deb00 | ACSJCA | JCA11.1.4300/W Library-x64 | research.3f (R4.1.i3 HF01:4938 | 2.1) 2018/08/24 11:08:00 | PROD-WS-116 | rq_3736418 | 9/28/2018 14:29:15 | 8 | JCA-DEFAULT Article pubs.acs.org/JACS 1 Construction of Challenging Proline−Proline Junctions via 2 Diselenide−Selenoester Ligation Chemistry †,⊥ †,⊥ †,∥ † 3 Jessica Sayers, Phillip M. T. Karpati, Nicholas J. Mitchell, Anna M. Goldys, ‡ ‡ § ,† 4 Stephen M. Kwong, Neville Firth, Bun Chan, and Richard J. Payne* † 5 School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia ‡ 6 School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia § 7 Graduate School of Engineering, Nagasaki University, Bunkyo 1-14, Nagasaki 852-8521, Japan 8 *S Supporting Information 9 ABSTRACT: Polyproline sequences are highly abundant in prokaryotic 10 and eukaryotic proteins, where they serve as key components of 11 secondary structure. To date, construction of the proline−proline motif 12 has not been possible owing to steric congestion at the ligation junction, 13 together with an n → π* electronic interaction that reduces the 14 reactivity of acylated proline residues at the C-terminus of peptides. 15 Here, we harness the enhanced reactivity of prolyl selenoesters and a 16 trans-γ-selenoproline moiety to access the elusive proline−proline 17 junction for the first time through a diselenide−selenoester ligation− 18 deselenization manifold. The efficient nature of this chemistry is 19 highlighted in the high-yielding one-pot assembly of two proline-rich 20 polypeptide targets, submaxillary gland androgen regulated protein 3B 21 and lumbricin-1. This method provides access to the most challenging of ligation junctions, thus enabling the construction of 22 previously intractable peptide and protein targets of increasing structural complexity. 23 ■ INTRODUCTION branched residue, e.g., valine (Val), threonine (Thr), or 50 11 isoleucine (Ile). Furthermore, thioesters bearing a C-terminal 51 24 Peptide ligation chemistry has revolutionized protein science by proline (Pro) residue have proven to be unreactive acyl donors 52 25 providing a means to access large polypeptide and protein 1−3 in traditional NCL reactions, including at Cys, selenocysteine 53 26 targets, including those that cannot be generated by 12,13 (Sec), and thiol/selenol amino acids. This lack of reactivity 54 27 recombinant expression technologies. This includes the ability has been attributed to a reduction in electrophilicity of the 55 28 to site-specifically incorporate nonproteinogenic amino acids, thioester due to a stabilizing n → π* electron donation from the 56 29 post-translational modifications, and/or isotopic or fluorescent 2 proximal amide carbonyl to the carbonyl of the thioester 57 30 labels. The most widely adopted method for the convergent 14,15 moiety. This stabilizing interaction is enabled by the rigid 58 31 assembly of peptide fragments to afford large polypeptides and trans-configured peptide bond induced by the cyclic Pro 59 32 proteins is undoubtedly native chemical ligation (NCL). This 14,15 4 residue. Attempts to overcome the challenge of performing 60 33 technology, first reported by Kent and co-workers, involves the 61 34 chemoselective reaction of a peptide possessing a C-terminal ligation chemistry at Pro thioesters have focused on enhancing 62 35 thioester functionality with a second fragment bearing an N- the reactivity of the acyl donor component. One approach has involved substituting the prolyl thioester with a more reactive 63 36 terminal cysteine (Cys) residue. While this technology has 16 1−3 37 enabled access to numerous protein targets to date, the selenoester functionality (Scheme 1A). These selenoester acyl 64 s1 38 conventional reaction is limited by the requirement of a Cys donors have been reported to react with N-terminal Cys- 65 39 residue at the ligation junction. This restriction has, however, containing peptides, albeit in the presence of a selenol catalyst 66 40 been recently addressed through the development of NCL at and a large molar excess of the acyl donor fragment. More 67 5 6−8 41 thiol-derived amino acids, selenocysteine, and a number of recently, Dong et al. have designed a prolyl thioester whereby 68 9 γ 42 synthetic selenol-derived amino acids that can serve as cysteine the -position of the Pro ring is functionalized with a thiol 69 17 fi 43 surrogates in reactions with peptide thioesters. Importantly, moiety (Scheme 1B). This modi ed Pro thioester reacts via a 70 44 following the ligation reaction, these thiol and selenol auxiliaries bicyclic thiolactone intermediate, which leads to activation of 71 45 can be removed through desulfurization or deselenization the carbonyl through the generation of a highly strained cyclic 72 γ 46 chemistry, respectively, to afford native polypeptides and thioester. While this is a very elegant strategy, the -thiol 73 10 47 proteins. One further limitation of the NCL manifold is that 48 reactions typically proceed slowly at sterically encumbered Received: July 25, 2018 49 thioesters, particularly those containing a C-terminal β- Published: September 21, 2018 © XXXX American Chemical Society A DOI: 10.1021/jacs.8b07877 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX Journal of the American Chemical Society Article Scheme 1. Ligation Reactions between (A) a Prolyl amino acid junctions containing Pro; we outline the first 110 Selenoester and Cysteinyl Peptide; (B) a Thiolated Prolyl example of the construction of challenging and hitherto 111 Thioester and Cysteinyl Peptide That Proceeds via a inaccessible Pro−Pro junctions through ligation chemistry 112 Thiolactone Intermediate; (C) Thioesters and Thio- or (Scheme 1D), as well as the application of this method in the 113 Selenoproline Peptides; (D) a Prolyl Selenoester and synthesis of two large Pro-rich polypeptide targets. 114 Selenoproline Peptides via Diselenide−Selenoester Ligation (DSL) ■ RESULTS AND DISCUSSION 115 Design and Synthesis of a γ-Selenoproline Building 116 Block. Before embarking on the development of the DSL 117 methodology at Pro, we first needed to prepare a suitably 118 protected selenol-derived Pro residue that would be compatible 119 with solid-phase synthesis protocols and the ensuing ligation 120 chemistry. Danishefsky and co-workers have previously reported 121 13 the preparation of a γ-selenoproline diselenide amino acid. We 122 initially replicated this synthetic route (see Supporting 123 Information), but as reported for other diselenide building 124 12 blocks, we observed that only one-half of the diselenide amino 125 acid was able to couple to a resin-bound peptide chain (i.e., the 126 other selenoproline unit within the diselenide dimer was unable 127 to couple to another peptide N-terminus). This is particularly 128 problematic in larger immobilized targets or when peptides are 129 more highly loaded on resin supports (>0.5 mmol/g). In order 130 to mitigate these issues, we sought to synthesize a monomeric 131 selenoproline building block 1, whereby the γ-selenol moiety 132 was orthogonally protected with a p-methoxy benzyl group 133 (PMB). The synthesis began with the C-terminal protection of 134 74 auxiliary must be removed via a postligation desulfurization commercially available trans-γ-hydroxyproline 2 as methyl ester 135 75 protocol and therefore lacks chemoselectivity in the presence of 3 (Scheme 2A). Conversion to cis-γ-iodoproline 4 was then 136 s2 76 native Cys residues found elsewhere in the sequence. performed under Mitsunobu conditions with inversion of 137 77 Danishefsky and co-workers have reported the use of both stereochemistry at the γ-position. Direct treatment of iodide 4 138 78 thioproline and selenoproline residues in NCL reactions with with 4-methoxybenzyldiselenide 5 in the presence of NaBH4 139 13,18 79 peptide thioesters (Scheme 1C). These ligation reactions then provided trans-γ-selenoproline methyl ester 6. Finally, 140 80 worked well at unhindered sites, but were inefficient at more 81 sterically encumbered C-terminal thioesters, e.g., Val, even with Scheme 2. (A) Synthetic Route to trans-γ-Selenoproline 1; 82 the enhanced nucleophilicity of the selenoproline moiety. (B) Incorporation of trans-γ-Selenoproline 1 Building Block 83 Importantly, when peptides bearing N-terminal thioproline or into Model Peptide 7 via Fmoc-SPPS 84 selenoproline residues were reacted with peptide thioesters 85 bearing a C-terminal Pro, no reaction was observed. This 86 inability to forge Pro−Pro was attributed to the deactivation of 87 the Pro thioester component through the n → π* interaction 88 from the adjacent carbonyl (vide supra). 89 We have recently reported the development of an additive- 90 free peptide ligation reaction between peptides bearing a C- 91 terminal selenoester and peptides containing an N-terminal 12 92 selenocystine residue (the oxidized form of Sec). Importantly, 93 these represent the fastest ligation reactions known, proceeding 94 efficiently in aqueous buffer without the addition of external 95 catalysts or reductants to afford selenopeptide products in 96 minutes (including at selenoesters bearing C-terminal β- 97 branched amino acids). Upon reaction completion, and without 98 purification, the selenocystine at the ligation junction can be 19,20 99 cleanly converted to alanine or serine through reductive or 21,22 100 oxidative deselenization, respectively. This diselenide− 101 selenoester ligation (DSL) manifold has been extended to 102 alternative selenol-derived amino acids including β-selenoas-