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) was neutralized 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
cyclen 4.5g (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) was dissolved in 40 ml acetonitrile and mixed with NaHCO3 (30 equiv.)
in ice bath, followed by dropwise addition of 3 equiv. (17.4mmol) tert-Butyl Bromoacetate dissolved
in 20 ml acetonitrile under nitrogen atmosphere. The mixture was stirred till completion of the
reaction as monitored by TLC. The mixture was then filtrated on a glass funnel through a pad. The
filtrate was vacuum-evaporated to afford the crude compound. The crude products were purified
through
recrystallization
in
toluene
to
afford
white
solid
1,4,7-Tri-Boc-1,4,7,10-tetraazacyclododecane 2.53 (S1) with a yield of 84.8%. 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
acetonitrile in the presence of K2CO3, and refluxed for 30 h at 100℃. After the completion of the
reaction, the mixture was filtrated, and the solvent was removed by rotating evaporation. The crude
products were purified by column chromatography on SiO2 to yield yellow brownish oily product
1,4,7-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 by addition of 0.1g 10% Pd/C and stirring for 48 h. After filtration and
evaporation of the solvent, 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): δ 3.90-1.90 (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
NH HN
NH HN
Figure S2: a. 1H NMR spectrum of cyclen
Intens.
x10 7
+MS, 0.2min #21
172.8
4
3
2
1
184.9
238.8
0
50
75
100
125
150
175
200
225
Figure S2: b. mass spectrum of cyclen
250
275
300 m/z
Bu tO2C
ButO 2C
H
N
N
N
N
CO2tBu
Figure S3: a. 1H NMR spectrum of S1
Intens.
x10 8
+M S, 0.5m i n #40
3.0
537.2
2.5
2.0
1.5
1.0
515.3
0.5
651.3
0.0
300
400
500
600
700
Figure S3: b. mass spectrum of S1
Figure S4: a. 1H NMR spectrum of S2
800
900
m /z
Intens.
x10 9
+MS, 0.3min #24
1.2
663.3
1.0
0.8
0.6
0.4
0.2
587.3
0.0
400
500
600
700
800
900
1000
1100
Figure S4: b. mass spectrum of S2
tBuO 2C
COOH
N
N
N
N
tBuO2C
CO 2tBu
Figure S5: a. 1H NMR spectrum of S3
Intens.
x10 8
+MS, 0.2min #15
595.2
5
4
3
2
1
573.3
0
100
200
300
400
500
600
700
Figure S5: b. mass spectrum of S3
2. Synthesis of peptide (P1 and P2)
800
900
m/z
m/z
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
Composition
P1
A dual targeting peptide
KFDGRGKGGCNGRC
P2
A single targeting peptide
KFDGRG
L1
A ligand, P1 binding to DOTA
KFDGRGK(-DOTA)GGCNGRC
L2
A ligand, P2 binding to DOTA
KFDGRGK(-DOTA)
CA1
A contrast agent, L1 combining 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