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Chemoselective Peptide Modification via Photocatalytic Tryptophan #-Position Conjugation Younong Yu, Li-Kang Zhang, Alexei V. Buevich, Guoqing Li, Haiqun Tang, Petr Vachal, Steven L. Colletti, and Zhi-Cai Shi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03973 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018
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Journal of the American Chemical Society
Chemoselective Peptide Modification via Photocatalytic Tryptophan β-Position Conjugation Younong Yu,‡ Li-Kang Zhang,§ Alexei V. Buevich,§ Guoqing Li,‡ Haiqun Tang,‡ Petr Vachal,‡ Steven L. Colletti,‡ and Zhi-Cai Shi*‡ ‡ §
Department of Discovery Chemistry, MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States Department of Process Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States
ABSTRACT: Targeting tryptophan is a promising strategy to achieve high levels of selectivity for peptide or protein modification. A chemoselective peptide modification method via photocatalytic tryptophan β-position conjugation has been discovered. This transformation has good substrate scope for both peptide and Michael acceptor, and has good chemoselectivity versus other amino acid residues. The endogenous peptides, glucagon and GLP-1 amide, were both successfully conjugated at the tryptophan βposition. Insulin was studied as a non-tryptophan control molecule, resulting in exclusive B-chain C-terminal-selective decarboxylative conjugation. This transformation provides a novel approach toward peptide modification to support the discovery of new therapeutic peptides, protein labeling and bioconjugation.
In recent years, peptides have gained increased interest as therapeutics.1 Understanding peptide and protein function is often critical to the discovery of new therapeutic drug targets. Whether the pursuit of a therapeutic or pathway biology, these areas of drug discovery can be enabled by basic research in peptide functionalization and protein bioconjugation.2 Concurrently, visible light-mediated photoredox catalysis has emerged as a powerful synthetic strategy that enables elusive bond constructions and challenging molecular transformations.3 The merger of photoredox catalysis with other modes of catalysis has led to unique bond forming reactions of high value to medicinal and process chemistry. 4 In our effort in developing novel peptide modification and labeling methods, we have been exploring efficient chemoselective photocatalytic reactions which would allow modification of specific amino acids in a peptide sequence. Tryptophan represents the lowest abundance of the 20 naturally occurring amino acids, with a frequency of 1.4% compared to the 5% average amino acid frequency.5 Yet tryptophan is included in the primary sequence of 90% of native proteins,6 and it plays important roles in protein stability and recognition.7 An analysis of protein secondary structures revealed that tryptophan is one of the hot spot residues with high prevalence across helices, strands and loops. 8 Due to its low abundance and important roles, targeting tryptophan is a promising strategy for peptide or protein modification. 9 Methods for tryptophan modification are limited to the C-2 or C-3 position of the indole ring.2f Selective tryptophan modifications on the benzo ring or β-position remain highly elusive.2f,9 Here we describe the discovery of a chemoselective peptide modification via photocatalytic tryptophan β-position conjugation. Tryptophan derived radicals have been extensively studied. These radicals have been detected in a number of enzymes and are catalytically relevant redox agents.10 In addition, tryptophan radicals or radical cations are believed to participate in electron transfer in cytochrome c peroxidase, DNA photolyase
and galactose oxidase.10 We speculated that the reactivity of the tryptophan radical could be extended to a practical method for peptide modification by accelerating the formation of the indolyl radical and/or the radical cation via modern photoredox catalysis. In principle, efficient trapping of the radical would render a useful transformation for peptide conjugation or modification. We began our study with a tryptophan containing tripeptide compound 1, which was subjected to photoredox conditions with methyl acrylate as the radical trapping agent (Table 1). The reaction gave compound 2 selectively in 45% conversion Table 1. Reaction optimization of the photocatalytic tryptophan β-position conjugation with methyl acrylate
Conversiona Entry
Catalyst 3h
6h
16 h
1
1.2 eq.
24%
32%
45%
2
3 eq.
36%
44%
54%
6 eq.
43%
51%
64%
4
10 eq.
50%
59%
81%
5
10 eq.
Without LED light:
6
3 eq.
30%
39%
50%
10 eq.
39%
48%
66%
3
0.10 eq.
0%
0.05 eq. 7
a conversion is determined by using the peak AUC of the UV 215 nm in LC-MS spectra of the reaction mixture
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using 10 mol% of Ir[dF(CF3)ppy]2(dtbbpy)+ PF6- and 1.2 equivalents of methyl acrylate after 16 hours using our integrated photoreactor11 (Table 1, entry 1). Optimization of the reaction conditions afforded 81% conversion by using 10 equivalents of methyl acrylate (Table 1, entry 4). The conjugated product 2 (two diastereoisomers with ca. 1:1 ratio) was purified using mass-directed reverse phase HPLC. The NMR and HRMS studies of both isomers revealed that the conjugation site was exclusively at the β-position of the tryptophan (see supporting information for details).
The reaction without LED light (Table 1, entry 5) did not proceed, confirming that this reaction is photoredox mediated. As control experiments, peptides 3 and 4 were also subjected to the photoredox conditions using 10 equivalents of methyl acrylate and 10 mol% of the photocatalyst. No phenylalanine or tyrosine β-position conjugation was observed under these conditions. We propose a possible mechanism for Trp βposition conjugation in Scheme 1. Via single-electron transfer (SET), the indole N is oxidized to form the radical cation A. Upon the formation of A, the benzylic proton (β-position) of the tryptophan becomes more acidic and can be extracted by K2HPO4 to form B,12 which collapses to form a more stable Trp-skatolyl radical C. DFT analysis of a model 2-acetamido3-(1H-indol-3-yl)pro-panamide neutral radical showed that the C-centered Trp-skatolyl radical is 4.55 kcal/mol more stable than the N1 Trp-indolyl radical, which was consistent with earlier reports13 (see supporting information for details). The Trp-skatolyl radical C can then undergo conjugation with methyl acrylate via Michael addition to form product 2. Scheme 1. Proposed mechanism of photocatalytic tryptophan β-position conjugation
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We first evaluated the scope of this reaction with respect to the Michael acceptors. Another six Michael acceptors, including ,β-unsaturated ester, amides, sulfones and ketone were reacted with the tripeptide 1 with 10 mol% of Ir[dF(CF3)ppy]2(dtbbpy)+ PF6- and 3 eq. of K2HPO4 for 16 hours. All six reactions proceeded smoothly to give moderate to good conversions of desired Trp β-position conjugated compounds 5 – 10 (Table 2). The respective products were successfully purified via mass-directed reverse phase HPLC. Compounds 5 – 10 were fully characterized by NMR and MS (see supporting information for details). The t-butyl ester also worked well to give conjugated product 5 which could be hydrolyzed to the corresponding acid for further derivatization. The alkyne containing ,β -unsaturated amide gave conjugated product compound 8, which could be further derivatized with azide-containing probes, peptides or proteins. The broad scope of the Michael acceptor suggests that this chemistry can be potentially used for either therapeutics or chemical biology. Table 2. The scope of Michael acceptor in the photocatalytic tryptophan β-position conjugation
Prod.
Conv. (Yielda)
Prod.
Conv. (Yielda)
5
83% (30%)
8
65% (51%)
6
96% (74%)
9
83% (28%)
7
59% (39%)
10
78% (30%)
a
Reaction yield is the combined isolated yield of isomers after the mass directed reverse phase HPLC purification To assess the substrate scope of the peptide for this transformation, we selected six linear or cyclic tryptophan containing peptides 11 – 16 (Figure 1). This set also tested the functional group compatibility of the reaction with other aromatic side chain containing amino acid residues (Phe, His and Tyr), and with other functional group containing amino acids (Met, Thr, Asp etc.). These six peptides were subjected to conjugation with methyl acrylate under the photoredox conditions described above (see
This transformation represents a novel method for tryptophan β-position C-H activation. A literature survey reveals that there is only one published method which converts Ltryptophan to β-Me-tryptophan via an enzymatic reaction.14 Thus a site-specific modification on the tryptophan β-position would be a new option for methods applied to peptide drug discovery and protein engineering. 15
supporting information for details, SI-Table-B). All six peptides gave desired Trp β-position conjugated products 17 - 21 and 25. We did not observe any conjugation on the benzylic positions of Phe, His or Tyr based on the results from substrates 12 - 16. There was also no decarboxylation of the Asp (substrate 13), no conjugation on the -OH of the threonine or tyrosine (substrates 14, 15 and 16) or S-conjugation on methionine (substrate 12 and 13). For compounds 15 and 16, we did observe Michael addition on N1 of the imidazole of the histidine due to the basicity of the imidazole. Both monoconjugated product 22 and 24 and bis-conjugated product on imidazole and β-position of tryptophan (23 and 26) were observed.
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Journal of the American Chemical Society Figure 1. Peptide substrate scope and functional group tolerability in the photocatalytic tryptophan β-position conjugation
Having assessed the functional group compatibility and substrate scope of this Trp β-position photocatalytic conjugationreaction, we next examined the applicability of this methodology to endogenous peptides. First we selected glucagon, an important tryptophan containing peptide hormone. In addition to Trp, glucagon contains 15 unique amino acids. We subjected glucagon to conjugation with methyl acrylate under the photoredox conditions (Scheme 3). After 2 hours, the reaction gave about 45% conversion. The reaction mixture was then purified via reverse phase HPLC to give conjugated product 29 (16% isolated yield). HRMS/MS studies of 29 revealed that the conjugation site was on the tryptophan and rigorous 2D NMR studies unambiguously elucidated that the conjugation was at the β-position of the tryptophan (see supporting information for details). We also observed a side product 30 in the reaction mixture (1:7 ratio compared to Compound 29), which was characterized via tandem MS analysis as tryptophan and C-terminal decarboxylative bis-conjugated product, though it was not isolated during the purification for further NMR studies. Remarkable chemoselectivity was observed with no conjugation on His, Phe, Tyr, Arg, Met, Ser, Lys and Thr. It is noteworthy that histidine conjugated products were not observed in contrast to the smaller peptides 15 and 16. One explanation is that the histidine in glucagon may not be conformationally accessible to the Michael acceptor.
a
Scheme 3. Photocatalytic conjugation of methyl acrylate with glucagon
Conversion is based on the peak area from LCMS spectra of the crude reaction. bYield is the combined isolated yield of isomers. cDid not obtain the product during purification.
The chemoselectivity of this method is further supported with insulin as a substrate (Scheme 2). In the absence of Trp, insulin contains 17 unique amino acids and 3 disulfide bonds. When insulin was subjected to conjugation with methyl acrylate or an alkyne-containing acrylamide under the conditions described above, it exclusively gave B-chain C-terminalselective decarboxylative conjugation products 27 and 28 (see supporting information for details). No conjugation on any other amino acids was observed. Interestingly under different photoredox conditions, A-chain selective C-terminal decarboxylative conjugation has been reported.16 Scheme 2. Insulin C-terminal-selective photoredox decarboxylative conjugate addition
We next selected GLP-1, a gut peptide hormone. Semaglutide, a Lys(26)-lipidated long-acting GLP-1 receptor agonist, was recently approved by the FDA as a once weekly treatment for type-II diabetes with CV benefit and weight loss.17 A novel conjugation method would provide additional options for the modification of GLP-1. In addition to Trp, GLP-1 contains 15 unique amino acids. We subjected GLP-1 (7-36) amide to the photocatalytic conjugation with methyl acrylate (Scheme 4). After 2 hours, the reaction proceeded to 30% conversion with the rest as mostly unreacted GLP-1 amide. The reaction mixture was purified via reverse phase HPLC to give a mixture of the desired tryptophan β-position conjugated product 31 and the lysine conjugated product 32 in a 1:2 ratio with 14% isolated yield. Both 31 and 32 were characterized via extensive HRMS-MS and NMR studies (see supporting information for details). We did not observe conjugation on any other amino acids in the sequence under these conditions. HRMS-MS confirmed that the conjugation site for compound 32 is on K34,
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which is closer to the C-terminus. These results suggest that the origins of chemoselectivity are potentially dependent upon Scheme 4. Photocatalytic conjugation of methyl acrylate with GLP-1 (7-36) amide
the conformation of the substrate and/or intramolecular hydrogen bonding, as well as functional group reactivity. In summary, we report a peptide modification method via photocatalytic tryptophan β-position conjugation, to further enable the discovery of therapeutic peptides and protein labeling. This transformation has general scope for Michael acceptors, as well as both simple and endogenous peptides with high chemoselectivity amongst amino acid residues. It provides access to C-H activation of the tryptophan β-position, and a new option for residue-specific peptide modification.
ASSOCIATED CONTENT Supporting Information Supporting figures, experimental procedures, and analytical data for new compounds. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author
[email protected] Notes The authors declare no competing financial interests. ORCID Zhi-Cai Shi: 0000-0001-9296-9895 Younong Yu: 0000-0002-3043-5908 Li-Kang Zhang: 0000-0003-3757-8116 Alexei V. Buevich: 0000-0002-5968-9151
ACKNOWLEDGMENT We would like to thank Prof. David W. C. MacMillan from Princeton University and Drs. Lin Yan, Ling Tong, Robert Garbaccio, Songnian Lin, Craig Parish, Abbas Walji, Thomas Williamson, Daniel DiRocco and Joseph Duffy from Merck & Co., Inc., Kenilworth, NJ, USA, for helpful discussions. The authors also would like to thank Zachary Brown and Dennis Feng for providing peptide 3 and 11, and Xiaohong Zhu, Cristina Grosanu, Kuanchang Chen, Min Liu and Xiao Wang for purification and analytical support.
REFERENCES
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Journal of the American Chemical Society
Table of Content
Chemoselective Peptide Modification via Photocatalytic Tryptophan β-Position Conjugation Younong Yu, Li-Kang Zhang, Alexei V. Buevich, Guoqing Li, Haiqun Tang, Petr Vachal, Steven L. Colletti, and Zhi-Cai Shi*
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