**2.4 Syntheses of ylidenebutenolide modified derivatives and the stereochemical and spectral characteristics of the synthesized derivatives**

Finally, the synthesis of ylidenebutenolide modified peridinin derivatives is described. The acetylene ester derivative was synthesized by the same protocol. Namely, the coupling between C17-allenic segment **10** and C20-acetylene methyl ester segment **16** by using the modified Julia olefination, which was followed by the isomerization gave all-*trans* **8** (Fig. 8 (A)) (Kajikawa et al., 2010).

On the other hand, there were some difficulities in the synthesis of the olefin ester derivative (Fig. 8). We obtained the only 9'E-olefin ester segment **17** by the similar synthetic process, which was not the desired 9'Z half-segment resulting from the contribution of the carbonyl group of the methyl ester. We then tried to connect the segments **10** and **17** by the modified Julia olefination and to obtain all-*trans* **9-3** by the isomerization. The anion derived from **10** was stirred with a mixture of stereoisomers of **17** under the same conditions used for the coupling of the previous peridinin derivatives. The reaction was completed within 5 min in the dark to produce the coupling products as a mixture of the stereoisomers in 46% amount, whose HPLC is shown in Fig. 8 (B). The major peak (peak 1) was estimated to be 45% of the mixture by HPLC analysis (other isomers were 16%, 14%, 5%, 5%, and others). Isomerization to the desired all-*trans* **9-3** was attempted under the same conditions previously used. After 5 days, the initially generated major peak (peak 1) changed to another peak (peak 2; 44% based on HPLC analysis) in an equilibrium state. We then isolated both compounds and elucidated their structures by NMR (400 and 750 MHz), and clarified that peak 1 was (13Z, 9'E)-isomer **9-1** and peak 2 was (13E, 9'E)-isomer **9-2**. Unfortunately, we could not obtain the desired all-*trans* (13E, 9'Z)- isomer **9-3**.

We investigated the stability of the synthesized ring opened derivatives **8** and **9**, and found the isolated all-*trans* acetylene ester derivative **8-1** was more labile than **8-2** (13Z-isomer). For instance, the isolated all-*trans* derivative **8-1** (13E-isomer) isomerized to the dihydrofuran derivative **8'** by a trace amount of hydrogen chloride in CDCl3, but the corresponding isomerization of **8-2** (13Z-isomer) was not observed (Fig. 8). In addition, the all-*trans* derivative **8-1** rapidly isomerized to give a mixture of Z-isomers upon illumination. This might occur due to the contribution of the carbonyl group of the methyl ester similar to the case of the 9'E-olefin ester derivative **9-2**.

In the PCP complex, peridinin exhibits an exceptionally high efficiency of energy transfer to Chl a. In order to make clear the effect of the ylidenebutenolide, we needed to measure the energy transfer efficiencies in peridinin derivatives. Futhermore, it was tried to construct the corresponding PCP derivatives using the synthesized peridinin derivatives **8** and **9** to compare with the energy transfer efficiencies of peridinin (private information from Dr. H. A. Frank). First, it was attempted to reconstitute the PCP apoprotein using the 9'E-olefin ester derivative **9-2** under the same conditions that were successful for peridinin (Ilagan et al., 2006), but the reconstitution was not observed. It was also tried to reconstitute the PCP apoprotein using the 13Z-isomer **8-2**, but it did not bind the protein either. The reason might be that these compounds were bent into a *cis* configuration, and hence they might not fit properly into the protein binding site. These results apparently showed that the ylidenbutenolide of peridinin at least contributes to the stereochemical stability of the

An Approach Based on Synthetic Organic Chemistry Toward Elucidation

AcO OH •

Allene function

AcO OH •

AcO OH •

AcO OH •

AcO

AcO

AcO

**peridinin** 

O

**2**: Acetylene Derivative

438.0 nm

450.0 nm

459.0 nm

**3**: Olefin Derivative

**4**: Diolefin Derivative

O

of Highly Efficient Energy Transfer Ability of Peridinin in Photosynthesis 145

C37-Peridinin (**1**)

454.0 nm

O O

**5**: C33-Peridinin Derivative

**6**: C35-Peridinin Derivative

**7**: C39-Peridinin Derivative

**3. Relationships between the unique structure and the special exited state of** 

From studies on peridinin and other carotenoids, it has been known that there are at least two important low-lying excited singlet states, denoted S1 and S2, which are related to the highly efficient energy transfer from peridinin to chlorophyll a (Fig. 10(A)). To elucidate the mechanism of this efficient energy transfer, it is important that we make clear the characteristics of these excited states and the energy transfer pathways such as those from S2 to QX and/or from S1 to QY. Recently, many researchers have tried to examine this particular mechanism. The conjugated double bonds of most carotenoids are symmetry and these double bonds can be regarded as polyenes described in terms of the idealized C2h point group in the spectroscopic fields (Hudson et al., 1973). The lowest excited singlet (S1) state is assigned to the 21Ag- state, and the second lowest singlet (S2) state is assigned to the 11Bu+ state. The excitation to S1 from the ground state is symmetry forbidden and is not directly accessible by one-photon processes in contrast to the allowed absorption to S2 state (Polivka et al., 2004). On the other hand, the conjugated double bond of peridinin and other carbonylcontaining carotenoids are asymmetric due to the presence of the conjugated carbonyl group, and these oxygenated carotenoids display a pronounced solvent dependence of its lowest excited singlet state lifetime (S1 lifetime). Namely, it has been proposed that the findings are consistent with the presence of an intramolecular charge transfer (ICT) state, which is uniquely formed in carotenoids containing the carbonyl group in conjugation with the -electron system of double bonds (Frank et al., 2000; Zigmantas et al., 2004). It has also been argued that changes in the position of the ICT state related to the S1 state rationalize the dependence on solvent polarity concerning S1 lifetime. In the case of peridinin, the relationship of these energy levels is well discussed based on the detailed experimental works. The ICT in the excited state manifold of peridinin is shown to be higher in energy than the S1 state in nonpolar solvents and shifts below S1 with increasing solvent polarity

Fig. 9. Structure of synthesized peridinin derivatives and result of UV spectra in hexane

**3.1 Measurement of ultrafast time-resolved optical absorption spectra** 

Allene-modified Derivatives Polyene chain-modified Derivatives Ylidenebutenolide-modified Derivatives

O O OH

O

OH

416.0 nm

436.5 nm

469.0 nm

Ylidenebutenolide ring

OH

OH

CO2Me

**8-1**: Acetylene Ester Derivative

**9-2**: (13E, 9'E)-derivative

O 9'

O

OMe O

OH

OH

410.0 nm

430.5 nm

O O

O

O

O O O

compound and would keep the all-*trans* conformer suitable for incorporation into the protein to form the PCP complex.

The maximum absorption wavelengths (max) in the electronic spectra of peridinin (**1**) and the synthesized derivatives **2~7, 8-1** and **9-2** in hexane were measured and are summerized in Fig. 9. Evidently, the diolefin derivative **4** and C39 peridinin derivative **7** showed the longer max than that of peridinin. The max value in polyene chain modified derivatives **5**-**7** increased almost 20 nm per one olefin unit added to the conjugated polyene. On the other hand, the olefin derivative **3**, having eight conjugated carboncarbon double bonds like peridinin, showed the shorter max than that of peridinin. The open-ring derivative also displayed a shorter max than peridinin (**1**) due to shorter effective -electron conjugated chain length, although the 9'E-olefin ester derivative **9-2** had the same conjugated carbon-carbon double bonds compared with peridinin. These results show that the allene and ylidenebutenolide group at least contribute to giving rise to the max value desirable for the marine organism to absorb light in the blue-green region of the visible spectrum.

Fig. 8. Stereochemical characteristics of ylidenebutenolide modified derivatives

compound and would keep the all-*trans* conformer suitable for incorporation into the

The maximum absorption wavelengths (max) in the electronic spectra of peridinin (**1**) and the synthesized derivatives **2~7, 8-1** and **9-2** in hexane were measured and are summerized in Fig. 9. Evidently, the diolefin derivative **4** and C39 peridinin derivative **7** showed the longer max than that of peridinin. The max value in polyene chain modified derivatives **5**-**7** increased almost 20 nm per one olefin unit added to the conjugated polyene. On the other hand, the olefin derivative **3**, having eight conjugated carboncarbon double bonds like peridinin, showed the shorter max than that of peridinin. The open-ring derivative also displayed a shorter max than peridinin (**1**) due to shorter effective -electron conjugated chain length, although the 9'E-olefin ester derivative **9-2** had the same conjugated carbon-carbon double bonds compared with peridinin. These results show that the allene and ylidenebutenolide group at least contribute to giving rise to the max value desirable for the marine organism to absorb light in the blue-green

Fig. 8. Stereochemical characteristics of ylidenebutenolide modified derivatives

protein to form the PCP complex.

region of the visible spectrum.

Fig. 9. Structure of synthesized peridinin derivatives and result of UV spectra in hexane
