Total synthesis of cis, cis -ceratospongamide

A total synthesis of cis, cis -ceratospongamide 1 was accomplished via 4+3 fragment condensation, macrolactamization and subsequent cyclodehydration. Macrolactamization of both linear peptides 4a & 4b produced the corresponding cyclopeptide 3 as a mixture of two conformational isomers ( cis, cis 3a and cis, trans 3b ). Further oxazoline ring closure furnished the cis, cis -ceratospongamide 1 which is identical to the natural product.


Introduction
A large number of natural products containing conformational constraints such as oxazole, thiazole and proline or N-alkyl amine have been isolated from bacteria, fungi, plants and marine organisms, over the last couple of decades.The cytotoxic and antineoplastic activities that they exhibit, as well as the possibility of their acting as metal chelating metabolites, have inspired a considerable number of structural and synthetic studies. 1 Recently, the isolation of two special conformationally stable cyclic heptapeptides, cis, cisand trans, trans-ceratospongamides (1, 2) have attracted our attentions. 2These two conformationally stable isomers were isolated from the marine red alga (Rhodophyta) ceratodictyon spongiosum by Gerwick and co-workers in the 2000.Both conformationally stable isomers of ceratospongamide contain two phenylalanine residues, one proline-thiazole amide unit, and one proline-isoleucine dipeptide further linked to an oxazoline segment.Amazingly, these two conformational isomers showed completely different bioactivities; the trans, transisomer exhibits potent inhibition of sPLA 2 expression in a cell-based model for anti-inflammation (ED 50 32 nM), whereas the cis, cis-isomer is inactive. 2Intrigued by the unusual molecular architecture of ceratospongamides, and the different bioactivity that the conformation caused, we have carried out the total synthetic studies.In an early communication, 3 we have reported our preliminary results towards the total synthesis of ceratospongamide. 4We now provide details of this synthesis involving two alternative macrocyclization protocols for the construction of the penultimate cyclopeptide 3, establishment of the conformational behavior of cyclopeptide 3, and cyclodehydration to furnish the final natural product.

Results and Discussion
The trans-oxazoline ring is an acid-sensitive and readily opened moiety. 5Furthermore, the propensity of the stereogenic centre adjacent to the oxazoline ring undergoes facile epimerization suggested that the oxazoline ring closure should be delayed until the final step. 6This consideration led us to target the penultimate cyclopeptide 3 as the most advanced intermediate and installation of the oxazoline ring was anticipated to be performed in the final step.At the outset of this work, we also proposed to prepare cyclopeptide 3 by macrocyclization of the linear peptides 4a and 4b (Scheme 1).This macrocyclization processes were planned as we believed these disconnections would minimize epimerization and steric hindrance at the C-terminus.In addition, the hydrogen-bond inducing turn-forming effect might occur in both linear precursors 4a and 4b, which should facilitate the macrocyclization process. 1,7Both peptides 4a and 4b can be prepared through a [4+3] fragment condensation of tetrapeptide 5 and a thiazole containing tripeptide 6, which can be further disconnected to give 2-(trimethylsilyl)ethyl carbamate (Teoc) protected phenylalanine methyl ester 7 and the thiazole methyl ester 8.

Scheme 1
Synthesis of the thiazole methyl ester 8 could be achieved by oxidation of the corresponding thiazoline to thiazole.Since the amino acid-derived thiazolines have propensity for epimerization under either basic or acidic conditions, so any cyclodehydration reaction leading to thiazoline and its further oxidation reaction to thiazole must be carried out under near-neutral conditions.Based on our previous experience obtained in the synthesis of amino acid-derived thiazolines and thiazoles, 8 we decided to employ the Wipf procedure for the preparation of Boc-L-Proline-derived thiazole 8 (Scheme 2).Thus, coupling of the the L-Boc-proline and L-serine methyl ester afforded dipeptide 12 in 89% yield.The hydroxy group in 12 was protected as its tertbutyldimethylsilyl ether affording dipeptide 13 which was smoothly converted into thioamide 14 with Lawesson's reagent. 9Subsequent removal of the silicon protecting group and reaction with Burgess reagent yielded thiazoline 16.Oxidation of thiazoline 16 by use of actived γ-manganese dioxide produced the thiazole derivative 8 in 44% overall yield. 10 Following removal of the Boc group in thiazole segment 8 using TFA in dichloromethane, this residue was then condensed with Teoc-L-phenylalanine (7) in the presence of EDCI and HOBt to provide tripeptide 6 in 84% yield.

Scheme 2
Synthesis of the fragment 5 is shown in Scheme 3. Dipeptide fragment 9 is easily prepared in good quantity according to Wipf's procedure. 11Thus, Cbz-protected L-isoleucine was condensed with L-threonine methyl ester in the presence of DCC and HOBt gave dipeptide 19 in 89% yield.This dipeptide was then treated with Burgess's reagent provided oxazoline 20 in 78% yield.Subsequent mild hydrolysis of the resulting peptidyl oxazoline with 0.3 M HCl gave the O-acyl amine which underwent in situ acyl migration to afford the desired dipeptide 9 in 51% yield over two-steps.The Cbz protecting group in this dipeptide was removed by hydrogenolysis over 10% Pd/C in methanol to give the corresponding amine 21 ready for use in next step.The complementary coupling partner 22 was prepared by condensing of L-Boc-phenylalanine with Lproline methyl ester using DCC and HOBt in 90% yield.Saponification of dipeptide 22 with lithium hydroxide furnished the necessary carboxylic acid 23 in 98% yield.The coupling of compounds 21 and 23 was then achieved using EDCI and HOBt to provide tetrapeptide 5 in 96% yield.With tetrapeptide 5 and tripeptide 6 in hand, we have explored two complementary routes for the assembly of linear heptapeptides (4a and 4b) to the macrocycle 3 (Scheme 4).After conversion of tripeptide 6 into its trifluoacetate salt 24 and tetrapeptide 5 into the corresponding carboxylic acid 25, coupling of these two segments 24 and 25 using EDCI and HOAt was accomplished to give linear peptide 4a in 90% yield. 12The linear heptapeptide 4a was then saponified using lithium hydroxide and acidolytic removal of the Boc group followed using TFA/CH 2 Cl 2 to provide the linear precursor.Macrocyclization with HATU in DMF under dilute conditions afforded the cyclopeptide 3 in 46% overall yield as a mixture of two conformational isomers (ratio: 3a:3b=1:1.3) 13An alternative route towards the synthesis of cyclopeptide 3 from linear heptapeptide 4b was also investigated (Scheme 4).Thus, segment condensation of the acid derived from tripeptide 6 and the corresponding amine derived from tetrapeptide 5 provided linear heptapeptide 4b in excellent yield.Removal of the methyl ester functionality by lithium hydroxide, and the Teoc protective group by TFA in 4b, gave the corresponding linear precursor which was then cyclized with FDPP 14 in DMF under dilute conditions to yield cyclopeptide 3 in poor yield (10%).Interestingly, the ratio of both conformational isomers (3a:3b) derived from this macrolactamization reaction was same as that obtained from the cyclization of heptapeptide 4a.Furthermore, no improvements were achieved by employing other macrocyclization reagents such as HBTU 15 or PyBOP. 16Macrocyclization of precursor 4a proceeded in more than twice the yield as obtained for the cyclization of precursoe 4b.Obviously, cyclization depends on the propensity of the linear precursor to adopt a conformation similar to the transition state required for cyclization. 18Therefore the yield of the macrocyclization step might vary dramatically with respect to the cyclization precursors.

Scheme 4
Finally, treatment of the pivotal penultimate cyclopeptide 3 with Deoxo-Flour 17 resulted in facile cyclodehydration to the oxazoline ring producing cis, cis-ceratospongamide structure 1 in 89% yield.This indicates the oxazoline formation is the conformer-determining step.The rigidity of the oxazoline ring presents in the macrocycle can enhance conformational stability (vide infra).The synthetic cyclopeptide showed 1 H and 13 C NMR spectra which were superimposable on those recorded for naturally derived cis, cis-ceratospongamide 1.
In order to understand why the cyclodehydration of the mixture of two conformational isomers (3a and 3b) produced cis, cis-ceratospongamide 1, we decided to detect the exact conformation of both isomers 3a and 3b.Extensive one-and two-dimensional NMR studies on the conformation of cyclopeptide 3 in chloroform indicated that there are two major conformational isomers (3a and 3b) present in the solution.The detailed NMR data, obtained from C-H COSY, H-H COSY, HMBC and ROESY experiments, allow us to assign the exact chemical shift of both conformational isomers 3a and 3b.The ratio of these two conformational isomers (3a:3b=1:1.3) was determined by integration of the NH proton of Phe-1 residue of each conformational isomer.It is known that the cis/trans conformational difference of the proline amide bonds correlates with differential values between proline β and γ carbons (∆δ βγ ).A characteristic feature of the cis X-Pro system is the large difference in chemical shift between the β and γ carbons (>8ppm) compared to the corresponding trans X-Pro isomers, where the difference is less (<6ppm). 19As can be seen from the Table 1, in conformational isomer 3a, ∆δ βγ of Pro-2 and Pro-1 are 12.8 and 9.4 ppm, respectively.Hence, both proline amides in 3a are cis.On the other hand, in conformational isomer 3b, ∆δ βγ of Pro-2 and Pro-1 are 11.8 and 3.8 ppm, respectively.This indicated that the proline amides in 3b are cis (Pro-2) and trans (Pro-1), respectively.These conformational assignments are further supported from the ROESY spectroscopic data (Figure 2), which showed a strong correlation between the α-protons of Pro-2/Phe-1 as well as Pro-1/Phe-2 in conformational isomer 3a.This is in agreement with the conclusion that 3a is the cis, cisisomer.On the other hand, the ROESY data showed a strong correlation between the α-protons of the Pro-2/Phe-1, but no such correlations between the αprotons of the Pro-1/Phe-2 in conformational isomer 3b.Therefore, 3b appears to be the cis, trans-isomer.Table 1. 13 C NMR spectral data (in ppm) for proline carbon and alpha carbons of amino acid residue of cyclopeptide 3a and 3b  Since the cyclization of both linear peptides 4a and 4b produced cyclopeptide 3 with the same ratio of conformational isomers (3a & 3b), this suggests that there is an inter-conversion between both conformational isomers 3a and 3b.This result is in accord with those obtained by the Deng and Taunton, 4a who observed that conformer interconversion of cyclopeptide 3 occurs at room temperature. 20Cyclodehydration of cyclopeptide 3 produced cis, cis ceratospongamide in excellent yield deserves further comments.Although the aforementioned NMR studies indicated there are two conformational isomers (3a & 3b) present in solution, the cis, cis ceratospongamide was derived from the corresponding cis, cis conformational isomer 3a.Since the conformer interconversion occurs at room temperature, conformational isomer 3b must equilibrate to the corresponding isomer 3a prior to taking part in the oxazoline formation process.Furthermore, the cyclodehydration reagent used for the oxazoline formation may interrupt the hydrogen bond associated with the Phe-2 in conformational isomer 3b, which would also facilitate the intramolecular trans/cis isomerization process.

Conclusions
A total synthesis of cis, cis-ceratospongamide 1 was accomplished via [4+3] fragment condensation, macrolactamization and subsequent cyclodehydration.Our results indicated that the yield of the macrocyclization step varied dramatically with respect to the cyclization precursors.Extensive NMR studies on cyclopeptide 3 revealed that there are two major conformational isomers present in the solution and the conformer interconversion facilitated the formation of cis, cis-ceratospongamide.

Experimental Section
General Procedures.All starting material and reagents were obtained from commercial sources and were used without further purification.Solvents were dried by distillation from sodium benzophenone ketyl (THF, Et 2 O) or CaH 2 (CH 2 Cl 2 ) under N 2 .Air-and/or moisture-sensitive reactions were performed in oven-dried (110 o C) glassware under N 2 .TLC was carried out on E. Merck pre-coated silica gel 60 GF 254 plates.Chromatography refers to flash chromatography on 230-400-mesh silica gel.Melting points are uncorrected.Specific rotations were measured with a Perkin Elmer 341 polarimeter at ambient temperature using 0.9998 dm cell with 1ml capacity.Infrared (IR) spectra were recorded with a Nicolet 5DXB FT-IR spectrometer. 1 H NMR and 13 C NMR spectra were recorded using either Bruker AC-300 MHz or Bruker AC-500MHz in CDCl 3 solution with tetramethylsilane as an internal standard.Electron impact (EI) mass spectra (HRMS and MS) were obtained with a Finnegan MAT 95.

N-Boc-L-Pro(C=S)-L-(OTBS)-Ser methyl ester (14).
To compound 13 (4.76 g, 11.423 mmol) in benzene (30 mL), a solution of Lawesson's reagent in benzene (30 mL) was added.After the reaction mixture was under refluxed for 6 hours, the solvent was evaporated and the residue was purified by flash chromatography on silica gel to give the title compound 14 (3.21 g, 65% yield).

N-Boc-L-Phe-L-Pro-L-Ile-L-aThr methyl ester (5). N-Boc-L-Phe-L-Pro methyl ester (22)
(330 mg, 1 mmol) was dissolved into the solution of lithium hydroxide (220 mg, 5 mmol) in THF (2 mL), methanol (2 mL) and H 2 O (1 mL).The reaction was followed by tlc until the starting material was consumed.The reaction solvent was then neutralized with diluted HCl solution and extracted with ethyl acetate (3x30 mL).The combined organic layers was dried and evaporated to give free acid (23) for use in next step with out further purification.N-Cbz-L-Ile-L-aThr methyl ester (9) (250 mg, 0.65 mmol) in methanol (10 mL) was added 10%Pd/C (10 mg).Hydrogen (1 atm) was then applied to the reaction system.The reaction mixture was stirred under hydrogen (1 atm) for 4 hours and then the Pd/C was filtered through a glass wool plug.After removing the solvent under reduced pressure, the residue was combined with acid 23 in THF (20 ml) at 0 o C and HOBt (135 mg, 1 mmol), EDCI (260 mg, 1.35 mmol) and NMM (0.5 mL, 4.55 mmol) were added.The reaction mixture was stirred at 0 o C for 1 hour and then at room temperature for 22 hours.The solvent was dried (Na 2 SO 4 ) and evaporated.The residue was purified by flash chromatography on silica gel to give title compound 5 (366 mg, 96% yield).[α] D 20 -48.04 (c 5.05, CHCl 3 );

N-Boc-L-Phe-L-Pro-L-Ile-L-aThr
Method two: Prepared from 4b.L-Boc-Phe-L-Pro-Thz-L-Phe-L-Pro-L-Ile-L-aThr methyl ester (4b) (30mg, 0.032 mmol) in THF (1 ml) was added a solution of lithium hydroxide (6.1 mg, 0.256 mmol), in 2:1 methanol/H 2 O (1.5 ml) and the solution mixture was stirred at room temperature for 4 hours, The solution was neutralized with diluted HCl solution (1 M) and then extracted by ethyl acetate (3x10 ml).The combined organic layers were dried (Na 2 SO 4 ) and concentrated.The residue was re-dissolved in 1:1 TFA/CH 2 Cl 2 (2 ml) at 0 o C and stirred at room temperature for 2 hours.The solvent was removed under reduced pressure and the residue was redissolved in CH 2 Cl 2 and reconcentrated repeatedly to remove excess TFA.The residue was redissolved in DMF (10 ml), and NMM (0.017 ml, 0.153 mmol) was added, followed by HATU (56 mg, 0.147 mmol).The reaction mixture was stirred at room temperature for 80 hours.The solvent was evaporated and the residue was purified by flash chromatography on silica gel to give cyclopeptides 3 (2.5 mg, 10% yield) as a mixture of conformational isomers (3a:3b=1:1.3
(127.6mg, 0.216 mmol) in THF (2 mL) was added a solution of lithium hydroxide (48.0 mg, 2.0 mmol) in 2:1 methanol/H 2 O (3 mL) and the reaction mixture was stirred at room temperature for 24 hours, acidified with 1M HCl at 0 o C, and extracted with ethyl acetate (3 x 30 mL).The organic extracts were dried (Na 2 SO 4 ) and concentrated to obtain the product acid, which was used without further purification.Cl 2 (2 mL) at 0 o C and stirred at room temperature for 2 hours.The solvent was removed under reduced pressure and the residue was redissolved in CH 2 Cl 2 and reconcentrated repeatedly to remove excess TFA.The dry sample was stored under high vacuum to give the TFA salt which was used without further purification.This salt was combined with the free acid derived from 5 in THF (10 mL) at 0 o C, and HOAt (123.9 mg, 0.91 mmol), EDCI (171.3 mg, 0.890 mmol) and NMM (0.35 mL, 2.273 mmol) were added.The reaction mixture was stirred at 0 o C for 1 hour and then stirred at room temperature for further 36 hours.Following dilution with CH 2 Cl 2 (20 mL) the solution was washed with saturated sodium hydrogen carbonate (5 mL), ammonium chloride (5 mL), brine (5 mL) and dried over sodium sulfate.Removal of the solvent in vacuo followed by chromatography on silica gel afforded the title compound 4a (174mg, 90% yield).
Cl 2 (2 mL) at 0 o C and stirred at room temperature for 2 hours.The solvent was removed under reduced pressure and the residue was redissolved in CH 2 Cl 2 and reconcentrated repeatedly to remove excess TFA.The dry sample was stored under high vacuum to give the TFA salt which was used without further purification.This salt was combined with the free acid derived from 6 in THF (10 mL) at 0 o C, and HOAt (0.188 g, 1.38 mmol), EDCI (250 mg, 1.3 mmol) and NMM (0.73 ml, 6.6 mmol) were added.The reaction mixture was stirred at 0 o C for 1 hour and then stirred at room temperature for further 36 hours.Following dilution with CH 2 Cl 2 (20 mL) the solution was washed with saturated sodium hydrogen carbonate (5 mL), ammonium chloride (5 mL), brine (5 mL) and dried over sodium sulfate.Removal of the solvent in vacuo followed by chromatography on silica gel afforded the title compound 4b (228 mg, 88% yield).