The shortest synthetic route to puromycin analogues using a modified robins approach

Article abstract of DOI:10.1021/jo102178h

We are reporting on the utility of commercial vinyl isocyanate for a practical synthetic route from adenosine to N⁶-bis-demethylpuromycin in seven steps and 65% overall yield. A clean one-pot conversion of 3′-bromo-2′-carbamoyl derivative 8 to

Full text of DOI:10.1021/jo102178h

NOTE
pubs.acs.org/joc
The Shortest Synthetic Route to Puromycin Analogues Using a
Modified Robins Approach
Kollappillil S. Krishnakumar, Sꢀebastien Goudedranche, Denis Bouchu, and Peter Strazewski**
Laboratoire de Synthꢁese de Biomolꢀecules (UMR 5246, ICBMS), Universitꢀe Claude Bernard Lyon 1, 43 boulevard du 11 novembre 1918,
69622 Villeurbanne Cedex, France
S Supporting Information
b
ABSTRACT: We are reporting on the utility of commercial
vinyl isocyanate for a practical synthetic route from adenosine
to N6-bis-demethylpuromycin in seven steps and 65% overall
yield. A clean one-pot conversion of 30-bromo-20-carbamoyl deriva-
tive 8 to 30-amino-30-deoxyadenosine derivative 10 is the main
feature of this synthetic pathway. This synthesis is the shortest
synthetic route toward 30-(aminoacylamido)deoxyadenosines
to date.
The synthesis of puromycin analogues has become a promi-
nent research area ever since the antibiotic puromycin 1 was
isolated from a culture broth of Streptomyces alboniger by Porter
et al.1 Even though the toxicity associated with puromycin
resulted in a very limited use as an antibiotic,2 puromycin, N6-
bis-demethylpuromycin 2 and other 30-(L-aminoacylami-
do)deoxyadenosines (Figure 1) have been extensively used as
very useful tools for mechanistic studies on protein biosynthesis3
because they mimic the nascent peptide accepting 30-terminus of
aminoacyl tRNA4 and they contain a stable amide bond in the 30-
position. Most of the synthetic routes toward puromycin analo-
gues suffer from numerous steps and low overall yield.5 In 2001,
Samano and Robins reported a synthetic pathway to puromycin
by extending the synthetic route to 30-amino-30-deoxyadenosine
3.6 Even though this method was considered to be the most
efficient synthetic route toward puromycin analogues, the very
sensitive final debenzylation reaction has been found to give
inconsistent results. In 2003 Strazewski and co-workers elabo-
rated on an alternative synthetic pathway to 30-amino-30-
deoxyadenosine 3 and other analogues of puromycin through
a well-known 30-oxidation/reduction/substitution procedure7
that helped to overcome the difficulty in the problematic final
N-debenzylation reaction of the Robins method.8 However, appli-
cation of this alternative method involves several disadvantages.
Lower initial yield of 20,50-bis-O-silylated adenosine and the not
very convenient separation from its 30,50-bis-O-silylated regioi-
somer, the usage of toxic yet irreplaceable chromium(VI) for the
oxidation reaction, the limited yield upon scaling up this reaction
due to the problematic chromatographic separation from resi-
dual chromium salts that may slowly degrade the product, and
consequently, the need to proceed with the subsequent reduc-
tion reaction within a minimum delay period of time are short-
comings that kept confronting us with the desire to search for
alternative routes. We thus turned our interest to the Robins
method once again and decided to use commercial vinyl iso-
cyanate, which facilitated the conversion of carbamoyl derivative
8, to obtain in one pot 30-amino-30-deoxyadenosine derivative 10
(Scheme 1).
Adenosine (4) was mixed with R-acetoxyisobutyryl bromide9
in moist acetonitrile to furnish a mixture of trans-20,30-bromo-
hydrin acetates that were converted into 20,30-anhydroadenosine
5 by using high vacuum-dried Dowex 1 ꢀ 2 (OH-) resin in
absolute methanol (91% from 4).6,10 The primary hydroxyl of 5
was quantitatively protected with a tert-butyldiphenylsilyl group
to obtain 6, which underwent, after treatment with synthesized
and freshly distilled dimethylboron bromide, clean conversion to
30-bromo xylo isomer 7 in 96% yield.11
The replacement of the N-benzyl function in the original
Robins approach by a more readily cleavable group should be a
key factor that could render the Robins strategy the fastest
reliable synthetic method toward puromycin analogues. In our
initial attempts to replace commercial benzyl isocyanate, N-tert-
butoxycarbonyl isocyanate seemed to be an attractive candidate
because the N-Boc group could be tracelessly cleaved by a
final acid treatment with volatile trifluoroacetic acid in 1,2-
dichloroethane (DCE). In our hands, unfortunately, the synth-
esis of N-tert-butoxycarbonyl isocyanate was troublesome,12
its crude purity as determined by IR spectroscopy in DCE
(ν NCO st 2236 cm-1; ν CN st 2252 cm-1; ν 2380 cm-1
)
and the isolated yields varied unreliably. This prompted us to
focus on two commercial isocyanates, allyl isocyanate and
vinyl isocyanate. Vinyl isocyanate turned out to be the best
reagent owing to the ideal combination of its high and chemo-
selective reactivity on secondary hydroxyl groups and the lability
Received: November 1, 2010
Published: March 01, 2011
r
2011 American Chemical Society
2253
dx.doi.org/10.1021/jo102178h J. Org. Chem. 2011, 76, 2253–2256
|

The Journal of Organic Chemistry
NOTE
of the N-vinyl group toward hydrolysis under mildly acidic
conditions.
methyl amine followed in situ by ammonium fluoride in warm
methanol, to obtain pure N6-bis-demethylpuromycin 2 in 92%
yield.10d,14
Treatment of 7 with vinyl isocyanate and triethylamine furn-
ished 9-[3-bromo-5-O-tert-butyldiphenylsilyl-3-deoxy-2-O-(N-
vinylcarbamoyl)-β-D-xylofuranosyl]adenine (8) in 96% isolated
yield.6 A one-pot conversion of carbamoyl derivative 8 through 9
to 30-amino-30-deoxyadenosine derivative 10 in 93% isolated yield
was accomplished by the addition of sodium hydride at -20 °C
followed by aqueous-methanolic NaOH and, ultimately, HCl.
The cyclized intermediate 9 that forms during the course of this
one-pot conversion was isolated and characterized by HRMS
(calcd for C29H33N6O4Si 557.2333 found 557.2315) and NMR
spectroscopy (SI). Thus, the use of vinyl isocyanate in this
synthetic pathway resulted in the shortest synthetic pathway for
the synthesis of 10 that can be used as a versatile intermediate for
the synthesis of different 30-(aminoacylamido)deoxyadenosines
by condensation with different amino acids.
10 was condensed with the oxybenzotriazolyl ester of
N-Fmoc-O-methyl-L-tyrosine, which was formed in situ by the
treatment of commercial N-Fmoc-O-Me-Tyr with HOBt and
DIC at 0 °C,13 to furnish compound 11 in an appreciable
91% yield without protecting the N6-amidine function. The
coupled compound was completely deprotected with ethanolic
In conclusion, a modified Robins approach consisting of seven
steps furnished N6-bis-demethylpuromycin in 65% overall yield.
The most notable feature of this approach is the one-pot
conversion of carbamoyl derivative 8 to 30-amino-30-deoxyade-
nosine derivative 10, which makes this synthetic pathway the
fastest synthetic route toward puromycin analogues with the
highest ever overall yield reported. Furthermore, this synthetic
route is well suited for the synthesis of other 30-(L-aminoacyla-
mido)deoxyadenosines that can be used as the 30-terminal mimic
of different aminoacyl tRNAs.
’ EXPERIMENTAL SECTION
All nonaqueous reactions were performed in oven-dried glassware
under nitrogen. All commercial chemical reagents were used as
supplied. The reactions were monitored by thin layer chromatography
(TLC), visualized by UV radiation (254 nm) and by spraying with
5% ethanolic H2SO4, soaking in ethanolic naphthoresorcinol (2%), and
subsequent warming with a heat gun. Column chromatography was
performed with flash silica gel (0.04-0.063 mm). High-resolution mass
spectrometry (HRMS) was conducted using the electrospray ionization
technique (ESI). Compounds were also characterized by 1H NMR and
13C NMR; the assignment of the signals was carried out with the help of
DEPT, COSY, and HSQC (see the Supporting Information). Com-
pounds 5, 6, and 7 were prepared as described in refs 6 and 10d and
characterized by 1H NMR, 13C NMR, DEPT, COSY, HSQC, and mass
spectrometry.
9-[3-Bromo-5-O-tert-butyldiphenylsilyl-3-deoxy-2-O-(N-
vinylcarbamoyl)-β-D-xylofuranosyl]adenine (8). Compound
7 (150 mg, 0.26 mmol) was coevaporated with THF (3 ꢀ 1 mL) then
dissolved in 1:1 anhydrous THF/MeCN (10 mL) under nitrogen.
Et3N (56 μL, 0.40 mmol) and vinyl isocyanate (39 μL, 0.53 mmol)
were added to the solution and the mixture was stirred for 48 h at
ambient temperature (further reagent was added if necessary
Figure 1. Puromycin (1), N6-bis-demethylpuromycin (2), and 30-amino-
30-deoxyadenosine (3).
Scheme 1. Synthetic Pathway to N6-Bis-demethylpuromycina
a Reaction conditions: (i) (1) Me2C(OAc)COBr, MeCN/H2O, rt; (2) DOWEX 1 ꢀ 2 OH-, MeOH, rt; (ii) TBDPSCl, pyridine, rt; (iii) Me2BBr,
Et3N, CH2Cl2, -78 °C; (iv) (CH2dCH)NCO, Et3N, THF/MeCN, rt; (v) (1) NaH, THF, -20 °C, (2) NaOH/MeOH/H2O; rt o/n, (3) aq HCl
pH 6 ∼ 6.5, 5min; (vi) Fmoc-Tyr(Me)-OH, HOBt, DIC, 0 °C to rt; (vii) (1) MeNH2/EtOH, rt. (2) NH4F, MeOH, 55 °C.
2254
dx.doi.org/10.1021/jo102178h |J. Org. Chem. 2011, 76, 2253–2256

The Journal of Organic Chemistry
NOTE
according to TLC). EtOH (500 μL) was added, and stirring was
continued for 30 min. Volatiles were evaporated and the residue was
chromatographed [SiO2, CH2Cl2/MeOH 100:0 to 93:7 step gradient]
to furnish 8 as a white amorphous solid (162 mg, 96%). Rf 0.46
(CH2Cl2/MeOH 9:1). HRMS (ESI) calcd for C29H34BrN6O4Si
637.1594, found 637.1584.
European research programme Synthcells FP6-NEST-Pathfinder
STREP No. 043359. P.S. thanks for the ongoing support through
the COST Action CM0703 Systems Chemistry.
’ REFERENCES
30-Amino-50-O-(tert-butyldiphenylsilyl)-30-deoxyadenosine
(10). NaH (60% in mineral oil; 9 mg, 0.23 mmol) in a round-
bottomed flask was rinsed with hexane (3 ꢀ 1 mL), anhydrous THF
(3 mL) was added, and the suspension was cooled to -20 °C under N2.
A solution of 8 (122 mg, 0.18 mmol, coevaporated with 3 ꢀ 1 mL of
THF) in anhydrous THF (2 mL) was added under N2 to the NaH
suspension, and stirring was continued for 12 h at -20 °C. NaOH (228
mg, 5.7 mmol) in MeOH/H2O 1:1 (4 mL) was added and the reaction
mixture was stirred at rt overnight and monitored by TLC. The mixture
was acidified to pH 6 ∼ 6.5 with HCl. After 5min, the solution was
filtered, the volatiles were evaporated and the residue was chromato-
graphed [SiO2, CH2Cl2/MeOH 100:0 to 92:8 step gradient] to yield 10
(102 mg, 93%) as a white amorphous solid. Rf 0.25 (CH2Cl2/MeOH
9:1). HRMS (ESI) calcd for C26H33N6O3Si 505.2384, found 505.2372.
50-O-(tert-Butyldiphenylsilyl)-30-[N-(9-fluorenyl)methoxy-
carbonyl-O-methyl-L-tyrosyl]amido-30-deoxyadenosine (11).
N-Fmoc-O-Me-L-Tyr (108 mg, 0.26 mmol) and HOBt (35 mg, 0.26 mmol)
were coevaporated with anhydrous THF (3 ꢀ 1 mL) and dissolved in
anhydrous THF (2 mL) and then the solution was cooled to 0 °C under N2
for 10 min. DIC (32 μL, 0.26 mmol) was added and the reaction mixture
was stirred at the same temperature for 15 min. This solution was added
slowly to a solution of 10 (95 mg, 0.19 mmol) in anhydrous THF (1 mL)
and the reaction mixture was stirred for 4 h at rt, taken up in EtOAc
(15 mL), and then washed with sat. NaHCO3 solution (15 mL) and H2O
(15 mL). The organic layer was dried with Na2SO4 and evaporated and the
residue underwent silica gel column chromatography [SiO2, CH2Cl2/
MeOH: 100:0 to 92:8 step gradient] to yield 11 (153 mg, 90%) as a white
amorphous solid. Rf 0.44 (CH2Cl2/ MeOH 9:1). HRMS (ESI) calcd for
C51H54N7O7Si 904.3854, found 904.3855.
(1) Porter, J. N.; Hewitt, R. I.; Hesseltine, C. W.; Krupka, G.; Lowery, J.;
Wallace, W. S.; Bohonos, N.; Williams, J. H. Antibiot. Chemother. 1952,
2, 409–410.
(2) Kmetec, E.; Tirpack., A. Biochem. Pharmacol. 1970, 19, 1493–1500.
(3) (a) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A.
Science 2000, 289, 905–920. (b) Nissen, P.; Hansen, J.; Ban, N.; Moore,
P. B.; Steitz, T. A. Science 2000, 289, 920–930. (c) Bayfield, M. A.;
Dahlberg, A. E.; Schulmeister, U.; Dorner, S.; Barta, A. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 10096–10101. (d) Hansen, J.; Schmeing, T. M.;
Moore, P. B.; Steitz, T. A. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 11670–11675. (e) Green, R.; Lorsch, J. R. Cell 2002, 110,
665–668. (f) Katunin, V.; Muth, G.; Strobel, S. A.; Wintermeyer, W.;
Rodnina, M. V. Mol. Cells 2002, 10, 339–346. (g) Parnell, K. M.; Seila,
A. C.; Strobel, S. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11658–11663.
(h) Rodnina, M. V.; Wintermeyer, W. Curr. Opin. Struct. Biol. 2003,
13, 334–340. (i) Sievers, A.; Beringer, M.; Rodnina, M. V.; Wolfenden,
R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7897–7901. (j) Weinger, J. S.;
Parnell, K. M.; Dorner, S.; Green, R.; Strobel, S. A. Nat. Struct. Mol. Biol.
2004, 11, 1101–1106. (k) Youngman, E. M.; Brunelle, J. L.; Kochaniak,
A. B.; Green, R. Cell 2004, 117, 589–599. (l) Schmeing, T. M.; Huang,
K. S.; Kitchen, D. E.; Strobel, S. A.; Steitz, T. A. Mol. Cells 2005,
20, 437–448. (m) Schmeing, T. M.; Huang, K. S.; Strobel, S. A.; Steitz,
T. A. Nature 2005, 438, 520–524. (n) Trobro, S.; Åqvist, J. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 12395–12400. (o) Huang, K. S.; Weinger,
J. S.; Butler, E. B.; Strobel, S. A. J. Am. Chem. Soc. 2006, 128, 3108–3109.
(p) Schroeder, G. K.; Wolfenden, R. Biochemistry 2007, 46, 4037–4044.
(q) Wohlgemuth, I.; Brenner, S.; Beringer, M.; Rodnina, M. V. J. Biol.
Chem. 2008, 283, 32229–32235. (r) Koch, M.; Huang, Y.; Sprinzl, M.
Angew. Chem., Int. Ed. 2008, 47, 7242–7245. (s) Lang, K.; Erlacher, M.;
Wilson, D. N.; Micura, R.; Polacek, N. Chem. Biol 2008, 15, 485–492.
(t) Kingery, D. A.; Pfund, E.; Voorhees, R. M.; Okuda, K.; Wohlgemuth,
I.; Kitchen, D. E.; Rodnina, M. V.; Strobel, S. A. Chem. Biol. 2008,
15, 493–500. (u) Voorhees, R. M.; Weixlbaumer, A.; Loakes, D.; Kelley,
A. C.; Ramakrishnan, V. Nat. Struct. Mol. Biol. 2009, 16, 528–533.
(4) Yarmolinsky, M. B.; Haba, G. L. Proc. Natl. Acad. Sci. U.S.A. 1959,
45, 1721–1729.
(5) (a) Baker, B. R.; Schaub, R. E.; Joseph, J. P.; Williams, J. H. J. Am.
Chem. Soc. 1955, 77, 12–15. (b)Suami, T.;Tadano, K.;Ayabe, M.;Emori, Y.
Bull. Chem. Soc. Jpn. 1978, 51, 855–861. (c) Okruszek, A.; Verkade, J. G. J.
Med. Chem. 1979, 22, 882–885. (d) Ozols, A. M.; Azhayev, A. V.; Dyatkina,
N. B.; Krayevsky, A. A. Synthesis 1980, 557–559. (e) Bouchu, D.;
Abou-Assali, M.; Grouiller, A.; Carret, G.; Pacheco, H. Eur. J. Med.
Chem.—Chim. Ther. 1981, 16, 43–47. (f) Lee, H.; Fong, K. L.; Vince, R.
J. Med. Chem. 1981, 24, 304–308. (g) Vince, R.; Daluge, S.; Brownwell, J.
J. Med. Chem. 1986, 29, 2400–2403. (h) McDonald, F. E.; Gleason, M. M.
J. Am. Chem. Soc. 1996, 118, 6648–6659. (i) Botta, O.; Strazewski, P.
Nucleosides Nucleotides 1999, 18, 721–724. (j) Chapuis, H.; Strazewski, P.
Tetrahedron 2006, 62, 12108–12115. (k) Takatsuki, K.; Ohgushi, S.;
Kohmoto, S.; Kishikawa, K.; Yamamoto, M. Nucleosides, Nucleotides Nucleic
Acids 2006, 25, 719–734. (l) Charafeddine, A.; Dayoub, W.; Chapuis, H.;
Strazewski, P. Chem.—Eur. J. 2007, 13, 5566–5584. (m) Okuda, K.; Hirota,
T.; Kingery, D. A.; Nagasawa, H. J. Org. Chem. 2009, 74, 2609–2612.
(6) (a) Robins, M. J.; Miles, R. W.; Samano, M. C.; Kaspar, R. L.
J. Org. Chem. 2001, 66, 8204–8210. (b) Samano, M. C.; Robins, M. J.
Tetrahedron Lett. 1989, 30, 2329–2332.
30-[O-Methyl-L-tyrosyl]amido-30-deoxyadenosine (2). Com-
pound 11 (137 mg, 0.15 mmol) was dissolved in 33% CH3NH2/EtOH
(12 mL). The reaction mixture was stirred at rt for 1 h. The solution was
concentrated under reduced pressure and coevaporated from CHCl3 (2 ꢀ
4 mL). The residue was dissolved in MeOH (5 mL) and ammonium
fluoride (30 mg, 0.81 mmol) was added to the solution. The reaction
mixture was warmed to 50-55 °C, stirred for 4 h, and monitored by TLC.
The volatiles were removed under reduced pressure. The residue was
purified by silica gel column chromatography [SiO2, EtOAc/MeOH/H2O
14:1:0.5, 12:1:0.5, 10:1:0.5, 8:1:0.5, 6:1:0.5, 4:1:0.5] to yield after evapora-
tion compound 2 (62 mg, 92%). Rf 0.10 (CH2Cl2/MeOH 9:1). HRMS
(ESI) calcd for C20H26N7O5 444.1996, found 444.1991.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR signal list-
S
b
ings of the compounds and 1H NMR, 13C NMR, DEPT, COSY,
and HSQC spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Email: strazewski@univ-lyon1.fr.
(7) Robins, M. J.; Hawrelak, S. D.; Hernꢀandez, A. E.; Wnuk, S. W.
Nucleosides Nucleotides 1992, 11, 821–834.
(8) Nguyen-Trung, N. Q.; Botta, O.; Terenzi, S.; Strazewski, P.
J. Org. Chem. 2003, 68, 2038.
(9) (a) Greenberg, S.; Moffatt, J. G. J. Am. Chem. Soc. 1973,
95, 4016–4025. (b) Russel, A. F.; Greenberg, S.; Moffat, J. G. J. Am.
Chem. Soc. 1973, 95, 4025–4030.
’ ACKNOWLEDGMENT
We acknowledge Bernard Fenet for NMR spectroscopic
analyses. K.K.S. is thankful for his graduate fellowship from the
2255
dx.doi.org/10.1021/jo102178h |J. Org. Chem. 2011, 76, 2253–2256

The Journal of Organic Chemistry
NOTE
(10) (a) Robins, M. J.; Hansske, F.; Low, N. H.; Park, J. I. Tetra-
hedron Lett. 1984, 25, 367–370. (b) Hansske, F.; Robins, M. J. Tetra-
hedron Lett. 1985, 26, 4295–4298. (c) Robins, M. J.; Wilson, J. S.; Madej,
D.; Low, N. H.; Hansske, F.; Wnuk, S. F. J. Org. Chem. 1995, 60,
7902–7908. (d) Michel, B. Y.; Krishnakumar, K. S.; Strazewski, P. Synlett
2008, 16, 2461–2464.
(11) (a) Guindon, Y.; Yoakim, C.; Morton, H. E. J. Org. Chem. 1984,
49, 3912–3920. (b) Guindon, Y.; Therien, M.; Girard, Y.; Yoakim, C.
J. Org. Chem. 1987, 52, 1680–1686. (c) Michel, B. Y. Synlett 2008,
2893–2894.
(12) (a) Le Goff, P.; Dewilde, D.; Tsuboniwa, N.; Urano, S. U.S.
Patent 5,386,057, January 31 1995. (b) Speziale, A. J.; Smith, L. R. J. Org.
Chem. 1965, 30, 4303–4305. (c) Speziale, A. J.; Smith, L. R. J. Org. Chem.
1963, 28, 1805–1811. (d) Speziale, A. J.; Smith, L. R. J. Org. Chem. 1962,
27, 4361–4365.
(13) K€onig, W.; Geiger, R. Chem. Ber. 1970, 103, 788–798.
(14) Krishnakumar, K. S.; Strazewski, P. Synlett 2010, 1055–1059.
2256
dx.doi.org/10.1021/jo102178h |J. Org. Chem. 2011, 76, 2253–2256