Supporting Information
Fig. S1 HPLC chromatogram of the separation of the two diastereoisomers V and VI.
Peptide purification was performed by reverse phase HPLC on a Delta Pak Waters C18-100Å silica column (solvent A: water + 0.1% TFA; solvent B: 90% acetonitrile + 0.1% TFA) with a linear gradient of 10-35% (v/v) B over 15 min. Peptide homogeneity (>95%) was determined by analytical HPLC on a Vydac C18 (218TP510) column using the same solvents with a linear gradient of 10-90% (v/v) B over 30 min.
I IV VII
II V VIII
III VI
Fig. S2 CD spectra of the entire series of analogues of PTH(1-11) in aqueous solution containing 20% TFE(v/v) at ~1 mM peptide concentration
Fig. S3 Secondary chemical shifts of the aCH protons of the inactive analogues, compared to the reference peptide.
L-Nle8-HarPTH (I)
Chemical Shift Values for [Aib1,3, Nle8, Gln10, Har11]-PTH(1-11)NH2, 1.50 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Aib / - / - / 1.776; 1.684 / -
2 / Val / 7.967 / 3.902 / 2.135 / gCH3= 1.005; 1.020
3 / Aib / 8.495 / - / 1.474 / -
4 / Glu / 8.370 / 4.001 / 2.130; 2.040 / gCH2= 2.522, 2.303
5 / Ile / 7.815 / 3.861 / 2.019 / gCH2= 1.682, 1.247 gCH3= 0.937
d CH3= 0.886
6 / Gln / 8.108 / 4.158 / 2.164 / gCH2= 2.461, 2.363 dNH2= 7.222, 6.748
7 / Leu / 8.060 / 4.193 / 1.810, 1.685 / gCH= 1.685 dCH3= 0.917
8 / Nle / 8.042 / 4.056 / 1.851; 1.813 / gCH2= 1.419; 1.296; ε CH3= 0.858
9 / His / 8.037 / 4.563 / 3.414, 3.302 / H2= 8.595 H4= 7.381
10 / Gln / 8.131 / 4.280 / 2.503, 2.451 / gCH2= 2.503; 2.451 dNH2= 7.439, 6.765
11 / Har / 8.070 / 4.249 / 1.910, 1.831 / gCH2= 1.625 dCH2= 1.528, 1.469
eCH2= 3.187 dN= 7.157
ωNH-term= 7.338, 7.046
D-Nle8-HarPTH (II)
Chemical Shift Values for [Aib1,3, D-Nle8, Gln10, Har11]-PTH(1-11)NH2, 1.50 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Aib / - / - / 1.700 / -
2 / Val / 7.923 / 4.026 / 2.142 / gCH3= 1.019
3 / Aib / 8.444 / - / 1.472 / -
4 / Glu / 8.053 / 4.154 / 2.081 / gCH2= 2.502, 2.397
5 / Ile / 7.883 / 4.034 / 1.977 / gCH2= 1.596, 1.245 gCH3= 0.940
d CH3= 0.890
6 / Gln / 8.108 / 4.275 / 2.127, 2.049 / gCH2= 2.406, 2.352 dNH2= 7.319, 6.737
7 / Leu / 8.049 / 4.257 / 1.705, 1.608 / gCH= 1.608 dCH3= 0.922
8 / D-Nle / 8.164 / 4.088 / 1.764 / gCH2= 1.258, 1.168 dCH2, ε CH3= 0.868
9 / His / 8.409 / 4.696 / 3.388, 3.217 / H2= 8.635 H4= 7.324
10 / Gln / 8.351 / 4.336 / 2.173, 2.111 / gCH2= 2.440 dNH2= 7.501, 6.776
11 / Har / 8.209 / 4.297 / 1.884, 1.778 / gCH2= 1.633 dCH2= 1.491, 1.442
eCH2= 3.196 dN= 7.158
ωNH-term= 7.477, 7.031
III
Chemical Shift Values for [Aib1, Ala3, L(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Aib / - / - / - / -
2 / Val / 7.929 / 4.099 / 2.128 / gCH3= 1.004
3 / Ala / 8.529 / 4.329 / 1.464 / -
4 / Glu / 8.427 / 4.143 / 2.119 / gCH2= 2.452, 2.510
5 / Ile / 7.908 / 3.971 / 1.933 / gCH2= 1.582, 1.249 gCH3= 0.945 dCH3= 0.945
6 / Gln / 8.065 / 4.192 / 2.18 / gCH2= 2.440, 2.389 NH2= 7.415, 6.756
7 / Leu / 7.923 / 4.142 / 1.724 / gCH2= 1.624, dCH2= 0.926
8 / L(aMe)Nle / 8.113 / (aMe)1.429 / 2.091, 1.705 / gCH2= 1.313, 1.248 dCH2= 1.105, eCH2= 0.877
9 / His / 7.947 / 4.559 / 3.439, 3.293 / H2= 8.603 H4= 7.400
10 / Gln / 8.202 / 4.289 / 2.22 / gCH2= 2.229, 2.479, NH2= 7.415, 6.756
11 / Arg / 8.025 / 4.329 / 1.985, 1.836 / gCH2= 1.727 dCH2= 3.223
eNH= 7.258 NH2-term= 7.365, 7.088
IV
Chemical Shift Values for [Aib1, Ala3, D(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Aib / - / - / - / -
2 / Val / 7.933 / 4.075 / 2.142 / gCH3= 1.017
3 / Ala / 8.555 / 4.304 / 1.468 / -
4 / Glu / 8.404 / 4.119 / 2.143 / gCH2= 2.544, 2.446
5 / Ile / 7.840 / 3.932 / 1.969 / gCH2= 1.616, 1.275 gCH3= 0.951 dCH3= 0.920, 0.902 (?)
6 / Gln / 8.075 / 4.158 / 2.220, 2.158 / gCH2= 2.457, 2.399 NH2= 7.386, 6.730
7 / Leu / 7.835 / 4.128 / 1.752, 1.691 / gCH2= 1.691 dCH2= 0.927
8 / D(aMe)Nle / 8.070 / (αMe) 1.438 / gCH2= 1.269 dCH2= 0.902 eCH2= 0.845
9 / His / 7.934 / 4.502 / 3.431, 3.303 / H2= 8.603 H4= 7.424
10 / Gln / 8.215 / 4.270 / 2.259 / gCH2= 2.495, 2.564 NH2= 7.317, 6.760
11 / Arg / 8.003 / 4.312 / 2.001, 1.862 / gCH2= 1.783 dCH2= 3.211
eNH= 7.244 NH2-term= 7.317, 7.072
V
Chemical Shift Values for [Aib1, 3, L(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1.0 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Aib / - / - / 1.671, 1.728 / -
2 / Val / 7.970 / 3.952 / 2.153 / gCH3= 1.041, 1.024
3 / Aib / 8.514 / - / 1.735, 1.465 / -
4 / Glu / 8.203 / 4.024 / 2.139. 2.086 / gCH2= 2.569, 2.390
5 / Ile / 7.758 / 3.863 / 2.005 / gCH2= 1.667, 1.247 gCH3= 0.944 dCH3= 0.892
6 / Gln / 7.985 / 4.159 / 2.150 / gCH2= 2.451, 2.354 NH2= 7.226, 6.743
7 / Leu / 8.034 / 4.107 / 1.731 / gCH2= 1.646 dCH2= 0.919
8 / L(aMe)Nle / 8.106 / (αMe) 1.445 / 2.126, 1.698 / gCH2= 1.275 dCH2= 1.110 eCH2= 0.879
9 / His / 7.902 / 4.527 / 3.441, 3.306 / H2= 8.591 H4= 7.412
10 / Gln / 8.222 / 4.272 / 2.227 / gCH2= 2.544, 2.475 NH2= 7.411, 6.752
11 / Arg / 8.012 / 4.320 / 1.985, 1.836 / gCH2= 1.726 dCH2= 3.208
eNH= 7.258 NH2-term= 7.335, 7.097
VI
Chemical Shift Values for Aib1, 3, D(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1.0 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / daltri1 / Aib / - / - / 1.727, 1.668 / -
2 / Val / 7.980 / 3.943 / 2.159 / gCH3= 1.038
3 / Aib / 8.544 / - / 1.454 / -
4 / Glu / 8.215 / 4.006 / 2.153, 2.083 / gCH2= 2.590, 2.398
5 / Ile / 7.736 / 3.854 / 2.029 / gCH2= 1.664, 1.274 gCH3= 0.943 dCH3= 0.881
6 / Gln / 7.965 / 4.149 / 2.164 / gCH2= 2.451, 2.350 NH2= 7.217, 6.740
7 / Leu / 7.916 / 4.092 / 1.743, 1.684 / gCH2= 1.684 dCH2= 0.912
8 / D(aMe)Nle / 8.078 / (αMe) 1.424 / 1.972, 1.803 / gCH2= 1.282 dCH2= 1.212 eCH2= 0.832
9 / His / 7.867 / 4.491 / 3.432, 3.307 / H2= 8.601 H4= 7.434
10 / Gln / 8.248 / 4.260 / 2.250 / gCH2= 2.571, 2.486 NH2= 7.388, 6.734
11 / Arg / 8.003 / 4.297 / 1.994, 1.851 / gCH2= 1.774, 1.705 dCH2= 3.200
eNH= 7.240 NH2-term= 7.298, 7.092
VII
Chemical Shift Values for [Ala1, Aib3, L(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Ala / 4.198 / 1.608 / -
2 / Val / 8.464 / 4.044 / 2.170 / gCH3= 1.053
3 / Aib / 8.479 / 1.477 / -
4 / Glu / 8.210 / 4.030 / 2.128 / gCH2= 2.441
5 / Ile / 7.703 / 3.889 / 2.028 / gCH2=1.618 g'CH2=1.282 gCH3=0.956 dCH3=0.917
6 / Gln / 7.789 / 4.172 / 2.144 / gCH2=2.430 g'CH2=2.358 NH2=7.268 / 6.478
7 / Leu / 8.037 / 4.104 / 1.723 / gCH2=1.649 dCH2=0.919
8 / L(aMe)Nle / 8.114 / (αMe) 1.445 / b=2.138 b'=1.700 / gCH2=1.296 g'CH2=1.121 dCH2,eCH2=0.883
9 / His / 7.885 / 4.525 / b=3.444 b'=3.311 / H2=8.596 H4= 7.424
10 / Gln / 8.221 / 4.278 / 2.232 / gCH2=2.543 g'CH2=2.484 NH2=7.405 / 6.748
11 / Arg / 8.005 / 4.323 / b=1.993 b'=1.843 / gCH2=1.730 dCH2= 3.215 eNH=7.261 NH2-term= 7.095 / 7.305
VIII
Chemical Shift Values for [Ala1, Aib3, D(aMe)Nle8, Gln10, Arg11]-PTH(1-11)NH2, 1 mM, H2O/TFE-d3 80/20, at T = 298 K.
AA / dNH / da / db / d1 / Ala / 4.194 / 1.604 / -
2 / Val / 8.464 / 4.039 / 2.170 / gCH3= 1.056
3 / Aib / 8.529 / b= 1.606 b'=1.476 / -
4 / Glu / 8.301 / 4.019 / 2.134 / gCH2=2.548 g'CH2=2.423
5 / Ile / 7.691 / 3.890 / 2.051 / gCH2=1.613 gCH3=1.306 dCH3=0.948
6 / Gln / 7.795 / 4.167 / 2.149 / gCH2=2.436 g'CH2= 2.357
NH2= 7.288 / 6.751
7 / Leu / 7.936 / 4.098 / 1.742 / gCH2=1.672 dCH2= 0.914
8 / D(aMe)Nle / 8.041 / (αMe) 1.431 / b= 1.947 b'=1.814 / gCH2= 1.276 dCH2= 1.217 eCH2= 0.843
9 / His / 7.858 / 4.508 / b= 3.433 b'=3.308 / H2=8.609 H4= 7.434
10 / Gln / 8.237 / 4.270 / 2.253 / gCH2=2.493 g'CH2=2.571 NH2= 7.396 / 6.739
11 / Arg / 7.999 / 4.303 / b= 1.997 b'=1.856 / gCH2= 1.705 g'CH2= 1.781 dCH2=3.206
eNH=7.242 NH2-term= 7.305 / 7.092
S4: Chemical shift Value for I-VIII analogues.
S5: Superimpositions of the ensembles of the lowest energy calculated structures for III-VIII analogues