Probing the binding requirements of modified nucleosides with the dna nuclease snm1a

molecules

Article

Probing the Binding Requirements of Modiﬁed Nucleosides

with the DNA Nuclease SNM1A

Eva-Maria Dürr and Joanna F. McGouran *

School of Chemistry and Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse St.,

D02 R590 Dublin, Ireland; durre@tcd.ie

* Correspondence: jmcgoura@tcd.ie

Abstract: SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its

inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However,

the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and

only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and

evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning

of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester,

carboxylate and hydroxamic acid malonate derivatives which were installed in the 5⁰-position or 3⁰-

position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant

SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration.

0

The most potent compound contains a hydroxamic acid malonate group at the 5 -position. Overall, our

studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and

indicate that groups containing a negatively charged group in close proximity to a metal chelator, such

as hydroxamic acid malonates, are promising structures in the design of inhibitors.

Keywords: nuclease; interstrand crosslink repair; SNM1A; nucleoside inhibitor; malonate; hydrox-

amic acid

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Dürr, E.-M.; McGouran, J.F.

Probing the Binding Requirements of

Modiﬁed Nucleosides with the DNA

Nuclease SNM1A. Molecules 2021, 26,

320. https://doi.org/10.3390/

molecules26020320

1. Introduction

The nuclease SNM1A (also known as DCLRE1A) is part of the interstrand crosslink

repair machinery in mammalian cells [ ]. Depletion of SNM1A increases sensitivity to

crosslinking agents [ ], although the precise mechanisms behind this are still under in-

1

2–5

vestigation. Current research suggests that SNM1A digests past crosslinks from an incision

Academic Editor: Derek J. McPhee

Received: 11 November 2020

Accepted: 31 December 2020

Published: 9 January 2021

5⁰to the site of the lesion; this role and other proposed pathways are summarised in a

recent review [

ase activity, although endonuclease activity requires a blocked 5⁰-end and stoichiometric

amounts of enzyme [

]. Exonuclease activity requires a 5⁰-phosphate group and is proces-

sive for high molecular weight substrates [ ]. The active site of SNM1A is postulated to

contain a di-metal centre, based on similarity with other members of the

However, only one metal ion has been observed in crystal structures of SNM1A to date [

1

]. SNM1A has been reported to have 5⁰-exonuclease as well as endonucle-

6

Publisher’s Note: MDPI stays neu-

tral with regard to jurisdictional clai-

ms in published maps and institutio-

nal afﬁliations.

7

β

-CASP family [

8

].

9

The active site is located within a wide binding groove, which permits the processing of

bulky substrates such as DNA containing interstrand crosslinks [8].

Like other DNA damage repair proteins [10], SNM1A is a potential target for overcom-

ing resistance to crosslinking agents in cancers [11]. Inhibitors reported to date are limited

to o-phenanthroline [12], cephalosporins [13], and a modiﬁed nucleoside inhibitor [14].

In our previous work designing nucleoside inhibitors, the hydroxamic acid modiﬁcation

censee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and con-

ditions of the Creative Commons At-

tribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

emerged as the most promising group [14]. Nucleosides

to inhibit SNM1A to varying degrees, and the more potent compound

have an IC₅₀value of 139 M. However, both compounds likely only target one of the two

postulated metal ions in the active site of SNM1A. Therefore, in this work, we expand the

1

and

2

(Figure 1a) were found

1

was found to

µ

Molecules 2021, 26, 320. https://doi.org/10.3390/molecules26020320

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 320

2 of 25

metal-binding group to a malonate group to probe whether an extended metal-binding

group achieves chelation to both postulated metal ions.

Figure 1. Design of modiﬁed nucleosides designed to bind to SNM1A. (a) Previously reported nucleoside inhibitors of

SNM1A; ( ) the proposed binding mode for hydroxamic acid malonate derivatives; (

b

c) structures of target (di)nucleosides.

In recent crystal structures of SNM1A, malonate was observed bound to the metal

ion in the active site [ ]. This highlights the suitability of the group as a metal chela-

9

tor. The malonate group has found application both as a scaffold for a hydroxamic acid

group [15–22] and as a phosphate bioisostere in many different contexts, including ana-

logues of phosphosugars [23–26] and nucleotides [27,28]. By combining the malonate

group with the hydroxamic acid group, binding of both metal ions is envisaged (Figure 1b).

The substituents of the malonate group were varied to include esters and carboxylate salts

to obtain further information about the binding mode.

Derivatives utilised either one carboxylic acid group of the malonate group to connect

the group to the nucleoside via an amide linkage (N-linked), or alkylation at the α-carbon

(C-linked) was employed to install the malonate group onto the nucleoside. The nucleobase

used for this family of modified nucleosides is thymine, allowing for direct comparison

with the previously evaluated compounds [14 ]. The 5⁰-C-linked series in particular is

directly related to the previously reported compound

2, as its malonate group is at the same

distance from the deoxyribose core as the hydroxamic acid group in compound

2. However,

the malonate modifications were introduced not only at the 5⁰-position, but also at the

3⁰-position and as a linker between two nucleosides (Figure 1 c) to probe the interactions

between the nucleoside and the active site of recombinant SNM1A in gel-based assays.

2. Results

2.1. Synthesis

The synthesis of the 5⁰-alkylated targets was adapted from Zlatev et al. [28] and is

shown in Scheme 1. Starting from 5⁰-deoxy-5⁰-iodothymidine ( ), protection of the 3⁰-

3

hydroxy group as the silyl ether and of the nucleobase with the benzyloxymethyl group

was carried out in 87% and 95% yield, respectively. This protected iodide then underwent

nucleophilic substitution with benzyl methyl malonate in 66% yield. Removal of the silyl

ether of malonate ester afforded alcohol in 71% yield, and catalytic hydrogenation

removed both the benzyloxymethyl group and the benzyl group to give carboxylic acid 8

in 86% yield. For use in assays, the sodium carboxylate salt was required, so ion exchange

was carried out following hydrogenation. Due to contamination with ion-exchange resin,

puriﬁcation by reversed-phase preparative TLC was employed and gave target in a

low yield of 22%. Carboxylic acid was used as an intermediate for targets 10 and 11

Following ester hydrolysis and ion exchange to furnish carboxylate salt 10, reversed-phase

5

6

7

9

8

.

Molecules 2021, 26, 320

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preparative TLC was again used to purify compound 10, which was obtained in 55% yield.

Carboxylic acid

8

was also employed in the synthesis of hydroxamic acid 11, which was

and 11 were

obtained in 15% yield via aminolysis with hydroxylamine [29]. Targets

9

isolated and used as mixtures of interconverting diastereomers. Unfortunately, synthesis

of a 1,3-dihydroxamic acid is not possible using aminolysis with hydroxylamine, as the

treatment of a 1,3-diester with hydroxylamine under basic conditions results in cyclisation

to form isoxazolidine-3,5-diones [30,31].

0

Scheme 1. Synthesis of 5 -modiﬁed C-linked targets 9, 10 and 11.

For the N-linked targets, amide coupling between 5⁰-amino-5⁰-deoxythymidine (12

and monomethyl potassium malonate was employed (Scheme 2) to furnish ester 13 in

)

54% yield. This target served as an intermediate in the synthesis of targets 14 and 15

Ester hydrolysis followed by ion exchange afforded sodium carboxylate 14 in 98% yield.

Hydroxamic acid 15 was obtained in 83% yield by treating ester 13 with hydroxylamine

under basic conditions [29].

.

0

Scheme 2. Synthesis of 5 -modiﬁed N-linked targets 13, 14 and 15.

Synthesis of the 3⁰-modiﬁed targets was attempted analogous to the 5⁰-modiﬁed

compounds. For the C-linked targets, initially nucleophilic substitution of a mesylate was

Molecules 2021, 26, 320

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envisaged (Scheme 3a). Inversion of the 3⁰-hydroxy group of protected thymidine 16 was

carried out by sequential mesylation and hydrolysis [32] in 90% yield. Mesylation [33

]

of the alcohol 17 proceeded in 90% yield. However, substitution of this mesylate 18 with

benzyl methyl malonate was unsuccessful, with the N-methylated elimination product

20 obtained in 46% yield instead of the desired product 19. An alternative strategy to

functionalise the 3⁰-position using protected 2,3⁰-anhydrothymidine [34

] 21 and methyl

triﬂate as an activating agent as reported by Saha et al. [35] was unsuccessful in our hands.

Only the hydrolysed product 23 was obtained after quenching the reaction, with none of

the desired product 22 observed (Scheme 3b).

0

Scheme 3. Attempted syntheses of 3 -modiﬁed targets. (

a) Attempted synthesis of C-linked targets

from mesylate 18. (

b

) Attempted synthesis of C-linked targets from protected anhydrothymidine 21

.

We then turned our attention to the N-linked targets. Commercially available AZT (24

)

was reduced to aminothymidine (25) by catalytic hydrogenation in 99% yield (Scheme 4).

Amine 25 was then used in an EDC-mediated amide coupling with monomethyl potassium

malonate, which afforded target 26 in 34% yield. Treatment of ester 26 with KOH followed

by ion exchange produced carboxylate salt 27 in 82% yield, while aminolysis of this ester

using the same conditions as for the 5⁰-modiﬁed nucleosides afforded hydroxamic acid 28

in 70% yield.

For the ﬁnal group of compounds, the malonate group forms a phosphodiester re-

placement and links two nucleosides. To this end, a 5⁰-modiﬁed carboxylic acid and a

0

3 -amino nucleoside were combined (Scheme 5). For the synthesis of the dinucleosides, pro-

0

tecting groups for the 5 - and 3 -hydroxy groups were used to aid solubility. The carboxylic

acid nucleoside 29 was obtained from protected malonate ester by catalytic hydrogena-

tion. The benzyloxymethyl group of was only partially removed in this step, but this

hemiaminal did not interfere with further reactions. The other nucleoside, 5⁰-protected

3⁰-aminothymidine 31, was synthesised from DMTr-protected AZT 30

36]. Reduction of

6

[

azide 30 furnished amine 31 in 95% yield. EDC-mediated coupling between nucleosides

29 and 31 afforded the fully protected dinucleoside 32 in 76% yield. Deprotection of the

3⁰- and 5⁰-hydroxy groups was achieved using TBAF and TFA, respectively, in 92% yield

for each step. This sequence afforded target 34, which was further derivatised. Sodium

carboxylate 35 was furnished from ester 34 in 89% yield by ester hydrolysis and subsequent

ion exchange, and hydroxamic acid 36 was obtained in 74% yield from ester 34 by aminol-

Molecules 2021, 26, 320

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ysis. As observed with the 5⁰-modiﬁed C-linked compounds

9

and 11, the dinucleosides

34–36 were also isolated and used as mixtures of interconverting diastereomers.

0

Scheme 4. Synthesis of 3 -modiﬁed N-linked targets 26–28.

Scheme 5. Synthesis of malonate-linked dinucleosides 34–36.

Molecules 2021, 26, 320

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The successful synthesis of the dinucleosides 34

–

36 completes a series of twelve

nucleosides. These can be divided into four groups: 5⁰-modiﬁed C-linked, 5⁰-modiﬁed N-

linked, 3⁰-modiﬁed N-linked, and dinucleosides. Each group consists of three compounds

each, with an ester, sodium carboxylate and a hydroxamic acid analogue.

2.2. Biological Evaluation

The twelve modiﬁed nucleosides (Figure 2a) were evaluated as inhibitors of recombi-

nant SNM1A to evaluate binding to the active site, using a previously reported gel-based

assay with the same 21-mer oligonucleotide substrate [14]. The nucleosides were incubated

with SNM1A for 5 min prior to the addition of the ﬂuorescently tagged 21-mer oligonu-

cleotide substrate and further incubation for 1 h. In the event of the nucleoside binding

to the active site, digestion of the substrate proceeds less efﬁciently (Figure 2b). SNM1A

0

removes nucleotide monomers from the 5 -end, generating a shorter oligonucleotide that in

turn is a substrate for SNM1A and can be digested further. The size of the oligonucleotide

products, analysed by gel electrophoresis, therefore indicates how strongly a compound

binds to the nuclease.

Figure 2. Evaluation of modiﬁed (di)nucleosides as inhibitors of SNM1A. (

a) Structures of the twelve (di)nucleosides

that were evaluated as inhibitors of SNM1A; ( ) schematic representation of assay; (

b

c) gel electrophoresis showing the

extent of digestion of ﬂuorescent 21-mer oligonucleotide substrate. SNM1A (2.5 nM) was pre-incubated with the modiﬁed

(di)nucleosides (1 mM) for 5 min before the oligonucleotide substrate (80 nM) was added and a further 60 min incubation.

nt = nucleotides.

Molecules 2021, 26, 320

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The results of this assay for the twelve modiﬁed nucleosides are shown in Figure 2c.

Lane 1 shows the oligonucleotide substrate in the absence of SNM1A, while lane 2 shows

maximal digestion in 60 min, in the absence of any modiﬁed nucleoside. Thymidine (T) was

used as a negative control to ensure that any inhibitory effects in lanes 4–15 can be attributed

to the nucleoside modiﬁcation, as thymidine has no effect on the hydrolysis of the substrate

(Figure 2c, lane 3). Encouragingly, nine out of twelve malonate-based nucleosides showed

some inhibition at 1 mM concentration (Figure 2c). Only three nucleosides (Figure 2c, lanes

7, 8, 10) had no impact on the activity of SNM1A.

Analysis of the size of oligonucleotide products remaining after the incubation for each

0

compound family revealed the following: the 5 -C-linked series produced three compounds

that led to almost full inhibition at 1 mM concentration, with malonate hydroxamic acid

11 emerging as the most potent overall (Figure 2c, lane 6). The dinucleoside series also

afforded three active compounds that impeded SNM1A₀activity (Figure 2c, lanes 13–15)

and was only slightly less effective in this assay than the 5 -C-linked series (Figure 2c, lanes

4–6). In contrast, both the 3⁰- and 5⁰-N-linked series contain compounds that did not have

any effect on SNM1A, with only nucleosides 13, 14 and 26 slowing the rate of hydrolysis

(Figure 2c, lanes 9, 11, 12). Within each series, the hydroxamic acid modiﬁcation was found

to lead to the strongest inhibition (Figure 2c, lanes 6, 9, 12, 15), with the exception of the

dinucleosides, where carboxylate 35 and hydroxamic acid 36 appear to have approximately

equal potency (Figure 2c, lanes 14 vs. 15). The carboxylate modiﬁcation resulted in

0

inhibition in all families except the 5 -N-linked series (Figure 2 c, lanes 5, 11, 14). The methyl

ester analogues were only active for the 5⁰-C-linked series, which contains an additional

carboxylate group, and the dinucleoside series (Figure 2c, lanes 4, 13).

To further investigate the potency of the different nucleosides, concentration-dependence

experiments were carried out for the nine nucleosides that showed inhibition (Figure 2

and Figures S1–S4), with concentrations ranging from 1 mM to 1

µM. Although partial

digestion of the substrate by SNM1A was observed at concentrations below 1 mM, the

strongest inhibitor 11 was found to impede hydrolysis at concentrations as low as 33 µM

(Figure 3, lanes 4–7) and its activity was found to be concentration dependent.

0

Figure 3. Concentration dependence of 5 -C-linked malonate nucleoside 11. Digestion of the phos-

phorylated oligonucleotide (80 nM) after incubation with SNM1A (2.5 nM) and nucleoside 11 (1000–

1

µ

M) or thymidine (

T

) (1 mM) for 60 min at 37 ^◦C, analy_◦sed by denaturing PAGE. SNM1A was

preincubated with the modiﬁed nucleoside for 5 min at 37 C prior to the addition of the substrate.

nt = nucleotides.

The results of identical assays for the other modiﬁed nucleosides are summarised

in Table 1 (for gel images see Figures S1–S4). All compounds that were active at 1 mM

concentration also have a partial inhibitory effect at 333

entry 10). Only the malonate hydroxamic acid 11 Table 1, entry 3) was effective at lower

concentrations. In three of the four series, the ester analogue is the weakest inhibitor

Table 1, entries 4, 7, 10; Figure 4). This result is consistent with the initial screen and can

µM, except dinucleoside 34 (Table 1,

(

be explained by the lack of an acidic proton compared to hydroxamic acids or the lack of

negatively charged atoms compared to carboxylate salts, making chelation unfavourable.

Molecules 2021, 26, 320

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This implies that the malonate group is not coordinating exclusively through the carbonyl

oxygen atoms but requires substituents with the potential to hold a negative charge to

0

achieve effective chelation. The 5 -C-linked malonate series (Table 1, entries 1–3) resulted in

the strongest binding. This ﬁnding is not surprising given the additional carboxylate group

that is present compared to all other derivatives. It is therefore likely that this carboxylate

participates in coordination to the metal centre. As observed in Figure 2c and Table 1, the

most potent compound is the hydroxamic acid malonate 11 (Table 1, entry 3).

Figure 4. Trends observed from the analysis of modiﬁed (di)nucleosides in this work. Potency of the modiﬁcations increases

in the following order: methyl ester malonamide < sodium carboxylate malonamide < hydroxamic acid malonamide

monomethyl sodium malonate < disodium malonate < hydroxamic acid sodium malonate. For the site of modiﬁcation, the

≈

0

observed trend is 5 -N-linked < 3 -N-linked < dinucleoside < 5 -C-linked.

In contrast to the C-linked malonates, of the N-linked 5⁰-modiﬁed nucleosides, only

one compound (15) binds SNM1A. Its lower activity (Table 1, entry 6 vs. 3) compared to

the corresponding C-linked hydroxamic acid 11 is possibly due to the increased distance

of the hydroxamic acid group from the deoxyribose core in 15, and/or the absence of the

0

additional carboxylate group. However, the 3 -modiﬁed nucleosides 26

–

28 (Table 1, entries

7–9) also contain a malonamide group, and two compounds in this series inhibited SNM1A

at 333 M concentration (Table 1, entries, 8, 9). This implies that the relative positioning of

µ

the group compared to the deoxyribose core is a causative factor in the lack of potency of

5⁰-N-linked nucleosides (Figure 4). For dinucleosides, the lowest concentrations at which

inhibition was observed for dinucleosides 35 and 36 are the same as for the 3⁰-modiﬁed

analogues (Table 1, entries 8 vs. 11, 9 vs. 12). However, the ester derivative 34 showed

some inhibition at 1 mM concentration (Table 1, entry 10), while the corresponding 3⁰-

derivative 26 was inactive (Table 1, entry 7). These results point towards the importance of

the second nucleoside as a recognition element. Nonetheless, the potency of the strongest

dinucleosides was signiﬁcantly lower than that of the strongest 5⁰-modiﬁed nucleoside

11 (Table 1, entry 3) which contained an additional carboxylate group. The beneﬁts of a

second nucleoside therefore do not counteract the negative effect of removing a carboxylate

group. The trends observed in these gel-based assays are summarised in Figure 4.

Comparison with the previously reported compounds

that compound 11 has a slightly lower potency than compound

vs. 8–11), which emerged as the best inhibitor in our previous work [14]. Structurally,

compound 11 is an analogue of hydroxamic acid , which differs only in the presence

1

and

2

(Figure 5a,b) shows

1

(Figure 5b, lanes 4–7

2

of an additional carboxylate group (Figure 5a). This additional group enhances binding

signiﬁcantly, as observed in Figure 5 b (lane 4 vs. 12), which points towards interactions

Molecules 2021, 26, 320

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between the carboxylate group and the active site and supports the presence of a second

metal ion. The binding mode of compound 11 to two metal ions shown in Figure 5c is

supported by the different activities of nucleosides

mode of the malonate anion in crystal structures of SNM1A [

2

and 11 and the observed binding

]. It is also consistent with

9

the requirement for groups with the potential to hold a negative charge and the enhanced

activity of the 5⁰-C-linked series, which contains an additional carboxylate group. It is,

however, important to note that these trends could also be the result of other interactions

such as hydrogen bonding with amino acid residues in the active site. The weaker binding

of the carboxylate 10 compared to the hydroxamic acid 11 (Table 1, entries 2 vs. 3) can be

attributed to the strong chelating ability of the hydroxamic acid group resulting from the

formation of a ﬁve-membered ring [37], as shown in Figure 5c.

Overall, our studies show that an extended metal-chelating group, such as malonate

hydroxamic acids, leads to enhanced binding to the nuclease SNM1A, consistent with the

presence of two metal ions in the active site. The synthesis and evaluation of methyl ester,

carboxylate and hydroxamic acid analogues of each point of installation revealed that a

group with the potential to hold a negative charge is required for binding. Variation in the

positioning of the malonate groups relative to the deoxyribose group revealed that of the

0

N-linked compounds, modiﬁcation of the 3 -position led to increased potency compared to

installation at the 5⁰-position. Interestingly, malonamide-linked dinucleosides were found

0

to be slightly superior to their 3 -modiﬁed analogues, which highlights the inclusion of two

nucleosides as recognition elements as a promising avenue for future nucleoside-based

inhibitors.

Figure 5. Comparison of previously reported compounds

binding mode. ( ) Structures of compounds and 11. (

and 11 at various concentrations. (

of SNM1A and the proposed binding mode of compound 11.

1

b

and

2 and compound 11 and the proposed

a

1

,

2

) Inhibition of SNM1A by compounds 1, 2

c

) The observed binding mode of malonate anion in the active site

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Table 1. Lowest concentrations at which inhibition was observed.

Lowest Inhibitory

Entry

Compound

Compound Family

Substituent

Concentration ¹(µM)

0

1

2

3

9

10

11

5 -C Malonate

ONa, OMe

ONa, ONa

ONa, NHOH

333

33

0

5 -C Malonate

0

5 -C Malonate

0

4

5

6

13

14

15

5 -N-Malonamide

OMe

ONa

NHOH

-

0

5 -N-Malonamide

0

5 -N-Malonamide

333

0

7

8

9

26

27

28

3 -N-Malonamide

OMe

ONa

NHOH

-

0

3 -N-Malonamide

333

0

3 -N-Malonamide

10

11

12

34

35

36

Dinucleoside

OMe

ONa

NHOH

1000

333

1

As observed in concentration gradient experiments, using approximately 3-fold dilutions.

3. Materials and Methods

3.1. General

¹H and ¹³C NMR spectra were recorded on Bruker 400 MHz or 600 MHz system

spectrometers in DMSO-d₆, CDCl₃, CD₃OD, acetone-d₆or D₂O relative to residual DMSO

(

δ_H= 2.50 ppm, δ_C= 39.52 ppm), CDCl₃, (δ_H= 7.26 ppm, δ_C= 77.16 ppm), CD₃OD

δ_H= 3.31 ppm, δ_C= 49.00 ppm), acetone-d₆(δ_H= 2.05 ppm, δ_C= 29.84 ppm) or D₂O

δ_H= 4.79 ppm) [38]. Chemical shifts are reported in ppm and coupling constants are

reported in Hertz (Hz) and accurate to 0.2 Hz. ¹³C NMR spectra are proton decoupled.

NMR spectra were assigned using HSQC, HMBC, DEPT and EXSY experiments. Modiﬁed

nucleosides are numbered according to standard nucleoside conventions. Mass spectrome-

try measurements were carried out on a Bruker ESI or APCI HRMS. Melting points were

measured using a Grifﬁn melting point apparatus and are uncorrected. Infrared (IR) spectra

were obtained on a Perkin Elmer spectrophotometer. Flash column chromatography was

carried out using silica gel, particle size 0.04–0.063 mm, purchased from Sigma Aldrich or

VWR. TLC analysis was performed on TLC Silica gel 60 F₂₅₄plates purchased from Merck

and visualised by UV irradiation (254 nm), ninhydrin stain (1.5 g ninhydrin, 5 mL AcOH,

500 mL 95% EtOH), anisaldehyde stain (9.2 mL p-methoxybenzaldehyde, 3.75 mL AcOH,

338 mL 95% EtOH, 12.5 mL conc. H₂SO₄) and iodine. Ion-exchange resin refers to Diaion

WT01S(H) resin, which was purchased from Alfa Aesar and activated by consecutive

washes with acetone, MeOH, 1 M NaOH (Na form only), H₂O and MeOH. Preparative

reversed-phase TLC was carried out on TLC silica gel 60 RP-18 F₂₅₄S plates purchased from

Merck. THF and CH₂Cl₂were dried using a PureSolv MD solvent puriﬁcation system.

Petroleum ether refers to the fraction of petroleum ether that boils at 40–60 ^◦C. Chemicals

were purchased from Acros Organics (Fair Lawn, NJ, US), Aldrich (Milwaukee, W, USA),

Alfa Aesar(Haverhill, MA, USA), Carbosynth (Compton, UK), Fisher Scientiﬁc (Waltham,

MA, USA), Fluorochem (Glossop, UK), Sigma Aldrich (St. Louis, MO, USA) and Merck

(Kenilworth, NJ, USA) and were used as purchased without further puriﬁcation.

3.2. Synthesis

3⁰-O-(tert-Butyldimethylsilyl)-5⁰-deoxy-5⁰-iodothymidine (4): Nucleoside

4

was prepared

according to a modiﬁed procedure [39]. Iodide

dry DMF (20 mL) under argon and cooled to 0 C. Imidazole (2.51 g, 36.9 mmol) and

TBDMS-Cl (5.56 g, 36.9 mmol) were added and the reaction mixture was stirred at 0 C for

3

(10.0 g, 28.4 mmol) was dissolved in

◦

6 h. After this time, TLC analysis (EtOAc) showed the consumption of starting material

(R_f= 0.5) and the formation of the product (R_f= 0.8). The reaction was quenched by the

addition of MeOH (5 mL), diluted with EtOAc (250 mL) and H₂O (100 mL). The layers

were separated and the organic layer was washed with brine (100 mL), dried over MgSO₄,

Molecules 2021, 26, 320

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ﬁltered and concentrated. The residue was recrystallised from EtOH to afford the desired

◦

4 as a white crystalline solid (11.59 g, 87%); mp 156–159 C. ν

_max/cm⁻¹(neat)

product

3155 (NH), 3024 (CH), 2930 (CH), 2892 (CH), 2857 (CH), 1691 (C=O), 1655 (C=O), 1470

(CH), 1426, 1402, 1369, 1309, 1291, 1274, 1197, 1130, 1100 (C-O), 1051, 1039, 829, 813, 777.

¹H NMR (400 MHz, CDCl₃):

δ

= 0.11 (s, 3 H, CH₃^TBDMS), 0.12 (s, 3 H, CH₃^TBDMS), 0.90 (s,

9 H, t-Bu^TBDMS), 1.95 (s, 3 H, CH₃^T), 2.15–2.22 (m, 1 H, H2⁰a), 2.29 (ddd, J₂₀_b,3₀= 3.9 Hz,

J

₁₀_,2₀_b= 6.6 Hz, J₂₀_a,2₀_b= 13.7 Hz, 1 H, H2⁰b), 3.39 (dd, J₄₀_,5₀_a= 4.6 Hz, J₅₀_a,5₀_b= 11.0 Hz, 1 H,

H5⁰a), 3.47 (dd, J₄₀_,5₀_b= 3.7 Hz, J₅₀_a,5₀_b= 11.0 Hz, 1 H, H5⁰b), 3.67–3.70 (m, 1 H, H4⁰), 4.26

(app. dt, J = 3.9 Hz, J = 7.0 Hz, 1 H, H3⁰), 6.27 (app. t, J = 6.6 Hz, 1 H, H1⁰), 7.47 (s, 1 H,

H6), 8.49 (bs, 1 H, NH) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ

=

−

4.5 (CH₃^TBDMS),

−

4.4

(CH₃^TBDMS), 7.3 (C5⁰), 12.8 (CH₃^T), 18.0 (qC, t-Bu^TBDMS), 25.8 (t-Bu^TBDMS), 40.6 (C2⁰), 75.2

0

−

(C3 ), 84.3 (C4 ), 84.8 (C1 ), 111.4 (C5), 136.0 (C6), 150.1 (C2), 163.6 (C4) ppm. HRMS (ESI ):

m/z calc. 465.0712 [M

H]⁻, found: 465.0704. The spectroscopic data are in agreement

−

with those reported in the literature [39].

N-Benzyloxymethyl-3⁰-O-(tert-butyldimethylsilyl)-5⁰-deoxy-5⁰-iodothymidine (5): Pro-

tected iodide

4 (9.33 g, 20.0 mmol) was dissolved in dry DMF (80 mL) under argon and

cooled to 0 ^◦C. DIPEA (17.5 mL, 97.6 mmol) was added, followed by the dropwise addi-

tion _◦of benzyl chloromethyl ether (8.4 mL, 57.7 mmol). The reaction mixture was stirred

at 0 C for 4 h. After this time, TLC analysis (petroleum ether-EtOAc, 2:1) showed the

consumption of starting material (R_f= 0.1) and the formation of the product (R_f= 0.4).

The reaction was quenched by the addition of MeOH (10 mL) followed by H₂O (100 mL).

The mixture was extracted with Et₂O (2

were dried over MgSO₄, ﬁltered, concentrated and coevaporated with toluene. Puriﬁcation

×

100 mL) and the combined organic extracts

by ﬂash column chromatography (EtOAc-petro₋le₁um ether, 3:1

→

2:1) afforded the desired

5 as a yellow oil (1.16 g, 95%). ν_max/cm (neat) 2954 (CH), 2929 (CH), 2857 (CH),

product

1710 (C=O), 1655 (C=O), 1463 (CH), 1361, 1275, 1253, 1069 (C-O), 1030, 833, 773, 733, 697.

¹H NMR (400 MHz, CDCl₃):

δ

= 0.11 (s, 3 H, CH₃^TBDMS), 0.12 (s, 3 H, CH₃^TBDMS), 0.90

(s, 9 H, t-Bu^TBDMS), 1.95 (d, J = 1.1 Hz, 3 H, CH₃^T), 2.13 (app. dt, J = 7.0 Hz, J = 13.7 Hz,

1 H, H2⁰a), 2.31 (ddd, J₂₀_b,3₀= 4.1 Hz, J₁₀_,2₀_b= 6.4 Hz, J₂₀_a,2₀_b= 13.7 Hz, 1 H, H2⁰b), 3.38 (dd,

J

₄₀_,5₀_a= 4.1 Hz, J₅₀_a,5₀_b= 10.8 Hz, 1 H, H5⁰a), 3.46 (dd, J₄₀_,5₀_b= 4.6 Hz, J₅₀_a,5₀_b= 10.8 Hz, 1 H,

H5⁰b), 3.70 (app. q, J = 4.1 Hz, 1 H, H4⁰), 4.25 (app. dt, J = 4.1 Hz, J = 7.0 Hz, 1 H, H3⁰),

4.70 (s, ₀2 H, O-CH₂-Ph), 5.49 (s, 2 H, N-CH₂-O), 6.29 (dd, J₁₀_,2₀_b= 6.4 Hz, J₁₀_,2₀_a= 7.0 Hz,

1 H, H1 ), 7.23–7.27 (m, 1 H, H^ar), 7.30–7.33 (m, 2 H, H^ar), 7.36–7.38 (m, 2 H, H^ar), 7.44 (app.

d, J = 1.1 Hz, 1 H, H6) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ

=

−

4.5 (CH₃^TBDMS),

−

4.4

(CH₃^TBDMS), 7.2 (C5⁰), 13.5 (CH₃^T), 18.0 (qC, t-B₀u^TBDMS), 25.8 (t-Bu^TBDMS), 40.7 (C2⁰), 70.7

0

(N-CH₂-O), 72.4 (O-CH₂-Ph), 75.2 (C3 ), 84.3 (C4 ), 85.4 (C1 ), 110.6 (C5), 127.77 (Ph), 127.81

(Ph), 128.4 (Ph), 134.7 (C6), 138.1 (qC, Ph), 150.9 (C2), 163.5 (C4) ppm. HRMS (APCI⁺): m/z

calc. 587.1433 [M + H]⁺, found: 587.1421.

C-(N³-Benzyloxymethyl-3⁰-O-(tert-butyldimethylsilyl)-5⁰-deoxythymidin-5⁰-yl) benzyl

methyl malonate (6): NaH (60% dispersion in mineral oil, 0.153 g, 3.82 mmol) was sus-

pended in dry DMF (10 mL) under argon and benzyl methyl malonate (2.1 mL, 11.7 mmol)

was added. BOM-protected iodide 5 (1.117 g, 1.91 mmol) was dissolved in dry DMF (10 mL)

and added dropwise, the reaction mixture was heated to 100 ^◦C and stirred for 24 h. After

this time, TLC analysis (CH₂Cl₂-EtOAc, 19:1) showed the consumption of starting material

(R_f= 0.4) and the formation of the product (R_f= 0.3). The reaction was cooled to r.t., diluted

with EtOAc (40 mL) and washed with H₂O (20 mL). The aqueous layer was extracted with

EtOAc (2

and concentrated. Puriﬁcation by ﬂash column chromatography (CH₂Cl₂-EtOAc, 19:1)

afforded the desired product as a yellow oil (0.838 g, 66%). Compound was isolated

as a 1:1 mixture of interconverting diastereoisomers.

_max/cm⁻¹(neat) 2953 (CH), 2930

×

40 mL) and the combined organic extracts were dried over MgSO₄, ﬁltered

6

ν

(CH), 2857 (CH), 1734 (C=O), 1710 (C=O), 1656 (C=O), 1464 (CH), 1361, 1253, 1151, 1087

1

(C-O), 1046 (C-O), 833, 773, 735, 696. H NMR (600 MHz, CDCl₃):

δ = 0.07–0.08 (m, 12 H,

4 × CH₃^TBDMS), 0.88–0.89 (2 × s, 18 H, t-Bu^TBDMS), 1.91 (d, J = 1.1 Hz, 3 H, CH₃^T), 1.95 (d,

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0

J = 1.1 Hz, 3 H, CH₃^T), 1.97–2.02 (m, 2 H, H2 a), 2.11–2.17 (m, 2 H, H5 a), 2.22–2.26 (m, 2 H,

H2⁰b), 2.36–2.40 (m, 2 H, H5⁰b), ₀3.62–3.66 (m, 2 H, H6⁰), ₀3.68 (s, 3 H, CO₂CH₃), 3.73 (s, 3 H,

CO₂CH₃), 3.80–3.84 (m, 2 H, H4 ), 4.05–4.08 (m, 2 H, H3 ), 4.70 (s, 4 H, 2

×

O-CH₂-Ph^BOM),

5.12–5.18 (m, 4 H, 2

(app. t, J = 6.5 Hz, 1 H, H1⁰), 6.18 (app. t, J = 6.6 Hz, 1 H, H1⁰), 7.04 (app. d, J = 1.1 Hz,

1 H, H6), 7.10 (app. d, J = 1.1 Hz, 1 H, H6), 7.24–7.38 (m, 20 H, 4

Ph) ppm. ¹³C NMR

4.61 (CH₃^TBDMS), 13.16

×

CO₂CH₂^Bn), 5.47 (s, 2 H, N-CH₂-O), 5.48 (s, 2 H, N-CH₂-O), 6.14

×

(151 MHz, CDCl₃):

δ

=

−

4.8 (CH₃^TBDMS),

−

4.63 (CH₃^TBDMS),

−

(CH₃^T), 13.20 (CH₃^T), 17.9 (qC, t-Bu^T₀^BDMS), 25.7 (t-Bu^TBDMS), 32.59 (C5⁰), 32.61 (C5⁰), 40.48

0

(C2 ), 40.55 (C2 ), 48.9 (C6 ), 49.1 (C6 ), 52.72 (CO₂CH₃), 52.78 (CO₂CH₃), 67.3 (CO₂C

H₂^Bn),

0

67.4 (CO₂CH₂^Bn), 70.5 (N-CH₂-O), 70.6 (N-CH₂-O), 72.2 (O- H₂-Ph^BOM), 74.95 (C3 ), 75.03

C

(C3⁰), 84.3 (C4⁰), 84.5 (C4⁰), 85.62 (C1⁰), 85.64 (C1⁰), 110.3 (C5), 110.4 (C5), 127.6 (Ph), 127.7

(Ph), 127.9 (Ph), 128.1 (Ph), 128.3 (Ph), 128.4 (Ph), 128.47 (Ph), 128.48 (Ph), 128.6 (Ph), 134.03

(C6), 134.05 (C6), 135.23 (qC, CO₂Bn), 135.24 (qC, CO₂Bn), 138.0 (qC, OBn^BOM), 150.77 (C2),

150.80 (C2), 163.37 (C4), 163.39 (C4), 168.5 (CO₂Bn), 169.1 (CO₂CH₃), 169.3 (CO₂Bn), 169.8

(CO₂CH₃) ppm. HRMS (APCI⁺): m/z calc. 667.3045 [M + H]⁺, found: 667.3054

C-(N³-Benzyloxymethyl-5⁰-deoxythymidin-5⁰-yl) benzyl methyl malonate (7): Protected

malonate

6

(100 mg, 150

µmol) was dissolved in THF (1.5 mL). Tetra-n-butylammonium

ﬂuoride trihydrate (85 mg, 270

µmol) was added and the solution was stirred at r.t. for

1 h. After this time, TLC analysis (CH₂Cl₂-EtOAc, 9:1) showed the complete consumption

of the starting material (R_f= 0.7) and product formation (R_f= 0.2). The yellow solution

was concentrated and puriﬁcation by ﬂash column chromatography (CH₂Cl₂-MeOH,

99:1

→

19:1) afforded the desired product

7

as a colourless wax (59 mg, 71%). Compound

7

was isolated as a 1:1 mixture of interconverting diastereoisomers.

ν

_max/cm⁻¹(CH₂Cl₂)

3458 (OH), 3033 (CH), 2955 (CH), 2928 (CH), 1732 (C=O), 1705 (C=O), 1641 (C=O), 1467

(CH), 1454 (CH), 1438, 1361, 1273, 1222, 1153, 1088 (C-O), 1074 (C-O), 1048, 1028, 774, 737,

697. ¹H NMR (400 MHz, acetone-d₆):

δ

= 1.83 (d, J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 1.87 (d,

J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 2.17–2.33 (m, 6 H, H2⁰a, H2⁰b, H5⁰a), 2.38 (ddd, J = 3.8 Hz,

J = 7.8 Hz, J = 14.2 Hz, 2 H, H5⁰b), 3.65 (s, 3 H, CO₂CH₃), 3.69 (s, 3 H, CO₂CH₃), 3.69–3.74

(m, 2 H, H6⁰), 3.83–3.89 (m, 2 H, H4⁰), 4.25–4.30 (m, 2 H, H3⁰), 4.53 (d, J = 4.4 Hz, 1 H, OH),

4.54 (d, J = 4.4 Hz, 1 H, OH), 4.65 (s, 2 H, O-CH₂-Ph^BOM), 4.66 (s, 2 H, O-CH₂-Ph^BOM), 5.16

(d, J = 3.5 Hz, 2 H, CO₂CH₂^Bn), 5.20 (d, J = 3.7 Hz, 2 H, CO₂CH₂^Bn), 5.44 (m, 2 H, N-CH₂-O),

5.45 (s, 2 H, N-CH₂-O), 6.26 (app. t, J = 6.8 Hz, 1 H, H1⁰), 6.28 (app. t, J = 6.8 Hz, 1 H, H1⁰),

7.22–7.38 (m, 21 H, 4

MHz, acetone-d₆):

= 13.1 (CH₃^T), 33.4 (C5⁰), 39.9 (C2⁰), 40.0 (C2⁰), 49.6 (C6⁰), 49.7 (C6⁰),

52.8 (CO₂H₃), 67.5 (CO₂C C

H₂^Bn), 71.26 (N-CH₂-O), 71.28 (N-CH₂-O), 72.4 (O- H₂-Ph^BOM),

×

Ph, H6), 7.41 (app. d, J = 1.1 Hz, 1 H, H6) ppm. ¹³C NMR (100

δ

C

74.79 (C3⁰), 74.85 (C3⁰), 84.9 (C4⁰), 85.1 (C4⁰), 86.1 (C1⁰), 110.35 (C5), 110.43 (C5), 128.2 (Ph),

128.68 (Ph), 128.73 (Ph), 128.9 (Ph), 129.0 (Ph), 129.2 (Ph), 129.3 (Ph), 135.84 (C6), 135.88

(C6), 136.90 (qC, CO₂Bn), 136.92 (qC, CO₂Bn), 139.6 (qC, OBn^BOM), 151.79 (C2), 151.81

(C2), 163.79 (C4), 163.81 (C4), 169.4 (CO₂Bn), 169.91 (CO₂CH₃), 169.94 (CO₂Bn), 170.4

(CO₂CH₃) ppm. HRMS (APCI⁺): m/z calc. 575.2000 [M + Na]⁺, found: 575.2015.

C-(5⁰-Deoxythymidin-5⁰-yl) malonic acid monomethyl ester (8): Protected malonate

7

(115 mg, 208 µmol) was dissolved in MeOH (10 mL) and added to a dried ﬂask containing

Pd/C (10%, 17 mg) and H₂was bubbled through the suspension while stirring at r.t. for

28 h. After this time, TLC analysis (CH₂Cl₂-MeOH, 9:1) showed the complete consumption

of starting material (R_f= 0.6) and the formation of the product (R_f= 0.2). The reaction

mixture was ﬁltered through celite and the ﬁltrate was concentrated to a colourless ﬁlm.

Puriﬁcation by ﬂash column chromatography (CH₂Cl₂-MeOH, 19:1

desired product as a white foam (61 mg, 86%). Compound was isolated as a 1:1 mixture

of interconverting diastereoisomers.

_max/cm⁻¹(neat) 3375 (OH, NH), 2955 (CH), 1686

→

MeOH) afforded the

8

ν

(C=O), 1665 (C=O), 1588 (C=O), 1474 (CH), 1437 (OH), 1371, 1269, 1198, 1168, 1086 (CO),

1

1049, 767. H NMR (600 MHz, DMSO-d₆):

δ

= 1.80 (2

×

s, 6 H, CH₃^T), 1.89–1.95 (m, 1 H,

H5⁰a), 1.98–2.06 (m, 4 H, H2⁰a, H5⁰a, H5⁰b), 2.08–2.16 (m, 3 H, H2⁰b, H5⁰b), 3.06 (app. t,

J = 6.9 Hz, 1 H, H6⁰), 3.13–3.15 (m, 1 H, H6⁰), 3.49 (s, 3 H, CO₂CH₃), 3.53 (s, 3 H, CO₂CH₃),

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3.59–3.62 (m, 1 H, H4⁰), 3.68–3.71 (m, 1 H, H4⁰), 4.00–4.02 (m, 1 H, H3⁰), 4.03–4.06 (m, 1 H,

H3⁰), 4.14 (bs, 2 H,₀OH), 5.53 (bs, 2 H, OH), 6.05 (app. t, J = 6.9 Hz, 1 H, H1⁰), 6.09 (app. t,

J = 6.9 Hz, 1 H, H1 ), 7.36 (s, 1 H, H6), 7.42 (s, 1 H, H6), 11.27 (bs, 2 H, NH) ppm. ¹³C NMR

(150 MHz, DMSO-d₆):

δ

= 12.12 (CH₃^T), 12.13 (CH₃^T), 33.4 (C5⁰), 33.8 (C5⁰), 38.7 (C2⁰), 50.9

(CO₂C

H₃), 51.4 (C6⁰), 52.1 (C6⁰), 73.1 (C3⁰), 73.4 (C3⁰), 83.38 (C1⁰), 83.42 (C1⁰), 84.2 (C4⁰),

85.4 (C4⁰), 109.5 (C5), 109.9 (C5), 136.0 (C6), 150.4 (C2), 150.5 (C2), 163.8 (C4), 172.3 (CO₂),

172.8 (CO₂) ppm. HRMS (APCI⁻): m/z calc. 341.0990 [M − H]⁻, found: 341.0990.

C-(5⁰-Deoxythymidin-5⁰-yl) monomethyl sodium malonate (9): Protected malonate

7

(250 mg, 452 µmol) was dissolved in MeOH (15 mL) and added to a dried ﬂask con-

taining Pd/C (10%, 50 mg) and H₂was bubbled through the suspension while stirring

at r.t. for 7 h. After this time, TLC analysis (EtOAc-MeOH, 2:1) showed the complete

consumption of starting material (R_f= 0.9) and the formation of the product (R_f= 0.2). The

reaction mixture was ﬁltered through celite and the ﬁltrate was concentrated to a colourless

ﬁlm. Puriﬁcation by ﬂash column chromatography (EtOAc-MeOH, 3:1

by elution through Diaion resin WT01S(H) (Na form) in MeOH. The resulting white solid

was puriﬁed by preparative reversed-phase TLC (H₂O-MeCN, 1:1), extracted with MeOH

→

2:1) was followed

and concentrated to afford the desired product

9

as a white solid (37 mg, 22%). Compound

9

was isolated as a 1:1 mixture of interconverting diastereomers.

ν

_max/cm⁻¹(neat) 3435

(OH, NH), 3210 (CH), 2951 (CH), 1665 (C=O), 1594 (C=O), 1473 (CH), 1436, 1369, 1269, 1195,

1

1165, 1084 (C-O), 1048, 768. H NMR (600 MHz, DMSO-d₆):

δ = 1.80 (d, J_CH3T,6= 0.9 Hz,

6 H, CH₃^T), 1.93–2.17 (m, 8 H, H2⁰a, H2⁰b, H5⁰a, H’5b), 3.18 (app. t, J = 6.9 Hz, 1 H, H6⁰),

3.23 (app. t, J = 9.4 Hz, 1 H, H6⁰), 3.52 (s, 3 H, CO₂CH₃), 3.56 (s, 3 H, CO₂CH₃), 3.59–3.62

(m, 1 H, H4⁰), 3.65–3.68 (m, 1 H, H4⁰), 4.02–4.07 (m, 2 H, H3⁰), 6.07 (app. t, J = 6.8 Hz, 1 H,

H1⁰), 6.10 (app. t, J = 6.9 Hz, 1 H, H1⁰), 7.37 (app. d, J = 0.9 Hz, 1 H, H6), 7.42 (app. d,

J = 0.9 Hz, 1 H, ₀H6), 11.27 (bs, 2 H, NH) ppm.₀¹³C NMR (150 MHz, DMSO-d₆):

δ

= 12.1

(CH₃^T), 33.0 (C5 ), 33.3 (C5 ), 38.5 (C2 ), 38.6 (C2 ), 50.4 (C6 ), 50.8 (C6 ), 51.3 (CO₂

CH₃), 73.1

0

(C3⁰), 73.3 (C3⁰), 83.4 (C1⁰), 84.0 (C4⁰), 84.9 (C4⁰), 109.6 (C5), 109.9 (C5), 136.05 (C6), 136.10

(C6), 150.42 (C2), 150.45 (C2), 163.7 (C4), 170.1 (CO₂Na), 170.3 (CO₂Na), 171.5 ( O₂CH₃),

171.8 (CO₂CH₃) ppm. HRMS (ESI⁺): m/z calc. 373.0618 [M + H]⁺, found: 373.0618.

C

C-(5⁰-Deoxythymidin-5⁰-yl) disodium malonate (10): KOH (20 mg, 356

µ

mol) was dis-

◦

solved in MeOH (0.5 mL) and cooled to 0 C. Ester

8 (30 mg, 88 µmol) was added and

the reaction was slowly warmed to r.t. and stirred for 3 h. A further portion of KOH

(20 mg, 356 mol) in MeOH (0.5 mL) was added and the reaction was stirred for a further

µ

4 h. After this time, TLC analysis (H₂O-i-PrOH-EtOAc, 1:2:2) showed the consumption of

starting material (R_f= 0.7) and the formation of a product (R_f= 0.6). The reaction mixture

was concentrated and the residue was taken up in H₂O and eluted through Diaion resin

WT01S(H) (H form) to remove excess KOH, followed by elution through Diaion resin

WT01S(H) (Na form). The white solid was puriﬁed by preparative reversed-phase TLC

(H₂O-MeCN, 1:1), extracted with H₂O and concentrated to afford the desired product 10

as a white solid (18 mg, 55%); mp 166–170 ^◦C dec. _max/cm⁻¹(neat) 3316 (OH, NH), 2937

ν

(CH), 2822 (CH), 1686 (C=O), 1587 (C=O), 1475 (CH), 1420, 1365, 1271, 1082 (C-O), 1024,

1

767. H NMR (600 MHz, D₂O):

δ

= 1.92 (s, 3 H, CH₃^T), 2.17–2.23 (m, 1 H, H5⁰a), 2.29–2.41

(m, 3 H, H2⁰a, H2⁰b, H5⁰b), 3.32–3.40 (m, 1 H, H6⁰), 3.97 (bs, 1 H, H4⁰), 4.34–4.36 (m, 1 H,

H3⁰), 6.24–6.26 (m, 1 H, H1⁰), 7.51 (s, 1 H, H6) ppm. ¹³C NMR (150 MHz, D₂O):

δ = 11.5

(CH₃^T), 33.2 (C5⁰), 37.8 (C2⁰), 55.2 (C6⁰), 73.6 (C3⁰), 84.8 (C1⁰), 85.0 (C4⁰), 111.5 (C5), 137.4

(C6), 151.7 (C2), 166.5 (C4), 176.5 (CO₂), 176.6 (CO₂) ppm. HRMS (ESI⁺): m/z calc. 373.0618

[M + H]⁺, found: 373.0618.

C-(5⁰-deoxythymidin-5⁰-yl) sodium N-hydroxymalonamide(11): Hydroxylamine hydrochlo-

ride (426 mg, 6.13 mmol) was suspended in MeOH (6 mL) and KOH (430 mg, 7.66 mmol)

was added. The suspension was warmed to 40 ^◦C to aid dissolution and the precipitate

was removed by ﬁltration. Ester 8 (93 mg, 272 µmol) was dissolved in the ﬁltrate and the

reaction was stirred at r.t. for 5 h. After this time, TLC analysis showed the consumption

of starting material (CH₂Cl₂-MeOH, 4:1, R_f= 0.8) and the formation of a product (H₂O-i-

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PrOH-EtOAc, 1:2:2, R_f= 0.3). The pH was adjusted to pH 7 using 1 M aq. HCl to quench

the reaction. The product was puriﬁed by ﬂash column chromatography (CH₂Cl₂-MeOH,

4:1

→

MeOH followed by H₂O-i-PrOH-EtOAc, 1:5:4

→

1:2:2), followed by elution through

Diaion resin WT01S(H) (Na form). The resulting white solid was puriﬁed by preparative

reversed-phase TLC (H₂O-MeCN, 1:1), extracted with MeOH and concentrated to afford

the desired product 11 as a white solid (15 mg, 15%). Compound 11 was isolated as a 1:1

mixture of interconverting diastereomers. ν

_max/cm⁻¹(neat) 3424 (OH, NH), 3213 (CH),

3045 (CH), 2936 (CH), 2818 (CH), 1655 (C=O), 1604 (C=O), 1579 (C=O), 1515, 1474 (CH),

1

1447, 1369 (C=O), 1272 (CO), 1126 (C-O), 1083 (C-O), 958, 770. H NMR (600 MHz, D₂O):

δ

= 1.92 (s, 3 H, CH₃^T), 1.93 (s, 3 H, CH₃^T), 2.08–2.15 (m, 2 H, H5⁰₀a), 2.27–2.33 (m, 2 H,

0

H’5b), 2.34–2.42 (m, 4 H, H2 a, H2 b), 3.19 (app. t, J = 7.2 Hz, 1 H, H6 ), 3.23 (dd, J = 4.6 Hz,

0

J = 10.4 Hz, 1 H, H6 ), 3.82 (app. dt, J = 4.0 Hz, J = 9.6 Hz, 1 H, H4 ), 3.92–3.94 (m, 1 H, H4 ),

4.31–4.34 (m, 2 H, H3⁰), 6.22–6.27 (m, 2 H, H1⁰), 7.48 (s, 1 H, H6), 7.50 (s, 1 H, H6) ppm.

¹³C NMR (150 MHz, D₂O):

δ

= 11.48 (CH₃^T), 11.52 (CH₃^T), 32.7 (C5⁰), 32.9 (C5⁰), 37.7 (C2⁰),

37.8 (C2⁰), 49.3 (C6⁰), 50.0 (C6⁰), 73.5 (C3⁰), 73.7 (C3⁰), 83.9 (C4⁰), 84.8 (C1⁰), 84.9 (C1⁰), 85.1

0

(C4 ), 111.5 (C5), 111.7 (C5), 137.3 (C6), 137.4 (C6), 151.69 (C2), 151.71 (C2), 166.5 (C4), 166.6

(C4), 170.0 (CONHO), 170.4 (CONHO), 175.5 (CO₂Na), 175.8 (CO₂Na) ppm. HRMS (ESI⁺):

m/z calc. 388.0727 [M + Na]⁺, found: 388.0731.

N-(5⁰-Deoxythymidin-5⁰-yl) amido methyl malonate (13): Aminothymidine 12 (500 mg,

2.07 mmol) was dissolved in dry DMF (15 mL), under argon. EDC

·

HCl (437 mg, 2.28 mmol)

◦

was suspended in dry DMF (20 mL) under argon and cooled to 0 C. Monomethyl

potassium malonate (356 mg, 2.28 mmol) was added and the suspension was stirred

at 0 ^◦C for 1 h. After this time, the aminothymidine solution was added followed by 4-

(dimethylamino)pyridine (25 mg, 0.21 mmol). The reaction was slowly warmed to r.t. and

stirred for 20 h. After this time, TLC analysis (CH₂Cl₂-MeOH, 4:1) showed the consumption

of amine starting material, (R_f= 0) and the formation of the product (R_f= 0.7). The reaction

mixture was concentrated and puriﬁcation by ﬂash column chromatography afforded the

desired product 13 as a white solid (385 mg, 54%); mp 68–72 ^◦C dec. _max/cm⁻¹(neat)

ν

3432 (OH, NH), 3290 (CH), 3098 (CH), 2955 (CH), 1755, 1708, 1655 (C=O), 1637 (C=O), 1567,

1473 (CH), 1402, 1365, 1259, 1218, 1198, 1153, 1100 (C-O), 1060, 1042, 1014, 955, 902, 852,

1

782. H NMR (600 MHz, DMSO-d₆):

δ

= 1.79 (d, J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 2.04 (ddd,

J = 3.3 Hz, J₁₀_,2₀_a= 6.3 Hz, J₂₀_a,2₀_b= 13.5 Hz, 1 H, H2⁰a), 2.13 (ddd, J = 6.5 Hz, J₁₀_,2₀_b= 7.9 Hz,

J₂₀_a,2₀_b= 13.5 Hz, 1 H, H2⁰b), 3.24–3.30 (m, 3 H, -CH₂, H5⁰a), 3.40 (ddd, J₄₀_,5₀_b= 4.6 Hz,

J_NH,5₀_b= 5.8 Hz, J₅₀_a,5₀_b= 14.0 Hz, 1 H, H5⁰b), 3.60 (s, 3 H, OCH₃), 3.73 (ddd, J = 3.6 Hz,

α

J

₄₀_,5₀_b= 4.6 Hz, J = 6.9 Hz, 1 H, H4⁰), 4.13–4.16 (m, 1 H, H3⁰), 5.30 (d, J = 4.0 Hz, 1 H, OH),

6.14 (dd, J₁₀_,2₀_a= 6.3 Hz, J₁₀_,2₀_b= 7.9 Hz, 1 H, H1⁰), 7.46 (app. d, J = 1.1 Hz, 1 H, H6), 8.28

(app. t, J = 5.8 Hz, CONH), 11.29 (bs, 1 H, NH^T) ppm. ¹³C NMR (150 MHz, DMSO-d₆):

0 0 0 0

= 12.0 (CH₃^T), 38.4 (C2 ), 41.0 (C5 ), 42.2 (

α-CH₂), 51.8 (OCH₃), 71.1 (C3 ), 83.7 (C1 ), 84.8

δ

(C4⁰), 109.8 (C5), 136.2 (C6), 150.5 (C2), 163.7 (C4), 165.5 (CONH), 168.4 (

COOCH₃) ppm.

HRMS (APCI⁻): m/z calc. 342.1296 [M + H]⁺, found: 342.1299.

N-(5⁰-Deoxythymidin-5⁰-yl) amido sodium malonate (14): KOH (164 mg, 2.92 mmol) was

dissolved in MeOH (6 mL). Ester 11 (100 mg, 293 mol) was added and the reaction was

µ

stirred for 2 h. After this time, TLC analysis (CH₂Cl₂-MeOH, 4:1) showed the complete

consumption of starting material (R_f= 0.8) and the formation of a product (R_f= 0.0). The

reaction mixture was eluted through Diaion resin WT01S(H) (H form) to remove excess

KOH, followed by elution through Diaion resin WT01S(H) (Na form) and concentrated to

afford the desired product 14 as a white foam (100 mg, 98%). ν

_max/cm⁻¹(neat) 3281 (OH,

NH), 2932 (CH), 1648 (C=O), 1595 (C=O), 1471 (CH), 1373 (C=O), 1316, 1270, 1087 (CH),

1

1049, 731. H NMR (400 MHz, D₂O):

δ

= 1.89 (d, J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 2.31–2.43

(m, 2 H, H2⁰a, H2⁰b), 3.21 (s, 2 H,

1 H, H4⁰), 4.43 (app. dt, J = 4.3 Hz, J = 6.3 Hz, 1 H, H3⁰), 6.28 (app. t, J = 6.9 Hz, 1 H, H1⁰),

7.45 (app. d, J = 1.1 Hz, 1 H, H6) ppm. ¹³C NMR (100 MHz, D₂O): = 12.0 (CH₃^T), 37.8

(C2⁰), 40.6 (C5⁰), 45.0 ( -CH₂), 71.2 (C3⁰), 85.0 (C1⁰), 84.1 (C4⁰), 111.7 (C5), 137.0 (C6), 154.6

α

-CH₂), 3.50–3.59 (m, 2 H, H5⁰a, H5⁰b), 4.02–4.06 (m,

δ

α

Molecules 2021, 26, 320

15 of 25

(C2), 170.3 (C4), 171.6 (CONH), 174.9 (

Na]⁺, found: 372.0791.

C

OONa) ppm. HRMS (ESI⁺): m/z calc. 372.0778 [M +

N-(5⁰-Deoxythymidin-5⁰-yl) amido N-hydroxymalonamide (15): Hydroxylamine hydro-

chloride (407 mg, 5.86 mmol) was suspended in MeOH (6 mL) and KOH (411 mg, 7.33 mmol)

was added. The suspension was warmed to 40 ^◦C to aid dissolution and the precipitate

was removed by ﬁltration. Ester 13 (100 mg, 293 µmol) was dissolved in the ﬁltrate and

the reaction was stirred at r.t. for 4 h. After this time, TLC analysis showed the complete

consumption of starting material (CH₂Cl₂-MeOH, 4:1, R_f= 0.8) and the formation of a

product (H₂O-i-PrOH-EtOAc, 1:5:4, R_f= 0.6). The pH was adjusted to pH 7 using 1 M aq.

HCl to quench the reaction. Puriﬁcation by ﬂash column chromatography (H₂O-i-PrOH-

EtOAc, 1:10:9) afforded the desired product 15 as a white foam (83 mg, 83%). ν

_max/cm⁻¹

(neat) 3205 (OH, NH), 3068 (CH), 2934 (CH), 2826 (CH), 1638 (C=O), 1551, 1475 (CH), 1414,

1

1368, 1270, 1088 (C-O), 1053. H NMR (600 MHz, DMSO-d₆):

δ = 1.80 (d, J_CH3T,6= 1.0 Hz,

3 H, CH₃^T), 2.02 (ddd, J = 3.1 Hz, J₁₀_,2₀_a= 6.2 Hz,₀J₂₀_a,2₀_b= 13.5 Hz, 1 H, H2⁰a), 2.15 (ddd,

J = 6.6 Hz, J₁₀_,2₀_b= 7.9 Hz, J₂₀_a,2₀_b= 13.5 Hz, 1 H, H2 b), 2.93 (d, J_αa,αb= 14.2 Hz, 1 H, α-CH_a),

2.97 (d, J_αa,αb= 14.2 Hz, 1 H, α

-CH_b), 3.29–3.34 (m, 2 H, H5⁰a, H5⁰b), 3.72–3.74 (m, 1 H,

H4⁰), 4.14–4.17 (m, 1 H, H3⁰), 5.33 (d, J = 4.3 Hz, 1 H, 3⁰-OH), 6.14 (dd, J₁₀_,2₀_a= 6.2 Hz,

0

J

₁₀_,2₀_b= 7.9 Hz, 1 H, H1 ), 7.54 (app. d, J = 1.0 Hz, 1 H, H6), 8.24 (app. t, J = 5.9 Hz, CONHC),

8.91 (s, 1 H, NOH), 10.60 (bs, 1 H, NHO), 11.28 (bs, 1 H, NH^T) ppm. ¹³C NMR (150 MHz,

DMSO-d₆):

= 12.1 (CH₃^T), 38.4 (C2⁰), 40.7 ( -CH₂), 40.9 (C5⁰), 71.0 (C3⁰), 83.5 (C1⁰), 84.8

δ

α

(C4⁰), 109.8 (C5), 136.3 (C6), 150.5 (C2), 163.8 (C4), 163.9 (

COONa), 166.7 (CONH) ppm.

HRMS (APCI⁺): m/z calc. 343.1248 [M + H]⁺, found: 343.1251.

1-[5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-deoxy-β-D-furanosyl]thymine (17): Alcohol 17 was pre-

pared according to a modiﬁed published procedure [32]. Protected thymidine 16 (5.00 g,

9.18 mmol) was dissolved in dry THF (40 mL) under argon, Et₃N (3.2 mL, 22.95 mmol) was

◦

added and the solution was cooled to 0 C. Methylsulfonate chloride (1.1 mL, 13.77 mmol)

was added dropwise and the resulting suspension was gradually warmed to r.t. and stirred

for 1 h. After this time, TLC analysis (EtOAc) showed the complete consumption of starting

material (R_f= 0.5) and the formation of the intermediate (R_f= 0.6). EtOH (20 mL) and aq.

◦

NaOH (50 mL, 10 M) were added to the reaction mixture and it was heated to 60 C for 17 h.

After this time, TLC analysis (CH₂Cl₂-MeOH, 19:1) showed the complete consumption

of the intermediate (R_f= 0.6) and the formation of the product (R_f= 0.1). The reaction

mixture was cooled to r.t. and the organic solvents were removed under reduced pressure.

The mixture was neutralised with aq. HCl and extracted with CH₂Cl₂(3

combined organic extracts were washed with H₂O (200 mL) and brine (200 mL), dried

×

150 mL). The

over MgSO₄, ﬁltered and concentrated to afford the desired product 17 as a yellow foam

(4.48 g, 90%). ν

_max/cm⁻¹(neat) 3396 (OH, NH), 3171 (OH, NH), 3004 (CH), 2954 (CH), 2932

(CH), 2836, 1682 (C=O), 1607 (C=O), 1508, 1464 (CH), 1445, 1271, 1247, 1175, 1065 (C-O),

1

1032, 903, 828, 755, 701. H NMR (400 ₀MHz, DMSO-d₆):

δ

= 1.64 (s, 3 H, CH₃^T), 1.86 (dd,

0

J

₁₀_,2₀_a= 2.0 Hz, J₂₀_a,2₀_b= 14.6 Hz, 1 H, H2 a), 2.52–2.57 (m, 1 H, H2 b), 3.19 (dd, J₄₀_,5₀_a= 2.8 Hz,

J₅₀_a,5₀_b= 10.3 Hz, 1 H, H5⁰a), 3.38 (dd, J₄₀_,5₀_b= 8.1 Hz, J₅₀_a,5₀_b= 10.3 Hz, 1 H, H5⁰b), 3.73 (s,

0 0

OCH₃), 4.08 (app. dt, J = 2.8 Hz, J = 8.1 Hz, 1 H, H4 ), 4.18–4.21 (m, 1 H, H3 ), 5.19

6 H, 2

×

(d, J₃₀_,OH= 3.3 Hz, 1 H, OH), 6.11 (dd, J₁₀_,2₀_a= 2.0 Hz, J₁₀_,2₀_b= 8.1 Hz, 1 H, H1⁰), 6.86–6.89

(m, 4 H, H^ar,DMTr), 7.20–7.24 (m, 1 H, H^ar,DMTr), 7.27–7.31 (m, 6 H, H^ar,DMTr), 7.42–7.43 (m,

2 H, H^ar,DMTr), 7.60 (s, 1 H, H6), 11.26 (s, 1 H, NH) ppm. ¹³C NMR (100 MHz, DMSO-d₆):

δ

= 12.4 (CH₃^T), 40.8 (C2⁰), 55.0 (OCH₃), 62.8 (C5⁰), 69.0 (C3⁰), 83.3 (C4⁰), 84.2 (C1⁰), 85.5

(qC, DMTr), 108.3 (C5), 113.1 (DMTr), 126.6 (DMTr), 127.75 (DMTr), 127.78 (DMTr), 129.7

(DMTr), 129.8 (DMTr), 135.5 (qC, DMTr), 135.7 (qC, DMTr), 136.8 (C6), 144.9 (qC, DMTr),

−

H] ,

150.5 (C2), 158.0 (qC, DMTr), 163.8 (C4) ppm. HRMS (APCI ): m/z calc. 543.2137 [M

−

found: 543.2141. Spectroscopic data are in agreement with the literature [32].

1-[5⁰-O-(4,4⁰-dimethoxytrityl)-3⁰-O-mesyl-2⁰-deoxy-β-D-furanosyl]thymine (18): Mesy-

lated thymidine 18 was prepared according to a published procedure [33]. Inverted

thymidine 17 (300 mg, 0.55 mmol) was dissolved in dry pyridine (5 mL) under argon

Molecules 2021, 26, 320

16 of 25

and the solution was cooled to 0 ^◦C. Methylsulfonate chloride (130

µL, 1.68 mmol) was

added dropwise and the reaction mixture was gradually warmed to r.t. and stirred for 18 h.

After this time, TLC analysis (CH₂Cl₂-MeOH, 19:1) showed the complete consumption

of starting material (R_f= 0.1) and the formation of the product (R_f= 0.6). The reaction

◦

mixture was cooled to 0 C and H₂O (0.4 mL) was added. After 5 min, the reaction mixture

was poured into ice-H₂O (60 mL) with vigorous stirring. The resulting precipitate was

collected by vacuum ﬁltration and washed with H₂O (10 mL) to afford the desired product

◦

18 as a white solid (309 mg, 90%); mp 88–92 C dec (lit. [40] 85.4–86.7 C) ν

_max/cm^–1

(neat) 3181 (OH, NH), 3058 (CH), 2955 (CH), 2930 (CH), 2856 (CH), 1689 (C=O), 1608,

1508, 1464 (CH), 1446, 1250, 1175, 1097 (C-O), 1033, 831, 779, 702. ¹H NMR (400 MHz,

acetone-d₆):

δ

= 1.73 (d, J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 2.45 (ddd, J₂₀_a,3₀= 1.0 Hz, J₁₀_,2₀_a= 3.2 Hz,

J₂₀_a,2₀_b= 15.7 Hz, 1 H, H2⁰a), 2.97 (ddd, J₂₀_b,3₀= 5.5 Hz, J₁₀_,2₀_b= 8.0 Hz, J₂₀_a,2₀_b= 15.7 Hz, 1 H,

H2⁰b), 3.02 (s, 3 H, SO₂CH₃), 3.39 (dd, J₄₀_,5₀_a= 4.9 Hz, J₅₀_a,5₀_b= 10.1 Hz, 1 H, H5⁰a), 3.61 (dd,

0

J₄₀_,5₀_b= 6.6 Hz, J₅₀_a,5₀_b= 10.1 Hz, 1 H, H5 b), 3.79 (s, 6 H, 2

5.41–5.44 (m, 1 H, H3 ), 6.29 (dd, J₁₀_,2₀_a= 3.2 Hz, J₁₀_,2₀_b= 8.0 Hz, 1 H, H1 ), 6.89–6.91 (m, 4 H,

H^ar,DMTr), 7.22–7.26 (m, 1 H, H^ar,DMTr), 7.30–7.34 (m, 2 H, H^ar,DMTr), 7.36–7.41 (m, 5 H, H6,

×

OCH₃), 4.43–4.46 (m, 1 H, H4 ),

0

4

×

H^ar,DMTr), 7.51–7.53 (m, 2 H, H^ar,DMTr), 9.96 (bs, 1 H, NH) ppm. ¹³C NMR (100 MHz,

acetone-d₆):

δ

= 12.6 (CH₃^T), 38.4 (SO₂CH₃), 40.0 (C2⁰), 55.5 (OCH₃), 62.5 (C5⁰), 80.4 (C3⁰),

82.1 (C4⁰), 84.6 (C1⁰), 87.5 (qC, DMTr), 110.8 (C5), 113.96 (DMTr), 113.99 (DMTr), 127.7

(DMTr), 128.7 (DMTr), 129.0 (DMTr), 131.01 (DMTr), 131.03 (DMTr), 136.2 (C6), 136.5 (qC,

DMTr), 136.6 (qC, DMTr), 145.9 (qC, DMTr), 151.3 (C2), 159.7 (qC, DMTr), 164.2 (C4) ppm.

HRMS (APCI⁻): m/z calc. 621.1912 [M

−

H]⁻, found: 621.1928. Spectroscopic data are in

agreement with the literature [33].

3⁰-Deoxy-2⁰,3⁰-didehydro-5⁰-O-(4,4⁰-dimethoxytrityl)-N³-methylthymidine (20): Comp-

ound 20 was obtained from the attempted synthesis of compound 19. NaH (60% dispersion

in mineral oil, 39 mg, 964

µmol) was suspended in dry DMF (1.0 mL) and benzyl methyl

malonate (175 L, 1.96 mmol) was added dropwise. Mesylate 18 (100 mg, 161

µ

µmol) was

dissolved in dry DMF (0.5 mL) under argon and added dropwise to the mixture of NaH

and benzyl methyl malonate. The reaction mixture was heated to 100 ^◦C for 120 h. After

this time, TLC analysis (petroleum ether-EtOAc, 2:1) showed the consumption of starting

material (R_f= 0.2) and the formation of a product (R_f= 0.6). The reaction mixture was cooled

to r.t. and H₂O (10 mL) was added. The mixture was extracted with EtOAc (3 × 20 mL

)

and the combined organic extracts were washed with brine (20 mL), dried over MgSO₄,

ﬁltered and concentrated. Puriﬁcation by ﬂash column chromatography (EtOAc-petroleum

ether, 1:3

white solid (40 mg, 46%).

→

EtOAc) did not afford the desired product 19 but afforded byproduct 20 as a

ν

_max/cm⁻¹(CH₂Cl₂) 3057 (CH), 2955 (CH), 2929 (CH), 2871

(CH), 2839 (CH), 1702 (C=O), 1667 (C=O), 1638 (C=O), 1608 (C=C), 1508, 1467 (CH), 1446,

1

1294, 1248, 1176, 1089 (C-O), 1033, 991, 829, 766, 733, 700. H NMR (600 MHz, acetone-d₆):

δ

= 1.33 (d, J_CH3T,6= 0.9 Hz, 3 H, CH₃^T), 3.27 (s, 3 H, N-CH₃), 3.33 (dd, J₄₀_,5₀_a= 2.7 Hz,

0

J₅₀_a,5₀_b= 10.4 Hz, 1 H, H5 a), 3.39 (dd, J₄₀_,5₀_b= 4.8 Hz, J₅₀_a,5₀_b= 10.4 Hz, 1 H, H5 b), 3.78 (2

×

s,

6 H, 2

×

OCH₃), 5.02–5.04 (m, 1 H, H4⁰), 6.02–6.03 (m, 1 H, H2⁰), 6.55 (app. dt, J = 1.6 Hz,

J =₀6.0 Hz, 1 H, H3⁰), 6.86-6.88 (m, 4 H, H^ar,DMTr), 7.06 (app. dt, J = 1.6 Hz, J = 3.5 Hz, 1 H,

H1 ), 7.23–7.25 (m, 1 H, H^ar,DMTr), 7.29–7.31 (m, 2 H, H^ar,DMTr), 7.33–7.34 (m, 4 H, H^ar,DMTr),

7.43 (d, J_CH3T,6= 0.9 Hz, 1 H, H6), 7.47–7.48 (m, 2 H, H^ar,DMTr) ppm. ¹³C NMR (150 MHz,

acetone-d₆):

δ

= 12.5 (CH₃^T), 27.8 (N-CH₃), 55.5 (OCH₃), 66.1 (C5⁰), 86.5 (C4⁰), 87.0 (qC,

DMTr), 91.2 (C1⁰), 110.0 (C5), 113.89 (DMTr), 113.90 (DMTr), 127.1 (C2⁰), 127.7 (DMTr),

128.6 (DMTr), 129.1 (DMTr), 131.02 (DMTr), 131.05 (DMTr), 135.0 (C6), 135.4 (C3⁰), 136.4

(qC, DMTr), 136.5 (qC, DMTr), 145.8 (qC, DMTr), 152.2 (C2), 159.70 (qC, DMTr), 159.73 (qC,

DMTr), 163.9 (C4) ppm. HRMS (APCI⁺): m/z calc. 563.2153 [M + Na]⁺, found: 563.2151

5⁰-O-(4,4⁰-Dimethoxytrityl)-N³-methyl thymidine (23): Compound 23 was obtained from

the attempted synthesis of compound 22. Anhydrosugar 21 (60 mg, 114

µ

mol) and 2,6-

mol) were dissolved in dry THF (2 mL) under

L, 115 mol) was added dropwise and

di-tert-butyl-4-methylpyridine (6 mg, 29

argon and cooled to 10 C. Methyl triﬂate (13

µ

◦

µ

Molecules 2021, 26, 320

17 of 25

the reaction was stirred for 10 min. NaH (60% dispersion in mineral oil, 6.7 mg, 168

µmol) was suspended in dry THF (0.5 mL) and benzyl methyl malonate (41 µL, 226 µmol)

was added dropwise. The malonate solution was added dropwise to the reaction and

it was stirred for 2.5 h. After this time, TLC analysis (EtOAc-MeOH, 19:1) showed the

complete consumption of starting material (R_f= 0.2) and the formation of a product

(R_f= 0.7). The reaction mixture was diluted with EtOAc (20 mL) and washed with brine

(

2 × 10 mL), dried over MgSO₄, ﬁltered and concentrated to an oil. Puriﬁcation by ﬂash

column chromatography (EtOAc-petroleum ether, 1:1 2:1) did not afford the desired

product 22 but afforded byproduct 23 as a white solid (20 mg, 31%).

_max/cm⁻¹(neat) 3429

→

ν

(OH), 3058 (CH), 2952 (CH), 2933 (CH), 2837, 1694 (C=O), 1625 (C=O), 1609 (C=O), 1508,

1475, 1445, 1296 (CH), 1247, 1175, 1157, 1105 (C-O), 1072 (C-O), 1031, 828, 766, 754, 734,

1

701. H NMR (400 MHz, acetone-d₆):

δ

= 1.75 (d, J_CH3T,6= 1.1 Hz, 3 H, CH₃^T), 2.02–2.07 (m,

0

1 H, H2 a), 2.68 (dd, J = 5.2 Hz, J₁₀_,2₀_b= 8.2 Hz, J = 13.4 Hz, 1 H, H2 b), 3.22 (s, 3 H, N-CH₃),

3.39 (dd, J = 3.6 Hz, J₅₀_a,5₀_b= 10.2 Hz, 1 H, H5⁰a), 3.61 (dd, J = 7.5 Hz, J₅₀_a,5₀_b= 10.2 Hz,

1 H, H5⁰b), 3.78 (s, 6 H, 2

×

OCH₃), 4.19–4.22 (m, 1 H, H4⁰), 4.40–4.45 (m, 2 H, H3⁰, OH),

6.23 (dd, J = 2.3 Hz, J₁₀_,2₀_b= 8.2 Hz, 1 H, H1⁰), 6.86–6.89 (m, 4 H, H^ar,DMTr), 7.20–7.24 (m,

1 H, H^ar,DMTr), 7.28–7.32 (m, 2 H, H^ar,DMTr), 7.37–7.42 (m, 4 H, H^ar,DMTr), 7.52–7.54 (m, 2 H,

H^ar,DMTr), 7.73 (d, J_CH3T,6= 1.1 Hz, 1 H, H6) ppm. ¹³C NMR (100 MHz, acetone-d₆):

δ = 13.5

(CH₃^T), 27.6 (N-CH₃), 42.2 (C2⁰), 55.5 (OCH₃), 63.7 (C5⁰), 70.8 (C3⁰), 84.7 (C4⁰), 86.7 (C1⁰),

87.0 (qC, DMTr), 108.7 (C5), 113.8 (DMTr), 113.9 (DMTr), 127.5 (DMTr), 128.5 (DMTr), 129.0

(DMTr), 130.98 (DMTr), 131.01 (DMTr), 136.1 (C6), 136.8 (qC, DMTr), 137.0 (qC, DMTr),

146.2 (qC, DMTr), 151.9 (C2), 159.6 (qC, DMTr), 164.0 (C4) ppm. HRMS (APCI⁺): m/z calc.

559.2439 [M + H]⁺, found: 559.2435.

3⁰-Amino-3⁰-deoxythymidine (25): Amine 25 was prepared according to a published

procedure [41]. AZT (24) (1.00 g, 3.74 mmol) was dissolved in MeOH (35 mL) and added to

a dried ﬂask containing Pd/C (10%, 150 mg) and H₂was bubbled through the suspension

while stirring at r.t. for 2 h. After this time, TLC analysis (EtOAc-MeOH, 4:1) showed the

complete consumption of starting material, (R_f= 0.8) and the formation of the product

(R_f= 0.1). The reaction mixture was ﬁltered through celite and the ﬁltrate was concentrated

to afford the desired product 25 as a white foam (893 mg, 99%). ν

_max/cm⁻¹(neat) 3363

(OH, NH), 3338 (OH, NH), 3290 (OH, NH), 2955 (CH), 2902 (CH), 2760 (CH), 1706 (C=O),

1663 (C=O), 1607 (C=O), 1477 (CH), 1441, 1421, 1370, 1360, 1331, 1295, 1245, 1207, 1092

1

(C-O), 1048, 1029, 955, 916, 869, 762. H NMR (400 MHz, DMSO-d₆):

δ = 1.76 (d, J = 1.1 Hz,

3 H, CH₃^T), 1.98 (dt, J₁₀_,2₀_a= 6.7 Hz, J₂₀_a,3₀= 6.7 Hz, J₂₀_a,2₀_b= 13.1 Hz, 1 H, H2⁰a), 2.08

(ddd, J₁₀_,2₀_b= 5.2 Hz, J₂₀_b,3₀= 7.1 Hz, J₂₀_a,2₀_b= 13.1 Hz, 1 H, H2⁰b), 3.37–3.42 (m, 1 H, H3⁰),

3.50–3.53 (m, 1 H, H4⁰), 3.56 (dd, J₄₀_,5₀_a= 3.8 Hz, J₅₀_a,5₀_b= 11.8 Hz, 1 H, H5⁰a), 3.65 (dd,

J₄₀_,5₀_b= 3.1 Hz, J₅₀_a,5₀_b= 11.8 Hz, 1 H, H5⁰b), 4.98 (bs, 1 H, OH), 6.08 (dd, J₁₀_,2₀_b= 5.2 Hz,

J₁₀_,2₀_a= 6.7 Hz, 1 H, H1⁰), 7.75 (app. d, J = 1.1 Hz, 1 H, H6) ppm₀. ¹³C NMR (100 MHz,

0 0 0 0

δ

= 12.3 (CH₃^T), 40.7 (C2 ), 50.8 (C3 ), 60.8 (C5 ), 83.5 (C1 ), 87.6 (C4 ), 108.9 (C5),

DMSO-d₆):

136.3 (C6), 150.4 (C2), 163.8 (C4) ppm. HRMS (ESI⁺): m/z calc. 264.0955 [M + Na]⁺, found:

264.0956. Spectroscopic data are in agreement with the literature [42].

N-(3⁰-Deoxythymidin-3⁰-yl) amido methyl malonamide (26): EDC

was suspended in dry DMF (8 mL) under argon and cooled to 0 C. Monomethyl potassium

malonate (285 mg, 1.66 mmol) was added and the suspension was stirred at 0 C for

·

HCl (318 mg, 1.66 mmol)

◦

1 h. After this time, aminothymidine 25 (400 mg, 1.66 mmol) was added followed by

4-(dimethylamino)pyridine (20 mg, 0.16 mmol) the reaction was slowly warmed to r.t.

and stirred for 26 h. After this time, TLC analysis (CH₂Cl₂-MeOH, 4:1) showed the

consumption of amine starting material, (R_f= 0.1) and the formation of the product

(R_f= 0.5). The reaction mixture was concentrated to an oil and puriﬁcation by ﬂash column

chromat_◦ography affo₋rd₁ed the desired product 26 as a colourless solid (190 mg, 34%); mp

153–154 C. ν_max/cm (neat) 3316 (OH, NH), 3247 (OH, NH), 3059 (CH), 2933 (CH), 1702

(C=O), 1655 (C=O), 1643 (C=O), 1551, 1476 (CH), 1438, 1272, 1101 (C-O), 1065 (C-O), 969,

768. ¹H NMR (400 MHz, DMSO-d₆): = 1.78 (d, J = 1.0 Hz, 3 H, CH₃^T), 2.05–2.11 (m,

δ

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1 H, H2⁰a), 2.19–2.27 (m, 1 H, H2⁰b), 3.25 (s, 2 H, COCH₂CO), 3.52–3.57 (m, 1 H, H5⁰a),

3.61–3.66 (m, 4 H, H5⁰b, CO₂CH₃), 3.75–3.78 (m, 1 H, H4⁰), 4.28–4.34 (m, 1 H, H3⁰), 5.09

(app. t, J = 5.2 Hz, 1 H, OH), 6.19 (app. t, J = 6.7 Hz, 1 H, H1⁰), 7.76 (app. d, J = 1.0 Hz,

1 H, H6), 8.57 (d, J = 7.2 Hz, 1 H, CONH), 11.29 (s, 1 H, NH^T) ppm. ¹³C NMR (100 MHz,

DMSO-d₆):

= 12.3 (CH₃^T), 36.8 (C2 ), 42.2 (CO

0 0 0

δ CH₂CO), 49.5 (C3 ), 51.9 (OCH₃), 61.4 (C5 ),

83.5 (C1⁰), 85.1 (C4⁰), 109.5 (C5), 136.1 (C6), 150.5 (C2), 163.8 (C4), 165.1 (CONH), 168.2

(CO₂CH₃) ppm. HRMS (ESI⁺): m/z calc. 364.1115 [M + Na]⁺, found: 364.1121.

N-(3⁰-Deoxythymidin-3⁰-yl) amido sodium malon_◦amide (27): KOH (25 mg, 446

µ

mol)

was dissolved in MeOH (1 mL) and cooled to 0 C. Ester 26 (50 mg, 147

µmol) was

added, the solution was slowly warmed to r.t. and stirred for 5.5 h. After this time,

TLC analysis (CH₂Cl₂-MeOH, 4:1) showed the consumption of starting material (R_f= 0.8)

and the formation of a product (R_f= 0.0). The reaction mixture was eluted through

Diaion resin WT01S(H) (H form) to remove excess KOH, then eluted through Diaion resin

WT01S(H) (Na form) and concentrated to a white solid. After puriﬁcation by ﬂash column

chromatography (CH₂Cl₂-MeOH, 4:1

eluted through Diaion resin WT01S(H) (Na form). The desired product 27 was obtained

as a white foam (42 mg, 82%); mp 202–205 C dec.

→

MeOH), the residue was taken up in MeOH and

◦

ν

_max/cm⁻¹(neat) 3431 (OH, NH),

3263 (OH, NH), 3071 (CH), 2954 (CH), 2926 (CH), 1642 (C=O), 1593 (C=O), 1473 (CH),

1

1441, 1379 (C=O), 1318, 1302, 1276, 1233, 1102 (C-O), 1073, 978, 927, 886, 785. H NMR

(600 MHz, DMSO-d₆):

δ

= 1.75 (d, J = 0.7 Hz, 3 H, CH₃^T), 2.05–2.09 (m, 1 H, H2⁰a), 2.19

(ddd, J₁₀_,2₀_b= 6.5 Hz, J = 8.0 Hz, J = 13.3 Hz, 1 H, H2⁰b), 2.70 (s, 2 H, COCH₂CO), 3.53 (dd,

J = 4.2 Hz, J₅₀_a,5₀_b= 11.9 Hz, 1 H, H5⁰a), 3.62 (dd, J = 2.9 Hz,₀J₅₀_a,5₀_b= 11.9 Hz, 1 H, H5⁰b),

0

3.70–3.73 (m, 1 H, H4 ), 4.27–4.31 (m, 1 H, H3 ), 5.06 (bs, 1 H, 5 -OH), 6.15 (app. t, J = 6.5 Hz,

1 H, H1⁰), 7.63 (app. s, 1 H, H6), 9.95–9.97 (m, 1 H, CONH) ppm. ¹³C NMR (150 MHz,

DMSO-d₆):

δ C

= 12.7 (CH₃^T), 37.4 (C2⁰), 44.8 (CO H₂CO), 48.5 (C3⁰), 61.4 (C5⁰), 83.5 (C1⁰),

0

85.0 (C4 ), 109.3 (C5), 135.6 (C6), 152.4 (C2), 166.4 (C4), 170.0 (CONH), 170.4 (

C

O₂Na) ppm.

HRMS (ESI⁺): m/z calc. 350.0959 [M + H]⁺, found: 350.0967.

N-(3⁰-Deoxythymidin-3⁰-yl) amido N-hydroxymalonamide (28): Hydroxylamine hydro-

chloride (407 mg, 5.86 mmol) was suspended in MeOH (6 mL) and KOH (411 mg, 7.33 mmol)

was added. The suspension was warmed to 40 ^◦C to aid dissolution and the precipitate

was removed by ﬁltration. Ester 26 (100 mg, 293 µmol) was dissolved in the ﬁltrate and

the reaction was stirred at r.t. for 2.5 h, during which a white precipitate formed. After

this time, TLC analysis (CH₂Cl₂-MeOH, 4:1) showed the complete consumption of starting

material (R_f= 0.7) and the formation of the product (R_f= 0.1). The reaction mixture was

cooled to 0 ^◦C and neutralised to pH 7 using aq. HCl (1 M). The white precipitate was

collected by vacuum ﬁltration to afford the desired product 28 as a white amorphous solid

◦

−1

(70 mg, 70%); mp 166–170 C dec.

ν

_max/cm (neat) 3253 (OH, NH), 3065 (CH), 2955 (CH),

1

2826 (CH), 1643 (C=O), 1599 (C=O),1552, 1431, 1274, 1101, 1072, 971, 883, 762. H NMR

(600 MHz, DMSO-d₆):

δ

= 1.77 (s, 3 H, CH₃^T), 2.08–2.12 (m, 1 H, H2⁰a), 2.18–2.23 (m, 1 H,

H2⁰b), 2.83 (s, 2 H, COCH₂CO), 3.54 (dd, J = 3.9 Hz, J₅₀_a,5₀_b= 12.0 Hz, 1 H, H5⁰a), 3.62 (dd,

J = 2.4 Hz, J₅₀_a,5₀_b= 12.0 Hz, 1 H, H5⁰b), 3.75–3.77 (m, 1 H, H4⁰), 4.28–4.32 (m, 1 H, H3⁰),

6.18 (app. t, J = 6.6 Hz, 1 H, H1⁰), 7.74 (s, 1 H, H6), 8.78 (bs, 1 H, N O-C) ppm. ¹³C NMR

H

(150 MHz, DMSO-d₆):

δ C

= 12.3 (CH₃^T), 37.0 (C2⁰), 40.8 (CO H₂CO), 49.1 (C3⁰), 61.4 (C5⁰),

83.5 (C1⁰), 85.0 (C4⁰), 109.4 (C5), 136.1 (C6), 150.8 (C2), 163.1 (CONHOH), 164.3 (C4), 167.6

(CONHC) ppm. HRMS (ESI⁻): m/z calc. 341.1103 [M − H]⁻, found: 341.1109.

C-(3⁰-O-(tert-Butyldimethylsilyl-5⁰-deoxy-N³-hydroxymethyl)thymidin-5⁰-yl) malonic acid

monomethyl ester (29): Malonate ester 6 (550 mg, 825 µmol) was dissolved in MeOH (20 mL)

and added to a dried ﬂask containing Pd/C (10%, 104 mg) and H₂was bubbled through the

suspension while stirring at r.t. for 1.5 h. After this time, TLC analysis showed the complete

consumption of starting material (CH₂Cl₂-EtOAc, 4:1, R_f= 0.8) and the formation of the

product (CH₂Cl₂-MeOH, 4:1, R_f= 0.5). The reaction mixture was ﬁltered through celite

and the ﬁltrate was concentrated to afford the desired product 29 as a yellow foam (380 mg,

95%). Compound 29 was isolated as a 1:1 mixture of interconverting diastereoisomers.

Molecules 2021, 26, 320

19 of 25

ν

_max/cm⁻¹(neat) 3188 (OH), 2955 (CH), 2929 (CH), 2858 (CH), 1705 (C=O), 1473 (CH),

1

1437, 1275, 1048 (C-O), 837, 779. H NMR (600 MHz, acetone-d₆):

δ = 0.13–0.14 (m, 12 H, 4

×

CH₃^TBDMS), 0.918–0.922 (2

×

s, 18 H, t-Bu^TBDMS), 1.86 (s, 3 H, CH₃^T), 1.88 (s, 3 H, CH₃^T),

2.15–2.25 (m, 4 H, H2⁰a, H5⁰a), 2.33–2.41 (m, 4 H, H2⁰b, H5⁰a), 3.60–3.63 (m, 2 H, H6⁰), 3.67

(s, 3 H, CO₂CH₃), 3.70 (s, 3 H, CO₂CH₃), 3.83–3.86 (m, 1 H, H4⁰), 3.88–3.91 (m, 1 H, H4⁰),

4.38 (m, 2 H, H3⁰), 5.38 (s, 4 H, N-CH₂-O), 6.27–6.30 (m, 2 H, H1⁰), 7.47 (s, 1 H, H6), 7.49 (s,

1 H, H6) ppm. ¹³C NMR (151 MHz, acetone-d₆):

δ

=

−

4.7 (CH₃^TBDMS),

−

4.5 (CH₃^TBDMS),

13.0 (CH₃^T), 18.48 (qC, t-Bu^TBDMS), 18.49 (qC, t-Bu^TBDMS), 26.1 (t-Bu^TBDMS), 33.3 (C5 ), 33.4

(C5⁰), 40.3 (C2⁰), 40.4 (C2⁰), 49.3 (C6⁰), 49.5 (C6⁰), 52.60 (CO₂

H₃), 52.64 (CO₂H₃), 65.4

(N-CH₂-O), 76.0 (C3⁰), 76.1 (C3⁰), 85.1 (C4⁰), 85.4 (C4⁰), 86.1 (C1⁰), 110.5 (C5), 110.6 (C5),

0

C

135.9 (C6), 151.5 (C2), 163.6 (C4), 170.3 (CO₂CH₃), 170.4 (CO₂H), 170.90 (CO₂CH₃), 170.93

(CO₂H) ppm. HRMS (ESI⁺): m/z calc. 509.1926 [M + Na]⁺, found: 509.1928.

3⁰-Amino-3⁰-deoxy-5⁰-O-(4,4⁰-dimethoxytrityl)thymidine (31): Amine 31 was prepared

according to a published procedure [36]. DMT-protected AZT 30 (3.00 g, 5.27 mmol) was

suspended in i-PrOH (100 mL) and NaBH₄(1.00 g, 26.43 mmol) was added. The suspension

was heated to reﬂux and stirred for 17 h. After this time, TLC analysis (CH₂Cl₂-MeOH,

9:1) showed the complete consumption of starting material (R_f= 0.7) and the formation of

the product (R_f= 0.4). The reaction mixture was diluted with CH₂Cl₂(200 mL), washed

with H₂O (200 mL) and brine (200 mL), dried over MgSO₄, ﬁltered and concentrated to

afford the desired product 31 as a white foam (2.73 g, 95%) which was used without further

puriﬁcation.

(C=O), 1508, 1463 (CH), 1445, 1299, 1247, 1175, 1031 (C-O), 826, 771, 756, 727, 701. ¹H

NMR (400 MHz, acetone-d₆):

= 1.55 (s, 3 H, CH₃^T), 2.29 (app. dt, J = 6.7 Hz, J = 13.2 Hz,

1 H, H2⁰a), 2.42 (ddd, J₁₀_,2₀_b= 5.5 Hz, J₂₀_b,3₀= 7.5 Hz, J₂₀_a,2₀_b= 13.2 Hz, 1 H, 2⁰-b), 3.24 (dd,

₄₀_,5₀_a= 3.7 Hz, J₅₀_a,5₀_b= 10.6 Hz, 1 H, H5⁰a), 3.43 (dd, J₄₀_,5₀_b= 2.9 Hz, J₅₀_a,5₀_b= 10.6 Hz, 1 H,

ν

_max/cm⁻¹(neat) 3164 (NH), 3057 (CH), 2930 (CH), 2836, 1682 (C=O), 1607

δ

J

H5⁰b), 3.79 (s, 6 H, 2

×

OCH₃), 4.03–4.08 (m, 1 H, H4⁰), 4.40–4.45 (m, 1 H, H3⁰), 6.31 (dd,

J₁₀_,2₀_b= 5.5 Hz, J₁₀_,2₀_a= 6.7 Hz, 1 H, H1⁰), 6.89–6.91 (m, 4 H, H^ar,DMTr), 7.22–7.26 (m, 1 H,

H^ar,DMTr), 7.31–7.39 (m, 6 H, H^ar,DMTr), 7.49–7.51 (m, 2 H, H^ar,DMTr), 7.74 (s, 1 H, H6), 10.02

(bs, 1 H, NH) ppm. ¹³C NMR (151 MHz, acetone-d₆):

δ

= 12.4 (CH₃^T), 39.8 (C2⁰), 55.5

(OCH₃), 60.0 (C3⁰), 64.0 (C5⁰), 85.7 (C1⁰), 85.9 (C4⁰), 87.3 (qC, DMTr), 110.6 (C5), 113.97

(DMTr), 114.03 (DMTr), 127.7 (DMTr), 128.7 (DMTr), 129.0 (DMTr), 130.97 (DMTr), 130.99

(DMTr), 131.01 (DMTr), 136.6 (C6), 136.7 (qC, DMTr), 146.0 (qC, DMTr), 151.2 (C2), 159.7

(qC, DMTr), 164.3 (C4) ppm. HRMS (ESI⁻): m/z calc. 542.2297 [M

−

H]⁻, found: 542.2305.

Spectroscopic data are in agreement with the literature [43].

C-(3⁰-O-(tert-Butyldimethylsilyl)-5⁰-deoxythymidin-5⁰-yl)-N-(3⁰-deoxy-5⁰-O-(4,4⁰-dime-

thoxytrityl)thymidin-3⁰-yl) amido methyl malonate (32): Carboxylic acid 29_◦(375 mg,

0.82 mmol) was dissolved in dry DMF (15 mL) under argon and cooled to 0 C. HOAt

(168 mg, 1.23 mmol) and EDC HCl (236 mg, 1.23 mmol) were added, followed by the

·

addition of aminothymidine 31 (469 mg, 0.86 mmol). The reaction mixture was warmed to

r.t and stirred at r.t. for 18 h. After this time, TLC analysis (CH₂Cl₂-MeOH, 9:1) showed

the consumption of starting materials (R_f= 0.2 and R_f= 0.5) and the formation of the

product (R_f= 0.8). The reaction was diluted with EtOAc (100 mL) and washed with H₂O

(50 mL), sat. aq. NaHCO₃(50 mL) and brine (50 mL). The combined organic extracts were

dried over MgSO₄, ﬁltered and concentrated. Puriﬁcation by ﬂash column chromatography

(EtOAc-petroleum ether, 9:1) afforded the desired product 32 as a white solid (614 mg, 76%).

−1

Compound 32 was isolated as 4:3 mixture of interconverting diastereoisomers. ν_max/cm

(neat) 2950 (CH), 2930 (CH), 2855 (CH), 1685 (C=O), 1608 (C=O), 1508, 1465 (CH), 1249, 1175,

1

1032 (C-O), 831, 777. Major diastereoisomer: H NMR (600 MHz, acetone-d₆):

δ

= 0.13–0.14

0

(2

×

s, 6 H, CH₃^TBDMS), 0.92 (s, 9 H, t-Bu^TBDMS), 1.43 (s, 3 H, 5 -CH₃^T), 1.84 (s, 3 H, 3 -CH₃^T),

2.09–2.20 (m, 2 H, 3⁰-H2⁰a, 3⁰-H5⁰a), 2.30–2.41 (m, 4 H, 5⁰-H2⁰a, 5⁰-H2⁰b, 3⁰-H2⁰b, 3⁰-H5⁰b),

3.37–3.45 (m, 2 H, 5⁰-H5⁰a, 5⁰-H5⁰b), 3.50–3.54 (m, 1 H, 3⁰-H6⁰), 3.56 (s, 3 H, CO₂CH₃), 3.79

(s, 6 H, 2

×

OCH₃), 3.88–3.91 (m, 1 H, 3⁰-H4⁰), 4.04–4.05 (m, 1 H, 5⁰-H4⁰), 4.33–4.36 (m,

1 H, 3⁰-H3⁰), 4.74–4.82 (m, 1 H, 5⁰-H3⁰), 6.18 (app. t, J = 7.0 Hz, 1 H, 3⁰-H1⁰), 6.27 (app. t,

Molecules 2021, 26, 320

20 of 25

J = 6.4 Hz, 1 H, 5⁰-H1⁰), 6.88–6.91 (m, 4 H, H^ar), 7.23–7.26 (m,₀1 H, H^ar), 7.31–7.39 (m, 6 H,

0

H^ar), 7.45 (s, 1 H, 3 -H6), 7.49–7.52 (m, 2 H, H^ar), 7.61 (s, 1 H, 5 -H6), 7.86 (d, J₃₀_,NH= 7.8 Hz,

1 H, CONH), 9.95–9.98 (m, 2 H, 5⁰-NH^T, 3⁰-NH^T) ppm. ¹³C NMR (151 MHz, acetone-d₆):

δ

=

−

4.63 (CH₃^TBDMS),

−

4.55 (CH₃^TBDMS), 12.15 (5⁰-CH₃^T), 12.54 (3⁰-CH₃^T), 18.54 (qC,

t-Bu^TBDMS), 26.16 (t-Bu^TBDMS), 33.4 (3⁰-C5⁰), 38.30 (5⁰-C2⁰), 39.8 (3⁰-C2⁰), 50.6 (5⁰-C3⁰), 50.7

(3⁰-C6⁰), 52.5 (CO₂H₃), 55.53 (OCH₃^DMT), 64.1 (5⁰-C5⁰), 76.6 (3⁰-C3⁰), 84.5 (5⁰-C4⁰), 84.8

C

(5⁰-C1⁰), 86.3 (3⁰-C4⁰), 86.6 (3⁰-C1⁰), 87.37 (qC, DMTr), 110.9 (3⁰-C5), 111.12 (5⁰-C5), 113.99

(CH, DMTr), 114.00 (CH, DMTr), 127.69 (CH, DMTr), 128.72 (CH, DMTr), 129.1 (CH, DMTr),

131.09 (CH, DMTr), 131.11 (CH, DMTr),₀136.2 (5⁰-C6, 136.5 (qC, DMTr), 136.7 (qC, DMTr),

0

137.6 (3 -C6), 145.95 (qC, DMTr), 151.3 (5 -C2, 3 -C2), 159.70 (qC, DMTr), 159.73 (qC, DMTr),

164.17 (5⁰-C4), 164.25 (3⁰-C4), 169.3 (CONH), 170.6 (CO₂) ppm. Minor diastereoisomer:

¹H NMR (600 MHz, acetone-d₆):

δ

= 0.09 (s, 3 H, CH₃^TBDMS), 0.11 (s, 3 H, CH₃^TBDMS),

0.88 (s, 9 H, t-Bu^TBDMS), 1.44 (s, 3 H, 5⁰-CH₃^T), 1.82 (s, 3 H, 3⁰-CH₃^T), 2.09–2.20 (m, 2 H,

3⁰-H2⁰a, 3⁰-H5⁰a), 2.30–2.41 (m, 3 H, 5⁰-H2⁰a, 3⁰-H2⁰b, 3⁰-H5⁰b), 2.48–2.53 (m, 1 H, 5⁰-H2⁰b),

3.37–3.45 (m, 2 H, 5⁰-H5⁰a, 5⁰-H5⁰b), 3.50–3.54 (m, 1 H, 3⁰-H6⁰), 3.66 (s, 3 H, CO₂CH₃),

3.74–3.76 (m, 1 H, 3⁰-H4⁰), 3.79 (s, 6 H, 2

×

OCH₃), 4.06–4.08 (m, 1 H, 5⁰-H4⁰), 4.33–4.36 (m,

1 H, 3⁰-H3⁰), 4.74–4.82 (m, 1 H, 5⁰-H3⁰), 6.22 (app. t, J = 6.9 Hz, 1 H, 3⁰-H1⁰), 6.32 (app. t,

J = 6.8 Hz, 1 H, 5⁰-H1⁰), 6.88–6.91 (m, 4 H, H^ar), 7.23–7.26 (m,₀1 H, H^ar), 7.31–7.39 (m, 6 H,

H^ar), 7.42 (s, 1 H, 3 -H6), 7.49–7.52 (m, 2 H, H^ar), 7.65 (s, 1 H, 5 -H6), 7.91 (d, J₃₀_,NH= 7.5 Hz,

0

1 H, CONH), 9.95–9.98 (m, 2 H, 5⁰-NH^T, 3⁰-NH^T) ppm. ¹³C NMR (151 MHz, acetone-d₆):

δ

=

−

4.58 (CH₃^TBDMS),

−

4.51 (CH₃^TBDMS), 12.13 (5⁰-CH₃^T), 12.46 (3⁰-CH₃^T), 18.53 (qC,

t-Bu^TBDMS), 26.17 (t-Bu^TBDMS), 33.3 (3⁰-C5⁰), 38.32 (5⁰-C2⁰), 40.1 (3⁰-C2⁰), 50.0 (3⁰-C6⁰), 51.3

(5⁰-C3⁰), 52.6 (CO₂H₃), 55.55 (OCH₃^DMT), 64.5 (5⁰-C5⁰), 76.3 (3⁰-C3⁰), 84.6 (5⁰-C4⁰), 84.9

C

(5⁰-C1⁰), 85.0 (3⁰-C4⁰), 85.5 (3⁰-C1⁰), 87.42 (qC, DMTr), 111.08 (3⁰-C5, 5⁰-C5), 114.01 (CH,

DMTr), 114.02 (CH, DMTr), 127.73 (CH, DMTr), 128.74 (CH, DMTr), 129.1 (CH, DMTr),

131.06 (₀CH, DMTr), 131.07 (CH, DMTr), ₀136.3 (5⁰-C6), 136.6 (qC, DMTr), 136.8 (qC, DMTr),

0

136.9 (3 -C6), 145.91 (qC, DMTr), 151.3 (3 -C2, 5 -C2), 159.72 (qC, DMTr), 159.74 (qC, DMTr),

164.18 (3⁰-C4), 164.20 (5⁰-C4), 168.4 (CONH), 171.2 (CO₂) ppm. HRMS (APCI⁺): m/z calc.

1004.4084 [M + Na]⁺, found: 1004.4068.

N-(3⁰-Deoxy-5⁰-O-(4,4⁰-dimethoxytrityl)thymidin-3⁰-yl)-C-(5⁰-deoxythymidin-5⁰-yl) amido

methyl malonate (33): Protected dimer 32 (580 mg, 0.59 mmol) was dissolved in THF (8 mL).

Tetra-n-butylammonium ﬂuoride trihydrate (316 mg, 1.06 mmol) was added and the solu-

tion was stirred at r.t. for 24 h. After this time, TLC analysis (EtOAc-MeOH, 9:1) showed

the complete consumption of the starting material (R_f= 0.7) and product formation as a

mixture of diastereomers (R_f= 0.3 and R_f= 0.4). The suspension was concentrated and pu-

riﬁcation by ﬂash column chromatography (EtOAc-MeOH, 19:1

→

9:1) afforded the desired

product 33 as a white solid (472 mg, 92%). ₋C₁ompound 33 was isolated as a 3:1 mixture of

interconverting diastereoisomers. ν_max/cm (neat) 3310 (OH, NH), 3073 (CH), 2950 (CH),

2930 (CH), 2839 (CH), 1655 (C=O), 1607 (C=O), 1508, 1466 (CH), 1248, 1175, 1031 (C-O), 828.

1

0

δ = 1.43 (d, J_CH3T,6= 1.0 Hz, 3 H, 5 -

Major diastereoisomer: H NMR (600 MHz, acetone-d₆):

CH₃^T), 1.86 (d, J_CH3T,6= 1.0 Hz, 3 H, 3 -CH₃^T), 2.10–2.23 (m, 2 H, 3 H2 a, 3 -H5 a), 2.27–2.38

0

(m, 4 H, 5⁰-H2⁰a, 5⁰-H2⁰b, 3⁰-H2⁰b, 3⁰-H5⁰b), 3.37–3.46 (m, 2 H, 5⁰-H5⁰a, 5⁰-H5⁰b), 3.52–3.56

(m, 4 H, 3⁰-H6⁰, CO₂CH₃), 3.78 (s, 6 H, 2

×

OCH₃), 3.90 (app. dt, J = 3.6 Hz, J = 10.2 Hz,

1 H, 3⁰-H4⁰), 4.05–4.07 (m, 1 H, 5⁰-H4⁰), 4.25–4.28 (m, 1 H, 3⁰-H3⁰), 4.57 (d, J₃₀_,OH= 3.3 Hz,

1 H, OH), 4.75–4.80 (m, 1 H, 5⁰-H3⁰), 6.21 (dd, J = 6.2 Hz, J = 7.6 Hz, 1 H, 3⁰-H1⁰), 6.27 (app.

0

t, J = 6.3 Hz, 1 H, 5 -H1 ), 6.88–6.91 (m, 4 H, H^ar), 7.22–7.25 (m, 1 H, H^ar), 7.30–7.38 (m, 6 H,

H^ar), 7.46 (app. d, J = 1.0 Hz, 1 H, 3⁰-H6), 7.49–7.52 (m, 2 H, H^ar), 7.63 (app. d, J = 1.0 Hz,

1 H, 5 -H6), 7.90 (d, J₃₀_,NH= 7.9 Hz, 1 H, CONH), 10.09–10.13 (m, 2 H, 5 -NH^T, 3 -NH^T) ppm.

0

¹³C NMR (151 MHz, acetone-d₆):

δ

= 12.2 (5⁰-CH₃^T), 12.54 (3⁰-CH₃^T), 33.6 (3⁰-C5⁰), 38.4

(5⁰-C2⁰), 39.5 (3⁰-C2⁰), 50.5 (5⁰-C3⁰), 50.7 (3⁰-C6⁰), 52.5 (CO₂

C

H₃), 55.53 (OCH₃^DMT), 64.0

(5⁰-C5⁰), 75.3 (3⁰-C3⁰), 84.4 (5⁰-C4⁰), 84.93 (5⁰-C1⁰), 86.0 (3⁰-C4⁰), 86.2 (3⁰-C1⁰), 87.36 (qC,

DMTr), 111.0 (3⁰-C5), 111.2 (5⁰-C5), 113.98 (CH, DMTr), 114.00 (CH, DMTr), 114.02 (CH,

DMTr), 127.68 (CH, DMTr), 128.71 (CH, DMTr), 129.1 (CH, DMTr), 131.07 (₀CH, DMTr),

0

131.09 (CH, DMTr), 136.3 (5 -C6), 136.5 (qC, DMTr), 136.63 (qC, DMTr), 137.6 (3 -C6), 145.92

Molecules 2021, 26, 320

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(qC, DMTr), 151.40 (5⁰-C2), 151.41 (3⁰-C2), 159.66 (qC, DMTr), 159.70 (qC, DMTr), 164.4

1

(5⁰-C4), 164.5 (3⁰-C4), 169.5 (CONH), 170.7 (CO₂) ppm. Minor diastereoisomer: H NMR

(600 MHz, acetone-d₆):

δ

= 1.44 (d, J_CH3T,6= 1.0 Hz, 3 H, 5⁰-CH₃^T), 1.83 (d, J_CH3T,6= 1.0 Hz,

3 H, 3⁰-CH₃^T), 2.10–2.23 (m, 2 H, 3⁰-H2⁰a, 3⁰-H5⁰a), 2.27–2.33 (m, 2 H, 3⁰-H2⁰₀b, 3⁰₀-H5⁰₀b),

0

2.39–2.42 (m, 1 H, 5 -H2 a), 2.49–2.54 (m, 1 H, 5 -H2 b), 3.37–3.46 (m, 2 H, 5 -H5 a, 5 -H5 b),

3.52–3.56 (m, 1 H, 3⁰-H6⁰), 3.65 (s, 3 H, CO₂CH₃), 3.76–3.78 (m, 7 H, 3⁰-H4⁰, 2

×

OCH₃),

4.08–4.10 (m, 1 H, 5⁰-H4⁰), 4.25–4.28 (m, 1 H, 3⁰-H3⁰), 4.51 (d, J₃₀_,OH= 4.2 Hz, 1 H, OH),

4.75–4.80 (m, 1 H, 5⁰-H3⁰), 6.24 (app. t, J = 6.9 Hz, 1 H, 3⁰-H1⁰), 6.33 (app. t, J = 6.6 Hz,

1 H, 5⁰-H1⁰), 6.88–6.91 (m, 4 H, H^ar), 7.22–7.25 (m, 1 H, H^ar), 7.30–7.38 (m, 6 H, H^ar), 7.42

(app. d, J = 1.0 Hz, 1 H, 3⁰-H6), 7.49–7.52 (m, 2 H, H^ar), 7.66 (app. d, J = 1.0 Hz, 1 H,

5⁰-H6), 7.98 (d, J₃₀_,NH= 7.5 Hz, 1 H, CONH), 10.09–10.13 (m, 2 H, 5⁰-NH^T, 3⁰-NH^T) ppm.

¹³C NMR (151 MHz, acetone-d₆):

δ

= 12.1 (5⁰-CH₃^T), 12.47 (3⁰-CH₃^T), 33.6 (3⁰-C5⁰), 38.3

H₃), 55.55 (OCH₃^DMT), 64.4

(5⁰-C2⁰), 39.7 (3⁰-C2⁰), 50.0 (3⁰-C6⁰), 51.2 (5⁰-C3⁰), 52.6 (CO₂

C

(5⁰-C5⁰), 75.1 (3⁰-C3⁰), 84.6 (5⁰-C4⁰), 84.87 (3⁰-C4⁰), 84.94 (5⁰-C1⁰), 85.3 (3⁰-C1⁰), 87.40 (qC,

DMTr), 111.1 (5⁰-C5, 3⁰-C5), 113.6 (CH, DMTr), 113.98 (CH, DMTr), 114.00 (CH, DMTr),

114.02 (CH, DMTr), 127.72 (CH, DMTr), 128.73 (CH, DMTr), 129.1 (CH, DMTr), 131.1 (CH,

DMTr), 136.44 (5⁰-C6), 136.55 (qC, DMTr), 136.66 (qC, DMTr), 136.9 (3⁰-C6), 145.93 (qC,

DMTr), 151.37 (3⁰-C2), 151.40 (5⁰-C2), 159.68 (qC, DMTr), 159.71 (qC, DMTr), 164.4 (5⁰-C4,

3⁰-C4), 168.7 (CONH), 171.2 (CO₂) ppm. HRMS (APCI⁻): m/z calc. 866.3254 [M

found: 866.3262.

−

H]⁻,

N-(3⁰-Deoxythymidin-3⁰-yl)-C-(5⁰-deoxythymidin-5⁰-yl) amido methyl malonate (34):

Protected dimer 33 (100 mg, 115 mol) was dissolved in CH₂Cl₂(980 L). Triethylsi-

lane (184 L, 1.15 mmol) and triﬂuoroacetic acid (20 L, 261 mol) were added and the

µ

solution was stirred at r.t. The solution turned bright red, followed by the formation of

a white precipitate and loss of the red colour after 10 min. After this time, TLC analysis

(EtOAc-MeOH, 4:1) showed the complete consumption of starting material (R_f= 0.6) and

the formation of the product (R_f= 0.4). The supernatant was removed and the precipitated

product was washed with toluene (2

bined and concentrated, and the resulting white solid was washed with CH₂Cl₂(3

×

1 mL). The washings and supernatant were com-

5 mL)

×

to remove impurities. The precipitates were combined to afford the desired product 34

as a white amorphous solid (60 mg, 92%). The product 34 was isolated as a 3:1 mixture

of interconverting diastereomers. ν

_max/cm⁻¹(neat) 3398 (OH, NH), 3261 (OH, NH), 3174

(OH, NH), 3050 (CH), 2955 (CH), 2819 (CH), 1742 (C=O), 1677 (C=O), 1655 (C=O), 1638

(C=O), 1547, 1473 (CH), 1369, 1273, 1203, 1097 (C-O), 1047 (C-O), 988. Major diastereoiso-

1

mer: H NMR (400 MHz, DMSO-d₆):

δ

= 1.77 (bs, 3 H, 5⁰-CH₃^T), 1.79 (bs, 3 H, 3⁰-CH₃^T),

1.88–1.96 (m, 1 H, 3⁰-H5⁰a), 1.99–2.23 (m, 5 H, 5⁰-H2⁰a, 5⁰-H2⁰b, 3⁰-H2⁰a, 3⁰-H2⁰b, 3⁰-H5⁰b),

3.42–3.45 (m, 1 H, 3⁰-H6⁰), 3.49–3.53 (m, 1 H, 5⁰-H5⁰a), 3.59–3.60 (m, 1 H, 5⁰-H5⁰b), 3.63 (s,

3 H, CO₂CH₃), 3.66–3.71 (m, 1 H, 3⁰-H4⁰), 3.73–3.76 (m, 1 H, 5⁰-H4⁰), 4.04–4.09 (m, 1 H,

3₀⁰-H3₀⁰), 4.23–4.29 (m, 1 H, 5⁰-H3⁰), 5.₀29 (bs, 2 H, 5⁰-OH, 3⁰-OH), 6.10 (app t, J = 7.0 Hz, 1 H,

0

3 -H1 ), 6.19 (app. t, J = 6.7 Hz, 1 H, 5 -H1 ), 7.38 (app. d, J = 0.9 Hz, 1 H, 3 -H6), 7.73 (app. d,

0

s, 2 H, 5 -NH^T,

J = 0.9 Hz, 1 H, 5 -H6), 8.63 (d, J₃₀_,NH= 7.4 Hz, 1 H, CONH), 11.25–11.27 (2

×

3⁰-NH^T) ppm. ¹³C NMR (100 MHz, DMSO-d₆):

δ

= 12.1 (3⁰-CH₃^T), 12.3 (5⁰-CH₃^T), 32.37

(3⁰-C5⁰), 36.5 (5⁰-C2⁰), 38.2 (3⁰-C2⁰), 49.0 (3⁰-C6⁰), 49.5 (5⁰-C3⁰), 52.1 (CO₂CH₃), 61.3 (5⁰-C5⁰),

73.3 (3⁰-C3⁰), 83.49 (5⁰-C1⁰), 83.69 (3⁰-C1⁰), 84.1 (3⁰-C4⁰), 84.9 (5⁰-C4⁰), 109.5 (5⁰-C5), 109.7

(3⁰-C5), 136.1 (5⁰-C6), 136.3 (3⁰-C6), 150.4 (5⁰-C2, 3⁰-C2), 163.7 (5⁰-C4, 3⁰-C4), 168.0 (CONH),

1

169.9 (CO₂) ppm. Minor diastereoisomer: H NMR (400 MHz, DMSO-d₆):

δ = 1.78 (d,

J

_CH3T,6= 1.1 Hz, 3 H, 5⁰-CH₃^T), 1.80 (d, J_CH3T,6= 1.1 Hz, 3 H, 3⁰-CH₃^T), 1.97–2.25 (m, 6 H,

5⁰-H2⁰a, 5⁰-H2⁰b, 3⁰-H2⁰a, 3⁰-H2⁰b, 3⁰-H5⁰a, 3⁰-H5⁰b), 3.41–3.45 (m, 1 H, 3⁰-H6⁰), 3.52–3.58

(m, 2 H, 5⁰-H5⁰a, 3⁰-H4⁰₀), 3.61–3.66 (m, 4 H, 5⁰₀-H5₀⁰b, CO₂CH₃), 3.7₀6–3.78 (m, 1 H, 5⁰-H4⁰),

0

4.04–4.09 (m, 1 H, 3 -H3 ), 4.30–4.35 (m, 1 H, 5 -H3 ), 5.29 (bs, 2 H, 5 -OH, 3 -OH), 6.15 (app

t, J = 7.1 Hz, 1 H, 3⁰-H1⁰), 6.20 (app. t, J = 6.7 Hz, 1 H, 5⁰-H1⁰), 7.42 (app. d, J = 1.1 Hz, 1 H,

0

3 -H6), 7.76 (app. d, J = 1.1 Hz, 1 H, 5 -H6), 8.71 (d, J₃₀_,NH= 7.3 Hz, 1 H, CONH), 11.29–11.30

(2

s, 2 H, 5⁰-NH^T, 3⁰-NH^T) ppm. ¹³C NMR (100 MHz, DMSO-d₆): = 12.1 (3⁰-CH₃^T),

12.3 (5⁰-CH₃^T), 32.42 (3⁰-C5⁰), 36.7 (5⁰-C2⁰), 38.0 (3⁰-C2⁰), 48.5 (3⁰-C6⁰), 49.7 (5⁰-C3⁰), 52.1

×

δ

Molecules 2021, 26, 320

22 of 25

0 0 0 0 0 0 0 0 0 0 0 0

(CO₂CH₃), 61.5 (5 -C5 ), 73.3 (3 -C3 ), 83.47 (3 -C4 ), 83.53 (5 -C1 ), 83.7 (3 -C1 ), 85.0 (5 -C4 ),

109.5 (5⁰₀-C5), 109.8 (3⁰-C5), 136.1 (5⁰-C6), 136.2 (3⁰-C6), 150.4 (5⁰-C2), 150.5 (3⁰-C2), 163.7

0

(5 -C4, 3 -C4), 167.5 (CONH), 170.3 (CO₂) ppm. HRMS (ESI⁺): m/z calc. 588.1912 [M + Na]⁺,

found: 588.1922.

N-(3⁰-Deoxythymidin-3⁰-yl)-C-(5⁰-deoxythymidin-5⁰-yl) amido sodium malonate (35):

◦

KOH (18 mg, 320

(30 mg, 53

µ

mol) was dissolved in MeOH (1 mL) and cooled to 0 C. Ester 34

µ

mol) was added, the solution was slowly warmed to r.t. and stirred for 7 h.

After this time, TLC analysis (H₂O-i-PrOH-EtOAc, 1:5:4) showed the consumption of

starting material (R_f= 0.8) and the formation of product (R_f= 0.3). The reaction mixture

was concentrated and eluted through Diaion resin WT01S(H) (H form) to remove excess

KOH, then eluted through Diaion resin WT01S(H) (Na form) and concentrated to afford

the desired product 35 as a white solid (27 mg, 89%). Compound 35 was isolated as a 1:1

mixture of interconverting diastereomers. ν

_max/cm⁻¹(neat) 3259 (OH, NH), 3063 (CH),

2925 (CH), 2820 (CH), 1655 (C=O), 1587 (C=O), 1474 (CH), 1366, 1270, 1090 (C-O), 1050

1

(C-O), 962,₀766. H₀NM₀R (600 MHz, CD₃OD₀):

δ

= 1.8₀9–1.91 (m, 12 H, 4

×

CH₃^T), 2.05–2.41

0

(m, 12 H, 3 -H2 a, 3 -H2 b, 3 -H5 a, 3 -H5 b, 5 -H2 a, 5 -H5 b), 3.23 (dd, J = 5.5 Hz, J = 8.0 Hz,

1 H, 3⁰-H6⁰), 3.31–3.35 (m, 1 H, 3⁰-H6⁰), 3.73–3.91 (m, 8 H, 3⁰-H4⁰, 5⁰-H4⁰, 5⁰-H5⁰a, 5⁰-H5⁰b),

0

4.14–4.19 (m, 2 H, 3 -H3 ), 4.46 (app. q, J = 7.2 Hz, 1 H, 5 -H3 ), 4.51 (app. q, J = 7.2 Hz, 1 H,

5⁰-H3⁰), 6.15–6.25 (m, 4 H, 3⁰-H1⁰, 5⁰-H1⁰), 7.48–7.50 (2

s, 2 H, H6), 7.88–7.90 (2 s, 2 H,

H6) ppm. ¹³C NMR (151 MHz, CD₃OD): = 12.45 (CH₃^T), 12.51 (CH₃^T), 12.53 (CH₃^T), 35.4

×

δ

0

(3 -C5 ), 35.5 (3 -C5 ), 38.5 (5 -C2 ), 38.8 (5 -C2 ), 39.9 (3 -C2 ), 40.1 (3 -C2 ), 49.7 (5 -C3 ), 50.0

(5 -C3 ), 53.6 (3 -C6 ), 55.1 (3 -C6 ), 62.0 (5 -C5 ), 62.3 (5 -C5 ), 75.4 (3 -C3 ), 75.3 (3 -C3 ), 85.9

(3⁰-C1⁰, 5⁰-C1⁰), 86.0 (3⁰-C1⁰, 5⁰-C1⁰), 86.15 (3⁰-C4⁰), 86.19 (5⁰-C4⁰), 86.6 (5⁰-C4⁰), 87.5 (3⁰-C4⁰),

111.3 (C5), 111.4 (C5), 111.7 (C5), 112.0 (C5), 137.8 (C6), 138.1 (C6), 138.2 (C6), 152.39 (C2),

152.43 (C2), 152.45 (C2), 152.54 (C2), 166.5 (C4), 166.6 (C4), 166.68 (C4), 166.70 (C4), 174.3

(CONH), 174.8 (CONH), 176.8 (CO₂), 177.1 (CO₂) ppm. HRMS (ESI⁺): m/z calc. 596.1575

[M + Na]⁺, found: 596.1594.

N-(3⁰-Deoxythymidin-3⁰-yl)-C-(5⁰-deoxythymidin-5⁰-yl) amido N-hydroxy malonamide

(36): Hydroxylamine hydrochloride (123 mg, 1.77 mmol) was suspended in MeOH (2 mL)

and KOH (124 mg, 2.21 mmol) was added. The suspension was warmed to 40 ^◦C to aid

dissolution and the precipitate was removed by ﬁltration. Ester 34 (50 mg, 88 µmol) was

dissolved in the ﬁltrate and the reaction was stirred at r.t. for 48 h, during which a white

precipitate formed. After this time, TLC analysis (H₂O-i-PrOH-EtOAc, 1:5:4) showed the

consumption of starting material (R_f= 0.8) and the formation of the product (R_f= 0.4).

The reaction mixture was neutralised to pH 7 using aq. HCl (1 M) and concentrated.

Puriﬁcation by ﬂash column chromatography (H₂O-i-PrOH-EtOAc, 1:5:4) afforded the

desired product 36 as a white amorphous solid (37 mg, 74%). Compound 36 was isolated

as a 5:4 mixture of interconverting diastereomers. ν

_max/cm⁻¹(neat) 3409 (OH, NH), 3162

(OH, NH), 2994 (CH), 2923 (CH), 1664 (C=O), 1543, 1474 (CH), 1364, 1271, 1089 (C-O), 1049

1

(C-O), 1019, 855, 765. Major diastereomer: H NMR (600 MHz, DMSO-d₆):

δ = 1.77–1.78

(m, 3 H, 5⁰-CH₃^T), 1.80 (d, J_CH3T,6= 1.0 Hz, 3 H, 3⁰-CH₃^T), 1.85–1.94 (m, 1 H, 3⁰-H5⁰a),

1.99–2.04 (m, 1 H, 3⁰-H2⁰a), 2.08–2.22 (m, 4 H, 5⁰-H’2a, 5⁰-H2⁰b, 3⁰-H2⁰b, 3⁰-H5⁰b), 3.18 (dd,

J = 6.0 Hz, J = 8.5 Hz, 1 H, 3⁰-H6⁰), 3.50–3.54 (m, 1 H, 5⁰-H5⁰a), 3.55–3.58 (m, 1 H, 3⁰-H4⁰),

3.59–3.64 (m, 1 H, 5⁰-H5⁰b), 3.77–3.80 (m, 1 H, 5⁰-H4⁰), 4.02–4.05 (m, 1 H, 3⁰-H3⁰), 4.26–4.33

(m, 1 H, 5⁰-H3⁰), 5.08–5.11 (m, 1 H, 5⁰-OH), 5.32 (d, J = 4.4 Hz, 1 H, 3⁰-OH), 6.08–6.11 (m,

1 H, 3⁰-H1⁰), 6.17–6.19 (m, 1 H, 5⁰-H1⁰), 7.44 (bs, 1 H, 5⁰-H6), 7.75 (app. d, J = 1.0 Hz,

1 H, 3⁰-H6), 8.37 (d, J₃₀_,NH= 7.3 Hz, 1 H, CONH), 8.96 (bs, 1 H, CONHO), 10.59 (bs, 1 H,

NH^Tor NHO ), 11.27 (bs, 1 H, NH^Tor NHO ) ppm. ¹³C NMR (151 MHz, DMSO-d₆):

H H

δ

= 12.08 (3⁰-CH₃^T), 12.3 (5⁰-CH₃^T), ₀32.4 (3⁰-C5⁰),₀36.9 (5⁰-C2⁰), 38.4 (53-C2⁰₀), 47.2 (3⁰₀-C6⁰₀),

0

49.3 (5 -C3 ), 61.2 (5 -C5 ), 73.2 (3 -C3 ), 83.5 (5 -C1 ), 83.6 (3 -C1 ), 83.9 (3 -C4 ), 84.8 (5 -C4 ),

109.4 (5⁰-C5), 109.77 (3⁰-C5), 136.19 (3⁰-C6), 136.21 (5⁰-C6), 150.39 (3⁰-C2), 150.44 (5⁰-C2),

163.7 (5⁰-C4, 3⁰-C4), 165.6 (CONHOH), 168.7 (

C

ONHC) ppm. Minor diastereomer: H

1

NMR (600 MHz, DMSO-d₆):

δ

= 1.77–1.78 (m, 3 H, 5⁰-CH₃^T), 1.81 (d, J_CH3T,6= 0.9 Hz, 3 H,

Molecules 2021, 26, 320

23 of 25

0

3 -CH₃^T), 1.85–1.94 (m, 1 H, 3 -H5 a), 1.99–2.04 (m, 1 H, 3 -H2 a), 2.08–2.22 (m, 4 H, 5 -H’2a,

5⁰-H2⁰b, 3⁰-H2⁰b, 3⁰-H5⁰b), 3.21 (dd, J = 5.6 Hz, J = 8.6 Hz, 1 H, 3⁰-H6⁰), 3.50–3.54 (m, 1 H,

5⁰-H5⁰a), 3.55–3.58 (m, 1 H, 3⁰-H4⁰), 3.59–3.64 (m, 1 H, 5⁰-H5⁰b), 3.77–3.80 (m, 1 H, 5⁰-H4⁰),

4.02–4.05 (m, 1 H, 3⁰-H3⁰), 4.26–4.33 (m, 1 H, 5⁰-H3⁰), 5.08–5.11 (m, 1 H, 5⁰-OH), 5.32 (d,

J = 4.4 Hz, 1 H, 3⁰-OH), 6.08–6.11 (m, 1 H, 3⁰-H1⁰), 6.17–6.19 (m, 1 H, 5⁰-H1⁰), 7.44 (bs, 1 H,

5⁰-H6), 7.77 (app. d, J = 1.0 Hz, 1 H, 3⁰-H6), 8.45 (d, J₃₀_,NH= 7.3 Hz, 1 H, CONH), 8.91 (bs,

1 H, CONHO), 10.59 (bs, 1 H, NH^Tor NHO ), 11.27 (bs, 1 H, NH^Tor NHO ) ppm. ¹³C

H H

NMR (151 MHz, DMSO-d₆):

38.3 (53-C2 ), 46.9 (3 -C6 ), 49.4 (5 -C3 ), 61.3 (5 -C5 ), 73.2 (3 -C3 ), 83.5 (5 -C1 ), 83.7 (3 -C1 ),

83.9 (3 -C4 ), 85.1 (5 -C4 ), 109.4 (5 -C5), 109.80 (3 -C5), 136.16 (5 -C6), 136.24 (3 -C6), 150.41

δ

= 12.12 (3⁰-CH₃^T), 12.3 (5⁰-CH₃^T), 32.5 (3⁰₀-C5⁰₀), 36.7 (5⁰₀-C2⁰₀),

0

(3 -C2), 150.44 (5 -C2), 163.7 (5 -C4, 3 -C4), 166.0 (CONHOH), 168.3 (CONHC) ppm. HRMS

(ESI⁻): m/z calc. 565.1900 [M − H]⁻, found: 565.1906.

3.3. Biological Evaluation

SNM1A (698-1040) was expressed and puriﬁed as described by Allerston et al. [8].

SNM1A (698-1040) was stored as a 1.0 M solution in reaction buffer (20 mM HEPES-

µ

KOH, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 0.05% Triton-X, 0.1 mg/mL BSA, 5% glycerol,

0.5 mM DTT). An oligonucleotide of the sequence 5⁰-TTA GCA GTC AGT CAG TCA

TCG-Cy3-3⁰was phosphorylated using T4 PNK (New England Biolabs) [44] and used

as the substrate. Modiﬁed nucleosides were added to the assay in 40% DMSO solution

(10 mM or as speciﬁed). Fluorescent oligonucleotides were protected from light during

incubations and gel electrophoresis.

Modiﬁed nucleosides (10 nmol or as speciﬁed) were mixed with SNM1A (698–1040)

(25 fmol) in reaction buffer containing 4% DMSO (10 µ

L) on ice and incubated at 37 ^◦C

for 5 min. Phosphorylated oligonucleotide (0.8 pmol) was added and the reaction was

incubated at 37 ^◦C for 60 min. The reaction was stopped by the addition of stop solution

◦

(2

µ

L, 95% formamide, 10 mM EDTA) followed by heating to 95 C for 3 min. The products

of the reaction were analysed using gel electrophoresis; 3 µL of each reaction was used.

Oligonucleotides were separated on a 15% acrylamide 6.5 M urea gel (2.9 g urea,

2.7 mL 40% acrylamide-bisacrylamide 25:1, 1.4 mL 5

×

TBE (0.45 M Tris, 0.45 M boric acid,

0.01 M EDTA pH 8.0), 0.6 mL H₂O) in 1X TBE at 150 V for 90 min alongside bromophenol

blue and xylene cyanol as markers for 8 and 28 nt, respectively, and imaged using Typhoon

FLA 9500.

Supplementary Materials: The following are available online, Figures S1–S4: Concentration-dependence

studies of compounds 9, 10, 15, 27, 28, 34–36; ¹H and ¹³C NMR spectra.

Author Contributions: Conceptualisation, E.-M.D. and J.F.M.; Methodology, E.-M.D. and J.F.M.;

Validation, E.-M.D.; Investigation, E.-M.D.; Writing—Original Draft Preparation, E.-M.D.; Writing—

Review and Editing, J.F.M.; Visualisation, E.-M.D.; Supervision, J.F.M.; Project Administration, J.F.M.;

Funding Acquisition, E.M.D. and J.F.M. All authors have read and agreed to the published version of

the manuscript.

Funding: This research was funded by Trinity College Dublin, the University of Dublin, with an

Ussher Postgraduate fellowship (E.-M.D.) and by The Wellcome Trust ISSF (J.F.M.).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in the Supplementary

Material.

Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design

of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or

in the decision to publish the results.

Sample Availability: Samples of the compounds are not available from the authors.

Molecules 2021, 26, 320

24 of 25

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Article Doi

Probing the binding requirements of modified nucleosides with the dna nuclease snm1a

DOI: 10.3390/molecules26020320

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Article abstract of DOI:10.3390/molecules26020320

Full text of DOI:10.3390/molecules26020320

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