A Fluorescent Sensor and Gel Stain for Detection of

Jun 10, 2015 - The design and synthesis of a fluorescent sensor of diphosphate esters along with its application for in-gel detection is described. Di...
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A Fluorescent Sensor and Gel Stain for Detection of Pyrophosphorylated Proteins Florence J. Williams, and Dorothea Fiedler ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00256 • Publication Date (Web): 10 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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A Fluorescent Sensor and Gel Stain for Detection of Pyrophosphorylated Proteins Florence J. Williams, Dorothea Fiedler* Department of Chemistry, Princeton University, Washington Road, Princeton, New Jersey 08544, United States Supporting Information Placeholder ABSTRACT: The design and synthesis of a fluorescent sensor of diphosphate esters along with its application for in-gel detection is described. Dinuclear zinc complex 1 selectively detects diphosphate esters in the presence of various other functional groups, including monophosphate esters. Complex 1 also constitutes a competent stain for visualization of pyrophosphorylated proteins in polyacrylamide gels. This reagent will facilitate the validation and exploration of protein pyrophosphorylation.

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modification and ion suppression. Therefore, we set out to develop a chemical tool which could be used to visualize this modification on proteins (Figure 1b).

a)

Post-translational modifications (PTMs) functionalize proteins to alter their activity, substrate specificity, and/or localization. PTMs dictate a variety of key decisions in the cell , and are altered in many disease states.1 2 Complementary to phosphorylation, a canonical PTM, protein pyrophosphorylation is a signaling mechanism which has only been characterized in vitro to date. Biochemical studies have established that pyrophosphorylation of proteins occurs through a non-enzymatic transfer of the β-phosphoryl group from an inositol pyrophosphate (PPIP) to a phosphoserine residue, resulting in the formation of , , , , a diphosphate ester (Figure 1a).3 4 5 6 7 Inositol pyrophosphates are generated in mammalian cells by either inositol hexakisphosphate kinase 1, 2 or 3 (IP6K 1,2,3), or by diphosphoinositol pentakisphosphate kinase 1 or 2 (PPIPK1,2), , through hyperphosphorylation of phytic acid.8 9 Deletion of IP6K genes, as well as mutations causing loss of kinase activity, result in prominent phenotypic changes at both the cellu, , , , , , lar and organismal level.10 11 12 13 14 15 16 These phenotypes include defects in telomere maintenance and height mis10a,h regulation. Remarkably, knockout of the IP6K1 gene in mice results in insulin hypersensitivity and resistance to 10d weight gain on a high fat diet. Future studies aimed at determining mechanistic rationale for these cellular responses would benefit from a conclusive determination whether protein pyrophosphorylation does indeed occur in living cells. While many methods exist to identify phosphorylated pro, , teins in complex samples,17 18 19 the detection of pyrophosphorylation has been limited to in vitro labeling with radioactive PP-IPs, and cannot be applied to the detection of in 3 vivo pyrophosphorylated proteins. Current mass spectrometric procedures may not readily detect pyrophosphorylated peptide fragments due to low stoichiometry of the

b)

Figure 1. (a) Current methods for detection and identification of phosphorylated or pyrophosphorylated peptides and proteins in complex samples. (b) A general design for detection of pyrophosphopeptides and proteins by fluorescence. Dizinc dipicolylamine frameworks have been well studied for their ability to bind phosphoryl-groups in aqueous environments, and have consequently found applications as spectroscopic reporters for phosphopeptides, multiply phosphorylated proteins, and pyrophosphate anion, among oth, , , , ,, , , , , , , ers.20 21 22 23 24 25 26 27 28 29 30 31 We recently described a dizinc(II) bis(dipicolylaminomethyl)phenol based complex which displayed a binding preference for diphosphate esters over monophosphate esters of approximately 100-fold.32 Additionally, this reagent showed minimal interactions with other anionic functional groups. We built upon this design with the intent to obtain a ratiometric fluorescent reagent for detection of diphosphate esters.

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Scheme 1. Synthesis of Ratiometric Fluorophore 1. DCE, HOAc allylamine

O N

Br

Br

N

NaHB(OAc) 3, 4 Å MS 83%

N

N

Br

Ns N

2) thiosalicyclic acid Pd(PPh3)4 (1 mol%) 23% over two steps

2

Bpin

1) Cu2O (2 eq), NsNHMe pyridine, reflux

OH

O

N

N H

N

Ns N

3

OH O

N N N

H2O2, NaOH

4

THF/H2O quant.

t-Bu

5 t-Bu

N

TFA, reflux 1 hr 38%

6 t-Bu

H N

NH HN NH HN N N N HO H2O N Zn Zn N O N

H N

OH N O

O Ns N

t-Bu

6

N

N H

N

Ns N

1) DCE, HOAc NaHB(OAc) 3, 4 Å MS 2) PhSH, DMF/H2O K2CO3 39% over 2 steps

OH

N

N

2X

ZnBr2 2H2O

N

N

NH

3

N

2+

K 2CO 3, MeOH 60%

HN

7

1

t-Bu t-Bu

Complex 1 was devised to incorporate a pyrene core as the fluorescent reporter group. The pyrene functionality allows for both excellent fluorescence properties,33 as well as a facile way to electronically link the anion binding pocket with the fluorescent moiety. The synthesis of 1 focused on a convergent strategy, separating the molecule into its fluorescent core and its zinc binding functionality (Scheme 1). The substituted dipicolylamine portion was rapidly accessed in three steps, involving reductive amination between commercial 6bromo-2-pyridinecarboxaldehyde and allylamine to give compound 2. Copper(I) catalyzed cross-coupling followed by deallylation provided secondary amine 3 . The pyrene core 6 was accessed by a Duff reaction performed on 7-(tertbutyl)pyren-2-ol (5), derived from boronic ester 4. Following a reductive amination to bring compounds 3 and 6 together, deprotection of the 2-nitrophenylsulfonyl groups furnished ligand 7. Zinc complex 1 was therefore obtained in six linear steps. Characterization of complex 1 revealed that the photometric properties changed in a ratiometric manner in response to diphosphate esters as compared to monophosphate esters. Fluorescence emission spectra corresponding to 355 nm and 370 nm excitation, respectively, showed characteristic differences when 1 was combined with AMP or ADP (Figures 2 and SI 2). These adenosyl phosphates were used as model compounds to confirm discrimination of the diphosphate ester from its corresponding monophosphate ester. These spectroscopic measurements were performed in a water:DMSO mixture at neutral pH, enabling the interrogation of biological samples. Because the mixed solvent system may perturb native protein structure, future work will focus on developing a derivative of complex 1 with increased water solubility. The ability of 1 to differentiate diphosphate esters from further functional groups was tested by examining model compounds and reagents that might interact with complex 1. Histidine is known to coordinate to related dizinc complexes,34 and citric acid was chosen as a mimic of polyacidic amino acid stretches contained in many proteins. While certain

functionalities elicited distinct changes in fluorescence (citrate, ADP-ribose, Figures 2 and SI 2), we were able to distinguish between different binding partners by evaluating the ratio of fluorescence emission intensities resulting from excitation at 370 nm and

c)

-

Pep1

H3N-Trp-Asn-Ala-Ser-Ala-Asn-Gly-COO

Pep2

AcHN-Lys-Ala-Asp-Asp-Phe-Gly-Pro-Ser-Ala-Trp-CONH 2

Figure 2. (a) Fluorescence spectra of complex 1 with different additives: (dashed) excitation spectra for emission at 435 nm; (solid) emission spectra following excitation at 370 nm; (b) Ratio of fluorescence emission intensity with different analytes at 438 nm resulting from 370 nm excitation over that resulting from 355 nm excitation. (c) Peptides utilized in fluorescence measurements. P- or PP- prefixes to the given peptide name indicate monophosphorylation or pyrophos-

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phorylation at the serine residue. All fluorescence measurements were carried out under the following conditions: 1:1

DMSO:HEPES (100 mM, pH = 7.16) with 3 µM 1 and 10 µM of the given analyte.

a) Protein-peptide conjugates synthesized for in-gel testing of staining with 1. b) SDS-PAGE gels showing successful ligation of phosphopeptides and discriminatory staining with 1. Bis-Tris (10%) gel, XT-MES running buffer. Loading: approx. 500 ng/lane. Loading was repeated and the gel was separated to treat one side with ProQ Diamond followed by Coomassie. The other side was treated with stain 1. (Top) Coomassie G stain (Middle) ProQ Diamond stain. (Right) Stain with 1, visualized at 365 nm excitation. c) Graphical representation of stain intensity of ProQ Diamond or upon treatment with 1, adjusted for protein loading as measured by integration of coomassie staining. Figure 3.

at 355 nm. As shown in Figure 2, diphosphate esters reproducibly presented a 0.60-0.65 ratio, while other analytes, such as citrate, displayed ratios of 0.55 or below. Encouraged by these observations, we examined a phosphopeptide, P-Pep1, and a pyrophosphopeptide, PP-Pep1 (monophosphate ester and diphosphate ester are denoted by prefixes P- and PP-, respectively). Complex 1 responded to these peptides similarly as to AMP and ADP. In order to further challenge the discriminatory properties of complex 1, P-Pep2 and PP-Pep2 were synthesized, incorporating two neighboring aspartic acid residues. As expected, complex 1 in the presence of P-Pep2 displayed a higher fluorescence ratio upon excitation at 370 nm and 355 nm than in the presence of P-Pep1. However, the fluorescence ratio is still markedly lower than the one observed when PP-Pep2 and PPPep1 are present. Additionally, we observed a bathochromic shift of 6-8 nm for the λmax,em when comparing fluorescence emissions with monophosphopeptides to that of pyrophosphopeptides. Further examination of the spectroscopic properties of 1 indicated that the complex may exhibit higher order interactions in aqueous solution due to the presence of a hydropho, bic pyrene core.35 36 However, we were able to obtain an ap–8 proximate binding dissociation value of 2 x 10 M when using a more organic solvent system [19:1 DMSO:HEPES(100 mM, pH = 7.16), Figure SI 8]. Despite these unusual properties, the binding affinity of 1 is sufficient to observe changes in analyte concentrations in the high nanomolar range (Figure SI 9). Next, we applied our fluorescent reagent for the visualization of proteins in SDS-PAGE gels. This objective is an ideal

application because it allows for detection of spatially separated protein mixtures with minimal manipulation of the proteins themselves. Additionally, as a commonly utilized technique, many laboratories already have the equipment and expertise necessary to perform in-gel fluorescence detection. To establish the distinction between pyrophosphorylated and monophosphorylated proteins in a gel, a selection of peptides were appended to Glutathione S-Transferase (GST) using glutaraldehyde ligation (Figure 3a).37 Because GST is known to self-associate, all ligated products were obtained as , , GST dimers (Figure SI 12).38 39 40 The peptides were chosen to 34 compare proteins containing histidines (vide supra), phos41 phoserines, proximal phosphoserines, and pyrophosphorylated serine residues. In addition, Pep2 was selected to test whether polyacidic stretches adversely affected dye recognition. The resulting protein-peptide conjugates were visualized on an SDS-PAGE gel with Coomassie-G blue, which confirmed successful ligation (as observed by an upward mobility shift). ProQ Diamond is a widely used reagent for phosphoprotein detection and also confirmed successful ligation of the phosphorylated and pyrophosphorylated peptides 19 (Figure 3b). However, ProQ Diamond exhibited no selectivity for monophosphate esters over diphosphate esters (Figure 3c). After optimization of the staining procedure, we were pleased to obtain a robust method for the visualization of pyrophosphorylated protein with complex 1. The procedure provided clear distinction of pyrophosphorylated proteins as compared to non-phosphorylated proteins as well as polyphosphoproteins (Figure 3b). Integration of the gel fluores-

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cence intensity revealed a 31-fold increase in staining of PPProt2 vs. P-Prot2 (Figure 3c). A difference of approximately 9-fold was also achieved in the presence of aspartic acid residues, between PP-Prot1 and P-Prot1. The latter observation is of significance, since acidic residues had exhibited minor interactions with 1 in fluorescence measurements in solution (Figure 2b). Staining with reagent 1 can be detected at the two most common wavelengths currently utilized in UV transilluminators, 302 nm and 365 nm, although 365 nm excitation is optimal (as depicted in Figure 3). Reagent 1 also exhibited bright in-gel fluorescence, requiring only ~400 ms exposure time for visualization.

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tion at 355 nm and 370 nm. Reagent 1 can thus function as a ratiometric sensor in solution. Importantly, 1 can be used to visualize pyrophosphorylated proteins in polyacrylamide gels, providing an important and easily utilized tool for applications towards in vivo investigations. The solubility properties of 1 require the use of a 50:50 DMSO:H2O mixture, which could result in significant protein denaturation for solution measurements. However, the application of 1 as a stain for denaturing gel electrophoresis is not adversely affected by this solvent system. Gel staining distinguishes pyrophosphorylated proteins from multiply monophosphorylated proteins in the presence of acidic amino acid stretches 3 and cell lysates. To date, the only method to identify pyrophosphorylated proteins has been an in vitro pyrophosphorylation reaction with radiolabeled PP-IPs, resulting in radiolabeling of the 3 protein. Gel staining with 1 provides an attractive alternative which can be used in comparative studies without the need for handling radioactive material. We envision that reagent 1 can also be applied to the detection of proteins pyrophosphorylated in vivo, for which there is currently no , , alternative technology.43 44 45 Due to the precision of fluorescence, intensity differences can be correlated to changes in pyrophosphorylation stoichiometry in protein samples. Future work will focus on the application of this technique in biological contexts.

Supporting Information

Figure 4. PP-Prot2 was combined with E. coli (DH5α) or S. cerevisiae (Y10) lysate and stained with Coomassie-G (for total protein) or with 1. 10% Bis-Tris gel, XT-MES running buffer. With the established SDS-PAGE staining protocol, we sought to validate the ability of 1 to detect pyrophosphoproteins in a complex mixture. PP-Prot2 was mixed with increasing amounts of E. coli and S. cerevisiae lysates. The mixture was resolved by SDS-PAGE and then stained with complex 1 (Figure 4). In all samples, the broad band for PP-Prot2 was readily observable despite the presence of functionally diverse E. coli and S. cerevisiae proteomes.42 Notably, in the lysate from S. cerevisiae, additional sharp bands become visible after staining with 1, and we are eager to determine the identity of these proteins in the future. In conclusion, dizinc complex 1 was developed for the detection of pyrophosphoproteins. The diphosphate ester functional group exhibits high affinity towards complex 1, with a dissociation constant in the low micromolar range. Binding of diphosphate esters to 1 results in a reproducible change in fluorescence emission intensity ratio upon excita-

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. Supporting information includes descriptions of all fluorescence experiments with corresponding spectra, including experiments not mentioned in text, a general protocol for polyacrylamide gel staining with 1, and experimental procedures and spectra for all new compounds.

Corresponding Author [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors would like to acknowledge L. Yates for her donation of P-Pep1 and PP-Pep1. Financial support from Princeton University and the National Institute of Health (DP2 CA186753) are gratefully acknowledged. D.Fiedler is a Rita Allen and Kimmel Scholar.

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Stebbing, J.; Lit, L. C.; Zhang, H.; Darrington, R. S.; Melaiu, O.; Rudraraju, B.; Giamas, G. (2014) The regulatory roles of phosphatases in cancer. Oncogene, 33, 939–953. 2 Walsh, C. (2005) Posttranslational Modification of Proteins: Expanding Nature’s Inventory,Vol. 1, Roberts and Company: Greenwood Village, CO. 3 Bhandari, R.; Saiardi, A.; Ahmadibeni, Y.; Snowman, A. M.; Resnick, A. C.; Kristiansen, T. Z.; Molina, H.; Pandey, A.; Werner, J. K., Jr.; Juluri, K. R.; Xu, Y.; Prestwich, G. D.; Parang, K.; Snyder, S. H. (2007) Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event. Proc. Nat. Acad. Sci., 104, 15305–15310. 4 Saiardi, A.; Bhandari, R.; Resnick, A.; Snowman, A. M.; Snyder, S. H. (2004) Phosphorylation of proteins by inositol pyrophosphates. Science, 306, 2101–2105. 5 Azevedo, C.; Burton, A.; Ruiz-Mateos, E.; Marsh, M.; Saiardi, A. (2009) Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc. Nat. Acad. Sci., 106, 21161−21166. 6 Szijgyarto, Z.; Garedew, A.; Azevedo, C.; Saiardi, A. (2011) Influence of inositol pyrophosphates on cellular energy dynamics. Science, 334, 802−805. 7 Burton, A.; Azevedo, C.; Andreassi, C.; Saiardi, A. (2013) Inositol pyrophosphates regulate JMJD2C-dependent histone demethylation. Proc. Nat. Acad. Sci, 47, 18970−18975. 8 These enzymes are also referred to as VIP1/2. 9 For a recent review, see: Wundenberg, T.; Mayr, G. W. (2012) Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis. Biol Chem., 393, 979–998. 10 Lachance, J., Vernot, B., Elbers, C. C., Ferwerda, B., Froment, A., Bodo, J. M., Lema, G., Fu, W., Nyambo, T. B., Rebbeck, T. R., Zhang, K., Akey, J. M., and Tishkoff, S. A. (2012) Evolutionary history and adaptation from high-coverage whole-genome sequences of diverse African hunter-gatherers. Cell, 150, 457−469. 11 Chakrabory, A.; Kim, S.; Snyder, S. H. (2011) Inositol pyrophosphates as mammalian cell signals. Science Signaling, , 4(188), re 1. 12 Chakrabory, A.; Koldobskiy, M. A.; Bello, N. T.; Maxwell, M.; Potter, J. J.; Juluri, K. R.; Maag, D.; Kim, S.; Huang, A. S.; Dailey, M. J.; Saleh, M.; Snowman, A. M.; Moran, T. H.; Mezey, E.; Snyder, S. H. (2010) Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain. Cell, 143, 897-910. 13 Bhandari, R.; Juluri, K. R.; Resnick, A. C.; Snyder, S. H. (2008) Gene deletion of inositol hexakisphosphate kinase 1 reveals inositol pyrophosphate regulation of insulin secretion, growth, and spermiogenesis. Proc. Nat. Acad. Sci., 105, 2349– 2353. 14 Alcazar-Roman, A. R.; Wente, S. R. (2008) Inositol polyphosphates: a new frontier for regulating gene expression. Chromosoma, 117, 1-13. 15 Illies, C.; Gromada, J.; Fiume, R.; Leibiger, B.; Yu, J.; Juhl, K.; Yang, S.-N.; Barma, D. K.; Falck, J. R.; Saiardi, A.; Barker, C. J.; Berggren, P.-O. (2007) Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells. Science, 318, 1299–1302. 16 Kamimura J.;Wakui, K.; Kadowaki, H.; Watanabe, Y.; Miyake, K.; Harada, N.; Sakamoto, M.; Kinoshita, A.; Yoshiura, K.-i.; Ohta, T.; Kishino, T.; Ishikawa, M.; Kasuga, M.; Fukushima, Y.; Niikawa, N.; Matsumoto, N. (2004) The IHPK1 gene is disrupted at the 3p21.31 breakpoint of t(3;9) in a family with type 2 diabetes mellitus. J. of Hum. Genet., 49, 360–365. 17 Generation of phosphotyrosine antibody: Ross, A. H.; Eisen, H. N. (1981) Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature, 294, 654−656. 18 Boersema, P. J.; Mohammad, S.; Heck, A. J. R. (2009) Phosphopeptide fragmentation and analysis by mass spectrometry. J. Mass Spec., 44, 861–878. 19 Seinberg, T. H.; Agnew, B. J.; Gee, K. R.; Leung, W. Y.; Goodman, T.; Schulenberg, B.; Hendrickson, J.; Beechem, J. M.; Haugland, R. P.; Patton, W. F. (2003) Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology., 3, 1128−1144. 20 For a review on anion recognition reagents based on the dizinc dipicolylamine framework see: Ngo, H. T.; Liu, X.; Jolliffe, K. A. (2012) Anion recognition and sensing with Zn(II)-dipicolylamine complexes. Chem. Soc. Rev., 41, 4928–4965. 21 Kraskouskaya, D.; Bancerz, M.; Soor, H. S.; Gardiner, J. E.; Gunning, P. T. (2014) An excimer-based, turn-on fluorescent sensor for the selective detection of diphosphorylated proteins in aqueous solution and polyacrylamide gels. J. Am. Chem. Soc., 136, 1234–1237. 22 Turkyilmaz, S.; Rice, D. R.; Palumbo, R.; Smith, B. D. (2014) Selective recognition of anionic cell membranes using targeted liposomes coated with zinc(ii)-bis(dipicolylamine) affinity units. Org. Biomol. Chem., 12, 5645.

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Wang, J.; Liu, B.; Liu, X.; Panzner, M. J.; Wesdemiotis, C.; Pang, Y. A (2014) binuclear Zn(II)-Zn(II) complex from a 2hydroxybenzohydrazide-derived Schiff base for selective detection of pyrophosphate. Dalton Trans., 43, 14142−14146. 24 Yang, S.; Feng, G.; Williams, N. H. (2012) Highly selective colorimetric sensing pyrophosphate in water by a NBDphenoxo-bridged dinuclear Zn(II) complex. Org. Biomol. Chem., 10, 5606–5612. 25 Pathberiya, L. G.; Barlow, N.; Nguyen, T.; Graham, B.; Tuck, K. L. (2012) Facile, divergent route to bisZn(II)dipicolylamine type chemosensors for pyrophosphate. Tetrahedron, 68, 9435–9439. 26 Lee, D.-N.; Jo, A.; Park, S. B.; Hong, J.-I. (2012) Colorimetric and orange light-emitting fluorescent probe for pyrophosphate in water. Tetrahedron Lett., 53, 5528–5530. 27 Jeong, Y.; Kim, S. Y.; Jin, G.-w.; An, S.; Lee, J. H.; Jeong, A.-r.; Chio, Y.; Hong, J.-I.; Park, J.-S. (2010) Spectrofluorimetric Determination of Bisphosphonateswith a Fluorescent Chemosensor, Zinpyr-1·2Zn2+. Bull. Korean Chem. Soc., 31(9), 2561–2564. 28 Kim, S. Y.; Hong, J.-I. (2010) Fluorescent Chemosensor for Detection of PPi Through the Inhibition of Excimer Emission in Water Bull. Korean Chem. Soc., 31(3), 716–719. (i) Lee, H. N.; Swamy, K. M. K.; Kim, S. K.; Kwon, J.-Y.; Kim, Y.; Kim, S.-J; Yoon, Y. J.; Yoon, J. (2007) Simple but effective way to sense pyrophosphate and inorganic phosphate by fluorescence changes. Org. Lett., 9, 243–246. 29 Ojida, A.; Mito-oka, Y.; Sada, K.; Hamachi, I. (2004) Molecular recognition and fluorescence sensing of monophosphorylated peptides in aqueous solution by bis(zinc(II)-dipicolylamine)-based artificial receptors. J. Am. Chem. Soc., 126, 2454– 2463. 30 Lee, D. H.; Kim, S. Y.; Hong, J.-I. (2004) A fluorescent pyrophosphate sensor with high selectivity over ATP in water. Angew. Chem Int. Ed., 43, 4777–4780. 31 Ojida, A.; Mito-oka, Y.; Inoue, M.-a.; Hamachi, I. (2002) First artificial receptors and chemosensors toward phosphorylated peptide in aqueous solution. J. Am. Chem. Soc., 124, 6256–6258. 32 Conway, J. H.; Fiedler, D. (2015) An affinity reagent for the recognition of pyrophosphorylated peptides. Angew. Chem. Int. Ed., 54, 3941–3945. 33 Lavis, L. D.; Raines, R. T. (2008) Bright ideas for chemical biology. ACS Chem. Bio., 3, 142–155. 34 Mito-oka, Y.; Tsukiji, S.; Hiraoka, T.; Kasagi, N.; Shinkai, S.; Hamachi, I. (2001) Zn(II) dipicolylamine-based artificial receptor as a new entry for surface recognition of alpha-helical peptides in aqueous solution. Tetrahedron Lett., 42, 7059– 7062. 35 The absorbance of 7 at 365 nm does not change in a linear fashion with changes in concentration (Figure SI 6). Similar observations of nonlinearity were observed in fluorescence measurements with decreasing amounts of PP-Pep1 or PP-Pep2, despite Job plot results indicating a 1:1 association between 7 and pyrophosphopeptide (Figure SI 7). 36 Concentrations changes did not affect the λmax,em values, making it improbable that the ratiometric properties of complex 1 arise simply from the presence or absence of these putative higher-order interactions. 37 The GST used was recombinantly expressed from E. Coli to avoid any prior phosphorylation of serine, threonine, or tyrosine residues. 38 Armstrong, R. (1991) Glutathione S-transferases: reaction mechanism, structure, and function. Chem Res. Toxicol., 4, 131– 140. 39 GST ligation was also performed in the absence of peptide in order to obtain a negative ligation control. 40 The ligation was also performed with insulin-like growth factor 1 (IGF1) expressed in E. coli. Pyrene Stain 1 successfully discriminated between the IGF1-peptide conjugate containing phosphorylation from that containing pyrophosphorylation. See SI. 41 The spacing of two phosphoserines separated by one amino acid is the most commonly observed spacing of two phosphoserines: Schweiger, R.; Linial, M. (2010) Cooperativity within proximal phosphorylation sites is revealed from largescale proteomics data. Biology Direct, 5(6), 1–17. 42 In both the E. coli and S. cerevisiae lysates we noted a significant amount of background fluorescence of high molecular weight proteins (> 100 kDa) after staining with 1. Presumably, these larger proteins contain extended hydrophobic areas, to which reagent 1 could bind non-specifically. 43 Small molecule sensors such as 1 also have an advantage over the development of antibodies, as antibodies often exhibit sequence specificity. In particular, obtaining a pan-specific antibody for recognition of phosphoserine/threonine proteins has historically been a challenge. 44 Zerweck, J.; Masch, A.; Schutkowski, (2009) M. Peptide Microarrays for Profiling of Modification State-Specific Antibodies in Methods in Molecular Biology, Vol. 524, pp 169−180, Humana Press, New York, NY. 45 Wang, Z.; Gucek, M.; Hart, G. W. (2008) Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc. Proc. Nat. Acad. Sci., 105, 13793−13798.

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