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PubMed1996

RecA-catalyzed, sequence-specific alkylation of DNA by cross-linking oligonucleotides. Effects of length and nonhomologous base substitutions.

Oligodeoxyribonucleotides (ODNs) bearing the reactive nitrogen mustard chlorambucil have been used as sequence-directed affinity labeling reagents to investigate the length and homology requirements for RecA-catalyzed alkylation of double-stranded DNA. The cross-linkage reaction, which takes place at the N-7 position of a targeted complementary strand guanine following strand exchange, was highly sequence specific with both a 272 bp DNA fragment and a linearized plasmid. Alkylation required the ODN to be at least 26 nucleotides long and to possess homology to the target in the vicinity of the modification site. The extent of alkylation was improved by using longer ODNs, with a 50-mer giving over 50% reaction. Mismatches inhibited alkylation when they perturbed the structure of the strand exchange product near the targeted guanine. Longer heterology also inhibited alkylation when it prevented strand exchange. Our inability to detect cross-linkage in stable synaptic complexes unable to undergo complete strand exchange is best explained by a model for homologous alignment in which the presynaptic filament approaches from the minor groove of the duplex. Since the N-7 position of guanine is in the major groove, it is inaccessible to the tethered chlorambucil group of the ODN during the search for homology. The reaction specificity of chlorambucil-bearing ODNs suggests that they may have general use as recombinase-mediated DNA targeting agents.

Affinity Labels↗
PubMed2001

Antibody recognition of melphalan adducts characterized using immobilized DNA: enhanced alkylation of G-Rich regions in cells compared to in vitro.

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

Alkylation↗
PubMed2002

Modified guanines representing O(6)-alkylation by the cyclophosphamide metabolites acrolein and chloroacetaldehyde: synthesis, stability, and ab initio studies.

Alkylation of DNA by acrolein and/or chloroacetaldehyde may result in the mutations that lead to the therapy-induced leukemia associated with cyclophosphamide (and ifosfamide) treatment. O(6)-(n-Propanalyl)guanine (O(6)-PAG) and O(6)-(ethanalyl)guanine (O(6)-EAG) were synthesized for use as authentic standards in investigations of DNA alkylation by acrolein and chloroacetaldehyde, respectively. Preparation of the O-methyl oximes of these aldehydes aided in confirming the structural assignments of O(6)-PAG and O(6)-EAG. HPLC was used to study the stability of O(6)-PAG under a variety of conditions. The decomposition of O(6)-PAG was attributed to an alpha,beta-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O(6)-PAG (1-10 mM) had a half-life of approximately 1 h (pH 7.4, 37 degrees C). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O(6)-PAG (1-10 mM) was approximately 16 h (pH 7.4, 37 degrees C). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O(6)-PAG to give a more stable oxazolidine. Under conditions similar to those that would be used for hydrolysis of DNA [0.1 M HCl-DMSO (98:2), pH 1.3, 70 degrees C, 30 min], there was an estimated 10-35% loss of O(6)-PAG. Under the same conditions, O(6)-EAG had apparent half-lives of 6.6 h (phosphate-DMSO) and 2.5 days (Tris-DMSO) and the estimated loss at pH 1.3 over 30 min (70 degrees C) was 15-20%. Ab initio quantum chemical calculations were used to understand the energy factors that underlie the occurrence of O- versus N-alkylations as well as possible, subsequent intramolecular cyclizations. Simulations of the free energies of reactions between acrolein and guanine indicated that N-alkylation was favored over O(6)()-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH(2) positions.

Acetaldehyde↗
PubMed1986

Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.

The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer- HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa O6-methylguanine-DNA methyltransferase. S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitro-sourea. This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer- cells.

Alkylating Agents↗
PubMed1998

Gene and human tumour cell line specific differencesin nitrogen mustard induced DNA alkylation and interstrand crosslinking frequencies.

The levels of N-alkyl purine and DNA interstrand crosslink formation, produced by the clinically used nitrogen mustard antitumour drug mechlorethamine (HN2), were quantitated at the level of specific genes in a panel of human tumour cell lines using modified Southern blotting methods. When purified genomic DNA was treated with HN2 in vitro, no significant difference in the extent of N-alkyl purine or interstrand crosslink formation in the N-ras, c-myc or CD3delta genes was observed. When the cell lines LS174T, Colo320HSR, J6 and U937 were treated with HN2, however, there was significant heterogeneity in the levels of N-alkyl purine formation in the three genes. The rank order of the extent of damage in the three genes was also different in the cell lines. The level of alkylation did not correlate with either the transcriptional activity of a gene or drug sensitivity. Crosslinks were not detectable in the N-ras or c-myc genes of LS174T, J6 or U937 cells treated with HN2, and only detectable in the amplified c-myc gene of the Colo320HSR cell line. In the related cell line Colo320DM, which has both native and translocated c-myc alleles which are both amplified and episomal, crosslinks were detected in the amplified native and rearranged c-myc alleles, and also in the N-ras gene which is also amplified in this cell line. For bifunctional alkylating agents such as HN2, therefore, heterogeneity of DNA damage can occur between different genes in human cells and can also vary for different lesions produced by the same agent. In addition, this heterogeneity can differ between human tumour cell lines.

Alkylation↗
PubMed1983

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs.

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.

Alkylating Agents↗
PubMed2006

Identification of candidate alkylator-induced cancer susceptibility genes by whole genome scanning in mice.

Secondary malignancies are a serious adverse consequence of alkylator chemotherapy. The risk of developing an alkylator-associated malignancy is influenced by genetic background, although the relevant genetic factors are poorly understood. To screen for novel susceptibility factors, we established a mouse model of alkylator-induced malignancy. We exposed mice from 20 inbred strains to the prototypical alkylating agent, N-nitroso-N-ethylurea (ENU). ENU was a potent carcinogen in many of the strains tested, inducing 140 tumors in 240 ENU-treated mice (66% incidence of at least one tumor in evaluable mice), compared with a background incidence of 8% spontaneous tumors in 240 strain-, age-, and sex-matched control mice (relative risk, 8.4; P < 0.0001). A wide variety of tumor histologies were noted, including epithelial carcinomas, soft tissue sarcomas, and hematopoietic tumors. Cancer susceptibility was a heritable trait for the most common tumor types, lung adenocarcinoma (H(2) = 0.25), T cell lymphoma (H(2) = 0.19), and myeloid malignancies (H(2) = 0.10). Quantitative trait locus mapping identified regions on chromosomes 3, 6, 9, and 15 containing candidate genes associated with lung adenoma, lung carcinoma, and lymphoma susceptibility. This novel mouse model recapitulates many features of human alkylator-associated cancer and supports the hypothesis that susceptibility to this syndrome is influenced by inherited polymorphisms that could be used to make informed clinical treatment decisions.

Alkylating Agents↗
PubMed2004

Design of novel antitumor DNA alkylating agents: the benzacronycine series.

Acronycine, a natural alkaloid originally extracted from the bark of the Australian ash scrub Acronychia baueri, has shown a significant antitumor activity in animal models. Acronycine has been tested against human cancers in the early 1980s, but the clinical trials showed modest therapeutic effects and its development was rapidly discontinued. In order to optimize the antineoplastic effect, different benzoacronycine derivatives were synthesized. Among those, the di-acetate compound S23906-1 was recently identified as a promising anticancer drug candidate and a novel alkylating agent specifically reacting with the exocylic 2-NH2 group of guanines in DNA. The study of DNA bonding capacity of acronycine derivatives leads to the identification of the structural requirements for DNA alkylation. In nearly all cases, the potent alkylating agents, such as S23906-1, were found to be much more cytotoxic than the unreactive analogs such as acronycine itself or diol derivatives. Alkylation of DNA by the monoacetate derivative S28687-1, which is a highly reactive hydrolysis metabolite of S23906-1, occurs with a marked preference for the N2 position of guanine. Other bionucleophiles can react with S23906-1. The benzacronycine derivatives, which efficiently alkylate DNA, also covalently bind to the tripeptide glutathione (GSH) but not to the oxidized product glutathione disulfide. Here we review the reactivity of S23906-1 and some derivatives toward DNA and GSH. The structure-activity relationships in the benzacronycine series validate the reaction mechanism implicating DNA as the main molecular target. S23906-1 stands as the most promising lead of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the acronycine skeleton.

Acronine↗
PubMed1984

Stereochemical properties of nucleosides alkylated by activated carcinogens.

An initial stage in the mechanism of chemical carcinogenesis by "activated" carcinogenic polycyclic aromatic hydrocarbons is believed to involve alkylation of DNA. However, very high (atomic) resolution studies of alkylated DNA are not technically feasible at this time, and therefore the detailed, high-resolution three-dimensional structures of portions of alkylated DNA have been determined. The initial phase of this study (reported here) has involved the preparation of a series of adenosines and 2'-deoxyadenosines substituted at N6 by related aralkyls of differing carcinogenic potential. We report here the crystal structure determinations of four of these compounds: Compound 1, N6-(anthracenyl-9-methyl)adenosine; Compound 2, N6-(10-methyl-anthracenyl-9-methyl)adenosine; Compound 3, N6-[12-methyl-benz(a)anthracenyl-7-methyl]adenosine; and Compound 5, N6-(10-methylanthracenyl-9-methyl)-2'-deoxyadenosine. Results are compared with those for a previously published analysis Compound 6, N6-[12-methylbenz(a)anthracenyl-7-methyl]-2'-deoxyadenosine. Several results of structural interest have emerged. All five compounds have the syn-conformational relationship between the sugar (ribose or 2'-deoxyribose) and the base (adenine), in contrast to the anti arrangement in B-DNA and in nonalkylated nucleosides. In four of the five compounds, there is an intramolecular hydrogen bond between the 5'-hydroxyl group and adenine. However, in the fifth molecule, this hydrogen bond is not found, and yet the conformation is syn. This indicates that formation of this internal hydrogen bond is not a prerequisite for the adoption of the syn-conformation. In general, the overall conformations of all five compounds are similar, the base lying approximately perpendicular to the polycyclic aromatic ring system. The packing of molecules in the unit cell is also of interest because it consists of alternations of adenine and polycyclic aromatic ring systems in columns through the crystal, indicating that this may serve as a model for the interaction with DNA. The oxygen atom of the sugar ring points towards the hydrocarbon ring system of another molecule. It is premature at this stage of our study to speculate as to the effects of alkylation on the conformational properties of either RNA or DNA. The only comment that appears justified is that the propensity of these adducts to adopt the syn-conformation may be indicative of a preference of alkylated DNA for the Z-conformation (even if the form that is initially attacked is B-DNA).(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed1999

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry for monitoring alkylation of beta-lactoglobulin B exposed to a series of N-substituted acrylamide monomers.

Delayed-extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, in both linear and reflectron modes, has been used to examine the alkylation of bovine beta-lactoglobulin-bound cysteines exposed to various molar concentrations (0.5-30 mM) of acrylamide and a number of its N-substituted monomers. These measurements were conducted at pH approximately 9.5, and showed that at 0.5 mM all monomers (except N-acryloylaminopropanol) resulted in a measurable alkylation of at least one cysteine out of five. At higher concentrations (15 mM) all investigated monomers resulted in substantial alkylation which, for some, involved all five cysteine residues. Reflectron MALDI-TOF measurements of a number of alkylated protein digests revealed that, at low molar ratios, the alkylation site is influenced by the identity of the monomer. For example acrylamide and N, N-dimethylacrylamide attacked Cys(160) as well as one of the three cysteines within the tryptic fragment [102-124], while other investigated monomers did not involve Cys(160). The implications of the present data for two-dimensional (2D) gel electrophoresis, and their eventual correlation to the toxicity of the investigated monomers, are discussed.

Acrylamides↗
PubMed1983

Cross-resistance to alkylating agents in multiple myeloma.

In order to ascertain whether multiple myeloma patients resistant to one alkylating agent would respond to a second one, high-dose intermittent cyclophosphamide was administered to 12 patients showing resistance to melphalan and prednisone. On the other hand, intermittent melphalan and prednisone treatment was employed in eight myeloma patients resistant to intermittent cyclophosphamide. Only one objective response was achieved among 12 patients on cyclophosphamide therapy, in spite of having employed high doses of this alkylating agent. No responses were achieved with melphalan in the group of cyclophosphamide-resistant patients. The median survival probability was 8.7 months (SD +/- 2.9) for all patients after starting the second alkylating agent. These results suggest cross-resistance between melphalan and cyclophosphamide in myeloma. The authors conclude that a single second alkylating agent cannot be recommended as a treatment of patients with multiple myeloma who are truly refractory to one alkylating agent.

Alkylating Agents↗
PubMed2002

In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.

Acrylamide↗
PubMed1991

Etoposide with lonidamine or pentoxifylline as modulators of alkylating agent activity in vivo.

In an effort to improve the additive anti-tumor efficacy of commonly used alkylating agents, the topoisomerase-II inhibitor etoposide was used in combination with either the mitochondrial poison and energy-depleting agent lonidamine or the hemorheologic agent and tumor-blood-flow-increasing agent pentoxifylline. In the FSaIIC murine fibrosarcoma system, these modulators were evaluated for modulation of whole-tumor cell killing vs. bone-marrow CFU-GM toxicity with the alkylating drugs CDDP, CTX, L-PAM or BCNU. Etoposide alone was essentially additive with the alkylating drugs for both tumor-cell and bone-marrow killing, except for BCNU, where a substantial increase in tumor-cell killing occurred (0.5 to 2.0 logs over the dose range of BCNU tested) without a significant increase in bone-marrow toxicity. Etoposide plus lonidamine was significantly more active than etoposide alone only with CTX and BCNU in tumor-cell vs. bone-marrow killing. Etoposide plus pentoxifylline was also most active with these two alkylating agents, where increases in tumor-cell killing of 0.5 to 1.0 log were observed. Hoechst-33342-defined tumor-cell sub-population studies revealed that etoposide significantly improved the killing of dim (putative hypoxic) cells by CDDP, but neither lonidamine nor pentoxifylline significantly improved killing of bright or dim cells together. With CTX, etoposide plus lonidamine or pentoxifylline substantially improved killing of dim cells over etoposide alone (each by about 0.8 logs). These data indicate that a therapeutic advantage may be achievable by combining etoposide with lonidamine or pentoxifylline for use with alkylating drugs.

Alkylating Agents↗
PubMed2001

Therapy-related leukemia associated with alkylating agents.

The leukemogenic potential of alkylating agents has been known for many years and almost all alkylating agents in clinical use have been shown to increase the risk of leukemia. With these drugs the risk of leukemia appears to increase with increasing patient age, as does the risk of de novo myeloid leukemia in the population. Susceptibility to alkylating agent-associated leukemia is influenced by the genetic constitution of the patient, and by the nature of the exposure. To illustrate the importance of these factors in etiology of leukemia, this paper discusses the contribution of disorders such as Fanconi anemia and neurofibromatosis to susceptibility to alkylating agent-associated leukemia. This paper also discusses the contribution of alkylating agents and other therapeutic exposures in the etiology of leukemias occurring after autologous bone marrow transplant.

Antineoplastic Agents, Alkylating↗
PubMed2003

Alkylating reactivity and herbicidal activity of chloroacetamides.

The relationship between S- and N-alkylating reactivity and herbicidal activity within a series of chloroacetamides, including several commercial herbicides and newly synthesised analogues was studied. The S-alkylating reactivity of selected chloroacetamides, as well as those of atrazine and chlorfenprop-methyl, was determined by in vitro GSH conjugation at a ratio of GSH to alkylating agent of 25:1. A spectrophotometric reaction using 4-(4-nitrobenzyl)pyridine was used to characterise the N-alkylating reactivity of the chemicals. Our results indicate that a reduced level of N-alkylating reactivity correlates with an improved herbicidal efficacy at a practical rate. However, the phytoxicity of the molecules is not simply dependent on chemical reactivities, but strictly related to the molecular structure, indicating that lipophilicity, uptake, mobility and induction of detoxifying enzymes may also be decisive factors in the mode of action.

Acetamides↗
PubMed1999

In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor.

Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.

Alkyl and Aryl Transferases↗
PubMed2000

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

Alkylating Agents↗
PubMed2000

The alkylated thiohydantoin method for C-terminal sequence analysis.

The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf. Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin. The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin. Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry. The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine. Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids. The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids. Sequencing stops at proline residues.

Alkylation↗