Ms. No. JA993387I

Neomycin–Acridine Conjugate: A Potent Inhibitor of Rev-RRE Binding

Sarah R. Kirk, Nathan W. Luedtke and Yitzhak Tor*

Department of Chemistry and Biochemistry, University of California, San Diego,

La Jolla, CA 92093-0358.

Supporting Information:

1. Synthetic procedures and characterization of neo–acridine (2) and its synthetic intermediates

•Figure S1. Synthetic scheme of neo–acridine

2. Synthetic procedures and characterization of Rev peptides and the RRE RNA

3. Experimental procedures and additional data for gel-mobility shift experiments

•Figure S2a. Gel-mobility shift for neo–acridine/RRE binding

•Figure S2b. Gel-mobility shift for Rev/RRE binding

4. Experimental procedures and conditions for fluorescence anisotropy measurements

5. Equations for calculating Kd and Ki values

•Figure S3a. Binding curve of RRE to Rev-Fl

•Figure S3b. Displacement of Rev off the RRE by neomycin B

6. Experimental procedures and additional data for enzymatic footprinting experiments

•Figure S4. Enzymatic footprinting on the RRE by RNase V1, RNase T1, and RNase A

•Figure S5 . Quantitative representation of RNase V1 cleavage of the RRE in the presence

of Rev

•Figure S6 . Quantitative representation of RNase V1 cleavage of the RRE in the presence

of neo–acridine

•Figure S7. Quantitative representation of RNase T1 and RNase A cleavage of the RRE in

the presence of Rev

•Figure S8. Quantitative representation of RNase T1 and RNase A cleavage of the RRE in

the presence of neo–acridine

Neo–acridine (2) Synthesis - experimental

Compound 1a and 1b have previously been reported (Michael, K.; Wang, H.; Tor, Y. Bioorg.

Med. Chem. 1999, 7, 1361-1371)

Compound 1c. Freshly cut sodium metal (0.56 g, 24.4 mmol) was dissolved in ethanol (25 mL,

degassed under argon). Aminoethanethiol·HCl (1.337 g, 11.8 mmol) was added to the solution at

room temperature and stirred for 15 min under argon. Compound 1b (0.40 g, 0.27 mmol) was

dissolved in ethanol (10 mL) and canulated into the thiolate salt solution. The reaction mixture was

stirred at room temperature for 5 hours. The reaction was quenched with cold CH2Cl2 (400 mL)

and sodium phosphate (50 mL, pH 5-6) on ice. The CH2Cl2 solution was then washed with brine

(2 × 50 mL). The organic layer was neutralized with sodium bicarbonate (2 × 50 mL), dried over

Na2SO4, and concentrated in vacuo. Flash column chromatography (9% CH3OH in CH2Cl2)

afforded the desired product as a white solid (0.228 g, 66%). Rf 0.24 (10% CH3OH in CH2Cl2);

1H NMR (500 MHz, methanol-d4, 25 °C): δ 5.42 (br, 1H), 5.18 (s, 1H), 4.95 (s, 1H), 4.25 (m,

1H), 4.05 (m, 2H), 3.88 (m, 2H), 3.75 (m, 2H), 3.46-3.56 (m, 6H), 3.24-3.34 (m, 7H), 3.02

(m, 2H), 2.88 (m, 2H), 2.80 (m, 2H), 1.97 (m, 1H), 1.60 (m, 1H), 1.42 (m, 54H); HRMS

(FAB) m/z [M+Na]+ 1296.6342, calcd for C68H116NaN6O27S 1296.6360.

9-phenoxyacridine. Prepared according to Dupre and Robinson J. Chem. Soc. 1945 , 549-

551.

Compound 1d. Phenol (0.422 g, 4.49 mmol) was heated to 60 °C. Upon melting, compound

1c (70 mg, 0.055 mmol) and 9-phenoxyacridine (45 mg, 0.165 mmol) were added. The reaction

mixture was stirred for 1 h at 60-70 °C. The mixture was cooled to room temperature and

precipitated with water (2 × 5 mL). The precipitate was dissolved in CH2Cl2 (15 mL), washed

with water, and concentrated in vacuo. Flash column chromatography (15% CH3OH in CH2Cl2)

afforded the desired product as a yellow solid (0.068 g, 85%). Rf 0.41 (15% CH3OH in CH2Cl2);

MS (FAB) m/z [M+H]+ 1452, calcd for C68H107N8O24S 1452.

Neo–acridine (2 ) . Compound 1d (0.030 g, 0.021 mmol) was dissolved in dioxane (2 mL,

treated over alumina). 1,2 ethanedithiol (0.007 mL, 0.080 mmol) was added followed by 4 M

HCl/dioxane (2 mL). The solution was swirled for 5 min by hand upon which a yellow precipitate

formed. Further precipitation was induced by adding ether and hexane (2 mL each). Precipitate

was recovered by centrifugation and several washes with ether and hexane. The yellow solid was

redissolved in water and lyophilized overnight. The product was HPLC purified on C-18

semiprep column. Isocratic conditions 0-15 min: 13% (0.1% TFA/CH3CN) / 87% (0.1%

TFA/H2O); 15-20 min: gradient up to 60% (0.1% TFA/CH3CN) / 40% (0.1% TFA/H2O). 1H

NMR (400 MHz, methanol-d4, 25 °C): δ 8.57 (m, 2H, Acr1, Acr1´), 8.00 (m, 2H, Acr3, Acr3´),

7.86 (m, 2H, Acr4, Acr4´), 7.61 (m, 2H, Acr2, Acr2´), 6.05 (d, J=3.7 Hz, 1H, 1´), 5.45 (d,

J=3.7 Hz, 1H, 1´´), 5.35 (b, 1H, 1´´´), 4.45 (m, 3H, AcrNCH2, 3´´), 4.37 (m, 2H, 2´´, 4´´),

4.29 (m, 1H, 5´´´), 4.16 (m, 2H, 6, 3´´´), 4.03 (m, 1H, 3´), 3.96 (bt, 1H, 5´), 3.88 (m, 1H, 5),

3.68 (b, 1H, 4´´´), 3.60 (m, 1H, 4), 3.35 (3.20–3.51) (m, 11H, 1, 2´´´, 6´eq, 2´, 6´´´ax, 4´,

AcrSCH2, 6´´´eq, 3, 5´´eq), 3.08 (d,d, J=8.1 Hz, 13.2 Hz, 1H, 6´ax), 2.85 (d,d, J=9.5 Hz, 13.6

Hz, 1H, 5´´ax), 2.44 (m, 1H, 2eq), 2.07 (m, 1H, 2ax); MS (FAB): m/z [M+Na]+ 873, calcd for

C38H58NaN8O12S 873; [M+H]+ 851, calcd for C38H59N8O12S 851. HRMS (MALDI) m/z [M+H]+

851.3967, calcd for C38H59N8O12S 851.3973. UV (water): λmax (nm) and ε (cm-1M-1): 222

(1.9x104), 266 (4.5x104), 412 (9.3x103), 434 (7.8x103).

Compound 3 was synthesized in a similar fashion as a model derivative. Its extinction coefficients

were used to verify the concentration of dilute neo–acridinesolutions. UV (10 mM phosphate

buffer, pH 7.5): λmax (nm) and ε (cm-1M-1): 222 (1.9x104), 266 (5.6x104), 412 (9.4x103), 434

(7.9x103).

N

HN

OH

3

HO

O

NH2

H2NO

HOHO

OO

H2N

OH

NH2

HOO

HOO

HO

NH2

H2N

Neomycin B (1)

HO

O

NHBoc

BocHNO

HOHO

OO

BocHN

OH

NHBoc

HOO

HOO

HO

NHBoc

BocNH

1a

(Boc)2O

DMF, H2O, Et3N60°C, 2h, 72% excess TPSCl

pyr, 20h, rt, 75%

TPSO

O

NHBoc

BocHNO

HOHO

OO

BocHN

OH

NHBoc

HOO

HOO

HO

NHBoc

BocNH

1b

NaOEt/EtOH 4.5h, rt, 80%

H2NCH2CH2SH

O

NHBoc

BocHNO

HOHO

OO

BocHN

OH

NHBoc

HOO

HOO

HO

NHBoc

BocNH

1c

N

O

9-phenoxyacridine

phenol, 60-75°C, 1h, 84%

SNH

N

O

NHBoc

BocHNO

HOHO

OO

BocHN

OH

NHBoc

HOO

HOO

HO

NHBoc

BocNH

1d

SNH

N

O

NH2

H2NO

HOHO

OO

H2N

OH

NH2

HOO

HOO

HO

NH2

H2N

Neo–acridine (2)

4M HCl/dioxane, HSCH2CH2SH,5 min., rt, 80%

Abbreviations

Boc = di-tert-butyldicarbonateDMF= dimethylformamideTPSCl= 2,4,6-triisopropylbenzenesulfonyl chloridepyr=pyridine

SH2N

Figure S1. Synthesis of Neo–acridine (2)

Synthesis of Rev Peptides

Peptide synthesis was carried out using standard Fmoc/HBTU chemistry on ABI Applied

Biosystems 431A peptide synthesizer using protected amino acids purchased from Calbiochem.

Protected amino acids used in the synthesis were: Fmoc-Arg-(Pbf)-OH, Fmoc-Asn-(Trt)-OH,

Fmoc-Gln-(Trt)-OH, Fmoc-Trp-(Boc)-OH, Fmoc-Thr-(tBu)-OH, Fmoc-Glu-(OtBu), and Fmoc-

Cys-(Trt)-OH. The first peptide coupling was done manually to avoid isomerization problems

associated with Cys. Fmoc-Cys-(Trt)-OH was converted into its symmetric anhydride by reaction

with 0.45 equivalents of DCC in dry CH2Cl2 for 10 min. The anhydride was separated from the

DCU precipitate by vacuum filtration and CH2Cl2 was subsequently removed in vacuo. The solid

anhydride was then dissolved in 30% N-methylpyrrolidone (NMP) in DMF and added to

deprotected Rink Amide MBHA resin. The coupling lasted 2.5 hours at 25°C and was monitored

by ninhydrin (Kaiser Test). Upon completion, the resin was washed with CH2Cl2 followed by

NMP, and loaded onto the ABI synthesizer. The remainder of the peptide synthesis was carried

out using standard HOBt/HBTU chemistry and monitored with a built-in conductivity meter. The

first round of automated coupling reactions (Ala) was done as a "double coupling" to ensure

efficiency. Upon completion of the automated synthesis, the resin was washed with DMF and

reacted with 0.3 M succinic anhydride, 0.3 M 1-hydroxybenztrizole hydrate, and 0.03 M DMAP in

DMF, for 1 h at room temperature. Succinylation of the peptide's N-terminus was monitored using

the Kaiser Test. Upon completion, the peptide was cleaved from its resin at room temperature for

2.5 h in a "cleavage cocktail" containing 88% TFA, 5% water, 5% phenol, and 2%

triisopropylsilane (v/v). The filtrate was then mixed with 15 volumes of 2% acetic acid, and

extracted 4 times with diethyl ether. The crude peptide was purified on a C-18 semiprep HPLC

column with an isocratic mixture of 14% acetonitrile (0.1% TFA) in water (0.1% TFA). The

purified peptide was lyophilized and reacted with 100 equivalents of 5-iodoacetamidofluorescein in

100 mM sodium phosphate (pH 8.0), 2 mM EDTA, and 30% (v/v) DMSO at room temperature in

the dark for 2 h. Separately, the purified peptide was also reacted with iodoacetamide (85 mM in

75% DMF/water (v/v) and 20 mM HEPES, pH 7.8) at 25°C in the dark for 2 h. The resulting

peptides ("Rev-Fl" and "Rev-IA", respectively) were then purified on a C-18 semiprep HPLC

column with an mixture of acetonitrile (0.1% TFA) and water (0.1% TFA); isocratic conditions

were 19% acetonitrile for Rev-Fl and 14% acetonitrile for Rev-IA. Electrospray mass

spectrometry confirmed exact masses for Rev-Fl (3,312 amu) and Rev-IA (2,982 amu). Analytical

HPLC confirmed a greater than 95% purity for each peptide. Molecular extinction coefficients

were taken as 77,000 cm-1 M-1 for Rev-Fl (498 nm), and 5,600 cm-1 M-1 for Rev-IA (280 nm).

Synthesis of 67-nt R R E

T7 RNA polymerase was utilized for "run-off" in vitro transcription with a complementary, 83-nt

DNA template as described by Uhlenbeck (Uhlenbeck, O.C.; Milligan, J.F. Methods in

Enzymology 1989 , 180, 51-62). Synthesis of the 83-nt DNA template was carried out using

standard phosphoramidite chemistry. The oligonucleotide was purified using denaturing

polyacrylamide gel electrophoresis (PAGE), followed by extraction and multiple rounds of ethanol

precipitation. The sequence and homogeneity of the template was confirmed using di-deoxy

sequencing techniques. Transcription products were also purified using denaturing PAGE,

extraction and multiple rounds of ethanol precipitation. The expected sequence of the 67-nt RNA

transcript was verified by 5' end labeling with 32P and subsequent enzymatic digestion (RNase T1,

A, and U2). The molecular extinction coefficient (260 nm) for the 67-nt RNA transcript was taken

as 741,400 cm-1 M-1.

Gel Mobility Shift Assay

RRE (25 nM with trace 5’-32P labeled RRE) in 10 mM HEPES (pH 7.5), 100 mM KCl, 0.5 mM

Na2EDTA, and 1 mM MgCl2 was heated to 90 ˚C for 90 sec and slowly cooled to room temperature

over 1h. 100 µg/mL BSA, 0.01% Nonidet P-40, 50 µg/mL tRNAmix (Sigma yeast type X) and 2

µM Rev (Rev-IA) were added to the cocktail and incubated on ice for 30 min. The cocktail was

aliquoted to individual tubes containing neomycin B (500 nM–25 µM), or neo–acridine (10

nM–2.5 µM) to a total volume of 10 µL. The samples were incubated for an additional 30 min on

ice. Loading buffer (3 µL of 30% glycerol with 0.14 nM bromophenol blue, and 0.19 nM xylene

cyanol FF) was added, and samples were loaded on a 10% or 17% native polyacrylamide gel and

run at 4 ˚C in a 1×running buffer (1× Tris–Borate, pH 7.5). The gel was exposed to a

phosphorimager screen and individual bands were quantified on a Molecular Dynamics

Phosphorimager™ 445 SI and analyzed with Imagequant™ software (Molecular Dynamics).

Figure S2a. Gel mobility shift of RRE in the presence of increasing concentrations of neomycin

B or neo–acridine . All lanes contained 25 nM RRE with trace 5’-32P labeled RRE and 2 µM

tRNAmix. Lanes 1 and 16, control; lanes 2–8, 5 µM, 50 µM, 500 µM, 1 mM, 2.5 mM, 5 mM, 10

mM neomycin B, respectively; lanes 9–15, 100 nM, 200 nM, 500 nM, 750 nM, 1.25 µM, 2.5

µM, 5 µM neo–acridine, respectively.

Rev-RRE Gel Shift

The relative affinity of RRE to Rev was also measured by gel-shift mobility assays under the

conditions reported above. Rev-IA was titrated into a solution containing 25 nM of folded,

radiolabeled RRE.

Figure S2b. Concentrations of Rev are 0 µM, 0.25µM, 1 µM, and 4 µM, respectively.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Neomycin B Neo–acridine

Fluorescence Anisotropy Measurements

A Rev-RRE complex was formed by mixing 10 nM Rev-Fl with 8.5 nM of the pre-folded 67-nt

RRE (folded as above except in 2 mM MgCl2).

Fluorescence anisotropy experiments were conducted at 22 °C in the presence of Rev-Fl (10 nM) in

a buffer containing 30 mM HEPES (pH 7.5), KCl (100 mM), sodium phosphate (10 mM),

NH4OAc (10 mM), guanidinium hydrochloride (10 mM), MgCl2 (2 mM), NaCl (20 mM), EDTA

(0.5 mM), and Nonidet P-40 (0.01%).

The Kd obtained for Rev-Fl binding to the RRE was used to calculate the K i values for

neo–acridine and Rev. See: Heyduk, T.; Lee, J.C. Proc. Natl. Acad. Sci. USA 1990 , 87, 1744-

1748; Sevenich, F.W.; Langowski, J.; Weiss, V.; Rippe, K. Nucleic Acids Res. 1998, 26, 1373-

1381

Upon binding the RRE, only minor changes in the emission spectrum of fluorescein were seen

(about 10% quenching of Rev-Fl). We have taken the change in anisotropy as being directly

proportional to the fraction of Rev-Fl bound by the RRE. At the starting point of each titration

there is 5 nM of unbound Rev-Fl, 5 nM Rev-Fl/RRE complex and 3.5 nM free RRE (see figure

S3a). Following complex formation, an inhibitor [I] is titrated into the thermocontrolled cuvette,

and a decrease in anisotropy is observed (see figure S3b for an example). At high concentrations

of inhibitor, the change in anisotropy saturates at 0.081 (the same value observed for the unbound

Rev-Fl peptide). Sufficient mixing time was always provided to allow for equilibrium to be

reached. At the concentration of inhibitor which disrupts half of the formed Rev-RRE complex,

there must be 2.5 nM of the Rev-Fl/RRE complex, 7.5 nM of free Rev-Fl, 5.23 nM of the

RRE/inhibitor complex, and 0.77 nM of free RRE (calculated from the Kd of Rev-Fl/RRE). From

a simple, three component, competitive binding equilibrium, the following equation can be derived:

The Kd of the Rev-RRE interaction is calculated by titration of the prefolded 67-nt RRE fragment

into a solution of 10 nM Rev-Fl.

[ RRE ] + [ RevFl ] [ RevFl - RRE ]

Kd = 2.3 nM+

[ I ]

Ki

[ I - RRE ]

[ RevFl ]

Kd [ RevFl-RRE ]

[ I - RRE ]Ki =

[ I ]

KD = 2.4 nM

0 50 100 150 200 250 300

0.080

0.085

0.090

0.095

0.100

0.105

0.110

0.115

0 2 4 6 8 10 12 14 160.080

0.082

0.084

0.086

0.088

0.090

0.092

0.094

0.096

0.098

Kd = 2.3 nMA

niso

trop

y

Concentration of Neomycin B (µM)

Ani

sotr

opy

Concentration of RRE (nM)

IC50 = 0.8 µM

a.

b.

A= anisotropy of the Rev-Fl A0= anisotropy of the Rev-Fl in the absence of RNA∆A=the total change in anisotropy at saturation of the Rev-Fl

Figure S3a. Both line shape and gel shift analysis indicate a 1:1 complex of the RRE to Rev-Fl. Given this, a Kd of 2.3 nM was calculated by nonlinear regression using the equation:

A =A0 + ∆A([RNA]total + [Rev-Fl]total + Kd) - ([RNA]total + [Rev-Fl]total + Kd)2 - 4[RNA]total [Rev-Fl]total ]

2[Rev-Fl]total

Figure S3b. Example of a displacement experiment where the Rev-RRE complex is disrupted with increasing concentration of inhibitor.

Enzymatic footprinting of RRE

RRE (25 nM with trace 5’-32P end labeled RRE) in 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and

1 mM MgCl2 was heated to 90 ˚C for 90 sec and slowly cooled to room temperature over 1 h. 100

µg/mL BSA, 0.01% Nonidet P-40, and 50 µg/ mL tRNAmix (approx. 2 µM) were added to the

cocktail and incubated at room temperature for 5 min. The cocktail was aliquoted out to individual

tubes containing Rev (500 nM–10 µM) or neo–acridine (500 nM–10 µM) and were incubated for 1

h at room temperature. RNases were added to individual tubes containing either Rev-IA peptide or

neo–acridine to a total volume of 10 µL. The solutions were incubated for 30 min at room

temperature and then quenched on ice with an equal volume of loading buffer (8 M urea, 50 mM

EDTA, 0.1×TBE, 0.14 nM bromophenol blue, and 0.19 nM xylene cyanol FF). The tubes were

heated to 90 ˚C for 90 sec and half of the sample was loaded on 13.5% polyacrylamide/7 M urea

gel while the other half was loaded on 20% polyacrylamide/7 M urea gel. Enzymes used include

1000 units/µL RNase T1 (cleaves primarily single stranded GpN after the 3’ phosphate), 7.2

units/µL RNase V1 (cleaves indiscriminately double stranded regions after the 3’ hydroxyl group)

or 1 × 10-5 units/µL RNase A (cleaves single stranded PypN after the 3’ phosphate). The gel was

exposed and individual bands were quantified on a Molecular Dynamics Phosphorimager™ 445 SI

and analyzed with Imagequant™ software (Molecular Dynamics).

Shown below is an enzymatic footprinting gel (Figure S4). Bar graphs (Figure S5–S8) show

quantitative representation of both suppressed and enhanced cleavage of the RRE in the prescience

of either the Rev-IA peptide or neo–acridine. Cleavage was normalized by dividing the amount of

cleavage observed in the experimental lane by the control lane. The normalized value obtained was

multiplied by 100 to give percent cleavage. To present footprinting data as negative numbers and

enhanced cleavage as positive values, 100 was subtracted from the percent cleavage (this procedure

brings all unaltered residues to a baseline value of zero). Different enzymes recognize different

structural features and, therefore, reveal structure as well as sequence dependent complexity.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Enzymatic Footprinting

Figure S4. Enzymatic cleavage of RRE by ribonuclease T1, ribonuclease A, and ribonuclease V1. All lanes contained 25 nM RRE with trace 5’-32P labeled RRE. Lane 1, control; Lanes 2–10 contain 1000 units ribonuclease T1; lane 3, 0.5 µM Rev; lane 4, 2 µM Rev; lane 5, 5 µM Rev; lane 6, 10 µM Rev; lane 7, 0.5 µM neo−acridine; lane 8, 2 µM neo−acridine; lane 9, 5 µM neo−acridine; lane 10, 10 µM neo−acridine; lane 11, T1 sequencing; lane 12, U2 sequencing; lane 13, base-generated ladder. Lanes 14–22 contain 1 × 10-5 units ribonuclease A; lane 15, 0.5 µM Rev; lane 16, 2 µM Rev; lane 17, 5 µM Rev; lane 18, 10 µM Rev; lane 19, 0.5 µM neo −acridine; lane 20, 2 µM neo−acridine; lane 21, 5 µM neo−acridine; lane 22, 10 µM neo−acridine. Lanes 23–31 contain 7.2 units ribonuclease V1; lane 24, 0.5 µM Rev; lane 25, 2 µM Rev; lane 26, 5 µM Rev; lane 27, 10 µM Rev; lane 28, 0.5 µM neo−acridine; lane 29, 2 µM neo−acridine; lane 30, 5 µM neo−acridine; lane 31, 10 µM neo−acridine; lane 32, base-generated ladder.