monitoring chemical reactions in real time with nmr spectroscopy
TRANSCRIPT
Monitoring Chemical Reactions in Real
Time with NMR Spectroscopy
Mark Zell,1 Brian Marquez,1 Dave am Ende,1 Pascal Dube,1 Eric Gorman,2 Robert
Krull,3 Don Piroli,3 Kimberly Colson,3 Michael Fey3
1. Pfizer Global Research and Development, 445 Eastern Point Road, Groton, CT 06340
2. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Drive, Lawrence, KS 66047
3. Bruker Biospin, 15 Fortune Drive, Billerica, MA 01821
Introduction
• Many analytical techniques (IR, UV, Raman) are used to monitor
chemical reactions. – For many reactions these techniques show important features of the reaction process
and therefore provide a great deal of information to apply to a process.
– Some features, such as structural isomers and low level impurities are not typically
observed.
– Utilization of NMR provides additional “molecular resolution” at the process development
stage to provide many details about the process (e.g. mechanistic details, kinetics of
impurity formation, etc…)
• On-line NMR is a very powerful tool for monitoring reaction progression as well as
providing mechanistic insight into the synthesis.
• We are exploring ways to add detailed, real-time chemical information to better
assess synthetic reactions and develop a better process understanding.
• This data should be acquired in a non-invasive way to better
understand the chemistry.
Introduction
Goals
Monitor chemical reactions via NMR Spectroscopy.
• Develop a system that can provide a real-time reaction
monitoring capability with value added information for
better process understanding.
Pictures of the “first” System in our lab (~2006)
NMR ProbeInlet and outlet
HPLC Pump
NMR Probe
ReturnReaction Vessel
Temperature controlSyringe Pump
NH2
NH2
HH
O
O
N
N
2 H2O
C12H24N2Mol. Wt.: 196.33
C2H2O2Mol. Wt.: 58.04
C14H22N2Mol. Wt.: 218.34
A Real Life Example: Flow NMR Semi-Batch Reaction
Profile for Alkylation of a Di-amine with Glyoxal
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Flow NMR Flow NMR –– Automatic Analysis in Automatic Analysis in
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Starting
Material
Product
Reaction Profile -- Intermediates
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Intermediate
Introduction
Goals
Monitor chemical reactions via NMR Spectroscopy.
• Develop a system that can provide a real-time
reaction monitoring capability with value added
information for better process understanding.
Next Generation
• Limitations with first system (HPLC/NMR flow probe):
– Fragile – 200 microliter small bore cell.
– If (When) flow cell broke had to be sent back for repair
– Flow rates had to be low to avoid pressure, easily plugged
– No temperature control to/from cell
– 1H observe only
• New Design in 2010:
Flow control unit coupled with a flow cell that will fit in a standard NMR
probe.
Easy to replace if flow cell is broken or plugged
Temperature control ~0 – 80° C (achieved -10° C to date), +/- ~2° C
Goal is to reach ~ -40° C
Standard 1H-19F-13C TXO NMR probe (low gamma probe on order)
5 Gauss Line
~1.5 m
Reactor
Thermostatted Supply
And Return Lines
Control
Unit
Fast Loop
(~ 25 ml/min)
Slow Loop
(~5 ml/min)
New and Improved!
1H, 13C, 19F Triple Resonance NMR Probe
Coming Soon!
ReactIR
Bruker IonTrap MS
New Flow Cell Design
9 8 7 6 5 4 3 2 1 ppm
First data acquired on new flow cell at Bruker. 2 mM sucrose sample.
Spectrum demonstrates good NMR lineshape even with tubing extended
Into flowcell.
5.35 ppm
Optimal Flow rate minimizes lag from
reaction vessel
Acetic Acid 1H Signal depends on the
flow rate through the NMR cell
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Peak A
rea (
arb
itra
ry u
nit
s)
Flow Rate (mL/min)
Effect of Flow Rate on Peak Area
Acetic Acid in D2O
Time to Reach Equilibrium at 5 mL/min
1 mL Acetonitrile in 100 mL D2O
Void volume ~5mL
Acetic Anhydride
O
O O
OH
O
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Time (min)Acetic Anhydride
Acetic Acid
NMR Flow Kinetics~20 ºC and 5 mL/min
Temp (ºC) 103 * kD (sec-1)
NMR tube 25 0.94
Literature ? 0.89
NMR flow ~20 0.82
Lit kH/kD = 2.9
O
O O D2O
OH
O
Quantitative NMR Spectroscopy
• Electronic REference To access In vivo Concentrations (ERETIC)
Method
– Prior to the ERETIC method
• Internal standards were necessary for absolute quantitation
• Potential problems included signal overlap, chemical interaction, and
errors in addition of the internal standard
– ERETIC
• Electronically generated signal injected into the spectrometer to
replace the internal standard
• Requires additional hardware and the reference signal can’t be moved
once the data is collected
– Digital ERETIC (QANTUS, Quantification by Artificial Signal, Upton,
et.al.)
• Synthetic signal added after data collection as part of the data
processing
• Reference signal can be moved anywhere in the spectrum without
recollecting the data and does not require additional hardware
Digital ERETIC Method
Analyte Spectrum (A)
ERETIC Spectrum (E)
Referenced Spectrum (A+E*x)
The ERETIC signal can be placed anywhere in
the spectrum without changing the calibration.
QNMR Potency Measurements
Compound Lot #
Theoretical
Potency
Traditional
QNMR
Digital ERETIC QNMR
Analyte Maleic acid
Benzoic acid NIST 39j 0.999996 --- 1.016 ---
Maleic acid 1296124 >0.99 0.990 --- 0.990
p-Fluorobenzoic acid NIST 2143 0.9995 0.987 1.024 0.994
PF-04971729-G3
L-Pyroglutamic acidGR02878
0.755
0.235
0.733
0.242
0.736
0.2410.996
3-TMS-1-propylsulfonic acid
sodium salt23021EB 1.000 0.917 0.868 0.941
1,4-Bis(TMS)benzene 04202KC 0.995 0.453* 0.460* 1.003
α-D-Glucose, anhydrous 12926AC 0.999 0.768 0.981 1.262
Digital ERETIC signal calibrated against the Maleic acid peaks in the Benzoic acid sample, assuming the purity determined by
the Traditional QNMR method
* = Sonicated several times but not all of the sample dissolved
Esterification reaction
O
OH
F
OHF
F
F
CDI
O
O
F
FF
F
Reaction Conditions:
First reaction:
20 mM para-fluorobenzoic acid circulating in reactor. CDI
and trifluoroethanol both added in excess. Acquired proton
data only (top spectra).
Second Reaction:
20 mM para-fluorobenzoic acid circulating in reactor. Added
CDI and allowed to stir over lunch. Added trifluoroethanol
and no reaction occurred. Added additional (non-
stoichiometric) amount of CDI and reaction proceeded.
Reaction did not go to completion. 19F (and 1H – not shown)
data acquired during this reaction (bottom spectra)
Reaction to be repeated again in the next couple of weeks
after pump variability is resolved.
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Time (min)
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Product
Impurity A
Impurity B
Product
Intermediate
StartingMaterialBenzoic acidMass Balance
Dilution due to additional trifluoroethanol
Esterification reaction
Applicability to Pfizer projects
• This technology is directly applicable to a wide range of internal synthetic projects.
• Previously showed the use of our early flow-NMR system to identify the presence of a transient intermediate in the synthesis of an active ingredient in a currently marketed product.
• The data shown in the following slides is from a current internal project, and is just one of many projects that could benefit from advances in the area of flow NMR technology. – Step 1: Formation of an aryl-metal species– Step 2: The addition of this aryl-metal to a cyclobutanone.
• These reactions are sensitive to moisture and require a careful control of the conditions. Our current understanding of the chemistry is limited by the use of off-line analytical tools.
Temperature • Reactor = -20 °C• Line temperature = -10 °C• NMR probe temperature = -10 °C
• The reaction mixture was circulated through the NMR probe at 5 mL/min.
• 1H, 19F, and 13C spectra were collected at various points during the reaction.
• Further steps were not investigated due to solvent-induced pump leak!
Applicability to Pfizer projects
Aryl bromide
Aryl bromide + isopropylmagnesium chloride
Aryl Bromide + IsopropylmagnesiumChloride + Butyl Lithium (during addition)
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (~10 min after addition)
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (~20 min after addition)
THF-p8
Applicability to Pfizer projects
Aryl bromide
Aryl bromide + isopropylmagnesium chloride
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (during addition)
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (~10 min after addition)
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (~20 min after addition)
19F Spectra (1H Decoupled)
Applicability to Pfizer projects
THF-p8
Aryl bromide
Aryl bromide + isopropylmagnesium chloride
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (during addition)
Aryl Bromide + Isopropylmagnesium Chloride + Butyl Lithium (~20 min after addition)
13C Spectra
Applicability to Pfizer projects
Before Addition
Time Since Cyclobutyl Ester Addition
During Addition
~5 min
~15 min
~25 min
10 min. interval
1H Spectra
Applicability to Pfizer projects
Next Steps
• Reduce the lag time to 30 seconds or less.
– Use of a pump with appropriate seals is critical. (so far using
slow loop at 5 ml/min through 1/16” tubing)
– Improve fast loop operation while keeping approx 5 ml/min
thru the slow loop.
• Further characterize the system at sub-ambient conditions
and ensure adequate thermostating of the lines.
• Extend to catalytic hydrogenations, follow reaction kinetics
without disrupting the gas-liquid equilibrium.
• Continue to extend to new chemistries, unit operations
(distillations, pKa determinations, extractions etc)
• Enable as facilitated walk-up platform