Int. J . Peptide Protein Res. 4, 1972, 395-404 Published by Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author(s)

AMINO ACIDS A N D PEPTIDES XXXIV. Synthesis of a Structure Related to Scotophobin

AKHTAR ALI, JURCEN H. R. FAESEL, DIMITRIOS SARANTAKIS, DAVID STEVENSON and BORIS WEINSTEIN

Department of Chemistry, University of Washington, Seattle, Washington 98/95, U.S.A.

Received 26 May 1972

A preparation is described for the pentadecapeptide L-seryl-L-aspartyl-L-asparaginyl- L-asparaginyl- L-glutamyl-L-glutaminylglycyl-L-lysyl-L-sc~ryl-L-alanyl-L-glutamyl-L- glutaminylglycylglycyl-L-tyrosinamide, which possesses the structure attributed to scotophobin, a possible “memory code word”. The biological activity of this com- pound is one-tenth of the natural product and the physical constants differ also. It is concluded that the proposed structure is in error, either in terms of sequence andlor in functionality.

The chemical transfer of learned behavior has been tried by many groups, but the results seem controversial and the interpretations remain sub- ject to challenge. Recently, fear of the dark, acquired by training, was claimed to be conveyed to naive animals by the injection of material extracted from the brains of trained donors ( I ) . The active factor was dialysable and appeared to be a basic protein of six to ten residues (2, 3). Later, with the aid of microdansylation, quanti- tative amino acid analysis, and mass spectroscopy studies, the material was characterized as a neutral tetradecapeptide and given the name “scoto- phobin” (4). Shortly afterwards, the structure was changed to an N-acetyl pentadecapeptide, Ac-ser- asp-asn-asn-glu-gln-gly-lys-ser-ala-glu-gln-gly-gly- tyr-NH, ( 5 ) . This compound was synthesized, but it differed from natural scotophobin both in terms of activity and Rf value (68). Subsequent- ly, scotophobin was reformulated as the corre- sponding desacetyl derivative, i.e., H-ser-asp-asn-

For the previous paper in this series, see ALI, A., COOK, R. M., and WEINSTEIN, B., Int. J . Peptide Pro- tein Res., 4, 177-180 (1972).

asn-glu-gln-gly-lys-ser-ala-glu-gln-gly-gly-tyr-N H, (9-1 I ) . On employing simple charge considera- tions, both of these compounds would be ex- pected to have acidic properties a t neutral pH.

In view of the unusual behavioral effects at- tributed to the natural material, a new synthesis was undertaken in order to establish the actual existence of a “memory code word” and to pro- vide a large quantity for other test purposes. Based on the earlier preparative experience de- veloped with N-acetylscotophobin, it was felt that a pyramid procedure would have several advantages over the original stepwise scheme. For example, the large, blocked peptides begin- ning with the subunit s&, were very insoluble and slow to react, while some fragments con- taining the S3.4 asn-asn sequence underwent facile hydrolytic cleavage (6, 7). Moreover, it seemed important in work of this type to employ a clas- sical preparative scheme in order to purify and to characterize each intermediate. In this fashion, one can be sure of the structure of the final pro- duct. Although a solid-phase procedure may be faster, the isolation of an impure product with a partial physiological effect contributes very little

395

A. ALI, J. H. R. FAESEL, D. SARANTAKIS, D. STEVENSON AND B. WEINSTEIN

to the understanding of the absolute relationship between structure and activity. With these con- siderations in mind, a synthesis of scotophobin involving the multiple use of the mixed anhydride procedure was attained in short order (Fig. 1). N-Benzyloxycarbonyl-0-t-butyl-tyrosine (I) on

treatment with N-methylmorpholine and isobutyl chloroformate, followed by the addition of am- monia, produced N-benzyloxycarbonyl-0-t-butyl- tyrosinamide (11). Removal of the protecting group by hydrogenation formed O-t-butyl- tyrosinamide (111) ; a coupling to N-benzyloxy- carbonyl-glycine (TV) by the mixed anhydride procedure gave N-benzyloxycarbonyl-glycyl-O-t- butyl-tyrosinamide (V). Hydrogenolysis then af- forded the S,,.,, amine, glycyl-0-t-butyl-tyrosin- amide (VI).

The sequence S,,.,, was prepared beginning with methyl glycinate (VII) and N-benzyloxy- carbonyl-glutamine (VIII), which on joining by the mixed anhydride procedure yielded methyl N-benzyloxycarbonyl-glutaminylglycinate (IX). Hydrogenolysis furnished the amine (X); a com- bination with N-benzyloxycarbonyl-y-t-butyl- glutamic acid (XI) by the mixed anhydride method produced the tripeptide (XII). Hydro- genation formed the amine (XIII) and a coupling to N-benzyloxycarbonyl-alanine (XIV) by a mixed anhydride step gave the tetrapeptide (XV). Hydrolysis by dilute alkali under mild conditions afforded N-benzyloxycarbonyl-alanyl-y-t-butyl- glutamyl-glutaminylglycine (XVI). No evidence for glutaminyl deamination was seen in this reac- tion.

The acid XVI was combined with amine VI using l-ethyl-3(3-N,N’-dimethylaminopropyl)- carbodiimide hydrochloride to yield the S,,.,, hexapeptide (XVII). Hydrogenolysis then fur- nished the amine (XVIII). A mixed anhydride coupling of N-benzyloxycarbonyl-NE-t-butyloxy- carbonyl-lysine (XIX) with methyl-0-t-butyl- serinate (XX) produced the dipeptide (XXI). Hydrolysis formed the corresponding acid (XXII), which was joined to the amine XVIII by a “water- soluble” carbodiimide coupling in the presence of N-hydroxysuccinimide to give the octapeptide (XXIII), without racemization at the serine resi- due (12, 13). Hydrogenolysis then afforded the S,,, fragment NE-r-butyloxycarbonyl-lysyl-O-t- butyl-seryl-alanyl-y-t- butyl-glutamyl-glutaminyl glycylglycyl-0-t-butyl-tyrosinamide (XXIV).

The preparation of the S3., (portion was achieved in a different fashion. N-t-Butyloxycar-

bonyl-glycine (XXV) was joined to N-benzyl- oxycarbonyl hydrazine (XXVI) via a mixed an- hydride intermediate to give the protected hydrazide (XXVII). Cleavage with trifluoro- acetic acid furnished the amine trifluoroacetate (XXVIII), which was combined with N-r-butyl- oxycarbonyl-glutamine (XXIX) by the mixed anhydride method to produce the dipeptide (XXX). Trifluoroacetic acid cleaved the t-butyl- oxycarbonyl group to form the amine salt (XXXI). A mixed anhydride coupling with N-t-butyloxy- carbonyl-y-benzyl-glutamic acid (XXXII) gave the tripeptide (XXXIII). Treatment with tri- fluoroacetic acid afforded the amine salt (XXXIV), which on the addition of p-nitrophenyl N-t-butyloxycarbonyl-asparagine (XXXV) yield- ed the tetrapeptide (XXXVI). Removal of the t-butyloxycarbonyl group by acid furnished the amine salt (XXXVII), which on coupling to XXXV produced the pentapeptide (XXXVIII). Addition of trifluoroacetic acid then formed asparaginyl-asparaginyl-y-benzyl-glutamyl-gluta- minylglycyl-N2-benzyloxycarbonyl-hydrazide tri- fluoroacetate (XXXIX).

The S1.? and S,., units were obtained in a simple manner. A mixed anhydride coupling between N-t-butyloxycarbonyl-0-t-butyl-serine (XL) and methyl /?-t-butyl-aspartate (XLI) gave the dipeptide (XLII). Hydrolysis with dilute base furnished N-t-butyloxycarbonyl-O-t-butyl-seryl- B-t-butyl-aspartic acid (XLIII), which was char- acterized as the dicyclohexylammonium salt. The acid XLIII was combined by the mixed anhydride method with the amine salt XXXIX to produce the Sl., heptapeptide (XLIV). Hydrogenolysis then formed N-t-butyloxycarbonyl-O-t-butyl- seryl- 8-t-butyl-aspartyl-asparaginyl-asparaginyl- glutamyl-glutaminylglycyl hydrazide (XLV).

An azide coupling between XLV and the amine XXIV gave the S,.,, pentadecapeptide (XLVI). Treatment with trifluoroacetic acid afforded the deblocked trifluoroacetate salt (XLVII) and pas- sage through an ion-exchange resin (AG-I -X2, acetate form) yielded scotophobin acetate (XLVIII). Similar procedures afforded the de- blocked peptides corresponding to the fragments 8-15 (XLIX) and 3-15 (L). These three com- pounds were then tested for the induction of a dark memory effect in untrained mice. (The pharmacology assays were furnished by Professor G. Ungar, Baylor College of Medicine, Texas Medical Center, Houston, Texas.) The first pos- sessed 5-10% of the activity of natural scoto-

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A. ALI, J . H . R. FAESEL, D. SARANTAKIS, D. STEVENSON A N D B. WEINSTEIN

phobin at a comparable dosage level, but the last two were inactive.

The lower biological behavior observed here for synthetic scotophobin, as well as a difference in Rf values (0.10 vs 0.57), means the postulated structure is still in serious error, either in terms of sequence or in amino acid functionality. It is sig- nificant that a compound of different sequence has a partial dark memory property, which suggests the alleged action may be due to other causes.

After this work was completed, the structure attributed to scotophobin was changed again based on a reinterpretation of the mass spectral data (the original mass spectral fragmentation pat- tern was produced by a pyrolysis technique and was considered to be nonreproducible; D. M. Desiderio, personal communication, January, 1971). As a result, eight possible pentadecapeptide structures were assigned to scotophobin (14). Each differed from one another only in terms of the precise functionality of the residues at posi- tions 2 (asp or asn), 5 and I1 (glu or gln). Two peptides from this revised group were made by a solid-phase synthesis, and on the basis of bio- logical and Rf comparison tests, a compound possessing the asp2, gln5,11 sequence was said to be identical with the natural product (1 5).

It should be noted on the basis of charge considerations that such a product would be ex- pected to possess basic properties. Moreover, the synthetic material has been reported to be un- stable in solution (16). This result is somewhat unusual, especially in view of the fact that the natural compound is itself isolated by an exten- sive chromatographic procedure. By contrast, the pentadecapeptides made by us are stable in solu- tion. In order to clarify these and other problems, a classical synthesis of the asp?, gln5,l1 structure is now underway in our laboratory.

EXPERIMENTAL SECTION

All melting points were determined on a Reichert "Thermopan" unit and are uncorrected. Evap- orations were performed under reduced pressure (water pump) with a rotatory apparatus at mini- mum pressure, while high-boiling solvents were removed at vacuum pressure (0.2-0.5 mm). Magnesium sulfate was used for drying purposes. Acetonitrite and N,N'-dimethylformamide were of spectroscopic quality; other solvents were reagent grade and petroleum ether had bp 30-60". Thin-layer chromatography employed silica gel

H as the support; chloroform-methanol (97:3, system A), acetic acid-n-butanol-ethanol-water ( I : 8 : 1 : 3, system B), acetic acid n-butanol- pyridine-water (3: 15: 10: 12, system C), or acetic acid n-butanol-water ( 1 :4: I , system D) for de- velopment, and iodine or ninhydrin for detection. Microanalyses were furnished by Galbraith La- boratories, Knoxville, Tenn., while amino acid analysis was carried out by AAA Laboratories, Seattle, Wa.

N n-B~nzylox~~carbonyl-O-tert-butyl-L-tyros~tiam~~~~~ (If). A solution of Nu-benzyloxycarbonyl-0-tprt- butyl-L-tyrosine (18.3 g, 49.4 mmol) and N- methylniorpholine (5.50 ml, 50.0 mmol) in tetra- hydrofuran (200 ml) a t -20" was treated with isobutyl chloroformate (5.95 ml, 50.0 mniol). After 10 min, a solution of 28% aqueous am- monia (3.05 ml) in tetrahydrofuran (30 ml) was added and the reaction allowed to warm to room temperature. After 3 hr, the solvent was evap- orated, the residue triturated with water, and the collected solid washed in turn with 10% citric acid solution, water, 10% sodium bicarbonate solution, and water. Crystallization from metha- nol-water gave a white solid (16.5 g, 90%): mp 135-136"; [u]:," -t 11.6" (c 2.0, chloroform); Rf 0.32, 0.87 (systems A, D).

Anal. Calcd. for C,lH,,N,O, (370.44): C , 68.09; H , 7.07; N, 7.56. Found: C, 68.06; H , 7.06; N. 7.53.

0-tert-Butyl-L-tyrosinamide (III). A solution of derivative 11 (11.70 g, 31.5 mmol) in methanol (200 ml) was hydrogenated overnight in the presence of 10% palladium-charcoal catalyst. After filtration, the solvent was evaporated to leave a solid, which was crystallized from ether- petroleum ether (7.00 g, 94%): Rf 0.23, 0.25 (systems A, D); ninhydrin positive.

N a-Benzyloxycarbonyl-glycyl-O- tert-butyl-l-tyro- sinamide ( V ) . A solution of Na-benzyloxycar- bonylglycine (6.20 g, 29.0 mmol) and N-methyl- morpholine (3.51 ml, 30.0 mniol) in tetrahydro- furan a t -20" was treated with isobutyl chloro- formate (3.77 ml, 30.0 mmol). After 5 min, a solution of 0-tert-butyl-L-tyrosinamide (7.00 g, 29.7 mmol) in tetrahydrofuran (100 ml) was added and the reaction was allowed to warm to room temperature. The reaction was worked-up in the usual fashion to give a solid, which was crystallized from methanol-water (7.50 g, 61 %):

398

AMINO ACIDS AND PEPTIDES. XXXIV

mp 144-146"; [a]:.' -2.6" (c 1.0, N,N'-dimethyl- formamide); Rf 0.50 (system A).

Anal. Calcd. for Cz,Hz9N,05. &HzO (436.50): C, 63.30; H, 6.93; N, 9.64. Found: C, 63.10; H, 6.65; N, 9.18.

Glycyl-0-tert-butyl-L-tyrosinarnide ( VI). A solu- tion of dipeptide V (4.27 g, 10.0 mol) in methanol (100 ml) in the presence of 10% palladium- charcoal catalyst (100 mg) was hydrogenated for 2 hr. After filtration, the solvent was removed to give a solid (2.94 g, 100%); Rr 0.40 (system D); ninhydrin positive.

Methyl Nu-Benzyloxycarbonyl-L-glutarninylglyci- nate ( IX) . A solution of Na-benzyloxycarbonyl- L-glutamine (28.0 g, 100 mmol) and N-methyl- morpholine (12.0 ml, 100 mmol) in N,N'-di- methylformamide (100 ml) and tetrahydrofuran (50 ml) at - 15" was treated with isobutyl chloro- formate (13.0 ml, 100 mmol). After 5 min a solution of methyl glycinate in N,N'-dimethyl- formamide (100 ml), freshly prepared by the addition of N-methylmorpholine (1 2.0 ml) to methyl glycinate hydrochloride (12.5g, 100mmol), was added and the reaction was stirred for 1 hr at -5", and at room temperature for 2 hr. It should be noted that N-methylmorpholine is a weak base and probably does not directly neu- tralize the methyl glycinate hydrochloride; in- stead, as the free ester is removed from the reac- tion, the equilibrium shifts to afford the corre- sponding morpholine salt. The solvent was re- moved and the residue was extracted with hot ethanol, then washed with 0.5 N aqueous hydro- chloric acid, water, 0.5 N aqueous potassium bi- carbonate, and water. The crude product was dried in vacuo over phosphorous pentoxide for several days to remove the absorbed water and crystallized from methanol-water (400 ml, 1 : 3) (21.0 g, 60%): mp 173-175"; Rf 0.13, 0.60 (sys- tems A, D).

Methyl L-Glutarninylglycinate ( X ) . A solution of dipeptide IX (2.01 g, 5 mmol) in methanol (50 ml) was hydrogenated in the presence of 10% pal- ladium-charcoal for 4 hr. Filtration and evapora- tion of the solvent left a solid (1.30 g, 87%); Rr 0.44 (system D); ninhydrin positive.

Methyl Nu-Benzyloxycarbonyl-y-tert-butyl-L-glu- tarnyl-L-glutarninylglycinate (XII) . A solution of Na-benzyloxycarbonyl-y-terr-butyl-L-glutamic

acid (13.60 g, 0.40 mol) and N-methylmorpholine (4.80 ml, 0.40 mmol) in tetrahydrofuran at -15" was treated with isobutyl chloroformate (5.20 ml, 0.40 mol). After 5 min, dipeptide amine X (7.50 g, 0.30 mol) in N,N'-dimethylformamide (35 ml) was added and the reaction was stirred until it reached room temperature. The solvent was evaporated and the oily residue was washed with 10 % citric acid solution, whereupon the result- ing solid was collected and washed with citric acid solution, water, 10 % sodium bicarbonate solution, and water. Crystallization from me- thanol-water gave a solid (13.0 g, 57%): mp 168- 169"; [a]:.' -8.4" (c 2.0, N,N'-dimethylform- amide); Rf 0.31, 0.65 (systems A, D).

Anal. Calcd. for CZ5H,,N4O9 (536.57): C, 55.97; H, 6.77; N, 10.50. Found: C, 55.98; H, 6.47; N. 10.43.

Methyl 7-tert-Butyl-L-glutarnyl-L-glutarninylglyci- nate (XIIZ). A solution of tripeptide XI1 (7.00 g, 0.012 mol) in methanol (200 ml) was hydrogenated in the presence of 10% palladium-charcoal cata- lyst (100 mg) for 2 hr. The reaction was filtered and evaporation of the solvent left a residue (4.80 g, 99%); Rf 0.01 (system A); ninhydrin positive.

Methyl Nu-Benzyloxycarbonyl-L-alanyl- y-tert-bu- tyl-L-glutarnyl-L-glutarninylglycinate ( X V ) . A so- lution of N u-benzyloxycarbonyl-L-alanine (3.35 g, 15 mmol) and N-methylmorpholine (1.80 ml, 15 mmol) in tetrahydrofuran (150 ml) at -20" was treated with isobutyl chloroformate (1.95 ml, 15 mmol). After 10 min, tripeptide amine XI11 (6.00 g, 15 mmol) in N,N'-dimethylformamide (50 ml) was added and the reaction was allowed to warm to room temperature for 3 hr. The solvent was evaporated and the residue was washed with 10% citric acid solution, water, 10 % sodium bicarbonate solution, and water. Crystallization from methanol-water, followed by another crystallization from methanol-ethyl ace- tate gave a solid (8.40 g, 92%): mp 202-204"; [a]:.' - 13.5" (c 1.5, N,N'-dimethylformamide); Ri 0.39, 0.75 (systems A, D).

Anal. Calcd. for CzsH4,N5010 (607.65): C, 55.26; H, 6.81; N, 11.55. Found: C, 55.26; H, 6.78; N, 11 50.

N a-Benzyloxycarbonyl-alanyl- y-tert-butyl-l- glutarnyl-L-glutarninylglycine (XVI) . A solution

399

A. ALI, J. n. R. FAESEL, D. SARANTAKIS, D. STEVENSON AND B. WEINSTEIN

of tetrapeptide XVI (11.4 g, 18.8 mmol) in methanol-acetone-water (300 mi: 60 ml: 50 ml) was treated with 1 N sodium hydroxide solution (27 ml, 27.0 mmol) and allowed to stand for 5 hr a t room temperature. Citric acid (1.03 g) was added as a buffer to the clear solution to neu- tralize most of the excess base, the solvent was evaporated to a small volume, the oily residue was triturated with 10% citric acid solution, and the resulting solid was collected and washed with water (5.10 g, 75%): mp 18C185"; [a]:.' -12.6' (c 1.0, 2 N sodium hydroxide solution); Rf 0.16, 0.72, 0.67 (systems A, C, D).

Anal. Calcd. for C,,H,,N,O,, (593.62): C, 54.64; H , 6.62; N, 11.89. Found: C, 54.42; H, 6.86; N, 11.68.

Na-Benzyloxycarbonyl-L-alanyl- y-tert-butyl-l- glutamyl-L-glutaminyIglycylglycy1-0-tert - butyl-l- tyrosinamide ( X V I I ) . A solution of the tetra- peptide acid XVI (7.40 g, 12.4 mmol) and N- methylmorpholine (1.495 ml, 12.4 mmol) in N,N'- dimethylformamide (100 ml) and tetrahydrofuran (100 ml) a t -15" was treated with isobutyl chloroformate (1.620 ml, 12.4 mmol). After 8 min, a solution of dipeptide amine VI (2.95 g, 10.1 mmol) in N,N'-dimethylformamide was added and the reaction was stirred for 1 hr a t -lo", then overnight a t room temperature. The solvent was evaporated and the residue was tri- turated with water. The resulting solid was col- lected, washed in turn with 10% citric acid solu- tion, water, saturated potassium bicarbonate solution, and water, and crystallized from me- thanol-water (1 : 1) (6.03 g, 70%): mp 218-220"; [a]:.' -7.4" (c 2.0, N,N'-dimethylformamide); Rf 0.66, 0.63 (systems C, D).

Anal. Calcd. for C,,H,,N,O,, (868.97): C, 57.90; H, 6.95; N, 12.90. Found: C, 57.82; H, 7.00; N, 12.76.

The same compound was prepared from VI and XVI with the aid of the "water-soluble'' carbodiimide and N-hydroxysuccinimide.

L-Alanyl-y - tert-butyl-L-glutamyl-L-glutaminylgly- cylglycyl-0-tert-butyl-1-tyrosinamide ( X V I I I ) . A solution of the hexapeptide XVIII (4.00 g, 3.22 mmol) in methanol (100 ml) was hydrogenated overnight in the presence of 10% palladium- charcoal catalyst (100 mg). Filtration and evap- oration of the solvent left a solid (2.70 g, 76%); Rf 0.35 (system D); ninhydrin positive.

Methyl Na-Benzyloxycarbonyl-NE-tert-butyloxy- carbonyl-L-lysyl-0-tert-butyl-L-serinate ( X X I ) . A solution of N a-benzyloxycarbonyl-NE-rrrt-butyl- oxycarbonyl-L-lysine (3.80 g, 10 mmol) and N- methylmorpholine (1.20 ml, 10 mmol) in tetra- hydrofuran (100 ml) a t -20" was treated with isobutyl chloroformate (1.30 ml, 10 mmol). After 10 min, a solution of methyl 0-tert-butyl-L- serinate (1.85 g, 10.6 mmol) in tetrahydrofuran (50 ml) was added and the reaction was allowed to reach room temperature. Removal of the solvent left a residue, which was taken into ethyl acetate and washed with 10% citric acid solution, water, 10% sodium bicarbonate, and water. Evaporation gave a solid that was crystallized from ether-petroleum ether (5.30 g, 99%): nip 73-75"; [a]:.' + 14.5" (c 2.0, chloroform); Rf 0.74 (system A).

Anal. Calcd. for C,,H,,N,O, (537.63): C, 60.22; H, 8.06; N, 7.82. Found: C, 59.52; H, 8.04; N, 7.31.

Na-Benzyloxycarbonyl-N € - tert-butyloxycarbonyl- L-lysyl-0-tert-butyl-L-serine ( X X I I ) . A solution of dipeptide XXI (10.76 g, 20 mmol) in methanol (100 ml) was treated with 1 N sodium hydroxide solution (25 ml, 25 mmol) and stirred at room temperature for 3 hr. The solvent was evaporated to a small volume, water was added, and the solution was extracted with ether. The aqueous phase was acidified with citric acid, then parti- tioned with ethyl acetate. The organic phase was washed with water, dried, and evaporated to leave a foam (10.2 g, 98%): Rf 0.87 (system A).

N a-Benzyloxycarbonyl- N & - tert - butyloxycarbonyl- L-lysyl- 0-tert-butyl-L-seryl-L-alanyl- y - tert- butyl- L-glutamyl-L-glutaminylglycylglycyl- 0-tert - butyl- L-tyrosinamide ( X X I I I ) . A solution of dipeptide acid XXII (4.80 g, 9.15 mmol) and N-methyl- morpholine (1.10 ml, 9.15 mmol) in tetrahydro- furan (100 ml) a t -10" was treated with isobutyl chloroformate (1.19 ml, 9.15 mmol). After 10 min, the hexapeptide amine XVlII (6.70 g, 9.15 mmol) in N,N'-dimethylformamide (200 ml) was added and the reaction was stirred overnight a t room temperature. The solvent was removed and the residue was treated in the usual manner to obtain a solid that was recrystallized from methanol containing some water (6.80 g, 60%): mp 245-248" (decomposition); [a]:." ~ 10.0" (c 1 .O, N,N'-dimethylformamide); Rt 0.74 (sys- tem D).

400

AMINO ACIDS AND PEPTIDES. XXXlV

Anal. Calcd. for C,,H,,N,,O,, (1240.48): C, 58.06; H, 7.55; N, 12.40. Found: C, 57.92; H, 7.75; N, 11.93.

The same compound was made in lower yield by the coupling of XXlI to XVlII with the presence of the "water-soluble" carbodiimide and N-hydroxysuccinimide.

NE-tert-Butj~loxycarbony/-L-/ysy/-O-tert-bufyI-L-se ryl- L-alanyl- y - tert-butyl-L-glutamyl-L-glutaminyl- glycylglycyl-0-tert-butyl-L-tyrosinamide (XXIV) . A suspension of the protected octapeptide XXIII (2.00 g, 1.61 mmol) in methanol (600 ml) was hydrogenated for 20 hr in the presence of 10% palladium-charcoal catalyst (0.500 g). After fil- tration, the solvent was removed to leave a white solid (1.77 g, 99%): Rf 0.32 (solvent D); nin- hydrin positive.

N2-Benzyloxycarbonylhydrazine Hydrochloride ( X X V I ) . A solution of benzyloxycarbonyl chloride (70 ml) in chloroform (100 ml) was added dropwise over a 30-minute period to a solution of 100% hydrazine hydrate (45.0 mi) in chloroform (500 ml) maintained at -10". After standing a t room temperature for 1 hr, a saturated solution of hydrogen chloride in ether (450 ml) was added and the precipitated solid was col- lected, redissolved by boiling with absolute ethanol (700 ml), refiltered, concentrated (200 ml), and allowed to stand overnight a t -20". The crystalline solid was collected and the mother liquid diluted with ether to yield a second drop (total, 22.60 9): Rf 0.71 (system D).

Na-tert-Butyloxycarbonylglycine N2-Benzyloxy- carbonylhydrazide (XXVII ) . A solution of Nu- tert-butyloxycarbonylglycine (17.50 g, 100 mmol) and N-methylmorpholine (12.0 ml, 10 mmol) in tetrahydrofuran (500 ml) a t -10" was treated with isobutyl chloroformate (13.0 ml, 10 mmol). After 30 min, a solution of N2-benzyloxycarbonyl- hydrazine in tetrahydrofuran (200 ml), previously prepared by mixing triethylamine (13.60 ml, 100 mmol) and N2-benzyloxycarbonylhydrazine hy- drochloride (20.26 g, 10 mmol), was added and the reaction was allowed to stand for 1 hr. The solvent was removed and the residue was distri- buted between ethyl acetate-water. The organic phase was washed with 10% citric acid solution, water, 10% sodium bicarbonate solution, and water. Removal of the solvent left a foam (24.9, g 75 %); Rr 0.43 (ethyl acetate-petroleum ether, 7:3).

Glycine N2-Benzyloxycarbonylhydrazide Trifluoro- acetate (XXVIII) . A solution of peptide XXVII (13.5 g, 41 mmol) in trifluoroacetic acid (50 ml) was allowed to stand a t room temperature for 1 hr. After evaporation of the solvent, the residue was triturated with ether to furnish a solid, which was crystallized from methanol-ether (10.0 g, 71 %): mp 174176".

Anal. Calcd. for CloH,,N,03. CF,COOH (317.24): C, 42.73; H, 4.18; F, 16.90; N, 12.43. Found: C, 42.58; H , 4.04; F, 17.07; N, 12.70.

N'-tert-BufyIoxycarbonyl-L-glutaminylglycine N2- Benzyloxycarbonylhydrazide ( X X X ) . A solution of Nu-fert-butyloxycarbonyl-L-glutamine (6.70 g, 27 mmol) and N-methylmorpholine (3.24 ml, 27 mmol) in tetrahydrofuran (200 ml) a t -10" was treated with isobutyl chloroformate (3.51 ml, 27 mmol). After 5 min, peptide XXVIII (9.10 g, 27 mmol) and N-methylmorpholine (3.24 ml, 27 mmol) in tetrahydrofuran (100 ml) were added and the reaction allowed to warm to room tem- perature. The reaction was worked-up in the usual fashion to leave a residue, which was crys- tallized from ethyl acetate containing a little petroleum ether (5.80 g, 57%): mp 145-146"; [a]:.' -4.5" (c 1.0, N,N'-dimethylformamide); Rf 0.31, 0.65 (systems A, D).

Anal. Calcd. for C,,H,,N,O, (451.47): C, 53.21 ; H, 6.47; N, 15.33. Found: C, 53.37; H, 6.52; N, 15.33.

L-Glutaminylglycine N2-Benzyloxycarbonylhydra- zide Trifluoroacetate (XXXI) . Dipeptide XXX (4.52 g, 1.0 mmol) was allowed to stand with trifluoroacetic acid (30 ml) for 1 hr. The solvent was evaporated and the residue was triturated with ether to yield a colorless, hygroscopic solid (4.52 g, 100%).

Na-tert-Butyloxycarbonyl-y-benzyl- L-glutamyl- L- glutaminylglycine N2-Benzyloxycarbonylhydrazide (XXXI I I ) . Nu-terf-butyloxycarbonyl-y-benzyl-L- glutamic acid (11.10 g, 32.8 mmol) was dissolved in tetrahydrofuran and treated in turn with N- methylmorpholine (3.95 ml, 32.3 mmol) and isobutyl chloroformate (4.25 ml, 32.8 mmol) a t - lo", then amine salt XXXl (1 5.60 g, 33.8 mmol) and N-methylmorpholine (4.00 mi, 33.8 mmol) in N,N'-dimethylformamide (100 ml) was added and the reaction was allowed to stand for 3 hr. The reaction was worked-up in the usual fashion to afford a solid (19.20 g, 87%): mp 178-179";

40 1

A. ALI, J . H. R. FAESEL, D. SARANTAKIS, D. STEVENSON A N D B. WEINSTEIN

[a]:.' -6.4" ( c 2.0, N,N'-dimethylformamide); Rf 0.19, 0.68 (systems A, D).

Anal. Calcd. for C,,H,,N,O,, (670.71): C, 57.23; H , 6.31; N , 12.53. Found: C, 57.13; H, 6.33; N , 12.36.

y-Benzyl-L-glutamyl-L-glutaminylglycine N2- Benzyloxycarbonylhydrazide Trifluoroacetate (XXXIV) . The tripeptide XXXIlI (1.00 g, 1.49 mmol) was dissolved in trifluoroacetic acid (10 ml) and allowed to stand a t room temperature for 1 hr. The solvent was removed and the residue was triturated with ether to give a solid (0.902 g, 100%): [a]:.' t10.1" ( c 1.0, N,N'-dimethyl- formamide); Rf 0.43 (system D).

N Q-tert-Butyloxycarbonyl-L-asparaginyl- 7-benzyl- L-glutamyl-L-glutaminylglycine N2-Benzyloxycar- bonylhydrazide (XXXVI) . A solution of the amine salt XXlV (5.10 g, 8.41 mmol) in N,N'-dimethyl- formamide (25 ml) was treated in turn with tri- ethylamine (1.170 ml, 8.41 mmol) and p-nitro- phenyl N a-tert-buty loxycarbonyl-L-asparagine (3.00 g, 8.50 mol) and acetic acid (3 drops). After 1 hr a t O", the reaction was allowed to stand a t room temperature for 5 days. Addition of ether precipitated a solid, which was collected and washed in turn with ether, methanol, 10% citric acid solution, and water (3.40 g, 52%): mp 2W203"; [a]:.' -17.4' ( c 0.5, N,N'-di- methylformamide); Rf 0.79 (system D).

Anal. Calcd. for C3BH48N8012 . H 2 0 (802.73): C, 53.85; H, 6.27; N, 13.94. Found: C, 53.77; H, 6.11; N , 13.75.

L- Asparaginyl- y-benzyl- L-glutamyl- L-glutaminyl- glycine N2-Benzyloxycarbonylhydrazide Trifluoro- acetate (XXXVII) . A solution of the protected tetrapeptide (XXXVI) (1.60 g, 2.10 mmol) was dissolved in trifluoroacetic acid (25 ml) and stirred a t 0" for 1 hr. The solvent was removed and the residue was triturated with ether to form a solid (1.60 g, 95%): [a]:.' -5.6" ( c 0.5, N,N'- dimethylformamide); Rf 0.40 (system D); nin- hydrin positive.

Anal. Calcd. for C33H41F3N8012 (798.73): C, 49.56; H, 5.18; F, 7.15; N , 14.08. Found: C, 50.04; H, 5.20; F, 7.06; N, 13.58.

Nu- tert-Bu tyloxycarbonyl-l-asparaginyl-l-nspara- ginyl- y-benzyl-L-glutamyl-L-glutaminylglycine N2- Benzyloxycarbonylhydrazide (XXX VIII). A solu-

tion of the amine salt XXXVll(3.00 g, 3.75 mmol) in N,N'-dimethylformamide (20 ml) was treated with triethylamine (0.560 nil, 3.95 mmol), then p-nitrophenyl Nu-tert-butyloxycarbonyl-L-aspa- ragine (1.35 g, 3.85 mmol) was added followed by acetic acid (3 drops), and the reaction was stirred for 5 days. The addition of ether precip- itated a solid, which was collected and washed with 5 % citric acid solution, water, and cold methanol (2.20 g, 66%): mp 204-206"; [a]:.' - 13.3" ( c 1.0, N,N'-dimethylformamide): Rf 0.60 (system D).

Anal. Calcd. for C40HS4N10014 (898.91); C , 53.45; H, 6.05; N, 15.60. Found: C, 53.57; H , 6.02; N , 15.42.

Amino acid ratio analysis: Asp (2.00, 2.04); Glu (2.00, 2.10); Gly (1.00, 1.00).

L-Asparaginyl-L-asparaginyl-y-b~~nzyl-L-glutamyl- L-glutaminylglycine N2-Benzyloxycarbonylhydra- zide Trifluoroacetate (XXXIX) . The protected pentapeptide XXXVlll (0.650 g, 0.72 mmol) was dissolved in trifluoroacetic acid (10 ml) and al- lowed to stand at room temperature for 30 min. The solvent was removed and the residue was triturated with ether to yield a solid (0.400 g, 61 %): mp 18CL181"; [u]:.' --10.2" ( c 0.5, N,N'- dimethylformamide); Rf 0.37 (system D); nin- hydrin test weak.

Anal. Calcd. for C3,H4,F,N,,Ol4 (912.83): C, 48.65; H , 5.19; F, 6.24; N , 15.35. Found: C, 48.64; H, 5.10; F, 6.09; N, 15.34.

Amino acid analysis: Asp (2.00, 2.06); Glu (2.00, 2.00); Gly (1.00, 1.00).

Methyl NQ-tert-Butyloxycarbonyl-0-tert-butyl-L- seryl-P-tert-butyl-L-aspartate (XLII) . A solution of NQ-tert-butyloxycarbonyl-0-tert-butyl-L-serine (1.05 g, 4 mmol) and N-methylmorpholine (0.41 g, 4.0 mmol) in tetrahydrofuran (60 ml) at -30" was treated with isobutyl chloroformate (0.55 g, 4 mmol). After 4 min, a solution of methyl P-tert- butyl-L-aspartate, freshly prepared from triethyl- amine (0.41 g, 4 mmol) and the corresponding peptide hydrochloride (0.96 g, 4 mmol) in tetra- hydrofuran (40 ml), was added and the mixture allowed to warm to room temperature. The solvent was removed and the residue was dis- solved in ethyl acetate and washed with 10% citric acid solution, water, 10% sodium bicar- bonate solution, and water. Evaporation gave an oil (1.89 g, 100%): Rf 0.70 (system A).

402

AMINO ACIDS AND PEPTIDES. XXXIV

N a-tert - Butyloxycarbonyl-0- tert - butyl-L-seryl-p- tert-butyl-L-aspartic Acid (XLIII). A solution of dipeptide XLlI (1.80 g, 4 mmol) in dioxane (5 ml) was treated with 1 N sodium hydroxide solution (4.50 ml). After 1 hr, water (30 ml) was added to the reaction mixture, and it was washed with ethyl acetate, then acidified with citric acid and extracted with ethyl acetate. Evaporation of the organic phase produced a foam (1.53 g, 88%): Rr 0.83 (system D). Addition of dicyclohexyl- amine furnished the crystalline salt: mp 108-1 10"; [a]:." $33.7" (c 1.0, chloroform).

Anal. Calcd. for C,,H,,N,O,.H,O (631.86): C, 60.83; H, 9.73; N, 6.55. Found: C, 60.94; H, 9.54; N, 6.85.

Na-tert - Butyloxycarbonyl-0-tert-butyl-L-seryl-p- tert - butyl-L-aspartyl-L-asparaginyl-L-asparaginyl- y-benzyl-L-glutamyl-L-glutaminylglycine N2-Ben- zyloxycarbonylhydrazide (XLI V). A solution of the partially deblocked dipeptide acid XLIII (0.433 g, 1 .O mmol) and N-methylmorpholine (0.12 ml, 1.0 mmol) in tetrahydrofuran (30 ml) at -15" was treated with isobutyl chloroformate (0.13 ml, 1.0 mmol). After 5 min, the reaction was treated with the amine salt XXXIX (0.913 g, 1 .O mmol) and N-methylmorpholine (0.12 ml, 1 .O mmol) in N,N'-dimethylformamide (10 ml) and then allowed to stand overnight. The solvent was removed and the oily residue was redissolved in N,N'-dimethylformamide (1 ml) and the ad- dition of ether formed a solid, which was col- lected and washed with methanol and water (1.00 g, 82%): mp 207-209"; [a]:.' -18.0" (c 0.25, N,N'-dimethylformamide); Rf 0.62 (sys- tem D).

Anal. Calcd. for C6sH,oN12019 (1213.29): C, 54.56; H, 6.65; N, 13.85. Found: C, 54.81; H, 6.51; N, 14.05.

N a-tert -Butyloxycarbonyl-0- tert- butyl-L-seryl-p- tert - butyl-L-aspartyl-L-asparaginyl-L-asparaginyl- L-glutamyl-L-glutaminylglycine Hydrazide (XL V ) . A solution of the protected heptapeptide hydra- zide XLIV (0.310 g, 0.257 mmol) in N,N'-di- methylformamide (10 ml) and acetic acid (10 ml) was diluted with methanol (300 ml) and hydro- genated overnight in the presence of 10% pal- ladium-charcoal catalyst (0.100 g). After filtra- tion of the catalyst, the solution was concen- trated to a small volume (5 ml) and treated with ether to produce a white solid (0.242 g, 100%):

mp 196-198"; [a]:.' -22.4" (c 0.25, N,N'-di- methylformamide): one spot (system D).

Anal. Calcd. for C,oH,8Nl,0,, (988.90); C, 48.59; H, 6.93; N, 17.00. Found: C, 48.41; H, 6.92; N, 16.81.

Nu-tert - Butyloxycarbonyl-0- tert - butyl-L-seryl-t% tert -butyl-L-aspartyl-L-asparaginyl-L-asparaginyl- L-glutamyl-L-glutaminylglycyl- N &- tert-butyloxy- carbonyl-L-lysyl-0- tert-butyl-L-seryl-L-alanyl-y- tert - butyl-L-glutamyl-L-glutarninylglycylglycyl-0- tert-butyl-L-tyrosinamide (XL VI) . A solution of the heptapeptide hydrazide XLV (0.350 g, 0.353 mmol) in N,N'-dimethylformamide (10 ml) was treated at -40" with a 2.62 N solution of hydro- gen chloride in tetrahydrofuran (0.405 ml, 1.059 nitrite (0.0848 ml, 0.706 mmol). After 30 min, triethylamine (0.25 ml, 1.765 mmol) was added and then a solution of octapeptide amine XXIV (0.392 g, 0.335 mmol) in N,N'-dimethylformamide (10 ml). The reaction was allowed to stand first at -10" for 24 hr, then at 0" for 48 hr, after which the solvent was evaporated to a very small volume and ether was added to precipitate a white solid. The product was washed in turn with saturated citric acid solution, water, and metha- nol to afford a powder (0.250 g, 34%): mp 213- 215"; [a]:.' - 10.4" (c 0.25, N,N'-dimethylform- amide); Rf 0.70 (system C).

Anal. Calcd. for C,,H,,,N,,O,, (2063.30): C, 53.55; H, 7.36; N, 14.28. Found: C, 53.15; H, 7.31 ; N, 14.65.

L-Seryl-L-aspartyl-L-asparaginyl-L-asparaginy1-L- glutamyl-L-glutaminyIglycyl-L-lysyl-L-seryl-L-ala- nyl-L-glutamyl-L-glutaminylglycylglycyl-L-tyrosin- amide Ditriguoroacetate (XL VII). A solution of the protected pentadecapeptide XLVI (0.206 g, 1 .O mmol) was dissolved in trifluoroacetic acid (5 ml) and was stirred at 0" for 1 hr. The solvent was evaporated and the residue was triturated with ether to give a solid (0.181 g, 100%): mp 155-160'; [ C Z I ~ , ~ . ~ -10.7" (c 0.15, N,N'-dimethyl- formamide); Rf 0.18 (system C).

Anal. Calcd. for C,,H,,N,,OZ8. 2CF,COOH (1810.63): C, 43.78; H, 5.40; F, 6.29; N, 16.22. Found: C, 43.39; H, 5.64; F, 6.75; N, 16.06.

L-Seryl-L-aspartyl-L-asparaginyl-L-asparaginy1-L- glutamyl-L-glutaminyIglycyl-L-lysyl-L-seryl-L-ala- nyl- L-glutamyl-L-glutaminylglycylglycyl-L-tyrosin- amide Diacetate (XL VIII). The aforementioned trifluoroacetate salt (XLVII) (0.181 g, 1.0 mmol)

403

A. ALI, J. H. R. FAESEL, D. SARANTAKIS, D. STEVENSON AND B. WEINSTEIN

was dissolved in water and passed through a n ion-exchange resin column ( 1 8 x 1.6 cm, AG I-X2 in the acetate form). Elution with acetic acid (1 %, 20 ml) gave after lyophilization a fluffy, white solid (0.113 g, 60%): m p 160-165" (de- composition); [u]:." - 1 1.4" (c 0.15, N,N'-di- methylformamide); R f 0.10 (system 9).

Anal. Calcd. for C,,H,,,N,,O,,. 10H,O (1882.79): C, 42.10; H,6.54; N, 15.60. Found : C, 41.75; H, 6.20; N, 16.07.

Amino acid ratio analysis: Ala (1.00, 1.00); Asp(3.00,2.87);Glu(4.00,4.20);Gly(3.00,3.04); Lys (1.00, 1.00); Ser (2.00, 1.74); Tyr (1.00,0.98). Digestion by leucine amino peptidase confirmed the optical homogeneity of the product.

ACKNOWLEDGEMEN1

We thank the National Institute of Mental Health (MH 19320) for the support of this work.

REFERENCES

1. UNGAR, G., GALVAN, L., and CLARK, R. H., Nature, 217, 1259-1261 (1968).

2. UNGAR, G., and FJERDINGSTAD, E. J.. in G. ADAM (Editor), Biology of memory, Plenum Press, New York, 1971, p. 137.

3. UNGAR, G., in A. LAJTHA (Editor), Protein meta- bolism of the nervous system, Plenum Press, New York, 1971, p. 571.

4. UNGAR, G., HO, I. K., GALVAN, L., and DESIDE-

RIO, D. M., Western Pharmacology Society, San Diego, California, February, 1970, Abstracts; Chem. Eng. N e w , February 9. 1970, p. I I .

5. UNGAR, G., personal communication, February 24, 1970.

6. A stepwise preparation of the N-acetylscotopho- bin sequence was initially discussed by B. Wein- stein at The Second American Peptide Symposium, Cleveland, Ohio, August, 1970; the biological re- sults for the tridecapeptide S,-,, were mentioned, also. Additional information on this synthesis was presented at the 160th National Meeting of the American Chemical Society, Chicago, Illinois, September, 1970, Abstracts ORGN 008.

7. ALI, A., FAESEL, J. H. R., SARANTAKIS, D., STEVEN- SON, D., and WEINSTEIN, B., in S. LANDE (Editor), Progress in peptide research, Gordon and Breach, New York, 1972, p. 37-1.

8. ALI, A., FAESEL, J. H. R., SARANTAKIS, D., STEVEN- SON, D., and WEINSTEIN, B., Experientia, 27, 1 I38 (1971).

9. UNGAR, G., Agents Actions, 1, 155-163 (1970). 10. UNGAR, G., in A. LAJTHA (Editor), Handbook of

neurochemistry, Vol. 6, Plenum Press, New York, 1971, p. 241.

11. UNGAR, G., in A. SCHWARTZ (Editor), Methods in pharmacology, Vol. I , Appleton-Century- Crofts, New York, 1971, p. 744.

12. SARANTAKIS, D., and WEINSTEIN, B., unpublished results.

13. WEINSTEIN, B., and PRITCHARD. A. E.. J. Chem. Soc., Perkin Trans. I , 1015-1020 (1972).

14. DESIDERIO, D. M., UNGAR, G., and WHITE, P. A., Chem. Commun., 432433 (1971).

15. PARR, W., and HOLZER, G., Z. Physiol. Chem., 352, 1043-1049 (1971).

16. UNGAR, G., Round Table, International Society for Neurochemistry, Hungary, July, 1971.

404