Transcript High-Field NMR Experiments in the Upper

High-Field NMR Experiments in the
Upper-Level Laboratory Courses at
Furman University
Tim Hanks, Moses Lee and
Larry Trzupek
Dept. of Chemistry, Furman University
Current NMR Instrumentation
Varian EM-360A (1981; $31,000) CW; H-1 only
Varian VXR-300S (1988; $329,000) FT; H-1/C-13
and multinuclear; VT; computer system updated to
Sun SPARCstation 5 in 1996
Varian Inova 500 (1996; $454,000) FT; indirect
detection probe, H-1 {N-15/P-31}; VT; Sun SPARC
station 5 computer system.
Calendar/Chemistry Curriculum at Furman
Fall (12 we e k s, 50 mi n .)
C h e m 11 (Prin cipl e s)
[Wint er t erm also]
C h e m 22 (Mol e cu l ar
S tru ctu re )
[Spring term also]
C h e m 33 (An al ytical)
C h e m 43 (Bi ological
C h em is try)
W in ter(8 wk s , 75 min .)
S pri n g (12 wee k s , 50 mi n.)
C h e m 12 (In organ ic/
Th ermo/Kin e ti cs)
C h e m 21 (O rgan ic)
[Spring term also]
[Fall t erm also]
C h e m 23 (Experi me n tal Te ch ni qu e s)
C h e m 31 (Ph ys ical)
C h e m 34 (P-C h e m IIExp. Te ch n iqu e s II)
C h e m 32 (S tru ctu re
an d Re acti vi ty)
C h e m 44 (Advan ced
Bi och e mi stry)
Note : C las se s typi cal ly me e t eve ryday of th e wee k , Mon day th ru Friday
Enantioselective Epoxidation (Hanks; Chem 23)
NMR Techniques:
- H-1 NMR analysis of fructose-based intermediates,
-methylstyrene, and epoxide product
- C-13 NMR analysis of the chiral catalyst and epoxide
product
- use of chiral lanthanide shift reagent for determination
of enantiomeric purity
Enantioselective Epoxidation (Hanks; Chem 23)
Background:
O
KHS O5 (as O XO NE)
C
H 3C
O
C
H 3C
CH 3
then,
O
O
CH 3
O
C
C
C
H 3C
O
CH 3
C
C
Enantioselective Epoxidation (Hanks; Chem 23)
Preparation of Precursor to a Chiral Dioxirane
H 3C
O
OH
O
OH
O
O
C
H 3C
HO
CH 3
CH 3
HClO4
OH
O
OH
OH
O
H 3C
CH 3
(D-fru ctos e )
H 3C
CH 3
O
O
O
O
O
O
H 3C
CH 3
O
(86%)
(PC C )
(33%)
Enantioselective Epoxidation (Hanks; Chem 23)
Enantioselective Epoxidation Using the Fructose-Based Dioxirane
H 3C
CH 3
O
O
O
O
H
HSO5
C
O
Ph
O
CH 3
H
(+ e nan tiome r)
O
H 3C
C
CH 3
H 3C
CH 3
O
O
O
H
HSO4
O
O
C
C
Ph
O
O
H 3C
CH 3
(reaction medium: 1:2 (v/v) CH3CN / CH 3 OCH2OCH3 ; H2O, 0.05 M in Na 2B4 O7
0.004 M in EDTA, and 0.004 M in (n-Bu) 4HSO4 .
CH 3
H
Enantioselective Epoxidation (Hanks; Chem 23)
H-1 NMR analysis, trans--methylstyrene, vinyl region
H
C
Ph
C
CH 3
H
6.6
6.4
6.2
Enantioselective Epoxidation (Hanks; Chem 23)
C-13 NMR analysis, chiral oxirane precursor
H 3C
CH 3
O
O
O
O
O
O
H 3C
CH 3
180
140
100
60
20 ppm
Enantioselective Epoxidation (Hanks; Chem 23)
NMR determination of enantiomeric purity using Eu(hfc)3
O
H
C
Ph
H 3C
CH 3
C
wi th
H
CH 3
CF2CF2CF3
Eu
O
H 3C
O
3
4.8
4.4
4.0
3.6
3.2 ppm
Enantioselective Epoxidation (Hanks; Chem 23)
NMR determination of enantiomeric purity; results
Typical yield of epoxide product: 60%
Typical enantiomeric excess: 84%ee
Reference: “Catalytic Asymmetric Epoxidation Using a
Fructose-Derived Catalyst”; Andy Burke,
Patrick Dillon, Kyle Martin and Tim Hanks,
J. Chem. Ed., accepted for publication, 1999.
Structure of a Tricyclic Compound (Lee; Chem 23)
Features:
- Microscale preparation
- Multi-step reaction sequence
- Use of basic 2-D NMR to establish structure
Initial Reactants:
cyclopentadiene, maleic anhydride
Target Compound:
endo-9-methoxycarbonyl-3-oxatricyclo[4,2,1,0]-2-nonane
Structure of a Tricyclic Compound (Lee; Chem 23)
Reaction Sequence
O
H
+
O
O
H
O
O
O
H2 O
H
H
H
H2 SO4
H CO 2H
CO 2H
CO 2H
O
O
1) S O C l2
H
2) C H3 O H
H CO 2CH 3
O
O
(yield: 40 - 70%)
References: W. J. Shepard, J. Chem. Ed., 40, 40-41 (1963);
L. F. Fieser and K. L. Williamson, Organic Experiments, 7th ed., D. C. Heath, pp. 283-294 (1992)
Structure of a Tricyclic Compound (Lee; Chem 23)
COSY spectrum of tricyclic product
7
6
5
4
1
8
3
O
9
H
H CO CH
2
3
2
O
Reference: “The Microscale Synthesis and the Structure Determination of Endo-9-methoxycarbonyl-3-oxatricyclo[4,2,1,0]-2-nonane”; M. Lee, J. Chem. Ed., 69, A172-A173 (1992)
Detailed NMR Characterization (Trzupek; Chem 34)
Goals:
to develop
- a basic understanding of 2D NMR methods
- the ability to carry out 2D experiments independently
- the ability to process 2D data productively
- a facility with the interpretation of such data
Requirements:
assignment of
- all H-1 resonances (chemical shift, mulitplet pattern)
- all C-13 resonances (chemical shift)
- all H-H coupling constant values
- all C-H coupling constant values
Detailed NMR Characterization (Trzupek; Chem 34)
NMR Techniques Available:
- simple H-1 spectrum
- resolution-enhanced H-1 spectrum
- proton decoupled H-1 spectrum
- use of lanthanide shift reagents
- relay COSY
- multiple quantum filtered COSY
- homonuclear 2D-J
- simple C-13 spectrum
- heteronuclear 2D-J
- HETCOR
-spectral simulation (H-1)
Detailed NMR Characterization (Trzupek; Chem 34)
Sample requirements:
- ready availability (commercial or easily prepared)
- good purity, solubility
- overlapping proton resonances
- complex splitting patterns
- manageable molecular size (5 to 8 types of H’s)
Typical candidates:
H
C
H
H
H
H
C
H 2C
H
C
CH 2
C
H
CH 3
C
H
H
C
H 2C
CH 2
OH
H
H
N
OH
O
(5-hexen-2-one)
H
(3,4-pentadien-1-ol)
(8-hydroxyquinoline)
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 3,4-pentadien-1-ol: COSY spectrum
HE HD
HD
C
H
D
HE
C
C
H 2C
B
C
CH 2
OH
A
HC
HB HA
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 8-hydroxyquinoline
HC
HD
HB
HA
HE
HF
N
OH
9
8
7 ppm
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 8-hydroxyquinoline: homonuclear 2D-J
HC
HD
HB
HA
HF
HE
HE
HB
HD
HF
N
OH
HC
HA
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 5-hexene-2-one: heteronuclear 2D-J
H
H
Ca C c
H
Hd
H 2C
d
Hf
He
e
CH 2
Cg
CH 3
f
O
(CDCl3)
Ha
Hc
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 3,4-pentadien-1-ol: H-1 simulation
H
C
H
D
HC
HE
D
C
HA
C
B
H 2C
C
CH 2
OH
A
HE
HD
HB
(simulated)
(actual)
5
4
3
2 ppm
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 5-hexene-2-one: HETCOR
HB
HA
Cf Cd
H
Cc
C
Ca C c
H 2C
D
d
Ce
Ca
e
CH E2
Cg
F
CH 3
f
O
HC
HA,B
HE
HF
Detailed NMR Characterization (Trzupek; Chem 34)
Results, 5-hexene-2-one: student report sheet
HB
HC
Ca C c
HA
H
e
E
H 2C d CH 2
D
Cg
O
F
CH 3
f
A
B
C
D
E
F
chem shift
multiplet
5.04
4.97
5.82
2.33
2.55
2.16
C
chem shift
a
c
d
e
f
g
114.9
136.7
27.5
42.5
29.7
207.8
tdd
tdd
ddt
tddd
t
s
J(x,y)
J(x,y)
J(x,y)
AB, 2.2 AC, 17.0
AB, 2.2
BC, 10.4
AC, 17.0 BC, 10.4
BD, 1.4
CD, 6.6
DE, 7.4
AD, 1.1
BD, 1.4
CD, 6.6
DE, 7.4
J(H)
160 (A,B)
156 (C)
122 (D,D)
124 (E,E)
128 (F,F,F)
---
(all chemical shifts in ppm; all J values in Hz)
3D structure of AZTMP by NMR (Lee; Chem 44)
Background:
- bioactivation of AZT:
AZT ---1---> AZTMP ---2----> AZTDP ---3---> AZTTP
- reaction rate of step 2 (thymidylate kinase) - v. slow
- consequence: build-up of AZTMP; imbalance in the
nucleoside pool (the basis of AZT toxicity)
Goal:
- to determine if the solution conformation of AZTMP is
significantly different from that of AMP and if that difference might be the basis for the sluggish kinase reaction
3D structure of AZTMP by NMR (Lee; Chem 44)
NMR techniques employed:
- H-1 spectrum
- P-31 spectrum
- COSY analysis
- homonuclear decoupling
(use of the above to assign proton chemical shifts and obtain H-H coupling constants throughout the molecule)
- determination of T1 relaxation time values for each H
- acquisition of NOE difference spectra
(use of the above to obtain non-bonded distances between
selected protons in the molecule)
3D structure of AZTMP by NMR (Lee; Chem 44)
AZTMP; H-1 spectrum in buffer (20:1 D2O/DMSO-D6)
H 3C
O
(DMSO-d5)
=O P-O
3
5’
O
4’
3’
N3
T-H6
1’
2’
NH
N
H
O
(HOD)
H1’
T-CH3
8
6
4
2 ppm
3D structure of AZTMP by NMR (Lee; Chem 44)
Peak assignments; COSY spectrum of AZTMP
H-1’
H 3C
O
=O P- O
3
5’
O
4’
3’
N3
1’
2’
N
H
NH
O
3D structure of AZTMP by NMR (Lee; Chem 44)
H 3C
O
J-values by homonuclear decoupling
=O P- O
3
5’
O
H-2’
4’
H-2”
3’
N3
1’
2’
N
H
NH
O
H-2”
H-2’
Decoouple
At H-3’
========>
2.40
2.36
//
2.24
2.20 ppm
2.40
2.36 // 2.24
2.20 ppm
3D structure of AZTMP by NMR (Lee; Chem 44)
Dihedral angles from the Karplus relationship
H 3C
O
=O P- O
3
5’
O
4’
3’
N3
1’
2’
N
H
NH
O
H-H coupling
J (Hz)
degrees
1’-2’
1’-2”
2’-3’
2”-3’
3’-4’
4’-5’
4’-5”
5’-5”
5’-P
5”-P
7.0
7.0
5.5
5.5
3.7
2.2
2.9
14.0
6.1
4.6
136
20
30
130
128
57
53
------
3D structure of AZTMP by NMR (Lee; Chem 44)
Additional conformational features from the J-values
H 3C
O
=O P-O
3
5’
O
4’
3’
N3
1’
2’
NH
N
O
H
(P-O5'-C5'-C4') torsional angle
PO 3=
H5'
H 5"
O
C 4'
(O5'-C5'-C4'-C3') torsional angle
t conformation (  = 180 o )]
O 4'
from J H5',P and J H5",P
population in t conformer
equals 70 +/- 10%
[+ conformation (  = 60 o )]
O 5'
C 3'
H5"
H5'
H4'
from J H4 ',H5 ' and J H4 ',H5 "
population in + conformer
equals 90 +/- 10%
3D structure of AZTMP by NMR (Lee; Chem 44)
Inversion-recovery method for the determination of T1’s
H 3C
O
=O P-O
3
5’
O
4’
3’
1’
2’
N3
N
H
NH
O
*
0.9
(* = H-1’)
0.7
*
0.5
*
0.3
*
0.1
*
3D structure of AZTMP by NMR (Lee; Chem 44)
Graphical use of inversion-recovery data to get T1 values
*
0.9
(* = H-1’)
0.7
*
0.5
*
0.3
*
0.1
*
3D structure of AZTMP by NMR (Lee; Chem 44)
Determination of the glycosidic torsional angle, 
- obtain NOE’s for irradiation at thymine H-6
- use known H-6/CH3 distance, NOE,
and T1 to obtain molecular correlation time, c
H 3C
O
H
O 3P-O
O
- use c thus determined, other NOE’s,
and other T1’s to get other distances
N
H
N3
NH
O
3D structure of AZTMP by NMR (Lee; Chem 44)
Results: solution-phase conformational structure of AZTMP
Comparison to structure of TMP: very similar; conclusion:
some other factor (steric bulk of azido group) responsible
for poor interaction with the thymidylate kinase.
M. Lee, J. Chem. Ed., 73, 184-187 (1996)
Acknowledgments
- National Science Foundation
- Keck Foundation
- Milliken Foundation
- Furman Chemistry Alumni
- Furman University