Efficient synthesis of s-adenosyl-L-homocysteine natural product analogues and their use to elucidate the structural determinant for cofactor binding of the DNA methyltransferase M·HhaI

Article abstract of DOI:10.1002/(sici)1099-0690(200002)2000:3<549::aid-ejoc549>3.0.co;2-7

5'-Acetylthio-5'-deoxy-2',3'-O-isopropylideneadenosine (8) was directly prepared from commercially available 2',3'-O-isopropylideneadenosine (7) and thioacetic acid under Mitsunobu conditions in almost quantitative yield. In situ cleavage of the acetylthio function of 8 followed by coupling with different alkyl bromides proceeded with high yields. Deprotection of the obtained 5'-thionucleosides yielded the S-adenosyl-L-homocysteine analogues decarboxylated AdoHcy (11), deaminated AdoHcy (14) and 5'- [3(cyano)propylthio]-5'-deoxyadenosine (16) in good overall yields. Direct deprotection of the thionucleoside 8 delivered 5'-thio-5'-deoxyadenosine (18) in excellent yield. In addition, binding constants of these AdoHcy analogues and the DNA methyltransferase M·HhaI were determined in a fluorescence assay.

Full text of DOI:10.1002/(sici)1099-0690(200002)2000:3<549::aid-ejoc549>3.0.co;2-7

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Efficient Synthesis of S-Adenosyl-L-Homocysteine
Natural Product Analogues and Their Use to Elucidate the Structural
Determinant for Cofactor Binding of the DNA Methyltransferase M·HhaI
Marc Pignot,[a][°] Goran Pljevaljcic,[a] and Elmar Weinhold*[a]
Keywords: Alkylations / Deaminated AdoHcy / Decarboxylated AdoHcy / Mitsunobu reaction / Nucleosides
5Ј-Acetylthio-5Ј-deoxy-2Ј,3Ј-O-isopropylideneadenosine (8)
was directly prepared from commercially available 2Ј,3Ј-O-
isopropylideneadenosine (7) and thioacetic acid under
Mitsunobu conditions in almost quantitative yield. In situ
cleavage of the acetylthio function of 8 followed by coupling
with different alkyl bromides proceeded with high yields.
Deprotection of the obtained 5Ј-thionucleosides yielded the
AdoHcy (11), deaminated AdoHcy (14) and 5Ј-[3-
(cyano)propylthio]-5Ј-deoxyadenosine (16) in good overall
yields. Direct deprotection of the thionucleoside 8 delivered
5Ј-thio-5Ј-deoxyadenosine (18) in excellent yield. In addition,
binding constants of these AdoHcy analogues and the DNA
methyltransferase M·HhaI were determined in
a
fluorescence assay.
S-adenosyl-
L
-homocysteine
analogues
decarboxylated
Introduction
Nucleosides containing 5Ј-sulfur substituents are found
in a number of natural products and serve important func-
tions in the cell. The most prominent representative is the
cofactor S-adenosyl--methionine (1, AdoMet), which is
the major methyl group donor in a myriad of methyltrans-
ferase-catalysed reactions (Scheme 1).[1] The transmeth-
ylation reaction yields the cofactor product S-adenosyl--
homocysteine (2, AdoHcy), which is a potent inhibitor of
methyltransferases and is removed by S-adenosyl--homo-
cysteine hydrolase.[2] In addition, 1 is involved in the polya-
mine biosynthesis. Decarboxylation of 1 by S-adenosyl--
methionine decarboxylase leads to decarboxylated AdoMet
(3), which serves as aminopropyl donor for the synthesis of
spermidine and spermine.[3]
Because of the biological importance and the variety of
the S-adenosyl--methionine metabolism, a great number
of nucleosides containing 5Ј-sulfur atoms were synthesized.
They could serve as pharmacologically valuable inhibitors
for S-adenosyl--homocysteine hydrolase,[4] S-adenosyl--
methionine decarboxylase,[5] and the aminopropyl transfer-
ases spermidine and spermine synthase (polyamine trans-
ferase I and II).[6] Among these compounds, structurally
related analogues of AdoHcy showed significant inhibitory
effects on S-adenosyl--methionine-dependent methyltrans-
ferases.[7] Recently, the human C5-cytosine DNA methyl-
transferase was suggested as a potential target for drug de- (1)
sign, because hypermethylation of upstream regulatory se-
quences leads to an inactivation of tumor suppressor genes
Scheme 1. Biological transformations of S-adenosyl--methionine
in numerous cancers.[8] Thus, inhibitors of the human DNA
methyltransferase could reverse the effects of DNA meth-
ylation and could have therapeutic value in the treatment
of cancer. For these important applications of AdoHcy ana-
logues, efficient synthetic routes leading to this class of
compounds are needed.
[°]
Present address:
Aventis Research & Technologies GmbH & Co KG,
Bioorganic Systems & Synthesis, Industriepark Höchst G 830,
D-65926 Frankfurt am Main, Germany
[a]
Max-Planck-Institut für molekulare Physiologie,
Abteilung Physikalische Biochemie,
Otto-Hahn-Straße 11, D-44227 Dortmund, Germany
Fax: (internat.) ϩ 49-(0)231/133-2399
In order to elucidate the structural determinant for cofac-
tor binding of DNA methyltransferases we revisited the
E-mail: elmar.weinhold@mpi-dortmund.mpg.de
Eur. J. Org. Chem. 2000, 549Ϫ555
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0203Ϫ0549 $ 17.50ϩ.50/0
549

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M. Pignot, G. Pljevaljcic, E. Weinhold
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synthesis of AdoHcy analogues carrying different deletions tected adenosine derivative 7 and thioacetic acid under
in the amino acid portion and report a very efficient syn- standard Mitsunobu conditions[18] without previous protec-
thetic strategy.
tion of the amino group of the adenine ring and activation
In general, there are two synthetic routes towards the for- of the 5Ј-hydroxy group (Scheme 3). This one-step pro-
mation of the 5Ј-thioether bond in AdoHcy and its ana- cedure is shorter than the reported syntheses of the 5Ј-
logues (Scheme 2). Route A starts from 2Ј,3Ј-O-isopropylid- thioacetates 6 or 8 and leads, after chromatographic purifi-
ene-5Ј-tosyladenosine (4)[9] or 5Ј-chloro-5Ј-deoxyadenosine cation, to a nearly quantitative yield.
(5),[10] in which the 5Ј-hydroxy group is converted into a
good leaving group. The adenosine 5Ј-thioether is then
formed by nucleophilic substitution with different thiols in
the presence of aqueous sodium hydroxide or formed in situ
with sodium in liquid ammonia from appropriate precur-
sors. However, this route is particularly problematic with
5Ј-tosylates as starting material, because cycloadducts can
form by an intramolecular attack of the N3 nitrogen atom
Scheme 3. One-step synthesis of the protected thionucleoside 8
of the adenine ring.[11] In addition, the reaction conditions
Coupling of the protected thionucleoside 8 with N-(3-
bromopropyl)phthalimide was achieved after in situ cleav-
age of the acetylthio function with NaOCH3 in MeOH un-
der rigorous exclusion of oxygen (Scheme 4). Exclusion of
can lead to other side reactions like depurination.[12] All
these factors result in mediocre yields in the coupling step.
Route B starts with 5Ј-acetylthio-5Ј-deoxy-N6-formyl-2Ј,3Ј-
O-isopropylideneadenosine (6), which is prepared from the
oxygen is necessary, because traces of oxygen can lead to
corresponding 5Ј-tosylate using potassium thioacetate.[13]
disulfide bond formation of the thiol produced in situ.[19]
The formation of the cycloadduct in this reaction is sup-
The protected AdoHcy analogue 9 was obtained in 82%
yield. Acid-catalysed hydrolysis of the isopropylidene acetal
pressed by acylation of the adenine amino group.[14] The
5Ј-thioacetate 6 is first cleaved in situ under basic con-
was accomplished with aqueous formic acid to yield the
ditions and then treated with an appropriate alkyl halide to
protected AdoHcy analogue 10. Decarboxylated AdoHcy
yield the desired adenosine 5Ј-thioether. If oxygen is rigor-
(11) was then obtained by hydrazinolysis of the phthalimide
function. Our overall yield of the AdoHcy analogue 11
from the protected thionucleoside 8 (72%) is considerably
higher than the yields reported for route A from the tosylate
ously excluded from the reaction mixture, excellent yields
can be obtained for the coupling reaction using this
route.[15] Unfortunately, route B is longer than route A, be-
cause the 5Ј-thioacetate 6 needs to be prepared from 2Ј,3Ј-
4 (48%)[20] or from the chloride 5 (37%),[13b] and is compar-
O-isopropylideneadenosine (7) in three steps.[16] Alterna-
able to the yield reported for route B starting from the
tively, 5Ј-acetylthio-5Ј-deoxy-2Ј,3Ј-O-isopropylideneadenos-
thioacetate 6 (80%).[15]
ine (8), which was prepared from the protected adenosine
The versatility of the protected thionucleoside 8 as an
intermediate in the synthesis of AdoHcy analogues is read-
ily demonstrated by the excellent coupling yields with
derivative 7 in two steps, can be used in route B.[17]
methyl 4-bromobutyrate and 4-bromobutyronitrile in ad-
Results and Discussion
dition to N-(3-bromopropyl)phthalimide (Scheme 4). The
We present a generally applicable synthesis for AdoHcy coupling products 12 and 15 were obtained in 89% and 98%
analogues modified in their amino acid portion, improving yield, respectively. Removal of the isopropylidene protecting
route B. The key step of the synthesis is the direct formation groups in aqueous formic acid delivered the nucleosides 13
of the thionucleoside 8 from commercially available, pro- and 16 in nearly quantitative yields. Deaminated AdoHcy
Scheme 2. Synthetic routes towards S-adenosyl--homocysteine (2) and analogues with different side chains
550
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S-Adenosyl-L-Homocysteine Natural Product Analogues
FULL PAPER
way with nearly quantitative overall yield. In our approach,
the acetonide group of the protected thionucleoside 8 was
first removed in aqueous formic acid, and the resulting 5Ј-
acetylthio-5Ј-deoxyadenosine (17) was then hydrolyzed un-
der oxygen-free conditions using a mixture of methanol and
water saturated with ammonia. After removal of the solvent
by lyophilisation, the thionucleoside 18 was obtained as a
white foam, which does not undergo disulfide bond forma-
tion under exposure to air, as verified by analytical re-
versed-phase HPLC.
In addition, the AdoHcy analogues 11, 14, 16 and 18
with different side chains were used to investigate the
thermodynamic contribution of the amino acid side chain
of AdoHcy to the cofactor binding of a methyltransferase.
Methyltransferases share a conserved catalytic domain
structure and bind the cofactor in a very similar way.[23]
For our cofactor binding studies, we chose the C5-cytosine
DNA methyltransferase from Haemophilus haemolyticus
(M·HhaI). This enzyme contains a single tryptophan resi-
due (Trp 41), which interacts with the adenine ring of the
bound cofactor.[24] Interestingly, its fluorescence intensity is
highly quenched upon binding of the cofactor, which offers
a convenient assay to measure cofactor binding of M·HhaI.
Fluorescence titrations with AdoHcy (2) and its truncated
analogues 11, 14, 16 and 18 are shown in Figure 1, and
the obtained binding constants are summarised in Table 1.
Decarboxylated AdoHcy (11) binds to M·HhaI with a
78-fold reduced affinity compared to AdoHcy (2). This
corresponds to a loss in free binding energy of 10.8 kJ
molϪ1 and indicates that the carboxylate group of AdoHcy
forms multiple hydrogen bonds with M·HhaI. In fact, three
hydrogen bonds were found between the carboxylate group
of the cofactor and M·HhaI in the three-dimensional struc-
ture of the binary complex.[24] In contrast to the decarb-
oxylated AdoHcy (11), deaminated AdoHcy (14) binds only
with a 17-fold reduced affinity compared to AdoHcy (2).
This smaller reduction in affinity is also in agreement with
Scheme 4. Synthesis of S-adenosyl--homocysteine analogues with
different side chains
(14) was then obtained quantitatively by hydrolysis of the the binary structure of M·HhaI in complex with the cofac-
methyl ester 13 in a mixture of aqueous ammonia and tor, where the amino group is only hydrogen-bonded to the
methanol. The overall yield of the AdoHcy analogue 14 enzyme through two water molecules.[24] Further replace-
from protected thionucleoside 8 was 85%, which is better ment of the carboxylate group by a nitrile group in the
than the yield obtained by route A from the tosylate 4 AdoHcy analogue 16 does lead to a similar binding affinity.
(72%).[20] An additional advantage of our strategy is that Since the nitrile group should be a weaker hydrogen bond
methyl 4-bromobutyrate is commercially available, while S- acceptor compared to the carboxylate group, this result is
benzyl-4-thiobutyric acid used in the preparation of the surprising. However, in contrast to deaminated AdoHcy
AdoHcy analogue 14 by route A has to be synthesized first. (14), the AdoHcy analogue 16 should not be charged under
The AdoHcy analogue 16 was prepared for the first time the assay conditions (pH ϭ 7.4) and thus the similar bind-
and can be regarded as an analogue of deaminated AdoHcy ing constants could result from a compensating hydro-
(14) in which the carboxylate group, a strong hydrogen phobic effect.[25] Most interestingly, the adenosine deriva-
bond acceptor, is replaced by a nitrile group, a weak hydro- tive 18 is also bound with quite good affinity. Although the
gen bond acceptor.
fact that its affininty is only 43-fold reduced, compared to
The protected thionucleoside 8 is also a very useful inter- AdoHcy (2), could also be partly explained by a hydro-
mediate for the synthesis of 5Ј-deoxy-5Ј-thioadenosine (18). phobic effect, this result suggests that the molecular anchor
The known synthesis procedures are either awkward[21] or for cofactor binding of M·HhaI is the adenosyl part of the
lead to poor yields of the thionucleoside 18.[22] The syn- cofactor. Due to the high structural homology of the cata-
thesis sequence presented in this publication (Scheme 4) al- lytic domains of methyltransferases, it is expected that the
lows for the synthesis of the thionucleoside 18 in a simple adenosyl part of the cofactor also functions as the struc-
Eur. J. Org. Chem. 2000, 549Ϫ555
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M. Pignot, G. Pljevaljcic, E. Weinhold
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according to literature procedures.[26] 2Ј,3Ј-O-Isopropylidene-
adenosine, N-(3-bromopropyl)phthalimide, 4-bromobutyronitrile
and methyl 4-bromobutyrate were purchased from Aldrich. Ϫ
NMR: Bruker AX 500 (500 MHz and 125.7 MHz, for 1H and 13C,
respectively). For 1H NMR, CDCl3 as solvent, tetramethylsilane
(TMS) as internal standard, D2O as solvent, 3-trimethylsilyl-
[2,2,3,3-D4]propionic acid sodium salt (TSP) as internal standard,
[D6]DMSO as solvent δH ϭ 2.49; for 13C NMR, CDCl3 as solvent,
TMS as internal standard, D2O as solvent, TSP as internal stand-
ard, [D6]DMSO as solvent δC ϭ 39.5. Assignment of 13C signals
tural determinant for cofactor binding of other methyl-
transferases.
1
are based on H,13C-correlated 2D-NMR and on 13C-DEPT spec-
tra. All NMR spectra were recorded at ambient probe temperature.
The nomenclature used to denote side chain atoms follows the no-
menclature of AdoHcy (2).[27] Ϫ Electrospray MS: Finnigan LCQ
connected to a nanoelectrospray ion source.[28] For measurements
in the positive ion mode samples were dissolved in CH3OH/water
(1:1 v/v) containing 5% formic acid; for measurements in the nega-
tive ion mode a mixture of CH3OH/water (1:1 v/v) was used. FAB-
MS: Finnigan MAT 8200 (70 eV, thioglycolic acid matrix). Ϫ TLC:
DCϪaluminia plates SilϪG60/UV254 (Merck). Compounds were
visualized by treatment with a solution of ammonium molybdate
(15 g), cerium(IV) sulfate (0.4 g) in 10% sulfuric acid (300 mL) and
heating with a hot air blower. Ϫ Flash chromatography: Merck
silica gel 60 (40Ϫ63 µm). Ϫ Reversed-phase HPLC: Analytical
HPLC analysis was carried out using a C-18 column (ODS Hyper-
Figure 1. Fluorescence titrations of the DNA methyltransferase
M·HhaI (1 µ) with increasing amounts of AdoHcy (2) (open tri-
angles) and the AdoHcy analogues 16 (open circles), 14 (closed
circles), 18 (open squares) and 11 (closed squares)
Table 1. Binding of AdoHcy (2) and its analogues 11, 14, 16, and
18 to the DNA methyltransferase M·HhaI
[a]
Ligand
Dissociation constant KD [µ]
∆∆G[b] [kJ/mol]
Ϫ1
˚
sil, 5 µm, 120 A, 250 ϫ 4.6 mm, Bischoff), flow rate 1 mL min
,
2
1.0 ± 0.1
15 ± 2.0
17 ± 0.3
43 ± 2.4
78 ± 3.0
UV detection at 259 nm. Preparative runs were performed with a
16
14
18
11
6.7
7.0
9.3
10.8
˚
C-18 column (ODS YMC-Pack, 5 µm, 120 A, 250 ϫ 20 mm,
YMC), flow rate 10 mL minϪ1, UV detection at 259 and 290 nm.
Standard buffer conditions: Buffer A [triethylammonium acetate
(0.1 , pH ϭ 7.0)] and buffer B [CH3CN containing buffer A
(30%)]. Standard gradient conditions: Gradient 1: 20% buffer B
(0Ϫ10 min), 20Ϫ100% buffer B (10Ϫ30 min); gradient 2: 40%
buffer B (0Ϫ10 min), 40Ϫ60% buffer B (10Ϫ15 min), 60% buffer
B (15Ϫ25 min), 60Ϫ100% buffer B (25Ϫ35 min); gradient 3: 10%
buffer B (0Ϫ10 min), 10Ϫ20% buffer B (10Ϫ30 min), 20Ϫ100%
buffer B (30Ϫ45 min); gradient 4: 0% buffer B (0Ϫ10 min), 0Ϫ20%
buffer B (10Ϫ30 min), 20Ϫ100% buffer B (30Ϫ45 min), 100%
buffer B (45Ϫ50 min); gradient 5: 20% buffer B (0Ϫ5 min),
20Ϫ40% buffer B (5Ϫ15 min), 40% buffer B (15Ϫ25 min),
40Ϫ100% buffer B (25Ϫ45 min); gradient 6: 7% buffer B (0Ϫ10
min), 7Ϫ40% buffer B (10Ϫ40 min), 40Ϫ100% buffer B (40Ϫ50
min), 100% buffer B (50Ϫ60 min). Ϫ Elemental analyses: LECO
CHNS-932. Since the accuracy of the standards (4-aminobenzene-
sulfonic acid, N-phenylacetamide; Merck) in the elemental analyses
was 0.1%, the analyses of compounds are given with the same accu-
racy. Ϫ Fluorescence titrations: Titrations were performed with an
SLM-Aminco 8100 fluorescence spectrometer. The excitation and
emission wavelengths were set to 290 nm and 350 nm, respectively,
and the excitation and emission bandwidths were adjusted to 1 and
16 nm, respectively.
[a]
Titration data from Figure 1 were fitted to the real solution of
the quadratic binding equation for one binding site and errors are
[b]
given as 2σ standard deviations. Ϫ
Calculated from ∆∆G ϭ
RTln[KD(AdoHcy analogue)/KD(AdoHcy)].
Conclusion
A variety of AdoHcy analogues with different side chains
was prepared in good overall yields from 5Ј-acetylthio-5Ј-
deoxy-2Ј,3Ј-O-isopropylideneadenosine (8), which is access-
ible from commercially available 2Ј,3Ј-O-isopropylidenead-
enosine (7) in a single step with excellent yield. The de-
scribed synthetic strategy of coupling a 5Ј-deoxy-5Ј-thioad-
enosine derivative with an alkyl halide (route B) results in
better yields compared to other strategies starting with 5Ј-
activated adenosine derivatives and alkylthiols (route A),
and should be most useful for the synthesis of AdoHcy ana-
logues, for which a corresponding halide of the side chain
component is commercially available. In addition, binding
of the synthesised AdoHcy analogues to the DNA methyl-
transferase M·HhaI was investigated using a fluorescence
assay. From these binding studies we conclude that the mo-
lecular anchor for cofactor binding of M·HhaI and most
likely of other methyltransferases as well is the adenosyl
part of the cofactor.
5Ј-Acetylthio-5Ј-deoxy-2Ј,3Ј-O-isopropylideneadenosine (8): To an
ice-cold solution of triphenylphosphane (5.7 g, 21.6 mmol) in ab-
sol. THF (30 mL), diethyl azodicarboxylate (3.4 mL, 21.6 mmol)
was added over 5 min. After stirring for 30 min, 2Ј,3Ј-O-isopropyl-
ideneadenosine (7) (3.0 g, 9.8 mmol) was added, and stirring was
continued for 10 min. To the resulting yellow suspension a solution
of thioacetic acid (1.6 mL, 21.6 mmol) in absol. THF (5 mL) was
added dropwise and stirring was continued for another 1 h at 0°C.
During this time the yellow suspension cleared, and an orange solu-
tion was obtained. At the end of the reaction the solvent was re-
moved under reduced pressure, and the resulting yellowish residue
was purified by flash chromatography on silica gel [350 g, CHCl3/
Experimental Section
General Remarks: All chemicals were of reagent quality and were
used without further purification. Solvents were dried and distilled
552
Eur. J. Org. Chem. 2000, 549Ϫ555