A convenient solid-phase synthesis methodology for preparing peptide-derived molecular imaging agents - Synthesis, characterization, and in vitro screening of Tc(I) - chemotactic peptide conjugates

Article abstract of DOI:10.1139/v05-224

A versatile solid-phase synthesis strategy for preparing peptide-chelate conjugates was developed. The methodology was optimized using a series of ligands, designed to bind Tc(I)/Re(I), and a chemotactic peptide fMFL, which was exploited as a model targeting vector. The peptide derivatives were prepared in parallel using a conventional automated peptide synthesizer in multi-milligram quantities, which provided sufficient material to perform complete characterization, radiolabelling, and in vitro screening studies. Because of the robust nature of the metal-chelate complexes, the Re complex of a chelate-peptide conjugate was prepared on the resin using the same methodology employed to prepare the free ligand conjugates. As such, the reported methodology is amenable to the preparation of libraries of novel Tc radiopharmaceutical ligands and their corresponding Re reference standards in which several factors, including peptide sequence, site of derivatization, and both the type and length of the spacer, can be easily varied.

Full text of DOI:10.1139/v05-224

2060
A convenient solid-phase synthesis methodology
for preparing peptide-derived molecular imaging
agents — Synthesis, characterization, and in vitro
screening of Tc(I) – chemotactic peptide
conjugates
Karin A. Stephenson, Sangeeta Ray Banerjee, Nicole McFarlane, Douglas
R. Boreham, Kevin P. Maresca, John W. Babich, Jon Zubieta, and John F. Valliant
Abstract: A versatile solid-phase synthesis strategy for preparing peptide–chelate conjugates was developed. The meth-
odology was optimized using a series of ligands, designed to bind Tc(I)/Re(I), and a chemotactic peptide fMFL, which
was exploited as a model targeting vector. The peptide derivatives were prepared in parallel using a conventional auto-
mated peptide synthesizer in multi-milligram quantities, which provided sufficient material to perform complete charac-
terization, radiolabelling, and in vitro screening studies. Because of the robust nature of the metal–chelate complexes,
the Re complex of a chelate–peptide conjugate was prepared on the resin using the same methodology employed to
prepare the free ligand conjugates. As such, the reported methodology is amenable to the preparation of libraries of
novel Tc radiopharmaceutical ligands and their corresponding Re reference standards in which several factors, including
peptide sequence, site of derivatization, and both the type and length of the spacer, can be easily varied.
Key words: radiopharmaceuticals, technetium, rhenium, peptides, solid-phase synthesis.
Résumé : On a développé une stratégie versatile de synthèse en phase solide de produits conjugués peptide–chélate.
On a optimisé la méthodologie en utilisant une série de ligands qui ont été développés pour se lier au Tc(I)/Re(I) et au
peptide chimiotactique fMFL qui a été exploité comme vecteur modèle de ciblage. On a préparé les dérivés peptidiques
en parallèle en faisant appel à un synthétiseur automatique conventionnel de peptides à des niveaux de plusieurs milli-
grammes, ce qui fournissait des quantités suffisantes de matériel pour en faire une caractérisation complète, un mar-
quage radiochimique et des études in vitro. En raison du fait que les complexes métal–chélate sont très robustes, un
complexe du Re d’un produit conjugué chélate peptide a été préparé sur la résine en faisant appel à la même méthodo-
logie qui avait été utilisée pour préparer les conjugués libres du ligand. De cette façon, la méthodologie qui a été mise
au point peut être amener à la préparation de librairies de nouveaux ligands radiopharmaceutiques du Tc et des pro-
duits de référence du Re dans lesquels il est possible de faire varier plusieurs facteurs, y compris la séquence pepti-
dique, le site de formation du dérivé ainsi que le type et la longueur du groupe d’écartement.
Mots clés : radiopharmaceutiques, technitium, rhénium, peptides, synthèse en phase solide.
[Traduit par la Rédaction] Stephenson et al. 2066
Introduction
thrombosis (1–8). Peptides are able to reach their target rap-
idly, thus avoiding the need to use radionuclides with exces-
sively long half-lives, and they typically clear from
nontarget tissues quickly, which improves image contrast
and quality (1, 9). Peptides can be prepared in a cost-effective
manner via automated solid-phase synthesis, and they can be
designed to target practically any receptor (10–12).
The traditional strategy for linking radiometals to peptides
involves appending a bifunctional chelate (BFC) to either the
N or C termini of the peptide. This was typically performed
in solution (1, 6), which can lead to numerous side products,
particularly if lysine residues are present in the peptide. To
avoid the poor regioselectivity of this approach, which often
results in the need to employ extensive HPLC purification to
isolate the desired product (13), several groups began to ex-
ploit the advantages of solid-phase synthesis (SPS) to pre-
pare peptide–ligand conjugates directly on polymer supports
Small peptides have been shown to be highly effective tar-
geting agents for delivering radionuclides to specific recep-
tors, including those associated with tumours, infection, and
Received 3 August 2005. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 3 February 2006.
K.A. Stephenson, N. McFarlane, D.R. Boreham, and
J.F. Valliant.1 Departments of Chemistry and Medical
Physics & Applied Radiation Sciences, McMaster University,
Hamilton, ON L8S 4M1, Canada.
S.R. Banerjee and J. Zubieta. Department of Chemistry,
Syracuse University, Syracuse, NY 13244-4100, USA.
K.P. Maresca and J.W. Babich. Molecular Insight
Pharmaceuticals Inc., Cambridge, MA 02142, USA.
1Corresponding author (e-mail: valliant@mcmaster.ca).
Can. J. Chem. 83: 2060–2066 (2005)
doi: 10.1139/V05-224
© 2005 NRC Canada

Stephenson et al.
2061
(14, 15). Using this approach, orthogonal protecting groups
can be employed, and large excesses of reagents can be used
to drive reactions to completion while byproducts are re-
moved by simple filtration (10, 15–17). SPS of this type is
amenable to automation and can therefore be used to prepare
libraries of compounds (18–21).
5 µm). The elution protocol for both analytical and
semipreparative experiments was a linear gradient of from
25% acetonitrile containing 0.1% TFA to 50% acetonitrile
containing 0.1% TFA over 15 min. The flow rates were
1.0 and 4.2 mL/min for the analytical and semipreparative
experiments, respectively. All HPLC experiments were
monitored at λ = 254 and 214 nm. Radio–HPLC experiments
were performed on a Varian Prostar HPLC equipped with an
autosampler (model 410), a UV–vis detector (model 345),
an NaI radiometric detector, Prostar Pumps (model 210),
and a Vydac C18 25 cm × 4.6 mm column (5 µm pore size)
fitted with a 2 cm guard column. The mobile phase con-
sisted of the following: solvent A = pH 2–2.5 triethyl-
ammonium phosphate buffer (7 mL NEt3, 4 mL concd.
H3PO4 in 1000 mL water); solvent B = CH3OH. The method
employed a gradient run over 30 min at a flow rate of
1 mL/min. The gradient ramped from 5% to 100% B, begin-
ning at 3 min and ending at 20 min.
Current SPS methods used to prepare peptide-targeted
radiotracers derived from 99mTc (the most widely used
radionuclide in diagnostic medicine (22–24)) typically in-
volve conjugating a ligand to the N-terminus of the resin-
bound peptide. For instance, Hoffman and co-workers (25)
prepared a series of bombesin derivatives using N3S triamide
chelates linked to the N-terminal amino acid through differ-
ent spacer groups. Egli et al. (26) attached a protected
histidine derivative to the N-terminus of neurotensin as a
way of tagging the peptide with the [99mTc(CO)3]+ core.
More recently, two strategies for incorporating ligands
within the backbone of the peptide have been reported.
Blower and co-workers (15) prepared a derivative of Fmoc-
lysine that contained a hydrazine substituent as a way of in-
corporating a 99mTc binding ligand within peptides. We re-
cently reported the synthesis of amino acid derived chelates
for Tc(I), which can also be incorporated regioselectively
into the backbone of a peptide (17, 27). A unique feature of
this system is that the Re complex can be prepared in paral-
lel with the free ligand, owing to the stability of the low spin
d6 Re(I) complex.
This paper describes a new approach for preparing
peptide–Tc ligand conjugates. The reported method is attrac-
tive in that a chelate can be attached at any position within
the backbone of the peptide and not simply to the terminal
amino or acid groups. Specifically, the approach involves us-
ing an on-resin orthogonal protecting group strategy, where
a Tc(I) chelate is linked to a specific lysine residue. This
methodology will ultimately be useful for creating libraries
of tracers as a means of rapidly identifying new radio-
pharmaceuticals.
Solid-phase peptide synthesis
Fmoc-glycine loaded SASRIN resin (150 mg/well,
0.69 mmol/g) was added to four wells of an Advanced
ChemTech 348 automated synthesizer, suspended in DMF
(2 mL/well), and shaken at 600 rpm for 1 min. The wells
were subsequently filtered, suspended in THF (2 mL/well),
shaken at 600 rpm for 1 min, and drained for 90 s. The THF
wash was repeated two more times. The DMF wash was
then repeated a final two times to complete the general wash
cycle. This procedure was used between every deprotection
and coupling step. Fmoc deprotection was accomplished
through the addition of a 20% v/v piperidine–DMF solution
to the active vessels (2 mL/well) and shaking for 5 min at
600 rpm. Following filtration, the process was repeated, but
with shaking for 10 min. The deprotected resin-bound amino
acid was washed using the general wash procedure and sub-
sequently coupled to the next Fmoc-protected amino acid us-
ing a standard HBTU coupling technique. A “cycle”,
according to Scheme 1, consists of Fmoc deprotection, a
wash, and coupling the specified amino acid, followed by an
additional wash.
Experimental section
Coupling reactions initially involved adding DMF
(200 µL) to the active vessels followed by a fivefold excess
of the protected amino acid as a 0.5 mol/L solution in DMF.
Five equivalents of HBTU as a 0.5 mol/L solution in DMF
were then added, followed by a 10-fold excess of DIPEA as
2.0 mol/L solution in DMF. The reaction block was subse-
quently shaken for 80 min at 600 rpm. Following filtration,
the resin was washed using the general washing procedure
prior to the start of the next cycle. Dde deprotection was ac-
complished through the addition of a 2% v/v hydrazine–
DMF solution to the active vessels (2 mL/well) and shaking
for 5 min at 600 rpm. Following filtration, the process was
repeated, with shaking for 10 min. The free ε amine was
then subjected to the general wash cycle and coupled to the
desired chelate using the HBTU protocol described above,
which was followed with a final wash.
Unless otherwise stated, all reagents and solvents were
ACS grade or higher and used without further purification.
Polystyrene-based N-α-9-fluorenylmethoxycarbonyl (Fmoc)-
glycine loaded SASRIN resin (0.69 mmol/g, 1% divinyl-
benzene, 200–400 mesh) was obtained from Bachem Inc.
Fmoc-protected amino acids were purchased from
NovaBiochem Inc., Bachem Inc., and Advanced ChemTech
Inc. Fluorescein-labelled fNLFNTK was purchased from
Molecular Probes Inc. (product No. F-1314).
Peptides were analyzed by electrospray mass spectrome-
try using a Micromass Quattro Ultima instrument in positive
ion mode. Samples were dissolved in 50:50 ACN–H2O prior
to analysis. FT IR spectra were acquired on a Bio-Rad FTS-
40 FT IR spectrometer. Analytical HPLC on nonradioactive
compounds was performed using a Varian Pro Star model
330 PDA detector, model 230 solvent delivery system, and a
C-18 Microsorb column (4.6 × 250 mm, 300 Å – 5 µm).
Semipreparative HPLC on nonradioactive compounds was
carried out using a Varian Pro Star HPLC fitted with a
model 320 UV detector, a model 215 solvent delivery sys-
tem, and a C-18 Microsorb column (10 × 250 mm, 300 Å –
Peptides were cleaved from the resin using a cocktail of
TFA containing (v/v/v) EDT (2%), water (2%), and TIS
(2%). The cleavage cocktail, cooled to 0 °C, was added to
the resin, and the mixture was allowed to warm to room
temperature over 60 min. The suspension was filtered into
© 2005 NRC Canada

2062
Can. J. Chem. Vol. 83, 2005
Scheme 1. Synthesis of compounds 5–8.
O
NHFmoc
O
N
H
1. 20% Piperidine
2. Wash
1. Cycle 2: Fmoc-Phe-OH
2. Cycle 3: Fmoc-Leu-OH
O
O
N
H
Fmoc
3. Fmoc-Lys(Dde)-OH (5.0 equiv.)
HBTU (5.0 equiv.), DIPEA (10.0 equiv.)
4. Wash
3. Cycle 4: For-Met-OH
4. 2% hydrazine–DMF
5. Wash
O
O
NH
O
(Cycle 1)
S
S
O
O
O
H
N
H
N
O
O
O
OH
H
N
H
N
H
N
H
N
H
N
H
1. Compound 1-4
HBTU, DIPEA
O
H
N
H
N
H
N
H
O
O
O
O
O
O
2. TFA, EDT,TIS, H 2O
N
NH
NH2
N
O
n
N
n = 1 (5)
n = 2 (6)
n = 3 (7)
[Re(CO)3]+ complex, n = 3 (8)
cold diethyl ether, and the resulting heterogeneous solution
was centrifuged at 3000 rpm and –5 °C for 10 min. The re-
sulting pellet was washed with cold ether (3 × 25 mL), dis-
solved in distilled–deionized water, and lyophilized, yielding
a white solid.
taining 0.1% BSA, 10 mmol/L HEPES, and 1.5 mmol/L
CaCl2 (HBSS+). The cells were spun for 7 min at 400 g at
5 °C, the supernatant was removed, and the pellet was re-
suspended in HBSS+. Cell counts were performed using a
hemacytometer and a Beckman Coulter Z2 Coulter particle
count and size analyzer. Cell concentrations were adjusted to
2 × 106 mL–1.
+
Preparation of fMLFK(4m-Tc(CO)3 )G (9)
Technetium-99m was obtained in saline from a commer-
cial 99Mo/99mTc generator (Cardinal Health), and
Flow cytometry
[
99mTc(CO)3(H2O)3]+ was prepared using the commercially
Samples were run on a Beckman Coulter EPICS XL,
equipped with an argon laser (488 nm excitation wave-
length). Fluorescence was measured using a 525 15 nm
band pass filter. The flow cytometer was calibrated with
fluorescein isothiocyanate (FITC)-labeled beads (Quantum
26 beads, Bangs Laboratories, Inc, Fishers, Indiana) prior to
sample analysis. The fluorescence intensity of the standard
was provided by the manufacturer in units of molecules of
equivalent soluble fluorochrome (MESF) and ranged from
5300 to 468 800. A linear fit of MESF vs. mean channel
number of the beads was used to convert all subsequent
mean channel values to MESF values.
available Isolink kits (Mallinckrodt). Compound 7 (0.1 mg,
0.11 µmol) and 370 MBq (10 mCi) [99mTc(CO)3(H2O)3]+
were combined and heated at 75 °C for 60 min. After cool-
ing to room temperature, the product was purified by HPLC
(Rt = 12.1 min).
Ligand challenge experiments
Compound 9 in methanol (50 µL) and PBS buffer (1 mL,
pH 7.2) was added to 0.1 mol/L solutions of cysteine in PBS
buffer (pH 7.2) and histidine in PBS buffer (pH 7.2). The so-
lutions were incubated at 37 °C for 24 h.
Leukocyte preparation
Equilibrium-binding assay
Whole human blood (10–15 mL) was collected in
vacutainer tubes (Becton Dickinson) containing sodium hep-
arin. The red blood cells were lysed using a modified ammo-
nium chloride method (1 part whole blood to 23 parts
0.145 mol/L ammonium chloride solution containing
1.5 mmol/L potassium bicarbonate and 0.1 mmol/L EDTA).
Tubes were incubated at room temperature for 15 min and
then centrifuged in a Beckman Coulter Allegra 6R centri-
fuge at 900 rpm (400 g) for 7 min at 5 °C. The supernatant
was removed, and the pellet of leukocytes were subsequently
resuspended in Hank’s Balanced Salt Solution (HBSS), con-
The equilibrium-binding assay was used to determine the
number of formyl peptide receptors (FPRs) on the surface of
the neutrophils and the dissociation constant (Kd) of the
fluorescein-labeled fNLFNTK (Molecular Probes) by pre-
paring a saturated binding curve of peptide concentrations
vs. fluorescence intensity (28, 29). Peptide stock solutions
were made by dissolving the peptide derivative in DMSO
and then diluting with HBSS+ to the desired concentrations,
ensuring the concentration of DMSO was less than 0.1%.
Cells at 1 × 106 mL–1 were equilibrated with 0.25, 0.50, 1.0,
3.0, 5.0, 10.0, and 20.0 nmol/L solutions of the peptide de-
© 2005 NRC Canada

Stephenson et al.
2063
Fig. 1. Tc(I)/Re(I) ligands and the Re(I) complex of 3.
rivative (L) in duplicate. The samples were incubated for 2 h
at 4 °C in the dark and then run on the flow cytometer.
Granulocytes were gated on the basis of forward and side
scatter parameters using Expo 32 ADC software (Beckman
Coulter). All samples were repeated in the presence of
30 µmol/L fMLF to account for nonspecific binding. The
number of receptor–ligand complexes per cell (B) was deter-
mined using the following equation, where Q is a normaliza-
tion factor used by Waller and F is the total fluorescence per
cell:
N
N
N
O
HO
N
N
HO
O
N
2
1
+
MESF
cell
⎛
⎜
⎞
⎟
N
N
B (in ligand bound per cell) = F
× Q
N
CO
O
⎝
⎠
O
N
HO
N
Re CO
CO
HO
The level of nonspecific binding did not vary significantly
with concentration of labeled peptide; thus, an average value
of nonspecific binding was subtracted from the total fluores-
cence per cell, F, when determining the number of receptor–
ligand complexes. The total number of N-formyl peptide
receptors, Rtot, and the equilibrium dissociation constant, Kd,
were evaluated by minimizing the squared residual of the
equilibrium solution using a one-site binding model. The
values of Rtot and Kd for each donor were solved simulta-
neously.
N
4
3
The peptide fMLFK(Dde)G was prepared on an Advanced
ChemTech 348 synthesizer using standard Fmoc–HBTU
protecting groups – coupling protocols (Scheme 1). The Dde
protecting group was removed by employing 2% hydrazine
in DMF, and the reaction time was optimized using
ninhydrin to ensure complete deprotection (48). Following
removal of the Dde protecting group, the bifunctional che-
lates (1–3) were coupled to the resin-bound peptides using
HBTU and DIPEA, and reaction times were again monitored
to ensure complete coupling (48). The time required
(80 min) for conversion to the desired product did not differ
from standard amino acid coupling procedures.
Peptide conjugates 5–7 were cleaved from the resin using
a cleavage cocktail containing 94% TFA, 2% triisopro-
pylsilane, 2% ethanedithiol, and 2% water. Nitrogen was
bubbled through the cocktail at 0 °C prior to and during use,
to help prevent oxidation of methionine. Peptides were pre-
cipitated by trituration with cold diethyl ether and centri-
fuged, and the resulting pellet was washed with cold ether to
remove any trace of the cleavage cocktail. Precipitates were
then dissolved in distilled water and lyophilized to yield
white solids.
Peptides 5–7 were analyzed using electrospray mass spec-
trometry (ESMS), and in each case the observed molecular
ion was consistent with the mass of the desired compound
(Table 1). Analytical HPLC and LC–MS studies of each
peptide indicated the presence of two peaks in a 3:1 ratio,
corresponding to the same m/z values. Previous studies in
our group and by others showed that the two peaks in the
HPLC chromatogram are the result of epimerization of
methionine, which is promoted by the electron-withdrawing
nature of the formyl group (17, 49). Notwithstanding, the
two isomers were readily separated using semipreparative
HPLC.
One of the advantages of using M(I) (M = Tc, Re) ligands
for methods involving solid-phase synthesis is the ability to
prepare the Re complex in parallel with compounds contain-
ing the free ligand. Preparation of Re complexes is vitally
important as they are needed for proper characterization of
the 99mTc-analogues and for performing in vitro screening
studies. For the method described here, the Re complex of 7
Results and discussion
The aim of this study was to develop a versatile bio-
conjugation strategy in which Tc ligands could be linked to
peptides using a conventional automated peptide synthesizer.
The method entailed incorporating lysine into the backbone
of a peptide in such a manner that the ε-nitrogen could be
selectively liberated and a Tc chelate added while the pep-
tide was still linked to the resin. Using this approach, a num-
ber of important structural features, including the position
and nature of the chelate, can be easily varied.
The blocking group selected for the ε-nitrogen of lysine
was the 1-(4,4-dimethyl-2,6-dioxocyclohexyldiene)ethyl (Dde)
protecting group, which is stable to the conditions used in
typical Fmoc solid-phase synthesis, and which can be selec-
tively liberated without affecting Boc protecting groups (30–
32). The planned approach is amenable to any bifunctional
chelating system containing a pendent acid group; however,
for the work described here a series of dipyridyl amine lig-
ands with linker arms of varying length were employed
(Fig. 1) (17). These ligands form stable complexes with
Tc(I), and their Re(I) complexes are sufficiently robust that
the metal complex can be incorporated into peptides using
the same methodology employed for the free ligand (33–37).
To develop the approach, the chemotactic peptide
fMLFKG, which binds to the formyl peptide receptor (FPR)
expressed on granulocytes, was used as a model system. La-
belled versions of fMLF have shown potential utility for im-
aging sites of infection and inflammation (38–47). fMLF
bioconjugates have traditionally been prepared in solution by
conjugating hydrazine-type ligands (HYNIC) to the ε amine
of fMLFK. This approach is not easily amenable to prepar-
ing libraries of fMLF derivatives, which would accelerate
the process of identifying the appropriate derivative for clini-
cal applications.
© 2005 NRC Canada

2064
Can. J. Chem. Vol. 83, 2005
Table 1. HPLC and ESMS data for compounds 5–8.
Compound number
Elution time (min)
Found m/z
Calcd. m/z
Yield (%)
νCO (cm–1)
876.4
890.5
904.5
876.1
890.1
904.1
5
6
7
8
6.6
6.5
6.9
8.7
34
35
33
30
—
—
—
1174.4
1174.3
1933, 2041
Fig. 2. Proposed mechanism for the degradation of an Re(I)–peptide conjugate.
S
S
O
O
O
H
N
H
N
O
O
O
OH
H
N
H
N
H
N
H
N
H
N
H
OH
H
N
H
N
H
N
H
O
O
O
O
O
O
CO
NH
N
NH
+
CO
CO
N
Re
O
O
N
H
N
N
N
Re
+
CO
CO
CO
+
(8) was prepared successfully in parallel with the synthesis
of compounds 5–7, without modifying the basic strategy.
After cleavage of the conjugate from the resin using the
same cocktail described above, the product was isolated in
comparable yields to the free ligands. Mass spectrometry
clearly indicated the presence of the desired product (8),
while IR showed two distinct metal CO stretches (1933 and
2041 cm–1), which are consistent with values reported for
analogous Re complexes (17, 33–37).
Scheme 2. Synthesis of fMLFK(4m-Tc(CO)3 )G (9).
S
O
O
O
H
N
H
N
OH
N
H
N
H
N
H
H
O
O
O
N
N
HN
N
As an aside, attempts to prepare the peptide containing the
Re(I) complex of 1 lead to a complex mixture of products
according to HPLC. MS analysis of the precipitate formed
after the addition of ether to the reaction mixture indicated
the presence of the target compound, along with several deg-
radation products. The likely cause of these impurities is
elimination to form an α,β-unsaturated amide, which con-
comitantly results in liberation of a neutral metal complex
(Fig. 2). By-products of this nature were not observed for
the other ligands.
O
7
[
99mTc(CO)3(OH2)3]+
To ensure the products from the solid-phase synthesis can
be radiolabeled, compound 7 (100 µg) was combined with
370 MBq (10 mCi) of [99mTc(CO)3(OH2)]+, which was pre-
pared using the commercially available Isolink kit
(Scheme 2). After 60 min at 75 °C, the desired product 9
was isolated by HPLC in greater than 98% radiochemical
purity in 63% radiochemical yield. Analytical HPLC of the
purified product is shown in Fig. 3. The stability of the puri-
fied peptide 9 towards metal substitution was tested using a
large excess of cysteine and histidine. The peptide and the
two amino acids were heated at 37 °C for 24 h, whereupon
HPLC analysis showed that the product was intact in both
cases and that the radiochemical purities remained above
95% (Fig. 4).
S
O
O
O
H
N
H
N
OH
N
H
N
H
N
H
H
O
O
O
CO
CO
CO
N
HN
N
Tc
+
O
9
N
© 2005 NRC Canada

Stephenson et al.
2065
Fig. 3. γ-HPLC radio chromatogram (mV vs. time (min)) for pu-
rified 9.
Table 2. Dissociation constants (Kd) for compounds 5–8.
Compound Number
Peptide sequence
Kd (nmol/L)
fMLF
32 3
21 6
52 3
55 8
67 6
5
6
7
8
fMLFK(2M)G
fMLFK(3M)G
fMLFK(4M)G
+
fMLFK(4M-Re(CO)3 )G
determined using flow cytometry. Flow cytometry is an at-
tractive tool for screening novel molecular imaging agents
because it is amenable to automation, which means that li-
braries can be evaluated rapidly, and it affords the opportu-
nity to investigate specific cell populations. To evaluate the
fMLF derivatives, human white blood cells were incubated
with 1.0 nmol/L fluorescein-labelled fNLFNTK and varying
amounts of the competitor in duplicate for 2 h at 0 °C in the
dark. fMLF, which was used as a reference standard, exhib-
ited a Kd that was in good agreement with the value reported
in the literature (28, 50). Conjugates 5–8 had dissociation
constants (Table 2) in the low nanomolar range, which is
similar to the value for fMLF, thus demonstrating that incor-
poration of the ligand or Re complex does not have a nega-
tive impact on the ability of the peptides to bind the FPR.
Fig. 4. γ-HPLC radio chromatograms (mV vs. time (min)) of the
(a) cysteine and (b) histidine challenges of 9 after 24 h at 37 °C.
Conclusions
The reported strategy offers a new alternative to existing
solid-phase synthetic methodologies for preparing peptide-
based radiotracers. The ligands and their rhenium complexes
employed in this study can be incorporated into the back-
bone of peptides using traditional automated solid-phase
synthesis methods. For instruments that have multiple reac-
tion wells, the free ligand and Re–peptide conjugates can be
prepared in parallel. The peptide derivatives described here
were prepared in parallel in multi-milligram quantities,
which provided sufficient material to allow for complete
characterization, radiolabelling, and in vitro screening stud-
ies to be performed from the products of a single run of the
synthesizer. The reported methodology can be used to pre-
pare libraries of compounds in which several factors, includ-
ing peptide sequence, site of derivatization, nature of the
chelate, and both the type and length of the spacer can be
varied, which will decrease the time and effort required to
identify lead compounds.
Acknowledgements
We would like to acknowledge the Ontario Research and
Development Challenge Fund (ORDCF) and the Natural
Sciences and Engineering Research Council of Canada
(NSERC) for funding and for providing a scholarship for
KAS.
With the above methodology in hand it is possible to gen-
erate diverse libraries of compounds. The next step is to se-
lect the appropriate in vitro screening method that will
indicate what compounds are the most promising for more
advanced biological studies. In the example presented here,
the ability of peptides 5–8 to bind to the FPR receptor was
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