Class-Ib Ribonucleotide reductases are manganese-containing enzymes suited for performing the conversion of ribonucleotides to deoxyribonucleotides, which are essential for DNA synthesis and repair.

1. 1
Anirban Sil
Reg. No. 20233224
Thesis Supervisor-Dr. Debangsu Sil
Dimanganese Cluster as a
Structural and Functional Model
for Class-Ib Ribonucleotide Reductases
Departmental Seminar
17th
September 2024

2. 2
Introduction
Image: Gyunghoon "Kenny" Kang
Newly discovered enzyme “square dance” helps generate DNA building blocks – MIT Departme
nt of Chemistry
Ribonucleotides Deoxyribonucleotides

3. Quaternary structure of the active holoenzyme complex in class I RNR
Boal et al. J. Biol. Chem. 2021, 297, 101137 3

4. 4
Proposed pathway for electron transfer from metallocofactor to active site of RNR
in E. Coli
Bennati et al. Chem. Sci., 2016, 7, 2170–2178

5. 5
Schematic of ribonucleotide reduction and radical translocation (RT) in class I
Ribonucleotide Reductase.
Boal et al. J. Biol. Chem. 2021, 297, 101137

6. 6
Five different subclasses of Type I Ribonucleotide Reductase
Boal et al. J. Biol. Chem. 2021, 297, 101137

7. 7
Mechanism for tyrosine radical formation by the enzymatic
cofactor of Class-Ib RNRs from E. Coli
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

8. Previously reported similar type of complexes
N N
O
N
N
N
N N
N
N
N
MnII
MnII
O O
2+
N N
N N
N
N
O
Mn Mn
O O
O
O
+
McDonald et al. Angew. Chem. Int. Ed. 2019, 58, 5718 –5722
N
N
S
MnII
S
N
N
S
S
Ph
Ph
Ph
Ph
Mn
II
N
N
S
MnII
SH
N
N
S
S
Ph
Ph
Ph
Ph
Mn
II
+
McDonald et al. Angew. Chem. Int. Ed. 2018, 57, 918 –922
Duboc et al. Angew. Chem. Int. Ed. 2017, 129, 8323-8327
8
Duboc et al. Angew. Chem. Int. Ed. 2017, 56, 8211-8215

9. 9

10. 10
OH
OH OH O
O OH O
O O O
O S
N
S
N
O
S
N
O
N
N
N
N
N
N
MnO2
Reflux
5 days
dry DMF
N2 atmos.
DABCO
BF3.Et2O
Toluene
inert atmos.
N
N
H N
S
S
N
N
N
N
N
N
N
N
N
N
N
N KOH in MeOH
Reflux 18h
I2
stirring 3h (r.t)
Cl
N
S
Yellow crystals
3/1 Toluene/H2O
stirring at r.t 18 h
stirring 18 h
(A) (B)
(C) (D)
(E)
(F)
Synthesis of ligand
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

11. 11
1
H-NMR and 13
C-NMR spectrum of ligand
S
S
N
N
N
N
N
N
N
N
N
N
N
N
1
H-NMR spectrum of ligand
13
C-NMR spectrum of ligand
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

12. Reaction scheme for synthesis of metal complex
S
S
N
N
N
N
N
N
N
N
N
N
N
N
MeCN/KC8
inert atmosphere (N2)
Mn(OAc)2·4H2O (4 eqv.)
NaClO4·H2O (3 eqv.)
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]ClO4
(complex 1)
+
average Mn-S = 2.59 Å
average Mn-S-Mn = 86.250
average Mn-Mn = 3.541 Å 12
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

13. 13
N
N
N
N
N
N
S
Mn Mn
O
O
O
O
[MnII
2(OAc)2(BPMT)]+
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
KO2/18-Crown-6
CH3CN/DMF
(00
C)
+ 2+
Reactivity of complex 1 with superoxide
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

14. 14
Reactivity of complex 1 with superoxide
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
N N
N
N
N
N
S
MnIII
MnII
O
O
[Mn(O2)Mn(BPMT)]2+
KO2/18-Crown-6
CH3CN/DMF
(00
C)
+ 2+
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

15. 15
Resonance Raman spectrum at -300
C
N N
N
N
N
N
S
MnIII
MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+
N N
N
N
N
N
S
MnIII
MnII
O18
O18
[Mn(O2)Mn(BPMT)]2+
2+
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

16. 16
DFT optimized structure of complex 2
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
N N
N
N
N
N
S
MnIII
MnII
O
O
[Mn(O2)Mn(BPMT)]2+
KO2/18-Crown-6
CH3CN/DMF
(00
C)
+ 2+
DFT optimized structure of complex 2
Complex 1 Complex 2
ν(O-O) stretching frequency (calculated) = 814 cm-1
ν(O-O) stretching frequency (experimental) = 791 cm-1

17. 17
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
X-Ray absorption spectroscopy
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
N N
N
N
N
N
S
MnIII
MnII
O
O
[Mn(O2)Mn(BPMT)]2+
KO2/18-Crown-6
CH3CN/DMF
(00
C)
+ 2+
Average oxidation state of Mn in complex 2
Shown in red is 2.6
Complex 2
Complex 1
Mn K-edge XAS spectra of 1 (black) and
Complex 2 (red)
MnO2
Mn2O3
MnO

18. 18
Comparison of Cyclic voltammogram of complex 1 and Zn complex
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
+
N
N
N
N
N
N
S
Zn Zn
O O
[ZnII
2(BPMT)(OAc)]2+
2+
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
0.38 V

19. 19
Kinetic studies on complex 2
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
N N
N
N
N
N
S
MnIII
MnII
O
O
[Mn(O2)Mn(BPMT)]2+
KO2/18-Crown-6
CH3CN/DMF
(00
C)
+ 2+
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

20. Catalytic activity of complex 2 on various substrates
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+
CHO
CHO
H
OH
2-PPA
CCA
2,6-DTBP
Complex 2
O16
CO2H
O
O
20
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

21. 21
Comparison of mass spectrum of product (red) with ESI-Mass spectrum of
acetophenone (blue)
m/z
m/z
N N
N
N
N
N
S
MnIII MnII
O
O
2+
O
CHO
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Abundance
100%

22. 22
Comparison of mass spectrum of product after isotopic labelling of complex 2
m/z
m/z
N N
N
N
N
N
S
MnIII MnII
O18
O18
2+
O18
CHO
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Abundance

23. Catalytic activity of complex 2 observed using UV-spectrum and kinetic studies
23
N N
N
N
N
N
S
MnIII MnII
O
O
2+
O
CHO
CHO
D
(a.)
(b.)
(a.) kobs(2-PPA) = 0.257 M-1
cm-1
(b.) kobs(α-[D1]-PPA) = 0.122 M-1
cm-1
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

24. EPR spectrum after addition of 2-PPA to solution of complex 1 and excess KO2
24
N
N
N
N
N
N
S
Mn Mn
O O
O
O
[MnII
2(OAc)2(BPMT)]+
+
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+
KO2
CHO
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

25. 25
Proposed mechanism for catalytic oxidation of 2-Phenylpropionaldehyde (2-PPA)
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

26. 26
Reaction of complex 2 with 2-PPA in presence of CBr4
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
m/z
N N
N
N
N
N
S
MnIII MnII
O
O
2+
Br
CHO
CHO
CBr4
Abundance

27. 27
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
m/z
m/z
N N
N
N
N
N
S
MnIII MnII
O
O
2+
CHO CO2H
Comparison of mass spectrum of obtained product (red) with ESI-Mass spectrum
of cyclohexane-carboxylic acid (blue)
Abundance
100%

28. Reactivity of complex 2 with 2,6-DTBP observed using UV-visible and kinetic
studies
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076 28
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+ O
O
t
Bu t
Bu
t
Bu t
Bu
OH
t
Bu t
Bu
53%

29. 29
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+
OH
OH
t
Bu
t
Bu
t
Bu
t
Bu
OH
t
Bu
t
Bu
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Reactivity of complex 2 with 2,4-DTBP observed using UV-visible and kinetic
studies
10%

30. 30
Conclusion
 The generation of a thiophenolate-bridged [Mn(II)O2Mn(III)]2+
core in 2
through activation of superoxide radical has been reported.
 Complex 2 exhibits a 16-line St=1/2 EPR signal at g=2, which is typically
associated with an unusual μ-η1
:η2
binding mode of the peroxo ligand.
 Complex 2 was capable of oxidation of 2-phenyl propionaldehyde, cyclohexane
carboxaldehyde and 2,6-di-tert-butyl-phenols.
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

31. 31
Thank you

32. 32
Boal et al. J. Biol. Chem. 2021, 297, 101137
Ribonucleotide Reduction
Proposed pathway showing how Ribonucleotide reduction
takes place in four basic steps

33. 33
UV-Vis spectrum of intermediate 2
UV-Vis spectrum of CmlI peroxo
intermediate
Comparison of UV-Vis spectrum of intermediate 2 with CmlI peroxo
intermediate
J. Am. Chem. Soc. 2015, 137, 1608−1617
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+

34. 34
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Analysis of plots obtained from cyclic voltammetry

35. 35
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
CHO
CHO
CHO
MeO
CHO
HO
Reaction of complex 2 with substituted benzaldehyde
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+

36. 36
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Hammet plot for reactivity of complex 2 with 4-substituted benzaldehydes
N N
N
N
N
N
S
MnIII MnII
O
O
[Mn(O2)Mn(BPMT)]2+
2+
Deformylated
products
CHO
X
(X=H,Me,OMe and OH)

37. 37
O
H/D
O
kH / kD = 1.22
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076
Reactivity of complex 2 with substituted phenols

38. 38
λmax (nm) ε (M-1
cm-1
)
380 nm 3875
510 nm 1470
560 nm 1050
695 nm 400

39. 39
Annu Rev Biochem. 2020; 89: 45–75.
N
N N
N
Co
O
OH
OH
A
CONH2
CONH2
CONH2
N
N
O
HO
O
OH
P
O
O-
O
NH
O
H2NOC
H2NOC
H2NOC
O
OH
OH
A
.
Fe S
Fe
S
S Fe
S
Fe
S
S
S
H2
N
O O
S
O
OH OH
A
Cys
Cys
Cys
N
H
O
.
5’-deoxyadenosyl
radical
Glycyl radical
Class II Ribonucleotide
Reductase
Class III Ribonucleotide
Reductase
Type II and Type III Ribonucleotide Reductase

40. 40
O
O S
N
O
(D)
S
N
O
N
N
N
N
N
N
(E)
1,2-dichloroethane
glacial acetic acid
N
N
H N
NaHB(OAc)3
Ray et al. Angew. Chem. Int. Ed. 2023, 62, 17076

41. 41
Mn2+
Mn3+ + e-
Ecell = -1.542 V
Dioxygen reduction is
unfavourable!
Chemistry of dioxygen reduction