# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 2 Dityptophanine
To examine the substrate tolerance of DtpC and to biosynthesize new dimeric products using DtpC in this study, brevianamide F (**4**) was provided to this enzyme as a substrate [9]. When adding only **4**, compound **5** was produced. Furthermore, adding both the natural substrate **2** and the substrate analog **4** provided compounds **6** as well as **5**. Using NMR and X-ray crystallographic data on **5** and **6**, the chemical structures of these compounds, including their absolute configurations, were determined. The homodimeric product **5** was named (\(-\))-dibreviianamide F, and the heterodimeric product **6** was named (\(-\))-tryprophenaline as new alkaloids (Figure 5). As in **1**, the stereochemistry of chiral carbon atoms in **5** and **6** was determined to be all in the \(S\) configuration by the Flack parameter value obtained from the crystallographic data. Thus, the biosynthetic mechanism for the formation of **5** and **6** is likely the same as that described above for the formation of **1** (Figure 4). Furthermore, as an example of engineered biosynthesis of non-native dimeric products, the heterologous host _Aspergillus niger_ A1179 modified [12] with the introduction of \(f\!m\!A\) (fumitremorgin NRPS [13]) genes was provided with the \(d\!tp\!C\) gene [14]. This engineered strain was able to isolate additional dimeric compounds **7** and **8** (Figure 6). These two compounds were analogs of the homodimeric product **5** previously produced (Figure 4). Elucidation of the chemical structures **7** and **8** showed **7** to be a new compound and **8** to be naseaseazine B isolated recently from a marine-dwelling streptomycete bacterium [15]. Dimerization in **7** and **8** did not form the C3-C3' linkage found in **1**, **5** and **6**. In **7**, dimerization occurred between C3 and N1', whereas positions C3 and C6 were involved in the dimerization that formed **8**. Those dimeric compounds are speculated also as products of a radical-mediated oxidative dimerization of tryptophan derivatives, where the linkages are formed at various different positions. DtpC is thought to extract the hydrogen from the diketopiperazine core amide nitrogen N10 of **2** to yield a radical. Then, the radical can migrate to C3 through the C2-N10 ring closure, and the subsequent coupling can proceed to form the C3-C3' linkage, giving the dimeric product **1**. However, when DtpC was provided with the unnatural substrate **4**, which lacks the bulky phenyl side chain group, the positioning of **4** within the active site of DtpC may be shifted from where **2** would be bound. This may cause an occasional extraction of the hydrogen from the indole nitrogen to yield a radical at N1, which can start migrating from the indole nitrogen to C6 and back to N1 (Figure 6, right column). Coupling of the radical at C3 in one monomer formed via the N10 extraction pathway (labeled as C3 in Figure 6, left column) with the radical at N1 in the other monomer formed via the N1 extraction pathway (labeled as N1 in Figure 6, right column) can produce the dimeric compound **7**. Similarly, coupling of the same C3 radical in one monomer with the C6 radical in the other monomer (labeled as C6N1 in Figure 6, right column) can produce **8**. The titer of these compounds was significantly lower than that of the native product **1** (approximately 350 ug/L of culture for **7** and **8** as compared to 2.3 mg/L of culture for **1**) [9; 14], presumably because of the low frequency of the N1 hydrogen extraction happening inside the active site of DtpC. There are other known natural products having similar dimerization patterns. For example, (\(-\))-asperazine A [16] and (\(+\))-pestalazine B [17; 18] have the same C3-N1 linkage as **7**, whereas (\(+\))-asperazine [19] and (\(+\))-pestalazine A [17; 20] have a C3-C7 linkage that was not observed in our study (Figure 6, boxed). Presumably, the biosynthesis of those dimeric diketopiperazine compounds is also catalyzed by P450 enzymes with relaxed substrate specificity, much like DtpC.