Chem. Res. Toricol. 1990, 3, 473-481
473
Structural Determination of Geometrical Isomers of Microcystins LR and RR from Cyanobacteria by Two-Dimensional NMR Spectroscopic Techniques Ken-ichi Harada,* Kiyoshi Ogawa, Kenji Matsuura, Hideaki Murata, and Makoto Suzuki Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan
Mariyo F. Watanabe Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku, Tokyo 160, Japan
Yoshiko Itezono and Noboru Nakayama Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247, Japan Received May 3, 1990
A nondestructive method using a combination of three 2D NMR techniques, DQF-COSY (double quantum filter correlation spectroscopy), HMQC (lH-detected multiple quantum coherence), and HMBC (heteronuclear multiple bond correlation), were developed for structural determination of microcystins, toxic heptapeptides produced by cyanobacteria. With this procedure we were able to assign all carbons and protons of microcystins LR (1) and RR (2), thus determining the constituent amino acid sequences. The procedure was also applied to the microcystin-associated nontoxic minor components, which have molecular weights and amino acid compositions similar to those of 1 and 2 and toxicities different from those of 1 and 2. From detailed analysis of these spectra we rapidly deduced that the minor components are geometrical isomers with respect to C-7 of the diene in Adda of the parent toxins. The structures were finally confirmed to be 3 and 4 by ROESY (rotating frame nuclear Overhauser and exchange spectroscopy) technique.
Introduction Several hepatotoxic peptides have been isolated from cyanobacteria such as Microcystis, Anabaena, and Oscillatoria (1-3), named microcystins, and characterized as monocyclic heptapeptides containing a common moiety composed of a novel 0-amino acid, 3-amino-9-methoxylO-pheny1-2,3,8-trimethyldeca-4,6-dienoic acid (Adda), N-methyldehydrdanine (Mdha), Dalanine (Ala), @-linked D-erythro-0-methylasparticacid (0-Me-Asp),and y-linked D-glutamic acid (Glu), plus two L-amino acids as the variants (4). We have established an analysis and isolation method for these peptides ( 5 6 ) . In our previous report two minor and structurally similar analogues of microcystins LR and RR were purified and characterized by our isolation method (7). Although they have the same molecular weights and amino acid compositions as those of the parent toxins, they do not possess similar toxicities. UV and 'H NMR spectral data for both minor components demonstrated that structural difference is clearly present compared with the parent toxins. This difference is probably responsible for the marked decrease in the observed toxicities of the minor components. Therefore, we were very interested in determining their structures. Botes et al. first determined the structure of microcystin LA, after successively determining the structures for microcystins YR, LR, YM, and YA (8,9). Their procedure for the structure determination, particularly the sequence determination of constituent amino acids, mainly involved
* To whom correspondence should be addressed. 0893-228x/90/2703-0473$02.50/0
partial hydrolysis of the peptides followed by Edman degradation and a mass spectrometric method of the resulting linearized products. Since microcystins are cyclic peptides containing nonprotein amino acids such as Adda and Mdha, we could encounter the following: (1)a partial hydrolysis yielding a mixture of linearized peptides that is relatively difficult to separate; (2) an enzymatic hydrolysis that does not succeed; (3) Edman degradation that does not proceed as usual because the compounds possess a @-aminoacid and a dehydroamino acid; (4) difficulty in distinguishing 0-Me-Asp from Glu by mass spectrometry because of the same molecular weight; and ( 5 ) the need for an additional chemical reaction to determine which carboxylic acid in the two acidic amino acids is bonded to other amino acid moieties (a-linkage or isolinkage). In order to overcome such problems, we planned to introduce a nondestructive method using a combination of recently developed 2D NMR spectroscopic techniques. Additionally, the established method was successfully applied to the full assignment of all protons and carbons of microcystins and the structure determination of the minor components associated with microcystins LR and RR.
Experimental Procedures Materials. We have described the purification of microcystins LR and RR and their minor Components in a previous paper (7). Instrumentation. NMR experiments were carried out on a JEOL JNM-GSX 400 and a GX-400spectrometers equipped with lH/13C dual probe. Microcystin LR and its minor component 0 1990 American Chemical Society
474 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
Harada et al.
were dissolved in 0.7 mL of CD,OD with tetramethylsilane as an internal standard. Microcystin RR and its minor component were dissolved in 0.7 mL of D 2 0 , and sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4was used as an external standard. The following experimental conditions were used for DQF-COSY, HMQC, HMBC, and ROESY experiments: (1) DQF-COSY. Pulse sequence 90°,-t,-900,-9008-Acq(t2), size l K , spectral width in F1 = 4000 Hz and F2= 4000 Hz, zero filling in Fl to 512, acquisition time for one scan 0.128 s, t 2 = 1.372 S.
(2) HMQC. Pulse sequence
size 2K, spectral width in F, = 25 000 Hz and F2= 5000 Hz, zero filling in F, to 512, acquisition time for one scan 0.205 s, A = 3.7 ms, t 2 = 1.285 s. (3) HMBC. Pulse sequence NH
H
2
size 2K, spectral width in Fl = 25000 Hz and F2= 4000 Hz, zero filling in Fl to 512, acquisition time for one scan 0.256 s, AI = 3.7 ms and A2 = 60 and 100 ms, t 2 = 0.7 s. (4) ROESY. Pulse sequence 9O0,-t1-spin lock,-Acq(t2), size l K , spectral width in F, = 5000 Hz and F2= 50oO Hz, zero filling in F, to 512, acquisition time for one scan 0.102 s, mixing time = 205 ms, t 2 = 1.872 s.
Figure 1. Structures of microcystins LR (1) and RR (2).
Results and Discussion Establishment of a Nondestructive Method for Structural Determination of Microcystins. Several 2D NMR spectroscopic techniques were used for structural determination of microcystins (3,10-12).To establish an appropriate method for structural determination using these techniques, the following objectives were put forth: (I) H-2 of each constituent amino acid must be found and assigned. (11)C-2 of each constituent amino acid must be assigned on the basis of the results of (I). (111)Carbonyl carbons of each constituent amino acid must be assigned. (IV) Finally, amino acid sequence must be determined from information based on (I), (II), and (111). First of all, our plan was applied to known microcystin LR (1) (Figure 1). Although D,O, DMSO-$, and CD3CN were employed for NMR in the spectral measurement of microcystins (3, 8-13), CDBODwas used in the present experiment because 1 is dissolved more easily in CDBOD than in other solvents (14). However, there is one disadvantage; two large solvent peaks appear at about 5 and 3.3 ppm in the spectra. Therefore, we used double quantum filter correlation spectroscopy (DQF-COSY) (15) for accomplishing objective I. The 400-MHz DQF-COSY spectrum of 1 in CDBODis reproduced in Figure 2. In the lower field region the olefinic and aromatic protons from Adda and Mdha appear. The two coupling networks, 6.24 ppm (Adda H-5)-5.48 (Adda H-41-4.56 (Adda H3)-3.05 (Adda H-2)-1.03 (Adda H-11) and 5.42 ppm (Adda H-7)-2.64 (Adda H-8)-1.01 (Adda H-13), 3.27 (Adda H9)-2.69, 2.81 (Adda H-10) are observed, enabling us to assign the Adda moiety resonances. The two signals at 5.89 and 5.43 ppm could be easily assigned to the exomethylene of Mdha. There are six signals of H-2 of the constituent amino acids at 4.70-4.10 ppm (H-3 signal in the only case of Adda). Figure 3 shows the enlarged DQF COSY spectrum
Figure 2. DQF-COSY spectrum (400 MHz) of microcystin LR in CDSOD obtained on a JEOL JNM-GSX 400 spectrometer. Coupling networks of Adda are shown.
(5.50.50 ppm) of 1. Each H-2 was assigned by connecting the protons of the side chain in the upper field region; for example, the H-2 of Glu has the following coupling network: 4.21 ppm (Glu H-2b2.12, 1.89 (Glu H-3)-2.55 (Glu H-4). Each H-2 was completely assigned as follows: Glu, 4.21 ppm; Ala, 4.59 ppm; Leu, 4.26 ppm; p-Me-Asp, 4.44 ppm; Arg, 4.36 ppm; and Adda (H-3), 4.56 ppm. 'H-detected multiple quantum coherence (HMQC) spectroscopic method has an advantage over C/H shift correlated spectroscopy (C/H COSY) in being more sensitive because it detects protons instead of carbons (16). Since sample amount was limited, we used HMQC for objective I1 and could readily assign each C-2 of the amino acid on the basis of the H-2s already assigned as follows:
Chem. Res. Toxicol., Vol. 3, NO.5, 1990 475
Geometrical Isomers of Microcystins
1
2
4
& Adda
:t
4
Add0 314
Adda 2 1 3
.:
e3
*: a8
Leu 311
g-Mc-Aap 3 1 2
:
:*=
e
A l a 312 Arg 312
Glu 312
.. a8
&M-Aap
*
213
1 .
Add.
Adda 1019
sa
Adda 8 1 9
-%33
312
-==
-
Arg 4 1 5
*e Adda
6-Me-Asp
513
t
Adda I l l 2
%8
SIlOft
Adda 1 3 1 8
Adda 9 1 8
: t' %?:
A r g 514
Gb B-*-Asp
315 Add.
-
88
Glu 413
2/11
Adda 8 / 1 3
Leu 5 , 6 1 4
Arg 3
Leu 4 1 5 . 6
Figure 3. Enlarged DQF-COSY spectrum ( 5 . 5 0 . 5 0 ppm) of microcystin LR in CD,OD. H-2 of each constituent amino acid is definitely assigned by the spectrum.
Figure 4. Sequence determination of peptide by HMBC technique. Carbonyl carbon is assigned by correlation via 'JcH, y d the relationship between carbonyl carbon and H-2of the neighboring amino acid is confirmed by 3 J ccoupling. ~
Glu, 55.43 ppm; Ala, 49.99 ppm; Leu, 55.37 ppm; @-MeAsp, 57.48 ppm; Arg, 52.98 ppm; Adda (C-3), 56.88 ppm. For accomplishing objectives I11 and IV the heteronuclear multiple bond correlation (HMBC) spectroscopic method was intorduced, which enabled us to sensitively detect the long-range coupling [usually two (2JcH)and three (3Jm)bond couplings] between carbon and hydrogen atoms (17). First, the carbonyl carbons were assigned by correlation of the carbonyl carbon and its own H-2, assigned via the 'JCH coupling. Next, the relationship between the carbonyl carbon and H-2 of the neighboring amino acid was confirmed by 3JcH coupling (Figure 4). There are nine carbonyl carbon signals at 180-166 ppm in the 13CNMR spectrum of 1. Figure 5 shows the HMBC spectrum (carbonyl carbon region) of 1. The seven cross peaks, a, b, c, f, g, i and j, due to 2JcHcouplings are observed in the spectrum, so that the carbonyl carbons of Glu, Leu, Arg, p-Me-Asp (C-1 and C-41, Adda, and Ala were definitely assigned together with that of Mdha (166.07
ppm). The correlations due to the 3JCHcouplings were also investigated, and cross peaks b (Ala C-1 and Leu H-2), d (p-Me-Asp C-4 and Arg H-2), e (Leu C-1 and 8-Me-Asp H-2), h (Arg C-1 and Adda H-3), and k (Mhda C-1 and Ala H-2) were observed. Additionally, the cross peak from the carbonyl carbon at C-5 of Glu to the N-methyl group of Mdha appeared, indicating that the sequence Glu-MdhaAla-Leu-@-Me-Asp-Arg-Addawas almost confirmed. However, no cross peak was found between Adda and Glu, and the cross peaks between Ala and Leu, and between Leu and 0-Me-Asp, are ambiguously overlapped. Kusumi et al. isolated a hepatotoxin named cyanoviridin RR [same as microcystin RR (2)] from Microcystis uiridis and determined its structure by use of 2D NMR techniques such as COLOC (correlation spectroscopy via long-range coupling), HOHAHA (homonuclear Hartmann-Hahn), and HMBC (10, 11). Furthermore, they reported that the addition of trifluoroacetic acid (TFA) makes only the H-2 (free carboxylic acids) of the acidic amino acids shift significantly to a lower field (10). As shown in Figure 6, the two H-2 signals of Glu and @-MeAsp are expectedly observed in the lower field of the spectrum measured in CD,OD and TFA, whereas the other H-2s had the almost same chemical shifts as those under the neutral conditions. The HMBC spectrum under acidic conditions showed clear cross peaks due to the 3JcHcouplings of the carbonyl carbons of Adda, Ala, and Leu with the H-2 of Glu, Leu, and 0-Me-Asp, respectively. Therer fore, the sequence Glu-Mdha-Ala-Leu-0-Me-Asp-Arg1 Adda was correctly determined for 1. From these results
Harada et al.
476 Chem. Res. Toxicol., Vol. 3, No. 5, 1990 N
3
4
2
I
I
1
vu
B I
Figure 5. HMBC spectrum of microcystin LR in CD30D. Seven cross peaks ( 2 J ~ H a,)b,, c, f, g, i, and j, and five cross peaks (3JCH)1 b, d, e, h, and k, are observed.
Adds Arg 2Leu 2Glu 2 Ala 2 B-Me-Asp 2 1
5
PPM
4
Figure 6. Partial 'H NMR spectra of microcystin LR in (a) CDBODand (b) CD,OD and TFA. H-2resonances of Glu and 8-Me-Asp are shifted to a lower field.
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 477
Geometrical Isomers of Microcystins
Table I. Chemical Shifts (ppm) of Carbons and Protons in Microcystin LR (1) and Its Minor Component (3) in CDSOD 3
1
Adda
carbon no. 1 2 3 4 5 6 7 8 9 10 11 12
Ala Ark7
13 14 15 16 17 18 19 20 1 2 3 1 2
3 4 5 Glu
6 1 2
3 4 5
Leu
1 2
@-MeAsp
5.42 d (10) 2.64 m 3.27 m 2.69 dd (14, 8) 2.81 dd (14, 4) 1.03 d (7) 1.61 s 1.01 d (7) 3.24 s 7.18 m 7.24 m 7.19 m 7.24 m 7.18 m 4.59 q (7) 1.32 d (7) 4.36 dd (9, 3.5) 1.58 m 2.01 m 1.54 m 3.13 m
29.45 26.76 42.00 158.59 177.44 55.43
4.21 t (7.5)
1.89 m 2.12 m 2.55 m
29.15 33.36 176.81 or 176.88 175.53 55.37
3 4
114.51 38.46
1
3.05 m 4.56 t (7.5) 5.48 dd (16, 10) 6.24 d (16)
38.98 15.65 or 16.11 12.98 16.58 58.76 140.56 130.59 129.25 127.11 129.25 130.59 175.20 49.99 17.35 172.19 52.98
2
1
2 3 4 5 Mdha
176.81 or 176.88 45.19 56.88 126.64 139.03 133.93 137.10 37.69 88.36
40.80 25.91 21.31 or 23.73 21.31 or 23.73 176.81 or 176.88 57.48 42.00 179.40 15.65 or 16.11 166.07 146.42
3 4 5 6
lH (mult) ( J , Hz)
'3C
4.26 dd (12, 4) 1.54 m 2.01 m 1.80 m 0.87 d (7) 0.89 d (7) 4.44 d (4) 3.14 m 1.05 d (7) 5.43 s 5.89 s 3.33 s
a combination of three 2D NMR techniques, DQF-COSY, HMQC, and HMBC, enabled us to fully assign all the carbons and protons of 1 as shown in Table I, and this procedure proved to be very effective for structure determination, especially the sequence determination of microcyst ins. In order to confirm the usefulness of this procedure, we determined the structure of another known microcystin RR (2) with it. From the DQF-COSY spectrum in DzO, the following H-2s were definitely confirmed: Glu, 3.87 ppm; Ala, 4.41 ppm; p-Me-Asp, 4.43 ppm; Arg-l,4.26 ppm; Arg-2,4.18 ppm; and Adda (H-3), 4.47 ppm. The C-2s of constituent amino acids were assigned by HMQC as follows: Glu, 58.7 ppm; Ala, 52.8 ppm; 6-Me-Asp, 59.1 ppm; Arg-l,54.4 ppm; Arg-2,58.2 or 58.3 ppm; Adda (C-3), 58.3 ~
13c
176.60 44.76 57.33 130.05 131.44 133.09 135.03 37.35 88.60 39.70 16.06 21.05 17.36 59.00 141.06 130.60 129.45 127.12 129.45 130.60 175.25 49.95 17.36 171.95 52.82 29.46 26.64 41.99 158.61 179.25 56.42 29.14 33.70 177.25 175.70 55.33 40.65 25.96 21.77 or 23.27 21.77 or 23.27 176.60 58.09 42.40 179.10 15.60 166.25 146.54 114.07 38.43
lH (mult) (J, Hz) 3.11 m 4.60 t (7.5) 5.62 dd (16, 10) 6.55 d (16) 5.28 d (10) 2.81 m 3.22 m 2.63 dd (14, 8) 2.77 dd (14, 4) 1.01 d (7) 1.83 s 1.03 d (7) 3.16 s 7.23 m 7.27 m 7.19 m 7.27 m 7.23 m 4.59 q (7) 1.34 d (7) 4.36 dd (9, 4) 1.45 m 2.02 m 1.48 m 3.05 m 4.12 t (7.5) 1.96 m 2.11 m 2.58 m 4.27 dd (12, 4) 1.55 m 2.05 m 1.79 m 0.87 d (7) 0.89 d (7) 4.37 d (4) 3.17 m 1.03 d (7) 5.42 d (
