Ala analogues of the cyclolinopeptide A

Ala Analogues of the Cyclolinopeptide A

GIANCARLO ZANOTTI, Centro di Chimica del Fannaco del CNR, Dtpartimento degli Studi Farmaceutici dePUniversith “La Sapienza,”

R o m ; TEODORICO TANCREDI, ICMIB del CNR, Arco Felice, Napoli; FILOMENA ROSSI, E?TORE BENEDE’ITI,

CARL0 PEDONE,* and PIERANDREA TEMUSSI, Diparthento di Chimica, Via Mezzocannone, 4 (80134) Napoli, I t a h

Synopsis

Analogues of cyclolinopeptide A, due to the replacement of each amino acid in the Prd-fig- Phe3-Phe4 sequences with an LAla residue, were synthesized by classical method in solution. Mixed anhydride and N,N-dicyclohexylcarbodiimide coupling methods have been used for the synthesis of both linear and cyclic peptides. The produds were characterized by Rf values and uv spectra, as well as by fast atom bombardment spectroscopy.

‘H-nmr studies on [Ah2] analogues are also reported. Preliminary data in CDCl, solution, at low temperature, seems more promising.

INTRODUCTION

Cyclolinopeptide A, an homodetic cyclic nonapeptide from linseed, belongs to a class of molecules that shows cytoprotective action against several poisonous substance^.^

While a considerable amount of structural and conformational information has been obtained by nmr and x-ray analysis on several peptides of this class, little is known on the conformational properties of cyclolinopeptide A. It has been suggested that the cytoprotective activity could be related to the presence of two adjacent aromatic amino acid residues, and that it could be increased if an pro or a D-PIU precedes or follows, respectively, the two aromatic residues. The conformation of these sequences also plays an impor- tant role in the activity; recent conformational analysis by x-ray difiaction2 and nmr spectroscopy has demonstrated for cyclolinopeptide A that the solid state structure is conserved at low temperature in CDCl,, while in other solvents an equilibrium among several conformations exists.

Further investigation on the conformational aspects of cytoprotective peptides is necessary to clear the structure-activity relationships and the role that the Pd-Pro2-Phe3-Phe4 sequence could play. To this purpose, a series of cyclolinopeptide A analogues has been synthe-

sized. These analogues differ from the natural product because each one of the four residues in the Pro-Pro-Phe-Phe sequence has been replaced with an L-Ala residue.

Here we report the synthesis of the analogues, carried out by conventional solution-phase procedures, and the preliminary conformational results in solution by nmr spectroscopy on the [Ala2] analogue.

*To whom correspondence should be addressed.

Biopolymers, Vol. 28,371-383 (1969) Q 1989 John Wiley & Sons, Inc. CCC oooS-3!525/89/010371-13$04.~

372 ZANOWI ET AL.

MATERIALS AND METHODS Analytical grade solvents were obtained and distilled prior to use. Amino

acids were purchased from Novabidem-Inalco Co. Precoated silica gel G-60 plates (Merck, F-254) were used for thin layer

chromatography (tlc). Solvent mixtures are indicated in the respective sec- tions. For the detection of the product, ninidrin reaction and iodine vapor staining were used. All the linear and cyclic peptides gave satisfactory amino acid analysis.

Fast atom bombardment (FAB) spectra were determined with a K r a b MS-50 double-focusing apparatus equipped with M-SCAN FAB source and FAB GUN (90 KeV, 20 PA). All nmr experiments have been performed by a Bruker AM-400 spectrome-

ter, equipped with a 400-MHz superconducting magnet and an Aspect 300 computer. One-dimensional (1D) spectra have been acquired in quadrature mode using 16K data points. Apodization, by using a Gaussian multiplication functi0n,~1~ and zero filling to 32K prior to transformation have been used throughout. Phase-sensitive two-dimemional spectra rotating frame nuclear Overhauser enhancement spectroscopy (ROESY),' double quantum filtered correlated spectroscopy (COSY DQF), and homonuclear Hartman-Hahn (HOHAHA)' have been acquired with quadrature detection in Fl.8 ROSEY and HOHAHA experiments have been performed using the low power trans- mitter of the spectrometer. The 90" pulse width was 70 ms and the sample concentration was always 6 mM.

Deuterated dimethyl sulfoxide (DMSO-d,) was purchased from C. Erba, Italy. Chemical shift values are in parts per million from internal tetramethyl- silane.

EXPERIMENTAL

Synthesis of [Ala'] Cyclolinopeptide A

2- Val-pro-OtBU (1) To a solution of 12.5 g of Z-Val(50 mmol) in 100 mL CH,Cl, were added at

O°C under stirring 10.3 g N,N-dicyclohexylcarbodijmide (DCCI; 50 mmol) and 8.55 g of Pro-tert-butyl ester (OtBu; 50 mmol) dissolved in 50 mL CH,Cl,. After stirring 1 h at 0°C and overnight at mom temperature, the solution was cleared from dicyclohexylurea by filtration and the filtrate extracted several times with aqueous 0.5M KHSO,, saturated NaHCO, and water, dried with Na,SO,, and evaporated to give 20 g of (1) (100%) as an oil pure on tlc. R, 0.8 in ether-petroleum ether (7 : 3).

Z-Leu- VaLh-OtBu (2) Twenty grams of (1) (50 mmol) were hydrogenated in 200 mL CH30H over

1.5 g of 10% Pd on charcoal. Hydrogenation was completed after 4.5 h. After removal of the catalyst, the filtrate was evaporated and the residue, dissolved in 60 mL CH,Cl,, was added at 0°C under stirring to the solution of 13.2 g of Z-Leu (50 mmol) and 10.3 g DCCI (50 mmol) in 100 mL CH2C1,. After 1 h at

ALA ANALOGUES OF CYCLOLINOPEPTIDE A 373

0°C and overnight at mom temperature, the reaction mixture was worked up as described above to give 22.7 g (88%) of (2) as an oil pure on tlc. R , 0.7 in ether-petroleum ether (6 : 4).

Z-Ik-Leu-Val-Pro-OtBu (3)

The amount of 22.7 g of (2) (44 -01) was hydrogenated over 1.1 g of 10% Pd on charcoal for 3 h at room temperature. After removal of the catalyst by filtration and evaporation of the filtrate, the residue, dissolved in 50 mL CH,Cl,, was added at 0°C under stirring to the solution of 11.6 g of Z-Ile (44 mmol) and 9.06 g DCCI (44 mmol) in 70 mL CH2C1,. After 1 h at 0°C and overnight at room temperature, the mixture was worked up as described in the preceding paragraphs. The crude residue was chromatographed in ether on a 5 X 100 cm column of silica gel, obtaining 4.9 g of (3) (17.5%) as a foam pure on tlc. R, 0.7 in CHC1,-CH,OH (95 : 5).

Z-Leu-Ik-OtBU (4)

The dipeptide (4) was prepared in a way analogous to that described for (1) from 7.07 g of Z-Leu (26.7 mmol), 5.5 g DCCI (26.7 mmol), and 5 g of Ile-OtBu (26.7 mmol) in 100 mL CH2C12, obtaining 9.5 g of (4) (82%) as an oil pure on tlc. R, 0.6 in ether-petroleum ether (6 : 4).

Z-Leu-Ik-Ik-Leu- Val-Pro-OtBu (5)

The OtBu group was removed from (4) by treatment with 1 N HCl in acetic acid for 3 h a t mom temperature. The solution was evaporated in good vacuo and the residue dried over KOH pellets. To the solution of 1.3 g Z-Leu-Ile (3.45 mmol) in 40 mL CH2C1, were added,

at - 10°C, 0.47 g isobutyloxycarbonylchloride (iBCC1; 3.45 mmol) and 0.38 g N-methylmorpholine (NMM; 3.76 -01). After 10 min the solution in 30 mL CH2Cl, of the amino component, which has been prepared as follows, was added: 2.17 g of (3) (3.45) mmol) were hydrogenated in 50 mL CH,OH for 3 h over 0.3 g of 10% Pd on charcoal. The catalyst was removed by filtration, the filtrate evaporated in vacuo, and the residue, dissolved in CH2C1,, added to the solution of the mixed anhydride prepared as described above. After 3 h at room temperature the reaction mixture was worked up as described in the preceding paragraphs. The crude residue was chromatographed in CHC1,- CH,OH (6 : 4) on a 2.5 X 50 cm column of silica gel yielding 1.73 g of (5) (58%) as a foam pure on tlc. R, 0.4 in CHC1,-CH,OH (96 : 4).

Z-Ph-Ph-OtBu (6)

In a way analogous to that deacribed for (1) the dipeptide ester was prepared from 3 g of Z-Phe (10 mmol), 2.2 g of Phe-OtBu (10 mmol) and 2.06 g of DCCI (10 mmol) in 100 mL CH,Cl,, obtaining 4.5 g of (6) (90%) as a foam pure on tlc. R, 0.7 in ether-petroleum ether (7 : 3).

Z-Ah-Ph-Ph-OtBu (7) The amount of 2.65 g of (6) (5.28 mmol) was hydrogenated in 70 mL CH,OH

over 0.5 g of 10% Pd on charcoal. After 6 h the hydrogenation was completed;

374 ZANOTTI ET AL.

the catalyst was removed by filtration, the filtrate was evaporated, and the residue, dissolved in 30 mL CH2C12, was added at 0°C under stirring to the solution of 1.17 g of Z-Ala (5.28 mmol) and 1.08 g DCCI (5.28 mmol) in 50 mL CH2C12. After 1 h a t 0°C and overnight at room temperature, the reaction mixture, worked up as described above, gave a residue that was chromatographed in ethyl acetate on a 5 X 50 cm column of silica gel. The amount of 1.7 g of (7) (56%), pure on tlc, was obtained as a foam. R, 0.6 in CHC13-CH3OH (95 : 5).

Z-Leu-Ilk-Ilk-Leu- Val-Pro-Ah-Phe-Phe-OtBu (8)

The amount of 1.61 g of (5) (1.9 mmol) was reacted in 10 mL of 1 N HCl in acetic acid a t room temperature for 3 h to remove the OtBu group. The solution was evaporated to dryness and the residue, dried in vacuo over KOH pellets, was dissolved in 50 mL CH2C12 and reacted a t - 10°C under stirring with 0.26 g iBCCl(l.9 -01) and 0.23 g NMM (2.27 mmol). After 10 min the precooled solution in 30 mL CH2C12 of the amino component, which has been prepared as follows, was added: 1.09 g of (7) (1.9 mmol) were hydrogenated in 50 mL CH,OH over 0.3 g of 10% Pd on charcoal. After 4 h the hydrogenation was completed, and the catalyst was removed by filtration, the filtrate evaporated to dryness, and the residue, dissolved in CH2C12, was added to the solution of the mixed anhydride described above. After 3 h at room temperature the reaction mixture was worked up as described in the preceding paragraphs and the residue chromatographed by eluting in CHC1,-CH,OH (95 : 5) on a 2.5 X 50 cm column of silica gel, obtaining 1.680 g of (8) (72%) as a foam, which gave a single spot on tlc. R, 0.45 in CHC1,-CH30H (95 : 5).

Mass spectral measurement (FAB) of (M-H)+ was 1222, as expected. The amino acid analysis was satisfactory.

Cy~lo(Pro'-Ah~-Phe~-Phe~-Leu~-I~~-Ilk~-Leu~-Val9) (9)

&protection of (8). The amount of 1.670 g of (8) (1.5) mmol) was hydrogenated in 100 mL of acetic acid over 0.3 g of 10% Pd on charcoal to remove the Z group. The hydrogenation was completed after 6 h. After removal of the catalyst, the filtrate was evaporated and the residue was reacted with 20 mL of trifluoroacetic acid (TFA) for 3 h for the removal of the OtBu group. The solution was evaporated to dryness and the residue was tritured with ether. The precipitate was filtered and dried in vacuo over KOH pellets to give 1.260 g of TFA salt (70%) pure on tlc. R, 0.85 n-butanol-acetic acid-water (4 : 1 : 1).

Cyclization

The amount of 1.14 g of TFA salt (10 mmol) was dissolved in 130 mL dimethylformamide (DMF) and 100 mL tetrahydrofurane (THF), and to the solution was added, at -15"C, 0.136 g iBCCl (10 mmol). After 10 min of stirring a t - 15°C a precooled solution of 0.2 mL NMM in 200 mL DMF and 100 mL THF was added. After standing 1 h a t 0°C and overnight a t room temperature, the solvent was evaporated under vacuum and the residue, dissolved in CHCl,, was extracted several times with aqueous 0.5M KHSO,,


saturated NaHCO, and water, dried with Na,SO,, and evaporated. The crude residue was chromatographed on 100 g of silica gel on a 2.5 X 50 cm column eluting with CHCl,-CH,OH (97 : 3). Fractions (15 mL) 27-30 afforded 80 mg (8%) of (9) pure on tlc. Rf 0.4 in CHCl,CH,OH (95 : 5). Mass spectral measurement (FAB) of (M-H)+ was 1014, as expected. The

amino acid analysis was satisfactory.

Synthesis of [Ald] cyclolinopeptide A

Z-Val-Ah-OtBu (10)

To a solution of 3.9 g of Z -Val (15 mmol) in 50 mL CH2C1 were added, at 0°C under stirring, 3.09 g DCCI (15 mmol) and 2.17 g of Ala-OtBu (15 mmol) in 20 mL CH2Cl,. After stirring 1 h at 0°C and overnight at mom temperature, the solution was worked up as previously d d b e d to give 4.8 g of (10) (85%) as an oil pure on tlc. R, 0.5 in ether-petroleum ether (6 : 4).

Z-Leu-Val-Ah-OtBu (11)

The amount of 3.8 g of (10) (10 -01) was hydrogenated in 50 mL CH,OH over 0.3 g of 10% Pd on charcoal. Hydrogenation was completed after 3 h. After removal of the catalyst, the filtrate was evaporated and the residue, dissolved in 20 mL CH2C12, was added, at 0°C under stirring, to the solution

After 1 h at 0°C and overnight at room temperature, the reaction mixture was worked up as described above to give 4.8 g of (11) (100%) as an oil pure on tlc. R, 0.7 in ether-petroleum ether (8 : 2).

Of 2.65 g Of Z-Leu (10 -01) and 2.06 g DCCI (10 -01) in 20 mL CH,C12.

Z-Ik-Leu- Val-Ah-OtBu (12)

The amount of 4.9 g of (11) (10 mmol) was hydrogenated over 0.3 g of 10% Pd on charcoal for 3 h at room temperature. After removal of the catalyst by filtration and evaporation of the filtrate, the residue, dissolved in 20 mL CH,Cl,, was added, at 0°C under stirring, to the solution of 2.65 g of Z-Ile (10 mmol) and 2.06 g DCCI (10 mmol) in 20 mL CH,Cl,. After 1 h at 0°C and overnight at room temperature, the mixture was worked up as described in the preceding paragraphs. The crude residue was chromatographed in ethyl acetate on a 5 X 70 cm column of silica gel, obtaining 6.1 g of (12) (100%) as a foam pure on tlc. R, 0.7 in CHCl,-CH,OH (97 : 3).

Z-Leu-Ik-&Leu- Val-Ah-OtBu (13)

The OtBu group was removed from (4), as previously described. To the solution of 3.8 g of Z-Leu-Ile (10 -01) in 100 mL CH,Cl, was added at, - 1O"C, 1.36 g iBCCl(l0 -01) and 1.2 g NMM (11 -01). After 10 min the solution in 30 mL CH,Cl, of the amino component, which has been prepared as follows, was added: 6.0 g of (12) (10 mmol) were hydrogenated in 150 mL CH,OH for 3 h over 0.9 R of 10% Pd on charcoal. The catalyst was removed by

376 ZANO'M'I ET AL.

atration, the filtrate evaporated in vacuo and the residue, dissolved in CH,Cl,, added to the solution of the mixed anhydride prepared as described above. After 3 h at room temperature the reaction mixture was worked up as described in the preceding paragraphs. The crude residue was recrystallized by methanol, yielding 4.8 g of (13) (58%) as a gel pure on tlc. R, 0.5 in CHCl,-CH,OH (96 : 4).

Z-Pro-Phe-Phe-OtBu (14)

The amount of 2.65 g of (6) (5.28 mmol) was hydrogenated in 70 mL CH30H over 0.5 g of 10% Pd on charcoal. After 6 h the hydrogenation was completed; the catalyst was removed by filtration, the filtrate was evaporated, and the residue, dissolved in 30 mL CH,Cl,, was added at 0°C under stirring to the solution of 1.3 g of 2-Pro (5.28 mmol) and 1.08 g DCCI (5.28 -01) in 50 mL CH,Cl,. After 1 h at 0°C and overnight at room temperature, the reaction mixture, worked up as described above, gave a residue that was chromatographed in ethyl acetate on a 5 X 50 an column of silica gel. Two grams of (14) (62%), pure on tlc, were obtained as a foam. R, 0.3 in ether-petroleum ether (8 : 2).

Z-Leu-Ile-Ile-Leu- Val-Ah-Pro-Phe-Phe-OtBu (15)

The amount of 2.4 g of (13) (2.89 mmol) was reacted in 20 mL of 1 N HCl in acetic acid at room temperature to remove the OtBu groups previously described. The residue, dissolved in 100 mL CH,Cl,, was reacted at -10°C under stirring with 0.40 g iBCCl (2.89 mmol) and 0.352 g NMM (3.46 -01). After 10 min the precooled solution in 60 mL CH,Cl, of the amino component, which has been prepared as follows, was added: 1.73 g of (14) (2.89 mmol) were hydrogenated in 60 mL CH,OH over 0.8 g of 10% Pd on charcoal. After 4 h the hydrogenation was completed, and the catalyst was removed by filtration, the filtrate evaporated to dryness, and the residue, dissolved in CH,Cl,, added to the solution of the mixed anhydride described above. After 3 h at room temperature the reaction mixture was worked up as described in the preceding paragraphs and the residue chromatographed by eluting in CHC1,- CH,OH (97 : 3) on a 2.5 X 50 cm column of silica gel, obtaining 1.4 g of (15) (39%) as a foam, which gave a single spot on tlc. R, 0.35 in CHCl,-CH,OH (9 : 1). Mass spectral measurement (FAB) of (M-H)+ was 1222, as expected. The


Cyclo(Ah1-Pro2- Phe3- Phe4--Leu5- Ile 6-Ik 7 - ~ u 8 - Val? (1 6)

Deprotection of (15). The amount of 1.0 g of (15) (0.8 mmol) was hydrogenated in 200 mL of acetic acid over 0.6 g 10% of Pd on charcoal to remove the Z group. The hydrogenation was completed after 6 h. After removal of the catalyst, the filtrate was evaporated and the residue was reacted with 20 mL of TFA for 3 h for the removal of the OtBu group. The solution was evaporated to dryness and the residue was tritured with ether. The precipitate was filtered and dried in vacuo over KOH pellets to give 0.700 g of TFA


salt (0.6 mmol) (75%), tlc pure product. R, 0.85 in n-butanol-acetic acid-water (4 : 1 : 1).

Cychation. The amount of 0.480 g TFA salt (0.42 mmol) was dissolved in 50 mL DMF and 35 mL THF, and to the solution was added, at -15"C, 0.057 g iBCCl (0.42 -01). After 10 mh at -15°C a precooled solution of 0.084 mL NMM in 70 mL DMF and 35 mL THF was added. After standing 1 h at 0°C and overnight at room temperature, the solvent was evaporated under vacuum, and the residue, dissolved in CHCl,, was extracted several times with aqueous 0.5M KHSO,, saturated NaHCO, and water, dried with Na,SO,, and evaporated. The crude residue was chromatographed on 50 g of silica gel on a 2.5 x 50 cm column eluting with CHC1,: CH,OH (97 : 3). Fractions (15 mL) 37-41 afforded 60 mg (14%) of (16), which gave a single spot on tlc. Rf 0.4 in CHCl,-CH,OH (95 : 5).

Mass spectral measurement (FAB) of (M-H)+ was 1014,as expected. The amino acid analysis was satisfactory.

Synthesis of [Alas] Cyclolinopeptide A

2-Leu-Ile-Ile-Leu- val-pro-pro-OtBU (1 7) The OtBu group was removed from 1.96 g of (5) (2.3 mmol) as described for

analogous reactions. The residue, dissolved in 30 mL CH,Cl,, was mixed at 0°C under stirring with the solution in 20 mL CH,Cl, of 0.475 g of DCCI (2.3 mmol) and 0.785 g Pro-OtBu (4.6 mmol). After 1 h of stirring at 0°C and overnight at room temperature, the solution was worked up as described in the preceding paragraphs, to give a residue that was purified by eluting in CHCl,-CH,OH (93:7) on 5 x 2.5 cm of silica gel, obtaining 2.2 g of tlc pure eptapeptide (17) (78%). R, 0.25 in CHC1,-CH,OH (93 : 7).

Z - L e ~ - I ~ - I l e - L e ~ - V a l - P r ~ - P r ~ - A h - O t B ~ (18)

The amount of 1.12 g of (17) (1.17 mmol) was deprotected in 10 mL of 1 N HCl in acetic acid for 3 h, as previously described. The residue, dissolved in 20 mL DMF : CHCl, (3 : l), was mixed at 0°C under stirring with the solution in 10 mL CH,Cl, of 0.240 g DCCI (1.17 mmol) and 0.340 g H-Ala-OtBu (2.34 mmol). After 1 h at 0°C and overnight at mom temperature, the solution was worked up as described above to give a residue that was purified by eluting in CHC1,-CH,OH (9 : 1) on a 2.5 x 40 cm column of silica gel, obtaining 1.0 g of tlc pure odapeptide (18) (98%). R, 0.4 in CHC1,-CH,OH (95 : 5).

Z-Leu-Ik-Ile-Leu- Val-Pro-Pro-Ah-Phe-OtBu (19)

One gram of (18) (0.97 mmol) was deprotected in 10 mL of 1 N HC1 acetic acid by adopting the usual procedure. The dried residue, dissolved in DMF : CHC1, (3 : l), was mixed at 0°C under stirring with the solution in 10 mL CH,Cl, of 0.200 DCCI (0.97 mmol) and 0.430 g of H-Phe-OtBu (1.94 mmol). After 1 h at 0°C and overnight at room temperature, the residue obtained by working up as described above was chromatographed on a 2.5 X 50 cm column of Sephadex LH-20 eluting with CH,OH to give 0.600 g of (19) (52%) pure on tlc. R, 0.5 in CHC1,-CH,OH (9 : 1).

378 ZANOTTI ET AL.

1) I-fPd 2)T A

Mass spectral measurement (FAB) of (M-H)+ was 1172, as expected. Amino acid analysis was satisfactory.

Cyclo(pro'-Pro2-Ala3- Phe 4-Leu5- ILe 6-Ile 7-Leu8- Val? (20)

Deprotection of (19). The amount of 0.300 g of (19) (0.26 -01) was hydrogenated in 50 mL acetic acid : methanol (8 : 2) over 0.060 g of 10% Pd on charcoal for 5 h. After removal of the catalyst, the residue was reacted with 10 mL TFA for 2 h. The solution was evaporated to dryness and the residue was tritured with ether. The precipitate was filtered and dried in vacuo over KOH pellets to give 0.230 g of TFA salt (80%) pure on tlc. R, 0.7 in n-butanol-acetic acid-water (4 : 1 : 1).

Cyclization. The amount of 0.230 g TFA salt (0.21 mmol) was dissolved in 24 mL DMF and 16 mL THF, and to the solution was added, at -15"C, 0.028 g iBCCl (0.21 mmol). After 10 min of stirring at -15°C a precooled solution of 0.04 mL NMM in 75 mL DMF and 25 mL THF was added. After 1 h at 0°C and overnight at mom temperature, the solvent was evaporated under vacuum and the residue, dissolved in CHCl,, was worked up as previously described for analogous cyclization reactions. The crude residue was chromatographed on 50 g of silica gel on a 2.5 X 50 cm column eluting with CHC1,-CH,OH (9 : 1). Fractions (15 mL) 19-26 afforded 25 mg (18%) of (20) pure on tlc. R, 0.4 in CHCl,-CH,OH (95 : 5). Mass spectral measurement (FAB) of (M-H)+ was 964, as expected. The


Cyc1ization:MA

X-Ala,Y=Pro Ile-Leu -Val-Al\a

I Ie-Leu-Ph-Phe (16)

1 X=Pro, Y=Ala

I 18- L e u-va 1- Prq

I le-Leu-Phe-Ph6 (9)

I 70 Ala I Scheme 1


Synthesis of [Ma4] cyclolinopeptide A

Z-Leu-Ile-Ile- Leu- Val-Pro-Pro-Phe-OtBu (21)

The OtBu group was removed from 1.71 g of (17) (2 mmol) with 10 mL of 1 N HC1 in acetic acid as described for analogous reactions. The dried residue was dissolved in 20 mL DMF : CHCl, (3 : 1) and reacted at 0°C with 0.855 g H-Phe-OtBu (4 mmol). After 1 h of stirring at 0°C and overnight at room temperature, the solution was worked up as described above to give a residue that was purified by eluting in CHC1,: CH,OH (97 : 3) on a 2.5 X 50 cm column of silica gel, obtaining 1.75 g (80%) of pure octapeptide (21). R, 0.7 in CHCl&H,OH (95 : 5).

Z-Leu-Ile- Ile-Leu- VaLPro-Pro-Phe-Ah- OtBu (22)

The amount of 1.75 g of (21) (1.6 mmol) was deprotected by treatment with 10 mL of 1 N HC1 in acetic acid, as described above. The residue of the deprotection reaction, dissolved in 20 mL DMF : CH2C12 (3 : l), was reacted with 0.47 g DCCI (1.6 mmol) and 0.4656 g H-Ala-OtBu (3.2 mmol). After 1 h of stirring at 0°C and overnight at room temperature, the crude residue was

1Cyclization:M A

X=Ale, V . P b

lk-Leu-Val-R-

l l ~ R o - A l a (20)

I 9"

1 1

I

.OtBu

0 t h

.OH

Scheme 2

380 ZANOTTI ET AL.

chromatographed on a 5 X 250 cm column of Sephadex LH-20 eluting with CH,OH. F'ractions (25 mL) 30-75 afforded 0.7 g (37%) of tlc pure nonapeptide (22). R , 0.5 in CHCl&H,OH (95 : 5). Mass spectra measurements (FAB) of (M-H)+ was 1172, as expected. The


C y ~ ~ k @ ? d - P r o ~ - P h e ~ - A h ~ - L e ~ '- ILe 6- ILe7-Leu '- Vd9) (23)

Deprotection of (22). The amount of 0.70 g of (22) (0.6 mmol) was hydrogenated in 30 mL of acetic acid over 0.1 g of 10% Pd on charcoal for 3 h. After removal of the catalyst, the dried residue was treated with TFA for 2 h. The residue was dried in vacuo on KOH pellets to give 0.50 g of TFA salt (95%) pure on tlc. R, 0.65 in n-butanol-acetic acid-water (4 : 1 : 1).

CycZizution. The amount of 0.5 g TFA salt(0.45 mmol) was dissolved in 50 mL DMF and 30 mL THF, and to the solution was added, at - 15OC, 0.060 g

I

bP.0

'3.0

T --- --- -- 7.8 7.4 p pm

Fig. 1. Phese-sensitive HOHAHA spectrum (400 MHz) of 6 mM [Maz] cyclolinopeptide A in DMSO-d6 at 320 K, showing the connedivities from NH to a and sidechain protons for the eight NH bearing residues. The lower insert shows the NH-CH, peaks from a phasesensitive COSYDQF spectrum in the same conditions. The HOHAHA spectrum is the r d t of 512 experiments of 96 scans each, acquired with a 2K time domain in F, and quadrature detection in Fl. The total mixing time was 37 ms. Gaussian-type windows were applied in both dimensions. Zero filling to 2K in Fl was performed prior to transformation.


iBCCl(0.45 mmol). After 10 min of stirring at - 15°C a precooled solution of 0.08 mL NMM in 150 mL DMF and 50 mL THF was added. After 1 h at 0°C and overnight at mom temperature, the solvent was evaporated under vacuum and the residue worked up as described in previous paragraphs for analogous cyclization reactions. The crude material was chromatographed on 75 g of silica gel on a 2.5 x 70 cm column eluting with CHC1,-CH,OH (9 : 1). Fractions (15 mL) 23-28 afforded 24 mg of cyclic peptide (23) (5.5%) pure on tlc. R , 0.45 in CHC1,-CH,OH (95 : 5).

Mass spectral measurement (FAB) of (M-H)+ was 964, as expected. The amino acid analysis was satisfactory. The general strategy of the synthesis is outlined in Schemes 1 and 2.

, . , . , . , . , . . . , . , , . . . , . , . 8 7 6

PPm Fig. 2. Phasesensitive ROESY spectrum (400 MHz) of 6 mM [Ah2] cyclolinopeptide in

DMSO-dB at 320 K. The contour plot shows only negative levels of the NH-NH region. It is possible to notice many correlations due to chemical exchange between the major and the minor isomers prosent in solution. The connectivitiea for V9 and A2 residues are shown. A mixing time of 200 m8 was used, for 512 experiments of 96 8~8118 each, acquired with a 2K time domain in F2 and quadrature detection in Fl. Gaussian-type windows were used in both dimensions prior to transformation with a zero filling to 2K in Fl.

382 ZANOTTI ET AL.

L

7 - 8 -

1 1 1 1 I

K 3 00 3 20

Fig. 3. Temperature dependence of 6 mM [Ahz] cyclolinopeptide A in DMSO-d,.

RESULTS AND DISCUSSION

From a preliminary nrnr study of the [Ala2] cyclolinopeptide A in DMSO-d, a t 320 K, it is clear that we face a complex system of exchanging isomers even if the substitution in the molecule of the Proz residue with Ala reduces the number of the possible conformers related to the cis-trans Xxx-Pro peptide bond isomerism. In fact, the amidic region of the ‘H-nmr spectrum shows, with the major eight NH resonances belonging to the nonproline residue, various smaller peaks due to the amidic protons of less populated isomer@) (Fig. 1). A combination of COSYDQF and HOHAHA spectra in DMSO-d, at 320 K shows the correlations from NHs to a and to side-chain protons. The eight prominent resonances are labeled according to the one-letter amino acid coding. The weaker correlations derive from minor isomefls). From ROESY and NOESY experiments at 320 K in DMSO-d, performed with a mixing time of 200 ms, it is not possible to extract useful data to build even a rough model of the structure of the [Ma2] cyclolinopeptide A. Figure 2 shows the amidic portion of the ROESY experiment; the cross peaks, in phase with the diagonal, are due to the chemical exchange between the different populated isomer@). The lack of NOE effects and the clear evidence of the coexistence of different slow-exchanging isomers are consistent with a high degree of mobil- ity of the peptide in DMSO-d,. Nevertheless, from a variable temperature study (Fig. 3), it should be possible to postulate the presence of two hydrogen bonds, involving the NHs of Val9 and one of the Leu residues.

The interpretation of preliminary data in CDCl, solution, at low temperature, seems more promising.


Attempts to obtain single crystals for the four analogues as well as details from nmr studies are currently being done in our laboratories.

We wish to thank Profs. B. Di Blasio and V. Pavone for helpful discussions. We would also like to thank Prof. Hero hcci and the Servizio di Spettrometria di Massa del

CNR di Napoli for the discussion of mass spectra (FAB). We acknowledge Mr. Franceaco Nappo for his technical assistance. This work was supported in part by the CNR (CTB 87.0588 and CTB 87.2460) and the Ministry of Education of Italy.

References 1. K d e r , H., Klein, M., Muller, A., Wagner, K., Bats, J. W., Ziegler, K. & Frimmer, M. (1986)

2. Di Blasio, B., Benedetti, E., Pavone, V., Pedone, C. & Goodman, M. (1987) Biopolymers 26, Awew. Chem. Int. Ed. Engl. 25,997-999.

2099-2101. 3. Emst, R. R (1966) Ah. Mugn. Reson. 2,l-135. 4. Lindon, J. C. & Ferrige, A. G. (1980) Prcg. NMR Spectrosc. 14,27-66. 5. Badenhausen, G., Kogler, H. & Emst, R. R. (1984) J. Mugn. Reson. 58,370-378. 6. Rance, M., Serensen, 0. W., Bodenhausen, G., Wagner, G., Emst, R. R. & Wuthrich, K.

(1983) Biochem. Bwphys. Res. Commun. 117,479-485. 7. Bax, A. (1985) J. Mugn. Reson. 65,355-360. 8. Marion, D. & Wuthrich, K. (1983) Biuchem. Biophys. Res. Commun. 113,967-974.

Received July 5, 1988 Accepted August 18,1988

Ala analogues of the cyclolinopeptide A

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