R. M. J. Liskamp et al.
runs were performed on an Alltech Alltima C8 column (250ꢁ
10 mm, pore size 90 ꢂ, particle size 10 mm, 4.8 mLminÀ1). The gra-
dient consisted of 100% solvent A for 5 min, to 100% solvent B in
120 min. The purity of all newly synthesized compounds was
judged by analytical HPLC and was found to be at least 98%.
MALDI-ToF analysis was performed on a Kratos Axima CFR appara-
tus with human ACTH(18–39) (2465.2 [M+H]+), or bovine insulin
oxidized B chain (3494.7 [M+H]+) as the external reference, and a-
cyano-4-hydroxycinnamic acid as matrix. High-resolution electro-
spray ionization (ESI) mass spectra were measured on a Micromass
LCT mass spectrometer calibrated with CsI by using nano-ESI at
1200 V capillary voltage and 50 V at the sample cone. All reported
mass values are monoisotopic.
2C(O)CH2CH2), 3.25–3.48 (m, 8H; 2OCH2CH2NH, 2CH2CH2C(O)CH2),
3.50–3.70 (m, 28H; 12OCH2, 2OCH2C(O)NHCH2), 3.83–3.84 (m, 9H;
3OCH3), 4.05 (s, 4H; 2C(O)CH2O), 4.10 (s, 4H; 2C(O)CH2O), 4.14 (t,
J=5.4 Hz, 4H; 2OCH2CH2NH), 4.34 (s, 2H; SCH2), 6.23 (s, 2H;
2CH3OCCH), 6.79 (t, J=2.2 Hz, 1H; CH2OCCH), 7.09 (d, J=2.5 Hz,
2H; 2C(O)CCH); 13C NMR (CDCl3): d=15.8 (CH2CCH), 23.6 (SCH2),
30.4 (CH2CH2CH2), 36.1 (C(O)CH2CH2), 37.7 and 37.8
(C(O)NHCH2CH2CH2), 39.6 (OCH2CH2NH), 55.9 and 56.4 (OCH3), 67.9
(OCH2CH2NH), 69.8 and 70.0 (C(O)CH2O), 70.4 (CH2CCH), 71.2 and
71.5 (OCH2), 83.7 (CH2CCH), 91.6 (CH3OCCH), 105.2 (SCH2C), 106.7
(CH2OCCH), 107.3 (SC(O)CCH), 140.7 (SC(O)C), 160.6 (CH3OC), 161.4
(CH2OC), 162.8 (CH3OC), 171.5, 172.0 and 174.0 (C(O)NH), 193.9
(SC(O)).
Chemistry: The syntheses of dihydroxybenzoic acid-based alkynes
2a–6a have been described previously.[18] The syntheses of 2b,
3b, and 15 have been reported earlier by Yim et al.[12] Details of
the synthesis and characterization of 7–11, 13, and 14 are de-
scribed in the Supporting Information.
Monomeric [Tyr3]octreotate peptide thio ester (12): Aqueous CuSO4
(47 mL, 4.8 mmol) and Na ascorbate (47 mL, 23 mmol) were added to
a solution of azide 1 (10.8 mg, 9.1 mmol) and alkyne 2b (3.5 mg,
9.5 mmol) in THF/H2O (3:1, v/v, 0.3 mL). The reaction mixture was
stirred for 16 h at room temperature. Then, the solvents were
removed under reduced pressure and the residue was taken up in
CH3CN/H2O and purified by semipreparative HPLC (C4). Com-
pound 12 was obtained in 66% yield (6.0 mmol, 9.4 mg). Rt(C4):
32.5 min. ESI-MS: m/z 1560.8 [M+H]+, 1582.8 [M+Na]+, 780.9
[M+2H]2+; calcd for C75H93N13O18S3: 1559.59.
Ahx-d-Phe-cyclo(Cys-Tyr-d-Trp-Lys-Thr-Cys)-Thr-OH (1): All reagents
and glassware were dried in vacuo for 18 h before use. Wang resin
(3.7 g (1.0 mmol), 0.27 mmolgÀ1) was swollen (DCM) and washed
(DMF) prior to loading of the first amino acid. Fmoc-Thr(tBu)-OH
(2.0 g, 5 mmol) was dissolved in DMF (5 mL), dried on 4 ꢂ molecu-
lar sieves, and added to the resin, followed by pyridine (0.7 mL,
8 mmol). The obtained slurry was gently swirled for 30 min until
the amino acid derivative dissolved completely. Then, 2,6-dichloro-
benzoyl chloride (DCBC: 0.7 mL, 5 mmol) was added, and the mix-
ture was gently swirled for 18 h at room temperature. The resin
was subsequently washed (DMF, DCM) and dried. The resin loading
was 68% (0.18 mmolgÀ1), as calculated from an Fmoc determina-
tion. The linear peptide sequence was synthesized according to
Fmoc/tBu solid phase peptide synthesis protocols. After coupling
of N-terminal 6-azido-hexanoic acid, the resin was washed (NMP,
DCM), dried, and suspended in TFA/H2O/TiPS (20 mL, 95:2.5:2.5,
v/v/v), and stirred for 3 h to cleave the peptide from the resin, and
to remove the side chain protecting groups. The crude peptide
was isolated by precipitation (3ꢁ) with cold (À208C) MTBE/hexane
(1:1, v/v). After centrifugation, the pellet was dissolved in acetoni-
trile/water (1:1, v/v), lyophilized, and purified by HPLC. This linear
peptide (45 mg, 38 mmol) was subsequently dissolved in acetoni-
trile/water (1:1, v/v), and DMSO (10%) was added to this solution.
The pH was adjusted to 7.5 with 5% aqueous ammonia. After stir-
ring overnight at room temperature, the solution was partially con-
centrated in vacuo, and the remaining DMSO was evaporated with
a rotational vacuum concentrator (Christ, Alpha-RVC). Following
preparative HPLC, the cyclic peptide was obtained in an overall
yield of 24% (36 mg). Rt: 20.07 min (C8); ESI-MS: m/z 1188.35
[M+H]+, 1210.40 [M+Na]+; calcd for C55H73N13O13S2: 1187.49.
DOTA-conjugated monomeric [Tyr3]octreotate sulfonamide (16): Pep-
tide thio ester 12 (3.7 mg, 2.3 mmol) was treated with TFA/TiPS
(95:5, v/v; 400 mL) for 3 h at room temperature. After concentration
in vacuo, the residue was dissolved in dry DMF (120 mL), then sulfo-
nyl azide 15 (1.7 mg, 2.4 mmol) followed by 2,6-lutidine (2.0 mL,
17 mmol) were added. The reaction mixture was stirred for 1 h at
room temperature and subsequently concentrated in vacuo. This
residue was treated with TFA/TiPS/H2O (95:2.5:2.5, v/v/v) for 3 h at
room temperature. Compound 16 was obtained in 65% yield
(2.8 mg, 1.51 mmol) after purification by semipreparative HPLC.
Rt(C8): 18.28 min; ESI-MS: m/z 1857.0 [M+H]+, 929.0 [M+2H]2+
619.6 [M+3H]3+; calcd for C83H113N19O24S3: 1855.74.
,
DOTA-conjugated dimeric [Tyr3]octreotate C9-spacer sulfonamide (17):
The synthesis of 17 was performed as described for 16, but start-
ing from thio ester 13 (1.7 mg, 0.6 mmol) and sulfonyl azide 15
(0.9 mg, 1.3 mmol). Compound 17 was obtained in 71% yield
(1.3 mg, 0.42 mmol) after purification. Rt(C8): 18.82 min; ESI-MS: m/z
1549.7 [M+2H]2+, 1033.5 [M+3H]3+, 775.4 [M+4H]4+; calcd for
C141H188N32O38S5: 3097.24.
DOTA-conjugated dimeric [Tyr3]octreotate C57-spacer sulfonamide
(18): The same procedure as described for 16 was used, but start-
ing from thio ester 14 (8.4 mg, 2.3 mmol) and sulfonyl azide 15
(1.7 mg, 2.4 mmol). Compound 18 was isolated after column chro-
matography in 22% yield (2.0 mg, 0.51 mmol). Rt(C8): 18.50 min;
ESI-MS: m/z 1953.0 [M+2H]2+, 1302.3 [M+3H]3+, 977.0 [M+4H]4+
781.8 [M+5H]5+; calcd for C177H254N38O52S5: 3903.70.
,
S-2,4,6-Trimethoxybenzyl
3,5-bis((4,8,24-trioxo-6,13,16,19-tetraoxa-
3,9,23-triazaoctacos-27-yn-1-yl)oxy)benzothioate (6b): Ethyl-3-(3-di-
methylaminopropyl)carbodiimide (EDCI, 47 mg, 0.245 mmol), 2,4,6-
trimethoxybenzylthiol (35 mg, 0.163 mmol) and a catalytic amount
of 4-dimethylaminopyridine (DMAP, 2 mg, 0.002 mmol) were added
to a solution of 6a (170 mg, 0.163 mmol) in dry DMF (10 mL). The
yellowish solution was stirred overnight under nitrogen atmos-
phere at room temperature. After evaporating the solvent, the
product was isolated by silica column chromatography by using a
gradient of DCM/MeOH (95:5 to 90:10, v/v). The 2,4,6-trimethoxy-
benzyl-protected dialkyne was obtained as a colorless oil in 58%
yield (117 mg, 94.9 mmol). Rf =0.40 (methanol/dichloromethane,
Cell line and culture conditions: The AR42J cell-line was kindly
provided by the Erasmus Medical Center (Rotterdam, The Nether-
lands). These cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco) supplemented with 10% fetal calf serum,
1% penicillin/streptomycin and 1% glutamine. The cultures were
maintained in a humidified atmosphere (5% CO2/95% air, 378C)
and routinely passed by using a trypsin-ethylenediaminetetraacetic
acid solution (Invitrogen).
Receptor binding studies: The half-maximal inhibitory concentra-
tion (IC50) of the peptides for binding to SSTR2 was determined on
AR42J tumor cells in a competitive binding assay with [111In-DO-
1
1:9); H NMR (CD3OD): d=1.74–1.83 (m, 8H; 4CH2CH2CH2), 2.30 (t,
J=2.6 Hz, 2H; 2CCH), 2.39 (m, 4H; 2CHCCH2), 2.47 (m, 4H;
758
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 750 – 760