Proline-Rich Proteins – Deriving a Basis for Residue-based … · 2008. 3. 4. · 1/12 Proline-Rich Proteins – Deriving a Basis for Residue-based Selectivity in Polyphenolic Binding

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Proline-Rich Proteins – Deriving a Basis for Residue-based Selectivity in Polyphenolic Binding M. K. Foley and A. K. Croft* Supplementary information

Contents

General Synthetic procedures 2

Synthesis of N-Acetyl-L-Amino acid-OMe derivatives 1a-e 2

Synthesis of N-Acetyl-pyrrolidine 3 5 1H NMR Analysis of N-Acetyl-proline methyl ester 2 5

Titration Curves 6

Titration of N-Acetyl-proline methyl ester 2 with phenol 4 6

Titration of N-Acetyl-pyrrolidine 3 with phenol 4 9

Titration of N-Acetyl-L-Amino acid-OMe derivatives 1a-e with phenol 4 10

Supplementary Material for Organic & Biomolecular ChemistryThis journal is (c) The Royal Society of Chemistry 2008

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General

Nuclear Magnetic Resonance (NMR) spectra were obtained either from a Bruker AC 250 NMR

spectrometer, operating at 250 MHz for 1H spectra and 62.9 MHz for 13C spectra or a Bruker

Avance 500 NMR spectrometer, operating at 500 MHz for 1H spectra, 125.0 MHz for 13C spectra,

50.7 MHz for 15N spectra and 470.6 MHz for 19F spectra. The spectra were processed with Bruker

XWIN NMR or WINNMR software. Infrared (IR) spectra were obtained using a Perkin Elmer IR

spectrometer, and the samples prepared in KBr discs. LC-MS analysis was carried out on an

Agilent 1100 series HPLC with a C18 reverse phase column and acetonitrile/water mobile phase,

coupled to a Bruker Micro-TOF MS.

Synthesis of N-Acetyl-L-Amino Acid-OMe Derivatives 1a-e

HN

R

OH

OR'

HN

R

OCH3

OR'

N

R

OCH3

OR'

HCl.

O

SOCl2

MeOH

AcCl/NEt3

CH2Cl2 4 5 1

Scheme 1. a R = H, R’ = H, b R = H, R’ = Me, c R = Me, R’ = H, d R = CH2Ph, R’ = H, e R = CH2Ph-4-O-Ph, R’ = H.

General Procedure: Synthesis of Amino Acid Methyl Ester Hydrochlorides 5a-e8,9

Thionyl chloride (0.8 cm3, 11 mmols) was added dropwise to methanol (25 cm3) at 0 ºC,

followed by addition of the L-amino acid or benzyl-protected-L-amino acid 4 (10 mmols, see

). The solution was heated to reflux for 6 hours, and then the methanol was removed under

reduced pressure. The yellow-coloured residue was recrystallised using methanol/diethyl ether

and left in a desiccator for 24 hours to afford the corresponding L-amino acid methyl ester

hydrochloride or benzyl-protected-L-amino acid methyl ester hydrochloride 5 as a cream coloured

powder.

HCl.H2NO

O NH

O

O

HCl.

HCl.H2N

O

O 5a 5b 5c

HCl.H2NO

O HCl.H2N

O

O

O

5d 5e


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5a; yield = 88%; m. p. 174-176 °C (lit.10 175 ºC); δH (500 MHz; CD3OD); 3.86 (3H, s,

C(O)OCH3), 3.90 (2H, s, NH2CH2); δC (125 MHz; CD3OD); 41.1 (CH2NH2), 53.6 (C(O)OCH3), 169.0

(C(O)OCH3).

5b; yield = 82%; m. p. 119-121 °C (lit.12 117-119 ºC); δH (500 MHz; CD3OD); 2.85 (3H, s,

NH(CH3)CH2), 3.91 (2H, d, J 7.3, NH(CH3)CH2), 4.07 (3H, s, C(O)OCH3); δC (125 MHz; CD3OD);

32.14 (N(CH3)H2ClCH2), 41.4 (N(CH3)HCH2), 52.3 (C(O)OCH3), 171.0 (C(O)OCH3).

5c; yield = 90%; m. p. 106-108 °C (lit.10 109-111 ºC); δH (500 MHz; CD3OD); 1.58 (3H, d, J

7.3, CH3CHNH2), 3.87 (3H, s, C(O)OCH3), 4.16 (1H, q, J 7.3, CH3CHNH2); δC (125 MHz; CD3OD);

16.28 (CH3CHNH2), 50.0 (CH3CHNH2), 53.8 (C(O)OCH3), 171.4 (C(O)OCH3).

5d; yield = 86%; δH (500 MHz; CD3OD); m. p. 155-156 °C (lit.10 158-162 ºC) 3.25 (1H, dd, J

7.1, 14.4, PhCH2CHNH2), 3.30 (1H, dd, J 6.6, 14.5, PHCH2CHNH2), 3.81 (3H, s, C(O)OCH3), 4.36

(1H, t, J 6.8, PHCH2CHNH2), 7.30-7.41 (5H, m, phenyl protons); δC (125 MHz; CD3OD); 37.4

(PhCH2CHNH2), 53.6 (C(O)OCH3), 55.4 (PhCH2CHNH2), 129.0 (Ar CH), 130.1 (ArCH), 130.5 (Ar

CH), 136.4 (ArC), 170.4 (C(O)OCH3).

5e; yield = 90%; m. p. 162-165 °C (lit.11 164-165 ºC); δH (500 MHz; d6-DMSO); 3.04 (1H,

dd, J 7.0, 14.1, 4-BzOPhCH2CHNH2), 3.12 (1H, dd, J 5.8, 14.1, 4-BzOPhCH2CHNH2), 3.67 (3H, s,

C(O)OCH3), 4.19 (1H, t, J 6.5, 4-BzOPhCH2CH), 5.09 (2H, s, PhCH2O), 6.97 (2H, d, Ar

CCHCHCO, J 8.70), 7.16 (2H, d, J 8.80, Ar CCHCHCO), 7.32-7.46 (5H, m, phenyl protons), 8.60

(2H, br s, -BzOPhCH2CHNH2); δC (125 MHz d6-DMSO); 35.0 (4-BzOPhCH2CHNH2), 52.5

(C(O)OCH3), 53.3 (4-BzOPhCH2CHNH2), 69.1 (PhCH2O), 114.9 (Ar CH), 126.6 (Ar C), 127.7

(ArCH), 127.8 (Ar CH), 128.4 (Ar CH), 130.5 (Ar CH), 137.0 (Ar C), 157.4 (Ar CO), 169.4

(C(O)OCH3).

General Procedure: Synthesis of N-Acetylated Amino Acid Methyl Esters 1a-e9,13

To a stirred suspension of L-amino acid methyl ester hydrochloride or benzyl-protected-L-

amino acid methyl ester hydrochloride 5 (24 mmols) under dry nitrogen in anhydrous

dichloromethane (100 cm3) at 0 °C was added triethylamine (3.2 cm3, 58 mmols), dropwise. The

solution was stirred at room temperature for 30 minutes and then acetyl chloride (1.9 cm3, 26

mmols) was added dropwise and the solution was stirred at room temperature for a further three

hours. The solvent was removed under reduced pressure, ethyl acetate (200 cm3) was added and

the mixture was filtered through a pad of silica gel. Removal of the solvent under reduced

pressure afforded a cream residue, which was recrystallised using dichloromethane/hexane to

afford the N-acetylated-L-amino acid methyl ester or benzyl-protected-N-acetylated-L-amino acid

methyl ester 1 as colourless crystals.

NH

O

O

O

N

O

O

O

NH

O

O

O

1a 1b 1c


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NH

O

O

O

NH

O

O

O

O

1d 1e

1a; yield = 70%; (500 MHz; CDCl3); m. p. 54-56 °C (lit.14 53-55 ºC); δH 1.99 (3H, s,

NHC(O)CH3), 3.70 (3H, s, C(O)OCH3), 3.98 (2H, d, J 5.1, CH2NH), 6.09 (1H, br s, CH2NH); δC

(125 MHz; CDCl3); 22.8 (NC(O)CH3), 41.2 (CH2NH), 52.2 (C(O)OCH3), 170.4 (NC(O)CH3), 170.5

(C(O)OCH3).

1b; yield = 45%; δH (500 MHz; CDCl3); m. p. 61-63 °C (lit.14 60-61 ºC); 1.99 (0.75H, s, cis-

N(CH3)C(O)CH3), 2.11 (3H, s, trans-N(CH3)C(O)CH3), 2.93 (0.75H, s, cis-N(CH3)C(O)CH3), 3.04

(3H, s, trans-N(CH3)C(O)CH3), 3.69 (3H, s, trans-C(O)OCH3), 3.74 (0.75H, s, cis-C(O)OCH3), 4.01

(0.5H, s, cis-CH2N) 4.09 (2H, s, trans-CH2N); δC (125 MHz; CDCl3); 21.3 (trans-N(CH3)C(O)CH3),

22.1 (cis-N(CH3)C(O)CH3), 34.7 (cis-(N(CH3)C(O)CH3), 37.2 (trans-(N(CH3)C(O)CH3), 45.2 (cis-

CH2N), 49.1 (trans-CH2N), 52.1 (trans-C(O)OCH3), 52.3(trans-C(O)OCH3), 169.8 (trans-

NC(O)CH3), 169.9 (cis-C(O)OCH3), 171.2 (trans-C(O)OCH3), 171.5 (cis-C(O)OCH3).

1c; yield = 68%; m. p. 38-39 °C (lit.15 36-37 ºC); δH (500 MHz; CDCl3); 1.32 (3H, d, J 7.3,

CH3CHNH), 1.94 (3H, s, NHC(O)CH3), 3.67 (3H, s, C(O)OCH3), 4.51 (1H, pent, J 7.20,

CH3CHNH), 6.06 (1H, br s, CH3CHNH); δC (125 MHz; CDCl3); 18.2 (CH3CHNH2), 22.9

(NC(O)CH3), 58.0 (CH3CHNH), 52.3 (C(O)OCH3), 169.9 (NC(O)CH3), 173.6 (C(O)OCH3).

1d; yield = 65%; δH (500 MHz; CDCl3); m. p. 88-89 °C (lit.16 87-88 ºC); 1.99 (3H, s,

NHC(O)CH3), (1H, dd, J 5.8, 13.9 PhCH2CH), 3.15 (1H, dd, J 6.0, 13.9, PhCH2CHNH), 3.73 (3H, s,

C(O)OCH3), 4.90 (1H, q, J 11.7 PhCH2CHNH), 5.88 (1H, br d, J 6.6, PhCH2CHNH), 6.92-7.28 (5H,

m, phenyl protons); δC (125 MHz; CDCl3); 24.2 (NC(O)CH3), 35.3 (PhCH2CHNH), 53.1

(C(O)OCH3), 53.2 (PhCH2CHNH), 115.0 (Ar CH), 127.5 (Ar CH), 130.3 (Ar CH), 137.0 (Ar C),

169.6 (NC(O)CH3), 172.2 (C(O)OCH3).

1e; yield = 68%; m. p. 121-124 °C (lit.17 120-121 ºC); δH (500 MHz; CDCl3); 1.98 (3H, s,

NC(O)CH3), 3.05 (1H, dd, J 5.5, 14.0, 4-BzlOPhCH2CHNH), 3.08 (1H, dd, J 5.1, 14.0, 4-

BzlOPhCH2CHNH), 3.72 (3H, s, C(O)OCH3), 4.84 (1H, dt, J 5.6, 7.9, 4-BzlOPhCH2CHNH), 5.03

(2H, s, PhCH2O), 5.91 (1H, br d, J 7.7, 4-BzlOPhCH2CHNH), 6.89 (2H, d, Ar CCHCHCO, J 8.5),

7.16 (2H, d, J 8.6, Ar CCHCHCO), 7.31-7.43 (5H, m, phenyl protons); δC (125 MHz; CDCl3); 23.2

(NC(O)CH3), 37.0 (4-BzlOPhCH2CHNH), 52.3 (C(O)OCH3), 53.2 (4-BzlOPhCH2CHNH), 70.0

(PhCH2O), 115.0 (Ar CH), 127.5 (Ar CH), 128.0 (Ar CH), 128.1 (Ar C), 128.6 (Ar CH), 130.3 (Ar

CH), 137.0 (Ar C), 158.0 (Ar CO), 169.6 (NC(O)CH3), 172.2 (C(O)OCH3).


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Synthesis of N-Acetyl Pyrrolidine 318

To a stirred solution of pyrrolidine (5.8 cm3, 70 mmol) in anhydrous diethyl ether (20 cm3) at

0 °C was added a solution of acetyl chloride (5.5 cm3, 77 mmol) in anhydrous diethyl ether

(20 cm3). The reaction mixture was warmed to room temperature and stirred for one hour. The

diethyl ether was removed under reduced pressure and the product 3 was recovered as a

colourless syrup without further purification.

3; yield = 75 %; δH (500MHz; CDCl3); b. p. 75-78 °C at 2 mmHg (lit.18 62 °C at 1 mmHg);

1.74-1.89 (4H, m, NCH2CH2), 1.95 (3H, s, NC(O)CH3), 3.34 (4H, q, J 12.0, NCH2CH2); δC (125

MHz; CDCl3); 21.9 (NC(O)CH3), 24.4 (NCH2CH2), 25.7 (NCH2CH2), 45.3 (NCH2CH2), 45.3

(NCH2CH2), 169.2 (C(O)CH3).

NMR Signal Assignment of N-Ac-Pro-OMe 2

The 1H NMR spectrum of the purchased proline derivative 2 was obtained using a 0.009 mol dm-3

solution, and 13C NMR using a 0.090 mol dm-3 solution.

2; δH (500 MHz; CDCl3); 1.91 (0.5H, m, cis-CαHCH2CH2CH2N), 1.99 (0.75H, s, cis-NC(O)CH3),

2.00 (2H m, trans-CαHCH2CH2CH2N), 2.06 (1H, m, trans-CαHCH2CH2CH2N), 2.10 (3H, s, trans-

NC(O)CH3), 2.14 (0.25H, m, cis-CαHCH2CH2CH2N), 2.19 (1H, m, trans-CαHCH2CH2CH2N), 2.29

(0.25H, m, cis-CαHCH2CH2CH2N), 3.51 (1H, m, (trans) CαHCH2CH2CH2N), 3.57 (0.25H, m, cis-

CαHCH2CH2CH2N), 3.65 (1.25H, trans/cis-CαHCH2CH2CH2N), 3.70 (3H, s, trans-C(O)OCH3), 3.77

(0.75H, s, cis-C(O)OCH3), 4.38 (0.25H, dd, J 8.5, 2.5, cis-CαHCH2CH2CH2N), 4.49 (1H, dd, J 8.5,

3.8, trans-CαHCH2CH2CH2N)); δC (125 MHz; CDCl3); 20.4 (cis-NC(O)CH3), 20.5 (trans-NC(O)CH3),

21.0 (cis-CαHCH2CH2CH2N), 23.0 (trans-CαHCH2CH2CH2N), 27.6 (trans-CαHCH2CH2CH2N), 29.6

(cis-CαHCH2CH2CH2N), 44.5 (cis-CαHCH2CH2CH2N), 45.9 (trans-CαHCH2CH2CH2N), 50.4 (trans-

C(O)OCH3), 50.8 (cis-C(O)OCH3), 56.7 (trans-CαHCH2CH2CH2N), 58.3 (cis-CαHCH2CH2CH2N),

167.6 (trans-NC(O)CH3), 167.7 (cis-NC(O)CH3), 170.8 (cis-C(O)OCH3), 171.6 (trans-C(O)OCH3).

N

O

O

O

!

2

N

O

3


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Titration Data

Titration of N-Acetyl-proline methyl ester 2 with phenol 4

Temperature dependent and H-bonding studies: trans-N-Acetyl-proline methyl ester

Figure 1. trans-N-Ac-Pro-OMe 1a α-1H NMR titration with addition of phenol 4 at -20 ºC.

Δδpredα-H 1 = 0.08 ppm ± 0.02; Ka

1 = 31.30 mol-1 dm3 ± 4.89 (○)

Δδpredα-H 2 = -0.07 ppm ± 0.01; Ka

2 = 5.32 mol-1 dm3 ± 1.67 (□)

Table 1. Δδobs values for both the trans- and cis-rotamers of N-Ac-Pro-OMe 1. Values in brackets are unreliable due to proton signal overlap.

trans-rotamer cis-rotamer

Proton

no.a Δδobs b Δδobs

1 c Δδobs

2 d

1 (α H) 0.069 0.021 -0.026

2 0.004 0.019 0.011

3/4 -0.015 (0.019) (0.036)

3/4 -0.083 0.012 (-0.038)

5/7 0.001 0.002 - e

6 -0.025 0.050 0.007

5/7 (-0.017) (-0.053) - e

8 (-0.036) (0.036) (-0.075) a See Scheme 1 of manuscript. b Addition of 0-0.9 M phenol 3a. c Addition of 0-0.27 M phenol 3a. d Addition of 0.27-1.8 M phenol 3a. e 1H NMR signal overlap with the trans-rotamer 1H NMR signals.


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Figure 2. trans-N-Ac-Pro-OMe 1a 1H NMR titration with addition of phenol 4 at 0 ºC

Δδpredα-H 1 = 0.07 ppm ± 0.02; Ka

1 = 21.44 mol-1 dm3 ± 3.64 (○)

Δδpredα-H 2 = -0.06 ppm ± 0.01; Ka

2 = 6.77 mol-1 dm3 ± 3.38 (□)

Figure 3. trans-N-Ac-Pro-OMe 1a 1H NMR titration with addition of phenol 4 at 40 ºC.

Δδpredα-H = 0.07 ppm ± 0.01; Ka = 11.48 mol-1 dm3 ± 4.06

Figure 4. trans-N-Ac-Pro-OMe 1a α-1H NMR titration with addition of phenol 4, in 0.1 M d6-DMSO.

Δδpred = 0.12 ppm ± 0.01; Ka = 0.43 mol-1 dm3 ± 0.06


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Temperature dependent and H-bonding studies: cis-N-Acetyl-proline methyl ester

Figure 5. cis-N-Ac-Pro-OMe 1a α-1H NMR titration with addition of phenol 4 at -20 ºC.

Δδpredα-H 1 = 0.02 ppm ± 0.01; Ka

1 = 39.23 mol-1 dm3 ± 10.83 (○)

Δδpredα-H 2 = -0.20 ppm ± 0.02; Ka

2 = 3.35 mol-1 dm3 ± 1.63 (□)

Figure 6. cis-N-Ac-Pro-OMe 1a 1H NMR titration with addition of phenol 4 at 0 ºC

Δδpredα-H 1 = 0.03 ppm ± 0.01; Ka

1 = 31.89 mol-1 dm3 ± 9.14 (○)

Δδpredα-H 2 = -0.27 ppm ± 0.18; Ka

2 = 2.15 mol-1 dm3 ± 4.54 (□)

Figure 7. cis-N-Ac-Pro-OMe 1a 1H NMR titration with addition of phenol 4 at 40 ºC.

Δδpredα-H 1 = 0.03 ppm ± 0.01; Ka

1 = 11.13 mol-1 dm3 ± 8.52 (○)

Δδpredα-H 2 = -3.09 ppm ± 0.08; Ka

2 = 0.02 mol-1 dm3 ± 12.22 (□)


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Figure 8. cis-N-Ac-Pro-OMe 1a α-1H NMR titration with addition of phenol 4, in 0.1 M d6-DMSO.

Δδpredα-H 1 = 0.04 ppm ± 0.01; Ka

1 = 0.93 mol-1 dm3 ± 0.30 (○)

Δδpredα-H 2 = -0.13 ppm ± 17.64; Ka

2 = 1.00 mol-1 dm3 ± 9221870.00 (□)

Titration of N-Acetyl-pyrrolidine 3 with phenol 4

Figure 9. N-Ac-Pyrr α-1H

NMR titration with addition of phenol 4

Δδpredα-H 1 = 0.04 ppm ± 0.02, Ka = 13.96 mol dm-1 ± 8.48 (○)

Δδpredα-H 2 = 0.64 ± 0.12; Ka

2 = 0.07 mol-1 dm3 ± 1.02 (□)


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Titration of N-Acetyl-L-Amino acid-OMe derivatives 1a-e with phenol 4

Figure 10. N-Ac-Gly-OMe 1a α-1H

NMR titration with addition of phenol 4 Δδpred

α-H 1 = 0.07 ± 0.05; Ka

1 = 3.25 mol-1 dm3 ± 2.21 (○)

Δδpredα-H

2 = 0.46 ± 0.21; Ka

2 = 0.03 mol-1 dm3 ± 1.12 (□)

Figure 11. N-Ac-Gly-OMe 1a NH-1H NMR titration with addition of phenol 4

ΔδpredNH = 0.61 ppm ± 0.04; Ka

NH = 2.40 mol-1 dm3 ± 0.59

Figure 12. N-Ac-Ala-OMe 1b NH-1H NMR titration with addition of phenol 4

ΔδpredNH = 0.55 ppm ± 0.05; Ka = 2.43 mol-1 dm3 ± 0.81


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Figure 13. trans-N-Ac-Sar-OMe 1c α-1H NMR titration with addition of phenol 4

Δδpredα-H = 0.06 ppm ± 0.01; Ka = 5.99 mol-1 dm3 ± 2.72 (○)

Figure 14. cis-N-Ac-Sar-OMe 1c α-1H NMR titration with addition of phenol 4

Δδpredα-H = 0.03 ppm ± 0.01; Ka = 6.92 mol-1 dm3 ± 1.14 (○)

Figure 15. N-Ac-Phe-OMe 1d NH-1H NMR titration with addition of phenol 4



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Figure 16. N-Ac-O-Bzl-Tyr-OMe 1e NH-1H NMR titration with addition of phenol 4



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Proline-Rich Proteins – Deriving a Basis for Residue-based … · 2008. 3. 4. · 1/12 Proline-Rich Proteins – Deriving a Basis for Residue-based Selectivity in Polyphenolic Binding