Self-assembled multivalent RGD-peptide arrays – Morphological … · 2013-04-04 ·...

Self-assembled multivalent RGD-peptide arrays – Morphological control and

integrin binding

Daniel J. Welsh,a Paola Posocco,b Sabrina Pricl*b,c and David K. Smith*a

5

a: Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK.

b: Molecular Simulations Engineering (MOSE) Laboratory, Department of Engineering and

Architecture (DEA), University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy

c: National Interuniversity Consortium for Material Science and Technology( INSTM), Research

Unit MOSE-DEA, University of Trieste E-mail:[email protected] 10

SUPPORTING INFORMATION

15

Contents

1 Synthetic Methods and Characterisation Data

2 Experimental Determination of Critical Aggregation Concentrations (CACs)

3 Fluorescence Polarisation Assay

4 Computational Methods 20

5 References

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013

1. Synthetic Methods and Characterisation Data

Materials and Methods. All reagents were commercially available and used as supplied without

further purification. Compound C12-RGD and protected RGD peptide H2N-Arg(Pbf)-Gly-

Asp(OtBu)-OtBu were synthesised as reported previously.1 Human integrin αvβ3 Triton X-100 5

formulation purified protein was purchased from Millipore and used without further purification.

Column chromatography was performed on silica using silica gel 60 provided by Fluka Ltd. (35-70

μm) while TLC was performed on Merck aluminium-backed plates, coated with 0.25 mm silica gel

60. Spots were visualised either by UV, or by use of an appropriate stain (ninhydrin solution 0.2%

(by mass) in ethanol, cerium molybdate stain: 180 ml H2O, 20 ml conc. H2SO4, 24 g ammonium 10

molybdate, 2 g cerium sulphate, or potassium permanganate stain: 1.5 g KMnO4, 10 g K2CO3, 1.25

ml 10% NaOH in 200 ml water). Preparative gel filtration chromatography was carried out using

Sephadex LH-20 purchased from Sigma Aldrich. Proton and carbon NMR chemical shifts (δ) are

reported in ppm using residual solvent as internal reference, as noted, and peak assignments were

deduced with DEPT-135 as well as 2D NMR experiments such as COSY and HSQC. All spectra 15

were recorded on either a JEOL ECX400 or a JEOL ECS400 (1H 400 MHz, 13C 100 MHz)

spectrometers or a JEOL EX270 (1H 270 MHz, 13C 68 MHz) spectrometer, as noted. ESI and HR

ESI mass spectra were recorded on a Thermo-Finnigan LCQ, and a Bruker Daltonics Microtoff

mass spectrometer respectively. Infrared spectra were recorded using a Shimadzu IRPrestige-21

FT-IR spectrometer. Melting points were measured on a Gallenkamp melting point apparatus and 20

are uncorrected. Optical rotation was measured as [α]D on a JASCO DIP-370 digital polarimeter.

Nile Red fluorescence was measured on a Hitachi F-4500 spectrofluorimeter. Fluorescence

polarisation data was collected on FluoroMax-3 and FluoroMax-4 spectrofluorimeters.

Synthesis of Protected Py-RGD (Py-Arg(Pbf)-Gly-Asp(OtBu)-OtBu). 1-Pyrenebutyric acid (81 25

mg, 0.28 mmol, 1.0 eq) was dissolved in DCM (4 ml) upon addition of DIPEA (0.1 ml, 0.57 mmol,

2.0 eq) and the reaction flask was cooled over an ice-water bath. TBTU (90 mg, 0.28 mmol, 1.0 eq)

was added as a solid and stirred for 10 min at 0°C before the protected RGD peptide (H2N-

Arg(Pbf)-Gly-Asp(OtBu)-OtBu) (200 mg, 0.28 mmol, 1.0 eq) was added as a solid and DCM (6

ml) was used to rinse any residual material into the reaction flask. The reaction was stirred for a 30

further 30 min at 0°C, then the ice-water bath was removed and the reaction stirred at rt for 21 h.

The organic phase was washed with 1.33 M NaHSO4 (3 × 50 ml), 1 M Na2CO3 (3 × 50 ml), water

(50 ml) and finally brine (50 ml). The organic phase was dried over MgSO4 and filtered before

removing the solvent in vacuo to yield the target compound as a yellow solid (280 mg, quantitative

yield). No further purification was required. Rf 0.43 (9:1 DCM/MeOH, UV and cerium stain). [α]D 35


= -0.5 (c = 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz) 8.18 (d, CH aromatic, J = 9.5 Hz, 1H); 8.07

(dd, CH aromatic, J = 7.5 Hz and 5.0 Hz, 2H); 7.98 (d, CH aromatic, J = 8.5 Hz, 2H); 7.93-7.86 (m,

overlapping CH aromatic × 3 and NH amide (Arg-Gly), 4H); 7.73 (d, CH aromatic, J = 8.0 Hz,

1H); 7.40 (d, NH amide (Gly-Asp), J = 7.5 Hz, 1H); 7.13 (app br d, NH amide (Py-Arg), 1H), 6.45

(br s, NH2 guanidine, 2H); 6.29 (br s, NH guanidine, 1H); 4.66 (dt, Asp α-H, J = 8.0 Hz and 5.0 Hz, 5

1H); 4.61-4.55 (m, Arg α-H, 1H); 3.99 (app br d, Gly CH2, 2H); 3.36-3.10 (br m, Arg CH2NH, 2H);

3.24 (t, CH2CH2CH2C(O)NH, J = 7.5 Hz, 2H); 2.79 (s, Pbf CH2, 2H); 2.76 (dd, Asp CHA, J = 17.0

Hz and 5.0 Hz, 1H); 2.68 (dd, Asp CHB, J = 17.0 Hz and 5.0 Hz, 1H); 2.57 (s, Pbf CH3Ar, 3H);

2.45 (s, Pbf CH3Ar, 3H); 2.35 (t, CH2CH2CH2C(O)NH, J = 7.0 Hz, 2H); 2.11 (quintet,

CH2CH2CH2C(O)NH, J = 6.5 Hz, 2H); 2.04 (s, Pbf CH3Ar, 3H); 1.96-1.83 (m, Arg CHCHA, 1H); 10

1.77-1.64 (m, Arg CHCHB, 1H); 1.64-1.48 (m, Arg CH2CH2NH, 2H); 1.36, 1.354, 1.347 (s × 3, Pbf

CH3 × 2 and C(CH3)3 × 2, calc 24H, found 23H). 13C NMR (CDCl3, 100 MHz) 173.58, 172.90,

169.98, 169.81, 169.22 (C(O)OtBu × 2, C(O)NH amide × 3); 158.65, 156.58 (Pbf aromatic C-O,

C=N guanidine); 138.28 (Pbf aromatic C), 135.87 (pyrene aromatic C), 132.90, 132.21 (Pbf

aromatic C × 2); 131.30, 130.82, 129.78, 128.60 (pyrene aromatic C × 4); 127.44, 127.28, 127.25, 15

126.54, 125.74 (CH aromatic × 5); 124.93, 124.87 (pyrene aromatic C × 2); 124.77, 124.69 (CH

aromatic × 2); 124.58 (Pbf aromatic C); 123.32 (CH aromatic); 117.42 (Pbf aromatic C); 86.30 (Pbf

CH2C(CH3)2O); 82.39, 81.45 (C(CH3)3 × 2); 52.85 (Arg α-CH); 49.38 (Asp α-CH); 43.08 (Pbf

ArCH2); 42.81 (Gly CH2); 40.33 (Arg CH2NH); 37.34 (Asp CH2); 35.73 (CH2CH2CH2C(O)NH);

32.75 (CH2CH2CH2C(O)NH); 29.48 (Arg CHCH2); 28.50 (Pbf CH2C(CH3)2O); 27.97, 27.80 20

(C(CH3)3 × 2); 27.36 (CH2CH2CH2C(O)NH); 25.37 (Arg CH2CH2NH); 19.31, 18.00, 12.48 (Pbf

ArCH3 × 3). max (cm-1) (solid): 3310w, 2932w, 2870w, 1728m, 1651m, 1620m, 1543s, 1450m,

1366m, 1242s, 1150s, 1096s, 988w, 910w, 841m, 810w, 779w, 733m. ESI-MS (m/z): Calc. for

C53H69N6O10S 981.4790; found: 981.4797 (100%, [M+H]+); Calc. for C53H68N6NaO10S 1003.4610;

found: 1003.4609 (32%, [M+Na]+). 25

Synthesis of Py-RGD. Protected Py-RGD (240 mg, 0.24 mmol) was dissolved in a mixture of

TFA, water and TIS (5 ml, 95:2.5:2.5) and shaken for 1.5 h, after which time TLC indicated that the

deprotection reaction was complete. The volatiles were removed in vacuo, then the residue was

dissolved in the minimum amount of hot MeOH, precipitated with cold Et2O, filtered and washed 30

with the minimum amount of cold Et2O to yield a tanish green solid (137 mg, 77% as TFA salt).

The sample was then dissolved in a mixture of water/tBuOH, filtered over a PTFE membrane filter

(0.2 μm), shell frozen and lyophilised to yield the product Py-RGD as a fluffy, pale green powder.

Rf 0.00 (9:1 DCM/MeOH, UV and cerium stain). [α]D = +2.6 (c = 0.5, DMSO). 1H NMR (3:1:1

CD3OD/CDCl3/D2O, 400 MHz) 8.23 (d, CH aromatic, J = 9.0 Hz, 1H); 8.08 (t, CH aromatic, J = 35


7.0 Hz, 2H); 8.03 (d, CH aromatic, J = 9.0 Hz, 2H); 7.95-7.90 (m, CH aromatic × 3, 3H); 7.82 (d,

CH aromatic, J = 8.0 Hz, 1H); 4.74 (t, Asp α-H, J = 5.5 Hz, 1H); 4.33 (app t, Arg α-H, J = 6.0 Hz,

1H); 3.96 (d, Gly CHA, J = 17.0 Hz, 1H); 3.91 (d, Gly CHB, J = 17.0 Hz, 1H); 3.30 (t,

CH2CH2CH2C(O)NH, J = 7.0 Hz, 2H, obscured by the CD3OD peak); 3.16 (t, Arg CH2NH, J = 6.5

Hz, 2H); 2.94-2.82 (m, Asp CH2, 2H); 2.45 (t, CH2CH2CH2C(O)NH, J = 7.0 Hz, 2H); 2.12 (quintet, 5

CH2CH2CH2C(O)NH, J = 7.0 Hz, 2H); 1.92-1.79 (m, Arg CHCHA, 1H); 1.79-1.56 (m, overlapping

Arg CHCHB and Arg CH2CH2NH, 3H). 13C NMR (3:1:1 CD3OD/CDCl3/D2O, 100 MHz) 176.07,

174.62, 174.29, 174.02, 171.11, 157.77 (C(O)OH × 2, C(O)NH amide × 3, C=N guanidine); 136.76,

132.11, 131.63, 130.64, 129.34, 128.13, 127.99, 127.29, 126.55, 125.64, 125.54, 125.43, 124.00

(aromatic C and CH); 54.22 (Arg α-CH); 49.75 (Asp α-CH); 43.16 (Gly CH2); 41.47 (Arg CH2NH); 10

36.53, 36.18 (CH2CH2CH2C(O)NH and Asp CH2); 33.44 (CH2CH2CH2C(O)NH); 29.51 (Arg

CHCH2); 28.22 (CH2CH2CH2C(O)NH); 25.54 (Arg CH2CH2NH). max (cm-1) (solid): 3179w,

3032w, 1751w, 1651m, 1520m, 1420m, 1319m, 1188s, 1042s, 872s, 748m, 702m. ESI-MS (m/z):

Calc. for C32H37N6O7 617.2718; found: 617.2722 (100%, [M+H]+).

15

Synthesis of Protected C22-RGD (C22-Arg(Pbf)-Gly-Asp(OtBu)-OtBu). Behenic acid (96 mg,

0.28 mmol, 1.0 eq) was dissolved in DCM (7 ml) upon addition of DIPEA (100 µl, 0.56 mmol, 2.0

eq) and the reaction flask cooled over an ice-water bath. TBTU (90 mg, 0.28 mmol, 1.0 eq) was

added as a solid and more DCM (3 ml) was used to rinse any residual TBTU into the reaction flask.

The reaction was stirred at 0°C for 10 min, then protected RGD peptide (H2N-Arg(Pbf)-Gly-20

Asp(OtBu)-OtBu) (200 mg, 0.28 mmol, 1.0 eq) was added as a solid and more DCM (2 ml) was

used to rinse any residual material into the reaction flask. The reaction was stirred for a further 30

min at 0°C, then the ice-water bath was removed and the reaction stirred at rt for 4 days. The

organic phase was washed with 1.33 M NaHSO4 (3 × 50 ml), 1 M Na2CO3 (3 × 50 ml), water (50

ml) and finally brine (50 ml). The organic phase was dried over MgSO4 and filtered before 25

removing the solvent in vacuo to yield the product as a white solid (280 mg, 96%). No further

purification was required. Rf 0.40 (9:1 DCM/MeOH, UV and cerium stain). [α]D = -2.6 (c = 1.0,

CHCl3). 1H NMR (CDCl3, 400 MHz) 7.86 (br t, NH amide (Arg-Gly), 1H); 7.34 (d, NH amide

(Gly-Asp), J = 8.0 Hz, 1H); 6.97 (br d, NH amide (C22-Arg), 1H), 6.39 (br s, NH2 guanidine, 2H);

6.24 (br s, NH guanidine, 1H); 4.64 (dt, Asp α-H, J = 8.5 Hz and 5.0 Hz, 1H); 4.54 (app q, Arg α-H, 30

1H); 4.03-3.87 (br m, Gly CH2, 2H); 3.36-3.12 (br m, Arg CH2NH, 2H); 2.93 (s, Pbf CH2, 2H);

2.78 (dd, Asp CHA, J = 17.0 Hz and 5.0 Hz, 1H); 2.68 (dd, Asp CHB, J = 17.0 Hz and 5.0 Hz, 1H);

2.55 (s, Pbf CH3Ar, 3H); 2.48 (s, Pbf CH3Ar, 3H); 2.19 (t, C22 CH2C(O)NH, J = 7.0 Hz, 2H); 2.06

(s, Pbf CH3Ar, 3H); 1.95-1.81 (m, Arg CHCHA, 1H); 1.76-1.62 (m, Arg CHCHB, 1H); 1.62-1.49

(m, Arg CH2CH2NH and C22 CH2 overlapping, 4H); 1.44 (s, Pbf CH3 × 2, 6H); 1.40, 1.39 (s × 2, 35


C(CH3)3 × 2, calc 18H, found 17H); 1.32-1.16 (m, C22 CH2’s, calc 36H, found 37H); 0.86 (t, C22

CH3, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) 174.11, 172.99, 170.08, 169.89, 169.28

(C(O)OtBu × 2, C(O)NH amide × 3); 158.76, 156.66 (Pbf aromatic C-O, C=N guanidine); 138.41,

132.95, 132.31, 124.61, 117.52 (Pbf aromatic C); 86.39 (Pbf CH2C(CH3)2O); 82.53, 81.59 (C(CH3)3

× 2); 52.70 (Arg α-CH); 49.43 (Asp α-CH); 43.32 (Pbf ArCH2); 42.88 (Gly CH2); 40.30 (Arg 5

CH2NH); 37.43 (Asp CH2); 36.46 (C22 CH2C(O)NH); 32.00, 29.79, 29.74, 29.68, 29.51, 29.44

(C22 CH2’s and Arg CHCH2, multiple overlapping peaks); 28.68 (Pbf CH2C(CH3)2O); 28.11, 27.94

(C(CH3)3 × 2); 25.76, 25.37, 22.77 (C22 CH2’s, Arg CH2CH2NH); 19.41, 18.05 (Pbf ArCH3 × 2);

14.21 (C22 CH3); 12.56 (Pbf ArCH3 × 1). max (cm-1) (solid): 3310w, 2924m, 2855w, 1728m,

1651s, 1543s, 1450m, 1366m, 1242s, 1150s, 1103s. ESI-MS (m/z): Calc. for C55H97N6O10S 10

1033.6981; found: 1033.6980 (100%, [M+H]+).

Synthesis of C22-RGD. Protected C22-RGD (250 mg, 0.24 mmol) was dissolved in a mixture of

TFA, water and TIS (10 ml, 95:2.5:2.5) and shaken for 1 h 20 min, after which time TLC indicated

that the deprotection reaction was complete. The volatiles were removed in vacuo, then the residue 15

was dissolved in the minimum amount of hot MeOH/water/tBuOH and the remaining orange/yellow

solid was filtered off. The filtrate was evaporated in vacuo to yield a white solid which was

redissolved in a mixture of hot water/tBuOH, precipitated with cold Et2O, filtered and washed with

the minimum amount of cold Et2O to yield a white solid. The sample was then dissolved in a

mixture of hot water/tBuOH with sonication, filtered over a PTFE membrane filter (0.2 μm), shell 20

frozen and lyophilised to yield the product as a fluffy white powder (120 mg, 63% as TFA salt).

Note: The compound forms a gel in DMSO-d6 and so NMR analysis was carried out at elevated

temperature to induce gel-sol transformation which resulted in better resolved NMR spectra. Rf

0.00 (9:1 DCM/MeOH, cerium stain). [α]D = +2.2 (c = 0.5, TFA). 1H NMR (DMSO-d6, 500 MHz,

temp = 70°C) 8.20-8.00 (br m, NH amide (Arg-Gly), Arg CH2NH, 2H); 7.83 (br d, NH amide 25

(Gly-Asp), J = 5.5 Hz, 1H); 7.71 (d, NH amide (C22-Arg), J = 7.5 Hz, 1H), 7.04 (br s, -NH2 and -

NH2+ of guanidine, 4H); 4.37 (br m, Asp α-H, 1H); 4.28 (app q, Arg α-H, J = 7.0 Hz, 1H); 3.83 (dd,

Gly CHA, J = 16.5 Hz and 6.0 Hz, 1H); 3.62 (dd, Gly CHB, J = 17.0 Hz and 5.0 Hz, 1H); 3.20-3.05

(br m, Arg CH2NH, 2H); 2.60-2.53 (m, Asp CH2, 2H); 2.14 (t, C22 CH2C(O)NH, J = 7.5 Hz, 2H);

1.86-1.73 (m, Arg CHCHA, 1H); 1.66-1.45 (m, overlapping Arg CHCHB, Arg CH2CH2NH and C22 30

CH2, 5H); 1.34-1.16 (m, C22 CH2’s, 36H); 0.87 (t, C22 CH3, J = 6.5 Hz, 3H). 13C NMR (DMSO-

d6, 125 MHz, temp = 70°C) 172.35, 172.05, 171.82, 171.55, 167.86, 156.79 (C(O)OH × 2,

C(O)NH amide × 3, C=N guanidine); 51.92 (Arg α-CH); 48.65 (Asp α-CH); 41.80 (Gly CH2);

40.32 (Arg CH2NH); 37.37 (Asp CH2); 34.89 (C22 CH2C(O)NH); 30.83, 29.09, 28.59, 28.58,

28.54, 28.53, 28.37, 28.34, 28.19 (C22 CH2’s and Arg CHCH2, multiple overlapping peaks); 24.78, 35


24.49, 21.58 (C22 CH2’s, Arg CH2CH2NH); 13.36 (C22 CH3). max (cm-1) (solid): 3287w, 3179w,

3102w, 3032w, 2916s, 2847m, 1751w, 1628s, 1535m, 1466w, 1396w, 1327w, 1188m, 1049m. ESI-

MS (m/z): Calc. for C34H65N6O7 669.4909; found: 669.4932 (100%, [M+H]+).

Synthesis of 1-ester. Lauric acid (2.00 g, 10 mmol, 2.0 eq) and L-lysine methyl ester 5

dihydrochloride (1.17 g, 5 mmol, 1.0 eq) were combined in DCM (40 ml) and stirred over an ice-

water bath. DIPEA (3.48 ml, 20 mmol, 4.0 eq) and TBTU (3.21 g, 10 mmol, 2.0 eq) were added

neat and the reaction was stirred for 64 h. The fine white solid was filtered off and washed with

DCM (25 ml). The organic fractions were combined and washed with hot 5% HCl (0.5 M, 3 × 100

ml), hot 1 M Na2CO3 (3 × 100 ml), hot 15% Na2CO3 (3 × 100 ml), hot 10% HCl (1 M, 3 × 100 ml), 10

and finally hot water (2 × 100 ml). The organic phase was dried over MgSO4, filtered and then the

filtrate was evaporated to yield the product as an off-white solid (2.34 g, 89%). No further

purification was required. Rf 0.53 (9:1, DCM/MeOH, cerium stain). [α]D = +5.6 (c = 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz) 6.40 (d, α-NH amide, J = 7.5 Hz, 1H); 6.01 (br t, ε-NH amide, J =

5.0 Hz, 1H); 4.50 (td, α-H, J = 8.0 Hz and 4.5 Hz, 1H); 3.68 (s, CH3OC(O), 3H); 3.18 (m, 15

CH2NHC(O), 2H); 2.19 (t, CH2C(O)NH, J = 7.0 Hz, 2H); 2.12 (t, CH2C(O)NH, J = 7.0 Hz, 2H);

1.85-1.13 (m, C12 and lysine CH2’s, calc 42H, found 45 H); 0.83 (t, 2 × C12 CH3, J = 7.0 Hz, 6H). 13C NMR (CDCl3, 100 MHz) 173.67, 173.44, 173.10 (C(O)NH amide × 2, C(O)OCH3); 52.33

(C(O)OCH3); 51.76 (α-CH); 38.63 (CH2NHC(O)); 36.81, 36.49 (CH2C(O)NH × 2); 31.94, 31.82,

29.68, 29.66, 29.58, 29.46, 29.43, 29.38, 29.35, 28.92, 25.91, 25.70, 22.72, 22.35 (C12 and lysine 20

CH2’s); 14.14 (C12 CH3 × 2). max (cm-1) (solid): 3302w, 2916m, 2855w, 1736m, 1643m, 1543s,

1458m, 1366m, 1242m, 1150s, 1103s. ESI-MS (m/z): Calc. for C31H61N2O4 525.4626; found:

525.4614 (100%, [M+H]+).

Synthesis of 1-acid. Compound 1-ester (2.24 g, 4.3 mmol, 1.0 eq) was dissolved in MeOH (50 ml) 25

with sonication and then cooled in an ice-water bath, upon which, the starting material precipitated

out of solution. 1 M NaOH (13 ml, 13 mmol, 3 eq) was added and the reaction was kept stirring at

0°C for ~2 h, then the ice-water bath was removed and the flask allowed to warm to rt. The mixture

was sonicated for 1 h and then more MeOH and 1 M NaOH (8.6 ml, 8.6 mmol, 2 eq, 5 eq in total)

were added for smoother stirring and to try to dissolve the starting material. The white suspension 30

was stirred vigorously at rt for 16 h, after which time the white suspension still remained. The

reaction was heated at 40°C for 2 h, after which time TLC indicated that the reaction was complete

(the white precipitate dissolved after ~30 min of heating) then the solvent was removed in vacuo.

The white residue was taken up in water (250 ml) and gently heated at 40°C to induce

solubilisation, then the solution was acidified to pH 1 using 1.33 M NaHSO4 (~30 ml) upon which a 35


white precipitate formed. This was taken up in DCM (250 ml), separated from the aqueous layer,

and then the aqueous layer was washed with more DCM (150 ml) and the organic fractions were

combined and washed with hot water (400 ml), then brine (400 ml), and then dried over MgSO4,

filtered, and the filtrate evaporated to yield the product as a white solid (~1.8 g, ~83%). Rf 0.04

(9:1, DCM/MeOH, cerium stain). [α]D = -72.2 (c = 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz) 5

10.17 (br s, CO2H, 1H); 6.81 (d, α-NH amide, J = 7.0 Hz, 1H); 6.10 (t, ε-NH amide, J = 5.5 Hz,

1H); 4.55 (dt, α-H, J = 7.0 Hz and 5.0 Hz, 1H); 3.35-3.27 (m, CH2NH, 2H); 3.22-3.14 (m, CH2NH,

2H); 2.25 (t, CH2C(O)NH, J = 7.0 Hz, 2H); 2.18 (t, CH2C(O)NH, J = 7.0 Hz, 2H); 1.93-1.18 (m,

C12 and lysine CH2’s, calc 42H, found 45 H); 0.87 (t, 2 × C12 CH3, J = 7.0 Hz, 6H). 13C NMR

(CDCl3, 100 MHz) 174.60, 174.47, 174.36 (C(O)NH amide × 2, C(O)OH); 52.25 (α-CH); 39.09 10

(CH2NHC(O)); 36.86, 36.61 (CH2C(O)NH × 2); 32.04, 31.68, 29.77, 29.67, 29.60, 29.47, 29.07,

25.98, 25.84, 22.78, 22.36 (C12 and lysine CH2’s); 14.17 (C12 CH3 × 2). max (cm-1) (solid): 3294w,

2970m, 2916m, 2855w, 1736m, 1643m, 1543s, 1458m, 1373m, 1242m, 1150s, 1103m. ESI-MS

(m/z): Calc. for C30H59N2O4 511.4469; found: 511.4470 (100%, [M+H]+).

15

Synthesis of 2-ester. Compound 1-acid (0.5 g, 0.98 mmol, 1.0 eq) dissolved in DCM (20 ml) upon

the addition of DIPEA (0.35 ml, 1.96 mmol, 2.0 eq). The reaction flask was cooled over an ice-

water bath, then TBTU (315 mg, 0.98 mmol, 1.0 eq) was added as a solid. The reaction mixture was

stirred at 0°C for 10 min, then methyl 6-aminohexanoate hydrochloride (179 mg, 0.98 mmol, 1.0

eq) was added as a solid. The reaction was stirred at 0°C for a further 30 min and then the ice-water 20

bath was removed and the reaction allowed to warm to rt. The reaction was refluxed at 40°C for 3

days, then diluted with DCM (50 ml) as material started precipitating out on cooling. The DCM

phase was washed with 1 M HCl (150 ml × 3), 1 M Na2CO3 (150 ml × 2), water (150 ml), and

finally brine (150 ml). The organic phase was dried over MgSO4, filtered and the filtrate evaporated

to yield the product as a waxy white solid (0.53 g, 85%). No further purification was required. Rf 25

0.44 (9:1, DCM/MeOH, KMnO4 stain). [α]D = -15.8 (c = 1.0, CHCl3). 1H NMR (CDCl3/CD3OD,

400 MHz) 4.27-4.22 (m, α-H, 1H); 3.64 (s, C(O)OCH3, 3H); 3.18-3.12 (m, 2 × CH2NH, 4H); 2.31

(t, CH2C(O)OCH3, J = 7.5 Hz, 2H); 2.20 (t, CH2C(O)NH, J = 7.0 Hz, 2H); 2.14 (t, CH2C(O)NH, J

= 7.0 Hz, 2H); 1.79-1.09 (m, C12 CH2’s, lysine CH2’s and C6 linker CH2’s, 48 H); 0.85 (t, 2 × C12

CH3, J = 7.0 Hz, 6H). 13C NMR (CDCl3/CD3OD, 100 MHz) 174.60, 174.41, 174.35, 172.28 30

(C(O)NH amide × 3, C(O)OCH3); 52.67 (α-CH); 51.21 (C(O)OCH3); 38.89, 38.53 (CH2NHC(O) ×

2); 36.16, 35.94 (CH2C(O)NH × 2); 33.55 (CH2C(O)OCH3); 31.61, 31.50, 29.31, 29.23, 29.04,

28.55, 28.44, 26.01, 25.68, 25.49, 24.19, 22.50, 22.36 (C12, lysine and C6 linker CH2’s); 13.62

(C12 CH3 × 2). max (cm-1) (solid): 3302w, 2916m, 2847w, 1736m, 1636m, 1543s, 1458m, 1373m,


1242m, 1150s, 1103m. ESI-MS (m/z): Calc. for C37H72N3O5 638.5466; found: 638.5465 (78%,

[M+H]+).

Synthesis of 2-acid. Compound 2-ester (0.5 g, 0.78 mmol, 1.0 eq) was dissolved in MeOH (20 ml)

with heating at 40°C. 1 M NaOH (2.35 ml, 2.35 mmol, 3 eq) was added and the reaction was kept 5

stirring at 40°C for 1 h, after which time TLC indicated presence of starting material, so heating

was increased to 50°C, after which time TLC still indicated presence of starting material, so heating

was increased to 60°C and the reaction refluxed for 3 h after a further addition of 1 M NaOH (2.35

ml, 2.35 mmol, 3 eq, 6 eq in total), after which time TLC did not indicate presence of starting

material. The solvent was then removed in vacuo and the residue was dissolved in water (50 ml) 10

and heated to 70°C until the sample became cloudy but had dissolved, then 1.33 M NaHSO4 was

added until pH ~2 was reached. The white precipitate was taken up in DCM with heating, and the

organic layer was separated and washed with hot water (250 ml), then hot brine (250 ml), dried over

MgSO4, filtered, and the filtrate evaporated to yield a white solid (~0.5 g). Purification by column

chromatography (SiO2, 9:1 DCM/MeOH) yielded the product as a waxy white solid (0.36 g, 73%). 15

Rf 0.33 (9:1, DCM/MeOH, KMnO4 stain). [α]D = -15.5 (c = 1.0, CHCl3). 1H NMR (CDCl3, 400

MHz) 7.32 (br s, NH amide, 1H); 7.12 (br s, NH amide, 1H); 6.38 (br s, NH amide, 1H); 4.48-

4.42 (m, α-H, 1H); 3.33-3.08 (m, 2 × CH2NH, 4H); 2.29 (t, CH2C(O)OH, J = 7.0 Hz, 2H); 2.18 (t,

CH2C(O)NH, J = 7.0 Hz, 2H); 2.13 (t, CH2C(O)NH, J = 7.0 Hz, 2H); 1.81-1.11 (m, C12 CH2’s,

lysine CH2’s and C6 linker CH2’s, calc 48 H, found 47 H); 0.83 (t, 2 × C12 CH3, J = 7.0 Hz, 6H). 20

13C NMR (CDCl3, 100 MHz) 177.27, 174.77, 174.52, 172.80 (C(O)NH amide × 3, C(O)OH);

52.81 (α-CH); 39.03, 38.90 (CH2NHC(O) × 2); 36.60, 36.31 (CH2C(O)NH × 2); 33.75

(CH2C(O)OH); 32.02, 31.76, 29.50, 29.47, 29.40, 29.26, 29.24, 29.19, 28.83, 28.54, 25.94, 25.68,

25.59, 24.09, 22.48, 22.42 (C12, lysine and C6 linker CH2’s); 13.87 (C12 CH3 × 2). max (cm-1)

(solid): 3302m, 2970m, 2916s, 2847m, 1736m, 1636s, 1543s, 1458m, 1373m, 1242m, 1157s, 25

1103m. ESI-MS (m/z): Calc. for C36H70N3O5 624.5310; found: 624.5293 (100%, [M+H]+); calc. for

C36H69N3NaO5 646.5129; found: 646.5103 (29%, [M+Na]+).

Synthesis of 3-alcohol. A solution of tetra(ethylene glycol) (27.2 g, 140 mmol) and triethylamine

(15 ml, 108 mmol) in dry THF (100 ml) was cooled to 0°C under nitrogen. To this was added p-30

toluenesulfonyl chloride solution (9.53 g, 50 mmol) in dry THF (10 ml) dropwise over 45 minutes.

The reaction mixture was allowed to warm to rt and stirred for 16 h. The reaction mixture changed

from a white to yellow coloured precipitate solution overnight. The solvent was removed in vacuo

and the yellow residue was dissolved in absolute ethanol (100 ml), then sodium azide (6.5 g, 100

mmol) was added as a solid and the mixture was refluxed for 22.5 h. The solvent was removed in 35


vacuo and the residue diluted with Et2O (250 ml) and washed with brine (50 ml). The organic layer

was separated and the aqueous layer extracted with DCM (3 × 100 ml). The organic fractions were

combined and dried over MgSO4, filtered and the filtrate evaporated to yield a crude, orange oil

(~15 g). Purification by column chromatography (SiO2, 1:1 cyclohexane/EtOAc to 100% EtOAc)

yielded the product as a clear, colourless oil (5.5 g, 50%). Rf 0.25-0.38 (EtOAc, cerium stain). 1H 5

NMR (CDCl3, 400 MHz) 3.73-3.59 (m, CH2O, 14H); 3.39 (t, CH2N3, J = 5.0 Hz, 2H); 2.64 (br s,

OH, 1H). 13C NMR (CDCl3, 100 MHz) 72.47, 70.62, 70.58, 70.51, 70.26, 69.98, 61.59 (7 ×

CH2O); 50.58 (CH2N3). max (cm-1) (oil): 3480w, 2909m, 2870m, 2099s, 1466w, 1350w, 1281m,

1103s, 1065s. ESI-MS (m/z): Calc. for C8H18N3O4 220.1292; found: 220.1294 (100%, [M+H]+).

10

Synthesis of 3-acid. Compound 3-alcohol (2.4 g, 10.96 mmol) and sodium hydride (0.88 g, 21.92

mmol, 2.0 eq) were solubilised in dry THF (~ 25 ml) and stirred at 0°C for 45 minutes. Bromoacetic

acid (1.52 g, 10.96 mmol, 1.0 eq) in dry THF (~ 25 ml) was then added. The reaction was stirred

under nitrogen at rt for 26.5 h. The solvent was removed in vacuo and the residue taken up in water

(100 ml), acidified to pH 2 with 1M HCl (20 ml) and then the aqueous phase was extracted with 15

DCM (3 × 200 ml). The organic fractions were combined, dried over MgSO4, filtered and the

solvent removed in vacuo to yield a crude, colourless oil (~3.2 g). This was purified by column

chromatography (SiO2, 9:1 DCM/MeOH) to yield the product as a colourless oil (~1.8 g, 59%). Rf

0.47 (65:25:4 CHCl3/MeOH/H2O, KMnO4 stain). 1H NMR (CDCl3, 400 MHz) 6.08 (br s,

C(O)OH, 1H); 4.12 (s, CH2C(O)OH, 2H); 3.73-3.59 (m, CH2O, 14H); 3.39 (t, CH2N3, J = 5.0 Hz, 20

2H). 13C NMR (CDCl3, 100 MHz) 172.93 (C(O)OH); 72.47, 70.62, 70.58, 70.51, 70.26, 69.98,

63.50, 61.59 (8 × CH2O); 50.58 (CH2N3). max (cm-1) (oil): 3588w, 2909m, 2870m, 2099s, 1751m,

1466w, 1435w, 1389w, 1350w, 1288m, 1111s. ESI-MS (m/z): Calc. for C10H19N3NaO6 300.1166;

found: 300.1165 (100%, [M+Na]+).

25

Synthesis of Compound 4. Compound 3-acid (78 mg, 0.28 mmol, 1.0 eq) was suspended in DCM

(2 ml) and DIPEA (100 μL, 0.56 mmol, 2.0 eq) was added. The reaction flask was cooled over an

ice-water bath, then TBTU (91 mg, 0.28 mmol, 1.0 eq) was added as a solid. The reaction mixture

was stirred at 0°C for 10 min, then protected RGD peptide (H2N-Arg(Pbf)-Gly-Asp(OtBu)-OtBu)

(200 mg, 0.28 mmol, 1.0 eq) was added as a solid and more DCM (4 ml) was used to rinse the 30

contents of the vial into the reaction flask. The reaction was stirred at 0°C for a further 20 min, then

the ice-water bath was removed and the reaction allowed to warm to rt and stirred for 5 days. The

DCM phase was washed with 1.33 M NaHSO4 (50 ml × 3), 1 M Na2CO3 (50 ml × 3), 1 M HCl (50

ml), water, and finally brine. The organic phase was dried over MgSO4, filtered and the filtrate

evaporated to yield a tacky white foam (0.26 g, 96%). Purification by column chromatography 35


(SiO2, 100% DCM, to 98:2 DCM/MeOH, to 95:5 DCM/MeOH) yielded the product as a white solid

(140 mg, 52%). Rf 0.53 (9:1 DCM/MeOH, UV and cerium stain). [α]D = -2.9 (c = 0.4, CHCl3). 1H

NMR (CDCl3, 400 MHz) 7.82 (app br t, NH amide (Arg-Gly), 1H); 7.44 (d, NH amide, J = 7.5

Hz, 1H); 7.16 (d, NH amide, J = 7.5 Hz, 1H); 6.31 (br s, NH2 guanidine, 2H); 6.18 (br s, NH

guanidine, 1H); 4.61-4.55 (m, Asp α-H and Arg α-H, 2H); 3.96 (s, OCH2C(O)NH, 2H); 3.94 (dd, 5

Gly CHA, J = 17.0 Hz and 5.5 Hz, 1H); 3.86 (dd, Gly CHB, J = 17.0 Hz and 5.5 Hz, 1H); 3.65-3.54

(m, TEG OCH2’s, 14H); 3.31 (t, CH2N3, J = 5.0 Hz, 2H); 3.28-3.20 (br m, Arg CHANH, 1H); 3.18-

3.07 (br m, Arg CHBNH, 1H); 2.89 (s, Pbf CH2, 2H); 2.76 (dd, Asp CHA, J = 17.0 Hz and 5.0 Hz,

1H); 2.63 (dd, Asp CHB, J = 17.0 Hz and 5.0 Hz, 1H); 2.53 (s, Pbf CH3Ar, 3H); 2.45 (s, Pbf CH3Ar,

3H); 2.02 (s, Pbf CH3Ar, 3H); 1.95-1.84 (br m, Arg CHCHA, 1H); 1.71-1.60 (br m, Arg CHCHB, 10

1H); 1.57-1.46 (br m, Arg CH2CH2NH, 2H); 1.40 (s, Pbf CH3 × 2, 6H); 1.359, 1.355 (s × 2,

C(CH3)3 × 2, calc 18H, found 17H). 13C NMR (CDCl3, 100 MHz) 172.20, 170.40, 170.01, 169.65,

169.09, 158.56, 156.52 (C(O)OtBu × 2, C(O)NH amide × 3, Pbf aromatic C-O, C=N guanidine);

138.27, 133.00, 132.21, 124.48, 117.34 (Pbf aromatic C); 86.27 (Pbf CH2C(CH3)2O); 82.37, 81.51

(C(CH3)3 × 2); 71.00, 70.52, 70.48, 70.43, 70.21, 69.91 (TEG OCH2’s and OCH2C(O)NH); 51.89 15

(Arg α-CH); 50.57 (CH2N3); 49.26 (Asp α-CH); 43.20, 42.71 (Pbf ArCH2, Gly CH2); 40.00 (Arg

CH2NH); 37.29 (Asp CH2); 29.66 (Arg CHCH2); 28.56 (Pbf CH2C(CH3)2O); 27.98, 27.81 (C(CH3)3

× 2); 25.30 (Arg CH2CH2NH); 19.28, 17.94, 12.45 (Pbf ArCH3 × 3). max (cm-1) (solid): 3325w,

2978w, 2916w, 2098m, 1736w, 1659m, 1543s, 1450w, 1366w, 1250m, 1142s, 1096s. ESI-MS (m/z):

Calc. for C43H72N9O14S 970.4914; found: 970.4893 (100%, [M+H]+). 20

Synthesis of Compound 5. Compound 4 (120 mg, 0.12 mmol) was dissolved in EtOH (10 ml)

followed by the addition of Pd/C catalyst (24 mg, 20%). The flask was subjected to several

vacuum/H2 purges and then stirred for 3 days under an atmosphere of H2. The catalyst was filtered

off over Celite, washed with MeOH, and the filtrate evaporated in vacuo to yield the product as a 25

clear, colourless oil (~120 mg, quantitative yield). No further purification was required. Rf 0.00 (9:1

DCM/MeOH, cerium stain). [α]D = -11.6 (c = 0.5, CHCl3). 1H NMR (CD3OD, 400 MHz) 4.66-

4.61 (br m, Asp α-H, 1H); 4.49-4.40 (br m, Arg α-H, 1H); 4.14-4.03 (br m, Gly CH2, 2H); 3.91 (s,

OCH2C(O)NH, 2H); 3.78-3.60 (m, TEG OCH2’s, 14H); 3.27-3.09 (br m, CH2NH2 and Arg CH2NH,

4H); 3.00 (s, Pbf CH2, 2H); 2.78-2.66 (m, Asp CH2, 2H); 2.58 (s, Pbf CH3Ar, 3H); 2.51 (s, Pbf 30

CH3Ar, 3H); 2.08 (s, Pbf CH3Ar, 3H); 1.95-1.84 (br m, Arg CHCHA, 1H); 1.80-1.66 (br m, Arg

CHCHB, 1H); 1.66-1.52 (br m, Arg CH2CH2NH, 2H); 1.45, 1.44 (s × 2, Pbf CH3 × 2, C(CH3)3 × 2,

24H). 13C NMR (CD3OD, 100 MHz) 172.76, 171.25, 171.23, 171.10, 159.82, 158.08 (C(O)OtBu

× 2, C(O)NH amide × 3, Pbf aromatic C-O, C=N guanidine); 139.35, 134.35, 133.48, 126.01,

118.41 (Pbf aromatic C); 87.69 (Pbf CH2C(CH3)2O); 83.28, 82.47 (C(CH3)3 × 2); 71.94, 71.83, 35


71.38, 71.27, 71.20, 71.10, 70.92, 70.77, 67.79, 66.84 (TEG OCH2’s and OCH2C(O)NH); 54.00

(Arg α-CH); 50.98 (Asp α-CH); 43.98, 43.25 (Pbf ArCH2, Gly CH2); 41.35 (Arg CH2NH); 40.55

(CH2NH2); 38.25 (Asp CH2); 30.26 (Arg CHCH2); 28.79 (Pbf CH2C(CH3)2O); 28.41, 28.24

(C(CH3)3 × 2); 26.79 (Arg CH2CH2NH); 19.68, 18.51, 12.61 (Pbf ArCH3 × 3). max (cm-1) (oil):

3742w, 2978m, 2886m, 1736w, 1667m, 1543m, 1458w, 1396w, 1366w, 1250m, 1088s. ESI-MS 5

(m/z): Calc. for C43H74N7O14S 944.5009; found: 944.4978 (100%, [M+H]+).

Synthesis of Protected (C12)2-Lys-RGD. Compound 2-acid (77 mg, 0.12 mmol, 1.0 eq) was

suspended in DCM (3 ml) and then DIPEA (50 μL, 0.25 mmol, 2.0 eq) was added. The reaction

flask was cooled over an ice-water bath, then TBTU (49 mg, 0.15 mmol, 1.2 eq) was added as a 10

solid. The reaction mixture was stirred at 0°C for 10 min, then compound 5 (120 mg, 0.12 mmol,

1.0 eq) was dissolved in DCM (2 ml) and added rapidly to the reaction flask, then more DCM (5

ml) was used to rinse the contents of the vial into the reaction flask. The ice-water bath was

removed; the reaction allowed to warm to rt and then refluxed at 40°C for 13 h. The reaction was

diluted with DCM (10 ml), washed with 1.33 M NaHSO4 (50 ml), 1 M Na2CO3 (50 ml), and finally 15

brine. The organic phase was dried over MgSO4, filtered and the filtrate evaporated to yield a brown

foam/solid (~180 mg, 92%). Purification by column chromatography (SiO2, 100% DCM, to 98:2

DCM/MeOH, to 95:5 DCM/MeOH, to 9:1 DCM/MeOH) yielded the product as a white solid (50

mg, 26%). Rf 0.33 (9:1 DCM/MeOH, UV and cerium stain). 1H NMR (CDCl3, 400 MHz) 7.99 (br

s, NH amide, 1H); 7.62 (br s, NH amide, 1H); 7.25, 7.23 (br s, NH amide × 2, 2H); 7.02 (br s, NH 20

amide, 1H); 6.93 (br s, NH amide, 1H); 6.45, 6.38 (br s × 2, NH2 guanidine, NH guanidine, NH

amide, 4H); 4.65-4.54 (m, Asp α-H and Arg α-H, 2H); 4.42-4.33 (m, Lys α-H, 1H); 4.04 (s,

OCH2C(O)NH, 2H); 4.01-3.86 (m, Gly CH2, 2H); 3.74-3.48 (m, TEG OCH2’s, 14H); 3.44-3.30,

3.29-3.08 (m × 2, CH2NHC(O) (Lys), CH2NHC(O) (C6 linker), C(O)NHCH2CH2O, Arg CH2NH,

2H+6H); 2.93 (s, Pbf CH2, 2H); 2.79 (dd, Asp CHA, J = 17.0 Hz and 5.0 Hz, 1H); 2.68 (dd, Asp 25

CHB, J = 17.0 Hz and 5.0 Hz, 1H); 2.56 (s, Pbf CH3Ar, 3H); 2.49 (s, Pbf CH3Ar, 3H); 2.21-2.10 (m,

CH2C(O)NH (C6 linker), CH2C(O)NH × 2 (C12 tails), 6H); 2.06 (s, Pbf CH3Ar, 3H); 1.98-1.04 (m,

C12 CH2’s, lysine CH2’s, C6 linker CH2’s, Arg CH2 × 2, Pbf CH3 × 2, C(CH3)3 × 2, calc 72H, found

75 H); 0.85 (t, 2 × C12 CH3, J = 7.0 Hz, 6H). 13C NMR (CDCl3, 100 MHz) 173.92, 173.88,

172.29, 172.23, 172.17, 170.72, 170.10, 169.73, 168.91, 158.67, 156.63 (C(O)OtBu × 2, C(O)NH 30

amide × 7, Pbf aromatic C-O, C=N guanidine); 138.32, 133.31, 132.23, 124.59, 117.48 (Pbf

aromatic C); 86.40 (Pbf CH2C(CH3)2O); 82.45, 81.64 (C(CH3)3 × 2); 77.47, 70.81, 70.35, 70.19,

70.00 (TEG OCH2’s and OCH2C(O)NH); 53.08, 52.44, 49.38 (Arg α-CH, Lys α-CH, Asp α-CH);

43.35, 42.88 (Pbf ArCH2, Gly CH2); 40.22 (Arg CH2NH); 39.24, 39.19, 38.95, 37.48, 36.79, 36.56,

36.15 (C(O)NHCH2CH2O, Asp CH2, CH2NHC(O) × 2 (Lys and C6 linker), CH2C(O)NH × 2 (C12 35


tails), CH2C(O)NH (C6 linker)); 32.21, 32.12, 31.99, 29.74, 29.71, 29.64, 29.53, 29.49, 29.43,

29.04, 28.90, 28.69, 28.11, 27.96, 26.37, 25.98, 25.86, 25.56, 25.18, 22.76, 22.67 (C12, lysine and

C6 linker CH2’s, Arg CHCH2, Arg CH2CH2NH, Pbf CH2C(CH3)2O, C(CH3)3 × 2); 19.40, 18.08

(Pbf ArCH3 × 2); 14.20 (C12 CH3 × 2); 12.57 (Pbf ArCH3). ESI-MS (m/z): Calc. for

C79H140N10NaO18S 1571.9960; found: 1571.9976 (12%, [M+H]+). Calc. for C79H142N10O18S 5

775.5107; found 775.5089 (100%, [M+2H]2+).

Synthesis of (C12)2-Lys-RGD. Protected (C12)2-Lys-RGD (49 mg, 31.6 μmol) was dissolved in

a mixture of TFA, water and TIS (2 ml, 95:2.5:2.5) and shaken for 1 h 15 min, after which time

TLC indicated that the deprotection reaction was complete. The volatiles were removed in vacuo, 10

then the residue was dissolved in the minimum amount of hot MeOH and precipitated with cold

Et2O, filtered and washed with the minimum amount of cold Et2O to yield a white solid. The

sample was then dissolved in a mixture of water/tBuOH, filtered over a PTFE membrane filter (0.2

μm), shell frozen and lyophilised to yield the product as a fluffy white powder (17 mg, 41% as TFA

salt). Rf 0.00 (9:1 DCM/MeOH, cerium stain). [α]D = -2.0° (c = 0.05, MeOH). 1H NMR (CD3OD, 15

400 MHz) 4.72 (t, Asp α-H, J = 5.5 Hz, 1H); 4.44 (dd, Arg α-H, J = 7.5 Hz and 6.0 Hz, 1H); 4.22

(dd, Lys α-H, J = 9.0 Hz and 5.5 Hz, 1H); 4.06 (s, OCH2C(O)NH, 2H); 3.97 (d, Gly CHA, J = 17.0

Hz, 1H); 3.87 (d, Gly CHB, J = 17.0 Hz, 1H); 3.74-3.56 (m, TEG OCH2’s, 14H); 3.52, 3.34, 3.22-

3.12 (t (J = 5.5 Hz), t (J = 5.0 Hz), m, CH2NHC(O) (Lys), CH2NHC(O) (C6 linker),

C(O)NHCH2CH2O, Arg CH2NH, calc 2H+2H+4H, found 2H+2H+6H); 2.83 (d, Asp CH2, J = 5.5 20

Hz, 2H); 2.25-2.13 (m, CH2C(O)NH (C6 linker), CH2C(O)NH × 2 (C12 tails), 6H); 2.01-1.20 (m,

C12 CH2’s, lysine CH2’s, C6 linker CH2’s, Arg CH2 × 2, calc 48H, found 52 H); 0.88 (t, 2 × C12

CH3, J = 7.0 Hz, 6H). max (cm-1) (solid): 3302m, 2940m, 2870m, 1636m, 1551m, 1450m, 1350m,

1273m, 1242m, 1219m, 1096s. ESI-MS (m/z): Calc. for C58H109N10O15 1185.8068; found:

1185.7971 (12%, [M+H]+). Calc. for C58H110N10O15 593.4071; found 593.4052 (100%, [M+2H]2+). 25

2. Experimental Determination of Critical Aggregation Concentration (CAC)

The Nile Red encapsulation experiment was adapted from literature methods.2 A 2.5 mM Nile Red

(technical grade, Sigma) stock solution was made in EtOH and diluted 1000-fold in the surfactant 30

system (i.e. 1 μL added to a 1 ml surfactant assay volume). A 1 mM stock solution of self-

assembling compound was made in PBS buffer. Aliquots of the stock were taken and diluted to the

desired concentration to make up a 1 ml assay volume in PBS buffer. Nile Red (1 μL) was added

with swirling and the fluorescence emission measured immediately after mixing. Nile Red

fluorescence was measured at room temperature using an excitation wavelength of 550 nm. 35


Fluorescence emission was monitored from 550 to 700 nm at 1 nm intervals. The critical

aggregation concentration (CAC) was calculated from plotting the absorption of Nile Red at 635 nm

(the mean of three independent measurements) against the log of the surfactant concentration,

setting the equations from the two trendlines as equal to one another and solving for at the turning

point. As is log concentration, 10 yields the CAC in mol dm-3. The graphs below present the 5

data for the compounds investigated in this study.

Fig. S1A. Nile Red fluorescence emission intensities at 635 nm plotted against log[C12-RGD]. 10

λex = 550 nm.

Fig. S1B. Nile Red fluorescence emission intensities at 650 nm plotted against log[Py-RGD].

λex = 550 nm. 15

y = 386.7x + 1,763.4

y = 7,638.4x + 27,341.0

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

‐5.00 ‐4.50 ‐4.00 ‐3.50 ‐3.00

I f/ a.u.

log[C12‐RGD] / log M

y = 412.9x + 2,164.3

y = 2290.4x + 9589.7

0

500

1000

1500

2000

2500

3000

‐5.00 ‐4.50 ‐4.00 ‐3.50 ‐3.00

I f/ a.u.

log[Py‐RGD] / log M


Fig. S1C. Nile Red fluorescence emission intensities at 640 nm plotted against log[C22-RGD].

λex = 550 nm.

5

Fig. S1D. Nile Red fluorescence emission intensities at 635 nm plotted against

log[(C12)2-Lys-RGD]. λex = 550 nm.

The photophysical properties of the pyrene unit itself were useful in investigating the phase

separation of pyrene lipids. The fluorescence spectra of various Py-RGD sample concentrations 10

were recorded using an excitation wavelength of 343 nm (Fig. S2) The fluorescence emission

spectra of Py-RGD decreases in intensity as the concentration of Py-RGD increases. This indicates

that self-assembly of the pyrenes in the core of the micelles results in quenching of the pyrene

fluorescence. We therefore hypothesised that the CAC could also be calculated by plotting just the

monomer emission (at 382 nm) against [Py-RGD] (Fig. S3). The CAC (122 μM) was calculated at 15

y = 705,320.81x + 3,993,376.75

y = 7,884,969.92x + 36,537,432.83

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1.40E+07

1.60E+07

1.80E+07

‐6.00 ‐5.00 ‐4.00 ‐3.00

I f/ a.u.

log[C22‐RGD] / log M

y = 731.0x + 4,495.4

y = 6,359.4x + 33,813.0

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

‐6.00 ‐5.50 ‐5.00 ‐4.50 ‐4.00

I f/ a.u.

log[(C12)2‐Lys‐RGD] / log M


the point where the two lines of best fit intersect. Using the direct fluorescence quenching of pyrene

as a fluorescent probe, intrinsic to the Py-RGD molecule, was therefore found to produce a CAC

value which matched quite favourably with that obtained from the Nile Red assay. We argue that

this confirms that using Nile Red as an indirect method is an accurate way of determining the

critical micelle/aggregation concentration of amphiphilic molecules. 5

Fig. S2. Averaged Py-RGD fluorescence emission spectra at different concentrations of Py-RGD.

λex = 343 nm. Experiments were run in triplicate.

10

Fig. S3 Py-RGD monomer emission (at 382 nm) versus [Py-RGD]. λex = 343 nm

0

500

1000

1500

2000

2500

3000

3500

4000

4500

350 400 450 500 550

I f/ a.u.

λ / nm

0mM

25μM

50μM

75μM

100μM

150μM

200μM

250μM

300μM

400μM

500μM

750μM

1mM

y = ‐4E+07x + 5833.9

y = ‐5E+06x + 1510.4

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0.00E+00 2.00E‐04 4.00E‐04 6.00E‐04 8.00E‐04 1.00E‐03 1.20E‐03

I f/ a.u.

[Py‐RGD] / M


3. Fluorescence Polarisation Assay

The FP competition experiment was adapted from the literature method.3 For the competition

assay, a solution of 187 μM cyclic peptide 5(6)-FL-c[RGDfK]1,3 in PBS buffer (0.01 M phosphate,

pH 7.4, 0.138 M NaCl, 0.0027 M KCl) was diluted with Tris buffer (50 mM TRIS, pH 7.4, 1 mM 5

CaCl2, 10 μM MnCl2, 1 mM MgCl2, 100 mM NaCl) to give a 100 nM stock. The assay mixture

(200 μl in a 100 μl volume microcuvette) was composed of 280 nM integrin αvβ3 (13.25 μg) and 10

nM 13 in TRIS buffer. The cuvette was incubated at 29°C for 5 min and then the single-point

fluorescence polarisation was obtained using λex = 485 nm, λem = 510 nm. 5(6)-FL-c[RGDfK]

alone (10 nM) served as control and all subsequent data with the protein present was normalised to 10

100 mP units using this value. All data points (Fig. 5, main paper) are presented as mean values ±

standard deviations from at least 5 independent scans. Competition experiments were performed

with the synthetic ligands, with PEG-GGG (previous work)1 and SDS serving as negative controls.

Stocks of these compounds were made in PBS and diluted in the TRIS assay buffer to the desired

concentration in the final stock titrant (100 μl) which also contained 280 nM integrin αvβ3 (6.625 15

μg) and 10 nM 5(6)-FL-c[RGDfK], then incubated at 29°C in a water bath for at least 5 min before

carrying out the titration experiment. The titrant was added to the assay mixture in microlitre

aliquots, the cuvette shaken and incubated at 29°C for at least 5 min, and then the single-point FP

value recorded, while incubation of the stock titrant was resumed at 29°C in between titrations. The

mP values were plotted against ligand concentration in Excel and the effective concentration at 20

which 50% displacement of the probe 5(6)-FL-c[RGDfK]’s binding to integrin αvβ3 was achieved

(EC50) was extrapolated at 50 mP units from the normalised data. As stated in the Results and

Discussion, this methodology is robust for the direct comparison of structurally related ligands, and

avoids the use of radiolabel assays.

25

4. Computational methods

Multiscale molecular modeling concepts. Scale integration in specific contexts in the field of

(bio)molecular modeling can be done in different ways. Any recipe for passing information from

one scale (usually lower) to another (higher) scale is based on the proper definition of many-scale 30

modeling which considers objects that are relevant at that particular scale, disregards all degrees of

freedom of smaller scales, and summarizes those degrees of freedom by some representative

parameters. Specifically, the multiscale modeling strategy developed by our group is based on the

systematic elimination of computationally expensive degrees of freedom while retaining implicitly

their influence on the remaining degrees freedom in the mesoscopic model. Accordingly, using the 35

information obtained from atomistic molecular dynamics (MD) simulations we parameterized the


coarse-grained (e.g., Dissipative Particle Dynamics (DPD), vide infra) models that incorporate all

essential physics/phenomena observed at the finer level. At the coarse-grained (mesoscopic) level,

we employed the corresponding most accurate and effective methods/simulation techniques

available to investigate physical properties of each system at that level. The outline of the general

strategy of our multiscale modeling approach is as follows: 1) extensive explicit solvent atomistic 5

MD calculations on model compounds are carried out. These simulations provide us with dynamic

properties/energies that help us to identify important interactions/correlations among system

components which are to be used per se and/or exploited to parameterize the next scale (mesoscale)

simulation models; (2) using conformational and structural properties obtained from MD

simulations at point (1) we parameterize the DPD model in which each segment represented as 10

single force centres (beads) and solvent is treated explicitly in the presence of ions and counterions.

Langevin dynamics are then conducted using the DPD representation of the system. These

simulations are orders of magnitude computationally less expensive than simulations with the

corresponding atomistic MD models, allowing us to simulate more realistic systems and to

significantly extend the accessible time scales. Most importantly, these type of simulations yield 15

information on the morphology of the systems under investigation in a length scale (L) range of 1

L 1000 nm; while extending the time scale up to seconds, that is, where most of the critical

energetic and structural phenomena involved in several aspects of the performance of these systems

take place.

20

Atomistic molecular dynamics simulations the RGD derivatives. All simulations and data

analysis were performed with the AMBER 11 suite of programs.4 All RGD compound models were

built and geometry optimized using the Antechamber module of AMBER 11 and the GAFF force

field.5 A suitable number of molecules for each compound were then solvated in a TIP3P6 water

box to generate a bulk system with concentration lower than the corresponding experimental CAC 25

value. Then, the required amount of Na+ and Cl- ions were added to neutralize the system and to

mimic an ionic strength of 150mM, removing eventual overlapping water molecules. The solvated

molecules were subjected to a combination of steepest descent/conjugate gradient minimization of

the potential energy, during which all bad contacts were relieved. The relaxed systems were then

gradually heated to 300 K in three intervals by running constant volume-constant temperature 30

(NVT) molecular dynamics (MD) simulation, allowing a 0.5 ns interval per each 100 K.

Subsequently, 10 ns MD simulations under isobaric-isothermal (NPT) conditions were conducted to

fully equilibrate each solvated RGD derivative system. The SHAKE algorithm7 with a geometrical

tolerance of 510-4 Å was imposed on all covalent bond involving hydrogen atoms. Temperature

control was achieved using the Berendsen et al. coupling algorithm8 with separate coupling of the 35


solute and solvent to the heat (0.5 ps time constant for heat bath and 0.2 ps pressure relaxation time)

and an integration time step of 2 fs. At this point, these MD runs were followed by other 10 ns of

NVT MD data collection runs, necessary for the estimation of the interaction energies (vide infra),

were carried out using a bath heat coupling of 1 ps. The particle mesh Ewald method9 was used to

treat the long-range electrostatics. For the calculation of the interaction energies, 1000 snapshots 5

were saved during the MD data collection period described above, one snapshot per each 10 ps of

MD simulation.

All of the production molecular dynamics (MD) simulations were carried out by using the Sander

and Pmemd module within the AMBER 11 platform working in parallel on 256 processors of the 10

IBM PLX calculation cluster of the CINECA supercomputer centre (Bologna, Italy). All energetic

analyses were performed by running the Ptraj module of AMBER 11 on the 10 ns MD trajectories

of each RGD system considered. The entire MD simulation and data analysis procedure was

optimized by integrating AMBER 11 in modeFRONTIER, a multidisciplinary and multi-objective

optimization and design environment. 15

(http://www.esteco.com/home/mode_frontier/mode_frontier.html).

At the atomistic level, the underlying procedure used to calculate the interaction energies between

all components of a given molecular system is well established.10 Accordingly, we started from the

concept that the total potential energy of a binary system composed, for instance, by salted water 20

molecules (W) and RGD derivatives (RGD) may be written as: E(W/RGD) = EW + ERGD + EW/RGD,

where the first two terms represent the energy of water and the RGD derivatives (consisting of both

valence and non-bonded energy terms), and the last term is the interaction energy between the two

component pair (made up of non-bonded terms only). By definition, the binding energy between

the RGD molecules and water is the negative of the corresponding interaction energy, i.e., 25

E / E / The binary binding energy EW/RGD values were directly obtained from the

corresponding equilibrated MD trajectories. Next, we deleted the water molecules, leaving the RGD

derivatives alone, and thus calculated the energy of the RGD molecules, ERGD. Similarly, we

deleted the RGD derivative molecules from the water/RGD compound system, and calculated EW.

Then, the binding energy E / could be calculated as: E / / . As the 30

MD frame choice is concerned, we decided to calculate the system energies at every 500 ps. All

data collected have then been averaged over 10 different model systems for each RGD molecule.

Dissipative Particle Dynamics (DPD) theory. The original DPD model11 consists of a collection

of soft repelling frictional and noisy balls. From a physical point of view, each dissipative particle 35


is regarded not as a single molecule of the fluid but rather as a collection of molecules that move in

a coherent fashion. In that respect, DPD can be understood as a coarse-graining of molecular

dynamics. There are three types of forces between dissipative particles. The first type is a

conservative force deriving from a soft potential that tries to capture the effects of the “pressure”

between different particles. The second type of force is a friction force between the particles that 5

wants to describe the viscous resistance in a real fluid. This force tries to reduce velocity differences

between dissipative particles. Finally, there is a stochastic force that describes the degrees of

freedom that have been eliminated from the description in the coarse-graining process. This

stochastic force will be responsible for the Brownian motion of (macro)molecules and colloidal

particles simulated with DPD. The postulated stochastic differential equations that define the DPD 10

model are:

1

∙ 2

In the above equations, ri and vi are the position and velocity of the dissipative particles (also called 15

beads), mi is the mass of particle i, is the conservative repulsive force between two DPD beads i

and j, rij = ri − rj , vij = vi − vj, and the unit vector from the jth particle to the ith particle is eij = (ri −

rj)/rij with rij = |ri − rj |. The friction coefficient governs the overall magnitude of the dissipative

force, and is a noise amplitude that governs the intensity of the stochastic forces. Finally, the

weight function (r) provides the range of interaction for the dissipative particles and renders the 20

model local in the sense that the particles interact only with their neighbors.12

It is essential to recall here the following, fundamental properties of DPD calculations: i) the above

DPD stochastic differential equations are translationally, rotationally and Galilean invariant, and ii)

most importantly, total momentum is conserved, ∑ / 0, because the three types of 25

forces satisfy Newton’s Third Law. Therefore, the DPD model captures the essentials of mass and

momentum conservation which are responsible for the hydrodynamic behavior of a fluid at large

scales.11e,f

30


DPD modeling. Molecules in DPD are built by tying DPD beads together using Hookean springs

with the potential given by:

, 112

3

where i, i+1 label adjacent beads in the molecule. The spring constant, kbb, and unstretched length 5

l0, are chosen so as to fix the average bond length to a desired value. Chain stiffness is modelled by

a three body potential acting between adjacent bead triples in a row:

1, , 1 1 4

in which the angle is defined by the scalar product of the two bonds connecting the pair of 10

adjacent beads i-1, i, and i+1.

Furthermore, in order to correctly derive the electrostatic interactions between charged beads (i.e.

present on the RGD compounds and ions), the electrostatic force between two charged beads i

and j was analyzed following the approach reported in Groot’s work.13 According to this study, the 15

electrostatic field is solved by smearing the charges over a lattice grid, the size of which is

determined by a balance between the fast implementation and the correct representation of the

electrostatic field.

The exact mapping from one bead to a group of atoms or molecules in DPD depends on the size of 20

the ensemble of atoms/molecules the bead is to represent. The physical masses of all beads (m) in a

DPD simulation are usually assumed to be identical, as are their size (rc). Note that this length rc is

the distance at which all nonbonded bead-bead forces vanish, and it corresponds to the diameter of a

bead if one pictures two beads at the extreme of their interaction range as two spheres the surfaces

of which just touch. The remaining physical unit required to convert dimensionless quantities to 25

physical quantities is a time scale. This can be extracted from the time scale of a relevant process in

the simulated system.

Concerning modeling the RGD derivatives mesoscale architectures, we modelled the different

compounds at a coarse-grained level using branched and flexible amphiphilic chains made up of 9 30

bead types: 4 neutral bead types C, C1, C2, and PY as the hydrophobic chains building block for

C12-RGD, Py-RGD, C22-RGD and (C12)2-Lys-RGD, another neutral bead P, as the chain


building block for the pegylated portion of (C12)2Lys-RGD, two neutral beads G and R for glycine

and the uncharged part of the side chain of arginine, a positively charged bead type Rc as the

terminal charged head of arginine, and a further negatively charged bead D for the anionic part of

aspartic acid. According to the chemical structures of the different molecules, and satisfying the

conditions of the matching between the pair-pair correlation functions obtained from the MD and 5

the DPD simulation for each compound,14 the coarse-grained models of each RGD derivative were

obtained, as shown in Figure S4.

Solvent molecules were simulated by single bead types W, and an appropriate number of

counterions of a charge of ± 1 were added to preserve charge neutrality and to account for the 10

experimental solution ionic strength. The inclusion of explicit counterions was necessary because

counterion condensation and the interactions between the counterions and the charged groups may

affect the complex structure to a certain extent.

15

Fig. S4. Schematic representation of the coarse-grained DPD models of the hydrophobically-

modified RGD derivatives considered in this work.

20

Having set the reference volume of one bead and the density of the system to a value of ρ = 3, then

the cut-off radius rc, which sets the length scale of the system (vide infra), corresponds

approximately to 0.66 nm. All simulations were performed in a 3D-periodic cubic/rectangular box,

equivalent to a maximum real volume of 79103 nm3. The appropriate number of RGD derivative

molecules was added to the simulation box in order to fit experimental concentrations. The 25

characteristic time scale for the present systems was then defined matching the experimental and


simulated diffusivity for water molecules.15 Accordingly, the physical time scale unit in the present

simulations was τ ≈ 0.009 ns. Up to 8 × 106 time steps were performed during DPD run, thus

allowing for a maximum physical time of 40 s.

Once all DPD beads are defined, they need to interact. The intra- and intermolecular interactions 5

between DPD particles are expressed by the conservative parameter aij, defined in the conservative

force expression:

1 5

which is for rij < rc and is zero otherwise. The range of is then set by rc, and aij is the maximum 10

force between beads of type i and j. rij is the distance between the centers of beads i and j, and rij is

the unit vector pointing from bead j to bead i. The fundamental DPD parameter aij can be estimated

by different approaches, mostly relying on the Flory-Huggins theory and the determination of the

Flory interaction parameter , or determined from chemical interaction energies. Since aij accounts

for the underlying chemistry of the system considered, in this work we employed a well-validated 15

strategy that correlates the interaction energies estimated from atomistic MD simulations to the

mesoscale aij parameter values.14 Following and adapting this computational recipe to the present

case, the interaction energies between the solvated RGD derivatives estimated using the MD-based

procedure described above were rescaled onto the corresponding mesoscale segments. The bead-

bead interaction parameter for water was set equal to aww = 25 in agreement with the correct value 20

of DPD density = 3.11g The maximum level of hydrophobic/hydrophilic repulsion was captured

by setting the interaction parameter aij between the water bead W and the hydrophobic aromatic

bead PY as 79. The counterions were set to have the interaction parameters of water. Once these

parameters were assigned, all the remaining bead-bead interaction parameters for the DPD

simulations were easily obtained, starting from the atomistic interaction energies values, as 25

described in our previous work.14 The entire set of DPD interaction parameters employed in this

work are summarized in Table S1.


Table S1. DPD bead-bead interaction parameters obtained for the hydrophobically-modified RGD

compounds in water.

C C1 C2 D G P Py R Rc W

C 25

C1 26 25

C2 - - 25

D 70 72 73 34

G 52 53 56 30 25

P 43 46 - 33 29 25

PY 27 - - 76 74 72 25

R 67 69 72 33 37 40 71 25

Rc 69 73 74 36 39 42 74 29 32

W 69 73 78 20 29 31 79 19 18 25

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13 R. D. Groot J. Chem. Phys. 2003, 118, 11265-11277.

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30


Protected Py-RGD (Py-Arg(Pbf)-Gly-Asp(OtBu)-OtBu) – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.08.0

0.00

0.50

1.00

1.50

2.008.

190

8.07

28.

065

8.05

57.

991

7.97

07.

927

7.90

97.

890

7.85

87.

742

7.72

27.

412

7.39

37.

260

7.13

57.

124

6.44

76.

292

5.25

84.

685

4.67

34.

665

4.66

14.

653

4.64

04.

605

4.58

74.

572

4.55

43.

992

3.98

03.

359

3.25

73.

238

3.21

93.

201

3.18

83.

173

3.10

22.

791

2.77

42.

758

2.74

52.

704

2.69

22.

661

2.64

92.

567

2.45

42.

372

2.35

42.

336

2.14

52

129

210

82

090

203

81

959

190

11

826

136

41

354

134

7

1.000.95

1.80

2.030.77

0.93

0.94

0.972.032.08

1.033.99

3.78

2.061.102.802.791.88

2.032.710.951.052.0022.79

CH2Cl 2

NH

HN

O

NH

NH2NS

O O

O

NH

O

O

O

OOOHAHB HA HB


Protected Py-RGD (Py-Arg(Pbf)-Gly-Asp(OtBu)-OtBu) – 13C NMR

5

ppm (t1)050100150200

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

173.

5817

2.90

169.

9816

9.81

169.

2216

5.69

158.

65

156.

5813

8.28

135.

8713

2.90

132.

2113

1.30

130.

8212

9.78

128.

6012

7.44

127.

2812

7.25

126.

5412

5.74

124.

9312

4.87

124.

7712

4.69

124.

5812

3.32

117.

4286

.30

82.3

981

.45

77.4

877

.16

76.8

4

53.5

052

.85

49.3

8

43.0

842

.81

40.3

338

.57

37.3

435

.73

32.7

529

.69

29.4

828

.50

27.9

727

.80

27.3

625

3719

3112

48


Py-RGD – 1H NMR

5

ppm (t1)1.02.03.04.05.06.07.08.0

0.0

1.0

2.0

3.0

4.0

8.18

88.

165

8.04

58.

024

8.00

27.

981

7.97

57.

956

7.87

97.

861

7.84

17.

773

7.75

3

4.77

1

4.35

94.

343

4.32

63.

993

3.95

13.

933

3.89

13.

314

3.31

03.

306

3.27

93.

261

3.24

03.

166

3.15

13.

137

2.90

12.

888

2.87

72.

451

2.43

42.

416

2.13

02.

111

2.09

32.

074

1.93

61

787

155

9

1.00

2.12

1.972.09

2.21

2.14

2.05

1.15

3.18

0.984.00

1.052.97

H2O

(CH3CH 2) 2O

(CH3CH2) 2O


Py-RGD – 13C NMR

5

ppm (t1)050100150200

0.0

1.0

2.0

3.0

4.0

5.0

176.

0717

4.62

174.

2917

4.02

171.

1115

7.77

136.

7613

2.11

131.

6313

0.64

129.

3412

8.13

127.

9912

7.29

126.

5512

5.64

125.

5412

5.43

124.

00

78.7

778

.45

78.1

354

.22

49.7

549

.64

49.4

349

.21

49.0

048

.79

48.5

748

.36

43.1

6

41.4

736

.53

36.1

833

.44

29.5

128

.22

25.5

4

(CH3CH

2) 2O

(CH3CH2) 2O


Protected C22-RGD (C22-Arg(Pbf)-Gly-Asp(OtBu)-OtBu) – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.08.0

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

7.85

57.

846

7.34

87.

328

7.26

06.

967

6.95

56.

392

6.23

54.

664

4.65

24.

643

4.63

94.

631

4.61

94.

567

4.54

84.

534

4.51

54.

032

3.96

63.

955

3.86

93.

358

3.11

52.

928

2.81

42.

801

2.77

92.

772

2.75

92.

705

2.69

32.

662

2.65

02.

552

2.48

22.

212

2.19

42.

174

2.06

21.

953

1.81

31.

756

1.62

21.

490

1.43

51.

397

1.39

31

318

121

80

856

083

9

1.000.97

1.88

1.81

1.922.841.072.863.04

1.882.940.961.283.725.6217.4137.10

3.27

0.84

0.86

0.87

2.78


Protected C22-RGD (C22-Arg(Pbf)-Gly-Asp(OtBu)-OtBu) – 13C NMR

5

ppm (t1)050100150

0.0

5.0

174.

1117

2.99

170.

0816

9.89

169.

2815

8.76

156.

66

138.

41

132.

9513

2.31

124.

61

117.

52

86.3

982

.53

81.5

977

.48

77.1

676

.84

52.7

049

.43

43.3

242

.88

40.3

038

.68

37.4

336

.46

32.0

029

.79

29.7

429

.68

29.5

129

.44

28.6

828

.11

27.9

425

.76

25.3

722

.77

1941

1805


5

C222-RGD – 1H NM

MR


C22-RGD – 13C NMR

5


1-ester – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.0

0.00

0.50

1.00

1.50

7.26

0

6.41

16.

392

6.02

26.

009

5.99

6

4.52

24.

511

4.50

24.

491

4.48

24.

470

3.69

93.

683

3.23

03.

213

3.19

73.

181

3.16

73.

151

3.13

42.

210

2.19

22.

171

2.13

52.

117

2.09

61.

846

1.73

31.

715

1.62

21.

529

1.50

91.

410

1.36

41.

245

1.23

11.

208

083

00

812

1.00

2.03

3.40

0.97

0.93

2.052.04

6.29

0.845.121.82

37.02


1-ester – 13C NMR

5

ppm (t1)050100150200

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0173.

671

173.

449

173.

106

77.4

7977

.160

76.8

42

52.3

4051

.768

38.6

35

36.8

1136

.499

31.9

4931

.820

29.6

8529

.663

29.5

8129

.464

29.4

3529

.387

29.3

5628

.927

25.9

1525

.708

22.7

2022

.354

14.1

49


1-acid – 1H NMR

5

ppm (t1)0.05.010.0

0.00

0.50

1.0010.1

71

7.26

06.

820

6.80

26.

110

6.09

66.

082

4.57

04.

558

4.55

24.

540

4.53

34.

521

3.35

33.

335

3.31

83.

301

3.28

43.

267

3.22

03.

206

3.19

13.

172

3.15

73.

148

3.14

32.

264

2.24

72.

226

2.20

22.

184

2.16

41.

928

1.82

81.

821

1.72

31.

670

1.55

91.

489

1.43

41

242

085

0

1.00

1.011.04

1.00

0.98

2.11

1.976.3736.88

6.18

2.10

0.28


1-acid – 13C NMR

5

ppm (t1)050100150

0.0

1.0

2.0

3.0

4.0

174.

6017

4.47

174.

36

77.4

777

.15

76.8

3

52.2

539

.09

36.8

636

.61

32.0

331

.67

29.7

729

.67

29.6

029

.46

29.0

6

25.9

725

.84

22.7

822

.36

14.1

7


2-ester – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.0

0.00

0.50

1.00

1.507.73

77.

734

7.72

47.

713

7.71

17.

596

7.57

9

4.67

9

4.27

34.

216

3.64

2

3.18

4

3.11

72.

326

2.30

72.

289

2.22

12.

203

2.18

42.

154

2.13

52.

116

1.78

8

1.09

20.

868

0.85

30.

836

1.00

2.98

3.96

1.931.941.87

1.096.783.99

35.61

5.72

0.780.81

NH

HN

O

NH

O

O O

O


2-ester – 13C NMR

5

ppm (t1)50100150

0.0

1.0

2.0

3.0

4.0

5.0

6.0

174.

6017

4.42

174.

3517

2.29

77.4

877

.16

76.8

452

.68

51.2

149

.02

48.8

148

.59

48.3

848

.17

47.9

547

.74

38.9

038

.53

36.1

635

.95

33.5

531

.62

31.5

129

.32

29.2

429

.05

28.5

528

.44

26.0

225

.69

25.5

024

1922

5022

3613

62

NH

HN

O

NH

O

O O

O


2-acid – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.0

0

10000

20000

30000

400007.32

0

7.26

07.

256

7.11

7

6.37

6

4.47

84.

460

4.44

24.

424

3.32

6

3.07

8

2.30

92.

291

2.27

42.

194

2.17

62.

155

2.15

12.

131

2.11

11.

810

1.23

11.

206

1.10

8

0.84

80.

831

0.81

4

0.90

1.102.82

0.80

0.73

0.75

1.923.75

0.52

10.06

35.70

6.00


2-acid – 13C NMR

5

ppm (t1)050100150200

0

50000

10000

15000

20000

25000

30000

177.

2717

4.77

174.

52

172.

81

77.4

877

.16

76.8

452

.81

39.0

338

.90

36.6

036

.32

33.7

5

32.0

331

.77

29.5

129

.48

29.4

129

.26

29.2

529

.20

28.8

428

.54

25.9

425

.69

25.5

924

.09

2249

2243

1387


3-alcohol – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.0

0

50000

10000

7.26

0

3.73

33.

518

3.34

63.

327

3.30

9

2.89

1

2.00

0.86

11.991.80


3-alcohol – 13C NMR

5

ppm (t1)050100150200

0

50000

10000

15000

20000

25000

77.6

3277

.160

76.6

8772

.471

70.6

1970

.577

70.5

1470

.260

69.9

7961

.590

50.5

78


3-acid – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.26

0

6.07

9

4.11

63.

730

3.71

93.

707

3.67

83.

667

3.66

03.

652

3.64

23.

609

3.59

73.

586

3.45

13.

399

3.38

53.

373

2.00

14.13

1.51

0.70

1.10


3-acid – 13C NMR

5


Compound 4 – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.08.0

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

3.00

7.80

1

7.44

67.

427

7.25

47.

166

7.14

76.

308

6.17

7

5.24

74.

612

4.60

04.

587

4.58

04.

567

4.55

13.

974

3.95

63.

932

3.91

83.

887

3.87

33.

845

3.83

13.

645

3.61

73.

607

3.59

53.

589

3.58

23.

539

3.32

63.

313

3.30

13.

284

3.20

23.

181

3.07

22.

893

2.78

72.

774

2.74

42.

732

2.65

32.

642

2.61

12.

599

2.52

72.

451

2.02

41.

952

160

11

570

145

81

397

135

91

355

2.00

0.85

0.88

0.90

1.980.82

2.901.00

14.07

2.160.801.06

2.361.011.032.902.97

2.871.02

1.022.055.7716.820.73

CH2Cl 2


Compound 4 – 13C NMR

5

ppm (t1)050100150200

0.0

1.0

2.0

3.0

4.0

5.0

172.

2117

0.41

170.

0116

9.66

169.

10

158.

5715

6.52

138.

28

133.

0113

2.21

124.

48

117.

34

86.2

782

.38

81.5

277

.48

77.1

676

.84

71.0

070

.52

70.4

970

.43

70.2

169

.91

53.5

0

51.8

950

.57

49.2

743

.20

42.7

140

.01

37.2

929

.67

28.5

727

.98

27.8

225

.30

1928

1245


Compound 5 – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.08.0

0.00

0.50

1.00

1.50

2.00

2.504.

856

4.66

34.

648

4.63

44.

628

4.62

14.

613

4.49

24.

453

4.44

54.

433

4.40

44.

138

4.09

84.

094

4.07

34.

065

4.02

73.

907

3.77

53.

757

3.74

53.

732

3.69

03.

668

3.65

13.

615

3.59

83.

314

3.31

03.

306

3.26

63.

220

3.20

13.

185

3.16

33.

150

3.12

63.

108

3.09

02.

997

2.77

82.

764

2.73

62.

720

2.70

32.

677

2.66

12.

576

2.51

22.

077

1.94

51.

837

179

81

664

151

71

445

144

0

1.111.09

24.002.34

1.181.28

3.02

3.013.06

2.14

2.064.15

2.082.04

13.93

0.59

0.890.40

CH2Cl 2


Compound 5 – 13C NMR

5

ppm (t1)050100150

0.0

1.0

2.0

3.0

4.0

172.

76

171.

2617

1.24

171.

10

159.

8315

8.08

139.

35

134.

3513

3.48

126.

02

118.

41

87.6

983

.28

82.4

871

.94

71.8

371

.38

71.2

771

.21

71.1

170

.93

70.7

767

.80

66.8

4

54.0

0

50.9

849

.69

49.4

849

.26

49.0

548

.84

48.6

248

.41

43.9

843

.25

41.3

640

.55

38.2

6

30.2

7

28.7

928

.41

28.2

426

7918

5112

62


Protected (C12)2-Lys-RGD – 1H NMR

5

ppm (t1)0.01.02.03.04.05.06.07.08.0

0.00

0.50

7.24

87.

227

7.02

06.

933

6.45

26.

378

4.65

34.

640

4.63

24.

628

4.62

04.

608

4.57

84.

565

4.54

44.

420

4.40

14.

386

4.36

64.

351

4.33

44.

042

4.01

34.

011

4.00

43.

994

3.96

13.

949

3.93

53.

918

3.90

53.

877

3.86

33.

742

3.69

93.

687

3.65

93.

648

3.62

93.

589

3.54

63.

533

3.52

13.

476

3.44

23.

390

3.37

73.

304

3.28

83.

202

3.18

83.

169

3.15

23.

080

2.92

72.

815

2.80

32.

773

2.76

02.

701

268

92

658

264

62

564

249

12

205

218

52

142

213

52

116

206

11

982

155

51

486

147

11

432

140

21

397

128

31

266

122

00

866

085

00

832

6.00

1.86

0.74

3.71

14.16

2.165.67

1.951.421.022.872.98

5.002.951.01

35.76

37.84

0.73

0.75

1.910.650.72

3.74

0.16


Protected (C12)2-Lys-RGD – 13C NMR

5

ppm (t1)050100150

0.00

0.50

1.00

1.50

2.00

2.50

173.

9317

3.88

172.

3017

2.24

172.

1717

0.73

170.

1016

9.74

168.

9215

8.68

156.

6313

8.33

133.

3113

2.23

124.

5911

7.48

86.4

082

.45

81.6

477

.48

77.3

777

.16

76.8

470

.81

70.5

770

.36

70.2

070

.01

53.0

952

.45

49.3

843

.35

42.8

940

.23

39.2

539

.20

38.9

6

37.4

936

.79

36.5

636

.16

32.2

232

.12

31.9

929

.74

29.7

129

.65

29.5

329

.50

29.4

329

.04

28.9

128

.69

2812

2796

2637

2599

2586

2518

2276

2268

1808

1421

1257


(C12)2-Lys-RGD – 1H NMR

ppm (t1)0.01.02.03.04.05.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

4.73

64.

439

4.43

54.

420

4.24

14.

227

4.21

94.

205

4.06

43.

986

3.94

43.

886

3.84

43.

735

3.72

03.

707

3.68

73.

677

3.65

33.

642

3.63

53.

629

3.61

73.

607

3.56

43.

534

3.52

03.

506

3.35

23.

339

3.32

53.

219

3.20

33.

186

3.15

63.

148

3.14

03.

123

2.84

32.

829

2.24

92.

231

2.21

12.

204

2.18

52.

166

2.14

72.

127

2.00

81.

907

185

41

460

143

41

201

089

70

880

5.61

1.16

1.09

0.98

2.301.191.23

14.00

2.18

2.03

6.32

2.12

5.73

1.25

15.71

35.12

H2O


Self-assembled multivalent RGD-peptide arrays – Morphological … · 2013-04-04 ·...

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