Suzuki-Miyaura cross-coupling catalyzed by protein-stabilized palladium nanoparticles under aerobic conditions in water: Application to a one-pot chemoenzymatic enantioselective synthesis of chiral biaryl alcohols

Article abstract of DOI:10.1039/b915184b

The preparation of palladium nanoparticles stabilized primarily within the protein cavity of a highly thermostable Dps protein (DNA binding protein from starved cells) and the use of this precatalyst system in Suzuki-Miyaura cross-coupling reactions under

Full text of DOI:10.1039/b915184b

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Supplementary Information
Suzuki-Miyaura cross-coupling catalyzed by protein-stabilized palladium
nanoparticles under aerobic conditions in water: application to a one-pot
chemoenzymatic enantioselective synthesis of chiral biaryl alcohols
Alessandro Prastaro,a Pierpaolo Ceci,b,c Emilia Chiancone,b,c Alberto Boffi,b,c Roberto Cirilli,d
Marisa Colone,e Giancarlo Fabrizi,a Annarita Stringaro,e and Sandro Cacchi*a
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza, Università di Roma, P.le A. Moro 5, 00185
Rome, Italy. Fax: +39 (06) 4991-2780; Tel: +39 (06) 4991-2795; E-mail: sandro.cacchi@uniroma1.it
b Dipartimento di Scienze Biochimiche, Sapienza, Università di Roma, P.le A. Moro 5, 00185 Rome, Italy.
Fax: +39 (06) 4440062; Tel: +39 (06) 49910990
c Istituto di Biologia e Patologia Molecolari CNR, P.le A. Moro 5, 00185 Rome, Italy. Fax: +39 (06)
4440062; Tel: +39 (06) 49910761; E-mail: emilia.chiancone@uniroma1.it
d Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
e
Dipartimento di Tecnologia e Salute, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome,
Italy
Experimental Section
General information
Melting points were determined with a Büchi B-545 apparatus and are uncorrected. All of the
reagents and solvents are commercially available and were used as purchased, without further
purification. Compounds were purified on axially compressed columns, packed with SiO2 25-40 µm
(Macherey Nagel), connected to a Gilson solvent delivery system and to a Gilson refractive index
detector, and eluting with n-hexane/AcOEt mixtures. 1H NMR (400.13 MHz) and 13C NMR (100.6
MHz) spectra were recorded with a Brüker Avance 400 spectrometer. Infrared (IR) spectra were
recorded on a JASCO FT/IR-430 spectrophotometer. Mass spectra were determined with a QP2010
Gas Chromatograph Mass spectrometer (EI ion source). UV-Vis absorption spectra were recorded
between 220-700 nm at rt with a spectrophotometer Varian Cary 50 and using a quartz cuvette with
0.1 cm path length. Transmission electron microscopy (TEM) analyses were made by Philips 208
transmission electron microscope (FEI Company) at 70 kV. The samples (10 µl) were applied to
carbon coated copper grids (200-mesh) and after 30 s on the grid the samples were dried and
visualized. In addition, some samples were negatively stained with (10 µl) phosphotungstic acid
(PTA) 2% w/v solution buffered at pH = 7.3 to visualize the protein shell (Figure S1).
The enantiomeric excess was determined by enantioselective HPLC on the Chiralpak AS-H chiral
stationary phase using the mixture n-hexane-2-propanol 95/5 (v/v) as a mobile phase.

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Cloning, protein expression and purification
Recombinant Thermosynechococcus elongatus Dps (TeDps) protein was obtained and purified as
described by Franceschini et al. (2006), removing the two ammonium sulfate cuts. Typically yield
was 200 mg of Dps protein per 1 L batch. Protein concentration were determined
spectrophotometrically on the basis of an ε molar of 19940 M-1 cm-1 (Mw 214 kDa) at 280 nm.
Preparation of of palladium nanoparticles stabilized by a thermophilic DNA binding protein
from starved cells (Pdnp/Te-Dps)
A Dps solution (0.23 µmol, 0.15 M in NaCl) was brought to pH 8.5 using 30 mM NaOH
(TITRINO, Metrohm AG). K2PdCl4 (969 µg, 29.7 µmol) was added to the protein solution under
stirring at room temperature for 30 min and then NaBH4 (156 µg, 41.4 µmol) for over 15 min. Any
possible aggregates of Dps and/or palladium particles produced during nanoparticles formation
were removed by filtration through 0.22 µm syringe filters. The solution was dialyzed against 0.15
M NaCl and concentrated for the following analyses. The samples containing Pdnp/Te-Dps was
checked using Supherose 6 gel-filtration column (GE Healthcare), equilibrated with 0.15 M NaCl.
Typical procedure for the Suzuki-Miyaura cross-coupling reaction using 0.05 mol% of
Pdnp/Te-Dps
4-Iodobenzoic acid (62 mg, 0.25 mmol), 0.05 mol% of Pdnp/Te-Dps, o-tolylboronic acid (34 mg,
0.25 mmol.) in Tris (2 mL, pH = 8.9, 100 mM) were orbitally stirred for 24 h at 100 °C with a
Heidolph Synthesis System. Then, after cooling, the liquid phase was extracted with CH2Cl2, the
organic layer was washed with deionized water and dried over Na2SO4. The solvent was removed
under vacuum and the residue was purified by chromatography (SiO2, 35 g, n-
Hexane/AcOEt/AcOH 96/3/1 v/v) to give 47.7 mg (90% yield) of 3a m.p: 187.5-188.8 °C; IR (KBr)
1
2949, 2905, 1673, 1608, 1427, 1321, 1291 cm-1; H NMR (DMSOd6) δ 8.02−7.99 (d, J = 8.6 Hz,
13
2H), 7.44-7.43(d, J = 8.0 Hz, 2H), 7.33-7.21 (m, 4H), 2.24 (s, 3H); CNMR (DMSOd6) δ 167.7,
146.24, 140.84, 135.17, 130.99, 129.86, 129.75, 129.73, 128.35, 126.56, 20.58. Anal Calcd for
C14H12O22 C, 79.22; H, 5.70; found C, 79.20; H, 5.68.
1
3b: m.p: 73.5-73.6 °C; IR (KBr) 2923, 2853, 1607, 1586, 1500, 1287, 1245, 806 cm-1; H NMR
13
(CDCl3) δ 7.68 (s, 4H), 7.58-7.56 (d, J = 8.0 Hz, 2H), 7.04-7.01 (d, J = 8.0 Hz, 2H); C NMR

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(CDCl3) δ 159.9, 144.35, 132.2, 128.4, 126.9, 125.77, 125.73, 125.7, 125.6, 123.1, 114.5, 55.4.
Anal Calcd for C14H11F3O C, 66.6; H, 4.4; found C, 66.7; H, 4.5.
3c: Oil; IR (neat) 2923, 2854, 2227, 1457 cm-1; 1H NMR (CDCl3) δ 7.74−7.72 (d, J = 8.0 Hz, 2H),
13
7.47-7.45 (d, J = 8.0 Hz, 2H), 7.34-7.28 (m, 3H), 2.28 (s, 3H); C NMR (CDCl3) δ 140.7, 137.2,
133.6, 132.7, 129.5, 128.6, 127.8, 127.6, 126.3, 115.7, 111.5, 21.7. Anal Calcd for C14H11N C,
87.01; H, 5.74; found C, 86.96; H, 5.01.
1
3d: Oil; IR (neat) 3030, 2925, 1901, 1611, 1484, 1438, 1330, 1258 cm-1; H NMR (CDCl3) δ 7.83
13
(s, 1H), 7.79-7.74 (m, 1H), 7.62-7.48 (m, 4H), 7.29 (d, J = 7.9 Hz, 2H), 2.42 (s, 3H); C NMR
(CDCl3) δ 141.9, 137.9, 131.1 (d, J = 32.1 Hz), 130.2, 129.7, 129.2, 127.0, 124.2 (d, J = 272.0 Hz),
123.6 (q, J = 4.1 Hz), 21.1. Anal Calcd for C14H11F3 C, 71.18; H, 4.69; found C, 71.12; H, 4.63.
3e: m.p.: 104-105 °C; IR (neat) 3060, 2355, 1677, 1477, 748 cm-1; 1H NMR (CDCl3) δ 8.29 (d, J =
8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.35 (m, 3H), 7.22(d, J = 7.2 Hz, 1H), 2.27 (s, 3H); 13C NMR
(CDCl3) δ 148.3, 143.7, 140.2, 135.6,133.6, 129.5, 128.6, 126.8, 125.6, 124.8, 21.7. Anal Calcd for
C13H11NO2, C, 73.23; H, 5.20; found C, 73.25; H, 5.25 (Known compound, see: W. Han, C. Liu,
and Z. Jina, Adv. Synth. Catal. 2008, 350, 501).
3f: m.p.: 110.6-111.3 °C; IR (KBr) 1687, 1348, 821 cm-1; 1H NMR (CDCl3) δ 8.22−8.21 (d, J = 4.0
Hz, 1H), 8.06-8.04 (d, J = 8.0 Hz, 2H), 7.77-7.67 (m, 3H), 7.45-7.43 (d, J = 8.0 Hz, 1H) 2.65 (s,
13
6H); C NMR (CDCl3) δ 197.7, 149.6, 142.8, 135.6, 141.8, 138.8, 136.5, 134.3, 133.5, 133.4,
133.2, 133.1, 131.4, 129.2, 129.0, 127.4, 127.0, 123.1, 26.7, 20.2. Anal Calcd for C15H13NO3, C,
70.58; H, 5.13; found C, 70.60; H, 5.17.
3g: m.p: 250.0-251.7 °C; IR (KBr) 2953, 2834, 2547, 1678, 1601, 1430, 1313, 1289 cm-1; 1H NMR
(DMSO-d6) δ 8.00−7.98 (d, J = 7.7 Hz, 2H), 7.76-7.68 (m, 4H), 7.07-7.05 (d, J = 6.4 Hz, 2H), 3.81
(s, 3H); 13C NMR (DMSO-d6) δ 167.7, 160.07, 144.46, 131.76, 130.46, 129.37, 128.64,
126.62, 115.02, 55.73. Anal Calcd for C14H12O3 C, 73.67; H,5.30; found C, 73.65; H,5.28
1
3h: m.p: 250.6 °C; IR (KBr) 2949, 2668, 2548, 1680 cm-1; H NMR (DMSO-d6) δ 8.03−8.01 (d, J
13
= 8.0 Hz, 2H), 7.81-7.75 (m, 4 H), 7.56-7.54 (d, J = 8.0 Hz, 2H); C NMR (DMSO-d6) δ 169.3,

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141.6, 133.7, 132.8, 130.8, 129.4, 128.8, 127.2. Anal. Calcd. for C13H9ClO2 C, 67.11; H, 3.90;
found C, 67.06; H, 3.84.
1
3i: mp: 218.0-219.4 °C; IR (KBr) 2949, 2668, 2548, 1680, 1607, 1422, 1318, 1288 cm-1; H NMR
(DMSO-d6) δ 12.9 (s, 1H), 8.04-8.02 (d, J = 8.0 Hz, 2H), 7.81-7.79 (d, J = 8.0 Hz, 2H), 7.74-7.72
13
(d, J = 8.0 Hz, 2H), 7.75-7.48 (t, 2 H), 7.44-7.42 (d, J = 8.0 Hz, 2H); C NMR (DMSO-d6)
δ 167.5, 144.7, 139.5, 130.4, 130.0, 129.5, 128.7, 127.4, 127.2. Anal Calcd for C13H10O2 C, 78.77;
H, 5.09 found C, 78.78; H, 5.10. (Known compound, see: K. M. Dawood, A. Kirschning
Tetrahedron 2005, 61, 12121).
3l: m.p: 97.9-98.4 °C; IR (KBr) 2923, 2360, 2341, 1673, 815 cm-1; 1H NMR (CDCl3) δ 8.06−8.0 (d,
J = 8.0 Hz, 2H), 7.68-7.66 (d, J = 8 Hz, 2H), 7.59-7.57 (d, J = 8.0 Hz, 2H), 7.47-7.45 (d, J = 8.0
13
Hz, 2H) 2.66 (s, 3H); C NMR (CDCl3) δ 197.8, 141.2, 136.6, 135.1, 134.2, 129.4, 127.6, 29.3.
Anal Calcd for C14H11ClO C, 72.89; H, 4.81; found C, 72.85; H, 4.78
1
3m: m.p: 120.6-121.4 °C; IR (KBr) 1685, 1605, 1396, 1356, 1322, 823 cm-1; H NMR (CDCl3)
13
8.09-8.07 (d, J = 8.0 Hz 2H), 7.75-7.70 (m, 6H), 2.67 (s, 3H); C NMR (CDCl3) δ197.6, 144.2,
143.4, 136.6, 129.1, 127.6, 127.5, 125.9, 26.73. Anal Calcd for C15H11F3O C, 68.19; H, 4.20 found.
C, 68.20; H, 4.21. (Known compound, see: B. H. Lipshutz et al. Org. Lett., 2008, 10, 4279).
3n: mp: 153.3-154.5 °C; IR (KBr) 1673, 1600, 1294, 1818 cm-1; 1H NMR (CDCl3) δ 8.03−8.01 (d,
J = 8.0 Hz, 2 H), 7.67-7.65 (d, J = 8.0 Hz, 2 H), 7.61-7.58 (d, J = 8.0 Hz, 2 H), 7.03-7.01 (d, J =
13
8.0 Hz 2H), 3.88 (s, 3H), 2.64 (s, 3H); C NMR (CDCl3) δ 197.7, 159.9, 145.4, 135.4, 132.3,
129.0, 128.4, 126.6, 114.4, 55.4, 26.6; Anal Calcd for C11H10OS C, 69.44; H, 5.30; found C, 69.50;
H, 5.35. (Known compound, see: C. C. Tzschucke et al, Angew.Chem. Int. Ed.. 2002, 41, 4500).
1
3o: m.p.:152.7-153.9 °C; IR (KBr) 1683, 1594, 1508, 1338, 1263 cm-1; H NMR (CDCl3)
δ 8.34−8.32 (d, J =8.0 Hz, 2H), 8.11-8.09 (d, J = 8.0 Hz, 2H), 7.80-7.83 (d, J =8.0 Hz, 2H), 7.75-
7.73 (d, J =8.0 Hz, 2H), 2.67 (s, 3H); 13C NMR (CDCl3) δ 197.4, 147.7, 146.2,143.1, 137.2, 129.2,
128.1, 127.7, 124.3, 26.5. Anal Calcd for C14H11NO3 C, 69.70; H, 4.60; found C, 69.73; H, 4.64.
3p: Oil °C; IR (neat) 3345, 3060, 1681, 1604, 1267 cm-1; 1H NMR (DMSO-d6) δ
13.23 (s, 1Η), 8.04−8.02 (d, J = 8.0 Hz, 1H), 7.99-7.92 (m, 3H), 7.73-7.71 (m, 1H), 7.50-7.25 (s,

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1H), 7.23-7.21 (s, 1H) 2.60 (s, 3H); C NMR (DMSO-d6) δ 198.6, 168.6, 140.9, 137.4, 134.7,
134.0, 132.8, 130.5, 128.6, 127.6, 127.4, 94.5, 27.3. Anal Calcd for C15H12O3, C, 74.99; H, 5.03;
found C, 74.97; H, 5.06.
1
3q: m.p.: 97.8-98.6 °C; IR (KBr) 2923, 2853, 1607, 1586, 1500, 1287, 1245, 806 cm-1; H
13
NMR(CDCl3) δ 7.25-7.21 (m, 6H), 6.95 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H), 2.27 (s, 3H); C NMR
(DMSO) δ 158.5, 141.5, 135.5, 134.4, 130.3, 130.2, 129.9, 126.9, 125.7, 113.5, 55.3, 20.5. Anal
Calcd for; found. C14H14O C, 84.81; H, 7.12; found C, 84.75; H, 7.07. (Known compound, see: Tao,
Bin; J. Org. Chem, 2004, 69, 4330).
3r: m.p.: 47.8-48.2: °C; IR (KBr) 3340, 1409, 1084 cm-1; 1H NMR (CDCl3) δ, 8.03 (d, J = 8.5 Hz,
2H), 7.69 (d, J = 8.5 Hz, 2H), 7.62–7.65 (m, 2H), 7.46–7.49 (m, 2H), 7.36–7.42 (m, 1H), 2.64 (s,
3H); 13C NMR δ 198.1, 146.1, 140.2, 136.2, 129.3, 129.2, 128.6, 127.6, 127.5, 27.0; MS (m/e) 196
(M+), 181, 152, 127, 102, 91, 76. Anal Calcd for; found. C14H14O C, 84.81; H, 7.12; found C, 84.75;
H, 7.07. (Known compound, see: G. E. Robinson, J. M. Vernon, J. Chem. Soc. C. 1971, 3363.).
Typical procedure for the one-pot chemoenzymatic synthesis of chiral biaryl alcohols
1-Chloro-4-iodobenzene (59.61 mg, 0.25 mmol), 0.05 mol% of Pdnp/Te-Dps, and 4-
acetylphenylboronic acid (40,9 mg, 0.25 mmol.) in TRIS (2 mL, pH = 8.9, 100 mM) were orbitally
stirred for 24 h at 100 °C with a Heidolph Synthesis System. After cooling the reaction to room
temperature, 500 µL of 2-propanol, (R)-LB-ADH (200 U/mg, 414 µL), and NADP+ (8.6 mg,
0.01mmol) were added and the mixture was stirred for 24h and at room temperature. After this
time, the liquid phase was extracted with CH2Cl2, the organic layer was washed with deionized
water and dried over Na2SO4. The solvent was removed under vacuum and the residue was purified
by chromatography (SiO2, 35 g, n-Hexane/AcOEt 70/30 v/v) to give 51.6 mg (89% yield) of
1
(R)-4a: m.p.:42.5-43.5 °C; IR (KBr) 3338, 1405, 1085, 898, 835 cm-1; H NMR (CDCl3) δ, 7.62-
13
7.60 (d, J = 8.5 Hz, 2H), 7.46-7.41 (m, 5H), 4.97–4.93 (m, 1H), 1.72 (s, 1H), 1.56 (s, 3H); C
NMR δ 144.8, 140.8, 140.5, 128.7, 127.3, 127.1, 125.8, 70.2, 25.1. Anal Calcd for; found. C14H14O
20
D
+50 (0.1, CHCl3), ee>99% ( 28 -43.7 for the (S)-
[
α
]
[
α
]
C, 84.81; H, 7.12; found C, 84.77; H, 7.09.
D
enantiomer, 99% ee; J.-i. Ito, S. Ujiie, and H. Nishiyama Organometallics 2009, 28, 630.)
1
(R)-4b: m.p: 97.5-96.5 °C; IR (KBr) 3313, 2973, 1486, 1390, 815 cm-1; H NMR (CDCl3) δ
7.58−7.52 (m, 4H), 7.48-7.46 (d, J = 8.0 Hz, 2H), 7.43-7.41 (d, J = 8.0 Hz, 2H), 4.98 (m, 1H), 1.84
13
(s, 1H), 1.52 (s, 3H); C NMR (CDCl3) δ 145.4, 139.4, 139.2, 133.4, 128.9, 128.3, 127.1, 126.0,

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20 +32 (0.1,
[
α
]
70.1, 25.2 Anal Calcd for C14H13ClO C, 72.89; H, 4.81; found C, 72.84; H, 4.77.
CHCl3), ee>99%
D
(R)-4c: m.p: 115.7-116.5 °C; IR (KBr) 3369, 2979, 2360, 1396, 1128, 823 cm-1; 1H NMR (CDCl3)
δ 7.71 (s, 4H), 7.62-7.60 (d, J = 8.0 Hz, 2H), 7.51-7.49 (d, J = 8.0 Hz, 2H), 5.00 (m, 1H), 2.08 (s,
13
1H), 1.58-1.56 (d, J = 8.0 Hz, 3H); C NMR (CDCl3) δ 145.9, 144.4, 138.9, 129.5, 129.2, 127.4,
127.3, 126.1, 125.7, 125.6, 122.9, 70.0, 25.2 Anal Calcd for C15H13F3O C, 67.66; H, 4.92; found C,
20 +27 (0.1, CHCl3), ee>99%.
[ ]
α
67.60; H, 4.94.
D
Typical procedure for the determination of the enantiomeric excess of 4a and 4c
The enantiomeric excess was determined by enantioselective HPLC on the Chiralpak AS-H chiral
stationary phase using the mixture n-hexane-2-propanol 95/5 (v/v) as a mobile phase. In these
conditions, the (R)-enantiomers of 4a-4c were eluted before the (S)-enantiomers. The enantiomeric
peak identification was carried out by spiking racemic forms of biaryl alchohols with the
enantiomers obtained from asymmetric reaction. The chromatographic data were: k1 (R)-4a = 1.25,
k2 (S)-4a = 1.51, α = 1.21, Rs = 2.63; k1 (R)-4b = 1.57, k2 (S)-4b = 1.97, α = 1.25, Rs = 3.34; (R)-4c
= 1.76, k2 (S)-4c = 2.08, α = 1.18, Rs = 2.58. k1: retention factor of the first eluted enantiomer,
defined as (t1 – t0)/t0 where t0 is the void time of the column; α: enantioselectivity factor defined as
k2/k1; Rs: resolution factor defined as 2(t2-t1)/(w1+w2) where t1 and t2 are retention times and w1 and
w2 are band widths at the baseline in time units. Other analytical chromatographic conditions: flow-
rate, 1.0 mL/min; temperature, 25 °C; detector: UV at 254 nm.
Figure S1. TEM image of Pdnp/Te-Dps
negatively stained with PTA and 2x
magnification in the inset.

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2
1
4c
0
-1
-2
2
1
4b
0
-1
-2
-3
2
1
4a
0
-1
-2
-3
225 240 255 270 285 300 315 330 345
Wavelength (nm)
Figure S2. CD spectra of 4a-4c in ethanol