Int. J. Peptide Protein Res. 14, 1979,68-79 Published by Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author(s)

SYNTHESIS, RESOLUTION AND CHARGE-DONOR PROPERTIES OF SIX TRYPTOPHAN ANALOGUES

HUBERTUS M. RAJH, JOSEPH H . UITZETTER, LAMBERTUS W. WESTERHUIS, CORNELIS L. VAN DEN DRIES and GODEFRIDUS I. TESSER

Department o f Organic Chemistry, Catholic University, Toernooiveld, Nijmegen, the Netherlands

Received 3 November, accepted for publications 27 November 1978

In order to investigate the special function o f tryptophan in peptide hormones, six tryptophan analogues have been synthesized, in which structural resemblance has been grossly retained and potentially essential properties have only partially been varied. The L-enantiomers have been isolated after enzymatic digestion o f racemic dipeptide derivatives, and charge-transfer properties of the compounds have been studied. Key words: charge-transfer properties; donor-acceptor complexes; enzymatic digestion; fluorinated indoles; peptide hormones; resolution; tryptophan-analogues.

Investigations into structure-activity relations of peptide hormones such as a-MSH and LHRH, or biologically active fragments of ACTH, gastrin and pancreozymin, have shown that a tryptophan residue, present in these sequences, is essential for the exertion of the complete set of biological actions of these compounds (Dedman el al., 1957; Morley et al., 1965; Gregory & Morley, 1968; Hofmann et al., 1970; Coy et al., 1974; Van Nispen et al., 1977b). In previous studies, aimed at the description of the precise role of the tryptophan residue in the interaction of these hormones with their receptors, special attention was paid to its charge-transfer capacity. Analogues in which tryptophan was replaced by pentamethyl-Lphenylalanine (Pmp), and having almost equal donor properties, or by L-phenylalanine but with very low donor strength, were synthesized 68

0367-8377/79/050068-12

and tested in appropriate biological assays (Van Nispen & Tesser, 1975; Van Nispen et al., 1977b). On the whole, the effects of these replacements appeared to be rather divergent. Exchange of Trp by Pmp in tetragastrin led to loss of activity (Van Nispen, 1974). Similarly, the same substitution in a-MSH and 0-corticotrophin (1 -24) caused loss oflipotropic activity in both hormones and loss of steroidogenesis in the ACTH-analogue. However, both analogues preserved a strong melanocyte-stimulating effect (Van Nispen, 1977a.b). Finally, in the releasing hormone LH-RH the substitution of Trp by Pmp resulted in a product that exhibited 34% of FSH- and 70% of LH-activity of the natural product (Coy el al., 1974). We realise that these divergent results may be caused by the rather imperfect structural variation which was introduced. Exchange of

$02.00/0 Q 1979 Munksgaard, Copenhagen

PROPERTIES OF SIX TRYPTOPHAN ANALOGUES

Trp for Pmp (or Phe) interferes with several other features of the tryptophan residue, which may equally well be involved in its special function. Both replacements lead to the loss of the possibility for hydrogen bonding, modify the lipophilic binding capacity, and may have steric consequences. Moreover, electron density is much more evenly distributed in the aromatic nucleus of Pmp than in the indole ring of Trp. The observation that the LH-RHanalogue, having the weak electron donor 3-(2-naphthyl)L-alanine instead of tryptophan in position 3, has a rather high LH-activity (Prasad et al., 1975; Yabe et al., 1976) may well be an indication that the contribution of the relevant residue to the activity is merely due to lipophilic interaction. The role of essential tryptophan residues in peptide chains can certainly not be attributed to its donor properties alone. This has also been demonstrated recently by Meyers et al. (1978) for the hormone somatostatin. Better insight into the special function of tryptophan may be expected from studies on analogues in which structural resemblance has been grossly retained and, as far as possible, only one of the characteristic features of the tryptophan residue has been changed.

FIGURE 1 Tryptophan analogues (la-f, a:X=O b:X=S c : X = N(CH,) d : X = NH, 5monofluoro e: X = NH, 6-monofluoro f: X = NH, 4,5,6,7-tetrafluoro

In this paper we report on the synthesis and resolution of a series of tryptophan analogues (Fig. 1) which seem to satisfy these conditions, and describe their properties in donor-acceptor complexes. In a subsequent paper their substitution for tryptophan in gastrin- and pancreozymin-fragments will be reported, and data of biological activities of the resulting compounds will be given. SYNTHESIS

In the synthesis of the oxygen-analogue of tryptophan (a) (Erlenmeyer & Grubemann, 1947), we found that akylation of the obvious malonic ester derivative is more complete when carried out in dimethylformamide. The same applied to the corresponding step in the synthesis of the sulfur-analogue (b), which was otherwise done as described (Chapman et al., 1969). For the synthesis of the N-methyl derivative (c) the procedure described for the preparation of N'"-methyltryptamine (Taborski et al., 1965) was carried out with L-tryptophan. The monofluorinated tryptophans (d) and (e) were obtained commercially as racemates. 4,5,6,7lreterafluoroindole (4), the starting compound for the preparation of tetrafluorotryptophan, was synthesized as described by Petrov (Petrov et al., 1968) with the exception that reduction of the intermediate nitro compound (2) was carried out with zinc in acetic acid instead of electrochemically (Scheme 1). After conversion O f 4 into the Mannich base (5) the alanyl sidechain was introduced with two differently protected a-amino malonic esters (Scheme 2). The protected dipeptide (15) is a convenient substrate for the resolution as will be shown below. It is remarkable that in the preparation of 10 the aminomalonate 8 does react with the hydroxysuccinimide ester but not with the p-nitrophenyl ester. The chosen strategy allowed mild conditions at several steps and the overall yield of the synthesis was higher than reported by Petrova (Petrova et al., 1971).

u: Benzofurylalanine, Bfa

b : Benzothienylalanine, Bta c : P-Methyl-tryptophan, Trp(Me) d : 5-Fluorotryptophan, Trp(5 F) e : 6-Fluorotryptopha11,Trp(6F) f: 4,5,6,7-Tetrafluorotryptophan,Trp(tF)

RESOLUTION

From the method for determination of the Cterminals of proteins (Ambler, 1972) we concluded that a simple resolution procedure for 69

H.M. RAJH ET AL.

SCHEME 1

S

F

F

neutral and aromatic amino acids could be devised by combination of an acylated glycyl residue (difficult to digest) and an aromatic or neutral amino acid (easily digestible). Thus for their resolution the racemates (a. b, d, e a n d n were converted into dipeptide derivatives Z-GlyX-OH or Boc-Gly-X-OH by acylation with ZGly-ONp or BocGly-ONSu, respectively. The dipeptides were digested with carboxypeptidase A (Scheme 3). The resulting Lenantiomers of b, d, e and f showed a negative Cotton effect between 350 and 600 nm; the sign of the

Cotton effect of a (Bfa) is opposite (Table 1). Proof of configurational purity was obtained through the method of Manning (Manning & Moore, 1968). The resolved analogues showed one peak after reaction with the N-carboxyanhydride of L-glutamic acid followed by ion exchange chromatography. The racemates gave two well-resolved peaks in this test. Physical constants and elemental analysis of the resolved tryptophan analogues are given in Table 2. The analogues a, b and f exhibit higher acid stability and resistance to air oxidation than tryptophan.

TABLE 1 Cotton-effects of L-enantiomers (a-f). Measurements were carried out with I % solution in 90% aqueous acetic acid

588 (D) 578 546 436 365

70

- 1.6" - 1.4" - 1.2" 2.3" + 15.5"

+

- 29.6" -31.0" -34.9" -S6.9" -83.0"

- 15.1" - 15.7" - 17.2" - 22.1" -

- 9.0" - 9.3" - 9.9" - 11.1" -

- 11.0" - 11.5" - 12.4" - 14.3" -

- 26.2" - 27.4" - 30.9" -49.7" - 69.0"

- 12.1"

- 13.0" - 15.5" - 27.3" -

82%

75%

12%

89%

H-BtaGH

H-Trp (SFFOH

H-Trp (6F)OH

H-Trp (tF>OH

0.46 (A) 0.46 (A)

2 66-26 8'

0.43 (A)

0.54 (A)

0.54 (A)

Rf

268-270'

263-264"

250" (dec.)

250" (dec.)

m.p.

upper values are calculated; the lower are the found contents.

85%

H-Bfa-OH

a The

Yield of digestion

Symbol

C,lH,F.lN,O,

CllHllFNPl

Cl IH 1,FN,O,H,O

CIlHllNO2S

CllHllNO3

Formula

216.20

222.21

240.23

221.27

205.21

Mol.wt.

Properties of the resolved enantiomers

TABLE 2

64.38 64.42 59.72 59.82 55.00 55.32 59.45 59.28 47.83 47.63

%C

5.37 5.54 4.91 5.16 5.41 5.24 4.99 4.96 2.92 3.09

%H

6.83 6.83 6.33 6.28 11.66 11.79 12.61 12.33 10.14 10.08

%N

Elemental analysisa

4

P

4

2

14.68 F:7.91 7.95 F:8.55 8.35 F:27.50 26.88

2

2

0

$

> z

$

- 3 S:14.48 g

-

Hetero

2 2

a P

H.M. RAJH ET AL. SCHEME 2

COOBzl CH/ hH'COOBzl I

CHO

(1)

route I1

route I

(5,

CHj MeS04@

. I

~;icoon21 I

CHO

(G)

COOBz 1 -C' &COOBzl {lY

(14)

Boc 1. H2/Pd 2. MeOH/pyridine

/COOH

P,L

R-CIIZ-C

rLH CllO

1. H2/Pd

(12)

1. HCl/THF 2. conc. HCl

72

2. PleOH/ pyridine

PROPERTIES OF SIX TRYPTOPHAN ANALOGUES SCHEME 3 Trp may be substituted as depicted in Fig. 1. Z or Boc may be used. enzyme ZGly-DL-TrpGH-(L) pH 8-8.5

H-Trp-OH

+ ZGly-D-TrpGH

They cannot be detected by Ehrlich's test. Benzothiophene showed a very broad maxiAnalogue c is very sensitive to acid and is easily mum between 480 and 530 nm. oxidized in the presence of tiny amounts of The CT-absorption band of the complexes heavy metals (Pd!). with indole, 5-fluoro-indole and 6-fluoroindole decreased slowly during the time of the measureDonor properties of the tyryptophan analogues ments, as was previously observed for indole by in charge-transfer complexes Foster (Foster & Hanson, 1965). For this For a comparison of the donor-properties of reason the measurements had to be carried out the analogues charge transfer complexation was fast. The rate of the reaction between TCNE studied in a series of simple indole analogues and N'"-methylindole was too rapid to obtain representing the heterocyclic moieties in the reproducible association constants. For benzocompounds a-f. The spectra of all indole ana- furan, benzothiophene and tetrafluoroindole no logues in dichloromethane in the presence of reaction was observed. tetracyanoethylene (TCNE) showed an absorption band, not due to either component alone. DISCUSSION These additional bands can be attributed to formation of donor-acceptor complexes as dis- The series contains a group of three compounds cussed by Foster (Foster & Hanson, 1965) and (a-c) in which the heterocyclic moiety cannot Cooper (Cooper et al., 1965). TCNE was used contribute to H-bonding, and three compounds as the acceptor because its absorption does not in which this ability has been retained (d-f). interfere with the CT-absorption band. The Apparently, the analogues a and b differ relative chargedonor properties were evaluated largely from tryptophan in donor strength as by determination of association constants (K) appears from the much lower values of ACT for and extinction coefficients (e) with the Foster the relevant chromophores; they also form equation (Foster et al.. 1953): weaker complexes. The donor strength of c could not be estid d = -K -+ K * E (Do S Ao) mated because the association constant of N'"Ao .Do A0 methylindole could not be measured with where Do,Ao = initial concentrations of donor TCNE as the acceptor, but Foster (Foster & and acceptor, respectively; d =measured, optical Hanson, 1964) determined the charge transfer bands of several methylindoles with chloranil density. and found #"-methylindole to be only slightly Extinctions (d) of solutions, containing a weaker as a donor than indole itself. The monofluorinated derivatives (d and e) constant concentration of TCNE and a varying excess of the donor compound, were measured; were included since they should be weak in all measurements a solution with an equal electron donors according to literature (Coy er donor concentration but without TCNE was aZ., 1974). Against expectation, however, they used as a blank. Results are given in Table 3. appear as good to very good donors in our test. Finally, d/Ao*Do was plotted agairist d/Ao at For that reason we added 4,5,6,74etrafluorothe wavelength of maximum absorption (hcT). indole to the series, whose association constant 73

H.M. RAJH ET AL. TABLE 3 Properties of the donor-acceptor complexes from some indole analogues and TCNE at mom temperature in dichloromethane

455 480-530

0.8a

1050

0.6a

2210

-

-

550

42

760

550

2.4

15 10

470

0.6

240

550

3.44b

not measurable Cli

2020

a Values

measured by Cooper el nl. (1965) for the TCNE-complexes with indole, benzofuran and benrothiophene in CHQ, at 22' are: K =4.8 E = 3000 lndole Benzofuran K = 1.07 e = 1100 Benzothiophene K = 1.08 E = 1700 Values measured by Foster et al. (1965)for the indole-TCNE complex in CH,Cl, are: 15" K = 3.3 c = 2230

25" K = 2.1

c = 2170

and charge transfer band differ more from those of indole. The absence of a simple relationship between K- and X,,,-values in a series of strongly related compounds has extensively been discussed in the literature (Dewar & Thompson, 1966; Paetzold, 1975). It implies that special binding forces as a consequence of charge transfer do not play a significant role in the complex formation in the ground state. However, the energies of the charge-transfer bands determined by the energies of the highestoccupied molecular orbital of the donor molecules are a measure for the ability to transfer electron-charge. Therefore, the series of spatially, very related compounds (a-fl seems appropriate to investigate if the special role of tryptophan can be attributed to H-bonding or charge-transfer, to neither or to both of them; in a and b both properties are absent, d and e are comparable to tryptophan in both aspects; 14

c is a good charge donor but cannot form a H-

bond; the reverse applies to f. EXPERIMENTAL PROCEDURES

Melting points were determined with a Totolli apparatus (Buchi) and are uncorrected. Specific rotations were measured with a Perkin-Elmer 241 Polarimeter. Thin-layer chromatography was on silica gel (TS; Merck F.254) using the following solvent systems: A = 1-butanol-acetic acid-water (4: 1 : 1) B = chloroform-methanol- acetic acid (5 :2 : 1) C = chloroform-merhanol-aceticacid (95 :20: 3) D = 1-butanol-pyridineacetic acid-water (4 : 1 :

1:2) Detection was with ninhydrin, the TDM reagent (Arx et al., 1976) and Ehrlich's reagent. The 'H-n .m.r . spectra were recorded with a Bruker WH-90 apparatus (TMS internal standard).

PROPERTIES OF SIX TRYPTOPHAN ANALOGUES

U.V. absorptions were determined with a off, washed with 50% ethanol, and dried to give Zeiss PMQ-3 spectrophotometer. 3.00 g (79%) of a white powder, m.p. 250252'. Diethyl a-(2-bromobenzolb]furyl3-methyI)-aacetamidomalonate

Diethyl a-acetamidomalonate (1 1.95 g, 55 mmol) in dimethyl formamide (40ml) was added to sodium methoxide (3 g, 55.2 mmol). The resulting clear solution was freed from methanol by partial evaporation. The concentrated solution was then treated with 16 g (55.2 mmol) of 2-bromo-3-bromomethylcumarone (Erlenmeyer & Grubemann, 1947). The reaction became exothermic and sodium bromide precipitated. The reaction mixture was left to cool, concentrated in vacuo, diluted with benzene, and extracted with water. The aqueous layer was discarded and the organic layer was washed with sodium hydrogen sulfate solution and with water. The dried extract was concentrated and the resulting oil was diluted with warm ethanol. Cooling gave the product (16.9 g, 72%);m.p. 116". Anal. calc. for ClaHz&IOd3r (426.26): C, 50.72; H, 4.73; N, 3.29. Found: C, 50.31; H, 4.71; N 3.14. P-(3-Benzolb]furyl)-DLalanine (k) was prepared from the above compound as described previously (Erlenmeyer & Grubemann, 1947). Diethyl a-(benzof bJ thienyl-3-methyl)*-formamidomalonate. On application of the above

prescription to diethyl a-formamidomalonate (Shaw & Nolan, 1957) and 3-chloromethylbenzo[b]thiophene (King & Nord, 1948) a yield of 81%was obtained. M.p. 104-105". Anal. calc. for Cl,Hl&IO$ (349.41): C, 58.44;

H, 5.48; N, 4.08. Found: C, 59.03; H, 5.60; N, 4.17. P-(J-Benzo/bJ thieny1)-Dlalanine (Ib). Diethyl

a a e n z o [b J thienyl-3-methy1)ill-formamidomalonate was deacylated, hydrolyzed, and decarboxylated by heating under reflux for 4 h with concentrated hydrochloric acid (6 g/100 ml). On cooling, a whte precipitate was obtained consisting of the hydrochloride of the racemic amino acid. The crude product was dissolved in 50% aqueous ethanol and the solution was neutralized with ammonia. The isoelectrically precipitated, zwitterionic product was filtered

Anal. calc. for CllHllNOzS (221.28): C, 59.71; H, 5.01 ; N, 6.33. Found: C, 59.70; H, 5.09;N, 6.40. ""-Me thy 1- L-trypto phan (Ic). L-Trypt ophan (5 g, 24.5 mmol) was dissolved in liquid ammonia (1 00 ml) and clean, finely divided sodium (1.1 5 g, 50 mmol) was then added. The characteristic, blue solution was decolorized by addition of 3.55 g (25 mmol) of methyliodide with stirring, and after 45 min the solvent was allowed to evaporate and the residue was dissolved in water. The pH was adjusted to 6-7 with hydrochloric acid and the zwitterionic product was collected by filtration, washed with water, and then dried with ethanol and ether. The product (4.1 g, 77%)was only chromatographically distinguishable from tryptophan in solvent system B. Rf 0.32 (B) (Trp Rf 0.18 (B)); [a]g= - 15.1" (C 1,9O%AcOH).

Anal. calc. for ClzHl&O2 (218.25): C, 66.03; H, 6.47;N, 12.84. Found: C, 65.82; H, 6.55; N, 12.73. Preparation o f digestible derivatives

The four racemates Ia,b, d and e were acylated with benzyloxycarbonylglycine p-nitrophenyl ester by the following general method: 100 mmol of the racemic amino acid were dissolved in 1000 ml of a mixture of acetonitrile and water (8 :2, vlv). The solution was treated with 1 15 mmol of Z-Gly-ONp and 2 10 mmol of triethylamine. The suspension was stirred at room temperature for 3-4 h. The clear, yellow solution was then concentrated in vacuo, and the residue dissolved in water after acidification to pH 5.5-6.0 with KHS04 solution; p-nitrophenol was extracted with ether. Further acidification of the water layer (to pH 2) precipitated the product which was isolated by filtration or, when incompletely precipitated, by extraction with ethyl acetate. Z-Gly-m-Bfa-OH: Yield 75%; m.p. 138-139' (from ethanol-water); Rf0.41 (C). Z-GZY-DL-B~U-OH: Yield 73%; m.p. 159-161' (from ethanol-water); Rf 0.43 (C). 75

H.M. RAJH ET AL.

Z - G Z ~ - D L - T ~ ~ ( S F ) - O HRecrystallization : from ethyl acetate-petroleum ether gave a chromatographically impure product, m.p. 137-140". Recrystallization from acetic acid/ water gave the pure product. Yield 70%; m.p. 143-145"; Rf 0.37 (C), Rf 0.66 (A). Z - Gly - D L- Trp( 6F)-OH: Yield 7 5 %; m .p . 166-167" (from acetic acid-water); Rf 0.33

((3. I -Pentafluorophenyl-2-aminoethanol(3) A mixture of acetic acid (70 ml), water (100 ml) and ethanol (35 ml) was heated to 60". A solution of 20 g (78 mmol) of l-pentafluorophenyl-:!-nitroethano1(2) (Petrov et al. 1968), dissolved in the same mixture, was added with stirring over 15 min. Meanwhile 16 g (240 mmol) of zinc-powder were added in small portions. The mixture was heated under reflux for 1 h , then cooled, and concentrated in vacuo. The residue was dissolved in ethanol and the insoluble zinc acetate was removed by filtration and washed with ethanol. The combined filtrates were evaporated, the residue was dissolved in water, the solution adjusted at pH 12 with 4 N sodium hydroxide, and then extracted with ether. The ethereal extract was washed with water, dried (Na2S04) and evaporated. The solid residue was recrystallized from carbon tetrachloride to give 3. M.p. 1 12-1 13" (Petrov et al., 1968, 116-1 17"); Yield 15.5 g (88%); Rf 0.18 (C), Rf 0.47 (B). The i.r.-spectrum was identical to that found by Petrov el al., 1968. 4 , 5 , 6 , 7-Tetrafluoroindole(4) The ringclosure of 3 t o give 4 was carried out in dimethylformamide as described by Petrov el al. (1968). The resulting product was purified by columnchromatography on aluminium oxide (neutral, Woelm). Compound 4 was eluted with hexaneether (3: 1). Yield 84%; m.p. 8990" (lit. 93-93.5"); Rf 0.66 (C), Rf 0.79 (A). The i.r.-spectrum was in agreement with that given in the literature (Petrov et al., 1968).

h. The mixture was then cooled, excess paraformaldehyde removed by filtration, and the fitrate evaporated in vacuo. The oily residue was dissolved in ethyl acetate and the extract washed with water, dried (Na2S04) and evaporated. The residue was recrystallized from ethanol-water to give the product (91%). M.p. 166-1 67" (lit. 168-1 69"); Rf 0.22 (C), Rf 0.43 (A). The 'H-n.m.r.-data were in agreement with previous values (Petrova et al., 1970). Quaternary salt of 4 , 5 , 6,7-tetrafluoro-3(Wpiperidinomethy1)indole ( 6 ) A solution ofdimethylsulfate (22 g, 175 mmol)

in THF (20 ml) was cooled to 10". The Mannich base 5 (10 g, 35 mmol), dissolved in THF (50 ml), was added dropwise over 30 min. The suspension was stored in the refrigerator overnight. Then the white solid was collected by filtration and washed with cold THF. Yield 10.3 g (74%). The product was used in the next step without further purification. Dibenzyl formamidomalonate ( 7 )

As described (Snyder & Matteson, 1957) for the preparation of the corresponding acetamidomalonate a solution of diethyl formamidomalonate (50 g, 246 mmol) in benzylalcohol (200 ml) was heated under nitrogen to 200", while 27 ml of ethanol distilled off (theoretical: 28.2 ml). Benzylalcohol was distilled off at reduced pressure, and the residue was crystallized from propanol-2. Yield 52 g (65%); m.p. 90-92"; Rf 0.75 (C), 'H-n.m.r. (CDC13): 8.24 s (lH), 7.29 s (lOH), 5 3 5 d (lH),5.19 s (4H). Anal. calc. for Cl$Il,NOs (327.34): C, 66.05; H, 5.23; N, 4.28. Found: C, 65.88; H, 5.25; N, 4.41. Dibenzyl aminomalonate-hydrochloride ( 8 ) Dibenzyl formamidomalonate (20 g, 61 mmol)

was dissolved in benzylalcohol (200 ml), which contained 2 mol hydrogen chloride per liter (dry hydrogen chloride was bubbled into benzylalcohol). The solution was stirred overnight, diluted with ether (500 ml), and the white pre4, 5 , 6, 7-Tetrafluoro-3-(N-piperidinornethyl) cipitate was collected by filtration and washed indole ( 5 ) with ether. Yield 17.4 g (85%); m.p. 148-150" Tetrafluoroindole (8 g, 43 mmol) and piperidine (83 ml, 84 mmol) were dissolved in pro- (dec.); Rf 0.70 (C), Rf0.63(A);n.m.r.(DMSOpanol-2 (150 d).Paraformaldehyde (3 g, 100 d6): 7.34 s (lOH), 5.27 two singulets coincide mmol) was added to the boiling solution over 1 at the same frequency (5H).

76

PROPERTIES OF SIX TRYPTOPHAN ANALOGUES

Anal. calc. for Cl7Hl$lC104(335.79): C, 60.81; H, 5.40;N, 4.17. Found: C, 60.01;H,5.80;N, 4.45. tert .-Butyloxycarbonylglycylaminomalonicacid dibenzyl ester (BOC-Cly-Ama(OBzl)J* ( 1 0) A solution of dibenzyl aminomalonate-hydrochloride (8) (14 g, 42 mmol) in DMF (50 ml) was treated with triethylamine (6.4 ml, 46 mmol) and BOC-Gly-ONSu ( 9 ) (10.8 g, 40 mmol) (Anderson et al., 1964). The solution was stirred overnight, evaporated in vacuo, the residue dissolved in ether and the extract washed with water, dried (Na2S04) and evaporated. The remaining oil was crystallized from ethyl acetate-petroleum ether. Yield 16.1 g (87%); m.p. 77-79"; 'H-n.m.r. (CDC13): 7.28 s (IOH), 7.16 d (lH), 5.28 d (lH), 5.17 s (4H), 3.87 d (2H), 1.45 s (9H).

in 9% acetic acid (200 ml) and hydrogenated in the presence of Pd/carbon. When hydrogen consumption was complete the catalyst was filtered off, and the filtrate was evaporated to dryness. Without further purification the product (8.7 g, Rf 0.38 (A)) was dissolved in 100 ml ofmethanol, which contained 1 ml pyridine. The solution was heated under reflux for 5 h and evaporated in vacuo, and the solid residue was washed with water. Yield 6 g (78%); Rf 0.16 (C), Rf 0.67 (A); m.p. 225" (dec.). Anal. calc. for cl2H&,&o,(304.21): c , 47.38; H, 2.65; N, 9.21. Found: C, 47.21;H, 2.46;N, 9.08. DL-4,

5 , 6 , 7-Tetrafluorotryptophan(1 3 )

Compound 12 (5.8 g, 19 mmol) was heated under reflux for 4 h in THF (100 ml), containAnal. calc. for CZ4HZ&l2Q(456.49): c, 63.15; ing 2 mol hydrogen chloride per liter. After H, 6.18;N, 6.14. Found: C, 63.02;H,6.25;N, removal of the solvent the solid residue was refluxed for another 4 h in conc. HCI (50 ml). On 6.24. cooling the product crystallized as its hydrochloride. The salt was dissolved in water, the Dib en zy I a-(4 , 5 , 6 , 7-te trafluoroindoly 1-3pH was adjusted to 7 with 4 N sodium hydroxide. methyl)*-formamidomalonate (1 1) The white precipitate was collected by fdtraA solution of dibenzyl formamidomalonate tion and washed with water. Yield 3.8 g (72%); (10.14 g, 3 1 mmol) in DMF (50 ml) was treated with 1.49 g of a sodium hydride emulsion, con- Rf 0.48 (A), Rf 0.56 (D); m.p. 258-260'. taining 50%NaH (3 1 mmol). When the hydrogen Anal. calc. for CllHsF4NzOZ(276.20): C, 47.84; evolution had ceased, 8.69 g (21 mmol) of the H, 2 9 2 ; N , 10.14. Found: C,47.72; H, 3.21;N, quaternary salt ( 6 ) and 10 ml of dioxan were 9.98. added at once. Within 15 min a clear solution was obtained. After 2 h the solvent was removed under reduced pressure and the oily residue Dibenzyla-(4, 5 , 6, 7-tetrafluoroindolyl-3dropped out in water. The white solid precipi- methyl)a-tert.-butybxycarbonylglycylamidotated and was collected by filtration and washed malonate (14) with water and methanol. Yield 12.4 g (95%); A solution of BOC-Gly-Ama(OBzl)z (20) (8.4 g, m.p. 148-151' (dec.); Rf 0.72 (C), Rf 0.81 18.4 mmol) in DMF (50 ml) was treated with (A); 'H-nm.r. (CD3COCD3): 8.39 s (lH), 8.16 0.9 g of a sodium hydride emulsion, containing s (lH), 7.48 s (lOH), 7.35 s (lH), 5.32 s(4H), 50% NaH (18.8 mmol). The further procedure was the same as described for the preparation 4.06 s (2H). Anal.Calc.for Cl7HleNC104(335.79): C,60.81; of (21). The resulting product was crystallized 61.37; H, 3.81; N, 5.30. Found: C, 61.i4; H, from methanol-water. Yield 6.7 g (8%); m.p. 141-143"; Rf 0.73 (C), Rf 0.85 (A); 'H-n.m.r. 4.26; N, 5.1 1. (CDClj): 8.73 s (lH), 7.24 s (lOH), 6.89 s (lH), 5.11 s (4H), 3.89 s (2H), 3.77 d (2H), 1.38 s (9H). N-Formyl-4, 5 , 6 , 7-tetrafluoro-~~-tryptophan (12) Anal. calc. for C3&31Fd307 (657.63): C, Compound 12 (13.4 g, 25 mmo1)was suspended 60.27; H, 4.75; N, 639. Found: C, 60.49; H, *The symbol Ama was chosen for aminomalonic acid. 5.11;N,6.32. 77

H.M. RAJH ET AL.

and elution with 3 N acetic acid. Ninhydrin positive fractions were pooled, evaporated in vacuo, and dried. The residues were washed with water, ethanol and finally ether. Physical the conversion of I 1 into 12. The product was constants and elemental analyses are given in crystallized from methanol-water. Yield 3.5 g Table 2. (79%); m.p. 165-166"; Rf 0.25 (C), Rf 0.76 (A); 'H-nmr. (CD3COCD3): 11.01 s (lH), 7.35 Charge-donor measurements s (lH), 4.71 m (lH), 3.73 d (2H), 3.33 m (2H), Indole and 4, 5 , 6,7-tetrafluoroindole (4) were purified as described for the latter by column 1.34 s (9H). chromatography on aluminium oxide (neutral, Anal. calc. for C18H1&OSF4 (433.37): C, Woelm) with n-hexaneether (3 : 1) as an eluent. 49.89; H, 4.42; N, 9.70. Found: C, 49.34; H, 5 - and 6-Fluorohdole (Brunschwig chemie) and 4.48; N, 9.79. N-methylindole (Fluka AG) were obtained b . By coupling of Boc-Gly-ONSu (9) and D L ~ , commercially and used without further purifica5 , 6. 7-tetrafluorotryptophan (1 3). A suspen- tion. Benzofuran (Aldrich) was freshly distilled. sion of compound 13 (3.4 g, 12.6 mmol) in Tetracyanoethylene was sublimated twice DMF (50 ml) was treated with triethylamine (125"/4 mm). (3.5 ml, 25.2 mmol) and BocCly-ONSu (3.5 g, The measurements were carried out at room 12.8 nunol). The mixture was stirred overnight temperature in dichloromethane (Merck), and the resulting solution evaporated in vacuo. which contained 0.3% ethanol as the solvent The residue was dissolved in ethyl acetate, and and in a closed quartz cell of 1 cm. the extract washed with water, dried (Na$04), In the experiments with indole, 5-fluoroand evaporated. The residue was crystallized indole and 6-fluoroindole varying amounts of from methanol-water. Yield 5.4 g (98%). The the solid donor were weighed in a cuvette. Then physical constants of the isolated product were 2 ml of a 5.104 M solution of TCNE in diidentical with those of the product described chloromethane were added. After dissolution under (a). the spectrum was traced from 400-600 nm as Enzymatic digestion fast as possible. Carboxypeptidase A (25 mg) was suspended in The amounts of the donors were chosen in 1 ml 0.1 N sodium hydrogen carbonate solu- such a way that their concentrations varied tion and dissolved by addition of 0.1 N sodium between 0.05 and 0.4 mol/l. The correction for hydroxide to pH 11. The solution was cooled the concomitant volume expansion was calcuand adjusted to pH 8.0 with 0.1 N hydro- lated from the formula Vy = Vo + y.O.0008 chloric acid. This quantity of enzyme was used (Overby & Ingersoll, 195I), where Vy = volume for the digestion o f 100 mmol of a racemic sub- after addition of y mg of the solid acceptor to strate. Each of the dipeptide derivatives were Vo ml of solution. The absorptions were dissolved in aqueous Nethylmorpholine (0.25 measured at eight donor concentrations. For M) and the solutions were made 0.2 M with 4 , 5 , 6 , 7-tetrafluoroindole the TCNE concenrespect t o the racemates. Sufficient glacial tration was 5.10-3 moll1 and the measurements acetic acid was added t o give pH 8.0. Then an were carried out at eight different donor conaliquot of the enzyme solution was added. centrations varying between 0.1 and 0.4 mol/l. Usually, the digestion was complete within 2 For benzothiophene and benzofuran (filled h ; the dipeptides derived from Ia, b and f gave a in the cuvette with a syringe) the TCNE concenheavy precipitate. A quantitative recovery of tration was 8.104 molll. Ten different donor the Lenantiomer was only possible by filtration concentrations were chosen, varying from 0.1 in the case of H-Bta-OH. to 0.8 mol/l. H-Bfa-OH and H-Trp(tF)-OH had to be, ACKNOWLEDGMENTS concentrated before filtration. The monofluorinated tryptophans (Id and e ) were isolated We thank Prof. Dr. R . J . F. Nivard for his kind interest after concentration of their digests, adsorption and clarifying comments, and Mrs. H.Wigman-Roeffen onto Biorad AG 1-X2 (acetate cycle), washing for her assistance in preparing the manuscript. Boc-Glycyl-(4,5 , 6, 7 - t e t r a f i u o r o ) - ~ ~ - t r y p t o phan (1 5) a. By consecutive hydrogenation and decarboxylation of 14. The procedure was the same as for

78

PROPERTIES OF SIX TRYPTOPHAN ANALOGUES

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Overby, L.R. & Ingersoll, A.W. (1951)J. Am. Chem. SOC.73,3363-3366 Paetzold, R. (1975)2. Chem. 15 (lo), 377-393 Petrov, V.P., Barkhash, V.A., Shchegoleva, G.S., Petrova, T.D., Savchenko, T.I. & Yakobson, G.G. (1968) Dokl. Akad. Nauk. SSSR 178, 864-867 Petrova, T.D., Savchenko, T.I., Shchegoleva, L.N., Ardyukova, T.F. & Yakobson, G.G. (1970)Khim. Geterotsikl. Soedin. 10,1344- 1347 Petrova, T.D., Savchenko, T.I., Ardyukova, T.F. & Yakobson, G.G. (1971)Khim. Geterosikl. Soedin.

2,213-214 Prasad, K.U., Roeske, R.W., Weitl, F.L., VilchezMartinez, J.A. & Schally, A.V. (1975)in Proceedings of the 4th American Peptide Symposium pp. 871876 (Walter, R. & Meienhofer, J., 4 s . ) Shaw, K.N.F. & Nolan, C. (1957)J. Org. Chem. 22,

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