applications of desorption electrospray ionization (desi)
TRANSCRIPT
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
Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.
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
Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.
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!!
Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.
Ru-amide + Ru-H
Relative –NH2 and –OH positions are necessary for reactivity
Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.
• 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
Perry et al. Angew. Chem. Int. Ed, 2011, 50, 250.
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
Perry et al. manuscript in preparation
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
Perry et al. manuscript in preparation
761.995 Da (0.2 ppm)
726.018 Da (0.7 ppm)
690.041 Da (0.4 ppm)
Summary
Perry et al. manuscript in preparation
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?
Perry et al. manuscript in preparation
• 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
Perry et al. manuscript in preparation
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
Perry et al. manuscript in preparation
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
Perry et al. manuscript in preparation
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
Perry et al. manuscript in preparation
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…
Perry et al. manuscript in preparation
• 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
MYC ON MYC OFF MYC ON
18:1/16:1 18:1/18:3
Down-regulation in the entire tissue
6 wks 4 mos 6 mos 2 mos
18:1/18:1 Diacylglycerophosphoinositol
Wild Type
Late Stage Tumor
MYC ON MYC OFF MYC ON
Early stage lipid species!
6 wks 4 mos 6 mos 2 mos
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