key: cord-0776579-j0rudy3i authors: Sulima, Agnieszka; Li, Fuying; Morgan, Jeffrey Brian; Truong, Phong; Antoline, Joshua F. G.; Oertel, Therese; Barrientos, Rodell C.; Torres, Oscar B.; Beck, Zoltan; Imler, Gregory H.; Deschamps, Jeffrey R.; Matyas, Gary R.; Jacobson, Arthur E.; Rice, Kenner C. title: Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines date: 2022-02-25 journal: Molecules DOI: 10.3390/molecules27051553 sha: ba25f5a7b5e1acdbb3d5b188026778cf97e813b4 doc_id: 776579 cord_uid: j0rudy3i In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)–hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT–hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT–hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness. Approximately 130,000 Americans died from drug overdose due to heroin from 1999 to 2019, an average of more than four deaths for every 100,000 people [1] . The combined Initially, the synthesis of N-((7R,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7ahexahydro-1H-4,12-methanobenzofuro [3,2-e] isoquinolin-11-yl)-3-(tritylthio)propanamide (1-AmidoMorHap epimer, 1, Figure 1 ) began with morphine as the starting material and followed the previously used route [19] . Briefly, the 6-amino compound 5 (Scheme 1) was prepared through 3-monoacetylation of morphine base using a modified Welsh procedure, followed by a Mitsunobu phthalimidation and removal of the phthalimide Initially, the synthesis of N-((7R,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7ahexahydro-1H-4,12-methanobenzofuro [3,2-e] isoquinolin-11-yl)-3-(tritylthio)propanamide (1-AmidoMorHap epimer, 1, Figure 1 ) began with morphine as the starting material and followed the previously used route [19] . Briefly, the 6-amino compound 5 (Scheme 1) was prepared through 3-monoacetylation of morphine base using a modified Welsh procedure, followed by a Mitsunobu phthalimidation and removal of the phthalimide protection [19] . Simultaneous acetylation of the 3-hydroxyl and 6-amino groups gave the intermediate 6 at an overall yield of 68% over the four steps (Scheme 1). protection [19] . Simultaneous acetylation of the 3-hydroxyl and 6-amino groups gave the intermediate 6 at an overall yield of 68% over the four steps (Scheme 1). Scheme 1. Synthesis of acetamido compound intermediate 6 from 6β-amino compound 5 [19] . With 6 in hand, we sought to introduce an amino group at the C1 position via nitration on an activated C1, followed by reduction of the nitro group. Many nitration conditions were attempted. Neither nitronium tetrafluoroborate in DMSO at room temperature nor sodium nitrate in acetic acid gave any nitration, while sodium nitrite in TFA decomposed the substrate 6. A classical HNO3/H2SO4 in nitromethane at −20 °C gave a mixture of the desired product 7, phenol 8, and over-nitrated product 9, likely the result of nitration of phenol 8 after loss of the hydrolytically unstable acetate (Scheme 2). Scheme 2. Over-nitration of intermediate 6. In an effort to avoid over-nitration, we opted to alter our C3 protection strategy (Scheme 3). Reasoning that the C2 position could be rendered sterically inaccessible using a bulky C3 protecting group, acetate 6 was hydrolyzed to free phenol 10 through basic methanolysis and re-protected as a TBS ether (11) . Nitration with bismuth nitrate gave the desired mono-nitrated compound 12 in moderate yield. After nitration, the TBS group was removed with TBAF to give phenol 8 at a yield of 92%, a 44% overall yield from phenol 10. With 6 in hand, we sought to introduce an amino group at the C1 position via nitration on an activated C1, followed by reduction of the nitro group. Many nitration conditions were attempted. Neither nitronium tetrafluoroborate in DMSO at room temperature nor sodium nitrate in acetic acid gave any nitration, while sodium nitrite in TFA decomposed the substrate 6. A classical HNO 3 /H 2 SO 4 in nitromethane at −20 • C gave a mixture of the desired product 7, phenol 8, and over-nitrated product 9, likely the result of nitration of phenol 8 after loss of the hydrolytically unstable acetate (Scheme 2). Molecules 2022, 26, x FOR PEER REVIEW 4 of 28 protection [19] . Simultaneous acetylation of the 3-hydroxyl and 6-amino groups gave the intermediate 6 at an overall yield of 68% over the four steps (Scheme 1). Scheme 1. Synthesis of acetamido compound intermediate 6 from 6β-amino compound 5 [19] . With 6 in hand, we sought to introduce an amino group at the C1 position via nitration on an activated C1, followed by reduction of the nitro group. Many nitration conditions were attempted. Neither nitronium tetrafluoroborate in DMSO at room temperature nor sodium nitrate in acetic acid gave any nitration, while sodium nitrite in TFA decomposed the substrate 6. A classical HNO3/H2SO4 in nitromethane at −20 °C gave a mixture of the desired product 7, phenol 8, and over-nitrated product 9, likely the result of nitration of phenol 8 after loss of the hydrolytically unstable acetate (Scheme 2). Scheme 2. Over-nitration of intermediate 6. In an effort to avoid over-nitration, we opted to alter our C3 protection strategy (Scheme 3). Reasoning that the C2 position could be rendered sterically inaccessible using a bulky C3 protecting group, acetate 6 was hydrolyzed to free phenol 10 through basic methanolysis and re-protected as a TBS ether (11) . Nitration with bismuth nitrate gave the desired mono-nitrated compound 12 in moderate yield. After nitration, the TBS group was removed with TBAF to give phenol 8 at a yield of 92%, a 44% overall yield from phenol 10. In an effort to avoid over-nitration, we opted to alter our C3 protection strategy (Scheme 3). Reasoning that the C2 position could be rendered sterically inaccessible using a bulky C3 protecting group, acetate 6 was hydrolyzed to free phenol 10 through basic methanolysis and re-protected as a TBS ether (11) . Nitration with bismuth nitrate gave the desired mono-nitrated compound 12 in moderate yield. After nitration, the TBS group was removed with TBAF to give phenol 8 at a yield of 92%, a 44% overall yield from phenol 10. While effective, we thought that a more stable protective group at the C3 position would aid the nitration step, and that codeine, rather than morphine, would be a better starting material. We found that nitration with methyl ether gave a much better yield than that of acetate and TBS ether-protected substrate. Compound 15 was synthesized at a yield of 74% over 3 steps following the procedure used to prepare 6 (Scheme 4) [23] . With compound 15 as the substrate, the nitration using the nitric acid and sulfuric acid mixture in nitromethane at −20 to −10 • C still gave an over-nitrated product, while nitration with bismuth nitrate yielded the desired nitro compound 16 at a yield of 88% without over-nitration. The absolute configuration of 16 was confirmed by X-ray crystallographic analysis ( Figure 2 ). The 1-nitro compound 16 was reduced with formamidinesulfinic acid (FSA) in basic solution to afford aniline 17. The methyl ether was cleaved with BBr 3 and the resulting hydrobromide salt was used directly without further purification due to its instability in solution. The resulting aminophenol was coupled with 3-(tritylthio)propanoic acid using O-(benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium tetrafluoroborate (TBTU) as the coupling reagent. Finally, the ester resulting from coupling Figure 2 . X-ray crystallographic structure determination of the absolute configuration of 16. Thermal ellipsoids in the plot are at the 50% probability level. The synthesis of hapten 2 (N-((7S,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide (1-AmidoMorHap, Figure 1 ) was accomplished in 8 steps from codeinone oxime (18, Scheme 5) [26] . Reduction of 18 with a five-fold excess of Red-Al in toluene at 70 °C afforded primary amine 19, which was acylated crude to give amide 20 at a yield of 75% over 2 steps. Using a smaller excess of Red-Al significantly diminished the yield of the final acylated product. While effective, we thought that a more stable protective group at the C3 position would aid the nitration step, and that codeine, rather than morphine, would be a better starting material. We found that nitration with methyl ether gave a much better yield than that of acetate and TBS ether-protected substrate. Compound 15 was synthesized at a yield of 74% over 3 steps following the procedure used to prepare 6 (Scheme 4) [23] . With compound 15 as the substrate, the nitration using the nitric acid and sulfuric acid mixture in nitromethane at −20 to −10 °C still gave an over-nitrated product, while nitration with bismuth nitrate yielded the desired nitro compound 16 at a yield of 88% without over-nitration. The absolute configuration of 16 was confirmed by X-ray crystallographic analysis ( Figure 2 ). The 1-nitro compound 16 was reduced with formamidinesulfinic acid (FSA) in basic solution to afford aniline 17. The methyl ether was cleaved with BBr3 and the resulting hydrobromide salt was used directly without further purification due to its instability in solution. The resulting aminophenol was coupled with 3-(tritylthio)propanoic acid using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) as the coupling reagent. Finally, the ester resulting from coupling with excess 3-(tritylthio)propanoic acid was hydrolyzed with NH4OH to give the desired hapten 1 at a yield of 31% (Scheme 4). The synthesis of hapten 2 (N-((7S,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7ahexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide (1-AmidoMorHap, Figure 1 ) was accomplished in 8 steps from codeinone oxime (18, Scheme 5) [26] . Reduction of 18 with a five-fold excess of Red-Al in toluene at 70 • C afforded primary amine 19, which was acylated crude to give amide 20 at a yield of 75% over 2 steps. Using a smaller excess of Red-Al significantly diminished the yield of the final acylated product. Following an analogous procedure to the synthesis of hapten 1, nitration of 20 with bismuth nitrate hydrate gave 21 in modest yield (Scheme 5). Utilizing a similar reduction/deprotection sequence that was used in the synthesis of 1 afforded a highly unstable amino alcohol that degraded upon standing and during purification. Attempts to take the crude alcohol through the acylation/hydrolysis sequence afforded the desired hapten 2, but the sequence was inconsistent, and the final hapten was extensively contaminated. Hypothesizing that the issue was oxidative instability and difficulty purifying the highly polar amino phenol, we sought to avoid having a free amino phenol present during the course of the synthesis, which necessitated replacing the methyl ether with a protecting group more amenable to late-stage removal. Cleavage of the C3-methyl (BBr 3 , −78-25 • C) followed by protection of the phenol as the tert-butyldiphenyl silyl ether gave 23 in good yield. Unfortunately, the silyl ether was labile under the strongly basic conditions of the FSA reduction. Switching to a tin chloride-mediated sodium borohydride reduction gave 24 followed by a TBTU coupling of the crude amine with the propionic acid linker affording amide 25 in moderate yield over two steps. Removal of the silyl group with tetrabutylammonium fluoride gave hapten 2 at a yield of 56%, a 6% overall yield from 18. Following an analogous procedure to the synthesis of hapten 1, nitration of 20 with bismuth nitrate hydrate gave 21 in modest yield (Scheme 5). Utilizing a similar reduction/deprotection sequence that was used in the synthesis of 1 afforded a highly unstable amino alcohol that degraded upon standing and during purification. Attempts to take the crude alcohol through the acylation/hydrolysis sequence afforded the desired hapten 2, but the sequence was inconsistent, and the final hapten was extensively contaminated. Hypothesizing that the issue was oxidative instability and difficulty purifying the highly polar amino phenol, we sought to avoid having a free amino phenol present during the course of the synthesis, which necessitated replacing the methyl ether with a protecting group more amenable to late-stage removal. Cleavage of the C3-methyl (BBr3, −78-25 °C) followed by protection of the phenol as the tert-butyldiphenyl silyl ether gave 23 in good yield. Unfortunately, the silyl ether was labile under the strongly basic conditions of the FSA reduction. Switching to a tin chloride-mediated sodium borohydride reduction gave 24 followed by a TBTU coupling of the crude amine with the propionic acid linker affording amide 25 in moderate yield over two steps. Removal of the silyl group with tetrabutylammonium fluoride gave hapten 2 at a yield of 56%, a 6% overall yield from 18. The dihydro analog of hapten 2 (N-((7S,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,5,6,7, 7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide, 1-AmidoDihydroMorHap, Figure 1 ) was synthesized from hydromorphone (Scheme 6). Reductive amination of hydromorphone with benzylamine gave secondary amine 26 in good yield. Hydrogenolysis of the benzyl group gave an intermediate (27) which underwent acylation to intermediate 28. Basic hydrolysis of the aryl acetate 28 gave phenol 29 in 70% over three steps. The phenol was protected as the TBS ether (30) and nitrated under acidic conditions (NaNO 2 /TFA) to give the C1-nitro compound 31 (Scheme 6). (tritylthio)propanamide, 1-AmidoDihydroMorHap, Figure 1 ) was synthesized from hydromorphone (Scheme 6). Reductive amination of hydromorphone with benzylamine gave secondary amine 26 in good yield. Hydrogenolysis of the benzyl group gave an intermediate (27) which underwent acylation to intermediate 28. Basic hydrolysis of the aryl acetate 28 gave phenol 29 in 70% over three steps. The phenol was protected as the TBS ether (30) and nitrated under acidic conditions (NaNO2/TFA) to give the C1-nitro compound 31 (Scheme 6). Removal of the phenolic protecting group gave 32, and the nitro moiety was reduced to the unstable amino compound 33 which was carried through a coupling/hydrolysis protocol to give hapten 3 in approximately 30% overall yield from hydromorphone. Removal of the phenolic protecting group gave 32, and the nitro moiety was reduced to the unstable amino compound 33 which was carried through a coupling/hydrolysis protocol to give hapten 3 in approximately 30% overall yield from hydromorphone. The epimer of 1-AmidoDihydroMorHap, N-((7R,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio) propanamide, Figure 1 ), was prepared in 7 steps from codeine at an overall yield of 19% (Scheme 7). Compound 16 (Scheme 4) served as the intermediate in the synthesis. Its absolute configuration was confirmed by X-ray crystallography ( Figure 2 ). The epimer of 1-AmidoDihydroMorHap, N-((7R,7aR)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide, Figure 1 ), was prepared in 7 steps from codeine at an overall yield of 19% (Scheme 7). Compound 16 (Scheme 4) served as the intermediate in the synthesis. Its absolute configuration was confirmed by X-ray crystallography ( Figure 2 ). Compound 16 was subjected to BBr3 demethylation of the C3-methoxy moiety to give 8, followed by formation of 34 by reduction of both the C-ring olefin and the C1-nitro to an amine using Pd/C. Coupling of the linker 3-(tritythio)propanoic acid and TBTU to the amine gave the desired compound hapten 4 at a yield of 36% over 3 steps from 16. The haptens were separately conjugated to tetanus toxoid (TT) carrier protein using the previously optimized method [18, 23, 24, 27] . Vaccines were adsorbed to aluminum hydroxide and mixed with Army Liposome Formulation (ALF43) adsorbed to aluminum hydroxide as an adjuvant. Immunogenicity was assessed by immunizing mice and collecting sera at weeks 0, 3, and 6. Antibodies to the immunizing haptens were measured using ELISA that used BSA-hapten conjugates as coating agents. Results showed that all of the haptens induced high antibody endpoint titers (>10 5 after the second vaccine dose) against their respective antigens ( Figure 3b ). This suggests that all of the vaccine candidates tested are immunogenic. Multiple reports have suggested that antibody endpoint titers may not predict efficacy of a vaccine against small molecules such as drugs of abuse [5, 23] . To this end, we assessed the efficacy of each vaccine candidate by in vivo hot plate assay. This assay measures the time it takes for the mouse to respond to a pain stimulus (heat). This has been used in the past to evaluate vaccines to drugs of abuse [27, 28] . Our results showed that among the conjugates tested, only TT-2 and TT-3 gave low % MPE values, suggesting that mice were protected from the antinociceptive effects of heroin ( Figure 3c ). The other Scheme 7. Synthesis of hapten 4 (1-AmidoDihydroMorHap epimer). Compound 16 was subjected to BBr 3 demethylation of the C3-methoxy moiety to give 8, followed by formation of 34 by reduction of both the C-ring olefin and the C1-nitro to an amine using Pd/C. Coupling of the linker 3-(tritythio)propanoic acid and TBTU to the amine gave the desired compound hapten 4 at a yield of 36% over 3 steps from 16. The haptens were separately conjugated to tetanus toxoid (TT) carrier protein using the previously optimized method [18, 23, 24, 27] . Vaccines were adsorbed to aluminum hydroxide and mixed with Army Liposome Formulation (ALF43) adsorbed to aluminum hydroxide as an adjuvant. Immunogenicity was assessed by immunizing mice and collecting sera at weeks 0, 3, and 6. Antibodies to the immunizing haptens were measured using ELISA that used BSA-hapten conjugates as coating agents. Results showed that all of the haptens induced high antibody endpoint titers (>10 5 after the second vaccine dose) against their respective antigens ( Figure 3b ). This suggests that all of the vaccine candidates tested are immunogenic. Multiple reports have suggested that antibody endpoint titers may not predict efficacy of a vaccine against small molecules such as drugs of abuse [5, 23] . To this end, we assessed the efficacy of each vaccine candidate by in vivo hot plate assay. This assay measures the time it takes for the mouse to respond to a pain stimulus (heat). This has been used in the past to evaluate vaccines to drugs of abuse [27, 28] . Our results showed that among the conjugates tested, only TT-2 and TT-3 gave low % MPE values, suggesting that mice were protected from the antinociceptive effects of heroin ( Figure 3c ). The other conjugates, TT-1 and TT-4, carrying epimeric haptens showed no significant difference with the unvaccinated controls ( Figure 3c ). While all TT-hapten conjugates induced high antibody endpoint titers to the targets, only TT-2 and TT-3 showed protection against heroin, suggesting that antibody titers may not predict the efficacy of heroin vaccines in vivo. This is consistent with previous reports [23, 29] . conjugates, TT-1 and TT-4, carrying epimeric haptens showed no significant difference with the unvaccinated controls ( Figure 3c ). While all TT-hapten conjugates induced high antibody endpoint titers to the targets, only TT-2 and TT-3 showed protection against heroin, suggesting that antibody titers may not predict the efficacy of heroin vaccines in vivo. This is consistent with previous reports [23, 29] . On week 10, mice received 1.0 mg/kg heroin (s.c.) and nociception was measured using the hot plate assay set at 54 °C. Response was reported in terms of %MPE. Results shown are the mean ± sem. One-way analysis of variance (ANOVA) with Dunnett's correction for multiple comparisons was used to determine statistical significance: ****, p < 0.0001, ***, p = 0.0001, vs. control group. Anti-heroin vaccines are thought to act by inducing antibodies that sequester the drugs in the periphery [4] [5] [6] . The sequestered drug is prevented from crossing the bloodbrain barrier due to increase in apparent size thus effectively blocking the drug's physiological effects. In vivo, heroin quickly hydrolyzes to 6-acetyl morphine (6-AM) and morphine. Studies suggested that the physiological effects of heroin are mainly due to heroin Balb/c mice (n = 10 per treatment group) were immunized with 50 µL of the TT-hapten formulation (10 µg TT-hapten containing ALF 50 mM MLV-PHAD and 30 µg Alhydrogel) at weeks 0, 3, and 6 and bled at weeks 0, 3, 6, and 8. (b) Anti-hapten ELISA of sera using BSA-hapten conjugates as coating agents. (c) Hot plate nociception assay. The color legends in (c) are the same as in (b). On week 10, mice received 1.0 mg/kg heroin (s.c.) and nociception was measured using the hot plate assay set at 54 • C. Response was reported in terms of %MPE. Results shown are the mean ± sem. One-way analysis of variance (ANOVA) with Dunnett's correction for multiple comparisons was used to determine statistical significance: ****, p < 0.0001, ***, p = 0.0001, vs. control group. Anti-heroin vaccines are thought to act by inducing antibodies that sequester the drugs in the periphery [4] [5] [6] . The sequestered drug is prevented from crossing the blood-brain barrier due to increase in apparent size thus effectively blocking the drug's physiological effects. In vivo, heroin quickly hydrolyzes to 6-acetyl morphine (6-AM) and morphine. Studies suggested that the physiological effects of heroin are mainly due to heroin and 6-AM with little contribution from other downstream metabolites such as morphine and the glycosylated metabolites [5, 30] . In vitro drug binding experiments using equilibrium dialysis and liquid chromatography tandem mass spectrometry (ED-LC-MS/MS) were performed to assess the drug sequestering potential of mice sera [22] . First, heroin and its two bioactive metabolites, 6-AM, and morphine were tested. Results showed that mice sera at week 8 after the first dose only TT-2 provided the highest binding to heroin (Figure 4a,b) . TT-1 and TT-3 showed slightly (fraction bound~0.4) but significantly higher than before the first dose (week 0). We arbitrarily defined a fraction bound value of <0.5 at low serum dilutions (1:200 or less) as weak binding. A similar trend was observed for the metabolite 6-AM where TT-2 and TT-3 showed the highest binding (fraction bound >0.4) (Figure 4c,d) . All conjugates tested did not bind morphine (Figure 4e,f) . These results are consistent with the in vivo efficacy data (Figure 3c) , where only TT-2 and TT-3 induced protective effects against heroin. In vitro drug binding experiments using equilibrium dialysis and liquid chromatography tandem mass spectrometry (ED-LC-MS/MS) were performed to assess the drug sequestering potential of mice sera [22] . First, heroin and its two bioactive metabolites, 6-AM, and morphine were tested. Results showed that mice sera at week 8 after the first dose only TT-2 provided the highest binding to heroin (Figure 4a,b) . TT-1 and TT-3 showed slightly (fraction bound ~0.4) but significantly higher than before the first dose (week 0). We arbitrarily defined a fraction bound value of <0.5 at low serum dilutions (1:200 or less) as weak binding. A similar trend was observed for the metabolite 6-AM where TT-2 and TT-3 showed the highest binding (fraction bound >0.4) (Figure 4c,d) . All conjugates tested did not bind morphine (Figure 4e We also tested the in vitro sequestration of drugs used to treat OUD, namely, buprenorphine, methadone, naloxone, and naltrexone. Results indicated that TT-1, TT-2, and TT-4 did not induce antibodies that can bind any of these drugs ( Figure 5 ). Only TT-3 induced antibodies that weakly bound buprenorphine (Figure 5b ). We also tested the in vitro sequestration of drugs used to treat OUD, namely, buprenorphine, methadone, naloxone, and naltrexone. Results indicated that TT-1, TT-2, and TT-4 did not induce antibodies that can bind any of these drugs ( Figure 5 ). Only TT-3 induced antibodies that weakly bound buprenorphine (Figure 5b ). The facial recognition hypothesis [16, 17] predicts that TT-2 and TT-3 would induce protection in vivo because of their resemblance to the target dug, heroin. Indeed, we observed that mice sera induced by these conjugates bound heroin and 6-AM (Figure 4a-d) . Interestingly, the epimers of haptens 1 and 4 failed to show binding to heroin or 6-AM. Heroin and 6-AM have an absolute configuration opposite that of the epimeric haptens 1 and 4 (Figure 4a-d) . This suggested that the configuration of the acetamide in the hapten at the C6 position is essential to induce antibodies that would bind heroin and 6-AM. This observation reinforced the facial recognition hypothesis that even subtle difference in the hapten structure, in this case, chirality at the C6 position, can vastly alter the specificity of the induced antibodies. The chirality of haptens and its effect on efficacy have been previously demonstrated for methamphetamine vaccines [5] . This was further visualized by constructing the Molecular Mechanics 2 (MM2)-optimized 3D models of the haptens and the target drugs ( Figure 6 ). It was observed that heroin and 6-AM resembled more closely the structures of haptens 2 and 3, than haptens 1 and 4, with emphasis on the substituent at the C6 position. The carbon-carbon double bonds in the morphinan ring did not significantly alter the 3D orientation of the haptens. The antibodies induced by TT-2 and TT-3 were notably weak (Figure 4 ). This may be because neither of these haptens exactly mimicked the three-dimensional orientation of the target drugs. Importantly, none of the conjugates induced antibodies that can bind morphine, methadone, naloxone, or naltrexone. This observation was expected because the structure of the haptens is distinct from the structure of these drugs [17] . Interestingly, we observed that TT-3 induced antibodies that weakly recognized buprenorphine (fraction bound~0.4 at 1:100 serum dilution). When modeled, the structure of buprenorphine has some resemblance to hapten 3 (Figure 1) , where the methoxy group in the C6 position of buprenorphine is projected away from the linker attachment site, and thus a part of the "face" recognized by the immune system. This structural feature is unique to buprenorphine among the therapeutics tested (buprenorphine, naloxone, naltrexone, and methadone). This difference may be attributed to the absence of the hydroxyl group at the C9 position that is present in naloxone and naltrexone. This hydroxyl group induced geometric change in those two molecules that cannot occur in buprenorphine, leaving the latter with a unique "face" that slightly mimicked that of hapten 3. Naloxone and naltrexone share the same epoxymorphinan ring structure as the haptens and buprenorphine, but their structures in three-dimensional space suggest no significant similarity to the "face" of the haptens exposed to the immune system for recognition. Collectively, these results suggested that linker attachment at the C1 position is feasible without altering the cross-reactivity of the antibodies against OUD therapeutics. It is important that anti-opioid vaccines induce antibodies that will not cross-react with therapeutic drugs since the ultimate goal of a vaccine is to complement existing treatments [4] [5] [6] . The present study has limitations. First, this study only evaluated a vaccine based on the core morphinan structure with the linker attachment site at the C1 position. The effect of chirality at C6 in the epoxymorphinan ring in the hapten on the quality of the vaccineinduced antibodies should also be investigated where the linker attachment is on another site in the epoxymorphinan nucleus that projects a distinctly different face of the hapten. Second, this study did not attempt to evaluate the functional changes in the vaccineinduced antibodies. Recently, we have demonstrated that affinity maturation takes place. Conducting this same study for the TT-hapten conjugates described here in a future study might provide valuable insights as to how hapten-specific antibody affinity changes with time. Third, since only an in-bred mouse strain was used in this study, it would be interesting to see how these vaccines behave in other animal models, such as rats, rhesus macaques, and rabbits. All reagents were obtained from commercial sources and used without further purification. Melting points were determined on a Mettler Toledo MP70 Melting Point System (Mettler Toledo, Columbus, OH, USA) and are uncorrected. Proton nuclear magnetic resonance ( 1 H NMR, 400 MHz) and carbon nuclear magnetic resonance ( 13 C NMR, 100 MHz) spectra were recorded on a Varian 400 wide-bore spectrometer (Varian, Palo Alto, CA, USA) in CDCl3 (unless otherwise noted) with the values given in ppm and All reagents were obtained from commercial sources and used without further purification. Melting points were determined on a Mettler Toledo MP70 Melting Point System (Mettler Toledo, Columbus, OH, USA) and are uncorrected. Proton nuclear magnetic resonance ( 1 H NMR, 400 MHz) and carbon nuclear magnetic resonance ( 13 C NMR, 100 MHz) spectra were recorded on a Varian 400 wide-bore spectrometer (Varian, Palo Alto, CA, USA) in CDCl 3 (unless otherwise noted) with the values given in ppm and J (Hz) assignments of 1 H resonance coupling. For 1 H NMR spectra (CDCl 3 ), the residual solvent peak was used as the reference (7.26 ppm) while the central solvent peak was used as the NMR reference (77.0 ppm in CDCl 3 ). The high-resolution mass spectra were obtained on a Waters (Waters Corp., Milford, MA USA) LCT Premier Time-of-Flight (TOF) mass spectrometer using electrospray ionization (ESI). Thin-layer chromatography (TLC) was performed on 0.25 mm Analtech GHLF silica gel and was used to determine the completion of the reaction depending on the polarity of the compounds, using CHCl 3 : MeOH: 28% NH 4 OH, 90:9:1. Gas chromatography (GC) was performed on an Agilent Technologies 6850 Series system (Agilent Technologies, Santa Clara, CA, USA) equipped with Agilent Technologies 7683B series injector and Agilent Technologies 5975C VL MSD Triple-Axis detector. Flash column chromatography was performed using Teledyne Isco prepacked silica gel (RediSep) columns (Teledyne Isco, Lincoln, NE, USA). Elemental analyses were performed by Micro-Analysis, Inc, Wilmington, DE, USA, and were within ±0.4% for C, H, and N. 1 N-((4R,4aR,7R,7aR,12bS)-7-Acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide (1). To a solution of 17 (0.728 g, 2.05 mmol) in CHCl 3 (40 mL) at −78 • C was added BBr 3 (2.56 g, 10.25 mmol) and the solution was stirred at room temperature overnight. The reaction was quenched with MeOH and the solvent was removed in vacuo. MeOH (20 mL) was added and the solvent was removed in vacuo. The procedure was repeated three times. The crude product was treated with CHCl 3 (50 mL) and 3-(tritylthio)propanoic acid (1.07 g, 3.07 mmol), Et 3 N (1.04 g, 1.42 mL, 10.2 mmol), O-(benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium tetrafluoroborate (TBTU, 1.32 g, 4.1 mmol) were added. After stirring overnight, the solvent was removed in vacuo and the residue was dissolved in THF (20 mL) and 28% NH 4 OH (5 mL) was added. The mixture was heated to 50 • C overnight. The solvent was removed in vacuo, the residue was treated with CHCl 3 (100 mL) and the solution was washed with saturated NaHCO 3 and brine and dried over anhydrous Na 2 SO 4 . After filtration and removal of solvent in vacuo, the residue was purified by flash chromatography (CHCl 3 :MeOH:NH 4 OH, 90:9:1) to afford hapten 1 (0.43 g, 31.1% over 3 steps) as yellow solid. N-((4R,4aR,7S,7aR,12bS)-7-Acetamido-9-hydroxy-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide (2) . To a solution of silanol 25 (0.116 g, 0.127 mmol) in THF (2 mL) was added 1.0 M tetrabutylammonium fluoride solution (0.510 mL, 0.508 mmol, 4 equiv). The solution was stirred for 16 h and then concentrated in vacuo to give a brown oil. The oil was purified by silica gel chromatography (CHCl 3 :MeOH:28% NH 4 OH, 99:0.9:0.1 to 90:9:1) to give hapten 2 as a clear oil (0.048 g, 56%). The oil was lyophilized in tert-butanol to give hapten 2 as a white powder suitable for biological studies. N-((4R,4aR,7S,7aR,12bS)-7-acetamido-9-hydroxy-3-methyl-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12methanobenzofuro[3,2-e]isoquinolin-11-yl)-3-(tritylthio)propanamide (3). To a solution of 32 (400 mg, 1.07 mmol) in MeOH (20 mL) was added 10% (w/w) Pd/C (200 mg). The reaction mixture was stirred under H 2 atmosphere (40 psi) for 5 h at room temperature. The mixture was filtered through a Celite pad, and the solvent was concentrated in vacuo to give the crude intermediate amine 3 (N-((4R,4aR,7S,7aR,12bS )-11-amino-9-hydroxy-3-methyl-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)acetamide) as a yellow solid that was used without purification. The intermediate 3 was dissolved in DCM (20 mL) and Et 3 N (4.2 mL). 3-(tritylthio)propanoic acid (1.12 g, 3.21 mmol, 3 equiv) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 1.20 g, 3.74 mmol, 3.5 equiv) were added to the reaction mixture, and the mixture was stirred at room temperature for 16 h. The reaction was quenched with saturated NaHCO 3 (20 mL) and extracted with DCM (3 × 25 mL). The combined organic layers were washed with brine, dried over MgSO 4 , filtered, and solvent removed in vacuo. The residual material was dissolved in THF (10 mL) and 28% NH 4 OH (6 mL) and heated at 60 • C for 5 h. After cooling to room temperature, H 2 O (15 mL) was added, and the mixture was extracted with CHCl 3 :MeOH (6:1) (3 × 30 mL). The combined organic layers were dried over MgSO4, filtered, concentrated in vacuo, and the product was purified through silica gel chromatography (CHCl 3 :MeOH:28% NH 4 OH, 90:9:1)) to give hapten 3 (495 mg, 69% yield) as a pale brown solid, mp 170-174 • C. (4R,4aR,7R,7aR,12bS)-7-Acetamido-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro [3,2-e]isoquinolin-9-yl acetate (6) . To a solution of the known phenolic amine 5 [23] in CHCl 3 (30 mL) at 0 • C was added a solution of Et 3 N (4.4 g, 6 mL, 43 mmol) and Ac 2 O (2.35 g, 2.2 mL, 23 mmol), and these were stirred at room temperature overnight, washed with a dilute solution of NaHCO 3 , H 2 O and brine, successively and dried over anhydrous Na 2 SO 4 . The crude product was purified by flash chromatography (CHCl 3 :MeOH:28% NH 4 OH, 90:9:1) to give the acetate 6 (2.42 g, 68% over 4 steps from morphine) as a white foam. 1 13 N-((4R,4aR,7R,7aR,12bS)-9-((tert-Butyldimethylsilyl)oxy)-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)acetamide (11) . To a solution of acetate 6 (1.33 g, 3.6 mmol) in MeOH (20 mL) was added K 2 CO 3 powder and the mixture was stirred for 1 h at 50 • C. The mixture was filtered and the filtrate was concentrated to give the crude intermediate 10 (N-((4R,4aR,7R,7aR,12bS )-9-hydroxy-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)acetamide). The crude phenol 10 was dissolved in CH 2 Cl 2 (50 mL). Imidazole (294 mg, 4.3 mmol) was added, followed by TB-SCl (650 mg, 4.3 mmol). The solution was stirred at room temperature for 2 h, washed with H 2 O and brine and dried over anhydrous Na 2 SO 4 . After filtration and removal of solvent in vacuo, the crude product was purified by purified by flash chromatography ( N-((4R,4aR,7R,7aR,12bS)-9-((tert-Butyldimethylsilyl)oxy)-3-methyl-11-nitro-2,3,4,4a,7,7ahexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)acetamide (12) . To a solution of TBS ether 11 (1.46 g, 3.3 mmol) in AcOH (20 mL) was added bismuth (III) nitrate pentahydrate (1.93 g, 4.2 mmol) and the mixture was stirred at room temperature overnight. The solvent was evaporated, and the residue was basified with NH 4 OH. The mixture was extracted with CH 2 Cl 2 (3 × 30 mL) and the combined extracts were washed with brine and dried over anhydrous Na 2 SO 4 . After filtration and removal of solvent in vacuo, the crude product was purified by flash chromatography (CHCl 3 :MeOH:28% NH 4 OH, 95:4.5:0.5) to give 12 (0.8 g, 50.0%) as a brown oil. [ N-((4R,4aR,7R,7aR,12bS)-9-Methoxy-3-methyl-11-nitro-2,3,4,4a,7,7a-hexahydro-1H-4,12methanobenzofuro[3,2-e]isoquinolin-7-yl)acetamide (16) : To a solution of methyl ether 15 (1.2 g, 3.5 mmol) in AcOH (60 mL) was added bismuth(III) nitrate pentahydrate (2.04 g, 4.2 mmol) and the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was basified with NH 4 OH. The mixture was diluted with CH 2 Cl 2 (100 mL) and filtered on a pad of celite. The two-layer filtrate was separated, and the aqueous layer was extracted with CH 2 Cl 2 (3 × 30 mL) and the combined extracts were washed with brine and dried over anhydrous Na 2 SO 4 . After filtration and evaporation, the crude product was purified by flash chromatography (CHCl 3 :MeOH:NH 4 All animal studies were conducted under an approved animal use protocol in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACI)-accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals. Experiments involving animals adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, 8th edition [34] . Female BALB/c mice, 6-7 weeks of age (n = 10 per group, for a total of 50 mice) were immunized intramuscularly (i.m.) on weeks 0, 3, and 6 using 50 µL of the vaccine formulation. This dose contained 10 µg of TT−hapten (based on the protein content of the protein−hapten conjugate), 20 µg of PHAD ® in ALF43 [18, 23] , and 30 µg of aluminum in aluminum hydroxide (Alhydrogel ® ) in DPBS pH 7.4. Mice were bled for ELISA assay at weeks 0, 3, 6, and 8. Challenge studies were performed at week 10. Mice received 1.0 mg/kg of heroin (s.c.), and this route has been used previously to evaluate anti-opioid vaccines [27, 28] . Antinociceptive effects were assessed 15 min after each heroin injection using the hot plate assay [35] . This assay involved placing the mouse on a hot plate set at 54 • C. The latency to a nociceptive response was recorded, defined as the time elapsed until the mouse licked or flicked its hind paw. The latency times were measured with a cutoff time of 30 sec to prevent injury. Antinociception, measured as %MPE, was calculated using Equation (1) Drug binding was measured using ED-LC-MS/MS as described previously [22, 27, 28] . Briefly, mice sera were diluted with 0.05% BSA in DPBS, pH 7.4 (ED buffer) containing 3-5 mg/mL NaF and 5 nM of a drug. The following drugs were tested: heroin, 6-A.M., morphine, methadone, naloxone, buprenorphine, and methadone. An aliquot (100 µL) was seeded into sample chambers of the ED plate and the buffer chamber was filled with 300 µL of ED buffer. The plate was incubated at 4 • C and 300 rpm for 24 h in a thermomixer. Aliquots (90 µL) from sample and buffer chambers were pipetted out and analyzed by LC-MS/MS. Graphical and statistical analyses were performed using Prism 9 (GraphPad Software, San Diego, CA, USA). All results were reported as the mean ± standard error of the mean (sem). The chemical structures and 3D optimization were made using ChemDraw 18.1. The built-in MM2 method was used for geometry optimization and energy minimization. In this study, we described the synthesis of haptens 1-4, (Figure 1 ), produced the individual TT-hapten conjugates and evaluated their efficacy both in vivo and in vitro. We showed that only haptens 2 and 3 can induce protection against the effects of heroin in vivo. The epimeric congeners of these haptens, haptens 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-AM. None of the TT-hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine. These results highlight the importance of judicious hapten design toward effective heroin vaccines. 97 (s, 1H), 2.61 (dd, J = 20.4, 6.0 Hz, 1H), 2.53 (dd 7R,7aR,12bS)-11-Amino-9-methoxy-3-methyl-2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro 8%) as an off-white foam. 1 H NMR (400 MHz To a stirred suspension of codeinone oxime [26] (18, 3.52 g, 11.27 mmol) in anhydrous toluene (225 mL) was added Red-Al solution 65% in toluene, 56.34 mmol, 17.18 mL, 5 equiv). 4 , filtered, and concentrated in vacuo to give intermediate 19 30 g, 22.53 mmol, 2.13 mL, 2 equiv) was added in one portion. The solution was stirred for 16 h, warming to 25 • C. The solution was washed with saturated aqueous NaHCO 3 (25 mL, H 2 O (25 mL), dried over Na 2 SO 4 , filtered, and solvent removed in vacuo to give a yellow oil. The oil was purified by CDCl 3 ) δ 6.60 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 6.35 (d, J = 8.7 Hz, 1H), 5.43 (d, J = 9 Hz, 1H), 1.99 (s, 3H), 1.77 (dd, J = 12.6, 1.6 Hz, 1H); 13 C NMR (100 MHz Amide 20 (2.97 g, 8.72 mmol) was dissolved in acetic acid (174 mL) under N 2 . The solution was cooled to 10 • C and freshly ground bismuth The organic layer was separated and the aqueous layer was extracted with 10% MeOH:CHCl 3 (4 × 50 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated in vacuo to give a yellow solid. The solid was triturated with 1:1 acetone: Et 2 O, filtered J = 8.7 Hz, 1H), 5.51 (dd, J = 9.7, 2.9 Hz, 1H), 5.44 (dt, J = 9 7S,7aR,12bS)-9-Hydroxy-3-methyl-11-nitro-2,3,4,4a 1 H NMR (400 MHz Phenol 22 (0.20 g, 0.54 mmol) was dissolved in CHCl 3 (5 mL) under N 2 , and imidazole (0.15 g, 2.15 mmol, 4 equiv) and tert-butyldimethylsilyl chloride (0.30 g, 1.10 mmol, 0.28 mL, 2 equiv) were added sequentially. The solution was heated to 50 • C until TLC indicated that the starting material was consumed. The solution was cooled, concentrated and purified via silica gel chromatography 400 MHz; CDCl 3 ) δ 7.70-7.63 (m, 5H), 7.45-7.41 (m, 2H), 7.39-7.34 (m, 4H), 5.41 (d, J = 8.9 Hz, 1H) 41 (s, 3H), 2.23 (td, J = 12.4, 3.5 Hz, 1H), 1.96 (td, J = 12.5, 5.1 Hz, 1H), 1.76 (s, 3H), 1.54 (dd, J = 12.7, 1.6 Hz, 1H), 1.14 (s, 9H); 13 C NMR (100 MHz M+H] calcd for C 35 H 40 N 3 O 5 Si 610.2737, found 610.2740. Anal calcd for C 35 H 39 N 3 O 5 Si•0 The aqueous solution was extracted with 10% MeOH:CHCl 3 (3 × 10 mL), and the combined organic extracts were dried over Na 2 SO 4 , filtered, and concd in vacuo to give the crude intermediate aniline (24) as clear oil that was used without purification in the subsequent reaction. The crude aniline 24 was dissolved in CHCl 3 (5 mL), and triethylamine (0.109 g, 1.08 mmol, 0.150 mL, 4 equiv) and 3-(tritylthio)propanoic acid (0.189 g, 0.541 mmol, 2 equiv) were added sequentially. Na 2 SO 4 , filtered, and concentrated in vacuo to give a yellow oil. The oil was purified via silica gel chromatography Hz, 5H), 6.86 (s, 1H), 6.76 (s, 1H), 5.59 (d, J = 8.8 Hz, 1H) To a solution of hydromorphone [31,32] (2.28 g, 8.0 mmol) in DCE (60 mL) was added benzylamine (0.96 mL, 8.8 mmol, 1.1 equiv), acetic acid (0.91 mL, 16.0 mmol, 2 equiv), and NaBH(OAc) 3 (2.54 g, 1.2 mmol, 1.5 equiv), respectively. The mixture was stirred at room temperature for 24 h. Water (20 mL) was added to the reaction mixture, and it was basified to a pH~8 with 28% NH 4 OH. The organic layer was separated, and the aqueous layer was extracted with CHCl 3 (3 × 25 mL). The combined organic layers were washed with brine, dried over MgSO 4 , filtered CDCl 3 ) δ 7.38-7.19 (m, 5H) 36 (s, 3H), 2.35-2.24 (m, 2H), 2.16-2.12 (m, 1H), 1.84 (td HRMS (ESI) m/z 377.2224 To the intermediate 27, DCM (25 mL), triethylamine (5.7, 41.1 mmol), and Ac 2 O (5.1 mL, 54 mmol) were added, respectively. The reaction mixture was stirred for 16 h at 40 • C, cooled to room temperature, and H 2 O (30 mL) was added to the reaction mixture. The mixture was basified to pH~8.5 with 28% NH 4 OH and extracted with CHCl 3 :2-propanol (9:1) (3 × 30 mL). The combined organic layers were concentrated in vacuo to give a thick viscous syrupy amide 28 2.03-1.93 (m, 4H), 1.73-1.64 (m, 2H), 1.51-1 To a solution of 29 (1.0 g, 3.0 mmol) in DCM (25 mL) was added 1H-imidazole (0.23 g, 3.3 mmol, 1.1 equiv) and tert-butyldimethylsilyl chloride (TBS-Cl, 0.60 g, 4.0 mmol, 1.3 equiv) and the mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaCO 3 H, and extracted with CHCl 3 (3 × 25 mL). The combined organic layers were washed with brine, dried over MgSO 4 , filtered, the solvent removed in vacuo, and the product was purified through silica gel chromatography 91% yield) as a white solid, mp 176-178 • C To a stirred solution of 30 (860 mg, 1.94 mmol) in trifluoroacetic acid (10 mL) at 0 • C was added NaNO 2 (174 mg, 2.52 mmol, 1.3 equiv). The reaction mixture was stirred for 30 min at 0 • C, slowly basified to pH~8.5 with 28% NH 4 OH, and extracted with CHCl 3 :2-propanol (8:1) (4 × 25 mL). The combined organic layers were dried over MgSO 4 , filtered Hz, 1H), 3.15 (dd, J = 6.1, 2.9 Hz, 1H), 2.77 (dd, J = 20.3, 6.2 Hz, 1H), 2.52 (dd 4R,4aR,7S,7aR,12bS The combined organic layers were dried over MgSO 4 , filtered, concd in vacuo, and purified through silica gel chromatography HRMS (ESI) m/z 374 CDCl 3 ) δ 9.63 (br, 1H), 7.75 (s, 1H) To a solution of acetamide 8 (0.25 g, 0.67 mmol) in MeOH (50 mL) was added 10% Pd/C (50 mg). The mixture was hydrogenated under a hydrogen balloon overnight 25 (s, 1H), 3.02 (s, 1H), 2.61 (d, J = 18.0 Hz, 1H), 2.39 (d, J = 11.2 Hz, 1H), 2.25 (s, 3H), 2.08 (t, J = 12.0 Hz, 1H), 1.98 (m, 2H), 1.81 (s, 3H), 1.62 (m, 2H), 1.52 (d, J = 12.0 Hz, 1H), 1.35 (d HRMS (ESI) m/z: calcd for C 19 H 26 N 3 O 3 344 Single-crystal X-ray diffraction data on compound 16 (deposition CCDC 1554589the SHELXL suite (Bruker, SHELXL v2014 Briefly, TT was incubated with the SM(PEG) 2 cross-linker at a 1:1600 molar ratio for 2 h. Excess linker was removed using a Zeba spin column, and the protein content of the flow-through was quantified using a BCA assay kit. The excess haptens were removed by repeated dialysis against PBS, pH 7.4, at 4 • C. The TT-hapten conjugates were sterile filtered and quantified using the BCA protein assay. The number of haptens attached per TT molecule in each conjugate was measured using matrix The economic burden of opioid use disorder and fatal opioid overdose in the United States COVID-19 and the Opioid Epidemic: Two Public Health Emergencies That Intersect With Chronic Pain Vaccines to treat opioid use disorders and to reduce opioid overdoses Conjugate Vaccine Immunotherapy for Substance Use Disorder Vaccines to combat the opioid crisis: Vaccines that prevent opioids and other substances of abuse from entering the brain could effectively treat addiction and abuse Countermeasures for Preventing and Treating Opioid Overdose Developing Translational Vaccines against Heroin and Fentanyl through Investigation of Adjuvants and Stability Development of a Clinically Viable Heroin Vaccine Synthesis and immunological effects of C14-linked 4,5-epoxymorphinan analogues as novel heroin vaccine haptens Combatting Synthetic Designer Opioids: A Conjugate Vaccine Ablates Lethal Doses of Fentanyl Class Drugs A Fentanyl Vaccine Alters Fentanyl Distribution and Protects against Fentanyl-Induced Effects in Mice and Rats Opioid Dose-and Route-Dependent Efficacy of Oxycodone and Heroin Vaccines in Rats Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats A morphine conjugate vaccine attenuates the behavioral effects of morphine in rats Facial recognition of heroin vaccine opiates: Type 1 cross-reactivities of antibodies induced by hydrolytically stable haptenic surrogates of heroin, 6-acetylmorphine, and morphine Synthesis of a deuterated 6-AmHap internal standard for the determination of hapten density in a heroin vaccine drug product A Stable Heroin Analogue That Can Serve as a Vaccine Hapten to Induce Antibodies That Block the Effects of Heroin and Its Metabolites in Rodents and That Cross-React Immunologically with Related Drugs of Abuse Synthesis and immunological effects of heroin vaccines Liposomes containing monophosphoryl lipid A: A potent adjuvant system for inducing antibodies to heroin hapten analogs Induction of Highly Specific Antibodies to a Hapten but not to a Carrier Peptide by Immunization A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse but Not Antibody Titer or Affinity, of a Heroin Hapten Conjugate Vaccine Correlates with Increasing Hapten Densities on Tetanus Toxoid, but Not on CRM197 Carriers Characterization and optimization of heroin hapten-BSA conjugates: Method development for the synthesis of reproducible hapten-based vaccines Heroin-HIV-1 (H2) vaccine: Induction of dual immunologic effects with a heroin hapten-conjugate and an HIV-1 envelope V2 peptide with liposomal lipid A as an adjuvant The Acid-catalyzed Conversion of Codeinone to 8-Hydroxydihydrocodeinone Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice Novel Vaccine That Blunts Fentanyl Effects and Sequesters Ultrapotent Fentanyl Analogues Effect of currently approved carriers and adjuvants on the pre-clinical efficacy of a conjugate vaccine against oxycodone in mice and rats mu Opioid receptor-mediated G-protein activation by heroin metabolites: Evidence for greater efficacy of 6-monoacetylmorphine compared with morphine Practical and High-Yield Syntheses of Dihydromorphine from Tetrahydrothebaine and Efficient Syntheses of (8S)-8-Bromomorphide Towards an Efficient Preparation of Hydromorphone Practical Considerations for the Development of Vaccines Against Drugs of Abuse Guide for the Care and Use of Laboratory Animals Methods Used to Evaluate Pain Behaviors in Rodents The authors express their gratitude to the Mass Spectrometry Facility NIDDK, for mass spectral data, and we also thank Klaus Gawrisch and Walter Teague (Laboratory of Membrane Biochemistry and Biophysics, NIAAA), for NMR spectral data. The authors thank Taeshia Washington, Courtney Tucker, Alexander Duval, and Alexander Anderson for outstanding technical assistance. There is no conflict of interest to declare.Sample Availability: Some of the compounds may be available in mg quantities upon request from the Corresponding Authors.