Structural characterisation and bioconjugation of an active ester containing oxorhenium(V) complex incorporating a thioether donor

Article abstract of DOI:10.1039/a804717k

A simple new 2,3,5,6-tetrafluorophenyl ester containing diamide-thioether-thiol bifunctional chelating agent LH₃, HS(CH₂)₂SCH₂C(O)NHCH₂C(O)NH(CH ₂)₃C(O)OC₆HF₄

Full text of DOI:10.1039/a804717k

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Structural characterisation and bioconjugation of an active ester
containing oxorhenium(V) complex incorporating a thioether donor
Matthias Glaser,a Mark J. Howard,a Kevin Howland,a Annie K. Powell,b Michael T. Rae,a
Sigrid Wocadlo,b Richard A. Williamsona and Philip J. Blower*a
a Biosciences Department, University of Kent, Canterbury, UK CT2 7NJ.
E-mail: P.J.Blower@ukc.ac.uk
b School of Chemical Sciences, University of East Anglia, Norwich, UK NR4 7TJ
Received 22nd June 1998, Accepted 3rd August 1998
A simple new 2,3,5,6-tetrafluorophenyl ester containing diamide–thioether–thiol bifunctional chelating agent
LH3, HS(CH2)2SCH2C(O)NHCH2C(O)NH(CH2)3C(O)OC6HF4, has been synthesised. The key intermediates
were prepared using standard peptide chemistry procedures. Reaction of LH3 with [Bu4N][ReOCl4] formed an
uncharged oxorhenium() complex, which was characterised by X-ray structural analysis. The five-co-ordinate
complex showed approximately square-pyramidal geometry with an apical oxo group and a basal ligand set
comprising a deprotonated thiol group, two deprotonated amide groups, and a thioether group. A second complex
of stoichiometry [ReO(LH2)2]Cl was formed by reaction of LH3 with a rhenium() gluconate intermediate in water
at pH 4.7. The 1:1 complex [ReOL] was conjugated with the small protein N-TIMP-2 by aminolysis at a lysine
residue, to form a 1:1 adduct as established by electrospray mass spectrometry.
and hydrazinonicotinamides (HYNIC).10 Some comparative
Introduction
reviews are available.2a,11
In the diagnosis and therapy of human diseases by use of target-
specific delivery of radiolabelled biomolecules, the development
of improved bifunctional chelating agents (BCA’s) has an
important role. These chelators should be capable of linking
small peptides, proteins, antibodies or their fragments with a
metallic radionuclide. Prominent among the metallic radio-
nuclides suitable for these purposes are technetium-99m and
rhenium-186/188. The benefits of these compared to other
radionuclides are well known.1 However, although several
designs have been used in practice with success, BCA’s for
Tc and Re that meet all criteria of an ideal radiopharmaceutical
do not yet exist. Some desirable properties are a high labelling
efficiency under mild conditions, minimal isomerism, and good
in vivo stability and pharmacokinetics. Also, the labelling
procedure should be as simple as is practically feasible so that
labelling can be performed quickly and with minimal handling
and purification, because of the hazard and cost associated
with the radionuclides.
Strategies for labelling a biological molecule using a bi-
functional chelator may be classified as (1) “pre-formed chelate”
approach (in which the metal is chelated first and the complex is
then conjugated to the biomolecule) and (2) “post-labelling”
or final-step-labelling (in which the metal is added to a
biomolecule–chelator conjugate).2 The latter is the theoretically
preferred approach because the number of steps involving
the radionuclide is minimised. Unfortunately, ligands which
complex the metal with the required speed and specificity in
the presence of the biomolecule (which itself contains strong
chelating groups), are not yet available and the preformed
chelate approach is required with the present generation of
chelators. New ligands suitable for post-labelling are now being
sought.
Tetradentate ligands containing a thioether donor have
recently been considered as a new type of chelator for
technetium or rhenium. Only a few examples (see below) have
been described hitherto. McBride and co-workers described the
synthesis and biological evaluation of a diamino–thioether–
thiol chelator 1 for 99mTc.12 Hom and co-workers developed
an amino–amido–thioether–thiol ligand 2 and prepared the
oxorhenium() complex that mimics the steroid 5α-dihydro-
testosterone (DHT).13 Scheunemann and co-workers14 and
Kelly and co-workers15 synthesised N-terminal derivatised
diamido–thioether–thiol ligand 3 and its structural variants 4–
6, though metal complexes of these have not been character-
ised. Technetium complexes of the amino–diamido–thioether
type 7 have been described by Wong and co-workers.2b Recently
Archer and co-workers16 and Hilger and co-workers17 prepared
several amido–thioether–dithiol ligands 8 and an amino-
analogue 9,16 and their Re and Tc complexes.
BCA’s contain an activated group that is able to link specific-
ally with corresponding reactive sites of the biomolecule. We
chose the 2,3,5,6-tetrafluorophenyl ester function because of
its well known behaviour towards peptides and proteins con-
taining primary amine residues.4,5b,5c This type of functional-
isation of multidentate ligands often needs time-consuming
multistep syntheses. The aim in this work was to produce an
easily synthesised BCA containing a thioether donor, and to
characterise its suitability for complexing Tc and Re. Here we
describe a simple synthesis (using standard procedures of
peptide chemistry) of such a ligand (LH3), the crystal structure
of its complex with rhenium(), and its bioconjugation with a
small protein, N-TIMP-2 (the active domain of human tissue
inhibitor of metalloproteinases-2).18
Several co-ordination systems have already been used in
the design of BCA’s for Tc and Re. These include the large
group of N2S2 ligands such as diaminedithiols,3 monoamide–
monoaminedithiols,4 diamidedithiols,4,5 and the N3S triamide-
thiols.6 Other examples are propylene amines (PnAO),7 tetra-
amines,8 boronic adducts of technetium dioximes (BATO’s),9
Results and discussion
Ligand synthesis
The commercially available dipeptide 10 was N-protected with
di-tert-butyl dicarbonate to give the N-BOC product 11
J. Chem. Soc., Dalton Trans., 1998, 3087–3092
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O
O
H
N
O
O
HO
NH2
NH
S
NH
S
O
O
NH HN
10
a
R
HS
O
HS
SH
S
R
R'
R
H
N
1
2
3
BOC
O
O
HO
N
H
O
11
NH
S
NH
S
NH
S
b
F
F
NH HS
NH HS
O
NH
HS
O
H
N
BOC
O
O
F
O
N
4
5
6
F
F
O
H
O
O
O
12
R
(CH2)n
O
O
NH HN
NH2
c
R'
S
F
(CH2)m
O
O
H
N
R''
R'''
N
S
HS
SH
F
O
NH3+ CF3COO–
CH3
7
8
F
O
13
S
HN
F
d
F
SH HS
O
H
N
O
9
S
SH
F
O
N
(Scheme 1). Coupling with 2,3,5,6-tetrafluorophenol to form
12 was done using 1,3-dicyclohexylcarbodiimide in tetrahydro-
furan. Deprotection of the amine with trifluoroacetic acid
produced the trifluoroacetate salt 13 in high yield. For this
deprotection the use of HCl in diethyl ether was also investi-
gated but the yields were unsatisfactory. The thioether–thiol
moiety was incorporated using the ring-opening reaction of
the amine group with 2-oxo-1,4-dithiane, an elegant method
previously used to prepare a multidentate thioether ligand
under mild conditions in one step.14 In this case generation of
the free amine and ring opening of 2-oxo-1,4-dithiane leading
to the desired thiol LH3 produced the best results by a one-pot
synthesis in a mixture of phosphate buffer (pH 6) and chloro-
form. Attempts to reduce the amide carbonyl group with BH3
to obtain an alternative, potentially more reactive chelator
containing an amine donor have not been successful. The
unprotected thiol LH3 could be stored under argon at 0 ЊC for
several months without decomposition, but in concentrated
F
O
H
LH3
e
f
O
S
S
S
O
Re+
S
N
N
S
S
O
Re
O
N
O
O
N
N
H H
O
O
H
H
N
O
F
O
O
O
F
F
F
F
O
O
1
dimethyl sulfoxide solution H NMR spectroscopy revealed
formation of 2,3,5,6-tetrafluorophenol over a period of hours
as a result of nucleophilic attack by the thiol group.
F
F
F
F
F
ReOL
F
F
[ReO(LH2)2]+
Preparation of rhenium(V) complexes
The complexation behaviour of LH3 towards rhenium was
investigated by preparing the neutral oxorhenium() complex
ReOL using [Bu4N][ReOCl4] and sodium acetate in methanol at
room temperature. TLC indicated the occurrence of two purple
species, a major one with Rf 0.5 and a minor one with Rf 0.4.
The latter has not been characterised but we surmise that it may
be a transesterified analogue of ReOL containing a methyl
group instead of the tetrafluorophenyl group. After recrystal-
lisation from ethyl acetate only one species (Rf 0.5) was found
Scheme 1 Synthesis of the ligand LH3 and its complex ReOL.
Reagents and yields (a) (BOC)2O, 67%; (b) 2,3,5,6-tetrafluorophenol,
1,3-dicyclohexylcarbodiimide, 78%; (c) trifluoroacetic acid, 91%; (d)
2-oxo-1,4-dithiane, phosphate buffer (pH 6, 1.0 M), 47%; (e) [Bu4N]-
[ReOCl4], NaO2CMe, 22%; (f) oxorhenium() gluconate, 70%.
Fig. 1 for atom numbering scheme). Co-ordination of the
ligand to rhenium() caused considerable downfield shifts of
the non-equivalent methylene hydrogen atoms of the five-
membered chelate rings.
1
by TLC and HPLC. Microanalysis, FABϩ–MS and H NMR
spectroscopy of this major product are consistent with a
mononuclear 1:1 complex ReOL, in which the reactive ester
Reaction of LH3 with an equimolar amount of oxorhe-
nium() gluconate19 in water (pH 4.7, room temperature)
afforded a brown product within 5 min. This has not been fully
characterised but elemental analysis and electrospray mass
spectrometry revealed a compound containing two ligand
units with the constitution [ReO(LH2)2]Cl (see Scheme 1). The
1
group is retained. Assignments of H NMR peaks were made
1
using two-dimensional correlated spectroscopy H/1H-COSY.
However, this analysis did not unequivocally allow assignment
of the signals for protons attached to C1,C2,C3 and C5 (see
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Table 2 Selected bond lengths (Å) and angles (Њ) for complex ReOL
Re(1)–O(1)
Re(1)–N(1)
Re(1)–N(2)
Re(1)–S(1)
Re(1)–S(2)
S(2)–C(2)
S(2)–C(3)
S(1)–C(1)
1.683(4)
1.988(5)
2.024(5)
2.285(2)
2.359(2)
1.820(7)
1.817(7)
1.839(7)
O(2)–C(4)
O(3)–C(6)
O(4)–C(10)
O(5)–C(11)
O(5)–C(10)
C(1)–C(2)
C(3)–C(4)
C(5)–C(6)
1.211(8)
1.220(7)
1.185(9)
1.387(10)
1.369(9)
1.511(10)
1.506(9)
1.517(9)
O(1)–Re(1)–N(1)
N(1)–Re(1)–N(2)
N(1)–Re(1)–S(1)
O(1)–Re(1)–S(2)
N(2)–Re(1)–S(2)
O(1)–Re(1)–N(2)
O(1)–Re(1)–S(1)
N(2)–Re(1)–S(1)
N(1)–Re(1)–S(2)
S(1)–Re(1)–S(2)
118.6(2)
78.5(2)
C(2)–S(2)–Re(1)
C(7)–N(2)–Re(1)
C(4)–N(1)–Re(1)
C(3)–S(2)–Re(1)
C(1)–S(1)–Re(1)
C(6)–N(2)–Re(1)
C(5)–N(1)–Re(1)
C(2)–C(1)–S(1)
C(4)–C(3)–S(2)
C(1)–C(2)–S(2)
104.2(2)
124.4(4)
125.7(4)
101.3(2)
106.2(3)
118.3(4)
118.0(4)
109.9(5)
112.8(5)
106.7(5)
127.2(2)
102.5(2)
150.9(2)
105.8(2)
114.2(2)
89.1(2)
Fig. 1 Structure of complex ReOL showing the atom labelling scheme.
Crystal data and structure refinement for complex
Table
1
ReOLؒMeCO2Et
82.3(2)
85.20(7)
Empirical formula
C16H15F4N2O5ReS2ؒC4H8O2
Formula weight
Crystal system
Space group
a/Å
b/Å
c/Å
729.72
Monoclinic
P21/n
14.093(2)
11.506(2)
17.288(3)
112.390(10)
2592.0(7)
4
0.171(2) Å below the plane, while N2 and S2 lie 0.218(5) Å and
0.187(2) Å above it. The rhenium atom lies 0.739(1) Å above the
plane. Bond lengths and angles are in the expected ranges with
the Re1–O1 distance at 1.683(4) Å and, unsurprisingly, the Re–
thioether linkage (Re1–S2) has the longest interaction at
2.359(2) Å. This distance is in accordance with other examples
of similar bonds.2b,16,20 The co-ordinated amido groups are close
to planar and are not likely to give rise to isomeric forms in
solution. The crystal data and methods of the structure
determination are summarised in Table 1. Selected bond
lengths and angles are given in Table 2.
β/Њ
U/Å3
Z
Dc/Mg mϪ3
µ/mmϪ1
F(000)
1.870
4.920
1424
Crystal size/mm, colour and habitus 0.20 × 0.20 × 0.10, red needle
θ Range for data collection/Њ
h, k, l Ranges
2.18–25.34
Ϫ16 to 16, Ϫ13 to 13, Ϫ20 to 20
Reflections collected
15143
4704
3416
0
Independent reflections
Reflections observed with F > 4σF
Standard decay correction (%)
Conjugation reaction of complex [ReOL] with N-TIMP-2
To obtain preliminary confirmation of the ability of this
complex to form bioconjugates by aminolysis of the active
ester group, purified ReOL was conjugated to the protein
N-TIMP-2. This protein is the active N-terminal domain of
human tissue inhibitor of metalloproteinases-2 consisting of
127 amino acid residues.18 Fig. 2 shows the electrospray mass
spectrum (ES-MS) of a “labelling” experiment where equi-
molar amounts of ReOL and protein were mixed at pH 8 for
1 h. The parent protein is detected together with a second ion
corresponding to an additional 475 mass units consistent with
1:1 conjugate formation with elimination of tetrafluorophenol.
This second peak confirms that the conjugate contains the
intact chelating group bound to the ReO3ϩ core as expected.
Assuming that the parent protein and the conjugate ionise
similarly in the mass spectrometer it is estimated that about
17% of the protein is conjugated. Increasing the complex:
protein ratio produced multiple labellings since N-TIMP-2
contains several reactive lysine residues.18 It should be noted
that the 1:1 ratio of protein to chelate complex in the present
reaction conditions is unrealistic as a model for radiolabelling
reactions with 99mTc or 188Re, where in practice the concen-
tration of protein will greatly exceed the concentration of
complex since the metal radionuclide is present at no-carrier-
added levels.
Weighting scheme, wϪ1
*
σ2(Fo ) ϩ (0.005P)2
2
Data, restraints, parameters
Goodness of fit F2
wR2 (all data)
4704, 0, 326
0.971
0.0965
0.0629
0.0393
R1 (all data)
R1 (observed data)
Largest difference peak and
hole/e ÅϪ3
1.730, Ϫ1.170
2
* P = [max(Fo ,0) ϩ 2Fc2]/3.
cationic character of this complex was confirmed by its binding
to a cation exchange column. Poor solubility properties pre-
vented full characterisation by NMR spectroscopy. It is not
possible to conclude with the present data whether this complex
has a cis- (as shown in Scheme 1) or trans-structure. Formation
of such 2:1 complexes has been hypothesised before for ligands
of the related type 4.16 Higher pH conditions may be needed for
efficient 1:1 complex formation as amide groups have to be
deprotonated. This is a general problem with chelators con-
taining amido–thiol groups. Inclusion of a thioether donor may
thus prove advantageous, allowing efficient complexation of
Re/Tc even at low pH to form an intermediate 2:1 complex
conjugate in the presence of protein, which may then be
converted to the 1:1 complex by raising the pH.
Conclusion
Crystallography
We have described a convenient synthesis of a new bifunctional
diamide–thioether–thiol ligand LH3 containing a tetrafluoro-
phenyl ester group. LH3 forms an uncharged oxorhenium()
complex ReOL, which may be used to form bioconjugates
with proteins by aminolysis at a lysine residue. Under acidic
conditions a stable 2:1 complex ion [ReO(LH2)2]ϩ can also be
formed. Further investigation of ligands of this type will
examine and optimise their use with 99mTc and 188Re using both
pre-formed chelate and post-labelling approaches.
Complex ReOL crystallises in the monoclinic space group
P21/n with the asymmetric unit containing one molecule of
the rhenium complex and one ethyl acetate of crystallisation
(Fig. 1). The complex is racemic with both enantiomers in the
crystal structure (centric space group). The geometry is best
described as distorted square-pyramidal with the oxo group
apical. In the least-squares plane defined by the nitrogen
and sulfur atoms, N1 and S1 lie respectively 0.234(6) Å and
J. Chem. Soc., Dalton Trans., 1998, 3087–3092
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Fig. 2 Deconvoluted electrospray mass spectrum of the products obtained from the conjugation reaction of ReOL with N-TIMP-2. Unmodified
N-TIMP-2 appears at m = 14080.5 and the 1:1 conjugate appears at m = 14555.2.
31.21 mmol in 25 cm3 ButOH) was added at 0 ЊC. After stirring
Experimental
General
at room temperature for 16 h KHSO4 (7.01 g, 51.50 mmol)
dissolved in water (50 cm3) was added. TLC (ethyl acetate,
iodine) revealed a yellow spot at Rf = 0.5. The reaction solution
was extracted with ethyl acetate (3 × 50 cm3) and then with
ethyl acetate–brine (50 cm3 :50 cm3). The combined organic
extracts were washed with brine and dried over MgSO4. After
evaporation of the solvent at 30–40 ЊC in vacuo the residue was
solidified by addition of diethyl ether (50 cm3) and storing at
Ϫ20 ЊC for 1 h to give a white powder which was collected by
filtration. Yield: 5.42 g (67%), mp 75–78 ЊC (Found: C, 50.75;
H, 8.00; N, 10.74. C11H20N2O5 requires C, 50.76; H, 7.75; N,
10.76%); IR: 1732, 1711, 1683, 1633, 1537, 1175 cmϪ1; 1H NMR
(270 MHz, CDCl3): δ 1.45 (9 H, s, 3 CH3), 1.85 (2 H, m, CH2),
2.40 (2 H, m, CH2,), 3.30 (2 H, m, CH2), 3.80 (2 H, m, CH2),
5.30–6.75 (1 H, m, NH), 7.01–7.20 (1 H, m, NH), 10.95 (1 H,
m, OH).
N-Glycyl-4-aminobutyric acid
1
was obtained from
Sigma. 2-Oxo-1,4-dithiane,21 oxorhenium() gluconate19 and
N-TIMP-222 were prepared according to the literature. All
other reagents and solvents were purchased from Aldrich,
Fisher, or Merck and were used as supplied. Tetrahydrofuran
was distilled from sodium–benzophenone ketyl immediately
prior to use. Thin-layer chromatography (TLC) was performed
using sheets from Riedel-de Haën (Silica gel 60 F 254). Thiol
groups were detected on TLC plates using Ellman’s reagent.23
Visualisation was also achieved by iodine vapour or UV
illumination. For column chromatography Silica gel 60 from
Fluka (0.035–0.07 mm) was used. Solid phase extraction was
performed with Sep-Pak Vac tC2 (6 cm3, 1 g) cartridges
(Waters). Ion exchange chromatography was done using
SAX and SCX disposable columns (Isolute SPE, 0.5 cm
diameter × 0.7 cm). Melting points were determined with a
Gallenkamp apparatus. IR spectra were recorded on a 2020
Galaxy Series FT-IR spectrometer (Mattson Instruments)
using KBr pellets. 1H and 13C NMR spectroscopy were
2,3,5,6-Tetrafluorophenyl N-BOC-glycyl-4-aminobutyrate 12.
To a stirred solution of 11 (3.0 g, 11.53 mmol) and 2,3,5,6-
tetrafluorophenol (2.91 g, 17.54 mmol) in thf (40 cm3) 1,3-
dicyclohexylcarbodiimide (3.09 g, 14.98 mmol) dissolved in thf
(70 cm3) was added. After stirring for 22 h at room temperature
the precipitate was filtered off and washed with thf (2 × 10
cm3). The filtrate was concentrated in vacuo at 40–45 ЊC. TLC
(ethyl acetate–n-hexane 2:3) showed ester 12 at Rf = 0.1. The
crude product was isolated by column chromatography (silica
gel). After elution with ethyl acetate–n-hexane (2:3) to
remove unreacted starting material ester 12 was obtained by
elution with ethyl acetate. Removal of the solvent in vacuo gave
a white solid immediately. Yield: 3.65 g (78%), mp 78–82 ЊC
(Found: C, 50.03; H, 5.13; N, 6.91. C17H20F4N2O5 requires: C,
50.00; H, 4.97; N, 6.86%); IR: 1773, 1676, 1652, 1528 cmϪ1; 1H
NMR (270 MHz, CDCl3): δ 1.45 (9 H, s, 3 CH3), 2.00 (2 H, m,
CH2), 2.70 (2 H, m, CH2), 3.40 (2 H, m, CH2), 3.80 (2 H, m,
CH2), 5.70 (1 H, m, NH), 6.50 (1 H, m, NH), 7.00 (1 H, m, aryl
H).
performed on a JEOL GSX (270 MHz) except for the H/1H
1
double quantum filtered COSY spectrum (DQF-COSY24) for
which a Varian UNITY Inova (600 MHz) spectrometer was
used. The standards were tetramethylsilane and 2,2-dimethyl-2-
silapentane-5-sulfonate. Fast atom bombardment mass spectra
(FABϩ-MS) were obtained using 3-nitrobenzyl alcohol as
matrix by the EPSRC Mass Spectrometry Centre, Swansea,
UK. Electrospray mass spectra were recorded on a Finnigan
MAT LCQ ion trap mass spectrometer by the Wellcome Trust
Protein Science Facility, University of Kent. Samples were
desalted on-line by reverse-phase HPLC on a C4 column
(2.1 × 50 mm) running on a Hewlett Packard 1100 HPLC
system. Mass spectra were acquired with the ion trap operating
in the high mass mode and spectra deconvoluted using Bio-
Explore to give protein masses. Microanalyses were performed
by the Microanalytical Service at the University of Kent.
N-Glycyl-4-amino-N-butyric acid 2,3,5,6-tetrafluorophenyl
ester trifluoroacetate 13. Selective acidolytic removal of an
N-tert-butyloxycarbonyl group in the presence of an ester func-
tion using trifluoroacetic acid has been described elsewhere.26
To a stirred solution of 12 (3.65 g, 8.94 mmol) in dichloro-
methane (60 cm3) trifluoroacetic acid (6 cm3) was added during
10 min at room temperature. After continued stirring at room
Preparations
N-BOC-Glycyl-4-aminobutyric acid 11. The preparation
of 11 was done by a modification of a known synthesis.25 To a
mixture of 10 (5.0 g, 31.21 mmol), NaOH (34 cm3, 1.0 N) and
water (25 cm3), a solution of di-tert-butyl dicarbonate (6.81 g,
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temperature for 4 h completion of the reaction was indicated by
TLC (ethyl acetate–n-hexane 2:3). The solvent was evaporated
in vacuo to give a yellow oil that was solidified by addition
of diethyl ether (60 cm3) and stirring for 5 min. The resulting
white solid was collected by filtration. Yield: 3.44 g (91%),
mp 95–100 ЊC (Found: C, 39.60; H, 3.09; N, 6.47. C14H13-
F7N2O5 requires: C, 39.82; H, 3.10; N, 6.63%); IR: 1780, 1674,
Reaction of LH3 with oxorhenium(V) gluconate
A stirred solution of ligand LH3 (40 mg, 0.0904 mmol) in
PriOH (8 cm3) was degassed with Ar for 10 min. An aqueous
(pH 4.7) solution of oxorhenium() gluconate19 (2.52 cm3,
0.0904 mmol) was added and the mixture stirred for 5 min at
room temperature. After addition of water (10 cm3) and ethyl
acetate (20 cm3) the orange-brown organic phase was separated,
washed twice with brine and dried over MgSO4. Evaporation of
the solvent and treatment with diethyl ether afforded a brown
solid insoluble in water and poorly soluble in chloroform.
Yield: 38 mg (70%), mp 95–100 ЊC (Found: C, 36.38; H, 3.43;
N, 5.19. C36H42ClF8N4O11ReS4 {for [ReO(LH2)2]ClؒEtOAc}
requires: C, 35.78; H, 3.50; N, 4.64%); IR: 1786, 1643, 1523
cmϪ1. ES-MS 1085.3 (Mϩ), 100%; 1107.3 (Mϩ Ϫ HϩNa), 25%.
The complex dissolved in acetonitrile (0.3 cm3) was trapped by
an SCX cation exchange column after elution with acetonitrile
(1 cm3) but was eluable using an SAX anion exchange column
and the same amount of eluting solvent.
1
1652, 1523 cm-1; H NMR (270 MHz, D2O): δ 2.00 (2 H,
quintet, J = 7.2, CH2CH2CH2), 2.85 (2 H, t, J = 7.2, CH2), 3.40
ϩ
(2 H, t, J = 7.2 Hz, CH2), 3.80 (2 H, s, CH2NH3 ), 7.33 (1 H,
m, aryl H).
N-(N-5-sulfanyl-3-thiapentanoyl)glycyl-4-aminobutyric acid
2,3,5,6-tetrafluorophenyl ester LH3. To a well-stirred solution of
2-oxo-1,4-dithiane (2 g, 14.90 mmol) in chloroform (100 cm3),
phosphate buffer (32 cm3, pH 6, 1.0 M) and water (18 cm3), the
trifluoroacetate salt 13 (2 g, 4.74 mmol) was added. Stirring at
room temperature was continued for 8 h. After addition of
water (50 cm3) and separation of the organic phase the aqueous
phase was extracted with chloroform (2 × 20 cm3). The com-
bined organic fractions were dried over MgSO4. The crude
product was concentrated in vacuo at 35 ЊC to give an oil and
purified by flash chromatography (ethyl acetate). Fractions con-
taining the thiol LH3 were combined. Evaporation of the sol-
vent in vacuo at 35 ЊC afforded a white solid that was stored
under argon at 5 ЊC. Yield: 979 mg (47%), mp 95–97 ЊC (Found:
C, 43.90; H, 4.22; N, 6.42. C16H18F4N2O4S2 requires: C, 43.44;
H, 4.10; N, 6.33%); IR: 1778, 1655, 1639, 1523 cmϪ1; 1H NMR
(270 MHz, CDCl3): δ 1.71 (1 H, t, J = 7.6, SH), 2.01 (2 H,
quintet, J = 6.6, CH2CH2CH2), 2.75 (6 H, m, CH2), 3.28 [2 H,
s, SCH2C(O)], 3.42 (2 H, q, J = 6.6, CH2CH2NH), 3.99 [2 H,
d, J = 5.6 Hz, C(O)CH2NH], 6.72 (1 H, m, NH), 7.01 (1 H,
m, aryl H), 7.58 (1 H, m, NH); 13C NMR (270 MHz, CDCl3):
δ 24.2, 24.5, 30.8, 35.6, 36.9, 38.7, 43.4, 103.0, 103.3, 103.6,
168.8, 169.1, 169.8; FABϩMS: m/z = 443 (MϩH), 465 (MϩNa).
Labelling of N-TIMP-2
In a typical experiment a solution of ReOL in dimethyl
sulfoxide (0.001 cm3, 0.001 mmol) was added to buffered N-
TIMP-2 (0.1 cm3, 0.001 mmol, borate buffer, pH 8.0, 100 mM).
After incubation for 1 h at room temperature the mixture
was stored at Ϫ80 ЊC until mass spectrometry could be per-
formed. Investigation by ES-MS revealed signals due to
unmodified N-TIMP-2 at m = 14080.5 (predicted mass 14084,
100%) and 1:1 conjugate at m = 14555.2 (protein ϩ 475,
18%).
Crystallography
Data collection. Intensity data were collected at 293(2) K on a
MSC/Rigaku Raxis-IIc diffractometer with monochromated
Mo-Kα radiation (0.71073 Å) using MSC data collection
software. Cell constants were obtained from least-squares
refinement of the setting angles of 349 centred reflections
(θ = 3.5–25Њ).
[2,3,5,6-Tetrafluorophenyl N-(N-5-sulfanyl-3-thiapentanoyl)-
glycyl-4-aminobutyrate(3؊)]oxorhenium(V) ReOL. LH3 (100
mg, 0.226 mmol) and sodium acetate (93 mg, 1.13 mmol) were
dissolved in methanol (50 cm3) under argon and cooled to 0 ЊC.
After addition of [Bu4N][ReOCl4] (130 mg, 0.226 mmol) in
methanol (5 cm3) during 5 min the mixture was allowed to
warm up and stir for 28 h at room temperature. A brownish
solution was obtained. The solvent was removed in vacuo
and the crude product purified using a small column of
silica gel and ethyl acetate containing 10% PriOH as eluent. A
purple product was collected and the solvent evaporated in
vacuo. Thin-layer chromatography at this point revealed two
purple spots, Rf 0.5 and 0.4. The crude product was dissolved
in acetonitrile (0.5 cm3) and applied to a tC2 cartridge
equilibrated with water. The column was eluted twice with
water containing MeCN (5 cm3, 10% MeCN; 5 cm3, 30%
MeCN). Elution with MeCN (5 cm3) and evaporation of the
solvent in vacuo gave the pure complex (TLC Rf 0.5). Yield:
32 mg (22%), mp 157–160 ЊC (decomp.) (Found: C, 30.45;
H, 2.86; N, 4.20. C16H15Fe4N2O5ReS2 requires: C, 29.95; H,
Structure analysis and refinement. The structure of ReOL
was solved by direct methods (SHELXS 86)27 and refined
2
on Fo by full-matrix least squares (SHELXL 93).28 All
non-hydrogen atoms were refined with anisotropic thermal
parameters. Hydrogen atoms were fixed on idealised positions.
CCDC reference number 186/1114.
See http://www.rsc.org/suppdata/dt/1998/3087/ for crystallo-
graphic files in .cif format.
Acknowledgements
We thank the BBSRC, the Association for International Cancer
Research, and the Arthritis and Rheumatism Council for grants
to M. T. R., M. G. and R. A. W. respectively. K. H., M. J. H. and
the core facilities within the Protein Science and Biomedical
NMR Laboratory are supported by the Wellcome Trust.
1
2.36; N, 4.37%); IR: 1786, 1659, 1639, 1523 cmϪ1; H NMR
(600 MHz, CDCl3; see Fig. 1 for atom numbering scheme):
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