applications of desorption electrospray ionization (desi)

Applications of Desorption Electrospray Ionization (DESI)

Richard H. Perry

Department of Chemistry

Stanford University Mass Spectrometry

September 26, 2011

Presented at the 2011 Stanford Mass Spectrometry Users’ Meeting

For personal use only.Please do not reuse or reproducewithout the author’s permission.

1

ReactantsProducts

Catalytzed Uncatalyzed E

I1 I2

Reaction Coordinate

DH

Ea

What are the identities of I1 and I2?

Identifying intermediates is critical for understanding chemical reactivity

A + B → I1 → I2 → P

Mass spectrometry (MS) overcomes many of these limitations

METHODS • Optical Spectroscopy

• NMR • Crystallography

• …

DISADVANTAGES OF THESE METHODS • Relatively long timescales

• Poor sensitivity • Require relatively pure samples

CHALLENGES • Short timescales

• Low concentrations • Complex systems

• Side reactions

GOAL Develop a simpler and cleaner approach for studying reaction mechanisms with

millisecond time resolution

DISADVANTAGES OF THESE METHODS • Not easily amenable to high-throughput

• Carry-over effects

Continuous-Flow Reaction times of 5 ms

MSESIA

B

Z. W. Takats et al. Science 2004, 306, 471-473. R. G. Cooks et al. Science 2006, 311, 1566-1570.

•Ionization in open environment → Ambient MS •No sample preparation

•High throughput •Simple

Desorption Electrospray Ionization (DESI)

R. H. Perry et al. Angew. Chem. Int. Ed. 2011, 50, 250-254.

+ +

+ + + +

+ + +

A

Phase Transfer

+ + + +

+ + + + A B

+

+ +

+

+

+ + +

+

A

+ +

+ + + +

+ + +

A

Reaction time of a few ms

100 m/s 4 mm

+ + + +

+ + +

+ + + +

+ + +

A+

B+

P+

I1+

I2+

4 m/s 2 mm

A B I2

P I1 A B

+ + + +

+ + + +

+ + + +

+ +

+ + + +

+ + + +

I1

Quenched

A + B → I1 → I2 → P

Using DESI to Access the Millisecond Timescale

Reaction occurs in solution!

R. Noyori and S. Hashiguchi, Acc. Chem. Res. 1997, 30, 97-102. M. Palmer et al. J. Org. Chem. 1997, 62, 5226-5228.

Ru (II)-Promoted Hydrogen Transfer

CATALYST

Kenny et al. Chem. Commun. 2000, 99 Haack et al. Angew. Chem. Int. Ed. 1997, 36, 285

Kenny et al. Chem. Commun. 2000, 99-100 Haack et al. Angew. Chem. Int. Ed. 1997, 36, 285

• Previous bulk ESI-MS studies identified these species Structures supported by x-ray crystallography results using diamines

200 400 600 800m/z

0

100 384

Ru-amide + Ru-H

Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.

MSESIb-Amino Alcohol

Ru Complex

Reaction completes in a few seconds

HVN2

45o

5 mm

2 mm


CTH by DESI

b-Amino Alcohol

Ru Complex

400 500 600 700 8000

100579

384 762

460420 634

m/z

Rel

ativ

e In

tens

ity

Ru+

Cl

OHCH3OHCH3

Ru

OHCH3Cl

Cl

H2O

H2O

NH3+

OH

ClRuCl

RuCl

ClNH3

+

HO

ClRu+

Cl

RuClCl

NH2

HOOH2OH2


Ru-amide + Ru-H

Ru-Cl

• First intermediate of the reaction!!

Orbitrap Mass Spectra m/Dm = 80,000

Mass accuracy = 0.3 ppm

0

50

100

755 758 761 764 767 770

Rel

ativ

e In

ten

sity

m/z

755 760 765 7700

100761.994

m/z

Ru+

Cl

OHCH3OHCH3

Ru

OHCH3Cl

Cl

H2O

H2O

NH3+

OH

ClRuCl

RuCl

ClNH3

+

HO

ClRu+

Cl

RuClCl

NH2

HOOH2OH2

Confirming Peak Assignments

Isotope Ratio %Error < 5%

Black: Calculated Grey: Experimental

Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250 Perry et al. Angew. Chem. Int. Ed. 2011, in preparation

400 500 600 700 8000

100579

750

372 448408634R

elat

ive

Inte

nsity

m/z

Ru-Cl Ru+

Cl

OHCH3OHCH3

Ru

OHCH3Cl

Cl

H2O

H2O

NH3+

OH

ClRuCl

RuCl

ClNH3

+

HO

ClRu+

Cl

RuClCl

NH2

HOOH2OH2

OH

NH2

Dm/z = 12 Da

Dm/z = 12 Da Another intermediate!!


Ru-amide + Ru-H

Relative –NH2 and –OH positions are necessary for reactivity


• Detected species are relevant to the CTH reaction • DESI can intercept intermediates on short timescales!!

579

750

448408

400 500 600 700 8000

100

634

m/z

Rel

ativ

e In

tens

ity

372

No CTH intermediates observed when NH2 is farther away from –OH

400 500 600 700 8000

100579

384 762

460420 634

m/z

Rel

ativ

e In

tens

ity


These species are present regardless of the

ligand.

576.934 Da 572.984 Da 569.033 Da

558.968 Da 555.017 Da

541.002 Da

565.082 Da

523.035 Da

Intermediates Formed from Reaction with the Solvent

Provides additional evidence that DESI can intercept rapid

reaction steps

Perry et al. manuscript in preparation

563 575

572.985

569 581

576.004

m/z

~3 Da Shift

629 638m/z

633.963

622 636m/z

630.937

Confirmation of the intermediates formed with methanol


512 530

523.044522.0 522.1

522.052522.039

522.0 522.1

522.039

512 530

522.039

m/z m/z

D2O

Confirmation of the intermediates formed with H2O


761.995 Da (0.2 ppm)

726.018 Da (0.7 ppm)

690.041 Da (0.4 ppm)

Summary


MILLISECONDS

• Indicates an associative mechanism • Observe step-wise breakdown of the dimer • Intermediates with CH3OH and H2O observed • Detected species agree with proposed catalytic

mechanisms

What can we learn about transfer hydrogenation catalysis?

350 450 550 650 7500

100

Rel

ativ

e A

bund

ance

576.9360

384.0902460.1064

763.9937

634.8921690.0424

726.0178550.0696

444.1118

420.0670

What are the identities of m/z 444 and m/z 460?


• Oxidative dehydrogenation of methanol to generate methyl formate

• Adventitious O2 oxidizes Ru (II) to Ru (IV)

Ru-Methyl Formate Intermediates

CH3OHO2 +

Ru(IV)

ON

H

O

H OO

H

Ru(II)

ON

H

O

HO

H

Ru(II)

ON

H


m/z 444.1118 (1.1 ppm)

m/z 460.1064 (0.3 ppm)

NMR

8.0 6.0 4.0 2.0 0.0 -2.0 -4.0 -6.0d (ppm)

H

OO

H3C

HO

OH3C

[Ru] ̶ H

8.05

3.68

-5.86


0

100 577

762460

384420

350 450 550 650 750m/z

0

100

Rel

ativ

e In

tens

ity

577

535 762

420

Rel

ativ

e In

tens

ity

535

384

a)

b)

Oxidation of Ru (II) to Ru (IV) by Adventitious O2

Normal

Ar-Degassed


100

Rel

ativ

e A

bund

ance

384

260 500m/z

100

Rel

ativ

e A

bund

ance

384

460

a) MS2 of m/z 444

b) MS2 of m/z 460

MS/MS


Ru(II)

ON

H

O

HO

H

Ru(IV)

ON

H

O

H OO

H

m/z 384

Normalized Collision Energy = 40%

Putting it all together…


• DESI can intercept intermediates on short timescales (millisecond regime)

• Detected new Ru (II) reaction intermediates in transfer hydrogenations catalyzed by (b-amino alcohol)(arene)Ru complexes

• Detected intermediates formed in the reaction with CH3OH and H2O

• Detected a Ru-methyl formate species that suggests formation of methyl formate in CTH

What we discovered…

Moving forward…

Combining the timescales of DESI and ESI provides a more complete picture of reaction pathways

Other Catalytic Reactions

Transfer Hydrogenation Water Oxidation C-H Amination

Many of the proposed intermediates of these catalytic systems short-lived and have not been directly observed

C-H Amination

12345789

Compound [M]+·

m/z (ppm)[M+Na]+

m/z (ppm)758.0808 (2.6)

1186.9057 (2.4)

781.0724 (0.1)

249.8863 (2.6)

344.9591 (1.1)

451.8144 (1.0)

383.9959 (1.6)

1006.9618 (0.7)

1615.7342 (0.3)

O

OO

O

RhO

O

Me

MeMe

Me

MeMe Me

MeRh

O

O

Rh2(esp)21

HNS

OCH2CCl3

Cl3CCH2OSO2NH2 PhI(O2CCH3)232

Cl3CCH2OSO2N=IPh4

5

6

PhI

9

8

H

7

CH2Cl2

O O

RhRhN

O O

RhRhN N

O O

RhRhN

O O

RhRhHNS

O

ORO

RO(O)2S

PhI

RO(O)2S

PhI S(O)2OR

IPh

Adamantane

O O

SO

ORO

Cahill, Perry, Roizen, Davis, Du Bois, and Zare. manuscript in preparation.

• Nitrenoid reacts through an H-atom abstraction pathway

DESI

Perry et al. manuscript in preparation.

High-Throughput Screening of Catalytic Mechanisms

Blank

Ru-amide + Ru-H

Ru-Cl

Catalyst Screening

Ligand Screening Air-sensitive reactions

Graphite electrode

Electrocatalysis 1.1 V – 1.2 V

Methanol Base

HVN2

45o

5 mm

2 mm

Solvent

DESI-MS and Electrocatalysis

K. R. Brownell et al. manuscript in preparation.

GOAL Probe catalytic reactions on surfaces in real-time

• This electrocatalytic reaction results in methanol oxidation • Transfer hydrogenation catalysts can store and release

energy for the electron economy

Another application of DESI…f Desorption

Biological Tissue Imaging MS

Deciphering the relationship between lipids and the c-MYC gene network…

The Many Faces of c-MYC

Vita and Henriksson, Semin. Cancer Biol. 2006, 16, 318

It is critical to understand the mode of action of MYC

Giuriato et al. Semin. Cancer Biol. 2004, 14, 3-11.

Studying c-MYC Expression in the Body Conditional Transgenic Mouse Models

tTA Tetracycline System

Doxycycline (dox)

MYC ON MYC OFF

Felsher, D. W.Cancer Res. 2008, 68, 3081

b) Complete reversion

a) Partial regression

c) Differentiation

e) Apoptosis

d) Arrest

f) Loss of signals to normal cells

Normal

Tumor

Outcomes of c-MYC Inactivation What are the factors and

mechanisms that lead to the these different outcomes?

MYC ON

MYC OFF

Felsher, Cancer Res. 2008, 68, 3081

Outcomes Depend on Tissue Type

How does the tissue location affect the mechanism of c-MYC-induced cancer progression and regression?

Lipids, the Warburg Effect, and Cancer

Menendez and Lupu, Nat. Rev. Cancer 2007, 7, 763

What is the relationship between lipids and the c-MYC oncogene network?

A simple approach to the problem…maybe…

Spatial Distribution Distinguish lipids that are specific

to tumors and normal tissue

c-MYC Expression • Wild Type • MYC ON • MYC OFF • MYC BACK ON

Tissue type Disease Stage

Lipid Composition CHEMICAL IMAGING Ideal to acquire simultaneously

• Dissecting tissue • Extraction • HPLC-MS

DESI Imaging

x

y

Rastering Direction

DESI Source

MS Inlet

Tissue or Cell

Ionization area

Ifa et al. Int. J. Mass Spectrom. 2007, 259, 8 Eberlin et al. Angew. Chem.-Int. Edit. 2010, 49, 873

MS DESI Source N2 Supply

Sample stage Extended

Capillary

Glass slide with tissue section

Previous studies show that DESI imaging is great for detecting lipids in tissues

What type of information can we obtain using DESI imaging MS?

m/z 215 m/z 511

m/z 583

Ion

Inte

nsity

Tumors ‘Normal’ (Adjacent)

Lipid Species Only Present in Normal Tissue Liver: c-MYC Activated

for 4 Months

• Images show that each species has a different intensity profile across the tissue

m/z 256 m/z 328 m/z 443 m/z 392

m/z 537 m/z 609 m/z 886

Lipid Species Present in Normal and Cancer Tissue

m/z 282 m/z 318 m/z 772 m/z 810

m/z 563 m/z 748 m/z 820 m/z 796

Lipid Species Present in Both Tumor Regions

m/z 320 m/z 499

m/z 513 m/z 466

Lipid Species Present in Only One Tumor Tissue

Different genotypes?

Lipid Species in Wild Type Tissue

m/z 215 m/z 511 m/z 583

m/z 772 m/z 796m/z 282

m/z 466 m/z 499m/z 320

Down-regulated in tumors

Up-regulated in tumors

Optical Image

200 300 400 500 600 700 800 900 1000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

885.75002

537.16668255.58334

561.16668511.33334810.58335327.33334

723.66668465.66668 909.41669

200 300 400 500 600 700 800 900 1000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

215.16667

885.50002269.33334 537.25001389.41667 794.66668 933.25002657.16668

Comparison of Wild Type and Adjacent Tissues

m/z 215 m/z 537 m/z 886

Adjacent

Wild Type Plotted on the same intensity scale

m/z 292 m/z 364 m/z 417

Wild Type

Adjacent

Plotted on the same intensity scale

• Cells in the adjacent tissue are affected by the tumors – i.e. not normal

• Species tumors release into the extracellular space?

Late Stage Tumor

m/z 772

m/z 563

m/z 320

Early Late

m/z 466

Early Late

Observe time course of each lipid

Identifying the detected species…

Just a reminder…

Fatty Acids Wild Type

Late Stage Tumor

MYC ON MYC OFF MYC ON 6 wks 4 mos 6 mos

Difference between wild type and adjacent tissue –

Warburg effect?

18:1

2 mos

Wild Type

Late Stage Tumor

MYC ON MYC OFF MYC ON

18:1 16:0 20:3

Monoacylglycerophosphoglycerols

6 wks 4 mos 6 mos 2 mos

Diacylglycerophosphoglycerols Wild Type

Late Stage Tumor

MYC ON MYC OFF MYC ON 6 wks 4 mos 6 mos

Different kinetics!

18:2/20:3 18:1/20:2 20:3/20:4 20:4/22:6

2 mos

Diacylglycerophosphoinositols Wild Type

Late Stage Tumor


18:1/16:1 18:1/18:3

Down-regulation in the entire tissue


18:1/18:1 Diacylglycerophosphoinositol

Wild Type

Late Stage Tumor


Early stage lipid species!


Prof. Richard N. Zare Dr. Konstantin Chingin Dr. Ali Ismail Thomas J. Cahill, III

The people who make anything possible…

Fe and Ru CTH Prof. Robert Waymouth Dr. Antonio De Crisci Kristen Brownell C-H Amination Prof. Justin Du Bois Jennifer Roizen MS Instrumentation Dr. Allis Chien Dr. Pavel Aronov

Ir WOC (Yale team) Prof. Robert Crabtree Prof. Mark Johnson Nathan Schley Biological Imaging Prof. Dean W. Felsher Dr. Emelyn Shroff Dr. Tahera Zabuawala Dr. David Bellovin

applications of desorption electrospray ionization (desi)

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