Peptidomimetic bond formation by DNA-templated acyl transfer

Article abstract of DOI:10.1039/c0ob00753f

The efficiencies of DNA-templated acyl transfer reactions between a thioester modified oligonucleotide and a series of amine and thiol based nucleophiles are directly compared. The reactivity of the nucleophile, reaction conditions (solvent, buffer, pH) a

Full text of DOI:10.1039/c0ob00753f

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Peptidomimetic bond formation by DNA-templated acyl transfer†
Mireya L. McKee,a Amanda C. Evans,b Simon R. Gerrard,c Rachel K. O’Reilly,*d Andrew J. Turberfield*a and
Eugen Stulz*c
Received 20th September 2010, Accepted 23rd November 2010
DOI: 10.1039/c0ob00753f
The efficiencies of DNA-templated acyl transfer reactions between a thioester modified oligonucleotide
and a series of amine and thiol based nucleophiles are directly compared. The reactivity of the
nucleophile, reaction conditions (solvent, buffer, pH) and linker length all play important roles in
determining the efficiency of the transfer reaction. Careful optimisation of the system enables the use of
DNA-templated synthesis to form stable peptide-like bonds under mild aqueous conditions close to
neutral pH.
use in biology and medicine. Acyl transfer reactions require an
Introduction
acyl donor group, such as an ester or activated ester, which
The synthesis of novel, biologically active peptidomimetic
can be attacked by a nucleophile which is commonly a nitrogen
based reagent. Liu and co-workers14,15 have used DTS to carry
out acyl transfer reactions between DNA-linked amino acids to
generate tripeptides. Native chemical ligation16 has been used by
Grossmann and Seitz17 to perform a DNA-catalyzed transfer of a
reporter group using iso-cysteine and thioester-modified peptide
nucleic acids, and by Stetsenko and Gait18 to conjugate peptides
to oligonucleotides. However, current synthetic systems lag behind
the natural systems for the synthesis of polypeptides by ribosomal
and non-ribosomal synthesis (NRPS).19,20 These natural systems
achieve efficient peptide bond formation with precise sequence
control by templated acyl transfer chemistries. The NRPS reaction
involves the nucleophilic attack of a primary amine on a thioester
within a controlled hydrophobic environment under physiological
conditions. Using DNA and peptide scaffolds, Joyce and co-
workers21 and Ghadiri and co-workers,22 respectively, have shown
that peptide-forming acyl transfer reactions can be performed,
without enzymes, in aqueous solvents at neutral or slightly basic
pH. This highlights the potential application of the acyl transfer
reaction as a tool for peptide synthesis in a synthetic NRPS
scheme.
Despite the many successful DNA templated acyl transfer
reactions achieved to date, there is no published study that
systematically compares alternative nucleophiles to the amine
using the same templates. The use of nucleophiles with tunable
reactivity could enable implementation of an adaptable system
in which a library of components could be linked using a
single set of reaction conditions. We hypothesized that stronger
nucleophiles would react more quickly and at lower pH than
regular amines and might therefore be suitable building blocks for
acyl transfer under physiological conditions. We have evaluated a
range of nucleophiles with different reactivities (amine, hydrazine,
hydrazone-nicotinate, benzylamine, aminooxy, hydrazide) under
the same reaction conditions and have varied temperature, pH
molecules normally requires sequential reactions of building
blocks to build up a specific sequence. Building blocks can be
based on natural and non-natural amino acids, or on molecules
that can be connected using specific ligation chemistries such as
amide formation, Wittig chemistry, Diels–Alder reaction or click
chemistry. A key goal of synthetic chemistry is to maintain a high
level of control of the sequence of reactions, in particular at low
reagent concentrations. Many advances have been made towards
this goal using the concept of DNA-templated synthesis (DTS),1–9
using the unique sequence-directed interactions of DNA to control
the relative positions of small-molecule reactants. DTS has been
used in nucleic acid sensing, sequence-specific DNA modifications,
the creation and assessment of DNA-encoded molecular libraries,
and the directed evolution of molecules linked to DNA.10–13 DTS
permits incorporation of natural and unnatural building blocks
into synthetic biomimetic oligomers, adding to its potential value
for drug discovery.
Acyl transfer reactions are of particular interest in peptide
chemistry and in the creation of peptidomimetic molecules for
aDepartment of Physics, University of Oxford, Clarendon Laboratory,
Parks Road, Oxford, United Kingdom, OX1 3PU. E-mail: a.turberfield@
physics.ox.ac.uk; Fax: (+44) 1865 272400; Tel: (+44) 1865 272200
bDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, United Kingdom, CB2 1EW
cSchool of Chemistry, University of Southampton, Highfield, Southampton,
United Kingdom, SO17 1BJ. E-mail: est@soton.ac.uk; Fax: (+44) 23 8059
3131; Tel: (+44) 23 8059 9369
dDepartment of Chemistry, University of Warwick, Gibbet Hill Road, Coven-
try, United Kingdom, CV4 7AL. E-mail: Rachel.OReilly@warwick.ac.uk;
Fax: (+44) 24 7652 4112; Tel: (+44) 24 7652 3236
† Electronic supplementary information (ESI) available: DNA sequences
used in this work, synthesis procedures and characterisation of ac-
tivated esters, HPLC chromatograms and ESI spectra of oligonu-
cleotide adapters and products, additional experimental results. See DOI:
10.1039/c0ob00753f
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Table 1 Characterisation data of modified oligonucleotides
DNA adapter
Modification
Expected Mass [Da]
Calculated Mass [Da]
%Yielda
T1-TAM
Thioester
Thioester
Thioester
Histamine/thioester
Benzylamine
Aminooxy
Hydrazide
Hydrazine
7961.1
9520.3
8173.5
8322.8
9653.4
9593.3
9682.4
9893.4b
9695.4b
9608.3
9894.5
7959.4
9519.1
8171.9
8320.9
9652.1
9590.1
9681.2
9892.0
9693.3
9607.8
9893.8
95.1
92.6
35.2
55.2
T2-TAM
T1-PEG-TAM
T1-HIS-TAM
N3
N4
N5
N6
N7
N8
N9
83.9 (>95)
31.5 (>95)
49.9
35.9 (c)
ca. 60 (c)
67.2 (>99)
41.0
Hydrazino-nicotinate
Thiol
Hydrazide
a Yields shown refer to activated ester couplings; data in parentheses refer to deprotection steps. b Mass of modified oligonucleotide with protecting
group measured before UV363 deprotection (N5) or at pH 11.0 (N6). c Oligonucleotide conjugates degraded over time and could not be isolated after
deprotection.
and solvent composition. In addition, both the linker to the DNA
template and the distance between the reacting groups on the
template have been varied, as the distance between reactive groups
has been shown to be a key factor in DTS chemistries.23 Reaction
yields were measured using PAGE and HPLC, and the products
were confirmed by mass spectrometry. As expected, lower pKa
values and a-effects favor acylation. However, we have found
that a careful balance between all the reaction parameters is
required to optimize acyl transfer from one DNA strand (acyl
donor) to another (acyl acceptor). The more nucleophilic modified
oligonucleotides are reactive at near-neutral pH and are promising
reagents for DNA-templated acyl transfer chemistry. We propose
that these transfer systems could serve as a platform for biomimetic
DTS based on the NRPS system.
The hydrolytic stability of thioester-modified oligonucleotide
T1-TAM was analyzed by PAGE and HPLC. The attached
fluorophore permits easy monitoring of the thioester bond because
the fluorescent tag is removed upon hydrolysis. After 48 h
incubation at 38 ◦C, the TAM-thioester oligonucleotide, the
thiolated oligonucleotide, and free TAMRA were separated by gel
electrophoresis and reverse-phase HPLC (Fig. 2). The results show
that T1-TAM is stable over a wide pH range in aqueous solutions.
The formation of free TAMRA and thiolated DNA indicated
that thioester hydrolysis occurs above pH 9.5. This confirms
that thioester-modified oligonucleotides may be used as activated
donors for DNA-templated acyl transfer reactions in aqueous
solutions,21 although high pH conditions should be avoided.
Results and discussion
pH stability of thioester-modified oligonucleotides
To investigate the use of thioesters as acyl donors in DTS, the
fluorescent dye TAMRA (TAM) was attached to thiol-modified
oligonucleotides, using its succinimide activated ester form, to
give the fluorescent thioesters T1-TAM, T1-PEG-TAM, T1-HIS-
TAM, and T2-TAM (Fig. 1) in 35–95% yields (Table 1). The
first three thioester-modified adapter oligonucleotides T1 have the
same nucleobase sequence (see ESI† for synthesis and sequences);
T2 is complementary to the nucleophile-modified adapters (see
below).
Fig.
2
Hydrolytic stability of the fluorescent TAMRA thioester,
T1-TAM: HPLC chromatograms of T1-TAM after 48 h incubation at
38 ◦C in universal buffer at indicated pH.
Nucleophile-modified oligonucleotides
The most commonly used nucleophiles in acyl transfer reactions
are primary amines, which upon acyl transfer form stable amide
bonds. However, without enzymatic catalysis, these reactions are
problematic under physiological conditions as amine protonation
greatly decreases reaction rates. It is therefore important to
investigate the use of nucleophiles with lower pKa values that
could potentially yield higher transfer efficiencies while still
generating stable linkages. Benzyl amine, aminooxy, hydrazide and
hydrazine moieties were chosen because they are more nucleophilic
Fig. 1 Structures of thioester-modified oligonucleotides (acyl donor).
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than simple amines at pH 7.0 as a result of either an a-effect
(neighbouring heteroatom) or the benzylic substitution, and all
form stable peptidomimetic bonds when reacting with carbonyl
compounds.24,25 The reactivities of the nucleophiles, based on their
pKa value, are estimated to be in the order; amine < benzylamine
< hydrazine < aminooxy < hydrazide < hydrazino-nicotinate.26
Acyl transfer reactions between these enhanced nucleophiles and
thioesters should generate benzylamides, alkoxyamides, and hy-
drazones. Several research groups have already modified oligonu-
cleotides with these moieties using solid phase synthesis27,28 or
post-synthetic modifications.29,30
A series of nucleophile-modified oligonucleotides were prepared
post-synthetically by coupling activated esters to amino-modified
oligonucleotides N1 and N2 (Fig. 3). Thiol oligonucleotide N8
was included as a positive control as it should effect partial
transfer of the fluorescent marker by trans-thioesterification. In
some cases, the nucleophilic groups prepared contained a photo-
cleavable protecting group, nitro-veratryloxycarbonyl (NVOC),31
to allow facile unmasking of the nucleophile by exposure to UV363
.
The hydrazine-modified oligonucleotides N6 and N7 partially
decomposed during HPLC purification after deprotection (see
ESI†). Decomposition of hydrazine conjugates has been reported
previously.32,33 Deprotection was therefore carried out immediately
before or immediately after the oligonucleotides were combined
to initiate an acylation reaction. Synthesis yields of nucleophilic
and thioester-modified oligonucleotides are shown in Table 1.
Scheme 1 DTS system for monitoring acyl transfer reactions.
template S5. Various experimental conditions were investigated to
determine factors that affect acyl transfer efficiency, as described
below.
Length of linker and adjacent residues affect acyl transfer
efficiency
It is possible that additional adapter length and flexibility could
favour interaction between nucleophile and thioester. To test this
hypothesis, a PEG spacer was incorporated in oligonucleotides T1-
PEG-TAM, N2 and N9. Virtually no fluorescent group transfer
was observed from PEG-containing thioesters (see ESI†). The
effects of incorporating a PEG spacer in the nucleophile adapter
were much smaller, but it reduced reaction yield with both
amine- and hydrazide-modified oligonucleotides. We infer that the
advantage of increased conformational freedom imparted by the
flexible linker is outweighed by the increased distance between
tethered reactants.
Functional groups adjacent to the reaction site may also influ-
ence reactivity. For example, NRPS makes use of catalytic residues,
particularly histidines, in close proximity to the transfer site. In
addition, it has been shown that histidines on peptide scaffolds can
catalyze acylation reactions.35,36 To explore this effect in our DTS
system, we designed strand T1-HIS-TAM (Fig. 1) and explored
its effectiveness in acyl transfer reactions. Contrary to what was
expected, when a histamine residue is added to the system, the
reaction yields were reduced significantly. The imidazole residue
promoted thioester hydrolysis rather than acylation, particularly
at higher pH values. Ester hydrolysis has also been reported in
synthetic peptide esterases, which use histidine residues.37,38
Fig. 3 Structures of the nucleophile-modified oligonucleotides.
Design of DNA-templated system to monitor transfer of
fluorescent tag
A simple DTS system was designed to allow the progress of
DNA-templated acyl transfer reactions to be monitored by
PAGE (Scheme 1).34 Thioester- and nucleophile-modified oligonu-
cleotides were designed with different lengths, which could easily
be separated by PAGE; the progress of the reaction could therefore
be monitored by observing the transfer of fluorescence between
gel bands. The reaction was triggered by annealing stoichiometric
quantities of both adapter oligonucleotides to a complementary
Effects of temperature, co-solvents and added base on acyl transfer
efficiency
Fig. 4 shows the results of a comparison between reactions
performed at room temperature and at 38 ◦C. The yields of
acyl transfer were significantly higher at 38 ◦C, but a further
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Fig. 4 Comparison between acyl transfer reactions at room temperature
(R.T.) and at 38 ◦C. Acyl transfer reactions were carried out for 24 h in
100 mM sodium phosphate buffer, pH 8.5, with (lanes 12–15) and without
(lanes 8–11) 10% THF at R.T. (a) and at 38 ◦C (b). Lane 1: Template
oligonucleotide S5; Lane 2: T1-TAM; Lane 3: blank; Lane 4: N1; Lane
5: N2; Lane 6: blank (a)/N5 (b) ; Lane 7: N9; lanes 8–15: acyl transfer
reactions with the nucleophiles as in lanes 4–7. Red channel: TAMRA
(Ex532/Em605 nm); green channel: SybrGold DNA stain (Ex488/
Em530 nm).
Fig. 5 DNA architectures and acyl transfer efficiency. Lane 1: S5
(template oligonucleotide); lane 2: T1-TAM; lane 3: N1; lane 4: T1-TAM +
N1 control (no template); lanes 5-13: T1-TAM + N1 in the presence of a
template oligonucleotide incorporating a 0–8 nucleotide single-stranded
gap (oligonucleotides 5–13, ESI†); lane 14: T2-TAM + N1 (end-of-helix
architecture); lane 15: T2-TAM. Reactions were carried out in 100 mM
sodium phosphate buffer, 100 mM NaCl, pH 9.0, 10% THF at 38 ◦C for
24 h.
increase to 45 ◦C or 55 ◦C promoted hydrolysis rather than higher
acylation yields. Co-solvents THF (10%), and DMF (10%, not
shown) were also added to the reaction mixtures to test their effects
on acylation. Thioester-activated acyl transfer experiments using
model small molecules showed an improvement in both efficiency
and purity when 10% THF was added as a co-solvent (see ESI†),
and improvements in rate and purity were also observed for the
DNA-templated acyl transfer system studied here. However, no
significant changes in conversion were observed using 10% DMF
as a co-solvent. Addition of bases such as 10% (v/v) DIPEA or
10 mM Mg2+-imidazole39 resulted in thioester hydrolysis rather
than enhanced acyl transfer, as observed with the histamine-
modified thioester T1-HIS-TAM. As a result of these observations,
all subsequent experiments were performed under optimized
conditions in the presence of 10% THF and at 38 ◦C without
additional base.
end-of-helix geometry, in which reactive groups are conjugated to
the 5¢ and 3¢ ends of complementary oligonucleotides, produced
the highest product yield, as recently observed with the Wittig-
based DTS synthesis of longer oligomers.42 The end-of-helix
configuration was therefore used in reactions presented below.
Effect of pH on acyl transfer efficiency
pH has an important effect on nucleophile reactivity, which
is reduced by protonation. The reactivities of amine (N1) and
hydrazide (N5) nucleophilic oligonucleotides over the pH range
7.0–9.5 were compared using the end-of-helix configuration.
Peptide bond formation was observed at the higher end of the
pH range for both nucleophiles, but thioester hydrolysis also
increased with pH as expected (see ESI†). Product formation near
neutral pH, with negligible hydrolysis, occurred with the hydrazide
oligonucleotide only.
The difference in reactivity between the amine and hydrazide
nucleophiles was assessed by measuring product formation as
a function of time. The reactions were quenched by adding a
displacer oligonucleotide complementary to the T2-TAM adapter
(see ESI†) at specific time points; products were analyzed by
HPLC. The results, which are summarized in Fig. 6, show that
hydrazide oligonucleotide N5 has a 3- to 10-fold faster acylation
rate at pH 7.5 than amine N1, while at pH 9.5 the rate of acylation
for both nucleophiles is similar. It should be noted that even
for the most reactive nucleophile N5 the acyl transfer yields are
surprisingly low.
Effect of DNA adapter design on acyl transfer efficiency
The architecture of the template has been shown to significantly af-
fect the rates of DNA-templated reactions.40,41 We have compared
acyl transfer efficiencies with the “across-the-nick” system, with
variable gap length, and the end-of-helix geometry (Fig. 5). As
expected, no product was observed in a control reaction using the
across-the-nick system from which the template oligonucleotide
was omitted (lane 4). A single-stranded DNA spacer of 0–8
nucleotides was introduced into the template oligonucleotide to
separate the reactive groups attached to the 5¢ and 3¢ ends of the
hybridized oligonucleotide adapters (lanes 5–13). The optimum
spacer was found to be a single unpaired nucleotide (lane 6). The
Acyl transfer reactions between nucleophiles N1–N6 and
thioester T2-TAM, using the end-of-helix configuration, were
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Table 2 Characterization data of acyl transfer products with N1–N9
product yields. Throughout the range of pH values studied, both
the hydrazide- (N5) and hydrazine- (N6) modified oligonucleotides
produced the greatest yields, as expected given the enhanced
nucleophilicity conferred by the neighboring heteroatom a-effect.
However, in the case of hydrazine N6, the fluorescent product
isolated had a mass corresponding to the combined mass of
modified oligonucleotides T2-TAM and N6, indicating that an
undesired cross-linking reaction, rather than the expected acyl
transfer, had occurred. Apparent yields corresponding to this
reaction are therefore not included in Fig. 7. Hydrazide N5
produced the stable fluorescent acyl transfer product N5-TAM as
designed, with no crosslinked products observed. For nucleophiles
N1–N4, the pattern of acylation varied significantly with pH, with
yield initially increasing then saturating as pH was increased. The
reaction yield with the amine (N1) saturates at higher pH than in
the case of the benzylamine- (N3) and aminooxy- (N4) modified
oligonucleotides, correlating with its higher pKa, as expected.
Unexpectedly, the aminooxy group N4 was the least effective
nucleophile studied under these conditions, and the amine gave
higher yields than both aminooxy and benzylamine groups at
pH 9.5. As discussed above, higher pH values increase thioester
hydrolysis and are therefore undesirable. At all pHs studied, but
especially at low pH, the hydrazide (N5) produced the highest yield
of transfer product. The hydrazide-modified oligonucleotide, N5-
TAM is therefore of particular interest for reactions to be carried
out at near-neutral pH.
DNA adapter
Expected Mass [Da]
Calculated Mass [Da]
N1
N2
N3
N4
N5
N6
N7
N8
N9
9933.5
10145.8
10066.5
10006.5
10095.5
10067.5
10068.5
10021.5
10307.4
9932.4
10144.4
10065.4
10003.2
10093.3
17450.5a
n.d.b
10019.2
10306.1
a Mass of acylation product was not observed; detected mass could cor-
respond to crosslinked product between the two reactive oligonucleotides.
b Not determined.
Fig. 6 Comparison between reactivities of amine- and hydrazide-mod-
ified oligonucleotides as functions of pH. Time courses of acylation
reactions, monitored by HPLC, using N1 and N5 nucleophiles in 100 mM
sodium phosphate buffer at the indicated pH.
Conclusions
We have investigated the use of a range of alternative nucleophilic
groups in acyl transfer reactions controlled by DNA-templated
synthesis. A thioester group was chosen as the acyl donor due to its
hydrolytic stability in aqueous buffers up to pH 9.5. The thioester-
modified oligonucleotide included a fluorescent tag which al-
lowed reaction progress to be readily monitored. Oligonucleotide
adapters modified with amine, benzylamine, aminooxy, hydrazide,
hydrazine, hydrazino-nicotinate, and thiol groups were synthesized
and used as nucleophiles in acyl transfer reactions in aqueous
buffers. The reactions yielded products with stable peptide-like
linkages, including amide, alkoxyamide, hydrazide and thioester
bonds.
carried out by incubating the modified oligonucleotides for
48 h at 38 ◦C (Fig. 7, Table 2). Thioester N8 was used as a
positive control (the trans-thioesterification product equilibrated
to approx 40–50% with T2-TAM under all conditions studied).
The reactions were quenched by adding a displacer oligonucleotide
S14. New peaks corresponding to the products were identified
from the chromatograms (see ESI†) and were characterized by
mass spectrometry. HPLC peak areas were used to compare
Several parameters were found to affect acyl transfer efficiency,
including pH and the architecture of the DNA adapters that
enforce proximity between nucleophile and thioester. Nucleophiles
with lower pKa values are particularly effective in buffers close
to neutral pH, where the hydrazide group displayed at least
one order of magnitude enhanced reactivity relative to simple
amines. Acyl transfer reactions at near-neutral pH values have the
significant advantage that the thioester is more stable to hydrolysis
than at higher pH values. The more reactive nucleophiles can
also be expected to react at higher rates with other activated
esters. This chemistry has the potential to enable biologically-
relevant reactions such as the formation of protein- or peptide-
oligonucleotide conjugates or peptidomimetic molecules, which
may require neutral conditions to be stable or active. We propose
that the DTS of peptidomimetic bonds in aqueous buffers could
be used to create macromolecule libraries and to select and evolve
novel bioactive compounds.43,44
Fig. 7 Comparison of yields of acyl transfer products, using nucleophiles
N1, N3–N5, determined from peak areas in HPLC chromatograms.
Acylation reactions were carried out in 100 mM sodium phosphate
buffer, 100 mM NaCl, at pH 6.5–9.5 for 48 h at 38 ◦C. Product peaks
were identified by simultaneous measurement of TAMRA fluorescence
and A260
.
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23 Z. J. Gartner, R. Grubina, C. T. Calderone and D. R. Liu, Angew.
Chem., Int. Ed., 2003, 42, 1370–1375.
Acknowledgements
24 J. Kalia and R. Raines, Angew. Chem., Int. Ed., 2008, 47, 7523–7526.
25 T. Zatsepin, D. Stetsenko, M. Gait and T. Oretskaya, Bioconjugate
Chem., 2005, 16, 471–489.
Funding for this work was provided by EPSRC IDEAS Factory
grants EP/F056605/1, EP/008597/1 and linked grants. We thank
Dr Nicky King (University of Exeter), Professor Andy Tyrrell
(University of York) and Dr Fred Currell (Queen’s University
Belfast) for helpful discussions, and acknowledge Diluar Khan
(Warwick) and Thomas J. Bandy (Southampton) for initial
thioester work. Some of the equipment used in this research
was obtained through Birmingham Science City with support
from Advantage West Midlands (AWM) and part funded by the
European Regional Development Fund (ERDF).
26 The pKa values have been calculated using ACD/I-Lab service and
were compared with literature values (given in brackets) for closest
match: amine 10.7 (10.6; from H. K. Hall, Jr., J. Am. Chem. Soc.,
1957, 79, 5441–5444); benzylamine 8.8 (9.3; ibid.); aminooxy 3.4 (4.6;
ibid.); hydrazide 2.9 (3.0; from; P. R. Campodo´nico, M. E. Aliaga, J.
G. Santos, E. A. Castro and R. Contreras, Chem. Phys. Lett., 2010,
488, 86–89); hydrazine 4.9 (5.2; from; J. B. Conant and P. D. Bartlett, J.
Am. Chem. Soc., 1932, 54, 2881–2889); hydrazino-nicotinate 1.6 (-0.5;
from; M. Mure, D. E. Brown, C. Saysell, M. S. Rogers, C. M. Wilmot,
C. R. Kurtis, M. J. McPherson, S. E. V. Phillips, P. F. Knowles and D.
M. Dooley, Biochemistry, 2005, 44, 1568–1582).
27 S. Raddatz, J. Mueller-Ibeler, J. Kluge, L. Wa¨ss, G. Burdinski, J. Havens,
T. Onofrey, D. Wang and M. Schweitzer, Nucleic Acids Res., 2002, 30,
4793–4802.
References
28 S. Antsypovich and G. von Kiedrowski, Nucleosides, Nucleotides
1 Y. Brudno and D. R. Liu, Chem. Biol., 2009, 16, 265–276.
2 L. J. Prins and P. Scrimin, Angew. Chem., Int. Ed., 2009, 48, 2288–2306.
3 M. F. Jacobsen, J. B. Ravnsbaek and K. V. Gothelf, Org. Biomol. Chem.,
2010, 8, 50–52.
Nucleic Acids, 2005, 24, 211–226.
29 T. Zatsepin, D. Stetsenko, M. Gait and T. Oretskaya, Nucleic Acids
Symp. Ser., 2005, 133–134.
30 G. Grimm, A. Boutorine and C. He´le`ne, Nucleosides, Nucleotides
Nucleic Acids, 2000, 19, 1943–1965.
4 K. Meguellati, G. Koripelly and S. Ladame, Angew. Chem., Int. Ed.,
2010, 49, 2738–2742.
5 Z. Gartner and D. Liu, J. Am. Chem. Soc., 2001, 123, 6961–6963.
6 X. Li and D. Liu, Angew. Chem., Int. Ed., 2004, 43, 4848–4870.
7 T. Grossmann, A. Strohbach and O. Seitz, ChemBioChem, 2008, 9,
2185–2192.
8 I. Boll, R. Kramer, J. Brunner and A. Mokhir, J. Am. Chem. Soc., 2005,
127, 7849–7856.
9 D. Summerer and A. Marx, Angew. Chem., Int. Ed., 2002, 41, 89–90.
10 M. Rozenman, B. McNaughton and D. Liu, Curr. Opin. Chem. Biol.,
2007, 11, 259–268.
31 B. Amit, U. Zehavi and A. Patchorn, J. Org. Chem., 1974, 39, 192–196.
32 A. Dirksen and P. Dawson, Bioconjugate Chem., 2008, 19, 2543–2548.
33 J. Vulterin and J. Zy´ka, Talanta, 1963, 10, 891–898.
34 It should be noted that it was not our intention to optimise the yields of
the acyl transfer reactions, but to directly compare the efficiency of the
various nucleophiles under different conditions. Therefore, the yields
stated do not reflect the maximum possible yields as the reactions were
stopped and evaluated after a certain time, irrespective of whether they
had gone to completion or not.
35 K. Broo, M. Allert, L. Andersson, P. Erlandsson, G. Stenhagen, J.
Wigstrom, P. Ahlberg and L. Baltzer, J. Chem. Soc., Perkin Trans. 2,
1997, 397–398.
11 J. Scheuermann, C. Dumelin, S. Melkko and D. Neri, J. Biotechnol.,
2006, 126, 568–581.
12 A. Silverman and E. Kool, Chem. Rev., 2006, 106, 3775–3789.
13 M. Hansen, P. Blakskjaer, L. Petersen, T. Hansen, J. Hjfeldt, K. Gothelf
and N. Hansen, J. Am. Chem. Soc., 2009, 131, 1322–1327.
14 Z. Gartner, M. Kanan and D. Liu, J. Am. Chem. Soc., 2002, 124,
10304–10306.
36 L. Leman, D. Weinberger, Z. Huang, K. Wilcoxen and M. Ghadiri, J.
Am. Chem. Soc., 2007, 129, 2959–2966.
37 K. Broo, L. Brive, P. Ahlberg and L. Baltzer, J. Am. Chem. Soc., 1997,
119, 11362–11372.
38 L. Baltzer, K. Broo, H. Nilsson and J. Nilsson, Bioorg. Med. Chem.,
1999, 7, 83–91.
39 N. Sakai, N. Sorde and S. Matile, Molecules, 2001, 6, 845–851.
40 Z. Gartner, R. Grubina, C. Calderone and D. Liu, Angew. Chem., Int.
Ed., 2003, 42, 1370–1375.
41 T. Grossmann, L. Ro¨glin and O. Seitz, Angew. Chem., Int. Ed., 2008,
47, 7119–7122.
15 T. Snyder and D. Liu, Angew. Chem., Int. Ed., 2005, 44, 7379–7382.
16 P. Dawson, T. Muir, I. Clark-Lewis and S. Kent, Science, 1994, 266,
776–779.
17 T. Grossmann and O. Seitz, J. Am. Chem. Soc., 2006, 128, 15596–15597.
18 D. Stetsenko and M. Gait, J. Org. Chem., 2000, 65, 4900–4908.
19 D. Schwarzer, R. Finking and M. A. Marahiel, Nat. Prod. Rep., 2003,
20, 275–287.
42 M. L. McKee, P. J. Milnes, J. Bath, E. Stulz, A. J. Turberfield and R. K.
O’Reilly, Angew. Chem., Int. Ed., 2010, 49, 7948–7951.
43 B. N. Tse, T. M. Snyder, Y. H. Shen and D. R. Liu, J. Am. Chem. Soc.,
2008, 130, 15611–15626.
44 R. E. Kleiner, C. E. Dumelin, G. C. Tiu, K. Sakurai and D. R. Liu, J.
Am. Chem. Soc., 2010, 132, 11779–11791.
20 C. E. Salomon, N. A. Magarvey and D. H. Sherman, Nat. Prod. Rep.,
2004, 21, 105–121.
21 R. Bruick, P. Dawson, S. Kent, N. Usman and G. Joyce, Chem. Biol.,
1996, 3, 49–56.
22 K. Wilcoxen, L. Leman, D. Weinberger, Z. Huang and M. Ghadiri, J.
Am. Chem. Soc., 2007, 129, 748–749.
1666 | Org. Biomol. Chem., 2011, 9, 1661–1666
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