Mio- and Dio-Fmoc - Two Modified Fmoc Protecting Groups Promoting Solubility

Abstract

Two novel Fmoc-derived protecting groups, Mio-Fmoc and Dio-Fmoc, were developed, which cause dramatically enhanced solubility of the corresponding derivatives in most organic solvents. Furthermore, the suitability of these groups in solid-phase peptide synthesis was highlighted.

protecting groups - solid-phase synthesis - solubility - peptides

References and Notes

1 Carpino LA. Han GY. J. Org. Chem.  1972,  22:  3404 

2 Grant GA. Synthetic Peptides: A User’s Guide   Oxford University Press; Oxford: 2002. 

3 Chan WC. White PD. Fmoc Solid Phase Peptide Synthesis. A Practical Approach   Oxford University Press; Oxford: 2004. 

4 Stigers KD. Koutroulis MR. Chung DM. Nowick JS. J. Org. Chem.  2000,  65:  3858 

5

Dtb-Fmoc-Cl is available from Sigma Aldrich.

6 Carpino LA. Wu A. J. Org. Chem.  2000,  65:  9238 

Selected applications:

7a Chi C. Im C. Wegner G. J. Chem. Phys.  2006,  124:  24907 

7b Egbe DAM. Nguyen LH. Hoppe H. Mühlbacher D. Sariciftci NS. Macromol. Rapid Commun.  2005,  26:  1389 

7c Bunz UHF. Chem. Rev.  2000,  100:  1605 

8 Carpino LA. Chao HG. Tien J. J. Org. Chem.  1989,  54:  4302 

9

We found that the sequence consisting of carboxylation of lithiated fluorenes¹⁴ followed by the reduction of the carboxylic acid with borane¹⁵ gives clearly better yields than the commonly employed treatment of the lithiated fluorenes with formaldehyde or paraformaldehyde.

10a Dess DB. Martin JC. J. Org. Chem.  1983,  48:  4156 

10b Dess DB. Martin JC. J. Am. Chem. Soc.  1991,  113:  7277 

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12 Tsunoda T. Suzuki M. Noyori R. Tetrahedron Lett.  1980,  21:  1357 

13

Friedel-Crafts Acylation of Fluorenes; General Procedure A To a stirred mixture of fluorene (3 or 5a, 60.2 mmol) and AlCl₃ (8.8 g, 66.0 mmol, 1.1 equiv) in anhyd CH₂Cl₂ (200 mL) 2-ethylhexanoyl chloride (11 mL) was added at 0 °C. After stirring overnight, ice and concd HCl were poured into the mixture until all the solids had been dissolved. The phases were separated, the aqueous phase was extracted twice with dichloromethane, the combined organic phases were dried, and concentrated. The residue was purified by flash chromatography (silica). 2-(2-Ethylhexanoyl)fluorene ( 4a)
Flash chromatography (PE-CH₂Cl₂, 1:1) gave 15.1 g of 4a (51.7 mmol, 86%) as a yellow solid; R _f 0.5 (PE-CH₂Cl₂, 1:1). ¹H NMR (CDCl₃, 300 MHz): δ = 0.81-0.93 (m, 6 H), 1.19-1.35 (m, 4 H), 1.46-1.64 (m, 2 H), 1.73-1.88 (m, 2 H), 3.36-3.45 (m, 1 H), 3.92 (s, 2 H), 7.25 (dd, ³ J = 6.8 Hz, ⁴ J = 1.0 Hz, 1 H), 7.36 (dt, ³ J = 7.4 Hz, ⁴ J = 1.5 Hz, 2 H), 7.80 (d, ³ J = 7.6 Hz, 2 H), 7.99 (dd, ³ J = 8.1 Hz, ⁴ J = 1.6 Hz, 1 H), 8.13 (d, ⁴ J = 0.8 Hz, 1 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 12.0, 13.9, 22.9, 25.6, 29.8, 31.9, 36.9, 47.7, 119.7, 125.2, 127.0, 127.5, 127.9, 136.3, 140.5, 143.3, 144.5, 146.1, 204.5. 2-(2-Ethylhexanoyl)-7-(2-ethylhexyl)fluorene ( 6)
Flash chromatography (PE-EtOAc, 10:1) gave 6.81 g of 6 (16.83 mmol, 95%) as a yellow oil; R _f 0.5 (PE-EtOAc, 20:1). ¹H NMR (CDCl₃, 300 MHz): δ = 0.77-0.89 (m, 12 H), 1.17-1.31 (m, 12 H), 1.39-1.60 (m, 3 H), 1.68-1.83 (m, 2 H), 2.49-2.61 (m, 2 H), 3.31-3.40 (m, 1 H), 3.86 (s, 2 H), 7.13 (dd, ³ J = 7.9 Hz, ⁴ J = 1.2 Hz, 1 H), 7.30 (s, 1 H), 7.67 (d, ³ J = 7.8 Hz, 1 H), 7.93 (dd, ³ J = 8.1 Hz, ⁴ J = 1.5 Hz, 1 H), 8.07 (s, 1 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 10.8, 12.0, 13.9, 14.1, 22.9, 23.0, 25.4, 25.6, 28.8, 29.8, 31.9, 32.3, 36.8, 40.4, 41.3, 47.6, 119.3, 120.4, 124.8, 126.0, 127.5, 128.1, 135.9, 138.1, 142.3, 143.3, 144.6, 146.4, 204.5. Reduction of 2-Ethylhexanoyl Groups; General Procedure B
AlCl₃ (12 g, 90.0 mmol, 1.7 equiv) was placed in a 500-mL round-bottom flask and anhyd Et₂O (300 mL) was added cautiously dropwise with external cooling with an ice bath and stirring. A solution of 4a (51.7 mmol) or 6 in anhyd Et₂O (100 mL) was added dropwise. After stirring for 30 min LiAlH₄ (3.42 g, 90.1 mmol, 1.7 equiv) was added portion-wise. The mixture was refluxed for 90 min and then allowed to cool to r.t. The excess of LiAlH₄ was destroyed with EtOAc and then dil. HCl was added until gas evolution ceased. The phases were separated, the organic phase was dried with MgSO₄, and evaporated. The residue was purified by flash chromatography (silica). 2-(2-Ethylhexyl)fluorene ( 5a)
Flash chromatography (PE-CH₂Cl₂, 100:3) gave 8.49 g of 5a (30.5 mmol, 59%) as a yellow solid; R _f 0.4 (PE-EtOAc, 100:3). ¹H NMR (CDCl₃, 300 MHz): δ = 0.99-1.09 (m, 6 H), 1.36-1.51 (m, 8 H), 1.69-1.80 (m, 1 H), 2.68-2.80 (m, 2 H), 3.98 (s, 2 H), 7.29 (d, ³ J = 7.8 Hz, 1 H), 7.39 (dt, ³ J = 7.4 Hz, ⁴ J = 1.2 Hz, 1 H), 7.45 (s, 1 H), 7.48 (t, ³ J = 6.9 Hz, 1 H), 7.64 (d, ³ J = 7.4 Hz, 1 H), 7.81 (d, ³ J = 7.8 Hz, 1 H), 7.87 (d, ³ J = 7.4 Hz, 1 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 10.8, 14.2, 23.1, 25.4, 28.9, 32.4, 36.8, 40.3, 41.3, 119.4, 119.5, 124.9, 125.8, 126.2, 126.6, 127.8, 139.2, 140.7, 141.8, 143.1, 143.3. 2,7-Bis(2-ethylhexyl)fluorene ( 5b)
Flash chromatography (PE) gave 4.47 g of 5b (11.44 mmol, 68%) as a colorless oil; R _f 0.6 (PE). ¹H NMR (CDCl₃, 300 MHz): δ = 0.85-0.93 (m, 12 H), 1.29-1.38 (m, 16 H), 1.62-1.64 (m, 2 H), 2.55-2.67 (m, 4 H), 3.85 (s, 2 H), 7.15 (d, ³ J = 7.7 Hz, 2 H), 7.32 (s, 2 H), 7.65 (d, ³ J = 7.7 Hz, 2 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 10.8, 14.2, 23.1, 25.4, 28.9, 32.3, 36.7, 40.3, 41.4, 119.0, 125.8, 127.7, 139.4, 140.1, 143.2. Fluorene-9-carboxylic Acids; General Procedure C1
Fluorene 5a or 5b (19.4 mmol) was dissolved in anhyd THF (30 mL) and cooled to -78 °C. n-BuLi (1.6 M, hexane; 15 mL, 24 mmol, 1.2 equiv) was added dropwise and then the solution was stirred for 1 h. A stream of carbon dioxide, obtained by evaporation of dry ice was passed through a drying jar, into the solution for 30 min, while warming to r.t. The mixture was acidified with dil. HCl to pH 1 and extracted with Et₂O. The solvent was evaporated and the residue was purified by flash chromatography. Fluorene-9-methanols; General Procedure C2
Substituted fluorene-9-carboxylic acid (17.24 mmol) was dissolved in anhyd THF (100 mL) and then BH₃·Me₂S (3.5 mL, 30.9 mmol, 2.1 equiv) was added dropwise at 0 °C. After warming to r.t. the solution was stirred for 2 h; crushed ice and an aq solution of tartaric acid were added cautiously until gas evolution ceased. The mixture was extracted several times with Et₂O, the organic phases were washed twice with a sat. aq solution of NaHCO₃, dried, and evaporated. The residue was purified by flash chromatography giving the fluorene-9-methanols. Fluorene-9-methyl Chloroformates; General Procedure C3
Fluorene-9-methanol (4.18 mmol) was stirred with a solution of phosgene (10% in toluene, 7.79 mmol, 1.9 equiv) in a sealed flask overnight. The solvent was removed in vacuo and the residue was purified by flash chromatography. 2-(2-Ethylhexyl)fluorene-9-methyl Chloroformate (Mio-Fmoc-Cl) ( 7a)
Instead of flash chromatography the residue of C1 was purified by dissolution in an aq solution of K₂CO₃ and extracted several times with Et₂O. After acidification with dil. HCl to pH 1 the product was extracted with CH₂Cl₂. The solution was dried and evaporated giving the pure fluorene-9-carboxylic acid. Yields: C1, 90%; C2, 55%; C3, 70%; R _f 0.6 (PE-EtOAc, 100:2). ¹H NMR (CDCl₃, 300 MHz): δ = 0.85-0.89 (m, 6 H), 1.20-1.34 (m, 8 H), 1.56-1.62 (m, 1 H), 2.52-2.64 (m, 2 H), 4.23 (t, ³ J = 7.6 Hz, 1 H), 4.44-4.57 (m, 2 H), 7.18 (d, ³ J = 7.8 Hz, 1 H), 7.27 (dt, ³ J = 7.4 Hz, ⁴ J = 1.0 Hz, 1 H), 7.33 (s, 1 H), 7.37 (t, ³ J = 7.4 Hz, 1 H), 7.53 (d, ³ J = 7.5 Hz, 1 H), 7.63 (d, ³ J = 7.8 Hz, 1 H), 7.70 (d, ³ J = 7.5 Hz, 1 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 10.8, 14.1, 23.0, 25.4, 28.8, 32.3, 40.3, 41.3, 46.0, 73.6, 119.8, 119.9, 125.0, 125.8, 126.8, 128.1, 129.2, 138.8, 141.4, 141.5, 142.4, 150.7. 2,7-Bis(2-ethylhexyl)fluorene-9-methyl Chloroformate (Dio-Fmoc-Cl) ( 7b)
Yields: C1, 89%; C2, 60%; C3, 97%. ¹H NMR (CDCl₃, 300 MHz): δ = 0.85-0.90 (m, 12 H), 1.26-1.35 (m, 16 H), 1.54-1.62 (m, 2 H), 2.53-2.66 (m, 4 H), 4.23 (t, ³ J = 7.6 Hz, 1 H), 4.52 (d, ³ J = 7.7 Hz, 2 H), 7.18 (d, ³ J = 7.8 Hz, 2 H), 7.33 (s, 2 H), 7.61 (d, ³ J = 7.8 Hz, 2 H). ¹³C NMR (CDCl₃, 75 MHz): δ = 10.8, 14.1, 23.0, 25.4, 28.8, 32.3, 40.3, 41.3, 45.9, 73.8, 119.5, 125.8, 129.2, 139.0, 141.0, 142.4, 150.7. Solid-Phase Peptide Synthesis
The assembly of the peptides was performed in a mechanical shaker with a fritted glass reactor. Wang resin¹⁶ (1 mmol/g, 200-400 mesh) is used as the solid support which is preloaded with Fmoc-Gly-OH.¹⁷ The preloaded resins (50 mg, loading 0.89 mmol/g) were swollen in DMF (0.5 mL) for 5 min, and than the excess solvent was removed by filtration. The Fmoc protecting group was removed by treating twice with 20% piperidine in DMF (5 min and 15 min) and then the resin was washed 5 times with DMF. The coupling step was performed by the Fmoc strategy using TBTU/HOBt¹⁸ as activation reagents. In a typical experiment, each Fmoc-dipeptide (Table [2] ) was introduced by a common coupling (60 min) using a two-fold excess of the dipeptide, activation of reagents, and a three-fold excess of DIPEA in DMF (0.5 mL). After washing five times with DMF and twice with CH₂Cl₂ the resin was dried in high vacuum. The substitution levels of the loaded resins were determined spectrophotometrically by Fmoc cleavage.³

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