Rapid Metal-Catalysis in Organic and Medicinal Chemistry

Uppsala, October 21, 2004

Mats LarhedUppsala University

Organic Pharmaceutical ChemistryDepartment of Medicinal Chemistry

Swedenmats.larhed@orgfarm.uu.se

Disposition

• Introduction• Metal-mediated microwave-heated organic reactions

– Instant chemistry– Carbonylations– Aminoindans– AT2 selective peptidomimetics– Heck reactions– Plasmepsin inhibitors– Pulsed microwave irradiation - PCR

• Acknowledgement• Extra material

Larhed et al. Drug Discovery Today 2001, 406Larhed et al. Acc. Chem. Res. 2002, 717Olofsson et al. Microwaves in Organic Chemistry, Ed.Loupy, Wiley VCH, 2002, 379Ersmark et al. Current Opinion in Drug Discovery & Development, 2004, 417

Microwave Flash-Heating. Pressurized System

• 2450 MHz• Two mechanisms:

– dipole oscillation – ionic conduction

• Rapid heating• Superheating• In situ heating• Non-thermal effects?

20

40

60

80

100

120

140

0 30 60 90 120 150 180 210 240 270 300 330Time (s)

Tem

pera

ture

(o C)

500 W x 15 s120W x 30s90 W x 60 s30 W x 120 s10 W x 300 s 5 W x 300 s

Kaiser et al. J. Organomet. Chem. 2000, 2Gabriel et al. Chem. Soc. Rev. 1998, 213

CH3CN bp 81-82 oC

Microwave-Assisted OrganicChemistry

• Pressurized system• Solvent-free (open vessel reactions)• Reflux system• Continuous flow system

Kaiser et al. Angew. Chem. 2000, 3595.

Instant Pd-Catalyzed Asymmetric Alkylation

Ph Ph

OAc

MeO OMe

O O PPh2

O

NtBu

Ph Ph

MeO OMe

O O

[Pd], Microwaves(Smith prototype)

+

30 s 120 W 99% eeYield 97% TOF 3500 h-1

• The reaction time with microwave-heating was15-60 s, as compared to 3 h at room temperature

• No detected decrease in enantioselectivity with microwave heating, 99% ee

Kaiser et al. J. Organomet. Chem. 2000, 2

Instant CarbonylationChemistry

RI Co2(CO)8

R

O

RMicrowaves 6-10 s, rt → 130 oC

• 6-10 s Irradiation time• 10 Examples• 57-97% Isolated yields• No product without heating Enquist et al. Org. Lett. 2003, 4875

Instant CarbonylationChemistry

• Temperature, pressure and powerprofiles for the 10 s synthesis ofbenzophenone with0.66 or 0.44 equiv Co2(CO)8

• The IR-thermometer lags behind

• Higher temperaturewith higher amountof Co2(CO)8

Enquist et al. Org. Lett. 2003, 4875

Instant Urea Synthesis

R NH2Co2(CO)8

Et3N, CH3CN Microwaves 10 s to 40 min

NH

NH

OR R2

NR

CO

• Isocyanate as key intermediate:

Enquist et al. In preparation

Instant Urea SynthesisENTRY STARTING

MATERIAL TIME

TEMP (OC)

UREA PRODUCT ISOLATED YIELD (%)

1 NH2

10 s

Flash N

HNH

O

83

2 NH2

10 s

Flash

HN

HN

O

83

3 NH2

10 s (10 min)

Flash N

HNH

O

70 (75)

4 NH2

O

40 min

150

HN

HN

OO O

46

5 NH2

20 min

120 N

HNH

O

61

6 NH2

20 min

120 N

HNH

O

38

7 NH2O

10 s

Flash N

HO N

H

O

O

86

8 NH2

10 s

Flash N

HNH

O < 5

Enquist et al. In preparation

Microwave-Assisted in situGeneration of Carbon Monoxide

In principle:

• Combinatorial chemistry applications limited to manipulations of solids and liquids

• Microwave-assisted organic transformations limited to manipulations of solids and liquids

Can controlled release of gases from solid or liquid reagents be achieved?

An Improved in situ Amino-Carbonylation Protocol – Chemical

CO-liberation• Cr(CO)6 80%• Mo(CO)6 84%• W(CO)6 77%• Fe3(CO)12 0%• Co2(CO)8 28%

• DBU mediated CO release

• Improved method with sluggish amines and amino acids

Wannberg et al. J. Org. Chem. 2003, 5751Wan et al. J. Comb. Chem. 2003, 82Wan et al. J. Org. Chem. 2002, 6232Kaiser et al. J. Comb. Chem. 2002, 109

FAB-MS

M(CO)6 Yield

X

Mo

O

O

OO

O

O

HNR'R''

Mo

N

N

OO

O

O

N

N

O

NR'R''

2 CO

[Pd], Mo(CO)6

Air, 15 minDBU, THF

+

10 equiv DBU+

R RX = I, Br23 examples35-95%

Fast Syntheses of Esters by in situCarbonylation

O

OBr

O

O

F3C

O

O

HOR2

O

O R2

Pd/C or palladacycle

Mo(CO)6

150-190 oC15-20 min

+X

R1

R1

23 Examples, 33-89% yield

OO

O

OSiBr

O

O

O

OOSi

O

O

O

O

O

O

O

O

O

OBr

O

O

F3C

OO

• Fast palladium-catalyzed ester synthesisfrom aryl iodides and bromides under air

• Focus on common protecting groupsO

O

OSi

O

O

F3C

SiO

OSi

Georgsson et al. J. Comb. Chem. 2003, 350

Solid Pase in situ CarbonylationReactions

O

NHS I

O O

O O

NH2S

NH

O

OO

O

NH

OI

O O

NH2O

OO

64%

1) Benzyl alcohol Mo(CO)6, DMAP Pd(OAc)2

150 °C, 15 minMicrowaves

2) TFA:DCM

71%

1) Benzylamine Mo(CO)6, DBU Pd(OAc)2

150 °C, 15 minMicrowaves

2) TFA:DCMWannberg et al. J. Org. Chem. 2003, 5751

Georgsson et al. J. Comb.Chem. 2003, 350

In situ Aminocarbonylation of Aryl Chlorides

ClR

NH

OR'

NH2R'

R+

[Pd]Mo(CO)6

170 oC, Air15-25 min

50-89%10 Examples

(SmithSynthesizer, Biotage AB)

• The combination of Fu-salt, [(t-Bu)3PH]BF4, and Herrmann’spalladacycle results in a highly active and thermostable catalytic system suitable for microwave chemistry

Lagerlund et al. In preparation

Catalyst Recycling in Carbonylation Chemistry with Fluorous Ligand

I

Pd(OAc)2, DBU,

Hydrazide, air P(PhCH2CH2C8F17)3

Mo(CO)6

THF, CF-84

NH

HN

OPh

O

Pd(P(PhCH2CH2C8F17)3)nMicrowaves, 5 min, 110 °C

+

NH

NH2

O

I

THF

micro-wave

P(Ph-C 2H 4

F 17) 3

Pd(OAc)2

FC-84

THF

Pd(P(P

hC 2H 4

C 8F 17

) 3) n

NH

HN

OO

FC-84

coolingRecycling 6 timeswith 64-79 % yield

Herrero et al. Synlett, 2004, 2335

Enamides in Reductive Heck Chemistry

OH

NHR

R

ROTf

Br

Br

NR' O

R''

NR' O

R''

Mo(CO)6Pd(OAc)2 Fu-salt Microwaves

Pd(OAc)2

R

NR'

O

R''

O

+dppp

8 examples26-63%

8 examples36-84%

P2 Indanol amine

• Carbonylative / reductive Heck cyclizations with in-situ CO iiirelease

Wu et al. J. Org. Chem. ASAP

Target Proteases

HIV-proteaseC2 symmetric dimer

Concern: Active mutants

Plasmepsin IIConcerns: 1) High flexibility

2) Similar to Cat D

Microwave-assisted synthesis of protease inhibitors:Alterman et al. J. Med. Chem. 1999, 3835; Schaal et al. J. Med. Chem. 2001, 155;Nöteberg et al. J. Med. Chem. 2003, 734; Nöteberg et al. J. Comb. Chem. 2003, 456;Ersmark et al. J. Med. Chem. 2004, 110; Ax et al. J. Med. Chem. AcceptedFor a summary see: Ersmark et al. Current Opinion in Drug Discovery & Development, 2004, 417

High-Speed Microwave-Assisted Library Generation

Wannberg et al. In preparation

NH

NHOH

OH

O

O

O

O

NH

ONH

O

I

I

NH

NHO

O

O

O

O

O

NH

ONH

O

I

IOO

NH

NHO

O

O

O

O

O

NH

ONH

O

(H)R'N

O

NR'(H)

O

R

R

NH

NHOH

OH

O

O

O

O

NH

ONH

O

(H)R'N

O

NR'(H)

O

R

R

10-CSA

CH2Cl2

AmineMo(CO)6

Pd(OAc)2DBU, THF

100 oC, 15 min

HCl /Diethylether

MeOH2 h

HIV-1 proteaseinhibitors

Biological Evaluation Against HIV-1 Protease and Work in Progress

NH

O

NH

O

NH

ON

S

NH

O

NH

O

NH

O

NH

O

NO

NO

O

NO

NH

ON

NH

O

N

653 nM

668 nM

1840 nM

863 nM

7.0 nM

16 nM

23 nM

>5000 nM

602 nM

392 nM

3.1 nM

678 nM

177 nM

65 nM

170 nM

1.6 nM

298 nM

7.2 nM

>5000 nM

ortho Ki meta Ki

O

HN N

H

HN

O

O

O

O

OH

OHNH

O

R1

R1

O

HN N

H

HN

O

O

O

O

OH

OHNH

O

R2

R2

R1

Metal-catalyzedintroduction ofmetabolically stableR-groups

Wannberg et al. In preparation

New previously “unknown” iiibinding site exploited?

High-Speed Microwave-Assisted Library Generation of HIV-1 Protease Inhibitors

Wannberg et al. In preparation

O

HN N

H

HN

O

O

O

O

OH

OHNH

O

O

HN N

H

HN

O

O

O

O

OH

OHNH

O

Microwaves15 min

I

IKi= 117 nM

NH

HN

O

O

O

O

OH

OHHO

OH

Ki= 4.1 nM

[Pd]

• Introduction of metabolicallyiiistable amide isosteres• Indanol amide optimal P2/P2´?

24 Examplesof coupling products

One-Pot Generation of Protected Aminoindanones

CHO

OTf

O

OH

NH

R''R'

N R''R'

OORR

NHR

OH

+ +

Pd(OAc)2 dppp, PMP

80 °C

R R' R''H4-methoxy6-methoxy

butylisoamylisobutylethylethylbenzyl

butylisoamylisobutylcyclohexylbenzylbenzyl

51-71%

NH

OOH

H

R' R''

+

Bengtson et al. J. Org. Chem. Accepted

• Three-component annulation• Immonium ion intermediate

Via

P2 Indanol aminein Indinavir

Dibenzylamine as an Ammonia Equivalent

CHO

OTf

O

OH NH2

OO

NH NH2

OO

+ +

Pd(OAc)2 dppp, PMP

90 °C

Pd/C, HCO2NH4 20 min, 100 °C

Microwaves

Bengtson et al. J. Org. Chem. Accepted

NH

OH

O

ONH

O

O

Compare

D29

D30

D29

D30

High-Speed Syntheses of ArylTriflates

R

OH O

H Tf2NPh, K2CO3R

OTf O

HMicrowaves 6.0 min, 120 oC

• 10 Different aryl triflates• 69-91% Isolated yields

Bengtson et al. Org. Lett. 2002, 1231

A New Route to 4-(2-Alkyloxyethyl)phenol by Terminal Arylation of Vinyl Ethers

Strategy:

NO2

Cl

OH

OR

NH2

OR

OHN CH(CH3)2

OH

OR

β1 selective β-blockers

Key Step

• Key Step - Regioselective Heck coupling with p-nitrophenyl f chloride and alkyl vinyl ethers followed by in situffhydrogenation? Datta et al. In preparation

The Development of Regioselective HeckArylations of Vinyl Ethers with Aryl

Chlorides

R

Cl

OBu R

OBuMicrowaves

[(t-Bu)3PH]BF4[Pd], Base

αβ

60-78% yields

• Key Methodology – Rapid reaction optimization withffautomated microwave reactor• More than 200 reaction conditions investigated• (t-Bu)3P both activates aryl chlorides and controls ffthe regioselectivity Datta et al. In preparation

Generation of Grignard Reagents from Aryl Chlorides by Microwave Heating

Nilsson et al. In preparation

Entry Ar-X Temp 1 Time 1 Temp 2 Time 2 Yield 2

Cl

150 °C

1 h

100 °C

30 min

80%

4

Cl

150 °C

1 h

100 °C

30 min

86 %

5

Cl

150 °C

1 h

100 °C

30 min

62%

6

Cl

150 °C

2 h

100 °C

30 min

Trace

7

Cl

O

150 °C

1 h

100 °C

30 min

73%

8

NCl

160 °C

2 h

100 °C

30 min

30%

ArCl + Mg 1) Iodine Ar-MgCl 2) Benzaldehyde Alcohol

Victor Grignard

Electron-rich aryl chlorides may serve as starting materials for theGrignard reaction!

Chelation-Assisted Heck Reactions

• Basic concept: Use the ability of intramolecularfactors to overcome the reluctance of substituted alkenes to participate in Heck couplings

• Chelation-accelerated presentation of the oxidative addition complex

PdX

L

LPd X

Z

IntramolecularHeck reaction

Chelation-acceleratedHeck reaction

Z

Nilsson et al. J. Am. Chem. Soc. 2001, 8217Nilsson et al. J. Am. Chem. Soc. 2003, 3430Svennebring et al. J. Org. Chem. 2004, 3345

Chelation-Controlled Heck Vinylation

PdPR3

R3P

Vinyl

Nx

O

PdPR3

NxVinyl

OONxPdPR3

PR3Vinyl

ONxVinyl

Pd(0) / PR3

Vinyl OTfONx

π-complex

σ-complex

β-elimination

chelation andreorganization

vinylpalladium(II) complex

Diels-Alderproducts

Dienophile

ONMe2 O

NEt2

ON

OTf OTf

OTf

OTf

MeO

N

OTf

Ph

OTf

Vinyl triflates

Chelating vinyl ethers

Stadler et al. Adv. Synth. Catal. Accepted

Microwave-Heated Terminal Heck Vinylations

Product ProductIsolated Yield Isolated Yield

ON

N

O NR2

Ph

ONR2

ONR2

ONR2

ONR2

ONR2

MeO

ON

R = MeR = Et

R = MeR = Et

R = MeR = Et

R = MeR = Et

R = MeR = Et

R = MeR = Et

30 %

28%

60%64%

59%65%

51%51%

47%45%

50%44%

46% 55%

Conditions: 1 mol% Pd(OAc)2, 3 mol% PPh3, DMSO, 120 °C, 30 min, 0.2 mmol scale. E/Z ~75:25

DFT-Calculations

• Calculated Coordination strengths

PdPR3

NxVinyl

O

PdPR3

Vinyl

ONx

dissociated π-complex B´

π-complex B

∆E

∆E [kcal/mol]

LigandPR3

ChelatingN-auxiliaries

PH3 PH3 PH3

22.7 18.9 17.128.6 25.0 23.2

ON O

NO

NN N

N

PH3 PH3 PH3

15.1 10.7 4.1

PPh3 PPh3 PPh3

Stadler et al. Adv. Synth. Catal. Accepted

Competitive Experiments• Experimental support for computational results

Comp. Alone

ONMe2

OTf

ONEt2

ONEt2

ONMe2

++

50 % (60 %)

17% (64 %)

ONMe2

OTf

ON

ON

ONMe2

+ +

11 %

---

(60 %)

(30 %)

ONEt2

OTf

ON

ON

ONEt2

+ +

10 % (64 %)

--- (30 %)

Generation of the StartingOrganopalladium(II) Intermediate

Pd XM X

Pd XH

Pd XX

[Pd(II)]

[Pd(II)]

[Pd(0)]

Transmetallation

Electrophilic palladation

Oxidative addition

Oxidativecoupling

Classiccoupling

• In the classic Pd(0)-catalyzed Heck coupling Pd(0) is regenerated after every turnover

• In order for the Pd(II)-mediated Heck couplings to be catalytic; areoxidant must be added to generate Pd(II) from Pd(0)

Larhed et al. Handbook of Organopalladium Chemistry for Organic Synthesis, E. Negishi, Ed.; John Wiley & Sons, Volume I, 2002, 1133

Oxidative Heck Couplings

Heck

Pd(0) Pd(II)

Pd(0)

R1

B(R2)2

OxidativeHeck

Ar1 XAr1

R1

Ar2

R1

Suzuki

Ar1 Ar2

Ar2

• Boronic acids readily undergoes transmetallation

• Microwave-assisted Oxidative Heck chemistry?

• Which reoxidant to use?

• Can we use ligands to improve the per-formance of the catalyst?

Relation between the classic Heck, the Oxidative Heck and the Suzuki coupling

Microwave-Assisted Oxidative Heck Couplings

O

O

O

O

O

MeO

O

O

O

OF

F F

FF

F F

FF

FF

FFO

O

O

O

O

F

F F

FF

F F

FF

FF

FF

B(OH)2

5-30 minMicrowavesSmith Synthesizer

+

45-85%R

EWG

R

EWG5% Pd(OAc)2200% Cu(OAc)2

Andappan et al. Mol. Div. 2003, 97

F

F F

FF

F F

FF

FF

FFO

O

Fe

O

O

OHC

O

O

O

OO2N

O

O

OMe

O

O

O

O

O

O

Medium-Scale Oxidative Heck Couplings with Microwaves

Andappan et al. J. Org. Chem. 2004, 5212

B(OH)2

N

ON

O

[Pd(II)]

5% Pd(OAc)2, 6% dmphen

αβ

~3 bar O2, EtCN + [Pd(0)]

Oxidation

dmphenNN

• Dmphen ligand enables direct reoxidation of Pd(0) by O2• Pressurized O2 gas used• Emrys Optimizer, 1 mmol, 80 %, α/β = 93/7 at 100 oC, 1 h• Emrys Advancer, 10 mmol, 66 %, α/β = 97/3 at 80 oC, 1 h

DFT Calculations - Regioselectivity

PdN N CH3H3C

+

NO

R

PdN N CH3H3C

+

R

PdN N CH3H3C

+

R

NO

NO

PdN N CH3H3C

+

R

NO

NO

NO

R

R

β-Product

α-Product

α

β

‡

‡

p-Substituent

∆Eπ ∆E*α ∆E*β ∆∆E*

-OCH3 -27.5 9.8 13.5 -3.7 -CH3 -27.9 12.4 14.8 -2.4 -H -29.0 13.9 15.3 -1.4

-CF3 -30.8 15.3 16.4 -1.1 -CHO -30.8 15.4 15.8 -0.5

-COCH3 -31.2 15.9 16.4 -0.5

Calculated π-complexation energies and insertion barriers for selected p-substituted phenyl rings [kcal/mol]

Andappan et al. J. Org. Chem. 2004, 5212

• Reduced dmphen model; the ...olefin and aryl fully represented• Electron donating groups favor …the α-product• High insertion barrier with electronn…withdrawing substituents

Geometries were optimized at the B3LYP/lanl2dz level

Plasmepsin I and II –Plasmodium falciparum

• Aspartic proteases (common target class in drugdiscovery, e.g. HIV-PR, renin)

• Relatively little research, although the crystal structure of Plasmepsin II has been solved

• Similar to human enzyme Cathepsin D (possible selectivity problem)

Plasmepsin II Cathepsin D

Plasmepsin Inhibitors

• A few lead substances:

1. Ki Plm II, 0.56 nM; Ki Cat D, 21 nM 1 2. Ki Plm II, 4.3 nM; Ki Cat D, 63 nM 2

Important for selectivity Important for activity1 Silva et al. Proc. Natl. Acad. Sci. USA. 1996, 93, 100342 Haque et al. J. Med. Chem. 1999, 42, 1428

3. Generic structure of new inhibitors

HN

OH

NH

ON

NH

O HN

ONH2

O

O

HN

OHN

O

NH

O

O

Cl

O

Basic nitrogen, will promote accumulation in the acidicfood vacuoleSites of variation to

improveactivity/selectivity

NNH2

OHN

OH

NH

R3

O

R1 R2O

R4

Diversity Strategy

HN

OH HN

NH2

O

NH O

R1

O

R2

Diversity byamide formation

Diversity by microwave-assistedPd-chemistry

• Selection of R1 and R2 based on the “maximum-dissimilarity” method

Nöteberg et al, J. Med. Chem. 2003, 734, Nöteberg et al, J. Comb. Chem. 2003, 456

Microwave-Assisted Decorations. R1.

HN

NH2

OHN

OH

NH O

R1

Br

HN

NH2

OHN

OH

NH O

R1

(HO)2B

Pd(PPh3)2Cl2140 °C, 20 min

28 - 46 %

FF

FF

F

OHN

O

NN

N

N

OO

HN O

OCF3

HN

N

O OO O

O O OO

OO

xNöteberg et al, J. Comb. Chem. 2003, 456

Ki (Plm I / nm) = 2Ki (Plm II / nm) = 120Ki (Cat D / nm) = 1400

Microwave-Assisted Lead-Optimization

(HO)2B

N

OHO

O

(HO)2B

(HO)2BCF3

CF3

(HO)2BNH2(HO)2B

HN O

OO

(HO)2B

(HO)2B

OHO

N

N

(HO)2BO

O S

(HO)2B

HN

NH2

OHN

OH

NH O

ON

Br

HN

NH2

OHN

OH

NH O

ON

R2

NO

(HO)2B

(HO)2B (HO)2B

O

(HO)2B

NH2

SH

O

BO

O

(HO)2B

BrZn

R2-B(OH)2

Pd(PPh3)2Cl2140 °C, 20 min

20 - 51 %

*

x xxxNöteberg et al, J. Comb. Chem. 2003, 456

Ki (Plm I / nm) = 13Ki (Plm II / nm) = 30Ki (Cat D / nm) = 1400

*Other reaction conditions: Pd(PPh3)2Cl2, THF, 130 °C, 30 min

Plasmepsin I and II Inhibitors

Not selective:

NNH O

HN

NH O

HN

NH2

O

O

Br

OH O O

S1

spacer

water P1

Improved inhibitors by extension of the P1’ arm ?

Ki (nM), Plm I = 0.5 Plm II 2.2, Cat D 4.9

S1’Attach spacer-P1’?

Johansson et al. J. Med. Chem. 2004, 3353

Decoration of Plasmepsin Inhibitors

H

H

NH

NOH

OHO

OO

OHO

OH

R''

R''

NH

NOH

OHO

OO

OHO

OH

Br

Br

O

O

NH

NOH

OHO

OO

OHO

OH

R'

R'

ON

O

F

OOS

N

N

O

O

NH

NOH

OHO

OO

OHO

OH

R

R

Si

KF

SONOGASHIRAPd(PPh3)4, CuI, HNEt2, DMFa) 120oC, 10 min, 91% yieldb) 90oC, 30 min, 46% yield

SUZUKIPd(PPh3)4, R'-B(OH)2, Na2CO3 DME:H2O:EtOH (12:4:3)90oC, 30 min, 33-63% yield

HECKHerrmann's palladacycle DIEA, DMF:H2O (17:3)a) 170oC, 25 min, 46% yieldb) 150oC, 30 min, 84% yield

R= R'=R''=

Ersmark et al. J. Med. Chem. 2004, 110

Ki (Plm I / nm) = 1.4Ki (Plm II / nm) = 29Ki (Cat D / nm) =2000

Selectivity!

Vinyl bromidesIndanol amide

Microwave-Assisted PCRM 1 2 3 4 5 M

Lane Microwave power pulses

Template DNA No. of cycles

Relative band intensity

1 75W/120W Plasmid 25 0.11 2 90W/120W Plasmid 25 0.42 3 100W/130W Plasmid 25 0.67 4 100W/130W Chromosomal 35 0.20 5 Conventional

PCR Plasmid 25 1.0

• Manual protocol iii“proof-of-principle”• Faster heating cycles

with microwaves • 25 cycles of

amplification in 1 h –lll50 × 15 s irradiationlllpulses• Large-scale ikautomated PCR?

Fermér et al, Eur. J. Pharm. Sci. 2003, 129

Polymerase chain reaction (PCR) is a technique which is used to amplify the number of copies of a specific region of DNA

Experimental PCR Set Up

• Automated milliliter-scale amplifications?Orrling et al, Chem. Commun. 2004, 790

Coolingairstream

Fluoroptictemperature measurement

IR-pyrometer

Controlledmicrowaves

Single-modemicrowaveapplicator

Coolingairstream

Fluoroptictemperature measurement

IR-pyrometer

Controlledmicrowaves

Single-modemicrowaveapplicator

Microwave-Assisted ThermocyclingMicrow ave-heated PCR, 2.5 mL

0

10

20

30

40

50

60

70

80

90

100

0 1000 2000 3000 4000 5000 6000Time (s)

Tem

p (o

C)

4 05 0

6 07 0

8 09 0

10 0

2 8 5 0 3 0 0 0 3 15 0 3 3 0 0

IR -s ens o r

F O -p ro b e

Microwave-Heated PCR, 2.5 mLTe

mp

(°C

) IR-pyrometer

FO-probe

• 2.5 mL scale in Emrys Optimizer EXP• Amplification efficiency over 95%• One microwave pulse per cycle Orrling et al, Chem. Commun. 2004, 790

Microwave-Assisted Thermocycling

Microwave-Heated PCR, 15 mL

40

50

60

70

80

90

100

4000 4200 4400 4600 4800 5000Time (s)

Tem

pera

ture

(°C

)

4 Cycles out of 33 cycles

• 15 mL microwave-assisted thermocycling• 33 cycles with more than 95% efficiency• No detected deactivation of the Taq polymerase• ~0.1 mg DNA – 150 times increased amplification scale

Orrling et al, Chem. Commun. 2004, 790

Acknowledgement

– Dr. Murugaiah Andappan– Dr. Daniel Nöteberg– Dr. Peter Nilsson– Dr. Karl Vallin– Jennie Georgsson– Johan Wannberg– Andreas Svennebring– Per-Anders Enquist– Gopal Datta– Kristina Orrling– Anna Bengtson– Maria Antonia Herrero– Olle Lagerlund

– Prof. Anders Hallberg– Prof. Christina Moberg– Prof. Bertil Samuelsson– Prof. Åke Pilotti– Dr. Mathias Alterman– Dr. Kristofer Olofsson– Dr. Yiqian Wan– Dr. Ulf Bremberg – Dr. Nils-Fredrik Kaiser– Dr. Xiongyu Wu– Dr. Alexander Stadler

Acknowledgement

• Swedish Foundation for Strategic Research• The Swedish Research Council• Knut och Alice Wallenbergs Foundation• Biotage AB• Medivir AB