Structure-guided design of a methyl donor cofactor that controls a viral histone H3 lysine 27 methyltransferase activity

Article abstract of DOI:10.1021/jm201000j

vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy.

Full text of DOI:10.1021/jm201000j

BRIEF ARTICLE
pubs.acs.org/jmc
Structure-Guided Design of a Methyl Donor Cofactor That Controls a
Viral Histone H3 Lysine 27 Methyltransferase Activity
Jiaojie Li,† Hua Wei,† and Ming-Ming Zhou*
Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1677, New York,
New York 10029, United States
S Supporting Information
b
ABSTRACT: vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the
effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To
control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-L-methionine (SAM) analogue as methyl donor
cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/
SAM analogue pairs using a “bump-and-hole” strategy.
’ INTRODUCTION
group is replaced with a substituent alkynyl group, are useful for
identifying enzyme substrates.11,12 However, no one has yet
reported a modified SAM analogue as an effective methyl donor
cofactor for a protein methyltransferase. Here, we describe the
design and synthesis of several substituted SAM molecules and
characterization of their methyl donor capability with vSET
H3K27 methylation activity. We succeeded in developing an
orthogonal vSET/SAM analogue pair that is very active, thereby
demonstrating the feasibility of this strategy for the protein
methyltransferases.
Epigenetic control of gene expression is responsible for
fundamental cellular processes such as genetic imprinting, stem
cell self-renewal, and differentiation, and it involves many types
of chemical modifications on chromatin, including DNA methyla-
tion, histone methylation, acetylation, phosphorylation, ubiqui-
tination, and ADP-ribosylation.1,2 The high complexity of this
regulatory system is made possible because different histone
modifications function in a context-dependent manner. It is
difficult to predict how different modifications work in concert
in gene regulation. A means to turn on or off histone modifica-
tions on demand would be an invaluable tool.
The polycomb repressive complex 2 (PRC2) methylates
histone H3 lysine 27 (H3K27), a mark for gene transcriptional
silencing.3 The PRC2 consists of three components: EZH2,
SUZ12, and EED in human.4,5 EZH2 of 751 amino acids contains
the catalytic SET domain lysine methyltransferase activity; its
optimal methyltransferase activity requires the other two sub-
units and other partners. It has proven very challenging to express
a functional PRC2 in situ. Notably, our recent studies show that a
SET protein (vSET) of 119 amino acids encoded by the
Paramecium bursaria chlorella virus can specifically methylate
H3K27 in eukaryotic cells.6ꢀ8 vSET is active on its own, making
it much easier to manipulate than the PRC2 complex.
We envision that vSET activity can be controlled in a variant
whose activity is dependent on a chemically engineered S-
adenosyl-L-methionine (SAM) derivative. The generation of
orthogonal vSET/SAM pairing may be accomplished using a
“bump-and-hole” strategy, pioneered by Shokat and colleagues in
the kinase field.9 Specifically, one would change a bulky residue in
vSET to alanine or glycine and add a substituent to SAM to fill
the space created in the protein. Given its function as a common
methyl donor cofactor, surprisingly, there are only a few reports
on the utilization of SAM analogues by protein methyltrans-
ferases. In one study, N6-substituted S-adenosyl-L-homocysteine
(SAH) analogues were designed as variant specific RMT1
(a yeast arginine methyltransferase) inhibitors.10 Other studies
reported that SAM analogues, in which the sulfonium methyl
’ STRUCTURE-GUIDED ENGINEERING OF ORTHOGO-
NAL VSET/SAM COFACTOR PAIR
We focused on chemical modifications of SAM at three
positions, the N6-amino of the adenine ring that is a site used
in other studies,10 and the 20- or 30-hydroxyl in the ribose moiety
that has yet to be explored. Close inspection of our crystal
structure of the vSET/SAH/H3K27me1 peptide complex
(Figure 1)8 reveals that the adenine ring of SAH forms aromatic
and hydrophobic interactions with Trp110 and the side chain
methyls of Leu13, and the N6-amino group is hydrogen-bonded
to the carboxylate of the C-terminal residue Asn119 and the main
chain carbonyl oxygen of His70. This set of interactions is
bolstered by a hydrogen bond formed between the side chain
amino of Asn119 and the backbone carbonyl oxygen of Leu13.
While pointing outward, the 20-hydroxyl is packed next to
Leu116. Further, the phenoxyl of Tyr109 forms two hydrogen
bonds, one with the 30-hydroxyl of SAH and another with the
main chain carbonyl of Asp49. Thus, we hypothesized that
change of Leu13, Asn119, Tyr109, or Leu116 to Ala or Gly
would create a cavity that could accommodate a substituent
group from a SAM analogue.
Received: July 26, 2011
Published: September 29, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 2a
a Reagents and conditions: (a) tosyl chloride, Py, 25 °C, 1 h; (b) NaNO2,
CH3COOH/Ac2O = 1:4, 0 f 25 °C, overnight; (c) NaOMe, MeOH/
Et2O = 1:6, reflux, 30 min; (d) N,N-dimethylformamide dimethyl acetal,
DMF, 25 °C, overnight; (e) (i) SnCl2 2H2O, 8, 1,2-dimethoxyethane,
3
25 °C, overnight; (ii) NH4OH/MeOH, 25 °C, 5 h; (f) SOCl2, MeOH,
25 °C, 16 h; (g) (CF3CO)2O, 60 °C, 1.5 h.
For the synthesis of 20- and/or 30-benzyl SAM, we generated
adenosine derivatives with dialkyl disulfides under Hata’s
condition.13,14 Our initial attempt at direct benzylation of unpro-
tected adenosine with phenyldiazomethane 7,15 prepared in three
stepsfrombenzylaminein44% yield(Scheme 2), resultedin a very
low yield (<10%).16 We found that protection of the amino at the
4-position of adenosine with N,N-dimethylformamide dimethyl
acetal improved the yield to 55%,17 producing the two regio-
isomers 9a and 9b (Scheme 2) that were separated by HPLC in a
ratioof3:2. The regiochemistry assignmentof9a and 9b was made
Figure 1. (A) Crystal structure of vSET complex (PDB code 3KMT).
SAH and key vSET residues are shown as sticks, and H3 peptide is in
orange: (top) stereoview; (bottom) mesh and solid forms of vSET
surface. (B) Pymol modeled surfaces of Y109G and L116A variants.
1
by comparison of the H NMR spectra with those reported
previously.18 The disulfide moiety N,N-bis(trifluoroacetyl)-L-
homocysteine dimethyl ester 1117 was obtained in 59% yield
after two steps from L-homocystine (Scheme 2).
Scheme 1a
However, treatment of 9a or 9b with 11 under Hata’s
condition, in the presence of tri-n-butylphosphine in pyridine
did not give the desired product. The coupling reaction did not
proceed in several solvent conditions tested (Supporting Infor-
mation Table 1). We noticed that the reaction of free adenosine
with 11 under the same conditions gave the desired product in
46% yield after 5 days (Supporting Information, Table 1, entry 3).
1
The H NMR spectrum of the product matches well with that
reported previously.19 Thus, we reasoned that introduction of
the bulky benzyl substituent near the coupling site likely inter-
feres with the reaction, and dialkyldisulfides are generally much
less reactive than diaryldisulfides in the phosphine-mediated
coupling reaction, which may also contribute to the failure of
the reaction.14,19
a Reagents and conditions: (a) benzylamine, EtOH, 70 °C, 16 h; (b)
SOCl2, Py, CH3CN, 0 f 25 °C, 6 h; (c) L-homocysteine thiolactone/
HCl, NaOH, KI, 100 °C, O/N; (d) MeI, HCOOH, 25 °C, 4 days.
We next introduced a good leaving group at the 50-position to
install the alkylthio substituent (Scheme 3). Selective conversion
of adenosine to 50-chloro derivative 12 was performed with
thionyl chloride in acetonitrile,20 followed by protection of the
amino group with N,N-dimethylformamide dimethyl acetal.17
Subsequent benzylation of 13 with phenyldiazomethane 7
afforded two regioisomers 14a and 14b in a ratio of 3:2. Their
’ SYNTHESIS OF SUBSTITUTED SAM ANALOGUES
We first synthesized (()-N6-benzyl-S-adenosyl-L-methionine
4 in a four-step synthetic route (Scheme 1). The reaction started
with condensation of 6-chloropurine riboside with benzylamine
in ethanol10 followed by replacement of the 50-hydroxyl of 1 with
chloride and subsequent coupling with L-homocysteine thiolac-
tone and methylation of the sulfur, affording the product of 4
with an overall yield of 6%. The intermediates and final product
were confirmed by mass spectrometry and NMR spectra.
1
regiochemistry assignment was done by comparison of the H
NMR spectra with those obtained from chlorination of 9a and
9b. In addition, the dibenzylated adenosine analogue 14c was
synthesized by increasing the amount of phenyldiazomethane in
the reaction (Scheme 3).
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 3a
Table 1. vSET/Cofactor Pairs with No Activity
vSET
cofactor
L13A
4, N6-MSAM, N6-IPSAM
4, N6-MSAM, N6-IPSAM
vSET (aa 1ꢀ118)
Y109H
19, SAM
19, SAM
19, SAM
19
Y109D
Y109N
WT
Table 2. kcat and Km of the vSET/Cofactor Pairs
kcat
(minꢀ1
Km
kcat/Km
(minꢀ1 Mꢀ1
)
a Reagents and conditions: (a) SOCl2, Py, CH3CN, 0 f 25 °C, 6 h;
(b) N,N-dimethylformamide dimethyl acetal, DMF, 25 °C, overnight;
vSET
cofactor
)
(μM)
WT
SAM
16a
3.9
126
86
30952
1628
2139
1512
7.7
(c) (i) SnCl2 2H2O, 7, 1,2-dimethoxyethane, 25 °C, overnight; (ii)
3
WT
0.14
0.37
0.13
NH4OH/MeOH, 25 °C, 5 h; (d) L-homocysteine thiolactone hydro-
chloride, NaOH, KI, 100 °C, O/N; (e) MeI, HCOOH, 25 °C, 4 days.
WT
16c
173
86
WT
16b
SAM
16a
Y109G
Y109G
Y109G
L116A
L116A
L116A
L116A
0.00048
0.00049
0.0024
7.4
62
2800
917
60
0.18
16c
2.6
SAM
16a
123333
1095
18056
938
0.15
137
72
16c
1.3
16b
0.12
128
was removed (Figure 1A). Almost no activity was detected after
an overnight reaction for both vSET variants in the presence of
N6-benzyl-SAM 4 (Figure 2B,A; Table 1). Similar results were
obtained when N6-methyl-SAM or N6-isopropyl-SAM was used
(data not shown). Note that the previously reported yeast
RMT1-E117G/N6-benzyl-SAM pair yielded only 0.2% arginine
methyltransferase activity compared to the WT-RMT1/SAM
pair.10 Given that the adenine ring forms a network of hydrogen
bonds with vSET residues Leu13, His70, and Asn119, our results
combined with those from the RMT1-E117G/N6-benzyl-SAM
pair argue that the N6-position of the adenine is not suitable for
generating SAM derivatives that would retain significant methyla-
tion activity with a paired vSET variant.
We next characterized Tyr109 variants with SAM or its
analogues. Mass spectrometry analysis shows that Y109G has
very little activity in methylating H3K27 with SAM compared to
the wild-type vSET (Figure 2A,C). The same is true with Y109A
(data not shown). We then tested the activity of Tyr109 variants
in the presence of 16a or 16b. The Y109G/16a pair was much
less active than wild-type/SAM pair as judged by the amount of
methylation signal generated in the mass spectrometry study
(Figure 2D). However, it was slightly more active than Y109G/
SAM and Y109G/16b pairs (Figure 2E). The activity of Y109A
was lower in the presence of SAM analogues than Y109G (data
not shown). Therefore, our further studies were carried out with
the Y109G variant. To better compare different enzyme/cofactor
pairs, we performed MichaelisꢀMenten enzyme kinetic study to
obtain kcat and Km (Table 2, Supporting Information Figure 1).
When SAM is used, change of Tyr109 to Gly causes a 8125-
fold drop in kcat but little change to Km (Table 2). It suggests that
the substitution at Tyr109 is most likely to directly interfere with
its possible function in substrate lysine binding. For Y109G,
changing the cofactor from SAM to 16a results in a small increase
Figure 2. Mass spectrometry data showing the activity of different
enzyme/cofactor pairs.
As expected (Scheme 3), coupling of 14a, 14b, or 14c with L-
homocysteine thiolactone proceeded successfully and led to 15a,
15b, and 15c, respectively.21 Because of a resulting sulfonium
chiral center,22 a diastereomeric mixture of (()-30-benzyl-S-
adenosyl-L-methionine 16a, (()-20-benzyl-S-adenosyl-L-methio-
nine 16b, and (()-20,30-dibenzyl-S-adenosyl-L-methionine 16c
was synthesized by methylating the sulfur atoms of 15a, 15b, and
15c with excess amount of methyl iodide in formic acid in the
dark for 4 days. The final products of the SAM derivatives 16aꢀc
were obtained as diastereomeric mixtures.
’ CHARACTERIZATION OF VSET H3K27 METHYLA-
TION WITH THE SAM ANALOGUES
We first tested the idea of adding a bulky group to the N6-
position of the adenine ring of SAM. To generate necessary extra
space, Leu13 was substituted with Ala or the C-terminal Asn119
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Journal of Medicinal Chemistry
BRIEF ARTICLE
of kcat but a much higher Km. The same cofactor change for the
wild-type vSET causes a 28-fold decrease of catalytic efficiency
but surprisingly a 1.5-fold decrease of Km. The decrease in Km
could be explained by the contribution of fortuitous interaction
between the benzyl group and some protein residues. The
extremely high Km for Y109G/16a might be associated with
the entropic penalty of fixing the benzyl group at a specific
orientation for its insertion into the artificial cavity. Thus, we
postulated that a second benzyl group at the 20-position might
restrict the conformation of 30-benzyl, securing its insertion into
the cavity. An additional 20-benzyl group in SAM (i.e., 20,30-
dibenzyl-SAM 16c) brings little change of the kcat/Km to the
wild-type vSET with an increase in kcat and Km. But a very
different scenario was observed for Y109G: a slight increase of
kcat but a much-improved Km (Table 2).
methyltransferase vSET and SAM. We selected the common N6-
position of the adenine ring and the novel 20- and 30-hydroxyl
groups of the ribose as the modification sites. We synthesized
seven SAM analogues and characterized their activity with the
wild-type and variant vSET proteins. We succeeded in engineer-
ing a paired vSET-L116A/16c, which is only slightly less active
than vSET-L116A/SAM but more active than the wild-type
vSET/16c. Given the presence of SAM in cells, further optimiza-
tion via chemical modifications of the enzyme and the cofactor is
needed to generate a paired vSET variant/modified SAM with
activity better than that of the variant with SAM. In summary, our
study demonstrates the feasibility of developing a methyl donor
cofactor that controls a histone lysine methyltransferase, which
could be a useful tool to study the effects of histone H3K27
methylation in vivo.
To mimic the hydrogen bond between Tyr109 and the 30-
hydroxyl of SAM, we synthesized another SAM analogue 19,23
which bears a carbamoylmethyl substituent at 20- and 30-oxygen
atoms, and tested its activity with Y109H, Y109D, or Y109N
(Supporting Information Scheme 1). We postulated that the
amide moiety of the carbamoylmethyl substituent of SAM could
form a hydrogen bond with the side chain on the residue
replacing Tyr109 in these variants. However, no activity was
detected for any of these vSET variants after an overnight
incubation and neither was any of these variants active with
SAM or the wild-type vSET with 19. These results emphasize the
importance of Tyr109 in substrate binding, and thus, change of
Tyr109 to His, Asp, or Asn is detrimental to vSET activity.
Inspection of residues around the ribose moiety of SAH in the
crystal structure suggests that changing Leu116 could create a
docking site for a benzyl group at the 20- or 30-position (Figure 1).
Indeed, most of the H3 peptide was converted to mono- or
dimethylated species after 19 h of reaction for L116A/16a
(Figure 2F). The variant was even more active with 16c, as
almost all the H3 peptide was converted to trimethylated species
(Figure 2G). Kinetic studies further revealed a kcat of 7.4 minꢀ1
and Km of 60 μM for L116A/SAM, which notably is even better
than the wild-type vSET/SAM pair (Table 2). When 16a was
used, the kcat of L116A was dropped to 0.15 minꢀ1, accompanied
by a slight increase of Km. Importantly, L116A/16c was more
active than WT/16c, with a 3.5-fold higher kcat and a 2.4-fold
lower Km. Comparison of the kcat/Km of L116A/16c with that of
L116A/16a argues that the presence of the benzyl groups at 20
and 30 positions has a synergistic effect on L116A catalytic
efficiency when compared to the single benzyl group analogues.
Since the presence of the 30-benzyl in SAM resulted in a
reduction in the enzymatic activity of the wild-type vSET and
L116A variant compared to SAM, we investigated whether 16b
would be more potent for L116A. The results show that for the
wild-type vSET, the removal of the 30-benzyl in 16c causes a
small decrease in kcat and Km with a net result of a 1.4-fold
decrease in the kcat/Km. However, for L116A, a 13-fold decrease
of kcat was seen with an overall 19-fold decrease in kcat/Km.
Interestingly, while adding a single benzyl to the 20- or 30-position
in SAM causes an ∼120-fold decrease in kcat/Km for L116A,
these two benzyl groups in 16c likely act synergistically, leading
to only a 7-fold decrease of kcat/Km.
’ EXPERIMENTAL SECTION
Chemical Synthesis. All compounds were synthesized using
commercially available starting materials without further purification
unless otherwise stated. 1H and 13C NMR spectra were recorded on a
Bruker 600 MHz NMR spectrometer using the residual signal of the
deuterated solvent as internal standard. Chemical shifts (δ) are reported
in ppm, coupling constants (J) in hertz. MS (ESI) analysis for new
compounds was performed on an Agilent G1969A high-resolution mass
spectrometer. All compounds that were tested in the biological assays
were analyzed by HPLC and LCMS to confirm the purity, which was
g95%. R and S indicate relative configurations.
(()-20,30-Dibenzyl-S-adenosylmethionine Iodide (16c). MeI
(0.03 mL, 0.5mmol) was added toa solution of15c (22.6mg, 0.04 mmol)
in HCOOH (1 mL). After the addition was completed, the mixture was
stirred at room temperature in the dark for 4 days. Isolation of 16c (6.0
mg, 21.3% yield) was achieved by HPLC on an Agilent Eclipse XDB-C18
9.4 mm ꢁ 250 mm column. 1H NMR (600 MHz, D2O) δ 2.22ꢀ2.26 (m,
1H), 2.31ꢀ2.35 (m, 1H), 2.83 (s, 3H), 2.95 (s, 2H), 3.45 (t, J = 9.0 Hz,
1H), 3.51ꢀ3.56 (m, 2H), 3.60ꢀ3.67 (m, 1H), 3.70ꢀ3.74 (m, 1H), 3.80
(t, J = 9.0 Hz, 1H), 3.82 (d, J = 12.0 Hz, 1H), 3.88 (d, J = 13.6 Hz, 1H),
4.14ꢀ4.23 (m, 2H), 4.34 (d, J = 13.6 Hz, 1H), 4.41 (s, 1H), 4.56ꢀ4.57
(m, 1H), 5.89 (d, J = 8.8 Hz, 1H), 6.72ꢀ6.78 (m, 1H), 6.86 (t, J = 7.9 Hz,
2H), 6.94 (d, J = 5.6 Hz, 2H), 7.42 (t, J = 5.3 Hz, 1H), 7.46 (t, J = 7.53 Hz,
2H), 7.50 (d, J = 3.9 Hz, 2H), 8.00 (s, 1H), 8.04 (s, 1H); HRMS (ESI)
calculated for C29H35N6O5S (M)+ 579.2384, found 579.2338.
Cloning, Expression, and Purification of vSET. vSET was
cloned into pET-22b and expressed as a nonfusion protein in BL21
(DE3) cells.8 The protein was purified with a heparin column and then a
Superdex 75 column.
Site-Directed Mutagenesis. Variants of vSET were generated
with the QuikChange mutagenesis kit (Stratagene). The presence of
appropriate amino acid change was confirmed by DNA sequencing. All
variants were purified as described for the wild type vSET.
Lysine Methyltransferase Reaction. The activity of a paired
vSET/cofactor was measured in a solution containing 1 μM enzyme,
100 μM H3 peptide (residues 13ꢀ33), and 1 mM methyl donor
cofactor. The reaction buffer consisted of 20 mM Tris at pH 8,
20 mM potassium chloride, and 10 mM magnesium chloride. The
reaction was performed at room temperature and stopped at a specified
time by the addition of trifluoroacetic acid (to 1%). The products were
analyzed by mass spectrometry.
Histone lysine methylation is a fundamental mechanism for
epigenetic control of gene expression in chromatin. Chemical
modulators capable of controlling the enzymatic activity of
specific lysine methyltransferases are extremely valuable tools.
In this study, we illustrated our approach with an H3K27-specific
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis procedures, spectro-
b
scopic details of the compounds, and enzyme kinetic analysis.
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Journal of Medicinal Chemistry
BRIEF ARTICLE
This material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Nakagawa, I.; Aki, K.; Hata, T. Synthesis of 50-alkylthio-50-
deoxynecleosides from nucleosides in a one-pot reaction. J. Chem. Soc.,
Perkin Trans. 1 1983, 1315–1318.
(15) Overberger, C. G.; Anselme, J. A convenient synthesis of
phenyldiazomethane. J. Org. Chem. 1963, 28, 592–593.
’ AUTHOR INFORMATION
(16) Christensen, L. F.; Broom, A. D. Specific chemical synthesis of
ribonucleoside O-benzyl ethers. J. Org. Chem. 1972, 37, 3398–3401.
(17) Chien, T. C.; Chen, C. S.; Yu, F. H.; Chern, J. W. Nucleosides
XI. Synthesis and antiviral evaluation of 50-alkylthio-50-deoxy quinazo-
linone nucleoside derivatives as S-adenosyl-L-homocysteine analogs.
Chem. Pharm. Bull (Tokyo) 2004, 52, 1422–1426.
(18) Kozai, S.; Fuzikawa, T.; Harumoto, K.; Maruyama, T. A new
method for the synthesis of 20-O-benzyladenosine using Mitsunobu
reaction. Nucleosides, Nucleotides Nucleic Acids 2003, 22, 145–151.
(19) Serafinowski, P. A convenient preparation of S-adenosylhomo-
cysteine and its analogues. Synthesis 1985, 926–928.
(20) van Tilburg, E. W.; von Frijtag Drabbe Kunzel, J.; de Groote,
M.; Vollinga, R. C.; Lorenzen, A.; IJzerman, A.P. N6,50-Disubstituted
adenosine derivatives as partial agonists for the human adenosine A3
receptor. J. Med. Chem. 1999, 42, 1393–1400.
(21) Miles, R. W.; Nielsen, L. P.; Ewing, G. J.; Yin, D.; Borchardt,
R. T.; Robins, M. J. S-Homoadenosyl-L-cysteine and S-homoadenosyl-L-
homocysteine. Synthesis and binding studies of non-hydrolyzed sub-
strate analogues with S-adenosyl-L-homocysteine hydrolase. J. Org.
Chem. 2002, 67, 8258–8260.
Corresponding Author
*Phone: 212-659-8652. Fax: 212-849-2456. E-mail: ming-ming.
zhou@mssm.edu.
Author Contributions
†These authors contributed equally to this work.
’ ACKNOWLEDGMENT
We thank L. Zeng and G. Gerona-Navarro for technical support
of NMR and LCMS analysis of chemical compounds, respectively,
and M. Mezei and M. Ohlmeyer for helpful discussions. This work
was supported by the grants from the National Institutes of Health
(to M.-M.Z.).
’ ABBREVIATIONS USED
PRC2, polycomb repressive complex 2; SAM, S-adenosyl-L-
methionine; SAH, S-adenosyl-L-homocysteine
(22) Samejima, K.; Nakazawa, Y.; Matsunaga, I. Improved synthesis
of decarboxylated S-adenosylmethionine and related sulfonium com-
pounds. Chem. Pharm. Bull (Tokyo) 1978, 26, 1480–1485.
(23) Milton, S.; Yeheskiely, R. E.; Stromberg, R. Synthesis of a 20-
O-(carbomoylmethyl)ribonucleoside H-phosphonate building block
and a model dinucleotide. Nucleosides, Nucleotides Nucleic Acids 2007,
26, 1495–1499.
’ REFERENCES
(1) Jenuwein, T.; Allis, C. D. Translating the histone code. Science
2001, 293, 1074–1080.
(2) Kouzarides, T. Chromatin modifications and their function. Cell
2007, 128, 693–705.
(3) Cao, R.; Wang, L.; Wang, H.; Xia, L.; Erdjument-Bromage, H.;
Tempst, P.; Jones, R. S.; Zhang, Y. Role of histone H3 lysine 27
methylation in polycomb-group silencing. Science 2002, 298, 1039–1043.
(4) Kuzmichev, A.; Nishioka, K.; Erdjument-Bromage, H.; Tempst,
P.; Reinberg, D. Histone methyltransferase activity associated with a
human multiprotein complex containing the Enhancer of Zeste protein.
Genes Dev. 2002, 16, 2893–2905.
(5) Montgomery, N. D.; Yee, D.; Chen, A.; Kalantry, S.; Chamberlain,
S. J.; Otte, A. P.; Magnuson, T. The murine polycomb group protein Eed
is required for global histone H3 lysine-27 methylation. Curr. Biol. 2005,
15, 942–947.
(6) Manzur, K. L.; Farooq, A.; Zeng, L.; Plotnikova, O.; Koch, A. W.;
Sachchidanand; Zhou, M.-M. A dimeric viral SET domain methyltrans-
ferase specific to Lys27 of histone H3. Nat. Struct. Biol. 2003, 10, 187–196.
(7) Mujtaba, S.; Manzur, K. L.; Gurnon, J. R.; Kang, M.; Van Etten,
J. L.; Zhou, M. M. Epigenetic transcriptional repression of cellular genes
by a viral SET protein. Nat. Cell Biol. 2008, 10, 1114–1122.
(8) Wei, H.; Zhou, M. M. Dimerization of a viral SET protein
endows its function. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 18433–18438.
(9) Knight, Z. A.; Shokat, K. M. Chemical genetics: where genetics
and pharmacology meet. Cell 2007, 128, 425–430.
(10) Lin, Q.; Jiang, F.; Schultz, P. G.; Gray, N. S. Design of allele-
specific protein methyltransferase inhibitors. J. Am. Chem. Soc. 2001,
123, 11608–11613.
(11) Islam, K.; Zheng, W.; Yu, H.; Deng, H.; Luo, M. Expanding
cofactor repertoire of protein lysine methyltransferase for substrate
labeling. ACS Chem. Biol. 2011, 6, 679–684.
(12) Wang, R.; Zheng, W.; Yu, H.; Deng, H.; Luo, M. Labeling
substrates of protein arginine methyltransferase with engineered en-
zymes and matched S-adenosyl-L-methionine analogues. J. Am. Chem.
Soc. 2011, 133, 7648–7651.
(13) Nakagawa, I.; Hata, T. A convenient method for the synthesis
of 50-S-alkylthio-50-deoxyribonucleosides. Tetrahedron Lett. 1975, 17,
1409–1412.
7738
dx.doi.org/10.1021/jm201000j |J. Med. Chem. 2011, 54, 7734–7738