A practical route to 3′-amino-3′-deoxyadenosine derivatives and puromycin analogues

Article abstract of DOI:10.1021/jo026627c

3′-Aminoacylamino-3′-deoxyadenosines, analogues of the antibiotic puromycin, have been synthesized from adenosine. They key 3′-azido derivative 10 was obtained through a 3′-oxidation/reduction/substitution procedure. A modified purification protocol on a

Full text of DOI:10.1021/jo026627c

A P r a ctica l Rou te to
′-Am in o-3′-d eoxya d en osin e Der iva tives
a n d P u r om ycin An a logu es
3
Nhat Quang Nguyen-Trung, Oliver Botta,
Silvia Terenzi, and Peter Strazewski*,†
Institute of Organic Chemistry, University of Basel, St.
J ohanns-Ring 19, CH-4056 Basel, Switzerland
peter.strazewski@unibas.ch
Received October 29, 2002
F IGURE 1. Puromycin (1) and 3′-amino-3′-deoxyadenosine
2).
(
Ab st r a ct : 3′-Aminoacylamino-3′-deoxyadenosines, ana-
logues of the antibiotic puromycin, have been synthesized
from adenosine. They key 3′-azido derivative 10 was ob-
tained through a 3′-oxidation/reduction/substitution proce-
dure. A modified purification protocol on a larger scale was
developed for the oxidation step using the Garegg reagent.
The coupling reaction between an Fmoc-L-amino acid and
the fully protected form of 3′-amino-3′-deoxyadenosine 11
furnished the aminoacylated compounds 12 in high yields.
The puromycin analogues were obtained in 10 steps and up
to 23% (14c) overall yield.
and a low overall yield. In 1989, Samano and Robins10
were the first to publish an efficient synthetic pathway
1
1
to puromycin. The latter recently reported details of
their nine-step synthesis to 3′-aminodeoxynucleoside 2
with an improved but, in our hands, still difficult
procedure for the problematic final N-debenzylation
reaction. We communicated our synthesis of 3′-alanyl-
1
2
amino-3′-deoxyadenosine from adenosine in 10 steps
and now report the optimized protocol for a general
synthesis of puromycin analogues 14.
Our synthetic pathway utilizes a protected form of 3′-
amino-3′-deoxyadenosine (2) synthesized from adenosine.
The 2′,5′-bis-O-TBDMS xylofuranosyladenine derivative
7 (Scheme 1) was synthesized in three steps as described
in the literature.13 Thus, the silylation furnished at most
60% of the desired 2′,5′-bis-O-silylated isomer 3. The
other products were 3′,5′-bis-O-silylated adenosine 4
The antibiotic puromycin (1, Figure 1), a metabolite
of Streptomyces alboniger, was first isolated by Porter and
co-workers in 1952. Puromycin has been and is being
1
extensively used for the elucidation of the mechanism of
2
3
the protein biosynthesis. Yarmolinsky and De la Haba
were the first to recognize close structural similarity
(34%) and 2′,3′,5′-tris-O-silylated adenosine 5 (traces); no
between puromycin and the aminoacyl end of aminoacyl-
tRNA. Further studies4-7 showed that puromycin inhibits
monosilylated product was isolated. To improve the yield
of 3, we isomerized compound 4 in a solution of 2.5%
protein synthesis by transferring the ribosomal nascent
(v/v) triethylamine in methanol, so 3 was obtained in 85%
peptide to the puromycin R-amino group. Five years ago,
yield after two isomerizations. The oxidation of the 3′-
a novel application of puromycin was introduced in the
1
3c
laboratories of Yanagawa and of Roberts and Szostak.8
hydroxyl group was achieved using the Garegg reagent.
The isolation of the ketone 6 on a >3 g scale, by
The principle, based on the peptidyl transfer ability of
puromycin, allows for the in vitro selection of proteins
or RNA strands via mRNA-peptide fusions using puro-
mycin as the point of covalent attachment. A number of
synthetic routes to puromycin and analogues have been
(
9) (a) Gerber, N. N.; Lechevalier, H. A. J . Org. Chem. 1962, 27,
1
1
731-1732. (b) Guarino, A. J .; Kredich, N. M. Biochim. Biophys. Acta
963, 68, 317-319. (c) Sowa, W. Can. J . Chem. 1968, 46, 1586-1589.
(d) Okruszek, A.; Verkade, J . G. J . Med. Chem. 1979, 22, 882-885. (e)
Ozols, A. M.; Azhayev, A. V.; Dyatkina, N. B.; Krayevsky, A. A.
Synthesis 1980, 557-559. (f) Ozols, A. M.; Azhayev, A. V.; Krayevsky,
A. A.; Ushakov, A. S.; Gnuchev, N. V.; Gottikh, B. P. Synthesis 1980,
559-561. (g) Saneyoshi, M.; Nishizaka, H.; Katoh, N. Chem. Pharm.
Bull. 1981, 29, 2769-2775. (h) Visser, G. M.; Schattenkerk, C.; van
Boom, J . H. Recl. Trav. Chim. Pays-Bas 1984, 103, 165-168. (i)
McDonald, F. E.; Gleason, M. M. J . Am. Chem. Soc. 1996, 118, 6648-
6659. (j) Baker, B. R.; J oseph, J . P.; Williams, J . H. J . Am. Chem. Soc.
1955, 77, 1-7. (k) Baker, B. R.; Schaub, R. E.; Williams, J . H. J . Am.
Chem. Soc. 1955, 77, 7-12. (l) Baker, B. R.; Schaub, R. E.; J oseph, J .
P.; Williams, J . H. J . Am. Chem. Soc. 1955, 77, 12-15. (m) Baker, B.
R.; Schaub, R. E.; Kissman, H. M. J . Am. Chem. Soc. 1955, 77, 5911-
5915. (n) Mengel, R.; Wiedner, H. Chem. Ber. 1976, 109, 433-443.
(10) Samano, M. C.; Robins, M. J . Tetrahedron Lett. 1989, 30, 2329-
2332.
9
reported, but most of them suffer from numerous steps
†
Present address: Laboratoire de Synth e` se de Biomol e´ cules,
B aˆ timent 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.
1) Porter, J . N.; Hewitt, R. I.; Hesseltine, C. W.; Krupka, G.; Lowery,
J . A.; Wallace, W. S.; Bohonos, N.; Williams, J . H. Antibiot. Chemother.
(
1
952, 2, 409-410.
2) (a) Suhadolnik, R. J . Nucleoside Antibiotics; Wiley: New York,
970; pp 1-50. (b) Suhadolnik, R. J . Nucleosides as Biological Probes;
(
1
Wiley: New York, 1979; pp 96-102.
3) Yarmolinsky, M. B.; De la Haba, G. L. Proc. Natl. Acad. Sci.
U.S.A. 1959, 45, 1721-1729.
4) Allen, D. W.; Zamecnik, P. C. Biochim. Biophys. Acta 1962, 55,
(
(
(11) Robins, M. J .; Miles, R. W.; Samano, M. C.; Kaspar, R. L. J .
Org. Chem. 2001, 66, 8204-8210.
(12) Botta, O.; Strazewski, P. Nucleosides Nucleotides 1999, 18,
721-723.
(13) (a) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault,
N. Y.; Sadana, K. L. Can. J . Chem. 1978, 56, 2768-2780. (b) Sung, W.
L.; Narang, S. A. Can. J . Chem. 1982, 60, 111-120. (c) Garegg, P. J .;
Samuelsson, B. Carbohydr. Res. 1978, 67, 267-270. (d) Robins M. J .;
Sarker, S.; Samano, V.; Wnuk, S. F. Tetrahedron 1997, 53, 447-456.
(e) Robins, M. J .; Samano, V.; J ohnson, M. D. J . Org. Chem. 1990, 55,
410-412.
8
65-874.
(
(
(
5) Nathans, D.; Neidle, A. Nature 1963, 197, 1076-1077.
6) Gilbert, W. J . Mol. Biol. 1963, 6, 389-403.
7) Symons, R. H.; Harris, R. J .; Clarke, L. P.; Wheldrake, J . F.;
Elliott, W. H. Biochim. Biophys. Acta 1969, 179, 248-250.
(
8) (a) Roberts, R. W.; Szostak, J . W. Proc. Natl. Acad. Sci. U.S.A.
1
997, 94, 12297-12302. (b) Nemoto, N.; Miyamoto-Sato, E.; Husimi,
Y.; 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-
9
3.
1
0.1021/jo026627c CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/06/2003
2
038
J . Org. Chem. 2003, 68, 2038-2041

SCHEME 1. Syn th etic P a th w a y to P u r om ycin An a logu esa
a
Key: (i) TBDMS-Cl, pyridine, rt; (ii) 2.5% (v/v) Et3N in MeOH, rt, 85%; (iii) CrO3, Ac2O, pyridine, rt, 85-91%; (iv) NaBH4, AcOH, 8
°
C, 84%; (v) 8 [) (CH3O)2HC-N(n-Bu)2], MeOH, rt, 97%; (vi) (1) 2 equiv of TfCl, DMAP, CH2Cl2, rt, (2) LiN3, DMF, rt, 86%; (vii) (1) PPh3,
18
dioxane, rt, (2) H2O, rt, 84%; (viii) Fmoc-L-amino acid, CBMIT, CH3NO2, N-methylimidazole, THF, rt, 12a (95%), 12b (84%), 12c (96%);
ix) pyridine-HF complex, THF, rt, 13a (75%), 13b (93%), 13c (98%); (x) 40% piperidine in MeOH (v/v), rt, 14a , 14b, 14c (54%).
(
precipitation with EtOAc as described in the literature,13d
resulted in low yields because part of the product was
trapped into the chromium-complex precipitate.
We therefore propose another purification procedure
according to which the crude material is directly loaded
in fact, during the activation of the 3′-hydroxyl group
with trifluoromethanesulfonyl chloride, the amino group
of 7 was derivatized as well. To protect the amino group,
1
5
we chose a base labile formamidine protecting group
using N,N-di(n-butyl)formamide dimethyl acetal (8),
1
6
onto a silica column and slowly eluted with CH
2
Cl
2
to
prepared as described by Froehler and Matteucci.
separate the product from the chromium complex. Then
a step gradient elution with EtOAc/hexane (1:1-1:0)
allows for the isolation of ketone 6. Under our conditions,
the product is already separated from the chromium
complex and will not be trapped in the precipitate. This
procedure increases the yield of the oxidation reaction,
as we obtained pure, colorless, chromium-free, and stor-
able 6 in 85-91% yield. The reduction of 6 (no traces of
Compound 8 reacts with 7 within 2 h at room tempera-
ture to furnish 9 in 97% yield. Derivative 10 was isolated
in 86% yield. The 3′-epimer of 10 that could have arisen
from small amounts of contaminating 3 was absent in 9,
as shown by NMR and in comparison with intentionally
synthesized material (same reaction sequence starting
from 3 instead of 7). The azido functionality was reduced
1
7
to the amine 11 (84%) using the Staudinger reaction.
3
) was performed at 12-13 °C using a cryostat. The xylo
Amine 11 should not be stored for too long as an oil, since
it contains a base-labile protection that is susceptible to
slow cleavage through nucleophilic attack by the alkyl-
amino function.
derivative 7 was isolated in 84% yield which contained
approximatively 3% of the ribo isomer 3 as determined
1
by H NMR. This mixture was not further purified, and
the contaminant was eliminated during the purification
of the next steps. The introduction of the azido function
into the 3′-position was performed through a nucleophilic
substitution of the triflate-activated 3′-xylo-hydroxyl
group with lithium azide.14 We first attempted these
reactions without protection of the 6-amino group of the
adenine moiety. The best yield we obtained was only 50%;
The key step in the reaction sequence is the coupling
of an N-protected L-amino acid to the sterically hindered
3′-amino group of 11. Saha, Schultz, and Rapoport
reported on a coupling method in which, via a highly
(15) McBride, L. J .; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H.
J . Am. Chem. Soc. 1986, 108, 2040-2048.
(16) Froehler B. C.; Matteucci, M. D. Nucleic Acids Res. 1983, 11,
8
031-8036.
(14) Robins, M. J .; Hawrelak, S. D.; Hern a´ ndez, A. E.; Wnuk, S. F.
(17) Vaultier, M.; Knouzi, N.; Carri e´ , R. Tetrahedron Lett. 1983, 24,
Nucleosides Nucleotides 1992, 11, 821-834.
763-764.
J . Org. Chem, Vol. 68, No. 5, 2003 2039

reactive acyl imidazolium intermediate, even secondary
amines are coupled in high yields without an additional
base and without racemisation of the carboxy compo-
nent.18
We synthesized three analogues 12 (Scheme 1) bearing
alanine, 3-(â-naphthyl)alanine, and p-methoxyphenyl-
alanine (O-methyltyrosine) in isolated yields of, respec-
tively, 95, 84, and 96% with respect to 11. The 5′- and
Isocratic conditions were used for the purification of the ana-
logues 14 (77% 0.65 mM aq NH OAc/23% HPLC-grade CH CN).
′,5′-Bis-O-(ter t-b u t yld im et h ylsilyl)-â-D-a d en osin e (3).
Compound 3 was prepared as described in the ref 15c with the
following reagents: adenosine (1 g, 3.74 mmol), TBDMS-Cl (1.7
g, 11.22 mmol), pyridine (8 mL). Isom er iza tion of 4. To a
suspension of a mixture of 3/4 with a high content of 4 (7.22 g,
14.5 mmol) in MeOH (90 mL) was added triethylamine (2.3 mL,
4
3
2
2
.5% v/v), and the solution was stirred at rt for 2 h. The solution
was evaporated to dryness, and purification by chromatography
EtOAc/hexane 7:3) furnished more pure 3 as a white solid. After
two isomerizations, the yield of 3 for the silylation reaction
increased to 85%: R
2
′-O-silyl groups were deprotected with the pyridine-
(
HF complex reagent in 75-98% yields, and the depro-
tection of the two amino groups was performed with a
solution of 40% piperidine in methanol. The analogues
were submitted directly to purification by preparative
reversed-phase HPLC furnishing target molecules 14a ,
f
3/4 ) 0.25/0.15 (EtOAc/hexane 7:3). Mp )
+
177-178 °C. MS (FAB) m/z: 496 [(M + H) , 100]. HRMS (CI):
+
m/z (calcd [M + H] ) 496.27753, m/z (found) 496.27778. Anal.
Calcd for C22
41 5 4 2
H N O Si : C, 53.30; H, 8.34; N, 14.13. Found:
C, 53.58; H, 8.37; N, 13.93.
1
4b, and 14c.
9
-(2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-er yth r o-p en to-
In conclusion, we describe here an efficient and reliable
route to puromycin analogues in 10 steps. Under opti-
mized and routine conditions, the protected compounds
fu r a n -3-u losyl)-9H-a d en in e (6). Compound 6 was prepared
as described in ref 13d with the following reagents: chromium-
(VI) oxide (CrO , 3.24 g, 32.4 mmol), acetic anhydride (3.0 mL,
3
1
3a ,b,c are isolated in 30-43% yields in nine steps
32.4 mmol), pyridine (5.2 mL, 64.8 mmol), 3 (5.35 g, 10.8 mmol),
and CH
as follows: the reaction mixture was directly poured onto the
chromatography column (800 g flash silica conditioned with CH
Cl in a 8 cm L column) and slowly eluted with CH Cl until a
2 2
Cl (57 mL). The purification procedure was modified
starting from adenosine. Usually, we prepared target
compounds 14a ,b,c on a 30 µmol scale without determin-
ing the yield of the final step; however, once we prepared
2
-
2
2
2
1
4
4c from 13c in 54% yield (excluding residual NH OAc)
yellow band reached the bottom of the column. Then a step
after RP-HPLC purification and repeated lyophilization,
i.e., in 23% overall yield with respect to adenosine. Our
synthetic pathway allows for a wide variety of amino acid
side chains to be introduced. Although, for in vivo
experiments, the 6-dimethylamino group is essential to
escape the activity of adenosine deaminases, it is not
needed for in vitro experiments on ribosomes as is the
focus of this work. Several studies19 have shown that
puromycin’s dimethylamino group is not necessary for
its biological activity. Experiments on the kinetics of the
ribosomal peptidyl transfer reaction using our puromycin
analogues are currently underway.
gradient elution from EtOAc/hexane 1:1 to 1:0 afforded 6 (4.95
g, 91%) as a white solid. R
f
) 0.46 (EtOAc). Mp ) 175-177 °C.
+
MS (FAB) m/z: 494 [(M + H) , 100]. HRMS (CI): m/z (calcd [M
+
+
22
H] ) 494.26190, m/z (found) 494.26225. Anal. Calcd for
Si : C, 53.52; H, 7.96; N, 14.18. Found: C, 53.36;
H, 7.78; N, 14.24.
-(2′,5′-Bis-O-(ter t-bu tyldim eth ylsilyl)-â-D-xylofu r an osyl)-
C
H
39
N
5
O
4
2
9
9H-a d en in e (7). Compound 7 was prepared as described in ref
13e with the following reagents: sodium borohydride (1 g, 39
mmol), acetic acid (48 mL), 6 (3 g, 6 mmol). A mechanical stirrer
was used throughout the synthesis. During the addition of
NaBH
4
to acetic acid, the internal temperature was kept at 15-
1
6 °C. During the addition and reaction of 6, the external cooling
bath temperature was kept close to 8 °C resulting in an internal
reaction temperature of 12-13 °C. After 2.5 days, the acetic acid
was evaporated under reduced pressure and the yellow residue
was taken up and extracted in an ice-cold mixture of saturated
NaHCO3 solution/EtOAc (1:3). The organic layer was washed
Exp er im en ta l Section
1H NMR (300 and 400 MHz) and 13C NMR (75 and 100 MHz)
spectra were obtained with solutions in CDCl
3
using tetra-
methylsilane as internal standard. H NMR spectra of the
analogues 14 were obtained from solutions in H O/5% D O at
00 MHz using sodium 3-(trimethylsilyl)propionate as internal
2 4
with brine, dried over anhyd Na SO , and evaporated. Com-
1
pound 7 (2.5 g, 84%) was obtained as a colorless foam. R ) 0.38
f
+
(EtOAc). Mp ) 65-69 °C. MS (FAB) m/z 496 [(M + H) , 100].
2
2
+
6
HRMS (CI): m/z (calcd [M + H] ) 496.27753, m/z (found)
1
13
496.27767. Anal. Calcd for C22 41 5 4 2
H N O Si : C, 53.30; H, 8.34; N,
standard. The signal listings of all H and C NMR spectra are
available in the Supporting Information. Mass spectra (MS and
HRMS) were obtained using fast atom bombardement (p-
nitrobenzyl alcohol) and chemical ionization (isobutane) methods
and electrospray ionization (ESI in MeOH for 12 and 13 and
14.13. Found: C, 53.15; H, 8.11; N, 14.03.
6-N-[(Di-n -b u t yla m in o)m et h ylen e]-9-(2′,5′-b is-O-(ter t-
bu tyldim eth ylsilyl)-â-D-xylofu r an osyl)-9H-aden in e (9). Com-
pound 7 (1.48 g, 2.98 mmol) was coevaporated three times with
absolute pyridine under reduced pressure and dissolved in 10
mL of methanol. After addition of N,N-di-n-butylformamide
dimethyl acetal16 (8, 1.21 g, 5.96 mmol), the solution was stirred
at rt for 2 h followed by evaporation. Purification by chroma-
tography (EtOAc/hexane 1:1) furnished 9 (1.84 g, 97%) as a
2
H O/MeOH 9:1 for the analogues 14). The melting points were
measured on a Kofler block and are uncorrected. TLC was run
on precoated silica gel F254 plates with fluorescent indicator.
Nucleosides were visualized on TLC plates by subsequent
2 4
spraying with concentrated H SO and 2% naphthoresorcin
solutions in ethanol, followed by heating. Column chromatog-
raphy was performed with flash silica gel 60 (40-63 µm).
yellowish oil. R
[(M + H) , 100].
3′-Azid o-6-N-[(d i-n -b u t yla m in o)m et h ylen e]-2′,5′-b is-O-
(ter t-bu tyld im eth ylsilyl)-3′-d eoxy-â-D-a d en osin e (10). Com-
pound 9 (2.00 g, 3.15 mmol) and DMAP (0.96 g, 7.87 mmol) were
f
) 0.55 (EtOAc/hexane 1:1). MS (FAB) m/z: 636
+
Reagent grade chemicals were used. LiN
NaN and LiCl (1:1) in EtOH (residual NaN
3 3
3
was prepared from
and LiCl were
filtered off after treatment with EtOH and then Et O). Ca u tion :
2
do not acidify; do not heat as a dry solid! Solvents were distilled
before use for extractions. Tetrahydrofuran was dried by distil-
lation from sodium/benzophenone. All other solvents were used
as purchased. Prep HPLC: 250 × 8 mm Eurospher 100/5 RP18
column (Knauer), flow rate: 2 mL/min; UV detection at 260 nm.
coevaporated with anhydrous CH
and then dissolved in anhydrous CH
was cooled to 0 °C, and trifluoromethylsulfonyl chloride (0.67
mL, 6.39 mmol) was added dropwise. After being stirred at 0
2
Cl
2
under reduced pressure
Cl (8 mL). The solution
2
2
°C for 30 min, the yellow solution was taken up in AcOH/H
1:99)/CH Cl and extracted twice. The organic layers were
washed with ice-cold saturated NaHCO solution and brine,
dried over anhyd Na SO , evaporated, and dried under high
vacuum (oil pump) resulting in a yellow oil. The residue was
taken up in DMF (30 mL) and added to solid lithium azide (LiN
2
O
(
2
2
3
(
18) Saha, A. K.; Schultz, P.; Rapoport, H. J . Am. Chem. Soc. 1989,
11, 4856-4859.
19) (a) Vince, R.; Isakson, R. J . J . Med. Chem. 1973, 16, 37-40. (b)
Vince, R.; Daluge, S. J . Med. Chem. 1977, 20, 930-934.
1
2
4
(
3
,
2
040 J . Org. Chem., Vol. 68, No. 5, 2003

0
.77 g, 15.75 mmol) under argon. The solution was stirred at rt
overnight and then taken up in saturated NaHCO solution/
EtOAc. After extraction, the organic layers were combined and
washed with brine, dried over anhyd Na SO , and evaporated.
Purification by chromatography (EtOAc/hexane 1:1) gave 10
6-N-[(Di-n -b u t yla m in o)m et h ylen e]-3′-[N-(9-flu or en yl)-
m e t h oxyca r b on yl-L-a m in oa cyl]-3′-d e oxy-â-D-a d e n osin e
(13). To a solution of 12 (0.05 mmol) in THF (0.5 mL) in an
Eppendorf tube was added pyridine-hydrogen fluoride (∼70%
HF) (11 µL, 0.12 mmol). The solution was stirred at rt for 4 h
3
2
4
(1.79 g, 86%) as a colorless oil. R
f
) 0.54 (EtOAc/hexane 1:1). IR
and then taken up in saturated NaHCO
extracted three times. The organic layer was washed with brine,
dried over anhyd Na SO , and evaporated. Purification by
3
solution/EtOAc and
-1
+
(KBr): 2107.5 cm (N
3
stretch). MS (FAB) m/z: 660 [(M + H) ,
00]. HRMS (FAB): (M + H) m/z (calcd) 660.42012; m/z (found)
60.42016.
′-Am in o-6-N-[(d i-n -bu tyla m in o)m eth ylen e]-2′,5′-bis-O-
ter t-bu tyld im eth ylsilyl)-3′-d eoxy-â-D-a d en osin e (11). To a
solution of 10 (483 mg, 0.732 mmol) in dioxane (14.5 mL, filtered
through Al to remove traces of peroxide) was added PPh (250
mg, 0.952 mmol), and the solution was stirred at rt overnight.
O was added (9 mL), and the solution was stirred for 2 h at
rt. The solution was taken up in saturated NaHCO solution/
EtOAc and extracted twice. The organic layers were combined,
washed with brine, dried over anhyd Na SO , and evaporated.
After chromatographic purification (EtOAc), 11 (390 mg, 84%)
was obtained as a colorless oil. R ) 0.36 (EtOAc). IR (KBr):
1
6
2
4
chromatography (step gradient from EtOAc to EtOAc/MeOH 9:1)
furnished 13 as an oil.
3
(
1
3a (“Ala ”). R
ESI) m/z: 700 [(M + H) , 100].
3b (“Na p h t”). R ) 0.66 (EtOAc/MeOH 9:1); 38.4 mg, 93%.
MS (ESI) m/z: 826 [(M + H) , 100].
3c (“O-MeTyr ”). R ) 0.55 (EtOAc/MeOH 9:1); 39.4 mg,
8%. MS (ESI) m/z: 794 [(M + H) , 100].
′-L-Am in oa cyla m in o-3′-d eoxy-â-D-a d en osin e (14). To 13
0.03 mmol) was added a solution (0.59 mL) of 40% (v/v)
f
) 0.42 (EtOAc/MeOH 9:1); 26.3 mg, 75%. MS
+
(
2
O
3
3
1
f
+
H
2
1
f
3
+
9
3
2
4
(
piperidine in MeOH. The mixture was stirred at rt for 2 h,
evaporated to dryness, and directly submitted to purification by
RP-HPLC. The fractions containing 14 were concentrated in a
f
-
1
+
3
1
6
2
678/3624 cm (NH stretch). MS (FAB) m/z: 634 [(M + H) ,
00]. HRMS (FAB): (M + H) m/z (calcd) 634.42962, m/z (found)
Si(CH
reduced pressure and then repeatedly lyophilized in Eppendorf
tubes under high-vacuum until NH OAc was present in lower
3 2 2
) Cl -treated glass flask on a rota-evaporator under
34.42919.
6
-N -[(D i-n -b u t y la m in o )m e t h y le n e ]-2′,5′-b is -O -(t er t -
4
bu tyld im eth ylsilyl)-3′-[N-(9-flu or en yl)m eth oxyca r bon yl-L-
a m in oa cyl]-3′-d eoxy-â-D-a d en osin e (12). To a solution of
carbonyl diimidazole (38.4 mg, 0.24 mmol) in absolute CH
than 5 molar equiv amounts with respect to the analogues 14,
1
as determined by H NMR. The analogues 14 were thus isolated
3 2
NO
as white fluffy solids.
(0.45 mL) at 0 °C was added methyl triflate (51.8 µL, 0.48 mmol),
and the solution was stirred for 10 min while warming to rt.
The solution was added to a suspension of N-(9-fluorenyl)-
methoxycarbonyl-L-amino acid (0.24 mmol) and N-methyl-
3′-L-Ala n yla m in o-3′-d eoxy-â-D-a d en osin e (14a ). t
R
(260
+
nm) ) 12-13 min. MS (ESI) m/z: 338 [(M + H) , 100].
3′-L-(3-(â-Na p h t h yl)a la n yla m in o)-3′-d e oxy-â-D-a d e n o-
imidazole (1.2 µL, 0.016 mmol) in absolute CH
resulting in a clear solution that was stirred at rt until the
production of CO had ceased. Then a solution of 11 (50 mg, 0.08
mmol) in THF (1 mL) was added under argon. After being stirred
at rt for 2 h, the solution was taken up in a CH Cl /H O and
extracted three times. The organic layers were combined, washed
with saturated NaHCO solution and H O, dried over anhyd Na
SO , and evaporated. After chromatographic purification (EtOAc),
compounds 12 were obtained as oils.
2a (“Ala ”). R ) 0.35 (EtOAc/hexane 7:3); 70.5 mg, 95%. MS
ESI) m/z: 928 [(M + H) , 100].
2b (“Na p h t”). R ) 0.31 (EtOAc/hexane 1:1); 70.8 mg, 84%.
MS (ESI) m/z: 1054 [(M + H) , 100].
2c (“O-MeTyr ”). R ) 0.51 (EtOAc/hexane 1:1); 79.3 mg,
6%. (Largest scale: 700 mg 12c ) 96% isolated yield.) MS (ESI)
3
NO
2
(0.6 mL)
sin e (14b). t
H) , 100].
R
(260 nm) ) 24-25 min. MS (ESI) m/z: 464 [(M +
+
2
3
′-L-(p -Met h oxyp h en yla la n yla m in o)-3′-d eoxy-â-D-a d e-
4
n osin e (14c). t (260 nm) ) 14-15 min (1.5 mg 14c‚2.3NH -
R
+
2
2
2
OAc ) isolated yield 54%). MS (ESI) m/z: 444 [(M + H) , 100].
3
2
2
-
Ack n ow led gm en t. We thank the Roche Research
Foundation and the Swiss National Science Foundation
for their financial support.
4
1
f
+
(
1
f
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra, 1H
+
13
and C NMR signal listings of the compounds. This material
1
f
is available free of charge via the Internet at http://pubs.acs.org.
9
+
m/z: 1034 [(M + H) , 100].
J O026627C
J . Org. Chem, Vol. 68, No. 5, 2003 2041