Synthesis and hydrolytic stability of 5′-aminoacylated oligouridylic acids

Article abstract of DOI:10.1016/S0040-4039(01)01076-0

Dimers and trimers of uridylic acid with a phenylalanine residue at their 5′-terminus were prepared in solution via intermediates with acid-labile protecting groups. A 5′-aminoacylated octanucleotide was prepared on solid support using a phosphoramidate linker and an allyloxycarbonyl-protected phenylalanine.

Full text of DOI:10.1016/S0040-4039(01)01076-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5681–5684
Synthesis and hydrolytic stability of 5%-aminoacylated
oligouridylic acids
Charles N. Tetzlaffa,b and Clemens Richerta,b,
*
aDepartment of Chemistry, University of Constance, Fach M 709, D-78457 Konstanz, Germany
bDepartment of Chemistry, Tufts University, Medford, MA 02155, USA
Received 10 May 2001; accepted 14 June 2001
Abstract—Dimers and trimers of uridylic acid with a phenylalanine residue at their 5%-terminus were prepared in solution via
intermediates with acid-labile protecting groups. A 5%-aminoacylated octanucleotide was prepared on solid support using a
phosphoramidate linker and an allyloxycarbonyl-protected phenylalanine. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
cleavage step under strongly basic conditions. To over-
come this difficulty, the current synthesis was
undertaken with acid-labile protecting groups for the
2%,3%- and 5%-hydroxyl groups of the ribonucleosides,
and initially also for the amino group of the aminoacyl
moiety. To make the approach attractive for routine
syntheses, we decided to focus on a route involving
commercially available RNA building blocks and sup-
ports only. This led to uridine derivative 1 with its
Aminoacylated ribonucleotides, in the form of charged
tRNAs, play a pivotal role in the translation of genetic
information. While tRNAs found in the ribosomal
machinery are exclusively 2%/3%-esterified nucleic acids, it
has recently been shown that 5%-aminoacylated ribonu-
cleotides can be formed in ribozyme-catalyzed reac-
tions,1 making it desirable to develop chemical
syntheses for such compounds. To our knowledge, pre-
vious synthetic work on 5%-aminoacylated RNA deriva-
tives has been limited to the aminoacylation of
nucleosides.2 Reports on 5%-aminoacylated 2%-deoxynu-
closide derivatives also exist.3 Out of an interest in
non-enzymatically catalyzed acyl transfer reactions and
expanding the synthetic chemistry of aminoacylated
RNA,4 we explored the chemical synthesis of 5%-
aminoacylated oligoribonucleotides. Here, we report
results from a study focused on phenylalanine-bearing
oligouridylic acids. Both a solution phase synthesis and
a synthesis on solid supports are being reported.
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl
(Fpmp)
group5 as the phosphoramidite building block and ace-
tonide 2 as the 3%-terminal residue (Scheme 1).
In a one-pot procedure, phosphoramidite 1, was con-
verted to dimer 3. Coupling with tetrazole activation,
followed by addition of oxidizer solution, and subse-
quent treatment with thiosulfate gave 3 in 92% yield
after chromatography on silica. Initially, solid Na2S2O3
was used in the last step of this one-pot procedure,
leading to a slow reduction of the excess iodine. Later
it was found that washing with a saturated thiosulfate
solution during the aqueous work-up gave a more rapid
conversion of the excess reagent. Dimer 3 was prepared
for aminoacylation by removal of the cyanoethyl
groups with triethylamine in acetonitrile (5:3 v/v) and
de-dimethoxytritylation with trichloroacetic acid in
CH2Cl2, giving 4 in 73% yield after precipitation and
washing with diethylether.6 The removal of the cya-
noethyl groups was performed, since published work on
the synthesis of peptide–DNA hybrids had shown that
free diesters give higher yields in acylation reactions
than triesters.7
2. Results and discussion
The key difficulty in preparing aminoacylated oligonu-
cleotides lies in the hydrolytic lability of the ester bond
that makes it impossible to use conventional syntheses
for oligonucleotides that involve a final deprotection/
Keywords: aminoacylated RNA; oligoribonucleotides; acylation reac-
tions.
Coupling of Boc-Phe-OH to 4 proceeded uneventfully
when the amino acid building block was activated with
* Corresponding author. Tel.: 49-(0)7531-88 4572; fax: 49-(0)7531-88
4573; e-mail: clemens.richert@uni-konstanz.de
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01076-0

5682
C. N. Tetzlaff, C. Richert / Tetrahedron Letters 42 (2001) 5681–5684
O
N
Ph
NH
O
O
H3N
O
O
O
Ph
NH
O
OH
O
O
O
O
N
O
O
N
P
O
1. tetrazole,
HO
N
O
N
NH
Boc-NH
NH
NH
O
O
R1-O
TFA
O
O
NH
O
N
O
O
Boc-Phe-OH,
HBTU, HOBT,
DIEA
DMT-O
O
O-Fpmp
O
O
O
O
O
N
O
2
O
O
P
O
NH
6
O-Fpmp
O
O
OH OH
O
N
O
P
2. I2, pyridine
3. Na2S2O3
O-Fpmp
OC2H4CN
R2-O
O
1
O
NH
O
O
P
O
O
(i-Pr)2N
5
3: R1 = DMT,
O
O
O
R2 = C2H4CN
1. NEt3
O
O
Ph
2. Cl3C-CO2H
4: R1 = H,
NH
R2
=
O
O
N
H3N
O
O
OH
O
O
O
O
P
NH
1. Cl3C-CO2H; 2. 1, tetrazole; 3. I2, pyridine; 4. Na2S2O3
O
O
N
3
O
5. NEt3 6. Cl3C-CO2H; 7. Boc-Phe-OH, HBTU, HOBT, DIEA; 8. TFA
O
OH
O
O
N
P
O
NH
O
O
O
7
OH OH
Scheme 1.
a mixture of the ‘uronium salt’ HBTU, hydroxybenzo-
triazole (HOBT), and Hu¨nig’s base (DIEA) in DMF.
Ester 5 was obtained in 86% yield after HPLC purifica-
tion, using a gradient of acetonitrile in water on a
C18-phase. In the final deprotection step, involving 80%
aqueous TFA at room temperature for 30 min, the Boc,
Fpmp, and isopropylidene groups were cleaved. Precip-
itation with ether gave aminoacylated dimer 6 in 82%
yield.8
b-cyanoethyl groups were removed with triethylamine,
followed by removal of the 5%-DMT group with
trichloroacetic acid (66% after chromatography for the
two steps). Then, Boc-protected L-phenylalanine was
coupled under the same conditions as described above
for the conversion of 4 to 5. Final full deprotection
with 80% TFA yielded aminoacylated trimer 7 in 9%
overall yield. The low yield was probably due to losses
during handling, since MALDI monitoring of the reac-
tion had shown 7 as the major peak in the crude.
Extension of the solution phase method to a nucleic
acid trimer was feasible, though giving a lower yield of
the fully assembled target molecule (Scheme 1). Pro-
tected dimer 3 was detritylated, and phosphoramidite 1
was coupled to the newly liberated hydroxyl group,
followed by oxidation with iodine/pyridine. From the
resulting intermediate, thus obtained in 90% yield, the
To allow for the synthesis of longer aminoacylated
strands, a solid phase synthesis was developed. One key
feature of this synthesis (Scheme 2) is the acid-labile
phosphoramidate linker to the solid support, adapted
from
a methodology reported by Gryaznov and
Letsinger for the synthesis of DNA oligomers.9 As a
O
N
O
Ph
Ph
NH
NH
O
O
O
O
N
Alloc-NH
H3N
O
O
O
O
O-Fpmp
OH
O
O
O
O
O
N
O
N
O
O
N
9
1. tetrazole,
DMT-O
PO
PO
NH
O
NH
NH
NH
DMT-O
O
O
O
O
O
O
N
O
O
1
O-Fpmp
OC2H4CN
O
O
1. [Pd(PPh3)4], PPh3,
[H2NEt2]+[HCO3]-
1. 7 RNA synthesis
cycles with 1
P
7
7
(i-Pr)2N
O-Fpmp
PO
OH
O-Fpmp
PO
NH
O
O
O
NH2
PO
OH
2. Ac2O, DMAP
3. I2, pyridine
NCC2H4O
O
O
2. Cl3C-CO2H
3. NH4OH
2. AcOH, H2O
NH
8
4. Alloc-Phe-OH, MSNT
10
Scheme 2.

C. N. Tetzlaff, C. Richert / Tetrahedron Letters 42 (2001) 5681–5684
5683
ligase,14 were available for this experiment from other
work on acyl transfer reactions. Solutions containing
the three aminoacylated species were exposed to pH
8.5, and the kinetics of the hydrolysis of the esters
linkages were monitored by MALDI-TOF MS under
conditions previously shown to allow quantitative
detection of oligonucleotides15 (Fig. 2).
consequence of using this linker, the RNA oligomers
prepared contained a 3%-terminal phosphate group. The
derivatized support was constructed by reacting phos-
phoramidite 1 with solid supports bearing amino
groups. The resulting immobilized phosphoramidate (8)
was extended to an octamer using a standard protocol
for phosphoramidites of ribonucleotides on an auto-
matic synthesizer. As expected,10 the yield of the
octanucleotide, as determined via release of the DMT-
cation, varied with the support. The best results were
obtained with long chain alkylamine (LCAA) CPG
The rates of ester hydrolysis under the pseudo-first
order conditions chosen were 7.0×10−4 s−1 for 10, 1.1×
10−3 s−1 for CUCCACCA-Trp-Boc, whose amino group
was protected, and 7.4×10−3 s−1 for CUCCACCA-Trp,
translating into half-life times of 103 min, 6×102 min,
and 90 min, respectively. The increased hydrolytic sta-
bility of 5%-aminocylated 10 suggests that if acyl transfer
from 2%/3%- to 5%-termini of RNA strands1 did take place
under prebiotic conditions, the 5%-aminoacylated
strands generated would have been more long-lived
than their 2%,3%-aminoacylated counterparts and there-
fore be potentially catalytically or metabolically useful
species.16
,
from Sigma with pore size 500 A and particle mesh size
of 80–120, which gave the octamer in 75% overall yield.
Polystyrene support from Novabiochem gave 23%,
TentaGel resin from Advanced ChemTech 62%, and
LCAA-CPG from CPG Inc. 64%.
After cleaving the cyanoethyl groups from the back-
bone phosphotriesters of the extended derivative of 8
with ammonium hydroxide, the terminal 5%-hydroxyl
group of the RNA octamer was coupled to Na-ally-
loxycarbonyl (Alloc) protected phenylalanine11 to pro-
duce 9. An Alloc, rather than Boc, protecting group
was chosen for the amino acid to introduce orthogonal-
ity, and thus allow for the assembly of oligopeptides on
the RNA terminus, if necessary. Since the HBTU/
HOBT coupling mixture gave only moderate yields
(53%), as determined by integrating peaks in the
MALDI-TOF mass spectra of crude 10, obtained after
deprotection with aqueous AcOH, a number of cou-
pling conditions were tested. A mixture of DCC and
HOBT gave 22% yield after 47 h reaction time and 8%
yield after 25 h. When DCC was employed with
DMAP, 25 h reaction time showed 64% of 10 in the
crude, whereas DPPA, PPh3/DIAD (Mitsunobu condi-
tions),2 cyanuric chloride, BOP-Cl, and Aldrithiol/
HBTU all gave <5% of the aminoacylated octamer.
In conclusion, the results from the exploratory synthetic
work presented here indicate that 5%-aminoacylated
The best coupling results were obtained with 1-(2-
mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT)
as the condensing agent. When employed with Hu¨nig’s
base as the co-reagent and DMF as solvent, 10 made
up 85% of the crude under optimized deprotecting
conditions.12 The optimized deprotection involved
treatment of the Alloc-deprotected derivative of 9 with
AcOH/water (19:1) at room temperature for 14 h,
rather than with AcOH/water (4:1) for 4 h, as recom-
mended for DNA oligomers,9 which gave increased
fragmentation. The amount of fragments was not
decreased further by rigorous exclusion of possible
contaminations with RNases or reducing the handling
prior to sampling for MALDI-TOF MS. A MALDI
mass spectrum of a typical crude product is shown in
Fig. 1.
Figure 1. Representative MALDI-TOF spectrum of crude 10,
as obtained after deprotection with AcOH/H2O (19:1).
An exploratory experiment was performed to compare
the stability of the 5%-ester in 10 towards hydrolysis
with that of esters in similar oligoribonucleotides
aminoacylated at the 2%/3%-position. The sequences
CUCCACCA-Trp, and CUCCACCA-Trp-Boc pre-
pared from the aminoacylated dinucleotides pdCpA-
Trp or pdCpA-Trp-Boc13 and 5%-CUCCAC-3% using T4
Figure 2. Kinetics of the hydrolysis of the ester linkages in
5%-aminoacylated 10 (squares), and 2%/3%-acylated RNA
octamers CUCCACCA-Trp (circles) and CUCCACCA-Trp-
Boc (diamonds) in a solution containing 300 mM LiCl, 20
mM HEPES buffer, and 0.2 mM EDTA, pH 8.5, as deter-
mined by MALDI-TOF MS.

5684
C. N. Tetzlaff, C. Richert / Tetrahedron Letters 42 (2001) 5681–5684
oligomers of uridylic acid can be prepared using a
methodology relying on acid-labile protecting groups
and a phosphoramidate linker. The successful employ-
ment of Alloc-Phe-OH as a building block in the syn-
thesis of 10 suggests that extension to mixed RNA
sequences should be feasible, if allyl-/allyloxycarbonyl-
protected phosphoramidites of adenosine, cytidine and
guanosine were to be employed.17 Finally, the synthetic
methodology for ester-containing oligonucleotides
might be of interest for the preparation of oligonucleo-
tide pro-drugs bearing 5%-appendages to be removed
by intracellular esterases after entry into cells.
4.42–4.46 (t, 1H); 4.71–4.75 (dd, 1H); 4.84–4.88 (dd, 1H);
5.60–5.63 (m, 1H), 1H); 5.76–5.79 (d, 1H); 5.92–5.95 (d,
1H); 6.92–6.97 (m, 4H); 7.53–7.56 (d, 1H); 7.74–7.77 (d,
1H); MALDI-TOF MS for C33H40FN5O15P: calcd 796.7,
found 796.7.
7. Peyrottes, S.; Mestre, B.; Burlina, F.; Gait, M. J. Tetra-
hedron 1998, 54, 12513–12522.
8. Brief protocol: A mixture of 4 (33.3 mg, 42 mmol),
Boc-Phe-OH (99 mg, 374 mmol), HOBT (50 mg, 370 mmol),
and HBTU (128 mg, 336 mmol) was dissolved in DMF (0.2
mL) and treated with DIEA (213 mL, 1.25 mmol). After
1 h, ammonium acetate solution (0.6 mL, 0.1 M) was added
and the solution HPLC purified (C18, CH3CN in water:
10% for 5 min, 10–50% in 30 min, elution at 23 min).
Product containing fractions was dried (37.4 mg, 86%) and
then resuspended in TFA (160 mL) and water (40 mL). After
30 min, Et2O (1 mL) was added. The precipitate was
washed with ether, lyophilized from water, and dried. Yield
References
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1
of 6: 24 mg, (34 mmol, 82%). H NMR (D2O, 300 MHz):
l 7.62 (d, 1H); 7.34 (d, 1H), 7.09 (m, 3H); 7.00 (m 2H);
5.67 (d, 1H); 5.62 (m, 2H); 5.51 (d, 1H); 4.98 (m, 1H); 4.83
(m, 1H); ca. 4.7, under HDO (m), 4.23 (m 2H); 4.01–4.02
(m, 4H); 3.91 (m, 2H); 2.90 (m, 2H); MALDI-TOF MS for
C27H31N5O15P: calcd 696.5, found 696.0.
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11. Prepared from L-phenylalanine and the N-hydroxysuccin-
imide derivative of Alloc-Cl in THF/water (1:2) containing
Na2CO3 (5.5 equiv.).
12. Abridged protocol: Solid support 8 (30 mg) was extended
to an octamer using the RNA program for ABI 381A
synthesizers (1 mmol scale, trityl off mode). The assembled
octamer was treated with NH4OH (1 mL) for 2 h at 55°C.
The solution was aspired, the support was washed with
water and CH3CN, and allowed to dry. A solution of
Alloc-Phe-OH (8 mg, 32 mmol), MSNT (10 mg, 33 mmol),
DIEA (50 mL, 0.29 mmol), and DMF (100 mL) was added
and the mixture bath-sonicated for 1 min. After 1 h the
supernatant was aspired, the support washed with CH3CN
and CH2Cl2, and dried. A solution of Pd(PPh3)4 (5 mg, 4.3
mmol) and diethylammonium bicarbonate (5 mg, 41 mmol)
in CH2Cl2 (0.4 mL) was added, followed by sonication for
2 min. After 2 h, the CPG was washed with CH2Cl2 and
CH3CN (1 mL) and dried. Then, AcOH (190 mL) and water
(10 mL) were added. After 14 h, the supernant was aspired,
filtered, and treated with Et2O (2 mL). The precipitation
was completed at −4°C for 1 h and centrifugation. The
resulting pellet gave 85–93% product, as determined by
integration of the MALDI-TOF mass spectrum. MALDI-
TOF MS for C81H98N17O66P8: calcd 2613.5, found 2613.5.
13. Prepared using a modification of the procedure reported
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6. Brief protocol: A solution of 1 (0.3 g, 0.32 mmol, from
Cruachem Ltd.) and 2 (98 mg, 0.62 mmol, from Sigma) in
CH3CN (0.75 mL) was treated with a solution of tetrazole
in CH3CN (8 mL, 0.45 M, 3.6 mmol). After 50 min, a
solution of I2 (12 mL, 0.2 M in water/THF/pyridine) was
added. After 45 min, Na2S2O3 (500 mg, 3.2 mmol) was
added, followed by stirring for 90 min and filtration. Then,
CH2Cl2 was added, the solution washed with satd
NaHCO3, dried, and chromatographed (silica, CH2Cl2/
MeOH/NEt3 82:10:8). The product was dissolved in
CH3CN (6 mL) and NEt3 (10 mL) under sonication, stirred
for 3.5 h, and dried. The residue was treated with
CCl3CO2H solution (2.5 mL, 0.18 M in CH2Cl2) for 3 min,
followed by precipitation with Et2O. The precipitate was
washed with Et2O, dried, and lyophilized from water. Yield
of 4: 185 mg (0.23 mmol, 73%). 1H NMR (CDCl3, 300
MHz): l 1.71 (s, 6H); 1.76–1.86 (m, 4H); 2.75–2.86 (m, 4H);
2.88 (s, 3H); 3.58–3.60 (m, 2H); 3.93–3.95 (m, 2H);
4.24–4.27 (m, 1H); 4.28–4.31 (m, 1H); 4.38–4.41 (m);
14. Prepared using conditions similar to those reported in:
Hohsaka, T.; Kajihara, D.; Ashizuka, Y.; Murakami, H.;
Sisido, M. J. Am. Chem. Soc. 1999, 121, 34–40.
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Nature Struct. Biol. 2000, 7, 28–33.
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