applications of bop reagent in solid phase peptide synthesis iii. solid phase peptide synthesis with...

I t i t . J . Peptide Protein Res. 33, 1989, 133-139

Applications of BOP reagent in solid phase peptide synthesis 111. Solid phase peptide synthesis with unprotected aliphatic and aromatic hydroxyamino acids using BOP reagent

ALAIN FOURNIER*. WALEED DANHO and ARTHUR M. FELIX

Peptide Research Department, Roche Research Center, Hoffmann-La Roche Inc.. Nutley, NJ, USA

Received 15 February, accepted for publication 1 July 1988

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate], which has been shown to be ideally suited for solid phase synthesis, has now been found to be useful for solid phase synthesis using a minimal side-chain protection scheme. This new application of the BOP reagent was exemplified by the successful synthesis of the CCK-7 analog, Ac-Tyr(S0, H)-Met-Gly-Trp-Met-Thr(S0, H)- Phe-NH,, using unprotected Boc(hydroxy)-amino acids [Boc-Thr-OH and Boc-Tyr-OH]. N-Terminal acetylation was achieved under mild conditions by using the BOP coupling reaction with acetic acid. This procedure provided the unprotected (Tyr2', Thr3,)-peptide-resin which is ready for the required sulfation on the solid support without selective side-chain deprotection of Ty2' and Thr3'. Solid phase sulfation was evaluated under a variety of conditions and it was determined that disulfation was optimal using pyridine acetyl sulfate (38 equiv.) in pyridine at 45" for 4 h. Shorter reaction times or milder conditions lead to the formation of the Thr3, monosulfated analog. Cleavage of the disulfated analog from the PAM-resin was achieved using liquid ammonia and the product was purified by preparative hplc and fully characterized. The advantages of the new procedure are compared with the reported synthesis of CCK-7.

Key words: BOP [benzotriazol- I-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate]; CCK-7 analogs; minimal side-chain protection; solid phase peptide synthesis; sulfation on the resin

In a previous communication from this laboratory (l), we described the BOP reagent (2) as ideally suited for solid phase peptide synthesis and the rate of coupling comparing favorably to other methods of activation. We also reported ( 3 ) the advantage of using BOP as a coupling reagent for solid phase side-chain to side- chain cyclizations, which were shown to proceed to completion more rapidly and to give purer peptides.

Our preliminary findings (1) also demonstrated that the BOP reagent could be used for the incorporation of unprotected Ser or Thr in solid phase synthesis. In spite of the serious danger of 0-acylation (4-6) we

~

* Present address: INRS-Sante, Universite du Quebec, 245, Boul. Hymus, Pointe-Claire, Quebec H9R 1G6, Canada. Abbreviations follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Biochern. J . 126, 773- 780 (1972)l. Additional abbreviations: DMF, dirnethylformamide; BOP, benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate; DJEA, diisopropylethylamine, DMS, di- methylsulfide; TFA, trifluoroacetic acid; MeOH, methanol [-OCH,-PAM]-resin [4-(oxymethyl)phenylacetamidomethyl]- polystyrene resin; PAS, pyridinium acetyl sulfate; PS, pyridine sulfur trioxide complex.

concluded that Boc(hydroxy)amino acids can be used with the BOP reagent, but the number of coupling cycles after its incorporation was somewhat limited. A successful synthesis of GRF(22-29)-NH2 was reported in which unprotected Ser was introduced by the BOP procedure at position 28 (1). We have now extended the utility of the BOP reagent to include the solid phase synthesis of sulfated CCK-7 analogs. A model compound, Ac-Tyr(S0, H)-Met-Gly-Trp-Met- Thr(S0,H)-Phe-NH, (7), was chosen since it contains both aliphatic (Thr) and aromatic (Tyr) amino acids and can be subsequently sulfated on the solid support without the necessity for selective side-chain deprotection. This class of acetyl-CCK-heptapeptides was reported by Penke et al. (7) to be among the most potent analogs.

Cholecystokinin (CCK) is a polypeptide hormone that was first isolated as a 33-amino acid peptide from the porcine gastrointestinal tract (8). A fragment of CCK, H-Asp-Tyr(S03 H)-Met-Gly-Trp-Met-Asp- Phe-NH, [cholecystokinin octapeptide (CCK-S)], has been shown to decrease food intake in lean and obese men (9). It is now accepted that CCK-8 has satiety-

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Boc-Thr-OH Boc-Met-OH Boc-Trp(X )-OH BOC-Gly-OH Boc-Met-OH

inducing effects and may be useful to reduce or sup- press food intake in man. The synthesis of CCK-8 presents some difficulties since it contains many sen- sitive amino acids (Met, Trp, Asp). In addition the introduction of the sulfate group into Tyr has been reported to cause many side reactions (10, 1 1).

Toth & Penke (12, 13) evaluated four different strat- egies for preparing CCK-8 peptides using solution phase methods and concluded that pyridine acetyl sulfate (PAS) was preferred for the final sulfation reaction since minimal side reactions were observed. Comstock & Rosamond (14) described a synthesis of CCK-8 by a solid phase procedure using PAM-resin and a Fmoc/t-Bu strategy in which sulfation of Tyr was carried out on the solid support using PAS and the peptide was cleaved from the resin by ammonolysis. Recently, Penke & Rivier (15) described a new route for the synthesis of CCK-8 utilizing 2,4-di- methoxybenzhydrylamine support in conjunction with the Fmoc/t-Bu strategy which utilized trifluoroacetic acid for cleavage of the peptide from the resin. All the above-mentioned syntheses utilized DCC, DCC/HOBt or symmetrical anhydride coupling procedures. These reagents have been reported to lead to side reactions including racemization and intra- molecular rearrangements (1 6) . In addition these methods of activation often require repetitive solid phase couplings for the complete introduction of a residue in the peptide chain which result in the con- sumption of protected amino acids and time-consum- ing cycles of synthesis.

The model CCK-7 analog, Ac-Tyr(S0, H)-Met- Gly-Trp-Met-Thr(S0,H)-Phe-NH, was first synthesized by Penke et al. (7) using the standard procedure described by Merrifield (17). The unsulfated heptapeptide analog was synthesized on a methylbenzhydrylamine-resin using Boc-protected

COUPLING [BOP ( 3 eq) -D IEA (6 O d - D H F ]

DEPROTECTION [ 50% TFA-CH2Cl 2]

NEUTRALIZATION [lo% DIEA-CH2C12]

benzyl side-chain protected amino acids and unprotected Trp. Penke also reported couplings with DCC, N-terminal acetylation on the resin using acetic anhydride and cleavage from the resin with HF. After purification by preparative hplc, a separate solution phase sulfation was required followed by an additional hplc purification of the final product. This communication describes the advantages of the BOP reagent for the solid phase synthesis of sulfated CCK- 7 analogs.

I AC-Tyr-!4et-G1y-Trp-Het-Thr-Phe-NH2

S03H

RESULTS AND DISCUSSION

UnsulJhted f Ac-Ty?,Thr'']-CCK-7 Starting with Boc-Phe-OCH,-PAM resin, the peptide was assembled in a stepwise manner as outlined in Fig. 1 using the protocol described in Table I . Thr and Tyr were introduced as Boc-Thr-OH and Boc-Tyr-OH, respectively. In addition comparative syntheses were carried out using either Boc-Trp-OH (unprotected) or Boc-Trp(For)-OH (protected). The acetylation of the N-terminal free amino group was carried out using 3 equiv. acetic acid and BOP reagent in the presence of 6equiv. DIEA according to the same protocol as shown in Table 1 for the Boc-amino acid residues. These mild conditions for solid phase acetylation offer important advantages over the usual acetic anhydride procedure. The recent report by Castro (18) using in situ neutralization after Boc-deprotection and that the use of trifluoroacetic acid with BOP reagent does not result in trifluoroacetylation (19) is probably due to the electron withdrawing effect of the trifluoromethyl- group and its inability to be activated by the BOP reagent.

Cleavage of a portion of the Trp-unprotected or Trp(For)-protected unsulfated peptide-resins was achieved with either liquid ammonia or ammonia in

NH3

FIGURE 1 k - T r-Het-Gly-Trp-C(et-Thr-Phe-NH2 T I Schematic representation for the

S03H S03H

Solid phase peptide synthesis

TABLE 1 Standard protocol for a synthetic cycle using BOP reagent"

Step Reagent Time

1 2 3 4 5 6 7 8 9

10 11 12 13

14 1s 16

1 Yo DMS/CH, CI, 50% TFA/CH,CI, + 1% DMS (v/v) 1 %a DMS/CH,CI, 50% TFA/CH,CI, + 1% DMS (v/v) CH, CI,

CH,CI,

CH,CI, MeOH CH, CI, DMF 3 equiv. Boc-AA-COOH/DMF + 3 equiv. BOP reagent/DMF + 6 equiv. DIEA DMF MeOH CH, CI,

10% DIEA/CH,Cl,

10% DIEA/CH,Cl,

1 x 1 min I x 1 min 1 x I min I x 20 min 4 x 1 min I x 5 min 1 x 1 min 1 x 5 min 1 x 1 min 2 x 1 min 1 x 1 min 2 x 1 min 1 x 120 min

2 x 1 min 2 x 1 min 3 x 1 min

"Solvents for all washings and couplings were measured to volumes of 10-20mL/g resin.

methanol. From comparison of the analytical hplc of the crude peptides it was concluded that slightly better yields of unsulfated peptides were achieved with Trp(For)-protection and that cleavage with liquid ammonia or ammonia in methanol gave similar re- coveries.

The unsulfated peptide was purified by preparative hplc and characterized by amino acid analysis, FAB mass spectrometry, and n.m.r. spectroscopy.

5 r 9 e 3w

/

C

TIME (minutes)

SulJhtion of peptide-resin Solid phase sulfation of the fully assembled peptide- resin was evaluated under a variety of conditions using PAS. Analytical hplc conditions were developed to separate the unsulfated, [Thr3*]-monosulfated and [TyrZ7 ,Thr32]-disulfated analogs (Fig. 2). Sulfation of either the Trp-unprotected or the Trp(For)-protected peptide-resins at 25" for 16 h followed by ammonolysis gave similar results. One major product was ob-

FIGURE 2 Analytical hplc of purified [Ac- Ty127 ,Thr"]-CCK-7 analogs. (a) Disulfated CCK-7 analog: [Ac- Tyr (SO, H)" ,Thr(SO, H)',]- CCK-7 (5 pg). (b) Monosulfated CCK-7 analog: [AC-TY~', Thr(S0, H)"]-CCK-7 (10 pg). (c) Unsulfated CCK-7 analog: [Ac- Ty37,Thr32]-CCK-7 (3 pg). (d) Admixture of unsulfated, monosulfated, and disulfated CCK-7 analogs. Conditions: Column, pBondapak C,,, (IOpm) Waters, (0.30 x 301x1); Eluant (A) 0.125% TFA, (B) 0.125% TFA/ CH,CN, Gradient: linear 20- 55% (B) in 25min; Flow rate: 2.5 mL/min; Detection: 215 nm; Sensitivity: 0.2 AUFS.

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Trp'O Admixture with Admiitwe with Thr32monosulf Unprotected 1

e

I 1

FIGURE 3 Analytical hplc of crude sulfated [Ac-Tyr",Thr3']-CCK-7 analogs using solid phase suIfation with PAS of 25" for 16 h. The peptides were cleaved from the resin using anhydrous ammonia. (a) Crude p e p t i d e u s i n g p r o t e c t e d Trp'" [Trp(For)]. (b) Crude peptide using unprotected Trp". (c) Admixture of crude peptide with unsulfated [ A c - T ~ ~ ' ~ ,Thr32]- CCK-7. (d) Admixture of crude peptide with monosulfated [Ac- Tyr",Thr (SO,H)"]-CCK-7. (e) Admixture of crude peptide with disulfated [Ac-Tyr(S0, H)27 , Thr(SO,H)"]-CCK-7. See Fig. 2 for column conditions.

E n N

W U z U

LL 0

U

m

m

J-

I 10

I 0 10 2 0 0 10 20 0 10 20 0 10 20

T I M E (minutes)

tained (Fig. 3a, b) that was found to co-elute with [Ac-Tyr" ,Thr(SO, H),']-CCK-7 (Fig. 3d). Minor components of unsulfated (Fig. 3c) and disulfated products (Fig. 3e) were also present in the reaction mixture. The major component was isolated by preparative hplc, characterized, and shown to be fully compatible with [Ac-Tyr2',Thr(S0, H)32]-CCK-7 (amino acid analysis, FAB mass spectrometry, i.r., u.v., and n.m.r. spectroscopy). A kinetic study of the solid phase sulfation was therefore carried out under more forcing conditions to obtain the disulfated CCK-7 analog.

Sulfation of the Trp-unprotected peptide-resin at 45" for 2h gave a major component of disulfated product with only minor amounts of [Thr32]-monosulfated and unsulfated product (Fig. 4a). The solid phase sulfation was optimized when the reaction pro- ceeded for 4 h at 45" (Fig. 4b). Longer reaction times resulted in a decrease in the disulfated product and an increase in [Thr32]-monosulfated component (Fig. 4c, d). Sulfation at higher temperatures (65" or SO") also resulted in a decrease in the amount of the disulfated product (data not shown). These observations are compatible with the earlier reports that Thr undergoes

Reaction Time 2h 4 h B h 18 h

c

w

C

/

I

FIGURE 4 Analytical hplc of crude sulfated [Ac-Tyr" ,Thr3']-CCK-7 analogs using unprotected Trp" and solid phase sulfation with PAS at 45" at different sulfation times. The peptides were cleaved from the resin using anhydrous ammonia. The arrow designates the peak corres- ponding to disulfated [Ac- Tyr(S0, H)27 ,Thr(SO, H)."]- CCK-7. (a) Crude reaction mixture at 2 h. (b) Crude reaction mixture at 4 h. (c) Crude reaction mixture at 8 h. (d) Crude reaction mixture at 18h. See Fig. 2 for column conditions.

E ?

W " z U

LL 0 v)

4

m

m

1 0 10 2 0

136

0 10 20 0 10 20 0 10 20

T I M E i minutes 1


carried out with 3 equiv. Boc-Thr-OH (1.18 g, 5.4mmol) and BOP reagent (2.39g, 5.4mmol), each reagent separately dissolved in 25mL DMF and introduced into the reactor. To that resin suspension 0.75 mL DIEA was added and the reaction carried out for 2 h at room temperature. After the coupling, the resin was washed (Steps 14-16) and ready for the next coupling cycle. The subsequent residues were sequen- tially linked to the growing peptide chain by the same procedure: Boc-Met (1.35 8); Boc-Trp (1.64 8); Boc-Gly (0.94g); Boc-Met (1.35g); and Boc-Tyr (1.52g). After the incorporation of the last Boc-amino acid, the peptide-resin was deprotected and neutralized (Steps I - 12) and the free amino group acetylated using 3equiv. of acetic acid (324mg, 310pL) and BOP reagent in the presence of 0.75mL DIEA, according to the same protocol as used previously for the Boc-amino acid residues (Table 1).

Isolation, purijication and characterization of the unsul- ,fated Ac-Tyr-Met-Gly-Trp-Met-Thr-Phe-NH,. A 250- mg portion of the dried peptide-resin was placed in a Wheaton pressure bottle (250-mL capacity). The con- tainer was purged with anhydrous ammonia, cooled to - 78", and ammonia (approximately 20 mL) con- densed into the reaction vessel. The bottle was tightly capped, the bath removed, and the resin stirred overnight. At the end of the reaction, the pressure bottle was cooled to - 78" before opening and the ammonia evaporated quickly by immersing the vessel in a water bath. The peptide was extracted with MeOH (3 x 15mL) and evaporated, and the crude Ac-Tyr- Met-Gly-Trp-Met-Thr-Phe-NH, was ready for purification.

A 25-mg portion of the crude peptide was purified by preparative hplc on a 2.3 x 30 cm pBondapak C,, column. Eluant: (A) H,O (0.0.22% TFA)-(B) CH,CN (0.022% TFA); the peptide was eluted with a linear gradient from 5 to 65% (B) in 4 h at a flow rate of 8mL/min with detection at 280nm. Fractions were collected at 3-min intervals and cuts were made after inspection by analytical hplc. Fractions, judged to be greater than 98% pure, were pooled and lyophilized. Characterization was achieved by amino acid analysis (3 N mercaptoethanesulfonic acid, 1 lo", 22 h): Thr 0.91, Gly 1.01, Met 1.95, Tyr 0.98, Phe 1.00, Trp 0.96; n.m.r. (400MHz in d, DMSO) confirmed the structure. FAB-MS: observed mass for (M + H)' = 976; calculated mass for (M + H)' = 976.

sulfation before Tyr (7). Subsequent disulfation of Tyr(S0, H) occurs at elevated temperature or over long periods of reaction. The disulfated product was purified by preparative hplc, characterized (amino acid analysis, ix., u.v., and n.m.r. spectroscopy) and found to be fully compatible with [Ac-Tyr(S0, H),', Thr(S0,H)32]-CCK-7.

The use of the BOP reagent in conjunction with unprotected aliphatic and aromatic hydroxyamino acids is clearly demonstrated in the synthesis of the CCK-7 analog, Ac-Tyr(S0,H)-Met-Gly-Trp-Met- Thr(S0, H)-Phe-NH, . This synthesis using the BOP reagent offers several important advantages over that previously reported (7): (a) use of unprotected aliphatic and aromatic hydroxyamino acids is cost efficient, which can be an important advantage in large-scale peptide synthesis; (b) direct sulfation on the resin eliminates the additional step of isolating and purify- ing the unsulfated precursor of the peptide analog; and (c) use of liquid ammonia or ammonia in methanol to cleave the peptide from the resin eliminates the HF reaction and side reactions associated with it.

EXPERIMENTAL PROCEDURES

Materials Boc-protected amino acids were purchased from Bachem Inc. and their purity was carefully checked by t.l.c., m.p., and optical rotation before use. ACS grade methylene chloride, dimethylformamide, and methanol were purchased from Fisher Scientific or Burdick and Jackson and trifluoroacetic acid (glass distilled) was obtained from Halocarbon and were used without further purification. Diisopropylethylamine was purchased from Pfaltz and Bauer Inc. and distilled from CaO and ninhydrin. BOP reagent was obtained from Richelieu Biotechnologies Inc. Boc-Phe-OCH,-PAM resin was purchased from Vega Biotechnologies, Inc., Tucson, Arizona, and has a substitution of 0.36 mmol/g.

Methods Solid phase synthesis was carried out manually by the protocol described in Table 1 using wrist-action shakers. High performance liquid chromatography (preparative and analytical) was carried out on an LDC Constametric IIG equipped with a gradient master and Spectromonitor I11 U.V. variable wavelength detector. Amino acid analyses were performed on the Beckman Model 121M Amino Acid Analyzer. The free peptides were hydrolyzed in 6~ HCl (Pierce Chemical Co.) in sealed, evacuated tubes at 110" for 24h or 3~ mercaptoethanesulfonic acid (Pierce Chemical Co.) at 1 10" for 22 h.

Solid phase synthesis. Boc-Phe-PAM resin (5 g, 1.8 mmol) was deprotected and neutralized according to the protocol described in Table 1 (Steps 1-8). After washing (Steps 9-12), the coupling (Step 13) was

Solid phase monosulfation: isolation, purlfication, and characterization of monosulfated Ac-Tyr-Met-Gly- Trp-Met-Thr(S0, H)-Phe-NH,. The monosulfate ester-containing peptide was prepared by sulfation of the side-chain of Thr3* using pyridinium acetyl sulfate reagent (PAS) or pyridine sulfur trioxide complex (PS). A typical sulfation was carried out as follows: PAS (218mg, 1 mmol, 38equiv.) or PS (162mg,

137

A. Fournier et al.

1 mmol, 38 equiv.) was dissolved in 20mL dry pyridine and 100 mg of the peptide-resin (approximately 0.0268 nmol of peptide) added. The suspension was shaken for 16 h at 20", filtered onto a sintered glass funnel and successively washed with pyridine, DMF, and MeOH. The resin was dried overnight in vacuo. The dried peptide-resin was placed in a Wheaton pressure bottle (250-mL capacity) and treated with anhydrous ammonia under the same conditions described for unsulfated Ac-Tyr-Met-Gly-Trp-Met-Thr- Phe-NH, above. From 100 mg peptide-resin, 30 mg of crude Ac-Tyr-Met-Gly-Trp-Met-Thr(S0, H)-Phe- NH, was obtained. Analysis by hplc indicated that the sample contained approximately 20 mg monosulfated peptide for an overall yield of 70%.

A 25-mg portion of the crude peptide was purified by preparative hplc on a 2.3 x 30cm pBondapak CLB column. Eluant: (A) H,O (0.022% TFA)-(B) CH,CN (0.022% TFA); the peptide was eluted with a linear gradient from 5 to 65% (B) in 4 h at a flow rate of 8mL/min with detection at 280nm. Fractions were collected at 3-min intervals and cuts were made after inspection by analytical hplc. Fractions, judged to be greater than 98% pure, were pooled and lyophilized. Characterization was achieved by amino acid analysis: (3 N mercaptoethanesulfonic acid, 1 lo", 22 h): Thr 0.95, GLy 1.00, Met 1.91, Tyr 0.99, Phe 1.02, Trp 1.02. Ultraviolet spectrum confirmed the free phenolic-OH group (0.1 N KOH). Infrared spectrum confirmed -OSO,H with a peak at 1250cm-' (KBr). N.m.r. (400MHz in d6 DMSO) confirmed the structure. FAB-MS: observed mass for (M + H)' = 1055; calculated mass for (M + H)+ = 1055.

Solid phase disulfation: isolation, purification and characterization of the disulfated Ac- Tyr (SO, H ) -Met- Gly- Trp-Met-Thr(S0,H)-Phe-NH,. The disulfate ester-containing peptide was prepared by disulfation of the side-chain of Ty8' and Thr3' using PAS or PS. A typical sulfation was carried out as described above for the monosulfation at 45" for 4 h and the resultant peptide-resin washed with pyridine, DMF, and MeOH, and dried overnight in vacuo. The dried peptide-resin was placed in a Wheaton pressure bottle and treated with anhydrous ammonia under the same conditions described for unsulfated Ac-Tyr-Met-Gly- Trp-Met-Thr-Phe-NH, above. From 100 mg sulfated peptide-resin, 27.2 mg of crude material was obtained after ammonolysis. Analysis by hplc indicated that the sample contained approximately 14.3 mg disulfated peptide for an overall yield of 50%.

An 8.2-mg portion of crude peptide was dissolved in 1 mL H,O, centrifuged and filtered through a 0.45 m Type HA Millipore filter. The filtrate was applied onto an ES Industries C-18 10 pm column (1.25 x 30 cm). The column was eluted at 5mL/min with a solvent system consisting of (A) 0.01 M ammonium acetate

and (B) CH,CN in a step gradient mode: 5-15% (B) in 6min followed by 15-45% (B) in 2 h with detection at 280 nm. Fractions were collected at 2-min intervals and fraction pooling was determined by analytical hplc using a Waters pBondapak C-18 10pm column (0.39 x 30cm), using a linear gradient of (A) 0.01 M ammonium acetate and (B) CH,CN (20-50% (B) in 30 min); flow rate, 2 mL/min and detection at 225 nm. Fractions 13-1 5 were pooled and lyophilized to give 2.2mg (25% overall yield) of pure material. Amino acid analysis (6 N HCI, 1 lo", 22 h): Thr 0.95, Gly 1 .OO, Met 2.01, Tyr 0.97, Phe 1.02. Ultraviolet spectrum A,,, 272 and 278 nm (0.1 N KOH). Infrared spectrum confirmed Tyr''(OS0,H) and Thr32(OS03H) with peaks at 1252-1232 and 1048 cm-' (KBr). N.m.r. (400 MHz in d, DMSO) confirmed the structure. The homoge- neity of the disulfated material was determined to be > 97% by analytical hplc using a pBondapak C-18, 10pm Waters column (0.30 x 30cm) using a linear gradient of (A) H,O (0.125% TFA) - (B) CH,CN (0.125% TFA) (20% to 55% (B) in 25 min); flow rate, 2.5 mL/min and detectionat 21 5 nm (0.2 AUFS) (Fig. 2).

ACKNOWLEDGMENTS

The authors thank Dr. F. Scheidl and his staff for the amino acid analyses, Dr. W. Benz for carrying out mass spectrometry, and Mr. J . Michalewsky for the preparative hplc purification.

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REFERENCES

Fournier, A,, Wang, C-T. & Felix, A.M. (1988) Int. J . Peptide Protein Res. 31, 86-97 Castro, B., Dormoy, J.R., Evin, G . & Selve, C. (1975) Te- trahedron Lpt t . , 1219-I222 Felix, A.M., Wang, C.-T., Heimer, E.P. & Fournier, A. ( I 988) Int. J . Pepride Protein Res. 31, 231-238 Stewart, J.M., Mizoguchi, T. & Wooley, D.W. (1967) Abstr., 153rd Natl. Meet., Am. Chem. SOC. No. 0-206 Ramachandran, J. & Li, C.-H. (1963) J . Org. Chem. 28, 173- 177 Paul, R. (1963) J . Org. Chem. 28, 236-237 Penke, B., Hajnal, F., Lonovics, J., Holzinger, G., Kador, T., Telegdy, G. & Rivier, J. (1984) J . Med. Chem. 27, 845-849 Mutt, V. & Jorpes, J. (1971) Biochem. J . 125, 57-58 Smith, G.P. (1984) Inr. J . Obesity 8, Suppl. 1, 35-38 Penke, B., Balaspiri, L., Zarandi, M., Kovacs, K. & Kovacs, L. (1979) in Peptides 1978 (Siemion, T.Z. & Kupryszewski, G., eds.), pp. 581-584, Wroclaw University Press, Wroclaw Penke, B., Toth, G., Zarandi, M., Nagy, A. & Kovacs, K. (1980) in Peptides (Brunfeldt, K., ed.), pp. 253-257, Scriptor, Copen hagen Toth, G., Penke, B., Zarandi, M. & Kovacs, K. (1985) Int. J . Peptide Protein Res. 26, 630-638 Penke, B., U.S. Patent 4102 878 (July 25, 1978) Comstock, J. & Rosamond, J. (1985) European Patent 0161 468 (Nov. 21, 1985) Penke, B. & Rivier, J. (1987) J . Org. Chem. 52, 1197-1200

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16. Bodanszky, M. (1984) Principles of Peptide Synthesis, p. 37, Springer-Verlag, New York

17. Merrifield, R.B. (1963) J . Am. Chem. SOC. 85, 2149-2154 18. Le-Nguyen, D. , Heitz, A. & Castro, B. (1987) J. Chem. SOC.

Perkin Trans. I, 1915 19. Castro, B. (1987) Japan Int. Pept. Symp., Kobe, Japan (Sept.

1987)

Address:

Dr. Arthur M. Felix Peptide Research Dept. Roche Research Center Hoffmann-La Roche Inc. Nutley, NJ 071 10 USA

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applications of bop reagent in solid phase peptide synthesis iii. solid phase peptide synthesis with...

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