Butyllithium

A series of eleven nicotine-like haptens were tested which differed in the site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to the carrier protein (Figure 1). All compounds were prepared by Pfizer, with the exception of Hapten 11 (trans-3̕-aminomethylnicotine, 3̕AmNic) which was purchased from Toronto Research Chemicals, North York, Ontario, Canada. The routes to synthesize all other haptens are summarized below. Additional synthetic procedures for all compounds can be found in Supplementary Information.
Haptens 1, 5, 6, 7 and 8 were all readily accessible via a common key intermediate, the boronate ester 2 (Figure 2); this intermediate could be readily synthesized as a single regioisomer from nicotine (1) utilizing the method of Hartwig [15 (link)] via iridium-mediated borylation. 2 was readily converted to bromide 3 using copper (II) bromide (CuBr₂) (Figure S1). Alcohol 6 was accessed from 2 via oxidative cleavage with hydrogen peroxide (H₂O₂) and acetic acid (AcOH); in this reaction, the presence of the acid was key to prevent the pyridine nitrogen being oxidized (Figure S16, Figure S17). 6 was then alkylated with bromide 7 and deprotected to provide Hapten 7 (Figure S18, Figure S19). The bromide 3 was used to synthesize a range of linking groups which could be readily appended via palladium coupling. Heck coupling of methyl acrylate followed by hydrogenation and hydrolysis allowed access to the sodium salt of Hapten 1 (Figure S2, Figure S3, Figure S4) while coupling with acrylonitrile and reduction allowed access to Hapten 6 (Figure S5, Figure S6, Figure S7).
Functionalisation at the 2- and 6-positions of nicotine was achieved using the methodology of Comins [16 (link)]. Lithiation with n-butyl lithium /lithium dimethylethanolamine (n-BuLi/LiDMAE) [17 (link)], followed by quenching into hexachloroethane gave a mixture of 2- and 6-substituted chloronicotines 13 and 14 (Figure S11) which were converted to Haptens 2 and 3 respectively by heating with the sodium alkoxide of ethanolamine (Figure S12, Figure S13). Lithiation at the 4-position was accomplished using Trimethylsilylmethyllithium (TMSCH ₂Li) [18 (link)]; quenching with hexachloroethane gave chloride 9 (Figure S14). Subsequent reaction with the sodium alkoxide of ethanolamine gave the 4-substituted Hapten 4 together with some undesired alcohol, 10 (Figure S15).
Ester 5 could be converted to the thioacetate Hapten 8 via reduction, mesylation and displacement with potassium thioacetate (Figure 3) (Figure S8, Figure S9, Figure S10). Cleavage of the acetate group was not carried out as the thiol was found to readily oxidise to the corresponding disulfide in situ and so the group was cleaved at the point of coupling to the carrier protein.
Palladium catalyzed coupling of zinc cyanide with bromide 3 allowed access to nitrile 17 which following hydrolysis, amide coupling and tert-butyloxycarbonyl (BOC) deprotection gave Hapten 5 (Figure 4) (Figure S24, Figure S25, Figure S26, Figure S27).
Hapten 9 was synthesized using a modification of the method of Ullrich [19 (link)].
Dibromopyridine 21 was metallated with lithium diisopropylamide (LDA) and quenched with methyl formate to give aldehyde 22 (Figure S28) which was converted through to ester 24 via Wittig reaction and hydrogenation over rhodium on alumina (Figure 5) (Figure S29, Figure S30). Ester 24 was reacted with the enolate of lactam 25 to give ketoamide 26 (Figure S31). Acidic hydrolysis with concomitant decarboxylation and condensation gave imine 27 (Figure S32) which was metallated with butyllithium leading to in situ ring-closure to give racemic spiro amine 28 (Figure S33). Methylation under reductive amination conditions gave 29 (Figure S34) while subsequent Heck coupling, hydrogenation and basic hydrolysis generated Hapten 9 as a racemic mixture (Figure S35, Figure S36, Figure S37).
Hapten 10 was synthesised via the commercially available bromide 32 utilising similar methodology to the previous compounds (Figure 6) (Figure S20, Figure S21, Figure S22, Figure S23).
The degree of hapten coupling to DT was measured for all haptens using a reverse-phase HPLC (RP-HPLC) method utilizing a Waters C-18 X-Bridge column with a gradient of 0.1% triethylamine (TEA): 0.1% TEA in methanol (Table 1). In this process, haptens were uncoupled from DT by acid hydrolysis and analysis of pre-hydrolysis and post-hydrolysis levels used to determine the degree of conjugation per unit loading of DT.

A solution of n-butyllithium in hexane (2.5 M, 8.8 ml, 22 mmol) was added dropwise over a period of 10 min to a solution of bis(2-bromo-4-(tert-butyl)phenyl)methane (4.38 g, 10 mmol) in anhydrous tetrahydrofuran (60 ml) at −78 °C under argon atmosphere. The solution was stirred for 1 h and then chlorodiphenylphosphine (4.84 g, 22 mmol) dissolved in anhydrous tetrahydrofuran (5 ml) was added dropwise. The mixture was continued to stir at −78 °C for 1 h and the system was heated to room temperature, and allowed to react overnight. The reaction was quenched with 2 N HCl solution. The mixture was extracted with ethyl acetate and water for 3 times, the combined organic phases were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel using EtOAc–petroleum ether mixture (1:50) as an eluent to afford the desired compound L11 as a white solid (4.2 g, 65% yield). Caution: The usage of n-butyllithium in hexane should be careful and protected by inert atmosphere owing to its easy flammability under air or moisture condition. Detailed synthetic procedures and reaction parameters for the other ligands were included in Supplementary Information.

Approximately 20 mg of Fe₃O₄ nanoparticles were added to a 1.6 M n-butyllithium (BuLi) anhydrous hexane solution to obtain various
nominal molar ratios of x = Li/Fe₃O₄ = 0, 0.5, 1, and 1.5. The mixtures were left to react for
3 days at room temperature under magnetic stirring in an argon-filled
glovebox environment, where the O₂ and H₂O levels
were kept below 1 ppm. After that, the remaining liquid was discarded,
and the particles were washed in hexane a couple of times to remove
any excess BuLi with the help of an external magnet to hasten the
sedimentation rate of the particles. The particles were then dried
to obtain powders for further analysis.