Naturally occuring depsipeptides exhibit interesting

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1 Simple Machine-Assisted Protocol for Solid-Phase Simple Synthesis Machine-Assisted of Depsipeptides Protocol for Solid-Phase Synthesis of Depsipeptides Jan Spengler, 1 Beate Koksch, 2 Fernando Albericio 1,3 1 Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, E Barcelona, Spain 2 Department of Chemistry and Biochemistry, Free University of Berlin, D Berlin, Germany 3 Department of Organic Chemistry, University of Barcelona, E Barcelona, Spain Received 31 July 2007; revised 15 September 2007; accepted 26 September 2007 Published online 5 October 2007 in Wiley InterScience ( DOI /bip ABSTRACT: A straightforward machine-assisted protocol for the synthesis of linear depsipeptides is reported. The synthesis was performed on a 433A Peptide Synthesizer (Applied Biosystems) using preprogrammed and optimized modules for Boc chemistry and without any need for hardware modification. The robustness of the protocol was demonstrated with 12 examples of 26-membered depsipeptides with single and multiple (up to 6) ester backbone substitutions. # 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: , Keywords: automated solid-phase depsipeptide synthesis; ester peptides; helical coiled coil peptides This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by ing the Biopolymers editorial office at biopolymers@wiley. com Correspondence to: Jan Spengler; jspengler@pcb.ub.es or Fernando Albericio; albericio@pcb.ub.es Contract grant sponsor: CICYT Contract grant number: BQU Contract grant sponsor: Generalitat de Catalunya Contract grant number: 2005SGR Contract grant sponsors: CIBER (nanomedicine), Institut for Research in Biomedicine, the Barcelona Science Park VC 2007 Wiley Periodicals, Inc. INTRODUCTION Naturally occuring depsipeptides exhibit interesting pharmacological activities. Typically, they are cyclic compounds comprised of 5 12 a-amino and a- hydroxy carboxylic acids, attached each other by amide and ester (depsi) bonds. 1 On the other hand, amide-to-ester substitutions in the backbone of peptides have proven to be a good tool for evaluating the role of backbone hydrogen bonds for protein structure formation. 2 The incorporation of an a-hydroxy acid into a peptide chain to give a depsipeptide can be advantageously performed on solid phase. However, ester bonds between the OH-function of an a-hydroxy acid and the next carboxylic acid often show limited stability for the basic conditions of Fmoc removal, which is the most convenient protecting group for a-amino function. 3 A low percentile of product loss in the course of each coupling cycle may be tolerable if the sequence to be synthesized is relatively short but it will impede the assembly of longer chains. Furthermore, the removal of the Fmoc group corresponding to the second residue after the hydroxyacid can lead to the formation of diketopiperazine (DKP). 4 Finally, repeated piperidine treatment throughout the synthesis process can also cause loss of chirality of the C-terminal component of the ester bond. 3b,5 In contrast, ester bonds are perfectly stable toward the conditions of Boc/benzyl solid phase peptide synthesis and other side-reactions are either minimized or suppressed. Consequently, the Boc/benzyl chemistry is the preferred strategy when many coupling cycles are to be performed after the formation of the ester bond. Especially, the in situ neutralization method is applied for the synthesis of long linear depsipeptides. This method takes advantage of the good secondary structure disrupting properties of TFA for protected peptide chains. 6 Aggregation occurs when the protonated a-ammonium peptide resin intermediate is neutralized. To avoid aggregation, the preacti- PeptideScience Volume 88 / Number 6 823

2 824 Spengler, Koksch, and Albericio vated amino acid is added together with a base to the protonated peptide resin. However, the machine-assisted variant of this protocol requires rather extensive hardware adaptations to the peptide synthesizer, such as removal of delivery line filters, replacement of tubes with larger diameters, exchange of transfer lines, and calibration of tube lengths among others. Most likely due to these complications, solidphase depsipeptide syntheses using the in situ neutralization method were performed manually. 7 In our investigation, several depsi analogues of the parent peptide had to be synthesized in order to study the effect of H-bond deletion on the backbone of a 26-membered helical coiled coil peptide. 8 A machine-assisted protocol would have been highly desirable for this purpose. The parent peptide, however, does not represent a difficult sequence. Furthermore, ester analogues of difficult sequences are in some cases easier to synthesize because the missing NH-protons of the ester bonds break the secondary structure formation which very often jeopardizes the synthetic process. This effect is employed in the so-called depsipeptide technique, which has been reported to overcome problems associated with the preparation of difficult sequences. 4,9 Thus, in order to perform an efficient depsipeptide synthesis, we have re-evaluated the solid-phase protocol and report now on an easily adaptable machine-assisted protocol that does not require hardware modifications. MATERIALS AND METHODS Chemicals and Apparatus Boc-Ala-OH, Boc-Glu(OBzl)-OH, Boc-Leu-OH, and Boc- Lys(2ClZ)-OH were purchased from Neosystem; leucic acid and lactic acid from Aldrich. H-OLys(2ClZ)-OH 10 and THP-OLeu-OH 3b were prepared according to reported procedures. PAM-resin preloaded with Leu (f ¼ 0.72 mmol/g) was purchased from Neo-MPS, N,N 0 -dicyclohexyl carbodiimide (DCC), 4-N,N 0 -dimethylaminopyridine (DMAP), and HOBt from Iris Biotech, trifluoroacetic acid (TFA, synthesis grade) from Fluorochem, N,N 0 -diisopropyl ethyl amine (DIEA) from Merck, dimethylformamide (DMF), dichloromethane (DCM) and diethyl ether were purchased from SDS. Solvents were used without prior drying. For cleavage, trifluoromethane sulfonic acid (TFMSA) and thioanisole from Fluka were used. Reverse phase analytical HPLCs were recorded with a HPLC Waters 1525, an automatic injector 717 plus, and an UV vis Waters 2487 detector at 220 nm. The Nucleosil C 18 column ( mm) was run with acetonitrile (0.036% TFA) and water (Millipore, 0.045% TFA). Data were managed with Breeze v3.20 software. Peptide purification was performed on the semipreparative HPLC model Waters 600 equipped with the automatic injector Waters 2700 and the detector UV vis Waters Samples were collected using the Waters Fraction Collector II. The Symmetry C 18 column ( mm) was run with acetonitrile (0.05% TFA) and water (Millipore, 0.1% TFA). Data were managed with MassLynux 3.5 software. For MALDI analysis, the samples were prepared with the ACH matrix (alpha-cyano-4-hydroxy cinnamic acid, Aldrich). The spectra were obtained with a 4700 Proteomics Analyzer (Applied Biosystems). Peptide Synthesis Peptide and depsipeptide syntheses were performed on a 433A Peptide Synthesizer (Applied Biosystems) using pre-programmed and optimized modules for Boc chemistry. In the following, we use the term reaction vessel to refer to the reactor into which the resin is placed. Activation vessel refers to the reactor where activation of amino acids takes place and with cartridge refers to the containers on which the amino acids are weighed. In a standard run, a HOBt solution is injected in the cartridge and the resulting solution is then transferred into the activation vessel, where a DCC solution is added. The solution with the amino-acid HOBt ester is filtered from the urea precipitate and transferred to the N-terminal deprotected resin (reaction vessel) where coupling proceeds under shaking (Scheme 1a). The coupling cycles are composed of modules. 11 The standard Boc chemistry for 0.10 mmol-synthesis (Boc/HOBt/DCC 0.10) involves preprogrammed modules a (activation of the amino acid in the activation vessel, 17 min), b (deprotection of the resin in the reaction vessel with TFA, 16 min), c (DCM-wash of the resin, 1.5 min), d (neutralization of the resin with DIEA and washing, 4.5 min), e (transfer of the amino-acid HOBt ester from the activation vessel to the reaction vessel, 5 min), f (coupling under shaking, 20 min), g (DMSO addition to the reaction vessel and capping of unreacted resin with Ac 2 O/DIEA, 16.5 min), and i (a 15 min wait without shaking,). Preprogrammed module G (DMSO and DIEA addition to the reaction vessel, no capping, 11.5 min) was also used later on in the described syntheses. Synthesis of Peptide H-L(1)EAKLKELEAKLAALEAK- LKELEAKL(26)-OH (pp) The parent peptide (pp) was synthesized on 140 mg (0.1 mmol) Boc-Leu-PAM-resin (f ¼ 0.72 mmol/g) by use of the previously described preprogrammed cycles (Table I). After complete synthesis, a 200 mg portion of resin was placed in a 50 ml round-bottom flask together with thioanisole (0.6 ml) as scavenger. At 08C, TFA (8 ml) followed by TFMSA (0.4 ml) were added with stirring. TFMSA allows the detachment of peptides from the PAM resin with the concomitant removal of all protecting groups except the tosyl of Arg. 12 After 2 h stirring, the ice-bath was renewed and the mixture was suspended by thoroughly stirring in dry diethyl ether (40 ml) for 30 min. The precipitate was retained in a filter of glass wool, washed with cold dry diethyl ether (20 ml), redissolved in 10% HOAc in water (30 ml) and lyophilized. Purification by semi-preparative HPLC and lyophilization gave the pure product (confirmed by HPLC and by MALDI, for yield, see Table 4). RESULTS AND DISCUSSION Differences Between Peptide and Depsipeptide Synthesis The difference between peptide and depsipeptide synthesis is that in the latter at least one ester bond has to be formed.

3 Machine-Assisted Protocol for the Synthesis of Depsipeptides 825 SCHEME 1 (a) Standard coupling of Boc-amino acids, (b) coupling of an a-hydroxy acid, and (c) O-acylation with a Boc-amino acid (or THP-hydroxy acid) on a resin. The couplings were performed with 10-fold excess of activated building block, respectively, to the resin functionalization. The distinct conditions for the formation and the particular chemical properties of esters require several modifications of the standard protocol of solid phase peptide synthesis. The low nucleophilic character of the a-hydroxy group permits the carboxy-activation of an a-hydroxy acid as HOBt-ester without the requirement of an a-hydroxy protecting group and can thus be coupled in the same way as Boc-amino acids (Scheme 1a). Consequently, neither deprotection nor capping need to be performed (Scheme 1b). 7 The acylation of the a-hydroxy function is a critical step. It requires more potent carboxyl-activating reagents than HOBt. Although a large repertoire of suitable activating reagents is known, for the purpose of a fully automated synthesis the activation should be optimally performed with a coupling reagent already present in the machine. The activation with N,N 0 -diisopropylcarbodiimide (DIC) in the presence of DMAP and a prolonged coupling time (1 h) is described and would meet this requirement (by replacing DIC for DCC). 7a An alternative is the generation of the symmetric anhydride and its coupling in the presence of DMAP (see the next paragraph and Scheme 1c). 13 A low level of epimerization is reported; 13 however, this low level can be tolerated if the resulting diastereomers are well separated by HPLC in the course of the final product purification. We found slightly better results with this method with respect to DCC/DMAP activation. If two hydroxy acids have to be incorporated consecutively, the first one can be coupled without an OH-protection scheme, but the second one has to be OH-protected. The THP group tolerates the OH-acylation conditions and can be cleaved under the same conditions as the Boc group with TFA (Scheme 1c). 3b Table I Programmed Synthesis for the Parent Peptide Using the Standard Protocol Cycle Repetitions Modules Remarks 1 1 c Washing and swelling of the unprotected resin H-Leu-PAM 2 1 aibcde Coupling of the first amino acid (K25) to the resin agcbcde Standard couplings of A24 until L fgc Final wash of the resin without N-terminal deprotection

4 826 Spengler, Koksch, and Albericio Table II Programmed Synthesis for L12k Cycle Repetitions Modules Remarks 1 1 c Washing and swelling of the unprotected resin H-Leu-PAM 2 1 aibcde Coupling of the first amino acid (K25) to the resin agcbcde Standard couplings of A24 until k12 (incl.) 16 1 AGcdeffffff Acylation with the symmetric anhydride of K11, prolonged coupling agcbcde Standard couplings without capping 27 1 fgc Final wash of the resin without N-terminal deprotection After acylation of the hydroxy acid, several chemical properties of the ester bond impinge on the further synthesis. First, the capping of unreacted functionalities with Ac 2 O/ DIEA is not recommended, because most probably an acyl transfer affects the ester bond. Second, another undesired side-reaction is the risk of DKP formation as it competes with the coupling of the third amino acid after the ester bond. The occurrence and degree of this reaction depend on the nature of the two amino acids which follow the ester bond. 14 For a combination which is prone to DKP formation, like -Pro-DXaa- (N-alkyl amino acids as Pro stabilize the amide bond cis-conformation and the presence of the sequence of both L and D residues stabilize the six-membered ring of the DKP), the interruption of the automated synthesis and manual performance of the in situ neutralization technique for the coupling of the third amino acid after the ester bond would be the best method. 15 In the following, we describe the detailed protocols for depsipeptide synthesis with a 433A Peptide Synthesizer (Applied Biosystems). This machine is often used for automated peptide synthesis. However, any other machine that offers the option to prolong coupling time, suppress HOBtaddition and acetylation (capping) would be adequate to perform depsipeptide synthesis in a fully automated manner. Synthesis of H-L(1)EAKLKELEAKk(12)AALEAKL- KELEAKL-OH (L12k) The synthesis of the depsi-analogue L12k (leucin 12 of the parent peptide is replaced with k, leucic acid; for nomenclature of depsipeptides see Ref. 2b). followed the standard protocol from the C-terminus until k12. During the cycle 15, leucic acid activated with HOBt (module a ) was transferred to the reaction vessel (module e ). The next cycle required modifications because the acylation step of the hydroxy group with K11 had to be performed. The K11 cartridge was filled with a double amount of amino acid (2 mmol) together with DMAP (10 mg). In module a the addition of HOBt was suppressed. For this purpose, the corresponding steps 17, 20, 21, 22, 23, and 24 of this module were deleted and the modified module a was saved as A. Upon addition of DCC in the activation vessel, the symmetric anhydride was formed and the urea precipitated. Module g for acetylation (capping) was replaced by G (see the section on peptide synthesizer). Module b for deprotection with TFA was not inserted. After modules cde six repetitions of the 20-min coupling module f (while the reaction vessel was shaking) were inserted. Thus, cycle 16 was programmed as: AGcdeffffff. The coupling of A11 to the N-terminal L1 followed the standard protocol, but without capping (module G instead of g ) (Table II). Synthesis of H-LEAKLKELEAKk(12)a(13)ALEAKL- KELEAKL-OH (L12kA13a) The synthesis of the depsi analogue L12kA13a (k ¼ leucic acid, a ¼ lactic acid) followed the previously described protocol from the C-terminus until a13. The acylation of a13 with k12 was performed with THP-protected leucic acid using the same cycle as for acylation with a Boc amino acid Table III Programmed Synthesis for L12kA13a Cycle Repetitions Modules Remarks 1 1 c Washing and swelling of the unprotected resin H-Leu-PAM 24 1 aibcde Coupling of the first amino acid (K25) to the resin agcbcde Standard couplings of A24 until a13 (incl.) 15 1 AGcdeffffff Acylation with the symmetric anhydride of THP-OLeu-OH (k12), prolonged coupling 16 1 AGcbcdeffffff THP-cleavage, acylation with the symmetric anhydride of K11, prolonged coupling agcbcde Standard couplings without capping 27 1 fgc Final wash of the resin without N-terminal deprotection

5 Machine-Assisted Protocol for the Synthesis of Depsipeptides 827 Table IV Compounds Synthesized Compound Purified Product in mg Obtained from 100 mg of Resin (yield in %) MALDI Found Exact Mass (Calculated) pp a 29 (40) M+H pp* b 25 (35) M+H L5k c 22 (30) M+H A10a d 16 (22) M+H K11j e 11 (15) M+H L12k f 22 (30) M+H L12k* g 16 (22) M+H A13a h 11 (15) M+H L22k i 21 (29) M+H L12kA13a j 7 (10) M+H L12kA14a k 8 (11) M+H L12kL15k l 18 (25) M+H L12kL15k* m 22 (30) M+H Hexa-k* n 5 (7) M+H Sequences (N-terminal NH 2, C-terminal CO 2 H): a LEAKLKELEAKLAALEAKLKELEAKL. b LEAKLKELEAKLKELEAKLKELEAKL. c LEAKkKELEAKLAALEAKLKELEAKL. d LEAKLKELEaKLAALEAKLKELEAKL. e LEAKLKELEAjLAALEAKLKELEAKL. f LEAKLKELEAKkAALEAKLKELEAKL. g LEAKLKELEAKkKELEAKLKELEAKL. h LEAKLKELEAKLaALEAKLKELEAKL. i LEAKLKELEAKLAALEAKLKEkEAKL. j LEAKLKELEAKkaALEAKLKELEAKL. k LEAKLKELEAKkAaLEAKLKELEAKL. l LEAKLKELEAKkAAkEAKLKELEAKL. m LEAKLKELEAKkKEkEAKLKELEAKL. n LEAKkKEkEAKkKEkEAKkKEkEAKL. as described before. The coupling cycle 16 involving K11 consequently contained THP-group cleavage with TFA (Table III). After completion of the synthesis, the depsipeptides were cleaved from the solid support and purified in the same manner as described for the parent peptide (pp). Table IV shows the yields of purified products. CONCLUSIONS In conclusion, we have demonstrated that linear depsipeptides can be synthesized in a straightforward machine-assisted manner with slight modifications in the programming of the peptide synthesizer. In all cases sufficient material for further investigation on structural properties was obtained. Therefore no further optimization, like control of reaction time during the acylation step or prevention of diketopiperazine formation as discussed above, was necessary. However, a decrease in yield respective to the all-amide pp, depending on the position and the number of ester substitutions, was observed. One reason for the decreasing yield can be the relative instability of the depsipeptide products, which contain free amino groups (side-chain of Lys). They decompose in aqueous solutions and probably also partially under the conditions of semi-preparative HPLC-separation (the depsipeptide L12kA13a decomposes in aqueous solutions within hours at room temperature). The authors thank Dr. Alberto Adeva (Technical and Scientific Services, Universitat de Barcelona). REFERENCES 1. (a) Hamada, Y.; Shioiri, T. Chem Rev 2005, 105, ; (b) Sarabia, F.; Chammaa, S.; Sánchez Ruiz, A.; Martín Ortiz, L.; López Herrera, F. J Curr Med Chem 2004, 11, (a) Yang, X.; Wang, M.; Fitzgerald, M. C. Bioorg Chem 2004, 32, ; (b) Powers, E. T.; Deechongkit, S.; Kelly J. W. Adv Protein Chem 2006, 72,

6 828 Spengler, Koksch, and Albericio 3. (a) Tulla-Puche, J.; Bayó-Puxan, N.; Moreno, J. A.; Francesch, A. M.; Cuevas, C.; Álvarez, M.; Albericio, F. J Am Chem Soc 2007, 129, ; (b) Kuisle, O.; Quiñoá, E.; Riguera R. J Org Chem 1999, 64, Coin, I.; Dölling, R.; Krause, E.; Bienert, M.; Beyermann, M.; Sferdean, C. D.; Carpino L. A. J Org Chem 2006, 71, Atherton, E.; Hardy, P. M.; Harris, D. E.; Matthews, B. H. In Peptides 1990, Proceedings of European Peptide Symposium, 21st; Giralt, E.; Andreu, D. Eds.; ESCOM Science Publications: Leiden, The Netherlands, 1991; p (a) Schnölzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent S. B. H. Int J Peptide Protein Res 1992, 40, ; (b) Schnölzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent S. B. H. Int J Peptide Protein Res Ther 2007, 13, (a) Blankenship, J. W.; Balambika, R.; Dawson P. E. Biochemistry 2002, 41, ; (b) Deechongkit, S.; Dawson, P. E.; Kelly J. W. J Am Chem Soc 2004, 126, ; (c) Beligere, G. S.; Dawson P. E. J Am Chem Soc 2000, 122, ; (d) Silinski, P.; Fitzgerald M. C. Biochemistry 2003, 42, Scheike, J. A.; Baldauf, C.; Spengler, J.; Albericio, F.; Pisabarro, M. T.; Koksch, B. Angew Chem Int Ed 2007, 46, (a) Sohma, Y.; Yoshiya, T.; Taniguchi, A.; Kimura, T.; Hayashi, Y, Kiso, Y. Biopolymers 2007, 88, ; (b) Tuchscherer, G.; Chandravarkar, A.,Camus, M.-S.; Berard, J.; Murat, K.; Schmid, A.; Mimna, R.; Lashuel, H. A.; Mutter, M. Biopolymers 2007, 88, (a) Deechongkit, S.; You, S.-L.; Kelly J. W. Org Lett 2004, 6, User Manual 433A Peptide Synthesizer, Applied Biosystems. 12. Jubilut, G. N.; Cilli, E. M.; Tominaga, M.; Miranda, A.; Okada, Y.; Nakaie, C. R. Chem Pharm Bull 2001, 49, Bianchi, E.; Steinkühler, C.; Taliani, M.; Urbani, A.; De Francesco, R.; Pessi, A. Anal Biochem 1996, 237, Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the Synthesis of Peptides and Proteins; CRC: Boca Raton, USA, Gairí, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron Lett 1990, 31,