A Conjugate Vaccine Using Enantiopure Hapten Imparts Superior

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A Conjugate Vaccine Using Enantiopure Hapten Imparts Superior Nicotine-Binding Capacity Jonathan W. Lockner,† Jenny M. Lively,† Karen C. Collins,† Janaína C. M. Vendruscolo,‡ Marc R. Azar,‡ and Kim D. Janda*,† †

Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States ‡ Behavioral Pharma Inc., 505 Coast Boulevard South, La Jolla, California 92037, United States S Supporting Information *

ABSTRACT: A leading nicotine conjugate vaccine was only efficacious for one-third of clinical trial participants, likely due in part to its use of racemic nicotine hapten, (±)-3′AmNic. Immunization of male Wistar rats with (+)-, (−)-, or (±)-3′-AmNicSucTT and subsequent antibody immunoassays suggest that a vaccine using enantiopure (−)-3′AmNic hapten imparts superior capacity to bind (−)-nicotine. Future nicotine vaccine clinical candidates must incorporate this design consideration (i.e., hapten enantiopurity) in order to maximize efficacy.

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INTRODUCTION According to the World Health Organization, there are over 1 billion smokers worldwide, and smoking is responsible for nearly 6 million deaths annually.1 The economic impact is also sobering: in the United States alone, smoking costs nearly $300 billion in medical expenses and lost productivity each year.2 The epidemiological link between chronic tobacco use and myriad diseases is well understood, and while many smokers wish to quit, currently available cessation aids do not help much. Synthetic small molecule agonists or antagonists target brain receptors involved in nicotine dependence.3−5 Acting centrally, these medicines have a variety of side effects.6 Meanwhile, we have been pursuing a pharmacokinetic (antibody-based) instead of a pharmacodynamic (drug-based) strategy to aiding smokers’ efforts to quit.7 Nicotine plays a central role in precipitating addiction to smoking tobacco. A nicotine vaccine stimulates the immune system to identify nicotine as a foreign antigen, eliciting antibodies that alter nicotine pharmacokinetics. Antinicotine antibodies reduce the concentration of free nicotine in the blood and prevent it from entering the central nervous system. Blocking the activation of brain reward systems can facilitate extinction of the addictive behavior, leading to better smoking cessation outcomes. A clinically approved nicotine vaccine would be a complementary addition to available therapies, which, when leveraged appropriately, could afford significantly better rates of sustained smoking abstinence. A leading nicotine conjugate vaccine, consisting of racemic hapten 3′-aminomethylnicotine conjugated to Pseudomonas aeruginosa exoprotein A ((±)-3′-AmNic-rEPA) and formulated with alum adjuvant, represents the most clinically advanced © XXXX American Chemical Society

nicotine vaccine to date, having progressed all the way through phase III.8−11 It was safe and well tolerated, but effective for only a fraction of clinical trial participants.12,13 Nevertheless, given the huge promise of a clinically approved nicotine vaccine, research continues unmitigated.14 Many design and formulation aspects have been scrutinized in recent years to furnish something better than (±)-3′-AmNic-rEPA. Efforts include boosting immunogenicity through the use of newer adjuvants,15−18 improving practicality through alternative routes of administration,19 and adopting multivalent strategies20−23 to increase antinicotine antibody binding capacity. For a vaccine aimed at conferring protective immunity against a specific small molecule such as nicotine, it is important that the vaccine displays adequate chemical epitope homogeneity.24−26 Other vaccines may be engineered to simultaneously target multiple prevailing epitopes, as in the case of diphtheria− tetanus−acellular pertussis (DTaP), measles−mumps−rubella (MMR), and 23-valent pneumococcal combination vaccines.27,28 Here, however, the aim is to specifically and selectively target only (−)-nicotine; thus, a vaccine should efficiently elicit antibodies capable of sequestering only (−)-nicotine. The notion that antibodies can enantiodifferentiate was first appreciated by Landsteiner nearly a century ago29,30 and continues to be exploited to this day. Such work includes enantioselective catalytic antibodies31−33 and stereospecific mAb to nicotine34 and cocaine.35−37 In the case of the nicotine mAb study, hybridomas were selected using (S)(−)-[3H]nicotine, thereby optimizing for antibodies specific for Received: April 7, 2014

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Scheme 1. Preparation of Nicotine Vaccine Conjugates Using Enantiopure Nicotine Haptensa

Reagents and conditions: (a) NH4HCO3, pyridine, Boc2O, CH3CN, H2O, rt, 4 h, 40 °C, 30 min, 84%; (b) Red-Al, toluene, rt, 18 h, 19%; (c) chiral SFC, ∼80% recovery of each enantiomer; (d) EDC, (−)-2 or (+)-2, pH 5.80 MES buffer, final dialysis against pH 7.4 PBS, hapten densities 41−44; (e) succinic anhydride, pH 8.65 Tris buffer, linker density 53. a

the naturally occurring isomer.34 The present study demonstrates that the capacity of antibodies to enantiodifferentiate should not be ignored in development of vaccines for drugs of abuse. Many of the nicotine vaccines that have undergone clinical evaluation began with manipulation of racemic trans-cotinine carboxylic acid ((±)-1, Scheme 1). To the best of our knowledge, no chiral separation step (nor asymmetric synthesis step) was included in the production of the hapten−protein conjugate that would become (±)-3′-AmNic-rEPA. Hence, we set out to examine the notion that a nonracemic, fully (−)-nicotine vaccine conjugate would be a superior immunogen, owing to the exquisite ability of antibodies to stereodifferentiate. For the present study, we chose to focus our attention on 3′-AmNic, the hapten used in (±)-3′-AmNicrEPA.

derived male rats (n = 10−12 per group, 250−300 g) were assigned randomly to (−)- or (+)-groups (first study), or (−)-, (+)-, and (±)-groups (second study). Rats were given free access to food and water during the immunization schedule (Figure 1), which consisted of a series of three (200 μL)

Figure 1. Rat immunization and bleed schedule.

intramuscular injections on days 0, 21, and 42. On days 28 and 49, plasma samples were obtained by tail vein bleed. On day 63, rats were anesthetized, bled by cardiac puncture, and euthanized. Enzyme-linked immunosorbent assays (ELISAs) were carried out using either (−)- or (+)-3′-AmNicSucBSA, prepared in a manner analogous to the TT conjugates above. For self-reactive ELISA, plasma samples were run against their respective haptens: rat plasma from the (−)-3′-AmNicSucTT group was assayed on (−)-3′-AmNicSucBSA plates, and rat plasma from the (+)-3′-AmNicSucTT group was assayed on (+)-3′AmNicSucBSA plates. For cross-reactive ELISA, plasma samples were run against their antipodes: rat plasma from the (−)-3′-AmNicSucTT group was assayed on (+)-3′-AmNicSucBSA plates, and rat plasma from the (+)-3′-AmNicSucTT group was assayed on (−)-3′-AmNicSucBSA plates. The (±)-group was assayed on both (−)- and (+)-plates. (−)-Nicotine-specific plasma antibody binding affinities and antibody concentrations were determined by competitive radioimmunoassay (RIA).47 First, the plasma dilution that binds ∼50% of 3H-labeled (−)-nicotine is determined. Then, the affinity constant is calculated by competition with unlabeled nicotine. Because the plasma samples were pooled for each vaccine group, the measured affinity constants are average affinities (Kd,avg) for each group.

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RESULTS First, racemic trans-3′-aminomethylnicotine (3′-AmNic, (±)-2) was prepared from commercially available racemic transcotininecarboxylic acid ((±)-1). Next, using chiral supercritical fluid chromatography (SFC), ∼600 mg of (±)-2 was separated into ∼250 mg of each enantiomer (Scheme 1). Given our prior experience38−40 coupling carboxylate-containing nicotine haptens to carrier proteins, we tried to do the same in the present context. Thus, each enantiomer of 2 was acylated with succinic anhydride. However, activation of succinylated haptens and mixing with TT gave conjugates with low hapten densities. Consequently, we employed an alternative choreography (Scheme 1, inset), in which the carrier protein (rather than the hapten) is first succinylated.18,41,42 First, TT was treated with succinic anhydride in pH 8.65 Tris buffer. Then, SucTT was treated with EDC and either (−)- or (±)-2 in pH 5.80 MES buffer, with final dialysis against pH 7.4 PBS. At this point, we had separate batches of (−)- and (+)-3′-AmNicSucTT (hapten densities ∼44 and ∼41 by MALDI-TOF MS), ready for formulation with adjuvants. For the second rat study, these procedures were repeated in order to furnish (−)-, (+)-, and (±)-3′-AmNicSucTT (hapten densities ∼56, ∼55, and ∼67). Each hapten−protein conjugate was formulated with phosphorothioated CpG ODN 182643−45 (Eurofins) and Alhydrogel 2% (InvivoGen). Vaccines prepared in this manner contained 100 μg of conjugate, 100 μg of CpG, and 100 μL of Alhydrogel per 200 μL of complete formulation.46 Wistar-

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DISCUSSION AND CONCLUSIONS The results of ELISA (first rat study) are summarized in Figure 2. By bleed 2 (day 49), titers were approximately 100000. Furthermore, cross-reactive ELISA results demonstrate an B

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Figure 2. Average ELISA titers for plasma from (−)-, or (+)-3′-AmNicSucTT groups (n = 12; first rat study). Each animal was assayed in duplicate; error bars are SEM. Left panel: Rat plasma on (−)-3′-AmNicSucBSA coated plates. Two-way repeated measures ANOVA (treatment: F1,22 = 17.29, ***P < 0.001). Right panel: Rat plasma on (+)-3′-AmNicSucBSA coated plates. Two-way repeated measures ANOVA (treatment: F1,22 = 15.29, ***P < 0.001).

Table 1. Anti-(−)-nicotine Antibody Binding Affinities and Concentrations from Radioimmunoassay (RIA) bleed 2

bleed 3

vaccinea

Kd,avg (nM)b

[Ab]avg (μg/mL)b

X (g/μmol)c

Kd,avg (nM)b

[Ab]avg (μg/mL)b

X (g/μmol)c

(−)-3′-AmNicSucTT (+)-3′-AmNicSucTT

23.9 ± 3.2 23.8 ± 3.2

47.5 ± 6.6 12.0 ± 1.2

1.99 0.50

250 ± 22 111 ± 9

118 ± 2.7 30.4 ± 2.6

0.47 0.27

a

Formulated with CpG ODN 1826 and Alhydrogel. bValues are average (with SEM) Kd and [Ab] determined by RIA using (−)-[N-methyl-3H]nicotine and pooled rat plasma samples (n = 12; first rat study) assayed in triplicate. cX is the quotient of [Ab]avg divided by Kd,avg. Units for X (μg/ mL/nM) have been reduced to g/μmol.

others,48 who reported that a human antinicotine mAb bound (+)-nicotine with slightly higher affinity than (−)-nicotine. Incidentally, this mAb (Nic12) was derived from another clinically evaluated nicotine vaccine that fell short in phase II.49 The seemingly counterintuitive result for the measured Kd,avg values may be rationalized by bearing in mind that nicotine (unlike cocaine and heroin) possesses greater conformational flexibility because it consists of two heterocyclic rings joined via a single carbon−carbon bond. Either enantiomer of nicotine can adopt an appropriate conformation suitable for making critical binding interactions with an antibody’s binding site. These include the pyridyl nitrogen serving as a hydrogen-bond acceptor and the pyrrolidinium nitrogen engaging in charge− charge and/or cation−π interaction(s). Linker attachment can also play a role in directing antihapten antibody quantity and quality. For instance, if morphine is coupled through its 3-position to a carrier protein, codeine (3methylmorphine) is a more effective inhibitor (than morphine) of the resultant morphine antiserum.50 In the present study, nicotine is linked to protein via the 3′-position on its pyrrolidine ring. This 3′-linkage may impose constraints on antibody elicitation such that the measured antinicotine antibodies, while being of lower quantity, nevertheless exhibit slightly higher affinity for free (−)-nicotine than antibodies elicited by the (−)-3′-AmNic conjugate. As a means for reconciling this seeming discrepancy between average antibody affinity and average antibody concentration, we propose the use of a composite parameter, X, defined as the ratio of [Ab]avg over Kd,avg for a given pool of antisera. Supposing that two ways for improving vaccine performance are to elicit higher [Ab]avg (antibody abundance) and lower Kd,avg (antibody utility) values, then as improvements are made in either/or/both of these terms, the ratio (X) will become larger. Thus, for bleed 3, (−)-group’s X = 0.47, while (+)-group’s X = 0.27. Conceptually speaking, the aim is to optimize protein design for a given ligand target (“antibody efficiency”); the inverse is widely applied in medicinal chemistry: optimizing a ligand design for a given protein target

approximately 3−5-fold difference in titers, suggesting that plasma antibodies produced by these two groups of rats possess a measurable level of enantiodifferentiation. Importantly, plasma from the (−)-3′-AmNicSucTT group has superior capacity to bind to natural (−)-nicotine displayed by (−)-3′AmNicSucBSA. Implications of this phenomenon will be discussed below. Radioimmunoassay provides a means for determining the average binding affinity and average antibody concentration for a soluble ligand. Because the ligand is soluble and free to associate/dissociate in the analysis milieu, it offers significant advantage over ELISA, in which the ligand is immobilized on the plate surface, not to mention conjugated to a carrier protein (e.g., BSA). Thus, the equilibrium environment simulated in a RIA experiment much more closely mimics that of free nicotine that would distribute in the blood and brain during tobacco use. It behooves researchers in the field to routinely incorporate RIA to evaluate the immunogenic efficacy of drug of abuse vaccine formulations. From each binding curve generated (see Supporting Information), average binding affinity (Kd,avg) and average antinicotine antibody concentration ([Ab]avg) were obtained (Table 1). By bleed 2, an approximately 4-fold difference in [Ab]avg was observed between the (−)-3′-AmNicSucTT group and the (+)-3′-AmNicSucTT group. The approximately 4-fold difference in antibody concentration observed in bleed 2 was maintained in bleed 3. It is interesting to note that ELISA results showed 3−5-fold difference in titers; the results of these two immunoassays (ELISA and RIA) correlate with one other. However, we were surprised to see higher affinities for (−)-nicotine in the (+)-group. Scrutiny of the ELISA results and the RIA results might suggest conflicting interpretations. In particular, it seems surprising that the binding affinity for nicotine is superior (lower Kd,avg at bleed 3) for the rats that received the (+)-vaccine. The (+)-group’s plasma antibodies (Kd,avg 111 nM) have 2-fold higher binding affinity for (−)-nicotine than the (−)-group’s plasma antibodies (Kd,avg 250 nM). This feature is congruent with the findings of C

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(e.g., “ligand efficiency”). That said, the ratio (X) is a means for assessing antibody efficiency and hence vaccine efficacy. Comparing X values for bleeds 2 and 3, we see the negative impact of decreasing K d,avg over the time course of immunization. While RIA affinity measurements to (−)-nicotine were never undertaken for the clinical vaccine, it is tempting to speculate that, besides antibody titer, untoward antibody affinity decrease may have also contributed to the vaccine’s inability to achieve the desired clinical outcomes. To validate our estimation (by algebraic extrapolation51) that racemic (±)-3′-AmNicSucTT would only be ∼63% as effective as enantiopure (−)-3′-AmNicSucTT, we conducted a second rat immunization study in which groups (n = 10) were administered (−)-, (+)-, or (±)-3′-AmNicSucTT in formulation with phosphorothioated CpG ODN 1826 and Alhydrogel. Average bleed 1 ELISA titers for the (−)-3′-AmNicSucTT group were similar (first study, ∼66000; second study, ∼60000). Importantly, however, the average bleed 1 ELISA titers (Figure 3) for the (±)-3′-AmNicSucTT group were only

Chart 1. Structure of Nicotine Hapten (−)-CNI

Recently, others have suggested that coadministration of two (“bivalent”) or three (“trivalent”) structurally distinct nicotine immunogens may be a viable means to compensate for suboptimal nicotine vaccine performance.20−23 The individual nicotine immunogens elicited antibodies with nonoverlapping specificities. If one accepts that (−)-3′-AmNic is structurally distinct from (+)-3′-AmNic, then a mixture of the two (racemic (±)-3′-AmNic) would itself constitute a “bivalent” nicotine vaccine. The data herein demonstrate that these two immunogens are not equally capable of eliciting anti-(−)-nicotine antibodies, and a mixture of the two would be inferior to pure (−)-3′-AmNic. Empirically, we find that the (+)-vaccine is only one-fourth as effective as the (−)-vaccine (4-fold lower [Ab]avg, Table 1) and the (±)-vaccine is only ∼60% as effective as the (−)-vaccine (Figure 3). Because structurally distinct immunogens activate distinct populations of antibody-producing B cells, we reckon the racemic hapten approach (as in (±)-3′-AmNic-rEPA) to be a readily remediable limitation. Instead, by using a vaccine consisting of a single optimized immunogen, bearing solely (−)-nicotine hapten, we target (with laser-like precision) a relatively tighter distribution of antibody-producing B cells. Serological analyses of groups of rats vaccinated with (−)-, (+)-, or (±)-3′-AmNic conjugates suggest that a vaccine using an enantiopure (−)-hapten may elicit plasma antibodies with superior capacity to bind natural (−)-nicotine. This will undoubtedly be a critical vaccine design consideration that is heeded in nicotine vaccines following (±)-3′-AmNic-rEPA. Future research in the nicotine vaccine field should incorporate this significant aspect in order to produce the most efficacious formulations capable of eliciting protective immunity against (−)-nicotine. A logical next step will be to correlate these serological data with in vivo efficacy data (for example, by measuring brain/plasma nicotine concentrations following acute nicotine administration). By taking full advantage of the capacity of antibodies to enantiodifferentiate, in combination with hapten conformational rigidity (e.g., via (−)-CNI), we anticipate a maximal impact on the ability of such a vaccine to elicit antibodies with even greater capacity to sequester (−)-nicotine. Additionally, for situations in which [Ab]avg and Kd,avg do not neatly correlate, use of the composite parameter, X, may be of benefit for ranking the effectiveness of vaccine formulations being tested. Put simply, one should seek to maximize X, by increasing [Ab]avg, decreasing Kd,avg, or both. The results of investigations along these lines will be reported in due course.

Figure 3. Average ELISA titers for plasma from (−)-, (+)-, or (±)-3′AmNicSucTT groups (n = 10; second rat study; bleed (1) on (−)- or (+)-3′-AmNicSucBSA coated plates. Each animal was assayed in duplicate; error bars are SEM. One-way ANOVA (F5,54 = 22.47, P < 0.001); Sidak test (***P < 0.001, ##P < 0.05).

∼61% as high as titers for the (−)-3′-AmNicSucTT group. All else with regard to vaccine composition and dose being equal, there is significant gain achieved by using enantiopure hapten. The present results validate the notion that one must factor chirality into vaccine design, taking care to mimic the natural stereochemistry of the small molecule, be it (−)-cocaine, (−)-heroin, (−)-nicotine, or any other intended target. The situation is more nuanced in the case of nicotine, which presumably has much to do with the conformational flexibility of nicotine itself, as well as linker placement. As a ligand, nicotine can bind in a variety of orientations within the binding site of an antinicotine antibody. By contrast, the additional structural constraints in cocaine or heroin impose greater conformational rigidity, and one observes unsurprising results for relative binding affinities of antibodies for natural versus unnatural enantiomers of these illicit drugs. With regard to nicotine, we have previously made the case that one may gain advantage by “building in” further conformational rigidity in hapten design, by recourse to ring fusion, as in CNI.39 We contend that an enantiopure version of CNI (Chart 1) would combine the benefits of conformational rigidity and optical purity, with synergistic effect for eliciting optimal antibody defense against (−)-nicotine in the bloodstream.

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EXPERIMENTAL SECTION

Chemistry. All reactions were carried out under an argon atmosphere with dry solvents using anhydrous conditions unless otherwise stated. Most chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. L-(−)-[N-Methyl-3H]-nicotine with specific activity of 81.7 Ci/mmol (product no. NET827250UC) was purchased from PerkinElmer (Boston, MA). BSA (product no. D

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column. Each cycle was 5 min. Peak 1 was collected by UV detection during 2.96−3.35 min, and peak 2 was collected during 3.78−4.30 min. All peak 1 and peak 2 collections were combined separately and rotary evaporated to dryness at 35 °C (5−10 mmHg). Peak 1: (−)-2, 2.712 min (2.875 min); 241.3 mg, >99% ee; [α]D22, −31.2 (c 0.25, MeOH). Peak 2: (+)-2, 3.664 min (3.860 min); 272.5 mg, >99% ee; [α]D22 +30.6 (c 0.15, MeOH). Hapten−Protein Conjugations. A solution of BSA (∼4 mg/mL) or TT (∼2 mg/mL) in pH 8.65 Tris buffer was treated with succinic anhydride (1.0 M in DMSO, 750−3000 equiv) at room temperature for 1 h. The bulk of the material was dialyzed against pH 5.80 MES buffer for the subsequent step (below), while a small portion of the material was dialyzed against PBS pH 7.4 for BCA assay and MALDITOF analysis. Succinyl group densities of 47 per BSA or 53 per TT were obtained. A solution of SucBSA (∼4 mg/mL) or SucTT (∼2 mg/mL) in pH 5.80 MES buffer was treated with (−)-2, (+)-2, or (±)-2 (0.2 M in H2O, 100−800 equiv) and EDC (2.0 M in H2O, 1000 equiv) at room temperature for 20−24 h. Final dialyses against PBS pH 7.4 removed excess reagents. Hapten densities of 67−68 per SucBSA, 41−44 per SucTT (first study), and 55−67 per SucTT (second study) were obtained. Concentrations of proteins and hapten−protein conjugates were determined using a BCA Protein Assay Kit (Thermo Scientific, product no. 23225) according to manufacturer’s directions. Biology. Each hapten−protein conjugate was mixed with phosphorothioated CpG ODN 1826 (Eurofins MWG Operon) and diluted to 1.0 mg/mL in pH 7.4 PBS. Then, an equal volume of Alhydrogel 2% (vac-alu-50, InvivoGen) was added dropwise, followed by 10 min of gentle inversion. Vaccines prepared in this manner contained 100 μg of conjugate, 100 μg of CpG, and 100 μL of Alhydrogel per 200 μL of complete formulation. All animal care and use was performed according to NIH guidelines and in compliance with protocols approved by the Institutional Animal Care and Use Committee at Behavioral Pharma, Inc. Wistar-derived male rats (n = 10−12 per group, 250−300 g) were purchased from Harlan (Indiana, USA) and assigned randomly to (−)-, (+)-, or (±)-groups. Rats were given free access to food and water during the immunization schedule, which consisted of three (200 μL) intramuscular injections on days 0, 21, and 42. On days 28 and 49, plasma samples were obtained by tail vein bleed. On day 63, animals were anesthetized, bled by cardiac puncture, and euthanized. ELISA. Plates (96-well Costar 3690) were coated with either (−)-3′-AmNicSucBSA or (+)-3′-AmNicSucBSA diluted in PBS pH 7.2 (5 μg/mL, 25 μL/well) and incubated at 37 °C overnight. Then, plates were fixed with MeOH (50 μL/well) at room temperature for 5 min, blocked with 5% nonfat powdered milk in PBS pH 7.2 (50 μL/well) 37 °C for 30 min, and charged with 2% BSA in PBS pH 7.2 (25 μL/ well). Plasma samples (diluted 1:100 in 1% BSA in PBS pH 7.2) were added to the first column and serially diluted across the plate. Plates were incubated at 37 °C for 90 min, washed with H2O, and treated with secondary antibody (goat-antirat Ig(H+L)-HRP, Southern Biotech, catalogue no. 3010-05, diluted 1:5000 in 2% BSA in PBS pH 7.2, 25 μL/well). Plates were incubated at 37 °C for 30 min, washed with H2O, treated with developing reagent (TMB Pierce Substrate Kit, 50 μL/well), and incubated at room temperature in the dark for 10−20 min. Color development was halted by addition of 2 M H2SO4 (50 μL/well), and plates were read on a SpectraMax M2e (Molecular Devices, Sunnyvale, CA) at 450 nm. Midpoint titers were obtained using Microsoft Excel and GraphPad Prism. RIA. Competitive RIA was carried out in a 5 kDa MWCO Equilibrium Dialyzer-96 (Harvard Apparatus, Holliston, MA) to allow easy separation of bound and free L-[N-methyl-3H]-nicotine tracer; specific activity = 81.7 Ci/mmol (PerkinElmer, Boston, MA). Pooled rat plasma were diluted in 2% BSA to a concentration that bound ∼50% of ∼30000 dpm of 3H-nicotine tracer. Each sample chamber was loaded with 75 μL of diluted plasma and 75 μL of radiolabeled tracer (∼30000 dpm), and each buffer chamber was loaded with 150 μL of unlabeled (−)-nicotine at varying concentrations in 1% BSA. Chamber contents were equilibrated at room temperature for at least 22 h. A 75 μL aliquot was removed from each sample/buffer chamber

77110) and EDC (product no. 22980) were purchased from Thermo Scientific Pierce Biotechnology (Rockford, IL). Tetanus toxoid (TT) was purchased from Statens Serum Institut (Copenhagen, Denmark). Yields refer to chromatographically (HPLC) and spectroscopically (1H NMR) homogeneous (≥95%) materials. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as the visualizing agent. Flash column chromatography was performed using E. Merck silica gel (60, particle size 0.040−0.063 mm). Organic solvents were concentrated on a rotary evaporator under reduced pressure, followed by further evacuation using a dual stage mechanical pump. NMR spectra were recorded on a Bruker Avance III HD with DCH CryoProbe (600 MHz) instrument or a Bruker BioSpin DRX (500 MHz) instrument and calibrated using residual undeuterated solvent as an internal reference (CD3OD @ δ 4.87 ppm 1H NMR, δ 49.00 ppm 13C NMR). The following abbreviations (or combinations thereof) are used to explain 1H NMR multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet. High-resolution mass spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization time-of-flight reflectron experiments. IR spectra were recorded on a Thermo Scientific Nicolet 380 FTIR spectrometer. (±)-trans-3′-Aminomethylnicotine ((±)-2). A suspension of (±)-trans-4-cotininecarboxylic acid (±)-1 (5.18 g, 23.5 mmol) and ammonium bicarbonate (2.60 g, 32.9 mmol) in CH3CN (104 mL) was treated with a solution of pyridine (0.95 mL, 11.8 mmol) in CH3CN (5 mL). The mixture was stirred at room temperature for 10 min, then treated with a solution of di-tert-butyl dicarbonate (7.57 mL, 32.9 mmol) in CH3CN (21 mL). The mixture was stirred at room temperature for 15 min, then treated with H2O (2.6 mL). The mixture was stirred at room temperature for 4 h, then at 40 °C for 30 min. The mixture was concentrated in vacuo. Purification by silica gel chromatography (10:1 CH2Cl2/MeOH) afforded (±)-S1 (4.33 g, 84%) as a white solid. Rf = 0.20 (silica gel, 10:1 CH2Cl2/MeOH). IR (neat) νmax 3304, 1651, 1393, 1299, 1128, 713 cm−1. 1H NMR (600 MHz, CD3OD) δ 8.57 (dd, J = 4.9, 1.6 Hz, 1 H), 8.53 (d, J = 2.0 Hz, 1 H), 7.82 (dt, J = 7.9, 1.9 Hz, 1 H), 7.54 (dd, J = 8.1, 4.7 Hz, 1 H), 4.87−4.86 (m, 3 H), 3.08 (ddd, J = 9.6, 7.9, 6.5 Hz, 1 H), 2.86 (dd, J = 17.0, 9.5 Hz, 1 H), 2.70 (dd, J = 17.1, 8.0 Hz, 1 H), 2.66 (s, 3 H). 13C NMR (150 MHz, CD3OD) δ 176.1, 175.8, 150.5, 149.4, 137.2, 136.9, 125.9, 66.5, 47.9, 35.5, 28.7. HRMS (ESI-TOF) calcd for C11H13N3O2H+ [M + H+], 220.1080; found, 220.1082. A solution of (±)-S1 (4.13 g, 18.8 mmol) in toluene (261 mL) was treated with a solution of Red-Al (65 wt % in toluene, 25.4 mL, 84.8 mmol). The mixture was stirred at room temperature for 18 h, then poured into a suspension of Celite (4.13 g) and Darco G60 charcoal (2.07 g) in H2O (41 mL). The quenched reaction mixture was filtered, and the filter cake was washed with H2O (2 × 8 mL). The filtrate was transferred to a separatory funnel, and layers were separated. The aqueous layer was concentrated in vacuo. Purification by silica gel chromatography (85:15:1 CH2Cl2/MeOH/NH4OH) afforded (±)-2 (0.68 g, 19%) as a pale-yellow syrup. Rf = 0.13 (silica gel, 85:15:1 CH2Cl2/MeOH/ NH4OH). IR (neat) νmax 3282, 1646, 1578, 1432, 1320, 1025, 713 cm−1. 1H NMR (500 MHz, CD3OD) δ 8.56 (d, J = 1.9 Hz, 1 H), 8.50 (dd, J = 4.9, 1.5 Hz, 1 H), 7.92 (dt, J = 8.0, 1.8 Hz, 1 H), 7.48 (dd, J = 7.8, 4.9 Hz, 1 H), 4.87 (s, 2 H), 3.29−3.25 (m, 1 H), 2.88 (d, J = 8.0 Hz, 1 H), 2.69 (dd, J = 12.7, 4.5 Hz, 1 H), 2.60 (dd, J = 12.7, 8.1 Hz, 1 H), 2.45 (q, J = 8.9 Hz, 1 H), 2.26−2.21 (m, 2 H), 2.16 (s, 3 H), 1.78−1.72 (m, 1 H). 13C NMR (125 MHz, CD3OD) δ 150.3, 149.5, 139.3, 137.9, 125.5, 74.2, 56.8, 51.8, 45.2, 40.6, 28.4. HRMS (ESITOF) calcd for C11H17N3H+ [M + H+], 192.1495; found, 192.1496. Chiral SFC Separation of (−)-2 and (+)-2. Column, Chiralpak AD-H, 21 mm × 250 mm; mobile phase, 85:15 CO2/MeOH (0.05% Et2NH); pressure, 120 bar; flow rate, 60.0 mL/min; injection amount, 0.8 mL (∼24 mg); temperature, 36 °C; wavelength, 260 nm; sample preparation, ∼600 mg of (±)-2 was dissolved in ∼20 mL MeOH; operational procedure, a preparative Berger MultiGram II SFC (Waters Inc., Milford, MA) instrument was equilibrated at conditions above. Dual Varian SD-1 pumps delivered CO2 (liquid) and MeOH (containing 0.05% Et2NH). Injections of 0.8 mL (∼24 mg compound, total 28 injections) onto a Chiralpak AD-H 21 mm ID × 250 mm E

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and added to 5 mL of scintillation fluid (Ecolite(+), MP Biomedicals, Santa Ana, CA). Radioactivity (dpm) was measured in a Beckman LS 6500 scintillation counter. Kd,avg and [Ab]avg values were obtained using Microsoft Excel and GraphPad Prism.

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ASSOCIATED CONTENT

S Supporting Information *

Tabular summary of nicotine vaccine clinical candidates, assay results, NMR spectra, HPLC chromatograms, and MALDITOF spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (858)-785-2516. Fax: (858) 784-2595. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jeff Elleraas at Pfizer La Jolla for separating (−)-2 and (+)-2 using chiral SFC, Mark Hixon at Takeda California for useful discussion regarding composite parameter X, and Nirvan Rouzbeh for technical assistance with rat injections and bleeds. This work was supported by Tobacco-Related Disease Research Program (TRDRP) grant no. 20XT-0156 (to K.D.J.). This is manuscript no. 27033 from The Scripps Research Institute.

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ABBREVIATIONS USED 3′-AmNic, 3′-aminomethylnicotine; BSA, bovine serum albumin; CpG ODN, cytosine-phosphorothioate-guanine oligodeoxynucleotide; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; MES, 2-(Nmorpholino)ethane-sulfonic acid; Suc, succinyl; TT, tetanus toxoid

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