A facile synthesis of α-amino-DOTA as a versatile molecular imaging probe

Article abstract of DOI:10.1016/j.tetlet.2006.08.019

An amino group has been introduced into one ligand of DOTA that can couple to peptidyl carboxylates by coupling α-brominated glycine to DO3A-^tBu (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid, tert-butylester)). α-Amino-DOTA was coupled

Full text of DOI:10.1016/j.tetlet.2006.08.019

Tetrahedron Letters 47 (2006) 7327–7330
A facile synthesis of a-amino-DOTA as a versatile
molecular imaging probe
Byunghee Yoo and Mark D. Pagel*
Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue Cleveland, OH 44106-7207, USA
Received 5 January 2006; revised 3 August 2006; accepted 9 August 2006
Available online 30 August 2006
Abstract—An amino group has been introduced into one ligand of DOTA that can couple to peptidyl carboxylates by coupling
a-brominated glycine to DO3A-tBu (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid, tert-butylester)). a-Amino-DOTA was
coupled to the carboxylate backbone terminus of a peptide to demonstrate the utility for derivatization.
Ó 2006 Elsevier Ltd. All rights reserved.
Macrocyclic metal chelates using DOTA are often
administered to in vivo patients and animal models to
create or enhance contrast in biomedical molecular
side chain amino group can compromise the utility of
the peptide for molecular imaging applications, because
peptide–biomolecule interactions critically depend on
the (native) peptide amino acid sequence.
imaging studies. Examples include paramagnetic Gd3+
-
DOTA for magnetic resonance imaging (MRI),1
radioactive 111In-DOTA for SPECT imaging,2 and
radioactive 64Cu-DOTA for PET imaging.3 More
recently, metal-DOTA chelates have been conjugated
to peptides to affect the pharmacokinetics of the me-
tal-DOTA imaging agent within in vivo systems, which
can be used to gain additional information about
biological processes at the molecular level. A variety of
peptidyl ligands4 have been employed for these molecu-
lar imaging studies, including peptides that bind to spe-
cific cell surface receptors,5 peptides that penetrate cell
membranes,6 peptides that nonspecifically interact with
the extracellular matrix,7 and very large peptide homo-
polymers that drastically alter renal clearance rates.8
To address these limitations, a facile methodology is re-
quired for conjugating DOTA to peptide carboxylates,
especially the C-terminus of a peptide. We have devel-
oped new a-amino-DOTA derivatives to conjugate
directly to the C-terminus of a peptide and used two
different glycine templates (BOC and CBZ protected)
to accommodate future applications via orthogonal pro-
tection strategies. BOC protection is more advantageous
in solution phase synthesis, and CBZ protection has
advantages in orthogonal synthesis and more complex
structures. We have also chelated lanthanide ions with
these DOTA derivatives. Finally, we have confirmed
that a-amino-DOTA-Gd3+ has good T1 MR relaxivity
to demonstrate that these DOTA derivatives may serve
as molecular imaging agents.
To synthesize these peptidyl imaging agents, the carb-
oxylates of DOTA have been conjugated to the amines
of peptides, including the N-terminus, the side chain of
lysine, and unnatural amino acid derivatives such as
p-NH2-phenylalanine.9 Other DOTA derivatives have
been devised for conjugation to peptide amino groups,
such as succinimide DOTA derivatives10 and isothiocy-
anate DOTA derivatives.11 However, coupling DOTA
only to peptide amines can limit synthesis methodolo-
gies.12 Also, modification of a peptide N-terminus or
Two different synthetic pathways for the a-bromination
of glycine templates are shown in Scheme 1. The com-
pound 213 was obtained by following previously
reported methods by use of N-bromosuccinimide and
filtered UV radiation (254 nm). The bromination showed
high efficiency in purity as determined by NMR.14 The
compounds 5–715 were synthesized according to a
previously reported synthetic pathway16 and acquired
with overall yield of 70% as determined by weight.
Keywords: DOTA; T1 relaxivity; MRI; Molecular imaging.
*
The compound 917 was synthesized from 2 and 8
Corresponding author. Tel.: +1 216 368 8519; fax: +1 216 368
and purified with a silica column. To continue further
4969; e-mail: mpagel@case.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.08.019

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B. Yoo, M. D. Pagel / Tetrahedron Letters 47 (2006) 7327–7330
O
O
NH2
O
+
N
O
OH
O
H
O
O
O
3
4
1
ii
i
OH
O
N
OH
Br
H
O
O
5
O
N
H
O
iii
O
O
2
O
O
N
O
H
O
O
6
iv
Br
O
N
O
H
O
O
7
Scheme 1. Synthesis of a-brominated glycine templates. Reagents and conditions: (i) N-bromosuccinimide, UV (254 nm, filtered), CCl4, 25 °C, 1.5 h,
97%; (ii) diethyl ether, rt, 6 days, 96%; (iii) MeOH, H2SO4, rt, 2 days, 95%; (iv) PBr3, CCl4, rt, 7 days, 75%.
experiments, the protecting groups of 9 were completely
removed with a cleavage cocktail (95% TFA/2.5%
water/2.5% thioanisole) for 30 min. The solution was
concentrated in vacuo and precipitated with ice-cooled
diethyl ether and purified with an Amberlite column,
yielding 1118 as a white solid (Scheme 2).
resin (Fluka, 0.75 mmol/g), HBTU and HOBt coupling
agents, and standard Fmoc chemistry protocols using an
Applied Biosystems 433 A peptide synthesizer.20 The
synthesized peptide sequence was selected to be a sub-
strate for the MMP-2 enzyme.21 The MMP-2 targeting
peptide sequence was Ac-Pro-Leu-Gly-Met-Trp-Ser-
Gly (Ac-PLG-MWSG) and the interaction of the pep-
tide-a-amino-DOTA complex with MMP-2 enzyme will
be the focus of a future report. The peptide was cleaved
from the resin with a 95% TFA/2.5% water/2.5% thio-
anisole cocktail for 30 min.22 The peptide was purified
by crystallization in dichloromethane/diethyl ether and
an Amberlite column (yield 90% by weight) and charac-
terized with a MALDI mass spectrometer (m/z: 811.90
(calcd 811.91) [M+Na]+).
To prepare 10,19 the compound 7 was coupled to 8 using
exhaustive alkylation conditions and purified with a silica
column. To remove the CBZ group by hydrogenolysis, 10
was dissolved in 10 mL of absolute ethanol, and 1,4-
hexadiene (0.94 mL, 10 mmol) and 10% Pd/C (0.55 g,
10 mmol) were added to the solution. The remaining
methyl and tert-butyl protecting groups were removed
by hydrolysis using a 1 N-NaOH solution. The aqueous
solution was lyophilized after purification with an Amber-
lite column, yielding a slightly yellowish solid of 11.18
To show the versatile application of coupling 11 to the C-
terminus of a peptide to synthesize peptidyl-DOTA
structure 13, the synthesized peptide (120 mg, 0.15 mmol)
was dissolved in NMP (5 mL) with 63 mg of HBTU and
26 mg of HOBt and stirred for 40 min to activate the
carboxyl group of the peptide. Compound 11 (80 mg,
0.15 mmol) and TEA (110 lL, 0.75 mmol) in 1 mL of
NMP was added slowly at room temperature. The reac-
tion mixture was stirred for 1 h. After removal of half
of the solvents under reduced pressure, the product 13
was precipitated by addition of diethyl ether (100 mL).
The obtained crude product was purified with an Amber-
lite column, yielding the product as a white solid (yield
71% by weight) and characterized with a MALDI-MASS
spectrometer (m/z: 1190.3 (calcd 1189.54) [M+H]+).
Due to the bulky protecting groups of 2 and 7, the reac-
tion yields were expected to be lower than conventional
exhaustive alkylation reactions,12 but the reactions
showed sufficiently high yields to be used in subsequent
synthesis steps without further purification. This demon-
strates the facile nature of the synthesis methodology.
The a-bromo glycine templates were produced as race-
mates so that (R)- and (S)-types were formed at approxi-
mately a 1 to 1 ratio, as evidenced by doublets at 5.20
and 5.46 ppm in the NMR spectrum. After the a-bromo
glycine templates were coupled to 8, the diastereomers in
9 and 10 were also determined to be in the same ratio as
the a-bromo glycine templates.
To demonstrate the coupling of 11 with the C-terminus
of a peptide, a peptide was synthesized with a Wang
To verify that 11 can serve as a molecular imaging
contrast agent, the T1 relaxivity of 12 was measured to

B. Yoo, M. D. Pagel / Tetrahedron Letters 47 (2006) 7327–7330
7329
Br
Br
O
O
N
N
H
O
O
O
O
H
N
+
+
N
O
N
N
O
N
N
O
H
H
O
O
O
N
O
O
O
N
O
O
O
O
7
2
8
8
O
ii
i
OH
O
O
O
O
O
O
O
HN
N
O
HN
N
OH
H2N
O
N
N
N
N
iv, v
O
iii
N
O
O
N
N
O
O
O
O
O
N
N
O
N
O
HO
O
O
O
O
O
OH
10
9
11
vi, vii
vi, viii, ix
OH
OH
O
O
OH
H2N
O
Ac-PLGMWSG-NH
OH
N
N
N
N
N
N
N
Gd3+
N
O
O
O
HO
HO
O
O
OH
13
OH
12
Scheme 2. Synthesis of a-amino-DOTA derivatives and coupling to a peptide backbone carboxylate group. Reagents and conditions: (i) K2CO3
(6 equiv), acetonitrile, 70 °C, 6 h, 95%; (ii) K2CO3 (6 equiv), acetonitrile, 70 °C, 6 h, 90%; (iii) 95%TFA/2.5%water/2.5%thioanisole, 30 min, 95%; (iv)
1,4-cyclohexadiene/10% Pd–C, EtOH; (v) 1 N-NaOH; (vi) Amberlite column; 71%; (vii) pH, 60 °C, GdCl3 (150 lL of 0.4 mM solution), 48 h; (viii)
Ac-PLGMWSG-OH (1 equiv), HBTU (1.1 equiv), HOBt (1.1 equiv), TEA (5 equiv), NMP, rt, 1 h; (ix) dialysis (MWCO-100); total yield 85% by
weight.
assess the efficiency of the chelate to alter T1-weighted
MR image contrast. Compound 11 (25.2 mg, 0.06
mmol) was dissolved in water at pH 6 and 60 °C, and
GdCl3 (150 lL of 0.4 mM solution) was added to
this solution and stirred for 1 h. The pH was adjusted
to 8 using 1 N-NaOH and stirred for 48 h. The chelation
was monitored using a standard Arsenazo III color
test.23 T1 measurements of 12 were conducted at 18 °C
and pH 7.1, with samples ranging from 5 to 50 mM.
The T1 inversion-recovery experiment was conducted
with a 600 MHz NMR spectrometer. The measured
relaxivity of 4.83 mMÀ1 sÀ1 compared very favorably
to the 3.83 mMÀ1 sÀ1 relaxivity of Gd3+-DOTA that is
used in clinical MRI studies.24
Acknowledgment
This work was supported by the US Army Medical
Research and Material Command under #W81XWH-
04-1-0731.
References and notes
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This report has demonstrated the preparation and appli-
cation of newly synthesized chelators for lanthanides,
11–13. Also, compound 11 was synthesized from two
different glycine templates to accommodate future solu-
tion phase and solid phase synthesis methods. The
incorporation of an amino group facilitated the conju-
gation of 11 to the C-terminus of a peptide backbone
with high efficiency and relatively high yield. Coupling
of 11 to peptide carboxylates greatly expands peptide–
DOTA synthesis strategies by complementing standard
methods that couple DOTA to peptide amines. Further-
more, the number of amino groups incorporated into
DOTA can be controlled from 1 to 4 by the selection
of the macrocyclic starting material, such as cyclen
(1,4,7,10-tetraazacyclododecane), DO1A-tBu, DO2A-
tBu, or DO3A-tBu.

7330
B. Yoo, M. D. Pagel / Tetrahedron Letters 47 (2006) 7327–7330
17. Compound (0.62 g, 2.0 mmol) in dry acetonitrile
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presence of K2CO3 (1.66 g, 12 mmol, 6 equiv). The reac-
tion mixture was heated to 70 °C for 6 h. After removal of
the undissolved solids by filtration, the solution was
concentrated in vacuo. The product was purified with
silica column using ethylacetate as an eluent, yielding the
product as a powdery solid (yield 95% by weight, with
purity confirmed using NMR). 1H NMR (300 MHz,
CDCl3) d 1.45 (s, 45H), 2.76 (t, 16H), 3.40 (s, 6H), 5.20
and 5.46 (split, 1H); 13C (125 MHz, DMSO-d6) d 171.45,
171.43, 171.37, 80.78, 80.65, 77.61, 57.45, 51.96, 46.93,
28.46, 28.30; MS-ESI m/z: 744.44 (calcd 743.50) [M+H]+.
18. Yield 90% by weight, with purity confirmed using NMR.
1H NMR (300 MHz, CDCl3) d 2.0 (s, 2H), 2.80 (t, 16H),
3.60 (s, 6H), 5.20 and 5.46 (split, 1H); 13C (125 MHz,
DMSO-d6) d 173.44, 172.87, 78.52, 53.33, 52.14, 47.93;
MS-ESI m/z: 459.31 (calcd 458.17) [M+K]+.
19. Compound 7 (0.6 g, 2 mmol) was coupled to 8 (1.1 g,
2.1 mmol) using the exhaustive alkylation conditions and
the product was purified with silica column using ethyl
acetate as an eluent (yield 90% by weight, with purity
confirmed using NMR). 1H NMR (300 MHz, CDCl3) d
1.45 (s, 27H), 2.78 (t, 16H), 3.40 (s, 6H), 3.80 (s, 3H), 5.20
and 5.46 (split, 1H), 7.20 (s, 5H); 13C (125 MHz, DMSO-
d6) d 171.43, 170.44, 170.10, 156.53, 136.71, 129.68,
128.22, 127.64, 79.06, 78.85, 78.63, 69.72, 65.51, 64.81,
57.96, 56.20, 55.75, 55.56, 51.89, 51.37, 49.76, 49.31, 48.35,
47.72, 47.39, 45.80, 27.75; MS-ESI m/z: 736.34 (calcd
735.44) [M+H]+.
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254 nm filtered UV lamp at 25 °C for 1.5 h (3.00 g, yield
97% by weight, with purity confirmed using NMR). 1H
NMR (300 MHz, CDCl3) d 1.45 (s, 9H), 1.50 (s, 9H), 5.90
(d, 1H), 6.20 (d, 1H); 13C (125 MHz, DMSO-d6) d 171.01,
156.29, 79.51, 63.72, 28.53; MS-ESI m/z: 310.02 (calcd
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6.20 and 6.45 (split, 1H), 7.55 (s, 5H); 13C (125 MHz,
DMSO-d6) d 170.71, 156.07, 137.37, 129.03, 128.57,
128.53, 73.78, 66.25; MS-ESI m/z: 304.07 (calcd 302.99)
[M+H]+.
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