Improved synthesis and biological evaluation of chelator-modified α-MSH analogs prepared by copper-free click chemistry

Article abstract of DOI:10.1016/j.bmcl.2011.08.017

Radionuclide chelators (DOTA, NOTA) functionalized with a monofluorocyclooctyne group were prepared. These materials reacted rapidly and in high yield with a fully deprotected azide-modified peptide via Cu-free click chemistry under mild reaction conditions (aqueous solution, room temperature). The resulting bioconjugates bind with high affinity and specificity to their cell-surface receptor targets in vitro and appear stable to degradation in mouse serum over 3 h of incubation at 37 °C.

Full text of DOI:10.1016/j.bmcl.2011.08.017

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5757–5761
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improved synthesis and biological evaluation of chelator-modified
analogs prepared by copper-free click chemistry
a-MSH
Nicholas J. Baumhover a,b, Molly E. Martin c, Sharavathi G. Parameswarappa d, Kyle C. Kloepping a,b
,
a,b,
M. Sue O’Dorisio c, F. Christopher Pigge d, , Michael K. Schultz
⇑
⇑
a Department of Radiology, Carver College of Medicine, The University of Iowa, 500 Newton Road, ML B180, Iowa City, IA 52242, USA
b Department of RadiationOncology, Free Radical Radiation Biology Program, Carver College of Medicine, The University of Iowa, 500 Newton Road, ML B180, Iowa City, IA 52242, USA
c Department of Pediatric Hematology/Oncology, Carver College of Medicine, The University of Iowa Research Park, B145 MTF, 2501 Crosspark Road, Coralville, IA 52241, USA
d Department of Chemistry, The University of Iowa, E557 CB, Iowa City, IA 52242, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Radionuclide chelators (DOTA, NOTA) functionalized with a monofluorocyclooctyne group were pre-
pared. These materials reacted rapidly and in high yield with a fully deprotected azide-modified peptide
via Cu-free click chemistry under mild reaction conditions (aqueous solution, room temperature). The
resulting bioconjugates bind with high affinity and specificity to their cell-surface receptor targets
in vitro and appear stable to degradation in mouse serum over 3 h of incubation at 37 °C.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 10 June 2011
Revised 30 July 2011
Accepted 2 August 2011
Available online 8 August 2011
Keywords:
Copper free click chemistry
Cyclooctyne
Peptide synthesis
Gallium-68
Copper-64
Melanocyte stimulating hormone
Molecular imaging
PET
Radionuclide therapy
PRRT
Radiolabeled peptides are increasingly recognized as effective
tools for targeted molecular imaging and therapy of cancer.1–4
high yields, and rapid kinetics, while being amenable to mild aque-
ous reaction conditions. Although these characteristics have been
investigated for the addition of polyamino-macrocyclic chelators
to molecular targeting vectors,9,11 use of the copper-catalyzed
reaction for this application is complicated by the need to remove
copper that is associated strongly with the chelator moiety prior to
radiolabeling.9,11,12
An alternative to the copper catalyzed cycloaddition reaction is
the use of cyclooctyne agents that undergo cycloaddition reactions
with azides spontaneously as a consequence of ring strain. In par-
ticular, fluoro-substituted cyclooctynes and dibenzocyclooctynes
have been shown to participate in [3+2] cycloadditions with azides
under mild conditions while exhibiting reaction profiles (chemose-
lectivity, yields, kinetics) comparable with their copper catalyzed
counterpart.13–16 Although synthesis of cyclooctyne derivatives
suitable for this application has been challenging,17 we recently
introduced a versatile monoflouro-substituted cyclooctyne (MFCO,
1) that can be conveniently prepared in only four steps from read-
ily available cyclooctanone in ꢀ40% overall yield (Scheme 1).18 We
also demonstrated that 1 could be coupled to a t-butyl protected
DOTA (t-DOTA) derivative for selective addition to a resin-bound
A
significant challenge in preparing these compounds is selective
attachment of chelators for coupling of radionuclides, while main-
taining the high affinity binding of the peptide for its in vivo
molecular target. A number of protection/deprotection schemes
are employed to enable selective installation of the chelator moi-
ety.5–7 Although these methods have proven effective, there is po-
tential for loss of chemical yield with each synthetic step. Thus,
simplified methods that enable selective attachment of the chela-
tor, while preserving radiometal coupling, serum stability, and
high affinity binding of the peptide for the molecular target can
be advantageous.
Click chemistry techniques that employ copper-catalyzed [3+2]
cycloaddition between azides and terminal alkynes have received
considerable attention recently for use in bioconjugation strate-
gies.8–10 These reactions are characterized by supreme selectivity,
⇑
Corresponding authors. Tel.: +1 319 356 3380 (M.K.S.); tel.: +1 319 335 3805
(F.C.P.).
E-mail addresses: chris-pigge@uiowa.edu (F.C. Pigge), michael-schultz@uiowa.
edu (M.K. Schultz).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.017

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N. J. Baumhover et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5757–5761
NDP-
receptors relative to the natural ligand. Since binding assays
involving NDP-
-MSH are well established,20–27 this ligand offers
a-MSH, which exhibits enhanced affinity for melanocortin
a
a means to test the effect of DOTA(NOTA)-MFCO conjugation via
fused triazole linkages on binding affinity to its cell-surface recep-
tor target (i.e., melanocortin receptor subtype 1; MC1R).
An azide-modified derivative of NDP-a-MSH (8) was prepared
by standard solid phase peptide synthesis. Incorporation of the
azide function was accomplished by coupling 6-azido hexanoic
acid to the N-terminus of the fully side-chain protected peptide
on resin, followed by deprotection and cleavage using routine pro-
cedures and purification/characterization by RP-HPLC and LC–MS.
DOTA-click-hex-NDP-a-MSH (9) was synthesized by reacting
Scheme 1. Synthesis of MFCO-amine (3).
100 nmol of azido-hex-NDP-
a
-MSH (8) with a 10-fold excess of
DOTA-MFCO (6) in 0.5 mL of H2O at rt (Scheme 3). The correspond-
ing NOTA analog was prepared similarly by treating 8 with a 20-
fold excess of 7. Reaction progress was monitored by observing
the disappearance of starting material using RP-HPLC (214 nm).
The Cu-free click reaction between DOTA-MFCO (6) and azido-
azide-modified peptide via copper-free click chemistry, followed
by deprotection and cleavage by standard methods.19 With success
in our initial proof of concept, we embarked to develop an im-
proved bioconjugation strategy that would allow direct attach-
ment of the chelator to bioligands under aqueous conditions
without the need for protecting groups. Toward this end, we report
here the preparation of fully-deprotected DOTA- and NOTA-MFCO
chelators that can be conveniently and selectively attached to fully
deprotected azide-modified peptides in aqueous solution at room
temperature. To evaluate the potential of the approach for molec-
ular imaging and radionuclide therapy, the DOTA- and NOTA-
MFCO were conjugated to an azide-modified variant of a well-
characterized peptide under investigation of molecular imaging
and therapy of metastatic melanoma (see below). The chelator
modified bioconjugates were efficiently radiolabeled with gal-
lium-68 (68Ga) and copper-64 (64Cu). Competitive binding assays
were conducted to evaluate the potential effect of the fused-tria-
zole linkage on the binding affinity and the stability of the chela-
tor-modified peptide conjugates was examined in mouse serum.
We further evaluated the effect of metallation on binding affinity
by labeling the chelator-modified peptides with stable Ga3+ and
comparing the resulting affinity to the unmetallated variant.
The synthesis of DOTA- and NOTA-MFCO began with conver-
sion of the acid (1) to the corresponding pentafluorophenyl ester
2 (Scheme 1). This material was previously prepared and used
without isolation in the synthesis of a biotin-MFCO conjugate.17
In the present case, however, it was found that 2 could be easily
isolated/purified and was stable for months at À20 °C. With the
purified MFCO-PFP ester in hand, amine-MFCO (3) was readily ob-
tained upon treatment of 2 with an excess of 1,4-diaminobutane in
CH2Cl2. A simple aqueous workup of the reaction mixture afforded
the desired MFCO-amine (3) as a pale yellow gummy solid in 92%
yield. Material obtained in this manner proved suitable for use in
subsequent coupling reactions without further purification.
The preparation of MFCO-functionalized DOTA and NOTA deriv-
atives (Scheme 2) proceeded smoothly by reaction of 3 with com-
mercially available DOTA and NOTA N-hydroxysuccinimide esters
(NHS esters) 4 and 5. These couplings were performed in DMF/
DMSO at room temperature, and each reaction proceeded to com-
pletion within 2 h as determined by HPLC monitoring. The desired
products 6 and 7 were isolated and purified by HPLC in good chem-
ical yield (85% and 47%, respectively) and their identities were con-
firmed by mass spectrometry.
hex-NDP-
a
-MSH (8) formed the desired product (9) in 2 h while
-MSH (10, not
complete formation of NOTA-click-hex-NDP-
a
shown in Scheme 3) required 5.5 h. The differences in reaction
kinetics under these conditions are unclear, but may be related
to differences in solubility of the NOTA- and DOTA-MFCO bifunc-
tional chelators. Further optimization of the reaction parameters
are the subject of ongoing research. No evidence of side reactions
was observed by monitoring the 214 nm absorbance of the HPLC
trace during the reaction period. The peptide conjugates were puri-
fied by HPLC, lyophilized, converted to the acetate form, and stored
at À20 or À80 °C (see Supplementary data). HPLC purification pro-
vided excellent isolated chemical yields and purity for these reac-
tions for both DOTA-click-hex-NDP-
observed 2370.4 amu; theoretical: 2370.7 amu); and NOTA-click-
hex-NDP- -MSH (10, 70.3%; ESI-MS: observed 2269.4 amu; theo-
a-MSH (9, 57.1%; ESI-MS:
a
retical: 2269.58 amu). While the formation of 1,4 and 1,5-triazole
regioisomers was expected, the species were not distinguishable
by radio- or UV-HPLC methods applied here (see Fig. 1; and Sup-
plementary Fig. 4). Although radioHPLC traces for 64Cu suggested
the presence of the expected regioisomers, efforts to separate these
species further by modification of HPLC parameters proved unsuc-
cessful (data not shown). On the other hand, mass spectral analysis
of the resulting bioconjugates indicated that the fluorine substitu-
ent is labile over a period of months despite storage at À20 and
À80 °C (see Supplementary Fig. 4). However, subsequent radiola-
beling and in vitro competitive binding assays and stability analy-
sis in serum indicate that loss of fluorine (as HF) neither degrades
the binding affinity of the peptides for their cognate receptor
target, nor promotes degradation of the chelator-peptide
bioconjugate.
The ability to incorporate radionuclides into the purified bio-
conjugates was next determined through radiolabeling with posi-
tron emitters 64Cu and 68Ga. Radiolabeling reactions were carried
out by methods described previously.18,28,29 Briefly, 64Cu (Wash-
ington University in Saint Louis, USA) was incubated for 45 min
at 70 °C with 5–10 nmol of peptide in 50 lL acetate buffer (pH
6.0). For 68Ga, radiolabeling was carried out through the use of a
68Ga/68Ge generator system IGG100 (Eckert Ziegler, GmbH, Berlin,
Germany) with a total 68Ge activity of approximately 900 MBq at
the time of the experiments presented here. 68Ga was eluted in
10 mL 0.1 M HCl, purified by cation exchange and incubated with
5–10 nmoles of chelator modified peptide at 100 °C for 12 min.
Preparation of 68Ga labeled variants required the insertion of a final
purification step using a disposable C-18 cartridge post-radiolabel-
ing to achieve suitable radiochemical purity (Fig. 1A and B). In each
case radioHPLC analysis demonstrated excellent radiochemical
purity (>98%) and specific activity (68Ga, 48 MBq nmoleÀ1 and
With 6 and 7 in hand, selective attachment of these new chela-
tors to an azide-modified peptide via Cu-free click chemistry was
next explored. The peptide chosen is a variant of
tin-stimulating hormone ( -MSH) in which two residues have
been replaced with unnatural amino acids (Nle4, -Phe7) to form
a-melanocor-
a
D
DOTA – 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; NOTA – 1,4,7-
triazacyclononane-1,4,7-triacetic acid.

N. J. Baumhover et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5757–5761
5759
Scheme 2. Synthesis of DOTA-MFCO (6) and NOTA-MFCO (7).
Scheme 3. Synthesis of DOTA-click-hex-NDP-
a
-MSH (9).
C
A
68
Ga Peptide
64
68
64
Cu Peptide
Free Ga
Free Cu
D
B
0
5
10
15
0
5
10
15
Retention Time (min)
Retention Time (min)
Figure 1. Radiochemical purity of 64Cu and 68Ga labeled DOTA- and NOTA-click-hex-NDP-a-MSH (9, 10): (A) [68Ga]DOTA-peptide; (B) [68Ga]NOTA-peptide; (C) [64Cu]DOTA-
peptide; (D) [64Cu]NOTA-peptide. Radiochemical purity in excess of 98% is achieved in all cases.
64Cu, 35 MBq nmoleÀ1). It is anticipated that the specific activity of
the 64Cu variant could be improved as no final purification step
was required to obtain >98% radiochemical purity at specific activ-
ity of 35 MBq nmoleÀ1 (Fig. 1C and D).

5760
N. J. Baumhover et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5757–5761
A, IC50 = 0.21 0.03 nM
A
B
120
100
80
60
40
20
0
120
B, IC50 = 0.25 0.07 nM
A, IC50 = 0.66 0.12 nM
B, IC50 = 2.1 0.75 nM
C, IC50 = 1.0 0.22 nM
C, IC50 = 0.59 0.12 nM
100
D, IC50 = 0.76 0.15 nM
E, IC50 = 0.82 0.21 nM
80
60
40
20
0
-12
-10
-8
-6
-12
-10
-8
-6
log [Unlabeled Peptide], M
log [Unlabeled Peptide], M
Figure 2. Competitive inhibition of [125I]-NDP-
correspond to the following: (A) NDP-
NDP- -MSH (10). (B) Peptides A through C in the figure legend correspond to the following: A—[Ga]DOTA-NDP-
click-NDP- -MSH.
a
-MSH binding to B16-F10 mouse melanocytes by
a
a
-MSH peptides. (A) Peptides A through E (n = 4) in the figure legend
-MSH; (D) DOTA-click-hex-NDP- -MSH (9); and (E) NOTA-click-hex-
-MSH, B—[Ga]DOTA-click-NDP- -MSH, and C—[Ga]NOTA-
a
-MSH; (B) azido-hex-NDP-
a
-MSH (8); (C) DOTA-amide-NDP-
a
a
a
a
a
Finally, the biological activities of DOTA and NOTA-click-hex-
NDP- -MSH peptide conjugates 9 and 10 were evaluated through
in vitro cell-binding assays. For these experiments, the effect of
the Ga3+ metallated DOTA-click-hex-NDP-
a-MSH conjugate
a
(2.1 0.75 nM) appeared somewhat higher than the unmetallated
counterpart (0.76 0.15), although the uncertainty obtained for
the metallated conjugate is rather large in comparison to other
experimental results suggesting the potential for an artifact that
requires further investigation. In general, these findings suggest
that the presence of fused triazole-MFCO linkages does not signif-
icantly alter the binding affinity or selectivity of the molecular tar-
geting vector in vitro. It is anticipated that this quality will be
preserved for conjugation of the MFCO-family of chelators to other
molecular targeting vectors as well.
the fused triazole-MFCO linkage on receptor binding was deter-
mined by comparison to binding affinities exhibited by
a-MSH
peptide derivatives lacking this motif. The effect of metallation
on receptor binding affinity was examined using stable Ga3+ la-
beled DOTA-click-hex-NDP-a-MSH. Specifically, the binding of 9
(metallated and unmetallated) and 10 to B16-F10 murine mela-
noma cells expressing MC1R was compared to binding affinities
obtained for NDP-a-MSH, azido-hex-NDP-a-MSH (8), and a
DOTA-NDP-
a
-MSH derivative prepared by standard N-amide bond
In summary, two novel chelator moieties (DOTA-MFCO 6 and
NOTA-MFCO 7) were successfully synthesized in high yield and
used for copper-free click conjugation to an azide-modified pep-
tide. Specifically, these MFCO-modified chelators were selectively
attached to an azide-modified fully-deprotected analog of NDP-
conjugation. By using stable Ga3+ for these experiments, we were
able to make this comparison using the same iodine-125 (125I) la-
beled native a-MSH competitor and melanoma cells, thus enabling
a fair comparison of observed binding affinities of the unmetallated
species conducted by us and according to methods described pre-
viously.30 Briefly, cell suspensions were incubated with a constant
a-MSH under aqueous conditions at room temperature within
2 h (DOTA-MFCO) and 5.5 h (NOTA-MFCO). The new NOTA and
DOTA peptide variants were each radiolabeled with 64Cu and
68Ga in excellent radiochemical purity and specific activity. In vitro
binding assays suggest that the fused-triazole conjugation strategy
provides a stable coupling and does not significantly alter the bind-
ing affinity of the peptide for its cognate MC1R receptor. Metalla-
tion with Ga3+ did not significantly alter the binding affinity of
quantity of 125I labeled NDP- -MSH ([125I]-[Nle4, -Phe7]-
a D a-MSH)
and increasing concentrations of peptide analog ranging from 10
À6
to 10À11 M. Following incubation, cells were pelleted by centri-
fugation, the media was aspirated, pellets were transferred to
12 Â 75 mm glass tubes, and the radioactivity of the cell pellet
was determined by gamma counter. Each experiment was per-
formed in quadruplicate and binding curves were plotted; IC50 val-
ues and their associated standard errors were calculated with
GraphPad Prism 5 curve-fitting software (GraphPad Prism version
5.01 for Windows, GraphPad Software, San Diego, CA). The ob-
the NOTA-click-hex-NDP-a-MSH or DOTA-click-hex-NDP-a-MSH
for their molecular target. The ‘‘click’’ bioconjugates were stable
in mouse serum when incubated at 37 °C for 3 h (a relevant time
frame for peptide-based molecular targeting), with evidence of a
served IC50 values for the DOTA- and NOTA-click-hex-NDP-
a
-
stability advantage over an amide-linked DOTA-NDP-a-MSH vari-
MSH (9 and 10) calculated in this way were less than 1 nM (0.82
and 0.76 nM, respectively, Fig. 2). No significant difference in bind-
ing affinity was observed for Ga3+ metallated NOTA- click-hex-
ant that is the subject of ongoing research (see Supplementary Figs.
5 and 6). Thus, the DOTA-MFCO (6) and NOTA-MFCO (7) entities
represent a new class of bifunctional chelator that enables selec-
tive attachment to fully-deprotected azide-modified peptides un-
der aqueous conditions at room temperature with excellent
chemical yields.
NDP-a-MSH (1.0 0.22 nM) and the unmetallated species
(0.82 0.21 nM). Comparison of affinity values for Ga3+ metallated
DOTA-amide-NDP (0.66 0.12 nM) and unmetallated conjugate
(0.59 0.12 nM) also yielded no significant difference associated
with metallation of the chelator modified peptide. The metallat-
ed-species affinity values are slightly elevated in comparison to
unmetallated conjugates and overall are slightly higher than stan-
dard N-terminal amide bond formation, although the difference is
not statistically significant. Furthermore, 9 and 10 display IC50 con-
Acknowledgments
The authors acknowledge support of the University of Iowa Hol-
den Comprehensive Cancer Center, the American Cancer Society
(IRG-77004-31), and the Roy J. Carver Charitable Trust (RJCCT 01-
224 and 09-3279).
centrations comparable to those of azido-hex-NDP-
a-MSH (8) and
NDP-a
-MSH.21,24,27,31–33 On the other hand, the binding affinity of

N. J. Baumhover et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5757–5761
5761
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.017.
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