# Patent

## Title
Nucleoside crystals, crystal nucleation and growth control with antifreeze proteins

## Abstract

The uses of antifreeze proteins (AFPs), including polypeptides, peptides, fragments of AFPs, and mimicries of AFPs, as highly efficient additives for crystal growth control are provided. AFPs are demonstrated to efficiently inhibit the nucleation of commercially and pharmaceutically important compounds (e.g., nucleosides, sugars, salts), increase the supersaturation of their solutions, modify their crystal habits, and result in novel morphologies.

## Background
FUNDING STATEMENT
This work was supported in full by NIH Grant GM086249, and in part by Research Corporation for Science Advancement Cottrell College Science Award CC10492.


BACKGROUND
Crystal growth control is essential in various fields of science and technology (e.g., chemistry, materials science, pharmaceutical development). For instance, changes in the size and shape of crystals of pharmaceutical compounds can impact their bioavailability, chemical stability, and production efficiency. Traditional methods to control crystallization usually include the alterations of temperature, solvent, supersaturation, and seeding conditions. A number of recent methods, such as using tailor-made additives, ledge-directed epitaxy, polymer microgel, polymer heteronuclei, capillaries, porous materials, and laser-induced nucleation have been developed and employed in the crystallization process of certain compounds to select favored and/or discover new crystalline forms of the compounds.
The concept of molecular recognition has been successfully used to elucidate the effects of additives (foreign ions or molecules) on crystal growth. Peptides and proteins are often used in vivo and in vitro to control the growth of minerals and produce new forms of solids with different physicochemical properties. For example, peptide additives in either α-helix or β-sheet arrangement designed to interact with calcite crystal faces have been demonstrated to control calcite crystal habit. Size and shape control of organic crystals, however, is more difficult due to their anisotropic properties (different atomic arrangements in three dimensions).
Although tremendous effort has been devoted to understanding the crystallization process and selective crystallization, the crystallization control process remains largely trial-and-error, experiencing substantial difficulties in exclusive production of the desired forms as well as the production of both thermodynamically and kinetically less favored forms. Moreover, much less progress has been made in additive-controlled organic crystallization than in additive-controlled inorganic crystallization, and the selective production of organic single crystals with defined crystal phase and morphology still remains an enigma. For example, nucleosides and their analogues, an important class of pharmaceutical compounds, have been used as viral mutagens, drugs for induction therapy, effective treatment of lymphoproliferative disorders, and spinal cord injury, however little is known about their size and shape control using additives.
Antifreeze polypeptides (AFPs) are a structurally diverse group of proteins found in many cold-adapted organisms to protect them from freeze damage through a noncolligative manner, providing an intriguing example of ice crystal growth control. AFPs can bind to specific faces of ice crystals and modify the habit of the ice crystals. Their affinity to ice depends on hydrogen bonding and hydrophobic interactions, unlike most protein-mineral interactions where ionic interactions often play a dominant role. Ice and clathrate hydrates (ice-like crystalline solids) are known to be inhibited and modified by AFPs. Although ice is known with many polymorphs, studies on ice morphs induced by AFPs are lacking. Until now, AFP studies focused on the isolation, antifreeze activity, structure determination, and ice or ice-like solid binding of AFPs. Detailed mechanisms of AFPs still remain unknown.
SUMMARY OF THE INVENTION
AFPs previously have been used for their antifreeze properties, largely to control ice and ice-like crystalline growth. We have found that AFPs can efficiently inhibit nucleation and modify single crystal growth of compounds having non-ice-like crystalline structures such as nucleosides, sugars, and salts (e.g., halides, carboxylates, phosphates, hydrogen/dihydrogen phosphates, sulfates, etc.), despite their being structurally different from ice and ice-like crystalline structures. This invention also presents novel examples of effective control of crystal growth of organic compounds by AFP additives. Moreover, AFPs can induce new crystalline forms with altered crystal shapes, e.g., via selective growth of hydrates and polymorphs of hydroxyl compounds.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1(a)-(c) show optical micrographs of the finally achieved m5U solids. (a) Needle-like orthorhombic m5U crystals, (b) reed-like amorphous m5U precipitates obtained in the presence of DAFP-1, (c) normal orthorhombic m5U crystals obtained in the presence of DAFP-1 and m5U seed crystals.
FIGS. 2(a)-(d) show optical micrographs of the finally achieved crystals or amorphous solids of m5U: (a) needle-shaped m5U crystals obtained in the presence of BSA, (b) needle-shaped m5U crystals obtained in the presence of the denatured DAFP-1 with completely reduced disulfide bonds, (c) gel-like amorphous m5U solids obtained in the presence of type III AFP, (d) normal orthorhombic m5U crystals obtained in the presence of type III AFP and seed m5U crystals in a saturated m5U solution.
FIGS. 3(a)-(g) show optical micrographs of the final achieved crystals or amorphous solids of C: (a) needle-like C crystals, (b) needle-like C crystals obtained in the presence of BSA, (c) needle-like C crystals obtained in the presence of the denatured DAFP-1 with completely reduced disulfide bonds, (d) powder-like amorphous C solids obtained in the presence of DAFP-1, (e) hair-like C crystals obtained in the presence of DAFP-1 and seed C crystals in a saturated C solution, (f) powder-like amorphous C solids obtained in the presence of type III AFP, (g) hair-like C crystals obtained in the presence of type III AFP and seed C crystals in a saturated C solution.
FIGS. 4(a)-(g) show optical micrographs of the final achieved crystals or amorphous solids of I: (a) hair-like I crystals, (b) hair-like I crystals obtained in the presence of BSA, (c) hair-like I crystals obtained in the presence of the denatured DAFP-1 with completely reduced disulfide bonds, (d) powder-like amorphous I solids obtained in the presence of DAFP-1, (e) plate-like I crystals obtained in the presence of DAFP-1 and seed I crystals in a saturated I solution, (f) powder-like amorphous I solids obtained in the presence of type III AFP, (g) plate-like I crystals obtained in the presence of type III AFP and seed I crystals in a saturated I solution.
FIG. 5 shows (a) The structure of the (100) face of m5U crystals (carbon, grey; oxygen, red; hydrogen, white; nitrogen, blue). The c-axis points into the plane of the paper. (b) Schematic representation: the growth rate along the a-axis is much faster than that along the b- or c-axis in a typical m5U crystal habit, while the growth along the a-axis is significantly inhibited in the presence of DAFP-1 becoming less than that along the b- or c-axis.
FIGS. 6(a)-(c) show representative powder XRD profiles of the samples from needle-like crystalline m5U (a), reed-like amorphous m5U precipitates obtained in the presence DAFP-1 (b), and normal orthorhombic crystalline m5U modified by DAFP-1(c), respectively. Major crystalline peaks in (a) are labeled with miller indexes (hkl).
FIGS. 7(a)-(c) show gel filtration HPLC analyses of the crystals of m5U monitored at 280 nm. FIG. 7(a) shows typical chromatograms of crystalline m5U obtained in the presence of denatured DAFP-1, FIG. 7(b) shows typical chromatograms of reed-like amorphous m5U precipitates obtained in the presence of DAFP-1, FIG. 7(c) shows typical chromatograms of normal orthorhombic crystalline m5U modified using DAFP-1 in the presence of m5U seed crystals. The retention times for m5U and DAFP-1 are 13.1 min and 11.5 min, respectively. Insets: enlarged portions (denoted by asterisks).
FIGS. 8(a)-(c) show representative PXRD profiles of the samples from needle-like C crystals (a), amorphous C (obtained in the presence DAFP-1) (b), and hair-like C crystals (modified by DAFP-1) (c), respectively. Major crystalline peaks in (a) are labeled with miller indexes (hkl).
FIGS. 9(a)-(c) show representative PXRD profiles of the samples from hair-like I crystals (a), amorphous I (obtained in the presence DAFP-1) (b), and plate-like I crystals (modified by DAFP-1) (c), respectively. Major crystalline peaks in (a) are labeled with miller indexes (hkl).
FIGS. 10(a)-(c) show optical micrographs of the final achieved thymine crystals in the absence of AFPs (a), in the presence of DAFP-1 (b), and in the presence of type III AFP (c), respectively.
FIG. 11 shows model of a Dendroides canadensis antifreeze protein (AFP) isoform aligned to the prism face of ice. The ribbon illustration of AFP isoform 1 from the beetle D. canadensis shows the ice-binding β sheet (green arrows) made up of aligned Thr-Cys-Thr motifs.
FIG. 12(a) shows view of the tertiary structure of DAFP-1 from Dendroides canadensis. DAFP-1 consists of seven repeat units. The putative ice-binding threonine residues are indicated as sticks. The average distance of the side-chain oxygen atoms in the threonine residues in the adjacent loops is 4.74 Å matching well to 4.80 Å, the unit-cell dimension along the c axis in m5U form I crystal.
FIG. 13 shows a synthesis strategy for 5-methyluridine
FIG. 14 shows 1H NMR spectrum of the product (m5U) in D2O (400 MHz)
FIG. 15 shows 13C NMR spectrum of the product (m5U) in D2O (100.64 MHz)
FIGS. 16(a)-(b) show optical micrographs of the finally achieved m5U crystals: (a) needle-shaped m5U hemihydrates (form I); (b) block-shaped m5U dihydrates (form II) obtained in the presence of DAFP-1.
FIGS. 17(a)-(b) show representative molecules in m5U form I (hemihydrate) (a) and m5U form II (dihydrate) (b).
FIG. 18 shows overlay of unique molecules in the crystal structure of m5U form I (all in gray) and form II (all in gray, except hydrogen atoms in light gray). Representative molecules in (a) m5U form I (m5U hemihydrate; all are in a red color) and (b) m5U form II (m5U dihydrate; all are in a blue color except that hydrogen atoms are in a gray color).
FIGS. 19(a)-(d) show packing diagrams of m5U form I (a and b) and form II (c and d). Dotted lines represent intermolecular hydrogen bonding in the crystals.
FIG. 20 shows DSC thermograms (exo up) of form I (a) and form II (b) of m5U.
FIG. 21 shows comparison of relative enthalpies for the phase transitions of form I and form II m5U.
FIG. 22 shows comparison of crystallization kinetics of form I and form II of m5U. Mass increase of crystals during the crystallization processes was used to estimate relative crystallinity.
FIGS. 23(a)-(b) show suggested mechanism for crystallization of m5U (a) and selective crystallization of m5U form II using DAFP-1 (b).
FIGS. 24(a)-(b) show optical micrographs of trehalose dihydrate crystals: (a) block-like trehalose dihydrate crystals; (b) trehalose dihydrate crystals with dramatically decreased sizes obtained in the presence of DAFP-1 (from 1-1.5 mm to 0.1-0.3 mm) The scale bars are the same for both graphs as shown in graph (a).
FIGS. 25(a)-(b) show optical micrographs of copper sulfate pentahydrate (CuSO4. 5 H2O) crystals: bright blue stone-like CuSO4.5 H2O crystals obtained in the absence of additives (a); bright blue stone-like CuSO4.5 H2O crystals with dramatically decreased sizes, which were obtained in the presence of DAFP-1 (b). The scale bars shown in white on top of the graphs are 1 mm



DETAILED DESCRIPTION
Throughout this specification, the terms “a” and “an” and variations thereof represent the phrase “at least one.” In all cases, the terms “comprising”, “comprises” and any variations thereof should not be interpreted as being limitative to the elements listed thereafter. Unless otherwise specified in the description, all words used herein carry their common meaning as understood by a person having ordinary skill in the art. In cases where examples are listed, it is to be understood that combinations of any of the alternative examples are also envisioned. The scope of the invention is not to be limited to the particular embodiments disclosed herein, which serve merely as examples representative of the limitations recited in the issued claims resulting from this application, and the equivalents of those limitations.
Throughout this specification, the following abbreviations and definitions apply: AFP=antifreeze protein; DAFP=Dendroides canadensis antifreeze protein; TmAFP=Tenebrio molito antifreeze protein; m5U=5-methyluridine; C=cytidine; I=inosine; MALDI-TOF=matrix-assisted laser desorption/ionization time-of-flight; HPLC=high-performance liquid chromatography; SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis; CD=circular dichroism; DSC=differential scanning calorimetry; PXRD=powder X-ray diffraction. The term “AFP” is defined herein by the cumulative group consisting of natural or engineered antifreeze proteins, antifreeze polypeptides and antifreeze peptides, active fragments of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, mimetics of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, their active mimetic fragments, and combinations thereof. The foregoing term “antifreeze” is defined as thermal hysteresis. The term “ice-like crystalline structures” is defined as ice and gas hydrates. The term “critical ratio” is the molar ratio of additive:compound required to completely inhibit the growth of crystals.
Control of crystallization by crystal nucleation and/or growth is essential in various fields of science and technology (e.g., chemistry, materials science, pharmaceutical development). The concept of molecular recognition has been successfully used to elucidate the effects of additives (foreign ions or molecules) on crystal growth. Peptides and proteins are often used in vivo and in vitro to control the growth of minerals and produce new forms of solids with different physicochemical properties. Size and shape control of organic crystals, however, is more difficult due to their anisotropic properties (different atomic arrangements in three dimensions).
Antifreeze polypeptides (AFPs) are a structurally diverse group of proteins found in many cold-adapted organisms (e.g., insect, plant, fungi, protist, bacteria) to protect them from freeze damage through a noncolligative manner, providing an intriguing example of ice crystal growth control. AFPs bind to specific faces of ice crystals and modify the habit of the ice crystals (FIG. 11). Their affinity to ice depends on hydrogen bonding and hydrophobic interactions, unlike most protein-mineral interactions where ionic interactions often play a dominant role. Ice and clathrate hydrates (ice-like crystalline solids) are known to be inhibited and modified by AFPs. Nevertheless, we have now discovered that the recognition ability of AFPs is beyond ice and ice-like crystalline solids.
AFPs have been found to produce inhibitory and habit-modifying effects on the stable nuclei formations and on non-ice-like compounds such as nucleoside crystals. Such effects of AFPs are analogous to their effects on ice despite the large structural differences. For example, ice Ih has a hexagonal structure, while nucleosides m5U, C, and I crystals have orthorhombic structures with different cell dimensions. Effects achieved with these non-ice-like crystals illustrate flexibility in AFP molecular recognition beyond ice and ice-like crystals. Moreover, the effects of AFPs on the nucleoside crystal growth are highly efficient (e.g., less than 1/1000 molar ratio of additive to compound), comparing with other additives on crystal growth control.
Use of AFPs in crystallization processes allows for control and selective crystal growth of polycrystalline ice and other important compounds, such as nucleosides, carbohydrates, salts, and their hydrate forms. Examples of nucleosides include, but are not limited to 5-methyluridine, cytidine, inosine and their analogs; deoxynucleosides and their analogs, including but not limited to adenosine, guanosine, 5-methyluridine, uridine, cytosine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, 5-ethyluridine, 5-iodouridine, and compounds having the following general structures:




Additional suitable compounds and their analogs comprise the following three general components: phosphate, sugar and amine base. Examples of the three components follow.
1. Examples of the phosphate component include, but are not limited to:




2. Examples of the sugar component include, but are not limited to: any mono-, di-, tri-saccharide/deoxysaccharide and their analogs.

3. Examples of the amine base component include, but are not limited to:





















    
    
        Base or OH or H.
        In addition, R and R1 can be any of the substituent groups including CH3, C2H5, F, Cl, Br, I, SMe, COO—, or any group from naturally modified bases.
    
    


Examples of carbohydrates include, but are not limited to monosaccharides, disaccharides, trisaccharides, and water (or alcohol) soluble multisaccarides. For example, sugars (trehalose, glucose, fructose, sucrose, lactose, maltose, galactose, 2-deoxy-galactose, a-methyl-D-mammoside, mannose, D-(+)-melibose, and D-(+)-raffinose).
Examples of salts include, but are not limited to Cl−, Br−, I−, F, OH−, COO−, phosphates, sulfates, and salt hydrates, including, but not limited to NaCl, KCl, CaCO3, Na2CO3, Na3PO4, Na2HPO4, NaH2PO4, and CuSO4.
Moreover, AFPs may be used as scaffolds to design novel, effective crystal growth inhibitors and modifiers. Most AFPs have at least one-dimensional repeats of the OH binding amino acids, while certain AFPs such as DAFP, TmAFP and others have two-dimensional repeats (adjacent loop and within the same repeat, 4.64 Å and 7.44 Å, respectively, In the case of TmAFP; FIGS. 12(a)-(c)). Thus, this scaffold pattern can be maintained, with selective exchange of amino acids (e.g., threonines in DAFP or TmAFP can be changed to other amino acids). The resulting engineered proteins may have a wider variety of uses and be designed for use with more diverse crystals forms.
It should be noted that while some of crystal nucleation and growth control appears to be affected at least in part by the “distance matching” dimensions between the non-ice-like compound and the AFP additive as discussed herein, we have obtained similar results for compounds having lattice spaces that do not correspond to AFPs, e.g., cytarabine, trehalose, glucose, xylitol, chloride, bromide, iodide, fluoride, phosphate, sulfate, and carboxylate salts. In the case of the salts, the anions are hydroxide or react similarly to hydroxides (e.g., fluorides, chlorides, bromides, iodides, phosphates, sulfates, and carbolxylates).
Crystal growth at the molecular level can be modeled as a process starting from prenucleation aggregate formation, and then followed by crystal nuclei evolution and macroscopic crystal development. The arrangements and shapes of at least some of these prenucleation aggregates and evolved nuclei are thought to resemble the final crystal structure, the control of which is a decisive step in producing the final crystalline form. Additives usually effect crystal growth by affecting nucleation (e.g., prevent, delay, promote) and/or modifying crystal habit (e.g., adsorbing onto specific crystal growing face(s) and changing the growth rate). Thus, AFPs may be used to produce new forms of compounds that have not been possible to obtain using prior methods.
In addition to the use of AFPs in the manner described herein, it is contemplated also that other parameters known in the art to affect crystal habit, such as temperature, pressure, and solvents may be used in combination with the AFPs in order to optimize desired results.
EXAMPLE 1
In this study, we demonstrate that AFPs can efficiently inhibit the nucleation and modify the single crystal growth of 5-methyluridine (m5U), cytidine (C), and inosine (I). m5U, C, and I are widely used nucleosides in the pharmaceutical industry, but little is known about their size and shape control using additives.












Molecular structures of (a) 5-methyluridine (m5U), (b) cytidine (C), and (c) inosine (I)

Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) at ACS grade or better, and were used without additional purification. Solvents and chemicals for the HPLC experiments were purchased at HPLC grade from Sigma-Aldrich. All of the aqueous solutions were prepared using Milli-Q water produced from a Synergy water system (Millipore) with a minimum resistivity of 18 MΩ·cm. All of the samples including the polypeptide samples were filtered through 0.2 μm filters before use unless otherwise indicated. 8 mL sample vials (National Scientific) were used for crystallization. All glassware and stir bars were first cleaned in a KOH/2-propanol bath. After rinsed with distilled water, the glassware and stir bars were soaked in 1 M HCl for 24 h and then rinsed with distilled water. Finally they were cleaned using RBS35 (Pierce), a surface-active detergent. After rinsed with distilled water and then with deionized water completely, the glassware and stir bars were air dried at room temperature before use.
AFP and Control Preparation
DAFP-1 was expressed and purified. The purified DAFP-1 was characterized using SDS-PAGE, MALDI-TOF mass spectrometer, CD spectrometry, and DSC and the identity of DAFP-1 was confirmed. The concentration of stock DAFP-1 solution was determined using a Cary 100 Bio UV-Vis spectroscopy (Varian) and the extinction coefficient of 5.47×103M−1 cm−1 at 280 nm was used.
BSA was purchased from Sigma-Aldrich (Item number A7030) and type III AFPs from fish were purchased from A/F Protein (Waltham, Mass.), which were used as received. The stock BSA and fish AFP solutions were prepared by weighing the solute and dissolving the solute in a known volume of water. The molecular weights, 66.5 kDa and 6.5 kDa, were used for BSA and type III fish AFPs, respectively. All of the weight measurements were carried out with an Ohaus Voyager Pro analytical and precision balance (Parsippany, N.J.).
Denatured DAFP-1 with completely reduced disulfide bonds was used as a second unknown and prepared following previously reported methods. To fully reduce all the disulfide bonds in DAFP-1, purified DAFP-1 ( ̃1 mM) was incubated in 0.10 M sodium citrate, pH 3.0, and 15.0 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 60° C. for 30 min. Then the denatured DAFP-1 was further purified using ÄKTA Purifier 10 (GE Healthcare) with a Sephacryl S-100 gel filtration column (GE Healthcare). In the denatured DAFP-1, all disulfide bonds were broken, resulting in the disruption of the structure of the conserved repeated threonine residues in the adjacent loops of DAFP-1 and thus the loss of the hydrogen bonding interactions with the m5U crystal surface.
Crystal Growth Procedure
We obtained the crystals of m5U, C, and I, respectively, by evaporation of their aqueous solutions at room temperature. m5U was known to be crystallized from aqueous ethanol solutions. We found that m5U can crystallize from its aqueous solution directly. On day 1, each sample vial was first added 600 μL of 400 mM m5U solution. Then 20 μL of water or polypeptide solutions at certain concentrations were added into each vial. The final m5U concentration was 387 mM in each vial. The additive/m5U molar ratios (×10−5) were varied from 0, 0.04, 0.4, 1.0, 1.2, 3.0, 4.0, 7.8, and 9.0. The vials were gently swirled after the addition and were left open in the air at the room temperature. At least three observations were recorded per day (every 8 hours) until the solutions in all the vials were dry. The experiments were repeated five times (Table 1).







TABLE 1







Sample results for crystal growth of m5U in the presence of AFPs 


in the absence of m5U seed crystals.











Polypeptide
Induction




concentration
time
Crystal


Samplea
(μM)
(day)b
qualityc













m5U
0
6
Yes


m5U + BSA/denatured DAFP-1
70.0
6
Yes


m5U + DAFP-1
70.0
13
No


m5U + DAFP-1
35.0
11
No


m5U + DAFP-1
30.0
10
No


m5U + DAFP-1
15.0
10
No


m5U + Type III AFP
1.50
12
No


m5U + Type III AFP
0.15
12
No


C
0
13
Yes


C + BSA/denatured DAFP-1
70.0
13
Yes


C + DAFP-1
20.0
21
No


C + DAFP-1
10.0
21
No


C + Type III AFP
20.0
20
No


C + Type III AFP
10.0
20
No


I
0
5
Yes


I + BSA/denatured DAFP-1
70.0
5
Yes


I + DAFP-1
2.0
15
No


I + DAFP-1
0.5
15
No


I + Type III AFP
20.0
14
No


I + Type III AFP
10.0
13
No





aEach sample contained 387 mM m5U, 252 mM C or 92 mM I on day 1. Results of each nucleoside alone and in the presence of each of the two polypeptide controls, BSA and denatured DAFP-1, were listed for comparison.


bThe day that the first appearance of solid was observed.


cThe suitableness of the resulting solids for single crystal x-ray diffraction. The identity and quality of single crystals of m5U, C, and I were examined using polarized microscope, PXRD, and single crystal x-ray diffraction.






In previous reports, crystals of C were prepared by evaporating of its aqueous ethanol solutions. Here we obtained C crystals by evaporation of its aqueous solution. It is known that fast evaporation from the aqueous solutions of I usually resulted in a mixture of its aggregates, α-, β-, and γ-form crystals. By slowly evaporating its aqueous solution at 20° C., the pure α-form I crystals were obtained. The experimental procedures for C and I were similar to those described above for m5U, while the concentrations of C and I solutions were 252 mM and 92 mM, respectively, in each sample vial on day 1 and the additive/C molar ratios (×10−5) were varied from 0, 0.04, 0.4, 1.0, 1.2, 3.0, 4.0, 7.9, and 10.0 and the additive/I molar ratios (×10−5) were varied from 0, 0.05, 0.5, 1.1, 2.2, 4.4, 8.1, 10.8, and 21.7. Photos of the vials were taken with a Canon EOS 30D camera during the crystallization process and when the process finished. Optical micrographs were taken under Nikon SMZ800 microscope with a Nikon Coolpix 5400 when the crystallization completed. The crystals were under Nikon SMZ800 microscope, and photos were taken by Nikon Coolpix 5400.
For the crystal habit study, crystallization conditions for the three nucleosides were used as described above. The formed nucleoside crystals in the absence of additives are homogeneous and reproducible with respect to their sizes and shapes. The same criterion was used for the study in the presence of each of the AFPs and controls. When the seed nucleoside crystals were first observed (less than 0.2 mg), DAFP-1 and type III AFP were added into the vials at final concentrations of 4.8 μM and 0.10 μM, respectively. Data were recorded as described above. The habit change can be stopped during the process. To stop the habit alteration, the mother liquor was removed from the vials and the seed crystals were washed quickly with cold water at 4° C. twice. The same volume of fresh saturated m5U solution as the removed mother liquor was added into the vial.
To make saturated thymine solution, an excess amount of thymine was first added into water and the resulting sample was shaken for two days and filtered. Then 20 μL of water or polypeptide solutions at certain concentrations were added into each vial with 1000 μL saturated thymine solution. The final thymine concentration was 2.94 mM in each vial and the final additive concentrations at the additive/thymine molar ratio of 1×10−4 or above. All the above mentioned experiments were repeated five times.
HPLC Analysis
The resulting solids, both crystals and precipitates, were analyzed by HPLC. The HPLC analysis were performed on a Waters HPLC system consisting of a Waters 1525 binary HPLC pump, a BioSuite 125, 4 μm UHR SEC HPLC column (4.6×300 mm), and a Waters 2998 photodiode array detector. The SEC buffer contained 0.10 M Na2SO4, 0.10 M NaPi (pH 7.00), and 0.02% NaN3. The solid m5U samples, each at a weight of 0.5±0.05 mg, were selected in at least four different locations in the same vial. The samples of precipitates were taken from the inside of solids. Each selected sample was dissolved in 100 μL HPLC buffer and diluted six-fold before HPLC analysis. For the solid thymine samples, the crystals were selected at three or more different locations in a vial. The selected crystals were dissolved in 1000 μL HPLC water and diluted to a final thymine concentration at about 0.600 mM. All the buffers and samples were filtered through a 0.1 μm filter and vacuum degassed before use. The flow rate was 0.30 mL/min at ambient temperature. The injection volume was 5.00 μL for all the HPLC experiments. Each experiment was repeated twice. Pure DAFP-1 and m5U were eluted at 11.5 and β 1 minutes, respectively. The HPLC analysis of all the thymine samples shows that only pure thymine was eluted at 14.5 minute.
X-ray Diffraction
All the resulting solids were sent to X-ray crystallography laboratory at the Beckman Institute of California Institute of Technology for analysis. The qualities of the solids for single crystal X-ray diffraction were determined (Table 1). The crystallographic data of m5U were from the m5U crystals grown in the presence of BSA. The resolution of the data was improved compared with that of previously reported data of m5U. The crystallographic data of m5U have been deposited in the Cambridge Database (CCDC) and the CCDC deposit number is 835399.
Powder X-ray diffraction (PXRD) data were collected at room temperature on a Rigaku Mini Flex II desktop diffractometer using Cu Kα radiation (30 kV, 15 mA) with a 2θ range of 5°-50°, a step size of 0.010°, and a step time of 2 s. Samples were lightly ground by hand using a mortar and pestle. The same amount of the resulting powders was then mounted and analyzed on a front loading sample holder.
The identity and quality of the final obtained nucleoside solids for single crystal X-ray diffraction and PXRD were determined (Table 1). The PXRD patterns of the crystalline thymine obtained in the presence of the AFPs are in agreement with the reported pattern of crystalline thymine.
The resulting crystals of the three nucleosides appear as orthorhombic needles (FIGS. 1(a)-4(a)). The effect of a beetle AFP from D. canadensis (DAFP-1) on m5U crystal growth was first investigated. DAFP-1 is a β-helical repeat protein with a size of 9 kDa containing 8 disulfide bonds, which are important for its structure and function as antifreeze. DAFP-1 was studied previously for its antifreeze enhancement effect by small molecular enhancers and interactions between DAFP-1 and reduced nicotinamide adenine dinucleotide in solution have been demonstrated recently. To completely inhibit single crystal growth, a reasonable additive/nucleoside molar ratio (the critical ratio) is needed. The critical ratio is the minimum molar ratio required to completely inhibit the growth of crystals for a specific compound. Preferably, this ratio is chosen such that the compound precipitates in a crystalline form under the operating conditions used while minimizing the amount of crystal growth inhibitor present that is still capable of modifying the crystal habit of the precipitated compound to display the desired physical and chemical characteristics.
Different amounts of DAFP-1 were added directly to m5U solution to determine the critical ratio of DAFP-1/m5U. The direct addition of DAFP-1 at all the tested concentrations delayed the first appearances of m5U precipitates in the solutions (Table 1), while higher additive/m5U molar ratios resulted in more significant delay. The critical ratio of DAFP-1/m5U was estimated to be 3.0×10−5, where the ratio is at 3.0×10−5 or higher, no single m5U crystal was detected, but reed-like amorphous m5U precipitates (Table 1 and FIG. 1(b)).
To further investigate how AFPs affect m5U crystal growth, DAFP-1 was added to the saturated m5U solutions in the presence of orthorhombic m5U seed crystals at the additive/m5U molar ratio of 1.2×10−5. The growth of m5U crystals was able to continue, but with an apparent change in the crystal habit, from needle-shaped to normal orthorhombic (FIG. 1(c)).
Examining by x-ray crystallography, the crystalline m5U samples in FIG. 1(a) and FIG. 1(c) (have the same orthorhombic crystal structure, while no samples in FIG. 1(b) are suitable for single crystal x-ray diffraction. In the absence of DAFP-1, the propagation of the needle-like m5U crystals is along the a-axis. The crystal habit is determined by the relative growth rates of different crystal faces, suggesting that the altered crystal habit in the presence of DAFP-1 was caused by selective interaction of DAFP-1 with the (100) faces of m5U crystals (FIG. 5). The distance between the repeated oxygen atoms in the ribose moieties on the (100) face of the m5U crystal, 4.80 Å (FIG. 5(a)), matches well to 4.74 Å, the average distance of the hydroxyl oxygen atoms in conserved repeated threonine residues in adjacent loops of DAFP-1, suggesting hydrogen bonding interactions between the hydroxyl groups of the ribose moieties in the m5U crystal and conserved threonines in DAFP-1 (FIG. 12(a)). Due to such interactions, m5U crystal growth rate along the a-axis decreased to be less than those along the b- or c-axis, resulting in normal orthorhombic m5U crystals (FIG. 5(b)).
The powder X-ray diffraction (PXRD) patterns (FIG. 6(a) and FIG. 6(c)) of the single crystalline m5U samples in FIG. 1(a) and FIG. 1(c) show the same characteristic peaks assigned to the same unit cell. In contrast, few peaks of very low intensity were observed in the PXRD pattern (FIG. 6(b)) of the reed-like amorphous m5U precipitates in FIG. 1(b), suggesting that these solids lack well-ordered structures, and the few weak peaks that cannot be assigned to any known crystalline phase of m5U might result from a local order in these m5U samples. The PXRD results are in good agreement with the single crystal X-ray diffraction data. The finally obtained solids were also analyzed using high performance liquid chromatography (HPLC) to identify whether DAFP-1 presents in the samples. The HPLC analysis (FIGS. 7(b) and 7(c))) indicated the presence of both m5U and DAFP-1 in the samples in FIG. 1(b) and FIG. 1(c), suggesting that DAFP-1 is occluded in the reed-like amorphous m5U precipitates and the normal orthorhombic m5U crystals, which supports interactions between DAFP-1 and m5U.
Under the same conditions, m5U crystal growth was also investigated in the presence of two controls, bovine serum albumin (BSA) and denatured DAFP-1 with complete reduction of its disulfide bonds, respectively. Neither BSA nor the denatured DAFP-1 inhibited or delayed the appearance of m5U precipitates at the control/m5U molar ratio of 1.8×104, a much higher ratio than those used for DAFP-1 (Table 1 and FIGS. 2(a)-(b))). The crystals were characterized to be m5U by X-ray crystallography and the results were confirmed by PXRD. HPLC analysis of these crystals revealed that only pure m5U presents, suggesting that the controls are simply excluded from the m5U crystals as impurities. These results indicate that the controls have no effect on the crystal growth of m5U.
To test the generality of the findings, the effect of DAFP-1 and the controls on the crystal growth of two other nucleosides, C and I, were examined. The space groups of m5U, C and I are all orthorhombic and repeated oxygen atoms in the hydroxyl groups in their ribose moieties are identified on their growing crystal faces, but the dimensions of the unit cells of these nucleosides are quite different. The two controls, BSA and the denatured DAFP-1, had no effects on the crystal growth of C and I. Interestingly, the effects of growth inhibition and habit modification were also observed for DAFP-1 on the crystal growth of C and I (FIGS. 3(d)-(g), FIGS. 4(d)-(g), FIGS. 8(b)-(c), and FIGS. 9(b)-(c)), suggesting that DAFP-1 can also interact with C and I crystals and such recognition can be flexible. The critical ratios of DAFP-1/C and /I were estimated to be 3.0×10−5 and 0.5×10−5.
To determine whether a different type of AFP can have similar effects, all the above experiments were carried out with a globular fish AFP, type III AFP. Similarly, direct additions of type III AFPs in the m5U, C, and I solutions effectively inhibited the crystal nucleation of m5U, C, and I, respectively (Table 1, FIGS. 2(c), 3(f), 4(f)). Additions of type III AFPs in the presence of the seed crystals of m5U, C, and I, altered the crystal habits of these nucleosides, respectively, and the resulting habits (FIGS. 2(d), 3(g) and 4(g) are similar to those caused by DAFP-1. However, we did not observe such inhibitory or crystal habit change effect of the AFPs on the crystal growth of thymine, the component base of m5U, under similar conditions (FIG. 10), suggesting that the hydroxyl containing ribose plays an important role in the recognition of m5U by AFPs.
Similarly, we crystallized trehalose dihydrate crystals by diffusing ethanol into the aqueous ethanol solution of trehalose directly. The protein:trehalose molar ratio (×10−5) was 9.0 in FIG. 24(b) The crystals formed in the presence of DAFP-1 show a dramatic decrease in size as compared to the crystals obtained without additive.
To further test the effect of AFP on different types of compounds, we tested copper sulfate pentahydrate. The crystal grows from copper sulfate pentahydrate aqueous solutions at room temperature. Seventy milligrams of copper sulfate pentahydrate were dissolved in one milliliter of deionized, milli-Q water. The solution was filtered and left in the air for evaporation. In the presence AFPs, the conditions were all the same except DAFP-1 was added to the solution at the molar ratio of 10−7 to copper sulfate. The presence of the AFP significantly delayed the appearance of the crystal for several days (from 7 days to 11 days) and the resulting crystals were found with dramatically decreased sizes (FIG. 25).
EXAMPLE 2
AFP can be used to control the crystallization of 5-methyluridine (m5U) and produce a new thermodynamically and kinetically less preferred crystal form, which is significantly less likely to be suitable for undesirable chemical and/or physical processes. We show selective nucleation and growth of a previously unknown, dihydrate crystalline form of 5-methylurudine (m5U) using a novel efficient protein additive, antifreeze protein (AFP). AFP can selectively inhibit the appearance of the hemihydrate form of m5U (form I), the only previously known crystalline form of m5U, and hence allows the growth of m5U dihydrate crystal (form II), the pure form of which cannot grow directly from m5U supersaturated solutions under the same conditions. The two forms obtained herein were characterized using X-ray crystallography and differential scanning calorimetry (DSC). Compared to the hemihydrate form, the block-like dihydrate crystalline form of m5U is thermodynamically and kinetically less preferred. A mechanism, supported by both experimental and theoretical results, was proposed for the AFP-induced selection process. The results suggest that crystallization processes using AFPs may be valuable for selective growth of hydrates and polymorphs of hydroxyl compounds. Furthermore, insights into the antifreeze mechanism of AFPs were provided.
The only previously known crystalline form of m5U is its hemihydrate crystal, designated form I, which was crystallized exclusively by evaporation from the supersaturated aqueous solutions. By using DAFP-1 as an additive, remarkably, we were able to control the crystallization of m5U and discover a new, less thermodynamically and kinetically preferred form, m5U dihydrate crystal, designated form II. Forms I and II crystals are readily distinguished from each other by their morphology and stability. Form I crystals are needle-like in appearance (FIG. 16(a)) and there was no change (in the appearance, single crystal X-ray diffraction pattern, or DSC profile) of the crystals after being left in air at room temperature for more than 24 months. Form II crystals are block-shaped (FIG. 16(b)) and became white opaque powders/blocks in 2 months. The resulting solids were not suitable for single-crystal X-ray diffraction.
Both thermodynamic analysis and kinetics analysis show the possibility of the AFP induced selective crystallization of the less thermodynamically and kinetically preferred m5U dihydrate crystal and gives insights into this process.
Thermodynamic Analysis.
According to the Gibbs-Volmer theory of homogeneous nucleation, the overall free energy change, ΔG, between a small nucleus of m5U and m5U in aqueous solution is equal to the sum of the surface excess free energy, ΔGs, and the volume excess free energy, ΔGv. Therefore,

ΔGi(ri)=ΔGsi+ΔGvi  (1)

where i=I or II, representing form I or form II of m5U. ΔGsi is a positive quantity, while ΔGvi is a negative quantity. For simplicity, a nucleus of m5U is assumed to be a sphere with a radius, r.
Then,

ΔGi(ri)=4πri2γi+(4πri3ΔGvi)/3  (2)

where, γi is the interfacial tension between the developing crystalline surface and the supersaturated solution where the nucleus is located. The critical nucleus, rci=−2γ/ΔGvi, is obtained at dΔGi(ri)/dri=0.
At the critical m5U form I nucleus, we get

ΔGcI=(4πγIrcI2)/3.  (3)

DAFP-1 can bind to m5U form I nucleus. For the AFP attached form I nuclei,

ΔGcI′=F(4πγIrcI2)/3  (4)

where I′ is m5U form I nucleus with AFP attachment and F is a correct factor resulting from the AFP attachment of m5U form I nucleus. To inhibit m5U form I nucleation and crystal growth, we need F>1 and hence ΔGcI′>ΔGcI.
At the critical m5U form II nucleus, we get

ΔGcII=(4πγIIrcII2)/3  (5)

To make the less preferred m5U form II nucleation and crystal growth occur before m5U form I nucleation and crystal growth, we need ΔGcI′>ΔGcII, that is F>(γIIrcII2)/(γIrcI2).
The hydrophilic face of the AFP can bind to the thermodynamically preferred, form I nuclei of m5U. The hydrophobic face of the AFP thus exposes to bulk solution, which results in large repulsive interactions against incoming m5U molecules and hence inhibits form I crystallization. In other words, enthalpic contribution leads F>1. However, DAFP-1 cannot bind to m5U form II nuclei. The total contributions from F, interfacial tension and the radius of nuclei, finally lead to the nucleation of the metastable crystalline. At this point, the free energy of form I nuclei with AFP attachment is still higher than that of crystalline form II. This finally allows the thermodynamically less preferred dihydrate crystalline form to fully grow.
Kinetic Analysis.
Ji is the nucleation rate of form I crystal. The kinetic coefficient for form i crystal growth is ki. For a system with two forms of potential crystals, i.e., m5U crystalline forms I and II, the following condition must be held to have m5U crystalline form II as the only crystallization product,

JIIkII3>>JIkI3  (6)

This suggests that the appearance of different structures may be influenced by additives designed to interfere selectively with either the nucleation, growth rates of a particular phase or both.
According to Arrhenius equation, the rate of nucleation can be expressed as

Ji=Aiexp(−ΔGci/kT)=Aiexp[−(16πγi3νi2)/(3k3T3{ln Si}2)]  (7)

where Ai is Arrhenius pre-exponential factor for species i, k is the Boltzmann constant. T is the temperature in kelvins, νi is the molecular volume for species i, Si is supersaturation of the species i.
In the absence of AFPs, only m5U form I crystals can form. Thus,

JIkI3>>JIIkII3  (8)

In the presence of AFPs, AFPs inhibit the form I nucleation. Therefore, JI′0 and JJII>>JI′, and we get

JIIkII3>>JI′kI′30  (9)

Under this kinetic condition described in the above equation, we can exclusively obtain the kinetically less preferred m5U crystalline form II.
Structure Aspects.
The structures of both forms were determined by single-crystal X-ray diffraction (FIGS. 17(a)-(b)). Both forms crystallize in the orthorhombic crystal system. Form I was solved in the space group P21212, whereas the new form is in P212121 (Table 2). A high degree of torsional freedom of the N1-C6 bond is in the m5U molecule.







TABLE 2







Crystallographie Data for the Hemihydrate (Form I) 


and Dihydrate (Form II) of 5-Methyluridine











parameter
form I
form II







formula
C10H14N2O6 · 
C10H14N2O6 ·




0.5H2O
2H2O



formula weight
267.24
294.26



temperature (K)
90(2)
100(2)



crystal system
orthorhombic
orthorhombic



space group
P21212
P212121



a/Å
13.953(2)
6.75400(10)



b/Å
17.201(3)
8.83730(10)



c/Å
4.8017(7)
20.9847(3)



α = β = γ
90° 
90° 



cell volume/Å3
1152.4(3)
1252.52(3)



calc density/g cm−3
1.540
1.560



Z
4
4



data/restraints/
1941/5/184
2199/10/206



parameters





final R indices
R1 = 0.0274
R1 = 0.0282



for I > 2σ(I)






wR2 = 0.0730
wR2 = 0.0741










The overlap of m5U molecules in form I and form II reveals a significant conformational difference in the ribose moiety (FIG. 18). Such change may allow optimization of hydrogen bonds between the hydroxyl groups in the ribose moiety and neighboring groups in m5U and water.
Water molecules are hydrogen bound to m5U molecules through N—H . . . O and O—H . . . O interactions. Comparing to those in form I, the number of strong intermolecular hydrogen bonds between two m5U molecules decreases, whereas the number of hydrogen bonds between m5U and water molecules increases in form II (Table 3). The strong intermolecular hydrogen bonds between two m5U molecules in form I are disrupted upon the introduction of additional water molecules. Consequently, the packing motifs of the two forms of m5U are different (FIGS. 19(a)-(d)).







TABLE 3







Geometrical Parameters for Hydrogen Bonding 


Interactions in Forms I and II of 5-Methyluridine












form
D-H...Aa
d(D-H) (Å)
d(H.,.A) (Å)
d(D...A) (Å)
<(DHA) (°)





form Ib
0(4)-11(40)...O(5)i
0.886(15)
1.881(15)
2.7658(15)
175.3(17)



O(5)-H(5O)...O(2)ii
0.840(15)
1.887(15)
2.7158(15)
168.7(18)



O(6)-H(6O)...O(1)iii
0.833(15)
1.890(15)
2.7097(14)
167.6(19)



O(7)-H(7O)...O(6)
0.860(14)
2.023(14)
2.8722(13)
169.2(18)



N(2)-H(2N)...O(7)iv
0.889(14)
2.106(15)
2.9764(13)
166.0(17)


form IIc
O(4)-H(4O)...O(8)
0.838(16)
1.853(16)
2.6902(15)
177(2)



O(5)-H(5)...O(7)i
0.86(2)
2.09(2)
2.8868(15)
154.2(19)



O(6)-H(6O)...O(2)ii
0.865(16)
1.957(17)
2.7954(16)
163(2)



O(7)-H(7WB)...O(5)iii
0.846(15)
2.073(15)
2.9103(17)
170.0(19)



O(8)-H(8WA)...O(5)iv
0.848(15)
2.041(16)
2.8510(16)
159(2)



O(8)-H(8WB)...O(1)v
0.862(15)
1.926(15)
2.7783(16)
169.9(19)



N(2)-H(2N)...O(7)
0.854(14)
2.049(15)
2.9016(16)
177.2(19)





aD = Donor, A = Acceptor.


bSymmetry transformations used to generate equivalent atoms:


i−x + 1, −y + 1, z;


ii−x + 1⁄2, y − 1⁄2, −z + 1;


iiix − 1⁄2 , −y + 3/2, −z;


iv−x + 1⁄2, y + 1⁄2, −z.


cSymmetry transformations used to generate equivalent atoms:


i−x + 3/2, −y + 1, z − 1⁄2;


ii−x, y + 1⁄2, −z + 1⁄2;


iii−x + 1, y + 1⁄2, −z + 1⁄2;


ivx + 1⁄2, −y + 1⁄2, −z;


v−x + 2, y − 1⁄2, −z + 1⁄2.






All the growing faces in form I crystal contain potential hydrogen bond donor or acceptor atoms (FIGS. 19(a)-(b)). On the (100) face, there are three oxygen atoms from the thymine moiety (O1), a hydroxyl group in the ribose moiety (O4), and water (O7), respectively, with the smallest possible repeating distance of 4.80 Å (or 17.20 Å) along c (or b) dimension (Table 2 and FIG. 19(a)-(b)). Two oxygen atoms from a hydroxyl group in the ribose moiety (O5) and water (O7), respectively, are on the face (O10) with the smallest possible repeating distance being 4.80 Å (or 13.96 Å) along c (or a) dimension (Table 2 and FIG. 19(a)-(b)). The oxygen atom in the thymine moiety (O1) is on the face of (001) repeating with the smallest possible distances of 13.95 Å (or 17.20 Å) along a (or b) dimension (Table 2 and FIG. 19(a)-(b)). In contrast to form I, the three growing faces of form II are more hydrophobic. No potential hydrogen bond donor or acceptor atom is observed on the face of (010) or (001); and the oxygen atoms (O7 and O8) of the two water molecule are on the (100) face both with the smallest repeating distance of 8.83 Å (or 20.98 Å) along b (or c) dimension, but the possible repeating distances are not even close to 4.80 Å (Table 2 and FIG. 19(c)-(d)).
Hydrogen bonding interactions are known to be essential in some AFPs to recognize ice crystals and the distance of the side chain hydroxyl groups in the conserved repeat residues in the AFPs matches that of repeating oxygen atoms on the prism face of the normal ice crystal. DAFP-1 is a repeat protein with conserved threonines as putative ice-binding residues in each repeat unit, and according to the modeled structure of DAFP-1, the average distance of the side-chain oxygen atoms in the threonines in the adjacent loops is 4.74 Å (FIG. 12 (a). It is remarkable that the distance matches well to the unit-cell dimension along the c axis in form I, 4.80 Å, with reasonable flexibility.
Oxygen atoms with a possible repeating distance of 4.80 Å are on the fast-growth (100) face and the (010) face in form I, suggesting that DAFP-1 can recognize these faces through hydrogen bonding interactions. However, DAFP-1 cannot interact with any face of m5U form II crystal due to the lack of match of possible hydrogen bond donor/acceptor atoms in DAFP-1 and the growing faces of form II. Therefore, DAFP-1 can selectively inhibit form I crystallization, but allow the growth of form II crystal.
The interactions between DAFP-1 and m5U are highly efficient since only a tiny amount of DAFP-1 as additives is needed (the additive/m5U molar ratio was 5×10−6). Moreover, such interactions need to be weak in order to guide the crystal growth. The selective crystallization of form II by using DAFP-1 cannot be obtained if the interactions between DAFP-1 and m5U are strong. This is because if the interactions between DAFP-1 and m5U are strong, some m5U molecules must bind to DAFP-1 completely in solution, leaving the rest pure m5U and finally resulting in form I only.
Thermodynamic Stability Analysis. Both forms of m5U were analyzed by DSC (FIG. 20). Form I displays two endothermic events upon heating. The first endotherm associated with water loss is at 139.07° C., whereas the second endotherm ascribed to the melting is at 185.65° C. Form II exhibited two endotherms at 137.04° C. and 153.72° C., respectively, for the loss of the two water molecules, followed by a recrystallization exotherm at 157.66° C. and then a melting endotherm at 183.41° C. Form II starts losing water and then melts at relatively lower temperatures than those of form I. The enthalpy profiles for both forms are compared in FIG. 21. Form I is more thermodynamically stable, which is in accordance with the previous observations.
The crystallization processes of both forms were carried out at ambient conditions. We assume that the reaction of form II becoming form I by losing water at room conditions, i.e., m5U form II→m5U form I+1.5 H2O. Heat is needed to cause the dihydate form II to lose water and become the hemihydrate form I (FIG. 21). Therefore, the AH for the reaction is positive, i.e.,

ΔH>0  (10)

At room temperature, T, we have

d(ΔG/T)dT=−ΔH/T2.  (11)

For a tiny temperature increase, dT, the resulting temperature, T′ can be expressed as T′=T+dT and

T′>T.  (12)

Assuming that ΔG is a constant for such a tiny change, we obtain

ΔG(d1/T)=−ΔH/T2dT.  (13)

By integrating the above equation, we get

ΔG(ln T′/T)=ΔH(1/T′−1/T).  (14)

Combining Eqs. 10, 12, 14, we obtain ΔG<0. Therefore, the free energy change of the form II to I transition at room containing is negative. That is form I crystal is more thermodynamically stable. The results are consistent with the observations that form I crystals were stable in air for at least 2 years, while form II crystals became white powders/blocks in 2 months in air.
Crystallization Kinetics Analysis.
In the absence of DAFP-1, form I starts growing on day 3 and the crystallization completes on day 9 (FIG. 22). DAFP-1 can inhibit the crystallization of form I. In the presence of DAFP-1, only form II can be grown and the crystallization starts on day 6 and finishes on day 15. The crystallization of form II cannot happen in the absence of DAFP-1, since the crystallization of form I has much faster kinetics than that of form II. However, the presence of DAFP-1 completely inhibits the fast growing form I and hence results in the exclusive formation of the new, slow growing form II. The average crystal growth rate of form II is estimated to be 10.5% per day, which is slower than that of form I, 13.3% per day.
Mechanism of AFP-Induced Selective Nucleation and Growth.
Our approach with regard to the mechanism of the AFP-induced control of crystallization rests on the assumption that supersaturated solutions contain nuclei adopting similar arrangements to the final crystal structure. This assumption has been successful in designing tailor-made inhibitors.
In the supersaturated solution of m5U, form I nuclei whose structure are similar to that of form I crystal are evolved around day 2 (FIG. 22-FIG. 23(a)). In the absence of DAFP-1, form I crystal grows. As suggested by the match between the hydroxyl groups in the conserved threonines in DAFP-1 and the hydrogen bond donor/acceptor atoms on the growing faces of (100) and (010) in form I of m5U, DAFP-1 can recognize the fast growing face of (100) and the (010) face of form I through hydrogen bonding interactions. Hence, form I nuclei can be completely inhibited in the presence of DAFP-1 and then the extent of supersaturation increases during the following days until the crystallization of form II around day 5 (FIG. 22-FIG. 23(b)). That is the higher degree of supersaturation attained in the presence of DAFP-1 is required for the formation of form II nuclei. In addition, due to the mismatch between the hydrogen bond donor/acceptor atoms in DAFP-1 and in the growing faces of form II, DAFP-1 cannot interact with the newly formed form II nuclei. Consequently, only form II grows, even though it is a thermodynamically and kinetically less preferred phase.
DAFP-1 can induce m5U to crystallize into a thermodynamically and kinetically less preferred dihydrate form and the structure analysis suggests that the mechanism of AFP-induced crystallization control of m5U is similar to that of antifreeze action of AFPs. One can view all ice polymorphs as “ice hydrates” since water molecule itself is the building unit of ice. Ice Ih is the most common form of ice, which is highly stable and the most dangerous to life. The action of AFPs on ice can be similar to that on m5U. AFPs may induce the crystallization of water into new ice polymorph(s), which are thermodynamically and kinetically less preferred ice polymorph(s) of ice comparing to the common, highly stable Ih. The AFP-induced new ice polymorph(s) readily melt, and hence the net effect is antifreeze.
Materials and Methods
Chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) at ACS grade or better and used as received Milli-Q water produced from a Synergy water system (Millipore) with a minimum resistivity of 18 MΩ·cm was used for making solutions. All the sample solutions were filtered through 0.2 μm filters before use. Sample vials (10 mL, National Scientific) were used for crystallization. Glassware and stir bars were cleaned as previously described.
Synthesis
The syntheses of o,o′-bis(trimethylsilyl)thymine and 5-methylurine used the following procedures modified from previous methods (FIG. 13). Trimethylchlorosilane (6.51 g, 0.06 mole) and thymine (3.78 g, 0.03 mole) were suspended in 100 mL dry benzene. The suspension was quickly stirred under N2 and 8.25 mL triethylamine in benzene was added dropwise. The mixture was then refluxed for 2 hours. O,O′-Bis(trimethylsilyl)thymine was crystallized upon standing at room temperature. After recrystallization for three times, the pure o,o′-bis(trimethylsilyl)thymine (7.55 g, yield 93%) appeared as slightly yellow crystals. Then, the obtained o,o′-bis(trimethylsilyl)thymine (11.90 g, 0.044 mole, 10% excess) and β-D-ribofuranose 1-acetate 2,3,5-tribenzoate (20.18 g, 0.04 mole) were first mixed in 80 mL CH3CN under N2. The mixture temperature was kept at 0° C. and stirred. SnCl4 (41.34 mL of 1.0 M) in methylene chloride was added dropwise. The reaction was run at 0° C. for 2 hours and warmed up to room temperature for another hour. KHCO3 (60 g) in 60 mL water was added to remove the trimethylsilyl protected group. The solvents were removed under reduced pressure at 60° C. The product was a white solid or crystals after silica gel chromatography using EtOAc in CH2Cl2 as eluent and the formation was revealed by TLC analysis (Rf=0.60) using 20% EtOAc in CH2Cl2. Sodium ethoxide in ethanol (668 mL, 21% w/v) was used to remove the Bz protecting group. The solvents were removed under reduced pressure at 50° C. The product was a white solid or crystals after silica gel chromatography using ethanol in CH2Cl2 as eluent and the formation was revealed by TLC analysis (Rf=0.70) using 25% EtOAc in CH2Cl2 and LC/ESI-MS and NMR (FIG. 14 and FIG. 15).
Preparation of Antifreeze Protein
DAFP-1 was expressed and purified as described previously. The purified protein was characterized using SDS-PAGE gel electrophoresis, MALDI-TOF mass spectrometer, circular dichroism (CD) spectrometry, and differential scanning calorimetry (DSC), respectively, as previously described and the identity of DAFP was confirmed. The concentration of stock DAFP-1 solution was determined using a Cary 100 Bio UV-Vis spectroscopy (Varian) and the extinction coefficient of 5.47×103 M−1 cm−1 at 280 nm was used.
Crystallization
Supersaturated m5U aqueous solutions (0.6 M) were made at 30° C. and filtered as soon as possible. Crystals were not grown after the temperature dropped to room temperature for a few days. On day 1, to each sample vial was added 600 μL of 0.6 M m5U solution, and then 5 μL of water or DAFP solution were added into the vial. The vials were gently swirled after the additions. The resulting m5U concentration was 595 mM in each vial and additive/m5U molar ratios (×10−6) were either 0 or 5. The sample vials were left open in the air at room temperature and three observations at least were recorded per day (every 8 hours) until the solutions in all the vials were dry. The above experiments were repeated five times. Optical micrographs were taken under Nikon SMZ800 microscope with a Nikon Coolpix 5400 when the crystallization completed.
Crystallization Kinetics
Under the above conditions, only form I crystalline m5U was able to grow in the absence of DAFP-1; however, in the presence of DAFP-1, form II crystalline m5U was grown exclusively. The kinetics of crystallization of m5U form I and form II, respectively, was estimated based on the rate of the weight of the occurring crystals and the initial weight of m5U in the specific vial. All the liquid in the vial was collected gently and the weight of the vial or the vial with crystals was measured. After the measurement, the collected liquid was put back into the vial and the crystallization continued. The weights were measured on an Ohaus Discovery semi-micro analytical balance at 9 am every day until the crystallization finished. The experiments were repeated on three vials and the average values were reported.
Single Crystal X-Ray Diffraction
Colorless crystal of m5U form I was mounted on a Cryoloop with Paratone-N oil and data was collected at 90K with a Bruker APEX I CCD using Cu K alpha radiation generated from a rotating anode. In a similar fashion, a colorless crystal of m5U form II was mounted on a Cryoloop with Paratone-N oil and data was collected at 100 K with a Bruker APEX II CCD using Cu K alpha radiation generated from a rotating anode. For both crystals data were corrected for absorption with SADABS and structures were solved by direct methods. All non-hydrogen atoms were refined anisotropically by full matrix least squares on F2. Hydrogen atoms on 0 and N atoms were found from Fourier difference maps and were refined isotropically with distances of O—H and 0.85 (0.02) or 0.86 (0.02) Å and N—H distance 0.87 (0.02) Å and at 1.20 or 1.50 Ueq parent atom. All other hydrogen atoms were placed in calculated positions and refined as riding models with C—H distances of 0.950 Å (CHar), 1.000 Å, (CH), 0.990 Å (CH2), 0.980 Å (CH3) and at 1.20 or 1.50 Ueq of parent C atom. Flack parameters for m5U forms I and II were −0.0187 and −0.1358. Bruker suite of X-ray data collection (APEX2 and SAINT), and Shelrick's processing and refinement programs (SHELXS97, SHELXL97, SHELXTL) were used for these structural determinations. The crystallographic data of m5U forms I and II have been deposited in the Cambridge Database (CCDC) and the CCDC deposit numbers are 916820 and 916088, respectively.
Differential Scanning Calorimetry (DSC)
All experiments were performed with a DSC 1 (Mettler Toledo, Ohio). An indium standard (Mettler Toledo, Ohio) was used to calibrate the instrument. Samples weighed 3-4 mg and were hermetically encapsulated in standard aluminum sample pans with pierced lids to release any pressure build-up during the experiments. The samples were heated from 25 to 200° C. at a rate of 5° C./min.

## Claims

1. A method of using an antifreeze protein (AFP) to inhibit the nucleation and crystallization of a nucleoside compound from a solution comprising:
obtaining a solution containing the nucleoside compound and an effective amount of at least one AFP, wherein the nucleoside compound can crystallize into an orthorhombic structure in the absence of AFP.

2. The method of claim 1, wherein the AFP is selected from the group consisting of natural or engineered antifreeze proteins, antifreeze polypeptides and antifreeze peptides, active fragments of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, mimetics of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, their active mimetic fragments, and combinations thereof.

3. The method of claim 1, wherein the nucleoside compound is selected from the group consisting of 5-methyluridine, cytidine, inosine, derivatives thereof, and combinations thereof.

4. The method of claim 1, wherein the AFP is either beetle D. canadensis AFP (DAFP-1) or globular fish AFP type III, wherein the nucleoside compound is selected from the group consisting of, 5-methyluridine, cytidine, inosine, derivatives thereof, and combinations thereof, wherein the ratio of the AFP to the nucleoside compound is about or less than about 0.05% by weight; and
wherein said method ensures the inhibited nucleation and crystallization affects about 100% of the total amount of the nucleoside compound.

5. A method of using an antifreeze protein (AFP) to alter the crystalline shape of a nucleoside compound formed from a solution comprising:
substantiating a first crystalline shape-of a nucleoside compound, wherein the nucleoside compound can crystallize into an orthorhombic structure in the absence of AFP, and wherein the nucleoside compound was crystallized into the first crystalline shape in the absence of AFP;
obtaining a solution containing the nucleoside compound;
confirming a seed crystal of the nucleoside compound is in the solution;
adding an effective amount of at least one AFP to the solution, wherein the seed crystal is present and wherein the ratio of AFP to the nucleoside compound is about or less than about 0.05% by weight;
verifying that the nucleoside compound forms solid crystals having a second crystalline shape that is different from the first crystalline shape; and
ensuring that about 100% of the formed solid crystals have the second crystalline shape.

6. The method of claim 5, wherein the AFP is selected from the group consisting of natural or engineered antifreeze proteins, antifreeze polypeptides and antifreeze peptides, active fragments of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, mimetics of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, their active mimetic fragments, and combinations thereof.

7. The method of claim 5, wherein the nucleoside compound excludes ice and gas hydrates.

8. A 5-methyluridine dihydrate crystal having an orthorhombic unit cell, wherein the unit cell has a first dimension measuring about 6.75 Å, a second dimension measuring about 8.84 Å, and a third dimension measuring about 20.98 Å, wherein the angle between the second dimension and the third dimension is about 90 degrees, wherein the angle between the third dimension and the first dimension is about 90 degrees, and wherein the angle between the first dimension and the second dimension is about 90 degrees.

9. Compositions comprising a 5-methyluridine dihydrate crystal, as claimed in claim 8.

10. A method of using an antifreeze protein (AFP) to produce a 5-methyluridine dihydrate crystal comprising:
obtaining a supersaturated 5-methyluridine aqueous solution with an effective amount of at least one AFP in the solution, wherein the ratio of AFP to 5-methyluridine is about or less than about 0.05%; and
ensuring the 5-methyluridine dihydrate crystal is a blocky shape, wherein the length of the longest side of each of the 5-methyluridine dihydrate crystal is no shorter than about 1000 micrometers, and wherein the length of the shortest side of the 5-methyluridine dihydrate crystal is no shorter than about 500 micrometers.

11. The method of claim 10, wherein the AFP is selected from the group consisting of natural or engineered antifreeze proteins, antifreeze polypeptides and antifreeze peptides, active fragments of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, mimetics of antifreeze proteins, antifreeze polypeptides and antifreeze peptides, their active mimetic fragments, and combinations thereof.