Direct solid-phase synthesis of octreotide conjugates: Precursors for use as tumor-targeted radiopharmaceuticals

Article abstract of DOI:10.1016/S0968-0896(99)00125-X

Somatostatin analogues, such as octreotide, are useful for the visualization and treatment of tumors. Unfortunately, these compounds were produced synthetically using complex and inefficient procedures. Here, we describe a novel approach for the synthesis of octreotide and its analogues using p-carboxybenzaldehyde to anchor Fmoc-threoninol to solid phase resins. The reaction of the two hydroxyl groups of Fmoc-threoninol with p-carboxybenzaldehyde was catalyzed with p-toluenesulphonic acid in chloroform using a Dean-Stark apparatus to form Fmoc-threoninol p-carboxybenzacetal in 91% yield. The Fmoc-threoninol p-carboxybenzacetal acted as an Fmoc-amino acid derivative and the carboxyl group of Fmoc-threoninol p-carboxybenzacetal was coupled to an amine-resin via a DCC coupling reaction. The synthesis of protected octreotide and its conjugates were carried out in their entirety using a conventional Fmoc protocol and an autosynthesizer. The acetal was stable during the stepwise elongation of each Fmoc-amino acid as shown by the averaged coupling yield (>95%). Octreotide (74 to 78% yield) and five conjugated derivatives were synthesized with high yields using this procedure, including three radiotherapy octreotides (62 to 75% yield) and two cellular markers (72 to 76% yield). This novel approach provides a strategy for the rapid and efficient large-scale synthesis of octreotide and its analogues for radiopharmaceutical and tagged conjugates.

Full text of DOI:10.1016/S0968-0896(99)00125-X

Bioorganic & Medicinal Chemistry 7 (1999) 1797±1803
Direct Solid-Phase Synthesis of Octreotide Conjugates:
Precursors for Use as Tumor-Targeted Radiopharmaceuticals
Hsing-Pang Hsieh, a,y Ying-Ta Wu, a,y Shui-Tein Chen a,*
and Kung-Tsung Wang a,b
aInstitute of Biological Chemistry, Academia Sinica, Taipei, 128, Yan-Chiu-Yuan Road, Sec II, Nankang, 115, Taiwan
bDepartment of Chemistry, National Taiwan University, Taipei, 100, Taiwan
Received 14 August 1998; accepted 25 February 1999
AbstractÐSomatostatin analogues, such as octreotide, are useful for the visualization and treatment of tumors. Unfortunately,
these compounds were produced synthetically using complex and inecient procedures. Here, we describe a novel approach for the
synthesis of octreotide and its analogues using p-carboxybenzaldehyde to anchor Fmoc-threoninol to solid phase resins. The reac-
tion of the two hydroxyl groups of Fmoc-threoninol with p-carboxybenzaldehyde was catalyzed with p-toluenesulphonic acid in
chloroform using a Dean±Stark apparatus to form Fmoc-threoninol p-carboxybenzacetal in 91% yield. The Fmoc-threoninol p-
carboxybenzacetal acted as an Fmoc-amino acid derivative and the carboxyl group of Fmoc-threoninol p-carboxybenzacetal was
coupled to an amine-resin via a DCC coupling reaction. The synthesis of protected octreotide and its conjugates were carried out in
their entirety using a conventional Fmoc protocol and an autosynthesizer. The acetal was stable during the stepwise elongation of
each Fmoc-amino acid as shown by the averaged coupling yield (>95%). Octreotide (74 to 78% yield) and ®ve conjugated deri-
vatives were synthesized with high yields using this procedure, including three radiotherapy octreotides (62 to 75% yield) and two
cellular markers (72 to 76% yield). This novel approach provides a strategy for the rapid and ecient large-scale synthesis of
octreotide and its analogues for radiopharmaceutical and tagged conjugates. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
labeled with iodine-123 ([123I]-Tyr3-octreotide) and
111In-labeled derivatives of octreotide ([111In]-diethyle-
netriaminepenta acetic acid [DTPA]-d-Phe1-octreotide)
have been developed as e€ective radiolabels for the
visualization of neuroendocrine tumors in humans using
single photon emission tomography (SPET).14±20 In
addition, FDA approval of the use of [111In]-DTPA-
octreotide has accelerated the development of somatos-
tatin-derived radiopharmaceuticals.21±32
Receptors for somatostatin, a cyclic tetradecapeptide
originally isolated from bovine hypothalamus,1±3 are
found to be expressed in approximately 90% of carci-
noid tumors.4±7 Consequently, somatostatin receptor
scintigraphy and radiotherapy have been applied to the
clinical visualization and treatment of tumors, particu-
larly for nonoperable acromegaly8 or gastroentero pan-
creatic tumors.9 The use of somatostatin itself for
medical treatment is limited, however, due to its short
half-life in vivo (2 to 3 min).10,11 This has led to the
development of several conformation-restricted cyclic
peptide analogues. For example, octreotide 1, consisting
of d-amino acids (d-Phe1, d-Trp4), amino alcohol
(threoninol), and a disul®de bond (Cys2-S-S-Cys7),
exhibits a prolonged half-life in vivo (60 to 90 min in
human)12 and is functionally 2000 times more e€ective
than somatostatin.13 Furthermore, [Tyr3]-octreotide
Large-scale synthesis of these analogues has been di-
cult because the existing methods of synthesis are ine-
cient and complex. The ®rst reported synthesis of
DTPA-d-Phe1-octreotide
2 by solid-phase peptide
synthesis33±35 (SPPS) had an overall yield of only 5%
based on the starting Cys(Acm)-resin.36 Field et al.36
concluded that the low yield was resulted from (1) the
inevitable undesired peptide bond formation between
octreotide and the two anhydride groups of DTPA and
(2) the poor solubility of DTPA in coupling solvents
such as DMF or N-methylpyrolindone (NMP). They
also isolated side chain protected [d-Trp(Boc),4 Lys(-
Boc),5 Thr(But)6]-octreotide with a 14% yield. This
indicated that the harsh cleavage conditions and poor
rate of peptide cleavage by aminolysis with threoninol
Key words: Octreotide; conjugates; peptide alcohol; radiotherapy.
* Corresponding author. Tel.: +886-2-2788-6230/+886-2-2785-5996
yext. 7071; fax: +886-2-2788-3473; e-mail: bcchen@gate.sinica.edu.tw
Hsing-Pang Hsieh and Ying-Ta Wu contributed equally to the
paper.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00125-X

1798
H.-P. Hsieh et al. / Bioorg. Med. Chem. 7 (1999) 1797±1803
were responsible for the low total yield. Recently,
Arano et al.37,38 and Schmidt et al.39 utilized modi®ed
resins, such as 2-chlorotrityl chloride resin and Rink
acid resin, to load Fmoc-Thr(But)-ol as the ®rst amino
acid residue to synthesize 2. The total yield of 2 reported
by Arano et al.37,38 was still relatively low (31.8% based
on the starting Fmoc-Thr(But)-ol-resin), while Schmidt
et al.39 did not mention the yield. Deferoxamine-con-
jugated octreotide (DFO-octreotide 4) has also been
synthesized with a relatively low yield, using a relatively
complex procedure.27,28
to form Fmoc-threoninol p-carboxybenzacetal. The
acetal derivative was used as an Fmoc-amino acid deri-
vative in SPPS. The feasibility of this reaction was
examined in the synthesis of 1 and ®ve conjugated
octreotides (2, 3, 4, 5, and 6). Scheme 1 shows the
synthesis of Fmoc-threoninol p-carboxybenzacetal B.
Fmoc-threonine was reduced with BH3/THF to produce
Fmoc-threoninol A with an 85% yield. B was prepared
with 91% yield from A and p-carboxybenzaldehyde (2.5
equiv amounts) in chloroform catalyzed by p-toluene-
sulfonic acid.
We have developed a method to facile synthesis of
octreotide.40,41 In this report we describe a novel proce-
dure for the rapid and the ecient synthesis of octreo-
tide conjugated derivatives: DTPA-octreotide 2,
1,4,7,10-tetraazacyclododecane-N,N0,N00,N000-tetraacetic
acid (DOTA)-octreotide 3, DFO-octreotide 4, and two
cell-visualization octreotide derivatives, biotin-octreo-
tide 5 and ¯uorescein-octreotide 6.42±50 The process uti-
lizes the Fmoc-chemistry of SPPS to rapidly synthesize
octreotide and its derivatives with high yields and purity.
Octreotide 1
The carboxyl group of B as the ®rst amino acid deriva-
tives was anchored onto the solid-phase resin using
DCC/HoBt in NMP. The peptide bond elongation was
carried out using an autosynthesizer with a standard
FastMoc protocol, as depicted in Scheme 2. After the
peptide bond formation was completed, the weight of
the elongated peptide on the resin was used to calculate
the average yields at each coupling step (Table 1). The
average coupling was higher than 95%. This was con-
sistent with the recorded coupling yield at each step on
the autosynthesizer. The results of the coupling yields
also indicated that the acetal linker was stable in the
stepwise elongation of each corresponding Fmoc-amino
acid. The cleavage of the side chain protecting groups of
Cys(Trt), Lys(Boc), d-Trp(Boc), Thr(But) and of the
elongated peptide from the resin was performed simul-
taneously using the TFA (tri¯uoroacetic acid) method.
After evaporation of the TFA solution under vacuum,
the resulting residue was washed with ice-cooled dry
ether and the reduced 1 was extracted with acetic acid
solution (20%). Figure 1 shows the chromatogram of
the reduced 1 directly after extraction with acetic acid
solution. The major peak with retention time of 30.1 min
was collected and identi®ed (refer to Experimental).
Results
In designing a simple method of synthesis of octreotide
and its conjugates, we considered the following: (i)
direct linkage between the Fmoc-threoninol and the
solid phase resin, (ii) the use of a dilute solution during
oxidation of the disul®de bond to prevent inter-
molecular cross linkage, and (iii) preparation of the
conjugated compounds to have a single free carboxyl
group that can directly couple to the octreotide in
advance.
Fmoc-threoninol p-carboxybenzacetal
It has been known that acetal51 resin can be pre-
pared by reacting benzaldehyde resin52 with glycerol
and can be used to generate a C-terminal diol deriva-
tive. In the synthesis of octreotide, our approach was to
utilize p-carboxybenzaldehyde to anchor Fmoc-threoni-
nol to solid-phase resins. The 1,3-dihydroxyl groups of
Fmoc-threoninol reacted with p-carboxybenzaldehyde
The formation of the disul®de bond was performed
using a dilute solution of octreotide (1 mM) at 4ꢀC, pH
7, in 5% ammonium acetate. The folding solution was
prepared by dilution of the extraction of acetic acid
solution (20%) to 1 mM octreotide followed by adjusting
the pH to 7 with ammonium hydroxide (25%). Progress
Scheme 1. The synthesis of Fmoc-threoninol p-carboxybenzacetal B.

H.-P. Hsieh et al. / Bioorg. Med. Chem. 7 (1999) 1797±1803
1799
Scheme 2. Synthetic procedure for octreotide and its derivatives.
Table 1. Data and yields of octreotide and conjugated derivatives
No.
Peptide
Calcd MW
FAB (M+H)+m/z
Peptide-resina (mg)
Average coupling b (%)
Isolated yieldsc (%)
1
2
3
4
5
6
Octreotide
1018
1393
1404
1756
1244
1322
1019.5
1394.6
1405.6
1756.5
1245.4
1323.4
899
394
393
1002
238
485
97
96
94
85
96
96
76
75
71
62
76
72
DTPA-octreotide
DOTA-octreotide
DFO-octreotide
Biotin-octreotide
Fluore-octreotide
a
b
c
Synthesis scale is (1) 0.24 mmol, (2) 0.1 mmol, (3) 0.1 mmol, (4) 025 mmol, (5) 0.062 mmol, and (6) 0.125 mmol, respectively.
Calculated from the increase in weight of the elongated petide-resin divided by the weight of protected peptide.
Obtained after RP-HPLC puri®cation using a Vydac C18 preparative column. Experimental procedures and conditions in text. Amino acid
analysis of octreotide after hydrolysis with 6 N HCl:TFA (4:1, v/v) for 3 h at 130ꢀC was listed as: amino acid/found/calcd=Lys/1.0/1; 1/2Cys/0.97/1;
Phe/1.94/2; Thr/0.65/1.
of the disul®de bond formation was monitored by high-
performance liquid chromotography (HPLC). Figure 2
shows the time course for folding of 1. The reduced
form of 1 had a retention time of 8.6 min and the oxi-
dized form of 1 had a retention time of 8.0 min. The
relative concentration of reduced 1 decreased gradually
with a corresponding increase in oxidized 1. Conversion
was complete after 48 h. Pure octreotide was isolated
using a preparative column. The fractions correspond-
ing to the desired peaks were collected and lyophilized.
The weight of 1 at this stage was 193 mg (approximately
76% yield, based on a 0.25 mmol scale). The peptide
was further characterized using amino acid composition
analysis and Fab-Mass (Table 1).
Conjugated derivatives
Direct coupling of the conjugated compounds on the
autosynthesizer was an e€ective procedure. Both ¯uor-
escein and biotin have a carboxyl group that can be

1800
H.-P. Hsieh et al. / Bioorg. Med. Chem. 7 (1999) 1797±1803
Figure 1. RP-HPLC analysis of reduced octreotide. Sample was taken
from the extraction (20% acetic acid solution). The retention time was
30.1 as monitored at UV 214 nm. The elution: A=5% acetonitrile
aqueous containing 0.1% TFA; B=90% acetonitrile aqueous con-
taining 0.1% TFA. See Experimental for HPLC conditions.
Scheme 3. The synthesis of N4-carboxypropionyl DFO-octreotide 4.
2±6 with yields of 65 to 72%. All analogues 2±6 were
characterized by amino acid composition and Fab-Mass
for con®rmation (Table 1).
Discussion
There is a growing interest in the use of octreotide con-
jugates as precursors in tumor-targeted radio-
pharmaceuticals. The present study demonstrates the
feasibility of the use of p-carboxybenzaldehyde to
anchor Fmoc-threoninol to solid-phase amine resin.
This linker is compatible with the Fmoc peptide synth-
esis protocol and can be easily applied to larger scale
preparation of octreotide derivatives. The ®rst residue B
can be easily and cost-e€ectively prepared from Fmoc-
threonine prepared via the direct reaction of threonine
with Fmoc-OSu.
Figure 2. The time course for folding of reduced octreotide 1. The
progress was followed by RP-HPLC monitored at 280 nm. The
reduced form had a retention time of 8.6 min and the oxidized form
had a retention time of 8.0 min. The elution: A=5% acetonitrile
aqueous containing 0.1% TFA; B=90% acetonitrile aqueous con-
taining 0.1% TFA. See Experimental for HPLC conditions.
Disul®de bond formation of the octreotide derivatives in
a dilute solution was e€ective and avoided intermolecular
disul®de bond cross linkage. Most importantly, DTPA,
DOTA, DFO, biotin, and ¯uorescein can be directly
incorporated with fully protected octreotide on the resin
without the necessity for protecting the e-amino group
of Lys5-octreotide as reported previously,36 thus avoid-
ing a more complex procedure. The method presented
in the present study provides a simpli®ed synthesis and
results in dramatically improved yields of conjugated
octreotide analogues. These results may aid in the
development of these compounds for use in the diag-
nosis and therapy of cancer.
coupled to 1 without modi®cation. The DTPA deriva-
tive (monoreactive DTPA derivative) was obtained with
a 24% total yield starting from diethylenetriamine
according to the procedures of Arano et al.38 The
DOTA derivative was prepared as previously descri-
bed53,54 by dissolving three equivalents of tetrabutyl-
ammonium hydroxide with DOTA in water and lyo-
philizing to yield the salt of the partially protected
DOTA. The ¯uorescein, biotin, DTPA and DOTA
derivatives were used as the ninth amino acid deriva-
tives and were successfully coupled to octreotide using
the autosynthesizer (Scheme 2). DFO has no carboxyl
group to be linked with 1. Scheme 3 shows the procedure
for synthesis of N4-carboxypropionyl DFO-octreotide
4. Protected octreotide was ®rst incubated with succinic
anhydride to produce 4-carboxypropionyl octreotide on
the autosynthesizer, followed by coupling DFO to 4-
carboxypropionyl octreotide using the DCC method.
Experimental
Fmoc protected amino acids were purchased from either
Bachem or Novabiochem (Germany). Rink Amide AM
resin (loading 0.56 mmol/g) was obtained from Nova-
biochem (Germany). Tri¯uoroacetic acid (TFA), tetra-
butyl ammonium hydroxide (20% solution in water),
and DOTA were from Merck Chemical Co. (Germany).
The same procedures for cleavage and disul®de formation
described above were used to complete the synthesis of

H.-P. Hsieh et al. / Bioorg. Med. Chem. 7 (1999) 1797±1803
1801
1 M BH3/THF, toluenesulfonic acid, diethylenetriamine-
pentaacetatic acid and p-carboxybenzaldehyde were
purchased from Aldrich Chemical Corporation (USA).
Solvents were obtained from ALPS Chemical Co.
(Taiwan).
of tri(tetrabutyl ammonium hydroxide) salt of DOTA.
The salt was used directly without further puri®cation.
Preparation of monoreactive DTPA derivative (D). The
monoreactive DTPA derivative was prepared as the
procedure reported by Arano et al.38 Using diethylene-
triamine as a starting material and following six reac-
tion steps to provide expected monoreactive DTPA
derivative as a yellowish oil in 24% total yield. FABMS
m/z 618 (M+1); 1H NMR (CDCl3) d 5.17 (1H, br,
COOH), 3.51 (2H, s, NCH2COOH), 3.50 (2H, s, NCH2
COO-But), 3.48 (2H, s, NH2COO-But), 3.45 (4H, s,
NH2COO-But), 2.83±3.01 (8H, m, NCH2CH2NCH2
CH2N), 1.46±1.48 (36H, s, C(CH3)3).
Synthesis of N-Fmoc-threoninol (A). To a solution of N-
Fmoc-l-threonine (N-Fmoc-l-Thr, 1.7 g, 5 mmol) dis-
solved in anhydrous THF (50 mL) was added dropwise
of 1 M BH3/THF (50 mL) through an additional fun-
nel over a period of 10 min. The resulting mixture was
then stirred at room temperature for 8 h and quenched
by the addition of 1 N HCl (50 mL). After stirring at
room temperature for 1 h, the mixture was diluted with
water (40 mL) and followed by the addition of a mix-
ture of THF:diethyl ether (1:1; 100 mL). The organic
layer was separated and the aqueous layer was extracted
with THF:ether (1:1; 2Â60 mL). The combined organic
layers were then washed with brine (2Â125 mL), 1.5 N
KOH (2Â125 mL), and brine (2Â125 mL), dried with
Na2SO4, ®ltered, concentrated under vacuum, and pur-
i®ed by ¯ash chromatography (ethyl acetate:n-hexane,
4:1) to a€ord white solid of N-Fmoc-l-threoninol (1.3 g,
80%; Rf 0.34 in ethyl acetate:n-hexane, 4:1). Mp 100±
Solid-phase peptide synthesis of the peptides. Octreotide
analogues were synthesized using FastMoc chemistry on
Applied Biosystems (ABI) 433A peptide synthesizer.
Fmoc protected amino acids were activated in situ in
AA cartridges with HoBt/HBTU/DIEA in NMP. Four
equivalent amounts of each amino acid derivative were
used. Coupling yield of each amino acid coupling cycle
was monitored by equipped conductivity sensor. The
average coupling yield was also calculated from the
increase in weight of the elongated peptide-resin divided
by the weight of protected peptide.
102ꢀC; FABMS: m/z 328 (M+1), 179, 178. H NMR
1
(CDCl3) d 7.73 (2H, d, 7.5 Hz), 7.57 (2H, d, 7.4 Hz),
7.37 (2H, t, 7.4 Hz), 7.28 (2H, t, 7.4 Hz), 5.58 (1H, d,
8.8 Hz), 4.43±4.37 (2H, m), 4.19±4.09 (2H, m), 3.73 (2H,
d, 4.2 Hz), 3.58±3.50 (1H, br), 1.16 (3H, d, 6.3 Hz,
CH3); 13C NMR (CDCl3) d 157.06, 143.75, 141.28,
141.24, 127.68, 127.03, 124.98, 119.95, 68.38, 66.71,
64.57, 56.05, 47.18, 20.22.
Synthesis of octreotide 1. The sequence was synthesized
on Rink Amide resin (loading 0.56 mmol/g resin). Resin
(446 mg, 0.25 mmol) was washed with DCM:DMF (1:1)
in reaction vessel for 20 min before starting the synthe-
sis programme of the peptide synthesizer. N-Fmoc-
threoninol p-carboxybenzacetal B (1 mmol, 459 mg) was
put in the ®rst AA cartridge followed by 1 mmol of each
of all the other protected amino acids. The resin-bound
sequence of this peptide was Fmoc-d-Phe-Cys(Trt)-Phe-
d-Trp(Boc)-Lys(Boc)-Thr(But)-Cys(Trt)-Thr-ol-acetal-
resin. After the ®nal Fmoc deprotection with 20%
piperidine and programmed wash procedure, the resin
complex was dried in vacuo and weighted to be 899 mg.
Its average coupling yield was computed to be 97%
which was consistent with the recorded coupling yield at
each step (higher than 95%) on the ABI 433A auto-
synthesizer. Peptide side chain deprotection and clea-
vage were carried out in TFA solution (containing TFA
9 mL, water 0.25 mL, thioanisole 0.5 mL, EDA 0.25
mL, and phenol 0.79 g). The dried side chain protected
peptide-resin (899 mg) was added to 10 mL TFA solu-
tion at 0ꢀC, and the mixture was stirred gently at room
temperature for 1.5 h. After evaporation of TFA under
vacuum, the peptide was precipitated by addition of ice-
cold dry ether. The precipitate was ®ltered, washed with
cold ether on a sintered glass funnel, extracted with
20% acetic acid solution, and dried by lyophilization to
a white powder. RP-HPLC analysis (detail HPLC con-
dition in the text) of the crude product at this stage
showed a major peak at 30.1 min as monitored at UV
214 nm (Fig. 1). Dilute the peptide product to about
1 mM with 5% ammonium acetate solution and adjust
pH to 7.0 with ammonium hydroxide (25%) to accom-
plish disul®de bond formation. During the folding pro-
cess, an aliquot of 0.1 mL sample was taken at di€erent
times and injected to a Rainin (C18, 5 mm, 100A,
Synthesis of N-Fmoc-threoninol p-carboxybenzacetal
(B). A mixture of N-Fmoc-threoninol (0.3 g, 0.9 mmol),
p-carboxybenzaldehyde (0.34 g, 2.25 mmol), a few
crystals of p-toluenesulfonic acid, and two drops of
dimethylsulfoxide in CHCl3 (30 mL) was heated over-
night under nitrogen with a Dean±Stark trap to remove
water. After the mixture cooled to room temperature,
the solvent was evaporated under reduced pressure to
provide yellowish oil. Flash chromatography of the
residue over silica gel using methylene chloride:ethyl
acetate (3:1) as eluent furnished the desired product (385
mg, 91%) (Rf ˆ 0:28 in methylene chloride:ethyl ace-
25
d
tate, 3:1). Mp 86±88ꢀC; ‰aŠ
33ꢀ (c 0.02, dichloro-
methane). FABMS m/z 460 (M+1), 179, 178, 154, 136.
1H NMR (CDCl3) d 10.11 (1H, COOH), 8.13 (2H, d,
8.3 Hz), 7.75 (2H, d, 7.5 Hz), 7.60±7.55 (4H, m), 7.41±7.28
(4H, m), 5.63±5.56 (2H, m), 4.47±4.42 (2H, m), 4.25±4.12
(4H, m), 3.71 (1H, d, 4.2 Hz), 1.27 (3H, m, CH3); 13C
NMR (CDCl3) d 171.13, 156.54, 143.81, 143.05, 141.30,
130.21, 129.87, 127.70, 127.04, 126.13, 125.02, 119.98,
100.73, 75.44, 71.81, 66.96, 48.70, 47.24, 17.52.
Preparation of tri(tetrabutylammonium hydroxide) salt
of 1,4,7,10-tetraazacyclo-dodecane N,N0,N00,N000-tetra-
acetic acid (C). To a solution of 1,4,7,10-tetraaza-
cyclododecane N,N0,N00,N000-tetraacetic acid (2 mmol,
0.808 g) dissolved in water (10 mL) was added a solu-
tion of tetrabutylammonium hydroxide (0.75 mL, 6
mmol) with stirring until homogeneous. The solution
was then lyophilized to a€ord a pale-yellowish oily residue

1802
H.-P. Hsieh et al. / Bioorg. Med. Chem. 7 (1999) 1797±1803
4.6Â250 mm) analytical column for monitoring the dis-
ul®de oxidation of reduced octreotide. HPLC analysis
showed the disul®de oxidation was above 90% complete
in 46 h. After 48 h the solution was loaded onto a Vydac
C18 preparative column and puri®ed to give a white
powder of octreotide 1 (193 mg). Amino acid analysis of
octreotide after hydrolysis with 6 N HCl:TFA (4:1; v/v)
for 3 h at 130ꢀC was listed as: amino acid/found/ calcd,
Lys/1.0/1; 1/2Cys/0.97/1; Phe/1.94/2; Thr/0.65/1.
FABMS m/z 1019.5 (M+H)+.
Puri®cation and characterization of peptides. Analytical
and preparative RP-HPLC were performed on a Hita-
chi L-6200A system equipped with L-6000 pump and L-
4250 UV±vis detector. The RP columns of Rainin (C18,
5 mm, 100A, 4.6Â250 mm) analytical column and
Vydac (C18, 22Â250 mm) preparative column was used.
For analysis of the synthesized octreotide derivatives by
RP-HPLC, the elution gradient of 0±100±100±0% B in
0±30±35±40 min at a ¯ow rate of 1.0 mL/min was uti-
lized, where A was 5% acetonitrile aqueous with 0.1%
TFA and B was 90% acetonitrile aqueous with 0.1%
TFA. For monitoring the folding of reduced octreotide,
the elution gradient of 50±60±60±50% B in 0±20±25±30
min at a ¯ow rate of 1.0 mL/min was used, where A was
5% acetonitrile aqueous with 0.1% TFA and B was
90% acetonitrile aqueous with 0.1% TFA. Peptide pur-
i®cation was conducted on a Vydac C18 preparative
column. The peptide solution was ®rst loaded to the
column with a ¯ow rate of 4 mL/min then collected by
¯owing 75% of acetonitrile aqueous through the col-
umn at a ¯ow rate of 8 mL/min, monitored at 280 nm.
Amino acid analysis and FABMS were performed at the
Precision Instrument Development center of the National
Science Council, Taiwan.
Synthesis of octreotide conjugated derivatives. Two
0.1 mmol scale and two 0.25-mmol scale of fully pro-
tected octreotide-resin was synthesized separately by the
above-described procedure and used for conjugation
experiments.
DTPA-octreotide 2. Coupling of DTPA to octreotide
was operated on ABI 433A as previous described
protocol. Fully side chain protected octreotide-resin
(0.1 mmol scale) was ®rst synthesized by the previous
method to give 364 mg of resin complex. Four equiva-
lent amount of DTPA derivative D (0.4 mmol, 247 mg)
was added into AA cartridges and was used as the ninth
AA residue. Using the same treatment, analysis and
puri®cation procedure for octreotide, 104 mg of pure
DTPA-octreotide was obtained. FABMS m/z 1394.6
(M+H)+.
Acknowledgements
Support for this research provided by the National Sci-
ence Council, Taiwan, is gratefully acknowledged.
Synthesis of DOTA-octreotide 3. Application of the
same procedure above, except using tri(tetrabutyl am-
monium hydroxide) salt of 1,4,7,10-tetraazacyclo-dode-
cane N,N0,N00,N000-tetraacetic acid C (200 mg) prepared
previously as the ninth amino acid residue. Using the
same treatment, analysis and puri®cation procedure for
octreotide, DOTA-octreotide 3 was obtained in 99 mg.
FABMS m/z 1405.6 (M+H)+.
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