Synthetic Histatin Peptides (Hst1, D-Hst1): Solid-Phase Peptide Synthesis and Mass Spectrometry Confirmation

This dataset documents the synthesis and mass spectrometry confirmation of histatin peptides (Hst1, D-Hst1, and cHst1).PEPTIDE SEQUENCES:- Linear Hst1 (Hst1): DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN (theoretical mass: ~4848 Da)- D-Hst1: D-amino acid analog of Hst1 (identical sequence, all D-amino acids)- Cyclic Hst1 (cHst1): Head-to-tail cyclic Hst1 (4830 Da, 18 Da less than linear due to lactam formation)SYNTHESIS METHOD:All peptides were manufactured by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry with a MilliGen 9050 peptide synthesizer (MilliGen/Biosearch, Bedford, MA, USA). Peptide synthesis-grade solvents were obtained from Biosolve (Valkenswaard, The Netherlands). N-Fmoc amino acids were obtained from Orpegen Pharma (Heidelberg, Germany).For Hst1 synthesis, preloaded solid-phase resin [Fmoc-L-Asn(Trt)-PEG-PS, 0.1 mmol, loading 0.16 mmol/g; Applied Biosystems, Foster City, CA, USA] in 1-methyl-2-pyrrolidone (NMP) was applied to the column and equilibrated at a flow rate of 4 ml/min. Fmoc removal was performed with piperidine (20% v/v) in NMP for 6 min. Fmoc amino acids were dissolved at 4 times molar excess in N,N-dimethylformamide (DMF) containing 0.6 mM 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate and 0.6 mM 1-hydroxybenzotriazole (HOBt), and coupled in the presence of 0.45 mM N,N-diisopropylethylamine (DIPEA) in NMP by recycling for 1.5 h. Washings between reaction steps were carried out with NMP.CLEAVAGE AND DEPROTECTION:The peptide was detached from the resin and deprotected with trifluoroacetic acid (TFA)/phenol/thioanisole/H2O (85:5:5:5) in a 25-ml syringe equipped with a frit, under gentle shaking for 2 h. N2 was flushed through the reaction mixture to reduce the volume to 2 ml. The reaction mixture was purged into ice-cold diethyl ether, followed by washings of the resin with TFA. The precipitated peptide was washed 4 times with ice-cold diethyl ether using a magnetically driven centrifuge (RVC 2-25; Christ, Osterode, Germany) at 230 g for 5 min, dissolved in H2O, flushed with N2 to remove excess diethyl ether, and lyophilized.CYCLIZATION (cHst1):Cyclization was performed using glutamic acid at position 4 as the starting point. Orthogonally protected Fmoc-Glu(Wang-resin)-ODmab (NovaBiochem, Läufelfingen, Switzerland) was used to allow on-resin head-to-tail ligation. After completion of the discontinuous sequence (K5-N38)(D1-E4), the N-terminal Fmoc was removed with 20% piperidine in NMP, and the C-terminal ODmab was removed by 2% hydrazine in DMF. On-resin head-to-tail cyclization was achieved by prolonged reaction (72 h) with 1 eq benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (Biosolve), 1 eq HOBt, and 1 eq DIPEA in DMF containing 20% dimethyl sulfoxide (DMSO; Biosolve) and 2% dichloromethane (DCM; Biosolve). After cleavage from the resin and purification by RP-HPLC, cyclization was confirmed by MALDI-TOF MS showing a molecular mass of 4830 Da (18 Da less than linear Hst1, indicating lactam formation). The overall yield was 1-3%.PURIFICATION:Peptides were purified by semipreparative RP-HPLC (Jasco Corp., Tokyo, Japan) on a Vydac C18-column (218MS510; Vydac, Hesperia, CA, USA). Peptides were dissolved in H2O containing 5% acetonitrile (AcN; Biosolve) and 0.1% TFA. Elution was performed with a linear gradient from 15 to 45% AcN containing 0.1% TFA in 20 min at a flow rate of 4 ml/min. The absorbance of the column effluent was monitored at 214 nm, and peak fractions were pooled and lyophilized. Reanalysis by RP-HPLC on an analytical Vydac C18-column (218MS54) developed with a similar gradient at a flow rate of 1 ml/min revealed a purity of ≥95%.MASS SPECTROMETRY CONFIRMATION:Mass spectra were recorded using two complementary systems:(1) Microflex LRF MALDI-TOF mass spectrometer equipped with an additional gridless reflectron (Bruker Daltonik, Bremen, Germany)(2) Thermo LTQ ion-trap mass spectrometer in nanospray configuration (Thermo Fisher Scientific, Hampton, NH, USA)Authenticity was routinely confirmed by mass spectrometry. Monoisotopic masses were assigned and compared to theoretical masses calculated from amino acid sequences.IMPORTANT NOTES:- These are chemically synthesized peptides, NOT isolated from biological samples.- No post-translational modifications are present.- No proteolytic digestion or database search was performed.- Mass spectrometry was performed for molecular weight confirmation only.- Only processed mass spectrometry documentation is provided; no raw instrument data files were generated or retained in exportable formats.FILES INCLUDED:- PDF documentation of synthesis methods and mass spectrometry results- Experimental protocols and confirmation data