (w/v)

PEG 3350.

Haemoglobin 10 20%-25% (w/v) PEG 3350, 0.2 M MgCI₂ and 0.1 M

Bis-Tris buffer, pH 5.5.

RoAb13 10 16-20%-16% (w/v) PEG 2 kDa.

Barbara 10 0.06M Magnesium Chloride and Calcium chloride,

0.1 M Bicine and Tris-8.5 20- 24% Glycerol and PEG

4 kDa.

Catalase was crystallised by samples 10, 11 , 12, 15 and 19.

Lysozyme was crystallised by samples 7, 11 and 15.

Trypsin was crystallised by samples 3, 9, 15, 17 and 19.

Thaumatin was crystallised by samples 1 , 4, 6-8, 10, 15, 17 and 19.

RoAb 13 was crystallised by samples 2, 7, 12, 15 and 19.

Unfunctionalised graphite (sample 13) and unfunctionalised SWNT (sample 16) failed to induce crystallisation of any of the proteins. In contrast, mPEG (5kDa) functionalised graphene (sample 15) crystallised all of the proteins tested, and mPEG (5kDa) functionalised SWNT (sample 17) crystallised trypsin and thaumatin. This indicates that functionalised carbon material is a better nucleant than unfunctionalised carbon material.

The functionalised carbon materials in Table 1 were also tested under the following conditions.

Solutions of 30 mg/ml Thaumatin containing 0.20 M NaK tartrate (NaKT) were found to be metastable. Controls at 0.25 M NaKT also remained clear in the large majority of the controls. Under these conditions, thaumatin was crystallised by samples 1 , 2, 4, 6, 7, 8, 9, 11 , 13, 14, 19 and 20.

Solutions of 20 mg/ml lysozyme containing 0.43 M sodium chloride were found to be metastable. Lysozyme was crystallised by samples 20, 2 (at 0.44 M NaCI) and for ungrafted MWNT(A) (at 0.46 M NaCI). Metastable conditions extended down to 0.40 M NaCI, at which conditions samples 8, 10, 14, 16, 19, and 23 yielded crystals.

Solutions of 60 mg/ml trypsin with between 16 and 18 %(w/v) PEG of mean molecular weight 8000 (PEG 8K) were found to be metastable. Samples 14, 17 and 19 produced crystals. Samples 3 and 9 yielded crystals at 18% PEG 8K.

Solutions of 15 mg/ml catalase with between 12 and 13 %(w/v) of the primary precipitant, PEG of mean molecular weight 3350 (PEG 3350) were found to be metastable. 14 %(w/v) PEG 3350 was a borderline condition where most, but not all controls remained clear for at least one week after setup. Trials containing samples 4 produced crystals at 13% PEG 3350. Samples 6, 8, and 1 1 gave crystals at 14% PEG 3350.

Solutions of 10 mg/ml RoAb13 with between 17 and 19 %(w/v) of the primary precipitant, PEG methyl ether of mean molecular weight 2000 (mPEG 2K), were found to be metastable. Crystals were obtained from sample 19 in 17 and 18% mPEG 2K trials within 10 days from setup. Solutions containing 19% mPEG 2K gave crystals with all samples except 16. Under these conditions, sample 19 was a potent nucleant as it was clearly effective with three model proteins (trypsin, thaumatin, lysozyme) and the antibody RoAb13. Furthermore, (i) it induced nucleation of lysozyme crystals quicker than any other functionalised carbon material and (ii) it remained effective in nucleating RoAb13 crystals at lower supersaturations than any other functionalised carbon material.

Samples 15 and 19, both of which are PEG functionalised, were studied using BET measurements to determine the specific surface area. The specific surface area of sample 15 was low (<10 m² g^"1) with an average pore width of 30 nm. Sample 19 had a surface area of 217 m² g^"1 with an average pore width of 13 nm. BET measurements are used to to determine the specific surface area. The measurements of adsorption and desorption isotherms of nitrogen at 77 K were carried out on 20-50 mg samples using Micromeritics ASAP 2010 apparatus. Specific surface areas were calculated according to the Brunauer, Emmett and Teller (BET) equation from the adsorption isotherms in the relative pressure range of 0.05-0.20p/p0.

PEGylated carbon materials

Further investigations were conducted to study PEGylated carbon materials as nucleants.

Five geometries of carbon, SWCNTs, MWCNTs, graphene, GNP and CB, were functionalised with methoxy(polyethylene glycol) (mPEG). The materials have different specific surface areas, curvature and porosity when in a network. The mPEG

(methoxy(polyethylene glycol)) functionalised carbon nanomaterials were synthesised using reduction chemistry as set out above. Carbon nanotubes, graphene and anionically charged carbon black solutions were functionalised with brominated mPEG (mPEG-Br). The hydroxyl terminated end of mPEG was brominated using tetrabromomethane. In short, the reduced nanocarbon solutions were prepared by mixing a premade sodium naphthalide solution (either in THF or DMAc) with the dried nanocarbon in a C/Na ratio of 12 (for flat sheet geometries) and 20 (for tubular and spherical geometries).

Thermogravimetric analysis (TGA) was utilised to determine the degree of

functionalisation by wt.%. The percentage of grafting with 5 kDa mPEG varied depending on carbon geometry (Fig. 1). Carbon black was functionalised with approximately 58 wt.% mPEG, as compared with graphene, GNPs, MWCNTs and SWCNTs which had grafting ratios between 8 and 22 wt.% respectively. From the grafting ratio the number of PEG chains per carbon can be estimated. In this way, the behaviour of the PEG chain on the different carbon surfaces can be considered. The specific surface area of the carbon materials will change with PEG chain coverage and this has been another important consideration. Crystallisation trials conducted were normalised to the PEG grafting ratio due to the large difference in grafting ratios observed from TGA the results of which have been discussed in the crystallisation sections. A summary of the characterisation data is shown in Table 3.

Table 3 - Summary of mPEG functionalised carbon nanomaterials

Material Grafting C : PEG Solubility Specific PEG I_D/IG (as I_D/IG

Ratio ratio (\ig ml^"1 Surface Area Function- received) function-

(%) in H₂0) (m²g^"1) alisation alised

%

MWNT-mPEG5k 8 4792 68 200 0.021 1 .080 1 .163 SWNT-mPEG5K 1 1 3371 51 700 0.030 0.052 0.383

GNP-mPEG5K 18 1885 200 680 0.053 0.807 0.927

G-mPEG5K 22 1477 20-35 1 100 0.068 0.046 0.473

CB-mPEG5K 58 302 100 350 0.331 1 .104 0.959

The Raman spectra of the grafted materials were considered and the results are shown in Fig. 2. The Raman spectra of graphene-mPEG showed mainly bi/tri-layer graphene and a symmetrical 2D peak indicative of few-layer graphenes. Single layer graphenes were also detected by Raman and observed by TEM as shown in Figure 3. This indicates that when dispersed in an appropriate solvent multi- and single- layer graphene are observed. This could either be due to the synthesis method or the stacking of graphene sheets upon drying. The SWCNTs and MWCNTs both show a similar percentage of grafting of ~ 10%. The Raman indicates an increase in D/G ratio with mPEG functionalisation. The Raman spectra of CB-mPEG show a large D peak and it is difficult to detect a significant difference from the Raman spectra between the as-received and functionalised material (see Fig. 5). XPS and TGA-MS was performed on graphene grafted mPEG 5kDa and this data can be found in Fig. 7 and 8 which confirms the grafted mPEG 5 kDa on the nanocarbon surface. There was a significant shift in the carbon and oxygen scans, as more CO single bonds were detected in the PEGylated graphene compared to the unmodified graphite surface (Fig. 7). Fragments of the PEG chain and the methoxy group, OMe, were also detected (Fig. 8).

The solubility of each functionalised nanocarbon was measured by UV-Vis absorbance after sonicating in water for 10-15 minutes and centrifuging at 1000-5000g and compared to starting material solubility. The solubility of the commercial as-received materials were tested, in some cases for example the as-received GNP solubility in water was already significant (value 200 μg ml^"1 with initial loading 1 mg ml^"1) but all materials showed improved solubility post-functionalisation. Despite the PEG-functionalisation, which improves the water compatibility, the relatively low grafting ratio and amphiphilic character caused some of the larger agglomerates to sediment over a period of 24-72 h as tracked by UV-Vis.

Protein Crystallisation with PEGylated Carbon Nanomaterials Lysozyme, thaumatin and trypsin were selected for crystallisation for determining the successful nucleant when using functionalised carbon geometries. The vapour diffusion hanging drop method was used for the crystallisation trials. The trials were set up in crystallisation plates containing wells which were filled with reservoirs containing 350-100 μΙ of the crystallising agent/precipitant solutions. To create crystallisation drops, 1 μΙ of protein solution was mixed with 1 μΙ of reservoir solution on a coverslip. A single grain of the nucleant was then inserted into each drop using fine forceps if the nucleant was in the solid state, or approximately 0.2 μΙ was dispensed into the drops using a standard micropipette when the nucleant was in liquid/aqueous formulation. The coverslips with the crystallisation drops were then inverted over the wells containing the reservoir solutions and the crystallisation plates were stored at 20°C in an incubator.

The metastable region was determined by varying condition and protein concentration. The addition of unbound 5 kDa mPEG was also investigated, especially as many conditions throughout protein crystallisation studies incorporate different PEG chain lengths in the conditions. Controls to ascertain the role of unbound mPEG chains, raw carbon nanomaterial and, the binary nature of the two (i.e. the optimal nucleant) were investigated. To assess the effect of the mPEG chain alone, an equivalent amount of mPEG (as that calculated on the nanocarbons) was inserted into the drop. Depending on the grafting ratio this was found to be between 0.1 and 1 % w/v. 1 % w/v is significant as the concentration of the conditions (for example 3 kDa PEG in trypsin) are normally reduced in 1 % w/v increments. Therefore controls of 5 kDa mPEG solutions were inserted into the protein/condition drop acting as the nucleant/additive at a concentration of up to 1 % w/v. This was completed for all three proteins and the drops were monitored > 28 days and no crystals were observed.

To control the amount of nucleant in each drop a measured amount of nucleant was added to the appropriate condition. This not only enabled an accurate concentration of the nucleant to be inserted into the drop for each crystallisation trial but it also created a liquid nucleant which is extremely valuable for the future development of these materials as nucleants involved in screening. The aim is to have as little nucleant present in the drop as possible - therefore in this way it is possible to dilute the nucleant concentration by adding more condition. As a result, it is possible to develop a plot where by optimal concentration of a nucleant in a drop can be determined. It was also of interest to normalise the quantity of nucleant by mPEG concentration. To do this each of the five nucleants were dispersed in the protein condition but this time all the drops contained equal amounts of mPEG. This way it could be certain that when crystals nucleate it is due to the nanocarbon/mPEG relationship and adds further control to the experiments. To control the amount of nucleant in each drop, a known concentration was added to the appropriate condition depending on the protein and dispersed.

Cone,

Protein Condition

mg ml^"1

Lysozyme 20 0.1M sodium acetate at pH 4.5 and 0.5-1.5M NaCI

Thaumatin 20 0.1 M Bis-tris propane at pH 6.8and 0.1-lM Na/K tartrate (NaKT)

Trypsin 50 0.2M ammonium sulphate, 0.1M cacodylate at pH 6.5 and 5-30%(w/v) PEG 8000

Catalase 10 0.1M tri-ammonium citrate at pH 6.8 and 10-20%(w/v) PEG 3350

Haemoglobin 10 20%-25% (w/V) PEG 3350, 0.2 M MgC12 and 0.1 M Bis-Tris buffer, pH 5.5.

RoAbl3 10 10 mM nickel chloride, 0.1 M TRIS at pH 8.5 andl6%-20 %(w/v) PEG2K MME

The dispersions were stable whilst the drops were monitored for crystals (over 24-72 h). The larger heavier agglomerates within the dispersions sediment first, as observed by optical microscopy. However, a large amount of functionalised material stays within solution and therefore cannot be observed by the optical microscope but can be observed in TEM (by drop casting the solution on a TEM grid). The aim in the crystallisation trials is to have as little nucleant present in the drop as possible - it is therefore advantageous to use the dispensing method, here, as it is possible to dilute the nucleant concentration by adding more condition if necessary. In comparing the effectiveness of different geometries, it is useful to normalise the experiments to the available PEG-modified surface. Since the mPEG grafting density was similar for most of the materials, the quantity of added nucleants was normalised to maintain a consistent grafted mPEG content.

Thaumatin crystallisation trials were performed in the metastable region (determined experimentally by systematically reducing the condition concentration) at a protein concentration of 20 mg ml^"1. The five nucleants were individually dispersed into 0.25 M sodium potassium tartrate, 0.1 M bis-trispropane at pH 6.8.

Lysozyme trials were conducted at 20 mg ml^"1 protein concentration. Trypsin trials were conducted at 50 mg ml^"1. The nucleants were again dispersed in appropriate conditions (0.4 M sodium chloride, 0.1 M sodium acetate, pH 4.5 for lysozyme and 15% w/v PEG 8 kDa, 0.2 M ammonium sulphate, 0.1 M sodium cacodylate, pH 6.5 for trypsin) using the method just described above.

These results indicated that functionalised flat based geometries, such as functionalised GNP and graphene, were faster in crystallising three proteins deep into the metastable region of three different proteins.

Crystallisation and Functionalised Graphene-mPEG The effect of the length of the PEG chain immobilized on flat geometry surfaces was studied. Chain length was varied between 0.2 - 10 kDa. These nucleants were synthesised using the method discussed above. However, shorter mPEG (< 5 kDa) chains were synthesised with PBr₃ rather than CBr₄. The mPEGs were grafted onto GNPs and graphene for a systematic chain length investigation. Fig. 4 shows the relationship between the percentage mass of polymer grafted and the number of mPEG chains per carbon and TGA plots. From TEM images of the nucleants it is possible to observe stacking of the graphene layers - the exfoliated and then functionalised sheets stack on top of one another in a misaligned manner. This creates a distribution of different sized 'nuclei pockets' that proteins can then sit in and begin nucleation.

The nucleants of functionalised GNPs and graphene were dispersed in appropriate conditions for lysozyme, thaumatin and trypsin at the same protein concentrations as discussed above. The nucleants were grafted with mPEGs of varying chain lengths 10, 5, 2, 0.75, 0.55 and 0.2 kDa and the same crystallisation trials performed. The drops were checked at 24, 48, 72 hours etc. The GNPs and graphene grafted with 5 kDa consistently crystallised the three proteins (also observed in the geometry study) within 48 hours and lysozyme after 24 hours. This is encouraging as these experiments were conducted over several weeks and repeated several times, consistently producing crystals deep into the metastable region with the graphene-mPEG 5 kDa nucleants. The graphene nucleants grafted with mPEGs of chain length between 200 Da and 2 kDA produced poorer crystallisation results with few drops nucleating crystals after 72 hours. The

immobilisation and low concentration of longer chain mPEG on a flat carbon surface is more favourable for nucleation than a flat carbon surface densely covered in shorter chains.

Haemoglobin and catalase and the target protein RoAb1329 were investigated. At 10 mg ml^"1 , haemoglobin produced crystals approximately 10-20 μηι long. RoAb13 protein also produced some single crystals. Catalase only crystallised with G-mPEG5kDa. It did not crystallise with any other nucleants tested, including GNP-mPEG5kDa and graphene oxide (GO). Catalase and haemoglobin crystallise by Oswald ripening which is a different crystallisation mechanism to thaumatin, lysozyme and trypsin. The fact that the G-mPEG 5kDa nucleant is able to crystallise proteins which crystallise from different mechanisms is promising as this material has the potential to be a more universal nucleant for protein crystallisation.

The nucleation mechanism was investigated using Ferritin and Graphene -mPEG 5kDa by transmission electron microscopy (TEM). The layers of functionalised graphene sheets show concentrated areas of ferritin accumulation on the flat face as well at the edges of the functionalised graphene sheets. Ferritin favourably interacts with the functionalised surface and clustering of ferritin in certain domains on the material was observed, as well as at folds and creases. These observations strongly support the hypothesis that both geometry and functionalization are important to encourage nucleation.

The combination of geometry and chemistry play separate but synchronised roles for successful nucleation.

Functionalised carbon nanomaterials can be employed as nucleants in protein

crystallisation trials. By changing nanocarbon geometry and mPEG chain length, it is possible to optimise the nucleants for protein crystallisation.

Claims

1. A method of crystallising a macromolecule comprising the step of adding a functionalised carbon material to a crystallisation sample comprising said macromolecule, wherein the functionalised carbon material comprises at least one chemical species covalently bound to the carbon material,

wherein the chemical species comprises a polymeric component.

2. A method according to any preceding claim, wherein the polymeric component comprises a polyacrylate, polyvinyl, polyamide, polyester, polyurethane, polysaccharide or polyether polymer, or a copolymer thereof.

3. A method according to claim 2, wherein the polymeric component comprises polyethylene glycol.

4. A method according to any preceding claim, wherein the polymeric component has a number average molecular weight M_n of at least about 0.2 kDa.

5. A method according to claim 4, wherein the polymeric component has a M_n of about 0.2 kDa to about 15 kDa, about 2 kDa to about 10 kDa, about 3 kDa to about 7 kDa, about 4 kDa to about 6 kDa, or about 5 kDa.

6. A method according to any preceding claim, wherein the carbon material comprises a nanomaterial.

7. A method according to claim 6, wherein the nanomaterial comprises graphene, graphite nanoplatelets, graphene oxide, single wall nanotubes, multi-wall nanotubes, or graphene nanoribbons.

8. A method according to claim 7, wherein the carbon material comprises graphite nanoplatelets or graphene.

9. A method according to any one of claims 1 to 5, wherein the carbon material is carbon black.

10. A method according to claim 1 , wherein the method is automated.

11. A method according to claim 1 , wherein the method is a method for screening for crystals or a method for optimisation of the crystallisation conditions.

12. Use of covalently functionalised carbon nanomaterial as defined in any one of claims 1 to 9 in the crystallisation of a macromolecule.

13. A nucleant for the crystallisation of a macromolecule comprising the functionalised carbon material as defined in claim 1 to 9.

14. A heterogenous liquid nucleant comprising a functionalised carbon material as defined in any one of claims 1 to 9 and a solvent.

15. A method of determining the structure of a macromolecule comprising the steps of:

(a) crystallising the macromolecule in the presence of a functionalised carbon material as defined in any one of claims 1 to 9;

(b) analysing the crystal structure of the crystal produced in step (a).

16. A kit of parts comprising functionalised carbon nanomaterial as defined in any one of claims 1 to 9.

17. An automated method of crystallising a macromolecule comprising adding a functionalised carbon material as defined in claims 1 to 9 to a crystallisation sample using an automated dispensing system.

18. A method according to claim 17 wherein the crystallisation is in a screen or optimisation.

19. A method according to claim 17 or 18 wherein the functionalised carbon material is added as a suspension in a liquid.

20. Use of an automated liquid dispensing system to dispense functionalised carbon material according to any one of claims 1 to 9.

21. A crystal obtainable or obtained by the method of any one of claims 1-20.

22. A method according to claim 1-11 , 15, 17-19 or a use according to claim 12, 20, wherein the macromolecule is a biological macromolecule.

23. A method or use according to claim 22, wherein the macromolecule is a protein.

24. A method or use as substantially described herein with reference to or as illustrated in any one or more of the examples or accompanying figures.