The hydrazide/hydrazone click reaction as a biomolecule labeling strategy for M(CO)3 (M = Re, 99mTc) radiopharmaceuticals

Article abstract of DOI:10.1039/c1cc15451f

Facile reactivity of hydrazides and aldehydes was explored as potential coupling partners for incorporation into M(CO)₃ (M = Re, ^99mTc) based radiopharmaceuticals. Both 'click, then chelate' and 'prelabel, then click' synthetic route

Full text of DOI:10.1039/c1cc15451f

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COMMUNICATION
The hydrazide/hydrazone click reaction as a biomolecule labeling
strategy for M(CO)3 (M = Re, 99mTc) radiopharmaceuticalsw
Tanushree Ganguly,a Benjamin B. Kasten,a Dejan-Kresimir Bucar,b
Leonard R. MacGillivray,b Clifford E. Berkmana and Paul D. Benny*a
Received 1st September 2011, Accepted 11th October 2011
DOI: 10.1039/c1cc15451f
Facile reactivity of hydrazides and aldehydes was explored as potential
coupling partners for incorporation into M(CO)3 (M = Re, 99mTc)
based radiopharmaceuticals. Both ‘click, then chelate’ and
‘prelabel, then click’ synthetic routes produced identical products
in high yields and lacked metal-hydrazide/-hydrazone inter-
actions, highlighting the potential of this click strategy.
hydrazide bifunctional chelator for complexing the diagnostic
molecular imaging radionuclide 99mTc (g, 140 keV (89%),
t1/2 = 6.0 h). The hydrazone moiety was selected for its high
stability at physiological pH, and lability under strongly acidic
and basic conditions as demonstrated by drug delivery agents
in tumor targeting.9–11 To our knowledge, such a click
approach with 99mTc has not yet been explored. Instead, the
paradigm from the literature indicates that hydrazine precursors
would be unavailable for hydrazone formation because they
interact with 99mTc metal centers. For instance, 99mTcV oxo
complexes react with hydrazines (i.e., 6-hydrazinonicotinic acid
(HYNIC)) to form complexes through nitrogen coordination
(Fig. 1, Route-1).12–17 Additionally, mixed observations of metal
coordination or combined oxidation/decarbonylation of the
metal are reported for hydrazines with the organometallic
fac-(M(OH2)3(CO)3)+ (M = Re, 99mTc) complex.12,18 Such
observations prompted our investigation of hydrazides as a
clickable moiety for hydrazone generation. We find that the model
hydrazide systems employed did indeed prefer hydrazone formation
over interactions with the metal centers (Fig. 1, Route-2).
The benefits of click reactions are well known, and their
applications have revolutionized numerous fields including
bioconjugate chemistry, material chemistry and polymer
sciences.1–4 However, the most widely used Huisgen 1,3-dipolar
cycloaddition click reaction has restricted usefulness in biological
applications due to the toxicity of copper in vivo.5 A sterically
strained cyclooctyne group alleviates the toxicity issue in a
copper-less click reaction. However, the installation of a bulky
functional group can significantly impact the pharmacodynamics
and biological efficacy of a clicked biomolecule. Alternatively,
the non-aldol addition reaction between a carbonyl and a
nucleophilic nitrogen as a click reaction bypasses both draw-
backs mentioned above. Key benefits of this click strategy are the
availability of amines for bioconjugation, rapid incorporation
into molecular frameworks, and a simple resultant click product
that minimally impacts the nature of the biomolecule.
The focus of this investigation was to prove the concept of
the hydrazide/hydrazone click reaction with a model aldehyde
for application with M(CO)3+ and to show its viable incorporation
into a biomolecule-related framework. To minimize possible
coordination of the hydrazide or hydrazone groups, a well-
known and efficient tridentate ligand, dipicolylamine (DPA),
Click reactions have become an integral feature in radio-
pharmaceutical chemistry in designing ligands for metal com-
plexation or for coupling pre-labeled radionuclide complexes
with a targeting biomolecule.6–8 The latter approach reduces the
exposure of biomolecules to harsh conditions (i.e., pH, temperature)
required for complexation and improves the site-specific labeling of
the metal. This ‘‘prelabel, then click’’ strategy allows efficient
incorporation of radiometals into sensitive biomolecules which
would be deactivated under standard radiolabeling conditions.
In this article, we pursue the non-aldol hydrazone click
reaction between an aldehyde-containing biomolecule and a
was utilized for selective coordination of M(CO)3 .
+ 19 The click-
able, bifunctional chelator was generated by functionalizing
a Department of Chemistry, Washington State University, Pullman
WA 99164, USA. E-mail: bennyp@wsu.edu; Fax: 509-335-8867;
Tel: 509-335-3858
b Department of Chemistry, University of Iowa, Iowa City,
IA 52242-1294, USA. E-mail: len-macgillivray@uiowa.edu;
Fax: 319-335-1270; Tel: 319-335-3504
w Electronic supplementary information (ESI) available. CCDC
829258. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1cc15451f
Fig. 1 General hydrazine/hydrazide reaction scheme: Route-1: M-HYNIC
type coordination and Route-2: Coupling with the aldehyde to give
hydrazone.
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center (Fig. S5w).22 1H NMR data of 4a and 4b also confirmed
the DPA coordination of Re(CO)3 indicated by the HA,B
splitting for the methylene protons adjacent to the pyridine
typically observed upon complexation. The minimal shift of the
hydrazone (CH) (7.86 ppm) upon complexation also confirmed
the non-coordination of the hydrazone with the Re center.
In Route 2 (prelabel, then click), the DPA-hydrazide ligands
(1a, 1b) were complexed with Re(CO)3. 1a was refluxed in
methanol with Re(CO)5OTf and 1b reacted with fac-
+
Re(CO)3(H2O)3 at 55 1C 30 min at pH 8 in a methanol/
water solution to yield 3a (46%) and 3b (87%), respectively.
1H NMR of 3a and 3b shows characteristic downfield shifts
and an HA,B type splitting of the methylene protons of DPA
+
indicative of Re(CO)3 coordination. Whereas, the methylene
protons adjacent to the hydrazide group do not shift signifi-
cantly from the free ligand as would be observed upon metal
coordination. This data supports the non-coordination of
hydrazide and the non-HYNIC behavior of these molecules.
The characteristic IR pattern of the fac-Re(CO)3 also confirmed
that oxidative decarbonylation to a Re(CO)2 species was not
observed under these conditions.
Scheme 1 Parallel synthesis approaches Route 1 (1 - 2 - 4, click,
then chelate) and Route 2 (1 - 3 - 4, prelabel, then click) for the
formation of M(CO)3+, (M = Re, 99mTc) DPA-hydrazone complexes.
the central amine of DPA with a hydrazide moiety. We
expected this to avoid undesirable interactions seen between
hydrazines and 99mTc metal centers while maintaining sufficient
reactivity for hydrazone formation. Our approach involved two
parallel synthetic routes to determine the influence of the
hydrazide and hydrazone products in the complexation of
In the second step, the Re(CO)3DPA-hydrazide complexes
(3a, 3b) were condensed with PNB at pH B 4 for 2–4 h at
50 1C in ethanol/water to yield the Re(CO)3DPA-hydrazone
complexes 4a (93%) and 4b (62%), respectively. The analytical
data collected of 4a and 4b in Route 2 was identical to the
results observed in Route 1. The reactivity of 3a and 3b towards
PNB was comparable in reaction conditions and yields observed
with the ligands (2a, 2b). In both Re(CO)3DPA-hydrazide
variations, the hydrazide exhibits preferential condensation with
the aldehyde over coordination. Re(CO)3DPA complexation does
not appear to impact the reactivity, through either electronic or
coordination effects, of the hydrazone formation. The lability of
the hydrazone linkage was explored, and the hydrazone bond is
stable at pH 3–12 and shows no decomposition on heating for over
10 h at 90 1C.
+
M(CO)3 (Scheme 1). Route 1: the DPA-linked hydrazide
ligand was reacted with an aldehyde to yield the corresponding
hydrazone clicked ligands followed by complexation with
M(CO)3+. Route 2: the DPA-hydrazide ligand was complexed
+
directly with M(CO)3 followed by hydrazone formation with
an aldehyde. Ideally, both routes would lead to the same
complex, indicating that neither the hydrazide in the prelabeled
approach nor the clicked hydrazone moiety interact with the
metal centers.
Two variations of the DPA-hydrazide ligands, 1a (n = 1)
and 1b (n = 5), were prepared in two steps with reasonable
overall yields: (1) alkylation of DPA with the corresponding
2-bromoacetic acid and 6-bromohexanoic acid ethyl esters in
the presence of triethylamine; (2) conversion of the ester to the
hydrazide by the addition of 35% hydrazine in ethanol.20
These two different linkers permitted the investigation of the
potential electrochemical or coordination interactions of the
hydrazide group with the M(CO)3 centers based on the proximity
of these groups. Linker 1a is more likely to coordinate the metal
center (5 or 6 member coordination ring) than the longer linker 1b.
In Route 1 (click, then chelate), the clicked hydrazone
analogues were prepared by the condensation of the hydrazide
(1a, 1b) with p-nitrobenzaldehyde (PNB) as a model system.
The reaction proceeded smoothly to completion at 50–55 1C,
30–45 min at pH 4.0 in an ethanol/water mixture to form the
hydrazone DPA ligands, 2a (81%) and 2b (62%). The clicked
hydrazone ligands (2a, 2b) were complexed with fac-
Re(OH2)3(CO)3+ in methanol/water at pH 8 (dropwise addition
of saturated NaHCO3 solution) at 55 1C for 2–3 h to yield a single
product 4a (45%) and 4b (57%), respectively. Confirmation of the
DPA coordination of 2a and 2b in the Re(CO)3 complexes were
elucidated from X-ray crystallographic and NMR data.w The
X-ray structure of 4a clearly delineates the DPA coordination to
Re(CO)3, while the hydrazone is orientated away from the metal
Radiolabeling studies with fac-99mTc(OH2)3(CO)3 were
+
conducted to evaluate the efficiency and efficacy of Routes 1
and 2 for comparison to the Re analogs. In Route 1, the
clicked DPA-hydrazone ligands (2a, 2b) in various concentra-
tions (10ꢀ4–10ꢀ5 M) were incubated for 30 min at 90 1C with
+
fac-99mTc(OH2)3(CO)3
to generate the corresponding
99mTc(CO)3 complexes 4a0 (97%) and 4b0 (91%) in excellent
yields. A single chromatographic peak for 4a0 (21.93 min) and
4b0 (21.68 min) was observed in radio (g) HPLC that correlated
with the tr of the Re analogs 4a (21.76 min) and 4b (21.82 min).
In Route 2, the DPA-hydrazide ligands (1a, 1b) were complexed
with fac-99mTc(OH2)3(CO)3 at 50 1C to generate fac-99m
-
+
Tc(CO)3DPA-hydrazide complexes 3a0 (46%) and 3b0 (78%)
in good yield. Observed as single peak on HPLC, the tr’s
(3a0, 16.98 min and 3b0, 17.95 min) of 99mTc species correlated
with the Re analogs (3a, 16.85 min and 3b, 17.89 min). The
hydrazone condensation of 3a0 and 3b0 with PNB at pH 4 was
found to be analogous to the Re complexes (3a, 3b) by comparison
of retention times by analytical HPLC. Comparative HPLC
results for compounds 3a (and 3a0) and 4a (and 4a0) can be seen
in Fig. S2w. To determine the optimal coupling conditions,
several variables such as temperature (60/90 1C), reaction time
(30/60 min), and [PNB] (10ꢀ4–10ꢀ6 M), were independently
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12846–12848 12847

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decarbonylation, HYNIC, or hydrazone coordination to the
metal center. The inclusion of the hydrazide/hydrazone system
into a peptide framework reported in this work can be readily
extended to a number of biomolecules for labeling with
99mTc(CO)3.
This research was funded in part by the Washington State
LSDF (08-012374880), Office of Science (BER), U.S. DOE
(DE-FG02-08-ER64672), NSF’s REU program at WSU
(#0851502) and NIH Biotechnology Training Program at
WSU (T32 GM008336) and NIH (5R01CA1406170).
Scheme 2 Glutamate model system for the hydrazide/hydrazone
Route 2 (1b - 3b - 6b, prelabel, then click).
Notes and references
evaluated (Table S1). Increased temperature and reaction
times were found to modestly improve the condensation
yields. However, PNB concentration had a significant effect
in the hydrazone formation. Excellent yields of 71–91% of the
hydrazone product were observed at 10ꢀ4 M PNB, while at
lower concentrations the product yields declined even with
increased temperature and reaction time.
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A one-pot synthesis was also carried out and it was proven
that addition of PNB to 3a (or 3b) without purification also
yielded 4a (or 4b). X-Ray quality crystals of 4a were isolated
by this method via repeated crystallization from methanol
(Fig. S5w). Labeling studies with 99mTc were also performed
to confirm the results from the Re studies. Details can be
found in supplementary information Table S1.w
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an important proof of concept for the Re/99mTc(CO)3DPA-
hydrazide/hydrazone click reaction and indicated its viable
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click reaction into
a biomolecule having free amines
(Scheme 2). 4-carboxybenzaldehyde provides a unique motif
to couple to free amines under standard peptide conditions,
while maintaining efficient reactivity with metal-hydrazides to
yield a single product.
Compound 5 was synthesized by coupling 4-carboxybenzal-
dehyde with H2N-Glu(OBn)(OBn) in the presence of EDC and
HoBt.21 Parallel synthetic approaches (Route 1 and 2) were
investigated with 5 under similar conditions (pH 4, 30–60 min)
as the PNB system. In Route 1 ‘‘click, then chelate’’, the clicked
ligand (7b) was complexed with fac-Re(OH2)3(CO)3+ to yield
6 (66%). In Route 2 ‘‘prelabel, then click’’, 6 was also obtained
in good yield (61%) by the coupling of 3b with 5 at pH 4 in
40 min. Characterization of the products from both routes con-
+
firmed the identical nature of the compounds. 99mTc(OH2)3(CO)3
studies performed according to the PNB conditions confirmed
product formation analogous to the Re compounds. A single peak
with a tr matching the Re analog 6 and similar labeling yields as
3b0 were obtained (60/90 1C, 10ꢀ4 M 5, 60/90 min; 70–80%).
In conclusion, we have presented here a novel hydrazide/
hydrazone click reaction which highlights the potential appli-
cation for labeling biomolecules with 99mTc(CO)3 as a fast and
efficient reaction. Either Route 1 or 2 takes advantage of the
strong chelator DPA to yield single, well-defined products that
differ considerably in behavior from previous reports of
21 S.-A. Poulsen, J. Am. Soc. Mass Spectrom., 2006, 17, 1074–1080.
22 Crystal data for 4a, C34H29F3N6O9ReS, Mr = 940.89 gmolꢀ1
,
%
triclinic, space group P 1, a = 10.9711(12) A, b = 11.6456(13) A,
c = 15.6740(17) A, a = 92.749(5)1, b = 109.576(5)1, g =
101.608(5)1,
V = T = 125(2) K, Z = 2,
1833.7(3) A3,
m(MoKa) = 3.447 mmꢀ1, 12 482 reflections measured, 6421
independent reflections (Rint = 0.0165). The final R1 values were
0.023 (I 4 2s(I)). The final wR(F2) values were 0.0572 (I 4 2s(I)).
The final R1 values were 0.0242 (all data). The final wR(F2) values
were 0.0578 (all data). The goodness of fit on F2 was 1.046. CCDC
deposition number: 829258.
c
12848 Chem. Commun., 2011, 47, 12846–12848
This journal is The Royal Society of Chemistry 2011