Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3′ cleavage reaction

Article abstract of DOI:10.1016/j.bmc.2013.12.006

The 3′ end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3′ untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3′ cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3′ cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine β-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200 μM to the 200 nM-100 μM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3′ end formation.

Full text of DOI:10.1016/j.bmc.2013.12.006

Bioorganic & Medicinal Chemistry 22 (2014) 834–841
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structurally diverse low molecular weight activators
of the mammalian pre-mRNA 30 cleavage reaction
⇑
Min Ting Liu , Nagaraja N. Nagre , Kevin Ryan
Department of Chemistry, The City College of New York, The City University of New York, New York, NY 10031, USA
a r t i c l e i n f o
a b s t r a c t
The 30 end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the
downstream boundary of the 30 untranslated region, which in many genes carries regulatory sequences. A
large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools
to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase nega-
tively regulates the 30 cleavage reaction, we have found a variety of new small molecule activators of
the in vitro reconstituted pre-mRNA 30 cleavage reaction. New activators include a cyclic peptide PPM1D
Article history:
Received 20 September 2013
Revised 23 November 2013
Accepted 3 December 2013
Available online 15 December 2013
Keywords:
RNA
inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved
nine b-naphthylamide analog. The minimal concentration required for in vitro cleavage has been
improved from 200 M to the 200 nM–100 M range. These compounds provide unexpected leads in
the search for small molecule tools able to affect pre-mRNA 30 end formation.
Ó 2013 Elsevier Ltd. All rights reserved.
L-argi-
RNA processing
Small molecule activators
Polyadenylation
30 end formation
Histone code
l
l
Naphthalene ring
1. Introduction
not yet available, but would enable new experimental inquiries
into the multi-protein complex that carries out 30 cleavage, and
Eukaryotic pre-messenger RNA (pre-mRNA) undergoes multiple
processing steps prior to translation, and each of these steps pro-
vides the cell with an opportunity for gene expression regulation.1
The pre-mRNA 30 cleavage step, a site-specific RNA hydrolysis reac-
tion that specifies where the polyadenylate (poly(A)) tail will be
added to the mRNA, can take place at different locations near the
end of a nascent mRNA, downstream of the translation stop codon.
This variability leads in many genes to alternative mRNA isoforms
with different 30 untranslated regions (30UTRs) between the stop
codon and the poly(A) tail.2,3 The pre-mRNA 30 cleavage step can
influence the stability of the mature mRNA because many 30UTRs
contain destabilizing sequences and miRNA binding sites, the loss
of which, when 30 cleavage occurs relatively close to the stop
codon, may slow turnover and allow escape from repression.2,4–6
Shorter 30UTRs resulting from alternative polyadenylation in some
oncogenes have been correlated with cellular proliferation and
may contribute to oncogenesis.7,8 The 30 cleavage and polyadenyl-
ation reactions are coupled in vivo but can be studied separately
in vitro. Natural product inhibitors of the pre-mRNA polyadenyla-
tion step have recently been found.9,10 However, low molecular
weight compounds that influence the 30 cleavage step in cells are
might provide
polyadenylation.
a
chemical tool to influence alternative
We previously found in vitro evidence that a kinase–phospha-
tase pair may exert influence over the 30 cleavage reaction.11 Con-
sidering this possibility led us to propose that creatine phosphate,
long known to stimulate the in vitro reaction at high concentra-
tion,12,13 might do so by acting as a serendipitous inhibitor of an
unknown 30 cleavage-suppressing protein phosphatase. This model
led to experiments identifying two protein phosphatase 2C (PP2C,
a.k.a. PPM1) family inhibitors that stimulated 30 cleavage in place
of creatine phosphate.14 In humans, the PPM1 superfamily consists
of at least eighteen different Mg2+- or Mn2+-dependent Ser/Thr
phosphatases defined by shared sequence homology.15–18 In
plants, the PPM1 family is greatly expanded, with at least 80 family
members in Arabidopsis.19 The in vitro 30 cleavage reaction is car-
ried out in the presence of excess EDTA, where free Mg2+ and
Mn2+ concentrations are very low. Thus, if a PPM1 family member
is involved in vitro, it would likely act in a manner that does not
require its metal-dependent enzymatic activity, perhaps acting
allosterically or by influencing the multi-protein 30 cleavage factor
complexes.
While progress in other major protein phosphatase families has
benefited from the identification of natural product inhibitors,20
inhibitor discovery for the PPM1 family has been slower. The inhib-
itors we previously identified as 30 cleavage activators had been
⇑
Corresponding author. Tel.: +1 2126508132; fax: +1 2126506107.
E-mail address: kr107@sci.ccny.cuny.edu (K. Ryan).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.006

M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
835
discovered using the PPM1A family member.21 They had micromo-
lar potency against PPM1A and only modest selectivity for the
PPM1 family over the other major protein phosphatase families.21
By analogy with the toxin inhibitors of the other protein phospha-
tase families, we expect PPM1 inhibitors to show a measure of
family-specific activity, enabling 30 cleavage activation even if
PPM1A is not the actual family member involved in pre-mRNA 30
cleavage. Here, we have tested more potent PPM1 inhibitors for
pre-mRNA 30 cleavage activity, and carried out structure–activity
relationships for two previously identified 30 cleavage activators.
Though we are not yet able to identify the protein targeted by
these small molecules, our results strengthen a model in which a
PPM1 family member or related protein acts to suppress 30 cleav-
age in vitro.
tyrosine-specific phosphatase, did not reduce activity. (Residual
CIP activity was inhibited11 before cp38 was added to the in vitro
reaction to prevent possible dephosphorylation of the cleavage fac-
tor proteins.) A repeat of this experiment is shown in Supplemen-
tary Figure S1. Cyclopeptide cp38 is therefore ꢀ1000-fold more
potent than the most potent 30 cleavage activator previously iden-
tified,14 and phosphorylation of its serine, which is required for
PPM1 inhibition,23 is required to activate pre-mRNA 30 cleavage.
PPM1D is present at very low levels in HeLa cells,27 an observation
that leads us to suppose that cp38 is likely not working through
this PPM1 family member, but may instead be a family-specific
inhibitor that activates 30 cleavage through another family mem-
ber, or homologous protein.
2.2. Pre-mRNA 30 cleavage activation by abscisic acid
2. Results and discussion
To further test the hypothesis that a PPM1 family member acts
to suppress 30 cleavage, we used the isoprenoid plant hormone (S)-
(+)-abscisic acid (ABA). This natural product is a potent inhibitor of
the group A plant PPM1 enzymes.28 It works by forming a ternary
complex with the PPM1 enzyme and a co-receptor from the PYR/
PYL/RCAR family of ABA receptors.28,29 For example, ABA inhibits
the plant PPM1s ABI1 and ABI2 with IC50 values of 60 and 70 nM,
respectively, when RCAR1 is present.29 ABA is bound mainly by
the PYR/PYL/RCAR receptor, but also makes contact with the
PPM1.30,31 In the absence of a PYR/PYL/RCAR co-receptor, ABA
can directly inhibit some plant PP2C/PPM1 enzymes in vitro,
though with much lower potency. For example, ABA reduces
ABI2 phosphatase activity in vitro by about 15% at ABA concentra-
2.1. Pre-mRNA 30 cleavage activation by a potent PPM1D
cyclopeptide inhibitor
Cyclic phosphopeptides based on substrates of the PPM1D fam-
ily member, also known as Wip1, are among the most potent
inhibitors of any PPM1 family phosphatase.22,23 We used one of
these, cyclic peptide 38 (cp38, Fig. 1A),22 in place of 50 mM crea-
tine phosphate in the in vitro pre-mRNA 30 cleavage reaction. This
peptide inhibits PPM1D with a Ki of 150 nM.22 In the 30 cleavage as-
say, a standard in vitro pre-mRNA 30 cleavage substrate adapted
from the simian virus 40 (SV40) late poly(A) signal24 is exposed
to partially purified 30 cleavage factors from HeLa cell nuclear
extract.11,13,25,26 Cleavage of the radiolabeled substrate into its 50
and 30 fragments can be observed and quantitated following reso-
lution on a denaturing polyacrylamide gel. In the absence of crea-
tine phosphate, or other activator, this reaction is inefficient. In all
experiments presented here, the amount of cleavage brought about
by an activator (50 fragment/(50 fragment + uncleaved SV40L RNA))
is normalized to that brought about by 50 mM creatine phosphate
(R.C., or relative cleavage in figures). As shown in Figure 1B, at
200 nM, cp38 activated a level of RNA cleavage comparable to that
produced by 50 mM creatine phosphate. Removal of cp38’s serine
and tyrosine phosphate groups with calf intestinal alkaline
phosphatase (CIP) prior to cleavage, led to complete loss of
activation, while removal of only the tyrosine phosphate, using a
tions above 3
when nanomolar concentrations of ABA were used in place of cre-
atine phosphate (not shown). However, at and above 100 M ABA,
l
M.29 We found no pre-mRNA 30 cleavage activation
l
approximately 10–15% of the normal 30 cleavage activity was ob-
served (cf. 50 mM creatine phosphate) (Fig. 1C). Two repeats of this
experiment are shown in Supplemental Figure S2. We consider two
possible interpretations of this result. First, the absence of activa-
tion at nanomolar ABA concentration may indicate that there are
no conserved mammalian proteins that can act in the manner of
the plant PYR/PYL/RCAR co-receptors, though ABA is found in ani-
mal cells.32 A BLAST search did not reveal any human RCAR homo-
logs. Second, homology between the plant and human PPM1
proteins may be sufficient to conserve the direct but much less
Figure 1. Pre-mRNA in vitro 30 cleavage activation by PP2C inhibitors. (A) Structure of cyclic peptide cp38 and (S)-(+)-abscisic acid (ABA). (B) Denaturing gel analysis of SV40L
pre-mRNA 30 in vitro cleavage reactions activated by cp38. Creatine phosphate (CP) or cp38 was added, with or without pre-treatment with CIP (calf intestinal alkaline
phosphatase), mock CIP (CIP buffer only) or tyrosine phosphatase (YOP). (Residual CIP activity was completely removed before mixing cp38 with cleavage factor proteins.11
)
Relative cleavage, R.C. = [50 fragment/(50 fragment + uncleaved RNA)] ꢁ 100, was normalized to CP, which is set to R.C. = 1.00. (C) Denaturing gel analysis of in vitro cleavage
using ABA in place of CP. DMSO lane corresponds to reaction without ABA.

836
M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
potent inhibition of PPM1 family members. In either case, though
its cleavage activation efficiency was low, compared to our first
generation compounds, ABA is a relatively potent 30 cleavage acti-
vator. This result adds to the accumulating though circumstantial
evidence that a PPM1 family member can influence pre-mRNA
processing.
in solubility, a point of practical importance due to the high activa-
tor concentrations needed for activity. In view of the poor results
changing the naphthyl and guanidino groups of lead compound
1, we decided to modify the a-amino group. To minimize a reduc-
tion in solubility we chose the morpholine group for its high water
solubility (compound 7, Fig. 3, and Scheme 1C). Unlike the other
changes, this modification retained activity.
A
representative
M (as
2.3. Structure–activity study of arginine b-naphthylamide: aryl
in vitro 30 cleavage comparison of 7 with 1 over 25–200
l
group and sidechain
usual, in place of, and compared to, creatine phosphate) is shown
in Figure 3B. Corroborating experiments are shown in Supplemen-
tary Figure S3. Compound 7 proved to be slightly more potent than
lead compound 1, and to have a similar maximum relative cleavage
(Fig. 3B). While only slightly more potent than 1, the activity of
compound 7 indicates that modifications of the alpha amine can
be tolerated, and that this part of the molecule merits a larger
SAR study beyond the scope of the present work.
Prior to the present work, compound 1 (Fig. 2A) was identified
as a 30 cleavage activator through a limited structure–activity rela-
tionship study undertaken using commercially available com-
pounds related to our initial lead, leucine b-naphthylamide, a
weak PPM1A inhibitor.14 To search for a more potent activator than
1, we synthesized a series of analogs in which the naphthalene ring
was altered (Fig. 2A, 2–4) or the guanidino group was alkylated or
replaced by another positively charged group (Fig. 2A, 5–6). The
synthesis of compounds 2–6 (Scheme 1A and B) was accomplished
by coupling the relevant aryl amine to the suitably protected
arginine, ornithine or lysine, followed by guanidinylation where
necessary, and finally deprotection. We evaluated each of the com-
pounds shown in Figure 2A for their ability to activate in vitro 30
cleavage at 1 mM in place of, and compared to, 50 mM creatine
phosphate. The results of duplicate reactions are shown in four
independent 30 cleavage reactions in Figure 2B–E. Changes to the
naphthalene ring and guanidino group of 1 led to loss of activity
(compounds 2, 3, 4, 5), and the guanidino group could not be re-
placed by trimethylammonium (compound 1 vs 6), even though
the two groups have a positive charge.
2.5. Structure–activity study on phosphocholine
We previously identified phosphocholine as a pre-mRNA 30
cleavage activator. Like creatine phosphate, it activates 30 cleavage
over the 10–50 mM range.14 Its ability to inhibit PPM1 family
proteins is unknown. Phosphocholine is an intermediate in the bio-
synthesis of phosphatidylcholines, and it is unlikely to be a physi-
ological cofactor in 30 cleavage.14 However, understanding the
determinants of 30 cleavage activity of this very simple structure
might help us to design more potent activators. To learn whether
all three methyl groups are necessary for activity, we synthesized
the mono- and dimethyl phosphocholine analogs, 9 and 10
(Scheme 2A). Their synthesis is shown in Scheme 2B. We also made
an analog of phosphocholine with an extra carbon between the
charged groups (compound 11, Scheme 2A, C). None of these com-
pounds showed detectable in vitro 30 pre-mRNA cleavage activity
(not shown). The lack of activity in these analogs showed that all
three methyls of the trimethylammonium group are required,
and the two-carbon spacing between this group and the phosphate
is critical.
2.4. Arginine b-naphthylamide modified at the a-position
retains potency
In previous work we found that acetylation of the
a-amino
group of leucine b-naphthylamide led to some loss of that
compound’s 30 cleavage activity, as well as a significant decrease
Figure 2. Structure–activity study of compound 1, previously found to activate pre-mRNA 30 cleavage in vitro. (A) Analogs included changes to the naphthyl group and the
arginine side-chain of 1. (B) In vitro 30 cleavage results for duplicate cleavage reactions using the indicated analogs in place of 1, all at 1 mM. R.C., relative cleavage, defined in
Figure 1, normalizes all compounds to activation by 50 mM CP.

M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
837
Scheme 1. (A and B) Synthesis of the arginine b-naphthylamide analogs used in Figure 2. (C) Synthesis of morpholine analog 7 used in Figure 3.
trimethyllysine and phosphoserine (or threonine) residues
(Fig. 4A). In an initial test of this idea, we replaced 50 mM crea-
tine phosphate in a 30 cleavage reaction with dipeptide 12, Ac-
Lys(Me)3-Ser(PO4)-CO2H (Fig. 4B). Peptide 12 may also be viewed
as a derivative of phosphoserine which, like creatine phosphate,
activates 30 cleavage in the 10–50 mM range.38 Peptide 12 acti-
vated in vitro 30 cleavage at ꢀ1 mM (Fig. 4C), while phosphoser-
ine and phosphocholine14 have no effect at this concentration.
This result shows that placing the two functional groups of phos-
phocholine in the structural context of a dipeptide, where they
can approach each other as closely as they do in phosphocholine,
reduced the concentration necessary for them to activate 30 cleav-
age. Viewed differently, placing phosphoserine in a dipeptide cou-
pled to N-(acetyl)trimethyllysine (to create 12) led to increased
potency. While far from directly implicating a histone protein in
the 30 cleavage reaction, this result indirectly supports the possi-
bility that phosphocholine works by mimicking such a cleavage-
promoting modified histone variant. We note that the partially
purified protein 30 cleavage factors used in our assay are fraction-
ated from soluble nuclear proteins, and not from the chromatin-
rich insoluble nuclear pellet, so a cleavage-enhancing chromatin
protein would be depleted or lost during cleavage factor prepara-
tion. Interestingly, the PPM1G family member plays a role in
histone H2A–H2B exchange into chromatin.39 PPM1G is also in-
volved in splicesomal splicing,40 a pre-mRNA processing step that
can be coupled to 30 cleavage.41 In view of this context, this result
justifies additional study of suitably modified lysine, serine and
threonine-containing peptide sequences taken from the heavily
modified histone protein tails.
Figure 3. Structure and 30 cleavage activity of a morpholine-modified arginine b-
naphthylamide analog. (A) Structure of 7. (B) Side by side comparison of 1 and 7 in
an SV40L pre-mRNA 30 in vitro cleavage reaction. R.C., relative cleavage as defined
in Figure 1, with activation by 50 mM creatine phosphate (CP) set to 1.00.
2.6. Phosphocholine as a possible modified histone protein
mimic
Trimethyllysine and phosphoserine residues are commonly
found among the histone proteins of chromatin,33 where in vivo
transcription takes place. Since several of the 30 cleavage factor
proteins associate with transcribing RNA polymerase II34,35 and
the 30 cleavage reaction is coupled to transcription and begins
co-transcriptionally,36,37 it is conceivable that a histone protein
could, within the chromatin, locally interact with RNA polymer-
ase II-associated cleavage factor proteins to play a role in 30
cleavage. In trying to understand how phosphocholine activates
30 cleavage in vitro, we have considered the possibility that it
may mimic a modified histone protein having closely situated

838
M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
Scheme 2. Phosphocholine (PhC) analogs. (A) Structures of the analogs. (B) Synthesis of analogs 9 and 10. (C) Synthesis of analog 11.
Figure 4. Phosphocholine (PhC) as a mimic of a modified histone protein tail. (A) Phosphocholine holds a trimethylammonium group close to a phosphate, as a histone
protein can do with trimethylated lysine and phosphoserine (or phosphothreonine). (B) Structure of dipeptide 12. (C) SV40L pre-mRNA 30 cleavage activation by peptide 12
compared to phosphocholine (PhC), creatine phosphate (CP) and phosphoserine (PS). R.C., relative cleavage, as defined in Figure 1.
(S)-(+)-Abscisic acid (ABA), phosphocholine (PhC), and phosphoser-
ine (PS) were purchased from TCI America. Creatine phosphate (CP)
was purchased from Calbiochem. Compound 1 was purchased from
Bachem. Compound 8 was purchased from Fluka. Compound 12
was purchased from Shanghai Apeptide Co., Ltd. Unless otherwise
stated, starting reagents were purchased from VWR. Analytical
TLC was performed on silica gel 60 F254 plates. Flash chromatogra-
phy was performed manually in glass columns on 230–400 mesh
silica gel (Alfa Aesar). Analytical samples were dried overnight in
vacuo over phosphorus pentoxide prior to testing. Melting points
were measured on a Laboratory Devices Melt-Temp apparatus. 1H
and 13C NMR spectra were recorded on a Varian Mercury 300 spec-
trometer or Varian Inova 500 spectrometer. High-resolution mass
spectra (HRMS) were measured using electrospray ionization
(ESI) on a Waters LCT XE (TOF) mass spectrometer. Infrared (IR)
spectra were recorded using a Thermo Nicolet 380 FT-IR spectrom-
eter or Thermo Nicolet 6700 FT-IR spectrometer.
3. Conclusions
Guided by a model proposing that a PPM1 phosphatase acts to
suppress or negatively regulate the pre-mRNA 30 cleavage reaction,
an obligatory step in the maturation of nearly all eukaryotic
mRNAs, we have assayed a group of structurally diverse PPM1
inhibitors and new synthetic small molecules for their ability to
activate this reaction in vitro. Our results lend further, though cir-
cumstantial, support to this model. The minimal concentration re-
quired for in vitro cleavage has been improved from 200
lM to the
200 nM-100 M range. In addition, our results point to future
l
experiments aiming to further the goal of developing membrane-
permeable small molecule activators of 30 cleavage sufficiently po-
tent for use in tissue culture experiments.
4. Experimental section
4.1. Materials and methods
4.2. General procedure for the synthesis of compounds 2–4
Unless otherwise stated, commercial reagents and solvents were
used without additional purification. Solvents were purchased from
VWR. Cyclic peptide 38 (cp38) was made as previously described.22
Z3-Arg-OH (Z, benzyloxycarbonyl) was dissolved in dichloro-
methane. 1.2 equiv of the amine was added followed by 2.5 equiv

M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
839
of 1-hydroxy-7-azabenzotriazole (HOAt). 1.5 equiv of 1-ethyl-3-(3-
dimethyllaminopropyl)carbodiimide hydrochloride (EDC) was dis-
solved in dichloromethane and slowly added to the reaction at
room temperature. Reaction progress was monitored by TLC (ethyl
acetate:dichloromethane). The solution was washed sequentially
with 0.5 M HCl solution, water, brine, dried, and concentrated.
The crude was purified by flash chromatography.42 The resulting
product was hydrogenated in methanol using a latex balloon with
catalytic amount (approximately a spatula tip) of palladium
hydroxide (Pd 20% on carbon, nominally 58% water) for 3 h. The
solution was then filtered through celite and concentrated to yield
the final product.
in dichloromethane (2 mL) and deprotected with trifluoroacetic
acid (2 mL) overnight. The solution was concentrated, suspended
in fresh dichloromethane, and extracted with water. The aqueous
layer was concentrated and dried overnight in a drying pistol with
phosphorus pentoxide to give 5 as a sticky, white solid in 68% yield.
1H NMR (300 MHz, D2O) d: 7.59–7.81 (m, 3H), 7.24–7.45 (m, 4H),
4.11–4.23 (m, 1H), 3.05 (m, 2H), 2.85 (q, J = 6.88 Hz, 2H), 1.85–
2.02 (m, 2H), 1.60 (m, 2H), 0.81 (t, J = 7.01 Hz, 3H). 13C NMR
(75 MHz, D2O) d: 169.49, 163.23, 162.76, 155.38, 133.99, 130.53,
128.39, 127.09, 126.75, 125.64, 123.90, 122.11, 118.25, 114.38,
53.35, 53.13, 40.15, 36.20, 28.26, 23.84, 13.02. IR (neat)
m
(cmꢂ1):
3208, 3051, 1659, 1542, 1181, 1131, 798, 721. HRMS (ESI)
[M+H]+: calcd for C18H25N5O m/z = 328.2137, found m/
z = 328.2121.
4.2.1. 2-Amino-5-guanidino-pentanoic acid phenylamide (2)
White solid (yield 72%, mp 110 °C dec). 1H NMR (300 MHz, D2O)
d: 7.17–7.32 (m, 4H), 7.10 (m, 1H), 3.89 (t, J = 6.33 Hz, 1H), 2.95–
3.11 (m, 2H), 1.70–1.87 (m, 2H), 1.43–1.61 (m, 2H). 13C NMR
(75 MHz, D2O) d: 169.49, 156.78, 135.98, 129.51, 129.35, 126.24,
4.2.5. [5-Amino-5-(naphthalen-2-ylcarbamoyl)-pentyl]-
trimethyl-ammonium (6)
Boc-Lys(Z)-OH (1 g, 2.6 mmol) was dissolved in dichlorometh-
ane (50 mL). 2-Naphthylamine44 (0.37 g, 2.6 mmol) was added fol-
lowed by HOAt (0.89 g, 6.54 mmol) and EDC (0.76 g, 3.96 mmol).
After 1.5 h, the solution was concentrated and purified by flash
chromatography, eluting with chloroform/ethyl acetate (4:1) to
give 6a (800 mg, 1.58 mmol), in 61% yield. Compound 6a
(288 mg, 0.57 mmol) was suspended in methanol (25 mL) and
hydrogenated with a catalytic amount of palladium hydroxide.
After 1.5 h, the solution was filtered through celite and concen-
trated to give 6b (212 mg, 0.57 mmol) in quantitative yield. Com-
pound 6b (205 mg, 0.55 mmol) was then dissolved in acetonitrile
(1 mL) and potassium carbonate (191 mg, 1.38 mmol) was added
followed by iodomethane (392 mg, 2.76 mmol). The solution was
stirred overnight, concentrated, suspended in dichloromethane,
and filtered. The filtrate was concentrated and the crude com-
pound was dissolved in dichloromethane (2 mL) and trifluoroacetic
acid (2 mL) was added. The solution was stirred for 1.5 h and then
concentrated, suspended in fresh dichloromethane, and extracted
with water. The aqueous layer was concentrated and dried over-
night over phosphorus pentoxide to give 6 as an orange, sticky so-
lid in 85% yield. 1H NMR (300 MHz, D2O) d: 7.88 (s, 1H), 7.66–7.78
(m, 3H), 7.28–7.40 (m, 3H), 3.99 (t, J = 6.60 Hz, 1H), 3.06–3.15 (m,
122.05, 121.83, 53.80, 53.51, 40.43, 28.65, 23.63. IR (neat)
m
(cmꢂ1): 3175, 1655, 1596, 1546, 1442, 1360, 1309, 1253, 752,
691. HRMS (ESI) [M+H]+: calcd for C12H19N5O m/z = 250.1668,
found m/z = 250.1655.
4.2.2. 2-Amino-5-guanidino-pentanoic acid naphthalen-1-
ylamide (3)
White solid (yield 62%, mp 166 °C dec). 1H NMR (300 MHz, D2O)
d: 7.57–7.75 (m, 3H), 7.21–7.41 (m, 4H), 4.18 (t, J = 6.33 Hz, 1H),
2.99 (t, J = 6.60 Hz, 2H), 1.79–1.99 (m, 2H), 1.47–1.73 (m, 2H). 13C
NMR (75 MHz, D2O) d: 169.52, 156.62, 133.93, 130.49, 128.71,
128.46, 128.05, 127.07, 125.63, 124.10, 121.95, 121.66, 53.35,
53.11, 40.34, 28.33, 23.86. IR (neat)
m
(cmꢂ1): 3149, 1658, 1536,
1497, 1348, 793, 770. HRMS (ESI) [M+H]+: calcd for C16H21N5O
m/z = 300.1824, found m/z = 300.1816.
4.2.3. 2-Amino-5-guanidino-pentanoic acid quinolin-3-ylamide
(4)
Yellow solid (yield 30%, mp 113 °C dec). 1H NMR (300 MHz,
D2O) d: 9.34 (s, 1H), 8.97 (s, 1H), 8.02 (d, J = 8.80 Hz, 1H), 8.07 (d,
J = 8.53 Hz, 1H), 7.91 (t, J = 7.70 Hz, 1H), 7.72–7.84 (m, 1H), 4.19
(t, J = 6.46 Hz, 1H), 3.11 (t, J = 6.74 Hz, 2H), 1.97 (m, 2H), 1.51–
1.73 (m, 2H). 13C NMR (75 MHz, D2O) d: 168.80, 156.79, 138.45,
138.08, 135.98, 135.76, 135.28, 134.63, 131.59, 130.88, 129.07,
2H), 2.84 (s, 9H), 1.83–1.94 (m, 2H), 1.66 (m, 2H), 1.34 (m, 2H). 13
C
NMR (125 MHz, D2O) d: 168.48, 162.78, 133.88, 133.41, 131.29,
129.43, 127.94, 127.84, 127.33, 126.38, 121.10, 119.01, 66.12,
120.31, 119.94, 53.65, 53.39, 40.37, 28.01, 23.66. IR (neat)
m
53.81, 53.05, 30.65, 22.31, 21.32. IR (neat) m
(cmꢂ1): 3049, 1671,
(cmꢂ1): 3159, 1663, 1558, 1491, 1466, 1365, 782, 747. HRMS
(ESI) [M+H]+: calcd for C15H20N6O m/z = 301.1777, found m/
z = 301.1786.
1198, 1128, 832, 799, 720. HRMS (ESI) [M]+: calcd for
[C19H28N3O]+ m/z = 314.2232, found m/z = 314.2223.
4.2.6. 5-Guanidino-2-morpholin-4-yl-pentanoic acid
naphthalen-2-ylamide (7)
4.2.4. 2-Amino-5-(N0-ethylguanidino)-pentanoic acid
naphthalen-1-ylamide (5)43
Boc-Orn(Z)-OH (1 g, 2.73 mmol) was dissolved in dichlorometh-
ane (50 mL). 2-Naphthylamine44 (470 mg, 3.28 mmol) was added
followed by HOAt (929 mg, 6.83 mmol) and EDC (785 mg,
4.09 mmol). Reaction progress was monitored by TLC. The result-
ing material was purified by flash chromatography, eluting with
dichloromethane/ethyl acetate (9:1) to give 7a (570 mg,
1.56 mmol, 49% yield). Compound 7a (320 mg, 0.65 mmol) was
dissolved in dichloromethane (2 mL) with trifluoroacetic acid
(2 mL) and stirred at room temperature overnight. The solution
was concentrated and used for the next step without further puri-
fication. The crude compound was dissolved in acetonitrile (6 mL)
and refluxed with potassium carbonate (360 mg, 2.6 mmol) and
bis(2-bromoethyl)ether (181 mg, 0.78 mmol) overnight. The next
day, the solution was filtered and purified by flash chromatography
(first 1:1 ethyl acetate/dichloromethane then 100% ethyl acetate)
to give 7b (100 mg, 0.22 mmol) as a yellowish solid in 33% yield.
Compound 7b was hydrogenated in methanol (12 mL) with cata-
lytic amount of palladium hydroxide. After 2 h, the solution was
Boc-ornithine(Z)-OH (500 mg, 1.36 mmol) was dissolved in
dichloromethane (25 mL). 1-Naphthylamine (234 mg, 1.64 mmol)
was added followed by HOAt (464 mg, 3.41 mmol) and EDC
(392 mg, 2.05 mmol). Reaction progress was monitored by TLC.
The resulting material was purified by flash chromatography, elut-
ing with dichloromethane/ethyl acetate (9:1) to give 5a (531 mg,
1.08 mmol, 79% yield). Compound 5a (531 mg, 1.08 mmol) was
hydrogenated for 2 h in methanol (50 mL) with a catalytic amount
of palladium hydroxide as described in Section 4.2. The solution
was filtered through celite and concentrated to give 5b. Compound
5b was dissolved in acetonitrile (7 mL) and ethylated N,N0-bis-tert-
butoxycarbonylpyrazole-1-carboxamidine (364 mg, 1.08 mmol)
was added followed by diisopropylethylamine (181 mg,
2.34 mmol). The solution was stirred overnight, concentrated and
purified by flash chromatography, eluting with dichloromethane/
ethyl acetate (4:1) to give 520 mg (0.83 mmol) of a white, sticky
solid. The resulting compound (170 mg, 0.27 mmol) was dissolved

840
M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
filtered through celite and concentrated. The resulting compound
was dissolved in acetonitrile (1 mL) and N,N0-bis-tert-but-
oxycarbonylpyrazole-1-carboxamidine45 (27 mg, 0.87 mmol) was
added followed by DIPEA (15 mg, 0.113 mmol). The reaction was
stirred overnight, concentrated and purified by flash chromatogra-
phy, eluting with ethyl acetate/hexanes (4:1) to give 7c (20 mg,
0.035 mmol) as a sticky, white solid in 41% yield. Compound 7c
was dissolved in dichloromethane (1 mL) and trifluoroacetic acid
(1 mL) was added. The solution was stirred overnight and then
concentrated, suspended in dichloromethane, and extracted with
water. The aqueous layer was concentrated and dried overnight
over phosphorus pentoxide to give 7 in 85% yield. 1H NMR
(300 MHz, D2O) d: 7.96 (s, 1H), 7.70–7.85 (m, 3H), 7.34–7.48 (m,
3H), 3.18–4.05 (m, 9H), 3.11 (t, J = 6.46 Hz, 2H), 1.86–2.15 (m,
2H), 1.45–1.72 (m, 2H). 13C NMR (125 MHz, D2O) d: 166.00,
162.95, 156.95, 133.31, 131.47, 129.47, 127.91, 127.36, 126.56,
120.97, 119.33, 117.67, 115.35, 69.27, 63.74, 40.48, 24.62, 23.86.
methanol (25 mL) with catalytic amount of palladium hydroxide
for 1 h. The solution was filtered through celite, concentrated, and
dried overnight over phosphorus pentoxide to give 10 (70 mg) as
a white solid (mp 155–157 °C) in 87% yield. 1H NMR (300 MHz,
D2O) d: 3.92 (m, 2H), 3.19 (m, 2H), 2.71 (s, 6H). 13C NMR (75 MHz,
D2O) d: 57.87, 42.95, 42.43. IR (neat)
m
(cmꢂ1): 3415, 2629, 2404,
1163, 1149, 1093, 1026, 993, 951, 931, 768. HRMS (ESI) [M+H]+:
calcd for C4H12NO4P m/z = 170.0582, found m/z = 170.0609.
4.2.9. Trimethyl-(3-phosphonooxypropyl)-ammonium iodide
(11)
3,3-Dimethylamino-1-propanol (500 mg, 4.846 mmol) was dis-
solved in ether (4.8 mL) and diphenyl chlorophosphate (1.56 g,
5.816 mmol) was added, followed immediately by triethylamine
(1 g, 9.692 mmol). After 1 h, the solution was concentrated and
purified by flash chromatography, eluting with ethyl acetate/tri-
ethylamine (19:1) to give 11a (1.45 g) in 92% yield. Compound
11a (1.45 g, 4.32 mmol) was added to a solution of sodium hydride,
57–63% in oil, (519 mg, 12.97 mmol) and excess benzyl alcohol in
diethyl ether (14.5 mL). After 1 h, the reaction was concentrated
and the crude was purified by flash chromatography, eluting with
ethyl acetate/triethylamine (19:1) to give 11b (246 mg) in 15%
yield. Compound 11b (246 mg, 0.68 mmol) was dissolved in aceto-
nitrile (1 mL) and iodomethane (192 mg, 1.35 mmol) was added.
After 1 h, the solution was concentrated to give 11c (337 mg),
which was used in the next step without further purification. Com-
pound 11c (337 mg, 0.67 mmol) was first hydrogenated in 50 mL
IR (neat)
1364, 1200, 800, 723, 707. HRMS (ESI) [M+H]+: calcd for
20H27N5O2 m/z = 370.2243, found m/z = 370.2224.
m
(cmꢂ1): 3360; 2877, 2718, 1668, 1563, 1508, 1435,
C
4.2.7. Phosphoric acid mono-(2-methylamino-ethyl) ester (9)
2-Methylaminoethanol (500 mg, 6.66 mmol) was stirred with
sodium bicarbonate (1.12 g, 13.31 mmol) in diethyl ether
(6.6 mL). Benzyl chloroformate, 30–35% in toluene, (3.6 g,
6.66 mmol) was slowly added at 0 °C. The solution was warmed
to room temperature after 15 min. After 1 h, the solution was con-
centrated and purified by flash chromatography, eluting with
dichloromethane/ethyl acetate (1:1) to give 9a in 63% yield. Com-
pound 9a (300 mg, 1.43 mmol) was dissolved in diethyl ether
(5 mL) and diphenyl chlorophosphate (578 mg, 2.15 mmol) was
added followed by triethylamine (290 mg, 2.87 mmol). After 2 h,
the solution was filtered and purified by flash chromatography,
eluting with dichloromethane/ethyl acetate (9:1) to give 9b
(633 mg) in 81% yield. Compound 9b (500 mg, 1.13 mmol) was
added to a solution containing sodium hydride, 57–63% in oil,
(86 mg, 2.15 mmol) and benzyl alcohol (368 mg, 3.42 mmol) in
diethyl ether (5 mL). After 1 h, the solution was purified by flash
chromatography, eluting with dichloromethane/ethyl acetate
(9:1) to give 9c (324 mg) in 60% yield. Compound 9c (324 mg,
0.69 mmol) was hydrogenated in methanol (25 mL) with catalytic
amount of Pd/C (10% Pd, 50% wet with water) for 1 h. The solution
was filtered through celite, and the celite pad was washed with
water. The solution was concentrated and dried overnight over
phosphorus pentoxide to give 9 (90 mg, 0.58 mmol) as a white so-
lid (mp 204–206 °C) in 84% yield. 1H NMR (300 MHz, D2O) d: 3.76–
3.94 (m, 2H), 2.97–3.13 (m, 2H), 2.49 (s, 3H). 13C NMR (75 MHz,
methanol/water (1:1) with
a catalytic amount of palladium
hydroxide. These conditions removed only one of the two benzyl
groups. The second benzyl group was removed by latex balloon
hydrogenation in 150 mL 30% (v/v) aqueous formic acid with a cat-
alytic amount of Pd/C. The reaction contents were filtered through
a celite pad, concentrated under reduced pressure, and washed
with dichloromethane. The aqueous layer was further evaporated
under reduced pressure, and the residue was exhaustively dried
overnight over phosphorus pentoxide to give 11 (80 mg) as a white
solid (mp 230 °C dec) in 35% yield. 1H NMR (300 MHz, D2O) d: 3.75
(m, 2H), 3.14–3.34 (m, 2H), 2.93 (s, 9H), 1.92 (m, 2H). 13C NMR
(75 MHz, D2O) d: 64.41, 60.60, 52.96, 24.29, 24.20. IR (neat)
m
(cmꢂ1): 3379, 3020, 2957, 2939, 2887, 2432, 1190, 1054, 932.
HRMS (ESI) [M]+: calcd for C6H17NO4P+ m/z = 198.0890, found m/
z = 198.0879.
4.3. Low molecular weight activator stock solutions
Cyclic peptide 38 (cp38), kindly provided by Appella,22 was dis-
solved in water (1 M) and then serially diluted with same. Dephos-
phorylation of cp38 with calf intestinal alkaline phosphatase (CIP;
Promega) and YOP Protein Tyrosine Phosphatase (NEB) were car-
ried out as described for other proteins.11 The possibility of expo-
sure of the cleavage factors to residual CIP activity was
eliminated by using an established and validated procedure.11
(S)-(+)-Abscisic acid (ABA) was dissolved in DMSO (500 mM) and
then serially diluted with water. Compounds 1–7 were placed in
water and treated with 1–3 equiv of 1.00 M HCl to dissolve the
compounds. Compound 1 was purchased as the HCl salt and dis-
solved in water directly. Compounds 8–11 were dissolved in water
and treated with NaOH until the pH reached 6–7.9. (1.1 equiv of
NaOH was added to compound 8 and 9; 1.0 equiv of NaOH was
added to compound 10; 1.5 equiv of NaOH was added to com-
pound 11; 0.25 equiv of NaOH was added to compound 12.)
D2O) d: 60.25, 49.11, 48.99, 32.77, 32.41. IR (neat)
m
(cmꢂ1):
3017, 2729, 2520, 1146, 1075, 1026, 923, 815. HRMS (ESI)
[M+H]+: calcd for C3H10NO4P m/z = 156.0426, found m/z
=
156.0424.
4.2.8. Phosphoric acid mono-(2-dimethylamino-ethyl) ester
(10)
2-Dimethylaminoethanol, 10a, (500 mg, 5.61 mmol) was dis-
solved in ether (5.6 mL) and diphenyl chlorophosphate (1.8 g,
6.73 mmol) was added followed by triethylamine (1.135 g,
11.22 mmol). After 1 h, the solution was concentrated and purified
by flash chromatography, eluting with dichloromethane/triethyl-
amine (9:1) to give 10b (1.37 g) in 76% yield. Compound 10b
(540 mg, 1.68 mmol) was added to a solution containing sodium
hydride, 57–63% in oil, (121 mg, 3.03 mmol) and benzyl alcohol
(545 mg, 5.04 mmol) in ether (5.4 mL). After 1 h, the solution was
concentrated and purified by flash chromatography, eluting with
ethyl acetate/methanol (19:1) to give 10c (340 mg) in 58% yield.
Compound 10c (166 mg, 0.48 mmol) was hydrogenated in
4.4. Cleavage factor fractionation from HeLa cell nuclear extract
HeLa cell pellets were purchased from the National Cell Culture
Center (Biovest International). Nuclear extract and 30 cleavage

M. T. Liu et al. / Bioorg. Med. Chem. 22 (2014) 834–841
841
factor DEAE-sepharose fractions (CPSF, CstF and CFm) were pre-
References and notes
pared as described in detail elsewhere.11,25 Active fractions from
each cleavage factor were pooled, concentrated by 70% ammonium
sulfate precipitation and dialyzed 2 ꢁ 3 h at 4 °C against Buffer
D50AS (20% glycerol, 20 mM Na-HEPES, pH 7.9, 0.2 mM EDTA,
0.2 mM PMSF, 0.5 mM DTT and 50 mM ammonium sulfate). Total
protein concentration of cleavage factor fractions was estimated
by Bio-Rad Bradford protein assay with bovine serum albumin as
the standard and determined to be: CPSF (3 mg/mL): CstF
(3.5 mg/mL): CFm (3 mg/mL). The amount of 30 cleavage factor
1. Darnell, J. E., Jr. RNA 2013, 19, 443.
2. Di Giammartino, D. C.; Nishida, K.; Manley, J. L. Mol. Cell 2011, 43, 853.
3. Tian, B.; Hu, J.; Zhang, H.; Lutz, C. S. Nucleic Acids Res. 2005, 33, 201.
4. Barreau, C.; Paillard, L.; Osborne, H. B. Nucleic Acids Res. 2005, 33, 7138.
5. Beisang, D.; Bohjanen, P. R. Wiley Interdiscip. Rev.-RNA 2012, 3, 719.
6. Matoulkova, E.; Michalova, E.; Vojtesek, B.; Hrstka, R. RNA Biol. 2012, 9, 563.
7. Mayr, C.; Bartel, D. P. Cell 2009, 138, 673.
8. Sandberg, R.; Neilson, J. R.; Sarma, A.; Sharp, P. A.; Burge, C. B. Science 2008, 320,
1643.
9. Jiang, B.; Xu, D.; Allocco, J.; Parish, C.; Davison, J.; Veillette, K.; Sillaots, S.; Hu,
W.; Rodriguez-Suarez, R.; Trosok, S.; Zhang, L.; Li, Y.; Rahkhoodaee, F.; Ransom,
T.; Martel, N.; Wang, H.; Gauvin, D.; Wiltsie, J.; Wisniewski, D.; Salowe, S.;
Kahn, J. N.; Hsu, M. J.; Giacobbe, R.; Abruzzo, G.; Flattery, A.; Gill, C.; Youngman,
P.; Wilson, K.; Bills, G.; Platas, G.; Pelaez, F.; Diez, M. T.; Kauffman, S.; Becker, J.;
Harris, G.; Liberator, P.; Roemer, T. Chem. Biol. 2008, 15, 363.
10. Overy, D.; Calati, K.; Kahn, J. N.; Hsu, M. J.; Martin, J.; Collado, J.; Roemer, T.;
Harris, G.; Parish, C. A. Bioorg. Med. Chem. Lett. 2009, 19, 1224.
11. Ryan, K. RNA Biol. 2007, 4, 26.
12. Hirose, Y.; Manley, J. L. J. Biol. Chem. 1997, 272, 29636.
13. Moore, C. L.; Sharp, P. A. Cell 1985, 41, 845.
14. Ryan, K.; Khleborodova, A.; Pan, J.; Ryan, X. P. RNA 2009, 15, 483.
15. Brautigan, D. L. FEBS J. 2013, 280, 324.
fractions used in a 12.5
lL cleavage reaction were: CPSF (0.8 lL);
CstF (0.6 L); CFm (3 L).
l
l
4.5. RNA substrate
SV40 late pre-mRNA (233 nt) was transcribed in vitro by SP6
RNA polymerase (Promega) from the pG3SVL-A plasmid linearized
at the DraI site.24 Substrate RNA was uniformly labeled by includ-
ing [a-
32P]-UTP (Perkin–Elmer Life Sciences) and 50 capped during
16. Chuman, Y.; Yagi, H.; Fukuda, T.; Nomura, T.; Matsukizono, M.; Shimohigashi,
Y.; Sakaguchi, K. Protein Pept. Lett. 2008, 15, 938.
transcription using the 50Me7G(50)ppp(50)G-Cap analog (NEB). The
transcript was purified on a 0.4 mm denaturing polyacrylamide
gel, located by shadowing on film, and extracted as previously
described.11
17. Lu, G.; Wang, Y. Clin. Exp. Pharmacol. Physiol. 2008, 35, 107.
18. Stern, A.; Privman, E.; Rasis, M.; Lavi, S.; Pupko, T. J. Mol. Evol. 2007, 64, 61.
19. Fuchs, S.; Grill, E.; Meskiene, I.; Schweighofer, A. FEBS J. 2013, 280, 681.
20. McCluskey, A.; Sim, A. T.; Sakoff, J. A. J. Med. Chem. 2002, 45, 1151.
21. Rogers, J. P.; Beuscher, A. E.; Flajolet, M.; McAvoy, T.; Nairn, A. C.; Olson, A. J.;
Greengard, P. J. Med. Chem. 2006, 49, 1658.
22. Hayashi, R.; Tanoue, K.; Durell, S. R.; Chatterjee, D. K.; Jenkins, L. M.; Appella, D.
H.; Appella, E. Biochemistry 2011, 50, 4537.
23. Yamaguchi, H.; Durell, S. R.; Feng, H.; Bai, Y.; Anderson, C. W.; Appella, E.
Biochemistry 2006, 45, 13193.
24. Takagaki, Y.; Ryner, L. C.; Manley, J. L. Cell 1988, 52, 731.
25. Ryan, K.; Murthy, K. G.; Kaneko, S.; Manley, J. L. Mol. Cell. Biol. 2002, 22, 1684.
26. Takagaki, Y.; Ryner, L. C.; Manley, J. L. Genes Dev. 1989, 3, 1711.
27. Belova, G. I.; Demidov, O. N.; Fornace, A. J., Jr.; Bulavin, D. V. Cancer Biol. Ther.
2005, 4, 1154.
28. Park, S. Y.; Fung, P.; Nishimura, N.; Jensen, D. R.; Fujii, H.; Zhao, Y.; Lumba, S.;
Santiago, J.; Rodrigues, A.; Chow, T. F.; Alfred, S. E.; Bonetta, D.; Finkelstein, R.;
Provart, N. J.; Desveaux, D.; Rodriguez, P. L.; McCourt, P.; Zhu, J. K.; Schroeder, J.
I.; Volkman, B. F.; Cutler, S. R. Science 2009, 324, 1068.
4.6. In vitro 30 cleavage reactions
Cleavage reactions were carried out as described in detail else-
where11,14 in 12.5
lL and contained, in addition to the cleavage
activators indicated in the figures, tRNA (0.1 mg/mL), pH 8 EDTA
(2 mM), 20-dATP (2 mM), DTT (0.41 mM), BSA (Roche; 40 ng/mL),
placental RNase inhibitor (Promega, 0.32 u/mL), polyvinyl alcohol
(2.5%), 10% glycerol, 10 mM HEPES-NaOH (pH 7.9), 25 mM ammo-
nium sulfate, the cleavage factor fractions described in Section 4.4,
and the RNA substrate (1–5 nM). The reactions were incubated at
30 °C for 2 h and then digested with Proteinase K, phenol–CHCl3
extracted, ethanol precipitated, and resolved on a 6% denaturing
29. Ma, Y.; Szostkiewicz, I.; Korte, A.; Moes, D.; Yang, Y.; Christmann, A.; Grill, E.
Science 2009, 324, 1064.
polyacrylamide gel. After drying, the gel was exposed to
a
30. Miyazono, K.; Miyakawa, T.; Sawano, Y.; Kubota, K.; Kang, H. J.; Asano, A.;
Miyauchi, Y.; Takahashi, M.; Zhi, Y.; Fujita, Y.; Yoshida, T.; Kodaira, K. S.;
Yamaguchi-Shinozaki, K.; Tanokura, M. Nature 2009, 462, 609.
31. Soon, F. F.; Ng, L. M.; Zhou, X. E.; West, G. M.; Kovach, A.; Tan, M. H.; Suino-
Powell, K. M.; He, Y.; Xu, Y.; Chalmers, M. J.; Brunzelle, J. S.; Zhang, H.; Yang, H.;
Jiang, H.; Li, J.; Yong, E. L.; Cutler, S.; Zhu, J. K.; Griffin, P. R.; Melcher, K.; Xu, H.
E. Science 2012, 335, 85.
32. Bassaganya-Riera, J.; Skoneczka, J.; Kingston, D. G.; Krishnan, A.; Misyak, S. A.;
Guri, A. J.; Pereira, A.; Carter, A. B.; Minorsky, P.; Tumarkin, R.; Hontecillas, R.
Curr. Med. Chem. 2010, 17, 467.
Molecular Dynamics Storm PhosphorImager screen and the bands
were quantitated using ImageQuant software. Relative cleavage
(R.C.) was calculated as [50 fragment/(50 fragment + uncleaved
RNA)] ꢁ 100. The total recovered RNA varied from lane to lane
due to losses during phenol–CHCl3 extraction and ethanol precipi-
tation. Use of the R.C. ratio standardizes the amount cleaved and
allows comparison of gel lanes with different amounts of total
recovered RNA.
33. Spotswood, H. T.; Turner, B. M. J. Clin. Investig. 2002, 110, 577.
34. Glover-Cutter, K.; Kim, S.; Espinosa, J.; Bentley, D. L. Nat. Struct. Mol. Biol. 2008,
15, 71.
Acknowledgments
35. Venkataraman, K.; Brown, K. M.; Gilmartin, G. M. Genes Dev. 2005, 19, 1315.
36. McCracken, S.; Fong, N.; Yankulov, K.; Ballantyne, S.; Pan, G.; Greenblatt, J.;
Patterson, S. D.; Wickens, M.; Bentley, D. L. Nature 1997, 385, 357.
37. Neugebauer, K. M. J. Cell Sci. 2002, 115, 3865.
38. Hirose, Y.; Manley, J. L. Nature 1998, 395, 93.
39. Allemand, E.; Hastings, M. L.; Murray, M. V.; Myers, M. P.; Krainer, A. R. Nat.
Struct. Mol. Biol. 2007, 14, 630.
40. Murray, M. V.; Kobayashi, R.; Krainer, A. R. Genes Dev. 1999, 13, 87.
41. Niwa, M.; Rose, S. D.; Berget, S. M. Genes Dev. 1990, 4, 1552.
42. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
43. Rossiter, S.; Smith, C. L.; Malaki, M.; Nandi, M.; Gill, H.; Leiper, J. M.; Vallance,
P.; Selwood, D. L. J. Med. Chem. 2005, 48, 4670.
This work was supported by Grant 5SC1GM083754 from the
National Institutes of Health (NIH). Additional infrastructural sup-
port at the City College of New York (CCNY) was provided by the
NIH National Center for Research Resources (2G12RR03060-
26A1) and the National Institute on Minority Health and Health
Disparities (8G12MD007603-27). We thank E. Appella and R. Hay-
ashi for providing cp38, and L. Yang (CCNY) for mass spectrometry.
44. Markiewicz, J. T.; Wiest, O.; Helquist, P. J. Org. Chem. 2010, 75, 4887.
45. Drake, B.; Patek, M.; Lebl, M. Synth.-Stuttg. 1994, 579.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.006.