Download PDF
Synlett 2025; 36(17): 2883-2889
DOI: 10.1055/s-0043-1775499
letter
Small Molecules in Medicinal Chemistry

Hydrophilic α-Aryl-α-Diazoamides for Protein Esterification

Authors

  • Aniekan Okon

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

  • Anton Morgunov

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

  • Jinyi Yang

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

  • Yana D. Petri

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

  • Henry R. Kilgore

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

  • Yanfeng Li

    b   Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA 01003, USA

  • Eric R. Strieter

    b   Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA 01003, USA

  • Ronald T. Raines  ∗

    a   Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

A.O. was supported by postdoctoral fellowship F32 CA247259 (National­ Institutes of Health, NIH). A.M. was supported by the Undergraduate Research Opportunities Program at the Massachusetts Institute of Technology. Y.D.P. was supported by a National Science Foundation (NSF) Graduate Research Fellowship. This work was supported by grants R35 GM148220, R35 GM149532, and P30 CA014051 (NIH).
Further Information
   Permissions and Reprints All articles of this category


Graphical Abstract

Preview

Abstract

Bioreversible protein esterification is a simple, customizable, and traceless strategy for the exogenous delivery of proteins into mammalian cells. Enabling this protein delivery strategy are α-aryl-α-diazoamides bearing a tolyl moiety. The aqueous solubility of the ensuing esterified protein is, however, often compromised, which can result in the loss of soluble esterified protein for downstream applications. Here, we undertook a structure–activity relationship campaign to generate hydrophilic diazoamides for use as protein esterification and cellular delivery agents. We find that the careful adjustment of the hydrogen-bond basicity of α-aryl-α-diazoamides is sufficient to engender soluble esterified proteins, as high hydrogen-bond basicity correlates with high aqueous solubility. Importantly, enhancing aqueous solubility of diazoamides should proceed pari passu with preserving their lipophilicity and reactivity towards esterification of carboxylic acids, as the best-performing diazoamide from our study contains an N-acetyl piperazine while retaining the tolyl moiety. Our efforts can inspire new generations of esterified proteins with better solubility.

Key words

cell - delivery - diazo compounds - esterification - hydrophilicity - hydrophobicity - proteins - solubility

Supporting Information



Publication History

Received: 02 March 2025

Accepted after revision: 17 June 2025

Article published online:
15 July 2025

© 2025. Thieme. All rights reserved

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

 

  • References and Notes

    • 1a Nagel YA, Raschle PS, Wennemers H. Angew. Chem. Int. Ed. 2016; 56: 122
      Search in Google Scholar
    • 1b Bolhassani A, Jafarzade BS, Mardani G. Peptides 2017; 87: 50
      Search in Google Scholar
    • 1c Sánchez-Navarro M. Adv. Drug. Delivery Rev. 2021; 171: 187
      Search in Google Scholar
    • 1d Saha A, Mandal S, Arafiles JV. V, Gómez-Gonzálex J, Hackenberger CP. R, Brik A. Angew. Chem. Int. Ed. 2022; 61: e202207551
      Search in Google Scholar
    • 2a Li W, Nicol F, Szoka FC. Jr. Adv. Drug Delivery Rev. 2004; 56: 967
      Search in Google Scholar
    • 2b Reshetnyak YK, Andreev OA, Lehnert U, Engelman DM. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6460
      Search in Google Scholar
    • 2c Thévenin D, An M, Engelman DM. Chem. Biol. 2009; 16: 754
      Search in Google Scholar
    • 2d Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, Saltzman WM, Slack FJ. Nature 2015; 518: 107
      Search in Google Scholar
    • 3a Rüssmann H, Shams H, Poblete F, Fu Y, Galán JE, Donis RO. Science 1998; 281: 565
      Search in Google Scholar
    • 3b Nishikawa H, Sato E, Briones G, Chen LM, Matsuo M, Nagata Y, Ritter G, Jäger E, Nomura H, Kondo S, Tawara I, Kato T, Shiku H, Old LJ, Galán JE, Gnjatic S. J. Clin. Invest. 2006; 116: 1946
      Search in Google Scholar
    • 3c Appelbaum JS, LaRochelle JR, Smith BA, Balkin DM, Holub JM, Schepartz A. Chem. Biol. 2012; 19: 819
      Search in Google Scholar
    • 3d Bachran C, Morley T, Abdelazim S, Fattah RJ, Liu S, Leppla SH. mBio 2013; 4: e00201
      Search in Google Scholar
    • 3e Schmit NE, Neopane K, Hantschel O. ACS Chem. Biol. 2019; 14: 916
      Search in Google Scholar
  • 4 Mitchell MJ, Billingsley MM, Haley RM, Wechsler ME, Peppas NA, Langer R. Nat. Rev. Drug Discovery 2021; 20: 101
    Search in Google Scholar
  • 5 Mecke A, Lee DK, Ramamoorthy A, Orr BG, Holl MM. Langmuir 2005; 21: 8588
    Search in Google Scholar
    • 6a Gasparini G, Bang EK, Molinard G, Tulumello DV, Ward S, Kelley SO, Roux A, Sakai N, Matile S. J. Am. Chem. Soc. 2014; 136: 6069
      Search in Google Scholar
    • 6b Gasparini G, Sargsyan G, Bang EK, Sakai N, Matile S. Angew. Chem. Int. Ed. 2015; 54: 7328
      Search in Google Scholar
    • 6c Du S, Liew SS, Zhang CW, Du W, Lang W, Yao CC. Y, Li L, Ge J, Yao SQ. ACS Cent. Sci. 2020; 6: 2362
      Search in Google Scholar
    • 6d Guo J, Wan T, Li B, Pan Q, Xin H, Qiu Y, Ping Y. ACS Cent. Sci. 2021; 7: 990
      Search in Google Scholar
  • 7 Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Front. Pharmacol. 2015; 6: 286
    Search in Google Scholar
  • 8 Sun Y, Lau SY, Lim ZW, Chang SC, Ghadessy F, Partridge A, Miserez A. Nat. Chem. 2022; 14: 274
    Search in Google Scholar
    • 9a Akishiba M, Takeuchi T, Kawaguchi Y, Sakamoto K, Yu HH, Nakase I, Takatani-Nakase T, Madani F, Gräslund A, Futaki S. Nat. Chem. 2017; 9: 751
      Search in Google Scholar
    • 9b Shinga K, Iwata T, Murata K, Daitoku Y, Michibata J, Arafiles JV. V, Sakamoto K, Akishiba M, Takatani-Nakase T, Mizuno S, Sugiyama F, Imanishi M, Futaki S. Bioorg. Med. Chem. 2022; 61: 116728
      Search in Google Scholar
    • 9c Giancola JB, Grimm JB, Jun JV, Petri YD, Lavis LD, Raines RT. ACS Chem. Biol. 2024; 19: 908
      Search in Google Scholar
  • 11 Ressler VT, Mix KA, Raines RT. ACS Chem. Biol. 2019; 14: 599
    Search in Google Scholar
  • 12 Jun JV, Petri YD, Erickson LW, Raines RT. J. Am. Chem. Soc. 2023; 145: 6615
    Search in Google Scholar
  • 13 Petri YD, Gutierrez CS, Raines RT. Angew. Chem. Int. Ed. 2023; 62: e202215614
    Search in Google Scholar
    • 14a Cheah KM, Jun JV, Wittrup KD, Raines RT. Mol. Pharm. 2022; 19: 3869
      Search in Google Scholar
    • 14b Maynard JR. J, Saidjalolov S, Velluz M.-C, Vossio S, Aumeier C, Moreau D, Sakai N, Matile S. ChemistryEurope 2023; 1: e202300029
      Search in Google Scholar
    • 15a Tanford C. In Physical Chemistry of Macromolecules . John Wiley & Sons; New York: 1961: 240
      Search in Google Scholar
    • 15b Arakawa T, Timasheff SN. Methods Enzymol. 1985; 114: 49
      Search in Google Scholar
    • 15c Shaw KL, Grimsley GR, Yakovlev GI, Makarov AA, Pace CN. Protein Sci. 2001; 10: 1206
      Search in Google Scholar
    • 15d Tokmakov AA, Kurotani A, Sato KI. Front. Mol. Biosci. 2021; 8: 775736
      Search in Google Scholar
    • 16a Zidovetzki R, Levitan I. Biochim. Biophys. Acta 2007; 1768: 1311
      Search in Google Scholar
    • 16b Kovacs T, Nagy P, Panyi G, Szente L, Varga Z, Zakany F. Pharmaceutics 2022; 14: 2559
      Search in Google Scholar
  • 17 Leo A, Hansch C, Elkins D. Chem. Rev. 1971; 6: 525
    Search in Google Scholar
  • 18 Laurence C, Brameld KA, Graton J, Le Questel JY, Renault E. J. Med. Chem. 2009; 52: 4073
    Search in Google Scholar
  • 19 Molinspiration Cheminformatics (Slovensky Grob, Slovakia) free web services (accessed July 7, 2025): https://www.molinspiration.com
  • 20 All cLogP values reported herein represent the cLogP of the product of an esterification reaction between an α-aryl-α-diazoamide and acetic acid. Such simplified ester products should approximate the cLogP of the pendant esters on an esterified protein.
  • 22 The conditions for GFP esterification and subsequent buffer exchange differed from those in ref. 10c.
  • 23 Jun JV, Raines RT. Org. Lett. 2021; 23: 3110
    Search in Google Scholar
  • 25 Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Nat. Biotechnol. 2006; 24: 79
    Search in Google Scholar
  • 26 This approximation represents a lower limit of the number of tolyl ester tags installed on the protein because the two esters have different contributions to the final observed mass of the esterified protein product and because of uncertainty in the number of pyridyl tags left on the protein at the end of the reaction workflow.
    • 27a Ni H, Hatit MZ. C, Zhao K, Loughrey D, Lokugamage MP, Peck HE, Cid AD, Muralidharan A, Kim Y, Santangelo PJ, Dahlman JE. Nat. Commun. 2022; 13: 4766
      Search in Google Scholar
    • 27b Kim M, Jeong M, Lee G, Lee Y, Park J, Jung H, Im S, Yang JS, Kim K, Lee H. Bioeng. Transl. Med. 2023; 8: e10556
      Search in Google Scholar
  • 28 Das B, Baidya AT. K, Mathew AT, Yadav AK, Kumar R. Bioorg. Med. Chem. 2022; 56: 116614
    Search in Google Scholar
    • 29a Hausig F, Sobotta FH, Richter F, Harz DO, Traeger A, Brendel JC. ACS Appl. Mater. Interfaces 2021; 13: 35233
      Search in Google Scholar
    • 29b Zhang D, Atochina-Vasserman EN, Maurya DS, Liu M, Xiao Q, Lu J, Lauri G, Ona N, Reagan EK, Ni H, Weissman D, Percec V. J. Am. Chem. Soc. 2021; 143: 17975
      Search in Google Scholar
  • 30 Tomoda H, Kishimoto Y, Lee YC. J. Biol. Chem. 1989; 264: 15445
    Search in Google Scholar
  • 31 Zheng N, Shabek N. Annu. Rev. Biochem. 2017; 86: 129
    Search in Google Scholar
  • 32 Wilkinson KD, Audhya TK. J. Biol. Chem. 1981; 256: 9235
    Search in Google Scholar
  • 33 Vijay-Kumar S, Bugg CE, Cook WJ. J. Mol. Biol. 1987; 194: 531
    Search in Google Scholar