VII. Supporting appendix
25. Known methods of N-methylation applied to macrocyclic peptides and depsipeptides
25
subsequently hydrolyzed to its N-methyl amino acid subunits. Use of Marfey’s reagent and 1H NMR analysis of the N-methyl amino acids confirmed their structure and stereochemistry, allowing for non-ambiguous structure elucidation.
The last example in Scheme 25 is N-dimethylation with CT3I for receptor binding studies of the natural product PF1022A44 (58). After over 70 different experiments, optimized conditions use a vast excess of CT3I and Ag2O in DMF to afford the radiolabeled product (59) in 7% yield.
PF1022A is a depsipeptide with alternating N-methyl amino acid and hydroxy acid residues, similar to (-)-verticilide. The difficulty to N-methylate dimethyl PF1022A precursor was accurate foreshadowing to optimization of the last N-tetramethylation step in this route to (-)-verticilide.
44 Pleiss, U.; Harder, A.; Turberg, A.; Londershausen, M.; Iinuma, K.; Mencke, N.; Jeschke, P.; Bonse, G. J. Label.
Compd. Radiopharm. 1996, 38, 61.
Table 7. N-Methylation optimization attempts: round 1
entry methylation conditions solvent temp (°C)
1 NaH, MeI THF/DMF (10:1) 0 to rt
2 Ag2O, MeI DMF 50
3 Ag2O, MeI THF rt
4 DIAD, PPh3, MeOH THF rt
Figure 8. Hypothesized solvent-dependent conformations of 53
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Initial attempts to N-methylate 53 were largely unsuccessful (Table 7), affording alcohol 60 as the major product. The same Ag2O conditions that successfully methylated the theonallapeptidolides43 and dimethyl PF1022A44 afforded 60 as the major product as well. What was most surprising about these trials is that the major product (60) was not N-methylated or epimerized, despite the large amount of methylating reagents used. It is hypothesized that N- methylation was not occurring in these reactions because of the conformation of 53 in solution (Figure 8).
Several literature examples,45,46,47,48 have demonstrated how cyclodepsipeptides have solvent-dependent conformations due to intramolecular hydrogen bonding. In-depth studies on the conformation of valinomycin,45,46 a cyclododecadepsipeptide natural product, in solution found that the depsipeptide exhibits a strong network of 4→1 intramolecular hydrogen bonds in non- polar to medium polar solvents (cyclic ether, hydrocarbon, DMSO, DMF), shielding the amide protons from the outside environment. In polar solvents (water, alcohols, DMF), the depsipeptide amide protons are exposed to the outside environment in a fast equilibrium between weak 3→1 intramolecular hydrogen bonds and hydrogen bonding with the solvent environment. With this
information, the lack of N-methylation in the initial attempts to methylate 53 makes sense because the solvents used were moderately polar, thus the amide hydrogens would be engaged in 4→1 intramolecular hydrogen bonding and less reactive (Figure 8). Unfortunately, polar solvents such as alcohols and water are not suitable to use in the basic conditions required for N-methylation.
However, according to several references,47,49,50,51,52,53 cyclic peptides and depsipeptides have the ability to coordinate ions in solution. The cyclic antibiotic, valinomycin, has been heavily studied in this field due to its ability act as a selective potassium ion carrier in biological membrane systems.X-ray crystal stuctures53 and NMR studies51,52 of the potassium ion-valinomycin complex have shown that the depsipeptide is in a bracelet-like structure: its ester carbonyls are coordinated
45 Patel, D. J.; Tonelli, A. E. Biochemistry 1973, 12, 486.
46 Grochulski, P.; Smith, G. D.; Langs, D. A.; Duax, W. L.; Pletnev, V. Z.; Ivanov, V. T. Biopolymers 1992, 32, 757.
47 Heitz, F.; Kaddari, F.; Heitz, A.; Raniriseheno, H.; Lazaro, R. Int. J. Pept. Protein Res. 1989, 34, 387.
48 Kato, T.; Mizuno, H.; Lee, S.; Aoyagi, H.; Kodama, H.; Go, N.; Kato, T. Int. J. Pept. Protein Res. 1992, 39, 485.
49 Pitchayawasin, S.; Kuse, M.; Koga, K.; Isobe, M.; Agata, N.; Ohta, M. Bioorg. Med. Chem. Lett. 2003, 13, 3507..
50 Kimura, S.; Imanishi, Y. Biopolymers 1983, 22, 2383.
51 Grell, E.; Funck, T. Eur. J. Biochem. 1973, 34, 415.
52 Patel, D. J. Biochemistry 1973, 12, 496.
53 Ovchinnikov, Y. A. FEBS Lett. 1974, 44, 1.
Figure 9. Hypothesized shift of hydrogen bond network in free and ion coordinated 53
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to the ion and the amide protons are exposed to the outside environment, partaking in weak H- bonding with the non-ion coordinated carbonyls. This bracelet-like conformation is extremely similar to valinomycin’s polar solvent conformation, but can be achieved in a non-polar solvent.
In order to achieve a “polar conformation” so that the amide protons would be reactive in N- methylation conditions, 53 was pre-stirred with KI in hopes that the cyclic substrate would coordinate to the potassium ion (Figure 9).
N-Methylation trials using an ion source to coordinate 53 are summarized in Table 8. Initial results of N-methylation using either KI or NaI as the ion source and NaH as the base in THF were slightly more promising in that the major products were N-methylated, but no desired N- methylated macrocyclic products were observed. Changing the base to Li2CO3 or K2CO3 did not produce any N-methylated side products. However, LiHMDS and Cs2CO3 did afford ring-opened and cleaved N-methylated side products; reactivity also appeared diminished relative to NaH.
Changing the solvent from THF to 2-Me-THF, MeCN, or DCM did not improve this. In fact, in
entry 11 (Table 8), LiHMDS in DCM did not react, but when the solvent was changed to THF in
Table 8. N-Methylation of ion-coordinated N-H 24-membered ring
entry ion source base solvent temp (°C) N-methylation? major productsa
1 KI NaH THF rt Yes 42, 61, + N-Me cleavage pdts
2 KI NaH THF 0 Yes 42, 61, + N-Me cleavage pdts
3 NaI NaH THF 0 Yes 42, 61, + N-Me cleavage pdts
4 KI NaH 2-Me THF 0 Yes 42, 61, + N-Me cleavage pdts
5 NaI NaH 2-Me THF 0 Yes 42, 61, + N-Me cleavage pdts
6 KI K2CO3 THF rt No 60
7 KI K2CO3 MeCN rt No 60
8 KI Cs2CO3 THF rt Yes 60, 42, 61, + N-Me cleavage pdts
9 KI Cs2CO3 + Sc(OTf)3 THF rt No 53 + ring opened 53
10 KI Li2CO3 THF rt No 53
11 KI LiHMDS DCM 0 No mostly 53
12 KI LiHMDS THF 0 Yes 60, 61, 42 + N-Me cleavage pdts
13 KI Ag2O + CaSO4 THF rt Yes 61
aMajor products were determined from the 1H NMR spectrum of the crude reaction mixture by comparison to 1H NMR
spectra from authentic samples, and verified by LCMS
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entry 12 (Table 8), N-methylation and cleavage of the macrocycle occurred. This may indicate that solvent has an effect on the stability of the potassium coordination complex.
In reviewing all trials to N-methylate 53, it is apparent that 53 must be in a “polar conformation” in order for N-methylation to occur. Although the valinomycin-potassium complex is very stable, valinomycin and 53 are structurally quite different. Valinomycin is a cyclododecadepsipeptide of [L-D-D-L]3 repeating units and 53 is a 24-membered depsipeptide of [L-D-L-D]2 repeating units. Cyclooctadepsipeptide valinomycin [L-D-D-L]2 ion complexes have been analyzed,53 but were found to be strained and unstable. Based on this, we can conclude that the ion complex of 53 did form in solution, but is too unstable for N-permethylation to go to completion. Additionally, solvent conformation studies of cyclooctadepsipeptide valinomycin analogs in DMF demonstrated that the macrocycles exhibited a more “polar conformation”
consisting of only two weak 3→4 H-bonds,50 unlike the cyclododecadepsipeptide natural product which exhibits a “nonpolar conformation” of four 4→1 H-bonds.52 With the hope that 53 would exhibit a similar “polar conformation” as the valinomycin analogs, N-methylation was attempted in DMF with NaH as the base. Much to our delight, these conditions successfully provided (-)- verticilide in 78% yield, and the total synthesis was completed in a 10-step longest linear sequence.
Furthermore, variation of the base and reaction time of the NaH/DMF methylation conditions afforded desmethyl analogs 62 and 63 in moderate selectivity (Table 9).
Table 9. N-Methylation conditions to afford (-)-verticilide and derivatives
entry base time major product yielda
1 NaH 25 m 21 78%
2 NaH 13 m 63 31%
3 LiHMDS 15 m 62 35%
aIsolated yield.
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