1. Synthesis of 1,4,7-Tri-Boc-10-(carboxymethyl)-1,4,7,10-tetraazacyclododecan
1.1 Synthesis of 1,4,7,10-tetraazacyclododecane
Cyclen hydrochloric salt 10g (0.031 mol) wasneutralized by 30% soudium hydroxide in distilled water,ice bath and magnetic stirring. Reaction was lasted one hour.After that, extracted with chloroform, organic phase was obtained. The solvent was removed through evaporation to yield cyclen4.5g (1,4,7,10-tetraazacyclododecane) in white powder (yield 84.3% ).IR(KBr, cm-1): 3296.23(νN-H), 1447.16(δN-H), 2926.07(νas(CH)), 2815.75(νs(CH)), 1446.57(δCH); 1H NMR (in CDCl3, 400 MHz): δ 2.69(s, 16H, CH2), 2.13 (s, 4H, N-H); ESI-MS( m/z): 172.8
1.2 Synthesis of 1,4,7-Tri-Boc-1,4,7,10-tetraazacyclododecane (S1)
1g cyclen (5.8 mmol) wasdissolved in 40 ml acetonitrile and mixed with NaHCO3 (30 equiv.) in ice bath,followed by dropwise addition of 3 equiv. (17.4mmol) tert-Butyl Bromoacetatedissolved in 20 ml acetonitrile under nitrogen atmosphere. The mixture was stirred tillcompletion of the reaction as monitored by TLC. The mixture was then filtrated on aglass funnel through a pad. The filtrate was vacuum-evaporated to afford the crudecompound. The crude products were purified through recrystallization in toluene toafford white solid 1,4,7-Tri-Boc-1,4,7,10-tetraazacyclododecane 2.53 (S1) with a yield of84.8%.IR(KBr, cm-1): 2977.01(νN-H), 2946.69, 2854.06, 2736.92(νCH), 1368.17, 1392.76(νc(CH3)3), 1724.38(νCO), 1150.02(νC-O-C); 1H NMR (CDCl3, 400 MHz): δ 10.05 (s, 1H, N-H), 3.38 (s, 4H, CH2), 3.30 (s, 2H, CH2), 3.11 (s, 4H, CH2), 2.93–2.88 (m, 12H, CH2), 1.46 (s, 27H, CH3); ESI-MS m/z: 537.2
1.3 Synthesis of 1,4,7-Tri-Boc-10-Cbz-1,4,7,10-tetraazacyclododecane (S2)
Benzyl bromoacetated 0.38g (2mmol)S1(1g 2mmol)in molar ratio of 1:1 were dissolved in acetonitrilein the presence of K2CO3, and refluxed for 30 h at 100℃. After the completion of thereaction, the mixture was filtrated, and the solvent was removed by rotatingevaporation. The crude products were purified by column chromatography on SiO2 toyield yellow brownish oily product1,4,7-Tri-Boc-10-Cbz-1,4,7,10-tetraazacyclododecane (S2) 0.83g and yield is 71.8%.1H NMR (in CDCl3, 400 MHz): δ 7.36-7.30 (m, 5H, CH), δ 5.13 (s, 2H), δ 3.50-2.41 (br, 24H), δ 1.46 (s, 27H).
1.4 Synthesis of the 1,4,7-Tri-Boc-10-(carboxymethyl)-1,4,7,10-tetraazacyclododecan
The benzyl group of S2 (1g 1.7 mmol)was removed by hydrogenolysis by dissolving S2 in 30 ml methanol, followed byaddition of 0.1g 10% Pd/C and stirring for 48 h. After filtration and evaporation of thesolvent, a yellowish powder product (0.96 g) is obtained in almost quantitative yield (~98%).The product was used in the next step without further purification.1H NMR (in CDCl3,
400 MHz): δ3.90-1.90 (br, 24H), δ 1.47 (s, 27H); ESI-Ms: 595.2(M+Na+1).
Figure S1: Synthesis of 1,4,7-Tri-Boc-10-(carboxymethyl)-1,4,7,10-tetraazacyclododecane
Figure S2: a.1H NMR spectrum of cyclen
Figure S2: b. mass spectrum of cyclen
Figure S3: a. 1H NMR spectrum of S1
Figure S3: b. mass spectrum of S1
Figure S4: a. 1H NMR spectrum of S2
Figure S4: b. mass spectrum of S2
Figure S5: a. 1H NMR spectrum of S3
Figure S5: b. mass spectrum of S3
2. Synthesisof peptide (P1 and P2)
Figure S6: a. HPLC eluting chart of P1; Figure S6: b. HPLC eluting chart of P2.
Eluted with a linear gradient of aqueous CH3CN (0~60%, 30 min) solution containing 0.1% (v/v) TFA and detected by UV absorbance at 220 nm.
Figure S7: a. MS chart of P1.
Figure S7: b. MS chart of P2
3.Synthesis of ligand (L1 and L2)
Figure S8: a. HPLC eluting chart of L1; Figure S8: b. HPLC eluting chart of L2.
Figure S9: a. MS chart of L1
Figure S9: b. MS chart of L2
4. Synthesis of contrast agent (CA1 and CA2)
Figure S10: a. Structure of CA1
Figure S10: b. Structure of CA2
Figure S11: a. MS chart of CA1
Figure S11: b. MS chart of CA2
Code name / Name / CompositionP1 / A dual targeting peptide / KFDGRGKGGCNGRC
P2 / A single targeting peptide / KFDGRG
L1 / Aligand, P1 binding to DOTA / KFDGRGK(-DOTA)GGCNGRC
L2 / Aligand, P2 binding to DOTA / KFDGRGK(-DOTA)
CA1 / A contrast agent, L1combining with Gd / KFDGRGK(Gd-DOTA)GGCNGRC
CA2 / A contrast agent, L2 combining with Gd / KFDGRGK(Gd-DOTA)
Table S1:the composition and name of compounds