Diketopiperazines produced by endophytic fungi found in association with two ... predictions suggest that the R-enantiomer (Figure 2B) for diketopiperazine 3.
Supplementary Information Diketopiperazines produced by endophytic fungi found in association with two Asteraceae species Denise O. Guimarãesa,#, Warley S. Borgesa,#, Noemi J. Vieiraa, Laiani F. de Oliveiraa, Carlos H. T. P. da Silvaa, Norberto P. Lopesb, Luis G. Diasc, Rosa Durán-Patrónd, Isidro G. Colladod, Mônica T. Pupoa
The extinction coefficients for left and right circularly polarized light are different for chiral compounds leading to a differential absorption. This effect was named circular dichroism (CD) and when the differential absorption of light is plotted against wavelength or wavenumber we have a CD spectrum. The presence of many Cotton effects in the experimental ECD spectra can allow a correct assignment of the absolute configuration. It is also expected that a reliable theoretical methodology can reproduce the sequence of Cotton effects in sign, position and intensity. But in practice, theoretical absorption bands are shifted relative to experimental ones and only an attempting assignment is possible. In the figure 1 is presented the experimental ECD (Figure 1A) and theoretical predictions based on the R-enantiomer (Figure 1B) and S-enantiomer (Figure 1C) for diketopiperazine 2 on the carbon 6 (*). Only a positive Cotton effect is observed in the experimental data. Theoretical predictions are shifted to longer wavelength. Discrimination is not possible by comparison between theoretical and experimental ECD spectra. In the figure 2, the experimental ECD (Figure 2A) shows a sequence of negative and positive Cotton effects of low intensity. By visual inspection, the theoretical predictions suggest that the R-enantiomer (Figure 2B) for diketopiperazine 3 has the better agreement with the data, but the rotational strengths appears shifted and their intensities are not correct. In the figure 3, the experimental ECD spectrum shows a positive Cotton effect at 311 nm ([θ]311 +1061) and a negative Cotton effect at 252 nm ([θ]252 -532) (Figure 3A). Theoretical calculations are shown for the four diketopiperazine 7 isomers: 3R;6Eisomer (Figure 3B), 3S;6E-isomer (Figure 3C), 3S;6Z-isomer (Figure 3D) and 3R;6Zisomer (Figure 3E). Although a very nice agreement is lacking between the experimental and theoretical ECD, it is still possible to assign the data to the 3R;6Zisomer for diketopiperazine 7. Theoretical absorption wavelength and optical rotatory strength for all diketopiperazines isomers are shown in Table 1.
1
A 10 5
ECD (mdeg)
0 -5207 227 247 267 287 307 327 347 367 387 -10 -15 -20 -25 -30
l (nm)
B 100 50
De
0 -50
170 190 210 230 250 270 290 310 330 350
HO
S
N
O
N *
-100
O
HO
-150
l (nm)
R-enantiomer
C 200 100
De
0 -100
170 190 210 230 250 270 290 310 330 350
-200
HO
S
N
O
N *
O
-300
HO
-400
l (nm)
S-enantiomer
Figure 1. Experimental (A) and theoretical ECD (B-C) for the diketopiperazine 2.
2
A 6
ECD (mdeg)
4 2 0 -2
210
220
230
240
250
260
270
-4 -6 -8
l (nm)
B 1500 1000
De
500 0 -500
180 190 200 210 220 230 240 250 260 270
-1000
l (nm)
C 1000
De
500 0 180 190 200 210 220 230 240 250 260 270 -500 -1000
l (nm)
Figure 2. Experimental (A) and theoretical ECD (B-C) for the diketopiperazine 3.
3
A 8 6
ECD (mdeg)
4 2 0 210 230 250 270 290 310 330 350 370 390 -2 -4
l (nm)
B 1200 1000 800
De
600 400 200 0 -200 170
200
230
260
290
320
350
380
410
440
470
-400
l (nm)
C 400 200
De
0 -200170 200 230 260 290 320 350 380 410 440 470 -400 -600 -800 -1000
l (nm)
4
D 400 200 0
De
-200
170
200
230
260
290
320
350
380
410
440
470
-400
S
N
O
N
O
-600 -800 -1000
O
-1200
l (nm)
3S;6Z-isomer
E 1200 1000
S
N
O
N
O
800
De
600 400 200 0 -200170
185
202
223
249
282
324
382
465
O
-400
l (nm)
3R;6Z-isomer
Figure 3. Experimental (A) and theoretical ECD (B-E) for the diketopiperazine 7.
5
Table 1 – Absorption wavelength and optical rotatory strength for theoretical ECD calculations for isomers of the diketopiperazines 2, 3 and 7. R-enantiomer for DKP 2
S-enantiomer for DKP 2
R-enantiomer for DKP 3
S-enantiomer for DKP 3
3R;6E-isomer for DKP 7
3S;6E-isomer for DKP 7
3S;6Z-isomer for DKP 7
3R;6Z-isomer for DKP 7
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
l (nm)
R(length)
283,9
17,0
291,0
32,1
249,1
8,6
249,0
-7,3
346,2
-33,9
349,9
47,3
333,2
-149,6
333,2
149,3
266,3
-2,2
268,5
-32,3
229,9
-40,0
229,5
40,8
304,9
17,7
307,7
-16,6
304,5
6,6
304,4
-6,6
258,6
-16,4
261,0
3,3
222,2
14,1
222,6
-14,2
274,1
-10,0
276,5
13,3
273,4
-40,0
273,3
40,2
235,1
3,0
244,1
3,2
219,7
-18,1
219,4
16,3
269,9
-12,9
271,1
5,1
268,0
-14,5
267,9
13,7
234,2
-6,5
238,0
-75,6
212,0
14,6
212,7
-8,1
261,9
7,2
263,7
-11,6
258,8
49,7
258,7
-49,4
231,3
10,4
237,2
40,4
211,2
-7,7
211,1
7,4
259,7
17,2
262,4
-2,9
256,3
-1,8
256,2
1,9
224,6
8,5
223,7
47,2
210,8
19,6
210,6
-22,5
250,5
25,7
251,5
-24,5
246,9
77,4
246,7
-78,1
220,2
-3,9
221,4
15,4
207,6
7,2
208,1
-1,6
244,1
57,9
244,2
-66,8
241,5
56,8
241,4
-41,4
218,1
-51,7
218,5
-13,0
207,2
-8,5
206,6
0,9
242,0
25,8
242,1
-3,0
241,2
-93,7
241,2
80,7
217,3
0,0
216,3
-39,5
201,0
-1,5
200,5
2,0
240,9
-51,1
241,1
65,9
237,3
-29,3
237,5
27,9
214,9
26,0
214,8
-7,9
198,9
46,7
198,8
2,6
233,6
29,6
231,9
-29,1
227,5
-10,3
227,5
10,5
212,6
15,9
214,3
-2,3
198,7
-5,6
198,7
-40,5
229,0
6,0
230,2
-13,0
225,4
6,9
225,5
-6,2
208,0
-4,3
209,1
-45,8
195,8
17,1
195,6
-16,6
224,9
41,2
226,1
-47,9
224,3
-0,2
224,4
-0,3
206,7
-6,1
205,6
13,6
195,2
-9,1
195,0
9,7
220,6
-0,4
222,2
2,6
217,7
-3,1
217,8
3,4
203,5
-1,5
203,1
24,8
193,7
1,2
193,7
6,7
214,8
6,2
214,8
-3,9
214,4
-28,2
214,5
28,3
R = Rotatory strength in length form (10-40 cgs).
6
Identification of the endophytic fungi The isolates VA17 and VR7 were identified based on their ribosomal DNA (rDNA) sequences as already described (Guimarães et al., 2008). The fungi were grown on YPD medium (1% [wt/v] yeast extract, 2% [wt/v] peptone, 2% [wt/v] dextrose) for 72 h at 30 °C on a shaking platform at 120 rpm, harvested by vacuum filtration, frozen in liquid nitrogen and stored at –70 °C. Genomic DNA was isolated using the phenolchloroform extraction technique and the pellets were allowed to dry. DNAs were then dissolved in 100 µL sterile water and 1 µL of DNA solution was used for amplification procedure. The DNAs were quantified with a fluorometer. Amplification of products of the correct size was verified on 1% agarose gels. The ITS1-ITS2 and ITS3-ITS4 primers pairs were used to amplify a large portion of the ITS1 region, 5.8S rDNA and the adjacent ITS2 region. PCR amplification was performed with a volume of 50 µL. One µL of genomic DNA (500 ng), 2.5 µL of 4 mM dNTP, 0.5 µL of 5U of Amplitaq platinum Taq polymerase per µL, 5 µL of 10X buffer (100 mM Tris-HCl [pH 8.3], 2 µL of 50 mM MgCl2, 1 µL of universal primers ITS1, ITS2, ITS3 and ITS 4 (21 pM) and 37 µL distilled water. Amplification was completed in a thermalcycle following cycling conditions: 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min, and a final step of 72 °C for 10 min. PCR products were purified in Qiagen columns. Full DNA sequence analysis of the PCR products obtained with universal fungal primers ITS3 – ITS4 specific for rDNA genes were used to confirm the species identification. Automated DNA sequencing was performed in both directions by using the initial amplifications primers, and the resulting DNA sequences aligned and analyzed with Sequencher software. Comparison of sequences from isolates and GenBank sequences was performed using advanced BLAST search (Henry et al., 2000) and Clustal W (Thompson et al., 1994). Identities higher than 98% were taken into account for the classification.
> VA17 its1and2 TCCGTAGGTGAAACCTGCGGAGGGATCATTACAGAGTTATCCAACTCCCAAACCC ATGTGAACTTATCTCTTTGTTGCCTCGGCGCAAGCTACCCGGGACCCAGCGCCCC GGGCGGCCCGCCGGCGGACAAACCAAAACTCTTGTGTATCTTAGTTGATCTATCT GAGCGTCTTATTTAATAAGTCAAAACTTTCAACAACGGTATCTCTTGGTTCTGGCAT CGAGGAAAGAACGC > VA17 its3and4 CGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAA CGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCA ACCCCTAAGCACAGCTTACTGTTGGGACTCTACGGCCCCCGTAGTTCCCCAAAGC CATTGGCGGAGTGGCAGTAGTCCTCTGAGCGTAGTAATTCTTTATCTCGCTTTTGT TAGGCGCTGCCCCCCCGGCCGTTAAACCCCCCAATTTTTTCTGGTTGACCTCGGA TCAGGTAGGAATACCCGCTGAACTTAA
7
> VR7 its1and2 GCCAGAACCAACAGAAATCGTTGTTAAAAGTTTTGMTTATTTTGCTTATGCCACTC AGAAGAAACGTCGTTACAATAGAGTTTGGTTATCCTCCGGCGGGCGCCGGGTCC GGTACCCGCGGGGGGTCCGGTCCGGGCCGGGAGGCGTCCTTTTCAGGGGACGG CCTACCCGCCGAAGCAACAGTTGTAGGTATGTTCACAAAGGGTTATAGAGCGGTA ACTCCAGTAATGATCCCTCCGCAGGTTCACCTACG > VR7 its3and4 CGAAAAATCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGA ACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTT CAACCCTCAAGCTCTGCTTGGTGTTGGGGCTCTACGGTCGACGTAGGCCCTCAAA GGTAGTGGCGGACCCTCCCGGAGCCTCCTTTGCGTAGTAACATTTCGTCTCGCAC TGGGATCCGGAGGGACTCTTGCCGTAAAACCCCCCAATTTTCCAAAGGTTGACCT CGGATCAGGTAGGAATACCCGCTGAACTTAA
VR7 taxonomy The isolate grown on oatmeal agar and malt agar 2% at 28oC exhibited colonies with light-grey aerial mycelium and salmon to pink sporodochia. Microscopic observations revealed hyaline mycelium, smooth conidiophores branched only at the base. Conidia hyaline, straight and attenuated at the ends, 11-15 x 4,5-6,5 um.
References Guimarães, D.O., Borges, W.S., Kawano, C.Y., Ribeiro, P.H., Goldman, G.H., Nomizo, A., Thiemann, O.H., Oliva, G., Lopes, N.P., Pupo, M.T., 2008. Biological activities from extracts of endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia. FEMS Immunol. Med. Microbiol. 52, 134-144. Henry T., Iwen P.C., Hinrichs S.H., 2000. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J. Clin. Microbiol. 38, 1510-1515.
Thompson J.D., Higgins D.G., Gibson T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 46734680.
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