Small-Molecule CO₂ Donors Accelerate Three-Component Peptide/Protein Bioconjugation with Alkylazides and Phosphines in Ionic Liquids

Abstract

Development of chemical modification techniques for peptides and proteins has been facilitated by a set of multicomponent reactions to date. This Letter demonstrates the use of small-molecule CO₂ donors to promote reaction efficiency of the coupling of alkylamine on peptides and proteins with azide-derived iminophosphorane and carbon dioxide.

Key words

multicomponent reaction - CO₂ donor - peptide - protein - amine - azide - phosphine

Publication History

Received: 14 September 2023

Accepted after revision: 16 November 2023

Accepted Manuscript online:
16 November 2023

Article published online:
18 December 2023

• References and Notes

• 1 Jacobi von Wangelin A, Neumann H, Gördes D, Klaus S, Strübing D, Beller M. Chem. Eur. J. 2003; 9: 4286
• 2 John SE, Gulati S, Shankaraiah N. Org. Chem. Front. 2021; 8: 4237
• 3 Touré BB, Hall DG. Chem. Rev. 2009; 109: 4439
• 4 Weber L. Curr. Med. Chem. 2002; 9: 2085
• 5 Levi L, Müller TJ. J. Chem. Soc. Rev. 2016; 45: 2825
• 6 Shaabani A, Shadi M, Mohammadian R, Javanbakht S, Nazeri MT, Bahri F. Appl. Organomet. Chem. 2019; 33: e5074
• 7 Kitadai N, Maruyama S. Geosci. Front. 2018; 9: 1117
• 8 Das T, Ghule S, Vanka K. ACS Cent. Sci. 2019; 5: 1532
• 9 Neochoritis CG, Zarganes-Tzitzikas T, Katsampoxaki-Hodgetts K, Dömling A. J. Chem. Educ. 2020; 97: 3739
• 10 Reguera L, Rivera DG. Chem. Rev. 2019; 119: 9836
• 11 Joshi NS, Whitaker LR, Francis MB. J. Am. Chem. Soc. 2004; 126: 15942
• 12 Chilamari M, Purushottam L, Rai V. Chem. Eur. J. 2017; 23: 3819
• 13 Dai Y, Weng J, George J, Chen H, Lin Q, Wang J, Royzen M, Zhang Q. Org. Lett. 2019; 21: 3828
• 14 Molla R, Joshi PN, Reddy NC, Biswas D, Rai V. Org. Lett. 2023; 25: 6385
• 15 Ohata J, Miller MK, Mountain CM, Vohidov F, Ball ZT. Angew. Chem. Int. Ed. 2018; 57: 2827
• 16 Sornay C, Hessmann S, Erb S, Dovgan I, Ehkirch A, Botzanowski T, Cianférani S, Wagner A, Chaubet G. Chem. Eur. J. 2020; 26: 13797
• 17 Munch HK, Rasmussen JE, Popa G, Christensen JB, Jensen KJ. Chem. Commun. 2013; 49: 1936
• 18 Wang Y, Czabala P, Raj M. Nat. Commun. 2023; 14: 4086
• 19 El-Shaffey HM, Gross EJ, Hall YD, Ohata J. J. Am. Chem. Soc. 2021; 143: 12974
• 20 El-Shaffey HM, Gross EJ, Hall YD, Ohata J. J. Am. Chem. Soc. 2022; 144: 9178
• 21 Ishizawa S, Tumurkhuu M, Gross EJ, Ohata J. Chem. Sci. 2022; 13: 1780
• 22 Ishizawa S, Tumurkhuu M, Gross EJ, Ohata J. Chem. Sci. 2022; 13: 6749
• 23 Hall YD, Uzoewulu CP, Nizam ZM, Ishizawa S, El-Shaffey HM, Ohata J. Chem. Commun. 2022; 58: 10568
• 24 Babin V, Sallustrau A, Loreau O, Caillé F, Goudet A, Cahuzac H, Del Vecchio A, Taran F, Audisio D. Chem. Commun. 2021; 57: 6680
• 25 Babin V, Sallustrau A, Molins M, Labiche A, Goudet A, Taran F, Audisio D. Eur. J. Org. Chem. 2022; e202200133
• 26 Green O, Finkelstein P, Rivero-Crespo MA, Lutz MD. R, Bogdos MK, Burger M, Leroux J.-C, Morandi B. J. Am. Chem. Soc. 2022; 144: 8717
• 27 Kong D, Moon PJ, Lui EK. J, Bsharat O, Lundgren RJ. Science 2020; 369: 557
• 28 Daptomycin Modification with Azide 2b To ionic liquid butylmethylpyrrolidinium (typically 30 μL), potassium carbonate aqueous solution (20 mM final concentration from 2 M stock aqueous solution), daptomycin (2.5 mM final concentration from 50 mM stock solution in DMSO), alkylazide (75 mM final concentration from 1 M stock solution in DMSO), and a CO₂ donor (80 mM final concentration from 2 M stock solution in DMSO) were added. After the solution was mixed well, triphenylphosphine (125 mM final concentration from freshly prepared 1 M stock solution in 1:1 DMSO/toluene) was added, and the reaction mixture was incubated at 50 °C for 1 h. For separation of the peptide from the reagents and ionic liquid, solution of a 1:1 acetone/toluene mixture (600 μL) was added. After the mixture was mixed well, the mixture was stored at –80 °C overnight. The precipitate was collected by centrifugation (15k rcf, 4 °C, 15 min). The precipitate was dried by placing the open container on bench at rt for 15 min and then redissolved in 1:1 water/acetonitrile (30 μL). The solution was analyzed by LC–MS. HRMS-ESI (m/z) Daptomycin modified with 2b: [M – 2 H]^2– calcd for C₉₀H₁₁₂N₁₈O₂₇: 937.38994; found: 937.3913. Daptomycin modified with 2c: [M + 2 H]²⁺ calcd for C₈₄H₁₂₃N₁₉O₃₁: 947.93890; found: 947.9420. Daptomycin modified with 2d: [M + 2 H]²⁺ calcd for C₇₇H₁₁₂N₁₈O₂₇Si: 875.39295; found: 875.3917. Daptomycin modified with 2e: [M + 2 H]²⁺ calcd for C₇₉H₁₁₁BrN₁₈O₂₉: 928.3537; found: 928.35466. Daptomycin modified with 2f: [M + 2 H]²⁺ calcd for C₈₃H₁₁₄N₂₀O₃₁: 944.40522; found: 944.4053.
• 29 α-Chymotrypsinogen A Modification with Azide 2g To ionic liquid butylmethylpyrrolidinium (typically 50 µL), α-chymotrypsinogen A (0.01 mM final concentration from 5 mM stock solution in 1 M K₂CO₃ aqueous solution), azide-biotin 2g (1 mM final concentration from 100 mM stock solution in DMSO), and a CO₂ donor (20 mM final concentration from 500 M stock solution in DMSO) were added. After the solution was mixed well, triphenylphosphine (3 mM final concentration from freshly prepared 150 mM stock solution in DMSO) was added, and the reaction mixture was incubated at 37 °C for 2 h. For separation of the peptide from the reagents and ionic liquid, solution of a 1:1 acetone/toluene mixture (1200 μL) was added. After the mixture was mixed well, the mixture was stored at –80 °C overnight. The precipitate was collected by centrifugation (15k rcf, 4 °C, 15 min). The precipitate was dried by placing the open container on bench at rt for 15 min and then redissolved in 5 mM ammonium bicarbonate aqueous solution (50 μL). 2.5 μL of the protein sample was mixed with LDS loading buffer and separated by SDS-PAGE. After the protein was transferred onto a PVDF membrane, the membrane was stained with Ponceau S (0.1% in 9:1 water/acetic acid) and imaged with imager IQ800. After destaining with TBST buffer, the membrane was blocked with 5% BSA in TBST for 30 min and blotted with streptavidin–Cy5 conjugate (1:2000 dilution) for 45 min. The membrane was imaged with Cy5 emission and filter after TBST wash processes.
• 30 Reslan M, Kayser V. Biophys. Rev. 2018; 10: 781
• 31 Mukhopadhayay A, Singh D, Sharma KP. Biomacromolecules 2020; 21: 867
• 32 Schindl A, Hagen ML, Muzammal S, Gunasekera HA. D, Croft AK. Front. Chem. 2019; 7: 347
• 33 Diamantis N, Banerji U. Br. J. Cancer 2016; 114: 362
• 34 Zhang H, Chen S. RSC Chem. Biol. 2022; 3: 18