O-N-Acyl migration in N-terminal serine-containing peptides: Mass spectrometric elucidation and subsequent development of site-directed acylation protocols

Tetrahedron Letters

Volumn 45, Issue 6, 2004, Pages 1173-1178

  Mouls, L ^a   Subra, G ^a   Enjalbal, C ^a   Martinez, J ^a   Aubagnac, J L ^a  

^a UNIVERSITÉ DE MONTPELLIER   (France)

Author keywords

O N Acyl shift; Peptide; Tandem mass spectrometry

Indexed keywords

HYDROXYL GROUP; PENTAPEPTIDE; PROTEOME; SERINE;

ACYLATION; AMINO TERMINAL SEQUENCE; ARTICLE; COLLISIONALLY ACTIVATED DISSOCIATION; FRAGMENTATION REACTION; LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY; MOLECULE; PALMITOYLATION; SYNTHESIS; VALIDATION PROCESS;

EID: 0742322008     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.tetlet.2003.12.008     Document Type: Article

References (9)

1. Benarek M.A., Feighner S.D., Pong S.S., McKee K.K., Hreniuk D.L., Silva M.V., Warren V.A., Howard A.D., Van Der Ploeg L.H., Heck J.V. J. Med. Chem. 43:2000;4370-4376.
2. Solid phase synthesis was performed on an automated synthesiser ACT496 Ω. The serine side chain was protected with a trityl group (Trt) and the arginine side chain was protected with a Pbf group (pentamethyldihydrobenzofuran-5-sulfonyl). Each well was filled with 150 mg of PS-Rink amide resin (Novabiochem). For the coupling step, three solutions were successively added to the reaction vessels: 400 μL of a 0.5 M solution of N-Fmoc protected amino acid (Senn Chemicals) in N-methyl pyrrolidone; 400 μL of a 0.5 M solution of N-methylmorpholine in DMF and 400 μL of a 0.5 M solution of HBTU in DMF. The coupling time was 90 min and deprotection (20 min) was carried out using 1.2 mL of DMF/piperidine 80/20 (v/v) solution. Five washes of 1.5 mL were used between steps including DMF (×2), DCM (×1), methanol (×1) and DMF (×1). Side chain deprotection and cleavage of peptides was performed for 2 h in the robot's 96-reactor block using 1.5 mL of cleavage cocktail (trifluoroacetic acid/water/ triisopropylsilane 95/2.5/2.5, v/v/v) per well. After removal of the resin by filtration, peptides were precipitated in diethyl ether and pelleted by centrifugation. The ether was decanted off and the crude peptide was dissolved in acetonitrile/water (50/50, v/v) containing 0.1% TFA.
3. On-resin trityl deprotection was performed by adding TFA/TIS/DCM (1/2.5/96.5, v/v/v) solution to the resin beads. After 30 min, the resin was washed three times with DCM and the deprotection solution was added for a further 30 min. This deprotection cycle was repeated until trityl removal was complete, as monitored by HPLC analysis of the deprotection filtrate solution.
4. On-resin palmitoylation was performed using pyridine (30 equiv, 1 M) and palmitoyl chloride (15 equiv, 0.5 M) in DCM for 90 min. The resin was then washed with (DCM (2×), DMF (1×), MeOH (1×) and finally DCM (1×)).
5. ESI mass spectra were recorded on a QTof I mass spectrometer (Waters-Micromass, Manchester, UK) fitted with a Z-spray ionisation source. Calibration was performed with an aqueous solution of phosphoric acid (0.1%) in the mass range of 50-1000 Da. Nitrogen was used as both nebulising and drying gas. The temperature of the source and of the drying gas was set at 80 and 150°C, respectively. The capillary voltage was set at 2900 V whereas the cone voltage was fixed at 30 V. Argon was chosen as the collision gas in MS/MS experiments. The collision energy was optimised for each sample. A Waters 2790 HPLC module equipped with an autosampler and a diode array UV detector PDA 996 (Waters-Micromass, Manchester, UK) was coupled to the mass spectrometer. Elution was performed in the HPLC module on a C-18 reversed phase column (Symmetry Shield, 50 × 2.1 mm, 3.5 μm) at a flow rate of 0.6 mL/min under gradient conditions from 100% solvent A (water 0.1% formic acid) to 100% solvent B (acetonitrile 0.1% formic acid) in 5 min. The flow was split prior to the mass spectrometer entrance allowing roughly 1/10 of the eluent in the ESI source. The MassLynx 4.0 software (Waters-Micromass, Manchester, UK) was used to control the equipment. All samples were dissolved in a solution composed of water/acetonitrile/formic acid (49/49/2, v/v) and 5 μL were injected for each experiment.
6. Sakakibara S., Shin K.H., Hess G.P. J. Am. Chem. Soc. 84:1962;4921-4928.
7. Chuan-Fa L., Tam J.P. Natl. Acad. Sci. U.S.A. Biochem. 91:1994;6584-6588.
8. Longevialle P., Girard J.P., Rossi J.C., Tichy M. Org. Mass Spectrom. 14:1979;414-416.
9. Special care must be taken during purification and storage. After purification by RP-preparative HPLC using acidic solvents, compounds separated must be quickly frozen and freeze-dried to avoid O-N-acyl transfer. This isomerisation was promptly observed in solution in the case of N-terminal serine-containing peptides but occurred very slowly if the serine was located in the peptide chain.