Stable organophosphorus analogues of S-adenosylmethionine and S-methylmethionine

Article abstract of DOI:10.1070/MC2003v013n06ABEH001856

The organophosphorus analogues of the biologically significant sulfonium compounds S-adenosylmethionine and S-methylmethionine are much more stable than their carboxylic prototypes.

Full text of DOI:10.1070/MC2003v013n06ABEH001856

, 2003, 13(6), 243–244
Stable organophosphorus analogues of S-adenosylmethionine and
S-methylmethionine
Kirill V. Alferov, Yurii N. Zhukov, Elena N. Khurs and Radii M. Khomutov*
V. A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russian Federation.
Fax: +7 095 135 1405; e-mail: khomutov@genome.eimb.relarn.ru
10.1070/MC2003v013n06ABEH001856
The organophosphorus analogues of the biologically significant sulfonium compounds S-adenosylmethionine and S-methyl-
methionine are much more stable than their carboxylic prototypes.
S-Adenosylmethionine [Met(Ado)] is of significance for cellular
metabolism; it participates in important reactions such as bio-
methylation and the synthesis of polyamines. In various cells
under pathological conditions considerable differences in these
processes have been observed. This allows us to consider the
metabolism of Met(Ado) as a perspective biochemical target
while searching for new physiologically active compounds
and antimicrobial medicines. S-Methylmethionine (vitamin U),
another natural sulfonium compound, is also a donor of methyl
groups, although of lesser importance than Met(Ado).
H
NH2
Ade
O
HOOC
S
Me
OH OH
Met(Ado)
1.5 < pH < 7
pH > 7
H
NH2
Ade
O
O
MeS
HOOC
S
OH
Me
OH OH
OH OH
The analogues of Met(Ado) obtained to the present time mostly
contain modified nucleoside moieties and have low affinities to
corresponding enzymes, while analogues with modified amino
acid moieties remain almost unstudied.1 These analogues, as
Met(Ado) itself, are unstable at neutral pH values, which is
caused by the presence of a reactive sulfonium moiety and an
appropriately placed carboxyl group within the same molecule.
Therefore, the analogues of Met(Ado) are not usually con-
sidered as effective tools for affecting Met(Ado)-dependent bio-
methylation processes, taking into account the transport of
charged compounds into the cell.
In this respect, organophosphorus analogues of Met(Ado), in
which the carboxyl group is replaced by an acidic phosphorus-
containing fragment, appeared promising. It was shown that a
phosphinic analogue of Met(Ado), 5'-{S-[3-amino-3-(hydro-
hydroxyphosphoryl)propyl]-S-methylthionia}-5'-deoxyadenosine
chloride 1 (Scheme 1), possesses high affinity to tRNA-methyl-
transferase and is a substrate of this enzyme2 (i.e. it can
participate in biomethylation reactions). Compound 1 is an
effective inhibitor of Met(Ado)-decarboxylase, a key enzyme of
polyamine biosynthesis, while the C–P bond of compound 1
remained stable to the action of the enzyme.2 Moreover, acid
1 displays biolological activity and suppresses the growth of
tumor cells L1210 affecting the intracellular level of Met(Ado).3
When cells were incubated with 1-amino-3-(methylthio)propyl-
phosphinic acid 2, the inhibition of the cell growth and forma-
tion and the intracellular accumulation of analogue 1 were
observed. This observation opens new possibilities to affect the
Met(Ado) metabolism.3 Thus, a comparative study of the sta-
bility of analogue 1 and related compounds seemed appropriate,
allowing us, in terms of reactivity, to estimate qualitative dif-
ferences between the organophosphorus analogues of sulfonium
compounds and their natural prototypes.
H
NH2
Ade
HOOC
OH
Scheme 2
phosphoryl)propyl]-S-methylthionia}-5'-deoxyadenosine acetate
3). At the same time, it was shown recently that a phosphonic
analogue of S-adenosylhomocysteine, the product of de-
methylation of Met(Ado), is an effective irreversible inhibitor
of S-adenosylhomocysteine hydrolase – the enzyme whose
activity determines the level of biomethylation in the cell.4
We studied the stability and decomposition of the phosphinic
analogues of biologically significant sulfonium compounds,
chloride 1 and iodide of 1-amino-3-(dimethylthionia)propyl-
phosphinic acid 4,† under different conditions.
Met(Ado) is a chemically unstable compound, which decom-
poses in aqueous solutions at pH > 1.5 to form S-methyl-
thioadenosine and homoserine according to the mechanism of
intramolecular alkylation [kH 6×10–6 s–1 (37 °C, pH 7.5) or
4×10–3 s–1 (100 °C, pH 4.5)], while at pH > 7 the hydrolysis
to adenine and S-pentosylmethionine becomes significant
[kH 3×10–6 s–1 (37 °C, pH 7.5)] (Scheme 2).7 Note that an
important catabolic pathway of Met(Ado) in the cells catalysed
by Met(Ado)-lyase consists of decomposition to S-methylthio-
adenosine and homoserine.8
First, we studied the stability of analogue 1 under conditions
described elsewhere.7 The progress of decomposition of acid 1
1
was monitored by H NMR spectroscopy. Therefore, the reac-
tion was carried out in a deuterated potassium phosphate buffer
(pD 7.1, which corresponds to pH 7.5). Under these conditions,
compound 1 remained stable for several days, whereas a half-
decomposition period of Met(Ado) was 28 h. Moreover, acid 1
remained stable in D2O upon the incubation of its solution for
few months at ambient temperature. While boiling in water at
pH 4.5, analogues 1 and 3‡ were found to be more stable than
Met(Ado) (kH 0.2×10–3 s–1 for compound 1).
Note that the phosphonic analogue of methionine [in which
the carboxylic group is replaced by the P(O)(OH)2 group] did
almost not affect the growth of tumor cells, as well as a phos-
phonic analogue of Met(Ado) (5'-{S-[3-amino-3-(dihydroxy-
Thus, at pH 4.5, compound 2 and phosphinic analogues of
homoserine and S-adenosylhomocysteine (Scheme 3, com-
pounds 6 and 7, respectively)§ were found among the products
of decomposition of 1 and no additional formation of adenine
and a phosphinic analogue of S-pentosylmethionine was ob-
served at pD 7.1 (cf. ref. 7).¶
NH2
X
Me
S
PO2YH
R
1 R = Ado, X = Cl, Y = H
R = Ado, X = OAc, Y = OH
4 R = Me, X = I, Y = H
5 R = Me, X = I, Y = OH
S-Methylmethionine is more stable than Met(Ado) and de-
composes with the formation of homoserine and dimethyl
3
†
Acid 1 (mixture of diastereomers), racemic acids 4, 5 and S-methyl-
methionine iodide were synthesised according to the published proce-
Scheme 1
dures.5,6
– 243 –

, 2003, 13(6), 243–244
logue 6 from compound 4 was carried out only under special
conditions: the heating of 4 in an AcOH–AcONa mixture
followed by acidic hydrolysis.6
NH2
PO2H2
Cl
Me
S
Note that S-adenosylhomocysteamine, which is free of the
COOH group, appeares to be more stable than Met(Ado).11
Therefore, different stability and ways of degradation of sulfo-
nium salts 1 and 4, as compared to their carboxylic prototypes,
may be explained by the low reactivity of the mesomeric phos-
phinic anion in intramolecular alkylation. The transformations
of this anion into ethers in case of aminoalkylphosphinates
were carried out only by methylation with diazomethane.12
Thus, the decomposition of sulfonium salts 1 and 4 proceeds
without the participation of acidic moieties of the molecules.
The stability of compounds 3 and 5 may be explained similarly.
Thus, the acidic phosphorus-containing group in the test ana-
logues of natural sulfonium compounds is capable not only to
carry out an ‘anchor’ function while binding to target enzymes,
likewise the carboxyl group, but also to increase considerably
the stability of the analogues, including the stability to the action
of Met(Ado) catabolic enzymes.
Ado
1
pH 4.5
MeS
PO2H2
AdoS
PO2H2
NH2
NH2
2
7
HO
PO2H2
NH2
6
Scheme 3
sulfide only upon heating in neutral and alkaline solutions10
(according to our data, kH 1.65×10–3 s–1 at 100 °C in 0.5 M
NaOH). Under these conditions, organophosphorus analogue 4
and 1-amino-3-(dimethylthionia)propylphosphonic acid iodide
5 were also found to be more stable (kH 0.3×10–4 or 1.1×10–4 s–1,
respectively), while the main product of the transformation of
compound 4 is methionine analogue 2, whereas acid 5 gave a
multicomponent mixture.†† The synthesis of homoserine ana-
This work was supported by the Russian Foundation for Basic
Research (grant no. 03-04-48949) and a grant for Scientific
School no. 1800-2003.4.
References
‡
1
2
3
R. T. Borchardt, in Enzymatic Basis of Detoxification, ed. W. B. Jakoby,
Academic Press, New York, 1980, vol. 2, p. 43.
V. I. Syrku, L. L. Zavalova and R. M. Khomutov, Bioorg. Khim., 1986,
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Kramer, J. T. Miller and K. V. Porter, Bioorg. Khim., 2000, 26, 718
(Russ. J. Bioorg. Chem., 2000, 26, 647).
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Int. Ed., 2001, 50, 316).
A 10% solution of I2 in EtOH was added dropwise with stirring to a
mixture of acid 1 (100 mg, 0.22 mmol), aqueous 0.5 M HI (2 ml) and
EtOH (1 ml) until a weak yellow colour was retained for 2–3 min. The
reaction mixture was lyophilised; the residue was dissolved in water,
applied onto FN-18 paper and chromatographed in the BunOH–AcOH–
H2O system (12:3:5). The strip of paper containing acid 3 was cut out
and eluted with water; the eluate was concentrated in vacuo to dryness.
The residue was dried in vacuo over P2O5 to give compound 3 (64%,
69 mg): mp 168 °C, Rf 0.03 (PriOH–25% NH4OH–H2O, 7:1:2); Rf 0.03
(BunOH–AcOH–H2O, 12:3:5). 1H NMR (400 MHz, D2O) d: 2.02 (s,
3H, MeCOO–), 2.07–2.54 (m, 2H, CH2CH), 2.99 (s, 3H, MeS+), 3.13–
3.30 (m, 1H, CHCH2), 3.45–3.72 (m, 2H, +SCH2CH2), 3.84–3.99 (m,
2H, 5'-CH2S+), 6.04 (d, 1H, H-1', J 4.7 Hz), 8.24 and 8.26 (2s, 1H, H-2
and 1H, H-8).
4
5
6
§
A solution of acid 1 (31 mg, 0.068 mmol) in 2 ml of a 0.05 M sodium
7
8
9
J. L. Hoffman, Biochemistry, 1986, 25, 4444.
acetate buffer (pH 4.5) was boiled for 5 h. The quantitative deter-
mination of acid 1 was performed by TLC.9 At known time intervals,
aliquot portions of the reaction mixture were applied to a Silufol plate.
The plate was developed by using BunOH–AcOH–H2O (12:3:5) and/or
PriOH–25% NH4OH–H2O (7:1:2), dried and treated with a ninhydrin
solution. Spots giving positive ninhydrin reactions were cut and extracted
with a 0.5% CdCl2 solution in 50% ethanol, and the absorbance of the
extracts was determined at 505 nm. To construct the calibration curve,
aliquot portions of an authentic 1 solution were subjected to the same
treatment. The identity of the products of decomposition with authentic
2, 6 and 7 was shown by TLC on a Silufol plate developed by the upper
layer of the BunOH–AcOH–H2O (12:3:5) and PriOH–25% NH4OH–H2O
(7:1:2) systems. Spots were visualised by UV and colour reactions with
ninhydrin and ammonium molybdate.
S. K. Shapiro and A. N. Hather, J. Biol. Chem., 1958, 233, 631.
N. G. Faleev, Y. N. Zhukov, E. N. Khurs, O. I. Gogoleva, M. V. Barbolina,
N. P. Bazhulina, V. M. Belikov, T. V. Demidkina and R. M. Khomutov,
Eur. J. Biochem., 2000, 267, 6897.
10 V. N. Bukin and G. N. Khuchua, in Vitamin U (S-metilmetionin):
priroda, svoistva, primenenie [Vitamin U (S-methylmethionine): Origin,
Properties, Applications], eds. V. N. Bukin and V. E. Anisimov, Nauka,
Moscow, 1973, p. 10 (in Russian).
11 V. Zappia, P. Galletti, A. Oliva and A. Santis, Anal. Biochem., 1977,
79, 535.
12 P. P. Gianousis and P. A. Bartlett, J. Med. Chem., 1987, 30, 1603.
The rate constants kH were obtained by fitting the data (concentrations
of acid 1 vs. time) to the rate equation of a first-order reaction.
Taking into account a considerable difference in the decomposition
rates for Met(Ado) and 1 and according to published data,7,11 we
assumed that the rate constants of hydrolysis of diastereomers of 1 are
equal.
Received: 2nd October 2003; Com. 03/2182
†† The solutions of S-methylmethionine and acids 4 and 5 (40 mg) in an
aqueous 0.5 M NaOH solution were boiled for 6 h. The quantitative
determination of S-methylmethionine and acids 4 and 5 and the identi-
fication of decomposition products with authentic homoserine, com-
pounds 2 and 6, were carried out as described above.
¶
A solution of acid 1 (20 mg, 0.04 mmol) in 0.7 ml of a 0.2 M
deuterated potassium phosphate buffer (pD 7.1) was incubated at 37 °C
for 144 h. 1H NMR spectra were recorded at known time intervals.
– 244 –