General
Trityl chloride polystyrene (TCP) resins were purchased from PepChem (Tübingen, Germany) and Sigma-Aldrich (Steinheim, Germany). 9-fluorenylmethyloxycarbonyl (Fmoc) and all other protected amino acid analogs were obtained from Iris Biotech (Marktredwitz, Germany) or Bachem (Bubendorf, Switzerland). Chelators were obtained from CheMatech (Dijon, France, or Macrocyclics (Dallas, USA)) while all other chemicals were bought from Sigma-Aldrich, Fluka, or Merck (Darmstadt, Germany) if not stated otherwise. Solvents and all other organic reagents were purchased from Sigma-Aldrich (Munich, Germany), CLN (Freising, Germany), and VWR (Darmstadt, Deutschland). Water for reversed phase (RP)-HPLC was filtered through a 0.2-μm filter (Thermo Scientific, Barnstead Smart2Pure, Niederelbert, Germany). Analytical RP-HPLC was performed on a Nucleosil 100 C18 (5 μm, 125 × 4.0 mm2) column (CS GmbH, Langerwehe, Germany) using a Sykam gradient HPLC System (Sykam GmbH, Eresing, Germany). For elution, linear gradients of acetonitrile (0.1 % (v/v) trifluoroacetic acid (TFA), solvent B) in water (0.1 % TFA (v/v), solvent A) at a constant flow of 1 mL/min over 15 min were used. UV detection was performed at 220 and 254 nm using a 206 PHD UV-vis detector (LinearTM Instruments Corporation, Reno, USA). Purities were determined at 220 nm using LabSolutions software by Shimadzu Corp. Preparative RP-HPLC was performed on a Sykam gradient HPLC System (Sykam GmbH, Eresing, Germany) equipped with a Multospher 100 RP 18-5 (250 × 20 mm2) column (CS GmbH, Langerwehe, Germany) at a constant flow of 10 mL/min using the same solvents as stated above (duration of gradient, 20 min). Electrospray ionization (ESI)-mass spectra were recorded on a Varian 500-MS IT mass spectrometer (Agilent Technologies, Santa Clara, USA).
General SPPS
Peptides were synthesized manually on solid TCP support using standard Fmoc strategy and an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). As side chain protecting groups, N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl] (Dde) for d-Orn and t-butyl and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) groups for d-Tyr and Arg, respectively, were utilized.
O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBt) were used as coupling reagents. N-alkylated amines were acylated using (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU) with 1-hydroxy-7-azabenzotriazole (HOAt) as racemization suppressant.
Cleavage of the peptide from the TCP support with retention of acid-labile protection groups was achieved by treating the peptidyl resin with a solution of dichloromethane (DCM):trifluoroethanol:acetic acid (6:1:3) (v/v/v); for deprotection of acid-labile groups, TFA:triisopropylsilane (TIPS):H2O (95:2.5:2.5) (v/v/v) was used (2× 30 min). Deprotection of Dde was carried out with 2 % hydrazine monohydrate in dimethylformamide (DMF) (v/v).
For Fmoc-deprotection, the peptide was treated with 20 % piperidine in N-methyl-2-pyrrolidone (NMP) (v/v) for 20 min.
For general coupling of amino acids, a solution of Fmoc-Xaa-OH (1.5 equiv), TBTU (1.5 equiv), HOBt (1.5 equiv), and N,N-diisopropylethylamine (DIPEA) (5 equiv) in NMP (1 mL/g resin) was added to the resin-bound peptide and shaken for 90 min at room temperature and washed six times with NMP.
Synthesis of A
The peptide was synthesized according to a previously published procedure [
12]. In short, synthesis was carried out using a standard Fmoc strategy using a TCP resin as solid support and HOBt/TBTU as coupling reagents. After selective
N-methylation of
d-Orn,
d-Tyr was coupled to the peptide with HATU/HOAt, cleaved from the resin, and finally cyclized.
Coupling AMB to the d-Orn side chain was carried out using Fmoc- or Boc-protected AMB (1.5 equiv). AMB was preactivated with DIC (N,N′-diisopropyl-carbodiimide) (1.5 equiv), HOAt (1.5 equiv), and DIPEA (4.5 equiv) in 5 mL DMF for 10 min. The d-Orn deprotected peptide precursor was dissolved in DMF, and the preactivated linker was added. After complete reaction (2 h), Fmoc deprotection and semi-preparative HPLC purification, appropriate chelators were coupled to the peptide. HPLC (50 to 100 % B in 15 min): t
R
= 8.0 min; ESI-mass spectra (MS): calculated for (C61H78N10O10S): 1142.5; found: m/z = 1143.6 [M+H]+.
A molar excess of the activated chelator was added to a free amino group of the peptide analog. Subsequent to successful coupling, the chelator-conjugated peptide was deprotected and purified. Metal complexation was performed in the presence of weakly chelating acetate buffers to reduce the likelihood of hydrolysis. Solutions for metal labeling comprised LuCl3 (20 mM), pH = 6.0; InCl3 (20 mM), pH = 4.5; and YCl3 (20 mM), pH = 5.9, each in ammonium acetate (0.1 M) and Ga(NO3)3 (2 mM) pH = 3.0; Cu(OAc)2 pH = 6.0; and ZrCl4 (20 mM) pH = 1.3, each in water. The chelator-conjugated peptide (250 μL, 2 mM, 1 equiv) was dissolved in H2O and DMSO up to 50 % (v/v), if necessary, and the metal (1–10 equiv) was added, pH adjusted, and heated for 30 min. Final metalated peptides were obtained in a purity ≥ 95 %, and used for in vitro studies without further purification, unless stated otherwise.
Coupling of DOTA or NOTA
For DOTA/NOTA derivatization, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-triacetic acid (NOTA) (1 equiv) was preactivated for 20 min using N-hydroxysuccinimide (NHS) (1.25 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (1.25 equiv), and DIPEA (2 equiv) dissolved in water (1 mL/0.3 mmol). The peptide
B (0.25 or 0.3 equiv, for DOTA or NOTA, respectively) was dissolved in DMF (1 mL per 0.15 mmol of peptide) and slowly added to the reaction mixture according to a previously published protocol [
13].
3: HPLC (30 to 55 % B in 15 min):
t
R
= 10.1 min; ESI-MS: calculated for (C56H73N13O12): 1119.6; found:
m/z = 1121.0 [M+H]
+, 1143.0 [M+Na]
+;
1: HPLC (30 to 55 % B in 15 min):
t
R
= 8.8 min; ESI-MS: calculated for (C60H80N14O14): 1220.6; found:
m/z = 1221.6 [M+H]
+, 1243.6 [M+Na]
+.
Coupling of DOTAGA-anhydride
2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-anhydride) (2 equiv) was dissolved in anhydrous DMF to obtain a white suspension and added to a solution of B (1 equiv) and triethylamine (10 equiv) in anhydrous DMF. 2: HPLC (30 to 55 % B in 15 min): t
R
= 9.8 min; ESI-MS: calculated for (C63H84N14O16): 1292.6; found: m/z = 1293.5 [M+H]+, 1315.5 [M+Na]+.
Coupling of NODAGA
4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triaza cyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid ((NODAGA)(tBu)3) (3 equiv) was coupled to B (1 equiv) using a solution of DIC (2 equiv), HOAt (2 equiv), and DIPEA (6 equiv) in anhydrous DMF. The product was fully deprotected and purified. HPLC (30 to 55 % B in 15 min): t
R
= 10.8 min; ESI-MS: calculated for (C59H77N13O14): 1191.6; found: m/z = 1193.0 [M+H]+, 1215.0 [M+Na]+.
Coupling of DTPA
3,6,9-tris(2-(tert-butoxy)-2-oxoethyl)-13,13-dimethyl-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oic acid (DTPA)(tBu)4 (2 equiv) was coupled to B (1 equiv) using a solution of DIC (1.5 equiv), HOAt (1.5 equiv), and DIPEA (4.5 equiv) in anhydrous DMF. After successful coupling, the product was deprotected and purified. HPLC (30 to 60 % B in 15 min): t
R
= 8.0 min; ESI-MS: calculated for (C58H75N13O16): 1209.6; found: m/z = 1210.8 [M+H]+, 1232.6 [M+Na]+.
Coupling of p-SCN-Bn-DTPA
2-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA) (21.0 mg, 32.4 μmol, 1.5 equiv) was coupled to B (18.0 mg, 21.6 μmol, 1 equiv) with DIPEA in anhydrous DMF to obtain pH ≈ 9. HPLC (30 to 60 % B in 15 min): t
R
= 11.2 min; ESI-MS: calculated for (C66H82N14O17S): 1374.6; found: m/z = 1375.8 [M+H]+.
Coupling of p-SCN-Bn-DFO
p-Isothiocyanatobenzyl-desferoxamine (DFO-Bn-NCS) (21.0 mg, 32.4 μmol, 1.5 equiv) was coupled to B (18.0 mg, 21.6 μmol, 1 equiv) in anhydrous DMF and adjusted with NEt3 to pH = 9.5. HPLC (30 to 60 % B in 15 min): t
R
= 14.0 min; ESI-MS: calculated for (C77H106N18O15S2): 1586.8; found: m/z = 1587.0 [M+H]+.
Coupling of NCS-MP-NODA
2,2′-(7-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NCS-MP-NODA) (1.9 mg, 5.4 μmol, 1.5 equiv) was coupled to A (3 mg, 3.6 μmol, 1 equiv) with DIPEA (1.1 μL, 3.2 μmol, 2 equiv) in anhydrous DMF to obtain pH ≈ 9. HPLC (20 to 50 % B in 15 min): t
R
= 9.0 min; ESI-MS: calculated for (C54H71N13O10S): 1093.5; found: m/z = 1095.1 [M+H]+, 1116.7 [M+Na]+.
Chelation of Ga3+ with DOTA, DOTAGA, NOTA, NODAGA conjugates
A solution of Ga(NO3)3 (250 μL, 2 mM, 1 equiv) in water, pH = 3.0, was added to the chelator-conjugated peptide, (250 μL, 2 mM, 1 equiv) dissolved in H2O and, if necessary, in DMSO up to 50 % (v/v). The final pH of the mixture was adjusted to 4–6 and heated at 90 °C for 30 min. [natGa]1: HPLC (30 to 55 % B in 15 min): t
R
= 9.0 min; ESI-MS: calculated for (C60H78GaN14O14): 1287.5; found: m/z = 1287.7 [M+H]+, 1311.7 [M+Na]+; [natGa]2: HPLC (30 to 55 % B in 15 min): t
R
= 9.8 min (Ga); ESI-MS: calculated for (C63H82GaN14O16): 1359.5; found: m/z = 1361.3 [M+H]+, 1383.2 [M+Na]+; [natGa]3: HPLC (30 to 55 % B in 15 min): t
R
= 9.3 min; ESI-MS: calculated for (C56H71GaN13O12): 1186.5; found: m/z = 1188.9 [M+H]+, 1209.9 [M+Na]+; [natGa]4: HPLC (30 to 55 % B in 15 min): t
R
= 10.8 min; ESI-MS: calculated for (C59H74GaN13O14): 1257.5; found: m/z = 1258.8 [M+H]+.
Chelation of In3+, Y3+, and Lu3+ with DOTA, DOTAGA, and DTPA conjugates
A solution of either YCl3, LuCl3, or InCl3 (250 μL, 20 mM, 10 equiv) in ammonium acetate (0.1 M, pH = 6.0) was added to the chelator-conjugated peptide (250 μL, 2 mM, 1 equiv). The pH was adjusted to 4–6, and the mixture heated at 90 °C for 30 min. [natLu]1 and [natY]1: HPLC (10 to 50 % B in 15 min): t
R
= 10.7 min each; ESI-MS: calculated for (C60H77LuN14O14): 1392.5; found: m/z = 1393.8 [M+H]+; ESI-MS: calculated for (C60H77N14O14Y): 1306.5; found: m/z = 1308.5 [M+H]+, 1329.7 [M+Na]+; [natLu]2 and [natY]2: HPLC (30 to 55 % B in 15 min): t
R
= 9.8 min each; ESI-MS: calculated for (C63H80LuN14O16): 1463.5; found: m/z = 1465.3 [M+H]+, 1488.1 [M+Na]+; ESI-MS: calculated for (C63H80N14O16Y): 1377.5; found: m/z = 1379.3 [M+H]+, 1402.2 [M+Na]+; [natIn]7: HPLC (30 to 55 % B in 15 min): t
R
= 10.3 min; ESI-MS: calculated for (C66H77InN14O17S): 1484.4; found: m/z = 1487.4 [M+H]+; [natLu]6, [natIn]6, and [natY]6: HPLC (30 to 60 % B in 15 min): t
R
= 9.1 min each; ESI-MS: calculated for (C58H71LuN13O16): 1380.5; found: m/z = 1382.8 [M+H]+, for (C58H71N13O16Y): 1294.4; found: m/z = 1296.7 [M+H]+, for (C58H71InN13O16): 1320.4; found: m/z = 1322.9, [M+H]+, 1344.9 [M+Na]+.
Chelation of Zr4+ with 8
To obtain [natZr]8, a solution of ZrCl4 in water (pH = 1.3) (20 mM) was added to the DFO-bearing peptide (2 mM). Quantitative complexation at room temperature occurred within a few minutes without forming any side products. HPLC (30 to 60 % B in 15 min): t
R
= 14.0 min; ESI-MS: calculated for (C77H103N18O15S2Zr): 1673.6; found: m/z = 1674.5 [M+H]+.
Chelation of Bi3+ with DOTA and DOTAGA conjugates
[natBi]1 and [natBi]2 were prepared by the dropwise addition of natBi(H3CCOO)3 (10 equiv) to a solution of DOTA or DOTAGA-peptide at pH ~ 5. [natBi]1: HPLC (30 to 55 % B in 15 min): t
R
= 8.8 min; ESI-MS: calculated for (C60H77BiN14O14): 1426.6; found: m/z = 1427.4 [M+H]+, 1451.5 [M+Na]+, 1465.1 [M+K]+; [natBi]2: HPLC (30 to 55 % B in 15 min): t
R
= 9.8 min; ESI-MS: calculated for (C63H80BiN14O16): 1497.6; found: m/z = 1499.3 [M+H]+, 1521.3 [M+Na]+, 1537.3 [M+K]+.
Chelation of Cu2+ with DOTA, DOTAGA, NOTA, NODAGA, and DTPA conjugates
A solution of Cu(OAc)2 (250 μL, 2 mM, 1 equiv) in water, pH = 6.0, was added to the chelator-conjugated peptide, (250 μL, 2 mM, 1 equiv). The mixture was incubated at room temperature for 30 min. HPLC revealed quantitative complexation. [natCu]1: HPLC (30 to 55 % B in 15 min): t
R
= 10.8 min; ESI-MS: calculated for (C60H77CuN14O14): 1280.5; found: m/z = 1282.8 [M+H]+, 1306.8 [M+Na]+.
[natCu]2: HPLC (30 to 55 % B in 15 min): t
R
= 11.0 min; ESI-MS: calculated for (C63H82CuN14O16): 1353.5; found: m/z = 1354.8 [M+H]+, 1377.8 [M+Na]+; [natCu]3: HPLC (30 to 55 % B in 15 min): t
R
= 10.5 min; ESI-MS: calculated for (C56H71CuN13O12): 1180.5; found: m/z = 1181.8 [M+H]+, 1203.6 [M+Na]+; [natCu]4: HPLC (30 to 55 % B in 15 min): t
R
= 10.9 min; ESI-MS: calculated for (C59H74CuN13O14): 1251.5; found: m/z = 1253.8 [M+H]+; [natCu]6: HPLC (30 to 55 % B in 15 min): t
R
= 9.8 min; ESI-MS: calculated for (C58H72CuN13O16): 1269.5; found: m/z = 1271.7 [M+H]+, 1232.8 [M+Na]+.
Chelation of AlF2+ with NOTA and NODA conjugates
The peptide was labeled with [natF]AlF by mixing AlCl3 (1.2 equiv, 0.468 μmol in 0.5 M NaOAc, pH = 4.0), NaF (1.2 equiv 0.468 μmol in 0.5 M sodium acetate, pH = 4.0) with 3 or 5 (1 equiv, 0.39 μmol). The [natF]AlF-labeled peptide was purified using RP-HPLC on a C18 (5 μm, 125 × 4.0 mm). [natF]AlF-3: HPLC (23 % B in 15 min): t
R
= 10.5 min; ESI-MS: calculated for (C56H71AlFN13O12): 1163.5; found: m/z = 1164.9 [M+H]+; [natF]AlF-5: HPLC (20 to 50 % B in 15 min): t
R
= 8.2 min; ESI-MS: calculated for (C54H69AlFN13O10S): 1137.5; found: m/z = 1139.0 [M+H]+, 1160.9 [M+Na]+.
Cell culture and determination of CXCR4 receptor affinity (IC50)
For in vitro experiments, the Jurkat T - cell line was used. The cells were maintained in RPMI 1640 medium (Biochrom) containing 10 % fetal calf serum (FCS) (Biochrom). The cell line was cultured at 37 °C in a humidified atmosphere with 5 % CO2 and passaged two to three times a week, depending on the cell count.
CXCR4 affinities were determined in competitive binding assays using Jurkat cells with [
125I]FC131 as the radioligand according to a protocol similar to previously published [
7]. FC131 (
cyclo(-
d
-Tyr-
l
-Arg-
l
-Arg-
l
-Nal-Gly-)) [
14] was synthesized and iodinated as described previously [
7]. Jurkat cells (4 × 10
5 cells per vial) were incubated with the respective peptide of interest at the final concentrations ranging from 10
−11 to 10
−5 M and app. 0.1 nM of [
125I]FC131. The total sample volume was 250 μL. After an incubation time of 120 min, the vials were centrifuged at 1300 rpm (Heraeus Megafuge, Thermo) for 3 min and the supernatant was removed. The cells were washed twice with 200 μL ice-cold Hank’s balanced salt solution (HBSS). After each washing step, the samples were centrifuged and the supernatant removed. Finally, the amount of displaced and bound radioligand in the combined fractions of the supernatant and the cell pellet was quantified. The half maximal inhibitory concentration (IC
50) values were determined using GraphPad Prism software.