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Supplementary Data
Optimization of RGD containing cyclic peptides against αvβ3 integrin
Y. Wang, W. Xiao, Y. Zhang, L. Meza, H. Tseng, Y. Takada, J. B. Ames, KS
Lam
General methods for library synthesis
S2
Table S1. MS and purity for cyclic peptide 1 derivatives
S2
Synthesis of focused libraries
S4
Table S2. Amino acids used in the synthesis of focused Library 1 and 2
S6
Table S3. Amino acids used in the synthesis of focused Library 1.
S7
Table S4. Amino acids used in the synthesis of focused Library 2
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NMR study of peptide 1 and 4
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Experimental Constraints and Structure Calculation
S14
Table S5. Chemical shifts of peptide 1 at 295K, DMSO-d6.
S6
Table S6. Chemical shifts of peptide 4 at 295K, DMSO-d6.
S15
Table S7. Structure statistics for the ensembles of 15 calculated structures
S16
of peptide 1 and peptide 4
Figure S1. Representative regions of the 2D NOESY spectrum of 1 and 4.
S17
Figure S2. Superimposed solution structures are the ensembles of 15
S18
structures for peptide 1 and 4.
In vivo and ex vivo optical imaging
S19
Abbreviations
S19
Reference
S21
S1
General methods for library synthesis
Coupling completeness and Fmoc deprotection were monitored by Kaiser test. For
Fmoc deprotection, beads were incubated with 20% 4-methyl piperidine solution in
DMF for twice (5mim, 15min) and then thoroughly washed with DMF, MeOH and
DMF three times each, respectively. For Alloc deprotection, the resulting beads were
incubated with (Pd(PPh3)4) (0.2 eqiv) and (PhSiH3) (20eqiv) in DCM for twice
(30min each time), and washed with 0.5% diethyldithiocarbamic acid sodium salt in
DMF (3 times) and DMF (8 times). For library synthesis, a typical synthetic cycle is
described as follows: beads were split into aliquots as desired, and each aliquot of
beads were coupled with a specific Fmoc-protected amino acid in the presence of DIC
and HOBt for 2 hours. All aliquots of beads were then mixed together and washed
with DMF five times.
Table S1. MS and purity for cyclic peptide 1 derivatives. (All derivatives were
cyclized through disulfide bond.)
Peptide No.
Puritya
MSb (calcd./found [M+1]+)
1
cGRGDdvc-NH2 99%
820.2951/ 821.3032
2
CGRGDdvc-NH2 99%
820.2956/821.4890
3
cGRGDdvC-NH2 99%
820.2956/821.5106
4
CGRGDdvC-NH2 99%
820.2956/821.3802
5
DPen-GRGDdvc-NH2 99%
848.3269/849.3022
6
cGRGDdv-DPen-NH2 99%
848.3269/849.3100
7
DPen-GRGDdv-DPen-NH2 99%
876.3582/877.6212
S2
8
caRGDdvc-NH2 99%
834.3113/835.2645
9
c-βala-RGDdvc-NH2 99%
834.3113/835.5603
10
cG-HoArg-GDdvc-NH2 99%
834.3113/835.4362
11
cG-Agb-GDdvc-NH2 99%
806.2800/807.2740
12
cG-Agp-GDdvc-NH2 99%
792.2643/793.3561
13
cGRGDd-DAbu-c-NH2 99%
876.3582/877.6212
14
cGRGDdic-NH2 99%
834.3113/835.7302
15
cGRGDd-DAgl-c-NH2 99%
818.2800/819.3598
16
cGRGDd-DPra-c-NH2 99%
816.2643/817.4833
17
cGRGDd-DBug-c-NH2 99%
834.3113/835.5552
18
Ac-cGRGDdvc-NH2 98%
863.2902/864.3522
19
R2-cGRGDdvc-NH2 99%
890.3375/891.4310
20
R3-cGRGDdvc-NH2 99%
904.3531/905.4380
21
R4-cGRGDdvc-NH2 99%
944.3844/945.4463
22
R5-cGRGDdvc-NH2 95%
992.2116/993.1967
23
R6-cGRGDdvc-NH2 99%
952.3531/953.4521
24
R7-cGRGDdvc-NH2 99%
972.2985/973.3254
25
R8-cGRGDdvc-NH2 95%
969.3069/970.5460
26
R9-cGRGDdvc-NH2 99%
956.3117/957.3084
27
R10-cGRGDdvc-NH2 99%
992.3092/993.3198
28
R11-cGRGDdvc-NH2 99%
945.2891/946.4421
S3
a
29
R12-cGRGDdvc-NH2 96%
925.3171/926.2625
30
R13-cGRGDdvc-NH2 96%
974.3375/975.2495
31
R14-cGRGDdvc-NH2 97%
1000.3531/1001.4553
32
c-Sar-RGDdvc-NH2 97%
834.3113/835.4168
33
cG-(NMe)Arg-GDdvc-NH2 97%
834.3113/835.2916
34
cGR-Sar-Ddvc-NH2 95%
834.3113/835.4901
35
cGRG-(NMe)Asp-dvc-NH2 96%
834.3113/835.5206
36
cGRGD-D(NMe)Asp-vc-NH2 95%
834.3113/835.2559
37
cGRGDd-D(NMe)Val-c-NH2 95%
834.3113/835.2916
38
cGRGDd-DIng-c-NH2 98%
908.3269/909.3024
39
cGRGDd-DBta-c-NH2 98%
926.2833/927.3051
40
cGRGDd-DNal1-c-NH2 98%
918.3113/919.3979
41
cGRGDd-DNal2-c-NH2 98%
918.3113/919.9768
Calculated from RP-HPLC. b MS of analogues were measured by MALDI-TOF.
Peptide 1 analogues and biotinylated peptide 40 have been synthesized using methods
as previously described.
Synthesis of focused libraries
TentaGel resin (4g, loading: 0.27mmol/g) were swollen in water for 24 h. After
filtration, a solution of Fmoc-OSu (0.043mmol), Alloc-OSu (0.17mmol) and DIPEA
(100μL) in DCM/diethyl ether (55:45) was added to the beads and the reaction was
done in 30 min under vigorously shaking. Then the inner lay of the beads was
protected by treatment with (Boc)2O (5.4mmol)/DIPEA (10.8mmol) for 2 h. After
S4
Alloc deprotection, a solution of Ac-Gly-OH (0.86 mmol), HOBt (0.86 mmol) and
DIC (0.86 mmol) in DMF was used to block the exposed N-terminus on bead surface.
Upon Fmoc deprotection and Boc deprotection, a solution of Fmoc-D-Cys(Trt)-OH
(3.24mmol), HOBt (3.24mmol) and DIC (3.24mmol) was added to the beads. The
coupling reaction was carried out at room temperature for 2 h. Then the focused
libraries were synthesized according to the standard “split-mix” approach at position
X6 and X7. To overcome sequencing misidentification due to similar retention times
using protein sequencer, the 71 building blocks for position X7 were divided into two
groups after coupling of Fmoc-D-Cys(Trt)-OH
(table S3 and S4; focused library 1
and 2, respectively). Then Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Gly-OH and Fmoc-D-Cys(Trt)-OH were coupled to the resin in the same
manner as described above, respectively. After removal of Fmoc, the beads were
sequentially washed with DMF, MeOH, DCM, and then dried under vacuum. The
beads were then dried under vacuum for 1 h before adding a TFA-based cleavage
cocktail (TFA: phenol: water: thioanisole: Tis, 10:0.5:0.5:0.5:0.25, v/w/v/v/v) for 3 h.
After neutralization with 10% DIPEA/DMF (twice), the resin was washed
sequentially with DMF, MeOH, DCM, DMF, DMF/water (60%/30%) and water,
three times each. Then the beads were transferred to a 1 liter bottle, to which was
added 500mL mixture of water, acetic acid and DMSO (75:5:20, pH=6). The beads
were shaken for two days until the Ellman test was negative. After filtration, the beads
were thoroughly washed with H2O. Finally, the bead library was stored in 75%
ethanol/waster and ready for screening.
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Table S2. Amino acids used in the synthesis of focused Library 1 and 2.
X6 (Focused library 1 and 2, at position 6)
DAsp
DGlu
DSer
DAsn
Aad
DBec
Bmc
Bmp
Phe(4-COOH)
DHyp
DHoser
DTha
Ahch
Actp
S6
Akch
Tyr(diI)
DTrp
DThz
D2Thi
D3Thi
DCit
DHoCit
Aib
Nglu
DFua
Table S3. Amino acids used in the synthesis of focused Library 1.
X7 (Focused library 1, at position 7)
DAsp
DSer
DAsn
Bmp
S7
DHoSer
Nglu
DHoCit
DBec
Aad
DHyp
Ahch
Phe(4-COOH)
Akch
Aecc
DAbu
DPhe(di-OCH3)
Cpa
D2Thi
DThz
DPhg
S8
DPhe(4-NO2)
DNle
DNMePhe
Aic
DChg
DBta
DBpa
DNal2
DNal1
DTic
Ppca
DCha
DBipa
Deg
Dpg
S9
Table S4. Amino acids used in the synthesis of focused Library 2.
X7 (Focused library 2, at position 7)
Acpc
Bmc
DCit
DGlu
Sar
DTha
DPra
Actp
Aib
DAgl
Acbc
DFua
DVal
DNva
D3Thi
DTrp
S10
DPhe
DIle
DBug
Ach
DNMeVal
DCpeg
DCɑMePhe
Tyr(2I)
DPhe(2-Cl)
DBua
DHPhe
DHLeu
D-Ala(styryl)
DIng
DSta
DPhe(4-CF3)
Oic
S11
DDpa
DPhe(4-tB
u)
DHoCha
DPhe(2Cl)
Natural amino acids are designated by the standard three-letter code. “D” stands for
D-configuration. Other abbreviations: Aad, 2-aminohexanedioic acid; DAbu,
D-α-aminobutyric acid; Acpc, 1-aminocyclopropane-1-carboxylic acid; Actp,
4-amino-4-carboxytetrahydropyran; Aecc, 8-amino-1,4-dioxaspiro[4.5]decane-8carboxylic acid; Ahch, 1-amino-1-(4-hydroxycyclohexyl) carboxylic acid; Aib,
2-aminoisobutyric acid; Aic, 2-aminoindane-2-carboxylic acid; Akch,
1-amino-1-(4-ketocyclohexyl) carboxylic acid; DAgl, D-allylglycine; DNMeAsp,
Nɑ-methyl-D-aspartic acid; NMeArg, Nɑ-methyl-arginine; Bec,
(R)-2-amino-3-(2-carboxyethylsulfanyl)propanoic acid; Bmc,
(R)-2-amino-3-(carboxymethylsulfanyl)propanoic acid; DBipa,
D-4,4’-biphenylalanine; Bmp, 4-carboxymethoxyphenylalanine; DBpa,
D-4-benzoylphenylalanine; DBta, D-3-benzothienylalanine; Bua, β-t-butyl-D-alaine;
DBug, D-ɑ-tert-butylglycine; DCha, β-cyclohexyl-D-alanine; DChg,
D-ɑ-cyclohexylglycine; DCit, D-citrulline; Deg, diethylglycine; Cpa,
β-cyclopropylalanine; DCpeg, D-cyclopentylglycine; DDpa, 3,3-diphenyl-D-alanine;
Dpg, di-n-propylglycine; DHCha, D-homocyclohexylalanine; DFua,
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2-furyl-D-Alanine; DHLeu, D-homoleucine; DHoCit, D-homocitrulline; DHPhe,
D-homophenylalanine; DHSer, D-homoserine; DHyp, hydroxyl-D-proline;
DIng, D-2-Indanylglycine; DNal1, D-3-(1-naphthyl)alanine;
DNal2, D-3-(2-naphthyl)alanine; Nglu, 3-(carboxymethylamino)propanoic acid;
DNle, D-norleucine; DNva, norvaline; Oic, L-octahydroindole-2-carboxylic acid;
DPen, D-penicillamine; DNMePhe, Nɑ-methyl-D-phenylalanine; DPhe(2-Cl),
2-chloro-D-phenylalanine; DCɑMePhe, ɑ-methyl-D-phenylalanine; DPhe(di-OCH3),
3,4-dimethoxy-D-phenylalanine; Phe(4-COOH), 4-carboxyphenylalanine;
DPhe(4-NO2), 4-nitrop-D-henylalanine; DPhe(4-CF3),
4-trifluoromethyl-D-phenylalanine; DPhe(4-tBu), 4-tert-butyl-D- phenylalanie;
DPhe(diCl), 3,4-dichloro-D-phenylalanie; DPhg, D-phenylglycine; Ppca,
(2S,5R)-5-phenyl pyrrolidine-2-carboxylic acid; DPra, propargylglycine; Sar, Nɑ
-methylglycine; DSta, 3-styryl-D-alanine; D2Thi, 3-(2-thienyl)-D-alanine; D3Thi,
3-(3-thienyl)-D-alanine; DTic, (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
DTha, 4-Thiazolyl-D-alanine; DThz, D-thiazolidine-4-carboxylic acid; Tyr(diI),
3,5-diiodotyrosine; DNMeVal, Nɑ-methyl-D-valine;
HArg, homoargine
NMR studies of peptides 1 and 4.
NMR Spectroscopy. All NMR experiments were performed at 295K on Bruker
Avance III 800 MHz spectrometer (Bruker, Karlsruhe, Germany) equipped with a
four-channel interface and triple-resonance probe with pulse field gradients, in
0.75mL DMSO-d6. Homonuclear two-dimensional NMR, NOESY (mix = 125 and
S13
250 ms), TOCSY ((mix = 70 ms), and DQF-COSY spectra were acquired using a
sweep width of 14423 Hz and 1024 complex points in F1. The transmitter carrier was
placed on the water resonance. Heteronuclear correlation experiments,
and
13
C-HMQC
13
C-HMBC, were performed to assign all carbon chemical shifts. 1H NMR and
13
C NMR chemical shifts are reported using DSS and DMSO-d6 as references. NMR
data were processed by NMRPipe (ref. 2) and analyzed with SPARKY (Goddard T.D.
and Kneller D.G., University of California at San Francisco).
Experimental Constraints and Structure Calculation. Distance constraints were
extracted from 2D NOESY with mixing times of 125ms and 250ms. NOE cross-peaks
with strong, medium and weak intensities were assigned to interproton distances of
2.9, 3.5 and 5 Å. For the NOEs involving methyl and methylene groups, the upper
bound distance constraints were modified using pseudoatom correction (Wuthrich K.,
1983 ref. 3). The backbone dihedral angle  was calculated from the Karplus Equation
using 3JHN coupling constants obtained from DQF-COSY spectrum (Wang A.C.,
1996 ref. 4). The 1 dihedral angles were defined according to the NOE intensities
between amino proton and two  protons in comparison with the NOE intensities
between  proton and two  protons in the same residue, as mentioned previously
(Assa-Munt N, 2001; Kessler H., 1994 ref. 5 and 6). The additional constraint data
from the chemical shifts of C, C and H were used in final structure refinement. The
NMR-derived distances and dihedral angles then served as constraints for calculating
the three-dimensional structures of two cyclic-peptides using distance geometry and
restrained molecular dynamics. Structure calculations were performed using the
S14
YASAP protocol within X-PLOR (Brunger A.T., 1992; Badger J., 1999 ref. 7), as
described previously (Bagby, S., 1994 ref. 8).
Table S5. Chemical shifts of peptide 1 at 295K, DMSO-d6.
Res# NH
H
H
c1
8.37
4.21
3.18
G2
8.83
4.03,3.79
R3
8.40
4.40
1.51,1.70
Others
1.48,1.42(H)
C
C
C
166.87
51.36
38.80
168.22
41.89
170.94
52.14
28.83
Others
24.82(C)
3.10(H)
40.00(C)
7.78(NH)
156.59(C)
7.45,7.08(NH)
G4
8.10
3.95,3.65
D5
8.51
4.51
2.48,2.61
d6
8.30
4.52
v7
7.62
4.14
168.69
41.57
12.48(COOH)
170.62
49.79
35.70
171.26(C)
2.58,2.76
12.48(COOH)
170.56
49.72
35.96
171.37(C)
2.00
0.88, 0.86
170.68
57.93
30.29
18.88,17.84
(CH3)
c8
8.41
4.50
2.91,3.11
(C)
7.55,7.25
171.36
51.20
39.90
C
C
C
166.80
51.45
39.56
(CONH2)
Table S6. Chemical shifts of peptide 4 at 295K, DMSO-d6.
Res#
NH
H
H
C1
8.39
4.20
3.19
Others
S15
Others
G2
8.70
3.97,3.84
R3
8.50
4.21
1.56,1.71
1.44,1.52(H)
168.34
41.79
171.46
52.81
28.00
24.87(C)
3.10(H)
40.03(C)
7.76(NH)
156.56(C)
7.46,7.06(NH)
G4
8.21
3.85,3.65
D5
8.25
4.59
2.51,2.66
d6
8.32
4.55
v7
7.41
4.20
168.46
41.81
12.49(COOH)
170.68
49.46
35.81
171.37(C)
2.60,2.78
12.49(COOH)
170.15
49.62
35.48
171.48(C)
1.98
0.85,0.82
170.80
57.64
30.27
19.00,17.59
(CH3)
C8
8.57
4.56
2.92,3.18
(C)
7.54,7.37
171.02
51.65
40.77
(CONH2)
Table S7. Structure statistics for the ensembles of 15 calculated structures of peptide
1 and peptide 4.
1
4
NOE restraints (total)
63
71
dihedral angle restraints
13
12
rmsd from ideal geometry
0.0062 
0.0096  0.00019
bond length (Å)
0.00059
S16
1.68  0.03
1.55  0.02
allowed region (%)
100
100
disallowed region (%)
0
0
0.35  0.072
0.52  0.19
bond angles (deg)
Ramachandran plot
rmsd of atom position from average structure
main chain (Å)
1.18  0.27
non-hydrogen (Å)
1.31 0.42
1
4
1
Figure S1. Representative regions of the 2D NOESY spectrum of 1 and 4.
S17
1
4
Figure S2. Superimposed solution structures are the ensembles of 15 structures for
peptide 1 and 4
S18
In vivo and ex vivo optical imaging
Peptide 40-SA-Cy5.5 (1.8 nmol), prepared by mixing 7.2 nmol of biotinylated peptide
40 with 1.8nmol of streptavidin-Cy5.5 in PBS overnight at 4°C, was injected via the
tail vein in an anesthetized mouse before imaging. Animals were placed on a
transparent sheet in the supine position. Images were acquired with a Kodak
IS2000MM Image station (Rochester, NY) with a 625/20 band pass excitation filter,
700WA/35 band pass emission filter, and 150 W quartz halogen lamp light source set
to maximum. Images were captured with a CCD camera set at F stop=0, FOV=150,
and FP=0. For ex vivo imaging, the mice were euthanized and their organs were
excised for imaging.
Abbreviations: All-OSu: N-allyloxycarbonyloxy succinimide HATU: 2 - (7 - Aza 1H - benzotriazole - 1 - yl) - 1,1,3,3 - tetramethyluronium hexafluorophosphate;
Boc2O: Di-t-butyl dicarbonate; DIC: 1, 3-Diisopropylcarbodiimide; DIPEA: N,
N’-diisopropylethylamine; EDT: 1, 2-ethanedithiol; Equiv: Equivlent; Fmoc-OSu:
N-(9-fluorenylmethyloxycarbonyloxy)-succinimide;
HBTU:
(O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HOBt:
1-Hydroxybenzotriazole; MeOH: Methanol; DCM: Dichloromethane; DMF: N,
N-Dimethylformamide; TFA: Trifluoro acetic acid; Tis: Triisopropylsilane;
Reference
1. Zhang S, Govender T, Norstrorm T, Arvidsson PI, An Improved Synthesis of
Fmoc-N-methyl-r-amino Acids. J Org Chem 2005; 70: 6918-6920.
S19
2.
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A, NMR Pipe: a
multidimensional spectral processing system based on UNIX pipes. J Biomol
NMR 1995; 6: 277-293
3. Wuthrich K, Billeter M, Braun W, Pseudo-structures for the 20 common
amino acids for use in studies of protein conformations by measurements of
intramolecular proton-proton distance constraints with nuclear magnetic
resonance. J Mol Biol 1983; 169: 949-961.
4. Wang AC, Bax A, Determination of the Backbone Dihedral Angles f in
Human Ubiquitin from Reparametrized Empirical Karlpus Equations. J Am
Chem Soc 1996; 118: 2483-2494.
5. Assa-Munt N, Jia X, Laakkonen P, Ruoslahti E, Solution structures and
integrin binding activities of an RGD peptide with two isomers. Biochemistry
2001; 40: 373-2378
6. Kessler H, and Seip S, NMR of peptides, in Two-dimensional NMR
spectroscopy: Applications for chemists and biochemists, New York: VCH
Publishers; 1994. p.642-643.
7. Brunger A T, X-PLOR, Version 3.1: A System for X-Ray Crystallography and
NMR, New Haven: Yale University Press; 1992.
8. Bagby S, Harvey TS, EagleSG, Inouye S and Ikura M, NMR-derived
three-dimensional solution structure of protein S complexed with calcium
Structure 1994; 2: 107– 122.
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