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: martinek@pharm.u-szeged.hu

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