Chemically Programmed Antibodies As HIV‑1 Attachment Inhibitors

52 Pages·2013·4.16 MB·English

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*
DepartmentofMolecularBiologyandChemistryandtheSkaggsInstituteforChemicalBiologyandDepartmentofImmunologyand
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
oftwosmall-moleculeanti-HIVcompoundsforthecreationof
chemically programmed antibodies. N-Acyl-β-lactam deriva-
tives 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
inhibitorsenablesitsapplicationinchemicallyprogrammedantibodiesandvaccinesandmayfacilitatethedevelopmentofnovel
bispecific antibodies and topical microbicides.
KEYWORDS: Bioconjugation, anti-HIV agent, chemically programmed antibody, microbicide, entry inhibitor
T
he retrovirus HIV-1, which causes acquired immune mAb could further enhance their activity in vivo through
deficiency syndrome (AIDS), has infected 34 million antibodyeffectorfunctionssuchasantibodydependentcellular
people worldwide, and this number is expected to increase by cytotoxicity (ADCC) and complement dependent cytotoxicity
2.5 million each year into the near future.1 Although the (CDC). Recently, we have described the development of
combination reverse transcriptase inhibitor/protease inhibitor chemically programmed antibodies based on the use of mAb
treatment known as HAART has proven successful,2,3 side 38C2,analdolaseantibodygeneratedbyreactiveimmunization
effects and viral escape are significant issues, and new by usinga 1,3-diketone hapten.22−24 Thisantibody possesses a
treatments are needed. The viral envelope protein gp120, the low pK lysine residue in its binding site that is key to its
a
primary target for antibody mediated viral neutralization, is an aldolaseactivitythatcanbesite-selectivelylabeledwithN-acyl-
emerging target for small molecule treatment of HIV β-lactams to produce a chemically programmed antibody.
infection.4,5 This protein is responsible for the entry of HIV Chemically programmed antibodies have duration times after
into host cells. In the initial step of entry, gp120 binds to the systemic dosing that depend on the properties of the antibody
CD4 glycoprotein expressed on the surface of human immune rather than on those of the conjugated small molecule,
cells. Bristol−Myers Squibb Pharmaceutical Research Institute providingforverysignificantextensionsinthepharmacokinetic
discoveredsmallmoleculesBMS-378806(1)andBMS-488043 profiles of the attached molecule.18,20 We have demonstrated
(2)thatbindtogp120(Figure1)andblockitsinteractionwith the utility of this approach by preparing mAb conjugates that
showpromisingactivityinavarietyofcancermodelsbutalsoin
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
TreatmentaswellasprophylaxisofHIV-1infectionrequires
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 small-
Figure 1. Chemical structures of gp120 inhibitors. molecule gp120 inhibitor would bind to gp120 and inhibit
CD4-mediatedentryofHIV-1intocells(Scheme2).Inrelated
CD4.6−11However,theshortpharmacokineticprofilesofthese work,Spiegelandco-workersrecentlyreportedthataderivative
of HIV-1 inhibitor 1 modified with a 1,3-dinitrophenyl hapten
small molecule inhibitors (half-lives after intravenous injection
moiety binds to HIV gp120.25 Their compound was designed
are 0.3 and 2.4 h, respectively) may limit their clinical
to bind noncovalently with polyclonal anti-1,3-dinitrophenyl
application.
Wehypothesizethatthepharmacokineticpropertiesofthese
small molecule gp120 inhibitors can be improved by Received: March 8, 2013
conjugation with a monoclonal antibody (mAb) (Scheme Accepted: April7, 2013
1).12−21 Furthermore, coupling of the small molecule to the
©XXXXAmericanChemicalSociety A dx.doi.org/10.1021/ml400097z|ACSMed.Chem.Lett.XXXX,XXX,XXX−XXX
ACS Medicinal Chemistry Letters Letter
Scheme 1. Chemoselective Modification of Aldolase Antibody 38C2 to Yield a Chemically Programmed Antibody
Scheme2.SchematicRepresentationoftheInhibitionofthe presence of CuSO , tris(3-hydroxypropyltriazolylmethyl)amine
4
HIV Entry by gp120 Inhibitor-Programmed mAb 38C2 (THPTA), and sodium-(L)-ascorbate proceeded smoothly to
yielddesiredcompound3withthelinkernowattheNorthern
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 1-
position also gave diminished biological activities, whereas the
piperazine of2wasalready optimized.Incontrast, substitution
wastoleratedatthe7-positionoftheazaindole.ONthebasisof
thesedata,wedesigned4bearingthelinkerat7-positionofthe
(DNP)antibodiesinsitu,withtheaimofenhancingtheactivity
azaindole (southern sector connection).
of 1. The activity of 1, however, was severely compromised
Target compound 4 was synthesized as shown in Scheme 4.
upontheadditionoftheDNPlinkerintheirreport.Parental1
Commercially available 2-hydroxy pyridine derivative 9 was
has HIV-1 neutralization activity in the nanomolar range,
subjected to bromination to afford 10 in good yield. The
whereas DNP linked 1 demonstrated micromolar activity in
hydroxygroupof10wasallylatedusingAg CO .Formationof
binding studies and was not shown to neutralize HIV-1. Our 2 3
conjugate strategy differs since we use a defined monoclonal the core azaindole structure was achieved by treatment of 11
with N,N-dimethylformamide dimethylacetal followed by
antibody covalently linked to 1. We hypothesized that our
reduction of nitro group in the presence of Fe in AcOH. The
strategy might allow us to recover the potent activity of 1
bromo group of 12 was replaced by a methoxy group, and 13
directly if the lack of activity of their DNP derivative of 1 was
was treated with borane-dimethylsulfide complex followed by
due to the noncovalent nature of attachment to antibody.
Alternatively,modificationofthelinkagestrategytothisfamily oxidationwithhydrogenperoxidetoreplacetheterminalolefin
with a primary alcohol. The reactivity of the substituent-free
of inhibitors might be key to restoring the activity of the small
nitrogen atom at the 1-position of the azaindole in 14 was
molecule.
To prepare derivatives of the Bristol−Myers Squibb problematic. After analysis of a number of protecting groups,
compoundsforconjugationtomAb,wefirstpreparedβ-lactam we found that the trimethylsilylethoxymethyl (SEM) group
3 (Figure 2) derived from BMS-378806 (1) from the known could be utilized.27 Protection of the reactive azaindole moiety
compound 5 (Scheme 3).7 Substitution of the nitro group by yielded 15, which was subjected to etherification with 1628 to
alcohol6followedbythetreatmentofPCl gaveBMS-378806 obtain 17. Removal of the SEM group was performed using
derivative7bearinganazidegroup.TheHu3isgenreactionof7 tetrabutylammonium fluoride (TBAF). A Friedel−Crafts
with β-lactam 8 possessing a terminal alkyne group in the reaction of 18 and methyl-2-chloro-2-oxoacetate was accom-
plished in the presence of an excess amount of AlCl .29 The
3
resultingcompound19washydrolyzedandcondensedwith1-
benzoylpiperazine 20 mediated by 3-(diethoxy-phosphory-
loxy)-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
mMPBS(pH7.4)atroomtemperaturefortwohours(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
keycatalyticlysineresidueshadreactedwiththelactam(Figure
3A). The MALDI-TOF mass analysis of 22a supported the
effective conjugation of 38C2 with 3 (Figure 3B). The
Figure 2. Synthetic targetsfor thisstudy. difference in mass between 38C2 and our preparation of 22a
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a
Scheme 3. Synthesis of the BMS-378806 Programming Agent 3
aReagentsandconditions:(a)NaH,DME,RT,2hthen50°C,3h.(b)PCl,EtOAc,RT,2.5h(37%intwosteps).(c)CuSO·5HO,THPTA,Na-
3 4 2
(L)-ascorbate, tBuOH, H2O,RT, 30 min (57%).
Scheme 4. Synthesis of the BMS-488043 Programming Supporting Information). Conjugate 22b was similarly
a
Agent 21 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
(Figure4).NeitherunconjugatedmAborconjugate22abound
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−activityrelationshipstudyofrelatedcompoundsthat
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
hypothesisthatthesubstituentatthenorthernsector4-position
of1disrupted gp120 binding,neither3nor7wereeffectivein
the assay (Figure 5A). The IC values of 4 and 21 with the
50
linker at southern 7-position were 67.5 and 25.4 nM,
respectively. The conjugate 22b also blocked infection with
an IC of 128 nM (Figure 5B). The unmodified mAb 38C2
aReagentsandconditions:(a)Br,AcOH,AcONa,RT,1h(75%).(b) 50
2 hadnorelevantanti-HIVactivity.Evidentfromthesestudiesis
AgCO,AllylBr, toluene, RT, 16 h(quant). (c)N,N-dimethylforma-
2 3 an impact on activity on linker attachment to the southern 7-
mide dimethylacetal, DMF, 130 °C, 2 h. (d) Fe, AcOH, 100 °C, 90
position; however, significant neutralization activity was
min(40%intwosteps).(e)CuI,MeONa,MeOH,DMF,RTto110
°C, 19 h (87%). (f) BH-MeS, THF, 0 °C to RT, 4 h then HO, preserved following linker addition at this site. We had
NaOH,HO,0°CtoRT3,152h(42%).(g)KOH,SEMCl,THF,2RT2, anticipated that conjugate 22b might exhibit significantly
2
30 min (88%). (h) NaH, DMF, RT, 19 h, (55%). (i) TBAF, enhanced activity over 4 and 21 given the bivalent display of
ethylenediamine, THF, RT to 70 °C, 21 h (85%). (j) AlCl, the compound on the antibody following conjugation as we
3
ClCOCO2Me, CH3NO2, CH2Cl2, RT, 4 h (40%). (k) NaOH, H2O, have noted with other antibody targeting agents. The lack of
MeOH, RT, 1 h. (l) DEPBT, DIPEA, RT, 10 h (38% in two steps).
enhanced activity following conjugation suggests that 22b is
(m)CuSO4·5H2O,THPTA,Na-(L)-ascorbate,tBuOH,H2O,RT,3h unable to engage the HIV-1 virion in a bivalent interaction.
(69%).
Monovalent binding of natural antibodies that react with the
CD4-binding site on gp120 has been suggested in the
literature.33Aspreviouslyreported,thechemicallyprogrammed
corresponded to two equivalents of the small molecule antibody strategy has been shown to significantly extend the
derivative of 3. ESI-MS analysis also indicated that both of half-life of the targeting molecule relative to the unconjugated
the two catalytic lysine moieties of 38C2 were modified (see molecule in studies concerned with small molecule, peptide,
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ACS Medicinal Chemistry Letters Letter
a
Scheme 5. Preparation of the gp120 Inhibitor Programmed Antibodies 22a and 22b
aReagents and conditions: (a)PBS (pH 7.4), RT, 2h.
Figure3.Analysisof22aand22b.(A)Catalyticactivityof22a,22b,andmAb38C2intheretro-aldolreactionofmethodol.(B)OverlayofMALDI
massspectraofmAb38C2(blue,MW =150357)and22a(green,MW =152932).(C)OverlayofMALDImassspectraofmAb38C2(blue,
av av
MW = 150357) and 22b (green, MW = 152946).
av av
and aptamer targeting molecules.18−21 Additional biological be seen in vivo for 22b such as ADCC and CDC activity, and
activities not accessible to the small molecule itself but rather these activities may be important to the activities of natural
characteristic of the antibody conjugate would be expected to 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
t■opical microbicides whose construction is hereby facilitated.
ASSOCIATED CONTENT
*
S Supporting Information
Syntheticprocedures,analyticaldata,andproceduresforELISA
andneutralizationassay.Thismaterialisavailablefreeofcharge
v■ia the Internet at http://pubs.acs.org.
Figure4.BindingofmAb38C2(200nM),22a(200nM),22b(200 AUTHOR INFORMATION
nM), and mAb b12 (2 nM) to JRFL gp120 as evaluated by ELISA.
Corresponding Author
PBS indicates the background control.
*(C.F.B.) Tel: [phone]. Fax: [phone]. E-mail:
[email protected].
Author Contributions
†
These authors contributed equally to this work.
Funding
This work was supported by NIH grant AI095038.
Notes
T■he authors declare no competing financial interest.
ACKNOWLEDGMENTS
WethankAngelicaCuevasforperformingHIV-1neutralization
a■ssays.
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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 KMnO . High
4
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 NH Cl was added to the reaction mixture. This was then extracted with AcOEt, and washed with H O
4 2
and brine. The combined organic layer was dried over MgSO , concentrated in vacuo, and purified by flash column
4
chromatography (Hex/AcOEt = 1/2) to afford compound S2 (1.31 g, 48%) as a colorless oil. 1H NMR (300 MHz, CDCl ) δ
3
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, CDCl ) δ 166.9, 143.6, 129.6, 129.2, 127.1, 79.6, 74.4, 72.5, 70.6, 70.55,
3
70.52, 70.3, 70.1, 69.8, 58.3, 52.0; HRMS: calcd for C H O (M+Na+) 403.1727, found 403.1725.
20 28 7

(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
CH Cl , dried over MgSO , concentrated in vacuo, and purified by flash column chromatography (AcOEt) to afford S3
2 2 4
(1.19 g, 94%) as a white solid. 1H NMR (500 MHz, DMSO-d ) δ 7.93 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 4.58 (s,
6
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-d ) 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;
6
HRMS: calcd for C H O (M+H+) 367.1751, found 367.1757.
19 26 7

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
SOCl (10 mL) and stirred at room temperature for 1 h. After completion of the reaction SOCl was removed by evaporation
2 2
in vacuo, and residue was dissolved in CH Cl , washed with sat. NaHCO , dried over MgSO , concentrated in vacuo to
2 2 3aq 4
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. NaHCO and brine, dried over MgSO , concentrated in vacuo, and purified by flash
3aq 4
column chromatography (Hex/AcOEt = 1/2) to afford 8 (225 mg, 41% over two steps) as a colorless oil. 1H NMR (400
MHz, CDCl ) 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,
3
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, CDCl ) δ 166.2, 164.2, 144.3,
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 C H NO
22 29 7
(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 NH Cl was slowly added, and the organic layer was extracted with CH Cl twice. Organic phase were combined,
4 2 2
dried over MgSO , concentrated in vacuo and the resulting crude brown residue was purified by flash column
4
chromatography (CH Cl : MeOH = 8:1) to afford a yellow oil (555 mg), which was then dissolved in AcOEt (20 mL). To
2 2
this solution was added PCl (0.794 mL, 9.10 mmol) and stirred at room temperature for 2.5h. The reaction was cooled to
3
0 ℃, quenched by sat.NaHCO until pH reached to 6. The mixture was extracted with AcOEt, dried over MgSO ,
3aq 4
concentrated in vacuo, and purified by flash column chromatography (CH Cl : MeOH = 15:1) to afford compound 7 (415
2 2
mg, 37% over two steps) as a colorless oil. 1H NMR (500 MHz, CDCl ) δ 8.33 (d, J = 5.0Hz, 1H), 8.10 (d, J = 17.2 Hz, 1H),
3
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, CDCl ) δ 184.71, 167.10, 161.34, 151.99, 146.51, 146.44, 139.80,
3
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 C H N O (MH+) 594.2671, found 594.2669.
29 35 7 7

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), CuSO -5H O (50 mM, 240 L) and Na-(L)-ascorbate (500 mM, 240 L). The reaction
4 2
mixture was stirred at room temperature for 30 min. Upon completion of the reaction CH Cl was added, and then washed
2 2
with H O and brine. Organic layer was dried over Na SO , concentrated in vacuo, and purified by preparative TLC (CHCl :
2 2 4 3
MeOH = 12:1) to give desired product 3 (13.9 mg, 57%) as yellow oil. 1H NMR (500 MHz, CDCl ) δ 8.36-8.00 (m, 1H),
3
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, CDCl ) δ 185.5, 171.4,
3
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,

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S4