Site-Selective Lysine Modification of Native Proteins and. Peptides via Kinetically Controlled Labeling

Transcription

1 ite-elective Lysine Modification of ative Proteins and Peptides via Kinetically Controlled Labeling Xi Chen a,b, Kasturi Muthoosamy b, Anne Pfisterer c, Boris euman d and Tanja Weil a,b,c * a Institute of rganic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, Ulm, Germany b Department of Chemistry, ational University of ingapore, 3 cience Drive 3, ingapore c Max Planck Institute for Polymer Research, Ackermannweg 10, D Mainz, Germany d Proteome Factory AG, Magnusstr. 11, aus 2, 1.G, Berlin, Germany * Corresponding author. Tel: (+) ; Fax: (+) ; tanja.weil@uni-ulm.de upporting Information Table of Contents Page o. upplementary cheme cheme 1 3 upplementary Figures Fig. 1~3: Interpretation of XIC Chromatographs Fig. 4: The MALDI-ToF-M spectrum of biotin-lc-lysozyme C 4 7 Biology Click Reaction Gel Filtration Affinity Chromatography BCA Assay

2 Rase A Functional Assay 10 Chemistry General tert-butyl 2-Propargylaminoacetate (10) Ethynyl Acid (11) -Boc-4,7,10-trioxa-1,13-tridecanediamine (13) Biotin-TE-(Boc) (14) Biotin-TE- 2 (15) Biotin-TE-Ethynyl-CtBu (16) Biotin-TE-Ethynyl-C (17) MR pectra

3 UPPLEMETARY CEME cheme 1: The general scheme for protein biotinylation and recovery of excess of unreacted protein. The bioconjugation step I: Addition of the biotinylation reagent into the protein solution in PB buffer (p 7.2) according to the kinetically controlled labeling protocol; the quenching step II: Addition of ethanolamine solution to quench any remaining unreacted biotinylation reagent; first biotin-removal step III: Gel filtration or dialysis to remove deactivated biotin-containing small molecules; protein recovery step IV: after loading the protein mixture into the monomeric avidin resin column, native protein is recovered by eluting the column with PB buffer; affinity elution step V: Mono-biotinylated protein is obtained by eluting the monomeric avidin column with biotin solution; second biotin-removal step VI: Gel filtration or dialysis to desalt and also to remove free (+)-biotin from solution. 3

4 UPPLEMETARY FIGURE Fig. 1. The XIC chromatographs (1+ and 2+) of unmodified KETAAAKF (1~8) of biotin-lc-(k1)rase A (3): both are false positive signals Top: XIC of unmodified KETAAAKF (1+) at m/z [M+] + ; bottom: XIC of unmodified KETAAAKF (2+) at m/z [M+2] 2+. The signals in the XIC chromatograph at t R min (above) and t R min (bottom) are false positives, see below. This suggests that unmodified K1 of Rase A does not exist. The M spectrum for the peak at t R min (1+): false positive signal pectrum of m/z 865 range at t R min, showing a charge state of +1: correct charge state but no alternate charge state at same t R which is uncommon. This indicates that this signal is a false positive. Additionally, the intensity of this signal is very low. 4

5 RM for the peak at t R min (2+) (bottom): false positive signal pectrum of m/z 433 range at t R min, showing a charge state of +1: wrong charge state. This indicates that this is a false positive signal. Fig. 2. The XIC chromatograph of modified and unmodified KETAAAKF (1~8) in biotin-te-ethynyl-(k1)rase A. From top to bottom: ethynylated KETAAAKF(2+) at m/z [M+2] 2+, ethynylated KETAAAKF(3+) at m/z [M+3] 3+, unmodified KETAAAKF (1+) at m/z [M+] + and unmodified KETAAAKF (2+) at m/z [M+2] 2+. Unmodified KETAAAKF (1-8) is not identified, proofing the full ethynylation at K1 of ethynyl-(k1)rase A (18). For details see below: 5

6 RM of the XIC peak at t R 12.7 min (2+) (Fig. 2d): false positive signal RM spectrum of the XIC peak at t R 12.7 min (bottom) indicates an unmatched charge of 3+, proofing a false positive. RM of the XIC peak at t R min (2+) (Fig. 2a): the 2+ charge is validated. RM of the XIC peak at t R min (3+) (Fig. 2b): the 3+ charge is validated. 6

7 All other remained tiny signals in Fig. 2c and Fig. 2d are all false positive signals. This is because, despite of their very weak intensity, no alternative charge state at the same t R are found. Fig. 3. The XIC chromatograph of mono-biotinylated T-14 mixture. The XIC chromatograph of mono-biotinylated T-14 (reduced form) (2+) showing a symmetric peak at t R min. Fig. 4. The MALDI-ToF-M spectrum of biotin-lc-lysozyme C (5). Biotin-LC-(K1)lysozyme C shows a mass peak of m/z [M+] +. A tiny peak at the right (m/z 14703) with Δ m/z 57.2 clearly suggests that this is not the bis-modified peak (for biotinylation, Δ m/z 339.5). In fact, this tiny peak correspondingly appears in the mass spectrum of the native lysozyme C. 7

8 BILGY Click Reaction a). cheme biotin biotin Cu(I) +, PB (p 7.2) RT, 24 h b). Procedure A solution of Cu(I) should be freshly prepared in advance by dissolving 25 mg of Cu and 100 mg sodium ascorbate in 1 ml of DI- 2. This solution quickly turns dark and further changes to bright yellow colour within a few minutes. ubsequently, ml ethynyl-rase A (19, 11 μm, 1.6 nmol), ml PB buffer (10, 1M), 0.04 ml TF and 1.6 μl azidocoumarin (20) solution in TF (10 mg ml -1, 0.08 μmol) were added into a 200 μl Eppendorf tube. Rapidly, 9.6 μl of the Cu (I) suspension (0.1 M, 0.96 μmol) was added to the reaction mixture as the catalyst. The Eppendorf tube was quickly flushed with argon, capped, sealed with parafilm, shaken and incubated at RT for 24 hours. Two controls in the absence of Cu (I) catalyst or azidocoumarin (20) were also set up using the sample procedure. Under a UV lamp, a strong fluorescence was detected while the controls showed almost no fluorescence (Figure 9d). This indicated that the ethynyl group of 19 is accessible for further bioorthogonal reactions and gel electrophoresis of the reaction solution confirms the formation of the click product coumarin-(k1)rase A (22, Figure 9d). Gel Filtration In a typical run, 1 ml protein solution can be purified by 4 g of ephadex G15 gel powder. First, 4 g of ephadex G15 gel needed to be incubated in 15 ml of MQ-water at RT overnight before use. Then, the gel was packed in a gel filtration column resulting in about 10 ml of activated gel. MQ-water was applied to wash the column continuously until the elution was completely free of any impurities as tested by UV determination. Later, 1 ml of protein solution was loaded on the column and eluted 8

9 with MQ-water. 0.5 ml per Fraction were collected and tested via UV absorption. Pure fractions were combined which is a protein solution in MQ-water free of any salts and small molecules. Water can be further removed via lyophilization to afford the lyophilized form of proteins. Affinity Chromatography A more detailed affinity chromatography procedure can be referred to published by Thermo cientific. Briefly, the Avidin bead column was first washed with PB buffer and then any irreversible binding sites were blocked by applying a biotin solution in PB buffer passing through the column. Later, the reversible binding sites were released by washing the column with glycine solution as the regeneration buffer. After washing out the regeneration buffer using PB, biotinylated protein solution was loaded onto the affinity column and native protein was recovered by washing the column with PB buffer. Finally, biotinylated protein was obtained via elution with biotin solution in PB as the elution buffer. BCA Assay The detailed procedure can refer to the protocol on the website published by ovagen. The only modification in our experiment is the usage of a solution constituted of protein and biotin in a 1:1 molar ratio as the standard rather than a pure protein solution. This is because biotin-lc-rase A, biotin-lc-lysozyme C and ethynyl-rase A all contain an extra biotin moiety that might contribute to the color development during the BCA assay. BCA assay of protein standards (three sets, from 25 μg ml -1 to 500 or 800 μg ml -1 ) and the sample (s) (three sets also) were assayed via BioTek microplate reader under 562 nm after incubation of them with BCA and Cu(II) solution at 37 C for 30 min. The standard curve was established using the averaged absorbance of standard solutions and the absorbance of the protein sample was plotted against the standard curve giving concentration of the modified protein. 9

10 Rase A Functional Assay Tris-EDTA buffer solution was freshly prepared from Tris buffer (10 mm) and EDTA (1 mm) and was adjusted to p µl of YBR dye (diluted to 1 concentration with Tris-EDTA buffer) was added into 9 wells in a sterilized 384-well plate. Then µl of yra Tris-EDTA buffer solution (10μg ml -1, keep in -20 C and thaw on ice before use) was added to each well. Quickly, 50 µl of blank Tris-EDTA buffer, native RaseA (25 ng ml -1 in Tris-EDTA buffer) and biotin-lc-rasea (25 ng ml -1 in Tris-EDTA buffer) were added to 3 charged wells, respectively, by a 10 station multi-channel pipette. The 384-well plate was subjected to BioTek ynergy 4 microplate reader immediately to determine the fluorescence change (excited at 485 nm; emitting at 535 nm). Parameters: one second of shaking with an interval of 13 seconds and monitored for seven minutes. The Relative Florescence Unit (RFU) of native Rase A, biotin-lc-rase A and the blank control were recorded. 10

11 CEMITRY General All chemical reagents were purchased from igma-aldrich, Merck or Regent and were used without further purification unless otherwise mentioned. Anhydrous DMF was dried over freshly activated 4 Å molecular sieve. 1 -MR, 13 C-MR, DEPT 135, DEPT 90 and MQC spectra were recorded with Bruker AC 300, AMX 500 or DRX500 MR spectrometers operating at 0 Mz for 1 and 75.48Mz for 13 C MR at 25 C. Chemical shifts were reported in ppm (δ scale) relative to the solvent signal (CDCl 3 : δ 7.26, δc 77.0; DM-d 6 : δ 2.50; MeD: δ 3.31, δc 49.0), and coupling constant (J) values were reported in hertz (z). igh-resolution mass spectra (RM) were recorded on a Finnigan MAT95XL-T mass spectrometer by direct infusion of the solution of each compound using electrospray ionization (EI) in positive mode. Low resolution EIM was determined using Finnigan LCQ quadrupole ion trap mass spectrometer. LC-EI-M analysis was achieved via chmadzu LC-20AD/PD-20A/IL-20AC/LCM-2010EV instrument equipped with a C-18 column using MeC/ 2 as eluent. tert-butyl 2-Propargylaminoacetate (10) K 2 C 3 Br 2 + DMF,RT % 10 Into a 25 ml chlenk flask equipped with a stir bar, K 2 C 3 (690 mg, 5.0 mmol) was added and it was dried under high vacuum with a strong heating by a heatgun for 10 min. After cooling down, the flask was charged with argon and dry DMF (16.5 ml, 0.3 M) was introduced followed by injection of -propargylamine (9, 550 mg, 10 mmol). The flask was placed in a RT water bath and tert-butyl bromoacetate (8, 975 mg, 5 mmol) was injected dropwise slowly to the stirring reaction mixture. After the injection completed, the reaction mixture was allowed to stir at RT for two days. Then, the reaction mixture was partitioned in EtAc/ 2 (v/v, 3/1) and the aqueous layer was drained out. The organic layer was washed with DI- 2, brine and removed of 11

12 EtAc under reduced pressure. The product was finally purified by silica gel chromatography (EtAc: exane 2: 5, Rf = 0.4, stained by I 2 ) to afford 561 mg brilliant light yellowish viscous liquid as the product 10 in a yield of 66%. 1 -MR (DRX500, CDCl 3 ): δ 3.45(d, J=2.6z, 2), 3.38 (s, 2), 2.20 (t, J=2.3z, 1), 1.46 (s, 9); 13 C-MR (AC300, CDCl 3 ): δ , 81.33, 71.75, 50.08, 37.61, 28.06; DEPT 135 (AC300, CDCl 3 ): (+) δ 28.06; (-) δ 50.08, 37.61; DEPT 90 (AC300, CDCl 3 ): o signal detected; EI-M (+): [M+] +, [2M+] + ; EA: C , calcd. C 63.88%, 8.93%, 8.28%, found C63.79%, 8.%, 8.42%. Ethynyl Acid (11) DMF, RT 10 79% 11 Into a dry 25 ml chlenk flask equipped with a stir bar, succinic anhydride (237 mg, 2.37 mmol) was added and the flask was evacuated then charged with argon. Dry DMF (5.9 ml, 0.2 M) was injected under a positive pressure of argon to form a solution. The flask was placed in a RT water bath and tert-butyl 2-propargylaminoacetate (10) was injected dropwise to the stirring solution. After the completion of the injection, the water bath was removed to allow the reaction to stir at RT overnight. Then, the reaction solution was dissolved in EtAc, washed with DI- 2 (5 ), brine, dried over anhydrous sodium sulphate, filtrated, concentrated and purified by silica gel chromatography (Me: CCl 3 1:5, Rf = 0.5, stained by I 2 ) to afford 250 mg light yellow sticky semi-solid as the product 11 in a yield of 79%. 1 -MR (AMX500, DM-d 6 ): δ (s, br, 1), 4.25(d, J=2.6z, 0.5 2), 4.18 (s, 0.5 2), 4.14 (d, J=2.6z, 0.5 2), 3.96 (s, 0.5 2), 3.34 (t, J=2.5z, 0.5), 3.18 (t, J=2.6z, 0.5), 2.67 (t, J=6.6z, 0.5 2), 2.47 (t, J=6.3z, 0.5 2), 2.43~2.(m, 2), 1.43 (s, 0.5 9), 1. (s, 0.5 9); 13 C-MR (AC300, CDCl 3 ): δ , , , , 82.79, 82.00, 78.02, 77.43, 77.37, 76.58, 73.43, 72.91, 48.72, 47.54, 38.05, 35.57, 29.05, 27.88, 27.84, 27.74; DEPT 135 (AC300, CDCl 3 ): (+) δ 28.01, 27.96; (-) δ 48.84, 47.66, 38.17, 35.57, 29.16, 27.86; RM (EI): 12

13 C a + calcd , found Boc-4,7,10-trioxa-1,13-tridecanediamine (13) (Boc) 2 2 Dioxane,RT 2 Boc 2 34% A solution of 4,7,10-trioxa-1,13-tridecanediamine (12, 7.5 g, 34.1 mmol) in 1,4-dioxane (100 ml) cooled by a RT water batch was treated with Boc anhydride (3.7 g, 16.9 mmol) and the reaction mixture was stirred at RT overnight. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (CCl 3 :Me: 3 (aq. 20% w/w) 6:1:0.21, Rf = 0.4, stained by I 2 ) to afford 1.86 g light yellowish oil as the product 13 in a yield of 34%. 1 -MR (AMX500, CDCl 3 ): δ 5.10 (s, br, 1), 3.62~3.65 (m, 4), 3.52~3. (m, 8), 3.22 (m, 2), 2.80 (t, J=6.8z, 2), 1.70~1.78 (m, 4), 1.43 (s, 9); EI-M: [M+a] +, [M+] +. Biotin-TE-(Boc) (14) 2 Biotin,EDC DMF, RT Boc 73% Boc Into a dry 25 ml chlenk flask, (+)-biotin (1.06 g, 4.35 mmol) and EDC Cl (1.11 g, 5.8 mmol) were combined and dry DMF (8.5 ml) was added to this mixture under argon atmosphere. The resulted suspension was allowed to stir for 10 min in an ice-water bath. Then, a solution of -Boc-4,7,10-trioxa-1,13-tridecanediamine (13, g, 2.9 mmol) in dry DMF (6 ml) was added to the suspension and the ice-water bath was removed to allow the reaction mixture to warm to RT and stir for two days. The reaction solution was partitioned in CCl 3 / 2 and the CCl 3 layer was separated. The aq. layer was extracted four times with CCl 3 and all organic layers were combined and washed with saturated a 2 C 3. The a 2 C 3 layer was extracted with CCl 3 and CCl 3 layers were combined again, washed with brine, dried over 13

14 anhydrous a 2 4, filtrated and removed of CCl 3 under reduced pressure. DMF was get ride of under high vacuum at 35 C with stirring and the resultant residue was finally purified by silica gel flash chromatography (CCl 3 :Me 6:1, Rf = 0.5, stained by I 2 ) to yield an off-white solid as the product 14 in a yield of 72.5%, 1.15g. 1 -MR (AMX500, CDCl 3 ): δ 6.61 (s, br, 1), 6.41 (s, br, 1), 5.67 (s, br, 1), 5.03 (s, br, 1), 4.48 (m, 1), 4.29 (m, 1), 3.~3.64 (m, 4), 3.53~3. (m, 6), 3.51 (t, J=5.7z, 2), 3.32 (q, J=6.1z, 2), 3.19 (m, 2), 3.13 (m, 1), 2.88 (dd, J 1 =12.6z, J 2 =4.4z, 1), 2.72 (d, J=12.6z, 1), 2.17 (t, J=7.6z, 2), 1.70~1.78 (m, 5), 1.~1.70 (m, 3), 1.42 (m, 2), (s, 9); 13 C-MR (AMX500, CDCl 3 ): δ , , 70.48, 70.44, 70.15, 70.00, 69.91, 69.46, 61.78,.15, 55.,.48, 38.43, 37.69, 36.00, 29.68, 28.93, 28.42, 28.22, 28.09, 25.65; DEPT 135 (AMX500,CDCl3): (+) δ 61.78,.15, 55.61, 28.42; (-) δ 70.48, 70.44, 70.15, 70.00, 69.90, 69.44,.48, 38.43, 37.67, 36.00, 29.68, 28.93, 28.22, 28.09, 25.65; RM (EI): C a + calcd , found Biotin-TE- 2 (15) 14 Boc 1. TFA/DCM, RT 2. 3 aq quantitative yeild 2 15 Dry DCM (15 ml) was added into a 100 ml RBF equipped with a stir bar containing biotin-te-(boc) (921.3 mg, 1.69 mmol) under argon to form a homogenous solution. The RBF was cooled in an ice-water bath and TFA (7.5 ml) was added under a flush of argon. The reaction solution was stirred for 12 min and TLC indicated the complete deprotection of the starting material. Reaction was terminated immediately in order to avoid further cleavage of the product by removal of DCM and TFA under reduced pressure. The obtained residue was evacuated overnight and further purified by silica gel flash chromatography (CCl 3 :Me: 3 (aq. 20% w/w) 1:0.25:0.1, Rf = 0.35) to afford a light yellow sticky solid as the product 15 in a quantitative yield, 974.3mg. 1 -MR (AMX500, DM-d 6 ): δ 7.75 (t, J=5.68z, 14

15 1), 6.41 (s, 1), 6.35 (s, 1), 4.30 (m, 1), 4.13 (m, 1), 3.51 (m, 4), 3.47 (m, 4), 3.43 (t, J=6.3z, 2), 3.38 (t, J=6.3z, 2), 3.20 (s, br, 2), 3.10 (m, 1), 3.07 (m 1), 2.82 (dd, J 1 =12.0z, J 2 =5.1z, 1), 2.62 (t, J=6.9z, 2), 2.57 (d, J=12.0z, 1), 2.04 (t, J=7.6z, 2), 1. (m, 5), 1.49 (m 3), 1.30 (m, 2); EIM: [M+] +. Biotin-TE-Ethynyl-CtBu (16) 3 BTU,DIEA 11, DMF,RT 2 86% BTU (526 mg, 1.39 mmol) was added into a dry 25 ml chlenk flask equipped with a stir bar. The chlenk flask was evacuated then charged with argon and cooled in an ice-water bath. A solution of 11 (245 mg, mmol) in dry DMF (4.34 ml) was added into chlenk flask and DIPEA (358 mg, 2.78 mmol) was injected. The reaction mixture was stirred at 0 C for tens of minutes and biotin-te- 2 (15, 387 mg, mmol) was introduced. The ice-water bath was removed and the chlenk flask was coated with aluminum foil. The reaction mixture was allowed to stir at RT for 2 days. DMF and DIPEA were removed under high vacuum overnight and the obtained residue was partitioned in CCl 3 /saturated a 2 C 3 (25 ml/25 ml). The CCl 3 layer was separated and the aq. layer was extracted twice with 8 ml of CCl 3. All organic layers were combined, washed with brine (10 ml), dried over anhydrous Mg 4, filtrated, concentrated and purified by silica gel flash chromatography (CCl 3 :Me 6:1, Rf = 0.4, stained by I 2 ) to afford a light yellow solid as the product 16 in a yield of 86.3%, 522.6mg. 1 -MR(AMX500, CDCl 3 ): δ 6.88 (t, J=5.4z, 0.5), 6.81 (t, J=5.1z, 0.5), 6.78 (m, 1), 6.32 (s, br, 1), 5.68 (s, br, 1), 4.47 (m, 1), 4.28 (m, 1), 4.24 (d, J=2.5z, 1), 4.17 (d, J=1.9z, 1), 4.12 (s, 1), 4.07 (d, J=5.1z, 1), 3.61 (m, 4), 3.57~5.50 (m, 8), 3.30 (m, 4), 3.12 (m, 1), 2.89 (dd, J 1 =12.7z, J 2 =8.2z, 1), 2.77 (m, 1), 2.72 (d, J=12.7z, 1), 2.57 (t, J=6.9z, 15

16 1), 2.47~2.52 (m, 2), 2.33 (t, J=2.5z, 0.5), 2.23 (t, J=2.5z, 0.5), 2.16 (t, J=7.3z, 2), 1.74 (m, 5), 1.64 (m, 3), 1.45 (s, 4.5), 1.42 (s, 4.5), 1.42 (m, 2); 13 C-MR(AMX500, CDCl 3 ): δ , , , , , , , 82.67, 81.89, 78.25, 77.57, 77.20, 73.43, 72.81, 70.41, 70.38, 70.00, 69.96, 69.81, 69.46, 61.81,.12, 55.62, 48.78, 47.55,.46, 38.54, 38.14, 37.57, 37.35, 35.90, 35.53, 31.10, 31.06, 29.02, 28.99, 28.90, 28.53, 28.51, 28.18, 28.04, 27.99, 27.96, 25.58; DEPT 135(AMX500, CDCl 3 ): (+) δ 61.81,.12, 55.63, 38.54, 27.99, 27.96; (-) δ 70.41, 70.38, 70.00, 69.95, 69.81, 69.46, 48.78, 47.55,.46, 38.14, 37.56, 37.35, 35.90, 35.53, 31.10, 31.06, 29.02, 28.99, 28.90, 28.51, 28.18, 28.04, 25.59; RM (EI): C a + calcd , found Biotin-TE-Ethynyl-C (17) 3 16 TFA DCM 70% 17 3 Into a 25mL RBF equipped with a stir bar containing 16 (522.6 mg, mmol), 10 ml dry DCM was added under a flush of argon and a homogeneous solution was formed. This RBF was cooled in an ice-water bath and TFA (5 ml) was added dropwise under a flush of argon. Ice-water bath was removed and the reaction mixture was allowed to stir at RT for five hours when TLC (CCl 3 :Me 4:1 Rf (M) = 0.5, Rf (P) = 0.35, stained by I 2 ) just indicated the completion of the reaction. DCM and TFA were removed immediately via a rotavap. The resultant residue was evacuated overnight and dissolved in DI- 2 to form a solution. This solution was washed with CCl 3 four times, filtrated and removed of water using a rotavap at C. The last trace of water in the product was get ride of under high vacuum to yield the targeted molecule 17 as light yellow sticky solid in a yield of 70.4% with a purity of 95.7%, mg. 1 -MR (MeD, DRX500): δ 4.51 (m, 1), 4.32 (s, 0.5 2), 4.32 (1), 4.30 (d, 16

17 J=2.1z, 0.5 2), 4.26 (d, J=2.9z, 0.5 2), 4.17 (s, 0.5 2), 3.63 (m, 4), 3.59 (m, 4), 3.52 (t, J=6.1z, 4), 3.26 (t, J=6.7z, 4), 3.21 (m, 1), 2.93 (dd, J1=12.6z, J2=4.8z, 1), 2.83 (t, J=6.9z, 0.5 2), 2.82 (0.5), 2.71 (d, J=12.8z, 1), 2.68 (0.5), 2.63 (t, J=6.4z, 0.5 2), 2.49 (m, 2), 2.20 (t, J=7.3z, 2), 1.76(m, 5), 1.56~1.70 (m, 3), 1.44 (m, 2); DEPT135 (AMX500, MeD): (+) δ 78.92, 78.66, 74.88, 74.29, 63.41, 61.67, 56.86, (-) δ 71.33, 71.04, 71.01, 69.80, 69.73, 48.95, 48.06,.97, 39.23, 37.82, 37.76, 36.74, 36.47, 31.75, 31.71, 30.22, 30.14, 29.67, 29.48, 29.45, 29.38, 26.79; 17

18 13 C-MR (AMX500, MeD): δ , , , , , , , 78.92, 78.65, 74.88, 74.29, 71.34, 71.04, 71.01, 69.81, 69.73, 63.41, 61.67, 56.86, 48.95, 48.06,.96, 39.23, 37.82, 37.77, 36.74, 36.47, 31.77, 31.71, 30.23, 30.14, 29.66, 29.48, 29.45, 29.38, 26.78; RM (EI): C a calcd , found: ; LC-EIM: (MeC / 2, gradient eluent, 214 nm, C-18 column): 95.7% purity, m/z 642 [M+] +. 18

19 Integral MR PECTRA 1 tert-butyl -MR Propargylamidoacetate (CDCl in CDCl3 3, DRX ): tert-butyl Propargylaminoacetate AME : CX0126~1 EXP : 1 PRC : 1 LCUC : 2 : 4 UCLEU : off 1 : z PULPRG : zg F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 0.10 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off C-MR (CDCl 3, AC 300): tert-butyl Propargylamidoacetate Propargylaminoacetate CDCl3 13C tandard AC300 AME : ja25cx EXP : 2 PRC : 1 LCUC : 2 : 352 UCLEU : off 1 : z PULPRG : zgpg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off

20 DEPT 135 (CDCl 3, AC 300): tert-butyl Propargylaminoacetate tert-butyl Propargylamidoacetate in CDCl3 DEPT135 AC300 AME : ja25cx EXP : 4 PRC : 1 LCUC : 2 : 68 UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off CY (CDCl 3, DRX 500): tert-butyl Propargylaminoacetate 20

21 Integral MR (CDCl 3, AMX 500): tert-butyl -(3-Carboxylpropyl)--Propargyl aminoacetate -CP--PP-Gly-tBu -PP-Gly-tBu salt in CDCl3 1 AMX500 AME : cx0202 EXP : 1 PRC : 1 LCUC : 2 : 4 UCLEU : off 1 : z PULPRG : zg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 0.30 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off C-MR (CDCl 3, AC 300): tert-butyl -(3-Carboxylpropyl)--Propargyl aminoacetate -Carboxylpropanoyl--propargylglycine tert-butyl ester in CDCl3 13C tandard AC300 AME : fe01chx EXP : 2 PRC : 1 LCUC : 2 : 158 UCLEU : off 1 : z PULPRG : zgpg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off

22 Integral DEPT 135 (CDCl 3, AC 300): tert-butyl -Carboxylpropanoyl--propargylglycine -(3-Carboxylpropyl)--Propargyl tert-butyl ester in CDCl3 DEPT135 AC300 aminoacetate AME : fe01chx EXP : 3 PRC : 1 LCUC : 2 : 30 UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off MR (CDCl 3, DRX 500): -Boc-4,7,10-trioxadodecanediamine in CDCl3 DRX500 AME : CX0130~1 EXP : 2 PRC : 1 LCUC : 2 : 4 UCLEU : off 1 : z PULPRG : zg F1 : Mz 2 Boc LVET : CDCl3 W : ppm TD : TE : K LB : 0.10 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off

23 Integral MR (CDCl 3, AMX 500): Biotin-TE--Boc Biotin-PEG--Boc in CDCl3 1 AMX500 Boc AME : cx0203 EXP : 1 PRC : 1 LCUC : 2 : 4 UCLEU : off 1 : z PULPRG : zg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 0.30 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off C-MR (CDCl 3, AMX 500): Biotin-PEG--Boc Biotin-TE--Boc in CDCl3 13C AMX500 AME : cx0203 EXP : 2 PRC : 1 LCUC : 2 : 150 UCLEU : off Boc 1 : z PULPRG : zgpg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off

24 DEPT 135 (CDCl 3, AMX 500): Biotin-TE--Boc Biotin-PEG--Boc in CDCl3 DEPT135 AMX500 AME : cx0203 EXP : 3 PRC : 1 LCUC : 2 : 36 UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off Boc CY (CDCl 3, DRX 500): Biotin-TE--Boc Boc 24

25 Integral MQC (CDCl 3, DRX 500): Biotin-TE--Boc Biotin-PEG--Boc MQC in CDCl3 ppm Boc Current Data Parameters AME cx0207drx EXP 6 PRC 1 F2 - Acquisition Parameters Date_ Time ITRUM spect PRBD 5 mm TXI 1-13 PULPRG inv4gstp TD 512 LVET Me 2 D 16 W z FIDRE z AQ sec RG DW usec DE 6.00 usec TE K D sec D sec D sec d sec D sec D sec D sec d sec d sec I sec ======== CAEL f1 ======== UC1 1 P usec P usec PL db F Mz 55 ======== CAEL f2 ======== CPDPRG2 garp UC2 13C P usec P4 25. usec PCPD usec PL db PL db F Mz ====== GRADIET CAEL ===== P usec ppm 1 -MR (CDCl 3, AMX 500): F1 - Acquisition parameters D0 4 TD 256 F Mz FIDRE z W ppm FnMDE undefined F2 - Processing parameters I 512 F Mz WDW QIE B 2 LB 0.00 z GB 0 PC 1. F1 - Processing parameters I 512 MC2 TPPI F Mz WDW QIE B 4 LB 0.00 z GB 0 Biotin-PEG-2 in DM 1 AMX500 Biotin-TE- 2 AME : cx0210 EXP : 4 PRC : 1 ITRUM : av500 2 LCUC : 2 : 4 UCLEU : off 1 : z PULPRG : zg30 F1 : Mz LVET : DM W : ppm TD : LB : 0.30 z *** 1D MR Plot Parameters *** UCLEU : off

26 C-MR (CDCl 3, AMX 500): Biotin-PEG-2 in DM 13C AMX500 Biotin-TE- 2 AME : cx0210 EXP : 5 PRC : 1 ITRUM : av500 2 LCUC : 2 : 324 UCLEU : off 1 : z PULPRG : zgpg30 F1 : Mz LVET : DM W : ppm TD : LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off DEPT 135 (CDCl 3, AMX 500): Biotin-TE- 2 Biotin-PEG-2 in DM DEPT135 AMX500 AME : cx0210 EXP : 6 PRC : 1 ITRUM : av500 LCUC : 2 2 : 37 UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : DM W : ppm TD : LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off

27 Integral MR (CDCl 3, AMX 500): Biotin-TE-Ethynyl-CtBu Biton-PEG-Ethynyl in CDCl3 1 AMX500 3 AME : cx0218 EXP : 1 PRC : 1 LCUC : 2 : 5 UCLEU : off 1 : z PULPRG : zg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 0.30 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off C-MR (CDCl 3, AMX 500): Biotin-PEG-Ethynyl in CDCl3 13C AMX500 Biotin-TE-Ethynyl-CtBu 3 AME : cx0218 EXP : 2 PRC : 1 ITRUM : av500 LCUC : 2 : 313 UCLEU : off 1 : z PULPRG : zgpg30 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off

28 DEPT 135 (CDCl 3, AMX 500): Biotin-TE-Ethynyl-CtBu Biotin-PEG-Ethynyl in CDCl3 DEPT135 AMX500 AME : cx0218 EXP : 3 PRC : 1 ITRUM : av500 LCUC : 2 3 : 67 UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : CDCl3 W : ppm TD : TE : K LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off MR (Me, AMX 500): Biotin-PEG-Ethy-C in MeD DRX500 Biotin-TE-Ethynyl-C 3 AME : CX0225~1 EXP : 31 PRC : 1 LCUC : 2 : 1 UCLEU : off 1 : z PULPRG : zg F1 : Mz LVET : MeD W : ppm TD : TE : K LB : 0.10 z F : Mz *** 1D MR Plot Parameters *** UCLEU : off

29 C-MR (Me, AMX 500): Biotin-TE-Ethynyl-C Biotin-Ethynyl-C in MeD13C AMX500 AME : cx0225 EXP : 4 PRC : 1 ITRUM : av500 LCUC : 2 : UCLEU : off 1 : z PULPRG : zgpg30 F1 : Mz LVET : MeD W : ppm TD : TE : K LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off DEPT 135 (Me, AMX 500): Biotin-TE-Ethynyl-C Biotin-Ey-C in MeD DEPT135 AMX500 AME : cx0225 EXP : 3 PRC : 1 ITRUM : av500 LCUC : 2 : UCLEU : off 1 : z PULPRG : dept135 F1 : Mz LVET : MeD W : ppm TD : TE : K LB : 1.00 z *** 1D MR Plot Parameters *** UCLEU : off

30 MQC (Me, DRX 500): Biotin-TE-Ethynyl-C Biotin-Ethy-C in MeD MQC 3 ppm ppm Current Data Parameters AME cx0225drx EXP 32 PRC 1 F2 - Acquisition Parameters Date_ Time ITRUM spect PRBD 5 mm TXI 1-13 PULPRG inv4gstp TD 512 LVET Me 5 D 16 W z FIDRE z AQ sec RG DW usec DE 6.00 usec TE K D sec D sec D sec d sec D sec D sec D sec d sec d sec I sec ======== CAEL f1 ======== UC1 1 P usec P usec PL db F Mz ======== CAEL f2 ======== CPDPRG2 garp UC2 13C P usec P4 25. usec PCPD usec PL db PL db F Mz ====== GRADIET CAEL ===== P usec F1 - Acquisition parameters D0 4 TD 10 F Mz FIDRE z W ppm FnMDE undefined F2 - Processing parameters I 512 F Mz WDW QIE B 2 LB 0.00 z GB 0 PC 1. F1 - Processing parameters I 512 MC2 TPPI F Mz WDW QIE B 4 LB 0.00 z GB 0 LCM: Biotin-TE-Ethynyl-C LC-EIM(himadu) C-18 column, 214nm, 0~100%MeC(0.1%TFA) T(R) = 8.571min mv(x1,000) Detector A Ch1:214nm smth 2.25 Inten.(x1,000,000) EI-M PLC Chromatograph m/z min Compound Table: Entry Ret. T Area eight Peak tart Peak top Area%