SUPPORTING INFORMATION
Structural similarity between 3-peptides synthesized
from 3-homo-amino acids or L-aspartic acid
monomers
Sahar Ahmed,1, † Tara Sprules,2 Kamaljit Kaur1, *
Table of Contents
Table S1. Characterization of 3-peptides 2-6............................................................................. S2
Figure S1. RP-HPLC traces of four crude β3-amido amino acid monomers. ............................. S3
Figure S2. RP-HPLC chromatograms of pure β3-peptides 2-6. .................................................. S4
Table S2: 1H NMR chemical shift assignments for β3-peptide 2 in CF3CD2OH. ....................... S5
Table S3: 1H NMR chemical shift assignments for 3-peptide 3a in CF3CD2OH. ...................... S6
Table S4: 1H NMR chemical shift assignments for 3-peptide 3b in CF3CD2OH....................... S7
Table S5: 1H NMR chemical shift assignments for 3-peptide 4a in CF3CD2OH. ...................... S8
Table S6: 1H NMR chemical shift assignments for 3-peptide 4b in CF3CD2OH. ...................... S9
Table S7: 1H NMR chemical shift assignments for 3-peptide 5a in CF3CD2OH. .................... S10
Table S8: 1H NMR chemical shift assignments for 3-peptide 5b in CF3CD2OH..................... S11
Table S9: 1H NMR chemical shift assignments for 3-peptide 6 in CF3CD2OH. ...................... S12
Figure S3. Circular dichroism (CD) spectra of β3-peptides 3-5 (200 μM) in TFE. .................. S13
Figure S4. 2D NOESY of β3-peptides 2, 3a, and 6 in TFE. ...................................................... S15
Table S10. Structure calculation statistics for β3-hexapeptide 2. .............................................. S16
Table S11. Structure calculation statistics for β3-hexapeptide 6. .............................................. S16
Figure S5. Solution structures of -peptides 1 and 2. .............................................................. S17
S1
Table S1. Characterization of 3-peptides 2-6.
Peptide
Mass [M+H]+
Obsd. (Calcd.)
HPLC Gradient
2
782.9 (782.6)
20-65% ACN/H2O
16.0
2
89
3a
699.8 (699.4)
12-20% ACN/H2O
29.5
2
62
3b
685.3 (685.4)
12-20% ACN/H2O
30.0
2
53
4a
885.8 (885.5)
12-45% ACN/H2O
21.0
2
72
4b
871.8 (871.4)
19-28% ACN/H2O
22.0
2
54
5a
1041.6 (1041.2)
19-28% IPA/H2O
23.0
1.5
81
5b
1027.9 (1027.6)
19-40% ACN/H2O
21.5
2
82
Elution Flow rate
time
(ml/min)
(min)
Yield
(%)
1100.4 (1100.6)
15-30% IPA/H2O
16.7
1.5
90
6
Vydac C18 semi-preparative (1 x 25 cm, 5 μm) HPLC column was used. A gradient of
ACN/water or IPA/water in 30 minutes with a flow rate of 1-5-2.0 mL/min was used.
S2
(a)
O
Absorbance (mV)
Fmoc
(c)
(b)
NH
O
COOH
N
H
Fmoc
NH
COOH
N
H
(d)
O
O
O
O
HN
O
O
Fmoc
N
H
NH
Fmoc
COOH
N
H
NH
COOH
Time (Min)
Figure S1. RP-HPLC traces of four crude β3-amido amino acid monomers, Fmoc-β3amV-OH
(a), Fmoc-β3amL-OH (b), Fmoc-β3amK(Boc)-OH (c) and Fmoc-β3amE(tBu)-OH (d). The HPLC
runs were performed using 30-100% IPA/H2O in 35 min with flow rate, 2 mL/min (insert shows
the structure of each monomer).
S3
Absorbance (mV)
Time (Min)
Figure S2. RP-HPLC chromatograms of pure β3-peptides 2-6 obtained using analytical Vydac
C18 analytical column (0.46 x 25 cm, 5 μm).
S4
Table 02: 1H NMR chemical shift assignments for β3-peptide 2 in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
2
3
4
5
7.39 (1H, H3N+),
3.57(1H,NHCHCH2),
2.89,2.68(2H,NHCHCH2)
8.34(d,J=9.4Hz,HN),
4.52(1H,NHCHCH2),
2.75,2.57(2H,NHCHCH2),
8.27(d,J=9.2Hz,HN),
4.36(1H,NHCHCH2),
2.46(2H,NHCHCH2)
7.47(d,J=9.4Hz,HN),
4.24(1H,NHCHCH2),
2.57,2.26(2H,NHCHCH2),
6.76 (d, J=8.9 Hz,HN),
4.38(1H,NHCHCH2),
2.53,2.34(2H,NHCHCH2),
6.54(d,J=9.5Hz,HN),
4.45(1H,NHCHCH2),
2.50,2.30(2H,NHCHCH2),
6.85, 5.82 (NH2)
C-terminus
6
Side chain shifts(δ)
2.09(1H,(CH3)2CH), 1.09(6H,(CH3)2CH)
7.29(3H,NH3+CH2CH2CH2CH2),
3.03,2.97(2H,NH3CH2CH2CH2CH2),
1.79,1.68(2H,NH3CH2CH2CH2CH2),
1.58(2H,NH3CH2CH2CH2CH2),
1.45,1.42(2H,NH3CH2CH2CH2CH2)
1.52(1H,(CH3)2CHCH2), 1.45,
1.28(2H,(CH3)2CHCH2), 0.94,0.89(6H,(CH3)2CH
CH2)
1.73(1H,(CH3)2CH), 0.91(6H,(CH3)2CH)
7.31(3H,NH3+CH2CH2CH2CH2),
2.97(2H,NH3CH2CH2CH2CH2),
1.68(2H,NH3CH2CH2CH2CH2),
1.58,1.45(2H,NH3CH2CH2CH2CH2),
1.38(2H,NH3CH2CH2CH2CH2)
1.58(1H,(CH3)2CHCH2), 1.42,
1.31(2H,(CH3)2CHCH2), 0.94(6H,(CH3)2CH CH2)
The final conc. of 2 in TFE-d2 was 5 mM. The chemical shifts were referenced to the TFE
methylene protons at 3.88 ppm and recorded at 15 ºC.
S5
Table 03: 1H NMR chemical shift assignments for 3-peptide 3a in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
2
3
4
7.77 (NH3+-terminal), 4.28
(1H,NHCHCH2), 3.08, 2.88
(2H,NHCHCH2),
8.21 (1H, HN), 4.88
(1H,NHCHCH2), 2.75,
2.64(2H,NHCHCH2)
7.63 (1H, HN),
4.79(1H,NHCHCH2), 2.80,
2.66(2H,NHCHCH2),
7.54 (1H, HN), 4.93
(1H,NHCHCH2),
2.77,2.67(2H,NHCHCH2),
Side chain shifts (δ)
7.57 (1H, NH). 3.04 , 3.18 (2H, (CH3)2CHCH2),
1.78 (1H, (CH3)2CH), 0.90(6H, (CH3)2CHCH2),
7.02 (1H, NH) 3.96 (1H, (CH3)2CH), 1.15 (6H,
(CH3)2CH),
7.29 (1H, NH), 7.20 (3H, NH3+) 3.25
(2H,NH3CH2CH2CH2CH2),
3.00(2H,NH3CH2CH2CH2CH2), 1.66
(2H,NH3CH2CH2CH2CH2), 1.57
(2H,NH3CH2CH2CH2CH2),
7.68 (1H, NH), 3.12 (1H (CH3)2CH CH2), 3.00
(1H, (CH3)2CH CH2), 1.80 (1H,(CH3)2CH), 0.92
(6H, (CH3)2CHCH2),
6.85, 6.52 (NH2)
C- terminus
3
The final conc. of  -peptide 3a in TFE-d2 was 0.5 mg/250 μL. The chemical shifts were
referenced to the TFE methylene protons at 3.88 ppm. NH2 terminal of residue 1 was not
completely assigned from NMR.
S6
Table 04: 1H NMR chemical shift assignments for 3-peptide 3b in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
2
3
4
Side chain shifts (δ)
7.71 (NH3+-terminal ), 4.27
(1H,NHCHCH2), 3.20, 2.99
(2H,NHCHCH2),
8.17 (1H, HN), 4.88
(1H,NHCHCH2),
2.93(2H,NHCHCH2)
7.61 (1H, HN),
4.82(1H,NHCHCH2), 2.89
(2H,NHCHCH2)
7.57 (1H, HN), 4.93
(1H,NHCHCH2), 2.77,
2.60(1H,NHCHCH2)
C- terminus
7.57 (1H, NH). 3.20, 2.99 (2H (CH3)2CHCH2),
1.8 (1H, (CH3)2CH), 0.91 (6H, (CH3)2CHCH2),
7.03 (1H, NH) 4.02 (1H, (CH3)2CH), 1.16 (6H,
(CH3)2CH)
3.45- 3.33(2H,NH3CH2CH2CH2), 2.60-2.77
(4H,NH3CH2CH2CH2)
7.69 (1H, NH) 2.60-2.77 (2H (CH3)2CHCH2),
1.80 (1H,(CH3)2CH), 0.91 (6H,
(CH3)2CHCH2),
6.86, 6.48 (NH2)
The final conc. of 3-peptide 3b in TFE-d2 was 0.5 mg/250 μL. The N-terminal NH3+ was not
completely assigned due to exchange with the solvent.
S7
Table 05: 1H NMR chemical shift assignments for 3-peptide 4a in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
Side chain shifts (δ)
1
4.27 (1H,NHCHCH2), 2.643.14 (2H,NHCHCH2)
2
8.57(1H, HN), 4.80
(1H,NHCHCH2), 2.64-3.14
(2H,NHCHCH2),
3
8.57 (1H, HN), 5.00
(1H,NHCHCH2), 2.64-3.14
(2H,NHCHCH2)
7.31 (1H, HN), 5.05
(1H,NHCHCH2), 2.64-3.14
(2H,NHCHCH2)
6.74 (1H, NH) 4.02 (1H, (CH3)2CH), 1.14 (6H,
(CH3)2CH)
7.29 (1H, HN), 4.27
(1H,NHCHCH2), 2.56
(2H,NHCHCH2)
8.34 (1H, NH), 3.32(2H,(CH3)2CHCH2), 1.78(1H,
(CH3)2CH), 0.91 (6H, (CH3)2CH)
4
5
C- terminus
7.79(NH), 3.72(1H,COOHCH2CH2NH)
3.64(1H,COOHCH2CH2NH), 2.54 (2H
COOHCH2CH2NH)
7.24 (1H, NH), 3.44(2H,(CH3)2CHCH2), 1.80 (1H,
(CH3)2CH), 0.91 (6H, (CH3)2CH)
7.96 (1H, NH), 7.20 (3H, NH3+) 3.47
(2H,NH3CH2CH2CH2CH2), 1.68-1.84
(2H,NH3CH2CH2CH2CH2)
6.76, 6.58 (NH2)
The final conc. of β3-peptide 4a in TFE-d2 was 0.6 mg/250 μL. The N-terminal NH3+ was not
observed due to exchange with the solvent. One of the CH2 protons of residue 4 were not
assigned from the NMR due to broadness of the peaks.
S8
Table 06: 1H NMR chemical shift assignments for 3-peptide 4b in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
4.26 (1H,NHCHCH2),
3.36,2.84 (2H,NHCHCH2)
2
8.50 (1H, HN), 4.80
(1H,NHCHCH2), 2.71,2.61
(2H,NHCHCH2)
3
8.50 (1H,HN), 5.00
(1H,NHCHCH2), 2.90,2.64
(2H,NHCHCH2)
7.44 (1H,HN), 5.03
(1H,NHCHCH2), 2.78,2.68
(2H,NHCHCH2)
4
Side chain shifts (δ)
7.78(NH),3.63(1H,COOHCH2CH2NH)
3.47(1H,COOHCH2CH2NH), 2.54 (2H
COOHCH2CH2NH)
7.27 (1H, NH), 3.05(2H,(CH3)2CHCH2), 1.77(1H,
(CH3)2CH), 0.89 (6H, (CH3)2CH CH2)
6.75 (1H, NH) 3.96 (1H, (CH3)2CH), 1.13 (6H,
(CH3)2CH)
8.12 (1H, NH), 7.20 (3H, NH3+) 3.38
(2H,NH3CH2CH2CH2), 3.03 (2H,NH3CH2CH2CH2),
1.95 (2H,NH3CH2CH2CH2),
7.28 (1H, NH), 4.26
8.25 (1H, NH), 3.11, 2.96(2H,(CH3)2CHCH2), 1.81(1H,
(1H,NHCHCH2), 2.72-2.64 (CH3)2CH), 0.91 (6H, (CH3)2CH)
(2H,NHCHCH2)
C- terminus
6.76, 6.56 (NH2)
5
The final conc. of β3-peptide 4b in TFE-d2 was 0.6 mg/250 μL. The N-terminal NH3+ was not
observed due to exchange with the solvent.
S9
Table 07: 1H NMR chemical shift assignments for 3-peptide 5a in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
2
3
4
5
6
Side chain shifts (δ)
5.15 (1H,NHCHCH2),
2.93,2.70 (2H,NHCHCH2)
8.89 (1H, (HN-terminal),
5.00 (1H,NHCHCH2),
3.16,2.57 (2H,NHCHCH2)
9.11 (1H, HN), 4.79
(1H,NHCHCH2), 3.06,2.57
(2H,NHCHCH2)
7.01 (1H, NH) 4.02 (1H, (CH3)2CH), 1.14 (6H,
(CH3)2CH)
7.56(NH), 3.52,3.44 (1H,COOHCH2CH2NH) 2.50 (2H
COOHCH2CH2NH)
8.50 (1H, HN), 5.08
(1H,NHCHCH2), 2.64,2.47
(2H,NHCHCH2)
7.30 (1H, HN), 5.30
(1H,NHCHCH2), 2.68
(2H,NHCHCH2)
7.75 (1H, NH) 4.00 (1H, (CH3)2CH), 1.20 (6H,
(CH3)2CH)
7.19 (1H, HN) 5.22
(1H,NHCHCH2), 2.70
(2H,NHCHCH2)
8.20 (1H, NH), 3.06, 3.02(2H,(CH3)2CHCH2), 1.80(1H,
(CH3)2CHCH2), 0.89 (6H, (CH3)2CH CH2)
C- terminus
6.93 (1H, NH), 3.06, 3.02(2H,(CH3)2CHCH2), 1.77(1H,
(CH3)2CH CH2), 0.87 (6H, (CH3)2CH CH2)
8.43 (1H, NH), 3.44,3.06(2H,NH3CH2CH2CH2CH2)
3.02(2H,NH3 CH2CH2CH2CH2),
1.63(2H,NH3CH2CH2CH2CH2),
1.70(2H,NH3CH2CH2CH2 CH2)
6.88, 6.73 (NH2)
The final conc. of 3-peptide 5a in TFE-d2 was 0.2 mg/250 μL. The N-terminal and side chain
NH3+ were not observed due to exchange with the solvent.
S10
Table 08: 1H NMR chemical shift assignments for 3-peptide 5b in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
1
2
3
4
5
2.93, 2.71
(2H,NHCHCH2)
8.81 (1H, (HN-terminal),
5.03 (1H,NHCHCH2),
3.09, 2.60
(2H,NHCHCH2)
9.01 (1H, HN), 4.81
(1H,NHCHCH2), 3.08,
2.60 (2H,NHCHCH2)
8.47 (1H, HN), 5.11
(1H,NHCHCH2), 2.65,
2.53 (2H,NHCHCH2)
7.46 (1H, HN), 2.71
(2H,NHCHCH2)
Side chain shifts (δ)
7.03 (1H, NH), 4.05 (1H, (CH3)2CH),1.15 (6H,
(CH3)2CH)
7.62(NH), 2.53(2H COOHCH2CH2NH) 3.55,3.47(2H
COOHCH2CH2NH)
6.97 (1H, NH),3.07, 3.04(2H, (CH3)2CH CH2), 1.78(1H,
(CH3)2CH CH2), 0.90 (6H, (CH3)2CH CH2)
7.74 (1H, NH)1.18 (6H, (CH3)2CH) 4.05 (1H, (CH3)2CH),
8.52 (1H, NH), 3.46, 3.30 (2H,NH3CH2CH2CH2) 3.03
(2H,NH3CH2CH2CH2) 1.96 (2H,NH3CH2CH2CH2)
7.25 (1H, HN) 5.22
8.16(1H,NH),3.07,2.99
(1H,NHCHCH2), 2.72
(2H,(CH3)2CHCH2)1.80(1H,(CH3)2CHCH2),0.90 (6H,
(2H,NHCHCH2)
(CH3)2CH CH2)
C- terminus
6.85, 6.76 (NH2)
The final conc. of 3-peptide 5b in TFE-d2 was 0.6 mg/250 μL. The N-terminal and side chain
NH3+ were not observed due to exchange with the solvent.
6
S11
Table 09: 1H NMR chemical shift assignments for 3-peptide 6 in CF3CD2OH (500 MHz).
Residue Backbone shifts (δ)
Side chain shifts (δ)
1
4.34 (1H,NHCHCH2),
3.49,2.89(2H,NHCHCH2)
2
8.67(d, J=9.0Hz, HN), 5.06
(1H,NHCHCH2),
3.182.58(2H,NHCHCH2)
3
9.08(d, J-8Hz, HN), 4.85
(1H,NHCHCH2),
3.09,2.60(2H,NHCHCH2)
6.94 (1H, NH), 3.07(2H,(CH3)2CHCH2), 1.77(1H,
(CH3)2CH), 0.89 (6H, (CH3)2CH)
4
8.52(d, J=7.9 Hz, HN), 5.03
(1H,NHCHCH2),
2.75,2.67(2H,NHCHCH2)
5
7.28 (1H, HN),
5.28(1H,NHCHCH2),
2.74,2.67(2H,NHCHCH2)
6
7.22(1H, HN), 5.23
(1H,NHCHCH2), 2.77(2H,
NHCHCH2).
8.08(1H, NH),7.30(3H,NH3+)
3.33(2H,NH3CH2CH2CH2CH2),
3.03(2H,NH3CH2CH2CH2CH2),
1.73(2H,NH3CH2CH2CH2CH2), 1.64
(2H,NH3CH2CH2CH2CH2)
8.39 (1H, NH),7.24(3H,NH3+)
3.33(2H,NH3CH2CH2CH2CH2),
3.03(2H,NH3CH2CH2CH2CH2),
1.72(2H,NH3CH2CH2CH2CH2), 1.64
(2H,NH3CH2CH2CH2CH2),
8.23 (1H, NH) 3.07, 2.99(2H (CH3)2CH CH2), 1.82
(1H, (CH3)2CH), 0.91 (6H, (CH3)2CHCH2),
C- terminus
7.79(NH), 3.66(1H,COOHCH2CH2NH)
3.49(1H,COOHCH2CH2NH), 2.58 (2H
COOHCH2CH2NH)
7.57(NH), 3.56,(1H,COOHCH2CH2NH)
3.45(1H,COOHCH2CH2NH), 2.56 (2H
COOHCH2CH2NH)
6.78, 6.72 (NH2)
The final conc. of 3-peptide 6 in TFE-d2 was 1.5 mg/250 μL. The N-terminal NH3+ was not
observed due to exchange with the solvent.
S12
0
2
-1
 x 10 (deg cm dmol )
(a)
-10
-3
5a
4a
3a
-20
210
225
240
(b)
-1
 x 10 (deg cm dmol )
195
2
0
-10
-3
5b
4b
3b
-20
195
210
225
240
Wavelength (nm)
Figure S3. Circular dichroism (CD) spectra of β3-peptides 3-5 (200 μM) in TFE.
S13
(a)
NH2
H
H2N
O
H
N
H
O
NH2
HO
H O
N
H
N
H
HO
N
H
H
NH2
N
H
1H-4H
2H-5H
3H-6H
2
S14
O
(b)
NH2
NH2
COOH
O
O
NH O
O
H
NH O
O
H
NH O
O
H
H2N
N
H
N
H
N
H
NHO
H
NH2
H2N
NH O
O
H
N
H
NH2
COOH
NH O
O
H
N
H
NH O
O
H
NH O
O
H
N
H
N
H
NH O
O
H
NHO
H
N
H
1H-4H
1H-4H
2H-5H
3H-6H
6
3a
Figure S4. 2D NOESY of β3-peptides 2, 3a, and 6 in TFE showing the long range NOEs,
CH(i)-CH(i+3) characteristic of 14-helix conformation.
S15
NH2
Table S10. Structure calculation statistics for β3-hexapeptide 2.
NOE upper distance limits
170
Intra-residue
68
Sequential
47
Medium range (I to i-2 or i+3)
53
Long range (I to i+4)
2
a
Final CYANA structures
CYANA target function
0.011±0.008
Average backbone RMSD
0.54±0.10 Å
Average heavy atom RMSD to mean
1.15±0.15 Å
Distance restraint violations
0
a
20 lowest energy structures of the 200 calculated.
Table S11. Structure calculation statistics for β3-hexapeptide 6.
NOE upper distance limits
167
Intra-residue
71
Sequential
42
Medium range (I to i-2 or i+3)
53
Long range (I to i+4)
1
a
Final CYANA structures
CYANA target function
0.0298±0.0287
Average backbone RMSD
0.50±0.12 Å
Average heavy atom RMSD to mean
1.44±0.14 Å
Distance restraint violations
0
a
20 lowest energy structures of the 200 calculated.
S16
Figure S5. Solution structures of -peptides 1 and 2 showing comparison of the right-handed
and the left-handed helices, respectively.
S17