Facile and rapid access to inosine puromycin analogues through the use of adenylate deaminase

Article abstract of DOI:10.1021/ol070818q

To study the ribosomal peptidyl transfer, puromycin analogues are of interest in which adenine has been replaced by hypoxanthine. We synthesized inosine puromycin analogues from 3′-azidodeoxyadenosine derivatives using adenylate deaminase for the quantita

Full text of DOI:10.1021/ol070818q

ORGANIC
LETTERS
2007
Vol. 9, No. 15
2787-2790
Facile and Rapid Access to Inosine
Puromycin Analogues through the Use
of Adenylate Deaminase
Adib Charafeddine, Hubert Chapuis, and Peter Strazewski*
Laboratoire de Synthe`se de Biomole´cules (UMR 5246, ICBMS), Baˆtiment Euge`ne
CheVreul (5ie`me e´tage), UniVersite´ Claude Bernard Lyon 1, 43 bouleVard du 11
noVembre 1918, F-69622 Villeurbanne Cedex, France
strazewski@uniV-lyon1.fr
Received April 5, 2007
ABSTRACT
To study the ribosomal peptidyl transfer, puromycin analogues are of interest in which adenine has been replaced by hypoxanthine. We
synthesized inosine puromycin analogues from 3 -azidodeoxyadenosine derivatives using adenylate deaminase for the quantitative transformation
of the N-heterocycle. The amino acid coupling was carried out under Staudinger Vilarrasa conditions in 94% yield starting from the protected
and in 82% using the unprotected azide, thus, in the presence of two hydroxyls and a lactam function.
′
−
Natural puromycin contains a dimethylamino group that
replaces the usual N-heterocyclic 6-amino group with its
adenine moiety. Whereas natural puromycin inhibits in vivo
the growth of bacterial colonies by interrupting the bacterial
ribosomal protein synthesis, which can be quantified in vitro
in protein synthesis inhibition assays, the 6-amino puromycin
analogue 3 is only active in vitro but completely inactive in
vivo. Adenosine deaminase (adenosine aminohydrolase,
ADA, EC 3.5.4.4) and adenylate deaminase (5′-adenylic acid
deaminase, AMP deaminase, AMPDA, EC 3.5.4.6) are
ubiquitously occurring metalloenzymes that belong to the
class of hydrolases and catalyze the irreversible hydrolytic
deamination of adenosine and its derivatives to the corre-
sponding inosines. The broad substrate specificity of these
enzymes on nucleosides modified either on the purine base
or in the ribose moiety has long been known.1 The ADA
reaction is thought to proceed via a tetrahedral intermediate
at C-6 of the base. A similar mechanism may account for
AMPDA as well.2-4 The use of enzymes in organic synthesis
(1) (a) Cory, J. G.; Suhadolnik, R. J. Biochemistry 1965, 4, 1729. (b)
Wolfenden, R.; J. Am. Chem. Soc. 1966, 88, 3157. (c) Cory, J. G.;
Suhadolnik, R. J. Biochemistry 1965, 4, 1733. (d) Minato, S.; Nakanishi,
K. J. Biochemistry 1967, 62, 21. (e) Zielke, C. L.; Suelter, C. H. In The
Enzymes; Boyer, P.D., Ed., Academic Press: New York, 1971; pp 47-78.
(2) Groziak, M. P.; Huan, Z. W.; Ding, H.; Meng, Z. Y.; Stevens, W.
C.; Robinson, P. D. J. Med. Chem. 1997, 40, 3336.
(3) Merkler, D. J.; Kline, P. C.; Weiss, P.; Schramm, V. L. Biochemistry
1993, 32, 12993.
* To whom correspondence should be addressed. Phone: +334 72 44
82 34. Fax: +334 72 43 13 23.
(4) Ciuffreda, P.; Buzzi, B.; Alessandrini, L.; Santaniello, E. Eur. J. Org.
Chem. 2004, 4405.
10.1021/ol070818q CCC: $37.00
© 2007 American Chemical Society
Published on Web 06/26/2007

is now widely accepted and will continue to gain momentum
as more synthetic research utilizes enzymes as alternative
and more desirable sources for specific biocatalysis. This is
particularly true for the field of nucleoside chemistry.5 ADA
and AMPDA are commercially available and have been used
as biocatalysts for chemoenzymatic transformations to
convert purine nucleoside derivatives into a variety of
compounds6 that often show interesting pharmacological
properties.7 Enzymes are also environmentally friendly
resources that function best under aqueous conditions, thus
decreasing the needed amount of toxic organic solvents.
Compared with ADA, the properties and the possible use
of AMPDA as a biocatalyst are much less explored.5c,8
Information available from the literature seems to indicate
that AMPDA is able to accept a wider range of substrates
but is less stereoselective.9 It was gratifying for us to find
that all tested compounds were substrates for the catalytic
action of AMPDA and that this enzymatic deamination
allowed a viable preparation of the hypoxanthine derivatives
1, 2, and 8 from the adenine derivatives 3, 4, and 7,
respectively (Figure 1 and Scheme 1).
EtOH (33%) followed 1 M TBAF/THF to give puromycin
derivative 3 in 70% yield which was enzymatically deami-
nated in the presence of AMPDA in phosphate buffer at pH
6.5 and 38 °C to furnish after 5 h the target inosine
puromycin analogue 1 in a quasi-quantitative isolated yield.
Stirring the reaction at 25 instead of 38 °C resulted in a much
prolonged deamination period of 48 h, yet still in quantitative
yield. No deamination whatsoever of 3 (well soluble in water)
was observed at 38 °C when AMPDA was replaced by ADA,
most probably owing to the steric hindrance at the 3′ position
of the ribofuranose moiety (vide supra).
The second route, pathway B, began with the total
deprotection of 5 in one pot using CH3NH2/EtOH (33%) fol-
lowed by 1 M TBAF/THF to give the unprotected 3′-azido-
deoxynucleoside 7 (Scheme 1). The enzymatic deamination
of 7 could be carried out with both ADA or AMPDA in
phosphate buffer (at pH 7.0 or 6.5, respectively). Both
enzymes were equally capable of deaminating this substrate
in a few minutes (∼20 min) at 25 °C and gave the resulting
3′-azidodeoxyinosine derivative 8 in close to quantitative
yield (>98% after chromatography).
The coupling of 8 with the 1-oxybenzotriazolyl ester of
N-Boc-O-methyltyrosine under the same Staudinger-Vilar-
rasa conditions resulted in compound 9 in a remarkably good
yield (82%) despite the presence of two hydroxyl groups
and a lactam function. Compound 9 was then deprotected
with CF3COOH in water (3:7) to give the inosine puromycin
analogue 1 in 80% yield after chromatography over silica
gel and lyophilization.
Previously synthesized10c compound 4 (Figure 1) was also
subjected to an enzymatic deamination with AMPDA in
phosphate buffer at pH 6.5 and 38 °C to give after 5 h the
inosine 2′-deoxyfluoropuromycin analogue 2 in a quantita-
tive yield. Like 3, compound 4 was no substrate for ADA at
38 °C.
(6) (a) Secrist, J. A., III; Montgomery, J. A.; Shealy, Y. F.; O’ Dell, C.
A.; Clayton, S. J. J. Med. Chem. 1987, 30, 746. (b) Vince, R.; Brownell, J.
Biochem. Biophys. Res. Commun. 1990, 168, 912. (c) Borthwick, A. D.;
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Seela, F.; Kaiser, K. Chem. Pharm. Bull. 1988, 36, 4153.
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Kelley, J. A.; Mitsuya, H.; Shirasaka, T. J. Med. Chem. 1995, 38, 1189.
(b) Zaitseva, G. V.; Zinchenko, A. I.; Barai, V. N.; Pavlova, N. I.; Boreko,
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M.; Ellis, M.; Klumpp, K.; Court, S.; Ford, M. Antimicrob. Agents
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Chim. Acta 1996, 79, 1451. (e) Seela, F.; He, Y.; Wei, C. Tetrahedron
1999, 55, 9481. (f) Sheppard, T. L.; Rosenblatt, A. T.; Breslow, R. J. Org.
Chem. 1994, 59, 7243.
(8) (a) Samejima, H.; Kimura, I.-C.; Noguchi, S.; Shimura, G. In Enzyme
Engineering; Pye, E. K., Weetall, H. H., Eds.; Plenum Press: New York,
1978; Vol. 3, pp 469-475. (b) Muzzarelli, R. A. A. Enzyme Microb.
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(9) (a) Ciuffreda, P.; Loseto, A.; Alessandrini, L.; Terraneo, G.; Sant-
aniello, E. Eur. J. Org. Chem. 2003, 4748. (b) Margolin, A. L.; Borcherding,
D. R.; Wolf-Kugel, D.; Margolin, N. A. J. Org. Chem. 1994, 59, 7214.
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Org. Chem. 2003, 68, 2038. (b) Chapuis, H.; Strazewski, P. Tetrahedron
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P. Chem. Eur. J. 2007, 13, 5566.
Figure 1. Puromycin analogues.
We previously reported the synthesis of compounds 3 and
4 from adenosine under optimized experimental conditions.10
Here, we present an alternative that gives direct and simple
access to the corresponding unprotected inosine puromycin
analogues. To obtain target compound 1 we proceeded via
two synthetic routes (Scheme 1).
The first one, pathway A, started from fully protected 2′-
azidodeoxynucleoside 5 that was first coupled with the
1-oxybenzotriazolyl ester of N-Fmoc-O-methyltyrosine to
give 6 in 94% yield under the recently published Staudinger-
Vilarrasa coupling conditions as developed for the synthesis
of nonfluorinated10b and fluorinated puromycin analogues.10c
Compound 6 was then deprotected in one pot with CH3NH2/
(5) (a) Ferrero, M.; Gotor, V. Monatsh. Chem. 2000, 131, 585. (b) Ferro,
M.; Gotor, V. Chem. ReV. 2000, 100, 4319. (c) Santaniello, E.; Ciuffreda,
P.; Alessandrini, L. Synthesis 2005, 509. (d) Gupta, M.; Nair, V. Collect.
Czech. Chem. Commun. 2006, 71, 769.
2788
Org. Lett., Vol. 9, No. 15, 2007

Scheme 1. Syntheses of Inosine Puromycine Analogue 1 Using Two Pathways
Earlier investigations already demonstrated that both
prolongued reactions times, replacement of the 2′-hydroxyl
group by fluorine, and most interestingly, much bulkier
substituents at the 3′ position impossible to handle by ADA,
all of which makes AMPDA a highly welcome alternative.
AMPDA is commercially available as a practical lyophilate
powder, and the cost per unit of reactivity may be some 30%
lower than that of ADA. The experimental workup of large
amounts of bulk enzyme poses absolutely no problems even
for as highly polar molecules as our target compounds. A
simple filtration of the reaction mixture over a short silica
gel column using a step gradient mixture of ethyl acetate-
methanol-water as the eluant provides, after lyophilization
from water, solid product material in high yields and
chemical purity (see the Experimental Part in the Supporting
Information).
Second, the recently published optimized Staudinger-
Vilarrasa coupling conditions,10b,c which provide high yield
access to amides directly from sterically demanding azides,
proved much more chemoselective than observed under other
modified Staudinger conditions.15 The activation of the Boc-
amino acid by HOBt does not lead to significant side
reactions when coupled to an iminophosphorane that contains
at least two competing nucleophilic centers in addition to
the one generated by the in situ addition of tributylphosphine
to 2′,5′-dihydroxyazide 8 (Scheme 1). The new synthetic
intermediates and target compounds were characterized
through MS and 1H, 19F, and 13C NMR (Supporting Informa-
tion) and will be subjected to biological assays. A more
systematic investigation of this minimal protection version
of the Staudinger-Vilarrasa coupling is currently underway
(such as, for instance, the direct conversion of 7 into 3).
enzymes, ADA and AMPDA, could tolerate some substitu-
tion at the 2′,3′-positions. However, AMPDA seemed a more
versatile biocatalyst, since it converted a larger number of
adenosine into the corresponding inosine derivatives in a time
well suitable for preparative purposes.9 Santaniello et al.’s
studies showed that solubilizing the substrate with a few
percent of a cosolvent like pyridine or DMSO reduced
somewhat the deamination rate of ADA, and the recovery
of the products often proved troublesome. However, the same
reaction at 50-60 °C rendered many substrates soluble in
water, and the ADA enzyme still showed good activity.11
Generally, a quite high tolerance of ADA for changes in the
N-heterocyclic positions 2 and 6 (to be hydrolyzed) is
observed, yet steric hindrance at the 2′- and 3′-positions, or
if bulky groups other than OH are present at the 5′-position,
disfavors the complete fitting of the substrate within the
active site of ADA, which leads to a slow reactivity or even
total inhibition.12-14
With this communication we wish to, first, add new
evidence for AMPDA’s tolerance toward higher temperature,
(11) (a) Ciuffreda, P.; Casati, S.; Santaniello, E. Tetrahedron 2000, 56,
3239. (b) Ciuffreda, P.; Loseto, A.; Santaniello, E. Tetrahedron 2002, 58,
5767-5771.
(12) (a) Bloch, A.; Robins, M. J.; McCarthy, J. R., Jr. J. Med. Chem.
1967, 10, 908. (b) Ciuffreda, P.; Loseto, A.; Santaniello, E. Tetrahedron:
Asymmetry 2002, 13, 239. (c) Ciuffreda, P.; Loseto, A.; Santaniello, E.
Tetrahedron: Asymmetry 2004, 15, 203. (d) Ciuffreda, P.; Alessandrini,
L.; Pavlovic, R.; Santaniello, E. Nucleosides, Nucleotides, Nucleic Acids
2007, 26, 121.
(13) (a) Maury, G.; Daiboun, T.; Elalaoui, A.; Ge´nu-Dellac, C.; Pe´rigaud,
C.; Bergogne, C.; Gosselin, G.; Imbach, J.-L. Nucleosides, Nucleotides 1991,
10, 1677-1692. (b) Nair, V.; Wiechert, R. J. Bioorg. Chem. 1980, 9, 423.
(14) (a) Marrone, T. J.; Straatsma, T. P.; Briggs, J. M.; Wilson, D. K.;
Quiocho, F. A.; McCammon, J. A. J. Med. Chem. 1996, 39, 277. (b) Wilson,
D. K.; Quiocho, F. A. Biochemistry 1993, 32, 1689.
(15) Kimmerlin, T.; Seebach, D. J. Peptide Res. 2005, 65, 229.
Org. Lett., Vol. 9, No. 15, 2007
2789

Supporting Information Available: Experimental pro-
tocols and chromatographic and spectroscopic characteriza-
tion of all new synthetic intermediates and final products.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL070818Q
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Org. Lett., Vol. 9, No. 15, 2007