Letter Cite This: Org. Lett. 2018, 20, 6526−6529
pubs.acs.org/OrgLett
N‑Methyl‑N‑phenylvinylsulfonamides for Cysteine-Selective Conjugation Rong Huang, Zhihong Li, Yao Sheng, Jianghui Yu, Yue Wu, Yuexiong Zhan, Hongli Chen,* and Biao Jiang* Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong, Shanghai 201210, China
Org. Lett. 2018.20:6526-6529. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/19/18. For personal use only.
S Supporting Information *
ABSTRACT: Use of N-methyl-N-phenylvinylsulfonamides to perform chemoselective modification of cysteine-containing peptides and proteins is reported. Probes linked to the drug were applicable to prepare antibody−drug conjugates (ADCs). The drug−antibody ratio for ADCs was controlled by rationally tuning the electron deficiency and linker hydrophilicity of the probes.
S
selectivity to different nucleophile residues. Herein, we apply vinylsulfonamides for development of an optimal cysteine modification method and explore its utilization in antibody conjugation. We previously reported a series of vinylsulfonamides to screen reagents for amino-selective modification.26b In this study, we first examined the use of those vinylsulfonamides for cysteine bioconjugation in a model peptide 1, which contained a free Nterminus and a variety of nucleophilic amino acid residues (lysine, tyrosine, histidine, serine, threonine, arginine, and glutamine) (Figure 1A). We found that N-methyl-N-phenylvinylsulfonamide (2), which was demonstrated as a lysineresidue-selective reagent in alkaline conditions in our previous work (pH >9 and in the presence of trimethylamine (Et3N) or 1,8-diazabicyclo[5.4.0]undec-7-ene),26a reacted with 1 efficiently and cleanly in PBS (pH 7.4) and CH3CN (2:1) at room temperature to afford a single product 3. It was assigned as a monomodified adduct based on the mass peak of m/z 963.4372 [M + 2H]2+, 642.6270 [M + 3H]3+, and 482.2225 [M + 4H]4+ (Figure 1B). The reaction was accelerated and the chemoselectivity was not affected by increasing the stoichiometry of 2 to 10 equiv (Figure S1). After the cysteine residue of 1 was captured by Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)), the corresponding product did not react with 2 under the same conditions as above. Also, modification for peptide 4, in which the cysteine of peptide 1 was mutated to alanine, was not observed. This suggests conjugation occurs selectively for cysteine over other potentially nucleophilic residues in 1 using vinylsulfonamide 2.
ite-selective protein modification plays an important role in the development of technologies for pharmaceutical and biological studies.1 A large number of methods have emerged for precise protein bioconjugation with natural and unnatural amino acids.2 Cysteine-based strategies have continued to attract much attention due to the unique reactivity of the sulfhydryl group and the low abundance of cysteine in natural proteins.3 Recently, site-directed mutagenesis to genetically install cysteine residues for proteins lacking them have been developed, allowing cysteine-selective approaches to amplify application for a wide range of proteins. Therefore, numerous electrophiles have been and continue to be designed to probe cysteine residues, such as maleimides,4 fluorobenzene,5 benzylic substitution,6 cyclopropenyl ketones,7 arenediazonium salts,8 5methylenpyrrolones,9 tetrafluoroethylation reagents,10 oxetanes,11 2-azidoacrylates,12 naphthalimide scaffolds,13 carbonylacrylic reagents,14 arylpropiolonitriles,15 allenamides,16 sulfone derivatives,17 azidopropylvinylsulfonamide,18 electrondeficient alkynes,19 chlorotetrazines,20 and palladium-mediated reagents.21 Michael addition of cysteine residues to maleimides is one of the most broadly used strategies, and important applications include the preparation of antibody−drug conjugates (ADCs).22 However, recent work has demonstrated that thio-maleimide conjugates are prone to retro-Michael additions and breakdown via thiol exchange reactions.23 Vinylsulfonamide-mediated Michael addition has emerged as an attractive strategy in the field of protein modification due to their increased stability and lack of byproducts.24 We have been studying vinylsulfonamides-based methods that can be employed for disulfide stapling 25 and selective amino modification in peptides and proteins,26 and we have demonstrated that rationally tuning the structure of probes and optimizing the reaction conditions can achieve good © 2018 American Chemical Society
Received: September 6, 2018 Published: October 4, 2018 6526
DOI: 10.1021/acs.orglett.8b02849 Org. Lett. 2018, 20, 6526−6529
Letter
Organic Letters
Figure 3. Selective modification of BSA with probe 5. (A) Reaction scheme. (B) MS and MS2 spectra of product 16. Figure 1. Selective cysteine modification. (A) Compound 2-mediated peptide modification. (B) Mass spectrum for modified peptide 3.
Figure 2. Selective incorporation of the functionalized probe 5 on peptide 1 for further conjugation with biologically relevant groups (7− 10).
Figure 4. Modification of trastuzumab and reduced trastuzumab. (A) Reaction scheme and structures of probes 18−20. (B) SDS-PAGE analysis. Lane 1: trastuzumab−18 conjugate. Lane 2: unmodified trastuzumab. Lane 3: negative control, trastuzumab (without free thiol groups). Left gel: fluorescence. Right gel: Coomassie staining.
LC-MS2 data clearly confirmed that the cysteine residue was selectively modified (Figure S2). A functionalized probe 5 containing a terminal alkyne group was synthesized for further bioconjugation via click chemistry (Scheme S1). Presence of the para-alkyne group in the phenyl had no effect on the selective modification of 1, which was labeled with 5 to generate a single alkyne-containing probe 6. Compound 6 can be further conjugated with a variety of important tags (7−10), including fluorescent probes (11, 12), a drug molecule (13), and a PEG polymer (14) (Figure 2). Products 11−14 were confirmed by LC-MS (Figure S3). We next evaluated the selective capability of 5 with a native protein, BSA (15) (Figure 3A), which has one free cysteine
residue (Cys34). Compound 5 (10 equiv) was added to a solution of BSA in PBS (pH 7.4), and then the mixture was incubated for 5 h at 37 °C. ESI-MS analysis showed that the main peak appeared at m/z 66690 Da. This indicated that monomodified BSA 16 was produced. After trypsin digestion, the modification site of 16 was determined as Cys34 based on the fragments in LC-MS2 (Figure 3B). CD spectra showed that the secondary structure of the modified BSA was retained (Figure S4). 6527
DOI: 10.1021/acs.orglett.8b02849 Org. Lett. 2018, 20, 6526−6529
Letter
Organic Letters
Figure 5. SDS-PAGE gels showing the stability of the fluorescent trastuzumab conjugate 22, (A) in pH 7.4 PBS buffer at room temperature, (B) with 100 equiv of GSH at room temperature, (C) in pH 7.4 PBS buffer at 37 °C, (D) with 100 equiv of GSH at 37 °C, (E) in human plasma at 37 °C. Top gels: fluorescence. Bottom gels: Coomassie staining.
Figure 7. Assessment of binding and internalization of the ADCs. (A) Binding of trastuzumab 17 and ADCs 31 and 32 on SK-BR-3 cells and MCF-7 cells as analyzed by flow cytometry. (B) Endocytosis of trastuzumab 17 and ADCs 31 and 32 in SK-BR-3 cells.
the wild-type antibody (Figure 4B). These results confirm high selectivity of the conjugation toward free cysteine residues. LCMS analysis was used to examine the coupling efficiency. The deconvoluted mass spectrum indicated, on average, 0.98 fluorophores were attached to the antibody (FAR = 0.98) (Figure S5). To improve the conjugation efficiency, 19, in which a para electron-withdrawing group and amide were introduced to phenyl instead of oxygen in 5, was designed and synthesized. A fluorescent moiety was installed on 19 to obtain probe 20 (Scheme S3). The reduced and wild-type trastuzumab were treated with 20 under the same conditions as listed above. SDSPAGE data indicated that enhanced fluorescent signals were found for the reduced antibody, and still no fluorescence signal appeared for the wild-type antibody (Figure S6). The ESI-MS spectrum confirmed more fluorophores were conjugated to the antibody (FAR = 2.40) (Figure S7). The stability of the fluorescent trastuzumab conjugate (22) was examined by incubating it in pH 7.4 PBS buffer for 72 h at room temperature and 37 °C. We observed that the fluorescence intensity remained almost constant as detected by SDS-PAGE at different incubation times (Figure 5A,C). When it was treated with 100 equiv of natural thiol nucleophile glutathione (GSH) at room temperature, with increased incubation temperature to 37 °C, no significant thiol exchange was observed, and it still maintained complete resilience to degradation (Figure 5B,D). The conjugate also showed superior stability when it was incubated in human plasma for 72 h at 37 °C (Figure 5E). Having observed the superior stability of 22, we next evaluated the application of the developed method on ADCs. The azide derivatives of monomethyl auristatin E 23 and 24 (Scheme S4) were reacted with compound 5 or 19, respectively, via azide−alkyne cycloaddition to afford drug linkers 25−28 (Schemes S5 and S6). Reduced trastuzumab was treated by 25− 28 in PBS buffer (pH 7.4) at 37 °C for 12 h to produce ADCs 29−32 with different drug−antibody ratios (DAR) (Figure 6). Consistent with the previous results, 19 attached the drug to the antibody more efficiently with significantly improved DAR compared to that with compound 5. In parallel, we explored the effect of linkers by increasing the hydrophilic coupling efficiency.
Figure 6. Synthesis of ADCs 29−32 with drug linkers 25−28. (A) Reaction scheme and structure of compounds 23−28. (B) DAR for ADCs 29−32.
Encouraged by these results, we further explored the application of this method to modify an antibody (trastuzumab, 17). First, a fluorescent derivative 18 was prepared (Scheme S2). Trastuzumab 17 was treated with tris(2-carboxyethyl)phosphine to reduce the disulfide bonds, releasing eight free cysteine residues. Both the reduced and wild-type antibodies were incubated with 18 (10 equiv) in PBS (pH 7.4) at room temperature for 24 h (Figure 4A). After filtration by a spindesalting column, SDS-PAGE analysis showed that the fluorescent bands (heavy chain and light chain) appeared for the reduced antibody, and no fluorescent labeling occurred for 6528
DOI: 10.1021/acs.orglett.8b02849 Org. Lett. 2018, 20, 6526−6529
Letter
Organic Letters
(7) Smith, N. J.; Rohlfing, K.; Sawicki, L. A.; Kharkar, P. M.; Boyd, S. J.; Kloxin, A. M.; Fox, J. M. Org. Biomol. Chem. 2018, 16, 2164−2169. (8) Naveen, N.; Sengupta, S.; Chandrasekaran, S. J. Org. Chem. 2018, 83, 3562−3569. (9) Zhang, Y.; Zhou, X.; Xie, Y.; Greenberg, M. M.; Xi, Z.; Zhou, C. J. Am. Chem. Soc. 2017, 139, 6146−6151. (10) Vaclavik, J.; Zschoche, R.; Klimankova, I.; Matousek, V.; Beier, P.; Hilvert, D.; Togni, A. Chem. - Eur. J. 2017, 23, 6490−6494. (11) Boutureira, O.; Martinez-Saez, N.; Brindle, K. M.; Neves, A. A.; Corzana, F.; Bernardes, G. J. L. Chem. - Eur. J. 2017, 23, 6483−6489. (12) Ariyasu, S.; Hayashi, H.; Xing, B.; Chiba, S. Bioconjugate Chem. 2017, 28, 897−902. (13) Zhou, P.; Yao, J.; Hu, G.; Fang, J. ACS Chem. Biol. 2016, 11, 1098−1105. (14) Bernardim, B.; Cal, P. M.; Matos, M. J.; Oliveira, B. L.; MartinezSaez, N.; Albuquerque, I. S.; Perkins, E.; Corzana, F.; Burtoloso, A. C.; Jimenez-Oses, G.; Bernardes, G. J. Nat. Commun. 2016, 7, 13128. (15) (a) Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J. S.; Strub, J. M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Bioconjugate Chem. 2014, 25, 202−206. (b) Kolodych, S.; Koniev, O.; Baatarkhuu, Z.; Bonnefoy, J. Y.; Debaene, F.; Cianferani, S.; Van Dorsselaer, A.; Wagner, A. Bioconjugate Chem. 2015, 26, 197−200. (16) Abbas, A.; Xing, B.; Loh, T. P. Angew. Chem., Int. Ed. 2014, 53, 7491−7494. (17) (a) Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R., Jr.; Xian, M. Org. Lett. 2012, 14, 3396−3369. (b) Toda, N.; Asano, S.; Barbas, C. F., 3rd Angew. Chem., Int. Ed. 2013, 52, 12592−12596. (18) Dadova, J.; Vrabel, M.; Adamik, M.; Brazdova, M.; Pohl, R.; Fojta, M.; Hocek, M. Chem. - Eur. J. 2015, 21, 16091−16102. (19) Shiu, H. Y.; Chan, T. C.; Ho, C. M.; Liu, Y.; Wong, M. K.; Che, C. M. Chem. - Eur. J. 2009, 15, 3839−3850. (20) Canovas, C.; Moreau, M.; Bernhard, C.; Oudot, A.; Guillemin, M.; Denat, F.; Goncalves, V. Angew. Chem., Int. Ed. 2018, 57, 10646− 10650. (21) (a) Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L.; Buchwald, S. L. Nature 2015, 526, 687−691. (b) Al-Shuaeeb, R. A.; Kolodych, S.; Koniev, O.; Delacroix, S.; Erb, S.; Nicolay, S.; Cintrat, J. C.; Brion, J. D.; Cianferani, S.; Alami, M.; Wagner, A.; Messaoudi, S. Chem. - Eur. J. 2016, 22, 11365−11370. (c) Zhang, C.; Welborn, M.; Zhu, T.; Yang, N. J.; Santos, M. S.; Van Voorhis, T.; Pentelute, B. L. Nat. Chem. 2016, 8, 120−128. (d) Lian, Z.; Bhawal, B. N.; Yu, P.; Morandi, B. Science 2017, 356, 1059−1063. (e) Rojas, A. J.; Pentelute, B. L.; Buchwald, S. L. Org. Lett. 2017, 19, 4263−4266. (22) (a) Cal, P. M.; Bernardes, G. J.; Gois, P. M. Angew. Chem., Int. Ed. 2014, 53, 10585−10587. (b) Rodrigues, T.; Bernardes, G. J. L. Angew. Chem., Int. Ed. 2018, 57, 2032−2034. (c) Joubert, N.; DenevaultSabourin, C.; Bryden, F.; Viaud-Massuard, M. C. Eur. J. Med. Chem. 2017, 142, 393−415. (23) (a) Lyon, R. P.; Setter, J. R.; Bovee, T. D.; Doronina, S. O.; Hunter, J. H.; Anderson, M. E.; Balasubramanian, C. L.; Duniho, S. M.; Leiske, C. I.; Li, F.; Senter, P. D. Nat. Biotechnol. 2014, 32, 1059−1062. (b) Kalia, D.; Malekar, P. V.; Parthasarathy, M. Angew. Chem., Int. Ed. 2016, 55, 1432−1435. (24) (a) Dadova, J.; Orsag, P.; Pohl, R.; Brazdova, M.; Fojta, M.; Hocek, M. Angew. Chem., Int. Ed. 2013, 52, 10515−10518. (b) Suh, E. H.; Liu, Y.; Connelly, S.; Genereux, J. C.; Wilson, I. A.; Kelly, J. W. J. Am. Chem. Soc. 2013, 135, 17869−17880. (c) Furman, J. L.; Kang, M.; Choi, S.; Cao, Y.; Wold, E. D.; Sun, S. B.; Smider, V. V.; Schultz, P. G.; Kim, C. H. J. Am. Chem. Soc. 2014, 136, 8411−8417. (25) Li, Z.; Huang, R.; Xu, H.; Chen, J.; Zhan, Y.; Zhou, X.; Chen, H.; Jiang, B. Org. Lett. 2017, 19, 4972−4975. (26) (a) Chen, H.; Huang, R.; Li, Z.; Zhu, W.; Chen, J.; Zhan, Y.; Jiang, B. Org. Biomol. Chem. 2017, 15, 7339−7345. (b) Huang, R.; Li, Z. H.; Ren, P. L.; Chen, W. Z.; Kuang, Y. Y.; Chen, J. K.; Zhan, Y. X.; Chen, H. L.; Jiang, B. Eur. J. Org. Chem. 2018, 2018, 829−836.
Not surprisingly, along with the increased hydrophilicity, DAR was enhanced. Furthermore, we confirmed that ADCs 31 and 32 retained excellent specificity and binding affinity to the Her2 antigen in SKBR3 (HER2+) cells, and weak binding was detected in MCF 7 cells (HER2−), as demonstrated by flow cytometry analysis (Figure 7A). We also evaluated the internalization of ADCs 31 and 32 by flow cytometry. The results demonstrated that 31 and 32 had an efficiency of internalization similar to that of trastuzumab 17 (Figure 7B). We have developed N-methyl-N-phenylvinylsulfonamides as simple and efficient handles for cysteine-selective modification. The method was successfully applied on protein and antibody modification and showed potential advantages in superior linker stability. DAR can significantly affect the efficacy of ADCs, and it is an important attribute used to measure the quality of ADCs. We demonstrated that DAR can be adjusted by rationally tuning the electron deficiency and linker hydrophilicity of vinylsulfonamide probes. We anticipate that the method will be useful for imaging and therapeutic conjugates.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02849. Experimental details, characterization data, and NMR spectra for all new compounds (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Hongli Chen: 0000-0002-9002-2603 Biao Jiang: 0000-0002-4292-7811 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Dr. Wenzhang Chen, Dr. Wei Zhu, Jiankang Chen, Dr. Chunchun Liu, Dr. Lixia Zhao, and Dr. Yan Nie (ShanghaiTech University, SIAIS Analytical Chemistry, Cell Sorting and Protein & Gene Platform) for technical assistance with LC-MS, MS2, flow cytometry, and CD experiments.
■
REFERENCES
(1) (a) Spicer, C. D.; Davis, B. G. Nat. Commun. 2014, 5, 4740. (b) Chen, X.; Wu, Y. W. Org. Biomol. Chem. 2016, 14, 5417−5439. (2) Boutureira, O.; Bernardes, G. J. Chem. Rev. (Washington, DC, U. S.) 2015, 115, 2174−2195. (3) (a) Koniev, O.; Wagner, A. Chem. Soc. Rev. 2015, 44, 5495−5551. (b) Gunnoo, S. B.; Madder, A. ChemBioChem 2016, 17, 529−553. (4) (a) Smith, M. E.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. J. Am. Chem. Soc. 2010, 132, 1960−1965. (b) Hatzakis, N. S.; Engelkamp, H.; Velonia, K.; Hofkens, J.; Christianen, P. C.; Svendsen, A.; Patkar, S. A.; Vind, J.; Maan, J. C.; Rowan, A. E.; Nolte, R. J. Chem. Commun. 2006, 2012−2014. (c) Renault, K.; Fredy, J. W.; Renard, P. Y.; Sabot, C. Bioconjugate Chem. 2018, 29, 2497−2513. (5) Embaby, A. M.; Schoffelen, S.; Kofoed, C.; Meldal, M.; Diness, F. Angew. Chem., Int. Ed. 2018, 57, 8022−8026. (6) Watanabe, K.; Ohshima, T. Chem. - Eur. J. 2018, 24, 3959−3964.
■
NOTE ADDED AFTER ASAP PUBLICATION Figure 4b was corrected on October 4, 2018. 6529
DOI: 10.1021/acs.orglett.8b02849 Org. Lett. 2018, 20, 6526−6529
