Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
  • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
  • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
  • A61B5/1459—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters invasive, e.g. introduced into the body by a catheter

Definitions

• BIOLOGICAL MOLECULES USING BORONATE BASED CHEMICAL AMPLIFICATION AND OPTICAL SENSORS by William Van Antwerp et al., filed on December 14, 1999, which is a Continuation of United States Patent Application Serial No. 08/749,366, now U.S. Patent No. 6,002,954 which claims the benefit of U.S. Provisional Patent Application Serial No. 60/007,515, filed November 22, 1995; and United States Patent Application Serial No. 09/078,392 "DETECTION OF BIOLOGICAL MOLECULES USING CHEMICAL AMPLIFICATION AND OPTICAL SENSORS", by William Van Antwerp et al., filed on November 21, 1999, which is a Continuation of U.S.
• This invention relates to the design of fluorescent compounds used as glucose sensors that have selected fluorescent properties, such as long fluorescence lifetimes, long absorption or emission wavelengths, or high quantum yields. More particularly, the invention relates to biomedical sensors for continuous transdermal optical transduction of tissue glucose concentration for the treatment of diabetes.
• U.S. Patent No. 5,503,770 to James et al. discloses a fluorescent compound used for the detection of saccharides or sugars such as glucose.
• the use of these fluorescent compounds was extended in U.S. Patent No. 6,002,954 to Van Antwerp et al., by incorporating the compounds in an implantable optical sensor for transduction of glucose concentration for the treatment of diabetes.
• the fluorescent transducer is implanted 1-3 mm below the surface of the skin and optically interrogated externally to determine the level of tissue glucose in diabetic patients.
• a minimally-invasive, continuous glucose sensor is of great benefit to patients in achieving tighter blood-glucose control when combined with existing insulin pumps.
• the fluorescent compound should operate at longer wavelengths than about 450 nanometers.
• the transmission through a few millimeters of skin increases logarithmically with wavelength - from 0.1% at about 400 nm to almost 100% at 850 nm.
• the longer the wavelength the greater the transmission through skin.
• An excitation and emission wavelength greater than about 600-650 nm is an enormous improvement over about 400-450 nm. Because of the significant increase in optical skin transmission at longer wavelengths, a practical glucose sensor can operate more efficiently, more accurately, and with a greater signal-to-noise ratio.
• This invention addresses the optical transmission problem and provides exemplary fluorescent compounds that have been demonstrated to exhibit the needed photochemical behavior and operate in a wavelength range that makes a subcutaneous fluorescent glucose sensor practical.
• the invention disclosed herein provides fluorescent analyte binding compounds defined by a specified formula and wherein substituent molecules of these compounds are selected to have complimentary molecular properties which can be determined via simplified version of the Rehm-Weller equation.
• the representative molecules of the invention have a number of exceptional activities that make them uniquely suited for incorporation into a sensor for analytes such as sugars. These activities include highly desirable fluorescent properties such as longer excitation and emission wavelengths, properties which are highly compatible with their use in minimally implantable systems for the continuous transdermal monitoring of blood glucose concentrations.
• Another object of the invention is to provide a sensor comprising a biocompatible polymer containing covalently bonded fluorescent molecules that specifically and reversibly bind glucose. Upon binding with the glucose, the fluorescence quantum yield of the molecules is increased, resulting in increased fluorescent emission at increasing glucose concentrations. Glucose transduction is achieved at wavelengths that allow the sensor to be implanted subcutaneously and permit improved optical transmission through skin.
• the invention basically involves a fluorescent compound having three functional components in one molecule: a substrate-recognition component (typically a substituted aryl boronic acid), a fluorescence "switch" that is mediated by a substrate recognition event (typically an amine), and a fluorophore.
• a substrate-recognition component typically a substituted aryl boronic acid
• a fluorescence "switch" that is mediated by a substrate recognition event (typically an amine)
• a fluorophore typically an amine
• the sensor molecule is designed so that the photo-excited fluorophore and the boron atom compete for the unbonded amine electrons. In the absence of glucose binding, electron transfer occurs predominantly with the fluorophore, causing fluorescent quenching and subsequently weak emission.
• glucose is bound to the boronate group, the average charge on the boron atom becomes more positive, which increases the attraction of the unbonded electrons, preventing electron transfer, thus
• Glucose transduction has been successfully achieved by three typical classes of candidate molecules that were investigated: transition metal-ligand boronate compounds and conjugated organic heterocyclic ring system compounds that are oxazine, oxazine-one, oxazone, and thiazine boronate compounds and anthracene boronate compounds.
• transition metal-ligand boronate compounds and conjugated organic heterocyclic ring system compounds that are oxazine, oxazine-one, oxazone, and thiazine boronate compounds and anthracene boronate compounds.
• these compounds are excited at wavelengths greater than about 400 nm.
• these compounds typically operate (emit) at wavelengths greater than about 450 nm.
• a glucose sensor according to the present invention can be incorporated into a minimally invasive, implantable system for continuous transdermal monitoring of glucose levels in diabetic patients.
• glucose sensors can be designed having other desired molecular properties, such as a longer fluorescence lifetime, specific excitation and emission wavelengths, high quantum yields, photostability, chemical stability, high water solubility, low temperature sensitivity, or low pH sensitivity.
• the present sensor/transducer scheme is useful more generally for the measurement of any cis-diol.
• the present sensor molecules have utility in the measurement of ethylene glycol contamination in boiler waters, where ethylene gycol contamination is an indication of heat exchanger tube degradation.
• These sensor molecules in addition to being used as glucose sensor molecules for diabetics, can be of use in industrial fermentation processes (e.g. beer and wine), or in any number of process points in the production of high fructose corn syrup (e.g. enzyme reactors).
• Figure 1 shows the generalized formula of the fluorescent compound of the present invention.
• Figure 2 shows examples of transition metal-ligand fluorophores according to the present invention.
• Figure 3 shows an example of a thiazine fluorophore.
• Figure 4 shows examples of oxazine fluorophores.
• Figure 5 shows examples of oxazine-one and oxazone fluorophores.
• Figure 6 shows the excitation and emission spectra of Ru(bistrifluoromethylbipyridine) in methanol/buffer.
• Figure 7 shows Ru(N-methyl benzyl boronate).
• Figure 8 shows a time history of Ru(N-methyl benzyl boronate) with additions of acid and base.
• Figure 9 shows a time history of Ru(N-methyl benzyl boronate) with additions of glucose.
• Figure 10 shows chloro-oxazine-5-one boronate.
• Figure 11 shows the excitation and emission spectra obtained for a sample of chloro-oxazine-5-one boronate in methanol/buffer.
• Figure 12 shows results of adding glucose to chloro-oxazine-5-one boronate.
• Figure 13 shows the results of a glucose reversibility experiment.
• Figure 14 shows an implantable glucose sensor system.
• Figure 15 shows exemplary saccharide sensing boronic acid derivatives of anthracene.
• Figure 16 shows a schematic drawing of PET sensors: a) in the absence of analyte, the electron donor (D) quenches the fluorescence of the electron acceptor/fluorophore (A); b) in the presence of analyte, electron transfer does not occur and A fluoresces.
• Figure 17 shows a scheme for the synthesis of typical boronate and benzyl bipyridine ligands of the invention.
• Figure 18 shows a scheme for the preparation of typical transition metal complexes of the invention.
• FIG. 20 shows a scheme pertaining to the synthesis of COB (Chloro- Oxazine Boronate) and is shown below as benzophenoxazinone 11.
• COB Chloro- Oxazine Boronate
• benzophenoxazinone 11 was constructed by coupling benzophenoxazinone 6a with phenyl boronate 10 via a methylene amine linkage.
• Figure 21 shows a scheme pertaining to the synthesis of COB (Chloro- Oxazine Boronate) and is shown below as benzophenoxazinone 11.
• Benzophenoxazinone 6a was synthesized by condensation of 3-amino-4- hydroxybenzyl alcohol 3 with 2,3-dichloro-l,4-napthoquinone 4.
• Figure 22 shows a scheme pertaining to the synthesis of COB (Chloro- Oxazine Boronate). After ring condensation, benzophenoxazinone 5 was then converted to the benzophenoxazinone bromide 6 using phosphorous tribromide in an ether/toluene solvent mixture at room temperature.
• COB Chloro- Oxazine Boronate
• Figure 23 shows a scheme pertaining to the synthesis of COB (Chloro- Oxazine Boronate).
• An amino boronate derivative 10 was synthesized by bubbling methylamine through a etheral solution of phenyl boronate 9. Methylaminophenyl boronate 10 was isolated cleanly in 99% yield.
• Figure 24 shows a scheme pertaining to the synthesis of COB (Chloro- Oxazine Boronate).
• coupling of the aminophenyl boronate 10 and benzophenoxazine 6a was preformed in refluxing tetrahydrofuran using potassium carbonate for four days.
• the target benzophenoxazine 11 was purified by chromatography and isolated as solid in 61% yield.
• Figure 25 shows a scheme pertaining to the synthesis of the napthalamide boronate transducer derivatives taught in Example 3.
• Figure 26 shows a scheme pertaining to the synthesis of the napthalamide boronate transducer derivatives as taught in Example 3.
• Figure 27 shows sample excitation and emission spectra of typical napthalamide boronate transducer derivatives as taught in Example 3 (3ay and 3cy).
• Figure 28 shows relative quantum yields of typical napthalamide boronate transducer derivatives as taught in Example 3.
• Figure 29 shows the fluorescence spectra (relative fluorescence vs.
• the present invention provides a composition comprising a fluorescent sensor molecule that can undergo intramolecular electron transfer which in turn modulates the fluorescence as a function of the concentration of an analyte such as glucose, galactose, or fructose.
• fluorescent compounds can be designed that have desired fluorescent properties, such as selected abso ⁇ tion and emission wavelengths, high quantum yields, and longer fluorescence lifetimes.
• emission wavelengths greater than about 450 nm, and thus can be used to sense glucose in media with high opacities in visible blue or ultraviolet light, such as skin.
• compounds have been created with excitation wavelengths greater than about 400 nm, and thus have the benefit of being excited at wavelengths above ultraviolet.
• compositions can be immobilized in a glucose permeable biocompatible polymer matrix to form a sensor that is implantable a few millimeters below the skin surface.
• the generalized compound comprises a substrate recognition component (a phenylboronic acid), a fluorescence switch component that acts as an electron donor (typically an amine), and a fluorophore that acts as an electron acceptor.
• fluorescence quenching of the excited state fluorophore by the fluorescence switch component leads to a reduced background fluorescence level.
• an analyte such as glucose
• the glucose-recognition component reversibly forms a glucose boronate ester, resulting in a more electron deficient boron atom that ultimately interacts with or coordinates to the fluorescence switch.
• the complexation of glucose by the boronate inhibits the transfer of lone-pair electrons to the excited fluorophore, leading to an increase in fluorescence emission intensity and fluorescent lifetime, either of which can be accurately correlated to the ambient glucose concentration.
• the fluorescent compound can be immobilized in a glucose permeable biocompatible polymer matrix to form an implantable sensor. Because of its long wavelength operating range, the sensor can be interrogated by applying excitation light through the skin and monitoring the intensity or lifetime of the emitted fluorescence externally. The measurement of emitted light thus allows quantification of glucose concentration.
• the generalized sensor compound is shown in Figure 1 and comprises three components: a fluorophore (F), a substrate-recognition site, and a fluorescence switch.
• the substrate recognition site is provided by a phenylboronic acid - (C 6 H 5 )B(0R') 2 , where R 1 is hydrogen or lower aliphatic and aromatic functional groups.
• R 1 is hydrogen.
• the phenylboronic acid is bonded through optional linkage L 1 to the fluorescence switch Z, which is typically nitrogen (amine), but could be another electron donor heteroatom such as sulfur, phosphorus, or oxygen.
• the switch Z is bonded through a second optional linkage L 2 to the fluorophore F.
• the linkages L 1 and L 2 are 0-4 contiguous atoms selected from carbon, oxygen, nitrogen, sulfur and phosphorus.
• Optional groups R , R , and R 4 are attached respectively to the phenyl group, the switch Z, and the fluorophore F.
• Groups R 2 , R 3 , and R 4 may be functional groups that can form covalent linkages to a polymer matrix.
• R 2 , R 3 , and R 4 may be hydrogen or a lower aliphatic or aromatic functional group.
• the present invention provides a reliable model for fluorescent sensor molecule development, which represents a tremendous advantage in the effort to create, for the first time, new glucose sensor molecules that have desired fluorescent properties, for example, to operate at more favorable wavelengths.
• This model allows a systematic build-up of a new molecule in small, relatively simple steps, rather than proceeding directly from a paper design of the molecule to a lengthy campaign to synthesize a single compound that may or may not prove viable. The likely multiple repetitions of the process required to eventually produce a satisfactory glucose sensor molecule are prohibitively time-consuming.
• a functioning glucose sensor molecule comprising the three functional components: a glucose-recognition component, a fluorescence on/off switch mediated by a glucose recognition event, and a fluorophore.
• the selection of fluorophore can be used to affect a number of molecular properties: excitation and abso ⁇ tion wavelength, quantum yield, fluorescence lifetime, photostability, chemical stability, solubility, temperature sensitivity, or pH sensitivity.
• excitation and abso ⁇ tion wavelength quantum yield, fluorescence lifetime, photostability, chemical stability, solubility, temperature sensitivity, or pH sensitivity.
• PET photoinduced electron transfer
• Serroni et al. Chem- EurJ1999, 5, 3523-3527; Grigg et al., JChem S Ch 1994, 185-187; Gust et al., Account Chem Res 1993, 26, 198-205; Kavarnos, G. J. In Fundamentals of photoinduced electron transfer; VCH Publishers: New York, NY, 1993; p Chapter 1; Wasielewski, M. R.
• FIG. 16 A schematic of a typical PET sensor is shown in Figure 16.
• an electron donor such as an amine
• A an electron acceptor
• FIG. 16 An electron donor (D)
• D quenches the fluorescence of an electron acceptor (A) in absence of an analyte.
• the oxidation potential of D is lowered and electron transfer does not occur, giving an increase in fluorescence.
• Other systems are known to operate by PET, such as the ruthenium and rhenium bipyridine complexes reported by Meyer (Meyer, T. J. Account Chem Res 1989, 22, 163-170) and Lakowicz (Murtaza et al., Anal
• the Examples include descriptions of the synthesis and fluorescence studies of new boronic acid derivatives of rhenium and ruthenium bipyridine complexes.
• Previously Yam has reported a rhenium bipyridyl complex for the transduction of saccharides by absorbance (see e.g. Yam et al., Chem Commun 1998, 109-110 and Mizuno et al., J Chem Soc Perkin Trans 1 2000, 407-413), and Shinkai has previously synthesized other types of organic and inorganic bipyridyl boronic acids (see e.g. Mizuno et al., JChem Soc Perkin Trans 2 1998, 2281-2288).
• PET photoelectron transfer
• E°(D + /D) is the oxidation potential of the donor
• E°(A/A " ) is the reduction potential of the acceptor
• ⁇ G 00 is the free energy corresponding to the equilibrium energy E 0 o (see e.g. Kavarnos, G. J. In Fundamentals of photoinduced electron transfer; VCH Publishers: New York, NY, 1993; p Chapter 1 and Rehm and Weller, Isr. J. Chem. 1970, 8, 259).
• the quantities w p and w T are Coulombic terms for the products and reactants, and are found to be small in polar solvents.
• E 00 the energy corresponding to [ ⁇ max (ex) + ⁇ m a x(em)]/2 for each fluorophore. While accurate E 00 values can also be found for example, in the literature for anthracene, [Ru(bipy) 3 ] 2+ , and a number of other compounds, we find this method of calculation useful for estimating equilibrium energies for new compounds that have not been previously reported.
• a compound with the desired fluorescent characteristics entails selecting a fluorophore F with the desired properties and simultaneously selecting a switch Z with an oxidation potential that results in a ⁇ Gp ET - hat is less than about 3.0 kcal mol " .
• the electrochemical potentials can be measured using the individual molecular groups before assembling the complete molecule.
• the invention typically entails selecting F and Z to satisfy the Rehm-Weller equation disclosed herein such that ⁇ G, the free energy for electron transfer is negative or at least only slightly positive.
• the PET mechanism can be active even with slightly positive ⁇ G values due, for example, to slight measurement variability and other minor effects.
• the ⁇ G should be less than about 3.0 kcal mol "1 . Consequently, in preferred embodiments of the fluorescent compounds described herein, F and Z are selected to satisfy the equation such the ⁇ G is less than about 3.0 kcal mol "1 In a more preferred embodiment, F and Z are selected to satisfy the equation such the ⁇ G is less than about 1.5 kcal mol "1 . In a highly preferred embodiment, F and Z are selected to satisfy the equation such that the ⁇ G is a negative value.
• Sensor molecules having a specific molecular formula as shown in Figure 1 where F and Z are selected to satisfy the energetic requirements of the simplified Rehm-Weller equation and where the excitation wavelength for F is greater than about 400 nm have a number of advantages over similar previously described molecules.
• such molecules have the advantage of being excited at a wavelength outside of the ultraviolet spectra, and therefore are particularly suited for use in, for example, subdermally implanted optical glucose monitoring systems (See e.g. U.S. Patent No. 6,011,984).
• ultraviolet light which has a spectrum that extends up to, but not beyond 400 nm, is known to be able to induce cumulative damage to human skin (see e.g. Lavker et al., J. Invest.
• Sensor molecules having a specific molecular formula as shown in Figure 1 where F and Z are selected to satisfy the energetic requirements of the simplified Rehm-Weller equation and where the emission wavelength for F is greater than about 450 nm have a number of advantages over similar previously described molecules, particularly in their ability to transmit a signal through a tissue such as skin. Specifically, the transmission through a few millimeters of skin increases logarithmically with wavelength - from 0.1% at 400 nm to almost 100% at 850 nm (see e.g. Optical-Thermal Response of Laser-Irradiated Tissue (A. J. Welch and M.J.C. van Gemert eds., Plenum Press) (1995); Francis A.
• the fluorophore (F) is a dye that has an excitation wavelength greater than 400 nm and an emission wavelength greater than 450 nm.
• Illustrative molecules provided herein fall into a variety of general categories including transition metal-ligand complexes, conjugated organic heterocyclic ring system compounds that are thiazines, oxazines, oxazine-ones, or oxazones, as well as anthracene complexes.
• transition metal-ligand fluorophores include ruthenium bistrifluoromethylbipyridine, chromium bipyridine, and rhenium tricarbonyl bipyridine.
• thermodynamic requirement Rehm-Weller equation
• Transition metals are those elements from Groups IIIA-IB in the periodic table, e.g., Co, Cr, Cu, Mo, Rh, Ru, W, Re, Os, Ir, and Pt.
• conjugated organic heterocyclic ring system fluorophores suitable for the present invention are discussed in Example 2 and shown in Figures 3-5.
• thiazine fluorophore is a thionine complex, as shown in Figure 3, but would also include substituted systems like 1,9-dimethyl-methylene blue.
• the oxazine fluorophores include oxazine 1, oxazine 170, or Nile Blue complex, as shown in Figure 4.
• An oxazine-one and an oxazone fluorophore are shown in Figure 5.
• Specific examples of anthracene fluorophores suitable for the present invention are discussed in Example 3 and shown in Figures 25-29.
• the fluorophore was selected to lengthen the fluorescence lifetime of the sensor molecule.
• the ruthenium complex Ru(N-methyl benzyl boronate)
• the ruthenium complex has a lifetime of 970 nanoseconds as contrasted with the COB (or anthracene-boronate) system with lifetimes of 10 ns or less.
• the substitution of this fluorophore has substantially altered the fluorescence lifetime of the sensor molecule.
• the compounds of the present invention may be used in solution in bioassay studies to detect concentrations of saccharides or sugars, such as glucose.
• the compounds may be immobilized in a biocompatible polymer matrix used for medical implants.
• the compounds are bound covalently to the polymer using techniques described in U.S. Patent No. 6,002,954 which is hereby inco ⁇ orated by reference. Basically, these methods involve adding a suitable tether to the molecule such that the tether can be used to covalently attach the compound to the matrix.
• the attachment point can be through R 2 or R 3 (see Figure 1) or alternatively through the fluorophore itself via R 4 .
• R could be a 5-hydroxypentyl tether or linker arm that could react with an isocyanate group to form an end capped urethane/compound on a polymeric matrix.
• the tether itself could be further modified (e.g., by reaction with methacrolyl chloride to form a methacrylate ester) to present a linker that is suitable for inco ⁇ oration in a free radical polymerized system such as an acrylic based hydrogel.
• U.S. Patent No. 5,628,310 to Rao et al. which is inco ⁇ orated herein by reference, describes an apparatus and method to enable minimally invasive transdermal measurements of the fluorescence lifetime of an implanted element without reagent consumption and not requiring painful blood sampling.
• U.S. Patent No. 5,476,094 to Allen et al. which is inco ⁇ orated herein by reference, disclosed membranes which are useful in the fabrication of biosensors, e.g., a glucose sensor, intended for in vivo use.
• the invention provided herein is directed to novel analyte detection systems based on more robust, small molecule transducers.
• These molecules can be used in a number of contexts including subcutaneously implantable membranes that provide a fluorescent response to, for example, increasing glucose concentrations. Once implanted, the membranes can remain in place for long periods in time, with glucose measured through the skin by optical excitation and detection.
• a number of similar systems have been published previously, largely from Shinkai's group and primarily involving detection by colorimetry and circular dichroism spectroscopy (see e.g.
• a typical implantable glucose sensor for use with the present invention is shown schematically in Figure 14.
• the fluorescent compounds 12 are inco ⁇ orated into the matrix 14 to form a small sensor 10, which is implanted about 1-3 mm below the skin surface.
• the sensor 10 is interrogated by an external instrument 16 that contains a light source 18 to excite the fluorescence and a detector 20 to measure the resultant emission.
• the detected optical signals are then converted into a glucose concentration.
• a calibration method is needed; either fluorescent lifetime measurement techniques are used or ratiometric methods using a second glucose insensitive fluorophore contained within the polymer.
• certain specific embodiments of the invention consist of fluorescent saccharide binding compounds defined by a specified chemical identity (for example the general formula shown in Figure 1), and wherein substituent molecules of these compounds are selected to have complimentary molecular properties which can be determined via simplified version of the Rehm- Weller equation and, additionally, have a specific desirable functional activity such as an excitation wavelength greater than 400 nm and/or an emission wavelength greater than 450 nm (e.g. the compound fluoresces at a wavelength greater than about 450 nm in the presence of an analyte such as glucose).
• Such embodiments are comparable to polypeptide chemicals of a specific formula (i.e. an amino acid sequence of a protein), selected to share a certain structural identity with a specified sequence (i.e.
• % identity and which have a defined and measurable function (e.g. catalyze a reaction of A -> B etc.).
• Functioning compounds defined by a specific chemical formula whose constituents are selected to meet thermodynamic requirements in order to obtain molecules having a certain activity can be, however, more easy to generate than functional polypeptides characterized as having a certain % of identity with a defined sequence because, unlike mere % identity, the simplified Rehm-Weller equation can be used to identify compounds likely to exhibit a specific desirable function before such compound is generated.
• the present sensor/transducer scheme is useful more generally for the measurement of any cis-diol.
• the present sensor molecules have utility in the measurement of ethylene glycol contamination in boiler waters, where ethylene gycol contamination is an indication of heat exchanger tube degradation as well as other uses in similar contexts (see e.g. U.S. Patent No. 5,958,192).
• These sensor molecules can be of use in industrial fermentation processes (e.g. beer and wine), or in any number of process points in the production of high fructose com syrup such as. enzyme reactors and the like (see e.g. U.S. Patent No. 5,593,868; U.S. Patent No.
• sensor molecules described herein exhibit characteristics which them particularly suited for uses such as the monitoring of industrial fermentation processes.
• the compounds described in the Examples below exhibit varying degrees of sensitivity to concentrations of analytes, properties which may be advantageous for use in the context of monitoring solutions of industrial fermentation processes where such solutions have analyte concentrations that exceed those observed, for example, in vivo.
• the compounds described in the Examples below function in a wide pH range and in the presence of high concentrations of alcohols such as methanol (see e.g. Figure 29), a property which can be advantageous in the context of monitoring fermentation processes.
• the simplified version of the Rehm-Weller equation can be employed to confirm that a more cost effective or easily synthesized compound maintains the thermodynamic properties that allow it to function as an analyte sensor. Consequently, by using the disclosure provided herein, one can reduce the amount of effort normally employed in the generation of such compounds.
• the disclosure provided herein teaches a number of embodiments of the invention.
• the invention consists of a fluorescent compound that emits a signal that can be correlated to an analyte (such as a saccharide) concentration and where the compound has the general specified formula shown in Figure 1.
• R 1 is typically selected from the group consisting of hydrogen and lower aliphatic and aromatic functional groups
• R 2 and R 4 typically are hydrogen, optional lower aliphatic or aromatic functional groups or functional groups that can form covalent bonds to the polymer matrix
• L 1 and L 2 typically are optional linking groups having from zero to four atoms selected from the group consisting of nitrogen, carbon, oxygen, sulfur, and phosphorus
• Z is a heteroatom selected from the group consisting of nitrogen, sulfur, oxygen and phosphorus
• R 3 typically is an optional group selected from the group consisting of hydrogen, lower aliphatic or aromatic functional groups; and groups that form covalent bonds to the polymer matrix
• F is a fluorophore with selected molecular properties.
• the fluorescent compound emits a signal that is correlated with the concentration of glucose.
• fluorophores (F) in the above formula include a variety of moieties or functional groups containing ⁇ -electron systems.
• Preferred fluorophores include transition metal-ligand complexes, oxazines, oxazine-ones, oxazones, thiazines, and naphtyl, anthryl, pyrenyl and phenanthryl compounds.
• the fluorophore- forming atomic or functional groups can be substituted ones as long as the substituent(s) do not alter the thermodynamic characteristics of the compounds in a way that adversely affect the fluorescence.
• a substituent R group such as R 1 , R 2 , or R 3 (combined with the nitrogen atom), denotes hydrogen, groups that form covalent bonds to the polymer matrix such as a 5-hydroxypentyl tether or linker arm that can react with an isocyanate group to form an end capped urethane/compound on a polymeric matrix, or are a lower aliphatic or aromatic functional group.
• R can be alkyl group having 1 to 6 carbon atoms, i.e. methyl, ethyl, propyl or butyl, or phenyl group.
• the phenyl group composing the phenylboronic acid may be substituted with an appropriate substituent or substituents as long as the subsitutions discussed herein do not adversely affect the fluorescence.
• candidate substituents include methyl, ethyl, propyl, butyl, phenyl, methoxy, ethoxy, butoxy, phenoxy, pyridyl, furanyl, thiophene and pyridone groups.
• F typically emits at a wavelength greater than about 450 nm. In preferred embodiments of the invention, F emits at a wavelength greater than about 500 nm, a wavelength greater than about 550 nm or a wavelength greater than about 600 nm. In highly preferred embodiments, the excitation wavelength for F is greater than about 400 nm or greater than about 450 nm. In this context, those skilled in the art understand that the excitation and emission wavelengths of such molecules are found over in a focused spectrum of wavelengths and do not occur at a single absolute point. Consequently, with molecules that, for example, have an emission maximum centered near 450 nm, it is therefore accurate to describe such molecules as typically emitting at a wavelength greater than about 450 nm.
• F is selected from the group consisting of transition metal-ligand complexes, oxazines, oxazine-ones, oxazones, thiazines and anthracenes.
• F comprises an oxazine-one boronate, an anthracene-boronate or a transition metal-ligand complex comprising a metal selected from the group consisting of ruthenium and chromium.
• the detection with the fluorescent compound of the present invention can be performed by adding the compound to the sample and by a photoscopic method, determining the increased intensity of the fluorescence due to the binding of the compound with the saccharide.
• the detection with the fluorescent compound of the present invention may be conducted by a chromatographic method where the compound of the present invention is supported on a supporting material through which the saccharide-containing sample is passed for the detection based on the increased fluorescent intensity due to the complex of the compound and the saccharide.
• another typical embodiment of the invention is a sensor comprising a polymer matrix containing a fluorescent compound as discussed above.
• the fluorescent compound is typically covalently bonded to a polymer matrix, and the matrix is biocompatible and implantable.
• inventions include a method for analyzing the concentration of an analyte in a sample by using a fluorescent compound as discussed above and introducing the fluorescent compound into a sample, measuring the fluorescence of the compound in the sample in the presence of the analyte and then determining the concentration of the analyte from the fluorescence measurement.
• the fluorescent compound is covalently bonded to a polymer matrix, wherein the matrix is biocompatible and implantable and the analyte comprises glucose.
• the measurement of fluorescence comprises measuring the intensity.
• the measurement of fluorescence comprises measuring the lifetime.
• the fluorescent saccharide binding compounds disclosed herein are defined by a specific formula, substituent molecules of which have complimentary molecular properties that can be determined via simplified version of the Rehm-Weller equation.
• the invention provided compounds of the specific formula shown in Figure 1, wherein F and Z are selected to satisfy the Rehm-Weller equation disclosed herein such that ⁇ G, the free energy for electron transfer, is less than about 3.0 kcal mol "1 .
• constituents such as the fluorophore F can be selected to satisfy this equation by so assessing the thermodynamic properties of any one of the wide variety of fluorophores known in the art that are capable of being inco ⁇ orated into this molecule, and in this way identify those with the appropriate characteristics.
• various permutations of this selection process are contemplated including the use of computer programs to efficiently test a wide variety of candidate molecules.
• ⁇ G the free energy for electron transfer, is a is less than about 3.0 kcal mol "1
• Figure 6 shows the excitation and emission spectra of Ru(bistrifluoromethylbipyridine) in buffer/methanol.
• the compound is excited at 460 nm, which is favorable for transdermal excitation, and emits well above 550 nm.
• Derivatives of this base fluorophore exhibit virtually the same excitation and emission spectra.
• 460 nm excitation is easily accomplished with low-cost, commercially available ultra-bright LED light sources.
• the excitation and emission wavelengths used were 460 and 560 nm, respectively, although operation at even higher wavelengths (red) is clearly possible.
• a glucose-sensing compound using the ruthenium fluorophore was synthesized: Ru(N-methyl benzyl boronate).
• the compound is shown in Figure 7. Samples of Ru(N-methyl benzyl boronate) were prepared at 10 ⁇ M concentration in distilled water.
• Figure 8 shows a time history of this sample with additions of acid and base. The behavior of this compound under acidic and basic conditions is a good indicator of whether or not the final compound maintains PET functionality.
• the Ru(N-methyl benzyl boronate) compound was tested for glucose sensitivity.
• a sample was prepared at a concentration of 10 ⁇ M in distilled water. Multiple additions of glucose were made as shown in Figure 9. Again, an intensity decay trend is superimposed on the data. Increases in the fluorescence corresponding to increases in the glucose concentration are clearly observed.
• the final glucose concentration is approximately 3000 mg/dL, clearly outside the clinical range, 50-500 mg/dL. Nevertheless, this increase gave rise to a fluorescence increase of approximately 50%. Although only a relatively small increase in fluorescence is expected for this compound over the clinical range of glucose concentrations, these results signify the first successful attempt to create a long-wavelength glucose-sensor molecule. Chemical modification of the molecule may permit one to maximize its response to changes in glucose concentration.
• Samples for fluorescence were prepared as 1.00 mM stock solutions in MeOH. A 30.0 ⁇ L aliquot of solution was then added to 3.000 mL of the appropriate solvent mixture (a combination of methanol and phosphate buffered saline - PBS) giving a final concentration of 10.0 ⁇ M for each complex. Changes in pH were carried out by the addition of small volumes of 1.0 M hydrochloric acid, 1.0 M sodium hydroxide, or glacial acetic acid. Glucose additions were performed by the addition of a concentrated solution of glucose in PBS to a stirred solution of fluorophore in a cuvette.
• (bpyMe)Re(CO) 3 Cl The preparations of (bpyMe)Re(CO) 3 Cl, (bpyN)Re(CO) 3 Cl, and (b ⁇ yNB)Re(CO) 3 Cl are analogous to that of (bpyCH 2 NEt. 2 )Re(CO) 3 Cl.
• [(bpyMe)Re(CO) 3 (py)](OTf), [(bpyN)Re(CO) 3 (py)](OTf), and [(bpyNB)Re(CO) 3 (py)](OTf) are analogous to that of [(bpyCH 2 NEt 2 )Re(CO) 3 (py)](OTf).
• a solution of AgOTf (263 mg, 1.02 mmol) in 5 mL of THF was added to a solution of (bpyMe)Re(CO) 3 Cl (499 mg, 1.02 mmol) in 50 mL of CH 2 C1 2 to immediately give a cloudy yellow mixture.
• Bipyridine Ligand Synthesis Typical compounds of the invention include the new boronate and benzyl bipyridine ligands which can be synthesized by the routes shown in Figure 17.
• the common intermediate to both sets of transition metal complexes prepared in this work is the bipyridyl boronate ligand bpyNB.
• Previous work by Meyer see e.g. Meyer, T. J. Account Chem Res 1989, 22, 163-170) and others has shown that compound bpyCH 2 Br provides the simplest entry into a variety of functionalized bipyridine compounds.
• the three ligand derivatives were prepared for both rhenium and ruthenium in order to aid in the inte ⁇ retation of the fluorescence and electrochemical data discussed below.
• the 1H and 13 C ⁇ 1 H ⁇ NMR spectra and MS data clearly confirm the identity of the compounds.
• ER spectra of the three chloro complexes, [(bpyX)Re(CO) 3 Cl] (bpyX bpyMe, bpyN, and bpyNB), each exhibit carbonyl stretches at 2022, 1917, at 1895 cm "1 ; CO resonances are observed at 2034 and 1931 cm “1 for each of the pyridium complexes [(bpyX)Re(CO) 3 (py)](OTf).
• the quantities w v and vv r are Coulombic terms for the products and reactants, and are found to be small in polar solvents.
• the compound's response to glucose in several solvent systems was characterized.
• a stock solution of the compound was prepared by dissolving 5.7 mg in 5 mL of methanol.
• Samples were prepared by adding 30 ⁇ L of this stock solution to 3 mL of solvent.
• Three solvent mixtures were used: (1) pure methanol (MeOH), (2) 66% MeOH by volume in pH 7.4 buffer, and (3) 25% MeOH by volume in pH 7.4 buffer.
• Figure 11 shows the excitation and emission spectra obtained for a sample of the oxazine-one compound in 66% MeOH/buffer.
• excitation and emission wavelengths of 450 nm and 560 nm respectively were chosen for subsequent steady-state fluorescence tests; however, emission wavelengths in excess of 600 nm would also be satisfactory for monitoring the fluorescence intensity.
• the 450 nm excitation band is well-matched to commercially available ultra-bright LED emitters.
• Glucose sensitivity testing was performed on the oxazine-one compound. Samples were prepared as described above and titrated with 30 ⁇ L aliquots of concentrated glucose solution (300 mg/mL). Each aliquot raised the glucose concentration of the sample by about 300 mg/dL.
• Figure 12 shows the results of an experiment performed with a 66% MeOH/buffer solvent.
• glucose transduction yields a 45% increase in the fluorescence intensity as the glucose concentration is increased from zero to 600 mg/dL. This is a switching fraction similar to that exhibited by anthracene boronate, but now the transduction has been achieved well within the green (for the emission), where a several-fold improvement in the optical transport efficiency of human skin is realizable.
• the reversibility of the glucose transduction was experimentally investigated. Samples, 1.5 mL in volume, were prepared as described above in 3.5 mL cuvettes (including stir bar volume).
• an aromatic boronic acid group was employed since it has been shown that they have selective recognition for saccharides. These two main components are attached via a methylene amine tether.
• the amine serves not only as a linker but is an integral part of the glucose sensing design.
• the target sensor molecule, 6-chloro-5H- benzo[a]phenoxazin-5-one boronate 11, is based on fluorescent signaling via photoinduced electron transfer.
• the PET process in this unique system is modulated by interaction of boronic acid and amine.
• COB Chloro- Oxazine Boronate
• benzophenoxazinone 11 The target molecule for glucose recognition is abbreviated as COB (Chloro- Oxazine Boronate) and is shown below as benzophenoxazinone 11.
• COB was constructed by coupling benzophenoxazinone 6a with phenyl boronate 10 via a methylene amine linkage (as shown in Figure 20).
• Benzophenoxazinone 6a was synthesized by condensation of 3-amino-4- hydroxybenzyl alcohol 3 with 2,3-dichloro-l,4-napthoquinone 4 ( Figure 21).
• the preparation of amino alcohol 3 required successive reductions from commercially available 4-hydroxy-3-nitrobenzoic acid 1.
• Reduction of benzoic acid 1 with borane-THF complex in tetrahydrofuran gave 4-hydroxy-3-nitrobenzyl alcohol 2 in 90%) yield.
• Subsequent reduction of nitro-alcohol 2 with sodium borohydride and 10% Pd/C catalyst in water provided 3-amino-4-hydroxybenzyl alcohol 3 in 97% yield.
• excitation and emission wavelengths were chosen for subsequent steady-state fluorescence tests. However, it should be mentioned that emission wavelengths in excess of 600nm would also be satisfactory for monitoring the fluorescence intensity and that the 450nm excitation band is quite well matched to currently available ultra-bright LED emitters.
• Samples, prepared as above, were titrated with 30 ⁇ L aliquots of concentrated glucose solution (300mg/mL). Roughly speaking, each aliquot raised the glucose concentration of the sample by about 300 mg/dL.
• Figure 12 shows the results of the experiment performed with a 66% MeOH/buffer solvent.
• naphthalimide derivatives studied in this project were prepared by the routes shown in Figures 25 and 26. These procedures are analogous to those previously reported for naphthalimide dye molecules, with some distinctions (see e.g. Alexiou et al., J. Chem. Soc, Perkin Trans. 1990, 837; de Silva et al., Angew. Chem. Int. Ed. Engl. 1995, 34, 1728; Kavarnos, G.J. Fundamentals of Photoinduced Electron Transfer; VCH: New York, 1993; pp 37-40. and Daffy et al. Chem. Eur. J. 1998, 4, 1810).
• naphthalimide framework has been shown to exhibit a wide range of spectral properties, depending on the alkyl groups appended to the imide nitrogen and the 4-position. Most work to date has used an n-butyl group off the imide nitrogen (e.g. lax)., generally giving rise to high quantum yields than shorter or unsaturated side chains.
• n-butyl group off the imide nitrogen (e.g. lax).
• lbx n-butyl group off the imide nitrogen
• the two compounds with secondary amino groups have quantum yields of 40-50 times that of the tertiary compounds, making them clearly superior choices for sensing pu ⁇ oses. Removal of the THP protecting group from compound 3cz shows no significant change in quantum yield or wavelength maximum.
• the fluorescence wavelengths for these compounds In addition to quantum yield changes, there are small but significant shifts in the fluorescence wavelengths for these compounds.
• the compounds with secondary amines have somewhat longer excitation wavelengths (435 nm) than the corresponding tertiary compounds (415 nm and 420 nm).
• the compounds functionalized with THP ethers have emission spectra shifted 10 nm towards the red of the butyl imide derivatives (535 nm vs. 525 nm).
• E°(D + /D) and E°(A/A " ) correspond to the reduction potential and oxidation potential of the electron donor (i.e. amine) and electron acceptor (i.e. fluorophore), respectively.
• the quantity ⁇ G is the equilibrium energy, which we estimate as the energy corresponding to the equilibrium wavelength ⁇ o 0 .
• /loo we use the average of the excitation and emission maxima for each fluorophore. Smaller solvent-dependent work terms for the Coulombic interaction between the reactants and products are left out for simplicity. Since the mechanism of fluorescence switching in these systems depends on PET, this gives us a rough estimate of which fluorophores should give the best response to glucose.
• Glucose additions were performed by the addition of a concentrated solution of glucose in PBS to a stirred solution of the fluorescent molecule in methanol/PBS. lax, lbx.
• a equimolar mixture of 4-chloro-l,8-naphthalic anhydride and either n-butylamine or 5-aminopentanol in ethanol was heated at reflux for 20 hours.
• the dark brown solution was filtered and cooled to -10 °C.
• a pure, tan powder was collected by filtration (90% yield). The identities of the pure products were confirmed by 1H and 13 C ⁇ 1H ⁇ NMR spectroscopy, as well as ESI/MS (electrospray ionization mass spectrometry). lex.
• Samples for fluorescence measurements were prepared as 1.00 mM stock solutions in MeOH. A 30.0 ⁇ L aliquot of solution was then added to 3.000 mL of the appropriate solvent mixture (a combination of methanol and phosphate buffered saline - PBS) giving a final concentration of 10.0 ⁇ M for each complex. Changes in pH were carried out by the addition of small volumes of concentrated hydrochloric acid, acetic acid, or sodium hydroxide. Glucose additions were performed by the addition of a concentrated solution of glucose in PBS to a stirred solution of fluorophore in a cuvette. Relative quantum yield measurements were carried out by measuring the relative intensities of equimolar solutions (10.0 ⁇ M) of two fluorophores. Compound 3ay was used as the reference.
• N-/ ⁇ -Butyl-4-chloronaphthalene-l,8-dicarboximide (lax) This compound has been reported previously. This preparation is similar to previously reports (see e.g. Daffy et al. Chem-Eur J 199S, 4, 1810-1815; de Silva et al., Chem Rev 1997, 97, 1515-1566). A portion of «-butylamine (5.00 mL, 50.6 mmol) was added to a suspension of 4-chloronaphthalene-l,8-dicarboximide (11.7 g, 50.5 mmol) in 160 mL of toluene. This mixture was brought to reflux for 17 hours to give a dark black-brown solution.
• N-(5'-Tetrahydropyranoxypentyl)-4-bromonaphthalene-l,8- dicarboximide (lex') The was analogous to literature procedures. A mixture of lbx' (4.681 g, 12.9 mmol), poly(4-vinylpyridinium hydrochloride) (202 mg, 1.31 meq), and 30 mL of 3,4-dihydro-2H-pyran (D ⁇ P) was heated at reflux for 23 hours to give an orange solution. The solvent was removed under vacuum, giving an orange oil. Purification was carried out by chromatography on silica gel using chloroform as the eluent.
• iV-w-Butyl-4-(N'-methylaminoethylene-N"-methylamino)naphthalene- 1,8-dicarboximide (2ay) A portion of NN'-dimethyl-l,2-diaminoethane (2.00 mL, 18.8 mmol) was added to a suspension of lax (1.02 g, 3.55 mmol) in 20 mL of 2-methoxyethanol, followed by triethylamine (0.495 mL, 3.55 mmol) to immediately give a dark brown mixture. This mixture was brought to reflux for 4 hours to give a dark brown solution.
• N-n-Butyl-4-( ⁇ '-methylaminoethylamino)naphthalene-l,8- dicarboximide (2az) This was prepared analogously to 2ay. The reaction of lax (1.67 g, 5.82 mmol), N-methyl-l,2-diaminoethane (2.56 mL, 29.1 mmol), and triethylamine (0.811 mL, 5.82 mmol) in 2-methoxyethanol, followed by crystallization from hot methanol, yielded 2az as a pure yellow powder (1.44 g, 76%).
• N-(5'-Hydroxypentyl)-4-(N'-methylaminoethylene-N"- benzylmethylamino)naphthalene-l ,8-dicarboximide (AP)(NI)(Meen)(Bn) A solution of lbx (0.292 g, 0.919 mmol), N-benzyl-N-methyl-l,2-diaminoethane (0.413 g, 2.51 mmol), triethylamine (0.175 mL, 1.26 mmol) in 20 mL of 2- methoxyethanol was heated at reflux for 41 hours.
• the procedure is analogous to that for the synthesis of 3ay.

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