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1
Supporting information
Peripheral cyclic ββββ-amino acids balance stability and edge-
protection of β-sandwiches
Gábor Olajos,a,c
Anasztázia Hetényi,b Edit Wéber,
a Titanilla Szögi,
b Lívia Fülöp
b and Tamás A.
Martineka,c
*
a Institute of Pharmaceutical Analysis, SZTE-MTA Lendület Foldamer Research Group, University of
Szeged, Somogyi u. 4., H-6720 Szeged, Hungary. Email: [email protected]
b Department of Medical Chemistry, University of Szeged, Dóm ter 8., H-6720 Szeged, Hungary.
c MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Dóm ter 8., H-6720 Szeged,
Hungary.
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2018
2
Table of contents
Optical density measurement ............................................................................................................................ 3
NMR spectra of peptides .................................................................................................................................. 4
Medium- and long-range interactions in the NOESY spectra of 6 and 8 ............................................................. 5
Diffusion ordered spectroscopy ......................................................................................................................... 6
Assigned chemical shifts .................................................................................................................................. 7
Peptide characterization ................................................................................................................................. 10
3
Fig. S1. Optical density at 280 nm measured for 6 at the beginning (blue) and the end (red) of the temperature-
dependent CD experiment.
4
Fig. S2. 2D NMR spectra of the dimeric BB14 and BB15 (compounds 5 and 6). NH-Hα region of overlaid
TOCSY (red) and NOESY (blue) spectra (left), Cα-Hα region of 13
C HSQC spectrum (right).
Fig. S3. 2D NMR spectra of the dimeric betabellin analogs (compounds 7 and 8). NH-Hα region of overlaid
TOCSY (red) and NOESY (blue) spectra (left), Cα-Hα region of 13
C HSQC spectrum (right).
5
Fig. S4. Detected aliphatic and aromatic side chain contacts showing medium- and long-range interactions in the
NOESY spectra of 6 and 8 (a and b, respectively).
6
Fig. S5. Diffusion ordered spectroscopy for BB15D and its analog. The obtained signal intensity plots as a
function of gradient strength compared for compounds 6 and 8 at low-salt conditions (a) and for compound 8 at
low- and high-salt conditions (b). Pulsed field-gradient spin echo (PFGSE) NMR measurements were performed
at 25 °C by using the stimulated echo and longitudinal eddy current delay (LED) pulse sequence with excitation
sculpting for water suppression. A time of 3 ms was used for the dephasing/refocusing gradient pulse length, and
300 ms for the diffusion delay. The gradient strength was changed quadratically (from 5% to 80% of the
maximum value); the number of steps was 16. Each measurement was run with 32 scans and 8K time domain
points. DOSY spectra were processed and evaluated by using the exponential fit implemented in Topspin 3.5.
7
Table S1. Assigned 1HN,
1Hα,
13Cα, and
13Cβ chemical shifts (ppm) for the dimeric peptides. ACHC
replacements are highlighted with grey.
Sequence
(BB14 and its analog) 5 7
Sequence
(BB15 and its analog) 6 8
NH H1 a a H1 a a
Hα a 4.32 a 4.10
Cα a 55.26 a 56.19
Cβ a 30.33 31.65 32.35
NH S2 8.38 a S2 a a
Hα a 4.57 4.53 4.53
Cα a 58.06 58.00 57.98
Cβ 64.69 63.78 63.73 63.77
NH L3 8.75 8.72 L3 8.79 8.71
Hα 4.46 4.50 4.46 4.48
Cα 55.11 55.25 55.36 55.14
Cβ 42.36 42.58 42.32 42.43
NH T4 8.15 8.24 T4 8.14 8.22
Hα 4.31 4.37 4.36 4.34
Cα 61.83 61.69 61.65 61.78
Cβ 69.69 69.91 69.79 69.7
NH A5 8.30 7.92 A5 8.28 7.82
Hα 4.38 2.77 4.33 2.71
Cα 52.48 a 52.27 47.54
Cβ 19.56 50.77 19.41 50.92
NH S6 8.31 8.14 K6 8.40 8.18
Hα 4.55 4.43 4.38 4.21
Cα 58.03 57.72 55.89 56.08
Cβ 63.78 63.86 32.89 33.06
NH I7 8.21 8.23 I7 8.20 8.11
Hα 4.22 4.22 4.55 4.55
Cα aa 61.30 58.26 58.24
Cβ 38.97 38.93 39.12 39.13
NH k8 8.48 8.50 p8 a a
Hα 4.32 4.32 4.41 4.41
Cα 56.2 56.27 62.96 62.92
Cβ 32.4 32.91 32.26 32.27
NH a9 8.29 8.32 k9 8.40 8.40
Hα 4.35 4.30 4.19 4.18
Cα 52.42 52.81 56.99 57.05
Cβ 19.45 19.36 33.09 33.02
NH L10 8.24 8.22 L10 8.27 8.26
Hα 4.48 4.47 4.42 4.41
Cα 55.39 54.76 54.69 54.74
Cβ 42.54 42.63 42.33 42.38
NH T11 8.38 8.31 T11 8.14 a
Hα 3.87 4.34 4.31 4.26
Cα 64.03 61.99 61.72 61.89
8
Cβ 72.32 69.77 69.95 69.88
NH I12 8.34 8.20 F12 8.30 8.26
Hα 4.35 4.20 4.72 4.72
Cα a 60.67 57.33 57.42
Cβ 39.76 38.97 40.14 a
NH H13 a 8.70 S13 8.32 8.31
Hα a 4.81 4.50 4.51
Cα a 55.01 57.73 57.82
Cβ 30.83 29.51 64.01 64.02
NH V14 8.35 8.36 I13 8.21 8.196
Hα 4.23 4.15 4.23 4.234
Cα 61.35 62.12 60.69 a
Cβ a 32.98 39.05 39.08
NH Q15 8.54 8.58 A15 8.30 8.3
Hα 4.36 4.37 4.61 4.6
Cα 55.82 55.78 50.45 50.5
Cβ 30.74 29.82 18.33 18.33
NH a16 8.29 8.41 p16 a a
Hα 4.36 4.36 4.41 4.41
Cα 52.39 52.27 63.30 63.31
Cβ 19.28 19.48 31.91 a
NH k17 8.40 8.45 h17 8.31 a
Hα 4.36 4.39 4.63 4.59
Cα 55.74 56.52 56.60 56.98
Cβ 33.03 33.24 a a
NH T18 8.15 8.20 T18 8.11 a
Hα 4.32 4.38 4.30 4.30
Cα 61.84 61.69 61.72 61.8
Cβ 69.68 69.81 69.66 69.96
NH A19 8.42 8.46 Y19 8.30 a
Hα 4.45 4.48 4.65 4.63
Cα a 52.41 57.82 57.9
Cβ 21.41 19.38 a a
NH T20 8.17 8.20 T20 8.13 a
Hα 4.35 4.41 4.31 4.32
Cα 61.06 61.50 61.40 61.82
Cβ 69.68 70.03 70.07 69.89
NH C21 6.37 8.52 C21 8.31 a
Hα 4.94 4.75 4.75 a
Cα 54.81 55.39 55.16 a
Cβ a a a a
NH Q22 8.46 8.53 A22 8.44 8.44
Hα 4.39 4.48 4.42 4.41
Cα 56.68 55.53 52.16 52.18
Cβ 31.32 30.03 19.51 19.53
NH V23 8.22 8.34 V23 8.11 8.11
Hα 4.35 4.10 4.45 4.44
9
Cα 61.05 62.63 59.55 59.56
Cβ a 32.88 32.99 33.02
NH k24 8.56 8.54 p24 a a
Hα 4.26 4.28 4.35 4.36
Cα 55.54 56.02 62.92 62.95
Cβ 32.44 32.88 32.15 32.12
NH a25 8.26 8.25 k25 8.27 8.23
Hα 4.36 4.22 4.15 4.14
Cα 52.4 52.57 56.52 56.46
Cβ 19.08 19.32 32.90 32.94
NH Y26 7.99 8.14 Y26 8.34 8.21
Hα 4.73 4.75 4.75 4.79
Cα 60.01 57.15 57.27 57.04
Cβ 41.94 39.13 38.74 a
NH T27 8.24 8.24 T27 8.21 8.26
Hα 3.84 4.33 4.29 4.27
Cα 64.11 61.97 61.87 62.22
Cβ 72.39 70.06 69.98 69.89
NH V28 8.16 7.85 A28 8.31 8.15
Hα 4.45 2.72 4.32 4.17
Cα 61.55 a 52.27 56.31
Cβ a 50.85 19.41 33.05
NH H29 8.68 8.35 K29 8.32 8.151
Hα 4.59 4.60 4.32 4.135
Cα 55.13 55.18 56.20 61.9
Cβ 29.41 29.08 33.04 33.06
NH I30 8.28 8.26 V30 8.23 8.15
Hα 4.12 4.22 4.14 4.14
Cα 62.63 61.02 61.99 61.90
Cβ 39.02 39.02 32.98 33.06
NH S31 8.47 8.49 S31 8.46 8.44
Hα 4.47 4.47 4.45 4.44
Cα 57.85 58.27 57.93 57.94
Cβ 63.77 63.69 63.78 63.76
NH E32 8.45 8.52 H32 a a
Hα 4.30 4.32 4.66 4.62
Cα 55.49 56.00 55.60 55.99
Cβ 31.35 30.41 30.28 31.01
a Missing resonance/the resonance could not be assigned due to chemical exchange.
10
Peptide Characterization
Lyophilized peptides were analyzed by ESI MS and by analytical HPLC measurements on an Aeris Peptide C18
2.5*25 mm column (Phenomenex) with a gradient from 5% A to 80% B over 25 min at a flow rate of 1.2
ml*min-1
. Eluents were: A: 0.1% TFA/water; B: 0.1% TFA/20% water/80% ACN. Peptide purity was >95%
according to analytical HPLC measurements.
Analytical HPLC chromatogram and ESI-MS spectrum of 2 (monomeric BB15); [MH22+]: 1756.25;
[MH33+
]:1171.58 [MH44+
]: 879.13; [MH55+
]: 703.49; [MH66+
]: 586.35; calculated MW: 3511
RT: 0.00 - 25.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
4.54
9.36
9.30
9.17
9.135.02
9.08
9.77
8.89 9.92 10.29 11.16 12.22 12.84 16.85 23.2013.69 20.60 21.06 22.2814.85 18.7018.13 24.9120.085.19 8.496.75 7.444.510.39 0.75 1.63 3.382.12
NL:2.31E8
TIC MS BB15S_re_2
BB15S_re_2 #2973 RT: 9.37 AV: 1 NL: 2.77E7T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
586.35
1171.58
703.49502.77
879.13
1756.25
595.77 1204.00553.20 713.32 1805.71808.71 901.13 1341.40489.27 1954.681029.70 1462.80 1580.371088.40 1632.05336.89225.95
11
Analytical HPLC chromatogram and ESI-MS spectrum of 3 (monomeric BB14 analog);
[MH33+
]:1177.61; [MH44+
]:883.94; [MH55+
]: 707.32; [MH66+
]:589.62; calculated MW: 3531
RT: 0.00 - 25.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
11.23
11.420.09 9.910.33 11.67 21.0318.1814.65 20.4116.9112.39 15.40 19.3613.88 21.2811.09 22.35 23.454.571.18 9.062.48 8.882.53 23.757.456.784.99 6.51
NL:3.10E7
TIC MS f14_150221205237
f14_150221205237 #3575 RT: 11.25 AV: 1 NL: 3.88E6T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
589.62
707.32
883.94
1177.61
1197.47910.98
677.92 742.45 1796.291243.08565.28221.60 338.56 1313.14505.39 1543.45989.96 1885.981468.96421.24 857.77 1143.35 1630.04 1721.10 1985.38
12
Analytical HPLC chromatogram and ESI-MS spectrum of 4 (monomeric BB15 analog); [MH22+]:
1811.18; [MH33+
]: 1207.62; [MH44+
]: 906.05; [MH55+
]: 724.97; [MH66+
]: 604.40; calculated MW:
3620
RT: 0.00 - 25.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
10.09
4.54
5.0210.91
23.1211.0611.91 24.2722.3318.6116.6012.45 20.9917.9515.84 20.5815.2813.64
9.175.34 8.876.77 8.025.76
NL:4.24E7
TIC MS BB15S_528_tiszt_p2_re
BB15S_528_tiszt_p2_re #3187 RT: 10.06 AV: 1 NL: 6.75E6T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
604.40
518.20
1207.62
724.97
906.05
1811.18
1225.39526.171239.55 1821.60613.95 919.34736.23 813.24453.56 534.14 1905.521285.551183.31 1632.521397.38 1750.041083.56 1478.75333.31283.39 1527.55
13
Analytical HPLC chromatogram and ESI-MS spectrum of 6 (dimeric BB15); [MH44+
]: 1756.33;
[MH55+
]: 1405.22; [MH66+
]: 1170.94; [MH77+
]: 1004.01; [MH88+
]: 878.68; [MH99+
]: 781.18; calculated
MW: 7020
RT: 0.00 - 25.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
9.02
9.04
9.24
9.354.55
9.4810.19 13.2711.27 12.56 13.754.66 14.45
15.12 15.83 17.99 18.10 19.00 20.15 22.47 24.6721.71 23.645.02 8.555.61 6.23 7.764.520.26 3.641.43 2.07
NL:1.26E8
TIC MS BB15D_tiszt_ell_151007174240
BB15D_tiszt_ell_151007174240 #2851 RT: 9.01 AV: 1 NL: 1.30E7T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
703.20
639.37781.18
1756.33
878.68
1004.01
1405.22
586.24
1170.94
541.19
791.90712.73 1437.84 1810.531039.58 1921.351229.57894.33 1560.42503.60 1716.121324.16394.22296.39
14
Analytical HPLC chromatogram and ESI-MS spectrum of 7 (dimeric BB14 analog); [MH55+]:
1412.61; [MH66+
]: 1177.81; [MH77+
]: 1009.70; [MH88+
]: 883.64; [MH99+
]: 785.56; calculated MW:
7060
RT: 0.00 - 25.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
10.85
11.15 17.1811.88 12.65 21.7618.99 21.2417.28 24.8919.54 22.1415.45 16.0114.28 23.930.09 10.730.32 10.258.198.092.49 4.571.22 3.57 7.285.894.90
NL:1.66E7
TIC MS f2
f2 #3416 RT: 10.83 AV: 1 NL: 1.75E6T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
785.56
883.64
707.16
1009.70
1177.81
1412.61
643.11
723.18 813.23 1770.95589.60 1455.73221.66 322.67 929.16 1825.92867.14417.76 1897.241051.69 1559.561395.94525.52 1682.131237.86
15
Analytical HPLC chromatogram and ESI-MS spectrum of 8 (dimeric BB15 analog); [MH44+
]:
1810.55; [MH55+
]: 1448.68; [MH66+
]: 1207.49; [MH77+
]: 1035.20; [MH88+
]: 905.85; [MH99+
]: 805.40;
calculated MW: 7238
The characterization of monomeric and dimeric BB14 (compounds 1 and 5) are described in the
supporting material of our previous work1 (designated as compounds 1 and 7, respectively).
1. G. Olajos, A. Hetenyi, E. Weber, L. J. Nemeth, Z. Szakonyi, F. Fulop and T. A. Martinek,
Chemistry, 2015, 21, 6173-6180.
RT: 0.00 - 15.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
7.99
4.54
7.84
8.14
8.197.53 8.424.56 10.858.79 11.167.42 12.0010.679.82 13.615.02 14.9614.4812.867.246.825.71
NL:8.53E7
TIC MS 528_ell_HPLC
528_ell_HPLC #2512 RT: 7.94 AV: 1 NL: 7.12E6T: ITMS + c ESI Full ms [200.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
1448.68
805.40
1035.20905.85
1207.49
724.99
659.15
1810.55
604.38
917.97558.02518.22 1237.881169.58877.83770.00 1579.84 1950.121322.97 1645.63 1768.03207.54 334.38 379.32