An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG)

Article abstract of DOI:10.1016/j.bmc.2020.115507

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.

Full text of DOI:10.1016/j.bmc.2020.115507

Bioorganic&MedicinalChemistry28(2020)115507
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Bioorganic & Medicinal Chemistry
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An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme
alkyladenine glycosylase (AAG)
Eduard Mas Clareta, Balqees Al Yahyaeia, Shuyu Chua, Ruan M. Elliottb, Manuel Imperatoa,
⁎
Arnaud Lopeza, Lisiane B. Meirac, Brendan J. Howlina, Daniel K. Whelligana,
b Department of Nutritional Sciences, School of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK
a Department of Chemistry, University of Surrey, Guildford GU2 7XH, UK
c Department of Clinical and Experimental Medicine, School of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK
A R T I C L E I N F O
A B S T R A C T
Keywords:
The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic
reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the
biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against
tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the
rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor
followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of
AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of
AAG.
Methylpurine DNA glycosylase (MPG)
Alkyladenine DNA glycosylase (AAG)
Base excision DNA repair
Ischaemic reperfusion
Small molecule inhibitor
Aza-nucleoside
1. Introduction
recruitment of neutrophils and macrophages leads to further production
of RONS.6 Both alkylation- and ischaemia reperfusion-induced AAG-
Alkyladenine glycosylase (AAG; also known as methylpurine DNA
glycosylase, MPG, and alkyl-N–purine glycosylase, ANPG, EC: 3.2.2.21)
is one of several DNA glycosylases that can initiate the base excision
repair (BER) pathway by hydrolysis of a range of alkylated, oxidised or
deaminated purine bases from the DNA backbone.1 AAG’s action gen-
erates apurinic (AP) sites, which are further processed by downstream
BER enzymes. Despite its beneficial role, AAG overactivity on DNA le-
sions can lead to cellular necrosis and tissue damage. Specifically, AAG
has been shown to drive alkylation-induced cytotoxicity in several
mouse tissues, namely the cerebellum, spleen, thymus, bone marrow,
pancreas and retina, with tissue damage reduced or absent in Aag-
knockout mice and dramatically increased in Aag-overexpressing
transgenic mice.2,3 Furthermore, in ischemia reperfusion mouse
models, Aag-knockout mice display reduced tissue necrosis in liver,
kidney and brain when compared to wild type.4 Ischemia reperfusion
models mimic ischaemic stroke, liver and kidney failure, and organ
transplantation. In these events the tissues are temporarily starved of
their blood supply before sudden reperfusion results in a burst of re-
active oxygen and nitrogen species (RONS). RONS can directly oxidise
DNA bases but also lead to lipid peroxidation products, which alkylate
DNA.5 Further to this, sterile inflammation is induced and the
dependent tissue necrosis is hypothesised to result from AAG activity on
substantial numbers of alkylated and oxidised DNA bases leading to the
accumulation of toxic repair intermediates in the DNA, which over-
whelm the repair capacity of downstream BER enzymes.3 Stable,
membrane-permeable inhibitors of AAG are required as chemical
probes to further investigate these mechanisms and to form leads to
potential chemoprotectives for patients on alkylative chemotherapy or
to generate rapid treatments for minimising tissue damage from stroke,
and organ failure and transplantation.
The only reported specific small molecule inhibitor of AAG is the
natural polyphenolic flavonol morin, which was shown to act directly
on AAG, and not through binding to DNA, with an IC50 of 2.6 μM
(measured using a gel-based biochemical assay).7 Although shown
through surface plasmon resonance and control experiments to be
specific for AAG and effective in cells, a myriad of other bioactivities
have been reported for morin resulting from both its antioxidant ac-
tivity and binding to biomacromolecules.8,9 These other bioactivities
may confound results when used as a probe in biological studies.
Therefore, we sought an alternative scaffold for chemical probe in-
hibitors through ligand-based design. Our specific requirements of a
final probe molecule match those of the Structural Genomics
⁎ Corresponding author.
E-mail address: d.whelligan@surrey.ac.uk (D.K. Whelligan).
https://doi.org/10.1016/j.bmc.2020.115507
Received 10 February 2020; Received in revised form 6 April 2020; Accepted 9 April 2020
Availableonline15April2020
0968-0896/©2020PublishedbyElsevierLtd.

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
backbone carbonyl of Val-262. Hydrogen bonding to this tightly bound
water molecule is considered integral to the strong affinity
(Kd = 23 pM) of this oligomer because the analogous uncharged tet-
rahydrofuran-containing oligomer showed
7 × weaker binding
(Kd = 160 pM).16 It is worthy of note that the pyrrolidine-containing
oligomer is an effective inhibitor despite lacking a damaged DNA base
mimic to occupy AAG’s base-binding pocket.16,17 Nevertheless, a base
mimic (adenine, joined to pyrrolidine via a CH2 linker for stability) has
been incorporated and a Kd < 1 pM against AAG reported.17 To the
best of our knowledge, no X-ray crystal structure for this complex has
been obtained and the lone nucleotide (or nucleoside) from this oli-
gomer has not been tested against AAG.
Scheme 1. Synthesis of ethenodeoxycytidine 1 and 5′-phosphorylated analo-
gues 2–4.
To inform the design of small molecule inhibitors, we overlaid the
published X-ray co-crystal structures of AAG in complex with the sub-
strate ethenoadenine (εA)-, inhibitory εC- and pyrr-containing duplex
oligomers (Fig. 1).18 [Note that the authors report that the εA-oligomer
remained un-hydrolysed in AAG’s active site through use of inhibitory
MgCl2, Mg2+ ions from which were not seen in the crystal structure.] In
the crystal structures, all of the DNA oligomers make several electro-
static/hydrogen bonding interactions between their phosphodiester
groups and basic residues on the surface of AAG. Any potent small
molecule inhibitor based on a single nucleotide would therefore have to
find further binding interactions within the active site to compensate
for the loss of the DNA chain (as exemplified by the lack of activity of
our εC lone nucleotides). Thus it would be necessary to include a base
mimic to find interactions in AAG’s base-binding pocket. To this end,
we proposed to synthesise hybrid inhibitors based on the immucillin
(discussed below) scaffolds containing the pyrrolidine moiety joined to
an alkylated DNA-base analogue. This could be achieved in two ways:
Consortium for their Target 2035 program to make chemical or anti-
body probes available for the entire human proteome.10,11 Specifically,
these are a biochemical assay Kd or IC50 < 100 nM, a cellular assay
IC50 or EC50 < 1 μM and > 30-fold selectivity against other mam-
malian BER enzymes and enzymes known to be inhibited by structu-
rally related molecules, such as purine nucleoside phosphorylase (PNP).
2. AAG small molecule inhibitor design
A duplex DNA 13-nucleotide oligomer containing an ethenodeox-
ycytidine (εC) nucleotide is reported to inhibit AAG with an IC50 of
39 nM, and has a Kd of 21 nM.12 This affinity is 2-fold greater than that
of a 13-mer substrate ethenodeoxyadenosine (εA)-containing oligomer
(Kd = 46 nM). Based on this, we first sought to test the lone εC-nu-
cleoside 1 and nucleotide 4 (Scheme 1) for any inhibitory activity with
a
view to subsequently optimising them into potent, membrane
permeable inhibitors if active. However, neither of these compounds,
nor the precursor di- or mono- benzylphosphodiesters 2 or 3 showed
any inhibition at up to 1 mM concentration.
1. in the form of inhibitor scaffold 5 where aromatic heterocycles are
attached via a C-atom (to deprive them of leaving group ability) to
C-1′ position of the pyrrolidine, a design which takes account of the
fact that in the overlay shown in Fig. 1, the εC-deoxyribose (pink)
overlaps well with the pyrrolidine group (green). Such molecules 5
are the subject of ongoing synthetic efforts in our group.
Another inhibitory duplex DNA oligomer, containing an abasic
pyrrolidine (pyrr) nucleotide mimic (pyrr-oligomer, Fig. 1), was de-
signed by the group of Verdine originally as an inhibitor of Escherichia
coli 3-methyladenine DNA glycosylase II (AlkA).13,14 It is proposed that
the protonated pyrrolidine mimics the oxocarbenium ion character of
the transition state (TS) (or formal oxocarbenium intermediate in an
SN1 mechanism 15), which involves cleavage of the N-glycosidic bond
between the base and C-1′ of the deoxyribose and attack by the nu-
cleophilic active site water molecule from the opposite side.
2. in the form of inhibitor scaffold 6 rationalised as follows: the overlay
in Fig. 1 shows that, compared to the εA-oligomer’s deoxyribose
group, the pyrrolidine is displaced towards the base-binding pocket.
This could be to maximize hydrogen bonding with the nucleophilic
water molecule or it could be due to the terminus of the εA base
pushing back against the end of its binding pocket. The latter is
evidenced by the εA-deoxyribose appearing displaced compared to
the εC-deoxyribose as well as the pyrrolidine. Either way, this
In the published co-crystal structure (green, Fig. 1),13 the proto-
nated pyrrolidine undergoes hydrogen bonding (2.8 Å) with the active
site nucleophilic water molecule, which is itself held in place and ac-
tivated by three hydrogen bonds with Glu125, Arg182 and the
Fig. 1. Overlay of published X-ray co-crystal structures of AAG complexed with DNA oligomers containing: an (substrate) ethenodeoxyadenosine (εA, cyan, PDB:
1F4R), an (inhibitory) ethenodeoxycytidine (εC, pink, PDB: 3QI5) and an (inhibitory) abasic pyrrolidine nucleotide mimic (pyrr, green, PDB: 1F6O). The O-atoms of
the overlayed, active site nucleophilic water molecules are coloured to match their crystal structures. Skeletal structures of the active-site bound nucleotides of these
DNA oligomers are shown on the right along with small molecule inhibitor scaffolds proposed in this paper.
2

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
flexibility in the binding mode may permit the N-atom to be moved
one position around the ring and itself to bear an alkylated base
mimic, which must be linked via a CH2 group to maintain the ba-
sicity (and therefore protonated form) of the pyrrolidine N-atom.
Importantly, this positioning of the N-atom has been shown, in Es-
cherichia
coli
5′-methylthioadenosine/S-adenosylhomocysteine
(MTAN), to better mimic a substrate transition state involving early,
SN1-like departure of the purine where most positive charge accu-
mulates at the anomeric carbon.19,20 To the best of our knowledge,
the “early-” or “late-ness” of the TS of AAG has not been determined
and may be substrate specific for this multi-substrate enzyme.
However, the comparison of both types of inhibitor in the future
may inform on the nature of the TS to some extent.
Both aza-nucleoside inhibitor scaffolds have been reported pre-
viously to mimic the transition states in enzymes which attack ribose-
and deoxyribose-N-glycosidic bonds.21 For example, they form the first
(5) and second (6) generation immucillins, which inhibit PNP by mi-
micking the TS of phosphorolysis of guanosine and inosine.22–24 Also,
compounds based on 6 inhibit the ADP-ribosylating action of the cho-
lera, pertussis and diphtheria toxins,25 and, as part of RNA-oligomers,
26
the ricin A-chain. Of most relevance to our work, the scaffolds have
been previously applied as inhibitors of other DNA glycosylases, either
as part of large DNA oligomers or as lone nucleosides. For example, a
DNA 11-mer containing the abasic form (lacking any arylmethylene
group) of nucleotide 6 showed a Kd of 0.11 nM against uracil DNA
glycosylase (UDG),27 and duplex DNA 30-mers containing nucleotides
based on 5 and 6 are nano- to picomolar inhibitors of Escherichia coli
MutY, bacterial Fpg, human 8-oxoguanine DNA glycosylase 1 (hOGG1)
and human Nei-like DNA glycosylase (hNEIL1).28,29
Herein, we report an investigation of the synthesis of five small
molecule, free aza-nucleoside analogues based on scaffold 6 and the
finding that one of them is a weak, but ligand efficient, inhibitor of AAG
worthy of further optimisation into a chemical probe.
3. Chemistry
The free nucleoside and nucleotides of εC were synthesised from
deoxycytidine as shown in Scheme 1. In our hands, ethenylation of 1 at
RT required 7 days at pH 3.5 and complete removal of methanol and
water from the purified product required extensive drying (100 °C
under vacuum for 5 h) which may explain the higher m.p. obtained by
us compared to others.30 Conversion of nucleoside 1 into the phosphate
was unsuccessful using POCl3 with no product being isolable by reverse
phase HPLC.31 To simplify purification, we opted for a dibenzylpho-
sphorylation, which would permit normal phase chromatography of the
Scheme 2. Synthesis of azanucleosides.
protected phosphate prior to hydrogenolysis of the benzyl protecting
which may be of interest to the synthetic chemistry community.42 The
route began with aza-Michael addition of glycine ethyl ester 7 to ethyl
acrylate (1 equiv.) to give the corresponding secondary amine 8 in up to
54% yield.43,44 In several instances the tertiary amine resulting from a
second aza-Michael addition of ethyl acrylate, was detected and re-
moved by column chromatography. This has not been reported in the
literature and it was not possible to prevent its formation by varying the
rate of addition of ethyl acrylate or the reaction time. Subsequent N-
benzylation of purified amine 8 afforded tertiary amine 9 in high yield.
The next step required a Dieckmann condensation to give pyrroli-
dinone 10. Pinto et al. showed that reaction of amine 9 with KOtBu in
THF at −78 °C chemoselectively produced pyrrolidinone isomer 10
(74% yield) over the alternative isomer resulting from formation and
attack by the enolate of the α-aminoester (13% yield).45 In our case,
formation of 10 was followed by GC–MS (m/z 247 [M+]) but, during
chromatographic purification of the reaction mixture the desired pro-
duct was oxidised to pyrrole 11 (characterised by GC–MS (m/z 245
[M+]) and NMR). The oxidation and aromatisation of oxo- and hy-
droxypyrrolidines to pyrroles in the presence of SiO2 and air has been
32,33
groups. Thus, the tribenzylphosphite-iodine coupling
was applied
to afford a mixture of di- and mono-benzylphosphates 2 and 3 in low
yield, along with recovery of starting material. To the best of our
knowledge, this is the first application of that coupling to a nucleoside,
albeit one bearing a non-nucleophilic base, and in a better-optimised
form it may be a useful method for the selective phosphorylation of the
primary alcohol of nucleosides without the need for secondary alcohol
protection.34 Subsequently, hydrogenation of dibenzylphosphate 2 af-
forded phosphate 4 in high yield, which was purified by reverse-phase
HPLC prior to biochemical assay.
For the synthesis of azanucleosides 6, several routes to the key di-
hydroxypyrrolidine 18 (and its enantiomer) have been published, in-
cluding those starting from sugars,35,36 those beginning with a 1,3-di-
polar cycloaddition and later employing enzymatic chiral
resolution,37–39 and those using an asymmetric 1,3-dipolar cycloaddi-
tion by means of chiral auxiliaries.40,41 Despite the latter two being 1–2
steps shorter, we chose to investigate the route based around the en-
zymatic chiral resolution of hydroxypyrrolidine 13 (Scheme 2), de-
scribed by Clinch, and we report here some nuances of each reaction
3

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
reported by Davis et al.46 A different purification procedure based on
crystallisation of the product at −4 °C for 18 h was attempted, giving
the desired pyrrolidine 10 in up to 60% yield, albeit with minor im-
purities. Due to the difficulties in purification of pyrrolidinone 10, al-
ternative, quantitative cyclisation conditions were sought to obviate the
need for chromatography. The use of LDA gave a mixture of products
and, again, only pyrrole 11 was isolated after chromatography. How-
ever, regioselective TiCl4-mediated Dieckmann condensation condi-
tions, published by Deshmukh et al. and reported to give pyrrolidinone
10 and related compounds in 40–60% yield after chromatography,47
proceeded in our hands to give 92% yield of 10 of sufficient purity to be
used in the next step without purification, thus avoiding oxidation to
the undesired pyrrole.
yield diol (+)-15 in 71% yield. This was followed by N-debenzylation
according to Clinch et al., who achieved quantitative yield for hydro-
genation over Pd/C in the presence of di-tert-butyl dicarbonate to give
the Boc-protected pyrrolidine (+)-17 which is hydrolysed with HCl to
give the pyrrolidine hydrochloride (+)-18·HCl.42 In our hands, this
procedure gave unsatisfactory yields of N-Boc pyrrolidine (+)-17 after
purification, perhaps due to concurrent O-tert-butoxycarbonylation (O-
Bocylation). In one instance, when more equivalents of Boc2O were
employed N-Boc-Bis-O-Boc-protected pyrrolidine 16 was isolated in
44% yield. Its characteristic features after 1H NMR analysis were
singlets at 1.49 ppm (18H) and 1.45 ppm (9H) corresponding to the
three tert-butyl groups. The presence of three Boc groups was consistent
with its 13C NMR spectrum which showed three carbonyl signals at
153.9, 153.0 and 152.4 ppm. O-Bocylation, despite being rare com-
pared to the analogous reaction on amines, has been reported in the
literature for protection of alcohols using catalysts such as DMAP, zinc
acetate, and perovskites (NaLaTiO4).54–56
The next step involved borohydride reduction of pyrrolidinone 10,
using the conditions reported by Zhang et al., and gave a mixture of
both β-hydroxyester diastereoisomers, in a ~1:2 ratio in favour of the
desired trans form 13, which was obtained in 37% yield after chro-
matography.48 This is comparable to the literature yield on the identical
substrate at large scale (42%)49 but a lot lower than that reported for
the reduction of the analogous N-Boc-protected (instead of Bn) pyrro-
lidinone (99%),48 and could perhaps be improved in the future by use
of the milder reducing agent NaBH3CN.50,51
As both Boc-protected pyrrolidines could be successfully hydrolysed
under the same conditions, a one-pot procedure was applied in later
attempts to give (+)-18·HCl in a yield of up to 78%. The specific optical
rotation measured for (+)-18·HCl ([ ]2D1 + 16.0) was in broad agree-
ment with that found in the literature ([ ]2D3 + 19.0).40
Finally, to access the proposed methylaryl inhibitors 6a-e, reductive
amination was applied to (+)-18·HCl and five heterocyclic aryl car-
baldehydes. Initially, NaBH(OAc)3 was chosen as reducing agent due to
its lower toxicity but this reagent requires the use of aprotic solvents
(such as DCE and THF) in which the salt (+)-18·HCl was insoluble.57
The alternative, but more toxic, reducing agent NaBH3CN can be used
in MeOH which effectively dissolved (+)-18·HCl and allowed the re-
action to proceed.58 No optimisation was carried out, but each small
scale reaction provided sufficient quantity of the proposed inhibitors
6a-e for biochemical testing against AAG.
Since fair amounts of the undesired cis diastereoisomer 12 were
accumulated during this work, its epimerisation into the desired trans
pyrrolidine 13 was studied on small scale (Table 1). Galeazzi et al.
achieved quantitative epimerisation of an analogous compound using
DBU in toluene at 70 °C for 12 h but this was ineffective on pyrrolidine
12 (entry 1) and gave some eliminated alkene 19 along with eliminated
and oxidised product pyrrole 20.53 The latter conversion 19 → 20 has
been previously described in dioxane at 90 °C.52 The alternative,
t
weaker base Et3N gave no reaction (entry 2) and use of BuOK gave
more of, or mostly, the unsaturated products in toluene or THF (entries
3–5). A balance was found using EtONa in protic solvent EtOH which
gave the desired diastereoisomer 13 as the largest component of the
mixture (entry 6). Unfortunately, a single attempt at scale-up of this
reaction gave none of the desired product, only an intractable mixture
of highly polar compounds presumed to include ester hydrolysis pro-
ducts.
4. Biochemical activity against AAG
The biochemical assay used to test inhibitor activity was based on
the colorimetric microplate assay, previously reported by two of the
authors, for determining base excision DNA repair enzyme activity.59
Briefly, it involves surface-bound DNA containing a substrate residue
for AAG (hypoxanthine) and terminal fluorescein. AAG’s action leaves
AP sites that render the DNA backbone prone to hydrolysis in the
subsequent alkaline hydrolysis step, thus releasing the fluorescein-
conjugated part of the DNA. The remaining ‘plate-bound’ fluorescein is
detected using an anti-fluorescein antibody conjugated to horseradish
peroxidase (HRP), incubation with a colour-changing substrate of HRP
and colorimetric quantification. The assay was shown to be effective by
testing published inhibitors εC-oligomer (lit. IC50 39 nM, Kd 21 nM)12
Enzymatic resolution of trans-pyrrolidine 13 was achieved ac-
cording to Clinch et al. by enantioselective acylation catalysed by lipase
B of Candida antarctica in tert-butyl methyl ether at 40 °C.42 After
chromatographic separation, the desired hydroxypyrrolidine (+)-13
was obtained in 83% yield with an [ ]2D2 of +19.5, comparable to the
literature value of +16.9. The acetylated form of the undesired en-
antiomer (−)-14 was isolated in 75% yield with [ ]2D2 − 40.7, consistent
with the literature value of −41.8.
LiAlH4 was used for the reduction of the ester moiety of (+)-13 to
Table 1
Epimerisation of cis-pyrrolidine 12 into trans-pyrrolidine 13.
Entry
Base
Solvent
Temp/°C
Time /h
Product ratio by NMRa
12
13
19
20
1
2
3
4
5
6
DBU
Toluene
Toluene
Toluene
THF
70
70
50
50
RT
RT
18
17
3
78
12
0
4
6
Et3N
100
51
0
0
tBuOK
tBuOK
tBuOK
EtONa
24
trace
0
17
62
75
23
8
2
trace
0
38
25
11
THF
17
18
EtOH
20
46
a
NMR peak integrals used to determine ratios: 12 (2.89 (1H, dd, J 10.0, 5.0 Hz, H-5a)); 13 (2.54 (1H, dd, J 9.5, 7.5 Hz, H-2b)); 19 (6.75 (1H, t, J H-4)); 20 (6.61,
6.62 (2H, 2 s, pyr-H) or 5.06 (1H, s, PhCH2-)).52 For example and reference spectra see ESI.
4

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
6. Experimental
6.1. General
Where used, solvents specified as “dry” were made so by being
passed through activated alumina columns, using a Pure Solv™ Micro
Solvent Purification System, and were stored over activated molecular
sieves (3 Å, 8 to 12 mesh).62 Flash column silica chromatography was
carried out using silica gel 40-63u 60 Å. Analytical thin layer chro-
matography (TLC) was performed using pre-coated aluminium-backed
plates (silica gel 60 F254) and visualised by UV radiation at 254 nm,
and/or by staining with basic potassium permanganate solution (K2CO3
(13.3 g), KMnO4 (2 g), water (200 mL), NaOH solution (10% w/v,
1.7 mL)). HPLC purity was assessed (compounds 1–4 only) on a Varian
920-LC Liquid Chromatography system and the conditions were as
follows: inj. vol. 20 μL, column Varian Pursuit XRs 5μ C18
Fig. 2. IC50 curve of aza-nucleoside 6b against AAG (0.05 U/μl). (U stands for
enzyme unit, defined as the amount of enzyme which catalyses the conversion
of 1 µmol of substrate per minute.) Error bars show standard deviation of three
replicates. Plotted using GraphPad Prism 7.04; curve fitted using equation:
“[Inhibitor] vs. response”
(150 mm × 4.6 mm), column temp. 40 °C, flow rate: 1 mL min−1
,
mobile phase (compounds 1–3) Solvent A, water, Solvent B, MeOH,
mobile phase (compound 4) Solvent A, water (0.1% formic acid), Sol-
vent B, MeOH (0.1% formic acid), gradient 0–10 min, 10–90% B;
10–15 min, 90% B; 15–17 min, 90–10% B; 17–20 min, 10% B, detec-
tion: UV 254 nm. Infra-red (IR) spectra were recorded in the range
600–4000 cm−1 using an Agilent Clary 600 FTIR spectrometer with
MKII Golden Gate Single Reflection ATR System. NMR spectra were
obtained on Bruker 500 MHz, 400 MHz or 300 MHz spectrometers. 1H
NMR spectra were referenced either to TMS at 0 ppm or to residual
(partially) protic solvent: 3.31 ppm for CHD2OD, 4.79 ppm for DOH or
7.26 ppm for CHCl3. The data is given as follows: chemical shift (δ) in
ppm, integration, multiplicity, coupling constants J (Hz), assignment
(where given, for non-trivial compounds, assignment was made using
DEPT, COSY, HSQC, HMBC and/or NOESY – see ESI – or by comparison
with literature in which assignments were made). 13C NMR spectra
were recorded at 126 MHz, 101 MHz or 75 MHz. They were referenced
to CDCl3 at 77.0 ppm or CD3OD at 49.0 ppm. 31P NMR were recorded at
202 MHz. The data is given as follows: chemical shift (δ) in ppm, as-
signment. All chemical shifts are expressed as parts per million relative
to tetramethylsilane (δH = 0.00 ppm) and coupling constants are given
in Hertz to the nearest 0.5 Hz. GC–MS spectra were recorded on an
Agilent Technologies 7890A GC system connected to an Agilent Tech-
nologies 5975C inert XL EI/CI mass selective detector (MSD) operating
in electron impact (EI) mode and the conditions were as follows: inj.
vol. 1 μL, inj. temp. 250 °C, column Agilent HP-5MS (30 m × 0.25 mm),
carrier gas (He) flow 1 mL min−1, oven temperature gradient 0–3 min,
50 °C; 3–23 min, 50–250 °C (10 °C ramp per minute), 23–25 min,
250 °C. HRMS spectra were recorded using either a Waters QTOF Pre-
mier using electrospray ionisation (compounds 1–4) or an Agilent 1260
Infinity II coupled to an Agilent 6550 iFunnel QTOF mass spectrometer
using electrospray ionisation (remaining compounds). LC conditions for
the latter were as follows: inj. vol. 1.00 µL, column Agilent Extend-C18,
column temp. 30 °C, flow rate 1.0 mL min−1, mobile phase Solvent A:
water (0.1% formic acid), Solvent B: MeCN (0.1% formic acid), gradient
0–3 min, 5–100% B; 3–3.5 min, 100% B; 3.5–4 min, 5% B. Specific
optical rotation was recorded using a Jasco P-2000 Polarimeter; con-
centration of the sample (c) is given in g/100 mL.
and morin (lit. IC50 2.6 μM)7 which gave a comparable IC50 value of
21 nM for εC-oligomer and a somewhat higher IC50 of 89 μM for morin.
The latter perhaps represents differences in the availability of morin
under our conditions, particularly in terms of the buffer components
used.
All five azanucleosides 6a-6e were tested in this assay, which re-
vealed that only 6b exhibited detectable inhibitory activity (Fig. 2).
Taking the mid-point between the top and bottom points of the curve,
the IC50 of 6b against AAG is 157 μM. While this is weak activity
against AAG, 6b has a molecular weight of only 197 g mol−1 and is
comprised of only 14 ‘heavy’ (non-hydrogen) atoms, making it akin to a
drug discovery fragment and,60 leaving plenty of scope for increasing
potency by the addition of further binding groups. In other words, 6b
shows a respectable, approximate (substituting IC50 for Kd) ligand ef-
ficiency of 0.37 kcal mol−1 per heavy atom.61
5. Summary and conclusions
We have shown that the small molecule aza-nucleoside mimic 6b
possesses weak inhibitory activity against the DNA base excision repair
enzyme AAG. Owing to its low molecular weight, 6b is a ligand-effi-
cient starting point for future optimisation into a more potent, selective
and membrane-permeable AAG inhibitor. This is in contrast to nu-
cleoside ethenodeoxycytidine 1 (and 5′-phosphates thereof), which we
have shown to exhibit no inhibition outside of a DNA oligomer. IC50 of
these small molecules was determined using our microplate-based
colorimetric assay as opposed to the more involved but established
radiolabel, gel-based assay.
In order to synthesise 6b and its aryl analogues, we made a detailed
study of one of the routes, largely reported by Clinch et al., and dis-
covered several nuances of the chemistry including: 1. improved access
to pyrrolidinone 10 using a TiCl4-mediated cyclisation which negated the
need for column chromatography which otherwise led to oxidation to the
pyrrole; 2. Isolation of the cis hydroxy-ester 12, as well as the desired
trans 13, from reduction of the pyrrolidinone 10, and the possibility (on
small scale) of its conversion to the trans using sodium ethoxide; 3.
Concurrent O-bocylation (along with desired N-bocylation) of pyrroli-
dine-diol 15 during hydrogenative removal of the N-benzyl group in the
presence of excess Boc2O; and, 4. The requirement to use NaBH3CN,
rather than NaBH(OAc)3, for reductive amination of pyrrolidinium salt
18.HCl due to its compatibility with MeOH, the solvent required for
dissolution of 18.HCl. In addition, during synthesis of ethenocytidine
phosphate, it was found that a tribenzylphosphite-iodine coupling of
ethenocytidine, previously never applied to nucleosides, followed by
hydrogenation, gave selectively the desired 5′-phosphate.
6.2. 3,N4-Etheno-2′-deoxycytidine (5,6-Dihydro-5-oxo-6-(β-D-2′-
deoxyribofuranosyl)imidazo[1,2-c]pyrimidine) (1)
A solution of 2′-deoxycytidine (0.10 g, 0.44 mmol) was dissolved in
50% w/w aqueous chloroacetaldehyde (1.1 mL, 8.8 mmol). The solu-
tion was adjusted to pH 3.5 by adding 1 M KOH aqueous solution and
stirred at room temperature for 7 days during which time the solution
was kept at pH 3.5. The reaction mixture was evaporated and purified
by flash column chromatography (MeOH in DCM 5–15%). After eva-
poration of the fractions, a white solid form was obtained which was
dissolved in a small amount of ethanol and precipitated by slowly
5

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
adding petroleum ether under ultrasound. The precipitate was filtered
and dried at 100 °C under vacuum for 5 h to give the title compound as
a white powder (76 mg, 69%); m.p. 149–151 °C (lit. 137–138 °C); purity
99.6% by HPLC; IR νmax (cm−1) 3456 (OeH), 3112 (Ar-H), 1657 (C]
O); 1H NMR (500 MHz, DMSO‑d6) δ 7.80 (1H, s, H-2), 7.74 (1H, d, J
8.0 Hz, H-7), 7.40 (1H, s, H-3), 6.73 (1H, d, J 8.0 Hz, H-8), 6.42 (1H, t, J
7.0 Hz, H-1′), 5.32 (1H, m, 3′-OH), 5.09 (1H, m, 5′-OH), 4.30 (1H, m, H-
4′), 3.86 (1H, m, H-3′), 3.61 (2H, m, H-5′), 2.23 (2H, m, H-2′); 13C NMR
(126 MHz, DMSO‑d6) δ 145.9, 144.1, 133.0, 128.4, 113.2, 99.0, 88.2),
85.7, 70.8, 61.7, 40.6; HRMS (ESI) m/z calcd for C11H13N3O4Na
[M + Na]+ 274.0803, found 274.0794; [ ]2D0 = +42.8 (c 2.0, MeOH);
NMR data agrees with that given in the literature.30,63
concentrated and purified by reverse-phase column chromatography
(Biotage® SNAP KP-C18-HS cartridge, MeOH/H2O 10–80%). After
evaporation of methanol under reduced pressure, the aqueous solution
was freeze-dried to give the title compound as a white powder (18 mg,
90%); purity 98.6% by HPLC; mp 142–144 °C (dec.); IR νmax (cm−1
)
3104 (Ar-H), 2942 , 1720 (C]O), 1630 (C]N), 930, 738; 1H NMR
(500 MHz, D2O) δ 8.32 (1H, d, J 8.0 Hz), 8.04 (1H, d, J 2.1 Hz), 7.75
(1H, d, J 2.1 Hz), 7.07 (1H, d, J 8.0 Hz), 6.51 (1H, t, J 6.5), 4.61 (1H,
m), 4.31 (1H, m), 4.14 (2H, m), 2.61 (1H, m), 2.44 (1H, m); 13C NMR
(126 MHz, D2O) δ 144.9, 144.1, 137.0, 122.2, 114.5, 93.6, 88.4, 87.2
3
2
(d, JP-C 8.6), 71.3, 65.0 (d, JP-C 4.8 Hz), 40.6; 31P NMR (202 MHz,
D2O) δ −0.01; HRMS (ESI) m/z: [M]− 330.0502, calc. 330.0491;
[ ]2D0 = +66.3 (c = 0.8, H2O).
6.3. 3,N4-Etheno-2′-deoxycytidine-5′-(dibenzyl)phosphate (6-(5′-O-
(dibenzyl)phosphate- β-D-2′-deoxyribofuranosyl)-5,6-dihydroimidazo[1,2-
c]pyrimidin-5-one) (2)
6.6. 3-[N-(Carboxymethyl)amino]propanoic acid bis(ethyl ester) (8)
To a solution of NaOH (2.00 g, 50 mmol) in water (21 mL) was
added glycine ethyl ester hydrochloride 7 (6.73 g, 50 mmol). The
mixture was cooled to 0 °C under N2 and ethyl acrylate (5.3 mL,
50 mmol) was added dropwise. The mixture was allowed to warm to RT
and stirred vigorously for 15 h.43 The reaction mixture was extracted
with DCM (3 × 20 mL). The combined organic layers were washed with
brine (2 × 20 mL), dried over anhydrous MgSO4, filtered, concentrated
and dried in vacuo. Flash column silica chromatography (DCM/EtOAc
[1:2]) gave the title compound as a colourless oil (5.29 g, 54%); Rf
(DCM/EtOAc [1:2]) 0.20; IR ν (cm−1) 3338 (NeH), 2982, 1730 (C]O),
1617, 1439, 1372, 1254, 1178 (CeO), 1096, 1026; NMR δH (300 MHz;
CDCl3) 4.19 (2H, q, J 7.0 Hz, CH2CH3), 4.15 (2H, q, J 7.0 Hz, CH2CH3),
3.41 (2H, s, NHCH2CO), 2.90 (2H, t, J 6.5 Hz, H-3), 2.51 (2H, t, J
6.5 Hz, H-2), 1.82 (1H, bs, H-1), 1.28 (3H, t, J 7.0 Hz, CH2CH3), 1.26
(3H, t, J 7.0 Hz, CH2CH3); δC (75 MHz; CDCl3) 172.4 (CO), 172.2 (CO),
60.8 (CH2CH3), 60.5 (CH2CH3), 50.9 (NHCH2CO), 44.8 (C-3), 34.9 (C-
2), 14.2 (CH2CH3), 14.2 (CH2CH3); GC–MS tR = 14.0 min, m/z (EI) 203
(M+, 11%), 157 (12%), 131 (19%), 130 (100%), 116 (100%), 84
(100%), 57 (10%), 56 (16%); 1H NMR and IR data agree with those
published in the literature.65
Iodine (0.15 g, 0.60 mmol) was added to a solution of tribenzyl-
phosphite (0.26 g, 0.72 mmol, prepared according to Wong et al. 64) in
dry DCM (3.0 mL) with stirring at −20 °C. A colourless solution was
obtained after iodine had dissolved and was slowly added to a solution
of ethenocytidine (0.15 g, 0.60 mmol) in dry pyridine (3.0 mL) with
stirring at −20 °C to give a pale brown solution. The reaction mixture
was stirred for 2 h at RT, then quenched by adding one drop of water.
After evaporation of solvent, a yellowish oil was obtained which was
subjected to flash column chromatography (MeOH in DCM 2–35%) to
give the title compound as a colourless oil (87 mg, 28%); Rf (DCM/
MeOH 10:1) 0.48; purity 96.6% by HPLC; IR νmax (cm−1) 1701 (C]O),
1627 (C]N), 1268 (CeO), 993; 1H NMR (500 MHz, CDCl3) δ 7.68 (1H,
s), 7.35–7.16 (12H, m), 6.49 (1H, t, J 6.6 Hz), 6.43 (1H, d, J 7.8 Hz),
5.02 (4H, m), 4.41 (1H, m), 4.20 (2 H, m), 4.07 (1H, m), 2.44 (1H, m),
2.10 (1H, m); 13C NMR (126 MHz, CDCl3) δ 145.7, 144.9, 135.3 (d, 3JP-
6.5 Hz), 132.6, 128.9, 128.8, 128.7, 128.7, 128.2, 128.1, 126.7,
C
3
2
112.8, 99.4, 85.7, 84.9 (d, JP-C 7.6), 70.6, 69.9 (d, JP-C 4.9), 69.8 (d,
2JP-C 4.9), 66.7 (d, JP–C 5.3,), 40.5; 31P NMR (202 MHz, CDCl3) δ
2
−0.52 (m); HRMS (ESI) m/z calcd for
C
25H27N3O7P [M+H]+
512.1586, found 512.1562; [ ]2D0 = +31.1° (c 0.8, MeOH).
6.7. 3-(benzyl-ethoxy-carbonylmethyl-amino)-propionic acid ethyl ester
(9)
6.4. (6-(5′-O-(monobenzyl)phosphate-β-D-2′-deoxyribofuranosyl)-5,6-
dihydroimidazo[1,2-c]pyrimidin-5-one) (3)
A mixture of 8 (4.767 g, 23.47 mmol) and NaHCO3 (2.225 g,
26.52 mmol) were dissolved in dry acetonitrile (26 mL). The system
was flushed with N2 and benzyl bromide (2.8 mL, 23.47 mmol) was
added dropwise while stirring. The reaction mixture was stirred under
N2 for 16 h.43 The reaction was quenched using water (40 mL) and
extracted with DCM (3 × 40 mL). The organic layer was washed with
water (100 mL), brine (2 × 100 mL), dried over anhydrous MgSO4,
filtered, concentrated and dried in vacuo. Flash column silica chroma-
tography of the resulting crude mixture (Pet. ether/EtOAc [9:1]) gave
the title compound as a clear oil (6.56 g, 95%); Rf (Pet. ether/EtOAc
[9:1]) 0.33; IR ν (cm−1) 2981, 2852, 1730 (C]O), 1453, 1370, 1247,
1181 (CeO), 1144, 1027; NMR δH (300 MHz; CDCl3) 7.34–7.21 (5H, m,
Ph), 4.15 (2H, q, J 7.0 Hz, CH2CH3), 4.13 (2H, q, J 7.0 Hz, CH2CH3),
3.82 (2H, s, PhCH2), 3.33 (2H, s, NCH2CO), 3.05 (2H, t, J 7.0 Hz, H-3),
2.49 (2H, t, J 7.0 Hz, H-2), 1.26 (3H, t, J 7.0 Hz, CH2CH3), 1.24 (3H, t, J
7.0 Hz, CH2CH3); δC (126 MHz, CDCl3) 172.5 (CO), 171.3 (CO), 138.8
(Ph), 128.8 (Ph), 128.3 (Ph), 127.2 (Ph), 60.4 (CH2CH3), 60.3
(CH2CH3), 57.8 (PhCH2), 53.9 (NCH2CO), 49.7 (C-3), 33.6 (C-2), 14.3
Further elution of the column described gave monobenzyl phos-
phate 3 which was further purified by reverse-phase HPLC (MeOH in
0.1% aqueous HCO2H 20–80%) to give a white solid (31 mg, 12%);
m.p. 122–124 °C; Rf (DCM/MeOH 10:1) 0.08; purity 99.8% by HPLC; IR
νmax (cm−1) 1721 (C]O), 1630 (C]N), 997; 1H NMR (500 MHz, D2O)
δ 8.15 (1H, d, J 7.9 Hz), 7.98 (1H, d, J 2.0 Hz), 7.72 (1H, d, J 2.0 Hz),
7.35–7.26 (10H, m), 6.89 (1H, d, J 7.9 Hz), 6.42 (1H, t, J 6.5 Hz), 4.90
(2H, m), 4.41 (1H, m), 4.23 (1H, m), 4.11 (1H, m), 4.02 (1H, m) 2.55
(1H, m), 2.30 (1H, m); 13C NMR (126 MHz, D2O) δ 144.6, 143.9, 137.8
3
(d, JP-C 6.7 Hz), 136.8, 129.1, 128.6, 128.0, 122.3, 114.5, 93.4, 88.5,
87.1 (d 3JP-C 9.0 Hz), 71.3, 68.2 (d, 2JP-C 5.5 Hz), 65.4 (d, 2JP-C 5.1 Hz),
40.5; 31P NMR [202 MHz, D2O] δ 0.27 (m); HRMS (ESI) m/z calcd for
C
18H19N3O7P [M−H]− 420.0961, found 420.0956; [ ]2D0 = +46.9 (c
1.7, H2O).
Further elution of the silica flash column gave recovered starting
material 1 (76 mg, 51%).
(CH2CH3), 14.2 (CH2CH3); GC–MS tR = 20.4 min. m/z (EI) 293 (M+
,
6.5. 3,N4-Etheno-2′-deoxycytidine-5′-phosphate ((6-(5′-O-phosphate-β-D-
2′-deoxyribofuranosyl)-5,6-dihydroimidazo[1,2-c]pyrimidin-5-one) (4)
1%), 221 (16%), 220 ([M−COOEt]+,100%), 206 (35%), 202 (10%), 91
(PhCH2+, 100%). Literature IR data (from the year 1965) does not
match.49
Pd/C (18 mg 10% (w/w) containing 50% H2O) was added to a so-
lution of dibenzylphosphate 2 (30 mg, 0.05 mmol) in methanol (5 mL).
The atmosphere was replaced with H2 (balloon) and the reaction mix-
ture was stirred 1 h. The mixture was filtered through Celite® and
washed with methanol (15 mL). The colourless filtrate was
6.8. Ethyl 1-benzyl-4-oxopyrrolidine-3-carboxylate (10)
To a solution of 1 M TiCl4 in dry DCM (3.43 mL) at −10 °C was
6

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
added diester 9 (1.00 g, 3.41 mmol) in dry DCM (20 mL) and the re-
sulting mixture was stirred at −10 °C for 0.5 h. Et3N (1.1 mL,
7.51 mmol) was added dropwise slowly and the mixture was stirred for
2.5 h.47 The reaction mixture was poured into saturated NaCl solution
(25 mL) and the pH was made basic (~8) with Et3N while stirring. The
precipitated salts were filtered through Celite® and washed with DCM.
After extraction with DCM (3 × 25 mL), the combined organic layers
were washed using saturated NaHCO3 solution (5 × 75 mL), dried over
anhydrous MgSO4, filtered, concentrated and dried in vacuo. The crude
material, a yellow oil, was used directly in the following step (0.77 g,
92%); Rf (Pet. ether/EtOAc [9:1]) 0.2; IR ν (cm−1) 2981, 2806, 1767
(C]O), 1725 (C]O), 1028 (CeO); NMR δH (500 MHz; CDCl3):
7.39–7.23 (5H, m, Ph), 4.24 (2H, qd, J 7.0, 1.0 Hz, OCH2CH3), 3.77
(1H, d, J 13.0 Hz, PhCHH-), 3.72 (1H, d, J 13.0 Hz, PhCHH-), 3.46 (1H,
t, J 8.5 Hz, H-2a), 3.35 (1H, t, J 9.5 Hz, H-3), 3.27 (1H, d J 17.0 Hz, H-
5a), 3.09 (1H, t, J 9.0 Hz, H-2b), 2.91 (1H, d, J 17.0 Hz, H-5b), 1.29
(3H, t, J 7.0 Hz, OCH2CH3); δC (101 MHz, CDCl3): 206.5 (CO), 167.4
(COOEt), 137.1 (Ph), 128.7 (Ph), 128.5 (Ph), 127.6 (Ph), 61.7
(OCH2CH3), 61.1 (C-5), 60.2 (PhCH2), 54.7 (C-3), 54.2 (C-2), 14.2
(CH3CH2). GC–MS tR = 18.8 min. m/z (EI) 247 (M+, 2%), 174.1
([M−COOEt]+, 65%), 117 (4%), 91 (100%), 65 (7%). No literature
data for this (ethyl diester) compound is available.42
6.11. Ethyl rel-(3R,4S)-1-benzyl-4-hydroxypyrrolidine-3-carboxylate
(( )-13)
Further elution, of the column described above, with Pet. Ether/
EtOAc [1:1] 1% Et3N gave the trans product ( )-13 as a yellow oil
(0.91 g, 37%); Rf (Pet. Ether/EtOAc [1:1], 1% Et3N) 0.10; IR ν (cm−1
)
3385 (OeH), 2979, 2932, 2799, 1727 (C]O), 1454, 1372, 1255, 1178
(CeO), 1027; NMR δH (400 MHz; CDCl3) 7.33–7.22 (5H, m, Ph), 4.51
(1H, dt, J 5.5, 3.0 Hz, H-4), 4.16 (2H, q, J 7.0 Hz, CH2CH3), 3.63 (2H, s,
PhCH2-), 3.12 (1H, t, J 9.5 Hz, H-2a), 2.95 (1H, dt, J 8.0, 3.5 Hz, H-3),
2.76 (1H, dd, J 10.0, 3.0 Hz, H-5a), 2.64 (1H, dd, J 10.0, 5.5 Hz, H-5b),
2.54 (1H, dd, J 9.5, 7.5 Hz, H-2b), 2.32 (1H, bs, OH, exchanged with
D2O), 1.26 (3H, t, J 7.0 Hz, CH3CH2); δC (101 MHz, CDCl3) 173.2 (CO),
138.3 (Ph), 128.7 (Ph), 128.3 (Ph), 127.1 (Ph), 74.3 (C-4), 61.8 (C-5),
60.9 (CH2CH3), 59.6 (benzyl CH2), 55.2 (C-2), 53.1 (C-3), 14.2
(CH3CH2); GC–MS tR = 20.1 min. m/z (EI) 249 (M+, 12%), 220 (10%),
204 (20%), 158 (79%), 133 (20%), 132 (26%), 91 (PhCH2+, 100%), 65
(14%); NMR data agrees with that reported in the literature.42
6.12. Epimerisation study 12 → 13 (Table 1, entry 6)
A 0.1 M solution of EtONa was prepared by the addition of sodium
(9 mg, 0.4 mmol) to anhydrous ethanol (4 mL) under nitrogen. After
complete consumption of the sodium, part of the resulting solution
(0.8 mL, 80 μmol) was transferred to a flask containing cis-pyrrolidine
12 (20 mg, 80 μmol) and the resulting solution was stirred at RT for
18 h. DCM (2 mL) was added to produce a precipitate which was re-
moved by filtration through Celite®. The filtrate was evaporated to
dryness and analysed by 1H NMR.
6.9. 1-benzyl-4-hydroxy-1H-pyrrole-3-carboxylate (11)
Flash column silica chromatography of a sample of ketone 10 in air
(Pet. Ether/AcOEt [9:1]) and secondly (DCM/Pet. Ether [3:1]) gave the
title compound as a colourless oil; Rf 0.45; NMR δH (400 MHz; CDCl3)
7.32–7.23 (3H, m, Ph), 7.03 (2H, d, J 7.5 Hz, Ph), 6.66 (1H, d, J 3.0 Hz,
5-H), 5.83 (1H, d, J 3.0 Hz, 2-H), 5.30 (2H, s, PhCH2), 4.25 (2H, q, J
7.0 Hz, CH3CH2), 1.55 (1H, bs, OH), 1.22 (3H, t, J 7.0 Hz, CH3CH2);
GC–MS tR = 20.2 min m/z (EI) 245 (M+, 49%), 200 (11%), 199 (35%),
198 (28%), 91 (PhCH2+, 100%); 1H NMR data is comparable to the
limited peaks reported in the literature.66
6.13. Ethyl (3R,4S)-4-(acetyloxy)-1-benzylpyrrolidine-3-carboxylate
((−)-14)
Vinyl acetate (2.18 mL) and lipase from Candida antarctica (1.37 g,
Sigma Aldrich, batch SLBG4222V) were added to a solution of ( )-8
(1.96 g, 7.88 mmol) in tert-butyl methyl ether (62 mL). The mixture was
stirred at 40 °C for 2.5 h and filtered through Celite®. The solids were
washed with EtOAc and the combined filtrates were washed using sa-
turated NaHCO3 solution, dried over anhydrous MgSO4, filtered, con-
centrated and dried in vacuo to afford a yellow oil. Flash column silica
chromatography of the resulting mixture (Pet. Ether/EtOAc [3:2]) gave
first acetate (−)-14 as a yellow oil (0.85 g, 75%); Rf (Pet. Ether/EtOAc
[3:2]) 0.73; IR ν (cm−1) 2979, 2799, 1732 (C]O), 1454, 1370, 1234
(CeO), 1194, 1174 (CeO), 1029; NMR δH (400 MHz; CDCl3) 7.33–7.25
(5H, m, Ph), 5.40 (1H, dt, J 6.5, 3.0 Hz, H-4), 4.17 (2H, q, J 7.0 Hz,
CH2CH3), 3.65 (1H, d, J 13.0 Hz, PhCHH-), 3.59 (1H, d, J 13.0 Hz,
PhCHH-), 3.16 (1H, t, J 8.5 Hz, H-2a), 3.06 (1H, td, J 8.0, 3.5 Hz, H-3),
2.82 (1H, dd, J 11.0, 2.5 Hz, H-5a), 2.76 (1H, dd, J 11.0, 6.0 Hz, H-5b),
2.49 (1H, dd, J 9.0, 8.5 Hz, H-2b), 2.05 (3H, s, COCH3), 1.26 (3H, t, J
7.0 Hz, CH3CH2); δC (126 MHz, CDCl3) 172.4 (CO), 170.7 (CO), 138.0
(Ph), 128.8 (Ph), 128.3 (Ph), 127.2 (Ph), 76.1 (C-4), 61.1 (CH2CH3),
59.7 (C-5), 59.6 (PhCH2-), 56.1 (C-2), 50.2 (C-3), 21.1 (COCH3), 14.2
(CH2CH3); GC–MS tR = 21.7 min. m/z (EI) 291 (M+, 0.5%), 231 (18%),
6.10. Ethyl rel-(3R,4R)-1-benzyl-4-hydroxypyrrolidine-3-carboxylate
(( )-12)
To a solution of oxopyrrolidine 7 (2.480 g, 10.03 mmol) in dry
degassed MeOH (20 mL) at 0 °C was added slowly NaBH4 (0.190 g,
5.01 mmol).48 The mixture was stirred for 2 h at 0 °C. After this time,
GC–MS analysis revealed that starting oxopyrrolidine was still present,
so 0.5 extra eq. of NaBH4 (0.190 g, 5.01 mmol) were added at 0 °C and
the mixture was allowed to warm to RT and stirred overnight. After
removal of solvents in vacuo, the residue was dissolved in water and
extracted with DCM/MeOH 9:1 (6 × 20 mL). The combined organic
layers were washed with brine (2 × 60 mL), dried over anhydrous
MgSO4, filtered, concentrated and dried in vacuo. Flash column silica
chromatography (Pet. Ether/EtOAc [4:1], 1% Et3N to Pet. Ether/EtOAc
[1:1], 1% Et3N) gave first the cis isomer ( )-12 as a yellow oil (0.57 g,
23%); Rf (Pet. Ether/EtOAc [1:1], 1% Et3N) 0.39; IR ν (cm−1) 3422
(OeH), 2805, 1727 (C]O), 1373, 1181 (CeO); NMR δH (400 MHz;
CDCl3) 7.38–7.22 (5H, m, Ph), 4.53–4.49 (1H, m, H-4), 4.20 (2H, q, J
7.0 Hz, CH2CH3), 3.72 (1H, d, J 13.0 Hz, PhCHH-), 3.67 (1H, d, J
13.0 Hz, PhCHH-), 3.19–3.10 (1H, m, H-3), 3.09–3.01 (1H, m, H-2a),
2.89 (1H, dd, J 10.0, 5.0 Hz, H-5a), 2.75 (1H, t, J 9.0 Hz, H-2b), 2.64
(1H, dd, J 10.0, 3.0 Hz, H-5b), 1.28 (3H, t, J 7.0 Hz, CH3CH2); δC
(101 MHz, CDCl3) 172.1 (CO), 138.5 (Ph), 128.7 (Ph), 128.3 (Ph),
127.1 (Ph), 71.7 (C-4), 61.7 (C-5), 60.9 (CH2CH3), 59.8 (PhCH2), 53.5
159 (13%), 158 (98%), 91 (100%); [ ]2D0 − 40.7 (c 1.40 CHCl3) (lit.42
,
[ ]2D0 − 41.8 (c 0.895, CHCl3)); HRMS (ESI) m/z calcd for C16H22NO4 [M
+H]+ 292.1543, found 292.1526. NMR and [ ]2D0 agrees with that
published in the literature.42
6.14. Ethyl (3S,4R)-1-benzyl-4-hydroxypyrrolidine-3-carboxylate
((+)-13)
(C-2), 48.8 (C-3), 14.2 (CH3CH2); GC–MS tR = 20.8 min. m/z (EI) 249
Further elution, of the column described above, with Pet. Ether/
EtOAc [1:4] gave hydroxypyrrolidine (+)-13 as a colourless gum,
which crystallised at −20 °C (0.82 g, 83%); m.p. 48–50 °C (lit.42
51–52 °C); Rf (Pet. Ether/EtOAc [3:2]) 0.20; IR ν (cm−1) 3414 (OeH),
2974, 2799, 1727 (C]O), 1454, 1372, 1242, 1178 (CeO), 1029; NMR
(M+, 4%), 204 (10%), 158 (50%), 133 (12%), 132 (17%), 91 (PhCH2
,
+
100%), 65 (10%); HRMS (ESI) m/z calcd for C14H20NO3 [M+H]+
250.1438, found 250.1427.
7

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
δH (400 MHz; CDCl3) 7.33–7.27 (5H, m, Ph), 4.51 (1H, dt, J 5.5, 3.0 Hz,
H-4), 4.16 (2H, q, J 7.0 Hz, CH3CH2), 3.68 (1H, d, J 13.0 Hz, PhCHH-),
3.64 (1H, d, J 13.0 Hz, PhCHH-), 3.16 (1H, t, J 9.0 Hz, H-2a), 2.97 (1H,
td, J 8.0, 3.0 Hz, H-3), 2.80 (1H, dd, J 10.0, 2.5 Hz, H-5a), 2.66 (1H, dd,
J 10.0, 5.5 Hz, H-5b), 2.57 (1H, dd, J 9.5, 7.5 Hz, H-2b), 2.07 (1H, bs,
OH, exchanged with D2O), 1.26 (3H, t, J 7.0 Hz, CH3CH2); δC
(126 MHz, CDCl3) 173.2 (C]O), 138.3 (Ph), 128.7 (Ph), 128.3 (Ph),
127.1 (Ph), 74.3 (C-4), 61.8 (PhCH2-), 60.9 (CH2CH3), 59.6 (C-5), 55.2
(C-3), 53.1 (C-2), 14.2 (CH2CH3); GC–MS tR = 20.8 min. m/z (EI) 249
(M+, 2%), 229 (11%), 184 (12%), 158 ([M−PhCH2]+, 25%), 91
(PhCH2+, 100%), 65 (11%); [ ]D20 + 19.5 (c 0.91 CHCl3) (lit.42
[ ]2D0 + 16.9 (c 0.71, CHCl3)); NMR and [ ]2D0 data agrees with that re-
ported in the literature.42
(+)-15 (0.050 g, 0.24 mmol) and di-tert-butyl dicarbonate (0.06 mL,
0.24 mmol) in MeOH (1 mL).42 The atmosphere was replaced with H2
(balloon) and the reaction mixture was stirred for 24 h. The mixture
was filtered through Celite®, the solvent was evaporated and the re-
sidue was subjected to flash column chromatography (AcOEt/MeOH
[35:1]) to afford N-Boc pyrrolidine (+)-17 as a colourless gum
(0.028 g, 54%); Rf (Pet. ether/EtOAc [2:1]) 0.24; NMR δH (500 MHz;
CDCl3) 4.29–4.22 (1H, m, H-3), 3.72–3.57 (4H, m, CH2O, H-2, H-5),
3.29–3.21 (1H, m, H-2), 3.12–3.09 (1H, m, H-5), 3.10 (0.5H, bs, OH,
exchanged to D2O), 2.94 (0.5H, bs, OH, exchanged to D2O), 2.69 (0.5H,
bs, OH, exchanged with D2O), 2.51 (0.5H, bs, OH, exchanged with
D2O), 2.35–2.28 (1H, m, H-4), 1.45 (9H, s, C(CH3)3); δC (126 MHz,
CDCl3) 154.7 (CO), 79.7 (C(CH3)3), (73.2, 72.3) (C-3), 63.0 (CH2O),
(52.7, 52.3) (C-2), (48.2, 47.6) (C-4), (46.6, 46.0) (C-5), 28.5 (C(CH3)3);
GC–MS tR = 19.2 min. m/z (EI) 217 (M+, 1%), 144 (19%), 112 (11%),
68 (15%), 57 (100%), 56 (46%), 55 (21%); [ ]2D0 + 23.2 (c 1.00 MeOH)
(lit.38 [ ]2D0 + 16 (c 0.8 MeOH)); NMR data agrees with that reported in
the literature.67
6.15. (3R,4R)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol ((+)-15)
Hydroxypyrrolidine (+)-13 (0.48 g, 1.91 mmol) was dissolved in
dry THF (15.6 mL) and cooled to 0 °C. LiAlH4 (0.29 g, 7.62 mmol) was
added slowly and the mixture was warmed to RT and stirred until LCMS
showed reaction completion (~2 h). The mixture was diluted with ether
and excess hydride was quenched at 0 °C by successive addition of
water (0.3 mL), aqueous NaOH solution (15%, 0.3 mL) and water
(0.9 mL). Then, the mixture was warmed to RT, stirred for 15 min, dried
over anhydrous MgSO4, filtered, concentrated and dried in vacuo. Flash
column silica chromatography (DCM/MeOH [9:1]) gave diol (+)-15 as
a colourless gum (0.28 g, 71%); Rf (DCM/MeOH [9:1]) 0.14; δH
(400 MHz; CD3OD) 7.36–7.27 (5H, m, Ph), 4.00 (1H, dt, J 6.0, 4.0 Hz,
H-3), 3.68 (1H, d, J 12.5 Hz, PhCHH-), 3.65 (1H, dd, J 10.5, 5.5 Hz,
–CHHOH), 3.58 (1H, d, J 12.5 Hz, PhCHH-), 3.51 (1H, dd, J 10.5,
7.5 Hz, –CHHOH), 2.92 (1H, dd, J 9.5, 8.0 Hz, H-5a), 2.75 (1H, dd, J
10.0, 6.0 Hz, H-2a), 2.59 (1H, dd, J 10.0, 4.0 Hz, H-2b), 2.37 (1H, dd, J
9.5, 6.5 Hz, H-5b), 2.24–2.16 (1H, m, H-4); δC (101 MHz, CDCl3) 137.8
(Ph), 128.9 (Ph), 128.4 (Ph), 127.3 (Ph), 74.2 (C-3), 64.6 (CH2O), 62.3
(C-2), 60.1 (PhCH2-), 55.9 (C-5), 50.0 (C-4); GC–MS tR = 20.1 min. m/z
(EI) 207 (M+, 3%), 133 (8%), 132 (11%), 91 (PhCH2+, 100%), 77
(Ph+, 5%), 65 (19%); [ ]2D0 + 31.1 (c 1.01 MeOH) (lit.42 [ ]2D0 + 33.0 (c
6.18. (3R,4R)-4-(Hydroxymethyl)pyrrolidin-3-ol hydrochloride
((+)-18·HCl)
6.18.1. Method 1
N-Boc pyrrolidine (+)-17 (0.026 g, 0.120 mmol) was dissolved in
MeOH (2 mL) and aqueous HCl (37%, 1 mL) was added at RT.42 The
mixture was stirred for 1 h and the solvent was evaporated to give the
title compound as a yellow oil (0.018 g, > 99%); NMR δH (400 MHz;
D2O) 4.44–4.35 (1H, m, H-3), 3.64–3.55 (3H, m, CH2O, H-2), 3.42 (1H,
dd, J 12.5, 4.5 Hz, H-5), 3.25 (1H, d, J 12.5 Hz, H-5), 3.15 (1H, dd, J
12.0, 5.5 Hz, H-2), 2.52–2.38 (1H, m, H-4); δC (101 MHz, D2O) 71.4 (C-
3), 60.4 (CH2O), 51.6 (C-5), 47.4 (C-4), 46.1 (C-2); [ ]2D0 + 16.0 (c 1.00
MeOH) (lit.40 [ ]2D0 + 19.0 (c 1.0, MeOH)); HRMS (ESI) m/z calcd for
C5H12NO2 [M+H]+ 118.0863, found 118.0861; all data agrees with
that reported in the literature.40,53
6.18.2. Method 2
0.75 MeOH)); HRMS (ESI) m/z calcd for
C
12H18NO2 [M+H]+
Pd/C (10% w/w, 0.091 g) was added to a stirred solution of diol
(+)-15 (0.45 g, 2.18 mmol) and di-tert-butyl dicarbonate (0.52 mL,
2.27 mmol) in MeOH (9.1 mL).42 The atmosphere was replaced with H2
(balloon) and the reaction mixture was stirred for 24 h. The mixture
was filtered through Celite® and the solvent was evaporated to give the
crude product (0.44 g) which was dissolved in MeOH (17.3 mL) and
aqueous HCl (37%, 8.7 mL) was added at RT. The mixture was stirred
for 30 min and the solvent was evaporated to give the title compound in
pure form as a yellow oil (0.24 g, 78%); NMR data was identical to that
from Method 1, above.
208.1332, found 208.1314; NMR data agrees with that reported in the
literature.42
6.16. tert-Butyl (3R,4R)-3-[(tert-butoxycarbonyl)oxy]-4-[[(tert-
butoxycarbonyl)oxy]methyl]pyrrolidine-1-carboxylate (16)
Pd/C (10% w/w, 0.057 g, 10%) was added to a stirred solution of
diol (+)-15 (0.28 g, 1.37 mmol) and di-tert-butyl dicarbonate (0.66 mL,
2.84 mmol) in MeOH (5.7 mL).42 The atmosphere was replaced with H2
(balloon) and the reaction mixture was stirred for 24 h. The mixture
was filtered through Celite®, the solvent was evaporated and the re-
sidue was subjected to flash column chromatography (EtOAc/MeOH
[2:1]) to afford firstly tris-Boc compound 16 as a colourless gum
(0.25 g, 44%); Rf (Pet. ether/EtOAc [2:1]) 0.72; IR ν (cm−1) 3321
(OeH), 2945, 2764, 1616 (C]O), 1454, 1400, 1065, 1047 (CeO);
NMR (400 MHz; CDCl3) 4.88 (1H, dt, J 5.5, 3.0 Hz, H-3), 3.93 (2H, dd, J
7.0, 2.5 Hz, CH2O), 3.61 (1H, dd, J 12.5, 5.5 Hz, H-2a), 3.52 (1H, dd, J
11.0, 7.5 Hz, H-5a), 3.42–3.31 (1H, m, H-2b), 3.25–3.16 (1H, m, H-5b),
2.60–2.57 (1H, m, H-4), 1.39 (18H, s, 2 × C(CH3)3), 1.36 (9H, s,
C(CH3)3); δC (101 MHz, CDCl3) 153.9 (CO), 153.0 (CO), 152.4 (CO),
82.5 (C(CH3)3), 82.2 (C(CH3)3), 79.4 (C(CH3)3), 75.9 (C-3), (65.4, 65.3)
(CH2O), (50.3, 50.1) (C-2), (46.4, 45.9) (C-5), (43.1, 42.2) (C-4), 28.2
(C(CH3)3), 27.5 (C(CH3)3), 27.5 (C(CH3)3); HRMS (ESI) Molecular ion
not detected.
6.19. General procedure for reductive amination to give test inhibitors 6a-6e
To a solution of pyrrolidine hydrochloride (+)-18.HCl (50 mg,
0.33 mmol) in anhydrous MeOH (2.2 mL) was added the appropriate
aryl aldehyde (1.07 equiv., 0.35 mmol) and the mixture stirred at RT
until complete dissolution. Then, NaBH3CN was added portionwise and
the mixture was left stirring under N2 for 18 h. General work-up in-
volved filtration of the mixture through Celite® and solvent evapora-
tion.
6.20. (3R,4R)-4-(Hydroxymethyl)-1-[(1H-imidazol-2-yl)methyl]
pyrrolidin-3-ol (+)-6a
The general procedure for reductive amination was followed. The
resulting crude product was adsorbed onto silica and flash column silica
chromatography was performed (DCM/MeOH [19:1] to [7:3]) to give
the title compound as a yellow oil (36 mg, 56%); Rf (DCM/MeOH [5:1])
0.15; NMR δH (400 MHz, MeOD) 7.02 (2H, s, Ar-H), 4.08–4.01 (1H, m,
H-3), 3.79 (1H, d, J 14.0 Hz, ArCHH-), 3.74 (1H, d, J 14.0 Hz, ArCHH-),
6.17. tert-Butyl (3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine-1-
carboxylate ((+)-17)
Pd/C (0.010 g, 10%) was added to a stirred solution of the diol
8

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
3.60 (1H, dd, J 11.0, 6.0 Hz, CHHO), 3.52 (1H, dd, J 11.0, 7.0 Hz,
CHHO), 3.01 (1H, t, J 9.0 Hz, H-5a), 2.77 (1H, dd, J 10.0, 6.0 Hz, H-2a),
2.68 (1H, dd, J 10.0, 3.5 Hz, H-2b), 2.38 (1H, dd, J 9.5, 7.0 Hz, H-5b),
2.26–2.17 (1H, m, H-4); δC 101 MHz, MeOD) 146.3 (Ar), 122.8 (Ar),
74.3 (C-3), 64.0 (CH2O), 62.9 (C-2), 57.1 (C-5), 52.8 (ArCH2-), 51.4 (C-
4); [ ]2D0 + 16.0 (c 4.85 MeOH); HRMS (ESI) m/z calcd for C9H16N3O2
[M+H]+ 198.1237, found 198.1235.
157.0 (Ar), 150.2 (Ar), 139.0 (Ar), 125.0 (Ar), 124.5 (Ar), 73.9 (C-3),
63.4 (CH2O), 63.1 (C-2), 61.7 (ArCH2-), 57.4 (C-5), 50.9 (C-4);
[ ]2D0 + 154.2 (c 1.00 MeOH); HRMS (ESI) m/z calcd for C11H17N2O2 [M
+H]+ 209.1285, found 209.1292.
6.24. (3R,4R)-4-(hydroxymethyl)-1-[(pyridin-4-yl)methyl]pyrrolidin-3-ol
((+)-6e)
6.21. (3R,4R)-4-(Hydroxymethyl)-1-[(1H-imidazol-4-yl)methyl]
pyrrolidin-3-ol ((+)-6b)
The general procedure for reductive amination was followed. Upon
addition of NaBH3CN, the mixture, initially an orange solution, turned
yellow. After 18 h the bright yellow suspension was filtered through
Celite® and the solvent was removed. In order to remove the boron salts
present in the crude product, the residue was taken up in water (5 mL)
and the pH was adjusted to 10 using aqueous NaOH solution (1 M). This
was extracted with DCM/MeOH (9:1, 3 × 10 mL) and the combined
organic layers were washed with brine (30 mL). Flash column silica
chromatography (DCM/MeOH [9:1]) gave the title compound and 4-
pyridinemethanol. Further chromatography (DCM/MeOH [3:1]) gave
the title compound as a yellow oil (10 mg, 16%); Rf 0.31 (DCM/MeOH
[3:1]); NMR δH (400 MHz, MeOD) 8.47 (2H, d, J 6.0 Hz, Ar-H), 7.46
(2H, d, J 6.0 Hz, Ar-H), 4.04 (1H, dt, J 6.0, 3.5 Hz, H-3), 3.76 (1H, d, J
14.0 Hz, ArCHH-), 3.66 (1H, d, J 14.0 Hz, ArCHH-), 3.63 (1H, dd, J
11.0, 6.0 Hz, CHHO), 3.53 (1H, dd, J 10.5, 7.5 Hz, CHHO), 2.96 (1H,
dd, J 9.0, 8.5 Hz, H-5a), 2.77 (1H, dd, J 10.0, 6.0 Hz, H-2a), 2.63 (1H,
dd, J 10.0, 3.5 Hz, H-2b), 2.39 (1H, dd, J 9.5, 6.5 Hz, H-5b), 2.26–2.18
(1H, m, H-4); δC (101 MHz, MeOD) 150.3 (Ar), 150.1 (Ar), 125.6 (Ar),
74.1 (C-3), 64.0 (CH2O), 63.1 (C-2), 59.9 (ArCH2-), 57.3 (C-5), 51.3 (C-
4); [ ]2D0 + 231.7 (c 1.00 MeOH); HRMS (ESI) m/z calcd for C11H17N2O2
[M+H]+ 209.1285, found 209.1297.
The general procedure for reductive amination was followed. As
soon as NaBH3CN was added, the mixture, initially a transparent so-
lution, turned cloudy. After stirring at RT for 22 h, LCMS revealed a
high concentration of unreacted starting material. For that reason, 1
extra equiv. of 1H-Imidazole-4-carbaldehyde was added (31 mg,
0.33 mmol) and the mixture was stirred for 1 h. It was then filtered
through Celite® and loaded onto a 2 g strong cation exchange column.
Elution with MeOH followed by NH3 solution (1 M in MeOH) gave the
title compound as a yellow oil (11 mg, 17%); Rf [DCM/MeOH (5:1)]
0.28; NMR δH (400 MHz, MeOD) 7.61 (1H, d, J 1.0 Hz, Ar-H), 6.97 (1H,
s, Ar-H) 3.98 (1H, dt, J 6.0, 4.0 Hz, H-3), 3.64 (1H, d, J 13.5 Hz,
ArCHH-), 3.61 (1H, dd, J 11.0, 6.0 Hz, CHHO), 3.57 (1H, d, J 13.5 Hz,
ArCHH-), 3.49 (1H, dd, J 10.5, 7.5 Hz, CHHO), 2.93 (1H, dd, J 9.5,
8.5 Hz, H-5a), 2.74 (1H, dd, J 10.0, 6.0 Hz, H-2a), 2.60 (1H, dd, J 10.0,
4.0 Hz, H-2b), 2.35 (1H, dd, J 9.5, 6.5 Hz, H-5b), 2.20–2.12 (1H, m, H-
4); δC (101 MHz, MeOD) 136.3 (Ar), 134.9 (Ar), 120.2 (Ar), 74.2 (C-3),
64.3 (CH2O), 62.8 (C-2), 57.0 (C-5), 52.3 (ArCH2-), 51.3 (C-4);
[ ]2D0 + 150.1 (c 1.00 MeOH); HRMS (ESI) m/z calcd for C9H16N3O2 [M
+H]+ 198.1237, found 198.1242.
6.25. AAG inhibitor biochemical assay
6.22. (3R,4R)-4-(Hydroxymethyl)-1-[(pyridin-2-yl)methyl]pyrrolidin-3-ol
((+)-6c)
6.25.1. Materials
Nunc® Immobiliser™ Amino 96-well plates were purchased from
ThermoFisher Scientific (Hemel Hemstead, UK). T4 DNA ligase was
purchased from Promega (Southampton, UK). It was supplied in a
storage buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM
dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA)
and 50% glycerol. Oligonucleotides HX02 and Loop01 were purchased
from Integrated DNA Technologies (Leuven, Belgium). They were
supplied lyophilised and were suspended in ultrapure (Milli-Q®) water
to 1 mM, and subsequently diluted to 10 µM solutions in the corre-
sponding buffer. Their sequences (5′ to 3′) are given below:‡
HX02: (P)CACGAAHCAACTCAGCAACTCCtt(NH2)
The general procedure for reductive amination was followed. The
crude product was purified by flash column silica chromatography
(DCM/MeOH [9:1]) to give the title compound as a yellow oil (39 mg,
61%); Rf 0.2 (DCM/MeOH [9:1]); NMR δH (400 MHz, D2O) 8.44–8.42
(2H, m, Ar-H), 7.80 (1H, ddd, J 8.0, 2.0, 1.5 Hz, Ar-H), 7.42 (1H, ddd, J
8.0, 5.0, 0.5 Hz, Ar-H) 4.05 (1H, dt, J 6.0, 4.5 Hz, H-3), 3.71 (1H, d, J
13.0 Hz, ArCHH-), 3.63 (1H, dd, J 11.0, 6.5 Hz, CHHO), 3.60 (1H, d, J
13.0 Hz, ArCHH-), 3.52 (1H, dd, J 11.0, 7.5 Hz, CHHO), 2.93 (1H, dd, J
10.0, 8.0 Hz, H-5a), 2.78 (1H, dd, J 10.5, 6.5 Hz, H-2a), 2.60 (1H, dd, J
10.5, 4.0 Hz, H-2b), 2.31 (1H, dd, J 10.0, 7.0 Hz, H-5b), 2.23–2.15 (1H,
m, H-4); δC (101 MHz, D2O) 149.3 (Ar), 147.8 (Ar), 138.6 (Ar), 133.1
(Ar), 124.1 (Ar), 72.4 (C-3), 62.4 (CH2O), 60.5 (C-2), 56.6 (ArCH2-),
55.2 (C-5), 48.5 (C-4); [ ]2D0 + 13.6 (c 1.00 MeOH); HRMS (ESI) m/z
calcd for C11H17N2O2 [M+H]+ 209.1285, found 209.1291.
Loop01: (flc)ttGGAGTTGCTGAGTTGATTCGTGAGCACCAACCGGT
GCT.
AAG enzyme (10,000 U/mL)§ was purchased from New England
Biolabs (Hitchin, UK). It was supplied in a storage buffer containing
100 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 0.5%
Tween® 20, 0.5% NP-40 and 50% glycerol.
6.23. (3R,4R)-4-(Hydroxymethyl)-1-[(pyridin-3-yl)methyl]pyrrolidin-3-ol
((+)-6d)
Goat antibody to fluorescein (goat anti-fluorescein) horseradish
peroxidase (1 mg/mL) was purchased from Abcam (Cambridge, UK). It
was supplied in a storage buffer containing 0.42% potassium phosphate
(pH 7.2), 0.87% sodium chloride, 1% BSA and 0.1% Gentamicin as a
preservative. It was diluted 10-fold into PBST containing 1% w/v BSA
and stored as single use aliquots at −20 °C. 3,3′,5,5′-
Tetramethylbenzidine (TMB) peroxidase substrate solution and perox-
idase substrate buffer were purchased from Insight Biotechnology Ltd
(Wembley, UK).
The general procedure for reductive amination was followed. The
crude product was purified by flash column silica chromatography
(DCM/MeOH [9:1]) to yield a yellow oil which was characterised as a
mixture of title compound and borohydride salts. Addition of MeOH
(1 mL) left an insoluble component which was removed by filtration
through Celite®. Evaporation of the filtrate gave the title compound
(28 mg, 44%); Rf 0.15 (DCM/MeOH [9:1]); NMR δH (400 MHz, MeOD)
8.54 (1H, d, J 4.5 Hz, Ar-H), 7.84 (1H, td, J 7.5, 1.5 Hz, Ar-H), 7.52 (1H,
d, J 8.0 Hz, Ar-H) 7.36 (1H, dd, J 7.5, 5.0 Hz, Ar-H), 4.15 (1H, dt, J 6.0,
3.5 Hz, H-3), 4.10 (1H, d, J 14.0 Hz, ArCHH-), 4.02 (1H, d, J 14.0 Hz,
ArCHH-), 3.64 (1H, dd, J 11.0, 5.5 Hz, CHHO), 3.57 (1H, dd, J 10.5,
6.5 Hz, CHHO), 3.26 (1H, dd, J 10.0, 8.5 Hz, H-5a), 3.06 (1H, dd, J
10.5, 6.0 Hz, H-2a), 2.91 (1H, dd, J 10.5, 3.0 Hz, H-2b), 2.73 (1H, dd, J
10.5, 6.5 Hz, H-5b), 2.36–2.26 (1H, m, H-4); δC (101 MHz, MeOD)
Bicarbonate buffer was prepared by dissolving 0.18 g of NaHCO3
and 0.04 g of Na2CO3 in 50 mL milliQ water (pH = 9.6). Phosphate
‡ DNA oligomer sequences: lower case letters indicate nucleotides linked via
phosphorothiate bonds; X = hypoxanthine; (P) = phosphate; (NH2) = amino
modifier group (CH2CH(OH)CH2O(CH2)3NH2); (flc) = fluorescein.
9

E. Mas Claret, et al.
Bioorganic&MedicinalChemistry28(2020)115507
buffered saline 0.1% v/v Tween® 20 (PBST) was prepared by dilution of
1 mL Tween® 20 (Sigma-Aldrich) in 1 L of ultrapure (Milli-Q®) water
which contained 10 PBS tablets (Sigma-Aldrich). AAG glycosylase
buffer was prepared by mixing 4 mL Tris 1 M (pH 7.8) (Sigma-Aldrich),
20 mL KCl 1 M, 4 mL EDTA 0.25 M (pH 8) (Sigma-Aldrich), 400 µL
egtazic acid (EGTA) 500 mM (Sigma-Aldrich) and 69.6 µL of β-mer-
captoethanol (Sigma-Aldrich) in 171.5 mL ultrapure (Milli-Q®) water.
Hybridisation buffer was prepared by mixing 30 mL 20xSSC (Sigma-
Aldrich), 0.1 mL Tween® 20, 1 mL EDTA 0.5 M and 68.9 mL ultrapure
(Milli-Q®) water. T4 ligase buffer was prepared by diluting 6 mL Tris
1 M (pH 7.8), 6 mL NaCl 1 M, 2 mL MgCl2 1 M in 186 mL ultrapure
(Milli-Q®) water. DNA ligase buffer was prepared by mixing
47.424 mL T4 ligase buffer, 480 µL DTT (1 M) and 96 µL ATP (Sigma-
Aldrich). Alkaline denaturation buffer was prepared by diluting 10 mL
NaOH 5 M and 2.5 20xSSC in 487.5 mL ultrapure (Milli-Q®) water.
well) and dried. A 1:1 mixture of TMB peroxidase substrate solution and
peroxidase substrate buffer was prepared. It was added to the plate
(100 µL/well). Once sufficient pale blue colour had developed (12 min),
phosphoric acid 1 M (100 µL/well) was added, and a colour change to
yellow was observed. The absorbance was read at 450 nm.
Step 7: Data processing
GraphPad Prism 7.04 was used to process the data. A standard curve
of absorbance vs. [AAG] was plotted and absorbances acquired from
wells containing inhibitor were interpolated into this to obtain an ap-
parent [AAG]. The values of apparent [AAG] were used to calculate the
% inhibition given that a real [AAG] of 0.05 U/100 μL was used. IC50
curves of the proposed inhibitors were fitted using equation:
“[Inhibitor] vs. response” which also generated IC50 values from the
mid-point between the top and bottom of the curve (not the y = 50%
mark).
6.25.2. Procedure
Declaration of Competing Interest
Step 1: Binding of HX02 substrate oligonucleotide to well surface A
0.5 nM solution of oligonucleotide HX02 was prepared by diluting
10 µM HX02 into bicarbonate buffer. It was added to the Nunc®
Immobiliser™ Amino plate (100 µL 0.5 nM, 0.05 pmol HX02 per well),
which was incubated overnight at 4 °C. Then, the liquid was decanted
from the wells and the plate was washed with PBST (3 × 150 µL/well)
and dried.
The authors declare that there are no conflicts of interest.
Acknowledgements
D. Whelligan and R. Elliott thank the Royal Society for a research
grant for part of this work. The EPSRC is gratefully acknowledged for
the “MILES” grant (EP/I000992/1), part of which helped fund devel-
opment of the biochemical assay and a summer research project for S.
Chu. E. Mas thanks the University of Surrey, U.K. for funding of a PhD
scholarship. B. Al Yahyaei thanks the Sultanate of Oman for funding of
a PhD scholarship. M. Imperato and A. Lopez thank the Erasmus+
scheme for funding of summer research projects.
Step 2: In situ hybridisation of HX02 and Loop01
A 0.5 nM solution of oligonucleotide Loop01 was prepared by di-
luting 10 µM Loop01 into hybridisation buffer. It was added to the plate
(100 µL 0.5 nM, 0.05 pmol Loop01 per well), which was heated to 95 °C
for 10 min. Then, it was cooled to 80 °C and kept at 80 °C for 10 min.
After this time, it was cooled to 70 °C, 60 °C, 50 °C, 40 °C and 30 °C each
for 10 min to promote annealing of the DNA strands. It was allowed to
cool to RT, the liquid was decanted, the plate was washed with PBST
(3 × 150 µL/well) and dried by vigorous tapping onto paper towel.
Step 3: Ligation reaction
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115507.
A 0.04 U/100 µL solution of T4 DNA ligase was prepared by diluting
3 U/µL T4 DNA ligase into ligase buffer. It was added to the plate
(100 µL/well), which was incubated at 37 °C for 2 h. The liquid was
decanted and the plate was washed with PBST (3 × 150 µL/well). The
final wash was left in the wells and the plates were frozen overnight.
Then, they were warmed to RT, emptied and dried by vigorous tapping
onto paper towel.
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11