Fungal infections are of continuous concern, especially with regard to immunocompromised patients. In an effort to develop new potential anti-fungal agents, we have begun synthesizing a library of potential inhibitors of the fungal Methionine Synthase (MetSyn) enzyme. Key differences between the B12-independant fungal MetSyn enzyme and the B12-dependant mammalian form can allow for an antifungal drug to be developed to exclusively bind the fungal enzyme and inhibit fungal growth while leaving the host (patient) unaffected. We are currently exploring the synthesis of various pterin and deazaguanine-based molecules as these mimic folate, an essential substrate for MetSyn function. We have begun testing these new molecules for activity in a fungal growth assay, as well as a fluorescent assay for monitoring MetSyn activity

Bennett, Zachary

Bockman, Anna

Burkhart, Grace

Moriarty, Noah

Pruet, Jeff

English

ValpoScholar

Valparaiso University 
ValpoScholar 
Symposium on Undergraduate Research and 
Creative Expression (SOURCE) 
Office of Sponsored and Undergraduate 
Research 
Spring 4-1-2020 
Synthetic Routes to a Library of Novel Methionine Synthase 
Inhibitors 
Zachary Bennett 
Valparaiso University, zachary.bennett@valpo.edu 
Grace Burkhart 
Valparaiso University, grace.burkhart@valpo.edu 
Anna Bockman 
Valparaiso University, anna.bockman@valpo.edu 
Noah Moriarty 
Valparaiso University, Noah.moriarty@valpo.edu 
Jeff Pruet 
Valparaiso University, jeffrey.pruet@valpo.edu 
Follow this and additional works at: https://scholar.valpo.edu/cus 
Recommended Citation 
Bennett, Zachary; Burkhart, Grace; Bockman, Anna; Moriarty, Noah; and Pruet, Jeff, "Synthetic Routes to a 
Library of Novel Methionine Synthase Inhibitors" (2020). Symposium on Undergraduate Research and 
Creative Expression (SOURCE). 885. 
https://scholar.valpo.edu/cus/885 
This Poster Presentation is brought to you for free and open access by the Office of Sponsored and Undergraduate 
Research at ValpoScholar. It has been accepted for inclusion in Symposium on Undergraduate Research and 
Creative Expression (SOURCE) by an authorized administrator of ValpoScholar. For more information, please 
contact a ValpoScholar staff member at scholar@valpo.edu. 
Figure 8. Kirby-Bauer tests against (A) S.cerevisiae, (B) and B. cereus
A
B
Figure 1. (Left) transformation of homocysteine to methionine byMetSyn
(Right) binding of methylated folate and methionine in MetSyn
Figure 9.MetSyn inhibition assay, showing comparitive levels of
inhibition by synthesized molecules
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Bu
ffe
r (
No
Hc
ys
)
Hc
ys
En
zy
m
e
Re
ac
tio
n
In
hi
bi
to
r A
In
hi
bi
to
r H
In
hi
bi
to
r K
In
hi
bi
to
r L
In
hi
bi
to
r N
In
hi
bi
to
r OF
lu
o
re
sc
e
n
c
e
In
te
n
si
ty
Preliminary Inhibitor Screening
Figure 4. Synthesis of deazaguanine and with linker
Cl
OEt
OO
HN
N NH2
O
H2N
NaOAc
H2O
70%
HN
N NH
O
H2N
OEt
O
1. NaOH
2. HCl
60%
HN
N NH
O
H2N
OH
O
HN
N NH
O
H2N
OH
O
O
H2N OMe
O EDC, HOBt
DMSO, NEt3
89%
HN
N NH
O
H2N
NH
O
O OMe
O
HN
N NH
O
H2N
NH
O
O OMe
O
HN
N NH
O
H2N
NH
O
O OH
O
1. NaOH
2. HCl
74%
Figure 5. Synthesis of protected amino acid tail
NH2
O
H2N
OH
O
Boc2O
NaCO3
H2O/dioxane
73%
NH2
O
N
H
OH
O
O
O
PIDA
EtOAc/MeCN/H2O
(2:2:1)
78%
NH3
N
H
O
O
O
O
NH3
N
H
O
O
O
O
CBZ-Cl (50% sol.)
K2CO3, KOH
THF/H2O, 0℃
88%
N-CBZ
N
H
OH
O
O
O
H
CCl3 O
NH
DCM
81%
N-CBZ
N
H
O
O
O
O
H
H2, Pd/C
MeOH
quant.
NH2
N
H
O
O
O
ON-CBZ
N
H
O
O
O
O
H
Figure 6. Coupling of amino acid tail to linker
O
O
O
O
HNH2N
HN
N NH
O
H2N
NH
O
O OMe
O
EDC, HOBt
DMSO, NEt3
62%
HN
N NH
O
H2N
NH
O
O N
H
O
O
O
O
O
HN
HN
N NH
O
H2N
NH
O
O N
H
O
O
O
O
O
HN
HCl
Dioxane
90%
HN
N NH
O
H2N
NH
O
O N
H
O O
OH
NH2
N
N OH2N
NH2
O H
N O
O
N
H H2N
O
OH
HN
N NH2N
O
S
O
N
H
H2N
O
OH
N
HN
O
H2N NH
N
S
O
O
N
H NH2
O
OH
HN
N NH2N
O
S
O
O
N
H
HN
N NHH2N
O
O
O
N
H
O
NH
NH2
O
OHHN
N NHH2N
O
O
H2N
O
OH
Folate Mimic Linker Amino Acid Tail
N
H
O
O
N
H NH2
O
OH
HN
N NHH2N
O
O
O
N
H
O
NH
NH2
HN
N NHH2N
O
O
N
H
O
O
N
H
NH2
Inhibitor A Inhibitor H
Inhibitor K Inhibitor L
Inhibitor M Inhibitor N
HN
N NHH2N
O
O
O
N
H
O
NH
Inhibitor O
CO2H
O
NH
CO2H
HO2C
Inhibitor P
Inhibitor R
N
HN
O
H2N NH
N
S
O
O
Inhibitor Q
N
H
CO2H
O
NH
CO2H
CO2H
Synthetic Routes to a Library of Novel Methionine Synthase Inhibitors
Zach Bennett, Grace Burkhart, Noah Moriarty, Anna Bockman, Jeff Pruet
Valparaiso University, IN
1. Pfaller, M. A.; Diekema, D. J. (2007). Epidemiology of Invasive
Candidiasis: A Persistent Public Health Problem. Clinical
Microbiology Reviews. 20 (1): 133–63.
2. Suliman, H.S.; Appling, D.R.; Robertus, J.D. (2007). The gene for
cobalamin-independent methionine synthase is essential in
Candida albicans: A potential antifungal target. Arch Biochem
Biophys. 467, 218-226.
3. Ubhi, D.; Kago, G.; Monzingo, A. F.; Robertus J. D. (2014).
Structural Analysis of a Fungal Methionine Synthase with
Substrates and Inhibitors. J. Mol. Bio. 426, 1839-1847.
We would like to thank Dr. Pruet for his guidance and direction on this
project, as well as Dr. Clark for his assistance in the protein purification of
methionine synthase, and Dr. Nunnely for her aid in Kirby-Bauer tests.
This work has been funded by the Indiana Academy of Sciences Senior
Research Grant (J.P.), EPIC scholarship (A.B.), and Eli Lilly (Z.B.).
References
Acknowledgements
Fungal infections are a common public health
concern in regards to immuno-compromised
patients who have a much higher mortality rate due
to their condition.1 Methionine Synthase (MetSyn),
is an enzyme which converts homocysteine to
methionine using a methylated folate molecule
(Figure 1). Due to the close binding of homocysteine
and folate in the fungal enzyme2,3, molecules can be
As MetSyn converts homocysteine to methionine,
MetSyn activity can be detected by homocysteine
concentration. The MeasureIT-thiol quantification
assay gives a fluorescent response in proportion to
Hcy concentration. High fluorescence shows large
concentration of Hcy, allowing the method to be
adapted to show enzyme activity. High fluorescence
compared to control shows inhibition of the enzyme
when mixed with our novel inhibitors, while low
fluorescence shows consumed Hcy, showing poor
inhibition.
Figure 7. Current library of inhibitors, and inhibitor design
synthesized to selectively
inhibit the fungal enzyme,
providing a safer anti-
fungal treatment (Figure 2).
Folate Mimic Linker Amino Acid Tail Potential MetSyn Inhibitors
Fungal infections are of continuous concern,
especially with regard to immunocompromised
patients. In an effort to develop new potential anti-
fungal agents, we have begun synthesizing a library
of potential inhibitors of the fungal Methionine
Synthase (MetSyn) enzyme. Key differences between
the B₁₂-independant fungal MetSyn enzyme and the
B₁₂-dependant mammalian form can allow for an
antifungal drug to be developed to exclusively bind
the fungal enzyme and inhibit fungal growth while
leaving the host (patient) unaffected. We are
currently exploring the synthesis of various pterin
and deazaguanine-based molecules as these mimic
folate, an essential substrate forMetSyn function.We
have begun testing these new molecules for activity
in a fungal growth assay, as well as a fluorescent assay
for monitoring MetSyn activity.
Multiple routes of synthesis to the folate mimic 7-
carboxymethyl pterin (7-CMP) were conceived of
and tested. Path A produced slightly better yields,
where Path B allowed for quick synthesis and easy
purification. This led to Path B to be our method of
choice for 7-CMP synthesis (Figure 3).
The linker is then coupled to the amino acid tail, and
deprotected, affording a potential inhibitor (Figure
6). A library of molecules has been prepared
(Figure 7) and tested on various fungi and bacteria
via Kirby-Bauer test, showing inhibitors H, N, and
A with large zones of inhibition in comparison to
the positive control (Figure 8).
In conclusion, our inhibitors show promise in
regards to inhibiting the enzyme methionine
synthase. More work is being done in testing the
enzymes directly on fungi and bacteria, as well as
cancer cell lines. Folate pathway inhibitors have also
been shown to be potent anti-cancer drugs, such as
methotrexate, so ours may show activity as well.
In a representative synthesis of an inhibitor, a
deazaguanine mimic is made, allowing the coupling
of a furan linker’s amine to the deazaguanine,
creating an amide (Figure 4). Synthesis of the
protected amino acid tail begins with a protected
asparagine, followed by the Hoffman rearrangement,
and the addition of CBZ and t-butyl ester (Figure 5).
Background
Figure 2. Cartoon of molecule binding to both binding sites of the
fungal MetSyn vs. failing to bind in the human enzyme
N
HN
O
H2N NH2
NH2
HN
N N
N
H2N
O
HN
N N
N
H2N
O
Path A Path B
FeSO4
H2SO4
H2O2
O O
OMe
HN
N N
N
H2N
O
CO2Me
1) KMnO4
NaOH
2) HCl
76%
HN
N N
N
CO2HH2N
O
MeOH
H2SO4 (cat)
Reflux
NaHCO3
90%
74%
OO
H H
OO
H
Na2SO3
60%
Figure 3. Various synthetic routes to 7-carboxymethyl pterin


Synthetic Routes to a Library of Novel Methionine Synthase Inhibitors

Pruet, Jeffrey

Valparaiso University

Valparaiso University ValpoScholar Symposium on Undergraduate Research and Creative Expression (SOURCE) Office of Sponsored and Undergraduate Research Spring 4-1-2020 Synthetic Routes to a Library of Novel Methionine Synthase Inhibitors Zachary Bennett Valparaiso University, zachary.bennett@valpo.edu Grace Burkhart Valparaiso University, grace.burkhart@valpo.edu Anna Bockman Valparaiso University, anna.bockman@valpo.edu Noah Moriarty Valparaiso University, Noah.moriarty@valpo.edu Jeff Pruet Valparaiso University, jeffrey.pruet@valpo.edu Follow this and additional works at: https://scholar.valpo.edu/cus Recommended Citation Bennett, Zachary; Burkhart, Grace; Bockman, Anna; Moriarty, Noah; and Pruet, Jeff, "Synthetic Routes to a Library of Novel Methionine Synthase Inhibitors" (2020). Symposium on Undergraduate Research and Creative Expression (SOURCE). 885. https://scholar.valpo.edu/cus/885 This Poster Presentation is brought to you for free and open access by the Office of Sponsored and Undergraduate Research at ValpoScholar. It has been accepted for inclusion in Symposium on Undergraduate Research and Creative Expression (SOURCE) by an authorized administrator of ValpoScholar. For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. Figure 8. Kirby-Bauer tests against (A) S.cerevisiae, (B) and B. cereusABFigure 1. (Left) transformation of homocysteine to methionine byMetSyn(Right) binding of methylated folate and methionine in MetSynFigure 9.MetSyn inhibition assay, showing comparitive levels ofinhibition by synthesized molecules0100020003000400050006000700080009000Buffer (NoHcys)HcysEnzymeReactionInhibitor AInhibitor HInhibitor KInhibitor LInhibitor NInhibitor OFluorescenceIntensityPreliminary Inhibitor ScreeningFigure 4. Synthesis of deazaguanine and with linkerClOEtOOHNN NH2OH2NNaOAcH2O70%HNN NHOH2NOEtO1. NaOH2. HCl60%HNN NHOH2NOHOHNN NHOH2NOHOOH2N OMeO EDC, HOBtDMSO, NEt389%HNN NHOH2NNHOO OMeOHNN NHOH2NNHOO OMeOHNN NHOH2NNHOO OHO1. NaOH2. HCl74%Figure 5. Synthesis of protected amino acid tailNH2OH2NOHOBoc2ONaCO3H2O/dioxane73%NH2ONHOHOOOPIDAEtOAc/MeCN/H2O(2:2:1)78%NH3NHOOOONH3NHOOOOCBZ-Cl (50% sol.)K2CO3, KOHTHF/H2O, 0℃88%N-CBZNHOHOOOHCCl3 ONHDCM81%N-CBZNHOOOOHH2, Pd/CMeOHquant.NH2NHOOOON-CBZNHOOOOHFigure 6. Coupling of amino acid tail to linkerOOOOHNH2NHNN NHOH2NNHOO OMeOEDC, HOBtDMSO, NEt362%HNN NHOH2NNHOO NHOOOOOHNHNN NHOH2NNHOO NHOOOOOHNHClDioxane90%HNN NHOH2NNHOO NHO OOHNH2NN OH2NNH2O HN OONH H2NOOHHNN NH2NOSONHH2NOOHNHNOH2N NHNSOONH NH2OOHHNN NH2NOSOONHHNN NHH2NOOONHONHNH2OOHHNN NHH2NOOH2NOOHFolate Mimic Linker Amino Acid TailNHOONH NH2OOHHNN NHH2NOOONHONHNH2HNN NHH2NOONHOONHNH2Inhibitor A Inhibitor HInhibitor K Inhibitor LInhibitor M Inhibitor NHNN NHH2NOOONHONHInhibitor OCO2HONHCO2HHO2CInhibitor PInhibitor RNHNOH2N NHNSOOInhibitor QNHCO2HONHCO2HCO2HSynthetic Routes to a Library of Novel Methionine Synthase InhibitorsZach Bennett, Grace Burkhart, Noah Moriarty, Anna Bockman, Jeff PruetValparaiso University, IN1. Pfaller, M. A.; Diekema, D. J. (2007). Epidemiology of InvasiveCandidiasis: A Persistent Public Health Problem. ClinicalMicrobiology Reviews. 20 (1): 133–63.2. Suliman, H.S.; Appling, D.R.; Robertus, J.D. (2007). The gene forcobalamin-independent methionine synthase is essential inCandida albicans: A potential antifungal target. Arch BiochemBiophys. 467, 218-226.3. Ubhi, D.; Kago, G.; Monzingo, A. F.; Robertus J. D. (2014).Structural Analysis of a Fungal Methionine Synthase withSubstrates and Inhibitors. J. Mol. Bio. 426, 1839-1847.We would like to thank Dr. Pruet for his guidance and direction on thisproject, as well as Dr. Clark for his assistance in the protein purification ofmethionine synthase, and Dr. Nunnely for her aid in Kirby-Bauer tests.This work has been funded by the Indiana Academy of Sciences SeniorResearch Grant (J.P.), EPIC scholarship (A.B.), and Eli Lilly (Z.B.).ReferencesAcknowledgementsFungal infections are a common public healthconcern in regards to immuno-compromisedpatients who have a much higher mortality rate dueto their condition.1 Methionine Synthase (MetSyn),is an enzyme which converts homocysteine tomethionine using a methylated folate molecule(Figure 1). Due to the close binding of homocysteineand folate in the fungal enzyme2,3, molecules can beAs MetSyn converts homocysteine to methionine,MetSyn activity can be detected by homocysteineconcentration. The MeasureIT-thiol quantificationassay gives a fluorescent response in proportion toHcy concentration. High fluorescence shows largeconcentration of Hcy, allowing the method to beadapted to show enzyme activity. High fluorescencecompared to control shows inhibition of the enzymewhen mixed with our novel inhibitors, while lowfluorescence shows consumed Hcy, showing poorinhibition.Figure 7. Current library of inhibitors, and inhibitor designsynthesized to selectivelyinhibit the fungal enzyme,providing a safer anti-fungal treatment (Figure 2).Folate Mimic Linker Amino Acid Tail Potential MetSyn InhibitorsFungal infections are of continuous concern,especially with regard to immunocompromisedpatients. In an effort to develop new potential anti-fungal agents, we have begun synthesizing a libraryof potential inhibitors of the fungal MethionineSynthase (MetSyn) enzyme. Key differences betweenthe B₁₂-independant fungal MetSyn enzyme and theB₁₂-dependant mammalian form can allow for anantifungal drug to be developed to exclusively bindthe fungal enzyme and inhibit fungal growth whileleaving the host (patient) unaffected. We arecurrently exploring the synthesis of various pterinand deazaguanine-based molecules as these mimicfolate, an essential substrate forMetSyn function.Wehave begun testing these new molecules for activityin a fungal growth assay, as well as a fluorescent assayfor monitoring MetSyn activity.Multiple routes of synthesis to the folate mimic 7-carboxymethyl pterin (7-CMP) were conceived ofand tested. Path A produced slightly better yields,where Path B allowed for quick synthesis and easypurification. This led to Path B to be our method ofchoice for 7-CMP synthesis (Figure 3).The linker is then coupled to the amino acid tail, anddeprotected, affording a potential inhibitor (Figure6). A library of molecules has been prepared(Figure 7) and tested on various fungi and bacteriavia Kirby-Bauer test, showing inhibitors H, N, andA with large zones of inhibition in comparison tothe positive control (Figure 8).In conclusion, our inhibitors show promise inregards to inhibiting the enzyme methioninesynthase. More work is being done in testing theenzymes directly on fungi and bacteria, as well ascancer cell lines. Folate pathway inhibitors have alsobeen shown to be potent anti-cancer drugs, such asmethotrexate, so ours may show activity as well.In a representative synthesis of an inhibitor, adeazaguanine mimic is made, allowing the couplingof a furan linker’s amine to the deazaguanine,creating an amide (Figure 4). Synthesis of theprotected amino acid tail begins with a protectedasparagine, followed by the Hoffman rearrangement,and the addition of CBZ and t-butyl ester (Figure 5).BackgroundFigure 2. Cartoon of molecule binding to both binding sites of thefungal MetSyn vs. failing to bind in the human enzymeNHNOH2N NH2NH2HNN NNH2NOHNN NNH2NOPath A Path BFeSO4H2SO4H2O2O OOMeHNN NNH2NOCO2Me1) KMnO4NaOH2) HCl76%HNN NNCO2HH2NOMeOHH2SO4 (cat)RefluxNaHCO390%74%OOH HOOHNa2SO360%Figure 3. Various synthetic routes to 7-carboxymethyl pterin

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Synthetic Routes to a Library of Novel Methionine Synthase Inhibitors

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