Oligonucleotide promoted peptide bond formation using a tRNA mimicking approach

Article abstract of DOI:10.1039/c7cc00831g

TransferRNA's role in protein translation is the prime example of an Informational Leaving Group (ILG). A simplified model produced oligophenylalanine with a modified uracil as an ILG in the presence of specific oligonucleotides. Our preliminary studies contribute to the importance of hybrid species in bridging the gap between peptides and nucleic acids.

Full text of DOI:10.1039/c7cc00831g

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Oligonucleotide promoted peptide bond formation using a tRNA
mimicking approach†
H.-P. Mattelaer,a,b C.-A. Mattelaer,a Nikolaos Papastavrou,a W. Dehaenb and P. Herdewijna*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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TransferRNA's role in protein translation is the prime example of functional groups of present-day amino acids. Such
an Informational Leaving Group (ILG). simplified model nucleobase-peptide hybrids could potentially increase the
A
produced oligophenylalanine with a modified uracil as ILG in catalytic functionality of nucleic acids.8 One of those uracil
presence of specific oligonucleotides. Our preliminary studies derivatives (5-(4-hydroxybenzyl)uracil) caught our eye, as the
contribute to the importance of hybrid species in bridging the gap phenolic moiety could activate carboxylic acids and thus
between peptides and nucleic acids.
initiate peptide synthesis.10 It can be considered as a uracil
bearing a tyrosine sidechain and as the chemical simplification
of tRNA, which acts as a leaving group (LG) and information
carrier during protein synthesis (Figure 1A, 1B). The compound
Translating genetic information into proteins plays a central
role in all vital functions and is operated by a complex, cellular
machinery. Simplified, this process represents an
oligonucleotide templated and catalysed production of
peptides. Templated synthesis has seen great advances over
the past years. Starting from non-enzymatic oligonucleotide
synthesis by prebiotic chemists1; oligonucleotides have been
used to catalyse many different type of chemistries, including
amide formation.2 However early attempts to catalyse peptide
formation only achieved dimerization and subsequent
cyclization of amino acids when using 2’(3’)-esters of
mononucleotides.3 More successful were enzyme mimicking
approaches for (amino) acyl transfer reactions by programmed
proximity4, with the artificial ribosome as prime example.5
However those designed molecular systems do not start from
a separate template. The search for a non-enzymatic
informational link between oligonucleotides and peptides
remains a challenge. A link that could be bridged by hybrids,
such as PNA.6 This oligonucleotide analogue combines a stable
peptide backbone with nucleobase pairing and has proven to
be a valuable example of templated amide bond formation.7
Another interesting take came from Robertson and Miller8
who drew their inspiration from tRNA which undergoes
extensive post-transcriptional modifications9 including
was loaded with an amino acid, N-protected with
a
photocleavable group (NVOC). This protection strategy is
known in amino acyl-tRNA synthesis11 as irradiation avoids the
use of harsh reagents incompatible with the oligonucleotide in
question. Here it allows us to start the oligomerization with
minimal interference. Furthermore, phenylalanine was chosen
for ease of follow-up during synthesis and analysis (Figure 1C).
Further synthetic details can be found in the Supplementary
Information (SI).
Figure 1. The Informational Leaving Group approach: A) aminoacyl-tRNA, depicted in
the typical cloverleaf representation with anticodon and 3’ amino acyl detailed; B) the
simplification of tRNA into Recognition, Linker and Amino Acid with Reactive Bond,
introducing
a Protecting Group as synthetic necessity; C) the model compound
envisaged based upon the prebiotic available 5-(4-hydroxybenzyl)uracil, using
phenylalanine as amino acid and N-(4,5-dimethoxy-2-nitrobenzyloxycarbonyl) as
photocleavable protecting group.
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Scheme 1. Studied model of the ILG approach: The green pathway describes the oligomerisation of free activated amino acid upon irradiation. By using an ILG
complementary to the used oligonucleotide (insert), the efficiency of this process could be increased (red). Expected side-reactions, competing with this process, are
depicted in blue. These consists of hydrolysis of the phenolic ester and side-products emanating from the photolytical removal of the amine protection group.
Before studying oligomerization reactions of the model seen in Figure 2, with an overlay of monomer elongation in the
compound (Scheme 1); any influence of the LG on amide absence or presence of (dA)10. The complex mixture seen is
formation was studied. Kinetic measurements were designed most likely caused by the photolytic deprotection of the
to compare 5-(4-hydroxybenzyl)uracil and p-cresol as LG; to amine, which results in reactive side-products (Scheme 1).13
determine any influence of the uracil moiety on the amide However, their low concentration and poor resolution limited
bond formation, as pyrimidinones are known for their catalytic the definitive identification. Fortunately, this was not the case
effect.12 Indications for a small catalytic effect were found but for the oligomerisation products. Oligomers of phenylalanine
the potential influence of LG is minimal in comparison to up to tetramers were identified, capped by the 5-(4-
solvent effects on peptide bond formation (SI, kinetics).
hydroxybenzyl)uracil LG. Furthermore, a few hydrolysis
Spontaneous formation of peptides, with or without uracil in product could be identified (Figure 3C). All products were
the LG, was studied by deprotecting a solution of monomer in identified using simultaneous MS spectra (LC-MS, Figure
DMSO by UV irradiation, followed by the addition of an equal 3A,3B) or with retention time derived from commercial
volume of either water, buffer and/or a solution of references (Figure 3D).
oligonucleotide. After a set time of incubation, the crude
reaction mixture was analysed by HPLC. An example can be
Figure 2. The photolytical cleavage of the model compound: A) representative example. Photolysis of the model compound of Figure 1C followed by incubating 1h at room
temperature, with either 5 mol% of oligodeoxyadenosine (red) or water (H2O). UV detection at 215 nm, zoom with identification of species. Full spectrum and other data in the SI.
B) AUC analysis of identified species. The mean Area Under the Curve (AUC) of peptide species with (red) or without (blue) added oligonucleotide, as identified in A, are plotted as
a relative percentage of the total AUC. Error values of three independent experiments; LG = 5-(4-hydroxybenzyl)uracil (insert), SM = starting material.
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Figure 3. Identification of investigated species: A) Ion extract chromatograms (IEC) of the main products: the LG and LG-capped amino acid/peptides. B) Normalized IEC of main
products illustrating increasing amounts of diasteroisomers with polymerisation and the increasing difficulty to observe higher polymerized species. C) Normalized IEC of side
products detected. D) Plot of retention times of LG capped oligomers (experimental data) versus uncapped, linear oligomers of phenylalanine (commercial references) illustrating
the
chemical
analogy
and
predictable
retention
times.
LG
=
(5-(4-hydroxybenzyl)uracil)
The mass spectrum in Figure 3B additionally revealed (dA)10 (Figure S8).A brief screening of different
secondary peaks with equal mass for oligomerized species at oligonucleotides can be seen in Figure 4. Oligodeoxythymidine
different retention times and with increasing relative intensity (dT)10 gave no significant improved oligomerisation compared
at greater chain length. This observation of presumed to control conditions, as did the alternating sequence of
diastereoisomers agrees with previously obtained results on deoxyadenosine and -cytosine d(CA)5. However, (dC)10 did
peptide formation of activated species. 14
increase oligomerisation with a similar effect on LGPhe3, but
Another interesting find is the relatively low amounts of with lower efficiency on producing LGPhe2 and LGPhe4 as
diketopiperazine (DKP) in all of the conducted analyses. compared to (dA)10.
Whether it be a with or without an oligonucleotide additive; In light of previous results, the influence of pH catalysis was
the cyclized product is only a minor side product of this system investigated. Unless specified, the reaction pH was
in contrary to other known systems3, even when using phenyl uninfluenced, resulting in an observed value of 6.1-6.5 after
esters.14 However, most important is the increase of trimer chromatographic analysis. A slight acidic pH (pH = 6) favoured
(LGPhe3) and tetramer (LGPhe4) by adding the oligonucleotide the product distribution towards oligomerized species (Figure
(Figure 2). This was confirmed by plotting the main products as S4, S5) in control conditions (without oligonucleotide). This
a relative percentage towards the total absorbance from three distribution is influenced by the acidic additive if we compare
independent experiments (with or without oligonucleotide) in both. Nevertheless, with (dA)10, higher oligomers (LGPhe3 and
Figure 2B. It should be noted that this remains a qualitative LGPhe4) followed the same increasing trend as seen in
confirmation against the control experiments as the growing aqueous solutions. Buffering at pH 7.5 follows this trend,
chain adds to the molecular extinction coefficient, leading to although the effects are more pronounced (Figure S6).
overestimation of larger species when comparing them
individually in one experiment. Furthermore, larger oligomers
(n > 4) were currently not found. Figure 3A shows the rapid
decay of MS signal intensity with larger species, probably due
to the combination of low concentration and difficult
ionisation under the conditions used e.g. supramolecular
assembly15 and gas-phase adducts might results in assemblies
too large to detect. Additionally, the extrapolation of Figure 3D
for LGPhe5 led to an area of several small peaks (tR ≈ 24min)
which might also overlap with the remaining starting material.
Figure 4. Testing different oligonucleotides: Overlay of standard photolysis experiment
By switching the LG to p-cresol, no noticeable change in
while using various oligonucleotides. UV detection at 215 nm, zoom with identification
of studied species.
reaction mixture composition was observed upon addition of
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An alkaline pH of 8.4 results in a distinctively different pattern H.P.M. is a doctoral fellow of FWO Vlaanderen (11B6313N),
with many new and unresolved peaks (Figure S7). The Belgium. This research received funding from FWO
oligomers could be determined, but no effect of the Vlaanderen, Belgium (G078014N). We thank Guy Schepers for
oligonucleotide was witnessed. Furthermore, incubating the providing the oligonucleotides and Chantal Biernaux for
samples for a prolonged period of time did not resulted in editorial help
higher yields of oligomers (Figure S8, S9). On the contrary, the
samples degraded and the mixtures became more complex
Notes and references
presumably caused by side reactions with the (previously
mentioned) reactive intermediates of the photolytical
deprotection.
1
2
T. Inoue and L. E. Orgel, Science, 1983, 219, 859-862.
K. Gorska and N. Winssinger, Angew. Chem. Int. Ed. Engl.,
2013, 52, 6820-6843.
3
4
(a) A. L. Weber and L. E. Orgel, J. Mol. Evol., 1981, 17, 190;
(b) A. L. Weber and L. E. Orgel, J. Mol. Evol., 1979, 13, 185.
(a) M. L. McKee, A. C. Evans, S. R. Gerrard, R. K. O'Reilly, A. J.
Our preliminary studies on peptide oligomerisation reactions
shows promise as implementation of an Informational Leaving
Group (ILG)16, inspired by the chemical simplification of tRNA.
However some issues still need to be addressed. First of all,
the in situ activation of the reaction by irradiation causes more
analytical problems than the issues we avoid in producing the
starting material. Secondly, the lipophilicity of the starting
material forced the use of DMSO/aqueous solutions. The
resulting oligomers are equally poorly soluble and prone to
Turberfield and E. Stulz, Org. Biomol. Chem., 2011, 9, 1661;
(b) K. M. Wilcoxen, L. J. Leman, D. A. Weinberger, Z. Z. Huang
and M. R. Ghadiri, J. Am. Chem. Soc., 2007, 129, 748; (c) W.
Meng, R. A. Muscat, M. L. McKee, P. J. Milnes, A. H. El-
Sagheer, J. Bath, B. G. Davis, T. Brown, R. K. O'Reilly and A. J.
Turberfield, Nat. Chem., 2016, 8, 542-548.
5
B. Lewandowski, G. De Bo, J. W. Ward, M. Papmeyer, S.
Kuschel, M. J. Aldegunde, P. M. Gramlich, D. Heckmann, S.
M. Goldup, D. M. D'Souza, A. E. Fernandes and D. A. Leigh,
Science, 2013, 339, 189.
supramolecular effects15, hampering analysis.
A different
method for reaction initiation (e.g. pH shift of the free
ammonium salt in solution) and a different amino acid could
help eliminating organic solvents, increase signal-to-noise and,
in result, should lead to witnessing longer oligomers. Thirdly,
the catalytic effect of the oligonucleotide is not solely
mediated by base pairing interactions. Although lacking an
uracil moiety in the LG does not produce any catalytic effect, it
is not only (dA)10 that influences the reaction. The presence of
an exocyclic amine seems to favour the peptide bond
formation17, which could be mediated by other interactions
with the ILG such as dipole-dipole interaction or salt-like
bridges. A similar oligomerisation effect is seen at acidic pH.
However, the oligonucleotide supplements the latter and acts
as organic catalyst at neutral pH. Additionally, it probably
provides a scaffold for directing the catalytic fragments, as an
alternating sequence of deoxyadenosine and -cytosine was not
as efficient. Therefore, NMR studies and titration experiments
will be part of future studies to confirm the specific nature of
the observed catalytic effect. Bridging the information gap
between nucleic acids and amino acid is not only important to
6
7
P. E. Nielsen, Chem. Biodivers., 2007, 4, 1996.
X. Piao, X. Xia, J. Mao and D. Bong, J. Am. Chem. Soc., 2015,
137, 3751-3754.
M. P. Robertson and S. L. Miller, Science, 1995, 268, 702.
J. A. McCloskey and P. F. Crain, Nucleic Acids Res., 1998, 26
196.
8
9
,
10 J. A. Walder, R. Y. Walder, M. J. Heller, S. M. Freier, R. L.
Letsinger and I. M. Klotz, Proc. Natl. Acad. Sci. USA, 1979, 76
51.
11 S. A. Robertson, J. A. Ellman and P. G. Schultz, J. Am. Chem.
Soc., 1991, 113, 2722-2729.
12 X.-Q. Liu, L. Jin, C. K. Kim and Y. Xue, J. Mol. Catal. A: Chem.,
2012, 355, 102.
13 A. Patchornik, B. Amit and R. B. Woodward, J. Am. Chem.
Soc., 1970, 92, 6333.
14 D. Beaufils, S. Jepaul, Z. Liu, L. Boiteau and R. Pascal, Orig.
Life Evol. Biosph., 2016, 46, 19.
15 Z. Shlomo, T. P. Vinod, R. Jelinek and H. Rapaport, Chem.
Commun., 2015, 51, 3154-3157.
16 (a) X.-P. Song, M. Maiti and P. Herdewijn, J. Syst. Chem.,
,
2011, 2, 3; (b) E. Bigan, H.-P. Mattelaer and P. Herdewijn, J.
Mol. Evol., 2016, 82, 93.
17 (a) F. M. Menger, A. V. Eliseev, N. A. Khanjin and M. J.
Sherrod, J. Org. Chem., 1995, 60, 2870; (b) D. N. Reinhoudt,
D. M. Rudkevich and F. de Jong, J. Am. Chem. Soc., 1996, 118
6880.
18 (a) B. H. Patel, C. Percivalle, D. J. Ritson, C. D. Duffy and J. D.
expand
chemistry
templated
and
catalysed
by
,
oligonucleotides, but contributes to the open-ended quest in
search of the origin of translation. Current ideas on the origin
of life emphasize the intertwining chemistry of both
biomolecules involved.18 Early attempts at peptide formation
on nucleic acid backbones, including PNA, were inefficient.3, 6,
Sutherland, Nat. Chem., 2015, 7, 301-307; (b) H. Griesser, P.
Tremmel, E. Kervio, C. Pfeffer, U. E. Steiner and C. Richert,
Angew. Chem. Int. Ed. Engl., 2017, 56, 1219-1223; (c) H.
Griesser, M. Bechthold, P. Tremmel, E. Kervio and C. Richert,
Angew. Chem. Int. Ed. Engl., 2017, 56, 1224-1228.
19
A more successful approach was improving self-replicating
peptides20 by introducing nucleobase side chains.21 By
changing the oligonucleotide in our model to RNA, our model
may become an example of the catalytic prowess modified
nucleobases and short oligonucleotides might have held in the
prebiotic world.
19 A. Bencheikh and L. E. Orgel, J. Mol. Evol., 1990, 30, 315.
20 D. H. Lee, J. R. Granja, J. A. Martinez, K. Severin and M. R.
Ghadiri, Nature, 1996, 382, 525.
21 S. Matsumura, T. Takahashi, A. Ueno and H. Mihara, Chem.
Eur. J., 2003, 9, 4829.
Acknowledgements
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