Synthesis of a DOTA-biotin conjugate for radionuclide chelation via Cu-free click chemistry

Article abstract of DOI:10.1021/ol100774p

A strain-induced copper-free click reaction mediated by a new and easily prepared cyclooctyne derivative was used to efficiently assemble a DOTA-biotin adduct capable of radionuclide (⁶⁸Ga) uptake. This synthetic strategy offers a potentially general and convenient means of preparing targeted radiolabeling and radiotherapeutic agents.

Full text of DOI:10.1021/ol100774p

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2398-2401
Synthesis of a DOTA-Biotin Conjugate
for Radionuclide Chelation via Cu-Free
Click Chemistry
Michael K. Schultz,*,† Sharavathi G. Parameswarappa,‡ and
F. Christopher Pigge*,‡
Departments of Radiology and Radiation Oncology, CarVer College of Medicine, and
Department of Chemistry, UniVersity of Iowa, Iowa City, Iowa 52242
michael-schultz@uiowa.edu; chris-pigge@uiowa.edu
Received April 2, 2010
ABSTRACT
A strain-induced copper-free click reaction mediated by a new and easily prepared cyclooctyne derivative was used to efficiently assemble a
DOTA-biotin adduct capable of radionuclide (68Ga) uptake. This synthetic strategy offers a potentially general and convenient means of
preparing targeted radiolabeling and radiotherapeutic agents.
Developing methods to efficiently construct bifunctional
materials featuring radionuclide chelators conjugated to
specific bioligands is an important objective in contemporary
bioconjugate chemistry. The impetus for much of this work
derives from the recognition that techniques such as positron
emission tomography (PET) and single photon emission
computed tomography (SPECT) offer unparalleled sensitivity
for in vivo diagnostic imaging of cancer and other diseases.
Additionally, targeted cell-specific delivery of radionuclides
presents opportunities to establish new radiopharmaceuticals
for cancer therapy.1
employed in radionuclide chemistry. Attachment of DOTA
to biomolecules is often accomplished through amide bond
formation via selective manipulation of one of the acetic acid
arms.2 Such preparative routes necessitate the extensive use
of protecting groups, resulting in added manipulation (pro-
tection/deprotection) and purification steps that decrease
overall synthetic efficiency. In this context, an attractive,
versatile, and modular approach toward DOTA bioconjuga-
tion involves application of high-yielding and bio-orthogonal
“click” reactions.
Derivatives of DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid, 1) are metal chelators commonly
(2) (a) Heppeler, A.; Froidevaux, S.; Ma¨cke, H. R.; Jermann, E.; Be´he´,
M.; Powell, P.; Hennig, M. Chem.sEur. J. 1999, 5, 1974. (b) Eisenwiener,
K.-P.; Prata, M. I. M.; Buschmann, I.; Zhang, H.-W.; Santos, A. C.; Wenger,
S.; Reubi, J. C.; Ma¨cke, H. R. Bioconjugate Chem. 2002, 13, 530. (c)
Wa¨ngler, B.; Beck, C.; Wagner-Utermann, U.; Schirrmacher, E.; Bauer,
C.; Ro¨sch, F.; Schirrmacher, R.; Eisenhut, M. Tetrahedron Lett. 2006, 47,
5985. (d) De Leo´n-Rodr´ıguez, L. M.; Kovacs, Z. Bioconjugate Chem. 2008,
19, 391.
† Departments of Radiology and Radiation Oncology.
‡ Department of Chemistry.
(1) (a) Volkert, W. A.; Hoffman, T. J. Chem. ReV. 1999, 99, 2269. (b)
Liu, S. Chem. Soc. ReV. 2004, 33, 445. (c) Shokeen, M.; Anderson, C. J.
Acc. Chem. Res. 2009, 42, 832.
10.1021/ol100774p  2010 American Chemical Society
Published on Web 04/27/2010

The prototypical click reaction advanced by Sharpless and
Meldal is a Cu(I)-catalyzed cycloaddition between azides and
terminal alkynes to afford 1,4-disubstituted triazoles.3 The
reaction offers a convenient means of conjoining two
molecules through functional groups (azide and alkyne) that
are easily incorporated into biomolecules and generally
compatible with existing biomolecular functionality (i.e.,
bioorthogonal). As a result, the use of Cu-catalyzed click
chemistry in biochemical settings is rapidly increasing.4
Several reports have appeared describing reactions of
alkyne-modified DOTA derivatives with various azide-
functionalized bioprobes (e.g., azido peptides) in the presence
of suitable Cu(I) catalysts.5 In these cases, the metal-chelating
ability of the DOTA macrocycle complicates the click cy-
cloaddition by sequestering the Cu catalyst. Additionally,
formation of DOTA-Cu adducts also interferes with subsequent
radionuclide uptake and binding. These problems have been
addressed by performing click reactions on DOTA derivatives
protected as tert-butyl esters (which exhibit attenuated metal
binding ability) and removing residual copper salts from triazole
products by precipitation with Na2S.
An alternative solution that has not been extensively
explored is the application of Cu-free variants of click
reactions to achieve DOTA bioconjugation.6 While thermally
activated cycloadditions between alkynes and azides are well-
established, these reactions require prohibitively high tem-
peratures and long reaction times.7 Strained alkynes, how-
ever, are known to be much more reactive in [3 + 2]
cycloadditions, and Bertozzi et al. have developed several
bifunctional cyclooctyne reagents for in vitro and in vivo
biomolecular applications (Figure 1).8 Inconveniently slow
that exhibits a kinetic profile well-suited for use in chemical
biology settings.8c The dibenzocyclooctyne 4 has also been
employed by Boons and co-workers to assemble glycocon-
jugates for live cell imaging studies.9 Finally, oxanorborna-
dienes (e.g., 5) have been found to react with azides under
physiological conditions via tandem [3 + 2] cycloaddition/
retro Diels-Alder sequences, thus providing another ap-
proach to Cu-free click chemistry.10
We are interested in preparing a variety of DOTA biocon-
jugates for use as novel radioimaging and radiotherapeutic
agents. Toward this end, it is envisioned that preparative
methods based on Cu-free click reactions will offer concise and
efficient synthetic routes to targeted DOTA derivatives. The
strained cycloalkyne and oxanorbornadiene reagents described
above, however, exhibit several shortcomings (for example, 4
possesses relatively hydrophobic arene rings, 5 gives rise to
unwanted byproduct in the click reaction, and 3 is prepared
through a long (∼12 step) synthesis) that may detract from their
use in certain settings. Consequently, we have designed and
synthesized a simpler bifunctional cyclooctyne derivative (9)
and demonstrate its utility as a bioconjugate linchpin through
construction of a DOTA-biotin assembly.
The synthesis of 9 begins with commercially available
cyclooctanone 6 (Scheme 1).11 Treatment of 6 with Select-
Scheme 1
fluor resulted in smooth fluorination, and 7 was obtained in
good yield. Exposure of 7 to excess KHMDS (∼2.2 equiv)
(3) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596. (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
(4) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48,
6974. (b) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba,
G.; Genazzani, A. A. Med. Res. ReV. 2007, 28, 278. (c) Moses, J. E.;
Moorhouse, A. D. Chem. Soc. ReV. 2007, 36, 1249.
(5) (a) Lebedev, A. Y.; Holland, J. P.; Lewis, J. S. Chem. Commun.
2010, 46, 1706. (b) Mindt, T. L.; Mu¨ller, C.; Stuker, F.; Salazar, J.-F.; Hohn,
A.; Mueggler, T.; Rudin, M.; Schibli, R. Bioconjugate Chem. 2009, 20,
1940. (c) Kno¨r, S.; Modlinger, A.; Poethko, T.; Schottelius, M.; Wester,
H.-J.; Kessler, H. Chem.sEur. J. 2007, 13, 6082. (d) Dijkgraaf, I.; Rijnders,
A. Y.; Soede, A.; Dechesne, A. C.; van Esse, G. W.; Brouwer, A. J.;
Corstens, F. H. M.; Boerman, O. C.; Rijkers, D. T. S.; Liskamp, R. M. J.
Org. Biomol. Chem. 2007, 5, 935.
Figure 1
chemistry.
. Examples of reagents (2-5) used in Cu-free click
cycloaddition rates were encountered between cyclooctyne
2 and azide-modified biomolecules,8a prompting development
of the difluorinated cyclooctyne 3 as a more reactive analogue
(6) For a review of Cu-free click reactions, see: Jewett, J. C.; Bertozzi,
C. R. Chem. Soc. ReV. 2010, 39, 1272.
Org. Lett., Vol. 12, No. 10, 2010
2399

Scheme 2
monofluoro analogues related to 2 but which lack a carboxyl
and Tf2NPh (1.05 equiv) at -78 °C afforded the correspond-
ing vinyl triflate in situ. Triflate elimination was found to
occur upon warming the reaction mixture to room temper-
ature, and cyclooctyne 8 was isolated in 60% yield.
Saponification of the methyl ester then gave 9 as a clear oil
after purification by column chromatography.
substituent (k ) 4.3 × 10-3 M-1 s-1).13
Having established the viability of 9 as a Cu-free click
reagent, we then turned our attention to the preparation of
potential radiolabeling agents. Biotin was chosen as a model
biological probe molecule, and cyclooctyne 9 was attached
to amine-functionalized biotin14 derivative 11 by first
converting the carboxyl moiety to an activated pentafluo-
rophenyl ester (Scheme 2). Without isolation, the PFE ester
was treated with a slight excess of 11 in DMF, and the
desired biotin-cyclooctyne derivative 12 was obtained in
49% isolated yield after purification by column chromatog-
raphy. This material was then treated with azide-modified
DOTA 13 in aqueous DMF.15 Click cycloaddition occurred
smoothly, and DOTA-biotin conjugate 14 was isolated in
44% yield afer HPLC purification.
The reactivity of 9 was first probed in a model click
reaction with benzyl azide. Gratifyingly, simply stirring an
equimolar mixture of these two compounds in MeOH at
room temperature afforded the expected triazole product 10
in excellent isolated yield (eq 1).12
(7) Huisgen, R. Angew. Chem., Int. Ed. 1963, 2, 565.
(8) (a) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046. (b) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard,
N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (c) Codelli, J. A.; Baskin,
J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486.
(d) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097. (e) Chang,
P. V.; Prescher, J. A.; Sletten, E. M.; Baskin, J. M.; Miller, I. A.; Agard,
N. J.; Lo, A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1821.
(f) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010,
132, 3688.
Next, the rate of cycloaddition was examined, again using
benzyl azide as a model reactant. Bertozzi has established the
beneficial effect of difluoro substitution in enhancing the rate
of azide cycloaddition to cyclooctynes.8c Given that 9 possesses
only a single fluoro group, we anticipated diminished rates of
cycloaddition with this compound relative to 3. Nonetheless,
monofluoro activation coupled with the presence of an electron-
withdrawing carboxyl group was still expected to deliver a more
reactive click reagent than parent cyclooctyne derivatives (e.g.,
2). The second-order rate constant for the reaction of 9 with
(9) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-j. Angew. Chem., Int.
Ed. 2008, 47, 2253.
(10) (a) van Berkel, S. S.; Dirks, A. J.; Debets, M. F.; van Delft, F. L.;
Cornelissen, J. J. L. M.; Nolte, R. J. M.; Rutjes, F. P. J. T. ChemBioChem
2007, 8, 1504. (b) van Berkel, S. S.; Dirks, A. J.; Meeuwissen, S. A.; Pingen,
D. L. L.; Boerman, O. C.; Laverman, P.; van Delft, F. L.; Cornelissen,
J. J. L. M.; Rutjes, F. P. J. T. ChemBioChem 2008, 9, 1805. (c) Laverman,
P.; Meeuwissen, S. A.; van Berkel, S. S.; Oyen, W. J. G.; van Delft, F. L.;
Rutjes, F. P. J. T.; Boerman, O. C. Nucl. Med. Biol. 2009, 36, 749. See
also: (d) Singh, I.; Vyle, J. S.; Heaney, F. Chem. Commun. 2009, 3276.
(11) This compound can also be prepared in high yield by treating
cyclooctanone with NaH and dimethyl carbonate: Frew, A. J.; Proctor, G. R.
J. Chem. Soc., Perkin Trans. 1 1980, 1245.
1
benzyl azide was determined by H NMR in CD3CN. A plot
of 1/[azide] vs time was analyzed using linear regression
methods, with the slope of the resulting line corresponding to
the rate constant (k) (see the Supporting Information). The rate
constant for reaction of 9 with benzyl azide under these
conditions was determined to be (1.47 ( 0.21) × 10-2 M-1
s-1. As expected, this reaction is slower than a similar
transformation involving difluorocyclooctyne 3 (k ) 4.2 ×
10-2)8c but is roughly 1 order of magnitude faster than reactions
using cyclooctyne 2 (k ) 2.4 × 10-3 M-1 s-1) as well as
(12) Compound 10 was produced as a ∼1:1 mixture of inseparable
triazole regioisomers.
(13) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R.
ACS Chem. Biol. 2006, 1, 644.
(14) Wilbur, D. S.; Hamlin, D. K.; Vessella, R. L.; Stray, J. E.; Buhler,
K. R.; Stayton, P. S.; Klumb, L. A.; Pathare, P. M.; Weerawarna, S. A.
Bioconjugate Chem. 1996, 7, 689.
(15) Compound 13 was purchased from Macrocyclics, Inc. (www.
macrocyclics.com). This material can be prepared from DOTA-mono-NHS-
tris-tert-butyl ester and 1-amino-3-azidopropane. Details of this synthesis
will be reported elsewhere.
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Org. Lett., Vol. 12, No. 10, 2010

A noteworthy consequence stemming from the use of Cu-
free click chemistry in the sequence described above is the
ability to bioconjugate the DOTA chelator without prior
protection of the carboxylic acid residues. This removes the
burden of deprotecting DOTA-biomolecular constructs and
allows for direct radiolabeling of click reaction products. In
the present study, proof-of-concept radiolabeling of the
DOTA-biotin conjugate demonstrates the utility of the
approach. An acetate buffer solution containing generator-
produced 68Ga (7.3 MBq; pH 3.8) was added to aliquots of
14 dissolved in the identical acetate buffer. The reaction was
carried out at 99 °C for 20 min. After cooling, aliquots of
the reaction mixtures were analyzed directly by radio-HPLC
without further purification (Figure 2). Two major peaks were
observed for the radiolabeled biotin-DOTA conjugate,
reflecting the formation of two regioisomers in the click
reaction leading to 14 as well as a minor peak that reflects
possible stereoisomer formation (see Supporting Informa-
tion). Significantly, the radiochemical purity in each solution
was >97% with specific activity up to 1.45 MBq nmol-1.
Figure 2.
Radio-HPLC trace of 68Ga-biotin-DOTA reaction
mixtures. Top: 68Ga labeling in the presence of 5 µmol of 14.
Bottom: 68Ga labeling in the presence of 5 nmol of 14. Peak
labeling: (1) “free” 68Ga; (2) 68Ga-14 complex as a mixture of
two regioisomers; (3) unknown trace impurity.
In conclusion, we have designed and synthesized a new
bifunctional cyclooctyne reagent suitable for use in Cu-free
click chemistry. Significantly, 9 is easily prepared in only
three steps from commercially available starting material in
high (47%) overall yield. Attachment of 9 to bioligands can
be accomplished through manipulation of the carboxyl
residue, and a biotin-cyclooctyne derivative 12 was prepared
to demonstrate this feature. The advantages of Cu-free click
reactions in the construction of potential radiolabeling agents
has been highlighted through formation of DOTA conjugate
14. This material was successfully labeled with 68Ga in >97%
radiochemical purity and specific activity of ∼1.5 MBq
nmol-1. This demonstrates the utility of 9 for the assembly
of radionuclide-chelated bioconjugates. It is anticipated that
high-specific activity agents can be prepared using this
strategy, and the synthesis of other DOTA bioconjugates for
targeted molecular imaging applications using this approach
is currently being examined in our laboratories.
Acknowledgment. We thank the American Cancer So-
ciety, The University of Iowa Holden Comprehensive Cancer
Center (M.K.S.), and the Carver Charitable Trust (F.C.P.,
S.G.P.) for generous financial support. We also thank Garry
Kiefer and Paul Jurek of Macrocyclics, Inc., for their
assistance in obtaining 13.
Supporting Information Available: Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL100774P
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