Synthesis of the ribosomal P-site substrate CCA-pcb

Article abstract of DOI:10.1021/ol052484f

(Chemical Equation Presented) CCA-pcb (cytidylyl-(3′5′)- cytidylyl-(3′5′)-3′(2′)-O-(N-(6-D-(+) -biotinoylaminohexanoyl)-L-phenylalanyl)adenosine), a ribosomal P-site substrate, was synthesized by phosphoramidite chemistry in 26 steps with an overall yield

Full text of DOI:10.1021/ol052484f

ORGANIC
LETTERS
2006
Vol. 8, No. 1
55-58
Synthesis of the Ribosomal P-Site
Substrate CCA-pcb
Minghong Zhong† and Scott A. Strobel*,†,‡
Department of Molecular Biophysics and Biochemistry, and Department of Chemistry,
Yale UniVersity, New HaVen, Connecticut 06520-8114
strobel@csb.yale.edu
Received October 13, 2005
ABSTRACT
CCA-pcb (cytidylyl-(3
synthesized by phosphoramidite chemistry in 26 steps with an overall yield of 18%, starting from biotin. The synthesis relies on the judicious
selection of orthogonal silyl protecting groups for the 5 -hydroxyls and acid-labile protecting groups (DMTr, AcE, and MeE) at other reactive
-esterification and nucleotide coupling were accomplished by in situ activation with
′5′)-cytidylyl-(3′5′)-3′(2′)-O-(N-(6-D-(+)-biotinoylaminohexanoyl)-L-phenylalanyl)adenosine), a ribosomal P-site substrate, was
′
sites to ensure the intactness of the labile ester. Both 3
imidazolium ions.
′
The catalytic mechanism of peptide bond formation by the
ribosome is an area of ongoing research. Proposed catalytic
strategies include general acid-base catalysis,1 substrate-
assisted catalysis,2 and catalysis derived solely from substrate
alignment.3 It is also possible that the reaction may follow
a pathway different from the stepwise mechanism involving
a tetrahedral intermediate that is observed for aminolysis
reactions in aqueous solution.4 Improved chemical and
enzymatic tools are needed to differentiate between these
mechanistic possibilities.
In addition to the reaction with full-sized tRNAs, the
ribosome can also catalyze peptide bond formation between
minimal A-site and P-site substrates. We previously reported
a modified fragment reaction that used the A-site substrate,
C-puromycin, and a P-site substrate with an extended
peptidyl-like chain, CCA-pcb.5 These substrates have played
a valuable role in the structural and biochemical character-
ization of the peptidyl transfer (PT) reaction.5,6 Studies have
included the use of N15-labeled C-puromycin for kinetic
isotope effect (KIE) analysis,6e a method that can be used to
characterize the transition-state structure of a chemical
reaction. Our goal is to determine intrinsic KIEs at several
† Department of Molecular Biophysics and Biochemistry.
‡ Department of Chemistry.
(1) (a) Muth, G. W.; Ortoleva-Donnelly, L.; Strobel, S. A. Science 2000,
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A. Science 2000, 289, 920-930.
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A. Nat. In press. (d) Hansen, J. L.; Schmeing, T. M.; Moore, P. B.; Steitz,
T. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 99, 11670-11675. (e) Seila, A.
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10.1021/ol052484f CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/09/2005

atomic positions in both the A-site and P-site substrates in
order to explore the transition state of the PT reaction. This
necessitates that isotopic substitutions be introduced into
CCA-pcb, but an efficient chemical synthesis of CCA-pcb
has not been reported.
Scheme 1. Synthesis of 6-(1-N-(4,4′-Dimethoxytrityl)-D-
(+)-biotinoyl)aminohexanoic Acid
The synthesis of CCA-pcb offers a synthetic challenge
because the ester linkage of aminoacylribonucleotide deriva-
tives is an activated, high energy bond with a free energy of
hydrolysis comparable to that of ATP.7 Fast and reversible
migration of the amino acyl occurs between the 2′- and 3′-
hydroxyl of the ribofuranose. This results in a mixture of
2′- and 3′-O-aminoacylated isomers. Most recent syntheses
of 2′/3′-O-aminoacyloligonucleotides utilized the cyano-
methyl ester of protected amino acids.8 The only synthetic
procedure reported for CCA-pcb was by direct coupling
between CpCpA and the cyanomethyl ester of pcb, which is
expected to provide low yields due to a mixture of mono-,
di-, and multiple aminoacylated byproducts.6a Here we report
an alternative route that provides an efficient synthesis of
CCA-pcb.
It was observed that the absence of a neighboring hydroxyl
stabilizes the ester bond of 2′/3′-aminoacylnucleotides against
hydrolysis and that the hydrolysis rate decreases dramatically
at low pH.9 A half-life of 250 h at pH 2.5 was observed for
a 2′/3′-O-L-phenylalanyladenosine methyl phosphate deriva-
tive.10 Therefore, we reasoned that the 2′-OH and the amino
groups should be protected by acid-labile protecting groups
such as ketal or ortho ester and trityl group, respectively. In
our synthesis, CCA-pcb was prepared in two different
schemes by phosphoramidite chemistry using acid-labile 2′-
bisacetoxyethoxymethyl (AcE), 2′-bismethoxyethoxymethyl
(MeE), and 4/6-(4,4′-dimethoxytrityl) (DMTr) as protecting
groups.
6-amino group of adenosine was protected with DMTr by
reaction with DMTrCl to give quantitatively compound 7.12
MeE was introduced into the 2′-position by reflux of
compound 7, with trismethoxyethoxy orthoformate, 4-tert-
butyldimethyl-siloxy-3-penten-2-one, and PPTS in DCM.
The derived syrup after chromatography was treated with
HF-TEMED/CH3CN to provide compound 9 (71%).13
Compound 9 was further protected by a sterically hindered
silyl group at the 5′-position by treatment with bis(trimeth-
ylsiloxy)cyclododecyloxylsilyl (DOD) chloride and imidazole
in cold THF to give 10 (64%) (Scheme 2). Compound 10 is
Synthesis of A-pcb Fragment 19. Biotin 1 is insoluble
in most organic solvents. Its solubility was enhanced by 1-N-
tritylation. Esterification of biotin by treatment with SOCl2
in methanol and tritylation with 4,4′-dimethoxytrityl chloride
(DMTrCl) in pyridine in the presence of Et3N,11 followed
by hydrolysis of methyl ester in 1 M NaOH/MeOH, gave
the tritylated biotin 2 (52%). This biotin derivative is highly
soluble even in DCM. DCC-mediated coupling between
compound 2 and methyl 6-aminohexanoate in pyridine and
hydrolysis of the methyl ester in 1 M NaOH/MeOH produced
compound 4 (77%) (Scheme 1). Significant loss of 2 during
purification was observed, and a crude separation followed
by amide formation gave 3 in much higher yield (83%).
Scheme 2. Synthesis of A and C Monomers
Adenosine 5 was converted into 3′,5′-O-(tetraisopropyl-
disiloxane-1,3-diyl)adenosine 6 (93%) via reaction with 1,3-
dichloro-1,1,3,3-tetraisopropyl-disiloxane in pyridine. The
(7) Preiss, J.; Berg, P.; Ofengand, E. J.; Bergman, F. H.; Dieckmann,
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Townsend, L. B., Ed.; Plenum Press: New York, 1994.
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2477-2503.
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unstable, and significant migration of the 2′-O-MeE to the
3′-position occurred during storage; thus, it was freshly
prepared and purified prior to the subsequent acylation
reaction.
(12) Kawana, M.; Takeuchi, K.; Ohba, T.; Kuzuhara, H. Bull. Chem.
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(13) Scaringe, S. A. Methods 2001, 23, 206-217.
56
Org. Lett., Vol. 8, No. 1, 2006

Aminoacylation of compound 10 was first attempted via
either the cyanomethyl ester8 or the acyl fluoride14 of
N-protected L-phenylalanine. Both gave low to moderate
yields probably due to significant steric hindrance caused
by DOD and MeE. Aminoacylation was then performed with
a stronger promoter. Compound 10 was treated with N-
FMOC-L-phenylalanine in the presence of mesitylenesulfonyl
tetrazole (MST)15,16 and excess N-methylimidazole in DCM
at ambient temperature to produce quantitatively the fully
protected derivative 16. The reaction involves the imidazo-
lium cation, as shown by 1H NMR, which was nucleophili-
cally displaced as a neutral leaving group by the 3′-hydroxyl
of 10. Removal of FMOC by treatment of 16 with 20%
piperidine in CH3CN resulted in 17 (97%). DCC mediated
amide formation with 4 (99%), followed by desilylation by
treatment with HF-TEMED in CH3CN, gave 19 in a yield
of 84% (Schemes 2 and 3).
was treated with HF-TEMED in CH3CN to provide
compound 14 (68%), which was further protected by DOD
(89%) (Scheme 2). The cytidylyl phosphoramidite 20 was
obtained by treatment of compound 15 with methyl tetra-
isopropylphosphorodiamidite (POMe) in the presence of
tetrazole (Tet) in DCM overnight (81%). Compound 20 was
a mixture of two diastereomers due to the chirality of the
1
phosphoramidite as shown by H NMR and 31P NMR.
Compound 20 was directly coupled to compound 14 in
the presence of PhImOTf 17 and molecular sieves in CH3-
CN/DCM (1:1), followed by oxidation with t-BuOOH in
toluene. The reaction selectively gave 21 in good yield (81%)
after chromatography. The diribonucleotide was transformed
into its phosphoramidite 22 as before (73%) (Scheme 4).
Scheme 4. Synthesis of C(3′5′)C Phosphoramidite
Scheme 3. Synthesis of A-pcb Derivative
Compound 22 is a mixture of four diastereomers due to the
chirality of phosphorus triester and phosphoramidite. Syn-
thesis of the dinucleotide with tetrazole and S-Et-tetrazole
only gave moderate yields, which might be caused by the
bulky silyl protecting group.
Synthesis of Protected CCA-pcb and Deprotection.
Diribonucleotide phosphoramidite 22 was coupled to 1 molar
equiv of compound 19 using PhImOTf and molecular sieves
as the promotor in CH3CN/DCM (1:1) to form the fully
protected CCA-pcb 23 in good yield (72%). Reactions with
tetrazole and S-Et-tetrazole gave only traces of products. The
success of the coupling reaction involves imidazolium cation,
which was displaced by the 5′-OH to release a neutral leaving
group.17 Desilylation of compound 23 by treatment with HF-
TEMED in CH3CN (73%) and cleavage of methyl phosphate
ester with S2Na2 (88%), followed by complete removal of
DMTr and orthoformate ester in 0.5 M HCOOH in MeOH-
Synthesis of C(3′5′)C Phosphoramidite. Cytidine was
treated with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane in
pyridine to afford 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-
cytidine (92%). Compound 12 was selectively tritylated at
the N4 to produce 13 (91%), which was further protected at
the 2′-position by reflux with a mixture of trisacetoxyethoxy
orthoformate, 4-tert-butyldimethylsiloxy-3-penten-2-one, and
PPTS in DCM. The syrup that resulted after chromatography
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R. J. Am. Chem. Soc. 2001, 123, 8165-8176.
Org. Lett., Vol. 8, No. 1, 2006
57

Scheme 5. Synthesis of Fully Protected CCA-pcb and Its
Scheme 6. Synthesis of CCA-pcb by Sequential Addition
Deprotection
of orthogonal silyl protecting groups for 5′-hydroxyls and
acid-labile protecting groups (DMTr, ACE, and MeE) at
other reactive sites to ensure the intactness of the labile ester.
Both 3′-esterification and nucleotide coupling were ac-
complished by in-situ activation with imidazolium ions. This
efficient synthesis will make it possible to prepare a series
of isotopically substituted P-site substrates for KIE analysis
of the PT reaction.
DCM (1:1) at 55 °C for 5 h gave compound 24 (80%) as a
mixture of two regiosiomers (2′/3′-ester) (Scheme 5). The
overall yield from biotin was 18%.
Compound 23 was also synthesized by sequential addition
of cytidylyl phosphoramidite 20 to A-pcb derivative 19
(Scheme 6). Coupling of 19 with 20 in the presence of
PhImOTf and molecular sieves gave fully protected com-
pound CpA-pcb, 25 (80%). Compound 25 was then desily-
lated (60%) and coupled to 20 a second time to afford a
white foam (72%), which was deprotected as described above
to produce a compound spectroscopically identical to com-
pound 24 in an overall yield of 9%.
Acknowledgment. We thank Stephen Scaringe and Amy
Seila for helpful suggestions. This research was supported
by an American Cancer Society Beginning Investigator Grant
to S.A.S.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
In conclusion, we have accomplished the first efficient
synthesis of CCA-pcb, which relies on the judicious selection
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Org. Lett., Vol. 8, No. 1, 2006