Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Str. 7, D-60438 Frankfurt am Main, Germany Institute of Biophysical Chemistry, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany Fmoc solid phase peptide synthesis formylation f-MLF magic-angle spinning Wang resin A mild synthetic method for N -formyl-Met-Leu-Phe-OH ( 1 ) is described. After Fmoc solid phase peptide synthesis, on-bead formylation and HPLC purification, more than 30 mg of the fully 13 C/ 15 N-labelled tripeptide 1 could be isolated in a typical batch. This peptide can be easily crystallised and is therefore well suited as a standard sample for setting up solid-state NMR experiments.

There appears to be a general lack of widely available and standardised samples for setting up new solid-state NMR experiments. Such a standard sample should show a small 13 C and 15 N linewidth and a short 1 H T 1 relaxation time. It should contain a number of molecular groups with different chemical shifts for setting up correlation spectra. The 13 C/ 15 N-labelled tripeptide N -formyl-Met-Leu-Phe-OH (f-MLF-OH) ( 1 ) has been shown in a number of solid-state NMR studies to fulfil these criteria. It has been used in great detail to examine spin dynamics in peptide and for distance measurements . MLF is a chemotactic peptide which plays an important role in antibody research . Although this tripeptide was briefly commercially available in the past, only one synthesis of f-MLF-OH ( 1 ) by solid phase methods has been published so far . Rigorous reaction conditions (EtOH, reflux, 24–65 h) were required to couple the first Boc-protected amino acid to the solid support (chloromethyl resin) and long reaction times (18 h) were necessary to attach further building blocks to the growing peptide chain. The formylation of the N -terminus with formic acid/acetic anhydride was carried out after cleavage from the resin with liquid hydrogen fluoride . No experimental procedures were given in all subsequent publications. In this work, we present for the first time in detail an improved practical synthesis for fully 13 C/ 15 N-labelled f-MLF-OH ( 1 ) based on the Fmoc-strategy.

The synthesis of the MLF tripeptide started with the immobilisation of 13 C/ 15 N-labelled Fmoc-Phe-OH to the solid support (Wang resin 2 ). This esterification step, leading to 3 quantitatively, was performed by activating the COOH group with MSNT under mild reaction conditions ( ) . Full conversion of the resin bound hydroxy groups could be demonstrated by a colourimetric test with Dabcyl-COOH . After removal of Fmoc with piperidine, the 13 C/ 15 N-labelled monomers Fmoc-Leu-OH and Fmoc-Met-OH were successively coupled to the solid support with DIC/HOBt . Quantitative conversion in each reaction was monitored by the Kaiser test . The resulting tripeptide 4 was formylated under mild conditions with 13 C-labelled ethyl formate to obtain 5 . The main advantage of using ethyl formate is the possibility of re-isolation of the expensive 13 C-labelled reagent for further formylation reactions, due to the absence of activating agents. To the best of our knowledge, formylation reactions of N -termini with ethyl formate have never been reported before in solid phase peptide synthesis. Other methods of formylation were not tested, since they require elevated reaction temperatures or acidic conditions , which might cause some premature removal of the peptide from the resin. The final step was the cleavage of f-MLF-OH ( 1 ) from the solid support with TFA . To prevent by-product formation from electrophilic intermediates present in the cleavage process, EDT and TIS were added as scavengers .

Compared to the solution phase synthesis of peptides, the solid phase synthesis offers a simplified purification of the intermediates. With the improved synthetic protocol based on Fmoc building blocks, where all reaction steps were carried out at room temperature, we were able to obtain the per- 13 C/ 15 N-labelled formylated Met-Leu-Phe tripeptide as carboxylic acid in acceptable yields [32.8 mg (23%) after HPLC purification]. Due to the selected reagents, i.e. MSNT for the esterification, DIC/HOBt for the peptide coupling and ethyl formate for the formylation, shorter reaction time and quantitative conversion could be accomplished when compared to the previous protocol from 1976 , where yields are not given. In contrast to the Boc-strategy, the Fmoc-method for solid phase peptide synthesis has the advantage of orthogonal conditions for the removal of N -protective groups and the cleavage from the Wang resin . Unintentional release of the growing peptide chain thus could be excluded. In addition, the use of liquid hydrogen fluoride is no longer required.

General: All reagents were obtained from commercial suppliers and were used without further purification. HPLC gradient: 0–5 min (0.1% TFA/MeCN 99:1), 5–20 min (0.1% TFA/MeCN 99:1 to 30:70), 20–25 min (0.1% TFA/MeCN 30:70), 25–30 min (0.1% TFA/MeCN 30:70 to 99:1), 30–40 min (0.1% TFA/MeCN 99:1). ESI-MS: Fisons VG Plattform II. NMR: Bruker AM 300 ( 1 H: 300 MHz; 13 C: 75.5 MHz). Immobilisation of the first monomer: Wang resin (485 mg, 0.315 mmol, capacity: 0.65 mmol/g) was swelled under argon with dry CH 2 Cl 2 (1.5 mL) in an oven dried reaction vessel for 15 min. In a second dry vessel, 13 C/ 15 N-labelled Fmoc-Phe-OH (250 mg, 0.63 mmol) was dissolved in dry CH 2 Cl 2 (3 mL) and MeIm (94 µL, 1.18 mmol). Afterwards MSNT (467 mg, 1.576 mmol) was added. The reaction mixture was shaken under argon until MSNT had completely dissolved. The amino acid solution was transferred via syringe to the resin and the suspension was flushed with argon. The sealed reaction vessel was gently agitated over night. The beads were transferred to a syringe with a filter and washed with dry CH 2 Cl 2 (5 ×). Some beads were dried in vacuo for the following colourimetric test with Dabcyl-COOH for the detection of polymer-supported OH groups . (See .) Coupling of further building blocks: In case of quantitative conversion, the resin was washed with N , N -dimethylformamide (DMF, 5 ×) and treated three times (15 min, 10 min, 5 min) with a piperidine solution (25% in DMF). Then the resin was washed with N -methylpyrrolidone (NMP, 5 ×) and a solution of 13 C/ 15 N-labelled Fmoc-Leu-OH (250 mg, 0.69 mmol), HOBt*H 2 O (145 mg, 0.945 mmol) and DIC (145 µL, 0.945 mmol) in NMP (2.5 mL) was aspirated into the syringe for the next coupling step. After 3 h the resin was washed with NMP (5 ×) and a few beads were tested for quantitative conversion by the Kaiser test . (See .) In case of a successful peptide extension, the Fmoc-protecting group was removed with piperidine (25% in DMF). Afterwards, the last coupling step was started by adding a solution of 13 C/ 15 N-labelled Fmoc-Met-OH (250 mg, 0.66 mmol), HOBt*H 2 O (145 mg, 0.945 mmol) and DIC (145 µL, 0.945 mmol) in NMP (2.5 mL). In case of quantitative coupling (Kaiser test), the Fmoc-protecting group was removed and the resin was washed with DMF (5 ×) and CH 2 Cl 2 (5 ×). The beads were dried in the syringe under reduced pressure. Formylation of the N -terminus: For the following formylation, 13 C-labelled ethyl formate (2 mL) was aspirated and the syringe was shaken over night at room temperature. The quantitative conversion was proved by the Kaiser test, after washing the resin with CH 2 Cl 2 (5 ×). Cleavage of the peptide from the solid support: For a successful cleavage, it is highly recommended to dry the beads in vacuo for at least 3 h. The cleavage solution [TFA (1.88 mL), H 2 O (50 µL), EDT (50 µL) and TIS (20 µL)] was added to the syringe. After shaking (3 × 90 min), the liberated peptide was filtered off into Millipore™ water. This solution was lyophilised and the residue was purified by HPLC. Yield: 32.8 mg (23%). HPLC conditions: Preparative: Reprosil AQ, 250 × 20, 10 µm, 0.1% TFA/MeCN (100:60), 10 mL/min; analytical: gradient: Reprosil AQ, 125 × 4.6 mm, 5 µm, 0.8 mL/min, t R = 21.08 min; isocratic: Lichrospher RP8 (Merck), 125 × 4.0 mm, 5 µm, 0.1% TFA/MeCN (66:34), 0.8 mL/min, t R = 5.07 min. 13 C NMR (δ[ppm] 75.5 MHz, MeCN-d 3 ): 173.5 (m, 1C), 172.6 (m, 1C), 171.8 (m, 1C), 162.8 (d, J = 12.8 Hz, 1C), 138.0 (m, 1C), 131.1-126.8 (m, 5C), 55.1-51.3 (m, 3C), 41.3 (t, J = 34.4 Hz, 1C), 37.8 (m, 1C), 32.4 (t, J = 35.5 Hz, 1C), 30.4 (m, 1C), 25.4 (m, 1C), 23.2 (m, 1C), 21.7 (m, 1C), 15.3 (s, 1C). MS (ESI): m / z (%) = 460.3 (100.0) [M–H] – , 13 C 21 H 31 15 N 3 O 5 S calcd. 461.26.

Synthesis of f-MLF-OH ( 1 ). a) Fmoc-Phe-OH, MSNT, MeIm, over night. b) 1. piperidine, 30 min; 2. Fmoc-Leu-OH, DIC, HOBt*H 2 O, 3 h. c) 1. piperidine, 30 min; 2. Fmoc-Met-OH, DIC, HOBt*H 2 O, 3 h; 3. piperidine, 30 min. d) HCOOEt, over night. e) TFA, EDT, H 2 O, TIS (94 : 2.5 : 2.5 : 1), 4.5 h. All reactions were carried out at room temperature. Abbreviations: MSNT = 1-(mesitylene-2-sulphonyl)-3-nitro-1,2,4-triazole, MeIm = N -methylimidazole, DIC = diisopropylcarbodiimide, HOBt = 1-hydroxybenzotriazole, TFA = trifluoroacetic acid, EDT = ethanedithiole, TIS = triisopropylsilane. PRQROPMIIGLWRP-BZSNNMDCSA-N InChI=1S/C21H31N3O5S/c1-14(2)11-17(23-19(26)16(22-13-25)9-10-30-3)20(27)24-18(21(28)29)12-15-7-5-4-6-8-15/h4-8,13-14,16-18H,9-12H2,1-3H3,(H,22,25)(H,23,26)(H,24,27)(H,28,29)/t16-,17-,18-/m0/s1 CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCSC)NC([H])=O OC(=O)[C@H](CC1=CC=CC=C1)NC([C@@H](NC(=O)[C@H](CCSC)NC(=O)[H])CC(C)C)=O |(279.45,-251.66,;269.47,-245.9,;269.47,-234.38,;259.49,-251.66,;259.49,-263.18,;269.47,-268.94,;279.45,-263.18,;289.42,-268.94,;289.42,-280.46,;279.45,-286.22,;269.47,-280.46,;249.52,-245.9,;239.54,-251.66,;229.56,-245.9,;219.59,-251.66,;209.61,-245.9,;209.61,-234.38,;199.63,-251.66,;199.63,-263.18,;209.61,-268.94,;209.61,-280.46,;219.59,-286.22,;189.66,-245.9,;179.68,-251.66,;179.68,-263.18,;169.7,-245.9,;229.56,-234.38,;239.54,-228.62,;239.54,-217.1,;251.06,-228.62,;239.54,-263.18,)| AuxInfo=1/1/N:27,28,31,23,22,24,21,25,15,29,11,5,17,26,20,14,8,4,12,7,2,16,10,6,18,13,9,1,3,30/E:(1,2)(5,6)(7,8)(28,29)/it:im/rA:31nOCOC.eCNCC.oONCCOC.eCNCOHCCCCCCCCCCSC/rB:s1;d2;s2;n4;s4;s6;s7;d7;s8;P8;s10;d12;s12;n14;s14;s16;d17;s17;s5;d20;s21;d22;s23;s20d24;s11;s26;s26;s15;s29;s30;/rC:279,4468,-251,6572,0;269,4702,-245,8971,0;269,4702,-234,3772,0;259,4935,-251,6572,0;259,4935,-263,1772,0;249,5169,-245,8971,0;239,5403,-251,6572,0;229,5637,-245,8971,0;239,5403,-263,1772,0;219,5871,-251,6572,0;229,5637,-234,3772,0;209,6105,-245,8971,0;209,6105,-234,3772,0;199,6339,-251,6572,0;199,6339,-263,1772,0;189,6573,-245,8971,0;179,6806,-251,6572,0;179,6806,-263,1772,0;169,7040,-245,8971,0;269,4702,-268,9371,0;279,4468,-263,1772,0;289,4234,-268,9371,0;289,4234,-280,4572,0;279,4468,-286,2172,0;269,4702,-280,4572,0;239,5403,-228,6172,0;239,5403,-217,0972,0;251,0603,-228,6172,0;209,6105,-268,9371,0;209,6105,-280,4572,0;219,5871,-286,2172,0; C21H31N3O5S CDK 04272620332D 0 0 0 0 0 999 V3000 M V30 BEGIN CTAB M V30 COUNTS 31 31 0 0 1 M V30 BEGIN ATOM M V30 1 O 279.44678 -251.65715 0 0 M V30 2 C 269.47015 -245.89714 0 0 M V30 3 O 269.47015 -234.37715 0 0 M V30 4 C 259.49353 -251.65715 0 0 CFG=1 M V30 5 C 259.49353 -263.17715 0 0 M V30 6 N 249.51694 -245.89714 0 0 M V30 7 C 239.54033 -251.65715 0 0 M V30 8 C 229.56371 -245.89714 0 0 CFG=2 M V30 9 O 239.54033 -263.17715 0 0 M V30 10 N 219.5871 -251.65715 0 0 M V30 11 C 229.56371 -234.37715 0 0 M V30 12 C 209.61049 -245.89714 0 0 M V30 13 O 209.61049 -234.37715 0 0 M V30 14 C 199.63387 -251.65715 0 0 CFG=1 M V30 15 C 199.63387 -263.17715 0 0 M V30 16 N 189.65726 -245.89714 0 0 M V30 17 C 179.68065 -251.65715 0 0 M V30 18 O 179.68065 -263.17715 0 0 M V30 19 C 269.47015 -268.93713 0 0 M V30 20 C 279.44678 -263.17715 0 0 M V30 21 C 289.4234 -268.93713 0 0 M V30 22 C 289.4234 -280.45715 0 0 M V30 23 C 279.44678 -286.21716 0 0 M V30 24 C 269.47015 -280.45715 0 0 M V30 25 C 239.54033 -228.61716 0 0 M V30 26 C 239.54033 -217.09715 0 0 M V30 27 C 251.06032 -228.61716 0 0 M V30 28 C 209.61049 -268.93713 0 0 M V30 29 S 209.61049 -280.45715 0 0 M V30 30 C 219.5871 -286.21716 0 0 M V30 31 H 169.70403 -245.89714 0 0 M V30 END ATOM M V30 BEGIN BOND M V30 1 1 1 2 M V30 2 2 2 3 M V30 3 1 2 4 M V30 4 1 5 4 CFG=3 M V30 5 1 4 6 M V30 6 1 6 7 M V30 7 1 7 8 M V30 8 2 7 9 M V30 9 1 8 10 M V30 10 1 8 11 CFG=1 M V30 11 1 10 12 M V30 12 2 12 13 M V30 13 1 12 14 M V30 14 1 15 14 CFG=3 M V30 15 1 14 16 M V30 16 1 16 17 M V30 17 2 17 18 M V30 18 1 17 31 M V30 19 1 5 19 M V30 20 2 19 20 M V30 21 1 20 21 M V30 22 2 21 22 M V30 23 1 22 23 M V30 24 2 23 24 M V30 25 1 24 19 M V30 26 1 11 25 M V30 27 1 25 26 M V30 28 1 25 27 M V30 29 1 15 28 M V30 30 1 28 29 M V30 31 1 29 30 M V30 END BOND M V30 END CTAB M END Procedures for colourimetric resin tests (including synthesis and analytical data of Dabcyl-COOH), crystallisation protocol, ESI, 13 C NMR and 13 C/ 15 N MAS-NMR spectra of f-MLF-OH ( 1 ) are available in the Supporting Information. A practical synthesis of the 13 C/ 15 N-labelled tripeptide N -Formyl-Met-Leu-Phe, useful as a reference in solid-state NMR spectroscopy. M.S. is grateful for a predoctoral fellowship from the Dr. Hilmer Foundation. J. Am. Chem. Soc. J. Am. Chem. Soc. Proc. Natl. Acad. Sci. U. S. A. J. Magn. Reson. J. Am. Chem. Soc. J. Am. Chem. Soc. J. Am. Chem. Soc. Curr. Opin. Struct. Biol. J. Biomol. NMR Bioorg. Med. Chem. Lett. J. Exp. Med. J. Am. Chem. Soc. Tetrahedron Lett. Tetrahedron Lett. Fmoc Solid Phase Peptide Synthesis: A Practical Approach Anal. Biochem. Anal. Biochem. J. Med. Chem. Anal. Chim. Acta Bull. Korean Chem. Soc. J. Am. Chem. Soc. J. Org. Chem. Chem.–Eur. J. Eur. J. Org. Chem. J. Org. Chem. This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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