Efficient Synthesis of Isotopically Pure Isotope-Coded Affinity

To permit a global characterization of the proteome with more rapid and ... The problems associated with the reported synthesis of “heavy” ICAT ...
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Bioconjugate Chem. 2004, 15, 224−230

TECHNICAL NOTES Efficient Synthesis of Isotopically Pure Isotope-Coded Affinity Tagging Reagents Anupama Patel and David M. Perrin* Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver, B. C. V6T 1Z1 Canada, and PENCE Inc. (Protein Engineering Network of Centers of Excellence), 750 Heritage Medical Research Center, Edmonton, Alberta T6G-2S2 Canada. Received June 3, 2003; Revised Manuscript Received July 23, 2003

Synthesis of an isotopically pure d8-ICAT linker, N-[(5,5,6,6,8,8,9,9-2H)-13-biotinamido-4,7,10-trioxatridecanyl] tert-butyloxy carbamide (12), has been achieved in seven steps with an overall yield of 33%. Conjugation of exchange-inert d4-starting materials by classic etherification reaction yielded a pure synthon, carrying eight deuteriums that remained exchange-inert throughout subsequent reactions. This modified synthesis constitutes a significant improvement to the reported syntheses of “heavy” ICAT reagent in terms of expense, yield, and isotopic retention. This synthesis is easily adapted to incorporate additional deuterium atoms and is equally applicable for incorporation of either 13C and/or 18O. In addition, this synthesis allows for the introduction of different orthogonal functionalities and provides for a high yielding series of differentially encoded ICAT tags.

INTRODUCTION

Proteomics is an exciting field in the study of proteins and particularly the study of complex systems involving multiple proteins where each may exert a subtle effect that synergistically gives rise to an identifiable global cellular or physiological state or process. The multiplicity of proteins and effectors increases the difficulty of assigning the cellular and physiological functions to individual proteins as well as complicates the process of drug discovery and target validation. In this growing research field of proteomics, various new approaches have become available to facilitate the identification of many of the proteins in a cell and to generate data that more closely correlate with protein activity levels. However, compared to genomics, proteomics studies face formidable technical challenges due to the structural complexity, the temporal nature of regulation, function, and abundance as well as vast structural variation of different proteins in a cell. To permit a global characterization of the proteome with more rapid and more detailed interrogation, various new strategies are being developed in the research field of proteomics (1-8). In this context, Aebersold described a creative alternative method dubbed isotope-coded affinity tagging (ICAT)1 methodology (9). Instead of relying on 2D PAGE and protein-staining methods for quantification, the ICAT method involves utilization of isotopically labeled chemical probes, (ICAT, Figure 1) (9) for the quantitative comparison of protein abundances between complex proteomes. Ultimately, this analysis is achieved by some form of mass spectrometry that is being interfaced with robotics and automated sample analysis so as to streamline the discovery process and eliminate human error. * To whom correspondence should be addressed. Phone: 001 604 822 0567; Fax: 001 604 822 2847; E-mail: dperrin@ chem.ubc.ca.

Figure 1. Cysteine-speciic ICAT-reagents for quantitative proteomics studies.9

Conceptually, the ICAT reagent is a trifunctional molecule, composed of (a) a specific reactive group capable of reacting covalently with an active site nucleophile (e.g. iodoacetamide for covalent attachment to a cysteinyl thiol); (b) an isotopically coded linker that occurs in a deuterated (isotopically “heavy”, X ) D) or nondeuterated (isotopically “light”, X ) H) form and provides the basis for accurate quantification; and (c) a biotin group that enables the isolation of the tagged peptides from complex peptide mixtures via avidin affinity chromatography. This approach makes use of the principle of isotope dilution for analysis. For the quantitative comparison between two proteomes in two different states (e.g. normal and disease state), the ‘light’ (nondeuterated) ICAT reagent is used to covalently tag proteins of one proteome and the ‘heavy’ (deuterated) ICAT reagent is used to covalently tag proteins of a second proteome. The two proteomes are then combined for quantitative analysis. Analysis often involves further steps such as digestion of the mixture with a protease (e.g. trypsin), isolation of the ICAT-labeled peptides by affinity purification on avidin, and analysis of the isolated labeled peptide mixture by liquid chromatography-mass spectrometry (LC/MS). Prior mixing of proteins that have been labeled with either light or heavy ICAT reagents eliminates much of the potential experimental error associated with postlabeling analysis. Since the two different physiologi-

10.1021/bc0300293 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/30/2003

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cal states are correlated with differential labeling, proteins that are present at very different levels in the two cell states will yield a pair of signals of unequal intensity in the LC-MS (each signal pair differing in mass by the number of deuterium atoms incorporated). Searching for peptide pairs of unequal intensity, followed by tandem MS sequencing, identifies proteins whose levels change as a consequence of the disease. Although many reports have highlighted the utility of the ICAT methodology for proteomic profiling (9-16), few have discussed the synthetic methodologies for generating ‘heavy’ ICAT reagents with respect to the isotopic purity and the cost of the synthesis. As initially reported for the synthesis of the ‘heavy’ ICAT reagent (9, 17), the d8-diamine linker was synthesized in two steps: first by Michael addition of diethylene glycol-d2 (i.e. O(CH2CH2OD)2) on the expensive d3-acrylonitrile, in the presence of catalytic NaOD solution in D2O, and then subsequent nitrile reduction using Raney nickel, providing an octadeuterodiamine linker. Reacting an excess molar equivalent of the d8-diamine linker with biotin-pentafluorophenyl ester (18) afforded the biotinylated d8- linkermonoamine, which was then treated with iodoacetic anhydride to give rise to the “heavy” ICAT reagent (Figure 1, X ) D). The problems associated with the reported synthesis of “heavy” ICAT reagent (9, 17) are as follows. (a) The Michael addition reaction results in significant (about 10%) loss of deuterium due to exchange with protium at the carbon R to the nitrile group. Consequently, d7 and d6 derivatives contaminate the desired d8-linker. This gives rise to an inseparable mixture of octa-, hepta-, and hexadeuterodinitrile linkers. (b) The subsequent nitrile reduction to amine groups using Raney nickel results in an increase in the percentage of d7 and d6 derivatives due to considerable exchange of the deuteriums with protiums at the R-carbon to the nitrile group. In addition, the yield in this step is very low due to a notable retroMichael type reaction (12). (c) The subsequent amide coupling reaction with biotin pentafluorophenyl ester necessarily requires a large molar excess of d8-diamine linker (9, 12) to ensure mono-biotinylation and avoid bisbiotinylation. The excess of unreacted diamine, being polar, is difficult to recover from the reaction mixture and results in a significant loss of the diamine. Recently Gelb and co-workers addressed these concerns, as they have described a different reaction strategy that improved the yield of the d8-diamine linker with respect to deuterium exchange (12). However, their process involves a larger number of steps, still requires the use of d3-acrylonitrile, and moreover does not address the problem of the loss of the diamine in the amide coupling reaction with biotin or other capture ligands. Addressing this problem not only applies to the deuterium-labeled approach, but to other, more recent approaches involving 13C-labeled linkers. EXPERIMENTAL SECTION

General. Methods And Materials. Flash chromatography was carried out using silica gel 60, 230-400 mesh, supplied by E. Merck Co. TLC was performed on silica gel 60 F254 on aluminum sheets (E. Merck, type 5554), and detection was carried out by staining with 8% ammonium molybdate tetrahydrate (Fluka) in 2 M sulfuric acid solution in methanol, and under UV light and by iodine adsorption when applicable. Nuclear magnetic resonance spectra (1H and 13C) were recorded at 298 K, at 400 MHz (for 1H) and 100.67 MHz for (13C

NMR) using a Bruker AV-400 instrument. Chemical shifts are given in ppm values relative to internal standard TMS (0.00 ppm). Solvent, signal multiplicity, coupling constants (in Hz), and integration ratios are indicated in the experimental procedures. Two-dimensional 1H-1H and 1H-13C COSY (HMQC) experiments were performed for complete signal assignments wherever necessary. ESI MS spectra were obtained on a Bruker Esquire-LC instrument. HRMS spectra were obtained for final compound 12 in the mass spectrometry laboratory of the Chemistry Department, UBC. Palladium hydroxide (20 wt. %), Raney nickel, n-Bu4NBr, formic acid, anhydrous DMF (99.8%), and DIPEA were purchased from Aldrich, and biotin from Sigma. Toluene was dried over Na metal, DIPEA was distilled over CaH2, and anhydrous DMF was stored on activated molecular sieves prior to use. Tetradeuterated precursors 1 (19) and 2 (20) were synthesized from the commercially available (Aldrich) and inexpensive compounds ethylene-d4 glycol and 2-bromoethanol-1,1,2,2-d4 according to a known literature procedure. Also following a literature procedure, compound 5 (22) from 3-chloropropylamine hydrochloride (Aldrich) and biotin pentaflourophenyl ester (18) from biotin have been prepared. As described in the following experimental procedures, all the compounds were easily purified by flash chromatographic methods, and structure of each compound has been characterized and confirmed by NMR and mass spectrometry. Synthesis. (1,1,2,2,4,4,5,5-2H)-5-Benzyloxy-1-tetrahydropyranyloxy-3-oxapentane (3). (1,1,2,2-2H)-2Benzyloxyethylene glycol (1) (1.1 g, 7.05 mmol) was suspended in NaOH solution (20 mL, 1 g/mL in H2O) containing n-Bu4NBr (0.1 g, 0.7 mmol) and heated at 80 °C for 30 min. To the hot reaction mixture (1,1,2,2-2H)1-tetrahydropyranyloxy-2-bromoethanol (2) (1.8 g, 8.46 mmol) was added, and the reaction mixture was further stirred at 80 °C for 3 h. Then the reaction mixture was cooled to room temperature and diluted with water (10 mL). The crude product was extracted with diethyl ether (2 × 15 mL) and ethyl acetate (10 mL). The organic layer was successively washed with water and brine and then dried over anhydrous Na2SO4. After filtration, the solvent was concentrated in vacuo, and the crude product obtained was purified by flash chromatography (petroleum ether-ethyl acetate, 6:1) to afford the title compound (3, 1.9 g, 6.6 mmol, 94%) as a colorless syrup. TLC (petroleum ether-ethyl acetate, 4:1): Rf ) 0.35 (UV, blue stain in ammonium molybdate). 1H NMR (400 MHz, CDCl3): δ ) 7.35-7.20 (m, 5H, 5 aryl-H), 4.61 (mc, 1H, OCHO), 4.55 (s, 2H, OCH2Ph), 3.85 (mc, 1H, OCHa), 3.47 (mc, 1H, OCHb), 1.91-1.40 (m, 6H, 3CH2) ppm; 13C NMR (100.61 MHz, CDCl3): δ ) 138.92 (aryl t-C), 128.97, 128.89, 128.26, 128.10 (5 aryl-C), 99.47 (OCHO), 73.74 (OCH2Ph), 70.29, 69.41, 66.43 (3 weak multiplets, 4 OCD2), 62.75 (OCH2), 31.14, 26.0, 20.04 (3 CH2) ppm. ESI-MS: m/z ) 311.21 [M + Na]+, 327.21 [M + K]+ observed for C16H16D8O4 (288.22). (1,1,2,2,4,4,5,5-2H)-5-Benzyloxy-3-oxapentan-1-ol (4). A solution of 3 (1.8 g, 6.25 mmol) in MeOH (10 mL) was treated with HCl solution (10% in MeOH, 0.5 mL) at 0 °C for 1 h. The acidic reaction mixture was neutralized with aqueous NaHCO3 solution and concentrated in vacuo. The crude product was purified by flash chromatography (petroleum ether-ethyl acetate, 7:3) to afford the alcohol derivative, (4, 1.15 g, 5.63 mmol, 90%) as colorless syrup. TLC (petroleum ether-ethyl acetate, 2:1): Rf ) 0.1 (UV, blue stain in ammonium molybdate). 1 H NMR (400 MHz, CDCl3): δ ) 7.35-7.20 (m, 5H, 5 aryl-H), 4.54 (s, 2H, OCH2Ph), 2.35 (bs, 1H, OH) ppm;

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NMR (100.61 MHz, CDCl3): δ ) 137.98 (aryl t-C), 128.39, 127.74 (5 aryl-C), 73.23 (OCH2Ph), 69.79, 68.28, 60.95 (3 weak multiplets, 4 OCD2) ppm. ESI-MS: m/z ) 205.18 [M + H]+, 227.20 [M + Na]+ observed for C11H8D8O3 (204.16). N-[(5,5,6,6,8,8,9,9-2H)-9-Benzyloxy-4,7-dioxanonanyl]-tert-butyloxycarbamide (6). Following the similar experimental procedure as in case of compound 3, the title compound 6 was prepared by reacting 4 (1.01 g, 4.95 mmol) and N-(3-chloropropyl)-tert-butyloxycarbamide (5) (1.92 g, 9.92 mmol) in NaOH solution (10 mL, 1 g/mL in H2O) containing n-Bu4NBr (0.1 g, 0.7 mmol) at 80 °C for 1 h. The crude product obtained was purified by flash chromatography (petroleum ether-ethyl acetate, 4:1) to afford the corresponding conjugate (6, 1.31 g, 3.62 mmol, 73%) as colorless syrup. TLC (petroleum ether-ethyl acetate, 2:1): Rf ) 0.3 (UV, blue stain in ammonium molybdate). 1H NMR (400 MHz, CDCl3): δ ) 7.36-7.20 (m, 5H, 5 aryl-H), 4.92 (bs, 1H, NHBoc), 4.55 (s, 2H, OCH2Ph), 3.52 (t, J ) 5.87 Hz, 2H, OCH2), 3.20 (mc, 2H, CH2NHBoc), 1.72 (m, 2H, OCH2CH2), 1.41 (s, 9H, t-Bu) ppm; 13C NMR (100.61 MHz, CDCl3): δ ) 156.32 (CONH), 138.56 (aryl t-C), 128.63, 128.0, 127.86 (5 aryl-C), 79.17 (C(CH3)), 73.46 (OCH2Ph), 69.81 (OCH2, weak multiplet for 4 OCD2), 38.90 (CH2NHBoc), 29.92 (OCH2CH2), 28.73 (C(CH3)) ppm. ESI-MS: m/z ) 362.29 [M + H]+, 384.3 [M + Na]+, 400.3 [M + K]+ observed for C19H23D8NO5 (361.27). N-[(5,5,6,6,8,8,9,9-2H)-9-Hydroxy-4,7-dioxanonanyl]tert-butyloxycarbamide (7). A mixture of 6 (1.14 g, 3.15 mmol) and Pd(OH)2 (20% on charcoal, 50 mg) in ethyl acetate (10 mL) was hydrogenated under atmospheric pressure for 3 h at RT. Then the reaction mixture was filtered through a thin Celite bed, and the residue was washed with MeOH (10 mL). The filtrate was concentrated in vacuo, and the residue was subjected to flash chromatographic purification on silica gel using neat ethyl acetate as the eluent, yielding the alcohol derivative (7, 0.74 g, 2.73 mmol, 87%) as a colorless syrup. For flash column chromatographic purification, neat ethyl acetate was needed to obtain the desired product 7 in pure form. The increased polarity of 7 may be accounted to the free hydroxyl group resulting from the hydrogenolysis. TLC (petroleum ether-ethyl acetate, 1:1): Rf ) 0.5 (white patch in ammonium molybdate). 1H NMR (400 MHz, CDCl ): δ ) 5.10 (bs, 1H, NHBoc), 3 3.52 (t, J ) 5.86 Hz, 2H, OCH2), 3.20 (t, J ) 6.37 Hz, 2H, CH2NHBoc), 2.78 (bs, 1H, OH), 1.71 (m, 2H, OCH2CH2), 1.41 (s, 9H, t-Bu) ppm; 13C NMR (100.61 MHz, CDCl3): δ ) 156.30 (CONH), 79.31 (C(CH3)), 69.73 (OCH2, weak multiplet for 3 OCD2), 61.18 (weak multiplet, CD2OH), 38.71 (CH2NHBoc), 29.90 (OCH2CH2), 28.69 (C(CH3)) ppm. ESI-MS: m/z ) 294.3 [M + Na]+, 310.25 [M + K]+ observed for C12H17D8NO5 (271.22). N-[(5,5,6,6,8,8,9,9- 2H)-12-Cyano-4,7,10-trioxadidecanyl]-tert-butyloxycarbamide (10). To a suspension of 7 (0.67 g, 2.47 mmol) and solid NaOH (10 mg, 0.24 mmol) in anhydrous toluene (10 mL) was added acrylonitrile (0.21 mL, 3.21 mmpl) under nitrogen atmosphere, and the reaction mixture was stirred for 3 h at RT in the dark. Then the reaction mixture was diluted with ethyl acetate and successively washed with water (5 mL) and brine. After drying over anhydrous Na2SO4, the organic layer was filtered and concentrated in vacuo. Purification of the crude product by flash chromatography (petroleum ether-ethyl acetate, 1:1) afforded the nitrile adduct (10, 0.77 g, 2.37 mmol, 96%) as colorless syrup. TLC (petroleum ether-ethyl acetate, 1:1): Rf ) 0.1 (iodine, white patch in ammonium molybdate). 1H 13C

NMR (400 MHz, CDCl3): δ ) 4.90 (bs, 1H, NHBoc), 3.70 (t, J ) 6.44 Hz, 2H, OCH2), 3.52 (t, J ) 5.97 Hz, 2H, OCH2), 3.18 (mc, 2H, CH2NHBoc), 2.58 (t, J ) 6.45 Hz, 2H, CH2CN), 1.73 (m, 2H, OCH2CH2), 1.41 (s, 9H, t-Bu) ppm; 13C NMR (100.61 MHz, CDCl3): δ ) 155.97 (CONH), 117.79 (CN), 78.93 (C(CH3)), 69.46 (OCH2, weak multiplet for 4 OCD2), 65.86 (OCH2), 38.68 (CH2NHBoc), 29.63 (OCH2CH2), 28.41 (C(CH3)), 18.84 (CH2CN) ppm. ESIMS: m/z ) 325.26 [M + H]+, 347.31 [M + Na]+, 363.31 [M + K]+ observed for C15H20D8N2O5 (324.25). N-[(5,5,6,6,8,8,9,9-2H)-13-Amino-4,7,10-trioxatridecanyl]-tert-butyloxycarbamide (11). A mixture of 10 (0.58 g, 1.79 mmol) and Raney nickel (30 mg) in MeOH (8 mL) was hydrogenated under atmospheric pressure for 12 h at RT. Then the reaction mixture was filtered through a thin Celite bed, and the filtrate was concentrated in vacuo which afforded the pure amine derivative 11 (0.6 g) as a colorless oil, in quantitative yield. After filtration of the catalyst, compound 11 was obtained in pure form without any further need for chromatographic purification. TLC (CH2Cl2-MeOH, 9:1): Rf ) 0.1 (iodine, white patch in ammonium molybdate). 1H NMR (400 MHz, d4-MeOH): δ ) 3.51 (t, J ) 6.1 Hz, 2H, OCH2), 3.46 (t, J ) 6.2 Hz, 2H, OCH2), 3.08 (t, J ) 6.76 Hz, 2H, CH2NHBoc), 2.52 (t, J ) 6.82 Hz, 2H, CH2NH2), 1.781.60 (m, 4H, 2 OCH2CH2), 1.38 (s, 9H, t-Bu) ppm; 13C NMR (100.61 MHz, d4-MeOH): δ ) 158.45 (CONH), 79.84 (C(CH3)), 70.36 (OCH2, weak multiplet for 4 OCD2), 69.81 (OCH2), 40.10 (CH2NHBoc), 38.71 (CH2NH2), 33.55, 30.92 (2 OCH2CH2), 28.80 (C(CH3)) ppm. ESI-MS: m/z ) 329.32 [M + H]+, 351.33 [M + Na]+ observed for C15H24D8N2O5 (328.28). N-[(5,5,6,6,8,8,9,9-2H)-13-Biotinamido-4,7,10-trioxatridecanyl]-tert-butyloxycarbamide (12). To a solution of 11 (obtained from the previous reaction without further purification, 0.6 g) in anhydrous DMF (5 mL) was added a solution of the biotin pentaflourophenyl ester (1.12 g, 2.74 mmol) in anhydrous DMF (5 mL) followed by DIPEA (0.3 mL, 1.61 mmol) addition under nitrogen atmosphere. Then the reaction mixture was stirred at RT for 12 h. The reaction mixture was concentrated in vacuo, and the crude product was purified by flash chromatography (CH2Cl2-MeOH, 19:1) using neutral silica gel that afforded the pure biotinylated derivative (12, 0.64 g, 1.15 mmol, 64% overall yield for two steps) in the form of a white solid (hygroscopic). Compound 12, being polar due to the presence of amide linkages, could not be purified on commercially available silica gel because of its slightly acidic nature. However, performing the flash column chromatography on neutral silica gel, compound 12 was obtained in pure form with out any loss. To neutralize the commercially available silica gel (230-400 mesh), it was loaded onto a column and washed with CH2Cl2 (300 mL), containing 0.1% triethylamine, prior to loading of the crude reaction mixture onto the same column. Then a solvent mixture of CH2Cl2 and MeOH in the ratio 19 to 1, without containing any triethylamine, was used for the chromatographic purification of 12. TLC (CH2Cl2-MeOH, 9:1): Rf ) 0.6 (iodine, blue stain in ammonium molybdate). 1 H NMR (400 MHz, d4-MeOH): δ ) 4.45 (mc, 1H, CH2CHNH), 4.26 (dd, J1 ) 4.47 Hz, J2 ) 7.87 Hz, 1H, CHCHNH), 3.51-3.42 (m, 4H, 2 OCH2), 3.20 (t, J ) 6.8 Hz, 2H, CH2CONH), 3.15 (mc, 1H, SCH), 3.06 (t, J ) 6.76 Hz, 2H, CH2NHBoc), 2.88 (dd, J1 ) 4.99 Hz, J2 ) 12.74 Hz, 1H, SCHa), 2.66 (d, J ) 12.72 Hz, 1H, SCHb), 2.16 (t, J ) 7.35 Hz, 2H, CH2NHCO), 1.77-1.50 (m, 6H, OCH2CH2, 2 CH2), 1.46-1.30 (m, 2H, CH2), 1.40 (s, 9H, t-Bu) ppm; 13C NMR (100.61 MHz, d4-MeOH): δ ) 175.93

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(CH2NHCOCH2), 166.07 (NHCONH), 158.44 (NHCO2tBu), 79.85 (C(CH3)), 69.88 (OCH2, weak multiplet for 4 OCD2), 69.82 (OCH2), 63.37, 61.62 (2 CH), 56.99 (SCH), 41.03 (SCH2), 38.73 (CH2NHBoc), 37.83 (CH2CONH), 36.85 (CH2NHCO), 30.94, 30.43, 29.80, 29.50, 28.81 (5 CH2), 26.87 (C(CH3)) ppm. ESI-MS: m/z ) 555.37 [M + H]+, 577.39 [M + Na]+, 593.38 [M + K]+ observed for C25H38D8N4O7S (554.36); HRMS: found 555.3668 [(M + H)+], C25H39D8N4O7S requires 555.3659. RESULTS AND DISCUSSION

To overcome all the problems associated with the synthesis of heavy-ICAT linker as discussed in the Introduction, as well as to expand the chemical functionality that may be introduced into ICAT reagents, here we describe an efficient synthesis of the “heavy” ICAT reagent. We contend that the synthesis reported herein is less expensive, facile, and high yielding with no loss of incorporated deuteriums throughout all the subsequent reaction conditions. The synthesis began with the conjugation of the two tetradeutero-precursors 1 (19) and 2 (20), by classical etherification reaction (21) at 80 °C using aqueous NaOH solution and phase-transfer catalyst (n-Bu4NBr), that gave rise to the orthogonally protected d8-diethylene glycol (3) in 94% yield with no loss of deuterium. Along with 1H NMR characterization, further evidence for the structure of 3 was obtained by ESI MS that showed signals at m/z ) 311.21 and 327.21 for [M + Na]+ and [M + K]+, respectively. In the 13C NMR spectrum of 3 instead of singlets, four multiplets with lower intensity have been observed for the four OCD2 groups arising due to the lack of 1H broad-band decoupling experiment. Unlike the reported literature method involving simultaneous introduction (9), this orthogonally protected glycol linker (3) provides for successive introduction of propylamine fragments by chemoselective deprotection of THP and benzyl groups. Thus, chemically differentiable functionalities, including at least one amine, could be further achieved for successive introduction of biotin and a chosen reactive group without the enormous loss of the precious d8-diamine linker. The THP group was hydrolyzed in the presence of the acid-stable benzyl group using mild acidic reaction conditions (methanolic HCl solution, 10%) to give the alcohol

derivative 4 in 90% yield (Scheme 1). Another etherification reaction between 4 and N-(3-chloropropyl)-tertbutyloxycarbamide (5) (22) under similar reaction conditions as used for 3, provided the Boc derivative 6 in 73% yield, thus installing a propylamine fragment on one end of the linker. In the 1H and 13C NMR spectra of compound 6, additional signals due to the extended chain were observed. In the 13C NMR spectrum of 6, the weak multiplets arising from the four OCD2 groups were still detectable even though they coincided with the strongly visible singlet corresponding to the newly introduced OCH2 group. To install the other propylamine fragment, compound 6, was subjected to hydrogenolysis to give the free alcohol 7 in high yield (87%). In the 13C NMR spectrum of 7, an upfield shift of the CD2OH signal (61.18 ppm) was observed, whereas the remaining OCD2 and OCH2 signals resonated together at 69.73 ppm. With an eye to preparing an orthogonally protected d8diamine linker 9, N-(3-chloro-propyl)-benzyloxy carbamScheme 1a

a Reagents and conditions: (a) aq NaOH, TBABr, 80 °C, 2 h, 94%; (b) HCl-MeOH (10%), RT, 1 h, 90%; (c) 5, aq NaOH, TBABr, 80 °C, 1 h, 73%; (d) Pd(OH)2, H2, ethyl acetate, 3 h, 87%.

Scheme 2a

a Reagents and conditions: (a) acrylonitrile, NaOH cat., absol toluene, RT, 3 h, 96%; (b) Ra/Ni, MeOH, RT, 12 h, quant.; (c) Biotin pfP ester, DIPEA, DMF, RT, 12 h, 64% overall yield for steps b and c.

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Figure 2. High-resolution mass spectrum of the d8-ICAT linker, N-[(5,5,6,6,8,8,9,9-2H)-13-biotinamido-4,7,10-trioxatridecanyl]tert-butyloxycarbamide (12).

ide (8) (23) was chosen for the etherification reaction with compound 7, which would then allow selective cleavage of the Cbz and the Boc groups successively in order to introduce biotin for the affinity purification on avidin and a reactive group to covalently tag the enzyme under study, respectively. Initially, the attempted etherification reaction led to the decomposition of the Cbz functionality in 9 due to the strongly basic medium and high reaction temperature. Thus a different approach was chosen wherein first, Michael addition on 7 gave the nitrile derivative 10 in 96% yield. NMR spectra of 10 showed additional signals corresponding to the newly introduced propylnitrile chain (at 117.79 ppm for CN carbon and a triplet at 2.58 ppm for CH2CN protons) and in the 13C NMR spectrum the OCD2 signals appeared as one broad multiplet and overlapping with one of the two OCH2 signals. Subsequently, Raney nickel reduction of the nitrile to amine 11 was achieved in greater than 80% yield (Scheme 2). A triplet corresponding to the newly formed CH2NH2 group at 2.52 ppm in the 1H NMR spectrum along with ESI mass spectrometry (m/z ) 329.32 and 351.33 for [M + H]+ and [M + Na]+) confirmed the amine formation. In this reaction we did not experience yield reduction due to formation of any side products resulting from the retro-Michael type reaction as reported by Gelb and co-workers (12). The monoamine linker 11, without further purification, was directly coupled to biotin using the activated biotin pentafluorophenyl ester in anhydrous DMF in the presence of DIPEA to give 12 in 64% overall yield for both steps (reduction and biotin coupling). New signals at 4.45 and 4.26 ppm, due to the CH protons of biotin, were well separated from all other signals in the 1H NMR spectrum of compound 12 and clearly confirmed the introduction of biotin moiety. As observed before, the 13C NMR spectrum revealed weak multiplets corresponding to OCD2 groups that overlapped one of the two OCH2 signals. An additional proof of structure and purity of

12 was obtained by HRMS which showed an [M + H]+ signal at m/z ) 555.3668 with a deviation of only -0.8 ppm from the required value (Figure 2). Having established the viability of this synthetic procedure, we envision utility for developing ICAT reagents of high isotopic purity. For instance, the d8-linker 12 can be readily elaborated into a cysteine specific d8ICAT reagent (Figure 1) by deprotection of the Boc group, followed by acylation of the resulting free amine with iodoacetic anhydride. Similarly, any similar d8-ICAT reagent (14, Scheme 2) can be prepared by introducing the reactive group of choice that can specifically tag an enzyme of interest. Unlike in the previously reported procedures, we incorporated eight deuteriums on the ICAT reagent within the diethylene glycol region in the very first step with no loss of deuteriums through etherification reaction of isotopically pure tetradeutero precursors 1 and 2. Those incorporated deuteriums are stable throughout the subsequent reaction conditions. Thus, the d8-ICAT reagent 12 was obtained in 100% isotopic purity and with 33% overall yield. The resultant d8-ICAT reagent 12 carries deuterium in different positions of the ICAT reagent and is a structural isoform of the original d8ICAT reagent (Figure 1). The slight compositional differences between the ICAT linker 12 reported herein, and previously reported ICAT reagents, should not affect the efficiency of the isotopically encoded protein quantification obtained in the ICAT method. CONCLUSION

In conclusion, we have described an inexpensive and high yielding synthetic strategy to prepare a d8-ICAT reagent with 100% isotopic purity. A salient aspect of this synthesis is that it allows the synthesis of a wide range of deuterated diamine linkers carrying at least four and potentially any multiple of four deuteriums as the

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etherification steps can be repeated to generate longer linkers with a substantially higher isotopic differential. Moreover, this work also enables the synthesis of a class of “heavy, medium, and light” reagents that might be useful for simultaneously probing three cell-based systems; for example (i) uninfected, (ii) infected without treatment, and (iii) infected with treatment. Longer linkers may easily give up to five differentially encoded tags. Whereas the work herein employed deuterated precursors, other isotopes can be introduced instead of deuterium with minimal alteration to the overall synthetic routes if desired. Indeed, recent reports have underscored slight, yet significant, chromatographic differences due to replacement of protium with deuterium (24). Consequently, other stable isotopes, namely 13C, have now been used instead of deuterium. These recent approaches have used 13C-labeled acrylonitrile to address this problem along with undesired exchange of D for H during Michael addition and hydrogenation. The synthetic strategy reported herein can be adapted for (13C or 18O) labeled ICAT reagents by using 13C- or 18O-labeled starting materials (25) in the etherification steps. Moreover, both carbon and oxygen isotopes could be used in tandem to generate an even higher isotopic differential on a relatively shorter linker arm. In general, this work suggests a modular approach to achieving a high-yielding synthesis of ICAT linkers to give defined isotopic mass differences. Finally, nitrile hydrolysis of 10 with appropriate amine protection, or alternately, Michael addition of 7 to acrylic acid esters is readily envisioned to generate “amino acid” ICAT linkers (carrying an amine at one end and a carboxylic acid at the other end) compatible with automated solidphase peptide synthesis. Furthermore, Michael addition of 7 to acrolein would give the corresponding aldehyde that could be alternately conjugated to amines, hydrazines, and oximes. In summary, (a) the orthogonal protection strategy obviates extensive loss or tedious recovery steps, (b) allows for effective economy at the level of monobiotinylation, (c) provides options for the introduction of a carboxylate/carbonyl functionality that can be handily converted to an activated ester for the direct attachment to amines, and (d) allows for multiple isotopic labelings through the simultaneous use of 13C or 18O to obtain shorter linkers with higher isotopic density if so desired. ACKNOWLEDGMENT

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(26) Abbreviations: ICAT, isotope coded affinity tagging; DIPEA, diisopropylethylamine; DMF, dimethylformamide; n-Bu4NBr, tetrabutylammonium bromide; THP, tetrahydropyran.

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