chemi-enzymic synthesis of isotopically labeled ...serlab03/ics_2016_nucleo.pdfmeαr 4.2 6.2 3.1 3.1...
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δH1 δH2 δH3 δH4 δH5a δH5b
αR1P 5.515 3.997 3.946 4.113 3.660 3.548
MeαR 4.99 4.11 4.03 4.09 3.73 3.66
δC1 δC2 δC3 δC4 δC5
αR1P 100.30 74.07 72.12 87.42 64.12
MeαR 106.2 74.1 72.8 87.5 64.2
3JH1,H2 3JH2,H3
3JH3,H4 3JH4,H5a
3JH4,H5b 2JH5a,H5b
αR1P 4.1 6.3 3.9 3.3 4.8 -12.4
MeαR 4.2 6.2 3.1 3.1 4.8 -12.3
2JC1,P 3JC2,P
4JC3,P 3JH1,P
4JH2,P
αR1P 5.2 6.8 br 6.3 1.5
Supported by NSF CHE 1402744 and NIH SBIR
HHSN261201500020C
Enzymic Route to Labeled Ribonucleosides
Formation of HX and UA when a 55 µM solution of I is treated with either PNPase
(νo = 10.3 ± 3.7 µM product/min) or a mixture of PNPase and XO (νo = 19.5 ± 0.6 µM product/min).
Enzymic Synthesis of α-D-[1-13C]R1P
Spectrophotometric Monitoring of Inosine Phosphorolysis
Less than 50% of I was phosphorolyzed in the absence of XO and
the reaction reached equilibrium after ~3 min, but complete
phosphorolysis was observed after 11 min when XO was added to
the reaction mixture. Inclusion of XO shifted the thermodynamic
equilibrium towards αR1P and increased the initial reaction velocity.
13C{1H} NMR Spectrum of [1’-13C]Inosine Phosphorolysis
(A) Reaction mixture without PNPase showing the C1’ signal of [1’-13C]inosine. (B) Reaction mixture after the addition of PNPase and
incubation, showing the C1 signal of α-D-[1-13C]R1P. Signal spitting
(inset) is caused by 2JC1,O,P. In (A) and (B), only signals from 13C-labeled
carbons are shown.
Isolation of αR1P The reaction scale was increased in an effort to isolate,
characterize and quantify the αR1P product: Inosine (65.5
mg), PNPase (4 units), XO (0.2 units), catalase (24,000 units)
in distilled deionized water (1.0 L).
The reaction mixture was incubated at 37 °C for three days.
Most of the sodium phosphate was removed by crystallization.
The αR1P was purified on a Bio-Gel P2 SEC column.
Partial 1H NMR Spectra (600 MHz) of αR1P
13C NMR Spectra (150 MHz) of αR1P
In D2O at 22 °C (pH 6.4), externally referenced to DSS.
In D2O at 22 °C (pH 6.4), externally referenced to DSS. (A) Full spectrum
with signal assignments. (B) Partial spectra showing signals split by JC,P.
NMR Chemical Shift Assignments
In D2O at 22 °C , externally referenced to DSS. MeαR denotes Methyl α-
Ribofuranoside .
NMR Coupling Constants in αR1P and MeαR
In D2O at 22 °C , externally referenced to DSS. MeαR denotes methyl α-
D-ribofuranoside. br denotes broaden signal (< 0.5 Hz).
O-Phosphorylation at O1 affects δH1 and δC1 significantly.
3JH1,H2 values in αR1P (4.1 Hz) and in MeαR (4.2 Hz) are
virtually identical, confirming the α-configuration at C1 of αR1P.
Similar 3JH,H values in αR1P and MeαR suggest similar ring and
exocyclic hydroxymethyl conformations in both compounds.
Staggered Rotamers About the C1-O1 Bond of αR1P
Rotamer III (C2 anti to P) is most abundant, based on the relatively
large experimental 3JC2,P (6.8 Hz) value and small 3JH1,P (6.3 Hz)
value. Observation of 4JH2,P (1.5 Hz) supports this assignment in that
this long-range coupling is only observed along H2-C2-C1-O1-P
pathways that adopt a “W-shaped” arrangement. This observation
also suggests that E1 is the preferred conformation of the ribose ring
of αR1P in which the C1-O1 bond is quasi-axial, allowing the “W-
shaped” arrangement along the H2-C2-C1-O1-P pathway.
Conversion of αR1P to Adenosine
1H NMR spectrum (600 MHz) of reaction mixture of αR1P with adenine in 10% D2O
after 4 h at 37 °C. The signals rising from the ribose residue of adenosine are
dominant from 3 ppm to 6 ppm (assignments in red) and the signals of unreacted
αR1P are weak. Signals A1 and A2 are assigned to the base residue of adenosine,
and signals B1 and B2 to unreacted adenine.
CHCl3 was added to the reaction mixture to inhibit microbial
growth.
Excess adenine (10 mg) was added as solid to drive the
conversion to completion.
Conversion of αR1P to adenosine (>90%) was observed after 4 h
at 37 °C.
Chemi-Enzymic Synthesis of Isotopically Labeled Nucleosides From a Common Labeled Precursor
Wenhui Zhang, Toby Turney, Ivana Surjancev and Anthony S. Serianni Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556-5670 USA
Abstract The synthetic challenges to prepare selectively stable isotopically
labeled ribo- and 2’-deoxyribonucleosides (e.g., with 13C, 2H and/or 15N) remain significant, partly because of the large number of
isotopomers that are possible when both their nitrogen base and
sugar constituents are considered. To address this problem,
milligram quantities of α-D-ribofuranosyl 1-phosphate (αR1P) were
prepared by PNPase-catalyzed phosphorolysis of inosine that was
driven to completion by enzymic coupling to xanthine oxidase and
catalase. αR1P was obtained in >50% isolated yield, and was
characterized by 1H and 13C NMR. To demonstrate feasibility, the
αR1P was recoupled to a different nitrogen base, adenine, to give
adenosine in high yield (~90%). The method was then applied to
[1’-13C]inosine to confirm its applicability in the preparation of α-D-
[1-13C]R1P. Simplicity, generality, high yields and convenient use
and removal of excess nitrogen base render practical the transfer of
a stable isotopically labeled α-D-ribofuranosyl ring of inosine to
other nitrogen bases (labeled or unlabeled) to prepare a wide range
of isotopically labeled ribonucleosides. With minor modification, the
same approach can be used to prepare labeled 2’-
deoxyribonucleosides. This work enables the selective labeling of
nucleosides with stable isotopes, thus promoting their incorporation
into oligonucleotides for structure-function studies
Summary. Milligram quantities of αR1P have been prepared, purified and characterized from PNPase-catalyzed phosphorolysis
of inosine, the latter reaction driven to completion by coupling to
xanthine oxidase and catalase. The isolated αR1P was recoupled to
a different nitrogen base, adenine, to give adenosine in high yield.
Simplicity, high yield and the convenient use and removal of excess
(solid) nitrogen base render practical the transfer of a stable
isotopically labeled β-D-ribofuranosyl ring of inosine to other
nitrogen bases to prepare labeled ribonucleosides. Application of
this strategy to the synthesis of labeled 2’-deoxyribonuclesides
using 2’-deoxyinosine as the 2-deoxy-β-D-erythro-pentofuranosyl donor is underway
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