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Supporting Information for:
Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit
Matthew G. Costales,1,4 Christopher L. Haga,2,4 Sai Pradeep Velagapudi,1,4 Jessica L. Childs-Disney,1 Donald G. Phinney,2 and Matthew D. Disney1,3,*
1Department of Chemistry, 2Department of Molecular Therapeutics, and 3Department of
Neuroscience, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458
4These authors contributed equally.
*Author to whom correspondence should be addressed: [email protected]
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SUPPLEMENTARY FIGURES & TABLE Figure S1. Binding assays of Targapremir-210 and Hoechst 33342 to pre-miR-210. A) Representative binding curve of Targapremir-210 with pre-miR-210. B) Representative binding curve of Hoechst 33342 with pre-miR-210. C) Secondary structure of pre-miR-210.
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Figure S2. Specificity of Targapremir-210 and miR-210 antagomir. MiRNA microarray analysis
of hypoxic MDA-MB-231 cells treated with either a miR-210 antagomir (50 nM) or Targapremir-
210 (200 nM). Dots represent individual miRNA gene counts (normalized intensity values).
Pearson r correlation coefficient = 0.9833.
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Figure S3. Studying the non-selective binding of Targapremir-210-CA-Biotin Chem-CLIP probe. A) Using a control Chem-CLIP probe that lacks the RNA-binding module, Control-CA-Biotin, we studied if highly abundant transcripts that were pulled down by Targapremir-210-CA-Biotin were due to non-specific effects from the chlorambucil (CA) and biotin modules. For three of the RNAs, there is no difference between enrichment from Targapremir-210-CA-Biotin and Control-CA-Biotin, suggesting that pull-down is due to non-selective reaction with CA. B) A C-Chem-CLIP experiment was completed to assess non-selective binding caused by addition of CA and biotin modules for hypoxia-associated miRNAs pulled down by Targapremir-210-CA-Biotin. Compared to the C-Chem-CLIP results with miR-210, the abundance of other miRNAs does not decrease as dramatically, indicating that some of the pull-down may be due to non-selective effects of the CA or biotin moieties. *, p<0.05, as compared to before pull-down fraction, as determined by a two-tailed Student t-test.
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Figure S4. Targapremir-210 (200 nM) selectively inhibits biogenesis of miR-210 in MDA-MB-231 cells cultured under hypoxic conditions while Targaprimir-96 (200 nM) selectively inhibits biogenesis of miR-96. * indicates p<0.05, as compared to the untreated sample, as determined by a two-tailed Student t-test.
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Figure S5. Studying Targapremir-210’s (200 nM) effect on in vitro topoisomerase activity. Targapremir-210 did not inhibit the formation of topoisomers (+Topo II) nor does it result in the formation of a linear product, unlike positive control VP16 (red box).
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Figure S6. Biological effect of Hoechst 33342. qPCR analysis of hypoxic MDA-MB-231 cells treated with 200 nM of either Hoechst 33342 or Targapremir-210. Targapremir-210 significantly affects biogenesis of miR-210 compared to the untreated sample. * indicates p<0.05, as determined by a two-tailed Student t-test.
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Figure S7. Tumor localization of Targapremir-210. Frozen resected tumor samples from either control, antagomir-treated, or Targapremir-210-treated mice showing localization of Targapremir-210 within the tumor mass as determined by fluorescent microscopy.
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Table S1. Sequences of qRT-PCR primers hsa-miR-210 FWD CTGTGCGTGTGACAGCGGCTGA
pre-miR-210 FWD GCAGCCCCTGCCCACCGCACACT
pre-miR-210 REV CCGCTGTCACACGCACAG
pri-miR-210 FWD GACTGGCCTTTGGAAGCTCC
pri-miR-210 REV ACAGCCTTTCTCAGGTGCAG
HIF1α FWD CGCGAACGACAAGAAAAAG
HIF1α REV AAGTGGCAACTGATGAGCAA
GPD1L FWD CCCCTGAAAGTGTGCATCGT
GPD1L REV GGCAGCTTGTGTCCAGGAA
hsa-miR-10b FWD TACCCTGTAGAACCGAATTTGTG
hsa-miR-21 FWD TAGCTTATCAGACTGATGTTGA
hsa-miR-23a FWD ATCACATTGCCAGGGATTTCC
hsa-miR-23b FWD ATCACATTGCCAGGGATTACC
hsa-miR-24 FWD TGGCTCAGTTCAGCAGGAACAG
hsa-miR-26a FWD TTCAAGTAATCCAGGATAGGCT
hsa-miR-26b FWD TTCAAGTAATTCAGGATAGGT
hsa-miR-27a FWD TTCACAGTGGCTAAGTTCCGC
hsa-miR-30a FWD TGTAAACATCCTCGACTGGAAG
hsa-miR-30b FWD TGTAAACATCCTACACTCAGCT
hsa-miR-30c FWD TGTAAACATCCTACACTCTCAGC
hsa-miR-30d FWD TGTAAACATCCCCGACTGGAAG
hsa-miR-93 FWD CAAAGTGCTGTTCGTGCAGGTAG
hsa-miR-103 FWD AGCAGCATTGTACAGGGCTATGA
hsa-miR-106a-5p FWD AAAAGTGCTTACAGTGCAGGTAG
hsa-miR-125a-5p FWD TCCCTGAGACCCTTTAACCTGTGA
hsa-miR-125b FWD TCCCTGAGACCCTAACTTGTGA
hsa-miR-152 FWD TCAGTGCATGACAGAACTTGG
hsa-miR-181a FWD AACATTCAACGCTGTCGGTGAGT
hsa-miR-192 FWD CTGACCTATGAATTGACAGCC
hsa-miR-193b FWD AACTGGCCCTCAAAGTCCCGCT
hsa-miR-195 FWD TAGCAGCACAGAAATATTGGC
hsa-miR-205 FWD TCCTTCATTCCACCGGAGTCTG
hsa-miR-206 FWD TGGAATGTAAGGAAGTGTGTGG
hsa-miR-224 FWD CAAGTCACTAGTGGTTCCGTT
hsa-miR-335 FWD TCAAGAGCAATAACGAAAAATGT
hsa-miR-339-5p FWD TCCCTGTCCTCCAGGAGCTCACG
hsa-miR-491-3p FWD CTTATGCAAGATTCCCTTCTAC
hsa-miR-497 FWD CAGCAGCACACTGTGGTTTGT
hsa-miR-466 FWD ATACACATACACGCAACACACAT
hsa-miR-505 FWD CGTCAACACTTGCTGGTTTCCT
hsa-miR-140 FWD TACCACAGGGTAGAACCACGG
hsa-miR-181a-2 FWD AACATTCAACGCTGTCGGTGAGT
hsa-miR-324 FWD ACTGCCCCAGGTGCTGCTGG
hsa-miR-648 FWD AAGTGTGCAGGGCACTGGT
hsa-miR-103a-1 FWD AGCAGCATTGTACAGGGCTATGA
hsa-miR-1273c FWD GGCGACAAAACGAGACCCTGTC
hsa-miR-4682 FWD TCTGAGTTCCTGGAGCCTGGTCT
hsa-miR-3120 FWD CACAGCAAGTGTAGACAGGCA
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hsa-miR-3174 FWD TAGTGAGTTAGAGATGCAGAGCC
hsa-miR-4446 FWD CAGGGCTGGCAGTGACATGGGT
hsa-miR-107 FWD AGCAGCATTGTACAGGGCTATCA
hsa-miR-3613 FWD ACAAAAAAAAAAGCCCAACCCTTC
RNU6 FWD ACACGCAAATTCGTGAAGCGTTC
Universal REV GAATCGAGCACCAGTTAC
18S Rrna-FWD GTAACCCGTTGAACCCCATT
18S Rrna-REV CCATCCAATCGGTAGTAGCG
28S rRNA-FWD AGAGGTAAACGGGTGGGGTC
28S rRNA-REV GGGGTCGGGAGGAACGG
5.8S rRNA-FWD ACTCGGCTCGTGCGTC
5.8S rRNA-REV GCGACGCTCAGACAGG
45S rRNA-FWD GAACGGTGGTGTGTCGTT
45S rRNA-REV GCGTCTCGTCTCGTCTCACT
5S rRNA-FWD GGCCATACCACCCTGAACGC
5S rRNA-REV CAGCACCCGGTATTCCCAGG
7SK-FWD CCCCTGCTAGAACCTCCAAAC
7SK-REV CACATGCAGCGCCTCATTT
7SL-FWD ATCGGGTGTCCGCACTAAGTT
7SL-REV CAGCACGGGAGTTTTGACCT
ACA16-FWD GGCCCTTATCGAAGCTGCA
ACA16-REV CGGCGACCGTCAAGGA
ACA44-FWD GTTTCCAAGGGCTGTGGCT
ACA44-REV TGTACTGACCTGCGCTGTCAA
ACA61-FWD CCTTTCCCATCGGATCTGAA
ACA61-REV CCACATGCCATATACCAGATTACAAC
BC200-FWD TGGCTCACGCCTGTAATCC
BC200-REV CCCAGGCAGGTCTCGAACT
HBI-36-FWD CAGCACTGCCAAGTGACCC
HBI-36-REV ATATGTACCCAGCTGCATGCAG
HBII-85-FWD TGGATCGATGATGAGTCC
HBII-85-REV TGGACCTCAGTTCCGATGAGA
HBII-420-FWD ACTGGTCCAGGATGAAACCTAATT
HBII-420-REV CCTAGGAGCTGGTCTCAGTCCC
U1-FWD CCATGATCACGAAGGTGGTTT
U1-REV ATGCAGTCGAGTTTCCCACAT
U2-FWD TTCTCGGCCTTTTGGCTAAG
U2-REV CTCCCTGCTCCAAAAATCCA
U4-FWD GCCAATGAGGTTTATCCGAGG
U4-REV TCAAAAATTGCCAATGCCG
U5-FWD TGGTTTCTCTTCAGATCGCATAAA
U5-REV CCAAGGCAAGGCTCAAAAAAT
U6-FWD CTCGCTTCGGCAGCACA
U6-REV AACGCTTCACGAATTTGCGT
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U12-FWD GCCCGAATCCTCACTGCTAA
U12-REV TCGCAACTCCCAGGCATC
U87-FWD ATGGGATCATGGAGCAGCTG
U87-REV TCACACCCATGACTGCCACT
U105-FWD CCCCTATCTCTCATGATGAACACATAT
U105-REV CCCCATCTCTTCTTCAGAGCG
tRNA(Lys)-FWD CGGCTAGCTCAGTCGGTAGA
tRNA(Lys)-REV CCCACGACCCTGAGATTAAG
tRNA(Ala)-FWD GGGGGTGTAGCTCAGTGGTA
tRNA(Ala)-REV AGGCCTCATACATGCAAAGC
tRNA(Cys)-FWD GCTCAGGGGTAGAGCATTTG
tRNA(Cys)-REV ACCGGGGACTTCTGGATCT
tRNA(His)-FWD GCAGTGACTGTATAGTGGTTAGCA
tRNA(His)-REV GTGGCCACAACACAGAGTG
tRNA(Ser)-FWD TAGTCGTGGCCGAGTGGTTA
tRNA(Ser)-REV GGAAACCCCAATGGATTTCTA
tRNA(Val)-FWD TGGTTATCACGTTCGCCTAA
tRNA(Val)-REV GTTTCGAACCGGGGACCT
tRNA(Arg)-FWD CAGTGGCGCAATGGATAAC
tRNA(Arg)-REV CAGGAGTCGAACCTGGAATC
tRNA(Gln)-FWD TGGTTAGCACTCTGGACTCTGA
tRNA(Gln)-REV AGGTTCCACCGAGATTTGAA
tRNA(Ile)-FWD CAGTTGGTTAGAGCGTGGTG
tRNA(Ile)-REV CCACGACCTTGGCGTTATTA
tRNA(Thr)-FWD TAGCTGGTTAAAGCGCCTGT
tRNA(Thr)-REV GAACCCAGGATCTCCTGTTTACT
tRNA(Asn)-FWD CAATGGGTTAGCGCGTTC
tRNA(Asn)-REV AACCACCAACCTTTCGGTTA
tRNA(Glu)-FWD CTGGTGGTCTAGTGGCTAGGA
tRNA(Glu)-REV CTGGCCGGGAATCGAAC
tRNA(Ini)-FWD CATAACCCAGAGGTCGATGG
tRNA(Ini)-REV TAGCAGAGGATGGTTTCGAT
tRNA(Phe)-FWD TCAGTTGGGAGAGCGTTACA
tRNA(Phe)-REV AGGGTTGAACCAGGGAACTT
tRNA(Trp)-FWD GCGCGTCTGACTCCAGAT
tRNA(Trp)-REV ACGTGATTTGAACACGCAAC
tRNA(Asp)-FWD TCTGCCTGTCATGTGGAGAC
tRNA(Asp)-REV CCTGTTGGGGACTCAAACTC
tRNA(Gly)-FWD GCATTGGTGGTTCAGTGGTA
tRNA(Gly)-REV ATTGGCCGGGAATTGAAC
tRNA(Leu)-FWD GGTCTAAGGCGCTGGATTAAG
tRNA(Leu)-REV CCCCCGAAGAGACTGGAG
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tRNA(Pro)-FWD GGGGTATGATTCTCGCTTAGG
tRNA(Pro)-REV ATTTGAACCCGGGACCTCT
tRNA(Tyr)-FWD CTGGTAGAGCGGAGGACTGT
tRNA(Tyr)-REV GGAATTGAACCAGCGACCTA
tRNA(Sec)-FWD GGCTTCAAACCTGTAGCTGTC
tRNA(Sec)-REV CCGAAATGGAATTGAACCAC
FTH1-FWD GGCAAAGTTCTTCAAAGCCA
FTH1-REV CATCAACCGCCAGATCAAC
GAPDH-FWD TGCACCACCAACTGCTTAGC
GAPDH-REV GGCATGGACTGTGGTCATGAG
ATP6-FWD TTCTGGAATGACTCCTTTGC
ATP6-REV TTGGCCAGAATGAACTTGAA
RPL8-FWD AGATGGGTTTGTCAATTCGG
RPL8-REV CAAGAAGACCCGTGTGAAGC
FLNA-FWD AGGGGACGGCCCTTTAAT
FLNA-REV GTCGCTCTCAGGAACAGCAG
RPS6-FWD CAAGAGAATGAAGGAGGCTAAGG
RPS6-REV AAGCTCGCAGAGAGGAAAGTCT
Rpl11-FWD ACTTCGCATCCGCAAACTCT
Rpl11-REV TGTGAGCTGCTCCAACACCTT
ENO1-FWD GCCTCCTGCTCAAAGTCAAC
ENO1-REV AACGATGAGACACCATGACG
COX2-FWD GCAGGGTTGCTGGTGGTAG
COX2-REV ATTTCATCTGCCTGCTCTGG
GNB2L1-FWD TGGCTAACTGCAAGCTGAAGAC
GNB2L1-REV ATCTGGAGAGACAGTCACCGTG
PKM2-FWD TACCATGCGGAGACCATCAA
PKM2-REV AGCAACGGGCCGGTAGAG
CLU-FWD CCAGTGGAAGATGCTCAAC
CLU-REV CGAGTCAGAAGTGTGGGAAGC
CANX-FWD GCAACCACTTCCCTTCCAT
CANX-REV TCCGCCTCTCTCTTTACTGC
RPS9-FWD AGACCCAGGTCTTCAAGCTG
RPS9-REV ATGAAGGACGGGATGTTCAC
GNAS-FWD CAGTGGAGATGGGCGTCACTA
GNAS-REV CGGCGGATGTTCTCAGTGT
COX1-FWD CGATGCATACACCACATGAA
COX1-REV AGCGAAGGCTTCTCAAATCA
B2M-FWD TGCTGTCTCCATGTTTGATGTATCT
B2M-REV TCTCTGCTCCCCACCTCTAAGT
RPS3-FWD TCCTCGGAGTTTCCCAGAC
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RPS3-REV TCCTAGGAGGGCTTGCTGT
EEF1A1-FWD TGCGGTTTTTGTCATCAAA
EEF1A1-REV AAGAGTGGGGTGGCAGGTATTAG
Rpl27-FWD TCCAAGGGGATATCCACAGA
Rpl27-REV CATGGGCAAGAAGAAGATCG
RPL37-FWD AAAACCAGAACATTTATTGCATGA
RPL37-REV TCCGTGAAGGAACAACACCT
RPL3-FWD CCTCCGTTCACCTGGATCT
RPL3-REV CCAAGTCATCCGTGTCATTG
EEF2-FWD GCACGTTCGACTCTTCACTG
EEF2-REV CTGGAGATCTGCCTGAAGGA
ND2-FWD GCCCTAGAAATAAACATGCTA
ND2-REV GGGCTATTCCTAGTTTTATT
TPT1-FWD CATGATTATCTACCGGGACCTCAT
TPT1-REV AACCCGTCCGCGATCTC
TMSB4X-FWD AGACCAGACTTCGCTCGTA
TMSB4X-REV CTGCTTCGTTCTCCTGTT
RPL4-FWD GCTCTGGCCAGGGTGCTTTTG
RPL4-REV ATGGCGTATCGTTTTTGGGTTGT
CCT5-FWD ACAGCCTTTGCTGCCATTAT
CCT5-REV GCCCTTTCCTCATCATCAAG
PRDX1-FWD GGGCACACAAAGGTGAAGTC
PRDX1-REV GCTGTTATGCCAGATGGTCAG
PFN1-FWD GATCACCGAACATTTCTGGC
PFN1-REV AAACGTTCGTCAACATCACG
Rps24-FWD AAGACCACACCGGATGTCATC
Rps24-REV TGCCAAAGCCAGTTGTCTTG
PPIA-FWD TTATTTGGGTTGCTCCCTTC
PPIA-REV AAGTGTGCCAAATCTGCAAG
LDHB-FWD TGGCGTGTGCTATCAGCATT
LDHB-REV GCTTATCTTCCAAAACATCCACAAG
LGALS1-FWD AAGCTGCCAGATGGATACGAA
LGALS1-REV CGTCAGCTGCCATGTAGTTGA
PGK1-FWD ATGGATGAGGTGGTGAAAGC
PGK1-REV CAGTGCTCACATGGCTGACT
BASP1-FWD CAATGCCAATCCTCCATTCT
BASP1-REV AACTACAGGTGCACCCAACC
YBX1-FWD TGATGGAGGGTGCTGACAAC
YBX1-REV CCTGCGGAATCGTGGTCTAT
KRT7-FWD TGGAGGACTTCAAGAATAAGTACGAA
KRT7-REV TCATGTAGGCAGCATCCACATC
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CYTB-FWD AATTCTCCGATCCGTCCCTA
CYTB-REV GGAGGATGGGGATTATTGCT
NONO-FWD CCCTTACAGTTCGAAACCTT
NONO-REV ATGACTACAGCCCTCTCTAC
ND1-FWD ATACCCCCCATTCCGCTACGAC
ND1-REV GTTTGAGGGGGAATGCTGGAGA
ALDOA-FWD CGGGAAGAAGGAGAACCTG
ALDOA-REV GACCGCTCGGAGTGTACTTT
P4HB-FWD GCAAACTGAGCAACTTCAAA
P4HB-REV TTCTTCAGGCCAAAGAACTC
LDHA-FWD ACCCAGTTTCCACCATGATT
LDHA-REV CCCAAAATGCAAGGAACACT
APP-FWD GCTGGCTGAACCCCAGATT
APP-REV CCCACTTCCCATTCTGGACAT
FOLR1-FWD GCACCACAAGGAAAAGCCAG
FOLR1-REV CATTCTTCCTCCAGGGTCGAC
ACTB-FWD CTGGAACGGTGAAGGTGACA
ACTB-REV AAGGGACTTCCTGTAACAATGCA
RPL13-FWD CCCGTCCGGAACGTCTATAA
RPL13-REV CTAGCGAAGGCTTTGAAATTCTTC
RPS21-FWD GGCGAGTTCGTGGACCTGTA
RPS21-REV GGATGGATGCGTGGTCCTT
S100A6-FWD CTGCAGGATGCTGAAATTGC
S100A6-REV GGAAGTTCACCTCCTGGTCCTT
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SUPPLEMENTARY METHODS
Determination of Binding Affinities of Hoechst 33342 and Targapremir-210.
Dissociation constants for the binding of pre-miR-210 RNA to Hoechst 33342 and Targapremir-
210 were determined using an in solution, fluorescent binding assay. RNA was folded in 1×
Binding Buffer (8 mM Na2HPO4, 190 mM NaCl, 1 mM EDTA, and 40 µg/mL BSA) at 60 °C for 5
min and then allowed to cool to room temperature for 10 min. Hoechst 33342 or Targapremir-
210 was added to a final concentration of 500 nM. Next, 1:2 serial dilutions were performed in
1× Binding Buffer supplemented with 500 nM of Hoechst 33342 or Targapremir-210. Solutions
were incubated for 30 min and then transferred to Corning non-binding surface 96-well black
plates. Fluorescence intensity was then measured on a Bio-Tek FLX-800 plate reader. Change
in fluorescence intensity was fit as a function of RNA concentration with equation 1:
𝐼 = 𝐼0 + 0.5∆𝜀([FL]0 − (([FL]0 + [RNA]0Kt)2 − 4[FL]0[RNA]0)
0.5) (1)
where I and I0 are the observed fluorescence intensity in the presence and absence of RNA,
respectively, is the difference between the fluorescence intensity in the absence and in the
presence of infinite RNA concentration, [FL]0 and [RNA]0 are the concentrations of compound
(Hoechst 33342 or Targapremir-210) and RNA, respectively, and Kt is the dissociation constant.
Microarray analysis of miRNA. MDA-MB-231 cells were treated with either a miRNA
antagomir against miR-210 or Targapremir-210 and placed under hypoxic conditions for 3 days.
Total RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research) and poly (A)-tailed
and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix). Biotin-labeled
RNA samples were hybridized to the GeneChip miRNA 4.0 microarray (Affymetrix). Following
hybridization, microarrays were washed and stained using the Fluidics Station 450 (Affymetrix)
and scanned using the GeneChip Scanner 3000 7G (Affymetrix). Expression analysis was
carried out using the Expression Console Software package (Affymetrix).
Tumor localization of Targapremir-210. Resected tumor samples were frozen using
O.C.T. Compound (Tissue-Tek) over dry ice. Frozen tissues were sectioned by cryostat and
transferred to microscope slides. Fluorescent microscopic analysis was carried out using a
Leica DMI3000 B upright fluorescent microscope.
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SYNTHETIC METHODS
Abbreviations: Boc, tert-butyloxycarbonyl; DCM, dichloromethane; DIC, N,N’-
Diisopropylcarbodiimide; DIEA, N,N-Diisopropylethylamine; DMF, N,N-dimethylformamide;
Fmoc, fluorenylmethoxycarbonyl chloride; HATU, 1-[Bis(dimethylamino)methylene-]1H-1,2,3-
trizaolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole;
HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser
desorption/ionization-time of flight; TFA, trifluoracetic acid
Targapremir-210
Targapremir-210 was synthesized as previously described.1
Scheme 1. Synthetic scheme for Control-CA-Biotin.
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Synthesis of Control-CA-Biotin (control Chem-CLIP probe lacking the RNA-binding
module). Rink amide resin (1 g, 0.6 mmol) was swollen in DCM for 5 min and then in DMF for
5 min. The resin was de-protected with a solution of 20% piperidine in DMF (5 mL, 2 x 20 min).
After washing with DMF (3 x 5 mL), the resin was treated with a solution of bromoacetic acid
(0.412 g, 3 mmol) and DIC (0.464 mL, 3 mmol) in DMF (5 mL) via microwave irradiation (3 x 15
s) using a 700 W microwave set to 10% power. The resin was washed with DMF (3 x 5 mL)
and then reacted with a solution of N-(4-aminoethyl)-biotin (344 mg, 1.2 mmol) and DIEA (0.3
mL, 0.6 mmol) in DMF (4 mL) via microwave irradiation (3 x 15 s) using a 700 W microwave set
to 10% power. The resin was washed with DMF (3 x 5 mL) and treated with a solution of Fmoc-
Dap(Boc)-OH (0.768 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), and DIC (0.464 mL, 3 mmol) via
microwave irradiation (3 x 15 s) using a 700 W microwave set to 10% power. The resin was
treated with a solution of 20% piperidine in DMF (4 mL), washed with DMF, and then treated
with a solution of Fmoc-β-alanine (0.56 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), DIC (0.464 mL,
3 mmol), and DIEA (0.3 mL, 1.8 mmol) in DMF (3 mL) via microwave irradiation (3 x 15 s) using
a 700 W microwave set to 10% power. The Boc protecting group was removed with a solution
of 30% TFA in DCM (4 mL) for 15 min at room temperature. The solution was concentrated in
vacuo and azeotroped with toluene three times. The resulting oil was purified by preparative
HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA over 60 min.
The purified compound (0.036 g, 0.05 mmol) was treated with a solution of acetic anhydride
(0.05 mL, 0.53 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (4 mL) via microwave irradiation (3
x 15 s) using a 700 W microwave set to 10% power. The reaction was concentrated under
vacuum and purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O
with 0.1% TFA over 60 min. Fractions containing the product were dissolved in 20% piperidine
in DMF (2 mL) and stirred for 20 min. The reaction mixture was concentrated under vacuum
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and purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1%
TFA over 60 min.
The resulting purified compound was treated with a solution of chlorambucil (5 mg, 0.01 mmol),
HOAt (2.72 mg, 0.02 mmol), HATU (7.6 mg, 0.02 mmol) and DIEA (0.016 mL, 006 mmol) at
room temperature for 3 h. The mixture was then concentrated under vacuum and purified via
preparative HPLC using a linear gradient of 0-100% CH3CN in H2O with 0.1% TFA over 60 min.
Isolated 533 nmol; 0.06% yield. Retention time: 38 min; hydrolyzed product retention time: 39
min. HRMS calculated: 828.3395 (M+H), mass found: 828.4584 (M+H). Hydrolyzed product,
HRMS calculated: 792.4078 (M+H), mass found: 792.4839 (M+H). Compound can hydrolyze
during purification due to reactive alkyl halogen groups.
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Figure S8. Characterization of Control-CA-Biotin. A) Analytical HPLC trace of Control-CA-
Biotin. A linear gradient with a flow rate of 1 mL/min from 0% to 100% CH3CN + 0.1% (v/v) TFA
in water + 0.1% (v/v) TFA was used. B) HRMS MALDI-TOF of Control-CA-Biotin.
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Scheme 2. Synthetic scheme for Targapremir-210-CA-Biotin.
Synthesis of Targapremir-210-CA-Biotin (Targapremir-210 Chem-CLIP probe). Rink amide
resin (1 g, 0.6 mmol) was swollen in DCM for 5 min and then with DMF for 5 min. The resin was
de-protected with a solution of 20% piperidine in DMF (5 mL, 2 x 20 min) and then washed with
DMF (3 x 5 mL). The resin was then treated with a solution of bromoacetic acid (0.412 g, 3
mmol) and DIC (0.464 mL, 3 mmol) in DMF (5 mL) via microwave irradiation (3 x 15 s) using a
700 W microwave set to 10% power. The resin was washed with DMF (3 x 5 mL) and treated
with a solution of N-(4-aminoethyl)-biotin (344 mg, 1.2 mmol) and DIEA (0.3 mL, 0.6 mmol) in
DMF (4 mL) via microwave irradiation (3 x 15 s) using a 700 W microwave set to 10% power.
The resin was washed with DMF (3 x 5 mL) and treated with a solution of Fmoc-Dap(Boc)-OH
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(0.768 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), and DIC (0.464 mL, 3 mmol) via microwave
irradiation (3 x 15 s) using a 700 W microwave set to 10% power. The resin was treated with a
solution of 20% piperidine in DMF (4 mL), washed with DMF, and treated with a solution of
Fmoc-β-alanine (0.56 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), DIC (0.464 mL, 3 mmol), and
DIEA (0.3 mL, 1.8 mmol) in DMF (3 mL) via microwave irradiation (3 x 15 s) using a 700 W
microwave set to 10% power. After washing with DMF, Boc was removed with a solution of
30% TFA in DCM (4 mL) for 15 min at room temperature. The solution was concentrated in
vacuo and azeotroped with toluene three times. The resulting oil was purified by preparative
HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA over 60 min.
The purified amine compound (0.036 g, 0.05 mmol) was treated with Hoechst carboxylate
(0.077 g, 0.15 mmol), HOAt (0.0204 g, 0.15 mmol), DIC (0.235 mL, 0.15 mmol) in DMF (3 mL)
in a microwave vial and reacted at 75 °C for 30 min in a Biotage Initiator. The reaction was
concentrated under vacuum and purified by preparative HPLC using a linear gradient of 20-
100% CH3OH in H2O with 0.1% TFA over 60 min.
Fractions containing the Hoechst-coupled product were re-suspended in 20% piperidine in DMF
(2 mL) and stirred for 20 min. The reaction mixture was concentrated under vacuum and
purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA
over 60 min.
Finally, the resulting compound was treated with a solution of chlorambucil (5 mg, 0.01 mmol),
HOAt (2.72 mg, 0.02 mmol), HATU (7.6 mg, 0.02 mmol) and DIEA (0.016 mL, 0.06 mmol) and
at room temperature for 3 h. The resulting mixture was concentrated under vacuum and
purified via preparative HPLC using a linear gradient of 0-100% CH3CN in H2O with 0.1% TFA
over 60 min. Isolated 230 nmol; 0.03% yield. Retention time: 40.5 min, HRMS calculated:
1278.5563 (M+H), mass found: 1278.7928 (M+H).
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Figure S9. Characterization of Targapremir-210-CA-Biotin. A) Analytical HPLC trace of
Targapremir-210-CA-Biotin. A linear gradient with a flow rate of 1 mL/min from 0% to 100%
CH3CN + 0.1% (v/v) TFA in water + 0.1% (v/v) TFA was used. B) HRMS MALDI-TOF of
Targapremir-210-CA-Biotin.
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REFERENCES [1] Velagapudi, S. P., Seedhouse, S. J., French, J., and Disney, M. D. J. Am. Chem. Soc.
2011; 133:10111.