*2.3. Peptide Synthesis*

Kisspeptin-8 (WNSFGLRF-NH2) was synthesized on a Rink Amide MBHA resin (Bachem, Bubendorf, Switzerland, subst.: 0.52 mmol/g) using *N*α-9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids (IRIS Biotech GmbH, Marktredwitz, Germany) by manual solid phase

peptide synthesis by the Department of Medical Chemistry (University of Szeged). The resin was swollen in dichloromethane (DCM). The Fmoc group was removed by treating the peptide-resin with 20% piperidine/*N*,*N*-dimethylformamide (DMF) solution twice (5 + 15 min). Solvents were purchased from VWR (Radnor, PA, USA).

The amino acids were activated with *N*,*N* -dicyclohexylcarbodiimide and 1 hydroxybenzotriazole in 50% DCM/DMF. The peptide-resin was incubated with this mixture for 3 h. The resin was washed with DMF (3×) and DCM (3×) after the deprotection and coupling steps.

The assembled peptides were cleaved from the resin by treating it with the following cleavage cocktail for 3 h: 90% trifluoroacetic acid (TFA) (Pierce, Rockford, IL, USA), 5% water, 2% dithiotreitol, 2% triisopropylsilane.

The peptides were precipitated with diethyl ether, dissolved in a mixture of acetonitrile (ACN) and water and lyophilized. The crude peptides were analyzed by HPLC (Hewlett-Packard Agilent 1100 system, column: Luna, c18 (2), 250 × 4.6 mm, 5 μm, 100 Å, Phenomenex, Aschaffenburg, Germany) and ESI-MS. The peptides were purified on a preparative HPLC column (Phenomenex Luna, c18 (2), 250 × 21.2 mm, 10 μm, 100 Å) using a Shimadzu 20-LC system. The fractions were analyzed on the above mentioned analytical HPLC system and measured by electrospray ionization mass spectrometry (ESI-MS) (see Appendix A Figures A1 and A2 for the results). The pure fractions were pooled and freeze-dried.
