Supporting Information
- S1 -
Space-filling model of a MM3 geometry optimization of Gd-TRENbisHOPO-TAM-Asp-Asp2-12OH (2) performed with CAChe.®
O
N
HN
NH
O
HO
HO
O
NH
O
O
N
H
OH O
H
N
HO
O
NH
O
H2O
O
ON
HN
HN
O
HO
HO
N
OH2
O
HN
O
O
O
Gd
OH OH
O HN
OH
OH
OH
OH
As opposed to other more loose dendrimers, every branch of (2) cannot rotate or oscillate
to a great extent because of the volume occupied by the neighboring branches to which it
is linked. The multitude of internal hydrogen bonding further limits the flexibility of the
dendrimer.
Supporting Information
- S2 -
Second sphere contribution to the relaxivity.
Relaxivity at 20 MHz, 25°C and pH = 7.2: r1p (mM-1 s-1) = 14.3
Relaxivity at 90 MHz, 25°C and pH = 7.2: r1p (mM-1 s-1) = 18.0
NMRD profile – 25°C, pH = 7.2
25
r1p (mM -1 s-1)
20
15
10
inner sphere
5
second sphere
0
0.01
0.1
1
10
100
1000
Proton Larmor Frequency (MHz)
second sphere: corresponds to the contribution of 2 water molecules at a
distance of 4 Å. It accounts for about 5% of the total relaxivity
Supporting Information
- S3 -
Experimental Procedure
Unless otherwise noted, starting materials were obtained from commercial
suppliers and used without further purification. All solvents were dried over activated
alumina and stored over 4Å molecular sieves. Water was distilled and further purified by
a Millipore cartridge system (resistivity 18 x 106 Ω). All organic extracts were dried over
anhydrous MgSO4 and solvents were removed with a rotary evaporator.
Flash
chromatography was performed on Merck Silica Gel (40-7 Mesh). Unless otherwise
stated, 1H and 13C NMR spectra were recorded on a Bruker DRX 500 at 500 MHz and
125 MHz, respectively; the residual solvent peak was used as an internal reference.
Elemental analysis and mass spectra (LR = low resolution; HR = high resolution; FAB
MS = fast atom bombardment mass spectroscopy; EI MS = electron ionization mass
spectrometry; ES MS = electrospray mass spectrometry) were performed by the
Microanalytical Laboratory and Mass Spectrometry Laboratory respectively at the
college of Chemistry at the University of California at Berkeley. Microanalytical analysis
of all complexes including the analysis of metal content were performed by Desert
Analytics, Tucson, Arizona. Matrix Assisted Laser Desorption Ionization mass spectra
(MALDI-MS) were recorded on an Applied Biosystems Voyager System 6322.
Supporting Information
t
BOC-N-Asp-6OBn
tetramethyluronium
- S4 (4)
-
hexafluorophosphate
O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’(HATU,
11.49
g,
30.2
mmol)
and
diisopropylethylamine (50 mL) were added to a solution of H2N-3OBn (3)11 (11.37 g,
29.0 mmol) and N-(tert-butoxycarbonyl)-L-aspartic acid (3.06 g, 13.1 mmol) in
anhydrous dimethylacetamide (60 mL). The solution was stirred at room temperature for
15 h. The reaction mixture was then evaporated to dryness in vacuo and the crude product
partitioned between methylene chloride (200 mL) and 1 M HClaq (2 × 200 mL). The
organic phase was further washed with brine (200 mL) and dried with anhydrous
magnesium sulfate. The product was further purified by flash chromatography over silica
eluting with a gradient of methylene chloride to 96% methylene chloride / 4% methanol.
(4) was obtained as a clear oil (11.6 g, 90.3 %) that was further dried under high vacuum
for 15 h at room temperature. 1H NMR (CD2Cl2) δ = 1.41 (s, 9H, C(CH3)3), 2.46 (m, 1H,
CH-N(H)), 2.76 (m, 1H, C(O)-CH2-CH), 3.07 (m, 1H, C(O)-CH2-CH), 3.82 (m, 12H, CCH2-O), 4.50 (s, 12H, O-CH2-Ar), 6.07 (s, 1H, C(O)-NH-C), 6.27 (s, 1H, C(O)-NH-C),
7.16 (s, 1H, NH-C(O)-O), 7.28-7.45 (m, 30 H, Ar-H); 13C NMR (CD2Cl2) δ = 28.4, 37.7,
38.7, 60.3, 60.4, 69.2, 69.3, 73.6, 75.0, 127.7,127.8, 127.9, 128.0, 128.5, 128.6,
138.7,138.9, 171.2, 174.0; LR FAB-MS m/z = 980.6 (MH+), (Calcd.980.5); HR FABMS for C59H70N3O10 m/z = 980.5033 (MH+) (Calcd. 980.5061 ); Anal. Calcd. (Found). C
72.30 (72.19), H 7.10 (7.30), N 4.29 (4.54).
Supporting Information
- S5 -
H2N-Asp-6OBn (5) - Trifluoroacetic acid (20 mL) was added dropwise over 20
min to a solution of tBOC-N-Asp-6OBn (4) (10.2 g, 10.4 mmol) in methylene chloride
(150 mL). The solution was further stirred at room temperature for 2 h. The reaction
mixture was then evaporated to dryness in vacuo and the crude product partitioned
between methylene chloride (200 mL) and 1 M KOHaq (150 mL). The organic phase was
further washed with 1 M KOHaq (2 × 150 mL) brine (1 × 150 mL) and dried with
anhydrous magnesium sulfate. Evaporation of the solvents in vacuo yielded (5) (8.00 g,
87.3 %) as a clear oil that was further dried under high vacuum for 15 h at room
temperature. 1H NMR (CD2Cl2) δ = 1.92 (bs, 2H, NH2), 2.45 (m, 1H, C(O)-CH(H)-CH),
2.64 (m, 1H, C(O)-CH(H)-CH), 3.55 (m, 1H, C(O)-CH2-CH-NH2), 3.83-3.90 (m, 12H,
C-CH2-O), 4.51 (s, 12H, O-CH2-Ar), 6.28 (s, 1H, C(O)-NH-C), 8.43 (s, 1H, C(O)-NHC); 13C NMR (CD2Cl2) δ = 38.7, 42.0, 59.8, 60.3, 69.3, 69.4, 73.6, 127.8, 127.9, 127.9,
128.0, 128.5, 128.6, 138.8, 138.9, 171.3, 174.0; LR FAB-MS m/z = 880.4 (MH+),
(Calcd. 880.5); HR FAB-MS for C54H62N3O8 m/z = 880.4539 (Calcd. 880.4937); Anal.
Calcd. (Found). C 73.70 (73.59), H 6.99 (7.11), N 4.77 (5.14).
Supporting Information
t
BOC-N-Asp-Asp2(12OBn)
- S6 (6)
O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate (HATU, 2.71 g, 7.12 mmol) and N-(tertbutoxycarbonyl)-L-aspartic acid (0.729 g, 3.126 mmol) were dissolved in anhydrous
dimethylacetamide (30 mL). The solution was stirred at room temperature for 30 minutes.
A solution of H2N-Asp(6OBn) (5) (5.01 g, 5.69 mmol) dissolved in anhydrous
dimethylacetamide (5 mL) and diisopropylethylamine (15 mL) was then added to the
aspartic acid solution. The reaction mixture was stirred at room temperature for 18 hours.
The solvents were then evaporated under reduced pressure and the crude product
partitioned between methylene chloride (100 mL) and 1 M HCl(aq) (100 mL). The organic
phase was further washed with 1 M HCl(aq) (1 × 100 mL) and brine (1 × 100 mL) and
dried with anhydrous MgSO4. The solvents were then removed under reduced pressure.
The crude product was further purified by flash chromatography eluting with a gradient
of 100 % CH2Cl2 to 94 % CH2Cl2 / 6% CH3OH. The protected dendron (6) was obtained
as a clear oil (4.25 g, 69.6 %) that was further dried under high vacuum at room
temperature for 18 hours. 1H NMR (CD2Cl2) δ = 1.44 (s, 9H, C(CH3)3), 2.46-2.97 (m,
2H, CH-CH2-C(O)), 2.66-2.97 (m, 1H, C(O)-CH2-CH), 3.81 (bm, 24H, C-CH2-O), 4.30
(b, 1H, CH-CH2-C(O)), 4.41 (s, 24H, O-CH2-Ar), 4.60 (b, 1H, CH-CH2-C(O)), 6.00 (s,
1H, C(O)-NH-C), 6.13 (s, 1H, C(O)-NH-C), 6.22 (s, 1H, NH-C(O)-O), 7.13 (s, 1H, NHC(O)-O), 7.28-7.45 (m, 60 H, Ar-H), 7.84 (s, 1H, NH-C(O)-O); 13C NMR (CD2Cl2) δ =
28.4, 37.7, 38.7, 60.3, 60.4, 69.2, 69.3, 73.6, 75.0, 127.7,127.8, 127.9, 128.0, 128.5,
128.6, 138.7,138.9, 171.2, 174.0; LR FAB-MS m/z = 1956.7 (MH+), (Calcd. 1956.9);
Anal. Found (Calcd.). C 71.50 (71.79), H 7.29 (6.85), N 4.60 (5.01).
Supporting Information
- S7 -
H2N-Asp-Asp2(12OBn) (7) Trifluoroacetic acid (20 mL) was added dropwise
over 20 min to a solution of tBOC-N-Asp-Asp2(12OBn) (6) (4.02 g, 2.05 mmol) in
methylene chloride (100 mL). The solution was further stirred at room temperature for 5
h. The solvents were then removed under reduced pressure and the crude product
partitioned between methylene chloride (100 mL) and 1 M KOHaq (100 mL). The organic
phase was further washed with 1M KOHaq (2 × 100 mL) and brine (1 × 100 mL), and
dried with anhydrous MgSO4. Evaporation of the solvent yielded a clear oil that was
further purified by flash chromatography over silica eluting with a gradient of 100 %
CH2Cl2 to 94 % CH2Cl2 / 6 % CH3OH. The dendron (7) was obtained as a clear oil (2.34
g, 64 %) that was dried under high vacuum at room temperature for 15 hours. 1H NMR
(CD2Cl2) δ = 2.46-2.97 (m, 2H, CH-CH2-C(O)), 2.66-2.97 (m, 1H, C(O)-CH2-CH), 3.82
(bm, 24H, C-CH2-O), 4.45 (s, 24H, O-CH2-Ar), 4.58 (b, 1H, CH-CH2-C(O)), 4.63 (b,
1H, CH-CH2-C(O)), 6.16 (b, 2H, C(O)-NH-C), 7.28-7.45 (m, 60 H, Ar-H), 8.33 (s, 1H,
NH-C(O)-O);
13
C NMR (CD2Cl2) δ = 37.7, 38.7, 60.3, 60.4, 69.2, 73.6, 75.0,
127.7,127.8, 127.9, 128.0, 128.5, 128.6, 138.7,138.9, 171.2, 174.0; LR FAB-MS m/z =
1856.8 (MH+), (Calcd. 1856.9); Anal. Found (Calcd.). C 72.13 (72.43), H 7.16 (6.78), N
4.90 (5.28).
Supporting Information
- S8 -
TAM-Bn2-Asp-Asp2(12OBn) (9) - All glassware were dried in the oven overnight. TAM-Bn2 (8)4 (1.00 g, 2.64 mmol) was suspended in freshly distilled toluene (15
mL) which was purged with N2(g). DMF (2 drops) followed by oxalyl chloride (3 mL)
was added to the suspension. The reaction mixture was stirred at room temperature under
N2(g) until complete dissolution of the starting material (ca. 2 hours). Solvents were
removed under reduced pressure and the resulting acid chloride (yellow oil) was
immediately dissolved in freshly distilled THF (4mL). A solution of H2N-AspAsp2(12OBn) (7) (420 mg, 0.226 mmol) in freshly distilled THF (15 mL) and anhydrous
diisopropylethylamine (4 mL) was then added dropwise over 1 hour to the acid chloride
solution. The reaction mixture was stirred under N2(g) at room temperature for 18 hours.
Solvents were then removed under reduced pressure and the crude product partitioned
between methylene chloride (100 mL) and 1 M KOH(aq) (100 mL). The organic phase
was further washed with 1 M KOH (1 × 100 mL) and brine (1 × 100 mL) and dried with
anhydrous MgSO4. The solvents were then removed under reduced pressure. The crude
product was further purified by flash chromatography eluting with a gradient of 100 %
CH2Cl2 to 94 % CH2Cl2 / 6% CH3OH. The protected dendron (9) was obtained as a clear
oil (252 mg, 50.3 %) that was further dried under high vacuum at room temperature for
18 hours. 1H NMR (CD2Cl2) δ = 2.46-2.97 (m, 2H, CH-CH2-C(O)), 2.66-2.97 (m, 1H,
C(O)-CH2-CH), 3.82 (bm, 24H, C-CH2-O), 4.45 (s, 24H, O-CH2-Ar), 4.58 (b, 1H, CHCH2-C(O)), 4.63 (b, 1H, CH-CH2-C(O)), 4.63 (b, 1H, CH-CH2-C(O)), 4.98 (b, 2H, TAM
O-CH2-Ar), 5.26 (b, 2H, TAM O-CH2-Ar), 6.16 (b, 2H, C(O)-NH-C), 7.28-7.45 (m, 70
H, Ar-H), 7.76 (d, 1H, TAM-Ar-H), 7.80 (m, 1H, NH-C(O)-O), 7.84 (d, 1H, TAM-ArH), 8.01 (s, 1H, NH-C(O)-O); 13C NMR (CD2Cl2) δ = 37.7, 38.7, 60.3, 60.4, 69.2, 73.6,
75.0, 120.7, 123.5, 125.3, 127.5, 127.7, 127.8, 127.9, 128.0, 128.5, 128.6, 138.7,138.9,
146.3, 149.3, 167.9, 172.0, 171.2, 174.0; LR FAB-MS m/z = 2218.2 (MH+), (Calcd.
2218.0); Anal. Found (Calcd.). C 72.26 (72.58), H 6.74 (6.41), N 4.18 (4.42).
Supporting Information
- S9 -
TREN-bis(HOPO-Bn)-TAM-Bn2-Asp-Asp2(12OBn)
(11)
-
O-(7-
Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU, 63
mg, 0.166 mmol) and TAM-Bn2-Asp-Asp2(12OBn) (9) (183 mg, 82.5 µmol) were
dissolved in freshly distilled methylene chloride (10 mL). The solution was stirred at
room temperature for 30 minutes. A solution of TREN-bis(HOPO-Bn) (10)4 (79 mg, 125
µmol) dissolved in anhydrous methylene chloride (5 mL) and diisopropylethylamine (2
mL) was then added to the dendron solution. The reaction mixture was stirred at room
temperature for 18 hours. The solvents were then evaporated under reduced pressure and
the crude product was purified by flash chromatography eluting with a gradient of 100 %
CH2Cl2 to 96 % CH2Cl2 / 4 % CH3OH. The protected dendrimer (11) was obtained as a
clear oil (91 mg, 40%) that was further dried under high vacuum at room temperature for
18 hours. 1H NMR (CD2Cl2) δ = 2.29 (b, 2H, CH-CH2-C(O)), 2.37 (b, 2H, CH-CH2C(O)), 2.54 (dd, 1H, CH-CH2-C(O)), 2.74 (dd, 1H, CH-CH2-C(O)), 2.29 (s, 6H, N-CH2CH2-N(H)), 3.11 (s, 6H, N-CH2-CH2-N(H)), 3.49 (s, 6H, ArN-CH3), 3.69-3.82 (m, 24H,
C-CH2-O), 4.35-4.41 (m, 24H, O-CH2-Ar), 4.61-4.64 (m, 2H, CH-CH2-C(O)), 4.77 (m,
1H, CH-CH2-C(O)), 4.96 (b, 2H, TAM O-CH2-Ar), 5.14-5.26 (dd, 2H, TAM O-CH2-Ar),
5.27 (bs, 4H, HOPO O-CH2-Ar), 6.07(s, 1H, NH-C(O)-O), 6.21 (s, 1H, NH-C(O)-O),
6.52 (d, 2H, HOPO Ar-H, J = 7), 7.02 (s, 1H, NH-C(O)-O), 7.05 (d, 2H, HOPO Ar-H, J
= 7), 7.06-7.38 (m, 80H, Ar-H), 7.56 (s, 1H, NH-C(O)-O), 7.60 (d, 1H, TAM Ar-H, J =
8), 7.72 (d, 1H, TAM Ar-H, J = 8), 7.76 (s, 2H, NH-C(O)-O), 7.94 (s, 1H, NH-C(O)-O);
13
C NMR (CD2Cl2) δ = 37.7, 38.7, 43.2, 43.3, 52.6, 55.5, 60.3, 60.4, 69.2, 73.6, 75.0,
120.7, 123.5, 125.3, 127.5, 127.7, 127.8, 127.9, 128.0, 128.5, 128.6, 138.7,138.9, 146.3,
149.3, 103.0, 125.9, 158.9, 163.9, 166.0, 167.9, 172.0, 171.2, 174.0; LR FAB-MS m/z =
2829.2 (MH+), (Calcd. 2829.3); Anal. Found (Calcd.). C 71.07 (71.34), H 6.79 (6.38), N
6.11 (6.44).
Supporting Information
- S10 -
TREN-bisHOPO-TAM-Asp-Asp2(12OH) (12) – TREN-bis(HOPO-Bn)-TAMBn2-Asp-Asp2(12OBn) (11) (150 mg, 53.0 µmol) was dissolved in glacial acetic acid (5
mL). Millipore water (1 mL), and 10 % wet Pd/C (32 mg) were then added to the
solution. The reaction mixture was stirred under H2 (80 atm) at room temperature for 48
hours. The Pd/C was then filtered, washed with acetic acid, and the filtrate was
evaporated to dryness. The resulting white solid was dissolved in methanol (50 mL) and
the solvent removed under reduced pressure. This last step was repeated until no more
acetic acid could be detected. The product was then redissolved in methanol (5 mL) and
added dropwise to diethyl ether (500 mL) under vigorous agitation. The resulting
precipitate was filtered, rinsed with diethyl ether, and dried under high vacuum at 50 oC
for 18 hours. The deprotected dendrimer (12) (71 mg, 97 %) was obtained as white
powder. 2.27 (b, 2H, CH-CH2-C(O)), 2.36 (b, 2H, CH-CH2-C(O)), 2.56 (b, 1H, CH-CH2C(O)), 2.73 (b, 1H, CH-CH2-C(O)), 2.31 (s, 6H, N-CH2-CH2-N(H)), 3.15 (s, 6H, N-CH2CH2-N(H)), 3.51 (s, 6H, ArN-CH3), 3.71-3.86 (m, 24H, C-CH2-O), 4.60-4.65 (m, 2H,
CH-CH2-C(O)), 4.73-4.77 (m, 1H, CH-CH2-C(O)), 6.09(s, 1H, NH-C(O)-O), 6.18 (s, 1H,
NH-C(O)-O), 6.46 (d, 2H, HOPO Ar-H, J = 7), 7.02 (s, 1H, NH-C(O)-O), 7.12 (d, 2H,
HOPO Ar-H, J = 7), 7. 26 (s, 1H, NH-C(O)) 7.31 (d, 1H, TAM Ar-H, J = 8), 7.34 (d,
1H, TAM Ar-H, J = 8), 7.62 (s, 2H, NH-C(O)), 7.87 (s, 2H, NH-C(O)), 8.10 (s, 1H, NHC(O)-O); LR FAB-MS m/z = 1386.5 (MH+), (Calcd. 1386.6); Anal. Found (Calcd.). C
48.10 (48.52), H 6.46 (6.03), N 12.77 (13.13).
Supporting Information
- S11 -
Gd-TREN-bisHOPO-TAM-Asp-Asp2-12OH (2) – A solution of Gd(acac)3
(15.3 mg, 30.1 µmol) in degassed methanol (1.5 mL) was added to a solution of TrenbisHOPO-TAM-Asp-Asp-12OH (12) (47.9 mg, 30.6 µmol) in degassed methanol (10
mL). The solution was purged with N2(g) and pyridine (4 drops) was added. The solution
was refluxed for 4 h under N2(g) during which time a small amount of white precipitate
appeared. The suspension was cooled to room temperature and precipitated in diethyl
ether (250 mL). The ether suspension was stirred at room temperature for 2 h. The
precipitate was then filtered, rinsed with diethyl ether (2 × 15 mL) and dried under high
vacuum for 15 h at room temperature. The Gd complex (2) was obtained as a beige
powder (47.7 mg, 95.0 %). ES-MS m/z = 770 (M2- Calcd. 770). The isotopic distribution
corresponded to that calculated for a doubly charged complex. MALDI-MS m/z = 1538
(M- Calcd. 1540). The isotropic distribution corresponded to that calculated for a mono
charged complex. Anal. Found (Calcd.) for (2)·5H2O C 40.11 (40.38), H 5.75 (5.63), N
10.60 (10.93), Gd 9.31 (9.44).