Versatile and Efficient Site-Specific Protein Functionalization by ...

German Edition: DOI: 10.1002/ange.201505456Protein ModificationInternational Edition: DOI: 10.1002/anie.201505456

Versatile and Efficient Site-Specific Protein Functionalization byTubulin Tyrosine LigaseDominik Schumacher, Jonas Helma, Florian A. Mann, Garwin Pichler, Francesco Natale,Eberhard Krause, M. Cristina Cardoso, Christian P. R. Hackenberger,* and Heinrich Leonhardt*

Abstract: A novel chemoenzymatic approach for simple andfast site-specific protein labeling is reported. Recombinanttubulin tyrosine ligase (TTL) was repurposed to attach variousunnatural tyrosine derivatives as small bioorthogonal handlesto proteins containing a short tubulin-derived recognitionsequence (Tub-tag). This novel strategy enables a broad rangeof high-yielding and fast chemoselective C-terminal proteinmodifications on isolated proteins or in cell lysates forapplications in biochemistry, cell biology, and beyond, asdemonstrated by the site-specific labeling of nanobodies, GFP,and ubiquitin.

Site-specific functionalization of proteins is crucial fora plethora of applications throughout the life sciences.Fluorescent proteins and self-labeling strategies like SNAP-[1] and HALO-[2] tagging have become indispensable tools forcell biologists to analyze intracellular activity and localizeproteins of interest. Genetic fusion with GFP or self-labelingprotein tags is straightforward; however, the size andbiochemical nature of the attachment may affect the proper-ties and application of the chimeric protein.[3] Protein trans-splicing, expressed protein ligation (EPL), and amber sup-pression and auxotrophic expression in combination withbioorthogonal labeling are prominent tools that allow theplacement of small tags and modifications within the proteinsequence.[4] However, low expression yields of geneticencoding systems[5] and challenges associated with impairedprotein folding with trans-splicing and EPL[6] can be limitingfactors when applying these techniques. As an alternative,chemoenzymatic approaches have attracted increasing atten-

tion for the site-specific modification of proteins by usingshort and specific recognition tags. A variety of enzymes, suchas trypsin,[7] Sortase A,[8] phosphopantetheinyl transferase(PPTase),[9] biotin ligase,[10] lipoic acid ligase,[11] AnkX,[12] andformylglycine generating enzyme,[13] open up broad possibil-ities for subsequent chemoselective and site-specific labeling.However, several chemoenzymatic approaches still need toovercome some limitations, including large and hydrophobicsubstrates for the enzymatic reaction that may affect theprotein of interest.[14] Moreover, the reversibility of someenzymatic reactions and product hydrolysis necessitatesextensive enzyme engineering and a high excess of catalystand substrate.[7, 14, 15]

Herein, we present a novel and fast method for the site-specific labeling of functional proteins that combines the useof small unnatural amino acids as bioorthogonal handles andthe technical advantages of chemoenzymatic labeling. Thetechnique, termed Tub-tag labeling, is based on the enzymaticligation of easy-to-synthesize small tyrosine derivatives to theC terminus of a fourteen amino acid hydrophilic recognitiontag (termed Tub-tag) by tubulin tyrosine ligase (TTL, Fig-ure 1a). In nature, TTL catalyzes the post-translationalattachment of tyrosine to the C terminus of a-tubulin, whichis involved in the regulation of microtubule homeostasis.[16]

Interestingly, it has been shown that TTL also utilizes tyrosinederivatives for the C-terminal modification of tubulin.[17]

We started our investigation by probing whether recombi-nant TTL can conjugate unnatural tyrosine derivatives to theisolated Tub-tag peptide, which mimics the C terminus oftubulin. For initial Tub-tag labeling experiments, we used 3-N3-l-tyrosine (1) and 3-formyl-l-tyrosine (2) as substratesand synthesized a 5,6-carboxyfluorescein-labeled Tub-tagpeptide (CF-Tub-tag) by standard solid-phase peptide syn-thesis (SPPS). The reaction process was analyzed by isocraticHPLC (Figure 1b for 1, Figure 1 c for 2, and Figure S1 in theSupporting Information). After 120 min of incubation (TTL/peptide 1:200) at 37 88C, we observed a conversion of 90 % and63% with 1 and 2, respectively. The initial reaction rate ofazide 1 surpasses that of aldehyde 2, resulting in anapproximately 2.6 times faster formation of the ligationproduct. However, extending the reaction in the case ofaldehyde 2 delivers equal amounts of labeled peptide, thusdemonstrating the broad substrate acceptance of tubulintyrosine ligase.

Encouraged by these results, we tested whether Tub-taglabeling can be applied to the site-specific modification ofproteins and whether the required C-terminal recognitionmotif influences the protein function. At the outset of ourstudies, we focused on camel-derived single-domain nano-

[*] D. Schumacher,[+] F. A. Mann, Dr. E. Krause,Prof. Dr. C. P. R. HackenbergerChemical Biology, Leibniz-Institut fír Molekulare PharmakologieBerlin (Germany)E-mail: [email protected]

Dr. J. Helma,[+] Dr. G. Pichler, Prof. Dr. H. LeonhardtDepartment of Biology II, Ludwig Maximilians Universit�t Mínchenand Center for Integrated Protein Science MunichMartinsried (Germany)E-mail: [email protected]

Dr. F. Natale, Prof. Dr. M. C. CardosoDepartment of Biology, Technische Universit�t DarmstadtDarmstadt (Germany)

D. Schumacher,[+] F. A. Mann, Prof. Dr. C. P. R. HackenbergerDepartment of Chemistry, Humboldt Universit�t zu BerlinBerlin (Germany)

[++] These authors contributed equally to this work.

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201505456.

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bodies,[18] which are used as analytical tools in biochemistry aswell as for the intracellular recognition and manipulation ofantigens in cell biology.[19] In this context, it was our particulargoal to access a site-specifically fluorescently-labeled nano-body, since these approximately 15 kDa antigen-recognizingproteins display superior properties in superresolution mi-croscopy owing to their exceptionally small size.[20]

We fused the Tub-tag sequence to the C terminus ofa GFP-specific nanobody (GBP4[21]) and performed TTL-mediated labeling with a TTL/GBP4 ratio of 1:5 at 37 88C for24 h. Tryptic digest followed by HPLC–MS/MS experimentsshowed the successful C-terminal addition of tyrosine, 3-N3-l-tyrosine (1), 3-formyl-l-tyrosine (2), 3-NH2-l-tyrosine (3),and 3-NO2-l-tyrosine (4 ; Figure S3 in the Supporting Infor-mation). Next, we combined the incorporation of 1 withsubsequent strain-promoted azide–alkyne cycloaddition(SPAAC)[22] for the conjugation of a dibenzocyclooctyne(DBCO)-biotin derivative (Figure 2a). For this, ligationswere performed using different ratios of TTL/GBP4 and thereactions stopped by cooling down fast to 4 88C after one orthree hours. A subsequent dialysis step was used to removeexcess tyrosine derivative 1, followed by SPAAC to DBCO-biotin. The yields were determined by SDS-PAGE analysis.Using a TTL/GBP4 ratio of 1:5 at 37 88C, we found that 82% ofthe GBP4 was converted after one hour, whereas a TTL/GBP4 ratio of 1:10 delivered 71% C-terminally modifiedGBP4. Extending the ligation time to three hours resulted in99% and 88% conversions at TTL/GBP4 ratios of 1:5 and1:10, respectively. The incorporation of 3-formyl-l-tyrosine(2) could be achieved with similar efficiency. To furthervalidate the modularity of the Tub-tag labeling concept, weperformed fluorescence labeling by SPAAC (Figure S7 in the

Supporting Information) and employed a variety of well-established bioorthogonal reactions, including Staudingerligation[23] (Figure S8 in the Supporting Information) andthe Staudinger-phosphite reaction[24] (Figure S9 in the Sup-porting Information), to 3-N3-l-tyrosine. In addition, hydra-zone- (Figure S10 in the Supporting Information) and oxime-(Figure S11 in the Supporting Information) forming reac-tions[3c] were applied on site-specifically incorporated 3-formyl-l-tyrosine (2). This allowed us to incorporate differentfunctional modules like biotin and fluorophores, and evenenabled the branched PEGylation of GBP4 (Figure S9 in theSupporting Information). To demonstrate the broad applic-ability of this chemoenzymatic modification, we additionallyapplied it to the site-specific functionalization of GFP,ubiquitin, and another GFP-specific nanobody (GBP1[21]).SDS-PAGE analysis showed similar yields to the modificationof GBP4 (Figure S12 and S13 in the Supporting Information).Moreover, the presence of the Tub-tag sequence did not affectthe fluorescence and fluorescence-modifying properties[21] ofGFP or GFP-specific nanobodies (Figures S4, S5 in theSupporting Information).

Having established the modular and high-yielding TTL-based modification method, we investigated the selectivity ofthe tyrosine transfer reaction within complex protein mix-tures. E. coli cell lysate containing overexpressed GFP-Tub-tag was incubated with 3-N3-l-tyrosine (1) and TTL for fourhours. A subsequent dialysis was used to remove excess azide,and SPAAC to DBCO-biotin showed selective C-terminalfunctionalization of GFP (Figure 3a, b and Figure S14 in theSupporting Information). Next, we probed the specificity andapplicability of the site-specifically modified nanobody GBP1for the enrichment and isolation of overexpressed GFP from

Figure 1. Tub-tag labeling of proteins. a) Chemoenzymatic labeling of proteins by tubulin tyrosine ligase (TTL). Unnatural tyrosine derivatives areligated to the C terminus of a short recognition tag (Tub-tag) to serve as bioorthogonal handles for the site-specific chemical modification ofa protein of interest (POI). b, c) C-terminal addition of b) 3-N3-l-tyrosine (1) and c) 3-formyl-l-tyrosine (2) to carboxyfluorescein-labeled peptide(CF-Tub-tag). HPLC fluorescence traces were taken at different time points of the TTL reaction and quantitation of substrate and product wasperformed through peak integration (Figure S1 in the Supporting Information). The red lines represents the consumption of the CF-Tub-tag, theblue and green lines the formation of C-terminally functionalized CF-Tub-tag-YN3/CHO. The mean values and standard deviation (SD) of threereplicate reactions are shown.

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cell lysate. Following 3-N3-l-tyrosine (1) incorporation viaTTL, GBP1 was biotinylated with DBCO-biotin as describedabove and immobilized on Streptavidin-coated magnetic

beads (Figure 3c). These beads were then used for theimmunoprecipitation of GFP from HEK cell lysates. Subse-quent western blot analysis demonstrated specific GFP

Figure 2. Principle and efficiency of TTL-mediated functionalization a) Illustration of TTL-mediated incorporation of azide 1 followed by strain-promoted azide–alkyne cycloaddition (SPAAC) with DBCO-biotin. Dark gray circle = biotin. b) Incorporation of azide 1 into the C terminus ofGBP4 by using different ratios of GBP4/TTL and reaction times (one and three hours), followed by SPAAC to a DBCO-biotin derivative. SDS-PAGEand western blot (Strep Ab-HRP) show efficient biotin labeling of GBP4 within one hour.

Figure 3. Lysate labeling and the application of chemoenzymatically functionalized nanobodies to protein enrichment and superresolutionmicroscopy. a) Schematic outline of Tub-tag labeling of GFP in complex protein mixtures (E. coli lysate). b) Coomassie staining and western blotanalysis showing the high selectivity of the tyrosine ligation and subsequent biotinylation. (+ : lysate treated with TTL, 3-N3-l-tyrosine (1) andDBCO-biotin; C1: no 3-N3-l-tyrosine (1) added; C2: lysate treated with DBCO-biotin; C3: lysate; C4: purified GFP). c) Outline of the site-specificbiotinylation of the GFP-binding nanobody GBP1 and subsequent immunoprecipitation of GFP. d) Coomassie staining and western blot analysisshowing efficient and specific GFP pulldown (Mock GBP1-biotin: lysate lacking overexpressed GFP; GFP GBP1-biotin: beads loaded with GBP1and lysate containing GFP; GFP control beads: beads without immobilized GBP1, I: input to streptavidin beads; FT: flowthrough; B: beads).e) Outline of the site-specific labeling of GBP1 with Alexa594 f) Immunofluorescence with GBP1-Alexa594. Shown is a fixed HeLa cell nucleuswith the lamina colabeled with LaminB1-GFP and GBP1-Alexa594. Scale bar:10 mm. g) expansion of the region highlighted in (d). h) Fluorescenceintensity profile along the dotted line shown in (g) demonstrates high colocalization accuracy at subdiffraction resolution.

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pulldown compared to controls with mock-transfected celllysate and non-functionalized beads (Figure 3d).

We subsequently studied whether site-specifically TTL-labeled GBP1 can be used to immunostain cellular structuresfor superresolution microscopy. The higher resolution ofsuperresolution microscopy imposes new requirements ondetection reagents, and using the smallest possible immuno-fluorescent binding reagents is thus important for unleashingthe full potential of superresolution microscopy.[20b]

Following 3-formyl-l-tyrosine (2) incorporation via TTL,GBP1 was labeled with the Alexa594 dye by using oximeformation (Figure 3e). In addition to the microscopy appli-cation, it was shown that both the azide and the aldehydetyrosine derivatives could be used to obtain functional proteinconjugates. HeLa cells expressing GFP-LaminB1, whichlocalizes at the interior of the nuclear envelope and formsthe nuclear lamina, were fixed and stained with GBP1-Alexa594 (ca. 1 mg per object slide). 3D-SIM superresolutionmicroscopy revealed laminar colocalization of the GBP1staining reagent and the GFP-LaminB1 at high resolution,thereby indicating functional binding to GFP in this cellularcontext (Figure 3 f–h). Similar results were obtained withGFP-PCNA and the detection of subnuclear DNA replicationsites (Figure S15 in the Supporting Information).

These studies demonstrate the applicability of Tub-tag-labeled nanobodies for functional studies in biochemistry andcell biology.

In summary, we introduce Tub-tag labeling for the simple,site-specific modification of functional and antigen-recogniz-ing proteins. The TTL-mediated chemoenzymatic ligation ofunnatural tyrosine derivatives like 3-N3-l-tyrosine (1) and 3-formyl-l-tyrosine (2) enabled bioorthogonal functionaliza-tion in a total yield of up to 99 % when using moderateenzyme concentrations and short reaction times. Thesemodified tyrosine residues then serve as bioorthogonalhandles for a variety of well-established chemoselectivelabeling reactions. The overall labeling efficiency under mildreaction conditions yields homogeneously modified andfunctional proteins, as demonstrated by the site-specificlabeling of nanobodies for immunoprecipitation and super-resolution microscopy. In summary, Tub-tag labeling endowsrecombinant antibodies—and proteins in general—with novelproperties for exploring and manipulating cellular functions,with possible applications in biotechnology and medicine.

Acknowledgements

This work was supported by grants from the DeutscheForschungsgemeinschaft (SPP1623) to M.C.C. (CA 198/8-1),C.P.R.H. (HA 4468/9-1) and H.L. (LE 721/13-1), the Nano-systems Initiative Munich (NIM) to H.L., the EinsteinFoundation Berlin (Leibniz-Humboldt Professorship) andthe Boehringer-Ingelheim Foundation (Plus 3 award) toC.P.R.H. and the Fonds der Chemischen Industrie (FCI) toC.P.R.H. and to D.S. (Kekul¦-scholarship). We thank RalfJacob for providing a plasmid with the TTL codingsequence.[25] We are grateful to Kristin Kemnitz-Hassanin,Heike Stephanowitz, and Andreas Maiser for technical

support and to the late Bij�n Mir-Montazeri for help in theinitial phase of this project. The authors declare competingfinancial interest: The technology described in the manuscriptis part of a pending patent application.

Keywords: antibodies · chemoenzymatic labeling · nanobodies ·site-specific functionalization · tubulin tyrosine ligase

How to cite: Angew. Chem. Int. Ed. 2015, 54, 13787–13791Angew. Chem. 2015, 127, 13992–13996

[1] A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K.Johnsson, Nat. Biotechnol. 2003, 21, 86 – 89.

[2] G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana,M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G.Vidugiris, J. Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit, K. V.Wood, ACS Chem. Biol. 2008, 3, 373 – 382.

[3] a) D. Schumacher, C. P. Hackenberger, Curr. Opin. Chem. Biol.2014, 18, 62 – 69; b) K. Lang, J. W. Chin, Chem. Rev. 2014, 114,4764 – 4806; c) E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int.Ed. 2009, 48, 6974 – 6998; Angew. Chem. 2009, 121, 7108 – 7133.

[4] a) C. P. Hackenberger, D. Schwarzer, Angew. Chem. Int. Ed.2008, 47, 10030 – 10074; Angew. Chem. 2008, 120, 10182 – 10228;b) L. R. Malins, R. J. Payne, Curr. Opin. Chem. Biol. 2014, 18,70 – 78; c) C. D. Spicer, B. G. Davis, Nat. Commun. 2014, 5, 4740;d) B. L. Nilsson, M. B. Soellner, R. T. Raines, Annu. Rev.Biophys. Biomol. Struct. 2005, 34, 91 – 118.

[5] a) L. Wang, P. G. Schultz, Angew. Chem. Int. Ed. 2005, 44, 34 –66; Angew. Chem. 2005, 117, 34 – 68; b) S. Milles, S. Tyagi, N.Banterle, C. Koehler, V. VanDelinder, T. Plass, A. P. Neal, E. A.Lemke, J. Am. Chem. Soc. 2012, 134, 5187 – 5195.

[6] a) G. Volkmann, H. D. Mootz, Cell. Mol. Life Sci. 2013, 70,1185 – 1206; b) A. Reif, S. Siebenhaar, A. Troster, M. Schmal-zlein, C. Lechner, P. Velisetty, K. Gottwald, C. Pohner, I. Boos, V.Schubert, S. Rose-John, C. Unverzagt, Angew. Chem. Int. Ed.2014, 53, 12125 – 12131; Angew. Chem. 2014, 126, 12321 – 12327.

[7] S. Liebscher, M. Schopfel, T. Aumuller, A. Sharkhuukhen, A.Pech, E. Hoss, C. Parthier, G. Jahreis, M. T. Stubbs, F. Bordusa,Angew. Chem. Int. Ed. 2014, 53, 3024 – 3028; Angew. Chem.2014, 126, 3068 – 3072.

[8] H. Mao, S. A. Hart, A. Schink, B. A. Pollok, J. Am. Chem. Soc.2004, 126, 2670 – 2671.

[9] J. Yin, P. D. Straight, S. M. McLoughlin, Z. Zhou, A. J. Lin, D. E.Golan, N. L. Kelleher, R. Kolter, C. T. Walsh, Proc. Natl. Acad.Sci. USA 2005, 102, 15815 – 15820.

[10] I. Chen, M. Howarth, W. Lin, A. Y. Ting, Nat. Methods 2005, 2,99 – 104.

[11] M. Fern�ndez-Su�rez, H. Baruah, L. Mart�nez-Hern�ndez, K. T.Xie, J. M. Baskin, C. R. Bertozzi, A. Y. Ting, Nat. Biotechnol.2007, 25, 1483 – 1487.

[12] K. Heller, P. Ochtrop, M. F. Albers, F. B. Zauner, A. Itzen, C.Hedberg, Angew. Chem. Int. Ed. 2015, 54, 10327 – 10330; Angew.Chem. 2015, 127, 10467 – 10471.

[13] P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee, C. R.Bertozzi, Proc. Natl. Acad. Sci. USA 2009, 106, 3000 – 3005.

[14] M. Rashidian, J. K. Dozier, M. D. Distefano, BioconjugateChem. 2013, 24, 1277 – 1294.

[15] M. W. Popp, J. M. Antos, G. M. Grotenbreg, E. Spooner, H. L.Ploegh, Nat. Chem. Biol. 2007, 3, 707 – 708.

[16] a) M. Rudiger, J. Wehland, K. Weber, FEBS J. 1994, 220, 309 –320; b) A. Szyk, A. M. Deaconescu, G. Piszczek, A. Roll-Mecak,Nat. Struct. Mol. Biol. 2011, 18, 1250 – 1258.

[17] A. Banerjee, T. D. Panosian, K. Mukherjee, R. Ravindra, S. Gal,D. L. Sackett, S. Bane, ACS Chem. Biol. 2010, 5, 777 – 785.

..AngewandteCommunications

13790 www.angewandte.org Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2015, 54, 13787 –13791

[18] C. Hamers-Casterman, T. Atarhouch, S. Muyldermans, G.Robinson, C. Hamers, E. B. Songa, N. Bendahman, R. Hamers,Nature 1993, 363, 446 – 448.

[19] a) U. Rothbauer, K. Zolghadr, S. Tillib, D. Nowak, L. Schermel-leh, A. Gahl, N. Backmann, K. Conrath, S. Muyldermans, M. C.Cardoso, H. Leonhardt, Nat. Methods 2006, 3, 887 – 889; b) L.Trinkle-Mulcahy, S. Boulon, Y. W. Lam, R. Urcia, F. M. Boisvert,F. Vandermoere, N. A. Morrice, S. Swift, U. Rothbauer, H.Leonhardt, A. Lamond, J. Cell Biol. 2008, 183, 223 – 239.

[20] a) J. Guizetti, L. Schermelleh, J. Mantler, S. Maar, I. Poser, H.Leonhardt, T. Muller-Reichert, D. W. Gerlich, Science 2011, 331,1616 – 1620; b) J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, H.Ewers, Nat. Methods 2012, 9, 582 – 584.

[21] A. Kirchhofer, J. Helma, K. Schmidthals, C. Frauer, S. Cui, A.Karcher, M. Pellis, S. Muyldermans, C. S. Casas-Delucchi, M. C.Cardoso, H. Leonhardt, K. P. Hopfner, U. Rothbauer, Nat.Struct. Mol. Biol. 2010, 17, 133 – 138.

[22] N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc.2004, 126, 15046 – 15047.

[23] E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007 – 2010.[24] a) N. Nischan, A. Chakrabarti, R. A. Serwa, P. H. Bovee-Geurts,

R. Brock, C. P. Hackenberger, Angew. Chem. Int. Ed. 2013, 52,11920 – 11924; Angew. Chem. 2013, 125, 12138 – 12142; b) R.Serwa, P. Majkut, B. Horstmann, J. M. Swiecicki, M. Gerrits, E.Krause, C. P. R. Hackenberger, Chem. Sci. 2010, 1, 596 – 602;c) R. Serwa, I. Wilkening, G. Del Signore, M. Muhlberg, I.Claussnitzer, C. Weise, M. Gerrits, C. P. R. Hackenberger,Angew. Chem. Int. Ed. 2009, 48, 8234 – 8239; Angew. Chem.2009, 121, 8382 – 8387.

[25] S. Zink, L. Grosse, A. Freikamp, S. Banfer, F. Muksch, R. Jacob,J. Cell Sci. 2012, 125, 5998 – 6008.

Received: June 14, 2015Revised: August 17, 2015Published online: September 25, 2015

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Supporting Information

Versatile and Efficient Site-Specific Protein Functionalization byTubulin Tyrosine LigaseDominik Schumacher, Jonas Helma, Florian A. Mann, Garwin Pichler, Francesco Natale,Eberhard Krause, M. Cristina Cardoso, Christian P. R. Hackenberger,* and Heinrich Leonhardt*

anie_201505456_sm_miscellaneous_information.pdf

1.#SUPPLEMENTARY#RESULTS#......................................................................................................................#1!1.1.#Fluorescence8HPLC#analysis#of#the!C8terminal#addition#of#1#and#2#.....................................................#1!1.2.#Dependency#of#the#initial#velocity#towards#substrate#concentration#of#38N38L8tyrosine#.............#2!1.3.#ESI8MS/MS#spectra#...............................................................................................................................................#3!1.4.#GBP1–Tub8tag#and#GBP1#induced#modulation#of#GFP#spectral#properties#.......................................#4!1.5.#Fluorescence#spectra#of#GFP#and#GFP–Tub8tag#..........................................................................................#4!1.6.#Labeling#reagents#.................................................................................................................................................#5!1.7.#SPAAC#labeling#of#GBP48N3#with#sulfo8Cy58DBCO#......................................................................................#6!1.8.#Staudinger8Ligation#with#biotin8phosphine#................................................................................................#7!1.9.#Staudinger8Phosphite#reaction#with#tris(PEG750)phosphite#S1#.........................................................#8!1.10.#Fluorescent#labeling#of#GBP4#by#hydrazone#forming#reaction#...........................................................#9!1.11.#Biotin#labeling#of#GBP4#by#oxime#forming#reaction.#...........................................................................#10!1.12.#Site8specific#functionalization#of#GBP1#....................................................................................................#11!1.13.#Site8specific#functionalization#of#GFP#and#ubiquitin#...........................................................................#12!1.14.#Selective#labeling#of#GFP#within#E.$coli$Cell#lysate#................................................................................#13!1.15.#Confocal#microscopy#......................................................................................................................................#13!

2.#Material#and#Methods#...............................................................................................................................#14!2.1.#General#Information#.........................................................................................................................................#14!2.2.#Experimental#Procedures#...............................................................................................................................#14!2.2.1.!TTL!expression!and!purification!...............................................................................................................................!14!2.2.2.!Determination!of!TTL!activity!using!CF–Tub?tag!...............................................................................................!15!2.2.3.!Nanobody–Tub?tag!expression!and!purification!...............................................................................................!15!2.2.4.!GFP–Tub?tag!expression!and!purification!............................................................................................................!15!2.2.5.!Ubiquitin–Tub?tag!expression!and!purification!.................................................................................................!15!2.2.6.!TTL!reaction!on!GBP4!followed!by!tryptic!digest!and!MSMS!analysis!.....................................................!15!2.2.7.!Chemoenzymatic!addition!of!1!and!2!to!nanobodies,!GFP!and!ubiquitin!................................................!16!2.2.8.!SPAAC!to!sulfo?Cy5?DBCO!or!biotin?DBCO!...........................................................................................................!16!2.2.9.!Staudinger?Ligation!........................................................................................................................................................!16!2.2.10.!Staudinger?Phosphite!reaction!...............................................................................................................................!16!2.2.11.!Hydrazone!and!oxime!forming!reactions!...........................................................................................................!16!2.2.12.!Chemoenzymatic!addition!of!1!to!GFP!in!E.#coli!lysate.!................................................................................!16!2.2.13.!Streptavidin!pulldown!assay!....................................................................................................................................!17!2.2.14.!Immunofluorescence!for!superresolution!microscopy!................................................................................!17!2.2.15!Transfection!for!confocal!microscopy!...................................................................................................................!17!2.2.16.!Immunofluorescence!for!confocal!microscopy!................................................................................................!17!2.2.17.!Confocal!Microscopy!and!Image!Analysis!...........................................................................................................!18!

2.3.#Chemical#Synthesis#............................................................................................................................................#18!2.3.1.!Synthesis!of!3?nitro?L?tyrosine!(4),!3?amino?L?tyrosine!(3)!and!3?azido?L?tyrosine!(1)!................!18!2.3.2.!Synthesis!of!3?formyl?L?tyrosine!(2)!.......................................................................................................................!20!2.3.3.!Synthesis!of!tris(PEG750)phosphite!S1!.................................................................................................................!21!2.3.4.!Synthesis!of!hydroxylamine?biotin!S5!....................................................................................................................!21!

2.4.#Synthesis#of#CF–Tub8tag#peptide#..................................................................................................................#23!

3.#NMR#Spectra#of#1,#2,#3,#4#and#S2#.............................................................................................................#25!3.1#38N38L8tyrosine#(1)#.............................................................................................................................................#25!3.2#38formyl8L8tyrosine#(2)#.....................................................................................................................................#26!3.3#38NH28L8tyrosine#(3)#..........................................................................................................................................#27!3.4#38NO28L8tyrosine#(4)#..........................................................................................................................................#28!3.5#Hydroxylamine#biotin#S2#.................................................................................................................................#29!

4.#References#....................................................................................................................................................#30!

S1

1.#SUPPLEMENTARY#RESULTS#1.1.#Fluorescence8HPLC#analysis#of#the!C8terminal#addition#of#1#and#2#

Figure'S1.'C,terminal'addition'of'(a)'3,N3,L,tyrosine'(1)'and'(b)'3,formyl,L,tyrosine'(2)'to"carboxyfluorescein'la,beled'peptide'(CF–Tub,tag)'catalysed'by'TTL.'HPLC,fluorescence'traces'were'taken'at'different'time'points'of'the'TTL'reaction.''

S2

1.2.# Dependency# of# the# initial# velocity# towards# substrate# concentration# of# 38N38L8tyrosine#

Figure'S2.'TTL'displays'Michaelis,Menten'kinetics'for'to'ligation'of'N3,L,tyrosine'to'carboxyfluorescein'labeled'peptide.'The'v0'values'were'determined'using'fluorescence'HPLC'analysis.'Substrate'and'product'was'quantitat,ed'by'peak'integration.'The'mean'value'and'standard'deviation'(SD)'of'three'replicate'reactions'is'shown.'One'should'note'that'due'to'limited'solubility'of'the'tyrosine'derivative,'substrate'saturation'couldn’t'be'reached.'Using'this'data,'vmax'and'Km'have'been'determined'by'Michaelis,Menten'fit'(Prism)'to'be'0.232'µM'!'s,1'±'0.007'and'2222'µM,'respectively.'

S3

1.3.#ESI8MS/MS#spectra#

Figure'S3.'ESI,MS/MS'spectra'of'C,terminal'modified'nanobodies.'Different'tyrosine'derivatives'have'been'add,ed'to'the'C,terminus'of'the'nanobody'GBP4'using'Tub,tag'labeling.'Tryptic'digest'followed'by'HPLC,MS/MS'ex,periments' revealed' successful' incorporation' of' (a)' L,tyrosine,' (b)' 3,N3,L,tyrosine,' (c)' 3,formyl,L,tyrosine,' (d)#3,NO2,L,tyrosine'and'(e)'3,NH2,L,tyrosine.'The'N,terminal'b,ions'and'the'C,terminal'y,ions'confirm'the'sequence'of' the'C,terminally'modified' tryptic'peptides.' In'particular,' the'mass'of' the'modified'y4' ions'(b,e)' in'combination'with'the'unmodified'b18,'b22'ions'prove'the'presence'of'the'tyrosine'modification'at'the'very'C,terminus.'

S4

1.4.#GBP1–Tub8tag#and#GBP1#induced#modulation#of#GFP#spectral#properties##

Figure'S4.'GBP1'induced'modulation'of'GFP'spectral'properties.'(a)'GFP'was'incubated'with'equimolar'amounts'of'GBP1'and'GBP1–Tub,tag'as'previously'described.[1]'Similar'increase'in'GFP'fluorescence'indicates'compara,ble'GFP,binding'capacities'of'GBP1'and'GBP1–Tub,tag.'The'mean'value'and'standard'deviation'(SD)'of'three'replicate'reactions'is'shown.'(b)'GFP'emission'spectra'upon'titration'of'GBP1–Tub,tag'from'0.0–0.5'µM'on'0.5'µM'purified'GFP.'

#1.5.#Fluorescence#spectra#of#GFP#and#GFP–Tub8tag#

Figure'S5.'Fluorescence'spectra'of'GFP'and'GFP–Tub,tag.'Similar'intensities'show'conserved'protein'functional,ity'upon'C,terminal'addition'of'the'Tub,tag'sequence.'

S5

1.6.#Labeling#reagents#

Figure'S6.'Molecules'used'for'bioorthogonal'addition'to'C,terminal'modified'nanobodies'

N

OHN

O

ONH

OS

HNNH

O

4HH

NH

ONH

OS

HNNH

O

4HH

O

P

O

O

N O N

CH2SO3-CH2SO3- COHO

O

HN

H2N

PO

O40 3

NO

NHO

N+

NH

SO

OO-

S OO OH

NH

O

S

NH

HN

O

ONH

OONH22

S2

H

H

DBCO-PEG4-biotin

S1

sulfo-Cy5-DBCO Alexa594-hydrazide

Phosphine-biotin

S6

1.7.#SPAAC#labeling#of#GBP48N3#with#sulfo8Cy58DBCO#

Figure'S7.'SPAAC' labeling'of'GBP4,N3'with'sulfo,Cy5,DBCO.'Shown' is' the' fluorescent' labeling'of'GBP4'with'sulfo,Cy5,DBCO.'3,N3,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP4'using'TTL.'A'follow,ing' incubation'with'30'eq.'sulfo,Cy5,DBCO'shows'selective' labeling'of'3,N3,L,tyrosine'containing'nanobody'by'strain'promoted'azide,alkyne'click'reaction'(SPAAC).'

S7

1.8.#Staudinger8Ligation#with#biotin8phosphine#

Figure'S8.'Staudinger,Ligation'with'biotin,phosphine.'Shown'is'the'labeling'of'GBP4'with'biotin,phosphine'using'Staudinger,Ligation.' 3,N3,L,tyrosine' was' enzymatically' incorporated' to' the' C,terminus' of' GBP4' using' TTL.'A'following'incubation'with'40'eq.'Biotin,phosphine'shows'selective'labeling'of'3,N3,L,tyrosine'containing'nanobody'by'Staudinger,Ligation.'

S8

1.9.#Staudinger8Phosphite#reaction#with#tris(PEG750)phosphite#S1#

Figure'S9.'Staudinger,Phosphite'reaction'with'tris(PEG750)phosphite'S1.'Shown'is'the'PEGylation'of'GBP4'with'tris(PEG750)phosphite'(S1)'by'Staudinger,Phosphite'reaction.'3,N3,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP4'using'TTL.'A'following'incubation'with'40'eq.'phosphite'shows'selective'labeling'of'3,N3,L,tyrosine'containing'nanobody'by'Staudinger'Ligation.'

S9

1.10.#Fluorescent#labeling#of#GBP4#by#hydrazone#forming#reaction##

#Figure'S10.'Fluorescent' labeling'of'GBP4'by'hydrazone' forming' reaction.'Shown' is' the' labeling'of'GBP4'with'Alexa594,hydrazide'using'hydrazone'forming'reaction.'3,formyl,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP4'using'TTL.'A'following'incubation'with'30'eq.'Alexa594,hydrazide'shows'selective'labeling'of'3,formyl,L,tyrosine'containing'nanobody'by'aldehyde'condensation.'

S10

1.11.#Biotin#labeling#of#GBP4#by#oxime#forming#reaction.#

Figure'S11.'Biotin'labeling'of'GBP4'by'oxime'forming'reaction.'Shown'is'the'labeling'of'GBP4'with'biotin'S2'us,ing'oxime'forming'reaction.'3,formyl,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP4'using'TTL.'A'following'incubation'with'30'eq.'S2'shows'selective'labeling'of'3,formyl,L,tyrosine'containing'nanobody'by'aldehyde'condensation.'

S11

1.12.#Site8specific#functionalization#of#GBP1#

Figure'S12.'Site,specific'functionalization'of'GBP1.'Shown'is'the'site,specific'labeling'of'the'GFP'specific'nano,body'GBP1'using'Tub,tag'labeling.'(a)'3,N3,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP1'using'TTL.'A'following'incubation'with'30'eq.'sulfo,Cy5,DBCO'or'30'eq.'biotin,DBCO'shows'selective'labeling'of'3,N3,L,tyrosine' containing' nanobody' by' strain' promoted' azide,alkyne' click' reaction' (SPAAC).' (b)' 3,formyl,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'GBP4'using'TTL.'A'following'incubation'with'30'eq.'Alexa594,hydrazide'shows'selective'labeling'of'3,formyl,L,tyrosine'containing'nanobody'by'aldehyde'condensa,tion.'

S12

1.13.#Site8specific#functionalization#of#GFP#and#ubiquitin#

Figure'S13.'Site,specific' functionalization'of'GFP'and'Ubiquitin.' (a)'3,N3,L,tyrosine'was'enzymatically' incorpo,rated'to'the'C,terminus'of'GFP'using'TTL.'A'following'incubation'with'30'eq.'biotin,DBCO'shows'selective'label,ing'of'3,N3,L,tyrosine'containing'GFP'by'strain'promoted'azide,alkyne'click'reaction'(SPAAC).'(b)'3,N3,L,tyrosine'was'enzymatically'incorporated'to'the'C,terminus'of'Ubiquitin'(Ub)'using'TTL.'A'following'incubation'with'30'eq.'biotin,DBCO'shows'selective'labeling'of'3,N3,L,tyrosine'containing'Ub'by'strain'promoted'azide,alkyne'click're,action'(SPAAC).'

S13

1.14.#Selective#labeling#of#GFP#within#E.$coli$Cell#lysate#

#Figure'S14.'Tub,tag'labeling'of'GFP'in'complex'protein'mixtures'(E.coli'lysate).'Coomassie'staining'and'western'blot'analysis'show'high'selectivity'of' the' tyrosine' ligation'and'subsequent'biotinylation.'3,N3,L,tyrosine'was'en,zymatically' incorporated' to' the' C,terminus' of' GFP' using' TTL.' A' following' incubation' with' 3' eq.' biotin,DBCO'shows' selective' labeling' of' 3,N3,L,tyrosine' containing' GFP' by' strain' promoted' azide,alkyne' click' reaction'(SPAAC).#

1.15.#Confocal#microscopy#

Figure'S15.'Confocal'micrographs'showing'co,localization'of'GBP1–Tub,tag–Alexa594'and'GFP,PCNA'fusions.'Confocal' micrographs' of' HeLa' cells' transfected' with' GFP,PCNA' fusions.' Cells' were' labeled' with' anti,GFP'(GBP1)'conjugated'to'the'fluorescent'dye'Alexa594'at'1:25.'The'DAPI,'GFP'and'Alexa594'channels,'as'well'as'the'overlay'are'shown.'Scale'bar:'5'µm.'

S14

2.#Material#and#Methods#2.1.#General#Information#

Analytical#HPLC#was'conducted'on'a'SHIMADZU'HPLC'system'(Shimadzu'Corp.,'Japan)'with'a'SIL,20A'autosampler,'2'pumps'LC2'AAT,'a'2489'UV/Visible'detector,'a'CTO,20A'column'oven'and'an'RF,10'A'X2'fluorescence'detector'using'an'Agilent'Eclipse'C18'5'µm,'250'x'4.6'mm'RP,HPLC,column'with'a'flow'rate'of'0.5'mL/min.'The'following'gradient'was'used:'Method'A:'(A'='H2O'+'0.1%'TFA,'B'='MeCN'+'0.1%'TFA)'35%'B,'0,15'min,'10,100%'B'15,17'min,'100%'B'17,22'min,'100,35%'B'22,25'min'and'35%'B'25,30'min.'UV'chromatograms'were'recorded'at'220'nm'and'fluorescence'spectra'with'Ex/Em'495/517'were'recorded.''Analytical#UPLC:'UPLC,UV'traces'were'obtained'on'a'Waters'H,class'instrument'equipped'with'a'Quater,nary'Solvent'Manager,'a'Waters'autosampler'and'a'Waters'TUV'detector'connected'to'a'3100'mass'detec,tor'with'an'Acquity'UPLC,BEH'C18'1.7'µm,'2.1'x'50'mm'RP'column'with'a'flow'rate'of'0.6'mL/min'(Water'Corp.,'USA).'The'following'gradient'was'used:'Method'B:'(A'='H2O'+'0.1%'TFA,'B'='MeCN'+'0.1%'TFA)'5,95%'B'0,3'min,'95%'B'3,5'min.'UPLC,UV'chromatograms'were'recorded'at'220'nm.'Preparative#HPLC#was'performed'on'a'Gilson'PLC'2020'system'(Gilson'Inc.,'WI,'Middleton,'USA)'using'a'Macherey,Nagel' Nucleodur' C18' HTec' Spum' column' (Macherey,Nagel' GmbH' &' Co.' Kg,' Germany).' The'following'gradient'was'used:'Method'C:'(A'='H2O'+'0.1%'TFA,'B'='MeCN'+'0.1%'TFA)'flow'rate'32'mL/min,'10%'B'0,5'min,'10,100%'B'5,35'min,'100'%'B'35,40'min.'Method'D:'(A'='H2O'+'0.1%'TFA,'B'='MeCN'+'0.1%'TFA)'10%'B'0,5'min,'10,100%'B'5,50'min,'100%'B'50,55'min.'Analytical#HPLC8MSMS:#Peptides'were'analyzed'by'a'Ultimate'3000'nanoLC'system'(Thermo'Scientific,'USA)'connected'to'an'LTQ'Orbitrap'XL'mass'spectrometer'(Thermo'Scientific,'USA).'LC'separations'were'performed'on'a'capillary'column'(Acclaim'PepMap100,'C18,'3'µm,'100'Å,'75'µm'i.d.'x'25'cm,'Thermo'Sci,entific,'USA)'at'an'eluent'flow'rate'of'300'nL/min.'The'following'gradient'was'used:'Method'D:'(A'='H2O'+'0.1%'formic'acid,'B'='MeCN'+'0.1%'formic'acid)'3,50%'B'0,50'min'Mass'spectra'were'acquired'in'a'data,dependent'mode'with'one'MS'survey'scan'with'a'resolution'of'30,000'(LTQ'Orbitrap'XL)'or'60,000'(Orbitrap'Elite)'and'MS/MS'scans'of'the'five'most'intense'precursor'ions'in'the'linear'trap'quadrupole,'respectively.''Column#chromatography#was'performed'on'silica'gel'(Acros'Silica'gel'60'Å,'0.035,0.070'mm).''High8resolution#mass#spectra'(HRMS)'were'measured'on'an'Acquity'UPLC'system'and'a'LCT'PremierTM'(Waters'Corp.,'USA)'time,of,flight'mass'spectrometer'with'electrospray'ionization'using'water'and'acetoni,trile'(10,90%'gradient)'with'0.1%'formic'acid'as'eluent.'NMR'spectra'were'recorded'with'a'Bruker'Ultrashield'300'MHz'spectrometer'(Bruker'Corp.,'USA)'at'ambi,ent' temperature.' The' chemical' shifts' are' reported' in' ppm' relative' to' the' residual' solvent' peak.' Product'yields'were'calculated'based'on'1H,NMR'spectra.'TFA'salt'content'was'determined'by'19F,NMR,'tetrafluo,roethylene'as'standard'and'considered'in'product'yield'calculation.''GFP#fluorescence#spectra'were'recorded'with'a'Jasco'FP,6500'spectrometer'(Jasco'Research'Ltd.,'Can,ada)'using'an'excitation'wavelength'of'488'nm.''Reagents#and#solvents#were,'unless'stated'otherwise,'commercially'available'as' reagent'grade'and'did'not'require'further'purification.'Resins'and'Fmoc,protected'amino'acids'were'purchased'from'IRIS'BioTEch'(Germany)'or'Novabiochem'(Germany).'SPPS'was'either'carried'out'manually'or'with'an'Activo,P11'automated'peptide'synthesizer'(Activotec,'UK)'via'standard'Fmoc,based'conditions'(Fast,moc'protocol'with'HOBt/HBTU'conditions).'

2.2.#Experimental#Procedures#2.2.1.#TTL#expression#and#purification#The'TTL'(Canis"lupus)'coding'sequence'was'amplified'from'a'mammalian'expression'vector[2],'cloned'into'

a'pET28,SUMO3' (EMBL,Heidelberg,'Protein'Expression'Facility)' and'expressed' in'E." coli'BL21(DE3)'as'Sumo,TTL'fusion'protein'with'an'N,terminal'His,Tag.'Cells'were'induced'with'0.5'mM'IPTG'and'incubated'at'18'°C'for'18'h.'Lysis'was'performed'in'presence'of'Lysozyme'(100'µg/mL),'DNAse'(25'µg/mL)'and'PMSF'(2'mM)'followed'by'sonification'(Branson®'Sonifiers'16'x'8sec,'20%'amplitude)'and'debris'centrifugation'at'20.000'g'for'30'min.'His,Sumo,TTL'was'purified'using'a'5'mL'His,Trap.'Purified'protein'was'then'desalted'on'a'PD10'column'(GE'Healthcare)s'buffer'was'exchanged'to'MES/K'pH'6.8'(20'mM'MES,'100'mM'KCl,'10'mM'MgCl2).'Protein'aliquots'were'shock,frozen'and'stored'at',80'°C'at'2.7'g/l.'

S15

2.2.2.#Determination#of#TTL#activity#using#CF–Tub8tag#Tyrosination'reactions'were'performed'in'a'250'µL'solution'consisting'of'20'mM'MES/K'pH'7.0,'100'mM'

KCl,'10'mM'MgCl2,'2.5'mM'ATP,'1'mM'tyrosine'derivative,'0.2'mM'CF–Tub,tag,'1'µM'TTL'and'5'mM'DTT'in'case'of'compound'2#or'5'mM'reduced'glutathione' in'case'of'compound'1,' respectively.'The'mixture'was'incubated'at'37'°C'and'several'aliquots'(25'µL)'were'taken,'mixed'with'equal'volumes'of'H2O'+'0.1%'TFA'and'subjected'to'isocratic'analytical'HPLC'equipped'with'a'fluorescence'detector'(Method:'A'='H2O'+'0.1%'TFA,'B'='MeCN'+'0.1%'TFAs'35%'B,'0,15'min,'10,100%'B'15,17'min,'100%'B'17,22'min,'100,35%'B'22,25'min'and'35%'B'25,30'min.).'For'v0'determination'of'TTL,'reactions'were'performed'using'different'concen,trations'of'1#(0.5'–'8'µM)'and'aliquots'taken'after'3,'6,'9'and'12'minutes.'Quantities'of'substrate'and'prod,uct' peptides' were' estimated' from' the' corresponding' peak,area' in' the' fluorescence' detection' spectrum'(Ex/Em:'495/517).'' 2.2.3.#Nanobody–Tub8tag#expression#and#purification#Nanobody–Tub,tag' fusion'expression'constructs'(pHen6'bacterial'expression'vector)'were'generated'by'

standard' molecular' biology' techniques' resulting' in' nanobodies' with' an' N,terminal' 6xHis' tag' and' a' C,terminal'alpha,Tubulin'derived'VDSVEGEGEEEGEE'peptide'(Tub,tag).'Proteins'were'expressed'in'E."coli'(JM109).'Cells'were' induced'with'0.5'mM'IPTG'and' incubated'at'18'°C' for'18'h.'Lysis'was'performed' in'presence'of'Lysozyme'(100'µg/mL),'DNAse'(25'µg/mL)' 'and'PMSF'(2'mM)' followed'by'sonication'(Bran,son®'Sonifiers'16'x'8sec,'20%'Amplitude)'and'debris'centrifugation'at'20.000'g'for'30'min.'The'protein'was'purified'with' an'Äkta'FPLC'system'using'a' 5'mL'His,Trap' (GE'Healthcare,'USA)' column,' peak' fractions'were'concentrated'to'2'mL'using'Amicon'filter'columns'(cut,off'3'kDas'(Merck'Millipore,'Germany)'and'sub,jected'to'size'exclusion'chromatography'using'a'Superdex'75'column'(GE'Healthcare,'USA).'Peak'fractions'were'pooled'and'protein'aliquots'were'shock,frozen'and'stored'at',80'°C.''

2.2.4.#GFP–Tub8tag#expression#and#purification#GFP–Tub,tag' fusion' expression' constructs' (pET28(c)' bacterial' expression' vector)' were' generated' by'

standard' molecular' biology' techniques' resulting' in' nanobodies' with' an' N,terminal' 6xHis' tag' and' a' C,terminal'alpha,Tubulin'derived'VDSVEGEGEEEGEE'peptide'(Tub,tag).'Proteins'were'expressed'in'E."coli'BL21(DE3).'Cells'were' induced'with'0.5'mM'IPTG'and' incubated'at'18'°C' for'18'h.'Lysis'was'performed'using'a'high'pressure'homogenizer'(Microfluidics'LM10'Microfluidizer)'and'debris'centrifugation'at'20.000'g'for'30'min.'The'protein'was'purified'with'an'Äkta'FPLC'system'using'a'5'mL'His,Trap'(GE'Healthcare,'USA)'column,'peak'fractions'were'concentrated'to'2'mL'using'Amicon'filter'columns'(cut,off'10'kDas'(Merck'Milli,pore,' Germany)' and' subjected' to' size' exclusion' chromatography' using' a' Superdex' 75' column' (GE'Healthcare,'USA).'Peak'fractions'were'pooled'and'protein'aliquots'were'shock,frozen'and'stored'at',80'°C.''

2.2.5.#Ubiquitin–Tub8tag#expression#and#purification#Ubiquitin–Tub,tag'fusion'expression'constructs'(pET28(c)'bacterial'expression'vector)'were'generated'by'

standard' molecular' biology' techniques' resulting' in' nanobodies' with' an' N,terminal' 6xHis' tag' and' a' C,terminal'alpha,Tubulin'derived'VDSVEGEGEEEGEE'peptide'(Tub,tag).'Proteins'were'expressed'in'E."coli'(JM109).'Cells'were'induced'with'0.5'mM'IPTG'and'incubated'at'18'°C'for'18'h.'Lysis'was'performed'using'a'high'pressure'homogenizer'(Micrufluidics'LM10'Microfluidizer)'and'debris'centrifugation'at'20.000'g'for'30'min.'The'protein'was'purified'with'an'Äkta'FPLC'system'using'a'5'mL'His,Trap'(GE'Healthcare,'USA)'col,umn,'peak'fractions'were'concentrated'to'2'mL'using'Amicon'filter'columns'(cut,off'3'kDas'(Merck'Millipore,'Germany)'and'subjected' to'size'exclusion'chromatography'using'a'Superdex'75'column'(GE'Healthcare,'USA).'Peak'fractions'were'pooled'and'protein'aliquots'were'shock,frozen'and'stored'at',80'°C.'

2.2.6.#TTL#reaction#on#GBP4#followed#by#tryptic#digest#and#MSMS#analysis##Tyrosination'reactions'were'performed' in'a'50'µL'solution'consisting'of'20'mM'MES/K'pH'7.0,'100'mM'

KCl,'10'mM'MgCl2,'2.5'mM'ATP,'1'mM'tyrosine'derivative,'1'µM'TTL,'5'µM'nanobody'and'5'mM'reduced'glutathione'in'case'of'compound'1'and'5'mM'DTT'in'case'of'compound'2,4'and'tyrosine,'respectively.'The'mixture'was'incubated'at'37'°C'for'24'h.'Proteins'were'separated'by'SDS,PAGE.'Protein'bands'of'interest'were'excised,'soaked'with'100'µL'50'mM'(NH4)2CO3/ACN'1:1'and'incubated'at'30'°C'for'10'min.'The'su,pernatant'was'removed'and'the'gel'pieces'were'incubated'in'50'mM'(NH4)2CO3'at'30'°C'for'further'10'min.'

S16

The'two'incubation'steps'were'repeated'until'the'pieces'were'colorless.'Hereafter,'the'gel'pieces'were'de,hydrated'by' the'addition'of'25'µL'ACN,' the'supernatant' removed'and' the'gels'were'dried'under' reduced'pressure.'In,gel'digest'was'performed'in'a'total'volume'of'20'µL'50'mM'(NH4)2CO3'at'37'°C'for'12'h'using'0.05'µg'trypsin'(Thermo'Fisher'Scientific,'USA).'20'µL'ACN'+'0.5%'TFA'was'added,'the'mixture'incubated'in' an' ultrasonic' bath,' the' supernatant' transferred' to' LC' glass' vials,' the' solvent' removed' under' reduced'pressure'and'the'residual'peptides'resuspended'in'6'µL'95%'H2O'+'0.1%'TFA,'5%'ACN'+'0.1%'TFA'solu,tion.'Peptides'were'separated'by'HPLC'and'analyzed'by'MSMS'experiments.'' 2.2.7.#Chemoenzymatic#addition#of#1#and#2#to#nanobodies,#GFP#and#ubiquitin#Tyrosination'reactions'were'performed'in'a'150'µL'solution'consisting'of'20'mM'MES/K'pH'7.0,'100'mM'

KCl,'10'mM'MgCl2,'2.5'mM'ATP,'1'mM'1'or'2,'0.1' ,'1'µM'TTL,'5'µM'nanobody/GFP/ubiquitin'and'5'mM'reduced'glutathione'in'case'of'compound'1'or'5'mM'DTT'in'case'of'compound'2,'respectively.'The'mixture'was'incubated'at'37'°C'for'1,3'h.'' 2.2.8.#SPAAC#to#sulfo8Cy58DBCO#or#biotin8DBCO#Tyrosination'reactions'were'performed'as'described'above'using'compound'1,'5'mM'reduced'glutathione'

and'a'ratio'of'10:1'GBP4/TTL'for'3'h.'The'reaction'mixtures'were'rebuffered'to'Dulbecco’s'PBS'pH'7.4#and#incubated'with'30'eq.'of'Sulfo,Cy5,DBCO'(Jena'Bioscience'GmbH,'Germany)'or'DBCO,PEG4,biotin'(Jena'Bioscience'GmbH,'Germany)#at'30'°C'for'4'h.'Proteins'were'separated'by'SDS,PAGE.'Biotinylated'nano,bodies'were'wet'blotted'onto'a'nitrocellulose'membrane'using'a'Bio,Rad'Mini,Protean'Tetra'System'(250'mA,'one'hour).'A'streptavidin'peroxidase'conjugate'(Merck'Millipore,'Germany)'was'used'for'detection.'Flu,orescently' labeled'nanobodies'were'visualized'by'a'Fuji'FLA,5000' laser' imager' (634'nm'excitation,'LPG,filter)'(Fujifilm,'Japan).'' 2.2.9.#Staudinger8Ligation#Tyrosination'reactions'were'performed'as'described'above'using'compound'1,'5'mM'reduced'glutathione'

and'a'ratio'of'10:1'GBP4/TTL'for'3'h.'The'reaction'mixtures'were'rebuffered'to'Dulbecco’s'PBS'pH'7.4#and#incubated'with'40'eq.'of'biotin,phosphine'(S3,'BIOMOL'GmbH,'Germany)'at'37'°C'for'24'h.'Proteins'were'separated'by'SDS,PAGE.'Biotinylated'nanobodies'were'wet'blotted'onto'a'nitrocellulose'membrane'using'a'Bio,Rad'Mini,Protean'Tetra' System' (250'mA,' 1' h).'A' streptavidin' peroxidase' conjugate' (Merck'Millipore,'Germany)'was'used'for'detection.' 2.2.10.#Staudinger8Phosphite#reaction#Tyrosination'reactions'were'performed'as'described'above'using'compound'1,'5'mM'reduced'glutathione'

and'a'ratio'of'10:1'GBP4/TTL'for'3'h.'The'reaction'mixtures'were'rebuffered'to'50'mM'Tris'pH'8.5,'100'mM'KCl#and#incubated'with'40'eq.'of'tris(PEG750)phosphite'(S2)'at'37'°C'for'24'h.'Proteins'were'separated'by'SDS,PAGE.'PEGylated'nanobodies'were'wet'blotted'onto'a'nitrocellulose'membrane'using'a'Bio,Rad'Mini,Protean'Tetra'System'(250'mA,'1h).'A'monoclonal'anti'PEG,B,47'antibody'(Abcam,'UK)'and'a'secondary'Goat'Anti,Rabbit'IgG'H&L'(HRP)'(Abcam,'UK)'were'used'for'detection.''

2.2.11.#Hydrazone#and#oxime#forming#reactions#Tyrosination' reactions'were'performed'as'described'above'with'compound'2,'5'mM'DTT'and'a' ratio'of'

10:1'GBP4/TTL'for'3'h.'The'reaction'mixtures'were'rebuffered'to'50'mM'MES/K'pH'6.0,'100'mM'KCl#and#incubated'with' 30' eq' of'Alexa594,hydrazide' (S5,' Thermo' Fisher' Scientific,' USA)' or' hydroxylamine,biotin'(S6)#at' 37' °C' for' 4' h.'Proteins'were' separated'by'SDS,PAGE.'Biotinylated'nanobodies'were'wet' blotted'onto'a'nitrocellulose'membrane'using'a'Bio,Rad'Mini,Protean'Tetra'System'(250'mA,'1'h).'A'streptavidin'peroxidase'conjugate'(Merck'Millipore,'Germany)'was'used'for'detection.'Fluorescently'labeled'nanobodies'were'visualized'by'a'Fuji'FLA,5000'laser'imager'(532'nm'excitation,'LPG,filter)'(Fujifilm,'Japan).'''

2.2.12.#Chemoenzymatic#addition#of#1#to#GFP#in#E.$coli#lysate.##Tyrosination'reactions'were'performed'in'250'µL'lysate'containing'overexpressed'GFP–Tub,tag,'2.5'mM'

ATP,'3'mM'1,'0.2'µM'TTL,'and'5'mM'reduced'glutathione.'The'mixture'was'incubated'at'30'°C'for'4'hours.'Excess'tyrosine'was'removed'using'dialysis'and'SPAAC'to'3'eq.'DBCO'was'performed'at'30°C'for'3'hours.'

S17

Proteins'were'separated'by'SDS,PAGE.'Lysate'samples'were'wet'blotted'onto'a'nitrocellulose'membrane'using'a'Bio,Rad'Mini,Protean'Tetra'System'(250'mA,'one'hour).'A'streptavidin'peroxidase'conjugate'(Merck'Millipore,'Germany)'was'used'for'detection.'''''

2.2.13.#Streptavidin#pulldown#assay#HEK'293T'cells'were'maintained'in'DMEM'supplemented'with'10%'fetal'calf'serum'and'gentamycin'at'50'

µg/mL'(PAA,'Germany).'For'pulldown'experiments'107'cells'were'seeded'in'p100'dishes.'Transfection'with'eGFP' encoding' plasmid' DNA' peGFP,C1' (Life' Technologies,' Germany)' was' performed' with' polyethyl,enimine'(PEI,'Sigma,'USA)'with'24'µg'DNA'per'dish.'Pulldown'reagent'was'prepared'by'using'biotinylated'GBP1' (Tub,tag'mediated).'For' this'purpose,'200'µL'slurry'of'Streptavidin,coated,'magnetic'beads' (Dyna,beads'MyOne'Streptavidin'T1)'were'washed'with'PBS'and' then' loaded'with'40'µg'biotinylated'GBP1'ac,cording' to' the'manufacturers’' instructions.'Functionalized'beads'were'equilibrated'with' IP'buffer' (0,5'mM'EDTA,'50'mM'Tris/Cl'pH'7.0,'150'mM'NaCl).'Whole'cell'lysates'were'prepared'using'200'µL'lysis'buffer'(0.5'mM'EDTA,'50'mM'Tris/Cl'pH'7.0,'150'mM'NaCl,'1%'NP40,'2'mM'PMSF,'1x'Mammalian'Protease'Inhibitor'Cocktail.'5%'of'lysate'supernatants'were'collected'as'input'samples.'The'remaining'sample'was'diluted'to'1'mL'using'IP'buffer'and'incubated'with'50'µL'bead'slurry'for'4'h.'After'magnetic'pulldown,'5%'supernatant'were' collected' as' flowthrough' samples.' For' Coomassie' staining' and' western' blotting' 2%' input,' 2%'flowthrough'and'20%'bead'fractions'were'boiled'with'Laemmli'buffer'at'95'°C'and'subjected'to'SDS,PAGE'and'transfer' to'a'nitrocellulose'membrane'(Bio,Rad,'USA).'A'monoclonal'anti,GFP'antibody'(Roche,'Swit,zerland)'and'HRP,conjugated'anti,mouse' IgG'secondary'antibody' (Jackson' ImmunoResearch,'USA)'was'used'for'detection.'

2.2.14.#Immunofluorescence#for#superresolution#microscopy#HeLa,Kyoto'cells'were'maintained'in'DMEM'supplemented'with'10%'fetal'calf'serum'and'gentamycin'at'

50'µg/mL'(PAA,'Germany).'For'immunofluorescence'experiments'106'cells'were'seeded'on'gridded'18×18'mm'coverslips'in'a'6,well'plate.'Transfection'with'LaminB1,GFP'encoding'plasmid'DNA'(kind'gift'from'Jan'Ellenberg)'was'performed'with'Lipofectamine'3000'(Life'Technologies,'USA)'according'to' the'manufactur,ers’' instructions.' 18' h' post' transfection,' cells'were'washed'with'PBST,' fixed'with' 2%' formaldehyde/PBS,'and'permeabilized'with'0.5%'Triton'X,100/PBS.'Next,' cells'were'blocked'with'2%'BSA/PBST.'Cells'were'then'incubated'with'Alexa594'Tub,tag'labeled'GBP1'(stock'solution'1'µg/µLs'dilution'1:50)'for'1'h,'washed'with'PBST,'DAPI'stained'(Life'Technologies,'USA)'and'mounted'in'Vectashield'anti,fading'reagent'(Vector'Laboratories,'USA)'on'object'slides.'High,resolution'microscopy'was'performed'with'an'DeltaVision'OMX'v3'(Applied' Precision,' Slovakia)' equipped' with' 405,' 488' and' 593' nm' laser' diodes,' a' 100×/1.4' NA' Plan,Apochromat'oil'objective'lens'(Olympus,'Japan)'and'Cascade'II:512'EM'CCD'cameras'(Photometrics,'USA)'as'described'previously[3].'Line'scan'fluorescence'intensity'analysis'was'performed'with'ImageJ.''

2.2.15#Transfection#for#confocal#microscopy##HeLa'cells'were'seeded'at'sub,confluent'concentration'on'glass'coverslips'(Carl'Roth,'Germany)'the'day'

before' PEI' transfection' (Sigma,Aldrich,' USA)'with' 2' µg' pENeGFP,PCNA[4].'After' transfection,' cells' were'incubated'for'18,24'h'at'37'°C,'5%'CO2.'

2.2.16.#Immunofluorescence#for#confocal#microscopy#Cells'were'fixed'in'3.7%'formaldehyde'in'PBS'for'10'min,'permeabilized'with'0.5%'Triton'X,100'(neoLab'

Migge' Laborbedarf,Vertriebs,' Germany)' for' 10' min,' and' blocked' in' 2%' bovine' serum' albumin' (Sigma,Aldrich,'UK)' for'60'min.'For'GFP' labeling,'cells'were' incubated' for'60'min'with'GBP1–Tub,tag–Alexa594'(1:25'or'1:50)'prior'to'extensive'washing'and'DNA'counterstain'with'1'µg/mL'DAPI'for'10'min.'All'steps'ex,cept' fixation'were' carried' out' in' PBS' supplemented'with' 0.02%'Tween' 20' (PBST,'Carl' Roth,' 9127.1)' at'room' temperature.'Glass' coverslips'were' then'mounted'with' anti,fade'Mowiol'mounting'medium' (Sigma,Aldrich,'UK).'

S18

2.2.17.#Confocal#Microscopy#and#Image#Analysis#Imaging' was' carried' out' with' a' Leica' SP5' II' confocal' point' scanner' (Leica' Microsystems,' Germany)'

equipped'with'two'HyD'hybrid'detectors.'Image'acquisition'was'performed'with'a'×60/1.4,0.6'NA'Planapo,chromat'oil' immersion'objective' lens.'To'visualize'DAPI,'GFP'and'GBP1–Tub,tag–Alexa594,'the'405,'488'and'561'nm'excitation'lasers'were'used,'respectively.'16bit'images'were'collected'and'analyzed'with'Fiji[5].'

2.3.#Chemical#Synthesis#2.3.1.#Synthesis#of#38nitro8L8tyrosine#(4),#38amino8L8tyrosine#(3)#and#38azido8L8tyrosine#(1)#'

Scheme#1.#Synthesis#of#tyrosine#derivatives#1,#3#and#4.#

38nitro8L8tyrosine#(4)#L,tyrosine'(2.00'g,'11'mmol)'was'added'to'10'mL'HOAc,'the'suspension'cooled'to'0'°C'and'HNO3'(1.47'

mL,'11'mmol,'7.5'N)'was'slowly'added.'After'4'h' the' reaction'was'diluted'with'H2O' (2.5'mL)' followed'by'neutralization'with'NH3'solution'(25%).'The'resultant'solution'was'filtrated,' the' filtrate' lyophilized'and'sub,jected' to' HPLC' purification' (method' C)' to' give' compound' 4' as' TFA' salt' (1.38' g,' 44%).'Analytical' data'matched'the'literature.[6]'

1H,NMR'(300'MHz,'D2O):'δ'7.89'(d,'J'='2.3'Hz,'1H,'CHphenyl),'7.43'(dd,'J'='8.7,'2.3'Hz,'1H,'CHphenyl),'7.03'

(d,'J'='8.7'Hz,'1H,'CHphenyl),'4.18'(t,'J'='6.6'Hz,'1H,'CH),'3.31,3.04'(m,'2H,'CH2)s'13C,NMR'(75'MHz,'D2O):'δ' 171.11,' 152.74,' 138.21,' 133.85,' 126.40,' 125.76,' 120.19,' 53.68,' 34.23s' ESI,HRMS' (m/z):[M]+' calcd.'for''C9H12N2O5,'227.0660s'found''227.0674.'

38amino8L8tyrosine#(3)#Compound'4' (1.38'g,'4.86'mmol)'was'dissolved' in'H2O'(100'mL)'and'conc.'HCl' (500'µL).'The'solution'

was'supplemented'with'Pd/BaSO4'(40'mg,'5%'catalyst'loading)'and'the'mixture'incubated'at'ambient'tem,perature'for'12'h'under'H2'atmosphere.'After'filtration'of'the'catalyst'and'removal'of'the'solvent'in'vacuo,"the'product'3'was'obtained'in'quantitative'yield'as'TFA'salt.'Analytical'data'matched'the'literature.[6]'

1H,NMR'(300'MHz,'D2O):'δ'7.42,7.15'(m,'2H,'CHphenyl),'7.05,6.89'(m,'1H,'CHphenyl),'4.11'(t,'J"="6.5'Hz,'

1H,'CH),' 3.24,3.06' (m,'2H,'CH2)s' 13C,NMR' (75'MHz,'D2O):' δ'='171.91,'149.37,'131.23,'126.32,'124.68,'117.84,'116.79,'54.47,'34.61s'ESI,HRMS'(m/z):[M]+'calcd.'for''C9H13N2O3,'197.0918s'found''197.0910.''

OH

OH

O

NH2

HNO3, HOAc

OH

OH

O

NH2

NO2

OH

OH

O

NH2

NH2

OH

OH

O

NH2

N3Pd/BaSO4H2, H2O

1. NaNO22. NaN3

0.5 M HCl41%

134

44% quant.

OH

OH

O

NH2

NO2

4

OH

OH

O

NH2

NH2

3

S19

38azido8L8tyrosine#(1)#3,amino,L,tyrosine'(3,'0.696'g,'3.21'mmol)'was'dissolved'in'0.5'M'HCl'(6'mL)'and'a'solution'of'NaNO2'

(0.221'g,'3.21'mmol)' in' ice,cold'H2O' (1'mL)'was'slowly'added'at'0' °C.'After'20'min,'a' solution'of'NaN3'(0.560'g,'8.62'mmol)'in'H2O'(3'mL)'was'added'within'30'min'and'stirred'at'0'°C'for'another'8'h.'The'grey'precipitate'was' isolated'and'purified'by'preparative'HPLC'(method'C)' to'give'pure'compound'1' (0.290'g,'41%).'

1H,NMR'(300'MHz,'D2O):'δ'6.95'(d,'J'='2.0'Hz,'1H,'CHphenyl),'6.89,6.79'(m,'2H,'CHphenyl),'4.06'(t,'J'='6.5'

Hz,'1H,'CH),'3.19,2.95'(m,'2H,'CH2)s'13C,NMR'(75'MHz,'D2O):'δ'172.04,'146.44,'127.24,'127.07,'126.58,'120.38,'116.86,'54.51,'34.83s'ESI,HRMS'(m/z):[M]+'calcd.'for''C9H11N4O3,'223.0823s'found''223.0830s'FT,IR:'νmax'(neat,'cm,1):'3074'(br),'2116,'1731,'1665,'1597,'1515,'1435,'1299,'1185,'1135,'1095,'813,'721,'655.''

OH

OH

O

NH2

N3

1

S20

2.3.2.#Synthesis#of#38formyl8L8tyrosine#(2)#The'synthesis'of'1'was'performed'according'to'a'known'procedure'in'literature.[7]'

Scheme#2.#Synthesis#of#38formyl8L8tyrosine#(2).#

N*[(1,18dimethylethoxy)carbonyl]8L8tyrosine#(S3)#To' a' solution' of' L,tyrosine' (1' g,' 5.5'mmol)' in' 1/1' dioxane/water' (50'mL),' triethylamine' (1.16'mL,' 8.28'

mmol)' was' slowly' added.' The' reaction' was' cooled' to' 0°C'with' an' ice/water' bath' and' di,tert,butyl' dicar,bonate'(1.32'g,'6.07'mmol)'was'added'in'two'steps.'After'1'h'at'0°C,'the'temperature'was'slowly'increased'to'ambient'temperature'and'the'mixture'was'stirred'for'further'24'h.'Dioxane'was'removed'under'reduced'pressure'and'the'aqueous'solution'mixed'with'saturated'NaHCO3'(25'mL),'washed'with'ethyl'acetate,'acidi,fied' to'pH'1'with'1'N'HCl,'extracted'with'ethyl'acetate'and' the'organic'extracts'were'washed'with'brine,'dried'over'MgSO4'and'evaporated'to'give'Boc'protected'tyrosine'S3'as'a'white'foam'(1.471'g,'95%)'which'was'used'in'the'next'step'without'further'purification.'Analytical'data'matched'the'literature[7a].''

1H,NMR'(300'MHz,'CDCl3):'δ'7.50,7.22'(m,'2H,'CHphenyl),'7.42'(dd,'J"='8.6,'2.3'Hz,'1H,'CHphenyl),'6.92'(d,'

J' =' 8.4'Hz,' 1H,'CHphenyl),' 5.11' (br,' 1H,'NH),' 4.73,4.28' (m,' 1H,'CH),' 3.32,2.90' (m,' 2H,CH2),' 1.42' (s,' 9H,'CH3).''

N8[(1,18dimethylethoxy)carbonyl]838(38formyl848hydroxyphenyl)8L8alanine#(S4)#To'a'suspension'of'S3'(2.00'g,'7.12'mmol)'in'chloroform'(30'mL)'and'H2O'(0.256'mL,'14.13'mmol)'pow,

dered'sodium'hydroxide'(1.71'g,'42.72'mmol)'was'added'and'the'mixture'was'refluxed'for'4'h.'Two'addi,tional'portions'of'powdered'sodium'hydroxide'(each'0.42'g,'10.68'mmol)'were'added'after'1'and'2'h.'After'8'h' at' reflux,' the' reaction'was' cooled' to' ambient' temperature,' diluted'with'water' and'ethyl' acetate' (15'mL'each),' the'organic' layer'discharged,' the'aqueous' layer'acidified' to'pH'1'with'1'N'HCl'and'back,extracted'with'ethyl'acetate.'The'organic'layers'were'washed'with'brine,'dried'over'MgSO4'and'concentrated.'Flash'column'chromatography'(silica'gel,'12/1'CHCl3/MeOH,'1%'acetic'acid)'gave'compound'S4'(0.49'g,'23%).'Analytical'data'matched'the'literature[7a].'

1H,NMR'(300'MHz,'CDCl3):'δ'9.85'(s,'1H,'CHO),'7.49,7.21'(m,'2H,'CHphenyl),'7.40'(dd,'J"='8.6,'2.3'Hz,'

1H,'CHphenyl),' 6.94' (d,'J' ='8.4'Hz,'1H,'CHphenyl),' 5.10' (br,' 1H,'NH),' 4.73,4.27' (m,'1H,'CH),' 3.30,2.89' (m,'2H,CH2),'1.40'(s,'9H,'CH3).'

OH

OH

O

NH2

H

OOH

OH

O

NH2

OH

OH

O

HNBoc

OH

OH

O

HNBoc

H

O

(tBuO2C)2O

Et3Ndioxane/H2O

95%

CHCl36eq NaOH

2 eq H2OΔ 8 h, 23%

TFA/CH2Cl2

OH

OH

O

HNBoc

S3

OH

OH

O

HNBoc

H

O

S4

S21

38formyl8L*tyrosine#(1)#Compound'S4'(0.49'g,'1.6'mmol)'was'dissolved'in'CH2Cl2.'TFA'(4'mL)'was'added'slowly'at'0°C'and'the'

mixture'was'warmed'to'ambient'temperature'within'2'h.'The'solvent'was'removed'at'high'vacuum.'Prepara,tive'HPLC'(method'C)'gave'compound'2'as'TFA'salt'(0.29'g,'80%).'Analytical'data'matched'the'literature[7a].'

1H,NMR'(300'MHz,'D2O):'δ'9.81'(s,'1H,'CHO),'7.52'(d,'J'='2.4'Hz,'1H,'CHphenyl),'7.40'(dd,'J"='8.6,'2.3'Hz,'

1H,'CHphenyl),'6.90'(d,'J'='8.6'Hz,'1H,'CHphenyl),'4.13'(t,'J'='6.6'Hz,'1H,'CH),'3.15'(m,'2H,CH2)s'13C,NMR'(75'MHz,'D2O):' δ' 197.18,' 171.68,' 159.21,' 138.07,' 134.02,' 126.03,' 120.97,' 117.73,' 54.18,' 34.48s'ESI,HRMS'(m/z):[M]+'calcd.'for''C10H12NO4,'210.0758s'found''210.0760.'

2.3.3.#Synthesis#of#tris(PEG750)phosphite#S1#S4'was'synthesized'based'on'a'protocol'by'Nischan'et'al[8].'Polyethylene'glycomethylether'was'carefully'

dried'at'70°C'under'high'vacuum.'Hexamethylphosphortriamide'(1'eq.,'0.135'mmol,'24.5'µL)'was'added'to'dry'polyethylene'glycomethylether'(3'eq.,'0.406'mmol,'0.314'g)'at'110°C'and'stirred'under'N2'stream'for'72'h.'The'product'was'recovered'as'a'white'paraffinic'solid'(0.133'mmol,'0.311'g).'In'order'to'avoid'hydrolysis'of'the'product,'no'purification'was'done.'Due'to'the'chemoselective'character'of'the'Staudinger,phosphite'reaction,' impurities' do' not' interfere' in' the' reaction' and' can' be' removed' easily' after' the' Staudinger,phosphite'reaction.'

1H,NMR'(400'MHz,'[D],acetonitrile):'δ'3.58,3.57'(m,'198.9H,'OCH2CH2O,),'3.32'(s,'9H,'OCH3)s'31P,NMR'

(400'MHz,'[D],acetonitrile):'δ'140.''

2.3.4.#Synthesis#of#hydroxylamine8biotin#S5#

Scheme#3.#Synthesis#of#hydroxylamine#biotin#S5.#

D8biotin#N8hydroxysuccinimide#ester#(S5)#1,ethyl,3,(3,dimethylaminopropyl)carbodiimide' (184'mg,'0.96'mmol)'was'added' to'a'solution'of'D,biotin'

(200'mg,'0.82'mmol)'and'N,hydroxysuccinimide'(102'mg,'0.89'mmol)'in'dry'DMF'(10'mL).'The'solution'was'stirred' for'12'h'at'ambient' temperature,'concentrated'and' the'product'crystallized' from'2,propanol' to'give'succinimide'ester'S5'(261mg,'80).'The'product'was'used'without'further'purification'and'analytical'data'are'in'accordance'with'those'reported'in'the'literature[9].'

OH

OH

O

NH2

H

O

2

PO

O40 3

S1

OH

S

NH

HN

O HOSuEDC • HClDMF, 80% NEt3, DMF, 93%

S6

NH

O

S

NH

HN

O

ONH

O

ONH2

1. TFA2. S8, NEt33. TFA

2

2

O

O

S

NH

HN

O

2

O

N

O

ONH

S

NH

HN

O

2

O

ONH

Boc

2

S5 S7

S2

71 %

H

H

H

H

H

H

H

H

S22

1H,NMR'(300'MHz,'DMSO):'δ'6.41'(s,'1H,'NH),'6.36'(s,'1H,'NH),'4.35,4.26'(m,'1H,'CH),'4.18,4.11'(m,'

1H,'CH),'3.14,3.05'(m,'1H,'CH),'2.89,2.75'(m,'5H,'2xCH2,'CH),'2.64'(t'J'='7.4'Hz,'2H,'CH2),'2.60,2.55'(m,'1H,'3.32,'CH),'1.72,1.32'(m,'6H,'3xCH2).''

28Amino83’[(tert8butoxycarbonyl)amino]ethylene#glycol#diethyl#ether#(S6)#To'a'solution'of'2,2’,(ethylenedioxy),bis(ethylamine)'(4.07'g,'27.46'mmol)'and'N,N,Diisopropylethylamine'

(1.56'mL,'9.17'mmol)' in'dry'CH2Cl2'(50'mL)'at'ambient' temperature'a'solution'of'di,tert,butyl'dicarbonate'(2.0'g,'9.16'mmol)'in'dry'CH2Cl2'(20'mL)'was'added'dropwise'within'20'min.'After'additional'stirring'for'1'h'at'ambient'temperature,'the'mixture'was'concentrated,'redissolved'in'20'mL'water'and'extracted'four'times'with'CH2Cl2'(10'mL).'The'organic'layers'were'combined,'washed'three'times'with'brine,'dried'with'MgSO4'and'concentrated'to'give'2.02'g'of'a'colorless'oil'of'S6'in'an'overall'yield'of'88.8%'containing'some'impuri,ties'of'double'protected'species'(20%'determined'from'1H,NMR).'The'analytical'data'are'in'accordance'with'those'reported'in'the'literature[10].'

1H,NMR'(300'MHz,'CDCl3):'δ'5.17'(br,'1H,'NHBoc),'3.63'(s,'4H,'OCH2CH2O),'3.56,3.47'(m,'4H,'CH2O),'

3.35,3.23'(m,'2H,'CH2NHBoc),'2.86'(t,'J'='5.2'Hz,'2H,'CH2NH2),'1.51'(s,'2H,'NH2),'1.42'(s,'9H,'CH3)s'13C,NMR'(75'MHz,'CDCl3):'δ'155.90,'79.03,'73.27,'70.08,'41.61,'40.21,'28.30.''

N*Boc8N’8D8biotinyl83,68dioxaoctane,1,88diamine#(S7)#To'a'solution'of'Boc,diamine'S6'(109'mg,'0.44'mmol)'and'NEt3'(81'µL,'0.59'mmol)' in'dry'DMF'(5'mL),'

D,biotin'N,hydroxysuccinimide'ester'(S5,'100'mg,'0.29'mmol)'was'added'and'stirred'for'12'h.''The'solvent'was' removed,' the' residue' resolved' in'CH2Cl2' (40'mL),'washed'with' 20'mL'brine,' dried'over'MgSO4'and'concentrated.'Flash'column'chromatography'(silica'gel,'CH2Cl2'/'MeOH:'99'/'1'�'93'/'7)'gave'compound'S7'(0.128'g,'93%).'Analytical'data'matched'the'literature[11].'

TLC'(CH2Cl2:MeOH,'90:10'v/v):'Rf"='0.37s"'1H,NMR'(300'MHz,'CDCl3):'δ'7.35,7.23'(br,'1H.'CONH),'6.77,

6.19' (br,' 2H,'NH),' 5.24,5.04' (br,' 1H,'CONH),' 4.59,4.48' (m,'1H,'CH),' 4.40,4.28' (m,'1H,CH),' 3.63' (s,' 4H,'OCH2CH2O),'3.58'(dt,'J1'='J2'='5.4'Hz,'4H,'CH2O),'3.46'(dt,'J1"='J2'=4.9'Hz,'2H,'CH2NH),'3.32'(dt,'J1'='J2'='5.4'Hz,' 2H,'CH2NHboc),' 3.22,3.12' (m,' 1H,'CH),' 2.98,2.89' (m,' 1H,'CHHexoS),' 2.76' (d,' J' =' 12.8'Hz,' 1H,'CHHendoS),'2.26'(t,'J'='7.4'Hz,'2H,'CH2CO),'1.82,1.61'(m,'4H,'CH2),'1.46'(s,'9H,'CH3),'1.26'(m,'2H,'CH2)s'13C,NMR'(75'MHz,'CDCl3):'δ'173.52,'165.20,'156.94,'79.10,'70.03'(4C),'61.78,'60.30,'55.30,'40.55,'40.31,'39.13,'35.64,'29.61,'28,35'(3C)'27.96,'25.46.''

N’8Boc8aminooxyacetyl8N8hydroxysuccinimide#ester#(S8)#1,ethyl,3,(3,dimethylaminopropyl)carbodiimide' (227'mg,' 1.8'mmol)' and'N,hydroxysuccinimide' (190'mg,'

1.65'mmol)'was'added'to'a'solution'of'N,Boc,aminooxyacetic'acid'(287'mg,'1.5'mmol)'in'dry'DMF'(10'mL)'and'stirred'at'ambient' temperature' for'12'h.'The'mixture'was'diluted'by' the'addition'of'H2O'(10'mL),'ex,tracted'twice'with'EtOAc,'the'organic'phase'dried'over'MgSO4'and'concentrated'under'vacuum.'The'yellow,ish'liquid'was'used'without'further'purification'(352'mg,'81%).'Analytical'data'matched'the'literature[12].'

S

NH

HN

OH

H

O

O

N

O

O

S5

OHN

O

OO

NH2

S6

NH

O

S

NH

HN

O

OO

HN O

OS7

H

H

S23

1H,NMR'(300'MHz,'CDCl3):'δ'7.84'(s,'1H,'NH),'4.61'(s,'2H,'CH2),'2.82'(s,'4H,'2x'CH2),'1.31'(s,'9H,'CH3)s'

13C,NMR'(151'MHz,'CDCl3)'δ'164.83,'162.92,'156.41,'82.17,'70.48,'27.88,'25.39.'

N*aminooxyacetyl8N’8D8biotinyl83,68dioxaoctane,1,88diamine#(S2)#Boc'protected'diamine'S7'(127'mg,'0.32'mmol)'was'dissolved'in'CH2Cl2'(4'mL),'TFA'(1'mL)'was'added'

and'the'solution'stirred'at'ambient'temperature'for'2'h.'TFA'was'removed'and'the'remaining'solid'was'dried'using'high'vacuum.'The'deprotected'diamine'was'dissolved' in'a'mixture'of'dry'DMF'(3'mL)'and'NEt3'(89'µL,' 0.64'mmol).'Hydroxysuccinimide' ester'S8' (152'mg,' 0.52'mmol)'was' dissolved' in' dry'DMF' (0.5'mL),'slowly'added'to'the'diamine'and'the'resulting'mixture'was'stirred'at'ambient'temperature'for'12'h.'The'sol,vent'was'removed'and'flash'column'chromatography'(silica'gel,'CH2Cl2:MeOH,''99:1'to'93:7)'gave'boc'pro,tected'hydroxylamine'S2'(TLC'='[CH2Cl2:MeOH,'90:10'v/v]:'Rf'='0.3).'A'final'deprotection'in'25%'TFA'solu,tion'(CH2Cl2)'followed'by'TFA'removal'gave'deprotected'hydroxylamine'S2'(102.2'mg,'71%).'

1H,NMR'(300'MHz,'D2O):'δ'4.50'(s,'2H,'COCH2O),'4.49,4.43'(m,'1H,'CH),'4.24,4.31'(m,'1H,CH),'3.54'(s,'

4H,'OCH2CH2O),'3.52,3.45'(m,'4H,'CH2O),'3.33'(dt,'J1"='J2'=5.3'Hz,'2H,'CH2NH),'3.24'(dt,'J1'='J2'='5.4'Hz,'2H,'CH2NHboc),'3.21,3.14'(m,'1H,'CH),'2.85'(dd,'J1'='13,'J2'='4.9,'Hz'1H,'CHHexoS),'2.62'(d,'J'='13'Hz,'1H,'CHHendoS),'2.13'(t,'J'='7.2'Hz,'2H,'CH2CO),'1.65,1.36'(m,'4H,'CH2),'1.32,1.20'(m,'2H,'CH2)s'13C,NMR'(151'MHz,'D2O)'δ'176.79,'168.56,'165.14,'71.52,'69.26,'69.23,'68.70,'68.44,'61.93,'60.09,'55.20,'39.52,'38.67,'38.50,'35.26,'27.68,'27.52,'24.97s'ESI,MS'(m/z):[M]+'calcd.'for''C18H34N5O6S,'448.22s'found''448.21.'''

2.4.#Synthesis#of#CF–Tub8tag#peptide#

CF–Tub,tag'peptide'was'synthesized'by'standard'Fmoc,based'chemistry'in'a'linear'synthesis'on'an'Ac,

tivotec' peptide' synthesizer' followed' by'manual' coupling' of' 5(6),carboxyfluorescein.' 0.1'mmol' of' Fmoc,L,Glu(tBu),Wang'resin'(subst:'0.58'mmol/g)'was'added'to'a'reaction'vessel'and'synthesis'was'performed'with'five' fold'amino'acid'excess.'Coupling'was'achieved'by'HOBt/HBTU/DIPEA'addition.'After' the' final'amino'acid' coupling,' the' fluorophore' was' coupled' in' a' double' coupling' procedure' with' 5' eq' of' 5(6),carboxyfluorescein,'HOBt,'HBTU'and'DIPEA'in'DMF'for'1'h.'The'peptide'was'cleaved'off'the'resin'by'addi,tion'of'TFA/DTT/Tis/thioanisol'(95/2/2/1)'within'4'h.'Subsequently,'the'cleavage'cocktail'was'evaporated'by'N2,flow'and'the'peptide'was'precipitated'by'the'addition'of'ice,cold'diethyl'ether.'The'precipitate'was'spun'down,'dissolved' in'water'and'purified'by'preparative'HPLC'(method'D).'The'peptide'was'obtained'with'a'yield'of'8%'(16'mg,'8'µmol)s'molar'mass'peptide'='1850.6'Das'ESI,HRMS'(m/z):'[M+2H]2+'calcd.'926.3165s'found'926.3065.'

O

O

ONH

BocN

O

O S8

NH

O

S

NH

HN

O

OO

HN

O

ONH2

S2

H

H

HOOC

O

O

HO

C OHVal Asp Ser Val Glu Gly Glu Gly Glu Glu Glu Gly Glu GluO

8

S24

Figure'S16.'LC,UV'at'220'nm,'10'to'100%'of'acetonitrile'in'water'containing'0.1%'TFA'on'a'RP,C18'column.'

S25

3.#NMR#Spectra#of#1,#2,#3,#4#and#S2#3.1#38N38L8tyrosine#(1)#

Figure'S17.'1H,NMR'(300'MHz,'D2O)'of'3,N3,L,tyrosine'(1)'

Figure'S18.'13C,NMR'(75'MHz,'D2O)'of'3,N3,L,tyrosine'(1)'

S26

3.2#38formyl8L8tyrosine#(2)#

Figure'S19.'1H,NMR'(300'MHz,'D2O)'of'3,formyl,L,tyrosine'(2)'

Figure'S20.'13C,NMR'(75'MHz,'D2O)'of'3,formyl,L,tyrosine'(2)'

S27

3.3#38NH28L8tyrosine#(3)#

Figure'S21.'1H,NMR'(300'MHz,'D2O)'of'3,NH2,L,tyrosine'(3)'

Figure'S22.'13C,NMR'(75'MHz,'D2O)'of'3,NH2,L,tyrosine'(3)'

'

S28

3.4#38NO28L8tyrosine#(4)#

Figure'S23.'1H,NMR'(300'MHz,'D2O)'of'3,NO2,L,tyrosine'(4)'

Figure'S24.'13C,NMR'(75'MHz,'D2O)'of'3,NO2,L,tyrosine'(4)'

S29

3.5#Hydroxylamine#biotin#S2##

Figure'S25.'1H,NMR'(300'MHz,'D2O)'of'hydroxylamine'biotin'S2'

Figure'S26.'13C,NMR'(75'MHz,'D2O)'of'hydroxylamine'biotin'S2' '

S30

4.#References#'

[1] A. Kirchhofer, J. Helma, K. Schmidthals, C. Frauer, S. Cui, A. Karcher, M. Pellis, S. Muyldermans, C. S. Casas-Delucchi, M. C. Cardoso, H. Leonhardt, K. P. Hopfner, U. Rothbauer, Nat. Struct. Mol. Biol. 2010, 17, 133-138.

[2] S. Zink, L. Grosse, A. Freikamp, S. Banfer, F. Muksch, R. Jacob, J. Cell Sci. 2012, 125, 5998-6008.

[3] J. Guizetti, L. Schermelleh, J. Mantler, S. Maar, I. Poser, H. Leonhardt, T. Muller-Reichert, D. W. Gerlich, Science 2011, 331, 1616-1620.

[4] H. Leonhardt, H. P. Rahn, P. Weinzierl, A. Sporbert, T. Cremer, D. Zink, M. C. Cardoso, J. Cell. Biol. 2000, 149, 271-280.

[5] J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, A. Cardona, Nat. Methods 2012, 9, 676-682.

[6] M. R. Seyedsayamdost, T. Argirevic, E. C. Minnihan, J. Stubbe, M. Bennati, J. Am. Chem. Soc. 2009, 131, 15729-15738.

[7] a) M. E. Jung, T. I. Lazarova, J. Org. Chem. 1997, 62, 1553-1555; b) A. Banerjee, T. D. Panosian, K. Mukherjee, R. Ravindra, S. Gal, D. L. Sackett, S. Bane, ACS Chem. Biol. 2010, 5, 777-785.

[8] N. Nischan, A. Chakrabarti, R. A. Serwa, P. H. Bovee-Geurts, R. Brock, C. P. Hackenberger, Angew. Chem. Int. Ed. 2013, 52, 11920-11924.

[9] E. Gerard, A. Meulle, O. Feron, J. Marchand-Brynaert, Bioorg. Med. Chem. Lett. 2012, 22, 586-590.

[10] M. Ishida, H. Watanabe, K. Takigawa, Y. Kurishita, C. Oki, A. Nakamura, I. Hamachi, S. Tsukiji, J. Am. Chem. Soc. 2013, 135, 12684-12689.

[11] M. Braun, X. Camps, O. Vostrowsky, A. Hirsch, E. Endress, T. M. Bayerl, O. Birkert, G. Gauglitz, Eur. J. Org. Chem. 2000, 1173-1181.

[12] K. K. Palaniappan, R. M. Ramirez, V. S. Bajaj, D. E. Wemmer, A. Pines, M. B. Francis, Angew. Chem. Int. Ed. 2013, 52, 4849-4853.