An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2009.05.061

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was

Full text of DOI:10.1016/j.tetlet.2009.05.061

Tetrahedron Letters 50 (2009) 4459–4462
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid
phase peptide synthesis
Byunghee Yoo a, Vipul R. Sheth b, Mark D. Pagel a,c,
*
a Biomedical Engineering Interdisciplinary Program, The University of Arizona, Tucson, AZ, USA
b Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA
c Department of Chemistry, The University of Arizona, Tucson, AZ, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional
solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was
used to synthesize a peptide–DOTA conjugate that was demonstrated to be a PARACEST MRI contrast
agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast
agents.
Received 17 April 2009
Revised 15 May 2009
Accepted 15 May 2009
Available online 23 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
DOTA
SPPS
Fmoc
PARACEST
MRI
Molecular Imaging
Molecular imaging has recently emerged as a powerful method
to provide medical information at the molecular level.1 Molecular
imaging contrast agents have been conjugated to many types of pep-
tides that target cell receptors and enzymes, in order to diagnose
pathological tissues and assess early therapeutic responses.2–5 Mac-
rocyclic metal chelates, such as metalated 1,4,7,10-tetraazacyclod-
odecane-N,N0,N00,N000-tetraacetic acid (DOTA) have been conjugated
to peptides and detected with MRI, PET, and SPECT imaging.6–8 As
a testament to their potential, 23% (187 of 820) of the journal publi-
cations that describe molecular imaging contrast agents with metal
chelates contain one or more peptidyl ligands. As a testament to
their demonstrated utility for biomedical imaging, 8% (46 of 594)
of the entries in the molecular imaging contrast agent database (MI-
CAD) consist of metal chelates with one or more peptidyl ligands.9
The facile synthesis of metal chelate contrast agents that in-
clude peptides is needed to accelerate the research and clinical
translation of molecular imaging. Previously reported methods
have conjugated the carboxylates of DOTA to the amines of pep-
tides, including the N-terminus, the side chain of lysine, and unnat-
ural amino acid derivatives.10 DOTA derivatives of succinimide and
isothiocyanate have also been conjugated to peptide amino
groups.11,12 However, coupling DOTA only to peptide amines can
limit synthesis methodologies.13,14
To resolve these limitations in the synthesis of peptidyl DOTA
conjugates, we previously developed an amine-derivatized DOTA
and used this product to couple DOTA to the C-terminus of a pep-
tide using solution-phase methods.15 We have also loaded an
amine-derivatized DOTA onto a polymeric support, and used this
product to couple DOTA to the peptide C-terminus and within
the peptide backbone using standard solid phase peptide synthesis
(SPPS) methods.16 These synthetic pathways used a CBZ group to
protect the
a-amino group of the DOTA derivative. Cleavage of
the CBZ group required harsh conditions using diethylaluminum
chloride/thioanisole at ꢀ78°.17 These harsh conditions also cleaved
the DOTA from the resin, which caused a lower concentration of
DOTA on the polymeric support and consequently reduced the fi-
nal yield of peptide–DOTA conjugates.
To address these limitations, we have converted the CBZ pro-
tecting group of an amine-derivatized DOTA to a Fmoc protecting
group before loading DOTA onto the resin. This report describes
the new synthetic pathway to prepare an Fmoc-protected,
amine-derivatized, DOTA-loaded polymeric support that is appli-
cable to conventional Fmoc-SPPS. This product was used to synthe-
size a peptide–DOTA conjugate with high yield and high purity.
Finally, this peptide–DOTA contrast agent was shown to have util-
ity as a PARACEST MRI contrast agent.
CBZ-Gly(Br)-OMe (methyl 2-(benzyloxycarbonylamino)-2-bro-
moacetate) was prepared with overall yield of 70% following a pre-
viously reported synthetic pathway.18,19 Compound
synthesized from 1 with tert-butyl bromoacetate in overall yield
2
was
* Corresponding author. Tel.: +1 520 404 7049; fax: +1 520 626 0395.
E-mail address: mpagel@u.arizona.edu (M.D. Pagel).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.05.061

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B. Yoo et al. / Tetrahedron Letters 50 (2009) 4459–4462
of 83%.20,21 Compound 3 was synthesized from CBZ-Gly(Br)-OMe
and 2 through exhaustive alkylation in the presence of K2CO3,
yielding a pale yellowish solid (83%).22 Subsequently, compound
4 was obtained quantitatively by the deprotection of the CBZ group
from compound 3 through hydrogenolysis using 10%-Pd/C.23 The
hydrogenolysis reaction continued for 4 h until there was no
change in H2 pressure, yielding product quantitatively as a pale
yellowish solid. Compound 5 was prepared from compound 4 by
use of Fmoc-Cl with zinc dust.24 In this step, a small portion of acti-
vated zinc dust was added until the reaction mixture attained neu-
tral pH and the mixture of Fmoc-Cl and zinc dust was added to the
reaction solution in one portion. The reaction time was 20 min. Un-
reacted Fulvene was washed with diethyl ether and the product
was dried to yield a white solid (87%).25 To prevent the formation
of a zinc chelate during this step, zinc ions were removed from the
zinc dust by thoroughly washing the dust with sequential treat-
ments of distilled water, EtOH, acetone, and MC. Furthermore,
the preparation of compound 5 was carried out in acetonitrile,
which minimized dissolution of zinc ions from zinc dust that is
insoluble in acetonitrile. The mass spectrum showed no evidence
that a zinc chelate was formed during this step. Compound 6
was obtained from compound 5 (4.1 g, 5 mmol) by the deprotec-
tion of tert-butyl ester using 50% TFA in MC for 2 h, yielding a pale
yellowish product quantitatively.26
was dispersed in MC and treated with acetic anhydride (Ac2O). The
concentration of the amino group was quantified as 0.41 mmol/g
using a Fmoc titration method29 and 0.44 mmol/g using picric acid
titration30 after correcting for the effect of tertiary amines on DOTA
from the results of Fmoc-NH-DOTA resin. These quantifications of
resin loading were based on the 1.61 g weight of the product 7, and
not the 1.00 g weight of the initial Wang resin, following the con-
vention in SPPS methodology. Compared with the initial concen-
tration of functional group of Wang resin, the loading efficiency
was 71–76%.
To demonstrate the synthesis of a peptide–DOTA conjugate, a
Wang resin 7 was then used to synthesize a peptide, CBZ-Gly-
Gly-Arg (ZGGR). This peptide is a substrate for urokinase-type
plasminogenequivvator (uPA), and the ability of the peptide–
DOTA conjugate to detect uPA is the focus of ongoing research
studies in our laboratory.31 Compound
7 was treated with
Fmoc-Arg(pbf)-OH/HBTU/HOBt/DIEA for 1 h and washed with
NMP. The same reaction was conducted a second time to double
couple the arginine amino acid to 7. The Fmoc group was depro-
tected with 20% piperidine in NMP for 40 min. Following the
same procedures, Fmoc-Gly and CBZ-Gly were double coupled
onto the resin. The peptide–DOTA conjugate was cleaved with
95% TFA in MC. The final product was precipitated in diethyl
ether and dried in vacuo, yielding white solids (88% as deter-
mined by weight, relative to the original concentration of the
amine on 7). The identity of the peptide–DOTA conjugate was
confirmed with mass spectrometry.
Compounds 3–6 have a geminal amine structure after coupling
CBZ-Gly(Br)-OMe to 2 in Step b (Fig. 1 and Scheme 1). To confirm
the successful synthesis of compounds 3–6, the chemical shifts of
the
thetic step. From 1D 1H and 13C NMR spectra, the peaks from H
and C were assigned to resonances at 5.53 ppm and 92.59 ppm
a
-proton and
a
-carbon, H
a
and C
a
, were tracked for each syn-
To chelate a lanthanide ion with this product, Z-Gly-Gly-Arg-
DOTA was first dissolved in acetonitrile. The solution was filtered
with a 0.2 lm PTFE membrane filter to remove undissolved solids
a
a
for compound 3, 4.23 ppm and 85.12 ppm for compound 4,
5.25 ppm and 84.83 ppm for compound 5, and 5.27 ppm,
84.56 ppm for compound 6, respectively. The 1H NMR spectra of
such as TFA salts. Tm-triflate was added to the solution and the
solution was stirred at 50 °C for 12 h. The pH was monitored for
4 h and DIEA was added as an acid scavenger to maintain a neutral
or weakly basic condition when a decrease in pH was detected. The
reaction mixture was concentrated and re-dissolved in water at pH
9 to remove excess Tm(III) that formed a Tm(OH)3 precipitate. The
pH was adjusted to 7 and the solution was lyophilized to yield 8 as
a white fluffy powder.
Ha
are shown in Figure 2.
Two equivalents of compound 6 (1.86 mmol) relative to the hy-
droxyl group content on the resin were used to modify the surface
of Wang resin (0.93 mmol/g) with HBTU (4 equiv)/HOBt
a
(1 equiv)/DIEA (10 equiv) for 1 day. After washing the resin with
NMP, the resin was treated with 50% tert-butanol in MC for
4 h.27,28 To cap un-reacted hydroxyl groups on the resin, the resin
The utility of the peptide–DOTA chelate for molecular imaging
studies of uPA was investigated by measuring the agent’s PARA-
Figure 1. NMR spectra of Ha in compounds 3–6. The peak from Ha is marked with an arrow.

B. Yoo et al. / Tetrahedron Letters 50 (2009) 4459–4462
4461
Scheme 1. Synthesis of a-amino-DOTA and a peptide–DOTA PARACEST MRI contrast agent. (a) tert-butylbromoacetate, K2CO3 (6 equiv), acetonitrile, 70 °C, 6 h, 83%; (b) CBZ-
Gly(Br)-OMe, K2CO3 (6 equiv), acetonitrile, 70 °C, 6 h, 83%; (c) H2, 10-Pd/C, ethanol; 2 h, quantitative; (d) Fmoc-Cl, zinc dust, acetonitrile, 87%; (e) 50% TFA in CH2Cl2,
quantitative; (f) (i) Wang resin, HBTU (4 equiv), HOBt (1 equiv), DIEA (10 equiv), NMP, rt, overnight; (ii) 50% tert-BuOH in CH2Cl2, 70%; (g) (i) 20% piperidine, NMP; (ii) SPPS of
Z-Gly-Gly-Arg(pbf) following standard Fmoc-chemistry (iii) 95% TFA, CH2Cl2, 4 h; (iv) Tm-triflate, acetonitrile, 50 °C, overnight.
Figure 2. Detection of urokinase Plasminogen Activator (uPA) with 8. (A) The proposed mechanism of uPA cleavage of 8 and the PARACEST spectra of 8 shows that an amide
proton (red) is converted to an amine (blue) after uPA cleaves the ZGGR peptide ligand from the agent. (B) The CEST spectrum showed a CEST effect from the amide at
ꢀ52 ppm before uPA was added (red). The disappearance of this CEST effect after uPA was added (blue) was used to detect uPA. An enzyme-unresponsive agent, Yb-DOTA-
Gly, shows a CEST effect at ꢀ16 ppm before and after uPA was added, which served as an internal control.
magnetic chemical exchange saturation transfer (PARACEST) effect.
A sample was prepared that consisted of 50 mM of 8 in HEPES buf-
fer at pH 7.2 and with 5% D2O, and was incubated at 37 °C over-
night with 100 U of uPA enzyme (EMD Biosciences Inc.) (Figure
2). A PARACEST agent that is unresponsive to uPA, Yb-DOTA-Gly4
was also included in the sample at 20 mM concentration as a con-
trol. Two CEST spectra were obtained before and after uPA incuba-
tion by acquiring a series of 1D NMR spectra with a 300 MHz
Varian NMR spectrometer at 37 °C and with frequency-selective
presaturation applied in 1 ppm increments between 70 ppm and
ꢀ70 ppm. This selective presaturation was performed with a con-
tinuous wave pulse applied for 4 s at 21 lT. Before incubation with
uPA, the CEST spectrum showed PARACEST effects at ꢀ16 ppm and
ꢀ52 ppm relative to water (which was referenced to 0 ppm to fol-
low the convention used for MRI studies).32 The PARACEST effect at
ꢀ16 ppm was assigned to Yb-DOTA-Gly4 and the PARACEST effect
at ꢀ52 ppm was assigned to 8 based on previous reports of the
PARACEST effects of Yb and Tm chelates of a peptide-(Tm-DOTA)
conjugate and Yb-DOTA-Gly4.33,34 The PARACEST effect at
ꢀ52 ppm disappeared after incubation with uPA, while the PARA-
CEST effect at ꢀ16 ppm was unchanged. Thus, 8 can be detected
using PARACEST MRI and can be used to detect uPA. However,

4462
B. Yoo et al. / Tetrahedron Letters 50 (2009) 4459–4462
14. De Leon-Rodriquez, L. M.; Kovacs, Z.; Dieckmann, G. R.; Sherry, A. D. Chem. Eur.
J. 2004, 10, 1149.
additional kinetics studies are warranted to prove that this change
in the PARACEST effect is due to the catalysis mechanism of the
uPA enzyme.
15. Yoo, B.; Pagel, M. D. Tetrahedron Lett. 2006, 47, 7327.
16. Yoo, B.; Pagel, M. D. Bioconjugate Chem. 2007, 18, 903.
17. Tsujimoto, T.; Murai, A. Synlett 2002, 8, 1283.
In conclusion, a new synthetic method has been demonstrated
that prepares an Fmoc-protected, amine-derivatized, DOTA-loaded
polymeric support for standard Fmoc SPPS. This polymeric support
can be used to conjugate DOTA to the C-terminus of a peptide with
high yield and high purity using conventional Fmoc-SPPS. Consid-
ering that a fully compatible Fmoc-SPPS method can facilitate
scale-up of a synthesis methodology, this method may be more
practical for commercial applications. Furthermore, Fmoc SPPS is
applicable to building peptide or chemical libraries, which may
further facilitate the development of diagnostic molecular imaging
contrast agents.
18. Methyl 2-(benzyloxycarbonylamino)-2-bromoacetate (CBZ-Gly(Br)-OMe). 1H
NMR (300 MHz, CDCl3) d 1.45 (s, 9H), 1.50 (s, 9H), 5.90 (d, 1H), 6.20 (d, 1H),
7.35 (m, 5H) ; 13C (125 MHz, CDCl3) d 170.71, 156.07, 137.37, 129.03, 128.57,
128.53, 73.78, 66.25; MS-ESI m/z : 304.07 [M+H]+ (calcd for C11H12BrNO4
302.99).
19. Williams, R. M.; Aldous, D. J.; Aldous, S. C. J. Org. Chem. 1990, 55, 4657.
20. Compound
2:
tert-Butyl
2,20,200-(1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetate (DO3A-tBu); 1H NMR (300 MHz, CDCl3) d 1.38 (s, 27H), 2.75
(m, 16H), 3.27 (s, 6H) ; 13C (125 MHz, CDCl3) d 172.59, 170.16, 169.08, 81.85,
57.90, 57.80, 55.48, 53.49, 51.13, 50.87, 49.25, 47.94, 47.15, 45.81, 28.04; MS-
ESI m/z : 515.41 [M+H]+ (calcd for C26H50N4O6 514.37).
21. Li, C.; Wong, W. T. Tetrahedron 2004, 40, 5595.
22. Compound 3: tert-butyl 2,20,200-(10-(1-(benzyloxycarbonylamino)-2-methoxy-
2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate
(CBZ-NH-
DOTA-tBu); 1H NMR (300 MHz, CDCl3) d 1.35 (s, 27H), 2.75 (t, 16H), 3.2 (s,
6H), 3.50 (s, 3H), 5.00 (s, 2H), 5.35 (d, 1H), 7.13 (m, 5H), 6.40 (d, 1H); 13C
(125 MHz, DMSO-d6) d 172.38, 170.19, 169.35, 156.68, 136.21, 128.08, 127.56,
126.45, 92.59, 81.15, 66.49, 69.72, 57.37, 55.19, 52.81, 52.24, 51.11, 50.77,
49.94, 47.70, 27.65; MS-ESI m/z : 758.43 [M+Na]+ (calcd. C37H61N5O10 735.91).
Acknowledgment
This work was supported by the National Cancer Institute
through NIH Grant R21 CA133455-01.
23. Compound
4:
tert-butyl
2,20,200-(10-(1-amino-2-methoxy-2-oxoethyl)-
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (NH2-DOTA-tBu); 1H
NMR (300 MHz, CDCl3) d 1.37 (s, 27H), 2.75 (t, 16H), 3.30 (s, 6H), 3.55 (s,
3H), 4.55 (s, 1H); 13C (125 MHz, DMSO-d6) d 170.24, 169.39, 163.21, 85.12,
81.98, 81.49, 57.86, 56.36, 55.44, 54.83, 52.72, 51.10, 49.03, 28.05, 27.92; MS-
ESI m/z : 602.43 [M+H]+ (calcd. C29H55N5O8 601.41).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.05.061.
24. Gopi, H. N.; Suresh Babu, V. V. J. Peptide Res. 2000, 55, 295.
25. Compound 5: tert-butyl 2,20,200-(10-(1-(((9H-fluoren-9-yl)methoxy)carbon-
ylamino)-2-methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetate (Fmoc-NH-DOTA-tBu); 1H NMR (300 MHz, CDCl3) d 1.40 (s, 27H),
2.85 (t, 16H), 3.55 (s, 6H), 3.90 (s, 3H), 4.15 (s, 2H), 4.4 (d, 1H), 5.36 (s, 1H), 7.5
(m, 8H); 13C (125 MHz, DMSO-d6) d 169.10, 163.42, 156.2, 144.41, 141.29,
127.34, 127.03, 124.73, 119.93, 84.83, 81.50, 65.01, 56.52, 55.00, 50.31, 47.50,
28.07; MS-ESI m/z : 846.40 [M+Na]+ (calcd for C44H65N5O10 823.47).
26. Compound 6: 2,20,200-(10-(1-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-
methoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid
(Fmoc-NH-DOTA); 1H NMR (500 MHz, CDCl3) d 2.85 (t, 16H), 3.55 (s, 6H), 3.90
(s, 3H), 4.15 (s, 2H), 4.4 (d, 1H), 5.35 (s, 1H), 7.5 (m, 8H), 13C (125 MHz, CDCl3) d
172.38, 170.19, 169.45, 169.35, 169.32, 156.68, 136.21, 128.08, 127.73, 84.56,
81.58, 80.99, 66.49, 61.71, 57.37, 55.19, 52.81, 48.94, 47.70, 46.70, 27.65, 17.94,
ESI-Mass m/z: 677.23 [M+Na]+ (calcd for C32H41N5O10 655.29).
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