Polyfluorophenyl Ester-Terminated Homobifunctional Cross-Linkers for Protein Conjugation

^◊Equal contributions from these authors

Published as part of the Cluster Recent Advances in Peptide and Protein Synthesis

Abstract

Along with N-hydroxysuccinimidyl, p-nitrophenyl, and phenylseleno esters, tetra- and penta-fluorophenyl esters were comparatively evaluated in term of their reactivity and hydrolytic stability. Their homobifunctional cross-linkers were prepared to conjugate proteins with small molecules, including carbohydrates, fluorescent dyes, and poly(ethylene glycol) monomethyl ether. The conjugations proceeded under mild conditions, affording the corresponding protein conjugates with good efficiency.

Key words

protein conjugation - cross-linking - carbohydrates - dyes - polyethylene glycol - linking group

• References and Notes

• 1 Means GE. Feeney RE. Bioconjugate Chem. 1990; 1: 2
• 2 Jones LH. Nat. Chem. 2015; 7: 952
• 3 Stephanopoulos N. Francis MB. Nat. Chem. Biol. 2011; 7: 876
• 4 Hermanson GT. Bioconjugate Techniques . Elsevier Academic; Amsterdam: 2013. 3rd ed. 275
• 5a Izumi M. Okumura S. Yuasa H. Hashimoto H. J. Carbohydr. Chem. 2003; 22: 317
• 5b Broecker F. Hanske J. Martin CE. Baek JY. Wahlbrink A. Wojcik F. Hartmann L. Rademacher C. Anish C. Seeberger PH. Nat. Commun. 2016; 7: 11224
• 5c Möginger U. Resemann A. Martin CE. Parameswarappa S. Govindan S. Wamhoff E.-C. Broecker F. Suckau D. Pereira CL. Anish C. Seeberger PH. Kolarich D. Sci. Rep. 2016; 6: 20488
• 6 Wu X. Ling C.-C. Bundle DR. Org. Lett. 2004; 6: 4407
• 7 Yin X.-G. Gao X.-F. Du J.-J. Zhang X.-K. Chen X.-Z. Wang J. Xin L.-M. Lei Z. Liu Z. Guo J. Org. Lett. 2016; 18: 5796
• 8 The ratios of aflatoxin B1/BSA are 25:1, 50:1, and 100:1; there are 3.9, 6.1, and 13.6 alfatoxin B1 molecules loaded per BSA molecule, respectively. For details, see the Supporting Information.
• 9a Du J.-J. Gao X.-F. Xin L.-M. Lei Z. Liu Z. Guo J. Org. Lett. 2016; 18: 4828
• 9b Gao X.-F. Du J.-J. Liu Z. Guo J. Org. Lett. 2016; 18: 1166
• 9c Gao X.-F. Sun W.-M. Li X.-M. Liu X.-J. Wang L.-S. Liu Z. Guo J. Catal. Commun. 2016; 73: 103
• 10 Yin X.-G. Chen X.-Z. Sun W.-M. Geng X.-S. Zhang X.-K. Wang J. Ji P.-P. Zhou Z.-Y. Baek DJ. Yang G.-F. Liu Z. Guo J. Org. Lett. 2017; 19: 456

For PFP esters used in peptide synthesis, see:
• 11a Kisfaludy L. Roberts JE. Johnson RH. Mayers GL. Kovacs J. J. Org. Chem. 1970; 35: 3563

For TFP esters used in peptide synthesis, see:
• 11b Hui KY. Holleran EM. Kovacs J. Int. J. Pept. Protein Res. 1988; 31: 205
• 12 Vetter D. Tumelty D. Singh SK. Gallop MA. Angew. Chem. Int. Ed. 1995; 34: 60
• 13 Lockett MR. Phillips MF. Jarecki JL. Peelen D. Smith LM. Langmuir 2008; 24: 69
• 14 Miermont A. Barnhill H. Strable E. Lu X. Wall KA. Wang Q. Finn MG. Huang X. Chem. Eur. J. 2008; 14: 4939
• 15 Bis(2,3,5,6-tetrafluorophenyl) Adipate (5); Typical Procedure Adipic acid (1.00 g, 6.84mmol) and EDC (3.15 g, 16.8 mmol) were dissolved in dry CH₂Cl₂ (50 mL), and the solution was allowed cool to 0 °C in an ice bath. 2,3,5,6-tetrafluorophenol (2.50 g, 15.1 mmol) was added, and the solution was warmed to rt and stirred for 6 h. After removal of the solvent under reduced pressure, the crude product was purified by flash column chromatography [silica gel, PE–CH₂Cl₂ (1:1)] to give a white solid; yield: 2.54 g (84%); mp 99.1–99.3 °C. ¹H NMR (600 MHz, CDCl₃): δ = 7.01 (t, J = 8.8 Hz, 2 H), 2.77 (s, 4 H), 1.94 (d, J = 4.8 Hz, 4 H). ¹⁹F NMR (376 MHz, CDCl₃): δ = –138.40 to –140.13 (m), –152.79 to –153.62 (m). ¹³C NMR (101 MHz, CDCl₃): δ = 168.90, 145.97 (dtd, J = 248.5, 11.9, 4.2 Hz), 140.53 (dddd, J = 250.4, 15.3, 4.8, 2.3 Hz), 129.80–129.23 (m), 103.19 (t, J = 22.8 Hz), 32.87, 23.84.
• 16a Steger J. Graber D. Moroder H. Geiermann A.-S. Aigner M. Micura R. Angew. Chem. Int. Ed. 2010; 49: 7470
• 16b Liu Y. Kang Y. Wang J. Wang Z. Chen G. Jiang M. Biomacromolecules 2015; 16: 3995

Cross-linkers that contain heterocyclic motifs proved to be immunodominant, leading to a reduced immune response to the haptens targeted, see:
• 17a Peeters JM. Hazendonk TG. Beuvery EC. Tesser GI. J. Immunol. Methods 1989; 120: 133
• 17b Buskas T. Li Y. Boons G.-J. Chem. Eur. J. 2004; 10: 3517
• 17c Yin Z. Chowdhury S. McKay C. Baniel C. Wright WS. Bentley P. Kaczanowska K. Gildersleeve JC. Finn MG. BenMohamed L. Huang X. ACS Chem. Biol. 2015; 10: 2364
• 18 Yildiz I. Deniz E. McCaughan B. Cruickshank SF. Callan JF. Raymo FM. Langmuir 2010; 26: 11503
• 19 Protein Conjugates of Half-Esters; General Procedure BSA or OVA (10 mg) was dissolved in PBS (pH 7.5, 2 mL), and the appropriate half-ester 11–14 was dissolved in DMF (100 μL). The ester solution was slowly injected into the protein solution and the mixture was left for 24 h at rt. The aqueous phase was then collected, diluted with deionized water, and dialyzed against five changes of deionized water. The solution was lyophilized to afford the protein conjugate as a white solid.
• 20 Patel MK. Vijayakrishnan B. Koeppe JR. Chalker JM. Doores KJ. Davis BG. Chem. Commun. 2010; 46: 9119
• 21a Harris JM. Chess RB. Nat. Rev. Drug Discov. 2003; 2: 214
• 21b Pelegri-O’Day EM. Lin E.-W. Maynard HD. J. Am. Chem. Soc. 2014; 136: 14323
• 22 Boccu E. Largajolli R. Veronese FM. Z. Naturforsch., C 1983; 38: 94
• 23 The degree of PEGylation on BSA as determined by the trinitrobenzenesulfonic acid method is generally higher than that verified by a more reliable method, see: Sartore L. Caliceti P. Schiavon O. Monfardini C. Veronese FM. Appl. Biochem. Biotechnol. 1991; 31: 213