experimental procedures solid phase peptide …1 experimental procedures solid phase peptide...
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1
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
13C-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
20000Io
n c
ou
nt
m/z
135827
500 1000 1500 2000 2500 3000
0
2000
4000
6000
8000
10000
m/z
Ion
co
un
t
135828
500 1000 1500 2000 2500 3000
0
6000
12000
18000
24000
30000
1882
m/z
Ion
co
un
t
29
500 1000 1500 2000 2500 3000
0
7000
14000
21000
28000
35000
m/z
Ion
co
un
t
183230
500 1000 1500 2000 2500 3000
0
5000
10000
15000
20000
m/z
Ion
co
un
t1398
31
500 1000 1500 2000 2500 3000
0
2000
4000
6000
8000
m/z
Ion
co
un
t
139832
500 1000 1500 2000 2500 3000
0
1000
2000
3000
4000
5000
2089
m/z
Ion
co
un
t
33
500 1000 1500 2000 2500 3000
0
3000
6000
9000
12000
m/z
Ion
co
un
t
1884
35
800 1600 2400 3200 4000
0
1500
3000
4500
6000
Ion
co
un
t
m/z
2841
500 1000 1500 2000 2500 30000
300
600
900
1200
1500
m/z
Ion
co
un
t
184437
500 1000 1500 2000 2500 3000
0
2000
4000
6000
8000
m/z
Ion
co
un
t
1668
39
500 1000 1500 2000 2500 3000
0
1500
3000
4500
6000
7500
2060
m/z
Ion
co
un
t
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
co
un
t
41
600 800 1000 1200
0
7000
14000
21000
28000
35000
661
m/z
Ion
co
un
t
43
600 800 1000 1200
0
3000
6000
9000
12000
15000
18000
m/z
Ion
co
un
t
83944
600 800 1000 1200
0
5000
10000
15000
20000
m/z
Ion
co
un
t
843
45
300 400 500 6000
5000
10000
15000
20000
Ion
co
un
t
m/z
416
46
600 900 1200 1500
0
3000
6000
9000
811
m/z
Ion
co
un
t
48
600 900 1200 1500
0
3000
6000
9000
12000
15000
18000
m/z
Ion
co
un
t
81149
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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
Inte
nsit
y (
mV
)
Retention time (min)
27
0 5 10 15 20 25 30
0
100
200
300
400
500
Retention time (min)
Inte
nsit
y (
mV
) 16.78
28
0 5 10 15 20 25 30
0
100
200
300
400
500
20.14
Retention time (min)
Inte
nsit
y (
mV
)
29
0 5 10 15 20 25 30
0
100
200
300
400
500
Inte
nsit
y (
mV
)
Retention time (min)
17.4830
0 5 10 15 20 25 30
0
50
100
150
200
250
Retention time (min)
Inte
nsit
y (
mV
)17.68
31
0 5 10 15 20 25 30
0
100
200
300
400
500
Retention time (min)
Inte
nsit
y (
mV
)
17.58
32
0 5 10 15 20 25 30
0
300
600
900
1200
1500
Retention time (min)
Inte
nsit
y (
mV
)
12.37
33
0 5 10 15 20 25 30
0
100
200
300
400
500
600
Retention time (min)
Inte
nsit
y (
mV
)
12.23
34
0 5 10 15 20 25 30
0
100
200
300
400
500
Retention time (min)
Inte
nsit
y (
mV
)
16.01
0 5 10 15 20 25 30
0
100
200
300
400
500
600
Retention time (min)
Inte
nsit
y (
mV
)
14.98
35
36
0 5 10 15 20 25 30
0
50
100
150
200
Retention time (min)
Inte
nsit
y (
mV
)
15.13
37
0 5 10 15 20 25 30
0
20
40
60
80
Retention time (min)
Inte
nsit
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
Inte
nsit
y (
mV
)
Retention time (min)
39
0 5 10 15 20 25 30
0
100
200
300
400
500
Retention time (min)
Inte
nsit
y (
mV
) 14.74
41
0 5 10 15 20 25 30
0
100
200
300
400
500
15.57
Retention time (min)
Inte
nsit
y (
mV
)
43
0 5 10 15 20 25 30
0
200
400
600
800
15.75
Retention time (min)
Inte
nsit
y (
mV
)
44
0 5 10 15 20 25 30
30
60
90
120
150
Retention time (min)
Inte
nsit
y (
mV
)23.70
45
0 5 10 15 20 25 30
0
50
100
150
200
250
300
Inte
nsit
y (
mV
)
Retention time (min)
19.31
48
0 5 10 15 20 25 30
0
50
100
150
200
250
300
Inte
nsit
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
+
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