High-yield immobilization of a puromycin analogue for the solid support synthesis of aminoacyl-tRNA fragments

Article abstract of DOI:10.1021/ol0346638

(Matrix presented) An efficient procedure for the immobilization of 3′-deoxy-3′-(O-methyltyrosyl)aminoadenosine was developed. A poly(ethylene glycol)-derived diacid linker/spacer was attached to aminomethyl polystyrene. Coupling of the 2′-hydroxy instead

Full text of DOI:10.1021/ol0346638

ORGANIC
LETTERS
2
003
Vol. 5, No. 15
603-2606
High-Yield Immobilization of a
Puromycin Analogue for the Solid
Support Synthesis of Aminoacyl-tRNA
Fragments
2
Nhat Quang Nguyen-Trung, Silvia Terenzi, Gerd Scherer, and Peter Strazewski*,†
Institute of Organic Chemistry, UniVersity of Basel, St. Johanns-Ring 19,
CH-4056 Basel, Switzerland
peter.strazewski@unibas.ch
Received April 18, 2003
ABSTRACT
An efficient procedure for the immobilization of 3′-deoxy-3′-(O-methyltyrosyl)aminoadenosine was developed. A poly(ethylene glycol)-derived
diacid linker/spacer was attached to aminomethyl polystyrene. Coupling of the 2′-hydroxy instead of the 2′-O-succinylated ribonucleoside
resulted in high immmobilization yields (over 80%) and allowed for the recovery of valuable unreacted material. This specific procedure
should be applicable to other ribonucleosides containing a bulky modification at the 3′-position and can be used for the stepwise construction
of 3′-aminoacyl- or 3′-peptidyl-RNA conjugates.
We recently published the synthesis of structural analogues
of puromycin, a natural nucleoside antibiotic broadly used
of oligonucleotides. Puromycyl-CPG can now be obtained
commercially; however, it is of great interest to develop this
original approach by synthesizing new molecules, analogues
of puromycin having, for instance, a better peptidyl transfer
ability. On that account a good immobilization procedure
for this class of molecules is needed. We synthesized some
analogues of puromycin and after fruitless attempts, as cited
in the literature, to attach our analogues in good yield on
the solid support, we developed a high-yield immobilization
procedure. This method enables the recovery of the unreacted
nucleosides, which is for us a very important point.
1
as a tool for molecular biologists. An innovative application
of puromycin was developed previously for the in vitro
formation of peptidyl-RNA “fusions”, which allows for the
2
in vitro selection of peptides and ribosomal peptidyl transfer
3
studies. To realize this fusion between peptide and RNA, it
4
,5
was necessary to synthesize oligoribonucleotides containing
puromycin at their 3′-end and thus adapt and anchor
puromycin to a solid support for the solid support synthesis
†
Present address: Laboratoire de Synth e` se de Biomol e´ cules, B aˆ timent
3′-Deoxy-3′-(L-p-methoxyphenylalanyl)aminoadenosine de-
Eug e` ne Chevreul (5 e` me e´ tage), Universit e´ Claude Bernard Lyon 1, Domaine
Scientifique de la Doua, 43 boulevard du 11 novembre 1918, F-69622
Villeurbanne Cedex, France.
rivative 1 is obtained on the synthetic pathway of puromycin
1
analogues. Modification of 1 was necessary to attach it onto
(
1) Nguyen-Trung, N. Q.; Botta, O.; Terenzi, S.; Strazewski, P. J. Org.
Chem. 2003, 68, 2038-41.
2) (a) Roberts, R. W.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A. 1997,
the solid support for the oligonucleotide synthesis. We first
(
9
4, 12297-12302. (b) Nemoto, N.; Miyamoto-Sato, E.; Husimi, Y.;
(4) Atkinson, T.; Smith, M. Oligonucleotide Synthesis: A Practical
Approach; IRL Press: Oxford, UK, 1984; pp 35-81.
(5) Kumar, P.; Ghosh, N. N.; Sadana, K. L.; Garg, B. S.; Gupta, K. C.
Nucleosides Nucleotides 1993, 12, 565-84.
Yanagawa, H. FEBS Lett. 1997, 414, 405-408. (c) Liu, R.; Barrick, J. E.;
Szostak, J. W.; Roberts, R. W. Methods Enzymol. 2000, 318, 268-93.
(3) Starck, S. R.; Roberts, R. W. RNA 2002, 8, 890-903.
1
0.1021/ol0346638 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/26/2003

decided to follow the current methodology for the im-
mobilization of nucleosides, which utilizes nucleosides
anchored on Long Chain Amino Alkyl-Controlled Pore Glass
Scheme 2. Immobilization of 3 on LCAA-CPGa
(
LCAA-CPG) by a succinyl linkage.4-8 So compound 1 was
protected at the 5′-position with dimethoxytrityl chloride and
the 2′-hydroxy group, in our case, was succinylated in good
yield to obtain the product 3 (Scheme 1).
Scheme 1. 5′-O-Protection and 2′-O-Succinylation of 1a
a
Reagents and conditions: (i) DCC, p-nitrophenol, dioxane,
DMF, LCAA-CPG. (ii) (a) DMAP (1.0 equiv), OCN(CH
2
)
6
NCO
NEt (1.0 equiv), LCAA-
O/C N (2:8 v/v), 2 h;
i
(
1.0 equiv), CH
CPG (0.5 NH ), 16 h; Et
N/NMI/CH
2
Cl
2
, rt, 10 min; (b) ( Pr)
O (wash); (c) H
Cl , 0.5 h.
2
2
2
2
5 5
H
(d) Ac
2
O/Et
3
2
2
Cl
2
and optical detection at 504 nm (ꢀ504 ) 72 678 µM‚cm;
reproducibility within 2-3%). Compared to the initial amino
loading value, the immobilization yield was about 10-12%.
But the steric hindrance of the Fmoc-aminoacyl group at the
a
Reagents and conditions: (i) DMTCl (1.5 + 0.5 equiv), N(n-
Bu)
extract; SiO
1.0 equiv), DMAP (0.5 equiv), 1,2-EtCl
acid extract; precipitation from Et O/hexane (1:1); quant.
4
NO
3
(0.3 equiv), C
5
H
5
N, rt/1 + 1 h; MeOH quench; NaHCO
3
i
2
; 81%. (ii) succinic anhydride (1.5 equiv), ( Pr)
2
NEt
3
′-position might be at the origin of the low yield, so
(
2
, 50 °C/0.5 h; 10% citric
intercalating a spacer between the succinylated ribonucleo-
side and the solid support should improve the reaction yield.
2
5
Gupta and co-workers developed an approach using alkyl
and aryl diisocyanates as spacers. In this procedure, succi-
nylate 3 is attached to one isocyanate function through the
action of DMAP and decarboxylation. Commercial hexam-
ethylene-1,6-diisocyanate was chosen, which reacted within
Succinylated derivative 3 was activated by using DCC and
p-nitrophenol and then reacted with LCAA-CPG (1000 Å
pore size). All immobilizations were carried out manually
with use of a Teflon syringe equipped with a polyethylene
filter. To obtain the best immobilization yield, 2 equiv of 3
were used with regard to the initial amino loading of LCAA-
CPG. The determination of the amino loading was performed
1
0 min. LCAA-CPG was added for the attachment of the
primary amino groups of the solid support to the second
isocyanate function (Scheme 2, ii).
9
The results we obtained were even less successful, since
a nucleoside loading of 6.7 µmol/g resulted. In addition to
the low immobilization yields, whatever the method we used,
we were confronted with the loss of 3. In fact compound 3
is obtained after an 11-step synthesis and for each im-
mobilization attempt 2 equiv of 3 were used and all the
unreacted material could not be recovered. To our knowl-
edge, no references referred to this problem. Although the
succinate linkage remains the most favored for solid support
oligonucleotide synthesis, some other activation procedures
have been developed; notably the activation of the solid
by using the ninhydrin test on untreated and on acid-
8
activated LCAA-CPG, which resulted in no significant
2
difference in loading (100-130 µmol NH /g). Following the
4
established literature procedure (Scheme 2, i), the best
nucleoside loading we obtained was only 12.7 µmol/g, as
+
determined by DMT cleavage with 3% Cl
3
CCOOH/CH
2
-
(
6) Pon, R. T.; Usman, N.; Ogilvie, K. K. BioTechniques 1988, 6, 768-
7
8
1
1
5.
(
7) Damha, M. J.; Usman, N.; Ogilvie, K. K. Can. J. Chem. 1989, 67,
31-39.
(8) Damha, M. J.; Giannaris, P. A.; Zabrylo, S. V. Nucleic Acids Res.
990, 118, 3813-20.
8
support, which can be directly condensed with the 2′ or 3′
(9) Sarin, V. K.; Kent, S. B.; Tam, J. P.; Merrifield, R. B. Anal. Biochem.
981, 117, 147-57.
hydroxy group of an appropriately protected ribonucleoside.
2604
Org. Lett., Vol. 5, No. 15, 2003

In this way the unreacted material could be recovered and
the loss of the precious ribonucleoside was limited. Earlier
works showed that this method applied to ribonucleosides
Scheme 3. Immobilization of 2 on AMPa
7,10
resulted mostly in low to medium yield and require longer
8
reaction times.
We decided to explore this strategy with some modifica-
tions. The LCAA-CPG solid support was replaced by a 50%
DVB-cross-linked aminomethyl polystyrene (AMP), whose
1
1
superiority has been shown by McCollum and Andrus. In
addition to the succinate linkage, we also used a com-
mercially available poly(ethylene glycol)-derived linker/
12
spacer: 3,6,9-trioxaundecanoic diacid. We have shown that
a similar spacer used for the solid support of oligonucleotides
proved to be superior in terms of both coupling yields and
homogeneity of the final product, when compared to several
1
3
other tested spacer molecules of up to double its length.
AMP with an amino loading of 28 µmol/g as determined
by the ninhydrin test (cf. Supporting Information) was
quantitatively derivatized, on one hand, with succinic
anhydride in the presence of (dimethylamino)pyridine (DMAP)
in pyridine (Scheme 3: i) and, on the other hand, with 3,6,9-
trioxaundecanoic diacid in the presence of HBTU ((O-
benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate) and N-methylmorpholine (NMM) in DMF
(Scheme 3: ii). The carboxylic functions of the derivatized
solid supports 6 and 8 were activated with oxalyl chloride
within 1 h at room temperature. After being washed with
absolute CH
for half an hour and submitted to the reaction with 2 in the
presence of DMAP in absolute CH Cl for 12 h, followed
2 2
Cl , the polymers were dried under high vacuum
2
2
by a capping procedure with acetic anhydride/N-methyl
imidazole (NMI)/pyridine in DMF (Scheme 3: iii). The
ribonucleoside immobilization yield was determined by the
trityl analysis and resulted in nucleoside loadings of 12.7
µmol/g for the succinate linkage and 24.1 µmol/g for the
poly(ethylene glycol) linkage, respectively. This procedure
was carried out several times and the lowest ribonucleoside
loading we obtained for the polyethylene linkage was 20
µmol/g, corresponding to immobilization yields of 80-87%
with respect to the initial amino loading. An excess of
compound 2 was worked up and chromatographed allowing
the recovery of pure starting material 2.
a
Abbreviation: PS ) polystyrene. Reagents and conditions: (i)
succinic anhydride (50 equiv), DMAP (3.0 equiv), C
5
H
5
N, AMP
; (ii) 3,6,9-trioxaunde-
canoic diacid (37 equiv), HBTU (18.5 equiv), NMM (18.5 equiv),
N, rt/15 min; AMP (1.0 NH ), rt, 16 h; wash: DMF, CH Cl
2
iii) (a) oxalyl chloride (100 equiv), CH Cl , rt/1 h; wash: CH Cl ;
(
2 5 5 2 2
1.0 NH ), rt/16 h; wash: C H N, CH Cl
C
(
5
H
5
2
2
2
;
2
2
2
(b) 2 (2.0 equiv), DMAP (10 equiv), CH Cl , rt/12 h; (c) Ac O/
NMI/C
regeneration of excess 2: extract solution from (b) with NaHCO
CH Cl ; SiO (CH Cl /MeOH 0-5%); 40-54%.
2
2
2
5
5
H N (each 1 M in DMF), rt/5 min; wash: CH
2
Cl
2
; (d)
3
/
To establish the usefulness of our immobilization method,
the charged polymer 9 was subjected to standard oligoribo-
nucleotide chain assembly on an automated DNA/RNA
synthesizer, using a phosphoramidite coupling protocol and
2
2
2
2
2
phosphate and A* corresponds to 3′-deoxy-3′-(L-p-methox-
yphenylalanyl)amino-â-D-adenosine. After the partial depro-
tection and cleavage from the solid support with 33%
ethanolic methylamine and evaporation, the crude compounds
5
-ethylthiotetrazole activation, a commercial 5′-O-phosphi-
tylating reagent, and 2′-O-TBDMS ribonucleoside mono-
mers. We synthesized, on a 1-µmol scale with average
stepwise yields of >98%, fragments of the invariant 3′-
terminal ACCA sequence of tRNA: pA*, CpA*, pCpA*,
CpCpA*, and ApCpCpA*, where p stands for a 5′-O-
were desilylated with Et
3
N‚3HF/DMF (3.3:1, 55 °C/1.5 h),
evaporated, and purified by reverse-phase HPLC (C18; buffer
A, H
2
O; buffer B, 90% CH
3
CN/10% 0.1 M NH
4
OAc). The
1
(
8.
10) Pon, R. T.; Ogilvie, K. K. Nucleosides Nucleotides 1984, 3, 485-
identity, purity, and assignments of the downfield H NMR
9
signals of the 3′-aminoacyl-tRNA analogues were established
(
(
11) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069-72.
12) Gunzenhauser, S.; Biała, E.; Strazewski, P. Tetrahedron Lett. 1998,
1
by electrospray mass spectroscopy, H NMR (600 MHz/
3
9, 6277-80.
31
inverse detection, in H
2
O/5% D
2
O, pH 7.5), and P NMR
(13) Katzhendler, J.; Cohen, S.; Rahamim, E.; Weisz, M.; Ringel, I.;
Deutsch, J. Tetrahedron 1989, 45, 2777-92.
(cf. Supporting Information).
Org. Lett., Vol. 5, No. 15, 2003
2605

In conclusion, we developed a high-yielding 2′-O-im-
mobilization method for 3′-L-R-aminoacylamino-3′-deoxy-
adenosines. We obtained over 80% immobilization yield
despite the presence of a bulky group in the 3′-position. This
procedure should be applicable to other ribonucleosides for
an anchorage through either the 2′- or the 3′-hydroxy group.
We also showed that the efficient recovery of the unreacted
ribonucleoside was possible. We demonstrated the ap-
plicability of our charged solid support 9 to an automated
oligoribonucleotide synthesis in preparing a number of short
we are convinced that it is possible to achieve, since we
synthesized several 3′-oligoalanyl-RNA conjugates on AMP
following a similar immobilization methodology using a
1
4
slightly longer spacer.
Acknowledgment. We thank the Roche Research Foun-
dation and the Swiss National Science Foundation for their
financial support.
Supporting Information Available: Procedures for the
synthesis of 2 and 3, the automatized oligonucleotide
3′-aminoacylated RNA strands. We did not yet explore the
1
31
synthesis, H NMR spectra and, where applicable, P NMR
of 2, 3, and the (oligo)ribonucleotides. This material is
available free of charge via the Internet at http://pubs.acs.org.
construction of 3′-peptidyl-RNA conjugates on 9, although
(14) Terenzi, S.; Biała, E.; Nguyen-Trung, N. Q.; Strazewski, P. Angew.
Chem., Int. Ed. 2003, 42, 2909-2912.
OL0346638
2606
Org. Lett., Vol. 5, No. 15, 2003