Improved radiosynthesis of 18f-labeled nucleoside analogues

Abstract

The disclosure provides for methods, including automated methods, of radio synthesizing certain 2'-deoxy-2'-[18F]fluoro-5-substituted-1-β-D-arabinofuranosyl-uracil and related compounds such as, for example, 2'-deoxy-2'-[18F]fluoro-5-methyl-1-β-D-arabinofuranosyl-uracil ([18F]FMAU).

Description

[0001]IMPROVED RADIOSYNTHESIS OF18F-LABELED NUCLEOSIDE ANALOGUES RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No.63 / 589,084 filed October 10, 2023, which is incorporated herein by reference. BACKGROUND OF THE INVENTION 2'-Deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil, or [18F]FMAU, is a nucleoside analog PET tracer that can incorporate directly into DNA, making it an effective marker for monitoring cell proliferation. Recent pilot studies of [18F]FMAU in patients have demonstrated its ability to visualize multiple types of tumors with high contrast, and it may prove valuable for cancer treatment monitoring as well. However, the conventional radiosynthesis of [18F]FMAU is very lengthy (~150 min), uses corrosive and unstable reagents that are not compatible with commercial automated synthesis modules and has a low overall activity yield (~5%), all of which significantly hinder clinical trials and further translation. [18F]FMAU analogues, such as 2’-deoxy-2’-[18F]fluoro-5-ethyl-1-^-D-arabinofuranosyluracil ([18F]FEAU) and 2’-deoxy-2’-[18F]fluoro-5-iodo-1-^-D-arabinofuranosyluracil ([18F]FIAU), are PET radiotracers for imaging reporter gene herpes virus type 1 thymidine kinase (HSV1- tk) expression. Hence, they have been used for gene-based therapy, transgenic models, and cell trafficking. Therefore, there is a need for a faster and more efficient method of synthesizing [18F]FMAU and its analogues whose radiosynthesis process is similar. The present disclosure satisfies this need. SUMMARY OF THE INVENTION The disclosure provides for methods of radio synthesizing certain 2'-deoxy-2'- [18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil or -cytosine compounds such as, for example, 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU). In some embodiments, a method for synthesizing a 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil may comprise the steps of: a) dispensing one or more droplets of a solution comprising: i) a [18F]fluoride radioisotope in a solvent; and ii) a phase transfer catalyst onto a first reaction site of one or more hydrophilic reaction sites using a first dispenser of a plurality of non-contact dispensers; b) thermally treating the [18F]fluoride radioisotope and the phase transfer catalysis on the first reaction site using a thermally controlled support to evaporate the solvent, providing a dried residue comprising a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) dispensing one or USC 2023-124-02 1 530.039WO1 more droplets of a ribose sugar precursor onto the first reaction site using a second dispenser of a plurality of non-contact dispensers to form a first mixture with the dried residue; d) thermally treating the first mixture on the first reaction site using the thermally controlled support to facilitate a fluorination reaction to fluorinate the ribose sugar, thereby providing a fluorinated ribose sugar; e) dispensing one or more droplets of a coupling reagent onto the first reaction site using a third dispenser of a plurality of non-contact dispensers to provide a coupling mixture, wherein the coupling reagent comprises a pyrimidine nucleobase, and thermally treating the coupling mixture to couple the fluorinated ribose sugar to the pyrimidine nucleobase to form a protected 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl- uracil; f) dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture using a fourth dispenser of a plurality of non-contact dispensers; g) thermally treating the deprotection mixture to form the 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil; h) removing the 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil using a collection tube by applying a vacuum to the collection tube, wherein a total volume of the combination of each reagent of the steps a) – h) is about 100 µL or less. In some embodiments, the phase transfer catalyst comprises tetrabutylammonium bicarbonate (TBAHCO3), wherein the (TBAHCO3) is present in an amount of about 0.05µmol to about 0.5 µmol. In some embodiments, the coupling reagent comprises the pyrimidine nucleobase in 1,4-dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS). In some embodiments, the pyrimidine nucleobase comprises a protected thymidine or thymidine, and the^pyrimidine nucleobase is present in an amount of about 0.1 µmol to about 1 µmol; the TMSOTf is present in an amount of about 1 µL to about 10 µL; and the HMDS is present in an amount of about 0.1 µL to about 5 µL. In some embodiments, the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil or -cytosine compound is: 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D- arabinofuranosyl-uracil ([18F]FMAU), 2'-[18F]fluoro-5-ethy1-1-^-D-arabinofuranosyluracil(FEAU), 2'-deoxy-2'-[18F]fluoro-5-fluoro-l-^-d-arabinofuranosyluracil ([18F]FFAU), 1-(2-deoxy-2-[18F]fluoro-^-D-arabinofuranosyl)-5-chlorouracil ([18F]FCAU), 1-(2-deoxy-2- [18F]fluoro-^-D-arabinofuranosyl)-5-bromouracil ([18F]FBAU), 1-(2-deoxy-2--[18F]fluoro-(3- D-arabinofuranosyl)uracil ([18F]FAU), 2'-[18F]fluoro-2'-deoxy-l-^-D-arabinofuranosyl-5- iodouracil ([18F]FIAU), 1-(2-deoxy-2-[18F] ^-D-arabinofuranosyl)cytosine ([18F]FAC), USC 2023-124-02 2 530.039WO1 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosylcytosine ([18F]FMAC), 2'- [18F]fluoro-5-ethyl-1-^-D-arabinofuranosyl-cytosine ([18F]FEAC), 2'-deoxy-2'-[18F]fluoro-5- fluoro-l-^-D-arabinofuranosyluracil ([18F]FFAC), 1-(2-deoxy-2-[18F]fluoro-^-D- arabinofuranosyl)-5-chlorocytosine ([18F]FCAC), 1-(2-deoxy-2-[18F]fluoro-^-D- arabinofuranosyl)-5-bromocytosine ([18F]FBAC), or 2'-deoxy-2'-[18F]fluoro-5- hydroxymethyl-1-^-D-arabino-furanosylcytosine ([18F]FHMAC). In some embodiments, a method of radio synthesizing certain 2'-deoxy-2'-[18F]fluoro- 5-substituted-l-^-D-arabinofuranosyl-uracil may be an automated method comprising the use of a radiosynthesis device, wherein the radiosynthesis device comprises the thermally controlled support configured to support a surface having the one or more reaction sites formed thereon; a fixture configured to support the plurality of non-contact dispensers and the collection tube, wherein the plurality of the non-contact dispensers is installed on the fixture positioned above the thermally controlled support and configured to respectively dispense one or more droplets of a respective reagent into the one or more reaction sites; the collection tube installed on the fixture above the support; and a motorized rotation stage operatively coupled to the support for controllably rotating the support, the motorized rotation stage configured to controllably rotate the support relative to the plurality of the non-contact dispensers to sequentially position the one or more reaction sites for dispensing respective reagent from the plurality of the non-contact dispensers into the one or more reaction sites, and to controllably rotate the support relative to the collection tube to sequentially position the one or more reaction sites for removing reaction product from the one or more reaction sites via the collection tube. In some embodiments, an automated method further comprises rotating the surface having the one or more hydrophilic reaction sites in order to perform another chemical reaction or to dispense another reagent, or both, by rotating the motorized rotation stage attached thereto in order to position the first reaction site in line with the second dispenser of the plurality of non-contact dispensers to dispense a second reagent. The method also may include rotating the surface by rotating the motorized rotation stage to position the first reaction site in line with the third dispenser of the plurality of non-contact dispensers to dispense a third reagent; optionally, after the step e), rotating the surface by rotating the motorized rotation stage to position the first reaction site in line with the fourth dispenser of the plurality of non-contact dispensers to dispense a fourth reagent; and optionally, after the step g), rotating the surface by rotating the motorized rotation stage to position the first reaction site in alignment with the collection tube. USC 2023-124-02 3 530.039WO1 In another embodiment, an automated method of using a radiosynthesis device to form 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU) compound comprising: a) dispensing one or more droplets onto a first reaction site of one or more hydrophilic reaction sites disposed on a surface using a first dispenser of a plurality of non- contact dispensers, wherein the one or more droplets comprise a solution comprising: i) [18F]fluoride radioisotope in a solvent, wherein the [18F]fluoride is present in an amount of about 1 mCi to about 500 mCi; and ii) a phase transfer catalyst comprising tetrabutylammonium bicarbonate (TBAHCO3) present in an amount of about 0.1 µmol to about 0.5 µmol; b) thermally treating the [18F]fluoride solution and the phase transfer catalysis on the first reaction site using a thermally controlled support coupled to the surface to evaporate the solvent, thereby providing a dried residue of a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) rotating the surface comprising the one or more hydrophilic reaction sites by rotating a motorized rotation stage attached to the thermally controlled support in order to position the first reaction site in line with a second dispenser of the plurality of non-contact dispensers, and dispensing one or more droplets of a ribose sugar precursor to the first reaction site to form a first mixture with the dried residue of the [18F]fluoride-phase transfer catalyst complex, wherein the ribose sugar precursor is present in an amount of about 0.1 µmol to about 10 µmol; d) thermally treating the first mixture on the first reaction site using the thermally controlled support for about 3 minutes at about 100 °C to about 105 °C to fluorinate the ribose sugar precursor to provide a fluorinate ribose sugar; e) rotating the surface to position the first reaction site at a third dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a coupling reagent onto the first reaction site to form a coupling mixture, and thermally treating the coupling mixture to couple the fluorinated ribose sugar for about 60 seconds at about 130 °C to form a protected [18F]FMAU compound, wherein the coupling reagent comprises a pyrimidine nucleobase in 1,4-dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS); f) rotating the surface to position the first reaction site at a fourth dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture, and thermally treating the deprotection mixturefor about 3 minutes at about 85 °C to form the [18F]FMAU compound, wherein the deprotectingreagent comprises potassium methoxide in methanol; and g) rotating the surface to position the first reaction site in alignment with a collection tube, and removing the [18F]FMAU compound using a collection tube by applying a vacuum to the collection tube, thereby providing the USC 2023-124-02 4 530.039WO1 [18F]FMAU compound, wherein a total volume of the combination of each reagent of the steps a) – g) is about 100 µL or less. Advantageously, the microscale synthesis methods and apparatus described herein reduce synthesis time of [18F]FMAU from 150 minutes to about 28 minutes, improves yield from 12±3% to 25±3%, and simplifies purification and quality control for cGMP production as compared to previous methods. These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention. Fig. 1. A schematic of an embodiment of a synthesis process flow on a droplet microreactor. Fig.2. FMAU-PET in cancer patients. Fig. 3A-G. Radio-HPLC chromatogram of crude [18F]FMAU (top: UV-254 nm, bottom: ^-ray) from microdroplet radiosynthesis using various purification methods. Detailed conditions are provided in Table 2. (A) = test 1; (B) = test 2; (C) = test 3; (D) = test 4; (E) = test 5; (F) = test 6; and (G) = test 7. Fig.4. Example of a radio-HPLC chromatogram of crude [18F]FMAU (top: UV-254 nm, bottom: ^-ray) from microdroplet radiosynthesis during purification. The mobile phase consisted of 96% PBS buffer and 4% EtOH (v / v) for the first 13 min, followed by a change to 15% PBS buffer and 85% MeCN (v / v) within 1 min, with an additional 6 min under the same conditions. Under these conditions, the retention time of [18F]FMAU (^-anomer) was 8.6 min, while the ^-anomer was 5.9 min. USC 2023-124-02 5 530.039WO1 Fig.5. Example of a radio-HPLC analysis of purified [18F]FMAU produced in a droplet reaction. The analytical mobile was 95% DI water and 5% MeCN (v / v) with 0.1% TFA. Under these conditions, the retention time of [18F]FMAU (^-anomer) was 5.0 min. Fig. 6. Example of a radio-HPLC analysis of a co-injection of purified [18F]FMAU produced in a droplet reaction and a reference standard. The analytical mobile phase was 95% DI water and 5% MeCN (v / v) with 0.1% TFA. Fig.7A-B. Automated microdroplet radio synthesizer setup for [18F]FMAU synthesis. (A) Diagram of “One-shot” tubing system which enables remote delivery of coupling reagents, complemented by piezoelectric dispensers for radioisotopes or other reagents. (B) Configuration of automated microdroplet system for [18F]FMAU preparation. Top view showing the arrangement of reagent dispensers and collection tubing positioned above the chip. Fig.8. A perspective view of an embodiment of a radiosynthesizer device that may be used to carry out the methods described herein. DETAILED DESCRIPTION Definitions. The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001. References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement USC 2023-124-02 6 530.039WO1 is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring. As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect. The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, and as part of a range. A recited range (e.g., USC 2023-124-02 7 530.039WO1 weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, … 9, 10. It also means 1.0, 1.1, 1.2.1.3, …, 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number10”, it implies a continuous range that includes whole numbers and fractional numbers less than number10, as discussed above. Similarly, if the variable disclosed is a number greater than “number10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number10. These ranges can be modified by the term “about”, whose meaning has been described above. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation. The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the or molecular level, for example, to bring USC 2023-124-02 8 530.039WO1 about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo. The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%. An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment). Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of” or “consisting essentially of” are used instead. As used herein, “comprising” is synonymous with "containing," or "characterized by," and is USC 2023-124-02 9 530.039WO1 inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein. The term “one-pot” is a term commonly used by ordinary persons skilled in the art referring to a strategy to improve the efficiency of a chemical reaction whereby a reactant is subjected to successive chemical reactions in just one reactor. The strategy avoids lengthy separation and purification steps of intermediate chemical compounds and saves time and resources while increasing chemical yield. A one-pot synthesis may require changing a solvent to a different solvent at one or more steps during the procedure, for example, by simply evaporation under reduced pressure. Alternatively, it may be possible to perform the synthesis with a single suitable solvent that can be used throughout the entire procedure without changing the solvent. Generally, a sequential one-pot synthesis is performed by adding reagents to a reactor one at a time and without work-up. As used herein, “PET” or “PET-scan” refers to positron emission tomography (PET) scanning using a molecular tracer. PET-scan is a nuclear medicine functional imaging technique that is widely used in the medical field to observe metabolic processes in the body as an aid to the diagnosis of disease. Embodiments of the Invention. The disclosure provides for methods of synthesizing 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compounds, and in particular, 2'-deoxy-2'- [18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU), and optionally using an apparatus for performing said methods. Generally, embodiments of a method for synthesizing a 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compound comprises: i) depositing a solution comprising [18F]fluoride and a phase transfer catalyst onto a hydrophilic reaction site, wherein both the [18F]fluoride and the phase transfer catalyst are dried onto the hydrophilic reaction site to form a dried residue; ii) adding a ribose sugar precursor in a 1,4-dioxane solvent to the hydrophilic reaction site to form a first with the dried [18F]fluoride and the phase transfer catalyst; iii) reacting the first mixture to fluorinate the ribose sugar precursor to form USC 2023-124-02 10 530.039WO1 a fluorinated ribose sugar; iv) coupling the fluorinated ribose sugar precursor to a pyrimidine nucleobase in a 1,4 dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS) to form a coupled 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compound; and v) deprotecting the coupled 2'- deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound in the presence of potassium methoxide in methanol to form the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound; wherein a total volume of each of the steps i) - v) is about 100 µL or less. In other embodiments, a method for synthesizing 2'-deoxy-2'-[18F]fluoro-5-substituted- l-^-D-arabinofuranosyl-uracil compounds may be partially or fully automated using a device or apparatus configured to perform microscale radiosynthesis reactions. For example, in some embodiments, an automated method for synthesizing a 2'-deoxy-2'-[18F]fluoro-5-substituted-l- ^-D-arabinofuranosyl-uracil compound may comprise the steps of: dispensing one or more droplets of reagent onto the one or more reaction sites of a surface using a first dispenser of a plurality of non-contact dispensers; heating and / or cooling the one or more droplets of reagent to form a first reaction product using a thermally controlled support coupled to the surface; optionally rotating the surface into position under a different non-contact dispensers by a motorized rotation stage operably coupled to the thermal support; optionally, dispensing one or more droplets of a second reagent onto the one or more reaction sites of a surface using a second dispenser of the plurality of non-contact dispensers to form a mixture; optionally, heating and / or cooling the mixture using the thermally controlled support to form a second reaction product; rotating the surface to place the one or more reaction sites containing the second reaction product thereon under the collection tube; and removing the second reaction product with the collection tube by applying a vacuum to the collection tube. In some embodiment, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different dispensers may be used to dispense various reagents necessary to complete the radiofluorination synthesis to produce the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound. For example, in one embodiment, a first dispenser may dispense the [18F]fluoride radioisotope and the phase transfer catalysis is a basic solvent, a second dispenser may dispense the ribose sugar precursor, a third dispenser may dispense 1,4-dioxane, a fourth dispenser may dispense one or more coupling reagents, a fifth dispenser may dispense one or more deprotection reagents, a sixth dispenser may dispense replenishment solution for the deprotection reagents (e.g., more deprotection reagents), and a dispenser may dispense a collection solution. USC 2023-124-02 11 530.039WO1 In some embodiments, the collection solution may comprise hexane and ethyl acetate (1:1, v / v). For example, in some embodiments, an automated method of using a radiosynthesis device to produce a 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compounds comprising the steps of: a) dispensing one or more droplets of a solution comprising: i) a [18F]fluoride radioisotope in a solvent; and ii) a phase transfer catalyst; onto a first reaction site of one or more hydrophilic reaction sites using a first dispenser of a plurality of non-contact dispensers; b) thermally treating the [18F]fluoride radioisotope and the phase transfer catalysis on the first reaction site using a thermally controlled support to evaporate the solvent, thereby providing a dried residue comprising a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) dispensing one or more droplets of a ribose sugar precursor onto the first reaction site using a second dispenser of a plurality of non-contact dispensers to form a first mixture with the dried residue; d) thermally treating the first mixture on the first reaction site using the thermally controlled support to facilitate a fluorination reaction to fluorinate the ribose sugar, thereby providing a fluorinated ribose sugar; e) dispensing one or more droplets of a coupling reagent onto the first reaction site using a third dispenser of a plurality of non-contact dispensers to provide a coupling mixture, wherein the coupling reagent comprises a pyrimidine nucleobase, and thermally treating the coupling mixture to couple the fluorinated ribose sugar to the pyrimidine nucleobase to form a protected 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound; f) dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture using a fourth dispenser of a plurality of non-contact dispensers; g) thermally treating the deprotection mixture to form the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound; h) removing the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound using a collection tube by applying a vacuum to the collection tube, wherein a total volume of the combination of each reagent of the steps a) – h) is about 100 uL or less. In some embodiments, the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compounds is FMAU. In some embodiments, the [18F]fluoride radioisotope in a solvent and the phase transfer catalyst may be dispensed onto a first reaction site of one or more hydrophilic reaction sites using a first dispenser of a plurality of non-contact dispensers as a single solution comprising both the [18F]fluoride radioisotope in a solvent and the phase transfer catalyst or as two separate solutions added to the first reaction site. USC 2023-124-02 12 530.039WO1 In some embodiments, a single dispenser may be used to dispense two or more reagents onto reaction site. In some embodiments, the amount of [18F]fluoride present in the reaction is in an amount of about 0.001 mCi to about 1 mCi. In some embodiments, the amount of [18F]fluoride present in the reaction is in an amount of about 0.1 mCi to about 1000 mCi. In some embodiments, the amount of [18F]fluoride present in the reaction is in an amount of about 1000 mCi to about 5000 mCi. In some embodiments, the amount of [18F]fluoride present in the reaction is in an amount of about 1 mCi to about 100 mCi, about 5 mCi to about 95 mCi, about 10 mCi to about 90 mCi, about 15 mCi to about 85 mCi, about 20 mCi to about 80 mCi, about 25 mCi to about 75 mCi, about 30 mCi to about 70 mCi, about 35 mCi to about 65 mCi, about 40 mCi to about 60 mCi, about 45 mCi to about 55 mCi, or about 50 mCi. In some embodiments, the [18F]fluoride is present in an amount of about 2 mCi to about 40 mCi. In some embodiments, the phase transfer catalyst comprises tetrabutylammonium bicarbonate, such as, for example, tetrabutylammonium hydrogen carbonate (TBAHCO3). Preferably, the phase catalyze (tetrabutylammonium bicarbonate or TBAHCO3) may be present in an amount of about 0.01 µmol to about 2 µmol, or about 0.05 µmol to about 1 µmol, or about 0.1 µmol to about 0.5 µmol. In some embodiments, the amount of tetrabutylammonium bicarbonate or TBAHCO3 in the reaction is about 0.5 µmol to about 0.25 µmol. In some embodiments, one or both of the [18F]fluoride solution and the phase transfer catalyst further comprise 1,4 dioxane as a solvent. In some embodiments, the ribose sugar precursor is present in the reaction in an amount of about 0.1 µmol to about 15 µmol, about 0.1 µmol to about 10 µmol, about 0.1 µmol to about 5 µmol, about 0.1 µmol to about 4 µmol, about 0.1 µmol to about 3 µmol, about 0.1 µmol to about 2 µmol, about 0.1 µmol to about 1 µmol, or about 0.1 µmol to about 0.5 µmol. In some embodiments, the ribose sugar precursor is present in the reaction in an amount of about 0.01 µmol to about 1 µmol, or about 0.5 µmol. In some embodiments, the ribose sugar precursor is 2-O-(trifluoromethanesulfonyl)-1,3,5-tri-O-benzoyl-^-D-ribofuranose. Optionally, the ribose sugar precursor is dissolved in 1,4-dioxane. In some embodiments, the coupling reagents comprise a pyrimidine nucleobase such as, but not limited to, a protected thymine (O, O’-bis(trimethylsilyl)thymine) or thymine, or a 5-substituted uracil. In some embodiments, the amount of the pyrimidine nucleobase in the reaction is about 0.1 µmol to about 1.5 µmol, about 0.1 µmol to about 1 µmol, or about 0.1 µmol to about 0.5 µmol. USC 2023-124-02 13 530.039WO1 In some embodiments, the coupling reagent further comprises trimethylsilyl trifluoromethanesulfonate (TMSOTf) that is present in an amount of about 1 µL to about 500 µL. In some embodiments, the TMSOTf is present in an amount of about 1 µL to about 100 µL. In some embodiments, the TMSOTf is present in an amount of about 1 µL to about 20 µL, about 1 µL to about 15 µL, about 1 µL to about 10 µL, about 1 µL to about 9 µL, about 1 µL to about 8 µL, about 1 µL to about 7 µL, about 1 µL to about 6 µL, about 1 µL to about 5 µL, about 1 µL to about 4 µL, about 1 µL to about 3 µL, or about 1 µL to about 2 µL. In some embodiments, the TMSOTf is present in an amount of about 1 µL to about 10 µL. In some embodiments, the coupling reagent further comprises hexamethyldisilane (HMDS) is present in an amount of about 0.1 µL to about 500 µL. In some embodiments, the HMDS is present in an amount of about 0.1 µL to about 100 µL. In some embodiments, the HMDS is present in an amount of about 0.1 µL to about 10 µL, about 0.1 µL to about 9 µL, about 0.1 µL to about 8 µL, about 0.1 µL to about 7 µL, about 0.1 µL to about 6 µL, about 0.1 µL to about 5 µL, about 0.1 µL to about 4 µL, about 0.1 µL to about 3 µL, about 0.1 µL to about 2 µL, or about 0.1 µL to about 1 µL. In some embodiments, the HMDS is present in an amount of about 0.1 µL to about 5 µL. In some embodiments, the deprotecting step comprises a use of about 10 µL to about 500 µL of the potassium methoxide in methanol, In some embodiments, the deprotecting step comprises a use of about 10 µL to about 100 µL of the potassium methoxide in methanol, about 20 µL to about 90 µL of the potassium methoxide in methanol, about 30 µL to about 80 µL of the potassium methoxide in methanol, about 40 µL to about 70 µL of the potassium methoxide in methanol, or about 50 µL to about 60 µL of the potassium methoxide in methanol. In some embodiments, the deprotecting step comprises a use of about 10 µL to about 40 µL of the potassium methoxide in methanol. In some embodiments, the total volume of the combination of each of the steps is about 100 µL to about 500 µL. In some embodiments, the total volume of each of the steps is about 100 µL or less; about 95 µL or less, about 90 µL or less, about 85 µL or less, about 80 µL or less, about 75 µL or less, about 70 µL or less, about 65 µL or less, about 60 µL or less, about 55 µL or less, about 50 µL or less, about 45 µL or less, about 40 µL or less, about 35 µL or less, about 30 µL or less, about 25 µL or less, about 20 µL or less, about 15 µL or less, about 10 µL or less, or about 5 µL or less. In some embodiments, the solution comprising a [18F]fluoride radioisotope in a solvent and a phase transfer catalyst may be thermally treated while the solution is in the reaction site USC 2023-124-02 14 530.039WO1 to evaporate the solvent, thereby providing a dried residue on the reaction site comprising a [18F]fluoride-phase transfer catalyst complex. In some embodiments, the radiofluorination step comprises a duration of about 1 to 5 minutes at about 100 °C to about 115 °C, during which time, the ribose sugar precursor is fluorinated to form a fluorinated ribose sugar. In some embodiments, the duration of the coupling step comprises a duration of about 0.5 minutes to about 3 minutes at about 120 °C to about 140 °C. In some embodiments, the duration of the deprotecting step comprises about 1 minute to about 5 minutes at about 75 °C to about 95 °C. In some embodiments, the duration of the radiofluorination step comprises about 3 minutes at about 100 °C to about 105 °C. In some embodiments, the duration of the coupling step comprises about 60 seconds at about 130 °C. In some embodiments, the duration of the deprotecting step comprises about 3 minutes at about 85 °C. In some embodiments, the method further comprises collecting the 2'-deoxy-2'- [18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound and mixing the same with a neutralizing acid. In some embodiments, the method further comprises purifying the 2'-deoxy- 2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound. In some embodiments, the method further includes purifying the synthesized compound, via, for example, high-performance liquid chromatography (HPLC). In some embodiments, the [18F]FMAU or other 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compounds may be collected and optionally purified using, for example, an analytic column. For example, For example, the compounds may be separated and purified using an analytical column and a gradient comprising the following parameters: about 0-13 minutes: about 90% to about 99% phosphate buffered saline (PBS) buffer and about 1% ethanol (EtOH) to about 10% EtOH; 13-14 min: changed to about 10% PBS buffer to about 20% PBS buffer and about 80% methyl cyanide (MeCN) to about 90% MeCN; about 14-20 min: about 10% PBS buffer to about 20% PBS buffer and about 80% MeCN to about 90% MeCN. In other embodiments, the compounds may be separated and purified using an analytical column and a gradient comprising the following parameters: about 0-20 min: about 90% PBS buffer to about 99% PBS buffer, about 1% MeCN to about 10% MeCN; about 20- 21 min: changed to about 25% PBS buffer to about 35% PBS buffer and about 65% MeCN to about 75% MeCN; about 21-30 min: about 25% PBS buffer to about 35% PBS buffer and about 65% to about 75%% MeCN. In some embodiments, that analytic column, can be for example, and Agilent ZORBAX Eclipse Plus C18, 95 Å, 3.5 µm, 100×4.6 some embodiments, the compounds may be USC 2023-124-02 15 530.039WO1 separated and purified using a gradient such as the following: Gradient: 0-13 min: about 96% PBS buffer, about 4% EtOH; 13-14 min: changed to about 15% PBS buffer and about 85% MeCN; 14-20 min: about 15% PBS buffer and about 85% MeCN. In another embodiment, the [18F]FMAU or other 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compounds may be purified using an analytic column such as Phenomenex Luna C18 (2), RP, 100 Å, 5 ^m, 250×4.6 mm. In some embodiments, the compounds may be separated and purified using a gradient such as the following: Gradient: 0-20 min: about 95% PBS buffer, about 5% MeCN; 20-21 min: changed to about 30% PBS buffer and about 70% MeCN; 21-30 min: about 30% PBS buffer and about 70% MeCN. In other aspects, the method further comprises formulating the compound into a pharmaceutical composition by adding, for example, a carrier, excipient, diluent, or a combination thereof to the purified compound. In some embodiments, the invention provides an18F-labeled nucleoside (uracil or cytosine) substituted at the 2'- and / or 5-position, wherein the 2'- and 5-position substituents are selected from hydrogen (H), hydroxyl (OH), halo (e.g., F, Cl, Br, or I), (C1-C6)alkyl, and hydroxy(C1-C6)alkyl (wherein the hydroxyl is located at any available carbon of the alkyl), and wherein at least one of the 2'- and 5-positions (preferably the 2'-position) is substituted by [18F]fluoro. Accordingly, the invention provides a 2'-18F-labeled 2'-deoxy-arabino–uracil optionally substituted at the 5-position of the uracil with a substituent selected from hydroxyl (OH), halo (e.g., F, Cl, Br, or I), (C1-C6)alkyl, and hydroxy(C1-C6)alkyl. In some embodiments, the18F-labeled 2'-deoxy-arabino-5-substituted or unsubstituted uracil or cytosine nucleoside is 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl- uracil ([18F]FMAU), 2'-[18F]fluoro-5-ethy1-1-^-D-arabinofuranosyluracil (FEAU), 2'-deoxy- 2'-[18F]fluoro-5-fluoro-l-^-D-arabinofuranosyluracil (-[18F]FFAU), 1-(2-deoxy-2--[18F]fluoro- ^-D-arabinofuranosyl)-5-chlorouracil ([18F]FCAU), 1-(2-deoxy-2-[18F]fluoro-^-D- arabinofuranosyl)-5-bromouracil ([18F]FBAU), 1-(2-deoxy-2--[18F]fluoro-(3-D- arabinofuranosyl)uracil ([18F]FAU), 2'-[18F]fluoro-2'-deoxy-l-^-D-arabinofuranosyl-5- iodouracil ([18F]FIAU), 1-(2-deoxy-2-[18F]fluoro-^-D-arabinofuranosyl)cytosine ([18F]FAC), 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosylcytosine ([18F]FMAC), 2'-[18F]fluoro-5-ethyl-1-^-D-arabinofuranosyl-cytosine (-[18F]FEAC), 2'-deoxy-2'-[18F]fluoro-5-fluoro-l-^-D-arabinofuranosyluracil ([18F]FFAC), 1-(2-deoxy-2-[18F]fluoro-^-D- arabinofuranosyl)-5-chlorocytosine ([18F]FCAC), 1-(2-deoxy-2-[18F]fluoro-^-D- arabinofuranosyl)-5-bromocytosine ([18F]FBAC), or 2'-deoxy-2'-[18F]fluoro-5- hydroxymethyl-1-^-D-arabino- ([18F]FHMAC). USC 2023-124-02 16 530.039WO1 In some embodiments, the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound comprises 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D- arabinofuranosyl-uracil ([18F]FMAU). FMAU shares many of in vivo characteristics of thymidine yet is not significantly catabolized in vivo. FMAU is a potential compound for PET imaging studies of cellular proliferation. In some embodiments, an automated method of using a radiosynthesis device to form 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU) compound comprises: a) dispensing one or more droplets onto a first reaction site of one or more hydrophilic reaction sites disposed on a surface using a first dispenser of a plurality of non- contact dispensers, wherein the one or more droplets comprise: i) a solution comprising a [18F]fluoride radioisotope in a solvent, wherein the [18F]fluoride is present in an amount of about 1 mCi to about 500 mCi; ii) a phase transfer catalyst comprising tetrabutylammonium bicarbonate (TBAHCO3); b) thermally treating the [18F]fluoride solution and the phase transfer catalysis on the first reaction site using a thermally controlled support coupled to the surface to evaporate the solvent, thereby providing a dried residue of a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) rotating the surface comprising the one or more hydrophilic reaction sites by rotating a motorized rotation stage attached to the thermally controlled support in order to position the first reaction site in line with a second dispenser of the plurality of non-contact dispensers, and dispensing one or more droplets of a ribose sugar precursor to the first reaction site to form a first mixture with the dried residue of the [18F]fluoride-phase transfer catalyst complex; d) thermally treating the first mixture on the first reaction site using the thermally controlled support to fluorinate the ribose sugar precursor to form a fluorinated ribose sugar; rotating the surface to position the first reaction site at a third dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a coupling reagent onto the first reaction site to form a coupling mixture, wherein the coupling reagent comprises a pyrimidine nucleobase in a 1,4 dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS), and thermally treating the coupling mixture to couple the ribose sugar precursor to the pyrimidine nucleobase to form a protected [18F]FMAU compound; f) rotating the surface to position the first reaction site at a fourth dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture, and thermally treating the deprotection mixture to form the [18F]FMAU compound, wherein the deprotecting reagent comprises potassium methoxide in methanol; and g) rotating the surface to position the first reaction site with a collection tube, and removing USC 2023-124-02 17 530.039WO1 the [18F]FMAU compound using the collection tube by applying a vacuum to the collection tube, thereby providing the [18F]FMAU compound, wherein a total volume of the combination of each reagent of the steps a-g is about 100 µL or less. In some embodiments, an automated method of using a radiosynthesis device to form 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU) compound comprising: a) dispensing one or more droplets onto a first reaction site of one or more hydrophilic reaction sites disposed on a surface using a first dispenser of a plurality of non- contact dispensers, wherein the one or more droplets comprise a solution comprising: i) [18F]fluoride radioisotope in a solvent, wherein the [18F]fluoride is present in an amount of about 1 mCi to about 500 mCi; and ii) a phase transfer catalyst comprising tetrabutylammonium bicarbonate (TBAHCO3) present in an amount of about 0.1 µmol to about 0.5 µmol; b) thermally treating the [18F]fluoride solution and the phase transfer catalysis on the first reaction site using a thermally controlled support coupled to the surface to evaporate the solvent, thereby providing a dried residue of a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) rotating the surface comprising the one or more hydrophilic reaction sites by rotating a motorized rotation stage attached to the thermally controlled support in order to position the first reaction site in line with a second dispenser of the plurality of non-contact dispensers, and dispensing one or more droplets of a ribose sugar precursor to the first reaction site to form a first mixture with the dried residue of the [18F]fluoride-phase transfer catalyst complex, wherein the ribose sugar precursor is present in an amount of about 0.1 µmol to about 10 µmol; d) thermally treating the first mixture on the first reaction site using the thermally controlled support for about 3 minutes at about 100 °C to about 105 °C to fluorinate the ribose sugar precursor to provide a fluorinate ribose sugar; e) rotating the surface to position the first reaction site at a third dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a coupling reagent onto the first reaction site to form a coupling mixture, and thermally treating the coupling mixture to couple the fluorinated ribose sugar for about 60 seconds at about 130 °C to form a protected [18F]FMAU compound, wherein the coupling reagent comprises a pyrimidine nucleobase in 1,4-dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS); f) rotating the surface to position the first reaction site at a fourth dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture, and thermally treating the deprotection mixture for about 3 minutes at about 85 °C to form the [18F]FMAU compound, wherein the deprotecting reagent comprises potassium methoxide in and g) rotating the surface to position the USC 2023-124-02 18 530.039WO1 first reaction site in alignment with a collection tube, and removing the [18F]FMAU compound using a collection tube by applying a vacuum to the collection tube, thereby providing the [18F]FMAU compound, wherein a total volume of the combination of each reagent of the steps a) – g) is about 100 µL or less. In some embodiments, the amount of 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound product is controlled by adjusting the amount of [18F]fluoride added to the reaction. In some embodiments, the total synthesis time is about 30 minutes or less. In some embodiments, the methods described herein may include the use of one or more apparatuses to facilitate the synthesis of the desired compounds. In some embodiments, the methods described herein may be partially or fully automated. In preferred embodiments, the apparatus comprises an apparatus as described in Van Dam et al., International Patent Pub. No. WO 2022 / 109244 and Wang et al., Lab Chip, 2019, 19, 2415-2424, incorporated herein by reference in their entirety. An exemplary apparatus is shown in Figure 8. A radiosynthesis device 100 that may be used in conjunction with the methods described herein that includes a support frame or housing 106 to hold the various components of the apparatus. The support frame 106 includes base 108 is oriented horizontally and has a flat bottom surface 109 such that it can stably sit on a supporting surface such a lab benchtop or table. Frame 106 includes support wall 110 and a support arm 112 extending therefrom. The support arm 112 is moveable up and down in the vertical direction. The support arm 112 is slidably coupled to the support wall 110 using a vertically oriented rail 114 attached to the support wall 110 which slidably receives a raceway of a slide 116 attached to the support arm 112. The support arm 112 has a plurality of dispenser receiving apertures 121 for receiving and holding dispensers 120. The dispenser receiving apertures 121 are arranged in angularly spaced apart relation along an arc of a circle having a center point. Dispenser receiving apertures 121 may be angularly spaced apart 45°, and two dispensers receiving apertures 121 do not have dispensers 120 installed in them. Depending on the particular radiosynthesis process being performed on the radiosynthesis device 100, more or fewer dispensers 120 may be required. A pneumatic cylinder 118 (e.g., a single-acting pneumatic cylinder) is attached to the support wall 110 and has an actuator rod 119 (e.g., a piston rod of a single-acting pneumatic cylinder) connected to the support arm 112. Actuator 118 is controllably actuatable to move the support arm 112 relative to the microfluidic chip 102. A plurality of non-contact dispensers 120 are installed on the support arm 112 of the frame 106 (inserted into and / or affixed to the dispenser receiving apertures 121), including dispenser 120 a, a second dispenser 120 b, USC 2023-124-02 19 530.039WO1 a third dispenser 120 c, a fourth dispenser 120 d and a fifth dispenser 120 e. The dispensers 120 extend downward from the support arm 112 above the microfluidic chip 102. The non- contact dispensers 120 are typically solenoid-based, non-contact fluid dispensers, but may be any suitable dispenser for dispensing the reagents utilized in a desired radiosynthesis process. The dispensers 120 may have metal components (nozzles), but such metal components may be susceptible to attack by acidic reagents. Hence, the metal nozzles may be cleaned and / or coated and / or made out of other materials (e.g., plastic) to improve the lifetime. In addition, disposable dispensers may be utilized, or dispensers having nozzles which are not degraded by the reagents, such as acidic reagents. The dispensers 120 are arranged in angularly spaced apart relation along an arc of a circle having a center point. In some embodiments, a typical arrangement of non-contact dispensers may include a first dispenser dispensing one more droplets of a [18F]fluoride reagent and a phase transfer catalyst; a second dispenser dispensing one more droplets of a ribose sugar precursor solution; a third dispenser dispensing one more droplets of a coupling reagent solution; a fourth dispenser dispensing one more droplets of a deprotection solution; optionally, a fifth dispenser dispensing one more droplets of a collection solution; and a collection tube. A collection tube 122 is also installed on support arm 112 of frame 106. The collection tube 122 inserts into and is affixed through a tube aperture 124 in the support arm 112. The collection tube 122 extends downward from the support arm 112 above the microfluidic chip 102, and terminates just above (e.g., about 0.5 mm or less) the surface of microfluidic chip 102. The collection tube 122 is also positioned angularly spaced apart in relation from the dispensers 120 along the same arc of a circle as the dispensers 120. In the illustrated embodiment of Fig. 8, the collection tube 122 is angularly spaced apart from the dispenser 102 e by 90° and by 45° from the directly adjacent dispenser receiving apertures 121 (there is one empty dispenser receiving apertures 121 between the collection tube 122 and the directly adjacent dispenser receiving apertures 121). The radiosynthesis device 102 also has a motorized rotation stage 124 mounted on the top of base 108. The motorized rotation stage 124 has a controllably rotatable platform 126. The motorized rotation stage 124 accurately rotates the rotatable platform 126 based on a control signal from a motor controller. A thermally controlled support 130 is coupled to the rotatable platform 126 of the motorized rotation stage 124, such that rotation of the rotatable platform 126 rotates the thermally controlled support 130. The thermally controlled support 130 includes a support base 132 which is mounted to the rotatable a plurality of risers 134 which are attached USC 2023-124-02 20 530.039WO1 to the base 132 and extending upward from the support base 132. The illustrated embodiment includes four risers 134, but any suitable number of risers 134 may be used. A support platform / heat sink 136 is mounted on top of the risers 134. A thermoelectric cooler 137 (e.g., a Peltier cooling device) is mounted on the top of the heatsink 136, and a fan 141 is mounted on the bottom surface of the heat sink 136. The thermoelectric cooler 137 is in thermal contact with the heat sink 136 and the microfluidic chip 102. The thermoelectric cooler 137, heatsink 136 and fan 141 may be integrated as an integrated cooling module 139. A heater element 138 (e.g., a ceramic heater) is mounted on top of the thermoelectric cooler 137, and the microfluidic chip 102 sits on the heater element 138, or on a chip holder mounted on the heater 138. The heater element 138 is also in thermal contact with the surface (e.g., microfluidic chip) 102. The heater element 138 may include positioning element(s), such as a recess, bumps, guides, etc., or a chip holder having such positioning element(s), for accurately positioning and / or securing the microfluidic chip 102 on the thermally controlled support 130. The thermally controlled support 130 may hold the microfluidic chip 102 such that the reaction site(s) 104 are off-center with respect to the axis of rotation of the motorized rotation stage 124 (and support 130, which has the same axis of rotation) so that the reaction site(s) 104 move through an arc when the support 130 is rotated (as opposed to a reaction site 104 position with its center on the axis of rotation in which case the reaction site 104 merely rotates about its center). A collection container holder 146 (e.g., a vial clip) is attached to the support arm 112 of the fixture for holding a collection container. A collection tube fluidly connects the collection tube 122 to the collection vial. In some embodiments, some reagents may be delivered through a different type of dispenser (pre-measured volume of solution delivered through inert tubing) instead of the typical piezoelectric dispensing valve. Other methods for delivering a desired reagent that are known in the art may be used with embodiments of the invention. In some embodiments, the apparatus is connected to a purification and formulations system. In some embodiments, the apparatus is connected to an isotope concentrator. In some embodiments, the surface is a Teflon-coated silicon chip patterned with a hydrophilic reaction site. In some embodiments, an automated method for synthesizing the [18F]FMAU or other 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compounds may be initiated by dispensing one or more droplets of a radioisotope stock solution comprising a radioisotope (e.g.,18F) in a solvent onto a site of the one or more reaction sites of USC 2023-124-02 21 530.039WO1 a surface using a first dispenser of a plurality of non-contact dispensers. In some embodiments, the radioisotope stock solution may include a base and phase transfer catalyst, which may be premixed into the stock solution, or introduced during upstream processing (e.g., by a radionuclide concentrator, or they can be dispensed into the reaction site (before or after the radioisotope stock solution is dispensed). The radioisotope stock solution on the first reaction site then may be thermally treated (e.g., heating and / or cooling) using the thermally controlled support to evaporate the solvent leaving a dried residue or residue of radioisotope complex on the first reaction site. The surface may then be rotated relative to the dispensers by rotating the motorized rotation stage to position the first reaction site at a second dispenser of the plurality of non-contact dispensers. One or more droplets of a ribose sugar precursor solution (e.g., 2- O-(trifluoromethanesulfonyl)-1,3,5-tri-O-benzoyl-^-D-ribofuranose) are dispensed onto the first reaction site using the second dispenser to dissolve the dried residue of radioisotope complex resulting in a solution of the ribose sugar precursor solution and radioisotope complex. The surface then may be rotated again by rotating the motorized rotation stage to position the first reaction site at a third dispenser of the plurality of non-contact dispensers. With the first reaction site positioned at the third dispenser, the solution of precursor solution and radioisotope complex on the first reaction site may be thermally treated (e.g., heated and / or cooled) using the thermally controlled support to perform a fluorination reaction thereby producing a fluorinated reaction product. Optionally, during the fluorination reaction, a replenishing reagent may be dispensed periodically onto the first reaction site using the third dispenser during the fluorination reaction. In the next step, the surface may be rotated by rotating the motorized rotation stage to position the first reaction site at a fourth dispenser of the plurality of non-contact dispensers. The fourth dispenser dispenses one or more droplets of a deprotection reagent onto the first reaction site containing the fluorinated reaction product. The deprotection solution and fluorinated reaction product on the first reaction site are thermally treated using the thermally controlled support to perform a deprotection reaction thereby producing a crude 2'-deoxy-2'- [18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound. The surface then again may be rotated by rotating the motorized rotation stage to position the first reaction site at a fifth dispenser of the plurality of non-contact dispensers. The fifth dispenser dispenses one or more droplets of a collection solution onto the first reaction site containing 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound to dilute the crude radiofluorinated compound. USC 2023-124-02 22 530.039WO1 The surface then may be rotated by rotating the motorized rotation stage to position the first reaction site at the collection tube. Then, the diluted crude radiofluorinated compound may be removed from the first reaction site using the collection tube through the application of a vacuum to the collection tube. Optionally the diluted crude radiofluorinated compound may be conveyed through the collection tube to a collection vial using a vacuum source connected to the collection vial. Optionally, the diluted crude radiofluorinated compound may be purified. In some embodiments, the process of collecting the diluted crude radiochemical product from the first reaction site may include repeating the dilution and removal steps multiple times For instance, the following process may be repeated a suitable number of times: rotating the surface by rotating the motorized rotation stage to position the first reaction site back to the fifth dispenser and dispensing one or more droplets of a collection solution onto the first reaction site containing crude radiochemical product; and rotating the surface by rotating the motorized rotation stage to position the first reaction site at the collection tube and removing the diluted crude radiochemical product with the collection tube by applying a vacuum to the collection tube. For instance, this collection process may be repeated two, three, four, five, or more times. In some embodiments, the surface may comprise a plurality of hydrophobic reactions sites for scalability. For example, 2 or more, 5 or more, 10 or more 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 75 or more, or 100 or more reactions may be run simultaneously on a single surface or multiple surfaces (e.g., one or more microfluidic chips). For example, a solution comprising [18F]fluoride and a phase transfer catalyst may be dispensed onto each of the hydrophilic reaction sites, then the entire surface or multiple surfaces may be simultaneously thermally treated. The method may continue in parallel until the final product is formed. The [18F]-labeled thymidine / uridine analogues disclosed herein can be used as a PET tracer for certain medical conditions, including, but not limited to, cancer, autoimmunity inflammation, tissue / organ repair, and bone marrow transplant. Results and Discussion. The radiosynthesis (Scheme I), comprising fluorination, coupling, and deprotection, was performed on a Teflon-coated silicon chip containing circular hydrophilic reaction sites (Figure 1). USC 2023-124-02 23 530.039WO1 Using starting activity of 18.5-1240 MBq of [18F]fluoride, the fluorination, coupling and deprotection reactions were optimized sequentially. A variety of solvents, phase transfer catalysts, amounts of precursor, and reaction temperatures were explored in the fluorination reaction. Coupling of the fluorinated intermediate with protected thymine via N-glycosylation was optimized by varying the coupling temperature / time and amounts of protected thymine, trimethylsilyl trifluoromethanesulfonate (TMSOTf), and hexamethyldisilane (HMDS). To optimize the deprotection step, we varied base amounts, solvent types and temperature / time. Reaction products were analyzed using TLC with Cerenkov luminescence imaging readout. We also optimized the purification and formulation protocols. The droplet synthesis performance is summarized in Table 1. Nearly quantitative fluorination conversion (95 ± 2%, n=4) was achieved in 3 min using 0.5 ^mol of precursor and 0.18 ^mol of tetrabutylammonium hydrogen carbonate (TBAHCO3) in 10 ^L of 1,4-dioxane at 100°C. High coupling efficiency (87 ± 4%, n=4) was achieved by performing the coupling reaction with 0.48 ^mol of protected thymine, 4 ^L of TMSOTf and 1 ^L of HMDS in 1,4- dioxane (10 ^L) at 130°C for 1 min. The optimal deprotection used 30 ^L of potassium methoxide (KOMe) (in 25% methanol) and ethanol (v / v, 2:1) at 85°C for 3 min. The purified [18F]FMAU was ready for injection after separation on an analytical-scale radio-HPLC system with a mobile phase of 5% (v / v) ethanol in 0.1 M phosphate-buffered saline (PBS) buffer (pH=6) (column: C18, 4.6×100 mm, 3.5 µm; flow rate: 1.2 mL / min; UV: 254 nm). The radiochemical yield was 25 ± 3% (n=4), 2x higher than that with the conventional method (12 ± 3%, n=4), and the activity yield was remarkably improved to 19 ± 2% (n=4) with excellent radiochemical purity (>99%). The overall preparation time was significantly shortened to 28 ± 1 min (n=4). The radiosynthesis of [18F]FMAU using a microdroplet reactor resulted in significant improvements in radiochemical yield, activity yield, and preparation time while utilizing 34- 200x fewer reagents. USC 2023-124-02 24 530.039WO1 Ta a oc e ca pu y , Ratio of anomers (^ / ^) 2.2 + / - 0.6 1.5 Total production time (min) 28 + / - 1 150 The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention. EXAMPLES Example 1. Improved Radiosynthesis of18F-Labeled Nucleoside Analogues. HPLC Purification Optimization The synthesis of [18F]FMAU involves multiple reaction steps with various reagents, resulting in increasing levels of UV impurities, radioactive side products, and residual reagents as the process progresses. To fully separate the product from these contaminants, a lengthy radio-HPLC purification time (25-35 min) using a semi-prep column is typically required for macroscale production (Chen et al., Nuclear Medicine and Biology, 39(7), pp. 1019–1025; Li et al. (2021) ACS Pharmacology & Translational Science, 4(1), pp.266–275). To optimize the purification of [18F]FMAU produced in the droplet reactor, the efficiency of three different analytical columns was evaluated, along with variations in the mobile phase, to achieve the final purified product ([18F]FMAU) in the shortest possible time. Detailed information on column types, mobile phases, flow rates, retention times of [18F]FMAU, volumes of collected USC 2023-124-02 25 530.039WO1 product from radio-HPLC, and individual radio-HPLC chromatograms of crude [18F]FMAU is provided in Table 2 and Figure 3. 7 o u , 100 Å, 3.5 • 13-14 min: changed to 15% PBS buffer and 1.2 12.1 ~1.8 µm, 150×4.6 85% MeCN mm • 14-20 min: PBS 15% buffer and 85% MeCN Initially, purification was performed using the reference conditions as described by Li et al. (2021), ACS Pharmacology & Translational Science, 4(1), pp. 266–275, but on an analytical column instead of semi-prep (i.e. Luna C18, RP, 100 Å, 5 ^m, 250 × 4.6 mm; mobile phase: 92% H2O, 8% MeCN, 0.1% TFA ; flow rate: 1 mL / min). However, in Test 1 (Table 2 and Figure 3), an impurity (visible in the UV chromatogram) co-eluted with USC 2023-124-02 26 530.039WO1 [18F]FMAU. To address this, Test 2 involved reducing the percentage of MeCN, replacing TFA with acetic acid (eliminating the risk of TFA residue in future product formulations), and increasing the flow rate slightly (1.2 mL / min vs. 1 mL / min). This adjustment successfully resolved [18F]FMAU from the UV impurity, albeit it resulted in a longer retention time (18.1 min in Test 2 vs. 11.0 min in Test 1). Additionally, the volume of the collected product increased from ~2 to ~5 mL, necessitating either a higher dilution volume for the SPE- formulation process or a longer evaporation time. In Test 3, utilizing PBS buffer (0.1 M concentration with pH = 6) instead of water and acetic acid yielded a slightly sharper product peak, though the retention time remained extended (19.1 min), and the collected volume was still large (~4 mL). Due to the late retention time of [18F]FMAU and the large volume of the purified product fraction when using a 250 mm long column, a shorter 100 mm analytical column (Agilent Zorbax Eclipse Plus C18, 95 Å, 3.5 µm, 100 × 4.6 mm) was evaluated. Using the same mobile phase as in Test 3, the retention time of [18F]FMAU was significantly reduced to 5.9 min (Test 4), providing a sharper product peak in a smaller fraction volume (~1.2 mL). In Test 5, the possibility of using a non-toxic injectable mobile phase was explored, eliminating the need for a reformulation step. By replacing MeCN with EtOH. A similar retention time of 6.0 min was achieved, maintaining excellent separation efficiency. To ensure complete separation of the [18F]FMAU peak from the UV impurity, the EtOH percentage was slightly reduced from 5% to 4% in Test 6, optimizing the retention time to 8.6 min. Another option, a 150 mm column (Symmetry C18 Column, 100 Å, 3.5 µm, 150 × 4.6 mm), was tested in Test 7. Using the same mobile phase as in Test 6, the retention time increased by 3.5 min, with a slightly higher collected volume (~1.8 mL). Ultimately, the radio-HPLC conditions from Test 6 were selected as the optimal purification method for [18F]FMAU synthesis. Examples of HPLC chromatograms are shown in Figures 4-6, including (1) crude [18F]FMAU from microdroplet radiosynthesis (Figure 4), (2) purified [18F]FMAU (Figure 5), and (3) co-injection of purified [18F]FMAU with the reference standard (Figure 6). Droplet-Based Automated Radiosynthesis Given that the coupling reagent TMSOTf used in the radiosynthesis process is highly corrosive to most materials, it is incompatible with the internal wetted components of the reagent dispensers and their nozzles integrated into the droplet-based synthesis module (Wang et al. (2017) Lab on a Chip, 17(24), pp.4342–4355; Wang et al., (2019) Lab on a Chip, 19(14), pp. 2415–2424.). To address this issue, 0.03”) was tested as an alternative to piezoelectric dispensers for the remote delivery of the coupling stock solution to the droplet USC 2023-124-02 27 530.039WO1 synthesizer and the subsequent coupling reaction (Figure 7). Specifically, a pre-measured bolus of the reagent was loaded into a small v-vial (0.3 mL) connected to the tubing, and nitrogen pressure was applied to push the bolus through the tubing and onto the chip as needed. During the automated implementation of certain reactions, reagents for prior steps were typically added manually to minimize the introduction of too many new variables simultaneously. In the initial attempt (Test 1 in Table 3), a volume of 12 ^L (slightly higher than the optimal 10 ^L needed for the optimized reaction conditions, to account for minor residual losses in the fluid path) of coupling stock solution was added to the v-vial. The loading process was initiated by applying 5 psi until all the liquid was dispensed onto the chip. However, only about 4 ^L of the solution (roughly measured with a micropipette) reached the chip, and no coupling product was observed after the reaction. This issue may be attributed to the viscosity of HMDS and TMSOTf, which caused significant adherence to the tubing as a thin residue before reaching the chip. To address this issue, coupling reagents were diluted with MeCN as a means to reduce residual losses of coupling reagents during dispensing and ensure more reliable delivery. In Test 2 (Table 3), 10 ^L of MeCN was added to the 12 ^L of coupling stock solution. A total of 22 ^L of the combined solution was manually loaded onto the chip using a micropipette. This adjustment led to improved coupling performance, with a high coupling percentage (71%, n = 1) and collection efficiency after coupling (81%, n = 1), resulting in a crude coupling product of 58% (n = 1). A similar performance was observed in Test 3 (Table 3) when the process was automated, with 22 ^L of diluted coupling solution loaded from the v-vial to the chip through tubing using 5 psi. The crude coupling product was slightly lower (51% compared to 58% with the manual method), and there was a reduction in coupling percentage (64% vs. 71% for the manual method) and collection efficiency after coupling (79% vs.81% for the manual method). This approach for loading TMSOTf, using vials of pre-measured reagents and tubing, can be broadly applied to other corrosive reagents or potentially all reagents. It provides a foundation for developing a disposable reagent loading system and full automation of droplet radiochemistry. USC 2023-124-02 28 530.039WO1 Coupling percentage (%) - 71 64 Crude coupling product (%) - 58 51aRadiofluorination solution was manually pipetted onto the chip, with radiofluorination carried out using 0.176 ^mol of TBAHCO3and 0.5 ^mol of precursor in 10 ^L of 1,4-dioxane at 100 °C for 3 min. Coupling stock solution were loaded either manually by pipetting or automatically by dispenser. Each 10 ^L of coupling stock solution contained 0.5 ^mol of protected thymine, 1 ^L of HMDS, 4 ^L of TMSOTf, and 5 ^L of 1,4-dioxane. To ensure adequate volume, 12 ^L of stock solution was used for dispensing. Coupling reactions were performed at 130 °C for 1 min. The chip collection solvent consisted of 4 x 20 ^L of hexane and ethyl acetate (1:1, v / v). The radio-TLC was developed using the same solvent system. All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference, and in particular, U.S. Patent Pub. Nos.2021 / 0009624 to Chen et al., 2012 / 0053337 to Zibo et al., 2022 / 0251025 to Van Dam et al., International Patent Pub. No. WO 2022 / 109244 to Chen et al; Li et al., Nucl Med Biol, ^^^^^^^^^^^^^^^^-206^^Chen et al., Nucl Med Biol^^^^^^^^^^^^^^^^^^^-1025^^and Li et al., ACS Pharmacol Trans Sci, ^^^^^^^^^^^^^^^-275. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims. USC 2023-124-02 29 530.039WO1

Claims

What is claimed is:

1. A method for synthesizing a 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound comprising the steps of: a) dispensing one or more droplets of a solution comprising: i) a [18F]fluoride radioisotope in a solvent; and ii) a phase transfer catalyst; onto a first reaction site of one or more hydrophilic reaction sites using a first dispenser of a plurality of non-contact dispensers; b) thermally treating the [18F]fluoride radioisotope and the phase transfer catalysis on the first reaction site using a thermally controlled support to evaporate the solvent, thereby providing a dried residue comprising a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) dispensing one or more droplets of a ribose sugar precursor onto the first reaction site using a second dispenser of a plurality of non-contact dispensers to form a first mixture with the dried residue; d) thermally treating the first mixture on the first reaction site using the thermally controlled support to facilitate a fluorination reaction to fluorinate the ribose sugar, thereby providing a fluorinated ribose sugar; e) dispensing one or more droplets of a coupling reagent onto the first reaction site using a third dispenser of a plurality of non-contact dispensers to provide a coupling mixture, wherein the coupling reagent comprises a pyrimidine nucleobase, and thermally treating the coupling mixture to couple the fluorinated ribose sugar to the pyrimidine nucleobase to form a protected 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound; f) dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture using a fourth dispenser of a plurality of non-contact dispensers; g) thermally treating the deprotection mixture to form the 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compound; and h) removing the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D-arabinofuranosyl-uracil compound using a collection tube by applying a vacuum to the collection tube, wherein a total volume of the combination of each reagent of the steps a) – h) is about 100 µL or less.

2. The method of claim 1, wherein the sugar precursor is present in an amount of about 0.1 µmol to about 10 µmol. USC 2023-124-02 30 530.039WO13. The method of claim 1 wherein the phase transfer catalyst comprises tetrabutylammonium bicarbonate (TBAHCO3), wherein the (TBAHCO3) is present in an amount of about 0.05 µmol to about 0.5 µmol.

4. The method of claim 1, wherein the coupling reagent comprises the pyrimidine nucleobase in 1,4-dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS).

5. The method of claim 4, wherein the pyrimidine nucleobase comprises a protected thymidine or thymidine, and the^pyrimidine nucleobase is present in an amount of about 0.1 µmol to about 1 µmol; the TMSOTf is present in an amount of about 1 µL to about 10 µL; and the HMDS is present in an amount of about 0.1 µL to about 5 µL.

6. The method of claim 1, wherein the deprotecting reagent comprises potassium methoxide in methanol.

7. The method of claim 1, wherein the total volume of the combination of each reagent of the steps a) – h) is about 50 µL or less.

8. The method of claim 1, further comprising neutralizing the 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compound with an acid.

9. The method of claim 1, wherein the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound is: 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU), 2'-[18F]fluoro-5-ethy1-1-^-D-arabinofuranosyluracil (FEAU), 2'-deoxy-2'-[18F]fluoro-5-fluoro-l-^-D-arabinofuranosyluracil ([18F]FFAU), 1-(2-deoxy-2--[18F]fluoro-^-D-arabinofuranosyl)-5-chlorouracil ([18F]FCAU),1-(2-deoxy-2-[18F]fluoro-^-D-arabinofuranosyl)-5-bromouracil ([18F]FBAU), 1-(2-deoxy-2--[18F]fluoro-(3-D-arabinofuranosyl)uracil ([18F]FAU), 2'-[18F]fluoro-2'-deoxy-l-^-D-arabinofuranosyl-5-iodouracil ([18F]FIAU), or 2'-deoxy-2'-[18F]fluoro-5-fluoro-l-^- ([18F]FFAC). USC 2023-124-02 31 530.039WO110. The method of claim 9, wherein the 2'-deoxy-2'-[18F]fluoro-5-substituted-l-^-D- arabinofuranosyl-uracil compound is 2'-deoxy-2'-[18F]fluoro-5-methyl-1-^-D- arabinofuranosyl-uracil ([18F]FMAU).

11. The method of claim 1, wherein the step d) is carried out for a duration of about 2 to 5 minutes at about 100 °C to about 115 °C.

12. The method of claim 1, wherein the step e) is carried out for a duration of about 0.5 minutes to about 2 minutes at about 120 °C to about 140 °C.

13. The method of claim 1, wherein the step g) is carried out for a duration of about 2 minutes to about 3 minutes at about 75 °C to about 95 °C.

14. The method of claim 1, further comprising purifying the 2'-deoxy-2'-[18F]fluoro-5- substituted-l-^-D-arabinofuranosyl-uracil compound.

15. The method of claim 1, wherein the method is an automated method comprising the use of a radiosynthesis device, wherein the radiosynthesis device comprises the thermally controlled support configured to support a surface having the one or more reaction sites formed thereon; a fixture configured to support the plurality of non-contact dispensers and the collection tube, wherein the plurality of the non-contact dispensers is installed on the fixture positioned above the thermally controlled support and configured to respectively dispense one or more droplets of a respective reagent into the one or more reaction sites; the collection tube installed on the fixture above the support; and a motorized rotation stage operatively coupled to the support for controllably rotating the support, the motorized rotation stage configured to controllably rotate the support relative to the plurality of the non-contact dispensers to sequentially position the one or more reaction sites for dispensing respective reagent from the plurality of the non-contact dispensers into the one or more reaction sites, and to controllably rotate the support relative to the collection tube to sequentially position the one or more reaction sites for removing reaction product from the one or more reaction sites via the collection tube. USC 2023-124-02 32 530.039WO116. The method of claim 15, further comprising, after the step b), rotating the surface comprising the one or more hydrophilic reaction sites by rotating the motorized rotation stage attached thereto in order to position the first reaction site in line with the second dispenser of the plurality of non-contact dispensers.

18. The method of claim 16, further comprising, after the step d), rotating the surface by rotating the motorized rotation stage to position the first reaction site in line with the third dispenser of the plurality of non-contact dispensers; optionally, after the step e), rotating the surface by rotating the motorized rotation stage to position the first reaction site in line with the fourth dispenser of the plurality of non- contact dispensers; and optionally, after the step g), rotating the surface by rotating the motorized rotation stage to position the first reaction site in alignment with the collection tube.

19. An automated method of using a radiosynthesis device to form 2'-deoxy-2'- [18F]fluoro-5-methyl-1-^-D-arabinofuranosyl-uracil ([18F]FMAU) compound comprising: a) dispensing one or more droplets onto a first reaction site of one or more hydrophilic reaction sites disposed on a surface using a first dispenser of a plurality of non- contact dispensers, wherein the one or more droplets comprise a solution comprising: i) [18F]fluoride radioisotope in a solvent, wherein the [18F]fluoride is present in an amount of about 1 mCi to about 500 mCi; and ii) a phase transfer catalyst comprising tetrabutylammonium bicarbonate (TBAHCO3) present in an amount of about 0.1 µmol to about 0.5 µmol; b) thermally treating the [18F]fluoride solution and the phase transfer catalysis on the first reaction site using a thermally controlled support coupled to the surface to evaporate the solvent, thereby providing a dried residue of a [18F]fluoride-phase transfer catalyst complex on the first reaction site; c) rotating the surface comprising the one or more hydrophilic reaction sites by rotating a motorized rotation stage attached to the thermally controlled support in order to position the first reaction site in line with a second dispenser of the plurality of non-contact dispensers, and dispensing one or more droplets of a ribose sugar precursor to the first reaction site to form a first mixture with the dried residue of the [18F]fluoride-phase transfer catalyst complex, wherein the ribose sugar precursor is present in an amount of about 0.1µmol to about 10 µmol; USC 2023-124-02 33 530.039WO1d) thermally treating the first mixture on the first reaction site using the thermally controlled support for about 3 minutes at about 100 °C to about 105 °C to fluorinate the ribose sugar precursor to provide a fluorinate ribose sugar; e) rotating the surface to position the first reaction site at a third dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a coupling reagent onto the first reaction site to form a coupling mixture, and thermally treating the coupling mixture to couple the fluorinated ribose sugar for about 60 seconds at about 130 °C to form a protected [18F]FMAU compound, wherein the coupling reagent comprises a pyrimidine nucleobase in 1,4-dioxane solvent in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and hexamethyldisilane (HMDS); f) rotating the surface to position the first reaction site at a fourth dispenser of the plurality of non-contact dispensers, dispensing one or more droplets of a deprotecting reagent onto the first reaction site to form a deprotection mixture, and thermally treating the deprotection mixture for about 3 minutes at about 85 °C to form the [18F]FMAU compound, wherein the deprotecting reagent comprises potassium methoxide in methanol; and g) rotating the surface to position the first reaction site in alignment with a collection tube, and removing the [18F]FMAU compound using a collection tube by applying a vacuum to the collection tube, thereby providing the [18F]FMAU compound, wherein a total volume of the combination of each reagent of the steps a) – g) is about 100 µL or less. ^ 20. The method of claim 19, wherein the pyrimidine nucleobase comprises a protected thymine or thymine, and the^pyrimidine nucleobase is present in a concentration of about 0.1 µmol to about 1 µmol; the TMSOTf is present in an amount of about 1 µL to about 10 µL; and the HMDS is present in an amount of about 0.1 µL to about 5 µL. USC 2023-124-02 34 530.039WO1

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