N(epsilon) functionalization of metal and organic protected L-histidine for a highly efficient, direct labeling of biomolecules with [Tc(OH2)3(CO)3]+.

Article abstract of DOI:10.1002/chem.200204445

Two different pathways for the introduction of an acetyl group at N(epsilon ) in a N(alpha), N(delta), and -COO protected histidine to afford N(epsilon)-(CH(2)COOH)-histidine derivative 7 b are presented. The purpose of this study is the coupling of 7 b t

Full text of DOI:10.1002/chem.200204445

FULL PAPER
e
N Functionalization of Metal and Organic Protected l-Histidine for a Highly
‡
Efficient, Direct Labeling of Biomolecules with [Tc(OH2)3(CO)3]
Jae Kyoung Pak, Paul Benny, Bernhard Spingler, Kirstin Ortner, and Roger Alberto*[a]
Abstract: Two different pathways for
the introduction of an acetyl group at Ne
in a Na, Nd, and -COO protected histi-
dine to afford Ne-(CH2COOH)-histidine
derivative 7b are presented. The pur-
pose of this study is the coupling of 7b to
amino groups in bioactive molecules
such as peptides. After full deprotection
of such a bioconjugate, histidine pro-
vides three coordination sites which
efficiently coordinate to [99mTc(OH2)3-
(CO)3] or [Re(OH2)3(CO)3] in a facial
geometry. This allows the development
of novel radiopharmaceuticals. Selective
derivatization at the Ne position has
conveniently been achieved by concom-
itant protection of Na and Nd with a
carbonyl group forming a six-membered
urea. Cyclic urea ring opening with Fm-
OH, coupling of phenylalanine as a
model to 7b through its primary amine
and removing of all protecting groups in
one step gave a histidine derivative of
phenylalanine which could be labeled at
10À5 m with 99mTc in very high yield and
even in about 50% yield at 10À6 m. The
X-ray structure of a complex with [Re-
in which a metal fragment is used as a
protecting group for organic function-
alities. The coordination to histidine
protects the Na, Nd and COO group in
one single step, subsequent alkylation
with BrCH2COOH(R) at Ne, coupling to
phenylalanine and oxidative deprotec-
‡
tion of [Re(CO)3] to [ReO4]À gave the
‡
(CO)3] in which anilin is coupled to 7b
corresponding bioconjugate in which
histidine is coupled to phenylalanine
through an acetylamide at Ne. Both
methods offer convenient pathways to
introduce histidine in a biomolecule
under retention of its three coordination
sites. The procedures are adaptable to
any biomolecule with pendant amines
and allow the development of novel
radiopharmaceuticals or inversed pepti-
des.
confirms the facial arrangement of his-
tidine. A second pathway applies direct-
‡
‡
‡
ly the [Re(CO)3] moiety as a protecting
group. This is one of the rare examples
Keywords:
chemistry
bioorganometallic
histidine
¥
¥
radiopharmaceuticals ¥ rhenium ¥
technetium
99mTc, ii) should be labeled at high specific activity (low vector
concentration) and finally retain its physicochemical proper-
ties and its affinity towards the corresponding receptor. For
routine use, the labeling process must be performed prefer-
entially in one single step. Different procedures are available
from literature and for peptides in particular the hynic
approach seems to be promising although it suffers from the
lack of a clearly defined compound which is required for
Introduction
The labeling of biologically active molecules with 99mTc for
radiopharmaceutical purposes is a field of intense research.[1
4]
The commercially available perfusion agents for radioimaging
have to be complemented by labeled vectors which will allow
a more precise targeting of various receptors expressed in
higher density on for example cancer cells. So far, a few
compounds are in pre-clinical trials[5 but none has found
clinical approval.[10
9]
15]
commercial application so far. Many chemical and biological
difficulties have to be overcome. Chemically, the targeting
vector has to be i) derivatized with an appropriate chelator for
A valuable extention of these ™classical∫ approaches is the
use of bioorganometallic compounds, the versatility of which
has recently been highlighted by Metzler-Nolte.[16] In that
context, we recently presented the one pot synthesis of the
‡
organometallic aqua ion [99mTc(OH2)3(CO)3] and showed the
versatility of using this complex fragment for the labeling of
[a] Prof. Dr. R. Alberto, Dr. J. K. Pak, Dr. P. Benny,
Dr. B. Spingler, Dr. K. Ortner
23]
various biomolecules and peptides in particular.[17 One of
the major advantages of the carbonyl approach is the
availability of a well defined complex with very high specific
activity only depending from the ligand type.[24] Naturally
occurring bidentate ligands such as N-terminal histidines in
Institute of Inorganic Chemistry, University of Z¸rich
Winterthurerstrasse 190, 8057 Z¸rich (Switzerland)
Fax : (‡41) 1 635 68 03
E-mail: ariel@aci.unizh.ch
Supporting information for this article is available on the WWW under
http://www.chemeurj.org or from the author: Ortep plots and atom
labeling schemes for non-hydrogen atoms of 6a and 11. X-ray
crystallographic tables of complexes 3, 6a, and 11.
peptide chains can efficiently be labeled with
‡
[
99mTc(OH2)3(CO)3] . An improvement in respect of specific
activity was the introduction of a terminal histidine through
Chem. Eur. J. 2003, 9, 2053 2061
DOI: 10.1002/chem.200204445
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2053

R. Alberto et al.
FULL PAPER
the a-amino group which allowed labeling at low ligand
concentration.[25] The relatively high lipophilicity of this type
of bifunctional chelator related to the difficulty of its synthesis
prompted us to seek for a differently derivatized histidine
which would allow its introduction into any peptide with a
minimum of synthetic work and a maximum of labeling
efficiency. Since the complex [99mTc(his)(CO)3] is hydrophilic
it is appropriate to derivatize the histidine at the e-nitrogen in
the imidazole ring. This functionalization leaves the highly
efficient tripodal coordination site untouched but still allows
the coupling to amine or carboxylic groups in biomolecules.
Finally, since the synthesis of [99mTc(his)(CO)3] can be
performed in one single step from [99mTcO4]À, histidine also
fulfils the requirement for a one pot labeling procedure
without affecting the ligand.
for Nd, Na and the carboxylate had to be introduced prior to
Ne derivatization. A major difficulty arose from the selective
protection of Ne and Nd since typical protecting agents tend to
react with both. In general, mixtures of Ne and Nd derivatives
are very difficult to separate even if the desired Ne-derivative
represents the main product. In addition, Na can also be very
competitive along such a derivatization. The key step of our
method to bypass these problems is represented by the ring
closure and the formation of a six-membered urea ring in
histidine which allows simultaneous and exclusive protection
of Na and Nd in histidine methyl ester. Subsequently, Ne was
alkylated to give the desired derivative of histidine. Amine
and carboxylate groups can be introduced in that way, we
focus in this paper only on the latter. These groups can then be
coupled with the C- or the N-terminus of a peptide through
amide formation. The approach is straightforward but de-
mands a multi-step synthesis. The full reaction scheme for the
introduction of a alkyl carboxylate at Ne is given in Scheme 2.
We want to present in this paper two different strategies for
the synthesis of Ne derivatized tripodal histidine, its conven-
ient coupling to an amino acid and its highly efficient labeling
‡
with 99mTc. One strategy employs the [Re(CO)3] moiety as a
novel organometallic protecting group for the three function-
alities, carboxylic acid, primary and aromatic Nd amine in
histidine. Alkylation at Ne in [Re(his)(CO)3][26] can be
performed in parallel to a procedure very recently described
for [Mo(his)(CO)3]À.[27 29] After coupling to an amino acid or
a peptide, the organometallic protecting group is released by
mild oxidation. Alternatively, the functionalities in histidine
were protected stepwise by organic groups. The simultaneous
protection of Nd and Na in one step by formation of a cyclic
urea ring is very convenient and is used to introduce an active
coupling group in Ne. The two methods are shown in
Scheme 1.
COOCH3
NH
NH2
COOR
HN
i
N
N
N
O
1: R = H
3
2: R = CH3
ii
O
C
COOH
COOCH3
NH
HOOC
iii
N
N
t BuO
N
N
NH2
5
O
4
iv
vi
O
COOCH3
NHPG
13
N
t BuO
N
6a: PG = Cbz
6b: PG = Fmoc
Results and Discussion
v
The coupling of histidine 1 through a carboxyl functionality
introduced at Ne to peptides is a multi-step procedure. The
acetyl linker attached to Ne has to be selectively deprotected
after the alkylation. Deprotection, activation and coupling to
peptides or amino acids should not affect the other protecting
groups as well. Deprotection of the coordinating function-
alities after the coupling process must occur under conditions
typically encountered in peptide synthesis to be generally
useful. Due to potential cross reactions, the protecting groups
O
COOCH3
NHPG
N
HO
N
7a: PG = Cbz
7b: PG = Fmoc
Scheme 2. i) 2, Im2CO, DMF, 708C, 6 h, 61%; ii) bromoacetic tert-butyl
ester, CH3CN, reflux, 24 h; iii) 6n HCl, reflux, 48 h; iv) 6a: BnOH, THF,
reflux, 16 h, 62% (from 3 in two steps); 6b: Fm-OH, CH3CN, RT, 14 h,
55% (from 3 in two steps); v) TFA/CH2Cl2 1:1, RT, 2.5 h; vi) [Et4N]2-
[ReBr3(CO)3], H2O, RT, 21 h, 68% (from 3 in three steps).
An approach to protect Na
and Nd in one step is the
reaction of l-histidine methyl
COOH
O
COOR
N
NH2
N
Peptide
Peptide
N
N
ester 2 with N,N'-carbonyldii-
midazole in DMF which led to
compound 3 by formation of a
six-membered urea ring com-
prising the Na and Nd protected
amino acid in very good
yield.[30] The structure of com-
pound 3 could be confirmed by
an X-ray structure and the mol-
ecule is depicted in Figure 1.[31]
HOOC
Peptide
Peptide
NH2
NHP
N
H
O
O
O
N
O
H N
N
N
2
HOOC
NH2
N
O
H
H2N
N
Re
CO
CO
Re
OC
CO
OC
CO
Scheme 1. Introduction of tripodal, Ne functionalized histidine into
a biomolecule for labeling with
‡
[
99mTc(CO)3] .
2054
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2053 2061

Tc Labeling of Histidine Peptides
2053 2061
Figure 2. ORTEP plot of 6a, ellipsoids drawn at 50% probability, showing
one of the two molecules in the asymmetric unit.
afford compounds 7a and 7b which can now be coupled to a
free amino group in the biomolecules. Deprotection of Na and
hydrolysis of the remaining ester will then give the histidine
derivatives retaining its tripodal mode of coordination. To
probe this process with an amino acid, we have chosen l-
phenylalanine ethyl ester (H-Phe-OEt). The reaction of 7a or
7b with H-Phe-OEt in the presence of BOP as a coupling
reagent and NEt3 as an organic base gave the compounds 8a
and 8b in 90 and 74% yield as white solids. The reaction was
strongly influenced by the pH of the solution and it proved to
be important to keep it neutral by repeatedly adding NEt3
during the reaction.
Figure 1. ORTEP plot of 3, ellipsoids drawn at 50% probability.
Alkylation of the urea compound 3 with bromoacetic tert-
butyl ester in refluxing acetonitrile for 24 h provided the
quaternary ammonium salt 4 in almost quantitative yield.
According to 1H NMR spectroscopy, the crude product 4 was
clean enough to proceed to the next step without further
purification.
Starting from compound 4, several options are feasible to
achieve a histidine derivative of a biomolecule. Refluxing 4 in
6m HCl for 48 h led to the fully deprotected compound 5
which can then be used for introduction into a biomolecule
‡[33]
To enable the coordination of the fac-[Re(OH2)3(CO)3]
‡
with the ™[Re(CO)3] ∫ moiety as a protecting group, as
complex to tripodal histidine in 8a or 8b (or in any other
derivatized biomolecule) or for labeling with the 99(m)Tc
discussed later in this text. Optionally, the urea ring can be
opened by refluxing 4 in neat methanol or ethanol. If
subsequent coupling with a biomolecule is attempted, the
resulting carbamates will not be very convenient since their
cleavage to give the amines requires drastic conditions. We
have therefore chosen other alcohols such as benzyl alcohol or
fluorenol (Fm-OH) which would result in a Cbz or a Fmoc
group easier to cleave. In neat benzyl alcohol, many side
products were found and only the reaction in acetonitrile or
THF gave a clear product. The Na-Cbz protected compound
6a was obtained from the reaction of benzyl alcohol in
refluxing THF in the presence of NEt3 overnight in an overall
yield of 62% relative to 3. Compound 6b was prepared from
acetonitrile since Fm-OH is poorly soluble in THF. Unex-
pectedly, the reaction with Fm-OH required much milder
conditions than the reaction with any other alcohol. We
observed that reflux conditions or even 408C were sufficient
to cause spontaneous deprotection of the once formed Fmoc
group. Thus, the ring opening reaction was performed at RT
for 14 h to give a 55% yield of 6b relative to 3. To obtain a
Boc protecting group, the reaction was performed with tert-
butanol; however even after 4 d the yield was less than 20%.
The X-ray structure of 6a could be elucidated and is shown in
Figure 2.[32]
‡
homologue [99(m)Tc(OH2)3(CO)3] ,[34] full deprotection is
required. In case of 8a which contains benzyloxycarbamate
(Cbz) as a protecting group, a two-step procedure was
employed to produce 9 (Scheme 3). In a first step, Cbz was
removed by hydrogenation with H2 and Pd/C under acidic
i
H
COOCH3
NHPG
7a,b
N
BOP
N
N
O
EtOOC
8a: PG = Cbz
8b: PG = Fmoc
H
COOH
NH2
ii
8a,b
iii
15
N
N
N
O
HOOC
9
Scheme 3. i) H-Phe-OEt, BOP, Et3N, CH2Cl2, RT, 1.5 2.5 d, 74 90%;
ii) deprotection: Cbz: H2, Pd/C, MeOH/H2O/AcOH (7:2:1), RT, 2 h;
Fmoc: piperidine, RT, 30 min; ester: 0.5m LiOHaq/MeOH (1:2), RT,
overnight; iii) [Et4N]2[ReBr3(CO)3], H2O, RT, 19 h, 40 50%.
Compounds 6a and 6b are fully protected with groups each
of which can independently be removed to receive a variety of
histidine derivatives. These can be employed in further
derivatizations for biomolecule coupling through Na, Nd, or
the carboxyl group of histidine. A free acetyl group at Ne
histidine is required for attaching 6a or 6b to the N-terminus
of a peptide after standard activation. Correspondingly, the
tert-butyl ester group in 6a and 6b were hydrolyzed to the
carboxylic group by reaction with TFA/CH2Cl2 1:1 for 2.5 h to
conditions. Clean deprotection occurred with a yield of 75
80% in MeOH/H2O/AcOH (7:2:1). Subsequent and quanti-
tative deprotection of the ester groups was performed by
alkaline hydrolysis in aqueous 0.5m LiOH at RT overnight. A
much more convenient one step deprotection is possible for
8b, containing Fmoc as a protecting group. Stirring these
compounds in piperidine for 40 min at RT was sufficient to
remove all the protecting groups and to produce 9 in one step
and in quantitative yield. Thus, for convenience the applica-
Chem. Eur. J. 2003, 9, 2053 2061
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2055

R. Alberto et al.
FULL PAPER
tion of 7b for the derivatization of biomolecules with tripodal
histidine is highly favoured.
significantly reduces the number of steps for derivatization
and coupling. The coordination of [Re(CO)3] with l-
‡
This fully deprotected derivative 9 was treated with
histidine gives quantitatively well characterized 12[26, 36] in
which Na, Nd, and the carboxylate are all protected in one
single step. The alkylation of 12 with ethyl bromoacetate or
tert-butyl bromoacetate in the presence of Cs2CO3 directly
produced the alkylated complex 14a or 14b, respectively, as
single products in very good yield (Scheme 5). A similar
reaction has recently been described for an organometallic
‡
[Re(OH2)3(CO)3] to yield the corresponding neutral com-
plex, that is, [(9)Re(CO)3] 15. Beside the coupling to H-Phe-
OEt, we linked 7b to a variety of other model compounds
containing an amino group in order to get suitable crystals to
confirm the structure of a rhenium complex by X-ray
structure analysis. Compound 7b was coupled to aniline to
yield the corresponding anilide 10 after deprotection with
piperidine (Scheme 4). Reaction of 10 with [Re
‡
(OH2)3(CO)3] in water or methanol gave the rhenium
O
O
ROOC
HN
O
N
i
N H2N
Re
O
H N
2
N
COOCH3
NHPG
O
Re
CO
CO
i
CO
CO
OC
7b
N
NH2
OC
N
H
N
12
13: R = H
14a: R = Et
14b: R = t Bu
ii
O
COOH
NH2
O
O
ii
14a,b
HOOC
N
N
O
N
H
N
N
H N
N
2
Re
10
CO
CO
OC
O
OH2
Re
iii
13
H2O
OC
10
OH2
CO
N
O
N
H
H N
2
Scheme 5. i) Ethyl/tert-butyl bromoacetate, Cs2CO3, CH3CN, 358C, 1.5 h,
90%; ii) deprotection: ethyl ester: 0.5m LiOH/MeOH (1:2), RT, overnight;
tert-butyl ester: TFA/CH2Cl2 1:1, RT, 2 h.
CO
Re
CO
CO
11
OC
Scheme 4. i) BOP, Et3N, CH2Cl2/DMF, RT, 2 d; ii) piperidine, RT, 30 min;
iii) [Et4N]2[ReBr3(CO)3], H2O, RT, overnight, 75%.
Mo complex with the purpose of attaching targeting mole-
cules to the new carboxylate but not for protecting histi-
dine.[37] It is imperative to note that alkylation at Na or Nd was
not observed at all during the alkylation reaction, illustrating
complex [(10)Re(CO)3] 11, its structure could finally be
elucidated,[35] and an ORTEP presentation is given in
Figure 3.
‡
the high kinetic and thermodynamic stability of [Re(CO)3] in
the histidine complex. With a labile complex, it might be
expected that alkylation also occurs at the other amines in 1
when in equilibrium they are shortly released from the
protecting metal.
The ester hydrolysis in 14a,b to the corresponding carbox-
ylic acid 13 was performed under different conditions depend-
ing on the type of ester. The ethyl ester of 14a was
deprotected by treatment with LiOH in MeOH/H2O mixture
and the tert-butyl ester of 14b was removed under acidic
conditions with trifluoroacetic acid in methylene chloride.
With each method, 13 was produced in high yield which can
now be coupled to a biomolecule by standard techniques.
Phenylalanine was chosen as a model compound and coupled
Figure 3. ORTEP plot of 11, ellipsoids drawn at 50% probability, showing
one of the two molecules in the asymmetric unit.
The molecular structure of 11 shows the tripodal histidine
‡
coordination to the [Re(CO)3] fragment occurring through
Nd, Na and the carboxylate. The complex is neutral and the
primary amino group is not deprotonated. The anilide group,
which is a model for a biomolecule, points away from the
metal complex. Bond lengths and angles are within the
expected range and comparable to the isoelectronic Mo
complex [Mo(his)(CO)3]À.[28]
with 13 to produce 15 (Scheme 6). The [Re(CO)3] acts as a
‡
versatile protecting group for 1 during the whole process.
Although the use of metal cations or complex fragments as
protecting groups is known in organometallic chemistry,
examples with Werner-type ligands are rare and only a
limited number of examples have been described at all.[38, 39]
In principle, a metal center could protect six functionalities
the same time. To successfully apply such a procedure, the
complex fragment must be robust, otherwise dynamic decom-
plexation/complexation would not allow selective derivatiza-
tion at non coordinated sites. The prerequisite limits the type
of metal centers for this purpose essentially to d6 systems as is
The method discussed with organic protecting groups is
convenient to introduce tripodal histidine in biomolecules.
Deprotection occurs under mild conditions and is compatible
with sensitive biomolecules. As a valid alternative to the pure
organic procedure, we have introduced the organometallic
‡
moiety [Re(CO)3] as a protecting motif which occupies the
‡
three reactive sites in histidine at the same time. This
the case with the [Re(CO)3] moiety.
2056
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2053 2061

Tc Labeling of Histidine Peptides
2053 2061
shorter than the fully organic approach discussed in the first
part. The only limitation for the choice of the biomolecule is
its sensitivity towards oxidative conditions. When the peptide
contains for example methionine, the organic method is
favorable since oxidative removal of rhenium would lead to
oxidation of the thio ether group. For many radiopharma-
ceutically interesting molecules such as central nervous
system receptor ligands, this sensitivity does not apply and
the method using an organometallic protecting group is much
more straightforward.
O
i
H
13
N
N
O
N H2N
Re
O
HOOC
15
CO
CO
OC
ii
H
N
15
COOH
NH2
N
N
O
HOOC
9
The tripodal histidine ligand is one of the most efficient
‡
chelating system for the [99mTc(CO)3] moiety, a crucial
ii
COOH
NH2
13
HOOC
N
prerequisite for the preparation of radiopharmaceuticals with
the highest possible specific activity. If high biomolecule
concentrations are required for complete labeling, the 99mTc
radiopharmaceutical has to be separated from unlabeled, cold
material to prevent blocking of the receptors with non-labeled
compounds. Ideally, the concentration of the derivatized
biomolecule is equal to 99mTc. Since the concentration of 99mTc
is very low (10À7 10À6 m), this is hardly achieved for kinetic
reasons but histidine allows complete labeling at concentra-
tions near stochiometry.[24] To assess this behaviour for the
derivatized histidines, we have performed labeling studies
under various, radiopharmaceutically useful conditions. Two
labeling procedures are possible. One is a two-step procedure
N
5
Scheme 6. i) H-Phe-OEt, BOP, Et3N, CH2Cl2/DMF, RT, overnight, 75%;
ester deprotection: 0.5m LiOH/MeOH (1:2), RT, overnight, quantitative;
ii) 10 30 equiv H2O2, H2O, HCl (TFA or AcOH), 508C, 4 48 h.
Instead of alkylating 12, ligand 5 can also directly be
coordinated to the [Re(CO)3] moiety. Compound 5 was
obtained analytically pure after reacting 4 in refluxing 6n HCl
for 2 d. Further reaction with [Et4N]2[ReBr3(CO)3] in water
afforded the complex 13 in 68% overall yield relative to 3 in
three steps (Scheme 2).
For subsequent labeling of a histidine derivatized biomo-
lecule with [99mTc(OH2)3(CO)3] , the protecting [Re(CO)3]
moiety must be removed to receive the final bioconjugate.
Due to the high kinetic and thermodynamic stability of
[Re(his)(CO)3], simple decomplexation at low pH is not
sufficient since recomplexation readily takes place.
‡
‡
consisting in initial preparation of [99mTc(OH2)3(CO)3] and
‡
‡
subsequent coordination to the compounds 5 and 9 to yield 16
and 17, respectively. Clinically more relevant, the labeling is
performed directly in one single step in one single vial from
[
99mTcO4]À in the presence of for example 9 and the reducing/
CO transfer reagent Na2[H3BCO2]. The two procedures are
depicted in Scheme 7.
‡
The [Re(CO)3] moiety must be ™deactivated∫ by oxidation
O
with H2O2 under acidic conditions to yield [ReO4]À and
provide the unprotected derivatized biomolecule. Experi-
mentally, the disappearance of the rhenium complex and the
appearance of [ReO4]À can conveniently be followed in the
HPLC trace at 250 nm wavelength.
OH2
HOOC
5
H2O
OC
[TcO4]-
OH2
CO
N
O
Tc
H2N
Tc
OC
N
CO
16
CO
CO
OH2
H2O
OC
Complexes 13 and 15 were examined as simple models for
more complicated biomolecules to assess the conditions
required for the complete oxidative removal of [Re(CO)3] .
9
OH2
CO
Tc
O
H
CO
N
‡
[TcO4]-
N
O
H2N
N
9
O
Conditions with variable equivalents of H2O2 relative to
rhenium and different pH values were screened with three
acids, HCl, TFA, and acetic acid. It was found that the H2O2
concentration (10, 20, or 30 equivalents) gradually improved
the conversion of 13 or 15 to [ReO4]À. The pH (0.5, 1, or 3) of
the solution greatly affected the reactivity of the rhenium
complex towards oxidation. 20 or 30 equivalents of H2O2
relative to Re at a pH of 0.5 with HCl were sufficient to
completely remove the Re after 8 h at 508C. At higher pH
values, the reaction proceeds with significantly slower con-
version rate to [ReO4]À. Similar results were also obtained
with trifluoroacetic acid whereas the reaction with acetic acid
and H2O2 showed only very slow oxidation. A low pH value is
crucial for a reasonable conversion rate and suggests that
HOOC
Tc
CO
CO
OC
17
Scheme 7. One- and two-step labeling of a histidine derivatized amino
acid.
The complex [99mTc(OH2)3(CO)3] was prepared as descri-
‡
bed in the literature[17, 40] or from commercially available
Isolink kits (available from Mallinckrodt). After buffering
with phosphate buffer of pH 7.4, an aliquot of a standard
solution of the ligands 5 or 9 was added in a final
concentration of 10À4, 10À5 or 10À6 m, in general in a total
volume of 1 mL. For each reaction, only the 99mTc complexes
16 and 17 were obtained. The two different synthetic routes
(the organic or the organometallic protection pathway) gave
similar labeling yields illustrating that compound 15 did not
decompose during the oxidative deprotection process. Slightly
lower yields found for the labeling of ligands obtained from
the oxidation process are likely to be due to lower effective
‡
oxidation occurs only when the [Re(CO)3] moiety is fully or
partially decomplexed from (protonated) histidine ligand but
‡
not directly in closed shell [Re(his)(CO)3] .
For the derivatization of a biomolecule with 5, the use of
‡
[Re(CO)3] as a protecting group is versatile and the method
Chem. Eur. J. 2003, 9, 2053 2061
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2057

R. Alberto et al.
FULL PAPER
concentrations. For characterization of the 99mTc compounds,
the retention times were compared to the fully characterized
rhenium analogues and proved to be the same. Labeling yields
under different conditions are summarized in Table 1.
peptide bond in 9 and no reduction or hydrolysis occurred.
This demonstrates the versatility of this one-pot process not
only for the model compounds presented herein but also for
other biologically active polypeptides or other receptor
binding molecules. The conditions employed in the one-pot
reaction corresponds exactly to what is required for routine
application, the quantitative formation of one single structur-
ally determined species at very high dilution. This product is
principally ready to be injected without purification or
separation from excess cold material or byproducts.
‡
Table 1. Labeling of Ligand 5 and 9 with [99mTc(CO)3] .
Method
No. of ligand c [m]
T [8C] t [min] Yield [%]
two-step labeling
5
5
5
9
9
9
9
9
9
9
10À4
10À5
10À6
10À4
70
70
70
90
30
30
30
30
30
30
30
20
20
20
quantitative
91[a]
20[b]
quantitative[c]
quantitative
quantitative[d]
41[e]
94
96
34[e]
5 Â 10À5 90
10À5
10À6
10À4
90
90
90
Conclusion
one-pot labeling
We have presented in this report two convenient methods for
the introduction of a tripodal Ne derivatized histidine into
model peptides. The organic method requires more steps but
can also be applied to biomolecules susceptible to oxidation
whereas the method using an organometallic moiety as
protecting group is straightforward but limited to biomole-
cules which are not very sensitive towards oxidation. Both
methods end up with the same compounds in which an acetyl
group at the imidazole ring can be coupled to an amino group
of any biomolecule. The functionality on the pendant arm
attached to Ne is variable and primary amines as well as
hydroxy groups can be introduced. After deprotection, the
histidines can be labeled with 99mTc to yield one single
radiopharmaceutical of very high specific activity without
purification. A full one-step procedure is possible without
preparing precursors or intermediates. Finally due to the high
hydrophilicity of the attached histidine complex, this method
is in particular suitable for hydrophilic biomolecules such as
most of the biologically active peptides. Currently, we are
studying a number of these peptides for their application as
novel radiopharmaceuticals.
5 Â 10À5 90
10À5
90
[a] The labeling was done quantitatively in 1 h. Ligand 5 from the
oxidation, at 758C for 30 min yield was 85%. [b] The labeling reached
64% yield after 1.5 h. [c] Ligand 9 from the oxidation showed 88% yield at
908C for 30 min. [d] Ligand 9 from the oxidation showed 73% yield at
908C for 30 min. [e] The labeling reached more than 65% yield after 1.5 h.
At a ligand concentration of 10À4 m both 5 and 9 were
quantitatively labeled at 708C after 30 min to provide the
‡
products 16 and 17. The yields relative to [99mTc(OH2)3(CO)3]
are better than 95%, the difference counts for [99mTcO4]À
small amounts of which were sometimes present after the
synthesis of [99mTc(OH2)3(CO)3] . In case of ligand 5, labeling
‡
was completed even at RT after 30 min at this concentration.
To afford complete complex formation, 10À5 m solutions
required longer reaction times or higher temperatures and
gave exclusively 16 and 17. In case of complex 17, quantitative
yield was achieved at 908C within 30 min. Since the concen-
tration of the ligand is reduced by a factor of 10 in this pseudo
first-order kinetics, higher temperature must compensate low
concentration. At 10À6 m (corresponds to 1 nmol of biomole-
cule), the concentrations of 99mTc and ligand become com-
parable and the kinetic law switches to second order slowing
down the rate further. The labeling reaction took longer and
the yields at 708C were 64% for 5 and 69% for 9 after 90 min,
Experimental Section
General techniques: All chemicals were purchased at highest commercial
quality and used without further purification, unless stated otherwise. All
the reactions were performed under nitrogen or argon and monitored by
thin-layer chromatography (TLC) carried out on 0.25 mm Merck silica gel
aluminium plates (60 F254) using UV light as visualizing agent. Silica gel
(particle size 0.040 0.063 mm) was used for flash column chromatography.
NMR spectra were recorded on Bruker, DRX-500 (500 MHz) or Varian
Gemini-2000 (300 MHz) instruments. Mass spectra were recorded on a
Merck, M-8000 LC/3DQMS under electrospray ionization(ESI) condition.
Yields refer to chromatographically and spectroscopically homogeneous
materials.
respectively. At this concentration the oxidation of
‡
[
99mTc(OH2)3(CO)3] by traces of O2 becomes a major side
reaction but still significant amounts of labeled compounds
were produced.
The one-pot reaction from [99mTcO4]À in the presence of 9
to compound 17 was investigated according to Scheme 7. The
[
99mTcO4]À generator eluate was now added to a vial contain-
ing the compounds required for the preparation of
Crystal data and experimental details are listed in the Supporting
Information. Suitable crystals were covered with ParatoneN oil, mounted
on top of a glass fibre and immediately transferred to a Stoe IPDS
diffractometer. Data was collected at 183(2) K using graphite-monochro-
mated MoKa radiation (l ˆ 0.71073 ä). Data was corrected for Lorentz and
polarisation effects as well as for absorption (numerical, but not for 3).
Structures were solved with direct methods using SHELXS-97[41] or
SIR97[42] and were refined by full-matrix least-squares methods on F2 with
SHELXL-97.[43] In case of the organic structures 3 and 6a, the configuration
of the histidine was set to match with the configuration of the used l-
histidine. The asymmetric units of 6a and 11 contain two molecules each. In
both cases with the exception of a torsion angle, the corresponding pair of
molecules possesses a practical identical geometry.
‡
[
99mTc(OH2)3(CO)3] and, in addition, a solution of ligand 9
to yield a final ligand in the concentration of 10À4, 5 Â 10À5 and
10À5 m, respectively. At 908C, for both concentrations 10À4
m
and 5 Â 10À5 m, almost quantitative formation of 17 was found
within 20 min. No side products were observed by radioactive
HPLC analysis. At 10À5 m the yield was 67% after 90 min.
Although heating at 958C was continued for much longer
time, no significant formation of side products was observed
proving the high thermal stability of the product 17. The one-
pot reaction under reductive conditions did not affect the
2058
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2053 2061

Tc Labeling of Histidine Peptides
2053 2061
[NEt4]2[ReBr3(CO)3],[33] fac-[Re(his)(CO)3],[36] and fac-[99mTc(H2O)3-
removed under reduced pressure and dried more in vacuo. The residue, the
crude compound 7a was dissolved in CH2Cl2 (10 mL) and neutralized by
adding Et3N dropwise. BOP (148 mg, 0.34 mmol) and Et3N (46 mL,
0.34 mmol) were added to the reaction mixture. After 45 min, a solution
of phenylalanine ethyl ester (84.6 mg, 0.37 mmol) and Et3N (51 mL,
0.37 mmol) in CH2Cl2 (10 mL) was added slowly by a syringe. The reaction
mixture was stirred for an additional 2.5 d at room temperature. The
solution was diluted with CH2Cl2 (30 mL) and extracted with 1n HCl
solution (20 mL), 1n NaHCO3 (20 mL), brine (20 mL). The organic layer
was dried over Na2SO4, concentrated under reduced pressure and purified
by flash column chromatography to afford 8a as a colorless oil (162 mg,
90%). Rf ˆ 0.2 (CH2Cl2/MeOH 40:1); 1H NMR (500 MHz, CD3CN, 258C):
d ˆ 7.37 7.28 (m, 9H; 2  4H-CHph, CHim), 7.16 (d, J ˆ 8.23 Hz, 2H; 2 Â
CHph), 6.77 (brd, J ˆ 7.65 Hz, 1H; NH), 6.69 6.66 (m, 2H; CHim, NH),
5.05 (s, 2H; CH2-Bn), 4.61 (dt, J ˆ 7.86 Hz, 1H; CH-Phe), 4.52 (s, 2H;
CH2Nim), 4.43 4.41 (m, 1H; CHCO), 4.11 (q, J ˆ 7.15 Hz, 2H; CH2CH3),
3.62 (s, 3H; OCH3), 3.10 (dd, J ˆ 8.19 Hz, 1H; CH2-Phe), 2.99 2.93 (m,
3H; CH2-Phe, CH2CH), 2.57 (s, N-CH3), 1.18 (t, J ˆ 7.13 Hz, 3H; CH3);
13C NMR (500 MHz, CD3CN, 258C): d ˆ 173.3, 172.0, 167.8, 157.0, 139.1,
138.3, 138.2, 137.8, 130.4, 129.5, 129.4, 129.0, 128.9, 127.9, 118.9, 67.2, 62.2,
‡[17]
(CO)3]
were prepared as previously reported.
5-Oxo-5,6,7,8-tetrahydroimidazo[1,5-c]pyrimidine-7-carboxylic methyl es-
ter (3): The compound was prepared according to literature with slight
modification.[30] Im2CO (1.88g, 11.61 mmol) was added at RT to a solution
of l-histidine methyl ester (2.73 g, 11.28 mmol) in DMF (80 mL). The
reaction mixture was heated to 708C for 6 h, then cooled down to RT and
poured slowly to 1m NaHCO3 aqueous solution (250 mL). Some solid
precipitated from the aqueous layer which was extracted with CH2Cl2.
During extraction the precipitate dissolved completely in CH2Cl2. The
combined organic extracts were dried over Na2SO4, concentrated under
reduced pressure, and purified by flash column chromatography to afford 3
as white solid (1.35 g, 61%). Rf ˆ 0.2 (EtOAc); 1H NMR (500 MHz,
CD3CN, 258C): d ˆ 8.01 (s, 1H; CHim), 6.77 (s, 1H; CHim), 6.61 (brs, 1H;
NH), 4.37 4.34 (m, 1H; CHCO), 3.67 (s, 3H; OCH3), 3.25 3.23 (m, 2H;
CH2CH); 13C NMR (500 MHz, CD3CN, 258C): d ˆ 172.1, 149.2, 135.4,
‡
126.9, 125.9, 53.6, 53.5, 23.7; MS (ESI): m/z (%): 195.73 (100) [M ], 167.8
(35), 135.8 (24); elemental analysis calcd (%) for C8H9N3O3 (195.18): C
49.23, H 4.62, N 21.54; found: C 49.32, H 4.77, N 21.24. Crystals suitable for
X-ray structure analysis were obtained by slow evaporation from EtOAc.
55.2, 54.8, 52.8, 49.9, 38.0, 30.3, 14.5; MS (ESI): m/z (%): 537.53 (100)
‡
2-(2-tert-Butoxy-2-oxoethyl)-7-methoxycarbonyl-5-oxo-5,6,7,8-tetrahy-
droimidazo[1,5-c]pyrimidine-2-ium bromide (4): Bromoacetic tert-butyl
[M ‡H];
elemental
analysis
calcd
(%)
for
C28H32N4O7
ˆ
‡ 0.5[N(CH3)2]3P O ‡ 0.5H2O (634.5): C 58.63, H 6.62, N 12.14; found:
ester (0.57 mL, 3.86 mmol) was added to
a solution of 3 (250 mg,
C 58.98, H 6.89, N 12.48.
1.28 mmol) in CH3CN (25 mL). The reaction mixture was heated under
reflux for 24 h, then cooled to RT and concentrated in vacuo. The residue
was washed with Et2O (2 Â 10 mL) and THF (2 Â 5 mL) and dried in vacuo
to afford 4 as a white sticky solid which was used in the next step without
any further purification. 1H NMR (300 MHz, D2O, 20 8C): d ˆ 9.39 (s, 1H;
CHim), 7.40 (s, 1H; CHim), 5.05 (s, 2H; CH2Nim), 4.78 4.61 (m, 1H;
CHCO), 3.63 (s, 3H; OCH3), 3.42 3.39 (m, 2H; CH2CH), 1.39 (s, 9H;
Methyl 1-{2-[(1-ethoxycarbonyl-2-phenylethyl)amino]-2-oxoethyl}-N-(9H-
fluoren-9-ylmethoxycarbonyl) histidinate (8b): The title compound was
prepared under the same condition as described for 8a. A solution of 7b
(13 mg, 0.03 mmol), prepared from compound 6b in TFA/CH2Cl2, BOP
(13 mg, 0.03 mmol), and Et3N (8 mL, 0.06 mmol) in CH2Cl2 was stirred for
1.5 d at room temperature. Flash column chromatography yielded 8b
(13 mg, 74%). Rf ˆ 0.15 (CH2Cl2/MeOH 60:1); 1H NMR (300 MHz,
CD3CN, 208C): d ˆ 7.83 (d, J ˆ 7.61 Hz, 2H; 2  CHfluoren), 7.63 (d, J ˆ
‡
‡
tBu); MS (ESI): m/z (%): 309.40 (13) [M À HBr], 253.80 (100) [M
À
ˆ
HBrÀ (CH2 C(CH3)2)].
7.44 Hz, 2H; 2 Â CHfluoren), 7.43 7.23 (m, 9H; 4 Â CHfluoren, 4 Â CHph
,
Methyl N-[(benzyloxy)carbonyl]-1-(2-tert-butoxy-2-oxoethyl) histidinate
(6a): DIPEA (0.52 mL, 3.01 mmol) and BnOH (2.1 mL, 20.08 mmol) were
added to a solution of crude 4 (390 mg) in THF (50 mL). After 16 h of
refluxing, the reaction solution was cooled down to room temperature,
concentrated under reduced pressure, and purified by flash column
chromatography to afford 6a as white solid (260 mg, 62% from 3). Rf ˆ
0.15 (CH2Cl2/MeOH 45:1); 1H NMR (500 MHz, CD3CN, 258C): d ˆ 7.39
7.32 (m, 6H; 5  CHph, CHim), 6.78 (s, 1H; CHim), 6.66 (brd, J ˆ 7.8 Hz, 1H;
NH), 5.06 (s, 2H; CH2-Bn), 4.58 (s, 2H; CH2Nim), 4.42 (q, J ˆ 2.6 Hz, 1H;
CHCO), 3.62 (s, 3H; OCH3), 2.96 (t, J ˆ 5.26 Hz, 2H; CH2CH); 13C NMR
(500 MHz, CD3CN, 258C): d ˆ 173.2, 168.3, 157.0, 139.1, 138.2, 137.8, 129.5,
129.0, 128.9, 119.2, 83.2, 67.2, 66.9, 55.2, 52.7, 49.3, 30.2, 28.2; MS (ESI): m/z
CHim), 7.14 (d, J ˆ 6.25 Hz, 1H; CHph), 6.71 6.69 (m, 2H; CHim, NH), 6.60
(brd, J ˆ 7.83 Hz, 1H; NH), 4.61 (dt, J ˆ 7.82 Hz, 1H; CH-Phe), 4.51 (s, 2H;
CH2Nim), 4.44 4.33 (m, 3H; CH2-fluoren, CH-fluoren), 4.22 (t, J ˆ
6.41 Hz, 1H; CHCO), 4.09 (q, J ˆ 7.15 Hz, 2H; CH2CH3), 3.61 (s, 3H;
OCH3), 3.09 (dd, J ˆ 5.64 Hz, 1H; CH2-Phe), 2.96 (dd, J ˆ 6.91 Hz, 3H;
CH2-Phe, CH2CH), 1.17 (t, J ˆ 7.12 Hz, 3H; CH3); 13C NMR (300 MHz,
CD3CN, 208C): d ˆ 173.5, 172.2, 168.0, 145.4, 142.4, 139.2, 137.9, 130.5,
129.4, 128.9, 128.4, 128.1, 126.3, 121.2, 119.1, 67.3, 62.3, 55.3, 54.8, 52.8, 50.0,
‡
48.1, 38.1, 30.9, 30.4, 14.4; MS (ESI): m/z (%): 625.68 (100) [M ‡H];
elemental analysis calcd (%) for C35H36N4O7‡H2O (642): C 65.42, H 5.92,
N 8.72; found: C 65.38, H 5.99, N 8.49.
Re complex 15
‡
(%): 417.53 (100) [M ]; elemental analysis calcd (%) for C21H27N3O6
(417.48): C 60.43, H 6.47, N 10.07; found: C 60.43, H 6.57, N 9.97. Crystals
suitable for X-ray structure analysis were obtained by vapor diffusion of
1-hexene into EtOAc.
From compound 8a: To remove the Cbz group, 8a (100 mg, 0.19 mmol) was
dissolved in a solution of MeOH/H2O/AcOH (7:2:1 v/v) (20 mL) which was
added dropwise by syringe into the reaction flask containing Pd/C
(100 mg). H2 gas was purged through the reaction solution for 2.5 h at
room temperature. The reaction mixture was filtered through Celite, rinsed
with the same solution (5 mL), concentrated in vacuo, and purified by
column chromatography to provide the methyl ester of 9 (59 mg, 79%).
Rf ˆ 0.15 (CH2Cl2/MeOH/25% NH4OH 10:1:0.1); 1H NMR (300 MHz,
CD3CN, 208C): d ˆ 7.33 7.14 (m, 6H; 5  CHph, CHim), 6.78 (brd, J ˆ
7.53 Hz, 1H; NH), 6.69 (s, 1H; CHim), 4.61 (dt, J ˆ 7.75 Hz, 1H; CH-
Phe), 4.53 (s, 2H; CH2Nim), 4.10 (q, J ˆ 7.14 Hz, 2H; CH2CH3), 3.70 3.63
(m, 4H; CHCO, OCH3), 3.13 3.07 (m, 3H; NH2, CH2-Phe), 2.97 (dd, J ˆ
7.69 Hz, 1H; CH2-Phe), 2.86 (dd, J ˆ 5.02 Hz, 1H; CH2CH), 2.73 (dd, J ˆ
7.06 Hz, 1H; CH2CH), 1.18 (t, J ˆ 7.13 Hz, 3H; CH3); 13C NMR (300 MHz,
CD3CN, 208C): d ˆ 176.3, 172.0, 167.9, 139.1, 138.8, 137.7, 130.4, 129.5, 127.9,
120.3, 62.3, 55.5, 54.8, 52.4, 49.9, 38.1, 33.8, 14.5; IR (THF): nÄ ˆ 1744, 1696,
Methyl 1-(2-tert-butoxy-2-oxoethyl)-N-(9H-fluoren-9-ylmethoxycarbonyl)
histidinate (6b): Crude 4 (400 mg), Fm-OH (543 mg, 2.77 mmol), and
DIPEA (0.24 mL, 1.39 mmol) were dissolved in acetonitrile (50 mL). After
16 h at RT the reaction solution was neutralized by adding 1n HCl solution
and concentrated under reduced pressure. The residue was dissolved in
CH2Cl2 (80 mL) and extracted with water (50 mL) and 1n HCl solution
(50 mL). The organic layer was dried over Na2SO4, concentrated in vacuo
and purified by flash column chromatography afforded 6b as a colorless oil
(233 mg, 55% from 3). Rf ˆ 0.1 (CH2Cl2/MeOH 60:1); 1H NMR (300 MHz,
CD3CN, 208C): d ˆ 7.83 (d, J ˆ 7.63 Hz, 2H; CHfluoren), 7.64 (d, J ˆ 7.66 Hz,
2H; CHfluoren), 7.44 7.31 (m, 5H; CHim, 4 Â CHfluoren), 6.78 (s, 1H; CHim),
6.63 (brd, J ˆ 7.83 Hz, 1H; NH), 4.56 (s, 2H; CH2Nim), 4.44 4.34 (m, 3H;
CHCO, CH2-fluoren), 4.23 (t, J ˆ 7.0 Hz, 1H; CH-fluoren), 3.61 (s, 3H;
OCH3), 2.94 (m, 2H; CH2CH), 1.43 (s, 9H; tBu); 13C NMR (300 MHz,
CD3CN, 208C): d ˆ 173.5, 168.6, 157.2, 145.4, 142.4, 139.3, 138.1, 128.9,
128.3, 126.3, 121.2, 119.4, 83.3, 67.3, 55.3, 52.8, 49.3, 48.1, 30.3, 28.2; MS
‡
1196 cmÀ1; MS (ESI): m/z (%): 403.13 (100) [M ‡H].
To remove the two ester groups, Cbz-deprotected phenylalanine-histidine
(16 mg, 0.04 mmol) was dissolved in MeOH (2 mL) and 0.5m LiOH
aqueous solution (1 mL) was added. After 17 h, the reaction solution was
neutralized with 1n HCl solution. The reaction mixture was concentrated in
vacuo and the crude product 9 was used in the next step without any further
purification.
‡
(ESI): m/z (%): 506.40 (100) [M ‡H]; elemental analysis calcd (%) for
C28H31N3O3‡0.5H2O (514.59): C 65.37, H 6.22, N 8.17; found: C 65.14, H
6.40, N 7.96.
Methyl N-[(benzyloxy)carbonyl]-1-{2-[(1-ethoxycarbonyl-2-phenylethyl)-
amino]-2-oxoethyl} histidinate (8a): A solution of 6a (140 mg, 0.34 mmol)
in CH2Cl2/TFA (2:2 mL) was stirred for 2.5 h at RT. The solvent was
For the complexation, the crude all-deprotected compound 9 was dissolved
in H2O (3 mL) and the pH of the solution was adjusted until 7 8 by adding
Chem. Eur. J. 2003, 9, 2053 2061
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2059

R. Alberto et al.
FULL PAPER
1n HCl or 0.5m LiOH solution. [Et4N]2[ReBr3(CO)3] (30 mg, 0.04 mmol)
was added immediately in one portion. A white solid precipitated at RT
which was filtered after 19 h and rinsed with cold water (1 mL). The filtrate
was concentrated in vacuo and purified by preparative HPLC to afford 15
Re complex 14b: The preparation is similar to compound 14a. To
compound 12, (25 mg, 0.059 mmol) and Cs2CO3 (20.4 mg, 0.065 mmol) in
acetonitrile (25 mL) was added tert-butyl bromoacetate (34.5 mg,
0.176 mmol). The reaction mixture was stirred at 358C for 1.5 h. The
reaction mixture was filtered, dried under vacuum and purified by silica gel
chromatography (EtOAc/EtOH 5:1) to yield complex 14b (29 mg, 90%).
1H NMR (300 MHz, CD3CN, 208C): d ˆ 7.93 (s, 1H; CHim), 6.90 (s, CHim),
4.65(s, 2H; CH2Nim), 3.94 3.25 (m, 1H; CHCO), 3.27 3.23 (q, 2H;
CH2CH), 1.45 (s, 9H; tBu); 13C NMR (CD3CN): d ˆ 181.3, 167.3, 143.1,
135.2, 120.6, 83.8, 52.3, 49.7, 28.5, 28.0, 27.8; IR (KBr): nÄ ˆ 2021, 1892, 1738,
(10 mg, 40%). HPLC gradient (%):
0
3 min ˆ 0.1% TFA aqueous
solution (100), 3.1 20 min ˆ 0.1% TFA/MeOH (75:25 0:100), 21
30 min ˆ MeOH (100). 1H NMR (500 MHz, CD3OD, 258C): d ˆ 7.93 (s,
1H; CHim), 7.26 7.17 (m, 5H; CHph), 6.84 (s, 1H; CHim), 4.67 (s, 2H;
COCH2), 4.55 4.52 (m, 1H; CH-Phe), 3.98 (q, J ˆ 4.54 Hz, 1H; CHCO),
3.34 3.26 (m, 2H; CH2-Phe), 3.24 3.10 (m, 2H; CH2CH); 13C NMR
(500 MHz, CD3OD, 258C): d ˆ 198.6, 197.5, 197.3, 185.3, 167.4, 143.6, 139.6,
135.6, 130.5, 129.4, 127.6, 121.0, 57.4, 53.4, 50.8, 39.4, 30.9, 28.8; IR (KBr):
nÄ ˆ 3436, 2024, 1904, 1677, 1636, 1434, 1381, 1203, 1138 cmÀ1; MS (ESI): m/z
‡
1635, 1155 cmÀ1; MS (ESI): m/z (%): 539.9 (100) [M ], 1076.8 (50);
elemental analysis calcd (%) for C15H18N3O7Re (538.5): C 33.43, H 3.34, N
7.80; found: C 33.30, H 3.85, N 7.68.
‡
(%): 631.07 (100) [M ‡H].
Re complex 13
From compound 13: BOP (7.4 mg, 0.02 mmol) and Et3N (2 mL, 0.02 mmol)
were added at room temperature to the solution of the complex 13 (8 mg,
0.02 mmol) in a solution of CH2Cl2/DMF (3:0.2 mL). After 30 min, a
solution of phenylalanine ethyl ester (4 mg, 0.02 mmol) and Et3N (2 mL,
0.02 mmol) in CH2Cl2 (2 mL) was added dropwise to the solution by
syringe. The reaction mixture was stirred overnight. The reaction solution
was concentrated in vacuo. The residue was treated with diethyl ether (2 Â
5 mL). The white solid was dissolved in THF (10 mL) and insoluble solid
was filtered off. The filtrate was concentrated in vacuo to provide the ethyl
ester of complex 15 (75%). 1H NMR (500 MHz, CD3CN, 258C): d ˆ 7.8 (s,
1H; CHim), 7.34 7.21 (m, 5H; CHph), 6.8 (s, 1H; CHim), 4.63 4.59 (m, 1H;
CHCO), 4.53 (d, 2H; CH2Nim), 4.08 (q, 2H; CH2CH3), 3.92 3.88 (m, 1H;
CH-His), 3.18 3.10 (2 Â dd, 2H; CH2-His, CH2-Phe), 3.05 2.96 (2 Â dd,
2H; CH2-His, CH2-Phe), 1.17 (t, 3H; CH3); 13C NMR (500 MHz, CD3CN,
258C): d ˆ 198.1, 196.6, 196.5, 180.4, 170.8, 165.5, 142.0, 136.7, 134.3, 129.4,
128.4, 126.8, 124.9, 120.3, 119.6, 61.2, 54.1, 51.4, 49.2, 37.2, 27.7, 13.4; IR
(KBr): nÄ ˆ 2020, 1886, 1733, 1636 cmÀ1; MS (ESI): m/z (%): 659 (100)
From compound 5: The crude 5 (300 mg) was dissolved in H2O (15 mL).
The pH of the solution was adjusted until 7 8 by adding 1n NaOH aqueous
solution. [Et4N]2[ReBr3(CO)3] (531 mg, 0.69 mmol) was added. After
stirring for 21 h at room temperature, the solution was concentrated in
vacuo. The residue was treated with CH2Cl2 (3 Â 10 mL) and diethyl ether
(2 Â 10 mL) and dried in vacuo. The sticky crude product was purified by
flash column chromatography to afford 13 as a white solid (240 mg, 68%
1
from 3 in three steps). Rf ˆ 0.1 (EtOH/CH2Cl2/AcOH 10:1:0.1); H NMR
(500 MHz, CD3CN/CD3OD, 258C): d ˆ 7.99 (s, 1H; CHim), 6.95 (s, 1H;
CHim), 4.78 (d, J ˆ 7.08 Hz, 2H; CH2Nim), 3.99 3.96 (m, 1H; CHCO),
3.24 3.20 (m, 1H; CH2CH), 3.10 3.05 (m, 1H; CH2CH); 13C NMR
(500 MHz, CD3CN/CD3OD, 258C): d ˆ 199.2, 197.7, 197.5, 183.8, 169.8,
143.7, 135.5, 121.2, 53.1, 28.8; IR (KBr): nÄ ˆ 3428, 2023, 1889, 1731, 1623,
‡
1521, 1437, 1181 cmÀ1; MS (ESI): m/z (%): 505.8 (23) [M ‡Na], 483.67
‡
(100) [M ‡H].
From complex 14a and 14b: To hydrolyze the ester groups, compound 14a
(30 mg, 0.057 mmol) was stirred in a solution of methanol (5 mL) and
LiOH (0.5m, 2 mL) overnight at room temperature and compound 14b
(15 mg, 0.028 mmol) was stirred in a solution of methylene chloride (2 mL)
and trifluoroacetic acid (2 mL) for 2 h at room temperature. Two crude
substances were purified by column chromatography (EtOH/THF/AcOH
10:1:0.1) to yield complex 15 (95% and 90%, respectively).
‡
[M ‡H]; elemental analysis calcd (%) for C22H24N4O8Re (658.6): C 40.12,
H 3.67, N 8.51; found: C 39.31, H 3.83, N 8.25.
The ethyl ester group of the complex was hydrolyzed by stirring the
complex overnight in solution of 0.5m LiOH and MeOH (1:2) at room
temperature, as mentioned above, to afford complex 15 quantitatively.
General procedure for the oxidation of rhenium in the complexes 15 and
13: A solution of compound 15 or 13 (5 mm in H2O, 500 mL) and acid (HCl,
TFA, or acetic) solution (1.0, 0.1, or 0.01m in H2O, 70 mL) were added to a
vial, which was sealed and then degassed with nitrogen (10 min). H2O2
(0.43, 0.86, or 1.29m in H2O, 60 mL) was added to the degassed vial,
followed by heating of the sample at 508C. Monitoring of the reaction
mixture was conducted by HPLC at 250 nm, where the reaction mixture
(10 mL) was injected on the HPLC at 4, 8, 24, and 48 h or until the rhenium
complex was not visible in the spectrum. The effectiveness of the reaction
condition was calculated by determining the peak area ratio of the rhenium
complex over the formation of perrhenate. When the rhenium complex was
no longer observed, the reaction mixture was treated with manganese
dioxide to remove residual H2O2 from the reaction mixture then filtered
with a Wattman 0.2 mm filter to yield the uncoordinated ligand in solution
to be used in 99mTc labeling.
Re complex 11: The compound 7b was treated with aniline, BOP, Et3N in a
solution of CH2Cl2/DMF under the same condition as described for 8a,
followed by deprotection of functional groups with piperidine to prepare
the compound 10. The compound 10 was coordinated with [Et4N]2[ReBr3(-
CO)3] to afford the complex 11 in the same manner as described for 8a in
overall yield 75%. IR (KBr): nÄ ˆ 3478, 2020, 1889, 1689, 1635 cmÀ1; MS
‡
(ESI): m/z (%): 558.2 (100) [M ‡H]; elemental analysis calcd (%) for
C17H18N4O6Re (557.35): C 36.6, H 2.71, N 10.05; found: C 36.98, H 3.03, N
9.95. Crystal structure is shown in Figure 3. Crystals suitable for X-ray
structure analysis were obtained from EtOH/toluene (1:1).
1-(Carboxymethyl)histidine (5): The crude compound 4 (300 mg) was
dissolved in 6n HCl aqueous solution (10 mL), heated under reflux for 2 d
and concentrated under reduced pressure. The sticky oily residue was
solidified in diethyl ether (20 mL) and the white solid was washed with
diethyl ether (2 Â 10 mL) and THF (5 mL) dried in vacuo and used in the
next step without further purification. 1H NMR (300 MHz, D2O, 20 8C):
d ˆ 8.69 (s, 1H; CHim), 7.38 (s, 1H; CHim), 4.96 (s, 2H; CH2Nim), 4.18 (t, J ˆ
6.3 Hz, 1H; CHCO), 3.31 (t, J ˆ 5.8 Hz, 2H; CH2CH); MS (ESI): m/z (%):
‡
General procedure for labeling 5 and 9 with [99mTc(H2O)3(CO)3] : A
solution of ligand (10À3 or 10À4 m in H2O, 100 mL) obtained from either
organic synthesis or through rhenium oxidation pathway was added to a
vial, which was then sealed and degassed with a stream of nitrogen gas for
‡
‡
213.82 (87) [M ‡H], 167.88 (100) [M À COOH].
‡
10 min. A solution of [99mTc(CO)3(H2O)3] (900 mL) was added to the vial
Re complex 14a: Ethyl bromoacetate (29.5 mg, 0.176 mmol) in acetonitrile
(5 mL) was added to a solution of complex 12, (25 mg, 0.059 mmol) and
Cs2CO3 (20.4 mg, 0.065 mmol) in acetonitrile (25 mL). The reaction
mixture was heated at 358C for 1.5 h. Glacial acidic acid was added to
the mixture to neutralized the pH. After standard work-up, the crude
substance was purified by a silica gel chromatography to provide complex
14a (30 mg, 90%). Rf ˆ 0.15 (EtOAc/EtOH 5:1); 1H NMR (300 MHz,
CD3CN, 208C): d ˆ 7.95 (s, 1H; CHim), 6.92 (s, 1H; CHim), 4.78 (s, 2H;
CH2Nim), 4.23 4.16 (q, J ˆ 7.1 Hz, 2H; CH2CH3), 3.91 3.87 (m, 1H;
CHCO), 3.21 2.98 (q, 2H; CH2CH), 1.28 1.23 (t, J ˆ 7.5 Hz, 3H; CH3);
13C NMR (300 MHz, CD3CN, 208C): d ˆ 199.5, 197.8, 197.8, 181.8, 168.8,
143.4, 135.7, 120.9, 63.0, 52.6, 49.4, 28.7, 14.4; IR (KBr): nÄ ˆ2020, 1883, 1741,
through a syringe and the vial was heated to 70 908C for 30 min to yield
‡
the corresponding [99mTc (CO)3] complexes, [(5)99mTc(CO)3] 16 and
[(9)99mTc(CO)3] 17 in high yield which was quantified by HPLC with
radioactive detection. All the results are described in Table 1.
Acknowledgement
We thank BBW and Mallinckrodt for financial support. B. Spingler thanks
the Swiss National Science Foundation for support.
‡
‡
1636 cmÀ1; MS (ESI): m/z (%): 511.8 (100) [M ‡H], 1020.7 (55) [2M ];
elemental analysis calcd (%) for C15H18N3O7Re (510.5): C 30.59, H 2.76, N
8.23; found: C 30.84, H 3.0, N 8.06.
[1] J. R. Dilworth, S. J. Parrott, Chem. Soc. Rev. 1998, 27, 43.
[2] S. S. Jurisson, J. D. Lydon, Chem. Rev. 1999, 99, 2205.
2060
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2053 2061

Tc Labeling of Histidine Peptides
2053 2061
[3] R. K. Hom, J. A. Katzenellenbogen, Nucl. Med. Biol. 1997, 24, 485.
[4] W. C. Eckelman, Eur. J. Nucl. Med. 1995, 22, 249.
[5] H. F. Kung, Y. Z. Guo, C. C. Yu, J. Billings, V. Subramanyam, J. C.
Calabrese, J. Med. Chem. 1989, 32, 433.
[6] M. P. Kung, D. A. Stevenson, K. Plossl, S. K. Meegalla, A. Beckwith,
W. D. Essman, M. Mu, I. Lucki, H. F. Kung, Eur. J. Nucl. Med. 1997, 24,
372.
[31] Crystallographic analysis for 3 (C8H9N3O3): colourless needles, 0.5 Â
0.1  0.1 mm3, orthorhombic, space group P212121, a ˆ 11.1523(15),
b ˆ 7.4112(8), c ˆ 10.7755(15) ä, Vˆ 890.6(2) ä3, Z ˆ 4, 1calcd
ˆ
1.456 gcmÀ3, R1(I ꢀ 2s(I)) ˆ 0.0417, wR2(F2, I ꢀ 2s(I)) ˆ 0.1056 (2644
independent, 2050 observed (I ꢀ 2s(I)) data), 163 parameters, no
absorption correction, m ˆ 0.114mmÀ1
.
CCDC-193892 (3), -193893 (6a) and -193894 (11) contain the
supplementary crystallographic data for this paper. These data can
be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrie-
ving.html (or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB21EZ, UK; (fax: (‡44)1223-336-033; or
e-mail: deposit@ccdc.cam.ac.uk).
[7] H. F. Kung, Nucl. Med. Biol. 2001, 28, 505.
[8] B. Johannsen, H. J. Pietzsch, Eur. J. Nucl. Med. Mol. Im. 2002, 29, 263.
[9] M. Langer, R. La Bella, E. Garcia-Garayoa, A. G. Beck-Sickinger,
Bioconjugate Chem. 2001, 12, 1028.
[10] S. Liu, S. J. Rettig, C. Orvig, Inorg. Chem. 1991, 30, 4915.
[11] S. Liu, D. S. Edwards, R. J. Looby, A. R. Harris, M. J. Poirier, J. A.
Barrett, S. J. Heminway, T. R. Carroll, Bioconjugate Chem. 1996, 7, 63.
[12] S. Liu, D. S. Edwards, A. R. Harris, Bioconjugate Chem. 1998, 9, 583.
[13] S. Liu, D. S. Edwards, Chem. Rev. 1999, 99, 2235.
[14] S. Liu, D. S. Edwards, A. R. Harrid, S. J. Heminway, J. A. Barrett,
Inorg. Chem. 1999, 38, 1326.
[15] C. Decristoforo, S. J. Mather, Nucl. Med. Biol. 1999, 26, 389.
[16] N. Metzler-Nolte, Angew. Chem. 2001, 113, 1072; Angew. Chem. Int.
Ed. 2001, 40, 1040.
[17] R. Alberto, K. Ortner, N. Wheatley, R. Schibli, A. P. Schubiger, J. Am.
Chem. Soc. 2001, 123, 3135.
[18] A. Amann, C. Decristoforo, I. Ott, M. Wenger, D. Bader, R. Alberto,
G. Putz, Nucl. Med. Biol. 2001, 28, 243.
[19] A. Egli, R. Alberto, L. Tannahill, R. Schibli, U. Abram, A. Schaffland,
R. Waibel, D. Tourwe, L. Jeannin, K. Iterbeke, P. A. Schubiger, J.
Nucl. Med. 1999, 40, 1913.
[20] R. La Bella, E. Garcia-Garayoa, M. Bahler, P. Blauenstein, R. Schibli,
P. Conrath, D. Tourwe, P. A. Schubiger, Bioconjugate Chem. 2002, 13,
599.
[21] R. Waibel, R. Alberto, J. Willuda, R. Finnern, R. Schibli, A.
Stichelberger, A. Egli, U. Abram, J. P. Mach, A. Pluckthun, P. A.
Schubiger, Nat. Biotechnol. 1999, 17, 897.
[22] J. Wald, R. Alberto, K. Ortner, L. Candreia, Angew. Chem. 2001, 113,
3152; Angew. Chem. Int. Ed. 2001, 40, 3062.
[23] J. Petrig, R. Schibli, C. Dumas, R. Alberto, P. A. Schubiger, Chem.
Eur. J. 2001, 7, 1868.
[24] R. Schibli, R. La Bella, R. Alberto, E. Garcia-Garayoa, K. Ortner, U.
Abram, P. A. Schubiger, Bioconjugate Chem. 2000, 11, 345.
[25] E. Garcia-Garayoa, L. Allemann-Tannahill, P. Blauenstein, M. Will-
mann, N. Carrel-Remy, D. Tourwe, K. Iterbeke, P. Conrath, P. A.
Schubiger, Nucl. Med. Biol. 2001, 28, 75.
[26] H.-J. Meder, W. Beck, Z. Naturforsch. 1986, 41b, 1247.
[27] D. R. van Staveren, T. Weyherm¸ller, N. Metzler-Nolte, Organome-
tallics 2000, 19, 3730.
[32] Crystallographic analysis for 6a (C21H27N3O6): colourless plates,
0.37  0.32  0.03 mm3, triclinic, space group P1 a ˆ 5.8798(6), b ˆ
10.8498(12), c ˆ 17.1745(16) ä, a ˆ 89.178(12), b ˆ 81.126(12), g ˆ
88.784(13)8, Vˆ 1082.21(19) ä3, Z ˆ 2, 1calcd ˆ 1.281 gcmÀ3
R1(I ꢀ
,
2s(I)) ˆ 0.0372, wR2(F 2, I ꢀ 2s(I)) ˆ 0.0662 (6242 independent, 3803
observed (I ꢀ 2s(I)) data), 549 parameters, 3 restraints, numerical
absorption correction, m ˆ 0.095mmÀ1, Tmin ˆ 0.9693, Tmax ˆ 0.9943.
[33] R. Alberto, A. Egli, U. Abram, K. Hegetschweiler, P. A. Schubiger, J.
Chem. Soc. Dalton Trans. 1994, 2815.
[34] R. Alberto, R. Schibli, U. Abram, R. Hubener, H. Berke, T. A. Kaden,
P. A. Schubiger, Chem. Commun. 1996, 1291.
[35] Crystallographic analysis for 11 (C17H12N4O6Re): colourless plates,
0.25  0.2  0.05 mm3, orthorhombic, space group P212121, a ˆ
18.9899(12), b ˆ 10.5834(7), c ˆ 19.3686(14) ä, Vˆ 3892.7(5) ä3,
Z ˆ 8, 1calcd ˆ 1.892 gcmÀ3
,
R1(I ꢀ 2s(I)) ˆ 0.0406, wR2(F 2, I ꢀ
2s(I)) ˆ 0.0680 (9371 independent, 6193 observed (I ꢀ 2s(I)) data),
505 parameters, 18 restraints, numerical absorption correction, m ˆ
6.284mmÀ1, Tmin ˆ 0.3107, Tmax ˆ 0.7483, Flack parameter: À0.012(11).
À
À
Selected bond lengths are: Re2 C15: 1.859(12), Re2 C13: 1.859(15),
À
À
À
Re2 C17: 1.891(13), Re2 O10: 2.157(6), Re2 N3: 2.202(7),
À
Re2 N12: 2.212(7).
[36] R. Alberto, R. Schibli, R. Waibel, U. Abram, A. P. Schubiger, Coord.
Chem. Rev. 1999, 192, 901.
[37] D. R. van Staveren, E. Bothe, T. Weyherm¸ller, N. Metzler-Nolte,
Chem. Commun. 2001, 131.
[38] S. Hanessian, G. Patil, Tetrahedron Lett. 1978, 1035.
[39] T. Tsuchiya, Y. Takagi, S. Umezawa, Tetrahedron Lett. 1979, 4951.
[40] R. Alberto, R. Schibli, A. Egli, U. Abram, T. A. Kaden, P. A.
Schubiger, J. Am. Chem. Soc. 1998, 120, 7987.
[41] G. M. Sheldrick, Acta Crystallogr. Sect. A 1990, 46, 467.
[42] A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giaco-
vazzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori, R. Spagna, J.
Appl. Crystallogr. 1999, 32, 115.
[43] G. M. Sheldrick, University Gˆttingen, 1997.
[28] D. R. van Staveren, E. Bill, E. Bothe, M. Buhl, T. Weyherm¸ller, N.
Metzler-Nolte, Chem. Eur. J. 2002, 8, 1649.
[29] D. R. van Staveren, N. Metzler-Nolte, Chem. Commun. 2002, 1406.
[30] R. Jain, L. A. Cohen, Tetrahedron 1996, 52, 5363.
Received: September 24, 2002 [F4445]
Chem. Eur. J. 2003, 9, 2053 2061
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2061