# Patent

## Title
Methods and systems for selective fluorination of organic molecules

## Abstract

A method and system for selectively fluorinating organic molecules on a target site wherein the target site is activated and then fluorinated are shown together with a method and system for identifying a molecule having a biological activity.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to and is a continuation-in-part of International Application No. PCT/US07/17409, filed Aug. 4, 2007, and a continuation-in-part of U.S. patent application Ser. No. 11/890,218, filed Aug. 4, 2007, which applications claim priority to U.S. Provisional Application Ser. No. 60/835,613 filed on Aug. 4, 2006. This application also claims priority to U.S. Provisional Application No. 61/126,829, filed May 7, 2008. The disclosures of each of the foregoing application are incorporated herein by reference in their entirety.


TECHNICAL FIELD
The present disclosure generally relates to the fields of synthetic organic chemistry and pharmaceutical chemistry. In particular, the present disclosure relates to methods and systems for the selective fluorination of organic molecules.
BACKGROUND
The importance of fluorine in altering the physicochemical properties of organic molecules and its exploitation in medicinal chemistry has been highlighted in recent reviews (Bohm, Banner et al. 2004). Although similar in size to hydrogen, HOOF substitutions can cause dramatic effects on several properties of organic molecules, including the lipophilicity, dipole moment, and pKa thereof. In addition, fluorine substitutions can dramatically alter the reactivity of the fluorinated site as well as that of neighboring functional groups.
In particular, in medicinal chemistry, there is a growing interest towards incorporating fluorine atoms in building blocks, lead compounds and drugs in that this may increase by many-fold the chances of turning these molecules into marketable drugs. Several studies have shown that potent drugs can be obtained through fluorination of much less active precursors. Some representative examples include anticholesterolemic Ezetimib (Clader 2004), anticancer CF3-taxanes (Ojima 2004), fluoro-steroids, and antibacterial fluoroquinolones.
The improved pharmacological properties of fluoro-containing drugs are often due to their improved pharmacokinetic properties (biodistribution, clearance) and enhanced metabolic stability (Park, Kitteringham et al. 2001). Primary metabolism of drugs in humans generally occurs through P450-dependent systems, and the introduction of fluorine atoms at or near the sites of metabolic attack has often proven successful in increasing the half-life of a compound (Bohm, Banner et al. 2004). A comprehensive review covering the influence of fluorination on drug metabolism (especially P450-dependent) is presented. (Park, Kitteringham et al. 2001).
In other cases, the introduction of fluorine substituents leads to improvements in the pharmacological properties as a result of enhanced binding affinity of the molecule to biological receptors. Examples of the effect of fluorine on binding affinity are provided by recent results in the preparation of NK1 antagonists (Swain and Rupniak 1999), 5HT1D agonists (van Niel, Collins et al. 1999), and PTB1B antagonists (Burke, Ye et al. 1996).
Over the past years, fluorination has been covering an increasingly important role in drug discovery, as exemplified by the development of fluorinated derivatives of the anticancer drugs paclitaxel and docetaxel (Ojima 2004).
However, only a handful of organofluorine compounds occur in nature and even those only in very small amounts (Harper and O'Hagan 1994). Consequently, any fluorine-containing substance selected for research, pharmaceutical, or agrochemical application has to be man-made.
Despite a few reports on the application of molecular fluorine (F2) for direct fluorination of organic compounds (Chambers, Skinner et al. 1996; Chambers, Hutchinson et al. 2000), this method typically suffers from poor selectivity and requires handling of a highly toxic and gaseous reagent. Several chemical strategies have been developed over the past decades to afford selective fluorination of organic compounds under friendlier conditions. These have been recently reviewed by Togni (Togni, Mezzetti et al. 2001), Cahard (Ma and Cahard 2004), Sodeoka (Hamashima and Sodeoka 2006), and Gouverneur (Bobbio and Gouverneur 2006). These strategies involve catalytic as well as non-catalytic methods. The latter comprise substrate-controlled fluorination methods, which generally make use of a chiral auxiliary, and reagent-controlled fluorination methods, which generally make use of chiral electrophilic N—F or nucleophilic fluorinating reagents.
These fluorination methods, however, need several chemical steps to prepare the chiral substrates (Davis and Han 1992; Enders, Potthoff et al. 1997) or the chiral reagents (Davis, Zhou et al. 1998; Taylor, Kotoris et al. 1999; Nyffeler, Duron et al. 2005) and have an applicability restricted to reactive C—H bonds (Cahard, Audouard et al. 2000; Shibata, Suzuki et al. 2000; Kim and Park 2002; Beeson and MacMillan 2005; Marigo, Fielenbach et al. 2005) in specific classes of compounds such as aldehydes (Beeson and MacMillan 2005; Marigo, Fielenbach et al. 2005) or di-carbonyls (Hintermann and Togni 2000; Ma and Cahard 2004; Shibata, Ishimaru et al. 2004; Hamashima and Sodeoka 2006).
Despite much progress in the field of organofluorine chemistry, the number of available methods for direct or indirect asymmetric synthesis of organofluorine compounds remains limited and additional tools are desirable. In particular, a general method to afford mono- or poly-fluorination of organic compounds at reactive and unreactive sites of their molecular scaffold is desirable.
SUMMARY
Provided herein are methods and systems for the selective fluorination of a target site of an organic molecule, which include the activation and subsequent fluorination of the target site. In the methods and systems herein disclosed, the target site is an oxidizable carbon atom of the organic molecule, the activation is performed by introducing an oxygen-containing functional group on the target site, and the fluorination of the activated site is performed by replacing the functional group introduced on the target site with fluorine The introduction of the oxygen-containing functional group and the replacement of the functional group with a fluorine can be performed by suitable agents.
Fluorination covers an increasingly important role in drug discovery and development. The disclosure provides a versatile strategy that combines cytochrome P450-catalyzed oxygenation with deoxofluorination to achieve mono- and polyfluorination of non-reactive sites in a variety of organic scaffolds. This procedure was applied for the rapid identification of fluorinated drug derivatives with enhanced membrane permeability.
The disclosure provides a method for fluorinating an organic molecule is disclosed, the method comprising providing an organic molecule comprising a target site; providing an Oxidizing agent that oxidizes the organic molecule by introducing an oxygen containing functional group on the target site, contacting the Oxidizing agent with the organic molecule for a time and under condition to allow introduction of the oxygen-containing functional group on the target site thus providing an oxygenated organic molecule, providing a fluorinating agent and contacting the fluorinating agent with the oxygenated organic molecule, for a time and under condition to allow replacement of the oxygen-containing functional group with fluorine.
The disclosure also provides a system for the fluorination of an organic molecule is disclosed, the system comprising an Oxidizing agent for introducing an oxygen-containing functional group in an organic molecule and a fluorinating agent for replacing the oxygen-containing functional group in the organic molecule with fluorine or a fluorine group. An oxygen-providing compound and/or fluorine-providing compound can also be included in the system.
The methods and systems of the disclosure allow the fluorination of organic molecules in one or more specific and predetermined target sites, including one or more target sites of interest, thus allowing a regioselective mono- and poly-fluorination.
The disclosure further allows introduction of fluorine in an fluorine unreactive site of a molecule, e.g. a site that, in absence of the oxygen-containing functional group is unlikely to undergo a chemical transformation such as a fluorination, as long as said site is oxidizable.
An advantage of the methods and systems herein disclosed is that by using a suitable agent, in particular a suitable oxidizing agent, it is possible to control the chirality of the final product and therefore produce a product molecule having a desired chirality (stereoselective fluorination).
Another advantage of the methods and systems herein disclosed is that the methods and system can provide fluorinated compounds wherein the fluorine is introduced in a predetermined site expected to be associated with a biological activity, which are therefore candidate compounds to be screened for the activity.
The disclosure provides a method for the identification of a molecule having a biological activity is disclosed, the method comprising, providing an organic molecule comprising a target site; providing an Oxidizing agent, contacting the Oxidizing agent with the organic molecule for a time and under condition to allow introduction of an oxygen-containing functional group on the target site thus providing an oxygenated organic molecule, providing a fluorinating agent; contacting the fluorinating agent with the oxygenated organic molecule, for a time and under condition to allow replacement of the oxygen-containing functional group with fluorine and testing the fluorinated organic molecule for the biological activity.
The disclosure also provides a system for identifying a molecule having a biological activity is disclosed. The system comprises an oxidizing agent capable of introducing an oxygen-containing functional group in a target site of an organic molecule, a fluorinating agent capable of replacing the oxygen-containing functional group in the organic molecule with fluorine, and an agent for testing the biological activity. An oxygen-providing agent and/or fluorine-providing-agent can also be included in the system.
A further advantage of the methods and systems for the identification of a molecule having a biological activity is the possibility to produce a broad spectrum of molecules that in view of the selected insertion of fluorine, already constitute promising candidates, thus shortening and improving the selection process.
An additional advantage of the methods and systems for the identification of a molecule having a biological activity is the possibility to confer new activities to a molecule that is already biologically active and/or to improve the biological activity of the original molecule by selective insertion of fluorine.
A still further advantage of the methods and systems of the identification of a molecule having a biological activity, is the possibility to derive molecules that have a biological activity that is pharmacologically relevant, or to improve the pharmacologically activity of a molecule that is already pharmacologically active. This in view of the known ability of fluorine to improve the pharmacological profile of drugs.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and implementations of the disclosure.
FIG. 1 is a schematic representation of the methods and systems for the selective fluorination of an organic molecule A according to an embodiment of herein disclosed;
FIG. 2 is a schematic representation of methods and systems for stereoselective fluorination of an organic molecule A according to an embodiment of what herein disclosed (chemo-enzymatic strategy), illustrated in comparison with methods and systems of the art (chemical strategy);
FIG. 3 is a graphic representation of the crystal structure of a P450 heme domain; helixes D, L, I and E in the domain are also indicated; the heme prosthetic group in the domain is indicated as “heme”; the cysteine in the heme-ligand loop is displayed in spheres (black).
FIG. 4 is a schematic representation of methods and system for identifying a molecule having a biological activity according to an embodiment disclosed in the present specification;
FIG. 5 illustrates exemplary results from the screening of a subset of pre-selected oxygenases for the identification of a suitable Oxidizing agent for the selective activation of the organic molecule dihydrojasmone. Panel A) is a diagram showing the conversion ratios for the reaction of activation of dihydrojasmone with wild-type P450BM3 and variants thereof, as determined by GC analysis; Panel B) is a diagram showing the product distribution obtained with wild-type P450BM3 and variants thereof in the reaction of activation of dihydrojasmone, as determined by GC analysis. Cpd 1 to cpd 9 indicate activated products 1 to 9;
FIG. 6 illustrates exemplary results from the screening of a subset of pre-selected oxygenases for the identification of a suitable Oxidizing agent for the selective activation of the organic molecule Menthofuran; Panel A) is a diagram showing the conversion ratios for the reaction of activation of menthofuran with wild-type P450BM3 and variants thereof, as determined by GC analysis. Panel B) is a diagram showing the product distribution obtained with wild-type P450BM3 and variants thereof in the reaction of activation of menthofuran, as determined by GC analysis. Cpd 1 to cpd 10 indicate activated products 1 to 10;
FIG. 7 illustrates exemplary results from the screening of a subset of pre-selected oxygenases for the identification of a suitable Oxidizing agent for the selective activation of the organic molecule dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline (MMPO). In particular, Panel (A) is a diagram showing the results from HTS screening of Oxidizing agent pool using calorimetric reagent Purpald; Panel (B) is a diagram showing the results from the re-screen of the positive hits identified with calorimetric HTS, where the regioselectivity of each oxygenase is determined by GC analysis (MMPOH is dihydro-4-hydroxymethyl-2-methyl-5-phenyl-2-oxazoline, that is the desired activated product); Panel (C) is a diagram showing the conversion ratios for the activation reactions of MMPO with each of the tested Oxidizing agents, as determined by GC analysis; and
FIG. 8 shows a diagram illustrating the time course for whole-cell activation of the organic molecule dihydrojasmone (DHJ) using batch culture of var3-expressing E. coli DH5α cells (0.5 L). The consumption of substrate (DHJ) and the accumulation of the desired activated product (oxDHJ) were monitored over time by GC analysis of aliquots of the cell culture.
FIG. 9 show test molecules (ordered according to their molecular weight) used in the methods of the disclosure. Arrows indicate the site(s) targeted by chemo-enzymatic fluorination.
FIG. 10A-C shows chemo-enzymatic fluorination of organic molecules. (a) Screening of P450 library in 96-well format. Reactions were carried out in the presence of the substrate, P450 enzyme from cell lysate, and a glucose-6-phosphate dehydrogenase-based NADPH regeneration system. TON: turnover number. Standard error is within 15%. WT=wild-type P450BM3. (b) Selective fluorination of cyclopentenone derivatives. Reagents and conditions: i) 1, 0.04 mol % var-H3, 88%; ii) DAST (1.2 equiv), CH2Cl2, 78° C., 12 h, 90% (20% ee); iii) 1, 0.04 mol % var-G6, 45%; iv) DAST (1.2 equiv), CH2Cl2, 78° C., 12 h, 85%; v) 2, 0.05 mol % var-H3, 85%; vi) DAST (1.3 equiv), CH2Cl2, 78° C., 12 h, 92% (78% ee); vii) 2, 0.05 mol % var-G4, 42%; viii) DAST (1.5 equiv), CH2Cl2, 78° C., 12 h, 89%; ix) 3, 0.05 mol % var-D10, 69%; x) DAST (1.2 equiv), CH2Cl2, 78° C., 3 h, 88% (dr 1:8.5, major: 5% ee, minor: 71% ee); xi) 3, 0.05 mol % var-G4, 62%; xii) DAST (1.2 equiv), CH2Cl2, 78° C., 5 h, 92% (dr 4:96, major: 0% ee, minor: 57% ee); xiii) 3, 0.07 mol % var-G5, 32%; xiv) DAST (1.2 equiv), CH2Cl2, 78° C., 5 h, 90% (dr not measurable). (c) Selective mono- and difluorination of prodrug ibuprofen methyl ester. Reagents and conditions: i) 0.1 mol % var-B4, 72%; ii) DAST (1.4 equiv), CH2Cl2, 78° C., 12 h, 86% (dr 1:3.2, major: 19% ee, minor: 44% ee); iii) 0.05 mol % var-G4, 88%; iv) DAST (1.4 equiv), CH2Cl2, 78° C., 12 h, 95%; v) 0.06 mol % var-B2, 93%; vi) DAST (1.2 equiv), CH2Cl2, 78° C., 12 h, 98% (dr 1:3.7, major: 9% ee, minor: 9% ee). The sequences of the P450 variants are described in a table below. Yields refer to the isolated products. Enantiomeric excess values were determined by chiral GC analysis.
FIG. 11A-C shows chemo-enzymatic —OCH3→—F transformation. (a) Screening of P450 demethylation activities using a Purpald-based assay for detection of formaldehyde formation in 96-well plate format. Standard error is within 15%. WT=wild-type P450BM3. (b) —OCH3→—F transformation in a 5-phenyl oxazoline derivative. Reagents and conditions: i) 0.1 mol % var-H1, KPi pH 8.0, room temperature, 48 h, 92%; ii) DAST (1.0 equiv), CH2Cl2, 0° C., 12 h, 40%. (c) —OCH3→—F transformation in Corey lactone derivatives. Yields refer to the isolated products.
FIG. 12 shows membrane permeability properties of 12 and its fluorinated derivatives. Effective permeability values (Pe) are calculated by monitoring accumulation of the organic molecule in a cross-membrane compartment. The correlation between Pe value and BBB-permeability in the absence of active transport has been previously established using a wide set of commercial drugs and is illustrated by the graded bar (black=not BBB-permeable; white=BBB-permeable). Caffeine served as control for assay validation (literature: 1.30×106 cm s−1; experimental: 1.24×10−6 cm s−1). M+/M− refers to the fraction of compound that crossed the BBB-mimic membrane as compared to system with no membrane after 16 hour incubation at room temperature. Values are means of three replicates.



DETAILED DESCRIPTION
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a species” includes a plurality of such species and reference to “the enzyme” includes reference to one or more enzymes and equivalents thereof, and so forth.
Unless defined 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 any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.
Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
Methods and systems for the selective fluorination of a predetermined target site of an organic molecule are herein disclosed. In these methods and systems, the predetermined target site is first activated by an Oxidizing agent that introduces an oxygen-containing functional group in the target site, and then fluorinated by a fluorinating agent that replaces the oxygen-containing functional group with fluorine or a fluorine group. In particular, activation and fluorination of an organic molecule can be performed as schematically illustrated in FIGS. 1 and 2, FIG. 2 also showing activation and fluorination of an organic molecule performed according to some embodiments herein disclosed, in comparison with chemical methods and systems of the art.
The term “target site” as used herein refers to an oxidizable C atom, i.e. a C atom in the organic molecule that bears an oxidizable bond. Examples of oxidizable bonds include but are not limited to a C—H bond, a C—C double bond, and a C—X bond, single or double, where X is an heteroatom independently selected from the group consisting of B (boron), O, (oxygen), P (phosphorous), N (nitrogen), S (sulphur), Si (silicium), Se (selenium), F (fluorine), Cl (chlorine), Br (bromine), and I (iodine).
The terms “activate” and “activation” as used herein with reference to a target site indicate a chemical reaction resulting in an enhanced reactivity of the C atom that forms the site, so that said C atom acquires or improves its ability to undergo a chemical transformation, more specifically a fluorination reaction. For example, the insertion of an oxygen atom in a target site bearing a C—H bond and resulting in the formation of a hydroxyl group (C—OH) on the site activates the target site for a deoxofluorination reaction. A further example is the insertion of an oxygen atom in a target site bearing a C═C double bond and resulting in the formation of an epoxy group activates the site for a ring-opening fluorination reaction. Accordingly, the wording “activated site” as used herein refers to a C atom of an organic molecule that, following activation, has acquired or improved its ability to undergo a chemical transformation and in particular a fluorination reaction when contacted with a fluorine.
The term “contact” as used herein with reference to interactions of chemical units indicates that the chemical units are at a distance that allows short range non-covalent interactions (such as Van der Waals forces, hydrogen bonding, hydrophobic interactions, electrostatic interactions, dipole-dipole interactions) to dominate the interaction of the chemical units. For example, when an oxygenase enzyme is ‘contacted’ with a target molecule, the enzyme is allowed to interact with and bind to the organic molecule through non-covalent interactions so that a reaction between the enzyme and the target molecule can occur.
The wording “chemical unit” identifies single atoms as well as groups of atoms connected by a chemical bond. Exemplary chemical units herein described include, but are not limited to fluorine atom, chemical groups such as oxygen-containing chemical group and fluorine-containing groups, organic molecules or portions thereof including target sites, chemical agents, including Oxidizing agents and fluorinating agents.
The term “agent” as used herein refers to a chemical unit that is capable to cause a chemical reaction specified in the identifier accompanying the term. Accordingly, an “Oxidizing agent” is an agent capable of causing an oxygenation reaction of a suitable substrate and a “fluorinating agent” is an agent capable of causing a fluorination reaction of a suitable substrate. An oxygenation reaction is a chemical reaction in which one or more oxygen atoms are inserted into one or more pre-existing chemical bonds of said substrate. A fluorination reaction is a chemical reaction in which a substituent connected to an atom in said substrate is substituted for fluorine.
The term “introducing” as used herein with reference to the interaction between two chemical units, such as a functional groups and a target site, indicates a reaction resulting in the formation of a bond between the two chemical units, e.g. the functional group and the target site.
The term “functional group” as used herein refers to a chemical unit within a molecule that is responsible for a characteristic chemical reaction of that molecule. An “oxygen-containing functional group” is a functional group that comprises an oxygen atom. Exemplary oxygen-containing functional groups include but are not limited to a hydroxyl group (—OH), ether group (—OR), carbonyl oxygen (═O), hydroperoxy group (—OOH), and peroxy group (—OOR).
The terms “replace” and “replacement” as used herein with reference to chemical units indicates formation of a chemical bond between the chemical units in place of a pre-existing bond in at least one of said chemical unit. In particular, replacing an oxygen-containing functional group on the target site with a fluorine or fluorine group indicates the formation of a bond between the target site and the fluorine or fluorine group in place of the bond between the target site and the oxygen-containing functional group.
Any organic molecule that includes at least one target site, i.e. at least one oxidizable C atom, and is a substrate of at least one Oxidizing agent, can be used as an organic molecule to be fluorinated according to the methods and systems herein disclosed.
In some embodiments, the Oxidizing agent is an enzyme such as an oxygenase or Oxidizing enzymes that is able to introduce an oxygen-containing functional group in the target site of the organic molecule using an oxygen source such as molecular oxygen (O2), hydrogen peroxide (H2O2), a hydroperoxide (R—OOH), or a peroxide (R—O—O—R′), including the oxidoreductases with an Enzyme Classification (EC) number typically corresponding to EC 1.13 or EC 1.14. Suitable oxygenases for the systems and methods herein described include but are not limited to monooxygenases, dioxygenases, peroxygenases, and peroxidases. In particular, monooxygenases and peroxygenase can be used to introduce on the target site an oxygen-containing functional group that comprises one oxygen atom, dioxygenases can be used to introduce on the target site an oxygen-containing functional group that comprises two oxygen atoms, and peroxydases can be used to introduce on the target site an oxygen-containing functional group that comprises one or two oxygen atoms.
In some embodiments, the oxygenases are wild-type oxygenases and in some embodiments the oxygenase is a mutant or variant. An oxygenase is wild-type if it has the structure and function of an oxygenase as it exists in nature. An oxygenase is a mutant or variant if it has been mutated from the oxygenase as it exists in nature and provides an oxygenase enzymatic activity.
In some embodiments, the variant oxygenase provides an enhanced oxygenase enzymatic activity compared to the corresponding wild-type oxygenase. In some embodiments, the variant oxygenases maintain the binding specificity of the corresponding wild-type oxygenase, in other embodiments the variant oxygenases disclosed herein are instead bindingly distinguishable from the corresponding wild-type and bindingly distinguishable from another. The wording “bindingly distinguishable” as used herein with reference to molecules, indicates molecules that are distinguishable based on their ability to specifically bind to, and are thereby defined as complementary to a specific molecule. Accordingly, a first oxygenase is bindingly distinguishable from a second oxygenase if the first oxygenase specifically binds and is thereby defined as complementary to a first substrate and the second oxygenase specifically binds and is thereby defined as complementary to a second substrate, with the first substrate distinct from the second substrate. In some embodiments, the variant oxygenase herein disclosed, has an increased enzyme half-time in vivo, a reduced antigenicity, and/or an increased storage stability when compared to the corresponding wild-type oxygenase.
In some embodiments, the oxygenase is a heme-containing oxygenase or a variant thereof. The wording “heme” or “heme domain” as used herein refers to an amino acid sequence within an oxygenase, which is capable of binding an iron-complexing structure such as a porphyrin. Compounds of iron are typically complexed in a porphyrin (tetrapyrrole) ring that may differ in side chain composition. Heme groups can be the prosthetic groups of cytochromes and are found in most oxygen carrier proteins. Exemplary heme domains include that of P450BM3 as well as truncated or mutated versions of these that retain the capability to bind the iron-complexing structure. A skilled person can identify the heme domain of a specific protein using methods known in the art. Exemplary organic molecules that can be oxidized by heme-containing oxygenases include C5-C22 alkanes, fatty acids, steroids, terpenes, aromatic hydrocarbons, polyketides, prostaglandins, terpenes, statins, amino acids, flavonoids, and stilbenes.
In particular, in some embodiments the “heme-containing oxygenase” is a cytochrome P450 enzyme (herein also indicates as CYPs or P450s) or a variant thereof. The wording “P450 enzymes” indicates a group of heme-containing oxygenases that share a common overall fold and topology despite less than 20% sequence identity across the corresponding gene superfamily (Denisov, Makris et al. 2005). In particular, the P450 enzymes share a conserved P450 structural core, which binds to the heme group and comprises a P450 signature sequence. The conserved P450 structural core is formed by a four-helix bundle composed of three parallel helices (usually labeled D, L, and I), and one antiparallel helix (usually labeled as helix E) (Presnell and Cohen 1989) and by a Cys heme-ligand loop which includes a conserved cysteine that binds to the heme group and the P450 signature. In particular, the conserved cysteine that binds to the heme group is the proximal or “fifth” ligand to the heme iron and the relevant ligand group (a thiolate) is the origin of the characteristic name giving 450-nm Soret absorbance observed for the ferrous-CO complex (Pylypenko and Schlichting 2004). The P450 signature sequence is the sequence indicated in the enclosed sequence listing as SEQ ID NO:1. FIG. 3 is a representation of the P450 structural core of bacterial P450BM3. In the illustration of FIG. 3, the prosthetic heme group (‘heme’) is located between the distal I helix (‘helix I’) and proximal L helix (‘helix L’) and is bound to the adjacent Cys heme-ligand loop containing the P450 signature sequence SEQ ID NO: 1. Helices D and E are also indicated in FIG. 3.
P450 enzymes are known to be involved in metabolism of exogenous and endogenous compounds. In particular, P450 enzymes can act as terminal oxidases in multicomponent electron transfer chains, called here P450-containing systems. Reactions catalyzed by cytochrome P450 enzymes include hydroxylation, epoxidation, N-dealkylation, O-dealkylation, S-oxidation and other less common transformations. The most common reaction catalyzed by P450 enzymes is the monooxygenase reaction using molecular oxygen (O2), where one atom of oxygen is inserted into a substrate while the other is reduced to water.
P450 monooxygenases can catalyze the monooxygenation of a variety of structurally diverse substrates. Exemplary substrates, that can be oxidized by naturally-occurring P450s include C5-C22 alkanes, cyclic alkanes, cyclic alkenes, alkane derivatives, alkene derivatives, C10-C20 fatty acids, steroids, terpenes, aromatic hydrocarbons, natural products and natural product analogues such as polyketides, prostaglandines, thromboxanes, leukotrienes, anthraquinones, tetracyclines, anthracyclines, polyenes, statins, amino acids, flavonoids, stilbenes, alkaloids (e.g. lysine-derived, nicotinic acid-derived, tyrosine-derived, tryptophan-derived, anthranilic acid-derived, histidine-derived, purine-derived alkaloids), beta-lactams, aminoglycosides, polymyxins, quinolones, synthetic derivatives such as aromatic heterocyclic derivatives (e.g. phenyl-, pyrimidine-, pyridine-, piperidine-, pyrrole-, furan-, triazol-, thiophene-, pyrazole-, imidazole-, tetrazole-, oxazole-, isoxazole-, thiazole-, isothiazole-, pyran-, pyridazine-, pyrazine-, piperazine-, thiazine-, and oxazine-derivatives), and the like.
Naturally-occurring P450 monooxygenases have been also mutated in their primary sequence to favor their activity towards other non-native substrates such as short-chain fatty acids, 8- and 12-pNCA, indole, aniline, p-nitrophenol, polycyclic hydrocarbons (e.g. indole, naphthalene), styrene, medium- and short-chain alkanes, alkenes (e.g. cyclohexene, 1-hexene, styrene, benzene), quinoline, steroid derivatives, and various drugs (e.g. chlorzoxazone, propranolol, amodiaquine, dextromethorphan, acetaminophen, ifosfamide, cyclophosphamide, benzphetamine, buspirone, MDMA).
P450 monooxygenases suitable in the methods and systems herein disclosed include cytochrome P450 monooxygenases (EC 1.14.14.1) from different sources (bacterial, fungi, yeast, plant, mammalian, and human), and variants thereof. Exemplary P450 monooxygenases suitable in the methods and systems herein disclosed include members of CYP102A subfamily (e.g. CYP102A1, CYP102A2, CYP102A3, CYP102A5), members of CYP101A subfamily (e.g. CYP101A1), members of CYP102e subfamily (e.g. CYP102E1), members of CYP1A subfamily (e.g. CYP1A1, CYP1A2), members of CYP2A subfamily (e.g. CYP2A3, CYP2A4, CYP2A5, CYP2A6, CYP2A12, CYP2A13), members of CYP1B subfamily (e.g. CYP1B1), members of CYP2B subfamily (e.g. CYP2B6), members of CYP2C subfamily (e.g. CYP2C8, CYP2C9, CYP2C10, CYP2C18, CYP2C19) members of CYP2D subfamily (e.g. CYP2D6), members of CYP3A subfamily (e.g. CYP3A4, CYP3A5, CYP3A7, CYP3A43), members of CYP107A subfamily (e.g. CYP107A1), and members of CYP153 family (e.g. CYP153A1, CYP153A2, CYP153A6, CYP153A7, CYP153A8, CYP153A11, CYP153D3, and CYP153D2, (van Beilen and Funhoff 2007)). Exemplary organic molecules oxidizable by P450 monooxygenases include C5-C22 alkanes, cyclic alkanes, cyclic alkenes, alkane derivatives, alkene derivatives, C10-C20 fatty acids, steroids, terpenes, aromatic hydrocarbons, natural products and natural product analogues such as polyketides, prostaglandines, thromboxanes, leukotrienes, anthraquinones, tetracyclines, anthracyclines, polyenes, statins, amino acids, flavonoids, stilbenes, alkaloids (e.g. lysine-derived, nicotinic acid-derived, tyrosine-derived, tryptophan-derived, anthranilic acid-derived, histidine-derived, purine-derived alkaloids), beta-lactams, aminoglycosides, polymyxins, quinolones, synthetic derivatives such as aromatic heterocyclic derivatives (e.g. phenyl-, pyrimidine-, pyridine-, piperidine-, pyrrole-, furan-, triazol-, thiophene-, pyrazole-, imidazole-, tetrazole-, oxazole-, isoxazole-, thiazole-, isothiazole-, pyran-, pyridazine-, pyrazine-, piperazine-, thiazine-, and oxazine-derivatives), and the like.
Other exemplary P450 monooxygenases suitable in the methods and systems herein disclosed include CYP106A2, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1, CYP8B1, CYP11A1, CYP1B1, CYP11B2, CYP17A1, CYP19A1, CYP20A1, CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1, CYP27A1, CYP27C1, CYP39A1, CYP46A1, CYP51A1.
In particular, in some embodiments P450 monooxygenases suitable in the methods and systems herein disclosed include CYP102A1 (also called P450BM3) from Bacillus megaterium (SEQ ID NO: 2), CYP102A2 from Bacillus subtilis (SEQ ID NO: 3), CYP102A3 from Bacillus subtilis (SEQ ID NO: 4), CYP102A5 from Bacillus cereus (SEQ ID NO: 5), CYP102E1 from Ralstonia metallidurans (SEQ ID NO: 6), CYP102A6 from Bradyrhizobium japonicum (SEQ ID NO: 7), CYP101A1 (also called P450cam) from Pseudomonas putida (SEQ ID NO: 8), CYP106A2 (also called P450meg) from Bacillus megaterium (SEQ ID NO: 9), CYP153A6 (SEQ ID NO: 54), CYP153A7 (SEQ ID NO: 55), CYP153A8 (SEQ ID NO: 56), CYP153A11 (SEQ ID NO: 57), CYP153D2 (SEQ ID NO: 58), CYP153D3 (SEQ ID NO: 59), P450cin from Citrobacter brakii (SEQ ID NO: 10), P450terp from Pseudomonas sp. (SEQ ID NO: 11), P450eryF from Saccharopolyspora erythreae (SEQ ID NO: 12), CYP1A2 (SEQ ID NO: 13), CYP2C8 (SEQ ID NO: 14), CYP2C9 (SEQ ID NO: 15), CYP2C19 (SEQ ID NO: 16), CYP2D6 (SEQ ID NO: 17), CYP2E1 (SEQ ID NO: 18), CYP2F1 (SEQ ID NO: 19), CYP3A4 (SEQ ID NO: 20), CYP153-AlkBurk from Alcanivorax borkumensis (SEQ ID NO: 60), CYP153-EB104 from Acinetobacter sp. EB104 (SEQ ID NO: 61), CYP153-OC4 from Acinetobacter sp. OC4 (SEQ ID NO: 62), and variants thereof. Exemplary organic molecules that can be oxidized by these P450 monooxygenases include branched and linear C10-C20 fatty acids, C6-C20 alkanes, cyclic alkanes, cyclic alkenes, alkane derivatives, alkene derivatives, steroids, terpenes, aromatic hydrocarbons, natural products and natural product analogues such as polyketides, prostaglandines, thromboxanes, leukotrienes, anthraquinones, tetracyclines, anthracyclines, polyenes, statins, amino acids, flavonoids, stilbenes, alkaloids (e.g. lysine-derived, nicotinic acid-derived, tyrosine-derived, tryptophan-derived, anthranilic acid-derived, histidine-derived, purine-derived alkaloids), beta-lactams, aminoglycosides, polymyxins, quinolones, synthetic derivatives such as aromatic heterocyclic derivatives (e.g. phenyl-, pyrimidine-, pyridine-, piperidine-, pyrrole-, furan-, triazol-, thiophene-, pyrazole-, imidazole-, tetrazole-, oxazole-, isoxazole-, thiazole-, isothiazole-, pyran-, pyridazine-, pyrazine-, piperazine-, thiazine-, and oxazine-derivatives), and the like.
In particular, in some embodiments P450 monooxygenases suitable for the methods and systems herein disclosed include CYP102A1 (SEQ ID NO: 2) and variants thereof, wherein none, one or more of the amino acids that are located within 50 Å from the heme iron are mutated to any other of the natural amino acids or mutated to an unnatural amino acid or modified in some way so to alter the properties of the enzyme. Examples of amino acid positions that can be modified in CYP102A1 to produce a P450 monooxygenase suitable in the methods and systems herein disclosed include without limitations: 25, 26, 42, 47, 51, 52, 58, 74, 75, 78, 81, 82, 87, 88, 90, 94, 96, 102, 106, 107, 108, 118, 135, 138, 142, 145, 152, 172, 173, 175, 178, 180, 181, 184, 185, 188, 197, 199, 205, 214, 226, 231, 236, 237, 239, 252, 255, 260, 263, 264, 265, 268, 273, 274, 275, 290, 295, 306, 324, 328, 353, 354, 366, 398, 401, 430, 433, 434, 437, 438, 442, 443, 444, and 446.
In particular, in some embodiments, P450 monooxygenases suitable in the methods and system herein disclosed are selected from the group consisting of CYP102A1 (SEQ ID NO:2) and variants thereof including CYP102A1var1 (SEQ ID NO: 21), CYP102A1var2 (SEQ ID NO: 22), CYP102A1var3 (SEQ ID NO: 23), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-3 (SEQ ID NO: 25), CYP102A1var3-4 (SEQ ID NO: 26), CYP102A1var3-5 (SEQ ID NO: 27), CYP102A1var3-6 (SEQ ID NO: 28), CYP102A1var3-7 (SEQ ID NO: 29), CYP102A1var3-8 (SEQ ID NO: 30), CYP102A1var3-9 (SEQ ID NO: 31), CYP102A1var3-5 (SEQ ID NO: 32), CYP102A1var3-6 (SEQ ID NO: 33), CYP102A1var3-12 (SEQ ID NO: 34), CYP102A1var3-13 (SEQ ID NO: 35), CYP102A1var3-14 (SEQ ID NO: 36), CYP102A1var3-15 (SEQ ID NO: 37), CYP102A1var3-16 (SEQ ID NO: 38), CYP102A1var3-17 (SEQ ID NO: 39), CYP102A1var3-18 (SEQ ID NO: 40), CYP102A1var3-19 (SEQ ID NO: 41), CYP102A1var3-20 (SEQ ID NO: 42) CYP102A1var3-21 (SEQ ID NO: 43), CYP102A1var3-22 (SEQ ID NO: 44), CYP102A1var3-23 (SEQ ID NO: 45), CYP102A1var4 (SEQ ID NO: 46) CYP102A1var5 (SEQ ID NO:47), CYP102A1var6 (SEQ ID NO: 48), CYP102A1var7 (SEQ ID NO: 49), CYP102A1var8 (SEQ ID NO:50), CYP102A1var9 (SEQ ID NO: 51), CYP102A1var9-1 (SEQ ID NO: 52)
The above variants are illustrated in particular in the following Table 1 wherein the respective sequences reporting in the enclosed Sequence Listing and the mutations of each variant with respect to the wild type (SEQ ID NO: 2) are listed.









TABLE 1





Name
Sequence
Mutation(s) with respect to CYP102A1







CYP102A1
SEQ ID NO: 2
—


CYP102A1var1
SEQ ID NO: 21
V78A, H138Y, T175I, V178I, A184V, H236Q,




E252G, R255S, A290V, A295T, L353V


CYP102A1var2
SEQ ID NO: 22
V78A, T175I, A184V, F205C, S226R, H236Q,




E252G, R255S, A290V, L353V


CYP102A1var3
SEQ ID NO: 23
R47C, V78A, K94I, P142S, T175I, A184V, F205C,




S226R, H236Q, E252G, R255S, A290V, L353V


CYP102A1var3-2
SEQ ID NO: 24
V78A, F81P, A82L, F87A, P142S, T175I, A180T,




A184V, A197V, F205C, S226R, H236Q, E252G,




R255S, A290V, L353V


CYP102A1var3-3
SEQ ID NO: 25
V78A, F81C, A82P, F87A, P142S, T175I, A180T,




A184V, A197V, F205C, S226R, H236Q, E252G,




R255S, A290V, L353V


CYP102A1var3-4
SEQ ID NO. 26
V78A, F81W, A82I, F87A, P142S, T175I, A180T,




A184V, A197V, F205C, S226R, H236Q, E252G,




R255S, A290V, L353V


CYP102A1var3-5
SEQ ID NO: 27
V78A, A82G, F87V, P142S, T175I, A184V, F205C,




S226R, H236Q, E252G, R255S, A290V, A328V,




L353V


CYP102A1var3-6
SEQ ID NO: 28
R47C, V78A, F87I, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




L353V


CYP102A1var3-7
SEQ ID NO: 29
R47C, V78A, F87A, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




L353V


CYP102A1var3-8
SEQ ID NO: 30
R47C, V78A, A82L, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




L353V


CYP102A1var3-9
SEQ ID NO: 31
R47C, V78T, A82G, F87V, K94I, P142S, T175I,




A184V, F205C, S226R, H236Q, E252G, R255S,




A290V, A328L, L353V


CYP102A1var3-10
SEQ ID NO: 32
R47C, L52I, V78F, A82S, K94I, P142S, T175I,




A184V, F205C, S226R, H236Q, E252G, R255S,




A290V, A328F, I366V, L353V, E464G, I710T


CYP102A1var3-11
SEQ ID NO: 33
R47C, L52I, V78F, A82S, K94I, P142S, T175I,




A184V, F205C, S226R, H236Q, E252G, R255S,




A290V, A328L, K349N, L353V, I366V, E464G,




I710T


CYP102A1var3-12
SEQ ID NO: 34
R47C, L52I, V78F, A82S, K94I, P142S, T175I,




A184V, L188P, F205C, S226R, H236Q, E252G,




R255S, A290V, A328L, I366V, L353V, E464G,




I710T


CYP102A1var3-13
SEQ ID NO. 35
R47C, V78T, A82G, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




A328L, L353V


CYP102A1var3-14
SEQ ID NO: 36
V78A, A82V, F81R, F87A, P142S, T175I, A180T,




A184V, A197V, F205C, S226R, H236Q, E252G,




R255S, A290V, L353V


CYP102A1var3-15
SEQ ID NO: 37
V78A, F81W, A82S, F87A, P142S, T175I, A184V,




A197V, F205C, S226R, H236Q, E252G, R255S,




A290V, L353V


CYP102A1var3-16
SEQ ID NO: 38
R47C, V78F, A82S, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




A328F, L353V, E464G, I710T


CYP102A1var3-17
SEQ ID NO: 39
V78A, F81V, A82T, F87A, P142S, T175I, A180T,




A184V, A197V, F205C, S226R, H236Q, E252G,




R255S, A290V, L353V


CYP102A1var3-18
SEQ ID NO: 40
R47C, L52I, V78F, A82S, K94I, P142S, T175I,




A184V, L188P, F205C, S226R, H236Q, E252G,




R255S, A290V, A328F, I366V, L353V, E464G,




I710T


CYP102A1var3-19
SEQ ID NO. 41
R47C, L52I, A74S, V78F, A82S, K94I, P142S,




T175I, L188P, F205C, S226R, H236Q, E252G,




R255S, A290V, A328F, I366V, L353V, E464G,




I710T


CYP102A1var3-20
SEQ ID NO: 42
R47C, V78A, A82V, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, A290V,




A328V, L353V


CYP102A1var3-21
SEQ ID NO: 43
R47C, V78A, F87V, K94I, P142S, T175I, A184V,




F205C, S226R, H236Q, E252G, R255S, I263A,




A290V, L353V


CYP102A1var3-22
SEQ ID NO: 44
R47C, V78A, A82F, K94I, P142S, T175I, A184V,




F205C, S226R, I263A, H236Q, E252G, R255S,




A290V, A328V, L353V


CYP102A1var3-23
SEQ ID NO: 45
R47C, V78A, K94I, P142S, T175I, A184V, F205C,




S226R, H236Q, E252G, R255S, A290V, A328V,




L353V


CYP102A1var4
SEQ ID NO: 46
F87A


CYP102A1var5
SEQ ID NO: 47
F87V


CYP102A1var6
SEQ ID NO: 48
F87V, L188Q


CYP102A1var7
SEQ ID NO: 49
A74G, F87V, L188Q


CYP102A1var8
SEQ ID NO: 50
R47L, F87V, L188Q


CYP102A1var9
SEQ ID NO: 51
F87A, T235A, R471A, E494K, S1024E


CYP102A1var9-1
SEQ ID NO: 52
F87A, A184K, T235A, R471A, E494K, S1024E









In some embodiments, the P450 monooxygenases listed in Table 1 or 6 are provided as oxygenating agents for the methods and systems herein disclosed, wherein the organic molecules, include branched and linear C6-C20 fatty acids, C2-C20 alkanes, cyclic alkanes, cyclic alkenes, alkane derivatives, alkene derivatives, steroids, terpenes, aromatic hydrocarbons, prostaglandines, aromatic heterocyclic derivatives such as phenyl-, pyrimidine-, pyridine-, piperidine-, pyrrole-, furan-, triazol-, thiophene-, pyrazole-, imidazole-, tetrazole-, oxazole-, isoxazole-, thiazole-, isothiazole-, pyran-, pyridazine-, pyrazine-, piperazine-, thiazine-, and oxazine-derivatives.
In some embodiments P450 monooxygenases suitable in the methods and systems herein disclosed include CYP102A2 from Bacillus subtilis (SEQ ID NO: 3), and variants thereof, wherein none, one or more of the amino acids that are located within 50 Å from the heme iron are mutated to any other of the natural aminoacids or mutated to an unnatural amino acid or modified in some way so to alter the properties of the enzyme.
In particular, in some embodiments, P450 monooxygenases suitable in the methods and system herein disclosed are selected from the group consisting of CYP102A2 (SEQ ID NO:3) and variants thereof including CYP102A2var1 (SEQ ID NO:63). The above variants are illustrated in particular in the following Table 2 wherein the mutations of each variant with respect to the wild type (SEQ ID NO: 3) are listed.











TABLE 2









Mutation(s) with



Name
Sequence
respect to CYP101A1









CYP102A2
SEQ ID NO: 3
—



CYP102A2var1
SEQ ID NO: 63
F88A










In some embodiments P450 monooxygenases suitable for the methods and systems herein disclosed include CYP102A3 from Bacillus subtilis (SEQ ID NO: 4), and variants thereof, wherein none, one or more of the amino acids that are located within 50 Å from the heme iron are mutated to any other of the natural amino acids or mutated to an unnatural amino acid or modified in some way so to alter the properties of the enzyme.
In particular, in some embodiments P450 monooxygenases suitable in the methods and systems herein disclosed are selected from the group consisting of CYP102A3 (SEQ ID NO:4) and variants thereof including CYP102A3var1 (SEQ ID NO: 64). The above variants are illustrated in particular in the following Table 3 wherein the mutations of each variant with respect to the wild type (SEQ ID NO: 4) are listed.











TABLE 3









Mutation(s) with



Name
Sequence
respect to CYP101A1









CYP102A3
SEQ ID NO: 4
—



CYP102A3var1
SEQ ID NO: 64
F88A










In particular, in some embodiments P450 monooxygenases suitable in the methods and systems herein disclosed include CYP101A1 (also called P450cam) from Pseudomonas putida (SEQ ID NO: 8) and variants thereof, wherein none, one or more of the amino acids that are located within 50 Å from the heme iron are mutated to any other of the natural aminoacids or mutated to an unnatural amino acid or modified in some way so to alter the properties of the enzyme.
In particular, in some embodiments, P450 monooxygenases suitable in the methods and system herein disclosed are selected from the group consisting of CYP101A1 (SEQ ID NO:8) and variants thereof including CYP101A1var1 (SEQ ID NO:65), CYP101A1var2 (SEQ ID NO:66), CYP101A1var2-1 (SEQ ID NO:67), CYP101A1var2-2 (SEQ ID NO:68), and CYP101A1 var2-3 (SEQ ID NO:69).
The above variants are illustrated in particular in the following Table 4 wherein the mutations of each variant with respect to the wild type (SEQ ID NO: 8) are listed.











TABLE 4









Mutation(s) with



Name
Sequence
respect to CYP101A1









CYP101A1
SEQ ID NO: 8
—



CYP101Avar1
SEQ ID NO: 65
Y96A



CYP101A1var2
SEQ ID NO: 66
Y96F



CYP101A1var2-1
SEQ ID NO: 67
Y96F, F87W



CYP101A1var2-2
SEQ ID NO: 68
Y96F, V247L



CYP101A1var2-3
SEQ ID NO: 69
F87W, Y96F, V247L










In some embodiments, the P450 enzyme is included in a P450-containing system, a system including a P450 enzyme and one or more proteins that deliver one or more electrons to the heme iron in the P450 enzyme. Natural P450-containing systems occur according to the following general schemes:
CYP reductase (CPR)/cytochrome b5 (cyb5)/P450 systems: typically employed by eukaryotic microsomal (i.e., not mitochondrial) CYPs, they involve the reduction of cytochrome P450 reductase (variously CPR, POR, or CYPOR) by NADPH, and the transfer of reducing power as electrons to the CYP. Cytochrome b5 (cyb5) can also contribute reducing power to this system after being reduced by cytochrome b5 reductase (CYB5R);
Ferrodoxin Reductase (FdxR) or Putidaredoxin Reductase (PdxR)/Ferrodoxin (Fdx) or Putidaredoxin (Pdx)/P450 systems, typically employed by mitochondrial and some bacterial CYPs. Reducing electrons from a soluble cofactor, typically NADPH or NADH, are transferred through the reductase to electron carrier, Fdx or Pdx, and transferred from the electron carrier to the P450 component;
P450-CPR fusion systems, where the CYP domain is naturally fused to the electron donating partners. An example of these systems is represented by cytochrome P450BM3 (CYP102A1) from the soil bacterium Bacillus megaterium; 
CYB5R/cyb5/P450 systems, where both electrons required by the CYP derive from cytochrome b5;
FMN/Fd/P450 systems, where a FMN-domain-containing reductase is fused to the CYP. This type of system was originally found in Rhodococcus sp;
P450 only systems, which do not require external reducing power. These include CYP5 (thromboxane synthase), CYP8, prostacyclin synthase, and CYP74A (allene oxide synthase).
In some embodiments, the oxidizing agent is a non-heme containing monooxygenases i.e. a monooxygenases that is able to function without a heme prosthetic group. These monooxygenases include, but are not limited to, flavin monooxygenases, pterin-dependent non-heme monooxygenases, non-heme diiron monooxygenases, and diiron hydroxylases. In these enzymes, oxygen activation occurs at a site in the enzyme's structural fold that is covalently or non-covalently bound to a flavin cofactor, a pterin cofactor, or a diiron cluster. Examples of non-heme containing monooxygenases include but are not limited to ω-hydroxylases (n-octane ω-hydroxylase, n-decane ω-hydroxylases, 9-α-hydroxylase, and AlkB), styrene monooxygenase, butane monooxygenases, propane monooxygenases, and methane monooxygenases. Non-heme containing monooxygenases catalyze the monooxygenation of a variety of structurally diverse substrates. Exemplary substrates accepted by progesterone 9-α-hydroxylase from Nocardia sp. include steroid derivatives. Exemplary substrates accepted by non-heme monooxygenases such as integral membrane di-iron alkane hydroxylases (e.g. AlkB), soluble di-iron methane monooxygenases (sMMO), di-iron propane monooxygenases, di-iron butane monooxygenases, membrane-bound copper-containing methane monooxygenases, styrene monooxygenase, xylene monooxygenase include C1-C24 linear and branched alkanes, alkenes, and aromatic hydrocarbons.
In some embodiments, the oxidizing agent is a dioxygenase or a variant thereof and in particular a dioxygenase involved in the catabolism of aromatic hydrocarbons. Dioxygenases are a class of oxygenase enzymes that incorporate both atoms of molecular oxygen (O2) onto the substrate according to the general scheme of reaction:




Dioxygenases are metalloprotein and activation of molecular oxygen is carried out in a site within the structural fold of the enzyme that is covalently or non-covalently bound to one or more metal atoms. The metal is typically iron, manganese, or copper. Example of dioxygenases are catechol dioxygenases, toluene dioxygenases, biphenyl dioxygenases. Catechol dioxygenases catalyze the oxidative cleavage of catechols and have different substrate specificities, including catechol 1,2-dioxygenase (EC 1.13.11.1), catechol 2,3-dioxygenase (EC 1.13.11.2), and protocatechuate 3,4-dioxygenase (EC 1.13.11.3). Toluene dioxygenase and biphenyl dioxygenases are involved in the natural degradation of aromatic compounds and typically introduce two oxygen atoms across a double bond in aromatic or non-aromatic compounds. Diooxygenases, e.g. toluene dioxygenase, can be engineered to accept substrates for which the wild-type enzyme shows only basal or no activity, e.g. 4-picoline (Sakamoto, Joern et al. 2001). Potentially suitable substrates for dioxygenase enzymes include but are not restricted to substituted or non-substituted monocyclic, polycyclic, and heterocyclic aromatic compounds. On these substrates, the diooxygenase can introduce one or more cis dihydrodiol functional group.
In some embodiments, the oxidizing agent is a peroxygenase. Natural peroxygenases are heme-dependent oxidases that are distinct from cytochrome P450 enzymes and peroxidases and that accept only peroxides, in particular hydrogen peroxide, as the source of oxidant. Natural peroxygenases are typically membrane-bound and can catalyze hydroxylation reactions of aromatics, sulfoxidations of xenobiotics, or epoxidations of unsaturated fatty acids. In contrast to cytochrome P450 monoxygenases, peroxygenases' activity does not require any cofactor such as NAD(P)H and does not use molecular oxygen. Examples are the plant peroxygenase (PXG) (Hanano, Burcklen et al. 2006), soybean peroxygenase (Blee, Wilcox et al. 1993), and oat seed peroxygenase.
In some embodiments, the peroxygenase is a cytochrome P450s can also use peroxides as oxygen donors. This constitutes the so-called ‘peroxide shunt pathway’ and the enzyme does not need a reductase and NAD(P)H to carry out catalysis. Normally, this peroxide-driven reaction in P450s is not significant. However, mutations in the heme domain of P450 enzymes can enhance their latent peroxygenase activity, as in the case of P450cam (Joo, Lin et al. 1999) and P450BM3 (Cirino and Arnold 2003). Using three engineered P450 enzymes, namely CYP102A1, CYP102A2 and CYP102A3, that are capable of peroxygenase activity, a library of  ̃6000 members peroxygenase chimeras was created by site-directed recombination (Otey, Landwehr et al. 2006).
Naturally-occurring P450 peroxygenases also exist. P450BSβ (CYP152A1) and P450SPα (CYP152B1), recently isolated from Bacillus subtilis and Sphingomonas paucimobilis (Matsunaga, Sumimoto et al. 2002; Matsunaga, Yamada et al. 2002), efficiently utilize H2O2 to hydroxylate fatty acids, prevalently in α and β positions.
Exemplary peroxygenases suitable in the methods and system herein disclosed include but are not limited to natural heme-containing peroxygenases, natural P450 peroxygenases, engineered P450s with peroxygenase activity, and P450 peroxygenase chimeras described in more details in the work of Arnold and co-workers (Otey, Landwehr et al. 2006). These peroxygenases show activity on a variety of substrates including fatty acids, 8- and 12-pNCA, indole, aniline, p-nitrophenol, heterocyclic derivatives (e.g. chlorzoxazone, buspirone), statins, naphtyl derivatives.
Other suitable Oxidizing agents for the systems and methods herein disclosed are peroxidases (EC number 1.11.1.x). Sequences of the peroxidase enzymes identified so far can be found in the PeroxiBase database. Peroxidases typically catalyze a reaction of the form: ROOR′+electron donor (2 e−)+2H+→ROH+R′OH. For most peroxidases the optimal oxygen providing compound is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or redox-active cysteine or selenocysteine residues. The nature of the electron donor is very dependent on the structure of the enzyme. For example, horseradish peroxidase can use a variety of organic compounds as electron donors and acceptors. Horseradish peroxidase has an accessible active site and many compounds can reach the site of the reaction. In contrast, cytochrome c peroxidase has a much more restricted active site, and the electron-donating compounds are very specific. Glutathione peroxidase is a peroxidase found in humans, which contains selenocysteine. It uses glutathione as an electron donor and is active with both hydrogen peroxide and organic hydroperoxide substrates.
In some embodiments the organic molecule has the structure of formula (I)




in which X═C atom is the target site, and R1, R2, and R3 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG) or are taken together to form a ring, such that the carbon atom is a secondary or tertiary carbon atom.

The term “aliphatic” is used in the conventional sense to refer to an open-chain or cyclic, linear or branched, saturated or unsaturated hydrocarbon group, including but not limited to alkyl group, alkenyl group and alkynyl groups. The term “heteroatom-containing aliphatic” as used herein refer to an aliphatic moiety where at least one carbon atom is replaced with a heteroatom.
The term “alkyl” and “alkyl group” as used herein refers to a linear, branched, or cyclic saturated hydrocarbon typically containing 1 to 24 carbon atoms, preferably 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl and the like. The term “heteroatom-containing alkyl” as used herein refers to an alkyl moiety where at least one carbon atom is replaced with a heteroatom, e.g. oxygen, nitrogen, sulphur, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur.
The term “alkenyl” and “alkenyl group” as used herein refers to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, preferably of 2 to 12 carbon atoms, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. The term “heteroatom-containing alkenyl” as used herein refer to an alkenyl moiety where at least one carbon atom is replaced with a heteroatom.
The term “alkynyl” and “alkynyl group” as used herein refers to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, preferably of 2 to 12 carbon atoms, containing at least one triple bond, such as ethynyl, n-propynyl, and the like. The term “heteroatom-containing alkynyl” as used herein refer to an alkynyl moiety where at least one carbon atom is replaced with a heteroatom.
The term “aryl” and “aryl group” as used herein refers to an aromatic substituent containing a single aromatic or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such as linked through a methylene or an ethylene moiety). Preferred aryl groups contain 5 to 24 carbon atoms, and particularly preferred aryl groups contain 5 to 14 carbon atoms. The term “heteroatom-containing aryl” as used herein refer to an aryl moiety where at least one carbon atom is replaced with a heteroatom.
The term “alkoxy” and “alkoxy group” as used herein refers to an aliphatic group or a heteroatom-containing aliphatic group bound through a single, terminal ether linkage. Preferred aryl alkoxy groups contain 1 to 24 carbon atoms, and particularly preferred alkoxy groups contain 1 to 14 carbon atoms.
The term “aryloxy” and “aryloxy group” as used herein refers to an aryl group or a heteroatom-containing aryl group bound through a single, terminal ether linkage. Preferred aryloxy groups contain 5 to 24 carbon atoms, and particularly preferred aryloxy groups contain 5 to 14 carbon atoms.
The terms “halo” and “halogen” are used in the conventional sense to refer to a fluoro, chloro, bromo or iodo substituent.
By “substituted” it is intended that in the alkyl, alkenyl, alkynyl, aryl, or other moiety, at least one hydrogen atom is replaced with one or more non-hydrogen atoms. Examples of such substituents include, without limitation: functional groups referred to herein as “FG”, such as alkoxy, aryloxy, alkyl, heteroatom-containing alkyl, alkenyl, heteroatom-containing alkenyl, alkynyl, heteroatom-containing alkynyl, aryl, heteroatom-containing aryl, alkoxy, heteroatom-containing alkoxy, aryloxy, heteroatom-containing aryloxy, halo, hydroxyl (—OH), sulfhydryl (—SH), substituted sulfhydryl, carbonyl (—CO—), thiocarbonyl, (—CS—), carboxy (—COOH), amino (—NH2), substituted amino, nitro (—NO2), nitroso (—NO), sulfo (—SO2—OH), cyano (—C≡N), cyanato (—O—C≡N), thiocyanato (—S—C≡N), formyl (—CO—H), thioformyl (—CS—H), phosphono (—P(O)OH2), substituted phosphono, and phospho (—PO2).
In particular, the substituents R1, R2 and R3 of formula I can be independently selected from hydrogen, C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, C5-C24 aryloxy, carbonyl, thiocarbonyl, and carboxy. More in particular, R1, R2 and R3 of formula I can be independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, C2-C14 alkoxy, C5-C14 aryloxy, carbonyl, thiocarbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (I) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxygenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be butane monooxygenase, CYP102A1 (SEQ ID NO:2), CYP102A1var4 (SEQ ID NO:46), CYP102A1var8 (SEQ ID NO: 50), CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-20 (SEQ ID NO:42), CYP102A var3-2 (SEQ ID NO:44), CYP102A1var3-3 (SEQ ID NO:25), CYP102A var3-4 (SEQ ID NO:26), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-7 (SEQ ID NO:29), CYP102A1var3-8 (SEQ ID NO:30), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var3-11 (SEQ ID NO:33), CYP102A1var3-13 (SEQ ID NO:35), CYP102A1var3-14 (SEQ ID NO:36), CYP102A1var3-15 (SEQ ID NO:37), CYP101A1 (SEQ ID NO:8), CYP101A1var1 (SEQ ID NO: 65), CYP101A1var2-3 (SEQ ID NO:69), CYP102A2 (SEQ ID NO:3), CYP102A2var1 (SEQ ID NO:63), CYP102A3 (SEQ ID NO:4), CYP102A3var1 (SEQ ID NO:64) and CYP153A6 (SEQ ID NO:54), CYP153A7 (SEQ ID NO:55), CYP153A8 (SEQ ID NO:56), CYP153A11 (SEQ ID NO:57), CYP153D2 (SEQ ID NO:58), CYP106A2 (SEQ ID NO:9) and/or CYP102A1var2 (SEQ ID NO:22) having mutations at F87 (e.g., F87I), P142 (e.g., P142S) or a combination thereof. In particular, in those embodiments at least one of said oxygenases or variants thereof is expected to activate the target site by introducing an oxygen-containing functional group in the form of a hydroxyl group. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be (R1R2R3CF), (R1R2CF2), (R1R3CF2), or (R2R3CF2).
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R1═H, —CH3 or ═O, and/or R2 and R2 are connected together through 4, 5, 6, or 7-methylene moiety to form a ring, the oxidizing agent can be an oxygenase, such as a P450 monooxygenase, and in particular CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-7 (SEQ ID: NO:29), CYP101A1 (SEQ ID NO:8), CYP101A1var1 (SEQ ID NO:65), and/or CYP101A1var2-3 (SEQ ID NO:69), and is expected to activate the target site of the corresponding compound of Formula (I) by introducing an oxygen-containing functional group in the form of a hydroxyl group.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R1=H, R2=-Me, -Et, —Pr, or -iPr, and/or R3=—(CH2)nCOOH with n between 9 and 15, the oxidizing agent can be an oxygenase such as a P450 monooxygenase, in particular CYP102A1 (SEQ ID NO:2), CYP102A1var4 (SEQ ID NO:46), CYP102A 1 var5 (SEQ ID NO:47), CYP102A2 (SEQ ID NO:3), CYP102A2var1 ((SEQ ID NO:63), CYP102A3 (SEQ ID NO:4), and/or CYP102A3var1 (SEQ ID NO:64), which is expected to activate the target site by introducing an oxygen-containing functional group in the form of a hydroxyl group.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R1=R2=-Me, R3=—CH2-o-substituted-Ph, activation can be performed by reacting the organic molecule with an oxygenase, such as a P450 monooxygenase, including CYP102A1 (SEQ ID NO:2), CYP102A1var3-4 (SEQ ID NO:26), CYP102A1var3-14 (SEQ ID NO:36), CYP102A1var3-15 (SEQ ID NO:37), CYP102A1var3-3 (SEQ ID NO:25), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var1 (SEQ ID NO:21), and/or CYP102A1var2 (SEQ ID NO:22), which introduce an hydroxyl group in the target site, as exemplified in Examples 11 and illustrated in corresponding scheme 11.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R1=R2=H, activation can be performed by reacting the organic molecule with an oxygenase such as a P450 monooxygenase, including CYP153A6 (SEQ ID NO:54), CYP153A7 (SEQ ID NO:55), CYP153A8 (SEQ ID NO:56), CYP153A11 (SEQ ID NO:57), CYP153D2 (SEQ ID NO:58), and/or CYP153D3 (SEQ ID NO:59), which are expected to introduce an hydroxyl group on the target site
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R3=n-C6-C10 alkyl (e.g. linear C6-C10 alkanes), activation can be performed by an oxygenase such as a butane monooxygenase, which is expected to introduce an hydroxyl group on the target site.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R3=cyclohexenyl (e.g. limonene), the oxidating agent can be an oxygenase, such as a P450 monooxygenase including CYP153A6 (SEQ ID NO:54), CYP153A7 (SEQ ID NO:55), CYP153A8 (SEQ ID NO:56), CYP153A11 (SEQ ID NO:57), CYP153D2 (SEQ ID NO:58), and/or CYP153D3 (SEQ ID NO:59) which are expected to introduce an hydroxyl group on the target site.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R3=n-C7, the oxidating agent can be a monooxygenase such as a P450 monooxygenases including CYP102A1var3-13 (SEQ ID NO: 35), which is expected to introduce an hydroxyl group on the target site.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), in which R1=H, R2 and R3 are connected through n methylene moieties, activation can be performed by reacting the substrate with monooxygenases such as P450 monooxygenases including CYP102A1var1 (SEQ ID NO: 21), CYP102A1var2 (SEQ ID NO: 22), CYP102A1var3-20 (SEQ ID NO: 42). In particular, when n=5, as in the case of cyclopentanecarboxylic acid derivatives, the compound of formula (I) can be activated with methods and systems herein disclosed wherein the oxidating agent is a monooxygenase CYP102A1var8 (SEQ ID NO: 50). When instead n=6, as in the case of camphor, cyclohexane and cyclohexene, the compound of formula (I) can be activated with methods and systems herein disclosed wherein the oxidating agent is a monooxygenase, such as a P450 monooxygenase including CYP101A1 (SEQ ID NO: 8), CYP153A6 (SEQ ID NO: 54), CYP153A7 (SEQ ID NO:55), CYP153A8 (SEQ ID NO: 56), CYP153A1 (SEQ ID NO: 57), CYP153D3 (SEQ ID NO: 59) or CYP153D2 (SEQ ID NO: 58). In those embodiments, activation is known or expected to result in the introduction of a hydroxyl group in the target site.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (I), wherein R1=H, R2 and R3 are connected through 5 or 6 methylene moieties, so to form a polycyclic unsaturated system, such as in steroids, activation can be performed by reacting the substrate with a monooxygenases such as a P450 monooxygenase including CYP106A2 (SEQ ID NO: 9), and the activation is expected to result in the introduction of an hydroxyl group in the target site.
In the compound of formula I, wherein R1=H, R2=—CH2COOH, R3=n-dodecyl, activation can be performed by reacting the substrate with peroxygenase P450BSβ (CYP152A1) (SEQ ID NO:70), resulting in the introduction of a hydroxyl group in the target site.
In some embodiments, the organic molecule has the structure of formula (II)




in which X is the target site C atom, and R4, R5, and R6 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG) or are taken together to form a ring, such that the carbon atom is a secondary or tertiary carbon atom.

In particular, the substituents R4, R5 and R6 of Formula (II) can be independently selected from hydrogen, C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, C5-C24 aryloxy, carbonyl, thiocarbonyl, carboxy, sulfhydryl, amino, substituted amino. More in particular, R4 can be independently selected from hydrogen, C2-C14 alkoxy, C5-C14 aryloxy, amino, substituted amino, sulfhydryl, substituted sulfhydryl, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, and C5-C14 substituted heteroatom-containing aryl, while R5 and R6 are independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, C2-C14 alkoxy, C5-C14 aryloxy, carbonyl, thiocarbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (II) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxugenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be a P450 monooxygenase or peroxygenase including CYP102A1 (SEQ ID NO:2), CYP102A1var4 (SEQ ID NO:46), CYP102A1var8 (SEQ ID NO:50), CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-7 (SEQ ID NO:9), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var3-14 (SEQ ID NO:36), CYP102A1var3-15 (SEQ ID NO:37), CYP102A1var3-17 (SEQ ID NO:39), CYP101A1 (SEQ ID NO:8), CYP101A1(Y96F), CYP101A1var2-1 (SEQ ID NO:67), CYP101A1var1 (SEQ ID NO:65), CYP101A1var2-2 (SEQ ID NO:68), CYP1A2 (SEQ ID NO:13), CYP2C9 (SEQ ID NO:15), CYP2C19 (SEQ ID NO:16), CYP2D6 (SEQ ID NO:17), CYP2E1 (SEQ ID NO:18), CYP3A4 (SEQ ID NO:20), P450BSβ (CYP152A1) (SEQ ID NO:70) and/or P450SPα (CYP152B1). In particular, in those embodiments at least one of said oxygenases or variants thereof is expected to activate the target site of a compound of Formula (II) by introducing an oxygen-containing functional group in the form of a hydroxyl group. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be (R5R6CF—(CO)—R4), (R5CF2—(CO)—R4), or (R6CF2—(CO)—R).
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (II) with R4=—OH, the Oxidizing agent can be a peroxygenase, such as a P450BSβ (CYP152A1) (SEQ ID NO:70) and/or or a peroxygenase P450SPα (CYP152B1), which are most expected activate the target site, in particular by introducing an oxygen-containing functional group in the form of a hydroxyl group.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (II), with R4=—OR, and R=a C1-C6 alkyl, the Oxidizing agent can be an oxygenase and in particular a P450 oxygenase such as CYP102A1(F87A), CYP102A1var3 (SEQ ID NO: 23), CYP102A1var3-7 (SEQ ID NO: 29), CYP102A1var3-14 (SEQ ID NO: 36), CYP102A1var3-15 (SEQ ID NO: 37), and/or CYP102A1var3-5 (SEQ ID NO: 27), which are most expected to activate the target site, in particular by introducing an oxygen-containing functional group in the form of a hydroxyl group.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (II), in which R4 is —OMe, —OEt, —OPr, —OBu, —OtBu, R5 is hydrogen, and R6 is benzyl, o-chloro-phenyl, p-chloro-phenyl, or m-chloro-phenyl, o-methyl-phenyl, p-methyl-phenyl, or m-methyl-phenyl, o-methoxy-phenyl, p-methoxy-phenyl, or m-methoxy-phenyl, the activation can be performed by reacting the substrate with oxygenase CYP102A1var4 (SEQ ID NO: 46), CYP102A1var3 (SEQ ID NO: 23), and CYP102A1var3-7 (SEQ ID NO: 29), as illustrated in Examples 1, 2, 3 and 4 and corresponding schemes 1, 2, 3, and 4.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (II), in which R4 is —OH, R5 is hydrogen, and R6 is a linear C12-16 alkyl chain, (for example a myristic acid), the activation can be performed by reacting the substrate with peroxygenases P450BSβ (CYP152A1) and P450SPα (CYP152B1), resulting in the introduction of an hydroxy group in the target site.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (II), in which R5 is -Me, and R5 and R6 are connected through a 6-methylene ring, (for example a α-thujone), the activation can be performed by reacting the substrate with monooxygenases CYP101A1 (SEQ ID NO: 8), CYP102A1 (SEQ ID NO: 2), CYP1A2 (SEQ ID NO: 13), CYP2C9 (SEQ ID NO: 14), CYP2C19 (SEQ ID NO: 16), CYP2D6 (SEQ ID NO: 17), CYP2E (SEQ ID NO: 18), and CYP3A4 (SEQ ID NO: 20), resulting in the introduction of an hydroxyl group in the target site.
In some embodiments, the organic molecule has the structure of formula (III)




in which X is the target site C atom, and R7, R5, R9, R10 and R11 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG) or are taken together to form a ring, such that the carbon atom is a secondary or tertiary carbon atom.

In particular, the substituents R7, R8, R9, R10, and R11, of Formula (III) can be independently selected from hydrogen, C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, C1-C24 alkoxy, C5-C24 aryloxy, carbonyl, thiocarbonyl, and carboxy. More in particular, R7, R8, R9, R10, and R11 are independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, C2-C14 alkoxy, C5-C14 aryloxy, carbonyl, thiocarbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (III) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxygenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be a P450 oxygenase including CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-6 (SEQ ID NO:28), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-8 (SEQ ID NO:30), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var3-11 (SEQ ID NO:33), CYP102A1var3-17 (SEQ ID NO:39), CYP102A1var5 (SEQ ID NO:47), CYP102A1var6 (SEQ ID NO:48), CYP102A1var7 (SEQ ID NO:49), CYP102A1var8 (SEQ ID NO:50), CYP101A1var1 (SEQ ID NO:65), CYP101A1var2-1 (SEQ ID NO:67), CYP101A1var2-3 (SEQ ID NO:69), CYP2C19 (SEQ ID NO:16) and/or CYP2D6 (SEQ ID NO:17). In particular, in those embodiments at least one of said oxygenases or variants thereof is expected to activate the target site of a compound of Formula III by introducing an oxygen-containing functional group in the form of a hydroxyl group. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be (R7R8CF—C(R9)═CR10R11), (R7CF2—C(R9)═CR10R11) or (R8CF2—C(R9)═CR10R11).
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (III) in which R7=H, R9=—CH3, R10=-n-C5H11, and R8 and R11 are linked to form a substituted 5-member ring, activation can be performed by reacting the substrate with oxygenases such as CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-6 (SEQ ID NO:28), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-8 (SEQ ID NO:30), CYP102A1var3-9 (SEQ ID NO:31), resulting in the introduction of an hydroxyl group in the target site as illustrated in Examples 5 and corresponding scheme 5.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (III), in which R7=H, R9=H, R10=—CH3, R8 and R11 are linked to form a substituted 6-member ring, activation can be performed by reacting the substrate with oxygenase CYP101A1var2-3 (SEQ ID NO:69), resulting in the introduction of an hydroxyl group in the target site as in the case of a-pinene.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (III), in which R7=R8=R10=H, R9 and R11 are connected through a substituted 5-member ring, activation can be performed by reacting the organic molecule with an oxygenase such as CYP102A1var3-2 (SEQ ID NO:24), resulting in the introduction of an hydroxyl group in the target site as illustrated by Example 6 and corresponding scheme 6.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (III), in which R10=R11=—CH3, R8=R9=H, and R7=substituted C5 alkenyl, activation can be performed by reacting the substrate with oxygenases such as CYP2C19 (SEQ ID NO:16) and CYP2D6 (SEQ ID NO:17), as in the case of linalool.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (III), in which R7=H, R9 and R11, are linked together to form a 6-membered aromatic ring, R7 and R10 are linked together to form a 5-carbon cyclic alkenyl, activation can be performed by reacting the substrate with oxygenases CYP102A1var5 (SEQ ID NO:47), CYP102A1var6 (SEQ ID NO:48), and/or CYP102A1var7 (SEQ ID NO:49), resulting in the introduction of an hydroxyl group in the target site as in the case of acenaphthene.
In some embodiments, the organic molecule has the structure of formula (IV)




in which X is the target site C atom, Ar can be a C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl or C5-C24 substituted heteroatom-containing aryl, while R12 and R13 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG) or are taken together to form a ring, such that the carbon atom is a secondary or tertiary carbon atom.

In particular, the substituent Ar of Formula (IV) can be C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, or C5-C14 substituted heteroatom-containing aryl, while R12 and R13 are independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, C2-C14 alkoxy, C5-C14 aryloxy, carbonyl, thiocarbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (IV) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxygenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be such as CYP102A1 (SEQ ID NO:2), CYP102A1var4 (SEQ ID NO:46), CYP102A1var5 (SEQ ID NO:47), CYP102A1var6 (SEQ ID NO:48), CYP102A1var7 (SEQ ID NO:49), CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-3 (SEQ ID NO:25), CYP102A1var3-4 (SEQ ID NO:26), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-7 (SEQ ID NO:29), CYP102A1var3-8 (SEQ ID NO:30), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var3-17 (SEQ ID NO:39), CYP102A1var8 (SEQ ID NO:50), CYP101A1var2-1 (SEQ ID NO:67), and/or CYP101A1var2-3 (SEQ ID NO:69). In particular, in those embodiments at least one of said oxygenases or variants thereof is expected to activate the target site of a compound of Formula IV by introducing an oxygen-containing functional group in the form of a hydroxyl group. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be R12R13ArC—F, R12ArCF2, or R13ArCF2 
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (IV), in which Ar=para-substituted phenyl R13=H, R12=-iPr, activation can be performed by reacting the organic molecule with an oxygenase such as a P450 monooxygenase including CYP102A1 (SEQ ID NO:2) and CYP102A1var5 (SEQ ID NO:47), which results in the introduction of an hydroxyl group in the target site as illustrated in Examples 10 and corresponding scheme 10.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (IV), in which Ar=para- or ortho or meta substituted phenyl (where substituent is halo, —CH3, or —OCH3), R12=H, R13=—COOR, where R is C1-C6 n-alkyl, activation can be performed by reacting the substrate with oxygenase CYP102A1var5 (SEQ ID NO:47), CYP102A1var3 (SEQ ID NO:23), and CYP102A1var3-7 (SEQ ID NO:29), as illustrated in Examples 1, 2, 3 and 4 and corresponding schemes 1, 2, 3, and 4.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (IV), in which R12═H, Ar is ortho substituted phenyl, R13 is linked to Ar through a phenyl moiety, activation can be performed by reacting the substrate with oxygenases CYP102A1var6 (SEQ ID NO:48) and CYP102A1var8 (SEQ ID NO:50), resulting in the introduction of an hydroxyl group in the target site as in the case of fluorene.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (IV), in which R12═H, Ar is ortho substituted phenyl, R13 is linked to Ar through a 2-methylene bridge, activation can be performed by reacting the substrate with oxygenase CYP102A1var5 (SEQ ID NO:47), resulting in the introduction of an hydroxyl group in the target site as in the case of indan.
In some embodiments the organic molecule has the structure of formula (V),




in which X is the target site C atom, R14, R15, R16, R17, R18 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy, and functional groups (FG) or are taken together to form a ring, such that the carbon atom is a secondary or tertiary carbon atom.

In particular, the substituents R14, R15, R16, R17, and R18 of Formula (V) can be independently selected from hydrogen, C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, carbonyl, thiocarbonyl, and carboxy. More in particular, R14, R15, R16, R17, and R18 is are independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, C2-C14 alkoxy, C5-C14 aryloxy, carbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (V) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxugenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be CYP102A1var8 (SEQ ID NO:50), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-3 (SEQ ID NO:25), CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-6 (SEQ ID NO:28), CYP102A1var3-9 (SEQ ID NO:31), CYP102A1var3-11 (SEQ ID NO:33), CYP102A1var3-16 (SEQ ID NO:38), CYP102A1var3-19 (SEQ ID NO:41, CYP102A1var3-18 (SEQ ID NO:40), CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-3 (SEQ ID NO:25), CYP102A1var3-14 (SEQ ID NO:36), CYP102A1var3-15 (SEQ ID NO:37), CYP102A1var3-17 (SEQ ID NO:39), CYP102A1var3-9 (SEQ ID NO:31), CYP101A1var2-3 (SEQ ID NO:69), and/or CYP3A4 (SEQ ID NO:20). In particular, in those embodiments, at least one of said oxygenases or variants thereof is expected to activate a compound of Formula V by affording a hydroxyl group at the target site. In these embodiments, the final product resulting from the application of the systems and methods herein disclosed can be R14R15R16C—F.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (V), in which R14, R15, R17, R18 are hydrogen, and R16 is 2-methyl-5-phenyl-4,5-dihydrooxazolyl, activation can be performed by reacting the substrate with oxygenases CYP102A1var3-5 (SEQ ID NO:27), CYP102A1var3-6 (SEQ ID NO:28), CYP102A1var3-11 (SEQ ID NO:33), CYP102A1var3-16 (SEQ ID NO:38), CYP102A1var3-19 (SEQ ID NO:41), CYP102A1var3-18 (SEQ ID NO:40), resulting in a hydroxyl group at the target site as illustrated in Examples 12 and corresponding scheme 12.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (V), in which R14, R15, R17, R18 are hydrogen, and R16 is 2,3,4,5-tetramethoxy-tetrahydro-2H-pyranyl, activation can be performed by reacting the substrate with oxygenases such as CYP102A1var3-2 (SEQ ID NO:24), CYP102A1var3-3 (SEQ ID NO:25), CYP102A1var3-14 (SEQ ID NO:36), CYP102A1var3-15 (SEQ ID NO:37), CYP102A1var3-17 (SEQ ID NO:39), CYP102A1var3-9 (SEQ ID NO:31), resulting in a hydroxyl group at the target site as illustrated in Examples 13 and corresponding scheme 13.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (V), in which R14═CN, R15=6-dimethylamino-naphtyl, R16═R17═H, R18═H, or hydrogen, activation can be performed by reacting the substrate with oxygenases such as CYP102A1var8 (SEQ ID NO:50) and CYP3A4 (SEQ ID NO:20), as in the case of α cyano-naphtyl ethers.
In some embodiments the organic molecule has the structure of formula (VI)




in which X is the target site C atom, and R19, R20, R21, R22 are independently selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, and functional groups (FG) or are taken together to form a ring representing in this case a cycloalkenyl, substituted cycloalkenyl, heteroatom-containing cycloalkenyl, or a substituted heteroatom-containing cycloalkenyl derivative.

In particular, the substituents R19, R20, R21 and R22 of formula VI are independently selected from hydrogen, C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 substituted heteroatom-containing alkyl, C1-C24 substituted heteroatom-containing alkyl, C2-C24 alkenyl, C2-C24 substituted alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C2-C24 substituted heteroatom-containing alkenyl, C5-C24 aryl, C5-C24 substituted aryl, C5-C24 substituted heteroatom-containing aryl, C5-C24 substituted heteroatom-containing aryl, carbonyl, thiocarbonyl, carboxy, and substituted amino. More in particular, R19, R20, R21 and R22 are independently selected from hydrogen, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 substituted heteroatom-containing alkyl, C1-C12 substituted heteroatom-containing alkyl, C2-C12 alkenyl, C2-C12 substituted alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C2-C12 substituted heteroatom-containing alkenyl, C5-C14 aryl, C5-C14 substituted aryl, C5-C14 substituted heteroatom-containing aryl, C5-C14 substituted heteroatom-containing aryl, carbonyl, and carboxy.
Oxidizing agents known or expected to react with the target site of a compound of Formula (VI) include but are not limited to oxygenases or variants thereof.
In some embodiments, the oxygenase can be a non-heme monooxygenase or a variant thereof, a heme-containing monooxygenase or a variant thereof, a peroxygenase or a variant thereof, such as any of the heme-containing monooxygenase, non heme-containing monooxygenases and peroxugenases herein disclosed. In particular, the oxygenase can be any of the P450 monooxygenases and P450 peroxygenases herein disclosed.
In some embodiments, the oxygenase or variant thereof can be CYP102A1 (SEQ ID NO:2), CYP102A1var1 (SEQ ID NO:21), CYP102A1var2 (SEQ ID NO:22), CYP102A1var3 (SEQ ID NO:23), CYP102A1var3-18 (SEQ ID NO:40), CYP102A1var5 (SEQ ID NO:47), CYP102A1var4 (SEQ ID NO:46), CYP102A1var3-21 (SEQ ID NO:43), CYP102A var3-22 (SEQ ID NO:44), CYP102A1var3-23 (SEQ ID NO:45), CYP102A var9 (SEQ ID NO:51), CYP102A1var9-1 (SEQ ID NO:52), and/or toluene dioxygenase. In particular, in those embodiments at least one of said oxygenases or variants thereof is expected to activate a compound of Formula VI by introducing an oxygen-containing functional group in the form of an epoxy group. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be (R19R20C(OH)—CFR21R22), (R19R20CF—C(OH)R21R22), or (R19R20CF—CFR21R22).
Additional Oxidizing agents that are expected to react with the target site of a compound of Formula (VI) include but are not limited to dioxygenases such as toluene dioxygenase. More specifically, dioxidizing agents are expected to activate a compound of Formula (VI) by introducing an oxygen-containing functional group in the form of a vicinal diol. In these embodiments, the final products resulting from the application of the systems and methods herein disclosed can be (R19R20C(OH)—CFR21R22), (R19R20CF—C(OH)R21R22), or (R19R20CF—CFR21R22).
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (VI), in which R19=R20=R21=H, R22=n-butyl, activation through epoxidation can be performed by reacting the substrate with oxygenases such as CYP102A1var1 (SEQ ID NO; 21), CYP102A1var3-21 (SEQ ID NO; 43), CYP102A1var3-22 (SEQ ID NO; 44), CYP102A1var3-23 (SEQ ID NO; 45), resulting in the introduction of an epoxide functional group at the target site as in the case of 1-hexene.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (VI), in which R19=R20=R21=H, R22=phenyl, activation through epoxidation can be performed by reacting the substrate with oxygenases var1, CYP1A2 (SEQ ID NO; 13), CYP102A1var9 (SEQ ID NO; 51) or CYP102A1var9-1 (SEQ ID NO; 52), resulting in the introduction of an epoxide functional group at the target site as in the case of styrene.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (VI), in which R19=R21=H, R21 and R22 are connected together through 4 methylene units so to form a 6-membered ring, activation through epoxidation can be performed by reacting the substrate with oxygenases of CYP153 family, such as CYP153A6 (SEQ ID NO; 54), CYP153A7 (SEQ ID NO; 55), CYP153A8 (SEQ ID NO; 56), CYP153A11 (SEQ ID NO; 57), CYP153D2 (SEQ ID NO; 58), resulting in the introduction of an epoxide functional group at the target site as in the case of cyclohexene.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (VI), in which R20=R21=H, =R19=n-pentyl, R22═C10C-alkenyl, activation through epoxidation can be performed by reacting the substrate with oxygenases CYP102A1 (SEQ ID NO; 2), resulting in the introduction of an epoxide functional group at the target site as in the case of linolenic acid.
In some embodiments of the methods and systems herein disclosed wherein the organic molecule is a compound of Formula (VI), in which R19=R21=H, R20 and R22 are linked together to form a 6-membered substituted or non-substituted aromatic ring, activation can be performed by reacting the substrate with toluene dioxygenase, resulting in the introduction of an oxygen-containing functional group in the form of a vicinal diol. In those embodiments, the oxygen-containing functional group will have the form of an epoxy group (C═(O)═C), that is an oxygen atom joined by single bonds to two adjacent carbon atoms so to form a three-membered ring.
In some embodiments, the oxidating agent suitable to activate an organic molecule including a target site with the methods and systems herein disclosed can be identified by (a) providing the organic molecule, (b) providing an Oxidizing agent, (c) contacting the Oxidizing agent with the organic molecule for a time and under condition to allow the introduction of an oxygen-containing functional group on the target site; (d) detecting the oxygen-containing functional group on the target site of the organic molecule resulting from step c), and repeating steps (a) to (d) until an oxygen containing functional group is detected on the target site. In particular, one or more oxidating agents can be provided under step b) of the method herein disclosed.
In particular, in embodiments wherein the organic molecule is a molecule of formula (I), (II), (III), and (IV), detecting the oxygen-containing functional group on the target site can be performed by: e) isolating of the organic molecule resulting from step c), for example by a separation method or a combination of separation methods, including but not limited to extraction, chromatography, distillation, precipitation, sublimation, and crystallization; and f) characterizing the isolated organic molecule resulting from step c) to identify the oxygen containing functional group, for example by a characterization method or a combination of methods, including but not limited to spectroscopic or spectrometric technique, preferably a combination of two or more spectroscopic or spectrometric techniques, including UV-VIS spectroscopy, fluorescence spectroscopy, IR spectroscopy, 1H-NMR, 13C-NMR, 2D-NMR, 3D-NMR, GC-MS, LC-MS, and MS-MS.
In particular, in embodiments wherein the organic molecule is a molecule of formula (V), detecting the oxygen-containing functional group on the target site can be performed by monitoring the removal of the —CHR17R18 moiety associated with the introduction of an oxygen containing functional group in the target site. In those embodiments, monitoring the removal of the —CHR17R18 moiety, can be performed by g) contacting the organic molecule resulting from step c) with a reagent that can react with an aldehyde (R—CHO), a ketone (R—C(O)—R), a dicarbonyl (R—C(O)—C(O)—R), or a glyoxal (R—C(O)—CHO) functional group; and h) detecting the formation of an adduct or a complex between an aldehyde, ketone, dicarbonyl, or glyoxal in the organic molecule, the aldehyde, ketone, dicarbonyl, or glyoxal resulting from the removal of the —CHR4R5 moiety.
Detecting the formation of an adduct or complex can be performed by spectroscopic (colorimetric, fluorimetric) or chromatographic methods and additional methods identifiable by a skilled person upon reading of the present disclosure.
Reagents that can react with an aldehyde, ketone, dicarbonyl, or glyoxal and suitable for the methods and systems described herein include but are not limited to 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole-4-amino-5-hydrazino-1,2,4-triazole-3-thiol (Purpald), (pentafluorobenzyl)-hydroxylamine, p-nitrophenyl-hydrazine, 2,4-dinitrophenyl-hydrazine, 3-methylbenzothiazolin-2-one hydrazone, diethyl acetonedicarboxylate and ammonia, cyclohexane-1,3-dione and ammonia, m-phenylenediamine, p-aminophenol, 3,5-diaminobenzoic acid, p-dimethylamino-aniline, m-dinitrobenzene, o-phenylenediamine, and the like.
In some embodiments, a plurality of oxidating agents can be provided to identify a suitable oxidating agent in the methods and systems herein disclosed. In particular, in some of those embodiments wherein the organic molecule is an organic molecule of general formula (I), (II), (III), (IV) and (V) a pool of Oxidizing agents, for example a library of engineered P450s, e.g. in a 96-well plate, can be provided. In particular, in embodiments wherein the organic molecule is an organic molecule of general formula (I), (II), (III) and (IV), isolating the organic molecule resulting from step c) can be performed by extracting the reaction mixture with organic solvent and characterizing the oxygen containing functional group in the organic molecule can be performed by GC analysis of the extraction solution. In some of those embodiments, selected mixtures of Oxidizing agent, and co-reagents (e.g. cofactors, oxygen) which gave rise to the largest amount of activated products for a given organic molecule, can be repeated at a larger scale. The activated products can be subsequently isolated by suitable technique including liquid chromatography and identified by 1H-, 13C-NMR, and MS and additional techniques identifiable by a skilled person. Examples of those embodiments is provided in the Examples section and illustrated in FIGS. 5 and 6.
In embodiments wherein the organic molecule is an organic molecule of general formula (V) wherein R1 is 2-methyl-5-phenyl-4,5-dihydrooxazolyl and R2═R3═R4═R5═H, upon contacting a library of engineered P450 monooxygenases (Oxidizing agents) the oxygen containing functional group can be detected using colorimetric reagent (e.g. Purpald) and measuring the change in absorbance (e.g. at 550 nm on a microtiter plate reader). In embodiments wherein the organic molecule is an organic molecule of general formula (V) wherein R1 is 2,3,4,5-tetramethoxytetrahydro-2H-pyranyl and R2═R3═R4═R5═H upon contacting a library of engineered P450 monooxygenases, the oxygen containing functional group can also using calorimetric reagent (e.g. Purpald) and measuring the change in absorbance (e.g. at 550 nm on a microtiter plate reader).
In some embodiments, the molecule is a therapeutic drug. For example, a common strategy for improving drugs' in vivo half-life involves blocking the sites in the molecule that are susceptible to attack by human P450s with fluorine substituents. The fluorination of position 2 in 15 and 18 (see below) would prevent one of the major P450-dependent routes of ibuprofen metabolism in humans. An added advantage of this approach is thus to enable fluorination of metabolically vulnerable sites in the target molecule. The methods of the disclosure find utility in the rapid identification of drug or lead compound derivatives with improved chemo-physical/biological properties as well as in the preparation of fluorinated synthons for chemical synthesis or fragment-based drug discovery programs.
In some embodiments, the isolated and characterized organic molecule that includes the oxygen-containing functional group at the target site can be used as authentic standard for high-throughput screening of other, more suitable Oxidizing agents, or improvement of reaction conditions for the activation reaction. In exemplary embodiments, high-throughput screening can be carried out performing the activation reaction in a multi-well plate, typically a 96-well or 384-well plate, each well containing the candidate organic molecule, the Oxidizing agent, and the co-reagents (e.g. cofactors, oxygen) required for the reaction to proceed, and detecting the activation of the target site using one of the following techniques, UV-VIS spectroscopy, fluorimetry, IR, LC, GC, GC-MS, LC-MS, or a combination thereof, according to the nature and properties of the candidate organic molecule and the activated product.
In some embodiments, an oxygenase that oxidizes a pre-determined organic molecule in a target site is provided by (i) providing a candidate oxygenase, (j) mutating the candidate oxygenase to generate a mutant or variant oxygenase, (k) contacting the variant oxygenase with the pre-determined organic molecule for a time and under condition to allow detection of an oxygen containing functional group on the target site, (l) detecting the introduction of the oxygen containing functional group on the target site and repeating steps (i) to (l) until formation of on oxygen containing functional group is detected.
In some embodiments, mutating the candidate oxygenase can be performed by laboratory evolutionary methods and/or rational design methods, using one or a combination of techniques such as random mutagenesis, site-saturation mutagenesis, site-directed mutagenesis, DNA shuffling, DNA recombination, and additional techniques identifiable by a skilled person. In particular, mutating a candidate oxygenase can be performed by targeting one or more of the amino acid residues comprised in the oxygenase's nucleotidic or amino acidic primary sequence to provide a mutant or variant polynucleotide or polypeptide.
In general, the term “mutant” or “variant” as used herein with reference to a molecule such as polynucleotide or polypeptide, indicates that has been mutated from the molecule as it exits in nature. In particular, the term “mutate” and “mutation” as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which a polynucleotide or polypeptide sequence is altered, as well as any detectable change in a cell wherein the mutant polynucleotide or polypeptide is expressed arising from such a mutation. Typically, a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation in a polynucleotide includes mutations arising within a protein-encoding region of a gene as well as mutations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a coding polynucleotide such as a gene can be “silent”, i.e., not reflected in an amino acid alteration upon expression, leading to a “sequence-conservative” variant of the gene. A mutation in a polypeptide includes but is not limited to mutation in the polypeptide sequence and mutation resulting in a modified amino acid. Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farmesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM (Humana Press, Towata, N.J.).
A mutant or engineered protein or enzyme is usually, although not necessarily, expressed from a mutant polynucleotide or gene. Engineered cells can be obtained by introduction of an engineered gene or part of it in the cell. The terms “engineered cell”, “mutant cell” or “recombinant cell” as used herein refer to a cell that has been altered or derived, or is in some way different or changed, from a parent cell, including a wild-type cell. The term “recombinant” as used herein with reference to a cell in alternative to “wild-type” or “native”, indicates a cell that has been engineered to modify the genotype and/or the phenotype of the cell as found in nature, e.g., by modifying the polynucleotides and/or polypeptides expressed in the cell as it exists in nature. A “wild-type cell” refers instead to a cell which has not been engineered and displays the genotype and phenotype of said cell as found in nature.
The term “engineer” refers to any manipulation of a molecule or cell that result in a detectable change in the molecule or cell, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the cell and mutating a polynucleotide and/or polypeptide native to the cell. Engineered cells can also be obtained by modification of the cell' genetic material, lipid distribution, or protein content. In addition to recombinant production, the enzymes may be produced by direct peptide synthesis using solid-phase techniques, such as Solid-Phase Peptide Synthesis. Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer
Variants of naturally-occurring sequences can be generated by site-directed mutagenesis (Botstein and Shortle 1985; Smith 1985; Carter 1986; Dale and Felix 1996; Ling and Robinson 1997), mutagenesis using uracil containing templates (Kunkel, Roberts et al. 1987; Bass, Sorrells et al. 1988), oligonucleotide-directed mutagenesis (Zoller and Smith 1983; Zoller and Smith 1987; Zoller 1992), phosphorothioate-modified DNA mutagenesis (Taylor, Schmidt et al. 1985; Nakamaye and Eckstein 1986; Sayers, Schmidt et al. 1988), mutagenesis using gapped duplex DNA (Kramer, Drutsa et al. 1984; Kramer and Fritz 1987), point mismatch, mutagenesis using repair-deficient host strains, deletion mutagenesis (Eghtedarzadeh and Henikoff 1986), restriction-selection and restriction-purification (Braxton and Wells 1991), mutagenesis by total gene synthesis (Nambiar, Stackhouse et al. 1984; Grundstrom, Zenke et al. 1985; Wells, Vasser et al. 1985)], double-strand break repair (Mandecki 1986), and the like. Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.
Additional details regarding the methods to generate variants of naturally-occurring sequences can be found in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methods for In vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) “Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods and Compositions for Cellular and Metabolic Engineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by Random Fragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “End Complementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;” WO 97/35966 by Minshull and Stemmer, “Methods and Compositions for Cellular and Metabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting of Genetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen Library Immunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine Vector Engineering;” WO 99/41368 by Punnonen et al. “Optimization of Immunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmer and Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;” EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by Recursive Sequence Recombination;” WO 99/23107 by Stemmer et al., “Modification of Virus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 by Apt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayre et al. “Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;” WO 98/27230 by Patten and Stemmer, “Methods and Compositions for Polypeptide Engineering;” WO 98/13487 by Stemmer et al., “Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection;” WO 00/00632, “Methods for Generating Highly Diverse Libraries;” WO 00/09679, “Methods for Obtaining in vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences;” WO 98/42832 by Arnold et al., “Recombination of Polynucleotide Sequences Using Random or Defined Primers;” WO 99/29902 by Arnold et al., “Method for Creating Polynucleotide and Polypeptide Sequences;” WO 98/41653 by Vind, “An in vitro Method for Construction of a DNA Library;” WO 98/41622 by Borchert et al., “Method for Constructing a Library Using DNA Shuffling;” WO 98/42727 by Pati and Zarling, “Sequence Alterations using Homologous Recombination;” WO 00/18906 by Patten et al., “Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre et al. “Evolution of Whole Cells and Organisms by Recursive Recombination;” WO 00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic Acid Recombination;” WO 00/42559 by Selifonov and Stemmer “Methods of Populating Data Structures for Use in Evolutionary Simulations;” WO 00/42560 by Selifonov et al., “Methods for Making Character Strings, Polynucleotides & Polypeptides Having Desired Characteristics;” WO 01/23401 by Welch et al., “Use of Codon-Varied Oligonucleotide Synthesis for Synthetic Shuffling;” and WO 01/64864 “Single-Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid Fragment Isolation” by Affholter.
In particular, in some embodiments, site-directed mutagenesis can be performed on predetermined residues of the oxygenase. These predetermined sites can be identified using the crystal structure of said Oxidizing agent if available or a crystal structure of a homologous protein that shares at least 20% sequence identity with said Oxidizing agent and an alignment of the polynucleotide or amino acid sequences of the Oxidizing agent and its homologous protein. The predetermined sites are chosen among the amino acid residues that are found within 50 Å, preferably within 35 Å from the oxygen-activating site of said Oxidizing agent. For example, when a cytochrome P450 monooxygenase is to be used as the Oxidizing agent, the predetermined site are chosen among the amino acid residues that are found within 50 Å, preferably within 35 Å from the heme iron. Mutagenesis of the predetermined sites can be performed changing one, two or three of the nucleotides in the codon that encodes for each of the predetermined amino acids. Mutagenesis of the predetermined sites can be performed in the described way so that each of the predetermined amino acid is mutated to any of the other 19 natural amino acids. Substitution of the predetermined sites with unnatural amino acids can be performed using methods established in vivo (Wang, Xie et al. 2006), in vitro (Shimizu, Kuruma et al. 2006), semisynthetic (Schwarzer and Cole 2005) or synthetic methods (Camarero and Mitchell 2005) for incorporation of unnatural amino acids into polypeptides.
In still further embodiments, libraries of engineered variants can be obtained by laboratory evolutionary methods and/or rational design methods, using one or a combination of techniques such as random mutagenesis, site-saturation mutagenesis, site-directed mutagenesis, DNA shuffling, DNA recombination, and the like and targeting one or more of the amino acid residues, one at a time or simultaneously, comprised in the Oxidizing agent's amino acid sequence. Said libraries can be arrayed on multi-well plates and screened for activity on the target molecule using a calorimetric, fluorimetric, enzymatic, or luminescence assay and the like. For example a method for making libraries for directed evolution to obtain P450s with new or altered properties is recombination, or chimeragenesis, in which portions of homologous P450s are swapped to form functional chimeras, can use used. Recombining equivalent segments of homologous proteins generates variants in which every amino acid substitution has already proven to be successful in one of the parents. Therefore, the amino acid mutations made in this way are less disruptive, on average, than random mutations. A structure-based algorithm, such as SCHEMA, can be used to identify fragments of proteins that can be recombined to minimize disruptive interactions that would prevent the protein from folding into its active form.
In some embodiments, activation of a target site in an organic molecule can be performed in a whole-cell system. To prepare the whole-cell system, the encoding sequence of the Oxidizing agent can be introduced into a host cell using a suitable vector, such as a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which the said sequence of the disclosure has been inserted, in a forward or reverse orientation. In some embodiments, the construct further comprises regulatory sequences, including, for example, a promoter linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
Accordingly, in other embodiments, vectors that include a nucleic acid molecule of the disclosure are provided. In other embodiments, host cells transfected with a nucleic acid molecule of the disclosure, or a vector that includes a nucleic acid molecule of the disclosure, are provided. Host cells include eucaryotic cells such as yeast cells, insect cells, or animal cells. Host cells also include procaryotic cells such as bacterial cells.
In other embodiments, methods for producing a cell that converts a target molecule into a pre-determined oxygenated derivative are provided. Such methods generally include: (a) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2 to SEQ ID NO: 70; (b) transforming a cell with an isolated nucleic acid molecule encoding a polypeptide of the disclosure; or (c) transforming a cell with an isolated nucleic acid molecule of the disclosure.
The terms “vector”, “vector construct” and “expression vector” as used herein refer to a vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
The terms “express” and “expression” refers to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
Polynucleotides provided herein can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.
Vectors can be employed to transform an appropriate host to permit the host to express a protein or polypeptide. Examples of appropriate expression hosts include: bacterial cells, such as E. coli, B. subtilis, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 br Bowes melanoma; or plant cells or explants, etc.
In bacterial systems, a number of expression vectors may be selected. depending upon the use intended for the Oxidizing polypeptide. For example, such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the Oxidizing agent-encoding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors; pET vectors; and the like.
Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used for production of the Oxidizing agent.
In some embodiments, the activation of the target site in an organic molecule by an oxidating agent can be performed using an immobilized Oxidizing agent. Immobilization of the Oxidizing agent can be carried out through covalent attachment or physical adsorption to a support, entrapment in a matrix, encapsulation, cross-linking of Oxidizing agent's crystals or aggregates and the like. Several immobilization techniques are known (Bomscheuer 2003; Cao 2005). The type of immobilization and matrix that preserves activity often depends on the nature and physical-chemical properties of the Oxidizing agent.
In any of the above mentioned embodiments, the oxygen-containing functional group introduced on a target site of any of the above molecules is then replaced by fluorine.
In some embodiments, the fluorination is performed by deoxofluorination of the oxygenated organic molecule.
The terms “deoxofluorination” and “deoxofluorination reaction” as used herein refer to a chemical reaction where an oxygen-containing chemical unit is replaced with fluorine. Accordingly, the terms “deoxofluorinating agent”, “deoxofluorinating agent”, and “deoxofluorination agent” as used herein refer to a chemical agent that is able to carry out a deoxofluorination reaction. The term “reagent” as used herein is equivalent to the term “agent”.
In some embodiments, the fluorination can be performed by ring-opening fluorination of the oxygenated organic molecule.
The terms “ring-opening fluorination” and “ring-opening fluorination reaction” as used herein refer to a chemical reaction where an epoxide is reacted with a nucleophile, specifically fluoride (F−) to afford a fluorohydrin (—CFR1—C(OH)R2—) or a vicinal difluoride-(—CR1F—CR2F—) containing derivative. Accordingly, the terms “ring-opening fluorination agent” and “ring-opening fluorinating agent” as used herein refer to a chemical agent that is able to carry out a ring-opening fluorination reaction.
In particular, the deoxofluorination reaction can be performed using commercially available, deoxofluorinating agents such as sulfur tetrachloride (SF4), DAST (diethylaminosulfur trifluoride, (Middleton 1975), U.S. Pat. No. 3,914,265; U.S. Pat. No. 3,976,691), Deoxo-Fluor (bis-(2-methoxyethyl)-aminosulfur trifluoride, (Lal, Pez et al. 1999), U.S. Pat. No. 6,222,064), DFI (2,2-difluoro-1,3-dimethylimidazolidine, (Hayashi, Sonoda et al. 2002), U.S. Pat. No. 6,632,949), or analogues and derivatives thereof. Other deoxofluorinating agents include XeF2, SiF4, and SeF4. The deoxofluorination reaction can be performed in the presence or in the absence of additional chemical agents that facilitate or enable the deoxofluorination to occur. These additional agents include but are not limited to hydrogen fluoride (HF), Lewis acids, fluoride salts (e.g. CsF, KF, NaF, LiF, BF3), crown-ethers, ionic liquids and the like.
In particular, the ring-opening fluorination reaction can be performed using nucleophilic fluoride-containing agents including without limitations metal fluorides (e.g. CsF, KF, NaF, LiF, AgF, BF3), potassium hydrogen difluoride (KHF2), Bu4NH2F3, R3N.nHF, Bu4NF.nHF, Py.9 HF (Olah's reagent), and the like. The ring-opening fluorination reaction can be performed in the presence or in the absence of additional chemical agents that facilitate or enable the deoxofluorination to occur. These additional agents include but are not limited to hydrogen fluoride (HF), Lewis acids, fluoride salts (e.g. CsF, KF, NaF, LiF), crown-ethers, ionic liquids and the like
Exemplary fluorinations of an organic molecule containing an oxygen-containing group include but are not limited to conversion of a hydroxyl group to a fluoride, a carboxylic acid group to a carbonyl fluoride, an aldehyde group to a gem-difluoride, a keto group to a gem-difluoride, an epoxide group to a fluorohydrin (also called vic-fluoro-alcohol), an epoxide group to a vic-difluoride.
Exemplary products produced by methods and systems herein disclosed comprise fluorinated derivatives of organic molecules which include 2-aryl-acetate esters, dihydrojasmone, menthofuran, guaiol, permethylated mannopyranoside, methyl 2-(4′-(2′′-methylpropyl)phenyl)propanoate and a 5-phenyl-2-oxazoline.
Specifically, the methods and systems herein disclosed have been applied to produce methyl 2-fluoro-2-phenylacetate, ethyl 2-fluoro-2-phenylacetate, propyl 2-(3-chlorophenyl)-2-fluoroacetate, propyl 2-fluoro-o-tolylacetate, and propyl 2-fluoro-p-tolylacetate starting from corresponding 2-aryl-acetate esters; 4-fluoro-3-methyl-2-pentylcyclopent-2-enone, 4,4-difluoro-3-methyl-2-pentylcyclopent-2-enone, and 3-(fluoromethyl)-2-pentylcyclopent-2-enone, starting from dihydrojasmone; methyl 2-(4′-(1′′-fluoro-2′′-methylpropyl)phenyl)propanoate and methyl 2-(4′-(2′′-fluoro-2′′-methylpropyl)phenyl)propanoate, starting from methyl 2-(4′-(2′′-methylpropyl)phenyl)propanoate; 6-fluoro-menthofuran-2-ol from menthofuran; 2-((3S,5S,8S)-4-fluoro-3,8-dimethyl-1,2,3,4,5,6,7,8-octahydroazulen-5-yl)propan-2-ol from (−)-guaiol; 6-fluoro-6-deoxy-1,2,3,4-tetramethyl-mannopyranoside starting from 1,2,3,4,6-pentamethyl-mannpyranoside; (4R,5S)-4-(fluoromethyl)-2-methyl-5-phenyl-4,5-dihydrooxazole, starting from (4S,5S)-4-(methoxymethyl)-2-methyl-5-phenyl-4,5-dihydrooxazole.
More specifically, the methods and systems herein disclosed have been applied to fluorinate a target site, namely a C carbon atom, in a highly regioselective manner despite the presence of other similar moieties in the molecule, as in the case of 1,2,3,4,6-pentamethyl-mannopyranoside.
Even more specifically, the methods and systems herein disclosed have been applied to fluorinate target organic molecules, namely 2-aryl-acetate esters, in a highly stereoselective manner, leading to the formation of the (R)-fluoro enantiomer in considerable excess over the (S)-fluoro enantiomer.
The above mentioned fluorinated products are or can be associated with a biological activity or can be used for the synthesis of chemical compounds that are or can be associated with a biological activity.
2-fluoro-2-phenylacetate derivatives find potential applications in the synthesis of prodrugs, in particular in the preparation of ester-type anticancer prodrugs with different susceptibility to hydrolysis, which can be useful in selective targeting of cancer cells (Yamazaki, Yusa et al. 1996). 2-(4′-(2′′-methylpropyl)phenyl)propionate also known as ibuprofen is a marketed drug of the class non-steroidal anti-inflammatory drugs (NSAIDs). This drug has ample application in the treatment of arthritis, primary dysmenorrhoea, fever, and as an analgesic, especially in the presence of inflammation process. Ibuprofen exerts its analgesic, antipyretic, and anti-inflammatory activity through inhibition of cyclooxygenase (COX-2), thus inhibiting prostaglandin synthesis. More recently, ibuprofen was found to be useful in the prophylaxis of Alzheimer's disease (AD) (Townsend and Pratico 2005). The anti-AD activity of ibuprofen is presumably due to its ability to lower the levels of amyloid-beta (A beta) peptides, in particular the longer, highly amyloidogenic isoform A beta 42, which are believed to be the central disease-causing agents in Alzheimer's disease (AD). There is therefore a growing interest towards the discovery of A beta 42-lowering compounds with improved potency and brain permeability (Leuchtenberger, Beher et al. 2006). Unlike other NSAIDs, ibuprofen was also found to be useful in protection against Parkinson's disease, although the underlying mechanism is not yet known (Casper, Yaparpalvi et al. 2000).
Dihydrojasmone incorporates a cyclopentenone structural unit. The cyclopentanone and cyclopentenone scaffolds are present in a wide range of important natural products such as jasmonoids, cyclopentanoid antibiotic, and prostaglandins. This type of compound has a broad spectrum of biological activities and important application in medicinal chemistry as well as in the perfume and cosmetic industry, and agriculture. Despite their relatively simple structures, the synthesis of these scaffolds is not trivial (Mikolajczyk, Mikina et al. 1999). Therefore, novel routes for functionalization (and specifically in the context of the disclosure, fluorination) of these scaffolds and compounds incorporating these scaffolds would be highly desirable.
Guaiol is a sesquiterpene alcohol having the guaiane skeleton, found in many medicinal plants. The essential oils of Salvia lanigera and Helitta longifoliata, which both contain guaiol as a major component, were found to possess pronounced antibacterial activity (De-Moura, Simionatto et al. 2002). Structural modification of naturally-occurring bioactive substances by conventional chemical methods is very difficult and often not feasible. Accessible methods to produce derivatives of these natural products (and specifically in the context of this disclosure, fluorinated derivatives) would be highly desirable.
Furans and 2-(5H)-furanones are attractive building blocks being present in a large number of natural products that display a wide range of biological activities, and being present in a number of drugs with biologically relevant properties, such as antifungal, antibacterial and anti-inflammatory activities (Knight 1994; De Souza 2005). Many methods are available for their synthesis. However, strategies for post-synthetic functionalization (and specifically in the context of the disclosure, fluorination) of these scaffolds and compounds incorporating these scaffolds would be highly desirable.
Embodiments, wherein methods for selective fluorination of protected hydroxyl groups in the form of R1—O—CHR2R3 are performed where the resulting product is R1—F is expected to expand our current synthetic capabilities and facilitate the synthesis of fluorinated compounds that bear multiple hydroxyl functional groups as well as the synthesis of compounds that incorporate chemical units or structural features that are uncompatible with the currently available methods for protection/deprotection of hydroxyl groups (Green and Wuts 1999). The protection of hydroxyl groups with alkyl groups different from methoxymethyl (MOM), tetrahydropyranyl (THP), allyl, and benzyl (Bn) is rarely used in practice, if ever, due to the requirement of harsh chemical reagents and conditions for their removal (e.g. strong Lewis acids in the case of a methoxy group). These chemical reagents are poorly chemoselective, reacting with any nucleophilic group of the molecule. Chemical methods for regioselective substitution, and more specifically fluorination, of a single protected hydroxyl functional group in the presence of multiple identically protected hydroxyl groups are not available.
In some embodiments, activation and fluorination of the organic molecules can be performed as it follows.
The activation reaction can be carried out in aqueous solvent containing variable amounts of organic solvents to facilitate dissolution of the organic molecule in the mixture. The co-solvents include but are not limited to alcohols, acetonitrile, dimethyl sulfoxide, dimethylformamide, acetone. The one or more Oxidizing agents can be present as free in solution or inside a cell where its expression has been achieved using a plasmid vector or other strategies as described earlier. The reaction can be carried out in batch, semicontinuously or continuously, in air or using devices to flow air or oxygen through the solution, at autogeneous pressure or higher. The reaction temperature will generally be in the range of 0° C. and 100° C., depending on the nature and stability of the biocatalysts and substrates, preferably in the range of about 4° C. and 30° C. The amount of biocatalyst is generally in the range of about 0.01 mole % to 10 mole %, preferably in the range of about 0.05 mole % to 1 mole %. The cofactor (NADPH) can be added directly, regenerated using an enzyme-coupled system (typically dehydrogenase-based), or provided by the host cell. Reducing equivalent to the biocatalysts can be provided though the use of an electrode or chemical reagents. Superoxide dismutase, catalase or other reactive oxygen species-scavenging agents, can be used to prevent biocatalyst inactivation and improve the yields of the activation reaction. Glycerol, bovine serum albumine or other stabilizing agents can be used to prevent biocatalyst aggregation and improve the yields of the activation reaction.
After the activation reaction, the activated products may or may not be isolated through any of the following methods or combination thereof: extraction, distillation, precipitation, sublimation, chromatography, crystallization with optional seeding and/or co-crystallization aids.
The activated products are then contacted with the fluorinating agent in the presence or the absence of an organic solvent under inert atmosphere. The activated products can be reacted in the form of isolated compound, purified compound, partially-purified mixtures or crude mixtures. No particular restriction is imposed upon the solvent of the reaction as long as the solvent does not react with the fluorination reagent, enzymatic product, or reaction product.
Solvents that can be used in the fluorination reaction include, but are not restricted to, dichloromethane, pyridine, acetonitrile, chloroform, ethylene dichloride, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, N-methylpyrrolidone, dimethylformamide, and 1,3-dimethyl-2-imidazolidinone, preferably dichloromethane or pyridine. The reaction temperature will generally be in the range of −80° C. to 150° C., preferably in the range of about −78° C. and 30° C. The amount of the fluorination reagent is preferably 1 equivalent or more for oxygen atom introduced in the molecular scaffold of the organic molecule during the enzymatic reaction. After completion of the reaction, the fluorinated products are isolated through any of the following methods or combination thereof: extraction, distillation, precipitation, sublimation, chromatography, crystallization with optional seeding and/or co-crystallization aids.
An advantage of the methods and systems is the possibility to perform fluorination of predetermined target sites in a candidate organic molecule. A further advantage is that subjecting the activated product or the fluorinated derivative to the action of the same Oxidizing agent used for its preparation or another Oxidizing agent, polyfluorination of the molecule at the same or another predetermined target site can be achieved. A further advantage is that the mono- and/or poly fluorination of predetermined target sites in a candidate organic molecule can be carried out under mild conditions (room temperature and pressure), with limited use of hazardous chemical and toxic solvents, in a chemoselective, regioselective, and stereoselective manner.
An additional advantage of the methods and systems herein disclosed is the possibility to carry out fluorination of nonreactive sites of a candidate organic molecule, that is sites that would could not be easily functionalized using chemical reagents or would react only after or concurrently to other, more reactive sites of the molecule.
A further advantage of the methods and systems herein disclosed is the possibility to produce fluorinated derivatives of candidate organic molecules with an established or potentially relevant biological activity in only two steps. This “post-synthetic” transformation represents a considerable advantage compared to synthesis of the same derivative or derivatives starting from fluorine-containing building blocks which may or may not be available, thus requiring numerous additional synthetic steps. For example, the described methyl 2-(4′-(1′′-fluoro-2′′-methylpropyl)phenyl)propanoate, methyl 2-(4′-(2′′-fluoro-2′′-methylpropyl)phenyl)propanoate, and methyl 2-(4′-(1′′,1′′-difluoro-2′′-methylpropyl)phenyl)propanoate prepared according to the methods and systems herein disclosed could be conceivably synthesized using (1-fluoro-2-methylpropyl)benzyl, (2-fluoro-2-methylpropyl)benzyl, (1,1-difluoro-2-methylpropyl)benzyl derivatives, which however are not commercially available and therefore need to be prepared from row material through several chemical steps.
A further advantage of the methods and systems herein disclosed is the possibility to produce fluorinated derivatives of a candidate organic molecule at a preparative scale, obtaining from a minimum of 10 up to hundreds milligrams of the final fluorinated product with overall yields (after isolation) of up to 80%. These quantities and yields enable the evaluation of the biological, pharmacological, and pharmacokinetic properties of said products as well as their use in further synthesis of more complex molecules.
An additional advantage of the methods and systems herein disclosed is the possibility to substitute protected hydroxyl groups in the form of R1—O—CHR2R3 for fluorine. A further advantage is that the substitution of protected hydroxyl group for fluorine can be carried out under mild conditions (room temperature and pressure), with limited use of hazardous chemical and toxic solvents, in a chemoselective and regioselective manner.
Classes of molecules that can be potentially obtained using the methods and systems herein disclosed include but are not limited to α-fluoro acid derivatives, fluoro-alkyl derivatives, fluoro-allyl derivatives, fluorohydrins, vic- and gem-difluoride derivatives.
Classes of molecules that can be potentially obtained in enantiopure form using the methods and systems herein disclosed include but are not limited to α-fluoro acid derivatives, fluoro-alkyl derivatives, fluoro-allyl derivatives, and fluorohydrins.
In general, the methods and systems herein disclosed, in contrast to previously known synthetic methods, provide a simple, environmentally benign, two-step procedure for regio- and stereospecific incorporation of fluorine in a wide variety of organic compounds both at reactive and non-reactive sites of their molecular scaffold. Particularly, it will be appreciated that methods and systems herein disclosed procedure gives access to organofluorine derivatives, whose preparation through alternative routes would require many more synthetic steps and much higher amounts of toxic reagents and organic solvents.
Accordingly, the methods and systems herein disclosed have utility in the field of organic chemistry for preparation of fluorinated building blocks and in medicinal chemistry for the preparation or discovery of fluorinated derivatives of drugs, drug-like molecules, drug precursors, and chemical building blocks with altered or improved physical, chemical, pharmacokinetic, or pharmacological properties.
In particular, in some embodiment of the methods and systems herein disclosed, the organic molecules are pre-selected among molecules of interest, such as drugs, drug precursors, lead compounds, and synthetic building blocks. The term “drug” as used herein refer to a synthetic or non-synthetic chemical entity with established biological and/or pharmacological activity, which is used to treat a disease, cure a disfunction, or alter in some way a physiological or non-physiological function of a living organism. Lists of drugs can be easily found in online databases such as www.accessdata.fda.gov, www.drugs.com, www.rxlist.com, and the like. The term “drug precursor” as used herein refers to a synthetic or non-synthetic chemical entity which can be converted into a drug through a chemical or biochemical transformation. The conversion of a drug precursor into a drug can also occur after administration, in which case the drug precursor is typically referred to as “prodrug”. Accordingly, any synthetic or semi-synthetic intermediate in the preparation of a drug can be considered a drug precursor. The term “lead compound” as used herein refers to a synthetic or non-synthetic chemical entity that has pharmacological or biological activity and whose chemical structure is used as a starting point for chemical modifications in order to improve potency, selectivity, or pharmacokinetic parameters. Lead compounds are often found in high-throughput screenings (“hits”) or are secondary metabolites from natural sources. Reports on the discovery and/or identification of lead compounds for various applications are widespread in the scientific literature and in particular in specialized journals such as Journal of medicinal chemistry, Bioorganic & medicinal chemistry, Current medicinal chemistry, Current topics in medicinal chemistry, European Journal of Medicinal Chemistry, Mini reviews in medicinal chemistry, and the like. The term “synthetic building blocks” as used herein refer to any synthetic or non-synthetic chemical entity that is used for the preparation of a structurally more complex molecule.
Upon fluorination of the target site of the pre-selected molecule, the fluorinated organic molecules produced can be further used in the synthesis of more complex molecules, or, in addition, or in alternative, being tested for biological activities.
In particular, in any embodiment, wherein identification of a an organic molecule having a predetermined biological activity is desired, the methods and systems herein disclosed further comprise testing the fluorinated organic molecule for the desired biological activity. Testing can in particular be performed by screening the products of the reaction by the methods and systems illustrated in FIG. 4 in form of mixture or as isolated compound for altered or improved metabolic stability, biological activity, pharmacological potency, and pharmacokinetic properties. This approach couples the exceptional ability of cytochrome P450 monooxygenases to selectively insert oxygen into non reactive C—H bonds with a deoxofluorination (DF) reaction in which the newly generated hydroxyl group is substituted by fluorine by means of a nucleophilic fluorinating reagent (FIG. 4B).
The wording “biological activity” as used herein refers to any activity that can affect the status of a biological molecule or biological entity. A biological molecule can be a protein or a polynucleotide. A biological entity can be a cell, an organ, or a living organism. The wording “pharmacological activity” as used herein refers to any activity that can affect and, generally but not necessarily, improve the status of a living organism.
In embodiments where identification of a molecule having pharmacological activity is desired, use of P450 as Oxidizing agents is particularly preferred, since Phase I drug metabolism in humans is mainly dependent on P450s. In this connection, one clear advantage of the methods and systems herein disclosed is that they allow for protection through fluorination of sites in the molecule that are sensitive to P450 hydroxylation attack.
A further advantage of the methods and systems herein described for the identification of a molecule having biological activity compared to corresponding strategies known in the art for producing fluorinated drugs (which mainly rely on the use of fluorinated building blocks), is that the methods herein disclosed can be carried out post-synthetically. As a consequence, the method herein disclosed can be broadly applied to produce oxygenated/fluorinated derivatives starting from marketed drugs, drugs in advanced testing phase, lead compounds, or screening hits.
Additionally, a pre-selection of organic molecules of interest and/or related fluorinated products can be made on the basis of the ability of fluorine atoms to improve dramatically the pharmacological profile of drugs. In particular, this can be done in view of several studies have shown that potent drugs can be obtained through fluorination of much less active precursors. Anticholesterolemic Ezetimib (Clader 2004), anticancer CF3-taxanes (Ojima 2004), fluoro-steroids, and antibacterial fluoroquinolones are only some representative examples. The improved pharmacological properties of fluoro-containing drugs are due mainly to enhanced metabolic stability (Park, Kitteringham et al. 2001). Primary metabolism of drugs in humans generally occurs through P450-dependent systems, and the introduction of fluorine atoms at or near the sites of metabolic attack has often proven successful in increasing the half-life of a compound (Bohm, Banner et al. 2004).
In some cases, the introduction of fluorine substituents leads to improvements in the pharmacological properties as a result of enhanced binding affinity of the molecule to biological receptors. Examples of the effect of fluorine on binding affinity are provided by recent results in the preparation of NK1 antagonists (Swain and Rupniak 1999), 5HT1D agonists (van Niel, Collins et al. 1999), and PTB1B antagonists (Burke, Ye et al. 1996).
Accordingly, with the methods and system herein disclosed production of various oxygenated/fluorinated products was expected starting from a given drug or a drug-like molecule, for example a ‘lead’ compound identified in a drug-discovery program.
In an embodiment of the methods and systems, an array of oxygenases (P450 monooxygenases, non-heme iron monooxygenases, dioxygenases and peroxygenases) can be used to produce various mono- and poly-oxygenated compounds. Some of these products can be isolated and subjected to fluorination, e.g. deoxo-fluorination, where all or a subset of the introduced oxygen-containing functional groups are substituted for fluorine. The resulting products can then be separated and tested for improved biological properties.
EXAMPLES
The present disclosure is further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
The following experiments have been carried out to perform chemo-enzymatic fluorination approach according to embodiments of the methods and systems herein disclosed.
First, a set of organic molecules has been selected, from which potentially useful fluorinated products can be obtained.
These compounds include: (a) 2-aryl acetic acid derivatives, as demonstrative examples of useful synthetic blocks, for example in the preparation of prodrugs with different susceptibility to hydrolysis. With the systems and methods herein described, stereoselective fluorination of the alpha position of these target molecules was achieved, affording 2-fluoro-2-aryl acetic acid derivatives in considerable enantiomeric excess; (b) ibuprofen methyl ester, as demonstrative example of a marketed drug, of which more potent and BBB (blood-brain-barrier)-penetrating derivatives are sought after for treatment of Alzheimer's and Parkinson's diseases. With the systems and methods herein described, regioselective fluorination of weakly reactive sites of this target molecule was achieved, affording various C—F derivatives; (c) dihydrojasmone, menthofuran, and guaiol, as demonstrative examples of various molecular scaffolds that are present in several natural, synthetic, and semisynthetic biologically active molecules. With the systems and methods herein described, regioselective fluorination of weakly reactive sites of these target molecules was achieved, affording various C—F derivatives; (d) dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline, as demonstrative example for chemoselective substitution of methoxygroup for fluorine. With the systems and methods herein described, fluorination of the methoxy protected group in the target molecules was achieved, affording a demethoxy-fluoro derivative; (e) permethylated mannopyranoside as demonstrative example for regioselective substitution of a specific methoxy group for fluorine in the presence of several other identical groups in the molecule. With the systems and methods herein described, regioselective fluorination of the methoxy protected group in position 6 of the target molecule was achieved, affording a 6-demethoxy-fluoro derivative.
A pool of Oxidizing agents, comprising wild-type P450BM3 (CYP102A1), variants of wild-type P450BM3 carrying one or more mutations at the positions 25, 26, 42, 47, 51, 52, 58, 64, 74, 75, 78, 81, 82, 87, 88, 90, 94, 96, 102, 106, 107, 108, 118, 135, 138, 142, 143, 145, 152, 172, 173, 175, 178, 180, 181, 184, 185, 188, 197, 199, 205, 214, 226, 231, 236, 237, 239, 252, 255, 260, 263, 264, 265, 267, 268, 273, 274, 275, 290, 295, 306, 324, 328, 354, 366, 398, 401, 430, 433, 434, 437, 438, 442, 443, 444, and 446, and a selection of the most active P450 chimera peroxygenases and monooxygenases from the libraries described in Otey et al. (Otey, Landwehr et al. 2006) and Landwehr et al. (Landwehr, Carbone et al. 2007) were arrayed on 96-well plates. Arrays were prepared by growing recombinant E. coli transformed with an expression plasmid encoding for the P450 sequence, inducing protein expression with IPTG, and preparing a cell lysate.
The activation reaction of the pre-selected organic molecules ibuprofen methyl ester, menthofuran, dihydrojasmone, and guaiol with the pool of pre-selected Oxidizing agents was tested at a 1-mL scale dissolving the organic molecule in phosphate buffer (1% ethanol) at a final concentration of 2 mM. The Oxidizing agent was then added to the solution at a final concentration of about 200-400 nM. The reaction was started by adding NADPH and a glucose-6-phosphate dehydrogenase cofactor regeneration system to the mixture. After 20 hrs incubation at room temperature, the reactions were extracted with chloroform and analyzed by gas chromatography. Total conversion ratios were calculated including in the experiment a sample containing no enzyme and adding an internal standard to the samples. The 20-30% most promising Oxidizing agents were re-tested at a larger scale (3 mL) to identify false positives and determine regioselectivity and product distribution. Exemplary results from the screening of the pool of P450s on dihydrojasmone and menthofuran are reported on FIGS. 5 and 6.
A group of about 5 to 10 most interesting Oxidizing agents were then selected based on the results from the re-screen, in particular based on their regioselectivity, conversion efficiency, or ability to produce “rare” activated product. Using the selected Oxidizing agents, conditions for the activation reaction were optimized, testing different co-solvents (e.g. ethanol, ethylacetate), additives (e.g. BSA, glycerol), ROS(Reactive oxygen species) scavengers (e.g. SOD, catalase), temperature, and target molecule:Oxidizing agent ratios. Once optimized, the activation reaction was scaled up to 100-300 mL reaction scale, where the Oxidizing agent concentration typically ranged from 0.5 to 15 μM, the target molecule concentration from 5 to 20 mM, and a cofactor regeneration system was used. The co-solvent was usually ethanol, typically at a final concentration of 0.5% to 2%.
Large scale reactions were incubated under stirring at room temperature for a period of time of up to 56 hours, during which target molecule conversion was monitored by extracting small aliquots of the reaction mixture and analyzing them by gas chromatography.
As the desired amount of activated product was produced, the reaction mixture was extracted with an organic solvent, typically chloroform, and the activated product was isolated by silica gel chromatography using hexane:ethyl acetate gradient. Purified products were identified using GC-MS, 1H-, and 13C-NMR.
Once the product with the activated target site was identified, the activated product was subjected to fluorination using the deoxo-fluorinating agent DAST in dichloromethane. Different reaction conditions were typically tested to optimize yield and possibly achieve quantitative conversion. During these tests, the conversion of the activated product to the corresponding fluorinated derivative was typically monitored by GC-MS.
After the fluorination reaction, the fluorinated product was isolated by silica gel chromatography using a hexane:ethyl acetate gradient. The identity of the purified product was confirmed by GC-MS, HR-MS, 1H-, 13C-, and 19F-NMR.
The pool of pre-selected Oxidizing agents and other selected variants from mutagenesis libraries of var3-10—i.e. libraries where positions 74, 82, 87, 88, and 328 position of var3-10 were subjected to saturation mutagenesis—were screened for activity towards activation of the pre-selected organic molecules dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline (MMPO) and 1,2,3,4,6-pentamethyl mannopyranoside using a calorimetric assay on a 96-well plate format. In the case of MMPO, for example, different Oxidizing agents were arrayed on a 96-well plate, each well containing about 150 μL phosphate buffer and about 1 μM Oxidizing agent. The target molecule was added to the solution from an ethanol stock to a final concentration of 2 mM (and 1% ethanol). After addition of 1 mM NADPH, the reaction mixture was incubated for 30 minutes at room temperature. After incubation, MMPO activation activity was determined using the calorimetric reagent Purpald (Sigma), which reacts with formaldehyde and serves in this case to detect the demethylation of the methoxy group in the target molecule. Positive ‘hits’ were re-tested on a 1-mL scale using 1 mM MMPO, 0.5 μM Oxidizing agent, 1 mM NADPH, and a cofactor regeneration system. After incubation at room temperature, the reaction mixtures were extracted with chloroform and analyzed by gas chromatography. In this way, the regioselectivity and conversion efficiency of each Oxidizing agent was established. The identity of the activated product was also confirmed by GC-MS. Most promising Oxidizing agents, that is those showing highest regioselectivity and/or conversion efficiency, were used for scale-up tests and for producing larger quantities of activated product for the fluorination reaction as described above. Representative results from the screening of the P450 pool for MMPO activation activity are reported on FIG. 7.
The activation of the target molecule dihydrojasmone was also carried out using a whole-cell system (FIG. 8). Specifically, the whole-cell system consisted of E. coli DH5a cells transformed with a pCWori vector that contains the sequence for var3. The whole-cell activation reaction was carried out growing a 0.5 L culture of the recombinant cells in TB medium, inducing the intracellular expression of var3 during mid-log phase by adding 0.5 mM IPTG, and growing the cells at 30° C. for further 12 hours. After that, 15 mL dodecane were added to the culture. Dihydrojasmone was then added to the culture at a final concentration of 30 mM. Formation of the activated product and consumption of the target molecule were monitored by gas chromatography for up to 36 hours. Conversion ratio at the end of the 36 hours amounted to  ̃10%. Higher conversion ratios (>90-95%) were achieved in vitro with the same variant using a cofactor regeneration system. The lower efficiency of the whole-cell system in the case of dihydrojasmone may be attributed to potential toxicity of this molecule or its activated product to the cells as well as their low membrane permeability. Nevertheless, this experiment demonstrates that the activation of the target molecule for the scope of the systems and methods herein described can also be performed using a whole-cell, especially in cases where the chemo-physical properties of the candidate molecule may make this option more favorable.
Chemical reagents, substrates and solvents were purchased from Sigma, Aldrich, and Fluka. Silica gel chromatography purifications were carried out using AMD Silica Gel 60 230-400 mesh. Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and an Agilent HP5 column (30 m×0.32 mm×0.1 μm film). Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and an Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film). GC-MS analyses were carried out on a Hewlett-Packard 5970B MSD with 5890 GC and a DB-5 capillary column. 1H, 13C, and 19F NMR spectra were recorded on a Varian Mercury 300 spectrometer (300 MHz, 75 MHz, and 282 MHz, respectively), and are internally referenced to residual protio solvent signal. Data for 1H NMR are reported in the conventional form: chemical shift (δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constant (Hz), integration, and assignment). Data for 13C NMR are reported in the terms of chemical shift (δ ppm). Data for 19F NMR are reported in the terms of chemical shift (δ ppm) and multiplicity. High-resolution mass spectra were obtained with a JEOL JMS-600H High Resolution Mass Spectrometer at the California Institute of Technology Mass Spectral facility.
Example 1
Stereoselective fluorination of methyl 2-phenyl acetate




Methyl 2-phenyl acetate was subjected to selective fluorination of the target site C (alpha position) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 90 mg methyl 2-phenyl acetate were dissolved in 500 μL ethanol and added to 240 mL potassium phosphate buffer pH 8.0. P450BM3 was added to the mixture at a final concentration of 2 μM. The mixture was split in 4 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL of a 5 mM NADPH solution were added to each vial and stirred for 2 minutes. 500 μL of a cofactor regeneration solution containing 300 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 4 hours, the reaction mixtures were joined together and extracted with chloroform (3×100 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded the activated product ((S)-methyl 2-hydroxy-2-phenylacetate, 40.5 mg). 40 mg (0.24 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 41 μL DAST (0.29 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate: 95% hexane) afforded the fluorinated product ((R)-methyl 2-fluoro-2-phenylacetate) (30 mg, 75% yield, pale yellow oil) in 74% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 3.75 (s, 3H, —OCH3), δ 5.77 (d, J=48 Hz, 1H, —CHF), δ 7.37-7.46 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ2.8, 89.5 (d, J=184.5 Hz), 126.8, 126.9, 129.0, δ 129.9, δ 134.4 (d, J=34.5 Hz), δ 169.0. 19F-NMR (282 MHz, CDCl3): δ −180.29 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C9H9FO2 requires m/z 168.0587, found 168.0594.
Example 2
Stereoselective fluorination of ethyl 2-phenyl acetate




Ethyl 2-phenyl acetate was subjected to selective fluorination of the target site C (alpha position) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 100 mg ethyl 2-phenyl acetate were dissolved in 500 μL ethanol and added to 250 mL potassium phosphate buffer pH 8.0. WT(F87A) was added to the mixture at a final concentration of 2 μM. The mixture was split in 4 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL of a 5 mM NADPH solution were added to each vial and stirred for 2 minutes. 500 μL of a cofactor regeneration solution containing 300 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 3 hours, the reaction mixtures were joined together and extracted with chloroform (3×100 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded the activated product ((S)-ethyl 2-hydroxy-2-phenylacetate, 66 mg). 66 mg (0.36 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 61 μL DAST (0.43 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded the fluorinated product ((R)-ethyl 2-fluoro-2-phenylacetate) (51 mg, 78% yield, pale yellow oil) in 93% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 1.24 (t, J=7.2 Hz, 3H, —CH3), δ 4.16-4.27 (m, 2H, —OCH2), δ 5.75 (d, J=48 Hz, 1H, —CHF), δ 7.37-7.46 (m, 5H); 13C-NMR (75 MHz, CDCl3): 14.2, 62.0, 81.2, 89.6 (d, J=184.5 Hz), 126.8, 126.9, 128.9, 129.8, 134.4 (d, J=34.5 Hz), δ 169.0. 19F-NMR (282 MHz, CDCl3): δ −180.27 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C10H11FO2 requires m/z 182.0743, found 182.0750.
Example 3
Stereoselective fluorination of propyl 2-(3-chlorophenyl)acetate




Propyl 2-(3-chlorophenyl)acetate was subjected to selective fluorination of the target site C (alpha position) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 95 mg propyl 2-(3-chlorophenyl)acetate were dissolved in 500 μL ethanol and added to 250 mL potassium phosphate buffer pH 8.0. Var3-7 was added to the mixture at a final concentration of 1 μM. The mixture was split in 4 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL of a 5 mM NADPH solution were added to each vial and stirred for 2 minutes. 500 μL of a cofactor regeneration solution containing 300 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 4 hours, the reaction mixtures were joined together and extracted with chloroform (3×100 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded the activated product ((S)-propyl 2-hydroxy-2-(3-chlorophenyl)acetate, 71 mg). 70 mg (0.3 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 64 μL DAST (0.45 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded the fluorinated product ((R)-propyl 2-fluoro-2-(3-chlorophenyl)acetate) (57 mg, 82% yield, colorless oil) in 89% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 0.85 (t, J=7 Hz, 3H, —CH3), δ 1.56-1.68 (m, 2H, CH2), δ 4.12 (t, J=6 Hz, 2H, —O—(CH2), δ 5.72 (d, J=48 Hz, 1H, —CHF), δ 7.32 (br, 3H), δ 7.44 (br, 1H); 13C-NMR (75 MHz, CDCl3): 10.3, 21.9, 67.7, δ 88.7 (d, J=186.5 Hz), 124.8, 126.9, 129.9, 130.3, 134.9. 19F-NMR (282 MHz, CDCl3): δ −182.8 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C11H12ClFO2 requires m/z 230.0510, found 230.0502.
Example 4
Stereoselective fluorination of propyl 2-(4-methylphenyl)acetate and propyl 2-(2-methylphenyl)acetate








Propyl 2-(4-methylphenyl)acetate and propyl 2-(2-methylphenyl)acetate were subjected to selective fluorination of the target site C (alpha position) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: stereoselective activation and fluorination of 2-(4-methylphenyl)acetate and propyl 2-(2-methylphenyl)acetate were carried out starting from 100 mg substrate according to the experimental protocol described in Example 3. The fluorinated product (R)-propyl 2-fluoro-2-(4-methylphenyl)acetate was obtained in 87% ee (54 mg, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 0.84-0.91 (m, 3H, —CH3), δ 1.57-1.68 (m, 2H, CH2), δ 2.37 (s, 3H, —CH3), δ 4.08-4.16 (m, 2H, —O—(CH2), δ 5.75 (d, J=48 Hz, 1H, —CHF), δ 7.18-7.27 (m, 2H), δ 7.27-7.44 (m, 2H); 13C-NMR (75 MHz, CDCl3): 10.40, 19.41, 22.08, 67.47, 126.57, 131.10. 19F-NMR (282 MHz, CDCl3): δ −178.5 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C12H15FO2 requires m/z 210.1056, found 210.1062. The fluorinated product (R)-propyl 2-fluoro-2-(2-methylphenyl)acetate was obtained in 87% ee (54 mg, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 0.83 (t, J=7.5 Hz, 3H, —CH3), δ 1.52-1.68 (m, 2H, CH2), δ 2.43 (s, 3H, —CH3), δ 4.12 (m, 2H, —O—(CH2), δ 5.96 (d, J=48 Hz, 1H, —CHF), δ 7.16-7.30 (m, 4H); 13C-NMR (75 MHz, CDCl3): 10.3, 19.3, 22.0, 29.9, 67.4, 87.4 (d, J=183 Hz), δ 126.5, δ 127.5, δ 129.8, δ 131.0. 19F-NMR (282 MHz, CDCl3): δ −180.1 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C12H15FO2 requires m/z 210.1056, found 210.1070.
Examples 1, 2, 3, and 4 illustrate the application of the systems and methods of the disclosure for stereoselective fluorination of a chemical building block, exemplified by 2-aryl acetic acid derivatives (Schemes 1-4).
Example 5
Regioselective fluorination of 3-methyl-2-pentylcyclopent-2-enone (dihydrojasmone) in position 4




Dihydrojasmone was subjected to selective fluorination of the target site C (position 4) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 270 μL dihydrojasmone were dissolved in 1.2 mL ethanol and added to 150 mL potassium phosphate buffer pH 8.0. Var2 was added to the mixture at a final concentration of 2 μM. The mixture was split in 4.8 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 600 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 600 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 36 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the activated product (4-hydroxy-3-methyl-2-pentylcyclopent-2-enone, 222 mg). 210 mg (1.15 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 215 μL DAST (1.5 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, 4-fluoro-3-methyl-2-pentylcyclopent-2-enone (193 mg, 92% yield, yellow oil). 1H-NMR (300 MHz, CDCl3): δ 0.88 (t, J=6.6 Hz, 3H, CH3), δ 1.25-1.40 (m, 6H, CH2), δ 2.10 (d, J=2.1 Hz, 2H, CH3), δ 2.20 (t, J=7.1 Hz, 2H), δ 2.44-2.60 (m, 1H), δ 2.70-2.82 (m, 1H), δ 5.47 (dd, J=54.2 Hz, J=5.8, 1H); 13C-NMR (75 MHz, CDCl3): δ 13.7, 14.2, 22.6, 23.1, 27.9, 29.9, 31.9, 41.4 (d, J=19.6 Hz), δ 91.2 (d, J=174 Hz): 19F-NMR (282 MHz, CDCl3): δ −179.08 (ddd, J=51.88 Hz, J=21.43 Hz, J=9.3 Hz). HRMS (EI+): exact mass calculated for C11H17FO requires m/z 184.1263, found 184.1255.
Example 6
Regioselective fluorination of 3-methyl-2-pentylcyclopent-2-enone (dihydroiasmone) in position 11




Dihydrojasmone was subjected to selective fluorination of the target site C (position 11) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 240 μL dihydrojasmone were dissolved in 1.1 mL ethanol and added to 130 mL potassium phosphate buffer pH 8.0. Var2 was added to the mixture at a final concentration of 2 μM. The mixture was split in 4.8 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 600 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 600 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 36 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the activated product (11-hydroxy-3-methyl-2-pentylcyclopent-2-enone, 35 mg). 30 mg (0.16 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 35 μL DAST (0.25 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, 11-fluoro-3-methyl-2-pentylcyclopent-2-enone (27 mg, 89% yield, yellow oil). 1H-NMR (300 MHz, CDCl3): δ 0.88 (t, J=6.6 Hz, 3H, CH3), δ 1.25-1.40 (m, 6H, CH2), δ 2.10 (d, J=2.1 Hz, 2H, CH3), δ 2.17 (t, J=7.6 Hz, 2H), δ 2.38-2.44 (m, 1H), δ 2.59-2.64 (m, 1H), δ 5.20 (d, J=48.8 Hz, 1H); 13C-NMR (75 MHz, CDCl3): δ 14.3, 22.8, 23.3, 28.4, 31.8, 29.9, 31.9, 34.8, 60.6, 80.3 (d, J=164 Hz), 87.9; 19F-NMR (282 MHz, CDCl3): δ −48.80 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C11H17FO requires m/z 184.1263, found 184.1263.
Examples 5 and 6 illustrate the application of the systems and methods of the disclosure for regioselective fluorination of an organic molecule at weakly reactive sites, exemplified by dihydrojasmone (Schemes 5 and 6).
Example 7
Regioselective difluorination of 3-methyl-2-pentylcyclopent-2-enone (dihydrojasmone) in position 4




Dihydrojasmone was subjected to selective difluorination of the target site C (position 4) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 4-fluoro-3-methyl-2-pentylcyclopent-2-enone was obtained according to the experimental described in Example 5. 180 mg 4-fluoro-3-methyl-2-pentylcyclopent-2-enone were dissolved in 900 μL ethanol and added to 120 mL potassium phosphate buffer pH 8.0. Var2 was added to the mixture at a final concentration of 2 μM. The mixture was split in 4.8 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 600 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 600 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 36 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the activated product (4-hydroxy-4-fluoro-3-methyl-2-pentylcyclopent-2-enone, 135 mg). 100 mg (0.54 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 100 μL DAST (0.7 mmol). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, 4,4-difluoro-3-methyl-2-pentylcyclopent-2-enone (85 mg, 85% yield, yellow oil). MS (EI+): m/z 202. Mw for C11H16F2O: 202.24.
Example 7 illustrates the application of the systems and methods of the disclosure for regioselective polyfluorination of an organic molecule at a weakly reactive site, exemplified by dihydrojasmone (Scheme 7).
Example 8
Regioselective Fluorination of Menthofuran




Menthofuran was subjected to selective fluorination of the target site C (position 6) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 112 mg menthofuran were dissolved in 0.6 mL ethanol and added to 125 mL potassium phosphate buffer pH 8.0. Var3-11 was added to the mixture at a final concentration of 0.7 μM. The mixture was split in 4 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 500 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 24 hours, the reaction nearly reached completion (95% substrate conversion). The reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. The resulting oil (53 mg) was subjected directly to deoxo-fluorination without purification of the activated product. 53 mg of the activation mixture ( ̃0.32 mmol) were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 150 μL DAST (1 mmol). The reaction was stirred in dry ice for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-10% ethyl acetate/hexane) afforded the fluorinated product, 6-fluoro-menthofuran-2-ol (12 mg, 22% yield, yellow oil). 1H-NMR (300 MHz, CDCl3): δ 1.13 (d, J: 75.6 Hz, 3H, —CH3), δ 1.2-1.3 (m, 2H, —CH2—), δ 1.84 (s, 3H, —CH3), 1.95-2.4 (dm, 2H, —CH2—), 2.4-2.6 (dm, 2H, —CH2—); 13C-NMR (75 MHz, CDCl3): δ 22.7 (d, J=209 Hz), 43.08, 45.36, 91.7 (d, J=215 Hz), 114.9, 127.1. 19F-NMR (282 MHz, CDCl3): δ −114.4 (m). HRMS (EI+): exact mass calculated for C10H13FO2 requires m/z 184.0909, found 184.0899.
Example 8 illustrates the application of the systems and methods of the disclosure for regioselective fluorination of an organic molecule at a weakly reactive site, exemplified by menthofuran (Scheme 8).
Example 9
Regioselective Fluorination of (−)-Guaiol




(−)-Guaiol was subjected to selective fluorination of the target site C (position 6) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 250 mg guaiol were dissolved in 2 mL ethanol and added to 210 mL potassium phosphate buffer pH 8.0. Var3-2 was added to the mixture at a final concentration of 3 μM. The mixture was split in 4.8 mL aliquots into 15 mL scintillation vials equipped with a stir bar. 600 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 600 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 48 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the activated product (6-hydroxy-guaiol, 30 mg, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.01 (d, J=6.9 Hz, 3H, —CH3), δ 1.22 (s, 3H, CH3), δ 1.28 (s, 3H, CH3), δ 1.25 (d, J=9 Hz, 3H, CH3), δ 1.42-1.45 (m, 2H,), δ 1.685 (bs, 2H), δ 1.74-1.183 (m, 2H), δ 1.95-2.03 (m, 2H), δ 2.15-2.24 (m, 2H), δ 2.54-2.72 (m, 3H), δ 2.97-3.06 (m, 1H), δ 3.67 (d, J=9 Hz, 1H); 13C-NMR (75 MHz, CDCl3): δ 11.20, 19.41, 26.08, 28.32, 31.07, 34.13, 35.33, 38.08, 42.42, 48.01, 72.12, 73.15, 178.94. 15 mg of the activated product ( ̃0.06 mmol) were dissolved in 1 mL dry dichloromethane (CH2Cl2) and a catalytic amount (3 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 18 μL DAST (0.12 mmol). The reaction was stirred in dry ice for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, 6-fluoro-guaiol (7 mg, 45% yield, pale yellow oil). MS (EI+): m/z 242. Mw for C15H25FO: 242.35.
Example 9 illustrates the application of the systems and methods of the disclosure for regioselective fluorination of an organic molecule at a weakly reactive site, exemplified by (−)-guaiol (Scheme 9).
Example 10
Regioselective fluorination of ibuprofen methyl ester (methyl 2-(4′-(2′′-methylpropyl)phenyl)propanoate) in 1′′ position




Ibuprofen methyl ester was subjected to selective fluorination of the target site C (position 1′′) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 150 mg ibuprofen methyl ester were dissolved in 1.4 mL ethanol and added to 150 mL potassium phosphate buffer pH 8.0. P450BM3 was added to the mixture at a final concentration of 10 μM. The mixture was split in 4 mL-aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 500 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 48 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5-40% ethyl acetate/hexane) afforded the activated product (methyl 2-(4′-(1′′-hydroxy-2′′-methylpropyl)phenyl)propanoate, 73 mg). 15 mg (0.06 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 11 μL DAST (0.72 mmol). The reaction was stirred in dry ice for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, methyl 2-(4′-(1′′-fluoro-2′′-methylpropyl)phenyl)propanoate (10 mg, 65% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 0.84 (d, J=6.9 Hz, 3H, CH3), δ 1.01 (d, J=6.9 Hz, 3H, —CH3), δ 1.49 (d, J=8.7 Hz, 3H, CH3), δ 2.05-2.08 (m, 1H), δ 3.66 (s, 3H, OCH3), δ 3.73 (q, J=7.5 Hz, 1H), δ 5.07 (dd, J=40.0, J=6.9 Hz, 1H, CHF), δ 7.25 (m, 4H); 13C-NMR (75 MHz, CDCl3): δ 15.5, 17.82 (d), 18.54 (d), 34.48 (d, J: 85.7 Hz), 45.37, 52.31, 99.3 (d, J=174 Hz), 175.6; 19F-NMR (282 MHz, CDCl3): δ −179.8 (m). HRMS (EI+): exact mass calculated for C14H19FO2 requires m/z 238.1369, found 238.1367.
Example 11
Regioselective fluorination of ibuprofen methyl ester (methyl 2-(4′-(2′′-methylpropyl)phenyl)propanoate) in 2′′ position




Ibuprofen methyl ester was subjected to selective fluorination of the target site C (position 2′′) according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 150 mg ibuprofen methyl ester were dissolved in 1.4 mL ethanol and added to 150 mL potassium phosphate buffer pH 8.0. Var3-4 was added to the mixture at a final concentration of 3 μM. The mixture was split in 4 mL-aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 500 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 48 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (5-40% ethyl acetate/hexane) afforded the activated product (methyl 2-(4′-(2′′-hydroxy-2′′-methylpropyl)phenyl)propanoate, 81 mg). 15 mg (0.06 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 11 μL DAST (0.72 mmol). The reaction was stirred in dry ice for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (0-30% ethyl acetate/hexane) afforded the fluorinated product, methyl 2-(4′-(1′′-fluoro-2′′-methylpropyl)phenyl)propanoate (7 mg, 45% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.28 (s, 3H, CH3), δ 1.35 (s, 3H, CH3), δ 1.48 (d, J=6.9 Hz, 3H, CH3), δ 2.87 (d, J: 20.4 Hz, 2H), δ 3.65 (s, 3H, OCH3), δ 3.70 (q, J=7.15 Hz, 1H), δ 7.17 (m, 4H); 13C-NMR (75 MHz, CDCl3): δ 18.80, 26.83 (d, J: 24.2 Hz), 45.24, 47.37 (d, J: 22.8 Hz), 52.25, 129.12 (d, J: 258.8 Hz), ca. 130, ca. 132, ca. 139, ca. 173; 19F-NMR (282 MHz, CDCl3): δ −137.7 (m). HRMS (EI+): exact mass calculated for C14H19FO2 requires m/z 238.1369, found 238.1370.
Example 10 and 11 illustrate the application of the systems and methods of the disclosure for regioselective fluorination of an organic molecule at weakly and non-reactive site, such as positions 1′′ and 2′′ of ibuprofen methyl ester (Schemes 10 and 11).
Example 12
Regioselective fluorination of dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline




Dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline was subjected to selective fluorination of the target site C atom carrying a methoxy group, according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 100 mg dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline ibuprofen methyl ester were dissolved in 1.2 mL ethanol and added to 160 mL potassium phosphate buffer pH 8.0. Var3-5 was added to the mixture at a final concentration of 3 μM. The mixture was split in 4 mL-aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 500 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 48 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (20% ethyl acetate/hexane) afforded the activated product (dihydro-4-hydroxymethyl-2-methyl-5-phenyl-2-oxazoline, 64 mg). 30 mg (0.16 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 22 μL DAST (0.32 mmol). The reaction was stirred in dry ice for 2 hours and then at −20° C. for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (20% ethyl acetate/hexane) afforded the fluorinated product, dihydro-4-fluoromethyl-2-methyl-5-phenyl-2-oxazoline (12 mg, 40% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.59 (s, 3H, CH3), δ 2.09 (dm, 1H, CH), δ 4.15-4.35 (m, 1H, CH), δ 5.66 (tm, 1H, CH), δ 7.37 (m, 5H, Ph); 19F-NMR (282 MHz, CDCl3): δ −114.14 (m). HRMS (EI+): exact mass calculated for C11H12FNO requires m/z 193.0903, found 193.0917.
In another aspect, example 12 illustrates the application of the systems and methods of the disclosure for selective fluorination of an organic molecule at a site carrying a protected hydroxyl group, such as in dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline (Scheme 12).
Example 13
Regioselective fluorination of 1,2,3,4,6-pentamethyl-α-D-mannopyranoside




1,2,3,4,6-pentamethyl-α-D-mannopyranoside was subjected to regioselective fluorination of the target site C in position 6, according to the systems and methods herein disclosed and, more specifically, according to the general procedure described above.
Experimental description: 50 mg of 1,2,3,4,6-pentamethyl-α-D-mannopyranoside were dissolved in 0.5 mL ethanol and added to 100 mL potassium phosphate buffer pH 8.0. Var3-6 was added to the mixture at a final concentration of 4 μM. The mixture was split in 4 mL-aliquots into 15 mL scintillation vials equipped with a stir bar. 500 μL 10 mM NADPH in KPi buffer were added to each vial and stirred for 2 minutes. 500 μL cofactor regeneration solution containing 500 mM glucose-6-phosphate and 10 units/mL glucose-6-phosphate dehydrogenase were then added to each vial. The resulting mixtures were stirred at room temperature. After 36 hours, the reaction mixtures were joined together and extracted with chloroform (3×50 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (10% ethyl acetate/hexane) afforded the activated product (1,2,3,4-tetramethyl-α-D-mannopyranoside, 30 mg). 15 mg (0.1 mmol) of activated product were dissolved in 2 mL dry dichloromethane (CH2Cl2) and a catalytic amount (4 drops) of ethanol was added to the solution. The solution was cooled to −78° C. (dry ice) and added with 85 μL DAST (0.6 mmol). The reaction was stirred in dry ice for 2 hours and then at room temperature for 16 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate (NaHCO3) and extracted with dichloromethane (3×15 mL). The organic phase was then dried over magnesium sulfate (MgSO4) and evaporated in vacuo. Purification of the resulting oil by silica gel chromatography (10% ethyl acetate/hexane) afforded the fluorinated product, 6-deoxy-6-fluoro-1,2,3,4-tetramethyl-α-D-mannopyranoside (4.5 mg, 30% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 3.35 (s, 3H, OCH3), 3.48 (s, 6H, OCH3), 3.53 (s, 3H, OCH3), 4.0-4.2 (m, 4H), 4.6 (dm, J: 47.5 Hz, 2H, CH2F); 13C-NMR (75 MHz, CDCl3): δ 55.24, 57.94, 59.24, 61.02, 71.12 (d, J: 18.4 Hz), 73.2, 75.5, 82.49 (d, J: 192 Hz), 98.26. 19F-NMR (282 MHz, CDCl3): δ −235.2 (m). ESI-MS: m/z calculated for Mw C10H19FO5: 238.2533, found 238.28.
In another aspect, example 13 illustrates the application of the systems and methods of the disclosure for regioselective fluorination of an organic molecule at a defined site carrying a protected hydroxyl group in the presence of other identical functional groups, such as in 1,2,3,4,6-pentamethyl-α-D-mannopyranoside (Scheme 13).
Example 14
Chemo-Enzymatic Fluorination of Unactivated Organic Compounds
To further test the validity of the methods and compositions of the disclosure, various classes of small molecules were targeted, including marketed pharmaceuticals (FIG. 9). For the enzymatic step, variants of the bacterial long-chain fatty acid hydroxylase P450BM3 from Bacillus megaterium were used. A panel of 96 P450s derived from a catalytically-promiscuous P450BM3 variant identified in the early stages of the directed evolution of a proficient alkane monoxygenase were used. These variants were found to exhibit good activity and various degrees of selectivity on alkanes and non-alkane substrates.
Enzyme Library Screening. Expression of P450BM3 variants in 96-well plates and preparation of cell lysates were performed as described.1 Screening of enzyme activity on cyclopentenone derivatives (1, 2, and 3) and ibuprofen derivatives (12 and 16) was carried out in 96-well microtiter plates mixing 700 μL KPi (100 mM, pH 8.0), 200 μL cell lysate, 10 μL 200 mM substrate in EtOH, and 100 μL of a cofactor regeneration solution containing 4 mM NADPH, 100 mM glucose-6-phosphate, and 10 U/mL glucose-6-phosphate dehydrogenase. After incubation for 16 hours at room temperature in orbital shaker, reactions were extracted with chloroform and analyzed by gas chromatography on a Shimadzu GC-17A GC using an Agilent HP5 column (30 m×0.32 mm×0.1 μm film), 1 μL injection, FID detector, and the following separation method: 300° C. inlet, 300° C. detector, 70° C. oven, 10° C./min gradient to 210° C., 50° C./min gradient to 260° C., and 260° C. for 2 min. Screening of enzyme activity on dihydro-4-methoxymethyl-2-methyl-5-phenyl-2-oxazoline (24) was carried out on microtiter plates by mixing 100 μL KPi (100 mM, pH 8.0), 50 μL cell lysate, 2 μL 200 mM 24 in DMSO, and 50 μL KPi 4 mM NADPH. Reaction mixtures were incubated for 30 minutes at room temperature and then added with 50 μL 150 mM Purpald in 2 M NaOH. Absorbance at 550 nm was recorded after 30 minutes. The most active variants identified in the colorimetric screen were re-tested for selectivity towards demethylation. 1-mL scale reactions were carried out using 1 mM 24, 0.5 μM P450, 400 μM NADPH, 10 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase. After stirring for 16 hours at room temperature, the reaction mixtures were extracted with chloroform and analyzed by GC-MS on a Hewlett-Packard 5970B MSD with 5890 GC using a DB-5 capillary column, 1 μL injection, 250° C. inlet, and the following separation program: 80° C. oven, 10° C./min gradient to 250° C., 250° C. for 3 min. Enzyme activity on 27a, 28a, and 29a was screened by mixing 50 μL cell lysate, 100 μL 100 mM KPi buffer (pH 8.0) containing 1 mM substrate (1% DMSO), and 50 μL 4 mM NADPH in microtiter plates. After 30 min, 50 μL 150 mM Purpald (Sigma) in 2 M NaOH were added to each well. Absorbance at 550 nm was measured after 60 minutes. The most active variants were re-tested at 1-mL scale using 1 mM substrate, 0.5 μM purified P450, and the cofactor regeneration system described above. After overnight incubation at 4° C., reactions were analyzed by HPLC-MS using an Agilent 1100 Series LC/MSD device and Kromasil 100, 5 μm-C18 column.
Membrane permeability assay. The membrane permeability assays were carried out adapting a described procedure and using 96-well MultiScreen-IP plates from Millipore. Filter plates were rinsed with 70% EtOH and double-distilled water prior to use. BBB-mimic membranes were prepared by coating filter wells with 5 μL porcine brain lipids (PBL, Avanti Polar Lipids) in dodecane (20 mg/mL). Control wells (PBL-free) were primed in the same way but not coated with PBL. Working solutions contained 0.5 mg/mL fluorinated compound in PBS pH 7.4 (5% EtOH). Assembled plates contained 150 μL working solution in the PBL-coated (or PBL-free) well, 300 μL PBS (5% EtOH) in the acceptor well, and were incubated at room temperature and 100% humidity. The change in organic compound concentration in the donor and acceptor wells was monitored over time by HPLC. Membrane permeability values (Pe) were calculated. All measurements were performed in triplicates. The assay was validated by comparing the experimental Pe value for caffeine with that reported in the art.
Synthetic Procedures:




Conversion of 1 to 5. 130 mg (1.18 mmol) 1 were dissolved in 1.5 mL ethanol and added to 200 mL potassium phosphate buffer pH 8.0. The solution was added with var-H3 (final conc.: 2.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 20% ethyl acetate in hexane) to afford 1a (130 mg, 88%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.71 (m, 3H, CH3), δ 2.08 (m, 3H, CH3), δ 2.27 (dd, J=18.3, 1.8 Hz, 1H), δ 2.77 (dd, J=18.3, 6.3 Hz, 1H), δ 4.73 (m, 1H). 13C-NMR (75 MHz, CDCl3): δ 8.18, δ 13.91, δ 44.44, δ 71.85, δ 138.37, δ 168.50, δ 205.96. HRMS (EI+): exact mass calculated for C7H10O2 requires m/z 126.0681, found 126.0687. Under argon, 50 mg (0.22 mmol) 1a and a catalytic amount (3 drops) of ethanol were dissolved in 3 mL dry dichloromethane. The solution was cooled to −78° C. and added with 38 μL DAST (0.26 mmol, 1.2 eq) in two aliquots. The reaction was stirred in dry ice for 12 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 10% to 50% dichloromethane in pentane) to afford 5 (25 mg, 90% yield, colorless oil). 1H-NMR (300 MHz, CD2Cl2): δ 1.68 (dm, J=4.2 Hz, 3H, CH3), δ 2.04 (m, 3H, CH3), δ 2.41 (tm, J=20.71H), δ 2.63-2.76 (m, 1H), δ 5.48 (dm, J=30.6 Hz, 1H). 13C-NMR (75 MHz, CD2Cl2): δ 7.83, δ 13.40, δ 41.21 (d, J=19.9 Hz), δ 91.59 (d, J=173.7 Hz), δ 128.03. 19F-NMR (282 MHz, CD2Cl2): δ −179.4 (dd, J=51.6, 24.2 Hz). HRMS (EI+): exact mass calculated for C7H9FO requires m/z 128.0637, found 128.0642. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 60° C. oven, 0.5° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 22% was estimated based on peak areas.




Conversion of 1 to 6. 130 mg (1.18 mmol) 1 were dissolved in 1.5 mL ethanol and added to 200 mL potassium phosphate buffer pH 8.0. The solution was added with var-G6 (final conc.: 2.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 20% ethyl acetate in hexane) to afford 1c (66 mg, 45%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.71 (m, 3H, CH3), δ 2.4 (dm, J=4.5 Hz, 2H), δ 2.65 (m, 2H), δ 4.58 (s, 2H). 13C-NMR (75 MHz, CDCl3): δ 8.23, δ 27.18, δ 34.10, δ 61.05, δ 136.15, δ 170.05, δ 210.61. HRMS (EI+): exact mass calculated for C7H10O2 requires m/z 126.0681, found 126.0685. Under argon, 50 mg (0.22 mmol) 1c and a catalytic amount (3 drops) of ethanol were dissolved in 3 mL dry dichloromethane. The solution was cooled to −78° C. and added with 38 μL DAST (0.26 mmol, 1.2 eq) in two aliquots. The reaction was stirred in dry ice for 12 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 10% to 50% dichloromethane in pentane) to afford 6 (24 mg, 85% yield, colorless oil). 1H-NMR (300 MHz, CD2Cl2): δ 1.67 (m, 3H, CH3), δ 2.36 (m, 2H), δ 2.56 (m, 2H), δ 5.23 (d, J=34.2 Hz, 2H). 13C-NMR (75 MHz, CD2Cl2): δ 7.95, δ 26.41 (d, J=4.3 Hz), δ 80.83 (d, J=164.0 Hz). 19F-NMR (282 MHz, CD2Cl2): δ −226.9 (t, J=42.3 Hz). HRMS (EI+): exact mass calculated for C7H9FO requires m/z 128.0637, found 128.0643.




Conversion of 2 to 7. 270 mg (1.62 mmol) 2 were dissolved in 2.5 mL ethanol and added to 250 mL potassium phosphate buffer pH 8.0. The solution was added with var-H3 (final conc.: 3 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 30% ethyl acetate in hexane) to afford 2a (250 mg, 85%, pale yellow oil). 1H-NMR (300 MHz, CDCl3): δ 0.87 (t, J=6.9 Hz, 3H, CH3), δ 1.25-1.39 (m, 6H), δ 2.08 (s, 3H, CH3), δ 2.17 (t, J=8.1 Hz, 2H), δ 2.27 (dd, J=18.3, 2.1 Hz, 1H), δ 2.77 (dd, J=18.3, 6.3 Hz, 1H), δ 4.72 (dm, J=5.1 Hz, 1H). 13C-NMR (75 MHz, CDCl3): δ 13.83, δ 14.22, δ 22.68, δ 23.16, δ 28.06, δ 28.06, δ 31.99, δ 44.60, δ 71.87, δ 142.73, δ 168.32, δ 205.59. HRMS (EI+): exact mass calculated for C11H18O2 requires m/z 182.1307, found 182.1311. Under argon, 200 mg (1.09 mmol) 2a and a catalytic amount (4 drops) of ethanol were dissolved in 5 mL dry dichloromethane. The solution was cooled to −78° C. and added with 200 μL DAST (1.4 mmol, 1.3 eq) in four aliquots. The reaction was stirred in dry ice for 12 hours. The reaction mixture was added with 7 mL saturated sodium bicarbonate and extracted with dichloromethane (3×20 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 30% ethyl acetate in hexane) to afford 7 (193 mg, 92% yield, pale yellow oil). 1H-NMR (300 MHz, CDCl3): δ 0.88 (t, J=6.6 Hz, 3H, CH3), δ 1.25-1.40 (m, 6H), δ 2.10 (d, J=2.1 Hz, 2H, CH3), δ 2.20 (t, J=7.1 Hz, 2H), δ 2.51 (tm, J=22.5 Hz, 1H), δ 2.70-2.82 (m, 1H), δ 5.47 (dd, J=53.7, 6.0, 1H). 13C-NMR (75 MHz, CDCl3): δ 13.75, δ 14.21, δ 22.66, δ 23.13, δ 27.90, δ 29.94, δ 31.96, δ 41.46 (d, J=19.6 Hz), δ 91.42 (d, J=174.9 Hz), δ 167.62. 19F-NMR (282 MHz, CDCl3): δ −179.1 (ddd, J=51.8, 21.4, 9.3 Hz). HRMS (EI+): exact mass calculated for C11H17FO requires m/z 184.1263, found 184.1255. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 70° C. oven, 1° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 78% was estimated based on peak areas.




Conversion of 2 to 8. 100 mg (0.60 mmol) 2 were dissolved in 1 mL ethanol and added to 150 mL potassium phosphate buffer pH 8.0. The solution was added with var-G4 (final conc.: 2 μM) and a cofactor regeneration system (final conc.: 500 □M NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (3×50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 30% ethyl acetate in hexane) to afford 2c (46 mg, 42%, pale yellow oil). GC-MS (EI+): m/z 182. Under argon, 46 mg (0.25 mmol) 2c and a catalytic amount (2 drops) of ethanol were dissolved in 2.5 mL dry dichloromethane. The solution was cooled to −78° C. and added with 54 μL DAST (0.38 mmol, 1.5 eq) in two aliquots. The reaction was stirred in dry ice for 12 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 30% ethyl acetate in hexane) to afford 8 (62 mg, 89% yield, pale yellow oil). 1H-NMR (300 MHz, CDCl3): δ 0.87 (t, J=6.5 Hz, 3H, CH3), δ 1.23-1.38 (m, 6H), δ 2.17 (t, J=7.5 Hz, 2H), δ 2.41 (m, 1H), δ 2.60 (m, 1H), δ 5.26 (d, J=47.1, 1H). 13C-NMR (75 MHz, CDCl3): δ 14.32, δ 22.86, δ 23.33, δ 28.46, δ 31.80, δ 31.93, δ 34.87, δ 80.37 (d, J=164.8 Hz). 19F-NMR (282 MHz, CDCl3): δ −227.9 (t, J=48.2 Hz). HRMS (EI+): exact mass calculated for C11H17FO requires m/z 184.1263, found 184.1263.




Conversion of 3 to 9. 230 mg (1.53 mmol) 3 were dissolved in 2 mL ethanol and added to 300 mL potassium phosphate buffer pH 8.0. The solution was added with var-D10 (final conc.: 2.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×80 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 0 to 20% ethyl acetate in hexane) to afford 3a (175 mg, 69%, colorless oil). GC-MS (EI+): m/z 166. Under argon, 100 mg (0.60 mmol) 3a and a catalytic amount (3 drops) of ethanol were dissolved in 5 mL dry dichloromethane. The solution was cooled to −78° C. and added with 103 μL DAST (0.72 mmol, 1.2 eq) in two aliquots. The reaction was stirred in dry ice for 3 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×20 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (10% ethyl acetate:90% hexane) to afford 9 (89 mg, 88% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.84 (d, J=6.6 Hz), δ 1.45-1.75 (m, 2H), δ 1.80-1.86 (m, 2H), δ 2.03 (m, 1H), δ 2.06 (dd, J=18.4, 1.8 Hz, 1H), δ 2.13-2.21 (m, 1H), δ 2.65 (dd, J=18.4, 6.9 Hz, 1H), δ 2.80 (m, 1H), δ 5.28 (dbr, J=50.1 Hz, 1H). 13C-NMR (75 MHz, CDCl3): δ 14.47, δ 18.78, δ 25.89, δ 28.67 (d, J=21.9 Hz), δ 36.29, δ 43.89, δ 79.93 (d, J=164.2 Hz), δ 135.71 (d, J=17.9 Hz), δ 183.76 (d, J=5.6 Hz), δ 206.26. 19F-NMR (282 MHz, CDCl3): δ −171.4 (m). HRMS (EI+): exact mass calculated for C10H13FO requires m/z 168.0950, found 168.0908. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 70° C. oven, 1° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. A diasteromeric ratio (dr) of 1:8.5, an enantiomeric excess of 5% for the major product, and an enantiomeric excess of 71% for the minor product were estimated based on peak areas.




Conversion of 3 to 10. 200 mg (1.33 mmol) 3 were dissolved in 1.5 mL ethanol and added to 200 mL potassium phosphate buffer pH 8.0. The solution was added with var-G4 (final conc.: 3.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 36 hours, the reaction mixture was extracted with dichloromethane (4×80 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (30% ethyl acetate in hexane) to afford 3b (137 mg, 62%, colorless oil). GC-MS (EI+): m/z 166. Under argon, 50 mg (0.30 mmol) 3b and a catalytic amount (3 drops) of ethanol were dissolved in 3 mL dry dichloromethane. The solution was cooled to −78° C. and added with 51 μL DAST (0.36 mmol, 1.2 eq) in two aliquots. The reaction was stirred in dry ice for 5 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (10% ethyl acetate:90% hexane) to afford 10 (46 mg, 92% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.27 (d, J=6.9 Hz, 3H), δ 1.71-1.79 (m, 3H), δ 2.02 (dbr, J=18.6, 1H), δ 2.03 (br, 1H), δ 2.10-2.13 (m, 1H), δ 2.23-2.35 (m, 1H), δ 2.67 (dd, J=18.6, 6.3 Hz, 1H), δ 2.92 (br, 1H), δ 5.20 (dbr, J=46.2 Hz, 1H). 13C-NMR (75 MHz, CDCl3): δ 17.55, δ 19.96, δ 20.18 (d, J=2.8 Hz), δ 29.80 (d, J=21.3 Hz), δ 36.12, δ 44.32, δ 86.48 (d, J=166.5 Hz), δ 142.79, δ 168.15 (d, J=13.9 Hz), δ 208.86. 19F-NMR (282 MHz, CDCl3): δ −174.8 (m). HRMS (EI+): exact mass calculated for C10H13FO requires m/z 168.0950, found 168.0913. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 70° C. oven, 1° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. A diasteromeric ratio (dr) of 4:96 and an enantiomeric excess of 57% for minor product were estimated based on peak areas.




Conversion of 3 to 11. 150 mg (1.0 mmol) 3 were dissolved in 1.5 mL ethanol and added to 200 mL potassium phosphate buffer pH 8.0. The solution was added with var-G5 (final conc.: 3.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 36 hours, the reaction mixture was extracted with dichloromethane (4×80 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (30% ethyl acetate in hexane) to afford 3c (53 mg, 32%, colorless oil). GC-MS (EI+): m/z 166. Under argon, 53 mg (0.32 mmol) 3c and a catalytic amount (3 drops) of ethanol were dissolved in 3 mL dry dichloromethane. The solution was cooled to −78° C. and added with 51 μL DAST (0.36 mmol, 1.2 eq) in two aliquots. The reaction was stirred in dry ice for 5 hours. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (10% ethyl acetate:90% hexane) to afford 11 (48 mg, 90% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.18 (d, J=7.2 Hz, 3H), δ 1.45-1.64 (m, 1H), δ 1.79-1.86 (m, 2H), δ 2.00 (dd, J=18.6, 3.9 Hz, 1H), δ 2.11-2.24 (m, 1H), δ 2.33-2.37 (m, 1H), δ 2.67 (dd, J=18.6, 6.3 Hz, 1H), δ 2.86 (m, 1H), δ 5.29 (dm, J=50.1 Hz, 1H). 13C-NMR (75 MHz, CDCl3): δ 17.36, δ 18.62, δ 26.58, δ 28.74 (d, J=20.7 Hz), δ 37.08, δ 43.67, δ 79.82 (d, J=164.2 Hz), δ 135.65, δ 208.57. 19F-NMR (282 MHz, CDCl3): δ −170.5 (m). HRMS (EI+): exact mass calculated for C10H13FO requires m/z 168.0950, found 168.0923. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film). Complete separation of all four diasteromers was not possible.




Conversion of 12 to 15. 1.4 mL DMSO containing 150 mg (0.68 mmol) 12 were dissolved in 150 mL 100 mM KPi pH 8.0. The solution was added with var-B4 (final conc.: 4.5 μM) and a cofactor regeneration system (final conc.: 1 mM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient from 5 to 40% ethyl acetate in hexane) to afford 13 (114 mg, 72%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 0.78 (d, J=6.9 Hz, 3H, CH3), δ 0.98 (d, J=6.9 Hz, 3H, CH3), δ 1.48 (d, J=6.9 Hz, 3H, CH3), δ 1.85-2.00 (m, 1H, CH), δ 3.65 (s, 3H, OCH3), δ 3.72 (q, J=6.9 Hz, 1H, CH), δ 4.33 (d, J=6.9 Hz, 1H, CH), δ 7.25 (m, 4H, CH). 13C-NMR (75 MHz, CDCl3): δ 18.43, δ 18.81, δ 19.26, δ 35.42, δ 45.32, δ 52.27, δ 79.99, δ 127.09, δ 127.52, δ 139.86, δ 142.77, δ 175.27. HRMS (EI+): exact mass calculated for C14H20O3 requires m/z 236.1412, found 236.1415. Under argon, 40 mg (0.17 mmol) 13 and a catalytic amount of ethanol were dissolved in 4 mL dry dichloromethane. The solution was cooled to −78° C. After 5 minutes, 29 μL DAST (0.24 mmol, 1.4 eq) were added to the mixture. The reaction was stirred in dry ice overnight. The reaction was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (5% ethyl acetate:95% hexane) to afford 15 (35 mg, 86% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 0.84 (d, J=6.3 Hz, 3H, CH3), δ 1.01 (d, J=5.7 Hz, 3H, CH3), δ 1.48 (d, J=6.9 Hz, 3H, CH3), δ 2.05-2.18 (m, 1H, CH), δ 3.66 (s, 3H, OCH3), δ 3.72 (q, J=7.5 Hz, 1H, CH), 5.07 (dd, J=40.0, J=6.9 Hz, 1H, CHF), δ 7.25 (m, 4H, CH). 13C-NMR (75 MHz, CDCl3): δ 17.78 (d, J=5.1 Hz), δ 18.58 (d, J=6.0 Hz), δ 18.81, δ 34.48 (d, J: 85.7 Hz), δ 45.37, δ 52.31, δ 99.3 (d, J=174 Hz), δ 126.65, δ 126.75, δ 127.56, δ 138.37, δ 140.6, δ 175.16. 19F-NMR (282 MHz, CDCl3): δ −179.8 (m). HRMS (EI+): exact mass calculated for C14H19FO2 requires m/z 238.1369, found 238.1367. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 70° C. oven, 1° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. A diasteromeric ratio (dr) of 1:3.2, an enantiomeric excess of 19% for the major product, and an enantiomeric excess of 44% for the minor product were estimated based on peak areas.




Conversion of 12 to 16. 2.5 mL DMSO containing 220 mg (1 mmol) 12 were dissolved in 250 mL 100 mM KPi pH 8.0. The solution was added with var-G4 (final conc.: 2 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×80 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient from 5 to 40% ethyl acetate in hexane) to afford 14 (208 mg, 88%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.21 (s, 6H, CH3), δ 1.48 (d, J=7.2 Hz, 3H, CH3), δ 2.73 (s, 2H, CH), δ 3.65 (s, 3H, OCH3), δ 3.70 (q, J=7.2 Hz, 1H, CH), δ 7.15 (d, J=8.1, 2H, CH), δ 7.23 (d, J=8.1, 2H, CH). 13C-NMR (75 MHz, CDCl3): δ 18.84, δ 29.41 (2C), δ 45.26, δ 49.51, δ 52.29, δ 71.00, δ 127.53 (2C), δ 130.98 (2C), δ 136.82, δ 138.92, δ 175.33. HRMS (EI+): exact mass calculated for C14H20O3 requires m/z 236.1412, found 236.1413. Under argon, 120 mg (0.51 mmol) 14 and a catalytic amount of ethanol (3 drops) were dissolved in 6 mL dry dichloromethane. The solution was cooled to −78° C. After 5 minutes, 87 μL DAST (0.61 mmol, 1.4 eq) were added to the mixture in three aliquots. The reaction was stirred in dry ice overnight. The reaction was then added with 8 mL saturated sodium bicarbonate and extracted with dichloromethane (3×20 mL). The organic phase was then dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (5% ethyl acetate: 95% hexane) to afford 16 (114 mg, 95% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.32 (d, J=23.6 Hz, 6H, CH3), δ 1.48 (d, J=6.9 Hz, 3H, CH3), δ 2.87 (d, J: 20.4 Hz, 2H), δ 3.65 (s, 3H, OCH3), δ 3.70 (q, J=7.1 Hz, 1H), δ 7.15 (d, J=8.1 Hz, 2H, CH), δ 7.21 (d, J=8.1 Hz, 2H, CH). 13C-NMR (75 MHz, CDCl3): δ 18.80, δ 26.83 (d, J: 24.2 Hz), δ 45.24, δ 47.37 (d, J: 22.8 Hz), δ 52.25, δ 98.37 (d, J=264 Hz), δ 129.12 (d, J: 258.8 Hz), δ 127.39, δ 130.85, δ 136.07, δ 138.92, δ 175.31. 19F-NMR (282 MHz, CDCl3): δ −137.7 (m). HRMS (EI+): exact mass calculated for C14H19FO2 requires m/z 238.1369, found 238.1370.




Conversion of 16 to 18. 2 mL DMSO containing 80 mg (0.33 mmol) 16 were dissolved in 200 mL 100 mM KPi pH 8.0. The solution was added with var-B2 (final conc.: 1 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 1 unit/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×80 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (5% ethyl acetate: 95% hexane) to afford 17 (78 mg, 93%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.31 (d, J=22.5 Hz, 3H, CH3), δ 1.32 (d, J=22.5 Hz, 3H, CH3), δ 1.48 (d, J=7.2 Hz, 3H, CH3), δ 3.65 (s, 3H, OCH3), δ 3.72 (q, J=7.2 Hz, 1H, CH), δ 4.69 (d, J=11.7 Hz, 1H, CH), δ 7.26 (d, J=8.1 Hz, 2H, CH), δ 7.34 (d, J=8.1 Hz, 2H, CH). 13C-NMR (75 MHz, CDCl3): δ 18.79, δ 21.55 (d, J=23.1 Hz), δ 24.08 (d, J=23.7 Hz), δ 45.31, δ 52.28, δ 79.29 (d, J=23.6 Hz), δ 98.14 (d, J=168 Hz), δ 127.42, δ 127.91, δ 138.28 (d, J=5.7 Hz), δ 140.55, δ 175.12. 19F-NMR (282 MHz, CDCl3): δ −146.8 (m). HRMS (EI+): exact mass calculated for C14H20FO3 requires m/z 255.1396, found 255.1394. Under argon, 30 mg (0.12 mmol) 17 and a catalytic amount of ethanol were dissolved in 3 mL dry dichloromethane. The solution was cooled to −78° C. After 5 minutes, 20 μL DAST (0.14 mmol, 1.2 eq) were added to the mixture. The reaction was stirred in dry ice overnight. The reaction was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (5% ethyl acetate:95% hexane) to afford 18 (30 mg, 98% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 1.32 (m, 6H, CH3), δ 1.51 (d, J=7.2 Hz, 3H, CH3), δ 3.66 (s, 3H, OCH3), δ 3.74 (q, J=7.2 Hz, 1H, CH), δ 5.29 (dd, J=45.0, 13.8 Hz, 1H, CHF), δ 7.31 (s, 4H, CH). 13C-NMR (75 MHz, CDCl3): δ 18.80, δ 23.01 (m), δ 45.38, δ 52.34, δ 96.80 (d, J=179 Hz), δ 96.52 (d, J=179 Hz), δ 127.50, δ 127.59, δ 134.8 (d, J=22.5), δ 141.28, δ 175.03. 19F-NMR (282 MHz, CDCl3): δ −150.7 (m), δ −187.8 (dd, J=45.6, 9.0 Hz). HRMS (EI+): exact mass calculated for C14H19F2O2 requires m/z 257.1353, found 257.1356. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 70° C. oven, 0.5° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. A diasteromeric ratio (dr) of 1:3.7, an enantiomeric excess of 9% for the major product, and an enantiomeric excess of 9% for the minor product were estimated based on peak areas.




Conversion of 19a to 19c. 90 mg (0.6 mmol) 19a were dissolved in 500 μL ethanol and added to 240 mL 100 mM KPi buffer pH 8.0. Var-A3 was added to the mixture at a final concentration of 2 μM. The mixture was added with a cofactor regeneration solution containing 0.5 mM NADPH, 30 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase (final concentrations). The reaction mixtures were stirred at room temperature and extracted with chloroform (3×100 mL) after 4 hours. The organic phase was then dried over anhydrous MgSO4 and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 19b (45 mg). 45 mg (0.27 mmol) of 19b and a catalytic amount (3 drops) of ethanol were dissolved in 2 mL dry dichloromethane under argon. The solution was cooled to −78° C. and added with 43 μL DAST (0.30 mmol, 1.2 eq). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were then dried over anhydrous MgSO4 and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 19c (34 mg, 75% yield, pale yellow oil) in 74% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 3.75 (s, 3H), δ 5.77 (d, J=48 Hz, 1H, —CHF), δ 7.37-7.46 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ 52.8, δ 89.5 (d, J=184.5 Hz), δ 126.8, 6 126.9, δ 129.0, δ 129.9, δ 134.4 (d, J=34.5 Hz), δ 169.0. 19F-NMR (282 MHz, CDCl3): δ −180.29 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C9H9FO2 requires m/z 168.0587, found 168.0594. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 100° C. oven, 2° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 74% was estimated based on peak areas.




Conversion of 20a to 20c. 100 mg (0.61 mmol) 20a were dissolved in 500 μL ethanol and added to 250 mL 100 mM KPi buffer pH 8.0. var-B4 was added to the mixture at a final concentration of 2 μM. The mixture was added with a cofactor regeneration solution containing 0.5 mM NADPH, 30 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase (final concentrations). The reaction mixtures were stirred at room temperature and extracted with chloroform (3×100 mL) after 3 hours. The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 20b (72 mg, 66%). 72 mg (0.4 mmol) of 20b and a catalytic amount (3 drops) of ethanol were dissolved in 2 mL dry dichloromethane under argon. The solution was cooled to −78° C. and added with 61 μL DAST (0.43 mmol, 1.2 eq). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous MgSO4 and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 20c (57 mg, 78% yield, pale yellow oil) in 72% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 1.24 (t, J=7.2 Hz, 3H, —CH3), δ 4.16-4.27 (m, 2H, —OCH2), δ 5.75 (d, J=48 Hz, 1H, —CHF), δ 7.37-7.46 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ 14.2, δ 62.0, δ 81.2, δ 89.6 (d, J=184.5 Hz) δ 126.8 δ 126.9 δ 128.9 δ 129.8 δ 134.4 (d, J=34.5 Hz), δ 169.0. 19F-NMR (282 MHz, CDCl3): δ −180.27 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C10H11FO2 requires m/z 182.0743, found 182.0750. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 100° C. oven, 2° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 72% was estimated based on peak areas.




Conversion of 21a to 21c. 95 mg (0.45 mmol) 21a were dissolved in 500 μL ethanol and added to 250 mL 100 mM KPi buffer pH 8.0. Var-C12 was added to the mixture at a final concentration of 1 μM. The mixture was added with a cofactor regeneration solution containing 0.5 mM NADPH, 30 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase (final concentrations). The reaction mixtures were stirred at room temperature and extracted with chloroform (3×100 mL) after 4 hours. The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 21b (71 mg, 75%). 70 mg (0.34 mmol) of 21b and a catalytic amount (3 drops) of ethanol were dissolved in 2 mL dry dichloromethane under argon. The solution was cooled to −78° C. and added with 64 μL DAST (0.45 mmol, 1.5 eq). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by flash chromatography (5% ethyl acetate:95% hexane) afforded 21c (63 mg, 82% yield, colorless oil) in 89% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 0.85 (t, J=7 Hz, 3H, —CH3), δ 1.56-1.68 (m, 2H, CH2), δ 4.12 (t, J=6 Hz, 2H, —OCH2), δ 5.72 (d, J=48 Hz, 1H, —CHF), δ 7.32 (br, 3H), δ 7.44 (br, 1H). 13C-NMR (75 MHz, CDCl3): δ 10.3, δ 21.9, δ 67.7, δ 88.7 (d, J=186.5 Hz) δ 124.8 δ 126.9 δ 129.9 δ 130.3 δ 134.9. 19F-NMR (282 MHz, CDCl3): δ −182.8 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C11H12ClFO2 requires m/z 230.0510, found 230.0502. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 100° C. oven, 2° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 89% was estimated based on peak areas.




Conversion of 22a to 22c. 100 mg (0.52 mmol) of 22a were dissolved in 500 μL ethanol and added to 250 mL 100 mM KPi buffer pH 8.0. Var-C12 was added to the mixture at a final concentration of 1 μM. The mixture was added with a cofactor regeneration solution containing 0.5 mM NADPH, 30 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase (final concentrations). The reaction mixtures were stirred at room temperature and extracted with chloroform (3×100 mL) after 4 hours. The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 22b (78 mg, 65%). 78 mg (0.37 mmol) of 22b and a catalytic amount (3 drops) of ethanol were dissolved in 4 mL dry dichloromethane under argon. The solution was cooled to −78° C. and added with 63 μL DAST (0.44 mmol, 1.5 eq). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by flash chromatography (5% ethyl acetate:95% hexane) afforded 22c (68 mg, 88%, colorless oil) in 85% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 0.83 (t, J=7.5 Hz, 3H, —CH3), δ 1.52-1.68 (m, 2H, CH2), δ 2.43 (s, 3H, —CH3), δ 4.12 (m, 2H, —OCH2), δ 5.96 (d, J=48 Hz, 1H, —CHF), δ 7.16-7.30 (m, 4H); 13C-NMR (75 MHz, CDCl3): δ 10.3, δ 19.3, δ 22.0, δ 29.9, δ 67.4, δ 87.4 (d, J=183 Hz) δ 126.5 δ 127.5 δ 129.8 δ 131.0. 19F-NMR (282 MHz, CDCl3): δ −180.1 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C12H15FO2 requires m/z 210.1056, found 210.1070. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 100° C. oven, 2° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 85% was estimated based on peak areas.




Conversion of 23a to 23c. 100 mg (0.52 mmol) of 23a were dissolved in 500 μL ethanol and added to 250 mL 100 mM KPi buffer pH 8.0. Var-C12 was added to the mixture at a final concentration of 1 μM. The mixture was added with a cofactor regeneration solution containing 0.5 mM NADPH, 30 mM glucose-6-phosphate and 1 U/mL glucose-6-phosphate dehydrogenase (final concentrations). The reaction mixtures were stirred at room temperature and extracted with chloroform (3×100 mL) after 4 hours. The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by silica gel chromatography (5% ethyl acetate:95% hexane) afforded 23b (70 mg, 65%). 70 mg (0.34 mmol) of 23b and a catalytic amount (3 drops) of ethanol were dissolved in 4 mL dry dichloromethane under argon. The solution was cooled to −78° C. and added with 58 μL DAST (0.4 mmol, 1.5 eq). The reaction was stirred in dry ice for 12 hours. The reaction mixture was then added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous MgSO4, and evaporated under reduced pressure. Purification of the resulting oil by flash chromatography (5% ethyl acetate:95% hexane) afforded 23c (59 mg, 83%, colorless oil) in 87% ee, as determined by chiral GC analysis. 1H-NMR (300 MHz, CDCl3): δ 0.84-0.91 (m, 3H, —CH3), δ 1.57-1.68 (m, 2H, CH2), δ 2.37 (s, 3H, —CH3), δ 4.08-4.16 (m, 2H, —OCH2), δ 5.75 (d, J=48 Hz, 1H, —CHF), δ 7.18-7.27 (m, 2H), δ 7.27-7.44 (m, 2H); 13C-NMR (75 MHz, CDCl3): δ 10.4, δ 19.4, δ 22.1, δ 29.9, δ 67.5, δ 87.2 (d, J=185 Hz) δ 126.5 δ 131.1. 19F-NMR (282 MHz, CDCl3): δ −178.5 (d, J=48.7 Hz). HRMS (EI+): exact mass calculated for C12H15FO2 requires m/z 210.1056, found 210.1062. Chiral GC analyses were carried out using a Shimadzu GC-17A gas chromatograph, FID detector, Agilent Cyclosilb column (30 m×0.52 mm×0.25 μm film) and the following separation program: 300° C. inlet, 300° C. detector, 100° C. oven, 2° C./min gradient to 200° C., 50° C./min gradient to 250° C., and 250° C. for 2 min. An enantiomeric excess of 87% was estimated based on peak areas.




Conversion of 24 to 26. 103 mg (0.5 mmol) 24 were dissolved in 2 mL DMSO and added to 200 mL potassium phosphate buffer pH 8.0. The solution was added with var-H1 (final conc.: 2.5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 2 units/mL glucose-6-phosphate dehydrogenase) and stirred at room temperature. After 48 hours, the reaction mixture was extracted with dichloromethane (4×70 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by flash chromatography (20% ethyl acetate: 80% hexane) to afford 25 (88 mg, 92%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 2.08 (s, 3H, CH3), δ 3.65 (dt, J=11.7, 3.6 Hz, 1H), δ 3.85-3.92 (m, 2H), δ 5.34 (d, J=7.8 Hz, 1H), δ 7.26-7.40 (m, 5H). 13C-NMR (75 MHz, CDCl3): δ 14.22, δ 56.83, δ 63.18, δ 83.27, δ 125.99, δ 128.50, δ 129.12, δ 140.58, δ 166.60. HRMS (EI+): exact mass calculated for C11H13NO2 requires m/z 191.0946, found 191.0943. 88 mg (0.46 mmol) 25 and a catalytic amount (3 drops) of ethanol were dissolved in 3 mL dry dichloromethane under argon. The solution was placed in ice. After ten minutes, 66 μL DAST (0.46 mmol, 1 eq) were added to the mixture in aliquots of 22 μL every 30 minutes. The reaction was stirred in ice overnight. The reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phase was then dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient 10 to 30% ethyl acetate in hexane) to afford 26 (35 mg, 40% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 2.12 (s, 3H, CH3), δ 4.2 (m, 1H), δ 4.57 (dm, J=47.4 Hz, 2H), δ 5.35 (d, J=7.2 Hz, 1H), δ 7.25-7.40 (m, 5H). 13C-NMR (75 MHz, CDCl3): δ 14.39, δ 60.64, δ 82.34 (d, J=19.6 Hz), δ 83.92 (d, J=155.7 Hz) δ 125.74 (2C), δ 128.72, δ 129.14 (2C), δ 140.34, δ 166.50. 19F-NMR (282 MHz, CDCl3): δ −230.8 (dt, J=24.2, 45.1 Hz). HRMS (EI+): exact mass calculated for C11H12FNO requires m/z 193.0903, found 193.0917.




Conversion of 27a to 27c. To a solution containing potassium phosphate buffer pH 8.0 and 0.5 mM 27a in 1% DMSO was added with var-G5 (final conc.: 5 μM), 1 mg/mL bovine serum albumin (BSA), 1000 U/mL catalase, 4.1 U/mL superoxide dismutase (SOD) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 2 units/mL glucose-6-phosphate dehydrogenase). The reaction was stirred for 48 hours at 4° C. then worked-up as described above (e.g. compound 16). The residue was purified by flash chromatography (33% ethyl acetate:67% hexanes, then 50% ethyl acetate:50% hexanes) to afford 27b (60%, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 2.2 (m, 3H), δ 2.51 (d J=17.1, 1H), δ 2.76 (m, 2H), δ 3.30 (s, 3H), δ 3.60 (d, J=5.7, 2H), δ 3.75 (q, J=4.8 Hz, 1H), δ 4.96 (m, 1H). 13C-NMR (75 MHz, CDCl3): δ 26.14, δ 27.4, δ 30.8, δ 48.73, δ 52.09, δ 60.40, δ 73.24, δ 85.57, δ 200.18. HRMS (EI+): exact mass calculated for C9H14O4 requires m/z 186.0892, found 186.0885. 14 mg (0.075 mmol) 27b were dissolved in 3 mL of dry dichloromethane and 2.5 μL of dry ethanol were added. The mixture was cooled to −80° C. and 15 μL DAST (0.11 mmol, 1.5 eq) were added. The reaction was allowed to come to room temperature slowly (16 h) and then quenched by addition of 3 mL of saturated NaHCO3 solution. The mixture was extracted with dichloromethane (4×15 mL), the organic phases were collected, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography (20% ethyl acetate:80% hexanes, then 33% ethyl acetate: 67% hexanes) to afford 27c (9.4 mg, 66% yield, colorless oil). 1H-NMR (300 MHz, CDCl3): δ 2.21 (m, 2H), δ 2.52 (m, 1H), δ 2.81 (m, 2H), δ 3.30 (s, 3H), δ 3.78 (q, J=7.2, 1H), δ 4.40 (ddd, J=47.4, 24.6, 9.6 Hz, 1H, CHF), δ 4.41 (ddd, J=47.4, 24.6, 9.6 Hz, 1H, CHF), δ 5.00 (m, 1H). 13C-NMR (75 MHz, CDCl3): δ 27.05, δ 35.68, δ 36.85, δ 39.28, δ 52.98 (d, J=18.3 Hz), δ 56.83, δ 83.18 (d, J=139 Hz), δ 84.51, δ 179.29. 19F-NMR (282 MHz, CDCl3): δ −225.8 (td, J=48.2, 27.1 Hz). HRMS (EI+): exact mass calculated for C9H13FO3 requires m/z 188.0849, found 188.0817.




Conversion of 28a to 28c. To a solution containing potassium phosphate buffer pH 8.0 and 1 mM 28a in 1% DMSO was added with var-B3 (final conc.: 5 μM) and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 2 units/mL glucose-6-phosphate dehydrogenase). The reaction was stirred for 48 hours at room temperature, then worked-up as described above. The residue was purified by flash chromatography (25% ethyl acetate:75% hexanes, then 33% ethyl acetate:67% hexanes) to afford 28b (75%, white powder). LC-MS (m/z): [M+H]+ 279.0. 1H and 13C NMR data were identical to those of the authentic standard. Two reaction vessels were prepared dissolving, under argon, 30 mg (0.11 mmol) 28b in 3 mL dry dichloromethane in each vessel. 18.5 μL DAST (0.13 mmol, 1.2 eq) were added each vessel in aliquots of 6 □L aliquots every 30 minutes. The reaction was stirred at room temperature. After 4 hours, the reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography (20% ethyl acetate:80% hexane) to afford 28c (33 mg, 55% yield, white powder). 1H-NMR (300 MHz, CDCl3): δ 2.50 (m, 4H), δ 2.96 (m, 2H), δ 4.54 (ddd, J=47.4, 21.0, 9.6 Hz, 1H, CHF), δ 4.55 (ddd, J=47.4, 21.0, 9.6 Hz, 1H, CHF), δ 5.12 (m, 1H), δ 5.41 (m, 1H), δ 7.44 (m, 2H), δ 7.56 (m, 1H), δ 8.01 (m, 2H). 13C-NMR (75 MHz, CDCl3): δ 36.20, δ 38.97, δ 40.30, δ 53.66 (d, J=18.5 Hz), δ 73.16, δ 83.62 (d, J=170 Hz), δ 84.92, δ 128.79, δ 129.89, δ 133.66, δ 166.22, δ 176.46. 19F-NMR (282 MHz, CDCl3): δ −226.7 (td, J=48.0, 30.1 Hz). HRMS (EI+): exact mass calculated for C15H16FO4 requires m/z 279.1033, found 279.1041.




Conversion of 29a to 29c. To a solution containing potassium phosphate buffer pH 8.0 and 0.5 mM 29a in 1% DMSO was added var-H1 (final conc.: 5 μM), 1 mg/mL BSA, 1000 U/mL catalase, 4.1 U/mL SOD and a cofactor regeneration system (final conc.: 500 μM NADPH, 30 mM glucose-6-phosphate, 2 units/mL glucose-6-phosphate dehydrogenase). The reaction was stirred for 48 hours at room temperature, then worked-up as described above (e.g. compound 16). The residue was purified by flash chromatography (25% ethyl acetate: 75% hexanes, then 50% ethyl acetate: 50% hexanes) to afford 29b (60%, colorless oil). LC-MS (m/z): [M+H]+ 355.0. 1H and 13C NMR data were identical to those of the authentic standard. Two reaction vessels were prepared dissolving, under argon, 20 mg (0.05 mmol) 29b in 2 mL dry dichloromethane in each vessel. 10 μL DAST (0.07 mmol, 1.2 eq) were added to each vessel in aliquots of 3 □L aliquots every 30 minutes. The reaction was stirred at room temperature. After 6 hours, the reaction mixture was added with 5 mL saturated sodium bicarbonate and extracted with dichloromethane (3×15 mL). The organic phases were collected, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography (20% ethyl acetate:80% hexane) to afford 29c (18 mg, 52% yield, white powder). 1H-NMR (300 MHz, CDCl3): δ 2.44 (m, 2H), δ 2.50-2.61 (m, 2H), δ 2.95-3.02 (m, 2H), δ 4.48 (ddd, J=47.1, 21.3, 11.7 Hz, 1H, CHF), δ 4.64 (ddd, J=47.1, 21.3, 11.7 Hz, 1H, CHF), δ 5.15 (m, 1H), δ 5.44 (m, 1H), δ 7.35-7.50 (m, 3H), δ 7.55-7.69 (m, 4H), δ 8.02-8.08 (m, 2H). 13C-NMR (75 MHz, CDCl3): δ 36.26, δ 39.00, δ 40.28 (d, J=4.27 Hz), δ 53.76 (d, J=18.2 Hz), 83.77 (d, J=183.9 Hz), δ 84.81, δ 127.45 (2C), δ 127.52 (2C), δ 128.35, δ 128.45, δ 129.17 (2C), δ 130.44 (2C), δ 140.08, δ 146.35, δ 166.11, δ 176.55. 19F-NMR (282 MHz, CDCl3): δ −226.6 (td, J=45.4, 30.2 Hz). HRMS (EI+): exact mass calculated for C21H20FO4 requires m/z 355.1346, found 355. 1332.
P450 expression and purification. For the chemo-enzymatic transformations, P450 enzymes were used in purified form. Enzyme batches were prepared as follows. Two liters Terrific Broth medium were inoculated with an overnight culture (10 mL) of recombinant E. coli DH5α cells harboring a pCWori plasmid encoding for the P450 variant under the control of Plac promoter. At OD600=1-1, cultures were added with 0.5 mM IPTG and 0.5 mM δ-aminolevulinic acid (ALA) and transferred to 25° C. Cells were harvested after 24 hours. The cell pellet was resuspended in 25 mM TRIS (pH 8.0) and cell membranes were disrupted by sonication. Cell lysate was loaded onto a Q resin and the column was washed with 3 column volumes of 25 mM TRIS (pH 8.0), 150 mM NaCl. Bound protein was eluted with 25 mM TRIS (pH 8.0), 340 mM NaCl and concentrated using Millipore Centricon. After buffer exchange with 100 mM KPi (pH 8.0), protein samples were frozen and stored at  ̃80° C. Protein concentration was determined in duplicate from CO-difference spectra. Yields typically ranged between 100 and 500 mg protein per liter depending on the variant.
The first group of test molecules (1-3; FIG. 10a,b) contains a cyclopentenone moiety found in several natural products (e.g. jasmonoids, cyclopentanoid antibiotic, and prostaglandins). The synthesis and functionalization of these scaffolds is not trivial. The activities of the enzymes towards these substrates were probed in multi-well format using GC and GC-MS (FIG. 10a). Depending on the substrate, approximately 30 to 50% of the 96 enzyme variants displayed useful activity (>800 turnovers), while 30 to 50% of this active subset showed moderate to excellent regioselectivity (50-100%). The most active and selective variants were applied in preparative scale reactions (100-300 mg) using  ̃0.05 mol % catalyst. Compared to 96-well plate reactions, three to four times higher turnover numbers could be obtained using purified enzyme and longer reaction times (24-48 hours). After flash chromatography purification, the hydroxylated products were subjected to deoxofluorination using the common nucleophilic fluorinating agent diethylaminosulfur trifluoride (DAST, 4). The identities and purities of the fluorinated products were established by GC-MS, HRMS, and 1H-, 13C- and 19F-NMR. Using this strategy, two to three different sites on each substrate were targeted with good to excellent regioselectivity (55-100%), affording the fluorinated derivatives 5-11 with yields of up to 80% over the two steps.
This fluorination strategy was then tested on a methyl ester pro drug of the anti-inflammatory drug ibuprofen (12; FIG. 10a,c). While preparation of α fluoro derivatives of this compound is straightforward, incorporating fluorine atoms in the poorly reactive isobutyl group is not. The methods above identified two chemo-enzymatic routes to achieve this goal in a selective (position 1: 75%; position 2: 100%) and efficient manner (yields over two steps for 15 and 16 were 62% and 84%, respectively) and at preparative scales (150-200 mg).
Two sequential chemo-enzymatic transformations were tested to fluorinate multiple sites of the same molecule. P450 variant B4 (var-B4)—which was used to convert 12 to 13—was found to retain comparable activity on 16, providing a possible route to the desired 17 intermediate. Re-screening of 12-active variants on 16, however, led to the identification of a more suitable candidate, var-B2, with higher activity than var-B4 towards 16 and excellent (100%) 2 regioselectivity. Using var-B2, the synthesis of fluoro-hydroxy derivative 17 was afforded in higher yields (93% vs. 72% for 1213) and required less catalyst (0.06 mol % vs. 0.1 mol % for 1213). 17 was then converted quantitatively to the desired difluoroderivative 18.
The value of the present approach as synthetic tool for asymmetric fluorination was also examined. In the absence of anchimeric group participation, the DF reaction generally preserves the enantiopurity of the enzymatic products through inversion of configuration. Chiral GC analysis showed appreciable stereoselectivity during preparation of 7 (78% ee), 9 (dr 1:8.5), 10 (dr 4:96), 15 (dr 1:3.2), and 18 (dr 1:3.7). The previous investigations on 2 phenyl acetic acid esters were extended, carrying out the asymmetric synthesis of the corresponding 2 fluoro-2-aryl acetic acid derivatives at 100 mg-scale (19a-23a; Table 5). In this case, up to 89% ee in up to 60% yield (two steps) were achieved.







TABLE 5













 


Stereoselective fluorination of substituted 2-aryl acetic acid esters.














Com-









pound
R1
R2
P450[a]
mol %
Yield (i)[b]
Yield (ii)[b]
ee[c]

















19a
H
Me
var-A3
0.1
45% (19b)
75% (19c)
74%


20a
H
Et
var-B4
0.1
66% (20b)
78% (20c)
72%


21a
m-Cl
Pr
var-C12
0.05
75% (21b)
82% (21c)
89%


22a
o-Me
Pr
var-C12
0.05
65% (22b)
83% (22c)
85%


23a
p-Me
Pr
var-C12
0.05
72% (23b)
88% (23c)
87%





[a]The sequences of the P450 variants are reported in Table 6.


[b] Yields refer to the isolated product.


[c]Enantiomeric excess determined by chiral GC analysis.













TABLE 6







Sequences of the P450BM3 variants.








Enzyme
Amino acid mutations compared to wildtype P450BM3





var-B2
V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A290V,



A295T, L353V


var-B3
R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G,



R255S, A290V, L353V


var-B4
F87A


var-C12
R47C, V78A, F87A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q,



E252G, R255S, A290V, L353V


var-D10
R47C, V78A, A82L, K94I, P142S, T175I, A184V, F205C, S226R, H236Q,



E252G, R255S, A290V, A328V, L353V


var-G4
V78A, A82G, F87V, P142S, T175I, A184V, F205C, S226R, H236Q,



E252G, R255S, A290V, A328V, L353V


var-G5
V78A, F87I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G,



R255S, A290V, L353V


var-G6
V78A, F81P, A82L, F87A, P142S, T175I, A180T, A184V, A197V, F205C,



S226R, H236Q, E252G, R255S, A290V, L353V


var-H1
R47C, L52I, V78F, A82S, K94I, P142S, T175I, A184V, F205C, S226R,



H236Q, E252G, R255S, A290V, A328L, K349N, L353V, I366V, E464G,



I710T


var-H3
V78A, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V









P450-catalyzed hydroxylation of methoxy groups leads to exposure of a free hydroxyl group through decomposition of the hemi-acetal produced and release of formaldehyde. This chemo-enzymatic strategy could be extended to substitute a methoxy group for fluorine, a challenging transformation for traditional chemical methods. This approach was first tested on the 5-phenyl oxazoline derivative 24 (FIG. 11a,b). The demethylation activities of the P450 variants could be easily assessed using a calorimetric screen (FIG. 11a). The most active variants from the screen were further analyzed with respect to the regioselectivity of oxidation using GC-MS. The highly selective P450 variant var-H1 (95%) was thus applied in combination to DF to afford the desired fluorine-containing compound 26.
The same approach was tested on a set of derivatives of the synthetically important building block Corey lactone (CL) (FIG. 11c). The use of various CLs (27a-29a) enabled investigation of the tolerance of the enzymatic transformation to structural variations within the target scaffold. Based on the colorimetric screen, 30 variants displayed activity on at least one CL (12 on 27a, 17 on 28a, 5 on 29a). Twelve variants were found to accept both 27a and 28a, five 28a and 29a, and five 27a and 29a. Interestingly, four variants ( ̃10% of the CL-active variants) could be used to activate the substrate for subsequent fluorination, regardless of the size of the variable substituent. Using the most active and selective enzymes towards each of the CLs, the desired fluorine-containing compounds 27c, 28c, and 29c were synthesized and isolated.
Ibuprofen has recently shown promising activity against amyloidogenic diseases. Anti-amyloidogenic drugs with high brain permeability are intensively sought after. Compounds 15, 16 and 18 were tested in a membrane permeability assay that mimics the composition of the blood-brain barrier (BBB). While 12 has only modest BBB-crossing potential, monofluorinated 15 and 16 and difluorinated 18 exhibit, respectively, very good and excellent membrane permeability properties (effective permeability value>10×10−6 cm s−1, FIG. 12), demonstrating how this procedure could be applied to rapidly screen various H→F substitutions in a target molecule for improvement in chemo-physical or biological properties.
The chemo-enzymatic approach of the disclosure has proven useful for fluorinating 13 of the 16 molecules tested (see FIG. 9). The molecular weight of these compounds ranges between 110 and 450 Da. About 75% of commercial drugs fall within this window. As the P450BM3 active site is largely hydrophobic, highly polar compounds may also be poor substrates. Difficulties were mostly associated with solubilisation of the substrate in aqueous media (30) or with the occurrence of side reactions—in particular elimination—during the deoxofluorination transformation (hydroxylated 31 and 32), which prevented isolation of the enzymatic and fluorinated products, respectively, in satisfactory yields. These issues could be addressed, however, by using P450BM3 variants with increased activity in the presence of organic co-solvent and applying milder nucleophilic fluorination methods.
The disclosure also provides compounds generated by the methods of the disclosure. Compounds developed by the methods include a set from Table 7 below.








TABLE 7





Compount



number
name







 1
2,3-Dimethyl-cyclopent-2-enone


 1a
4-Hydroxy-2,3-dimethyl-cyclopent-2-enone


 1c
3-Hydroxymethyl-2-methyl-cyclopent-2-enone


 2
3-Methyl-2-pentyl-cyclopent-2-enone


 2a
4-Hydroxy-3-methyl-2-pentyl-cyclopent-2-enone


 2c
3-Hydroxymethyl-2-pentyl-cyclopent-2-enone


 3
3-Methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


 3a
7-Hydroxy-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


 3b
4-Hydroxy-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


 3c
5-Hydroxy-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


 4
Diethylaminosulfur trifluoride


 5
4-Fluoro-2,3-dimethyl-cyclopent-2-enone


 6
3-Fluoromethyl-2-methyl-cyclopent-2-enone


 7
4-Fluoro-3-methyl-2-pentyl-cyclopent-2-enone


 8
3-Fluoromethyl-2-pentyl-cyclopent-2-enone


 9
7-Fluoro-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


10
4-Fluoro-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


11
5-Fluoro-3-methyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one


12
2-(4-Isobutyl-phenyl)-propionic acid methyl ester


13
2-[4-(1-Hydroxy-2-methyl-propyl)-phenyl]-propionic acid methyl ester


14
2-[4-(2-Hydroxy-2-methyl-propyl)-phenyl]-propionic acid methyl ester


15
2-[4-(1-Fluoro-2-methyl-propyl)-phenyl]-propionic acid methyl ester


16
2-[4-(2-Fluoro-2-methyl-propyl)-phenyl]-propionic acid methyl ester


17
2-[4-(2-Fluoro-1-hydroxy-2-methyl-propyl)-phenyl]-propionic acid methyl ester


18
2-[4-(1,2-Difluoro-2-methyl-propyl)-phenyl]-propionic acid methyl ester


19a
Phenyl-acetic acid methyl ester


19b
(S)-Hydroxy-phenyl-acetic acid methyl ester


19c
(R)-Fluoro-phenyl-acetic acid methyl ester


20a
Phenyl-acetic acid ethyl ester


20b
(S)-Hydroxy-phenyl-acetic acid ethyl ester


20c
(R)-Fluoro-phenyl-acetic acid ethyl ester


21a
(3-Chloro-phenyl)-acetic acid propyl ester


21b
(S)-(3-Chloro-phenyl)-hydroxy-acetic acid propyl ester


21c
(R)-(3-Chloro-phenyl)-fluoro-acetic acid propyl ester


22a
o-Tolyl-acetic acid propyl ester


22b
(S)-Hydroxy-o-tolyl-acetic acid propyl ester


22c
(R)-Fluoro-o-tolyl-acetic acid propyl ester


23a
p-Tolyl-acetic acid propyl ester


23b
(S)-Hydroxy-p-tolyl-acetic acid propyl ester


23c
(R)-Fluoro-p-tolyl-acetic acid propyl ester


24
(4S,5S)-4,5-Dihydro-4-(methoxymethyl)-2-methyl-5-phenyl-1,3-oxazole


25
(4S,5S)-4,5-Dihydro-2-methyl-5-phenyl-4-oxazolemethanol


26
(4R,5S)-4-Fluoromethyl-4,5-dihydro-2-methyl-5-phenyl-1,3-oxazole


27a
(3aR,4S,5R,6aS)-Hexahydro-5-methoxy-4-(methoxymethyl)-2H-cyclopentafuran-2-one


27b
(3aR,4S,5R,6aS)-Hexahydro-4-(hydroxymethyl)-5-methoxy-2H-cyclopentafuran-2-one


27c
(3aR,4R,5R,6aS)-4-Fluoromethyl-hexahydro-5-methoxy-2H-cyclopentafuran-2-one


28a
(3aR,4S,5R,6aS)-5-(Benzoyloxy)hexahydro-4-(methoxymethyl)-2H-cyclopentafuran-2-one


28b
(3aR,4S,5R,6aS)-5-(Benzoyloxy)hexahydro-4-(hydroxymethyl)-2H-cyclopentafuran-2-one


28c
(3aR,4R,5R,6aS)-5-(Benzoyloxy)4-(fluoromethyl)-hexahydro-2H-cyclopentafuran-2-one


29a
(3aR,4S,5R,6aS)-[1,1′-Biphenyl]-4-carboxylic acid, hexahydro-4-(hydroxymethyl)-2-oxo-2H-



cyclopentafuran-5-yl ester


29b
(3aR,4S,5R,6aS)-[1,1′-Biphenyl]-4-carboxylic acid, hexahydro-4-(methoxymethyl)-2-oxo-2H-



cyclopentafuran-5-yl ester


29c
(3aR,4R,5R,6aS)-[1,1′-Biphenyl]-4-carboxylic acid,4-(fluoromethyl)-hexahydro-2-oxo-2H-



cyclopentafuran-5-yl ester


30
Nabumetone


31
Phenylbutazone


32
Glipizide









The disclosure thus provides a method and system, and in particular a chemo-enzymatic method and system for selectively fluorinating organic molecules on a target site wherein the target site is activated and then fluorinated are present together with a method and system for identifying a molecule having a biological activity. In particular, A chemo-enzymatic method for preparation of selectively fluorinated derivatives of organic compounds with diverse molecular structures is presented together with a system for fluorination of an organic molecule and a method for identification of a molecule having a biological activity.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.
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## Claims

1. A method for fluorinating an organic molecule, the method comprising
contacting an organic molecule comprising a structure of formula I:





wherein,
X is the target site carbon;
R1 is selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, and functional group;
R2 is selected from the group consisting of a hydroxyl, alkoxy, or aryloxy; and
R3 is a Corey lactone or Corey lactone derivative;
with an oxygenase comprising a polypeptide having oxygenase activity, wherein the polypeptide has at least 95% identity to a polypeptide selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52; SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO:68, and SEQ ID NO: 69; and
contacting a fluorinating agent with the oxidized organic molecule, for a time and under condition to allow replacement of the oxygen-containing functional group with fluorine.

2. The method of claim 1, wherein the oxygenase is a variant P450BM3 oxygenase enzyme.

3. The method of claim 1, wherein R1 is a hydrogen.

4. The method of claim 1, wherein the organic molecule of formula I is a Corey lactone selected from the group consisting of:

5. The method of claim 1, wherein the Corey lactone or Corey lactone derivative is a prostaglandin precursor.

6. The method of claim 1, wherein the oxygenase is selected from the group consisting of CYP102A1, CYP102A2, CYP102A3, CYP102A5, CYP102E1, CYP102A6, CYP101A1, CYP106A2, CYP153A6, CYP153A7, CYP153A8, CYP153A11, CYP153D2, CYP153D3, P450cin, P450terp, P450eryF, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP153-AlkBurk, CYP153-EB104, CYP153-OC4, P450BSβ (CYP152A1), P450SPα (CYP152B1) and variants of any of the foregoing having oxygenase activity.

7. The method of claim 1, wherein the oxygenase is a monooxygenase or a peroxygenase.

8. The method of claim 1, wherein the organic molecule has the structure of formula (V)




in which X is the target site C atom, R14, is selected from the group consisting of hydrogen, aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, and functional group, R15 is hydrogen, R16 is a Corey lactone or Corey lactone derivative, R17 is selected from the group consisting of hydrogen, alkyl, and aryl, and R18 is hydrogen.

9. The method of claim 8, wherein the oxygenase is a monooxygenase or a peroxygenase.

10. The method of claim 9, wherein the oxygenase or variant thereof is selected from the group consisting of CYP3A4 and an oxygenase having a sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:40, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:31 and SEQ ID NO:69.

11. The method of claim 1, wherein the fluorinating agent is a nucleophilic fluorination reagent.

12. The method of claim 11, wherein the fluorinating agent is selected from the group consisting of diethylaminosulfur trifluoride, bis-(2-methoxyethyl)-aminosulfur trifluoride, and 2,2-difluoro-1,3-dimethylimidazolidine (DFI).