Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State

Article abstract of DOI:10.1021/acs.biochem.8b00031

Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5′-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic pK_a, consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2′-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2′-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2′-OH deprotonation exceeds the extent of P-O bond formation.

Full text of DOI:10.1021/acs.biochem.8b00031

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Article
Evidence that nucleophile deprotonation exceeds
bond formation in the HDV ribozyme transition state
Jun Lu, Selene C. Koo, Benjamin Weissman, Michael E. Harris, Nan-sheng Li, and Joseph A. Piccirilli
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00031 • Publication Date (Web): 07 May 2018
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Evidence that nucleophile deprotonation exceeds bond formation in the HDV ribozyme
transition state
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Jun Lu‡ a, Selene C. Koo‡ a, Benjamin P. Weissman‡ a, Michael E. Harrisb, Nan-Sheng Li*a and
Joseph A. Piccirilli*a
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aDepartment of Biochemistry and Molecular Biology and Department of Chemistry, University
of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
bDepartment of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida 32611,
USA.
‡ These authors contributed equally to this work.
*Corresponding Authors: nli@uchicago.edu, jpicciri@uchicago.edu
Abstract: Steric constraints imposed by the active sites of protein and RNA enzymes pose
major challenges to the investigation of structure-function relationships within these systems. As
a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized
phosphoramidites from propanediol derivatives and incorporated them at the 5´ termini of RNA
and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed
electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular
transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution
reduces the maximal rate and the kinetic pKa, consistent with the expected electron-withdrawing
effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo
transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed
by a ribofuranose ring are not a strict requirement for ribozyme catalysis. In contrast to the
nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction
rate consistent with a mechanism in which (1) the 2´hydroxyl nucleophile exists predominantly in
its neutral, protonated form in the ground state and (2) the 2¢ hydroxyl bears some negative charge
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in the rate-determining step, consistent with a transition state in which the extent of 2´-OH
deprotonation exceeds the extent of P-O bond formation.
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Introduction
The hepatitis delta virus (HDV) ribozyme serves as an outstanding model system for
foundational studies of RNA catalysis due to its small size1, the availability of high-resolution
crystal structures2-4 and wealth of biochemical and kinetic data5. The RNA genome of the hepatitis
delta virus encodes two forms of the HDV ribozyme, genomic and antigenomic, which mediate an
essential step of viral replication by cis-cleavage6. Both forms of the ribozyme adopt similar
overall structure, are likely to employ similar catalytic mechanisms and can be divided into trans-
cleaving constructs though separation of 5¢-terminus from the rest of the ribozyme. Like the other
small endonucleolytic ribozymes, the HDV ribozyme facilitates cleavage by catalyzing the
nucleophilic attack of the 2´ hydroxyl on the adjacent phosphate to form a 2¢, 3¢-cyclic phosphate
and 5¢-hydroxyl termini (Figure 1A).
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A
5´RNA
‡
5´RNA
5´RNA
O
O
O
N
N
N
O
O
O
P
O
-
OH B!
O
P
OH
O
O
O
-
H
B
!O
P
O
-
!
B
O
O
O
O
O
-
HO
A
!
N
N
N
A
H
A
H
O
O
O
O
O
OH
O
P
OH
O
P
OH
O
P
O
O
O
O
O
O
O
3´RNA
3´RNA
3´RNA
R:
B
CH3
CH2F
CF3
‡
R
R
R
-
O
OH
B!
N
O
OH
O
O
O
-
H
B
!O
P
O
P
O
P
-
!
O
B
O
O
O
-
HO
!
N
N
A
H
A
H
O
O
O
A
O
OH
O
P
OH
O
P
OH
O
P
O
O
O
O
O
O
O
O
3´RNA
3´RNA
3´RNA
Figure 1 A general mechanism of ribozyme-mediated cleavage of standard RNA oligonucleotides (A) and
propanediol tethered RNA substrates (B). Cleavage occurs with attack of a 2¢ hydroxyl group on the adjacent
phosphate to give 2¢, 3¢ cyclic phosphate and 5¢ hydroxyl containing products. A–H represents a general acid, which
protonates the 5¢ leaving group; B- represents a general base, which deprotonates the 2¢ nucleophile. For simplicity,
we show the reaction occurring via a concerted mechanism. B) A general mechanism for cleavage of tethered-
propanediol substrates
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A significant body of functional7-10, spectroscopic11, 12, and crystallographic2-4 data support
the role of C76 in the antigenomic form as a general acid that stabilizes the leaving group through
proton transfer. Perhaps the strongest evidence was generated through observation of C76 mutants
with substrates containing a 5¢-bridging phosphorothioate at the cleavage site9. In the 5¢-
phosphorothioate background, activation of the leaving group is not required for cleavage because
of the highly labile thiolate leaving group, therefore, the insensitivity of the phosphorothioate to
C76 mutation provides evidence for the leaving group activation by the mutated group.
Subsequently, similar experiments have been employed to interrogate leaving group activation in
other ribozymes13-15. However, an analogous experimental approach to investigate nucleophile
activation has remained elusive since no chemical system with a pre-activated nucleophile has
been devised.
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Consequently, nucleophilic activation arguably represents the least understood feature of
ribozyme (and enzyme) catalysis 16. Phosphoryl transfer to oxygen nucleophiles shows a strong
dependence on the nucleophile pKa17-19, which suggests that reactivity is highly sensitive to the
protonation state of the nucleophile. Ground state interactions with metal ions, hydrogen bond
donors, or solvent can also influence reactivity20. In HDV ribozyme, the mechanism of
nucleophile activation remains poorly understood. However, crystallographic3 and biochemical21,
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data are consistent with a well-positioned magnesium ion with inner-sphere coordination to
the pro-Rp oxygen of the scissile phosphate, which could play a role in nucleophile activation7
The features of nucleophile activation have profound significance for understanding
catalysis, yet limited strategies exist to probe nucleophile function. Analogous to substrates with
leaving group modifications, a series of substrates that perturb the nucleophilic 2´-hydroxyl
group might provide a complementary strategy to investigate catalytic mechanism, e.g. identify
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charge development on the nucleophilic oxygen through the transition state, estimate the degree
of bonding to the nucleophile in the transition state, and identify possible interaction partners
with the nucleophile. In particular, we aimed to develop substrates that differ in nucleophilicity
and basicity from the hydroxyl group in the native ribozyme and uncover ribozyme
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modifications whose effects on catalysis are suppressed or enhanced in the presence of these
mutant substrates. Optimally, these substrates should maintain the 2´-hydroxyl group, but
modulate its basicity of that group via inductive effects.
A series of oligonucleotides containing modifications (R: CH3, CH2F, CHF2, CF3, and
wild-type [H]) at the 2¢-β-position of the ribose ring were developed previously in our laboratory
to perturb the pKa and nucleophilicity of the 2¢-hydroxyl group18; Brønsted analysis of these
substrates in non-enzymatic, hydroxide-catalyzed cleavage reactions yield an apparent βnuc value
of -0.75, indicating a significant degree of bonding to the nucleophile in the transition state,
consistent with recent results from kinetic isotope effect analysis23. However, introduction of these
modified nucleotides into HDV ribozyme substrates blocked cleavage, presumably because the
ribozyme active site could not accommodate substituents at the 2¢-β-position, thereby precluding
efforts to understand similarities and differences in nucleophile bonding between solution and
ribozyme-catalyzed reactions. Since the HDV ribozyme has no strict sequence requirements
upstream of the cleavage site 24-26, we anticipated that the ribozyme might catalyze cleavage of
substrates lacking a nitrogenous base and/or the ribose ring at the cleavage site. Thus, we
considered the use of pKa-perturbing modifications in the context of substrates possessing greater
flexibility and occupying less volume at the cleavage site relative to natural nucleotides.
Specifically, we were inspired by 2-(hydroxylproyl)aryl phosphate model compounds (herein
tethered to oligonucleotides to generate “tethered substrates”) initially developed over 50 years
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ago27. Here we report the incorporation of 1,2-propanediol, 3-fluoro-1,2-propanediol and 3,3,3-
trifluoro-1,2-propanediol into the 5¢-terminus of DNA and RNA oligonucleotides and the analysis
of their hydroxide-catalyzed and HDV-ribozyme catalyzed cleavage reactions, respectively
(Figure 1B).
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Materials and Methods
Synthesis of phosphoramidites (4a-c)
All reactions were carried out in anhydrous solvents under a positive pressure of Argon.
Commercially available reagents and anhydrous solvents were used without further purification.
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1H, 13C, F and P NMR spectra were recorded on a Bruker 500 or Bruker 400 MHz NMR
1
spectrometer. H chemical shifts are reported in δ (ppm) relative to tetramethylsilane and 13C
chemical shifts δ (ppm) relative to the solvent used. High resolution mass spectra were obtained
from the Department of Chemistry, University of California at Riverside.
1,2-Bis(tert-butyldimethylsiloxy)propane (2a). 1H-imidazole (1.97 g, 29.0 mmol) and t-
BuMe2SiCl (4.22 g, 28.0 mmol) were added to a solution of 1a (0.76 g, 10.0 mmol) in dry DMF
(10 mL). The mixture was stirred overnight at room temperature, then evaporated to a syrup under
vacuum. The residue was partitioned between CH2Cl2 and H2O, the organic layer was washed with
water followed by brine, and dried over anhydrous Na2SO4. The sodium sulfate removed by
filtration and the solvent was subsequently removed under vacuum. The residue was purified by
silica gel chromatography, eluting with 2% ethyl acetate in hexane, to give product 2a as an oil:
2.90 g (95% yield). 1H NMR (CDCl3) 3.80 (m, 1H), 3.52 (m, 1H), 3.33 (m, 1H), 1.11 (d, 3H, J
= 6.1 Hz), 0.90 (s, 9H), 0.89 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR
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(CDCl3) 69.6, 69.2, 26.2, 26.1, 20.8, 18.6, 18.4, -4.4, -4.6, -5.1, -5.2. HRMS (FAB+): m/z calc
for C15H37O2Si2, [M+H+]: 305.2332; found: 305.2325.
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1,2-Bis(tert-butyldimethylsiloxy)-3-fluoropropane (2b). Compound 2b was prepared from 1b
(1.00 g, 10.6 mmol), 1H-imidazole (2.95 g, 30.7 mmol), and t-BuMe2SiCl (4.48 g, 29.7 mmol) as
described for 2a. Silica gel chromatography (2% ethyl acetate in hexane) of the residue yielded
3.09 g (90% yield) of 2b as an oil. 1H NMR (CDCl3) 4.18-4.46 (m, 2H), 3.79-3.88 (m, 1H), 3.44-
3.53 (m, 1H), 0.82 (s, 9H), 0.81 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H), -0.02 (s, 6H); 13C NMR (CDCl3)
85.9, 84.2, 72.4, 72.2, 63.9, 63.8, 26.0, 25.9, 18.4, 18.3, -4.7, -5.30, -5.36, -5.4, 19F NMR (CDCl3)
-109 (t). HRMS (FAB+): m/z calc for C15H36FO2Si2, [M+H+]: 323.2238; found: 323.2238.
1,2-Bis(tert-butyldimethylsiloxy)-3,3,3-trifluoropropane (2c). Compound 2c was prepared
from 1c (1.00 g, 7.7 mmol), 1H-imidazole (1.52 g, 22.3 mmol), and t-BuMe2SiCl (3.25 g, 21.5
mmol) as described for 2a. Silica gel chromatography (2% ethyl acetate in hexane) of the residue
yielded 1.99 g (72% yield) of 2c as an oil. 1H NMR (CDCl3) 3.97-4.03 (m, 1H), 3.81-3.84 (m,
1H), 3.63-3.66 (m, 1H), 0.91 (s, 9H), 0.90 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H), 0.072 (s, 3H), 0.071
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(s, 3H); C NMR (CDCl3) 124.6.0 (q), 73.0 (q), 63.3, 26.0, 25.7, 18.5, 18.2, -4.6, -5.2, -5.4, -
5.5, 19F NMR (CDCl3) -77.6 (d). HRMS (FAB+): m/z calc for C15H34F3O2Si2, [M+H+]: 359.2049;
found: 359.2044.
2-tert-Butyldimethylsiloxy-1-propanol (3a). HF·pyridine (890 µL, 6.84 mmol; Aldrich) was
carefully diluted with pyridine (1.0 mL) and then added dropwise to a solution of 2a (1.53 g, 5.02
mmol) in THF (10 mL). After overnight stirring at room temperature, thin-layer chromatography
of the mixture showed that no remaining starting material. The mixture was partitioned between
CH2Cl2 and H2O; the organic layer was washed with 5% aq. NaHCO3 followed by brine and dried
over anhydrous Na2SO4. The sodium sulfate removed by filtration and the solvent was
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subsequently removed under vacuum. The residue was purified by silica gel chromatography,
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eluting with 17% ethyl acetate in hexane, to give product 3a as an oil: 516 mg (54% yield). H
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NMR (CDCl3) 3.91 (m, 1H), 3.49 (m, 1H), 3.37 (m, 1H), 2.02 (m, 1H), 1.12 (d, 3H, J = 6.2 Hz),
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0.91 (s, 9H), 0.09 (s, 6H); C NMR (CDCl3) 69.2, 68.3, 25.9, 19.9, 18.2, -4.3, -4.7. HRMS
(FAB+): m/z calc for C9H23O2Si, [M+H+]: 191.1467; found: 191.1467.
2-tert-Butyldimethylsiloxy-3-fluoro-1-propanol (3b). Compound 3b was prepared from 2b
(2.89 g, 8.96 mmol) and HF·pyridine (1.20 mL, 9.21 mmol) as described for 3a. Silica gel
chromatography (10% ethyl acetate in hexane) of the residue yielded 0.99 g (53% yield) of 3b as
an oil. 1H NMR (CDCl3) 4.28-4.47 (m, 2H), 3.95-4.00 (m, 1H), 3.56-3.67 (m, 1H), 0.92 (s, 9H),
0.11 (s, 6H); 13C NMR (CDCl3) 84.6, 83.3, 71.4, 71.3, 63.3, 63.2, 25.9, 18.2, -4.6, -4.8; 19F NMR
(CDCl3) -106. HRMS (FAB+): m/z calc for C9H22FO2Si, [M+H+]: 209.1373; found: 209.1367.
2-(tert-Butyldimethylsiloxy)propyl cyanoethyl N,N-diisopropylphosphoramidite (4a). To a
solution of 3a (441 mg, 2.16 mmol) in dry dichloromethane (5 mL) under argon were added N,N-
diisopropylethylamine
(1.85
mL,
10.75
mmol),
2-cyanoethyl
N,N-
diisopropylchlorophosphoramidite (1.00 g, 4.23 mmol), and 1-methylimidazole (173 µL, 0.136
mmol). After 1 hour stirring at room temperature, thin-layer chromatography of the mixture
showed that no remaining starting material. The reaction mixture was then quenched with MeOH
(1 mL) and stirred for an additional 5 minutes. After the solvent was removed under vacuum, the
residue was purified by silica gel chromatography, eluting with 5% ethyl acetate in hexane
containing 0.5% triethylamine, to give the corresponding phosphoramidite 4a as an oil: 689 mg
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(82% yield): P NMR (CD3CN) 150.8, 150.3. HRMS (FAB+): m/z calc for C18H40N2O3SiP,
[M+H+]: 391.2546; found: 391.2551.
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2-tert-Butyldimethylsiloxy-3-fluoropropyl cyanoethyl N,N-diisopropylphosphoramidite
(4b). Compound 4b was prepared from 3b (441 mg, 2.12 mmol), N,N-diisopropylethylamine (2.29
mL, 13.31 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.00 g, 4.23 mmol), and
1-methylimidazole (200 µL, 0.157 mmol) as described for 4a. Silica gel chromatography (5% ethyl
acetate in hexane containing 0.5% triethylamine) purification of the residue yielded 625 mg (72%
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yield) of 4b as an oil. P NMR (CD3CN) 151.9, 151.4; F NMR (CDCl3) -107.7 (d), 108.5
(d). HRMS (FAB+): m/z calc for C18H39N2O3FSiP, [M+H+]: 409.2452; found: 409.2454.
2-tert-Butyldimethylsiloxy-3,3,3-trifluoropropyl
cyanoethyl
N,N-
diisopropylphosphoramidite (4c). 2-tert-Butyldimethylsiloxy-3,3,3-trifluoro-1-propanol (3c)
was prepared from 2c (1.81 g, 5.04 mmol) and HF·pyridine (680 µL, 5.18 mmol) as described for
3a. Silica gel chromatography (10% ethyl acetate in hexane) of the residue yielded 441 mg (36%
yield) of 3c as an oil. 1H NMR (CDCl3)
4.03-4.07 (m, 1H), 3.77-3.81 (m, 1H), 3.70-3.75 (m,
1H), 0.93 (s, 9H), 0.15 (s, 6H); 13C NMR (CDCl3) 124.4 (q), 71.8 (q), 61.9, 25.6, 18.2, -4.9, -
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5.2; F NMR (CDCl3)
-77.4 (d). Compound 4c was then prepared from 3c (387 mg, 1.58
mmol), N,N-diisopropylethylamine (1.71 mL, 9.94 mmol), 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (1.0 g, 4.23 mmol), and 1-methylimidazole (160 µL, 0.126
mmol) as described for 4a. Silica gel chromatography (5% ethyl acetate in hexane containing 0.5%
triethylamine) purification of the residue yielded 590 mg (84% yield) of 4c as an oil. 31P NMR
(CD3CN)
152.3, 151.5; 19F NMR (CDCl3)
-77.6 (t). HRMS (FAB+): m/z calc for
C18H37N2O3F3SiP, [M+H+]: 445.2263; found: 445.2262.
Experiments with substrates that perturb the 2¢-hydroxyl nucleophile pKa to study nucleophile
activation by the HDV ribozyme
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RNA substrates were 3¢-end radiolabeled as follows: 10 pmol RNA was incubated with 10
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pmol 5¢-32P-pCp (cytidine 3¢,5¢-bisphosphate), 6 µM ATP, 1x RNA ligase buffer, 3.3 mM DTT, 1
µL DMSO, and 20 U T4 RNA ligase in a 10 µL reaction volume at 4 °C overnight. The reaction
was then quenched by the addition of 8 µL stop solution (95% formamide, 15mM EDTA, 0.01%
w/v bromophenol blue and xylene cyanol), gel purified on a 20% denaturing polyacrylamide gel,
eluted into 1 mL TE overnight, and finally ethanol precipitated with glycogen as a carrier.
DNA substrates were 3¢-end radiolabeled as follows: 10 pmol DNA was incubated with 10
pmol α-32P-ddATP (dideoxyadenosine triphosphate), 5 µg BSA, 1x TdT (terminal
deoxytransferase) buffer, and 15 U TdT in a 10 µL reaction volume at 37 °C for 1 hr. The reaction
was then quenched by the addition of 8 µL stop solution, gel purified on a 20% denaturing
polyacrylamide gel, eluted into 1 mL TE overnight, and finally ethanol precipitated with glycogen
as a carrier.
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To measure the rate of base-catalyzed cleavage of DNA substrates, the following high-pH
reaction conditions were employed: 19.5 µL reaction buffer at the appropriate pH was added to
0.5 uL 3¢-end radiolabeled substrate and incubated at 25 °C. Between pH 12.0 and 14.59, reaction
conditions were set by mixing appropriate concentrations of KOH (3.89 M, pH 14.59) and KCl
(3.89M) to achieve the desired hydroxide concentration while maintaining a constant ionic
strength, and the pH of the reaction solutions were confirmed by colorimetric strips. In solutions
with high ionic strength, pH defined as the negative log of activity of hydronium ions, can deviate
from the expected pH in terms of hydronium concentration10, however, The pH values reported
herein refer the concentration of hydronium. Reaction aliquots of 1 µL were removed at
appropriate time-points, quenched by adding 6 µL 1 M Tris-HCl pH 7.5 (final pH’s of quenched
reaction aliquots are typically no higher than 8) with 7 µL stop solution, and rapidly chilled on dry
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ice. Hydrolysis products were separated from unhydrolyzed substrate on a denaturing 20%
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polyacrylamide / 7M urea gel and quantified using a PhosphorImager with ImageQuant software
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obst
y = 1− yo − Ae−k
(Molecular Dynamics). Data for cleavage reactions was fit to the equation
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using Origin 7.0 (OriginLab), where kobs is the first-order rate constant for substrate cleavage, and
A is the extent of substrate cleavage; for reactions that did not reach completion (>80% reacted)
after more than 20 days (reaction rate < 3 x 10-5 min-1), data were fit assuming A = 0.8 to obtain
kmax
k . The reaction pK was determined by fitting reaction rate data to the equation
k =
obs
a
1+10pKa− pH
.
9
HDV ribozyme-catalyzed reactions were conducted as previously described . Briefly,
ribozyme (final concentration 1 µM) was preincubated with 10 mM MgCl2 at 70 °C for 2 min,
then 25 °C for 14 min; buffer was added, and reaction was initiated by the addition of trace
radiolabeled substrate (10 µL reaction volume). Buffers used contained 25 mM acetic acid, 25
mM MES, and 50 mM Tris (pH 4.0-8.0), or 50 mM MES, 25 mM Tris, and 25 mM 2-amino-2-
methyl-1-propanol (pH 7.5-10.0). Reaction aliquots were removed at appropriate times, quenched
by addition 9uL stop solution, and rapidly chilled on dry ice. Cleavage products were separated
from uncleaved substrate on a denaturing 20% polyacrylamide / 7M urea gel and quantified using
a PhosphorImager with ImageQuant software (Molecular Dynamics). Data for cleavage reactions
was fit to the equation y = 1- yo - Ae-k t using Origin 7.0 (OriginLab), where kobs is the first-
obs
order rate constant for substrate cleavage, and A is the extent of substrate cleavage. Reaction pKa´s
(pKa1, pKa2) were determined by fitting reaction rate data to the equation
kmax
.
k =
1+10 pH- pKa1 +10 pKa2- pH +10 pKa2- pKa1
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1.
Synthesis and incorporation of phosphoramidites (4a-c) into RNA/DNA substrates
Commercially available, racemic 1,2-propanediol (1a), 3-fluoro-1,2-propanediol (1b) and
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3,3,3-trifluoro-1,2-propanediol (1c) were converted into the corresponding phosphoramidites (4a-
c) in three steps, as shown in Figure 2. Both primary and secondary hydroxyl groups of 1 were
first protected as tert-butyldimethylsilyl (TBS) ethers in 72-95% yield (Figure 2). The primary
TBS ethers were selectively cleaved using HF-Pyridine complex to give alcohols 3a-c in 36-54%
yield
(Figure
2).
Phosphitylation
of
3a-c
with
2-cyanoethyl
N,N-
diisopropylchlorophosphoramidite gave phosphoramidites (4a-c) in good yields (72-84%; Figure
2). All phosphoramidites were prepared as racemic mixtures with the exception of the 1,2-
propanediol derivative which was prepared as both a racemic mixture and a enantiopure S isomer.
Ribozyme-catalyzed-cleavage assays showed that both enatiopure and racemic versions of the
resulting oligonucleotides display identical kinetic behavior (data not shown). All subsequent
assays were performed with oligomers derived from racemic phosphoramidites.
CN
(i-Pr NP(OCH CH CN)Cl
2
2
2
OTBS
1-methylimidazole,
i-Pr NEt
OH
OTBS
OH
OTBS
OTBS
TBSCl
DMF
HF-Py
O
O
2
R
P
OH
R
R
R
N(i-Pr)
2
4a: R = CH (82%)
3
4b: R = CH F (72%)
3a: R = CH (54%)
3
3b: R = CH F (53%)
2a: R = CH (95%)
3
2b: R = CH F (90%)
1a: R = CH
1b: R = CH F
3
2
2
4c: R = CF (84%)
2
3c: R = CF (36%)
2
2c: R = CF (72%)
1c: R = CF
3
3
3
3
Figure 2 Synthesis of propanediol phosphoramidites from corresponding diols.
To investigate non-enzymatic base-catalyzed cleavage of these modified linkages, we
incorporated the phosphoramidites (4a-c) into DNA oligonucleotides (5¢-Xgg gtc ggc-3¢). To
investigate HDV ribozyme-catalyzed cleavage of these linkages, we incorporated 4a-c into RNA
oligonucleotides (5¢-XGG GUC GGC-3¢; Figure 1). We used a modified 1-µmole RNA protocol
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with double coupling of the phosphoramidites (4a-c) (~75 mg) in anhydrous acetonitrile (0.75
mL). After the synthesis, the solid support was treated with a 3:1 (v/v) mixture of concentrated
aqueous ammonium hydroxide and ethanol at 55 °C for 4 hours and then desilylated with
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triethylamine trifluoride complex at 65 °C for 25 min . We purified the oligonucleotides on a
denaturing polyacrylamide gel and confirmed their identities by MALDI-TOF MS (Table S1).
2.
Base-catalyzed cleavage of tethered DNA oligonucleotides
Under alkaline conditions, the 5¢-propanediol phosphate tethers [5¢-Xgg gtc ggc-3¢; X:
RCH(OH)CH2OPO(OH)O-5¢; R: CH3, CH2F, CF3] release themselves from DNA via
intramolecular transphosphorylation to the neighboring hydroxyl group, analogous to alkaline
cleavage of RNA by 2´-O-transphosphorlyation (Figure 1B). To assess the effect of increasing
fluorine substitution on reactivity of the nucleophilic hydroxyl group, we observed the rate of
tether release over a range of pH’s (25 °C, KCl 3.90 M, pH 12.0 to 14.6; Table 1 and Figure S1).
Our expectation was that the inductive effects from fluorination would reduce both the
nucleophilicity and the pKa of the 2′ hydroxyl nucleophile, causing the fluorinated substrates to
react more slowly at pH values above pKa, when the substrates react from an oxyanionic ground
state, but react with a similar – or even enhanced – rate at pH values below the pKa. For
comparison, ethanol and trifluoroethanol, primary alcohols that have a -CH3 or -CF3 group geminal
to the hydroxyl group, respectively, ionize with pKa values of 15.2 and 13.2, respectively29. Indeed,
at the highest pH tested (pH 14.6), the methyl (R: CH3) releases itself from the tether about 10-
fold faster than the trifluoromethyl (R: CF3) substrate (Table 1 and Figure S1). At pH 13, the two
substrates react at similar rates, and at lower pH values the trifluoromethyl substrate reacts
somewhat faster than its methyl counterpart (Table 1 and Figure S1; however, we emphasize that
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observations for reactions occurring below pH 13 should be interpreted with caution as they
correspond to rates with half-lives on the order of weeks and are thus subject to more significant
errors than faster reactions (Table 1). Together, these observations suggest that the electron-
withdrawing effect of fluorination reduces both the pKa and the intrinsic rate of reaction. Fitting
of these data to a single-ionization model suggests a ∆pKa ~ 1 from the methyl substrate (pKa =
14.5) to the trifluoromethyl substrate (pKa = 13.5) (Figure S1). Given a ∆pKa ~ 1 between the two
substrates, comparison of the maximal, observed rates offers a crude estimate of the Brønsted
coefficient (βnuc ~ 1), consistent with the value of ~0.75 measured previously18, suggesting a that
the bond between the 2´-oxygen and the phosphorus is nearly or completely formed in the
transition state.
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Table 1 Rates for uncatalyzed tether release under basic conditions. Relative rates compared to the R: CH3 substrate
at the given pH. The pH values reported here reflect the calculated concentration of hydronium rather than the
hydronium activity (see methods), whereas the pH values in Figure S1 reflect hydronium activity.
R: CH3
R: CF3
rate (min-1)
pH
rate (min-1)
krel
0.1
0.3
0.4
0.8
2.9
14.6
14.0
13.5
13.0
12.5
(8.4 ± 0.2) × 10-4
(3.7 ± 0.5) × 10-4
(1.3 ± 0.4) × 10-4
(5.0 ± 3.0) × 10-5
(6.8 ± 4.7) × 10-6
(1.1 ± 0.4) × 10-4
(1.0 ± 0.2) × 10-4
(5.0 ± 0.3) × 10-5
(4.0 ± 2.0) × 10-5
(2.1 ± 1.5) × 10-5
The monofluoromethyl substrate (R: CH2F) reacted at rates and with apparent pKa similar
to the methyl substrate, suggesting minimal perturbation from the single fluorine substitution
(Figure S1). However, based upon our prior experience synthesizing an analogous β-branched
ribonucleotide30, the monofluoromethyl substrate may release itself through a more complex
mechanism (Figure 3). Possibly, the oxyanion initially attacks the vicinal carbon to displace
fluoride, generating the corresponding epoxide (Figure 3). Subsequent base-catalyzed opening of
the epoxide would result in conversion to a glycerol tether which could undergo release to form
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product by attack at the phosphorous (Figure 3). Formation of the glycerol tether could account
for the modestly higher reactivity of the monofluoromethyl substrate relative to the methyl
substrate at high pH values. Further work is necessary to determine whether this alternative
pathway occurs; therefore, our subsequent analysis largely focuses on the methyl and
trifluoromethyl substrates, which likely follow the simpler, expected reaction path. We do not
expect this alternative mechanism to be operative in the ribozyme catalyzed reaction where the pH
is closer to neutrality.
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OH
OH
OH
O
OH
O
O
O
O
O
Ka,2
k4
P
+
O
P
O
O
P
O
HO
O
O
DNA
DNA
HO DNA
k3[OH]
OH
O
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DNA
k2
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F
O
OH
O
O
O
O
O
Ka,1
k1
P
+
O
P
O
O
P
O
HO
O
O
DNA
DNA
HO DNA
Figure 3 Proposed mechanism of cleavage by monoflouromethylpropanediol tethered DNA oligonucleotide under alkaline
conditions.
For comparison with the tethered substrates, we also determined the relationship between
pH and rate for DNA oligonucleotides containing either a uridine or abasic residue upstream of
the cleavage side under alkaline conditions. The 2´-hydroxyl of the uridine substrate ionized with
pKa ~13.5 and exhibited a maximum cleavage rate of 0.090 min-1 (Figure S2), consistent with
previous observations31. The abasic ribose ionized with a higher pKa, ~14.5 and maximal rate of
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0.84 min-1 – about ten-fold faster than uridine – presumably reflecting the electron-withdrawing
effect of uracil heterocycle versus a hydrogen atom at the 1´ position (Figure S2). Compared to
the cleavage rate of the abasic ribose under similar conditions, the methyl-tethered (R: CH3)
substrate, which should have a comparably nucleophilic 2´-hydroxyl, reacts about 500-fold
slower (Table 3), reflecting the structural preorganization imparted by the ribose ring. This
comparison suggests that conformational restriction around the C2´-C3´ bond, which is freely
rotatable in the tethered substrates but restricted in the ribofuranosyl context, imparts a rate
enhancement of two to three orders of magnitude.
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3.
HDV ribozyme-catalyzed cleavage of RNA oligonucleotides
Next, we tested the tethered RNA oligonucleotides (5¢-XGG GUC GGC-3¢; X:
RCH(OH)CH2OPO(OH)O5¢-; R: CH3, CH2F, CF3; Figure 1) as substrates for HDV ribozyme-
catalyzed cleavage at various pH values. To ensure that differences in reactivity between tethered
and ribonucleotide substrates arose from differences in the cleavage step rather than substrate
binding, we examined rate as a function of ribozyme concentration. The rate was insensitive to
ribozyme concentration over the range of 50-2000 nM (Figure S3). These data set an upper limit
of 100 nM for Km and suggest that at the ribozyme concentrations employed in our subsequent
experiments (1000nM) the reaction proceeds exclusively from a bound ES complex ground-state
for both substrates (Figure S3).
Qualitatively, the tethered substrates reacted from the ES complex with a pH-dependence
similar to that of the wild-type substrate, showing a log-linear increase with pH in the acidic limb
of the profile that transitions to a pH-independent regime and finally to an inverse dependence at
pH values greater than 9.0 (Figure 4, Table 2). The log-linear increase with pH reflects a rate-
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stimulating deprotonation. The identity of the titrating group remains uncertain but has been
attributed to deprotonation of a water molecule coordinated to an active site metal ion to generate
the catalytic base32. The transition to the pH independent regime reflects the onset of deprotonation
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,
of the general acid9, 11 33, In the log-linear region of pH, the ES complexes for the tethered
substrates react about two orders of magnitude slower than the wild type, ribonucleotide substrate,
consistent with the importance of the conformational constraint imposed by the ribose ring. (Table
3).
Figure 4 Ribozyme-catalyzed cleavage of modified substrates that perturb the nucleophile pKa. pH-rate profile for
HDV ribozyme-catalyzed cleavage of RNA substrates. Filled squares and solid line correspond to R = CH3; half-
filled triangles and dashed line correspond to R = CH2F; open circles and dotted line correspond to R = CF3. Data
were fit to a double ionization model (see methods).
Table 2 Summary of fits to pH-titration curves for HDV ribozyme-catalyzed cleavage of tethered substrates (Figure
4).
kmax (min-1)
pKa1
pKa2
R: CH3
R: CH2F
R: CF3
(1.27 ± 0.06) x 10-3
(1.59 ± 0.07) x 10-3
(2.07 ± 0.07) x 10-3
9.5 ± 0.1
5.3 ± 0.1
10.1 ± 0.1 5.2 ± 0.2
10.1 ± 0.1 5.1 ± 0.1
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The tethered substrates react more slowly than the standard, ribose-containing substrates
in both the presence and absence of ribozyme (Table 3). However, the difference in reactivity
between the two sets of substrates is reduced in the presence of the ribozyme, presumably due to
conformation restriction imposed by the ribozyme active site on the tethered substrates (Table 3).
Comparison between substrate pairs with similar observed solution pKa’s (WT/CF3 and
Abasic/CH3) allows for evaluation of the energetic contribution from conformational restriction
while controlling for differences in nucleophile pKa. For each pair of substrates, there is a ~5-fold
difference between the ratio of rates in the solution reaction compared to the ribozyme reaction
(Table 3).
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Table 3 Comparison of the cleavage rates of the modified DNA/RNA ribonucleotide and tethered substrates in the
presence of ribozyme (pH 5) or absence of ribozyme (pH 14). aRates extrapolated from best-fit curve to pH 5. bBase-
catalyzed reactions measured with N-1 = U and ribozyme catalyzed reaction observed with N-1 = C. Previous
investigation suggests that nucleophile pKa is insensitive to the identity of the N-1 nucleobase31. cRatios of rates
given for sterically distinct substrates with similar nucleophile pKa’s.
pKa
Base-Catalyzeda
6.9 x 10-2
Ribozyme-catalyzed
1.7 x 10-1
Ribonucleotide - WT
13.5 ± 0.1b
Ribonucleotide -Abasic 14.5 ± 0.1
2.0 x 10-1
4.4 x 10-2
R: CF3
R: CH3
13.5 ± 0.1
14.5 ± 0.2
3.7 x 10-4
1.3 x 10-3
1.1 x 10-4
4.9 x 10-4
c
Abasic/CH3
540
627
90
c
WT/CF3
133
While it remains possible that a non-chemical step masks the true rate of the ribofuranosyl
substrates, a more compelling explanation is that the constrained environment of the ribozyme
active site modestly accelerates the tethered substrates by reducing their conformational entropy.
Possibly ribozyme docking confers some restriction around the C2´-C3´ bond but not as much as
the ribose ring. Moreover, this result provides a plausible explanation for our prior observation
that 2¢-β-substituted ribonucleotide substrates were inactive. The same features within the active
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site that restrict rotation around the C2´-C3´ bond, conferring a rate enhancement on the tethered
substrates, might clash sterically with a substituent in the 2¢-β-position preventing catalysis.
The reactivity of the methyl-tethered substrate relative to trifluoromethyl-tethered substrate
provides insight into the protonation state of the nucleophilic hydroxyl group in the starting ground
state of the ES complex. In the non-enzymatic reactions, when the reaction starts from an
oxyanionic ground state (pH > pKa), the electron-withdrawing effect of –CF3 manifests in the
nucleophilic attack step, resulting in lower maximal rate. However, when the reaction starts from
the protonated ground state (pH < pKa), the electron-withdrawing effect on the pKa increases the
fraction of oxyanion present, offsetting the reduced nucleophilicity. In the HDV reaction, the
trifluoromethyl substrate reacts moderately faster than the methyl substrate (1.6 fold) and the
monofluoromethyl substrate reacts with an intermediate rate (Table 2). This effect from fluorine
substitution more closely resembles the effect observed for non-enzymatic reactions at low pH
rather than at high pH, consistent with the ES complex starting from the protonated ground state
rather than the deprotonated oxyanion ground state. Moreover, the faster reaction with increasing
electron-withdrawing power suggests that the effective charge on the 2´-oxygen increases as the
reaction progresses towards the transition state, implying that the extent of O–H bond cleavage
exceeds the extent of O–P bond formation. The pathway to achieve this condition could involve a
concerted process, in which deprotonation and nucleophilic attack occur simultaneously, or a
stepwise process involving pre-equilibrium deprotonation of the 2´-OH to form the corresponding
oxyanion followed by O–P bond formation to an extent that a significant degree of the effective
charge on the 2´-oxyanion is lost in the transition state.
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Comparison between HDV-catalyzed reactions of the abasic and wild type ribonucleotide
substrates reinforces the trend observed for the tethered substrates that the substrate bearing the
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higher pKa nucleophile reacts more slowly. The abasic substrate reacts ~4-fold slower than the
wild type substrate (Table 3), consistent with prior observations26. Given that HDV is known to be
largely insensitive to the identity of the sequence upstream of its cleavage site, it seems plausible
that increase in pKa upon removal of the electron-withdrawing nucleobase could explain some, if
not all, of the difference in reactivity between the abasic and wild-type substrates.
Several alternative explanations could account for the rate stimulation from fluorine
substitution observed in the ribozyme reaction: (I) The transition state for the HDV-ribozyme
reaction has relatively little bonding between the nucleophilic oxygen and the phosphorous atom,
so perturbation of the geminal hydroxyl’s nucleophilicity by fluoride substitution has a negligible
impact on reactivity. However, the non-enzymatic reaction occurs with a large effective charge
change on the nucleophile18. Moreover, the established importance of general acid catalysis in the
HDV ribozyme implicates significant bond breaking to the leaving group in the transition state9,
which, in the case of phosphodiesters, accompanies significant bond formation to the
nucleophile34. (II) Differential ground state interactions such as solvation of the nucleophile could
attenuate the relative reactivity of the methyl-tethered substrate relative to the trifluoromethyl-
tethered substrate. Stronger solvation of the former substrate in the ground state could engender a
greater energetic penalty for nucleophile desolvation en route to the chemical transition state. It is
important to note, however, that the tethered substrates react with nearly identical Km values in
kinetic experiments, suggesting that the substrates bind similarly in the ground state. Lastly, we
cannot rule out the possibility that a non-chemical step limits the reaction rates of the tethered
substrate and masks the true chemical effect of fluorine substitution. However, the significantly
lower reactivity of the tethered substrates compared to the wild-type substrate argues against this
possibility.
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Conclusions and Implications. In an effort to obtain substrates that subtly perturb the
basicity of the 2´-hydroxyl nucleophile in the HDV ribozyme reaction, we prepared the
phosphoramidite derivatives of 1,2-propanediol, 3-fluoro-1,2-propanediol and 3,3,3-trifluoro-1,2-
propanediol and successfully incorporated them into the 5¢-terminus of RNA and DNA
oligonucleotides. These oligonucleotides undergo hydroxide and HDV ribozyme-catalyzed
transphosphorylation to release the tethers. The hydroxide-catalyzed reactions show that the
nucleophilic hydroxyl group ionizes about one pKa unit lower and reacts about 10-fold slower for
the trifluoromethyl substrate relative to the methyl substrate. This relative reactivity suggests an
apparent βnuc value around one, consistent almost complete loss of negative charge from the
oxyanion nucleophile in the transition state due to substantial bond formation between the oxygen
nucleophile and the scissile phosphate18. Comparison between the base-catalyzed and RNA-
catalyzed reactions of these suggest that that (I) the ribozyme´s catalytic apparatus engages without
the cleavage site nucleobase or the conformational constraints imposed by the ribofuranose ring,
(II) the nucleophilic hydroxyl group predominantly populates the neutral form in the starting
ground state for the enzyme-substrate complex, and (III) in the transition state, the 2¢ hydroxyl
bears some negative charge consistent with a transition state in which extent of proton loss from
the 2¢-OH exceeds the extent of nucleophilic attack.
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The tethered substrates described here provide a simple and accessible context in which to
test structure activity relationships for both solution and ribozyme-catalyzed transphosphorylation.
Insights gleaned from these substrates could help to interpret additional biochemical data gathered
on ribozyme transition states including linear free-energy relationships for general acid-base
catalysis and kinetic isotope effects. Specifically, the qualitative information derived here
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regarding the protonation of the ribozyme substrate ground state could inform future heavy atom
kinetic isotope measurements for the 2´-O nucleophile35.
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Acknowledgments. This work was supported by the National Institute of Health (1R56AI081987
and 1R01AI081987) to J.A.P. and GM096000 to M.E.H. S.C.K. was partially supported by a
National Institute of General Medical Sciences Medical Scientist Research Service Award (5T32
GM07281) and B.P.W was partially supported by the Chemistry and Biology Interface Training
Program (T32GM008720).
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OH B!
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-
O
OH
O
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-
H B
!O
P
1
O P O
P
-
!
O
B
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!
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OH
OH
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OH
O P O
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O P O
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O P O
O
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3´RNA
3´RNA
3´RNA
Increasing reactivity from hydroxyl ground state
R: CH3
R: CH2F
R: CF3
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