Supplementary information
Cellulose chiral induction during the synthesis of cellulose N-phthaloyl-amino acid esters
Nicolas Vanthuyne, Christian Roussel
1) General information
Infrared spectra (KBr pellets) were recorded on a Perkin-Elmer 1720X FT-IR. The frequencies are in cm-1.
NMR spectra were recorded on a Bruker Avance DPX-200 MHz or a Bruker Avance DPX-300 MHz. The chemical shifts are expressed in ppm and the coupling in Hz. The spectra were calibrated from the residual chloroform signal.
Element analysis were performed either by the “Service commun de microanalyse of the faculty of Sciences St Jérôme, Aix-Marseille University on a Thermo Finnigan FA 1112 or by the Central analysis Service of the CNRS in Vernaison (France)
Optical rotations were recorded on a Perkin-Elmer 241 polarimeter at 589 nm (Sodium D line). A 10 cm cell was used and the temperature was set at 25°C.
Chiral HPLC analyses were performed on a Merck-Hitachi chromatographic unit: Merck-Hitachi L-6000 pump, Rheodyne injection valve, Merck-Hitachi L-4200 UV-detector, with HSM software.
The different analytical columns (250x4.6 mm) tested are ChiralcelÒ OD-H, ChiralpakÒ AS, AD columns from Chiral Technology Europa (Illkirch, France), (R,R)-Whelk-O1 and (S,S)- Ulmo from Regis Technologies (Morton Grove, USA).
Hexane, 2-PrOH and ethanol, HPLC grade, from Hipersolv Chromanorm (VWR), were degassed and filtered on a 0.45 mm millipore membrane before use. Retention times Rt in minutes, retention factors ki = (Rti-Rt0)/Rt0 and enantioselectivity factor a = k2/k1 are given. Rt0 was determined by injection of tri-tertio-butyl benzene.
Chemicals were purchased from Merck-Clevenot (Microcristalline Cellulose Avicel), Roquette Freres (Sulfonic resin Purolite C150), Aldrich, Fluka, Acros or Lancaster for the racemic or optically pure amino acids. HPLC solvents were from SDS (Peypin, France), they were filtered on Millipore HV 0.45µm and degassed before use.
2) N-phthaloyl amino acids: synthesis and enantioselective chromatography
General procedure: The method already described (Calmès et al. 1997) was employed for the preparation of racemic or optically pure N-phthaloyl amino acids. In a 350 ml flask equipped with a Dean-Stark, AA (0.1 mole) and finely powdered phthalic anhydride (0.1 mole) were dissolved in toluene (150 mL) and triethylamine (1.3 mL). The mixture was refluxed during 2-4 hrs and then the solvent was evaporated in vacuo. The solid residue was stirred in water (200 mL) and concentrated HCl (2 mL) to yield a dispersed powder. The powder was filtered and washed with water ( 3 x 50 mL). After drying in vacuo, the N-pht-AA were obtained as white powders.
The yields and ees of the obtained N-pht-AA and the chromatographic data are reported in Table 1.
N-Pht-Gly. 1H-nmr (200 MHz, CDCl3): 4.49 (s, 2H), 7.7-8.0 (m, 4H)
N-Pht-Phg. 1H-nmr (200 MHz, CDCl3): 6.10 (s, 1H), 7.20-7.90 (m, 9H), 9.35 (s, 1H, OH)
N-Pht-Ala. 1H-nmr (200 MHz, CDCl3): 1.70 (d, 3H, J=7.4), 5.02 (q, 1H, J=7.4), 7.65-7.95 (m, 4H), 8.59 (s, 1H, OH)
N-Pht-Val. 1H-nmr (200 MHz, CDCl3): 0.91 (d, 3H, J=6.8), 1.16 (d, 3H, J=6.8), 2.75 (m, 1H), 4.63 (d, 1H, J=8.6), 7.65-7.95 (m, 4H)
N-Pht-Phe. 1H-nmr (200 MHz, CDCl3): 3.58 (d, 2H, J=8.4), 5.21 (t, 1H, J=8.4), 7.10-7.20 (m, 5H), 7.60-7.85 (m, 4H)
N-Pht-Leu. 1H-nmr (200 MHz, CDCl3): 0.91 (d, 3H, J=6.4), 0.94 (d, 3H, J=6.4), 1.49 (m, 1H), 1.93 (m, 1H), 2.35 (m, 1H), 4.97 (dd, 1H, J=4.4 and 11.4), 7.65-7.95 (m, 4H)
N-Pht-Ileu. 1H-nmr (200 MHz, CDCl3):0.85 (t,0.5 CH2CH3, J=7), 0.91 (d,0.5 CHCH3, J=6.8), 0.97 (t,0.5 CH2CH3, J=7), 1.12 (d,0.5 CHCH3, J=6.8), 0.9-1.8 (m, 2H), 2.51 (m, 1H), 4.73 (dd, 1H, J=8.0 and 13.2), 7.65-7.95 (m, 4H), 9.01 (s, 1H, OH)
Table 1. Yields, ee’s and enantioselective chromatography data for N-phthaloyl amino acids
N-Pht-AA / Yield (ee) (%) / Mp (mp Lit)°C / Rt (min) / k(c) / a(c) / Rs
N-Pht-Gly / 71 / 196 (195-198)
rac-N-Pht-Ala / 93 (0(a)) / 160 ( 152-154) / 8.96 / 10.15 / 4.85 / 5.64 / 1.16 / 1.70
D-N-Pht-Ala / 87 (91(a)) / 146 / 10.15 / 5.64
L-N-Pht-Ala / 92 (89(a)) / 145 / 8.96 / 4.85
rac-N-Pht-Phg / 83 (0(b)) / 170(164-166) / 9.77 / 11.01 / 8.05 / 9.19 / 1.14 / 1.28
D-N-Pht-Phg / 87 (0(b)) / id
rac-N-Pht-Val / 88 (0(a)) / 101 (98-100) / 7.37 / 8.87 / 3.82 / 4.80 / 1.26 / 2.92
D-N-Pht-Val / 89 (97(a)) / 115 / 8.87 / 4.80
L-N-Pht-Val / 88 (92(a)) / 116 / 7.37 / 3.82
rac-N-Pht-Phe / 98 (0(b)) / 158 (172-174) / 7.46 / 9.07 / 5.17 / 6.50 / 1.26 / 2.24
D-N-Pht-Phe / 97 (100(b)) / 180 / 9.07 / 6.50
L-N-Pht-Phe / 97 (99(b)) / 181 / 7.46 / 5.17
rac-N-Pht-Leu / 98 (0(a)) / 142 (138-140) / 6.41 / 7.44 / 3.19 / 3.86 / 1.21 / 2.04
L-N-Pht-Leu / 83 (99.5(a)) / 120 / 6.41 / 3.19
rac-N-Pht-Ileu / 70 (0(a)) / 121 / 7.08 / 8.35 / 3.63 / 4.46 / 1.23 / 2.46
L-N-Pht-Ileu / 85 (93.5(a)) / 124 / 7.08 / 3.63
(a) (S,S)-Ulmo, hexane/2-PrOH/tfa 98/2/0.1, 2mL/min, 254nm
(b) (R,R)-Whelk-O1, hexane/2-PrOH/tfa 95/5/0.1, 2mL/min, 254nm
(c) ki=ti-t0/t0; α=k2/k1
3) N-Phthaloyl amino acid chlorides.
N-Phthaloyl amino acid (20 mmole) and methylenechloride (100 mL, stabilized with amylene) were placed in a 250 mL flask equipped with a condenser and a CaCl2 guard. Thionyl chloride (14.6 mL, 10 eq) was added dropwise at room temperature. At the end of addition, the mixture was refluxed for 2 hrs. The solvent was evaporated in vacuo and the acid chlorides were obtained as yellow powders or orange oils which solidified on standing. 1H-nmr was used to check the total conversion of the amino-acids. The acid chlorides were used without further purification for the acylation of cellulose.
N-Pht-Gly chloride. 1H-nmr (200 MHz, CDCl3): 4.82 (s, 2H), 7.7-8.0 (m, 4H)
N-Pht-Phg chloride. 1H-nmr (200 MHz, CDCl3): 6.19 (s, 1H), 7.30-7.95 (m, 9H)
N-Pht-Ala chloride. 1H-nmr (200 MHz, CDCl3): 1.78 (d, 3H, J=7.2), 5.16 (q, 1H, J=7.2), 7.70-8.0 (m, 4H)
N-Pht-Val chloride. 1H-nmr (200 MHz, CDCl3): 0.91 (d, 3H, J=6.8), 1.16 (d, 3H, J=6.8), 2.75 (m, 1H), 4.74 (d, 1H, J=8.4), 7.75-8.00 (m, 4H)
N-Pht-Phe chloride. 1H-nmr (200 MHz, CDCl3): 3.60 (d, 2H, J=8.4), 5.32 (t, 1H, J=8.4), 7.10-7.20 (m, 5H), 7.65-7.90 (m, 4H)
N-Pht-Leu chloride. 1H-nmr (200 MHz, CDCl3): 0.94 (d, 3H, J=6.6), 0.96 (d, 3H, J=6.6), 1.5 (m, 1H), 2.04 (m, 1H), 2.37 (m, 1H), 5.12 (dd, 1H, J=4.4 and 11.0), 7.70-8.00 (m, 4H)
N-Pht-Ileu chloride. 1H-nmr (200 MHz, CDCl3):0.86 (t,0.5 CH2CH3, J=7.2), 0.90 (d,0.5 CHCH3, J=7.0), 0.98 (t,0.5 CH2CH3, J=7.2), 1.12 (d,0.5 CHCH3, J=6.6), 0.9-1.8 (m, 2H), 2.51 (m, 1H), 4.73 (dd, 1H, J=8.0 and 13.2), 7.65-7.95 (m, 4H), 9.01 (s, 1H, OH)
4) Synthesis and characterization of cellulose triesters 1-15
General procedure: Cellulose was previously dried in vacuo at 120°C for 12 hrs and pyridine was kept on KOH pellets after distillation. Cellulose (500 mg, 3.09 mmol) and pyridine (50 mL) were placed in a three necked 100 mL flask equipped with a condenser (water guard), a septum and a funnel. The mixture was heated at 80°C for 1 hr and let to cool down at room temperature. Solid N-pht-AA chloride ( 6 eq.) was added under nitrogen flow and then the mixture was heated at 80°C for 40 hrs. After the mixture has been cooled to room temperature, it was poured in MeOH (600 mL) and the resulting solid was filtered. The solid was dissolved in CH2Cl2 (300 mL) and the resulting solution was slowly added to MeOH (800 mL), the cellulose ester precipitated. The filtration, the dissolution and the precipitation steps were performed another time to afford the cellulose esters as white or slightly colored powders after filtration and drying ( 50°C, in vacuo). (Table 2)
Cellulose tris-(N-phthaloyl-alaninate) 1 (and 2, 3)
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1756 (C=O, ester); 1714 (C=O, Phthaloyl); 1382 (C-N)
1H-nmr (300 MHz, CDCl3): 1.0-2.0 (broad, 9H), 3.0-6.5 (broad, 10H), 7.0-8.5 (broad, 12H)
Cellulose tris-(N-phthaloyl-phenylalaninate) 4 (and 5, 6)
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1766 (C=O, ester); 1710 (C=O, Phthaloyl); 1380 (C-N)
1H-nmr (300 MHz, CDCl3): 3.0-6.0 (broad, 16H), 6.5-8.5 (broad, 27H)
Cellulose tris-(N-phthaloyl-valinate) 7 (and 8, 9)
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1755 (C=O, ester); 1717 (C=O, Phthaloyl); 1386 (C-N)
1H-nmr (300 MHz, CDCl3): 0.4-1.5 (broad, 18H), 2.2-3.2 (broad, 3H), 3.5-6.5 (broad, 10H), 7.0-8.5 (broad, 12H)
Cellulose tris-(N-phthaloyl-leucinate) 10 (and 11)
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1761 (C=O, ester); 1713 (C=O, Phthaloyl); 1383 (C-N)
1H-nmr (300 MHz, CDCl3): 0.2-3.0 (broad, 24H), 3.0-6.0 (broad, 10H), 7.0-8.5 (broad, 12H)
Cellulose tris-(N-phthaloyl-isoleucinate) 12 (and 13)
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1763 (C=O, ester); 1715 (C=O, Phthaloyl); 1386 (C-N)
1H-nmr (300 MHz, CDCl3): 0.2-3.0 (broad, 24H), 3.0-6.0 (broad, 10H), 7.0-8.5 (broad, 12H)
Cellulose tris-(N-phthaloyl-phenylglycinate) 14
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1764 (C=O, ester); 1719 (C=O, Phthaloyl); 1384 (C-N)
Cellulose tris-(N-phthaloyl-glycinate) 15
IR (KBr) υmax (cm-1): 3100-2800 (C-H); 1776 (C=O, ester); 1712 (C=O, Phthaloyl); 1380 (C-N)
Degrees of substitution (DS)
The degrees of substitution reported in Table 2 were calculated from % of nitrogen and % of carbon according to the following equations:
%C = [72+12DS(10+x)] / [162+DS(186+12x+y)]
DS(C)= [72 – 162(%C)] / [(186+12x+y)(%C)-12(10+x)]
%N = 14DS / [162+DS(186+12x+y)]
DS(N) = [(-162)(%N)] / [(186+12x+y)(%C)-14]
Table 2. Element analysis, degree of substitution and yield for cellulose esters 1-15
Cellulose ester / Engaged N-Pht-AA / %C / %H / %N / DS(N) / DS(C) / Yield (%)N-Pht-Ala / 61.18 / 4.05 / 5.49 / 3.00 / 3.00
1 / rac-N-Pht-Ala / 61.10 / 4.06 / 5.47 / 2.95 / 2.94 / 99
2 / L-N-Pht-Ala / 61.08 / 4.12 / 5.47 / 2.95 / 2.92 / 96
3 / D-N-Pht-Ala / 61.12 / 4.12 / 5.51 / 3.05 / 2.95 / 99
N-Pht-Phe / 68.88 / 4.33 / 4.23 / 3.00 / 3.00
4 / rac-N-Pht-Phe / 68.78 / 4.31 / 4.25 / 3.09 / 2.92 / 94
5 / L-N-Pht-Phe / 68.75 / 4.37 / 4.22 / 2.96 / 2.90 / 61
6 / D-N-Pht-Phe / 68.28 / 4.43 / 4.12 / 2.58 / 2.60 / 99
N-Pht-Val / 63.60 / 5.06 / 4.95 / 3.00 / 3.00
7 / rac-N-Pht-Val / 63.52 / 5.09 / 4.92 / 2.92 / 2.93 / 81
8 / L-N-Pht-Val / 63.40 / 5.11 / 4.89 / 2.83 / 2.84 / 75
9 / D-N-Pht-Val / 63.45 / 5.09 / 4.91 / 2.89 / 2.88 / 88
N-Pht-Leu / 64.65 / 5.50 / 4.71 / 3.00 / 3.00
10 / rac-N-Pht-Leu / 64.54 / 5.55 / 4.68 / 2.89 / 2.92 / 88
11 / L-N-Pht-Leu / 64.52 / 5.54 / 4.72 / 3.02 / 2.90 / 93
N-Pht-Ileu / 64.65 / 5.50 / 4.71 / 3.00 / 3.00
12 / rac-N-Pht-Ileu / 64.56 / 5.52 / 4.73 / 3.06 / 2.93 / 83
13 / L-N-Pht-Ileu / 64.50 / 5.48 / 4.68 / 2.89 / 2.88 / 77
N-Pht-Phg / 68.14 / 3.89 / 4.42 / 3.00 / 3.00
14 / rac-N-Pht-Phg / 68.03 / 3.88 / 4.43 / 3.06 / 2.92 / 99
N-Pht-Phg / 59.75 / 3.46 / 5.81 / 3.00 / 3.00
15 / N-Pht-Phg / 59.71 / 3.47 / 5.82 / 3.03 / 2.96 / 98
5) Total hydrolysis of the cellulose esters 1-15 and amino acids recovery.
General procedure: In a 50mL flask, cellulose tris(N-phthaloyl-amino acid esters) (0.2 mmole) and lithium hydroxide mono hydrate (162 mg, 20 eq.) were placed in THF (20 mL) and water (10 mL). The reaction medium was stirred at room temperature for two days. Then the mixture was filtered on a membrane to collect native cellulose in quantitative yield (32 mg). THF was evaporated and the residual water acidified (pH=1). The aqueous solution was passed on a sulfonic resin Purolite C150 (30 mL) which had been washed with water (150 mL). The resin was washed with water (200 mL) and then with aqueous ammonia (5M, 150 mL) to liberate the amino acids. Water elimination yielded the free amino acids in quantitative yield.
The amino acids were either N-fmoc- or N-cbz protected before enantioselective chromatography to determine the enantiomeric ratio. (Table 3) The orders of elution were assigned using authentic samples.
Table 3. Enantioselective chromatography data for the analysis of amino acids resulting from the hydrolysis of the cellulose esters 1-15
L/D ratio / HPLC conditions / Rt (min) / Retention factork / a / Rs
rac-Cbz-Ala / 65 / 35 / (R,R)-Whelk-O1 Hexane / 2-PrOH / tfa (95/5/0.1)
2 mL/min / 6.91 / 8.35 / 3.55 / 4.49 / 1.26 / 1.85
D-Cbz-Ala / 65 / 35 / 8.35 / 4.49
L-Cbz-Ala / 64 / 36 / 6.91 / 3.55
rac-Cbz-Phg / 54 / 46 / Chiralpak AS Hexane / 2-PrOH / tfa (80/20/0.1)
1 mL/min / 9.09 (L) / 24.08 (D) / 2.01 / 7.03 / 3.50 / 6.23
rac-Fmoc-Val / 63 / 37 / (R,R)-Whelk-O1 Hexane / 2-PrOH / tfa (95/5/0.1)
1 mL/min / 15.36 / 17.49 / 4.12 / 4.83 / 1.17 / 1.67
D-Fmoc-Val / 61 / 39 / 17.49 / 4.83
L-Fmoc-Val L / 63 /37 / 15.36 / 4.12
rac-Cbz-Phe / 75 / 25 / Chiralpak AS Hexane / 2-PrOH / tfa (80/20/0.1)
1 mL/min / 8.48 / 15.87 / 1.83 / 4.29 / 2.33 / 3.06
D-Cbz-Phe / 74 / 26 / 15.87 / 4.29
L-Cbz-Phe / 73 / 27 / 8.48 / 1.83
rac-Cbz-Leu / 64 / 36 / Chiralpak AS Hexane / 2-PrOH / tfa (80/20/0.1)
1 mL/min / 6.04 / 17.24 / 1.01 / 4.75 / 4.70 / 6.12
L-Cbz-Leu / 62 / 38 / 6.04 / 1.01
rac-Cbz-Ile / 60 / 40 / Chiralpak AS Hexane / 2-PrOH / tfa (80/20/0.1)
1 mL/min / 5.86 / 16.17 / 0.95 / 4.39 / 4.62 / 5.87
L-Cbz-Ile / 65 / 35 / 5.86 / 0.95
6) Enantioselective adsorption
Three solutions of hexane / 2-PrOH (99:1 v:v) containing respectively 45µg / mL of Pirkle’s alcohol, 40µg / mL of Tröger’s base and 300µg / mL of benzoine were prepared and added with 0.5-2mg / mL of tri-tertiobutyl benzene (TTB). TTB was used as an internal standard to monitor the enantioselective adsorptions of the samples. These solutions were analyzed by enantioselective chromatography: Tröger’s base on Chiralcel OD-H, benzoin on Chiralpak AD and Pirkle’s alcohol on Chiralpak AS according to literature (Roussel et al. 2001). The esters 1-15 (10mg) were placed in separated vials with 1mL of hexane / 2-PrOH (99:1 v/v). The mixtures were sonicated for 5 min and kept at 10°C for 24 hrs. Then, the test sample solution (500µL) containing TTB was added and the resulting mixture was sonicated for 10 min and kept at 10°C for 2hrs. After that period, the supernatants were analyzed by enantioselective chromatography: Tröger’s base on Chiralcel OD-H, benzoin on Chiralpak AD and Pirkle’s alcohol on Chiralpak AS.