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Emerging Utility of Fluorosulfate Chemical Probes Lyn H. Jones*
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Jnana Therapeutics, 50 Northern Avenue, Boston, Massachusetts 02210, United States ABSTRACT: Aryl fluorosulfates are finding widespread utility in chemical biology and medicinal chemistry. The contextdependent engagement of tyrosine, lysine, serine, and histidine amino acid residues in functional protein sites has enabled chemogenomic and chemoproteomic techniques that demonstrate considerable promise for drug discovery and biomedical research. KEYWORDS: Fluorosulfate, sulfonyl fluoride, sulfur(VI)-fluoride, chemoproteomics, protein labeling
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rotein labeling chemistry has a myriad of applications in chemical biology and drug discovery. Biomolecule crosslinking is applied to the advancement of biotherapeutic and vaccine modalities, while functionalized chemical tool molecules have found utility as reporters of protein occupancy and as targeted covalent inhibitors. Cysteine labeling has been the go-to approach for developing specific covalent binders, but the amino acid is under-represented in functional protein sites, and the high nucleophilicity of the side chain hinders the creation of residue-selective probes. Aryl sulfonyl fluorides have been developed to target a number of nucleophilic residues beyond cysteine, including tyrosine, lysine, serine, threonine, and histidine.1,2 The sulfonyl fluoride electrophilic warhead is ideally suited to the elaboration of contextdependent chemical probes because its reactivity is sensitive to the microenvironment of the binding site. Hydrogen bonding to the fluorine activates the sulfur−fluorine bond, and neighboring residues may increase the nucleophilicity of the side chain functionality. The electrophilicity of sulfur is relatively high, and under somewhat forcing conditions aryl sulfonyl fluorides react nondiscriminately with nucleophilic amino acid side chains. As expected, aryl fluorosulfates possess lower reactivity due to ameliorated electrophilicity of the sulfur atom (oxygen-mediated resonance stabilization, Figure 1a) but similar chemoselectivity to the sulfonyl fluoride (see below). By way of illustration, we showed that benzene fluorosulfate (BFS), even at high concentrations and extended reaction times, does not react appreciably with albumin (a model protein substrate), while benzenesulfonyl fluoride (BSF) under the same conditions labels albumin with multiple copies (Figure 1b).3 Similarly, para- and meta-carboxyl benzene fluorosulfates were found to be stable to hydrolysis over 24 h at pH 7.5, while the sulfonyl fluorides were readily hydrolyzed.4 Although studies of fragments such as these provide some information regarding the intrinsic chemical reactivity of sulfur(VI)-fluoride reagents, it is important to bear in mind that the rate of reaction with proteins may be considerably different, particularly when equilibrium binding interactions are considered. Several examples are now emerging that © XXXX American Chemical Society
Figure 1. (a) Electrostatic potential maps of energy-minimized structures of benzenesulfonyl fluoride and benzene fluorosulfate (MolView). The fluorosulfate electrophile is less reactive. (b) Intact protein MS analysis of the reaction of human serum albumin (HSA, 1 μM) with the sulfur(VI)-fluoride fragment (300 μM) after 24 h.3 (c) Aryl fluorosulfate publication count by year (Scifinder).
describe the design, synthesis, and application of aryl fluorosulfates in various research areas, (including chemical
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DOI: 10.1021/acsmedchemlett.8b00276 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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occupancy to be assessed in living cells. A nanomolar fluorosulfate DcpS inhibitor (FS-p1) was prepared that surprisingly reacted with a noncatalytic serine in a different location of the same binding site (Figure 2b).3 Basic amino acid residues lie in close proximity to the serine that are presumed to reduce the hydroxyl group pKa, increasing its reactivity toward the fluorosulfate electrophile. Although the rate of reaction with DcpS was slower, optimization of the equilibrium binding interaction would likely enhance the rate of adduct formation. Small molecule-mediated engagement of a noncatalytic serine residue is extremely rare, the only other example being aspirin-COX2. Importantly, FS-p1 was found to be metabolically and chemically stable (10 μL/min/mg turnover in human liver microsomes; no degradation after 24 h incubation in phosphate buffered saline pH 7.4), establishing the suitability of the aryl fluorosulfate motif in drug design for the first time. The special features of fluorosulfate protein reactivity were recently harnessed in the development of a chemical biology approach termed “inverse drug discovery”.7 In this strategy, drug-like clickable compounds were armed with fluorosulfate warheads, exposed to whole proteomes, and affinity enrichment followed by quantitative mass spectrometry analysis identified protein−probe adducts. Several hits were validated using site-directed mutagenesis, peptide mapping, and X-ray crystallography. Tyrosine and lysine side chains were identified as fluorosulfate-reactive nucleophiles, which appear to be the preferred targets of sulfur(VI)-fluoride reagents more generally; for example, FS-Probe1 labeled HSDL2, an emergent glioma target for which no inhibitors were known (Figure 2c). In a similar study, intracellular lipid binding proteins (iLBPs) were identified as targets of biaryl fluorosulfates (Figure 2c). In this work, a clickable fluorosulfate probe was found to react with a tyrosine residue in the binding site of cellular retinoic acid binding protein 2 (CRABP2).8 As observed in other cases,6 proximal amino acids bearing basic side chains likely reduce the pKa of the tyrosine phenol group thus enhancing its reactivity. Unbiased chemogenomic explorations such as these enable chemical probes to be matched with protein targets that become the starting points for medicinal chemistry optimization. Chemoproteomic mapping also has the potential to unearth alternative therapeutic modalities such as allosteric inhibitors and modulators of protein−protein interactions. Broader application of this technology will result in the generation of target−ligand interaction maps that may influence therapeutic target selection and expedite hit generation for drug discovery programs in the future. The excellent biocompatibility and templated reactivity of aryl fluorosulfates makes the functionality well-suited to covalent protein cross-linking chemistry. Fluorosulfate-Ltyrosine (FSY) was genetically encoded into proteins in Escherichia coli and mammalian cells resulting in efficient intraprotein bridging and interprotein cross-linking via proximity-enabled tyrosine, lysine, and histidine labeling.9 For instance, based on the known structure of the interacting proteins PAPS reductase and Trx1, FSY was incorporated into E. coli Trx1 at a site proximal to a tyrosine residue in PAPS reductase. Desired cross-linking was confirmed using tandem MS analysis. This proof-of-principle study illustrates the potential for fluorosulfate chemical biology to map the architecture of protein interaction networks in living cells. The advances in fluorosulfate chemical biology have been possible due to the innovative synthetic strategies that were
biology and medicinal chemistry, Figure 1c), some of which are highlighted below. Fluorosulfate inhibitors of transthyretin (TTR, a transporter of thyroxine and holo-retinol-binding protein) were prepared recently to assess their ability to covalently engage the target. The meta-isomer TTR-mFS-Probe labels TTR more readily than the para-isomer TTR-pFS-Probe due to optimal positioning of the electrophile with a previously identified pKaperturbed, and thus reactive, lysine ε-amino group, that forms a salt bridge with a neighboring glutamate in the binding site (Figure 2a).5 The product of labeling was the sulfamated TTR-
Figure 2. (a) Covalent transthyretin modulators TTR-mFS-Probe (labels the TTR Lys15 residue) and the fluorogenic ligand TTR-pFSProbe. (b) Cocrystal structure of SF-p1 (blue) with DcpS (gray ribbon) showing reaction with Tyr143 (pink) overlaid with a model of FS-p1 (gold) showing proximity to the noncatalytic Ser272 (neighboring histidine triad and Arg294 are highlighted). (c) Fluorosulfates Biaryl-FS-Probe and FS-Probe1 used for chemoproteomic profiling experiments. (d) Sulfurylation of phenols for the preparation of aryl fluorosulfates.
SO3− due to protein-mediated hydrolysis of the initial adduct. Interestingly, TTR-pFS-Probe was used as an environmentsensitive fluorogenic reporter of target engagement in vivo due to the fluorescence of the TTR·Probe complex (the fluorescence enhancement was significantly lower for TTRmFS-Probe or sulfonyl fluoride congeners that also react with the lysine). TTR misfolding and aggregation is a causative factor in amyloid diseases and chemical probes such as those described provide novel ways to modulate and explore this clinically important target. Inhibitors of the mRNA decapping scavenger enzyme (DcpS) were developed in our group for the treatment of spinal muscular atrophy. A sulfonyl fluoride covalent inhibitor SF-p1 was rationally designed to react with a specific tyrosine residue in the binding pocket (Figure 2b).6 A click handle was also incorporated into the probe to enable drug−target B
DOI: 10.1021/acsmedchemlett.8b00276 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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established recently to create fluorosulfate chemical probes. Work from Sharpless, Finn, and co-workers showed that click functionalization of phenols using sulfuryl fluoride gas readily furnished a variety of fluorosulfate probes (Figure 2d).2 A liquid-based, in situ protocol suitable for library chemistry in 96-well plates was also developed using sulfuryl fluoride dissolved in acetonitrile.10 Thirty-nine anticancer phenolic compounds were converted to fluorosulfates, three of which possessed superior cytotoxicity compared to the parent. However, sulfuryl fluoride is a toxic gas that is strictly regulated and quite difficult to operate in the laboratory. Therefore, new reagents were designed recently to facilitate fluorosulfate preparation. A shelf-stable, though hygroscopic, fluorosulfuryl imidazolium triflate salt, itself prepared from SO2F2, delivered the SO2F− functionality efficiently to phenol (and amine) nucleophiles (Figure 2d).11 A stable and crystalline reagent, [4-(acetylamino)phenyl]imidodisulfuryl difluoride (AISF) enabled click fluorosulfurylation of phenols.12 Substrate scope was exemplified using electron rich/ poor and sterically congested phenols, and the tyrosine residue in desmopressin (a macrocyclic peptide antidiuretic) was also chemoselectively derivatized. In conclusion, the chemical toolkit continues to expand, and the utility of the fluorosulfate electrophilic motif illustrates the advances that can be made in chemical biology using innovative synthetic techniques. Future work in the area should include the development of additional sulfur(VI)fluoride warheads that assist the rational design of reactive chemical probes dictated by the features of the binding sites being targeted. Additionally, pioneering chemoproteomic and chemogenomic research in this field is enabling a new paradigm in drug discovery, whereby medicinal chemistry increasingly affects the exploration of biology.
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(2) Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) fluoride exchange (SuFEx): another good reaction for click chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 9430−48. (3) Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 2015−2020. (4) Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A study of the reactivity of S. Org. Biomol. Chem. 2017, 15 (45), 9685−9695. (5) Baranczak, A.; Liu, Y.; Connelly, S.; Du, W. G.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans. J. Am. Chem. Soc. 2015, 137 (23), 7404−14. (6) Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes. ACS Chem. Biol. 2015, 10 (4), 1094−8. (7) Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. ″Inverse Drug Discovery″ Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200−210. (8) Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 7353−64. (9) Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 4995−4999. (10) Liu, Z.; Li, J.; Li, S.; Li, G.; Sharpless, K. B.; Wu, P. SuFEx Click Chemistry Enabled Late-Stage Drug Functionalization. J. Am. Chem. Soc. 2018, 140 (8), 2919−2925. (11) Guo, T.; Meng, G.; Zhan, X.; Yang, Q.; Ma, T.; Xu, L.; Sharpless, K. B.; Dong, J. A New Portal to SuFEx Click Chemistry: A Stable Fluorosulfuryl Imidazolium Salt Emerging as an ″F-SO. Angew. Chem., Int. Ed. 2018, 57 (10), 2605−2610. (12) Zhou, H.; Mukherjee, P.; Liu, R.; Evrard, E.; Wang, D.; Humphrey, J. M.; Butler, T. W.; Hoth, L. R.; Sperry, J. B.; Sakata, S. K.; Helal, C. J. Am Ende, C. W., Introduction of a Crystalline, ShelfStable Reagent for the Synthesis of Sulfur(VI) Fluorides. Org. Lett. 2018, 20 (3), 812−815.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Lyn H. Jones: 0000-0002-8388-5865 Notes
The author declares the following competing financial interest(s): L.H.J. is an employee and shareholder of Jnana Therapeutics.
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ACKNOWLEDGMENTS I thank O. Fadeyi, J. Kelly, B Sharpless, R. Robinson, L. Roberts, A. Gopalsamy, R. Kyne, E. Hett, H. Xu, J. Li, J. Majmudar, and C. am Ende for useful discussions.
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ABBREVIATIONS AISF, [4-(acetylamino)phenyl]imidodisulfuryl difluoride; BFS, benzene fluorosulfate; BSF, benzenesulfonyl fluoride; CRABP2, cellular retinoic acid binding protein 2; DcpS, decapping scavenger enzyme; FSY, fluorosulfate-L-tyrosine; HSDL2, hydroxysteroid dehydrogenase-like protein 2; iLBP, intracellular lipid binding protein; PAPS, phosphoadenosine phosphosulfate; TTR, transthyretin.
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REFERENCES
(1) Narayanan, A.; Jones, L. H. Sulfonyl fluorides as privileged warheads in chemical biology. Chem. Sci. 2015, 6, 2650−2659. C
DOI: 10.1021/acsmedchemlett.8b00276 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
