ARTICLE https://doi.org/10.1038/s41467-019-10503-7 OPEN Single-site glycine-specific labeling of proteins Landa Purushottam1, Srinivasa Rao Adusumalli1, Usha Singh2, V.B. Unnikrishnan1, Dattatraya Gautam Rawale1, Mansi Gujrati2, Ram Kumar Mishra2 & Vishal Rai 1 Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from 1234567890():,; N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed. 1 Department of Chemistry, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal 462066, India. 2 Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal 462066, India. Correspondence and requests for materials should be addressed to V.R. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2539 | https://doi.org/10.1038/s41467-019-10503-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10503-7 ingle-site labeling of proteins facilitates investigation of sev- targeting of N-terminus Gly poses a prominent complexity as Seral biological processes through attachment of biophysical there is no side-chain residue to assist. probes, imaging probes, and toxins. Such labeling emerges Here, we demonstrate that an aldehyde with appropriately through pre-engineered protein equipped with unnatural amino designed hydrogen bond acceptor can result in the exclusive acids1, an amino acid sequence recognized by enzymes2, and single labeling of N-terminus Gly residue. The remarkable selectivity of cysteine3,4 or a cysteine placed in a π-clamp5. The understanding C–C bond formation extends from an amino acid to structurally of organic chemistry with proteins has nurtured the growth of diverse proteins. Besides, the protocol operates conveniently in a bioconjugation. These biomolecules can be perceived as a macro- mixture of proteins or cell lysate. The residue-specific installation molecular substrate with multiple nucleophilic functional groups of an orthogonal aldehyde group renders analytically pure tagged (NuP). The obvious route for bioconjugation would involve its proteins in high yields through the late-stage single-site intro- reaction with an electrophile (Fig. 1a). For single-site labeling in duction of the desired probe. such cases, a functional group has to compete with all the other nucleophilic residues (chemoselectivity) and its additional copies Results (site-selectivity). The chemoselective labeling of low-frequency residues (Cys6,7,Tyr8, and Trp9) can address the latter to some We hypothesized that a multi-step transformation could provide extent. For other cases, the single-site labeling of side chain func- a roadmap to address the challenge of chemoselectivity and tionalities could be driven through ligand–protein interaction10, residue selectivity. The initial generation of the EL with capability linchpin directed modification11 or targeting the ε-amine of most to generate another reactive center (latent nucleophile, NuL, path ε 12–16 d) could provide the platform. While the first step can address reactive Lys residue (N -NH2) . Alongside, the N-terminus α α chemoselectivity, the latter can give us an opportunity to explore -amine (N -NH2) has established its place as a notable reactivity fi hotspot. In a remarkable example, the biomimetic transformation the residue speci city. Also, the NuL generated with a protein will be required to display orthogonal reactivity in the presence of of N-terminus residue delivers a carbonyl group primed for sub- fi sequent orthogonal reactions17. proteinogenic nucleophiles (Fig. 1c). At rst, the challenge was to α design an imine that can generate the NuL under physiological The kinetic labeling of N -NH2 can be achieved through its chemoselective modification with an electrophile as determined conditions. We hypothesized that H-bond acceptors in the imine by a few groups including us (Fig. 1a)18–30. The alternate route group (Fig. 1b) could assist proton shuttling and stabilize the NuL. involves a latent electrophile (E ) generated in the form of imine We anticipated that the intramolecular H-bond acceptors might α L overcome the competing intermolecular interactions with protein from N -NH2 and aldehyde (Fig. 1b). It gives us the opportunity to address the two challenges in two separate steps. If we can and solvent. Subsequently, we can explore the reactivity of NuL generate E chemoselectively, the nucleophile is only required to towards an external electrophile (Fig. 1c). Also, we anticipated L that the unsubstituted amino acid (Gly) could potentially render address the site-selectivity. This approach delivers reductive fi alkylation in a chemoselective transformation (path a, Fig. 1b)31. a reactivity pro le different from all the other substituted However, the site-selectivity gets compromised very early in the amino acids. reaction in the presence of competing imines. On the contrary, the nucleophilic attack of backbone amide to imine generated by Aminoalcohol formation with Gly under ambient conditions. α N -NH2 and 2-pyridinecarboxaldehyde can render isolable imi- We selected ortho-substituted benzaldehyde to garner the dazolidinone with high selectivity (path b, Fig. 1b)32. Unfortu- advantage of geometrical constraint imposed by the aromatic nately, none of these methodologies can distinguish one N- ring. We placed one H-bond acceptor ortho to the aldehyde in the terminus residue from the other. In this perspective, N-terminal form of a methoxy group (2a, Fig. 2a). The reaction of Gly amide Cys containing proteins can render unique reactivity to form 1a with reagent 2a fails to result in any adduct (Fig. 2a). Next, we thiazolidine with an aldehyde or 2-cyanobenzothiazole (path c, designed the reagent 2b that proffers a carbonyl group as H-bond – Fig. 1b)33 35. However, it is a challenge to identify unique reac- acceptor. It was exciting to note the formation of isolable product tivity for other N-terminus residues. In particular, selective (3ab, 27% conversion, LC-MS). Encouraged by this result, we a Selectivity landscape in labeling of 'Nup' in proteins E R H E E Reaction H Nup N Nu Nup H N p Protein: nucleophile (Nu ) 2 Protein H N Protein Nu p 2 p E O E External: electrophile (E) Nup Nup Nup E E E R′ – CHO b N-terminus labeling of proteins enabled by 'EL' Path c Path d Nup Nu R H Nu p Reaction E H p L N EL Protein Nup R′ Protein: latent electrophile (EL) N Protein Nu Nu Nu p Nu p O p External/internal: nucleophile (Nu) Nu Path b Path a Path d c This work: 'NuL' enables residue-specific labeling (R = H) E Nu p Nu Reaction R Nup p H N NuL Protein Nup Protein: latent nucleophile (Nu ) R′ L N Protein E Nu Nu External: electrophile (E) p Nu p O p NuL H Fig. 1 Residue-specificity in labeling of proteins. a Nucleophilic addition of protein residues to an electrophile. b A latent electrophile, imine, renders the N-terminus labeling of proteins (path a–path c). c Latent nucleophile enables single-site N-terminus Gly labeling 2 NATURE COMMUNICATIONS | (2019) 10:2539 | https://doi.org/10.1038/s41467-019-10503-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10503-7 ARTICLE a R OH OH CHO O O n 2 (50 mM) O O NaHCO buffer O 3 R (0.1 M, pH 7.8) NH2 CHO CHO CHO H2N ° NH2 5 mM, 25 C, 24 h NH2 2a 2b n = 3 (2c) (3ac, 33%) 1a (3aa, 0%) (3ab, 27%) n = 2 (2d) (3ad, 63%) OH O n = 1 (2e) (3a, > 95%) 3 b OH OH OH O O O O O NaHCO3 buffer O O (0.1 M, pH 7.8) 1 O H2N + R NH2 + NH2 5 mM, 25 °C, 24 h NH2 N R1 CHO NH2 Amino acid 1 2e (50 mM) OH O R1 3 4 Amide derivative of amino acids: R1 = H (3a) > 95% < 5% Gly (1a), Ala (1b), Arg (1c), Asn (1d), Asp (1e) R1 ≠ H (3b–3t) 0% Variable/reversible Cys (1f), Glu (1g), Gln (1h), His (1i), llel (1j) Leu (1k), Lys (1I), Met (1m), Phe (1n), Prol (1o) Ser (1p), Thr (1q), Trp (1r), Thr (1s), Val (1t) c O 2e, NaHCO3 buffer O O (0.1 M, pH 7.8) H2N H N H2N NH 2 NH + NH 2 5 mM, 25 °C, 24 h 2 2 Ph Ph Ph 1n 100% 0% d NuL OO OO HO NH2 HO H NH2 R1 = H R1 ≠ H 1 –H2O O N R 1a 1b–1t O N 1 + 2e O EL O O HO O O NH2 2e OH H2O 3a + 2e O N OH O O OH Fig. 2 Stable aminoalcohol formation with glycine. a Aldehyde with H-bond acceptors forms a stable aminoalcohol with 1a (also see Supplementary Fig. 72). b Selective formation of aminoalcohol with 1a. c Stereo-stability of substituted amino acid (1n) under the reaction conditions. d Plausible mechanism for glycine specific formation of aminoalcohol investigated the effect of two oxygen centers as H-bond acceptors remained inert towards any subsequent transformation contrary in the reagent 2c.