Covalent Modification of a Cysteine Residue in the XPB Subunit of The

Angewandte Chemie

DOI: 10.1002/anie.201408817 Covalent Inhibitors Covalent Modification of a Cysteine Residue in the XPB Subunit of the General Transcription Factor TFIIH Through Single Epoxide Cleavage of the Transcription Inhibitor Triptolide** Qing-Li He, Denis V. Titov, Jing Li, Minjia Tan, Zhaohui Ye, Yingming Zhao, Daniel Romo, and Jun O. Liu*

Abstract: Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to Figure 1. Structures of triptolide and triptolide analogues under clinical triptolide, thus validating XPB as the physiologically relevant development. Potential sites of attack by a nucleophile from a protein target of triptolide. Together, these results deepen our under- are marked with red arrows. Sections for which the analogues differ in standing of the interaction between triptolide and XPB and structure from triptolide are highlighted in blue. have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs. (XPB)/ERCC3 subunit of TFIIH as a new molecular target of triptolide.[4] We showed that triptolide forms a covalent Triptolide (1, TPL), a diterpene triepoxide (Figure 1), was complex with XPB and inhibits its DNA-dependent ATPase isolated from Trypterygium Wilfordii Hook F (Lei Gong Teng activity without affecting its DNA helicase activity. or Thunder God Vine), which has been used in traditional Several analogues of triptolide have been developed as Chinese medicine for centuries.[1] Numerous cellular activities potential anticancer and immunosuppressive drug leads have been found for triptolide, including inhibition of the (Figure 1). They include PG490-88 and WilGraf for treating activity of a number of unrelated transcription factors and graft rejection after organ transplantation, LLDT8 for treat- global inhibition of mRNA synthesis.[2] A number of putative ing rheumatoid arthritis, and Minnelide for treating cancer.[5] cellular targets of triptolide have been reported to date. Among these analogues, Minnelide is currently undergoing Among them are the calcium channel polycystin-2, the Phase I clinical trial for cancer.[6] It is noteworthy that all membrane protease ADAM10, the dCTP pyrophosphatase analogues of triptolide in clinical development contain the (DCTPP1), and the kinase-regulating protein TAB1.[3] By intact core structure of triptolide. using a systematic top-down approach with the inhibitory Triptolide is decorated with four potentially reactive effect of triptolide on de novo RNA synthesis as the starting chemical groups that may covalently react with XPB: the point, we recently identified the Xeroderma Pigmentosum B butenolide moiety in the five-membered lactone or one of the

[*] Dr. Q.-L. He,[+] Dr. D. V. Titov,[$] [+] Prof. J. O. Liu [$] Current address: Department of Molecular Biology, Massachusetts Department of Pharmacology, Johns Hopkins School of Medicine General Hospital, Boston, MA 02114 (USA) 725 North Wolfe Street, Hunterian Building, Room 516 [+] These authors contributed equally to this work. Baltimore, MD 21205 (USA) [**] This work was supported in part by a discretionary fund and FAMRI E-mail: [email protected] (J.O.L.), NIGMS training grant (D.T), Robert A. Welch Foundation Dr. J. Li, Prof. D. Romo (A-1280 to D.R.) and NIH (R37 GM 069874 to D.R.), NIH Natural Products LINCHPIN Laboratory and Dept. of Chemistry GM105933 and CA160036 (Y.Z.) and Maryland Stem Cell Research Texas A&M University Fund (2011-MSCRFE-0087 to Z.Y.). Support of the Natural Products P.O. Box 30012, College Station, TX 77843 (USA) LINCHPIN Laboratory from the Vice President for Research, College Prof. M. Tan, Prof. Y. Zhao of Science, and the Department of Chemistry at Texas A&M Ben May Department for Cancer Research, University of Chicago University is gratefully acknowledged. Chicago, IL 60637 (USA) Supporting information for this article (including experimental Prof. Z. Ye details) is available on the WWW under http://dx.doi.org/10.1002/ Institute for Cell Engineering, Johns Hopkins University anie.201408817. School of Medicine, Baltimore, MD 21205 (USA)

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three epoxide groups (Figure 1). There have been disagree- TFIIH, albeit to different degrees (Figure 2b). Among the ments in the literature as to which of the epoxide groups is the four analogues, only 5, which lacks the 12,13-epoxide, most reactive electrophile for thiols. One group reported that completely lost its activity at the highest soluble concentra- the 9,11-epoxide of triptolide is opened by propanethiol to tions, while the remaining analogues retained partial anti- form an adduct at C9 (2; Figure 2a).[7] Years later, another proliferative activity. To further determine the ability of the group reported that the same reaction led to the opening of analogues to covalently bind XPB, we incubated each the 12,13-epoxide of triptolide at the C12 position (3; analogue with purified TFIIH for 2 h before subjecting the Figure 2a).[8] To distinguish between those two alternative incubation mixture to extensive dialysis. Only if an analogue paths, we reacted N-acetyl-l-cysteine methyl ester (10 mm) is covalently bound to XPB, would there be no recovery of

with triptolide (0.1 mm) in a PBS buffer (pH 7.4, 1 mm MgCl2 XPB activity after dialysis. Significant recovery of the ATPase and 1% DMSO) at room temperature for 72 h. Only one activity of XPB was observed with analogue 5, while the product was detected and isolated in 55% yield, with 45% remaining analogues 6–8 retained the ability to covalently unreacted triptolide recovered (Figure S1a in the Supporting bind XPB and caused irreversible inhibition similar to that Information). By means of LC–MS (Figure S1b) and 2D- caused by triptolide (Figure 2c, and Figure S3). Together, NMR (H-H COSY, HMBC, NOE; Figure S2), the structure these observations indicate that only the 12,13-epoxide of of the product was determined to be the C12 adduct of N- triptolide mediates covalent modification of XPB, while the acetyl-l-cysteine methyl ester (4; Figure 2a). These results other two epoxide groups, as well as the butenolide moiety, suggest that the 12,13-epoxide of triptolide is intrinsically the are dispensable for covalent binding of triptolide to XPB. most reactive toward thiols. Having identified the epoxide group in triptolide that is To assess which of the four potentially reactive groups in involved in the covalent modification of XPB, we turned to triptolide is involved in covalent modification of XPB, we the reciprocal question of which amino acid residue in XPB is synthesized four analogues of triptolide in which the bute- covalently modified by triptolide. HeLa cell nuclear extract none unit and each of the three epoxide groups were was incubated with triptolide to allow the formation of the eliminated either individually or in combination (Figure 2b). triptolide–XPB covalent complex, which was then immuno- We then assessed the cellular activity as well as the ability of precipitated using an anti-XPB antibody and subsequently each of the four analogues to covalently bind to XPB by using subjected to SDS-PAGE. The XPB protein band, as judged by a “binding-dialysis-activity recovery” sequence. All four its molecular mass, was excised from the gel and subjected to analogues suffered from a significant loss in activity for in-gel digestion with trypsin. The tryptic peptide mixture was inhibition of cell proliferation and the ATPase activity of extensively analyzed by using high-resolution mass spectrometry. With this approach, 82% coverage of the XPB protein sequence was achieved (Fig- ure S4). All identified tryptic peptides gave the expected molecular masses with one exception. Further MS/MS sequencing of the peptide with a mismatch in molecular mass revealed that it spans residues 335–346 of XPB (335SGVIVLPCGAGK346) and there is a mass shift of + 360.1573 Da for this peptide at Cys342, which corresponds to the exact mass of triptolide (Figure 3a). These results suggest that Cys342 is likely the residue in XPB that is covalently modified by triptolide. Phylogenetic sequence alignment revealed that Cys342 is conserved among eukaryotes but is changed to either a threonine or a valine in Figure 2. The C12,13-epoxide of triptolide (TPL) forms a covelant bond with XPB. A) The C12,13-epoxide various archaeal species (Fig- of triptolide reacts with N-acetyl-l-cysteine methyl ester to form the adduct 4. B) Inhibition of cell ure S5).[9] To verify that Cys342 proliferation and the ATPase activity of TFIIH by triptolide analogues. Mean values SEM from two Æ independent experiments are shown. C) Covalent binding of triptolide analogues to XPB as determined by mediates the covalent binding the lack of recovery of XPB ATPase activity upon dialysis of the analogue–XPB complex. Mean values SD of triptolide to XPB, we Æ from two independent experiments are shown. DMSO= dimethyl sulfoxide. mutated it to Ser, Thr, and

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knock-out of XPB is lethal, we used a combination of CRISPR/Cas9 and piggyBac transposase methods to generate a single-allele knock-in mutant line.[10] We introduced a point mutation (C342T) to one XPB allele with a piggyBac transposon based homologous recombi- nation donor that facilitates foot- print-free gene editing, followed by knock-out of the remaining wild type allele of XPB by using CRISPR/Cas9, thereby generating a HEK293T cell line (T7115) that only expresses the XPB C342T mutant (Figure S13). The knock-out of one XPB allele and mutation of the other (C342T) were verified by PCR/restriction digestion with AflII and sequencing (Figures S9,S10). As expected, the resultant T7115 line had a slower growth rate in comparison with wild-type HEK293T cells as judged by cell density, which likely Figure 3. Triptolide binds covalently to C342 of XPB. A) MS/MS spectrum of a peptide from human reflects the lower enzymatic activity XPB (TFIIH) treated with triptolide. Insets: MS/MS spectrum of m/z 730.8895, which led to the of the XPB C342T mutant (Fig- identification of a mass shift of + 360.1573 Da at the Cys342 residue of 335SGVIVLPCGAGK346. The ure S11). Importantly, the XPB- labels (b) and (y) designate N- and C-terminal fragments of the peptide, respectively. The label D C342T-expressing T7115 line was 3 designates (b) or (y) ions with water and/or ammonia loss. B) [ H]-triptolide does not bind completely resistant to triptolide covalently to recombinant C342A, C342S, or C342T XPB mutant proteins. at concentrations up to 30 mm, while the proliferation of wild-type HEK293T cells was completely Ala. Each of the mutants, as well as the wild-type XPB inhibited at 100 nm (Figure 4b). It is noteworthy that the protein, was produced through baculovirus-driven overex- T7115 cells were still sensitive to two other inhibitors of pression in insect cells, followed by purification. Although transcription, namely actinomycin D and a-amanitin,[11] thus wild-type XPB bound covalently to [3H]-triptolide, none of suggesting that the resistance conferred by the C342T mutant the Cys342 mutants were capable of forming a covalent of XPB is highly specific to triptolide (Figure S12). complex with [3H]-triptolide (Figure 3b). In comparison to The intrinsic electrophilicity of epoxide groups led to an wild-type XPB, all three mutants have lower intrinsic ATPase earlier hypothesis that triptolide likely reacts with thiol activity (Figure S6). When the mutant proteins were assayed groups in enzymes to elicit its antitumor activity.[7] Indeed, in the presence of triptolide, none were inhibited by up to 100 mm of triptolide. These results support the hypothesis that Cys342 is the residue that is covalently modified by triptolide and that this covalent modification is essential for the inhibition of XPB by triptolide. In addition to assessing the intrinsic ATPase activity of the Cys342 mutant XPB proteins, we also purified their corresponding TFIIH complexes (Fig- ure S7). Of the three mutants, the C342T mutant TFIIH complex possessed the highest enzymatic activity and remained resistant to 100 mm triptolide (Figure 4a, and Figure S8). The retention of signifi- Figure 4. The C342T XPB mutant is resistant to triptolide in vitro and confers cant enzymatic activity of the C342T mutant in the high-level triptolide resistance to HET293T cells. A) C342A, C342S, and C342T context of TFIIH and its insensitivity to triptolide XPB mutant TFIIH complexes were resistant to triptolide at concentrations up to 100 mm and C342T-TFIIH has the highest ATPase activity. Mean values SD from offered a precious opportunity to assess the impor- Æ two independent experiments are shown. B) A mutant knock-in cell line (T7115) tance of XPB as a mediator of the effect of triptolide expressing only the C342T XPB mutant is selectively resistant to triptolide in cell on cell proliferation by replacing endogenous wild- proliferation assay. Mean values SEM from four independent experiments are Æ type XPB with this mutant. Since the complete shown.

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several epoxide-containing natural products, such as fuma- toxicity. The identification and verification of the 12,13- gillin, trapoxin B, and E-64, are known to work through epoxide group as the only group involved in the covalent covalent modification of key residues, including cysteine, of modification of XPB suggests that the other epoxide groups target proteins.[12] We showed that the a, b-unsaturated and the butenolide are likely to be dispensable for the lactone, 7,8-epoxide, and 9,11-epoxide groups are all dispen- anticancer activity of triptolide through inhibition of tran- sable for the covalent binding of triptolide to XPB. A 9,11- scriptional initiation. It would thus be logical to design and deoxytriptolide analogue has been previously shown to be synthesize novel triptolide analogues devoid of the other slightly more potent than triptolide for the inhibition of epoxide and the butenolide groups as the next generation of cancer cell proliferation,[13] which is consistent with our triptolide analogues to determine whether they have reduced finding. The identification of the 12,13-epoxide of triptolide toxicity while maintaining their anticancer activity. as the sole functional group required for the covalent modification of XPB suggests that the reaction of the cysteine Received: September 4, 2014 thiol from XPB takes advantage of the intrinsic chemical Revised: October 26, 2014 reactivity of the 12,13-epoxide of triptolide.[7] Published online: December 12, 2014 The identification of Cys342 as the sole residue of XPB to mediate the formation of a covalent complex with triptolide is Keywords: inhibitors · medicinal chemistry · natural products · .target validation · transcription factors in agreement with the earlier hypothesis that triptolide works by modifying cysteine thiols in enzymes.[7] It is interesting to note that the same cysteine residue was also detected by using a clickable iodoacetamide probe in conjunction with mass [1] a) S. M. Kupchan, W. A. Court, R. G. Dailey, Jr., C. J. Gilmore, spectrometry to profile active cysteine residues in proteins.[14] R. F. Bryan, J. Am. Chem. Soc. 1972, 94, 7194 – 7195; b) X.-M. That the same cysteine is not absolutely conserved in all Zhao, Supplement to Materia Medica, Zhang’s Jie Xing Tang Pulishing House, Qian Tang, 1765. organisms suggests that it is not essential for the intrinsic [2] a) D. Qiu, G. Zhao, Y. Aoki, L. Shi, A. Uyei, S. Nazarian, J. C. [9] enzymatic activity of XPB. Indeed, we found that the Ng, P. N. Kao, J. Biol. Chem. 1999, 274, 13443 – 13450; b) S. D. mutation of Cys342 to alanine, serine, or threonine did not Westerheide, T. L. Kawahara, K. Orton, R. I. Morimoto, J. Biol. completely abolish its ATPase activity (Figure 4a, and Chem. 2006, 281, 9616 – 9622; c) K. Y. Lee, W. Chang, D. Qiu, Figures S6,S8). The inability of any of the three cysteine P. N. Kao, G. D. Rosen, J. Biol. Chem. 1999, 274, 13451 – 13455; mutants to form a covalent complex with [3H]-triptolide d) S. J. Leuenroth, C. M. Crews, Cancer Res. 2008, 68, 5257 – 5266; e) C. McCallum, S. Kwon, P. Leavitt, W. Shoop, B. indicates that Cys342 is the only residue that undergoes Michael, T. Felcetto, D. Zaller, E. O’Neill, B. Frantz-Wattley, covalent modification by triptolide. C. Thompson, G. Forrest, E. Carballo-Jane, A. Gurnett, Therapy To date, several putative target proteins of triptolide have 2005, 2, 261 – 273; f) S. Vispe, L. DeVries, L. Creancier, J. Besse, been reported, including polycystin-2, ADAM10, DCTPP1, S. Breand, D. J. Hobson, J. Q. Svejstrup, J. P. Annereau, D. TAB1, and XPB, thus raising the question of which targets are Cussac, C. Dumontet, N. Guilbaud, J. M. Barret, C. Bailly, Mol. physiologically relevant for its antitumor activity.[3] In a recent Cancer Ther. 2009, 8, 2780 – 2790. study to establish an elegant new target validation method [3] a) S. J. Leuenroth, D. Okuhara, J. D. Shotwell, G. S. Markowitz, Z. Yu, S. Somlo, C. M. Crews, Proc. Natl. Acad. Sci. USA 2007, involving a combination of in vitro selection of drug-resistant 104, 4389 – 4394; b) R. Soundararajan, R. Sayat, G. S. Robertson, cancer cell lines followed by next-generation sequencing to P. A. Marignani, Cancer Biol. Ther. 2009, 8, 2054 – 2062; c) T. W. identify responsible mutations, Smurnyy et al. found multiple Corson, H. Cavga, N. Aberle, C. M. Crews, ChemBioChem 2011, triptolide-resistant mutations in XPB (ERCC3) and its 12, 1767 – 1773; d) Y. Lu, Y. Zhang, L. Li, X. Feng, S. Ding, W. partner protein GTF2H4.[15] Importantly, no triptolide-resist- Zheng, J. Li, P. Shen, Chem. Biol. 2014, 21, 246 – 256. ant mutations were found in polycystin-2, ADAM10, [4] D. V. Titov, B. Gilman, Q. L. He, S. Bhat, W. K. Low, Y. Dang, M. DCTPP1, or TAB1. As described above, we took advantage Smeaton, A. L. Demain, P. S. Miller, J. F. Kugel, J. A. Goodrich, J. O. Liu, Nat. Chem. Biol. 2011, 7, 182 – 188. of the triptolide-resistant C342T mutant and generated [5] Z. L. Zhou, Y. X. Yang, J. Ding, Y. C. Li, Z. H. Miao, Nat. Prod. a mutant HEK293T cell line (T7115) that is dependent on Rep. 2012, 29, 457 – 475. a single C342T mutant allele of XPB for growth and survival. [6] R. Chugh, V. Sangwan, S. P. Patil, V. Dudeja, R. K. Dawra, S. The XPB C342T mutant renders the T7115 cell line nearly Banerjee, R. J. Schumacher, B. R. Blazar, G. I. Georg, S. M. completely resistant to triptolide. It is noteworthy that the Vickers, A. K. Saluja, Sci. Transl. Med. 2012, 4, 156ra139. level of resistance conferred by the C342T mutation is [7] S. M. Kupchan, R. M. Schubert, Science 1974, 185, 791 – 793. [8] D. Q. Yu, D. M. Zhang, H. B. Wang, X. T. Liang, Acta Pharma- approximately 100-fold higher than the most triptolide- [15] col. Sin. 1992, 27, 830 – 836. resistant mutants previously identified. Together, these [9] L. Fan, A. S. Arvai, P. K. Cooper, S. Iwai, F. Hanaoka, J. A. results validate XPB as the key target that is responsible for Tainer, Mol. Cell 2006, 22, 27 – 37. the antiprolifeative activity of triptolide. [10] a) K. Yusa, L. Zhou, M. A. Li, A. Bradley, N. L. Craig, Proc. One of the major hurdles for triptolide to becoming Natl. Acad. Sci. USA 2011, 108, 1531 – 1536; b) P. Mali, L. Yang, a clinically useful drug is its toxicity, which has been attributed K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, in part to the presence of multiple reactive electrophiles, G. M. Church, Science 2013, 339, 823 – 826; c) L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. including epoxide groups and the butenolide moiety.[16] Most, Jiang, L. A. Marraffini, F. Zhang, Science 2013, 339, 819 – 823; if not all, analogues of triptolide in preclinical and clinical d) S. M. Choi, Y. Kim, J. S. Shim, J. T. Park, R. H. Wang, S. D. development to date have all three epoxide and the buteno- Leach, J. O. Liu, C. Deng, Z. Ye, Y. Y. Jang, Hepatology 2013, 57, lide groups intact (Figure 1), which likely contributes to their 2458 – 2468.

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[11] a) T. J. Lindell, F. Weinberg, P. W. Morris, R. G. Roeder, W. J. Kembhavi, K. Hanada, Acta Biol. Med. Ger. 1981, 40, 1513 – Rutter, Science 1970, 170, 447 – 449; b) E. Reich, R. M. Franklin, 1517. A. J. Shatkin, E. L. Tatum, Science 1961, 134, 556 – 557. [13] B. Zhou, Z. Miao, G. Deng, J. Ding, Y. Yang, H. Feng, Y. Li, [12] a) E. C. Griffith, Z. Su, B. E. Turk, S. Chen, Y.-W. Chang, Z. Wu, Bioorg. Med. Chem. Lett. 2010, 20, 6217 – 6221. K. Biemann, J. O. Liu, Chem. Biol. 1997, 4, 461 – 471; b) N. Sin, [14] E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare, M. B. L. Meng, M. Q. W. Wang, J. J. Wen, W. G. Bornmann, C. M. Dillon, D. A. Bachovchin, K. Mowen, D. Baker, B. F. Cravatt, Crews, Proc. Natl. Acad. Sci. USA 1997, 94, 6099 – 6103; c) M. Nature 2010, 468, 790 – 795. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, J. Biol. [15] Y. Smurnyy, M. Cai, H. Wu, E. McWhinnie, J. A. Tallarico, Y. Chem. 1993, 268, 22429 – 22435; d) J. Taunton, C. A. Hassig, S. L. Yang, Y. Feng, Nat. Chem. Biol. 2014, 10, 623 – 625. Schreiber, Science 1996, 272, 408 – 411; e) A. J. Barrett, A. A. [16] a) B. J. Chen, Leuk. Lymphoma 2001, 42, 253 – 265; b) F. Du, Z. Liu, X. Li, J. Xing, J. Appl. Toxicol. 2014, 34, 878 – 884.

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69451 Weinheim, Germany

Covalent Modification of a Cysteine Residue in the XPB Subunit of the General Transcription Factor TFIIH Through Single Epoxide Cleavage of the Transcription Inhibitor Triptolide** Qing-Li He, Denis V. Titov, Jing Li, Minjia Tan, Zhaohui Ye, Yingming Zhao, Daniel Romo, and Jun O. Liu* anie_201408817_sm_miscellaneous_information.pdf 1

Experimental Procedures

Synthesis of Triptolide analogs

General'Experimental'!

All!non&aqueous!reactions!were!performed!under!a!nitrogen!atmosphere!in!oven&

dried!glassware.!Toluene,!benzene,!diethyl!ether,!acetonitrile,!and!methylene!chloride!were! purified! by! passing! through! commercially! available! pre&dried,! oxygen&free! formulations! through!activated!alumina!columns.!Tetrahydrofuran!was!freshly!distilled!over!sodium!and! benzophenone.! Methanol! was! freshly! distilled! from! Mg! turnings.! Triethylamine! was! distilled!from!calcium!hydride!and!i&Pr2NEt!was!distilled!from!potassium!hydroxide!prior! to!use.!Yields!refer!to!chromatographically!and!spectroscopically!(1H!NMR)!homogeneous! materials,! unless! otherwise! stated.! Reagents! were! purchased! at! the! highest! commercial! quality! and! used! without! further! purification,! unless! otherwise! stated.! Reactions! were! monitored!by!LC/MS!and!TLC.!LC/MS!was!carried!out!on!an!ion!trap!LC/MS!using!a!C18!50! x! 2.10! mm! 3! micron! column,! eluting! with! a! solvent! gradient! of! 5:95! →! 100! acetonitrile/water! in! 15.8! min,! detecting! with! UV! 250! nm,! PDA! 190&400! nm! and! MS! ion! trap!(ionization!modes!are!APCI!(+)!and!(&)!or!ESI!(+)!and!(&),!scan!range!100&2100).!Thin& layer!chromatography!(TLC)!was!carried!out!on!0.25!mm!silica!gel!plates!(60F&254)!using!

UV!light!as!visualizing!agent!and!an!ethanolic!solution!of!phosphomolybdic!acid!and!cerium! sulfate!or!an!ethanolic!solution!of!para&anisaldehyde!and!heat!as!developing!agents.!Flash! column!chromatography!was!performed!with!60Å!Silica!Gel!(230&400!mesh)!as!stationary! phase! using! a! gradient! solvent! system! (EtOAc/hexanes! as! eluent! unless! indicated! otherwise).! Preparative! thin&layer! chromatography! (PTLC)! separations! were! carried! out! on!0.25!or!0.50!mm!silica!gel!plates!(60F&254).!1H!NMR!chemical!shifts!were!measured!at!

! 2

300!or!500!MHz!and!referenced!relative!to!trace!amounts!of!chloroform!(7.26!ppm)!and!

are!reported!in!parts!per!million.!Coupling!constants!(!J')!are!reported!in!Hertz!(Hz),!with!

multiplicity! reported! following! usual! convention:! s,! singlet;! d,! doublet;! t,! triplet;! q,!

quadruplet;!dd,!doublet!of!doublets;!ddd,!doublet!of!doublet!of!doublets;!dddd,!doublet!of!

doublet!of!doublet!of!doublets;!dt,!doublet!of!triplets;!dq,!doublet!of!quartets;!m,!multiplet;!

br!s,!broad!singlet.!13C!NMR!spectra!were!measured!at!75!MHz!or!125!MHz!and!referenced!

relative!to!residual!chloroform!(77.16!ppm)!and!are!reported!in!parts!per!million!(ppm).!

High! resolution! mass! spectra! (HR&MS)! were! obtained! through! the! Center! for! Chemical!

Characterization! and! Analysis! (Texas! A&M! University)! on! a! mass! spectrometer! using!

MALDI!(Matrix&assisted!laser&desorption!ionization)!or!electrospray!ionization.!!

Synthesis of Analog 4:

O O H O N O OH O O S O OH H PBS(PH=7.4) O 1mM MgCl2 1% DMSO O OH O SH O O H O O N H O 4

Triptolide(15 mg, 0.0416mmol, 0.1mM, 1.0 equiv) was dissolved in DMSO (4.16mL) and PBS

(420ml,including 1mM MgCl2) under argon and to this was added N-acetyl-L-cysteine methyl

ester(0.7375g, 4.16mmol, 10mM, 100.0 equiv). After 72hrs, the reaction mixture was extracted

with ethyl acetate(5X200ml), and the organic layer was washed with brine (1 x 200 mL), dried

over Na2SO4, and concentrated in vacuo. The residue was purified by HPLC (Agilent pursuit

! 3

1 XRs 5 C18 250X10.0mm, 55%CH3CN/H2O) to give Analog 6(12.4mg, 55%). H-

NMR((CD3)2CO,500MHz), δ 4.76(m, 3H), 3.99(d, J=5.5Hz, 1H), 3.73(s, 3H), 3.47(d, J=5.0Hz,

1H), 3.40(d, J=6.0Hz, 1H), 3.31(dd, J=14.0, 5.0Hz, 1H), 3.04(s, 1H), 2.94(dd, J=13.5, 8.0Hz,

1H), 2.85(m, 1H), 2.26(m, 2H), 2.20(m, 1H), 1.95-2.09(m,8H), 1.56(dd, J=12.5, 4.5Hz), 1.39 (m,

13 1H), 1.09(s, 3H), 1.01(d, J=6.5Hz, 3H), 0.84(d, J=6.5Hz, 3H). C-NMR ((CD3)2CO,125MHz),

δ 174.3, 172.6, 162.9, 125.7, 79.1, 77.8, 71.4, 70.8, 62.4, 62.1, 60.0, 53.3, 53.2, 48.2, 41.1, 37.2,

34.7, 31.7, 24.3, 23.3, 23.2, 18.4, 16.8, 16.7, 14.8.

MS (ESI) Calcd for (M+H+) 537.2, Found, 537.3

Synthesis of Analog 5:

O O

O O OH OTMS O O O O H H O O triptolide(1) S13 !

(5bS,6aS,7aR,8R,8aS,9aS,9bS,10aS,10bS)&8a&isopropyl&10b&methyl&8&((trimethylsilyl)oxy)&

1,5,5b,6,6a,8,8a,9a,9b,10b&decahydrotris(oxireno)!

[2',3':4b,5;2'',3'':6,7;2''',3''':8a,9]phenanthro[1,2&c]furan&3(2H)&one (S13):! Triptolide! (1,!

o 20.0!mg,!56!µmol)!was!dissolved!in!dry!CH2Cl2!(0.8!mL)!and!cooled!to!0! C!under!N2!and!

Et3N! (23! µL,! 166! µmol,! 3! equiv)! was! added.! A! solution! of! TMSOTf! in! CH2Cl2! (0.3! mL!

solution!containing!15 µL!of!TMSOTf)!was!added!dropwise!and!the!mixture!was!stirred!at!0!

oC!for!1!h.!Upon!completion!of!the!reaction,!the!mixture!was!quenched!with!PBS!buffer!(0.1!

M,!1!mL),!extracted!with!CH2Cl2!(2!x!10!mL).!The!organic!layer!was!washed!with!brine!(5!

! 4

mL),!dried!over!MgSO4!and!concentrated!to!give!the!crude!material,!which!was!purified!by! flash! chromatography! on! silica! gel! (gradient! elution,! 30→! 40%,! EtOAc/hexane)! to! give! desired!TMS&protected!alcohol!S13!as!a!white!solid!(25!mg,!~100%!yield):! 1H!NMR!(500!

MHz,!CDCl3)!δ!4.66!(d,!J'=!3.0!Hz,!2H),!3.74!(d,!J'=!3.0!Hz,!1H),!3.51!(s,!1H),!3.48!(d,!J'=!3.5!Hz,!

1H),!3.22!(d,!J'=!6.0!Hz,!1H),!2.69&2.65!(m,!1H),!2.31&2.26!(m,!1H),!2.22&2.08!(m,!3H),!1.91!

(dd,!J'=!15.0,!13.5!Hz,!1H),!1.58!(dd,!J'=!13.0,!4.5!Hz,!1H),!1.26&1.23!(m,!1H),!1.21&1.15!(m,!

1H),!1.06!(s,!3H),!0.94!(d,!J'=!7.0!Hz,!3H),!0.82!(d,!J'=!6.5!Hz,!3H),!0.16!(s,!9H);!13C!NMR!(125!

MHz,!CDCl3)!δ!173.5,!160.5,!125.7,!74.1,!70.1,!65.3,!63.8,!61.1,!60.2,!55.1,!55.0,!40.6,!35.9,!

+ 29.6,!27.7,!23.9,!17.6,!17.2,!16.7,!13.6,!0.48!(3C);!HRMS!(ESI+)!calcd.!for!C23H33O6Si!(M+H )!

433.2046,!found!433.2040.!

O O O

O MeO OMe O OTMS 10 eq. N OTMS O 2 O O O Rh (OAc) , benzene, H 2 4 H O reflux O S13 S14 !

(5aS,5bS,6aS,9R,9aR,10aS,11aS)&8&isopropyl&5a&methyl&9&((trimethylsilyl)oxy)&

4,5,5a,6a,9,10a,11,11a&octahydrobis(oxireno)[2',3':4b,5;2'',3'':8a,9]phenanthro[1,2&

c]furan&3(1H)&one!(S14):! Tris&epoxide! S13!(8!mg,!20.4!µmol)!was!mixed!with!Rh2(OAc)4!

catalyst! (0.9! mg,! 2! µmol,! 0.1! equiv),! vacuum! dried! for! 5! min! and! purged! with! N2.! Dry! benzene! (1.3! mL)! was! added! to! give! a! clear! green! solution,! which! was! heated! to! 80! oC.!

Diazo!reagent!(32!mg,!204!µmol,!10!equiv)!was!dissolved!in!anhydrous!benzene!(0.65!mL)!

and! added! slowly! (0.1! mL/h! via! syringe! pump)! to! the! reaction! mixture! at! 80! oC.! Upon! complete!addition,!the!mixture!was!stirred!at!80!oC!for!2!h!before!slowly!being!cooled!down!

! 5

to! ambient! temperature! and! stirred! overnight.! The! mixture! was! filtered! through! a! short!

silica!gel!pad,!rinsed!with!40%!EtOAc/Hexanes!(5!x!1!mL).!The!filtrate!was!concentrated!to!

give!the!crude!material,!which!was!purified!by!flash!chromatography!on!silica!gel!(gradient!

elution,!20→!50%,!EtOAc/hexane)!followed!by!prep.!TLC!purification!(eluent:!2%!MeOH/!

1 CH2Cl2)!to!give!desired!des&epoxy!S14!as!a!colorless!oil!(1.0!mg,!13%!yield):! H!NMR!(500!

MHz,!CDCl3)!δ!5.81!(d,!J'=!4.5!Hz,!1H),!4.68!(br!s,!2H),!3.53!(d,!J'=!4.5!Hz,!1H),!3.51!(s,!1H),!

3.28!(d,!J'=!5.5!Hz,!1H),!2.68!(d,!J'=!13.0!Hz,!1H),!2.34!(p,!J'=!7.0!Hz,!1H),!2.30&2.15!(m,!1H),!

2.17&2.01!(m,!1H),!2.09!(dt,!J'=!15.0,!6.0!Hz,!1H),!1.99!(t,!J'=!13.5!Hz,!1H),!1.71!(dd,!J'=!12.0,!

5.0!Hz,!1H),!1.23&1.18!(m,!1H),!1.13!(s,!3H),!1.08!(d,!J'=!7.0!Hz,!3H),!1.01!(d,!J'=!7.0!Hz,!3H),!

+ 0.16!(s,!9H);!HRMS!(ESI+)!calcd.!for!C23H32O5SiNa!(M+Na )!439.1917,!found!439.1904.!

O O OTMS TBAF/HF O OH O O THF H O O quantitative yield H O S14 5 !

(5aS,5bS,6aS,9R,9aS,10aS,11aS)&9&hydroxy&8&isopropyl&5a&methyl&4,5,5a,6a,9,10a,11,11a&

octahydrobis(oxireno)[2',3':4b,5;2'',3'':8a,9]phenanthro[1,2&c]furan&3(1H)&one!(5):!A!stock!

solution!of!1M!TBAF/HF!in!THF!was!prepared!by!mixing!1M!TBAF/THF!(1!mL)!with!HF/Py!

(70!wt%,!25!µL).!24!µL!of!such!solution!was!further!diluted!in!1!mL!THF!and!0.2!mL!of!

diluted!solution!(containing!4.8!µmol!of!TBAF!and!4.8!µmol!HF)!was!mixed!with!S14!(0.5! mg,! 1.2! µmol).! The! mixture! was! stirred! at! ambient! temperature! for! ~7! h! until! full! consumption! of! starting! material! as! evidenced! by! TLC.! The! reaction! mixture! was! then! extracted!with!CH2Cl2!(5!x!2!mL).!The!organic!layer!was!washed!with!sat.!NH4Cl!(2!mL),!sat.!

! 6

NaHCO3! (2! mL)! and! brine! (2! mL),! dried! over! MgSO4! and! concentrated! to! give! the! crude!

material,!which!was!purified!by!flash!chromatography!on!silica!gel!(gradient!elution,!30→!

70%,!EtOAc/hexane)!to!give!the!desired!des&epoxy!alcohol!5!as!a!colorless!oil!(0.4!mg,!99%!

1 yield):! H!NMR!(500!MHz,!CDCl3)!δ!5.81!(d,!J'=!4.5!Hz,!1H),!4.68!(br!s,!2H),!3.70!(d,!J'=!4.5!Hz,!

1H),!3.40!(d,!J'=!5.5!Hz,!1H),!3.30!(d,!J'=!11.5!Hz,!1H),!2.69&2.64!(m,!1H),!2.52!(d,!J'=!11.5!Hz,!

1H),!2.43!(p,!J'=!7.0!Hz,!1H),!2.36&2.32!(m,!1H),!2.18&2.13!(m,!2H),!2.03!(t,!J'=!13.5!Hz,!1H),!

1.67!(dd,!J'=!12.0,!5.0!Hz,!1H),!1.28&1.22!(m,!1H),!1.18!(s,!3H),!1.10!(d,!J'=!7.0!Hz,!3H),!1.04!(d,!

+ J'=!7.0!Hz,!3H)!!;!HRMS!(ESI+)!calcd.!for!C20H25O5!(M+H )!345.1702,!found!345.1715.!

Synthesis of Analog 6:

O O

O O SOCl , Et N OAc 2 3 OAc OH O CH2Cl2, 0°C O H 84% H O O S11 S12 !

(5bS,8S,8aR,9aS,9bS,10aR,10bS)&8a&isopropyl&10b&methyl&3&oxo&2,3,5,5b,6,8,8a,9a,9b,10b& decahydro&1H&bis(oxireno)[2',3':4b,5;2'',3'':6,7]phenanthro[1,2&c]furan&8&yl!acetate!(S12):!

o Acetate!S11!(2.0!mg,!5.0!µmol)!was!dissolved!in!dry!CH2Cl2!(0.3!mL)!and!cooled!to!0! C!and! then!Et3N!(21!µL,!149!µmol,!30!equiv)!was!added.!A!stock!solution!of!SOCl2!in!CH2Cl2!was! prepared!by!mixing!SOCl2!(18!µL)!in!CH2Cl2!(1!mL)!and!100!µL!of!this!solution!(containing!

o 1.8 µL!of!SOCl2,!25!µmol,!5!equiv)!was!added!to!the!reaction!mixture!at!0! C.!The!mixture!

was!stirred!at!0!oC!for!1!h.!Upon!completion!of!the!reaction,!the!mixture!was!quenched!with!

sat.! NaHCO3! solution! (2! mL),! extracted! with! CH2Cl2! (4! x! 2! mL).! The! organic! layer! was!

! 7

washed!with!brine!(2!mL),!dried!over!MgSO4!and!concentrated!to!give!the!crude!material,! which! was! purified! by! flash! chromatography! on! silica! gel! (gradient! elution,! 20→! 66%,!

EtOAc/hexane)!to!give!desired!alkene!S12!as!a!colorless!oil!(1.6!mg,!84%!yield):! 1H!NMR!

(500!MHz,!CDCl3)!δ!6.32!(dd,!J'=!5.5,!1.5!Hz,!1H),!5.76!(s,!1H),!4.71!(d,!J'=!19.0!Hz,!1H),!4.67!

(d,!J'=!18.5!Hz,!1H),!3.72!(d,!J'=!3.0!Hz,!1H),!3.55!(d,!J'=!3.5!Hz,!1H),!2.78!(d,!J'=!12.0!Hz,!1H),!

2.37!(d,!J'=!16.0!Hz,!1H),!2.27!(dt,!J'=!17.5,!5.5!Hz,!1H),!2.19&2.09!(m,!2H),!2.11!(s,!3H),!1.98!

(p,!J'=!7.0!Hz,!1H),!1.64!(dd,!J'=!13.0,!5.0!Hz,!1H),!1.30!(dt,!J'=!12.5,!6.5!Hz,!1H),!0.99!(d,!J'=!

+ 7.0!Hz,!3H),!0.90!(s,!3H),!!0.88!(d,!J'=!7.0!Hz,!3H)!!!;!HRMS!(ESI+)!calcd.!for!C22H27O6!(M+H )!

387.1808,!found!387.1815.!

O O

O O HOCH2CH2NH2 OAc OH O Et3N, MeOH, 23°C H O 85% H O O S12 6 !

(5bS,8S,8aR,9aS,9bS,10aR,10bS)&8&hydroxy&8a&isopropyl&10b&methyl&

5,5b,6,8,8a,9a,9b,10b&octahydro&1H&bis(oxireno)[2',3':4b,5;2'',3'':6,7]phenanthro[1,2&

c]furan&3(2H)&one!(6):! Alkene!S12!(4.6!mg,!11.9!µmol)!was!dissolved!in!MeOH!(0.3!mL)!

and!mixed!with!Et3N!(100!µL,!717!µmol,!60!equiv).!2&hydroxylethylamine!(120!µL,!1994!

µmol,!168!equiv)!was!added!to!the!reaction!mixture!and!the!resulting!solution!was!stirred!

at! ambient! temperature! for! 24! h.! Upon! completion! of! the! reaction,! the! mixture! was! extracted!with!CH2Cl2!(3!x!5!mL).!The!organic!layer!was!washed!with!sat.!NH4Cl!solution!(2!

mL),!brine!(2!mL),!dried!over!MgSO4!and!concentrated!to!give!the!crude!material,!which! was! purified! by! flash! chromatography! on! silica! gel! (gradient! elution,! 20→! 66%,!

! 8

EtOAc/hexane)!to!give!desired!alcohol!6!as!a!white!solid!(2.6!mg,!63%!yield):!1H!NMR!(500!

MHz,!CDCl3)!δ!6.06!(dd,!J'=!5.5,!2.0!Hz,!1H),!4.71!(br!s,!2H),!4.34!(dd,!J'=!12.0,!1.0!Hz,!1H),!

3.83!(d,!J'=!3.0!Hz,!1H),!3.52!(dd,!J'=!3.0,!1.0!Hz,!1H),!2.89!(d,!J'=!12.0!Hz,!1H),!2.81!(d,!J'=!

12.0!Hz,!1H),!2.39!(ddd,!J'=!18.0,!4.0,!2.0!Hz,!1H),!2.29!(dd,!J'=!12.5,!5.0!Hz,!1H),!2.27&2.25!(m,!

1H),!2.21&2.12!(m,!2H),!1.61!(dd,!J'=!12.0,!5.0!Hz,!1H),!1.30!(dt,!J'=!12.0,!6.0!Hz,!1H),!1.05!(d,!

J'=!7.0!Hz,!3H),!0.98!(s,!3H),!!0.91!(d,!J'=!7.0!Hz,!3H)!!!;!HRMS!(ESI+)!calcd.!for!C20H25O5!

(M+H+)!345.1702,!found!345.1691.!

Synthesis of Analog 7:

O O

O O LiBH4, BF3OEt2 OH OH O OH O 87% O H H O O S10 triptolide(1)

(5bS,7aR,8S,8aR,9aS,9bS,10aS,10bS)&7a,8&dihydroxy&8a&isopropyl&10b&methyl&

5,5b,6,7,7a,8,8a,9a,9b,10b&decahydro&1H&bis(oxireno)[2',3':4b,5;2'',3'':6,7]! phenanthro[1,2&c]furan&3(2H)&one!(S10):!To!a!solution!of!triptolide!(1,!9!mg,!28!µmol)!in! dry!THF!(2!mL)!was!added!a!LiBH4!solution!(2!N,!28!µL,!56!µmol)!followed!by!BF3•Et2O!

(neat!solution,!7!µL,!56!µmol).!The!mixture!was!stirred!under!N2!at!ambient!temperature!

for!3!h.!!Upon!completion!of!the!reaction,!the!mixture!was!quenched!with!aq.!HCl!solution!

(1!N,!1!mL)!and!stirred!for!additional!10!min.!The!mixture!was!extracted!with!CH2Cl2!(3!x!5! mL),! the! organic! layer! was! washed! with! NaHCO3! solution,! brine,! dried! over! MgSO4! and! concentrated.! The! residue! was! purified! by! flash! chromatography! on! silica! gel! (gradient! elution,!20→!66%,!EtOAc/hexane)!to!give!diol!S10!as!a!colorless!oil!(7.1!mg,!78%!yield):!1H!

! 9

NMR!(500!MHz,!CDCl3)!δ!4.76&4.67!(m,!2H),!3.84!(d,!J'=!2.0!Hz,!1H),!3.83!(d,!J'=!3.5!Hz,!1H),!

3.55!(d,!J'=!12.0!Hz,!1H),!3.44!(dd,!J'=!3.0,!1.0!Hz,!1H),!2.57!(d,!J'=!12.5!Hz,!1H),!2.53&2.50!(m,!

1H),!2.36&2.19!(m,!3H),!2.15!(tq,!J'=!13.0,!2.0!Hz,!1H),!1.96!(dq,!J'=!13.0,!3.5!Hz,!1H),!1.67&

1.57!(m,!3H),!1.28!(dt,!J'=!11.5,!7.0!Hz,!1H),!1.23!(s,!3H),!1.03!(d,!J'=!7.0!Hz,!3H),!0.85!(d,!J'=!

+ 7.0!Hz,!3H)!!!;!HRMS!(ESI+)!calcd.!for!C20H27O6!(M+H )!363.1808,!found!363.1815.!

! (5bS,7aR,8S,8aS,9aS,9bS,10aS,10bS)&7a&hydroxy&8a&isopropyl&10b&methyl&3&oxo&

2,3,5,5b,6,7,7a,8,8a,9a,9b,10b&dodecahydro&1H&bis(oxireno)[2',3':4b,5;2'',3'':6,7]! phenanthro[1,2&c]furan&8&yl! acetate (S11):! A! stock! solution! of! DMAP! in! CH2Cl2! was! prepared! by! dissolving! 2! mg! DMAP! in! 16! mL! of! CH2Cl2.! 0.8! mL! of! this! DMAP/CH2Cl2!

solution!was!then!added!to!S10!(7.0!mg,!25!µmol)!to!give!a!clear!solution,!to!which!Ac2O!

(4.7!µL,!50!µmol)!and!Et3N!(14!µL,!100!µmol)!were!added!respectively.!The!mixture!was!

stirred! at! ambient! temperature! for! 24! h! to! give! 70%! conversion.! The! mixture! was! then!

concentrated!and!the!residue!was!purified!by!flash!chromatography!on!silica!gel!(gradient!

elution,!20→!66%,!EtOAc/hexane)!to!give!acetate!S11!as!colorless!oil!(5.0!mg,!63%!yield):!

1 H!NMR!(500!MHz,!CDCl3)!δ!5.20!(s,!1H),!4.71!(dd,!J'=!28.0,!17.0!Hz,!2H),!3.83!(d,!J'=!3.0!Hz,!

1H),!3.44!(d,!J'=!3.0!Hz,!1H),!2.54!(d,!J'=!12.5!Hz,!1H),!2.35&2.32!(m,!1H),!2.27&2.23!(m,!1H),!

2.26!(d,!J'=!2.0!Hz,!1H),!2.20!(s,!3H),!2.12&2.05!(m,!1H),!1.95&1.83!(m,!2H),!1.61&1.54!(m,!3H),!

1.31!(dt,!J'=!12.0,!6.5!Hz,!1H),!1.17!(s,!3H),!0.99!(d,!J'=!7.0!Hz,!3H),!0.79!(d,!J'=!7.0!Hz,!3H)!!!;!

+ HRMS!(ESI+)!calcd.!for!C22H29O7!(M+H )!405.1913,!found!405.1924.!

! 10

O O O Cl Cl O SOCl2, Et3N OAc OAc + OH OAc CH2Cl2, 23°C O O Cl O Cl H H H O O O S11 S9, 35% yield 7, 53% yield !

Acetate!11!(1.6!mg,!4.4!µmol)!was!dissolved!in!dry!CH2Cl2!(0.4!mL)!and!Et3N!(58!µL,!417!

µmol,! 95! equiv)! was! added.! A! stock! solution! of! SOCl2! in! CH2Cl2! was! prepared! by! mixing!

SOCl2!(65!µL)!in!CH2Cl2!!(1!mL)!and!100!µL!of!this!solution!(containing!6.5 µL!of!SOCl2,!88.8!

µmol,!20!equiv)!was!added!to!the!reaction!mixture!and!stirred!at!ambient!temperature!for!

20!min.!Upon!completion!of!the!reaction!as!determined!by!TLC,!the!mixture!was!quenched! with!sat.!NaHCO3!solution!(1!mL),!extracted!with!CH2Cl2!(2!x!5!mL).!The!organic!layer!was! concentrated,!and!the!residue!was!purified!by!flash!chromatography!on!silica!gel!(gradient! elution,! 20→! 66%,! EtOAc/hexane)! to! give! dichloride! 9! (0.6! mg,! 35%! yield)! and! diastereomeric!dichloride!7!(0.9!mg,!53%!yield)!both!as!colorless!oils.!

!(3bR,5S,6S,6aR,7aR,8S,8bS)&5,8&dichloro&6a&isopropyl&8b&methyl&1&oxo&

1,3,3b,4,5,6,6a,7a,8,8b,9,10&dodecahydrooxireno[2',3':6,7]phenanthro[1,2&c]furan&6&yl!

1 acetate!(S9):! H!NMR!(500!MHz,!CDCl3)!δ!6.04!(s,!1H),!4.97!(d,!J'=!3.0!Hz,!1H),!4.80!(d,!J'=!3.5!

Hz,!1H),!4.80!(d,!J'=!17.5!Hz,!1H),!4.65!(d,!J'=!18.0!Hz,!1H),!3.53!(br!s,!1H),!3.08!(d,!J'=!12.5!

Hz,!1H),!2.52!(d,!J'=!15.5!Hz,!1H),!2.36&2.33!(m,!1H),!2.28!(dt,!J'=!14.5,!5.0!Hz,!1H),!2.18&2.12!

(m,!2H),!2.14!(s,!3H),!2.11&2.05!(m,!1H),!1.63&1.57!(m,!1H),!1.15!(s,!3H)!1.05!(d,!J'=!7.0!Hz,!

! 11

+ 3H),!0.95!(d,!J'=!7.0!Hz,!3H)!!!;!HRMS!(ESI+)!calcd.!for!C22H27Cl2O5!(M+H )!441.1236,!found!

441.1250.!

5,8&dichloro&6a&isopropyl&8b&methyl&1&oxo&!

1,3,3b,4,5,6,6a,7a,8,8b,9,10&dodecahydrooxireno[2',3':6,7]phenanthro[1,2&c]furan&6&yl!

1 acetate!(7) (stereochemistry at C7,C11 unconfirmed):! H!NMR!(500!MHz,!CDCl3)!δ!5.66!(s,!1H),!

4.93!(d,!J'=!4.5!Hz,!1H),!4.81!(d,!J'=!16.5!Hz,!1H),!4.70!(d,!J'=!4.0!Hz,!1H),!4.65!(d,!J'=!17.0!Hz,!

1H),!3.52!(d,!J'=!4.5!Hz,!1H),!3.30!(d,!J'=!12.5!Hz,!1H),!2.53!(d,!J'=!20.5!Hz,!1H),!2.32!(dd,!J'=!

13.0,!6.0!Hz,!1H),!2.21!(dt,!J'=!13.0,!4.5!Hz,!1H),!2.17!(s,!3H),!2.26&2.14!(m,!2H),!2.07&2.02!(m,!

2H),!0.97!(d,!J'=!7.0!Hz,!3H),!0.96!(s,!3H),!0.88!(d,!J'=!7.0!Hz,!3H);!HRMS!(ESI+)!calcd.!for!

+ C22H27Cl2O5!(M+H )!441.1236,!found!441.1224.!!

Synthesis of Analog 8 (Furan-Triptolide):

O O Dibal-H (4.5 equiv), Me O CH2Cl2, -78 , 10 min, Me O OH OH O O O H H O O 8

Triptolide (20 mg, 0.0555mmol, 1.0 equiv) was dissolved in anhydrous CH2Cl2 (2mL) and

cooled to -78 °C under argon and to this was added Dibal-H (1.0 M in Hexanes, 250µL,

0.250mmol, 4.5 equiv) dropwise. The solution was stirred for an additional 10 minutes at -78 °C,

then the reaction mixture was partitioned between EtOAc(30 mL) and 1M Rochelle’s salt (20

mL). The biphasic mixture was warmed to room temperature and stirred vigorously for 1 hr, and

the aqueous layers were separated and the organic layer was washed with H2O (3 x 10 mL) and

! 12

brine (1 x 15 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash chromatography (Hexanes-20% EtOAc/Hexanes) to give Analog 2 (7.5mg, 39%). 1H-NMR

(CDCl3,400MHz) δ 7.14(S, 2H), 3.93(d,J=2.8Hz,1H), 3.53(d, J=2.4Hz,1H), 3.41(d,

J=10.8Hz,1H ),3.36(d,J=5.2Hz,1H), 2.82(d, J=10.8Hz, 1H), 2.70(dd, J=12.0, 8.0Hz, 1H),

2.58(dd, J=16.0, 8.0Hz, 1H), 2.44 (m, 1H), 2.28 (m,1H), 2.00 (m,1H), 1.47 (dd, J=12.0,4.0Hz,

13 1H),1.19(m, 1H),1.10(s,3H), 1.02(d, J=6.8Hz, 3H), 0.89(d, J=6.8Hz, 3H). C-NMR(CDCl3,

100MHz), δ 137.7, 137.4, 124.3, 119.0, 73.8, 67.0, 65.7, 60.7, 60.6, 56.9, 54.7, 37.6, 35.9, 31.0,

28.0, 26.1, 17.7, 16.8, 15.5, 12.9. MS (ESI) Calcd. for (M+H+) 345.2, Found, 345.2

Materials. Triptolide was purchased from various commercial sources including Sigma, and 3H-

triptolide was prepared by AmBios Labs, Inc. with specific activity of 4.0 Ci/mmol. General

chemicals were purchased from Sigma and Fisher. 32γ-ATP, [3H]-thymidine and [3H]-uridine

were purchased from Perkin Elmer.

Cell proliferation assay. HEK293T cells were seeded at 10,000 cells per well in 96-well plates

0 and cultured in DMEM plus 10% FBS at 37 C with 5% CO2. Twenty four hours after seeding, drugs were added at indicated concentrations and incubation was continued for an additional 24 h (unless indicated otherwise). An aliquot of 1 µCi of [3H]-thymidine (Perkin Elmer) was added per well and incubation was continued for an additional 6 h (unless indicated otherwise). Cells were harvested onto a Printed Filtermat A glass fiber filter (Perkin Elmer) using Tomtec

Harvester 96 Mach III and filters were immersed in Betaplate Scint (Perkin Elmer) scintillation fluid, followed by scintillation counting on 1450 Microbeta JET (Perkin Elmer).

! 13

Determination of triptolide modification site on XPB. Six hundred microliters of HeLa

nuclear extract containing 4 mg of total protein was incubated with 10 µM triptolide in 1.2 ml of

10 mM HEPES (pH 8.0), 50 mM KCl, 5 mM MgCl2, 10% glycerol, 0.1 mM EDTA, 0.5 mM

DTT and 50 µM PMSF for 1 h at 30 0C. Fifteen micrograms (15 µl) of affinity purified rabbit anti-XPB antibody (A301-337A, Bethyl Laboratories) was added and the mixture was incubated for 1 hour at 4 0C. The mixture was added to 100 µl of Dynabeads Protein A (100.01D,

Invitrogen, storage solution removed) and further mixed by rotation for 15 min at room

temperature. The supernatant was aspirated and the beads were washed 3 times with 500 µl

PBST and one time with 500 µl PBS. The beads were resuspended in 40 µl of sample buffer,

boiled for 5 min and subjected to SDS-PAGE. The protein band of interest was exercised and in-

gel digested. The resulting tryptic digest was dissolved in 3 µL of HPLC solvent A (0.1% formic

acid in water, v/v) and injected into an Eksigent NanoLC-1D plus HPLC system (Eksigent

Technologies) equipped with an in-house packed 360 µm OD × 75 µm ID reversed phase C12

column. Peptides were eluted from 5% to 70% HPLC solvent B (0.1% formic acid in acetonitrile,

v/v) in solvent A at a flow rate of 200 nl/min using a 2-hour cycle. Mass spectrometric analysis

was performed on a LTQ Orbitrap Velos mass spectrometer (ThermoFisher Scientific). Precursor

ion masses were acquired in the Orbitrap mass spectrometer with a resolution of 60,000 at m/z

400, followed by data-dependent MS/MS fragmentation of the 20 most intense precursor ions in

the linear ion trap mass spectrometer. PTMap software was used for unrestrictive protein post-

translational modification analysis.5

! 14

[3H]-triptolide binding to recombinant his-XPB or TFIIH. Wild type or mutant his-XPB (469

ng) was incubated with or without 50 µM cold triptolide in 40 µl binding buffer [20 mM Tris

(pH 7.9), 4 mM MgCl2, 1 µM ATP, 100 µg/ml BSA, and 100 nM Adenosine Major Late

Promoter (AdMLP)] for 1 h at 30 0C. One micromole per liter of [3H]-triptolide (4 Ci/mmol) was

added and the mixture was incubated for 1 h 30 min at 300C. Samples were boiled in sample buffer and subjected to 12% SDS-PAGE. Wild type or mutant TFIIH (70 nM) was incubated with or without 200 µM cold triptolide in 30 µl binding buffer [20 mM Hepes (pH 7.9), 4 mM

MgCl2, 1 mM TCEP, indicated concentrations of ATP and AdMLP for 1 h at RT. One

micromole per liter of [3H]-triptolide (4 Ci/mmol) was added and the mixture was incubated for

1 h 30 min at RT. Samples were boiled in sample buffer and subjected to 8% Phosphate SDS-

PAGE. After electrophoresis, the gel was soaked in En3hance solution (Perkin Elmer) according

to manufacturer instructions and exposed to pre-flashed x-ray film for 2 or 3 weeks prior to

development.

Binding-dialysis-activity recovery assay. A 50-µl reaction mixture including 20 mM Hepes

(pH 7.9), 4 mM MgCl2, 1mM TCEP, 1mg/ml BSA, 20 nM TFIIH and 10/100 µM triptolide or its analogs was incubated for 2 h at RT. Then the reactions were dialyzed in 2 x 250ml dialysis buffer [20 mM Hepes (pH 7.9), 4 mM MgCl2] overnight (Slide-A-Lyzer MINI Dialysis Units,

Thermo, 10000MWCO, No.69570). The resultant reaction was diluted to 10 nM TFIIH to detect

their ATPase activities by adding 200 nM AdMLP, 1mM TCEP and 1 µM [32P]- γ-ATP as

previously reported.1

! 15

Protein expression and purification. The full length human XPB (wild type, C342A, C342S or

C342T XPB) was PCR amplified using the following primers(Table S1): XPB-for: TATTCT

AGA GCC ACC ATG GGC AAA AGA GAC CGA G, XPB-rev: TAT ATA CTC GAG TTT

CCT AAA GCG CTT GAA GAG. Wild type and mutant his-XPB proteins were purified from

baculovirus driven insect cells as previously described.1 Wild type TFIIH(XPB with STAP tag)

and mutant C342 TFIIH were over-expressed in HEK293T as previously described, then the cell

lysates was incubated with IgG beads for 2hrs and digested with TEV protease overnight at 4°C,

and the resulting supernatant were incubated with Steptavidin beads, then washed with 2mM

biotin to get the proteins.

Radioactivity based TFIIH and XPB ATPase assay. The DNA-dependent ATPase assay was

performed as previously described.1 Briefly, a 10-µl reaction mixture contained 20 mM Tris (pH

32 7.9), 4 mM MgCl2, indicated concentration of ATP, 1 µCi [γ- P]ATP (3000 Ci/mmol), 100

µg/ml BSA, indicated concentration of AdMLP, indicated concentration of TFIIH or his-XPB and indicated concentrations of triptolide. The reactions were started by either addition of

TFIIH/his-XPB or ATP and incubated at 37 0C for indicated time. The reactions were stopped by

addition of 2 µl of 0.5 M EDTA and dilution up to 100 µl with TE buffer. An aliquot of 1 µl

reaction mixture was spotted on PEI-cellulose and the chromatogram was developed with 0.5 M

LiCl and 1 M HCOOH. The percent of ATP hydrolysis was quantified using a PhosphorImager.

Knock-in C342T XPB in HEK293T cell line.2-4 The overall workflow is outlined in Figure

S13. A gRNA cloning vector and Cas9-D10A were purchased from Addgene (Plasmid 41824:

gRNA_Cloning Vector, Plasmid 41816: hCas9_D10A). pCMV-hyPBase and pMCS-AAT-

! 16

PB:PGKpuroΔtk were from Wellcome Trust Sanger Institute. In-fusion HD cloning kit (Cat No.

639648) was from Agilent. Primers (Supplementary Table S2) were synthesized by IDT. The

gRNA-XPB plasmid was constructed as previously described.3 Briefly, the XPB-gRNA-for and

XPB-gRNA-rev primers were annealed and extended by AccuPrime Pfx DNA polymerase

(Invitrogen), then the 100bp fragment was cloned into Afl II linearized gRNA cloning vector using In-fusion HD cloning kit. XPB right arm fragments was obtained by XPB-R-F and XPB-

R-R primers and cloned into pGEM-T vector, resulting the pGEM-XPB-R. The pGEM-XPB-R was mutated by XPB-mutant-for and XPB-mutant-rev primers to get pGEM-XPB-R-Mutant in which Cys342(TGC) was mutated to Thr342(ACC). Puromycin resistance gene, XPB left arm and right mutant arm fragments were obtained separately using Puro-for, Puro-rev, XPB-L-F,

XPB-L-R, XPB-R-F and XPB-R-R primers from pMCS-AAT-PB:PGKpuroΔtk, genomic DNA and pGEM-XPB-R-Mutant, and cloned into EcoR V linearized pBluscript SK plasmid using In- fusion HD cloning kit to get donor-XPB plasmid.

100000 HEK293T cells in 24-wells plate were transfected with 1 µg Cas9-D10A plasmid, 1µg gRNA-XPB and 1 µg donor-XPB plasmid using lipofectamine 2000 as per manufacturer’s instructions. Three days later, the cells were screened with 0.5 µg/ml puromycin. 7-10 days later, single clone was picked and cultured for another one or two weeks. Then total DNA was extracted and the clones were screened using PCR. Primers Endo-XPB-for2 and Endo-XPB-rev were used to confirm that one XPB allele was disrupted, and primers HR-Puro-for and Endo-

XPB-rev were used to show that the puromycin gene was inserted in the correct position in the

HEK293T genome. Disruption of one XPB allele in clone T7 was further confirmed by southern blot (Figure S9a).

! 17

For transposon excision, cell line T7 was transfected with pCMV-hyPBase and cultured for 4 days. Cells were then seeded at one cell per well in 96-wells plate. After one week, the clones were split into another 96-wells plate, and screened with 0.5µg/ml puromycin. Puromycin sensitive clone T71 was cultured sequentially and the total DNA was extracted. Primers HR-

Puro-for and Endo-XPB-rev were used to show that the puromycin gene was removed from the

HEK293T genome. Primers Endo-XPB-for2 and Endo-XPB-rev were used to confirm that one

XPB copy was wild type and one XPB copy was C342T-XPB knock-in. The resultant PCR products were digested with AflII. Wild type XPB(1266bp) can’t be digested by Afl II, but the

Knock-in C342T-XPB(1266bp) can be digested into 260bp and 1006bp fragment (Figure S9b).

100000 T71 cells per well of 24-well plate were transfected with 1 µg Cas9 plasmid, 1µg gRNA-

XPB and 1 µg donor-XPB plasmid using lipofectamine 2000 as per manufacturer’s instructions.

Three days later, the cells were screened with 0.5 µg/ml puromycin. 7-10 days later, single clone was picked and cultured for another one or two weeks. Then total DNA was extracted and the clones were screened using PCR. Primers HR-Puro-for and Endo-XPB-rev were used to show that the puromycin gene was inserted in the correct position in the HEK293T genome. Primers

Endo-XPB-for2 and Endo-XPB-rev were used to confirm that one XPB allele was disrupted, then the PCR products were digested with Afl II. If the PCR products can’t be digested, it means that the cell clone only has one wild type XPB allele. Otherwise, the PCR products can be digested into 260bp and 1006bp fragment, it means that the cell clone only has the C342T-XPB knock-in cell line (Figure S9C, T7115). The PCR products were sequenced to confirm that the

T7115 clone has the desired XPB configuration (Figure S10).

! 18

Table S1. XPB Mutant Primers

Primer Name Sequence

XPB-C342A -for GTC ATT GTT CTT CCC GCT GGT GCT GGA AAG TCC

XPB-C342A -rev GGA CTT TCC AGC ACC AGC GGG AAG AAC AAT GAC

XPB-C342S -for GTC ATT GTT CTT CCC TCC GGT GCT GGA AAG TCC

XPB-C342S -rev GGA CTT TCC AGC ACC GGA GGG AAG AAC AAT GAC

XPB-C342T-for GTC ATT GTT CTT CCC ACC GGT GCT GGA AAG TCC

XPB-C342T-rev GGA CTT TCC AGC ACC GGT GGG AAG AAC AAT GAC

XPB-for: TAT TCT AGA GCC ACC ATG GGC AAA AGA GAC CGA G

XPB-rev: TAT ATA CTC GAG TTT CCT AAA GCG CTT GAA GAG

! 19

Table S2. XPB-C342T knock-in primers used in this work with a combination of CRISPR- Cas and piggyBac transposase technology

Primer name Sequence XPB-gRNA- TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAAACGGGCGTGCACGTTCG for: XPB-gRNA- GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC CGAACGTGCACGCCCGTTTC

rev: XPB-mutant- GGTCATTGTTCTTCCCACCGgtaagtggtaccag

for: XPB-mutnat- ctggtaccacttacCGGTGGGAAGAACAATGACC

rev: XPB-L-F: GTATCGATAAGCTTGAT tggttctaccctttacaacttc

XPB-L-R: GCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAAGGTCAATGTTGATATCAGGG

XPB-R-F: CGTACGTCACAATATGATTATCTTTCTAGGGTTAAGCCCACAGCTGTCCTCAG

XPB-R-R: GGCTGCAGGAATTCGATccattaaacagagatccttcc

Puro-for: AGTCTGCGTAAAATTGACGC

Puro-rev: GATAATCATATTGTGACGTACG

HR-Puro-for: CGTCAATTTTACGCATGATTATCTTTAAC

Endo-XPB-for- ctttatcccggttgttgactgagca

2: Endo-XPB-rev: tcggcaaaagaccactattattattacaca

! 20

Triptolide*(1)*

O OH O O H O C20H24O6 Exact Mass: 360.1573 Mol. Wt.: 360.401 *

DAD1 B, Sig=218,4 Ref=360,100 (QLI\QH2014-10-12 2014-10-12 17-38-35\1AC-0101.D) 1 400 DMSO HPLC 200

0 2.5 5 7.5 10 12.5 15 17.5 20 MSD1 TIC, MS File (D:\DATA\QLI\QH2014-10-12 2014-10-12 17-38-35\1AC-0101.D) ES-API, Pos, Scan, Frag: 200, "POS" 3000000 2500000 TIC 2000000 1500000 1000000 500000 0 0 2.5 5 7.5 10 12.5 15 17.5 20 PMP1, PMP1D, Solvent Ratio B

Solvent ratio 60

0 2.5 5 7.5 10 12.5 15 17.5 20 *

* 21

Major*Peak*(positive)*

*MSD1 SPC, time=13.448 of D:\DATA\QLI\QH2014-10-12 2014-10-12 17-38-35\1AC-0101.D ES-API, Pos, Scan, Frag: 200, "POS

100 Max: 148672 383.0

60 361.1

20 362.1 744.1 721.1 0

200 400 600 800 1000 *

* 22

Analog*4*

O H N O O S OH

O OH O O H O C26H35NO9S Exact Mass: 537.2033 Mol. Wt.: 537.6224 *

DAD1 B, Sig=218,4 Ref=360,100 (QLI\QH2014-10-12 2014-10-12 17-38-35\1AD-0201.D) DMSO HPLC 4 400 300 200 100 0 2.5 5 7.5 10 12.5 15 17.5 20 MSD1 TIC, MS File (D:\DATA\QLI\QH2014-10-12 2014-10-12 17-38-35\1AD-0201.D) ES-API, Pos, Scan, Frag: 200, "POS" TIC 2500000 2000000 1500000 1000000 500000 0 2.5 5 7.5 10 12.5 15 17.5 20 PMP1, PMP1D, Solvent Ratio B

Solvent ratio 60

2.5 5 7.5 10 12.5 15 17.5 20 * 23

*********Major*Peak*(positive)*

*MSD1 SPC, time=10.904 of D:\DATA\QLI\QH2014-10-12 2014-10-12 17-38-35\1AD-0201.D ES-API, Pos, Scan, Frag: 200, "POS

100 Max: 113176 560.1

40 561.1

20 144.1 562.1 502.1 176.0 538.2 343.1 0

200 400 600 800 1000 * 24

Analog*5:*

OH O

O H O

Chemical Formula: C20H24O5 Exact Mass: 344.1624 Molecular Weight: 344.4070 *

Intens . JL-441-A.D: TIC +All MS x107

4 2 0 Intens . JL-441-A.D: TIC -All MS x107 2

0 Intens . JL-441-A.D: UV Chromatogram, 208-212 nm [mAU]

500

0 Intens . JL-441-A.D: EIC 345.0 +All MS x107

0.5

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Time [min] *

Major*peaks*(+*mode):*

200 225 250 275 300 325 350 375 Wavelength [nm] Intens . UV, 12.0-12.7min #(1795-1912), [mAU]

-20 Intens . +MS, 12.3-13.0min #(541-575) x106 0.8 345.2

0.6

0.4 475.1 0.2 147.2 700.5 580.9630.5 801.4 947.6 1163.5 0.0 250 500 750 1000 1250 1500 1750 2000 m/z * 25

Analog*6%

O H O

Chemical Formula: C20H24O5 Exact Mass: 344.1624 Molecular Weight: 344.4016 *

Intens . JL-425-A.D: TIC +All MS x109

Intens . JL-425-A.D: TIC -All MS x108

0.50 0.25 0.00 Intens . JL-425-A.D: UV Chromatogram, 252-256 nm [mAU] 200 100

0 Intens . JL-425-A.D: UV Chromatogram, 208-212 nm [mAU] 2000

1000

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Time [min] *

Major*peaks*(positive*mode):*

200 220 240 260 280 300 320 340 360 380 Wavelength [nm] Intens . UV, 12.4-13.1min #(1855-1969), [mAU] 1000

750

500

250

0 Intens . +MS, 12.7-13.4min #(577-609) x107 345.2 6

398.2 4

2 121.0 196.2 2077.9 2166.9 256.2 576.0 651.2 799.3 1908.8 0 250 500 750 1000 1250 1500 1750 2000 m/z * 26

Analog*7:*

OAc Cl

H O Cl O

Chemical Formula: C22H26Cl2O5 Exact Mass: 440.1157 Molecular Weight: 441.3450 *

Intens . JL-421-B.D: TIC +All MS x108

Intens . JL-421-B.D: TIC -All MS x107

Intens . JL-421-B.D: UV Chromatogram, 208-212 nm [mAU]

2000

0 Intens . JL-421-B.D: UV Chromatogram, 190-400 nm [mAU] x104

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Time [min] *

Major*peaks*(positive*mode):*

200 225 250 275 300 325 350 375 Wavelength [nm] Intens . UV, 14.4-15.1min #(2157-2268), [mAU] 600

400

200

Intens . +MS, 14.4-15.1min #(654-686) x107 441.0 1.5

1.0

382.1 0.5 185.2 553.3 243.1 612.9 850.8 2149.8 150.2 707.1 768.1 2048.9 0.0 250 500 750 1000 1250 1500 1750 2000 m/z * 27

Analog*8%

O OH O

H O C20H24O5 Exact Mass: 344.1624 Mol. Wt.: 344.4016 *

DAD1 B, Sig=218,4 Ref=360,100 (QLI\LCMS000007.D) DMSO HPLC 300 8 200 100 0 -100 -200 2.5 5 7.5 10 12.5 15 17.5 20 MSD1 TIC, MS File (D:\DATA\QLI\LCMS000007.D) ES-API, Pos, Scan, Frag: 200, "POS" TIC 3000000

2000000

1000000

0 2.5 5 7.5 10 12.5 15 17.5 20 PMP1, PMP1D, Solvent Ratio B Solvent ratio

2.5 5 7.5 10 12.5 15 17.5 20 *

* 28

Major*Peak*(positive)*

*MSD1 SPC, time=19.539 of D:\DATA\QLI\LCMS000007.D ES-API, Pos, Scan, Frag: 200, "POS"

100 Max: 216832 367.0

40 368.1 20 711.2 671.2

200 400 600 800 1000 *

* 29

1 H(500!MHz)!NMR!of!4!in!((CD3)2CO

13 C(125!MHz)!NMR!of!4!in!((CD3)2CO

DEPT7135!!NMR!of!4!in!((CD3)2CO

1 1 H7 H!Cosy!(500!MHz)!NMR!of!4!in!((CD3)2CO

1 13 H7 C!HMBC!NMR!of!4!in!((CD3)2CO

1 1 H7 H!NOESY(500!MHz)!NMR!of!4!in!((CD3)2CO

! 32

OH OH

O H O

O S1

ppm 8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR of S10 in CDCl3

OAc OH

O H O

S11 O

ppm 8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR of S11 in CDCl3 33

OAc Cl

H O Cl O S

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR of S9 in CDCl3

OAc Cl

H O Cl O

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR! of 7 in CDCl3 34

OAc

O H O

O S1

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR of S12 in CDCl3

O H O

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR! of 6 in CDCl3 35

OTMS O

O H O

O S1

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR! of S13 in CDCl3

220ppm 210220 200210 190200 180190 170180 160170 150160 140150 130140 120130 110120 100110 90100 8090 7080 6070 5060 4050 3040 2030 1020 010

!0 13 C (125 MHz) NMR of S13 in CDCl3 36

OTMS O

O H O

O S14

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR of S14 in CDCl3

OH O

O H O

5 O

8.0ppm 7.58.0 7.07.5 6.57.0 6.06.5 5.56.0 5.05.5 4.55.0 4.04.5 3.54.0 3.03.5 2.53.0 2.02.5 1.52.0 1.01.5 0.51.0 0.00.5

!0.0 1 H (500 MHz) NMR! of 5 in CDCl3 37

1 H(400!MHz)!NMR!of!8!in!CDCl3! 38

13 C(100!MHz)!NMR!of!8!in!CDCl3!

! 39

References

1. D. V. Titov, B. Gilman, Q. L. He, S. Bhat, W. K. Low, Y. Dang, M. Smeaton, A. L.

Demain, P. S. Miller, J. F. Kugel, J. A. Goodrich, J. O. Liu, Nat Chem Biol 2011, 7, 182-

188.

2. L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L.

A. Marraffini, F. Zhang, Science 2013, 339, 819-823.

3. P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, G. M.

Church, Science 2013, 339, 823-826.

4. K. Yusa, L. Zhou. M. A. Li, A. Bradley, N. L. Craig, Proc Natl Acad Sci U S A 2011, 108,

1531-1536.

5. Y. Chen, W. Chen, M. H. Cobb, Y. Zhao, Proc Natl Acad Sci U S A 2009, 106, 761-766.

! Adduct&4& A TPL# 40

B 16# 18# 20# 22# 24# 537.9# 361.0# 310.9#

16# 18# 20# 22# 24# Fig. S1. HPLC and MS analysis of the products of reaction between TPL and N-acetyl-L-cysteine methyl ester. (A) HPLC analysis of the reaction products of TPL and N-acetyl-L-cysteine methyl ester 4. (B) LC-MS (ESI, pos) analysis of the reaction products. 41

O H O N H H H-H Cosy O H C N 3 HMBC O O S O H NOE OH S CH H H 3 H H O OHCH3 OH H3C O O O H O

Fig. S2. The 2D-NMR analysis of the triptolide-cysteine adduct 4. 42

Fig. S3. Binding-dialysis-activity recovery assay. Different Triptolide analogs (100 µM) were incubated with TFIIH complex for 2 h. The reaction mixture were dialyzed to remove unbound small molecules and the remaining ATPase activities were determined using TLC assay.

Sequence#Coverage:#82%# ## Matched#pepBdes#shown#in#Bold&Red# ## ##### ##1 MGKRDRADRD KKKSRKRHYE DEEDDEEDAP GNDPQEAVPS AAGKQVDESG 51 TKVDEYGAKD YRLQMPLKDD HTSRPLWVAP DGHIFLEAFS PVYKYAQDFL 101 VAIAEPVCRP THVHEYKLTA YSLYAAVSVG LQTSDITEYL RKLSKTGVPD 151 GIMQFIKLCT VSYGKVKLVL KHNRYFVESC HPDVIQHLLQ DPVIRECRLR 201 NSEGEATELI TETFTSKSAI SKTAESSGGP STSRVTDPQG KSDIPMDLFD 251 FYEQMDKDEE EEEETQTVSF EVKQEMIEEL QKRCIHLEYP LLAEYDFRND 301 SVNPDINIDL KPTAVLRPYQ EKSLRKMFGN GRARSGVIVL PCGAGKSLVG 351 VTAACTVRKR CLVLGNSAVS VEQWKAQFKM WSTIDDSQIC RFTSDAKDKP 401 IGCSVAISTY SMLGHTTKRS WEAERVMEWL KTQEWGLMIL DEVHTIPAKM 451 FRRVLTIVQA HCKLGLTATL VREDDKIVDL NFLIGPKLYE ANWMELQNNG 501 YIAKVQCAEV WCPMSPEFYR EYVAIKTKKR ILLYTMNPNK FRACQFLIKF 551 HERRNDKIIV FADNVFALKE YAIRLNKPYI YGPTSQGERM QILQNFKHNP 601 KINTIFISKV GDTSFDLPEA NVLIQISSHG GSRRQEAQRL GRVLRAKKGM 651 VAEEYNAFFY SLVSQDTQEM AYSTKRQRFL VDQGYSFKVI TKLAGMEEED 701 LAFSTKEEQQ QLLQKVLAAT DLDAEEEVVA GEFGSRSSQA SRRFGTMSSM 751 SGADDTVYME YHSSRSKAPS KHVHPLFKRF RK

Fig. S4. Sequence coverage of XPB by MS experiment. The matched peptides are colored in red. 44 Walker#A#moBf# G * G K S/T Homo sapiens Mus musculus Danio rerio Drosophila melanogaster Geodia cydonium Saccharomyces cerevisiae Leptosphaeria maculans Pyrococcus furiosus Archaeoglobus fulgidus Haliangium ochraceum Thermococcus kodakarensis

Fig. S5. Sequence alignment of XPB orthologues from different organisms. Arrow points to Cys342 in human XPB. 45

7%#

6%#

5%#

wtXPB# C342AUXPB## C342SUXPB# C342TUXPB# 4%#

3%#

#ATP#Hydrolysis,#%# 2%#

1%#

0%# no# 0# 3# 10# 30# 100# 300# no# 0# 3# 10# 30# 100# 300# no# 0# 3# 10# 30# 100# 300# no# 0# 3# 10# 30# 100# 300# DNA# DNA# DNA# DNA# Triptolide,#μM#

Fig. S6. TPL does not inhibit the ATPase activity of recombinant C324A, C342S,C342T-XPB mutants. 46

XPB& XPD#

p62& p52& p44&

CDK7# CyclinH# p34& #MAT1#

Fig. S7. Protein composition of representative TFIIH complexes with XPB mutant purified using STAP tag affinity pulldown. 47

TPL(μM)# TPL(μM)# 0.1##0.3##1###3###10##100# 0.1##0.3##1###3###10##100#

wtTFIIH# C342AUTFIIH#

TPL(μM)# TPL(μM)# 0.1##0.3##1###3###10##100# 0.1##0.3##1###3###10##100#

C342SUTFIIH# C342TUTFIIH#

Fig. S8. Effects of triptolide on the ATPase activity of the C324A, C342S,C342T-XPB TFIIH mutants. A B C 48 T7115# T7# T71#

EcoR#I:#7053bp# BglI:#5340bp#

Fig. S9. Verification of the knock-in cell line T7115. (A) Southern blot analysis of T7 cell line that has one wild-type XPB with the other allele knocked out. (B) PCR-Afl II digestion analysis of T71 cell line that has one wild-type XPB and one C342T knock-in XPB. (C) PCR-Afl II digestion analysis of T7115 cell line with one XPB allele knocked out and the other replaced by C342T mutant. The XPBUpuro#lane#shows#that#the#puromycinUresistant#gene#was#inserted#the#right#posiBon. 49 Cys342Thr# TTAA#posiBon# mutaBon#

Wild##

MutaBon##

Fig. S10. Sequence confirmation of HEK293T and T7115 cell lines at the C342 coding loci. The TAAA was changed to TTAA transposon-excised site without changing the amino acid, and Cys342(TGC) was mutated to Thr342(ACC). 50

Fig. S11. The T7115 cell line has a slower proliferation than the wild type HEK293T cells. The cell proliferation was detected by Alamar Blue after three days of seeding the two different cell lines with 1000 cells per well in 96 wells plate. 51

A B

Fig. S12. The T7115 cell line is equally sensitive to Actinomycin D and α-Amanitin as wild type HEK293T cells. 52

gRNA#targeted#sequence# !!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTAAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCTGCGUUU#

!!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTAAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCTGCGUUU#

Donor# !!!!TGATATCAACATTGACC# TTAA...5' PB PGK-puro¦¤T K 3' PB... TTAA GCCCACAGUUUUUUUUUUUUUUUUUGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCACCGUUU# CAS9UD10A,#gRNA.#Screened#the#Puromycin#resistant#cell#lines#

!!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTAAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCTGCGUUU#

!!!!TGATATCAACATTGACC# TTAA...5' PB PGK-puro¦¤T K 3' PB... TTAA GCCCACAGUUUUUUUUUUUUUUUUUGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCACCGUUU#

pCMVUhyPBase,#Screened#the#puromycin#sensiBve#cell#lines#

!!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTAAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCTGCGUUU#

!!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTTAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCACCGUUU#

!!!!TGATATCAACATTGACC# TTAA...5' PB PGK-puro¦¤T K 3' PB... TTAA GCCCACAGUUUUUUUUUUUUUUUUUGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCACCGUUU# !!!!GGAATGATTCTGTCAACCCTGATATCAACATTGACCTTAAGCCCACAGCTGTCCTCAGACCCTATCAGGAGAAGAGCTTGCGAAAGATGTTTGGAAACGGGCGTGCACGTTCGGGGGTCATTGTTCTTCCCACCGUUU#

Fig. S13. The workflow to generate the C342T knock-in cell line using a#combinaBon#of#CRISPR/Cas9#and# piggyBac4transposase#technology