puromycin serves as an aminoacyl acceptor.4,6 At the A site,
CCA-Phe and CACCA-Phe revealed similar binding activi-
ties.7 Therefore, only nucleotides of the CCA terminus play
an important role in the binding of aminoacyl-tRNA to the
A site. In most fragment reactions, puromycin was used as
a peptidyl acceptor and did not exhibit full acceptor activity
but required addition of alcohol. Recently, Starck and Roberts
reported that puromycin-oligonucleotides revealed steric
restrictions for ribosome entry and multiple modes of
translation inhibition.8a It was reported that tRNA bearing
3′-amino-3′-deoxyadenosine in the final sequence retained
full acceptor activity.8b Thus, the synthesized pCpCpA-NH-
Phe might likely exhibit full activity for the peptidyl
transferase reaction in the ribosome. Here, we report the first
synthesis of the new compound of CpCpA-3′-deoxy-3′-N-
phenylalanine and the enzymatic activity of 32pCpCpA-NH-
Phe.
Scheme 1. Synthesis of
2′-O-Benzoyl-N,N-dibenzoyl-3′-deoxy-3′-N-(Boc-phenylalanyl)
Adenosine with 5′-TBDPS Protectiona
The target compound was synthesized from 3′-amino-3′-
deoxy-adenosine 1 by phosphoramidite chemistry. We have
modified Robins’ nine-step route for the synthesis of
3′-amino-3′-deoxyadenosine 1 into a seven-step process with
55% overall yield.9 Briefly, adenosine was protected with
tert-butyldiphenylsilyl at the 5′-position and then treated with
R-acetoxyisobutyryl bromide to yield 2′-O-acetyl-3′-bromo-
3′-deoxy-5′-O-tert-butyldiphenylsilyl-adenosine. This was
treated with 0.5 N ammonia in methanol and then reacted
with benzylisocyanate to yield 3′-(benzylamino)-5′-O-(tert-
butyl)diphenylsilyl-3′-N,2′-O-carbonyl-3′-deoxyadenosine. This
product was reacted with sodium hydride and then with 1.0
N NaOH and finally deprotected by hydrogenation with
Pd-C (10%) to yield 3′-deoxy-3′-amino-3′-deoxyadenosine
1. Since compound 1 contains multiple functional groups,
finding a suitable protection group for each functional group
is critical toward the synthesis of CpCpA-NH-Phe.
Boc-L-phenylalanine was first introduced into the 3′-
position of 3′-amino-3′-deoxyadenosine 1, which also acted
as a protection group of the 3′-amino group. The reaction in
DMF was not successful because of the poor solubility of 2
in DMF and a high racemization of L-phenylalanine. When
N-(tert-butyloxycarbonyl)-L-phenylalanine N-hydroxy suc-
cinimide ester was stirred with 3′-amino-3′-deoxyadenosine
1 in anhydrous dimethyl sulfoxide (DMSO) at room tem-
perature for 4 h, an optically pure 3′-(N-tert-butyloxycarbo-
nyl-L-phenylalanine)amido-3′-deoxyadenosine 2 was ob-
tained in 95% yield.10 The tert-butyl-diphenylsilyl (TBDPS)
group was first used to protect the 5′-hydroxyl group of 2 to
yield 5′-O-(tert-butyl-diphenylsilyl)-3′-(N-tert-butyloxycar-
bonyl-L-phenylalanine) amido-3′-deoxy-adenosine 3 in 85%
yield (Scheme 1). This reaction was highly regioselective
a Reaction conditions: (a) Boc-Phe-NHS ester, DMSO, rt; (b)
TBDPS-Cl, pyridine, 2 days; (c) benzoyl chloride, pyridine, 0-25
°C; (d) TBAF, THF, 0-25 °C.
to form the 5′-protected compound. After benzoylation, the
fully protected 3′-amino-3′-deoxyadenosine (98%) was treated
with tert-butylammonium fluoride (TBAF) in anhydrous
THF to remove the 5′-O-TBDPS group. Unfortunately, the
deprotection of TBDPS produced the desired compound 5
(57%) as well as 6-N-monobenzoyl compound 6 (13%) and
6-N,N-2′-O,5′-O-tetrabenzoyl compound 7 (8%), which was
formed via benzoyl migration.
We have also investigated the DMTr protection of the 5′-
hydroxy of 2. Reaction of 2 with 4,4′-dimethoxyltrityl
chloride in dry pyridine yielded a mixture of components
(Scheme 2). The mixture was separated by a flash column
of silica gel to give the desired product 3′-(N-tert-butyloxy-
carbonyl-L-phenylalanine)amido-3′-deoxy-5′-O-(4,4′-dimeth-
oxytrityl)-adenosine 10 in 47% yield,11 unreacted 2 (14%),
3′-(N-tert-butyloxycarbonyl-L-phenylalanine)amido-3′-deoxy-
2′-O-(4,4′-dimethoxytrityl)-adenosine 8, and 2′-O,5′-O-bis-
(4,4′-dimethoxytrityl)-3′-(N-tert-butyloxycarbonyl-L-phen-
ylalanine)amido-3′-deoxyadenosine 9. Compounds 8 and 9
were treated with 80% acetic acid at room temperature to
regenerate the starting material 2 without the cleavage of
(6) (a) Cerna, J.; Rychlik, I.; Krayevsky, A. A.; Gottikh, B. P. Febs Lett.
1973, 37, 188-191. (b) Moazed, D.; Noller, H. F. Cell 1989, 57, 585-
597. (c) Mercer, T. F. B.; Symons, R. H. Eur. J. Biochem. 1972, 28, 38-
45. (d) Parlato, G.; Guesnet, J.; Crechet, J.-B.; Parmeggiani, A. FEBS Lett.
1981, 125, 257-260.
(7) (a) Lessard, J. L.; Pestka, S. J. Biol. Chem. 1972, 247, 6901-8. (b)
Bhuta, P.; Kumar, G.; Chladek, S. Biochim Biophys Acta 1982, 696, 208-
11.
(8) (a) Starck, S. R.; Roberts, R. W. RNA 2002, 8, 890-903. (b) Fraser,
T. H.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2671-5.
(9) (a) Samano, M. C.; Robins, M. J. Tetrahedron Lett. 1989, 30, 2329-
2332. (b) Zhang, L.; Zhang, B. Unpublished results.
(10) Compound 2: TLC (85:15 chloroform/methanol), Rf ) 0.52; 1H
NMR (DMSO-d6) δ 8.39 (s, 1H), 8.14 (s, 1H), 8.02 (d, J ) 7.7 Hz, 1H),
7.32 (s, 2H), 7.28-7.15 (m, 5H), 6.93 (d, J ) 8.4 Hz, 1H), 6.04 (d, J )
3.3 Hz, 1H), 5.94 (d, J ) 2.9 Hz, 1H), 5.17 (t, J ) 5.5 Hz, 1H), 4.51 (br,
1H), 4.46 (m, 1H), 4.25 (m, 1H), 3.90 (m, 1H), 3.66-3.43 (dm, 2H), 2.98-
2.71 (dm, 2H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ 168.1, 152.2, 151.3,
148.7, 145.0, 135.3, 134.2, 125.4, 124.1, 122.3, 115.2, 85.4, 79.7, 74.2,
69.1, 57.2, 51.9, 46.5, 33.9, 24.2, 21.2. ESI-MS (m/z) calcd for C24H31N7O6
513.2, found 536.3 [M + Na]+.
3616
Org. Lett., Vol. 4, No. 21, 2002