Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 Experimental procedures Solid phase peptide synthesis (SPPS) Solid phase peptide synthesis (SPPS) was performed using a microwave-assisted peptide synthesizer (CEM) or in a standard manual reaction vessel under argon. Rink-amide MBHA resin and Wang resin were purchased from Sigma-Aldrich. DMF, DMSO, NMP, DCM, MeOH, ACN and DIEA were dried and distilled using standard protocols. The peptides were purified on a Merck Hitachi HPLC using a reverse-phase C8 semi-preparative column (Vydac) with a gradient of 5% to 6% acetonitrile in water (both containing.1% (v/v) trifluoroacetic acid). The 1 H-NMR spectra were measured on 4 MHz or 6 MHz spectrometer (Bruker) and the 13 C-NMR spectra were measured at a 1 MHz frequency using CDCl 3 as solvent. The peptides identity was tested using MALDI-TOF Mass spectrometry on a PerSeptive Biosystems Voyager- DE PRO Biospectrometry workstation (Fig. S1). The peptides purity was confirmed using analytical HPLC (Fig. S2). Fmoc deprotection: The peptidyl-resin was treated twice with 2% piperidine in NMP for 3 minutes using microwave irradiation. The product was washed 4 times using NMP. HBTU/HOBt coupling of protected amino acids: Fmoc-protected amino acid (1.5 equiv), 1- hydroxybenztriazole hydrate (HOBt, 1.5 equiv), o-benzotriazole-1-yl-n,n,n,ntetramethyluronium hexafluorophosphate (HBTU, 1.5 equiv) and diisopropyl-ethylamine (DIEA) (2 equiv) were dissolved in dry DMF. The solution was mixed with the resin-bound peptide and then irradiated in microwave for 5 minutes in the peptide synthesizer. The process was repeated twice and the resin was then washed with DMF, NMP and DCM. HBTU coupling of malonic acid: Malonic acid (1 equiv) was activated by adding HBTU (1.2 equiv) and DIEA (1.5 equiv) in DMF or DMSO. The mixture was stirred for 1 minutes at o C and then added into a vessel containing pre-swollen resin-bound peptide in DMF or DMSO. The reaction was then allowed to continue for another 1 hr. The completion of the reaction was monitored by the Kaiser-ninhydrin and chloranil tests. Negative response in these color tests indicated the completion of the reaction. The resin was then washed thoroughly with DMF, DCM, Ethanol and diethyl ether. 1
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 Cleavage of the peptide from the resin: A solution (1 ml) of trifluoroacetic acid (TFA)/TDW/triisopropylsilane (TIS) (92:4.5:3.5) was cooled to o C and incubated with 2mg resin-bound peptide for 2h. The cleaved peptide was precipitated with ice cold diethyl ether, the solution centrifuged and the peptide washed twice more with ether. Then minimum volume of ACN/TDW (3:2) was used to dissolve the crude peptide. The solution was lyophilized before purification in HPLC. Optimization of the acetylation reaction conditions The activation of malonic acid (1 equiv) by HBTU (1.2 equiv) was optimized in presence of different bases (1.5 equiv) in different organic solvents as shown in Table S1. The mixture was stirred for 1 minutes at o C and then added to pre-swollen resin-bound peptide 4 in the different solvents. The reaction was then allowed to continue for another 1 hr. The completion of the reaction was monitored by the Kaiser-ninhydrin and chloranil tests. The resin was then washed thoroughly with DMF, DCM, Ethanol and diethyl ether. A solution (1 ml) of trifluoroacetic acid (TFA)/TDW/triisopropylsilane (TIS) (92:4.5:3.5) was cooled to o C and incubated with 2mg resin-bound peptide 4 for 2h. The cleaved peptide 3 was precipitated with ice cold diethyl ether, the solution centrifuged and the peptide washed twice more with ether. Then minimum volume of ACN/TDW (3:2) was used to dissolve the crude peptide and purified in HPLC. After optimization of bases and solvent system, we concluded that DIPEA/Et 3 N in DMF/DMSO gave best yield in the shortest time (Table S1). Table S1: Optimization of the acetylation reaction conditions Reactants Solvent Base Time (mins) Yield (%) 1 DMF DIPEA 16 98 2 DMF Et 3 N 16 98 3 KILNPEEIEKYVAEI - DMF DBU 18 72 4 resin, 4 DMF NMM 26 73 5 + DMF Pyridine 22 84 6 Malonic acid, 55 DMSO DIPEA 17 96 7 NMP DIPEA 25 75 2
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 2 15 27 1358 1 8 28 1358 3 24 29 1882 1 5 6 4 2 18 12 6 5 1 15 2 25 3 5 1 15 2 25 3 5 1 15 2 25 3 35 28 3 1832 2 15 31 1398 8 6 32 1398 21 14 7 1 5 4 2 5 1 15 2 25 3 5 1 15 2 25 3 5 1 15 2 25 3 5 4 3 33 289 75 6 45 34 26 12 9 35 1884 2 3 6 1 15 3 5 1 15 2 25 3 5 1 15 2 25 3 5 1 15 2 25 3 6 45 36 2841 15 12 37 1844 8 6 39 1668 3 9 4 6 15 2 3 8 16 24 32 4 5 1 15 2 25 3 5 1 15 2 25 3 Figure S1: MALDI-TOF Mass of acetylated peptides as listed in table 1. 3
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 16 12 8 4 41 1172 35 28 21 14 7 661 43 18 15 12 9 6 3 44 839 6 9 12 15 6 8 1 12 6 8 1 12 2 45 843 2 46 416 9 48 811 15 15 6 1 5 1 5 3 6 8 1 12 3 4 5 6 6 9 12 15 18 15 12 49 811 9 6 3 6 9 12 15 Figure S1 (continued): MALDI-TOF Mass of acetylated peptides as listed in table 1. 4
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 5 4 27 16.28 5 4 28 16.78 5 4 29 2.14 3 2 1 5 1 15 2 25 3 3 2 1 5 1 15 2 25 3 3 2 1 5 1 15 2 25 3 5 4 3 17.48 25 2 31 17.68 5 4 32 17.58 3 15 3 2 1 5 1 15 2 25 3 1 5 5 1 15 2 25 3 2 1 5 1 15 2 25 3 15 12 33 12.37 6 5 34 12.23 5 4 35 16.1 9 6 3 5 1 15 2 25 3 4 3 2 1 5 1 15 2 25 3 3 2 1 5 1 15 2 25 3 6 5 4 36 14.98 2 15 37 15.13 8 6 38 1.5 3 1 4 2 1 5 1 15 2 25 3 5 5 1 15 2 25 3 2 5 1 15 2 25 3 Figure S2: Reverse phase HPLC trace (in 5-6 Acetonitrile / Water) of acetylated peptides as listed in table 1. 5
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 6 5 4 3 2 39 12.53 5 4 3 2 41 14.74 5 4 3 2 43 15.57 1 1 1 5 1 15 2 25 3 5 1 15 2 25 3 5 1 15 2 25 3 8 6 44 15.75 15 12 45 23.7 3 25 2 48 19.31 4 9 15 6 2 3 5 1 15 2 25 3 5 1 15 2 25 3 1 5 5 1 15 2 25 3 3 25 2 49 19.13 15 1 5 5 1 15 2 25 3 Figure S2 (continued): Reverse phase HPLC trace (in 5-6 Acetonitrile / Water) of acetylated peptides as listed in table 1. 6
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 t=21 min t= 18 min t= 15 min 67 t=12 min t= 9 min minutes t= 6 min minutes t= 3 min minsmin 58 59 61 54 Figure S3: 1 H NMR kinetics of the acetylation of anisidine (53) in d 6 -DMSO The plot of the integral of the peak at 1.86 ppm (acetylated anisidine, 54) relative to the peak at 2.7 ppm (tetramethyl urea, 59) versus the reaction time shows a sigmoidal nature. This indicates that the formation of the N-acetyl anisidine (54) from the ketene (61) in scheme 2 (main text) is irreversible. The process is fast and highly spontaneous since it took only 2 minutes to reach saturation. 7
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is The Royal Society of Chemistry 214 A B C D E F G H + I J K L Figure S4: Optimized structures of compounds (58-64) for the three different possible pathways of acetylation as shown in figure 2 in the main text. A. Minimized Structure of the reactant (58) for path A. B. Optimized transition state structure of path A (TS1) for the self-dissociation process. C. Optimized transition state structure of path B (TS2). D. Minimized structure of reactant complex for the first step of path C (59 62) where the attack of 58 by HOBt anion takes place. E. Optimized transition state of first step of path C (TS3a). F. Minimized structure of the reactant complex for path C (63). G. Optimized transition state of path C (TS3b), which represents the selfdissociation. H. Minimized structure of the product for path A (59--61). I. Minimized intermediate product for path B (64) from where no self-dissociation is possible. J. Minimized structure of the product for the first step of path C (63 59) K. Minimized structure of the final product for path C (6 + [61 62]). L. Minimized structure of the product ([6.62] + CO 2 ) where it was considered that the CO 2 already left the reaction medium. 8