Enzymatic radiosynthesis of deoxy-[18f]fluorocellobiose and deoxy-[18f]fluorocellotriose

Abstract

Enzymatic radiosyntheses of deoxy-[18F]fluorocellobiose and deoxy-[18F]fluorocellotriose are disclosed. Deoxy-[18F]fluorocellobiose is synthesized in 1-2 hours from (i) deoxy-[18F]fluoroglucose, glucose-1-phosphate, and a cellobiose phosphorylase (CBP), or (ii) deoxy-[18F]fluoroglucose-1-phosphate, glucose, and a CBP. Deoxy-[18F]fluorocellotriose is synthesized in 1-2 hours from (i) deoxy-[18F]fluoroglucose, glucose-1-phosphate, CBP, and a CBP variant, (ii) deoxy-[18F]fluorocellobiose, glucose-1-phosphate, and a CBP variant, or (iii) deoxy-[18F]fluoroglucose-1-phosphate, cellobiose, and a CBP variant.

Description

4239-109758-02 E-080-2023-0-PCT-02ENZYMATIC RADIOSYNTHESIS OF DEOXY-[18F]FLUOROCELLOBIOSE AND DEOXY-[18F]FLUOROCELLOTRIOSE FIELD

[0001] This disclosure concerns enzymatic radiosyntheses of deoxy-[18F]fluorocellobiose and / or deoxy-[18F]fluorocellotriose. CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of the earlier filing date of U.S. Provisional Application No.63 / 492,302, filed March 27, 2023, which is incorporated by reference in its entirety herein. SEQUENCE LISTING

[0003] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence.xml” (4,322 bytes), which was created on March 13, 2024, which is incorporated by reference herein. BACKGROUND

[0004] The recent global incidence of invasive fungal infections has sharply increased, resulting in high mortality and morbidity mainly in immunocompromised and immunodeficient patients. Of these, Aspergillus fumigatus is one of the most common causative agents of invasive fungal diseases. One of the biggest hurdles to treating fungal infections remains that lack of timely definitive diagnosis which is complicated by the non-specific symptoms and the use of non-specific diagnostic methods. Therefore, the development of fungal-specific positron emission tomography (PET) imaging agents could be useful in the early detection of infection.18F-labeled deoxycellobiose is a promising candidate for detecting fungal infections, such as Aspergillus, Candida¸ Cryptococcus, or Mucormycetes infections (e.g., as disclosed by Hammoud et al., US 2022 / 0273829 A1). However, half-life of fluorine-18 is just 109.77 minutes. Thus, a need exists for a rapid method to synthesize18F-labeled deoxycellobiose for use in PET imaging techniques. PET imaging techniques can distinguish infectious lesions of fungal origin from other differentials such as tumors, tuberculous granulomas or bacterial / viral pneumonia, and may be useful for monitoring treatment response. SUMMARY

[0005] Enzymatic radiosyntheses of deoxy-[18F]fluorocellobiose and deoxy-[18F]fluorocellotriose are disclosed. Deoxy-[18F]fluorocellobiose is synthesized in 1-2 hours from (i) deoxy- [18F]fluoroglucose ([18F]FDG), glucose-1-phosphate (Glc-1P), and a cellobiose phosphorylase4239-109758-02 E-080-2023-0-PCT-02(CBP), or (ii) deoxy-[18F]fluoroglucose-1-phosphate ([18F]FGlc-1P), glucose, and a CBP. Deoxy- [18F]fluorocellotriose is synthesized in 1-2 hours from (i) [18F]FDG, Glc-1P, a CBP and a CBP variant, (ii) deoxy-[18F]fluorocellobiose, glucose-1-phosphate, and a CBP variant, or (iii) deoxy- [18F]fluoroglucose-1-phosphate, cellobiose, and a CBP variant.

[0006] In aspects of a first synthesis, deoxy-[18F]fluorocellobiose is prepared by (i) combining deoxy-[18F]fluoroglucose ([18F]FDG) with glucose-1-phosphate (Glc-1P) and a cellobiose phosphorylase (CBP) to provide a first solution having at least a 10-fold molar excess of the Glc-1P relative to the [18F]FDG, and (ii) incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a second solution comprising deoxy- [18F]fluorocellobiose.

[0007] In aspects of a second synthesis, deoxy-[18F]fluorocellobiose is prepared by (i) phosphorylating [18F]FDG to form deoxy-[18F]fluoroglucose-1-phosphate ([18F]FGlc-1P), (ii) combining the [18F]FGlc-1P with glucose and a solution comprising a CBP to provide a first solution having at least a 10-fold molar excess of the glucose relative to the [18F]FGlc-1P, and (iii) incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a second solution comprising deoxy-[18F]fluorocellobiose.

[0008] In any of the foregoing or following aspects, the second solution comprising deoxy- [18F]fluorocellobiose may further comprise (A) CBP and unreacted Glc-1P (first synthesis) or (B) CBP and unreacted glucose (second synthesis), and the method further comprises separating (A) the CBP and the unreacted Glc-1P or (B) the CBP from the second solution to provide a deoxy- [18F]fluorocellobiose solution. In certain aspects, the method further includes purifying the deoxy- [18F]fluorocellobiose solution.

[0009] In aspects of a third synthesis, deoxy-[18F]fluorocellotriose is prepared by (i) combining [18F]FDG with Glc-1P, a CBP, and a CBP variant to provide a first solution having at least a 10- fold molar excess of the Glc-1P relative to the [18F]FDG, and (ii) incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a subsequent solution comprising deoxy-[18F]fluorocellotriose.

[0010] In aspects of a fourth synthesis, deoxy-[18F]fluorocellotriose is prepared by combining the deoxy-[18F]fluorocellobiose of the first or second synthesis with Glc-1P and a CBP variant to provide a third solution having at least a 10-fold molar excess of the Glc-1P relative to the deoxy- [18F]fluorocellobiose; and incubating the third solution at a temperature of from 35 °C to 40 °C for a time of from 1 hour to 2 hours to provide a subsequent solution comprising deoxy- [18F]fluorocellotriose. In some aspects, the subsequent solution further comprises unreacted Glc-1P4239-109758-02 E-080-2023-0-PCT-02and the CBP variant, the method further comprises separating the unreacted Glc-1P and the CBP variant from the subsequent solution to provide a deoxy-[18F]fluorocellotriose solution. In certain aspects, the method further includes purifying the deoxy-[18F]fluorocellotriose.

[0011] In any of the foregoing aspects, the process may be an automated process performed by an automated system.

[0012] In aspects of a fifth synthesis, deoxy-[18F]fluorocellotriose is synthesized by (i) phosphorylating [18F]FDG to form [18F]FGlc-1P; (ii) combining the [18F]FGlc-1P with cellobiose and a CBP variant to provide a first solution having at least a 10-fold molar excess of the cellobiose relative to the [18F]FGlc-1P; and (iii) incubating the first solution at a temperature of from 35 °C to 40 °C for a time of from 1 hour to 2 hours to provide a subsequent solution comprising deoxy-[18F]fluorocellotriose. In some implementations, the subsequent solution further comprises unreacted cellobiose and the CBP variant, the method further comprising separating the unreacted cellobiose and the CBP variant from the deoxy-[18F]fluorocellotriose to provide a deoxy- [18F]fluorocellotriose solution. The method may further include purifying the deoxy- [18F]fluorocellotriose.

[0013] The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS.1-E are flow charts showing steps in exemplary syntheses of [18F]fluorocellobiose (FIGS.1A, 1B) and [18F]fluorocellotriose (FIGS.1C-1E).

[0015] FIGS.2A-2C are flow charts showing steps in exemplary automated syntheses of [18F]fluorocellobiose (FIG.2A) and [18F]fluorocellotriose (FIGS.2B, 2C).

[0016] FIG.3 is a high-performance liquid chromatography (HPLC) spectrum of a reaction mixture comprising 2-deoxy 2-[18F]fluorocellobiose.

[0017] FIGS.4A-4D are HPLC of 2-deoxy 2-[18F]fluorocellobiose in whole human serum at 37 °C after 0 h (FIG.4A), 2 h (FIG.4B), 3 h (FIG.4C), and 4 h (FIG.4D).

[0018] FIG.5 is a graph showing in vitro uptake of 2-deoxy 2-[18F]fluorocellobiose by Aspergillus fumigatus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans cells.4239-109758-02 E-080-2023-0-PCT-02

[0019] FIG.6 shows PET / CT imaging of in vivo uptake of 2-deoxy 2-[18F]fluorocellobiose by mice inoculated with A. fumigatus, E. coli, and S. aureus.

[0020] FIGS.7A-7C are graphs of T / NT ratios for SUV mean (FIG.7A) and SUV mean (FIG.7B) for mice inoculated with live or heat-killed A. fumigatus, E. coli, and S. aureus, and SUV mean for mice inoculated with live A. fumigatus or sterile LPS (FIG.5C).

[0021] FIGS.8A and 8B are HPLC chromatograms of CBP (FIG.8A) and purified 2-deoxy 2- [18F]fluorocellobiose ([18F]FCB) (FIG.8B).

[0022] FIGS.9A and 9B are an HPLC chromatogram of 2-deoxy 2-[18F]fluorocellotriose synthesized in a one-step reaction (FIG.9A) and an LCMS spectrum showing the 2-deoxy 2- [18F]fluorocellotriose co-eluting with a non-radioactive standard (FIG.9B); solid line = in-line radio detector, dotted line = UV detector at 220 nm.

[0023] FIGS.10A-10D show HPLC chromatograms of 2-deoxy 2-[18F]fluorocellobiose in whole human serum at 37 °C after 0 h (FIG.10A), 1 h (FIG.10B), 2 h (FIG.10C), and 4 h (FIG.10D). SEQUENCE LISTING

[0024] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0025] SEQ ID NO: 1 is a cloned amino acid sequence for a cellobiose phosphorylase (CBP) enzyme

[0026] SEQ ID NO: 2 is the amino acid sequence for Cellumonas uda cellobiose phosphorylase (CuCBP) found in GenBank under accession number AAQ20920.1

[0027] SEQ ID NO: 3 is the amino acid sequence for a CBP variant His_OCP2-52R (OCP2-52R). DETAILED DESCRIPTION

[0028] Aspects of methods for enzymatic radiosynthesis of deoxy-[18F]fluorocellobiose and / or deoxy-[18F]fluorocellotriose are disclosed. In some aspects, the method is an automated method. Chemical synthesis of18F-labeled cellobiose and cellotriose is challenging due to the multi-step preparation of precursors, which are unstable. Moreover, chemical radiolabeling requires multi steps. Briefly, chemical radiolabeling comprises incorporation of fluorine-18 to prepare a fluorine- 18 labeled intermediate; time consuming high-performance liquid chromatography (HPLC) purification of the intermediate and deprotection of the protecting group, resulting in poor radiochemical yield (1-3%) of deoxy-[18F]fluorocellobiose and / or deoxy-[18F]fluorocellotriose. The4239-109758-02 E-080-2023-0-PCT-02overall reaction time is 2.5 hours or more.18F has a half-life of just 109.77 minutes, resulting in losses of the radioactivity when syntheses are lengthy. Advantageously, some aspects of the disclosed method allow synthesis of deoxy-[18F]fluorocellobiose in 2 hours or less, such as in 1.5 hours or less. This method is a simple one step process, precursors are commercially available, and there is no need for time-consuming HPLC purification. The radiochemical yields are high (60- 70%). I. Terms, Abbreviations and Numerical Ranges

[0029] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0030] The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and / or perform equally well. Nor does it mean that the alternatives are listed in a preferred order unless stated otherwise.

[0031] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley’s Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 2016 (ISBN 978-1-118-13515-0).4239-109758-02 E-080-2023-0-PCT-02

[0032] CBP: cellobiose phosphorylase

[0033] Eluent: A fluid used to elute a substance from a chromatography column or cartridge; a mobile phase that moves analytes through the chromatography column or cartridge.

[0034] [18F]FCB: deoxy-[18F]fluorocellobiose

[0035] [18F]FCT: deoxy-[18F]fluorocellotriose

[0036] [18F]FDG: deoxy-[18F]fluoroglucose

[0037] [18F]FGlc-1P: deoxy-[18F]fluoroglucose-1-phosphate

[0038] Glc-1P: glucose-1-phosphate (or glucose-1-phosphate disodium salt)

[0039] SPE: solid-phase extraction II. Enzymatic Radiosynthesis A. Enzymatic Radiosynthesis of Deoxy-[18F]fluorocellobiose

[0040] In some aspects, deoxy-[18F]fluorocellobiose ([18F]FCB) is enzymatically synthesized using commercially available deoxy-[18F]fluoroglucose ([18F]FDG) as a starting material. The [18F]FDG may have a structure according to Formula I: where one or more of R1, R2, R3andof R1, R2, R3and R4are -OH. In some implementations, one of R1, R2, R3and R4is18F and the [18F]FDG ,thereof. In certainaspects, the [18F]FDG is 2-deoxy-2-[18F] . 0041] [1[8F]FCB synthesized from [18F]FDG may have a structure according to Formula II or Formula III:4239-109758-02 E-080-2023-0-PCT-02. In some ishasany combination thereof.the [18F]FDG is 2-deoxy-2-[18F]fluoroglucose, and the or2′-deoxy-2′-[18F]fluorocellobiose.some as in FIG.1A, a first synthesis method includes (i) combining [18F]FDG with glucose-1-phosphate (Glc-1P) and a solution comprising a cellobiose phosphorylase (CBP) to provide a first solution having at least a 10-fold molar excess of the Glc-1P relative to the [18F]FDG (step 1), and (ii) incubating the first solution at a temperature of from 25 °C to 45 °C, such as a temperature of from 35 °C to 40 °C, for a time of from 1 hour to 2 hours to provide a4239-109758-02 E-080-2023-0-PCT-02second solution comprising [18F]FCB (step 2). In such aspects, R3is -OH, and the method produces [18F]FCB having a structure according to Formula II: .FDG. In some aspects, the first solution has at least a 25-fold molar excess, at least a 50-fold molar excess, at least a 100-fold molar excess, or at least a 200-fold molar excess of the Glc-1P relative to the [18F]FDG. In certain implementations, the first solution has a 10-fold to 1000-fold molar excess of Glc-1P relative to the [18F]FDG, such as a 25-fold to 500-fold, or 50-fold to 250-fold molar excess of Glc-1P relative to the [18F]FDG.

[0045] In some implementations, an initial amount of the [18F]FDG is from 1 mCi to 200 mCi, such as an initial amount in a range having endpoints selected from 1 mCi, 5 mCi, 10 mCi, 25 mCi, 50 mCi, 75 mCi, 100 mCi, 125 mCi, 150 mCi, 175 mCi, and 200 mCi. An initial concentration of the Glc-1P in the first solution may be from 50 mM to 200 mM, such as an initial concentration in a range having endpoints selected from 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, and 200 mM.

[0046] Without wishing to be bound by a particular theory of operation, the large molar excess of Glc-1P may be a driving factor in the synthesis, thereby reducing the reaction time and / or improving the reaction yield compared to other synthetic methods. In one example, when 100 mCi of [18F]FDG, 75 mM Glc-1P, and 1 mg CBP in 1.9 mL of saline were incubated for 1.25 hours at 40 °C, a 60-70% yield of [18F]FCB with a radiochemical purity of > 98% was obtained.

[0047] In any of the foregoing aspects, the method may be an automated method (FIG.2A). The automated method may utilize a commercial synthesis apparatus to perform the method steps. One exemplary apparatus is the Trasis AllInOne (AIO) radiosynthesis module (Trasis, Tucker, GA). In some implementations, an automated method includes transferring [18F]FDG to a syringe (step 1), transferring Glc-1P from a vial to a reactor containing a CBP enzyme (step 2), transferring the [18F]FDG from the syringe to the Glc-1P vial to rinse excess Glc-1P from the vial (step 3), and then transferring the [18F]FDG from the vial to the reactor (step 4). The first solution is incubated at a temperature of from 25 °C to 45 °C, such as a temperature of from 35 °C to 40 °C, for a time of from 1 hour to 2 hours to provide a second solution comprising [18F]FCB (step 5).4239-109758-02 E-080-2023-0-PCT-02

[0048] In some aspects (FIG.1B), a second synthesis method includes (i) phosphorylating [18F]FDG to form deoxy-[18F]fluoroglucose-1-phosphate ([18F]FGlc-1P) (step 1), (ii) combining the [18F]FGlc-1P with glucose and a solution comprising a CBP to provide a first solution having at least a 10-fold molar excess of the glucose relative to the [18F]FGlc-1P (step 2), and (iii) incubating the first solution at a temperature of from 25 °C to 45 °C, such as from 35 °C to 40 °C, for a time of from 1 hour to 2 hours to provide a second solution comprising [18F]FCB (step 3). In such aspects, the method produces [18F]FCB having a structure according to Formula III: .1P. In some aspects, the first solution has at least a 25-fold molar excess, at least a 50-fold molar excess, at least a 100-fold molar excess, or at least a 200-fold molar excess glucose relative to the [18F]FGlc-1P. In certain implementations, the first solution has a 10-fold to 1000-fold molar excess of glucose relative to the [18F]FGlc-1P, such as a 25-fold to 500-fold, or 50-fold to 250-fold molar excess of glucose relative to the [18F]FGlc-1P. Without wishing to be bound by a particular theory of operation, the large molar excess of glucose may be a driving factor in the synthesis, thereby reducing the reaction time and / or improving the reaction yield compared to other synthetic methods.

[0050] In some implementations, an initial amount of the [18F]FDG is from 1 mCi to 200 mCi, such as an initial amount in a range having endpoints selected from 1 mCi, 5 mCi, 10 mCi, 25 mCi, 50 mCi, 75 mCi, 100 mCi, 125 mCi, 150 mCi, 175 mCi, and 200 mCi. An initial concentration of the glucose in the first solution may be from 50 mM to 200 mM, such as an initial concentration in a range having endpoints selected from 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, and 200 mM.

[0051] In any of the foregoing or following aspects, the first solution may be prepared in saline (e.g., normal saline) or a buffer, such as a 4-(2-hydroxyethyl-1-piperazineethansulfonic acid (HEPES) buffer, pH 7-7.5. In any of the foregoing or following aspects, the time may be from 1 hour to 1.5 hours, or from 70 minutes to 80 minutes. In certain implementations, the temperature is from 37 °C to 40 °C.4239-109758-02 E-080-2023-0-PCT-02

[0052] In any of the foregoing or following aspects, the first solution may comprise from 0.5 mg to 2 mg CBP, such as from 1.0 mg to 1.5 mg CBP. In some aspects, the CBP is a CBP synthesized in a bacterial expression system and having a sequence according to SEQ ID NO: 1 (infra).

[0053] In any of the foregoing or following aspects, the second solution comprising [18F]FCB may further comprise (A) CBP and unreacted Glc-1P (first synthesis method) or (B) CBP and unreacted glucose (second synthesis method), and the method may further comprise separating the CBP from the second solution to provide a [18F]FCB solution (FIGS.1A, 1B). Separating the CBP from the second solution may include adding a C1-C3 alkanol to the second solution to precipitate (A) the CBP and unreacted Glc-1P or (B) the CBP, and removing the precipitated components to provide the [18F]FCB solution (FIG.1A, step 3, step 4; FIG.1B, step 4, step 5). In some implementations, the C1-C3alkanol is ethanol. Removing (A) the precipitated CBP and the precipitated unreacted Glc-1P or (B) the precipitated CBP can be performed by any suitable method, including but not limited to filtration. In some aspects, the precipitated reactants are removed by flowing the solution comprising the C1-C3 alkanol, [18F]FCB, and (A) precipitated CBP and precipitated unreacted Glc- 1P (FIG.1A, step 4) or (B) precipitated CBP through a filter (FIG.1B, step 5), whereby the precipitated reactants are retained on the filter and the [18F]FCB solution flows through the filter. In some aspects, the filter is a 5 μm filter. Alternatively, the second solution may be heated at a temperature of 80 °C to 120 °C for 5 minutes to 15 minutes, such as at a temperature of 100 °C for 5 minutes, to precipitate the CBP (FIG.1A, step 3; FIG.1B, step 4). The precipitated CBP is removed by flowing the solution through a filter (FIG.1A, step 4; FIG.1B, step 5), whereby the precipitated CBP is retained on the filter. In some aspects, the filter is a 5 μm filter.

[0054] In some aspects, the method is an automated method and separating the CBP may include incubating the second solution at a temperature of 80 °C to 120 °C, such as a temperature of 95 °C to 105 °C, for an effective amount of time (e.g., 5-15 minutes) to precipitate the CBP, followed by filtering the second solution to remove the precipitated CBP (FIG.2A, step 6). In some aspects, the filter is a 5 μm filter.

[0055] The [18F]FCB solution may be further purified. In some aspects, the [18F]FCB solution is flowed through a solid-phase extraction (SPE) cartridge comprising a solid phase that retains the [18F]FCB (FIG.1A, step 5; FIG.1B, step 6; FIG.2A, step 7). Any unreacted Glc-1P in the solution is separated from the [18F]FCB as the solution flows through the SPE cartridge. In some implementations, the solid phase comprises a silica support with a weakly basic surface, such as a surface comprising amino groups (e.g., aminopropyl groups). In other implementations, the solid phase may comprise cation exchange resin, anion exchange resin, neutral aluminum oxide, and4239-109758-02 E-080-2023-0-PCT-02porous adsorptive resin. The [18F]FCB is eluted from the SPE cartridge by flowing an eluent through the SPE cartridge (FIG.1A, step 6; FIG.1B, step 7; FIG.2A, step 8). Suitable eluents include alkanols, aqueous alkanol solutions, and saline. In on example, the solid phase comprised silica with surface aminopropyl groups, and the eluent was 70% ethanol in water. In some implementations, the [18F]FCB solution is flowed through a series of SPE cartridges to provide purified [18F]FCB. The SPE may also be a FDG purification cartridge (filled with cation exchange resin, anion exchange resin, neutral aluminum oxide and porous adsorptive resin). This anion exchange resin will remove Glc-1P.

[0056] The second synthesis method includes an initial step of phosphorylating [18F]FDG to form deoxy-[18F]fluoroglucose-1-phosphate ([18F]FGlc-1P) (FIG.1B, step 1). Phosphorylation may be performed by any suitable method, including chemical and / or enzymatic methods.

[0057] In some aspects, the phosphorylation process includes preparing acetylated [18F]FDG, phosphorylating the acetylated [18F]FDG by MacDonald phosphorylation, and deacetylating the phosphorylated acetylated [18F]FDG to provide [18F]FGlc-1P, e.g., as described by Prante et al. (J Label Compd Radiopharm 2007, 50:55-63). An exemplary phosphorylation and deacetylation of acetylated 2-deoxy-2-[18F]fluoroglucose is shown below: .

[0058] In otherusing a galactokinase (GalK) or N-acetylhexosamine kinase (NahK) in combination with adenosine triphosphate (ATP) and a source of Mg2+ions, e.g., as described by Kempa et al. (JACS Au 2021, 1:508-516). An exemplary phosphorylation is shown below: , 14where R -R are as previously defined, and ADP is adenosine diphosphate.4239-109758-02 E-080-2023-0-PCT-02B. Enzymatic Radiosynthesis of Deoxy-[18F]fluorocellotriose

[0059] This disclosure also concerns aspects of methods for making deoxy-[18F]fluorocellotriose ([18F]FCT). The [18F]FCT may have a structure according to any one of Formulas IV-VI, where R1, R2, R3, and R4are as previously defined. ,is ,,or any combination thereof. In some aspects, theVI and the [18F]FCT is ,4239-109758-02 E-080-2023-0-PCT-02ororis synthesized from [18F]FCB by combining the [18F]FCB with Glc-1P and a CBP variant to provide a third solution having at least a 10-fold molar excess of the Glc-1P relative to the [18F]FCB (step 1), and incubating the third solution at a temperature of from 25 °C to 45 °C, such as from 35 °C to 40 °C, for a time of from 1 hour to 2 hours to provide a subsequent solution comprising [18F]FCT (step 2). In any of the foregoing or following aspects, the time may be from 1 hour to 1.5 hours, or from 70 minutes to 80 minutes. In certain implementations, the temperature is from 37 °C to 40 °C.

[0062] In some aspects, combining the [18F]FCB with Glc-1P and a CBP variant to provide the third solution comprises adding the Glc-1P and the CBP variant to the [18F]FCB solution obtained after removing (A) the CBP and the unreacted Glc-1P (first synthesis method) or (B) the CBP (second synthesis method) from the initial [18F]FCB synthesis, e.g., after step 4 of FIG.1A or step 5 of FIG.1B. In certain aspects, the Glc-1P and the CBP variant are added to [18F]FCB that has been purified, e.g., after step 6 of FIG.1A, step 7 of FIG.1B, or step 6 of FIG.2A. In an independent aspect, combining the [18F]FCB with Glc-1P and the CBP variant to provide the third solution comprises adding the CBP variant to the second solution obtained from the initial synthesis of the [18F]FCB, wherein the second solution comprises [18F]FCB, unreacted Glc-1P, and the CBP, e.g., after step 2 of FIG.1A (prior to separating and / or purifying the [18F]FCB. If the second solution does not comprise sufficient Glc-1P to provide at least a 10-fold molar excess of Glc-1P relative to the [18F]FCB, additional Glc-1P is added to provide the third solution.4239-109758-02 E-080-2023-0-PCT-02

[0063] If the [18F]FCB has a structure according to Formula II, then the synthesized [18F]FCT has a structure according to Formula IV: .synthesized [18F]FCT has a structure according to Formula V: .[18F]FCB. In some aspects, the third solution has at least a 25-fold molar excess, at least a 50-fold molar excess, at least a 100-fold molar excess, or at least a 200-fold molar excess of Glc-1P relative to the [18F]FCB. In certain implementations, the third solution has a 10-fold to 1000-fold molar excess of Glc-1P relative to the [18F]FCB, such as a 25-fold to 500-fold, or 50-fold to 250-fold molar excess of Glc-1P relative to the [18F]FCB. Without wishing to be bound by a particular theory of operation, the large molar excess of Glc-1P may be a driving factor in the synthesis, thereby reducing the reaction time and / or improving the reaction yield compared to other synthetic methods.

[0066] In some implementations, an initial concentration of the Glc-1P in the third solution of FIG.1C may be from 50 mM to 200 mM, such as an initial concentration in a range having endpoints selected from 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, and 200 mM. An initial amount of the [18F]FCB may be from 1 mCi to 200 mCi, such as an initial amount in a range having endpoints selected from 1 mCi, 5 mCi, 10 mCi, 25 mCi, 50 mCi, 75 mCi, 100 mCi, 125 mCi, 150 mCi, 175 mCi, and 200 mCi.

[0067] In any of the foregoing or following aspects, the third solution may be prepared in saline (e.g., normal saline) or a buffer, such as a 4-(2-hydroxyethyl-1-piperazineethansulfonic acid (HEPES) buffer, pH 7-7.5. In any of the foregoing or following aspects, the time may be from 1 hour to 1.5 hours, or from 70 minutes to 80 minutes. In certain implementations, the temperature is from 37 °C to 40 °C.

[0068] In any of the foregoing or following aspects, the third solution may comprise from 0.5 mg to 2 mg of the CBP variant, such as from 1.0 mg to 1.5 mg of the CBP variant. In some aspects, the4239-109758-02 E-080-2023-0-PCT-02CBP variant is a variant of a Cellumonas uda cellobiose phosphorylase (CuCBP) found in GenBank under accession number AAQ20920.1 (SEQ ID NO: 2, infra). In certain examples, the CBP variant is OCP2_M52R, which includes 6 mutations relative to SEQ ID NO: 2, specifically : M ^R at position 52, N ^D at position 156, N ^D at position 163, T ^I at position 508, E ^G at position 649, and N ^A at position 667 (Ubiparip et al., Appl. Microbiol. and Biotech.2020, 104:8327-8337). In other examples the CBP variant is His_OCP2-52R (SEQ ID NO: 3, infra; referred to hereinafter as OCP2-52R). OCP2_M52R and OCP2-52R may be used interchangeably in the syntheses disclosed herein.

[0069] In some aspects, the subsequent solution comprising [18F]FCT further comprises unreacted Glc-1P and the CBP variant, and the method further includes separating the CBP variant from the subsequent solution to provide a [18F]FCT solution. In some implementations, separating the CBP variant from the subsequent solution includes adding a C1-C3 alkanol to the subsequent solution to precipitate the CBP variant and unreacted Glc-1P (step 3, FIG.1C), and removing the precipitated CBP variant and the precipitated unreacted Glc-1P to provide the [18F]FCT solution. In certain implementations, the C1-C3alkanol is ethanol. Removing the precipitated CBP variant and the precipitated unreacted Glc-1P can be performed by any suitable method, including but not limited to filtration (step 4, FIG.1C). In some aspects, the precipitated reactants are removed by flowing the solution through a 5 μm filter, whereby the precipitated reactants are retained on the filter and the [18F]FCT solution flows through the filter.

[0070] Alternatively, separating the CBP variant from the subsequent solution may comprise heating the subsequent solution at a temperature of from 80 °C to 120 °C for 5 minutes to 15 minutes, such as at a temperature of 100 °C for 5 minutes, to precipitate the CBP variant (FIG.1C, step 3). The precipitated CBP is removed by flowing the solution through a filter (FIG.1C, step 4), whereby the precipitated CBP is retained on the filter. In some aspects, the filter is a 5 μm filter.

[0071] The [18F]FCT may be further purified. In some aspects, the [18F]FCT solution is flowed through a SPE cartridge comprising a solid phase that retains the [18F]FCT (step 5, FIG.1C). In some implementations, the solid phase comprises a silica support with a weakly basic surface, such as a surface comprising amino groups (e.g., aminopropyl groups), or a solid phase suitable for retaining fluorodeoxyglucose. The [18F]FCT is then eluted from the SPE cartridge by flowing an eluent through the SPE cartridge (step 6, FIG.1C). Suitable eluents include alkanols, aqueous alkanol solutions, and saline. In some implementations, the [18F]FCT solution is flowed through a series of SPE cartridges to provide purified [18F]FCT.4239-109758-02 E-080-2023-0-PCT-02

[0072] In some aspects, as shown in FIG.1D, [18F]FCT according to Formula VI, are as previously defined, is synthesized to provide a third solution (step 1). Themay an FDG to form [18F]FGlc-1P, as previously described with respect to FIG.1B, step 1. In FIG.1D, step 1, [18F]FGlc-1P is combined with cellobiose and a CBP variant to provide a third solution having at least a 10-fold molar excess of the cellobiose relative to the [18F]FGlc-1P. In some aspects, the first solution has at least a 25- fold molar excess, at least a 50-fold molar excess, at least a 100-fold molar excess, or at least a 200- fold molar excess cellobiose relative to the [18F]FGlc-1P. In certain implementations, the first solution has a 10-fold to 1000-fold molar excess of cellobiose relative to the [18F]FGlc-1P, such as a 25-fold to 500-fold, or 50-fold to 250-fold molar excess of cellobiose relative to the [18F]FGlc-1P. Without wishing to be bound by a particular theory of operation, the large molar excess of cellobiose may be a driving factor in the synthesis, thereby reducing the reaction time and / or improving the reaction yield compared to other synthetic methods. In some implementations, an initial concentration of the cellobiose in the third solution may be from 50 mM to 200 mM, such as an initial concentration in a range having endpoints selected from 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, and 200 mM. An initial amount of the [18F]FGlc-1P may be from 1 mCi to 200 mCi, such as an initial amount in a range having endpoints selected from 1 mCi, 5 mCi, 10 mCi, 25 mCi, 50 mCi, 75 mCi, 100 mCi, 125 mCi, 150 mCi, 175 mCi, and 200 mCi. Steps 2-6 are as previously described for FIG.1C. Step 2 is as previously described for FIG.1C and provides a subsequent solution comprising the [18F]FCT according to Formula VI: .3, then filtering in step 4 will remove the CBP variant and excess [18F]FGlc-1P.

[0073] In some aspects, as shown in FIG.1E, [18F]FCT according to Formula IV is directly synthesized in a “one-pot” process by combining [18F]FDG, Glc-1P, CBP, and a CBP variant to provide a solution having at least a 10-fold molar excess of the Glc-1P relative to the [18F]FDG4239-109758-02 E-080-2023-0-PCT-02(step 1). Steps 2-6 are as previously described for FIG.1C. In step 3, both CBP and the CBP variant are precipitated.

[0074] In another aspect (not shown), [18F]FCT may be synthesized by combining [18F]Glc-1P, glucose, Glc-1P, CBP, and a CBP variant to provide a solution. Steps 2-6 are as previously described for FIG.1C. In step 3, both CBP and the CBP variant are precipitated. In this method, the [18F]FCT is a mixture of [18F]FCT according to Formula V and Formula VI. The solution has at least a 10-fold molar excess of Glc-1P and / or glucose relative to the [18F]Glc-1P.

[0075] In some aspects, [18F]FCT is synthesized via an automated method, as shown in FIG.2B. In some implementations, an automated method includes transferring [18F]FCB to a syringe (step 1), transferring Glc-1P from a vial to a reactor containing a CBP enzyme (step 2), transferring the [18F]FCB from the syringe to the Glc-1P vial to rinse excess Glc-1P from the vial (step 3), and then transferring the [18F]FCB from the vial to the reactor (step 4) to form the third solution. The third solution is incubated at a temperature of from 25 °C to 45 °C, such as a temperature of from 35 °C to 40 °C, for a time of from 1 hour to 2 hours to provide a subsequent solution comprising [18F]FCT (step 5). In certain aspects the method further includes separating the unreacted Glc-1P and the CBP variant from the subsequent solution to provide a [18F]FCT solution. The unreacted Glc-1P and CBP variant may be separated from the subsequent solution by incubating the subsequent solution at a temperature of 80 °C to 120 °C, such as a temperature of 95 °C to 105 °C, for an effective amount of time (e.g., 5-15 minutes) to precipitate the CBP, followed by filtering the subsequent solution to remove the precipitated CBP (FIG.2B, step 6). In some aspects, the filter is a 5 μm filter. The [18F]FCT solution may be further purified. In some aspects, the [18F]FCT solution is flowed through a solid-phase extraction (SPE) cartridge comprising a solid phase that retains the deoxy-[18F]fluorocellobiose (FIG.2B, step 7). In some implementations, the solid phase comprises a silica support with a weakly basic surface, such as a surface comprising amino groups (e.g., aminopropyl groups). The SPE cartridge may be washed with saline to remove unbound components. The [18F]FCT is eluted from the SPE cartridge by flowing an eluent through the SPE cartridge (FIG.2B, step 8). Suitable eluents include alkanols, aqueous alkanol solutions, and saline. In on example, the solid phase comprised silica with surface aminopropyl groups, and the eluent was 70% ethanol in water. In some implementations, the [18F]FCT solution is flowed through a series of SPE cartridges to provide purified [18F]FCT.

[0076] In an independent aspect, [18F]FCT is synthesized via an automated method, as shown in FIG.2C. The automated method includes transferring [18F]FDG to a syringe (step 1), transferring Glc-1P from a vial to a reactor containing a CBP enzyme and a CBP variant enzyme (step 2),4239-109758-02 E-080-2023-0-PCT-02transferring the [18F]FDG from the syringe to the Glc-1P vial to rinse excess Glc-1P from the vial (step 3), and then transferring the [18F]FDG from the vial to the reactor (step 4) to form the third solution. Steps 5-8 are as described for FIG.2B.

[0077] In any of the foregoing or following aspects, the first solution may be prepared in saline (e.g., normal saline) or a buffer, such as a 4-(2-hydroxyethyl-1-piperazineethansulfonic acid (HEPES) buffer, pH 7-7.5. In any of the foregoing or following aspects, the time may be from 1 hour to 1.5 hours, or from 70 minutes to 80 minutes. In certain implementations, the temperature is from 37 °C to 40 °C.

[0078] In any of the foregoing or following aspects, the first solution may comprise from 0.5 mg to 2 mg of the CBP variant, such as from 1.0 mg to 1.5 mg of the CBP variant. In some aspects, the CBP variant is a variant of a Cellumonas uda cellobiose phosphorylase (CuCBP) found in GenBank under accession number AAQ20920.1 (SEQ ID NO: 2). In certain examples, the CBP variant is OCP2_M52R or OCP2-52R, as described supra.

[0079] In some aspects, the subsequent solution comprising [18F]FCT further comprises unreacted cellobiose and the CBP variant, and the method further includes separating the unreacted cellobiose and the CBP variant from the subsequent solution to provide a [18F]FCT solution. In some aspects, separating the CBP variant from the subsequent solution includes adding a C1-C3alkanol to the second solution to precipitate the CBP variant, and removing the precipitated CBP variant. In some implementations, the C1-C3alkanol is ethanol. Removing the precipitated CBP variant can be performed by any suitable method, including but not limited to filtration. In some implementations, the [18F]FCT and unreacted cellobiose are separated, e.g., by flowed the solution of [18F]FCT and unreacted cellobiose through a SPE cartridge comprising a solid phase that selectively retains the [18F]FCT, and then eluting the retained [18F]FCT.

[0080] The [18F]FCT may be further purified. In some aspects, the [18F]FCT solution is flowed through a SPE cartridge comprising a solid phase that retains the [18F]FCT. In some implementations, the solid phase comprises a silica support with a weakly basic surface, such as a surface comprising amino groups (e.g., aminopropyl groups), or a solid phase suitable for retaining fluorodeoxyglucose. The [18F]FCT is then eluted from the SPE cartridge by flowing an eluent through the SPE cartridge. Suitable eluents include alkanols, aqueous alkanol solutions, and saline. In some implementations, the [18F]FCT is flowed through a series of SPE cartridges to provide purified [18F]FCT.

[0081] Some aspects of the disclosed enzymatic radiosyntheses provide significant advantages compared to other chemical and / or enzymatic methods. For example, the disclosed enzymatic4239-109758-02 E-080-2023-0-PCT-02radiosyntheses may reduce the reaction time and / or improve the reaction yield compared to other synthetic methods. In one example, when 100 mCi of [18F]FDG, 60 mM Glc-1P disodium salt, and 1 mg CBP in 1.9 mL of saline were incubated for just 1.25 hours at 40 °C, a 60-70% yield of [18F]FCB with a radiochemical purity of > 98% was obtained. In comparison, a similar (but non- radioactive) enzymatic method with equimolar amounts of glucose-1-phosphate and deoxy-fluoro- glucose required an incubation time of 16 hours with a yield of 80% (Peterson et al., Chem. Eur. J. 2021, 27:1374-1382). A 16-hour incubation time is entirely unsuitable for a radionuclide having a half-life of just 109.77 minutes. In another comparison, a chemical method starting with water containing18F (obtained by irradiating [18O]H2O with protons to produce18F), may have a yield of just 1-3% with a synthesis time of 2.5 hours (US 2022 / 0273829 A1). Additionally, some aspects of the disclosed enzymatic radiosyntheses include a one-step incubation followed by simple filtration and solid phase extraction, whereas the chemical method requires multiple steps, including a multi- step synthesis of precursors and laborious, time-intensive HPLC purification of an intermediate product, prior to solid-phase extraction. Moreover, some aspects of the disclosed enzymatic radiosyntheses may be carried out via an automated method as described herein, thereby reducing the labor requirements as well as achieving the above-mentioned advantages. III. Uses of Deoxy-[18F]fluorocellobiose and Deoxy-[18F]fluorocellotriose

[0082] A main barrier to effectively treating fungal infections is the inability of current clinical diagnostic methods to provide an accurate and timely diagnosis of infection. Standard methods of diagnosis involve using non-specific imaging modalities such as computed tomography (CT), molecular methods relying on testing galactomannan presence in blood, or culturing fungi from bronchoalveolar lavage (BAL) and biopsy specimens obtained using invasive procedures. The use of positron emission tomography (PET) techniques for diagnosis of deep seated infections has the advantage of being a non-invasive method that can provide highly sensitive functional information and if combined with a fungal-specific radiotracer, can distinguish infectious lesions of fungal origin from other differentials such as tumors, tuberculous granulomas or bacterial / viral pneumonia.

[0083] [18F]fluorodeoxyglucose (FDG) is useful as an imaging agent for magnetic resonance imaging (MRI) and computed tomography (CT) scanning. However, FDG is metabolized by most living cells and therefore is a nonspecific imaging agent. A more selective imaging agent is needed for detection and imaging of fungal infections. Aspergillus and other fungal species are known to have a unique sugar diet that does not overlap much with other microbes and mammalian cells due at least in part to their ubiquitous presence in the environment outside the human host. Thus, these4239-109758-02 E-080-2023-0-PCT-02fungi have evolved different mechanisms to hydrolyze complex polysaccharides to produce simple sugars to be used as nutrients. For example, cellobiose and cellotriose are among the breakdown products generated from fungal enzymatic degradation of plant-based lignocellulosic biomass, and subsequently are converted into glucose. Because the fungi have a unique diet, radiolabeled fluorocellobiose and / or fluorocellotriose are useful as selective imaging agents to detect and image fungal infections. Radiolabeled fluorocellobiose and / or fluorocellotriose may be used in conjunction with several imaging techniques, including MRI, CT, and positron emission tomography (PET).

[0084] This disclosure encompasses in vivo and ex vivo / in vitro methods of using one or more of the radiolabeled compounds provided herein, such as deoxy-[18F]fluorocellobiose or deoxy- [18F]fluorocellotriose, to detect a fungus. The radiolabeled compound may be provided in a composition further comprising a pharmaceutically acceptable carrier, such as water or saline. In some aspects, the composition is a liquid composition, for example suitable for injection into a subject. In some implementations, the composition is frozen or freeze-dried. The composition may be provided in a container, such as a glass or plastic vial.

[0085] The methods can detect any fungus of interest, such as an Aspergillus, Candida, Cryptococcus, or Mucormycetes. In some examples, the method detects Aspergillus, such as A. fumigatus, A. flavus, A. terreus, or A. niger. In some examples, the method detects Candida, such as C. albicans. In some examples, the method detects Cryptococcus, such as C. neoformans or C. gattii. In some examples, the method detects Mucormycetes, such as a Rhizopus, Mucor, Rhizomucor, Syncephalastrum, Cunninghamella bertholletiae, Apophysomyces, or Lichtheimia. The method can include contacting the fungus in vivo with one or more compounds or compositions provided herein, thereby detecting the fungus.

[0086] In some examples, the method is an in vivo method of detecting a fungal infection in a subject (such as any fungus provided above, or listed in Table 1 below; thus in some examples, the subject is one having a disease listed in Table 1). In some examples the subject is a mammal or bird or fish, such a human or veterinary subject. In some examples, the subject is immunocompromised, such as a cancer patient (e.g., one undergoing chemo and / or radiation therapy), a subject who has received a transplant (e.g., transplant of at least one of a stem cell or solid organ such as a lung, heart, liver, kidney, pancreas, or intestine), a subject having a primary immunodeficiency (examples of primary immunodeficiency diseases include those listed in Al- Herz et al. (Frontiers in Immunology, volume 5, article 162, April 22, 2014, herein incorporated by reference in its entirety), e.g., T-B+ SCID, T-B- SCID, WHIM syndrome, IL-7 receptor severe4239-109758-02 E-080-2023-0-PCT-02combined immune deficiency (SCID), Adenosine deaminase deficiency (ADA) SCID, Purine nucleoside phosphorylase (PNP) deficiency, Wiskott-Aldrich syndrome (WAS), Chronic granulomatous disease (CGD) , Leukocyte adhesion deficiency (LAD), Duchenne muscular dystrophy, Glycogen storage disease type IA, Retinal Dystrophy, and X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia (XMEN)) or a subject with HIV. In such examples, the contacting includes administering one or more compounds or compositions provided herein (such as 1, 2, 3, 4 or 5 compositions) to a subject, and the method further includes subsequently performing diagnostic imaging (such as nuclear imaging) of the subject, thereby detecting the fungal infection in the subject. For example, the diagnostic imaging can be performed at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, or at least 120 minutes, such as 15 to 30, 15 to 60, 30 to 60, 30 to 120, or 60 to 120 minutes after administering the one or more compounds to the subject. In some examples, administering includes injection into the subject, such as IV administration. In some examples, depending on the size and weight of the subject, a least 1 millicurie, at least 2 millicuries, at least 3 millicuries, at least 4 millicuries, at least 5 millicuries, at least 10 millicuries, such as1-3, 1-5, 1-10, 1-20, 5-20 or 5-10 millicuries of the one or more compounds is administered to the subject.

[0087] In some examples, diagnostic imaging of the subject includes nuclear imaging of the brain, lungs, heart, sinuses, and / or abdomen of the subject. In some examples, positron emission tomography (PET) nuclear imaging technology is used. PET enables visualization of metabolic processes in vivo. PET imaging detects pairs of gamma rays emitted indirectly by a positron- emitting radionuclide (such as18F in [18F]FCB or [18F]FCT). PET systems have sensitive detector panels to capture gamma ray emissions from inside the body and use software to plot and triangulate the source of the emissions, creating 3-D computed tomography images of the tracer concentrations within the body.

[0088] The in vivo methods can be used to detect a fungal infection in a subject, such as in the blood, kidney, heart, esophagus, lungs, sinuses, gastrointestinal tract, and / or central nervous system (e.g., brain, spinal cord). Detection of the administered radiolabeled compound(s) provided herein, such as [18F]FCB or [18F]FCT, indicates the presence (and location) of a fungal infection. In some examples, such methods are used to monitor treatment of a fungal infection. Thus, in some examples, the subject is one who has previously been treated with one or more anti-fungal compositions.

[0089] Also provided are ex vivo or in vitro methods of detecting a fungus, for example by incubating or contacting the one or more radiolabeled compounds with a sample containing the4239-109758-02 E-080-2023-0-PCT-02fungus, for example a biological sample obtained from a subject, thereby detecting the fungal infection. The method can further include detecting the uptake of the radiolabeled compound(s) provided herein, such as [18F]FCB or [18F]FCT, in the sample, for example by using a beta counter, radioTLC or autoradiography.

[0090] Table 1: Fungal diseases affecting patients with weakened immune system Fungus Disease manifestations Method of treatment Aspergillosis Most common is Aspergillus Allergic aspergillosis: e4239-109758-02 E-080-2023-0-PCT-02IV. Examples

[0091] Materials and Methods

[0092] Cellobiose phosphorylase_His (CBP, custom synthesized) was obtained from GenScript (Piscataway, NJ 08854). The lyophilized whole human serum was obtained from MP Biomedicals, LLC (Solon, OH, USA) and dissolved in 2 mL saline. This serum solution was directly used without inactivation for the stability study of 2-deoxy 2-[18F]fluorocellobiose. All other chemicals and reagents were purchased from MilliporeSigma (St. Louis, MO, USA) and used without further purification. Fluorine-18 was received from the National Institutes of Health cyclotron facility (Bethesda, MD, USA). [18F]FDG was obtained from CardinalHealth (Silver Spring, MD, USA). FDG Purification Cartridge (Base Hydrolysis) (KT-100) was obtained from ISOFLEX (San Francisco, CA, USA) and conditioned with 12 mL ethanol followed by 30 mL water. Acrodisc®Sterile Syringe Filters (5 µm) with Supor®Membrane were purchased from VWR (Radnor, PA, USA). Other columns and Sep-Pak®NH2 cartridges used in this synthesis were obtained from Agilent Technologies (Santa Clara, CA, USA) and Waters (Milford, MA, USA), respectively. Analytical high-performance liquid chromatography (HPLC) analyses were performed on an Agilent®1200 Series instrument equipped with a multi-wavelength UV detector connected in series with a Bioscan flow count radio detector. Mass spectrometry (MS) was performed on a 6130 Quadrupole LCMS, Agilent Technologies instrument equipped with a diode array detector. LC- MS / MS analysis was performed on Agilent®6460C triple quadrupole mass spectrometry with an ESI source and a flow count radio detector (Eckert & Ziegler, B-FC-3500 diode). The LC inlet was an Agilent®1200 series chromatographic system equipped with a 1260 binary pump, 1290 thermostatted column compartment and 1260 high-performance autosampler. Instrument control and data processing were performed using Agilent®’s MassHunter®software.

[0093] Enzyme specification: A cellobiose phosphorylase (CBP) enzyme was custom- synthesized in a bacterial expression system “cellobiose phosphorylase_His.” Protein Name: cellobiose phosphorylase_His Sequence Name: cellobiose phosphorylase_His Target Vector:pET30a;Cloning Sites:NdeI(CATATG),HindIII(AAGCTT) Final sequence (SEQ ID NO: 1): MRYGHFDDEAREYVITTPHTPYPWINYLGSEQFFSLLSHQAGGYSFYRDAKMRRLTRYRYNNIPADAGG RYLYVNDGGDVWTPSWLPVKADLDHFEARHGLGYSTITGERNGVRVETLFFVPVGENAEVQKVTVTNT SDSYKSLTLFSFVEFCLWNAQDDQTNYQRNLSIGEVEVEQESPHGSAIYHRTEYRERRDHYAVFAVNTQ AEGFDTDRDTFVGAYNSLGEAAVPLKGESANSVASGWYPIGSHSVAVSLAPGESRELVYVLGYVENPDE4239-109758-02 E-080-2023-0-PCT-02EKWADDAKQVVNKERAHALLSRFATSEQTDAAFAALKDYWTDLLSTYSVSSNDEKLDRMVNIWNQY QCMVTFNMSRSASFFETGIGRGMGFRDSNQDLLGFVHLIPERARERIIDIASTQFADGSAYHQYQPLTKR GNNDIGSGFNDDPLWLIAGTAAYIKETGDFSILDEPVPFDNEPGSEVPLFEHLTRSFEFTVTHRGPHGLPLI GRADWNDCLNLNCFSTTPGESFQTTENQAGGVAESTFIAAQFVLYGEQYAELAARRGLADVADRARGH VAEMRDALLTDGWDGSWFLRAYDYYGNPIGTDAHDEGKIWIEPQGFAVMAGVGVGEGPQDTDAPAIK ALDSVNEMLATDHGMVLQYPAYTTYQVHMGEVSTYPPGYKENGGIFCHNNPWVIIAETVVGRGGRAF DYYKRITPAYREDISDVHRLEPYVYAQMIAGKEAVRHGEAKNSWLTGTAAWNFVTVSQYLLGVRPEYD GLVVDPQIGPDVPSFTVTRVARGATYEITVTNSGTDGSRGRLVVDGTPVEGNLVPYAPAGSTVRVDVTL HHHHHH**

[0094] OCP2_M52R CBP is a variant of Cellumonas uda cellobiose phosphorylase (CuCBP) found in GenBank under accession number AAQ20920.1 with 6 mutations: M ^R at 52, N ^D at 156, N ^D at 163, T ^I at 508, E ^G at 649, and N ^A at 667 (Ubiparip et al., Appl. Microbiol. and Biotech.2020, 104:8327-8337). CuCBP (SEQ ID NO: 2) 1 MRYGHFDDEA REYVITTPHT PYPWINYLGS EQFFSLLSHQ AGGYSFYRDA KMRRLTRYRY 61 NNIPADAGGR YLYVNDGGDV WTPSWLPVKA DLDHFEARHG LGYSTITGER NGVRVETLFF 121 VPVGENAEVQ KVTVTNTSDS YKSLTLFSFV EFCLWNAQDD QTNYQRNLSI GEVEVEQESP 181 HGSAIYHRTE YRERRDHYAV FAVNTQAEGF DTDRDTFVGA YNSLGEAAVP LKGESANSVA 241 SGWYPIGSHS VAVSLAPGES RELVYVLGYV ENPDEEKWAD DAKQVVNKER AHALLSRFAT 301 SEQTDAAFAA LKDYWTDLLS TYSVSSNDEK LDRMVNIWNQ YQCMVTFNMS RSASFFETGI 361 GRGMGFRDSN QDLLGFVHLI PERARERIID IASTQFADGS AYHQYQPLTK RGNNDIGSGF 421 NDDPLWLIAG TAAYIKETGD FSILDEPVPF DNEPGSEVPL FEHLTRSFEF TVTHRGPHGL 481 PLIGRADWND CLNLNCFSTT PGESFQTTEN QAGGVAESTF IAAQFVLYGE QYAELAARRG 541 LADVADRARG HVAEMRDALL TDGWDGSWFL RAYDYYGNPI GTDAHDEGKI WIEPQGFAVM 601 AGVGVGEGPQ DTDAPAIKAL DSVNEMLATD HGMVLQYPAY TTYQVHMGEV STYPPGYKEN 661 GGIFCHNNPW VIIAETVVGR GGRAFDYYKR ITPAYREDIS DVHRLEPYVY AQMIAGKEAV 721 RHGEAKNSWL TGTAAWNFVT VSQYLLGVRP EYDGLVVDPQ IGPDVPSFTV TRVARGATYE 781 ITVTNSGTDG SRGRLVVDGT PVEGNLVPYA PAGSTVRVDV TL

[0095] Pseudomonas aeruginosa (P. aeruginosa) isolate was obtained from Chronic Granulomatous Disease and Autosomal Dominant Hyper-IgE Syndrome patients from the Department of Microbiology at the Clinical Center of the National Institutes of Health, Bethesda, MD, USA (Peterson KL et al., Nucl Med Biol 2013, 40(5):638-642). Escherichia coli (E. coli, strain ATCC 25922), Staphylococcus aureus (S. aureus, strain 29213), J774A.1 murine macrophage cell line (strain TIB-67) and Aspergillus niger (strain ATCC 1015) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Aspergillus fumigatus B- 5233 is a clinical isolate obtained from an invasive aspergillosis patient at the National Institutes of4239-109758-02 E-080-2023-0-PCT-02Health (NIH) (kindly donated by Dr. K.J. Kwon-Chung from the National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, MD, USA). Candida albicans (C. albicans, strain SC5314) was kindly donated by Dr. Michail Lionakis from NIAID, NIH.

[0096] Aspergillus cultures were plated on Malt extract agar (MEA) slants for 3–5 days at 37°C. The conidia were collected in sterile PBS with 0.1% Tween 20 (PBST) and passed through 40-µm cell strainers, washed twice and spore counts were obtained from a hemocytometer. Hyphal cultures were grown either in yeast glucose or minimal media (Tsai et al., J Biol Chem, August 3, 2001, 276(31):29292-8). Candida cultures were grown in yeast glucose media and maintained at 30°C. All bacterial cultures were grown in LB media (Sigma Aldrich) at 37°C. The J774 cells were grown in DMEM media with 10% fetal bovine serum and 5% penicillin-streptomycin (Gibco).

[0097] Animals: All experimental procedures, including handling and care of the animals, were approved by the Animal Care and Use Committee of the Clinical Center of the NIH and performed in accordance with relevant NIH policies. Healthy female CD-1 mice (ages 6-8 weeks, Charles River, Charleston, SC, USA) were used for AF myositis and sterile inflammation models. All mice were housed with 12 hour light / dark cycle with free access to food and water. Female BALB / c mice (ages 6-9 weeks, Charles River, Charleston, SC, USA) were used to develop bacterial myositis models.

[0098] In Vitro Uptake Assays with Radiolabeled Sugars: Bacterial and Candida cells as well as Aspergillus hyphae were used to perform in vitro uptake assays with the radiolabeled sugars- 3H-2-deoxyglucose (3H-2DG), and 3H-cellobiose (Moravek Inc, CA, USA) as detailed before (Lai et al., J Fungi (Basel), December 28, 2021, 8(1):25). Briefly, live and heat killed cultures were incubated with 0.5 μCi / 2.5 mL culture at 37°C and samples were collected at 30, 60, and 120 min time-points.

[0099] For J774 cells, cells were plated in 6 well plates overnight. On the experiment day the growth media was replaced with DMEM without glucose. Radiolabeled sugars were added at the same concentration as above and incubated for 10, 30, and 120 min. All samples underwent 3 washes with cold PBS and pellets were resuspended in scintillation fluid (Ultima Gold XR, PerkinElmer, Chicago, IL, USA) and read on the Microbeta2 counter (Perkin Elmer). The counts were normalized to pellet weights and to 3H-2DG uptake.

[0100] Uptake assays with18F-deoxycellobiose were performed as described above with minor differences. The activity added was 0.5 μCi / mL of culture. After 30, 60, and 120 mins of incubation, 1 mL samples were collected in triplicates, washed with cold PBS and the pellets were4239-109758-02 E-080-2023-0-PCT-02immediately read using an automated gamma counter (PerkinElmer). Counts were corrected for background and decay and normalized to pellet weight.

[0101] Animal Models:

[0102] Aspergillus myositis model: CD-1 mice were immunosuppressed with cyclophosphamide injections (IP), 4 days (150mg / kg) and 1 day (100mg / kg) before infection. A 100 µL suspension containing 5×107live (right thigh) and 5×107heat-killed Aspergillus fumigatus (AF) spores (left thigh) were injected intramuscularly (IM) in the thighs. PET / CT imaging was done 2 days after infection. For heat killed spores, the inoculum was autoclaved for 30 minutes and the absence of growth was routinely verified by plating on slants.

[0103] Bacterial myositis model: Immunocompetent BALB / c mice were injected intramuscularly with either E. coli (live- 5x108and heat killed- 1x1010) or S. aureus (live- 1x108and heat killed- 1x1010) inoculum in 100 µL suspensions. The cultures were incubated at 95 °C for 1 h for heat killing and the absence of growth was routinely verified by plating. PET imaging was performed 4 to 5h after inoculation. BALB / c mice were used to induce bacterial infections since this mouse background provided us with the most reliable and reproducible myositis model.

[0104] Lipopolysaccharide (LPS) myositis / sterile inflammation model: CD1 mice were intramuscularly injected with 75µg of LPS (Invivogen, CA, USA) in a 50 µl volume, into the left thighs to induce sterile inflammation. The mice were scanned 18-20 hours after inoculation with LPS.

[0105] PET / CT imaging: The imaging with 18F-deoxycellobiose was conducted using the nanoScan®PET / CT (Mediso, Budapest, Hungary). Mice were intravenously injected with the radiotracer (~9 MBq) via the tail vein. In some cases, half the dose was given as a bolus and the rest as a slow infusion over a period of 45 mins to prolong the circulation time of the ligand. The animals were first anesthetized with 3–4% isoflurane and kept warm using a heating pad during the scan. Dynamic PET images for up to 60 mins were acquired and the animals were injected 10s after the start of the scan. For static scans, the animals were imaged at either 30, 60 or 120 mins time- points after radiotracer injection. The emission sinograms were corrected for scatter,18F-decay, random, and dead time. The resulting histograms were then reconstructed applying Fourier rebinning and 3D ordered subject expectation maximization algorithm (OSEM-3D). The CT images were obtained for attenuation correction and later provided anatomical localization for drawing regions of interest for quantitative analysis. The images were analyzed with Fusion4239-109758-02 E-080-2023-0-PCT-02software (Mediso Ltd., Budapest, Hungary) or MIM 7.1.6 (MIM software Inc., OH, USA). The ligand uptake is expressed as SUVmean and SUVmax target-to-nontarget ratios (T / NT).

[0106] GMS Staining: After the PET / CT scans, Af infected mice were first transcardially perfused with sterile saline and 4% PFA. The thigh muscles were further soaked in 4% PFA for 15- 20 mins and then embedded in OCT (Tissue-Tek) and stored at −80 °C until they were ready to be sectioned. GMS (Grocott’s methenamine silver) staining (ScyTek Laboratories Inc., West Logan, UT, USA) was performed on 8–12 µm frozen sections to confirm the presence of fungi in the infected areas. Images of stained sections were collected using VS200 slide scanner (Olympus, MA, USA) or Eclipse E200 (Nikon, Melville, NY, USA).

[0107] Statistics: GraphPad Prism®9 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. Statistical significance was determined by paired or unpaired two- tailed t-test based on the experimental design (longitudinal versus cross-sectional). A p value < 0.05 was considered statistically significant. Quantitative data are expressed as mean ± standard error of the mean (SEM).

[0108] Example 1 – Synthesis and stability evaluation of 2-deoxy-2-[18F]fluorocellobiose

[0109] The half-life of fluorine-18 is 109.77 minutes, making a short synthesis time desirable. To determine whether 2-deoxy-2-[18F]fluorocellobiose could be made in a short time using trace amounts of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) and a large excess of glucose-1-phosphate (Glc-1P), a test radiolabeling reaction was performed with 1 mCi of [18F]FDG, 1 mg of cellobiose phosphorylase (CBP), and 37 mg of Glc-1P. The synthesis reaction is shown below:chromatography (HPLC) resulting in >90% consumption of [18F]FDG in 1 h. HPLC conditions: Agilent®AdvanceBio®glycan column (250 × 6.4 mm), 2.7 μm (Santa Clara, CA). Eluent 80% - 65% D in 10 min; flow rate 0.5 mL / min. C: acetonitrile; D: 50 mM ammonium formate. Solid line, in-line radio detector; dotted line, UV detector at 220 nm. A nearly quantitative consumption of [18F]FDG was observed in 1.25 h (FIG.3).4239-109758-02 E-080-2023-0-PCT-02

[0111] The reaction was performed again with 100 mCi (~1.5 mL saline) of [18F]FDG, 37 mg of Glc-1P (100 µL), and CBP (1 mg in 300 µL saline) were incubated for 1 h at 40 °C to produce 2- deoxy-2-[18F]fluorocellobiose. After the completion of the reaction, ethanol (6 mL) was added to the reaction mixture precipitate out the CBP and Glc-1P, and the resulting mixture was passed through a 5 µm filter and then a stack of four solid-phase extraction (Sep-Pak®-NH2) cartridges where the product was retained on the cartridges. The retained product was eluted with 2 mL of 70% ethanol in water and diluted with saline for injection. Alternatively, FDG purification cartridges #KT-100 (Huayi Isotopes Co., Jiangsu Province, China) may be used, with the product eluted by saline. The identity of the product was confirmed using the liquid chromatography-mass spectrometry method (LCMS) by coeluting with the non-radioactive standard and matching the mass peak with the radiation peak. The overall yield of the reaction was 60-70% (n =6, decay corrected) in 2 h synthesis time with a radiochemical purity of > 98%.

[0112] To the solution of 2-deoxy 2-[18F]fluorocellobiose (1 mCi in 100 µL) was added 500 µL of whole human serum and incubated at 37 °C. An aliquot of the reaction mixture (5 µL) was directly injected into the HPLC in 1 h intervals to determine the stability. In a typical reaction, starting with 100 mCi of [18F]FDG, 32 mCi of 2-deoxy 2-[18F]fluorocellobiose was obtained in 2 h.2-deoxy 2- [18F]fluorocellobiose is stable as determined by analytical HPLC for at least up to 4 h in whole human serum at 37 °C (FIGS.4A-4D). HPLC condition: Agilent®AdvanceBio®glycan column (250 × 4.6 mm), 2.7 µm. Eluent 80% -65% D in 10 min; flow rate 0.5 mL / min. C: acetonitrile; D: 50 mM ammonium formate. Solid line, in-line radio detector; dotted line, UV detector at 220 nm.

[0113] Example 2 – In vitro uptake of cellobiose

[0114] Uptake assays were performed to compare the intake of tritiated cellobiose by A. fumigatus, gram-positive bacteria (S. aureus), gram-negative bacteria (E.coli), P. aeruginosa and C. albicans. The uptake was normalized to pellet weights and is represented as becquerel / mg of culture after accounting for the beta counter efficiency. The uptake of 3H-cellobiose in AF was much higher when compared to all other cultures tested and the levels progressively increased with each time- point up to 120 mins. Similar results were observed with the uptake of18F-2-fluoro-2-deoxy-L- rhamnose, which was found to be much higher in AF than E.coli. The uptake of 18F- deoxycellobiose in AF steadily increased over time (FIG.5). While a small increase in uptake by S. aureus over time was observed, the levels of18F-deoxycellobiose uptake in AF was about 4 times higher by 2 hours when normalized to weight (FIG.5).4239-109758-02 E-080-2023-0-PCT-02Example 3 – PET / CT imaging

[0115] Serum stability assays were performed to assess in vitro stability of18F-deoxycellobiose before its use for PET imaging. The ligand was found to be stable for up to 4 hours (FIGS.4A-4D). Uninfected control mice underwent dynamic PET / CT imaging for up to 60 mins and an additional static scan at 120 mins with18F-deoxycellobiose. The ligand was found to be stable (no defluorination) and cleared quickly such that there was no background uptake seen by 2 hours. In the AF myositis mice, increased ligand uptake was observed in the right thigh with the live infection when compared to the left thigh with the heat killed fungi. At 2 hours after radiotracer injection, the target to non-target uptake ratio (T / NT) was found to be quite high owing to the extremely low background. On the other hand, ligand retention was found to be much lower in the bacterial myositis models. Infected tissues from bacterial myositis mice were excised to confirm the presence of infection by enumerating the CFUs while GMS staining was done AF infected tissues. The plasma half-life of the ligand was calculated to be 7.3 minutes in control mice.

[0116] More impressive results were obtained with bolus-infusion (B / I) model of imaging with AF myositis mice showing even higher levels of uptake in the areas of live infection compared to heat killed, bacterial myositis and sterile inflammation LPS myositis (FIG.6). T / NT ratios for SUV mean and max were significantly higher in AF myositis compared to those of bacterial infection at 2-2.5 hour imaging time point (FIGS.7A, 7B). SUV mean values were also higher at 2-2.5 hour imaging time point compared to LPS myositis (FIG.7C).

[0117] The results demonstrate that18F-deoxycellobiose is a promising candidate for selective fungal infection imaging. The Aspergillus family includes a conserved cellulose metabolic component that is usually not present in most bacteria as they cannot assimilate cellobiose. This is the presumed basis for selective uptake of18F-deoxycellobiose in AF infection foci compared to sterile inflammation and bacterial infection.

[0118] Additionally, the18F-deoxycellobiose demonstrated quick clearance and low background noise which are good characteristics of an imaging ligands since immediate imaging can be performed without having to wait for background clearance.

[0119] Example 5 – Automated Synthesis of 2-deoxy-2-[18F]fluorocellobiose

[0120] Manual synthesis was performed. Briefly, 100 mCi (~1.2 mL saline) of [18F]FDG), 37 mg of Glc-1P (100 µL of water and 100 µL of 1 M HEPES buffer pH 7.6) and CBP (1.5 mg in 100 µL 50 mM Tris-HCl, 500 mM NaCl, 10 % Glycerol, pH 8.0, final concentration 1 mg / mL) were incubated for 1h at 40 °C followed by heating at 100 °C for 5 min. The mixture was filtered through a 5 µm filter and slowly passed through the system FDG purification cartridge. The4239-109758-02 E-080-2023-0-PCT-02cartridge was flushed with 3 mL saline. The product was eluted with 4 mL saline to obtain [18F]fluorocellobiose ([18F]FCB).

[0121] A fully automated synthesis was performed using the Trasis AllInOne (AIO) radiosynthesis module (Trasis, Tucker, GA). The same amounts of materials and solvents as mentioned in the manual method were used for this automated synthesis. The reagent vials consisted of (a) [18F]FDG; (b) Glc-1P; and (c) CBP in a reactor vial. Glc-1P was transferred to the reactor under a vacuum. [18F]FDG solution was transferred to a syringe. The GLc-1P vial was rinsed with [18F]FDG solution and transferred into the reactor. The reaction mixture was incubated for 1 h at 40 °C and the reaction temperature then was increased to 100 °C. After 5 min, the precipitated enzyme was filtered using a 5µm filter. [18F]FCB was retained in the FDG cartridge by passing the solution through the cartridge. The cartridge was washed with 3 mL saline followed by the elution of [18F]FCB with 4 mL saline into the product vial. A flow diagram of key steps is depicted in FIG.2A.

[0122] Implementing automated PET radiopharmaceutical production can reduce the possibility of human errors, leading to enhanced reproducibility and reduction of radiation exposure to the researchers. The primary modification in the automated synthesis occurs in the purification step. In the manual synthesis, after incubating the reaction mixture of [18F]FDG, Glc-1P, and cellobiose phosphorylase at 40 °C for 1 h, ethanol was added to precipitate out the enzyme. In this automated procedure, the reaction temperature was raised to 100 °C and kept at this temperature for 5 min. The precipitated enzyme was filtered off using a 5 µm filter and the solution was slowly passed through a FDG purification cartridge. This purification method effectively eliminated the enzyme as confirmed by high-performance liquid chromatography (HPLC) using a size-exclusion column (FIGS.8A-8B). HPLC conditions: column, TSKgel®SuperSW3000 (4.6 mm ID x 30 cm, 4µm; Tosoh Corporation, Japan); eluent, 0.1 M sodium phosphate, 0.1 M sodium sulfate, 0.05% sodium azide in water, 10% iso-propyl alcohol (pH 6.8), flow rate 0.35 mL / min; dotted line UV detector (FIG.8A, FIG 8B), solid line radio detector (FIG.8B).

[0123] A fully automated cassette-based synthesis of [18F]FCB was performed in the Trasis AIO module using the process outlined in FIG.2A. The overall yield of the reaction was 50-70% (n=6, decay corrected) in 1.5 h synthesis time with a radiochemical purity of > 98%. The radiochemical yield and purity were comparable to the manual synthesis. The identity of the product was confirmed using the liquid chromatography-mass spectrometry method (LCMS) by coeluting with the non-radioactive standard and matching the mass peak with the radiation peak. ESI Mass Spec Calculated for C12H21FO10Na 367.10 found 367.20 (M + Na). HPLC conditions: XBridge™4239-109758-02 E-080-2023-0-PCT-02amide column (4.6 mm x 150 mm, 3.5 µm; Waters Corp., Milford, MA); eluent, 90-50% B in 16 min; A= 95:5 water : acetonitrile + 0.1% NH4OH; B= 5:95 water : acetonitrile + 0.1% NH4OH; flow rate 0.5 mL / min.

[0124] The results demonstrated that the cassette-based synthesis offers enhanced reproducibility and compliance with current good manufacturing practice (cGMP) standards. The radiochemical yield (RCY) was 50-70% with a high radiochemical purity (> 98%). In a typical reaction starting with 100 mCi of [18F]FDG, 36 mCi of the product was obtained in 1.5 h (64% RCY, decay corrected). Overall, the protocol reliably provides a [18F]FCB suitable for clinical research.

[0125] Example 6- Enzymatic Radiosynthesis of 2-deoxy-2-[18F]fluorocellotriose

[0126] Materials and Methods

[0127] Materials: Cellobiose phosphorylase_His (CBP) and His_OCP2-52R (OCP2-52R, a CBP variant) were obtained from GenScript (Piscataway, NJ 08854). The lyophilized whole human serum was obtained from MP Biomedicals, LLC (Solon, OH, USA) and dissolved in 2 mL saline. This serum solution was directly used without inactivation for the stability study of 2-deoxy 2- [18F]fluorocellotriose. All other chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Fluorine-18 was received from the National Institutes of Health cyclotron facility (Bethesda, MD, USA). Columns and Sep-Pak cartridges used in this synthesis were obtained from Agilent Technologies (Santa Clara, CA, USA) and Waters (Milford, MA, USA), respectively. Analytical high-performance liquid chromatography (HPLC) analyses were performed on an Agilent®1200 Series instrument equipped with a multi- wavelength UV detector connected in series with a Bioscan flow count radio detector. Mass spectrometry (MS) was performed on a 6130 Quadrupole LCMS, Agilent Technologies instrument equipped with a diode array detector.

[0128] Enzyme specification: The CBP enzyme has a sequence according to SEQ ID NO.: 1 (supra). The CBP variant, OCP2-52R has a sequence according to SEQ ID NO.: 3:

[0129] MHHHHHHRYGHFDDEAREYVITTPHTPYPWINYLGSEQFFSLLSHQAGGYSFYRD AKRRRLTRYRYNNIPADAGGRYLYVNDGGDVWTPSWLPVKADLDHFEARHGLGYSTITG ERNGVRVETLFFVPVGENAEVQKVTVTNTSDSYKSLTLFSFVEFCLWDAQDDQTDYQRNL SIGEVEVEQESPHGSAIYHRTEYRERRDHYAVFAVNTQAEGFDTDRDTFVGAYNSLGEAA VPLKGESANSVASGWYPIGSHSVAVSLAPGESRELVYVLGYVENPDEEKWADDAKQVVN KERAHALLSRFATSEQTDAAFAALKDYWTDLLSTYSVSSNDEKLDRMVNIWNQYQCMVT FNMSRSASFFETGIGRGMGFRDSNQDLLGFVHLIPERARERIIDIASTQFADGSAYHQYQPL TKRGNNDIGSGFNDDPLWLIAGTAAYIKETGDFSILDEPVPFDNEPGSEVPLFEHLTRSFEFT4239-109758-02 E-080-2023-0-PCT-02VTHRGPHGLPLIGRADWNDCLNLNCFSTTPGESFQTIENQAGGVAESTFIAAQFVLYGEQY AELAARRGLADVADRARGHVAEMRDALLTDGWDGSWFLRAYDYYGNPIGTDAHDEGKI WIEPQGFAVMAGVGVGEGPQDTDAPAIKALDSVNEMLATDHGMVLQYPAYTTYQVHMG GVSTYPPGYKENGGIFCHANPWVIIAETVVGRGGRAFDYYKRITPAYREDISDVHRLEPYV YAQMIAGKEAVRHGEAKNSWLTGTAAWNFVTVSQYLLGVRPEYDGLVVDPQIGPDVPSF TVTRVARGATYEITVTNSGTDGSRGRLVVDGTPVEGNLVPYAPAGSTVRVDVTL**

[0130] Synthesis and Results

[0131] Synthesis of fluorine-18 labeled cellotriose was first prepared in two steps (Method A); i) production of 2-deoxy 2-[18F]fluorocellobiose by incubating commercially available 2-deoxy 2- [18F]fluoroglucose ([18F]FDG) with glucose 1-phosphate (Glc-1P) and cellobiose phosphorylase (CBP) at 37 °C for 1h, ii) conversion of 2-deoxy 2-[18F]fluorocellobiose to 2-deoxy 2- [18F]fluorocellotriose by incubating the reaction mixture for additional 1 h with another enzyme OCP2-52R (de Andrade et al., Chem. Eur. J.2021, 27:1374-1382; Ubiparipl et al., Applied Microbiology and Biotechnology 2020, 104:8327-8337). The reaction was performed with 100 mCi of [18F]FDG (in ~1.5 mL saline), 37 mg (100 μL) of Glc-1P, 1 mg of CBP (in 300 μL saline), and 1 mg of OCP2-52R (in 300 μL saline).chromatography (HPLC) resulting in the consumption of [18F]FDG with the appearance of a new peak (FIG.9A). HPLC conditions: Agilent AdvanceBio glycan (250 x 4.6 mm), 2.7 µm. Eluent 80% -65% D in 10 min; flow rate 0.5 mL / min. C: acetonitrile; D: 50 mM ammonium formate. The identity of the final radiolabeled product was confirmed by liquid chromatography mass spectrometry by co-eluting with a non-radioactive standard (FIG.9B). The production of 2-deoxy 2-[18F]fluorocellotriose was then tested in one step (Method B) by mixing both enzymes (CBP and4239-109758-02 E-080-2023-0-PCT-02OCP2-52R) with [18F]FDG and Glc-1P. HPLC reveals a similar result, >95% production of 2- deoxy 2-[18F]fluorocellotriose, after 1 h incubation. The reaction time was 1 h shorter than the two- step reaction.

[0133] After the completion of the reaction, ethanol was added to precipitate out the CBP, Glc-1P and OCP2-52R. The precipitate was filtered off using a 5 µm filter and the solution was slowly passed through the FDG purification cartridge to retain the product on the cartridge. The product was eluted with saline for injection. The identity of the product was confirmed using the liquid chromatography-mass spectrometry method (LCMS) by coeluting with the non-radioactive standard and matching the mass peak with the radiation peak. The overall yield of the reaction was 60-70% (n =2, decay corrected) in 1.5 h synthesis time with a radiochemical purity of > 95%. In a typical reaction, starting with 100 mCi of [18F]FDG 32 mCi of 2-deoxy 2-[18F]fluorocellotriose in 1.5 h.

[0134] Stability was evaluated by adding 500 μL of whole human serum to the solution of 2-deoxy 2-[18F]fluorocellotriose (1 mCi in 100 µL), and incubating at 37 °C. An aliquot of the reaction mixture (5 µL) was directly injected into the HPLC in 1 h intervals to determine the stability. The 2-deoxy 2-[18F]fluorocellobiose was stable as determined by analytical HPLC for at least up to 4 h in whole human serum at 37 °C (FIGS.10A-10D). HPLC conditions: Agilent®AdvanceBio®glycam column (250 x 4.6 mm), 2.7 µm. Eluent 80% -65% D in 10 min; flow rate 0.5 mL / min. C: acetonitrile; D: 50 mM ammonium formate.

[0135] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

4239-109758-02 E-080-2023-0-PCT-02We claim:

1. A method for enzymatic radiosynthesis of deoxy-[18F]fluorocellobiose, deoxy- [18F]fluorocellotriose, or both, the method comprising: (A) (i) combining deoxy-[18F]fluoroglucose ([18F]FDG) with glucose-1-phosphate (Glc- 1P), a cellobiose phosphorylase (CBP), and optionally a CBP variant to provide a first solution having at least a 10-fold molar excess of the Glc-1P relative to the [18F]FDG, and (ii) incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a second solution comprising deoxy- [18F]fluorocellobiose if the CBP variant is absent or a subsequent solution comprising deoxy-[18F]fluorocellotriose if the CBP variant is present; or (B) (i) phosphorylating [18F]FDG to form deoxy-[18F]fluoroglucose-1-phosphate ([18F]FGlc-1P), (ii) combining the [18F]FGlc-1P with glucose and a solution comprising a CBP to provide a first solution having at least a 10-fold molar excess of the glucose relative to the [18F]FGlc-1P, and (iii) incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a second solution comprising deoxy- [18F]fluorocellobiose, wherein the [18F]FDG has a structure according to (I), where one or more of R1, R2, R3, and R4is18F and the others of R1, R2,and wherein, in step (A)(i), R3is -OH.

2. The method of claim 1, wherein: (i) an initial amount of the [18F]FDG is from 1 mCi to 200 mCi; or (ii) in step A(i) an initial concentration of the Glc-1P is from 50 mM to 200 mM, or in step (B)(ii) an initial concentration of the glucose is from 50 mM to 200 mM; or (iii) an amount of the CBP is from 0.5 mg to 2 mg; (iv) an amount of the CBP variant, if present in step (A)(i), is from 0.5 mg to 2 mg; or (v) any combination of two or more of (i), (ii), (iii), and (iv).4239-109758-02 E-080-2023-0-PCT-023. The method of claim 1 or claim 2, wherein the [18F]FDG ,combination thereof.

4. The method of claim 3, wherein the [18F]FDG .

5. The method of any one of claims 1-4, wherein the method is an automated method and steps (A)(i) and (A)(ii) comprise: (A) (i) using an automated system to combine the GLC-1P with the CBP, and optionally the CBP variant, in a reactor and to add the ([18F]FDG to the reactor; and (A) (ii) using the automated system to incubate the first solution at the temperature of from 25 °C to 45 °C for the time of from 1 hour to 2 hours.

6. The method of any one of claims 1-5, wherein the second solution comprising deoxy-[18F]fluorocellobiose further comprises (A) CBP and unreacted Glc-1P or (B) CBP and unreacted glucose, the method further comprising separating the CBP from the second solution to provide a deoxy-[18F]fluorocellobiose solution.

7. The method of claim 6, wherein separating the CBP from the second solution comprises: adding a C1-C3alkanol to the second solution to precipitate (A) the CBP and unreacted Glc- 1P or (B) the CBP; and removing (A) the precipitated CBP and the precipitated unreacted Glc-1P or (B) the precipitated CBP to provide the deoxy-[18F]fluorocellobiose solution.

8. The method of claim 6, wherein separating the CBP from the second solution comprises: heating the second solution at a temperature of from 80 °C to 120 °C for 5 minutes to 15 minutes to precipitate the CBP; and removing the CBP to provide the deoxy-[18F]fluorocellobiose solution.4239-109758-02 E-080-2023-0-PCT-029. The method of claim 6, wherein the method is an automated method and separating (A) the CBP and the unreacted Glc-1P from the second solution comprises: using the automated system to incubate the second solution at a temperature of 80 °C to 120 °C for an effective amount of time to precipitate the CBP; and using the automated system to filter the second solution to remove the precipitated CBP from the second solution to provide a deoxy-[18F]fluorocellobiose solution.

10. The method of any one of claims 7-9, further comprising purifying the deoxy- [18F]fluorocellobiose solution by: (i) flowing the deoxy-[18F]fluorocellobiose solution through a solid-phase extraction (SPE) cartridge, whereby the deoxy-[18F]fluorocellobiose solution is retained in the SPE cartridge, and eluting the deoxy-[18F]fluorocellobiose from the SPE cartridge by flowing an eluent through the SPE cartridge; or (ii) using the automated system to flow the deoxy-[18F]fluorocellobiose solution through a solid-phase extraction (SPE) cartridge, whereby the deoxy-[18F]fluorocellobiose solution is retained in the SPE cartridge, and using the automated system to elute the deoxy-[18F]fluorocellobiose from the SPE cartridge by flowing an eluent through the SPE cartridge.

11. The method of any one of claims 1-10, wherein: (i) the CBP variant is absent and following step (A)(ii), the deoxy-[18F]fluorocellobiose has a structure according to Formula II or (ii) following stepa structure according to Formula III .4239-109758-02 E-080-2023-0-PCT-0212. The method of claim 11, wherein: (a) the deoxy-[18F]fluorocellobiose has a structure according to Formula II and the deoxy- [18F]fluorocellobiose is , (b) the and the deoxy- 18[ F]13. The method of claim 12, wherein the deoxy-[18F]fluorocellobiose is:

14. The method of claim 1, wherein: steps (A)(i) and (A)(ii) are performed; the CBP variant is present; and incubating the first solution at the temperature of from 25 °C to 45 °C for the time of from 1 hour to 2 hours provides the subsequent solution comprising deoxy-[18F]fluorocellotriose, wherein the deoxy-[18F]fluorocellotriose has a structure according to Formula IV .4239-109758-02 E-080-2023-0-PCT-0215. The method of claim 14, wherein the deoxy-[18F]fluorocellotriose is: ,or an further comprising: using an automated system to combine the [18F]FDG, Glc-1P, CBP and CBP variant to form the first solution; and using the automated system to incubate the first solution at the temperature of from 25 °C to 45 °C for the time of from 1 hour to 2 hours to provide the subsequent solution comprising deoxy- [18F]fluorocellotriose.

17. The method of any one of claims 14-16, wherein the subsequent solution further comprises CBP and the CBP variant, the method further comprising separating the CBP and the CBP variant from the deoxy-[18F]fluorocellotriose to provide a deoxy-[18F]fluorocellotriose solution.

18. The method of claim 17, wherein separating the CBP and the CBP variant from the subsequent solution comprises: adding a C1-C3alkanol to the subsequent solution to precipitate the CBP, the CBP variant, and unreacted Glc-1P; and removing the precipitated CBP, the precipitated CBP variant, and the precipitated unreacted Glc-1P to provide the deoxy-[18F]fluorocellotriose solution.

19. The method of claim 17, wherein separating the CBP variant from the subsequent solution comprises: heating the subsequent solution at a temperature of from 80 °C to 120 °C for 5 minutes to 15 minutes to precipitate the CBP and the CBP variant; and removing the CBP and the CBP variant to provide the deoxy-[18F]fluorocellotriose solution.4239-109758-02 E-080-2023-0-PCT-0220. The method of claim 17, wherein the method is an automated method and separating the CBP and the CBP variant from the subsequent solution comprises: using the automated system to incubate the subsequent solution at a temperature of 80 °C to 120 °C for an effective amount of time to precipitate the CBP and the CBP variant; and using the automated system to filter the subsequent solution to remove the precipitated CBP and CBP variant from the subsequent solution to provide a deoxy-[18F]fluorocellotriose solution.

21. The method of any one of claims 1-13, further comprising: combining the deoxy-[18F]fluorocellobiose with Glc-1P and a CBP variant to provide a third solution having at least a 10-fold molar excess of the Glc-1P relative to the deoxy- [18F]fluorocellobiose; and incubating the third solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a subsequent solution comprising deoxy-[18F]fluorocellotriose.

22. The method of claim 21, wherein the method is an automated method further comprising: using an automated system to combine the deoxy-[18F]fluorocellobiose with the Glc-1P and the CBP variant in a reactor; and using the automated system to incubate the third solution at the temperature of from 25 °C to 45 °C for the time of from 1 hour to 2 hours to provide the subsequent solution comprising deoxy-[18F]fluorocellotriose.

23. The method of claim 21 or claim 22, wherein the subsequent solution further comprises unreacted Glc-1P and the CBP variant, the method further comprising separating the CBP variant from the subsequent solution to provide a deoxy-[18F]fluorocellotriose solution.

24. The method of claim 23, wherein separating the CBP variant from the subsequent solution comprises: adding a C1-C3 alkanol to the subsequent solution to precipitate the CBP variant and unreacted Glc-1P; and removing the precipitated CBP and the precipitated unreacted Glc-1P to provide the deoxy- [18F]fluorocellotriose solution.4239-109758-02 E-080-2023-0-PCT-0225. The method of claim 23, wherein separating the CBP variant from the subsequent solution comprises: heating the subsequent solution at a temperature of from 80 °C to 120 °C for 5 minutes to 15 minutes to precipitate the CBP variant; and removing the CBP variant to provide the deoxy-[18F]fluorocellotriose solution.

26. The method of claim 23, wherein the method is an automated method and separating the CBP variant from the subsequent solution comprises: using the automated system to incubate the subsequent solution at a temperature of 80 °C to 120 °C for an effective amount of time to precipitate the CBP variant; and using the automated system to filter the subsequent solution to remove the precipitated CBP variant from the subsequent solution to provide a deoxy-[18F]fluorocellotriose solution.

27. The method of any one of claims 21-26, wherein: the deoxy-[18F]fluorocellobiose has a structure according to Formula II , wherein one or more of R1, R2, and R3is18F and the others of[18F]fluorocellotriose has a structure according to Formula IV or the deoxy-III , wherein R3is -OH, one or more of R1, R2, and R4is18F and theand the deoxy-[18F]fluorocellotriose has a structure according to Formula .4239-109758-02 E-080-2023-0-PCT-0228. The method of claim 27, wherein: (a) the deoxy-[18F]fluorocellotriose has a structure according to Formula IV and the deoxy- [18F]fluorocellotriose is: ,any combination thereof; or (b) to Formula V and the deoxy- 18[ F] ,or any combination thereof.

29. The method of claim 28, wherein the deoxy-[18F]fluorocellotriose is:

30. The method of any one of claims 17-20 or 23-29, further comprising purifying the deoxy-[18F]fluorocellotriose by: (i) flowing the deoxy-[18F]fluorocellotriose solution through a solid-phase extraction (SPE) cartridge, whereby the deoxy-[18F]fluorocellotriose solution is retained in the SPE cartridge, and eluting the deoxy-[18F]fluorocellotriose from the SPE cartridge by flowing an eluent through the SPE cartridge; or4239-109758-02 E-080-2023-0-PCT-02(ii) using the automated system to flow the deoxy-[18F]fluorocellotriose solution through a solid-phase extraction (SPE) cartridge, whereby the deoxy- [18F]fluorocellotriose solution is retained in the SPE cartridge, and using the automated system to elute the deoxy-[18F]fluorocellotriose from the SPE cartridge by flowing an eluent through the SPE cartridge.

31. The method of any one of claims 14-30, wherein an amount of the CBP variant is from 0.5 mg to 2 mg.

32. A method for enzymatic radiosynthesis of deoxy-[18F]fluorocellotriose, comprising: phosphorylating [18F]FDG to form [18F]FGlc-1P; combining the [18F]FGlc-1P with cellobiose and a CBP variant to provide a first solution having at least a 10-fold molar excess of the cellobiose relative to the [18F]FGlc-1P; and incubating the first solution at a temperature of from 25 °C to 45 °C for a time of from 1 hour to 2 hours to provide a subsequent solution comprising deoxy-[18F]fluorocellotriose, wherein the [18F]FDG has a structure according to (I), where one or more of R1, R2, R3, and R4is18F and the others of R1, R2,and wherein the deoxy-[18F]fluorocellotriose has a structure according to Formula VI, .

33. The method of claim 32, wherein an initial concentration of the cellobiose is 50 mM to 200 mM.

34. The method of claim 32 or claim 33, wherein the subsequent solution further comprises unreacted cellobiose and the CBP variant, the method further comprising separating the unreacted cellobiose and the CBP variant from the deoxy-[18F]fluorocellotriose to provide a deoxy- [18F]fluorocellotriose solution.4239-109758-02 E-080-2023-0-PCT-0235. The method of any one of claims 32-34, wherein the deoxy-[18F]fluorocellotriose is: or36. The method of claim 35, wherein the deoxy-[18F]fluorocellotriose is: .

37. The method of any one of claims 32-36, wherein: (i) an initial amount of the [18F]FDG is from 1 mCi to 200 mCi; or (ii) an amount of the CBP variant is from 0.5 mg to 2 mg; or (iii) both (i) and (ii).

38. The method of any one of claims 14-37, wherein the CBP variant is a Cellulomonas uda OCP2_M52R variant or a Cellulomonas uda His_OCP2-52R variant.

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