Available online at www.sciencedirect.com

ScienceDirect More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins Dominik Schumacher and Christian PR Hackenberger The quest to enlarge the molecular space of functional biomolecules has led to the discovery of selective, mild and high-yielding chemical reactions for the modification of peptides and proteins. These conjugation methods have recently become even more advanced with the advent of modern biochemical techniques such as unnatural protein expression or enzymatic reactions that allow the site-specific modification of proteins.Within this overview, we will highlight recent examples that describe the site-specific functionalization of proteins. These examples go beyond the straightforward attachment of a given functional moiety to the protein backbone by employing either an innovative linkerdesign or by novel conjugation chemistry, where the modification reaction itself is responsible for the (altered) functional behaviour of the biomolecule. The examples covered herein include ‘turn-on’ probes for cellular imaging with low levels of background fluorescence, branched or cleavable polymer–protein conjugates of high stability within a cellular environment, the installation of natural occurring posttranslational modifications to help understand their role in complex cellular environments and finally the engineering of novel antibody drug conjugates to facilitate target specific drug release. Addresses Department of Chemical Biology II, Leibniz-Institut fu¨r Molekulare Pharmakologie (FMP), Robert-Ro¨ssle-Str. 10, Berlin, Germany Corresponding author: Hackenberger, Christian PR ([email protected])

Current Opinion in Chemical Biology 2014, 22:62–69 This review comes from a themed issue on Synthetic biomolecules Edited by Paul F Alewood and Stephen B Kent For a complete overview see the Issue and the Editorial Available online 3rd October 2014 http://dx.doi.org/10.1016/j.cbpa.2014.09.018 1367-5931/# 2014 Elsevier Ltd. All right reserved.

Introduction Demands for the understanding of biological processes and for the treatment of complex molecular dysfunction are constantly rising. This trend is even further accelerated by the need for personalized medicine to cope with individual physiological variation. The chemical modification of functional biomolecules is one way to help address these challenges. Despite the many advances Current Opinion in Chemical Biology 2014, 22:62–69

in this area within recent years, there is still a great need to develop effective probes for protein labelling, in order to advance the understanding of naturally occurring posttranslational protein modifications and to introduce moieties for intracellular stabilization. Furthermore, methods for the specific targeting of bioactive peptides, proteins and other biologically relevant molecules need to be enhanced for successful biological and in particular intracellular applications. Over the years, mostly unselective coupling methods to natural amino acids and reactive side chains have been expanded by the incorporation of unnatural functional groups into biomolecules. Techniques including solid phase peptide synthesis, expressed protein ligation, genetic code expansion, or metabolic engineering allow the site-specific placement of unique chemical handles in a polypeptide sequence or even in a complex cellular surface. These bioorthogonal functionalities can then be further addressed by appropriate chemical reactions [1,2]. From the many successful chemoselective attachments of functional entities to peptides and proteins, this review will highlight recent examples in several areas of modern chemical biology, that either take advantage of the specific molecular composition of the formed conjugate or that install multifunctional linkers into a protein architecture.

‘Turn-on’ fluorescent labels Since it was shown that the green fluorescent protein (GFP) from Aequorea victoria remains active when expressed in eukaryotic and prokaryotic cells, it has intensively been used for labelling and localization of proteins in living organisms [3]. Although fusion of GFP to a protein of choice is straightforward, its photobleaching properties and the unknown resulting changes of the treated system due to the size of the GFP protein restrict its applications. Organic compounds on the contrary are small and often have better photophysical properties [4]. One major drawback in the use of classical fluorophores for live cell and fixed cell imaging is the high background fluorescence and consequent low signal-to-noise ratio. Therefore, a lot of effort has been made to develop ‘turn-on’ fluorophores that are non-fluorescent until they are conjugated to the molecule of interest. A phosphine probe that has been modified with a fluorescence resonance energy transfer (FRET) quencher was one of the first examples suitable for live cell experiments. Upon Staudinger reaction with azides, the quencher gets cleaved and the fluorescence quantum yield increases 170-fold [5]. More recently, unsymmetrical aryl-tetrazines have been used in fluorogenic labelling reactions. When conjugated to certain fluorophores, the tetrazine www.sciencedirect.com

Synthesis of functional peptides and proteins Schumacher and Hackenberger 63

Figure 1

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last decade, many new (semi-)synthetic methods have been developed to access functional PTMs and mimics of PTMs in protein molecules [9–11].

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Turn on probes. (a) Fluorescent turn-on after inverse-electron-demand Diels–Alder tetrazine cycloaddition of trans-cyclooctenol and different tetrazine fluorophores. Turn-on ratios of up to 1600-fold can be applied in live-cell imaging where washing steps are unfavourable. (b) Transcyclooctene-lysine was incorporated into a protein using amber suppression methods. Rapid labelling with tetrazine conjugated fluorophores was performed in living cells [6,7].

moiety can play an important double role by acting as a bioorthogonal reactant for selective biomolecule functionalization and by simultaneously quenching the fluorophore, which is then turned on by cycloaddition to strained alkenes [6]. Furthermore, boron dipyromethene tetrazine fluorophores show up to 1600-fold fluorescence increase when coupled to trans-cyclooctene (TCO) derivatives (Figure 1) [7]. These properties are of huge advantage for live cell imaging by making intensive probe wash steps unnecessary. The unique chemistry of tetrazine probes enabled Lemke et al. to perform a pulsechase dual labelling of the insulin receptor and virus-like particles in mammalian cells using two cyclooctynyl derivatives and selective tetrazine coupling [8]. This strategy allows the rapid labelling of cellular processes at two specific moments in time.

Chemical installation of posttranslational modifications (PTMs) PTMs such as phosphorylation, glycosylation and lipidation are nature’s way of regulating protein function, transport and degradation. However, only for a few PTMs the biological functions are fully understood since their selective replication remains highly challenging. Over the www.sciencedirect.com

One of the frequently occurring PTMs is the ubiquitination of proteins, which is used by eukaryotes as a marker before protein degradation by the proteasome [12]. In nature, ubiquitin is enzymatically conjugated to the protein via an isopeptide bond between the e-NH2 of specific lysine residues and the C-terminal carboxy group of ubiquitin. Ubiquitin itself has seven lysine residues which facilitates the formation of defined linear and branched ubiquitin chains, making the study of this complex PTM even more challenging. Many isopeptide variants of ubiquinated peptides and proteins have been synthesized using disulfide bonds, oxime bonds and triazoles to link the ubiquitin to the target protein [13– 16]. However, generating proteins that carry site-specific native ubiquitinations is indispensible to mimic cellular processes. Native ubiquitination of histone H2 by expressed protein ligation revealed a direct correlation to an increased methylation of H3 [17]. A combination of genetic code expansion, orthogonal amino acid protection and final aminolysis of ubiquitin thioester furnished a native di-ubiquitin-conjugate [18]; alternatively, peptide synthesis and sequential native chemical ligations provided access to ubiquitinated peptides with varying lengths of specific ubiquitin chains [19]. These studies showed that deubiquitinases have a higher activity towards shorter chains as well as Lys29-linked and Lys48-linked poly-ubiquitins. The genetically directed site-specific incorporation of d-thiol-L-lysine and dhydroxy-L-lysine enables the generation of isopeptide bonds at flexible positions within proteins (Figure 2a) [20]. Strieter et al. used an enzymatic reaction to generate allylamine containing ubiquitin and obtained a K48Clinked di-ubiquitin by thiol-ene coupling (Figure 2a) [21]. Glycosylation and phosphorylation have a high impact on protein function, localization and cellular signalling processes [22,23]. Davis developed two complementary methods for the chemical glycosylation of proteins by utilizing glycosyl thiols. The first strategy relies on the auxotrophic incorporation of L-homoallylglycine into a protein molecule, followed by a hydrothiolation reaction with a 1-glycosyl thiol [24]. In the second case, naturally occurring cysteines within a protein are quantitatively transformed into dehydroalanine (Dha) by incubation with O-mesitylenesulfonylhydroxylamine. The resulting alkene is used to generate S-linked glycoproteins by addition of nucleophilic glycosyl thiols [25,26]. Furthermore, the Dha-approach has been used to install thiophosphates as phosphoserine mimics into different proteins like histone H3 [27]. In our own group, we have used the chemoselective Staudinger-phosphite reaction for the site-specific Current Opinion in Chemical Biology 2014, 22:62–69

64 Synthetic biomolecules

Figure 2

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Posttranslational modification. (a) Evolution of pyrrolysyl-tRNA synthetase/tRNACUA allowed the site-specific incorporation of d-thiol-lysine into ubiquitin. Native chemical ligation to a C-terminal thioester containing ubiquitin in combination with desulfurization gave native di-ubiquitin in 1 [20]. An allylalanine-containing ubiquitin derivative was generated enzymatically. Thiol-ene coupling using the radical initiator lithium acyl phosphinate (LAP) to a [K48C]ubiquitin cysteine mutant gave K48C-linked di-ubiquitin in 2 [21]. (b) Generation of phosphorylated peptides. Staudinger reaction of azides with phosphites followed by light initiated hydrolysis gave phosphorylated tyrosine analogues and naturally occurring phosphorylated lysine peptides in 1 and 2 [28,30]. High yielding and selective synthesis of pyrophosphopeptides in 3 [32].

chemical phosphorylation of proteins by installing phosphor-Tyr mimetics as well as for the acquisition of glycosylated phosphopeptides, which were found in the parasite Leishmania mexicana (Figure 2b) [28,29]. Another recent utilization of this reaction in the area of intrinsically labile PTMs allowed us to obtain and characterize the properties of site-specifically phosphorylated lysine-peptides for the first time (Figure 2b) [30]. Reports describing access to other forms of phosphorylated peptides and proteins included the synthesis of phospho-Hismimetics by Muir and coworkers using Ru-catalyzed cycloaddition reactions (Figure 2b) [31] and the use of phosphorimidazolide reagents to generate pyrophosphorylated peptides by Fiedler et al., which represents the first example of synthetic pyrophosphorylation and facilitated the in vitro characterization of this recently described PTM (Figure 2b) [32]. Current Opinion in Chemical Biology 2014, 22:62–69

Protein-PEG conjugates The high selectivity and low toxicity of peptide and protein based drugs make them promising candidates for future therapeutic treatments. However, the frequent low effectiveness of these classes of drugs due to physical instability and proteolysis as well as strong immune responses and fast kidney clearance pose major challenges. Shielding peptides and proteins by conjugation to natural and synthetic (bio-)polymers is a promising strategy to address these drawbacks. To date, the most commonly used polymer is polyethylene glycol (PEG). Its hydrophilicity, low cytotoxicity and immunogenicity have resulted in nine approved PEG-protein drugs so far [33]. Early PEGylation strategies were based on the uncontrolled conjugation of lysine residues to electrophilic PEG derivatives [34]. The thiol moiety of the amino acid cysteine served to be a more promising point of www.sciencedirect.com

Synthesis of functional peptides and proteins Schumacher and Hackenberger 65

Figure 3

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In our own group, we have incorporated both stable and light-cleavable PEGs by the Staudinger-phosphite reaction into p-azido-phenylalanine-containing biomolecules with excellent chemoselectivity and yields in aqueous and cell-lysate conditions (Figure 3b) [40,41]. Using this approach, inexpensive linear PEGs can be used as starting materials to produce branched PEG-chains in a one-pot reaction. Small branched PEG-chains have the advantage of stabilizing bioconjugates with similar or higher protective effects compared to large linear PEGchains, while maintaining bioavailability and activity in vivo and in vitro (the ‘umbrella effect’). This concept was demonstrated for the Staudinger-phosphite PEG-conjugates in the rarely addressed intracellular distribution and biological activity of a PEGylated peptide inside a cell. Attaching just two small sized PEG 750 chains to a 22 amino acid BH3-peptide led to a significant stabilization in cell lysates and in cells as well as a concentration dependent increase of intracellular activity [40]. www.sciencedirect.com

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The high impact of the PEGylation site on the mode of action of a protein has been shown by Cho et al. using the example of the human growth hormone (hGH) [39]. hGH is effectively used as a treatment for agromegaly and other growth-associated dysfunctions, but has yet to be applied on a daily basis. To increase the drug’s efficiency, nineteen surface exposed amino acids were selected, and each of these residues were substituted one at a time with p-acetylphenylalanine (pAcPhe) using amber suppression recombinant DNA expression and attached to a 30 kDa amino-oxy PEG using oxime-forming ligation. With this strategy they were able to identify a PEG-hGH conjugate that displayed similar in vivo pharmacology with weekly doses when compared to daily-dosed unPEGylated hGH.

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attachment for PEG, since its rare occurrence in proteins and its low pKa value and high nucleophilicity allows for a rapid and selective conjugation reaction under physiologic conditions. Typically, any natural Cys residues can be replaced, and a new Cys introduced wherever desired, by site directed mutagenesis. Novel linker systems have recently been developed to address unwanted hydrolysis and retro-Michael reactions in maleimide–cysteine conjugations [35]. Thereby, the use of methylsulfonyl functionalized phenyloxadiazoles for the chemoselective functionalization of thiols increase the linkage half-life of PEGylated proteins drastically when incubated in human serum albumin [36]. Additionally, for the PEGylation of disulfide-containing proteins a technique called ‘disulfide bridging’ was developed, which allowed the selective and reversible coupling of PEG chains to disulfides, while retaining the protein’s structure [37]. In this, dibromomaleimides have been shown to be efficient bridging reagents to PEGylate proteins in less than 15 min using equimolar amounts of the PEG reagent, with little or no need for purification (Figure 3a) [38].

Current Opinion in Chemical Biology

Shielding proteins and peptides by PEGylation. (a) Dibromomaleimides have been inserted into disulfide bonds while retaining the proteins structure and biological activity of the peptide or protein. The disulfidebond of salmon calcitonin (sCT) was reduced by TCEP. A slight excess of 1.1 equivalents 2,3-dibromomaleimide 1 led to full conversion and gave PEGylated cross-linked peptide after 15 min reaction time [38]. (b) Starting from linear polyethylene building blocks and hexamethylphophortriamide (HMPT), PEG-phosphites were generated. A subsequent Staudinger phosphite reaction with p-azido-phenylalanine gave PEGylated peptide. The chemoselective reaction led to a promising branching point by simultaneous attachment of two PEG chains. The impact of the attached PEGchains on proteolysis has been probed. Two PEG 2000 chains led to a 57-fold increase in the peptides half-life in Jurkat cell lysate and a significant increase of intracellular activity [40].

Another example for a photocleavable PEGylation strategy was published by the Deiters group [42]. Using an ortho-nitrobenzyl linked PEG, they showed reversible caging of proteins and peptides, and most importantly the timed regulation of biomolecules activity.

A new hope for Pharma: antibody–drug conjugates Antibodies are well-established tools to probe the recognition and biological behaviours of cellular proteins [43]. The ability of engineered monoclonal antibodies to selectively interfere with receptor binding and signal transduction into cells led to the clinical trial investigation and Current Opinion in Chemical Biology 2014, 22:62–69

66 Synthetic biomolecules

approval of several therapeutic antibodies [44]. More recently, significant progress for the production of antibody–drug conjugates (ADCs) and antibody–fluorophore conjugates (AFCs) has been made [45]. Similar to PEGconjugation, conventional coupling methods address reduced disulfides as well as genetically engineered cysteine and lysine residues, yielding heterogeneous and often irreproducible mixtures [46]. However, selectively modified antibodies have shown superior efficiency, pharmacokinetics and an improved therapeutic index compared to their unselective ADC counterparts, supporting the need for highly selective antibody conjugation [47]. Furthermore, the conjugation site influences the antibody-conjugate stability and its chemical and structural surroundings can be used to control the hydrolysis of ADCs and AFCs [48]. The group of Dario Neri has recently developed two ‘traceless’ cleavable

linkers for the site-specific attachment of cytotoxic Cemadotin (Cem) to C-terminal or N-terminal cysteine residues in IgGs, diabodies and small immunoproteins (SIP) (Figure 4a) [49,50,51]. In the first case they selectively reduced the C-terminal cysteine involved in an interchain disulfide bond of a diabody using tris(2-carboxyethyl)phosphine hydrochloride (TCEPHCl) without affecting other disulfide bridges present in the antibody. The resulting free thiol was either coupled to a maleimidecontaining 1,2-aminothiol linker followed by a thiazolidine formation with a benzaldehyde-containing Cem analogue (Cem-CHO) or the resulting cysteine residues were activated with 5,50 -dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) and then coupled to a sulfhydryl group containing CemCH2-SH derivative within 3 min in very high yields (>95%) [49]. To perform N-terminal coupling, they introduced an N-terminal cysteine into

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Antibody–drug conjugates that allow target-specific drug release. (a) Two cleavable linkers for antibody–drug conjugates [49,50,51]. After disulfide reduction of a F8 antibody in small interfering protein (SIP) or diabody (Db) format free thiols were either coupled to a maleimide-cysteine linker followed by thiazolidine-formation to the drug Cemadotin-CHO (Cem-CHO) in example 1, or activated by Ellman’s reagent and coupled to CemCH2SH in 2. The thiazolidine-conjugated drug is released after 70 hours when incubated in PBS, pH 7.4. The disulfide-linked drug is cleaved in the presence of glutathione and other thiols. An N-terminal cysteine containing Db was produced by a suitable protease cleaving design. Thiazolidine formation with Cem-CHO resulted in the antibody–drug conjugate in 3. (b) p-Acetylphenylalanin was incorporated into trastuzumab, a full length IgG1 antibody. The antineoplastic agent auristatin was coupled to the monoclonal antibody using oxime-forming ligation [56]. Current Opinion in Chemical Biology 2014, 22:62–69

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Synthesis of functional peptides and proteins Schumacher and Hackenberger 67

antibodies using a suitable protease cleavage design that generated a free 1-amino-2-thiol functionality and thus allowed direct thiazolidine formation with Cem-CHO. Both conjugation methods allowed the cleavage of the attached moiety. The thiazolidine heterocycles hydrolyze over time (half-life: 45 hours in PBS 378C), whereas the disulfide bond is cleaved in the presence of glutathione or other thiols, which are released during tumor cell lysis. Genentech developed another site-specific cysteine conjugation method based on the introduction of an additional cysteine into antibodies using genetic engineering followed by maleimide coupling (THIOMAB) [47,52]. Recently also dibromomaleimides were used for the modification of cysteine (resp. disulfide) containing antibodies [37,53,54]. The anti-cancer drug doxorubicin was conjugated to a trastuzumab Fab fragment using the aforementioned disulfide bridging approach [53]. This thiomaleamic acid linker is stable at physiological pH, but is cleaved in the acidic lysosome and under thiol treatment. Furthermore, partial hydrolysis and ring opening at pH 8.0 generates thiol-stable bioconjugates and extends the applicability of these linkers [55]. The need to engineer cysteine residues, reduce disulfide bonds and perform reoxidization steps limits the applicability of cysteine-based couplings and causes challenges in scalability. One way to circumvent these challenges is the use of genetically encoded unnatural amino acids. Schultz et al. conjugated the monoclonal antibody trastuzumab to an alkoxy-amine auristatin derivative by incorporation of pAcPhe in the constant region of the IgG, to receive an ADC with improved pharmacokinetics and high efficacy (Figure 4b) [56]. In a similar manner, they conjugated small interference RNAs (siRNAs) to HER2 Fab and full-length IgG to selectively deliver siRNAS to target cells [57].

Conclusion and future Optimization and further development of chemoselective conjugation methods have led to a broad variety of siteselectively functionalized peptides and proteins. Furthermore, the clever usage of these functional groups by conjugation and the advancement of highly functional linkers has enabled the application of bioconjugates in complex cellular surroundings and has taken bioorthogonal chemistry to the next level. Considerable efforts are taking place to further expand the scope of methods and make bioorthogonal and chemoselective functionalization of proteins and peptides more efficient. This will lead to an even better understanding of biological processes and enable a highly targeted intervention to treat illnesses caused by cellular dysfunction.

Acknowledgements The authors acknowledge financial support from the German Science Foundation (SPP 1623 and SFB 765), the Einstein Foundation Berlin (Leibniz-Humboldt Professorship), the Boehringer-Ingelheim Foundation (Plus 3 award) and the Fonds der Chemischen Industrie (FCI). The authors thank Alec Michels and Charlotte Wittig for proofreading. www.sciencedirect.com

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Carlson JC, Meimetis LG, Hilderbrand SA, Weissleder R: BODIPYtetrazine derivatives as superbright bioorthogonal turn-on probes. Angew Chem Int Ed Engl 2013, 52:6917-6920.

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Chalker JM, Bernardes GJ, Davis BG: A ‘‘tag-and-modify’’ approach to site-selective protein modification. Acc Chem Res 2011, 44:730-741.

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18. Virdee S, Ye Y, Nguyen DP, Komander D, Chin JW: Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat Chem Biol 2010, 6:750-757. 19. Bavikar SN, Spasser L, Haj-Yahya M, Karthikeyan SV, Moyal T, Kumar KS, Brik A: Chemical synthesis of ubiquitinated peptides with varying lengths and types of ubiquitin chains to explore the activity of deubiquitinases. Angew Chem Int Ed Engl 2012, 51:758-763. 20. Virdee S, Kapadnis PB, Elliott T, Lang K, Madrzak J, Nguyen DP, Riechmann L, Chin JW: Traceless and site-specific  ubiquitination of recombinant proteins. J Am Chem Soc 2011, 133:10708-10711. In this work, the authors ubiquitinated recombinant proteins by the use of amber suppression methods in combination with desulfurization. d-thiollysine was incorporated into SUMO and ubiquitin by an evolved pyrrolysyl-tRNA synthetase/tRNACUA pair. Native chemical ligation to a ubiquitin-thioester led to a ubiquitin dimer and ubiquitinated SUMO. 21. Valkevich EM, Guenette RG, Sanchez NA, Chen YC, Ge Y, Strieter ER: Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases. J Am Chem Soc 2012, 134:6916-6919. 22. Unverzagt C, Kajihara Y: Chemical assembly of Nglycoproteins: a refined toolbox to address a ubiquitous posttranslational modification. Chem Soc Rev 2013, 42:44084420. 23. Hunter T: Why nature chose phosphate to modify proteins. Philos Trans R Soc Lond B Biol Sci 2012, 367:2513-2516. 24. Floyd N, Vijayakrishnan B, Koeppe AR, Davis BG: Thiyl glycosylation of olefinic proteins: S-linked glycoconjugate synthesis. Angew Chem Int Ed 2009, 48:7798-7802. 25. Bernardes GJL, Chalker JM, Errey JC, Davis BG: Facile  conversion of cysteine and alkyl cysteines to dehydroalanine on protein surfaces: versatile and switchable access to functionalized proteins. J Am Chem Soc 2008, 130:5052. The authors developed an easy method for the transformation of cysteine to dehydroalanine on the protein surface, giving fast access to the conjugation of functional moieties to biomolecules. 26. Grayson EJ, Bernardes GJL, Chalker JM, Boutureira O, Koeppe JR, Davis BG: A coordinated synthesis and conjugation strategy for the preparation of homogeneous glycoconjugate vaccine candidates. Angew Chem Int Ed 2011, 50:4127-4132.

34. Lecolley F, Tao L, Mantovani G, Durkin I, Lautru S, Haddleton DM: A new approach to bioconjugates for proteins and peptides (‘‘pegylation’’) utilising living radical polymerisation. Chem Commun (Camb) 2004:2026-2027. 35. Baldwin AD, Kiick KL: Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjug Chem 2011, 22:1946-1953. 36. Toda N, Asano S, Barbas CF 3rd: Rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: a serumstable alternative to maleimide-based protein conjugation. Angew Chem Int Ed Engl 2013, 52:12592-12596. 37. Smith ME, Schumacher FF, Ryan CP, Tedaldi LM, Papaioannou D, Waksman G, Caddick S, Baker JR: Protein modification,  bioconjugation, and disulfide bridging using bromomaleimides. J Am Chem Soc 2010, 132:1960-1965. Dibromomaleimides have been used to conjugate PEGchains into peptidic disulfide bonds without affecting the peptide’s structure. The conjugation proceeded in less than 15 min with equimolar ratios of peptide and dibromomaleimide. 38. Jones MW, Strickland RA, Schumacher FF, Caddick S, Baker JR, Gibson MI, Haddleton DM: Polymeric dibromomaleimides as extremely efficient disulfide bridging bioconjugation and pegylation agents. J Am Chem Soc 2012, 134:1847-1852. 39. Cho H, Daniel T, Buechler YJ, Litzinger DC, Maio Z, Putnam AM, Kraynov VS, Sim BC, Bussell S, Javahishvili T et al.: Optimized clinical performance of growth hormone with an expanded genetic code. Proc Natl Acad Sci U S A 2011, 108:9060-9065. 40. Nischan N, Chakrabarti A, Serwa RA, Bovee-Geurts PH, Brock R,  Hackenberger CP: Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains. Angew Chem Int Ed Engl 2013, 52:11920-11924. The Staudinger-phosphite reaction was used to install a branching point for two linear PEG-chains attached to a biomolecule. These PEG peptide conjugates displayed excellent stabilization in cell lysates and a concentration-dependent intracellular activity. 41. Serwa R, Majkut P, Horstmann B, Swiecicki JM, Gerrits M, Krause E, Hackenberger CPR: Site-specific PEGylation of proteins by a Staudinger-phosphite reaction. Chem Sci 2010, 1:596-602. 42. Georgianna WE, Lusic H, McIver AL, Deiters A: Photocleavable polyethylene glycol for the light-regulation of protein function. Bioconjug Chem 2010, 21:1404-1407.

27. Chalker JM, Lercher L, Rose NR, Schofield CJ, Davis BG: Conversion of cysteine into dehydroalanine enables access to synthetic histones bearing diverse post-translational modifications. Angew Chem Int Ed Engl 2012, 51:1835-1839.

43. Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC et al.: Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 2006, 3:887-889.

28. Serwa R, Wilkening I, Del Signore G, Muhlberg M, Claussnitzer I,  Weise C, Gerrits M, Hackenberger CPR: Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem Int Ed 2009, 48:82348239. This study represents the first example for a site-specific chemical phosphorylation of proteins and peptides using the Staudinger-phosphite reaction. Analogues of phosphorylated Tyr residues in proteins were generated by this method and were recognized by p-Tyr antibodies.

44. Page DB, Postow MA, Callahan MK, Allison JP, Wolchok JD: Immune modulation in cancer with antibodies. Annu Rev Med 2014, 65:185-202.

29. Jaradat DMM, Hamouda H, Hackenberger CPR: Solid-phase synthesis of phosphoramidate-linked glycopeptides. Eur J Org Chem 2010:5004-5009. 30. Bertran-Vicente J, Serwa R, Schuemann M, Schmieder P, Krause E, Hackenberger CP: Site-specifically phosphorylated lysine peptides. J Am Chem Soc 2014 http://dx.doi.org/10.1021/ ja507886s. 31. Kee JM, Villani B, Carpenter LR, Muir TW: Development of stable phosphohistidine analogues. J Am Chem Soc 2010, 132:1432714329. 32. Marmelstein AM, Yates LM, Conway JH, Fiedler D: Chemical pyrophosphorylation of functionally diverse peptides. J Am Chem Soc 2014, 136:108-111. 33. Pasut G, Veronese FM: State of the art in PEGylation: the great versatility achieved after forty years of research. J Control Release 2012, 161:461-472. Current Opinion in Chemical Biology 2014, 22:62–69

45. Alley SC, Okeley NM, Senter PD: Antibody–drug conjugates: targeted drug delivery for cancer. Curr Opin Chem Biol 2010, 14:529-537. 46. Ducry L, Stump B: Antibody–drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem 2010, 21:5-13. 47. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS et al.: Site-specific  conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 2008, 26:925-932. In this work the authors performed for the first time a site-specific conjugation of a drug to an antibody and showed that site-specificity improves the therapeutic index of the drug. 48. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, ParsonsReponte KL, Tien J, Yu SF, Mai E et al.: Conjugation site modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat Biotechnol 2012, 30:184-189. 49. Bernardes GJ, Casi G, Trussel S, Hartmann I, Schwager K,  Scheuermann J, Neri D: A traceless vascular-targeting antibody–drug conjugate for cancer therapy. Angew Chem Int Ed Engl 2012, 51:941-944. www.sciencedirect.com

Synthesis of functional peptides and proteins Schumacher and Hackenberger 69

The authors engineered a traceless site-selective modification of a small interfering protein with a cytotoxic drug showing that a noninternalizing vascular targeting antibody–drug-conjugate mediates a strong antitumour activity.

54. Schumacher FF, Sanchania VA, Tolner B, Wright ZV, Ryan CP, Smith ME, Ward JM, Caddick S, Kay CW, Aeppli G et al.: Homogeneous antibody fragment conjugation by disulfide bridging introduces ‘spinostics’. Sci Rep 2013, 3:1525.

50. Bernardes GJ, Steiner M, Hartmann I, Neri D, Casi G: Site-specific chemical modification of antibody fragments using traceless cleavable linkers. Nat Protoc 2013, 8:2079-2089.

55. Ryan CP, Smith ME, Schumacher FF, Grohmann D, Papaioannou D, Waksman G, Werner F, Baker JR, Caddick S: Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem Commun (Camb) 2011, 47:5452-5454.

51. Casi G, Huguenin-Dezot N, Zuberbuhler K, Scheuermann J, Neri D: Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery. J Am Chem Soc 2012, 134:5887-5892. 52. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen T, Dugger DL, Li G et al.: Engineered thiotrastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2positive breast cancer. Clin Cancer Res 2010, 16:4769-4778. 53. Castaneda L, Maruani A, Schumacher FF, Miranda E, Chudasama V, Chester KA, Baker JR, Smith ME, Caddick S: Acidcleavable thiomaleamic acid linker for homogeneous antibody–drug conjugation. Chem Commun (Camb) 2013, 49:8187-8189.

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56. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA,  Halder R, Forsyth JS, Santidrian AF, Stafin K et al.: Synthesis of site-specific antibody–drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 2012, 109:16101-16106. This is the first example for the site-specific generation of a full-length antibody–drug-conjugate using amber suppression methods. This approach allows the flexible conjugation of a drug (or other functionalities) to a surface exposed residue of choice within an antibody. 57. Lu H, Wang D, Kazane S, Javahishvili T, Tian F, Song F, Sellers A, Barnett B, Schultz PG: Site-specific antibody–polymer conjugates for siRNA delivery. J Am Chem Soc 2013, 135:13885-13891.

Current Opinion in Chemical Biology 2014, 22:62–69