Int. J. Peptide Protein Res. 16,1980,267-279 SYNTHESIS OF ANALOGS OF THE SERUM THYMIC NONAPEPTIDE, "FACTEUR THYMIQUE SERIQUE" (FTS) Part 11 JEAN MARTINEZ,* DIDIER BLANOT, GENEVIEVE AUGER, ANDRE SASAKI and EVANGHELOS BRICAS Research Unit 15, CNRS, Peptide Laboratory, Institute of' Biochemistry, University o f Paris-South, Orsay, France Received 5 February, accepted for publication 7 April 1980 New analogs of FTS (Facteur Thymique Serique), <Glu-Ala-Lys-Ser-Gln-Gly- Gly-Ser-Asn . OH, a circulating thymic factor, were prepared by replacing the amino acid residues in positions 1, 3, 4, 5, 6 and 3 and 6 together. Five other analogs tjf C-terminal heptapeptide were prepared by replacing the amino acid residues in position 3 or 6. These peptides were synthesized using conventional synthesis in solution. Key words: analogs; circulating thymic factor; "Facteur Thyrnique Skrique" FTS; peptide 1 2 3 4 5 6 7 8 9 synthesis. In the first part of this work (Blanot ef al., 1979a), we described the synthesis of 14 analogs of a peptide designated FTS, "facteur thymique serique", having the structure: <GIu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH 1 2 3 4 5 6 7 8 9 This nonapeptide has been isolated by Bach et al. (1976, 1977) and is defined by its ca- pacity to induce @-antigen on 0-negative rosette- forming spleen cells. Syntheses of this com- pound have been carried out by the con- ventional method in solution (Bricas et al., 1977) and by the solid phase method (Fukuda et al., 1978; Strachan et al., 1979; Wan & Folkers, 1979). In the second part of this work, we wish to report the preparation of: (1) 17 nonapeptide analogs modified in one of the positions 1,3,4, 5, or 6: ID-< Glu'] -FTS** [D-Gh' ] -FTS [Gh' ] -FTS [Pro' ] -FTS [D-LyS3] -FTS [orn3 1 -FTS [ Arg3 ] -FTS [N"-Ac-Lys3 ] -FTS [ Har3 ] -FTS [Hep3] -FTS [D-Ser4 ] -FTS [Ma" ] -FTS [Asn'] -FTS [Was ] -FTS [~-Gln~] -FTS ~ ~ 1 ~ 5 1 -FTS [ D-da6 ] -FTS (2) one nonapeptide analog modified in [iV-Ac-Lys3,D-Ala6 ] -FTS *Present address: Equipe de Recherche NO. 19.5 du both positions 3 and 6: CNRS, Ecole Nationale SupCrieure de Chimie, 34075 ~fontpellier, France. 0367-8377/80/040267-13 $02.00/0 0 1980 Munksgaard, Copenhagen
Transcript
Int. J. Peptide Protein Res. 16,1980,267-279
S Y N T H E S I S OF A N A L O G S O F T H E S E R U M THYMIC N O N A P E P T I D E , " F A C T E U R T H Y M I Q U E S E R I Q U E " (FTS)
Part 11
JEAN MARTINEZ,* DIDIER BLANOT, GENEVIEVE AUGER, ANDRE SASAKI and EVANGHELOS BRICAS
Research Unit 15, CNRS, Peptide Laboratory, Institute of' Biochemistry, University o f Paris-South, Orsay, France
Received 5 February, accepted for publication 7 April 1980
New analogs of FTS (Facteur Thymique Serique), <Glu-Ala-Lys-Ser-Gln-Gly-
Gly-Ser-Asn . OH, a circulating thymic factor, were prepared by replacing the
amino acid residues in positions 1 , 3, 4 , 5, 6 and 3 and 6 together. Five other analogs tjf C-terminal heptapeptide were prepared by replacing the amino acid residues in position 3 or 6. These peptides were synthesized using conventional synthesis in solution.
In the first part of this work (Blanot ef al., 1979a), we described the synthesis of 14 analogs of a peptide designated FTS, "facteur thymique serique", having the structure:
This nonapeptide has been isolated by Bach et al. (1976, 1977) and is defined by its ca- pacity to induce @-antigen on 0-negative rosette- forming spleen cells. Syntheses of this com- pound have been carried out by the con- ventional method in solution (Bricas et al., 1977) and by the solid phase method (Fukuda
et al., 1978; Strachan et al., 1979; Wan & Folkers, 1979).
In the second part of this work, we wish to report the preparation of:
(1) 17 nonapeptide analogs modified in one of the positions 1 , 3 , 4 , 5 , or 6:
ID-< Glu'] -FTS** [D-Gh' ] -FTS [Gh' ] -FTS [Pro' ] -FTS [D-LyS3] -FTS [orn3 1 -FTS [ A r g 3 ] -FTS
[N"-Ac-Lys3 ] -FTS [ Har3 ] -FTS
[Hep3 ] -FTS [D-Ser4 ] -FTS [Ma" ] -FTS
[Asn'] -FTS
[Was ] -FTS
[ ~ - G l n ~ ] -FTS
~ ~ 1 ~ 5 1 -FTS
[ D - d a 6 ] -FTS
( 2 ) one nonapeptide analog modified in
[iV-Ac-Lys3, D-Ala6 ] -FTS
*Present address: Equipe de Recherche NO. 19.5 du both positions 3 and 6 : CNRS, Ecole Nationale SupCrieure de Chimie, 34075 ~fontpellier, France.
and (3) five C-terminal heptapeptide analogs modified in position 3 or position 6:
(des-<Glu’ , Ala2)[iVE-Ac-Lys3] -FTS (des-<Glu’ , Ala2)[Orn3]-FTS (des-<Glu’ , Ma2)[D-Lys3] -FTS (des-<Glu’, Ala2)[Hep3] -FTS (des-<Glu’, MaZ)[D-Ma6] -FTS The fragment condensation route was
chosen in association with a stepwise synthesis of the fragments by conventional methods in solution.
Analogs modified in position I were pre- pared according to the scheme of synthesis of the [D-Ala2]-FTS and the shorter-chain analogs, described in Part I (Blanot et al., 1979a), using ~-<Glu-OTcp, or the mixed anhydrides of Z-D-Gln(Mbh)-OH, Boc-Pro-OH or Z-Glu(OBut)-OH.
A scheme related to the preceding one was used for the preparation of analogs modi- fied in position 3: in this variation, Gln’ was not protected. The synthesis was carried out by the following steps:
(a) saponification of Z-Ser(But)Gln-Gly-Gly- OMe (Blanot et al., 1 9 7 9 ~ ) followed by coup- ling, by the mixed anhydride procedure, with the partially protected dipeptide derivative H-Ser(But)-Asn-OBut (Blanot el al., 1979a),
**Abbreviations used: the customary L-indication for amino acid residues is omitted, only enantiomers are indicated by D. Standard abbreviations for amino acids and derivatives are those recommended by the IUPAC-IUB Commission on Biochemical Nomen- clature (1972) Biochemistry 11, 1726-1732. Other abbreviations used are: Ac, acetyi; AcOEt, ethyl acetate, Boc, ferf.-butyloxycarbonyl, But, lert.- butyl, DCHA, dicyclohexylamine; DMF, dimethyl- formamide; <Glu, L-pyroglutamic acid; Har, homo- arginine; Hep, heptyline (2-aminoheptanoic acid); HFTLC, high performance thin-layer chromatography; Mbh, 4,4’dimethoxybenzhydryl; Me, methyl; MeOH, methanol; Nva, norvaline; Nps, o-nitrophenylsulfenyl; Pcp, pentachlorophenyl; Tcp, trichlorophenyl; THF, tetrahydrofuran; Z, benzyloxycarbonyl.
The analogs of FTS obtained by replacing, for instance, the L-glutamine in position 5 by L-asparagine or L-glutamic acid are abbreviated as [Asn’l-FTS and [Glu’ 1 -FTS, whereas the shorter chain analogs obtained by omission of one or several amino acid residues are abbreviated as (des-<Glu’ , Ala’)[D- Ala6 ] -FTS for the heptapeptide H-Lys-SerCln- D- AlaCly-Ser-Asn-OH.
268
(b) coupling of the mixed anhydrides of Z-Lys(Ac)-OH, Z-Hep-OH, Nps-~-Lys(Boc)-0H or Nps-Orn(Boc)-OH with the partially protected hexape tide derivative H-Ser(But)-Gln-G1y -GI?.
(c) removal of the h@-protecting group by catalytic hydrogenolysis for the Z group, or pyridinium hydrogen bromide/indole for the ~ p s group (Konig, 1971 ; Klostermeyer & Sch- wertner, 1973) and coupling of <Clu-Ala-N, with the partially protecled heptapeptide derivatives thus obtained;
(d) purification of the protected nona- peptide derivatives by preparative t.1.c. and removal of all the protecting groups by CF3COOH/anisole in order to obtain [W- Ac-Lys3 ] -FTS, [ Hep3 ] -FTS, [ D-Lys3 ] -FTS and [Om3 ] -FTS. The corresponding hepta- peptide analogs were obtained after the same acidolytic treatment of the partially protected heptapeptide derivatives obtained in (c).
Analogs modified in position 4 were pre- pared, without protection of G h S , by the following steps:
(a) synthesis of Z-Gln-Gly-Gly-OMe, hydra- zinolysis of the methyl ester and coupling, by the azide method, with the partially pro- tected dipeptide derivative H-Ser(But)-Asn- OBut (Blanot er al., 1979~) ;
(b)coupling of Z-D-Ser-OPcp or of the mixed anhydride of Z-Ala-OH with the par- tially protected pentapeptide derivative H-Gln-Gly -Gly-Ser(But)-Asn-OBut,
(c) coupling of the tripeptide azide deriva- tive <Glu-Ma-Lys(Boc)-N, with the partially prctected hexapeptide derivatives thus ob- tained;
(d) removal of all the protecting groups by CF3COOH/anisole in order to obtain [ D -Ser4 ] -FTS and [ Ma4 ] -FTS.t
Analogs modified in position 5 were ob- tained by the following steps:
(a)coupling, by the mixed anhydride pro-
Ser(Bu P )-Asn-OBut;
~
tThe absence of racemization of the Lys3 residue in [Ah4 ] -FTS was proved by the complete splitting of the Lys3-Ala4 bond by trypsin. In the case of [D-Ser4]-FTS, a faint quantity of nonapeptide was not split by trypsin: this is probably due to the slower hydrolysis of the Lys3-D-Ser4 bond by this enzyme.
SYNTHESIS OF FTS ANALOGS
TABLE 1 Physicochemical properties of protected fitial nonapeptide derivatives modified in positiorl I
Compound Melting point [a] ,,(c) Yield Elemental analysis (" C) in DMF (upper: calc.; lower: found)
cedure, of Z-Gly-Gly-OH with the partially protected dipeptide derivative H-Ser(But)-Asn- OBut (Blanot et al., 1979a),
(b) coupling, by the mixed anhydride procedure, of Z-D-Gln(Mbh)-OH, Z-Glu(OBut)- OH, Z-Asn(Mbh)-OH or Z-Nva-OH with the partially protected tetrapeptide derivative H-Gly-Gly-Ser(But)-Asn-OBut ;
(c) synthesis of the protected nonapeptide derivatives by stepwise elongation using Z- Ser(But)-OH, Nps-Lys(Boc)-OH, Z-Ala-OH (mixed anhydrides) and <Glu-OTcp;tf
(d) removal of all the protecting groups by CF3COOH/anisole in order to obtain [D- Gin5 ] -FTS, [Glu' ] -FTS, [AmS ] -FTS and [ h a S ] -FTS
The analog modified in position 6 [D- d a 6 ] - F T S was obtained by the following steps:
(a) stepwise synthesis of Z-Lys(Boc)-Ser (But )-Gln(Mbh)-D-Ma-Gly-OMe 1
f 4 The poor yields obtained for the coupling of ':Glu-OTcp with the partially protected octapeptide derivatives modified in position 5 may be due to the formation of a byproduct from the amino component. Such a side-reaction has already been observed during the synthesis of [B-Ala-NH; ] -FTS (Blanot et a[., 1979~).
L L -
(b) saponification of the methyl ester and coupling, by the mixed anhydride procedure. with the partially protected dipeptide deriva- tive H-Ser(But)-Asn-OBu' (Blanot eral.. 197%).
(c) synthesis of the protected nonapeptide derivative by stepwise elongation using Z-Ala- OH (mixed anhydride) and <Glu-OTcp;
(d) removal of all the protecting groups by CF,COOH/anisole in order to obtain [ D - d a 6 ] -FTS. The corresponding heptapeptide analog was prepared by the same acidolytic treatment of the partially protected hepta- peptide derivative H-Lys(Boc)-Ser(Bu')-Gln (Mbh)-D-Ala-Gly-Ser(But)-Asn-OBut.
The double-modified arialogs [iVE-Ac-Lys3 , D - d a 6 ] -FTS were prepared by acetylation of [D-da6 I -FTS according to the method of Reboud-Ravaud & Ghelis (1976) using I%'- acetyl-benzotriazole. The product was purified by preparative t.1.c.
The analogs [A$ f -FTS and [Ha? J-FTS were obtained by guanidination of synthetic [Om3] -FTS and FTS, respectively, using 0-methylisourea. The guanidinated products. which gave Sakaguchi reaction, could be distinguished from the starting peptides by high voltage paper electrophoresis. The yield of the reaction, as judged by the intensities
269
J. 51.4RTINEZ ET AL.
TABLE 2 Phyricochemical properties of peptide derivatiives modified in position 3
-
Compound Melting point [ajD(c) Yield Elemental ad;.ds to C ) in DMF (upper: calc.; lower: foun5
Recrystallization solvents: (a) MeOH/ether; (b) MeOH/AcOEt; (c) H,O; (d) MeOH; (e) DMP/ether. *Calculated with 1 mol of MeOH. **Calculated with 1 mol of H,O. ?See amino acid analyses of the corresponding deprotected nonapeptide in Table 7. Homogeneous by t.1.c. in three different solvent systems.
IX <Glu-Ala-Om(Boc)-Ser(But)-Gln- dec. without -2.7" (0.62) 58% - - -t
-.
of coloration of the products with the modi- fied chlorine reagent (von Arx e t al., 1976), was about 70%. However, the yield of recovery of the guanidinated products, purified by preparative high voltage paper electrophoresis, was much lower because of partial overlapping of the spots of the starting and guanidinated products.
The characterization and the purity of the synthesized derivatives were established by elemental analysis, melting point, optical rotation an3 t.1.c. in different solvent systems (Tables 1-6) and those of the final peptides by amino acid analysis of acid hydrolysates, optical rotation, HPTLC in several solvent
270
systems and high voltage electrophoresis (Tables 7 and 8).
The biological and immunological activities of the FTS analogs described in the two parts of this paper have been determined with the collaboration of J.F. Bach, M. Dardenne and J.M. PlCau and are reported elsewhere (Bach et al., 1978; Blanot eral., 1979b).
EXPERIMENTAL PROCEDURES
General procedures are the same as those already described in Part I (Blanot et ul.,
In the following, we describe two examples 19794.
SYNTHESIS OF FTS ANALOGS
TABLE 3 Physicochemical properties of peptide derivatives modified in position 4
Compound Melting point [aID(c) Yield Elemental analysis (" C) in DMF (upper: calc.; lower: found)
C H N
VIII <Glu-Ala-NHNHBoc (a) broad m.p. -72.1"(0.37)* 51% 48.54 7.57 16.17** from 79" 48.54 7.33 16.55
XVI <Glu-Ma-Lys(Boc)NHNH-Z (b) broad m.p. -18.5"(0.8) 767c 56.24 6.99 14.57 from 130" 55.84 6.97 14.14
XVII <Glu-Ala-Lys(Boc)-DSer-Gln- semi-solid -2.0" (1) 70% GIYGly-Ser(But)-Asn-OBut product
- - XIV Z-D-Ser-GlnGlyGly-Ser(But)- semi-solid -1.2" (1) 65% -
.L - . - -
Recrystallization solvents: (a) MeOH/ether; (b) Ethanol/ether; (c ) MeOH; (d) DMF/ether; (e) H,O. *In MeOH. **Calculated with 1 mol of MeOH. :**Calculated with 1 mol of H,O. fSee amino acid analyses of the corresponding deprotected nonapeptide in Table 7. Homogeneous by t.1.c. in three different solvent-sy stems.
of the synthesis of FTS analogs, as well as the acetylation and guanidination procedures. Yields and physical constants of the intermediate Protected compounds are given in Tables 1-6, and physical constants and amino acid analyses of the FTS analogs are given in Tables 7 and 8.
Synthesis of [ O m 3 J-FTS
Z-Ser(But)-Gln-Giy-Gly-OH (I). 6.1 g (12.1 mmol) of Z-Ser(But)-Gln-Gly-Gly-OMe (Blanot et al., 1979a) were dissolved in 670ml of 90% MeOH. 25.3ml of N NaOH were added in two portions. After continuous stirring for 3 11, the mixture was neutralized by 25.3 ml of N HCl and evaporated in vacuo. The residue was taken up in MeOH. After filtration, ether
was added to the solution: the product precipi- tated; 6.1 g were obtained; Rf(B) = 0.58, Rf(E) = 0.07.
Z-Ser(Buf)-Gin-Gly-Gly-Ser(Bu')-Asn-OBu' (II). 3.14g (5.85mmol) of (I) were coupled with 2.92 g (7.46 mmol) of H-Ser(But)-Asn-OBu'. CH,COOH (Blanot et al., 1979~) by the mixed anhydride procedure in DMF using isobutyl chlorocarbonate and N-methylmor- pholine. After filtration, the reaction mixture was evaporated and the residue was triturated in ether. The product was filtered, triturated in hot chloroform and recrystallized in MeOH/ ether, to yield 3.05g of (11); Rf(A) = 0.68, Rf(B) = 0.84, Rf(C) = 0.43.
27 1
J. MARTINEZ ET AL.
TABLE 4 Physicochemical properties of' peptide derivatives modified in position 5
.____ - Compound Melting point [aID(c) Yield Elemental analysis
Recrystallization solvents: (a) THF/petroleum ether; (b) MeOH/H,O; (c) DMF/ether. *In MeOH. t S : calc. 2.42, found 2.32. t f S : calc. 2.57, found 2.79.
H-Ser(But)-Gln -Gly -Gly -Ser(Bu') -Am -OBut . CH3C00H (IIZ) 1.5 g (1.76mmol) of (11) were dissolved in a mixture of 200ml MeOH, 2ml acetic acid and 2ml water, and hydro- genated for 4 h at atmospheric pressure with 5% Pd/C as catalyst. The catalyst was fitered off and the filtrate was evaporated in vacuo. The residue was taken up several times with benzene and dried. After trituration in ether, 1.19g of (111) were obtained (yield: 87%). Rf(A) = 0.42.
Nps-Om(Boc)-OH. DCHA (I V). This derivative was prepared according to the method of Barral & Savrda (1973), by the action of
212
o-nitrophenylsulfenyl-thiocyanate (Lecher & Simon, 1921) on the copper complex of N 6 -Boc-ornithine (Marchiori et al., 1967). Recrystallization in ethanol. Yield: 61%, m.p. 183-184" (dec.), [a] D = -34.6' (c 1, CHC13).*
*The same procedure, with the copper complex of NE-Boc-D-lysine, was used for the preparation of a-Nps-~-Boc-D-Lys-OH. DCHA: yield, 58%; m.p. 189-
OH-DCHA was prepared according to Benoiton (1963). Z-Hep-OH was prepared by benzyloxycar- bonylation of L-hepryline obtained by enzymatic resolution of DL-heptyline according to Sanborn & Hein (1968): yield, 94%; oil; Rf(E) = 0.65.
194" ; [a] D = + 34.7" (C 1.48, CHCI, ). Z-LyNAcF
SYNTHESIS OF FTS ANALOGS
TABLE 5 Physicochemical properties of peptide derivatives modified in position 5
Compound Melting point [a ID ( c ) Yield Elemental analysis (" C) in DMF (upper: calc., lower: found)
Recrystallization solvents: (a) hleOH/H,O; (b) DMF/ether; (c) MeOH. *In MeOH. tS: calc. 2.45, found 2.45. ttS: calc. 2.99, found 2.86.
i'Vps-Om (Boc) -Ser (But) -Gln -Gly -Gly -Ser(But) - Asn-OBut (V). 85mg (0.15mmol) of (IV) were converted into free acid by the method of Spangenberg et al. ( 1 971). Nps-Orn(Boc)- OH was then coupled with 90 rng (0.1 2 mrnol) of (111) by the mixed anhydride procedure as previously described. The residue, resulting from the evaporation of the solvent, was triturated in ether, then in water. 83mg of (v) were obtained; Rf(A) = 0.66, Rf(E) = 0.68.
H-Om (Boc~-Ser(But)-Gln-Gl.~Gl~~-Ser(But)-As~- OBut- Hbr (LY) 65mg (0.06mmol) of ( v ) were dissolved in DMF. 14mg (0.12mrnol) of indole and 10 ing (0.06 mmol) of pyridinium bromide were added. and the reaction mixture
was stirred for 14 h. After evaporation of DMF, the residue was triturated in ether to yield 60mg of (VI) (loo%), RdA) = 0.51, Rf(E) = 0.06.
H-Ala-NHNHBoc. CH,COOH (VII)- 2.32 g (6.9 mmol) of Z-Ala-NHNHBoc (Yanaihara et al., 1973) were hydrogenated as previously de- scribed. 1.81 g were obtained (yield: low), Rf(A) = 0.47.
<Glu-Ala-NHNHBoc (YIIIj 1.81 g (6.9 mmol) of (VII) were dissolved in 2Oml of DMF. 2.16g (7 mmol) of <Glu-OTcp (Anderson et al., 1967) and 0.98ml (7mmol) of tri- ethylamine were added. After stirring over- night at room temperature. the reaction mix-
113
J. MARTINEZ ET AL.
TABLE 6 Phjlsicochernical properties of pepride derivatives modiBed in position 6
Compound Melting point [(Y],,(c) Yield Elemental analysis
Recrystallization solvents: (a) acetone/hexane; (b) MeOH; (c) DMF/H,O. *Calculated with 2 mol of H,O. ?M.p. of the L-isomer 94-96' (Poroshin et ul., 1961) and 98-99" (Zervasel al. 1963).
ture was poured into 200ml water and washed three times with AcOEt in order to remove trichlorophenol. The aqueous phase was then concentrated to cu. 8Oml, it was saturated with NaCl and extracted with 1 liter of AcOEt. After drying and evaporation of AcOEt, ether was added: 1.13g of a slightly hygroscopic solid were obtained; Rf(A) = 0.64, Rf(E) = 0.63.
< Glu-Ala-Om (Bocj-Ser(Bu f)-Gln-Gly-Gly-Ser- (Buf)-Asn-03uf (ZX). 28.3 mg (0.09 mmol) of (VIII) were treated by 0.8ml anhydrous CF3COOH for 30min. After evaporation of the acid without heating, the residue was triturated in ether. After drying for 2-3h on P, O5 and KOH, 29.5 mg <Glu-Ala-NHNH, . CF3COOH were obtained (yield: 100%) as a very hygroscopic white powder, Rf(A) = 0.26. This unstable compound was used im- mediately for the next operation: <Glu-Ala-
274
NHNHz -CF3COOH (0.09mmol) was dissolved in 5 ml DMF. After cooling at -5", 0.1 17ml M NaNO, (0.117mmol) and 0.45ml N HCI (0.45 mmol) were added and, after lOmin, followed by 0.1 26 ml (0.9 mmol) of triethyla- mine, and 59 mg (0.06 mmol) of (VI) dissolved in 2 ml DMF. The reaction mixture was stirred for 1 h at -5" and 6 0 h at +4". It was evapor- ated in vacuo, and the residue was triturated in ether. The crude product thus obtained was purified by preparative t1.c. (solvent system D) to yield 39mg of (IX), homogeneous by t.l.C.; %(A) = 0.44, Rf(C) = 0.13, Rf(D)= 0.35. [Om3]-FTS. 12.5 mg (0.011 mmol) of (K) were dissolved in 4 ml of a 10 : 1 mixture of trifluoroacetic acid and anisole. After 3h at room temperature, the acid was evaporated in vacuo without heating. The residue was taken up in 20ml water and washed with AcOEt ( l O m l ) and ether (2 x l O m l ) . After
TA
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0.40
) I
Asp,.,,Ser,.,,Glu,.,,Gl~,.,,Ala,.,,L~s,.,,
Hig
h pe
rfor
rnan
ce t
hinh
yer
chro
mat
ogra
phie
s w
ere
carr
ied
out o
n M
erck
HPT
LC p
late
s of
sili
ca g
el 6
01~,
,, (1
0 x
10) u
sing
the
follo
win
g so
lven
t sys
teiii
s:
I, e
than
ol-M
am
mon
ium
ace
tate
7:3
. v/
v; J
, pr
opan
-1-0
1-w
ater
2:
1,
v/v;
K,
chlo
rofo
rm-m
etha
nol-
conc
. am
mon
ium
hyd
roxi
de 2
:2: 1
, V/V
; Id, c
hlor
ofor
m-
met
hano
l-co
nc. a
mm
oniu
m h
ydro
xide
I :2
: 1, v
/v.
*Ele
ctro
phor
etic
mob
ility
at p
H 1
.9, 3
7.5
V/c
m, 4
th;
MA^
^: di
stan
ce r
un b
y th
e pe
ptid
e/di
stan
ce r
un b
y as
part
ic a
cid.
**
Ele
ctro
phor
etic
mob
ility
at p
tl 4
.0,
33 V
/cm
, 3 11
. $C
orre
cted
with
the
ave
rage
rec
over
y of
pep
tide
give
n by
the
am
ino
acid
ana
lysi
s.
0.18
I
0.01
0.
33
0.60
I
--
I -
I -7
8.7"
(c
0.6
9)
I A
sp,.,
,Ser
,.,,
Glu
,.,,
LY
S~
.~,(
~~
Y~
.~~
A
la,.,
, Pr
o,.,,
j 0.
31
I 0.
08 1
0.24
I
0.40
I
0.83
,
-
[ Ild
] -FT
S I
0.36
I
0.12
I0.
18
0.25
0.
80
1 -~
I
~~
' A
spl.O
l Ser
l.yl G
1uZ.
07G
1Y2.0
0 H
3r1.
03
LYsO
.OS
--L
-.-.l
-I---L
TAB
LE 8
Pl
iysi
coch
emic
al p
rope
rtie
s an
d am
ino
acid
ana
lyse
s of
FTS
ana
logs
I I
I I i &(I
)
Com
poun
d I
HPT
LC v
alue
s i
I I
Am
ino
acid
ana
lysi
s (6
N H
CI, 1
05",
24
h)
Rf(
J) R
t(K
) R
f(L)
M
~sp
* j M
ASP
**~ [
QID
(in %
Olt
[ D-G
ln'
] -FT
S '0
.31
0.08
0.
24
0.39
I
-
I -
48.0" (c
0.65
)tt I
Asp
,.,,
Ser,
.,,G
lu,.,
,G~~
,.,,
Ala
,.,,
LYS,
.,,
[Glu
' 1-F
TS
:0.2
8 0.
35
0.27
0.
52
I -
I -
, -59.
5" (
c 0.
55)'f
t I
Asp
,.,,
Ser,
.,,G
lu,.,
,Gl~
,.,, M
a,.,9
LY
S,.,,
:0
.30
0.07
0.
23
0.37
4
-
I -
, -50.
lo(c
0.68
)tt 1
A~P,.,,S~~,.,,G~~,.,,~~Y,.,,A~~,.,,LY~,.,,
[Am
' ] -F
TS
[ Nva
' ] -
FTS
l 0.4
6 0.
23
0.39
0.
62
-
[ D-A
la6 ]
-FT
S 1 0
.36
0.12
0.
32
0.47
0.
83
I 1.1
5 -5
5.30
(~
0.3
9)
1 Asp,.,,Ser,.,,Glu,.,,~~~,.,,Ala,.,,
LYS,
.,,
(des
-iG
lu',
Ala
z)[N
f-A
c-L
ys3]
-FT
S I 0.
38
0.23
0.
55
0.68
-
I -
1 -2
6.3"
(c0.
57)
Asp,,,,Ser,.,,Glu,.,,Gl~,.,,L~s,.,,
(des
-<G
lu*,
Ala
')[D
-Lys
']-FT
S 10
.18
0 0.
16
0.30
-
i -
1 48
.9"(
c0.6
2)
I A
sp,.,
, S
er,.
,,~
lu,.
,,~
l~,.
~,
LYS,
.,, (d
es-<
Glu
', A
la2)
[Om
3]-F
TS
10.1
5 0
0.14
0.
29
I -
I -
{ -34
.7"(
c 0.
50)
I A
sp,,,
, Se
r,.,,
Glu
,.,,
~~
~,.
,,O
r~,.
,,
(des
-<C
lu',
Ala
2)[H
ep3]
-FT
S 10
.54
0.45
0.
60
0.81
-
- -3
0.7"
(c 0
.79)
{
Asp
,.,,
Ser,.
,,Glu
,.,,G
lY,.,
, H
e~0.
98
(des
-<C
lu',
Ala
Z)[
D-A
la']-
FTS
10.2
0 0
0.22
0.
35
-
I -
, -15.
9"(c
0.
49)
I A
sp,,,
,Ser
,.,,G
lu,.,
,~ly
,.,,
Ma,
.,,
LY
S,.,
~
HPT
LC as
thos
e of
Tab
le 7
. *,
**E
lect
roph
oret
ic m
obili
ties a
s th
ose
in T
able
7.
?Cor
rect
ed w
ith t
he a
vera
ge re
cove
ry o
f pe
ptid
e gi
ven
by t
he am
ino
acid
ana
lysi
s.
tt No
t cor
rect
ed.
I I
I
-
63
.3" (
C 0.
60)t
t 1
ASP
,.,,
Ser,
.6,G
1~o.
99
Gly
,.,,
Ala
,.,,
LYS,
.,, N
va,.,
, I
I [N
E-A
c-Ly
s3, D
-Ala
6 ] -F
TS
10.5
6 0.
38
0.63
0.
82
I -
1-
I
-
l A
sp,.,
, Se
r,.,,,
GIu
,.9~
GlY
i.05 M
ai.9
0 LY
Si.o
o
1 I L1.- -,
SYNTHESIS OF FTS ANALOGS
fitration and lyophdization of the aqueous phase, 9.7mg (yield 91%) were obtained. The product was homogeneous by t.l.c., HPTLC and high voltage electrophoresis.
Synthesis of [D-Ser4 J-FTS
Z-Gln-Gly-Gly-OMe (X). 3.9 g (9.7 mmol) of Z-Gln-ONp (Fluka), 2 g (9.7 mmol) of H-Gly- Gly-OMe CH3 COOH [obtained by catalytic hydrogenolysis of Z-Gly-Gly-OMe (Albertson & McKay, 1953)] and 1.1 ml (9.7mmol) of N-methylmorpholine were dissolved in DMF (25 ml) and cooled in an ice-water bath. After stirring overnight at +4", the solvent was evaporated, and the residue was taken up in water. After washings with ether in order to remove p-nitrophenol, the aqueous phase was saturated with NaCl and extracted with chloro- form. The volume of the organic solution was reduced by evaporation. After a few hours of clulling, the precipitated product was filtered and rinsed with ether. 3.8g of (X) were ob- tained; Rf (A) = 0.57, Rf (E) = 0.70.
Z-Gln-GZy-GZy-NHNH2 (XZ). 408 mg (1 mmol) of (X) were dissolved in lOml MeOH, and 0 . 5 d (10mmol) of hydrazine hydrate were added. After 3 h at room temperature, the solution was cooled. The precipitated product was filtered and rinsed with ether. From concentrated mother liquors, two other crops were recovered. In total, 385mg were ob- tained, R&) = 0.25.
Z-G In -Gly-Ciy -Ser(Bu ')-Am -OBu (XU) . 89 8 mg ('.?lnmol) of (XI) were dissolved in 15ml DMF and converted into the corresponding azide by the method of Mazur & Schlatter ( 1964). The tripeptide azide was coupled with 955 mg (2.44mmol) of H-Ser(But)- Asn-OBut . CH3 COOH (Blanot et ul., 19790). After filtration and evaporation of the reaction inixture, the residue was taken up in water. The pH was brought to cu. 4 by addition of KHSO,, and the product was extracted with chloroform. After drying on MgSO4, the organic solution was reduced by evapora- tion. By addition of ether and cooling, the product precipitated (950mg), &(A) = 0.72, R,(E) = 0.59.
H-Gln-Gly-Gly-Ser (Bu')-Asn-OBut. CH, COOH (XZZI). 400mg (0.566mmol) of (XII) were hydrogenated as previously described ('yield: loo%, 358 mg), Rf(A) = 0.36.
Z-D-Ser-Gln-Gly-Gly-Ser (But)-Asn-OBut(XILr). 202 mg (0.32 mmol) of (XIII) were mixed with 146 mg (0.32 mmol) of Z-D-Ser-OPcp (Kovacs et ul., 1967) and 0.036ml (0.32mmol) of N- methylmorpholine in 8ml DMF and cooled in an ice-water bath. The reaction mixture was stirred for 1 h at 0" and 40h at room temperature. After evaporation in vacuo. the residue was taken up in 30ml hot chloro- form and triturated. The product was filtered and rinsed with ether. 145mg were obtained as a semi-solid product; Rf(A) = 0.68, Rf(E) = 0.41.
H - D - Ser-Gln-Gly-Gly-Ser(Bu')-Asn-OBu' *
CH3COOH (XV). 80mg (0.1 mmol) of (XIV) were hydrogenated as previously described (yield: 100%,52 mg), Rf (A) = 0.39.
<Glu-Ala-Lys(Boc)-NHNHZ ( X VI). 220 Ing (0.67 mmol) of <Glu-Ala-NHNH2 . CF, COOH (prepared from (VIII) as indicated in the preparation of (IX)) are dissolved in 20nd DMF and converted to the corresponding azide by the method of Medzihradszky (,cf. Zaoral, 1965). The dipeptide azide was coupled with 266 mg (0.56 mmol) of H-Lys(Boc)- NHNHZ. HBr (Sakarellos et ul., 1976). After evaporation of the solvent. the residue was taken up in water. The pH was brought to cu. 4 by addition of KHSO,, and the product was extracted with chloroform. After drying on MgS0, , the organic layer was evaporated, and the residue was triturated in ether to yield 246mg of compound (XVI); Rf(A) = 0.64, Rf(E) = 0.74.
< G l u - A l a - l , y s ( B o c ) - D - S e r - G l n - ~ l ~ - ~ ~ l ~ ~ - S ~ ~ ~ ~ ~ ' ) - Am-0Bu' (Xi 'II) . S8mg (0.1 rnmol) o f 'mI) were hydrogenated for 4: h in 3ml DMF (the use of MeOH or acetic acid as solvents led to the appearance of byproducts during the hydrogenolysis). After filtration of the catalyst, the DMF was evaporated. The residue of < Glu-Ala-Lys(Boc)-NHNH, (R,(E) = 0.29) was dried in vucuo for 3 3 h. This unstable
217
J. MARTINEZ ET AL.
compound was immediately dissolved in 3ml DMF and transformed into the corres- ponding azide by the method of Mazur & Schlatter (1 963). The tripeptide azide was coupled with 52 mg (0.1 mmol) of (XV). The reaction mixture was filtered, the solvent was evaporated and the residue was triturated in hot chloroform. The product was filtered and recrystallized from MeOH. 75mg were obtained as a semi-solid product, Rf(A)= 0.07, R,(C) = 0.06.
iD-Ser4/-FTS. It was obtained from 18.4mg (0.017mmol) of (XVII) as described for [Om3]-FTS (yield: 87%, 14.6mg). It was homogeneous by t.l.c., HF'TLC and high voltage electrop.horesis.
Preparation of the doublemodified analog [NE-Ac-Lys3 , D-Ala6/-FTS by acetylation of [D-Ala6]-FTS. [D-da6] -FTS was acetylated by the method of Reboud-Ravaud & Ghelis (1976): to a solution of 1.3pmol of [D-Aki6] - FTS in 0.8ml of 0.05 M borate buffer, pH 8.0, 5 0 4 of a 0.48mM solution of acetyl- benzotriazole (24pmol) (Staab, 1957) in dioxane were added. After stirring for 45min at room temperature, the pH was brought to 8 by addition of 2 0 d of N NaOH, and the same amount of acetyl-benzotriazole was added. After 30min, the reaction mixture was washed with ether in order to eliminate benzotriazole and lyophilized. The product was purified by preparative t.1.c. in solvent system J. 0.65pmol (yield: 50%) were re- covered, homogeneous by t.1.c. and HPTLC.
Preparation of [Har3]-FTS by guanidination of FTS 0-methylisourea sulfate was con- verted to an aqueous solution (0.25 M ) of the free base with barium hydroxide. loo$ (25 pmol) were added to 2 . 4 3 ~ m o l of synthetic FTS dissolved in 1.4ml of 0.025 M carbonate- bicarbonate buffer pH 10.5. After 6 days at room temperature, two products appeared by high voltage electrophoresis (PH 1.9, 37.5 v/cm, 44 h): a major one, chlorine and Saka- guchi positive, corresponding to the guanidi- nated product, and a minor one, ninhydrin and chlorine positive, corresponding to the starting material (these two spots partially overlapped).
The reaction mixture was lyophilized, and the guanidinated product was purified by pre- parative high voltage electrophoresis in the above conditions. 0.62pmol of a product homogeneous by high voltage electrophorem were obtained (yield: 25%).*
For the preparation of [Arg3 J -FTS from [Orn3]-FTS by the same procedure, two preparative high voltage electrophoreses were necessary to obtain a pure product (yield: 15%).
ACKNOWLEDGMENTS
This investigation was supported by grants from the Centre National de la Recherche Scientitique, France and from the Dtltgation GBnBrale la Recherche Scientilique et Technique, France.
REFERENCES
Albertson, N.F. & McKay, F.C. (1953) J. Am. Chem.
Anderson, J.C., Barton, M.A., Hardy, P.M., Kenner, G.W., Preston, J. & Sheppard, R.C. (1967) J. Chem. SOC. (C), 108-113
Bach, J.F., Dardenne, M., Pltau, J.M. & Rosa, J. (1976) Compt. Rend. Acud. Sci. 283D, 1605- 1607
Bach, J.F., Bach, M.A., Blanot, D., Bricas, E., Char- reire, J., Dardenne, M., Fournier, C. & Plkau. J.M. (1978) Bull. Inst. Pasteur 76, 325-398
Barral, I. & Savrda, J. (1973) Synthesis, 795-796 Benoiton, L. (1963) Om. J. Chem. 41, 1718-1721 Blanot, D., Martinez, J., Auger, G. & Bricas, E.
(197Ya)Znt. 1 PeptideProtein Res. 14,41-56 Blanot, D., Martinez, J., Sasaki, A., Auger, G., Bricas,
E, Dardenne, M. & Bach, J.F. (1979b) inpeptides, Proc. 6th American Peptide Symposium (Gross, E. & Meienhofer, J., eds.), p. 551, Pierce Chemical Co., Rockford, IL
Bricas, E., Martinez, J., Blanot, D., Auger, G., Dar- denne, M., Pltau, J.M. & Bach, J.F. (1977) in Peptides, Proc. 5th American Peptide Symposium (Goodman, M. & Meienhofer, J., eds.), pp. 564- 567, J. Wiley and Sons, New York
*This product, obtained by guanidination of FTS, has been shown by t.1.c. and high voltage electro- phoresis to be identical with [Har3 ] -FTS obtained by Dr. P. Lefrancier and his colleagues, at the Institut Choay, by a stepwise synthesis in which the homo- arginine residue was introduced directly.
SOC. 75,5323-5326
278
SYNTHESIS OF FTS ANALOGS
Fukuda, T., Kitada, C. & Fujino, M. (1978) J. Chem. SOC. Chem. Comm., 220-221
Klostermeyer, H. & Schwertner, E. (1973) Z. Natur- forsch. 28b, 334-338
Konig, W. (1971) Hoppe-Seylers Z. Physiol. Chem. 352,2
Kovacs, J . , Ceprini, M.Q., Dupraz, C.A. & S c h d t , G.N. (1967) J. Org. Chem. 32,3696-3698
Lecher, H. & Simon, K. (1921) Ber. 54,632-638 Marchiori, F., Rocchi, R., Vivaldi, G., Tamburro,
A. & Scoffone, E. (1967) J. Chem. SOC. (C),
Mazur, R.H. & Schlatter, J.M. (1964) J. Org. Chem.
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