Letter pubs.acs.org/OrgLett
Reversible Macrocyclization of Peptides with a Conjugate Acceptor Amber M. Johnson and Eric V. Anslyn* Department of Chemistry, University of Texas, 1 University Station A1590, Austin, Texas 78712, United States S Supporting Information *
ABSTRACT: Macrocyclic peptides are an increasingly important class of biopharmaceuticals. A new method of macrocyclization is reported that involves reaction of the N-terminal amine and thiol side chain of cysteine with a Meldrum’s acid derived conjugate acceptor. This reaction, which utilizes naturally occurring amino acids and requires no orthogonal protection of side chains, can also be reversed to yield the original linear peptide as desired. Our group recently reported a new type of “click” chemistry where components can be coupled to a Meldrum’s acid derived conjugate acceptor via an amine and a thiol.17 The reaction is reversible upon the addition of dithiothreitol (DTT) and returns both components to their original states (Scheme 1a).
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s biological systems become increasingly understood, pharmaceuticals improve to more adequately treat disease states. Biomolecules are being increasingly utilized as potential drugs, as they have unique and more complex mechanisms of action in comparison to traditional small molecule therapeutics.1 Peptides are a promising class of biotherapeutics, especially as they can target protein−protein interactions, which is a challenging target for small molecule drugs.2 Macrocyclic peptides are of particular interest,3 especially in the area of antibiotic development,4 and have commercial uses including the antibiotic vancomycin and the immunosuppressant cyclosporine A.5 In comparison to their linear analogues, macrocyclic peptides show greater stability to proteolytic degradation,6 among other effects.7 Several methods of macrocyclization exist. Those that utilize reactivity between side chains found in the canonical amino acids include amidation8 and disulfide formation.9 Noncanonical amino acids can also be incorporated to increase the functionality available for macrocyclization. Side chains can be derivatized to incorporate different reactive functionality10 or cyclize by reaction with bifunctional linker molecules.11 Organometallic techniques such as copper-catalyzed click chemistry,12 olefin metathesis,13 and palladium-catalyzed cross coupling14 are also common. Enzymatic techniques15 and multicomponent reactions16 have also been reported. However, most of these later methods require the introduction of an unnatural amino acid. In this work, we report a new method of peptide macrocyclization based on addition of the N-terminal amine and a cysteine side chain thiol to a Meldrum’s acid derived conjugate acceptor. This technique uses naturally occurring amino acids, has no specific sequence requirements for the peptide, and is a one-pot reaction under mild conditions. Additionally, the macrocyclization is reversible, which allows for the regeneration of the original linear peptide. © XXXX American Chemical Society
Scheme 1. Addition of Amines and Thiols to Conjugate Acceptor 1 and Reversal with DTT: (a) General Scheme; (b) Peptide Macrocyclization
This reaction can be extended to intramolecular systems to form macrocycles, and Scheme 1b shows this concept applied to peptides where the N-terminus or lysine (Lys) side chain contains an amine and the cysteine (Cys) side chain contains a thiol. Received: February 14, 2017
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DOI: 10.1021/acs.orglett.7b00451 Org. Lett. XXXX, XXX, XXX−XXX
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membered ring at the N-terminus rather than a larger macrocycle. To determine the reactive sites in the peptide (Cys vs Lys vs N-terminus), groups were selectively blocked before cyclization was attempted. When the peptides were derivitized with iodoacetamide, a thiol-selective reagent,20 an amine added to the conjugate acceptor but no cyclization was observed even upon sparging with nitrogen. This shows that the thiol is involved in cyclization, which is consistent with our previous work in which only one amine adds to 1 in a predominantly aqueous solvent system. To determine whether the reactive amine is the N-terminus or the Lys side chain, acetylated peptides 15 and 16 were synthesized (Table 2). In comparison to their nonacetylated
To determine how cyclization proceeds to form macrocycles of varying size, a library of peptides was synthesized with Cys and Lys in variable positions (Table 1). Lys was included to Table 1. Peptide Library for Initial Cyclization Studies
peptide
sequence
yield (%)
7 8 9 10 11 12 13 14
GCTGKYD YGCKTYD YKTCYTD KTGYCTD KGTGYCD CGTYGYKD KTGYTYGCD CGYTGYTGKD
n/aa 63 79 93 97 50 95 99
Table 2. Secondary Peptide Library
a
Cyclized peptides could not be isolated due to unfavorability of the cyclization reaction.
determine whether macrocycles could be formed with the side chain of Lys or whether reaction would be limited to the Nterminus. Several amino acids not participating in cyclization were chosen to yield peptides with good solubility, and the composition was kept consistent to minimize the potential effects of other side chains on macrocyclization. It should be noted that Cys could not be placed at the C-terminus due to a side reaction during solid-phase synthesis that resulted in a loss of the thiol in the side chain.18 Cyclization was performed simply by mixing the peptide, conjugate acceptor 1, and tris(2-carboxyethyl)phosphine (to prevent disulfide formation) in a 4:1 buffer (100 mM phosphate, pH 7.8)/acetonitrile mixture, and the crude reactions were characterized by LCMS. Conjugate acceptor 1 readily reacted with an amine on each of the peptides to form structures similar to 5, followed by cyclization to 6 with Cys under some conditions. For example, cyclization did not typically proceed on a small scale (0.5 mg/100 μL). This was attributed to the reversibility of thiol addition, as such a small volume could not be sparged with nitrogen to liberate the methyl mercaptan byproduct of the cyclization reaction. When the reactions were performed at 10 mg or larger (typically in 2 mL solvent), and sparged with nitrogen, cyclization was observed with all peptides, and no evidence of oligomerization was detected. This makes for an incredibly simple method for peptide cyclization. The reactivity depended on the distance between the reactive amine of the N-terminus and the Cys side chain. Cyclization did not proceed cleanly with peptides 7 and 8 and is consistent with the reported difficulty of forming small peptide macrocycles.19 In 9 and 10, a mixture of complete and incomplete cyclization was observed with nitrogen sparging, but prolonged sparging and replenishing the solvent could drive the cyclization to near completion. However, cyclization proceeded cleanly even without nitrogen sparging with 11 and 13. This suggests that at least four amino acids are required between reactive sites for cyclization to be favorable. An exception to this trend was with peptides 12 and 14, which contain an Nterminal Cys. A reaction proceeded extremely cleanly with these peptides but instead led to the formation of a small, five-
peptide
sequence
yield (%)
15 16 17 18 19 20 21 22 23 24
Ac-CGTYGYKD Ac-KGYTGYTGCD CGTYGYAD AGTYGYCD KFNIYRVSCD KALTHGPYCD KGYMQEWRCD KGTYGYCD CTGYTYGKD KGYTGYTGCD
n/aa 38 99 95 46 91 87 67 95 83
a
Cyclized peptides could not be isolated due to unfavorability of cyclization reaction.
analogues, cyclization was not complete after overnight reaction, and the major products observed by LCMS were unreacted peptide and conjugate acceptor. This suggests that the initial amine addition proceeds faster for the N-terminal amine than the Lys side chain. This is consistent with its lower pKa, where less of the amine is protonated and therefore more available for reactivity. Our previous studies showed that amines do not undergo exchange once reacted with 1, and thus reaction occurs at the more available amine. Reaction at the Nterminus was further supported in cyclization experiments with 17 and 18, which contain a Cys but not a Lys. While cyclization was expected in 17 because of the proximity of the amine and the thiol, cyclization in 21 also occurred which shows the favorability of cyclization with the N-terminus. Cyclization is compatible with all the remaining 18 canonical amino acids, which are embodied in peptides 19−21. Studies are currently underway to determine the effects of noncanonical amino acids. For a direct comparison between the two possible amines that can be involved in cyclization, 25 was synthesized, which mimics an N-terminal Lys. Equimolar amounts of 1 and 25 were mixed, and the reaction was monitored by 1H NMR (Figure 1). Addition proceeded rapidly, and the α-proton shifted downfield, while the side-chain methylenes did not shift to any significant extent, further supporting reaction with the N-terminus. This macrocyclization strategy is easily reversed, which allows one to use standard sequencing methods, such as Edman degradation, if desired. As reported previously, addition of DTT “declicks” the reactions of 1 with amines and thiols.17 Thus, upon addition of DTT to the peptide macrocycles, the original peptide is regenerated. Initial studies with 7−14 resulted in B
DOI: 10.1021/acs.orglett.7b00451 Org. Lett. XXXX, XXX, XXX−XXX
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cyclization reversal in all peptides except 12 and 14, those with an N-terminal Cys where the 5-membered ring with 1 occurs. Switching the positions of the Cys and Lys in 12−14 (22−24) again resulted in macrocycles that could only be linearized if the N-terminal amino acid was not a Cys. For example, 14, with an N-terminal Cys, could be cyclized but not linearized, but linearization was possible with 24 where the Cys was removed from the N-terminus. In summary, we have demonstrated a new method of forming macrocyclic peptides by reacting the N-terminus and Cys side chain thiol with a conjugate acceptor (1). The reaction proceeds almost quantitatively provided the reactive sites are at least four amino acids apart. This is a one-pot reaction that occurs under mild, aqueous conditions, has no specific sequence requirements, and is compatible with all canonical amino acids. The cyclization is also reversible allowing the original peptide to be recovered. Future studies include screening macrocycles for antimicrobial activity, incorporating noncanonical functional groups such as boronic acids, and generating more complex structures by incorporating multiple reactive sites in each peptide.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00451. Peptide characterization and LCMS spectra of cyclization experiments (PDF)
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REFERENCES
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Figure 1. 1H NMR monitoring of competing amine reactions: (a) 25; (b) 25 + 1 (400 MHz, 4:1 buffer/CD3CN).
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Eric V. Anslyn: 0000-0002-5137-8797 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge financial support from DARPA (N66001-14-2-4051), NSF (CHE-1212971), and the Welch Regents Chair (F-0046). C
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