CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos.

62/261,621 filed December 1, 2015 and 62/278,126 filed January 13, 2016, which are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Chemical sensors are analytical tools that provide information on real-time processes through the measurement of a specific compound, molecule, ion, or energy change in a complex system. Fluorescent chemical sensors are optical devices that provide information through a signal transduction that arises from a physical or chemical interaction with a sample (analyte) in a solution. The signal is transmitted to a fluorophore (a molecular dye) that then emits an electromagnetic signal within the ultraviolet to visible light spectrum. The strength of the signal corresponds directly to the sample concentration. Fluorescent sensors are usually very sensitive, with little required to detect even trace levels of the emitted radiation. As they continuously operate without the need for an external power source, they are ideally suited for non-invasive, low-cost in vivo monitoring of biologically relevant processes. Of considerable interest has been the development of in vivo biosensors for continuous glucose monitoring. In 2012, the global revenue generated from glucose sensing devices was nearly $200 million, and the value for this market is predicted to nearly triple by 2020, reaching $568.5 million at a CAGR of 14.8% from 2013 to 2018. A continuous glucose sensor that is non-invasive, cheap, and user friendly stands to capitalize on this market.

DESCRIPTION OF FIGURES

[0003] FIG. 1 describes the general di-acid/fluorophore unit, wherein the bis-boronic acids are separated by a carbon chain of six-units in length. The reporter pyrene is located on the amine terminus.

[0004] FIG. 2 shows the present invention, wherein the glucose-sensing unit(s) are appended to arms of a polyoxazoline palmer chain. The aryl-boronic acids have an electronically controlled group para to the boron to allow for optimization of the device to applications in which the pH, concentration, and required selectivity can change.

[0005] FIG. 3 illustrates an overall synthetic route to the device. The oxazoline monomers are microwave irradiated with an appropriate initiating and terminating species. In the present example, methyl triflate and a propenamide are used, respectively.

Deprotection of the carbonate arms, then reductive amination allows for the introduction of the fluorescent reporting units and aryl-boronates.

[0006] FIG. 4 illustrates an overall synthetic route to the device.

DESCRIPTION OF THE INVENTION

[0007] Issues in biosensor design for glucose monitoring may stem from physical limitations of a molecular sensor in a physiological system. Improvements on sensor solubility and ligand selectivity may provide benefits. Further opportunities for

improvement may lead to a clinically suitable dye. Two relevant issues are: 1) the covalent grafting of the sensor to a polymer matrix and what effect tethering will have on analyte binding and selectivity; and 2) the means by which an accurate, quantifiable signal will be sent upon analyte recognition, whether through fluorescence or some other absorbance based methods. While fluorescence detection is both more selective and sensitive than other absorption measurements, direct fluorescence is prone to interference, photo bleaching, and molecular instability from other light sources. The elimination of many of these issues can be resolved using referenced detection techniques, which are independent of fluctuations in light intensity, detector sensitivity, leaching, photo bleaching,

concentration of the luminophore, and changes in the optical alignment. These methods often increase the number of steps needed in the synthesis, making them lengthier, reducing overall yield, increasing costs, and modifying the sensor's solubility. Additionally, the fluorophores selected for ratiometric measurements need to possess similar photostabilities as, otherwise, drift will be observed caused by two different photo- bleaching rates, limiting the types of fluorescent dyes that can be used. Regardless of the technique, sensitivity and selectivity are key to accurate analyte recognition. Ideally, the fluorophore has a high extinction coefficient, minimizes photobleaching effects and self- quenching, has a large Stokes shift to reduce signal interference, and the emitted signal is able to penetrate tissue without being absorbed or inducing autofluorescence of biological molecules.

[0008] Synthesis of an optical sensor may benefit from a synthetic plan that minimizes product loss, toxic byproducts, and costs, as well as beneficially accounts for influences each environment will have on the dye's properties. Each application may place constraints on the type of fluorescent dye. An effective in vivo fluorescent sensor may be selected based on several considerations. The fluorescent biosensor may be compatible with hydrophilic systems and may be chemically, mechanically, and thermally stable. This may be beneficial for stability in vivo as well as during deposition and chemical

modifications by which the sensor is incorporated. The ability to covalently bind the sensor to prevent dye migration and leaching is useful for sensor longevity and biomedical applications. The sensor may operate at a pH suitable for the target application, ideally over a wide range. It follows that the sensor may also possess an appropriate pK _a , which prevents the use of many well-known and widely available chemical functionalities for these applications. Selective interactions with a predetermined analyte is also useful, especially so for glucose recognition where the problem is compounded by glucose equilibration to other isomeric forms, the prevalence of other similar biological saccharides, and that saccharides possess hydroxyl functionalities that make them hydromimetic with bulk water.

[0009] Towards the goal of achieving a biologically functional in vivo glucose sensor, we have developed compounds of formula (I),

(I) which is a poly(oxazoline) derived brush-copolymer with glucose-selective receptor and fluorescent reporting side chain.

[0010] ¹ of formula (I) may be any functional handle which allows for the introduction of crosslinking moieties for substrate specific immobilization. In an alternative embodiment, R ¹ is one or more substituents selected from the group consisting of

, and

where the arrow points to the remainder of the compound.

[0011] R ² of formula (I) is hydrogen, an electron-withdrawing group, and/or electron-donating group. In an alternative embodiment, R ² is hydrogen. An electron- donating group may be, for example, -O ^" , -NR ₂ , -NH ₂ , -OH, OR, NH(C=0)R, -COOR (where R is an alkyl group in the preceding examples) or other group known to one of skill in the art. An electron-withdrawing group (also called "electron-attracting" or "negative" group) may be, for example, -CN, -COOR, -COOH (but not -COO ^" salts), -CHO, -(C=0)R, -S0 ₃ H, -N0 ₂ , -NO, - ONO, halogen, -N R3 ⁺ , -NH3 ⁺ (but not the neutral amines) (where R is an alkyl group in the preceding examples), or other group known to one of skill in the art.

[0012] In formula (I), x is an integer in a range from 0 to 50. In an alternative embodiment, x is an integer in a range from 0 to 25. In another alternative embodiment, x is 0.

[0013] In formula (I), y is an integer in a range from 1 to 50. In an alternative embodiment, y is an integer in a range from 20 to 40. In another alternative embodiment, y is 25.

[0014] In formula (I), m is an integer in a range from 2 to 10. In an alternative embodiment, m is an integer in a range from 3 to 8. In another alternative embodiment, m is in an integer in a range from 4 to 7. In yet another alternative embodiment, m is 5 or 6.

[0015] In formula (I), n is an integer in a range from 2 to 30. In an alternative embodiment, n is an integer in a range from 2 to 20. In another alternative embodiment, n is an integer in a range from 4 to 10. In yet another alternative embodiment, n is 6. [0016] In formula (I), Fl represents a fluorophore, which is a fluorescent moiety, preferably a polyaromatic hydrocarbon or a polyheteroaromatic moiety (where at least one heteroatom is nitrogen or oxygen), and is optionally substituted with a substituent that does not interfere with fluorescence, such as alkyl. Suitable fluorophores include xanthene and derivatives thereof, cyanine and derivatives thereof, naphthalene and derivatives thereof, coumarin, oxadiazole-type (such as pyridyloxazole, nitrobenzoxadiazole, and

benzoxadiazole), anthracene and derivatives thereof, pyrene and derivatives thereof, oxazine-type, acridine and derivatives thereof, arylmethine-type, and tetrapyrrole and derivatives thereof. A preferred polyaromatic hydrocarbon is pyrene attached at any chemically reasonable ring-carbon.

[0017] In an embodiment, the fluorophore of the compound of formula (I) is pyrene, as shown in formula (la),

[0018] When synthesizing a compound according to formula (I), starting reagents may include

1

(where m is an integer in a range from 2 to 10 and where this starting reagent, as a result of synthesis, may ultimately be incorporated in a compound of formula (I) as

where y, m, n, ² , and Fl are as previously defined), and

o ·····-···-

(which, as a result of synthesis, may ultimately be incorporated in a compound of formula

as where x is as previously defined).

[0019] In an embodiment, starting reagents are

where m is 5 or 6, and

These starting reagents may be mixed together or may be added separately and

consecutively.

[0020] The fluorescent property of the dye is highly dependent upon the interactions between the amine and the boronic acid. Increasing either the Lewis acidity of the boronic acid, or the Lewis basicity of the amine results in a stronger interaction between the pair. The increase in this interaction leads to increased fluorescence emission intensity as well as the ability to operate at lower pHs. The inclusion of electron-withdrawing groups on the phenylboronic acid or electron-donating groups at the amine may enhance this effect (it may be noted that this is desirable only to a point, as too strong of a B-N interaction would decrease the binding affinity of the dye to glucose). The nature of the acid-amine pair in saccharide sensing has been studied extensively, and the N-alkyl-o-(aminomethyl)phenyl boronic acid fragment has been shown to be very effective in facilitating the fluorescent signal upon substrate binding. For this reason, the electronics of the fragment are to be optimized as described above.

[0021] The diboronic acid moiety is both glucose specific and has an affinity ~22x greater than the mono-acid, and the spacing between these residues has been shown to be best suited for glucose binding when it is 6 carbon units in length. Changing this spacing will decrease glucose specificity, but can increase the affinity for other types of saccharides. The fluorophore is proximal to one amine to allow for quenching to be unambiguous. The nature of the fluorophore is dependent on several factors, including the surface area of the π- system, hydrophobicity, and sterics. For diboronic systems, the greatest stability binding constant was observed with pyrene (K ₀ bs= 962 ± 70, compare to anthracene K ₀ bs= 441 ± 76).

[0022] Despite pyrene having a larger area and being more hydrophobic than anthracene, anthracene has more per/ ^' -hydrogens that prevent effective binding of the saccharide upon complexation. Further studies have shown that glucose, despite being more hydrophilic than other congeners, paradoxically has stronger affinities for more hydrophobic pockets. As pyrene is more hydrophobic than anthracene, this effect further amplifies the discrepancy between the two flourophore binding stabilities.

[0023] While polyoxazolines have gained considerable attention as PEG alternatives for biological applications due to their ease of synthesis, their highly tunable nature, and their biocompatibility, they have not yet been used for in vivo optical biosensing applications. Their dual-solubility in both chlorinated and aqueous solvents makes them versatile polymers that are readily modifiable for a variety of applications. In the present example, the polyoxazoline chain is end-capped with an acrylamide handle, but this can be any functional handle to allow for the introduction of crosslinking moieties for substrate specific immobilization.

[0024] A compound according to the invention can be used to determine saccharide level in biological fluids, including, but not limited to, blood, urine, sweat, tears and interstitial fluid. In an embodiment, a compound according to the invention can be used to determine glucose level in interstitial fluid.

[0025] Methods of incorporating a compound and/or composition according to the invention may be continuous or discrete and include, but are not limited to, use of an incorporation device, such as minimally invasive devices; an implantable device; a device that interfaces with blood, such as from a finger prick or a device capable of sampling blood; and/or a device that interacts with tears on a patient's cornea.

[0026] In an embodiment, a compound and/or composition according to the invention is incorporated via a minimally invasive device that interfaces with blood and is accessible from the top of a patient's skin. In another embodiment, a compound and/or composition according to the invention is incorporated via an implantable device that interfaces with blood from within blood vessels. In another embodiment, a compound and/or composition according to the invention is incorporated via a device that interfaces with blood, such as a finger prick or a device capable of sampling blood. [0027] In an embodiment, a compound and/or composition according to the invention is incorporated via a minimally invasive device that interfaces with urine, such as a test strip.

[0028] In an embodiment, a compound and/or composition according to the invention is incorporated via a minimally invasive device that interfaces with sweat on the skin.

[0029] In an embodiment, a compound and/or composition according to the invention is incorporated via a minimally invasive device that interfaces with tears on the cornea. In another embodiment, a compound and/or composition according to the invention is incorporated via an implantable device that interfaces with tears from within the body such as tear ducts. In another embodiment, a compound and/or composition according to the invention is incorporated via a device that interacts with tears on the cornea.

[0030] In an embodiment, a compound and/or composition according to the invention is incorporated via a minimally invasive device that is accessible from the top of skin, such as optically transparent microneedles. In another embodiment, a compound and/or composition according to the invention is incorporated via an implantable device that is not accessible from the top of a patient's skin. In another embodiment, a compound and/or composition according to the invention is incorporated via a device that interfaces with interstitial fluid extracorporeal^. In another embodiment, a compound and/or composition according to the invention is incorporated via a device that interfaces with interstitial fluid intradermally.

[0031] Methods of detection fluorescence using a compound and/or composition according to the invention include, but are not limited to, using a minimally invasive and wearable fluorescence detection device to measure fluorescence intensity or fluorescence lifetime; using an implantable or non-implantable fluorescence detection device to measure fluorescence intensity or fluorescence lifetime; and/or using a fluorescence detection device to measure fluorescence intensity or fluorescence lifetime. EXAMPLES

[0032] Synthesis of (1):

Di-tert-butyl dicarbonate (25.0 g, 114 mmol) was added to a stirred mixture of 15.0 g (114 mmol) of 6-aminohexanoic acid and 20.6 mL (150 mmol) of triethyl amine (TEA) in 160 mL dry THF. After 3 days, 300 mL water was added. The pH was adjusted to 5 with 1 M aqueous KHSC -solution, and the product was separated by extraction with ethyl acetate, which was subsequently removed under reduced pressure. The solid residual was identified as the desired protected amino acid (yield 1/4 22.4 g (85%)).

[0033] 5.2 g (22.5 mmol) of Boc-amino acid were dissolved in 70 mL of dry THF and 3.13 mL triethylamine (22.5 mmol). At 0°C, 2.9 mL (22.5 mmol) of iso-butyl chloroformate was added under nitrogen atmosphere and the mixture was stirred for 5 min at 0 °C, and a further 10 min at room temperature. After cooling to 0 °C, 2.6 g (22.5 mmol) of 2- chloroethyl amine hydrochloride, dissolved in 10 mL of dry DMF and 3.13 mL of TEA (22.5 mL), was added, and the mixture was stirred for 1 h at room temperature. The solvent was evaporated and the residue dissolved in DCM. The solution was extracted with 10% (w/w) Na ₂ C03 and saturated aqueous NaCI solutions. The solvent was evaporated and the remaining oil dissolved in diethyl ether. The product was precipitated in n-hexane, 4.8 g (yield 1/4 73%) of the product was collected.

[0034] 5.5 g (18.7 mmol) of tert-Butyl 5-(2-Chloroethylcarbamoyl) Pentyl Carbamate were dissolved in 40 mL of dry DMF and 5.1 g (37.4 mmol) of dry K2CO3 was added. The solution was stirred for 5 h at 70 °C under a nitrogen atmosphere. The solvent was evaporated under reduced pressure and the remaining solid was dissolved in DCM and successively filtered to remove the excess K2CO3. The solvent was evaporated and 4.1 g (yield 1/4 86%) of 1 was collected.

[0035] Synthesis of (3):

Poly[(2-methyl-2-oxazoline)-co-(N-Boc-5-aminopentyl-2-oxazol ine)], P(EtOx45-co- NBocOx ₅ )stat. MeOTf (50 mg, 0.3 mmol, 1.0 equiv), and EtOx (13.7 mmol, 45 equiv) were added to 7.5 mL of ACN. Heating to 135 °C for 40 min was performed by a microwave system (150 W). The reaction mixture was cooled to T, and 1 (1.52 mmol, 5 equiv) was syringed into the vial. Mixture was reheated for 20 minutes, after which, the solution was cooled to RT, benzyl alcohol (0.94 mmol, 3.2 equiv) was added, and was stirred O/N for termination. The solution was precipitated 3x into diethyl ether and filtered. The collected solid was freeze-dried, and a colorless solid was obtained (94%).

[0036] Synthesis of (4):

To a stirred solution of 5.3 mL (7.71 g, 60.7 mmoles 1.1 eq) oxalyl chloride in 100 mL methylene chloride (2mL/mmol) at -70° C. under argon was added 8.6 mL (10.23 g, 121.5 mmoles 2.2 eq) dimethyl sulfoxide in 30 mL methylene chloride (0.25 mL/mmol DMSO). After 2 minutes a solution of 12 g (55.2 mmoles) of N-Boc-6-amino-l-hexanol in 50 mL methylene chloride was added over 5 min. After 20 min. 38.5 mL (27.9 g, 276 mmoles 5 eq) triethylamine was added. After 10 minutes more, the mixture was allowed to warm to 20° C. and 250 mL (4.5mL/mmol) water was added. After separation, the aqueous layer was extracted again with methylene chloride and the combined organic layers were washed with brine and dried over sodium sulfate. After evaporation in vacuo, the residue was chromatographed (Flash, hexane-ethyl acetate, 7:3) to provide 11.36 g of a pale yellow oil (95.6%).

[0037] Synthesis of (5):

The Boc protected polymer (200-500 mg) was dissolved in 2 mL of trifluoroacetic acid and DCM (1:1) and the solution was stirred for 15 h. After solvent evaporation, the residue was precipitated with diethyl ether and isolated by centrifugation.

[0038] A solution of deprotected 3 (2.42 mmol) and 4 (2.90 mmol 1.2 eq) in THF and methanol was stirred at room temperature for 20 hours under a nitrogen atmosphere. The solvent was removed under vacuum, and the residue was washed with methanol. The precipitate was filtered off, the filtrate was removed and dried under vacuum to give a yellow oil (940mg, 93%). A solution of N-benzyl-N'-pyren-lyl methylene hexane-l,6-diamine (420 mg, 1.00 mmol) and sodium borohydride (190 mg, 5.00 mmol 5 eq) in methanol (10.0 ml) was stirred at room temperature for 3 hours under a nitrogen atmosphere. The solvent was removed under vacuum, and the residue was dissolved in chloroform and washed with water, and dried over magnesium sulphate. The solvent was removed and the residue was dried under vacuum to give a yellow oil (92%).

[0039] Synthesis of (6):

Deprotection of the terminal amine performed as previously described. A solution of pyrenecarboxaldehyde (614.8 mgs, 2.67 mmol, 1 eq.) in THF (0.2 mol dm ³ ) was added with heating at reflux to a solution of hexamethylenediamine (1.55g, 13.3 mmol, 5 eq.) and toluene-p-sulfonic acid (2.53 g, 13.3 mmol, 5 eq.) in ethanol (1 mol dm ³ ) and then the mixture was heated to reflux for 3 h under a nitrogen atmosphere. After cooling to room temperature, sodium borohydride (303.0 mg, 8.01 mmol, 3 eq.) was added to the solution which was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and the residue was dissolved in chloroform. The chloroform phase was washed with water, and dried over magnesium sulfate and the solvent was then removed under reduced pressure to yield a yellow oil (78%).

[0040] Synthesis of (7):

A solution of 6 (291 mg, 0.69 mmol), 2-(2-bromomethyl-phenyl)- [l,3,2]dioxaborinane (422 mg, 1.66 mmol, 2.4 eq), and potassium carbonate (380 mg, 2.76 mmol 4 eq) in dry acetonitrile (10 ml 15 mL/mmol) was heated at reflux for 20 hours under nitrogen atmosphere. The solvent was removed under vacuum, the residue was dissolved in chloroform and washed with water. The solvent was dried over magnesium sulphate and removed under vacuum (172 mg, 35%).

[0041] The remaining steps are carried out by methods known in the art.

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