Formation of a Flavin-Linked Peptide

In a previous study, we showed that formylmethylflavin (FMF) can bind to cysteine. In this study, FMF was reacted with native peptides (CG and CKLVFF) containing an N-terminal cysteine. The formation of flavin-CG and flavin-CKLVFF was confirmed using HPLC and ESI-MS. Storage of flavin-CKLVFF in DMSO at −30 °C for 7 days resulted in no detectable deposition. In contrast, flavin-CKLVFF formed deposits when stored in water at −30 °C for 1 day, but no deposit was observed in the aqueous solution of flavin-CKLVFF after 7 days storage in the presence of 0.1% Triton X-100.


Introduction
Flavins are biological oxidation reagents that can photooxidize tryptophan and tyrosine [1], thereby introducing hydroxyl groups into peptides and increasing their hydrophilicity. For example, we previously suggested that the photooxidation of amyloid beta peptides (Aβ) by flavins may result in the hydroxylation of aggregated Aβ fibrils and disruption of the aggregated fibrils [2]. The suppression of aggregation is likely to inhibit Aβ toxicity, since the deposition of Aβ in the brain parenchyma and cerebro-vasculature is a critical step in the pathogenesis of Alzheimer's disease [3,4]. Furthermore, Aβ toxicity is linked to the assembly state of the Aβ peptides [5][6][7].

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A previous report showed that a short Aβ fragment (KLVFF; Aβ 16-20 ) binds full-length Aβ [8]. When the KLVFF sequence binds oxidation reagents, the product may alter the Aβ aggregation pathway and inhibit Aβ toxicity. We previously proposed that flavin-CKLVFF would likely disrupt aggregated Aβ fibrils [2].
2-Aminoethanethiol derivatives, such as cysteine, react with aldehydes and form a five-membered heterocyclic ring via an imine [9][10][11]. Therefore, an aldehyde group is likely to react with cysteine located at the N-terminus of a peptide. Formylmethylflavin (FMF) [12,13] contains an aldehyde group and can be used to introduce a flavin into a peptide containing an N-terminal cysteine. Using this approach, we attempted the synthesis of flavin-CKLVFF. Previously, we reported the reaction between FMF and cysteine [2]. One month after our report [2], another group revealed that a flavin-linked peptide synthesized from FMF and a hydroxylamine derivative can photooxidize Aβ, and that the oxygenated Aβ exhibits decreased aggregation and cytotoxicity [14]. However, the hydroxylamine derivative used was not a native peptide. In this study, flavin-CKLVFF (1) was synthesized using the native peptide, CKLVFF (Scheme 1). We anticipate that flavin-CKLVFF (1) will exhibit decreased aggregation and cytotoxicity, comparable to that of the hydroxylamine derivative. Scheme 1. Synthesis of flavin-CKLVFF (1) by reaction between FMF and CKLVFF.

Synthesis of Flavin-CG (2)
As a proof-of-concept experiment, we first determined whether a short peptide, CG, can be covalently linked to FMF. CG was reacted with FMF at 65 °C. Analysis of the reaction solution by HPLC ( Figure 1) showed two major peaks, at 19.9 and 20.5 min (Figure 1). The ratio of the peak areas at 19.9 and 20.5 min in Figure 1 was determined as 35:65 and defined as the product 2a and 2b, respectively. Then, the UV-vis spectra of the peaks were shown in Figure 2. These peaks were isolated and analyzed using ESI-MS in negative-ion mode ( Figure 3) and shown to be due to flavin-CG (2).

Synthesis of Flavin-CKLVFF (1) Using the Native Peptide
The reaction between FMF and cysteine [2] and the synthesis of flavin-CG (2) (Section 2.1) were performed in water. However, CKLVFF is poorly soluble in water, so to obtain high concentrations of CKLVFF, the solvent was changed to DMSO.
FMF and CKLVFF in DMSO were reacted at 65 °C for 1 h, and then the solvent was replaced with water using a Sep-Pak cartridge. Analysis of the reaction solution by HPLC provided the profiles shown in Figure 4. FMF and its degradation product, lumichrome (LC), were detected at 2.5 and 4.4 min, respectively. Two major peaks were detected at 18.1 and 19.0 min. The ratio of the peak areas at 18.1 and 19.0 min in Figure 4 was determined as 60:40 and defined as the product 1a and 1b, respectively. Then, the UV-vis spectra of the peaks were shown in Figure 5.
Next, the time course of the reaction between FMF and CKLVFF was determined. The reaction was carried out at 65 °C for 0-3 h; samples were withdrawn periodically and analyzed using HPLC. The time-course profiles of the 18.1 and 19.0 min peaks are shown in Figure 6. The intensities of both peaks increased between 0-30 min, and then decreased after 45 min. In contrast, the amount of LC increased throughout the 3 h experiment, showing that flavin-CKLVFF (1) gradually degraded to LC at 65 °C.
The products providing the peaks at 18.1 and 19.0 min in Figure 4 were isolated and analyzed using electrospray ionization-mass spectrometry (ESI-MS) in negative-ion mode (Figure 7). Both peaks were identified as flavin-CKLVFF (1). Since flavin-CKLVFF (1) also contains the five-membered heterocyclic ring, the peaks in Figure 4 are likely diastereomers of flavin-CKLVFF (1).

Stability of Flavin-CKLVFF (1)
To determine the stability of flavin-CKLVFF (1) in DMSO or water at −30 °C, FMF and CKLVFF in DMSO were reacted at 65 °C for 1 h, and then the solution was left for 1 day, 3 days or 7 days in DMSO at −30 °C and analyzed using HPLC (Figure 8a-d). The increase in the amount of LC after 1 day was less than 4%, whereas the amount of flavin-CKLVFF (1) after 1 day of storage was 1.4 times higher than without storage. Furthermore, no significant increase in the amount of LC was detected after 7 days, and the amount of flavin-CKLVFF (1) after 7 days was almost the same as after 1 day. The mechanism behind these findings remains unclear, but unreacted FMF and unreacted CKLVFF might gradually react during the first day of storage.
Next, FMF and CKLVFF in DMSO were reacted at 65 °C for 1 h, and then the solvent was replaced with water using a Sep-Pak cartridge. After 1 day in water at −30 °C, a yellowish deposit was observed in the sample. Since LC is poorly soluble in water, LC might be formed from flavin-CKLVFF (1) and deposited in water. However, because LC solid is green, the yellowish deposit is likely to be not LC. Then, flavin-CKLVFF (1) might be aggregated and deposited in water as another possible mechanism. To suppress formation of this deposit, 0.1% Triton X-100 was added to flavin-CKLVFF (1) in water and the mixture was left for 1 day, 3 days or 7 days, and then analyzed by HPLC (Figure 8e-h). The amount of flavin-CKLVFF (1) did not decrease, indicating that flavin-CKLVFF (1) should be stored in DMSO at −30 °C, and that it is essential that the DMSO be replaced with water prior to use. If flavin-CKLVFF (1) must be stored in water, 0.1% Triton X-100 should be added prior to storage at −30 °C.