Chemically Programmed Antibodies As HIV‑1 Attachment InhibitorsShinichi Sato,† Tsubasa Inokuma,† Nobumasa Otsubo, Dennis R. Burton, and Carlos F. Barbas, III*

Department of Molecular Biology and Chemistry and the Skaggs Institute for Chemical Biology and Department of Immunology andMicrobial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States

*S Supporting Information

ABSTRACT: Herein, we describe the design and applicationof two small-molecule anti-HIV compounds for the creation ofchemically programmed antibodies. N-Acyl-β-lactam deriva-tives of two previously described molecules BMS-378806 andBMS-488043 that inhibit the interaction between HIV-1gp120 and T-cells were synthesized and used to program thebinding activity of aldolase antibody 38C2. Discovery of asuccessful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entryinhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novelbispecific antibodies and topical microbicides.

KEYWORDS: Bioconjugation, anti-HIV agent, chemically programmed antibody, microbicide, entry inhibitor

The retrovirus HIV-1, which causes acquired immunedeficiency syndrome (AIDS), has infected 34 million

people worldwide, and this number is expected to increase by2.5 million each year into the near future.1 Although thecombination reverse transcriptase inhibitor/protease inhibitortreatment known as HAART has proven successful,2,3 sideeffects and viral escape are significant issues, and newtreatments are needed. The viral envelope protein gp120, theprimary target for antibody mediated viral neutralization, is anemerging target for small molecule treatment of HIVinfection.4,5 This protein is responsible for the entry of HIVinto host cells. In the initial step of entry, gp120 binds to theCD4 glycoprotein expressed on the surface of human immunecells. Bristol−Myers Squibb Pharmaceutical Research Institutediscovered small molecules BMS-378806 (1) and BMS-488043(2) that bind to gp120 (Figure 1) and block its interaction with

CD4.6−11 However, the short pharmacokinetic profiles of thesesmall molecule inhibitors (half-lives after intravenous injectionare 0.3 and 2.4 h, respectively) may limit their clinicalapplication.We hypothesize that the pharmacokinetic properties of these

small molecule gp120 inhibitors can be improved byconjugation with a monoclonal antibody (mAb) (Scheme1).12−21 Furthermore, coupling of the small molecule to the

mAb could further enhance their activity in vivo throughantibody effector functions such as antibody dependent cellularcytotoxicity (ADCC) and complement dependent cytotoxicity(CDC). Recently, we have described the development ofchemically programmed antibodies based on the use of mAb38C2, an aldolase antibody generated by reactive immunizationby using a 1,3-diketone hapten.22−24 This antibody possesses alow pKa lysine residue in its binding site that is key to itsaldolase activity that can be site-selectively labeled with N-acyl-β-lactams to produce a chemically programmed antibody.Chemically programmed antibodies have duration times aftersystemic dosing that depend on the properties of the antibodyrather than on those of the conjugated small molecule,providing for very significant extensions in the pharmacokineticprofiles of the attached molecule.18,20 We have demonstratedthe utility of this approach by preparing mAb conjugates thatshow promising activity in a variety of cancer models but also inthe area of anti-infectives through the preparation of CCR5blocking mAbs that inhibit HIV-1 entry and neuraminidaseinhibitors that neutralize influenza.18−20

Treatment as well as prophylaxis of HIV-1 infection requiresthe development of a cocktail of inhibitors. In order tocomplement our anti-CCR5 blockade based on this strategy,18

we envisioned that the conjugate of mAb 38C2 and the small-molecule gp120 inhibitor would bind to gp120 and inhibitCD4-mediated entry of HIV-1 into cells (Scheme 2). In relatedwork, Spiegel and co-workers recently reported that a derivativeof HIV-1 inhibitor 1 modified with a 1,3-dinitrophenyl haptenmoiety binds to HIV gp120.25 Their compound was designedto bind noncovalently with polyclonal anti-1,3-dinitrophenyl

Received: March 8, 2013Accepted: April 7, 2013

Figure 1. Chemical structures of gp120 inhibitors.

Letter

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(DNP) antibodies in situ, with the aim of enhancing the activityof 1. The activity of 1, however, was severely compromisedupon the addition of the DNP linker in their report. Parental 1has HIV-1 neutralization activity in the nanomolar range,whereas DNP linked 1 demonstrated micromolar activity inbinding studies and was not shown to neutralize HIV-1. Ourconjugate strategy differs since we use a defined monoclonalantibody covalently linked to 1. We hypothesized that ourstrategy might allow us to recover the potent activity of 1directly if the lack of activity of their DNP derivative of 1 wasdue to the noncovalent nature of attachment to antibody.Alternatively, modification of the linkage strategy to this familyof inhibitors might be key to restoring the activity of the smallmolecule.To prepare derivatives of the Bristol−Myers Squibb

compounds for conjugation to mAb, we first prepared β-lactam3 (Figure 2) derived from BMS-378806 (1) from the knowncompound 5 (Scheme 3).7 Substitution of the nitro group byalcohol 6 followed by the treatment of PCl3 gave BMS-378806derivative 7 bearing an azide group. The Huisgen reaction of 7with β-lactam 8 possessing a terminal alkyne group in the

presence of CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine(THPTA), and sodium-(L)-ascorbate proceeded smoothly toyield desired compound 3 with the linker now at the Northernsector of the molecule as suggested by Spiegel et al.26

Inhibitor 2 presented us with opportunities to explore thesouthern sector of the molecule for attachment. Structure−activity relationship studies of 29−11 found that bulkysubstituents at the 4-position of the azaindole unit decreasedthe inhibition activity of the compound. Thus, a northernsector connection would be ill-advised. Protection at the 1-position also gave diminished biological activities, whereas thepiperazine of 2 was already optimized. In contrast, substitutionwas tolerated at the 7-position of the azaindole. ON the basis ofthese data, we designed 4 bearing the linker at 7-position of theazaindole (southern sector connection).Target compound 4 was synthesized as shown in Scheme 4.

Commercially available 2-hydroxy pyridine derivative 9 wassubjected to bromination to afford 10 in good yield. Thehydroxy group of 10 was allylated using Ag2CO3. Formation ofthe core azaindole structure was achieved by treatment of 11with N,N-dimethylformamide dimethylacetal followed byreduction of nitro group in the presence of Fe in AcOH. Thebromo group of 12 was replaced by a methoxy group, and 13was treated with borane-dimethylsulfide complex followed byoxidation with hydrogen peroxide to replace the terminal olefinwith a primary alcohol. The reactivity of the substituent-freenitrogen atom at the 1-position of the azaindole in 14 wasproblematic. After analysis of a number of protecting groups,we found that the trimethylsilylethoxymethyl (SEM) groupcould be utilized.27 Protection of the reactive azaindole moietyyielded 15, which was subjected to etherification with 1628 toobtain 17. Removal of the SEM group was performed usingtetrabutylammonium fluoride (TBAF). A Friedel−Craftsreaction of 18 and methyl-2-chloro-2-oxoacetate was accom-plished in the presence of an excess amount of AlCl3.

29 Theresulting compound 19 was hydrolyzed and condensed with 1-benzoylpiperazine 20 mediated by 3-(diethoxy-phosphory-loxy)-3H-benzo[d][1.2.3]triazine-4-one (DEPBT)30 to affordthe derivative of BMS-488043 21. As the final step, a Huisgenreaction was performed under conditions described forsynthesis of 3 to obtain the desired compound 4.Conjugation of agent 3 with mAb 38C2 to form 22a was

carried out by incubating 38C2 with six equivalents of 3 in 10mM PBS (pH 7.4) at room temperature for two hours (Scheme5). We evaluated the conjugation by measuring the catalyticactivity of retro-aldol reaction of methodol as per the standardmethod.15 Once a conjugate is formed, the antibody cannotcatalyze the retro-aldol reaction of methodol. Compound 22ahad undetectable catalytic activity indicating that each of thekey catalytic lysine residues had reacted with the lactam (Figure3A). The MALDI-TOF mass analysis of 22a supported theeffective conjugation of 38C2 with 3 (Figure 3B). Thedifference in mass between 38C2 and our preparation of 22a

Scheme 1. Chemoselective Modification of Aldolase Antibody 38C2 to Yield a Chemically Programmed Antibody

Scheme 2. Schematic Representation of the Inhibition of theHIV Entry by gp120 Inhibitor-Programmed mAb 38C2

Figure 2. Synthetic targets for this study.

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corresponded to two equivalents of the small moleculederivative of 3. ESI-MS analysis also indicated that both ofthe two catalytic lysine moieties of 38C2 were modified (see

Supporting Information). Conjugate 22b was similarlyprepared from 4 and 38C2 and characterized (Figure 3A,C).Initially, the binding of antibody conjugates 22a and 22b to

gp120 was evaluated using an ELISA with gp120-coated plates(Figure 4). Neither unconjugated mAb or conjugate 22a boundto gp120 at 200 nM. Signal in these cases was similar to thenegative control of buffer alone (PBS). In contrast, the 22bbound strongly to gp120 at this concentration as did thepositive control broadly neutralizing antibody b12.31 The lackof binding by 22b is consistent with the results of thestructure−activity relationship study of related compounds thatthe bulky substituent at 4-position of the azaindole 1diminished the biological activity.9−11 Loss of binding activityat this concentration is consistent with the reported lowbinding activity of the DNP conjugate study and indicates thatthe northern site of the linker attachment is likely responsiblefor the loss in binding, not the fact that DNP conjugates withantibodies are reversibly formed.The anti-HIV activities of the conjugates 22a and 22b were

measured in neutralization assays with a single round ofinfectious virus (JRFL) as described previously.32 Conjugate22a showed very weak neutralization activity, consistent withthe low gp120 binding activity observed. Confirming ourhypothesis that the substituent at the northern sector 4-positionof 1 disrupted gp120 binding, neither 3 nor 7 were effective inthe assay (Figure 5A). The IC50 values of 4 and 21 with thelinker at southern 7-position were 67.5 and 25.4 nM,respectively. The conjugate 22b also blocked infection withan IC50 of 128 nM (Figure 5B). The unmodified mAb 38C2had no relevant anti-HIV activity. Evident from these studies isan impact on activity on linker attachment to the southern 7-position; however, significant neutralization activity waspreserved following linker addition at this site. We hadanticipated that conjugate 22b might exhibit significantlyenhanced activity over 4 and 21 given the bivalent display ofthe compound on the antibody following conjugation as wehave noted with other antibody targeting agents. The lack ofenhanced activity following conjugation suggests that 22b isunable to engage the HIV-1 virion in a bivalent interaction.Monovalent binding of natural antibodies that react with theCD4-binding site on gp120 has been suggested in theliterature.33 As previously reported, the chemically programmedantibody strategy has been shown to significantly extend thehalf-life of the targeting molecule relative to the unconjugatedmolecule in studies concerned with small molecule, peptide,

Scheme 3. Synthesis of the BMS-378806 Programming Agent 3a

aReagents and conditions: (a) NaH, DME, RT, 2 h then 50 °C, 3 h. (b) PCl3, EtOAc, RT, 2.5 h (37% in two steps). (c) CuSO4·5H2O, THPTA, Na-(L)-ascorbate, tBuOH, H2O, RT, 30 min (57%).

Scheme 4. Synthesis of the BMS-488043 ProgrammingAgent 21a

aReagents and conditions: (a) Br2, AcOH, AcONa, RT, 1 h (75%). (b)Ag2CO3, AllylBr, toluene, RT, 16 h (quant). (c) N,N-dimethylforma-mide dimethylacetal, DMF, 130 °C, 2 h. (d) Fe, AcOH, 100 °C, 90min (40% in two steps). (e) CuI, MeONa, MeOH, DMF, RT to 110°C, 19 h (87%). (f) BH3-Me2S, THF, 0 °C to RT, 4 h then H2O2,NaOH, H2O, 0 °C to RT, 15 h (42%). (g) KOH, SEMCl, THF, RT,30 min (88%). (h) NaH, DMF, RT, 19 h, (55%). (i) TBAF,ethylenediamine, THF, RT to 70 °C, 21 h (85%). (j) AlCl3,ClCOCO2Me, CH3NO2, CH2Cl2, RT, 4 h (40%). (k) NaOH, H2O,MeOH, RT, 1 h. (l) DEPBT, DIPEA, RT, 10 h (38% in two steps).(m) CuSO4·5H2O, THPTA, Na-(L)-ascorbate, tBuOH, H2O, RT, 3 h(69%).

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and aptamer targeting molecules.18−21 Additional biologicalactivities not accessible to the small molecule itself but rathercharacteristic of the antibody conjugate would be expected to

be seen in vivo for 22b such as ADCC and CDC activity, andthese activities may be important to the activities of naturalanti-HIV-1 antibodies.34

Scheme 5. Preparation of the gp120 Inhibitor Programmed Antibodies 22a and 22ba

aReagents and conditions: (a) PBS (pH 7.4), RT, 2 h.

Figure 3. Analysis of 22a and 22b. (A) Catalytic activity of 22a, 22b, and mAb 38C2 in the retro-aldol reaction of methodol. (B) Overlay of MALDImass spectra of mAb 38C2 (blue, MWav = 150 357) and 22a (green, MWav = 152 932). (C) Overlay of MALDI mass spectra of mAb 38C2 (blue,MWav = 150 357) and 22b (green, MWav = 152 946).

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In conclusion, synthesis of 3 and 4 allowed for theexploration of two linkage strategies for the BMS seriesattachment inhibitors 1 and 2 and their conjugation to mAb38C2 to create chemically programmed antibodies 22a and22b. Compound 4 and its antibody conjugate 22b possessedgood biological activity and effectively neutralized HIV-1,validating a southern site for linkage of this family ofattachment inhibitors. The northern linkage site explored in 3and 22a destroyed biological activity. We anticipate thatconjugation to the antibody should improve the bioactivity andpharmacokinetic properties significantly, and therefore, 22bwarrants further testing in anti-HIV models. While the

discovery of a viable site of conjugation for this promisingfamily of attachment inhibitors35 has allowed us to establishgood antiviral activity in the case of a chemically programmedantibody, active conjugation to this family of inhibitors shouldalso facilitate their application in chemically programmedvaccines,36 chemical approaches to bispecific antibodies,37 andtopical microbicides whose construction is hereby facilitated.

■ ASSOCIATED CONTENT*S Supporting InformationSynthetic procedures, analytical data, and procedures for ELISAand neutralization assay. This material is available free of chargevia the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*(C.F.B.) Tel: 858-784-9098. Fax: 858-784-2583. E-mail:carlos@scripps.edu.Author Contributions†These authors contributed equally to this work.FundingThis work was supported by NIH grant AI095038.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Angelica Cuevas for performing HIV-1 neutralizationassays.

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Figure 4. Binding of mAb 38C2 (200 nM), 22a (200 nM), 22b (200nM), and mAb b12 (2 nM) to JRFL gp120 as evaluated by ELISA.PBS indicates the background control.

Figure 5. Evaluation of small molecule gp120 inhibitors and mAbconjugates in a single-round neutralization assay usingU87.CD4.CCR5 cells and HIV-1 JRFL: (A) 1 (IC50 1.05 nM), 3(IC50 > 200 nM), 7 (IC50 > 200 nM), 22a (IC50 > 1000 nM), andmAb 38C2 (IC50 > 1000 nM); (B) 2 (IC50 1.98 nM), 4 (IC50 67.50nM), 21 (IC50 25.41 nM), 22b (IC50 128.6 nM), and mAb 38C2 (IC50>1000 nM).

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Supporting Information

Chemically Programmed Antibodies AS HIV-1 Attachment Inhibitors

Shinichi Sato‡, Tsubasa Inokuma‡, Nobumasa Otsubo, Dennis R. Burton and Carlos F. Barbas III*

Contents

General procedure page 2

Synthesis of the -lactam hapten 8 page 2-3

Synthesis of 3 page 4-5

Synthesis of 4 page 5-9

Bioconjugation of 38C2 and -lactam page 10-12

ELISA assay of the BMS conjugates 22 page 13

Neutralization assay of the gp120 inhibitors page 14 1H and 13C NMR page 15-46

S1

General procedure 1H NMR and 13C NMR spectra were recorded on Bruker DRX-600 (600 MHz), DRX-500 (500 MHz), Varian Inova-400

(400 MHz), or Varian MER-300 (300 MHz) spectrometers in the stated solvents using tetramethylsilane as an internal

standard. Chemical shifts were reported in parts per million (ppm) on the δ scale from an internal standard (NMR

descriptions: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad). Coupling constants, J, are reported in Hertz.

Mass spectroscopy was performed by the Scripps Research Institute Mass Spectrometer Center. Analytical thin-layer

chromatography and flash column chromatography were performed on Merck Kieselgel 60 F254 silica gel plates and Silica

Gel ZEOprep 60 ECO 40-63 Micron, respectively. Visualization was accomplished with anisaldehyde or KMnO4. High

performance liquid chromatography (HPLC) was performed on SHIMADZU GC-8A using VYDAC HPLC Column. LCMS

ESI analysis was performed on Agilent 1100 with SB C-18 column, using 1-100% acetonitrile gradient for 20 min method.

Protein deconvolution was performed using TOF Protein Confirmation Software. Unless otherwise noted, all the

materials were obtained from commercial suppliers, and were used without further purification. All solvents

were commercially available grade. All reactions were carried out under nitrogen atmosphere unless

otherwise mentioned. Amide starting materials, tyrosine 1, histidine, tryptophan, serine, cystein, lysine and

(Ile3)-pressionoic acid 6, were commercially available compounds or prepared according to published

procedures1). All proteins were obtained from commercial sources: chymotrypsinogen A (ImmunO), BSA and

myoglobin from equine heart (Sigma), Herceptin (Genentech). Cyclic RGD peptide was purchased from

Peptides International Inc and stored at -20 ℃. Zeba spin desalting columns (7k MWCO, product #89882)

and mini slyde-a-lyzer dialysis units (3.5k MWCO, product # 69550) were obtained from Pierce. Structural

analysis of chymotrypsinogen A (entry 2CGA), myoglobin (entry 1DWR) were based on information from the

Protein Data Bank. Sequence information for BSA was obtained from Swiss-PROT database (P02769).

Synthesis of the -lactam hapten 8

Methyl 4-(2,5,8,11,14-pentaoxaheptadec-16-yn-1-yl)benzoate (S2): To a solution of 3,6,9,12-tetraoxapentadec-14-yn-1-ol

(S1)1 (1.66 g, 7.15 mmol) in THF (45 mL) was added 57% NaH (361 mg, 8.58 mmol), and stirred at 0 ℃ for 15 min.

Methyl 4-(bromomethyl)benzoate (1.97 g, 8.58 mmol) was added and stirred at room temperature for 4 h, and then saturated

aqueous solution of NH4Cl was added to the reaction mixture. This was then extracted with AcOEt, and washed with H2O

and brine. The combined organic layer was dried over MgSO4, concentrated in vacuo, and purified by flash column

chromatography (Hex/AcOEt = 1/2) to afford compound S2 (1.31 g, 48%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ

8.01 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 4.63 (s, 2H), 4.20 (d, J = 2.4 Hz, 2H), 3.90 (s, 3H), 3.73-3.65 (m, 16H),

2.45 (t, J = 2.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 166.9, 143.6, 129.6, 129.2, 127.1, 79.6, 74.4, 72.5, 70.6, 70.55,

70.52, 70.3, 70.1, 69.8, 58.3, 52.0; HRMS: calcd for C20H28O7 (M+Na+) 403.1727, found 403.1725.

(1) Sun, X.-L.; Stabler, C.L.; Cazalis, C. S.; Chaikof, E. L.; Bioconjugate Chem. 2006, 17, 52-57.

S2

4-(2,5,8,11,14-Pentaoxaheptadec-16-yn-1-yl)benzoic acid (S3): To a solution of S2 (1.31 g, 3.44 mmol) in EtOH (35 mL)

was added 2M NaOH aqueous solution (17.2 mL, 34.4 mmol), and stirred at room temperature for 2 h. EtOH was

evaporated in vacuo and remained solution was neutralized with 2M HClaq. Reaction mixture was then extracted twice with

CH2Cl2, dried over MgSO4, concentrated in vacuo, and purified by flash column chromatography (AcOEt) to afford S3

(1.19 g, 94%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 7.93 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 4.58 (s,

2H), 4.14 (d, J = 2.4 Hz, 2H), 3.61-3.58 (m, 4H), 3.56-3.49 (m, 12H), 3.41 (t, J = 2.4 Hz, 1H); 13C NMR (126 MHz,

DMSO-d6) δ 167.2, 143.9, 129.7, 129.3, 127.1, 80.3, 77.0, 71.4, 69.9, 69.81, 69.79, 69.78, 69.75, 69.5, 69.4, 68.5, 57.5;

HRMS: calcd for C19H26O7 (M+H+) 367.1751, found 367.1757.

1-(4-(2,5,8,11,14-Pentaoxaheptadec-16-yn-1-yl)benzoyl)azetidin-2-one (8): S3 (500 mg, 1.36 mmol) was dissolved in

SOCl2 (10 mL) and stirred at room temperature for 1 h. After completion of the reaction SOCl2 was removed by evaporation

in vacuo, and residue was dissolved in CH2Cl2, washed with sat. NaHCO3aq, dried over MgSO4, concentrated in vacuo to

afford the corresponding acid chloride (505 mg). This compound was used for next reaction without further purification. To

a solution of 2-azetidinone (103 mg, 1.44 mmol) in THF (35 mL) was added nBuLi (2.88 M in hexane solution, 0.501 mL,

1.44 mmol) at -78 ℃, and stirred for 10 min. The above obtained acid chloride (505 mg, 1.31 mmol) in THF (5 mL) was

added at -78 ℃, and stirred at 0 ℃ for 1 h. 10% Citric acid aqueous solution was added, and then extracted with AcOEt.

Organic layer was washed with sat. NaHCO3aq and brine, dried over MgSO4, concentrated in vacuo, and purified by flash

column chromatography (Hex/AcOEt = 1/2) to afford 8 (225 mg, 41% over two steps) as a colorless oil. 1H NMR (400

MHz, CDCl3) δ 7.96 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 4.63 (s, 2H), 4.20 (d, J = 2.4 Hz, 2H), 3.78 (t, J = 5.3 H,

2H), 3.11 (t, J = 5.3 H, 2H), 3.72-3.64 (m, 16H), 2.44 (d, J = 2.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 166.2, 164.2, 144.3,

131.1, 130.1, 127.0, 79.9, 74.8, 72.7, 70.9, 70.80, 70.77, 70.6, 70.1, 69.3, 58.6, 37.0, 35.2; HRMS: calcd for C22H29NO7

(M+H+) 420.2017, found 420.2010.

S3

Synthesis of 3

To a stirred mixture of 57% NaH (401 mg, 9.53 mmol) in DME (50 mL) was added 62 (2.09 g, 9.53 mmol) and the

resulting yellow solution was stirred for 2 h, then 5 (835 mg, 1.91 mmol) in DME (25 mL) was added to the mixture and

stirred at 50 ℃ for 3 h. After completion of the reaction, the mixture was allowed to cool to room temperature, a saturated

solution of NH4Cl was slowly added, and the organic layer was extracted with CH2Cl2 twice. Organic phase were combined,

dried over MgSO4, concentrated in vacuo and the resulting crude brown residue was purified by flash column

chromatography (CH2Cl2 : MeOH = 8:1) to afford a yellow oil (555 mg), which was then dissolved in AcOEt (20 mL). To

this solution was added PCl3 (0.794 mL, 9.10 mmol) and stirred at room temperature for 2.5h. The reaction was cooled to

0 ℃, quenched by sat.NaHCO3aq until pH reached to 6. The mixture was extracted with AcOEt, dried over MgSO4,

concentrated in vacuo, and purified by flash column chromatography (CH2Cl2 : MeOH = 15:1) to afford compound 7 (415

mg, 37% over two steps) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.33 (d, J = 5.0Hz, 1H), 8.10 (d, J = 17.2 Hz, 1H),

7.57-7.37 (m, 5H), 6.81 (m, 1H), 5.17-2.90 (m, 15H), 4.51-4.34 (m, 2H), 4.17-3.97 (m, 2H), 3.94-3.80 (m, 2H), 3.42 (t, J =

4.9 Hz, 2H), 1.56-1.18 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 184.71, 167.10, 161.34, 151.99, 146.51, 146.44, 139.80,

135.90, 135.75, 135.40, 130.37, 128.97, 127.31, 114.16, 108.23, 102.04, 71.31, 71.04, 70.92, 70.85, 70.26, 69.54, 68.88,

68.82, 50.94, 45.06; HRMS: calcd for C29H35N7O7 (MH+) 594.2671, found 594.2669.

To a solution of 7 (14.2 mg, 23.9 mol) and 8 (20.5 mg, 48.9 mol) in tert-BuOH (1.2 mL) were added aqueous solutions

of THPTA3(50 mM, 240 L), CuSO4-5H2O (50 mM, 240 L) and Na-(L)-ascorbate (500 mM, 240 L). The reaction

mixture was stirred at room temperature for 30 min. Upon completion of the reaction CH2Cl2 was added, and then washed

with H2O and brine. Organic layer was dried over Na2SO4, concentrated in vacuo, and purified by preparative TLC (CHCl3 :

MeOH = 12:1) to give desired product 3 (13.9 mg, 57%) as yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.36-8.00 (m, 1H),

8.09-8.02 (m, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.73 (s, 1H), 7.53-7.45 (m, 7H), 6.84-6.78 (m, 1H), 4.74-4.66 (m, 4H), 4.68 (s,

2H), 4.54 (t, J = 4.0 Hz, 2H), 4.48-4.39 (m, 2H), 4.14-4.05 (m, 2H), 3.92-3.84 (m, 4H), 3.85 (t, J = 5.5 Hz, 2H), 3.79-3.64

(m, 24H), 3.66-3.59 (m, 5H), 3.18 (t, J = 5.5 Hz, 2H), 1.38-1.30 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 185.5, 171.4,

166.0, 163.9, 160.9, 144.8, 144.0, 135.4, 135.13, 135.11, 131.0, 130.0, 129.9, 128.7, 127.1, 127.0, 126.9, 123.8, 114.0, 72.5,

71.0, 70.7, 70.62, 70.56, 70.54, 70.53, 70.50, 70.46, 70.4, 69.9, 69.6, 69.4, 69.32, 69.30, 68.64, 68.59, 64.5, 50.1, 44.7, 36.8,

(2) (a) Kohn, H. L.; Park, K. D. Patent WO 2010014236. (b) Wang, T.; Zhang, Z.; Wallace, O. B.; Deshpande, M.; Fang, H.; Yang, Z.; Zadjura, L. M.; Tweedie, D. L.; Huang, S.; Zhao, F.; Ranadive, S.; Robinson, B. S.; Gong, Y-F.; Ricarrdi, K.; Spicer, T. P.; Deminie, C.; Rose, R.; Wang, H-G. H.; Blair, W. S.; Shi, P-Y.; Lin, P-F.; Colonno, R. J.; Meanwell, N. A. J. Med. Chem. 2003, 46, 4236-4239. (3) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853-2855.

S4

35.0, 30.9, 29.7; HRMS: calcd for C51H65N8O14 (M+H+) 1013.4615, found 1013.4624.

Synthesis of 4

To a solution of 9 (3.03 g, 19.7 mmol) in AcOH (90 mL) was added AcONa (3.50 g, 42.7 mmol) and Br2 (0.753 mL, 29.2

mmol) in AcOH (15 mL) and stirred at room temperature for 1 h. After completion of the reaction, the mixture was added

H2O, resulting insoluble powder was collected by filtration, washed with H2O, and dried in vacuo to afford 10 as a yellow

powder (1.69 g, 75%). ,1H NMR (500 MHz, DMSO-d6) δ 8.01 (s, 1H), 2.22 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ

154.6, 144.4, 142.7, 139.6, 100.5, 19.1; HRMS: calcd for C6H579BrN2O3 (M+H+) 232.9556, found 232.9554, calcd for

C6H581BrN2O3 (M+H+) 234.9541, found 234.9535.

To a solution of 10 (1.6 g, 6.72 mmol) in toluene (60 mL) was added Ag2CO3 (9.0 g, 32.6 mmol) and allyl bromide (6.0 mL,

70.9 mmol) and it was stirred at room temperature for 16 h. Then, the reaction mixture was filtered through celite,

concentrated in vacuo, and purified by flash column chromatography (hexane / EtOAc = 20 / 1) to afford 11 (1.87 g, quant.)

as a pale yellow crystal. 1H NMR (500 MHz, CDCl3) δ 8.30 (s, 1H), 6.00 (ddt, J = 17.2, 10.6, 5.4 Hz, 1H), 5.37 (dq, J =

17.2, 1.5 Hz, 1H), 5.26 (dq, J = 10.5, 1.2 Hz, 1H), 4.91 (dt, J = 5.4, 1.4 Hz, 2H), 2.37 (s, 3H); 13C NMR (125 MHz, CDCl3)

δ 153.8, 149.2, 141.4, 132.1, 118.7, 115.0, 68.2, 18.2; HRMS: calcd for C9H979BrN2O3 (M+H+) 272.9875, found 272.9860,

calcd for C9H981BrN2O3 (M+H+) 274.9854, found 274.9840.

To a solution of 11 (1.87 g, 6.72 mmol) in DMF (20 mL) was added N,N-dimethylforamide dimethylacetal (20 mL) and

stirred at 130 ℃ for 2 h. After completion of the reaction, the mixture was quenched with slow addition of H2O, and the

organic layer was extracted with Et2O twice. Organic phase were combined, dried over Na2SO4, concentrated in vacuo. The

resulting crude red residue was dissolved in AcOH (30 mL), added Fe powder (1.60 g, 28.7 mmol) and stirred at 100 ℃

for 90 min. Then, the reaction mixture was filtered through celite, washed with H2O, and quenched with sat.NaHCO3aq,

extracted with AcOEt, dried over Na2SO4 and purified by flash column chromatography (Hex / AcOEt = 10 / 1) to afford 12

(674 mg, 40% over two steps) as a brown solid. 1H NMR (500 MHz, CDCl3) δ 8.81-8.67 (br, 1H), 7.82 (s, 1H), 7.32 (t, J =

2.8 Hz, 1H), 6.57 (dd, J = 3.1, 2.3 Hz, 1H), 6.15 (ddt, J = 17.2, 10.4, 5.6 Hz, 1H), 5.43 (dq, J = 17.2, 1.5 Hz, 1H), 5.29 (dq,

J = 10.4, 1.1 Hz, 1H), 5.00 (dt, J = 5.6, 1.2 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 150.0, 136.0, 134.8, 133.6, 126.8, 121.2,

118.3, 105.9, 103.6, 67.0; HRMS: calcd for C10H979BrN2O (M+H+) 252.9977, found 252.9971. calcd for C10H9

81BrN2O

S5

(M+H+) 254.9956, found 254.9952.

To a solution of 12 (259 mg, 1.02 mmol) in DMF (7.0 mL) was added CuI (275 mg, 1.44 mmol) and 25% NaOMe/MeOH

solution (6.7 mL) and stirred at 110 ℃ for 19 h. Then the reaction mixture was quenched with H2O, filtered through celite,

extracted with Et2O, dried over Na2SO4 and purified by flash column chromatography (Hex / AcOEt = 1 / 5) to afford 13

(184 mg, 87%) as a pale brown solid. 1H NMR (500 MHz, CDCl3) δ 8.73-8.61 (br, 1H), 7.28 (s, 1H), 7.21 (t, J = 2.8 Hz,

1H), 6.65-6.60 (m, 1H), 6.15 (ddt, J = 17.2, 10.4, 5.6 Hz, 1H), 5.41 (dq, J = 17.2, 1.6 Hz, 1H), 5.26 (dq, J = 10.4, 1.2 Hz,

1H), 4.97 (dt, J = 5.7 Hz, 1.4 Hz, 2H), 3.95 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 146.6, 146.0, 134.1, 126.5, 125.7, 122.0,

117.7, 115.3, 100.8, 66.6, 56.4; HRMS: calcd for C11H12N2O2 (M+H+) 205.0971, found 205.0973.

A solution of the 13 (230 mg, 1.13 mmol) in THF (6.0 mL) was added BH3-SMe2/THF (0.2 mL/1 mL) at 0 ℃ by

controlling the rate of dropwise addition and stirred at the same temperature for 1 h, then allowed to warm to room

temperature. After stirring for 4 h, the reaction mixture was added H2O (2.5 mL), 3N NaOH aq. (2.5 mL) and H2O2 (2.5

mL) in a stepwise manner at 0 ℃ and stirred at room temperature for 15 h. Then the reaction mixture was extracted with

AcOEt, dried over Na2SO4, and purified by flash column chromatography (Hex : AcOEt = 1/2 to AcOEt 100%) to afford 14

(106 mg, 42%) as a pale brown solid. 1H NMR (400 MHz, CDCl3) δ 9.09-8.93 (br, 1H), 7.23 (t, J = 2.8 Hz, 1H), 7.20 (s,

1H), 6.63 (m, 1H), 4.65 (t, J = 5.8 Hz, 2H), 3.94 (s, 3H), 3.69 (t, J = 5.8 Hz, 2H), 1.99 (p, J = 5.8 Hz, 2H); 13C NMR (125

MHz, CDCl3) δ 147.0, 146.6, 126.7, 126.0, 121.9, 114.8, 100.9, 62.9, 58.6, 56.4, 33.3; HRMS: calcd for C11H14N2O3

(M+H+) 223.1077, found 223.1078.

To a solution of 14 (100 mg, 0.450 mmol) in THF (10 mL) were added [2-(chloromethyl)ethyl]trimethylsilane (87.3L,

0.495 mmol) and crushed KOH (152 mg, 271 mmol) at room temperature and stirred at the same temperature for 30 min.

Then the reaction mixture was quenched with H2O, extracted with AcOEt, dried over Na2SO4 and purified by flash column

chromatography (Hex / AcOEt = 1 / 1) to afford compound 15 (140 mg, 88%) as a white solid. 1H NMR (400 MHz, CDCl3)

δ 7.23 (s, 1H), 7.18 (d, J = 3.1 Hz, 1H), 6.59 (d, J = 3.1 Hz, 1H), 5.71 (s, 2H), 4.65 (t, J = 5.6 Hz, 2H), 3.93 (s, 3H), 3.73 (t,

J = 5.6 Hz, 2H), 3.50-3.43 (m, 2H), 2.02 (p, J = 5.6 Hz, 2H), 0.90-0.82 (m, 2H), -0.08 (s, 9H); 13C NMR (100 MHz,

CDCl3) δ 147.2, 146.4, 130.8, 128.4, 121.6, 115.2, 100.7, 77.3, 66.0, 63.4, 59.3, 56.4, 33.2, 18.1, -1.2; HRMS: calcd for

S6

C17H28N2O4Si (M+H+) 353.1891, found 353.1890.

To a solution of 15 (84.0 mg, 0.239 mmol) in DMF (5.0 mL) was added 164 (393 mg, 1.20 mmol) and 57% NaH (30.2 mg,

0.717 mmol) at room temperature and it was stirred at the same temperature for 6 h. In order to complete the reaction, same

amount of 16 and 57% NaH were added again, and stirred at the same temperature for 13 h. The reaction mixture was

quenched with a sat.NH4Claq, extracted with AcOEt, deried over Na2SO4, evaporated in vacuo and purified by flash column

chromatography (Hex / AcOEt = 1 / 2) to afford 17 (72.4 mg, 55%) as a colorless oil. At the same time, starting material 15

was recovered (34.3 mg, 41 %). 1H NMR (400 MHz, CDCl3) δ 7.24 (s, 1H), 7.17 (d, J = 3.1Hz, 1H), 6.58 (d, J = 3.1 Hz,

1H), 5.71 (s, 2H), 4.50 (t, J = 6.4 Hz, 2H), 3.92 (s, 3H), 3.71-3.58 (m, 18H), 3.51-3.43 (m, 2H), 3.39-3.31 (m, 2H), 2.21 (p,

J = 6.4 Hz, 2H), 0.87-0.79 (m, 2H), -0.08 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 146.6, 146.2, 130.2, 127.9, 121.8, 115.4,

100.7, 77.2, 71.0, 71.0, 70.93, 70.91, 70.7, 70.3, 56.4, 51.0, 29.9, 18.1, -1.2; HRMS: calcd for C25H43N5O7Si (M+H+)

554.3004, found 554.3007.

To a solution of the compound 17 (84.0 mg, 0.152 mmol) in THF (6.6 mL) was added 1M tetra-n-butylammonium floride /

THF solution (1.50 mL, 1.50 mmol) and ethylenediamine (375 L, 5.62 mmol) at room temperature and stirred at 70 ℃

for 21 h. After completion of the reaction, the reaction mixture was quenched with a sat.NH4Claq, extracted with AcOEt,

dried over Na2SO4, and purified by flash column chromatography (AcOEt 100%) to afford 18 (54.9 mg, 85%) as a colorless

oil. 1H NMR (400 MHz, CDCl3) δ 9.48-9.40 (br, 1H), 7.25 (s, 1H), 7.23 (t, J = 2.7 Hz, 1H), 6.60 (m, 1H), 4.52 (t, J = 6.2 Hz,

2H), 3.94 (s, 3H), 3.71-3.57 (m, 16H), 3.38-3.31 (m, 2H), 2.09 (p, J = 6.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 146.5,

146.4, 126.4, 125.8, 122.2, 115.3, 100.5, 70.92, 70.89, 70.8, 63.4, 56.4, 50.9, 29.8; HRMS: calcd for C19H29N5O6 (M+H+)

424.2190, found 424.2189.

To a solution of 18 (5.2 mg, 0.0123 mmol) in CH3NO2 (0.2 mL) and CH2Cl2 (2 mL) was added AlCl3 (19.7 mg, 0.148

mmol) and stirred at room temperature for 5 min. Then methyloxalyl chloride (13.6 L, 0.148 mmol) was added and stirred

(4) Ban, H.; Gavrilyuk, J.; Barbas, C. F., III. J. Am. Chem. Soc. 2010, 132, 1523-1525.

S7

at room temperature for 4 h. After that the reaction was quenched with MeOH (0.3 mL) and water, extracted with CH2Cl2,

dried over Na2SO4, evaporated in vacuo and purified by preparative TLC (CH2Cl2 / MeOH = 10 / 1) to afford 19 (2.5 mg,

40%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 3.3 Hz, 1H), 7.43 (s, 1H), 4.55 (t, J = 5.7 Hz, 2H),

3.94 (s, 3H), 3.92 (s, 3H), 3.76-3.59 (m, 16H), 3.38-3.30 (m, 2H), 2.08 (p, J = 5.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ

181.5, 165.2, 146.9, 146.5, 135.7, 123.7, 123.1, 119.8, 115.0, 71.1, 70.88, 70.86, 70.76, 70.75, 70.3, 57.3, 52.8, 50.9, 29.5;

HRMS: calcd for C22H31N5O9 (M+H+) 510.2194, found 510.2194.

To a solution of 19 (12.5 mg, 0.0245 mmol) in MeOH (5 mL) was added 0.1N NaOHaq. (2.5 mL) and stirred

at room temperature for 1 h. After checking the completion of the reaction by LC-MS, the mixture was quenched with

1N HCl (0.5 mL), extracted with AcOEt, dried over Na2SO4, and concentrated in vacuo. The resulting crude brown residue

was dissolved in DMF (2.0 mL), added 205 (7.5 mg, 0.0268 mmol), DEPBT (5.4 mg, 0.0268 mmol) and DIPEA (9.6 L,

0.0538 mmol) and stirred at room temperature for 10 h. After completion of the reaction, the mixture was quenched with a

sat.NH4Claq, extracted with AcOEt, dried over Na2SO4, and purified by preparative TLC (CH2Cl2 / MeOH = 20 / 1) to

afford 21 (6.8 mg, 38% over two steps) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.07 (s, 1H), 7.49-7.36 (m,

6H), 4.54 (t, J = 5.7 Hz, 2H), 3.92 (s, 3H), 3.89-3.41 (m, 8H), 3.72-3.60 (m, 16H), 3.35 (dd, J = 6.1, 3.9 Hz, 2H), 2.08 (p, J

= 5.7 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 186.2, 171.0, 147.0, 146.5, 136.0, 135.4, 130.5, 129.0, 127.4, 123.5, 123.2,

120.4, 115.7, 71.1, 70.86, 70.85, 70.80, 70.3, 69.6, 57.6, 51.0, 29.5; HRMS: calcd for C32H41N7O9 (M+H+) 668.3038, found

668.3040.

To a solution of 21 (5.3 mg, 7.94 mol) and 8 (3.6 mg, 8.73 mol) in tert-BuOH (400 L) were added aqueous solutions of

THPTA (50 mM, 100 L), CuSO4‧5H2O (50 mM, 100 L) and Na-(L)-ascorbate (500 mM, 100 L). The reaction mixture

was stirred at room temperature for 3 h. Upon completion of the reaction CH2Cl2 was added, and then washed with H2O and

brine. Organic layer was dried over Na2SO4, concentrated in vacuo, and purified by preparative TLC (CH2Cl2 : MeOH =

20:1) to give desired product 3 (60. mg, 69%) as yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 3.0 Hz, 1H), 7.95 (d,

(5) Wang, T.; Yin, Z.; Zhang, Z.; Bender, J. A.; Yang, Z.; Johnson, G.; Yang, Z.; Zadjura, L. M.; D’Arienzo, C. J.; DiGugno Parker, D.; Gesenberg, C.; Yamanaka, G. A.; Gong, Y. F.; Ho, H. T.; Fang, H.; Zhou, N.; McAuliffe, B. V.; Eggers, B. J.; Fan, L.; Nowicka-Sans, B.; Dicker, I. B.; Gao, Q.; Colonno, R. J.; Lin, P. F.; Meanwell, N. A.; Kadow, J. F. J. Med. Chem., 2009, 52, 7778-7787.

S8

J = 8.3 Hz, 2H), 7.81 (s, 1H), 7.47-7.40 (m, 8H), 4.70-4.64 (m, 2H), 4.63-4.58 (m, 2H), 4.52 (t, J = 5.1 Hz, 2H), 4.50 (t, J =

5.9 Hz, 2H), 3.93 (s, 3H), 3.90-3.45 (m, 8H), 3.85 (t, J = 5.1 Hz, 2H), 3.77 (t, J = 5.5 Hz, 2H), 3.72-3.54 (m, 30H), 3.11 (t, J

= 5.5 Hz, 2H), 2.06-1.98 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 186.24, 171.03, 166.34, 164.30, 146.96, 146.57, 144.40,

136.30, 135.46, 131.33, 130.49, 130.25, 129.01, 127.45, 127.26, 124.56, 123.41, 123.25, 120.33, 115.65, 72.89, 71.05,

70.97, 70.93, 70.91, 70.89, 70.80, 70.77, 70.39, 70.22, 70.04, 69.88, 69.07 64.82, 64.02, 57.66, 50.67, 37.14, 35.38, 29.56;

HRMS: calcd for C54H70N8O16 (MH+) 1087.4982, found 1087.4980.

S9

Bioconjugation of 38C2 and -lactam

A mixture of 47.8 L of 38C2 (55.8 M PBS solution), 14.4 L of PBS (pH 7.4) and 1.6 L of the hapten 3 (10 mM DMSO

solution) was incubated at 23 ℃ for 2 h. Complete conversion of the reaction was verified by loss of catalytic activity

mAb 38C2 as monitored by methodol-based assay.6 The reaction mixture was purified by gel filtration using Micro

Bio-Spin column (BIO-RAD) to remove excess hapten to obtain the conjugate 22a (37.6 M). The increasing of molecular

weight of antibodies were detected by MADLI-TOF and ESI-MS analysis.

○Result of the methodol assay

(6) Sinha, S. C.; Das, S.; Li, L. S.; Lerner, R. A. Barbas III, C. F. Nat. Protoc. 2007, 2, 449-456.

S10

○MALDI-TOF analysis

Overlay of MALDI mass spectra of mAb 38C2 (blue, MWav = 150357) and 22a (green, MWav = 152932)

Overlay of MALDI mass spectra of mAb 38C2 (blue, MWav = 150357) and 22b (green, MWav = 152946).

S11

○ESI-MS analysis

ESI-MS spectra of mAb 38C2

ESI-MS spectra of 22a (exact mass of 3 is 1012.45)

ESI-MS spectra of 22b (exact mass of 4 is 1086.49)

S12

ELISA assay of the BMS conjugates 22

96 well plates were coated with JR-FL gp120 (5 g/mL in PBS, pH 7.4, 50 L/well) at 4 ℃ overnight. Plates were washed

with Buffer A (1% nonfat milk and 0.1% Tween 20 in PBS, pH 7.4, 150 L/well, three times) and then blocked with 150 L

of 5% nonfat milk in PBS (pH 7.4) at 37 ℃ for 4 h. After removing the gp120 solution by decantation, varying

concentration of the conjugates were added in Buffer A (100 L/well) and incubated at 37 ℃ for 2 h. Then washing with

Buffer A (150 L/well, three times) and incubated with AP-conjugated anti-mouse (-selective, 100 L/well) (1:1000

dilution in Buffer A, pH 7.4) at 37 ℃ for 1 h. Then washing with Buffer A (150 L/well, three times) followed by washing

with PBS (pH 7.4, 150 L/well, three times), a solution of AP substrate (two tablets) in AP developer (10% diethanolamine,

0.01% MgCl2, 3 mM NaN3) was added (50 L/well) and monitored the optical density after 120 min by Mark microplate

reader (405 nm) (N = 3).

S13

Neutralization assay of the gp120 inhibitors

Replication-incompetent HIV-1 enveloped pseudovirus was generated by cotransfection of 293T cells with JR-FL HIV-1

Env-expressing plasmid and pSG3ΔEnv as previously described.7 Serial dilutions of samples (50 l) along with wt b12,

2D7, 2G12 and an isotype control antibody, DEN3, were added to TZM-bl target cells (50 l) and preincubated at 37 ℃

for 1 h. Following incubation 250TCID50 of pseudovirus (100 l) was added to each well and incubated at 37 ℃.

Luciferase reporter gene expression was evaluated 48 h post infection. The percentage of virus neutralization at a given

antibody concentration was determined by calculating the reduction in luciferase expression in the presence of antibody

relative to virus-only wells. The antibody dilution causing 50% reduction (50% inhibitory concentration [IC50]) was

calculated by regression analysis using GraphPad Prism (N = 2).

(7) Zwick, M. B.; Labrijn, A. F.; Wang, M.; Spenlehauer, C.; Saphire, E. O.; Binley, J. M.; Moore, J. P.; Stiegler, G.; Katinger, H.; Burton. D. R.; Parren, P. W. H. I. J. Viol. 2001, 75, 10892-10905.

S14

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

S19_Note. I-049/Compound S19_1H.fidC-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

1.00

18.6

8

3.29

2.30

2.17

2.17

2.14

0.00

0.00

2.43

2.43

2.44

3.66

3.66

3.67

3.68

3.69

3.69

3.70

3.91

4.19

4.20

4.63

7.28

7.41

7.43

8.00

8.02

S15

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-4000

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

32000S19_Note. I-049/Compound S19_13C.fidC-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

51.9

9

58.3

269

.03

69.8

070

.33

70.5

270

.55

70.6

072

.53

74.4

576

.75

77.0

077

.00

77.2

679

.59

127.

1212

9.23

129.

59

143.

60

166.

86

S16

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600S20_Note. I-050/Compound S20_1H.fid

1.00

12.9

54.

17

2.18

2.04

2.05

2.04

0.00

0.00

3.41

3.41

3.42

3.51

3.52

3.53

3.54

3.55

3.59

4.13

4.58

7.44

7.45

7.92

7.94

S17

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-1000

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

S20_Note. I-050/Compound S20_13C.fidC-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

39.5

039

.50

57.4

768

.50

69.4

869

.76

69.7

769

.79

69.8

4

77.0

4

80.3

1

127.

1212

9.28

129.

72

143.

70

167.

16

S18

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-200

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300S21_Note. I-053/Compound S21_1H.fid-600H-1 Routine 1D, DCH CryoProbe, 1-13-2006

1.00

2.26

18.8

12.

36

2.26

2.30

2.26

2.17

0.00

0.00

2.43

2.44

2.44

3.10

3.11

3.12

3.66

3.67

3.68

3.69

3.70

3.78

4.20

4.63

7.44

7.45

7.96

7.97

S19

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

S21_Note. I-053/Compound S21_13C.fid-600C-13 Routine 1D, DCH CryoProbe, 10-26-2006

34.9

336

.68

58.2

969

.00

69.7

970

.29

70.5

170

.57

72.4

574

.45

76.7

977

.00

77.0

077

.21

79.5

7

126.

8012

9.79

130.

86

143.

97

163.

8516

5.87

S20

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

shinsato500_03052012_2h/180DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.54

2.53

9.60

2.42

24.9

22.

51

2.29

0.98

4.96

0.96

1.00

0.06

240.

1385

0.98

46

1.32

101.

3599

HD

O

3.21

833.

4066

3.41

633.

4262

3.55

423.

7057

3.71

633.

7342

3.86

774.

0978

4.44

404.

6302

4.87

21

6.80

86

7.34

18 C

DC

l37.

4748

8.08

228.

1167

8.32

688.

3367

S21

20253035404550556065707580859095100105110115120125130f1 (ppm)

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

3.0E+08

3.5E+08

4.0E+08

shinsato500_03052012_2c/180C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

45.0

610

50.9

363

68.8

186

68.8

755

69.5

444

70.2

592

70.8

514

70.9

182

71.0

376

71.3

128

77.1

059

77.3

595

77.6

140

102.

0416

108.

2341

114.

1632

127.

3055

128.

9736

130.

3675

S22

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

0

50

100

150

200

250

300

350

400

450334-proton2/334DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

4.39

29.2

5

7.38

2.81

2.63

2.69

11.8

54.

88

1.17

5.82

7.51

1.00

3.16

0.80

-0.0

00.

00

1.27

1.33

3.09

3.10

3.12

3.56

3.64

3.66

3.77

3.81

4.03

4.37

4.46

4.60

6.74

7.27

7.41

7.43

7.66

7.94

7.96

8.01

8.26

S23

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-20000

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000334-carbon2/334C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

29.6

830

.91

35.0

236

.79

44.7

3

50.1

364

.49

68.5

968

.64

69.3

069

.32

69.3

869

.59

69.8

670

.40

70.4

670

.50

70.5

370

.54

70.5

670

.62

70.6

971

.04

72.5

176

.75

77.0

077

.00

77.2

077

.25

113.

9612

3.78

126.

8812

7.02

127.

0512

8.66

129.

8613

0.04

130.

9613

5.11

135.

13

144.

0314

4.77

160.

8616

3.94

165.

95

171.

43

185.

54

S24

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08

3.0E+08

3.2E+08

3.4E+08

3.6E+08

3.8E+08shinsato500_03062012_2h/180DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.07

1.00

-0.0

003

2.21

90

2.51

05

8.01

41

S25

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08

2.20E+08

2.30E+08shinsato500_03062012_2c/180C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

19.0

505

39.8

926

DM

SO

40.0

594

DM

SO

40.2

264

DM

SO

40.3

935

DM

SO

40.5

606

DM

SO

40.7

278

DM

SO

40.8

947

DM

SO

100.

4532

139.

5696

142.

6746

144.

3624

154.

5444

S26

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.00E+07

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08

2.20E+08

2.30E+08

2.40E+08

shin500_0808/54DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.27

2.19

1.12

1.12

1.06

1.00

-0.0

001

1.57

67 H

DO

2.36

302.

3681

4.90

374.

9066

4.90

964.

9144

4.91

744.

9203

5.25

325.

2716

5.27

435.

3857

5.95

995.

9706

5.98

115.

9917

5.99

436.

0025

6.00

506.

0156

6.02

616.

0368

7.26

41 C

DC

l3

8.30

13

S27

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-2.00E+07

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08shinsato500_01102012_c/180C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

18.1

716

68.2

273

77.1

058

77.3

596

77.6

145

114.

9556

118.

7340

132.

0947

141.

4141

149.

1805

153.

8145

S28

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

shin500_0813up/54DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

2.33

1.17

1.16

1.14

1.07

1.08

1.00

0.97

-0.0

068

-0.0

016

-0.0

001

0.00

63

1.60

83 H

DO

4.98

974.

9921

4.99

485.

0009

5.00

355.

0061

5.27

725.

2979

5.30

065.

4388

5.44

206.

1135

6.13

456.

1479

6.16

89

6.56

686.

5713

6.57

296.

5774

7.26

06 C

DC

l37.

3092

7.31

467.

3204

7.82

26

8.74

33

S29

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

shinsato500_01116012_c/180C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

67.0

287

77.1

061

77.3

603

77.6

139

103.

6309

105.

8885

118.

2823

121.

2033

126.

7751

133.

6362

134.

7698

136.

0362

150.

0105

S30

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.00E+07

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08

2.20E+08

2.30E+08

shin500_0813down/54DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.23

2.13

1.03

1.05

1.00

0.99

1.01

0.96

0.87

0.00

04

1.68

40 H

DO

3.95

43

4.96

814.

9710

4.97

384.

9794

4.98

214.

9849

5.25

485.

2576

5.40

135.

4327

5.43

58

6.12

386.

1345

6.14

466.

1582

6.17

91

6.62

256.

6272

6.63

31

7.20

327.

2086

7.21

427.

2612

CD

Cl3

7.27

31

8.67

53

S31

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.00E+07

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08

2.20E+08

2.30E+08

shin500_0813down/54DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.23

2.13

1.03

1.05

1.00

0.99

1.01

0.96

0.87

0.00

04

1.68

40 H

DO

3.95

43

4.96

814.

9710

4.97

384.

9794

4.98

214.

9849

5.25

485.

2576

5.40

135.

4327

5.43

58

6.12

386.

1345

6.14

466.

1582

6.17

91

6.62

256.

6272

6.63

31

7.20

327.

2086

7.21

427.

2612

CD

Cl3

7.27

31

8.67

53

S32

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08

3.0E+08

3.2E+08

3.4E+08

3.6E+08

3.8E+08

4.0E+08

4.2E+08400shinsato_0815/60H-1 Routine 1D experiment. BBO Probe, 9-13-2007

2.39

0.81

2.17

3.29

2.25

1.00

0.97

1.11

0.95

0.00

030.

0748

1.24

111.

2589

1.27

061.

2768

1.28

661.

3990

1.96

361.

9780

1.99

232.

0068

2.02

112.

0482

2.98

37

3.35

25

3.67

933.

6937

3.70

803.

9352

4.11

454.

1327

4.63

524.

6497

4.66

40

6.61

736.

6244

6.63

02

7.20

527.

2160

7.22

307.

2297

7.26

63 C

DC

l3

9.02

86

S33

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-4.0E+07

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08

3.0E+08

3.2E+08

3.4E+08

3.6E+08

3.8E+08shinsato500_01092012_c/180C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

33.3

228

56.4

265

58.5

619

62.9

087

77.1

063

77.3

599

77.6

135

100.

9190

109.

5777

114.

8298

121.

8873

125.

9549

126.

7190

146.

6176

146.

9833

S34

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.00E+07

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08

1.70E+08

1.80E+08

1.90E+08

2.00E+08

2.10E+08

2.20E+08shinsato400_120411_h/150H-1 Routine 1D experiment. BBO Probe, 9-13-2007

8.97

2.16

2.14

2.04

2.04

3.07

2.09

2.00

0.96

0.99

1.00

-0.0

976

-0.0

923

-0.0

838

-0.0

759

-0.0

677

-0.0

647

-0.0

598

-0.0

202

0.83

830.

8447

0.84

720.

8587

0.86

120.

8705

0.87

310.

8794

0.88

85

1.99

132.

0055

2.01

982.

0342

2.04

382.

0481

3.44

853.

4547

3.46

653.

4691

3.47

133.

4832

3.48

963.

7045

3.71

853.

7326

3.74

603.

9202

3.92

854.

2265

4.63

304.

6474

4.66

16

5.70

97

6.58

586.

5935

7.17

187.

1796

7.23

067.

2604

S35

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

3.0E+08

3.5E+08

4.0E+08

4.5E+08

shinsato400_120411_c/150C-13 Routine 1D experiment. BBO Probe, 9-13-2007

-1.1

749

18.0

534

33.2

181

56.3

821

59.3

070

63.4

343

66.0

328

77.0

425

CD

Cl3

77.3

190

77.3

598

CD

Cl3

77.6

779

CD

Cl3

100.

6872

115.

1612

121.

6171

128.

4218

130.

7504

146.

4380

147.

1746

S36

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

shinsato400_120711_H/150H-1 Routine 1D experiment. BBO Probe, 9-13-2007

8.91

2.21

2.06

2.38

2.22

17.5

1

3.07

1.92

1.95

0.93

1.00

0.91

-0.0

996

-0.0

913

-0.0

832

-0.0

764

-0.0

264

-0.0

186

0.05

570.

8153

0.83

550.

8493

0.85

59

1.19

211.

2208

1.23

871.

2565

1.37

04

2.02

49 H

DO

2.09

212.

1082

2.12

432.

1404

2.15

653.

3575

3.45

073.

4712

3.49

123.

6130

3.62

553.

6310

3.63

543.

6415

3.64

393.

6520

3.65

613.

6687

3.91

91

4.48

494.

5009

4.51

70

5.70

055.

7167

6.57

346.

5812

7.16

647.

1742

7.22

077.

2457

7.26

00

S37

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08shinsato400_120711_c/150C-13 Routine 1D experiment. BBO Probe, 9-13-2007

-1.2

075

-1.1

784

-1.1

724

-1.1

578

-1.1

412

18.0

835

29.9

364

50.9

540

56.3

615

70.3

024

70.6

662

70.9

075

70.9

282

70.9

518

70.9

663

77.0

416

CD

Cl3

77.1

499

77.3

603

CD

Cl3

77.6

776

CD

Cl3

100.

7105

115.

4327

121.

8140

127.

9099

130.

2412

146.

2250

146.

5683

S38

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

shinsato400_120911_h/150H-1 Routine 1D experiment. BBO Probe, 9-13-2007

2.16

2.22

17.6

1

3.41

2.11

0.94

1.07

1.00

0.82

0.00

02

1.25

67

2.05

872.

0743

2.08

982.

1053

2.12

073.

3487

3.36

163.

6067

3.61

253.

6181

3.63

143.

6466

3.65

083.

6553

3.66

173.

6776

3.93

333.

9452

4.50

474.

5200

4.53

544.

6463

5.11

91

6.59

116.

5971

6.60

366.

6147

7.20

227.

2196

7.22

617.

2332

7.25

137.

2730

CD

Cl3

9.44

309.

4435

S39

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

-4.0E+07

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08

3.0E+08

3.2E+08

3.4E+08shinsato400_120911_c/150C-13 Routine 1D experiment. BBO Probe, 9-13-2007

29.7

592

50.9

391

56.4

380

63.3

904

70.8

424

70.8

942

70.9

217

77.0

415

CD

Cl3

77.3

602

CD

Cl3

77.6

774

CD

Cl3

100.

4554

115.

2938

122.

2205

125.

7951

126.

3775

146.

4371

146.

4782

S40

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

shinsato400_121211_h/150H-1 Routine 1D experiment. BBO Probe, 9-13-2007

2.62

2.43

17.7

52.

873.

04

2.20

1.06

1.00

-0.0

087

-0.0

003

0.00

790.

0708

0.83

380.

8442

0.85

230.

8617

0.87

180.

8800

0.88

781.

2547

2.04

752.

0502

HD

O2.

0646

HD

O2.

0791

HD

O2.

0935

2.10

782.

2904

3.33

073.

3429

3.35

223.

3559

3.62

223.

6344

3.66

113.

6672

3.70

903.

9232

3.93

58

4.53

834.

5528

4.56

71

5.11

86

7.26

57 C

DC

l37.

4295

7.55

63

8.20

988.

2180

9.79

29

S41

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

-1.00E+07

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

1.10E+08

1.20E+08

1.30E+08

1.40E+08

1.50E+08

1.60E+08shinsato400_121411_2c/150C-13 Routine 1D experiment. BBO Probe, 9-13-2007

29.4

469

50.9

250

52.7

628

57.2

717

70.2

909

70.7

484

70.7

617

70.8

559

70.8

794

71.0

749

77.0

419

CD

Cl3

77.3

598

CD

Cl3

77.6

773

CD

Cl3

115.

0331

119.

8442

123.

1246

123.

6886

135.

7136

146.

4787

146.

8534

165.

1890

181.

5082

S42

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-5.0E+07

0.0E+00

5.0E+07

1.0E+08

1.5E+08

2.0E+08

2.5E+08

3.0E+08

3.5E+08

4.0E+08

4.5E+08

5.0E+08

5.5E+08

6.0E+08

6.5E+08shinsato500_121711_h/145DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

2.41

2.43

3.14

18.1

33.

493.

59

2.10

6.27

1.00

-0.0

064

-0.0

009

0.00

010.

0054

0.07

15

0.86

610.

8798

0.89

341.

2554

1.31

241.

3213

1.32

641.

3357

2.04

482.

0565

2.06

782.

0793

2.09

072.

1021

2.20

422.

3208

3.35

593.

6254

3.63

483.

6454

3.65

703.

6695

3.67

333.

6813

3.68

523.

6876

3.69

643.

7078

3.92

45

4.52

764.

5390

4.55

02

5.11

74

7.26

71 C

DC

l37.

2682

7.42

427.

4336

7.47

37

8.07

28

S43

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-5.0E+06

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

3.5E+07

4.0E+07

4.5E+07

5.0E+07

5.5E+07

6.0E+07

6.5E+07shinsato500_121711_c/145C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

29.4

689

50.9

559

57.6

249

69.6

216

70.3

026

70.7

981

70.8

640

71.0

500

77.1

065

77.3

600

77.5

645

77.6

137

115.

6757

120.

3477

123.

1540

123.

4475

127.

4207

129.

0126

130.

5014

135.

3948

136.

0362

146.

5122

146.

9531

171.

0472

186.

2315

S44

-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)

-2.0E+07

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

1.6E+08

1.8E+08

2.0E+08

2.2E+08

2.4E+08

2.6E+08

2.8E+08shinsato500_121911_h/145DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe

3.10

2.16

41.1

73.

652.

973.

89

4.42

2.33

2.38

8.32

0.90

2.04

1.00

-0.0

070

-0.0

005

0.00

56

1.20

391.

2152

1.21

631.

2550

1.27

36

2.01

292.

0249

2.03

68

3.09

593.

1066

3.11

793.

4877

3.61

513.

6246

3.63

293.

6394

3.64

563.

9250

4.48

374.

4954

4.50

724.

5134

4.52

374.

5336

4.60

864.

6681

7.26

257.

4161

7.43

13

7.81

257.

9438

7.96

038.

1028

8.10

87

S45

-20-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

-2000000

-1000000

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

11000000

12000000

13000000

14000000

15000000

16000000

17000000

18000000

19000000

20000000

21000000

22000000

23000000

24000000

25000000shinsato500_121911_c/145C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05

0.32

100.

3361

29.5

620

35.3

834

37.1

432

50.6

736

57.6

583

64.0

191

64.8

211

69.0

726

69.8

838

70.0

385

70.2

245

70.3

918

70.7

674

70.8

035

70.8

873

70.9

090

70.9

728

71.0

545

72.8

874

77.1

062

77.3

597

77.5

648

77.6

133

115.

6474

120.

3286

123.

2520

123.

4069

124.

5590

127.

2589

127.

4525

129.

0170

130.

2503

130.

4866

131.

3310

135.

4621

136.

3050

144.

4015

146.

5591

146.

9553

164.

2999

166.

3399

171.

0340

186.

2423

S46