Int. J . Peptide Protein Res. 40, 1992, 363-369

Synthesis of specific peptide substrates for HIV-1 proteinase BALRAJ K. HANDA and CORINNE KAY

Departments of

I

Physical and Medicinal Chemistry, Research Division, Roche Products Limited, Welwyii Garden City, Hertfordshire, England

Received 9 September 1991, accepted for publication 26 January 1992

Two small peptide substrates for HIV- 1 proteinase were synthesised. The sequences chosen were basically from that of the gag-pol protein, which is the natural substrate for the proteinase. To protect these peptides from the attack of exopeptidases, the N- and C-termini were suitably protected, which also makes these substrates specific to HIV-proteinase and eliminates the requirement for highly purified enzyme. Key words: colorimetric assay for HIV-proteinase; gag-pol protein; HIV-proteinase; synthetic substrates for HIV-proteinase

Retroviral proteinases are essential for retrovirus maturation and replication. Murine leukemia virus variants mutated in the proteinase region produced noninfectious virions (1, 2). Human immunodeficiency virus (HIV), the causative agent of AIDS, is a retrovirus (3, 4). Molecular organization of the HIV-1 genome resembles that of other retroviruses and comprises gag, pol and env as genes necessary for viral replication. During its replication cycle the polyprotein products of the gag and gag-pol genes are processed by the virally encoded proteinase to provide, respectively, the structural proteins of the virus core and essential enzymes including proteinase itself (2, 5). This key enzyme, therefore, represents a potential chemotherapeutic target. HIV-proteinase mediated cleavage of the gag and gag-pol polypeptides is the target against which therapeutic agents must be directed, but these large proteins

are not convenient substrates for quantitative highthroughput assays. HIV-proteinase cleaves a number of specific peptide bonds within the gag and gag-pol polyproteins (6). The ability of the enzyme to cleave Tyr.Pro and Phe.Pro sequences is of particular interest in the context of drug development as peptide bonds N-terminal to prolyl residues are normally resistant to cleavage by mammalian endopeptidases. This property also provides a rational basis for the design of substrates selective for the viral proteinase. The amino acid sequences spanning subsites P4-P2' for the Phe.Pro and Tyr.Pro cleavage sites within the gag and gag-pol polyproteins are well conserved and changes within this region are conservative (Fig. 1). This implies that a hexa- or heptapeptide should serve as an adequate specific substrate for the enzyme. Synthetic peptide substrates and a colorimetric assay have been devised.

Cleavage site

Cleavage sequence

gag 132-133 pol 68-69 pol 167-168

P1'

P2'

P3 '

P4'

Asn

1 Tyr- -Pro

Ile

V a1

Gln

Asn

1 . Phe- -Pro

Gln

Ile

Thr

Asn

-1 Phe- -Pro

Ile

S er

Pro

PI

P6

P5

P4

P3

P2

Ser

Gln

Val

S er

Gln

GlY

Ile

TYr GlY

Val cys

Ser Thr

Phe Leu

P1

FIGURE 1 Cleavage sites of the proteinase in the gag and gag-pol precursor proteins of HIV-1

363

B.K. Handa and C. Kay Cleavage of peptide substrates at the X.Pro bond releases N-terminal prolyl peptides that are reacted with isatin to form a blue product, which is measured spectrophotometrically. The details of this assay have been published elsewhere (8). In this paper we provide the Ofthe synthesis O f t w o peptide substrates (Fig. 2, which were extensively used in the assay.

MATERIALS AND METHODS Nuclear magnetic resonance spectra were recorded on a Bruker WM 300 instrument. FAB/MS spectra were obtained on a Finnigan MAT 8400 mass spectrometer with the SS 300 datasystem. Microanalyses were performed on a Perkin Elmer 240C elemental analyser. Analytical HPLC was performed with the ABI model 151A on an Aquapore butyl reverse phase column (7 p, 30 x 4.6 mm). The elution gradient comprised 950; TFA in water A-95% B over 15 min, where A = 0.1 ";

Succ.Ser.Leu.Asn.Tyr.Pro.Ile.NHiBu 1

Succ.Val.Ser.Gln.Asn.Phe,Pro.Ile.NHiBu 2

FIGURE 2 Structures of the two synthetic substrates for HIV-protcinase

and B = 0.085% TFA in 70% acetonitrile. TLC was performed on Merck Kieselgel 60 F254 plates using the following systems (v/v); (A) CHCL/MeOH (19:1), (B) C H C13/ MeOH/CH3COO H / H 2 0 ( 120: 15 :3 :2), (C) C H C 1 3 / M e O H / C H 3 C O O H / H 2 0 (60: 18:2:3), (D) CHCh/MeOH/CH3COOH/H20 (30:20:4:6). The compounds were visualised directly under UV light (254 nm), by spray solution of 0.5x ninhydrin in 1-butanol and heating at 100" for 5 min and by chlorination followed by spray with a mixture of 1% KI-1 % starch solution.

TABLE 1 Atial\.rical duru of peprides 1 , 2 and rhe main biterniediures

Compound

Yield [M

t

MS H I - or [ M I -

Molecular formula (mol 1r.t.)

'4nalysis

found (calc.)

C

li

N

5

77

418

66.12 (66.16)

8.69 (8.45)

9.88 (10.06)

7

50

637

68.19 (67.90)

8.49 (8.23)

8.79 (8.80)

9

63

751

64.10 (63.98)

7.50 (7.78)

11.14 (11.19)

15

77

430

58.45 (58.58)

8.68 (8.90)

6.22 (6.50)

16

85

1029

60.11 (60.10)

8.35 (X.36)

10.25 10.52 0.2 M CHCI:,

17

XX

-

61.93 (68.06)

7.90 (7.85)

9.92 (9.52)

19

77

679

62.97 (62.8 7)

7.32 (7.47)

12.24 (12.21) 0.5 M HzO

22

58

424

56.48 (56.73)

6.94 (6.90)

10.02 9.92

24

68

-

56.55 (56.49)

7.18 (7.39)

10.57 (10.54) 0.5 M H20

26

59

545

53.53 (53.36)

7.90 (7.88)

10.18 (9.96) 1 M H2O

27

88

107 1

58.11 (58.15)

7.89 (8.16)

12.89 (12.79) 1.3 M HrO

1

67

86 1

55.75 (55.55)

7.17 (7.02)

12.35 (12.09) 1.4 M H2O

2

94

959

55.66 (55.73)

7.29 (7.40)

14.22 (14.44) 0.6 M H20

364

Peptide substrates for HIV- 1 protease Peptide synthesis Amino acid derivatives were prepared using standard procedures described in the literature. HBTLJ (2-( 1Hbenzotriazol- 1-y1)-1,1,3,3-tetramethyluroniumhexafluorophosphate was a gift from Dr R. Knorr (F. Hoffmann La Roche, Basle). All other reagents were purchased from either Aldrich or Fluka. The - ONSu active esters were obtained by reacting suitably protected amino acids with N-hydroxysuccinimide (HONSu) and dicyclohexylcarbodiimide (DCCI) in dimethoxyethane and were mostly crystalline, The mixed anhydride reactions were carried out in tetrahydrofuran at - 15O using isobutylchloroformate or pivaloylchloride and N-ethylmorpholine (NEM). The reaction work-up procedure involved the usual acidbase wash in ethylacetate or chloroform. The analytical data of peptides 1, 2 and their main intermediates are provided in Table 1. Z.Pro.Ile.NH.iBu (5). Z.Ile.NH.iBu (obtained by mixed anhydride coupling ofZ.Ile.OH and isobutylamine) was hydrogenolysed in methanol for 18 h in the presence of 5% Pd/C. The catalyst was removed by filtration and solvent evaporated to give H.Ile.NH.iBu (3) as an oil in quantitative yield. A solution of (3) (23 g, 124 mmol) andZ.Pro.ONsu(4)(43 g, 124 mmol)inDMF(200 mL) was stirred for 1 h at 0" followed by 18 h at room temperature. The solid was collected by filtration and washed with DMF, water, 0.5 M HCl, water and dried to obtain 40 g of (5). Rf (B) = 0.35.

Z. TyqBu).Pro.Ile.NH.iBu(7). Z.Pro.Ile.NHiBu (5) was hydrogenolysed in methanol to give H.Pro.Ile.NH.iBu (6) as a solid. Z.Tyr('Bu).OH (5.5 g, 14.8 mmol) and (6)(4.2 g, 14.8 mmol) were dissolved in D M F (100 mL) and cooled in an ice-salt bath and HONSu (3.4g, 29.6 mmol) and DCCI (3.05 g, 14.8 mmol) were added. After overnight stirring at room temperature DCU was removed and the solvent evaporated. The residue was partitioned between ethyl acetate and water. The organic layer was washed with 10% citric acid, saturated sodium bicarbonate, water, brine and dried over anhydrous sodium sulphate. The crude product obtained after solvent evaporation was purified by silica gel column with chloroform to give 4.7 g of (7). Rf (A) = 0.35. Z.Asn. TyeBu).Pro.Zle. NHiBu (9). Z.Tyr('Bu).Pro.Ile. NHiBu (7) (4.7 g, 7.39 mmol) was hydrogenolysed and coupled with Z.Asn.OH (1.97 g, 7.39 mmol) in D M F using HOBt (1 g, 7.39 mmol) and DCCI (1.52 g, 7.39 mniol) as described for (7). The crude product was purified by silica gel column using 5% methanol in chloroform to yield 3.5 g of (9) as a solid. Rf (B) = 0.38. "u. 0.Succ.SeeBu).Leu. OH (15). Monomethyl succinate was converted to methyl-tert.-butyl ester (9) and then hydrolysed with sodium hydroxide in methanol to mono tert.-butyl succinate as a crystalline solid. Z.Ser

(tBu).Leu.OMe (11) was obtained by mixed anhydride coupling of Z.Ser( 'Bu).OH and H.Leu.OMe hydrochloride in 97% yield (oil) and hydrogenolysed to Tos - H; .Ser(tBu).Leu.OMe (12). tBu.O.Succ.ONSu (2.35 g, 8.68 mmol) and (12) (4g, 8.68 mmol) were coupled in D M F in the presence of N-ethylmorpholine and the reaction worked up as described for (7) to obtain 1.6 g oftBu.O.Succ.Ser(tBu).Leu.OMe after column purification (ethylacetate-hexane, 1:1). Ester hydrolysis in methanol with 1 M NaOH gave (15). Rf (B) = 0.67. Succ, Ser.Leu.Asn. Tyr.Pro.Ile.NHiBu (1). Z .Asn. Tyr (tBu).Pro.Ile.NHiBu (9) (4.23 g, 5.63 mmol) was hydrogenolysed and coupled with (15) (2.43 g, 5.63 mmol) in D M F using HONSu (1.3 g, 11.28 mmol) and DCCI (1.16 g, 5.63 mmol). The crude product was purified by flash column chromatography (5 % methanol in chloroform) to obtain 5 g of (16), which was treated with trifluoroacetic acid (50 mL) containing anisole (5 mL). After 2 h the solvent was evaporated and the crude product purified by flash column with CHCh/MeOH/ AcOH/H20 (90:15:3:2) to give 2.8 g of (1) as a white solid. Rf (C) = 0.45. HPLC: Fig. 5(a). Z.Asn.Phe.Pro.Ile.NHiBu (19). Using the procedure for the preparation of (5), Z.Phe.ONSu (28 g, 70.7 mmol) and H.Pro.Ile.NH.iBu (6) (20 g, 70.6 mmol) gave 35 g of Z.Phe.Pro.Ile.NH.iBu (17), which was hydrogenolysed and coupled (8.5 g, 19.8 mmol) with Z.Asn.OH (5.36 g, 19.8 mmol) using HOBt (2.67 g, 19.8 mmol), HBTU (7.5 g, 19.8 mmol) and NEM (2.27 g, 19.8 mmol). After overnight stirring at room temperature the solid was filtered and washed with DMF, 0.5 M HCI, water, sodium bicarbonate, water and ether to give 10.3 g of (19) as a white solid. Rf (B)= 0.51. Z.SefBu).Gln.OH (22). A solution of Z.Ser('Bu).ONSu (39.9 g, 101.6 mmol) in D M F (250 mL) was added to H.Gln.OH (14.9g, 101.6mmol) in 1 M NaOH (25.4 mL) at 0" under stirring. Sodium bicarbonate (21 g) was added and the reaction mixture stirred overnight at room temperature. Any solid was removed by filtration and the solvent evaporated, the residue dissolved in water (150 mL) and acidified to pH -4 with 2 M HCl at 0 O . The product was extracted into ethyl acetate and washed with water, brine, dried and the solvent evaporated. 25g of (22) was obtained after crystallisation from ethylacetate-hexane. Rf (C) = 0.58.

Z. Val.Se@Bu).Gln.OH (24). (22) (25 g, 59 mmol) was hydrogenolysed and coupled with Z.Val.ONSu (19 g, 55.4 mmol) in D M F in the presence of 4 M NaOH (13.8 mL) and sodium bicarbonate (12 g) as described for (22) to give (24) as a white solid. It was hydrogenolysed in methanol-water (4:l) to yield H.Val. Ser('Bu).Gln.OH (25) quantitatively. Rf (D) = 0.76. 365

B.K. Handa and C. Kay er

n

Yr

!Bu

Pro

I

e NHiBu

3 NHiBu

Z-

Z

TOSH+,

'Bu.0.Succ

'Bu.0. Succ

'Bu.0.Suc

NHiBu

*Bu

14 !Bu

15

'

-NHiBu

3H Tos.H+~

i

iv

OMe

1

OH Tos-H+

?Bu

1

iii

-NHiBu

I

-NHiBu

- NHiBu

ii

:Bu

SUCl

>Bu

- NHiBu

I -NHiBu

Reagents and conditions: i. H:, Pd/C, 6 h, ii. DCCI/HONSu, iii. H2, Pd C, p-tolucnc sulphonic acid. 4h. iv. DCCI/HOBt, v. pivaloylchloridelN-ethylmorpholine in THF at - IS', vi. 1 M sodium hydroxide in methanol, 4 h. vii. trifluoroacetic acid,anisole.

FIGURE 3 Synthetic route for the synthesis of hcxapeptide 1

[Bu.O.Succ.Val.Se('Bu). Gln.OH (26). 'Bu.O.Succ. ONSu (8 g, 29.5 mmol) and (25) (1 1.5 g, 29.6 mmol) in D M F (200mL), 4~ NaOH (7.5 mL) and water (10 mL) were reacted as described for (22) in the presence of sodium bicarbonate (8.4 g) to obtain 9.5 g of (26) as a white solid. Rf (C) = 0.65.

Succ. Va1.Ser.Gln.Asn.Phe.Pro.ile.NHiBu (2). H .Am. Phe.Pro.1le.NH.iBu (5.45 g, 10 mmol), obtained after overnight hydrogenolysis 'of (19) in methanol-DMF ( 2 :l), was coupled with LBu.Succ.Val.Ser(tBu).Gln.OH (26) (5.45 g, 10 mmol) in the presence of HOBt (1.35 g, 10 rnmol), HBTU (3.79 g, 10 mmol) and NEM (1.15 g, 10 mmol) and the reaction worked up as described for (19). The white solid so obtained was suspended in water (1 50 mL), heated at 50 ', filtered and triturated with ether to give 9.5 g of (27), which was treated with trifluoroacetic acid (95 mL) containing phenol ( 5 8). After 2 h the solvent was evaporated and the resulting 366

solid washed with ether several times. It was then suspended in isopropanol-water (1:3), heated at 70°, allowed to cool, filtered and further washed with ethylacetate and ether to obtain 8 g of (2) as a white solid. Rf (C) = 0.32. HPLC: Fig. 5(b). RESULTS AND DISCUSSION A major problem frequently encountered with peptide substrates for proteinases, particularly in crude enzyme preparations, is the degradation of the substrates by other endo and exo peptidases. Two ways of overcoming this problem are to block N - and C-termini against exopeptidase attack and to keep the size of the peptide as small as possible. Isobutylamide was chosen to protect the C-terminus against carboxypeptidase action as this also provides a mimic of the side chain of P3' valine. A succinyl residue was considered suitable lo block the N-terminus against aminopeptidase attack

Peptide substrates for HIV- 1 protease coupling methods therefore, played an important role in obtaining the pure products. Pre-activated amino acids in the form of crystalline N-hydroxysuccinimide esters were used. The dipeptide derivative H.Pro.Ile.NHiBu (6) was coupled with Z.Phe.ONSu to give Z.Phe. Pro.Ile.NHiBu which upon hydrogenolysis and coupling with Z.Asn.OH gave (19), one of the two main fragments required. For this coupling, HBTU was preferred over DCCI/HOBt as the resulting urea is easily removed from the highly insoluble compound (19) by solvent washings. The second fragment tBu.O.Succ.Val.Ser(tBu) .Gln.OH (26) was synthesised by successive salt couplings with - ONSu activated amino acids. This strategy eliminated the necessity of using a C-terminal protecting group for Gln. Once again, HBTU served exceedingly well to obtain the fully protected heptapeptide (27) in pure form. Finally, peptide 2 was obtained from (27) by trifluoroacetic acid treatment using phenol as scavenger. Throughout the synthesis of peptide 2, extensive use of solvent washings was made to remove the impurities. All of the intermediates and final products during the syntheses of 1 and 2 were characterized by elemental analysis, H1-NMR and FAB/MS. Final purity of the

and to enhance water solubility. Synthesis of peptides 1 and 2 is schematically described in Figs. 3 and 4. The hexapeptide 1 was synthesised by DCCI/HOBt coupling of the two major fragments (15) and (lo), which in turn were prepared from suitably protected amino acids in a stepwise coupling manner. Z.Pro.Ile. HNiBu was obtained from Z.Pro.ONSu and H.1le.NHiBu and further elongation to tetrapeptide Z.Asn. Tyr('Bu).Pro.Ile.NHiBu (9) was accomplished by coupling with Z.Tyr('Bu).OH and Z.Asn.OH respectively requiring silica gel column chromatography in each step. To obtain (15), Z.Ser(tBu).OH and cl H; .Leu.OMe were coupled via the mixed anhydride method. Hydrogenolysis followed by coupling with tBu.O.Succ.ONSu (13) gave (14), which was hydrolysed to free acid tBu.O.Succ.Ser(tBu).Leu.OH (15). At each step column chromatography was required to obtain pure products. The final deblocking of the fully protected hexapeptide (16) was effected by trifluoroacetic acid-anisole mixture and the product purified by flash column to obtain peptide 1. The presence of Val, Gln and Phe in peptide 2 conferred low solubility thereby making silica gel column chromatography unsuitable for purification of this compound and many of its intermediates. The choice of

Val

Ser

Gln

e

NHiBu NHiBu

NHiBu

NHiBu

NHiBu

tBu.O.Succ

I liii1,fBu I

261

II OH

Iii

27

v

-NHiBu

NHiBu

2 Reagents and conditions: i. H1 PdjC in methanol/DMF (2:1), 36 h, ii. HBTU/HOBt, N-ethylmorpholine, iii. salt coupling, iv. Hz, Pd/C in methanol/HIO (l:l), 6 h, v. TFAjphenoL FIGURE 4 Synthetic route for the synthesis of heptapeptide 2.

367

B.K. Handa and C. Kay (a) Peptide 1

(b) Peptide 2

Conditions: i. Aquapore butyl reverse phase column (7 p, 30 x 4.6 mm) ii. 95"" A-95'" B over 15 min, A = 0.1 %, B = 0.085% TFA in 70% acetonitrile. FIGURE 5 Purity check of peptides 1 and 2 b) analktical reverse phase HPLC.

two peptides was checked by reverse phase analytical HPLC (Fig. 5). A colorimetric assay for HIV- 1 proteinase using the peptide substrates 1 and 2 has been devised (8) where the released dipeptide H.Pro.Ile.NHiBu is reacted with isatin to form a blue product which is measured spectrophotometrically. Although peptide 1 is, as predicted, an adequate substrate, the heptapeptide 2 was hydrolysed I0 times more rapidly (when assayed using equimolar substrate and enzyme concentrations). Km values for peptides 1 and 2 were approximately 1.42 and 0.79 respectively (8). Several spectrometric assays have been recently described including the fluorescence based assays of Geoghegan (10) and Matayoshi et a/. ( I I ) and the chromogenic assays of Nashed et al. (12), Tomaszek et 01. (13) and Richard etal. (14). The major advantage of these assays is that they allow continuous measurements to be made. However, there may be problems such as low water solubility of substrates (1 1) or fluorescence quenching (10). The colorimetric assay utilizing peptides 1 and 2 was designed to be suitable for use in a drug screening program. Although a stopped time assay, it has the advantage of being simple, cheap, sensitive and the use of peptides blocked at both amino and carboxy termini makes these substrates specific for 368

HIV proteinase, thus the requirement for highly purified enzyme is eliminated. Approximately 750 potential proteinase inhibitors have been evaluated utilizing these peptide substrates in the colorimetric assay (15). ACKNOWLEDGEMENT We thank the Department of Physical Methods for the spectral and anal).tical data.

REFERENCES 1. Katoh, J . . Yoshinaka, Y., Rein, A,, Shibuya, M., Okada, T. & Oroszlan, S . (1985) Virology 145, 280-292 2. Kohl, N.E., Emini, E.A., Schleif, W.A., Davis, J., Hcimbuch, J.C.. Dixon, R.A.F., S c h i c k , E.J. & Sigal, J.S. (1988) Proc. Yatl. Acad. Scr. USA 85, 4686-4690 3. Barre-Sinoussi, F., Cherman, G.C.. Rey, G . , Nugeyre, M.T., Chamaret, S . , Gruest, G . , Dauguet, C . , Axler-Blin, C., VezinetBrun. F.. Rouzioux, C., RoLernbaum, W. & Montagnier, L. (1983) Science 220, 868-870 4. Gallo. R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M., KaRedfield, R., Oleske, G., plan, M., Haynes, B.F., Palker, T.G., Safai, B., White, G., Foster, P. & Markham, P. (1984) Science 224, 500-502 5. Ratner, L., Haseltine, W., Paiarca, R., Livak, K.J., Starlich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A.,

Peptide substrates for HIV-1 protease

6.

7. 8. 9. 10. 11.

12.

13.

Baumiester, K., Ivanoff, L., Petteway, S.R., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. & Wong-Staal, F. (1985) Nature 316, 277-284 Henderson, L.E., Copeland, T.D., Sowder, R.C., Schnltz, A.M. & Oroszlan, S. (1988) in Human Retroviruses, Cancer and AIDS: Approaches to Prevenlion and Therapy, pp. 135-147, Liss, New York Norbeck, W.D. (1990) in Annual Reports in Medicinal Chemistry (Plattner, ed.) pp. 149-158, Academic Press, New York Broadhurst, A.V., Roberts, N.A., Ritchie, A.J., Handa, B.K. & Kay, C. (1991) Anal. Biochem. 193, 280-286 Anderson, G.W. & Callahan, F.M. (1960)J. Am. Chem. SOC.82, 3359 Geoghegan, K.F., Spencer, R.W., Danley, D.E., Contillo, L.C. & Andrews, G.C. (1990) FEBS Lett. 262, 119-122 Matayoshi, E.D., Wong, G.T., Krafitt, G.A. & Erickson, J . (1 990) Science 247, 954-957 Nashed, N.T., Louis, J.M., Sayer, J.M., Wondrak, E.M., Mora, P.T., Oroszlan, S. & Jerina, D.M. (1989) Biochem. Biophys. Res. Commun. 163, 1079-1085 Tomaszek, T.A., Magaard, V.W., Bryan, H.G., Moore, M.L. & Meek, T.D. (1990) Biochenz. Biophys. Res. Comnzun. 168, 274280

14. Richard, A.D., Phylip, L.H., Farmerie, W.G., Scarborough, P.E., Aleurez, A., Dunn, B.M., Hirel, Ph.-H., Konvalinka, J., Strop, P., Pavlickova, L., Kosta, V. & Kay, J. (1 990) J . Biol. Chem. 265, 7733-7736 15. Roberts, N.A., Martin, J.A., Kinchington, D., Broadhurst, A.V., Craig, J.C., Duncan, I.B., Galpin, S.A., Handa, B.K., Kay, J., Krohn, A., Lambed, R.W., Merrett, J.M., Mills, J.S., Parkes, K.E.B., Redshaw, S., Ritchie, A.J., Taylor, D.L., Thomas, G.J. & Machin, P.J. (1990) Science 248, 358-361

Address: Dr B . K . Handa Department of Physical Methods Research Division Roche Products Limited P 0 Box 8 Welwyn Garden City Herts AL7 3AY, UK Tel. (0707)328128 Ext 2451 Fax (0707)373504

369