Thiazolidine-Masked α-Oxo Aldehyde Functionality for Peptide and

Communication pubs.acs.org/bc

Thiazolidine-Masked α‑Oxo Aldehyde Functionality for Peptide and Protein Modification Xiaobao Bi, Kalyan Kumar Pasunooti, Julien Lescar, and Chuan-Fa Liu* School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore S Supporting Information *

ABSTRACT: α-Oxo aldehyde-based bioconjugation chemistry has been widely explored in peptide and protein modifications for various applications in biomedical research during the past decades. The generation of α-oxo aldehyde via sodium periodate oxidation is usually limited to the N-terminus of a target protein. Internal-site functionalization of proteins with the α-oxo aldehyde handle has not been achieved yet. Herein we report a novel method for site-specific peptide and protein modification using synthetically or genetically incorporated thiazolidine-protected α-oxo aldehyde. Efficient unmasking of the aldehyde was achieved by silver ion-mediated hydrolysis of thiazolidine under mild conditions for the first time. A model peptide and a recombinant protein were used to demonstrate the utility of this new method, which were site-specifically modified by oxime ligation with an oxyamine-functionalized peptide labeling reagent. Therefore, our current method has enriched the α-oxo aldehyde synthetic tool box in peptide and protein bioconjugation chemistry and holds great potential to be explored in novel applications in the future.

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strategies are also quite useful to install an aldehyde group into a peptide or protein. The oxidative cleavage of an N-ter Ser or Thr residue with sodium periodate can efficiently produce a unique aldehyde handle.19−21 However, the aldehyde can only be introduced at the N termini of proteins and very careful control of the reaction should be performed to prevent overoxidation of methionine and cysteine residues during the reaction.19,22 To overcome this limitation, protected α-oxo aldehyde derivatives have been introduced into peptides during solid phase peptide synthesis (SPPS). These derivatives were compatible with the acidic resin cleavage and side-chain deprotection conditions, but can be deprotected to reveal the aldehyde in a subsequent step.23 Examples are the glyoxylic acid dimethyl acetal protected aldehyde developed by Tam and coworkers,24 the diisopryl thioacetal derivative developed by Qasmi and co-workers,25 and the α,α′-diaminoacetic acid derivatives developed by Melnyk and co-workers.26,27 These derivatives either require harsh conditions to liberate the aldehyde group or can only be introduced into short peptides using standard coupling procedures. Therefore, new methods that allow for the introduction of suitably protected α-oxo aldehyde and its mild deprotection are still highly desired in protein bioconjugation chemistry.

hemical modification of peptides and proteins is an important strategy to endue them with tailor-made properties for various applications.1−5 Certain proteinogenic amino acids, such as cysteine, tyrosine, and lysine, can be exploited to functionalize a peptide or protein with other chemical moieties.6−8 However, site-selectivity is a major challenge when more than one copy of such an amino acid is present within a protein.9 To overcome this problem, bioorthogonal chemical functionalities such as azide, alkyne, alkene, or halides have been introduced into peptides and proteins to achieve site-specific modification.10−14 Besides the bioorthogonal azide and alkyne groups, aldehyde and ketone are preferable choices as chemical handles for the functionalization of peptides and proteins due to their unique reactivity in many types of chemical transformations.15,16 There are now a variety of strategies to incorporate an aldehyde handle into peptides and proteins. For instance, an enzymatic method relies on the use of a specific enzyme, formylglycine-generating enzyme (FGE), which has the ability to oxidize the side chain of a cysteine residue to the aldehyde functional group.17 This strategy requires a pentapeptide consensus sequence in the target protein for FGE recognition. Genetic incorporation of unnatural amino acids is shown to be a straightforward way to directly install a benzaldehyde moiety into proteins for fast labeling.18 However, direct genetic incorporation of an alkyl aldehyde-carrying unnatural amino acid into proteins has not been achieved yet, possibly due to its higher reactivity and instability in living cells. Many chemical © XXXX American Chemical Society

Received: November 18, 2016 Revised: December 14, 2016

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DOI: 10.1021/acs.bioconjchem.6b00667 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Herein we aim to develop a novel method to install protected α-oxo aldehyde into proteins in a site-specific manner using the genetic code expansion technology. The Pyrrolysyl-tRNA synthetase (PylRS)/tRNA (pylT) pairs from Methanosarcina barkeri (Mb) and M. mazei (Mm) can co-translationally insert the 22nd natural amino acid pyrrolysine 1 (Scheme 1A) into Scheme 1. (A) Structures of Pyrrolysine (1), Nε-LThiaprolyl-L-lysine Methyl Ester (2), Nε-D-Proline-L-lysine (3), Nε-(Thiazolidine-2-carbonyl)-L-lysine Methyl Ester (4). (B) General Procedure for the Genetic Installation of Aldehyde into Proteins for Site-Specific Modificationa

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(i) Genetic incorporation of 4 into proteins in E. coli cells transformed with genes encoding for the Pyrrolysyl-tRNA synthetase (PylRS)/tRNA (pylT) pair and a target protein containing an amber codon mutation (TAG); (ii) deprotection of thiazolidine on the incorporated unnatural amino acid residue to unmask the α-oxo aldehyde; (iii) site-specific modification of protein via oxime ligation with oxyamine-functionalized labeling reagents.

Figure 1. (A) Procedure for the chemical synthesis of 11. (i) pyridine, ethanol, 92%; (ii) BOC anhydride, NaHCO3, THF, 95%; (iii) 8, EDC, DMAP, DCM, 91%; (iv) Pd (0), phenylsiliane, DCM, quantitative. (B) Procedure for the synthesis and modification of peptide 13. (i) DIEA, PyBOP; (ii) TFA/TIS/H2O (95/2.5/2.5); (iii) silver(I) acetate, H2O (0.045% TFA); (iv) biotinylated peptide bearing an oxyamine nucleophile H2NOCH2CO-GIGGIRK(Biotin), sodium acetate buffer, pH 4.5. (C) C18 analytical HPLC and mass analysis of peptide 12 treated by silver acetate after 20 min. Peak a corresponds to the starting material 12 with the observed mass 1346.74 Da, calcd 1346.60 Da, and peak b corresponds to the product 13 with the observed mass 1287.7 Da, calcd 1287.47 Da. (D) C18 analytical HPLC and mass monitoring of oxime ligation between peptide 13 and H2NOCH2CO-GIGGIRK(Biotin). Peak b corresponds to the peptide 13, peak c corresponds to the peptide H2NOCH2CO-GIGGIRK(Biotin) with observed mass 998.69 Da, calcd 998.20 Da, and peak d corresponds to the ligated product 14 with the observed mass 2267.1 Da, calcd 2267.66 Da.

proteins in response to the in-frame amber stop codon located in the mRNA.28 These pairs are orthogonal to endogenous tRNAs and aminoacyl-tRNA synthetases in Escherichia coli, yeast, and mammalian cells.29−32 Wild type PylRS/pylT pairs and their artificially evolved mutants have become an outstanding genetic code expansion tool to incorporate many unnatural amino acids, such as 2 and 3 (Scheme 1A), into proteins.21,33 In our previous study, the genetically incorporated 2 allowed us to site-specifically modify proteins via thiazolidine ring formation under mild conditions.21 In the presence of a large excess of methoxylamine in the buffer, the thiazolidine ring of 2 can be deprotected efficiently to release the 1,2aminothiol functionality while the formaldehyde is quenched by the methoxylamine. Inspired by this work, we envision that the 2-N-alkylcarboxamide-1,3-thiazolidine moiety in the unnatural amino acid 4 may be a novel useful protected aldehyde handle for site-specific protein modification. If 4 could be incorporated into a protein, then the deprotection of the thiazolidine ring will allow us to introduce a unique aldehyde group into a protein at a predetermined site. Finally, the protein could be further modified via oxime ligation with oxyamine-functionalized labeling reagents (Scheme 1B).34 To investigate the feasibility of introducing the α-oxo aldehyde into proteins through a thiazolidine-protected derivative, an initial model study was undertaken on a small peptide. The new amino acid derivative 11 with the masked aldehyde was synthesized according to the procedure shown in Figure 1A. Starting from the glyoxylic acid 5, the thiazolidine-2carboxylic acid 7 was prepared using known literature procedures.35 The secondary amine of 7 was protected with Boc to afford 8. (S)-Fmoc-Lys-O-allyl 9 was coupled with 8 to give 10, followed by deprotection of O-allyl group with

palladium (0) and phenylsilane to afford the desired product 11. Previous work showed that phenylsilane can work as a mild coupling reagent for amidation reaction,36 so after palladiummediated O-allyl deprotection of 10 and subsequent removal of palladium through filtration, 11 in the deprotection reaction mixture was directly loaded onto Rink amide resin using PyBOP as coupling reagent. Following standard Fmoc SPPS, 11 was successfully added at the C-terminal side of a model peptide (ACILGHSDWC), which is a reported HIV integrase inhibitor,37 and peptide 12 was obtained after final cleavage with TFA for 1 h (Figure 1B, step ii). Brik and co-workers reported that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine.38 Encouraged by this finding, we wondered if other heavy metal ions could be used to deprotect thiazolidine in aqueous solution. Silver ion was explored in our study. C18 analytical HPLC and electrospray ionization mass spectrometry (ESIMS) analysis showed that about 80% peptide 12 could be converted to the desired peptide 13 (Figure 1C) within 20 min B

DOI: 10.1021/acs.bioconjchem.6b00667 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry under the tested conditions: 100 μM peptide 12 and 500 μM silver acetate in H2O containing 0.045% TFA. To further test the existence of the α-oxo aldehyde functional group on peptide 13, a biotinylated peptide bearing an oxyamine nucleophile was synthesized (Supporting Information). Dawson and co-workers reported that aniline could serve as excellent catalyst to speed up the oxime ligation under acidic and neutral pH condition.39 Therefore, imine formation was performed in the sodium acetate buffer (pH 4.5, 10 mM aniline) in which 150 μM oxyamine-functionalized peptide and 50 μM peptide 13 was mixed together for 2 h. C18 analytical HPLC result showed that about 85% peptide 13 was converted to the ligated product 14. These results show that the newly designed amino acid building block 11 is fully compatible with Fmoc-based SPPS and can be efficiently deprotected using silver acetate in the acidic solution. Here we also propose a mechanism for deprotection of the thiazolidine ring to unmask aldehyde by silver acetate (Scheme 2). The attack on the sulfur atom of thiazolidine by silver ion Scheme 2. Proposed Mechanism for Ag+-Mediated Thiazolidine Ring Deprotection

Figure 2. (A) Chemical synthesis of 4. (i) 8, EDC, DMAP, DCM, 90%; (ii) TFA. (B) SDS-PAGE analysis of the expression of ubiquitin in the absence or presence of 4. (C) ESI-MS analysis of ubiquitin 17 with the observed mass 9503.2 Da, calcd 9502.91 Da, and ubiquitin 18 with the observed mass 9461.7 Da, calcd mass of hydrated form of aldehyde 9461.78 Da.

structural similarity of the two amino acids. This indicates that subtle structural changes in an amino acid substrate can have profound effects on its ability to be recognized by PylRS and hence its incorporation efficiency. The final yield of ubiquitin 17 incorporating 4 at position 63 is about 1.5−2 mg/L after purification by Ni-NTA chromatography. Because 4 has two isomers as confirmed by NMR analysis (Supporting Information), it is not clear which isomer was recognized by the PylRS/ pylT pair in our present study. Interestingly, a previous study showed that the isomer of 3 (i.e., L-prolyl-lysine) was not a substrate for the wild type PylRS/pylT pair, indicating that the C-5 stereocenter in the ring of 1 was very important for substrate recognition by the synthetase.33 So, it is possible that the equivalent configuration of 4 may be recognized by the synthetase. To reveal the aldehyde functional group in the ubiquitin 17, the protein was dissolved in 10% acetic acid aqueous solution and treated with 50 equiv of silver acetate for 30 min. Quantitative conversion of ubiquitin 17 into 18 was confirmed by ESI-MS analysis (Figure 2C). Then, to further confirm that ubiquitin 18 carried the aldehyde functional group, 50 μM ubiquitin 18 obtained above was used directly for reaction with 250 μM biotinylated peptide carrying an oxyamine nucleophile in the phosphate buffer (pH 7) containing 100 mM aniline for 2.5 h (Figure 3A). The reaction was completed and confirmed by the ESI-MS analysis (Figure 3B). The biotinylated ubiquitin 19 was also confirmed by Western blot using avidin-HRP conjugate (Figure 3C). These results showed that the novel unnatural amino acid 4 could be efficiently recognized by the wild type PylRS/pylT pair and serve as a useful handle for introduction of an α-oxo aldehyde into proteins for site-specific modification. In summary, we have presented a novel method to incorporate a newly designed protected α-oxo aldehyde into peptides and proteins using thiazolidine-2-carboxyl derivatives. This method allows us to introduce an alkyl aldehyde, which

first causes C−S bond rupture and ring opening to form intermediate III. Then the immonium intermediate III quickly undergoes hydrolysis to release the aldehyde IV. Next, to test whether the aldehyde can be installed into a protein using genetic code expansion technology, 4 was prepared in a similar way (Figure 2A). (S)-Boc-Lys-OMe 15 was coupled with 8 followed by deprotection of Boc group with TFA to give the required 4 which was used directly for incorporation experiments since the ester would be hydrolyzed inside the cell.40 Given the structural similarity between 2, 3, and 4, we anticipated that the PylRS/pylT pair that can recognize 2 or 3 may also accept 4 as its substrate. So we tested the incorporation efficiency of 4 into proteins using Methanosarcina barkeri (Mb) mutant and wild type PylRS/ pylT pairs, which can recognize 2 and 3, respectively.21,33,41 E. coli BL21 (DE3) cells that harbored two different plasmids, pEVOL-pylT-ThzKRS or pEVOL-pylT-PylRS and pETDuetUbiK63TAG, were employed for the investigation. Plasmids pEVOL-pylT-ThzKRS and pEVOL-pylT-PylRS contain genes coding mutant and wild type PylRS/pylT pairs, respectively. Plasmid pETDuet-UbiK63TAG carries a codon-optimized ubiquitin protein gene with an amber mutation at its K63 position. The transformed E. coli cells were cultured in LB medium to reach OD = 0.6 then supplemented with 5 mM 4 and 1 mM IPTG to induce for 8 h at 37 °C. Interestingly, our results showed that 4 can only be efficiently recognized by wild type PylRS/pylT pair and its incorporation into ubiquitin was confirmed by the SDS-PAGE and ESI-MS analysis (Figure 2B and C). The finding that 4 is not recognized by the same mutant PylRS that recognizes 2 is surprising given the high C

DOI: 10.1021/acs.bioconjchem.6b00667 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00667. Materials and reagents; Detailed procedure for the chemical synthesis of compounds and peptide; Protein thiazolidine deprotection with Ag(OAc); NMR profile (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: cfl[email protected]. Fax: +65 67953021. ORCID

Chuan-Fa Liu: 0000-0001-7433-2081 Notes

The authors declare no competing financial interest.

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Figure 3. (A) Strategy for the site-specific modification of ubiquitin 18 via oxime ligation with oxyamine-functionalized labeling reagent. (B) ESI-MS analysis of ubiquitin 19 with the observed mass 10 424 Da, calcd 10 423.97 Da. (C) SDS-PAGE (up panel) and Western blot analysis (down panel) of ubiquitin 18 and 19.

ACKNOWLEDGMENTS This work is supported by the A*STAR of Singapore (ETPLQP-19-06 to CFL) and Nanyang Technological University. Dr Pasunooti was partly supported by a Tier 1 complexity grant (RGC2/14 to JL).

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has a higher reactivity than the reported benzaldehyde,18 at any position of a synthetic peptide or recombinant protein. We first found that silver ion is an excellent reagent to deprotect thiazolidine ring to release the aldehyde under mild conditions, which is fully compatible with the side-chain functional groups of all natural amino acids. The newly synthesized building block 11 will be a useful reagent for site-specific installation of an aldehyde group into synthetic peptides using standard Fmocbased SPPS in future applications. The functionalization of a protein with an aldehyde through a genetically incorporated unnatural amino acid at an internal site will have minimal effects on the overall structure and function of the protein compared with the previously reported enzymatic method.17 The choice of thiazolidine for protecting the highly reactive αoxo aldehyde also avoids the potential side reaction with other aminothiol-containing compounds after its cellular uptake in living organisms and the novel unnatural amino acid 4 is not toxic to the E. coli cells at relatively high concentrations (5 mM in the current study). An aldehyde group is a useful handle not only for oxime ligation but also for thiazolidine ligation and hydrazone ligation as shown in previous studies.42−47 Combining these robust bioconjugation chemistries with our current method will provide new opportunities to choose a proper site for peptide and protein modification in novel applications. Therefore, our current study further expands the available repertoire for peptide and protein modification using aldehyde-based bioconjugation chemistry. Nevertheless, a limitation of this method is that the acidic solution that contains an excess of silver ion for thiazolidine deprotection will cause protein denaturation and a refolding step will be needed for certain studies. A recent study shows that palladium complexes can be used to rescue protein activities in living cell contexts.48 So, our future work will be directed to screening stable and water-soluble metal complexes for the deprotection of thiazolidine ring at neutral pH condition, which can be used on proteins under their native states. Overall, our method adds to the exciting latest developments on chemical and enzymatic bioconjugation methods for peptide and protein manipulation.49−51

ABBREVIATIONS BOC, tert-butyloxycarbonyl; Fmoc, fluorenylmethyloxycarbonyl; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid; EDC, 1-ethyl3-(3-(dimethylamino)propyl)carbodiimide; DMAP, 4-dimethylaminopyridine; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; TIS, triisopropylsilane

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REFERENCES

(1) Spicer, C. D., and Davis, B. G. (2014) Selective chemical protein modification. Nat. Commun. 5, 4740. (2) Wang, P., Zhang, S., Meng, Q., Liu, Y., Shang, L., and Yin, Z. (2015) Site-specific chemical modification of peptide and protein by thiazolidinediones. Org. Lett. 17, 1361−1364. (3) Gentilucci, L., De Marco, R., and Cerisoli, L. (2010) Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Curr. Pharm. Des. 16, 3185−3203. (4) Yang, R., Bi, X., Li, F., Cao, Y., and Liu, C. F. (2014) Native chemical ubiquitination using a genetically incorporated azidonorleucine. Chem. Commun. 50, 7971−7974. (5) Bi, X., Yang, R., Feng, X., Rhodes, D., and Liu, C. F. (2016) Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes. Org. Biomol. Chem. 14, 835−839. (6) Baslé, E., Joubert, N., and Pucheault, M. (2010) Protein chemical modification on endogenous amino acids. Chem. Biol. 17, 213−227. (7) Boutureira, O., and Bernardes, G. J. L. (2015) Advances in chemical protein modification. Chem. Rev. 115, 2174−2195. (8) Müller, M. M., and Muir, T. W. (2015) Histones: at the crossroads of peptide and protein chemistry. Chem. Rev. 115, 2296− 2349. (9) Gauthier, M. A., and Klok, H. A. (2008) Peptide/protein− polymer conjugates: synthetic strategies and design concepts. Chem. Commun., 2591−2611. (10) Lau, Y. H., de Andrade, P., Wu, Y., and Spring, D. R. (2015) Peptide stapling techniques based on different macrocyclisation chemistries. Chem. Soc. Rev. 44, 91−102. (11) Chen, X., and Wu, Y. W. (2016) Selective chemical labeling of proteins. Org. Biomol. Chem. 14, 5417−5439.

D

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Communication

Bioconjugate Chemistry (12) Lang, K., and Chin, J. W. (2014) Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764−4806. (13) Yang, M., Li, J., and Chen, P. R. (2014) Transition metalmediated bioorthogonal protein chemistry in living cells. Chem. Soc. Rev. 43, 6511−6526. (14) Li, X., Fekner, T., Ottesen, J. J., and Chan, M. K. (2009) A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem., Int. Ed. 48, 9184−9187. (15) Lang, K., and Chin, J. W. (2014) Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16−20. (16) Spears, R. J., and Fascione, M. A. (2016) Site-selective incorporation and ligation of protein aldehydes. Org. Biomol. Chem. 14, 7622−7638. (17) Carrico, I. S., Carlson, B. L., and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat. Chem. Biol. 3, 321−322. (18) Tuley, A., Lee, Y. J., Wu, B., Wang, Z. U., and Liu, W. R. (2014) A genetically encoded aldehyde for rapid protein labeling. Chem. Commun. 50, 7424−7426. (19) Geoghegan, K. F., and Stroh, J. G. (1992) Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. Bioconjugate Chem. 3, 138−146. (20) Zhang, L., and Tam, J. P. (1996) Thiazolidine formation as a general and site-specific conjugation method for synthetic peptides and proteins. Anal. Biochem. 233, 87−93. (21) Bi, X., Pasunooti, K. K., Tareq, A. H., Takyi-Williams, J., and Liu, C. F. (2016) Genetic incorporation of 1,2-aminothiol functionality for site-specific protein modification via thiazolidine formation. Org. Biomol. Chem. 14, 5282−5285. (22) Liu, B., Burdine, L., and Kodadek, T. (2006) Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins. J. Am. Chem. Soc. 128, 15228−15235. (23) El-Mahdi, O., and Melnyk, O. (2013) α-Oxo Aldehyde or glyoxylyl group chemistry in peptide bioconjugation. Bioconjugate Chem. 24, 735−765. (24) Shao, J., and Tam, J. P. (1995) Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone, and thiazolidine linkages. J. Am. Chem. Soc. 117, 3893−3899. (25) Qasmi, D., René, L., and Badet, B. (1994) Synthesis of “cho-copeptides” by n-terminal acylation with a glyoxylyl equivalent. Tetrahedron Lett. 35, 4343−4344. (26) Far, S., and Melnyk, O. (2004) A novel a,a′-diaminoacetic acid derivative for the introduction of the alpha-oxo aldehyde functionality into peptides. Tetrahedron Lett. 45, 1271−1273. (27) Far, S., and Melnyk, O. (2005) Synthesis of glyoxylyl peptides using an Fmoc-protected a,a′-diaminoacetic acid derivative. J. Pept. Sci. 11, 424−430. (28) Dumas, A., Lercher, L., Spicer, C. D., and Davis, B. G. (2015) Designing logical codon reassignment-expanding the chemistry in biology. Chem. Sci. 6, 50−69. (29) Mukai, T., Kobayashi, T., Hino, N., Yanagisawa, T., Sakamoto, K., and Yokoyama, S. (2008) Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818−822. (30) Neumann, H., Peak-Chew, S. Y., and Chin, J. W. (2008) Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232−234. (31) Hancock, S. M., Uprety, R., Deiters, A., and Chin, J. W. (2010) Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a Pyrrolysyl-tRNA synthetase/trna pair. J. Am. Chem. Soc. 132, 14819−14824. (32) Wan, W., Tharp, J. M., and Liu, W. R. (2014) Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta, Proteins Proteomics 1844, 1059− 1070.

(33) Polycarpo, C. R., Herring, S., Bérubé, A., Wood, J. L., Söll, D., and Ambrogelly, A. (2006) Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 580, 6695−6700. (34) Rose, K. (1994) Facile synthesis of homogeneous artificial proteins. J. Am. Chem. Soc. 116, 30−33. (35) Engel, W., and Schieberle, P. (2002) Identification and quantitation of key aroma compounds formed in maillard-type reactions of fructose with cysteamine or isothiaproline (1,3thiazolidine-2-carboxylic acid). J. Agric. Food Chem. 50, 5394−5399. (36) Ruan, Z., Lawrence, R. M., and Cooper, C. B. (2006) Phenylsilane as an active amidation reagent for the preparation of carboxamides and peptides. Tetrahedron Lett. 47, 7649−7651. (37) Desimmie, B. A., Humbert, M., Lescrinier, E., Hendrix, J., Vets, S., Gijsbers, R., Ruprecht, R. M., Dietrich, U., Debyser, Z., and Christ, F. (2012) Phage display-directed discovery of LEDGF/p75 binding cyclic peptide inhibitors of HIV replication. Mol. Ther. 20, 2064−2075. (38) Jbara, M., Maity, S. K., Seenaiah, M., and Brik, A. (2016) Palladium mediated rapid deprotection of n-terminal cysteine under native chemical ligation conditions for the efficient preparation of synthetically challenging proteins. J. Am. Chem. Soc. 138, 5069−5075. (39) Dirksen, A., Hackeng, T. M., and Dawson, P. E. (2006) Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Angew. Chem., Int. Ed. 45, 7581−7584. (40) Liu, Q. Y. (2014) Synthesis of Photo/Chemical-Activated Nucleosides and Unnatural Amino Acids, PhD thesis, University of Pittsburgh, Chapter 6, p 180. (41) Nguyen, D. P., Elliott, T., Holt, M., Muir, T. W., and Chin, J. W. (2011) Genetically encoded 1,2-aminothiols facilitate rapid and sitespecific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J. Am. Chem. Soc. 133, 11418−11421. (42) Liu, C. F., and Tam, J. P. (1994) Chemical ligation approach to form a peptide bond between unprotected peptide segments. concept and model Study. J. Am. Chem. Soc. 116, 4149−4153. (43) Liu, C. F., and Tam, J. P. (1994) Peptide segment ligation strategy without use of protecting groups. Proc. Natl. Acad. Sci. U. S. A. 91, 6584−6588. (44) Rao, C., and Tam, J. P. (1994) Synthesis of peptide dendrimer. J. Am. Chem. Soc. 116, 6975−6976. (45) King, T. P., Zhao, S. W., and Lam, T. (1986) Preparation of protein conjugates via intermolecular hydrazone linkage. Biochemistry 25, 5774−5779. (46) Dirksen, A., Dirksen, S., Hackeng, T. M., and Dawson, P. E. (2006) Nucleophilic Catalysis of Hydrazone Formation and Transimination: Implications for Dynamic Covalent Chemistry. J. Am. Chem. Soc. 128, 15602−15603. (47) Xu, Y., Xu, L., Xia, Y., Guan, C. J., Guo, Q. X., Fu, Y., Wang, C., and Li, Y. M. (2015) Fast and catalyst-free hydrazone ligation via ortho-halo-substituted benzaldehydes for protein C-terminal labeling at neutral pH. Chem. Commun. 51, 13189−13192. (48) Li, J., Yu, J., Zhao, J., Wang, J., Zheng, S., Lin, S., Chen, L., Yang, M., Jia, S., Zhang, X., et al. (2014) Palladium-triggered deprotection chemistry for protein activation in living cells. Nat. Chem. 6, 352−361. (49) Wright, T. H., Bower, B. J., Chalker, J. M., Bernardes, G. J., Wiewiora, R., Ng, W. L., Raj, R., Faulkner, S., Vallée, M. R. J., Phanumartwiwath, A., et al. (2016) Posttranslational mutagenesis: a Chemical strategy for exploring protein side-chain diversity. Science 354, aag1465−1−aag1465−11. (50) Nguyen, G. K.T., Qiu, Y., Cao, Y., Hemu, X., Liu, C. F., and Tam, J. P. (2016) Butelase-mediated cyclization and ligation of peptides and proteins. Nat. Protoc. 11, 1977−1988. (51) Cao, Y., Nguyen, G. K. T., Chuah, S., Tam, J. P., and Liu, C. F. (2016) Butelase-mediated ligation as an efficient bioconjugation method for the synthesis of peptide dendrimers. Bioconjugate Chem. 27, 2592−2596.

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