Chemoselective Synthesis of N-Terminal Cysteinyl Thioesters via β,γ

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Chemoselective Synthesis of N‑Terminal Cysteinyl Thioesters via β,γ‑C,S Thiol-Michael Addition Rita Petracca,† Katherine A. Bowen,† Lauren McSweeney,† Siobhan O’Flaherty,‡ Vito Genna,§ Brendan Twamley,† Marc Devocelle,‡ and Eoin M. Scanlan*,†

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†

School of Chemistry and Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland ‡ Department of Chemistry, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland § Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Joint IRB-BSC Program in Computational Biology, Baldiri-Reixac 10-12, 08028 Barcelona, Spain S Supporting Information *

ABSTRACT: Dehydroalanine (ΔAla) is a highly electrophilic residue that can react efficiently with sulfur nucleophiles to furnish cysteinyl analogues. Herein, we report an efficient synthesis of N-terminal cysteinyl thioesters, suitable for S,Nacyl transfer, based on β,γ-C,S thiol-Michael addition. Both ionic and radical-based methodologies were found to be efficient for this process. he thiol-Michael reaction is a widely utilized “click” process with applications in organic synthesis,1−3 bioconjuga4−6 drug discovery,7,8 and polymer functionalization.9−11 tion, Robust thioether bonds are formed with fast reaction kinetics, high regioselectivity, and often quantitative yields across a broad substrate range.9 The versatility of the thiol-Michael reaction has rendered it one of the most widely utilized methods for siteselective modification of therapeutic proteins, in particular, for the synthesis of antibody drug conjugates.12 Dehydroalanine (ΔAla) is a naturally occurring13 unsaturated amino acid residue that has emerged as an important “chemical tag” for the chemical mutagenesis of proteins.14,15 ΔAla derivatives function as highly efficient Michael acceptors for the synthesis of substituted peptide and protein substrates under physiological conditions.6,16 Ligation reactions with ΔAla forge robust covalent bonds and have been applied to the introduction of a range of important post-translational modifications onto protein substrates, including lipidation, glycosylation, phosphorylation, and lysine methylation/acetylation.17,18 More recently, ΔAla has been exploited as an electrophilic residue for regioselective amination reactions19 and for Cβ−Cγ bond-forming reactions through a radical-mediated process.20,21 However, a known limitation of Michael addition to ΔAla is a lack of stereoselectivity and the formation of diastereomeric mixtures from chemical ligations using this method.14 In the context of chemoselective modification of peptides and proteins via thiolMichael addition, we became interested in thioacids as substrates for ΔAla addition to furnish N-terminal thioester products, a synthetic strategy that has not previously been explored. N-Terminal peptide thioester fragment formation is a critical feature of protein chemical synthesis strategies including native chemical ligation (NCL)22 and expressed protein ligation.23 Peptide thioesters are typically formed through

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© XXXX American Chemical Society

dynamic, reversible processes including transthioesterification or N,S-acyl transfer, where the presence of a free thiol and/or reactive thioester is required.24 Herein, we investigate Nterminal dehydroalanine as a substrate for peptide thioester synthesis via β,γ-C,S thiol-Michael addition of peptide thioacids. This novel approach for N-terminal cysteinyl thioester formation offers an alternative synthetic disconnection for the synthesis of biologically and synthetically important peptide thioester derivatives (Figure 1).

Figure 1. General scheme for ΔAla-mediated β,γ-C,S thiol-Michael addition.

Thioacids are known substrates for Michael addition reactions,25,26 but their reactivity with ΔAla to furnish cysteinyl thioesters remains unexplored. Studies involving ΔAla as an Nterminal electrophilic residue are relatively rare, primarily due to the propensity for rapid spontaneous hydrolysis of the free amino ΔAla monomer under aqueous acidic conditions.27 To avoid competing hydrolysis and promote 1,4-addition, we focused on N-substituted ΔAla derivatives which are stable under a wide range of conditions. Monosubstituted ΔAla Received: March 22, 2019

A

DOI: 10.1021/acs.orglett.9b01013 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. Optimization Table for ΔAla-Mediated Thioester Preparation via β,γ-C,S Thiol-Michael Addition

entry

ΔAla

2 (equiv)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1a 1a 1a 1a 1a 1a 1b 1b 1b 1b 1b 1c 1c 1d 1d 1e 1d

4.0 4.0 1.2 4.0 10 4.0 10 20 4.0 6.0 4.0 4.0 4.0 10 4.0 4.0 4.0

base (equiv) K2CO3 (4) TEA (0.1) TEA (4)

TEA (0.1) TEA (6) TEA (0.1) TEA (0.1) TEA (0.1) TEA (0.1) TEA (0.1)

solvent

time (h)

temp (°C)

yield (%)

CHCl3 CHCl3 DMF H2O toluene DMF/buffer pH 8 toluene toluene DMF H2O THF DMF DMF/buffer pH 8 toluene THF THF DMF/buffer pH 8

12 24 96 96 16 4 16 20 16 16 5 16 4 16 5 5 4

rt rt rt rt refl 37 refl refl rt rt rt rt 37 refl rt rt 37

no conva no conva no conva no conva no convb 47d 50c 87c 84c 53c 89c no convb 98c 90d 76d no convb 98d

a Crude 1H NMR shows only the presence of the starting ΔAla. bCrude 1H NMR shows the conversion of the starting ΔAla into a complex mixture of degradation products. c1H NMR conversion of the purified thioester as an inseparable mixture with starting material. dIsolated yield.

To further probe the reactivity of the N-substituted ΔAla derivative in thiol-Michael 1,4-conjugate addition, computational studies were carried out. Three different ΔAla derivatives were modeled: (i) Boc-ΔAla 1a, (ii) bis-Boc-ΔAla 1d, and (iii) acetyl-ΔAla 1c. Structures were first minimized at force-fieldbased level using GAFF33 and then further optimized using an all-electron quantum approach (Supporting Information). The fully optimized molecular geometries were identified as global minima in the potential energy surface. Concomitantly, the electrostatic surface potentials were modeled through computational studies (Figure 2a). The acetyl-ΔAla 1c system presents a highly negative region (red area) close to the double bond; here, the presence of the small acetyl group allows the electron cloud to occupy a large portion of the molecule. In contrast, the Boc protecting group in the Boc-ΔAla 1a derivative imposes a structural conformation that constrains electron density, thus

derivatives are reported to be poorly electrophilic under certain conditions, whereas disubstituted derivatives are reported to function as efficient Michael acceptors.28−30 To explore the influence of nitrogen substitution on the reactivity of N-terminal ΔAla toward thioacid conjugate addition, we first evaluated the reactivity of several ΔAla monomers (1a−e) as Michael acceptors for thioacetic acid (Table 1). No thioester products were formed with the monosubstituted ΔAla derivatives 1a or 1c, in the presence of organic solvents or water (Table 1, entries 1−5 and 12). However, in the presence of DMF/phosphate buffer at pH 8, a moderate 47% conversion was found for N-Boc derivative 1a (entry 6) with full conversion to the thioester for 1c (entry 13). In the case of the disubstituted ΔAla derivative 1b (R1 = Boc and R2 = Acetyl), the desired thioester 3b was formed as the major product in high yield (Table 1, entries 7 and 8), although with the requirement for a large excess of thioacid and increased temperatures to achieve full conversion. Addition of a catalytic amount of triethylamine (TEA) resulted in a marked acceleration of the conjugate addition process at room temperature, consistent across the range of solvents screened, including water (Table 1, entries 9− 11). For the N-bis-Boc-substituted ΔAla derivative 1d, use of catalytic TEA resulted in high-yielding formation of thioester 3d in two different solvent systems (Table 1, entries 14 and 15). Azido derivative 1e, which functioned as a “masked” amine, furnished none of the desired thioester product (entry 16). Finally, thiol-Michael addition to 1d in DMF/phosphate buffer furnished 3d in quantitative yield (Table 1, entry 17), suggesting a potentially significant solvent effect to enhance the overall yield.31,32 After initial screening studies (and owing to the low reactivity of 1a), the N-bis-Boc-substituted ΔAla derivative 1d emerged as the most promising candidate for further investigation.

Figure 2. (a) Minimized structures of ΔAla derivatives 1a, 1c, and 1d and corresponding electrostatic potential maps. Blue corresponds to low electron densities, whereas red corresponds to high electron densities. (b) Crystallographic structures of 1c (top) and 1d (bottom). B

DOI: 10.1021/acs.orglett.9b01013 Org. Lett. XXXX, XXX, XXX−XXX

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screened (Table 2, entries 1−4). 1c proceeded with full conversion under aqueous buffered conditions (Table 2, entry 6). Interestingly, the azido derivative 1e furnished the thioester product with complete conversion, whereas this substrate failed to react under ionic conditions (Table 2, entry 10). Radicalmediated addition of a glycine thioacid derivative furnished the desired thioester in 61% yield, highlighting the potential scope to extend this process to longer peptides chains (Table 2, entry 11). Having established both ionic and radical-mediated conditions for conjugate addition of thioacetic acid onto the model substrate, bis-Boc-ΔAla 1d, we set out to investigate the scope of the methodology for a range of C-terminal thioacid derivatives and N-terminal ΔAla peptides. C-Terminal thioacids have been synthesized and used extensively in organic synthesis and chemical biology.26 Thioacid derivatives of Fmoc-amino acids were prepared, starting from the corresponding Fmoc-aminoS(trityl)thioacid, and reacted with various ΔAla derivatives (Table 3). The pH of the buffer solution was adjusted to 8.

generating a more negative electrostatic potential near to the electrophilic double bond. In both cases, the effect of the marked electron density reduces the double bond electrophilicity, consequently reducing reactivity for the conjugate addition. In the case of the bis-Boc-ΔAla 1d, the presence of two Boc groups in the system generates a geometry that allows a more relaxed electronic distribution around the unsaturated moiety, thereby enhancing its reactivity as a Michael acceptor (Figure 2a). The crystal structure of bis-Boc-ΔAla 1d was obtained (Figure 2b, bottom) and clearly shows the presence of a highly conjugated π-system formed by the two carbonyl moieties of the Boc groups. The planarity of the molecule allows the formation of the conjugated π-system, which enhances an optimal electron distribution, thus increasing double bond electrophilicity. The absence of this extended π-system in the acetyl-ΔAla 1c renders the double bond less electrophilic as carbonyl electrons are delocalized toward the amidic bond (Figure 2b, top). Having established conditions for efficient 1,4-conjugate addition of a thioacid onto 1d, we set out to investigate if a radical-mediated process could be employed. Radical-mediated thiol−ene ligation conditions are known to be highly compatible with peptides and proteins and occur under extremely mild conditions.34−36 We have recently established acyl thiol−ene methodology as an efficient method for thiolactone synthesis; however, radical-mediated thiol−ene addition onto ΔAla remains unexplored.37 Davis and co-workers reported mild carbon-centered radical addition onto internal ΔAla residues for post-translational mutagenisis of proteins.20 Thiol−ene reaction conditions were screened for conjugate addition of thioacetic acid onto 1a−e (Table 2). A synergistic photosensitizer−

Table 3. ΔAla-Mediated Peptide Ligation: Scope Expansiona

Table 2. Optimization Table for ΔAla-Mediated Thioester Preparation via a Radical Mechanisma

entry

ΔAla

R

solvent

yield (%)

1 2 3 4 5 6 7 8 9 10 11

1a 1a 1a 1b 1c 1c 1c 1d 1d 1e 1d

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 Gly

DMF EtOAc H2O/NH4OOCH3 DMF DMF H2O/NH4OOCH3 H2O DMF H2O/NH4OOCH3 H2O/NH4OOCH3 H2O/NH4OOCH3

no convb no convb no convb no convb no convb 98c no convb 36c 99c 99c 61c

entry

Michael acceptors

Fmoc-aa-COSH

aa-SCOR1

1 2 3 4 5 6 7 8 9 10 11 12

ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla, 1d ΔAla-I, 4 ΔAla-W, 5 ΔAla-WLVKGRd, 16

G A V I H Y F L AG AG I I

6, 81%b 7, 68%b 8, 72%c 9, 70%b 10, 61%b 11, 91%c 12, 56%b 13, 64%b 14, 38%b 15, 58%b no conversion 17, 48%e

a

All reactions were conducted using 1.5 equiv of thioacid in DMF/ buffer pH 8 for 10 h at 37 °C. bIsolated yields. c1H NMR conversion. d Peptide was synthesized using manual solid phase peptide synthesis (SPPS). eYield calculated as HPLC conversion.

Under the optimized ionic conditions, N-terminal cysteinyl thioesters 6−15 were obtained in high yield for all of the thioacid derivatives screened (Table 3, entries 1−8). When bisBoc-ΔAla 1d was reacted with a dipeptide thioacid, a decrease in the yield of the conjugate addition product was observed, possibly as a result of increased steric hindrance (Table 3, entry 9). However, a good conversion was obtained when a dipeptide−dipeptide coupling was carried out using the same substrate (Table 3, entry 10). Introduction of a bulky tryptophan residue adjacent to the bis-Boc-ΔAla residue 5 completely disabled its electrophilic character (Table 3, entry 11). Computational investigations were performed in order to rationalize this finding (Supporting Information). These revealed that the tryptophan aromatic moiety imposes an overall geometry that profoundly impacts the local electrostatic distribution. Indeed, the observed increase of the local electron density reduces the electrophilic nature of the bis-Boc-ΔAla compound, thus no longer favoring conjugate addition. Finally,

a

All reactions were conducted using 4 equiv of thioacid and 0.2 equiv of photoinitiator and sensitizer. b1H NMR of the crude reaction mixture shows none of the thioester product. cIsolated yield.

photoinitiator pairing of 4′-methoxyacetophenone and 2,2dimethoxy-2-phenylacetophenone, respectively, in aqueous buffer at pH 6 at 25 °C was employed, irradiating at 365 nm. Overall, the findings were similar to those of the ionic process, with 1d found to function as the most efficient substrate for the thioester synthesis under radical-mediated conditions (Table 2, entry 9). 1a and 1b did not react under any of the conditions C

DOI: 10.1021/acs.orglett.9b01013 Org. Lett. XXXX, XXX, XXX−XXX

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potentially useful as a chemical ligation methodology for peptides. This methodology may have significant applications for peptide thioester synthesis and convergent chemical protein synthesis ligations. Further studies on larger peptide substrates are ongoing in our laboratory.

on-resin example 7-mer peptide acceptor 16 on a Rink amide resin furnished the thioester 17 in moderate yield, again highlighting the potential scope to extend this process to longer peptide chains (Table 3, entry 12). Having investigated thiol-Michael addition for the formation of N-cysteinyl peptide thioesters, we set out to explore the sequential N-deprotection and S,N-acyl transfer to furnish a native peptide bond, in a manner analogous to native chemical ligation.38 Following optimization studies on thioester 3a to furnish the corresponding N-acetyl cysteine derivative (Supporting Information), we selected peptide thioesters 15 and 17 to investigate peptide bond formation. Cysteinyl thioester 15 was treated with TFA (20% in DCM) for 1 h; the residue was stirred in DCM containing Amberlyst A21 to remove the residual TFA, and the resulting peptide 18 was recovered in 51% after only 20 min (Figure 3a). Similar conditions (Figure 3b) were applied to

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01013. Experimental procedures, crystallographic data, computational data, and spectral data (PDF) Accession Codes

CCDC 1904489−1904490 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. ORCID

Vito Genna: 0000-0002-4664-8086 Marc Devocelle: 0000-0001-7641-1306 Eoin M. Scanlan: 0000-0001-5176-2310 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Science Foundation Ireland (SFI) under Grant Nos. 15/CDA/3310 (E.M.S.) and 16/RI/3737 (S.O.F. and M.D.), and the Irish Research Council (R.P. and K.A.B.). The authors thank Dr. John O’Brien (School of Chemistry, Trinity College Dublin) for NMR support.

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REFERENCES

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Figure 3. Thioester synthesis and S,N-acyl shift of 15 (a) and 17 (b).

thioester 17 to furnish peptide 19, which was found by mass spectrometric analysis. Moreover, a further desulfurization step was performed to furnish the final alanine analogue 20 in a process analogous to NCL desulfurization (Figure 3b). In conclusion, for the first time, conjugate β,γ-C,S thiol-Michael addition has been explored for the formation of N-terminal cysteinyl peptide thioesters. The methodology differs from traditional transthioesterification protocols in that it is fast and irreversible. Both ionic and radical-mediated conditions offer efficient access to N-terminal cysteinyl thioesters, with an azido derivative reacting exclusively under radical conditions. The steric and electronic environment around the electrophilic center is critical to successful conjugate addition. A chemical ligation approach for introduction of ΔAla through SPPS is introduced for the first time. The methodology is compatible with aqueous buffered conditions at neutral pH, rendering it D

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DOI: 10.1021/acs.orglett.9b01013 Org. Lett. XXXX, XXX, XXX−XXX