Solid-phase synthesis of an A-B loop mimetic of the Cε3 domain of human IgE: Macrocyclization by Sonogashira coupling

Article abstract of DOI:10.1021/jo026693e

The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B loop of the Cε3 domain of human immunoglobulin E has been carried out. The key macrocyclization step to form the 65-membered ring is achieved in ～15% yield vi

Full text of DOI:10.1021/jo026693e

Solid -P h a se Syn th esis of a n A-B Loop Mim etic of th e CE3 Dom a in
of Hu m a n IgE: Ma cr ocycliza tion by Son oga sh ir a Cou p lin g
Alan C. Spivey,*,† J ohn McKendrick, and Ratnasothy Srikaran
Department of Chemistry, University of Sheffield,
Brook Hill, Sheffield S3 7HF, U.K.
Birgit A. Helm
Department of Molecular Biology and Biotechnology, University of Sheffield,
Western Bank, Sheffield S10 2UH, U.K.
a.c.spivey@imperial.ac.uk
Received November 12, 2002
The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B
loop of the Cꢀ3 domain of human immunoglobulin E has been carried out. The key macrocyclization
step to form the 65-membered ring is achieved in ∼15% yield via an “on-resin” Sonogashira coupling
reaction which concomitantly installs a diphenylacetylene amino acid conformational constraint
within the loop.
In tr od u ction
Human allergic disorders (type I hypersensitivity re-
sponses) ranging from hay fever, excema, and food aller-
gies to potentially life threatening asthma and anaphy-
lactic shock are increasing worldwide.1 Central to the
cascade of events that lead to these clinical allergic
manifestations are protein-protein binding events be-
tween human immunoglobulin E (hIgE) and its class-
specific Fc receptors (FcꢀRI and FcꢀRII) on effector
cells.2-4 Disruption of these interactions represents one
possible therapeutic strategy for treatment of these aller-
gic disorders.5-10 We have previously described a 21-
residue disulfide-constrained cyclic peptide, 1, containing
residues Leu340-Thr357 of native hIgE that inhibits
hIgE-triggered 5-hydroxytryptamine secretion in a ge-
netically engineered rat basophilic leukemia cell line
transfected with the extracellular domain (R-chain) of
human FcꢀRI with an IC50 of ∼12 µM (Scheme 1).11
F IGUR E 1. Schematic representation of the antiparallel
A-B â-strand and Ω-loop regions in the Cꢀ3 domain of hIgE.
Boxed residues Tyr339, Cys358, and Leu359 are those mutated
in disulfide-constrained cyclic peptide 1 [i.e., Tyr339Cys,
Cys358Ser, and Leu359Cys, Scheme 1).
This antagonist was designed to crudely mimic an
exposed Ω-loop12,13 in hIgE terminating the antiparallel
A-B â-strand in the Cꢀ3 domain which had been im-
plicated as a binding “hot spot” 14 for the interactions with
both receptors (Figure 1).15
† Present address: Department of Chemistry, South Kensington
campus, Imperial College, London, SE7 2AZ.
(1) Holgate, S. T. Nature 1999, 402 (Nov 25, Suppl. B2-4).
(2) Turner, H.; Kinet, J .-P. Nature 1999, 402 (Nov 25, Suppl. B24-
30).
1
Although H NMR studies indicated that, in aqueous
solution, cyclic peptide 1 displayed no discernible second-
ary structure, its ability to inhibit hIgE-FcꢀRI complex
formation did display strong pH dependence. We consid-
ered that this could be indicative of a pronounced
conformational binding dependence and initiated a pro-
(3) Sutton, B. J .; Gould, H. J . Nature 1993, 366, 421-428.
(4) Sayers, I.; Helm, B. A. Clin. Exp. Allergy 1999, 29, 585-94.
(5) Helm, B. A.; Carroll, K.; Housden, J . E. M.; Sayers, I.; Spivey,
A. C.; Carey, E. M. In Proceedings of the International Symposium on
Recent Advances in Molecular Biology, Allergy and Immunology;
Ramchand, C. N., Nair, P. N., Pilo, B., Eds.; Allied Publishers Ltd.:
New Dehli, 2001; pp 65-80.
(6) Garman, S. C.; Sechi, S.; Kinet, J .-P.; J ardetszky, T. S. J . Mol.
Biol. 2001, 311, 1049-1062.
(11) Helm, B. A.; Spivey, A. C.; Padlan, E. A. Allergy 1997, 52, 1155-
1169.
(7) Wurzburg, B. A.; Garman, S. C.; J ardetszky, T. S. Immunity
2000, 13, 375-385.
(8) Barnes, P. J . Nature 1999, 402 (Nov 25, Suppl. B31-38).
(9) Garman, S. C.; Kinet, J .-P.; J ardetzky, T. S. Cell 1998, 95, 951-
961.
(10) Helm, B. A.; Sayers, I.; Padlan, E. A.; McKendrick, J . E.; Spivey,
A. C. Allergy 1998, 53 (S45), 85-90.
(12) Olsen, G. L.; Bolin, D. R.; Bonner, M. P.; Bos, M.; Cook, C. M.;
Fry, D. C.; Graves, B. J .; Hatada, M.; Hill, D. E.; Kahn, M.; Madison,
V. S.; Rusiecki, V. K.; Sarabu, R.; Spinwall, J .; Vincent, G. P.; Voss,
M. E. J . Med. Chem. 1993, 36, 3039-3049.
(13) Leszczynski, J . F.; Rose, G. D. Science 1986, 234, 849-855.
(14) Bogan, A. A.; Thorn, K. S. J . Mol. Biol. 1998, 280, 1-9.
10.1021/jo026693e CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/29/2003
J . Org. Chem. 2003, 68, 1843-1851
1843

Spivey et al.
SCHEME 1. Str u ctu r es a n d Bin d in g Ch a r a cter istics of th e Wild -Typ e a n d Mu ta ted Lin ea r P ep tid es
Com p r isin g th e Tyr 339-Leu 359 Ep itop e of h IgE a n d of th e Disu lfid e-Con str a in ed Cyclic P ep tid e 1a
a
For details of the binding assay see Helm et al., ref 15.
gram to prepare more conformationally rigid analogues
of this lead structure.16
Conformationally restricted mimetics of peptide ligands
often exhibit enhanced specificity, affinity, and oral
activity against a given receptor.17 Such peptidomimetics
range in sophistication from peptides incorporating minor
structural modifications to increase their rigidity and
decrease their proteolytic susceptibility, through scaffold-
constrained peptide loops (e.g., containing â-turn mimet-
ics),18 to totally nonpeptidic compounds that mimic the
key structural elements of the peptide.19,20
F IGURE 2. General structure of tolan amino acids I, with
approximate dimensions, and schematic representation of
generalized peptidomimetic II incorporating such a constrain-
ing element.
(15) When we initiated this investigation in 1997, there was no
experimental high-resolution structural information for hIgE and our
modeling used a structure of hIgE (Brookhaven PDB code 2IGE)
derived from sequence homology with an X-ray structure of human
Our plan was first to replace the flexible and labile
IgG1-Fc [Brookhaven PDB code 1FC1, 2.9 Å resolution (Padlan, E.
A.; Davies, D. R. Mol. Immunol. 1986, 23, 1063-1075)]. The evidence
that the Leu340-Thr357 A-B Ω-loop targeted in this study constituted
a binding hot spot for the “high-affinity” hIgE-FcꢀRI interaction came
from studies with truncated IgE-derived peptides (summarized by
Helm et al. in ref 11). However, site-directed mutagenesis studies
reported in 1998 failed to identify specific residues in the A-B loop
responsible for this binding (Sayers, I.; Cain, S. A.; Swan, J . R. M.;
Pickett, M. A.; Watt, P. J .; Holgate, S. T.; Padlan, E. A.; Schuck, P.;
Helm, B. A. Biochemistry 1998, 37, 16152-16164). Moreover, in 2000
an X-ray structure of human IgE bound to the extracellular R-subunit
of the FcꢀRI receptor was disclosed [Brookhaven PDB code 1F6A, 3.5
Å resolution (Garman, S. C.; Wurzburg, B. A.; Tarchevskaya, S. S.;
Kinet, J .-P.; J ardetzky, T. S. Nature 2000, 406 (J uly 20), 259-266)].
In this structure the A-B loop region of hIgE did not form part of the
contact surface with FcꢀRI. A direct role for the A-B loop in the FcꢀRI
interaction therefore appears doubtful at the present time, and studies
are ongoing in our laboratories to gain further understanding of the
dynamics of the binding at this protein-protein interface. However, a
direct role for the Leu340-Thr357 A-B Ω-loop in the binding of hIgE
to its “low-affinity” receptor FcꢀRII/CD23 (Sayers, I.; Helm, B. A. Clin.
Exp. Allergy 1999, 29, 585-594) is more clear-cut. Indeed, we have
recently obtained clear evidence for the involvement of Lys352 in this
interaction (Sayers, I.; Housden, J . A.; et al. Manuscript in prepara-
tion). Since this interaction has a role in maintaining and modulating
the level of mediator release initiated by hIgE, the use of peptidomi-
metics such as 22 as the basis of novel vaccines is an attractive prospect
(cf. the following two references: Shakib, F.; Hooi, D. S. W.; Smith, S.
J .; Furmonaviciene, R.; Sewell, H. F. Clin. Exp. Allergy 2000, 30, 1041-
1046. Robinson, J . A. Synlett 1999, 429-441).
disulfide constraint present in compound 1 with a more
rigid and robust scaffold and then to explore progressively
shorter loop sequences. In the expectation that significant
conformational changes would accompany binding, we
were looking to develop a semirigid scaffold that would
be amenable to solid-phase parallel synthesis of small
libraries of peptidomimetics for screening. To this end
we were attracted to the use of diphenylacetylene (tolan)
amino acids I as scaffolds (Figure 2).
We envisaged that generic chemistry could be devel-
oped to allow access to a series of these tolans in which
the amine and carboxylate moieties could occupy o-, m-,
or p-positions in the A and B rings, respectively. These
tolan amino acids should be capable of spanning a range
of relative separations and orientations encompassing
those found between the N- and C-termini of target hIgE
epitopes, the appropriate tolan for a given epitope being
selected with the aid of molecular modeling. A similar
family of biphenyl amino acids has been described by
Neustadt et al.21 Moreover, Kemp and Li22 have described
a synthesis of the di-o-tolan amino acid and its use as a
â-turn mimetic.
(16) Cochran, A. G. Curr. Opin. Chem. Biol. 2001, 5, 654-659.
(17) J ackson, S.; DeGrado, W.; Dwivedi, A.; Parthasarathy, A.;
Higley, A.; Krywko, J .; Rockwell, A.; Markwalder, J .; Wells, G.; Wexler,
R.; Mousa, S.; Harlow, R. J . Am. Chem. Soc. 1994, 116, 3220-3230.
(18) Souers, A. J .; Ellman, J . A. Tetrahedron 2001, 57, 7431-7448.
(19) Liskamp, R. M. J . Recl. Trav. Chim. Pays-Bas 1994, 113, 1-19.
(20) Marshall, G. R. Tetrahedron 1993, 49, 3547-3558.
(21) Neustadt, B. R.; Smith, E. M.; Lindo, N.; Nechuta, T.; Bron-
nenkant, A.; Wu, A.; Armstrong, L.; Kumar, C. Bioorg. Med. Chem.
Lett. 1998, 2395-2398.
(22) Kemp, D. S.; Li, Z. Q. Tetrahedron Lett. 1995, 36, 4175-4178.
1844 J . Org. Chem., Vol. 68, No. 5, 2003

Solid-Phase Sonogashira Macrocyclization
SCHEME 2. Over view of Ou r Str a tegy for th e
Solid -P h a se Syn th esis via Son oga sh ir a
Ma cr ocycliza tion of P ep tid om im etic IIa
In cor p or a tin g a Di-o-tola n Am in o Acid
Con str a in in g Elem en t
SCHEME 3 a
a
Reagents and conditions: (i) ICl, HOAc; (ii) FmocCl, pyridine,
CH2Cl2; (iii) TFA, concd HCl; (iv) BzCl, AgCN, CH2Cl2, MeCN; (v)
(S)-Fmoc-Ser(t-Bu)-Cl, AgCN, CH2Cl2.
coupling with an aryl iodide having an o-amide group.29
Conseqently, we decided to perform exploratory studies
in solution.
Here we present a novel method for the solid-phase
synthesis of cyclic tolan amino acid-constrained peptides
exemplified by the synthesis of a direct analogue of cyclic
peptide 1 in which the disulfide bridge has been replaced
by a di-o-tolan amino acid. The method features an “on-
resin” Sonogashira macrocyclization. Although the Heck
reaction has been used previously for macrocycliza-
tion,23-26 this is the first example, to our knowledge, of
macrocyclization by Sonogashira coupling.27
For the preparation of the ring-A-containing unit III
(Scheme 2), we required access to iodobenzoic acid deriv-
ative 4.30 Of various reagent combinations that have
been reported to effect iodination of methyl 4-amino-
benzoate,31-37 we found that use of ICl in AcOH38 was
particularly convenient. This reaction proceeded smoothly
on a preparative scale to give methyl 4-amino-3-iodoben-
zoate (2) in 74% yield. Protection of the aniline nitrogen
of iodobenzoate 2 with 9-fluorenylmethoxycarbonyl chlo-
ride (Fmoc-Cl) gave methyl ester 3 in 89% yield. Subse-
quent hydrolysis using HCl-TFA39 then gave the re-
quired acid 4 (61% yield, Scheme 3).
To examine the reactivity of aniline 2 toward amide
formation, we performed exploratory coupling with ben-
zoic acid. All the standard peptide coupling reagents that
we examined failed to provide any coupled product,
starting materials being returned in all cases.40 Benzoyl
Resu lts a n d Discu ssion
The approach we envisaged for the synthesis of our
peptidomimetic is shown in Scheme 2. First, a 2-iodo-
aniline moiety would be immobilized to the solid support.
The aniline nitrogen of this unit, which would form the
A-ring of the tolan, would then be used as the anchor
point for building up the peptide. The C-terminus of the
peptide would then be capped off with 2-ethynylbenzoic
acid, which would form the B-ring of the tolan unit,
setting the stage for the on-resin Sonogashira macrocy-
clization. Finally, cleavage by acid from what was
anticipated to be a Rink amide-type linker would provide
the macrocyclic peptidomimetic in solution. Although this
strategy leaves a primary amide function at the position
where the tolan was attached to the solid support, we
did not anticipate that this group, which should remain
unprotonated at physiological pH, would adversely affect
the performance of the peptidomimetic. Furthermore, we
reasoned that it would be possible to use a traceless
linker28 in a “second-generation” approach.
(29) Dyatkin, A. B.; Rivero, R. A. Tetrahedron Lett. 1998, 39, 3647-
3650.
(30) A 4-amino-3-iodobenzoate, linked via an amide bond to Rink
amide AM resin (cf. ring-A-containing unit III, Scheme 2) has been
prepared previously for the solid-phase synthesis of 2,3-disubstituted
indoles via a Pd(0)-mediated heteroannulation of nonterminal alkynes
(see Zhang et al., ref 31). An analogous resin having an ester linkage
to TentaGel-S resin has also been employed for the solid-phase
synthesis of 2-substituted indoles via a Pd(0)/Cu(I)-mediated “one-pot”
Sonogashira/5-endo-dig coupling/cyclization protocol (see Fagnola et
al., ref 33).
(31) Zhang, H.-C.; Brumfield, K. K.; Maryanoff, B. E. Tetrahedron
Lett. 1997, 38, 2439-2442.
(32) Sy, W.-W. Synth. Commun. 1992, 22, 3215-3219.
(33) Fagnola, M. C.; Giuseppina, I. C.; Visentin, G.; Cabri, W.; Zarini,
F.; Mongelli, N.; Bedeschi, A. Tetrahedron Lett. 1997, 38, 237-2310.
(34) Kajigaeshi, S.; Kakinami, T.; Yamasaki, H.; Fujisaki, S.; Kondo,
M.; Okamoto, T. Chem. Lett. 1987, 2109-2112.
At the outset, we had two concerns regarding the
implementation of this strategy: the expected low reac-
tivity of the aniline nitrogen toward peptide coupling,22
and the expected difficulty of performing Sonogashira
(35) Ezquerra, J .; Pedregal, C.; Lamas, C. J . Org. Chem. 1996, 61,
5804-5812.
(23) Gibson, S. E.; Middleton, R. J . Contemp. Org. Synth. 1996, 3,
447-471.
(36) Barluenga, J .; Gonzalez, J . M.; Garcia-Martin, M. A.; Campos,
P. J .; Asensio, G. J . Org. Chem. 1993, 58, 2058-2060.
(37) Barluenga, J .; Rodriguez, M. A.; Campos, P. J . J . Org. Chem.
1990, 55, 2104-3106.
(38) Hill, M. L.; Raphael, R. A. Tetrahedron 1990, 46, 4587-4594.
(39) Bunin, B. A.; Ellman, J . A. J . Am. Chem. Soc. 1992, 114,
10997-10998.
(40) Since Kemp and Li (ref 22) have reported that they employed
“standard peptide coupling reactions” to attach N-blocked amino acids
to the aniline nitrogen of the di-o-tolan amino acid methyl ester, the
failure of the aniline nitrogen to couple in our system can probably be
attributed to the influence of the adjacent iodine atom.
(24) Akaji, K.; Teruya, K.; Akaji, M.; Aimoto, S. Tetrahedron 2001,
57, 2293-2303.
(25) Akaji, K.; Kiso, Y. Tetrahedron Lett. 1997, 38, 5185-5188.
(26) Hiroshige, M.; Hauske, J . R.; Zhou, P. J . Am. Chem. Soc. 1995,
117, 11590-11591.
(27) For the formation of a 10-membered carbocycle by intramo-
lecular Sonogashira coupling, see: Dai, W.-A.; Wu, A. Tetrahedron Lett.
2001, 42, 81-83.
(28) Comley, A. C.; Gibson, S. E. Angew. Chem., Int. Ed. 2001, 40,
1012-1032.
J . Org. Chem, Vol. 68, No. 5, 2003 1845

Spivey et al.
SCHEME 4 a
SCHEME 5 a
a
Reagents and conditions: (i) 2-TMS-acetylene, PPh3, CuI,
Et3N, Pd(PPh3)2Cl2, THF; (ii) KF‚H2O, MeOH; (iii) 2 M NaOH,
MeOH; (iv) 13, PPh3, CuI, Et3N, Pd(PPh3)2Cl2, THF; (v) 13, PPh3,
CuI, piperidine, K2CO3, Pd(PPh3)2Cl2, MeOH, THF.
a
Reagents and conditions: (i) CH2dCHCH2OH, Ti(O-i-Pr)4; (ii)
RhCl(PPh3)3, EtOH, H2O; (iii) NaOH, MeOH; (iv) SOCl2, DMF,
CH2Cl2; (v) 2,2,2-TBE-OH, i-Pr2EtN, DMAP, CH2Cl2; (vi) (S)-Fmoc-
Ser(t-Bu)-Cl, AgCN, CH2Cl2; (vii) Zn powder, HOAc; (viii) MeNH2‚
HCl, Et3N, CH2Cl2.
required; Pd(PPh3)4-catalyzed conditions45-47 failed to
exhibit any chemoselectivity, whereas Rh(PPh3)3Cl-
catalyzed conditions48 furnished a mixture of the desired
acid 8 and the deiodinated acid 9 in 50% and 15% yields,
respectively (Scheme 4). Since these products were dif-
ficult to separate, and we were unable to delineate
conditions under which deoiodination could be completely
suppressed, we abandoned allyl ester protection in favor
of 2,2,2-tribromoethyl (TBE) ester protection.49 The TBE
ester could not be introduced by transesterification of
methyl ester 6, so it was prepared from acid 10 via
conversion to the acid chloride, reaction with TBE alcohol
to give TBE ester 11 (93% yield), and then AgCN-
mediated coupling with (S)-Fmoc-Ser(t-Bu)-Cl to give
serine amide 12 in 93% yield. Deprotection of the TBE
ester using powdered Zn in AcOH gave the desired acid
8 smoothly in 97% yield (Scheme 4).
For the preparation of the ring-B-containing unit (see
IV, Scheme 2), we required access to 2-ethynylbenzoic
acid (16). Thus, Sonogashira coupling of methyl 2-iodo-
benzoate with 2-(trimethylsilyl)acetylene [2-(TMS)acety-
lene] using Pd(PPh3)2Cl2, CuI, and Et3N in THF50 gave
the diprotected derivative 14 in 92% yield. Treatment of
this ester with KF in MeOH effected desilylation to give
ester 15 in 82% yield. Alternatively, treatment of ester
14 with NaOH in MeOH gave the desired acid 16 as a
colorless oil in 48% yield after rapid flash chromatogra-
phy over SiO2 (Scheme 5).
chloride (BzCl) was also unreactive even in the presence
of a variety of additives [e.g., 1-hydroxybenzotriazole
(HOBt) or 4-(dimethylamino)pyridine (DMAP)] but was
eventually found to couple almost quantitatively, albeit
slowly, in the presence of AgCN.41,42
Although Carpino43 and others41,42 have shown that
many amino acid chlorides can be coupled with little or
no racemization, we thought it would be prudent to verify
this for the coupling of aniline 2 with Fmoc-Ser(t-Bu)-
Cl, as required for our A-B loop peptidomimetic. Thus,
(S)-Fmoc-Ser(t-Bu)-Cl was coupled to aniline 2 to give
serine amide derivative 6 in 81% yield. The antipodal
serine amide ent-6 was similarly prepared using (R)-
Fmoc-Ser(t-Bu)-Cl. Analysis of both amides by chiral
stationary-phase (CSP) HPLC (Chiralcel OD) indicated
that there was less than 0.5% racemization during
coupling (Scheme 3).
Unfortunately, we were unable to translate this success
in solution to the solid phase. Although acid 4 could be
readily coupled to Rink amide-functionalized polystyrene
(PS) and Rink amide-functionalized polyethylene glycol
(PEG)-grafted PS (NovaSynTGR), and the Fmoc group
could be removed quantitatively (piperidine, DMF) from
either resin, the resulting resin-bound anilines would not
couple with (S)-Fmoc-Ser(t-Bu)-Cl under the AgCN-
mediated conditions. This failure can probably be at-
tributed to the heterogeneity of the reaction conditions;
AgCN has very low solubility in CH2Cl2-MeCN. To
circumvent this impasse, we opted to attach the “pre-
coupled” serine amide 6 directly to the resin. This
necessitated a change of ester protecting group to one
orthogonal to both tert-butyl ether and Fmoc groups. Our
initial choice was the allyl ester. Thus, transesterification
of methyl ester 6 with allyl alcohol and Ti(Oi-Pr)4 gave
allyl ester 7 quantitatively (Scheme 4).44 Conditions for
deallylation in the presence of the aryl iodide were
With both fragments in hand we turned our attention
to the key Sonogashira reaction. Initial attempts to
couple iodoanilines 11 and 12 with 2-ethynylbenzoate 15
under a range of conditions failed to furnish more than
trace amounts (<10%) of the desired tolans and further-
more resulted in very poor recovery of the iodoanilines.
Suspecting that the TBE ester was unstable to the
coupling conditions, we prepared the methylamide ana-
logue of TBE ester 11 (i.e., 13) from acid 10 (Scheme 4).
This resulted in a striking improvement in coupling
(45) Deziel, R. Tetrahedron Lett. 1987, 28, 4371.
(46) Zhang, H. X.; Guibe, F.; Balavoine, G. Tetrahedron Lett. 1988,
29, 623-626.
(47) Bardaji, J . E.; Torres, J . L.; Xaus, N.; Clapes, P.; J orba, X.; de
la Torre, B. G.; Valencia, G. Synthesis 1990, 531-532.
(48) Kunz, H.; Waldmann, H. Helv. Chim. Acta 1985, 68, 618-622.
(49) Woodward, R. B.; Heusler, K.; Gosteli, J .; Naegeli, P.; Oppolzer,
W.; Ramage, R.; Ranganathan, S.; Vorbruggen, H. J . Am. Chem. Soc.
1966, 88, 852.
(41) Takimoto, S.; Inanga, J .; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. J pn. 1976, 49, 2335-2336.
(42) Suresh Babu, V. V.; Gayathri, K. Indian J . Chem. 1998, 37B,
1109-1113.
(43) Carpino, L. A.; Chao, H. G.; Beyermann, M.; Bienert, M. J . Org.
Chem. 1991, 56, 2635-42.
(44) Oppolzer, W.; Lienard, P. Helv. Chim. Acta 1992, 75, 2572-
2582.
(50) Thorand, S.; Krause, N. J . Org. Chem. 1998, 63, 8551-8553.
1846 J . Org. Chem., Vol. 68, No. 5, 2003

Solid-Phase Sonogashira Macrocyclization
SCHEME 6 a
a
Reagents and conditions: (i) Rink amide resin (0.58 mmol g-1), PyBOP, i-Pr2EtN, CH2Cl2; (ii) NovaSynTGR resin (0.23 mmol g-1),
PyBOP, i-Pr2EtN, CH2Cl2; (iii) piperidine, DMF; (iv) automated, sequential coupling and deprotection of Fmoc-Pfp/Dhbt amino acid esters
[Ser(t-Bu), Thr(t-Bu), Ile, Pro, Lys(Boc), Arg(Pmc), Phe, Leu, Asp(t-Bu)]; (v) 16, PyBOP, i-Pr2EtN, CH2Cl2; (vi) PPh3, CuI, Et3N, Pd(PPh3)2Cl2,
DMF; (vii) TFA-H2O-i-Pr3SiH (95:4:1 v/v/v).
efficiency with ethynylbenzoate 15, giving tolan 17 in
65% yield. Moreover, the mass balance in this reaction
could now be accounted for by recovered iodoaniline. By
employing TMS-protected ethynylbenzoate 14 as the
coupling partner, and employing in situ deprotection,51
tolan 17 could be obtained in 90% yield (Scheme 5). Since
we planned to use Rink amide-type attachment to the
resin, these were pleasing developments.
We were now in a position to check out our strategy
on the solid phase and opted to investigate two resins in
parallel: Rink PS and NovaSynTGR, since significant
differences in coupling efficiencies between the two
resins have been noted previously for certain solid-phase
Sonogashira couplings (vide infra).29 Thus, benzoate 8
was coupled to Rink PS and NovaSynTGR resins using
benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa-
fluorophosphate (PyBOP) to give functionalized resins
18R and 18N, respectively (Scheme 6).
and 20N. Treatment of analytical samples of both resins
with acid effected cleavage from the respective resins
with concomitant side-chain deprotection to afford soluble
peptide derivatives that were homogeneous by RP-HPLC
and had the expected molecular weight by ES-MS (MH+
m/z 2534).
The stage was now set to investigate macrocyclization
by Sonogashira coupling. Using the condtions optimized
for the coupling of iodobenzamide 13 with ethynylben-
zoate 15, we were pleased to note that cyclization
occurred using the Rink resin 20R. The cyclized product
22 was purified by RP-HPLC (yield ∼15%) and its
identity confirmed by high-resolution MALDI-MS (MH+
m/z 2406.2).
We investigated the use of a variety of alternative
macrocyclization conditions to try to improve the ef-
ficiency of the macrocyclization, including the use of
Pd(PPh3)4, Pd2(dba)3, and Pd(OAc)2 as alternative pal-
ladium sources. However, despite considerable experi-
mentation, we were unable to improve upon these origi-
nal solution-optimized conditions. Moreover, we did not
observe any cyclization when using NovasynTGR resin
20N. This was somewhat surprising since previous solid-
phase Sonogashira coupling reactions have successfully
been carried out on PS-based,31,54-59 PEG-based,33,60-62
The syntheses of the linear peptides 19R and 19N
having the IgE Cꢀ3 sequence 358-34052 were carried out
on an automated peptide synthesizer using the Fmoc-
pentafluorophenyl (Pfp)/3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazinyl (Dhbt)-activated ester method.53 Fol-
lowing Fmoc deptotection of the N-terminal Leu residue,
16 was coupled manually using PyBOP to give resins 20R
(51) Schultz, D. A.; Gwaltney, K. P.; Lee, H. J . Org. Chem. 1998,
63, 4034-4038.
(52) The sequence prepared incorporates a Cys358Ser “mutation”
as in disulfide-cyclized peptide 1 (Scheme 1 and Figure 1).
(53) Atherton, E.; Holder, J . L.; Meldal, M.; Sheppard, R. C.; Valerio,
R. M. J . Chem. Soc., Perkin Trans. 1 1988, 2887-2894 and references
therein.
(54) Liao, Y.; Fathi, R.; Reitman, M.; Zhang, Y.; Yang, Z. Tetrahe-
dron Lett. 2001, 42, 1815-1818.
(55) Huang, S.; Tour, J . M. J . Am. Chem. Soc. 1999, 121, 4908-
4909.
(56) Berteina, S.; Wendeborn, S.; Brill, W. K.-D.; De Mesmaeker,
A. Synlett 1998, 676-678.
J . Org. Chem, Vol. 68, No. 5, 2003 1847

Spivey et al.
dures.67 Flash chromatography (FC) was carried out using
Kieselgel 60 F254 (230-400 mesh) silica gel. Only distilled
solvents were used as eluents. Thin-layer chromatography
(TLC) was performed on DC-Alufolien plates precoated with
silica gel 60 F254 which were visualized either by quenching
of ultraviolet fluorescence (λmax ) 254 nm) or by charring with
aqueous KMnO4 in 5% K2CO3. All reaction solvents were
distilled before use and stored over activated 4 Å molecular
sieves, unless otherwise indicated. Anhydrous CH2Cl2, Et3N,
piperidine, and i-Pr2EtN were obtained by distillation from
CaH2. Anhydrous THF and Et2O were obtained by distillation,
immediately before use, from sodium/benzophenone ketyl
under an atmosphere of nitrogen. Anhydrous DMF was
obtained by distillation from MgSO4 under reduced pressure.
Petrol refers to the fraction of light petroleum boiling between
40 and 60 °C. High-resolution mass spectrometry (HRMS)
measurements are valid to(5 ppm.
and other63 types of solid supports. In particular, the
success of this cyclization on Rink PS and not on
NovasynTGR is contrary to the findings of Dyatkin and
Rivero, who reported that the latter was superior for the
preparation of a library of propargylamines by solid-
phase Sonogashira coupling from a resin-bound 3-iodo-
benzoate.29 Corroborative evidence for a decisive role for
the resin in the cyclization comes from our inability
also to obtain any cyclized material via attempted off-
resin macrocyclization under a variety of conditions [e.g.,
Pd(PPh3)2Cl2, CuI, and THF, Pd(OAc)2, CuI, P(C6H4-o-
SO3Na)3, and H2O-MeCN (1:1), and Pd(OAc)2, P(C6H4-
o-SO3Na)3, and H2O-MeCN (1:15)].64,65
Con clu sion s
Amino acids, coupling reagents, and resins were obtained
commercially. Peptide syntheses were performed on a peptide
synthesizer using an Fmoc-Pfp/Dhbt-activated ester strategy.
All HPLC was carried out using an HPLC system with a
diode-array UV detector monitoring at 219 nm. Normal-phase
analytical CSP HPLC was performed using a Chiralcel OD
column (4.6 mm × 25 cm), flow rate 1 mL min-1. Reversed-
phase analytical HPLC was performed using a C18, 5 µm
column (4.6 mm × 25 cm), flow rate 1 mL min-1. Reversed-
phase preparative HPLC was performed using a C18, 10 µm
We have demonstrated that Sonogashira coupling can
be used to cyclize resin-bound peptide derivative 20R to
give peptidomimetic 22, a process that concomitantly
incorporates a tolan amino acid conformational constraint
within the macrocycle. We are currently exploring this
strategy for the synthesis of a focused library of related
tolan-constrained peptidomimetics having shorter loop
sequences.
Compound 22 was designed as a peptidomimetic of an
exposed Ω-loop in hIgE that has been implicated as
having a role in the binding of hIgE to both Fc receptors,
interactions that play a pivotal role in type I hypersen-
sitivity responses.15 Initial studies into the inhibition by
peptidomimetic 22 of IgE-triggered 5-hydroxytryptamine
secretion in our rat basophilic leukemia cell line trans-
fected with human FcꢀRI R-chain indicate that it is not
a significantly more potent antagonist than disulfide-
constrained cyclic peptide 1 (vide supra).66 These inhibi-
tion studies and the results of surface plasmon resonance
SPR studies of the binding of peptidomimetic 22, and
related peptidomimetics, with recombinent soluble FcꢀRI
R-chain will be reported in full elsewhere.
column (2.2 cm × 25 cm), flow rate 5 mL min-1
.
Disu lfid e-Con st r a in ed Cyclic P ep t id e 1. A portion of
Fmoc-L-Cys(Tr)-PEG-PS resin (1.1 g, nominal loading level
0.18 mmol g-1) was treated with piperidine-DMF (1:4 v/v) for
30 min to deprotect the terminal Fmoc group. After being
washed with DMF, the resin was subjected to automated
coupling cycles to build up the linear peptide sequence
required. The amino acid derivatives employed were Fmoc-L-
Ser(t-Bu)-ODhbt, Fmoc-L-Thr(t-Bu)-ODhbt, Fmoc-L-Ile-OPfp,
Fmoc-L-Pro-OPfp, Fmoc-L-Lys(Boc)-OPfp, Fmoc-L-Arg(Pmc)-
OH, Fmoc-L-Phe-OPfp, Fmoc-L-Leu-OPfp, Fmoc-L-Asp(t-Bu)-
OPfp, and Fmoc-L-Cys(Tr)-OPfp. A standard 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium tetrafluorobarate (TBTU; 4
equiv)-HOBt (4 equiv)/60 min coupling cycle was employed
for the two Fmoc-L-Arg(Pmc)-OH residues, all the other
residues were coupled using standard activated ester coupling
cycles (30-80 min). Residues Pro359, Phe334, Pro335, Ser336,
Pro337, and Arg338 were double coupled. A portion of the
resulting resin (250 mg) was treated with piperidine-DMF
(1:4 v/v) for 30 min to deprotect the terminal Fmoc group.
Cleavage of the linear peptide from the resin and global side-
chain deprotection were effected by treatment with a mixture
of TFA-PhOH-H2O-PhSMe-1,2-ethanedithiol (EDT) (20:
0.7:0.5:0.5:0.25 v/w/v/v/v) for 30 min. Following filtration, the
filtrate was concentrated in vacuo, precipitated from cold Et2O
(4×), collected by filtration, and lyophilized from 70% MeCN-
H2O (v/v) with 0.1% TFA (v/v) to yield 90 mg of the crude
peptide. This was purified by preparative reversed-phase
HPLC, gradient 0-70% MeCN-H2O (v/v) with 0.1% TFA (v/
v) over 60 min, to give the linear peptide as a white solid (67
mg, 63%): MS (ES+) m/z calcd for C105H171N28O30S2 (MH+)
2368.2, found 2368.
Exp er im en ta l Section
Gen er a l Meth od s. All synthetic reactions were performed
under anhydrous conditions and an atmosphere of nitrogen
in oven-dried glassware. Yields refer to chromatographically
and spectroscopically (1H NMR) homogeneous materials, un-
less otherwise indicated. Reagents were used as obtained from
commercial sources or purified according to known proce-
(57) Collini, M. D.; Ellingboe, J . W. Tetrahedron Lett. 1997, 38,
7963-7966.
(58) J ones, L.; Schumm, J . S.; Tour, J . M. J . Org. Chem. 1997, 62,
1388-1410.
(59) Yu, K.-L.; Deshpande, M. S.; Vyas, D. M. Tetrahedron Lett.
1994, 35, 8919-8922.
(60) Tan, D. S.; Foley, M. A.; Stockwell, B. R.; Shair, M. D.;
Schreiber, S. L. J . Am. Chem. Soc. 1999, 121, 9073-9087.
(61) Tan, D. S.; Foley, M. A.; Shair, M. D.; Schreiber, S. L. J . Am.
Chem. Soc. 1998, 120, 8565-8566.
The pH of an aqueous solution of this crude peptide (0.1
mg mL-1) was adjusted to 8.5 using NH4HCO3, and the
resulting solution was stirred in air for 90 h at 4 °C. The 90 h
incubation time was determined empirically by a time course
taking aliquots for ES-MS at 24, 48, 72, and 90 h. The residue
was lyophilized to give disulfide-constrained cyclic peptide 1
as a white solid (65 mg, 61%): MS (ES+) m/z calcd for
(62) Fancelli, D.; Fangola, M. C.; Severino, D.; Bedeschi, A. Tetra-
hedron Lett. 1997, 38, 2311-2314.
(63) Kahn, S. I.; Grinstaff, M. W. J . Am. Chem. Soc. 1999, 121, 1,
4704-4705.
(64) Amatore, C.; Blart, E.; Genet, J . P.; J utand, A.; Lemaire-
Audoire, S.; Savignac, M. J . Org. Chem. 1995, 60, 6829-6839.
(65) Genet, J . P.; Savignac, M. J . Organomet. Chem. 1999, 576, 305-
317.
C
105H169N28O30S2 (MH+) 2366.2, found 2367.
4-Am in o-3-iod oben zoic Acid Meth yl Ester (2).38 A solu-
(66) The fact that the IC50 exhibited by tolan-constrained peptide
22 does not appear to exceed that of disulfide-constrained cyclic peptide
1 is not surprising given the limited degree of conformational control
that the tolan amino acid could be expected to exert over such an
extended peptide loop.
tion of ICl (11.0 g, 67.7 mmol) in AcOH (500 mL) was added
(67) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988.
1848 J . Org. Chem., Vol. 68, No. 5, 2003

Solid-Phase Sonogashira Macrocyclization
dropwise over a period of 15 min to a stirred solution of methyl
4-aminobenzoate (10.0 g, 66.0 mmol) in AcOH (500 mL) and
the resulting solution stirred at rt for a further 1 h. Evapora-
tion of the solvent left a brown solid which was suspended in
H2O-CH2Cl2 (1:1, 500 mL), and solid NaHCO3 was added until
a clear solution was obtained. The organic layer was then
separated and further washed with saturated NaHCO3 (100
mL) and brine (100 mL). The organic layer was dried (MgSO4)
and evaporated to dryness in vacuo to leave a brown oil which
solidified on standing. Purification by FC (EtOAc/hexane, 1:4)
gave iodobenzoate 2 as a brown solid (13.6 g, 74%): mp 89-
(138 mg, 81%, >99.5% ee by HPLC; see the Supporting
Information): mp 121-122 °C; 1H NMR (CDCl3) δ 1.20 (s, 9H),
3.55 (dd, J ) 9.3, 6.2 Hz, 1H), 3.9 (s, 3H), 3.94 (br m, 1H),
4.25 (t, J ) 6.2 Hz, 1H), 4.45-4.6 (br m, 3H), 5.8 (br s, 1H),
7.25-7.45 (br m, 4H), 7.6 (br s, 2H), 7.77 (d, J ) 6.8 Hz, 2H),
8.00 (dd, J ) 7.5, 1.9 Hz, 1H), 8.40 (d, J ) 7.5 Hz, 1H), 8.45
(d, J ) 1.9 Hz, 1H), 8.70 (br s, 1H); 13C NMR (CDCl3) δ 27.5,
47.1, 52.3, 56.4, 61.5, 67.4, 74.4, 88.7, 120.1, 120.3, 125.0, 127.1,
127.2, 127.8, 130.7, 140.3, 141.4, 141.7, 143.6, 156.1, 165.2,
169.2; MS (ES+) m/z 665 (MNa+, 100), 643 (MH+, 20); HRMS
(ES+) m/z calcd for
C
30H32IN2O6 (MH+) 643.1305, found
1
90 °C [lit.38 mp 85-87 °C (EtOAc)]; H NMR (CDCl3) δ 3.85
643.1290.
(s, 3H), 4.58 (br s, 1H), 6.68 (d, J ) 8.4 Hz, 1H), 7.80 (dd, J )
8.4, 1.9 Hz, 1H), 8.38 (d, J ) 1.9 Hz, 1H).
The enantiomer of this compound was also prepared in 84%
yield and >99.5% ee by HPLC (see the Supporting Informa-
tion) using the same procedure but employing (R)-Fmoc-Ser-
(t-Bu)-Cl.
4-(9H-F lu or en -9-ylm eth oxyca r bon yla m in o)-3-iod oben -
zoic Acid Meth yl Ester (3). To a solution of iodobenzoate 2
(250 mg, 0.9 mmol) and pyridine (88.0 µL, 1.2 equiv) in
CH2Cl2 (5 mL) at 0 °C was added Fmoc-Cl (258 mg, 1.1 equiv).
The solution was stirred at 0 °C for a further 15 min before
the ice bath was removed and stirring continued at rt for 1 h.
CH2Cl2 (10 mL) was added and the solution washed with 1 M
NaHSO3 (2 × 10 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo to leave a white solid which was
recrystallized (CH2Cl2/petrol) to give iodobenzoate 3 as a white
solid (400 mg, 89%): mp 183-185 °C; 1H NMR (CDCl3) δ 3.91
(s, 3H), 4.33 (t, J ) 6.2 Hz, 1H), 4.51 (d, J ) 6.2 Hz, 2H), 7.25-
7.47 (m, 5H), 7.63 (d, J ) 7.5 Hz, 2H), 7.78 (d, J ) 7.5 Hz,
2H), 7.98 (dd, J ) 9.3, 3.75 Hz, 1H), 8.13 (br d, J ) 9.3 Hz,
1H), 8.46 (d, J ) 2.5 Hz, 1H); 13C NMR (CDCl3) δ 47.0, 52.3,
67.8, 87.6, 118.8, 120.2, 125.0, 126.4, 127.2, 128.0, 130.9, 140.4,
141.4, 142.1, 143.5, 152.8, 165.2; MS (EI+) m/z 499 (M+, 10),
178 (100); HRMS (EI+) m/z calcd for C23H18INO4 (M+) 499.0281,
found 499.0270.
4-(9H-F lu or en -9-ylm eth oxyca r bon yla m in o)-3-iod oben -
zoic Acid (4). A solution of iodobenzoate 3 (200 mg, 0.4 mmol)
in TFA (9 mL) and concd HCl (5 mL) was heated at 80 °C for
36 h. H2O (25 mL) was added and the precipitate collected
via filtration. The solid was triturated with CH2Cl2 (10 mL)
and collected by filtration to give iodobenzoic acid 4 as a pale
yellow solid (119 mg, 61%): mp 174-176 °C; 1H NMR (DMSO)
δ 4.31 (t, J ) 7 Hz, 1H), 4.43 (d, J ) 7 Hz, 2H), 7.25-7.48 (m,
6H), 7.72 (d, J ) 7.3 Hz, 2H), 7.86 (d, J ) 1.5 Hz, 1H), 7.89 (d,
J ) 7.6 Hz, 2H), 8.34 (m, 1H), 9.27 (s, 1H); 13C NMR (DMSO)
δ 47.0, 66.8, 95.7, 120.6, 125.8, 126.2, 127.6, 128.2, 129.5, 130.2,
140.4, 141.2, 143.9, 144.1, 154.2, 166.1; MS (EI+) m/z 485 (M+,
5), 178 (100); HRMS (EI+) m/z calcd for C22H16INO4 (M+)
485.0124, found 485.0131.
(S)-4-[3-ter t-Bu toxy-2-(9H-flu or en -9-ylm eth oxyca r bo-
n yla m in o)p r op ion yla m in o]-3-iod oben zoic Acid Allyl Es-
ter (7). Methyl ester 6 (50.0 mg, 0.80 mmol) and Ti(Oi-Pr)4
(443 mg, 2.0 equiv) were dissolved in allyl alcohol (5 mL), and
the resulting solution was sealed in a pressure tube under
nitrogen. The solution was heated at 120 °C for 1 h, allowed
to cool to rt, and quenched by the addition of saturated
NH4Cl (5 mL). CH2Cl2 (10 mL) was added and the precipitate
removed by filtration through Celite. The organic phase was
separated, dried (Na2SO4), and concentrated in vacuo to leave
an oily residue, which was purified by FC (EtOAc/hexane, 1:4)
to give allyl ester 7 as a colorless solid (51.0 mg, 98%): mp
1
71-73 °C; H NMR (CDCl3) δ 1.22 (s, 9H), 3.55 (dd, J ) 7.5,
5.6, 1H), 3.96 (br d, J ) 6.2 Hz, 1H), 4.25 (t, J ) 6.3 Hz, 1H),
4.40-4.55 (m, 3H), 4.81 (m, 2H), 5.25-5.45 (m, 2H), 5.78 (br
s, 1H), 5.95-6.06 (m, 1H), 7.26-7.48 (m, 4H), 7.63 (br d, J )
6.8 Hz, 2H), 7.78 (br d, J ) 7.5 Hz, 2H), 8.05 (br d, J ) 8.1 Hz,
1H), 8.37-8.43 (m, 1H), 8.46 (d, J ) 1.2 Hz, 1H), 8.70 (br s,
1H); 13C NMR (CDCl3) δ 21.9, 27.5, 47.1, 61.5, 65.2, 65.8, 67.4,
68.8, 74.4, 88.7, 118.6, 120.1, 125.0, 127.2, 127.6, 127.8, 130.8,
132.0, 140.3, 141.4, 141.8, 143.6, 164.3, 169.2; MS (FAB+) m/z
669 (MH+, 100); HRMS (FAB+) m/z calcd for C32H33IN2O6I
(M+) 668.1383, found 668.1320.
(S )-4-[3-t er t -Bu t oxy-2-(9H -flu or e n -9-ylm e t h oxyca r -
b on yla m in o)p r op ion yla m in o]-3-iod ob en zoic Acid (8).
Meth od 1. Allyl ester 7 (380 mg, 0.56 mmol) was dissolved in
a mixture of EtOH and H2O (10 mL, 9:1). To the solution was
added Wilkinson’s catalyst [RhCl(PPh3)3, 60.0 mg, 11 mol %],
and the solution was heated at reflux for 2 h with rapid
stirring. Upon cooling, the solution was concentrated in vacuo
and the residue purified by FC (EtOAc/hexane/AcOH, 4:1:0.01)
to give iodobenzoic acid 8 as a white solid (179 mg, 50%) and
(S)-4-[3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-
propionylamino]benzoic acid (9) as a pale brown solid (42.0
mg, 15%).
4-Ben zoyla m in o-3-iod oben zoic Acid Meth yl Ester (5).
To a solution of iodobenzoate 2 (50.0 mg, 0.18 mmol) in
CH2Cl2 (3 mL) were added BzCl (32.0 µL, 2.0 equiv) and a
suspension of AgCN (24.0 mg, 1.0 equiv) in MeCN (3 mL). The
suspension was stirred for 48 h and filtered through Celite.
The filtrate was partitioned between CH2Cl2 (10 mL) and 5%
NaHCO3 (10 mL). The organic layer was further washed with
1 M citric acid (10 mL) and brine (10 mL), dried (MgSO4), and
concentrated in vacuo to leave a pale brown solid. Purification
by FC (EtOAc/hexane, 1:10) gave iodobenzoate 5 as a white
solid (68.0 mg, 99%): mp 144-146 °C; 1H NMR (CDCl3) δ 3.82
(s, 3H), 7.51-7.67 (m, 3H), 7.96-8.03 (m, 2H), 8.08 (dd, J )
8.1, 1.8 Hz, 1H), 8.50 (d, J ) 1.8 Hz, 1H), 8.52 (br s, 1H), 8.14
(d, J ) 7.5 Hz, 1H); 13C NMR (CDCl3) δ 52.3, 88.8, 120.0, 127.2,
129.1, 131.0, 132.6, 134.1, 140.2, 142.1, 165.2; MS (ES+) m/z
404 (MNa+, 100), 382 (MH+, 40); HRMS (ES+) m/z calcd for
Da ta for 8: mp 124-126 °C; 1H NMR (CDCl3) δ 1.15 (s,
9H), 3.50 (dd, J ) 7.5, 6.2 Hz, 1H), 3.92 (br d, 1H), 4.17 (t, J
) 6.2 Hz, 1H), 4.40-4.51 (m, 3H), 5.85 (br s, 1H), 7.19-7.40
(m, 4H), 7.55 (br s, 2H), 7.69 (br d, J ) 6.5 Hz, 2H), 7.99 (br
d, J ) 7.5 Hz, 1H), 8.36 (d, J ) 7.5 Hz, 1H), 8.42 (d, J ) 1.9
Hz, 1H), 8.64 (br s, 1H); 13C NMR (CDCl3) δ 27.5, 47.1, 57.2,
61.5, 69.3, 74.6, 88.6, 120.1, 125.0, 126.3, 127.1, 127.8, 131.4,
140.9, 141.4, 142.3, 143.6, 156.2, 169.2; MS (EI+) m/z 628 (M+,
15), 178 (100); HRMS (EI+) m/z calcd for C29H29IN2O6 (M+)
628.1070, found 628.1058.
15H13INO3 (MH+) 381.9940, found 381.9955.
Da ta for 9: mp 113-115 °C; H NMR (CDCl3) δ 1.26 (s,
1
C
9H), 3.48 (t, J ) 8.5 Hz, 1H), 3.92 (br s, 1H), 4.24 (t, J ) 6.7
Hz, 1H), 4.35-4.55 (m, 3H), 5.88 (br d, J ) 4.6 Hz, 1H), 7.24-
7.43 (m, 4H), 7.60 (br s, 4H), 7.76 (d, J ) 7.3 Hz, 2H), 8.09 (d,
J ) 7.3 Hz, 2H), 9.00 (br s, 1H); 13C NMR (CDCl3) δ 27.5, 47.1,
62.3, 118.9, 120.1, 125.0, 127.1, 127.8, 131.6, 141.3, 142.3,
143.7, 156.2, 168.9, 170.6; MS (FAB+) m/z 503 (MH+, 10), 179
(100); HRMS (FAB+) m/z calcd for C29H31N2O6 (MH+) 503.2182,
found 503.2196.
(S)-4-[3-ter t-Bu toxy-2-(9H-flu or en -9-ylm eth oxyca r bo-
n yla m in o)p r op ion yla m in o]-3-iod oben zoic Acid Meth yl
Ester (6). To a solution of iodobenzoate 2 (73.0 mg, 0.26 mmol)
in CH2Cl2 (6 mL) were added (S)-Fmoc-Ser(t-Bu)-Cl (96.0 mg,
1.0 equiv) and AgCN (35.0 mg, 1.0 equiv). The suspension was
stirred for 48 h and filtered through Celite. The filtrate was
concentrated in vacuo to leave an off-white solid. Purification
by FC (EtOAc/hexane, 1:4) gave iodobenzoate 6 as a white solid
J . Org. Chem, Vol. 68, No. 5, 2003 1849

Spivey et al.
4-Am in o-3-iod oben zoic a cid (10).68 To a solution of
methyl ester 2 (2.0 g, 7.2 mmol) in MeOH (6 mL) was added
2 M NaOH (12 mL). The solution became turbid and was
stirred for 24 h, during after which time the solution became
clear. The mixture was concentrated in vacuo, the residue was
suspended in H2O (25 mL), and 1 M HCl was added until a
pH of 4 was reached. The aqueous phase was extracted with
EtOAc (2 × 10 mL), and the combined organic washings were
dried (MgSO4) and concentrated in vacuo to give acid 10 as a
pale brown solid (1.5 g, 79%): mp 201-203 °C [lit.68 mp 193-
chloride (750 mg, 2.80 mmol) in CH2Cl2 (10 mL) were added
MeNH2‚HCl (250 mg, 1.75 equiv) and Et3N (0.90 mL, 2.30
equiv). The resulting solution was stirred for 0.5 h, diluted
with EtOAc (25 mL), and washed with 5% NaHCO3 (50
mL). The organic layer was then washed with brine (2 × 25
mL), dried (MgSO4), and concentrated in vacuo. Purification
by FC (EtOAc/hexane, 4:6) gave methylbenzamide 13 as a
white solid (432 mg, 56%): mp 64-67 °C; 1H NMR (CDCl3) δ
2.90 (2 s, 3H), 4.46 (br s, 2H), 6.35 (br s, 1H), 6.63 (d, J ) 6.9
Hz, 1H), 7.52 (dd, J ) 7.5, 1.8 Hz, 1H), 8.05 (d, J ) 1.8 Hz,
1H); 13C NMR (CDCl3) δ 26.9, 113.5, 118.7, 125.6, 128.4, 138.2,
149.6, 166.7; MS (ES+) m/z 299 (MNa+, 100), 277 (MH+, 50);
HRMS (ES+) m/z calcd for C8H10IN2O (MH+) 276.9838, found
276.9839.
1
194 °C (EtOH-H2O)]; H NMR (d6-DMSO) δ 6.00 (br s, 2H),
6.58 (d, J ) 7.5 Hz, 1H), 7.66 (dd, J ) 7.5, 1.2 Hz, 1H), 8.18
(d, J ) 1.2 Hz, 1H), 12.32 (br s, 1H).
4-Am in o-3-iod oben zoic Acid 2,2,2-Tr ibr om oeth yl Ester
(11). A solution of iodobenzoic acid 10 (1.40 g, 5.32 mmol) in
SOCl2 (8.0 mL, excess) was heated to reflux for 1.5 h. The
solution was then concentrated in vacuo and the residue
dissolved in toluene (15 mL) and then reconcentrated in
vacuo to give the crude acid chloride as a yellow oil. This oil
was added to a solution of 2,2,2-TBE-OH (1.58 g, 1.05 equiv),
i-Pr2EtN (1.12 mL, 1.50 equiv), and DMAP (20.0 mg, catalytic)
in CH2Cl2 (10 mL) and the resulting solution stirred for 1 h.
The reaction mixture was then diluted with CH2Cl2 (50 mL)
and washed with 5% NaHCO3 (30 mL) and brine (2 × 30 mL).
The organic layer was dried (MgSO4) and concentrated in
vacuo to leave a brown solid. Purification by FC (EtOAc/
hexane, 1:4) gave TBE ester 11 as a pale brown solid (2.61 g,
93%): mp 126-127 °C; 1H NMR (CDCl3) δ 4.52 (br s, 2H), 5.11
(s, 2H), 6.75 (d, J ) 6.9 Hz, 1H), 7.95 (dd, J ) 6.9, 1.2 Hz,
1H), 8.43 (d, J ) 1.2 Hz, 1H); 13C NMR (CDCl3) δ 36.4, 77.0,
113.2, 119.5, 131.8, 141.7, 151.5, 163.4; MS (EI+) m/z 525, 527,
529, 531 (M+, 15, 50, 50, 15), 246 (100); HRMS (CI+) m/z calcd
for C9H879Br3INO2 (MH+) 525.7150, found 525.7140.
2-Tr im eth ylsila n yleth yn ylben zoic Acid Meth yl Ester
(14).69 To a solution of methyl 2-iodobenzoate (1.0 g, 4.65
mmol), 2-TMS-acetylene (986 µL, 1.5 equiv), PPh3 (30.0 mg,
2.5 mol %), CuI (22.0 mg, 2.5 mol %), and Et3N (978 µL, 1.5
equiv) in THF (10 mL) was added Pd(PPh3)2Cl2 (163 mg, 5
mol %) with stirring. The solution was stirred at rt for 18 h,
concentrated in vacuo, and then purified by FC (EtOAc/hexane,
1:100) to give benzoate 14 as a colorless oil (993 mg, 92%): 1H
NMR (CDCl3) δ 0.25 (s, 9H), 3.85 (s, 3H), 7.33 (td, J ) 7.7, 1.5
Hz, 1H), 7.41 (td, J ) 7.6, 1.5 Hz, 1H), 7.55 (ddd, J ) 7.6, 1.5,
0.5 Hz, 1H), 7.87 (ddd, J ) 7.6, 1.5, 0.6 Hz, 1H).
2-Eth yn ylben zoic Acid Meth yl Ester (15).70 To a solution
of 14 (500 mg, 2.1 mmol) in MeOH (10 mL) was added KF‚
H2O (1.0 g, excess). The suspension was stirred for 36 h,
filtered, and partitioned between EtOAc (20 mL) and 0.1 M
HCl (20 mL). The organic layer was further washed with brine
(2 × 25 mL), dried (MgSO4), and concentrated in vacuo to leave
a brown oil. Purification by FC (EtOAc/hexane, 1:50) gave
ethynylbenzoate 15 as a colorless oil (281 mg, 82%): 1H NMR
(CDCl3) δ 3.40 (s, 1H), 3.96 (s, 3H), 7.34-7.49 (m, 2H), 7.57-
7.64 (m, 1H), 7.88-7.96 (m, 1H).
(S)-4-[3-ter t-Bu toxy-2-(9H-flu or en -9-ylm eth oxyca r bo-
n yla m in o)p r op ion yla m in o]-3-iod oben zoic Acid 2,2,2-Tr i-
br om oeth yl Ester (12). To a solution of (S)-Fmoc-Ser(t-Bu)-
OH (140 mg, 0.42 mmol) in CH2Cl2 (4 mL) were added SOCl2
(55.0 µL, 2.0 equiv) and DMF (10 µL, catalytic). The solution
was stirred for 3 h and then concentrated in vacuo to give the
crude acid chloride as an oil. This oil was added to a solution
of aminobenzoate 11 (200 mg, 0.38 mmol) in CH2Cl2 (5 mL)
before AgCN (51.0 mg, 1.0 equiv) was added with stirring. The
resulting suspension was stirred for 48 h and then filtered
through a pad of Celite. The filtrate was concentrated in vacuo
and the residue purified by FC (EtOAc/hexane, 1:4) to give
iodobenzoate 12 as a colorless solid (315 mg, 93%): mp 88-
2-Eth yn ylben zoic a cid (16).71 To a solution of 14 (250 mg,
1.05 mmol) in MeOH (5 mL) was added a 2.0 M NaOH solution
(5 mL). The resulting solution was stirred for 2 h, EtOAc (40
mL) was added, and the organic layer was discarded. The
aqueous layer was acidified to pH 1 by the addition of 1 M
HCl and extracted with EtOAc (2 × 25 mL). The combined
organic extracts were dried (MgSO4) and concentrated in vacuo
to leave a pale brown foam. Purification by FC (EtOAc/hexane,
1:4) gave acid 16 as a yellow solid (75.0 mg, 48%): 1H NMR
(CDCl3) δ 3.46 (s, 1H), 7.41-7.61 (m, 2H), 7.68 (dd, J ) 7.5,
1.2 Hz, 1H), 8.10 (dd, J ) 6.2, 1.2 Hz, 1H).
1
2-(2-Am in o-5-m e t h ylca r b a m oylp h e n yle t h yn yl)b e n -
zoic Acid Meth yl Ester (17). Meth od 1. To a solution of
iodobenzamide 13 (175 mg, 0.63 mmol), ethynylbenzoate 15
(100 mg, 0.63 mmol), PPh3 (16.0 mg, 10 mol %), CuI (12.0 mg,
10 mol %), and Et3N (131 µL, 1.5 equiv) in THF (5.0 mL) was
added Pd(PPh3)2Cl2 (22 mg, 5 mol %). The solution was stirred
at rt for 72 h, diluted with EtOAc (25 mL), and washed with
0.1 M HCl (20 mL) and brine (25 mL). The organic layer was
dried (MgSO4), concentrated in vacuo, and then purified by
FC (EtOAc/hexane, 7:3) to give tolan 17 as a pale yellow solid
90 °C; H NMR (CDCl3) δ 1.20 (s, 9H), 3.58 (dd, J ) 7.9, 2.2
Hz, 1H), 3.95 (br d, J ) 7.9 Hz, 1H), 4.25 (t, J ) 5.6 Hz, 1H),
4.40-4.57 (m, 3H), 5.15 (s, 2H), 5.75 (br d, J ) 4.5 Hz, 1H),
7.20-7.48 (m, 4H), 7.61 (br s, 2H), 7.74 (d, J ) 9.0 Hz, 2H),
8.13 (dd, J ) 9.0, 1.2 Hz, 1H), 8.45 (d, J ) 10.1 Hz, 1H), 8.54
(d, J ) 1.2 Hz, 1H), 8.68 (s, 1H); 13C NMR (CDCl3) δ 27.5,
35.6, 47.1, 57.2, 61.5, 67.4, 74.4, 77.1, 88.8, 120.1, 120.3, 124.9,
125.8, 127.1, 127.8, 131.3, 140.8, 141.4, 142.5, 143.6, 157.0,
162.8, 169.3; MS (FAB+) m/z 891, 893, 895, 897 (MH+, 30,
100, 100, 30); HRMS (ES+) m/z calcd for C31H3179Br3IN2O6
(MH+) 890.8777, found 890.8741.
1
(132 mg, 65%): mp 60-62 °C; H NMR (CDCl3) δ 2.99 (2 s,
3H), 3.93 (s, 3H), 4.40 (br s, 2H), 6.15 (br s, 1H), 6.68 (d, J )
8.5 Hz, 1H), 7.34-7.43 (m, 1H), 7.50-7.68 (m, 3H), 7.88 (d, J
) 1.2 Hz, 1H), 8.01-8.09 (m, 1H); 13C NMR (CDCl3) δ 26.8,
52.4, 91.7, 94.0, 106.5, 113.4, 122.9, 124.3, 127.6, 129.3, 130.0,
130.6, 131.1, 132.2, 133.7, 152.3, 165.2, 167.6; MS (EI+) m/z
308 (M+, 40), 277 (100); HRMS (EI+) m/z calcd for C18H16N2O3
(M+) 308.1161, found 308.1165.
2-(2-Am in o-5-m e t h ylca r b a m oylp h e n yle t h yn yl)b e n -
zoic Acid Meth yl Ester (17). Meth od 2. A solution of
iodobenzamide 13 (100 mg, 0.38 mmol), ethynylbenzoate 14
(S )-4-[3-t er t -Bu t oxy-2-(9H -flu or e n -9-ylm e t h oxyca r -
b on yla m in o)p r op ion yla m in o]-3-iod ob en zoic Acid (8).
Meth od 2. 2,2,2-Tribromoethyl ester 12 (100 mg, 0.11 mmol)
was dissolved in AcOH (5 mL), and Zn powder (200 mg, excess)
was added. The suspension was stirred for 0.5 h, filtered
through Celite, and concentrated in vacuo. The residue was
purified by FC (EtOAc/hexane/AcOH, 4:1:0.01) to give acid 8
as a white solid (68.0 mg, 97%). The spectroscopic data are
given above.
4-Am in o-3-iod o-N-m eth ylben za m id e (13). The acid chlo-
ride of iodobenzoic acid 10 was prepared as detailed in the
preparation of compound 11. To a solution of this crude acid
(69) Erdelyi, M.; Gogoll, A. J . Org. Chem. 2001, 66, 4165-4169.
(70) Padwa, A.; Krumpe, K. E.; Kassir, J . M. J . Org. Chem. 1992,
57, 4940-4948.
(71) Brandsma, L.; Hommes, H.; Verkruijsse, H. D.; De J ong, R. L.
P. Recl. Trav. Chim. Pays-Bas 1985, 104, 226-230.
(68) Hirschfeld, J .; Buschauer, A.; Elz, S.; Schunack, W.; Ruat, M.;
Traiffort, E.; Schwartz, J . C. J . Med. Chem. 1992, 32, 2231-2238.
1850 J . Org. Chem., Vol. 68, No. 5, 2003

Solid-Phase Sonogashira Macrocyclization
(177 mg, 2.0 equiv), PPh3 (28.0 mg, 28 mol %), CuI (20.0 mg,
28 mol %), and piperidine (753 µL, 20 equiv) in THF (15 mL)
was purged with nitrogen for 15 min. Solid K2CO3 (116 mg,
2.2 equiv) and Pd(PPh3)2Cl2 (28 mg, 14 mol %) were added,
and the resulting suspension was heated to reflux, during
which time MeOH (2 mL) was added dropwise (to desilylate
14 in situ).51 After 15 h, saturated NH4Cl and Et2O (100 mL,
1:1) were added, and the aqueous layer was discarded. The
organic layer was dried (MgSO4) and concentrated in vacuo
to leave a brown oil. Purification by FC (EtOAc/hexane, 1:1)
gave tolan 17 as a yellow oil (100 mg, 90%). The spectroscopic
data are given above.
DMF, the resin was suspended in CH2Cl2 (5 mL) and allowed
to swell for 5 min before addition of acid 16 (58 mg, 0.40 mmol),
PyBOP (205 mg, 0.40 mmol), and i-Pr2EtN (78 µL, 0.45 mmol).
The resulting mixture was allowed to stand for 3 h with
occasional swirling before the resin was separated by filtration
and washed with DMF (3 × 15 mL), CH2Cl2 (3 × 15 mL),
MeOH (3 × 15 mL), and Et2O (3 × 15 mL). The resin was
then dried under high vacuum to give 20R as an orange resin
(260 mg). Analytical cleavage/side-chain deprotection as above
gave a crude sample of the Fmoc-terminally-protected pep-
tide: MS (ES+) m/z calcd for C115H170IN28O29 (MH+) 2534.2,
found 2534; analytical reversed-phase HPLC, gradient 0-70%
MeCN-H2O (v/v) with 0.1% TFA (v/v) over 60 min, Rf 14.5
min.
Resin 18R. A portion of Fmoc-protected Rink amide resin
(750 mg, nominal loading level 0.58 mmol g-1) was treated
with piperidine-DMF (1:4 v/v) for 30 min to deprotect the
Fmoc group. After being washed with DMF, the resin was
suspended in CH2Cl2 (10 mL) and allowed to swell for 5 min
before addition of acid 8 (311 mg, 0.49 mmol), PyBOP (300
mg, 0.58 mmol), and i-Pr2EtN (175 µL, 1.0 mmol). The
resulting mixture was allowed to stand for 3 h with occasional
swirling before the resin was separated by filtration and
washed with DMF (3 × 25 mL), CH2Cl2 (3 × 25 mL), MeOH
(3 × 25 mL), and Et2O (3 × 25 mL). The resin was then
resuspended in DMF (8 mL) and Ac2O (2.0 mL, excess) and
i-Pr2EtN (2.0 mL, excess) added to cap off any remaining
unreacted amino groups. The resulting mixture was allowed
to stand for 3 h with occasional swirling before the resin was
separated by filtration and washed with DMF (3 × 25 mL),
CH2Cl2 (3 × 25 mL), MeOH (3 × 25 mL), and Et2O (3 × 25
mL). The resin was then dried under high vacuum to give 18R
as a pale orange resin [825 mg, 0.34 mmol g-1 (by Fmoc test)].
Resin 21R a n d Tola n -Con str a in ed Cyclic P ep tid e 22.
A suspension of resin 20R (25.0 mg, assumed ∼0.1 mmol g-1),
PPh3 (1.0 mg), CuI (1.0 mg), and Et3N (5.0 µL) in THF (1 mL)
was purged with nitrogen for 15 min. Pd(PPh3)2Cl2 (1.2 mg)
was added, and the resulting suspension was shaken at rt.
After 15 h, the resin was separated by filtration and washed
with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL),
and Et2O (3 × 5 mL). The resin was then dried under high
vacuum to give 21R as an orange resin (20.1 mg). This resin
sample was treated with TFA-H2O-i-PrSiH (95:4:1 v/v/v) for
2 h. Following filtration, the filtrate was concentrated in vacuo,
precipitated from cold Et2O, and lyophilized from 70% MeCN-
H2O (v/v) with 0.1% TFA (v/v). The resulting solid (3.8 mg)
was purified by analytical reversed-phase HPLC, gradient
0-70% MeCN-H2O (v/v) with 0.1% TFA (v/v) over 60 min, to
give peptidomimetic 22 as a white solid (0.7 mg, ∼15%), Rf
12.4 min: MS (ES+) m/z calcd for C115H169N28O29 (MH+)
2406.2, found 2406.2.
Resin 19R. A portion of resin 18R (220 mg, 0.34 mmol g-1
)
was treated with piperidine-DMF (1:4 v/v) for 30 min to
deprotect the terminal Fmoc group. After being washed with
DMF, the resin was subjected to automated coupling cycles to
build up the linear peptide sequence using the method
described above for the preparation of cyclic peptide 1. The
resin was then dried under high vacuum to give 19R as an
orange resin [320 mg, 0.12 mmol g-1 (by Fmoc test)]. Analytical
cleavage/side-chain deprotection of 10 mg of this resin with
TFA-PhOH-H2O (95:2.5:2.5 v/w/v) for 2 h, then precipitation
from Et2O, and lyophilization from 70% MeCN-H2O (v/v) with
0.1% TFA (v/v) gave a crude sample of the Fmoc-terminally-
protected peptide: MS (ES+) m/z calcd for C121H176IN28O30
(MH+) 2628.2, found 2628.
Ack n ow led gm en t. The generous support of this
work by The Wellcome Trust (Grant GR051392FR) is
gratefully acknowledged. We are also grateful to Dr.
A. J . G. Moir (Department of Molecular Biology and
Biotechnology, University of Sheffield) for performing
the automated peptide syntheses and Mr. N. Rhodes
and Dr. I. Sayers for initial studies.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compounds 3-9, 11-13, and 17 and ES-MS spectra
of compounds 1 and 22 and samples cleaved from resins 19
and 20. This material is available free of charge via the
Internet at http://pubs.acs.org.
Resin 20R. A portion of resin 19R (300 mg, 0.12 mmol g-1
)
was treated with piperidine-DMF (1:4 v/v) for 30 min to
deprotect the terminal Fmoc group. After being washed with
J O026693E
J . Org. Chem, Vol. 68, No. 5, 2003 1851