Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide phosphates, and sugar nucleotides

Article abstract of DOI:10.1016/j.carres.2019.107893

Nucleosides and nucleotides are a group of small molecule effectors and substrates which include sugar nucleotides, purine and pyrimidine-based nucleotide phosphates, and diverse nucleotide antibiotics. We previously reported that hydrogenation of the nucleotide antibiotic tunicamycin leads to products with reduced toxicity on eukaryotic cells. We now report the hydrogenation of diverse sugar nucleosides, nucleotide phosphates, and pyrimidine nucleotides. UDP-sugars and other uridyl and thymidinyl nucleosides are quantitatively reduced to the corresponding 5,6-dihydro-nucleosides. Cytidyl pyrimidines are reduced, but the major products are the corresponding 5,6-dihydrouridyl nucleosides resulting from a deamination of the cytosine ring.

Full text of DOI:10.1016/j.carres.2019.107893

Journal Pre-proof
Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide
phosphates, and sugar nucleotides
Neil P.J. Price, Michael A. Jackson, Karl E. Vermillion, Judith A. Blackburn, Trina M.
Hartman
PII:
S0008-6215(19)30413-6
DOI:
https://doi.org/10.1016/j.carres.2019.107893
CAR 107893
Reference:
To appear in: Carbohydrate Research
Received Date: 12 July 2019
Revised Date: 26 November 2019
Accepted Date: 13 December 2019
Please cite this article as: N.P.J. Price, M.A. Jackson, K.E. Vermillion, J.A. Blackburn, T.M. Hartman,
Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide phosphates, and sugar
nucleotides, Carbohydrate Research (2020), doi: https://doi.org/10.1016/j.carres.2019.107893.
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GRAPHICAL ABSTRACT
HIGHLIGHTS
•
•
•
Quantitative catalytic hydrogenation of pyrimidines.
Nucleosides, nucleotide phosphates, and sugar nucleotides.
Ring opening of 5,6-DH-uridyl and 5,6-DH-thymidyl bases.
1

Rhodium-catalyzed reductive modification of pyrimidine nucleosides,
nucleotide phosphates, and sugar nucleotides.
Neil P. J. Price*, Michael A. Jackson, Karl E. Vermillion,
Judith A. Blackburn, Trina M. Hartman
Agricultural Research Service, U.S. Department of Agriculture, National Center for
Agricultural Utilization Research, Peoria, IL, U.S.A.
*Correspondence: Dr. N. P. J. Price, Agricultural Research Service, U. S. Department of
Agriculture, USDA-ARS, National Center for Agricultural Utilization Research, 1815 N
University Street, Peoria, IL, 61604, U.S.A.
E-mail: neil.price@ars.usda.gov
2

ABSTRACT
Nucleosides and nucleotides are a group of small molecule effectors and substrates which
include sugar nucleotides, purine and pyrimidine-based nucleotide phosphates, and diverse
nucleotide antibiotics. We previously reported that hydrogenation of the nucleotide antibiotic
tunicamycin leads to products with reduced toxicity on eukaryotic cells. We now report the
hydrogenation of diverse sugar nucleosides, nucleotide phosphates, and pyrimidine nucleotides.
UDP-sugars and other uridyl and thymidinyl nucleosides are quantitatively reduced to the
corresponding 5,6-dihydro-nucleosides. Cytidyl pyrimidines are reduced, but the major products
are the corresponding 5,6-dihydrouridyl nucleosides resulting from a deamination of the cytosine
ring.
Keywords: 5,6-dihydropyrimidines, catalytic hydrogenation, pyrimidines, sugar nucleotides.
3

1. Introduction
Nucleoside antibiotics are a diverse group of small metabolites that include antibacterial,
antiviral, and antifungal agents [1–3]. Many of these are either antibacterial (e.g., caprazamycin,
capuramycin, liposidomycin, tunicamycin, muraymycin, and mureidomycin), or antifungal (e.g.,
polyoxins and nikkomycins) [4–7]. Other nucleosides and nucleotides are small molecule
effectors and substrates such as sugar nucleotides, uridine monophosphate (UMP), adenosine
monophosphate (AMP), cyclic AMP, S-adenosylmethionine (SAM), and purines involved in
sulfur metabolism (adenosine phosphosulfates, APS, and (P)APS). The binding, and hence the
activity, of several of these inhibitors and substrates is at least partially determined by
interactions between the target protein and the nucleotide base motif, often via a planar, pseudo-
aromatic π-π stacking complex [8]. Hydrogenation of the carbon-5 – carbon-6 double bond in
the uridyl motif of tunicamycin, for example, leads to a modified, non-planar 5,6-dihydrouridyl
substituent, and with a concomitant loss of biological activity and toxicity on eukaryotic cells [9,
10]. The rhodium-catalyzed hydrogenation of the uridyl base is of interest because the product
5,6-dihydrouridyl base is non-aromatic and assumes a non-planar chair configuration [8, 9]. This
is also seen for the streptovirudins, a group of tunicamycin structural variants that have a
naturally-occurring 5,6-dihydrouracil group replacing the more common uracil moiety [11, 12].
In the current paper, we have investigated the breadth of this catalytic reduction as
applied to three pyrimidine nucleosides (uridine, thymidine, and cytidine), four nucleotide
phosphates (UMP, UDP, UTP, and CMP), and four sugar nucleotides (UDP-Glc, UDP-Gal,
UDP-GlcNAc, and UDP-glucuronic acid). Similar approaches have been investigated previously
[13–16]. Especially of interest is the applicability of the hydrogenation to diverse nucleoside
bases, the effect of the multiple phosphate groups, and the overall stability, especially with
4

respect to the generally labile sugar nucleotides. Moreover, this work provides access to a group
of reduced nucleotides suitable for biological testing as potential antibacterial or antifungal
agents.
2. Results and Discussion
2.1. Catalytic hydrogenation of sugar nucleotides. Sugar nucleotides may differ in both the
nucleotide and monosaccharide motifs and are notoriously susceptible to hydrolysis during
handling. Under the conditions described in the Experimental section 3.2., various sugar
nucleotides were hydrogenated as exemplified by UDP-N-acetylglucosamine (UDP-GlcNAc,
Fig. 1). NMR analysis of the 5,6-dihydro-UDP-GlcNAc (5,6-DHU-GlcNAc) showed two
carbohydrate anomeric signals are apparent for the β-ribosyl H-1̍ (5.84 ppm, 87.3 ppm) and the
α-N-acetylglucosaminyl H-1 (5.44 ppm, 94.8 ppm). The completeness of the reaction is evident
̍
from the absence of uridyl alkene signals (Fig. 1.A). The product 5,6-DHU-GlcNAc was also
apparent from the 2 Da mass increase in the MALDI/MS spectra ([M+H]+ = m/z 654.5; [M+Na]+
= m/z 676.5) (Fig. 1.B and C) due to hydrogenation of the uridyl 5,6-double bond. The
analogous uridine MALDI/MS ions were observed at m/z 652.5 and m/z 674.4, respectively (Fig.
1.B).
Catalytic hydrogenation of three other uridyl sugar nucleotides was also undertaken.
HSQC NMR analysis showed that the UDP-Glc, UDP-Gal and UDP-GlcA are cleanly reduced to
5,6-dihydro-UDP-Glc, 5,6-dihydro-UDP-Gal and 5,6-dihydro-UDP-GlcA, respectively (Fig. 2,
supplementary Table 1). The β-ribosyl H-1
̍
anomeric protons were observed at 5.85, 5.85 and
5

5.84 ppm, respectively, with corresponding 13C-NMR chemical shifts at 86.7, 86.9, and 86.9
ppm. The α-anomer NMR signals for the corresponding hexose sugars were apparent at
5.52/94.6, 5.50/94.8, and 5.52/94.8 ppm, respectively. The hydrogenated C5 and C6 positions in
the base moieties were apparent at 30.2 and 36.0 ppm, respective, with corresponding H5 and
1
H6-a,b H-NMR signals at 2.73 ppm (overlapped) and 3.51, 3.61 ppm (Fig. 2). That the
hydrogenations had proceeded to completion is evident from the complete disappearance of H5
and H6 alkene proton signals (7.9 and 5.9 ppm, respectively) or the corresponding C5 and C6
13C-NMR signals at 142 and 102 ppm.
2.2. Hydrogenation of nucleotide phosphates. The effect of phosphate groups on the
hydrogenation reaction was investigated next, as exemplified by the reduction of UMP, UDP,
and UTP (Fig. 3). MALDI/MS analysis showed the expected 2 Da mass increase due to
reduction of the uridyl 5,6-double bonds. Hence, the [M+H]+, m/z 347.3 and [M+Na]+, m/z
369.3 ions are increased by 2 Da to m/z 349.3 and 371.3, respectively, for 5,6-DH-UMP.
Similarly, m/z 427.2, 449.2, 471.2, and 493.1 are increase by 2 Da to m/z 429.2, 451.2, 473.2,
and 495.1 for 5,6-DH-UDP; and m/z 507.3, 529.2, 551.3, and 573.2 increase by 2 Da to m/z
509.3, 531.3, 553.3, and 575.3 for 5,6-DH-UTP. NMR analysis shows an absence of 5,6-alkene
signals, confirming the complete reduction of these products. Each has a single ribosyl-1
anomeric signal in the HSQC spectra at 5.82 ppm, 86.5 – 87.1 ppm, and the uridyl 5 and 6 a,b
signals are also apparent at 2.72 ppm, 29.9 – 30.6 ppm and 3.52 – 3.61 ppm, 35.8 ppm,
respectively (Fig. 2, supplementary Table 1).
̍
6

2.3. Catalytic hydrogenation of other pyrimidine nucleosides. The most common pyrimidine
nucleosides, uridine, cytidine, and thymidine, differ structurally in the base substituent and the
pentose sugar motif. Under the catalytic conditions described uridine is quantitatively
hydrogenated to 5,6-dihydrouridine (5,6-DHU), as shown by both MALDI/MS and NMR
analysis (supplementary Fig. S.1). The MS molecular ions for uridine ([M+H]+ = m/z 245.2,
[M+Na]+ = m/z 267.2) are converted to 5,6-DHU ([M+H]+ = m/z 247.3, [M+Na]+ = m/z 269.3),
with the mass increase of 2 Da resulting from reduction of the uridyl double bond. NMR
analysis also showed that the reaction is quantitative, the resulting 5,6-DHU lacking any residual
NMR signals in the alkene region typical of uridine. A single ribosyl-1
apparent at 5.80 ppm/87 ppm, and the reduced uridyl H5 and H6 signals at 3.7 and 3.5 ppm,
respectively (supplementary Fig. S.1.). Thymidine is similarly converted to 5,6-
dihydrothymidine (5,6-DHT), with an analogous mass increase of 2 Da, with the characteristic
anomeric ribosyl-1 signals at 6.25 ppm and 82 ppm, respectively (supplementary Fig. S.1,
̍ anomeric signal is
̍
supplemental Table 1). Hence, the hydrogenation is unaffected by the 5-methyl substituent on
the thymine ring, and both thymidine and uridine are quantitively reduced to the corresponding
5,6-anhydronucleosides. Note that the 5,6-DHT is assumed to contain a new chiral carbon-6,
although this has not been confirmed by the current methods.
2.4. Cytidine nucleosides. It was noted that the hydrogenation of the cytidine-based nucleoside
and nucleotides (cytidine, CMP, CDP, and CTP) did not proceed as for the respective uridyl or
thymidyl analogs. Catalytic reduction of cytidine, for example, resulted in an almost complete
1
de-amination of the cytidyl ring, with the major product characterized by H-NMR and
MALDI/MS as 5,6-dihydrouridine (Fig. 4). The anticipated product, 5,6-dihydrocytidine, was
7

only produced at about 10% yield during this reaction (Fig. 4). In analogous reactions with
CMP, CDP, and CTP de-amination also occurred, and the major products obtained were the
analogous 5,6-dihydrouridyl phosphate nucleotides (data not shown). The deamination of
cytidine and cytosine at acidic pH is well known [17, 18], and indeed also occurs enzymatically
by cytidine deaminase during pyrimidine salvaging [19]. However, the observed de-amination
of the cytidine nucleotides during catalytic hydrogenation was unexpected, and this therefore
precludes the production of 5,6-dihydrocytidine nucleosides by this route.
2.5. Base-catalyzed hydrolytic ring opening.
Base-catalyzed hydrolytic ring opening of the 5,6-dihydrouridyl ring of hydrogenated
tunicamycins, and some other nucleotides, is known [9, 10], but not for other pyrimidine
nucleosides, nucleotide phosphates, or nucleotide sugars. Hence, the hydrogenation products
obtained from above were hydrolyzed with aqueous sodium hydroxide under conditions
described previously [10]. The products were analyzed by MALDI/MS, 1H-NMR, 13C-NMR and
HSQC-NMR (supplementary Table 1, supplementary Fig. S.2). Under these mild conditions
(0.25 M aqueous sodium hydroxide for 18 h at ambient temperature) the uridyl and thymidyl
nucleosides were quantitatively hydrolyzed, resulting in opening of the base ring without
cleavage of the ribosyl or 2-deoxyribosyl N-glycosidic bonds (Scheme 1). This was evident
from MALDI/MS due to the 18 Da mass increase which occurs during the hydrolysis of the
amide bond in the reduced nucleotide base ring, and from the NMR analysis of the nucleoside 1ʹ,
5, and 6 protons (supplementary Fig. S.2., supplementary Table 1). Noticeably, the often labile
sugar nucleotides were hydrolytically base opened but the rest of the molecule was unaffected,
with no hydrolysis of the glycosidic or phosphate bonds (e.g. 5,6-DHU-GlcNAc, supplementary
8

Fig. S.2.C). The corresponding non-hydrogenated nucleosides were resistant to base hydrolyzed,
and were completely stable under these alkaline conditions (data not shown).
3. Experimental Section
3.1. Materials and Instrumentation
All chemicals and solvents, except for the catalyst (5 wt% Rh/Al2O3 from BASF
Engelhard, Iselin, NJ) were obtained from Sigma-Aldrich Inc., St Louis, MO, USA. NMR
spectra were run in D2O at 27 ºC using a Bruker Avance III instrument (Bruker BioSpin,
Billerica, MA, USA). MALDI-TOF mass spectra were recorded using a Bruker-Daltonics
Microflex instrument (Bruker-Daltonics, Billerica, MA, USA) in reflectron mode. The matrix
used was 2,5-dihydrobenzoic acid.
3.2. Hydrogenation conditions
The hydrogenations were undertaken as described previously [10]. Briefly, the
nucleoside starting material (30 mg) in aqueous solution (3 mL) were reacted for 3 hours at room
temperature (21 ͦ C). Catalyst (5 wt% Rh/Al2O3; 20 mg) was introduced at the start of the
reactions, and hydrogen gas was bubbled through at atmospheric pressure. The products were
recovered by filtration to remove the catalyst, followed by lyophilization to remove the water.
MALDI/MS spectra were recorded on the aqueous solutions immediately after removal of the
catalyst. The lyophilized product residues were re-dissolved in deuterated water for analysis by
NMR (1H-NMR, 13C-NMR, HSQC) without further purification. For the base-catalyzed ring
opening reactions, the hydrogenated nucleosides were treated at room temperature for 18 h using
9

aqueous sodium hydroxide (0.25 M). After neutralization with HCl (0.25M) the samples were
analyzed by NMR and MALDI/MS.
Acknowledgements
The authors thank C. D. Skory (ARS-USDA, Peoria, IL, USA) for the initially review of
the manuscript. Mention of any trade names or commercial products is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the US
Department of Agriculture. USDA is an equal opportunity provider and employer.
References
1. M. Serpi, V. Ferrari, F. Pertusati J. Med. Chem., 59 (2017), pp. 10343−10382
2. G. Niu, H. Tan Trends Microbiol., 23 (2015), pp. 110−119
3. W. Chen, J. Qi, P. Wu, D. Wan, J. Liu, X. Feng, Z. Deng Z J. Ind. Microbiol. Biotechnol., 43
(2016), pp. 401−417
4. W. Winn, R. J. M. Goss, K. Kimura, T. D. H. Bugg Nat. Prod. Rep., 27 (2010), pp. 279−304
5. S. Rachakonda, L. Cartee Curr. Med. Chem., 11 (2004), pp. 775−793
6. K. Kimura, T. D. H. Bugg Nat. Prod. Rep., 20 (2003), pp. 252−273
7. K. Isono Pharmacol. Ther., 52 (1991), pp. 269−286
8. K. M. Ivanetich, D. V. Santi Prog. Nucleic Acid Res. Mol. Biol., 42 (1992), pp. 127−156
9. N. P. Price, T. M. Hartman, J. Li, K. K. Velpula, T. A. Naumann, M. R. Guda, B. Yu, K. M.
Bischoff J. Antibiot., 70 (2017), pp. 1070−1077
10. N. P. Price, M. A. Jackson, K. E. Vermillion, J. A. Blackburn, J. Li, B. Yu J. Antibiot., 70
(2017), pp. 1122−1128
10

11. K. Eckardt, W. Ihn, D. Tresselt, D. Krebs J. Antibiot., 34 (1981), pp. 1631−1632
12. K. Eckardt, H. Thrum, G. Bradler, E. Tonew, M. Tonew J Antibiot., 28 (1975), pp. 274−279
13. W. E. Cohn, D. G. Doherty J. Am. Chem. Soc., 78 (1956), pp. 2863–2866
14. N. K. Kochetkov, E, I. Budovskii, V. N. Shibaev, G. I Eliseeva Russ. Chem. Bull. 14 (1965),
pp. 884–885
15. P. Cerutti, K. Ikeda, B. Witkop J. Am. Chem. Soc. 87 (1965), pp 2505–2507
16. P. Cerutti, Y. Kondo, W. R. Landis, B. Witkop J. Am. Chem. Soc. 90(1968), pp. 771–775
17. R. Shapiro, R. S. Klein Biochemistry, 5 (1966), pp. 2358–2362
18. V. Labet, C. Morell, T. Douki, J. Cadet, L. A. Eriksson, A. Grand J. Phys. Chem., 114
(2010), pp. 1826-1834
19. S. Vincenzetti, G. De Sanctis, S. Costanzi, G. Cristalli, P. Mariani, G. Mei, J. Neuhard, P.
Natalini, V. Polzonetti, A. Vita Protein Eng., 16 (2004), pp. 1055–1061
11

List of Table, Figures, and Schemes
Figure 1.
HSQC-NMR spectrum of 5,6-dihydro-UDP-N-acetylglucosamine (A), and
MALDI/MS spectra of UDP-N-acetylglucosamine before (B) and after hydrogenation (C). The
molecular adduct ions [M+H]+, m/z 652 and [M+Na]+, m/z 674 are increase by 2 mass units due
to quantitative hydrogenation of the 5,6-uridyl double bond.
Figure 2. 1H-NMR, 13C-NMR, and HSQC spectra of other hydrogenated sugar nucleotides. (A)
5,6-DHU-Glc; (B) 5,6-DHU-Gal; and (C) 5,6-DHU-glucuronic acid.
Figure 3. Effects of phosphate groups. HSQC-NMR spectra of (A) 5,6-dihydrouridine
monophosphate; (B) 5,6-dihydrouridine diphosphate; and (C) 5,6-dihydrouridine triphosphate.
Corresponding MALDI/MS spectra before (D, E, F) or after (G, H, I) hydrogenation. Molecular
+
adduct ions ([M+H]+ and [M+Na]n ) are increase by 2 Da after hydrogenation.
Figure 4. De-amination of 5,6-dihydrocytidine nucleosides during hydrogenation. 1H-NMR
spectra of (A). 5,6-dihydrouridine from hydrogenation of uridine; (B). hydrogenated cytidine.
Both samples are predominantly converted to 5,6-dihydrouridine. The anticipated 5,6-
dihydrocytidine is shown as a minor product indicated by the black arrows.
Scheme 1. Catalytic hydrogenation and alkaline-catalyzed ring opening of pyrimidine sugar
nucleosides, nucleotide phosphates, and nucleosides.
12

Supplementary Table 1. NMR Chemical Shifts for the Characteristic Ribosyl-1ʹ and
Nucleotide Base-5,6 Positions.
Supplementary Figure S.1. Catalytic hydrogenation products 5,6-dihydrouridine (top) and 5,6-
dihydrothymidine (lower). Shown are HSQC-NMR spectra (A, B), and MALDI/MS spectra
either before (C, D) or after (E, F) hydrogenation. Molecular ions ([M + H]+ and [M + Na]+) are
increased by 2 Da after hydrogenation due to reduction of the 5,6-double bond in the base.
Supplementary Figure S.2. HSQC-NMR spectra of the alkaline hydrolyzed ring-opened
pyrimidine nucleotides. (A) 5,6-dihydrouridine diphosphate; (B) 5,6-dihydro-thymidine; and (C)
5,6-dihydrouridine-UDP-N-acetylglucosamine.
13

B
C
A
Figure 1
.
HSQC-NMR spectrum of 5,6-dihydro-UDP-
N-acetylglucosamine (A), and
MALDI/MS spectra of UDP-
N
-acetylglucosamine before (B) and after hydrogenation (C). The
molecular adduct ions [M+H]+, m/z 652 and [M+Na]+, m/z 674 are increase by 2 mass units due
to quantitative hydrogenation of the 5,6-uridyl double bond.
14

A
B
C
Figure 2. 1H-NMR, 13C-NMR, and HSQC spectra of other hydrogenated sugar nucleotides. (A)
5,6-DHU-Glc; (B) 5,6-DHU-Gal; and (C) 5,6-DHU-glucuronic acid.
15

D
G
A
B
C
E
H
F
I
Figure 3. Effects of phosphate groups. HSQC-NMR spectra of (A) 5,6-dihydrouridine
monophosphate; (B) 5,6-dihydrouridine diphosphate; and (C) 5,6-dihydrouridine triphosphate.
Corresponding MALDI/MS spectra before (D, E, F) or after (G, H, I) hydrogenation. Molecular
+
adduct ions ([M+H]+ and [M+Na]n ) are increase by 2 Da after hydrogenation.
16

Figure 4. De-amination of 5,6-dihydrocytidine nucleosides during hydrogenation. 1H-NMR
spectra of (A) hydrogenated uridine, and (B) hydrogenated cytidine. Both samples are
predominantly converted in 5,6-dihydrouridine. The anticipated 5,6-dihydrocytidine is shown as
a minor product indicate by the black arrows.
17

O
OH
O
O
R1
R1
R1
NH
NH2
NH
N
O
N
O
N
O
R3O
R3O
R3O
O
O
O
Rh/alumina
H2, r.t., 3 h
aq. NaOH
0.25M, 18 h, r.t.
HO
HO
HO
R2
R2
R2
Scheme 1: Catalytic hydrogenation and alkaline-catalyzed ring opening of pyrimidine sugar
nucleosides, nucleotide phosphates, and nucleosides. R1 is H or CH3, R2 is H or OH, R3 is H,
phosphate, pyrophosphate, or a monosaccharide-1-pyrophosphate.
18

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: