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