Ketoxime peptide ligations: oxidative couplings of alkoxyamines to N-aryl peptides

Ketoxime peptides are readily accessible from oxidative couplings between N-aryl peptides and alkoxyamines under catalyst-free conditions.


Table of Contents of the Supporting Information
Title, authors and address S1

Experimental Procedures and Characterization Data
Fmoc-based SPPS: Fmoc deprotection and HBTU couplings S4 Incorporation of N-terminal N-aryl amino acid residues S4 Synthesis of aminooxyacetyl-GRGDSGG S5 Cleavage test of resin-bound peptides S5 Full cleavage S5 Analysis and purification of peptides 2a-j, 3a-d and aminooxyacetyl-GRGDSGG 16 S5 Table S1. Characterization data for N-aryl amino acid-terminated peptides and aminooxyacetyl-GRGDSGG 16 S6 Oxime ligation reactions procedures S6-S12 Table S2. Comparison of oxidative coupling conversions in different buffers S9 at pH 8.5. Table S3. Control studies to compare buffer composition while keeping pH S9 constant. Table S4. Comparative reactivity of 2a-b with varying potassium S10 phosphate buffer concentration at pH 7. Table S5. Comparative reactivity for peptide 2b in varying % MeCN vs EtOH S11 in phosphate buffer pH 7. Table S6. Characterization data for N-aryl A-XYRAG peptides S12 Solution Phase Synthesis Procedures S13 Table S7. N-(p-NMe 2 -Ph)glycine tert-butyl ester oxidation in organic solvents S14

General methods
General: Polystyrene Rink Amide resin (0.78 mmol/g) was purchased from Protein Technology, Inc™, and the manufacturer's reported loading of the resin was used in the calculation of the yields. Solid phase peptide synthesis (SPPS) was performed using an automated Biotage Syro Wave TM peptide synthesizer in 10 mL parallel reactors with PTFE frits. Incorporation of Bocaminooxy acetic acid and N-aryl amino acids were performed manually in disposable filter columns with 20 µM PE frit filters and caps purchased from Applied Separations (cat # 2413 and 2416 for 3 mL and 6 mL filter columns, respectively) with gentle agitation on a Thermo Fisher vortex mixer equipped with a microplate tray. Solution draining and washing of the resin was done by connecting the filter columns to a water aspirator vacuum via a waste trap. All solution phase reactions were performed in oven-dried glassware sealed with microwave caps or rubber septa and were stirred with Teflon-coated magnetic stir bars. Tetrahydrofuran (THF) was dried by passage over a column of activated alumina (JC Meyers Solvent System). Thin layer chromatography (TLC) was performed using Silicycle silica gel 60 F-254 precoated plates (0.25 mm) and were visualized by exposure to ultraviolet light (UV) and/or submersion in aqueous ninhydrin solution. Samples were purified using a Biotage® Isolera One, employing polypropylene cartridges preloaded with silica gel (25 micron) and were eluted with UV detection (254, 280 nm). Nuclear magnetic resonance (NMR) spectra ( 1 H, 13 C) were recorded on a 600 MHz Bruker spectrometer at 24 ºC for compounds 6-8 and a 700 MHz Bruker spectrometer for ketoximes 4bc and 17-19. Chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to residual protium in the NMR solvent (CHCl 3 , δ 7.26). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant in Hertz, and integration. Chemical shifts for 13 C NMR spectra are recorded in parts per million (ppm, δ scale) and are referenced to the central peak of deuterochloroform (δ 77.16). Ketoximes 4b-d and 17-19 were prepared as 1-3 mM solutions in D 2 O or CD 3 OD. 1 H NMR for the ketoximes 4b-d and 17-19 were run with water suppression using Bruker's noesygppr1d experiment method. All spectra were obtained with complete proton decoupling. Infrared (IR) spectra were collected on a Thermo Scientific Nicolet iS5 FTIR instrument using attenuated total reflectance (ATR) mode and signals are reported in reciprocal centimeters (cm -1 ). Only selected IR frequencies are reported. Melting points were obtained on a Mettler Toledo MP50 One Click Melting Point System.

Fmoc-based SPPS: Fmoc deprotection and HBTU couplings
Peptide syntheses were performed on Polystyrene Rink Amide resin (0.78 mmol/g) using standard manual solid phase peptide synthesis protocols (SPPS) on an automated shaker or using a Biotage Syro Wave TM peptide synthesizer. Couplings of amino acids (3 equiv) were performed in DMF using HBTU (3 equiv) as coupling reagent and DIEA (6 equiv) as base. Fmoc deprotections were performed by treating the resin with 20% piperidine in DMF (v/v) for 5 minutes, followed by treatment with a fresh solution of 20% piperidine in DMF (v/v) for 15 minutes. Resin was washed after each coupling and deprotection reaction with DMF (3 x 1 mL). Prior to cleavage from the resin or storage, resin was washed with CH 2 Cl 2 (3 x 1 mL). Peptides 2a and 3a (R 1 = H) were synthesized based on previous methods. 1 Peptides 2b-j and 3b-d were synthesized using slightly modified procedures. Specifically, resin-bound Fmoc-LYRAG (1, 100 mg, 0.078 mmol) was transferred to a 3 mL disposable filter column, treated with 1 mL of a 20% piperidine in DMF solution (v/v) and gently agitated for 5 minutes, followed by another 15 minutes incubation with fresh reagent (1 mL). The resin was washed with DMF (5 x 1 mL) and treated sequentially with a 0.6 M solution of the corresponding racemic a-substituted bromoacetic acid derivatives in DMF (1 mL, 0.6 mmol) and 86 μL of N,N-diisopropylcarbodiimide (DIC, 0.56 mmol) for 25 minutes. The bromoacetylation procedure was repeated a second time with fresh reagent and the resin was washed with DMF (5 x 1 mL). The resin was then treated with a 2 M solution of 4-(dimethylamino)aniline or 4-methoxyaniline in DMF for 3 hours at room temperature. For peptides 2d and 3d (R 1 = CH 2 Ph), an additional 3 hours with fresh reagents at Guthrie, Q. A. E.; Young, H. A.; Proulx, C.

Incorporation N-terminal N-aryl amino acid residues
S5 room temperature was required. For peptide 2j (R = CH(CH 3 ) 2 ), the displacement required heating with sonication to 60°C for 24 h.
Note: As previously reported, care must be taken to avoid addition of base during activation and coupling of (Boc-aminooxy)acetic acid to prevent overacylation. 3 Cleavage from the resin should be done using TFA/EDT/H 2 O/TIPS (94:2.5:2.5: Once cleaved from solid support, aminooxyacetyl-terminated peptides should be manipulated in an acetone-free laboratory to avoid side reactions, and pooling and freezing of HPLC fractions must be done immediately. 4
S6 immediately and lyophilized. Characterization data for all peptides can be found in Table S1 below. LCMS chromatograms can be found starting on p.S13.

B. Oxime ligation conditions for N-(p-MeO-Ph)-amino acid-terminated peptide analogs
The following procedures were used to generate the data presented in Table 1, entries 5-8 of the manuscript.

At pH 4.5:
The ligation conditions used for these analogs were based on previously optimized procedures for N-(p-MeOPh)glycine-LYRAG 3a. 1  S9 before sealing the reaction mixture under an oxygen balloon. After oxidation to the aketoamides (5b-d) was achieved (~24 h), 1 mL of the O-benzylhydroxylamine hydrochloride stock solution (10 mM) was mixed with the peptide to give a final concentration of peptide and O-benzylhydroxylamine hydrochloride of 1 mM and 5 mM, respectively. Samples were tested at different time points by taking aliquots and analyzing them immediately by LCMS without prior quenching.

E. Buffer salt effect with peptide analogs 2a-b.
The studies on the effect of buffer salt described in Figure 2b of the manuscript were conducted after noticing a significant decrease in reactivity upon changing from a phosphate buffer to a glycine-NaOH buffer at pH 8.5 (Table S2). Glycine NaOH 0 2b Tris 88 2b Britton-Robinson 80 Studies described in Figure 2b of the manuscript: Returning to a of pH 7.5, further studies on buffer salt composition were performed using the same procedure as described in A, using ten different buffers (Tris, TEA, HEPES, TES, PIPES, phosphate, MOPS, Tricine, Bicine, imidazole) with a final buffer concentration of 0.1 M. Premade buffers were purchased from Alfa Aesar through Fisher scientific at concentrations between 0.2-1.0 M and diluted to 0.1 M using ultrapure water from a Synergy UV water purification system (EMD Millipore). The Britton-Robinson "universal buffer" was made using phosphoric acid (85% in water), glacial acetic acid, and o-boric acid and varying amounts of sodium hydroxide following literature procedure to make buffers at pH 4.6, 7 and 8. 5. 7 Control studies were also done at pH 4.5 and pH 7 using the Britton-Robinson "universal buffer" to confirm reactivity despite changing the buffer composition (Table S3).  Table S3. Control studies to compare buffer composition while keeping pH constant.

F. Effect of phosphate buffer salt concentration with peptide analogs 2a-b.
To study the effect of buffer salt concentration using potassium phosphate buffer, a stock solution of 100 mM phosphate buffer pH 7 was diluted with ultrapure water to afford final concentration of 50 mM, 25 mM, 10 mM, 5 mM and 1 mM potassium phosphate in water. These solutions were used to monitor oxidative coupling reactions (Table S4). Table S4. Comparative reactivity of 2a-b with varying potassium phosphate buffer concentration at pH 7.

G. Oxime ligation studies with 2a-b, h-i in buffer/EtOH solvent mixtures.
The following procedure was used to generate data presented in Table 2 and Figure 4 of the manuscript.
The studies were performed using the same procedure as described in A, but preparing the 2 mM stock solution of peptide 2a-b, h-j using a mixture of ethanol and 0.  (Table S5).

4-(p-dimethylamino-phenyl)alanine tert-butyl ester (8)
4-Dimethylamino-aniline (600.0 mg, 4.41 mmol, 2.00 equiv) and sodium acetate (180.7 mg, 2.20 mmol, 1.00 equiv) were added to a 10-mL round-bottom flask prior to the addition of ethanol (1.47 mL) and tert-butyl 2-bromopropanoate (294.9 μL, 2.20 mmol, 1.00 equiv). The round-bottom flask was capped with a septum and flushed with argon. The solution was allowed to stir under an atmosphere of argon at room temperature overnight. The crude solution was condensed to dryness before the product was isolated by flash-column chromatography eluted with a 10% ethyl acetate-hexanes to 40% ethyl acetate-hexanes gradient. This afforded a brown solid (386.9 mg, 84% yield).  General procedures used to generate the data in Table S7:

tert-butyl 2-((4-(dimethylamino)phenyl)amino)-2-oxoacetate (7)
4-(p-dimethylamino-phenyl)glycine tert-butyl ester 7 (25.0 mg, 0.10 mmol, 1.00 equiv) was added to a 4-mL vial with a stir bar. Solvent (1.00 mL) was added to the vial and the solution was sparged with oxygen for 30 seconds. The solution was allowed to stir under an oxygen atmosphere for 24 hours, unless otherwise noted. The crude solution was condensed to dryness before the product was isolated by flash-column chromatography (eluted with 5% ethyl acetate-hexanes to 40% ethyl acetates-hexanes gradient) affording a yellow oil (11.1 mg, 45 % yield The following is numerical data that pertains to Scheme 5 in the manuscript. The minor peaks that indicate further oxidation are unidentified at this time.