Efficient preparation of 2,4-diaminopyrimidine nucleosides: Total synthesis of lysidine and agmatidine

Article abstract of DOI:10.1021/ol301769j

An efficient route for the synthesis of 2,4-diaminopyrimidine ribosides from cytidine is described consisting of six steps with overall yields >50% and only one chromatographic step. The key amine addition step utilizes LiCl and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to ensure clean conversion to a single tautomeric product. This route has been used to prepare the modified tRNA nucleosides lysidine and agmatidine in quantities suitable for structural characterization.

Full text of DOI:10.1021/ol301769j

ORGANIC
LETTERS
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Vol. XX, No. XX
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Efficient Preparation of 2,
4-Diaminopyrimidine Nucleosides: Total
Synthesis of Lysidine and Agmatidine
Brett J. Kopina and Charles T. Lauhon*
Pharmaceutical Sciences Division, School of Pharmacy,
University of Wisconsin;Madison, Madison, Wisconsin 53705, United States
clauhon@wisc.edu
Received June 27, 2012
ABSTRACT
An efficient route for the synthesis of 2,4-diaminopyrimidine ribosides from cytidine is described consisting of six steps with overall yields >50%
and only one chromatographic step. The key amine addition step utilizes LiCl and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to ensure clean
conversion to a single tautomeric product. This route has been used to prepare the modified tRNA nucleosides lysidine and agmatidine in
quantities suitable for structural characterization.
2,4-Diaminopyrimidine ribosides are one of the poorest
studied classes of nucleosides capable of WatsonꢀCrick
pairing. Only a handful of derivatives have been described,
and a general large scale method for their preparation has
yet to be reported. Two members of this class of nucleo-
sides havebiological significance. Lysidine (1, Figure 1) is a
modified nucleoside found in bacterial tRNA and is nearly
universally conserved.1 The similarly structured agmati-
dine (2) is exclusive to tRNA in archea where it serves a
similar purpose.2 These N1-alkylated 2,4-diaminopyrimi-
dines are unique in that they are stable despite their
unusually high basicity, with pKa values close to 13.3 In
addition, theyhave accesstoa number oftautomeric forms
for which direct experimental evidence is lacking.
The biosynthesis of lysidine4 and agmatidine5 occurs
post-transcriptionally from tRNA at cytidine 34, the first
position of the anticodon. This single modification
changes not only the amino acid attached to the tRNA
(from methionine to isoleucine) but also its codon recogni-
tion from 50-AUG to 50-AUA.6 The altered pairing pre-
ference of lysidine and agmatidine for A over G is
proposed to be the result of a combination of steric bulk
at position 2 and protonation at N3.1 However, there is to
date no experimental evidence to support this hypothesis.
Thus, it is useful to have an efficient route to prepare these
nucleosides as well as analogs for both structural and
biological study.
(1) Muamatsu, T.; Yokoyama, S.; Horie, N.; Matsuda, A.; Ueda, T.;
Yamaizumi, Z.; Kuchino, Y.; Nishimura, S.; Miyazawa, T. J. Biol.
Chem. 1988, 263, 9261.
(2) (a) Mandal, D.; Kohrer, C.; Su, D.; Russell, S. P.; Krivos, K.;
Castleberry, C. M.; Blum, P.; Limbach, P. A.; Soll, D.; Raj Bhandary,
U. L. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2872. (b) Ikeuchi, Y.;
Kimura, S.; Numata, T.; Nakamura, D.; Yokogawa, T.; Ogata, T.;
Wada, T.; Suzuki, T.; Suzuki, T. Nat. Chem. Biol. 2010, 6, 277.
(3) Brown, D. J.; Jacobsen, N. W. J. Chem. Soc. 1962, 3172.
(4) (a) Soma, A.; Ikeuchi, Y.; Kanemasa, S.; Kobayashi, K.; Ogasawara,
N.;Ote, T.;Kato, J.;Watanabe, K.;Sekine, Y.;Suzuki, T.Mol. Cell 2003, 12,
689. (b) Ikeuchi, Y.; Soma, A.; Ote, T.; Kato, J.; Sekine, Y.; Suzuki, T. Mol.
Cell 2005, 19, 235.
(5) (a) Terasaka, N.; Kimura, S.; Osawa, T.; Numata, T.; Suzuki, T.
Nat. Struct. Biol. 2011, 18, 1268. (b) Osawa, T.; Satoshi, K.; Terasaka,
N.; Inanaga, H.; Suzuki, T. Nat. Struct. Biol. 2011, 18, 1275.
(6) Muramatsu, T.; Nishikawa, K.; Nemoto, F.; Kuchino, Y.;
Nishimura, S.; Miyazawa, T.; Yokoyama, S. Nature 1988, 336, 179.
r
10.1021/ol301769j
XXXX American Chemical Society

Scheme 1. Synthetic Route toward 2,4-Diaminopyrimidine
Nucleosides
Figure 1. N4 amino (A) and imino (B) tautomeric structures for
the protonated forms of lysidine 1 and agmatidine 2.
Previously, lysidine and agmatidine have been prepared
in micromole amounts for initial identification of the
biologically produced nucleosides.1,2 These routes involve
reacting protected lysine or free agmatine with S-alkylated
2-thiocytidine salts using the methodology of Ueda et al.7
Since 2-thiocytidine is not readily available in large
amounts, we looked for a more practical and efficient
route from cytidine. We report herein a general synthetic
route that can provide a number of N2-alkylated-2,4-
diaminopyrimidine nucleosides in high yield with only
one chromatographic step. In addition, we present the first
experimental data on the structure and tautomeric state of
these nucleosides.
Our synthesis (Scheme 1) begins with cytidine and
utilizes 4-bromobenzoyl and 20,30-isopropylidene protec-
tion to give intermediate 4 in 91% overall yield. The
cyclization of N4-benzoylcytidine to the 2,50-anhydrocyti-
dine derivative under Mitsonobu conditions has been
previously reported in modest yield.8 Our use of p-bromo-
benzoyl protection at N4 gave a more crystalline product 5
that could be cleanly isolated from the reaction byproducts
in 73% yield on a multigram scale.
The next step involved addition of an amine to the 2,50-
anhydrocytidine 5 specifically at C-2. In our hands reac-
tion of the anhydrocytidine derivatives with alkylamines
alone was sluggish, providing low yields of complex pro-
duct mixtures. However, addition of LiCl with THF as
solvent gave good conversion to a small number of amine
addition products. NMR and LC-MS analysis of the
products was consistent with a mixture of tautomeric
hydrochloride salts. Interestingly, these isomers appear
to interconvert slowly enough to be partially separable.
However, conditions in which the purported tautomers
can be reliably separated or interconverted have proven
elusive.
single product by TLC. After silica gel chromatography
the desired products were isolated as hydrochloride salts.
As shown in Table 1 (entries 1ꢀ7), the optimized reaction
conditions gave high yields under mild conditions for a
variety of basic primary amines. Less basic amines, such as
anilines or hydroxylamines (entries 8 and 9), and second-
ary amines (not shown) did not react under these condi-
tions. Unprotected agmatine appeared to react smoothly,
but we were unable to isolate the resulting product.
The use of LiCl in these reactions was intended to
increase the reactivity of the electrophile via coordination
to either the acyl group or the 50-oxygen. We observed that
LiCl increased both the solubility of the reactants and the
rate of reaction. It also provided a counterion for efficient
purification of the product by silica gel chromatography.
Of the many Lewis acids tested, only LiCl and LiBF4 gave
satisfactory results. Reactions containing only DBU gave
conversion to a product that could only be isolated in low
yield.
To confirm the structural characteristics of these mole-
cules, the ethanolamine adduct 6b was crystallized from
propanol and an X-ray structure was obtained. The struc-
ture (Figure 2) clearly shows the product as a hydrochlor-
ide salt in which the chloride is found in close proximity to
C-2 of the pyrimidine ring. This is consistent with NMR
evidencefor protonation at N2. The ethanol side chain is in
the trans configuration relative to the glycosidic bond to
To resolve the issue of tautomeric mixtures, 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) was added to the
amine addition reactions to keep the product in a neutral
form. This modification resulted in the production of a
(7) Ueda, T.; Ohtsuka, H. Chem. Pharm. Bull. 1973, 21, 1530.
(8) Kimura, J.; Yagi, K.; Suzuki, H.; Mitsunobu, O. Bull. Chem. Soc.
Jpn. 1980, 53, 3670.
B
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Deprotection of the amine addition products 6 by
sequential treatment with ammonia in methanol, followed
by trifluoroacetic acid at 0 °C, gave the desired target
molecules 8b (R = CH2CH2OH), lysidine (1), and agma-
tidine (2) in high yield and >95% purity. Overall yields for
the synthesis of both lysidine and agmatidine from cytidine
ranged from 50 to 60%.
Table 1. Addition of Amines to 2,50-Anhydrocytidine 5
While the usual convention is to draw the cationic
derivatives of lysidine and agmatidine as imino tautomers
with the N2, N3, and N4 nitrogens each protonated
(Structure B in Figure 1), NMR analysis is consistent with
the N4 amino tautomer A. Figure 3 shows 15N HSQC data
for agmatidine in DMSO-d6. The two NH protons at 8.1
and 8.3 ppm both correlate to a single nitrogen at 109 ppm,
assigned as N4. The 15N signal at 97 ppm is assigned as N2
based on gCOSY data, in which the N2 proton correlates
to the adjacent methylene protons. The 15N signal at 137 ppm
is assigned to the guanidine of the agmatine side chain
based on gCOSY data and its significant 15N chemical shift
in the HSQC spectrum of Boc-protected precursor 7g.11
Lysidine exhibits a similar HSQC spectrum under these
conditions, except that the 15N signal at 137 ppm is
absent.11
Although our NMR datasupportthe amino tautomer in
DMSO, this does not preclude involvement of the imino
tautomer in base pairing. We have been unable thus far to
determine the tautomeric structures of lysidine and agma-
tidine in water at pH 7, as the heteroatom protons ex-
change rapidly with solvent. Thus, in aqueous media at
physiological pH it is possible that the tautomeric equili-
brium is shifted toward the imino form. Alternatively, the
tautomers may interconvert rapidly enough to provide the
imino tautomer for base pairing to adenosine.
a Yields are for the isolated products after chromatography. b No
product detectable.
avoid interaction with the H10 proton as previously pre-
dicted for lysidine itself1 using computational methods.9
NMR analysis of 6b is consistent with the tautomer shown
in Figure 2 in which the N2 and N4 nitrogens are proto-
nated. Although the positive charge is likely greatest
among the N1ꢀC2ꢀN2 atoms of the ring, a true repre-
sentation is likely that of a highly resonant cation in which
electrons are delocalized throughout the ring as well as the
exocyclic nitrogens.10
Figure 2. X-ray structure of intermediate 6b crystallized from n-
propanol. Two populated conformations of the isopropylidene
are shown.
Figure 3. 15N HSQC data for agmatidine in DMSO-d6 consis-
tent with tautomeric structure 2A (Figure 1). The 15N signal at
137 ppm is absent in the lysidine 15N HSQC data.
Org. Lett., Vol. XX, No. XX, XXXX
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two hydrogen bonds can only be obtained if the N2-alkyl
group is in the unfavorable cis orientation. Pairing with a
single hydrogen bond and the N2-alkyl group trans will
incur steric interaction with the adenine base. The imino
tautomer (Figure 4b) is able to form a WatsonꢀCrick base
pair with two hydrogen bonds and the more favorable
trans N2-alkyl group due to protonation at N3. However,
there remains a possible steric interaction with H-2 of the
adenine base. Thus, it is likely that some deviation in the
geometry of the base pairing accompanies the lysidine/
agmatidine interaction in the anticodon. Such deviation
may be more readily achieved for the amino tautomer
which is less constrained. This possibility should not be
discounted, as wobble pairings with altered geometry and
hydrogen bonding patterns are common.13
In summary, we have developed an efficient synthetic
route to the biologically important 2,4-diaminopyrimidine
ribosides lysidine and agmatidine. We have also presented
the first experimental evidence describing their conforma-
tion and tautomeric state. Current efforts are underway to
expand our initial structural studies and to incorporate
these nucleosides and their analogs into RNA to fully
understand the mechanism of their binding specificity.
Figure 4. Potential base pairing interaction between adenosine
(A) and (a) the amino tautomer of lysidine and agmatidine or
(b) the imino tautomer.
The predominance of the N4-amino tautomer in the 15
N
HSQCspectra of lysidine and agmatidine isconsistent with
previous work on arabino diaminopyrimidines, in which
the authors favored protonation on the exocyclic nitrogens
based on UV spectral analysis.10 Interestingly, this tauto-
merprecludesdonation ofa hydrogen bond from N3toN1
of adenosine during base pairing. As shown in Figure 4,
thishydrogen bondiscrucialconsidering thestericdemand
imposed by the alkylamino substituent at C-2.1,9 Thus, for
a GꢀU wobble type of pairing12 (Figure 4a) between the
amino tautomer of lysidine/agmatidine and adenosine,
Acknowledgment. The authors acknowledge Dr. Victor
Young at the University of Minnesota for determining the
crystal structure of intermediate 6b; Tom Stringfellow,
Chenxi Jia, and Cameron Scarlett of the UW;Madison
School of Pharmacy Analytical Instrumentation Center;
and Richard Hsung for discussion and comments on the
manuscript. We also gratefully acknowledge the University
of Wisconsin School of Pharmacy for financial support.
Supporting Information Available. Experimental pro-
cedures, NMR and mass spectrometry data, and the
crystallographic information file for compound 6b. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(9) Sonawane, K. D.; Tewari, R. Nucleosides, Nucleotides Nucleic
Acids 2008, 27, 1158.
(10) Doerr, I. L.; Fox, J. J. J. Chem. Soc. 1967, 1462.
(11) See Supporting Information.
(12) Varani, G.; McClain, W. H. EMBO Rep. 2000, 1, 18.
(13) Agris, P. F.; Vendeix, A. P.; Graham, W. D. J. Mol. Biol. 2006,
366, 1.
The authors declare no competing financial interest.
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