549 TERPENE CONJUGATION terpenes (small lipophilic molecules) as new moieties to increase peptide lipophilicity while having lowest possible change in molecular weight. Terpenes are herbal organic compounds that are used as chemical penetration enhancers (10–12). Some terpenes can increase permeation of hydrophilic compounds (such as fluorouracil) through skin by different mechanisms, including lipid fluidization and complexation such effects depend on their structure (13). We hypothesized that the terpene enhancement effects might help to increase permeation of the conjugated systems as well. For our study, we selected a cyclic terpene (perillic acid) and a linear terpene (citronellic acid) (Figure 1). These terpenic enhancers were covalently attached to KTTKS, and KTTKS and Pal-KTTKS were synthesized as controls. We then investigated peptide permeation through a lipophilic membrane model of n-hexadecane and then studied their permeation through the human epidermal membrane. MATERIAL AND METHODS MATERIALS We purchased 2-chlorotrityl chloride resin (1.2 mmol/g), Fmoc-protected amino acids, and 2-(7Aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) from GL Biochem (China). GmbH (Germany) provided piperidine and trifluoroacetic acid, and Sigma Aldrich (United Kingdom) and Merck (Germany) supplied citronellic acid, perillic acid, and all the other chemicals. We applied all chemicals without further purification. METHODS Synthesis and characterization of peptides. KTTKS, Pal-KTTKS, Cit-KTTKS, and Per- KTTKS were synthesized by the Fmoc (standard fluorenylmethyloxycarbonyl) strategy, using 2-chlorotrityl chloride resin (14). Fmoc amino acids including Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)−OH, and Fmoc-Lys(Boc)−OH were used to synthesize the peptides. The solution of 20% piperidine in dimethylformamide (DMF) was employed for 30 min at ambient temperature to remove the Fmoc protection groups. Activation of each Fmoc was performed using solutions of N,N-diisopropylethylamine (DIEA) in N-methyl-2-pyrrolidone (NMP) (1 M) and HATU in dimethylformamide (DMF) (0.3 M) these two solutions were added to the amino acid powder in order to prepare a solution. This process protected the amino acid for coupling purposes. Then, the amino acid solution was added to the glass reaction vessel containing swollen resin at ambient temperature and reaction continued for 60 min. After completing the synthesis, a cleavage cocktail containing trifluoroacetic acid, triisopropylsilane, and water (99:0.5:0.5 v/v %) were used to cleave the peptide from resin. The resin was then filtered, and the solution containing peptide was subjected to the cold methyl tert-butyl ether (MTBE) to produce a white suspension. This suspension was then centrifuged and the MTBE subsequently decanted. The remaining solid was dried by lyophilizer and stored at −20°C. To synthesize the derivatives, we added the solution of conjugating moiety (palmitic acid or citronellic acid or perillic acid) to the reaction vessel containing swollen resin- bound KTTKS as the last stage of coupling. The coupling conditions were as described

550 JOURNAL OF COSMETIC SCIENCE previously—1 M DIEA/NMP, 0.3 M HATU/DMF, 60-min reaction time, and ambient temperature. The cleavage and other processes were as described previously. In order to confirm peptide synthesis, we used an Agilent Triple Quad LC/MS 6410 mass spectrometer with an electrospray ionization interface. Peptide assay. A LC-MS/MS method for assay of KTTKS and Pal-KTTKS has been reported by Choi et al. (4). Using this approach, we developed different methods for assay of KTTKS and the conjugates by LC-MS. The concentrations of KTTKS and its derivatives were measured by a selected ion chromatogram method using Agilent Triple Quad LC/MS 6410. Capital C8-Optimal column (250× 4.6 mm, i.d. 5 µm) was used for all peptides. The mobile phase of acetonitrile and ammonium acetate solution (20 mM) containing glacial acetic acid (0.05% w/v) under isocratic elution mode was employed for all four peptides, with the proportion of 40:60 (v/v) for KTTKS, Cit-KTTKS, and Per-KTTKS and 60:40 (v/v) for Pal-KTTKS. The flow rate was 0.6 mL/min for KTTKS and Cit-KTTKS, 0.5 mL/min for Per-KTTKS, and 0.3 mL/min for Pal-KTTKS. Gas temperature and gas flow were adjusted at 350°C and 10 L/min respectively. The injection volume was 25 µL. Mass to charge (m/z) ratios of 564, 716, 712, and 802 were used for KTTKS, Cit-KTTKS, Per-KTTKS, and Pal-KTTKS respectively. Stability studies. Aqueous solutions of Cit-KTTKS and Per-KTTKS were individually prepared at a concentration of 100 µg/mL. Each solution was divided into three glass vials and placed in a 32°C water bath for 48 h. After this period of time, the concentrations of peptides were measured by LC-MS (as described previously) and they were compared to the initial concentrations. Permeation studies Membrane model permeation study. To perform this study, a lipophilic barrier was prepared by deposition of n-hexadecane into a filter using a solution of n-hexadecane in n-hexane (5% v/v) (15). PVDF (polyvinylidene fluoride) filters with pore size of 0.45 µm were mounted on the static Franz-type diffusion cells (effective surface of 3.8 cm2) and 63 µL aliquots of n-hexadecane in n-hexane solution were distributed to each filter using sampler. Diffusion cells were then placed under the fume hood, in order to evaporate the n-hexane for 1 h. After that, the aqueous solutions of different permeants (100-1000 µg/mL) were placed in the donor compartments separately. The receptor compartments were filled with deionized water. The diffusion cells were then placed for 24 h at 37°C in a water bath to have a 32°C temperature on the surface (skin temperature). Finally, the peptide concentrations in the donor and receptor phases were measured by LC-MS as described previously. We performed these experiments in triplicate. Epidermal membrane permeation study. The epidermis used in the permeation study was obtained from female donors who underwent abdominoplasty. This experiment was performed under supervision and according to ethics committee regulations of Shahid Beheshti University of Medical Sciences. After removing subcutaneous fat, skin samples were frozen at −20°C. The heat separation method was used to separate the epidermis from dermis (16). The detached epidermis was then mounted on side-by-side horizontal diffusion cells (effective surface of 1.5 cm2). The donor compartments were individually filled with aqueous solutions of peptides with concentration of 100−500 µg/mL and the receptor compartments were filled with 10%