Chemical Mutagenesis of an Emissive RNA Alphabet

Article abstract of DOI:10.1021/jacs.5b10420

An evolved fluorescent ribonucleoside alphabet comprising isomorphic purine (^tzA, ^tzG) and pyrimidine (^tzU, ^tzC) analogues, all derived from isothiazolo[4,3-d]pyrimidine as a common heterocyclic core, is describ

Full text of DOI:10.1021/jacs.5b10420

Communication
pubs.acs.org/JACS
Chemical Mutagenesis of an Emissive RNA Alphabet
Alexander R. Rovira, Andrea Fin, and Yitzhak Tor*
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States
S Supporting Information
*
ABSTRACT: An evolved fluorescent ribonucleoside
tz
tz
alphabet comprising isomorphic purine ( A, G) and
tz
tz
pyrimidine ( U, C) analogues, all derived from
isothiazolo[4,3-d]pyrimidine as a common heterocyclic
core, is described. Structural and biochemical analyses
illustrate that the nucleosides, particularly the C-
nucleosidic purine analogues, are faithful isomorphic and
isofunctional surrogates of their natural counterparts and
show improved features when compared to an RNA
alphabet derived from thieno[3,4-d]-pyrimidine. The
restoration of the nitrogen in a position equivalent to
the purines’ N7 leads to “isofunctional” behavior, as
illustrated by the ability of adenosine deaminase to
tz
deaminate A as effectively as adenosine, the native
substrate.
Figure 1. Evolution of an emissive RNA alphabet.
luorescent nucleoside analogues, which have been devel-
“isofunctional”, illustrated by the ability of adenosine deaminase
(ADA) to deaminate the adenosine analogue as effectively as
adenosine, the native substrate.
F
oped to address the nonemissive nature of the nucleobases
found in DNA and RNA, have found great utility in biophysical
1
analysis and discovery assays. In addition to favorable
While standard conditions provided the isothiazole uridine
tz
tz
6
photophysical features, a key element that dictates their utility
and ultimate use is their structural similarity to the native
counterparts. Characterized as their isomorphic nature,
researchers have tried to maximize this trait by minimizing
changes to the Watson−Crick (WC) pairing face while2
introducing fluorescence-enhancing electronic perturbations.
The impact of these alterations on the physical properties (e.g.,
tautomerization) and photophysics (excited state level and
dynamics) are, however, frequently unpredictable and ultimately
empirically assessed.
and cytidine analogues U and C, respectively, common
approaches to forming the carbon−carbon glycosidic bonds of
the adenosine and guanosine analogues failed under a wide
7
,8
variety of conditions. A less common approach was required to
9
form the desired connectivity. A strategy starting from a
ribofuranose-derived precursor as opposed to late-stage
glycosylation with the nucleobase and desired sugar was
conceived. To construct the C-glycosylated isothiazole ring, a
ribofuranose derivative substituted with a primary thiol 3 was
synthesized (Scheme 1), starting from a known benzyl-protected
precursor, which was subjected to Swern oxidation conditions to
We have previously completed the first emissive RNA
3
10
alphabet, comprising pyrimidine and purine analogues, all
give 1. Treatment of 1 with diiodomethane and methyllithium
11
fundamentally derived from thieno[3,4-d]-pyrimidine as a
furnished the primary halide 2, which was reacted with
potassium thioacetate and reduced to give the primary thiol 3
(Scheme 1).
common heterocyclic nucleus (Figure 1). While highly emissive
4
and valuable as illustrated by several applications, a limitation of
the previously reported thieno analogues is the lack of the basic
a
nitrogen, corresponding to N7 in the purine skeleton (Figure
Scheme 1. Synthesis of the Sugar Precursor
5
1
). As many biomolecular interactions of purine nucleosides and
nucleotides rely on the basicity and coordinating ability of this
position, we have sought to reinstall this functionality into a new
isomorphic RNA alphabet, with higher structural and electronic
similarity to the native purines. Herein we report the synthesis,
photophysical analysis, and performance of a next generation
alphabet based on an isothiazolo[4,3-d]pyrimidine core. This
atomic mutagenesis reinstates the basic moiety at the native
position yielding analogues that better mimic a purine (Figure 1).
This chemically evolved isomorphic alphabet was found to not
only have unique photophysical features but also to be highly
a
Reagents and conditions: (a) CH I , MeLi, toluene, −78 °C, 1 h,
2 2
6
0
7%. (b) Potassium thioacetate, DMF, rt, 6 h, 74%. (c) Et O, LiAlH ,
2
4
°C to rt, 1 h, >90%.
Received: October 5, 2015
Published: November 2, 2015
©
2015 American Chemical Society
14602
DOI: 10.1021/jacs.5b10420
J. Am. Chem. Soc. 2015, 137, 14602−14605

Journal of the American Chemical Society
Communication
a
Scheme 2. Synthesis of Purine Analogues
Figure 2. X-ray crystal structures of isothiazolo[4,3-d]pyrimidine
tz
tz
tz
tz
analogues: (a) A, (b) G, (c) U, and (d) C.
a
Reagents and conditions: (a) 3, EtOH, morpholine, 5 h, 0 °C to rt,
6
6
7% (5a), 79% (5b). (b) Et SiH, BF ·OEt , DCM, −78 °C to rt, 4 h,
3
3
2
3% (6a), 79% (6b). (c) i. EtOC(O)NCS, CH CN, rt, 4 h; ii. EDCl,
3
HMDS rt, 48 h, 63% (d) i. 1 M NaOH, MeOH, 65 °C; ii.
HSCH CH SH, BF ·OEt , DCM, rt, 48 h, 59%. (f) CH(OEt) , Ac O,
2
2
3
2
3
2
1
7
20 °C, 16 h, 69%. (g) P S , pyridine, 65 °C, 2 h. (h) i. NH , MeOH,
2
5
3
0 °C, 16 h; ii. HSCH CH SH, BF ·OEt , DCM, rt, 72 h, 32% (for
2 2 3 2
steps g and h).
Reactions of 3 with both ester and amide-substituted N-tosyl
derivatives 4a and 4b furnished the cyclized 5a and 5b,
1
2
respectively, in good yields (Scheme 2). To set the stereocenter
at the anomeric carbon, reduction of 5a and 5b with triethylsilane
and BF ·OEt was found to be the most effective, yielding key
Figure 3. Comparison of intermolecular H-bonding seen in the crystal
structures of (a) G and (b) G.
tz
3
2
13
precursors 6a and 6b, respectively (Scheme 2). Only one
diastereomer was isolated and found to be the desired one (see
below). With this key substrate in hand, the synthesis of the
protected guanosine analogue 7 was accomplished using a mild
two-step, one-pot reaction with an isothiocyanate precursor.
After cleavage of the carbamate, deprotection using 1,2-
ethanedithiol and BF ·OEt yielded the final nucleoside
Synthesis of the adenosine analogue was accomplished
via initial construction of the inosine analogue 8 using triethyl
orthoformate. This was subsequently converted to the thioamide
by treatment with P S in pyridine followed by methanolic
pK values of the modified nucleoside analogues are listed in
a
Table 1. The ground-state absorption spectra in aqueous solution
displayed bathochromic-shifted maxima compared to the
corresponding native nucleosides ranging from 312 to 338 nm
14
tz
tz
for U to A, respectively (Figure 4a). Visible emission maxima
tz
tz
ranging from 392 nm (for U) to 459 nm (for G) were
observed for all modified nucleosides upon excitation at their
maxima. The emission quantum yield of the purine analogues,
3
2
tz 15,16
G.
tz
tz
0.25 for G and 0.05 for A, was higher than the pyrimidine
tz
tz
analogues, with 0.01 for U and 0.05 for C in water.
9
The absorption spectra taken in dioxane showed batho and
2
5
tz
tz
tz
ammonia to give the protected final product, which was then
hypochromic shifts for A, C, and G in comparison to the
aqueous solutions, while no significant variations were observed
tz
subjected to the same deprotection conditions to afford
Scheme 2).
Crystal structure determination confirmed the proposed
structure and anomeric configuration of the modified ribonu-
cleosides (Figure 2 and Tables S1−S4). In the solid state, the
purine analogues A and G showed an anti-orientation at the
glycosidic linkages, while the pyrimidines U and C were found
to be in the syn-orientation (Figure 2). Rewardingly, analysis of
G’s crystal packing pattern shows pairing through both the WC
and Hoogsteen faces, which is identical to the pattern seen for
A
tz
(
for U. The emission intensity is sensibly lower in dioxane. The
fluorescence maxima displayed a remarkable hypsochromic shift
tz
tz
tz
for G and U, a slight blue-shift for A, and a bathochromic shift
tz
for C. This suggests a charge-transfer character of their excited
tz
tz
states (Figure S5). This is manifested for all nucleosides, albeit to
different extents, in their responsiveness toward polarity changes,
as seen by the linear correlations between the measured Stokes
shifts and microscopic solvent polarity parameters (Figure 4b,
Table 1).
tz
tz
tz
17
guanosine in the solid state (Figure 3). This intermolecular
All new nucleosides display sensitivity toward pH variations,
hydrogen bonding arrangement, illustrating the restoration of a
thus facilitating the extraction of pK values (Figure 4c,d and
Table 1). The deprotonation of U between pH 8 and 11 is
a
tz
tz
“functional” Hoogsteen face and “N7”, suggests that G is likely
to share G’s H-bonding and tautomeric preferences.
The fundamental spectroscopic properties, the sensitivity
toward environmental polarity, and the spectroscopically derived
characterized by a red shift of its absorption maximum yielding a
pK value of 8.9, which is comparable to the values reported for
a
18
tz
N3 deprotonation in uridine (pK 9.20−9.25). C is the least
a
1
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DOI: 10.1021/jacs.5b10420
J. Am. Chem. Soc. 2015, 137, 14602−14605

Journal of the American Chemical Society
Communication
Table 1. Photophysical Properties of Isothiazolo[4,3-d]pyrimidine Nucleoside Analogues
c
pKa
a
a
a
b
solvent
λabs (ε)
λem (Φ)
Φε
Stokes shift
polarity sensitivity
27.7
abs
4.25
em
tzA
tzC
tzG
tzU
tzI
water
338 (7.79)
342 (7.42)
325 (5.45)
333 (5.03)
333 (4.87)
339 (4.65)
312 (5.17)
314 (5.20)
316 (7.63)
315 (6.62)
410 (0.05)
409 (0.03)
411 (0.05)
419 (0.04)
459 (0.25)
425 (0.17)
392 (0.01)
377 (<0.01)
377 (0.01)
372 (<0.01)
413
193
289
181
1203
539
41
5.23
4.76
6.42
6.14
8.27
6.01
6.53
5.36
5.13
3.29
dioxane
water
10.5
102.0
45.4
12.7
2.94
2.46, 10.38
9.88
dioxane
water
3.55, 8.51
2.25, 8.88
9.26
dioxane
water
8.94
dioxane
water
21
46
7.83
dioxane
26
4.79
a
3
−1
−1
3
−1
λ , ε, λ , and Stokes shift are reported in nm, 10 M cm , nm, and 10 cm respectively. All photophysical values reflect the average of at least
abs
em
b
−1
−1
three independent measurements. Sensitivity to solvent polarity reported in cm /(kcal mol ) is equal to the slope of the linear fit in Figure 4b.
c
pK values reflect the average over three independent measurements and are equal to the inflection point determined by the fitting curves in Figure
a
4c,d. See Supporting Table S5 for expanded data including experimental errors.
th
tz
Figure 5. (a) Deamination of A and A. (b) Enzymatic deamination of
tz
tz
th
th
A to I (black), A to I (red), and A to I (gray) with ADA monitored
by real-time absorption at 260, 340, and 340 nm, respectively. Inset:
zoomed in region between 0 and 300 s. (c) Enzymatic deamination of
Figure 4. (a) Absorption (dashed lines) and emission (solid lines)
tz
tz
tz
tz
spectra of A (red), C (green), G (orange), and U (purple) in water.
b) Stokes shift correlation versus solvent polarity (E (30)) of water/
tz
tz
A to I by ADA monitored by real-time absorption (red) at 340 nm
(
T
tz
tz
tz
tz
and real time emission (light purple) at 410 nm (excitation at 322 nm).
dioxane mixtures for A (red), C (green), G (orange), and
U
tz
Inset: HPLC relative peak area variation at different time-points for A
(
purple). (c) Absorption maxima and (d) emission maxima variation
tz
tz
tz
tz
tz
(
red) and I (blue) monitored at 340 nm.
versus pH for A (red), C (green), G (orange), and U (purple).
responsive to pH changes, displaying minor blue shifts in both
the absorption and emission spectra (Figure 4c,d), and yielding
two pKa values (Table 1). A was characterized by a
deamination of adenosine to inosine. This important metabolic
21
transformation is catalyzed by adenosine deaminase (ADA).
tz
We have previously reported the ability of ADA to recognize and
th
bathochromic shift both in its absorption and emission maxima
deaminate A, the thieno[3,4-d]-pyrimidine-based analogue of
th
upon deprotonation of N1 (pK = 4.25 and 3.29, respectively) in
the naturally occurring adenosine, to I (Figure 5a), the
a
close proximity to the reported values for adenosine (pK 3.6−
corresponding inosine derivative. While of significance in and
a
1
9
tz
th
th
4
.2). pH titration of G showed two distinct red-shifted
of itself, the enzymatic conversion of A to I was, however,
4b
transitions of the absorption maximum and two different
approximately 20-times slower compared to that of adenosine.
isosbestic points at 325 and 333 nm (Figure S2), assigned to
Since ADA has been crystallographically shown to form a H-
21a,b
deprotonation of N7 and N1 (pK = 3.55 and 8.51, respectively).
bond to the N7 of its substrate,
restoration of this functionality in A, the new adenosine
surrogate, should facilitate its deamination compared to A.
we hypothesized that
a
tz
These values correlate well with the reported values for
20
th
guanosine (pK = 3.2−3.3 and 9.2−9.6, respectively). Taken
a
together, these observations not only illustrate the responsive-
ness of these nucleosides, but further indicate the ability of the
isothiazole ring to mimic the basic imidazole moiety in the native
purines.
Steady state absorption and emission spectra, taken at defined
tz
time-intervals upon addition of ADA to A, showed a fast and
tz
tz
efficient conversion of A to I (Figure S6). Real-time
continuous measurements, relying on the photophysical differ-
tz
tz th
th
4b,22
To demonstrate the utility of the new analogues and the
impact of restoring the basic N7 in evolving the thieno alphabet
into the isothiazolo one, we selected to probe the enzymatic
ences between A and I, A and I, and A and I,
show that,
th
while the deamination reaction of A is indeed sluggish, ADA
deaminates A to I at the same rate as it deaminates A to I
tz
tz
1
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DOI: 10.1021/jacs.5b10420
J. Am. Chem. Soc. 2015, 137, 14602−14605

Journal of the American Chemical Society
Communication
tz
(
Figure 5b). The reaction half-life for A deamination, calculated
2012, 18, 5987−5997. (d) Dumat, B.; Bood, M.; Wranne, M. S.;
́
Lawson, C. P.; Foller Larsen, A.; Preus, S.; Streling, J.; Graden, H.;
assuming a pseudo-first order reaction, was comparable to the
one observed for adenosine, the native substrate (t1/2 = 39 and 57
s, respectively) and substantially shorter than the one obtained
for A (t = 818 s). This remarkable initial deamination rate of
A by ADA, substantiating our hypothesis, was confirmed by
HPLC analyses (Figure 5c inset, Figure S8). Taken together,
these observations highlight the improved functionality of the
isothiazolopyrimidine over the thienopyrimidine core.
In summary, we introduce a second-generation family of
emissive nucleoside analogues, based on an isothiazolopyrimi-
dine scaffold. This atomic mutation results in higher
isomorphicity and significantly improved functionality when
compared to the thienopyrimidine-based RNA alphabet. The
presence of the isothiazole core with its nitrogen in the
equivalent position to N7 of the native purines, restores the
Hoogsteen face, as well as the basicity and native H bonding
ability of these surrogates, as illustrated structurally (for G) and
biochemically (for A deamination by ADA). These observa-
tions, indicating that the newly introduced purine surrogates
display improved structural and functional characteristics, are of
significance since very few emissive, isomorphic, and non-
perturbing purine analogues have so far been made and
biophysically exploited.
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th
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8
716.
tz
(6) The synthesis of the pyrimidine analogues is described in the
Supporting Information (Scheme S5).
tz
(7) (a) Cote, G. L.; Flitsch, S.; Ito, Y.; Kondo, H.; Nishimura, S.; Yu, B.;
́
Fraser-Reid, B. O.; Tatsuta, K.; Thiem, J. Glycoscience: Chemistry and
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(
chemistry, grignard formation, alkyllithium chemistry, and standard
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(9) Wamhoff, H.; Berressem, R.; Nieger, M. J. Org. Chem. 1993, 58,
5181−5185.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b10420.
■
*
S
(10) See Supporting Information (Scheme S1).
(
(
11) Bessieres, B.; Morin, C. Synlett. 2000, 1691−1693.
Synthetic details, photophysical data, enzymatic protocols
and HPLC traces (PDF)
X-ray crystallographic data for G (CIF)
X-ray crystallographic data for C (CIF)
X-ray crystallographic data for U (CIF)
12) Gewald, K.; Bellmann, P. Liebigs Ann. Chem. 1979, 1979, 1534−
1
546.
tz
(
13) van Rijssel, E. R.; van Delft, P.; Lodder, G.; Overkleeft, H. S.; van
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tz
́
tz
tz
(
2
(
X-ray crystallographic data for A (CIF)
AUTHOR INFORMATION
Corresponding Author
ytor@ucsd.edu
Notes
■
*
1661−1664.
(16) Seley, K. L.; Zhang, L.; Hagos, A.; Quirk, S. J. Org. Chem. 2002, 67,
3365−3373.
(17) (a) Additionally, an overlay of the purine analogues and natural
nucleosides may be found on Figure S1. (b) Marsh, R. E.; Bugg, C. E.;
Thewalt, U. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem.
The authors declare no competing financial interest.
1
970, B26, 1089−1101.
ACKNOWLEDGMENTS
We thank the National Institutes of Health for generous support
GM 069773), the Chemistry and Biochemistry MS Facility, and
the UCSD X-ray crystallography Facility.
■
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̈
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DOI: 10.1021/jacs.5b10420
J. Am. Chem. Soc. 2015, 137, 14602−14605