towards new gramicidin S-based bactericides
Knaap, M. van der
Citation
Knaap, M. van der. (2010, September 8). Site-selective incorporation of alpha- and beta- amino acid derivatives : towards new gramicidin S-based bactericides. Retrieved from https://hdl.handle.net/1887/15935
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Summary and Future Prospects
The molecule around which this Thesis revolves is the antibiotic cyclic peptide gramicidin S (GS). Several modifications are presented, with the goal to improve the biological profile, to design and synthesise structurally novel peptides, or both. Chapter 1, the General Introduction, presents the subject of natural cyclic peptides with antibiotic activity in general and gramicidin S in particular. The pressure from society to invent new antibiotics has been an incentive to take GS as a lead compound, but to be clinically broadly applicable the toxicity of GS towards mammalian cells is a first hurdle to be taken. This may be achieved by preparing GS derivatives in which the structure and/or the amino acid composition have been altered. From a structural point of view GS is interesting due to its rigid β-hairpin. The effect of a specific modification may be probed and compared to the parent peptide, as detailed Röntgen diffraction structures and NMR data are available. The remainder of this Chapter focuses on the modifications of GS that have appeared in the literature in the past five decades. Arranged by amino acid residue substitution, an overview of GS derivatives is given, with a focus on the effect of a particular modification on the membrane disrupting activity.
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The question was raised what the best position would be for the attachment of a conjugation-, or polymerisation handle. Previous research had shown that proline was not the best position for modifications, thus attention was directed at the D-phenylalanine residue.
Chapter 2 describes the synthesis of Fmoc-D-p-NO2-Phe-OH by chiral phase transfer catalysis using a Chinchona alkaloid derivative, which resulted in high yield and high enantiopurity of the desired amino acid. This building block was subsequently incorporated in gramicidin S, as replacement of one of the DPhe residues. With the protecting groups still in place on the side chains of ornithine, the nitro functionality could be reduced by the action of palladium on charcoal in the presence of ammonium formate. It was shown that the resulting aniline derivative could be acylated or alkylated with a variety of aliphatic and aromatic acyl chlorides and halides, respectively. The peptides that were obtained were analysed by NMR spectroscopy to probe the structure, which in general was not significantly affected by the modification. The products were also assessed for their antibacterial and hemolytic activity. It appeared that most of the modifications resulted in a decrease in activity against bacterial cell membranes, but two acylated derivatives, adamantylcarbonyl and pivaloyl, appeared to be comparably active against various bacterial strains. Unfortunately
Scheme 1 Preparation of oligomeric GS constructs.
Summary and Future Prospects
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there was no dissection in the correlation between the antibacterial and hemolytic activity observed. Altogether it may be concluded that the D-phenylalanine residue is a good position to introduce modifications and a practical synthetic route was presented.
It would be interesting to prepare oligomeric GS derivatives, as a synergistic effect of the close proximity of several membrane active peptides may possibly occur. Dimers that were covalently linked at the DPhe and Pro positions showed good activities against bacteria, as measured in a broth medium.[1] Besides, GS and some derivatives form six-membered cyclic entities in crystal structures, possibly resembling pores that may be formed in biological membranes.[2] Scheme 1 presents two potential templates for the attachment of GS derivatives via the D-phenylalanine aromatic ring, leading to tri-, tetra- or hexameric GS constructs.
These kinds of molecules may give more insight into the cooperativity between several closely spaced gramicidin S molecules.
In Chapter 3 the question is addressed whether a triazole would be an adequate mimic for the phenyl ring in the phenylalanine residue. Both moieties are aromatic, which characteristic appears to be beneficial for the activity of GS on bacterial membranes. Towards this goal a GS peptide was synthesised in which one of the D-phenylalanine residues was replaced by a D- propargylglycine residue. The resulting alkyne was engaged in copper(I)-catalysed conjugation reactions with several aromatic azides. This modification did not have a detrimental effect on the secondary structure and the resulting peptides showed high activity against bacteria. The 1H-triazole derivative, however, showed marked decrease in the antibacterial activity, whereas the histidine derivative (imidazole) showed good activity, presumably due to the higher basicity of the imidazole compared to the triazole. Whereas the Cu(I)-catalysed cycloaddition reaction delivers 1,4-substituted triazoles, the Ru(II)-catalysed reaction is known to yield 1,5-substituted triazoles and this reaction was also engaged.
Surprisingly, it appeared that after deprotection, the resulting triazoles displayed identical spectral characteristics as the corresponding 1,4-triazoles. The cause of this observation is at this moment not exactly clear, but this result is unprecedented in the literature. Though it can not be excluded yet that the 1,4-triazoles are obtained already after the ruthenium catalysed
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reaction, it seems most likely that an acid catalysed regioisomeriation occurs during the Boc- deprotection step. In order to verify this hypothesis, and ultimately obtain the 1,5-triazoles, alternative protective groups on the amines of the ornithines may be necessary, Fmoc for example, though these need to be introduced postsynthetically. Alternatively, the Boc-group may be removed under non-acidic thermal conditions (DMSO, > 100 °C).[3,4] As the acid catalysed regioisomerisation possibly involves a relatively stable benzyl cation, alkyl substituted triazoles may be prepared, as it is less likely that this type of substituent is released as cation from the triazole.
It is interesting to note that the 1H-triazole amino acid presented in this Chapter as part of a cyclic decameric peptide, has never been prepared before in enatiomerically pure form.[5,6]
Triazolalanine is an isostere of the natural amino acid histidine, which is particularly relevant in the context of peptidases and esterases. These enzymes often contain a catalytic triad,
consisting of an aspartate/glutamate, a histidine and a nucleophilic serine, or cysteine residue (Scheme 2A). As the basicity of a triazole is lower than that of an imidazole, it would be interesting to see the effect of the replacement of the imidazole by a triazole on the enzyme kinetics (Scheme 2A). Candida antarctica strains are available that are histidine auxotrophe, meaning that they are unable to synthesise histidine and may substitute that residue for an Scheme 2 A) Catalytic triad; B) Reagents and conditions i) 2,4-dimethoxybenzylazide, CuSO4 (10 mol%), sodium ascorbate (15 mol%), DMF (90%); ii) TFA/DCM, μW (9%); iii) o-nitrobenzylazide, copper(I), H2O/tBuOH; iv) 300 nm light, H2O.
Summary and Future Prospects
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other amino acid, triazolalanine for example. This residue was synthesised as depicted in Scheme 2B (see experimental section). Commercially available Boc-propargylglyine (6) was first reacted with 2,4-dimethoxybenzylazide under the agency of in situ generated copper(I), yielding triazole 7 in good yield. The protective groups were removed under acidic conditions in TFA/DCM followed by purification by crystallisation from MeOH/H2O. The low yield was mainly due to difficulties during purification. The resulting amino acid (8) may be added to histidine auxotrophic cells. Alternatively, a C. antarctica able to exchange methionine for propargylglycine and in which the active site histidine is substituted for a methionine, is available as well. Thus, it is possible to incorporate a propargylglycine in the catalytic triad of CalB (Candida antarctica lipase B). ‘Click’ chemistry under conditions that are compatible with the enzyme may yield a protected triazole, which needs to be deprotected under biocompatible circumstances. The o-nitrobenzyl may be suitable for this purpose, as it is known to be labile under irradiation with light at 300 nm.[7,8] The enzyme (9) therefore needs to be exposed to o-nitrobenzylazide and copper(I) in water, probably with small amounts of an organic co-solvent to obtain a homogeneous mixture. The protected triazole 10 may then be liberated by irradiation with light. The thus obtained enzyme 11 may be used in enzyme kinetics experiments.
Two series of peptides based on three known antibiotic peptides with various ring sizes were presented in Chapter 4. On one end of the spectra an octameric peptide was designed based on GS and on cyclic peptides with alternating D- and L-peptides, a class of peptides originally designed in the laboratory of Ghadiri. Also hybrid peptides between the octamer and GS were synthesised by introducing one or two additional residues and by changing the stereochemistry of ornithine. A second independent series of peptides was prepared as well in which GS and the known antibiotic cyclic dodecameric peptide gratisin were central. Gratisin is known to have high antibacterial, but low hemolytic activity. In addition the three- dimensional structure of this peptide is unknown, making it an interesting target. A hybrid peptide between GS and gratisin was obtained by omitting one of the DTyr residues from gratisin. All the peptides were analysed by NMR and it was found that gratisin did not yield a
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clearly resolved spectrum, indicating that it does not adopt one single structure in solution.
The hybrid peptide indicated however that the DPhe-Pro-DTyr sequence adopted an α-turn type of conformation. None of the peptides approached the antibacterial activity of GS.
Curiously, known antibiotic gratisin did not display any biocidal activity in the assay used.
Gratisin contains two additional D-tyrosine residues compared to gramicidin S. These two residues have marked influence on the structure and activity of the cyclic peptide. To investigate the influence of DTyr and the other aromatic residue in the turn, D-phenylalanine, a series of peptide is proposed. Starting from gratisin 12, one (13) or two DPhe residues are removed to give the cyclic peptide 14. Here a turn is formed by the Pro-DTyr dipeptide, likely resulting in a type II β-turn, which is the mirror image of the type II´ β-turn with respect to the dihedral angles. Removing the last two remaining D-amino acids, nona- (15) and octamers (16) are obtained. Proline might be able to form a turn on its own, resulting in turn- sheet motif, comparable to GS. If these peptides are able to form the β-sheet conformation, they should be rendered with amphiphilicity and thus antibacterial activity.
As the structure of gratisin is not exactly known and structure activity relationships are lacking it would be interesting to investigate this in further detail. It seems obvious that Val- Orn-Leu forms a strand, comparable to GS. Therefore, it would be most interesting to examine the remainder of the molecule. This might be done by substituting D-phenylalanine,
Scheme 3 Series of peptides to investigate the structure of gratisin 12.
Summary and Future Prospects
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proline and D-tyrosine individually for glycine, alanine, or D-alanine, respectively and probe the effect on the structure. The balance in hydrophobicity and hydrophilicity and the effect of cationic residues may be investigated by replacing one of the residues by serine or lysine.
Chapter 5 describes a new route towards α-substituted β-amino acids (β2-amino acids).
Acylated derivatives of the Evans’ chiral oxazolidinone were aminomethylated using the trifluoroacetate salt of dibenzylmethyleneiminium to give the desired β-amino acid precursor in good yield and enantioselectivities. The chiral auxiliary was efficiently removed by displacement with the anion of ethanethiol. The resulting thioester could be hydrolysed under mild conditions using mercury salts in wet THF. The conditions outlined above work well, even on sterically encumbered amino acids and this method may be applied to the synthesis of various amino acids. All β-amino acids with proteinogenic side chains may be obtained using this method, probably with the exception of methionine and cysteine.
As α-methylbenzylamine is a cheap chiral compound it would be an interesting source of chiral induction in the synthesis of β-amino acids. Benzylated α-methylbenzylamine (17) has already been used in asymmetric Michael additions to yield β3-amino acids in high yield and enantiopurity. Scheme 4 outlines a synthetic scheme to synthesise β2-amino acids. The route is based on the aminomethylation reaction described in Chapter 5. The enolates of tert-butyl esters 20 are reacted with chiral 19. The iminium ion may be synthesised from the symmetric aminal of benzyl α-methylbenzylamine (17) in two synthetically simple steps. The amino
Scheme 4 Reagents and conditions i) formalin; ii) (CF3CO)2O, THF; iii) a) LDA, THF, -78 °C; b) 19, -78 °C; iv) H2, Pd/C, AcOH/MeOH; v) FmocCl, NaHCO3, H2O/dioxane; vi) TFA/DCM.
NH Ph Ph
N N
Ph Ph
Ph Ph
ii
i N Ph
Ph
17 18 19
O
R O iii
O R O Ph N
Ph
iv, v O
O R FmocHN
vi O
OH R FmocHN
20 21 22 23
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methylated product 21 can be debenzylated and protected with the fluorenylmethyl carbamate and liberated from its ester using TFA in DCM to give amino acids 23.
Chapter 6 and Chapter 7 of this Thesis, deal with the application of α-, β2- and β3-amino acids in cyclic decameric peptides based on gramicidin S. In Chapter 6 the leucine and valine residues in GS were replaced by β3hLeu and β2hVal, respectively. It was shown that by positioning of the β-amino acids in these specific positions, the four hydrogen bridges in GS were maintained. This resulted in the formation of an antiparallel β-sheet and two type IIȁβ- turns. All these results were confirmed by NMR spectroscopy. The peptides were designed to be amphiphilic and thus possess membrane activity, but unfortunately none of the peptides displayed any significant activity against bacteria. The reason for this inactivity is not completely clear at the moment, but it might be due to the bend in the β-sheet, observed in a crystal structure from one of the peptides, which is much more pronounced than in gramicidin S. The fact that both β2- and β3-amino acid have a high propensity to form helices might be reflected by this observation. trans-Disubstituted β-amino acids on the other hand are known to fold into pleated sheets. By substituting the β2- and β3-amino acids by β2,3- amino acids a more extended conformation may be adopted.[9] This has the added advantage that more functionalities may be introduced, leading to fine-tuning of the hydrophobic/hydrophilic ratio. Keeping in mind the design presented in Chapter 6, this would mean that the functionality to be introduced needs to be hydrophobic and a peptide 26
Scheme 5 Proposed β-GS peptides with extended conformation and peptide 27 with inverted polarity
NH N
H
HN H
N O
O O
O HN HN
NH NH
O
O O
O N
N O
O
H2N
H2N
R1 R2
R1
R2
24: R1= (CH2)3NH2; R2= H 25: R1= H; R2= (CH2)3NH2
26: R1= R2= (CH2)3NH2
NH N
H
HN H
N O
O O
O HN HN
NH NH
O
O O
O N
N O
O
R1
R2 R1
R2 H2N
H2N NH2
NH2
27: R1=iPr; R2=iBu
Summary and Future Prospects
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as in Scheme 5 is proposed. This peptide contains β-amino acids with a hydrophobic and a hydrophilic side chain, with a trans-relationship between each other. Peptides 24 and 25 are included to see whether all β-amino acids need to be disubstituted or that a more extended configuration is already attained with two disubstituted β-amino acids. Alternatively, the polarity of β-GS may be inverted by swapping the hydrophobic and hydrophilic residues (27).
In Chapter 7 the introduction of D-amino acids in the βαβ-strand of β-GS was shown.
Compared to the design of β-GS in Chapter 6, not only the stereochemistry of the ornithine was inverted, but also the side chains on the β-amino acids were shifted to the adjacent carbon. This did not yield β-sheets with very strong hydrogen bonding and appeared to be very sensitive to the number and position of the side chains. In addition it was shown that these peptides were devoid of antibacterial activity.
In Chapter 6 and Chapter 7 the α-amino acid ornithine in the middle of the strand was retained. However, also the position of the α-amino acid may be shifted, while keeping the hydrogen bonds and pleated sheet structure. In Scheme 6 new peptides are proposed in which
the position of the α-amino acid in the strand is varied, to give αββ and ββα patterns. In this Scheme side chains are positioned in such a way that amphiphilicity should be attained, but alternative side chain positionings are possible as well. Peptide 28 contains six hydrophobic and four hydrophilic residues and in 29 this is the other way round.
Scheme 6
NH
HN H
N H
N O
HN HN
HN
NH O
N N O
O
NH N
H N
H HN O
HN NH NH
NH O
N N O
O
O O O
O
O O
O O
O
O O O
R R
R R
NH2 NH2 NH2
H2N H2N
H2N R R
R R
28: R = (CH2)3NH2 29:iBu
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EXPERIMENTAL SECTION
(S)-2-(tert-butoxycarbonylamino)-3-(1-(2,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)pro- panoic acid (7)
N-Boc-propargylglycine (500 mg, 2.34 mmol) and 2,4-dimethoxybenzyl azide (1.5 eq, 3.52 mmol, 679 mg) were dissolved in DMF (10 mL) and CuSO4 (1M in H2O, 0.1 eq, 0.23 mmol, 230 μL) and sodium ascorbate (1M in H2O, 0.15 eq, 0.35 mmol, 350 μL) were added. After 45 min the mixture was diluted with water and extracted with EtOAc (2 × 100 mL). The organics were washed with water and brine, dried over MgSO4, filtered and evaporated. The crude product was purified by column chromatography (50 → 100% EtOAc in PE) to yield the pure title compound in 90% yield (857 mg).
1H NMR (400 MHz, CDCl3) δ 12.14 (brs, 1H), 7.35 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.47-6.44 (m, 2H), 5.57 (d, J
= 6.7 Hz, 1H), 5.41 (s, 2H), 4.49 (dd, J = 10.8, 5.1 Hz, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.32 (d, J = 3.3 Hz, 2H), 1.40 (s, 9H); 13C (100 MHz, CDCl3) δ 172.76, 161.61, 158.31, 155.31, 141.90, 131.43, 122.92, 114.74, 104.48, 98.66, 79.60, 55.37, 55.30, 52.85, 49.12, 28.17, 27.43; IR neat (cm-1): 2976.2, 2362.1, 1711.8, 1614.9, 1590.3, 1508.6, 1458.0, 1366.4, 1290.1, 1209.6, 1028.6, 833.6, 783.3; [α]D +44.8° (c = 1.0, CHCl3); LC/MS: Rt = 6.96 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 406.93 [M + H]+; HRMS: calculated for C19H27N4O6 m/z 407.19251;
found: m/z 407.19231.
(S)-2-amino-3-(1H-1,2,3-triazol-4-yl)propanoic acid (8)
Triazole 7 (144 mg, 0.35 mmol) was dissolved in CH2Cl2 (0.5 mL) and TFA (0.5 mL) and anisole were added (1 eq). The solution was subsequently heated for 5 min under microwave irradiation at 70 °C. The product was concentrated in vacuo and the product was purified by crystallisation from MeOH/H2O (4.67 mg, 4% for di-TFA salt).
1H NMR (400 MHz, D2O) δ 7.81 (s, 1H), 4.07 (dd, J = 6.7, 5.3 Hz, 1H), 3.37 (dt, J = 15.9, 7.1 Hz, 2H).
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