Letter
pubs.acs.org/acsmedchemlett
Chemically Programmed Antibodies As HIV‑1 Attachment Inhibitors
Shinichi 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 and
Microbial 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 application
of two small-molecule anti-HIV compounds for the creation of
chemically programmed antibodies. N-Acyl-β-lactam derivatives of two previously described molecules BMS-378806 and
BMS-488043 that inhibit the interaction between HIV-1
gp120 and T-cells were synthesized and used to program the
binding activity of aldolase antibody 38C2. Discovery of a
successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1
gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry
inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel
bispecific antibodies and topical microbicides.
KEYWORDS: Bioconjugation, anti-HIV agent, chemically programmed antibody, microbicide, entry inhibitor
T
mAb could further enhance their activity in vivo through
antibody effector functions such as antibody dependent cellular
cytotoxicity (ADCC) and complement dependent cytotoxicity
(CDC). Recently, we have described the development of
chemically programmed antibodies based on the use of mAb
38C2, an aldolase antibody generated by reactive immunization
by using a 1,3-diketone hapten.22−24 This antibody possesses a
low pKa lysine residue in its binding site that is key to its
aldolase activity that can be site-selectively labeled with N-acylβ-lactams to produce a chemically programmed antibody.
Chemically programmed antibodies have duration times after
systemic dosing that depend on the properties of the antibody
rather than on those of the conjugated small molecule,
providing for very significant extensions in the pharmacokinetic
profiles of the attached molecule.18,20 We have demonstrated
the utility of this approach by preparing mAb conjugates that
show promising activity in a variety of cancer models but also in
the area of anti-infectives through the preparation of CCR5
blocking mAbs that inhibit HIV-1 entry and neuraminidase
inhibitors that neutralize influenza.18−20
Treatment as well as prophylaxis of HIV-1 infection requires
the development of a cocktail of inhibitors. In order to
complement our anti-CCR5 blockade based on this strategy,18
we envisioned that the conjugate of mAb 38C2 and the smallmolecule gp120 inhibitor would bind to gp120 and inhibit
CD4-mediated entry of HIV-1 into cells (Scheme 2). In related
work, Spiegel and co-workers recently reported that a derivative
of HIV-1 inhibitor 1 modified with a 1,3-dinitrophenyl hapten
moiety binds to HIV gp120.25 Their compound was designed
to bind noncovalently with polyclonal anti-1,3-dinitrophenyl
he retrovirus HIV-1, which causes acquired immune
deficiency syndrome (AIDS), has infected 34 million
people worldwide, and this number is expected to increase by
2.5 million each year into the near future.1 Although the
combination reverse transcriptase inhibitor/protease inhibitor
treatment known as HAART has proven successful,2,3 side
effects and viral escape are significant issues, and new
treatments are needed. The viral envelope protein gp120, the
primary target for antibody mediated viral neutralization, is an
emerging target for small molecule treatment of HIV
infection.4,5 This protein is responsible for the entry of HIV
into host cells. In the initial step of entry, gp120 binds to the
CD4 glycoprotein expressed on the surface of human immune
cells. Bristol−Myers Squibb Pharmaceutical Research Institute
discovered small molecules BMS-378806 (1) and BMS-488043
(2) that bind to gp120 (Figure 1) and block its interaction with
Figure 1. Chemical structures of gp120 inhibitors.
CD4.6−11 However, the short pharmacokinetic profiles of these
small molecule inhibitors (half-lives after intravenous injection
are 0.3 and 2.4 h, respectively) may limit their clinical
application.
We hypothesize that the pharmacokinetic properties of these
small molecule gp120 inhibitors can be improved by
conjugation with a monoclonal antibody (mAb) (Scheme
1).12−21 Furthermore, coupling of the small molecule to the
© XXXX American Chemical Society
Received: March 8, 2013
Accepted: April 7, 2013
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Scheme 1. Chemoselective Modification of Aldolase Antibody 38C2 to Yield a Chemically Programmed Antibody
presence of CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine
(THPTA), and sodium-(L)-ascorbate proceeded smoothly to
yield desired compound 3 with the linker now at the Northern
sector of the molecule as suggested by Spiegel et al.26
Inhibitor 2 presented us with opportunities to explore the
southern sector of the molecule for attachment. Structure−
activity relationship studies of 29−11 found that bulky
substituents at the 4-position of the azaindole unit decreased
the inhibition activity of the compound. Thus, a northern
sector connection would be ill-advised. Protection at the 1position also gave diminished biological activities, whereas the
piperazine of 2 was already optimized. In contrast, substitution
was tolerated at the 7-position of the azaindole. ON the basis of
these data, we designed 4 bearing the linker at 7-position of the
azaindole (southern sector connection).
Target compound 4 was synthesized as shown in Scheme 4.
Commercially available 2-hydroxy pyridine derivative 9 was
subjected to bromination to afford 10 in good yield. The
hydroxy group of 10 was allylated using Ag2CO3. Formation of
the core azaindole structure was achieved by treatment of 11
with N,N-dimethylformamide dimethylacetal followed by
reduction of nitro group in the presence of Fe in AcOH. The
bromo group of 12 was replaced by a methoxy group, and 13
was treated with borane-dimethylsulfide complex followed by
oxidation with hydrogen peroxide to replace the terminal olefin
with a primary alcohol. The reactivity of the substituent-free
nitrogen atom at the 1-position of the azaindole in 14 was
problematic. After analysis of a number of protecting groups,
we found that the trimethylsilylethoxymethyl (SEM) group
could be utilized.27 Protection of the reactive azaindole moiety
yielded 15, which was subjected to etherification with 1628 to
obtain 17. Removal of the SEM group was performed using
tetrabutylammonium fluoride (TBAF). A Friedel−Crafts
reaction of 18 and methyl-2-chloro-2-oxoacetate was accomplished in the presence of an excess amount of AlCl3.29 The
resulting compound 19 was hydrolyzed and condensed with 1benzoylpiperazine 20 mediated by 3-(diethoxy-phosphoryloxy)-3H-benzo[d][1.2.3]triazine-4-one (DEPBT)30 to afford
the derivative of BMS-488043 21. As the final step, a Huisgen
reaction was performed under conditions described for
synthesis 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 10
mM PBS (pH 7.4) at room temperature for two hours (Scheme
5). We evaluated the conjugation by measuring the catalytic
activity of retro-aldol reaction of methodol as per the standard
method.15 Once a conjugate is formed, the antibody cannot
catalyze the retro-aldol reaction of methodol. Compound 22a
had undetectable catalytic activity indicating that each of the
key catalytic lysine residues had reacted with the lactam (Figure
3A). The MALDI-TOF mass analysis of 22a supported the
effective conjugation of 38C2 with 3 (Figure 3B). The
difference in mass between 38C2 and our preparation of 22a
Scheme 2. Schematic Representation of the Inhibition of the
HIV Entry by gp120 Inhibitor-Programmed mAb 38C2
(DNP) antibodies in situ, with the aim of enhancing the activity
of 1. The activity of 1, however, was severely compromised
upon the addition of the DNP linker in their report. Parental 1
has HIV-1 neutralization activity in the nanomolar range,
whereas DNP linked 1 demonstrated micromolar activity in
binding studies and was not shown to neutralize HIV-1. Our
conjugate strategy differs since we use a defined monoclonal
antibody covalently linked to 1. We hypothesized that our
strategy might allow us to recover the potent activity of 1
directly if the lack of activity of their DNP derivative of 1 was
due to the noncovalent nature of attachment to antibody.
Alternatively, modification of the linkage strategy to this family
of inhibitors might be key to restoring the activity of the small
molecule.
To prepare derivatives of the Bristol−Myers Squibb
compounds for conjugation to mAb, we first prepared β-lactam
3 (Figure 2) derived from BMS-378806 (1) from the known
compound 5 (Scheme 3).7 Substitution of the nitro group by
alcohol 6 followed by the treatment of PCl3 gave BMS-378806
derivative 7 bearing an azide group. The Huisgen reaction of 7
with β-lactam 8 possessing a terminal alkyne group in the
Figure 2. Synthetic targets for this study.
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Scheme 3. Synthesis of the BMS-378806 Programming Agent 3a
Reagents 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%).
a
Supporting Information). Conjugate 22b was similarly
prepared 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 bound
to gp120 at 200 nM. Signal in these cases was similar to the
negative control of buffer alone (PBS). In contrast, the 22b
bound strongly to gp120 at this concentration as did the
positive control broadly neutralizing antibody b12.31 The lack
of binding by 22b is consistent with the results of the
structure−activity relationship study of related compounds that
the bulky substituent at 4-position of the azaindole 1
diminished the biological activity.9−11 Loss of binding activity
at this concentration is consistent with the reported low
binding activity of the DNP conjugate study and indicates that
the northern site of the linker attachment is likely responsible
for the loss in binding, not the fact that DNP conjugates with
antibodies are reversibly formed.
The anti-HIV activities of the conjugates 22a and 22b were
measured in neutralization assays with a single round of
infectious virus (JRFL) as described previously.32 Conjugate
22a showed very weak neutralization activity, consistent with
the low gp120 binding activity observed. Confirming our
hypothesis that the substituent at the northern sector 4-position
of 1 disrupted gp120 binding, neither 3 nor 7 were effective in
the assay (Figure 5A). The IC50 values of 4 and 21 with the
linker at southern 7-position were 67.5 and 25.4 nM,
respectively. The conjugate 22b also blocked infection with
an IC50 of 128 nM (Figure 5B). The unmodified mAb 38C2
had no relevant anti-HIV activity. Evident from these studies is
an impact on activity on linker attachment to the southern 7position; however, significant neutralization activity was
preserved following linker addition at this site. We had
anticipated that conjugate 22b might exhibit significantly
enhanced activity over 4 and 21 given the bivalent display of
the compound on the antibody following conjugation as we
have noted with other antibody targeting agents. The lack of
enhanced activity following conjugation suggests that 22b is
unable to engage the HIV-1 virion in a bivalent interaction.
Monovalent binding of natural antibodies that react with the
CD4-binding site on gp120 has been suggested in the
literature.33 As previously reported, the chemically programmed
antibody strategy has been shown to significantly extend the
half-life of the targeting molecule relative to the unconjugated
molecule in studies concerned with small molecule, peptide,
Scheme 4. Synthesis of the BMS-488043 Programming
Agent 21a
a
Reagents and conditions: (a) Br2, AcOH, AcONa, RT, 1 h (75%). (b)
Ag2CO3, AllylBr, toluene, RT, 16 h (quant). (c) N,N-dimethylformamide dimethylacetal, DMF, 130 °C, 2 h. (d) Fe, AcOH, 100 °C, 90
min (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%).
corresponded to two equivalents of the small molecule
derivative of 3. ESI-MS analysis also indicated that both of
the two catalytic lysine moieties of 38C2 were modified (see
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Scheme 5. Preparation of the gp120 Inhibitor Programmed Antibodies 22a and 22ba
a
Reagents 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 MALDI
mass 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).
and aptamer targeting molecules.18−21 Additional biological
activities not accessible to the small molecule itself but rather
characteristic of the antibody conjugate would be expected to
be seen in vivo for 22b such as ADCC and CDC activity, and
these activities may be important to the activities of natural
anti-HIV-1 antibodies.34
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discovery of a viable site of conjugation for this promising
family of attachment inhibitors35 has allowed us to establish
good antiviral activity in the case of a chemically programmed
antibody, active conjugation to this family of inhibitors should
also facilitate their application in chemically programmed
vaccines,36 chemical approaches to bispecific antibodies,37 and
topical microbicides whose construction is hereby facilitated.
■
ASSOCIATED CONTENT
* Supporting Information
S
Synthetic procedures, analytical data, and procedures for ELISA
and neutralization assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
Figure 4. Binding of mAb 38C2 (200 nM), 22a (200 nM), 22b (200
nM), and mAb b12 (2 nM) to JRFL gp120 as evaluated by ELISA.
PBS indicates the background control.
AUTHOR INFORMATION
Corresponding 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.
Funding
This work was supported by NIH grant AI095038.
Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS
We thank Angelica Cuevas for performing HIV-1 neutralization
assays.
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Chien, C.; Grasela, D. M.; Zheng, M.; Lin, P.-F.; Meanwell, N. A.;
Kadow, J. F. Inhibitors of human immunodeficiency virus type 1 (HIV1) attachment. 12. Structure−activity relationships associated with 4fluoro-6-azaindole derivatives leading to the identification of 1-(4benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248). J. Med. Chem.
2013, 56, 1656−1669.
(36) Popkov, M.; Gonzalez, B.; Sinha, S. C.; Barbas, C. F., III. Instant
immunity through chemically programmable vaccination and covalent
self-assembly. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 4378−4383.
(37) Gavrilyuk, J.; Ban, H.; Uehara, H.; Sirk, S. J.; Saye-Francisco, K.;
Cuevas, A.; Zablowsky, E.; Oza, A.; Seaman, M. S.; Burton, D. R.;
Barbas, C. F., III. Antibody conjugation approach enhances breadth
and potency of neutralization of anti-HIV-1 antibodies and CD4-IgG.
J. Virol. 2013, DOI: 10.1128JVI.03146-12.
F
dx.doi.org/10.1021/ml400097z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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
Synthesis of 3
Synthesis of 4
page 2-3
page 4-5
page 5-9
Bioconjugation of 38C2 and -lactam
ELISA assay of the BMS conjugates 22
Neutralization assay of the gp120 inhibitors
1
H and 13C NMR
page 10-12
page 13
page 14
page 15-46
S1
General procedure
1
H NMR and
13
C 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);
13
C 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);
13
C 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);
13
C 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 C10H981BrN2O
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 (87.3L,
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);
13
C 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.
1
H 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
0.00
0.00
2.44
2.43
2.43
4.20
4.19
3.91
3.70
3.69
3.69
3.68
3.67
3.66
3.66
4.63
7.43
7.41
7.28
8.02
8.00
S19_Note. I-049/Compound S19_1H.fid
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
700
650
600
550
500
450
400
350
300
250
200
150
100
50
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S15
4.0
-50
1.00
18.68
3.29
2.30
2.17
2.17
2.14
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
51.99
79.59
77.26
77.00
77.00
76.75
74.45
72.53
70.60
70.55
70.52
70.33
69.80
69.03
58.32
129.59
129.23
127.12
143.60
166.86
S19_Note. I-049/Compound S19_13C.fid
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
32000
30000
28000
26000
24000
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
-2000
-4000
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S16
80
70
60
50
40
30
20
10
0
-10
-20
0.00
0.00
4.13
3.59
3.55
3.54
3.53
3.52
3.51
3.42
3.41
3.41
4.58
7.45
7.44
7.94
7.92
S20_Note. I-050/Compound S20_1H.fid
1600
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S17
4.0
-100
4.17
12.95
1.00
2.18
2.04
2.05
2.04
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
39.50
39.50
69.84
69.79
69.77
69.76
69.48
68.50
57.47
80.31
77.04
129.72
129.28
127.12
143.70
167.16
S20_Note. I-050/Compound S20_13C.fid
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
8000
7500
7000
6500
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
-500
-1000
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S18
80
70
60
50
40
30
20
10
0
-10
-20
2300
0.00
0.00
2.44
2.44
2.43
3.78
3.70
3.69
3.68
3.67
3.66
3.12
3.11
3.10
4.20
4.63
7.45
7.44
7.97
7.96
S21_Note. I-053/Compound S21_1H.fid-600
H-1 Routine 1D, DCH CryoProbe, 1-13-2006
2200
2100
2000
1900
1800
1700
1600
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
10.0
9.5
9.0
8.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S19
4.0
3.5
3.0
1.00
2.26
2.36
18.81
7.5
2.26
8.0
2.30
2.26
10.5
2.17
-100
2.5
-200
2.0
1.5
1.0
0.5
0.0
-0.5
36.68
34.93
79.57
77.21
77.00
77.00
76.79
74.45
72.45
70.57
70.51
70.29
69.79
69.00
58.29
130.86
129.79
126.80
143.97
165.87
163.85
S21_Note. I-053/Compound S21_13C.fid-600
C-13 Routine 1D, DCH CryoProbe, 10-26-2006
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S20
80
70
60
50
40
30
20
10
0
-10
-20
0.1385
0.0624
1.90E+08
0.9846
1.3599 HDO
1.3210
4.0978
3.8677
3.7342
3.7163
3.7057
3.5542
3.4262
3.4163
3.4066
3.2183
4.8721
4.6302
4.4440
6.8086
7.4748
7.3418 CDCl3
8.3367
8.3268
8.1167
8.0822
shinsato500_03052012_2h/180
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S21
4.0
3.5
3.54
-1.00E+07
2.53
2.51
24.92
2.42
9.60
2.29
0.98
4.96
0.96
1.00
0.00E+00
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
45.0610
50.9363
71.3128
71.0376
70.9182
70.8514
70.2592
69.5444
68.8755
68.8186
77.6140
77.3595
77.1059
102.0416
108.2341
114.1632
130.3675
128.9736
127.3055
shinsato500_03052012_2c/180
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
4.0E+08
3.5E+08
3.0E+08
2.5E+08
2.0E+08
1.5E+08
1.0E+08
5.0E+07
0.0E+00
130
125
120
115
110
105
100
95
90
85
80
75
70
f1 (ppm) S22
65
60
55
50
45
40
35
30
25
20
450
0.00
-0.00
1.33
1.27
3.12
3.10
3.09
4.03
3.81
3.77
3.66
3.64
3.56
4.60
4.46
4.37
6.74
7.66
7.43
7.41
7.27
8.26
8.01
7.96
7.94
334-proton2/334
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
400
350
300
250
200
150
100
50
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S23
4.0
3.5
4.39
29.25
7.38
2.81
4.88
11.85
2.69
2.63
1.17
7.51
5.82
1.00
3.16
0.80
0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S24
80
70
60
50
36.79
35.02
30.91
29.68
44.73
77.25
77.20
77.00
77.00
76.75
72.51
71.04
70.69
70.62
70.56
70.54
70.53
70.50
70.46
70.40
69.86
69.59
69.38
69.32
69.30
68.64
68.59
64.49
50.13
135.13
135.11
130.96
130.04
129.86
128.66
127.05
127.02
126.88
123.78
113.96
144.77
144.03
165.95
163.94
160.86
171.43
185.54
334-carbon2/334
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
40
30
260000
240000
220000
200000
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
-20000
20
10
0
-10
-20
3.8E+08
-0.0003
2.2190
2.5105
8.0141
shinsato500_03062012_2h/180
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
3.6E+08
3.4E+08
3.2E+08
3.0E+08
2.8E+08
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
11.0
10.5
10.0
9.5
9.0
8.5
8.0
-2.0E+07
3.07
1.00
0.0E+00
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S25
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
2.30E+08
2.20E+08
19.0505
40.8947 DMSO
40.7278 DMSO
40.5606 DMSO
40.3935 DMSO
40.2264 DMSO
40.0594 DMSO
39.8926 DMSO
100.4532
144.3624
142.6746
139.5696
154.5444
shinsato500_03062012_2c/180
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
2.10E+08
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
-1.00E+07
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S26
80
70
60
50
40
30
20
10
0
-10
-20
1.5767 HDO
2.40E+08
-0.0001
2.3681
2.3630
5.3857
5.2743
5.2716
5.2532
4.9203
4.9174
4.9144
4.9096
4.9066
4.9037
6.0368
6.0261
6.0156
6.0050
6.0025
5.9943
5.9917
5.9811
5.9706
5.9599
8.3013
7.2641 CDCl3
shin500_0808/54
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
2.30E+08
2.20E+08
2.10E+08
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S27
3.27
2.19
1.12
1.12
1.06
1.00
-1.00E+07
4.0
3.5
3.0
2.5
-2.00E+07
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
2.10E+08
18.1716
68.2273
77.6145
77.3596
77.1058
118.7340
114.9556
132.0947
141.4141
149.1805
153.8145
shinsato500_01102012_c/180
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
-1.00E+07
-2.00E+07
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S28
80
70
60
50
40
30
20
10
0
-10
-20
2.6E+08
0.0063
-0.0001
-0.0016
-0.0068
1.6083 HDO
6.1689
6.1479
6.1345
6.1135
5.4420
5.4388
5.3006
5.2979
5.2772
5.0061
5.0035
5.0009
4.9948
4.9921
4.9897
6.5774
6.5729
6.5713
6.5668
7.3204
7.3146
7.3092
7.2606 CDCl3
7.8226
8.7433
shin500_0813up/54
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
2.33
1.16
1.17
1.14
1.07
1.08
1.00
0.97
0.0E+00
5.0
4.5
f1 (ppm) S29
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
67.0287
77.6139
77.3603
77.1061
105.8885
103.6309
121.2033
118.2823
126.7751
136.0362
134.7698
133.6362
150.0105
shinsato500_01116012_c/180
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-2.0E+07
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S30
80
70
60
50
40
30
20
10
0
-10
-20
1.6840 HDO
2.30E+08
0.0004
3.9543
6.1791
6.1582
6.1446
6.1345
6.1238
5.4358
5.4327
5.4013
5.2576
5.2548
4.9849
4.9821
4.9794
4.9738
4.9710
4.9681
6.6331
6.6272
6.6225
8.6753
7.2731
7.2612 CDCl3
7.2142
7.2086
7.2032
shin500_0813down/54
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
2.20E+08
2.10E+08
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S31
3.23
2.13
1.05
1.03
1.00
0.99
0.96
1.01
0.87
-1.00E+07
4.0
-2.00E+07
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
1.6840 HDO
2.30E+08
0.0004
3.9543
6.1791
6.1582
6.1446
6.1345
6.1238
5.4358
5.4327
5.4013
5.2576
5.2548
4.9849
4.9821
4.9794
4.9738
4.9710
4.9681
6.6331
6.6272
6.6225
8.6753
7.2731
7.2612 CDCl3
7.2142
7.2086
7.2032
shin500_0813down/54
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
2.20E+08
2.10E+08
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S32
3.23
2.13
1.05
1.03
1.00
0.99
0.96
1.01
0.87
-1.00E+07
4.0
-2.00E+07
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
4.0E+08
0.0748
0.0003
2.0482
2.0211
2.0068
1.9923
1.9780
1.9636
1.3990
1.2866
1.2768
1.2706
1.2589
1.2411
2.9837
3.3525
4.1327
4.1145
3.9352
3.7080
3.6937
3.6793
4.6640
4.6497
4.6352
4.2E+08
6.6302
6.6244
6.6173
7.2663 CDCl3
7.2297
7.2230
7.2160
7.2052
9.0286
400shinsato_0815/60
H-1 Routine 1D experiment. BBO Probe, 9-13-2007
3.8E+08
3.6E+08
3.4E+08
3.2E+08
3.0E+08
2.8E+08
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S33
4.0
3.5
3.0
-2.0E+07
2.39
0.81
2.17
3.29
2.25
1.00
1.11
0.97
0.95
0.0E+00
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
3.8E+08
33.3228
62.9087
58.5619
56.4265
77.6135
77.3599
77.1063
100.9190
109.5777
114.8298
126.7190
125.9549
121.8873
146.9833
146.6176
shinsato500_01092012_c/180
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
3.6E+08
3.4E+08
3.2E+08
3.0E+08
2.8E+08
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-2.0E+07
-4.0E+07
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S34
80
70
60
50
40
30
20
10
0
-10
-20
0.8885
0.8794
0.8731
0.8705
0.8612
0.8587
0.8472
0.8447
0.8383
-0.0202
-0.0598
-0.0647
-0.0677
-0.0759
-0.0838
-0.0923
-0.0976
2.0481
2.0438
2.0342
2.0198
2.0055
1.9913
4.2265
3.9285
3.9202
3.7460
3.7326
3.7185
3.7045
3.4896
3.4832
3.4713
3.4691
3.4665
3.4547
3.4485
4.6616
4.6474
4.6330
5.7097
6.5935
6.5858
7.2604
7.2306
7.1796
7.1718
shinsato400_120411_h/150
H-1 Routine 1D experiment. BBO Probe, 9-13-2007
2.20E+08
2.10E+08
2.00E+08
1.90E+08
1.80E+08
1.70E+08
1.60E+08
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S35
4.0
3.5
3.0
2.5
2.0
1.5
1.0
8.97
2.16
2.14
2.04
2.04
3.07
2.09
2.00
0.96
1.00
0.99
-1.00E+07
0.5
0.0
-2.00E+07
-0.5
-1.0
77.6779 CDCl3
77.3598 CDCl3
77.3190
77.0425 CDCl3
-1.1749
18.0534
33.2181
4.5E+08
66.0328
63.4343
59.3070
56.3821
100.6872
115.1612
121.6171
130.7504
128.4218
147.1746
146.4380
shinsato400_120411_c/150
C-13 Routine 1D experiment. BBO Probe, 9-13-2007
4.0E+08
3.5E+08
3.0E+08
2.5E+08
2.0E+08
1.5E+08
1.0E+08
5.0E+07
0.0E+00
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S36
80
70
60
50
40
30
20
10
0
-10
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S37
4.0
3.5
3.0
2.5
2.0
1.5
1.0
8.91
2.21
2.06
17.51
2.22
2.38
3.07
1.92
1.95
0.93
0.91
1.00
0.8559
0.8493
0.8355
0.8153
0.0557
-0.0186
-0.0264
-0.0764
-0.0832
-0.0913
-0.0996
1.3704
1.2565
1.2387
1.2208
1.1921
3.9191
3.6687
3.6561
3.6520
3.6439
3.6415
3.6354
3.6310
3.6255
3.6130
3.4912
3.4712
3.4507
3.3575
2.1565
2.1404
2.1243
2.1082
2.0921
2.0249 HDO
4.5170
4.5009
4.4849
5.7167
5.7005
6.5812
6.5734
7.2600
7.2457
7.2207
7.1742
7.1664
shinsato400_120711_H/150
H-1 Routine 1D experiment. BBO Probe, 9-13-2007
0.5
0.0
-0.5
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
-1.00E+07
-1.0
-1.1412
-1.1578
-1.1724
-1.1784
-1.2075
18.0835
29.9364
50.9540
2.8E+08
70.9663
70.9518
70.9282
70.9075
70.6662
70.3024
56.3615
77.6776 CDCl3
77.3603 CDCl3
77.1499
77.0416 CDCl3
100.7105
115.4327
121.8140
130.2412
127.9099
146.5683
146.2250
shinsato400_120711_c/150
C-13 Routine 1D experiment. BBO Probe, 9-13-2007
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-2.0E+07
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S38
80
70
60
50
40
30
20
10
0
-10
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S39
4.0
3.5
2.16
2.22
17.61
3.41
2.11
0.94
1.00
1.07
0.82
3.0
2.5
2.0
1.5
0.0002
1.2567
3.6776
3.6617
3.6553
3.6508
3.6466
3.6314
3.6181
3.6125
3.6067
3.3616
3.3487
2.1207
2.1053
2.0898
2.0743
2.0587
3.9452
3.9333
4.6463
4.5354
4.5200
4.5047
5.1191
7.2730 CDCl3
7.2513
7.2332
7.2261
7.2196
7.2022
6.6147
6.6036
6.5971
6.5911
9.4435
9.4430
shinsato400_120911_h/150
H-1 Routine 1D experiment. BBO Probe, 9-13-2007
1.0
0.5
0.0
2.5E+08
2.0E+08
1.5E+08
1.0E+08
5.0E+07
0.0E+00
-0.5
-1.0
29.7592
50.9391
56.4380
3.4E+08
70.9217
70.8942
70.8424
63.3904
77.6774 CDCl3
77.3602 CDCl3
77.0415 CDCl3
100.4554
115.2938
126.3775
125.7951
122.2205
146.4782
146.4371
shinsato400_120911_c/150
C-13 Routine 1D experiment. BBO Probe, 9-13-2007
3.2E+08
3.0E+08
2.8E+08
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-2.0E+07
-4.0E+07
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S40
80
70
60
50
40
30
20
10
0
-10
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S41
4.0
3.5
2.62
2.43
3.04
2.87
17.75
2.20
1.06
1.00
3.0
2.5
2.0
1.2547
0.8878
0.8800
0.8718
0.8617
0.8523
0.8442
0.8338
0.0708
0.0079
-0.0003
-0.0087
2.2904
2.1078
2.0935
2.0791 HDO
2.0646 HDO
2.0502 HDO
2.0475
3.9358
3.9232
3.7090
3.6672
3.6611
3.6344
3.6222
3.3559
3.3522
3.3429
3.3307
4.5671
4.5528
4.5383
5.1186
7.5563
7.4295
7.2657 CDCl3
8.2180
8.2098
9.7929
shinsato400_121211_h/150
H-1 Routine 1D experiment. BBO Probe, 9-13-2007
1.5
1.0
0.5
0.0
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-2.0E+07
-0.5
-1.0
29.4469
1.60E+08
71.0749
70.8794
70.8559
70.7617
70.7484
70.2909
57.2717
52.7628
50.9250
77.6773 CDCl3
77.3598 CDCl3
77.0419 CDCl3
123.6886
123.1246
119.8442
115.0331
135.7136
146.8534
146.4787
165.1890
181.5082
shinsato400_121411_2c/150
C-13 Routine 1D experiment. BBO Probe, 9-13-2007
1.50E+08
1.40E+08
1.30E+08
1.20E+08
1.10E+08
1.00E+08
9.00E+07
8.00E+07
7.00E+07
6.00E+07
5.00E+07
4.00E+07
3.00E+07
2.00E+07
1.00E+07
0.00E+00
-1.00E+07
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S42
80
70
60
50
40
30
20
10
0
-10
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S43
4.0
3.5
2.41
3.59
3.49
18.13
3.14
2.43
2.10
6.27
1.00
3.0
2.5
2.0
1.5
1.0
0.0715
0.0054
0.0001
-0.0009
-0.0064
1.3357
1.3264
1.3213
1.3124
1.2554
0.8934
0.8798
0.8661
3.9245
3.7078
3.6964
3.6876
3.6852
3.6813
3.6733
3.6695
3.6570
3.6454
3.6348
3.6254
3.3559
2.3208
2.2042
2.1021
2.0907
2.0793
2.0678
2.0565
2.0448
4.5502
4.5390
4.5276
5.1174
7.4737
7.4336
7.4242
7.2682
7.2671 CDCl3
8.0728
shinsato500_121711_h/145
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
6.5E+08
0.5
0.0
6.0E+08
5.5E+08
5.0E+08
4.5E+08
4.0E+08
3.5E+08
3.0E+08
2.5E+08
2.0E+08
1.5E+08
1.0E+08
5.0E+07
0.0E+00
-5.0E+07
-0.5
-1.0
29.4689
50.9559
77.6137
77.5645
77.3600
77.1065
71.0500
70.8640
70.7981
70.3026
69.6216
57.6249
6.5E+07
136.0362
135.3948
130.5014
129.0126
127.4207
123.4475
123.1540
120.3477
115.6757
146.9531
146.5122
171.0472
186.2315
shinsato500_121711_c/145
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
6.0E+07
5.5E+07
5.0E+07
4.5E+07
4.0E+07
3.5E+07
3.0E+07
2.5E+07
2.0E+07
1.5E+07
1.0E+07
5.0E+06
0.0E+00
-5.0E+06
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S44
80
70
60
50
40
30
20
10
0
-10
-20
2.8E+08
0.0056
-0.0005
-0.0070
1.2736
1.2550
1.2163
1.2152
1.2039
2.0368
2.0249
2.0129
3.9250
3.6456
3.6394
3.6329
3.6246
3.6151
3.4877
3.1179
3.1066
3.0959
4.6681
4.6086
4.5336
4.5237
4.5134
4.5072
4.4954
4.4837
7.4313
7.4161
7.2625
8.1087
8.1028
7.9603
7.9438
7.8125
shinsato500_121911_h/145
DQF-COSY (States-TPPII) using Gradient Pulse, DRX-500, BBO Probe
2.6E+08
2.4E+08
2.2E+08
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm) S45
4.0
3.5
3.0
-2.0E+07
3.10
2.16
3.89
2.97
3.65
41.17
2.38
2.33
4.42
8.32
1.00
2.04
0.90
0.0E+00
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
25000000
0.3361
0.3210
29.5620
37.1432
35.3834
50.6736
77.6133
77.5648
77.3597
77.1062
72.8874
71.0545
70.9728
70.9090
70.8873
70.8035
70.7674
70.3918
70.2245
70.0385
69.8838
69.0726
64.8211
64.0191
57.6583
146.9553
146.5591
144.4015
136.3050
135.4621
131.3310
130.4866
130.2503
129.0170
127.4525
127.2589
124.5590
123.4069
123.2520
120.3286
115.6474
171.0340
166.3399
164.2999
186.2423
shinsato500_121911_c/145
C-13-APT, BBO Probe, DRX-500, using deptq-135 pulse, 5-2-05
24000000
23000000
22000000
21000000
20000000
19000000
18000000
17000000
16000000
15000000
14000000
13000000
12000000
11000000
10000000
9000000
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
-1000000
-2000000
220
210
200
190
180
170
160
150
140
130
120
110
100
90
f1 (ppm) S46
80
70
60
50
40
30
20
10
0
-10
-20