Total solid-phase synthesis of dehydroxy fengycin derivatives

A rapid and efficient solid-phase strategy for the synthesis of dehydroxy fengycins derivatives is described. This synthetic approach involved the linkage of a Tyr to a Wang resin via a Mitsunobu reaction and the elongation of the peptide sequence followed by subsequent acylation of the N-terminus of the resulting linear peptidyl resin, esterification of the phenol group of a Tyr with an Ile, and final macrolactamization. The amino acid composition as well as the presence of the N-terminal acyl group significantly influenced the stability of the macrolactone. Cyclic lipodepsipeptides with a l-Tyr3/d-Tyr9 configuration were more stable than those containing the Tyr residues with an opposite configuration. This work constitutes the first approach on the total solid-phase synthesis of dehydroxy fengycin derivatives.


Introduction
Cyclic lipodepsipeptides are a structurally diverse group of nonribosomal metabolites typically produced by bacterial and fungal species, such as Pseudomonas or Bacillus strains. [1][2][3][4] This class of compounds have raised interest due to their wide range of biological activities and to their structural complexity. The main structural feature of these cyclic peptides is the occurrence of an ester bond between the side-chain of an amino acid and the C-terminal end of the sequence. The fatty acid moiety is commonly attached to the N-terminus of this macrolactone. Moreover, the complexity of the structure of these lipodepsipeptides is increased by the combination of L-and D-amino acids as well as of other unnatural residues present in their sequence. 5,6 Fengycins are a family of natural cyclic lipodepsipeptides isolated from Bacillus strains. 5,6 They consist of an eight-residue macrocycle with a phenyl ester linkage between the phenol group of a tyrosine (Tyr) and the α-carboxylic group of an isoleucine (Ile) (Figure 1). This cyclic depsipeptide bears at its N-terminus a dipeptidyl tail acylated with a β-hydroxy fatty acid. Different fengycin isoforms have been described, being fengycins A and B the most common. [7][8][9][10][11][12][13][14] These two isoforms differ on the amino acid at position 6, which is a D-alanine (D-Ala) in fengycin A and a D-valine (D-Val) in the B isoform ( Figure 1). Sang-Cheol et al. isolated from Bacillus amyloliquefaciens fengycin S. 15 Compared to fengycin B, this isoform bears a D-serine (D-Ser) at position 4 instead of a D-allothreonine (D-allo-Thr). The configuration of Tyr residues at position 3 and 9 of fengycins has been a controversial issue. Fengycins were first reported to incorporate a D-Tyr 3 and an L-Tyr 9 whereas the opposite configuration (L-Tyr 3 /D-Tyr 9 ) was assigned to another family of related peptides named as plipastatins. 7,16 However, it has been recently confirmed that the configuration of Tyr 3 and Tyr 9 in fengycins and plipastatins is identical, being L-Tyr 3 /D-Tyr 9 . [17][18][19][20] Since the term fengycins is most 5 widely used than plipastatins, herein we employed the former term for the peptides of this study, but using the correct configuration. Fengycin lipopeptides have attracted considerable interest due to their strong antifungal activity and their low hemolysis compared to other lipopeptides isolated from Bacillus spp. 7,21,22 Their significant antifungal activity has been ascribed to their propensity to form stable membrane-bound aggregates. 23 Moreover, they have been described as elicitors of plant defence responses. 24,25 The use of compounds with such activity is considered as a promising alternative approach for the protection of crops against phytopathogens. 2,26,27 Despite their interest, up to now and to the best of our knowledge, the synthesis of fengycins has only been accomplished by the group of Marahiel and Essen. Their strategy comprised the solid-phase preparation of the linear precursor and its enzymatic cyclization in solution. 5,28,29 The lack of suitable synthetic protocols for fengycins could be attributed to the presence of a highly labile phenyl ester function in their structure. In fact, the synthesis of cyclic depsipeptides, either in solution or on solid 6 phase, has only been reported for those peptides bearing an ester bond involving the ß-hydroxyl group of a Ser or a Thr residue. [30][31][32][33][34] Recently, we have established a convenient solid-phase strategy for the preparation of the macrolactone of eight amino acids present in fengycins. 35 The key steps of the synthesis were the formation of the phenyl ester bond and the on-resin head-to-side-chain cyclization.
In view of these previous results, we decided to study the application of this methodology to the total solid-phase synthesis of dehydroxy fengycin derivatives with the general structure I (Figure 2).
These derivatives are fengycin A, B and S analogues that contain a D-Thr 4 instead of a D-allo-Thr 4 and a dehydroxy fatty acid chain. Synthesis of analogues containing both an L-Tyr 3 /D-Tyr 9 or a D-Tyr 3 /L-Tyr 9 configuration was studied. This approach would benefit from the advantages inherent to the solidphase methodology and would allow rapid access to a variety of fengycin analogues.

Synthesis of Cyclic Lipodepsipeptides Bearing a L-Tyr 3 /D-Tyr 9
Our investigations started with the solid-phase synthesis of the cyclic lipodepsipeptide BPC838 containing D-Thr 4 , D-Val 6 , D-Tyr 9 and an octanoyl group (Scheme 1). This synthesis was chosen as a model to check the feasibility of our approach. Based on our previous experience on the preparation of cyclic depsipeptides, the retrosynthetic route that we envisaged involved as main steps: (i) the preparation of the heptapeptidyl resin 1 with the N-terminal L-Tyr 3 protected with a pnitrobenzyloxycarbonyl (pNZ) group, (ii) the ester bond formation between L-Tyr 3 and Ile 10 , (iv) the simultaneous deprotection of Ile 10 and D-Tyr 9 followed by the on-resin cyclization and (vi) the elongation of the peptide sequence and its final acylation. As protecting groups, allyl (All) and allyloxycarbonyl (Alloc) were selected for Tyr 9 and Ile 10 , respectively, because they can be simultaneously removed in presence of palladium and are orthogonal with the 9fluorenylmethoxycarbonyl (Fmoc) and tert-butyl ( t Bu) groups. The pNZ group was chosen to protect Tyr 3 because it has been reported to be orthogonal to Fmoc as well as to t Bu and allyl-based protecting groups, and its removal can be achieved under neutral conditions using SnCl 2 in presence of catalytic amounts of acid. 36 Scheme 1. Retrosynthetic analysis of BPC838.
According to this route, Fmoc-D-Tyr-OAll was anchored to a Wang resin by treatment with PPh 3 and diisopropylazodicarboxylate (DIAD) in anhydrous tetrahydrofuran (THF) under microwave irradiation for 30 min at 60º C (Scheme 2). 35 The loading of the resulting resin Fmoc-D-Tyr(Wang)-OAll was 0.33 mmol/g as determined by the Fmoc method. 37 In addition a Marfey's test confirmed that the D-Tyr residue did not racemise under these conditions. 38 Then, the peptidyl resin pNZ-Tyr 3 -D- (1) was prepared following a Fmoc/ t Bu/allyl (All) strategy. Fmoc group removal was performed using piperidine/DMF (3:7) and the coupling of the corresponding amino acids was mediated by N,N'-diisopropylcarbodiimide (DIPCDI) and 2-cyano-2-(hydroxyimino)acetate (Oxyma) in DMF. All the amino acids, except for Tyr 3 , were incorporated as Fmoc-derivatives with the side-chain group protected with trityl (Tr) for Gln and t Bu for Glu and Thr. Tyr 3 was introduced as pNZ-Tyr-OH to allow the required esterification of the phenol group. This tyrosine derivative was prepared from commercially available H-Tyr(O t Bu)-OH by treatment with pNZ-N 3 , 36 followed by side-chain deprotection under acidic conditions (68% overall yield). After completion of the peptide sequence, acidolytic cleavage with trifluoroacetic acid (TFA)/H 2 O/triisopropylsilane (TIS) (95:2.5:2.5) of an aliquot of resin 1 afforded the expected peptide pNZ-Tyr-D-  Then, Alloc-Ile-OH was incorporated to 1 using the conditions previously described for the preparation of cyclic depsipeptides analogous to the cyclic moiety of fengycins (Scheme 2). 35 Therefore, esterification of 1 with Alloc-Ile-OH was achieved employing DIPCDI, 4dimethylaminopiridine (DMAP) and N,N-diisopropylethylamine (DIEA) in DMF. Two treatments of 24 h were necessary to obtain a complete conversion. An aliquot of the resulting resin pNZ-Tyr 3 (O- corresponding to [M + Na] + was observed (& symbol indicates the location of the ester linkage 39 ). The linear peptide precursor was not detected either by HPLC or mass spectrometry. To confirm the structure of 4, the crude reaction mixture resulting from acidolytic cleavage was treated with CH 3 OH/H 2 O/NH 3 (4:1:1), conditions that are known to hydrolyze ester bonds. [40][41][42] HPLC and mass spectrometry analysis of the resulting crude revealed only the presence of the linear peptide resulting from the hydrolysis of the ester bond in 4, pNZ-Tyr 3 -D-Thr-Glu-D-Val-Pro-Gln-D-Tyr 9 -Ile 10 -OH, and of the corresponding methyl ester pNZ-Tyr 3 -D-Thr-Glu-D-Val-Pro-Gln-D-Tyr 9 -Ile 10 -OMe. Therefore, this result demonstrated the formation of the cyclic depsipeptide 4.
We then attempted the pNZ group removal of resin 3 using SnCl 2 (6 M) in HCl/dioxane (1.6 mM) and DMF for 1 h at room temperature. Unexpectedly, this deprotection resulted to be troublesome and even after repeating this treatment 5 more times, the deprotected cyclic peptide BPC822 was obtained together with an important amount of the pNZ-protected cyclic depsipeptide 4 as shown by mass spectrometry.
Based on these results, we envisaged an alternative route to obtain the cyclic lipodepsipeptide BPC838 (Scheme 3). In this approach, instead of building the macrolactone and then incorporating the tail, we planned the synthesis of the linear peptidyl resin 5a already bearing the acylated dipeptidyl tail.
Then, this resin would be esterified at Tyr 3 with Ile 10 and finally cyclized to render the desired cyclic lipodepsipeptide.

Scheme 3. Alternative retrosynthetic analysis of BPC838.
For this purpose as depicted in Scheme 4, we first synthesized the linear peptidyl resin Fmoc- through side-chain anchoring of Fmoc-D-Tyr-OAll to a Wang resin followed by sequential Fmoc removal and coupling steps using the conditions mentioned above for resin 1.

Synthesis of Cyclic Lipodepsipeptides Bearing D-Tyr 3 /L-Tyr 9
Taken into account the different configuration reported for Tyr 3 and Tyr 9 for natural fengycins, we decided to prepare cyclic lipodepsipeptides incorporating a D-Tyr 3 and an L-Tyr 9 ( Figure 4). In particular, we attempted the synthesis of BPC846 (   In our previous studies on the synthesis of the macrolactone of eight amino acids present in fengycins, compounds bearing the same configuration at the Tyr residues than the cyclic lipodepsipeptides depicted in Figure 4 were obtained together with a dimeric product and the linear precursor prior to cyclization. 35 The results obtained herein point out, on the one hand, that the presence of the fatty acid tail in the linear precursor may favour its cyclization and, on the other hand, that the resulting macrolactone is less stable than the one containing the Tyr residues with an opposite configuration (i.e. BPC846 vs. BPC838). Moreover, these findings further support the recent hypothesis of Honma and co-workers that postulated L-Tyr 3 /D-Tyr 9 as the correct fengycin configuration. 19

Conclusion
In summary, here we describe an efficient solid-phase approach for the total synthesis of dehydroxy derivatives of fengycins A, B and S. The synthesis was accomplished using a Fmoc/ t Bu/allyl strategy, being the key steps the formation of a phenyl ester and the final macrolactamization. This study revealed the significance of the configuration of the Tyr residues on the stability of the macrolactone and showed that the most stable compounds were those containing an L-Tyr 3 and a D-Tyr 9 . This study represents the first approach on the total solid-phase synthesis of fengycin derivatives and would allow rapid access to a large variety of analogues. 43 20

Experimental Section General Methods
Peptide synthesis was performed manually in a polypropylene syringe fitted with a polyethylene porous disc. Solvents and soluble reagents were removed in vacuo. Wang resin was purchased from Fluka.
Amino acids derivatives and other chemical reagents were purchased from IrisBiotech, Sigma-Aldrich or Panreac and were used without further purification, unless otherwise noted. Solvents were purchased from Sigma-Aldrich, Sharlau, VidraFoc, SDS or VWR international. All organic solvents were synthesis grade except for CH 3 CN which was multisolvent HPLC grade. Solvents were purified and dried by an activated alumina purification system (mBraun SPS-800) or by conventional distillation techniques. H 2 O was deionised and filtered by a COT Millipore Q-gradient system (COT < 3ppb) with a resistivity of 18 MΩ·cm -1 .
Microwave-assisted reactions were carried out on a Discover® S-Class CEM Corporation Microwave equipped with an Explorer-Autosampler and controlled with the Synergy TM software. The equipment was provided with a multispeed magnetic stirring system with adjustable speed and an automated power control system based on temperature feedback through a volume-independent noninvasive infrared sensor control which ranges from 0 to 300 Watts. IR spectra were recorded with a Bruker Alpha FT-IR spectrometer equipped with a Bruker platinum ATR adaptador and wavenumbers (ν) are expressed in cm -1 .
The "&" symbol was used as indicator of the ester chemical bond to facilitate the view of cyclic depsipeptide formulas. On the one-line formula, the "&" symbol indicates both the location of one end of a chemical bond and the point to which this bond is attached. This symbol has already used in the nomenclature of other cyclic depsipeptides. 39 Allyl N α -(9-fluorenylmethyloxycarbonyl)-D-tyrosinate, allyl N α -(9-fluorenylmethyloxycarbonyl)-Ltyrosinate and N α -allyloxycarbonyl-L-isoleucine were prepared according to previously reported procedures. 35

24
The resulting residue was digested in pentane to yield Fmoc-D-Tyr-OH as a white powder (0.58 g, 97% yield    The coupling of the corresponding protected amino acid (4 equiv) was carried out in presence of ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma) (4 equiv) and N,N'-diisopropylcarbodiimide (DIPCDI) (4 equiv) in DMF for 3 h under stirring at room temperature. The completion of each coupling was monitored by a Kaiser test 45 or a chloranil test. 46 The Fmoc protecting group was removed by treating the resin with piperidine/DMF (3:7, 1×2 + 3×10 min). After each coupling and deprotection step, the resin was washed with DMF (6×1 min) and CH 2 Cl 2 (3×1 min), and air-dried.

Solid-Phase Synthesis of Cyclic Lipodepsipeptides
To obtain the lipopeptidyl resins, each peptidyl resin was subjected to N-terminal Fmoc removal as described above. After washings, the resin was treated with the corresponding fatty acid (3 equiv), Oxyma (3 equiv) and DIPCDI (3 equiv) in DMF under overnight stirring at room temperature. The resin was then washed with DMF (6×1 min) and CH 2 Cl 2 (3×1 min), and air-dried. Completion of the reaction was checked with the Kaiser test. 45 An aliquot of each resulting lipopeptidyl resin was treated with trifluoroacetic acid (TFA)/H 2 O/triisopropylsilane (TIS) (95:2.5:2.5) for 2 h at room temperature.
Following TFA evaporation, the resulting crude lipopeptide was precipitated with diethyl ether and then decanted to give a white solid that was taken up in H 2 O/CH 3 CN (1:1), lyophilized, and analyzed by HPLC and mass spectrometry.

Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5a).
This peptidyl resin was prepared from 6 following the above procedure using octanoic acid as fatty acid. Acidolytic cleavage of an aliquot of 5a afforded C

Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5b).
This peptidyl resin was prepared from 6 following the above procedure using lauric acid as fatty acid. Acidolytic cleavage of an aliquot of 5b afforded C 11

Pro-Gln(Tr)-D-Tyr(Wang)-OAll (5c).
This peptidyl resin was prepared from 6 following the above procedure using palmitic acid as fatty acid. Acidolytic cleavage of an aliquot of 5c afforded C 15
Following TFA evaporation and diethyl ether extraction, the crude peptide was dissolved in H 2 O/CH 3 CN, lyophilized, analyzed by HPLC and characterized by mass spectrometry. (

Glu(O t Bu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OAll (7c).
Starting from 5c, this depsipeptidyl resin was prepared according to the general procedure described above. Acidolytic cleavage of an aliquot of
Following TFA evaporation and diethyl ether extraction, the crude peptide was dissolved in H 2 O/CH 3 CN, lyophilized, analyzed by HPLC and characterized by mass spectrometry.

Glu(O t Bu)-D-Val-Pro-Gln(Tr)-D-Tyr(Wang)-OH (8a). This peptidyl resin was obtained from 7a
following the above general procedure. Acidolytic cleavage of an aliquot of 8a afforded C
The resin was then washed with DMF (6×1 min), CH 2 Cl 2 (3×1 min) and diethyl ether (3×2 min), and dried in vacuo. The completion of the cyclization was checked with the Kaiser test. 45 The resulting peptide was cleaved from the resin with TFA/H 2 O/TIS (95:2.5:2.5) for 2 h at room temperature.
Following TFA evaporation and diethyl ether extraction, the crude peptide was dissolved in H 2 O/CH 3 CN, lyophilized and analyzed by HPLC and mass spectrometry.

Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx Spectral data of amino acids HPLC and ESI-MS of linear peptides, depsipeptides and lipodepsipeptides HPLC, ESI-MS, HRMS, 1D and 2D NMR of cyclic lipodepsipeptides