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Cite this: Chem. Commun., 2014, 50, 12534
Solid phase chemical ligation employing a rink amide linker for the synthesis of histone H2B protein†
Received 18th August 2014, Accepted 30th August 2014
Muhammad Jbara, Mallikanti Seenaiah and Ashraf Brik*
DOI: 10.1039/c4cc06499b www.rsc.org/chemcomm
Presented here is a solid phase chemical ligation strategy employing native chemical ligation and the commercially available Rink-amide linker as a key element in our approach. The method was applied for the synthesis of histone H2B, which sets the ground for the rapid preparation of posttranslationally modified analogues of this protein.
Solid phase chemical synthesis revolutionized organic synthesis in terms of yield, convenience and speed of process. Solid phase peptide synthesis (SPPS),1 for example, has made it possible, even for non-experts, to rapidly prepare a library of peptides of various lengths (typically of 30–50 amino acids) and compositions. Yet, the assembly of proteins from peptide fragments on a solid support, employing state of the art chemoselective ligation methods, e.g. native chemical ligation (NCL), has not matured to the level of SPPS.2 Success in such a strategy would significantly facilitate protein synthesis, as purification and lyophilization steps, after each ligation, will be eliminated leading to the rapid synthesis of target proteins in high yield. While one pot synthesis could also achieve some of these goals as documented in several excellent examples,3 the attractiveness of solid phase chemical ligation (SPCL) stems from the possibility of performing a higher number of chemical steps and with a greater diversity of chemical transformations. In SPCL an obvious demand, in addition to the high efficiency of the applied chemistry, is the suitability of the polymeric support and the linker, bridging temporally the polypeptide to the support, to the ligation conditions. For example, if NCL is the method of choice, such a support should be water compatible and the linker stable along the synthetic process such as removal of the Cys protecting group in the middle fragments and desulfurization.4 Since the first report by Kent and coworkers on the development and use of SPCL in protein synthesis, employing the base labile linkers and NCL,5 several other studies have attempted to
Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures and analytical data for peptide fragments. See DOI: 10.1039/c4cc06499b
12534 | Chem. Commun., 2014, 50, 12534--12537
devise alternative approaches based on different linkers or ligation chemistries. These include the use of safety catch linkers,6 a peptide sequence that can be cleaved enzymatically7 and His6-Tag8 to facilitate the isolation and handling of intermediate products during the synthetic process. Recently, the use of the alkylsulfonylethyloxycarbamate linker has been revisited, combined with the triazole ligation strategy9 or NCL employing a bis(2-sulfanylethyl)amide thioester surrogate.10 In addition, the Wang linker combined with oxime ligation was also investigated.11 These studies highlight the importance of finding a novel linker, which can be easily introduced and cleaved efficiently to release the final polypeptide product. Here we report that the widely used Rink amide linker in SPPS,12 which is commercially available and can be cleaved under relatively mild acidic conditions, meets these requirements for SPCL as we demonstrate in model peptides and the synthesis of full length histone H2B. Our strategy for SPCL employing the Rink-amide linker is depicted in Scheme 1. PEGA resin, which exhibits excellent swelling properties in water,13 was chosen to serve as a solid support. A spacer of three Ala residues will be included between the resin and the linker. To enable the first ligation, 1,3-thiazolidine-4-carboxo (Thz) will be coupled to the Rink amide linker, which can be quantitatively converted to free Cys using methoxylamine at a pH of 4. Subsequently, NCL can be applied to mediate the assembly of multiple peptides combined with other synthetic steps if required. At the final stage, treatment of the bound polypeptide-resin with TFA would release the peptide from the solid support. This strategy would generate a C-terminal Cys residue (or Ala if desulfurization is applied) instead of the native one, which we believe is a tolerable modification in many proteins and might also serve as a handle for further modifications, e.g. with fluorophores. If desired, one could also introduce a modified Rink amide (e.g. with an alkyne functionality) to the C-terminal of unprotected peptide thioester via direct aminolysis, which would enable its attachment to the resin bearing a complementary functional group (e.g. azide). Although this would generate the polypeptide with the native C-terminal residue,
This journal is © The Royal Society of Chemistry 2014
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Scheme 1
ChemComm
Principles of SPCL based on the Rink-amide linker. Scheme 2
the extra synthetic steps coupled with the less important role of the C-terminal amino acid in the structure and function of proteins made this option less favorable to us. To examine the above described strategy, we first chose a model peptide 1, Thz-LYRAGLYRL, which was prepared using the N-acylurea method developed by Dawson and Blanco.14 To the Cys-Rink-amide PEGA resin, peptide 1 was ligated employing NCL, followed by Thz to Cys conversion using methoxylamine, at a pH of 4. After washing with GnHCl, this step was repeated with another portion of peptide 1 and subsequent methoxylamine treatment (Scheme 2). An important aspect of SPCL is the removal of extra reagents such as 4-mercaptophenylacetic acid (MPAA), which is known to interfere with the desulfurization step.15 In solution phase ligation, extra efforts are needed to get rid of aromatic thiols to enable one pot ligation and desulfurization.16 In SPCL, a washing step enabled radical based desulfurization17 on the solid support, for the first time, in which the free Cys residues were converted quantitatively to their corresponding Ala within 12 h. Finally, cleavage of the polypeptide by treatment of the polypeptide bond resin with 95% TFA for 1 h afforded the desired product in 40% isolated yield for the six synthetic steps (Scheme 2). Our success with this model peptide prompted us to apply this strategy to a more complex synthetic protein target. Our group is interested in devising synthetic strategies for accessing highly homogenous posttranslationally modified histones.18 The diverse and large number of posttranslational modifications
This journal is © The Royal Society of Chemistry 2014
Examining our SPCL strategy in model peptide 1.
(PTMs), which histones are known to undergo, requires efficient synthetic methods for rapid assembly of these proteins.19 Thus, SPCL could be of great use in these synthetic endeavors and assist the ongoing studies aiming at dissecting the roles of PTMs in the chromatin context. One such histone is H2B, which is known to undergo several modifications such as ubiquitination and glycosylation. Such analogues are currently being prepared by employing semi- or total chemical synthesis from multiple fragments via NCL combined with desulfurization.18,19 Our strategy for the synthesis of a (HA-tag)-H2B protein using SPCL is depicted in Scheme 3A and B. According to our experience with the total chemical synthesis of the H2B protein in solution,18b we divided the polypeptide into four fragments, H2B(1–20) labeled with the N-terminal HA-tag, H2B(21–57), H2B(58–96), and H2B(97–124). In peptides H2B(21–57), H2B(58–96), and H2B(59-124), Ala 97, 58, and 21 were substituted with Thz to enable NCL. The fragments (HA)-H2B(1–20) and H2B(21–57) were prepared using a C-terminal Lys protected with 6-nitroveratryloxycarbonyl (Nvoc) at e-amine to prevent lactamization during the ligation step.18b The assembly of the polypeptide started with the ligation of fragment H2B(97–124)Nbz (Nbz: N-acyl-benzimidazolinone), followed by conversion of Thz to free cysteine to enable ligation with H2B(58–96)-Nbz. Washing and subsequent treatment with methoxylamine enabled ligation with H2B(21–57), which was followed by another step of methoxylamine and a final ligation with (HA)-H2B(1–20)-Nbz. Subsequently, the polypeptide resin was exposed to UV to remove
Chem. Commun., 2014, 50, 12534--12537 | 12535
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Scheme 3 SPCL applied to the synthesis of the histone H2B. (A) The sequence of H2B showing the ligation sites and different fragments. (B) The ligation strategy employing NCL and desulfurization that was carried on the solid support. (C) HPLC analysis of the crude product with the major peak corresponding to the H2B protein, also showing the observed mass of 14766.2 Da (calculated 14 766 Da). (D) CD analysis of purified H2B.
the two Nvoc protecting groups, which showed complete ligation and protecting group removal based on analytical cleavage (ESI†). Finally, the resin was subjected to the desulfurization conditions (14 h), which after cleavage with 95% TFA afforded the desired polypeptide corresponding to the full length H2B as the major product with acceptable purity (Scheme 3C). Nevertheless, we performed a final HPLC purification to give HA–H2B(1–125) in 10% isolated yield for the 10 steps. The whole process was completed within 4 days. Circular dichroism (CD) analysis showed the expected helical structure of this histone (Scheme 3D).
12536 | Chem. Commun., 2014, 50, 12534--12537
In summary, a new SPCL approach was introduced featuring the use of a Rink-amide linker, which is easily installed and cleaved. The strategy was applied to the synthesis of H2B from four fragments employing NCL combined with desulfurization. Notably, 10 steps were carried out on the solid support with only one final purification step. We are currently applying this method for the synthesis of modified histones to assist the ongoing studies in unraveling the histone code. This research was partially supported by a Grant from the GIF, the German-Israeli Foundation for Scientific Research and Development.
This journal is © The Royal Society of Chemistry 2014
View Article Online
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Published on 08 September 2014. Downloaded by New York University on 19/10/2014 09:23:22.
Notes and references 1 R. B. Merrifeld, J. Am. Chem. Soc., 1963, 85, 2149. 2 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science, 1994, 266, 776. 3 (a) D. Bang and B. H. Kent Stephen, Angew. Chem., Int. Ed., 2004, 43, 2534; (b) J. Li, Y. Li, Q. He, Y. Li, H. Li and L. Liu, Org. Biomol. Chem., 2014, 12, 5435; (c) N. Ollivier, J. Vicogne, A. Vallin, H. Drobecq, R. Desmet, O. El Mahdi, B. Leclercq, G. Goormachtigh, V. Fafeur and O. Melnyk, Angew. Chem., Int. Ed., 2012, 51, 209; (d) B. Fauvet, S. M. Butterfield, J. Fuks, A. Brik and H. A. Lashuel, Chem. Commun., 2013, 49, 9254; (e) K. Sato, A. Shigenaga, K. Kitakaze, K. Sakamoto, D. Tsuji, K. Itoh and A. Otaka, Angew. Chem., Int. Ed., 2013, 52, 7855. 4 P. E. Dawson, Isr. J. Chem., 2011, 51, 862. 5 L. E. Canne, P. Botti, R. J. Simon, Y. Chen, E. A. Dennis and S. B. H. Kent, J. Am. Chem. Soc., 1999, 121, 8720. 6 A. Brik, E. Keinan and P. E. Dawson, J. Org. Chem., 2000, 65, 3829. 7 G. J. Cotton and T. W. Muir, Chem. Biol., 2000, 7, 253. 8 D. Bang and S. B. H. Kent, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 5014. 9 V. Aucagne, I. E. Valverde, P. Marceau, M. Galibert, N. Dendane and A. F. Delmas, Angew. Chem., Int. Ed., 2012, 51, 11320. 10 L. Raibaut, H. Adihou, R. Desmet, A. F. Delmas, V. Aucagne and O. Melnyk, Chem. Sci., 2013, 4, 4061.
This journal is © The Royal Society of Chemistry 2014
ChemComm 11 I. E. Decostaire, D. Lelievre, V. Aucagne and A. F. Delmas, Org. Biomol. Chem., 2014, 12, 5536. 12 H. Rink, Tetrahedron Lett., 1987, 28, 3787. 13 M. Meldal, Tetrahedron Lett., 1992, 33, 3077. 14 J. B. Blanco-Canosa and P. E. Dawson, Angew. Chem., Int. Ed., 2008, 47, 6851. 15 P. Siman, O. Blatt, T. Moyal, T. Danieli, M. Lebendiker, H. A. Lashuel, A. Friedler and A. Brik, ChemBioChem, 2011, 12, 1097. 16 (a) K. M. Cergol, R. E. Thompson, L. R. Malins, P. Turner and R. J. Payne, Org. Lett., 2014, 16, 290; (b) T. Moyal, H. P. Hemantha, P. Siman, M. Refua and A. Brik, Chem. Sci., 2013, 4, 2496; (c) R. E. Thompson, X. Liu, N. Alonso-Garcia, P. J. Pereira, K. A. Jolliffe and R. J. Payne, J. Am. Chem. Soc., 2014, 136, 8161. 17 Q. Wan and S. J. Danishefsky, Angew. Chem., Int. Ed., 2007, 46, 9248. 18 (a) M. Haj-Yahya, N. Eltarteer, S. Ohayon, E. Shema, E. Kotler, M. Oren and A. Brik, Angew. Chem., Int. Ed., 2012, 51, 11535; (b) P. Siman, S. V. Karthikeyan, M. Nikolov, W. Fischle and A. Brik, Angew. Chem., Int. Ed., 2013, 52, 8059; (c) L. Long, J. P. Thelen, M. Furgason, M. Haj-Yahya, A. Brik, D. M. Cheng, J. M. Peng and T. T. Yao, J. Biol. Chem., 2014, 289, 8916. 19 (a) C. Chatterjee and T. W. Muir, J. Biol. Chem., 2010, 285, 11045; (b) R. K. McGinty, J. Kim, C. Chatterjee, R. G. Roeder and T. W. Muir, Nature, 2008, 453, 812.
Chem. Commun., 2014, 50, 12534--12537 | 12537
Published on 08 September 2014. Downloaded by New York University on 19/10/2014 09:23:22.
COMMUNICATION
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Cite this: Chem. Commun., 2014, 50, 12534
Solid phase chemical ligation employing a rink amide linker for the synthesis of histone H2B protein†
Received 18th August 2014, Accepted 30th August 2014
Muhammad Jbara, Mallikanti Seenaiah and Ashraf Brik*
DOI: 10.1039/c4cc06499b www.rsc.org/chemcomm
Presented here is a solid phase chemical ligation strategy employing native chemical ligation and the commercially available Rink-amide linker as a key element in our approach. The method was applied for the synthesis of histone H2B, which sets the ground for the rapid preparation of posttranslationally modified analogues of this protein.
Solid phase chemical synthesis revolutionized organic synthesis in terms of yield, convenience and speed of process. Solid phase peptide synthesis (SPPS),1 for example, has made it possible, even for non-experts, to rapidly prepare a library of peptides of various lengths (typically of 30–50 amino acids) and compositions. Yet, the assembly of proteins from peptide fragments on a solid support, employing state of the art chemoselective ligation methods, e.g. native chemical ligation (NCL), has not matured to the level of SPPS.2 Success in such a strategy would significantly facilitate protein synthesis, as purification and lyophilization steps, after each ligation, will be eliminated leading to the rapid synthesis of target proteins in high yield. While one pot synthesis could also achieve some of these goals as documented in several excellent examples,3 the attractiveness of solid phase chemical ligation (SPCL) stems from the possibility of performing a higher number of chemical steps and with a greater diversity of chemical transformations. In SPCL an obvious demand, in addition to the high efficiency of the applied chemistry, is the suitability of the polymeric support and the linker, bridging temporally the polypeptide to the support, to the ligation conditions. For example, if NCL is the method of choice, such a support should be water compatible and the linker stable along the synthetic process such as removal of the Cys protecting group in the middle fragments and desulfurization.4 Since the first report by Kent and coworkers on the development and use of SPCL in protein synthesis, employing the base labile linkers and NCL,5 several other studies have attempted to
Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures and analytical data for peptide fragments. See DOI: 10.1039/c4cc06499b
12534 | Chem. Commun., 2014, 50, 12534--12537
devise alternative approaches based on different linkers or ligation chemistries. These include the use of safety catch linkers,6 a peptide sequence that can be cleaved enzymatically7 and His6-Tag8 to facilitate the isolation and handling of intermediate products during the synthetic process. Recently, the use of the alkylsulfonylethyloxycarbamate linker has been revisited, combined with the triazole ligation strategy9 or NCL employing a bis(2-sulfanylethyl)amide thioester surrogate.10 In addition, the Wang linker combined with oxime ligation was also investigated.11 These studies highlight the importance of finding a novel linker, which can be easily introduced and cleaved efficiently to release the final polypeptide product. Here we report that the widely used Rink amide linker in SPPS,12 which is commercially available and can be cleaved under relatively mild acidic conditions, meets these requirements for SPCL as we demonstrate in model peptides and the synthesis of full length histone H2B. Our strategy for SPCL employing the Rink-amide linker is depicted in Scheme 1. PEGA resin, which exhibits excellent swelling properties in water,13 was chosen to serve as a solid support. A spacer of three Ala residues will be included between the resin and the linker. To enable the first ligation, 1,3-thiazolidine-4-carboxo (Thz) will be coupled to the Rink amide linker, which can be quantitatively converted to free Cys using methoxylamine at a pH of 4. Subsequently, NCL can be applied to mediate the assembly of multiple peptides combined with other synthetic steps if required. At the final stage, treatment of the bound polypeptide-resin with TFA would release the peptide from the solid support. This strategy would generate a C-terminal Cys residue (or Ala if desulfurization is applied) instead of the native one, which we believe is a tolerable modification in many proteins and might also serve as a handle for further modifications, e.g. with fluorophores. If desired, one could also introduce a modified Rink amide (e.g. with an alkyne functionality) to the C-terminal of unprotected peptide thioester via direct aminolysis, which would enable its attachment to the resin bearing a complementary functional group (e.g. azide). Although this would generate the polypeptide with the native C-terminal residue,
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 08 September 2014. Downloaded by New York University on 19/10/2014 09:23:22.
Communication
Scheme 1
ChemComm
Principles of SPCL based on the Rink-amide linker. Scheme 2
the extra synthetic steps coupled with the less important role of the C-terminal amino acid in the structure and function of proteins made this option less favorable to us. To examine the above described strategy, we first chose a model peptide 1, Thz-LYRAGLYRL, which was prepared using the N-acylurea method developed by Dawson and Blanco.14 To the Cys-Rink-amide PEGA resin, peptide 1 was ligated employing NCL, followed by Thz to Cys conversion using methoxylamine, at a pH of 4. After washing with GnHCl, this step was repeated with another portion of peptide 1 and subsequent methoxylamine treatment (Scheme 2). An important aspect of SPCL is the removal of extra reagents such as 4-mercaptophenylacetic acid (MPAA), which is known to interfere with the desulfurization step.15 In solution phase ligation, extra efforts are needed to get rid of aromatic thiols to enable one pot ligation and desulfurization.16 In SPCL, a washing step enabled radical based desulfurization17 on the solid support, for the first time, in which the free Cys residues were converted quantitatively to their corresponding Ala within 12 h. Finally, cleavage of the polypeptide by treatment of the polypeptide bond resin with 95% TFA for 1 h afforded the desired product in 40% isolated yield for the six synthetic steps (Scheme 2). Our success with this model peptide prompted us to apply this strategy to a more complex synthetic protein target. Our group is interested in devising synthetic strategies for accessing highly homogenous posttranslationally modified histones.18 The diverse and large number of posttranslational modifications
This journal is © The Royal Society of Chemistry 2014
Examining our SPCL strategy in model peptide 1.
(PTMs), which histones are known to undergo, requires efficient synthetic methods for rapid assembly of these proteins.19 Thus, SPCL could be of great use in these synthetic endeavors and assist the ongoing studies aiming at dissecting the roles of PTMs in the chromatin context. One such histone is H2B, which is known to undergo several modifications such as ubiquitination and glycosylation. Such analogues are currently being prepared by employing semi- or total chemical synthesis from multiple fragments via NCL combined with desulfurization.18,19 Our strategy for the synthesis of a (HA-tag)-H2B protein using SPCL is depicted in Scheme 3A and B. According to our experience with the total chemical synthesis of the H2B protein in solution,18b we divided the polypeptide into four fragments, H2B(1–20) labeled with the N-terminal HA-tag, H2B(21–57), H2B(58–96), and H2B(97–124). In peptides H2B(21–57), H2B(58–96), and H2B(59-124), Ala 97, 58, and 21 were substituted with Thz to enable NCL. The fragments (HA)-H2B(1–20) and H2B(21–57) were prepared using a C-terminal Lys protected with 6-nitroveratryloxycarbonyl (Nvoc) at e-amine to prevent lactamization during the ligation step.18b The assembly of the polypeptide started with the ligation of fragment H2B(97–124)Nbz (Nbz: N-acyl-benzimidazolinone), followed by conversion of Thz to free cysteine to enable ligation with H2B(58–96)-Nbz. Washing and subsequent treatment with methoxylamine enabled ligation with H2B(21–57), which was followed by another step of methoxylamine and a final ligation with (HA)-H2B(1–20)-Nbz. Subsequently, the polypeptide resin was exposed to UV to remove
Chem. Commun., 2014, 50, 12534--12537 | 12535
View Article Online
Communication
Published on 08 September 2014. Downloaded by New York University on 19/10/2014 09:23:22.
ChemComm
Scheme 3 SPCL applied to the synthesis of the histone H2B. (A) The sequence of H2B showing the ligation sites and different fragments. (B) The ligation strategy employing NCL and desulfurization that was carried on the solid support. (C) HPLC analysis of the crude product with the major peak corresponding to the H2B protein, also showing the observed mass of 14766.2 Da (calculated 14 766 Da). (D) CD analysis of purified H2B.
the two Nvoc protecting groups, which showed complete ligation and protecting group removal based on analytical cleavage (ESI†). Finally, the resin was subjected to the desulfurization conditions (14 h), which after cleavage with 95% TFA afforded the desired polypeptide corresponding to the full length H2B as the major product with acceptable purity (Scheme 3C). Nevertheless, we performed a final HPLC purification to give HA–H2B(1–125) in 10% isolated yield for the 10 steps. The whole process was completed within 4 days. Circular dichroism (CD) analysis showed the expected helical structure of this histone (Scheme 3D).
12536 | Chem. Commun., 2014, 50, 12534--12537
In summary, a new SPCL approach was introduced featuring the use of a Rink-amide linker, which is easily installed and cleaved. The strategy was applied to the synthesis of H2B from four fragments employing NCL combined with desulfurization. Notably, 10 steps were carried out on the solid support with only one final purification step. We are currently applying this method for the synthesis of modified histones to assist the ongoing studies in unraveling the histone code. This research was partially supported by a Grant from the GIF, the German-Israeli Foundation for Scientific Research and Development.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Communication
Published on 08 September 2014. Downloaded by New York University on 19/10/2014 09:23:22.
Notes and references 1 R. B. Merrifeld, J. Am. Chem. Soc., 1963, 85, 2149. 2 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science, 1994, 266, 776. 3 (a) D. Bang and B. H. Kent Stephen, Angew. Chem., Int. Ed., 2004, 43, 2534; (b) J. Li, Y. Li, Q. He, Y. Li, H. Li and L. Liu, Org. Biomol. Chem., 2014, 12, 5435; (c) N. Ollivier, J. Vicogne, A. Vallin, H. Drobecq, R. Desmet, O. El Mahdi, B. Leclercq, G. Goormachtigh, V. Fafeur and O. Melnyk, Angew. Chem., Int. Ed., 2012, 51, 209; (d) B. Fauvet, S. M. Butterfield, J. Fuks, A. Brik and H. A. Lashuel, Chem. Commun., 2013, 49, 9254; (e) K. Sato, A. Shigenaga, K. Kitakaze, K. Sakamoto, D. Tsuji, K. Itoh and A. Otaka, Angew. Chem., Int. Ed., 2013, 52, 7855. 4 P. E. Dawson, Isr. J. Chem., 2011, 51, 862. 5 L. E. Canne, P. Botti, R. J. Simon, Y. Chen, E. A. Dennis and S. B. H. Kent, J. Am. Chem. Soc., 1999, 121, 8720. 6 A. Brik, E. Keinan and P. E. Dawson, J. Org. Chem., 2000, 65, 3829. 7 G. J. Cotton and T. W. Muir, Chem. Biol., 2000, 7, 253. 8 D. Bang and S. B. H. Kent, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 5014. 9 V. Aucagne, I. E. Valverde, P. Marceau, M. Galibert, N. Dendane and A. F. Delmas, Angew. Chem., Int. Ed., 2012, 51, 11320. 10 L. Raibaut, H. Adihou, R. Desmet, A. F. Delmas, V. Aucagne and O. Melnyk, Chem. Sci., 2013, 4, 4061.
This journal is © The Royal Society of Chemistry 2014
ChemComm 11 I. E. Decostaire, D. Lelievre, V. Aucagne and A. F. Delmas, Org. Biomol. Chem., 2014, 12, 5536. 12 H. Rink, Tetrahedron Lett., 1987, 28, 3787. 13 M. Meldal, Tetrahedron Lett., 1992, 33, 3077. 14 J. B. Blanco-Canosa and P. E. Dawson, Angew. Chem., Int. Ed., 2008, 47, 6851. 15 P. Siman, O. Blatt, T. Moyal, T. Danieli, M. Lebendiker, H. A. Lashuel, A. Friedler and A. Brik, ChemBioChem, 2011, 12, 1097. 16 (a) K. M. Cergol, R. E. Thompson, L. R. Malins, P. Turner and R. J. Payne, Org. Lett., 2014, 16, 290; (b) T. Moyal, H. P. Hemantha, P. Siman, M. Refua and A. Brik, Chem. Sci., 2013, 4, 2496; (c) R. E. Thompson, X. Liu, N. Alonso-Garcia, P. J. Pereira, K. A. Jolliffe and R. J. Payne, J. Am. Chem. Soc., 2014, 136, 8161. 17 Q. Wan and S. J. Danishefsky, Angew. Chem., Int. Ed., 2007, 46, 9248. 18 (a) M. Haj-Yahya, N. Eltarteer, S. Ohayon, E. Shema, E. Kotler, M. Oren and A. Brik, Angew. Chem., Int. Ed., 2012, 51, 11535; (b) P. Siman, S. V. Karthikeyan, M. Nikolov, W. Fischle and A. Brik, Angew. Chem., Int. Ed., 2013, 52, 8059; (c) L. Long, J. P. Thelen, M. Furgason, M. Haj-Yahya, A. Brik, D. M. Cheng, J. M. Peng and T. T. Yao, J. Biol. Chem., 2014, 289, 8916. 19 (a) C. Chatterjee and T. W. Muir, J. Biol. Chem., 2010, 285, 11045; (b) R. K. McGinty, J. Kim, C. Chatterjee, R. G. Roeder and T. W. Muir, Nature, 2008, 453, 812.
Chem. Commun., 2014, 50, 12534--12537 | 12537
