1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins

Article abstract of DOI:10.1002/anie.202105126

Linear or branched 1,3-diketone-linked thymidine 5′-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

Full text of DOI:10.1002/anie.202105126

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Accepted Article
Title: 1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking
with Arginine-Containing Peptides and Proteins
Authors: Denise-Liu' Leone, Martin Hubalek, Radek Pohl, Veronika
Sykorova, and Michal Hocek
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202105126
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1
,3-Diketone-Modified Nucleotides and DNA for Cross-Linking
with Arginine-Containing Peptides and Proteins
[a,b]
[a]
[a]
[a]
[a,b]
Denise-Liu' Leone,
Martin Hubálek, Radek Pohl, Veronika Sýkorová and Michal Hocek*
Dedicated to the memory of Dr. Dana Hocková.
[
a]
D.-L. Leone, Dr. R. Pohl, Dr. M. Hubálek, Dr. V. Sýkorová, Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
E-mail: hocek@uochb.cas.cz
Homepage: http://www.uochb.cas.cz/hocekgroup
D.-L. Leone, Prof. Dr. M. Hocek
[
b]
Department of Organic Chemistry, Faculty of Science,
Charles University in Prague,
Hlavova 8, CZ-12843 Prague 2, Czech Republic
Supporting information for this article is given via a link at the end of the document.
Abstract: Linear or branched 1,3-diketone-linked thymidine 5'-O-
mono- and triphosphate were synthesized through CuAAC click
reaction of diketone-alkynes with 5-azidomethyl-dUMP or –dUTP.
The triphosphates were good substrates for KOD XL DNA polymerase
in primer extension synthesis of modified DNA. The nucleotide
bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with
arginine-containing peptides to form stable pyrimidine-linked
conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group
was not reactive. Reaction with Lys or terminal amino group formed
enamine adducts that were prone to hydrolysis. This reactive HDO
modification in DNA was used for bioconjugations and cross-linking
with Arg-containing peptides or proteins (e.g. histones).
there has been only one example of Arg targeting with DNA using
4,6-dioxoheptylamido group attached to DNA postsynthetically to
a 2'-amino group of an 2'-amino-2'-deoxyuridine which was
chemically incorporated to the oligonucleotide (ON) through
phosphoramidite chemistry.[18]
bioconjugations, the most common reactive groups used to target
In affinity labelling and
Arg are glyoxals[ and other 1,2-dioxo derivatives. Since these
highly reactive species were difficult to attach to dNTPs for
polymerase synthesis of reactive DNA, we focused on 1,3-
diketones and report here the synthesis of dNTP building blocks
bearing 2,4-pentandione (either linear linked to C1 or branched
linked through C3), their use in the enzymatic synthesis of
reactive DNA probes and comparison of reactivity with Arg-
containing peptides and proteins.
19]
[20]
Protein-DNA interactions are important in many biological
processes[ and therefore identification of these interactions is
urgently needed.[3] Covalent cross-linking of the binding proteins
with reactive DNA, mostly driven by proximity effect, is one of the
most promising methods for their studies. In addition, covalent
protein-DNA conjugates are also useful for other applications in
chemical biology, biosensing[4] or targeting of therapeutic nucleic
acids[5] and therefore new efficient bioconjugation methods are
highly desirable.[6] There are some widely-used non-specific
cross-linking methods, typically based on photochemical
1,2]
generation of reactive species, i.e. radicals or carbenes[8] in
DNA that cross-link randomly to any amino acids. More useful but
also challenging are amino acid-specific cross-linking reactions
and there were only handful of them reported for DNA-protein
cross-linking. DNA bearing free thiol group cross-linked with Cys
through disulfide formation.[9] Vinylsulfonamide group was also
used to cross-link with Cys through conjugate addition,[10]
whereas chloroacetamide alkylated Cys or His in proteins.[11] Most
commonly used was cross-linking of aldehyde-linked DNA with
Lys either through reversible imine formation[12] or through
irreversible reductive amination[13,14] that requires additional
[7]
3
NaBH CN as stoichiometric reductant. Recently we have reported
squaramate-linked DNA that reacted with Lys-containing peptides
and proteins without any additional reagent.[15] As most of these
reactive groups are not compatible with phosphoramidite
synthesis, the polymerase synthesis using reactive-labelled 2'-
deoxyribonucleoside triphosphates (dNTPs) is advantageously
used for the construction of the reactive DNA probes.[16]
Scheme 1. Synthesis of HDO- and PDO-modified dNMPs and dNTPs and
reactions with Arg and Arg- or Lys-containing peptides. Conditions:
NaHCO /Na CO buffer (0.5 M, pH 10), 37°C, 18 h.
3 2 3
Arginine is another relevant nucleophilic amino acid often
present in DNA-binding proteins.[17] To the best of our knowledge,
1
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Figure 1. a) Enzymatic synthesis of 19DNA_THDO and 30DNA_TPDO.b) Cross-linking of 19/30DNA_THDO with Arg-containing tri-, hepta- and decapeptide c) Cross-
linking of 30DNA_TPDO with Arg-containing tri-, hepta- and decapeptide d) Cross-linking of 30DNA_THDO with Lys-containing or N-unprotected or negative control
peptides. e) PAGE analysis of the PEX reactions with dTHDOTP or dTPDOTP using KOD XL DNA polymerase, primer primA and templates Temp
19_1T
or Temp
30_1T
.
P: primer, (T+): Natural dNTPs,(T-):natural dNTPs without dTTP,(THDO/PDO): dTHDOTP or dTPDOTP, dCTP,dGTP, dATP;.f) PAGE analysis of cross-linking of
1
9DNA_THDO and 30DNA_THDO with N-BocArg, and arginine containing tri- and decapeptide (SI, part 2.3).
2
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HDO10pept
HDO10pept
30DNA_THDOARA
We designed and synthesized modified thymidine nucleotides
bearing the 1,3-diketone attached at position 5 to ensure that the
modification points out to the major groove of DNA and that the
nucleotides can be substrates for DNA polymerases. We chose
pentan-2,4-dione as the suitable 1,3-diketone moiety and
attached it to the nucleotide either through the terminal or internal
carbon and planned to attached them through the Cu-catalyzed
alkyne-azide cycloaddition (CuAAC)[21] to 5-azidomethyl-2'-
deoxyuridine nucleotides, that are easily available through radical
19DNA_T
30DNA_T
(35%),
(16%). Only for 30DNA_T
(13%)
no difference
and
HDOArg
in mobility was observed in the PAGE analysis due to minute
difference in size and resolution limit of the 20% PAGE gel. Most
of the products of conjugation were also successfully
characterized by MALDI (Table S4 in SI).
Furthermore, we wanted to test the reactivity of DNA_T
HDO
toward
Lys-containing peptides which could form reversible enamine-
bonds under the reaction conditions (Figure 1d). The reactions of
HDO
bromination
of
22]
protected
thymidine,
azidation
and
30DNA_T
and Lys-containing peptides, with N-unprotected
phosphorylation.[ The building blocks were the corresponding
terminal alkynes and oct-7-yne-2,4-dione (1) and 3-(prop-2-yn-1-
yl) pentane-2,4-dione (2) that were easily prepared according to
published procedures.[23] The CuAAC click reaction of alkynes 1
or 2 with azido-linked thymidine monophosphate dTN3MP or
triphosphate dTN3TP proceeded smoothly (Scheme 1) to give the
desired products dTHDOMP (40% yield), dTPDOMP (26% yield),
dTHDOTP (18% yield), dTPDOTP (34% yield). The isolated yields
were lowered by difficult purification of the nucleotides and partial
hydrolysis of the triphosphates, but the procedure was
straightforward and enabled the preparation of sufficient
tripeptide and with two negative control peptides containing all
amino acids except for Arg were conducted using the same
conditions as for the arginine derivatives either in absence or in
3
the presence of NaBH CN. In all cases we have observed only
traces (<0.03%) of conjugation products with the exception of the
reactions with peptides containing two or more Lys that gave 6-
11% conversion probably due to formation of double enamine
adducts that were more stable toward hydrolysis (Table S5 in SI).
These experiments clearly show the HDO group reacts with Lys
2
or terminal NH only reversibly and the enamine adducts are
unstable under denaturating or hydrolysis conditions.
X
quantities of the modified nucleotides. The modified dT MP were
then tested in model reactions with N-BocArginine or Arg-
containing tripeptide (3 equiv.). The reactions proceeded in
NaHCO
3
/Na
2
CO
3
buffer (0.5 M, pH 10) at 25 °C for 24 h. While
HDO
the linearly-linked dT MP reacted well to give the
corresponding conjugates dTHDOArgMP (42% yield) or
dTHDOARAMP (35% yield), the branched dT MP did not give any
reaction even with large excess of the Arg or peptide (20 equiv.).
PDO
HDOArg
HDOARA
The conjugates dT
MP and dT
MP were isolated by
reverse phase HPLC in pure form and fully characterized by NMR
and MS to confirm the covalent binding and formation of the stable
pyrimidine ring from the guanidine group and diketone. They did
not undergo any hydrolysis in water at r.t. in 20 h (Figure S45 SI).
The reactions of dTHDOMP with excess of N-terminal amino
group of AGA tripeptide or with Lys-containing tripeptide gave
enamine adducts dTHDOAGAMP (13%) and dTHDOAKAMP (35%),
that were prone to hydrolysis in water (77-87% at r.t. in 20 h).
Then we tested the modified triphosphates dTHDOTP and
dTPDOTP as substrates for KOD XL DNA polymerase in primer
extension experiments (PEX) (Figure 1a). Both of them were good
substrates and gave full-length 19- or 30-mer PEX products
containing 1, 2 or 4 modifications as demonstrated by PAGE
analysis (Figure 1e, for the ON sequences see Table S1 and for
gels with 2 or 4 modifications, see Figure S1 in SI) and all the
modified DNAs were also characterized by MALDI-TOF analysis
(
Table S3 and Figures S20-S27 in SI).
Next we used 19-bp or 30-bp double-stranded DNAs
(
19DNA_THDO, 19DNA_TPDO, 30DNA_THDO and 30DNA_TPDO
)
containing one modification to test cross-linking reaction with N-
BocArg and Ac-Arg-containing tripeptide and decapeptide (Figure
1
b,c). The reactions were conducted in presence of an excess of
6
amino acid or peptide (10 equiv.) and at 37°C for 18 h. Similarly
to the reactions of model nucleotides, DNA bearing branched
PDO gave only traces of conjugated products on PAGE analysis
(
Figures S4-S7 in SI), whereas the modified DNA bearing linear
HDO group reacted well as shown in the PAGE analysis (Figure
f) confirming the formation of the desired cross-linked
conjugates with slower mobility in conversions 10-35%:
9DNA_THDOArg 19DNA_THDOARA (10%),
(10%),
1
Figure 2. Cross-linking of 1,3-diketone-modified DNA with proteins: a) reaction
scheme. Conditions: NaHCO
17.5 % SDS-PAGE analysis of cross-linking of 30_DNA_T
3
/Na
2
CO
3
buffer (0.05 M, pH 10), 37°C, 18 h: b)
HDO
1
with histones (5
equiv. of protein) and BSA.
3
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Then we approached the ultimate goal of this study, to test
This work was supported by the Czech Academy of Sciences
(Praemium Academiae award to M. Ho.), by the Czech Science
Foundation (18-03305S to D.-L. L. and M. Ho.) and by European
the reactivity of the HDO-modified DNA (19DNA_THDO and
3
HDO
[15]
0DNA_T ) with proteins. Following our previous work, we
used bovine serum albumin (BSA) as a negative control of a
protein containing 26 Arg which does not interact with DNA, GST-
tagged core domain of p53 protein (GSTp53CD)[24] as an example
of a DNA-binding protein containing arginine but not in the
proximity to the modification in the binding site,[25] and finally a set
of Arg-rich histones (H2A, H2B, H3.1 and H4) that strongly bind
DNA and their arginines participate on the interaction with DNA.
Unlike the model-studies with peptides (that required a large
excess of Arg-containing peptides to observe any cross-linking),
the cross-linking reactions were performed using 5 equiv. or even
Regional
Development
Fund;
OP
RDE
(No.
CZ.02.1.01/0.0/0.0/16_019/0000729 to M.Hu.). The authors
thank Robert Rampmaier for help in preparation of peptides.
Keywords: DNA • proteins • bioconjugations • cross-linking
reactions • nucleotides • DNA polymerases
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DNA probes. The HDO moiety reacts with arginine to form a
stable aromatic pyrimidine ring and the covalent adducts are
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5
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Arginine-specific 1,3-diketone-linked reactive nucleotide for enzymatic construction DNA probes for cross-linking with peptides and
proteins.
Institute and/or researcher Twitter usernames: @hocekm @IOCBPrague
6
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