Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.

Patients with non-small cell lung cancer (NSCLC) harboring common mutations of the epidermal growth factor receptor (EGFR) are sensitive to EGFR-tyrosine kinase inhibitors (TKI). Although forms of EGFR harboring single uncommon mutations such as G719X or L861Q are thought to be less sensitive to EGFR-TKI, the efficacy of these drugs in patients with double uncommon mutations has remained unclear. We here present an NSCLC patient found to be positive for double uncommon EGFR mutations (G719X and L861Q) by clinical genomic sequencing analysis of a pleural effusion specimen who showed a durable response to the EGFR-TKI afatinib. The sensitivity of EGFR with single or double uncommon mutations to afatinib and the EGFR-TKI erlotinib was also evaluated in vitro with a visual assay based on HEK293 cells transiently transfected with expression plasmids for yellow fluorescent protein (YFP)-tagged fragments of the EGFR intracellular domain (ICD). Whereas forms of EGFR with double uncommon mutations were more sensitive to erlotinib than were those with single uncommon mutations, those with single or double uncommon mutations were similarly sensitive to afatinib, consistent with the patient's clinical outcome. Our data support the notion that afatinib is the most suitable EGFR-TKI for NSCLC harboring uncommon mutations of EGFR. Furthermore, the YFP-EGFR-ICD assay is potentially applicable to prediction of EGFR-TKI efficacy in patients with such mutations.

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

α3/4 Fucosyltransferase 3-dependent synthesis of Sialyl Lewis A on CD44 variant containing exon 6 mediates polymorphonuclear leukocyte detachment from intestinal epithelium during transepithelial migration.

Polymorphonuclear leukocyte (PMN) migration across the intestinal epithelium closely parallels disease symptoms in patients with inflammatory bowel disease. PMN transepithelial migration (TEM) is a multistep process that terminates with PMN detachment from the apical epithelium into the lumen. Using a unique mAb (GM35), we have previously demonstrated that engagement of the CD44 variant containing exon 6 (CD44v6) blocks both PMN detachment and cleavage of CD44v6. In this article, we report that PMN binding to CD44v6 is mediated by protein-specific O-glycosylation with sialyl Lewis A (sLe(a)). Analyses of glycosyltransferase expression identified fucosyltransferase 3 (Fut3) as the key enzyme driving sLe(a) biosynthesis in human intestinal epithelial cells (IECs). Fut3 transfection of sLe(a)-deficient IECs resulted in robust expression of sLe(a). However, this glycan was not expressed on CD44v6 in these transfected IECs; therefore, engagement of sLe(a) had no effect on PMN TEM across these cells. Analyses of sLe(a) in human colonic mucosa revealed minimal expression in noninflamed areas, with striking upregulation under colitic conditions that correlated with increased expression of CD44v6. Importantly, intraluminal administration of mAb GM35 blocked PMN TEM and attenuated associated increases in intestinal permeability in a murine intestinal model of inflammation. These findings identify a unique role for protein-specific O-glycosylation in regulating PMN-epithelial interactions at the luminal surface of the intestine.

A Nanozyme-Based Electrode for High-Performance Neural Recording.

Implanted neural electrodes have been widely used to treat brain diseases that require high sensitivity and biocompatibility at the tissue-electrode interface. However, currently used clinical electrodes cannot meet both these requirements simultaneously, which hinders the effective recording of electronic signals. Herein, nanozyme-based neural electrodes incorporating bioinspired atomically precise clusters are developed as a general strategy with a heterogeneous design for multiscale and ultrasensitive neural recording via quantum transport and biocatalytic processes. Owing to the dual high-speed electronic and ionic currents at the electrode-tissue interface, the impedance of nanozyme electrodes is 26 times lower than that of state-of-the-art metal electrodes, and the acquisition sensitivity for the local field potential is ≈10 times higher than that of clinical PtIr electrodes, enabling a signal-to-noise ratio (SNR) of up to 14.7 dB for single-neuron recordings in rats. The electrodes provide more than 100-fold higher antioxidant and multi-enzyme-like activities, which effectively decrease 67% of the neuronal injury area by inhibiting glial proliferation and allowing sensitive and stable neural recording. Moreover, nanozyme electrodes can considerably improve the SNR of seizures in acute epileptic rats and are expected to achieve precise localization of seizure foci in clinical settings.

Oligosaccharide analysis in urine by maldi-tof mass spectrometry for the diagnosis of lysosomal storage diseases.

BACKGROUND: There are 45 known genetic diseases that impair the lysosomal degradation of macromolecules. The loss of a single lysosomal hydrolase leads to the accumulation of its undegraded substrates in tissues and increases of related glycoconjugates in urine, some of which can be detected by screening of free oligosaccharides (FOS) in urine. Traditional 1-dimensional TLC for urine oligosaccharide analysis has limited analytical specificity and sensitivity. We developed fast and robust urinary FOS and glycoaminoacid analyses by MALDI-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the diagnosis of oligosaccharidoses and other lysosomal storage diseases. METHODS: The FOS in urine equivalent to 0.09 mg creatinine were purified through sequential passage over a Sep-Pak C18 column and a carbograph column and were then permethylated. MALDI-TOF/TOF was used to analyze the permethylated FOS. We studied urine samples from individuals in 7 different age groups ranging from 0-1 months to ≥ 17 years as well as urine from known patients with different lysosomal storage diseases. RESULTS: We identified diagnostic urinary FOS patterns for α-mannosidosis, galactosialidosis, mucolipidosis type II/III, sialidosis, α-fucosidosis, aspartylglucosaminuria (AGU), Pompe disease, Gaucher disease, and GM1 and GM2 gangliosidosis. Interestingly, the increase in urinary FOS characteristic of lysosomal storage diseases relative to normal FOS appeared to correlate with the disease severity. CONCLUSIONS: The analysis of urinary FOS by MALDI-TOF/TOF is a powerful tool for first-tier screening of oligosaccharidoses and lysosomal storage diseases.

Serum N-glycan and O-glycan analysis by mass spectrometry for diagnosis of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDGs) are caused by defects in genes that participate in biosynthetic glycosylation pathways. To date, 19 different genetic defects in N-glycosylation, 17 in O-glycosylation, and 21 in multiple glycosylation are known. Current diagnostic testing of CDGs largely relies on indirect analysis of glycosylation of serum transferrin. Such analysis alone is insufficient to diagnose many of the known glycosylation disorders. To improve the diagnosis of these groups of CDGs, we have developed serum or plasma N- and O-glycan profiling using a combination of MALDI-TOF/MS and LC-MS/MS technologies. Using this approach, we analyzed samples from nine patients with different known multiple glycosylation disorders, including three with COG deficiencies, one with TMEM165-CDG, two with PGM1-CDG, and three with SLC35A2-CDG, and one patient with combined type I and type II of unknown molecular etiology. Measurement of the relative quantities of various N- and O-glycan species clearly differentiates patients and controls. Our study demonstrates that structural analysis and quantitation of combined N- and O-glycan profiles are reliable diagnostic tools for CDGs.

The dominating role of N-deacetylase/N-sulfotransferase 1 in forming domain structures in heparan sulfate.

Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains sulfated domains known as N-S domains and low sulfate domains known as N-Ac domains. The distribution of the domain structures is likely governed by the action of glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here, we sought to determine the substrate specificity of NDST using model substrates and recombinant NDST protein. We discovered that NDST-1 carries out the modification in a highly ordered fashion. The enzyme sulfates the substrate from the nonreducing end toward the reducing end consecutively, leading to the product with a cluster of N-sulfo glucosamine residues. Furthermore, a preexisting N-sulfo glucosamine residue prevents the action of NDST-1 at the residues immediately located at the nonreducing end, allowing the formation of an N-Ac domain. Our results provide the long sought evidence for understanding the formation of sulfated versus nonsulfated domains in the HS isolated from cells and tissues. The study demonstrates the regulating role of NDST-1 in mapping the sulfation patterns of HS.

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach.

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3 nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.

Enzymatic placement of 6-O-sulfo groups in heparan sulfate.

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups.

Chemoenzymatic design of heparan sulfate oligosaccharides.

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate.

Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non-small cell lung cancer patients treated with osimertinib.

OBJECTIVES: The epidermal growth factor receptor (EGFR, also known as Her1) is a member of the human epidermal growth factor receptor (HER) family of proteins and a target of tyrosine kinase inhibitors (TKIs) in the treatment of non-small cell lung cancer (NSCLC) positive for activating mutations ofEGFR. Signal transduction by HER family proteins is dependent on their homo- or heterodimerization, but little is known of the relation between the relative proportions of such dimers of Her1 and sensitivity to EGFR-TKIs. We here investigated the feasibility of assessing this relation with the in situ proximity ligation assay (PLA) technique, which is able to detect the interaction of two proteins of interest when they are in close proximity. MATERIALS AND METHODS: In situ PLA was applied to detect Her1 homodimers and Her1 heterodimers in NSCLC cell lines and tissue specimens positive for EGFR activating mutations. RESULTS: In situ PLA allowed visualization and quantitative assessment of Her1 homodimers as well as of Her1 heterodimers with Her2, Her3, or Her4 not only in NSCLC cell lines but also in NSCLC tissue specimens obtained from various anatomic sites and by different collection methods. Treatment of NSCLC cell lines with EGFR-TKIs resulted in a decrease in the number of Her1 dimers, with the effect on homodimers being greater than that on heterodimers. A high ratio of Her1 heterodimers to homodimers was associated with poor progression-free survival in NSCLC patients treated with osimertinib. CONCLUSION: In situ PLA allows the detection of HER family dimers in NSCLC tissue, and quantitative assessment of Her1 homo- and heterodimers may prove informative for prediction of the response of NSCLC patients to EGFR-TKI treatment.

Preactivation-based, one-pot combinatorial synthesis of heparin-like hexasaccharides for the analysis of heparin-protein interactions.

Heparin (HP) and heparan sulfate (HS) play important roles in many biological events. Increasing evidence has shown that the biological functions of HP and HS can be critically dependent upon their precise structures, including the position of the iduronic acids and sulfation patterns. However, unraveling the HP code has been extremely challenging due to the enormous structural variations. To overcome this hurdle, we investigated the possibility of assembling a library of HP/HS oligosaccharides using a preactivation-based, one-pot glycosylation method. A major challenge in HP/HS oligosaccharide synthesis is stereoselectivity in the formation of the cis-1,4-linkages between glucosamine and the uronic acid. Through screening, suitable protective groups were identified on the matching glycosyl donor and acceptor, leading to stereospecific formation of both the cis-1,4- and trans-1,4-linkages present in HP. The protective group chemistry designed was also very flexible. From two advanced thioglycosyl disaccharide intermediates, all of the required disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified building-block preparation. Furthermore, the reactivity-independent nature of the preactivation-based, one-pot approach enabled us to mix the building blocks. This allowed rapid assembly of twelve HP/HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. The speed and the high yields achieved in glycoassembly without the need to use a large excess of building blocks highlighted the advantages of our approach, which can be of general use to facilitate the study of HP/HS biology. As a proof of principle, this panel of hexasaccharides was used to probe the effect of backbone sequence on binding with the fibroblast growth factor-2 (FGF-2). A trisaccharide sequence of 2-O-sulfated iduronic acid flanked by N-sulfated glucosamines was identified to be the minimum binding motif and N-sulfation was found to be critical. This provides useful information for further development of more potent compounds towards FGF-2 binding, which can have potential applications in wound healing and anticancer therapy.

Immune checkpoint protein and cytokine expression by T lymphocytes in pleural effusion of cancer patients receiving anti-PD-1 therapy.

OBJECTIVES: Pleural effusion (PE) occasionally develops in cancer patients during treatment with antibodies to programmed cell death-1 (PD-1) or to its ligand PD-L1 (hereafter, αPD-1 therapy). Such effusion often contains infiltrated mononuclear cells, although the types of immune cell present as well as the outcome of such patients have remained unclear. MATERIALS AND METHODS: We performed a multi-institutional, observational study to examine the clinical outcome of patients who develop PE after the onset of αPD-1 therapy. We compared the immune cell profiles and the immune status of lymphocytes in PE as determined by flow cytometry between nine patients who developed effusion during αPD-1 therapy (αPD-1 group) and 15 patients who developed PE during treatment with other anticancer agents (control group). RESULTS: Most mononuclear cells in PE were lymphocytes in both the αPD-1 and control groups. The frequency of both CD4+ and CD8+ T lymphocytes expressing the immune checkpoint proteins TIM-3 or TIGIT as well as that of CD8+ T lymphocytes expressing PD-L1 were increased in the αPD-1 group compared with the control group. αPD-1 therapy continued for a substantial period after the emergence of PE in six of the nine patients in the αPD-1 group, and the frequency of CD4+ T lymphocytes in PE expressing the immune checkpoint protein LAG-3 or the cytokine interkeukin-17 was lower for these patients than for those who did not receive a sustained treatment benefit. CONCLUSION: Our results suggest a clinical benefit of continuing αPD-1 therapy in some patients who develop PE. We found that infiltrating T lymphocytes in PE manifest a more exhausted phenotype during αPD-1 therapy than during treatment with other cancer drugs, with subpopulations of these cells characterized by specific immune checkpoint protein and cytokine expression profiles possibly contributing to the antitumor immune response.

Expression of brain-derived neurotrophic factor and its receptor TrkB is associated with poor prognosis and a malignant phenotype in small cell lung cancer.

OBJECTIVES: TrkB is a receptor for brain-derived neurotrophic factor (BDNF) and is highly expressed in various cancers, with BDNF-TrkB signaling having been implicated in tumor progression and metastasis. The role of the BDNF-TrkB system in small cell lung cancer (SCLC), a neuroendocrine cancer, has remained unclear, however. We examined BDNF and TrkB expression in SCLC patients as well as the function of BDNF-TrkB signaling in SCLC cell lines. MATERIALS AND METHODS: BDNF and TrkB expression in tumor specimens of 58 SCLC patients and 20 non-small cell lung cancer (NSCLC) patients was examined by immunohistochemistry and was scored on the basis of the distribution and intensity of staining. TrkB-overexpressing SCLC (SBC5TrkB) cells were established by retrovirus transduction and were examined for the effects of BDNF on intracellular signaling, cell proliferation, and cell migration in vitro. RESULTS: The staining score for TrkB in NSCLC and SCLC specimens was 2.80 ±â€¯0.19 and 3.60 ±â€¯0.15, respectively, whereas that for BDNF was 1.95 ±â€¯0.32 and 2.76 ±â€¯0.14, respectively. High levels of both TrkB and BDNF expression in SCLC tumors were significantly associated with poor overall survival in multivariate analysis (hazard ratio = 1.821, P = 0.036). BDNF activated AKT and ERK signaling pathways in and promoted the migration of SBC5TrkB cells, and these effects were attenuated by the pan-Trk inhibitor GNF-5837. GNF-5837 also inhibited the proliferation of SBC5TrkB cells in the presence of BDNF. CONCLUSION: Coexpression of BDNF and TrkB was associated with poor prognosis in SCLC patients, and BDNF promoted the migration of TrkB-overexpressing SCLC cells. TrkB is thus a potential therapeutic target for SCLC.

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry.

Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.

Evaluation of a glycoengineered monoclonal antibody via LC-MS analysis in combination with multiple enzymatic digestion.

Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.

Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins.

Ultralow molecular weight (ULMW) heparins are sulfated glycans that are clinically used to treat thrombotic disorders. ULMW heparins range from 1500 to 3000 daltons, corresponding from 5 to 10 saccharide units. The commercial drug Arixtra (fondaparinux sodium) is a structurally homogeneous ULMW heparin pentasaccharide that is synthesized through a lengthy chemical process. Here, we report 10- and 12-step chemoenzymatic syntheses of two structurally homogeneous ULMW heparins (MW = 1778.5 and 1816.5) in 45 and 37% overall yield, respectively, starting from a simple disaccharide. These ULMW heparins display excellent in vitro anticoagulant activity and comparable pharmacokinetic properties to Arixtra, as demonstrated in a rabbit model. The chemoenzymatic approach is scalable and shows promise for a more efficient route to synthesize this important class of medicinal agent.

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal.

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.

A potent, water-soluble and photoinducible DNA cross-linking agent.

Water-soluble DNA cross-linking phenol and biphenol derivatives 3 and 6 have been synthesized by a Mannich reaction. Both of them can cross-link DNA by photoactivation using visible light (wavelength > 400 nm). Compound 6 can cross-link DNA at pH 5.0 and 7.7, whereas no cross-link was observed at pH 10.0. Density functional theory (DFT) calculation indicated that 6 displays a twist structure. Therefore, it could bind to DNA naturally and has potent property to cross-link DNA after photoactivation.