Bioconjugate Chem. 1996, 7, 271−273
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TECHNICAL NOTES Synthesis of Norbiotinamine and Its Derivatives Wojciech Szalecki* Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402. Received April 11, 1995X
Synthesis of norbiotinamine (II) and its reactive derivatives, isothiocyanates (III and IV), iodoacetamide (V), and maleimide (VI), are described. In addition, N-norbiotinyl-γ-L-glutamylamide (VII) was prepared as a potential alternative to the fixable polar tracer biocytin (biotinoyllysine).
The strong (Kd) ) 10-15 M-1) specific binding of biotin1 (I) to avidin has found widespread application in biochemical research, analyses, and separations. Many reagents are available for the coupling of biotin to different functional groups (1). All of these reagents are made by conversion of the carboxylic acid of biotin to an activated moiety, with the most widely used one being the N-hydroxysuccinimidyl ester. However, NHS esters have some limitations; for example, they do not react with aromatic amines. On the other hand, it is impractical to use biotinoyl chloride (even in situ), because the imidazole ring of biotin is acetylated. The goal of this work was to find alternatives to the above methods of biotinylation which would not have the limitation described. Since an isothiocyanate is more reactive than an NHS ester but less reactive than an acid chloride, we hypothesized that a biotin derivative with a terminal amino group in place of the carboxylic acid (compound II) could be used to prepare the isothiocyanate (III) and other reactive derivatives. Norbiotinamine (II) was obtained from biotin in 55% overall yield by modified Curtius rearrangement (2), using diphenylphosphoryl azide in t-BuOH, followed by hydrolysis of the intermediate tert-butoxycarbamate. Norbiotin isothiocyanate (III) was obtained in ca. 50% yield, after a reaction of the amine II with CS2, followed by oxidation of the thiouronium salt with Pb(NO3)2 (3). An alternative reaction using a less reactive TCDP substitute of thiophosgene (4, 5) gave the product III in 85% yield. We were also interested in making an extended version of the isothiocyanate. Thus, norbiotinamine (II) was treated with the NHS ester of Cbz-aminododecanoic (aminolauric) acid to form, after deprotection, N-norbiotinyl-12-aminododecanoamide (VIII). The reaction of the amine VIII with * Author to whom correspondence should be addressed. Telephone (503) 465-8300. Fax: (503) 344-6504. X Abstract published in Advance ACS Abstracts, February 1, 1996. 1The Chemical Abstracts name for biotin (I) is 1H-thieno[3,4d]imidazole-4-pentanoic acid, hexahydro-2-oxo-, [3aS(3aR,4β,6aR)]-. The following nomenclature was used in the paper: norbiotinamine (II) for the norbiotinylamine obtained from Curtius rearrangement of biotin, biotinoyl for the acyl residue of biotin, and biotinyl for the alkyl residue obtained by reduction of the carboxylic acid group of biotin. Other abbreviations: Boc, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl; DPPA, diphenylphosphoryl azide; NHS, N-hydroxysuccinimide; DCC, dicyclohexylcarbodiimide; SIA, succinimidyl iodoacetate; TCDP, 1,1′-thiocarbonyldi-2,2′-pyridone; DMF, dimethylformamide.
1043-1802/96/2907-0271$12.00/0
TCDP yielded the isothiocyanate IV, containing a 13atom aliphatic spacer. The iodoacetyl (V) and maleimide (VI) derivatives of norbiotinamine (II) were prepared, since reagents of this type are commonly used for alkylation, especially of protein thiols. The iodoacetamide (V) was made from the amine (II) using succinimidyl iodoacetate. Attempts to prepare biotinylmaleimide using the Mitsunobu reaction (6) on biotinol (7) gave a mixture of many unidentified products which precluded the possibility of isolation of the maleimide in a significant yield. A maleimide derivative was eventually synthesized in 63% yield by reaction of norbiotinamine with succinimidyl β-maleimidopropionate to obtain N-norbiotinyl-β-maleimidopropionylamide (VI). Norbiotinamine (II) is an interesting alternative to biotin. In order to couple biotin to amino acids, reaction has to be with an amino group. A popular example is biocytin (biotin lysine). However, norbiotinamine could be coupled with a carboxylic group of amino acids to give inverse peptides, having the amide linkage oriented in the opposite direction. As an example, norbiotinamine was coupled with the NHS ester of N-Boc-glutamic acid γ-benzyl ester. The resulting norbiotinylamide of γglutamic acid (VII), obtained after deprotection, is similar to biocytin, but it has a shorter aliphatic chain and an inverted amide bond. The structures of all products were established by NMR analyses and derivatization. However, norbiotinamine (II), the title product and starting material for the reactive derivatives, had its purity confirmed by elemental analysis. This analysis showed that norbiotinamine (II) forms a stable carbonate in the presence of air (as seen with cadaverine). The reactions and purifications were easily monitored by thin layer chromatography (TLC) analyses. Usually components of even complex reaction mixtures were readily identified on a single fluorescent TLC plate by first checking under a UV lamp, next exposed to iodine vapors (I2 on SIO2), and, after evaporation of the iodine, dipped in a biotin sensitive reagent (or in ninhydrin solution) and heating. EXPERIMENTAL PROCEDURES 1H
NMR spectra were recorded on a 400 MHz Bruker FT NMR spectrometer. Chemical shifts are given in δ relative to the residual protons in a deuterated solvent. J values are in hertz. Melting points were not corrected. Elemental analysis was performed by Desert Analytics, Tucson, AZ. TLC utilized Merck 60 F-254, 0.2 mm precoated silica gel aluminum sheets. Biotin derivatives © 1996 American Chemical Society
272 Bioconjugate Chem., Vol. 7, No. 2, 1996
Szalecki
Scheme 1a
a
a, DPPA, NEt3, t-BuOH; b, HCl, dioxane; c, NaOH; d, TCDP; e, HBr/AcOH; f, SIA; g, N-Boc-L-glutamic acid, R-Bz, γ-NHS ester.
were visualized by a dip consisting of a 0.4% solution of 4-(dimethylamino)cinnamaldehyde in 0.6 N HCl containing 90% 2-propanol. Primary amines were identified with a 0.3% solution of ninhydrin in 3% AcOH/BuOH. Column chromatography used silica gel 60, Merck “Geduran”, 40-63 µm. All operations involving iodoacetyl compounds were run under subdued light. Biotin and SIA were from Molecular Probes, Inc. DCC was from Fluka Chemical Corp. Diphenylphosphoryl azide was from Lancaster Synthesis, Inc. N-Boc-L-glutamic acid and R-Bz ester were from Sigma Chemical Corp. TCDP was from Aldrich Chemical Corp. Succinimidyl esters were made using NHS and DCC. Norbiotinamine (II). Molecular Formula, C9H17N3OS. Formula Weight, 215.31 (277.34 for the carbonate). A mixture of biotin (I) (9.8 g, 40 mmol), diphenylphosphoryl azide (12.1 g, 44 mmol), and triethylamine (6 mL, 4.35 g, 43 mmol) in 200 mL of t-BuOH was heated to reflux for 18 h. Solvents were evaporated, and the residue was dissolved in 6 N HCl in 50% MeOH. After the solution was stirred overnight, the pH of the solution was adjusted to 12 using 50% NaOH. Inorganics were filtered off and washed with MeOH. The filtrates were evaporated, and the residue was then dissolved in water and poured on a column containing 300 mL of strongly acidic Dowex 50-X4 (200-400 mesh), H+ form. Impurities were removed with a 5% solution of triethylamine in 20% MeOH. The product II was eluted with 5% NaOH. A concentrated solution of product was purified on an LH-20 column. Lyophilization yielded 4.75 g (55%) of fluffy, white powder of II. The melting point of the amine is dependent on traces of CO2 and H2O; the melting point of the carbonate is 265-268 °C dec [TLC, concd NH3/H2O/PrOH (3:1:6), Rf 0.67]: 1H NMR (D2O) δ 4.62 (m, 1H), 4.45 (m, 1H), 3.37 (m, 1H), 3.02 (dd, 1H, J ) 12.9, 4.9), 2.79 (d, 1H, J ) 12.9), the triplet of CH2-
NH2 is at 2.55 for the “free” amine, 2.78 for the carbonate, or 2.88 for the hydrochloride, 1.7-1.2 (mm, 6H). Anal. Calcd for C9H17N3OS + CO2 + H2O: C, 43.30; H, 6.90; N, 15.15. Found: C, 43.53; H, 6.47; N, 15.35. Norbiotinyl Isothiocyanate (III). Molecular Formula, C10H15N3OS. Formula Weight, 257.38. A solution of TCDP (465 mg, 2 mmol) in 20 mL of CHCl3 was added to a stirred mixture of norbiotinamine (II) carbonate (554 mg, 2 mmol) in 100 mL of MeCN and 20 mL of CHCl3. After 15 min, 1 drop of NEt3 was added. The yelloworange color of TCDP almost disappeared after an additional 15 min. The reaction mixture was flash chromatographed on 120 g of SiO2, using 10% MeOH in CHCl3 as eluent. The major fractions were evaporated. The residue was crystallized with EtOAc/hexanes to give 436 mg (85%) of light beige crystals of III: mp 185-186 °C; 1H NMR (DMSO-d6) δ 6.43 (s, 1H), 6.36 (s, 1H), 4.31 (m, 1H), 4.15 (m, 1H), 3.66 (t, 2H, J ) 6.6, CH2NCS), 3.13 (m, 1H), 2.84 (dd, 1H, J ) 12.4, 5.1), 2.59 (d, 1H, J ) 12.4), 1.7-1.3 (mm, 6H). N-Norbiotinyl-12-aminododecanoamide (VIII). Succinimidyl 12-Cbz-aminododecanoate (2.68 g, 6 mmol) was added to a mixture of norbiotinamine (II) carbonate (1.1 g, 4 mmol), 10 mL of DMF, and 200 mL of CHCl3. After 18 h, excess NEt3 (1.4 mL, 10 mmol) was added. After another 20 h, the reaction mixture was poured on a wide suction column with 200 g of dry SiO2. The Cbzamide was eluted with 5% MeOH/CHCl3 and, after evaporation, deprotected by dissolving in 80 mL of 15% HBr/AcOH. After 6 h, the VIII hydrobromide was precipitated with ether and then flash chromatographed on 200 g of SiO2, starting with 1:10:89 and finishing with 1:15:84 solution of AcOH/MeOH/CHCl3. After evaporation, 1.55 g (80%) of a salt of VIII was obtained: 1H NMR (DMSO-d6) δ 7.73 (t, 1H, J ) 5.4), 6.42 (s, 1H), 6.36 (s, 1H), 4.31 (m, 1H), 4.13 (m, 1H), 3.09 (m, 1H), 3.02 (m,
Bioconjugate Chem., Vol. 7, No. 2, 1996 273
Technical Notes
2H, CH2NHCO), 2.82 (dd, 1H, J ) 12.4, 5.1), 2.76 (t, 2H, J ) 7.5, CH2NH2), 2.58 (d, 1H, J ) 12.4), 2.03 (t, 2H, J ) 7.4, CH2CONH), 1.91 (s,
