Visual methods for the nanomolar detection of electrophilic reagents

Visual methods for the nanomolar detection of electrophilic reagents

Journal of Biochemical and Biophysical Methods, 8 (1983) 213-222 Elsevier 213 Visual methods for the nanomolar detection of electrophilic reagents *...

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Journal of Biochemical and Biophysical Methods, 8 (1983) 213-222 Elsevier

213

Visual methods for the nanomolar detection of electrophilic reagents * Kenneth A. Jacobson and A b r a h a m Patch0rnik Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel (Received 18 May 1983) (Accepted 20 June 1983)

Summa~ A series of highly colored nitrophenolates and nitrothiophenolates has been tested as spray reagents for the detection of electrophilic species of the types commonly used in pepfide and protein chemistry. Sensitive TLC detection of agents for alkylation, acylation, sulfonylation and phosphorylation was demonstrated, In addition, the thiophenolate sprays were sensitive for oxidizing agents in nanomolar quantities. Selective TLC detection of acylating and phosphorylating agents was accomplished by subsequent alkali treatment resulting in the restoration of color. Key words: protein reagent; alkylation; acylation; peptide coupling; chromatographic detection; oxidizing agent.

Introduction

The chemical modification of proteins is carried out frequently with electrophilic reagents, such as alkylating agents (e.g. iodoacetate, halomethyl ketones, mustards), conjugated olefins (e.g. N-ethylmaleimide), acylating agents (e.g. fluoresceinisothiocyanate), phosphorylating agents (e.g. diisopropylfluorophosphate) or sulfonyl halides (e.g. dansyl chloride). Affinity labeling requires the synthesis of reactive derivatives of ligands, in some cases bearing groups which react with specific nucleophilic amino acid residues. During peptide synthesis, reactive acylating agents, such as N-hydroxysuccinimide esters of N-protected amino acids, often are isolated. A previously reported method for the colorimetric determination of alkylating agents in solution involves prolonged heating at 100°C with 4-(4'-nitrobenzyl)pyridine [1]. For the chromatographic detection of electrophilic reagents, such as those used in protein and peptide chemistry, we suggest a class of rapidly reacting nitroaromatic c o m p o u n d s , I , as s e n s i t i v e s p r a y r e a g e n t s . S t r u c t u r e I r e a c t s as a s u l f u r o r o x y g e n

* Dedicated to Professor E. Lederer on the occasion of his 75th birthday. 0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.

214 nucleophile and is highly colored in the ionic state prior to reaction. Thus on a TLC plate or on paper a reagent or modified substrate containing an alkylating, H, or acylating, IV, group appears as a white spot on a colored background after spraying at room temperature or with mild heating. The high extinction coefficients [2,3] for I (e420(MeOH) for 2,4-dinitrothiophenolate is 16 100) make this detection method highly sensitive. In addition to the reactions shown in Fig. 1, there are other reactions of thiols [4I such as Michael addition, cyanylation, or oxidation (A = S) through which the colored species, I, might be consumed. The lability of active nitroaryl esters, V, to alkaline hydrolysis provides a means for distinguishing between alkylating and acylating reagents. Cleavage of the product under strongly basic conditions, causing the return of color, is expected generally with acylation products, V, but not with alkylation products, III, unless fl-elimination or aromatic nucleophilic substitution occurs [2].

O2N-~

) ) - - A e Et 3NH* + R - CH 2 - L --+

B

I

//

O2N--~

~V--ACH= - R + Et3N.HL

B

III 0

H

I

+R-C-L~

IV

OeN A=SorO B = H, NO 2, COO-, etc. L = leaving group

A C I

t-IN-

V

Fig. 1. Alkylafionand acylationof nitrophenolatesand pStrothiophenolates.

R + Et3N.HL

215 Materials and Methods

PicryI chloride (8c) was prepared by a modification of Okon [5]. Picric acid (5.4 g, 16 mmol) was dissolved in dry benzene (150 ml) and evaporated to near dryness. Pyridine (10 ml) and phosphorous oxychloride (1.5 ml. 16 mmol) were added, and the solution was heated for 2 h at 100°C. Toluene (100 ml) was added, and the solution was extracted with water and sodium phosphate buffer (0.1 M. p H = 7). The organic layer was dried and evaporated to a light yellow oil. which crystallized. Yield after drying in vacuo 3.6 g (95%), m.p. 82-83°C (Ref. 5 reported 82 83°C). Mass spectrum molecular ion peak at 247 m/e. analysis: calc. for C6H2N306C] 29.11% C, 0.81% H, 14.3% C1, 16.97% N; found 28.95% C, 0.83% H, 13.8% C1, 16.95% N. Spray reagents A. Basic medium: acetone/triethylamine (9:1, by volume); B. Hydrolysis reagent: ethanolic NaOH, 2.5 M; C. Solutions containing the triethylamine salts of the following: p-Nitrothiophenol (1, Aldrich), 2,4-dinitrothiophenol (3, Chemical Procurement Laboratories), p-nitrophenol (5), 2,4-dinitrothiophenol (6) and picric acid (7), were prepared by dissolving the neutral compound in solution A at a concentration of 5 raM. 3-Carboxy-4-nitrothiophenol (2): Sodium borohydride (0.1 g in 2 ml H 2 0 ) was mixed with 10 ml of a 25 m M solution of Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), in methanol, and the volume was adjusted to 100 ml with solution A. 2,4-Dinitrothiophenol (3) was also prepared as follows: Sodium sulfide (Na2S. 9HaO, Merck, 0.5 g, 2 mmol) was dissolved in 100 ml of a mixture of water/acet o n e / 1 M sodium bicarbonate (2:1:1). Chloro-2,4-dinitrobenzene (0:1 M in acetone, 5 ml) was added and the solution stirred for 1 h. n-Butanol and saturated aqueous sodium chloride (25 ml each) were added, and the phases were mixed and separated. The upper phase was washed three times with saturated sodium chloride solution to remove residual sodium sulfide and diluted to 100 ml with solution A. 2,4,6-Trinitrothiophenot (4): From picryl chloride (0.1 M in acetone, 5 ml) and sodium sulfide solution (as above, 100 ml) by the method used to prepare D N P S (3). The reaction took place immediately. Thiophenolate solutions 1-4 were prepared daily because of instability.

Detection of electrophilic reagents Solutions of 1-5 were sprayed on TLC plates (silica, cellulose, or polyamide) or on filter paper. Reagents to be identified were spotted in approximately micromolar quantities. A positive reaction was indicated by a white or light yellow spot on a deep yellow or orange background. With T N P S - after heating positive spots were orange on a grey background. Some compounds reacted immediately and others were heated to 80°C (heat gun) for 1 rain. The background faded over a period of hours. To prove the structures of alkylation and acylation products compounds 13, 22 and 26 (Table 2) were chromatographed, and the bands which reacted positively

216 after spraying products (i.e. cinimide, and Finnigan 4021

with P N P S - were extracted from the T L C plate. The expected benzyl 4-nitrophenyl thioether, 3-(4'-nitrophenylthio)-N-ethyl suc4-nitrophenyl thiobenzoate, respectively) were identified using a G C - M S (245, 280 and 259 m/e, respectively).

Selective detection of acylating reagents After the above treatment with solution 1, 2, 3 or 5 the T L C plate was sprayed with solution B, and heated (80°C). The restoration of color to roughly the intensity of the b a c k g r o u n d indicated an acylating reagent. Then, spraying with ninhydrin (0.3% in methanol) and heating tended to increase the contrast for thiophenolate sprays, except with polyamide T L C plates. Results and Discussion Basic solutions in acetone (5 raM) of the c o m p o u n d s shown in TaMe 1 were tested for reaction with a variety of electrophilic reagents. A n excess of triethylamine was used to prevent loss of color due to protonation. The electrophiles on a chromatographic m e d i u m were immersed or sprayed with one of the solutions. D N P O - , 6, and picrate, 7, were not sufficiently nucleophilic to result in loss of color even in the presence of strong electrophiles, such as tosyl chloride or iodoacetate, and thus the useful spray reagents were limited to the thiophenolates (compounds ]-4) and P N P O - , 5. In several cases, solutions of nitrothiophenolate reagents which are not readily available, were generated as needed from c o m m o n biochemical reagents. The 2,4-dinitrothiophenolate ion, 3, was formed by treatment of a 1-halo-2,4-dinitrobenzene, 8a, b, with excess hydrosulfide ion, and subsequently was extracted from other salts into n-butanol. N o further purification was necessary; Picryl chloride (chloro-2,4,6trinitrobenzene), 8c, was treated in a similar manner to give 2,4,6-trinitrothiophenolate, 4. TABLE 1 COLORED ANIONS TESTED AS REAGENTS FOR ELECTROPHILIC SPECIES Z OaN~AeE~3NH X

~

Y

Compound

X

1 2 3 4 5 6 7

H - C O O - Et3NH + H H H H H

Y H H NOa NOa H NO2 NO2

Z H H

A S S

H

S

NOa H

S O

H

O

NOz

O

TriethylarvAnesalt of: 4-nitrothiophenol (PNPS-) 3-carboxy-4-nitrothiophenol (CNPS-) 2,4-dinitrothiophenol (DNPS) 2,4,6-trinitrothiophenol (TNPS) 4-rStrophenol(PNPO -) 2,4-dinitrophenol (DNPO) picric acid

217

•NO 2 O2N--~ /

B=H

.NO 2

\)--'Hal

a c e t o n e /.w a t e r

Hal=C1

= H

= F

= NO 2

=

8a 8b

C1

~

S-

O2N

B = H = NO 2

3

4

8c

5-Mercapto-2-nitrobenzoic acid was synthesized first by Degani and Patchornik [6]. We prepared 3-carboxy-4-nitrothiophenolate, 3, in situ from a solution of Ellman reagent, 9, by reduction of the disulfide bond.

O2N

S

methanol' O2N

_

_

9

Se

O2C 2

A variety of compounds which were tested for reaction with DNPS spray are listed in Table 2. Some similar compounds, for example benzyl chloride, 13, and the benzyl bromide derivative, 14, could be differentiated by the rate of reaction on the TLC plate. The list includes compounds which alkylate, 10-17, or arylate, 19, through nucleophilic substitution, a-Halo-carbonyl compounds were highly reactive. Less reactive alkyl halides, such as 1-bromododecane, 17, could be detected weakly. As predicted, the products of alkylation were not cleaved by alkali treatment. Reactions with fl-chloroalanine and similar compounds (e.g. 12), however, are expected to be alkali reversible due to elimination [2]. Tribenzyl phosphate, I8, reacted with D N P S - in a manner that was not reversible in strong alkali. This could be explained by the alkylation of sulfur by a benzyl group. 4-Nitrophenyl cyanate and similar cyano-transfer reagents have been investigated by Kohn and Wilchek [7] as less toxic, more stable substitutes for cyanogen bromide for the activation of agarose-based resins. Phenyl cyanate, 20, was detectable with spray reagents 1-5, but 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, 21, reacted only with the most nucleophilic PNPS-.

O~C

- N: + I ~ O2N (A = S)

20

_@_

SCN

B

218

TABLE 2 IDENTIFICATION OF ELECTROPHILIC AND OXIDIZING REAGENTS SPOTTED ON SILICA T L C PLATES a USING DNPS - SPRAY Disappearance of color b

Compound

at ambient temp.

10. 11. 12. 13. 14. 15. 16.

17. 18. 19. 20, 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

a b c d

Return of color after NaOH spray

at 80°C

+ + +

Bromoacetic acid Iodoacetamide ]~-Chloropropionic acid Benzyl chloride 4-Bromomethyi benzoic acid N-Chloromethyl phthaiimide Methyl tosylate 1-Bromododecane Tribenzyl phosphate 2,4- Dinitrochlorobenzene Pheny~ cyanate 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate N-Ethylmaleimide Methyl cinnamate Benzalacetone 4- Phenyl- 1,2,4-triazolin-3,5-dione Benzoic anhydride Succinic anhydride N-t-Butyloxycarbonyl phenylalanine N-hydroxysuccinimide ester Phenyl isothiocyanate p-ToluenesulfonyI chloride Diisopropylfluorophosphate Phospholine iodide 2-Hydroxyethyl disulfide N-Bromosuccininfide Benzoyl peroxide o-Nitrophenylsulfenyl chloride 2-Nitrobenzyl alcohol Benzaldehyde

--

+d +

+ --

+

+ _

+d

--

+

--

+

+

+ --

+ d

--

+ d

--

+

--

+

--

+

--

+

+ + + -4+ +c +d

+d + + --

+ d

+ + + + --

+

+d

Kiese!gel 60, approximately 1 ffmol applied. Within several minutes. See text, Weak reaction.

The lesser reactivity of 20 is consistent with the expectation that the positive charge is shared by two nitrogens with resonance stabilization.

I

G H 3 ~ N ~ N ~ _

CN

CN -~ CHs

CH3

21

1

219 Compounds which undergo Michael addition, 22-25, generally reacted weakly with the thiophenolates and were best detected with P N P S - sprays. The diacylazo compound [8], 25, is assumed to react with two equivalents of thiol in a two-step process [9] as shown below. Thiol adducts of acyclic azodicarboxylic acid derivatives analogous to 39 have been isolated and applied to the synthesis of asymmetric disulfides [9]. Such intermediates are expected to be detectable using the thiophenolate sprays. N--'N

/

ArS\N--NH

\

O=C

Ar-S

C=O

~

~N /

/

NH--NH

\

O=C

/

Ar-S ~

C=O

\

' O=C

C=O

+ Ar-S-S-Ar

\N /

\N /

I 18 25

39

40

Acylating agents, 26-29, reacted with compounds 1 - 5 with heating. The products were cleavable with alkali treatment as in Fig. 1, resulting in orange spots in the case of D N P S - . This selective detection of acylating agents was best observed with PNPS and D N P S - sprays: Phosphorylating agents, 31 and 32, also reacted with thiophenolate sprays, probably with phosphorylation of the sulfur anion, and the products were alkali labile. Spray reagents 1 through 5 were also very sensitive towards sulfonyl chlorides, e.g. 30, but reversibility was seen only when relatively minute quantities, i.e. less than 10 nmol, were applied. The resulting thiolsulfonic ester, 42, is base labile [10], but with larger quantities applied unconsumed sulfonyl chloride was persistent even after alkali treatment.

ArSO2C1 + I

ArSO2-S-~/

~ X - - - N O 2 + Et3N.HC1 /-,.,,

\

(A = S) 41

42

Various carboxyl-activated amino acid derivatives used in peptide synthesis were detected. The detection limit with D N P S - of N-hydroxysuccinimide active esters *, 43, of N~-t-butyloxycarbonyl (Boc) amino acids (Ala, Asp(OBzl), Leu, Phe, Thr, and Tyr(OBzl)) was 10 nmol. The Val and Ile derivatives were visibly slower reacting and were detectable only at 30 nmol. This may be due to greater hindrance of the carbonyl as a result of the branched B-carbon. Symmetric anhydrides of protected amino acids were also detected. The anhydride of Boc-Phe-OH synthesized by the method of Wieland et al. [11] was detected with P N P S - after TLC (R F 0.46, 70% ethyl acetate/hexane). When Boc-Phe-OH in

* The reaction is shown as reversible, since the product is an active thiolester. The forward direction is favored due to excesshydroxysuccinimideester (estimated > 10-fold).

220 0

II C~CH , 1 43

0

2

+

I~_CH2 "V+HO--N t

C~CH 2

II

C~CH 2

O

O

+ EtBN

II

44

dichloromethane reacted with one half molar equivalent of dicyclohexylcarbodiimide the expected symmetrical anhydride [12] was detected. Another active intermediate (R F 0.63), which reacted faster than the anhydride with PNPS-, presumably the 2-t-butoxy-5(4H)-oxazolone derivative [13], was observed in the presence of excess carbodiimide. The mixed carboxylic-carbonic anhydride method of peptide bond formation requires the activation of the carboxylate component with an alkyl chloroformate. Fuller et al. [14] reported that the generation of the mixed anhydride may not be complete after 4 min at - 15°C and suggested warming to room temperature for up to one hour. If the activation step is incomplete then residual chloroformate causes undesirable acylation of the amine component. We were able to follow by TLC (silica, 20% ethyl acetate/hexane) the reaction of isobutyl chloroformate with Boc-protected amino acids in tetrahydrofuran (as in Ref. 14) with detection of both isobutyl chloroformate (R F 0.67) and the mixed anhydride ( R e 0.42 for Boc-Val©CO-O-Bu!). The chloroformate was sufficiently stable under the chromatography conditions such that 10 nmol or more couid be detected using PNPS spray. With a volume corresponding to 1 ~mol of starting materials applied for each TLC determination the method was sensitive for 1% of unreacted isobutyl chloroformate. Detection of the chromatographed mixed anhydride was less sensitive by an order of magnitude. By sampling periodically and following changes in the TLC profiIe we were abte to compare the rates of activation of Boc-Ala-OH and Boc-Vat-OH. Boc-Val-OH was slower and was predominantly unreacted after 4 rain at - t5°C. Both required room temperature for up to 20 rain to react completely with isobutyl chloroformate. It is interesting to note that generation of some symmetric anhydride accompanied the formation of the mixed anhydride, presumably from the attack of the amine salt of the Boc amino acid on the mixed anhydride° Thiols are oxidized readily to symmetric and mixed disulfides or beyond, and thus the thiophenolates also detected oxidizing agents, 33-35. The detection limit for N-bromosuccinimide with P N P S - or T N P S - was one nanomole. Thus, the sensitivity to oxidizing agents was not diminished in the less nucleophilic thiophenotates. o-Nitrophenylsulfenyl chloride, 36, wkich is used to introduce the NPS amino-protecting group [15]i reacted with D N P S - spray, probably also with the formation of a disulfide, 45. Inorganic oxidizing agents such as ammonium persulfate and sodium nitrate (weak) also gave positive reactions with thiophenolates. Some nitroarornatics, such as 37, reacted weakly possibly through reduction of the nitro group. P N P O - is stable

221

0~

NO 2

-C1 + 3

0 2 ~ .

~

, ~\/S-S

NO2

36

45

to oxidation, and thus may be used to distinguish between certain oxidizing agents and other reactive species. For example, compounds 34-37 (Table 2) were detectable with the thiophenolates, but completely unreactive towards P N P O - . The disulfide, 33, gave a weak reaction with PNPO-. A comparison of reactivities and sensitivities of compounds 1-5 is given in Table 3. P N P S - being the most nucleophilic anion of the series is also the most sensitive in general. Detection on the order of 1 nmol (e.g. alkylating agents, sulfonyl halides) corresponds to 0.1-1 /zg and is roughly one order of magnitude less sensitive than the detection of amino acids with ninhydrin spray [161. The detection limit was higher for acylating agents (10 nmol), which generally gave less contrast between the developed spot and the background. PNPS-, C N P S - and D N P S - reacted immediately with the more reactive compounds tested, such as tosyl chloride and iodoacetamide. The less nucleophilic T N P S - and P N P O - required heating under similar conditions. The detection limits reported in Table 3 were determined by spraying immediately after spotting the compound on the TLC plate because of possible decomposition during chromatography. However it was noted for compounds 11, 13, 22, 26, 30, and 35 (Table 2) that the detection limits were generally in agreement to the same order of magnitude before and after chromatography in mixtures of ethyl acetate and hexane. T N P S - also reacted with various monoanions such as the nucleophilic fluoride ion. Fluoride applied as KF, or triethylammonium fluoride, was detectable to a limit of 1 nmol. Thus, the high sensitivity of T N P S - for DFP may be a result of

TABLE 3 DETECTION LIMITS FOR ELECTROPHILIC REAGENTS IN NANOMOLES (ON SILICA GEL TLC PLATES, SPOTS 0.5 CM IN DIAMETER) Electrophilic reagent Diisopropylfluorophosphate Tosyl chloride Iodoacetamide N-Ethylmaleimide Benzoic ~mhydride

PNPO

PNPS

CNPS

DNPS

TNPS

a

2 1 2 2 10

2 1 2 3 10

1b 1 4 20 10

1c 8 20 100 100

20 100 a 40

a Not detectable. b Initially turns dark brown, then light slowly. ° See text.

222 h y d r o l y s i s o f D F P to l i b e r a t e F - r a t h e r t h a n p h o s p h o r y I a f i o n o f sulfur. P N P S - , C N P S - a n d D N P S - d i d n o t d e t e c t f l u o r i d e in q u a n t i t i e s of 10 n m o l . T N P S - aiso r e a c t e d w i t h o t h e r a n i o n s , s u c h as chloride, b r o m i d e a n d acetate.

S i m p l i f i e d d e s c r i p t i o n of t h e m e t h o d and its a p p l i c a t i o n s Nitrothiophenol derivatives in basic sotution react rapidly on thin-layer chromatography plates with compounds bearing reactive electrophilic groups, such as alkylating agents (e.g. iodoacetamide, benzyl broraide) and activated carbonyl species (e.g. active esters, anhydrides) or with oxidizing agents (e.g. peroxides, N-bromosuccinimides). The detection principle is based on a negative color reaction in which the anionic chromophore is effectively consumed forming a colorless product. The reaction occurs at room temperature or with gently heating. By comparing rates of disappearance of color one may distinguish reactivifies of different groups. The series of easily prepared spray reagents is suggested for the detection of reagents which label biopolymers, including modified iigands for affinity chromatography. Active acyl intermediates for peptide synthesis also are detectable by this method.

Acknowledgements W e w i s h to t h a n k D r . J. K o h n for p r o v i d i n g s a m p l e s o f c y a n y l a t i n g r e a g e n t s a n d M r . I. J a c o b s o n f o r p r o t e c t e d a m i n o a c i d a c t i v e esters. K e n n e t h J a c o b s o n is g r a t e f u l f o r the f i n a n c i a l s u p p o r t o f the M y r o n B a n t r e l l F e l l o w s h i p .

References 1 2 3 4

Preussman, R., Schneider, H. and Epple, F. (1969) Arzneim.-Forsch. 19, 1059-1073 Sokolovsky, M., Sadeh, T. and Patchornik, A. (1964) J. Am. Chem. Soc. 86, 1212-1217 Sokolovsky, M., Wilchek, M. and Patchornik, A. (1962) Bull. Res. Council Israel. 11A, 79-80 Friedman, M. (1973) The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins. Pergamon Press, Oxford 5 0 k o n , K. (1959) Roczniki Chem. 33, 45-49 6 Degani, Y. and Patchornik, A. (1971) J. Org. Chem. 36, 2727-2728 7 Kohn, J. and Wilchek, M. (1983) Appl. Biochem. Biotechnol., in press 8 Cookson, R.C., Gilani, S.S.H. and Stevens, LD.R. (1962) Tetrahedron Lett., 615-618 9 Wunsch, E., Moroder, L. and Romani, S. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1461-1464 10 Hookway, H.T. (1950) J. Chem. Soc. 1932-1934 11 Wieland, T., Flor, F. and Birr, C. (1973) Liebigs An~. Chem. t595-1600 12 Rebek, J. and Feitler, D. (1974) J. Am. Chem. Soc. 96, 1606-1607 13 Benoiton, N.L. and Chen, F.M.F. (1981) Can. J. Chem. 59, 384-389 14 Fuller, W.D., Marr-Leisy, D., Chatnrvedi, N.C, Sigler, G.F. and Verlander, M.S. (I981) in Peptides, Synthesis, Structure, Function, Proceedings of the Seventh American Pepfide Symposium (Rich, D.H. and Gross, E., eds.), pp. 201-204. Pierce, Rockford, Ill. t5 Zervas, L., Borovas, D. and Gazis, E. (1963) J. Am. Chem. Soc. 85, 3660-3666 16 Fahmy, A.R., Niederwieser, A., Pataki, G. and Brenner, M. (196!) Helv. Chim. Acta 44, 2022-2026