1. Preparation of Isotopically Labeled Ascorbic Acid Precursors to
Better Understand Ascorbic Acid Biosynthesis in vitro and in planta
Patrick L. Cavins1, Susan Z. Lever1,2
1Department of Chemistry, University of Missouri, Columbia MO 65211
2University of Missouri Research Reactor (MURR), University of Missouri, Columbia MO 65211
OBJECTIVES
Nuclide
Symbol
Half-Life
Decay
Mode
Nuclear Spin
Percent
Abundance
14C 5,730 years b- 0+ Trace
13C Stable 1/2+ 1.109%
12C Stable 0+ 98.93%
11C 20.3 mins b+ 3/2+ N/A
• Carbon-13, a stable isotope with favorable mass spectrometry (MS) and nuclear
magnetic resonance spectroscopy (NMR) properties, can be utilized as a surrogate
for C-11 or C-14.
• The Kiliani-Fischer reaction extends the carbon backbone via addition of cyanide; the
reaction produces epimeric carbons.1 These ratios are known to be pH dependent.2
• Optimal conditions where used to produce the preferred epimer of lactones 13C-3
and 13C-6.
This work was supported by the
Plant Imaging Consortium (PIC,
http://plantimaging.cast.uark.edu/),
funded by the National Science
Foundation Award Numbers IIA-
1430427 and IIA-1430428. Any
opinions, findings, and conclusions
or recommendations expressed in
this material are those of the
author(s) and do not necessarily
reflect the views of NSF.
SYNTHESIS OF L-GALACTONO-1,4-LACTONE
INTRODUCTION
ACKNOWLEDGEMENTS
plc.ii.sgr_5LX.1
plc.ii.sgr_5LX.1.1
0.000
0.001
0.002
0.003
−500
0
500
1000
0 5 10 15
0 5 10 15
time
response
channel
plc.ii.sgr_5LX.1
plc.ii.sgr_5LX.1.1
Response(AU)
Time (Min)
L-Xylose and 3
plc.ii.sgr_11_GalLLyx_only
plc.ii.sgr_11_GalLLyx_only.1
0.000
0.001
0.002
0.003
0.004
0
200
400
600
0 5 10 15
0 5 10 15
time
response
channel
plc.ii.sgr_11_GalLLyx_only
plc.ii.sgr_11_GalLLyx_only.1
Response(AU)
Time (Min)
L-Lyxose and 6
Name Retention Time
L-Xylose 8.2 - RI
3 6.9 - RI
L-Lyxose 7.0 - RI
6 5.9 - RI
2 Silent
• Synthesis, purification, and characterization of carbon-13 labelled gulono-1,4-lactone
(13C-3) and galactono-1,4-lactone (13C-6) from the respective pentoses L-Xylose and L-
Lyxose.
• Optimization and characterization of reaction intermediates (1, 2, 4, and 5) required
for successful carbon-11 implementation.
SYNTHESIS OF L-GULONO-1,4-LACTONE
L-Xylose
O
C
OH
OH
OH
HO
*
1
C
OH
OH
OH
HO N
OH
*
K13
CN
H2O, 48 hrs, RT
Ba(OH)2 • 8H2O
2hrs, 75°C
C
OH
OH
OH
HO
OH
*
O
OH
2
*
C
O
OH
HO OOH
OH
13
C-3
18N H2SO4,
2hrs, 75°C
• Epimeric carbons are indicated by bonds; the initial epimeric purity was 82%
(Gulo) and 18% (Ido).
• Two successive methyl cellosolve recrystallizations removed the unwanted epimer.
Response(AU)
CHARACTERIZATION OF L-GULONO-1,4-LACTONE
*
C
O
OH
HO OOH
13
C-6
L-Lyxose
O
C
OH
OH
OH
HO
*
4
*
OH
NHO
OH
OH
OH
CK13
CN
H2O, 48 hrs, RT
Ba(OH)2 • 8H2O
2hrs, 75°C
C
OH
OH
OH
HO
OH
*
O
OH
5
18N H2SO4,
2hrs, 75°C
OH
• Epimeric carbons are indicated by bonds; the initial epimeric purity was 88% (Gal) and 12%
(Tal).
• The presence of 13C-6 lactone is also indicated by the 13C-NMR Spectra (A), HPLC injection (B),
and analytical TLC using 20% H2O in ACN (C).
PREPARATION FOR 11C SYNTHESIS
180 160 140 120 100 80 60 40 20 ppm
62.10
67.20
70.18
70.70
71.23
71.60
81.95
81.97
175.56
178.57
Current Data Parameters
NAME plc.i.sgr.99.2Recry
EXPNO 2
PROCNO 1
F2 - Acquisition Parameters
Date_ 20160504
Time 11.24
INSTRUM spect
PROBHD 5 mm CPTCI 1H/
PULPROG zgpg30
TD 65536
SOLVENT D2O
NS 256
DS 4
SWH 42613.637 Hz
FIDRES 0.650232 Hz
AQ 0.7689557 sec
RG 188.21
DW 11.733 usec
DE 20.00 usec
TE 298.0 K
D1 2.00000000 sec
D11 0.03000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 150.8012132 MHz
NUC1 13C
P1 11.00 usec
PLW1 94.00000000 W
======== CHANNEL f2 ========
SFO2 599.6623986 MHz
NUC2 1H
CPDPRG[2 waltz16
PCPD2 80.00 usec
PLW2 6.30959988 W
PLW12 0.06309600 W
PLW13 0.04038100 W
F2 - Processing parameters
SI 32768
SF 150.7845288 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40
plc.i.sgr.99.2Recry || 04 May 2016
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 ppm
62.10
67.20
70.18
70.70
71.23
71.60
81.95
81.97
Current Data Parameters
NAME plc.i.sgr.99.2Recry
EXPNO 2
PROCNO 1
F2 - Acquisition Parameters
Date_ 20160504
Time 11.24
INSTRUM spect
PROBHD 5 mm CPTCI 1H/
PULPROG zgpg30
TD 65536
SOLVENT D2O
NS 256
DS 4
SWH 42613.637 Hz
FIDRES 0.650232 Hz
AQ 0.7689557 sec
RG 188.21
DW 11.733 usec
DE 20.00 usec
TE 298.0 K
D1 2.00000000 sec
D11 0.03000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 150.8012132 MHz
NUC1 13C
P1 11.00 usec
PLW1 94.00000000 W
======== CHANNEL f2 ========
SFO2 599.6623986 MHz
NUC2 1H
CPDPRG[2 waltz16
PCPD2 80.00 usec
PLW2 6.30959988 W
PLW12 0.06309600 W
PLW13 0.04038100 W
F2 - Processing parameters
SI 32768
SF 150.7845288 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40
plc.i.sgr.99.2Recry || 04 May 2016
3.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.84.9 ppm
3.681
3.682
3.690
3.691
3.702
3.710
3.711
3.749
3.750
3.788
3.794
3.809
3.814
4.056
4.062
4.064
4.066
4.070
4.075
4.076
4.078
4.079
4.085
4.504
4.508
4.518
4.522
4.535
4.536
4.541
4.542
4.544
4.549
4.550
4.554
4.555
4.557
4.558
4.562
4.563
4.733
4.740
4.748
1.11
1.01
0.94
0.99
1.00
1.00
Current Data Parameters
NAME plc.i.sgr.99.2Recry
EXPNO 1
PROCNO 1
F2 - Acquisition Parameters
Date_ 20160504
Time 11.13
INSTRUM spect
PROBHD 5 mm CPTCI 1H/
PULPROG zg30
TD 65536
SOLVENT D2O
NS 16
DS 2
SWH 12019.230 Hz
FIDRES 0.183399 Hz
AQ 2.7262976 sec
RG 32.25
DW 41.600 usec
DE 30.00 usec
TE 298.0 K
D1 1.00000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 599.6637031 MHz
NUC1 1H
P1 8.35 usec
PLW1 6.30959988 W
F2 - Processing parameters
SI 65536
SF 599.6599586 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00
plc.i.sgr.99.2Recry || 04 May 2016
• Carbon-13 (A, B, D) and proton (C) NMR Spectra of 13C-3 containing 0.03% percent dioxane.
• The 13C-DEPT (D) and proton (C) both show unique spectral properties (arrows).180 160 140 120 100 80 60 40 20 ppm
60.79
62.11
67.20
70.18
70.19
70.70
70.71
71.22
71.61
81.96
81.97
178.57
Current Data Parameters
NAME plc.i.sgr.99.2Recry
EXPNO 3
PROCNO 1
F2 - Acquisition Parameters
Date_ 20160504
Time 11.40
INSTRUM spect
PROBHD 5 mm CPTCI 1H/
PULPROG deptsp135
TD 65536
SOLVENT D2O
NS 256
DS 4
SWH 36231.883 Hz
FIDRES 0.552855 Hz
AQ 0.9043968 sec
RG 188.21
DW 13.800 usec
DE 20.00 usec
TE 298.0 K
CNST2 145.0000000
D1 2.00000000 sec
D2 0.00344828 sec
D12 0.00002000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 150.7997054 MHz
NUC1 13C
P1 11.00 usec
P13 2000.00 usec
PLW0 0 W
PLW1 94.00000000 W
SPNAM[5] Crp60comp.4
SPOAL5 0.500
SPOFFS5 0 Hz
SPW5 17.37800026 W
======== CHANNEL f2 ========
SFO2 599.6619178 MHz
NUC2 1H
CPDPRG[2 waltz16
P3 8.00 usec
P4 16.00 usec
PCPD2 80.00 usec
PLW2 6.30959988 W
PLW12 0.06309600 W
F2 - Processing parameters
SI 32768
SF 150.7845284 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40
plc.i.sgr.99.2Recry || 04 May 2016
180 160 140 120 100 80 60 40 20 ppm
60.79
62.11
67.20
70.18
70.19
70.70
70.71
71.22
71.61
81.96
81.97
178.57
Current Data Parameters
NAME plc.i.sgr.99.2Recry
EXPNO 3
PROCNO 1
F2 - Acquisition Parameters
Date_ 20160504
Time 11.40
INSTRUM spect
PROBHD 5 mm CPTCI 1H/
PULPROG deptsp135
TD 65536
SOLVENT D2O
NS 256
DS 4
SWH 36231.883 Hz
FIDRES 0.552855 Hz
AQ 0.9043968 sec
RG 188.21
DW 13.800 usec
DE 20.00 usec
TE 298.0 K
CNST2 145.0000000
D1 2.00000000 sec
D2 0.00344828 sec
D12 0.00002000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 150.7997054 MHz
NUC1 13C
P1 11.00 usec
P13 2000.00 usec
PLW0 0 W
PLW1 94.00000000 W
SPNAM[5] Crp60comp.4
SPOAL5 0.500
SPOFFS5 0 Hz
SPW5 17.37800026 W
======== CHANNEL f2 ========
SFO2 599.6619178 MHz
NUC2 1H
CPDPRG[2 waltz16
P3 8.00 usec
P4 16.00 usec
PCPD2 80.00 usec
PLW2 6.30959988 W
PLW12 0.06309600 W
F2 - Processing parameters
SI 32768
SF 150.7845284 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40
plc.i.sgr.99.2Recry || 04 May 2016
Response(AU)
Time (Min) Time (Min)
Response(AU)
Response(AU)
Time (Min)
First
Void
Volume
Void
Volume
Void
Volume
Epimers: 18%/82% Epimers: 5.5%/94.5%
• A Phenomenex Luna-NH2 (5µ x 250 x 4.6 mm) column paired with a RI detector (flow
cell temperature = 35°C) was used for all HPLC separations. The eluent was 20% H2O
in ACN. A: Crude mixture 13C-3, flow rate = 2.0 mL/min. B: First recrystallization with
methyl cellosolve, flow rate = 1.0 mL/min. C: Second recrystallization with methyl
cellosolve, flow rate = 1.0 mL/min. D: Crystal structure of 13C-3.
A B
C D
A
B
C
D
C
O
OH
HO OOH
OH
13C-3
1
23
4
56
plc.ii.sgr.11.prd4
plc.ii.sgr.11.prd4.1
0.000
0.005
0.010
0.015
0.020
0
500
1000
1500
2000
0 5 10 15
0 5 10 15
time
response
channel
plc.ii.sgr.11.prd4
plc.ii.sgr.11.prd4.1
Void
Volume
Response(AU)
Time (Min)
BA
C
Optimal Radiochemical
Yield
Optimal Chemical
Yield
3 Days10 Min
Time (Min)
2
Response(AU)
• Carbon-11’s short half life presents unique synthetic considerations:
See Katie Foley’s Poster for More Information About in vitro and in planta work
• Separation of intermediates is key for successful carbon-11 labeling.
References: (1) Serianni, A.; Nunez, H.; Barker, R. Cyanohydrin synthesis: studies with carbon-13-labeled cyanide. J. Org. Chem. 1980, 45, 3329-3349. (2) Zhu, P.; Zacizek, J.; Serianni, A. Acyclic forms of [1-13C]aldohexoses in aqueous solution: Quantitation by 13C NMR and deuterium isotope effects on tautomeric equilibria. J. Org. Chem. 2001, 66, 6244-6251
0
0
50
100
PercentProductYield
20 40 60
Radiochemical
Yield
Reaction Time (minutes)
Radioactive
Decay
Chemical
Yield