Download METAL BINDING TO MODIFIED BASES AND NUCLEOSIDES Iskra

METAL BINDING TO MODIFIED
BASES AND NUCLEOSIDES
Iskra Muhamedagic
North Carolina Agricultural and Technical State University
PRESENTATION OUTLINE
• INTRODUCTION
-tRNA and importance of metals in tRNA
-Pharmacological/medical applications of modified nucleosides
• METHODS
-UV/VIS and Job’s plot method
-LC/MS
-1D/2D NMR
-Restrained Molecular Dynamics of U-S4U-U oligonucleotide
• RESULTS AND DISCUSSION
-UV spectra, Job’s plots, and LC/MS data discussion
-1D NMR of monomers
-1D/2D NMR and RMD of U-S4U-U
• CONCLUSIONS AND FUTURE WORK
INTRODUCTION
• tRNA is polynucleotide chain of 75 to 90 units long that folds
•
•
into native L shape
Most common modifications are S4U and S2U at positions 8 and
34, respectively
S4U controls tRNA folding, S2U provides binding site for metal
ions
Bases and nucleosides
modifications can take place
in sugar or base
C1’―N1 is b-glycosyl bond
there are five metal binding sites
S4
OH
O
P
5
6
-
O
5'
O
Torsion Angle Atoms involved

(n-1)O3’-P-O5’-C5’
b
P-O5’-C5’-C4’

O5’-C5’-C4’-C3’

C5’-C4’-C3’-O3’

C4’-C3’-O3’-P

C3’-O3’-P-O5’(n+1)

O4’-C1’-N1-C2
0
C4’-O4’-C1’-C2’
1
O4’-C1’-C2’-C3’
2
C1’-C2’-C3’-C4’
3
C2’-C3’-C4’-O4’
4
C3’-C4’-O4’-C1’
4
1
N
3NH
2
S4U
O2
O
1'
4'
O
2'
3'
O
OH
NH
nucleotide unit
•
•
•
•
O
P
-
O
O
O
O
S
N
S2U
O
O
P
-
O
OH
OH
Sugar puckering and base’s orientation
• relative to sugar base can
•
adopt anti or syn orientation
S2U puckers into C3’endo/anti form; S4U prefers
C2’-endo/syn conformation
3E
3
2E
2T
3T
2
• sugar can pucker into
anti
syn
C3’-endo or C2’-endo
form with either twist
or envelope
conformation
METHODS
• UV/VIS (Ultraviolet/ Visible Spectroscopy)
-absorbance was monitored in 200 – 400 nm range
-for S2U and 2TU, lmax = 272 nm
-for S4U and 4TU, lmax = 332 nm
-binding stoichiometry was determined by modified Job’s plot
• LC/MS (Liquid Chromatography/ Mass Spectroscopy)
-1 mM solutions of ligands and mercury acetate were
prepared in deionized H20 and in 50% H20/50% ACN
-binding stoichiometry and charge is obtained from
m/z ratio
• NMR (Nuclear Magnetic Resonance)
-NMR data (1D and 2D DQFCOSY and ROESY)
were acquired on 500 MHz DRX spectrometer at
25°C at The School of Pharmacy, University of
Connecticut or at North Carolina State University,
Department of Chemistry by Dr. Mufeed Basti
Restrained Molecular Dynamics (RMD) of U-S4U-U
-energy minimization and molecular dynamics
was performed using Discover (Accelrys)
-distance constraints generated from ROESY NMR
data were used in molecular dynamics simulation
-force constant: 1000 kcal mol-1 deg-2 at 298 K
RESULTS AND DISCUSSION
• UV spectra of ligands and complexes
0.90
0.70
0.50
2TU-Hg 10th step
2TU-Hg 15th step
0.30
0.10
Absorbance
Absorbance
1.10
2TU
2TU-Hg 5th step
-0.10
200 225 250 275 300 325 350 375 400
1.50
1.30
1.10
0.90
0.70
0.50
0.30
0.10
-0.10
200 225 250 275 300 325 350 375 400
Wavelength (nm)
0.90
0.70
0.50
S2U
S2U-Hg 5th step
S2U-Hg 10th step
S2U-Hg 15th step
0.30
0.10
-0.10
Wavelength (nm)
Absorbance
Absorbance
1.10
4TU
4TU-Hg 5th step
4TU-Hg 10th step
4TU-Hg 15th step
1.70
1.50
1.30
1.10
0.90
0.70
0.50
0.30
0.10
-0.10
S4U
S4U-Hg 5th step
S4U-Hg 10th step
S4U-Hg 15th step
200 225 250 275 300 325 350 375 400
200 225 250 275 300 325 350 375 400
Wavelength (nm)
Wavelength (nm)
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
0.0
Hg-4TU
Hg-S4U
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Aobs - (G x [g])
Aobs - (G x [g])
Job’s plots and Kequilibrium
Hg-2TU
Hg-S2U
-0.1
-0.2
-0.3
-0.4
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
[h]t/([h]t + [g]t)
3.0
2.3
1.5
0.8
K 0.0
-0.8
-1.5
-2.3
-3.0
[h]t/([h]t + [g]t)
0.020
0.015
0.010
0.005
K 0.000
-0.005
-0.010
-0.015
-0.020
0 2 4 6 8 10 12 14 16 18 20
0 2 4 6 8 10 12 14 16 18 20
Titration step
Titration step
LC/MS data analysis
S2U-Hg in H2O #26-45 RT: 0.65-1.14 AV: 20 NL: 3.50E5
T: + c ESI Full ms [ 100.00-2000.00]
457.0
100
95
90
85
455.0
80
75
70
Relative Abundance
65
60
454.1
55
50
45
40
35
453.1
30
459.0
25
20
464.1
15
10
5
471.2
447.1
430.7
0
430
434.2 436.5
435
440.2 442.7 445.3
440
445
448.1
461.1
451.3
450
455
460
465.1
465
m/z
469.3
470
472.4
476.0 478.8 481.0
475
480
486.2 488.3
485
490
492.0 493.5
495
1D NMR analysis of S2U and S4U
H6
H1'
H2'
H5
H6
H3'
H6
H2'
8.0
7.9
Chemical Shift (ppm)
H2'
S2U and S2U-Hg
-C3’-endo
-N3 involvement
6.00
5.75
4.25
Chemical Shift
(ppm) Shift (ppm)
Chemical
H5'
H5"
H5'
H5"
H4'
H1' H5
H3'
H5"
H4'
H1' H5
H3'
H5'
H4'
4.00
3.75
Chemical Shift (ppm)
3.50
Proposed model for Hg-ligand complexes
H
O
Hg
N
S
N
S
N
S
N
H
N
Hg
H
N
O
N
O
N
O
S
H
OH
HO
OH
HO
O
O
N
HO
N
S
N
Hg
S
O
S
OH
N
N
O
HO
N
O
HO
O
OH
HO
N
O
OH
Hg
O
N
S
OH
1D NMR of U-S4U-U
O
U1
H
H
N
HH
HO
H
N
O
O
H
H
H
7.5
HO P O
O
H
7.0
6.5
Chemical Shift (ppm)
H
H
OH
O
6.0
N
H
H
N
O
O
H
H
O
H
H
OH
H
HO
S
H
P
O
H
4.6
O
H
N
O H
H
N
O
O
H
H
H
U3
H
OH
OH
4.5
4.4
4.3
4.2
4.1
Chemical Shift (ppm)
4.0
3.9
3.8
2D NMR of U-S4U-U
U 1
H 4 '/ H 5 "
U 1
H 4 '/ H 5 '
3.8
3.9
U 3
4.0
H 4 '/ H 3 '
H 2 '/H 3 '
4.1
4.2
4
4.3
U
H 3 '/ H 2 '
4.4
U 1
U 1
H 4 '/ H 3 '
4.5
H 2 '/H 3 '
U
S
1
4
H 2 '/ H
U
H
100  J1'2'
%S 
J1'2'  J 3'4'
1 '
2 '/ H
U
H 2 '/ H
5.80
5.85
1 '
3
4.6
ppm
S
ppm
U 3
1 '
5.90
5.95
4.50
4.25
4.00
ppm
3.75
7.65
S 4U H3'/H6
S4U H1'/H6
7.70
7.80
U1 H2'/H6
U1 H3'/H6
ppm
7.75
S 4 U H2'/H6
U3 H3'/H6
U3 H2'/H6
7.85
7.90
U3 H1'/H6
U1 H1'/H6
7.95
5.95
5.90
5.85
ppm
5.80
5.75
5.70
4.6
4.5
4.4
ppm
4.3
4.2
0.5
0.6
H 3 '/ P 1
S
S
4
U
4
U
H 5 " /P 1
0.7
H 5 '/ P 1
U 3
H 5 '/ P 2
0.8
U 3
H 5 " /P 2
0.9
1.0
1.1
S
4
U
H 3 '/ P 2
1.2
4.50
4.25
ppm
4.00
p p m
U 1
RMD and U-S4U-U
-A-stacking of bases
-observed equilibrium
between C3’-endo/
C2’-endo and anti/syn
CONCLUSIONS AND FUTURE WORK
• UV/VIS in combination with the Job’s plot method
•
•
can be used to calculate the binding constant and
stoichiometry of binding of mercury to modified
nucleosides and bases
method is not suitable for zinc and cadmium
complexes because of lower affinity of ligand to
metal as well as the formation of multiple types of
complexes
future studies are directed towards finding effective
ligands for zinc and cadmium metal ions
THANK YOU
My advisors Dr. Mufeed Basti and Robert
Gdanitz
Dr. Nadja Cech from UNC-Greensboro