Bioconjugate Chem. 1991, 2, 38-43
38
Diazo- and Azido-Functionalized Glutaraldehydes as Cross-Linking Reagents and Potential Fixatives for Electron Microscopy Sui Xiong Cai and John F. W. Keana* Department of Chemistry, University of Oregon, Eugene, Oregon 97403. Received October 16, 1990
The synthesis of diazo and perfluorophenyl azide (PFPA)functionalized glutaraldehydes 7 and 13a-d as new cross-linkingreagents for bioconjugationand potential fixatives for electron microscopy is reported. A key step is the generation of the 1,5-dialdehyde structures by oxidative cleavage of the corresponding cyclopentene epoxide using HI04 in aqueous tetrahydrofuran. A model reaction between 3-substituted glutaraldehyde 14 and 6-aminohexanoic acid resulted in the formation of pyridinium ion containing products with UV spectra comparable to those observed with glutaraldehyde itself. Thus modification of glutaraldehyde in the 3-position most probably did not significantly change its reactivity with amines under chemical-fixation conditions. Fixation of red blood cells by 7 demonstrates that as a fixative, 7 is comparable to glutaraldehyde.
Bifunctional reagents are widely used for the crosslinking of biomolecules such as membrane components (1,2). Glutaraldehyde (1) has been used as a cross-linking
""3
OHC
1
3-Cyclopentene-1-methanol 2'-Diazo-3',3',3'-trifluoropropionate (6). (a) A solution of 1.54g of acyl chloride 5 (12) (8.93 mmol) in CHzClz (10 mL) was added dropwise to a stirred solution of 0.767 g (7.8 mmol) of alcohol 3a (13) and 1.22 g (12 mmol) of Et3N in ether (5 mL). It was stirred for 1h then filtered. The filtrate was washed with water (2 X 15 mL), dried, and evaporated and the residue was distilled to give 1.14 g of 6 as a yellow oil: bp 52-53 "C (0.8 mm); 'H NMR 6 2.10 (m, 2), 2.50 (m, 2), 2.65 (m, l ) , 4.18 (d, 2, J = 7.2 Hz), 5.65 (9, 2); IR 2140, 1720 cm-l; = 236 nm, log E = 3.92. Anal. Calcd for UV A,, C ~ H ~ F ~ NC, Z 46.16; O ~ : H, 3.87; N, 11.96. Found: C, 45.94; H, 3.84; N, 11.72. (b) A solution of 57.3 mg (0.192 mmol) of anhydride 8 and 18.2 mg (0.186mmol) of alcohol3a with 39.4 mg (0.498 mmol) of pyridine in THF (2 mL) was heated at 50 "C for 12 h. It was diluted by ether (3 mL), washed by 5% Na2COS(1mL) and water (2 X 2 mL). The organic phase was dried and evaporated to leave 32.4 mg of 6 as a pale yellow oil (74 96 ). 1,5-Dioxopentane-3-methanol 2'-Diazo-3',3',3'-trifluoropropionate (7). To a stirred mixture of 111 mg (0.475 mmol) of 6 in water (1.5 mL) and ether (1.5 mL) was added 350 pL of 2.5 wt 5% Os04 (6.82 mg, 0.026 "01)tert-butyl alcohol solution, followed by addition of 236 mg (1.20 mmol) of NaI04 portionwise and the mixture was stirred for 2 h. The mixture was filtered and the filtrate was extracted with ethyl acetate (2 X 1.5 mL). The extract was dried and evaporated to leave 147 mg of crude 7 as a dark-brown oil: 'H NMR 6 2.60 (m), 2.98 (m), 4.28 (m), 9.75 (s), also 1.8, 2.5, 4.1, 5.5-5.0 (integral 5.5-5.0:9.75 = 11:2) [After the sample in the NMR tube had been kept at 25 "C in the dark for 2 days, retaking of the 'H NMR showed a relative increase in the peaks at 6 2.60 (m), 2.98 (m),4.28(d),and 9.75 (s) (5.5-5.09.75 = 3.5:2).];MS m / z 274 (M++ 2H20 - N2), 238 (M+- N2); IR 2130,1720 cm-'; UV A,, = 239 nm, log E = 3.92. On one occasion, a 22-mg sample was separated by preparative TLC (1:2:2 acetoneether-CHzCld to give 2 mg of quite pure dial 7 (Rf= 0.450.60): 'H NMR 6 2.60 (m, 4), 2.98 (m, 11, 4.28 (d, 2, J = 5.4 Hz), 9.77 (s, 2). The structure assignment was supported by proton decoupling. Irradiation at 6 2.98 transformed the multiplets at both 6 4.28 and 2.60 to singlets.
oHCxH202cx
OHC
2a,R=H 2b, R C H & C X
reagent for protein-protein (3) and drug-antibody conjugation ( 4 ) as well as a fixative (5) of biological specimens for electron microscopy (EM) (6). Glutaraldehyde fixation chemistry is thought to involve a reaction with free amino groups of proteins resulting in the formation of pyridinium and polypyridinium ions (7, 8). Glutaraldehyde generally does not react with lipids (9)and no reagent has been reported to be an efficient fixative for saturated lipids (IO). Herein we report the development of a family of photoactive glutaraldehydes 2 as new cross-linking reagents and potential fixatives for EM preparations. The 3-position of glutaraldehyde was chosen for modification since the protons in the 3-position are not involved in the condensation reaction leading to the formation of pyridinium ions in the proposed mechanism (7,8). Photolysis of the diazo or azido group X generates a highly reactive carbene or nitrene intermediate which is capable of insertion into unreactive CH bonds with concomitant formation of a stable covalent linkage (11). EXPERIMENTAL PROCEDURES General Chemical Procedure. Solvents and chemicals were reagent grade and used as received unless otherwise specified. Red blood cells were from fresh human blood of a healthy volunteer. 'H NMR spectra were measured on a QE-300 NMR spectrometer in CDC13. IR spectra were recorded on a Nicolet 5DXB FTIR spectrometer in CDC13. UV spectra were measured on a Beckman DU-7 spectrometer. All reactions involving diazo or azido compounds were run under subdued light by wrapping the flasks with aluminum foil. Reactions were routinely monitored by TLC or 'H NMR. MgS04 was used as the drying agent for organic solutions. Photolysis was carried out in a Rayonet photochemical reactor with 254-nm lamps and quartz reaction tubes.
0 1991 American Chemical Society
Diazo- and Azido-Functionallzed Glutaraldehydes
Bioconjugate Chem., Voi. 2, No.
1, 1991
39
2-Diazo-3,3,3-trifluoropropionic Anhydride (8). WaNa2C03 (2 X 10 mL) and water (2 X 10 mL), dried, and ter (0.2mL, ll mmol) was added dropwise to a vigorously evaporated to leave a liquid which was purified by preparative TLC (5:5:1CH2C12-hexane-methanol) to give stirred solution of 1.0 g (5.8mmol) of acyl chloride 5 in 206 mg (48%) of 12a as a colorless solid (mp 38-40 "C): CH2C12 (10mL), dioxane (5 mL), and pyridine (1.4mL). The mixture was stirred for 0.5 h, then poured into a 'H NMR 6 1.516 (m, 2),2.220 (m, 3), 3.514 (s,2),4.328 (d, mixture of 2 N HCl(35 mL) and ice (10g). It was stirred 2,J = 4.78 Hz); and 1.948 (m, 4), 2.49 (m, l),3.532 (9, 2), for 10 min and extracted with CHzCl2 (3 X 25 mL). The 4.213 (d, 2,J = 8.10 Hz); IR 3000,2129,1733,1648,1490, extract was dried and evaporated to leave 0.62 g (73%) of = 264 nm, log E = 4.27. Anal. Calcd 1421 cm-'; UV ,A, solid which was purified by chromatography (silica gel, for C13HgF4N303: C, 47.14;H, 2.73;N, 12.69. Found: C, 1:4ether-hexane) followed by crystallization (CHCl3-hex47.29;H, 2.61; N, 12.53. ane) to give 8 as large colorless needles: mp 55-56 "C; IR 1,5-Dioxopentane-3-methanol4'-Azido-2',3',5',6'-tet2148,1789,1720cm-'. Anal. Calcd for C6F6N403: C, 24.84; rafluorobenzoate (13a). A solution of 17.7 mg (0.053 N, 19.31. Found: C, 24.43,N, 19.81. mmol) of 12a and 20 mg (1.0mmol) of HI04 in water (0.5 3-Cyclopentane- lYl-bis[2-[2-( 2-methoxyet hoxy)mL) and THF (0.5 mL) was heated at 50 "C for 1 h. The solution was extracted with CHCl3 (3 X 1 mL) and the ethoxylethanol] (3d). A mixture of 0.64 g (0.081 mol) of LiH with 4.0 g (0.031mol) of diol 3b (14) in di-n-butyl extract was dried and evaporated to leave 18.6 mg of 13a ether (80 mL) was refluxed overnight. To the mixture as a pale yellow oil (100%): 'H NMR 6 2.67 (m, 4),3.021 (m, l), 4.386 (d, 2,J = 5.1 Hz), 9.775(s,2);also 5.5-5.0 (m), was added 19.4 g of 2-[2-[2-(2-chloroethoxy)ethoxy]ethoxyltetrahydropyran (9) (15)at room temperature and it 4.2 (m) [lH NMR of the sample in the NMR tube after was heated at 100 "C for 10 days. To the mixture was being kept at 25 "C in dark for 6 h showed an increase of then added 0.45 g of LiH and it was heated at 100 "C for the signals for 13a and a decrease of the 6 5.5-5.0signals.]; 7 days. The mixture was filtered and the filtrate was cm-l; UV ,A, = 267 IR 2957,2129,1734,1648,1490,1420 washed with water (3 X 60 mL), dried, and evaporated to nm, log = 4.25;MS m/z 347 (14,M+), 319 (3,M+ - Nz), leave a red oil which was dissolved in MeOH (60mL) and 276 (25,M+ - Nz - CHzCHO), 218 (30,N&eF4CO), 190 CHC13 (60mL),then 3 mL of concentrated HC1was added. (5,NCGF~CO), 162 (80,NC6F4), 69 (100);high-resolution The mixture was refluxed for 2 h, then 17 g of NaHC03 MS calcd for C13H9F4N304 347.0526,found 347.0531. was added portionwise at room temperature. The mixture 3,4-Epoxycyclopentane-1 l-dimethanol Bis (4-azidowas stirred for 15 min and filtered. The filtrate was 2,3,5,6-tetrafluorobenzoate)(12b). To a solution of 38.6 evaporated to leave an oil which was distilled (0.005 mm, mg (0.301mmol) of diol 3b and 130 mg (1.29 mmol) of 90.0 "C) to give 9.1 g of yellow oil residue. The residue Et3N in 10 mL of ether was added 213 mg (0.840mmol) was purified by chromatography (silica gel, ether-ethanol of 10 and the mixture was stirred for 8 h. It was filtered = 20:1,20:2,20:3,and 20:4,400 mL of each) to give 6.47 and the filtrate was evaporated to leave 1 lb as a liquid: g (537; ) of 3d as a pale yellow oil: 'H NMR 6 2.185 (s, 4), 5.663 (s, 2). The liquid 'H NMR 6 2.376 (s,4),4.371 (s,4), 3.001 (s, 2), 3.393 (s, 4),3.621 (m, 12), 3.665 (s, 8), 3.714 was dissolved in CHzCl2 (15mL) and to the solution was (d, 4, J = 3.6 Hz), 5.568 (s, 2). Anal. Calcd for added 400 mg of m-chloroperbenzoic acid. The solution C19H360~0.5HzO:C, 56.83;H, 9.28. Found: C, 56.72;H, was heated at 50 "C for 2.5 h then 3 mL of 10% aqueous 9.38. Na2S03 was added and the mixture stirred for 30 min. It 2-[2-[2-(3-Cyclopentenylmethoxy)ethoxy]ethoxy]was washed with 5 % aqueous NaHC03 (3 X 10 mL) and ethanol (3c). Alcohol 3c was prepared from 3a in a water (2X 10 mL), dried, and evaporated to leave 260 mg manner similar to that for 3d. From 3.04 g (31.0 mmol) of residue which was separated by preparative TLC (1: of 3a and 18.2 g (72.2mmol) of 9 there was obtained 5.70 1:0.4CHC13-hexane-acetone) to give 60 mg (34%) of 12b g (809; ) of 3c as an almost colorless thick oil: 'H NMR as a pale yellow oil: 'H NMR 6 1.800 (d, 2,J = 15.14 Hz), 62.0-2.5(m,6),3.362(d,2,J=7.2Hz),3.67(m,12),5.6362.183 (d, 2,J = 15.14 Hz), 3.598 (s, 2),4.236 (s, 2), 4.339 (s, 2). Anal. Calcd for ClzH220~0.1H20:C, 62.09;H, 9.64. cm-l. Anal. Calcd (s,2);IR, 3000,2129,1737,1647,1490 Found: C, 61.96;H, 9.28. for C Z ~ H ~ ~ F ~C, N 43.61; ~ O F H, ~ : 1.74;N, 14.53. Found: C, 43.82;H, 1.64;N, 14.53. 3-Cyclopentene-l-methanol4'-Azido-2',3',5',6'-tetrafluorobenzoate (1 la). Ester 1 la was prepared from 1,5-Dioxopentane-3,3-dimethanol Bis(4-azid0-2,3,5,6tetrafluorobenzoate) (13b). The oxidation of 12b by alcohol 3a and acyl chloride 10 (16) in a manner similar to that of 6. From 250 mg (0.985mmol) of 10 and 42.0mg HI04 was carried out in a manner similar to that of 12a. (0.428mmol) of 3a there was obtained 120 mg (89%) of From 24 mg (0.042mmol) of 12b and 40 mg (0.21mmol) 1 la as a yellow oil after purification by preparative TLC of HI04 there was obtained 24.2 mg (98%) of dial 13b as (1:2 ether-hexane): 'H NMR 6 2.16 (m, 2), 2.52 (m, 2), a thick oil: 'H NMR 6 2.960 (m),4.512 (m), 9.759 (s); also 1.5-2.0,2.80,4.32,5.0-6.0; IR 3000,2129,1737,1647,1490 2.73 (m, 11, 4.289 (d, 2, J = 7.2 Hz), 5.67 (s, 2); IR 2935, 2119, 1732, 1648, 1490 cm-'. A neat sample of l l a was cm-l. stored at 25 "C in the dark for 1day. An lH NMR of the 2-[ 242-[ (3,4-Epoxycyclopentanyl)methoxylethoxy1resulting yellow solid showed a complicated spectrum in ethoxy]ethanol4'-Azido-2',3',5',6'-tetrafluorobenzoate which the 6 5.67 singlet due to l l a had decreased. The (12c). Epoxide 12c was prepared from alcohol 3c and 10 intensity of the 2119 cm-l peak (azide) in the IR spectrum in a manner similar to that of epoxide 12a. From 152 mg also decreased. The sample in the NMR tube (CDCl3) (0.663mmol) of 3c and 176 mg (0.697mmol) of 10 there however showed no change after 10 days in the dark. was obtained l l c as a colorless oil: 'H NMR 6 2.065 (m, 3,4-Epoxycyclopentane-l-methanol4'-Azido-2',3',5',6'2),2.441 (m, 3),3.355 (d, 2,J = 7.2Hz), 3.587 (m, 2),3.649 tetrafluorobenzoate (12a). To a solution of 1 la [freshly (m, 2),3.676 (s,4),3.813 (t, 2,J = 4.80 Hz),4.515 (t, 2,J prepared from 330 mg (1.30mmol) of 10 and 127 mg (1.29 = 4.80 Hz),5.638 (s, 2); IR 2868, 2129, 1737, 1648, 1491 mmol) of 3a] in ether (15 mL) was added 352 mg (1.73 cm-l. Epoxidation of the colorless oil gave 278 mg (94%) mmol) of 85 % m-chloroperbenzoic acid and it was stirred of 12c as a pale yellow oil after purification by preparative TLC (5:5:1CHC13-hexane-methanol): 'H NMR 6 1.856 at 25 "C for 1 h. To the solution was added 0.5 g of anhydrous Na2C03 and the mixture was stirred for 1 h (m, 4),2.340 (m,l),3.263 (d, 2,J = 7.80 Hz), 3.459 (5, 2), and filtered. The filtrate was washed with 5% aqueous 3.659 (m, 8),3.808 (t, 2,J = 4.80 Hz), 4.509 (t, 2,J = 4.80
40
Bioconjugate Chem., Vol. 2, No. 1, 1991
Hz); and 1.430 (m, 2), 2.096 (m, 3), 3.380 (d, 2, J = 5.40 Hz), 3.451 (s, 2), 3.659 (m, 8), 3.808 (t, 2, J = 4.80 Hz), 4.509 (t, 2, J = 4.80 Hz); IR 2871, 2129, 1736, 1648, 1490 cm-'; UV,,,A = 264 nm, log t = 4.30. Anal. Calcd for C19H21F4N306: C, 49.25; H, 4.57; N,9.07. Found: C, 49.11; H, 4.68; N, 9.25. 2-[2-[2-[(1,5-Dioxopentan-3-yl)methoxy]ethoxy]ethoxy ]ethanol 4'-Azido-2',3',5',6'-tetrafluorobenzoate (13c). The oxidation of epoxide 12c by HI04was carried out in a manner similar to that of 12a. From 40 mg (0.086 mmol) of 12c and 40 mg (0.21 mmol) of HI04 there was obtained 41 mg (99%) of dial 13c as an colorless oil: 'H NMR (fresh sample in CDCl3) 6 2.5 (m, 4), 2.85 (m, l),3.42 (m, 2), 3.6 (m, 8), 3.809 (m, 2), 4.519 (m, 2), 9.751 (s, 2); also 3.33, 2.70; IR 2129, 1735, 1648, 1490 cm-'. 'H NMR of the sample as a CDC13 solution after three days showed increasing intensity of 2.5 (m), 2.85 (m), 3.42 (d), and 9.75 (s) and decreasing intensityof 3.33 and 2.70; IR 2129,1733, = 267 nm, log t = 1726, 1652, 1647, 1490 cm-l; UV,,,A 4.31. (3,4-Epoxycyclopentane-1,l-diyl)bis[2-[2-(2-methoxyethoxy)ethoxy]ethanol] Bis(4-azido-2,3,5,6-tetrafluorobenzoate) (12d). Ester 12d was prepared from 3d and 10 in a manner similar to that of 12a. From 265 mg (1.05 mmol) of 10 and 195 mg (0.498 mmol) of diol 3d there was obtained 1 Id as a colorless oil: lH NMR 6 2.174 (s,4), 3.364 (s,4), 3.58 (m, 8),3.662 (s,8), 3.182 (t, 4),4.509 (t, 4), 5.552 (s, 2). Epoxidation of the colorless oil gave 206 mg (49%)of 12d as a pale yellow oil: 'H NMR 6 1.694 (d, 2, J = 15.0 Hz), 1.862 (d, 2, J = 15.0 Hz), 3.277 (s, 2), 3.289 (s,2), 3.454 (s, 2), 3.7-3.5 (m, 16),3.813(m, 4), 4.513 (t,4, J = 4.63 Hz); IR 3000, 2129, 1736, 1648, 1490 cm-'. Anal. Calcd for C & . ~ F ~ N ~C, O47.04; ~ I : H, 4.07; N,9.97. Found: C, 47.10; H, 4.06; N, 9.57. (1,5-Dioxopentane-3,3-diyl)bis[2-[ 2-(2-methoxyethoxy)ethoxy]ethanol] Bis(4-azido-2,3,5,6-tetrafluorobenzoate) (13d). Epoxide 12d was oxidized in a manner similar to that of 12a. From 48 mg (0.057 mmol) of 12d and 40 mg (0.21 mmol) of HI04there was obtained 48 mg (99%)of dial 13d as an oil: 'H NMR 6 2.0-1.6 (m, 4), 3.5-3.2 (m, 4), 3.505 (s,2),3.7-3.6 (m, 16),3.806 (m,4), 4.511 (t, 4), 9.777 (s, 2); also 6 5.0 and 1.8; IR 2876, 2129, 1735, 1649, 1490 cm-'. 1,5-Dioxopentane-3-methanolPropionate (14). A solution of 0.392 g (4.00 mmol) of alcohol 3a in 1.50g (11.5 mmol) of propionic anhydride was heated at 100 OC for 15 h. It was cooled to 25 OC and diluted with ether (15 mL). The solution was washed with 5% aqueous Na~C03 (2 X 6 mL) and water (2 X 10 mL), dried, and evaporated to give 0.507 g (82% ) of a pale yellow oil which was purified by chromatography (silica gel, 1:l hexane-ether) to give 0.48 g of ester as colorless liquid: 'H NMR 6 1.14 (t, 3, J = 7.50 Hz),2.08 (m, 2), 2.33 (4, 2, J = 7.50 Hz), 2.48 (m, 2), 2.60 (m, l), 3.99 (d, 2, J = 6.90 Hz), 5.65 (s, 2). Anal. Calcd for CgH140~0.3H20:C, 67.72; H, 9.22. Found: C, 67.77; H, 9.08. The ester was oxidized by Os04/NaI04 in a manner similar to that of ester 6. From 201 mg of the ester there was obtained 180 mg (74%)of dial 14 as a pale brown oil: 'H NMR 6 1.14 (m), 2.37 (m), 2.56 (m), 2.95 (m), 4.063 (m), 9.759 (s); also 2.50, 3.90, and 5.0-6.0. Reaction of Glutaraldehyde (1) and 1,5-Dioxopentane-3-methanol Propionate (14) with 6-Aminohexanoic Acid (15). To a solution of 0.1 mL of 0.1 M glutaraldehyde in phosphate buffer (pH = 7.4) was added 1.8mL of buffer and 0.1 mL of 0.05 M amine 15 in buffer. The UV absorption of the solution was monitored at 265 nm (absorption maximum). A straight line absorption increase (AOD/min = 0.061) was observed. The reaction
Cai and Keana
Table I. Fixation of Red Blood Cells by Dialdehyde 7 and Glutaraldehyde 1 at 25 "C in PBS Buffer (pH = 8.0) dial [dial] incuba photob color' 7 0.05 15 45 colorless 1 0.05 15 45 co1or1ess none 15 45 red 7 0.05 60 no colorless 1 0.05 60 no colorless none 60 no red 7 0.002 51 9 colorless 1 0.002 51 9 colorless 1 0.002 60 no colorless 7 0.0004 55 4.5 red 1 0.0004 55 4.5 red 1 0.0004 60 no red
Incubation time (min). Photolysis time (min). Color of the supernatant solution, water + buffer (1:l). Scheme I
3a
6
5
52.60
54.28
, 69.75
7
62.9%
of dialdehyde 14 with amine 15 was carried out in the same manner under the same conditions and a similar absorption increase was observed (AOD/min = 0.059). Fixation of Red Blood Cells by Dialdehydes 1 and 7. Red blood cells were prepared according to Steck (17). A sample (10 mL) of fresh blood from a healthy donor was mixed with pH 8.0 phosphate-buffered saline (PBS) (10 mL) and centrifuged at 4 "C for 5 min. The supernatant solution and "buffy coat" were removed by aspirator through a Pasteur pipet. The pelleted red cells were washed by PBS (10 mL). The pellet was then diluted with PBS (2 mL) and kept at 4 "C. A mixture of 1 mL of 0.05 M of 7 (PBS) and 0.1 mL of the above red blood cells was incubated at 25 "C for 15 min. The sample was photolyzed (Rayonet reactor, 254 nm) in a quartz tube for 45 min. The sample was then mixed with 5 mL of PBS and centrifuged. The pellet was mixed with 1 mL of PBS and 1 mL of water and centrifuged. The supernatant solution was clear and colorless. The pellet was mixed with 1 mL of PBS and observed under an optical microscope and photographed. Other experiments were carried out in the same manner. Representative conditions and results are collected in Table I. RESULTS AND DISCUSSION
Our plan called for the masking of the glutaraldehyde moiety in the form of the cyclopentenyl group in alcohol 3a (13) (Scheme I) and the attachment of photoactive agents to the hydroxyl group. Two photoactive acylating agents, 2-diazo-3,3,3-trifluoropropionylchloride (5) and 4-azido-2,3,5,6-tetrafluorobenzoyl chloride (10) were selected for these studies. The strongly electron-withdrawing CF3 group in 5 both stabilizes the diazo function and suppresses Wolff rearrangement of the corresponding carbene (12). Another advantage of this photoactive group is its relatively small size, which should not cause drastic reduction of the aqueous solubility of the modified glutaraldehyde.
Bioconjugate Chem., Vol. 2, No. 1, 1991
Diazo- and Azido-Functlonalized Glutaraldehydes
Acylation of alcohol 3a by 5 gave ester 6, which was subjected to oxidation of the cyclopentene moiety with osmium tetroxide and sodium periodate (18). A strong diazo absorption peak at 2130 cm-l in the IR spectrum of the product mixture indicated that the diazo function was stable under the oxidation conditions. An lH NMR spectrum of the products showed the presence of the expected dialdehyde 7, as evidenced by characteristic peaks at 6 9.75, 4.28, 2.98, and 2.60, together with other peaks at 6 5.0-5.5, 4.1,2.5, and 1.8. Korn et al. (19) reported a similar complicated lH NMR spectrum for glutaraldehyde in water. They proposed that aqueous glutaraldehyde exists as a mixture of free monomer, cyclic hydrate, and oligomers. The signals of 7 increased at the expense of the other signals after the sample was kept as a CDC13 solution in the dark, corresponding to a relative increase of the dial form. A pure (by NMR) sample of 7 was obtained by preparative TLC. The solubility of crude 7 in aqueous buffer was measured by UV absorption at 239 nm (Amm) to be 0.17 M, which is in the range of concentrations (1-4 wt%, 0.1-0.4 M) of glutaraldehyde employed for EM applications. Anhydride 8 was prepared from 5 via reaction with water (eq 1). Acylation of alcohol 3a by 8 gave ester 6. Since CF3CCOCl
II
--
3a
(CF3
5
$OCI
3
I
a n=O, R=H b n=O, R=CH2OH c n=3, R=H d n=3, R=CH2(0CH2CH&OH
NZ 8
acyl chloride 5 is a low-boiling liquid, crystalline anhydride 8 constitutes a convenient alternative reagent for introduction of the diazotrifluoropropionyl group into another molecule. 4-Azido-2,3,5,6-tetrafluorobenzoyl chloride (10)is among the functionalized perfluorophenyl azides (PFPAs) recently developed in our laboratory as photolabeling agents showing improved CH insertion efficiency when photolyzed in hydrocarbon solvents (16). Before the acylation reaction, three ethyleneoxy units were inserted into alcohol 3a and diol 3b, serving both to increase the aqueous solubility of the final products and as a spacer arm connecting the dial function and the azido function. The chain extension was carried out by coupling of diol 3b with an excess of THP-protected chloro ether 9 in the presence of LiH. Hydrolysis of the THP protecting group (eq 2) then gave alcohol 3d. Alcohol 3c was similarly prepared.
3a, R = H 3b, R = CH20H
I N3
10
11 X, X = HC=CH E
1
2 X, X = HCTFH
I n F
RF
a n=O,R=H b n=O, R=CH202CC6F4N3 c n=3. R=H 1 d n=3; RICH2(0CH2CH2)302CCC6F4N3
(1)
Pyridine
NZ
Scheme I1
11, 12, 13 HzO
41
9
v
3c,R=H
36,R = CHz(OCHzCH&OH
The mono PFPA modified dialdehyde 13a was prepared first. Reaction of alcohol 3a with acyl chloride 10 gave ester lla (Scheme 11). The 'H NMR and IR spectra suggested that possibly an azido olefin [3 + 21 cycloaddition (20)had taken place during storage of a neat sample at room temperature in the dark for 1day. Since a dilute solution of freshly prepared 1 la in CDC13was stable under
the same conditions, the cycloaddition reaction was probably intermolecular. Dialdehydes in the glutaraldehyde series are generally difficult to obtain in analytical pure form due to the presence of different hydrated forms and oligomers (19). For example, several 3,3-disubstituted glutaraldehydes such as 3,3-dimethylglutaraldehyde(21) and 3,3-diphenylglutaraldehyde (22) were reported without elemental analysis characterization. Therefore, it was important that the precursor to dialdehyde 13a should be fully characterized. Owing to the tendency of the cyclopentene double bond and the azido group to undergo a cycloaddition reaction, alkene lla was converted to epoxide 12a with m-chloroperbenzoic acid (MCPBA). Epoxide 12a was isolated as a colorless low-melting solid and was fully characterized. The 'H NMR spectrum of 12a revealed the presence of two stereoisomers in a ratio of about 2:l. Epoxidation of alcohol 3a under similar conditions was reported to produce the corresponding epoxide as a mixture of cis-trans isomers (14). Treatment of 12a with HI04 (23)gave dialdehyde 13a as a mixture of hydrated forms which behaved similarly to that of dialdehyde 7. The 'H NMR spectrum showed that the signals corresponding to 13a (6 2.67,3.021,4.386, 9.775) increased after the sample was allowed to stand as a solution in CDC13. High-resolution mass spectrum confirmed the molecular ion to be C13HgFqN304. The solubility of dialdehyde 13a in aqueous solution was found M) than that of dialdehyde 7 (0.17 to be lower (4.4 X M). Diester 1 lb was prepared from diol 3b and acyl chloride 10. Epoxidation of llb by MCPBA was carried out at 50 OC to give epoxide 12b,which was oxidized with HI04 to give dialdehyde 13b as a thick oil. The 'H NMR spectrum of 13b showed peaks at 6 2.960 and 9.759 for the CHzCHO moiety and 4.512 for the CHzOzC moiety, as well as signals around 6 1.5-2.0 and 5.0-6.0. This can be compared with the reported 1H NMR spectrum for 3,3-dimethylglutaraldehyde which showed 6 2.6 and 10.0 for the CHzCHO moiety as well as 6 5.1 and 1.7 for the hydrate forms (21). Acylation of alcohol 3c by 10 gave ester llc which was unstable in the pure form (cf. lla). Epoxidation of llc
42
Cai and Keana
Bioconlugate Chem., Vol. 2, No. 1, 1991
by MCPBA produced a stereoisomeric mixture of epoxides 12c which gave a satisfactory elemental analysis. Oxidative cleavage of epoxide 12c by HI04 gave a mixture containing dialdehyde 13c and various hydrated forms as observed from the 'H NMR spectrum. The signals corresponding to 13c increased after the sample was kept in CDCl3 for several days. The IR spectrum of a freshly prepared sample of 13c in CDC13 showed a single peak in the carbonyl region at 1735 cm-' corresponding to the carbonyl of the ester group, whereas after 3 days the IR spectrum of the solution showed a new peak a t 1726 cm-' corresponding to the aldehyde group and 1733 cm-l for the ester. The solubility of 13c in aqueous solution was found to be 0.010 M, a value significantly higher than that of 13a. Bis-PFPA-modified dialdehyde 13d was prepared similarly. Esterification of diol 3d by 10 gave diester lld, which was epoxidized by MCPBA to give 12d. Oxidation with HI04 then gave dialdehyde 13d, which showed spectral properties similar to those of 13c. The next series of experiments were designed to determine whether the new 3-substituted glutaraldehydes behaved similarly to glutaraldehyde under conditions related to EM fixation. Hardy et al. (8) reported that glutaraldehyde reacted with amines in aqueous solution to form polypyridinium type polymers which were characterized by a UV maximum at 265 nm. However, dialdehydes 7 and 13a-d all had strong absorption at 265 nm due to the diazo and azido groups, respectively, thus making it difficult to monitor their reaction with amines in aqueous solution by UV spectroscopy. Therefore dialdehyde 14 was synthesized from alcohol 3a and propionic anhydride followed by oxidation as a representative 3-substituted glutaraldehyde for our model studies. Parallel reactions of dialdehyde 14 and glutaraldehyde with 6-aminohexanoic acid (8) in aqueous buffer (pH = 7.4) were monitored at 265 nm (eq 3). The absorption
oHc3
