Cyclization of Tetra-L-alanine Induced by Complexation of Technetium

Article abstract of DOI:10.1021/ic951030j

Complexation of technetium with tetraalanine yields an unstable complex that converts to a monooxotechnetium(V) complex of cyclic tetraalanine. The structure of the latter was determined by single-crystal X-ray analysis. In this negatively charged complex, the technetium atom is coordinated to one oxygen and four amide nitrogen atoms. The more polar complex that is initially formed is assumed to be an oxotechnetium(V) complex of tetraalanine, with the oxotechnetium moiety coordinated to three amide nitrogen atoms and the amine nitrogen atom. In this complex, the carboxyl group would be forced in the vicinity of the free amine group, thereby facilitating amide formation and cyclization. Cyclization of tetra-L-alanine occurs on both no carrier added (technetium-99m) and carrier added (technetium-99) scale.

Full text of DOI:10.1021/ic951030j

6240
Inorg. Chem. 1996, 35, 6240-6244
Cyclization of Tetra-L-alanine Induced by Complexation of Technetium
G. Bormans,† O. M. Peeters,‡ H. Vanbilloen,† N. Blaton,‡ and A. Verbruggen*,†
Laboratory for Radiopharmaceutical Chemistry, FFW, KU Leuven, Herestraat 49, B-3000 Leuven,
Belgium, and Laboratory for Analytical Chemistry and Medicinal Physicochemistry, FFW, KU Leuven,
Van Evenstraat 4, B-3000 Leuven, Belgium
ReceiVed August 4, 1995X
Complexation of technetium with tetraalanine yields an unstable complex that converts to a monooxotechnetium(V)
complex of cyclic tetraalanine. The structure of the latter was determined by single-crystal X-ray analysis. In
this negatively charged complex, the technetium atom is coordinated to one oxygen and four amide nitrogen
atoms. The more polar complex that is initially formed is assumed to be an oxotechnetium(V) complex of
tetraalanine, with the oxotechnetium moiety coordinated to three amide nitrogen atoms and the amine nitrogen
atom. In this complex, the carboxyl group would be forced in the vicinity of the free amine group, thereby
facilitating amide formation and cyclization. Cyclization of tetra-L-alanine occurs on both no carrier added
(technetium-99m) and carrier added (technetium-99) scale.
Introduction
Technetium-99m is the most intensively used radioisotope
in diagnostic nuclear medicine. It owes its popularity to its
excellent physical characteristics (T1/2 ) 6.02 h; photon energy
of 140 keV; no corpuscular radiation) and chemical properties
(transition metal) and to its availability from a 99Mo/99mTc
generator. Only very small amounts of Tc (typically about
1-40 ng) are present in radiopharmaceutical preparations, which
necessitates the use of milligram amounts of the long-lived 99Tc
(T1/2 ) 2.12 × 105 years) for the structure determination of
99mTc-labeled agents by X-ray crystallography. The oxotech-
netium(V) complex of mercaptoacetyltriglycine (99mTc-MAG3),
in which the technetium atom is coordinated to an oxygen atom,
a thiol sulfur atom, and three nitrogen amide atoms,1 is an
established compound for the evaluation of renal function.2 To
assess the involvement of the thiol group for efficient renal
extraction of this type of tracer agent, we have synthesized
technetium-99m complexes with tetrapeptides that can be
Figure 1. Comparison of the structures of mercaptoacetyltriglycine
(MAG3) and tetrapeptides.
from plasma, whereas 99mTc-A4-A is cleared by both the
considered derivatives of mercaptoacetyltriglycine in which the
thiol group has been replaced by an amino group (Figure 1).3
In contrast to MAG3, which forms a single complex with
hepatobiliary and renal systems in almost equal proportions.3
technetium-99m in standard conditions, labeling of the tetrapep-
Electrophoresis experiments showed that 99mTc-A4-A is
tide tetra-L-alanine (A4) with technetium-99m yields two
negatively charged at alkaline pH, but neutral in acidic condi-
different complexes (99mTc-A4-A and 99mTc-A4-B) that can be
tions, suggesting the presence of an ionizable carboxylic acid
separated by HPLC.3 Variable amounts of the two complexes
group. The behavior of 99mTc-A4-B in electrophoresis clearly
were obtained depending on the labeling conditions, but the
is different as migration does not vary as a function of pH.3
more hydrophilic complex 99mTc-A4-A appears to be less stable
Prompted by the diverging biological characteristics and the
as it slowly converts to 99mTc-A4-B. This conversion also takes
puzzling results of electrophoresis, we have conducted com-
place after isolation of 99mTc-A4-A with HPLC. This indicates
plexation experiments with long-lived technetium-99 to be able
that the original technetium complex (99mTc-A4-A) is modified
to determine the exact structure of the different complexes.
and that technetium is not simply transferred to another ligand.
Biological evaluation in mice revealed that 99mTc-A4-B is
characterized by an almost exclusive hepatobiliary clearance
Experimental Section
NH499TcO4 as a black solid was acquired as a gift from Mallinckrodt
Medical (Petten, The Netherlands). Na99mTcO4 was eluted from an
Ultratechnekow 99mTc generator (Mallinckrodt Medical). Commercially
available reagents and solvents were of pro analysi quality and were
used without further purification. Fab mass spectra were recorded by
using a m-nitrobenzyl alcohol matrix on a VG 30-250 spectrometer
(VG Instruments Inc.) at the probe temperature. Xenon was used as
the primary beam gas, and the ion gun was operated at 8 keV and 100
µA. Data were collected over the mass range 100-1000 Da at 0.7
s/scan.
* Author to whom correspondence should be addressed.
† Laboratory for Radiopharmaceutical Chemistry.
‡ Laboratory for Analytical Chemistry and Medicinal Physicochemistry.
X Abstract published in AdVance ACS Abstracts, September 15, 1996.
(1) Grummon, G.; Rajagopalan, R.; Palenik, G. J.; Koziol, A. E.; Nosco,
D. Inorg. Chem. 1995, 34, 1764.
(2) Eshima, D.; Taylor, A. Semin. Nucl. Med. 1992, 22, 61.
(3) Vanbilloen, H.; Bormans, G.; De Roo, M.; Verbruggen, A. Eur. J.
Nucl. Med. 1995, 22, 325.
S0020-1669(95)01030-5 CCC: $12.00 © 1996 American Chemical Society

Tc-Induced Cyclization of Tetra-L-alanine
Inorganic Chemistry, Vol. 35, No. 21, 1996 6241
Table 1. Crystal Data
chemical formula C36H36As1N4O5Tc1 V (Å3)
Warning: Technetium-99 is a long-lived (T1/2 ) 2.12 × 105 years)
weak â- emitter (Emax ) 292 keV) and requires special radioprotective
measurements during handling.4
1637(2)
d(measd) (Mg m-3
) 1.5
fw
777.61
P1
Preparation of TcOCl4(Ph)4As.5 NH499TcO4 (90 mg 0.5 mmol)
was dissolved in 10 mL of 12 N HCl and spiked with 200 MBq of
Na99mTcO4 in 2 mL of generator eluate. Tetraphenylarsonium chloride
(420 mg, 1 mmol) was added and the solution was stirred for 30 min
at room temperature. The precipitate was filtered off and washed with
two portions of 8 mL of 12 N HCl and two portions of a 2-propanol
diethyl ether (40:60) mixture. The precipitate was dissolved in 30 mL
of methanol.
space group
a (Å)
b (Å)
d(calcd) (Mg m-3
Z
)
1.577
2
9.502(6)
13.154(9)
13.889(13)
89.07(6)
88.48(6)
70.64(1)
λ (Å)
0.71073
1.490
792
c (Å)
µ (mm-1
F(000)
)
R (deg)
â (deg)
γ (deg)
R, wR(F2)
0.042, 0.094
Table 2. Fractional Atomic Coordinates and Equivalent Isotropic
Displacement Parameters (Å2) of the (Cyclotetraalanyl)-
technetium(V) Anion
Preparation of 99mTc/99Tc-tetra-L-alanine (Tc-A4). Sodium (15.84
mg, 0.66 mmol) was dissolved in 20 mL of methanol, and 100 mg
(0.33 mmol) of tetra-L-alanine was added to this solution. TcOCl4(Ph)4As
(225 mg, 0.33 mmol) in 10 mL of methanol was added via a dropping
funnel to the latter solution under stirring at room temperature. The
solution immediately turned black due to the precipitation of TcO2.
TcO2 was filtered off and the volume of the filtrate was reduced by
x
y
z
Ueq
Tc1
O1
-0.16686(4)
0.03170(3)
-0.0284(3)
0.0126(3)
-0.0743(4)
-0.1811(5)
-0.0953(5)
-0.1452(3)
-0.0526(4)
-0.0383(5)
-0.1266(5)
0.0689(5)
0.1085(4)
0.1115(4)
0.2279(5)
0.2624(5)
0.2649(5)
0.3554(3)
0.1782(4)
0.1944(4)
0.2099(5)
0.0919(5)
0.0888(3)
0.49051(3) 0.0276(2)
0.0129(5)
0.4817(3)
0.3824(4)
0.3916(5)
0.3396(5)
0.5002(5)
0.5297(3)
0.5526(4)
0.6590(5)
0.7161(5)
0.6820(5)
0.7599(3)
0.6088(4)
0.6087(5)
0.6528(5)
0.4986(5)
0.4747(3)
0.4375(3)
0.3417(4)
0.2668(5)
0.3179(5)
0.2494(3)
0.037(1)
0.034(1)
0.033(2)
0.058(3)
0.037(2)
0.046(2)
0.034(1)
0.044(2)
0.068(3)
0.037(2)
0.048(2)
0.035(1)
0.042(2)
0.046(2)
0.038(2)
0.037(1)
0.034(1)
0.037(2)
0.046(2)
0.039(2)
0.055(2)
N111 -0.2833(5)
C112 -0.3497(6)
C113 -0.2583(9)
C114 -0.3603(6)
O115 -0.4406(4)
N121 -0.2788(5)
C122 -0.3052(7)
C123 -0.1937(9)
C124 -0.2924(6)
O125 -0.3300(5)
N131 -0.2347(5)
C132 -0.2557(7)
C133 -0.1400(7)
C134 -0.2714(6)
O135 -0.3124(5)
N141 -0.2402(5)
C142 -0.2897(7)
C143 -0.1941(7)
C144 -0.3305(7)
O145 -0.4092(5)
evaporation under reduced pressure to a final volume of 4 mL.
A
fraction of the concentrated solution was stored in an open vial at room
temperature to enable a slow evaporation of methanol, yielding bright
yellow crystals.
HPLC Analysis. The compounds were analyzed on a 250 × 4.6
mm column filled with Hypersil ODS 5 µm (Shandon Scientific,
England) eluted at a flow rate of 1 mL/min with a ternary gradient
mixture of ethanol (A)-0.025 M phosphate buffer (pH 5.85) (B)-
water (C): T ) 0 min, B ) 100%; T ) 15 min, A ) 20%, B ) 80%;
T ) 20 min, A ) 50%, B ) 50%; T ) 20.1 min, A ) 50%, C ) 50%;
T ) 25 min, A ) 90%, C ) 10%.
The column effluent was monitored for UV absorbance at 215 nm
and for radioactivity using a 2 in. NaI(Tl) scintillation detector.
Preparation of No Carrier Added 99mTc-A4 and Kinetic Analysis
of the Conversion Rate. Tetra-L-alanine (1 mg) was dissolved in 0.1
mL of 0.5 M phosphate buffer (pH 11). SnCl2‚2H2O (40 µg) dissolved
in 20 µL of 0.05 N HCl was added immediately followed by 2 mL of
-
99mTcO4 (740 MBq) solution in saline obtained from a technetium
Tc2
O2
0.24952(4)
0.0657(5)
0.3488(5)
0.3705(7)
0.2429(7)
0.3594(6)
0.3920(6)
0.3120(4)
0.3381(7)
0.2052(8)
0.3627(6)
0.4045(6)
0.3335(5)
0.3885(7)
0.285(1)
0.4288(7)
0.5182(5)
0.3710(5)
0.4238(8)
0.3452(8)
0.4293(7)
0.4885(6)
0.45826(3) -0.02245(3) 0.0256(1)
generator (Ultratechnekow, Mallinkrodt Medical).
0.5193(4)
0.4031(4)
0.2923(5)
0.2828(6)
0.2353(5)
0.1388(4)
0.3045(3)
0.2709(5)
0.2464(6)
0.3630(5)
0.3554(4)
0.4538(4)
0.5408(5)
0.6297(6)
0.5800(5)
0.6311(4)
0.5508(4)
0.5535(5)
0.6493(5)
0.4534(5)
0.4295(4)
-0.0143(4)
0.047(2)
0.034(1)
0.038(2)
0.041(2)
0.032(2)
0.033(2)
0.045(1)
0.036(2)
0.047(2)
0.037(2)
0.051(2)
0.031(1)
0.035(2)
0.061(3)
0.034(2)
0.043(2)
0.028(1)
0.037(2)
0.044(2)
0.034(2)
0.049(2)
The solution was neutralized with 0.5 M H3PO4, and an aliquot of
500 µL was subjected to reversed-phase HPLC as described earlier.
The peak with a retention time corresponding to 99mTc-A4-A was
collected and immediately divided into four fractions of 200 µL that
were added to 0.5 mL of 0.5 M phosphate buffer with pH’s of 4, 6, 8,
and 10, respectively.
The solutions were analyzed at intervals of 40 min over 280 min by
RP-HPLC as described earlier, but using isocratic elution at a flow
rate of 1 mL/min with 0.025 M phosphate buffer (pH 5.85)-EtOH
(70:30) instead of gradient elution.
N211
C212
C213
C214
O215
N221
C222
C223
C224
O225
N231
C232
C233
C234
O235
N241
C242
C243
C244
O245
0.0971(4)
0.1221(5)
0.1966(5)
0.0251(4)
0.0234(4)
-0.0454(3)
-0.1443(5)
-0.1861(6)
-0.2047(5)
-0.2875(3)
-0.1512(3)
-0.1775(5)
-0.2313(5)
-0.0854(5)
-0.0852(4)
-0.0069(3)
0.0868(5)
Linear regression was applied to the plots of the logarithm of the
residual fraction of 99mTc-A4-A vs time (seven observations) to obtain
the k values. R2 values of the regression varied between 0.9981 and
0.9997.
Esterification and Hydrolysis. A neutralized solution (500 µL)
of 99mTc-A4-A obtained as described earlier was applied to a Sep-Pak
C18 column (Waters, Milford, MA). The column was rinsed twice
with 5 mL of H2O and dried by a stream of nitrogen for 10 min. The
Sep-Pak column was eluted with 2 mL of methanol, and 2 mmol of
diazomethane27 in 3.5 mL of ether was added to the eluate, which was
then analyzed by HPLC (isocratic elution as described earlier). The
previous solution (0.5 mL) was added to 0.5 mL of 1 N NaOH. After
10 min of incubation at room temperature, the solution was neutralized
and analyzed by HPLC (isocratic elution as described earlier).
Crystal Structure. The crystal structure was determined on a
yellow, plate-shaped crystal (0.32 × 0.20 × 0.04 mm) isolated from
the methanolic mixture. Data were collected at room temperature on
a Stoe STADI4 automated four-circle diffractometer (program: DIF4)6
equipped with graphite-monochromated Mo KR radiation (see Table
0.1421(5)
0.1431(4)
0.2175(3)
1). Accurate lattice parameters were derived by automatic centering
and least-squares analysis of 25 reflections in the range 16 e 2θ e
24°. The ω-2θ scan technique (scan width ) 1.20° plus R1-R2
divergence) was used to collect 6028 intensities with indices -11 e h
e 11, -15 e k e 15, 0 e l e 16. Of these measured reflections,
6028 were unique and 3595 were considered observed (I g 2σI). Three
reflections (002, 020, 232) monitored periodically (120 min) showed
an average decay of <2%. The intensities were corrected for Lp and
linear decay (program: REDU4).7 Empirical absorption corrections8
(4) Davison, A.; Orvig, C.; Trop, H. S.; Sohn, M.; De Pamphilis, B. V.;
Jones A. G. Inorg. Chem. 1980, 19, 1988.
(5) Cotton, F. A.; Davison, A.; Day, V. W.; Gage, L. D.; Trop, H. S.
Inorg. Chem. 1979, 18, 3024.
(7) REDU4. Data reduction program. Version 7.03; Stoe & Co.: Darm-
stadt, Germany, 1992.
(6) DIF4. Diffractometer control program. Version 6.2; Stoe & Co.:
Darmstadt, Germany, 1988.
(8) North, A. C. T.; Phillips, D. C.; Mathews, F. S. Acta Crystallogr.
1968, A24, 351.

6242 Inorganic Chemistry, Vol. 35, No. 21, 1996
Bormans et al.
Table 3. Selected Bond Lengths and Bond Angles
Bond Lengths (Å)
Tc1-O1
1.629(4)
1.957(5)
1.951(5)
1.941(5)
1.958(5)
Tc2-O2
1.665(4)
1.940(5)
1.939(5)
1.932(5)
1.953(5)
Tc1-N111
Tc1-N121
Tc1-N131
Tc1-N141
Tc2-N211
Tc2-N221
Tc2-N231
Tc2-N241
Bond Angles (deg)
80.4(3)
129.1(4)
78.9(3)
78.7(3)
129.3(4)
79.5(3)
115.6(4)
114.6(4)
115.3(4)
116.1(4)
N131-Tc1-N141
N121-Tc1-N141
N121-Tc1-N131
N111-Tc1-N141
N111-Tc1-N131
N111-Tc1-N121
O1-Tc1-N141
O1-Tc1-N131
O1-Tc1-N121
O1-Tc1-N111
N231-Tc2-N241
N221-Tc2-N241
N221-Tc2-N231
N211-Tc2-N241
N211-Tc2-N231
N211-Tc2-N221
O2-Tc2-N241
O2-Tc2-N231
O2-Tc2-N221
O2-Tc2-N211
79.7(3)
129.3(4)
80.2(3)
78.5(3)
129.3(4)
79.4(3)
115.8(4)
114.5(4)
114.9(4)
116.2(4)
Figure 2. HPLC chromatograms: (A) analysis of the labeling reaction
mixture of 99mTc-A4 with the presence of 99mTc-A4-A and 99mTc-A4-B
(radioactivity detection); (B) analysis immediately after the preparation
of 99Tc-A4 showing the presence of 99mTc-A4-A (radioactivity detec-
tion); (C) analysis of a sample of the crystals obtained from the 99Tc-
A4 preparation [(cyclotetra-L-alanyl)oxotechnetium(V)] exhibiting a
retention time identical to that of 99mTc-A4-B (UV detection 215 nm).
based on ψ scans were applied (program: EMPIR).9 Reflections were
measured in 10° intervals. The minimum and maximum transmission
values ranged from 0.601 to 0.732. The structure was solved by using
a combination of the Patterson method and direct methods on the
difference structure (program: DIRDIF).10 The model was refined on
F2 by full-matrix least-squares (program: SHELXL-93).11 Displace-
ment parameters were anisotropic for all non-hydrogen atoms. H-atoms
were located on ideal molecular geometries and refined by using a riding
model. Analysis of the final difference map revealed -0.68 e Å-3 as
the deepest hole and 0.70 e Å-3 as the highest peak. General
calculations were performed using PARST.12 The absolute configu-
ration was determined according to Flack.13
Final fractional coordinates and equivalent isotropic displacement
parameters with estimated standard deviations (esd) of the (cyclotetra-
L-alanyl)oxotechnetium(V) anions are listed in Table 2. Table 3
contains selected bond lengths and bond angles. A complete report is
included in the supporting information.
of the reaction mixture immediately after preparation showed
the presence of only one compound with a retention time
identical to that of 99mTc-A4-A, obtained by labeling tetra-L-
alanine with technetium-99m in alkaline conditions (Figure 2).
HPLC analysis of the mixture was repeated 24 h after synthesis
and yielded a chromatogram that shows the presence of two
peaks, the retention times of which correspond to 99mTc-A4-A
and 99mTc-A4-B, respectively. The conversion of Tc-A4-A to
Tc-A4-B, which was observed on the technetium-99m scale,
also appeared to proceed for the carrier-added technetium-99
complex that was initially obtained. Subsequent HPLC analyses
showed that this conversion to Tc-A4-B proceeded further as a
function of time and was complete after 14 days.
Yellow crystals started to form about 20 days after synthesis,
and, as could be expected, the retention time on HPLC of the
crystallized compound was identical to that of 99mTc-A4-B
(Figure 2). Negative ion Fab mass spectroscopy of the crystal
surprisingly showed a molecular ion with a mass of 385 Da,
corresponding to a Tc complex in which an oxotechnetium core
is bound to the four amide nitrogen atoms of cyclic tetra-L-
alanine, of which four protons have been withdrawn [(cyclotetra-
L-alanyl)oxotechnetium(V)]. This structure was unexpected as
we were unsuccessful in previous attempts to chelate techne-
tium-99m with cyclic tetrapeptides.
Results and Discussion
99Tc pertechnetate was spiked with 99mTc to facilitate the
detection of any contaminations caused during handling and
synthesis and to enable radiometric detection with a NaI(Tl)
detector during HPLC analysis.
The technetium complex was synthesized in two steps with
an initial reduction of technetium(VII) pertechnetate to oxo-
technetium(V) tetrachloride in concentrated hydrochloric acid.5
Oxotechnetium(V) tetrachloride was isolated by precipitation
with tetraphenylarsonium chloride, and the salt was added to a
solution of the ligand tetra-L-alanine in sodium methanolate/
methanol. Previous experiments on the technetium-99m scale
indicated that complexation of technetium by tetrapeptides
proceeds with a higher yield in an alkaline environment.3
Accordingly, we used an alkaline solution to facilitate the
deprotonation of the amide groups, which is necessary for
chelation of technetium by tetra-L-alanine. The colloidal
precipitate of TcO2, which was also formed during this reaction,
was easily removed by filtration to yield a clear yellow solution,
the color typical for Tc(V) complexes.1,14-21 HPLC analysis
The structure derived from the results of mass spectroscopy
was confirmed by single-crystal X-ray analysis. An ORTEX22
plot is shown in Figure 3. The asymmetric unit consists of two
tetraphenylarsonium cations and two (cyclotetra-L-alanyl)-
oxotechnetium(V) anions. Apart from the peptide bond atoms,
the structure is pseudocentrosymmetric. The (cyclotetra-L-
alanyl)oxotechnetium(V) anions have a square pyramidal ar-
(9) EMPIR. Empirical absorption correction program. Version 1.2; Stoe
& Co.: Darmstadt, Germany, 1992.
(16) DePamphilis, B. V.; Jones, A. G.; Davis, M. A.; Davison, A. J. Am.
Chem. Soc. 1978, 17, 5570.
(17) Kawashima, M.; Koyama, M.; Fujinaga, T. J. J. Inorg. Nucl. Chem.
1976, 38, 801.
(18) Davison, A.; Orvig, C.; Trop, H.; Sohn, M.; DePamphilis, B. V.; Jones,
A. Inorg. Chem. 1980, 19, 1988.
(19) Smith, J.; Byrne E.; Cotton, F.; Sekutowski, J. C. J. Am. Chem. Soc.
1978, 16, 5570.
(10) Beurskens, P. T.; Admiraal, G.; Beurskens, G.; Bosman, W. P.; Garcia-
Granda, S.; Gould, R. O.; Smits, J. M.; Smykalla, C. The DIRDIF
program system, Technical Report of the Crystallography Laboratory;
University of Nijmegen: Nijmegen, The Netherlands, 1992.
(11) Sheldrick, G. M. SHELXL93. Program for Crystal Structure Refine-
ment; Univ. of Go¨ttingen: Go¨ttingen, Germany, 1993.
(12) Nardelli, M. J. Comput. Chem. 1983, 7, 95.
(13) Flack, H. D. Acta Crystallogr. 1983, A39, 876.
(14) Jones, A.; Davison, A.; LaTegola, M.; Brodack, J.; Orvig, C.; et al. J.
Nucl. Med. 1982, 23, 801.
(15) Smith, J.; Byrne, E.; Cotton, F.; Sekutowski, J. Inorg. Chem. 1978,
17, 5571.
(20) Jurisson, S.; Schlemper E.; Troutner, D.; Canning, L.; Nowotnik, D.;
Neirinckx R. Inorg. Chem. 1986, 25, 543.
(21) Rao, T. N.; Adhikesavalu, D.; Camerman, A.; Fritzberg, A. R. J. Am.
Chem. Soc. 1990, 112, 5798.
(22) McArdle, P. ORTEX2.1b. J. Appl. Crystallogr. 1994, 27, 438.

Tc-Induced Cyclization of Tetra-L-alanine
Inorganic Chemistry, Vol. 35, No. 21, 1996 6243
Figure 4. HPLC analysis of 99mTc-A4 (A) prior to and (B) after
reaction with diazomethane and (C) after attempted alkaline hydrolysis
yielding 99mTc-A4-B.
Figure 3. ORTEX drawing of the anion (cyclotetra-L-alanyl)oxotech-
netium(V).
Table 4. k-Values (min-1) of the Conversion of 99mTc-A4-A to
99mTc-A4-B
pH
k
4
6
8
4.91 × 10-3
10.01 × 10-3
3.03 × 10-3
10
no conversion observed
rangement around the Tc atom with the oxo group occupying
the apical position. The maximum deviation from the least-
squares plane through the four nitrogen atoms is 0.0026 and
0.0020 Å, respectively. All alanyl methyl substituents adopt
the syn configuration with respect to the oxotechnetium group.
Technetium bond lengths are given in Table 3 and are very
similar to those reported in the literature for other technetium(V)
oxo complexes.23-25
Figure 5. Proposed structure of Tc-A4-A and its conversion to Tc-
A4-B.
The conversion of 99mTc-A4-A to 99mTc-A4-B follows first-
order kinetics, and the rate is largely dependent on the pH. The
conversion proceeds with the highest rate at pH 6, whereas at
pH 10 no conversion was observed (Table 4).
99mTc-A4-B, probably due to the reactivity of the free primary
amino group toward the methyl ester (Figure 4).
Electrophoresis of 99mTc-A4-A further showed that the
complex has no charge at pH < 3, but migration distance to
the anode increases as a function of pH with a steep rise between
pH 4 and 6. This observation also confirms the assumption
that the carboxylic acid group is not involved in the complex-
ation of tetraalanine.3
As the initially formed complex (Tc-A4-A) is not stable, it
is difficult to determine its exact structure, but different
observations indicate that it is the oxotechnetium(V) complex
of tetraalanine in which the oxotechnetium group is bound to
the amine nitrogen atom and the three amide nitrogen atoms.
The yellow color of the Tc-A4-A solution is strongly
indicative for an oxotechnetium(V) core as such a yellow color
is typical for oxotechnetium(V) complexes. The migration
distances of 99mTc-MAG3 and 99mTc-A4-A in size exclusion
chromatography are identical (results not shown), which indi-
cates that the structure of 99mTc-A4-A could be a 1:1 technetium:
ligand complex, as is the case for 99mTc-MAG3.1
To confirm whether the carboxylic acid group indeed is not
involved in the complexation of technetium, we have reacted
99mTc-A4-A with diazomethane, a reagent known to convert
carboxylic acids rapidly to their methyl esters. Analysis of the
reaction mixture showed the presence of a new radiolabeled
compound (probably 99mTc-A4-A-methyl ester) with a retention
time intermediate between those of 99mTc-A4-A and 99mTc-A4-B
(Figure 4). This conversion supports the theory that the
carboxylic acid is not involved in the chelation of the peptide
as in a Tc-tetraalanine complex only the carboxylic acid group
can be expected to react with diazomethane. Attempts to
hydrolyze the supposed methyl ester in alkaline conditions to
again obtain 99mTc-A4-A always resulted in the formation of
All of the former observations are compatible only with a
structure of Tc-A4-A in which the oxotechnetium(V) core is
coordinated by three amide nitrogen atoms and the lone pair of
the amine nitrogen atom (Figure 5). In this structure, the
carboxylic acid group can be forced in the vicinity of the amine
group, facilitating amide formation and subsequent cyclization
of the tetraalanine ligand, resulting in the formation of (cy-
clotetra-L-alanyl)oxotechnetium(V) (99Tc-A4-B). To our knowl-
edge, a similar conversion of a ligand induced by complexation
of technetium has not been reported before.
(23) Faggiani, R.; Lock, C. J. L.; Epps, L. A.; Kramer, A. V.; Brune, D.
Acta Crystallogr. 1988, C44, 777.
(24) Mahmood, A.; Halpin, W. A.; Baidoo, K. E.; Sweigart, D. A.; Lever,
S. Z. Acta Crystallogr. 1991, C47, 254.
(25) Fair, C. K.; Troutner, D. E.; Schlemper, E. O.; Murman, R. K.; Hoppe,
M. L. Acta Crystallogr. 1984, C40, 1544.
Cyclization of tetrapeptides normally requires specialized
conditions that provide only low yields.26 It therefore will be
interesting to investigate whether complexation of a stable

6244 Inorganic Chemistry, Vol. 35, No. 21, 1996
Bormans et al.
transition metal (e.g., Re) with tetraalanine also induces a similar
cyclization of the tetrapeptide. On the condition that the metal
can be removed in an appropriate way, this could provide a
potential new synthesis procedure for cyclic tetrapeptides.
Acknowledgment. The authors thank Dr. Lihsueh Lee
(University of Cincinnati) for recording the Fab mass spectra.
Supporting Information Available: Tables of positional param-
eters, displacement parameters, bond distances, bond angles, and torsion
angles (17 pages). Ordering information is given on any current
masthead page.
(26) Schwyzer, R.; Iselin, B.; Rittel, W.; Sieber, P. HelV. Chim. Acta 1956,
39, 872.
(27) Hudlicky, M. J. Org. Chem. 1980, 45, 5377.
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