Supplementary Information

Predicting and Investigating the Physicochemical Properties of Macrocyclic Peptides

Praew Thansandote, Robert M. Harris, Hannah L. Dexter, Graham L. Simpson,* Sandeep Pal, Richard J. Upton, Klara Valko

1. Synthesis: experimental procedures.................................................................................1-8

2. Appendix: Fmoc counting, LCMS and HRMS of compounds........................................9-27

3. Assays: experimental details..............................................................................................28

4. Computational modelling..............................................................................................28-29

5. NMR Exchange Data....................................................................................................29-36

6. Summary Table of Results.................................................................................................37

7. References.........................................................................................................................38

Synthesis

General. 2-chlorotrityl-chloride resin was purchased from Novabiochem (1.7 meq/g). All Fmoc-protected amino acids and all reagents were purchased from PTI, Novabiochem, Chem-Impex or Sigma-Aldrich. Oasis-HLB cartridges were purchased from Waters. UPLC analysis was conducted on a Waters Acquity UPLC CSH C18 column (50mm x 2.1mm i.d. 1.7μm packing diameter) at 30 degrees centigrade equipped with both a photodiode array detector and an evaporative light scattering detector to determine the identity, purity and ratios of peptide products. Peptides were purified using a preparative HPLC system (Waters) equipped with an XSELECT CSH prep OBD column, 5 µ 150x30 mm.

Abbreviations. ACN, acetonitrile; DMF, N,N-dimethylformamide; DIPEA, N,N-diisopropylethylamine, DCM, dichloromethane; NMP, N-Methyl-2-pyrrolidone; THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide; HCTU, O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HATU, 1-[Bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate; TFA, trifluoroacetic acid; LCMS, liquid chromatography mass spectrometry; ELSD, evaporative light scattering detector; SPPS, solid phase peptide synthesis; MDAP, mass directed autopurification.

1

Loading of Fmoc-Tyr-OAllyl on Trichloroacetimidate Resin:

Synthesized according to the procedure of Albericio et. al.1a In a centrifuge tube, trichloroacetimidate resin (521 mg, 1.40 mmol) was swollen using dry DCM (approx 10 ml) under a nitrogen atmosphere. A solution of Fmoc-Tyr-Oallyl ester1b (1.28 g, 2.80 mmol) in a minimum amount of dry THF (approx 15 mL) was added. The resin was shaken gently for 5 minutes. Boron trifluoride diethyl etherate (35.0 μL, 0.285 mmol) was added and the resin was shaken for 4 hours at room temperature. The resin was washed with THF (5 x 1 minute), DMF (5 x 1 minute) and DCM (5 x 1 minute) and treated with a 1:1 DMF:water solution (3 x 15 minutes) with DMF washes in between, in order to hydrolyse the remaining trichloroacetimidate. The resin was then washed with DMF (5 x 1 minute), dry THF (5 x 1 minute) and EtOAc (5 x 1 minute). The resin was again swollen using DCM (approx 10 mL) then acetylated by treatment with acetic anhydride (5.71 g, 0.285 mmol) and DMAP (0.684 g, 5.60 mmol) in DMF (10 mL) for 45 minutes. The resin was transferred to a filter tube and washed with DMF (5 x 1 minute) then DCM (5 x 1 minutes). The resin was dried for 40 minutes under vacuum using a glass chamber vacuum manifold.

Determination of resin loading. According to the procedure of Lokey et.al.2 A 15 mg sample of the resin was weighed into a 3 mL polypropylene tube. The resin was swollen three times using DCM, followed by washing with DMF (5 x 30 seconds). Next, the resin was treated with 0.5 mL of a 20% piperidine in DMF solution for 5 minutes, draining into a 100 mL volumetric flask. The resin was treated again with 0.5 mL of the 20% piperidine in DMF solution for 10 minutes, collecting the washings into the same volumetric flask. The resin was washed with 0.5 mL of DMF (5 x 30 seconds) and the washings collected in the same volumetric flask. DMF was added until the 100 mL line on the volumetric flask, and the resulting sample was used for a UV/vis absorbance reading at λ = 301 nm. The piperidine-dibenzofulvene adduct that was formed upon fmoc deprotection has a molar extinction coefficient of ε = 7800 L•mol-1•cm-1 and using Beer’s Law the loading value was determined.

General SPPS procedures for room temperature synthesis. Linear peptides were synthesized on the Protein Technologies Symphony Multiplex peptide synthesizer. Preloaded resin (0.1 mmol) was weighed directly into a Symphony reactor vessel. The peptide synthesis was then synthesized on the machine using the following conditions (single coupling): Fmoc-AA-OH (2.5 mL, 5 equiv., 0.2M in NMP), HCTU (1.25 mL, 5 equiv., 0.4M in DMF), DIPEA (1.25 mL, 0.8 M in NMP) at room temperature. DMF washing steps occurred after each step in the coupling. All couplings were repeated twice (double couplings). After each coupling, Fmoc deprotection proceeded with a 20% piperidine/DMF solution followed by successive washes with DMF.

General SPPS procedures for microwave synthesis. Linear peptides were synthesized on the CEM Liberty 1 microwave peptide synthesizer. Preloaded resin (0.1 mmol based on

2

loading) was weighed directly into a 30 mL microwave reaction vessel or transferred using DCM. The peptide synthesis was then synthesized on the machine using the following conditions (single coupling): Fmoc-AA-OH (2.5 mL, 5 equiv., 0.2M in NMP), coupling reagent (1.0 mL, 5 equiv., 0.5M in DMF), DIPEA (1 mL, 1.0M in NMP), 20 W (power) and 75 oC for 10 minutes. DMF washing steps occurred after each step in the coupling. Single or double (two successive) couplings were completed as specified for each amino acid. After each coupling, Fmoc cleavage proceeded with 20% piperidine/DMF solution followed by successive washes with DMF.

Deallylation and on-resin cyclisation. According to the procedure of Lokey et. al.2 Linear peptide on resin was transferred to 20 mL centrifuge tube. Enough 10% piperidine/DMF (approx 5 mL) was added to swell the resin. The centrifuge tube was then purged with nitrogen and tetrakis (triphenylphosphine)palladium(0) (1 equiv.) was added. The tube was purged again with nitrogen, sealed and shaken for 3 hours. The resin was then transferred to a filter tube and washed with the following solutions: THF (5 x 30 seconds), DMF (5 x 30 seconds), 5% DIPEA (volume/volume)/0.05% sodium diethyldithiocarbamate (mass/volume) in DMF (3 x 15 minutes) with DMF (5 x 30 seconds) between each treatment. The resin was then rinsed with DCM and dried for 40 minutes under vacuum before being transferred to a 20 mL centrifuge tube. DMF was added to swell the resin and HOAt (5 equiv.), PyBOP (5 equiv.), and DIPEA (7.5 equiv.) added before being shaken at room temperature overnight. The resin was then transferred to a filter tube and rinsed with DMF (5 x 1 minutes) and DCM (3 x 1 minute) before being dried for 30 minutes under vacuum using a glass chamber vacuum manifold.

Resin cleavage. The dry resin was added to a centrifuge tube. In a second centrifuge tube, 9.5 mL TFA (95%), 0.25 mL TIPS (2.5%) and 0.25 mL of water (2.5%) was added. The solution in the second centrifuge tube was added to the first containing the resin, followed by agitation for 1 hour at room temperature. The solution was then filtered and the eluent collected. The eluent was evaporated under a stream of nitrogen to remove the majority of the TFA. The peptide was purified by MDAP-HPLC and the purified fractions were passed through an Oasis-HLB cartridge to remove TFA and then freeze-dried in 1,4-dioxane to yield a white lyophilisate.

Final compounds are reported with overall yields based on the preloaded resin (0.1 mmol). For example, an overall yield of 5% for a hexa- or hepta-mer is an average of 70-75% per step in the 9-10 step route detailed above. In the case of further transformations (eg. N-methylation on resin) the overall yield is inclusive of an additional step.

Synthesis of cyclic Leu.NMe-D-Leu.NMe-Leu.Leu.D-Pro.Tyr (2b)

3

Compound 2b was synthesized from Fmoc-Tyr-OAllyl on trichloroacetimidate resin according to the general procedure. D-Proline was double coupled, Leucine was single coupled, NMe-Leucine was single coupled, D-NMe-Leucine was double coupled and the terminal Leucine was single coupled. Single couplings used HCTU and double couplings used HATU to afford compound 2b as a white lyophilisate (3.0 mg, 15% yield). HRMS (ESI) for C40H65N6O7 [M+H]+ calculated 741.4916, measured 741.4910 (1.4 ppm error); 1H NMR (400 MHz, chloroform-d) δ = 7.50 (d, J = 8.0 Hz, 1H), 7.13 - 6.94 (m, 3H), 6.74 (d, J = 8.3 Hz, 1H), 6.13 (d, J = 8.0 Hz, 1H), 5.31 (dd, J = 4.0, 11.6 Hz, 1H), 5.19 - 5.07 (m, 1H), 4.97 - 4.87 (m, 1H), 4.84 (dd, J = 4.0, 8.0 Hz, 1H), 4.80 - 4.68 (m, 1H), 4.03 (dd, J = 4.7, 7.5 Hz, 1H), 3.70 (m, 1H), 3.63 - 3.50 (m, 1H), 3.34 - 3.28 (m, 1H), 3.25 (s, 3H), 3.02 (s, 3H), 2.24 - 1.76 (m, 8H), 1.76 - 1.48 (m, 11H), 1.00 - 0.82 (m, 24H).

Synthesis of cyclic Leu.D-Leu.Leu.Leu.D-Pro.Tyr on Wang resin (precursor to 1b)

4

Synthesized according to general SPPS procedure for room temperature synthesis. The cyclic peptide was not cleaved from the resin or purified as it was used for subsequent N-methylation studies.Synthesis of cyclic Leu.D-Leu.Leu.Leu.D-Pro.Tyr compound 1b

Synthesized according to general SPPS procedures for room temperature peptide synthesis, deallylation, cyclisation and cleavage. Compound 1b was isolated as a white lyophilisate (9.3 mg, 4.7% yield). HRMS (ESI) for C38H61N6O7 [M+H]+ calculated 713.4596, measured 713.4592 (0.6 ppm error); 1H NMR (600 MHz, Chloroform-d) δ = 7.57 (d, J = 9.2 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.1 Hz, 2H), 6.70 (d, J = 8.0 Hz, 1H), 6.02 (d, J = 6.6 Hz, 1H), 5.77 (d, J = 8.8 Hz, 1H), 5.10 (brs, 1H), 4.72 (q, J = 7.3 Hz, 1H), 4.64 (ddd, J = 3.7, 8.8, 12.1 Hz, 1H), 4.51 - 4.44 (m, 1H), 4.42 - 4.35 (m, 1H), 4.28 - 4.20 (m, 1H), 3.92 (t, J = 6.8 Hz, 1H), 3.74 - 3.64 (m, 1H), 3.55 (dt, J = 6.6, 10.3 Hz, 1H), 3.22 (dd, J = 7.3, 14.7 Hz, 1H), 3.11 (dd, J = 4.8, 14.7 Hz, 1H), 2.31 - 2.21 (m, 1H), 2.14 - 1.99 (m, 12.9 Hz, 2H), 1.99 - 1.85 (m, 3H), 1.80 - 1.50 (m, 6H), 1.50 - 1.38 (m, 4H), 1.00 - 0.85 (m, 24H).

5

Optimisation of Solvent for On-resin N -methylation

General Method for On-resin N -methylation Cyclized peptide-bound resin 11 (236 mg, 0.071 mmol) was added to a 15 mL centrifuge tube and placed under nitrogen. THF (4 mL) was added to swell the resin followed by lithium tert-butoxide (1.42 mL, 3.12 mmol). The centrifuge tube was flushed with nitrogen before being capped and agitated for 40 minutes at room temperature. The resin was transferred to a filter tube using THF and drained. Solvent (as specified, 4.00 mL) was added to the filter tube along with iodomethane (1.00 mL, 16.1 mmol) without draining. The filter tube was securely stoppered and capped on both ends and the solution was agitated again for 1 hour. The resin was rinsed with MeOH (3 x 1 min), and DCM (3 x 1 min) and then dried under vacuum for 5 minutes. The dried resin was then transferred to a 15 mL centrifuge tube and cleaved under the general resin cleavage procedure (95% TFA/2.5% TIPS/2.5% H2O). The crude peptide was purified by MDAP-HPLC and passed through Oasis-HLB cartridge to remove TFA then freeze-dried in 1,4-dioxane to yield a white lyophilisate.

6

Entry Solventa Unreacted Peptideb

Selectivity (%)b

Mono Di Tri

1 THF 10 90 - -

2 DMSO 5 - - 95

3 MeCN 30 70 - -

4 1,4-Dioxane 50 50 - -

5 DMF 10 - - 90

aDielectric constants: THF 7.5; DMSO 46.7; MeCN 36.6; 1,4-dioxane 2.3; DMF 38.3; bBy LCMS

Synthesis of 2c

Synthesized according to general procedure for on-resin N-methylation. The solvent used in the methylation step was DMSO. Compound 2c was isolated as a white lyophilisate (1.4 mg, 2.6% yield) as a white lyophilisate. HRMS (ESI) for C41H67N6O7 [M+H]+ calculated 755.5066, measured 755.5062 (0.5 ppm error); 1H NMR (600 MHz, DMSO-d6) δ = 8.96 (s, 1H), 7.41 (d, J = 9.5 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.8 Hz, 1H), 6.61 (d, J = 8.5 Hz, 2H), 5.39 - 5.24 (m, 1H), 5.09 - 4.86 (m, 3H), 4.78 - 4.67 (m, 1H), 4.54 - 4.44 (m, 1H), 3.55 (m, 2H), 3.26 (dd, J = 4.5, 15.3 Hz, 1H), 3.06 (s, 3H), 3.01 (s, 3H), 2.89 (s, 3H), 2.74 (m, 1H), 2.08 - 1.92 (m, 2H), 1.91 - 1.71 (m, 6H), 1.67 - 1.30 (m, 12H), 1.30 - 1.22 (m, 6H), 1.05 (d, J = 6.0 Hz, 2H), 0.97 (d, J = 6.3 Hz, 3H), 0.94 - 0.84 (m, 6H), 0.80 (d, J = 6.5 Hz, 3H).Note: if the solvent used in the methylation step was DMF, compound 2c was isolated as a white lyophilisate (1.2 mg, 2% yield).

7

Compound 2a

Synthesized according to general procedure for on-resin N-methylation with 260 mg of resin (0.078 mmol). After the deprotonation step the resin was not transferred to a filter tube and the iodomethane was added directly to the centrifuge tube. After methylation, the resin was transferred to a filter tube using THF. Compound 2a was isolated as a white lyophilisate (2.3 mg, 4% yield). HRMS (ESI) for C39H63N6O7 [M+H]+ calculated 727.4753, measured 727.4764 (1.5 ppm error); 1H NMR (600 MHz, DMSO-d6) δ = 9.01 (brs, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 9.3 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.3 Hz, 2H), 4.91 - 4.77 (m, 2H), 4.68 (td, J = 8.7, 4.0 Hz, 1H), 4.25 - 3.99 (m, 3H), 3.63 - 3.38 (m, 3H), 2.94 (s, 3H), 2.77 - 2.65 (m, 1H), 1.96 - 1.71 (m, 4H), 1.67 - 1.32 (m, 12H), 0.93 - 0.79 (m, 24H);

8

2. Appendix

a) Determination of resin loading for Fmoc-Tyr(Oallyl) on trichloroacetimidate Wang resin

UV/vis Spectrum compound Fmoc-Tyr(OAllyl) on resin

9

N26191-10.SampleName

Praew N26191-10 fmco countingDescription

200 300 400 500 600 700

-0.15-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

nm

A

300.99nm, 0.14448A

Determination of resin loading:

Resin loading calculation:

loading (mmol/g) = (1000*absorbance)/(M*7800*D)

M= mass of original resin which has been functionalized by AA (mg)M= (10 mg mini-cleavage sample* 521 mg original resin) / 435 mg functionalized resinM= 12.0 mg

D= 1/V = 1/100 mL = 0.01 mL^(-1) (as Fmoc collected was diluted to 100mL, therefore V= 100 mL

Loading (mmol/g) = (1000*0.14448)/(12.0 mg*7800 L/mol*0.01 mL^(-1)

Loading (mmol/g) = 0.15

b) LCMS of peptides 1a-c, 2a-c, 3, 4a-b, 5-8, 9a-b, 10a-b

Conditions:

The UPLC analysis was conducted on an Acquity UPLC CSH C18 column (50mm x 2.1mmi.d. 1.7μm packing diameter) at 30 degrees centigrade.The solvents employed were:A = 0.1% v/v solution of Trifluoroacetic Acid in Water.B = 0.1% v/v solution of Trifluoroacetic Acid in Acetonitrile.The flow rate was 0.9 (mL/min)The gradient employed was:

Time (min) Method 1 (CSH general)%A %B

10

0.0 98 20.2 98 22.5 3 972.9 3 973.0 98 2

Time (min) Method 2 (CSH focused)%A %B

0.0 60 400.2 60 402.0 40 602.4 3 972.9 3 973.0 98 2

The UV detection was a summed signal from wavelength of 210nm to 350nm.Injection volume : 0.3ul

MS Conditions

MS : Waters ZQIonisation mode : Alternate-scan Positive and Negative ElectrosprayScan Range : 100 to 1000 AMUScan Time : 0.27 secondsInter scan Delay : 0.10 seconds

Peptide 1a

LC/MS : Method 2

11

HRMS

Peptide 1b

LC/MS : Method 1

12

HRMS

Peptide 1c

LC/MS : Method 2

13

HRMS

Peptide 2a

LC/MS: Method 1

14

HRMS

Peptide 2b

LC/MS: Method 1

15

HRMS

Peptide 2c

LC/MS: Method 1

16

HRMS

Peptide 3

LC/MS: Method 1

17

HRMS

Peptide 4a

LC/MS: Method 1

18

HRMS

Peptide 4b

LC/MS: Method 1

19

HRMS

Peptide 5

LC/MS: Method 1

20

HRMS

Peptide 6

LC/MS: Method 1

21

HRMS

Peptide 7

LC/MS: Method 2

22

HRMS

Peptide 8

LC/MS: Method 2

23

HRMS

Peptide 9a

LC/MS: Method 1

24

HRMS

Peptide 9b

LC/MS: Method 1

25

HRMS

Peptide 10a

LC/MS: Method 1

26

HRMS

Peptide 10b

LC/MS: Method 1

27

HRMS

3. Assays

Chemi-Luminescent Nitrogen Detection (CLND) solubility assay

GSK in-house kinetic solubility assay: 5 µL of 10 mM DMSO stock solution of the compound was diluted to 100 µL with pH 7.4 phosphate buffered saline, equilibrated for 1 hour at room temperature and filtered through Millipore MultiscreenHTS-PCF filter plates (MSSL BPC). The

28

filtrate was quantified by a suitably calibrated flow injection Chemi-Luminescent Nitrogen Detection.3 The standard error of the CLND solubility determination was ±30 µM, and the upper limit of the solubility was 500 µM when working from a 10 mM DMSO stock solution.

Artificial Membrane Permeability (AMP) assay

The donor cell contained 2.5 µL of 10 mM sample solution in pH 7.4 phosphate buffer. To enhance solubility, 0.5% hydroxypropyl-cyclodextrin (encapsin) was added to the buffer. The artificial membrane was prepared from 1.8% phosphatidylcholine and 1% cholesterol in decane solution. The sample concentration in both the donor and acceptor compartments were determined by LC-MS after 3 h incubation at room temperature.4 The permeability (logPapp) measuring how fast molecules pass through the black lipid membrane was expressed in nm/s. The average standard error of the assay was around ±30 nm/s which could be higher at the low permeability range.

This AMP assay is the GSK version of the PAMPA published by Kansy et al.5 using phosphatidylcholine and cholesterol for the preparation of the membrane on the filter. The membrane is much thinner (~10 µm instead of 100 µm), thus the experiment takes only 3 hours. Cyclodextrin is used to enhance the solubility of the compounds and HPLC-MS or CLND technology (see description above) is used for the concentration determination of the compounds in the donor and acceptor cells. Both the donor and acceptor cells contain the same pH phosphate buffer (pH 7.4) modeling cellular permeability rather than pH 6.5 that is used for modeling intestinal permeability.

ChromlogD assay

The Chromatographic Hydrophobicity Index (CHI)6 values were measured using a reversed phase HPLC column (Luna C18 (2), Phenomenex, UK) with a fast acetonitrile gradient at a starting mobile phase of pH = 7.4. CHI values were derived directly from the gradient retention times by using a calibration line obtained for standard compounds. The CHI value approximates to the volume % organic concentration when the compound elutes. CHI was linearly transformed into ChromlogD by a least-square fitting of experimental CHI values to calculated ClogP values for over 20,000 research compounds using the following formula: ChromlogD = 0.0857CHI-2.00. The average error of the assay was ±3 CHI units which was equivalent to ±0.25 ChromlogD.

4. Computational modeling

All the cyclic peptides were protonated at pH7.4 using MOE 2012. The pKa of all the compounds were determined using ChemAxons’s Marvin pKa calculator (http:/www.chemaxon.com). The atomic charges of each compound in the dataset were calculated using the MMFF94x force field in MOE. This was followed by minimizing each compound in the MOE database using the MMFF94x force-field. AM1 charges were derived for the minimized conformations of the molecule. This step was followed by generating multiple conformations of each molecule using LowModeMD7 at 300K using Berendsen thermostat. The conformational search was done in vacuum and all the conformations within 20 kcal/mol of the lowest energy conformer were retained. Using this conformational analysis approximately 10,000 conformations were generated for each peptide. The cyclic peptide conformation with the lowest energy was retained for solvation energy calculations. The polar (or the electrostatic) part of the solvation energy (polar solvation energy) was

29

calculated by numerically solving the Poisson Boltzmann (PB) equation. SVMChromlogD for each compound was predicted by using a model obtained using a Support Vector Regression (SVM) method (GSK’s internal model for predicting ChromlogD).

5. NMR experiments

NMR experiments were carried out on a Bruker AVII+ 600 spectrometer equipped with a 5mm TCI cryoprobe running TOPSPIN 3.2 Software, using standard Bruker pulse sequences.

For the deuterium exchange experiments, a stock solution of 0.77 µL of TFA (trifluoroacetic acid) in 600 µL of D2O was prepared. Aliquots of this stock solution (50 µL) were then added to the NMR samples (1 mg of peptide in 500 µL of CDCl3). The resulting mixture was then immediately shaken for 1 minute and sonicated for 20 seconds. NMR spectra were then recorded at timepoints up to 19 hours with the solutions left at ambient laboratory temperatures (~296K) between the NMR measurements (at 303K).

The CDCl3 NH exchange rates after 19 hours of exchange study were classified on this basis: Fast < 20% intensity remaining. Medium = 20-70% intensity remaining.Slow >70% intensity remaining.

30

31

Water exchange rate studies were carried out in a 90% H2O/10% D2O solution at pH 7.6 (50 mM phosphate buffer) and were based on the exchange broadening observed on the NH resonances. The NH assignments were initially carried out at pH 3.8 (20 mM acetate buffer) using ROESY and DIPSI spectra.

The water NH exchange rates were classified on their linewidths in 1H WATERGATE spectra at 303K (1H presaturation with excitation sculpting spectra were used to confirm that the broadenings observed were due to solvent exchange): Fast NH's severely broadened by exchange (linewidths >10Hz).Medium NH's broadened by exchange (linewidths 5-10Hz).Slow NH's relatively unaffected by exchange (linewidths <5Hz).

Peptide 1b: NH exchange broadening with change in pH

32

Peptide 1a: 1H WATERGATE at pH 7.6

Peptide 1b: 1H WATERGATE at pH 7.6

33

Peptide 1c: 1H WATERGATE at pH 7.6

NH Exchange Rates

1. Peptide 1a CDCl3 Water pH 7.6Leu1NH Fast MediumLeu2NH Fast MediumLeu3NH Fast Medium/SlowLeu4NH Medium MediumTyr6NH Medium Medium/Slow

2. Peptide 1b

CDCl3 Water pH 7.6Leu1NH Slow SlowLeu2NH Medium Medium/SlowLeu3NH Fast FastLeu4NH Medium SlowTyr6NH Fast Fast

3. Peptide 1c

CDCl3 Water pH 7.6Leu1NH Slow Medium/FastLeu2NH Medium Medium/FastLeu3NH Slow Medium/SlowLeu4NH Slow Medium

34

Tyr6NH Slow Slow

Peptide 1a: ROESY with excitation sculpting in water at pH 3.8

35

Peptide 1b: ROESY with excitation sculpting in water at pH 3.8

Peptide 1c: ROESY with excitation sculpting in water at pH 3.8

36

6. Summary Table of Results

Compound

Mean CLND

Solubility (uM)

CLND Solubility

Values (uM)

CLND Solubility

Number of Runs

Mean ChromLogD

ChromLogD Range

ChromLogD Number of

Runs

Mean AMP Permeability

(nm/s)

AMP Permeability

Range

AMP permeability Number of

Runs

Number of Rapidly

Exchanging NHs (CDCl3)

1b 170 151 - 189 2 5.53 5.35 - 5.71 2 111 92 - 130 2 21a 324 307, 340 2 5.07 4.81 - 5.24 3 41 40 - 42 2 31c 248.5 247 - 250 2 5.57 5.35 - 5.79 2 415 400 - 430 2 02b 103 101 - 105 2 7.52 7.46 - 7.58 2 346.667 290 - 380 3 3 51.67 12-74 3 5.98 5.93-6.03 3 130 110 - 150 2

10a 372a378, 352,

385 3 4.65 4.57 - 4.76 3 <6.5b 3-10b 2 4a 78 60 - 96 2 6.56 6.49 - 6.63 2 350 320 - 380 2 9a 485a 449, 520 2 4.75 4.74 - 4.76 2 13.5b 10-17b 2 9b 451a 483, 419 2 4.45 4.41 - 4.51 3 <6.5b 3-10b 2

10b 371a 357, 385 2 4.2 4.19 - 4.2 2 10.5b 3-18b 2 5 102.5 94 - 111 2 6.65 6.58 - 6.72 2 303.333 300 - 310 3 27 28.5 17-40 2 7.21 7.12 - 7.35 3 300 270 - 330 4 12c 53.67 48 - 62 3 8.32 8.28 - 8.37 3 363 340-390 3 2a 192.33 158 - 216 3 6.19 6.16 - 6.22 3 144 66 - 200 4 6 122.5 88 - 157 2 6.88 6.83 - 6.92 2 185 180 - 190 2

4b 148.5 130 - 167 2 6.11 6.04 - 6.17 2 170 160 - 180 2 8 205.5 194 - 217 2 6.05 5.9 - 6.19 2 200 190 - 210 2

a Approaching upper detection limit of solubility assay

b Approaching lower detection limit of permeability assay

37

7. References

1. (a) Isidro-Llobet, A.; Boas, U.; Jensen, K. J.; Álvarez, M.; Albericio, F. J. Org. Chem. 2008, 73, 7342; (b) Rezai, T.; Yu, B.; Milhauser, G. L.; Jacobson, M. P.; Lokey, R. S. J. Am. Chem. Soc. 2006, 128, 2510.

2. Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lokey, R. S.; Jacobson, M. P. J. Am. Chem. Soc. 2006, 128, 14073.

3. Bhattachar, S. N.; Wesley, J. A.; Seadeek, C. J. Pharm. Biomed. Anal. 2006, 41, 152.

4. Veber, D. F.; Johnson, S.R.; Chen, H- Y.; Smith, B. R.; Ward, K. W.; Kopple, D. J. Med. Chem. 2002, 45, 2615.

5. Kansy, M.; Senner, F.; Gubernator, K. J. Med. Chem. 1998, 41, 1007.

6. Valko, K.; Bevan, C.; Reynolds, D. Anal. Chem. 1997, 69, 2022.

7. Molecular Operating Environment (MOE), 2013.08, Chemical Computing Group Inc.: 2013

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