Antibody-catalyzed anaerobic destruction of methamphetamine

Abstract

Methamphetamine [(+)-2] abuse has emerged as a fast-rising global epidemic, with immunopharmacotherapeutic approaches being sought for its treatment. Herein, we report the generation and characterization of a monoclonal antibody, YX1-40H10, that catalyzes the photooxidation of (+)-2 into the nonpsychoactive compound benzaldehyde (14) under anaerobic conditions in the presence of riboflavin (6). Studies have revealed that the antibody facilitates the conversion of (+)-2 into 14 by binding the triplet photoexcited state of 6 in proximity to (+)-2. The antibody binds riboflavin (K_d = 180 μM), although this was not programmed into hapten design, and the YX1-40H10-catalyzed reaction is inhibited by molecular oxygen via the presumed quenching of the photoexcited triplet state of 6. Given that this reaction is another highlight in the processing of reactive intermediates by antibodies, we speculate that this process may have future significance in vivo with programmed immunoglobulins that use flavins as cofactors to destroy selectable molecular targets under hypoxic or even anoxic conditions.

Abuse of drugs such as amphetamine (1) and its congeners, methamphetamine (2), ephedrine (3), methylenedioxyamphetamine (4), and methylenedioxymethamphetamine (5) (Fig. 1), is a serious global problem (1–3). Immunopharmacotherapy that utilizes antibody-based therapeutics is being studied as an innovative approach to controlling the abuse of these psychoactive agents (4, 5). In this regard, antibody catalysts that can convert drugs of abuse into inactive forms are viewed potentially as more efficient immunotherapeutic agents than immunoglobulins that simply bind and either sequester or eliminate the drug. Our group and others have developed antibodies with cocaine esterase activity (6–8). More recently, we have disclosed catalytic antibodies that, in conjunction with the naturally occurring photosensitizer riboflavin, can oxidatively degrade nicotine into putatively nonpsychoactive substances (9). Here we report a viable, cofactor-based catalytic antibody system for the destruction of psychoactive substances. To aid in developing efficient systems for decomposing drugs of abuse, we have generated a murine monoclonal antibody (mAb), YX1-40H10, which, in the presence of white light and riboflavin (6), is able to photodegrade the psychoactive enantiomer of methamphetamine [(+)-2] into a nonpsychoactive product via a type I photooxidation process.

Fig. 1.

Drugs of abuse targeted for catalytic antibody degradation. (A) (±)-Amphetamine (1) and its congeners (2–5). (B) Riboflavin (6) and tetrahydroisoquinoline hapten 7.

Results and Discussion

Hapten 7 was designed to elicit antibodies that recognize the panel of amphetamine (1) and amphetamine-like psychoactive agents 2–5, and it contains a number of key structural features (Fig. 1). First, it possesses the substituted β-phenethylamine pharmacophore, which is common to this family of drugs. Second, hapten 7 was prepared without imposition of stereocontrol at C-3 of the tetrahydroisoquinoline nucleus. Agents 2–5 all have a chiral center at the analogous carbon in their structure; however, they are not all abused in enantiomerically pure form. The use of rac-7 is not simply a product of synthetic expediency, rather we have shown previously that immunization with racemic haptens such as rac-7 elicits panels of mAbs that contain members with exquisite enantioselectivity against one of the two enantiomers of their corresponding substrate (10). Finally, the hapten 7 contains a constrained tetrahydroisoquinoline moiety, whereas the substrates 1–5 all have a conformationally free substituted aminoethyl aromatic side chain. This apparent departure from structural simile between hapten and substrate, wherein the conformationally free component of a target molecule is mimicked by locking the isostructural locus within the hapten into a constrained conformation, has previously been shown by us to increase the immune response against the substrate (11).

Hapten 7 was synthesized in seven steps from 3,4-(dimethylenedioxy)phenylacetic acid (8) (Scheme 1). In brief, acid 8 was converted into methyl ketone 10 by addition of a methyl Gringard reagent to Weinreb amide 9 in an overall yield of 60% (for two steps). Subsequent reductive amination of 10 afforded amine 11 in low yield (32%). A Pictet–Spengler reaction between 11 and formaldehyde installed the tetrahydroisoquinoline core of 12 in 64% yield. The spacer was then extended by coupling of an additional β-alanine unit to give amide 13, which was then converted into hapten 7 by hydrogenolysis (78% yield for two steps).

Scheme 1.

Synthesis of hapten 7: Reagents and conditions. a, HNMe(OMe), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and triethylamine, 74% yield; b, MeMgCl and THF, 0°C, 80% yield; c, H-β-Ala-OBn (Bn, benzyl), NaBH(OAc)₃, and AcOH, 32% yield; d, CH₂O, HCO₂H, reflux, 64% yield; e(1), H₂, Pd(OH)₂, and MeOH; e(2), H-β-Ala-OBn, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and triethylamine, 78% yield in two steps; f, H₂ and Pd(OH)₂, 48% yield.

Hapten 7 was conjugated to carrier proteins [BSA and keyhole limpet hemocyanin (KLH)], and the KLH-7 conjugate was used to immunize mice, using standard protocols. After hybridoma production, 12 murine mAbs were generated, and ELISA studies with the BSA-7 conjugate revealed that each antibody bound hapten 7. These mAbs were then further examined for their ability to recognize substrates 2–5. Competition ELISA between BSA-7 and the panel of substrates (2–5) revealed that a single mAb, YX1-40H10, could bind to (+)-2 with an apparent binding constant, K_dapp[(+)-2], of 75 μM.

In a preliminary screen for catalysis, the 12 mAbs with affinity for BSA-7 (see above) were tested for their ability to degrade the amphetamines 1–5 in the presence of riboflavin (6) and visible light in aqueous buffer. Thus, 6 (60 μM), the mAb (20 μM), and 1–5 (1 mM) in PBS (pH 7.4) were incubated in the presence of white light (3.4 mW·cm⁻²) under ambient aerobic conditions at 4°C. Activity was initially assessed by simply monitoring the loss of 1–5 by HPLC. From this screen, only mAb YX1-40H10, which binds (+)-2, was found to accelerate the photochemical degradation of (+)-2. No other antibody-mediated photodegradation of 1–5 by the 12 mAbs was detected.

YX1-40H10-Mediated Photodegradation of (+)-2.

RP-HPLC analysis was conducted on the reaction mixture, after photoirradiation (400–700 nm, 3.4 mW·cm⁻²), of antibody YX1-40H10 (20 μM), riboflavin (60 μM), and (+)-2 (1 mM) in PBS (pH 7.4) at 4°C. This analysis revealed two peaks generated in the HPLC (peak 1, retention time R_T ≈ 8.9 min; peak 2, R_T ≈ 12.5 min; Fig. 2A). To identify these two products, a large-scale (1 ml), long-duration (24 h) photochemical reaction was performed in the presence or absence of mAb YX1-40H10 (Fig. 2 A and B, respectively). This comparison revealed that peak 1 (R_T ≈ 8.9 min) was generated in almost the same amount in the presence or absence of YX1-40H10; that is, this product is not antibody-dependent. In contrast, peak 2 (R_T ≈ 12.5 min) was produced in significantly elevated amounts in the antibody-mediated photochemical process relative to the noncatalyzed reaction.

Fig. 2.

RP-HPLC analysis of the photooxidation of (+)-2 by riboflavin (60 μM). The analytical HPLC was performed on a Hitachi L-7400 instrument using a Grace Vydac reversed-phase C₁₈ column. The column was eluted with an isocratic solvent system, 20% MeCN (0.1% TFA). All of the HPLC runs were monitored at 254 nm. See Results for a description of runs A–D.

The diethyl ether extract of the YX1-40H10-catalyzed large-scale reaction (Fig. 2 A) was then concentrated and analyzed by ¹H NMR. This analysis demonstrated that benzaldehyde (14) was formed as a major product in this reaction [see supporting information (SI) Materials and Methods]. This conclusion was further strengthened when a coinjection of the YX1-40H10-catalyzed reaction with 14 (20 μM) revealed that the unknown peak 2 has the same retention time as authentic benzaldehyde (R_T ≈ 13.0 min; Fig. 2C). Further support for 14 as being a major photoproduct in the YX1-40H10-catalyzed process came from chemical derivitization of the assay mixture with dinitrophenylhydrazine (DNPH). When DNPH was added to the diethyl ether extract of the YX1-40H10-catalyzed reaction, a yellow precipitate formed. Analysis of this precipitate by ¹H NMR also clearly established its identity as the DNPH hydrazone of benzaldehyde (see SI Materials and Methods).

Given that peak 1 formation occurs independently of YX1-40H10 and that this same peak is observed upon photoirradiation of riboflavin (6) alone in PBS (pH 7.4) (data not shown), it was surmised that the unknown peak was originating as a result of the known photochemical decomposition of riboflavin. This supposition was confirmed by coinjection of the YX1-40H10-catalyzed photodecomposition assay with an authentic sample of lumichrome (15), a major photodecomposition product of riboflavin (12, 13). This coinjection with 15 resulted in an increase in the area of peak 1, confirming its constitution (Fig. 2D).

Characterization of YX1-40H10-Catalyzed Photodegradation of (+)-2.

Given that benzaldehyde (14) is a known product of the YX1-40H10-mediated process, kinetic studies were undertaken to measure the catalytic parameters, K_m (Michaelis–Menten constant) and k_cat (catalytic rate constant), for production of 14. Conditions of saturating riboflavin (60 μM) were determined, and the initial rates of benzaldehyde formation were obtained at varying (+)-2 concentrations in the presence of YX1-40H10 (10 μM). Under such conditions, saturation kinetics with respect to (+)-2 are observed (Fig. 3A).

Fig. 3.

Kinetics of the YX1-40H10-catalyzed photooxidation of (+)-2. (A) Initial rate v of YX1-40H10-catalyzed benzaldehyde (14) formation vs. substrate (+)-2 concentration, all determined at saturating riboflavin concentration (60 μM). (B) Initial rate, v, of YX1-40H10-catalyzed benzaldehyde (14) formation vs. riboflavin (6) concentration as a function of YX1-40H10 concentration, all determined at saturating methamphetamine concentration and varied antibody concentrations [10 μM antibody (circles), 20 μM antibody (squares), and 40 μM antibody (diamonds)]. (C) Inhibition of the YX1-40H10-catalyzed photodegradation of (+)-2 by hapten 7. Graph shows the time-dependent formation of benzaldehyde (14) during the photoirradiation of riboflavin (60 μM) and (+)-2 (300 μM) in PBS (pH 7.4) at 4°C, with the following additions: 40H10 (40 μM sites) (inverted triangles), YX1-40H10 (40 μM sites) and 7 (10 μM) (triangles), YX1-40H10 (40 μM sites) and 7 (40 μM) (diamonds), and no addition (background reaction) (squares). Each point is the mean of at least duplicate experiments, and the connecting lines are point-to-point.

The high concentration of YX1-40H10, relative to substrate concentration, used in these kinetic studies invalidates the usual Michaelis–Menten approximation that free and total substrate should be essentially equal. Therefore, the reaction velocity (v) for the YX1-40H10-catalyzed formation of 14 was derived from Eq. 1, wherein the free concentration of (+)-2 is explicitly solved for. Fitting of the experimental kinetic data of initial rate v vs. (+)-2 (under conditions of saturating riboflavin) to Eq. 1 yields a k_cat of 3.7 ± 0.1 × 10⁻² min⁻¹ and a K_mapp[(+)-2] of 27 ± 3 μM. The combination of these two parameters gives an apparent second-order specificity constant for YX1-40H10 (k_cat/K_mapp) of 23.3 ± 2 M⁻¹·s⁻¹.

where S_T is the total (+)-2 concentration and A_T is the total YX1-40H10 binding site concentration (40 μM).

Initial rates of benzaldehyde (14) formation were also measured under conditions of saturating (+)-2 (300 μM) as a function of varying riboflavin 6 concentration (Fig. 3B). Saturation of the initial rate v with increasing 6 was again observed, and unexpectedly the K_mapp(6) determined from fitting the experimental data to Eq. 1 was found to be 10 μM. This value is substoichiometric with respect to antibody concentration. We next examined the dependence of riboflavin saturation on YX1-40H10 kinetics at several concentrations of antibody (10, 20, and 40 μM) (Fig. 2B). The experimental initial rate data were then fit to Eq. 2, wherein the critical parameter K′_m represents the ratio of riboflavin to antibody concentration at 0.5 V_max. This system then reveals a best-fit solution of k_cat = 5.4 ± 0.2 × 10⁻² min⁻¹ and a K′_m of 0.26 ± 0.03.

where [R] is the riboflavin concentration; A_T is the antibody concentration; and K′_m is the ratio of [6] to [40H10] that produces one-half the velocity at saturation.

The YX1-40H10 k_cat value of ≈5.4 × 10⁻² min⁻¹ calculated under conditions of saturating (+)-2 and varying photosensitizer 6 is almost identical to the k_cat determined under conditions of riboflavin saturation with varying concentrations of (+)-2 (≈3.7 × 10⁻² min⁻¹). Importantly, the equivalency in the k_cat values in both cases offers strong support for the formation of a kinetically viable ternary complex between YX1-40H10 and (+)-2 and 6.

After administration of a typical dose of 2 (20 mg), the plasma level of 2 has been shown to be ≈0.5 mM, with a clearance half-life of ≈12 h (k_cl = 0.0096 min⁻¹). Such a systemic concentration is below the K_m of YX1-40H10, and therefore the antibody would be functioning in vivo under substrate-limiting conditions. Under such a scenario, the rate, v, of destruction of 2 by YX1-40H10 at a plasma concentration (E_total) of 0.5 mM is given by Eq. 3 and is ≈0.00070 min⁻¹.

This additional rate of destruction of 2 supplied by YX1-40H10 should result in a drop of the clearance half-life to 6.8 h, showing that in principle the antibody may significantly impact the normal in vivo clearance of this drug.

To assess whether the YX1-40H10-mediated process is occurring within the antibody complementarity-determining regions, the effect of hapten 7 on the initial rate of YX1-40H10-catalyzed 14 formation was investigated. Thus, the photoirradiation (white light, 400–700 nm, 3.4 mW·cm⁻²) experiments were performed with YX1-40H10 (20 μM and 40 μM sites), riboflavin (60 μM), and (+)-2 (500 μM) at 4°C in the presence of different concentrations of hapten 7 (0, 10, and 40 μM) (Fig. 3C). Under such conditions, addition of 10 μM 7 results in a 25% reduction in the initial rate of benzaldehyde (14) formation, and addition of 40 μM 7 inhibits the YX1-40H10-mediated process to an initial rate just slightly above the photooxidation rate in the absence of antibody. Thus, hapten 7 is a stoichiometric inhibitor of the YX1-40H10-catalyzed reaction, supporting the notion that this process is taking place within the antibody-binding sites.

Oxygen Quenches the YX1-40H10-Catalyzed Photodegradation of (+)-2.

Given that this overall photochemical process involves an apparent photooxidation of (+)-2 to yield benzaldehyde (14), the origin of the oxygen atom in 14 was studied. Thus, the YX1-40H10-catalyzed formation of 14 was investigated in the presence of various oxygen concentrations (Fig. 4). The initial rate data reveal that the YX1-40H10-catalyzed process is quenched by oxygen and that the oxygen atom in 14 does not originate from molecular oxygen, because benzaldehyde formation occurs facilely under anaerobic conditions. The oxygen quenching of the YX1-40H10-mediated process is typical for a process that involves triplet excited riboflavin (³6) (14) and suggests that this overall process is a type I photooxidation process with the origin of the oxygen atom in 14 being a water molecule.

Fig. 4.

Oxygen dependence of the YX1-40H10-catalyzed photodegradation of (+)-2. Conditions were pure oxygen (inverted triangles), ambient aerobic (triangles), or anaerobic (squares). The reaction mixtures were composed of 1 mM (+)-2, 60 μM riboflavin (6), and 20 μM antibody YX1-40H10 in PBS (10 mM phosphate buffer/100 mM NaCl, pH 7.4). The reaction was irradiated (3.4 mW·cm⁻²) at 4°C. Aliquots of the reaction were taken and analyzed by RP-HPLC over a period of 24 h.

Given that ³6 was seen as a critical intermediate in this photooxidation process, hematoporphyrin-IX and methylene blue were studied as alternative triplet dye sources for the YX1-40H10-mediated process (15). However, when these dyes were used as photosensitizers, there was no enhancement in the rate of benzaldehyde formation above that observed in buffer alone (data not shown). These findings suggest that recognition of riboflavin by YX1-40H10 may be a key aspect in this overall process.

No specific antibody recognition of riboflavin had been programmed during hapten 7 design; however, it is known that a fraction of immunoglobulins bind flavins in vivo (16–18). Therefore, the dissociation constant, K_d, of 6 for YX1-40H10 was measured by equilibrium dialysis with [³H]riboflavin. These binding studies reveal that riboflavin is a weak ligand for YX1-40H10 (K_d = 180 μM). Interestingly, the B_max value (14.9 μM for 13.4 μM antibody concentrations) reveals that the stoichiometry of riboflavin binding is one molecule per antibody molecule. Although the location of riboflavin binding on YX1-40H10 is unknown, given that this process is a photooxidation process, the substrate 2 and riboflavin (6) would have to be in relatively close proximity for efficient energy/electron transfer.

Reaction Mechanism Studies.

GC-MS analysis of organic extracts of the riboflavin-mediated photodegradation reaction of (+)-2 in aqueous buffer under anaerobic conditions revealed unreacted (+)-2 with trace amounts of benzaldehyde and the previously undetected component phenylacetone (16) (Fig. 5). In the YX1-40H10-mediated process, 14 is the major component observed in the GC-MS analysis, but trace amounts of 16 are also observed (Fig. 5).

Fig. 5.

GC-MS analysis of the diethyl ether extract of YX1-40H10-mediated degradation of (+)-2 under anaerobic conditions. GC-MS analyses were run at 70 eV on a 6850 Network GC system (Agilent Technologies, Palo Alto, CA) attached to a 5973 inert-mass selective detector (Agilent Technologies). The system was equipped with an Agilent 19091A-002 column (25 m × 0.20 mm; 0.11 μm film thickness). Helium flow rate was 1.2 ml·min⁻¹. The oven temperature program was 50°C from 0 to 5 min; 50–300°C from 5 to 25 min, 20°C·min⁻¹; and 300°C from 25 to 27.5 min. X's mark solvent peaks. (Inset) Analysis of riboflavin-mediated photodegradation.

To investigate whether phenylacetone (16) observed in the GC-MS arises as an intermediate or byproduct of the photooxidation of (+)-2, phenylacetone (16, 1 mM) was photoirradiated (400–700 nm, 3.4 mW·cm⁻²) with 6 (60 μM) in PBS, pH 7.43, at 4°C in the presence or absence of YX1-40H10 (20 μM) under anaerobic conditions. At times during the photoirradiation, aliquots of the reaction were removed and analyzed for benzaldehyde content by RP-HPLC. Benzaldehyde was indeed formed in the aqueous buffer background reaction (2.6 ± 0.5 nM·min⁻¹), suggesting that 16 could be an intermediate (rather than a byproduct) in this process. Interestingly, in the YX1-40H10-mediated process, the rate of benzaldehyde formation was significantly enhanced (17.4 ± 0.2 nM·min⁻¹) over the reaction in aqueous buffer (Fig. 6A). This ability to process 16 into 14 by YX1-40H10 constrains the mechanism for the process (Scheme 2).

Fig. 6.

Riboflavin-photosensitized aqueous reaction. Shown are phenylacetone (16) (A) and ephedrine (3) (B) converted into benzaldehyde (14) under anaerobic conditions. The reaction mixtures were composed of 1 mM phenylacetone (16) [or ephedrine (3)], 60 μM riboflavin, and 20 μM antibody YX1-40H10 in PBS. The reaction was irradiated (3.4 mW·cm⁻²) at 4°C. Aliquots of the reaction were taken and analyzed by RP-HPLC over a period of 24 h.

Scheme 2.

Proposed mechanism for the YX1-40H10-catalyzed conversion of (+)-2 into benzaldehyde (14) via either phenylacetone (16) or ephedrine (3). RF, riboflavin; B, general base.

Although the mechanism is still far from resolved, guided by the GC-MS data and the fact that the oxygen in 14 originates from water and not molecular oxygen (see above), it seems reasonable to propose a process that proceeds via a stepwise photooxidation of (+)-2 by ³6, leading to the putative N-methyliminium species 17. Although there are no reports of this specific process, there is clear precedent for photooxidation of amines by riboflavin that proceeds via radical cations on nitrogen (19, 20). The iminium species 17 has two clear fates. (i) It may be trapped by water, with subsequent elimination of methylamine that yields phenylacetone (16) and from there onto 14. (ii) Alternatively, the N-methyliminium 17 may isomerize into the benzylic carbonium ion 18, which can then be trapped by a water molecule, yielding ephedrine (3), and can then be further photooxidized to 14.

The potential involvement of 3 in the YX1-40H10-mediated process was investigated by anaerobic photoirradiation (400–700 nm, 3.4 mW·cm⁻²) of a solution of (+)-3 (1 mM) and 6 (60 μM) in PBS, pH 7.43, at 4°C in the presence or absence of YX1-40H10 (20 μM). At times during the photoirradiation, aliquots of the reaction were analyzed by RP-HPLC. These studies reveal that 3 is rapidly converted into 14 in aqueous buffer, suggesting that if this β-aminoalcohol is generated as an intermediate in the aqueous background reaction, it would not accumulate and probably would not be detected (Fig. 6A). In fact, we do not detect, by either GC-MS or RP-HPLC, the formation of 3 in either the YX1-40H10-catalyzed or buffer-only photooxidation of (+)-2. Importantly, there is no rate enhancement in the photooxidation of 3 above the aqueous buffer background in the presence of YX1-40H10, suggesting that the antibody cannot utilize 3 as an intermediate (Fig. 6B) and strengthening the potential for the YX1-40H10-mediated pathway to proceed via phenylacetone (16).

Methamphetamine abuse continues to spread across the United States at an ever-increasing rate. This fact, coupled with the potent neurotoxicity of (+)-2, leads to a clear and urgent need for development of effective therapies to treat the addiction associated with (+)-2. Although catalytic antibody approaches have proven effective in the hydrolysis of the benzoyl ester of cocaine to generate nonpsychoactive metabolites, examination of the methamphetamine structure yields no such sites for catalytic-antibody development. Consequently, we have turned to a cofactor-based approach predicated on the intrinsic ability of all antibodies to oxidize their cognate antigen when presented with singlet dioxygen (¹O₂^*) (21–25). Analogous to our previous studies into antibody-catalyzed nicotine oxidation (9, 11), methamphetamine oxidation is indeed a facile process in the presence of specific antibodies and the innate photosensitizer riboflavin. However, this work has revealed that under appropriate conditions of light flux, efficient photochemical destruction of antigens can be antibody-catalyzed, even in the absence of oxygen, providing alternative chemical routes to those reported previously. In addition, the discovery of this photooxidation process has additional implications for immune defense, autoimmune disease, and the treatment of cancer in cases where one is dealing with sites of inflammation that are hypoxic and even anoxic. In total, this study hints that, under appropriate conditions of riboflavin, light, and antibody, antigen photooxidation, and hence destruction, is a real possibility.

Materials and Methods

Experimental procedures and spectroscopic data for the preparation of hapten 7 are available in SI Materials and Methods. Hyperimmunization of a KLH-7 conjugate was performed as described previously (26). All HPLC analyses were performed on a Hitachi (San Jose, CA) L-7400 instrument equipped with a Vydac reversed-phase C₁₈ column and an isocratic mobile phase of 20% MeCN/80% H₂O (0.1% TFA) at 1 ml/min with UV detection at 254 nm.

Standard Method of Photooxidation of (+)-2.

A solution of methamphetamine (+)-2 (1 mM) and riboflavin (6) (60 μM) in PBS (pH 7.4) was irradiated on a white light transilluminator (400–700 nm, 3.4 mW·cm⁻² Fischer-Biotech transilluminator) at 4°C. Throughout the irradiation, aliquots (25 μl) were removed and analyzed by RP-HPLC for formation of benzaldehyde (14) as described above. Data were collected from at least duplicate samples and are reported as mean ± SEM.

Oxygen Dependence of the YX1-40H10-Mediated Photooxidation of (+)-2.

Solutions of both (+)-2 (1 mM), YX1-40H10 (20 μM) in PBS (pH 7.4), and riboflavin (60 μM) in water were lyophilized (Labconco) in duplicate. The lyophilized riboflavin was then dissolved in rigorously degassed water (200 μl) under ambient air, oxygen, or argon, and the resultant solution was transferred into the lyophilized solid containing (+)-2 and YX1-40H10 under argon. This mixture was then photoirradiated on a white light transilluminator (400–700 nm, 3.4 mW·cm⁻²) at 4°C. Aliquots were taken and subjected to RP-HPLC analysis to measure the formation of 14.

Measurement of the YX1-40H10–Riboflavin Dissociation Constant, K_d.

All experiments were performed in a Spectrum equilibrium dialysis apparatus with samples (1 ml) introduced into both the ligand and receptor chambers. In all cases, the receptor chambers were filled with YX1-40H10 in PBS (2 mg/ml). The ligand chambers were filled with solutions of 6 (5–160 μM) containing a fixed concentration of tritiated 6 (0.04 nmol/ml) in PBS in duplicate. The receptor and ligand were equilibrated overnight in the dark. Samples (150 μl) from the receptor/ligand chamber were added into scintillation fluid (5 ml) and analyzed (Beckman scintillation counter). The membranes were also analyzed by the same method to allow a calculation of total ligand in counts per minute. Analysis was carried out by calculating free and bound ligand concentrations and fitting to a single binding saturation model, using Prism 4 software (GraphPad, San Diego, CA).

Inhibition of YX1-40H10 with Hapten 7.

In a typical experiment, YX1-40H10 (20 μM) was preincubated at room temperature for 5 min with 7 (10 μM or 40 μM, final concentration) in PBS (pH 7.4). Then, (+)-2 (1 mM) and 6 (60 μM) were added to this mixture, and the resultant solution was irradiated on a white light transilluminator at 4°C. Aliquots were taken from the reaction mixture and analyzed by RP-HPLC, as detailed above. Reactions were run in duplicate and were reported as the mean ± SEM of benzaldehyde (14) formation.

Kinetic Analysis of the YX1-40H10-Mediated Photooxidation of (+)-2.

Initial rate of benzaldehyde (14) formation vs. (+)-2 concentration.

All of the reaction mixtures contained antibody YX1-40H10 (20 μM), 6 (60 μM), and (+)-2 (at various concentrations ranging from 25 to 300 μM). The reactions were performed with photoirradiation at 4°C, and three time points were taken in the first 30 min of the reaction. It was confirmed that <5% of the (+)-2 was consumed during this period. The initial rate was obtained from the linear part (r² > 0.985) of a plot of 14 formation vs. time. Data were corrected for background. Data analysis was performed using KaleidaGraph for the PC (Synergy Software, Reading, PA). This kinetic characterization was repeated in at least duplicate, with interassay data showing a low variance (<15%).

Initial rate of benzaldehyde (14) formation vs. concentration of riboflavin (6).

Initial rates of formation of 14 were measured with assay mixtures consisting of (+)-2 (300 μM) and 6 (at various concentrations ranging from 5 to 60 μM). The initial rates, v, of 14 formation were then plotted against the concentration of 6. Data were corrected for background, and data analysis was performed using KaleidaGraph for the PC.

Photooxidation of Ephedrine (3) or Phenylacetone (16) with Riboflavin Under Anaerobic Conditions.

A solution of racemic ephedrine (3) (1 mM; Sigma–Aldrich) or phenylacetone (16) plus or minus antibody YX1-40H10 (20 mM) and 6 (60 μM) in PBS (pH 7.43) was lyophilized. To the resulting reaction mixture was added deoxygenated water under argon. The reaction solution was then photoirradiated with white light at 4°C. Aliquots of the reaction were taken and analyzed by RP-HPLC for benzaldehyde (14) formation over a period of 24 h. The same assay was then repeated in the presence of YX1-40H10 (20 μM). Photodegradation of phenylacetone (16) was performed under the same conditions, replacing 3 with 16.

Abbreviation

DNPH

dinitrophenylhydrazine.