self-assembled multivalent rgd-peptide arrays – morphological … · 2013-04-04 ·...
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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:Sabrina.Pricl@di3.units.it 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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
= -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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
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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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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,
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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
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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
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(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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
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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
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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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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.
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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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30
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
5
C222-RGD – 1H NM
MR
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
C22-RGD – 13C NMR
5
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
3-acid – 13C NMR
5
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
(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
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013
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