A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins

Abstract

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.

Figure 1.. The conjugation of oxidized selenocysteine to electron-rich small molecules is a one-pot chemoselective reaction for the covalent attachment of natural products and pharmaceuticals to polypeptides and proteins.

(a) Site-selective conjugation of vancomycin to polypeptides and proteins. (b) Examples of bioconjugates prepared through our conjugation reaction.

Figure 2.. Selenocysteine-based conjugation of vancomycin to antibacterial peptides enables the discovery of potent Gram-positive and Gram-negative antibacterial agents.

(a) Conjugation of antimicrobial-peptides to vancomycin. (b) ROESY and HMBC correlations for vancomycin in peptide-vancomycin conjugate 13i. (c and d) Two vancomycin-antibacterial peptide conjugates (19v and 23v) showed lower minimum inhibition concentration (MIC) compared to the controls including vancomycin only (van), antibacterial peptide only (19p and 23p), antibacterial peptide with a p-methoxybenzyl protected selenocysteine and a glycine on the N-termini (19ep and 23ep), and the mixtures of vancomycin and antibacterial peptides. MIC values are from a single activity screening experiment (Supplementary Section 14).

Figure 3.. Conjugation of vancomycin to affibody generates conjugates with retained structure and binding affinity.

(a) Conjugation of affibody to vancomycin. The affibody variant 27 containing activated selenocysteine was generated in situ from variant 25 (Supplementary Section 23). (b) Deconvolved mass spectrum of purified affibody-vancomycin conjugate. (c) Far-UV Circular dichroism (CD) spectrum of affibody, affibody-selenocysteine variant 25, and affibody-vancomycin conjugate 27. The CD spectrum of 27 was obtained by subtracting the measured spectra with that of vancomycin only (Supplementary Section 24). (d) Sensorgrams from BioLayer interferometry analysis of affibody-vancomycin conjugate binding to trastuzumab antibody. Retained binding was observed compared to the native affibody (K_D = 2.0 ± 0.2 nM; the final K_D is the average of the K_D obtained from three different concentrations, the error is standard error.)

Figure 4.. Conjugation of genistain to antibody leads to conjugates with retained target binding affinity.

(a) A two-step strategy of conjugating an antibody to genistein. Genistein was first modified with a polyglycine peptide through the selenocysteine-mediated conjugation (step 1); the obtained product was then conjugated to antibody through sortase-mediated ligation reaction (step 2). (b) Deconvolved mass spectra of the reduced trastuzumab-genistein conjugate 30. The N-linked glycans on antibody heavy chains were removed by treatment with PNGase F. (c) Sensorgrams from BioLayer interferometry analysis of trastuzumab-genistein conjugate 30 binding to recombinant HER2 protein. Similar binding affinity was observed compared to native trastuzumab (dissociation constant K_D = 0.3 ± 0.2 nM; the final K_D is the average of the K_D obtained from three different concentrations, the error is standard error.)

Figure 5.. Selenocysteine-based conjugation of peptides to (hetero)arenes, natural products, and pharmaceuticals proceeded in moderate to high yields.

Amino acids are shown in one-letter code, U – selenocysteine; Yields were determined by integration of the total ion currents (TICs) from LC−MS analyses of the crude reaction mixtures (Supplementary Section 8). *Yield in parentheses run with 1 mM CuSO₄, 1 mM 4,4’-di-tert-butyl-2,2’-dipyridyl (L1). ^fl100 μM 8 and 1 mM small molecule. ^§4 mM CuSO₄, 4 mM L1, 4 mM small molecule. ^‡4 mM small molecule. ^#2 mM CuSO₄, 2 mM L1.