Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate

Article abstract of DOI:10.1016/j.bmc.2018.03.032

Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet synthetic reports on these materials are lacking. Here we report two strategies to prepare the exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic thymidylate biosynthesis and a novel nucleotide analog. Our most effective strategy involves preserving the existing glyosidic bond of thymidine and manipulating the base to generate the exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-aromatic nucleobase isomer of thymine, which provides an additional strategy to access nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The strategies reported here are straightforward and extendable towards the synthesis of various pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme reaction mechanisms or serve as templates for rational drug design.

Full text of DOI:10.1016/j.bmc.2018.03.032

Accepted Manuscript
Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine mono-
phosphate
Dibyendu Mondal, Eric M. Koehn, Jiajun Yao, David F. Wiemer, Amnon Kohen
PII:
S0968-0896(18)30227-X
https://doi.org/10.1016/j.bmc.2018.03.032
BMC 14270
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2018
15 March 2018
21 March 2018
Please cite this article as: Mondal, D., Koehn, E.M., Yao, J., Wiemer, D.F., Kohen, A., Chemo-enzymatic synthesis
of the exocyclic olefin isomer of thymidine monophosphate, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.03.032
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chemo-enzymatic synthesis of the exocyclic olefin
isomer of thymidine monophosphate
Dibyendu Mondal, Eric M. Koehn,† Jiajun Yao, David F. Wiemer,* and Amnon Kohen*
Department of Chemistry University of Iowa, Iowa City, Iowa 52242-1294, United States
E-mail: david-wiemer@uiowa.edu
RECEIVED DATE:
KEYWORDS: thymidylate, metabolism, nucleotide analogue, synthesis.
† Current address: Department of Chemistry and California Institute for Quantitative Biosciences,
University of California Berkeley, Berkeley, California 94720-3220, United States
Draft: March 14, 2018
1

Abstract.
Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction
intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet
synthetic reports on these materials are lacking. Here we report two strategies to prepare the
exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic
thymine biosynthesis and a novel nucleotide analog. Our most effective strategy involves
preserving the existing glyosidic bond of thymidine and manipulating the base to generate the
exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-
aromatic nucleobase isomer of thymine, which provides an additional strategy to access
nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The
strategies reported here are straightforward and extendable towards the synthesis of various
pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme
reaction mechanisms or serve as templates for rational drug design.
2

Introduction.
Nucleotides are essential molecules commonly referred to as the building blocks of life
because they make up DNA and RNA as well as cell machinery for expression, regulation, and
generation of gene products. Thus, structural analogs of nucleotides have seen a variety of uses
including, but not limited to, cancer therapeutics, mechanistic bioreagents, and in vivo probes for
metabolic studies.1-3 Unnatural and noncanonical nucleotides have been produced through
modification of the phosphate,4-7 the sugar,8-11 and the nucleobase3,12-15 moieties resulting in a
range of biologically relevant compounds with a variety of activities and functions.9 The focus
of this report involves nucleobase modifications that result in a new structural variant of the
thymidyl moiety. In the past nucleobase analogs that mimic thymidyl moieties have produced
antiviral agents (e.g., trifluorothymidine16,17), antitumor agents (e.g., 5-F-uridine18,19), and
biological flourophores (e.g., 5-aminoallyluridine20). These past examples showcase the
importance of modifications of this pyrimidine nucleotide in particular, and the targeting of
thymidyl biological processes in general.
Thymine is unique among the DNA nucleosides. Deoxyguanosine, deoxyadenosine, and
deoxycytosine can be produced directly from their respective ribosides through the action of
ribonucleotide reductases, but thymidyl moieties must be produced de novo by methylation of
uridine.21 Several enzymes catalyze this reductive methylation, including thymidylate synthase
(TSase),22 flavin-dependent thymidylate synthase (FDTS),23 and methylenetetrahydrofolate-
tRNA-(uracil-5-)-methyltransferase (TrmFO),24 to provide the thymidine that is essential for
DNA production and cell proliferation.22 As a result, thymidyl biosynthetic enzymes are often
the target of chemotherapeutic agents19 and they have shown potential as antibacterial25 and
antiviral26 drug targets as well. However, while the well-studied “classical” thymidylate
synthase (TSase) has been the focus of cancer drug development (e.g., 5-F-dU together with
3

leucovorin), less is known regarding the inhibition of alternative thymidyl biosynthesis enzymes,
which limits the development of antibacterial and antiviral compounds that target these
enzymes.23 For example, the enzyme FDTS, which is present in several important human
pathogens, functions by a remarkably different chemical mechanism from classical TSase. At
this time there are no known inhibitors that selectively target FDTS.27 Compounds that mimic
the unique intermediates for this alternative thymidylate biosynthesis may advance our ability to
exploit this attractive target.
All of the known biosynthetic pathways that lead to thymidylic acid (1) by reductive
methylation, whether catalyzed by FDTS (from 2) or by TRMFO or TSase (from 3), have in
common an exoxyclic methylene group at the C5-C7 position of the pyrimidine ring (Figure 1).
The existence of exocylic methylene intermediates has been supported by nucleophilic trapping
experiments which disrupt the enzymatic turnover and allow isolation of derivatives of the
reaction intermediates.25,28,29 Many of these putative intermediate species can only be
hypothesized based on these chemical trapping experiments along with the known reactivity of
an α,β-unsaturated system, and therefore direct access to this analog of dTMP would greatly
facilitate mechanistic study of thymidyl biosynthesis.29 Exocyclic olefin analogs of thymidine
have appeared in studies by Greenberg and his group (e.g. 4), but all the currently known
compounds are also substituted at the C-6 position. Nevertheless, these doubly-modified
thymidine analogues have been used to demonstrate the formation of interstrand cross-linking
and biocongugates in RNA and DNA via a mechanism involving exocyclic methylene-modified
thymidines.12,13,30,31 Thus there has been substantial interest in designing synthetic routes that can
generate modified nucleotides,9,32 and one modified only by transposition of the olefin to an
exocyclic position (i.e. compound 2) would be especially valuable in studies of enzyme
4

mechanism(s). Unfortunately, due to the various functional groups present in the starting
materials, formation of an exocylic methylene moiety is challenging and traditional reactions
proceeding by installation and removal of orthogonal blocking groups ultimately impacts
synthetic feasibility.9
Figure 1: Thymidylic acid and some olefin analogues of biological significance.
To the best of our knowledge, only transient olefin isomers of thymidine or thymidylate
have been proposed,23 occurring either as intermediates in enzyme reactions or along the
pathway to DNA labeling via photolysis. Furthermore, all of these examples have a heteroatom
substituent at C-6 of the pyrimidine ring and therefore have a different C-6 oxidation state than
the dihydro species proposed as an intermediate during alternative thymidylate biosynthesis (i.e.
compound 2).23,33 In 1973 Klötzer34 reported the synthesis of an exocyclic methylene nucleobase
5

isomer of thymine, but synthesis of the exocyclic methylene isomer of thymidine or thymidylate
has not been reported. Herein we present two different synthetic routes to achieve the unique C-
5 isomer of dTMP (1) bearing an exocyclic olefin. This isomer has been proposed25,29,35,36 as an
intermediate during the FDTS catalyzed reaction and therefore represents an important structural
motif in pathogenic dTMP biosynthesis. We also report on the chemical and pH stability of this
compound, along with derivatization at the C-7 methylene terminus that results in novel
nucleophilic addition products and may serve as a possible gateway to novel compounds with
antibacterial or antiviral activities.23 Finally, the reactivity reported herein suggests that
compound 2 may have potential use in reaction with nucleophilic residues of thymidyl binding
proteins or in DNA.
Results and Discussion.
Two complementary synthetic approaches have been developed to prepare the target
compound 2. Although the approaches are orthogonal in strategy, both afford the exocyclic C-5
olefin isomer of thymidine (10) and then follow the same phosphorylation pathway via an
enzymatic kinase. Exploration of these two orthogonal synthetic strategies paves the way for
future synthetic modifications that can easily afford novel isotopic labeling patterns in compound
2.
The first approach began with thymidine and applied the general synthetic strategy of
functionalizing C-7, followed by dearomatization via reduction, and finally an elimination
reaction to create the exocyclic methylene group and generate compound 2 (Scheme 1). More
specifically, the protected nucleoside 5 was prepared from commercial thymidine using a
previously published procedure.37 Subsequent protection of the C-7 primary hydroxyl group of
compound 5 was required to prevent side products during the reduction. Introduction of the
6

triethylsilyl group (6) allowed selective deprotection later in the sequence, even in the presence
of two TBDMS groups. Reduction of compound 6 gave a mixture of diastereomers (7), of little
long-term consequence given the intent to generate the exocyclic olefin. This mixture was then
deprotected selectively at C-7 to obtain compound 8. Attempted tosylation of the primary
alcohol 8 resulted in a mixture of compounds and isolation of the individual components was not
pursued. Remarkably, optimized conditions allowed tosylation followed by elimination in a
single flask, furnishing compound 9 in high yield (91%). Removal of the TBS groups was
achieved upon treatment with TEA·3HF buffered with pyridine to prevent tautomerization of the
exocyclic double bond to thymidine. Phosphorylation of compound 10 proved to be challenging
with POCl3 or (H3CO)3P39 reagents due to varying degrees of isomerization under different
38
reaction conditions. It is possible that strategies based on other P(III) reagents40 still could be
employed to accomplish a chemical formation of the desired monophosphate, but we turned to
enzymatic methods instead. When an enzymatic reaction was attempted, phosphorylation of
compound 10 was achieved by a pyrimidine kinase to obtain the desired compound 2 in high
yield.41 While isolation of compound 2 was complicated by the various components necessary
for the enzymatic reaction, it was possible to isolate milligrams of material by HPLC. The NMR
spectra of this product were clearly consistent with the bis(triethylammonium) salt of compound
2. Of special significance were resonances attributable to the exocyclic methylene group at 6.22
and 5.89 ppm, both of which correlated to a carbon resonance at 126.4 ppm in an HSQC
experiment, as well as those at 4.15 and 4.25 ppm, which correlated to the C-6 resonance at 41.6
ppm.
7

Scheme 1. Synthesis of the new thymidylate analogue 2.
Through an orthogonal synthetic strategy, compound 2 was prepared by initial synthesis
of the thymine isomer using procedures based on a known synthetic route (Scheme 2).34 In
brief, commercial diethyl bis(hydroxymethyl)malonate 11 was converted to the dibromide 12 as
previously described34 providing the desired starting material. After formation of the acid
chloride, reaction with ammonia gave the amide 13, which eliminates HBr to give the olefin 14
through a procedure slightly modified from the original synthesis. Condensation of the olefin 14
with trichloroacetyl isocyanate gives imide 15, which then can be cyclized and deacylated to
give the methylene hexahydropyrimidine dione 16 in low yield. Glycosylation of compound 16
then was accomplished by enzymatic means, through the use of commercial E. coli thymidine
phosphorylase (subjected to anaerobic dialysis) and freshly synthesized deoxyribose-1-phosphate
(dR-1-P).42 Although it is unstable, it was advantageous to use dR-1-P directly as opposed to
8

generation in situ as often reported43,44 because this allows one to drive the reaction with an
excess of the sugar phosphate (e.g. 10-fold). The final 5’-phosphorylation with a pyrimidine
kinase was performed in situ with glycosylation to shift the equilibrium in favor of the desired
product. Overall conversion in this last step was limited by the glycosylation efficiency and
typically was between 10-20%, perhaps because of substantial product inhibition from inorganic
phosphate. Nevertheless it should be possible to isolate a milligram quantity from these
reactions. However the earlier strategy would be more easily extended to provide larger
quantities of the desired product because only one enzymatic transformation is required in that
sequence.
Scheme 2. Preparation of thymidine analogue 16 and its conversion to thymidylate analogue 2.
The synthetic strategies described above are complementary and could be employed to
obtain a range of products with various isotopic labeling patterns. For example, the sequence
described in Scheme 1 could be used to introduce isotopic labels at the nucleobase, while
Scheme 2 is attractive for introduction of radiolabeled sugar moieties with minimal downstream
workup and by-product formation.
9

During the course of these syntheses, compound 2 was found to be unstable under
various conditions. For example, at high pH (>10) it decomposes to multiple products that were
not characterized. In contrast, at 14 °C it was sufficiently stable in 0.5% formic acid in water to
allow LCMS analysis but at lower pH (<5) and room temperature rearrangement of the double
bond is observed and compound 2 isomerizes slowly to produce dTMP. Partial isomerization to
dTMP also was observed during lyophilization of compound 2, although this could be minimized
by addition of equimolar salt (NaCl or KCl) during concentration. The use of triethylammonium
bicarbonate buffer (pH 7.2) or triethylammonium acetate (pH 5.6) during HPLC purification also
minimized isomerization. While conditions that allow or promote isomerization should be
avoided during preparation of compound 2, the observed isomerization serves as an additional
structure proof because the dTMP formed by isomerization is indistinguishable from commercial
dTMP.
It is notable that the exocyclic olefin 16 is much more stable than its glycosylated
counterparts 10 and 2. Incubation of 16 under acidic (<2) or basic (>10) conditions provided no
noticeable change in structure after 24 hours. Storage of compound 16 under conditions that
caused derivatization of compound 2 (vide infra), gave no detectable addition products. These
observations are consistent with the original report on the synthesis and stability of compound
16.34 While mechanistic studies on the precise nature of this isomerization are beyond the scope
of this report, this instability should be taken as a note of caution during preparation of
compounds 10 and 2.
Compound 2 also proved to be reactive with nucleophiles. For example, incubation of
the α,β-unsaturated compound 2 in a buffer solution of tris(hydroxymethyl)aminomethane
(TRIS) or in the presence of dithiothreitol (DTT), led to its disappearance. Analysis of these
10

reaction mixtures by LCMS revealed new materials which were 121 and 154 AMU higher in
molecular weight than the starting material 2, which suggests the addition of TRIS and DTT,
respectively (cf. SI). Further analysis by MSMS revealed the parent peak for compound 2, which
also supports formation of addition products. Due to the presence of an α,β-unsaturated system,
it would not be surprising if compound 2 represents a good Michael acceptor as shown in Figure
2. The potential for addition of nucleophiles to C-7 also could represent a strategy to produce a
variety of addition products leading to new nucleotide analogs based on the dihydrothymine
skeleton .
Figure 2: Nucleophilic addition to the α,β-unsaturated system 2.
One clear application of the exocyclic olefin isomers of thymidine (10) and dTMP (2), is
for direct exploration of thymidyl biosynthetic pathways. In the past, modified pyrimidines that
can trap nucleophiles by reaction at C-6 have been explored as a strategy for inhibitor design and
drug development targeting classical TSase. These efforts have resulted in the discovery of key
chemotherapeutics.19 They also have been crucial in rationalizing the activity of compounds like
5-F-dU, which inhibit TSase enzymes by a mechanism based on covalent addition to the
especially electrophilic C-6 position of the pyrimidine. Because compounds 10 and 2 do not
11

have an electrophile site at C-6 they would not be expected to covalently inhibit classical TSase
enzymes. However, compounds 10 and 2 closely resemble intermediates of alternative thymidyl
biosynthesis pathways (e.g., FDTS), which means that these materials may be used to illuminate
dTMP production in organisms relying on these alternative biosynthetic pathways. The
strategies for isotopic labeling and derivatization of compound 2 enabled by the studies reported
here should add to the available tools for mechanistic biochemists to differentiate classical
TSases reactions from alternative thymidyl-biosynthesizing enzymes. This in turn should aid in
the development of potential antibiotics with low side reactions and limited human toxicity.
Conclusions.
Two different synthetic approaches to the C-5 olefin isomer of thymidylate have been
developed. For preparation of large amounts of material, the more attractive approach presented
here uses commercially available thymidine as the starting material and yields compound 2 in
just 6 steps. This route is scalable and the initial steps can be performed on gram scale with the
final step on a milligram scale. At the same time, a complementary synthetic approach also has
been developed where the key glycosylation step is enzymatic and can be performed on a
milligram or sub-milligram scale, presumably with radiolabeled carbohydrates. The synthetic
routes described herein are amenable to introduction of isotopic labels at a variety of sites, as
well as derivatization or functionalization that provides a gateway to other novel nucleotide
analogs. While the most direct application of compound 2 may be in the exploration of
alternative thymidylate biosynthesis pathways, applications can be envisioned wherever the
common nucleotide thymidylate is found in metabolism.
12

EXPERIMENTAL SECTION
General. All chemical reagents and solvents were obtained from commercial suppliers except
α-hydroxythymidine (5),37 3-bromo-2-(bromomethyl)-propanoic acid (12),34 and deoxyribose-1-
phosphate,42 which were prepared by known procedures. Reactions in nonaqueous solvents were
performed under an argon atmosphere and monitored by analytical thin-layer chromatography on
plates coated with 0.25 mm silica gel 60 F254. The TLC plates were visualized under UV light
1
at 254 nm. All H NMR spectra were obtained on 300, 500, or 600 MHz instruments with
chemical shifts reported in ppm referenced to residual solvent in CDCl3 (1H, 7.26 ppm; 13C
13
13
NMR; 77.0 ppm), (CD3)2SO (1H, 2.50 ppm; C NMR; 39.5 ppm), CD3OD (1H, 3.31 ppm; C
NMR; 49.0 ppm), and D2O (1H, 4.80 ppm). High-resolution mass spectra were obtained with a
QTOF mass spectrometer and reported as m/z. LCMS traces were recorded on a Acquity UPLC
BEH C18 column (2.1 × 50 mm, 1.7µm) [flow rate = 0.2 mL/min]; Line A, 0.5% formic acid
and Line B, acetonitrile; run time 24 min.
3’,5’-Di-t-butyldimethylsilyl-7-triethylsilyl-2′-deoxyuridine (6). In a round bottom flask, the
primary alcohol 537 (1.00 g, 2.06 mmol, 1.0 eq), imidazole (200 mg, 2.94 mmol, 1.5 eq), and
TESCl (370 mg, 2.45 mmol, 1.2 eq) were dissolved in DMF (10 mL) and the solution was stirred
at rt overnight. The reaction was quenched by addition of water and extracted with EtOAc (2 x
300 mL). The combined organic phase was washed with water (3 x 300 mL), dried (MgSO4),
and filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/EtOAc; 9:1 to 4:1) to yield compound 6 (1.10 g, 89%) as a
1
white solid: H NMR (500 MHz, CDCl3) δ 0.07–0.08 (m, 12H), 0.63–0.71 (m, 6H), 0.88–0.93
(m, 18H), 0.95–1.02 (m, 9H), 1.98–2.07 (m, 1H), 2.28–2.36 (m, 1H), 3.64–3.70 (m, 1H), 3.75–
3.81 (m, 1H), 3.94–4.00 (m, 1H), 4.37–4.41 (m, 1H), 4.43–4.52 (m, 2H), 6.24–6.35 (m, 1H),
13

7.50–7.56 (m, 1H), 8.57–8.68 (m, 1H); 13C NMR ( 125 MHz, CDCl3): δ −5.4 (2C), −4.7, −4.8,
4.4 (3C), 6.8 (3C), 18.0, 18.4, 25.7 (3C), 25.9 (3C), 40.5, 57.7, 63.3, 72.6, 85.4, 87.8, 114.3,
136.0, 150.0, 162.1; HRMS (ESI-QTOF) calcd for C28H56N2O6Si3Na (M + Na)+ 623.3344,
found, 623.3349.
3’,5’-Di-t-butyldimethylsilyl-5,6-dihydro-7-triethylsilyl-2′-deoxyuridine (7). Compound 6
(1.60 g, 2.66 mmol) was dissolved in a mixture of methanol (40 mL) and water (8 mL). To this
solution was added the solid catalyst (Rh/Al2O3, 1.5 g, 5%) and the mixture was subjected to
hydrogenation at rt and 170 psi for 18 h. The solution then was filtered and the filtrate was
concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane
1
/EtOAc; 9:1 to 7:3) to yield compound 7 (1.20 g, 75%) as a colorless oil: H NMR (500 MHz,
CDCl3) δ 0.07–0.08 (m, 12H), 0.59–0.64 (m, 6H), 0.90–0.91 (m, 18H), 0.94–0.98 (m, 9H), 1.97–
2.02 (m, 2H), 2.66–2.74 (m, 1H), 3.40–3.47 (m, 2H), 3.59–3.65 (m, 1H), 3.68–3.74 (m, 1H),
3.77–3.80 (m, 1H), 3.84–3.89 (m, 1H), 3.97–4.03 (m, 1H), 4.33–4.39 (m, 1H), 6.27–6.31 (t, 1H),
7.64–7.73 (s, 1H); 13C NMR (125 MHz, CDCl3) δ −5.4 (2C), −4.8 (2C), 4.3 (3C), 6.7 (3C), 18.0,
18.4, 25.8 (3C), 25.9(3C), 37.3, 38.7, 43.4, 59.8, 63.1, 72.1, 83.8, 86.2, 152.4, 170.0; HRMS
(ESI-QTOF) calcd for C28H58N2O6Si3Na (M + Na)+, 625.3500, found 625.3514.
3’,5’-Di-t-butyldimethylsilyl-5,6-dihydro-5-hydroxymethyl-2′-deoxyuridine (8).
Compound 7 (1.0 g, 1.66 mmol) was dissolved in anhydrous methanol (15 mL). To this solution
was added solid Pd/C (~100 mg, 10%) and the mixture was stirred at rt under a hydrogen
atmosphere for 20 minutes. The solution then was filtered through a celite bed and the filtrate
was concentrated in vacuo. The initial product was purified by silica gel column chromatography
1
(hexane/EtOAc; 7:3 to 1:1) to yield the primary alcohol 8 (710 mg, 88%) as a colorless oil: H
NMR (500 MHz, MeOD) δ 0.10–0.15 (m, 12H), 0.94 (d, J =3.4 Hz, 18H), 1.94 (ddd, J = 13.2,
14

6.0, 2.5 Hz, 1H), 2.14–2.22 (m, 1H), 2.70–2.78 (m, 1H), 3.46–3.56 (m, 2H), 3.68–3.78 (m, 2H),
13
3.78–3.87 (m, 3H), 4.39–4.48 (m, 1H), 6.27–6.30 (dd, J = 8.6, 5.9 Hz, 1H); C NMR (125
MHz, MeOD) δ −8.2, −8.1, −7.4, −7.3, 16.0, 16.4, 23.4 (3C), 23.6 (3C), 35.0, 36.6, 41.5, 57.4,
61.7, 71.2, 82.4, 85.1, 152.0, 170.1; HRMS (ESI-QTOF) calcd for C22H44N2O6Si2Na (M + Na)+
511.2636, found 511.2636.
3’,5’-Di-t-butyldimethylsilyl-5-methylene-6-hydro-2′-deoxyuridine (9). In a round bottom
flask the primary alcohol 8 (710 mg, 1.45 mmol, 1.0 eq), tosyl chloride (555 mg 2.91 mmol, 2
eq), triethylamine (294 mg, 2.91 mmol, 2 eq) and a catalytic amount of DMAP were dissolved in
CH2Cl2 (10 mL) and the resulting solution was stirred at room temperature overnight. The
reaction mixture then was quenched by addition of water and extracted with EtOAc (3 x 50 mL).
The combined organic phase was washed with water (2 x 50 mL), dried (Na2SO4), and filtered,
and the filtrate was concentrated in vacuo. The initial product was purified by silica gel column
chromatography (hexane/EtOAc, 9:1 to 7:3) to yield compound 9 (622 mg, 91%) as a white
1
solid: H NMR (500 MHz, CDCl3) δ 0.09–0.11 (m, 12H), 0.91–0.94 (m, 18H), 1.95–2.01 (m,
1H), 2.02–2.09 (m, 1H), 3.72–3.79 (m, 2H), 3.82–3.84 (m, 1H), 3.97–4.00 (d, 1H), 4.28–4.31
13
(d, 1H), 4.39–4.43 (m, 1H), 5.65 (s, 1H), 6.32–6.35 (t, 1H), 6.39 (s, 1H) 7.50 (br s, 1H); C
NMR (125 MHz, CDCl3) δ −5.5, −5.4, −4.8, −4.6, 18.0, 18.4, 25.8 (3C), 25.9 (3C), 37.3, 41.6,
63.0, 72.0, 83.9, 86.5, 124.7, 131.4, 152.00, 163.0; HRMS (ESI-QTOF) calcd for
C22H42N2O5Si2Na (M + Na)+ 493.2530, found 493.2531.
5-Methylene-6-hydro-2′-deoxyuridine (10). In a plastic vial, compound 9 (100 mg, 0.21
o
mmol) was dissolved in pyridine (1.5 mL) and the solution was cooled to 0 C. To this cold
solution, TEA·3HF (0.42 mmol, 2 eq) was added dropwise and the reaction was stirred for 5 h.
The reaction mixture then was diluted with pyridine (5 mL) and concentrated in vacuo. The
15

initial product was purified by silica gel column chromatography (CH2Cl2/MeOH, 9:1 to 8.5:1.5)
to yield compound 10 (48 mg, 94%) as a white solid: 1H NMR (600 MHz, D2O) δ 2.04–2.11 (m,
1H), 2.26–2.34 (m, 1H), 3.62–3.66 (m, 1H), 3.70–3.74 (m, 1H), 3.85 (br d, J = 5.5 Hz, 1H),
4.14– 4.23 (m, 2H), 4.32–4.35 (m, 1H), 5.81–5.84 (m, 1H), 6.20–6.24 (m, 1H), 6.26–6.29 (m,
1H); 13C NMR (126 MHz, D2O) δ 35.0, 41.8, 61.6, 70.8, 84.2, 85.3, 126.4, 130.5, 153.8, 165.9;
HRMS (ESI-QTOF) calcd for C10H14N2O5Na (M + Na)+ 265.0800, found 265.0796. DEPT135
and HSQC data are included in the SI.
5′-Monophospho-5-methylene-6-hydro-2′-deoxyuridine (2). Method A. HSV Thymidine
kinase (200 nM) was incubated at 34 °C in the presence of compound 10 (18 mg, 18.6 mM in 4
mL final volume), 20 mM HEPES (pH 7.8), 3 mM MgCl2, 3 mM KCl, 0.8 mM ATP, 59 mM
creatine phosphate and a trace amount of creatine phosphokinase (CPK). The reaction was done
on a 4 ml scale. After 20 h, the reaction mixture was filtered through Amicon Ultra-4 4 mL
centrifugal filters (MWCO 10 KDa) at 4°C. The product was purified by HPLC (semi-prep
column) using a linear gradient of MeOH (5-100%) in 4 mM TEAB pH 6.8 at 3.2 ml/min. The
product peak was detected spectrophotometrically at 210, 240, 260, and 290 nm, manually
collected, and the center cut fractions were combined to give ~3 mg of the pure product: 1H
NMR (500 MHz, D2O) δ 1.97–2.02 (m, 1H), 2.28–2.34 (m, 1H), 3.76–3.78 (t, 1H), 3.90–3.91
(m, 1H), 4.15 (d, 1H, J = 15.0), 4.25 (d, 1H, J = 15.0), 4.41–4.44 (m, 1H), 5.89 (s, 1H), 6.18–
31
6.31 (m, 1H), 6.22 (s,1H); P NMR (500 MHz, D2O) δ 3.88; HRMS (ESI-QTOF) calcd for
C10H15N2O8P (M – H)- 321.0488, found 321.0486. The COSY and HSQC data, and LCMS
data, are included in the SI.
Method B: In a plastic vial, bis(cyclohexylammonium) deoxyribose-1-phosphate (21 mg, 0.05
mmol) and compound 16 (1.5 mg, 0.012 mmol) were added to a degassed buffer (5 mL, 20 mM
16

HEPES, 100 mM KCL, 5 mM MgCl2). To this mixture 500 units of previously dialyzed
commercial thymidine phosphorylase (TP) was added and the solution was incubated at rt for 20
h. Conversion to the thymidine analogue 10 was monitored by HPLC. When >20% conversion
was observed, 12.5 mg of ATP were added followed by addition of 500 ng of mutant dCK (≥ 50
units). The resulting mixture was incubated for 20-48 h at rt until quantitative conversion of the
nucleoside 10 to the nucleotide monophosphate 2 was observed. The reaction mixture was
filtered through a 10,000 MWCO Millipore membrane prior to HPLC purification.
3-Bromo-2-(bromomethyl)propionamide (13).34 Through a modification of the reported
procedure, compound 12 (46 g, 190 mmol) was dissolved in anhydrous benzene (300 mL) and
the solution was cooled to 0 °C. After PCl5 (48 g, 230 mmol) was added to the cold solution in
four portions (to avoid a vigorous reaction), the reaction mixture allowed to warm to rt slowly,
and then heated at reflux for 2 h. The solvent was removed in vacuo, petroleum ether (60-80 °C)
was added to the resulting residue, and the mixture was filtered. Anhydrous NH3 was bubbled
through the filtrate to give a white precipitate, which was isolated by filtration, washed with
1
water, and dried under vacuum desiccation to give compound 13 (31.8 g, 69%): H NMR (300
MHz, D3CS(O)CD3) δ 3.0–3.12 (m 1H) 3.5–3.7 (m, 4H) 7.32 (s, 1H) 7.71 (s, 1H).
2-Bromomethylacrylamide (14).34 1,4-Dioxane (27 mL) was added to a 250 mL Schlenk flask,
diisopropylethylamine (6.5 mL) was added, and the mixture was degassed by several freeze-
pump-thaw cycles. The resulting solution was transferred by cannula to a 100 mL Schlenk flask
that contained the dibromide 13 (8.0 g, 33 mmol) and hydroquinone (30 mg, 0.27 mmol). The
resulting solution was stirred for 9 hours at 50–60 °C. After concentration by vacuum
distillation, ice water (5 mL) and HCl (1 N, 1 drop) were added along with ethyl acetate (20 mL).
This solution was extracted with ethyl acetate and the combined extracts were concentrated in
17

1
vacuo to give the acrylamide 14 (2.5 g, 47%) as a cream-colored solid: H NMR (300 MHz,
(CD3)2SO) δ 4.75 (s, 2H), 5.85 (s, 1H), 5.92 (s, 1H), 7.31 (s, 1H), 7.73 (s, 1H); HRMS (ESI-
QTOF) calcd for C4H6 BrNO 162.9633 (M+), found (EI+) 162.9633.
N-Trichloracetyl-N’-2-bromomethacroyl-urea (15).34 Compound 14 (1.76 g, 11 mmol) and
benzene (38 mL) were added to a 250 mL Schlenk flask, followed by the addition of
trichloroacetyl isocyanate (2.0 g, 11 mmol). This solution was heated at reflux for one h,
allowed to cool, and gravity filtered, and the filtrate was concentrated to ~ 11 mL in vacuo.
Dropwise addition of petroleum ether (40–60 °C) yielded compound 15 as a white crystalline
solid (3.32 g, 89%): 1H NMR (300 MHz, D3CS(O)CD3) δ 4.42 (s, 1H), 4.5 (s, 1H) 6.2–6.45 (m,
2H) 11.57 (s, 1H), 12.52 (s, 1H).
Dihydro-5-methylene-2,4(1H,3H)-pyrimidinedione (16).34 A solution of NaHCO3 (0.86 g, 10
mmol) in water (7.2 mL) was stirred while bromide 15 (1.5 g, 4.3 mmol) was added to produce a
suspension. Ethanol (13.2 mL) was added and the reaction was heated quickly to 55-60 °C.
After 2 minutes the solution was gravity filtered, the filtrate was allowed to cool to rt for 1 hour,
and then concentrated in vacuo at 30 °C until a precipitate was observed. A minimum amount of
1N NaOH was added at 0 °C resulting in a clear solution that was filtered and then neutralized by
addition of glacial acidic acid. Crystallization occurred upon dropwise addition of EtOH at 0 °C
to give the initial product. Recrystallization from water and ethanol gave the desired product 16
1
(54 mg, 10%) as a white crystalline solid: H NMR (300 MHz, D3CS(O)CD3) δ 3.87 (s, 2H)
5.53 (s, 1H) 5.97 (s, 1H) 7.41 (s, 1H) 10.0 (s, 1H); HRMS (ESI-QTOF) calcd for C5H6N2O2 (M+)
126.0430, found (EI+) 126.0432.
18

1
Supporting Information Available: Both H and 13C NMR spectra and LCMS data. This
material is available free of charge via the Internet at:
Acknowledgment. The enzyme dCK was provided as a generous gift by Prof. Arnon Lavie of
the University of Illinois – Chicago. Financial support from the NIH (5 R01 GM110775 to A.
K.), the predoctoral Training Program in Biotechnology (T32 GM008365 to E. M. K.), the
National Science Foundation Graduate Research Fellowship Program (to E. M. K.), and the Roy
J. Carver Charitable Trust through its Research Program of Excellence (#01-224 to D. F. W.) is
greatly appreciated.
Notes.
The Authors declare no competing financial interest.
References.
(1)
(2)
Robertson, K. D. Nature Reviews Genetics 2005, 6, 597.
Lakshminarasimhan, R.; Liang, G. In DNA Methyltransferases - Role and
Function; Jeltsch, A., Jurkowska, R. Z., Eds.; Springer International Publishing: Cham, 2016, p
151.
(3)
Rode, A. B.; Kim, B. M.; Park, S. H.; Hong, I. S.; Hong, S. H. Bioorg. Med.
Chem. Lett. 2011, 21, 1151.
(4)
(5)
Chen, X. M.; Wiemer, D. F. J. Org. Chem. 2003, 68, 6108.
Chen, X. M.; Wiemer, A. J.; Hohl, R. J.; Wiemer, D. F. J. Org. Chem. 2002, 67,
9331.
19

(6)
8, 2501.
(7)
Jung, K. Y.; Hohl, R. J.; Wiemer, A. J.; Wiemer, D. F. Biorg. Med. Chem. 2000,
Ni, F.; Kung, A.; Duan, Y. K.; Shah, V.; Amador, C. D.; Guo, M.; Fan, X. G.;
Chen, L.; Chen, Y. H.; McKenna, C. E.; Zhang, C. J. Am. Chem. Soc. 2017, 139, 7701.
(8)
(9)
Varga, A.; Lionne, C.; Roy, B. Current Drug Metabolism 2016, 17, 237.
Roy, B.; Depaix, A.; Perigaud, C.; Peyrottes, S. Chem. Rev. 2016, 116, 7854.
(10) Abraham, T. W.; Kalman, T. I.; McIntee, E. J.; Wagner, C. R. J. Med. Chem.
1996, 39, 4569.
(11) Abraham, T. W.; McIntee, E. J.; Iyer, V. V.; Schinazi, R. F.; Wagner, C. R.
Nucleosides Nucleotides 1997, 16, 2079.
(12) Ding, H.; Majumdar, A.; Tolman, J. R.; Greenberg, M. M. J. Am. Chem. Soc.
2008, 130, 17981.
(13) Peng, X. H.; Hong, I. S.; Li, H.; Seiclman, M. M.; Greenberg, M. M. J. Am.
Chem. Soc. 2008, 130, 10299.
(14) Wagstaff, B. A.; Rejzek, M.; Pesnot, T.; Tedaldi, L. M.; Caputi, L.; O'Neill, E. C.;
Benini, S.; Wagner, G. K.; Field, R. A. Carbohydr. Res. 2015, 404, 17.
(15) Wagner, G. K.; Pesnot, T.; Field, R. A. Nat. Prod. Rep. 2009, 26, 1172.
(16) Lalau, C.; Oosterhuis, J. A.; Versteeg, J.; Vanrij, G.; Delavalette, J.; Craandijk,
A.; Lamers, W. Br. J. Ophthalmol. 1982, 66, 506.
(17) Boon, R. Antivir. Chem. Chemother. 1997, 8, 5.
(18) Taddia, L.; D'Arca, D.; Ferrari, S.; Marraccini, C.; Seveni, L.; Ponterini, G.;
Assaraf, Y. G.; Marverti, G.; Costi, M. P. Drug Resistance Updates 2015, 23, 20.
20

(19) Wilson, P. M.; Danenberg, P. V.; Johnston, P. G.; Lenz, H. J.; Ladner, R. D.
Nature Reviews Clinical Oncology 2014, 11, 282.
(20) Dittmar, K. A.; Sorensen, M. A.; Elf, J.; Ehrenberg, M.; Pan, T. Embo Reports
2005, 6, 151.
(21) Motorin, Y.; Seidu-Larry, S.; Helm, M. In DNA Methyltransferases - Role and
Function; Jeltsch, A., Jurkowska, R. Z., Eds.; Springer International Publishing: Cham, 2016, p
19.
(22) Islam, Z.; Strutzenberg, T. S.; Gurevic, I.; Kohen, A. J. Am. Chem. Soc. 2014,
136, 9850.
(23) Mishanina, T. V.; Yu, L. P.; Karunaratne, K.; Mondal, D.; Corcoran, J. M.; Choi,
M. A.; Kohen, A. Science 2016, 351, 507.
(24) Hamdane, D.; Argentin, M.; Cornu, D.; Golinelli-Pimpaneau, B.; Fontecavet, M.
J. Am. Chem. Soc. 2012, 134, 19739.
(25) Koehn, E. M.; Fleischmann, T.; Conrad, J. A.; Palfey, B. A.; Lesley, S. A.;
Mathews, II; Kohen, A. Nature 2009, 458, 919.
(26) Irwin, C. R.; Hitt, M. M.; Evans, D. H. Frontiers in Oncology 2017, 7.
(27) Mishanina, T. V.; Koehn, E. M.; Kohen, A. Bioorg. Chem. 2012, 43, 37.
(28) Barrett, J. E.; Maltby, D. A.; Santi, D. V.; Schultz, P. G. J. Am. Chem. Soc. 1998,
120, 449.
(29) Mishanina, T. V.; Koehn, E. M.; Conrad, J. A.; Palfey, B. A.; Lesley, S. A.;
Kohen, A. J. Am. Chem. Soc. 2012, 134, 4442.
(30) Hong, I. S.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 10510.
(31) Sloane, J. L.; Greenberg, M. M. J. Org. Chem. 2014, 79, 9792.
21

(32) Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Leukemia 2001, 15, 875.
(33) Tomkuvienė, M.; Kriukienė, E.; Klimašauskas, S. In DNA Methyltransferases -
Role and Function; Jeltsch, A., Jurkowska, R. Z., Eds.; Springer International Publishing: Cham,
2016, p 511.
(34) Klötzer, W. Monatshefte Fur Chemie 1973, 104, 415.
(35) Stull, F. W.; Bernard, S. M.; Sapra, A.; Smith, J. L.; Zuiderweg, E. R. P.; Palfey,
B. A. Biochemistry 2016, 55, 3261.
(36) Conrad, J. A.; Ortiz-Maldonado, M.; Hoppe, S. W.; Palfey, B. A. Biochemistry
2014, 53, 5199.
(37) Sun, Q.; Sun, J.; Gong, S. S.; Wang, C. J.; Pu, S. Z.; Feng, F. D. RSC Adv. 2014,
4, 36036.
(38) Kore, A. R.; Xiao, Z.; Senthilvelan, A.; Charles, I.; Shanmugasundaram, M.;
Mukundarajan, S.; Srinivasan, B. Nucleosides, Nucleotides and Nucleic Acids 2012, 31, 567.
(39) Van Daele, I.; Munier-Lehmann, H.; Froeyen, M.; Balzarini, J.; Van Calenbergh,
S. Journal of Medicinal Chemistry 2007, 50, 5281.
(40) Pahnke, K.; Meier, C. Chembiochem 2017, 18, 1616.
(41) Waldman, A. S.; Haeusslein, E.; Milman, G. J. Biol. Chem. 1983, 258, 1571.
(42) Friedkin, M.; Roberts, D. J. Biol. Chem. 1954, 207, 245.
(43) Ghanem, M.; Murkin, A. S.; Schramm, V. L. Chem. Biol. 2009, 16, 971.
(44) Schwartz, P. A.; Vetticatt, M. J.; Schramm, V. L. Biochemistry 2011, 50, 1412.
22

Graphical abstract
23