Metal complex catalysis
in Biological conditions
Literature Seminar
2013.12.2 (Mon.)
Takushi Araya
1

Contents
1. Introduction
2. Redox catalyst
3. Pd catalyst
4. Others
5. Summary

Biological condition・buffer (pH7.4)
・protein (many functional groups)
・cell (surface or inside)2

Good points

1. Introduction

・Modification in Living cell・Site specific prodrug activation(・Eco-friendly & safe)Ar

Low H2O & O2 tolerence
Temperature, hv sensitive

Just mixing

3Problematic points

1. Introduction

・Low functional group tolerance
O2
NH2
OHC

CO2H

M-Ln
OH

・Toxicity

H2O
HS

Cl-

e.g.) Fenton reaction

・Low water solubility

J. A. Imalay et al., J. Bacteriol. 2003, 185, 1942-1950

Pd(PPh3)4, Hoveyda-Grubbs 2nd cat. etc…

Ligand must be・protect active site
・water soluble4

Contents
1. Introduction
2. Redox catalyst ・・・

R-SH oxication
Reduction by HCO2-

3. Pd catalyst

Reduction by NADH

4. Others
5. Summary5

2-1. R-SH oxidation (1)-1

2. Redox

Cytotoxic [Ru] complex oxidize GSH to GSSG

24 h

30 min1H NMR spectra of GSH after incubation (37℃) with 4 for(a) 30 min and (b) 24 h

GSSG (mM)(A2780 ovarian and A549 lung cancer cell lines)

Catalytic oxidation of GSH (10 mM) to GSSG by
4 (100 µM; blue) or 5 (100 µM; red) for 24 h, 37℃

6
P. J. Sadler et al. Proc. Natl. Acad. Sci. USA 2008, 105, 11628-11633

2-1. R-SH oxidation (1)-2

2. Redox(Generated ROS amount)[Ru] increases cell ROS level([(ηη6-bip)Ru(en)Cl]PF6)([(ηη6-tha)Ru(en)Cl]PF6)

Incubation of A549 cancer cells with Ru complex (25 µM)

Thiol cancels [Ru] cytotoxicity
After 24 h exposure to ruthenium compounds,
Lighter : 96 h recovery for A549 lung cancer cells
Darker : same but pretreated to increase intracellular thiol level(retreated 5 mM N-Ac-Cys for 2 h)Cisplatin
(5 M)

(5 M)

(1 M)

(5 M)

(5 M)
7
P. J. Sadler et al. Proc. Natl. Acad. Sci. USA 2008, 105, 11628-11633

2-1. R-SH oxidation (1)-3

2. Redox

Ru-X is replaced to Ru-SG

Proposed catalytic cycle

I-

HPLC-analysis
Reaction of GSH (5 mM) with 4 (Ru-I) (a)and corresponding chlorido complex (50 M) (Ru-Cl) (b)(10 mM phosphate buffer (pH 7.9), 95% H2O, 5% MeOH)
After 1 h incubation at 37℃, GS- adduct is major peak
and peaks of starting material have disappeared.

*X is initially I-, which is displaced GS- during the early stage**I--Catalyzed Decomposition of H2O2
J. C. Hansen, J. Chem. Educ., 1996, 73, 728-732
8P. J. Sadler et al. Proc. Natl. Acad. Sci. USA 2008, 105, 11628-11633

2-1. R-SH oxidation (2)-1

2. Redox

Allylcarbamate cleavage by Ru(cod) complex(0.2 M)

yield determined by GC-MS*Presence of excess thiol, no negative influence observed
yield determined by GC-MS
Obtain as byproduct PhS-SPh
9
E. Meggers et al. Angew. Chem., Int. Ed. 2006, 45, 5645-5648

2-1. R-SH oxidation (2)-2

2. Redox

Reaction occurred in cell extract, HeLa cell and E. coli
[Ru], RSH
2.5 h, 37℃

Entry 1: 4 (0.5 mM) in DMSO/H2O (1:1)
Entry 2: 4 (0.5 mM) in DMSO/cell extract (1:1), and GSH (3.5 mM), pH 7.0
Entry 3: Same as entry 2, but with [Cp*Ru(cod)Cl] (100 mµM)
Entry 4: Same as entry 2, but with [Cp*Ru(cod)Cl] (100 mµM) and PhSH (3.5 mM)*Yields determined by fluorescence intensity

Fluorescence imaging of HeLa cellsa) and b): preincubated with 4 (100 mµM) for 30 min, washed with PBS
buffer, then treated with [Cp*Ru(cod)Cl] (20 mµM) and PhSH (500 mµM)a) right after this additionb) after 15 min[Cp*Ru(cod)Cl], NaCl (300 mM)
20 mM phosphate buffer (pH 7.8)c)–f): preincubated with 4 (100 mµM) for 30 min and at same time withmembrane carbocyanine dye DiIC18(5). After washing with PBS buffer,cells were treated with [Cp*Ru(cod)Cl] (40 mµM) and PhSH (100 mµM)c)right after this additiond)–f) after the indicated times.

3 h, rtHistone H2B-Lys27(N-alloc)Lys in E. coli
P. G. Schultz et al, Chem. Commun., 2010, 46, 5506-5508

No cytotoxicity observed (no10figure)
E. Meggers et al. Angew. Chem., Int. Ed. 2006, 45, 5645-5648

2-1. R-SH oxidation (3)-1

2. Redox

Photoactivatable catalyst

2 (0.1 mM)
DMSO:1 mM phosphatebuffer (pH 7.25) = 1:1

hv
5 (0.5 mM)(non-fluorescent)

37℃, 10 min, hv (λ ≥330 nm)
6 (fluorescent)β-mercaptoethanol (ME), PhSH, and cysteine (Cys) were all 5 mM
Yields determined by fluorescence intensity(rhodamine 110: λex 488 nm, λem 520 nm)

Reaction dynamics
2 (0.1 mM), 5 (0.5 mM), β-ME (5 mM), PhSH
(5 mM), DMSO/H2O (1/1), hv (λ ≥330 nm)
Yields determined by fluorescence intensity(pyrene, λex 319 nm, λem 390 nm;
rhodamine 110, λex 488 nm, λem 520 nm)

11E. Meggers et al. Organometallics, 2012, 31, 5968−5970

2-1. R-SH oxidation (3)-2

2. Redox

Inside HeLa cells

after wash

[Ru][Ru], PhSH

[Ru], hv 10 min[Ru], PhSH, hv 5 min[Ru], PhSH, hv 10 min

12
E. Meggers et al. Organometallics, 2012, 31, 5968−5970

2-1. R-SH oxidation (4)2. Redox

Ar-N3 reduction by FeTPP and R-SH

13
E. Meggers et al. ChemBioChem 2012, 13, 1116-1120

2-1. R-SH oxidation (4)Reductant scope

2. Redox
Ar-N3 were reduced in cell

Fluorescence were observed in vivo, due to reductive metabolism of Ar-N3

nametode

zebrafish

14
E. Meggers et al. ChemBioChem 2012, 13, 1116-1120

2-2. Reduction by HCO2H (1)2. Redox

Reductive alkylation by [Ir-H] and HCO2-

1b was best
(1a afford small amount of 2a,
1c gave no product(data not shown))

15
M. B. Francis et al. J. Am. Chem. Soc. 2005, 127, 13490-13491

2-2. Reduction by HCO2H (1)2. Redox

Aldehyde substrate scope

59% overall conversion

16
M. B. Francis et al. J. Am. Chem. Soc. 2005, 127, 13490-13491

2-2. Reduction by HCO2H (2)-0

2. Redox

Precedents

E. Steckhan et al. J. Chem. Soc., Chem. Commun. 1988, 1150-1151

R. H. Fish et al. Angew. Chem. Int. Ed. 1990, 38, 1429-1432
R. H. Fish et al. Inorg. Chem. 2001, 40, 6705-6716

Y. Watanabe et al. Organometallics, 2002, 21, 2964-2969*Ir, Ru complex also reduce NAD+ to NAD catalytically17
P. Stepnicka et al. Eur. J. Inorg. Chem. 2007, 4736-4742

2-2. Reduction by HCO2H (2)-1

2. Redox+[Ru] complex reduces NAD+
Ru
HN

Cl
NH

18
P. J. Sadler et al. J. Biol. Inorg. Chem. 2006, 11, 483-488

2-2. Reduction by HCO2H (2)-2

2. Redox
Turnover frequency (h-1)

Ligand effects
+

H

O

H H
NH2

[Ru] cat. (0.5 eq), HCO2- (25 eq)

O

0.85

NH2
Ru

N+

D2O, pD 7.2, 37°C

R

N

Cl

HN
NH

R

+

0.16
Ru
HN

Cl
NH
+

1:NAD+:HCO2-=1:2:25

0.18
Ru
HN

Cl
NH

0.05

A549 (lung cancer cells)were tolerant to 2.5 mM HCO2(highest concentration in this test, data not shown)

19
P. J. Sadler et al. J. Biol. Inorg. Chem. 2006, 11, 483-488

2-3. Reduction by NADH (1)-1

2. Redox[Ru-H] (also [Ir-H]) reduces pyruvate
+

2+

+

hmb

R

R

Ru
Ru
N

N

Cl

Cl
N

OH2
N

N

hmb

N

Ir

thn

Cp*

p-cym

Cpxph

Ability to form hydride complex
N

N

N

N

N

phen

N

bpm

2+

Ir
N
N

OH2

20
P. J. Sadler et al., Angew. Chem. Int. Ed. 2012, 51, 3897 –3900

2-3. Reduction by NADH (1)-2

2. Redox

Proposed mechanism of hydride migration

Ring slippage
η6 to η4

Ring slippage
η5 to η3

Ring Slip Reaction of Cp*Rh complex

A. D. Selmeczy et al. Organometallics 1991, 10, 1577-1586.

21
P. J. Sadler et al., Angew. Chem. Int. Ed. 2012, 51, 3897 –3900

2-3. Reduction by NADH (2)-1

2. Redox[Ir-H] reduces quinones to…?
2+

R
Ir
N
N

OH2

+ NADH//

broad peak

22
P. J. Sadler et al., Angew. Chem. Int. Ed. 2013, 52, 4194-4197

2-3. Reduction by NADH (2)-2

2. Redox[Ir-H] reduces quinones to semiquinone radical
2+

Ir
N
N

OH2

stable 20 h~

1 NADH reduces 2 quinone
menadione (VK3)
duroquinone

yield 93%
(menadione)

23
P. J. Sadler et al., Angew. Chem. Int. Ed. 2013, 52, 4194-4197

2-3. Reduction by NADH (2)-3

2. Redox

Proposed raction mechanisma) One electron transfer×2 (proposed)b) Two electron transfer

XX/YY is each potential-energy/free-energy difference (kcal・mol-1)
by DFT calculation

24
P. J. Sadler et al., Angew. Chem. Int. Ed. 2013, 52, 4194-4197

Contents
1. Introduction
2. Redox catalyst
3. Pd catalyst ・・・

Mizorogi-Heck
Sonogashira4. Others

Suzuki-Miyaura
Tsuji-Trost

5. Summary25

3-0. History

MizorogiHeck

3. Pd

Yokoyama et al.
proteinbuffer (pH 8.3)
Yield: 2%

Sonogashira
2005

2006

SuzukiTsuji-Trost

Lin et al.
proteinbuffer (pH 8.0), inside E.coli
Yield: ～90%

Yokoyama et al.
proteinbuffer (pH 8.3)
Yield: 25%

Chen et al.
proteinbuffer (pH 7.6)
Yield: ～95%

2009

2011

2012

2013

Davis et al.
proteinbuffer (pH 8.0)
Yield: >95%

Bradley et al.
Small molecule
Inside HeLa cell
Yield: ---Davis et al.
proteinon E.coli surface
Yield: ---Davis et al.
peptidebuffer (pH 8.0)
Yield: <48%Francis et al.
protein
Buffer (pH 8.5)
Yield: 50-65%

26

3-1. Mizorogi-Heck reaction

3. Pd(4 mM)
Pd(OAc)2 (1 mM)
TPPTS (2 mM)
0.1 M TAPS buffer (pH 8.3)
1.6 M DMSO (12% v/v)
5℃, 50 h, w/o O2(p-I-Phe)32-Ras-His (13 μM)

Yield 2%
(28% : dehalogenated,
70% : starting material)by protease→LC-MS/MS

27
S. Yokoyama et al. ChemBioChem 2006, 7, 134-139

3-2. Sonogashira coupling (1)3. Pd(15 mM)
Pd(OAc)2 (1.7 mM)
TPPTS (8.3 mM)
CuOTf (0.7 mM)
0.09 M TAPS buffer (pH 8.3)
2.3 M DMSO (18% v/v)
6℃, 80 min, w/o O2(p-I-Phe)32-Ras-His (13 μM)

Yield 25%
(13%:dehalogenated)by SDS-PAGE,
protease→LC-MS/MS analysis

Detected by Streptoavidin-HRP

28
S. Yokoyama et al. ChemBioChem 2007, 8, 232-238

3-2. Sonogashira coupling (2)-1

3. Pd

Good Suzuki-coupling Ligand in water*
I

+
BocHN

CO2H

Ph

Pd(OAc)2 (1 mol%)
L1 (2 mol%)(HO)2B
1.5 eq

0.2 M phosphate-HCO2H buffer
37°C, 4 h, open air

BocHN

CO2H

95%

And Good Cu-free Sonogashira coupling Ligand
in organic solvent***Described more detail in “3-3. Suzuki coupling (1)“B. G. Davis et al. J. Am. Chem. Soc. 2009, 131, 16346-16347**Y. –X. Xie et al. Eur. J. Org. Chem. 2005, 4256–4259

29
Q. Lin et al. J. Am. Chem. Soc. 2011, 133, 15316-15319
bHPLC yield3-2. Sonogashira coupling (2)-2

3. Pd*Mixture of Ar-I, Pd complex, and sodium ascorbate was stirred at 37℃ for 60 min to activate

Ar-I =

HPG:homopropargylglycine
1a (100 µM)
Pd(OAc)2・L2 (1 mM)
5 mM sodium ascorbate
2 mM phosphate buffer (pH 8)
DMSO (0.1 mM), 37℃, 4 hCentrifugation(Figure a)

fluorescence(Figure b, left)+
Coomassie blue staining(Figure b, right)

HPG-Ub overexpressing E. coli
1 : control (w/o Pd complex)
2 : add 1a and Pd
3 : add preactivated 1a and Pd(stirred at 37℃, 1 h before adding)30
Q. Lin et al. J. Am. Chem. Soc. 2011, 133, 15316-15319
bLC-MS yield3-2. Sonogashira coupling (3)-1

3. Pd

Ligand-free, Pd(NO3)2 shows best conversion
Pd source (100 µM)
w or w/o Ligand (200 µM)
sodium ascorbate (1 mM)+
GFP-alkyne (10 µM)

2 mM phosphate buffer (pH 7.6)
25℃, 1 h
p-I-Phe-FL525 (100 µM)

bcalculated from sample florescence on SDS-PAGE(Pd(NO3)2)

31
P. R. Chen et al. J. Am. Chem. Soc. 2013, 135, 7330−7338

3-2. Sonogashira coupling (3)-2

3. Pd

Pd source (100 µM)
sodium ascorbate (1 mM)
p-I-Phe-Fluor 525 (100 µM)+

1×PBS buffer (pH 7.6)
25℃, 1 hor
CuSO4 (100 µM)
BTTAA (500 µM)
sodium ascorbate (5 mM)

GFP-alkyne (10 µM)

N3-Fluor 525 (100 µM)

1×PBS buffer (pH 7.6)
25℃, 1 h

Pd vs Cu
Protein deactivation

Reaction dynamics

Acute toxicity(100 µM)(100 µM)*Same reaction condition as above*GFP (10 µM) +Pd(NO3)2 (100 µM) or Cu(l) (100 µM)*Pd(NO3)2 (200 µM) or Cu(l) (200 µM)
32
incubate 25℃, 1 h

P. R. Chen et al. J. Am. Chem. Soc. 2013, 135, 7330−7338

3-3. Suzuki coupling (1)

3. Pd

?

33
B. G. Davis et al. J. Am. Chem. Soc. 2009, 131, 16346-16347

3-3. Suzuki coupling (2)3. Pd

Cell-surface labeling (E. coli)

control

p-I-Phe mutant

Fluorescence observed

34
B. G. Davis et al. J. Am. Chem. Soc. 2012, 134, 800-803

3-3. Suzuki coupling (3)-1

3. Pd

A(p-I-F)VNTANST(p-I-F)VNTANSTVNTANST sequence :Comprehensive carcinoma homing peptide
35
B. G. Davis et al. J. Am. Chem. Soc. 2013, 135, 13612-13615

3-3. Suzuki coupling (3)-2

3. Pd

18F・・・PET tracer, t1/2=109 min<1 h total reaction time is desired for PET

18F-DG whole body PET imaging

Source:wikipedia, ポジトロン断層法

36
B. G. Davis et al. J. Am. Chem. Soc. 2013, 135, 13612-13615

3-3. Suzuki coupling (4)-1

3. Pd

Pd0 microsphere synthesis

Powder XRD patterns

Pd0 microsphere
Pd0 powder
Naked microsphere
TEM of Pd0 nanoparticle
37
M. Bradley et al. Nat. Chem. 2011, 3, 241-243

3-3. Suzuki coupling (4)-2

3. Pd

Pd microspheres are taken into cells (24 h, 75%~)

Mitochondria(red)Synthesized compound 5(green)mergedAllow: Pd microsphere(not localized within mitochondria)

38
M. Bradley et al. Nat. Chem. 2011, 3, 241-243

3-4. Tsuji-Trost Reaction

3. Pd
Also see Mr Suzuki’s Literature Seminar (2013)

Y146, Y171

50-65% (by HPLC)
Mainly Y171-alkylated productin chymotrypsinogen A(most accessible)(secondaly accessible)

Chymotrypsinogen A

Rhodamine Dye

Lys-NH2, Cys-SH and other residue were intact

39
M. B. Francis et al. J. Am. Chem. Soc. 2006, 128, 1080-1081

Contents
1. Introduction
2. Redox catalyst
3. Pd catalyst
4. Others ・・・

Photo Cross Linking
OH・ generator

5. Summary

Metathesis
Rh-carbenoid404-1. Photo Cross Linking-1+ MMPP :Ni(lll)4. Others

Ru(lll)Pd(lll)APS : ammonium persulfate((NH4)2S2O8)

41
J. -C. Bonnafous et al. Trends Pharmacol. Sci. 26, 210−217

4-1. Photo Cross Linking-2

4. Others

hv

× ×

(kDa)

+ APS

×× ×
×
(kDa)

・・・

Ru(bipy)32+ (50 µM), APS (1 µM)

・・・

0.5 mM phosphate buffer (pH 7.4)
hv, rt, <1sec

Cross-Linked AβAβ(freshly isolated, low MW)(kDa)(number of Aβ sequence)
42
D. B. Teplow et al. Proc. Natl. Acad. Sci. USA 2003, 100, 330-335

4-2. OH・ generator

4. Others+
Ferrocene
antitumor compound
estrogen receptor antagonist(anti-breast cancer)

compoundoestradiol(Z) TAM-OH(Z)-Fc TAM-OH(E)-Fc TAM-OH

binding affinity (%)
100
107
40
12

IC50 (μM)
6.4
3.4
4.9

G. Jaouen et al. Chem. Commun. 1996, 955-956

More active derivatives are reported

Ferrocene derivatives generate ROS and DNA damage
G. Cavigiolio et al. Inorg. Chim. Acta. 2000, 42-48
in this tissue, relation of OH・ and DNA damage is not associated
But there are many reports to relate them.
c.f.) D. MacNamee et al. J. Biol. Chem. 1974, 249, 2447-2452 43

4-3. Olefin metathesis (1)-1

4. Others

Ester chelates to [Ru] center(M=Ru)
A. Fürstner et al. J. Am. Chem. Soc. 1997, 119, 9130-9136

Chalcogen assisted Cross-Metathesis

44
B. G. Davis et al. J. Am. Chem. Soc. 2008, 130, 9642-9643

4-3. Olefin metathesis (1)-2

4. Others

Minor MS signal of 1+17 ・・・strong chelation?
by LC-MS

MgCl2 disrupts nonproductive chelation of Ru + protein(SBL-S156XXX have no Cys-SH)

45
B. G. Davis et al. J. Am. Chem. Soc. 2008, 130, 9642-9643

4-3. Olefin metathesis (2)-1

4. Others

Less reactive partner→cross-metathesis(entry 1~7)

More reactive partner→self-metathesis(entry 10)

Much less reactive partner→no reaction(entry 8,9,11~14)(Only self-metathesis)

46
B. G. Davis et al. J. Am. Chem. Soc. 2010, 132, 16805-16811

4-3. Olefin metathesis (2)-2

4. OthersConv.
(hindered)>95

Less hindered olefin(RT, 2 h)

30

30

30
65
>95
0
0

0
(Only self-metathesis)
0
0
0

47
B. G. Davis et al. J. Am. Chem. Soc. 2010, 132, 16805-16811

4-3. Olefin metathesis (2)-3

4. Others
Conv.
(S)
>95

S→Se

(RT, 30 min)

>95

>95

>95
>95
>95

S vs Se

29
53
Self-metathesis0(Only self-metathesis)
0
Cross-metathesis
0048
B. G. Davis et al. J. Am. Chem. Soc. 2010, 132, 16805-16811

4-3. Olefin metathesis (2)-4

4. Others

49
B. G. Davis et al. J. Am. Chem. Soc. 2010, 132, 16805-16811

4-3. Olefin metathesis (3)4. Others

Water soluble Metathesis catalysts

PEG : Poly Ethylene Glycol, MW≒2000

50
R. H. Grubbs et al., J. Am. Chem. Soc. 2010, 132, 16805-16811 2006, 128, 3508-3509

4-4. Rh carbenoid

4. Others
Also see Mr Suzuki’s Literature Seminar (2013)

51% (5:6 = 1:1.4)(also same at 10 µM)

Subtilisin Carlsberg (100 µM)

These have no Cys・・・Selective?

51
M. B. Francis et al., J. Am. Chem. Soc. 2004, 126, 10256-10257

Contents
1. Introduction
2. Redox catalyst
3. Pd catalyst
4. Others
5. Summary52

5. Summary[Ru] [Ir] [Rh] (2-2(2))
NAD+

NADH
HCO2[Ir] (2-3(1,2))

O
O
OO

R1

R3

R2

R4

or

O

O-

O
OOH

NAD+

1

R

R3

R2

R4

or

O

53
Figure: http://biologica.concord.org/webtest1/view_3D_cells.htm

Appendix54

2-1. R-SH oxidation (4)Appendix
Ar-N3 is reduced in Liver, microsome

MZP

MAP

: no reduction occured
R. J. Griffin et al. Xenobiotica 1991, 21, 935-943

J. A. Slack et al. Xenobiotica 1988, 18, 1157-1164

Fluorescence observed in vivo, due to reductive metabolism of Ar-N3

in vivo
nametode and zebrafish
nametode

zebrafish

55
E. Meggers et al. ChemBioChem 2012, 13, 1116-1120

2-3. Reduction by NADH (1)
2+

+

R

R

[Ru-H] (also [Ir-H]) complex reduce pyrvate

Ir

Ru
N

Appendix

N

Cl

OH2
N

N

Time course
hmb

thn

Cp*

p-cym

Cpxph

Ability to form hydride complex
N

N

phen

N

N

N

N

bpm

Ability to form [Ru-H]

56
P. J. Sadler et al., Angew. Chem. Int. Ed. 2012, 51, 3897 –3900

2-3. Reduction by NADH (1)Appendix[Ir-H] complex formed by NADH

57
P. J. Sadler et al., Angew. Chem. Int. Ed. 2012, 51, 3897 –3900

2-3. Reduction by NADH (1)Appendix[Ir-H] complex reduces pyrvate
2+

Ir
N
N

・

OH2

・

58
P. J. Sadler et al., Angew. Chem. Int. Ed. 2012, 51, 3897 –3900

2-3. Reduction by NADH (2)Appendix2+

Few semiquinone radical anions (M・-) forms [Ir] adducts

Ir
N
N

OH2

yield 93%
(menadione)

S. Fukuzumi et al. ChemPhysChem 2006, 7, 942-954

59
P. J. Sadler et al., Angew. Chem. Int. Ed. 2013, 52, 4194-4197

3-2. Sonogashira coupling (1)Appendix

Detection by SDS-PAGE

Yield 25%
(13%:dehalogenated)

Condition
in TAPS buffer (0.09 M, pH 8.3)
2.3 M DMSO (18% v/v)
iF32-Ras-His (13 μM)
propagylated biotin (15 mM)
Pd(OAc)2 (1.7 mM)
TPPTS (8.3 mM)
CuOTf (0.7 mM)
6℃, 80min, w/o O260

3-2. Sonogashira coupling (2)E. coli cytotoxicity of Pd complex

Appendix
Each treatment→incubate 4 h→dilute 106→overnight incubation 37℃→measurement

Brank : DMSO 1 %Pd complex : DMSO 1 %, Pd complex 1 mM
w/o Preactivation : DMSO 1 %, Pd complex 1 mM, 1a 1 mM, sodium ascorbate 8 mM
w Preactivation : DMSO 1 %, preactivated [Pd complex 1 mM, 1a 1 mM, sodium ascorbate 8 mM] (stirred 37℃, 1 h before addition)
Ar-I : DMSO 1%, 1a 1 mM

61
Q. Lin et al. J. Am. Chem. Soc. 2011, 133, 15316-15319

3-2. Sonogashira coupling (2)Appendix

The conversions were calculated based on the disappearance of aryl iodides,
whose amounts were quantified by their absorption at 254 nm (red line).

Reagents (except alkene) are mixed then stirred 37℃ 1 h,
separate each half and added twice in all reaction.

Many doubtful points…?

62
Q. Lin et al. J. Am. Chem. Soc. 2011, 133, 15316-15319

3-2. Sonogashira coupling (3)Pd(NO3)2 has no acute toxicity to E. colicf) SDS-PAGE fluorescence yield

AppendixPd(NO3)2 gives few membrane damage

Yield <95% by LC-MS

value of C7 as (r.f.i/r.c.i)max
yield = RF×95 (%)
r.f.i. : relative fluorescence intensity
r.c.i. : relative coomassie stain intensity

Shigella shows almost same results
63
P. R. Chen et al. J. Am. Chem. Soc. 2013, 135, 7330−7338

3-2. Sonogashira coupling (3)2Appendix???

64
P. R. Chen et al. J. Am. Chem. Soc. 2013, 135, 7330−7338

3-3. Suzuki coupling (1)Appendix

Free Cys-SH inhibits coupling65
B. G. Davis et al. J. Am. Chem. Soc. 2009, 131, 16346-16347

3-3. Suzuki coupling (3)Appendix

Toxicity of Guanidine salt
LD50: 500 mg/kg (domestic rabbit)
月刊ファインケミカル 2008年6月号 72-75

Kryptofix 2.2.2
K+ trap66

4-2. Olefin metathesis (3)Appendix

Other water soluble Olefin metathesis catalysts

67
K. Grela et al., Catal. Sci. Technol., 2012, 2, 2424-2427

4-4. Rh carbenoid

4. Others

S-h insertion of Rh Carbenoid

P. Ratananukul et al. Tetrahedron Lett. 1982, 23, 2509-2512

Q-. L. Zhou et al. Chem. Sci. just accepted

Acknowledgement
Dr. Kuninobu and Dr. Nishi
Thanks for kind lecture68