Eleclruphuresis 1990. I I . 6 17-620

Marcella Chiari' Pier Giorgio Righetti' Patrizia Ferraboschi, Tikam Jab3 Robert Shorr3 'Department of Biomedical Sciences and Technologies, University of Milano ,Institute ofEndocrinology, Faculty of Pharmacy, University of Milano 3ATBiochem, Malvern, PA, USA

Syntesis ofthiomorpholino buffers

6 17

Synthesis of thiomorpholino buffers for isoelectric focusing in immobilized pH gradients The two commercially available Immobilines having a pK of 6.2 (2-morpholino ethyl acrylamide) and 7.0 (3-morpholinopropylacrylamide) have been modified and two new buffers have been synthesized: 2-thiomorpholinoethylacrylamide,pK 6.6, and 3-thiomorpholinopropyl acrylamide, pK 7.4. The replacement of an oxygen with a sulfur atom in the morpholino ring is thus seen to shift the pKvalues of these two bases by +0.4 p H units. In formulations in which the two new bases replaced the standard morpholino derivatives, identical pH profiles and protein patterns wereobtained. The reason for this work was to try to close the gap between the pK 7.0 and 8.5 species and to provide the users of immobilized p H gradients with more buffers in the neutral pH region. The two new thiomorpholino derivatives are an important step in this direction.

1 Introduction The technique of isoelectric focusing (IEF) in immobilized p H gradients (IPG) I11 is now well standardized and trouble-free. The last problems remaining, (i) instability of the alkaline Immobilines 121 and (ii) oxidation by persulfate during the polymerization step 131, have now found a solution. In recent work, we have also decoded the structure and proposed the synthesis of 4 acidic acrylamide buffers (disregarding the pK 4.4 species) 141 and of 6 basic counterions 151. With these chemicals, it is possible to extend the fractionation capability of IPGs to cover the entire p H 2.5-1 1 range 161. However, it has been known for a long time that there are two major gaps in the pK distribution of the Immobiline chemicals along the pH scale: a gap between pH 4.6 and pH 6.2 (i. e., between the pK values of the weakest acidic and weakest basic Imrnobilines) and a second gap between pH 7.0 and pH 8.5 (i. e., between the pK values of 3-morpholinopropylacrylamide and N,N-dimethylaminoethylacrylamide). The presence ofthese gaps is a nuisance because, in principle, it is difficult to arrange for linearpHgradients(f0rthat thegoldenruleApK= 1 applies)17]. By computer simulations, we have been able to optimize all recipes and thus to smooth even the p H gradients encompassing these two gaps 181; nevertheless, the availability of additional acrylamide-buffers would render the technique more user-friendly and extend its flexibility. In the present report, we have addressed one of the two problems, namely how to bridge the gap in the p H 7.0-8.5 region. We report here the synthesis of two morpholino derivatives, one of which fulfills this requirement.

2 Materials and methods 2.1 Materials

power supply and Pharmalyte carrier ampholytes, pH 6.5-9, were purchased from Pharmacia-LKB Biotechnologies AB, Bromma, Sweden. Acrylamide, N,N'-methylenebisacrylamide (Bis), N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate, and Coomassie Brilliant Blue R-250 were from Bio-Rad, Richmond, CA. The 2-bromoethylamine hydrobromide, 3-bromopropylamine hydrobromide, thiomorpholine and acryloyl chloride were from Aldrich (Steinheim, FRG). The hemoglobin mutants were a gift from Dr. A. Mosca (Milano), while horse heart myoglobin was purchased from Sigma (St. Louis, MO).

2.2 IEF in IPGs Separations were in 0.5 mm thick gels, of 4 %T, 4 %C composition, in I P G pH 6.5-8.5 intervals (see Table 2 for the recipes, simulations according to 191). After standard polymerization (1 h at 50 "C) [ 101 the gel slabs were washed (3 x 500 mL) in distilled water and dried in the air. They were then rehydrated in 0.5 % carrier ampholytes (CA) in a pH 6-9 interval [ 111. Samples were applied anodically in Paratex strips (20 pL for a total of ca. 50 kg protein). Focusing was continued up to 4 h at 10 "C and 2000 V (after an initial low voltage run of 2 h at 400 V) [12]. The gels were stained in Coomassie Brilliant Blue R-250 containing copper sulfate [ 131.

2.3 Thin-layer chromatography Thin-layer chromatography was carried out on silica gel plates 60F254 from Merck, developed for 10 min with chloroform-methanol (9 : 1). The plates were stained with phosphomolibdic acid in ethanol. Column chromatography was performed on a silica gel Merck 60 (230-400 mesh).

Immobiline 11, Repel- and Bind-Silane, GelBond PAG, the Multiphor 2 chamber, Multitemp thermostat, Macrodrive ~ _ _ _ _ _ Correspondence:Prof. P. C I~i~liclli.-~iiicersity of Milano, Via Celoria 2, Milano 20 133, Italy Abbreviations: % C, g cross-linker/%T; CA, carrier ampholytes; Hb, hemoglobin; IEF, isoelectric focusing; IPG, immobilized pH gradient; % T: (g acrylamide i- g Bis)/100 mL; TEMED, N,N,N',N'-tetramethylethylenediamine; TMEA, 2-thiomorpholinoethylacrylamide; TMPA, 3-thiornorpholinopropy lacr y lamide. 0VCH Verlagsgesellschaft mbH, D 6940 Weinheim, 1990

2.4 Titration curves Both thiomorpholino derivates were titrated manually in a thermostated vessel (25 "C) under anaerobic conditions to prevent CO, adsorption. Of a 10 mM solution of each amine, 6 mL were titrated with 6 mL of 10 mM HCI. An independent evaluation of the inflection point (pK value) was also obtained by measuring thepH ofa 2: 1 molar solution amine : titrant. 01 73-0835/90/0808-06 17 %3.50+.25/0

6 18

M. Chiari el a(.

Eleclrophoresis 1990. 1I , 6 17-620

2.5 NMR analysis

Proton magnetic resonance analyses were carried out with a 500 MHz spectrometer, model A h - 5 0 0 , from Bruker (Rheinstetten, FGR)for solutions in CDCI,. Using MeSi as internal standard, 60 MHz 'H-NMR spectra were recorded with a Varian EM260-L spectrometer (Palo Alto, CA) for solutions in CDCI,.

with chloroform/methanol (9: 1, v/v). A white, crystalline powder is recovered (0.3 g, 32.5 % yield). The 'H-NMR (CDCl,) data were 6: 2.5, m, -N-CH,, 2H; 2.6-2.8, m. N ' ' S, 8H;3,4, m, C a 2 - N H ,2H; 5.6, d.=CH, 1H; 6.1, m, CH=, I H ; 6.3, d. CHA, 1H. C,H,,N,OS (200.17 Da): Calcu1ated:C: 53.99;H: 7.99,N: 13.99;Found:C:53.85;H: 7.99; N: 14.10.

2.8 Synthesis of 3-thiomorpholinopropylamine 2.6 Synthesis of 2-thiomorpholinoethylamine

T o a suspension of 3-bromopropylamine (7.4 g, 34 mmol) in absolute ethanol (6.6 mL), 2.9 mL (29 mmol) of thiomorpholine are added. After 30 h reaction at the boiling point, the solution is cooled and a precipitate is recovered and dissolved in 15 % Na,CO,. After eliminating insoluble material by filtration, the water is removed by evaporation in w c u o and 3.5 gof crude product are recorered. The product is dissolved in chloroform and filtered. The solvent is first desiccated over N a sulfate and then evaporated in vacuo, thus yielding 2.7 g of product (60 96yield). The 'H-NMR (CDCl,) data were 6: 2.2, m, -CH,-, 2.5-3, m, CH,-N, 8H; 3.1-3.5, m, CH,-S, 4H.

To a suspension of 2-bromoethylamine (6.9 g, 34 mmol) in absolute ethanol (6.6 mL), 2.9 mL of thiomorpholine (29 mmol) are added. The solution is brought to the boiling point and kept at that temperature for 30 h. After cooling, a precipitate is recovered by filtration and dissolved in 15 % Na,CO,. After filtering away insoluble material, 3 g of product are obtained upon solvent evaporation. The crude product is dissolved in chloroform and filtered, and then the solvent is eliminated by evaporation in v~zcuo.Two g of aliquid product are recovered (46 % yield). The unreacted thiomorpholine is eliminated by evaporation at 169 "C (1 atm). The 'H-NMR (CDCl,) data were 6: 2.5-3, m, CH,-N, 8H, 2.9 Synthesis of 3-thiomorpholinopropylacrylamide 3.1-3.5, m, CH,-S, 4H. 2.8 g (1 7 mmol) of 3-thiomorpholinopropylamine (synthesized as described above) are dissolved in anhydrous chloro2.7 Synthesis of 2-thiomorpholinoethylacrylamide form (20 mL) and added dropwise to a solution of acryloyl chloride (1.8 mL. 23 mmo1)in anhydrouschloroform(20 mL), 0.7 g (4.7 mmoi)of2-thiomorpholinoethylamineare dissolved thermostated at 0 "C. The reaction is continued for 1 h; in 5 mL anhydrous toluene and added dropwise to a solution of thereafter, 2.3 g (17 mmol) of K,CO3 are added and the acryloyl chloride (534 FL, 6.5mol) in anhydrous toluene mixture is stirred for 10 min. After filtering and solvent (4 mL)thermostated at0"C. Thereactionisallowedtoproceed evaporation, a crude product is recovered, redissolved in for 1 h; the precipitate formed is recovlered by filtration and chloroform and washed with a 15 % solution of Na,CO,. redissolved in 5 mL of chloroform. To this solution are added After desiccation over Na sulfate and solvent evaporation, 0.65 g(4.7 mmol) of K,CO, and afew drops ofwater. After 10 1.5 g of a yellowish, oily product are recovered. This material min the solid is removed by filtration and 0.4 g of crude is purified by chromatography on silica gel (1230 ratio of product are recovered after evaporation of chloroform in product:silica gel) and eluted with methylene chloride/methvacuo. The crude product is purified by silica gel column anol (8:2, v/v). A liquid product of 1.09 g is thus recovered chromatography (1:SO ratio of product:silica gel) and eluted (30 o/o yield). The purity of 2-thiomorpholinoethylacrylamide

4

9 2-thiomorpholino ethyl acrylamide

9 3-thiomorpholino propyl acrylamide

PH 8 7

pK 1.4

6 5 4

3 0

2

ml 10 mM HCI

4

6

0

2

4

6

ml 10 mM HCI

Figure I . Titration of the two thiomorpholino derivatives. Six mL each of a I 0 m M solution ofthe thiomorpholinos, thermostated at 25 "C and kept under argon, were titratcd manually with 6 m L o f I0rnr.r HCI with a Radiometer pH-meter. Model PHM-64. fitted with a combination microelectrode. Lef't:titra~ tion of'ttie pK 6.6 derivative; right: titration of the pK 7.4 species.

Electrophoresis 1990. 11, 6 17-620

Syntesis of thiomorpholino buffers

(TMPA), as well as of 2 thiomorpholinoethylacrylamide (TMEA), was checked by thin-layer chromatography as described in Section 2.3. The ‘H-NMR (CDC1,) data were 6: 1.7, m, -CH,-, 2H; 2.5, m. - C H , - N , 2H; 2.6-2.8, m, N --yS, 8H; 3.4, m.=,-NH, 2H; 5.6,d, =CH, 1H; 6.0, m, CH=, 1H; 6.2, d, CHA, 1H. C,,H,8N,0S (214.18 Da): Calc.: C: 56.33;H: 8.40;N: 13.07;Found:C:56.20;H: 8.40; N: 13.19.

3 Results Figure 1 gives the titration curves of the two new derivatives, TMEA and TMPA. Upon measurement of the inflection point, it is seen that in both cases the pK values ofthe two thioderivatives are shifted by +0.4 pH units (pK 6.6 andpK 7.4) as compared with the two corresponding, commercially available morpholino-chemicals (Immobilines with pK’s 6.2 and 7.0, respectively). The pK values were obtained under strictly anaerobic conditions, so as to avoid absorption of atmospheric CO, into these slightly alkaline solutions. In addition, the same values were obtained by carefully measuring the pH of a 2:l molar solution of base:titrant, the pH of which, by definition (Henderson-Hasselbalch equation), should correspond to the pK value. The properties of these two novel acrylamide weak bases are summarized in Table 1. Table 1. Basic acrylamide buffers pKa) Formula

Name

6.6

CH,=CH-CO-NH-(CHJ2-N

a

7.4

CHz=CH-CO-NH-(CH,)3-N

c> S 3-thiomorpholino-

Mr

S 2-thiomorpholino- 200.17 eth ylacrylamide

propylacrylamide 2 14.18 a) All pK values determined at 25 “C under anaerobic conditions

6 19

In order to assess the correct behavior of these two novel chemicals, IPG gels encompassing the pH 6.5-8.5 range have been run with standard, available recipes and with recipes recalculated by substituting, in the formulations, pK 6.2 with the new pK 6.6 chemicals, and pK 7.0 with the new pK 1.4. The corresponding recipes are given in Table 2. Two approaches were used: in one (Table 2, central part), a new molarity for thiomorpholino (pK 6.6) was calculated, while leaving constant the values of all other Immobilines. In this approach. one will notice that, owing to the fact that thiomorpholino is a slightly stronger base, a correspondingly lower amount as compared with the pK 6.2 species has to be used ifthe same pH Table 2. IPG recipes with morpholino and thiomorpholino derivatives for pH 6.5-8.5 CAa’

Control PK

CRC’

360 pL w 129 pL 86 pL 251 pL

3.6 6.2 7.0 8.5

90 pLW 71 pL 130 pL 169 pL

360 pL 55 pL 86 pL 251 pL

pK 6.6 PK 3.6 6.6 7.0 8.5

CR 90 pL 71 pL 130 pL 169 pL

CA 397 pL 139 pL 92 pL 268 pL

pK 7.4 PK 3.6 6.2 7.4 8.5

Cn 85 pL 79 pL 111 pL 154 pL

CA

a) Acidic, dense solution (pH 6.5 extreme) b) Volume of lmmobiline to be added to a 7.5 mL solution c) Basic, light solution (pH 8.5 extreme)

Figure2. Control and IPG gel incorporating the pK6.6thiomorpholinoderivative.Two IPG gcls(pH 6.5-8.5.4 ‘hT.4 ‘%tC)\\ereprepared.onewithacontrol recipe(Ctrl.,leftgel. upper Cormulation in Table 2) and one with the pK 6.6thiomorpholinoderivativc(rightgel,middleforinulationiiiTable 2).Thegels wererunsidebysideinaMultiphorIIchamber at 10 OCfor3 h at 2000V(afteraninitialrunfor 2 h at400V).StainingwithCoomassieBrilliantBlucR~250 in CU++.Twenty pL(cu. 50 pgtotal protein)samplewere applied anodically in stripsofparatex. Samplcs: (1).(2)hemoglobin(Hb)A.SandE;(3).(4)HbA and D; (5) H b A, S and C. The extensive heterogeneity is due to autooxidation and sample aging.

620

Electrophoresis 1990, 11, 617-620

M. CIiiari el a[.

Figure 3. Control and IPG gel incorporating the p K 7.4 thiomorpholino derivative. All conditionsas in Fig. 2,except that, in addition to the control gel, another gel was made with the pK 7.4 compound, utilizing the formulation of Table 2 (bottom recipe). Samples: (1) Hb A/S Paris:(2) Hb A/C:(3) Hb A/Lepore; (4) Hb A h ; (5) horse heart myoglobin.

6.5-8.5 interval is to be obtained. In the other (Table 2, last recipe) the entire recipe containing the new pK 7.4 buffer was recalculated by computer simulation, so as to optimize the linearity and buffering capacity. The results ofthese gels show, in Fig. 2 (pK 6.6 chemical) andin Fig. 3 (pK 7.4 chemical), that the new formulations with the two new chemicals give essentially identical results with the control formulations, thus suggesting that the two new chemicals are interchangeable with the commercial morpholino derivatives.

4 Discussion In our recent work 14, 51 we describedl 9 weak acrylamide acids and bases and two strong titrants for use in IPGs. With this set of 1 1 chemicals (10 in reality, sirice the pK 4.4 Immobiline was withdrawn), it is possible to generate any desired pH gradient in the pH 2.5-11 interval. TI-ius, with the correct choice of a few chemicals, it is possible to obtain much finer data and more reproducible results than with carrier ampholytes, which are thought to consist of perhaps a few thousand different species [ 141. Today, the IPG technique is performing extremely well, yet in our c’omputer simulations we had become aware of two major “gap’s”in these chemicals: one in the acidic region (pH 4.6 to 6.2) and one in the alkaline zone (pH 7.0 to 8.5). At present, there is no data on chemicals which cover these regions. While in extended pH gradients we have overcome these problems by arranging for linear recipes by computer optimization [9], there remains the problem that, in narrow and ultranarrow ranges, the lack of suitable buffers in these “gaps” produces gradients which are difficult to control in terms of buffering power and liinearity. Clearly, the availability of new buffers would be advantageous. We have addressed in the present research the problem of the p H 7.0 to 8.5 “gap”. One obvious solution was to try to modify the commercially available Immobilines bordering this pH hole. We thus focused our efforts on the two morpholino derivatives (the pK 6.2 and pK 7.0 species). By substituting, in the morpholino ring, an oxygen atom with a sulfur, both bases become stronger and shift their pK values by + 0.4 pH units. The two new thiomorpholino chemicals we have introduced appear to produce perfectly functioning, 1PG patterns, fully interchangeable with the standard morpholino species already available. In addition, the pK 7.4 compound appears particularly interesting as it is a step towards closing one of the

two “gaps” in the commercial chemicals. The search, naturally, is continuing.

Supported in part by a grantfrom Agenzia Spaziale Italiana (Roma).MCthanksATBiochemf o r afellowshipenablingher to carry through thepresent study. We thank Dr. E. Gianazza for the computer simulations of Table 2 and Professors E. Santaniello and A . Manzocchi(UniversilyofMilano, Dept. di Chimica e Biochimica Medica)forproviding laboratory space and facilities and for critical discussions. Received March 5, 1990

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6- 317-339. 121 Gaveby, B. M., Pettersson, P., Andrasko, J., Ineva-Flygare, L., Johannesson, U., Gorg, A.. Postel, W., Domscheit, A,, Mauri, P. L., Pietta, P., Gianazza, E. and Righetti, P. G., J . Biochem. Biophys. Methods 1988, 16, 141-164. 131 Righetti, P. G., Chiari, M., Casale, E. and Chiesa, C.. Theor. Appl. Electrophoresis 1989, I , 115-121. 141 Chiari, M.,Casale,E.,Santaniello,E.andRighetti,P.G.. Theor.App1. Electrophoresis 1989, I , 99- 102. I51 Chiari, M., Casale,E., Santaniello,E. and Righetti, P. G., Theor.App1. Electrophoresis 1989, I , 103-107. 161 Righetti, P. G., Gianazza, E., Gelfi, C., Chiari, M. and Sinha, P. K., Anal. Chem. 1989,61, 1602-1612. [71 Celentano, F., Gianazza, E., Dossi, G. and Righetti, P. G.. Chemomeir. Intel. Lab. Systems 1987, I , 349-358. [81 Righetti, P. G. and Gianazza, E., in: Glick, D. (Ed.), Methods of Biochemical Analysis, Wiley, lnterscience 1987, Vol. 32, pp. 215-278. 191 Gianazza, E., Celentano, F., Dossi, G., Bjellqvist, B. and Righetti, P. G., Electrophoresis 1984.5, 88-97. [ l o ] Righetti, P. G., Ek, K. and Bjellqvist, B., J . Chromatogr. 1984,291, 3 1-42. 111 Rimpilainen, M. and Righetti, P. G., Electrophoresis 1985, 6 , 4 19-422. 121 Righetti, P. G., Chiari, M. and Gelfi, C., Elecirophoresis 1988, 9. 65-73. 131 Righetti, P. G. and Drysdale, J. W., J. Chromatogr. 1974, 98, 27 1-321. 14 I Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Applications, Elsevier, Amsterdam. 1983, pp. 3 1-75.