Radioiodine labelling of a small chemotactic peptide, utilizing two different prosthetic groups: A comparative study

Article abstract of DOI:10.1002/jlcr.1471

The use of iodobenzoates as pre-labelled prosthetic groups in the radioiodination of peptides is becoming increasingly popular. The utilization of an iodovinyl ester unit as an alternative conjugation agent for the preparation of a radioiodinated peptide

Full text of DOI:10.1002/jlcr.1471

Research Article
Received 21 August 2007,
Revised 8 October 2007,
Accepted 9 October 2007
Published online 3 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1471
Radioiodine labelling of a small chemotactic
peptide, utilizing two different prosthetic
groups: A comparative study
Ã
Daniel D. Rossouw
The use of iodobenzoates as pre-labelled prosthetic groups in the radioiodination of peptides is becoming increasingly
popular. The utilization of an iodovinyl ester unit as an alternative conjugation agent for the preparation of a
radioiodinated peptide conjugate (peptide-[123I]I-PEA) was investigated in this study. A pre-labelled unit, containing a
tetrafluorophenyl ester group, was purified on a small C18 column and the dimethylformamide eluate was used directly in
the conjugation step. Similar methodology was applied for a comparative radiosynthesis of an iodobenzoate analogue
(peptide-[123I]IB), using a succinimidyl ester. The influences of various reaction parameters on conjugation yields were
investigated while maintaining a fixed amount of peptide. At high activity levels (more than 200 MBq) the conjugation yield
of peptide-[123I]I-PEA was very sensitive to relatively large reaction volumes and increased amount of base. By optimizing
these parameters, the formation of radiochemical impurities was minimized. Under similar conditions, peptide-[123I]I-PEA
formed much faster than peptide-[123I]IB. Sep-Pak C18 purification afforded conjugates with radiochemical purities both in
excess of 98% and free from unreacted peptide. Recovered conjugation yields of peptide-[123I]I-PEA in 50% ethanol were in
excess of 60%, while those for peptide-[123I]IB were less than 40%.
Keywords: 123I; peptide; conjugates; iodovinyl
used by Hadley and Wilbur4 to prepare iodovinyl antibody
conjugates, but has, to this author’s knowledge, never been
Introduction
Radiolabelled monoclonal antibodies and peptides have be-
come useful non-invasive diagnostic tools to detect malignant
tumours and various types of infectious and inflammatory
lesions. The presence of the tyrosine amino acid unit in many of
their structures makes direct electrophilic radioiodination highly
feasible. The lack of such a unit, as well as in vivo deiodination
properties shown by directly labelled proteins, has prompted
radiochemists to make use of pre-labelled prosthetic groups,
such as the succinimidyl-iodobenzoates (SIB). The latter have
been utilized especially in the labelling of monoclonal anti-
bodies, but also recently in the labelling of a small chemotactic
peptide.1,2 The general consensus is that radioiodinated peptide
conjugates are radiochemically more stable in vivo. There is,
however, little clarity on the influence of the prosthetic groups
on the biological behaviour of the peptide. Some investigators
claimed that a 18F-labelled peptide–succinimidyl-fluorobenzoate
conjugate had a somewhat lower affinity than the parent
peptide.3 Changes observed in receptor-binding properties and
the increase in hepatobiliary excretion could be the result of the
increased lipophilicity of the labelled peptide, although
biological activity is preserved.2 The nature of the prosthetic
group might also influence the overall biological behaviour of
the conjugate.1 The most suitable prosthetic group should
therefore be selected in order to minimize alteration of the
biological behaviour of the parent peptide.
used to prepare iodovinyl peptide conjugates. The chemical and
biological stabilities of vinyl iodides are comparable to those of
aryl iodides. These features would therefore most likely not be
sacrificed. One of the iodovinyl compounds prepared by Hadley
and Wilbur4 was selected for this study. The methodology to
prepare labelled iodovinyl peptide conjugates was investigated,
and
a similar methodology was applied to prepare an
iodobenzoate peptide conjugate in parallel. The effects of
various conjugation reaction conditions on the different
conjugation yields were compared. An alternative purification
method for labelled peptide conjugates was also investigated.
The biological evaluation of the conjugates will be described in
a later study.
The small peptide model chosen for this study is
a
chemotactic peptide, N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fNleLFN-
leYK). It has been labelled in a few other studies with various
radioisotopes such as 99m-technetium,5 111-indium,6 18-fluor-
ine,3 131-iodine2 and 123-iodine.1 Radiolabelled chemotactic
peptides generally show promise as imaging agents for bacterial
infection.
Radionuclide Production Group, iThemba LABS, Faure, South Africa
*Correspondence to: Daniel D. Rossouw, Radionuclide Production Group,
iThemba LABS, Faure, South Africa.
E-mail: niel@tlabs.ac.za
The main purpose of this study was to investigate the
utilization of the iodovinyl unit in peptide labelling. It has been
J. Label Compd. Radiopharm 2008, 51 48–53
Copyright r 2008 John Wiley & Sons, Ltd.

D. D. Rossouw
columns from which the pure product was eluted with
methanol. Evaporation of the methanol solutions resulted in
up to 70% loss in activity. Dimethylformamide (DMF), also being
the solvent in the peptide conjugation step, was subsequently
selected as eluant. Despite the much less volatile nature of
Results and discussion
The reaction sequences for the radiosyntheses of the radi-
olabelled peptide conjugates are shown in Schemes 1 and
Scheme 2. The first step comprises the radiosyntheses of the
123I-labelled prosthetic groups or intermediates from their
respective precursors. Reaction conditions were not deliberately
or systematically optimized. It was found, however, that under
the stated conditions of labelling, chloramine-T (CAT) proved to
be a superior oxidant than N-bromosuccinimide (NBS) to
synthesize N-succinimidyl-4-[123I]iodobenzoate ([123I]SIB) from
its tributylstannyl precursor (N-suc-TBS-BA, 1). Amounts of
20–40 mg of precursor 1 generally resulted in good yields of
[
123I]SIB, it was isolated similarly. It was presumed that labelling
precursors could co-elute with the labelled intermediates and
could consequently compete with them for coupling to the
peptide. High-performance liquid chromatography (HPLC)
purification of pre-labelled intermediates prior to their coupling
to proteins is advisable,3 especially when using an N-halosucci-
nimide as oxidant7 or small quantities of protein (less than
100 mg).8 The volatility of TFP-[123I]I-PEA would also eliminate
the possibility of HPLC purification of the labelled intermediate,
and mini-column purification was therefore regarded as the only
option. The results presented in Table 1 suggest that first-step
labelling reactions containing up to 40 mg precursor did not
have any significant effect on subsequent conjugation yields.
Using excessive amounts, as illustrated for 100 mg TFP-TBS-PEA,
could reduce conjugation yields significantly. Precursor contents
in DMF eluates were not determined.
[
123I]SIB (50–75%). Owing to occasional erratic yields obtained
with 20 mg precursor, no experiments were carried out using less
than this amount. NBS proved to be a suitable oxidant for the
synthesis of 2,3,5,6-tetrafluorophenyl 5-[123I]iodo-4-pentenoate
(TFP-[123I]I-PEA) from 2,3,5,6-tetrafluorophenyl 5-(tri-n-butylstan-
nyl)-4-pentenoate (TFP-TBS-PEA) 2. Amounts of 5–40 mg of
precursor 2 resulted in fair yields of TFP-[123I]I-PEA (results not
presented).
The work-up of TFP-[123I]I-PEA reaction mixtures was slightly
adapted from conventional methods due to the high volatility of
the labelled product. (Hadley and Wilbur4 only referred to
volatile radioiodide.) Initially, they were purified on C18 mini-
Conjugation reactions are normally conducted in small volumes
(approximately 5 ml).2,3 A mini-column containing a small amount
of C18-packing material was therefore required, enabling a
selective and quantitative washout of free radioiodide with water
Scheme 1
Scheme 2
Table 1. Effect of mass of pre-labelled precursors on radiochemical yields (RCY) of peptide conjugates
RCYa conjugate 3
Mass precursor 1
(mg)
Mass precursor 2
(mg)
RCYb conjugate 4
(%)
(%)
20
40
64.770.6 (n 5 3)
65.074.4 (n 5 3)
5
10
40
100
83 (n 5 1)
81 (n 5 1)
82 (n 5 1)
28 (n 5 1)
aRCY as assessed by analytical HPLC, ex-overnight. For peptide-[123I]IB conjugation reactions: 40 mg peptide and 5 ml DIPEA per
experiment. Reaction volume: 280–400 ml. Activity content: 96–388 MBq.
bRCY as assessed by analytical HPLC. For peptide-[123I]I-PEA conjugation reactions: 40 mg peptide and 2 ml DIPEA per experiment.
Reaction volume: 180–250 ml. Activity content: 300–400 MBq.
J. Label Compd. Radiopharm 2008, 51 48–53
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D. D. Rossouw
and optimal recovery of the labelled intermediate in the smallest base, generate higher amounts of the more polar impurity
possible volume of DMF. A mini-column containing 100 mg C18 (entries 8, 10 and 11). This becomes more pronounced at higher
was selected for this purpose. Elution of the main fraction was activity levels (entry 12). At higher activity levels, the optimal
preceded by a pre-elution with 130 or 150 ml DMF in order to formation of 4 is favoured by reaction volumes of 250 ml or less
displace water. A minimum volume of 180–200ml DMF was and a DIPEA content of 2 ml (entries 4, 5 and 6). Less than 2 ml
required for optimal elution of the labelled intermediate. The DIPEA could result in a too slow reaction rate, as suggested by
average radiochemical recovery yields of [123I]SIB and TFP-[123I]I- entry 2. The nature of the formed impurities is debatable.
PEA were quite similar (63 and 65%, respectively). On average, 10
The reaction parameters that dictate peptide-[123I]I-PEA
and 20% free radioiodide was eluted from mini-columns during conjugation yields do not have a similar strong impact on
purification of TFP-[123I]I-PEA and [123I]SIB, respectively, suggesting peptide-[123I]IB 3 yields (Table 3). The [123I]SIB-containing DMF
that more of the former was formed during labelling. Its lesser eluates were dried with anhydrous MgSO4 prior to peptide
recovery was due to its higher lipophilicity, as also indicated by its conjugation as inconsistent conjugation yields were obtained,
stronger retention by the C18.
under similar reaction conditions, using undried eluates. This
Conjugation reaction conditions were optimized by using a suggests that the reaction is sensitive to the presence of
fixed, relatively small amount of peptide throughout and moisture (results not presented). Peptide-[123I]I-PEA 4 conjuga-
varying other reaction parameters. The rationale behind this tion yields are unaffected by the drying of eluates (results not
was that peptides are often very expensive and/or difficult to presented). Peptide-[123I]IB conjugation reactions proceeded
remove from reaction mixtures, and that the use of excessive slower than peptide-[123I]I-PEA reactions under similar condi-
amounts (more than 50 mg) should be avoided. The authenti- tions. Traces of unreacted [123I]SIB were still present after 1 h,
cities of radiolabelled peptide conjugates were confirmed by whereas TFP-[123I]I-PEA was completely consumed within
comparing their HPLC retention times with those of authentic 30 min. The reason for this is not entirely clear, given the higher
compounds, using UV detection. The effects of the reaction reactivity of N-hydroxysuccinimide esters.4 (The TFP ester
parameters on the formation of various radiochemical species in analogue of N-suc-TBS-BA was also synthesized, but this
peptide-[123I]I-PEA 4 conjugation reaction mixtures are pre- precursor yielded very poor radioiodination and subsequent
sented in Table 2. Other than 4, two unidentified, relatively more peptide conjugation yields.) Unlike peptide-[123I]I-PEA conjuga-
polar impurities, A and B, are formed, neither of the latter being tion reactions, varying amounts of free radioiodine were also
the hydrolysed counterpart of TFP-[123I]I-PEA. The size of the formed during the course of peptide-[123I]IB reactions, together
activity incorporated into conjugation reaction mixtures, as well with p-[123I]iodobenzoic acid and another unknown impurity.
as the reaction volume, which determines the peptide Extended reaction times led to increased formation of peptide-
concentration, both have an impact on the formation of these
[
123I]IB with simultaneous consumption of p-[123I]iodobenzoic
species. At relatively low activity levels (74–137 MBq), less acid. This suggests that the latter is sufficiently reactive for
impurities are formed (entries 1, 3 and 9). At activity levels in conjugation purposes, albeit very slow. Extended reaction
excess of 200 MBq and volumes larger than 250 ml, higher periods for peptide-[123I]I-PEA reactions did not result in the
amounts of especially the more polar impurity, A, are formed formation of significantly more product.
(entries 2, 7 and 8). Under these conditions increased amounts Conjugation reaction mixtures were purified on a Sep-Pak
of N,N-diisopropylethylamine (DIPEA), used as the conjugation mini-column. The radiochemical yields of the conjugates are
Table 2. Effects of various reaction parameters on the formation of various radiochemical species in peptide-[123I]I-PEA
conjugation reaction mixtures
Radiochemical species in reaction mixturea
(%)
Activity
(MBq)
Reaction volume
DIPEA
Entry no.
(ml)
(ml)
A
B
C
1
2
3
4
5
6
7
8
9
111
296
137
296
300
333
322
226
74
300
300
200
200
250
180
290
300
250
300
250
250
1
1
2
2
2
2
2
3
5
5
5
5
4
24
6
9
1
5
3
5
6
6
5
4
7
6
3
86
66b
86
81
83
80
64
59
86
52
62
41
15
11
12
28
34
7
37
30
53
10
11
12
226
259
474
40 mg peptide per experiment. Reaction times: 25–50 min. A, unknown radiochemical impurity, Rt 6 min; B, unknown
radiochemical impurity, Rt 7.5 min; C, peptide-[123I]I-PEA 4, Rt 12.7 min.
aAs assessed by analytical HPLC, balances made up by small amounts of unknown radiochemical impurities.
bContains 9% unreacted TFP-[123I]I-PEA.
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D. D. Rossouw
Table 3.
conjugation reaction mixtures
Effects of various reaction parameters on the formation of various radiochemical species in peptide-[123I]IB
Radiochemical species in reaction
mixtureb (%)
Entry no. Activity (MBq) Reaction volumea (ml) DIPEA (ml) Reaction time (min)
A
B
C
D
E
1
293
107
130
250
389
238
240
270
390
295
370
300
2
30
60
12
13
15
2
3
3
2
3
4
6
6
9
8
9
11
4
4
8
7
9
40
27
7
33
20
6
17
18
6
4
8
13
2
1
21
7
1
27
9
0
13
2
1
14
4
0
2
1
47
58
66
24
48
74
25
46
65
44
59
70
29
50
64
45
64
67
Overnight
25
2
5
55
150
25
10
6
3
5
55
Overnight
25
11
15
17
20
6
6
9
7
4
5
6
5
14
7
60
Overnight
25
55
Overnight
25
70
10
33
18
11
27
8
5
10
15
20
21
Overnight
4
4
0
40 mg peptide per experiment. A, free radioiodine, Rt 3.1 min; B, p-[123I]iodobenzoic acid, Rt 8.9 min; C, unknown impurity, Rt
10.7 min; D, unreacted [123I]SIB, Rt 13.7 min; E, Peptide-[123I]IB 3, Rt 18.8 min.
aMgSO4-dried DMF eluates.
bAs assessed by analytical HPLC, balances made up by varying small amounts of other unknown radiochemical impurities.
Table 4. Radiochemical yields (RCY) of peptide conjugates after Sep-Pak C18 purification
RCYb
Final HPLC yielda
50% EtOH eluant
(ml)
Remaining activity on C18c
Combined RCYbc
(%)
Conjugate
(%)
(%)
(%)
Peptide-[123I]IB 3
48
62
59
80
80
76
75
5
2.5
5
2.5
2.5
2.2
3.0
36
39
35
63
68
64
62
13
12
22
2
4
4
49
51
57
65
72
68
64
Peptide-[123I]I-PEA 4
2
aAs assessed by analytical HPLC.
bActivity in 50% EtOH, expressed as a percentage of starting activity in conjugate reaction mixture.
cExpressed as a percentage of starting activity in conjugate reaction mixture.
presented in Table 4. The more polar radiochemical impurities compound 4 was still generally higher than that of 3. This can
as well as unreacted peptide were quantitatively and selectively be ascribed to the lesser amounts of radiochemical impurities
eluted with mixtures of 0.1% trifluoroacetic acid (TFA) and formed under optimal conditions during the peptide-[123I]I-PEA
acetonitrile (ACN). The conjugation products were eluted with reaction. The radiochemical purities of all the conjugates after
50% aqueous ethanol, thereby making it possible to fairly Sep-Pak purification were in excess of 98%.
quickly convert the eluate into a radiopharmaceutical product.
An added advantage of using this eluant was that lipophilic
precursors, if still present, would not co-elute. UV-HPLC analyses
Experimental
of these eluates revealed the absence of any traces of precursor
or unreacted peptide. The more lipophilic nature of peptide-
The chemotactic peptide was obtained from Sigma and
GenScript Corporation (USA). All other reagents and chemicals
were obtained from either Aldrich or Fluka. Analytical HPLC was
performed on the following system: HPLC pump (Agilent 1100
Series, equipped with a HP 1100 Series Control Module for
[
123I]IB 3 resulted in a higher proportion of activity retained by
C18 than in the case of peptide-[123I]I-PEA 4. The sum totals of
activity, however, show that the overall radiochemical yield of
J. Label Compd. Radiopharm 2008, 51 48–53
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D. D. Rossouw
binary gradient elution), analytical C18 column (Phenomenex (18.5 min). The product peak fraction was collected during two
Luna, 250 Â 4.6 mm, 5 mm), injector (Rheodyne Model 7725), UV separate runs (2 Â 20 ml) and concentrated at 451C under
detector (Spectra Series UV100, set at 254 nm), radiation reduced pressure to approximately half of the original volume.
detector (Carroll&Ramsey Model 105S-1 with a CsI(Tl) detector The concentrate was loaded on to a Sep-Pak C18 mini-column
probe). Chromatograms were recorded on a Chromatopac C- (500 mg C18), the column was washed with water (4–5 ml) and
R8A from Shimadzu. HPLC-grade acetonitrile was used. Binary the peptide-IB was eluted with methanol (0.8 ml). The structure
gradient elution was carried out for the analyses of all reaction was confirmed by mass spectral analysis. MS, m/z 1054.4 (M1H),
mixtures and purified reaction products, using mixtures of 0.1% 1076.4 (M1Na).
TFA and ACN as mobile phase. Elution was carried out at 1 ml/
fNleLFNleYK-5-iodo-4-pentenoate (peptide-I-PEA), 4: This refer-
min, starting with 50% ACN over 15 min, followed by a 50–90% ence compound was synthesized from fNleLFNleYK and 2,3,5,6-
linear gradient of ACN over a period of 5 min and finally a tetrafluorophenyl 5-iodo-4-pentenoate (TFP-I-PEA) [prepared
90–100% gradient of ACN over 5 min. Radioactivity measure- from TFP-TBS-PEA 2 according to the method of Hadley and
ments were carried out in a Vinten Isocal II Radionuclide Assay Wilbur4]. In short, a solution of the peptide in DMF (10 mM, 12 ml,
Calibrator. Electrospray ionization mass spectrometry was 0.12 mmol) was mixed with a solution of TFP-I-PEA in DMF
performed on a Waters API Q-TOF Ultima instrument. Strata (53.5 mM, 2.0 ml, 0.11 mmol) and a solution of DIPEA (0.4 ml) in
C18 and Sep-Pak C18 mini-columns were obtained from DMF (1 ml) was added. The mixture was stirred at room
Phenomenex and Waters, respectively.
temperature for approximately 2.5 h, after which it was diluted
N-succinimidyl 4-tri-n-butylstannylbenzoate (N-suc-TBS-BA), 1: with 10 ml methanol. The product 4 was isolated by HPLC, using
This precursor was synthesized analogously to the method of isocratic elution with 0.1% TFA/ACN (50:50) at 1 ml/min. The
Wilbur et al.,8 except for using 4-bromobenzoic acid instead of retention time of 4 was in the region of 12.1–12.5 min. Isolation
the corresponding methyl ester as the starting material for the of the product from the HPLC eluate was carried out as
metal–halogen exchange reaction. Reaction intermediates and described for peptide-IB 3. The structure for 4 was confirmed by
final product were purified by means of silica gel chromato- mass spectral analysis. MS, m/z 1032.4 (M1H), 1054.4 (M1Na).
graphy as described.
2,3,5,6-Tetrafluorophenyl 5-(tri-n-butylstannyl)-4-pentenoate (TFP-
TBS-PEA), 2: This precursor was synthesized analogously to the
Radiochemical syntheses
No-carrier-added [123I]iodine was produced by means of the
127I(p,5n)123Xe-123I route, using a 66 MeV proton beam, and
recovered in 0.01 M NaOH solutions. These solutions were
reduced in volume by a factor of 3–5 by means of evaporation,
method of Hadley and Wilbur,4 with minor modifications. Methyl
4-pentynoate was synthesized by means of esterification of 4-
pentynoic acid in refluxing methanol containing a catalytic
amount of concentrated sulphuric acid. In short, a solution of 4-
pentynoic acid (510 mg, 5.2 mmol) in methanol (2 ml) containing
concentrated H2SO4 (35 ml) was refluxed for about 6 h. Diethyl
ether (8 ml) was added and further work- up was conducted
according to the described method. Methyl 5-(tri-n-butylstannyl)-
4-pentenoate was also synthesized according to the described
method, but the purification step was slightly modified by diluting
the final reaction mixture with approximately 2.3 volumes of
hexane prior to silica gel chromatography instead of evaporating
the solvent. Hydrolysis of the methyl ester was conducted with
NaOH instead of KOH, and the tetrafluorophenyl ester was
synthesized as described. Purification of the ester was started by
diluting the filtered reaction mixture with 2.3 volumes of hexane,
followed by silica gel chromatography as described. This precursor
was sometimes also further purified by means of HPLC, using the
C18 column and 100% acetonitrile as eluant at a flow rate of 1 ml/
min. The precursor appeared as a double peak (cis/trans mixture)
with a retention time of 20–21 min. The fraction containing the
peak was collected and evaporated to dryness under a flow of
nitrogen.
fNleLFNleYK-p-iodobenzoate (peptide-IB), 3: This reference
compound was synthesized from fNleLFNleYK and N-succinimi-
dyl-4-iodobenzoate (SIB) [prepared from 4-iodobenzoic acid
according to the method of Garg et al.9]. In short, a solution of
the peptide in DMF (10 mM, 20 ml, 0.2 mmol) was mixed with a
solution of SIB in DMF (145 mM, 1.4 ml, 0.2 mmol) and a solution
of DIPEA (0.5 ml) in DMF (1 ml) was added. The mixture was stirred
at room temperature for approximately 10 h, after which it was
diluted with 16 ml methanol. The product peptide-IB was
isolated by HPLC, using the elution conditions as described
earlier. Retention times of the various components under these
conditions were as follows: 4-iodobenzoic acid (8.3 min),
unknown impurity (9.8 min), SIB (12.7 min), peptide-IB
therefore effectively rendering
0.03–0.05 M.
a NaOH concentration of
Peptide-[123I]I-PEA, 4: The radiosynthesis of the pre-labelled
intermediate, TFP-[123I]I-PEA, was based on a literature method,4
with few modifications. In short, to a solution of precursor 2
(5–40 mg) in 10% acetic acid/methanol (125–300 ml) was added a
solution of 123I in 0.03–0.05 M NaOH (50–80 ml, up to 600 MBq). A
solution of NBS (30 mg) in methanol (30 ml) was added and the
mixture was stirred at room temperature. After 10 min the
reaction was quenched with a solution of Na2S2O5 in water
(0.72 mg/ml, 24 ml). The reaction mixture was diluted with water
(0.5–1.0 ml, depending on the volume of the reaction mixture),
and loaded on a pre-conditioned C18 mini-column (Strata C18-E,
100 mg, 55 mm, 1 ml). The C18 was washed with water (6 ml) and
subsequently eluted with DMF (150 ml), followed by another
portion of DMF (180–300 ml). The second DMF eluate, containing
TFP-[123I]I-PEA, was mixed with a solution of the peptide in DMF
(10 mM, 5 ml), DIPEA (1–5 ml) was added and the mixture stirred
at room temperature. After complete consumption of TFP-[123I]I-
PEA, the reaction mixture was purified on a pre-conditioned
Sep-Pak C18 (500 mg) column after dilution with water (2 ml).
Free radioiodide as well as the more polar radiochemical
impurities were eluted with 0.1% TFA/acetonitrile 5 70:30 (v/v)
(14 ml). Subsequent elution with water (8 ml) and water/ethanol
(50:50) (v/v) (1.5 ml) was followed by a final elution of 4 with
water/ethanol (50:50) (v/v) (2.2–3 ml).
Peptide-[123I]IB, 3: The radiosynthesis of the pre-labelled
intermediate, [123I]SIB was essentially similar to that of TFP-
[
123I]I-PEA, but CAT was used as an oxidant instead of NBS. In
short, to a solution of precursor 1 (20 or 40 mg) in 10 or 30%
acetic acid/methanol (100–125 ml) was added a solution of 123I in
0.03–0.05 M NaOH (50–60 ml, up to 600 MBq). A solution of CAT
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D. D. Rossouw
(96 mg) in methanol (96 ml) was added and the mixture was iodopentenoate conjugation reaction proceeds faster than its
stirred at room temperature. After 10 min the reaction was iodobenzoate counterpart. Under optimal conjugation reaction
quenched with a solution of Na2S2O5 in water (0.72 mg/ml, conditions radiochemical yields of the peptide–iodopentenoate
48 ml). The reaction mixture was diluted with water (0.5) and conjugate were also higher than those of its iodobenzoate
loaded on a pre-conditioned C18 Strata C18-E. The C18 was analogue. These features could make a radiolabelled iodovinyl
washed with water (6 ml) and subsequently eluted with DMF intermediate an attractive alternative for the preparation of a
(130 ml), followed by another portion of DMF (200–300 ml). The radiolabelled peptide conjugate, provided that its biological
second DMF eluate, containing [123I]SIB, was filtered through a properties remains intact.
small column containing anhydrous MgSO4 (50 mg, pre-mois-
tened with DMF). The column was washed with small amounts Acknowledgement
of DMF (40–80 ml). The combined dried eluate was mixed with a
solution of the peptide in DMF (10 mM, 5 ml), DIPEA (2–10 ml) was
added and the mixture stirred at room temperature. Reaction
mixtures were purified on Sep-Pak C18 as described before,
using the following eluants: 0.1% TFA/acetonitrile 5 70:30 (v/v)
(6 ml); 0.1% TFA/acetonitrile 5 60:40 (v/v) (5 ml). Subsequent
elution with water (8 ml) and water/ethanol (50:50) (v/v) (2 ml)
was followed by a final elution of 3 with water/ethanol (50:50)
(v/v) (2.5–5 ml).
The author wishes to thank Ms G. Visser for preparing some of
the cold precursors used in this work.
References
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