Int. J . Peptide Protein Res. 40, 1992. 54-61

Synthesis and biological activity of the mono- and di-galactosyl-vespulakinin 1 analogues M. GOBBO. L. BIONDI. F. FILIR.4. B. SCOLARO. R. ROCCHI and T. PIEK*

Biopolvmer Reseurch Centre, C .A'. R . , Depciriineiir of Orgunic Cheiiiisr~.,Uiii~~ersiy of Poduu, Paduo, Italy; *Department of Phuri~iucologj~, Vriiversirj.of Aimrerdurii. Anisrerduni. The Netherlands

Received 30 December 1991, accepted for publication 8 March 1992

Syntheses are described of some mono- and di-glycosylated analogues of vespulakinin 1. The solid phase procedure, based on the Fnioc chemistry. lvas used to prepare (Galr)Thr3-vespulakinin 1, (GalP)Thr3vespulakinin 1 and the di-glycosylated analogue ((Gal z)Thr3. (Gal r)Thr?-vespulakinin 1. The 8-glycosylated derivative was also prepared by the continuous flow variant of the Fmoc polyamide method. The synthesized glycopeptides were purified and characterized by amino acid analysis. optical rotation, analytical HPLC, 'Hand I3C-NMR and FAB-MS. Preliminary pharmacological experiments showed that the carbohydrate-free vespulakinin 1 is less active than bradykinin (about 0.3 times on a molar basis) when tested by guinea pig rectum contraction, and the tuo monoglycosylated analogucs are equiactive (about 0.9 times the bradykinin activity). The most active derivative, the (Gal x)Thr3, (Gal r)ThrJ-\.espulakinin 1 analogue, was about 2.5 times as active as bradykinin. K e r IwordJ: glycopeptides; gl>cos! laled bcspulakinin analogues; solid phase gllcopeptidc aynthesis

Vespulakinin 1, a glyco-heptadecapeptide possessing high bradikinin-like activity in lowering rat blood pres-

sure, contains two oligosaccharide units O-glycosidically linked to threonine residues in positions 3 and 4 (1). Synthesis, conformation and biological activity of the carbohydrate-free heptadecapeptide corresponding A preliminary report on the synthesis of gl!cos>lated vespulakinin to the amino acid sequence of vespulakinin 1 have been analogues has been presented at the Japan S>niposium on Pepride already described (2, 3). We report in this cornrnuniChemistry, Kobe, Japan, September 28-October 2. 1987 (Gobbo. cation the solid phase synthesis of vespulakinin 1 anM., Biondi, L. Cavaggion, F., Filira. F. & Rocchi. R. (1988) in Pepride alogues containing either one or two D-galactopyranosyl Cheniisrry 1987 (Shiba. T. & Sakakibara, S.. eds.) pp. 327-330. Proresidues 0-glycosidically linked to the hydroxy side tein Res. Foundation. Osaka. 1988). The amino acid residues are of chain of threonine residue in position 3 (either u or /? L-configuration. Standard abbreviations for amino acid derivatives. linkage) or in positions 3 and 4 ( x linkage). The ungpeptides, and glycopeptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1984) lycosylated heptadecapeptide was also prepared to control the efficiency of the automatic synthetic procedure Europe017 J . Biocheni. 138. 9-37 and (3985) J . B i d . Cheru. 262. 13and to establish the mildest conditions for an efficient 18. Other abbreviations: DCC. ,V,:\' -dic)clohex>Icarbodiimide: HODhbt, 3-h~droxy-2,3-dihydro-4-oxo-benzo-1.2.3e: DCM. final cleavage from the resin and side chain deprotecI\,h'dimethS.lformamide; DMAP. dichloromethane; DMF. tion. The difficulties often associated with removal of 4-dimethylaminopyridine; FAB-MS. fast atom bombardment mass the side chain protection become more apparent with spectroscopy; Fmoc. 9-fluorenylmethyloxycarbon~1: Gal. ~ - g a l a c t o - peptides containing multiple arginine residues. In the pyranosy!; Gal(Ac)i, 2.3,4,6-tetra-o-acet!I-D-&alactop!rano~!l; case of VSK 1 sequence the presence of five arginines HOBt. 1-h~droxybenzotriarolc;HMP. 4-hSdrox>mcth!I-phcnox!may require a prolonged reaction time to cleave the side methyl-copo1)st)rcne- 1 divinllbenzene resin: HPLC, high perforchain protecting groups. It is thus important to minimance liquid chromatography: Mtr. ?-mcth!ox~-2.3.6-trimcth! I mize the exposure to acid conditions of the chemically benzcnesulfon>!; NMP. .V-niethS.lpyrroiidone; NMR. nuclear magnetic resonance; Pmc, 2,2,~,7,8-pcntameth)I-chro1nan-h- sensitive 0-glycosidic linkagc present in thc glycosysulfonyl; Pfp. pcntafluorophenyl: tBu, tert -but!l: TFA. trifluoroacc- lated VSK 1 analogues. For the sake of comparison the tic acid: VSK 1, vespulakinin I . p-glycosylated VSK 1 analogue was also prepared by O0

54

Mono- and di-galactosyl-vespulakinin 1 the continuous flow variant of the solid phase procedure (4). Solid phase syntheses of the carbohydrate-free, mono-, and di-glycosylated VSK 1 were performed by the Fmoc strategy on hydroxymethylphenoxymethyl resin. To reduce possible difficulties during the acid cleavage from the resin and the side chain deprotection, and possible damage to the chemically sensitive glycosidic linkage, the Pmc group, possessing an increased acid lability over Mtr ( 9 , was preferred as guanidino protecting group for arginine. Serine and threonine were protected on their side chain functions as tert.-butyl ethers. Fmoc-(Gal a)Thr-OH (6) and Fmoc-(Gal j)ThrOH (6) were unprotected at the sugar hydroxyl functions. For a better evaluation of the problems possibly involved in the side chain deprotection of a medium size glyco-peptide, Fmoc-Arg(Mtr)-OH and Fmoc[ Gal(Ac)4b]Thr-OH (6,7) were used in the continuous flow solid phase synthesis (4) of (Gal P)Thr3-VSK 1. Hydrazine hydrate was used for deacetylation (8). EXPERIMENTAL PROCEDURES CM 52 was obtained from Whatman. The standard NMP-HOBt solvent activation strategy was used in the solid phase peptide syntheses performed on the Applied Biosystems Model 43 1 A Peptide Synthesizer. The chain assemblies on the HMP resin (capacity 0.94 mmol/g, Applied Biosystems) were identical for all syntheses and consisted of 4 equiv. Fmoc-amino acidOBt esters preactivated in NMP, followed by coupling to the growing peptide chain. Coupling yields were determined by ninhydrin analysis (9) of a small aliquot of peptide resin (3-5 mg) removed automatically after each amino acid addition. The final peptide resins were N" deprotected with 20% piperidine in NMP for 20 min, followed by thorough washing and drying of the resin prior to cleavage, deprotection, HPLC and amino acid analysis. All chemicals for the solid phase syntheses, with the exception of Fmoc-(Gal a)Thr-OH (6), Fmoc-(Gal P)Thr-OH (6), and Fmoc-Arg(Pmc)-OH (Novabiochem) were supplied by Applied Biosystems. The continuous flow solid phase synthesis of (Gal b)Thr3-VSK 1 was performed on a manually controlled Pepsynthesizer apparatus (Cambridge Research Biochemicals Ltd.) by the same procedure previously described for preparing the carbohydrate-free VSK 1 (2). Polydimethylacrylamide-Kieselguhr supported resin functionalized as sarcosine methyl ester (Pepsyn K), Fmoc-Arg(Mtr)-OH, Fmoc-Thr(tBu)-OH, Fmocamino acid pentafluorophenyl esters, and pentafluorophenyl 4-hydroxymethylphenoxyacetate were Cambridge Research Biochemicals Ltd. products. Pepsyn K was converted to the 4-hydroxymethylphenoxyacetylnorleucyl derivative (Pepsyn KA) as previously described (lo). Fmoc-Ser(tBu)-ODhbt (1 l), FmocThr(tBu)-ODhbt (1 1) and Fmoc[ Gal(Ac)4 j]Thr-OH (6, 7) were prepared according to the literature. Inter-

mediate Fmoc-peptide resins were deprotected with 20 % piperidine-DMF solution. Samples were removed at each acylation step for qualitative ninhydrin (12) and trinitrobenzenesulfonic acid (13) tests. Resin samples for amino acid analyses were taken after removal of the Fmoc-protecting group. Optical rotations were determined with a Perkin Elmer model 241 polarimetcr. Amino acid analyses were carried out with a Carlo Erba model 3A 29 amino acid analyzer equipped with a Perkin Elmer Sigma 10 Chromatography Data Station following hydrolysis of peptide samples for 22 h at 110" in sealed, evacuated vials in constant boiling hydrochloric acid. HPLC separations were performed, under the indicated conditions, on a Perkin Elmer series 3B liquid chromatograph equipped with a Perkin Elmer 561 recorder. NMR spectra were recorded at 298°K on a Bruker AM-400 at 400 MHz ('H) or 100.6 MHz ( 13C)equipped with an Aspect 3000 computer. Concentration of the samples was in the range 5-10 mM in D20 (99.96%, pD 6.0). Chemical shifts (6) are expressed relative to H O D set at 4.8 ppm (IH) or to dioxane set at 67.8 ppm ("C) as internal standard. Positive fast atom bombardment mass spectra were obtained at 5kV, under xenon, with a VG-ZAB 2F mass spectrometer. Nitrobenzyl alcohol was used as matrix. CD measurements were performed at 25" using a Jasco-500 A Spectropolarimeter equipped with a DP501 N Data Processor. The number of accumulated scans ranged from 26 to 32, depending on the intensity of the CD signal. The spectra reported in this paper are original computer-drawn DC curves; [O]M and [O]R represent molar ellipticity values (deg cm2 dmol - l ) per mole of peptide and per mole of peptide residues, respectively. In the aromatic absorption region the spectra are reported in [O]M ellipticity units, while in the peptide absorption region the spectra are reported in [ O]R ellipticity units. All solvents were freshly distilled. Evaporations were carried out under reduced pressure at 40-45" using a rotary evaporator. Yield are based on the weight of vacuum dried product. SOLID PHASE PEPTIDE SYNTHESIS Assemblies ofthe carbohydrate-free VSK 1, (Gala)Thr3VSK 1, (GalP)Thr3-VSK 1, and (Galx)Thr3, (Gala)Thr4-VSK 1 on the Applied Biosystems Model 431 A Peptide Synthesizer were performed on a 0.25 mmol scale. Loading of the HMP resin (267 mg, 0.25 mmol) with Fmoc-Arg(Pmc)-OH (0.663 g, 1 mmol) was accomplished automatically through the conventional DCC activation in the presence of DMAP. In the case of the mono- and di-glycosylated VSK 1 a double acylation was used to increase the efficiency of loading. Quantitation of this first coupling (59% for the carbohydrate-free vespulakinin 1 and 72-75 % for the 55

M. Gobbo et al. glycosylated derivatives) was determined by spectrophotometric measurement of the Fmoc-piperidine adduct on two samples of resin (2.1-2.3 mg) with identical results. For chain elongation the prepackaged Fmoc-amino acids (1 mmol) were automatically dissolved with NMP (2.1 mL) and 1 M HOBt in N M P (1 mL), and activated in a separate vessel by addition of 1 M DCC in NMP (1 mL). Before packaging Fmoc(Gal a)Thr-OH (6) and Fmoc-(Gal fl)Thr-OH (6) were dissolved in N M P (2.2 mL) and filtered. The resulting clear solutions were automatically combined with the HOBt solution and transferred from the cartridge to the activation vessel. The Fmoc-amino acid activated esters were transferred into the reaction vessel for acylation. All coupling yields were higher than 99.2",. Prior to each coupling the peptide-resin was deprotected with 20% piperidine in N M P (4.5 mL). Before proceeding with the TFA cleavage and side chain deprotection, the Fmoc group was removed from the N-terminal amino acid and the final peptide-resin was washed with DCM and dried under high vacuum. In the case of the carbohydrate-free VSK 1, deblocking experiments ranging from 1 h to 3.5 h showed that an acid treatment of 2 h was sufficient for a complete cleavage and deprotection reaction. Typically 30-50 mg samples were suspended in TFA (1 mL), containing phenol (50 pL), thioanisole (25 pL) and water (25 pL), and stirred at room temperature. After the desired reaction time, the resin was removed by filtration and washed with TFA (2 x 1 mL), the filtrate and washings were combined, evaporated at 25 ', and the oily residue triturated with tert.-butyl methyl ether (20 mL). The resulting precipitate was collected by centrifugation, redissolved in TFA (1 mL), and reprecipitated with [err.butyl methyl ether (10 mL). This procedure was repeated three times and the product was finally dried in vacua in the presence of phosphorous pentoxide and potassium hydroxide pellets. The crude carbohydratefree VSK 1 trifluoroacetate (15-23 mg) was purified by semipreparative HPLC (3) (purification yield 60-70",); [ x ] ~ '-74.3' (c 0.93, water); amino acid ratios: Thr 2.96, Ser 1.03, Gly 2.00, Ala 1.00, Phe 1.85, Pro 2.98, Arg 5.10. 'H NMR (DzO, 400 MHz): 1.24 (d, 3H, JB,;. 6.5 Hz, Thl-4-CH3); 1.25 (d, 3H, J0,;.6.5 Hz, Thr3-CH3); 1.37 (d, 3H, Jp,? 6.4 Hz, Thrl-CH3); 1.47 (d, 3H, JZ,B 7.1 Hz, Ala-CH3); 7.25-7.43 (m, 10H, ZPhe, H-arom.). 13CNMR (D20, 100.6 MHz): 17.70 (Ala-CP); 19.93 (3 Thr-Cy); 25.11, 25.43, 25.58, 25.88 (3 Pro-C;: 5 ArgCy); 28.82, 29.28, 29.41, 30.45 (3 Pro-CP, 5 Arg-CP); 37.92, 38.38 (2 Phe-CB); 41.71 ( 5 Arg-Cd); 43.21,43.42 (2 Gly-Cr); 48.96, 49.20 (3 Pro-Cd); 5 1.00 (1 Ala-Cx); 67.41, 68.18(3 Thr-Cfi; 128.44, 129.97, 130.30, 137.38 (2 Phe C-arom.); 157.92 ( 5 Arg-CG). Lit. [ r ] 2 ^-78.6' (c 1.0, water) (2) [ r ] g ' -74.6" (c 1.0, water) (2), [ r ] $ -76.5" (c 0.65, water) (3). The analytical HPLC elution profile is superposable to those of the carbohydrate-free vespulakinin 1 previously prepared by different synthctic strategies (2, 3). In the case 56

of (Galr)Thr3-VSK 1, the crude peptide (85 mg) obtained by TFA treatment (2 h) of an aliquot of the final peptide-resin (160 mg) was precipitated twice with tert.butyl methyl ether and purified by semipreparative HPLC (Fig. 1). Yield 52 mg (62%); [ a ] g ' -50.5" (c 0.51, water); amino acid ratios: Thr 3.02, Ser 0.85, Gly 1.86, Ala 1.02, Phe 2.00, Pro 3.03, Arg 5.18. 'H NMR (DzO, 400 MHz) 7.43-7.25 (m, 10H, 2 Phe H-arom.); 5.08(d, lH,J1,24.0Hz,H-l); 1 . 4 7 ( d , 3 H , J , ~ 7 . 1 H ~ , Ala-CH3); 1.37 (d, 3H, Jp,, 6.4 Hz, Thrl-CH3); 1.29 (d, 3H, Jp,, 6.4 Hz, Thr3-CH3); 1.24 (d, 3H, Jp,? 6.4 Hz, Thr4-CH3). 13C-NMR (D20, pD 6.0, 100.6 MHz): 17.80 (Ala-Cp); 18.66, 19.93 (3 Thr-Cy); 25.11, 25.40, 25.58, 25.88 (3 Pro-Cy, 5 Arg-Cy); 28.81, 29.26, 29.36, 30.45 (3 Pro-CP, 5 Arg-CB); 37.92, 38.38 (2 Phe-CB); 41.73 ( 5 Arg-Cd); 43.19,43.42 (2 Gly-Ca); 48.96,49.12 (3 Pro-Ca); 67.31,68.36 (2 Thr-CB); 69.62 (C-2); 70.46, 70.60 (C-3. C-4); 72.62 (C-5); 75.80 (Thr-CB); 100.57 (C-1); 128.43, 129.96, 130.30, 137.11, 137.33 (2 Phe C-arom.), 157.91 ( 5 Arg-CG). FAB-MS: m/z 2120 (calc. for C~H148N32027:2122).

- 90 I

/ / - 80

I

-

/

-70

I

E

/

0

rn

nI

-60

I I

4 I

u u c

,/

'/a

B

- 50

4

n

- 40

L 0

vl

n

a

- 30 - 20

I

I I

I

I

5

10

I

I

15 20 T i m e (min)

I

I

25

FIGURE 1 Semiprcparative HPLC elution profile of (Galr)Thr3-VSK 1 (solid phase synthesis). Load 5 mg. Column Vydac C 18 (22 x 250 nm, 10 pm. Alltech). Eluant A, aqueous 0.10); TFA; B, 90% acetonitrile in A. Elution: isocratic 15": B over 5 min, linear gradient 15-50% B over 20 min and 50-90"" B over 5 min. Flow rate 9.5 mL/min.

Mono- and di-galactosyl-vespulakinin 1

*> - 90 0.1 m

E

- 60

0

FIGURE 2

0

3

a./

/

0.1 u

0 c

I a L

0 yl

4 0

0n

Analytical HPLC elution profiles of glycosylated vespulakinin 1 derivatives. Column: Aquapore octyl R P 300 (4.6 x 220 mni, 7 pm, Browlee Labs.). Eluants as in Fig. 1. Elution: isocratic 1004, B over 2 min, linear gradient 10-90x B over 20 min, isocratic 90% B over 2 min. Flow rate 1.5 mL/min a) (Gal x)Thr3-VSK 1, load 31 pg; b)(Gal x)Thr3, (Gal a)Thr4-VSK 1, load 25pg; c)(Gal B)Thr3-VSK 1, load 25 pg; d) (Gal B)Thr3-VSK 1, continuous flow procedure, load 35 pg.

B

- 50

- 40 - 30 - 20

I

I

1

1

I

10

15

20

10

15

I

10

1J I

20 10 Time (min)

I

15

The analytical HPLC elution profile of the mono-aglycosylated heptadecapeptide is shown in Fig. 2a. In the case of (Gala)Thr3, (Galcc)Thr4-VSK 1 the TFA treatment (2 h) of the final peptide resin (72 mg) yielded 40 mg (55 %) of glycopeptide trifluoroacetate. The analytical HPLC elution profile of the crude product indicated the presence of one main peak accompanied by several contaminants. Attempts to improve the quality of the product by increasing the deblocking time up to 4.5 h failed. The crude deblocked glycopeptide (24.8 mg) was purified, in portions, by semipreparative HPLC (Fig. 3). Four fractions (1 to 4) were separately collected, pooled, and evaporated to dryness, yielding respectively: Fraction 1: traces of material. Fraction 2: 13.9mg (56%); [cc]g" -27.1' (c 0.66, water); amino acid ratios: Thr 3.00, Ser 1.06, Pro 2.93, Gly 2.00, Ala 1.06, Phe 1.86, Arg 5.15. 'H NMR (D20, 400 MHz) 7.43-7.25 (m, 10H, 2 Phe, H-arom.); 5.08, 5.09 (2d, 2H, J1,2= J'1,24.2 Hz, H-1 & H-1'); 1.47 (d, 3H, Jl,p 7.1 Hz, Ala-CH3); 1.37 (d, 3H, Jay 6.4 Hz, Thr1-CH3); 1.30 (d, 3H, Jp,,6.4 Hz, Thr3-CH3; 1.27 (d, 3H, Jp,? 6.3 Hz, Thr4-CH3). Fruction 3: 4.3 mg (17%); amino acid ratios: Thr 0.89, Ser 0.99, Pro 3.04, Gly 2.00, Ala 0.92, Phe 1.94, Arg 5.16. Fraction 4: less than 1 mg. Only fraction 2 gave the correct amino acid composition and an analytical HPLC elution profile corresponding to a single component as shown in Fig. 2b. In the case of the fi-glycosylated analogue, TFA treatment of the peptide resin (160 mg) yielded 88 mg of a crude product, which was precipitated twice from TFA with tert.-butyl methyl ether and further purified by semipreparative HPLC under the conditions re-

portedinFig. l.Yield53 mg(60Z); [ u ] ~ -67" ~ " (cO.5, water); amino acid ratios: Thr 3.04, Ser 1.01, Pro 3.09, Gly 2.00, Ala 0.98, Phe 2.00, Arg 5.16. ' H NMR (D20, 400 MHz): 7.43-7.25 (m, 10H, 2 Phe H-arom.); 1.47 (d, 3H, J,,p 7.1 Hz, Ala-CH3); 1.37 (d, 3H, Jij,;. 6.4 Hz,

I

I

I

I

I

5

10

15

20

Time

I

(rnin)

FIGURE 3 Semipreparative HPLC elution profile of (Galr)Thr', (Ga1x)ThrlVSK 1. Load 4 mg. Column Aquapore octyl R P 300 (250 x 10 rnm, 7 pm, Browlee Labs.). Eluants as in Fig. 1. Elution: isocratic 18y0 B over 5 min, linear gradient 18-32% b over 10 min, and 32-90"; B over 5 min. Flow rate 5 mL/min.

51

M. Gobbo et al. Thrl-CH3); 1.28 (d, 3H,Jp., 6.3 Hz, Thr3-CH3); 1.24 Mtr)-symmetrical anhydride (1.0 mmol) prepared im(d, 3H, Jp., 6.4 Hz, Thr"CH3); 13C-NMR (D20. mediately before use and dissolved in D M F (2 mL) in 100.6 MHz): 17.76 (Ala-Cp); 16.48, 19.96 (3 Thr-Cy); the presence of DMAP catalyst (24.5 mg, 0.2 mmol). 25.14, 25.42, 25.59, 25.88 (3 Pro-C;; 5 Arg-C;)); 28.82, After 90 rnin recirculation the column was washed with 29.40, 30.46 (3 Pro-Cfl, 5 Arg-Cb); 41.73 (5 Arg-C6); D M F for 20 min and residual hydroxy groups on the 43.20,43.42 (2 Gly-Cr); 48.96,49.19 (3 Pro-C6); 67.41, resin were blocked by circulating for 45 min a solution 68.40 (2 Thr-Cj); 69.88 (C-4); 71.98 (C-2); 73.80,75.20 of acetic anhydride (0.038 mL, 0.4 mmol) and DMAP (Thr-Cp, C-3); 76.50 (C-5); 102.13(C-1); 128.44,129.96, (24.5 nig, 0.2 mmol) in D M F (2 mL) followed by D M F 130.30, 137.13, 137.38 (2 Phe C-arom.); 157.91 (5 Arg- for 20min. The Fmoc group was then cleaved with Cc). FAB-MS: m/z 2120 (calc. for C91H1-18N~027: 20°, piperidine-DMF solution (10 min) and the resin washed with D M F (20 min). Amino acid analysis of a 2122). The analytical HPLC elution profile of the mono-p- sample of resin gave the results shown in Table 1 (column 1). glycosylated heptadecapeptide is shown in Fig. 2c. The synthesis was continued using the suitable FmocCONTINUOUS FLOW SOLID PHASE PEPTIDE amino acid derivatives (0.8 mmol) and repeated twice. In the first synthesis Fnioc-[ Gal(Ac)4j]Thr-OH (6, 7) SYNTHESIS and the five arginine residues were coupled as symmetThe Pepsyn KA resin (2 g, 0.2 meq) was swollen in rical anhydrides, and Thr 1, Thr 4, and Ser 14 were D M F for 2 h, packed in the reaction column (1 x 10 cm) activated as esters of the 3-hydroxy-2,3-dihydro-4-0~0of the Pepsynthesizer and esterified with Fmoc-Arg- 1,2,3-benzo-triazine(14). All other amino acid residues TABLE 1 Amino acid unu(v.resfor intennediute uridfrnul peptide resins crnd .f.r {Gal fi~Tht-'-V S K I . Wl7ei7 t11.0fjgure.7 ure given for the same frugment the fjr.rr column refer.r to the rynthesis curried o i t t h!. utilizing Fi~toc-SentBir~-ODhbr arid Ft~ioc-Thr(tBu)-ODhbt. * compound 2; * * compound 1

Residues

Nle '4%

1 17 1.06 1.00

1.00 1.00

Phe Pro Ser GlY Thr Ala Residues

Nle Ar€! Phe Pro Ser Gly Thr Ala

Pro

Ser Gly Thr Ala

58

3 14-17

4 11-17

5 10-17

1.11 1.04 0.99 0.97

1.10 1.03 0.96 0.98 1.03

1.12 1.10 2.00 1.95 0.95 1.03

1.12 1.10 2.00 3.00 0.97 1.00

8 7-17 1.16 3.02 2.03 2.93 0.98 2.03

Residues

Nle Arg Phe

2 15-17

12 3-17 1.06 4.80 2.00 3.20 1.06 2.03 1.50

1.16 4.00 2.10 2.91 0.98 2.05

1.09 3.91 2.01 3.07 0.97 2.01

1 .oo

2.09 1.73

1.11 5.00 2.01 3.20 I .OO 2.07 1.73 0.88

1.06 2.05 1.98 2.99 0.96 1.02

1.15 4.82 2.10 3.00 0.98 2.10

1.12 4.80 2.20 3.10 1.06 2.03 2.40 0.87

1.07 4.82 1.97 3.15 0.98 2.07

1.08 4.80 2.03 3.17 1.OO 2.05 0.75

15 1-17 1.13 4.98 2.05 3.23 1.06 2.06 2.67 0.91

1.15 2.10 2.06 2.90 0.97 2.02

11 4- 17

I4 1-17

13

2-17 1.12 4.93 2.02 3.19

1.12 2.00 2.00 3.04 0.9s 1.03

7 8-17

10 5-17

9

6-17 I .07 3.01 1.98 3.05 0.97 1.98

6 9-17

5.15* 1.95 3.02 1.02 2.05 2.90 1.02

4.98** 1.98 3.02 1.00 2.10 2.91 1.06

1.12 4.85 2.04 3.20 1.02 2.05 0.s2

Mono- and di-galactosyl-vcspulakinin 1 were introduced as pentafluorophenyl esters. Alternatively Thr 1, Thr 4, and Ser 14 were also introduced as symmetrical anhydrides with very similar results. Negative ninhydrin (12) and trinitrobenzenesulfonic acid (13) tests for residual amine were normally obtained within the first 70 min of the acylation reaction period but in the case of arginine complete acylation required up to 12 h. Samples for amino acid analysis were removed after addition and deprotection of residues 15 (proline), 14 (serine), 11 (proline), 10 (proline), 9 (arginine), 8 (glycine), 7 (arginine), 6 (arginine), 5 (arginine), 4 (threonine), 3 (threonine), 2 (alanine), and 1 (threonine). The results obtained are shown in Table 1 (columns 2-14). The final a-amino free peptide resin was washed on a synthered glass funnel with DMF, 2-methyl-butan-201, acetic acid, 2-methyl-butan-2-01, DMF, and ethyl ether and dried in vacuo. A sample of resin (0.7 g) was treated with trifluoroacetic acid (70 mL) containing 5 % thioanisole. After 8 h stirring under nitrogen the resin was filtered, washed with trifluoroacetic acid (3 x 5 mL) and dichloromethane (3 x 5 mL), and dried. Amino acid analysis of a sample of resin after cleavage indicated 1.9% residual peptide. The filtrate and washings were combined and evaporated to dryness. The residue was taken up in water (50 mL) and extracted with ethyl ether (5 x 30 mL) and chloroform (5 x 30 mL). The organic phases were separately combined, extracted with water (2 x 30 mL) and the aqueous phase and washings were combined and lyophilized (130 mg, 80 % with respect to the Nle content on the resin). The crude material was dissolved in methanol (0.55 mL) and hydrazine hydrate (0.055 mL, 1.14 mmol, about 5 to 1 molar excess with respect to each acetyl group present in the molecule) was added (18). A precipitate was formed, the mixture stirred overnight at room temperature, and acetyl acetone (0.14 mL, 1.14 mmol) was added. After 1 h the mixture was diluted with water (20 mL), extracted with ether (6 x 20 mL) and chloroform (6 x 20 mL) and lyophilized (117 mg, 96%). The crude product was dissolved in 0.05 M ammonium acetate and applied to a CM 52 column (3.5 x 50 cm) previously equilibrated with the same buffer. Elution was carried out by a linear gradient of ammonium acetate prepared from 750 mL each of equilibrating buffer and 1.0 M ammonium acetate (flow rate 27 mL/h, fraction volume 4.5 mL). Elution was monitored by UV absorption at 254 nm, and fractions corresponding to a broad main peak and some minor peaks were separately collected, pooled, lyophilized, and tested by amino acid analysis and analytical HPLC. Only two peaks corresponding to fractions 194- 198 (compound 1,3 mg) and fractions 217-221 (mean peak, compound 2, 20 mg) gave correct amino acid ratios as shown in Table 1 (column 15). The faster moving peak on the ion exchange chromatography (compound 1) became the slower moving on the reverse phase HPLC column suggesting that it could correspond to a less

basic and more hydrophobic product, possibly the glycoheptadecapeptide still partially protected at the arginine side chain. Compound 2 was purified by semipreparative HPLC, in the conditions indicated in Fig. 1. The isolated product (15 mg, 75%, Fig. 2d) showed proton NMR data comparable to those of the compound previously prepared by solid phase synthesis but the optical rotation value was significantly lower: [ u]’,”’ -53.1 ( c 0.5, water). O

PRELIMINARY PHARMACOLOGICAL SCREENING The pharmacological activity of the four synthetic VSK 1 analogues, and of mammalian bradykinin, was screened by using a cascade of smooth muscle preparation: the rat stomach fundus trip, the rat duodenum, the guinea pig ileum, the rat colon, and the guinea pig rectum (15). Agonists were added to the superfusion fluid in single doses of 1.3 and l o n g in 50pL. The contractions, or relaxations, of the smooth muscles were plotted as the percentage of the maximum contraction, or relaxation, produced by 100 ng of bradykinin, on a probit scale against log doses. The results were linear plots and the effective 50% dose (ED50) was found by interpolation. On a molar base the carbohydrate-free VSK 1 was less active than bradykinin (about 0.3 times) and the (Gal a)Thr3- and (Gal /?)Thr3-VSK 1 analogues were equiactive, both showing about 0.9 times the bradykinin activity. The most active derivative was the diglycosylated analogue which was about 2.5 times more active than bradykinin when tested on the contraction of the guinea pig rectum. Comparable results were obtained with the other smooth muscle preparations. Bradykinin derives its name because of the slow contraction and relaxation induced in mammalian smooth muscles, in comparison with contractions evoked by other agonists like histamine, acetylcholine, or serotonin. All four VSK 1 analogues showed a very slow relaxation, after the contraction, with a half-decay time as long as twice that shown by bradykinin. The diglycosylated VSK 1 analogue might thus be about 5 times more effective than bradykinin. DISCUS SION The heptadecapeptide corresponding to the amino acid sequence of VSK 1 and its mono-a, mono-/?, and di-rglycosylated analogues have been prepared by the standard solid phase procedure. The (Gal p)Thr3-VSK 1 was also prepared by the continuous flow variant (4). Both procedures gave products showing the correct amino acid composition and identical HPLC elution profiles (Fig. 2) but the optical rotation value of the compound obtained by the standard procedure was significantly higher. Preliminary experiments carried out 59

M. Gobbo et al. on the carbohydrate-free vespulakinin 1 established that a 2 h exposure to trifluoroacetic acid is sufficient for the final cleavage of the peptide from the resin and removal of the side chain protecting groups. The protecting group strategy based on the use of Fmoc group for the N"-protection in combination with tert.-butyl and Pmc for side chain protection led to these mild conditions and possible damage to the chemically sensitive 0-glycosidic linkage involving an unprotected sugar moiety (16, 17) was thus reduced. Further reduction of the time of exposure to trifluoroacetic acid of the peptide-resin yielded a product whose analytical HPLC profile indicated the presence of a larger number of contaminants. In the case of the P-glycosylated analogue prepared by the continuous flow variant, final deacetylation of the sugar moiety was satisfactorily achieved by treatment with a methanolic solution of hydrazine hydrate (8) but difficulties have been encountered in the final cleavage from the resin and removal of the side chain protecting groups. In spite of the prolonged trifluoroacetic acid treatment needed for a satisfactory extent of cleavage from the resin, several peaks were eluted from the CM 52 column after deacetylation (8) and separately collected. Only two products showed the expected amino acid composition and their chromatographic behavior suggested that, in one of them, an uncomplete removal of the Mtr groups used for protecting the arginine side chain probably occurred. The chemical shift for the anomeric proton(s) of (Gal-a)Thr3-VSK 1 (5.08, d, 1H) and (Galr)Thr3 (Galcr)ThF-VSK 1 (5.08, d, 1H and 5.09, d, 1H) and

:i

thevaluesforthe Hl-H2couplingconstants ( 5 1 ~ 4 . 0Hz and 51.2 = J ' 1.2 4.2 Hz respectively) define the CI configuration of the glycosidic linkages. In the proton NMR spectrum of (GalP)Thr3-VSK 1 the anomeric proton signal is probably masked by other signals and was not identified. However the presence of the sugar moiety is evidentiated by the 13C-NMR (102.7, C-1) and by FAB-MS. The CD spectra of (Gal r)Thr3-vespulakinin 1 in 5x M Tris buffer, pH 6.93, and in trifluoroethanol, reported in Fig. 4, are similar to those determined for the carbohydrate-free vespulakinin 1 (2, 3). In the far UV region the C D pattern of the glycopeptide in aqueous medium is typical of a random structure, with a negative band in the 200 nm region. In the fluorinated solvent there is a negative shoulder around 220 nm indicating that a small amount of ordered structure is formed. In the near UV the C D pattern exhibits the characteristic vibronic structure of Lb transitions (18). As occurred in the carbohydrate-free heptadecapeptide (2) the intensity of the spectrum is higher in trifluoroethanol, suggesting the presence of a certain degree of conformational constraint of the aromatic moieties in this medium. Comparable results were obtained for the other glycosyltated analogues. The fact that the carbohydrate-free VSK 1 is less active than bradykinin, and the recovery from contraction slower, indicates that on the test system used the considerable increase in the size of the bradykinin molecule decreases the penetration rate. It is therefore possible that the di-glycosylated VSK 1 analogue could be much more active than found in this preliminary inves-

4l

A

B

3

2

2-

1

1-

0

y'

.P-

/

N

P

V

-8

-9

9 - 10-

-11

-.

I

I

I

- o 11 l

I

250

260

270

280

290

300

310

nm

Far and near ultraviolet CD spectra of (Gal r)Thr3-vespulakinin 1 prepared by the solid phase synthesis: A, 30 pm in (a) 5 x 10 Tris buffer, pH 6.93, (b) trifluoroethanol. B, 300 pm in (a) 5 x 10 Tris buffer, pH 6.93, (b) trifluoroethanol. The optical path length was 0.1 cm. ~

~

60

Mono- and di-galactosyl-vespulakinin 1 tigation. Since kinins have neurotoxic actions in the insect central nervous system (19) a more detailed study on the glycosylated VSK 1 analogues is underway.

ACKNOWLEDGMENTS Financial support by the “Progetto Chimica Fine 11” of the National Research Council of Italy is gratefully acknowledged. The authors wish to thank Dr. Stefano Mammi for the helpful discussion on the interpretation of the NMR spectra, Dr. Umberto Vettore, Centro di Studio sulla Stabilita e Reattivita dei Composti di Coordinazione del C.N.R. di Padova for the FAB-MS spectra, and Mr. F. Cavaggion for skillful technical support.

REFERENCES 1. Yoshida, H., Geller, P.G. & Pisano, J.J. (1976) Biochemistry 15, 61-64 2. Rocchi, R., Biondi, L., Filira, F. & Scolaro, B. (1987) Int. J . Peptide Protein Res. 30, 240-256 3. Biondi, L., Cavaggion, F., Filira, F. & Rocchi, R. (1988) Gazz. Chim. It. 118, 427-433 4. Dryland, A. & Sheppard, R.C. (1986) J. Chem. SOC.Perkin. Trans. I, 125-137, and references cited therein 5. Ramage, R. & Green, J. (1977) Tetrahedron Lett. 28,2287-2290 6. Filira, F., Biondi, L., Cavaggion, F., Scolaro, B. & Rocchi, R. (1990) Int. J. Peptide Protein Res. 36, 86-96 7. Filira, F., Biondi, L., Scolaro, B., Foffani, M.T., Mammi, S., Peggion, E. & Rocchi, R. (1990) Int. J . Biol. Macroniol. 12,41-49 8. Schulteiss-Reiman, P. & Kunz, H. (1983)Angew. Chem. Int. Ed. Engl. 12, 62-63 9. Sarin, V.K., Kent, S.B., Tam, J.P. & Merrifield, R.B. (1981) Anal. Biochem. 117, 147-157 10. Dryland, A. & Sheppard, R.C. (1988) Tetrahedron Lett. 44,859876

11. Altherton, E., Cameron, L., Meldal, M. & Sheppard, R.C. (1986)

J. Chem. SOC.Chem. Commun., 1763-1765 12. Kaiser, E., Colescott, R.C., Bossinger, C.D. & Cook, P.(year)J. Anal. Biochem. 34, 595-598 13. Hancock, W.S. & Battersby, J.E. (1976) Anal. Biochem. 21,260264 14. Cameron, L., Meldal, M. & Sheppard, R.C. (1987)J. Chem. Soc. Chem. Commun., 270-272 15. Piek, T. (1991) in Pesticide Chemistry (Frehse, H., ed.), pp. 7585, VCH Verlagsgesellschaft, Weinheim 16. Kunz, H. & Waldmann, H. (1985)Angew. Chem. Inr. Ed. Engl. 24, 883-885 17. Kunz, H. & Unverzagt, C. (1988) Angew. Chem. Inr. Ed. Engl. 27, 1697-1699 18. Strickland, E.H. (1974) CRC Crit. Rev. Biochem., 113-195 19. Piek, T. (1991) Toxicon 29, 139-149

Address: Dr. M . Gobbo Biopolymer Research Centre C.N.R. Department of Orgaiic Chemistry University of Padua Via Marzolo, 1 1-35131 Padua, Italy T. Piek Department of Pharmacology University of Amsterdam Meibergdreef, 15 1105 AZ Amsterdam, The Netherlands

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