New S-adenosyl-L-methionine analogues: Synthesis and reactivity studies

Article abstract of DOI:10.1021/ol9009859

Two new and complementary synthetic strategies for 5'-N-chloroethylamino- 5'-deoxyadenosines are presented. Additionally, the reaction kinetics of their conversion into aziridines under typical enzyme assay conditions is reported using time-resolved NMR spectroscopy. A stable photocaged derivative of 5'-N-chloroethylamino-5'-deoxyadenosine has also been synthesized, and its stability and activation in aqueous solution at physiological pH have been examined.

Full text of DOI:10.1021/ol9009859

ORGANIC
LETTERS
2009
Vol. 11, No. 14
2976-2979
New S-Adenosyl-L-methionine
Analogues: Synthesis and Reactivity
Studies
Andrew P. Townsend,† Stefanie Roth,† Huw E. L. Williams,† Eleni Stylianou,‡ and
Neil R. Thomas*,†
UniVersity of Nottingham, School of Chemistry, Centre for Biomolecular Sciences,
UniVersity Park, Nottingham, NG7 2RD, United Kingdom, and School of Biomedical
Sciences, UniVersity of Nottingham Medical School, Queen’s Medical Centre,
Nottingham NG7 2UH, United Kingdom
neil.thomas@nottingham.ac.uk
Received May 5, 2009
ABSTRACT
Two new and complementary synthetic strategies for 5′-N-chloroethylamino-5′-deoxyadenosines are presented. Additionally, the reaction kinetics
of their conversion into aziridines under typical enzyme assay conditions is reported using time-resolved NMR spectroscopy. A stable photocaged
derivative of 5′-N-chloroethylamino-5′-deoxyadenosine has also been synthesized, and its stability and activation in aqueous solution at
physiological pH have been examined.
S-Adenosyl methione (SAM) is the second most abundant
coenzyme in the human body and often referred to as
“mother nature’s methyl iodide”. Methyltransferases (MTs)
transfer the activated methyl group from the sulfur center to
specific positions in a variety of substrates, e.g., DNA, RNA,
proteins, and secondary metabolites.1 In recent years, in-
creasing interest in these enzymes has led to the design of
two classes of SAM analogues: Aziridinoadenosines (A1) and
double-activated SAM analogues (B) (Figure 1).2 These
compounds have been used to label a variety of biopolymers
and secondary metabolites using MTs. Other interesting
applications of these coenzyme analogues include their use
as inhibitors of specific enzymes3 or their use as chemical
tools for the identification of methylation targets.4 Aziridi-
noadenosines are highly reactive and unstable compounds;
therefore, 5′-N-halogenoethylamino-5′-deoxyadenosines (A0)
have been proposed as synthetic precursors, as they can form
the aziridine ring in situ (Figure 1).5 To date, only iodo
compounds have been synthesized and successfully reported
to alkylate DNA molecules via intermediate aziridine forma-
tion in reactions catalyzed by Thermus aquaticus (MTaq.1)
and E. coli (EcoR1) DNA methylases,5,6 while the 5′-N-
(3) For selected references, see: (a) Borchardt, R. T.; Wu, Y. S.; Huber,
J. A; Wycpalek, A. F. J. Med. Chem. 1976, 19, 1104–1110. (b) Guerard,
C.; Breard, M.; Courtois, F.; Drujon, T.; Ploux, O. Bioorg. Med. Chem.
Lett. 2004, 14, 1661–1664. (c) Byers, T. L.; Wechter, R. S.; Hu, R.-H.;
Pegg, A. E. Biochem. J. 1994, 303, 89–96. (d) Benghiat, E.; Crooks, P. A.
J. Med. Chem. 1983, 26, 1470–1477.
† University of Nottingham.
(4) For a review on the elucidation of biological pathways using chemical
probes, see: Shogren-Knaak, M. A.; Alaimo, P. J.; Shokat, K. M. Annu.
ReV Cell DeV. Biol. 2001, 17, 405–433. (a) Schmidt, F. H.-G.; Hueben,
M.; Gider, B.; Renault, F.; Teulade-Fichou, M.-P.; Weinhold, E Bioorg.
Med. Chem. 2008, 16, 40–48. (b) Dalhoff, C.; Lukinavicius, G.; Klima-
sˇauskas, S.; Weinhold, E. Nature Prot. 2006, 1, 1879–1886. (c) Dalhoff,
C.; Lukanivicius, G.; Klimasˇauskas, S.; Weinhold, E. Nature Chem. Biol.
2006, 2, 31–32. (d) Comstock, L. R.; Rajski, S. R. J. Org. Chem. 2004, 69,
1425–1428.
‡ University of Nottingham Medical School.
(1) For reviews, see: (a) Fontecave, M.; Atta, M.; Mulliez, E. Trends
Biochem. Sci. 2004, 29, 243–249. (b) Chiang, P. K.; Gordon, R. K.; Tal,
J.; Zeng, G. C.; Doctor, B. P.; Pardhasaradhi, K.; McCann, P. P. FASEB J.
1996, 10, 471–480. (c) Frey, P. A.; Magnusson, O. T. Chem. ReV. 2003,
103, 2129–2148.
(2) (a) Klimasˇauskas, S.; Weinhold, E. Trends Biotechnol. 2007, 25,
99–104. (b) Dalhaff, C., Weinhold, E. In Modified Nucleosides; Herdewijn,
P., Ed.; Wiley-VCH: Weinheim, Germany, 2008; pp 223-247.
(5) Weller, R. L.; Rajski, S. R. Org. Lett. 2005, 5, 2141–2144.
10.1021/ol9009859 CCC: $40.75
Published on Web 06/24/2009
 2009 American Chemical Society

Figure 1. S-Adenosylmethionine and currently available analogues. A0: Halogenoethylamino precursor. A1: Aziridinoadenosine. B: Double-
activated analogues.
homoalanine derivative of A0 has been shown to alkylate
the arginine side chain of a peptide corresponding to the
N-terminal of the H4-histone in a PRMT1-mediated reac-
tion.7
quantitative yields as the corresponding acetonide 2.11 The
5′-nitrogen was introduced using diphenylphosphoryl azide
(dppa) and subsequent treatment with sodium azide, followed
by reduction to give amine 4 in 89% yield over the three
steps. Amine 4 was then subjected to a direct reductive
amination with chloroacetic aldehyde, and pleasingly, the
corresponding N-chloromustard was installed in 73% yield.
Subsequent acid-mediated deprotection of the acetonide gave
the desired compound (6) in five steps and 64% overall yield
(Scheme 1). This provides the first reported synthesis of this
These compounds have been used increasingly as biologi-
cal tools; however, their take up is hampered by the long,
inflexible, and low-yielding syntheses that have been reported
to date even following some optimization.5,6 Even more
surprising, currently there are no substantive studies on the
reaction kinetics for the aziridine formation and breakdown
of these nucleosides.8 Previous studies on simple alkyl
nitrogen mustards suggest that the iodo compounds are highly
reactive electrophiles themselves9 and could therefore com-
pete with the in situ formed aziridines for available nucleo-
philes, opening the opportunity for undesired nonspecific and
nonenzyme mediated alkylations.
Scheme 1. Synthesis of
5′-N-Chloroethylamino-5′-deoxyadenosine (6)
Here, we report two new and efficient routes to N-
chloromustard-substituted adenosines, the synthesis of a
photocaged version, and kinetic studies on the formation and
chemical behavior of the aziridine compounds in a phosphate
buffer at physiological pH. In previously reported syntheses,
the installation of the 5′-nitrogen proved to be a major
difficulty which led to lengthy and inefficient synthetic
routes.
We proposed that the introduction of the nitrogen in a
simpler fashion into standard protected adenosine derivatives
would decrease the length and improve the yield of a
synthetic route immensly. We therefore chose reductive
amination10 as the key reaction to introduce the complete
N-chloromustard substituent on the 5′-position in a single
step. Starting from commercially available adenosine (1), the
2′- and 3′-hydroxyl groups were selectively protected in
(6) (a) Pignot, M.; Siethoff, C.; Linscheid, M.; Weinhold, E. Angew.
Chem., Int. Ed. 1998, 37, 2888–2891. (b) Pljevaljcic, G.; Schmidt, F.;
Weinhold, E. ChemBioChem 2004, 5, 265–269. (c) Comstock, L. R.; Rajski,
S. R. Nucleic Acids Res. 2005, 33, 1644–1652. (d) Weller, R. J.; Rajski,
S. R. ChemBioChem 2006, 7, 243–245.
compound and is one of the shortest and highest yielding
syntheses of a nitrogen-containing SAM analogue to date.
In order to gain a deeper insight into the reactivity and
chemical behavior of these compounds, we investigated the
kinetics of the transformation of the chloromustard adenosine
6 as well as the in situ formed aziridinoadenosine 7 by time-
resolved 1H NMR spectroscopy (Figure 2). As expected, the
conversion of the chloromustard side chain into the aziridine
(7) Osborne, T.; Weller Roska, R. L.; Rajski, S. R.; Thompson, P. R.
J. Am. Chem. Soc. 2008, 130, 4574–4575.
(8) Note that there has been an investigation with respect to the in situ
formation of the aziridine by Rajski et al.; however, this was just a
preliminary investigation and did not lead to the determination of a half-
life: Petersen, S. G.; Rajski, S. R. J. Org. Chem. 2005, 70, 5833–5839.
(9) Jenkins, T. C.; Naylor, M. A.; O’Neill, P.; Threadgill, M. D.; Cole,
S.; Stratford, I. J.; Adams, G. E.; Fielden, E. M.; Suto, M. J.; Stier, M. A
J. Med. Chem. 1990, 33, 2603–2610.
(10) For a review on reductive aminations, see: Baxter, E.; Reitz, A. B.
Org. Reactions 2002, 59, 1–714.
(11) Note that 2′,3′-O-isopropylideneadenosine is also commercially
available, i.e., from Sigma-Aldrich.
Org. Lett., Vol. 11, No. 14, 2009
2977

before.14 For the synthesis, we decided to apply a second
approach to install the 5′-nitrogen, a nucleophilic displace-
ment of a leaving group at the 5′-position, which would be
complementary to the reductive amination route.
The leaving group was introduced by transforming the free
5′-hydroxy group of the 2′,3′-O-isopropylideneadenosine (2)
into a mesylate (8). Subsequent nucleophilic displacement
could only be achieved using 2-aminoethanol, both as
reactant and as solvent. All other reaction conditions and
nucleophiles tested led primarily to a previously described
intramolecular cyclization product.15
Pleasingly, using 2-aminoethanol provided the correspond-
ing 5′-hydroxyethylaminoadenosine 9 in 53% yield without
the formation of the cyclized side product.16 The NVOC-
protecting group was installed as its carbamate using the
corresponding commercially available chloroformate, and the
free hydroxy group was transformed subsequently into the
chloride by an Appel chlorination. After acid-catalyzed
deprotection, the photocaged nucleoside 12 was obtained in
36% overall yield in six steps from adenosine (Scheme 2).
Figure 2. Half-life studies of 6. Peaks: 8.31 (H-2, 7), 8.28 (H-2,
6), 8.26 (H-8, 6), 8.25 (H-8, 7). (Note that the 5′-nitrogen of 7
would be partially protonated.)
Scheme 2. Synthesis of a Photocaged SAM Analogue (12)
ring followed typical first-order reaction kinetics, and the
half-life (t1/2) of the parent compound was determined to be
t1/2 ) 390 min in 25 mM sodium phosphate buffer at pH )
7.4 and 25 °C.
Furthermore, we observed that the concentration of aziri-
dine 7 formed, reached a maximum, and then began to
decrease after approximately half of the chloromustard had
been converted to it, producing several products. Investiga-
tion of the NMR solution with electrospray mass spectrom-
etry, revealed the major components to be products of
aziridine ring-opening by nucleophiles present in the buffer
system (hydroxide and phosphate ions). Based on these
findings, we concluded that there seems to be an “optimal
time-frame” for the use of N-chloromustard-substituted SAM
analogues in biological assays, and ideally, a mainly non-
nucleophilic buffer system should be employed.12 Due to
the low stability of the chloromustard-substituted compounds
in aqueous solutions and on prolonged storage at -20 °C,
we decided to synthesize a photocaged derivative of the
parent chloromustard-substituted adenosine 6. Photocleavable
protecting groups have previously been used in various
biological applications and have proved to be extremely
useful in controlling the reactivity of highly reactive probes,
thus allowing the storage of the compounds not only in pure
form on the bench but even in the buffer necessary for the
biological assays without any reaction occurring.13 We chose
the 6-nitroveratryl group (NVOC), as this group can be
cleanly removed by exposure of the “caged” compound to
UV radiation of wavelength of >350 nm. NVOC has been
used for the protection of nucleoside-based drugs and probes
To our surprise, the photocaged compound (12) could be
stirred unchanged at room temperature in daylight for 12 h
(13) For a review on photolabile protecting groups, see: Bochet, C. G.
J. Chem. Soc., Perkin Trans. 1 2002, 125–142.
(14) (a) Wei, Y.; Yan, Y.; Pei, D.; Gong, B. Bioorg. Med. Chem. Lett.
1998, 8, 2419–2422. (b) Furuta, T.; Watanabe, T.; Tanabe, S.; Sakyo, J;
Matsuba, C. Org. Lett. 2007, 9, 4717–4720.
(15) The intramolecular cyclization product results of an attack of N-9
on the 5′-carbon to form a tetracyclic salt; see: (a) Liu, F.; Austin, D. J. J.
Org. Chem. 2001, 66, 8643–8645. (b) Liu, F.; Austin, D. J. Tetrahedron
Lett. 2001, 42, 3153–3154. (c) Ashton, T. D.; Scammells, P. J. Bioorg.
Med. Chem. Lett. 2006, 42, 4564–4566.
(16) It should be noted that this yield is that after a double purification
by column chromatography. The overall yield can be increased by submitting
the crude product directly to the next step.
(12) Kinetic studies involving N-chloromustards bearing a second
nitrogen substituent are ongoing and will be reported in due course.
2978
Org. Lett., Vol. 11, No. 14, 2009

was possible with just 2 min irradiation with a standard
laboratory Mercury UV lamp (Scheme 3).
Scheme 3
.
Activation and Reactivity Studies of Photocaged
In conclusion, we presented two new, highly efficient,
versatile, and complementary approaches for the synthesis
of 5′-N-chloroethylamino-5′-deoxyadenosines for applica-
tions as nitrogen-containing SAM analogues. For the first
time, a detailed investigation of the reaction kinetics of the
aziridine formation and the chemical behavior of the aziridine
has been conducted using time-resolved NMR spectroscopy
and electrospray mass spectrometry. In addition, a photo-
caged derivative has been synthesized and found to be stable
in solid form, in aqueous buffer solution, and in methanol,
in which its activation by UV irradiation was studied.
Analogue 12
Acknowledgment. This work was funded by the BBSRC
SCIBS initiative (BBE0140891) to N.R.T. and E.S. and a
BBSRC strategic research studentship to N.R.T. to support
A.P.T. (BBSSH200511988).
Supporting Information Available: Experimental pro-
cedures, characterization data, 1H and 13C NMR spectra for
new compounds as well as details on the kinetic studies. This
material is available free of charge via the Internet at
http://pubs.acs.org.
without any reaction in the same buffer system as previously
used for the NMR studies of compound 6. Activation of the
compound by removal of the photocaging protecting group
OL9009859
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2979