Biomimetic protecting-group-free 2′, 3′-selective aminoacylation of nucleosides and nucleotides

Article abstract of DOI:10.1039/c0ob00795a

Aminoacyl phosphate monoesters can be prepared free of an amino-protecting group and used directly in lanthanum-promoted selective monoacylation of either the 2′ or 3′-hydroxyl of nucleosides and nucleotides. For example, phenylalanyl ethyl phosphate rapi

Full text of DOI:10.1039/c0ob00795a

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Biomimetic protecting-group-free 2¢, 3¢-selective aminoacylation of
nucleosides and nucleotides†
Sohyoung Her and Ronald Kluger*
Received 28th September 2010, Accepted 18th November 2010
DOI: 10.1039/c0ob00795a
Aminoacyl phosphate monoesters can be prepared free of
an amino-protecting group and used directly in lanthanum-
promoted selective monoacylation of either the 2¢ or 3¢-
hydroxyl of nucleosides and nucleotides. For example, pheny-
lalanyl ethyl phosphate rapidly forms esters with either of
the 2¢ or 3¢-hydroxyls of ribonucleosides and nucleotides in
the presence of lanthanum ions in aqueous buffer. Oligomer-
ization of the aminoacyl phosphate is much slower than
ester formation and is not a competitive process. Competing
hydrolysis of the reagent is slow. By extension, this route
should provide a simplified general route to synthetically
aminoacylated derivatives of tRNA.
Chemical aminoacylation of the 2¢ or 3¢-hydroxyls of nucleosides,
nucleotides, and oligonucleotides is normally a multistep process
involving protection and deprotection of numerous functional
groups in the initial reaction components.1–3 In contrast, the cor-
responding enzymatic reactions occur directly, utilizing enzyme-
bound aminoacyl adenylates.4–6 This biological intermediate is
Scheme 1 Lanthanum coordination and aminoacylation.
generated from the reaction of ATP and a specific amino
acid, which activates the aminoacyl group toward substitution.
unnatural amino acids into proteins.15 However, the required
It also provides a complex entity whose binding interactions
deprotection prior to ribosomal utilization adds a step where
with the enzyme permit precise orientation for the required
direct utilization is clearly preferable. Furthermore, the procedure
reaction.7–9 Methods for direct chemical aminoacylation have been
as reported required a 1000 fold excess of reagents to obtain the
investigated utilizing a biomimetic approach, where N-protected
aminoacylated tRNA in any useful quantity.
aminoacyl phosphate monoester and lanthanum ion serve as
The use of synthetic routes that are free of protecting groups
functional analogues of an aminoacyl adenylate and enzyme.10
at critical stages has become an area of significant interest and
In this system, it is proposed that binding interactions between
has been illustrated with compelling examples.16–18 The use of an
the enzyme and the substrate are mimicked by the formation of
acylating agent with a free amino group raises obvious concerns.
a bis-bidentate complex of the reactants and lanthanum. This
First, is the amino group capable of reacting with the activated
model is based on the analysis of related reactions by Clarke and
carboxyl of a second aminoacyl phosphate as these compounds
coworkers as well as the observed specificity for monoacylation of
react readily with other amines?19 Second, will the unprotected
diols (Scheme 1).11–14
compound selectively form an ester with a terminal diol as do their
The recent report by Duffy and Dougherty of aminoacylation
N-protected analogues? A promising precedent can be found in the
of the 3¢-terminal of tRNA and the utilization of the deprotected
earliest examples of synthetic aminoacyl adenylates. These were
product in an expression system provides an important demonstra-
reported to be produced without protecting groups; however, the
tion of the feasibility of direct aminoacylation for incorporating
materials were not analyzed and were assumed to be unstable.20,21
Where such species are formed transiently, they have been used to
Davenport Laboratories, Department of Chemistry, University of Toronto,
80 St. George Street, Toronto, Ontario, Canada M5S 3H6. E-mail: rkluger@
chem.utoronto.ca; Tel: +1 416-978-3582
† Electronic supplementary information (ESI) available: Materials and
methods, synthetic procedures for unprotected acyl phosphates, analytical
and spectroscopic data, chromatograms. See DOI: 10.1039/c0ob00795a
test theories of the origin of peptide formation in living systems
and appear to function productively.22–24
We now report the preparation and characteri-
zation of protecting-group-free aminoacyl phosphate esters as
well as their successful use in lanthanum-directed 2¢, 3¢-selective
676 | Org. Biomol. Chem., 2011, 9, 676–678
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monoaminoacylation of nucleosides and nucleotides. The
optimization of these methods now presents an opportunity to
extend the process to reaction with tRNA where isolation or
spectroscopic detection of products remains a challenge.
Further analysis of the isolated aminoacyl esters was carried
out with H NMR (Fig. 2). The chemical shift for the signal of
1
the 1¢-proton of the ribofuranoside depends on the location of the
ester: the doublet is further downfield for the 2¢-ester than for the
3¢-ester due to the interaction of the 2¢-ester with the aminoacyl
moiety. Therefore, the site of aminoacylation can be determined
from the relative chemical shifts. The doublet corresponding to
the 1¢-proton of the ribofuranoside appears at d 6.06 for the 2¢-
ester while the 3¢-ester signal appears at d 5.88. The same trend is
observed for the chemical shifts of the 6¢ proton of the pyrimidine
ring. The signals appear at d 8.03 and d 7.92 for the 2¢ and
3¢-esters, respectively. Based on this analysis, the two peaks in
the HPLC corresponding to the Phe monoesters of uridine were
assigned as the 2¢-ester (22 min) and 3¢-ester (37 min). Integration
of the chromatograms establishes that the ratio [2¢-ester] : [3¢-ester]
is approximately 1 : 2, which reflects the lower energy of the 3¢-
ester. These observations are consistent with the results reported
for N-t-BOC-aminoacyl ethyl phosphates.10,25,26 In addition, the
NMR spectrum of either ester after separation by HPLC results
in peaks for both esters being observed. This requires that the two
mono-esters equilibrate, consistent with well-known equilibration
between 2¢ and 3¢-aminoacyl esters.27–29
Aminoacyl phosphate monoesters were produced by deprotec-
tion of N-t-BOC-aminoacyl ethyl phosphates, compounds that
are formed by the coupling of the protected amino acid and
ethyl phosphate.25 The protecting group is needed to direct the
precursor to form the anhydride rather than an amide. Ethyl
dichlorophosphate is converted to ethyl phosphoric acid by
addition to water and then neutralized with tetraethylammonium
hydroxide to obtain bis(tetraethylammonium) ethyl phosphate. N-
t-BOC-phenylalanyl ethyl phosphate (BOCPheEP) was prepared
by DCC-promoted coupling of bis(tetraethylammonium) ethyl
phosphate and N-t-BOC-phenylalanine. The amino group was
liberated by addition of a small amount of trifluoroacetic acid.
The product was precipitated with added acetone to give PheEP
as a white powder (30% yield). The stereochemical integrity of the
1
sample is maintained, evidenced by H NMR spectra indicating
no exchange of the a-proton of PheEP in D2O during the course
of the reactions.19
We then tested the reactivity and regiospecificity of com-
binations of ribonucleosides (adenosine, cytidine, uridine) and
ribonucleotides (5¢-AMP, 5¢-CMP) with PheEP in the presence
of lanthanum triflate. Reactions were conducted with equimolar
concentrations of reactants in 100 mM, pH 6 MES (2-(N-
morpholino)ethanesulfonic acid) buffer at room temperature.
These were quenched with a saturated solution of EDTA to remove
1
lanthanum ions. H NMR, high resolution MS-ESI and HPLC
(C18 reversed phase column) were used to monitor the reaction
and characterize products (Fig. 1).
Fig. 2 1H NMR of two phenylalanine monoesters of uridine separated by
reversed phase HPLC (A: Ester 1, B: Ester 2) Note that the 2¢ and 3¢-esters
equilibrate.
Rapid mono-aminoacylation was also achieved in reactions of
PheEP with adenosine, cytidine, 5¢-AMP and 5¢-CMP. Analysis
with HPLC and MS-ESI confirmed the formation of PheEP-
monoesters of adenosine (calculated m/z = 415.1724, found m/z =
415.1711, 415.1720) cytidine (calculated m/z = 391.1612, found
m/z = 391.1599, 391.1605), 5¢-AMP (calculated m/z = 493.1242,
found m/z = 493.1197, 493.1263), and 5¢-CMP (calculated m/z =
469.1129, found m/z = 469.1143, 469.1156).
Control reactions without added lanthanum ion were performed
with equimolar concentrations of ribonucleosides and PheEP in
MES (pH 6, 100 mM) buffer. The reactants and the hydrolysis
product (Phe) were observed by HPLC with no ester or amide
peaks. The requirement for the cis-diol, which in principle
establishes selectivity for the 3¢-terminal of any RNA, was tested
with 2¢-deoxycytidine as a reactant in the presence of lanthanum
ion. After 60 min, only the starting materials and the hydrolysis
products were observed via HPLC. This is consistent with the
proposed mechanism of aminoacylation proceeding exclusively
Fig. 1 Reversed phase HPLC chromatogram of lanthanum-catalyzed
aminoacylation of uridine with PheEP after 1 h. Reaction conditions:
[La(OTf)3] = [uridine] = [PheEP] = 10 mM in MES buffer (pH 6, 100 mM)
at 25 ◦C.
The reaction of uridine with PheEP in the presence of lanthanum
triflate is complete in less than one minute, giving a mixture of
monoaminoacylation products (HPLC elution times of 22 min and
37 min) as well as the hydrolysis product, phenylalanine (Phe, 12
min). The product peaks were isolated and characterized by high-
resolution ESI(+)-MS as phenylalanyl monoesters of uridine (M +
H+ calculated m/z = 392.1452, found m/z = 392.1454, 392.1444).
The yield of esters is 60–70% based on the integrated areas on
chromatograms. HPLC analysis also showed that the products
were formed within the first minute following addition of the
reactants. The resulting aminoacyl ester is resistant to hydrolysis,
being unchanged in neutral solution for hours.
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via bis-bidentate coordination of lanthanum ion by an aminoacyl
phosphate and the 2,3-diol of terminal ribosyl derivative.
assist in developing a chemical catalytic protocol for conveniently
synthesizing aminoacyl-tRNA esters for use in ribosomal forma-
tion of proteins with amino acids that are not specified by the
genetic code.15,30–32 We are currently investigating this process in
order to expand the scope of the methodology.
We thank NSERC Canada for support of this research through
a Discovery Grant and for a Postgraduate Scholarship to SH. Ash-
ley Trang assisted in the laboratory and Tara Andrusiak provided
preliminary studies in this system.
An aminoacyl ethyl phosphate is unusual in that it contains both
a free amino group and a reactive carboxyl group. Since a free
amine would normally be a more reactive nucleophile than water
or the hydroxyl groups of ribose ring, the free amino group should
react as a nucleophile toward the acyl group of another aminoacyl
phosphate ester to form an amide. At pH higher than the pKA of
the PheEP amino group (pKA = 7.8),25 this process would compete
with aminoacylation or hydrolysis. Thus, PheEP polymerizes to
form oligopeptides when incubated in 250 mM pH 8 HEPES
buffer. However, this is suppressed where the solution’s pH is below
the pKA of the amino group. At a lower pH, the N-protonated form
of PheEP is the major species and is not a nucleophile. When the
lanthanum-catalyzed reactions are carried out in pH 6 MES buffer,
polymerization is very slow compared to the rapid monoacylation.
In effect, the proton is a mobile protecting group (Scheme 2).
Notes and references
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In conclusion, we have demonstrated that efficient lanthanum-
promoted aminoacylation of ribonucleosides and ribonucleotides
can be achieved with an aminoacyl phosphate ester with a free
amino group. The methods outlined here overcome the problem
of low yields as seen in previous reports. These results also predict
that the protecting-group-free aminoacyl phosphate esters can
be utilized in the direct and selective aminoacylation at the 3¢-
terminal hydroxyl of oligonucleotides and tRNA. This should
678 | Org. Biomol. Chem., 2011, 9, 676–678
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The Royal Society of Chemistry 2011
©