ANALYTICAL BIOCHEMISTRY 74,477-483 (1976)

Evaluation of the Fluorescamine Test for Monitoring Completion of Coupling on

Nitrochloromethylated Resin

JOHN A. SMITH

Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia

Received January 27, 1976; accepted April 15, 1976

The fluorescamine test was found to be unsuitable for monitoring the completion of coupling for solid phase peptide synthesis when nitrochloro- methylated polystyrene-divinylbenzene resin was employed as a solid support. This limitation of the normally useful fluorescamine test resulted from the nitrated polymer completely quenching the fluorescence from the fluorophor-resin. The ninhydrin color test, which was unaffected by quench- ing effects, was found to be a suitable method by which reaction complete- ness can be monitored on a nitrochloromethylated resin.

Fluorescamine’ has been shown to be a suitable reagent for fluoro- metric detection of primary amino groups (1). More recently, this reagent was used to detect the completeness of coupling in solid phase peptide synthesis (2), and the fluorescamine test was compara- tively more sensitive than the ninhydrin color test (3). However, the evaluation of both tests was done only with the routinely used chloro- methylated polystyrene-divinylbenzene resin, introduced by Merri- field (4).

When a nitrochloromethylated polystyrene-divinylbenzene resin was used in our laboratory for the synthesis of an octapeptide be- tween the A and B helices of sperm whale myoglobin, it was ob- served that the fluorescamine test gave consistently false-negative tests, although the ninhydrin test allowed monitoring of completeness of coupling at each stage of the synthesis (unpublished). Although the nitrochloromethylated resin has not been used often since the intro- duction of the Boc2 amino-protecting group, the resin is useful in synthetic schemes requiring removal of side chain-protecting groups while leaving the proximal benzyl ester linkage intact (unpublished).

I Fluorescamine, Fluram, 4-phenylspiro[furan2(3H), l’-phthalanl-3,3’-dione. * Abbreviations used: Cbz, benzyloxycarbonyl; Boc, tert-butyloxycarbonyl; Acp, e-amino-

caproic acid; NO*, nitro.

477 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

478 JOHN A. SMITH

I investigated in a comparative manner both the fluorescamine test and the ~nhyd~n test when these tests were used with the nitro- chloromethylated resin. The fluorescamine test had no practical value in monitoring reaction completeness, because the nitrated resin com- pletely quenched the fluorophor-resin fluorescence. In contrast, the ninhydrin color test was satisfactory.

METHODS

The 1% cross-linked chloromethylated polystyrene-divinylbenzene (1.29 mequiv of Cl/g), Bio-Beads S-Xl, was purchased from Bio- Rad. Boc-glycine and arginine (NO,), Cbz-Acp, Acp, and arginine monohydrochloride were obtained from the Protein Research Founda- tion (Minoh, Osaka, Japan), Fox Chemical Co., Calbiochem, and Roth (Karlsruhe), respectively. Ethyl acetate, dichloromethane, and dioxane were obtained from Fluka. Pyridine, toluene, fuming nitric acid, ethanol, and chloroform were from Analar. The reagents used were as fol- lows: HBr-glacial acetic acid (British Drug Houses), trifluoroacetic acid and ninhydrin (Pierce Chemical Co.), phenol (Analar), fluorescamine (Hoffman-LaRoche), and ethylamine and m-nitrotoluene (Kodak). The tuberculin syringes were obtained from Jintan Terumo, and the poly- ethylene disks were from Konte Biomedical Products.

All the solvents were analytical grade and were distilled before use. The reagents were used as supplied.

The 1% cross-linked c~oromethylated polystyrene-divinylbenzene resin was nitrated to yield 6.35 mM N/g, according to Stewart and Young (5). The materials coupled to the resin were protected at their amino groups by either Cbz or Boc groups. They were coupled by a benzyl ester linkage to the resin by refluxing in ethyl acetate for 24 hr, as outlined by Stewart and Young (5). The Cbz group was removed by treatment with 2 N HBr-glacial acetic acid for 60 min at ambient temperature, according to Merrifield (4). The Boc group was cleaved with 30% trifluoroacetic acid in dichloromethane for 30 min at ambient temperature, as suggested by Gutte and Merrifield (6). The coupled materials were hydrolyzed from the resin by refluxing in 6 N HCl in dioxane for 24 hr, as described by Stewart and Young (5). After filtration on a coarse sintered-glass funnel, the hydrolysate was concentrated by evaporation, and an amino acid analysis was carried out with a Beckman Model 120B amino acid analyzer. Acp was found to run at the same time as glycine during amino acid analysis.

The procedure for the fluorescamine test was that previously pub- lished by Felix and Jimenez (2), except that plastic tuberculin syringe barrels fitted with polyethylene disks were used instead of a medium sintered funnel.

FLUORESCAMINE TEST ON NITROCHLOROMETHYLATED RESIN 479

TABLE 1

SUMMARY OF EXPERIMENTS USED TO EVALUATE THE APPLICABILITY OF THE FLUORESCAMINE TEST FOR MONITORING THE COMPLETENESS OF

COUPLING REACTIONS ON NITROCHLOROMETHYLATED RESIN: A COMPARISON OF THE FLUORESCAMINE

AND NINHYDRIN TESTS

Test

Compound” Fluorescamine Ninhydrin

Acp (free in solution) (0.05 mM) Nitrochloromethylated resin with coupled Cbz-Acp

(0.068 mmolig) Nitrochloromethylated resin with coupled Acp (0.068

mmol/g) Nitrochloromethylated resin with coupled glycine

(0.086 mmol/g) Acp hydrolyzed from. nitrochloromethylated resin Glycine hydrolyzed from nitrochloromethylated resin Chloromethylated resin with coupled glycine

Positive

Negative

Negative

Negative Positive Positive Positive

Positive

Negative

Positive

Positive - -

Positive

a Abbreviations used: Acp, l -aminocaproic acid: Cbz, benzyloxycarbonyl.

For materials free in solution, the fluorescence was monitored at 475 nm with the excitation at 390 nm in the manner suggested in the Hoffman-LaRoche technical data for using fluorescamine for the estimation of free amino groups. A Perkin-Elmer MPF-3 spectro- fluorimeter was employed.

The ninhydrin test was as outlined by Kaiser ef al. (3). Fluorescamine (approximately 1 mg) was reacted with ethylamine

(20 ~1) in chloroform (2.5 ml). The excitation was at 350 nm, and the emission was observed at 475 nm. The m-nitrotoluene (diluted 1:5 with chloroform) was added in l-p1 aliquots. The effect on the emission spectrum was observed after the m-nitrotoluene was added. An identi- cal experiment was completed with toluene.

Arginine (NO,) methyl ester hydrochloride (13.5 mg), prepared according to Hofmann et al. (7), was dissolved in 0.2 M borate buffer, pH 9.0 (5 ml). A fluorescamine assay for free amino groups in solution was completed on an aliquot (2 ml) of this solution. An identical estimation of free amino groups was done with arginine hydro- chloride (10.5 mg) for comparison of arginine with and without nitro protection of its guanidino group.

RESULTS AND CONCLUSIONS

Table 1 summarizes the comparative results for the fluorescamine and ninhydrin tests, when nitrochloromethylated resin was used as

480 JOHN A. SMITH

(at tb)

FIG. 1. Structures of substituted nitrochloromethylated resin and an analogous compound used for the quenching of fluorescence experiment: (a) nitrated polystyrene- divinylbenzene resin with an amino acid coupled via a benzyi ester linkage; (b) m- nit~toluene.

the solid support. Acp (free in solution) gave positive fluorescamine and ninhydrin tests. As expected, when either Cbz-Acp or Boc-glycine, lacking free amino groups, was coupled to the nitrated resin, both tests were negative. However, after the amino-protecting groups were removed, only the ninhydrin test became positive. When these resins were hydrolyzed, the free amino groups of Acp and glycine were readily detected by the fluorescamine test. These results implied that the ni~ochloromethylated resin was quenching the fluorescence in the fluorescamine test. This conclusion was further substantiated by the observation that free amino groups were detected on a chloromethylated resin with coupled glycine by the fluorescamine as well as the ninhydrin test.

A comparison of the structures shown in Fig. 1 shows that m- nitrotoluene has a structure similar to that of the nitrated polystyrene- divinylbenzene polymer. Thus, any quenching of fluorescence attributed to Pn-nitrotoluene may, by analogy, be attributed to the nitrated resin.

As shown in Fig. 2, when m-nitrotoluene was added to a fluorescing solution of ethylamine and fluorescamine in chloroform, the fluorescence was quenched, and the efficiency of this quenching increased with continued addition of the quencher. However, there was no quench- ing attributable to the added toluene, which was chosen as a model for a chloromethylated resin.

In solution, the fluorescence quenching is expected to follow the Stern-Volmer relationship (8): Z,/Z - 1 = KQ-r.[Q], where IO = in- tensity of fluorescence emission without quencher, Z = intensity of fluorescence emission with quencher, Kc = Stern-Volmer quenching

FLUORESCAMINE TEST ON NITROCHLOROMETHYLATED RESIN 481

[F’uoro~hor ~[Quencherl 0.9 045 0.30

T”‘uene I I

7.3 LR

[m-Nitrotoluenel (mzees/liter x;> ) or [Toluenel

FIG. 2. Quenching of fluorescamine-ethylamine fluorescene by m-nitrotoluene and toluene. The ratio of fluorophor to quencher concentration is shown on the upper scale. The chloroform solution contained ethylamine (1.2 x IO-‘M) and fluorescamine (1.44 x lo-$M). The fluorophor concentration was assumed equal to the fluorescamine concentration.

constant, r = lifetime of excited state of the fluorophor, and [Q] = con- centration of the quencher. According to Stern and Volmer (8), dynamic quenching, which is a diffusion-controlled, collisional quench- ing, should be seen as a linear relationship between the amount of quenching and the concentration of the quencher added. However, as discussed by Lakowicz and Weber (9), static quenching, result- ing from sustained contact between the fluorophor and the quencher, would lead to a positive deviation of a Stern-Volmer plot.

Figure 2 is a Stern-Volmer plot of the quenching experiments done with m-nitrotoluene and toluene. As discussed above, the non- linearity of this plot for m-nitrotoluene was attributed to a combina- tion of dynamic and static quenching of fluorescence. At low Con- centrations of the quencher, dynamic quenching dominated the quench- ing, and linearity was approached. At higher quencher concentrations, the quenching was probably dominated by complex formation between the ftuorophor and the quencher, resulting in static quenching. As evidenced by the increased slope of the Stem-Volmer plot in this

482 JOHN A. SMITH

higher concentration range, static quenching was more effective than dynamic quenching and dominated the quenching by m-nitrotoluene.

In the case of the nitrated resin, only static quenching can occur, and this was consistently observed to quench the fluorophor-resin fluorescence. Although such quenching is distance dependent (lo), no fluorescence was seen after successively coupling eight amino acids to the nitrated resin in the case of the peptide from sperm whale myoglobin mentioned previously. However, rather than a dis- tance effect, this may have been due to the conformation of the pep- tide which had its free amino group close to the nitrated resin quencher. In addition, the nitrated resin has a lOO-fold excess of nitro to fluorescamine groups estimated by dividing the number of nitro groups per gram of resin by the number of amino groups coupled per gram of resin. Thus, fluorescence from a positive fluorescamine test would be expected to be completely quenched by the nitrated resin, be- cause of the large excess of quencher and the high efficiency of static quenching by the resin.

Although the fluorescamine test has been shown to be more sensi- tive than the ninhydrin test (2) for a chloromethylated resin, in the case of a nitrochloromethylated resin the fluorescamine test does not allow completeness of coupling to be evaluated. The ninhydrin method, which is a calorimetric test and unaffected by quenching effects, was entirely satisfactory. Other methods, discussed by Hit-t et al. (ll), might also be suitable alternatives to the ninhydrin test, although any that are fluorometric may be fraught with difficulty due to quenching by the solid support.

I would now like to examine the question of whether or not nitro- protected, argininyl guanidino groups can interfere with the fluores- camine test. When reacted in solution with fluorescamine, equimolar concentrations of either free arginine or nitroarginine methyl ester have similar fluorescence intensities (i.e., 80 and 72, respectively). This similarity indicates that the nitrated guanidino group did not effectively quench fluorescence. By analogy, in syntheses involving nitroarginine- containing peptides coupled to a chloromethylated resin, the nitro group would not interfere with the fluorescamine test. Thus, the nitro group as a guanidino-protecting group can be used in syntheses, which will be monitored by the fluorescamine test, if desired.

ACKNOWLEDGMENTS

The author was the recipient of a National Health and Medical Research Council Post-Graduate Medical Scholarship from the National Health and Medical Re- search Council of Australia. I wish to thank Professor S. J. Leach, Dr. W. Sawyer, and Dr. G. Tregear for their helpful suggestions and discussions. This work was carried out in the laboratory of Professor S. J. Leach with the support of a grant

FLUORESCAMINE TEST ON NITROCHLOROMETHYLATED RESIN 483

from the Australian Wool Corporation. The fluorescence-quenching experiments were carried out with the help of Miss E. Haigh.

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Biochem. 34, 595-598. 4. Merrifield, R. B. (1%3)J. Amer. Chem. Sot. 85, 2149-2154. 5. Stewart, J. M., and Young, J. D. (l%9) Solid Phase Peptide Synthesis, Freeman,

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78, 238-242. 8. Stem, O., and Volmer, M. (1919)Physik. Z. 20, 183-188. 9. Lakowicz, J. R., and Weber, G. (1973) Biochemistry 12, 4161-4179.

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11. Hirt, J., de Leer, E. W. B., and Beyerman, H. C. (1973) in The Chemistry of Polypeptides (Katsoyonnis, P. G., ed.), pp. 363-387, Plenum, New York.