S1
SUPPORTING INFORMATION
Title of manuscript: THE FLUORESCENCE AND INFRARED ABSORPTION PROBE PARACYANOPHENYLALANINE: EFFECT OF LABELING ON THE BEHAVIOR OF DIFFERENT
MEMBRANE-INTERACTING PEPTIDES
Authors: Sara Bobone, Marta De Zotti, Annalisa Bortolotti, Barbara Biondi, Gema Ballano,
Antonio Palleschi, Claudio Toniolo, Fernando Formaggio, Lorenzo Stella
I.
Conditions at Each Step of Peptide Synthesis and Purification Methods
II.
The Synthesis of the AlaP Analogs in Solution
III.
The Solid-Phase Synthesis of the TM Analogs
S2
I. Purification Methods and Conditions at Each Step of Peptide Synthesis
Peptide Synthesis in Solution
Synthetic strategy. The general strategy of our solution-phase synthesis (segment condensation
approach) exploited for the preparation of the three PheCN-containing AlaP analogs is shown in
Scheme 1 of the main text. It follows the successful strategy we already reported for AlaP itself
[S1]. Briefly, for the synthesis of the Ala9 and Ala15 analogs, we first synthesized the Boc/OBzl
(Boc, tert-butyloxycarbonyl; OBzl, benzyloxy) terminally-protected segment A, as reported
previously [S2]. Conversely, for the synthesis of Ala4, segment A was C-terminally protected as
OAll (allyloxy) to avoid catalytic hydrogenation in the presence of the sensitive PheCN probe.
Similarly, while segment C employed in the synthesis of Ala4 and Ala9 was obtained as previously
reported [Boc/Phol(Bzl) terminally protected] [S2], for Ala15 the C-terminal benzyl ether group
was removed one step before the introduction of the PheCN residue. Finally, to avoid any acidic
treatment in the presence of the acid labile Aib-Pro bonds, the N-terminal Boc protecting group of
segments A and B was replaced by Fmoc (9-fluorenylmethyloxy). The step-by-step synthesis of
each segment was performed through the EDC/HOAt C-activation method. The segment
condensation step was carried out by activating the C-terminal free carboxyl function (characterized
by the non-epimerizable Aib residue at its C-terminus) with EDC, to give the corresponding, mildly
reactive, 5(4H)-oxazolone [S3].
Purification methods. Each peptide segment was purified by either recrystallization from ethyl
acetate/petroleum ether or by flash chromatography. In the latter case, mixtures of
dichloromethane/methanol at different ratios were employed as eluant. The crude, final products
were purified by preparative RP-HPLC using a Shimadzu (Kyoto, Japan) LC-8A pump system
equipped with a SPD-6A UV-detector, on a Phenomenex C4 column (22x250 mm, 10μ, 300Å). The
binary elution system used was: A, H2O; B, CH3CN/H2O (9:1 v/v). The presence of three acid labile
Aib-Pro bonds prevents the use of acidic eluants. Gradient, 50%-90% B in 30 min (flow rate 15
ml/min); spectrophotometric detection at λ=216 nm.
Solid-Phase Peptide Synthesis
Assembly of peptides on the Advanced ChemTech (Louisville, KY) 348Ω peptide synthesizer was
performed on a 0.05 mmol scale, by the FastMoc methodology (HBTU, HOBt, DIPEA, double
acylation protocol, 45 min coupling time, N-methylpyrrolidin-2-one as solvent), starting with the
Rink Amide MBHA LL resin. The peptide cleaved from the resin was filtered and collected. The
crude peptides were purified by preparative RP-HPLC on a Vydac C18 column (10x250 mm, 5 μ,
300 Å) using a Shimadzu (Kyoto, Japan) LC-8A pump system equipped with a SPD-6A UVdetector (flow rate 15 ml/min, λ=216 nm) and a binary elution system: A, 0.1% TFA in H2O; B,
0.1% TFA in CH3CN/H2O (9:1 v/v); gradient 50%-65% B in 15 min. The purified fractions were
characterized by analytical RP-HPLC on a Phenomenex Jupiter C18 column (4.6x250 mm, 5 μ, 300
Å); gradient 60%-80% B. in 20 min. Electrospray ionization (ESI-MS) was performed by using a
PerSeptive Biosystem Mariner instrument (Framingham, MA).
References
[S1] Peggion, C.; Coin, I.; Toniolo, C. Biopolymers (Pept Sci) 2004, 76, 485-493.
[S2] De Zotti, M.; Ballano, G.; Jost, M.; Salnikov, E. S.; Bechinger, B.; Oancea, S.; Crisma, M.;
Toniolo, C.; Formaggio, F. Chem Biodivers 2014, 11, 1163-1191.
[S3] Carpino, L. A. J Am Chem Soc 1993, 115, 4397-4398.
S3
II. The Synthesis AlaP Analogs in Solution
SEGMENT A (Ala4)
Boc-PheCN-Aib-OAll
Yield: 80%. M.p.: 127°C. Rf1: 0.78, Rf2: 0.90, Rf3: 0.60. []D20= 4.4° (c=0.34, MeOH). IR (film):
3301, 2227, 1742, 1656, 1532 cm-1. 1H NMR (400MHz, CDCl3):  7.61 [d, 2H, PheCN Ar, J = 7.8
Hz], 7.37 [d, 2H, PheCN Ar, J = 8.0 Hz], 6.47 [s, 1H, Aib NH], 5.90 [m, 1H, Allyl CH], 5.34 [m, 1H,
Allyl CH trans], 5.28 [m, 1H, Allyl CH cis], 5.03 [m, 1H, PheCN NH], 4.64 [d, 2H, Allyl CH2, J =
5.5 Hz], 4.44 [m, 1H, PheCN α-CH], 3.78 [dd, 1H, PheCN β-CH2, J = 7.1 Hz, J = 13.8 Hz], 3.08 [dd,
1H, PheCN β-CH2, J = 6.7 Hz, J = 13.8 Hz], 1.53 [s, 3H, Aib β-CH3], 1.51 [s, 3H, Aib β-CH3], 1.43
[s, 9H, OtBu 3 CH3]. ESI-MS: [M+H]+exp.= 416.21; [M+H]+calcd= 416.23.
Boc-Aib-PheCN-Aib-OAll
Yield: 80%. M.p.: 69°C. Rf1: 0.73, Rf2: 0.81, Rf3: 0.51. []D20= 34.1° (c=0.27, MeOH). IR (film):
3340, 2228, 1741, 1694, 1660, 1506 cm-1. 1H NMR (400MHz, CDCl3):  7.61 [d, 2H, PheCN Ar, J
= 8.2 Hz], 7.37 [d, 2H, PheCN Ar, J = 8.2 Hz], 7.13 [s, 1H, Aib NH], 6.53 [d, 1H, PheCN NH, J = 7.4
Hz], 5.93 [m, 1H, Allyl CH], 5.34 [dd, 1H, Allyl CH trans, J = 1.5 Hz, J = 17.3 Hz], 5.27 [dd, 1H,
Allyl CH cis, J = 1.2 Hz, J = 10.5 Hz], 4.75 [s, 1H, Aib NH], 4.70 [m, 1H, PheCN α-CH], 4.65 [m,
2H, Allyl CH2], 3.25 [m, 2H, PheCN β-CH2], 1.54 [s, 3H, Aib -CH3], 1.50 [s, 3H, Aib -CH3], 1.45
[s, 3H, Aib -CH3], 1.40 [s, 9H, OtBu 3 CH3], 1.31 [s, 3H, Aib -CH3]. ESI-MS: [M+H]+exp.=
501.26; [M+H]+calcd.= 501.32.
Fmoc-Pro-Aib-PheCN-Aib-OAll
Yield: 78%. M.p.: 89°C. Rf1: 0.70, Rf2: 0.83, Rf3: 0.50. []D20= 58.4° (c=0.31, MeOH). IR (KBr):
3304, 2227, 1740, 1671, 1531 cm-1. 1H NMR (400MHz, CDCl3):  7.84 [d, 2H, PheCN Ar, J = 7.5
Hz], 7.60-7.22 [m, 10H, PheCN Ar, Fmoc Ar], 7.21 [s, 1H, Aib NH], 7.14 [d, 1H, PheCN NH, J = 8.4
Hz], 6.36 [s, 1H, Aib NH], 5.92 [m, 1H, Allyl CH], 5.33 [dd, 1H, Allyl CH trans, J = 1.4 Hz, J =
17.2 Hz], 5.20 [m, 1H, Allyl CH cis], 4.66 [m, 1H, Fmoc CH], 4.63 [m, 2H, Allyl CH2], 4.36 [m,
1H, Fmoc CH2], 4.54 [dd, 1H, α-CH, J = 6.6 Hz, J = 10.0 Hz], 4.29 [m, 1H, Fmoc CH2], 4.10 [m,
1H, α-CH], 3.58-3.46 [m, 3H, 2H Pro δ-CH2, 1H PheCN β-CH2], 3.00 [dd, 1H, PheCN β-CH2, J =
10.8 Hz, J = 14.6 Hz], 2.25-2.13 [m, 1H, Pro γ-CH2], 2.11-1.89 [m, 3H, Pro β-CH2, γ-CH2], 1.57 [s,
3H, Aib β-CH3], 1.52 [s, 3H, Aib β-CH3], 1.45 [s, 3H, Aib β-CH3], 1.12 [s, 3H, Aib β-CH3]. ESIMS: [M+H]+exp.= 720.32; [M+H]+calcd.= 720.32.
Fmoc-Pro-Aib-PheCN-Aib-OH
Yield: 82%. M.p.: 121°C. Rf1: 0.27, Rf2: 0.77, Rf3: 0.21. []D20= 55.2° (c=0.29, MeOH). 1H NMR
(400MHz, CDCl3): δ 7.84 [d, 2H, PheCN Ar, J = 7.4 Hz], 7.60-7.18 [m, 12H, PheCN Ar, Fmoc Ar,
Aib NH, PheCN NH], 6.42 [s, 1H, Aib NH], 4.66 [m, 2H, Fmoc CH, α-CH], 4.31-4.22 [m, 2H, Fmoc
CH2], 4.07 [m, 1H, α-CH], 3.58-3.43 [m, 3H, 2H Pro δ-CH2, 1H PheCN β-CH2], 3.04 [dd, 1H, PheCN
β-CH2, J = 10.9 Hz, J = 14.6 Hz], 2.32-2.17 [m, 1H, Pro γ-CH2], 2.09-1.90 [m, 3H, Pro β-CH2, γCH2], 1.55 [s, 6H, Aib β-CH3], 1.42 [s, 3H, Aib β-CH3], 1.17 [s, 3H, Aib β-CH3]. ESI-MS:
[M+H]+exp.= 680.31; [M+H]+calcd.= 680.32.
S4
SEGMENT B (Ala9)
Boc-PheCN-Aib-Gly-Leu-Aib-OAll
Yield: 51%. M.p.: 83°C. Rf1: 0.58, Rf2: 0.79, Rf3: 0.42. []D20= 11.6° (c=0.30, MeOH). IR (KBr):
3327, 2228, 1741, 1662, 1528 cm-1.
Boc-Aib-PheCN-Aib-Gly-Leu-Aib-OAll
Yield: 61%. M.p.: 218°C. Rf1: 0.63, Rf2: 0.81, Rf3: 0.43. []D20= 39.8° (c=0.28, MeOH). IR
(KBr): 3393, 3356, 3317, 3270, 2224, 1744, 1688, 1666, 1656, 1647, 1532 cm-1. 1H NMR
(400MHz, CDCl3):  7.64 [d, 2H, PheCN Ar, J = 8.1 Hz], 7.54 [s, 1H, Aib NH], 7.44-7.33 [m,
5H,pCNPhe NH, Leu NH, Gly NH, PheCN e Ar], 6.72 [s, 1H, Aib NH], 5.88 [m, 1H, Allyl CH],
5.29 [dd, 1H, Allyl CH trans, J = 1.2 Hz, J = 17.2 Hz], 5.20 [dd, 1H, Allyl CH cis, J = 0.9 Hz, J =
10.4 Hz], 4.90 [s, 1H, Aib NH], 4.56 [m, 2H, Allyl CH2], 4.52-4.44 [m, 2H, PheCN α-CH, Leu αCH], 4.18 [dd, 1H, Gly α-CH2, J = 7.2 Hz, J = 17.6 Hz], 3.76 [dd, 1H, Gly α-CH2, J = 5.1 Hz, J =
17.1 Hz], 3.28-3.17 [m, 2H, PheCN β-CH2], 2.00-1.93 [m, 1H; 1H Leu β-CH2], 1.84-1.65 [m, 2H;
1H Leu β-CH2, Leu γ-CH], 1.58 [s, 3H, Aib β-CH3], 1.57 [s, 3H, Aib β-CH3], 1.52 [s, 3H, Aib βCH3], 1.46 [s, 3H, Aib β-CH3], 1.37 [s, 3H, Aib β-CH3], 1.37 [s, 9H, OtBu, 3 CH3], 1.25 [s, 3H, Aib
β-CH3], 0.99 [d, 3H, Leu δ-CH3, J = 6.4 Hz], 0.94 [d, 3H, Leu δ-CH3, J = 6.3 Hz].
ESI-MS: [M+H]+exp.= 756.39; [M+H]+calcd.= 756.49.
Boc-Glu(OMe)-Aib-PheCN-Aib-Gly-Leu-Aib-OAll
Yield: 85%. M.p.: 105°C. Rf1: 0.59, Rf2: 0.79, Rf3: 0.39. []D20= 39.9° (c=0.31, MeOH). IR
(KBr): 3332, 2228, 1739, 1660, 1532 cm-1. 1H NMR (400MHz, CDCl3):  7.65 [s, 1H, NH], 7.60
[d, 2H, PheCN Ar, J = 8.2 Hz], 7.59 [s, 1H, NH], 7.53 [m, 1H, Gly NH], 7.47 [d, 1H, PheCN NH, J =
7.9 Hz], 7.35 [d, 2H, PheCN Ar, J = 8.2 Hz], 6.92 [s, 1H, Aib NH], 6.68 [s, 1H, Aib NH], 5.88 [m,
1H, Allyl CH], 5.77 [m, 1H, Glu(OMe) NH], 5.30 [dd, 1H, Allyl CH trans, J = 1.5 Hz, J = 17.2 Hz],
5.19 [dd, 1H, Allyl CH cis, J = 1.3 Hz, J = 10.5 Hz], 4.56 [m, 2H, Allyl CH2], 4.47-4.36 [m, 2H,
PheCN α-CH, Leu α-CH], 4.02 [dd, 1H, Gly α-CH2, J = 6.2 Hz, J = 17.1 Hz], 3.92-3.82 [m, 2H, Gly
α-CH2, Glu(OMe) α-CH], 3.74 [s, 3H, Glu(OMe) CH3], 3.41 [m, 1H, PheCN β-CH2], 3.14 [dd, 1H,
PheCN β-CH2, J = 10.8 Hz, J = 15.1 Hz], 2.61-2.44 [m, 2H; 2H Glu γ-CH2], 2.12-1.91 [m, 2H; 2H
Glu β-CH2], 1.90-1.80 [m, 1H; 1H Leu β-CH2], 1.76-1.69 [m, 2H; 1H Leu β-CH2, Leu γ-CH], 1.56
[s, 3H, Aib β-CH3], 1.54 [s, 3H, Aib β-CH3], 1.53 [s, 3H, Aib β-CH3], 1.51 [s, 3H, Aib β-CH3], 1.42
[s, 3H, Aib β-CH3], 1.39 [s, 9H, OtBu, 3 CH3], 1.34 [s, 3H, Aib β-CH3], 0.92-0.88 [m, 6H, Leu δCH3]. ESI-MS: [M+H]+exp.= 899.46; [M+H]+calcd.= 899.54.
Fmoc-Ala-Glu(OMe)-Aib-PheCN-Aib-Gly-Leu-Aib-OAll
Yield: 90%. M.p.: 93°C. Rf1: 0.59, Rf2: 0.77, Rf3: 0.38. []D20= 27.9° (c=0.33, MeOH). IR (KBr):
3331, 2228, 1738, 1660, 1532cm-1. 1H NMR (400MHz, CDCl3):  7.65 [m, 1H, NH], 7.83-7.28 [m,
17H, PheCN Ar, Fmoc Ar], 7.10 [s, 1H, Aib NH], 5.88 [m, 1H, Allyl CH], 5.36 [m, 1H, Ala NH],
5.31 [dd, 1H, Allyl CH trans, J = 1.4 Hz, J = 17.3 Hz], 5.18 [dd, 1H, Allyl CH cis, J = 1.3 Hz, J =
10.6 Hz], 4.62-4.52 [m, 3H, Fmoc CH, Allyl CH2], 4.44-4.36 [m, 3H, α-CH, Fmoc CH2], 4.26 [m,
1H, α-CH], 4.03 [dd, 1H, α-CH, J = 5.5 Hz, J = 11.4 Hz], 3.98-3.88 [m, 3H, α-CH], 3.56 [s, 3H,
Glu(OMe) CH3], 3.41 [dd, 1H, PheCN β-CH2, J = 3.9 Hz, J = 14.5 Hz], 3.10 [dd, 1H, PheCN β-CH2,
J = 13.3 Hz, J = 14.4 Hz], 2.61-2.50 [m, 2H; 2H Glu γ-CH2], 2.15-1.99 [m, 2H; 2H Glu β-CH2],
1.92-1.82 [m, 1H; 1H Leu β-CH2], 1.80-1.68 [m, 2H; 1H Leu β-CH2, Leu γ-CH], 1.56 [s, 3H, Aib
β-CH3], 1.54 [s, 9H, Aib β-CH3], 1.49 [d, 3H, Ala β-CH3, J = 7.2 Hz], 1.45 [s, 3H, Aib β-CH3], 1.16
[s, 3H, Aib β-CH3], 0.96 [d, 3H, Leu δ-CH3, J = 5.9 Hz], 0.93-0.87 [m, 3H, Leu δ-CH3].
ESI-MS: [M+H]+exp.= 1092.52; [M+H]+calcd.= 1092.53.
S5
Fmoc-Ala-Glu(OMe)-Aib-PheCN-Aib-Gly-Leu-Aib-OH
Yield: 76%. M.p.: 130°C. Rf1: 0.13, Rf2: 0.75, Rf3: 0.10. IR (KBr): 3408, 2228, 1734, 1659, 1529
cm-1. 1H NMR (400MHz, CDCl3):  8.53 [s, 1H, NH], 7.83-7.32 [m, 16H, PheCN Ar, Fmoc Ar],
7.23 [d, 1H, NH, J = 7.5 Hz], 7.18 [s, 1H, NH], 5.44 [m, 1H, Ala NH], 4.54 [dd, 1H α-CH, J = 6.4
Hz, J = 10.4 Hz], 4.49-4.35 [m, 2H, α-CH, CH Fmoc], 4.31-4.23 [m, 1H, α-CH], 4.08-3.94 [m, 2H,
α-CH], 3.84 [dd, 1H, α-CH, J = 4.7 Hz, J = 17.3 Hz], 3.56 [s, 3H, Glu(OMe) CH3], 3.50-3.41 [m,
1H, PheCN β-CH2], 3.07-2.96 [m, 1H, PheCN β-CH2], 2.58-2.50 [m, 2H; 2H Glu γ-CH2], 2.14-2.06
[m, 2H; 2H Glu β-CH2], 1.86-1.70 [m, 3H; Leu β-CH2, Leu γ-CH], 1.62 [s, 3H, Aib β-CH3], 1.57 [s,
3H, Aib β-CH3], 1.54 [s, 3H, Aib β-CH3], 1.52 [s, 3H, Aib β-CH3], 1.48 [d, 3H, Ala β-CH3, J = 7.2
Hz], 1.44 [s, 3H, Aib β-CH3], 1.11 [s, 3H, Aib β-CH3], 0.96 [d, 3H, Leu δ-CH3, J = 6.3 Hz], 0.92 [d,
3H, Leu δ-CH3, J = 6.2 Hz]. ESI-MS: [M+H]+exp.= 1052.49; [M+H]+calcd.= 1052.51.
SEGMENT C (Ala15)
Boc-PheCN-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol
Yield: 49%. M.p.: 84°C. Rf1: 0.56, Rf2: 0.79, Rf3: 0.26. IR (KBr): 3303, 2228, 1738, 1654, 1534
cm-1. 1H NMR (400MHz, CDCl3):  7.82 [m, 1H, NH], 7.68 [d, 1H, Ar PheCN, J = 7.9 Hz], 7.42 [d,
1H, Ar PheCN, J = 8.0 Hz], 7.31-6.97 [m, 9H, Phol Ar, 4NH], 5.48 [m, 1H, NH], 4.30-4.16 [m, 3H,
α-CH], 4.10 [m, 1H, α-CH], 3.68-3.65 [m, 1H, Phol CH (CH2OH)], 3.69 [m, 6H, Glu(OMe) CH3],
3.63-3.53 [m, 1H, Phol CH (CH2OH)], 3.23 [dd, 1H, PheCN β-CH2, J = 4.3 Hz, J =14.3 Hz], 3.00
[dd, 1H, PheCN β-CH2, J = 9.4 Hz, J =14.1 Hz], 2.94-2.81 [m, 2H, Phol β-CH2], 2.61-2.49 [m, 2H, 2
Glu(OMe) β-CH2], 2.40-2.13 [m, 5H, Glu(OMe) γ-CH2 e Glu(OMe) β-CH2], 2.11-1.99 [m, 1H, 1
Glu(OMe) γ-CH2 o 1 Glu(OMe) β-CH2], 1.57 [s, 3H, Aib CH3], 1.49 [s, 3H, Aib CH3], 1.47 [s, 3H,
Aib CH3], 1.45 [s, 9H, Boc CH3], 1.42 [s, 3H, Aib CH3].
ESI-MS: [M+H]+exp.= 880.41; [M+H]+calcd.= 880.41.
Boc-Pro-PheCN-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol
Yield: 57%. M.p.: 87°C. Rf1: 0.58, Rf2: 0.79, Rf3: 0.27. IR (KBr): 3319, 2228, 1739, 1658 cm-1.
1H NMR (400MHz, CDCl ): 7.78 [m, 1H, NH], 7.72-7.64 [m, 2H, Ar Phe , J = 7.9 Hz], 7.343
CN
7.15 [m, 7H, Phol CH, 4NH], 7.04 [m, 1H, NH], 7.02 [d, 1H, NH, J = 8.5 Hz], 6.36 [d, 1H, NH, J =
6.4 Hz], 4.51 [dd, 1H, α-CH, J = 4.9 Hz, J =14.2 Hz], 4.34 [m, 1H, α-CH], 4.22-4.10 [m, 3H, α-CH],
3.76-3.73 [m, 1H, Phol CH (CH2OH)], 3.69 [m, 3H, Glu(OMe) CH3], 3.67 [m, 3H, Glu(OMe) CH3],
3.58-3.54 [m, 2H, Phol CH (CH2OH), Pro γ-CH2], 3.42-3.36 [m, 1H, Pro γ-CH2], 3.26 [dd, 1H,
PheCN β-CH2, J = 6.9 Hz, J =14.2 Hz], 3.18 [dd, 1H, PheCN β-CH2, J = 4.9 Hz, J =14.2 Hz], 2.982.86 [m, 2H, Phol -CH2], 2.63-2.46 [m, 2H, 2 Glu(OMe) -CH2], 2.46-2.24 [m, 5H, Glu(OMe) γCH2 e Glu(OMe) β-CH2], 2.14-2.03 [m, 1H, 1 Glu(OMe) -CH2 o 1 Glu(OMe) β-CH2], 2.01-1.81
[m, 4H, 1 Pro γ-CH2 e Pro β-CH2], 1.58 [s, 3H, Aib CH3], 1.52 [s, 3H, Aib CH3], 1.47 [s, 3H, Aib
CH3], 1.43 [s, 3H, Aib CH3], 1.41 [s, 9H, Boc CH3]. ESI-MS: [M+H]+exp.= 977.47; [M+H]+calc=
977.52.
S6
Segment [B-C] (Ala9)
Fmoc-Ala-Glu(OMe)-Aib-PheCN-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)Phol
Yield: 40%. ESI-MS: [M+H]+exp.= 1838.83; [M+2H]++exp.= 919.91, [M+2H]++calcd. = 919.93.
Segment [B-C] (Ala15)
Fmoc-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-PheCN-Aib-Aib-Glu(OMe)-Glu(OMe)Phol
Yield: 56%. ESI-MS: [M+H]+exp.= 1838.81; [M+2H]++exp.= 919.91, [M+2H]++calcd.= 919.93.
Segment [A-B-C] [(Ala4)
Fmoc-Pro-Aib-PheCN-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-AibGlu(OMe)-Glu(OMe)-Phol
Yield: 54%. ESI-MS: [M+H]+exp.= 2205.18; [M+2H]++exp. = 1103.07, [M+2H]++calcd.= 1103.05.
Ala4
Ac-Aib-Pro-Aib-PheCN-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-AibGlu(OMe)-Glu(OMe)-Phol
Yield: 33%. HPLC: tR =17.5 min (flux: 1ml/min; 50-100% B over 30 min; C4 Vydac column).
ESI-MS: [M+2H]++exp.= 1055.54. [M+2H]++calcd.= 1055.58.
Ala9
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-PheCN-Aib-Gly-Leu-Aib-Pro-Val-Aib-AibGlu(OMe)-Glu(OMe)-Phol
Yield: 58%. HPLC: tR =12.0 min (flux: 1ml/min; 50-100% B over 30 min; C4 Vydac column).
ESI-MS: [M+H]+exp.= 2082.14; [M+2H]++exp.= 1041.05; [M+2H]++calcd.= 1041.52.
Ala15
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-PheCN-Aib-AibGlu(OMe)-Glu(OMe)-Phol
Yield: 40%. HPLC: tR =15.8 min (flux: 1ml/min; 50-100% B over 30 min; C4 Vydac column).
ESI-MS: [M+H]++exp.= 1041.54; [M+2H]++calcd= 1041.52.
S7
III. The Solid-Phase Synthesis of the TM Peptides
min
Mariner Spec /7:8 (T /0.52:0.61) ASC=>DECONV [BP = 2413.6, 446]
2413.5690
100
446.0
2414.5808
90
80
% Intensity
70
60
2415.6139
2412.5820
50
40
2416.5704
30
20
2418.5383
10
0
2400
2406
2412
2418
2424
0
2430
Mass (m /z)
Mariner Spec /7:8 (T /0.52:0.61) ASC[BP = 805.5, 261]
805.5310
100
261.3
90
80
70
% Intensity
604.3963
60
50
40
30
20
1207.7953
10
0
99.0
879.2
1659.4
2439.6
3219.8
0
4000.0
Mass (m /z)
Fig. S1. Analytical HPLC of TM (elution conditions: 50-80% B over 30 min) and related ESI-MS
spectrum (MWcalcd=2412.63).
S8
min
Mariner Spec /7:9 (T /0.53:0.71) ASC=>DECONV [BP = 2472.5, 11700]
2472.5681
100
1.2E+4
90
80
2473.5935
% Intensity
70
2471.5586
60
50
2474.6057
40
30
2475.6153
20
10
0
2450
2460
2470
2480
2490
0
2500
Mass (m /z)
Mariner Spec /7:9 (T /0.53:0.71) ASC[BP = 619.1, 6130]
619.1532
100
6130.0
825.1923
90
80
% Intensity
70
60
50
40
30
20
10
0
99.0
434.7939
654.4402
1237.3028
879.2
1659.4
2439.6
3219.8
0
4000.0
Mass (m /z)
Fig. S2. Analytical HPLC of TM3 (elution conditions: 60-80% B over 20 min) and related ESI-MS
spectrum (MWcalcd=2471.61).
S9
min
Mariner Spec /7:8 (T /0.53:0.62) ASC=>DECONV [BP = 2472.5, 10398]
2472.5730
100
1.0E+4
90
80
2473.5960
% Intensity
70
2471.5628
60
50
2474.6077
40
30
2475.6158
20
10
0
2450
2460
2470
2480
2490
0
2500
Mass (m /z)
Mariner Spec /7:8 (T /0.53:0.62) ASC[BP = 825.2, 5565]
825.1935
100
5565.1
619.1546
90
80
% Intensity
70
60
50
40
30
20
10
0
99.0
434.7942
801.5349
1237.3167
879.2
1659.4
2439.6
3219.8
0
4000.0
Mass (m /z)
Fig. S3. Analytical HPLC of TM11 (elution conditions: 60-80% B over 20 min) and related ESIMS spectrum (MWcalcd=2471.61).
S10
Acronym
Ala4
Ala9
Ala15
TM
TM3
TM11
MW calc
2109.16
2081.04
2081.04
2412.63
2471.61
2471.61
MW exp
2109.08
2081.14
2081.08
2412.58
2471.55
2471.54
tR
17.5*
12.0*
15.8*
15.0**
8.5#
8.4#
Table S1. Chemical characterizations of the AlaP and TM analogs
Column: Vydac C4, 300 Å, 5 μ; 50-100% B over 30 min
*
Column: Phenomenex C18, 300 Å, 5 μ; 50-80% B over 20 min
**
#
Column: Phenomenex C18, 300 Å, 5 μ; 60-80% B over 20 min