The use of picric acid as a simple monitoring procedure for automated peptide synthesis

ANALYTICAL

BIOCHEMISTRY

69, 497-503 (1975)

The Use of Picric Procedure

Acid

as a Simple

for Automated

Peptide

Monitoring Synthesis

W. S. HANCOCK, J. E. BATTERSBY, AND D. R. K. HARDING Department

of Chemistry, Biochemistry, Palmerston North,

and Biophysics, New Zealand

Massey

University,

Received March 21, 1975: accepted June 19, 1975 The picric acid method has been shown to provide a simple procedure for monitoring the progress of an automated peptide synthesis. A change in properties of the polymer was observed as the protein content of the matrix increased, which resulted in increased difficulty in eluting bound picric acid. An extended washing procedure was used to overcome this problem.

A key advantage of the Merrifield solid phase method (1) is that the synthetic procedure can be automated (2). As well as allowing a great reduction in labour, automation allows the synthesis to be carried out under carefully controlled conditions.’ The increased rate of an automated synthesis, however, places stringent requirements on any method used to monitor the progress of the synthesis. Such monitoring is essential as knowledge of the individual coupling yields allows one to evaluate the success of the synthesis and plan subsequent purification steps. The monitoring procedure must be inexpensive, rapid, easily automated, and be suitable for use on the whole resin batch. To date, two procedures have been developed to monitor an automated synthesis by measurement of the free amino groups present on the polymer at various stages of the synthesis. The perchloric acid titration method of Brunfeldt (3) requires extensive modification of the synthesiser, and acetic acid used in the titration can interfere in subsequent coupling reactions. The adaption of the Dorman method, using 36C1 (4), requires expensive radiochemicals and sophisticated scintillation counters. As neither of these procedures satisfies the previous conditions, the picric acid method recently described by Gisin (5) was evaluated for this purpose. This paper will show that the method can be readily introduced into a commercial peptide synthesiser, and allows a rapid measurement of the total free amino groups present at different stages of the synthesis. 1 For example, the exclusion of oxygen can lead to an improvement in recovery of Met and Trp peptides. 497 Copyright AU rights

0 1975 by Academic Press. Inc. of reproduction in any form reserved.

498

HANCOCK,

BATTERSBY

AND

HARDING

MATERIALS

The 2% cross-linked chloromethylated Polystyrene-divinylbenzene resin (3.5 mmole/g) was purchased from Fluka Chemical Company. All amino acids were protected at the o-amino position with the BOC2 group, and the side chains by the following groups: Arg(N0,) and Glu(OBz1). These amino acid derivatives were obtained from the SchwarzlMann Chemical Company. All other reagents and solvents were analytical grade and used without further purification unless specified. Amino acid analyses were performed on a Beckman 12OC amino acid analyser. METHODS General Solid Phase Synthetic Procedures

Polystyrene resin was esterified with BOC-glycine (0.59 mmole/g) using the method of Merrifield (1). Peptides were prepared by the solid phase method (1) using a Schwarz/Mann peptide synthesiser and a program of washes which has been described previously (5). The coupling step was carried out with a threefold excess of the appropriate amino acid (0.2 M) and DCC (0.2 M, Fluka Chemical Company) as the coupling reagent. All coupling reactions were carried out twice, with a total reaction time of 4 hr. The BOC groups were removed by one 30-min treatment of the resin with 50% (v/v) trifluoroacetic acid in methylene chloride. The synthesiser was modified to allow the collection of two series of washes in separate reservoirs, in addition to the normal waste reservoir. This modification was simply achieved by the connection of two valves in series after the waste valve. The valves were connected to spare solenoid drivers which could be programmed by wiring the correct address code on the patch panel. The machine was then programmed to use these valves with one of the spare function numbers. Procedure for the Automated Picric Acid Assay

The following series of washes were used for: (A) conversion of an amine-resin to the picrate: 3 X CH&l,, 2 X 0.3 M picric acid/CH,Cl, (10 min), 3 X CH,Cl,, 3 X C,H,OH, 5 X CH,Cl, (all these washes were collected in the waste reservoir); 2 The abbreviations recommended by the IUPAC-IUB Commission Nomenclature (J. Biol. Chem. 241, 2491 (1967)) have been used. 3 All washes were for 1 min and 20 ml unless otherwise specified.

on Biochemical

PEPTIDE

SYNTHESIS

WITH

PICRIC

499

ACID

(B) elution of the bound picrate from an amino acid-resin: 3 X (GH,LN" (5 min), 3 X CH2C12, 3 x C,H50H, 3 x CHyCl, (collected in a separate vessel); (C) elution of bound picric acid from a peptide-resin: 3 X (C2HS)JN” (5 min), 3 X CHJX, 3 x C2H50H, 3 x CH,Cl, (collected in a separate vessel), 3 x (C2H&N (5 min), 3 x CH&l,, 3 X C,H,OH, 3 X CH,Cl, (collected in the second vessel). The amount of picric acid present in the eluant was measured spectrophotometrically as described by Gisin (6). The molar absorptivity of triethylamine picrate (eas8) was found to be 14,500. RESULTS

AND

DISCUSSION

It was found that the picric acid method provided a convenient and inexpensive method for monitoring an automated synthesis. Since the determination is carried out on the total sample, the picric acid method was found to be more accurate than amino acid analysis for determination of the substitution of an amino acid or peptide-resin. The agreement between the two procedures for determination of the substitution of a series of BOC-amino acid-resins is shown in Table 1. The yield of a synthesis can be accurately determined if a picric acid assay is performed on both the amino acid and peptide-resin. This simple determination contrasts with the tedious procedure necessary for obtaining accurate samples for amino acid analysis. The result of a picric acid assay on the deprotected peptide-resin gives a clear indication of the success of a synthesis. In poor syntheses the amount of picric acid bound to the peptide-resin is usually significantly TABLE DETERMINATION

OF THE

ACID-RESINS

1

SUBSTITUTION

BY THE

PICRIC

OF ACID

BOC-AMINO

METHOD

Substitution (wmoleslg resin) BOC-amino acid-resin

Amino acid analysis”

Picric acid method

Ala Arg(NO,) GlY Thr(Bzl) Ile

660 245 690 167 1178

643 260 666 167 1203

U A IO-mg sample of peptide-resin was dried to constant weight and then hydrolysed with 1 ml of HChpropionic acid (1: 1) at 130°C for 2 hr (13). 4 A 10% solution of triethylamine

in dichloromethane

was used.

500

HANCOCK,

BATTERSBY TABLE

AND 2

DETERMINATION OF THE YIELD BY THE PICRIC ACID

Peptide synthesised Somatostatin Angiotensin II Cys-Try-Phe-Gln-Asn CYS hd’Q)-Arg(NO,) Be-Ala-Val-Gly Cys-Asn-Asp-Gly-Arg(N0,) I Gly- Pro-Thr

HARDING

OF A SYNTHESIS METHOD

Yield of peptide-resin (pmolesp

Yield of cleaved peptide (I*moles)6

220 (9%) 1068 (95%) 209 (97%)

208 (94%) 844 (7%) 153 (73%)

524 (94%) 431 (57%) 560 (81%)

415 (7%) 165 (38%) 3 14 (6%)

L1The picric acid assay was performed on both the amino acid and peptide-resin, thus allowing determination of the yield of the synthesis. * The peptide was cleaved from the resin by stirring the resin with HBr and acetic acid for 2 hr. The extracted peptide was quantitated by amino acid analysis and the Lowry protein assay (14).

lower than the initial substitution value (see peptides 5 and 6 in Table 2). This observation was confirmed by low yields of peptide material obtained by cleavage of these peptides from the resin. Apparently the same factors which cause incomplete coupling reactions, e.g. steric hindrance, poor solvation of the matrix, or loss of peptide chains during the synthesis, also decrease the binding of pick acid. A serious problem in monitoring the progress of a solid phase peptide synthesis is a change in properties as the matrix is transformed from a predominately polystyrene to a peptide-resin. The solvation of the resin can be significantly altered as the peptide content of the polymer increases (4,7). This change in solvation can retard the elution of materials trapped within the resin matrix. For example, a significant amount of 36C1 present in an octapeptide-polymer could only be displaced by extended washing of the resin (4). To ensure that all of the bound picric acid was eluted, a second series of triethylamine washes was included in all analyses of peptide-resins (see Methods). The extra washes always removed additional bound picric acid (2-17% of the first washes), and as the protein content of the matrix increased the bound pick acid became more difficult to elute (Fig. 1).5 For even small peptides extensive washing is necessary for an accurate estimation of bound picric acid, 5 As might be expected, there is considerable variation in the amount of picric acid retained by different peptide-polymers of the same number of residues. The overall trend of increased “stickiness” of the resin is, however, quite clear.

PEPTIDE

SYNTHESIS

WITH

PICRIC ACID

501

NUMBER OF RESIDUES. FIG. 1. The effect of peptide content of the polymer on the difficulty of eluting bound picric acid. O.D., refers to the optical density at 358 nm of the first set of picric acid washes and O.D., to the second set (see Methods).

while the use of this assay for the analysis of larger peptides is questionable. As picric acid is a relatively strong acid with a pK, of 0.8 (81, it was decided to investigate the stability of the a-amino BOC protecting group to this reagent. A single l-g sample of BOC-lle-polymer was shaken with 0.3 M picric acid for increasing time intervals, and at each stage the extent of deprotection was determined by measuring the amount of picrate bound to the resin. Although the rate of deprotection was slow (1.5% of the total protected amino groups per hour; see Fig. 2) any premature removal of a-amino protection would be deleterious to the synthesis.6 The slow increase in the amount of bound picric acid is paralleled by the appearance of free amino groups on the resin, as measured by the bromocresol purple (9) and ninhydrin assays (10). The binding of picric acid must, therefore, be due to deprotection of the a-amino group

2

FIG. 2. The rate of deprotection methane.

4

6

8

TIME (hours) of BOC-Ile-resin

la

12

by 0.3

14

M

pick

acid in dichloro-

6 It was observed that the rate of loss of the BOC group was much greater for several peptide-resins than for BOC-Be-resin, so that the use of picric acid m these cases would cause serious side-reactions.

502

HANCOCK,

BATTERSBY

6

5

RESIDUE

4

AND

3

2

HARDING

1

NUMBER

FIG. 3. The monitoring of a synthesis of the peptide H-Glu(OBzl)-Glu(OBzl)Arg(NO,)-Met-Phe-Gly-OH by picric acid and amino acid analysis. Samples of peptideresin (1 mg) were removed after each coupling reaction and hydrolysed with HCI and propionic acid. The yield of addition of each amino acid was determined by amino acid analysis (x). The amount of picric acid bound was measured using the procedure described in the Methods section (a).

and not other side reactions such as introduction of quaternary ammonium sites. The picric acid method, can, however, be used to monitor the individual steps of a synthesis by measurement of the extent of deprotection after the addition of each residue. Such measurements do not necessarily give an indication of the success of the previous coupling reaction. The extreme situation would occur when the coupling yield was zero but the binding of picric acid to the deprotected a-NH, group was 100%. In most cases, however, one would expect that a low coupling yield would be accompanied by some reduction in the amount of picric acid bound. An example of the use of this procedure is shown in Fig. 3, in which the synthesis of a hexapeptide was followed both by the picric acid method and amino acid analysis. The amino acid analysis suggested that the synthesis occurred in good yield, except for some 20% of the initial glycine esterified to the resin, which apparently did not couple during the synthesis. This lack of reactivity was confirmed by the isolation of glycine (90 pmoles, 15% of total esterified to the resin) in the peptide material cleaved from the resin with HBr and acetic acid. A similar observation was made by Losse and Ulbrich in a study on the steric accessibility of amino acid residues esterified to the polymer (11). The large amount of unreacted glycine observed in these studies can be explained by the lack of steric bulk of glycine, which allows the amino acid to be attached to areas of the matrix which can not be penetrated by the more bulky residues. Picric acid analysis, however, shows a slow decline from the initial

PEPTIDE

SYNTHESIS

WITH

PICRIC

ACID

503

substitution value of 690 to 580 pmoles at the end of the synthesis. This decrease is probably caused by a loss of peptide chains by side reactions such as transesterification (41, chain loss due to deprotection steps (71, intramolecular aminolysis (121, or by reaction of the amino groups with impurities in solvents.7 The picric acid procedure was found to provide a reliable and convenient method for monitoring an automated peptide synthesis and can provide valuable information as to any side reactions occurring during the synthesis. The method can be used to determine the yield of peptide resins and follow the progress of deprotection reactions. Picric acid should be used with caution, however. to determine the yield of coupling reactions as the BOC-protecting group was found to be slowly cleaved by this reagent. ACKNOWLEDGMENTS The authors thank Miss M. Porter, Miss R. Christiansen, and Mr. M. Schmidt for technical assistance and Dr. G. Midwinter for the generous running of numerous amino acid analyses. The authors acknowledge Grants No. 741126 from the Medical Research Council of New Zealand, and No. 73/94 from the University Research Grants Committee, which supported this work.

REFERENCES I. 2. 3. 4.

9. 10.

Merrifield, R. B. ( 1963) J. Amer. Chem. Sot. 85, 2149. Merrifield, R. B., Stewart, J. M., and Jernberg, N. (1966) Anal. Chem. 38, 1905. Brunfeldt, K., Christensen, T., and Villemoes, P. (1972) FEES Let?. 22, 238. Hancock, W. S., Prescott, D. J.. Vagelos, P. R., and Marshall, G. R. (1973). J. Org. Ckem. 38, 77 1. Hancock, W. S., Prescott, D. J., Marshall, G. R., and Vagelos. P. R. (1972). J. Biol. Chem. 247, 6224. Gisin, B. F. (1972) Anal. Chim. Acta. 58, 248. Marshall. G. R., and Merrifield. R. B. (1971) Biochemical Aspects of Reactions on Solid Supports, Academic Press, New York. Fieser. L. F., and Fieser, M. (1957) Introduction to Organic Chemistry, pp. 428, D.C. Heath, Boston. Beyerman, H. C., (1971) Peptides 1969. p. 145, North-Holland, New York. Kaiser, E., Colescott, R. L., Bassinger, C. D.. and Cook, P. 1. (1970)Ana[. B&hem.

I I. 12. 13. 14.

Losse. G., and Ulbrich, R., (I 973) Peptides 1972, p, 152, North-Holland, New York. Gisin, B. F.. and Merrifield, R. B. (1972) J. Amer. Chem. Sot. 94, 3102. Westall, F. C., and Robinson, A. B. (I 970) J. Org. Chem. 35, 2842. Lowry, 0. H.. Rosebrough, N. J., Farr. A. L.. and Randall, R. J. (195 I ) J. Bid. Chern.

5. 6. 7. 8.

34, 595.

193, 265.

15. Brunfeldt. K., and Christensen, T. (1972) FE&S Let?. 19, 345.

7 Brunfeldt observed that aldehyde impurities in dichloromethane caused a drop in free amino groups during the course of a synthesis monitored by a HCIO., titration (15).