Solid-phase synthesis and applications of N-(S-acetylmercaptoacetyl) peptides

187,3‘%354

ANALYTICALBIOCHEMISTRY

(1990)

Solid-Phase Synthesis and Applications A/-(S-Acetylmercaptoacetyl) Peptides Jan Wouter

Drijf’hout,*,l

Willem

Bloemhoff,-f’

of

Jan T. Poolman,$

and Peter

Hoogerhout$’

*Department of Medical Microbiology, State University of Groningen, The Netherlands; TGorlaeus Laboratories, of Leiden, The Netherlands; and $.RIVM (National Institute of Public Health and Environmental Protection), Laboratory of Bacterial Vaccines, P.O. Box I,3720 BA Bilthoven, The Netherlands

Received

November

State University

lo,1989

The reagent pentafluorophenyl S-acetylmercaptoacetate was used to modify the N-terminus of resin-bound side-chain-protected peptides. The modification was carried out in an automated cycle in the final stage of fluorenylmethoxycarbonyl (Fmoc)/polyamide-mediated solid-phase synthesis. Side-chain deprotection and cleavage from the resin with aqueous trifluoroacetic acid gave the IV-(S-acetylmercaptoacetyl) peptides. The S-acetylmercaptoacetyl peptides were transformed into reactive thiol-containing peptides by incubation with hydroxylamine at neutral pH. The S-deacetylation was performed in the presence of a sulfhydryl-reactive compound (or intramolecular group) to enable immediate capture of the sensitive thiol. Three applications were investigated. An S-acetylmercaptoacetyl peptide, containing a sequence of a meningococcal membrane protein, was incubated with hydroxylamine in the presence of 5-(iodoacetamido)fluorescein to give the corresponding fluorescein-labeled peptide in 62% yield. The same peptide was also S-deacetylated in the presence of bromoacetylated poly-L-lysine to afford a peptidefpolylysine conjugate. Finally, a peptide corresponding to a sequence of herpes simplex virus glycoprotein D was prepared. This peptide, containing an N-terminal S-acetylmercaptoacetyl group and an additional C-terminal S-(3-nitro-2-pyridinesulfenyl)cysteine residue, was converted into a cyclic disulfide peptide (20%). 0 1990 Academic Press, IIW.

Synthetic peptides play an important role in current biochemical, pharmacological, and immunological research and are widely prepared using solid-phase meth’ Present address: Merrell Dow Research Institute, kara, Strasbourg Cedex, France. ’ To whom correspondence should be addressed. 0003.2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

Inc. reserved.

16, Rue d’An-

odology. After deprotection and cleavage from the solid support, further processing of the peptides is often required, for example, coupling with marker substances (labels) or proteins. Numerous homo- and heterobifunctional-crosslinking reagents have been used for such purposes. However, it is often difficult to achieve selectivity in the coupling reaction since most peptides contain several reactive groups. At present, introduction of a group of specific reactivity into peptides during solid-phase synthesis, before the final deprotection steps, is rather uncommon. Reported methods are N-terminal chloroacetylation (1) and bromoacetylation (2) or coupling of an S-(3-nitro-2-pyridinesulfenyl)cysteine derivative (3). The modified peptides, obtained after complete deprotection, contain good leaving groups and can be coupled with nucleophilic compounds, including sulfhydryl-containing proteins.3 On the other hand, modification of peptides with a nucleophilic group seems to be useful. In cysteine-devoid peptides this amino acid can be added to the sequence of interest. However, cysteine-containing peptides may require laborious additional deprotection and purification steps and are difficult to handle due to their susceptibility to (air) oxidation. As an alternative, we have been using N-terminal S-acetylmercaptoacetylation of peptides in solid-phase synthesis. The reagent N-succinimidyl S-acetylmercaptoacetate has been proposed for protein modification in solution (5). In solid-phase synthesis, according to the Fmoc*/polyamide method (6),

’ An interesting special case is solid-phase synthesis of biotinyl peptides (4), which can be coupled noncovalently with (streptfavidin. 4 Abbreviations used: tBu, tert-butyl; DMF, N,N-dimethylformamide; FAB, fast atom bombardment; Fmoc, Auorenylmethoxycarbonyl; HSV gD, herpes simplex virus glycoprotein D; Nle, norleucine; Npys, 3-nitro-2-pyridinesulfenyl; Pfp, pentafluorophenyk RP, reversed phase; SAMA, S-acetylmercaptoacetyl. 349

350

DRIJFHOUT

we have used the corresponding pentafluorophenyl ester (7). The S-acetylmercaptoacetyl (SAMA) group is stable under the acidic conditions necessary for side-chain deprotection and cleavage of the peptides from the solid support. The SAMA peptides can be transformed rapidly and quantitatively into the corresponding thiol derivatives by mild treatment with hydroxylamine at about neutral pH. It is most convenient to perform the S-deacetylation in the presence of a sulfhydryl-reactive compound. In this way a successive conjugation reaction will occur immediately. In this paper we illustrate the versatility of this approach by presenting three examples. A peptide corresponding to the sequence 176-185 of a class 1 outer membrane protein of Neisseriu menilzgitidis (8) containing an N-terminal SAMA-Gly-Gly spacer was prepared. This peptide was either coupled with a fluorescence label or conjugated with bromoacetylated poly-L-lysine. Furthermore, a peptide corresponding to the sequence 9-21 (9) of glycoprotein D (gD) from mature herpes simplex virus (HSV) type 1 (10) and containing an N-terminal SAMA group and an additional C-terminal cysteine residue was prepared and, thereafter, converted into a cyclic disulfide peptide. MATERIALS

AND

METHODS

Fmoc-protected amino acid esters, l-hydroxybenzotriazole, and solvents for peptide synthesis were purchased from Pharmacia/LKB, Cambridge, UK. 3-Nitro2-pyridinesulfenyl chloride (Npys-Cl) was prepared (11) starting from 2-chloro-3-nitropyridine (Aldrich). NSuccinimidyl bromoacetate was prepared according to published procedures (12,13) and crystallized from isopropanol (91%). Poly-L-lysine hydrochloride (1M, 30,000-70,000; av 38,000; Lot 107F-50151) was obtained from Sigma Chemical Co. (St. Louis, MO) and 5(iodoacetamido)fluorescein from Molecular Probes, Inc. (Eugene, OR) NMR spectra were recorded on a JEOL-FXBOO spectrometer. Chemical shifts are given in ppm (6) relative to internal tetramethylsilane. HPLC. Two systems were used for RP-HPLC analysis. An Applied Biosystems Model 151A separation system, equipped with a Prep-10 Aquapore Octyl, Cs, 300A pore size, 20-pm spherical silica column (10 X 100 mm cartridge), was used in experiments with the meningococcal peptide. A liquid chromatograph 7000 (Micromeretics Instrument Corp., Norcross, GA) was used for analysis of synthetic HSV gD peptides. Analytical and semipreparative runs were performed on Merck Lichrocart 5C,, (0.46 X 25 cm) and polygosil 10Cls (MachereyNagel, Diiren, FRG) (0.9 X 25 cm) columns, respectively. FAB mass spectra were recorded in the positive ion mode on a JEOL HX llO/HX 110 mass spectrometer,

ET

AL.

equipped with a standard JEOL FAB source operated 3 kV. Spectra were obtained using a magnet scan rate 40 s from m/z 0 to 2000. The peptide 6 was deposited an acid-etched stainless-steel probe tip with glycerol matrix.

at of on as

Amino acid analysis. Samples were hydrolyzed with 5.7 N HCl in uacuo at 110°C in sealed glass tubes for 24 h. The amino acid content of the hydrolysates was determined with a Kontron Liquimat III analyzer. Pentafiuorophenyl S-acetylmercaptoacetate (SAMAOPfp) (7). A solution of N,N’-dicyclohexylcarbodiimide (4.50 g, 21.8 mmol) in 40 ml dichloromethane was added dropwise to a solution of S-acetylmercaptoacetic acid (2.68 g, 20 mmol) (14) and pentafluorophenol (3.75 g, 20.4 mmol) in 20 ml dichloromethane. After stirring for 17 h, the reaction mixture was filtered and evaporated to dryness in vacua. The residue obtained was dissolved in 25 ml dichloromethane and applied to a column of 25 g kieselgel60,230-400 mesh (Merck). The effluent obtained on elution with dichloromethane (100 ml) was evaporated to dryness in vacua. The residue was crystallized from pentane (80 ml). Yield: 4.51 g (75%). Mp: 4041°C. ‘H NMR (CDC&): 6 2.45 (s, 3 H), 4.02 (s, 2 H). i3C NMR (CDCl& 29.5 (CH,), 30.3 (CH,), 124.7 (C-l, m, arom.), 134.9-143.7 (5 X C-F, m), 165.0 (C=O, ester), 192.7 (C=O, acetyl). Peptide synthesis (general). The C-terminal residue was attached to the polymer by using the symmetrical anhydride, which was previously prepared from the Fmoc-protected amino acid by treatment with N,N’-dicyclohexylcarbodiimide according to standard procedures (6). A solution of 0.5 mmol of the anhydride in 4 ml DMF and 24 mg (0.2 mmol) of (N,N-dimethylamino)pyridine was added to 1 g of ultrosyn A (a physically supported polyamide, containing an acid-labile handle to the extent of 81 pmol/g; Pharmacia/LKB), suspended in a minimum volume of DMF. After gentle shaking for 2 h at room temperature, the resin was collected by filtration and washed with DMF. A Biolynx 4170 automated peptide synthesizer (Pharmacia/LKB) was used for continuous-flow solid-phase synthesis (6). Fmoc deprotection (10 min) was effected with piperidine/DMF, 2/8 (v/v). Coupling reactions (45 min) were performed with Fmoc-protected amino acid pentafluorophenyl esters (0.5 mmol) in the presence of 1-hydroxybenzotriazole (0.5 mmol) or-in the case of Arg and Thr-with esters of 3,4-dihydro-3-hydroxy-4oxo-1,2,3-benzotriazine (0.5 mmol). The following sidechain-protecting groups were used: tBu, tert-butyl (thio)ethers (Cys, Thr, Tyr) or esters (Asp); Boc, tertbutyloxycarbonyl (Lys); Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Arg). In the last cycle of the synthesis SAMA-OPfp (0.5 mmol) was coupled in the presence of 1-hydroxybenzotriazole (0.5 mmol) for 30 min, using a standard protocol with omission of the piperi-

SYNTHESIS

OF

N-(S-ACETYLMERCAPTOACETYL)

a

FIG. 1. (a) Synthesis and deprotection of S-acetylmercaptoacetyl peptide 1, containing sequence 176-185 of a Neisseria meningitidis class 1 outer membrane protein. (b) Conjugation of the meningococcal peptide (1) with &(iodoacetamido)fluorescein (3).

dine treatment (i.e., the Fmoc-deblocking step, which in this case would cause undesirable S-deacetylation). SAMA-Gly-Gly-Tyr-Tyr-Thr-Lys-Asp-Thr-Asn-AsnAsn-Leu (1, Fig. la). The protected peptide resin (0.55 g) was washed with DMF, tert-amyl alcohol, acetic acid, tert-amyl alcohol, DMF, and diethyl ether (3 X 5 ml each) and suspended in trifluoroacetic acid/water, 9515 (v/v). After standing at room temperature for 1.5 h with occasional shaking, the resin was removed by filtration and washed with trifluoroacetic acid (3 X 5 ml), diethyl ether (5 ml), trifluoroacetic acid (5 ml), and diethyl ether again (2 X 5 ml). The combined filtrate and washings were concentrated in vacua at 30°C. The residue was precipitated by addition of diethyl ether (25 ml), collected by centrifugation, and dried in vacua over sodium hydroxide. Yield: 92 mg. HPLC: Fig. 2a. Amino acid analysis: Asx 4.00 (ref), Thr 1.86 (2), Gly 1.90 (2), Tyr 1.37 (2), Leu 0.93 (l), Lys 0.92 (1); peptide content 67%.

351

PEPTIDES

A quantity (20 mg) of the material was purified by RPHPLC on the Prep-10 column. Amounts of 1 mg were injected under the conditions described in the legend to Fig. 2. Fractions corresponding to the main peak were combined, evaporated to dryness in vacua at 35”C, and lyophilized from acetic acid, to give 14.7 mg of 1 (peptide content not determined). Coupling of 1 with 5-(iodoacetamido)fluorescein. A solution of 3.1 mg (6 pmol) &(iodoacetamido)fluorescein (3) in 500 ~1 DMF was mixed with 7.4 mg of (HPLCpurified) peptide 1 and 625 ~10.1 M sodium phosphate, containing 5 mM EDTA, pH 6.1. Figure 3a shows the HPLC elution profile. A solution (125 ~1) of 0.2 M hydroxylamine (in the phosphate buffer, pH 6.1) was added and the formation of the fluorescein-labeled peptide (4) was monitored by RP-HPLC analysis (Fig. 3). After 18 h, 4 was separated from the excess 5-(iodoacetamido)fluorescein (3) by semipreparative HPLC (samples of 125 ~1 were injected under the conditions described in the legend to Fig. 2). The appropriate fractions were combined and evaporated to dryness in vacua at 35°C. The residue was taken up in 20 ml of 0.01 M ammonium acetate (stock solution for immunofluorescence experiments). The concentration of 4 was found to be 1.4 X 1O-4M by uv measurement (based on c492= 62.7 X lo3 of the fluorescence label at pH 7.3), corresponding with a yield of 5.1 mg (62%, including the HPLC purification of 1). Conjugation of 1 withpoly(Lys). Crude peptide 1 was dissolved in acetonitrile/O.l M sodium phosphate, containing 5 mM EDTA (pH 6.1), l/l (v/v), in a concentration of 7.5 mg (5 pmol)/ml. Poly(Lys) hydrochloride (11 mg, av mol wt 38,000) was dissolved in 100 ~1water and diluted with 0.85 ml 0.1 M sodium phosphate (pH 7.8) and a solution of 5.9 mg (25 pmol) N-succinimidyl bromoacetate in 50 ~1 DMF was added. The reaction mixture was stirred at room temperature and, after 1 h, subjected to gel filtration using a Sephadex PD-10 column (Pharmacia) equilibrated in 0.1 M sodium phosphate, O.lOOA214

+ a

0.075-

b

0.050-

0.025

O-

/ 15

,LL 0

5

10

15

FIG. 2. RP-HPLC of meningococcal peptide 1 before (a) and after (b) purification. Column: Prep-lo, Aquapore Octyl. Solvent A: 0.1% trifluoroacetic acid in water. Solvent B: 0.09% trifluoroacetic acid in acetonitrile/water, 2/l (v/v). A linear gradient from 10 to 100% B in 20 min and a flow rate of 2.5 ml/min were used.

352

DRIJFHOUT

ET

AL.

0.100 &JO 4

b 0.075

C

!

-

3

0.025 i ii 1

,

0

5

10

I

I

15

0

5

-w min

IO

15

6

5

10

15

FIG. 3. Preparation of fluorescein-labeled meningococcal peptide 4, monitored by RP HPLC. (a) Reaction mixture (10 ~1) before addition of hydroxylamine (see Materials and Methods); (b and c) 2.5 and 18 h after addition of hydroxylamine, respectively. 1, SAMA peptide; 2, thiol peptide; 3,5-(iodoacetamido)fluorescein; 4, fluorescein-labeled peptide (see also Fig. lb). The chromatographic conditions were identical with those described in the legend to Fig. 2.

containing 5 mM EDTA (pH 6.1). The bromoacetylated poly(Lys) was eluted with the same buffer and collected in 3.0 ml. Figure 4a shows the HPLC pattern, The solution of bromoacetylated poly(Lys) was mixed with 1.2 ml of the solution of peptide 1 (Fig. 4b) and deaerated with helium. Next, 50 ~1 of 0.2 M hydroxylamine (in the pH 6.1 buffer) was added. The reaction mixture was examined by HPLC analysis after 5 and 30 min (Figs. 4c and d, respectively). After 60 min, 10 mg

0.100 A 214 t 0.075

a

0.050

0.025

0 0.100

li; b

0.075

0.050

0.025

0

5

10

15

FIG. 4. HPLC-monitoring of the conjugation of (crude) meningococcalpeptide 1 with bromoacetylatedpoly(Lys). (a) Bromoacetylated poly(Lys) (15 pl of the fraction obtained after PD-10 gel filtration; see Materials and Methods). (b) Reaction mixture (20 ~1) before addition of hydroxylamine. (c and d) Reaction mixture (22.5 ~1) 5 and 30 min after addition of hydroxylamine, respectively. [The arrow in (c) indicates the retention time of thiol peptide 2.1 The HPLC conditions are described in the legend to Fig. 2.

(88 pmol) 2-aminoethanethiol hydrochloride was added. After a further period of 60 min (HPLC analysis remained as depicted in Fig. 4d), the conjugate was purified (in two portions) over a PD-10 column using water as the eluant. The appropriate fractions were combined and lyophilized to give 20 mg of a hygroscopic white solid. Since the peptide content of crude 1 was 67% and the conjugation with poly(Lys) was complete, the peptide content of the conjugate was estimated to be 30% by weight. Synthesis of the cyclic [Nle”] HSV gD (9-21) derivative (6, Fig. 5). Resin-bound [Nle”] SAMA HSV gD (9-21)-Cys(tBu) (36 pmol) was washed with DMF. Subsequent treatment with 3-nitro-2-pyridinesulfenyl chloride in DMF (0.2 M, 8 eq) for 90 min gave protected [Nle’l] SAMA HSV gD (9-21)-Cys(Npys)-polymer. Cleavage from the resin and removal of all acid-labile protecting groups were performed by treatment with trifluoroacetic acid/water, 9515 (v/v), for 1 h at room temperature, followed by 1 h at 50°C. [Nlell] SAMA HSV gD (9-21) -Cys( Npys) -OH (5) was purified by semipreparative RP-HPLC, on the polygosillOC1s column. A linear gradient of water/acetonitrile/trifluoroacetic acid, 94/6/0.1 to 40/60/0.1 in 30 min, and a flow rate of 4.0 ml/min were used. The yield was 22 mg. Figures 6a and b show analytical HPLC before and after the purification, respectively. An aliquot (5 mg) of purified 5, dissolved in 5.0 ml 0.1 M sodium phosphate (pH 7.5), was cyclized upon addition of 200 ~1 of a solution of hydroxylamine (0.2 M) in 0.1 M sodium phosphate (pH 6). The cyclization was complete in 1 h, as indicated by analytical RP-HPLC analysis (Fig. 6~). The cyclic disulfide peptide (6) was purified by semipreparative RP-HPLC under the conditions described above. The yield was 2.8 mg. Amino acid analysis: Arg 2.09 (2), Asx 3.14 (3), Gly 0.95 (l), Ala 1.00 (ref), Pro 1.09 (l), Phe 1.07 (l), Leu 1.00 (l), Nle 1.04

SYNTHESIS CI+3.CO-S-CH2-(Pr~leCted

peptide)-NH_

OF ,CO-

N-(S-ACETYLMERCAPTOACETYL)

(Polymer)

and tert-butyl groups withstand the usual deprotection conditions (i.e., trifluoroacetic acid) and must be removed in a separate step with mercury salts or iodine. Thereafter, purification of the peptides is often troublesome. Removal of the S-trityl group is possible with trifluoroacetic acid, but suffers from the fact that an equilibrium is established between cleaved trityl ion and protected cysteine. In contrast, deprotection of SAMA peptides is easily achieved by treatment with hydroxylamine at about neutral pH and, more importantly, this step can be performed in the conjugation stage. In this way experimental handling of sensitive thiol peptides is circumvented. As outlined in Fig. lb, a fluorescence label was coupled with SAMA peptide 1. Thus, a solution of peptide 1 and an excess of 5-(iodoacetamido)fluorescein (3) in N,Ndimethylformamide/sodium phosphate buffer (pH 6) was incubated with a solution of hydroxylamine. The progress of the reaction was monitored by RP-HPLC (Fig. 3). As shown in Fig. 3b, the intermediate thiol peptide (2) could hardly be detected, indicating a very fast subsequent reaction of 2 with the iodoacetamido compound 3. (The thiol peptide could easily be detected in the absence of 3). The conversion of SAMA peptide 1 into fluorescein-labeled peptide 4 was complete after 18 h. The labeled peptide was purified by RP-HPLC and is

CH

CH$O-S-C~(Protected

peptide).NY

,CO-

(Polymer)

CH

CF3COOH

C~-Co-SCHi(Peplide)-NH

,COOH -CH CH I2 (5)

NVH

C%-(Peptide)-NH

,COOH

Y

‘CH

Peptide

FIG. 5.

= Leug-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg~ly-Lys.A~p2t

Synthesis

of the cyclic

[Nle”]

HSV

gD (9-21)

derivative.

(l), Cys 0.20 (l), Lys 1.59 (2). FAB MS showed MH’ m/z 1704.9 (calculated: m/z 1704.86).

at A214C4

RESULTS

AND

353

PEPTIDES

DISCUSSION

In solid-phase peptide synthesis, according to the Fmoc/polyamide method (6), S-acetylmercaptoacetyl peptides are conveniently prepared by coupling pentafluorophenyl S-acetylmercaptoacetate (7) in a fully automated cycle at the end of the program. Side-chain deprotection and cleavage of the peptides from the solid support are performed with trifluoroacetic acid in the presence of scavengers (usually 5% water or thioanisole), which leave the S-acetylmercaptoacetyl (SAMA) group intact. A typical example (Fig. la) is presented by the synthesis of a peptide containing the sequence 176-185 of a class 1 outer membrane protein of N. meningitidis. After removal of the Fmoc group from the N-terminal Gly-Gly spacer, the SAMA residue was incorporated. Deprotection was effected with trifluoroacetic acid/water, 95/5, for 1.5 h at room temperature and afforded peptide 1 in good yield. The use of SAMA peptides for conjugation with sulfhydryl-reactive compounds offers advantages over the application of cysteine-containing peptides. In Fmocmediated solid-phase synthesis, the side chain of cysteine residues is typically protected with an acetamidomethyl, tert-butyl, or trityl group. The acetamidomethyl

a

b

t

!?

0.4

d

s 0.2

0

Ami"

FIG. 6.

L-

L

IO

20

30

0

10

20

-2

Analytical RP-HPLC chromatograms of HSV gD peptides. (a) [Nle”] SAMA HSV gD (9-21)-Cys(Npys)-OH [5, crude, 10 ~1 of a solution of 0.5 mg/ml in acetonitrile/water, l/l (v/v)]. (b) 5, purified by RP semipreparative HPLC (see Materials and Methods). (c) Reaction mixture (30 ~1) 1 h after incubation of 5 with hydroxylamine. 6, cyclic disulfide peptide; 7, 3-nitro-2-thiopyridone. (d) 6, purified by semipreparative RP HPLC. Column: Merck LiChrocart 5C,s (0.46 X 25 cm). Gradient: water/acetonitrile/trifluoroacetic acid, 94/6/0.1 to 40/60/0.1, in 30 min. Flow rate: 1.0 ml/min.

354

DRIJFHOUT

currently being used in immunofluorescence experiments (unpublished data). Peptide 1 was also used for conjugation with poly-Llysine. First, poly(Lys) was bromoacetylated in a sodium phosphate buffer (pH 7.8). Since fully bromoacetylated poly(Lys) was expected to be insoluble in aqueous solutions, a rather small amount of N-succinimidyl bromoacetate was used for the modification. Bromoacetylated poly(Lys) was purified by gel filtration in sodium phosphate buffer (pH 6). The purified material showed a broad elution profile when examined by RP-HPLC (Fig. 4a). An excess of the solution of bromoacetylated poly(Lys) was mixed with a solution of crude SAMA peptide 1 in acetonitrile/sodium phosphate buffer (pH 6) (Fig. 4b), and the mixture was incubated with hydroxylamine. As shown in Figs. 4c and d, S-deacetylation of peptide 1, followed by conjugation of the liberated thiol peptide 2 with bromoacetylated poly(Lys), proceeded rapidly and quantitatively. Remaining bromoacetyl groups were then blocked by addition of an excess 2-aminoethanethiol, and the peptide-poly(Lys) conjugate was purified by gel filtration in water and lyophilized. The material obtained has been used successfully as a coating antigen in enzyme-linked immunosorbent assays (unpublished data). The final example (Fig. 5) of the use of SAMA peptides concerns the preparation of a cyclic disulfide peptide (6) containing sequence 9-21 of herpes simplex virus glycoprotein D. The peptide sequence was modified in two ways. For convenience, that is, to avoid problems caused by possible oxidation or alkylation of methionine-11, this residue was replaced by isosteric norleutine. More importantly, SAMA and a protected cysteine residue were added to the sequence at the N- and the Cterminus, respectively, to enable the cyclization to occur. In the first step of the synthesis Fmoc-Cys(tBu)-OH was esterified with the solid support, followed by assembly of the protected sequence in the usual manner. The resin-bound Cys(tBu)-peptide was then treated with 3nitro-2-pyridinesulfenyl chloride (Npys-Cl) to give the corresponding (sulfhydryl-reactive) Cys(Npys)-peptide. Cleavage of the peptide from the resin and removal of all acid-labile protecting groups proved to be troublesome. Since the peptide contains two arginine residues protected at their side chains with 4-methoxy-2,3,6-trimethylbenzenesulfonyl groups, deprotection with trifluoroacetic acid in the presence of thioanisole seemed to be the method of choice. Several arginine-containing SAMA peptides have been deprotected successfully under these conditions. In this particular case, however, peptide 5 could not be obtained in acceptible purity. For-

ET

AL.

tunately, treatment of the peptide-resin with aqueous trifluoroacetic acid at 50°C gave a better result (Fig. 6a) and peptide 5 was obtained in moderate yield after semipreparative RP-HPLC. On incubation of a very dilute solution of peptide 5 in a sodium phosphate buffer (pH 7.5) with hydroxylamine, two distinct products readily formed as detected by HPLC (Fig. 6~). The faster eluting (yellow) compound was easily identified as 3-nitro-2-thiopyridone (7). The slower eluting compound was purified by HPLC and, thereafter, subjected to FAB MS analysis. The FAB mass spectrum showed a signal of the protonated molecular ion (MH+) at m/z 1704.9, which is in agreement with the structure of cyclic peptide 6. In conclusion, it has been demonstrated that S-acetylmercaptoacetyl peptides can be prepared conveniently by solid-phase synthesis according the Fmoc/polyamide method. S-Deacetylation of the peptides, followed by reaction with a sulfhydryl-reactive group, can be performed by a facile procedure. ACKNOWLEDGMENTS The synthesis 21), is part of a lands’ Technology pices of Dr. S. State University). for the FAB MS

of the cyclic peptide, research program on Foundation, Grant Welling-Wester and We thank E. Evers analysis.

containing [Nle”] HSV gD (9HSV (supported by the NetherGGN37.0506) under the ausDr. G. W. Welling (Groningen and G. van de Werken (RIVM)

REFERENCES 1. Lindner,

W., and Robey,

F. A. (1987)

Znt. J. Peptide

Protein

Res.

30,794-800. 2. Robey, F. A., and Fields, R. L. (1989) Anal. Biochem. 177, 373377. 3. Drijfbout, J. W., Perdijk, E. W., Weijer, W. J., and Bloemhoff, W. (1988) Znt. J. Peptide Protein Res. 32,161-166. 4. Lavielle, S., Chassaing, G., Beaujouan, J. C. Torrens, quet, A. (1984) Znt. J. Peptide Protein Res. 24,480-487.

Y., and Mar-

5. Duncan, R. J. S., Weston, P. D., and Wrigglesworth, R. (1983) Anal. Biochem. 132,68-73. 6. Dryland, A., and Sheppard, R. C. (1986) J. Chem. Sot., Perkin Trans. 1,125-137. 7. Drijfbout, J. W. (1989) Ph.D. Thesis, Leiden, The Netherlands. 8. Barlow,

A. K., Heckels,

J. E., and Clarke,

I. N. (1989)

Mol.

Micro-

biol. 3, 131-139. 9. Eisenberg, R. J., Long, D., Hogue-Angeletti, (1984) J. Viral. 49,265-268.

R., and Cohen,

G. H.

10. Watson, R. J., Weis, J. H., Salstrom, J. S., and Enquist, (1982) Science 218,381-384. 11. Matsueda, R., Higashida, S., Ridge, R. J., and Matsueda, (1982) Chem. Z&t. 921-924. 12. Hora, J. (1973) Biockem. Biophys. Acta 310,264-267. 13. Sepal, D. M., and Hurwitz, E. (1976) Biochemistry 160,

L. W.

5258. 14. Benary,

E. (1913)

Ber. Dtsch.

Chem.

Ges. 46,2103-2107.

G. R.

5252-