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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 60% acetonitrile in water (both containing 0.001% (v/v) trifluoroacetic acid). The 1H-NMR spectra were measured on 400 MHz or 600 MHz spectrometer (Bruker) and the
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C-NMR spectra were measured at a 100 MHz frequency using CDCl3 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 20% piperidine in NMP for 30
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,N-tetramethyluronium 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 10 minutes at 0 oC 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.
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Cleavage of the peptide from the resin: A solution (10 ml) of trifluoroacetic acid
(TFA)/TDW/triisopropylsilane (TIS) (92:4.5:3.5) was cooled to 0 oC and incubated with 200mg 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 10 minutes at 0 oC 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 (10 ml) of trifluoroacetic acid (TFA)/TDW/triisopropylsilane (TIS) (92:4.5:3.5) was cooled to 0 oC and incubated with 200mg resin-bound peptide 4 for 2h. The cleaved peptide 30 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/Et3N 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 KILNPEEIEKYVAEI -resin, 4 + Malonic acid, 55 DMF DIPEA 16 98 2 DMF Et3N 16 98 3 DMF DBU 18 72 4 DMF NMM 26 73 5 DMF Pyridine 22 84 6 DMSO DIPEA 17 96 7 NMP DIPEA 25 75
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Figure S1: MALDI-TOF Mass of acetylated peptides as listed in table 1.
500 1000 1500 2000 2500 3000 0 5000 10000 15000 20000 Ion cou nt m/z 1358 27 500 1000 1500 2000 2500 3000 0 2000 4000 6000 8000 10000 m/z Ion cou nt 1358 28 500 1000 1500 2000 2500 3000 0 6000 12000 18000 24000 30000 1882 m/z Ion cou nt 29 500 1000 1500 2000 2500 3000 0 7000 14000 21000 28000 35000 m/z Ion cou nt 1832 30 500 1000 1500 2000 2500 3000 0 5000 10000 15000 20000 m/z Ion cou nt 1398 31 500 1000 1500 2000 2500 3000 0 2000 4000 6000 8000 m/z Ion cou nt 1398 32 500 1000 1500 2000 2500 3000 0 1000 2000 3000 4000 5000 2089 m/z Ion cou nt 33 500 1000 1500 2000 2500 3000 0 3000 6000 9000 12000 m/z Ion cou nt 1884 35 800 1600 2400 3200 4000 0 1500 3000 4500 6000 Ion cou nt m/z 2841 500 1000 1500 2000 2500 3000 0 300 600 900 1200 1500 m/z Ion cou nt 1844 37 500 1000 1500 2000 2500 3000 0 2000 4000 6000 8000 m/z Ion cou nt 1668 39 500 1000 1500 2000 2500 3000 0 1500 3000 4500 6000 7500 2060 m/z Ion cou nt 34 36
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Figure S1 (continued): MALDI-TOF Mass of acetylated peptides as listed in table 1.
600 900 1200 1500 0 4000 8000 12000 16000 1172 m/z Ion cou nt 41 600 800 1000 1200 0 7000 14000 21000 28000 35000 661 m/z Ion cou nt 43 600 800 1000 1200 0 3000 6000 9000 12000 15000 18000 m/z Ion cou nt 839 44 600 800 1000 1200 0 5000 10000 15000 20000 m/z Ion cou nt 843 45 300 400 500 600 0 5000 10000 15000 20000 Ion cou nt m/z 416 46 600 900 1200 1500 0 3000 6000 9000 811 m/z Ion cou nt 48 600 900 1200 1500 0 3000 6000 9000 12000 15000 18000 m/z Ion cou nt 811 49
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Figure S2: Reverse phase HPLC trace (in 5-60 Acetonitrile / Water) of acetylated peptides as
listed in table 1. 0 5 10 15 20 25 30 0 100 200 300 400 500 16.28 Inten sit y ( mV )
Retention time (min) 27 0 5 10 15 20 25 30 0 100 200 300 400 500
Retention time (min)
Inten sit y ( mV ) 16.78 28 0 5 10 15 20 25 30 0 100 200 300 400 500 20.14
Retention time (min)
Inten sit y ( mV ) 29 0 5 10 15 20 25 30 0 100 200 300 400 500 Inten sit y ( mV )
Retention time (min) 17.48 30 0 5 10 15 20 25 30 0 50 100 150 200 250
Retention time (min)
Inten sit y ( mV ) 17.68 31 0 5 10 15 20 25 30 0 100 200 300 400 500
Retention time (min)
Inten sit y ( mV ) 17.58 32 0 5 10 15 20 25 30 0 300 600 900 1200 1500
Retention time (min)
Inten sit y ( mV ) 12.37 33 0 5 10 15 20 25 30 0 100 200 300 400 500 600
Retention time (min)
Inten sit y ( mV ) 12.23 34 0 5 10 15 20 25 30 0 100 200 300 400 500
Retention time (min)
Inten sit y ( mV ) 16.01 0 5 10 15 20 25 30 0 100 200 300 400 500 600
Retention time (min)
Inten sit y ( mV ) 14.98 35 36 0 5 10 15 20 25 30 0 50 100 150 200
Retention time (min)
Inten sit y ( mV ) 15.13 37 0 5 10 15 20 25 30 0 20 40 60 80
Retention time (min)
Inten sit y ( mV ) 10.05 38
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Figure S2 (continued): Reverse phase HPLC trace (in 5-60 Acetonitrile / Water) of acetylated
peptides as listed in table 1.
0 5 10 15 20 25 30 0 10 20 30 40 50 60 12.53 Inten sit y ( mV )
Retention time (min) 39 0 5 10 15 20 25 30 0 100 200 300 400 500
Retention time (min)
Inten sit y ( mV ) 14.74 41 0 5 10 15 20 25 30 0 100 200 300 400 500 15.57
Retention time (min)
Inten sit y ( mV ) 43 0 5 10 15 20 25 30 0 200 400 600 800 15.75
Retention time (min)
Inten sit y ( mV ) 44 0 5 10 15 20 25 30 30 60 90 120 150
Retention time (min)
Inten sit y ( mV ) 23.70 45 0 5 10 15 20 25 30 0 50 100 150 200 250 300 Inten sit y ( mV )
Retention time (min) 19.31 48 0 5 10 15 20 25 30 0 50 100 150 200 250 300 Inten sit y ( mV )
Retention time (min) 19.13 49
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Figure S3: 1H NMR kinetics of the acetylation of anisidine (53) in d6-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 20 minutes to reach saturation.
67 54 61 58 59 t= 3 min minsmin t= 6 min minutes t= 9 min minutes t=12 min t= 18 min t= 15 min t=21 min
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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 self-dissociation. 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 (60 + [61…62]). L. Minimized structure of the
product ([60….62] + CO2) where it was considered that the CO2 already left the reaction medium.
A B C D E F G H I J K L I L +