9-Fluorenylmethyl Chloroformate1

[28920-43-6]  · C15H11ClO2  · 9-Fluorenylmethyl Chloroformate  · (MW 258.71)

(base-labile protecting group for primary and secondary amines;3 used in peptide synthesis;5 used for derivatization of amines and amino acids prior to HPLC analysis and fluorescence detection13)

Alternate Names: 9-fluorenylmethoxycarbonyl chloride; FMOC-Cl.

Physical Data: mp 62-64 °C.

Solubility: sol organics (CH2Cl2, THF, dioxane); reacts with alcohols, amines, and water.

Form Supplied in: white crystalline solid; widely available.

Handling, Storage, and Precautions: FMOC-Cl is an acid chloride, and should be protected from moisture and heat. The reagent can evolve CO2 (pressure!) upon prolonged storage; therefore bottles should be opened carefully. FMOC-Cl is harmful if swallowed, inhaled, or absorbed through skin, is corrosive, and is a strong lachrymator. Inhalation may be fatal. Use in a fume hood.

N-Terminus Blocking in Peptide Synthesis.

Solid-phase peptide synthesis based on base-labile FMOC protection1 is rapidly augmenting and/or replacing older methods based on Boc and Z (Cbz) protection. The major advantage offered by the use of FMOC protection is that repetitive treatment with HF or CF3CO2H is no longer required for the deprotection steps. Most FMOC-protected amino acids are commercially available, although the search for orthogonal side-chain functional group protection continues.2 FMOC protection is currently the method of choice for the synthesis of glycopeptides which carry acid-labile O-glycosidic bonds (eq 1).3,5b,7

FMOC protection of amino acids is accomplished with Shotten-Bauman conditions (e.g. NaHCO3/dioxane/H2O or NaHCO3/DMF), or with anhydrous conditions (e.g. pyridine/CH2Cl2) at room temperature or below (eq 1).3a,4 A typical synthetic protocol for solid-phase peptide synthesis would involve coupling (eq 2), followed by deprotection with 20-30% Piperidine in DMF (eq 3), although other amines (e.g. 2% DBU, 10% Et2NH, or 50% morpholine in DMF) have been used. Many workers no longer use the chloroformate ester FMOC-Cl (1), preferring the more shelf-stable carbonate FMOC-OSu (2) [82911-69-1],5 or FMOC-pfp (3) [88744-04-1]. When used in the standard way,4 FMOC-Cl (1) has been shown to promote the formation of FMOC-dipeptides in 3-7% yield, presumably via the mixed anhydride intermediate.5a

Use of FMOC-OSu (2) provides N-protected amino acids in comparable yields without the formation of the dipeptide impurities.5 FMOC-pfp (3) has been used to prepare either FMOC-protected amino acids, or the corresponding pfp esters in one pot. Advantages of this reagent include supression of the dipeptide byproducts, as well as the production of the activated pfp esters using the same pentafluorophenol released during the N-protection step (eq 4).6 This same N-protection/C-activation procedure has been used to produce serine glycosides (eq 5),3b and for the solid-phase synthesis of human intestinal mucin fragments (eq 6).7 The use of FMOC-N3 (4) is also claimed to reduce dipeptide formation during the protection step,8 but the storage or use of potentially explosive oxycarbonyl azides cannot be recommended by this reviewer. Alternatively, the azide (4) may be generated in situ from (1).4

Fluorenylmethyl esters have been used for C-terminus protection in combination with FMOC N-terminus protection for solution-phase coupling of protected amino acids and peptide fragments. This permits simultaneous deprotection of both termini with Et2NH/DMF (eq 7).9

Amine and Hydroxyl Protection for Nucleosides, Carbohydrates, and Natural Products.

FMOC has been used to protect the amino groups on various nucleoside bases (adenosine, cytosine, guanine, and their 2-deoxy derivatives) for solid-phase oligonucleotide synthesis in a one-pot procedure.10 Chlorotrimethylsilane in dry pyridine was used for transient protection of the 2,3,5- or 3,5-hydroxyls, followed by the addition of FMOC-Cl, and hydrolytic workup of the TMS ethers to produce crystalline FMOC-protected nucleosides and 2-deoxynucleosides in good yield (eq 8). The FMOC group was removed from the heterocyclic bases with excess Triethylamine in dry pyridine.

FMOC-Cl has been used for 3,5-hydroxyl protection of deoxyribose for the introduction of modified bases such as azacytidine using the classical Vorbrüggen reaction.11 While the carbonate protecting groups do not alter the stereoselectivity of the N-glycoside formation, the FMOC groups can be removed under much milder conditions (Et3N/pyridine) than the typical 4-methylbenzoyl group (eq 9).

Perhaps the most impressive use of the FMOC group in the synthesis of natural products is provided by the key tetrasaccharide intermediate to calicheamicin.12 The robust nature of this particular FMOC group is amply demonstrated by the fact that it survives 15 discrete reaction steps, including exposure to cat. NaH/HOCH2CH2OH, Br2, EtN3, K-Selectride, i-Bu2AlH, and NaSMe (eq 10). Clearly, the use of FMOC protection is not limited to peptide synthesis.

FMOC Derivatization for Fluorescence Detection and Analysis.

Derivatization of amino acids and other primary and secondary amines with (1) for HPLC analysis has been described. The chief advantage of this methodology is the high sensitivity due to the intense fluorescence of the fluorenyl moiety, but the FMOC also reduces polarity for faster elution from the column. Amino acids have been detected in concentrations as low as 26 femtomolar using a precolumn derivatization scheme.13 FMOC derivatization of lyso-gangliosides in a two-phase Et2O-H2O system followed by chromatographic purification with fluoresence detection has also been reported.14 Optically active 1-(9-fluorenyl)ethyl chloroformate (FLEC®) (5) is commercially available in both (+) [107474-79-3] and (-) forms for the analysis of racemates.

1. For illustrative procedures using FMOC and other protection schemes, see The Practice of Peptide Synthesis; Bodanszky, M.; Bodanszky, A., Eds.; Springer: Berlin, 1984.
2. (a) Histidine: Fischer, P. M. et al. Int. J. Pept. Protein Res. 1992, 40, 19. (b) Glutamate: Handa, B. K.; Keech, E. Int. J. Pept. Protein Res. 1992, 40, 66.
3. (a) Polt, R.; Szabo, L.; Treiberg, J.; Li, Y.; Hruby, V. J. JACS 1992, 114, 10249. (b) Lüning, B.; Norberg, T.; Tejbrant, J. CC 1989, 1267.
4. Carpino, L. A.; Han, G. Y. JOC 1972, 37, 3404.
5. (a) Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. S 1983, 671. (b) Bardaji, E.; Torres, J. L.; Clapes, P.; Albericio, F.; Barany, G.; Rodriguez, R. E.; Sacristin, M. P.; Valencio, G. JCS(P1) 1991, 1755. For an alternate synthesis of FMOC-OSu (2) from fluorenylmethyl alcohol and phosgene: (c) Ten Kortenaar, P. B. W. et al. Int. J. Pept. Protein Res. 1986, 27, 398.
6. Schön, I.; Kisfalady, L. S 1986, 303.
7. (a) Jansson, A. M.; Meldal, M.; Bock, K. TL 1990, 31, 6991. (b) Bielfeldt, T.; Peters, S.; Meldal, M.; Paulsen, H.; Bock, K. AG(E) 1992, 31, 857.
8. Tessier, M. et al. Int. J. Pept. Protein Res. 1983, 22, 125.
9. (a) Bednarek, M. A.; Bodanszky, M. Int. J. Pept. Protein Res. 1983, 21, 196. (b) Bodanszky, M. et al. Int. J. Pept. Protein Res. 1981, 17, 444.
10. Heikkila, J.; Chattopadhyaya, J. ACS 1983, B37, 263.
11. Ben-Hattar, J.; Jiricny, J. JOC 1986, 51, 3211.
12. Nicolaou, K. C.; Groneborg, R. D.; Miyazaki, T.; Stylianides, N.; Schulze, T. J.; Stahl, W. JACS 1990, 112, 8193.
13. (a) Einarsson, S.; Folestad, S.; Josefsson, B. Anal. Chem. 1986, 58, 1638. (b) Einarsson, S. J. Chromatogr. 1985, 348, 213. (c) Veuthey, J.-L. J. Chromatogr. 1990, 515, 385. (d) For a review: Betner, I.; Foeldi, P. In Modern Methods in Protein Chemistry; Tschesche, H., Ed.; de Gruyter: Berlin, 1988; Vol. 3, p 227.
14. Neuenhofer, S.; Schwarzmann, G.; Egge, H.; Sandhoff, K. B 1985, 24, 525.

Robin L. Polt

University of Arizona, Tucson, AZ, USA

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