Profiling base excision repair glycosylases with synthesized transition state analogs

Article abstract of DOI:10.1016/j.bmcl.2011.05.085

Two base excision repair glycosylase (BER) transition state (TS) mimics, (3R,4R)-1-benzyl (hydroxymethyl) pyrrolidin-3-ol (1NBn) and (3R,4R)- (hydroxymethyl) pyrrolidin-3-ol (1N), were synthesized using an improved method. Several BER glycosylases that repair oxidized DNA bases, bacterial formamidopyrimdine glycosylase (Fpg), human OG glycosylase (hOGG1) and human Nei-like glycosylase 1 (hNEIL1) exhibit exceptionally high affinity (K _d～pM) with DNA duplexes containing the 1NBn and 1N nucleotide. Notably, comparison of the K_d values of both TS mimics relative to an abasic analog (THF) in duplex contexts paired opposite C or A suggest that these DNA repair enzymes use distinctly different mechanisms for damaged base recognition and catalysis despite having overlapping substrate specificities.

Full text of DOI:10.1016/j.bmcl.2011.05.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4969–4972
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Profiling base excision repair glycosylases with synthesized transition
state analogs
⇑
Aurea M. Chu, James C. Fettinger, Sheila S. David
Department of Chemistry, University of California, Davis, Building 143, One Shields Avenue, Davis, CA 95616, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two base excision repair glycosylase (BER) transition state (TS) mimics, (3R,4R)-1-benzyl (hydroxymethyl)
pyrrolidin-3-ol (1NBn) and (3R,4R)-(hydroxymethyl) pyrrolidin-3-ol (1N), were synthesized using an
improved method. Several BER glycosylases that repair oxidized DNA bases, bacterial formamidopyrim-
dine glycosylase (Fpg), human OG glycosylase (hOGG1) and human Nei-like glycosylase 1 (hNEIL1) exhibit
exceptionally high affinity (KdꢀpM) with DNA duplexes containing the 1NBn and 1N nucleotide. Notably,
comparison of the Kd values of both TS mimics relative to an abasic analog (THF) in duplex contexts paired
opposite C or A suggest that these DNA repair enzymes use distinctly different mechanisms for damaged
base recognition and catalysis despite having overlapping substrate specificities.
Received 7 April 2011
Revised 20 May 2011
Accepted 23 May 2011
Available online 30 May 2011
Keywords:
Base excision repair
DNA glycosylase
Pyrrolidine analogs
Ó 2011 Elsevier Ltd. All rights reserved.
Transition state analogs
8-Oxo-7,8-dihydro-2-deoxyguanosine
hOGG1
Fpg
Nei
hNEIL1
Chemical tools developed to study enzymes that act upon
oligosaccharides provide inspiration for the use of similar strate-
gies with functionally similar enzymes that act on nucleic acids.1
Indeed, DNA glycosylases initiate base excision repair (BER) at
damaged bases within DNA by catalyzing N-glycosidic bond cleav-
age2,3 in a manner mechanistically analogous to oligosaccharide
hydrolysis catalyzed by a large and diverse class of glycoside
hydrolases.4 Chemically modified sugars have played key roles in
elucidating the structural and mechanistic properties of glycoside
hydrolases and hold promise as powerful tools to visualize and
tag glycosidases in cellular contexts.5–7
hydrolysis that proceeds through two oxacarbenium ion-like transi-
tion states (TS1 and TS2,Fig. 1B).11,12 The pyrrolidines 1N and 4N mi-
mic the positive charge on the 2’-deoxyribose oxacarbenium ion and
may more closely approximate TS2.13 Pyrrolidine homonucleosides
(Fig. 1C) that contain an appended base or base mimic would be ex-
pected to more closely mimic the first oxacarbenium ion-like TS
(TS1). These TS mimics incorporated into RNA have been shown to
be potent inhibitors of ricin A-chain (RTA) that hydrolyzes adeno-
sine within ribosomal RNA.14–17 Consistent with similar mecha-
nisms for base excision, an adenine pyrrolidine homonucleotide,
phA incorporated into a DNA duplex (Fig. 1B) has been used for bind-
ing studies with MutY.8
Among the myriad of modified DNA bases, oxidative modifica-
tions are particularly mutagenic and are contributors to human
diseases, including cancer and neurological disorders.18–20 A cadre
of DNA glycosylases play crucial roles in excising oxidatively dam-
aged DNA bases as the first step in BER.20,21 In mammalian cells,
the human OG glycosylase (hOGG1) removes primarily 8-oxo-
7,8-dihydroguanine (OG) and 2,6-diamino-4-hydroxy-5-formami-
dopyrimidine (FapyG), while the human endonuclease III enzyme
(hNTH) removes oxidized thymines, such as thymine glycol (Tg).
The human nei-like (NEIL1) glycosylase has a broad substrate
specificity in removing damaged pyrimidines, such as Tg and
5-hydroxyuracil (5-OHU) as well as the oxidized purines FapyG,
guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) but not
OG.20,22–26 In contrast, in Escherichia coli, the formamidopyrimidine
There is a particularly rich history of the use of azasugars as high
affinity inhibitors of glycosidase enzymes and many of these inhib-
itors have shown promise as pharmaceuticals.5 The efficacy of inhib-
itors such as Nojirimycin (Fig. 1A) has been attributed to mimicry of
the glycosidase ‘oxacarbenium ion-like’ transition state providing
for favorable electrostatic interactions within the active site.5 The
pyrrolidine analogs, (3R,4R)-3-hydroxymethyl-2-(hydro xymethyl)
pyrrolidinium (4N) and (3R,4R)-(hydroxymethyl) pyrrolidin-3-ol
(1N) (Fig. 1A) incorporated within DNA duplexes have been found
to have subnanomolar affinity for several DNA glycosylases.8–10 Ki-
netic isotope effects studies of uracil-DNAglycosylase (UDG) and the
adenine glycosylase MutY indicate an SN1 mechanism of base
⇑
Corresponding author.
E-mail address: ssdavid@ucdavis.edu (S.S. David).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.085

4970
A. M. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4969–4972
NH2
N
N
N
A
B
OH
H
H
H
H
H
N
N
N
N
OH
HO
DNAO
DNAO
DNAO
NH2 DNAO
DNAO
H
OH
OH
DNAO
phA
1N
B-
4N
δ−
B
δ+
O
δ+
O
DNAO
DNAO
DNAO
DNAO
DNAO
B
H2O
O
O
O
δ+
OH
OH
ODNA
TS2
ODNA
ODNA
ODNA
ODNA
TS1
NH2
C
H
N
N
RO
RO
RNAO
RNAO
N
N
H
N
H
DA-14
1NBn :R=DNA
Bz-10: R=RNA
D
*5'-TGTTCATCATGGGTCXTCGGTATATCCCAT-'3
3'-ACAAGTAGTACCCAGYAGCCATATAGGGTA-5'
X= 1NBn, 1N, 4N, THF Y= C, A
Figure 1. (A) Nojirimycin and aza-ribose transition state mimics. (B) SN1 mechanism for monofunctional DNA glycosylases (such as uracil-DNA glycosylase) based on KIE
studies.3 Note that the enzymes studied herein are bifunctional glycosylase/lyase enzymes that utilize an amine nucleophile (Lys or N-terminal proline) rather than a water
molecule in the base displacement process. (C) Structure of 1NBn, Bz-10 and DA-14. (D) Sequence context of the 30-base pair duplex used in this study.
glycosylase (Fpg) is able to remove a broad array of oxidized bases,
primarily those derived from purines, including OG, FapyG, and the
hydantoins27,28 while the Nei glycosylase removes primarily oxi-
dized pyrimidines (e.g., thymine glycol), Gh, Sp and 4,6-diamino-
5-formamidopyrimidine (FapyA).29 An additional important fea-
ture of these oxidized-base specific glycosylases is the ability to
catalyze associated b- (hOGG1) or b and d-lyase (hNEIL1, Fpg,
Nei) strand scission reactions.
Due to the overlapping but distinct substrate specificity of this
group of DNA glycosylases, we reasoned that each glycosylase may
exhibit distinct binding preferences for pyrrolidine analogs that
mimic TS1 versus TS2. An inhibitor of RTA that attracted our atten-
tion was Bz-10 (Fig. 1C). Bz-10 was reported to exhibit a Ki of
Bn
N
Bn
Bn
a
Bn
b
O
N
H
1
NHOH.HCl
2
71 %
97 % EtO2C
3
CO2Et
Bn
N
Bn
N
Bn
N
c
+
d
O
O
O
98 %
EtO2C
EtO2C
HO2C
5
4
12
OH
34 %
OH
OH
e
H3B
Bn
H
N
Bn
0.099
lM, establishing it as a far better inhibitor than DA-14
N
g
N
f
(Fig. 1C, Ki value of 0.280
lM) which has a 9-deazaadenyl substitu-
75 %
80%
ent and closely resembles adenine. The N1 atom of Bz-10 has a pKa
around 8.5 making it cationic at physiological pH.15 Herein, we re-
port modified and improved syntheses of 1N and 1NBn phospho-
ramidites and evaluate the utility of these analogs within duplex
DNA as TS mimics for the BER glycosylases, Fpg, hOGG1, NEIL1
and Nei.10,30 An expected feature of TS mimicry is high enzyme
affinity and therefore we determined dissociation constants (Kd)
of this group of BER glycosylases with a 30 bp duplex containing
centrally located synthetic transition state mimics (1N, 4N,
1NBn) relative to the product analog (THF) using electrophoretic
mobility shift assays (EMSA).31–34
HOH2C
HOH2C
HOH2C
OH
6
8
7
OH
OH
i
h
NR
DMTrOH2C
CN
Fmoc
N
R
N
i
j
O
N
P
O
HOH2C
11
DMTrOH2C
9
10
OH
OH
61 %
R = Fmoc, 81%
= Bn, 63%
R = Fmoc, 82%
= Bn, 78%
1NBn (7) has recently been published as an intermediate product
in the synthesis of (3R,4R)-N-tert-butoxycarbonyl-4-hydroxy-
methyl.35 We modified this procedure to start from an economical
precursor (1) and provide higher yielding syntheses of 1NBn (7),
1N (8) and the corresponding phosphoramidite monomers (Scheme
1). Briefly, dibenzylamine (1) was used to make the N-benzylhydr-
oxylamine (2).36 The reaction time was lengthened for steps c–d to
obtain lactam (5): the cleavage of the N–O bond of isoxazolidine
(3) and the hydrolytic resolution of the racemic ester (4) using the
immobilized form of lipase B of Candida antarctica (Cal-B).35 The
reduction of the lactam (5) to benzylamine (6) was accomplished
Scheme 1. Reagents and conditions: (a) (1) Na2WO4ꢂ2H20, H2O2, MeOH, rt 18 h, (2)
20% HCl; (b) diethymaleate, CH2O, EtOH, reflux, 2.5 h; (c) Zn, AcOH, 8 h; (d) Cal-B
lipase, pH 7.5, 30 °C,16–24 h; (e) (1) BH3.DMS, THF, reflux, 11 h, (2) H2O; (f) MeOH,
10% Pd/C, H2, 24 h, rt, 80%; (g) MeOH, 10% Pd/C,10 days, rt, 75%; (h) FmocCl,
dioxane, 10% aq NaHCO3, rt; (i) DMTrCl, Hunig’s base, DMAP, DCM, rt, 1 h; (j) ClPNi-
Pr2(OCH2CH2CN), DCM, iPr2NEt, rt, 2 h.
using borane dimethyl sulfide.37 The latter reaction produced a sta-
ble amine-borane complex (6) as confirmedby HR-MS analysis and a
signal at ꢁ9.6 ppm in 11B NMR, which is known to arise from a ter-
tiary-amine–borane complex (Supplementary data).38 Decomplex-

A. M. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4969–4972
4971
Table 1
ation to generate 1NBn (7) was accomplished by stirring with meth-
Dissociation constants (Kd, nM)a for various DNA repair glycosylases
anol for 24 h in the presence of 10% Pd/C catalyst under H2 gas
atmosphere. The increased time of methanolic cleavage resulted in
tandem decomplexation and debenzylation to generate 1N (8).
HR-MS, 1H NMR, and 13C NMR for 1N (8) and additional X-ray
crystallography for intermediates (5), (12), and 1NBn (7) (Supple-
mentary data, Fig. 2) confirmed the identity and absolute stereo-
chemistry of the products. Fmoc protection of 1N was performed
using the procedure of Filichev et al.39 Tritylation (10) and phosph-
itylation (11) for both nucleosides were carried out with minor mod-
ifications of the previous methods17 to provide an overall yield of the
1NBn and 1N phosphoramidites of 9% and 4%, respectively. 31P NMR
(CDCl3) indicated formation of the phosphoramidite linkage with
peaks at ꢀ148–9 ppm for both 1NBn and IN phosphoramidite (11)
(Supplementary data). These phosphoramidites were incorporated
into oligonucleotides with >95% efficiency using standard auto-
mated phosphoramidite synthesis and were deprotected using stan-
dard conditions. HR-MS analysis of an 11-nucleotide fragment
containing 1NBn [d(50TGTCCA1NBnGTCT-30)] gave a strong molecu-
lar ion peak at the expected molecular weight of m/z 3261.77 (Sup-
plementary data) indicating that 1NBn is stable under the conditions
used for the DNA synthesis and deprotection. The 1NBn nucleoside
was also generated from 1N by reductive amination.40
Electrophoretic mobility shift assays (EMSA) were used to mea-
sure the relevant dissociation constants of Fpg, hOGG1, hNEIL1 and
Nei in a manner analogous to previous reports from our laboratory
with MutY.33 Briefly, these experiments entailed titration of the
relevant enzyme into a solution of 50-[32P-phosphate]-end-labeled
DNA duplex and analysis on native PAGE to separate and detect
both the free DNA duplex and protein–DNA complex. Quantitation
via storage phosphor autoradiography and fitting of the data to a
one binding isotherm provide the Kd values listed in Table 1. In-
deed, inspection of these Kd values reveal distinct differences be-
tween the various enzymes with respect to their preference for
the TS mimics over the abasic site analog, the influence of the base
opposite and relative non-specific and specific DNA binding affin-
ity. Interestingly, Fpg exhibits the highest affinities while Nei
exhibits the lowest affinities for all of the DNA duplexes examined.
Remarkably, Fpg shows exceptionally high affinity for all of the
TS analogs (1N, 4N, and 1NBn) and the THF product analog when
paired with C in the 30 bp duplex. Using the lowest concentration
of the analog-containing duplex that may be practically detected
via storage phosphor autoradiography (5 pM duplex), the DNA du-
plex was observed completely bound at all concentrations of Fpg
used (Supplementary data). On this basis of the DNA concentration
used in the EMSA, an upper limit for the Kd of 5 pM was estimated.
Although Fpg and hNEIL1 have significant sequence homology,
hNEIL1 exhibits a significantly reduced affinity for all of the TS ana-
logs and the THF-containing duplexes. Moreover, like Fpg, hNEIL1
exhibits a 5-fold higher affinity for the duplexes containing posi-
tively charge analogs 1N, 4N, 1NBn over the uncharged THF prod-
uct analog when the opposite base is C (Table 1). In contrast, the
bacterial Nei enzyme exhibited weak affinity for all the analogs
and did not show a significant preference for positively charged
TS analogs over the THF analog.
Central base pair Fpg
hOGG1d
hNEIL1
Nei
32 12
1NBn:C
1N:C
4N:C
THF:C
1NBn:A
1N:A
THF:A
G:C
<0.005b
<0.005b (72%), 32 (20%) 0.2 0.1
<0.01 (36%), 5 2 (50%) 0.2 0.1
b
b
b
<0.005
<0.005
<0.005
ꢀ0.02c
ꢀ0.01c
ꢀ0.05c
>150f
8
3
<0.01 (50%), 5 2 (20%) 0.1 0.05 NDe
b
<0.005
1
2
4
4
1
1
1
3
24
22
9
8
4
4
1
400 300
500 400
400 200
>1200f
8
44 5f
>2000f
a
Dissociation constant measured were performed at 25 °C, 100 mM NaCl and pH
7.5. The DNA duplex concentration was 5 pM for all experiments except those with
X:A-duplexes in binding titrations with hOGG1 where 20 pM was used. Errors
reported in dissociation constants are standard deviation of the average of at least
three trials. The concentrations of enzyme are active enzyme concentrations.
Upper limit estimation; too tight to measure accurately, Kd is estimated to be
lower than the [DNA] used.
b
c
Tight apparent Kd estimated from the plot. The data fits better using a two-site
binding isotherm with the second weaker binding capacity representing 20% for 1N
and 1NBn, and 50% for THF duplex and providing for a similar Kd value with all three
duplexes of approximately 6 nM.
d
The data with hOGG1 fits best using a two-site binding isotherm that provides
two Kd values with relative capacities indicated in closed parentheses. The per-
centages of the two binding fractions differ between duplexes with the 1NBn giving
the highest fraction of the high affinity Kd (see Supplementary data for represen-
tative data).
ND–not determined.
G:C represents non-specific binding. The lower limit estimates Kd are based on
the highest enzyme concentration used that still provided less than 50% bound
DNA.
e
f
Surprisingly, hOGG1, a functional human homolog of bacterial
Fpg in preventing mutations associated with OG, has a very high
affinity for both THF:C and 1NBn:C, with Kd values that are similar
to those for Fpg with the same duplexes (Table 1). Curiously, the
hOGG1 binding titration curves with the duplexes containing the
1N, 4N and 1NBn analogs paired with C are biphasic and are best
fit with a two-site binding isotherm yielding two distinct Kd values.
Notably, the 1NBn:C duplex exhibited the largest fraction with the
extremely tight Kd compared to the corresponding 1N and 4N con-
taining DNA. This behavior does not appear to be due to the pres-
ence of ds and ss DNA in the initial DNA duplex sample after
annealing (Supplementary data). Indeed, EMSA analysis of hOGG1
binding to the 30 nt ss oligonucleotide containing 1N and
1NBn-containing DNA provided Kd values that are significantly
higher (76 12 and 83 15 nM, respectively) than either of the
Kd values observed with duplex DNA. Moreover, if the presence
of ss DNA is a factor, the fraction representing binding to the du-
plex (the tighter Kd) would be expected to follow the duplex stabil-
ities with THF > 1N > 1NBn (Chu, A., David, S. S., unpublished
results). The fact that hOGG1 binds 1NBn more efficiently than
1N suggests that the two-site binding data reflects an enzyme-
dependent effect on the DNA duplex, such as hOGG1 providing sta-
bility for the duplex form. An alternative intriguing possibility is
that the biphasic curves are capturing two mutually exclusive
DNA binding sites on the enzyme. Indeed, hOGG1 been shown to
have two base-binding sites.20 Further investigation of this inter-
esting feature of hOGG1 binding with these TS mimics is in pro-
gress using alternative binding methods.
Interestingly, the EMSA data with Fpg fit more accurately to a
two-site binding isotherm in the case of the analogs paired with
the A-containing duplex, with the proportion representing the
tighter Kds being greater with the 1N and 1NBn analog compared
to THF. Comparison of the tighter Kd values indicates a reduction
in binding affinity of Fpg for the TS analogs paired with A relative
to C, consistent with the known ability of Fpg to select against re-
moval of OG paired with A.28,41 Notably, these results also show
that by pairing with A, there is a slightly higher preference of
Fpg for the presence of the positively charged 1N and 1NBn analogs
Figure 2. X-ray crystal structure of 1NBn (7) CCDC no: 825694.

4972
A. M. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4969–4972
over the neutral THF analog consistent with published data that
indicated an 8-fold tighter binding of Fpg to a DNA duplex contain-
ing a central 4N:T over the corresponding THF:T containing du-
plex.10 Similarly, the relative affinity of hNEIL1 for the 1N and
1NBn TS analog containing duplexes when paired with A is less
than for C, as was observed with Fpg, indicating a similar prefer-
ence for C opposite the lesion, TS or product analog site. In contrast,
Nei glycosylase shows a slight preference for base pair context
with A, similar to the base specificity that has been reported
previously.29
Of this group of glycosylases, hOGG1 is distinct in showing a
marked preference for the uncharged product analog (THF) over
the positively charged abasic/TS mimic (1N, 4N). Moreover, when
the analogs were paired with A, the high affinity and specificity
of hOGG1 for the 1NBn and uncharged THF analog is completely
lost. Consistent with these results, cleavage of an abasic site by
hOGG1 is dependent on the opposite base.42 The reduced binding
affinity observed when the analogs were base paired to A is also
consistent with the large degree of selectivity of hOGG1 for re-
moval of OG or FapyG when paired with C over A, especially com-
pared to Fpg.43 The results with the 1NBn:C duplex show that the
presence of a base mimic (Bn) and C are needed for high affinity.
This suggests that both the damaged base and its opposite partner
C are utilized in TS stabilization. Indeed, recent structural studies
of an inactive hOGG1 mutant (Q315F) bound to an OG:C duplex
shows that the OG lesion is almost completely and properly in-
serted into the OG binding pocket.44 This suggests that there are
strict steric requirements for positioning of the base within the ac-
tive site to reach the proper TS required for hOGG1 mediated base
excision. The ability of the simple benzyl substituent to provide for
improved recognition of the positively charged azaribose suggests
that further elaboration of pyrrolidine nucleotides with base-like
moieties and screening may provide for derivatives with even
higher affinity for hOGG1 over other glycosylases.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.085.
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Overall, the results suggest that even though the BER glycosy-
lases studied herein have similar and overlapping substrate speci-
ficities, there are distinct differences in the damaged base
recognition and excision process within this group. This may be re-
lated to their biological function and the particular demand for
accuracy versus efficiency when removing damaged bases. The dif-
ferent features of the base excision reaction coordinate for each en-
zyme are suggested by the distinct recognition patterns for the
series of TS and product analogs studied herein. This data indicates
that 1NBn, in addition to 4N and 1N, is an useful TS mimic for
studying the catalytic mechanism of BER glycosylases and may
serve as general inhibitors of these enzymes. These new chemical
tools will provide avenues for additional structural studies of
DNA glycosylases as well as potential chemical biology tools to
identify and probe BER glycosylases and associated protein part-
ners in a cellular milieu. Moreover, such studies provide a starting
point for developing high affinity inhibitors of BER glycosylases
that may have chemotherapeutic applications.
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We thank Dr. Sheng Cao, Dr. Micheal Leipold and Ms. Paige
McKibbin for providing the purified enzymes used in this work.
We also thank Jennifer Mason for useful discussions on the syn-
thetic work. Andrew Ferreira and Dr. Jerry Dallas of the UCD
NMR facility helped obtain the 11B NMR. This work was supported
by grants from the National Cancer Institute of the National Insti-
tutes of Health (CA67985 and CA090689). We also acknowledge
NSF (#0840444) for the department dual source diffractometer.
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