1/12 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 1 H 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 Chemistry This journal is (c) The Royal Society of Chemistry 2008
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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
Supplementary Material for Organic & Biomolecular ChemistryThis journal is (c) The Royal Society of Chemistry 2008
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5a; yield = 88%; m. p. 174-176 °C (lit.10 175 ºC); δH (500 MHz; CD3OD); 3.86 (3H, s,
Supplementary Material for Organic & Biomolecular ChemistryThis journal is (c) The Royal Society of Chemistry 2008
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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.