Synthesis of ketone-containing peptides

Abstract

Peptide synthesis is performed by synthesizing a ketone-containing peptide backbone via O to C acyl shift, including ribosomal, solid phase, and chemical synthesis.

Description

Synthesis of Ketone-containing Peptides

[0001] Government Support Clause

[0002] This invention was made with government support under grant number 2002182 awarded by the National Science Foundation. The government has certain rights in the invention.

[0003] Introduction

[0004] Ribosomes have evolved over billions of years to catalyze a single reaction, formation of an amide bond between two α-amino acids. Recent work has shown that in addition to canonical and non-canonical α-amino acids (1, 2), under cellular conditions ribosomes also promote reactions of α-hydroxy acids (3) as well as certain β2-hydroxy (4) and β3-amino acids (5). Under optimized in vitro conditions and using chemically pre-acylated tRNAs, ribosomes also support reactions of α-thio acids (6) and a variety of non-α-amino acids, including N-terminal aramids and 1,3-dicarbonyls (7, 8), α-aminoxy and α-hydrazino acids (9), and cyclic β-amino acids (10). Despite these advances, there are no reports of a ribosomal product containing a newly formed backbone C–C bond as a ketone, due largely to the challenges of generating and maintaining a carbon nucleophile in water (Fig.1A). Enzymes can install backbone ketones via recognition of an 11-amino acid tag (11, 12) or biosynthetically to the terminus of peptides,(13) and chemical synthesis can ligate molecules containing a ketone onto the C-terminus or side chain (14–16). However, there is no general strategy to build C–C bonds as ketones internally within a genetically encoded polypeptide backbone.

[0005] Acyl shifts are well known to alter the peptide backbone. Intramolecular acyl shifts occur spontaneously and rapidly in the context of the native chemical ligation (NCL) (17) or during the final step of intein splicing (18). Recent work has shown that α-amine nucleophiles generated in close proximity to a reactive acyl group promote O to N acyl shifts that establish β2-peptide linkages within proteins in cells (19). In these cases, the acyl shifts make use of a side chain nucleophile and generate a thermodynamically stabilized amide product. Intramolecular acyl shifts are also initiated by the amide nitrogen during isoaspartate formation (Fig.1B) (20). Although pseudo-intramolecular decarboxylative Claisen reactions generate new C–C bonds via S to C acyl shifts during polyketide biosynthesis, in this case the carbon nucleophile is generated transiently and in close proximity to the electrophile (21). Intramolecular Claisen-type rearrangements and O to C acyl shifts to form C–C bonds have great precedent in chemical synthesis, but we know of no examples in which this reactivity has been used to edit the peptide backbone (22, 23). 1 B24-105-2WO

[0001] Summary of the Invention

[0002] We disclose that peptides containing a dehydrolactic acid motif rapidly isomerize to generate a backbone-embedded α,γ-diketoamide via a spontaneous O to C acyl shift. The dehydrolactic acid motif can be introduced into peptides ribosomally or via solid-phase synthesis using α-hydroxy phenylselenocysteine followed by oxidation. Subsequent incubation at physiological pH produces a α,γ-diketoamide that can be diversified using a variety of nucleophiles including hydrazines and hydroxylamines to form pyrazoles and oximes, respectively, all embedded directly within the polypeptide backbone. This general strategy, predicated on an intricate cascade of acyl rearrangements, incorporates a C–C bond as a ketone into the backbone of chemically and ribosomally synthesized peptides, yielding genetically encoded, new-to-nature biopolymers whose structures are one step closer to pharmacologically privileged natural products.

[0003] This invention is broadly utilized, as the hydroxy acid monomer can be incorporated anywhere within a peptide and does not have any sequence or motif constraints. One practical application of the invention is to expand the diversity and drug-like properties of cyclic peptide libraries prepared for mRNA display, where peptide products are screened for binding to protein targets and / or for cell permeability. Our invention permits incorporation of ketones, pyrazoles, and oximes into the backbone of these peptides, installing motifs that are more akin to small molecule drugs than the typical polyamide backbone.

[0004] The invention provides methods, compounds and compositions relating to synthesis of ketone-containing peptide backbone via O to C acyl shift, substantially as disclosed herein.

[0005] Aspects and embodiments of the invention include:

[0006] 1. A method of peptide synthesis, comprising synthesizing a ketone-containing peptide backbone via O to C acyl shift, substantially as disclosed herein, including ribosomal, solid phase, and chemical synthesis.

[0007] 2. The method of claim 1 comprising incorporating into the peptide backbone an α-L- hydroxy acid that can serve as a substrate for an aminoacyl tRNA synthetase to be incorporated into a peptide, and transform chemically into a dehydrolactic acid moiety.

[0008] 3. The method of claim 2, according to the reaction scheme: 2 B24-105-2WO, including alkyl, alkenyl, alkynyl, including cyclics and heterocyclics, and aryl and heteroaryl, such as substituted or unsubstituted: phenyl, cyclohexyl, furan, thiophene or azole;

[0012] (a) chemical synthesis, in vitro translation, cellular synthesis, etc.

[0013] (b) elimination via oxidation, alkylation, enzymes, etc.

[0014] 4. The method of claim 3 wherein:

[0015] the R substituents are independently C0-C6: aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl, alkyloxy, alkyl, alkenyl, alkynyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido, carboxyl, cyanyl, ester, haloformyl, hydroperoxyl, hydroxyl, imine, isocyanide, iscyante, N-tert-butoxycarbonyl, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, sulfhydryl, thiol, thiocyanyl,

[0016] 5. The method of claim 1 comprising using an enzyme that forms dehydroalanine from Cys or Ser, and also functions on the analogous ester to catalytically generate a dehydrolactic acid moiety.

[0017] 6. The method of claim 2 wherein the α-L-hydroxy acid is α-L-hydroxy- phenylselenocysteine (OH-SecPh) or an analog thereof:as described herein.

[0020] 7. The method of claim 2 comprising incorporating the α-L-hydroxy acid into the peptide backbone (e.g. by solid-phase peptide synthesis or ribosomal translation) to form an incorporated dehydrolactic acid, under conditions wherein the dehydrolactic acid monomer 3 B24-105-2WOisomerizes via a spontaneous O to C acyl shift under physiological conditions to form a peptide containing a 1,3-diketo amide.

[0021] 8. The method of claim 2, further comprising chemically diversifying the 1,3- diketoamide; for examples, using hydrazines to form pyrazoles and hydrazones, or hydroxylamines to form oximes, or nitronates to form tertiary alcohols.

[0022] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0023] Brief Description of the Drawings

[0024] Figs.1A-E. Acyl shifts represent a strategy to edit the polypeptide backbone. (A) Known and unknown reactions within the ribosome peptidyl transferase center (PTC). Amines are excellent and canonical nucleophiles for peptide bond formation, and alcohols also react within the PTC. While carbanions can act as nucleophiles, they are difficult to control at neutral pH. As a result, C–C bonds have yet to be formed directly within the PTC. (B) Established heteroatom-to-heteroatom acyl shifts that alter the peptide backbone, including native chemical ligation and isoaspartate formation. Recently reported backbone extension acyl rearrangements (BEAR) exploit O to N acyl shifts to post-translationally install β2-amino acid linkages into polypeptides and proteins. (C) Dehydroalanine (DHA) allows for post-translational installation of diverse side chains. (D) Dehydrolactic acid (DHL) permits post-translational backbone editing. DHL resembles DHA but contains a reactive ester once introduced into a polypeptide. Unlike DHA, DHL first undergoes an O to N acyl shift to reveal a pendant enol, which undergoes a subsequent N to C acyl shift, generating an α,γ-diketoamide product embedded within the polypeptide backbone. (E) The α,γ-diketoamide can be diversified using hydrazines to form pyrazoles and hydrazones, and with hydroxylamines to form oximes.

[0025] Figs 2A-B. DFT studies suggest that dehydrolactic acid isomerizes via an overall O to C acyl shift. (A) Shown is an energy landscape illustrating the calculated enthalpies (ΔHo) of intermediates along the proposed pathway between DHL 1 and enol 9. The proposed pathway begins with intramolecular cyclization of DHL tautomer 2 to form succinimide 3. Release of the ester bond in the tetrahedral intermediate generates the crucial enol-containing intermediate 4, which can tautomerize into unproductive intermediate 5 or cyclize with the acetamide carbonyl to generate the new C–C bond in 6. Final ring opening installs the second keto group in 7, and subsequent tautomerization ultimately leads to low-energy enol 9. (B) The transition state calculated for the key C–C bond forming step is characterized by an energy barrier (ΔH ) of 14.3 kcal / mol. 4 B24-105-2WO

[0026] Figs.3A-F. Tripeptide 12 containing a dehydrolactic acid monomer (DHL-peptide 12) isomerizes into an α,γ-diketoamide upon base treatment. (A) Scheme illustrating the structure of the DHL precursor OH-SecPh (10), its incorporation into tripeptide 11, and oxidation to produce DHL-peptide 12. (B) (Left) LC-HRMS chromatogram and spectrum of DHL-peptide 12, which is stable in methanol. (Right) Section of HMBC NMR spectrum of DHL-peptide 12 in methanol-d4, with three cross-peaks apparent in the amide carbonyl range. (C) (Left) LC-HRMS chromatogram and spectrum of DHL-peptide 12 following 4 hour incubation in 50 mM NaPi pH 8. (Right) Section of HMBC NMR spectrum of DHL-peptide 12 in methanol-d4after addition of 1 equivalent of TMG. Apparent are two cross-peaks in the amide carbonyl range, and one peak shifted downfield to the ketone carbonyl range. (D) Incubation of DHL-peptide 12 in 50 mM NaPi pH 7 followed by 20 mM hydrazine in 50 mM NaPi pH 6 generates a product containing a backbone pyrazole, 12a. (E) LC-HRMS of 12a showing the EIC overlaid on the TIC following reaction in (D). (F)1H NMR confirms the pyrazole structure of peptide 12a.

[0027] Figs.4A-C. DHL rearrangements proceed in multiple contexts and react with multiple α-nucleophiles. (A) Six additional tripeptides 13-17 containing the DHL precursor OH-SecPh were prepared and oxidized to generate DHL-peptides 18–22. (B) DHL-containing tripeptides first isomerize in buffer at pH 7 and are subsequently reacted with either hydrazine to form pyrazoles or O-methyl hydroxylamine to form oximes. (C) LC-HRMS chromatograms showing the EIC (red) corresponding to pyrazole-peptides 18a, 20a, and 22a (top) and oxime- peptides 19b, 18b, and 21b (bottom), and remaining DHL (gray) overlaid on the total ion chromatogram (TIC).

[0028] Figs.5A-C. DHL-containing peptides prepared via solid-phase peptide synthesis can be diversified into oximes and pyrazoles following an O to C acyl shift. (A) Structures of DHL-peptides 23–26. (B) Structures of α-nucleophiles a–i used to make pyrazole- and oxime- peptides. (C) Structures of pyrazole- and oxime-peptides and corresponding EICs and mass spectra from LC-HRMS following reactions with the α-nucleophiles, including thalidomide- peptide 23i, oxime 23e, pyrazole-peptide 25a, fluoro-benzyl pyrazole-peptide 24h, phenyl- pyrazole 26c, and benzoic acid-pyrazole 26g.

[0029] Figs.6A-E. Genetic encoding of a C–C bond. (A) Scheme illustrating the acylation of tRNAPylValwith OH-SecPh 10 using the M. alvus PylRS variant FRS1. Shown is the LC-MS total ion chromatogram (TIC) of the product mixture overlayed with the extracted ion chromatograms (EIC) corresponding to unreacted tRNAPylVal(non-acyl tRNA), mono-acylated tRNAPyl(m Pyl Valono-acyl) and diacylated tRNAVal(di-acyl) following incubation with OH-SecPh 10 and FRS1. This mixture was added to an in vitro translation reaction along with cDNA 5 B24-105-2WOcoding for peptide MALAVNA (S11), WT translation machinery, and the amino acids Ala, Leu, and Asn to generate OH-SecPh-containing peptide 25. (B) Scheme illustrating the oxidation of OH-SecPh-containing peptide 25 into DHL-peptide 24 and its diversification into pyrazole 24a. (C–E) Structures and mass spectra of (C) IVT-generated OH-SecPh-containing peptide 25; (D) DHL-peptide 24; and (E) pyrazole 24a.

[0030] Figs.7A-C. General method and examples of generating DHL within polypeptide. (A) A hydroxy acid containing a beta-selenium, sulfur, or oxygen can be incorporated into a peptide via chemical synthesis, in vitro ribosomal translation, or synthesis in live cells. Once in the peptide, the monomer side chain can be eliminated to produce an alpha / beta unsaturated amide and enol ester, which comprises the unique structure of DHL. Elimination could proceed using oxidation, alkylation, or enzymes. (B) The monomer most thoroughly investigated so far is OH- SecPh, which can be incorporated into peptides chemically and using the ribosome. Oxidation leads to DHL. (C) An analog, OH-BocSeK, can be incorporated into protein in live cells. Oxidation leads to DHL.

[0031] Figs.8A-D. OH-SeBocK can be incorporated into protein by cellular translation machinery using Amber codon suppression. (A) Protein gel displaying GFP protein (red box) in the expected mass range only when cells were supplemented with OH-SeBocK. GFP protein has stop codon (TAG) in the 2ndposition. Synthetase PylRS can acylate OH-SeBocK onto Pyl-tRNA coding for the TAG codon. (B) LC-MS chromatogram (left) and deconvoluted mass (right) of GFP-TAG protein after expression with OH-SeBocK. The observed mass matches the calculated mass of GFP-2TAG containing OH-SeBocK. (C) Protein gel displaying GFP protein (red box) in the expected mass range only when cells were supplemented with OH-SeBocK. GFP protein has stop codon (TAG) in the 200thposition. Synthetase PylRS can acylate OH-SeBocK onto Pyl-tRNA coding for the TAG codon. (D) LC-MS chromatogram (left) and deconvoluted mass (right) of GFP-TAG protein after expression with OH-SeBocK. The observed mass matches the calculated mass of GFP-200TAG containing OH-SeBocK.

[0032] Figs.9A-D. DHL-peptide 12 isomerizes under basic conditions. (A) LC-HRMS trace (left) and mass spectrum (right) of purified DHL-peptide 12, as seen in Fig.3. (B) LC-HRMS trace (left) and mass spectrum (right) following incubation of DHL-peptide 12 with 1 equivalent TMG in methanol for 15 minutes. (B–C) LC-HRMS trace (left) and mass spectrum (right) following incubation of DHL-peptide 12 with 50 mM NaPi pH 8 for (C) 4 hours (as seen in Fig. 3) and (D) 16 hours.

[0033] Fig.10. Extracted ion chromatograms (EIC) and total ion chromatograms (TIC) following reaction with DHL tripeptides (12,18-22) and hydrazine to form pyrazole-peptides 6 B24-105-2WO12a, 18a-22a. the EIC of pyrazole-peptide is shown as red, and the EIC of remaining DHL- peptide is shown as gray.

[0034] Fig.11. Extracted ion chromatograms (EIC) and total ion chromatograms (TIC) following reaction with DHL tripeptides (12,18-22) and O-methyl hydroxylamine to form oxime-peptides 12b, 18b-22b. the EIC of oxime-peptide is shown as red, and the EIC of remaining DHL-peptide is shown as gray.

[0035] Figs.12A-C. HO-SecPh peptides more rapidly form DHL in MeCN than in buffer. (A) OH-SecPh-peptide S7 was treated with 20 eq. H2O2 in either (B) acetonitrile or (C) 50 mM NaPi pH 6.5 for 1h or 22h. (B–C) Extracted ion chromatograms (EIC) corresponding to OH- SecPh-peptide S7 (gray) and DHL-peptide 23 (red) are overlaid on the total ion chromatogram (TIC). In water, removal of the acetyl group from the N-terminus is also observed (yellow).

[0036] Figs.13A-C. HO-SecPh peptides more rapidly form DHL in MeCN than in buffer. (A) OH-SecPh-peptide S8 was treated with 20 eq. H2O2 in either (B) acetonitrile or (C) 50 mM NaPi pH 6.5 for 1h or 22h. (B–C) Extracted ion chromatograms (EIC) corresponding to OH- SecPh-peptide S8 (gray) and DHL-peptide 24 (red) are overlaid on the total ion chromatogram (TIC). In water, cleavage of the ester bond in 24 is observed (blue).

[0037] Figs.14A-C. HO-SecPh peptides more rapidly form DHL in MeCN than in buffer. (A) OH-SecPh-peptide S9 was treated with 20 eq. H2O2 in either (B) acetonitrile or (C) 50 mM NaPi pH 6.5 for 1h or 22h. (B–C) Extracted ion chromatograms (EIC) corresponding to OH- SecPh-peptide S9 (gray) and DHL-peptide 26 (red) are overlaid on the total ion chromatogram (TIC). In water, cleavage of the N-terminal acetyl-Proline is also observed (yellow).

[0038] Figs.15A-D. DHL-peptide isomerization to the reactive diketone is pH and time- dependent. (A) DHL-peptide 23 was incubated in NaPi buffer pH 7, 8, or 9 between 15 – 75 mins, and then diluted in buffer containing hydrazine at pH 6 and incubated for 15 min before analysis by LC-HRMS. Mass spectrum of pyrazole-peptide 23a (right) (B) Time course from pH 7 condition. (C) Time course from pH 8 condition. (D) Time course from pH 9 condition. (B-D) Extracted ion chromatograms (EIC) corresponding to DHL-peptide (gray) and pyrazole-peptide (red) are overlaid on the total ion chromatogram (TIC).

[0039] Figs.16A-B. DHL-peptide isomerization to the reactive diketone is time-dependent. (A) DHL-peptide 25 was incubated in NaPi buffer pH 8 between 15 – 75 mins, and then diluted in buffer containing hydrazine at pH 6 and incubated for 15 min before analysis by LC-HRMS. (B) Time course from pH 8 condition. Extracted ion chromatograms (EIC) corresponding to DHL- peptide (gray) and pyrazole-peptide (red) are overlaid on the total ion chromatogram (TIC). Mass spectrum of pyrazole-peptide 25a (lower right).

[0040] Figs.17A-C. DHL-peptide isomerization to the reactive diketone is time-dependent. (A) 7 B24-105-2WODHL-peptide 26 was incubated in NaPi buffer pH 9 between 15 mins – 4h, and then diluted in buffer containing hydrazine at pH 6 and incubated for 1 hour before analysis by LC-HRMS. (B) Time course from pH 9 condition. Extracted ion chromatograms (EIC) corresponding to DHL- peptide (gray) and pyrazole-peptide (red) are overlaid on the total ion chromatogram (TIC). (C) Mass spectrum of pyrazole-peptide 26a.

[0041] Fig.18. DHL-peptide 23 reacts with a variety of alpha-nucleophiles to form substituted pyrazoles and oximes. Extracted ion chromatograms (EIC) corresponding to DHL-peptide (gray) and pyrazole- or oxime-peptide (red) are overlaid on the total ion chromatogram (TIC).

[0042] Fig.19. DHL-peptide 24 reacts with a variety of alpha-nucleophiles to form substituted pyrazoles and oximes. Extracted ion chromatograms (EIC) corresponding to DHL-peptide (gray) and pyrazole- or oxime-peptide (red) are overlaid on the total ion chromatogram (TIC).

[0043] Fig.20. DHL-peptide 25 reacts with a variety of alpha-nucleophiles to form substituted pyrazoles and oximes. Extracted ion chromatograms (EIC) corresponding to DHL-peptide (gray) and pyrazole- or oxime-peptide (red) are overlaid on the total ion chromatogram (TIC).

[0044] Fig.21. DHL-peptide 26 reacts with a variety of alpha-nucleophiles to form substituted pyrazoles and oximes. Extracted ion chromatograms (EIC) corresponding to DHL-peptide (gray) and pyrazole- or oxime-peptide (red) are overlaid on the total ion chromatogram (TIC).

[0045] Figs.22A-C. In vitro translation of peptide S10. (A) Total ion chromatogram (TIC) following in vitro translation with cDNA coding for peptide MALAVNA, including Val and excluding Met. (B) Extracted ion chromatogram (EIC) of the mass corresponding to peptide S10. (C) Mass spectrum of peptide S10.

[0046] Figs.23. In vitro translation of peptide 24 and subsequent reactions. (A) Total ion chromatogram (TIC, left) and extracted ion chromatogram (EIC, right) following in vitro translation with cDNA coding for peptide MALAVNA, including excluding both Val and Met. (B) Total ion chromatogram (TIC, left) and extracted ion chromatogram (EIC, right) following removal of translational machinery via MWCO filtration and desalting. (C) Total ion chromatogram (TIC, left) and extracted ion chromatogram (EIC, right) following 3h incubation with 20 mM H2O2 in MeCN. (D) Total ion chromatogram (TIC, left) and extracted ion chromatogram (EIC, right) following 1 hour incubation in 50 mM NaPi pH 8 followed by 30 minutes incubation in 20 mM hydrazine in 50 mM NaPi pH 6. (E–F) Mass spectrum of (E) peptide 27, (F) oxidized peptide S11, (G) DHL peptide 24, (H) pyrazole peptide 24a.

[0047] Description of Particular Embodiments of the Invention

[0048] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is 8 B24-105-2WOunderstood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0049] Installation and use of dehydrolactic acid to generate diketone-containing peptides and their derivatives

[0050] We sought to install a C–C linkage in a ribosomal product by generating a carbon nucleophile transiently and in close proximity to a reactive ester using a dehydrolactic acid (DHL) monomer. DHL is the ester analog of dehydroalanine (DHA) (24, 25), a species that may be generated within a polypeptide either biosynthetically (26, 27) or synthetically via oxidation of a selenocysteine analog (28) or elimination of serine or cysteine (29–31). While DHA reacts readily in an intermolecular fashion at the electrophilic β-carbon (24, 32) (Fig. 1C), we envisioned that DHL would react preferentially in an intramolecular fashion via a O to N acyl shift to reveal an imide with a pendant enol nucleophile (Fig.1D). Subsequent attack of the enol on the imide would generate an α,γ-diketoamide, formally replacing a C–N bond with a C–C bond. In a manner reflective of classic tricarbonyl chemistry (33), we envisioned that the newly formed α,γ-diketoamide could be subsequently diversified to embed substituted pyrazoles and oximes within the polypeptide backbone, generating ribosomal products whose structures begin to capture the diversity of synthetic materials (Fig.1E).

[0051] Computational evidence supports the feasibility of DHL rearrangements to form new C–C bonds

[0052] While O to C acyl shifts have precedent, the transformation proposed above does not. We suspected that the amide nitrogen initiates isomerization by reacting with the backbone ester, as seen during isoaspartate formation (20). A second acyl shift onto a carbon nucleophile could then be feasible, as seen during intramolecular Claisen rearrangements of allylic alcohols used to stereoselectively produce γ,δ-unsaturated esters (23), or the O to C acyl shift employed in the synthesis of benzofurans (22). The pathway we envisioned begins with conversion of DHL 1 into the high-energy tautomer (2) (Fig. 2A). Subsequent proximity-driven cyclization generates intermediate 3 and ultimately succinimide 4, revealing the nascent enol nucleophile in equilibrium with keto tautomer 5. Proximity-driven attack of the enol generates pyrrolidine- dione 6 in the C–C bond forming step. The second ring opening forms the diketone species 7, which tautomerizes to the low energy diketoamide (8) and enol (9).

[0053] We found that the transition state for this key C–C bond forming step was 14.3 kcal / mol, suggesting this step should be spontaneous and fast at room temperature. Final ring 9 B24-105-2WOopening installs the α,γ-diketoamide in 16, and tautomerization reveals the energy sink of enol 18, which is about 8 kcal / mol lower in enthalpy than the starting DHL 10 (Fig.2B).

[0054] Experimental evidence supports DHL rearrangements to form new C–C in model tripeptide

[0055] Encouraged by the computational data described above, we designed a model tripeptide to establish experimental evidence for the O to C DHL rearrangement. DHL was installed using an α-L-hydroxy-phenylselenocysteine (OH-SecPh) (10) precursor, which was synthesized via a known epoxide ring opening reaction (34) followed by hydrolysis and introduced into tripeptide 11. Treatment of tripeptide 11 with H2O2in MeOH for 1 hr afforded DHL-peptide 12, which was purified to homogeneity using RP-HPLC and characterized by NMR and LC-HRMS (Fig. 3A). Although purified DHL-peptide 12 was stable in MeOH (Fig.3B), addition of tetramethyl guanidinium (TMG) base for 15 min at RT led to the appearance of several chromatographically distinct species which possessed the same mass as DHL-peptide 12 when analyzed by LC- HRMS (Fig.9A-D). These isomeric species also arose from treatment of DHL-peptide 12 with 50 mM NaPi pH 8 and persisted for at least 8 hours (Fig.3C). When isolated, each peak re- equilibrated into the same set of multiple peaks, suggesting that base treatment of DHL-peptide 12 resulted in a set of at least three interconverting isomers.

[0056] Although the isomers could not be isolated, they could be partially characterized by NMR. The Heteronuclear Multiple Bond Correlation (HMBC) spectrum of purified DHL- peptide 12 in methanol-d4contains 3 carbonyl cross-peaks in the range expected for an amide, an ester, and a carbamate (170–180 ppm) (Fig. 3B). Treatment of DHL-peptide 12 with 1 equivalent of tetramethyl guanidinium (TMG) shifts one of these three cross-peaks downfield into the range expected for a ketone (198 ppm) (Fig.3C).

[0057] We confirmed the structure of α,γ-diketoamide isomer 12’ by characterizing its conversion into pyrazole 12a. DHL-peptide 12 was treated first with 50 mM NaPi pH 7 for 1 hour at RT and then with excess hydrazine at pH 6, as appropriate for a classic Knorr pyrazole synthesis (Fig.3D) (35). Within minutes after the addition of hydrazine, the chromatographically distinct but unisolable peaks that are evident in Fig.3C coalesced into a single chromatographic peak whose mass corresponded to pyrazole 12a (Fig.3E). NMR characterization of the product confirmed the structure of the pyrazole, further elucidating that intermediate 12’ is a α,γ-diketoamide as hypothesized (Fig.3F).

[0058] O to C rearrangements are efficient in tripeptides

[0059] Next we explored whether the two-step O to C rearrangement to generate a reactive α,γ- diketoamide would proceed in diverse ⍺-amino acid contexts. We synthesized five analogs of tripeptide 11 in which the N-terminal Boc-Ala was replaced by either Boc-Gly (13), Boc-Pro 10 B24-105-2WO(14) Boc-Glu(OtBu) (15), Boc-Phe (16), or Boc-Val (17) (Fig. 4A). Treatment of each tripeptide with H2O2in MeOH afforded the corresponding DHL-peptides 12 and 18–22 within an hour as determined by LC-HRMS. New DHL-peptides 18–22 were purified by RP-HPLC and their structures were confirmed by NMR. Each DHL-peptide was incubated at pH 7 for 1 hour and then combined at pH 6 with hydrazine (a) or O-methyl hydroxylamine (b) (Fig. 4B). Reactions were incubated for 6 hours at RT and analyzed by LC-HRMS.

[0060] All DHL-peptides were transformed into pyrazoles and oximes under these conditions. Pyrazole formation was quantitative for DHL-peptides containing an N-terminal Ala, Gly, and Glu(OtBu) residue, while reactions of DHL-peptides containing an N-terminal Pro, Phe, or Val contained a small amount of remaining DHL-peptide as determined by LC-HRMS (Figs.10-11). Unsurprisingly, these observations suggest that rearrangement is most efficient with reduced steric hindrance near the ester carbonyl. Example chromatograms are shown for pyrazole- peptides 18a, 20a, and 22a, and oxime-peptides 19b, 18b, and 21b (Fig. 4C). We observed little double addition of hydroxylamines to the model peptides, although we suspect that oxime formation can occur on either ketone, and that each oxime can exist as the E or Z isomer, as indicated by the multiple chromatographic peaks observed for the product.

[0061] DHL permits polypeptide backbone editing on peptides made on the solid phase

[0062] The unique reactivity of an α,γ-diketoamide would be of great importance within a peptide because of its propensity to be orthogonally labeled and diversified into drug-like motifs. Incorporation of ketones into polypeptides (>5 amino acids) is challenging on the solid phase, and is often limited to coupling protected ketones on the termini or side-chains (14–16). We found that OH-SecPh could be installed as in any depsipeptide by SPPS, and that the resultant peptides could be rearranged and diversified with many α-nucleophiles. We used a depsi-dipeptide block containing α-OH-SecPh preceded by Gly (S5) or Ala (S6) to prepare a series of hexa- or heptapeptides with a variety of natural side chains (SM section 2). Following resin cleavage, deprotection, and purification, these peptides were treated with H2O2to generate DHL-peptides 23–26 (Fig.5A). Although oxidative elimination to generate peptides 23–26 proceeded slowly in water (>1 hour), the reaction was accelerated in aprotic solvents such as acetonitrile (Figs.12A-C; Figs.13-A-C; Figs.14A-C) (36). DHL-peptides 23–26 formed within 1 h in MeCN and H2O2and were dried via lyophilization.

[0063] DHL peptides 23–26 were then isomerized in 50 mM NaPi pH 7–9 for varying times and derivatized with a variety of substituted hydrazines and hydroxylamines (a–i) (Fig. 5B). We first investigated the isomerization of DHL-peptides 23–26 by incubating in pH 7, 8, or 9 for 15 minute intervals before diluting in NaPi pH 6 containing excess hydrazine. While higher pH enhanced the rate of isomerization, the majority of the sequences isomerized fully within 1h at 11 B24-105-2WOpH 8 and converted to the corresponding pyrazole-peptide after 15 minutes (Figs. 15A-D; Figs.16A-B). The exception was Pro-containing DHL-peptide 26 which required a 4h incubation in NaPi pH 9 and then excess hydrazine in NaPi pH 6 for 1h before fully converting to pyrazole peptide 26a (Figs. 17A-C).

[0064] In addition to hydrazine, the peptides were diversified with a variety of other α- nucleophiles. The highest yielding reactions occurred with electron-rich hydrazines, such as 2- hydrazine-benzoic acid (g) and 2-fluoro-benzyl hydrazine (h), while the electron-poor 4-cyano- phenyl hydrazine (d) showed less conversion of DHL-peptide. O-benzyl hydroxylamine (f) and O-methyl hydroxylamine (b) also efficiently formed their respective oximes. Finally, the thalidomide hydrazine (i), an analog of a known binder to E3 ligase CBRN, also led to a significant amount of substituted pyrazole formation. Example chromatograms are shown for synthesis of thalidomide-peptide 23i, oxime 23e, pyrazole-peptide 25a, fluoro-benzyl pyrazole- peptide 24h, phenyl-pyrazole 26c, and benzoic acid-pyrazole 26g are shown in Fig.5C, with the remainder shown in the supplemental information (Figs. 18-20). These labeling reactions demonstrate the ease with which DHL-peptides made via SPPS can be diversified into diverse heterocycles or bioconjugated materials.

[0065] DHL permits polypeptide backbone editing on peptides made by the ribosome

[0066] Encouraged by the reactivity described above, we sought to establish conditions appropriate for DHL rearrangements and modifications in the context of genetically encoded peptides prepared using in vitro translation. We tested a variety of synthetase / tRNA pairs, including PhSeRS-K4 which was engineered to incorporate SecPh,(37) and found efficient activity with FRS1, an analog of M. alvus PylRS that acylates tRNAPylwith several α-hydroxy phenylalanine derivatives (38). FRS1 acylated tRNAPylValwith OH-SecPh within 4 h at 37 °C to produce a mixture of mono- and diacylated tRNAPylValproducts in 76% yield (Fig. 6A). The acylated tRNAPylValproducts were added to a commercial in vitro translation system composed of purified components (PureExpress, NEB) (39) lacking Met and Val, along with cDNA encoding MALAVNA (S11), with Val as the recoded position (Fig 22A-C). After 2 h at 37oC, the peptide products were isolated and desalted; LC-HRMS revealed efficient biosynthesis of peptide 25 (Fig. 6B). We found desalting to be necessary before DHL formation, likely due to excess reducing agent in the translation mixture.

[0067] The ribosomal product 25 was then oxidized, isomerized, and subjected to Knorr pyrazole synthesis. Desalted translation mixture containing 25 was first treated with 20 mM H2O2in MeCN for 45 min, and monitored by LC-HRMS where DHL peptide 24 was detected (Fig.6D). The peptide was then lyophilized to remove oxidant and reconstituted in 50 mM NaPi pH 8 to isomerize for 1h. Subsequent addition of 20 mM hydrazine in 50 mM NaPi pH 6 12 B24-105-2WOafforded the pyrazole-peptide 24a (Fig. 6F, Figs.23A-H). DHL-peptide 24 was made and could be derivatized into ribosomal products that resemble chemically synthesized molecules.

[0068] Discussion

[0069] While the ribosome catalyzes bond formation largely by induced proximity, it has thus far been limited–in vitro or in vivo–to O, N, and S nucleophiles. C–C bonds have yet to be formed within the PTC because of the challenges associated with stabilizing and maintaining a carbanion in water. Here we overcome this limitation by generating the reactive carbon nucleophile only when needed and poised for nucleophilic attack on a reactive carbonyl. The reaction we describe involves two successive acyl shifts to effectively convert a backbone ester into a ketone by forming a new C–C bond. The first O to N acyl shift is reminiscent of the spontaneous isomerization of aspartate into isoaspartate via an imide intermediate. The second N to C acyl shift generates a new C–C bond and the final α,γ-diketoamide product. This second acyl shift, in which a transient enol attacks a proximal acyl group, is akin to decarboxylative Claisen reactions used to form C–C bonds in polyketide natural products. Like a polyketide, the final α,γ-diketoamide can be subsequently diversified to generate genetically encoded materials with diverse and valuable heterocycles embedded within the peptide backbone.

[0070] The incorporation of DHL significantly enlarges the ability to incorporate non-peptidic elements into genetically encoded peptides or proteins, in vitro or in cells. This advancement provides otherwise non-existent opportunities to expand protein and polypeptide structure and function. For example, cyclic peptides are emerging as powerful therapeutic candidates, but often suffer from proteolysis and poor cell permeability due to solvent-exposed amide protons (40) and backbone N-methylation is a hallmark of bioactive cyclic peptides and natural products (41). Unnatural peptide backbones, such as additional methylenes or heterocycles, can enhance membrane permeability while adding chemical diversity (42, 43). While recent years have seen notable advances in the ribosomal synthesis of multiple types of amide bonds, still no general strategy exists to embed genetically encoded C–C bonds as ketones internally within the polypeptide backbone.

[0071] References

[0072] 1. C. C. Liu, P. G. Schultz, Adding New Chemistries to the Genetic Code. Annual Review of Biochemistry 79, 413–444 (2010).

[0073] 2. C. Noren, S. Anthony-Cahill, M. Griffith, P. Schultz, A general method for site- specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

[0074] 3. S. Fahnestock, A. Rich, Ribosome-Catalyzed Polyester Formation. Science 173, 340–343 (1971).

[0075] 4. N. X. Hamlish, A. M. Abramyan, B. Shah, Z. Zhang, A. Schepartz, Incorporation 13 B24-105-2WOof Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase. ACS Cent. Sci.10, 1044–1053 (2024).

[0076] 5. C. Melo Czekster, W. E. Robertson, A. S. Walker, D. Söll, A. Schepartz, In Vivo Biosynthesis of a β-Amino Acid-Containing Protein. J. Am. Chem. Soc.138, 5194–5197 (2016).

[0077] 6. R. Takatsuji, K. Shinbara, T. Katoh, Y. Goto, T. Passioura, R. Yajima, Y. Komatsu, H. Suga, Ribosomal Synthesis of Backbone-Cyclic Peptides Compatible with In Vitro Display. J. Am. Chem. Soc.141, 2279–2287 (2019).

[0078] 7. O. Ad, K. S. Hoffman, A. G. Cairns, A. L. Featherston, S. J. Miller, D. Söll, A. Schepartz, Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro. ACS Cent. Sci.5, 1289–1294 (2019).

[0079] 8. I. J. Knudson, T. L. Dover, D. A. Dilworth, C. Paloutzian, H. Cho, A. Schepartz, S. J. Miller, Chemo-ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded quinolines. ChemRxiv [Preprint] (2024). https: / / doi.org / 10.26434 / chemrxiv-2024- kvdpq.

[0080] 9. T. Katoh, H. Suga, Consecutive Ribosomal Incorporation of α-Aminoxy / α- Hydrazino Acids with l / d-Configurations into Nascent Peptide Chains. J. Am. Chem. Soc.143, 18844–18848 (2021).

[0081] 10. T. Katoh, T. Sengoku, K. Hirata, K. Ogata, H. Suga, Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic β-amino acids. Nat. Chem.12, 1081–1088 (2020).

[0082] 11. B. I. Morinaka, E. Lakis, M. Verest, M. J. Helf, T. Scalvenzi, A. L. Vagstad, J. Sims, S. Sunagawa, M. Gugger, J. Piel, Natural noncanonical protein splicing yields products with diverse β-amino acid residues. Science 359, 779–782 (2018).

[0083] 12. D. Richter, E. Lakis, J. Piel, Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous β-amino acid dienophiles. Nat. Chem.15, 1422–1430 (2023).

[0084] 13. D. T. Nguyen, L. Zhu, D. L. Gray, T. J. Woods, C. Padhi, K. M. Flatt, D. A. Mitchell, W. A. van der Donk, Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino- α-keto Acid Groups by Three Different Metalloenzymes. ACS Cent. Sci.10, 1022–1032 (2024).

[0085] 14. A. Citarella, N. Micale, Peptidyl Fluoromethyl Ketones and Their Applications in Medicinal Chemistry. Molecules 25, 4031 (2020).

[0086] 15. L. A. Marcaurelle, C. R. Bertozzi, Direct incorporation of unprotected ketone groups into peptides during solid-phase synthesis: Application to the one-step modification of peptides with two different biophysical probes for FRET. Tetrahedron Letters 39, 7279–7282 (1998). 14 B24-105-2WO

[0087] 16. P. Marceau, C. Buré, A. F. Delmas, Efficient synthesis of C-terminal modified peptide ketones for chemical ligations. Bioorganic & Medicinal Chemistry Letters 15, 5442– 5445 (2005).

[0088] 17. P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Synthesis of Proteins by Native Chemical Ligation. Science 266, 776–779 (1994).

[0089] 18. N. H. Shah, T. W. Muir, Inteins: Nature’s Gift to Protein Chemists. Chem Sci 5, 446–461 (2014).

[0090] 19. L. T. Roe, C. K. Schissel, T. L. Dover, B. Shah, N. X. Hamlish, S. Zheng, D. A. Dilworth, N. Wong, Z. Zhang, A. Chatterjee, M. B. Francis, S. J. Miller, A. Schepartz, Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2- peptide linkages. bioRxiv [Preprint] (2023). https: / / doi.org / 10.1101 / 2023.10.03.560714.

[0091] 20. D. W. Aswad, M. V. Paranandi, B. T. Schurter, Isoaspartate in peptides and proteins: formation, significance, and analysis. Journal of Pharmaceutical and Biomedical Analysis 21, 1129–1136 (2000).

[0092] 21. A. M. Soohoo, D. P. Cogan, K. L. Brodsky, C. Khosla, Structure and Mechanisms of Assembly-Line Polyketide Synthases. doi: 10.1146 / annurev-biochem-080923- 043654 (2024).

[0093] 22. G. Kretzschmar, V. Kraft, T. Olpp, K. Rossen, Process for the production of benzofurans (2010). https: / / patents.google.com / patent / WO2010136500A1 / en?oq=WO+2010136500.

[0094] 23. C. Agami, F. Couty, G. Evano, Claisen Rearrangements of Allylic and Propargylic Alcohols Prepared by an N-Boc-2-acyloxazolidine Methodology − Application to the Synthesis of Original Chiral Building Blocks. European Journal of Organic Chemistry 2002, 29–38 (2002).

[0095] 24. J. Dadová, S. R. Galan, B. G. Davis, Synthesis of modified proteins via functionalization of dehydroalanine. Current Opinion in Chemical Biology 46, 71–81 (2018).

[0096] 25. X.-P. Fu, Y. Yuan, A. Jha, N. Levin, A. M. Giltrap, J. Ren, D. Mamalis, S. Mohammed, B. G. Davis, Stereoretentive Post-Translational Protein Editing. ACS Cent. Sci.9, 405–416 (2023).

[0097] 26. L. M. Repka, J. R. Chekan, S. K. Nair, W. A. van der Donk, Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes. Chem. Rev.117, 5457–5520 (2017).

[0098] 27. J. M. Gavaret, J. Nunez, H. J. Cahnmann, Formation of dehydroalanine residues during thyroid hormone synthesis in thyroglobulin. J Biol Chem 255, 5281–5285 (1980).

[0099] 28. N. M. Okeley, Y. Zhu, W. A. van der Donk, Facile Chemoselective Synthesis of Dehydroalanine-Containing Peptides. Org. Lett.2, 3603–3606 (2000). 15 B24-105-2WO

[0100] 29. H. Weiner, W. N. White, D. G. Hoare, D. E. Koshland, The formation of anhydrochymotrypsin by removing the elements of water from the serine at the active site. J Am Chem Soc 88, 3851–3859 (1966).

[0101] 30. T. J. Holmes, R. G. Lawton, Cysteine modification and cleavage of proteins with 2-methyl-N1-benzenesulfony-N4-bromoacetylquinonediimide. J Am Chem Soc 99, 1984–1986 (1977).

[0102] 31. J. M. Chalker, S. B. Gunnoo, O. Boutureira, S. C. Gerstberger, M. Fernández- González, G. J. L. Bernardes, L. Griffin, H. Hailu, C. J. Schofield, B. G. Davis, Methods for converting cysteine to dehydroalanine on peptides and proteins. Chemical Science 2, 1666–1676 (2011).

[0103] 32. M. Liu, M. Sovrovic, H. Suga, S. A. K. Jongkees, Phosphine addition to dehydroalanine for peptide modification. Org. Biomol. Chem.20, 3081–3085 (2022).

[0104] 33. H. H. Wasserman, J. Parr, The Chemistry of Vicinal Tricarbonyls and Related Systems. Acc. Chem. Res.37, 687–701 (2004).

[0105] 34. P. A. Bartlett, P. M. Chouinard, Stereocontrolled synthesis of (E)- and (Z)-3- deuteriophosphoenolpyruvate. J. Org. Chem.48, 3854–3855 (1983).

[0106] 35. L. Knorr, Einwirkung von Acetessigester auf Phenylhydrazin. Berichte der deutschen chemischen Gesellschaft 16, 2597–2599 (1883).

[0107] 36. H. J. Reich, S. Wollowitz, J. E. Trend, F. Chow, D. F. Wendelborn, Syn elimination of alkyl selenoxides. Side reactions involving selenenic acids. Structural and solvent effects of rates. J. Org. Chem.43, 1697–1705 (1978).

[0108] 37. J. Wang, S. M. Schiller, P. G. Schultz, A Biosynthetic Route to Dehydroalanine- Containing Proteins. Angewandte Chemie 119, 6973–6975 (2007).

[0109] 38. R. Fricke, C. V. Swenson, L. T. Roe, N. Hamlish, O. Ad, S. Smaga, C. L. Gee, A. Schepartz, “Orthogonal synthetases for polyketide precursors” (preprint, Synthetic Biology, 2022); https: / / doi.org / 10.1101 / 2022.02.28.482149.

[0110] 39. Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Cell-free translation reconstituted with purified components. Nat Biotechnol 19, 751–755 (2001).

[0111] 40. G. Bhardwaj, J. O’Connor, S. Rettie, Y.-H. Huang, T. A. Ramelot, V. K. Mulligan, G. G. Alpkilic, J. Palmer, A. K. Bera, M. J. Bick, M. Di Piazza, X. Li, P. Hosseinzadeh, T. W. Craven, R. Tejero, A. Lauko, R. Choi, C. Glynn, L. Dong, R. Griffin, W. C. van Voorhis, J. Rodriguez, L. Stewart, G. T. Montelione, D. Craik, D. Baker, Accurate de novo design of membrane-traversing macrocycles. Cell 185, 3520-3532.e26 (2022).

[0112] 41. M. A. Abdalla, L. J. McGaw, Natural Cyclic Peptides as an Attractive Modality 16 B24-105-2WOfor Therapeutics: A Mini Review. Molecules 23, 2080 (2018).

[0113] 42. J. R. Frost, C. C. G. Scully, A. K. Yudin, Oxadiazole grafts in peptide macrocycles. Nature Chem 8, 1105–1111 (2016).

[0114] 43. P. J. Salveson, A. P. Moyer, M. Y. Said, G. Gӧkçe, X. Li, A. Kang, H. Nguyen, A. K. Bera, P. M. Levine, G. Bhardwaj, D. Baker, Expansive discovery of chemically diverse structured macrocyclic oligoamides. Science 384, 420–428 (2024).

[0115] Supplementary Information

[0116] General Synthetic Details

[0117] All reactions were carried out under ambient atmosphere unless otherwise noted. Room temperature (rt or RT) is defined as 21–23 °C. All reagents were obtained from commercial sources and used without further purification, unless otherwise noted. Deionized water was used for reactions, extraction solutions, and reverse phase chromatography. Acetonitrile and ethyl acetate used for chromatography were High-Performance Liquid Chromatography (HPLC) grade, while hexanes and methanol used for chromatography was certified ACS grade. Analytical thin-layer chromatography (TLC) was performed using 60 Å Silica Gel F254 pre- coated plates (0.25 mm thickness). TLC plates were visualized by irradiation with a UV lamp or with Hanessian’s stain (cerium ammonium molybdate). Flash chromatography was performed using a Teledyne ISCO Combiflash NextGen 300+ equipped with a 4, 12, or 24 g RediSep columns for normal phase column chromatography. Reverse phase HPLC purification was performed on an Waters LC Prep 150 system equipped with a Waters 2998 UV photodiode array detector, a Waters 2707 autosampler, and an Waters Fraction Collector III using a preparative reverse phase C18 column (CSH C1819 x 150 mm OBD Column 5 μm). The mobile phase for HPLC was water with 0.1% (v / v) trifluoroacetic acid (solvent A) and acetonitrile with 0.1% (v / v) trifluoroacetic acid (solvent B), at a flow rate of 20 mL / min. Analytes were collected based on their absorbance at 280 nm or 214 nm. Routine1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500, or 600 MHz spectrometers at ambient temperature unless otherwise stated. Chloroform-d was purchased from Cambridge Isotope Laboratories and used without further purification. Methanol-d4was purchased from Sigma Aldrich. Spectra were processed using MestReNova 14.2.0 using the automatic phasing, polynomial baseline correction capabilities, and zero-filling. Splitting was determined using the automatic multiplet analysis function with intervention as necessary. Spectral data are reported as follows: chemical shift (multiplicity [singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), pentet (p), multiplet (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplet of doublets (dtd), doublet of doublet of doublet of doublets (dddd), doublet of triplets (dt), triplet of 17 B24-105-2WOdoublets (td), etc.], coupling constant (Hz), integration). The abbreviation “app” denotes an apparent multiplicity (e.g., “app t” denotes an apparent triplet). Chemical shifts are reported in parts per million (ppm, δ), and coupling constants are reported in Hz.1H resonances are referenced to solvent residual peaks for CDCl3(7.26 ppm) or to methanol-d4(3.31 ppm)1. Routine13C NMR spectra were recorded on a Bruker 500 (126) or 600 (151) MHz spectrometers with protons fully decoupled.13C Resonances are reported in ppm relative to solvent residual peaks for CDCl3(77.16 ppm) or DMSO-d6(39.52 ppm) or methanol-d4(49.00 ppm)

[0118] Synthesis of α-hydroxy-phenylselenocysteine and derivatives

[0120] Methyl (R)-2-hydroxy-3-(phenylselanyl)propanoate (S2)

[0121] (Following a procedure adapted from Bartlett et al., 1983) (1). Hexanes (180 mL) and methyl (2S)-glycidate (6.1 g, 0.06 mol) were added to a dried flask under nitrogen and cooled to -78 °C. In a separate flask, diphenyldiselenide (9.4 g, 0.03 mol) was added to a dried flask under nitrogen, and a solution of DIBALH (60 mL, 0.06 mol) was added with stirring. Once this mixture had fully dissolved and the evolution of gas ceased, it was transferred dropwise with stirring to the flask containing 16. The mixture was stirred for 2 h at -78 °C before being allowed to warm slowly to room temperature; stirring was continued for an additional 16 h. Concentrated phosphate buffer (500 mL, pH 3) was then added until the mixture reached pH 3. Saturated Rochelle’s salt (500 mL) was added, and the aqueous phase was extracted three times with EtOAc (200 mL). Combined organic layers were dried over Na2SO4, concentrated under vacuum, and purified by flash chromatography (0-100% EtOAc) to afford S2 as a yellow oil (9.4 g, 60%).

[0122] 1H NMR (600 MHz, CDCl3): δ 7.54 – 7.57 (m, 2H), 7.24 – 7.28 (m, 3H), 4.47 (dd, J = 5.4, 4.4 Hz, 1H), 3.58 (s, 3H), 3.34 (dd, J = 13.1, 4.4 Hz, 1H), 3.25 (dd, J = 13.1, 5.4);

[0123] 13C NMR (150 MHz, CDCl3): δ 173.4, 133.6, 129.3, 127.6, 69.6, 52.6, 32.8

[0124] LC-HRMS (ESI) m / z calcd. for C10H12NaO3Se+[MNa]+: 282.9844, found: 282.9839 18 B24-105-2WO

[0125]

[0126] (R)-2-hydroxy-3-(phenylselanyl)propanoic acid (OH-SecPh) (10)

[0127] To a vial containing S2 (3.7 g, 14 mmol) was added a 2 M solution of LiOH in THF (36 mL, 71 mmol) with stirring. After 30 minutes, 2 M HCl (20 mL) was added, and the aqueous mixture was extracted three times with EtOAc. The combined organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to yield 10 as a pale yellow solid (3.5 g, 99%).

[0128] 1H NMR (600 MHz, CDCl3): δ 7.54 – 7.57 (m, 2H), 7.23 – 7.27 (m, 3H), 4.45 (dd, J = 6.5, 4.2 Hz, 1H), 3.39 (dd, J = 13.2, 4.2 Hz, 1H), 3.22 (dd, J = 13.2, 6.5 Hz, 1H);

[0129] 13C NMR (150 MHz, CDCl3): δ 176.6, 133.7, 129.4, 128.7, 127.9, 69.4, 32.5;

[0130] LC-HRMS (ESI) m / z calcd. for C9H10NaO3Se+[MNa]+: 268.9687, found: 268.9670

[0131] Synthesis of DHL-tripeptides

[0134] To a vial containing 10 (500 mg, 2.0 mmol) was added NHS (235 mg, 2.0 mmol) and anhydrous THF (5 mL) with stirring. Then, DIC was added (319 µL, 2.0 mmol) and the reaction was stirred until completion as monitored via LC-MS. The solution was then purified directly by flash chromatography (0-100% EtOAc / Hexanes) to yield S3 as a white solid (547 mg, 80%).

[0135] 1H NMR (500 MHz, CDCl3): δ 7.52 – 7.56 (m, 2H), 7.30 – 7.35 (m, 2H), 7.23 – 7.29 (m, 6H), 6.98 – 7.06 (s, 1H), 4.40 (dd, J = 5.9, 1.6 Hz, 2H), 4.19 (dd, J = 8.4, 3.9 Hz, 1H), 3.52 (dd, J = 13.2, 3.9 Hz, 1H), 3.20 – 3.33 (m, 1H), 3.13 (dd, J = 13.2, 8.4 Hz, 1H),

[0136] 13C NMR (125 MHz, CDCl3): δ 171.6, 137.8, 133.3, 129.6, 128.9, 128.1, 127.9, 127.8, 70.2, 43.4, 34.0

[0137] LC-HRMS (ESI) m / z calcd. for C16H17NNaO2Se+[MH]+: 336.0497, found: 336.0536 19 B24-105-2WOprop-1-en-2-yl (tert-butoxycarbonyl)-L-alaninate (12) , . l) was added to a solution of S3 (150 mg, 0.5 mmol) and Boc- Ala-OH (212 mg, 1.1 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 11 and Boc-Ala. The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (395 µL of 50% solution, 6.7 mmol) was then added with stirring. Upon completion as determined by LC-MS, the mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield the DHL-containing peptide 12 as a white solid (76 mg, 51% over 2 steps).

[0141] 1H NMR (500 MHz, CDCl3): δ 7.73 (s, 1H), 7.30 (d, J = 4.4 Hz, 4H), 7.23 (q, J = 4.4, 1H), 6.21 (s, 1H), 5.31 (s, 1H), 5.01 (d, J = 5.7 Hz, 1H), 4.56 (dd, J = 14.9, 6.3 Hz, 1H), 4.45 (dd, J = 14.9, 5.7 Hz, 1H), 4.23 (qd, J = 7.2, 5.7 Hz, 1H), 1.46 (d, J = 7.2 Hz, 3H), 1.31 (s, 9H)

[0142] 13C NMR (125 MHz, CDCl3): δ 171.4, 161.1, 156.3, 146.8, 138.0, 128.7, 127.9, 127.4, 111.7, 81.2, 50.0, 43.7, 28.3, 17.0

[0143] LC-HRMS (ESI) m / z calcd. for C18H24N2NaO5+[MNa]+: 371.1577, found: 371.1585-1H-pyrazol-3-yl)ethyl)carbamate (12a)

[0146] To a solution of DHL-peptide 12 (10 mg, 0.03 mmol) was added 50 mM NaPi pH 7 (850 µL) and MeOH (575 µL), which was incubated with stirring at room temperature for 1 h. Then, hydrazine (21 µL, 0.4 mmol) in 50 mM NaPi pH 5 (850 µL) was added to the mixture and stirred for 1 h. The solution was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield the pyrazole-containing peptide 12a as a white solid (7.3 mg, 74%).

[0147] 1H NMR (500 MHz, CDCl3): δ 7.31 – 7.34 (m, 4H), 7.26 – 7.29 (m, 1H), 6.69 (s, 1H), 4.89 (s, 1H), 4.81 (p, J = 6.9 Hz, 1H), 4.58 – 4.65 (m, 2H), 1.56 (d, J = 7.0 Hz, 3H), 1.44 (s, 9H);

[0148] 13C NMR (125 MHz, CDCl3): δ 162.4, 156.8, 148.7, 145.9, 138.1, 128.9, 127.9, 127.7, 102.9, 80.9, 43.5, 42.1, 28.5, 18.5; 20 B24-105-2WO

[0149] LC-HRMS (ESI) m / z calcd. For C18H25N4O3+[MH]+: 345.1921, found: 345.1918 1-en-2-yl (tert-butoxycarbonyl)glycinate (18)added to a solution of S3 (79 mg, 0.2 mmol) and Boc-Gly- OH (91 mg, 0.5 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 13 and Boc-Gly. The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (152 µL of 50% solution, 3 mmol) was then added with stirring. Upon completion as determined by LC-MS, the mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield DHL-peptide 18 as a white solid (24 mg, 42% over 2 steps).

[0153] 1H NMR (500 MHz, CDCl3): δ 7.47 (s, 1H), 7.23 – 7.34 (m, 5H), 6.19 (s, 1H), 5.37 (s, 1H), 5.18 (d, J = 5.9 Hz, 1H), 4.51 (d, J = 5.9 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 1.35 (s, 9H)

[0154] 13C NMR (125 MHz, CDCl3): δ 168.0, 161.2, 156.9, 146.5, 137.8, 128.7, 128.0, 127.6, 111.7, 81.3, 43.8, 43.2, 28.3

[0155] LC-HRMS (ESI) m / z calcd. for C17H22N2NaO5+[MNa]+: 357.1421, found: 357.1421

[0156]

[0157] 1-(3-(benzylamino)-3-oxoprop-1-en-2-yl) 5-(tert-butyl) (tert-butoxycarbonyl)-L- glutamate (19)

[0158] NMI (60 µL, 0.75 mmol) was added to a solution of S3 (50 mg, 0.15 mmol) and Boc- Glu(OtBu)-OH (113 mg, 0.4 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 14 and Boc- Glu(OtBu). The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (105 µL of 50% solution, 1.8 mmol) was then added with stirring. Upon completion as determined by 21 B24-105-2WOLC-MS, the mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield DHL-peptide 18 as a white solid (19 mg, 34% over 2 steps).

[0159] 1H NMR (500 MHz, CDCl3): δ 7.88 (s, 1H), 7.29 (d, J = 3.8 Hz, 4H), 7.20 – 7.25 (m, 1H), 6.23 (s, 1H), 5.44 (d, J = 5.9 Hz, 1H), 5.36 (s, 1H), 4.56 (dd, J = 14.9, 6.3 Hz, 1H), 4.46 (dd, J = 14.9, 5.8 Hz, 1H), 4.20 (dt, J = 8.2, 5.8 Hz, 1H), 2.34 – 2.46 (m, 2H), 2.04 – 2.18 (m, 2H), 1.44 (s, 9H), 1.31 (s, 9H)

[0160] 13C NMR (125 MHz, CDCl3): δ 172.4, 170.5, 161.3, 156.6, 146.6, 137.9, 128.7, 127.9, 127.4, 112.2, 81.7, 81.1, 54.2, 43.8, 31.7, 28.3, 28.2, 25.9

[0161] LC-HRMS (ESI) m / z calcd. for C24H34N2NaO7+[MNa]+: 485.2258, found: 485.2261 1-en-2-yl) 1-(tert-butyl) (S)-pyrrolidine-1,2-

[0164] NMI (60 µL, 0.75 mmol) was added to a solution of S3 (50 mg, 0.15 mmol) and Boc- Pro-OH (80 mg, 0.4 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 15 and Boc-Pro. The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (145 µL of 50% solution, 2.5 mmol) was then added with stirring. Upon completion as determined by LC-MS, the mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield the DHL-peptide 20 as a white solid (30 mg, 49 % over 2 steps).

[0165] 1H NMR (500 MHz, CDCl3): δ 8.24 (s, 1H), 7.27 – 7.30 (m, 4H), 7.19 – 7.24 (m, 1H), 6.24 (s, 1H), 5.27 (s, 1H), 4.57 (dd, J = 15.0, 6.4 Hz, 1H), 4.44 (dd, J = 15.0, 5.8 Hz, 1H), 4.35 (dd, J = 8.2, 5.4 Hz, 1H), 3.41 – 3.54 (m, 2H), 2.24 – 2.33 (m, 1H), 2.01 – 2.14 (m, 2H), 1.88 – 1.98 (m, 1H), 1.31 (s, 9H)

[0166] 13C NMR (125 MHz, CDCl3): δ 171.3, 161.3, 155.5, 146.9, 138.1, 128.6, 127.8, 127.3, 112.3, 81.1, 59.1, 47.0, 43.7, 30.2, 28.4, 24.9

[0167] LC-HRMS (ESI) m / z calcd. for C20H26N2NaO5+[MNa]+: 397.1734, found: 397.1741 22 B24-105-2WO

[0168]

[0169] 3-(benzylamino)-3-oxoprop-1-en-2-yl (tert-butoxycarbonyl)-L-phenylalaninate (21)

[0170] NMI (60 µL, 0.75 mmol) was added to a solution of S3 (50 mg, 0.15 mmol) and Boc- Phe-OH (99 mg, 0.4 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 16 and Boc-Phe. The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (136 µL of 50% solution, 2.3 mmol) was then added with stirring. Upon completion as determined by LC-MS, the mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield DHL-peptide 21 as a white solid (29 mg, 45% over 2 steps).

[0171] 1H NMR (500 MHz, CDCl3): δ 7.59 (s, 1H), 7.19 – 7.36 (m, 10H), 6.14 (s, 1H), 5.00 (s, 1H), (dd, J = 5.5 Hz, 1H), 4.53 (dd, J = 4.5 Hz, 1H), 4.39 – 4.44 (m, 1H), 3.11 (d, J = 7.4 Hz, 2H), 1.29 (s, 9H)

[0172] 13C NMR (125 MHz, CDCl3): δ 170.6, 160.9, 156.3, 146.7, 138.1, 135.2, 129.3, 129.1, 128.6, 127.8, 127.3, 111.9, 81.4, 55.7, 43.6, 37.3, 28.3

[0173] LC-HRMS (ESI) m / z calcd. for C24H28N2NaO5+[MNa]+: 447.1890, found: 447.1902

[0174]

[0175] 3-(benzylamino)-3-oxoprop-1-en-2-yl (tert-butoxycarbonyl)-L-valinate (22)

[0176] NMI (60 µL, 0.75 mmol) was added to a solution of S3 (50 mg, 0.15 mmol) and Boc- Val-OH (81 mg, 0.4 mmol) in DCM (2 mL) at room temperature. The reaction was stirred until completion as monitored by TLC. The crude reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of intermediate 17 and Boc-Val. The resulting mixture was then dissolved in MeOH (3 mL). Hydrogen peroxide (59 µL of 50% solution, 1.2 mmol) was then added with stirring. Upon completion as determined by LC-MS, 23 B24-105-2WOthe mixture was purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min) to yield DHL-peptide 22 as a white solid (32 mg, 59 % over 2 steps).

[0177] 1H NMR (500 MHz, CDCl3): δ 7.66 (s, 1H), 7.28 – 7.31 (m, 4H), 7.21 – 7.26 (m, 1H), 6.21 (s, 1H), 5.26 (s, 1H), 4.97 (d, J = 6.3 Hz, 1H), 4.59 (dd, J = 14.9, 6.5 Hz, 1H), 4.42 (dd, J = 15.0, 5.6 Hz, 1H), 4.01 (t, J = 6.3 Hz, 1H), 2.09 – 2.19 (m, 1H), 1.32 (s, 9H), 1.05 (dd, J = 11.6, 6.8 Hz, 6H)

[0178] 13C NMR (125 MHz, CDCl3): δ 170.8, 161.0, 156.7, 146.9, 138.1, 128.6, 127.8, 127.4, 111.7, 81.1, 60.0, 43.7, 30.3, 28.3, 19.2, 18.7

[0179] LC-HRMS (ESI) m / z calcd. for C20H28N2NaO5+[MNa]+: 399.1890, found: 399.1899

[0180] Ester block synthesisa was added K2CO3(1.7 g, 12.2 mmol) and PMB-Cl (1.7 mL, 12.2 mmol) with stirring. The reaction was heated to 50 °C and determined to be complete after 4 hours as determined by TLC. The mixture was quenched with sat. NH4Cl (30 mL) and extracted with EtOAc (3 x 30 mL). Combined organic layers were dried over Na2SO4, concentrated under vacuum, and purified by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford the PMB ester S4 as a white solid (1.9 g, 64%).

[0184] 1H NMR (500 MHz, CDCl3): δ 7.52 – 7.57 (m, 2H), 7.24 – 7.27 (m, 3H), 7.18 – 7.22 (m, 2H), 6.84 – 6.89 (m, 2H), 5.06 (d, J = 11.8 Hz, 1H), 4.83 (d, J = 11.8 Hz, 1H), 4.49 (q, J = 5.0 Hz, 1H), 3.81 (s, 3H), 3.35 (dd, J = 13.0, 4.4 Hz, 1H), 3.23 (dd, J = 13.0, 5.3 Hz, 1H), 3.16 (d, J = 6.1 Hz, 1H);

[0185] 13C NMR (125 MHz, CDCl3): δ 173.0, 160.0, 133.6, 130.5, 129.5, 129.3, 127.55, 127.1, 114.1, 69.8, 67.6, 55.4, 32.8;

[0186] LC-HRMS (ESI) m / z calcd. For C17H18NaO4Se+[MNa]+: 389.0263, found: 389.0296 24 B24-105-2WO-9-yl)methoxy)carbonyl)glycyl)oxy)-3- p y y p p d (S5)

[0189] To a solution of S4 (630 mg, 1.4 mmol) in DCM (2 mL) was added Fmoc-Gly-OH (2.1 g, 6.9 mmol), NMI (775 µL, 9.7 mmol), and DIC (1.1 mL, 6.9 mmol) with stirring and monitored by TLC. Upon completion, the solution was purified directly by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of PMB-protected ester block and Fmoc-Gly. To the isolated residue in DCM (10 mL) was added trifluoroacetic acid (1.3 mL, 16.7 mmol) with stirring. The reaction was monitored by TLC and concentration under vacuum upon completion. The resulting residue was purified directly by flash column chromatography on silica gel (0-10% MeOH / DCM 1% AcOH) to afford a white solid (710 mg, 97%).

[0190] 1H NMR (500 MHz, CDCl3): δ 7.75 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.49 – 7.52 (m, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 7.21 – 7.25 (m, 3H), 5.37 (dd, J = 8.2, 3.8 Hz, 1H), 5.17 (t, J = 5.9 Hz, 1H), 4.36 – 4.44 (m, 2H), 4.19 (t, J = 6.7 Hz, 1H), 3.84 (qd, J = 18.2, 5.9 Hz, 2H), 3.35 (dd, J = 13.6, 3.9 Hz, 1H), 3.22 (dd, J = 13.6 Hz, 8.2 Hz, 1H);

[0191] 13C NMR (125 MHz, CDCl3): δ 171.5, 169.5, 156.9, 143.8, 141.5, 133.8, 129.4, 129.1, 127.9, 127.3, 125.2, 120.2, 73.0, 67.4, 47.2, 42.5, 27.5;

[0192] LC-HRMS (ESI) m / z calcd. For C26H23NNaO6Se+[MNa]+: 548.0583, found: 548.0536methoxy)carbonyl)-L-alanyl)oxy)-3- (phenylselanyl)propanoic acid (S6)

[0195] To a solution of S4 (1.1 g, 2.2 mmol) in DCM (2 mL) was added Fmoc-Ala-OH (3.5 g, 11.2 mmol), NMI (1.3 mL, 15.7 mmol), and DIC (1.8 mL, 11.2 mmol) with stirring and monitored by TLC. Upon completion, the solution was purified directly by flash column chromatography on silica gel (0-100% EtOAc / Hexanes) to afford a mixture of PMB-protected ester block and Fmoc-Ala. To this residue was added DCM (10 mL) and trifluoroacetic acid (1.6 mL, 20.9 mmol) with stirring. The reaction was monitored by TLC and concentration under vacuum upon completion. The resulting residue was purified directly by flash column 25 B24-105-2WOchromatography on silica gel (0-10% MeOH / DCM 1% AcOH) to afford a white solid (1.0 g, 84%).

[0196] 1H NMR (500 MHz, CDCl3): δ 7.77 (d, J = 7.7 Hz, 2H), 7.60 (t, J = 7.0 Hz, 2H), 7.49– 7.54 (m, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.24 (d, J = 7.0 Hz, 3H), 5.42 – 5.48 (m, 1H), 5.06 (d, J = 7.8 Hz, 1H), 4.41 – 4.55 (m, 2H), 4.28 – 4.33 (m, 1H), 4.30 (t, J = 6.9 Hz, 1H), 3.38 (dd, 13.5, 4.0 Hz, 1H), 3.26 (dd, J = 13.5, 7.7 Hz, 1H), 1.36 (d, J = 7.2 Hz, 3H);

[0197] 13C NMR (125 MHz, CDCl3): δ 172.0, 156.1, 143.9, 143.8, 141.5, 133.8, 129.4, 129.0, 128.0, 127.3, 125.2, 125.1, 120.2, 72.5, 67.2, 49.6, 47.2, 27.5, 18.3, 2.0;

[0198] LC-HRMS (ESI) m / z calcd. for C27H25NNaO6Se+[MNa]+: 562.0739, found: 562.0749

[0199] Computational methods

[0200] Methods to Support Figure 2

[0201] For each compound that was studied, molecular mechanics methods (Macromodel, OPLS4 force field) were used to minimize a starting population of 10,000 conformers that was initially generated using the Conformational Search tool in the Schrödinger Maestro 2022-2 environment. The geometries of all unique conformers that were found to be within 5 kcal / mol of the global minimum were then optimized again using DFT at the B3LYP-D3 / 6-31G** level. The global minima resulting from this round of computation were subjected to a second geometry optimization and vibrational spectrum calculation in QChem v.6.02 (B3LYP-D3 / 6- 31G**) to determine the zero-point energies, enthalpy corrections, and the internal entropy values at 298.15 K. All compounds were confirmed to have no imaginary vibrational frequency modes. For all species, refined electronic energy calculations were performed on the optimized geometries using an expanded basis set (ωB97M-V / 6-311++G(3df, 3pd)). For calculations including solvation, a CPCM model was used with Bondi van der Waals radii and a SAS solvent probe radius of 1.4 Å. In these cases, solvation was introduced only into the final electronic energy calculations. For transition state analysis…Tabulated energy and entropy values appear in Figure S1 along with atomic coordinates of the optimized structural minima used for each calculation.

[0202] General Peptide Synthesis & Labeling Methods

[0203] Methods to Support Figure 3

[0204] Purified DHL-containing peptides in MeOH (10 mM, 15 µL) were transferred to PCR tubes. The peptide was either diluted with 50 mM NaPi pH 8, or supplemented with 1 equivalent of tetramethyl guanidinium and analyzed after 4 and 16 hours, and 15 minutes, respectively. Each reaction was then analyzed on a Zorbax Eclipse Plus C18 column (1.8 µm, 1.2 x 50 mm, room temperature, Agilent) using a linear gradient from 5 to 95% acetonitrile over 7.5 min with 26 B24-105-2WO0.1% formic acid as the aqueous mobile phase after an initial hold at 95% 0.1% formic acid for 0.5 min (0.7 mL / min) using an 1290 Infinity II UHPLC (G7120AR, Agilent).

[0205] DHL-peptide 12 (3 mg) was analyzed by NMR in methanol-d4before and after addition of 1 eq. tetramethyl guanidinium (TMG). Specifically, Heteronuclear Multiple Bond Correlation (HMBC) spectroscopy was used in order to observe proton coupling to carbonyl carbons. Analysis was performed on a Bruker 700 MHz instrument.

[0206] Methods to Support Figure 4

[0207] Purified DHL-containing peptides in MeOH (10 mM, 15 µL) were transferred to PCR tubes. Then, NaPi pH 7 (50 mM, 7.5 µL) was added to each tube and incubated for 1 h at room temperature. Then, solutions of each alpha-nucleophile (75 mM, 7.5 µL) dissolved in 50 mM NaPi pH 6 were added to each respective tube and incubated for 6 h. Each reaction was then analyzed on a Zorbax Eclipse Plus C18 column (1.8 µm, 1.2 x 50 mm, room temperature, Agilent) using a linear gradient from 5 to 95% acetonitrile over 7.5 min with 0.1% formic acid as the aqueous mobile phase after an initial hold at 95% 0.1% formic acid for 0.5 min (0.7 mL / min) using an 1290 Infinity II UHPLC (G7120AR, Agilent).

[0208] Solid-phase peptide synthesis methods to support Figure 5

[0209] Peptides were synthesized on 2-chlorotrityl chloride resin (0.6 mmol / g) on a 0.1 mmol scale. The resin was first swelled with DCM (5 mL). The first Fmoc-amino acid (5 eq.) was coupled with DIPEA (7.5 eq.) in DCM (3 mL) for 30 min. Fmoc-amino acids (5 eq.) were coupled to the resin along with HATU (4.9 eq.), and DIPEA (7.5 eq.) in DMF (2 mL) for 30 min, followed by Fmoc deprotection with 20% piperidine in DMF (1 mL). Each ester block (1.1 eq.) was coupled in DCM along with DIC (1.1 eq.) and NHS (1.1 eq.) for 2 – 16 h. Peptidyl resins were cleaved in a solution of 97.5% TFA, 1.25% water, and 1.25% thioanisole to yield peptides with a C-terminal carboxylic acid. If no side chain protecting groups were present, the resin was cleaved with 5% TFA in DCM (3 mL). Peptides were purified by RP-HPLC (5-95% acetonitrile / water with 0.1% TFA over 30 min).

[0210] Methods for labeling SPPS-synthesized peptides (to support Figure 5)

[0211] Purified OH-SecPh-containing peptides in MeCN (5 mM) were transferred to PCR tubes. Then, H2O2in MeCN (50 mM) was added to each tube. Oxidation and elimination were monitored by LC-HRMS. When elimination was completed, the tubes were lyophilized to remove excess H2O2. Then, peptides were resuspended in MeCN and diluted into 50 mM NaPi at the pH indicated (7–9) to 5 mM concentration. For time and pH-dependent experiments, isomerization was monitored by addition of hydrazine (20 mM) in 50 mM NaPi pH 6, incubated for 15 min and analyzed by LC-HRMS. Peptide 26 required 4 hours of isomerization at pH 9, and then 1 hour incubation with hydrazine before analysis by LC-HRMS. Once the time and pH 27 B24-105-2WOneeded for isomerization was determined, peptides were then treated accordingly before incubation with other substituted hydrazine labels (40 mM) in 50 mM NaPi pH 6 for 2 hours. Each reaction was then analyzed on a Zorbax Eclipse Plus C18 column (1.8 µm, 1.2 x 50 mm, room temperature, Agilent) using a linear gradient from 5 to 95% acetonitrile over 7.5 min with 0.1% formic acid as the aqueous mobile phase after an initial hold at 95% 0.1% formic acid for 0.5 min (0.7 mL / min) using an 1290 Infinity II UHPLC (G7120AR, Agilent).

[0212] General Biochemistry Methods

[0213] Methods to Support Figure 6A

[0214] DNA templates used for transcribing cDNA for in vitro translation as well as E. coli tRNAValAsnE2were prepared using polymerase chain reactions (PCR) by annealing and extending overlapping primers. The templates were then purified from protein components with a 1:1 (v / v) phenol / chloroform solution and precipitated in 3 volumes of 75% (v / v) ethanol. T7 HiScribe RNA synthesis kit (New England Biolabs (NEB)) was used to generate each tRNA in 200 µL reactions containing 10 µg of DNA template. Transcription reactions were incubated at 37° C for 4 h, and then treated with 100 U of RNAse-free DNAse I (Sigma-Aldrich) for an additional 2 h to digest template DNA. Sodium acetate pH 5.2 was added to a final concentration of 200 mM and isolated using a Quick-RNA miniprep kit (Zymo Research).

[0215] In vitro tRNA acylation was performed with the synthetase FRS1. cDNA templates and tRNA were made in vitro as previously described.(2) HEPES pH 7.5 (100mM), MgCl2 (10 mM), DTT (4 mM), ATP (10 mM), substrate (10 mM), iPPase (0.004 U / µL), tRNA (25 µM), and FRS1 (5 µM) were combined and incubated for 2 – 6 hours.(3) tRNA was isolated from the mixture using a Quick-RNA miniprep kit, and used directly in in vitro translation experiments following intact RNA LC-MS analysis as previously described.(3)

[0216] Methods to Support Figure 6B–E

[0217] In vitro transcription / translation of short peptides were carried out using the PureExpress (∆tRNA, ∆aa (E6840S)) kit by New England Biolabs with minor modifications. For each reaction on a 12.5 µL scale: Solution A ((∆tRNA, ∆aa) (2.5 µL), an amino acid mixture containing only the encoded residues at 33 mM (0.25 µL), tRNA solution (1.25 µL), Solution B (3.75 µL), 500 ng dsDNA template (0.25 µL), and water (to 12.5 µL). Met was omitted from the reaction mixture. When using precharged tRNA, Valine was also omitted from the reaction mixture. The reactions were then incubated for 2 h at 37ºC. The translated peptides were first passed through a molecular cutoff filter (Pall Corporation) to remove protein components. The mixture was first analyzed on a Zorbax Eclipse Plus C18 column (1.8 µm, 1.2 x 50 mm, room temperature, Agilent) using a linear gradient from 0 to 55% acetonitrile over 6.5 min with 0.1% formic acid as the aqueous mobile phase after an initial hold at 95% 0.1% formic acid for 0.5 28 B24-105-2WOmin (0.7 mL / min) using an 1290 Infinity II UHPLC (G7120AR, Agilent). Peptides were identified using LC-HRMS with an Agilent 6530 QTOF AJS-ESI (G6230BAR). The mixture was then desalted using an analytical RP-HPLC, using a Zorbax Eclipse Plus C18 column (1.8 µm, 1.2 x 150 mm, room temperature, Agilent) using a linear gradient from 5 to 65% acetonitrile over 30 minutes with 0.1% trifluoroacetic acid as the aqueous mobile phase (1 mL / min) using a 1260 Infinity II LC system (G7110B, Agilent). Fractions containing the peptide were identified using LC-HRMS.

[0218] Desalted reaction mixture was removed of solvent by lyophilization, and then reconstituted in 20 mM H2O2in MeCN and incubated at room temperature for 2 hours. Oxidant was removed by lyophilization, and the mixture was then dissolved in 50 mM NaPi pH 8 for 1 hour. The solution was then diluted to 20 mM hydrazine in 50 mM NaPi pH 6 for 30 minutes before analysis by LC-HRMS.

[0219] References

[0220] 1. P. A. Bartlett, P. M. Chouinard, Stereocontrolled synthesis of (E)- and (Z)-3- deuteriophosphoenolpyruvate. J. Org. Chem.48, 3854–3855 (1983).

[0221] 2. O. Ad, K. S. Hoffman, A. G. Cairns, A. L. Featherston, S. J. Miller, D. Söll, A. Schepartz, Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro. ACS Cent. Sci.5, 1289–1294 (2019).

[0222] 3. R. Fricke, C. V. Swenson, L. T. Roe, N. X. Hamlish, B. Shah, Z. Zhang, E. Ficaretta, O. Ad, S. Smaga, C. L. Gee, A. Chatterjee, A. Schepartz, Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo. Nat. Chem.15, 960–971 (2023). 29 B24-105-2WO

Claims

CLAIMS 1. A method of peptide synthesis, comprising synthesizing a ketone-containing peptide backbone via O to C acyl shift by incorporating into the peptide backbone an α-L-hydroxy acid that can serve as a substrate for an aminoacyl tRNA synthetase to be incorporated into a peptide, and transform chemically into a dehydrolactic acid moiety.

2. The method of claim 1, wherein the synthesizing is effected by chemical synthesis, in vitro translation, or cellular synthesis.

3. The method of claim 1, comprising elimination via oxidation, alkylation or enzyme.

4. The method of claim 1 comprising using an enzyme that forms dehydroalanine from Cys or Ser, and also functions on the analogous ester to catalytically generate a dehydrolactic acid moiety.

5. The method of claim 1, comprising incorporating the α-L-hydroxy acid into the peptide backbone (e.g. by solid-phase peptide synthesis or ribosomal translation) to form an incorporated dehydrolactic acid, under conditions wherein the dehydrolactic acid monomer isomerizes via a spontaneous O to C acyl shift under physiological conditions to form a peptide containing a 1,3-diketo amide.

6. The method of claim 1, further comprising chemically diversifying the 1,3-diketoamide; for examples, using hydrazines to form pyrazoles and hydrazones, or hydroxylamines to form oximes, or nitronates to form tertiary alcohols.

7. A method of peptide synthesis that incorporates a C–C bond as a ketone into the backbone of chemically or ribosomally synthesized peptides, the method comprising: incorporating a dehydrolactic acid moeity into a peptide ribosomally or via solid-phase synthesis using α-hydroxy phenylselenocysteine followed by oxidation.

8. The method of claim 7, further comprising incubating the peptide under conditions wherein the dehydrolactic acid moeity isomerizes to generate a backbone-embedded α,γ-diketoamide moiety via a spontaneous O to C acyl shift, preferably wherein the conditions comprises physiological pH. 30 B24-105-2WO9. The method of claim 8, further comprising diversifying the α,γ-diketoamide moiety with a nucleophile.

10. The method of claim 8, further comprising diversifying the α,γ-diketoamide moiety with a nucleophile, that is hydrazines or hydroxylamines to form pyrazoles or oximes, respectively.

11. The method of any of claims 1-10, according to the reaction scheme:R is C1-C18 or C1-C10 substituted or unsubstituted hydrocarbyl or substituted or unsubstituted heterohydrocarbyl, including alkyl, alkenyl, alkynyl, including cyclics and heterocyclics, and aryl and heteroaryl.

12. The method of claim 11, wherein R is substituted or unsubstituted: phenyl, cyclohexyl, furan, thiophene or azole.

13. The method of claim 11, wherein: the R substituents are independently aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl, alkyloxy, alkyl, alkenyl, alkynyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido, carboxyl, cyanyl, ester, haloformyl, hydroperoxyl, hydroxyl, imine, isocyanide, iscyante, N-tert-butoxycarbonyl, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, sulfhydryl, thiol, and thiocyanyl.

14. The method of any of claims 1-10, wherein the α-L-hydroxy acid is α-L-hydroxy- phenylselenocysteine (OH-SecPh) or an analog thereof: 31 B24-105-2WOr O, and R is C1-C18 or C1-C10 optionally substituted hydrocarbyl or heterohydrocarbyl, including alkyl, alkenyl, alkynyl, including cyclics and heterocyclics, and aryl and heteroaryl, such as substituted or unsubstituted: phenyl, cyclohexyl, furan, thiophene or azole.

15. The method of claim 14, wherein: the R substituents are independently aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl, alkyloxy, alkyl, alkenyl, alkynyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido, carboxyl, cyanyl, ester, haloformyl, hydroperoxyl, hydroxyl, imine, isocyanide, iscyante, N-tert-butoxycarbonyl, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, sulfhydryl, thiol, and thiocyanyl. 32 B24-105-2WO

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