Cyclic-FMN Is a Detectable, Putative Intermediate of FAD Metabolism

Luxene Belfleur; Juha P. Kallio; Wito Richter; Natalie R. Gassman; Mathias Ziegler; Marie E. Migaud

doi:10.3390/biom16010175

Cyclic-FMN Is a Detectable, Putative Intermediate of FAD Metabolism

Luxene Belfleur
, Juha P. Kallio
, Wito Richter
, Natalie R. Gassman
, Mathias Ziegler
, Marie E. Migaud

Research output: Contribution to journal › Article › peer-review

Abstract

Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4′,5′-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO₄ and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.

Original language	English
Article number	175
Journal	Biomolecules
Volume	16
Issue number	1
DOIs	https://doi.org/10.3390/biom16010175
Publication status	Published - Jan 2026

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10.3390/biom16010175Licence: CC BY

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@article{5824abd1e3354ae19f51e21c60722ba3,

title = "Cyclic-FMN Is a Detectable, Putative Intermediate of FAD Metabolism",

abstract = "Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4′,5′-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO4 and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.",

keywords = "cyclic nucleotide, cyclic-FMN, divalent cations, FAD, riboflavin, vitamin B2",

author = "Luxene Belfleur and Kallio, \{Juha P.\} and Wito Richter and Gassman, \{Natalie R.\} and Mathias Ziegler and Migaud, \{Marie E.\}",

note = "Publisher Copyright: {\textcopyright} 2026 by the authors.",

year = "2026",

month = jan,

doi = "10.3390/biom16010175",

language = "English",

volume = "16",

journal = "Biomolecules",

issn = "2218-273X",

publisher = "MDPI Multidisciplinary Digital Publishing Institute",

number = "1",

}

TY - JOUR

T1 - Cyclic-FMN Is a Detectable, Putative Intermediate of FAD Metabolism

AU - Belfleur, Luxene

AU - Kallio, Juha P.

AU - Richter, Wito

AU - Gassman, Natalie R.

AU - Ziegler, Mathias

AU - Migaud, Marie E.

PY - 2026/1

Y1 - 2026/1

N2 - Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4′,5′-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO4 and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.

AB - Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4′,5′-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO4 and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.

KW - cyclic nucleotide

KW - cyclic-FMN

KW - divalent cations

KW - FAD

KW - riboflavin

KW - vitamin B2

UR - https://www.scopus.com/pages/publications/105028845870

U2 - 10.3390/biom16010175

DO - 10.3390/biom16010175

M3 - Article

C2 - 41594715

AN - SCOPUS:105028845870

SN - 2218-273X

VL - 16

JO - Biomolecules

JF - Biomolecules

IS - 1

M1 - 175

ER -

Cyclic-FMN Is a Detectable, Putative Intermediate of FAD Metabolism

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