A p-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides

Peptides. Vol. 2, pp. 45-50, 1981.Printed in the U.S.A.

A p-Methylbenzhydrylamine Resin for Improved Solid-Phase Synthesis of Peptide Amides G A R Y R. M A T S U E D A 2 A N D J O H N M. S T E W A R T z

Department of Biochemistry, Biophysics and Genetics, University o f Colorado School o f Medichle Denver, CO 80262 Received 19 January 1981 MATSUEDA, G. R. AND J. M. STEWART. A p-methylbenzhydrylamhze reshzfor improved solid-phase synthesis of peptide amides. PEPTIDES 2(1) 45-50, 1981.--A p-methylbenzhydrylamine-resin, a-(p-tolyl)-a-aminomethyl-functionalized copoly(styrene-l%-dvinylbenzene),was prepared for the improved solid-phase synthesis of peptide a-carboxamides. Following a Friedel-Crafts acylation of polymer beads, the resulting ketone resin was reductively aminated via the Leuckart reaction. A comparison of the relative acid stability of the p-methylbenzhydrylamine(MBHA)-, benzhydrylamine(BHA)-, and a-phenyethylamine-resins indicated that the p-methylbenzhydrylamine-resinprovided the best yields of the model peptide carboxamides. Methylbenzhydrylamine resin

Solid-phasesynthesis

THE CONTINUING need for the synthesis of biologically interesting peptide carboxyl-terminal amides has focused our attention on the chemistry of the amine resin supports introduced by Pietta and Marshall [13]. These benzhydrylamine (BHA) resin supports permit the direct synthesis of peptide a-carboxamides using the standard methods of solid-phase peptide synthesis as introduced by Merrifield [7] and described by Stewart and Young [19]. Since the introduction of the BHA resin, many successful solid-phase syntheses of peptide amides have been reported, notably thyroliberin [14,16], luteoliberin [15], thyrocalcitonin [24], substance P [20] and apamin [22]. Use of the amine-support is an especially convenient synthetic strategy, because a single treatment of the protected peptide-resin with anhydrous hydrogen fluoride provides the desired a-carboxamide productand can simultaneously remove all side-chain blocking groups. In addition to the growing number of naturally occurring carboxyl-terminal peptide amides which can be synthesized by using these amine-polymers, one might anticipate an increasing use of amine-resins for the synthesis of protein fragments with interesting biological properties. One would be justified, for example, to synthesize the 1-34 fragment of parathyroid hormone [21] or the 1-24 fragment of adrenocorticotropic hormone as a a-carboxamide, because a free carboxyl group is not present in the intact hormone.

Peptideamides

Furthermore, the a-carboxamide derivatives should provide analogs which are resistant to degradation by carboxypeptidases and hence should have prolonged hz vivo lifetimes. Several large fragments of human growth hormone containing up to 128 amino acid residues have been synthesized on the BHA-resin [11]. In one instance, the BHA-resin proved to be invaluable. For the solid-phase synthesis of [chlorambucill]-luteoliberin, ammonolysis of the peptide from the standard benzyl ester resin to yield the a-carboxamide would have surely destroyed the alkylating capacity of the bis(chloroethyl)amino functional group. The use of the amine-resin strategy provided a convenient direct route for the complete solid-phase synthesis of the peptide a-carboxamide with an intact, functional alkylating group, which remained protonated during the HF cleavage step. Use in our laboratory of the BHA-resin for pe~tides with carboxyl-terminal Phe, Val or Tyr residues has shown that yields of such peptide amides may be extremely low due to incomplete cleavage of the peptide from the resin by HF at 0~ For example, an attempted synthesis of the a-carboxamide of angiotensin II (with A. Paiva) yielded no product. This limitation in the chemistry of the BHA-resin prompted the synthesis of p-methylbenzhydrylamine (MBHA)-resin and a study of the relative stabilities of these amine-resins. Independently, Orlowsky et al. [10] pursuing

~Supported by Grant HL-12325 from the NHLBI-NIH. Nomenclature is in accordance with recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, J. biol. Chem. 250: 3215, 1975. Other abbreviations used: Boc, tert-butyloxcycarbonyl; DCC, N,N'dicyclohexylcarbodiimide;TFA, trifluoroacetic acid; TEA, triethylamine; BHA, benzhydrylamine;MBHA,p-methylbenzhydrylamine; PEA, a-phenylethylamine; MOBHA, p-methoxybenzhydrylamine and DCM, dichloromethane. *Present address: Massachusetts General Hospital, Boston, MA 02114. 3"1"owhom reprint requests should be sent.

Copyright 9 1981 A N K H O International Inc.--0196-9781/81/010045-06501.10/0

MATSUEDA AND STEWART

46

SCHEME

Friedel-eraft s

1 ROC0-Cl :AICI3

NH--C

CH3

O

BHA: R=H 1. PEA

Leuckart

2. BHA

~i HCO2NH4:HCO2H

~

3

HN-CHO 3. MBHA

Hydrolysis

4. MOBHA

~ HC't:EtOH CH3

NH29HCI

5. PEA Ether FIG. 1. Structures of the amine-resins examined, in order of increasing liability toward acidolytic cleavage. The encircled "P" and attached phenylcne ring represent the crossIinked polystyrene matrix.

the same goal have introduced the p-methoxybenzhydrylamine-resin. Some work has also been done with aminopolymers containing the phenylethyl [18] or the p-alkoxybenzyl [14], rather than the benzhydryl-type of substitution. The structures of the various amine resins are given in Fig. I. METHOD Infrared spectra were recorded on a Beckman Acculab-4 spectrophotometer. Resin samples for IR were dried over P2Os hi vocuo, ground, and pressed into KBr pellets (4 mg resin/100 mg KBr). Amino acid analyses were performed with a Beckman 120(2 amino acid analyzer. Peptides were synthesized automatically on a Beckman 990 Peptide Synthesizer. Boc amino acids were of the L-configuration except glycine, and were purchased from Bachem or Beckman.

Acylation of Poly(styrene-l%-divinylbenzene) Copoly(styrene-l%-divinylbenzene) resin beads, 200-400 mesh (Lab Systems, San Marco, CA) (25 g) and 250 ml CICH~CH2CI were placed in a 500 ml 3-neck round bottom flask fitted with a thermometer, a CaCI., drying tube and a 200 ml dropping funnel. The acylating reagent was pre-

formed in the dropping funnel by mixing 50 mmol acyl chloride and 50 mmol anhydrous AICI~ in 150 ml CICH2CH2Ci. After chilling the resin suspension to - 15~ the aeylating reagent was added carefully, with stirring, so that the temperature of the resin suspension remained below 5~. After addition was complete, the ice bath was removed and the mixture stirred for four hours at room temperature. The resin was then collected in a sintered glass funnel and washed successively with C2HsOH, DCM, C2HsOH and CHaOH. Under 400x magnification the product was visually indistinguishable from the starting material. Typically, the benzoyl and toluoyl resins have a strong iR band at 1650 cm-' with an intensity greater than the 1600 cm - ' band of polystyrene.

O.rhne Derivative of Acylated Polymers To determine the degree of acylation of polymers, the oxime derivative was prepared. Ketone resin (500 mg), 500 mg hydroxylamine HCI and 2 ml pyridine were heated overnight at 105~ in a closed tube. The resin was then collected and washed successively with DCM, C2HsOH, H20, C2HsOII and MeOH, and dried. The IR spectrum showed complete loss of the C=O band. The degree of aeylation was determined by combustion analysis for N.

47

MBHA RESIN FOR SYNTHESIS OF PEPTIDE AMIDES

Reductive Amination of Acylated Polymers by the Leuckart Reaction Ketone resin (24 g) and 75 g HCO.,NH4 were mixed with 90 ml HCOlqH2, 60 m188% HCOzH and 300 ml CrHs-NO2 in a 1 1. 3 neck round bottom flask fitted with a thermometer and a Dean-Stark trap. The magnetically stirred contents of the reaction flask were heated to provide a reaction temperature of 165~ for 22 hr. Approximately 40 ml of aqueous phase was collected in the trap during the initial heating period. Unless this phase was removed, the reaction temperature would not reach 165~. Very little additional aqueous distillate was obtained once an inner temperature of 165~was attained. After the reaction the resin was collected by filtration and washed with CzHsOH, DCM and C2HsOH. The resin was deformylated by hydrolysis for 60 min in 300 ml of 12 M HCI:C2HzOH (1: I) at reflux temperature. The amine hydrochloride resin was collected by filtration, washed with C2HsOH, DCM and MeOH, and dried. The amine hydrochloride resin showed a decreased IR carbonyl band and a new band at 3600 cm -1. When subjected to the Kaiser ninhydrin test [5], the amine resin gave a blue color after 60 see at 25~ and an intense blue color after 60 sec at 115~ The Volhard chloride procedure was performed on the amine HC1 resin to obtain quantitative substitution data. Phenylethylamine and phenylethylamine-ether resins were prepared as previously described [18].

Preparation of N-acetylamhloacyl Reshls A mixture of Boc-amino acids was coupled manually to the amine resins according to the general procedures described by Stewart and Young [19]. In one such experiment, 1.7 g MBHA resin (0.60 mEq NH2/g) was washed with DCM, neutralized with triethylamine (TEA)/DCM (1:19) for 5 min, washed with DCM and subsequently coupled in DCM for 60 min with a mixture containing 0.4 mmol each of BocGlu(Bzl), Boc-Gly, Boc-Leu, Boc-Phe and Boc-Met (2 mmol total) and 2 mmol DCC. The resulting mixed Boc-aminoacyl polymer was ninhydrin negative by the Kaiser test. The aminoacyi resin was deprotected for 30 min in TFAIDCM (1:3), neutralized with TEA/DCM and acetylated in DCM for 60 min with 10 mmol Ac20/TEA (1:1). The resin was washed exhaustively with DCM and MeOH and dried hz vactto over

P205. Determination of Secondary Amine Content of Reshls The amine resin hydrochloride was neutralized and then coupled exhaustively with Boc-Gly (2.5 equivalents per equivalent of amine) by the procedure described in the preceding paragraph. At the end of the coupling reaction, the resin was ninhydrin negative by the Kaiser test [5]. The resin was washed with DCM, neutralized, washed with DCM and charged with chloride by treatment with an excess of 0.1 M pyridine hydrochloride in DCM, followed by thorough washing with DCM. Volhard chloride determination revealed the amount of basic groups remaining on the resin which could not be coupled with Boc-Gly activated by DCC.

Cleavage of N-acetylamhzoacyl Reshzs by HF Acetylaminoacyl-resin (300 mg) was stirred for 30 min at 0~ with 5 ml anhydrous HF and 0.5 ml anisole. Since the HF was collected at - 7 6 ~ additional time (15 min) was allowed for the reaction temperature to rise from - 7 6 ~ to 0~ HF was then removed by distillation under reduced pressure. The

HF-treated resin was washed with ether to remove residual anisole and with 1 M HOAc and glacial HOAc to remove the cleaved N-acetylamino acid amides. The resin was then washed thoroughly with MeOH and dried hl vacuo over

P2Os. Analysis of Resin-Bound Amhzo AcMs The substitution of resin bound amino acids was determined by hydrolysis with propionic acid/12 M HCI (1"1) for 18 hr at 130~ in N2-flushed sealed tubes. The 2--4 hr hydrolysis time [23] used with standard Merrifield resin was not adequate for amino acids and peptides on amine resins. It was necessary to wash resin samples before hydrolysis with TFA/DCM (1:3) in order to remove all amino acids not covalently attached.

Stability of the N-acetyhunhtoacyI-Reshz Bond to TFA Portions of 50 mg of resin were suspended in 2 ml anhydrous TFA and agitated for 24 hr. The resin was filtered and washed with HOAc, HzO and MeOH before drying h~ vacuo over P20~. Dry resin samples were then subjected to propionic:HCI hydrolysis and amino acid analysis as described above.

Preparation of Gly-Trp-Met-Asp(Bzl)-Phe-Ntl-Reshls The C-terminal pentapeptide fragment of gastrin was assembled on BHA- and MBHA- resins by the following procedure. Amine resin (1.0 g; 0.5 mEq amine/g) was swelled with DCM for 5 min (all wash and reagent volhmes were 20 ml). The resin was prewashed and then 9 with TEA/DCM (1:19) for5 min. After six DCM washes, the resin was coupled with Boc-Phe (0.5 mmol) for 1 hr and then washed six times with DCM. The resin was neutralized as before, washed six times and acetylated for 15 min with 10 mmol Ac20/TEA (1:1) in DCM. The picric acid test [2] performed at this point did not indicate the presence of any basic groups, and the Kaiser test was negative. The aminoacyl resin (0.4 mmol Phe/g) was transferred to the vessel of the Beckman 990 Synthesizer for automatic coupling of the remaining 4 residues (Boc-Gly, -Trp, -Met, and -Asp(Bzl) each at a 2.5 fold excess. The coupling cycle for each amino 9acid was as follows (all washes were 20 ml for 1.5 min): wash 3• prewash with TFA/DCM (1:3) containing 1 mg/ml indole, 30 min deprotection with the same reagent, wash 6• prewash with TEA/DCM (1:9) followed by 5 min neutralization with the same basic reagent, wash 6• mix 5 min with Boc amino acid (1 mmol) in 11 ml DCM, add DCC (1 mmol in 4 ml DCM) and couple 2 hr; wash 3• At the completion of four coupling cycles, the peptide resin was deprotected as before, washed 3 times each with DCM and EtOH, and dried. Treatment with HF of pentagastrin-BHA resin or -MBHA resin was performed as described above. Peptidyl-resin aliquots were taken before and after HF treatment and hydrolyzed with propionic acid-HCI to determine amino acid (and hence peptide) content. RESULTS AND DISCUSSION

Preparation of Ketone Reshzs (See Scheme) Acylation of polystyrene beads proceeds reasonably well under several published conditions [3, 10, 12, 14]. Using CICHzCH2CI as solvent, reproducible acylation at a useful substitution has been obtained without de-

MATSUEDA AND STEWART

48

tectable discoloration of the initial resin. Benzoylation and toluoylation of the styrene-l%-divinylbenzene copolymer under the conditions described herein produced substitutions of 1.0-1.3 mmol keto groups/g resin, as determined by nitrogen analysis of the corresponding oxime resins. When the reactions were carried out for 1 hr, 50-65% of the 4 hr substitution was obtained. When the reaction temperature was elevated to the reflux temperature of CICHzCHzCI and the time reduced to 15 min, the resulting ketone resin was no longer in bead form, but was completely soluble, indicating that depolymerization had taken place. Although stable complexes of polystyrene beads with AICI3 have been described by Neckers et al. 19], chloride determinations of resin samples indicated that the protocol of washes described in the Method successfully removed all chloride which might have come from residual AICI3 or its salts.

TABLE 1 COMPARISONOF AMINE-FUNCTIONALIZED-POLYMERSFOR THE SOLID-PHASESYNTHESISOF AMINOACIDa-CARBOXAMIDES Initial Substiiution (mmol/g) NHz-$ Gly

Removed by HF* Removed by TFA'I percent percent Phe

PEAw 0 . 4 5 0.27 0.17 BHA 0.43 0.21 0.10 MBHA 0.48 0.34 0.16

Gly"

Phe

Gly

Phe

70 95 98

6 50 96

0 14 60

0 I0 1

*HF treatment: HF:anisole (9:1) for 30 min at 0~ tNeat TFA treatment: 24 hr at 24~ :~Amine group substitution on starting resins. w resin was coupled with an equimolar mixture of Boc-Gly and Boc-Phe, deprotected with TFA and acetylated.

Preparation of Benzhydrylamhze and p-Methylbenzhydrylamhte Reshts (See Scheme) The procedures described are similar to those used by Rivaille et al. [15] and Hruby et al. [4], but provide amine resins with a minimal amount of residual keto groups as detected by IR spectroscopy. Volhard chloride determinations on the HCI salt of amine polymers indicated that the yields in the reductive amination were 45-55% even when the substitution of benzoyl or toluoyl groups was as low as 0.34 mmol/g resin. Nitrobenzene was used as a solvent to avoid aggregation of the resin during the Leuckart reaction. When Leuckart reaction temperatures greater than 180~ were used, the resulting resin products were strongly colored. Although the yellow-brown color could be completely bleached by a 15 min reduction with sodium borohydride in refluxing dimethylformamide, this was avoided to prevent reduction of residual ketone groups. In addition to discoloration these amine resin products from high temperature Leuckart reactions contained secondary amines on the polymer. This side reaction, which has also been observed in Leuckart reaction products starting with low molecular weight ketones [8], was avoided by keeping the reaction temperature below 170~. It is not certain that the secondary amine side products would be detrimental to the outcome of a solid phase synthesis, since secondary amine groups on the resin did not couple detectably with dicyclohexylcarbodiimide (DCC)-activated amino acids. However, they bind acidic compounds which may interfere with the synthesis in ways such. as chain, t.e_rna.ination by acylation [1]. Based upon our experience with a-phenethylamine (PEA)-resin, we have adopted an operational test for the detection of secondary amines generated on the resin by the Leuckart reaction. The amount of secondary amine was assumed to be the difference between the amount of chloride held in salt linkage by the resin before and after DCC coupling with Boc-gly, which did not acylat e the secondary amines on the resin. C. Birr (unpublished observation) has also observed the resistance of a polymer secondary amine to standard DCC-mediated coupling. When BHA and MBHA-resins synthesized by the procedure described below were subjected to this test for secondary amines, no detectable amount of chloride could be charged to either polymer after DCC coupling with Boc-Gly. By contrast, a PEA-resin which was prepared via the Leuckart reaction at 180~ ~ contained 20-40% "secondary" amine. The important and most significant result of the above experiments is that there are no remaining basic groups on the BHA or MBHA resins

after a single DCC-mediated coupling when the Leuckart reaction temperature was kept below I70 ~. Chemistry of Amine Resins Any resin which produced a mixture of C-terminal amides and acids upon treatment with anhydrous HF was considered unacceptable. Accordingly, a resin function test was developed to demonstrate that the Leuckart reaction gave resins which yielded only a-carboxamides. A mixture of Boo-amino acids was coupled to the amine-resins, deprotected with trifluoroacetic acid in dichloromethane, dried, and treated with anhydrous HF. The eluates from the HF-treated resin contained no detectable amount of free amino acids when analyzed directly. However, upon acid hydrolysis of the HF cleavage products prior to amino acid analysis, 95-100% recoveries of the amino acids were obtained. On the other hand, free amino acids were detected (3-5%) in the HF- product using a BHA-resin which was prepared by reduction of the acyl-resin oxime (gift of Dr. R. Orlowski). The use of such a resin poses additional problems in purification of products. This problem can be avoided by using the Leuckart reaction as described in the Experimental Section. Two experiments were designed to test the relative reactivities of the PEA, BHA and MBHA resins. In the first, the three resins were coupled with 1.5 equivalents (total) of an equimolar mixture of Boc-Phe and Boc-Gly. After deprotection with trifluoroacetic acid/dichloromethane, the aminoacyl resins were N-acetylated, washed and dried. Acetylation was important, since it gave a resin having an acid lability similar to that of a peptide-resin. Portions of the resin were treated with anhydrous HF for 30 minutes at 0 ~ or with anhydrous TFA for 24 hr. After extraction of cleaved amino acid amides, samples of the resins were hydrolyzed with propionic acidrHCI for determination of amino acids remaining on the resin. The results are shown in Table 1. The PEA resin was judged unsuitable for general solid-phase peptide synthesis, although thyroliberin,
49

M B H A RESIN FOR S Y N T H E S I S O F PEPTIDE A M I D E S TABLE 2 SUSCEPTIBILITY OF VARIOUSACETYLAMINOACYLAMINO RESINS TO HF CLEAVAGE

TABLE 3 CLEAVAGE OF THE GASTRIN PENTAPEPTIDE AMIDE FROM AMINE RESINS

AcetyI-AminoAcid Removed~omResinby HFTreatment, Pcrcentt Resin* Glu Gly Met Leu Phe His Thr Pro Val

Amino Acid Substitutions

Asp

Gly

BHA MBHA

BHA Resin

0.20 0.20 0.17 0.07

0.20 0.19 0.15 0.07

89 96

92 97

79 91

70 87

54 83

97 99

92 97

81 94

74 92

*Each resin was coupled with Boc-amino acid mixtures, deprotected with TFA and acetylated, then cleaved with ttF. tDetermined by hydrolysis before and after HF treatment (see text). HF treatment: HF:anisole (9:1) for 30 min at 0~

which have a C-terminal glycine residue. The PEA-ether resin, in which the removal o f the peptide amide from the resin is greatly facilitated by the presence of the ether link para to the peptide amide, was found not to be compatible with the use of Boc a-protection, since T F A readily removed amino acid amides from this resin. This resin may be ideal for syntheses using much more labile a-blocking groups such as the a,2,4,6,-tetramethylbenzyloxycarbonyl protecting group [6] or Bpoc groups. Table 2 presents the data from an experiment designed to compare further the relative H F reactivities o f the BHA and M B H A resins. A two-fold excess mixture o f Boc amino acids was coupled to each polymer, and the product Was deprotected, acetylated, and treated with H F as before. Amino acid analyses of the hydrolyzed resin samples taken before and after H F treatment are shown in Table 2. The M B H A resin provided much better yields of acetyl amino acids from the H F cleavage reaction than did the BHA resin.

Comparative Synthesis o f Gly-Trp-Met-Asp-Phe-Ntl2 The C-terminal pentapeptide fragment ofgastrin was synthesized on BHA and M B H A resins to determine the relative yields of peptide obtainable by H F cleavage. Propionic acid-HCl hydrolysis was used to determine the amount of peptide still attached to the polymers after treatment of peptide-resins with anhydrous HF. Because Trp was destroyed partially during the rigorous hydrolysis, (only 70% recovery even when mercaptoethanol was added) data for Trp are not included. See Table 3. Although the hydrolysis data would suggest that this pentapeptide-BHA resin bond is completely stable to H F , an amount o f p e p t i d e corresponding to a yield of 0.001 mmole/g resin was obtained after hydrolysis of the combined eluates. On the other hand, 55% o f the pentapeptide a-carboxamide of gastrin synthesized on the M B H A was released by the standard H F procedure. Undoubtedly, increased yields could have been obtained by raising the temperature or prolonging the time o f H F treatment. We have observed that [IleS]-angiotensen II amide synthesized on PEA resin was cleaved almost quantitatively by H F at 20-23 ~ As a general solid phase procedure, room temperature H F cleavage is unacceptable, due to side reactions [17].

Before HF After HF MBHA Resin Before HF After HF

Met (mmol/g) 0.t8 0.18 0.13 0.06

Phe

0.20 0.20 0.17 0.08

Some questions about peptide amine resins are still unanswered. The results reported here on BHA resins were typical of those obtained with BHA resins prepared in this laboratory or obtained commercially from Beckman Instruments. In contrast, a BHA resin purchased from L a b Systems, Inc., appeared to have a reactivity more like our MBHA resins. We know o f no explanation for this large rate difference. Orlowski et al. [I0] reported that a glycine-pmethoxy-BHA resin was sufficiently stable to allow the synthesis of at least a dipeptide without significant loss o f amino acid from the resin during T F A deprotection. This resin would be expected to have a lability similar to that of our PEA ether resin, which lost acetyl glycine.readily upon treatment with T F A (18% loss in 30 min treatment with 50% T F A in DCM). The anticipated lability o f the methoxy-BHA resin was confirmed in this laboratory on a sample of resin provided by Dr. Orlowski (25% loss o f a c e t y l glycine after 17 hr treatment with TFA). These apparent inconsistencies remain to be explained. The results suggest that no single type of amine resin will be ideal for the synthesis of all peptide amides. It is clear that the peptide-resin bond is too stable in BHA resins to allow synthesis of peptides with C-terminal amino acids which are relatively stable to HF, such as phenylalanine and valine. F o r synthesis of such peptides, the MBHA resin should be used. The results on removal of acetyl amino acids from resins by T F A suggest that long peptides or proteins terminating in glycine might not be adequately stable on the MBHA resin i f a - B o c protection is used, and for these peptides the BHA resin may be preferable. In choosing a resin for use in the synthesis of a particular peptide, one should also take into consideration the fact that peptide-resin bonds have been found to be significantly more stable than N-acetyl-aminoacyl-resin bonds. For this reason, the losses of peptide from a relatively labile resin may be much less than the losses of single amino acids indicated in these experiments. The MBHA resin is probably the best choice Of resins available for general peptide amide synthesis, in conjunction with a-Boc protection. With more labile a-protecting groups, use of H F can be avoided altogether by use of an ether-linked amine resin.

REFERENCES 1. Brunfeldt, K. and T. Christensen. Process monitoring in solidphase peptide synthesis. Amino group blocking effect of impure methylene chloride. FEBS Lett. 19: 345-346, 1972.

2. Gisin, B. F. Monitoring of reactions in solid-phase peptide synthesis with picric acid. Analytica chim. Acta 58: 248-249, 1972.

50 3. tlraby, V. J., F. Muscio, C. M. Groginsky, D. M. Gitu and D. Saba. Solid-phase synthesis of (2-isoleucine,4-1eucine)oxytocin and (2-phenylanine,4-1eucine)oxytocin and some of their pharmacological properties. J. Med. Chem. 16: 624--629, 1973. 4. Hruby, V. J., D. A. Upson and N. S. Agarwal. Comparative use of benzhydrylamine and chloromethylated resins in solid-phase synthesis of carboxamide terminal peptides. Synthesis of oxytocin derivatives. J. org. Chem. 42" _3552-3555, 1977. 5. Kaiser, E., R. L. Colescott, C. D. Bossinger and P. I. Cook. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Analyt. Biochem. 34: 595598, 1970. 6. Matsueda, G. R. and J. M. Stewart. Phenylethyl group containing resins for the synthesis ofpeptides. In: Peptides: Chemistry, Structure attd Biology, edited by R. Walter and J. Meienhofer. Ann Arbor, MI: Ann Arbor Science Publishers, Inc., 1975, pp. 333--339. 7. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. chem. Soc. 85: 2149-2154, 1963. 8. Moore, M. L. The Leuckart reaction. Org. React. 5: 301-330, 1950. 9. Neckers, D. C., D. A. Kooistra and G. W. Green. Polymerprotected reagents. Polystrene-aluminum chloride. J. Am. chem. Soc. 94: 9284-9285, 1972. 10. Orlowski, R. C., R. Walter and D. Winkler. Study of benzhydrylamine-type polymers. Synthesis and use of p-methoxybenzhydrylamine resin in the solid-phase preparation of peptides. J. org. Chem. 41: 3701-3705, 1976. 11. Pena, C., J. M. Stewart, A. C. Paladini, J. M. Dellacha and J. A. Santome. Conformati0nal analysis ofgrowth hormones and synthesis of a fragment of human growth hormone. In: Peptides: Chemistry, Structure and Biology, edited by R. Waiter and J. Meienhofer. Ann Arbor, MI: Ann Arbor Science Publishers, Inc., 1975, pp. 523-528. 12. Pietta, P. and O. Brenna. P-(Aminomethyl)phenoxymethyl polymer for solid phase synthesis of protected peptide amides. J. org. Chem. 40: 2995--2996, 1975. 13. Pietta, P. G. and G. R. Marshall. Amide protection and amide supports in solid-phase peptide synthesis. Chem. Commun. 650--65 I, 1970.

MATSUEDA AND STEWART 14. Pietta, P. G., P. F. Cavallo, K. Takahashi and G. R. Marshall. Preparation and use of benzhydrylamine polymers in peptide synthesis. 11. Synthesis of thyrotropin releasing hormone, thyrocalcito.nin 26--32, and eledoisin. J. org. Chem. 39:44 48, 1974. 15. Rivaille, P., A. B. Robinson, M. Kamen and G. Milhaud. Synthese en phase solide de I'hormone de liberation de I'hormone luteotrophique, tteh', chim. Acta 54: 2772-2775, 1971. 16. Rivier, J., W. Vale, bl. Monahan, W. Ling and R. Burgus. Synthetic thyrotropin-releasing factor analogs. 3. Effect of replacement or modification of histidine residue on biological activity. J. Med. Chem. 15: 479--482, 1972. 17. Sano, S. and S. Kawanishi. Hydrogen fluoride-anisole catalyzed reaction with glutamic acid containing peptides. J. Am. chem. Soc. 97: 3480--3484, 1975. 18. Stewart, J. M. and G. R. Matsueda. New urethane protecting groups: the optically active l-arylethoxycarbonyl group. U.S. Potent 3,954,709, May 4, 1976. 19. Stewart, J. M. and J. D. Young. Solid Phase Peptide Synthesis. San Francisco: W. H. Freeman and Co., 1969. 20. Tregear, G. W., H. D. Niall, J. T. Potts, Jr., S. E. Leeman and M. M. Chang. Synthesis of substance P. Nature, New Biol. 232: 87-89, 1971. 21. Tregear, G. W., J. Van Rietschoten, E. Greene, H. D. Niall, H. T. Keutmann, J. A. Parsons, J. L. H. O'Riordan and J. T. Potts, Jr. Solid phase synthesis of the biologically active N-terminal 1-34 peptide of human parathyroid hormone. Hoppe-Seyler's Z. physiol. Chem. 355: 415-421, 1974. 22. Van Rietschoten, J., F. Granier, H. Rochat, S. Lissitzky and F. Miranda. Synthesis of apamin, a neurotoxic peptide from bee venom. Eur. J. Biochem. 56: 35-40, 1975. 23. Westfail, F. C., J. Scotchler and A. B. Robinson. The use of propionic acid-hydrochloric acid hydrolysis in Merrifield solid phase peptide synthesis. J. org. Chem. 37: 3363-3369, 1972. 24. Rivaille, P. and C. Milhaud. Synthese en phase solide de la thyrocaicitonine humaine, llelv, chhn. Acta 55: 1617-1619, 1972.