Synthesis and Self-Alkylation of Isotope-Coded Affinity Tag Reagents

Feb 23, 2005 - A pair of ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d8) and ...
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Bioconjugate Chem. 2005, 16, 458−464

458

Synthesis and Self-Alkylation of Isotope-Coded Affinity Tag Reagents Zhidong Zhang, Patrick J. Edwards, Roger W. Roeske, and Lili Guo* Department of Biochemistry and Molecular Biology, School of Medicine, Indiana University, Indianapolis, Indiana 46202. Received September 16, 2004

A pair of ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d8) and N-(13-iodoacetamido-4,7,10-trioxa-tridecanyl)biotinamide (8c, ICATd0), and an alternative pair of ICAT reagents, N-(10-iodoacetamido-2,5,5,6,6,9-hexadeutero-4,7-dioxadecanyl)biotinamide (8b, s-ICAT-d6) and N-(10-iodoacetamido-4,7-dioxa-decanyl)biotinamide (8a, s-ICAT-d0), were successfully synthesized. A mixture of sodium borohydride and cobalt(II) chloride reduced the intermediate dinitrile to the diamine without loss of the deuterium labels, which occurred when Raney nickel was the reducing agent. The problem caused by unsymmetrical biotinylation of the intermediate diamine was solved by using the solid-phase method in which one end of the diamine was attached to a chlorotrityl chloride resin, followed by biotinylation of the resin-bound amine. The self-alkylation of ICAT reagents that accounted for their instability and their limitations in the applications was also studied.

INTRODUCTION

Quantitative proteomics has become very important because of its potential to determine properties of biological systems that are not determined by DNA or mRNA sequence analysis alone, but related to protein expression, subcellular location, posttranslational modification, and interactions, as well as the rate of change of these properties under specific conditions. This information can aid in the discovery of diagnostic or prognostic protein markers, therapeutic targets in understanding the initiation and progression of disease states (1-4). One of the approaches in quantitative proteomics is based on stable isotope labeling of proteins or peptides and automated tandem mass spectrometry (MS/MS) (5, 6). The isotope-coded affinity tag (ICAT) method developed by Aebersold and co-workers (7) is a pioneering step in this field. The ICAT reagents have three elements: an affinity tag (biotin), a linker containing stable isotopes (either eight H atoms (d0) or eight deuterium atoms (d8)), and a reactive moiety to react with sulfhydryl groups of cysteines in proteins. The protein mixtures from two sets of cell states (or tissue extracts) are independently labeled with the d0 and d8 ICAT reagents; the samples are combined, and then proteolytically cleaved. The ICAT labeled peptides are isolated by affinity chromatography on an avidin column and analyzed by microcapillary liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Peptide sequence information is obtained by tandem mass spectrometry and computer searches of protein data banks. Quantification of proteins in two cell states is performed by comparing the intensity of the identical peptide peak pair from the two samples defined by the isotopic difference in the light and heavy reagent. Although the ICAT reagent has been widely used in quantitative proteomics (8-13), synthesis of the ICAT reagent was not adequately described in the original * To whom correspondence should be addressed. Phone: (317) 274-7507. Fax: (317) 274-4686. E-mail: [email protected].

papers (7, 14). Our first attempt to reproduce the published synthesis resulted in considerable loss of the deuterium labels. Another problem was biotinylation of the diamines, in which a large excess of ether diamine was required to avoid formation of bis-biotinylation. Recently, a seven-step alternative synthesis of ICAT-d8 was published by Perrin (15). Their orthogonal protection strategy overcame deuterium loss and allowed effective mono-biotinylation. We also like to report here our successful resolution of these problems to obtain the pair of pure ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)bio tinamide (Scheme 1, 8d, ICAT-d8) and N-(13-iodoacetamido4,7,10-trioxa-tridecanyl)biotinamide (Scheme 1, 8c, ICATd0), in reasonable yields. The synthetic scheme was also used to synthesize alternative ICAT reagents, N-(10iodoacetamido-2,5,5,6,6,9-hexadeutero-4,7-dioxa-decanyl)biotinamide (Scheme 1, 8b, s-ICAT-d6) and N-(10iodoacetamido-4,7-dioxa-decanyl)biotinamide (Scheme 1, 8a, s-ICAT-d0). We also studied the self-alkylation of ICAT reagents, which accounted for their instability and their limitations in the applications. EXPERIMENTAL PROCEDURES

General. Most of the reagents and solvents were obtained from Sigma-Aldrich (MO) or Fisher Scientific (NJ). 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIEA) were purchased from Applied Biosystems (Warrington, UK). 2-Chlorotrityl chloride resin was from Novabiochem (CA). Trifluoroacetic acid (TFA) was obtained from Halocarbon (NJ) and distilled before use. Triisopropylsilane (TIS) was obtained from Sigma-Aldrich (MO). Analytical reverse-phase HPLC was performed on a Varian model 5000 liquid chromatograph with a UV-100 detector unless otherwise specified. A Vydac C18 column (218TP54, 4.6 × 250 mm) was used at a flow rate of 1 mL/min. Preparative reverse-phase HPLC was carried out on a Waters-600E HPLC system fitted with a 486

10.1021/bc049777y CCC: $30.25 © 2005 American Chemical Society Published on Web 02/23/2005

Technical Notes

Bioconjugate Chem., Vol. 16, No. 2, 2005 459

Scheme 1

tunable absorbance detector using a Vydac C18 column (218TP101550, 50 × 250 mm) at a flow rate of 15 mL/ min. The absorbance was recorded at 214 nm wavelength, and a solvent system of A, 0.1% TFA in water, and B, 70% acetonitrile in water containing 0.09% TFA, was used both for analytical and for preparative HPLC. Flash chromatography was performed on a 5 × 60 cm column packed with Baker silica gel (40 µm average particle

diameter) using 2% MeOH in CHCl3 as eluting solvent. Electrospray ionization mass spectra (ESI-MS) were obtained on a Finigan LCQ LC/MS system. TLC was carried out on Merck Kieselgel 60 precoated plates and visualized by iodine absorption. 1-Butanol/acetic acid/water/pyridine (4/1/2/1 vol) was used as development solvent. For study of self-alkylation of ICAT compounds, the solid samples of ICAT compounds were flushed with

460 Bioconjugate Chem., Vol. 16, No. 2, 2005

nitrogen and stored at -20 °C. The solution samples were made in a concentration of 1 mg/mL solvent mixture and stored at room temperature. The reaction of self-alkylation was monitored by HPLC and ESI-MS. HPLC analysis was conducted on a Varian Prostar 210 system fitted with a Prostar 320 UV/vis detector using a Vydac C18 218TP54 column at a flow rate of 1 mL/min. Solvent gradient was an isocratic 5% B for 5 min, then a linear gradient 5% to 60% B for 25 min. Synthesis of Ether Dinitriles. 2,2,3,3,11,11,12,12Octadeutero-4,7,10-trioxa-1,13-tridecanedinitrile (3d). To a stirred solution of di(ethylene glycol-d2) (1d) (2.54 mL, 26.7 mmol) in benzene (3 mL) was added sodium deuterioxide (4 drops of 40% solution in D2O), followed by dropwise addition of a solution of acrylonitrile-d3 (2b) (3.52 mL, 53.5 mmol) in benzene (3 mL) at room temperature over 20 min. After being stirred for 1 h, the reaction mixture was filtered and the solvent was removed by rotary evaporation. The residue was purified by flash chromatography, and a clear oil (4.85 g) was obtained in 82% yield. TLC: only one spot, Rf ) 0.65. ESI-MS m/z ) 221.1 [M + H]+ (calc. 221.2). Ether dinitriles 3a, 3b, and 3c were prepared as 3d from the appropriate glycol and acrylonitrile. The crude products were obtained in 96% or higher yields, and TLC showed only one spot with Rf at 0.65. Reduction of Ether Dinitriles. Synthesis of 2,2,3,3,11,11,12,12-Octadeutero-4,7,10-trioxa-1,13-tridecanediamine (4d) with NaBH4-Raney Nickel Catalyst. To a stirred solution of 3d (4.8 g, 21.8 mmol) in MeOH (20 mL) was added Raney 2800 nickel (8.5 g) followed by dropwise addition of a solution of NaBH4 (1.76 g 46.5 mmol) in 8 M NaOH (40 mL) over 2.5 h. The reaction temperature was kept between 15 and 25 °C with a water bath, and the reaction was stopped 5 min after the H2 bubbles ceased. The catalyst was filtered off and washed with MeOH (50 mL × 2). Washing and mother liquor were combined and concentrated in a vacuum. Solid KOH (19 g) was added to the residue, and the diamine product was salted out of solution as a light yellow oil. Crude product was separated and purified by vacuum distillation. Diamine (4d) (2.61 g, yield 52%) was collected at 150-152 °C/0.75 mmHg. TLC Rf ) 0.22. ESI-MS m/z ) 227.3 (main peak) [M + H]+ (calc. 229.2). 4,7,10-Trioxa-1,13-tridecanediamine 4c was prepared as for 4d using 3c as starting ether dinitrile and NaBH4Raney nickel as reducing reagent. Crude product was obtained in 79% yield. TLC Rf ) 0.22. ESI-MS m/z ) 221.2 [M + H]+ (calc. 221.2). Synthesis of 2,2,3,3,11,11,12,12-Octadeutero-4,7,10-trioxa-1,13-tridecanediamine (4d) with NaBH4-CoCl2‚6H2O Catalyst. To a stirred mixture containing 3d (1.32 g, 6 mmol), CoCl2‚6H2O (5.71 g, 24 mmol), and MeOH (60 mL) was added NaBH4 (4.54 g, 120 mmol) in portions at room temperature over 15 min. The mixture was allowed to stir for 1 h. 6 N hydrochloric acid (15 mL) was added to dissolve the black precipitate formed (Co2B), and then the mixture was made basic with 8 N NaOH (8 mL). The Co(OH)2 precipitate and MeOH were removed by filtration and rotary evaporation, respectively. To the residue was added solid KOH (16 g), and the slurry was extracted with ether (40 mL × 4). The combined extracts were dried over Na2SO4 and evaporated to remove ether. A light yellow oil (1.0 g, 73%) was obtained. TLC Rf ) 0.22. ESIMS m/z ) 229.3 [M + H]+ (calc. 229.2). There were two impurity peaks, m/z ) 212.3 and 440.6 [M + H]+, which corresponded to the cyclic amine O(CH2CH2OCD2CD2CH2)2NH (calc. 212.2), and the dimer of ether diamine

Technical Notes

4d [H2NCH2CD2CD2O(CH2CH2O)2CD2CD2CH2]2NH (calc. 440.5). The crude product was used for the next step of synthesis without vacuum distillation because the sample was too small to distill. Ether diamines 4c, 4a, and 4b were prepared by the same procedure as 4d using NaBH4-CoCl2‚6H2O catalyst in yields 61-65%. TLC showed all three diamines had similar Rf values (0.22). ESI-MS m/z: for 4c, 221.2 [M + H]+ (calc. 221.2); for 4a, 177.2 [M + H]+ (calc. 177.2); for 4b, 182.8 [M + H]+ (calc. 183.2). ESI-MS also showed two impurities in the reduction products, which were cyclic amines and dimers of the ether diamine. Solid-Phase Synthesis of N-(Amino-oxa-alkyl)biotinamide. N-(13-Amino-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxatridecanyl)biotinamide (7d). A solution of 4d (0.99 g, 4.3 mmol) in 4 mL of CH2Cl2 was added to 254 mg, 0.43 mmol of 2-chlorotrityl chloride resin in a 30 mL reaction vessel having a glass frit (Peptides International, Inc., KY). The vessel was flushed with N2 and then shaken at room temperature for 2 h. The reaction mixture was filtered, and the diamine-loaded resin was washed with CH2Cl2, MeOH, 20%DIEA in NMP, and NMP (15 mL × 4, each). The filtrate and CH2Cl2 washings were collected and treated with solid KOH followed by rotary evaporation to recover the unreacted 4d. To the washed resin were added 2 M DIEA in NMP (1.29 mL, 2.58 mmol), 0.45 M HBTU/HOBt in DMF (4.5 mL, 2 mmol), and biotin (0.525 g, 2.15 mmol) in a mixed solvent of DMSO and NMP (vol ratio 1:1, 12 mL). The reaction vessel was flushed with N2 and shaken at room temperature for 24 h, after which the Kaiser test was negative. The resin was separated by filtration and washed with a 1:1 (vol) solution of DMSO and NMP, NMP and CH2Cl2 (15 mL × 3, each). Compound 7d was cleaved from resin with 20 mL of a cocktail solution containing TFA, CH2Cl2, and TIS in a volume ratio of 16:4:1. The reaction mixture was flushed with N2 followed by shaking at room temperature for 2 h. The resin was removed by filtration, and the solvents were evaporated. The residue was extracted with ether (100 mL × 2), dissolved in H2O (50 mL), and lyophilized. The crude 7d was purified by preparative HPLC, using isocratic 5% solvent B for 30 min, then a linear gradient from 5% to 30% B in 330 min at a flow rate of 15 mL/min. After lyophilization, a colorless viscous liquid (57 mg) was obtained in 24% yield. HPLC showed only one peak at retention time (Rt) 18.65 min using isocratic 5% solvent B for 2.5 min, then a linear gradient from 5% to 30% B in 27.5 min at a flow rate of 1 mL/min. TLC Rf ) 0.36. ESI-MS m/z ) 455.4 [M + H]+ (calc. 455.3). N-(Amino-oxa-alkyl)biotinamides 7c, 7a, and 7b were also prepared as 7d with the above procedure using diamines 4c, 4a, and 4b as starting materials, respectively. For 7c: yield 18%, HPLC Rt ) 18.65 min, TLC Rf ) 0.38. ESI-MS m/z ) 447.4 [M + H]+ (calc. 447.3). For 7a: yield 22%, HPLC Rt )16.88 min (obtained on a 130A Separation System of Applied Biosystems using Vydac 218TP52 column; running isocratic 10% B for 16 min and then a linear gradient 10-70% B for 30 min at a flow rate of 0.2 mL/min). TLC Rf ) 0.37. ESI-MS m/z ) 403.3 [M + H]+ (calc. 403.2). For 7b: yield 16%, HPLC Rt ) 16.71 min (as done previously for 7a), TLC Rf ) 0.36. ESI-MS m/z ) 409.5 [M + H]+ (calc. 409.3). Iodoacetylation of N-(Amino-oxa-alkyl) Biotinamide. N-(13-Iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d8). To a solution of 7d (55 mg, 0.097 mmol) in DMF (1 mL) was added DIEA (37 µL, 0.213 mmol) and a solution of

Technical Notes

iodoacetic anhydride (37.7 mg, 0.107 mmol) in DMF (2 mL). The mixture was flushed with N2 and stirred at room temperature for 3 h. The solvent was removed by rotary evaporation. The crude product was purified by preparative HPLC using isocratic 5% B for 50 min, then a linear gradient of 5-50% solvent B in 340 min at a flow rate of 15 mL/min. A light yellow solid was collected in a yield of 63% after lyophilization of the peak fractions. Analytical HPLC showed only one peak with a Rt ) 15.55 min using isocratic 5% solvent B for 2.5 min, then a linear gradient of 5-60% solvent B for 27.5 min at a flow rate of 1 mL/min. TLC Rf ) 0.60. ESI-MS m/z ) 623.3 [M + H]+ (calc. 623.2). 8c (ICAT-d0), 8a (s-ICAT-d0), and 8b (s-ICAT-d6) were also prepared as 8d (ICAT-d8) with the above procedure using 7c, 7a, and 7b as starting materials, respectively. For 8c: yield 57%, HPLC Rt ) 15.61 min, TLC Rf ) 0.60, ESI-MS m/z ) 637.3 [M + Na]+ (calc. 637.2). For 8a: yield 55%, HPLC Rt ) 22.78 min (obtained on a 130A Separation System of Applied Biosystems using a Vydac 218TP52 column; running isocratic 10% B for 16 min and then a linear gradient 10-70% B for 30 min at a flow rate of 0.2 mL/min). TLC Rf ) 0.61, ESI-MS m/z ) 571.3 [M + H]+ (calc. 571.1). For 8b: yield 50%, HPLC Rt ) 22.56 min (as done previously for 8a), TLC Rf ) 0.61, ESI-MS m/z ) 599.4 [M + Na]+ (calc. 599.2). RESULTS AND DISCUSSION

Synthesis of ICAT Compounds. As shown in Scheme 1, two pairs of ICAT compounds were prepared: one is compounds 8c and 8d, the classic ICAT-d0 and ICAT-d8 (7); the ICAT-d8 was prepared from acrylonitrile-d3 and di(ethylene glycol-d2). Another pair is compounds 8a and 8b, termed s-ICAT-d0 and s-ICAT-d6 as they have a shorter linker than the classic one. The s-ICAT-d6 was prepared with nondeuterated acrylonitrile and fully deuterated ethylene glycol as starting materials. The preparation of s-ICAT-d0 and s-ICAT-d6 was intended to lower the cost of the procedure. The use of acrylonitrile instead of its fully deuterated analogue may reduce the cost of starting materials by about 5-fold. Synthesis of ICAT compounds began with the reaction of ethylene glycol or di(ethylene glycol) 1a, 1b, 1c, and 1d with acrylonitrile 2a and 2b to form ether dinitrile 3a, 3b, 3c, and 3d by Michael addition in benzene (16). After removal of solvent, the desired compounds were obtained in yield of 96% or higher and with good purity as shown on TLC (only one spot with Rf values around 0.65). These ether dinitriles were used in the next reaction without further purification. Reduction of deuterated ether dinitrile to diamine is one of the crucial reactions in the synthesis because it may involve loss of deuterium by exchange with hydrogen. Gerber et al. prepared ether diamine 4d by hydrogenation of ether dinitrile 3d with Raney Ni catalyst (14). They found that the product was obtained in very low yield and with considerable exchange of deuterium (17). We also tried a mixture of Raney Ni and NaBH4 to reduce 3d to 4d. The product was obtained in 53% yield after vacuum distillation. However, ESI-MS showed the main peak was diamine-d6 (m/z ) 227.3 [M + H]+); about onefourth of deuterium on the molecule was replaced by hydrogen during reduction. When the ratios of Ni and NaBH4 to dinitrile were increased by 50%, the main peak in ESI-MS was diamine-d4; the deuterium loss increased to 35%. These results of reduction and hydrogenation catalyzed by Raney Ni indicated that nickel may be responsible for deuterium loss in the reactions. Horner

Bioconjugate Chem., Vol. 16, No. 2, 2005 461

(18) investigated the H-D exchange process on Raney nickel in toluene. When Raney nickel was shaken for 16 h with a solution of toluene in cyclohexane under a deuterium atmosphere, hydrogen atoms both in the aromatic ring and in the methyl group of toluene could be exchanged by deuterium. To overcome the problem, we tried several non-nickelcatalyzed reduction systems such as LiAlH4 (19), AlH3 (20), H3B‚S(CH3)2 (21), and NaBH4-CoCl2 (22). We found that the best result was obtained with NaBH4-CoCl2. Satoh et al. first reported that nitriles could be easily reduced to primary amines with sodium borohydridetransition metal salts (e.g., CoCl2) systems in MeOH (22). Ganem et al. studied the mechanism of NaBH4-CoCl2 reduction and concluded that the Cobalt boride formed in situ served as a true catalyst, strongly coordinating nitriles and activating them toward reduction by NaBH4 (23, 24). Dinitriles 3a, 3b, 3c, and 3d were reduced to diamines 4a, 4b, 4c, and 4d with NaBH4-CoCl2 in MeOH at room temperature. The reaction gave the desired products in 61-74% yields, and ESI-MS showed appropriate molecular weight with little loss of incorporated deuterium from deuterated diamines. Two impurities in the crude products were identified by ESI-MS as cyclic amines and dimers of diamine. These secondary amine byproducts are often seen in the reduction of nitriles when the reactions proceed stepwise (19, 25). The crude diamines were used in the next step of synthesis without distillation. Biotinylation of the symmetrical diamines is another crucial step in ICAT synthesis. Wilbur et al. prepared N-(13-amino-4,7,10-trioxatridecanyl)biotinamide, compound 7c, by acylation of ether diamine 4c with biotin tetrafluorophenyl ester in DMF in the presence of triethylamine (26). Aebersold et al. used this method to prepare its deuterated analogue 7d in their study of ICAT reagent (7). A large excess of ether diamine must be used in this method to minimize formation of bis-biotin derivative of diamine. The excess of unreacted diamine is difficult to recover from the reaction mixture and results in a significant loss of diamine. Recently, Patel and Perrin described a new method to prepare 7d (15). An orthogonally protected di(ethylene glycol)-d8 linker was first prepared, and the linker was then extended by successive introduction of protected propylamine fragments. A monoamine was obtained by chemoselective deprotection and coupled with biotin pentafluorophenyl ester in DMF in the presence of DIEA. This method overcame the problem associated with the bis-biotinylation of diamine. However, the process involved a large number of reaction steps. We wish to report a solid-phase biotinylation of diamine compounds. A 2-chlorotrityl chloride resin is loaded with 10 equiv of diamine relative to the resin in CH2Cl2. The reaction mixture was filtered, and the unreacted diamine was recovered by treatment of the filtrate with solid KOH to remove HCl followed by rotary evaporation to remove CH2Cl2. Because the loading reaction involved only resin, diamine, and CH2Cl2, the diamine could be recovered in quantitative yield. Biotinylation of the resin-bound amine was effected with 5 equiv of biotin relative to the resin in a solvent mixture of NMP, DMF, and DMSO in the presence of DIEA and HBTU/HOBt. The biotinylated amine was cleaved from resin with a cocktail containing TFA, CH2Cl2, and TIS in a volume ratio of 16:4:1. The crude product was purified by preparative HPLC. The biotin derivative of diamines 7a, 7b, 7c, and 7d were prepared by this

462 Bioconjugate Chem., Vol. 16, No. 2, 2005

Technical Notes

Scheme 2

Table 1. HPLC and ESI-MS Analysis of Aged ICAT Reagentsa ICAT HPLC ICAT-d8 ICAT-d0 s-ICAT-d6 s-ICAT-d0 a

dimeric sulfonium salt ESI-MS [M + H]+, m/z

cyclic sulfonium salt

ESI-MS [M + H]+, m/z

HPLC

ESI-MS [M + H]+, m/z

Rt, min

%

exp.

calc.

Rt, min

%

exp.

calc.

exp.

calc.

22.4 22.4 21.6 21.6

59.9 63.3 52.3 67.4

623.1 615.1 577.1 571.3

623.2 615.2 577.1 571.1

23.3 23.2 22.2 22.1

40.1 36.7 47.7 32.6

1117.3 1101.1 1025.1 1013.1

1117.5 1101.5 1025.3 1013.3

495.3 487.1 449.3 443.4

495.3 487.3 449.2 443.2

The samples were stored under a nitrogen atmosphere at -20 °C for 1 year.

method from 4a, 4b, 4c, and 4d, respectively, in overall yields of 16-24% for the three steps of solid-phase synthesis. The low yield was caused by use of crude diamine in the synthesis. Compound 7c was obtained in 43% yield if reagent diamine (Aldrich) was used. Analysis by HPLC, TLC, and ESI-MS showed the compounds to be quite pure and to have the correct molecular weight. Solid-phase synthesis overcame the problem of formation of bis-biotin derivative and difficult recovery of diamine and made the synthetic process simpler and easier. Iodoacetylation of mono-biotinylated amine, the last step of ICAT synthesis, was carried out with iodoacetic anhydride in DMF in the presence of DIEA (7). After purification by HPLC, compounds 8d (ICAT-d8), 8c (ICAT-d0), 8b (s-ICAT-d6), and 8a (s-ICAT-d0) were obtained from 7d, 7c, 7b, and 7a, respectively, in yields of 50-63%. ESI-MS gave appropriate molecular weights with little loss of incorporated deuterium. TLC and HPLC analysis showed good purity for 8d and 8c and a considerable amount of dimeric sulfonium salts in 8a and 8b, which were formed during HPLC purification. Self-Alkylation of ICAT Compounds. There are two functional groups in an ICAT molecule, dialkyl sulfide of biotin on one end and iodoalkyl of iodoacetamide on another end. Dialkyl sulfides are sufficiently nucleophilic to react with primary alkyl halides under mild conditions,

frequently simply by mixing the reagents at room temperature (27). Thus, ICAT compounds may undergo selfalkylation between the dialkyl sulfide and iodoalkyl group, to give dimeric sulfonium salt by intermolecular alkylation and cyclic sulfonium salt by intramolecular alkylation (Scheme 2). The self-alkylation of ICAT compounds results in a stability problem of the ICAT reagent. No dimeric or cyclic sulfonium salts were found by HPLC and ESI-MS in the crude product, most likely because the last step of ICAT synthesis was carried out in a nonaqueous medium and in the presence of a base (DIEA). Some dimeric sulfonium salts were formed during HPLC purification in a mixed solvent of acetonitrile and water with 0.1% TFA. The two s-ICAT compounds formed dimeric sulfonium salts more readily than the longer one, and it was difficult to obtain a pure product by HPLC in this solvent system. The undesired self-alkylation reaction proceeded not only in an aqueous solution but also in their solid state. Table 1 shows the analysis of ICAT samples which were stored at -20 °C for about 1 year. The HPLC and ESI-MS data demonstrated that about one-third to onehalf of ICAT compounds were converted into dimeric sulfonium salts and a tiny amount of cyclic sulfonium salts. The stability of ICAT compounds in different solvent systems was examined using s-ICAT-d0 as an example.

Bioconjugate Chem., Vol. 16, No. 2, 2005 463

Technical Notes Table 2. Effect of Different Solvent Systems on Stability of s-ICAT-d0

dimeric sulfonium salt %, HPLC days of storage at room temp

water 75% CH3CN 25% TFA 0.1%

water 75% CH3CN 25% NH4OAc 0.01 M

water 67% MeOH 33% NH4OAc 0.01 M

water 67% MeOH 33%

MeOH

0 4 14

32.1 39.3 57.4

32.4 37.9 54.9

34.3 36.7 46.3

33.1 36.5 47.9

32.6 20.0 11.5

Table 3. Regeneration of ICAT Compounds from Their Dimeric Sulfonium Salts (DSS) in MeOH composition %, HPLC

days of storage at room temp

ICAT

DSS

ICAT

DSS

s-ICAT-d6 ICAT DSS

s-ICAT-d0 ICAT DSS

0 5 11 19 34 70

59.9 78.0 83.4 85.4 88.8 93.0

40.1 22.0 16.6 14.6 11.2 7.0

63.3 86.1 89.1 91.5 93.7 95.6

36.7 13.9 10.9 8.5 6.3 4.4

52.3 69.0 75.3 78.8 85.5 89.6

67.4 83.2 87.2 90.7 92.9 94.4

ICAT-d8

ICAT-d0

The results are in Table 2. The content of dimeric sulfonium salt was increased by 12.0-25.3% in all of the solvent systems containing water during 2 weeks of storage at room temperature. Neither the acidic buffer nor the neutral buffer showed any significant effect on reaction rates of self-alkylation in the mixed solvents used. The most interesting observation was the behavior of s-ICAT-d0 in pure MeOH. Instead of self-alkylation, its dimeric sulfoniun salt was dealkylated into its monomer form at a quite rapid rate. The content of dimeric sulfonium salt was decreased from 32.6% to 11.5% in 2 weeks of storage in MeOH. This fact demonstrates that the self-alkylation reaction of ICAT compounds is reversible; the sulfonium salt can be dealkylated by the iodide ion, leading to the regeneration of ICAT compounds. Both self-alkylation of ICAT compounds and dealkylation of its sulfonium salt are nucleophilic substitution reactions. The solvent that is used in a nucleophilic substitution reaction can have a profound effect on the rate of the reaction (28). In reactions in which product formation is favored by charge development in the transition state, solvents of high dielectric constant favor the process; conversely, in reactions in which reduction or dispersal of charge in the transition state favors product formation, solvents of low dielectric constant will favor the process. Thus, self-alkylation of ICAT compounds is greatly accelerated by solvents of high dielectric constant because charge is developed in the transition state and product; the dealkylation of ICAT sulfonium salts involving negatively charged nucleophiles, on the other hand, takes place more rapidly in solvents of low dielectric constant. Water has a much higher dielectric constant than MeOH (78 vs 33). Therefore, the equilibrium of the reversible reaction of ICAT self-alkylation will shift in favor of formation of the sulfonium salt in the solvent systems containing water and shift in favor of dealkylation in MeOH. The conversion of ICAT sulfonium salts into their monomer forms in MeOH was further demonstrated by all four ICAT compounds studied (Table 3). The contents of dimeric sulfonium salt in the aged ICAT samples were reduced from 32.6% to 47.7% to about 5-10% in 70 days of storage in MeOH at room temperature. The regeneration treatment by MeOH or other solvents of low dielectric constant may provide a useful method for purification of ICAT compounds contaminated by their sulfonium salts.

47.7 31.0 24.7 21.2 14.5 10.4

32.6 16.8 12.8 9.3 7.1 5.6

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