Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.     

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9‐methylated histone H3 tails

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin‐associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host–guest system between protein and protein. About 10‐ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono‐, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen‐bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen‐bonded interactions associated with aromatic cage in Me3K9 H3‐HP1 complex were observed to be much more stable than those in Me1K9 H3‐HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein–protein interactions through a trimethylated post‐translational modification. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Peptide mass mapping of acetylated isoforms of histone H4 from mouse lymphosarcoma cells treated with histone deacetylase (HDACs) inhibitors

The acetylated isoforms of histone H4 from mouse lymphosarcoma cells treated with HDAC inhibitors trichostatin A (TSA) and depsipeptide (DDP) were separated by acetic acid urea-polyacrylamide gel electrophoresis (AU-PAGE), in-gel digested, and analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The acetylation pattern of histone H4 in mouse lymphosarcoma cells induced by TSA was established in which acetylation initially occurred at K16 followed by K12 and then K8 and/or K5. An identical order of acetylation was found for cells treated with DDP.     

Structural and magnetic properties of iron(II) complexes with 1,4,5,8,9,12-hexaazatriphenylene (HAT)

Reactions of Fe(II) salts with the ligand 1,4,5,8,9,12-hexaazatriphenylene (HAT) led to the isolation and characterization of four new compounds: [Fe3(HAT)(H2O)12](SO4)3.3.3H2O (1), [Fe2(HAT)(SO4)(H2O)5](SO4).2H2O.CH3OH (2), [Fe2(HAT)(SO4)(H2O)5](SO4).3H2O (3), and [Fe3Cl5(HAT)(CH3OH)4(H2O)]Cl (4). Compound 1 crystallizes as a trinuclear cluster in which HAT acts as a tris-chelating ligand. Compounds 2 and 3 are two polymorphs of an infinite one-dimensional structure in which the Fe atoms are coordinated to HAT and then connected into the chain through bridging sulfate anions. Compound 4 exhibits a similar chain structure, but with bridging chloride ligands. The magnetic behavior of the new compounds is indicative of weak antiferromagnetic coupling between the Fe(II) centers through the HAT ligand.     

Selective Inhibition of Lysine‐Specific Demethylase 5A (KDM5A) Using a Rhodium(III) Complex for Triple‐Negative Breast Cancer Therapy

Lysine‐specific demethylase 5A (KDM5A) has recently become a promising target for epigenetic therapy. In this study, we designed and synthesized metal complexes bearing ligands with reported demethylase and p27 modulating activities. The Rh(III) complex 1 was identified as a direct, selective and potent inhibitor of KDM5A that directly abrogate KDM5A demethylase activity via antagonizing the KDM5A‐tri‐/di‐methylated histone 3 protein–protein interaction (PPI) in vitro and in cellulo. Complex 1 induced accumulation of H3K4me3 and H3K4me2 levels in cells, causing growth arrest at G1 phase in the triple‐negative breast cancer (TNBC) cell lines, MDA‐MB‐231 and 4T1. Finally, 1 exhibited potent anti‐tumor activity against TNBC xenografts in an in vivo mouse model, presumably via targeting of KDM5A and hence upregulating p27. Moreover, complex 1 was less toxic compared with two clinical drugs, cisplatin and doxorubicin. To our knowledge, complex 1 is the first metal‐based KDM5A inhibitor reported in the literature. We anticipate that complex 1 may be used as a novel scaffold for the further development of more potent epigenetic agents against cancers, including TNBC.     

LSD1: more than demethylation of histone lysine residues

Lysine-specific histone demethylase 1 (LSD1) represents the first example of an identified nuclear protein with histone demethylase activity. In particular, it plays a special role in the epigenetic regulation of gene expression, as it removes methyl groups from mono- and dimethylated lysine 4 and/or lysine 9 on histone H3 (H3K4me1/2 and H3K9me1/2), behaving as a repressor or activator of gene expression, respectively. Moreover, it has been recently found to demethylate monomethylated and dimethylated lysine 20 in histone H4 and to contribute to the balance of several other methylated lysine residues in histone H3 (i.e., H3K27, H3K36, and H3K79). Furthermore, in recent years, a plethora of nonhistone proteins have been detected as targets of LSD1 activity, suggesting that this demethylase is a fundamental player in the regulation of multiple pathways triggered in several cellular processes, including cancer progression. In this review, we analyze the molecular mechanism by which LSD1 displays its dual effect on gene expression (related to the specific lysine target), placing final emphasis on the use of pharmacological inhibitors of its activity in future clinical studies to fight cancer.Subject terms: Epigenetics, Histone post-translational modifications     

Single-molecule magnets: a new class of tetranuclear manganese magnets

The preparation, X-ray structure, and detailed physical characterization are presented for a new type of single-molecule magnet [Mn4(O2CMe)2(pdmH)6](ClO4)2 (1). Complex 1.2MeCN.Et2O crystallizes in the triclinic space group P1, with cell dimensions at 130 K of a = 11.914(3) A, b = 15.347(4) A, c = 9.660(3) A, alpha = 104.58(1) degree, beta = 93.42(1) degree, gamma = 106.06(1) degree, and Z = 1. The cation lies on an inversion center and consists of a planar Mn4 rhombus that is mixed-valent, MnIII2MnII2. The pdmH- ligands (pdmH2 is pyridine-2,6-dimethanol) function as either bidentate or tridentate ligands. The bridging between Mn atoms is established by either a deprotonated oxygen atom of a pdmH- ligand or an acetate ligand. The solvated complex readily loses all acetonitrile and ether solvate molecules to give complex 1, which with time becomes hydrated to give 1.2.5H2O. Direct current and alternating current magnetic susceptibility data are given for 1 and 1.2.5H2O and indicate that the desolvated complex has a S = 8 ground state, whereas the hydrated 1.2.5H2O has a S = 9 ground state. Ferromagnetic interactions between MnIII-MnII and MnIII-MnIII pairs result in parallel spin alignments of the S = 5/2 MnII and S = 2 MnIII ions. High-frequency EPR spectra were run for complex 1.2.5H2O at frequencies of 218, 328, and 436 GHz in the 4.5-30 K range. A magnetic-field-oriented polycrystallite sample was employed. Fine structure is clearly seen in this parallel-field EPR spectrum. The transition fields were least-squares-fit to give g = 1.99, D = -0.451 K, and B4 degrees = 2.94 x 10(-5) K for the S = 9 ground state of 1.2.5H2O. A molecule with a large-spin ground state with D < 0 can function as a single-molecule magnet, as detected by techniques such as ac magnetic susceptibility. Out-of-phase ac signals (chi' M) were seen for complexes 1 and 1.2.5H2O to show that these complexes are single-molecule magnets. A sample of 1 was studied by ac susceptibility in the 0.4-6.4 K range with the ac field oscillating at frequencies in the 1.1-1000 Hz range. A single peak in chi' M vs temperature plots was seen for each frequency; the temperature of the chi' M peak varies from 2.03 K at 995 Hz to 1.16 K at 1.1 Hz. Magnetization relaxation rates were evaluated in this way. An Arrhenius plot gave an activation energy of 17.3 K, which, as expected, is less than the 22.4 K value calculated for the thermodynamic barrier for magnetization direction reversal for an S = 8 complex with D = -0.35 K. The 1.2.5H2O complex with an S = 9 ground state has its chi' M peaks at higher temperatures.     

Single-molecule magnets: Jahn-Teller isomerism and the origin of two magnetization relaxation processes in Mn12 complexes

Several single-molecule magnets with the composition [Mn12O12(O2CR)16(H2O)x] (x = 3 or 4) exhibit two out-of-phase ac magnetic susceptibility signals, one in the 4-7 K region and the other in the 2-3 K region. New Mn12 complexes were prepared and structurally characterized, and the origin of the two magnetization relaxation processes was systematically examined. Different crystallographic forms of a Mn12 complex with a given R substituent exist where the two forms have different compositions of solvent molecules of crystallization and this results in two different arrangements of bound H2O and carboxylate ligands for the two crystallographically different forms with the same R substituent. The X-ray structure of cubic crystals of [Mn12O12(O2CEt)16(H2O)3]. 4H2O (space group P1) (complex 2a) has been reported previously. The more prevalent needle-form of [Mn12O12(O2CEt)16(H2O)3] (complex 2b) crystallizes in the monoclinic space group P2(1)/c, which at -170 degrees C has a = 16.462(7) A, b = 22.401(9) A, c = 20.766(9) A, beta = 103.85(2) degrees, and Z = 4. The arrangements of H2O and carboxylate ligands on the Mn12 molecule are different in the two crystal forms. The complex [Mn12O12-(O2)CC6H4-p-Cl)16(H2O)4].8CH2Cl2 (5) crystallizes in the monoclinic space group C2/c, which at -172 degrees C has a = 29.697(9) A, b = 17.708(4) A, c = 30.204(8) A, beta = 102.12(2) degrees, and Z = 4. The ac susceptibility data for complex 5 show that it has out-of-phase signals in both the 2-3 K and the 4-7 K ranges. X-ray structures are also reported for two isomeric forms of the p-methylbenzoate complex. [Mn12O12(O2CC6H4-p-Me)16(H2O)4]. (HO2CC6H4-p-Me) (6) crystallizes in the monoclinic space group C2/c, which at 193 K has a = 40.4589(5) A, b = 18.2288(2) A, c = 26.5882(4) A, beta = 125.8359(2) degrees, and Z = 4. [Mn12O12(O2CC6H4-p-Me)16(H2O)4].3(H2O) (7) crystallizes in the monoclinic space group I2/a, which at 223 K has a = 29.2794(4) A, b = 32.2371(4) A, c = 29.8738(6) A, beta = 99.2650(10) degrees, and Z = 8. The Mn12 molecules in complexes 6 and 7 differ in their arrangements of the four bound H2O ligands. Complex 6 exhibits an out-of-phase ac peak (chi(M)' ') in the 2-3 K region, whereas the hydrate complex 7 has a chi(M)' ' signal in the 4-7 K region. In addition, however, in complex 6, one Mn(III) ion has an abnormal Jahn-Teller distortion axis oriented at an oxide ion, and thus 6 and 7 are Jahn-Teller isomers. This reduces the symmetry of the core of complex 6 compared with complex 7. Thus, complex 6 likely has a larger tunneling matrix element and this explains why this complex shows a chi(M)' ' signal in the 2-3 K region, whereas complex 7 has its chi(M)' ' peak in the 4-7 K region, i.e., the rate of tunneling of magnetization is greater in complex 6 than complex 7. Detailed 1H NMR experiments (2-D COSY and TOCSY) lead to the assignment of all proton resonances for the benzoate and p-methyl-benzoate Mn12 complexes and confirm the structural integrity of the (Mn12O12) complexes upon dissolution. In solution there is rapid ligand exchange and no evidence for the different isomeric forms of Mn12 complexes seen in the solid state.     

Hydrogen atom transfer reactions of a ruthenium imidazole complex: hydrogen tunneling and the applicability of the Marcus cross relation

The reaction of Ru(II)(acac)2(py-imH) (Ru(II)imH) with TEMPO(*) (2,2,6,6-tetramethylpiperidine-1-oxyl radical) in MeCN quantitatively gives Ru(III)(acac)2(py-im) (Ru(III)im) and the hydroxylamine TEMPO-H by transfer of H(*) (H(+) + e(-)) (acac = 2,4-pentanedionato, py-imH = 2-(2'-pyridyl)imidazole). Kinetic measurements of this reaction by UV-vis stopped-flow techniques indicate a bimolecular rate constant k(3H) = 1400 +/- 100 M(-1) s(-1) at 298 K. The reaction proceeds via a concerted hydrogen atom transfer (HAT) mechanism, as shown by ruling out the stepwise pathways of initial proton or electron transfer due to their very unfavorable thermochemistry (Delta G(o)). Deuterium transfer from Ru(II)(acac)2(py-imD) (Ru(II)imD) to TEMPO(*) is surprisingly much slower at k(3D) = 60 +/- 7 M(-1) s(-1), with k(3H)/k(3D) = 23 +/- 3 at 298 K. Temperature-dependent measurements of this deuterium kinetic isotope effect (KIE) show a large difference between the apparent activation energies, E(a3D) - E(a3H) = 1.9 +/- 0.8 kcal mol(-1). The large k(3H)/k(3D) and DeltaE(a) values appear to be greater than the semiclassical limits and thus suggest a tunneling mechanism. The self-exchange HAT reaction between Ru(II)imH and Ru(III)im, measured by (1)H NMR line broadening, occurs with k(4H) = (3.2 +/- 0.3) x 10(5) M(-1) s(-1) at 298 K and k(4H)/k(4D) = 1.5 +/- 0.2. Despite the small KIE, tunneling is suggested by the ratio of Arrhenius pre-exponential factors, log(A(4H)/A(4D)) = -0.5 +/- 0.3. These data provide a test of the applicability of the Marcus cross relation for H and D transfers, over a range of temperatures, for a reaction that involves substantial tunneling. The cross relation calculates rate constants for Ru(II)imH(D) + TEMPO(*) that are greater than those observed: k(3H,calc)/k(3H) = 31 +/- 4 and k(3D,calc)/k(3D) = 140 +/- 20 at 298 K. In these rate constants and in the activation parameters, there is a better agreement with the Marcus cross relation for H than for D transfer, despite the greater prevalence of tunneling for H. The cross relation does not explicitly include tunneling, so close agreement should not be expected. In light of these results, the strengths and weaknesses of applying the cross relation to HAT reactions are discussed.     

Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes

Nucleosomes carry extensive post‐translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysine 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27‐specific lysine methyltransferase (KMT) polycomb repressive complex 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3‐mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.     

Switchover of the Mechanism between Electron Transfer and Hydrogen‐Atom Transfer for a Protonated Manganese(IV)–Oxo Complex by Changing Only the Reaction Temperature

Hydroxylation of mesitylene by a nonheme manganese(IV)–oxo complex, [(N4Py)Mn^IV(O)]²⁺ ( 1 ), proceeds via one‐step hydrogen‐atom transfer (HAT) with a large deuterium kinetic isotope effect (KIE) of 3.2(3) at 293 K. In contrast, the same reaction with a triflic acid‐bound manganese(IV)‐oxo complex, [(N4Py)Mn^IV(O)]²⁺‐(HOTf)₂ ( 2 ), proceeds via electron transfer (ET) with no KIE at 293 K. Interestingly, when the reaction temperature is lowered to less than 263 K in the reaction of 2 , however, the mechanism changes again from ET to HAT with a large KIE of 2.9(3). Such a switchover of the reaction mechanism from ET to HAT is shown to occur by changing only temperature in the boundary region between ET and HAT pathways when the driving force of ET from toluene derivatives to 2 is around ?0.5 eV. The present results provide a valuable and general guide to predict a switchover of the reaction mechanism from ET to the others, including HAT.     

Recognition of Dimethylarginine Analogues by Tandem Tudor Domain Protein Spindlin1

Epigenetic readout of the combinatorial posttranslational modification comprised of trimethyllysine and asymmetric dimethylarginine (H3K4me3R8me2a) takes place via biomolecular recognition of tandem Tudor-domain-containing protein Spindlin1. Through comparative thermodynamic data and molecular dynamics simulations, we sought to explore the binding scope of asymmetric dimethylarginine mimics by Spindlin1. Herein, we provide evidence that the biomolecular recognition of H3K4me2R8me2a is not significantly affected when R8me2a is replaced by dimethylarginine analogues, implying that the binding of K4me3 provides the major binding contribution. High-energy water molecules inside both aromatic cages of the ligand binding sites contribute to the reader–histone association upon displacement by histone peptide, with the K4me3 hydration site being lower in free energy due to a flip of Trp151.     

Wheel-shaped [Mn12] single-molecule magnets

The reaction of [Mn(12)O(12)(O(2)CCH(3))(16)(H(2)O)(4)].4H(2)O.2CH(3)COOH with n-methyldiethanol amine (H(2)mdea), n-ethyldiethanol amine (H(2)edea), or n-butyldiethanol amine (H(2)bdea) leads to the formation of wheel-shaped Mn(III)(6)Mn(II)(6) complexes with the general formula [Mn(12)(R)(O(2)CCH(3))(14)] (1, R = mdea; 2, R = edea; and 3, R = bdea). Complex 1 crystallizes in the triclinic space group P1, whereas complex 3 crystallizes in the monoclinic space group C(2/c). Complex 1a has the same molecular structure as complex 1 but crystallizes in the monoclinic space group P2(1/n). Complex 3a has the same molecular structure as complex 3 but crystallizes in the triclinic space group P1. Variable-temperature magnetic susceptibility data collected for complexes 1, 2, and 3 indicate that antiferromagnetic exchange interactions are present. The spin ground states of complexes 1, 2, and 3 were determined by fitting variable-field magnetization data collected in the 2-5 K temperature range. Fitting of these data yielded the spin ground-state parameters of S = 8, g = 2.0, and D = -0.47 cm(-1) for complex 1; S = 8, g = 2.0, and D = -0.49 cm(-1) for complex 2; and S = 8, g = 2, and D = -0.37 cm(-1) for complex 3. The ac magnetic susceptibility data were measured for complexes 1, 2, and 3 at temperatures between 1.8 and 10 K with a 3 G ac field oscillating in the range 50-1000 Hz. Slow kinetics of magnetization reversal relative to the frequency of the oscillating ac field were observed as frequency-dependent out-of-phase peaks for complexes 1, 2, and 3, and it can be concluded that these three complexes are single-molecule magnets.     

Slow hydrogen atom self-exchange between Os(IV) anilide and Os(III) aniline complexes: relationships with electron and proton transfer self-exchange

Hydrogen atom, proton and electron transfer self-exchange and cross-reaction rates have been determined for reactions of Os(IV) and Os(III) aniline and anilide complexes. Addition of an H-atom to the Os(IV) anilide TpOs(NHPh)Cl(2) (Os(IV)NHPh) gives the Os(III) aniline complex TpOs(NH(2)Ph)Cl(2) (Os(III)NH(2)Ph) with a new 66 kcal mol(-1) N-H bond. Concerted transfer of H* between Os(IV)NHPh and Os(III)NH(2)Ph is remarkably slow in MeCN-d(3), with k(ex)(H*) = (3 +/- 2) x 10(-3) M(-1) s(-1) at 298 K. This hydrogen atom transfer (HAT) reaction could also be termed proton-coupled electron transfer (PCET). Related to this HAT process are two proton transfer (PT) and two electron transfer (ET) self-exchange reactions, for instance, the ET reactions Os(IV)NHPh + Os(III)NHPh(-) and Os(IV)NH(2)Ph(+) + Os(III)NH(2)Ph. All four of these PT and ET reactions are much faster (k = 10(3)-10(5) M(-1) s(-1)) than HAT self-exchange. This is the first system where all five relevant self-exchange rates related to an HAT or PCET reaction have been measured. The slowness of concerted transfer of H* between Os(IV)NHPh and Os(III)NH(2)Ph is suggested to result not from a large intrinsic barrier but rather from a large work term for formation of the precursor complex to H* transfer and/or from significantly nonadiabatic reaction dynamics. The energetics for precursor complex formation is related to the strength of the hydrogen bond between reactants. To probe this effect further, HAT cross-reactions have been performed with sterically hindered aniline/anilide complexes and nitroxyl radical species. Positioning steric bulk near the active site retards both H* and H(+) transfer. Net H* transfer is catalyzed by trace acids and bases in both self-exchange and cross reactions, by stepwise mechanisms utilizing the fast ET and PT reactions.     

Single-molecule magnets: a reductive aggregation route to new types of Mn12 complexes

Three dodecanuclear Mn clusters [Mn12O10(OMe)3(OH)(O2CC6H3F2)16(MeOH)2].8MeOH (1), [Mn12O10(OMe)4(O2CBu(t))16(MeOH)2] (2), and [Mn12O12(O2CBu(t))16(MeOH)4] (3) synthesized by reductive aggregation reactions are reported. Clusters 1 and 2 possess a central alkoxide-bridged planar Mn4 topology, whereas 3 is a new high-symmetry member of the normal Mn12 family. Complexes 1 and 2 crystallize in the monoclinic space groups C2/c and P2(1)/n, respectively. Both consist of four Mn(IV) and eight Mn(III) ions held together by 10 mu3-O2- ions, and either (i) one mu-OH- and three mu-MeO- groups for 1 or (ii) four mu-MeO- groups for 2. Complex 3 crystallizes in the orthorhombic space group Aba2 and possesses the normal Mn12 structure but with terminal MeOH molecules. The cyclic voltammogram (CV) of 1 exhibits no reversible redox processes. Variable-temperature, solid-state dc and ac magnetic susceptibility measurements on 1 and 2 reveal that they possess S = 5 and 9 ground states, respectively. In addition, ac susceptibility measurements on complex 1 in a zero dc field in the temperature range 1.8-10 K and in a 3.5 G ac field oscillating at frequencies in the 5-1488 Hz range display a nonzero frequency-dependent out-of-phase (chi(M)') signal at temperatures below 3 K, with the peak maxima lying at temperatures below 1.8 K. For complex 2, two frequency dependent chi(M)' signals are seen, one in the higher temperature range of 3-5 K and a second at lower temperatures with its peak maxima at temperatures below 1.8 K. Single-crystal magnetization vs dc field scans down to 0.04 K for 1.8MeOH and 2 show hysteresis behavior at <1 K, confirming that both complexes are new examples of SMMs.     

A Genetically Encoded Allysine for the Synthesis of Proteins with Site‐Specific Lysine Dimethylation

Using the amber suppression approach, N^ϵ‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.