On-resin native chemical ligation for cyclic peptide synthesis

Article abstract of DOI:10.1021/jo049839d

A novel cysteine derivative, N^α -trityl-S-(9H-xanthen-9-yl)-L-cysteine [Trt-Cys(Xan)-OH] has been introduced for peptide synthesis, specifically for application to a new strategy for the preparation of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i) side-chain anchoring of Fmoc-Asp-OAl via its free β-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii) stepwise chain elongation of the peptide by standard Fmoc/tBu solid-phase chemistry; (iii) removal of the N-terminal Fmoc group; (iv) coupling of Trt-Cys(Xan)-OH; (v) selective Pd(0)-promoted cleavage of the C-terminal allyl ester; (vi) coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii) selective removal of the N ^α-Trt and S-Xan protecting groups under very mild acid conditions; (viii) on-resin cyclization by native chemical ligation in an aqueous milieu; and (ix) final acidolytic cleavage of the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR), and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the desired cyclic product was obtained in 76-86% overall yield with initial purities of ～70%, whereas for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native chemical ligation/cyclization methodology appears to have advantages of convenience and specificity, which make it promising for further generalization.

Full text of DOI:10.1021/jo049839d

On -Resin Na tive Ch em ica l Liga tion for Cyclic P ep tid e Syn th esis1,2
J udit Tulla-Puche and George Barany*
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E.,
Minneapolis, Minnesota 55455
barany@umn.edu
Received J anuary 27, 2004
A novel cysteine derivative, NR-trityl-S-(9H-xanthen-9-yl)-L-cysteine [Trt-Cys(Xan)-OH] has been
introduced for peptide synthesis, specifically for application to a new strategy for the preparation
of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide
cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i) side-chain anchoring of Fmoc-Asp-OAl via
its free â-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii) stepwise chain elongation of the
peptide by standard Fmoc/tBu solid-phase chemistry; (iii) removal of the N-terminal Fmoc group;
(iv) coupling of Trt-Cys(Xan)-OH; (v) selective Pd(0)-promoted cleavage of the C-terminal allyl ester;
(vi) coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii) selective
removal of the NR-Trt and S-Xan protecting groups under very mild acid conditions; (viii) on-resin
cyclization by native chemical ligation in an aqueous milieu; and (ix) final acidolytic cleavage of
the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-
dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR),
and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the
desired cyclic product was obtained in 76-86% overall yield with initial purities of ∼70%, whereas
for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native
chemical ligation/cyclization methodology appears to have advantages of convenience and specificity,
which make it promising for further generalization.
In tr od u ction
The constraint imposed by cyclization may result in en-
hanced biological activity, as well as increased resistance
to proteolysis, by comparison with linear analogues.3b,d,4
Moreover, cyclic peptides may be used as mimics for
proteins in folding studies and to study ligand-receptor
interactions and structure-activity relationships.3e,4a,b,5
Numerous strategies, both solution and solid-phase,
have been reported for the synthesis of “head-to-tail”
cyclic peptides.3b,d,6 Cyclization needs to be carried out
either under high dilution conditions in solution3b,6b,c or
on a solid support to exemplify the “pseudodilution”
principle.7 In each case, the rationale is to favor intramo-
lecular cyclization over unwanted yield-diminishing dimer-
ization and oligomerization side reactions.
A useful solid-phase approachswhich has the added
benefit of avoiding tedious purification steps after each
individual chemical stepsrelies on anchoring of the
starting amino acid residue to the support via its side
chain. This culminates with deprotection steps that free
the N- and C-termini so that they can be joined to each
Cyclic peptides, be they from natural sources or
designed, are important synthetic targets because of their
potential uses as antibiotics and other therapeutic agents.3
* To whom correspondence should be addressed: phone 612-625-
1028, fax 612-626-7541.
(1) Portions of this work were presented at the Eight International
Solid-Phase Synthesis & Combinatorial Libraries Symposium, London,
U.K., September 2-5, 2003.
(2) Abbreviations: Boc, tert-butyloxycarbonyl; Bzl, benzyl; CLEAR,
cross-linked ethoxylate acrylate resin, or poly[trimethylolpropane
ethoxylate (14/3 EO/OH) triacrylate-co-allylamine]; DIEA, N,N-diiso-
propylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMAP,
4-(N,N-dimethylamino)pyridine; DME, 1,2-dimethoxyethane; DMF,
N,N-dimethylformamide; ESMS, electrospray mass spectroscopy; EtOAc,
ethyl acetate; Et2O, diethyl ether; Et3SiH, triethylsilane; FABMS, fast
atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxy-
carbonyl; Gdn‚HCl, guanidine hydrochloride; HATU, N-[(dimethyl-
amino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium
hexafluorophosphate N-oxide; HOAt, 1-hydroxy-7-azabenzotriazole (3-
hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine); HOBt, 1-hydroxybenzotria-
zole; HMPA, hydroxymethylphenoxyacetic acid; HPLC, high-pressure
liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/
ionization time-of-flight (mass spectrometry); NMR, nuclear magnetic
resonance; PAC, peptide acid linker; PEGA, poly[N,N-dimethacryl-
amide-co-poly(ethylene glycol)]; PEG-PS, poly(ethylene glycol)-
polystyrene (graft resin support); PhSiH3, phenylsilane; Pmc, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl; tBu, tert-butyl; TFA, trifluoroacetic
acid; tR, retention time; Trt, triphenylmethyl or trityl; Xan, 9H-
xanthen-9-yl. Amino acid symbols denote the L-configuration. Ab-
breviations used for amino acids follow the IUPAC-IUB Commission
of Biochemical Nomenclature in J . Biol. Chem. 1972, 247, 977-983.
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10.1021/jo049839d CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/15/2004
J . Org. Chem. 2004, 69, 4101-4107
4101

Tulla-Puche and Barany
SCHEME 1. Solid -P h a se Na tive Ch em ica l Liga tion /Cycliza tion
other and thereby effect the required cyclization. Amino
acids used for solid-phase side-chain anchoring to initiate
synthetic routes toward cyclic peptides include Asx/ Glx,8
Lys/Orn,8e,9 Ser/Thr,8e,10 Tyr,8e,10,11 His,12 Phe,13 and Cys.14
It should be noted in passing that the most general
approach to cyclic peptides, which does not require side-
chain functionality, is the backbone anchor linkage (BAL)
approach.15 Indeed, some of the transformations needed
for the current study have already been demonstrated
for BAL;15a,c conversely, cyclizations on BAL follow logi-
cally from a seminal side-chain anchoring precedent.8d
A further relevant theme for the present work is the
use of native chemical ligation,16 a method that under
aqueous conditions serves to join two essentially unpro-
tected peptide chains, one with a cysteine residue at the
N-terminus and the other with a thioester moiety at the
C-terminus (Scheme 1). As elegantly shown both in
solution17 and on the solid phase,18 when the N-terminal
Cys and the C-terminal thioester are within the same
chain, native chemical ligation becomes intramolecular,
and cyclic peptides (or even proteins) can form, especially
when there is a conformational bias to do so.19 Native
chemical ligation/cyclization has been generalized by
using selenocysteine in place of Cys and relying on an
ultimate deselenation step to produce an Ala residue at
the cyclization site.20 We reasoned that side-chain an-
choring as described in the previous paragraph, together
with intramolecular native chain ligation, could be
elaborated into an advantageous solid-phase method to
synthesize cyclic peptides. In this sense, our proposed
approach differs from the sole literature precedent18 for
on-resin native chemical ligation/cyclization, where the
thioester was part of the linkage to the support, with the
consequence that cyclization was concomitant with re-
lease from the support.
The present work was motivated by our interest in pre-
paring cyclic analogues of proteins, specifically sequences
related to bovine pancreatic trypsin inhibitor (BPTI). Due
to our commitment to mild orthogonal Fmoc/tBu solid-
phase chemistry,21 a modified protection scheme needed
to be worked out to manage the key N-terminal Cys.22
In addition, the required C-terminal thioester had to be
introduced at a late stage of the synthesis, due to the
base lability of thioesters (vide infra). The overall side-
chain anchoring/native ligation strategy was illustrated
on the model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-
Pro-Asp-Phe), which in the native protein encompasses
residues 53-58, continues in a circle to 1-4, and includes
two modifications [Cys55 to Abu and Arg53 to Cys].
Because native chemical ligation occurs under aqueous
conditions, poly[N,N-dimethacrylamide-co-poly(ethylene
glycol)] (PEGA)23 and cross-linked ethoxylate acrylate
resin (CLEAR)24 were expected to be useful supports due
to their known excellent swelling in water; this was borne
out experimentally. Poly(ethylene glycol)-polystyrene
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4102 J . Org. Chem., Vol. 69, No. 12, 2004

Solid-Phase Native Chemical Ligation/ Cyclization
(PEG-PS) graft resin supports,25 which also swell with
a range of hydrophilic and hydrophobic solvents but
perhaps not as well in water, were included to complete
the comparison.
a residue with a C-terminal allyl ester, selective depro-
tection, and direct on-resin modification with isothio-
uronium salts, or the solution counterpart in which the
partially protected peptide is modified after cleavage from
a chlorotrityl resin.27c A number of the aforementioned
approaches have been reported to give low or modest
yields,27c,30 and in some cases specific side reactions such
as aspartimide formation,28b hydrolysis/spontaneous cy-
clization,27c thioester migration,27c and racemization27c,28b
have been mentionedsall the more reason to favor the
“backing-off” approach. Second, the issue of providing
selectively removable protection simultaneously to the
NR-amino and the â-thiol groups of cysteine has been
tackled before, for example by use of thiazolidine deriva-
tives that are labile to acid, metals, and electrophiles.31
(In some tandem ligations, the sulfhydryl is allowed to
remain unprotected, because thiol-disulfide exchange
equilibrations continue until the thermodynamic end
point of the desired native ligation is reached.16b,c,e) In
the present work, we take advantage of two highly acid-
sensitive protecting groups, S-9H-xanthen-9-yl (Xan)32 for
a sulfhydryl function and N-trityl (Trt) for the NR-
amine.33
P r ep a r a t ion of Lin ea r P r ot ect ed R esin -Bou n d
P ep tid e-Th ioester . The overall specific procedure is
delineated in Scheme 2. First, a p-alkoxybenzyl alcohol-
type handle34 was loaded onto the free amine functional-
ity of a commercially available CLEAR support. Standard
N,N′-diisopropylcarbodiimide (DIPCDI)/1-hydroxy-7-aza-
benzotriazole (3-hydroxy-3H-1,2,3-triazolo-[4,5-b]pyri-
dine) (HOAt) activation, in DMF, was applied. Mean-
while, HMPA-PEGA was already available. Following
that, Fmoc-Asp-OAl was attached via its free â-carboxyl
side chain to both HMPA-CLEAR and HMPA-PEGA,
by use of DIPCDI-mediated esterifications catalyzed by
4-(N,N-dimethylamino)pyridine (DMAP). For PEG-PS,
a corresponding sequence of steps8d has been carried out
to provide a commercial product which is a chemically
equivalent starting point for the synthesis.
Resu lts a n d Discu ssion
Sid e-Ch a in An ch or in g/Na tive Ch em ica l Liga tion
Str a tegy. Based on considerations outlined in the In-
troduction to this paper, the overall plan for cyclic peptide
synthesis entailed: (i) side-chain anchoring of Fmoc-Asp-
OAl via its free â-carboxyl as a p-alkoxybenzyl ester to a
solid support [PEGA, CLEAR, and PEG-PS were inves-
tigated and compared]; (ii) stepwise chain elongation by
standard Fmoc/tBu solid-phase chemistry; (iii) removal
of the N-terminal Fmoc group; (iv) coupling of NR-trityl-
S-(9H-xanthen-9-yl)-L-cysteine [Trt-Cys(Xan)-OH]; (v)
selective Pd(0)-promoted cleavage of the C-terminal allyl
ester; (vi) coupling of the C-terminal residue, i.e., H-Phe-
SBzl, preactivated as a thioester; (vii) selective removal
of the NR-Trt and S-Xan protecting groups under very
mild acid conditions; (viii) on-resin cyclization by native
chemical ligation in an aqueous milieu; and (ix) final
acidolytic cleavage of the cyclic peptide from the resin.
Several aspects of this plan require commentary, by
way of comparison to related literature approaches. For
one, C-terminal peptide thioesters are usually made by
Boc chemistry and C f N chain assembly, due to the
lability of the thioester moiety to nucleophilic displace-
ment by piperidine, the base generally used for repetitive
removal of the NR-amino 9-fluorenylmethyloxycarbonyl
(Fmoc) protecting group. To prepare thioesters by Fmoc
chemistry, approaches include (i) use of carefully attenu-
ated Fmoc removal conditions, e.g., a cocktail of 2%
(w/v) 1-hydroxybenzotriazole (HOBt) in hexamethylen-
imine-1-methylpyrrolidine-1-methyl-2-pyrrolidinone-
dimethyl sulfoxide (1.6:20:40:40);26 (ii) use of a “safety-
catch” type handle that is first activated (e.g., alkylation
reaction) and then cleaved by the appropriate thiolate
nucleophile;27 (iii) use of standard [e.g., 4-(hydroxymeth-
yl)phenylacetamidomethyl (PAM)] resins during chain
assembly, followed by organoaluminum compound-cata-
lyzed transthioesterification for cleavage;28 (iv) use of an
indirect “backing-off” procedure15c [featuring one step in
the N f C direction], as shown previously with BAL and
chosen in the present work with side-chain anchoring;
(v) an acid-labile BAL strategy [to date demonstrated
only for C-terminal glycine] wherein the C-terminal resi-
due from the start is a base-stable, acid-labile trithio-
ester;29 and (vi) a strategy with side-chain anchoring of
The linear sequence was assembled by standard Fmoc
solid-phase peptide synthesis, via N-[(dimethylamino)-
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(c) Moroder, L.; Musiol, H.-J .; Schaschke, N.; Chen, L.; Hargittai, B.;
Barany, G. In Synthesis of Peptides and Peptidomimetics (Houben-
Weyl E22a: Methods of Organic Chemistry); Goodman, M., Felix, A.
M., Moroder, L., Toniolo, C., Eds.; Georg Thieme Verlag: Stuttgart
and New York, 2002; pp 384-424.
(23) (a) Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080. (b)
Meldal, M. Methods Enzymol. 1997, 289, 83-104.
(24) Kempe, M.; Barany, G. J . Am. Chem. Soc. 1996, 118, 7083-
7093.
(25) Zalipsky, S.; Chang, J . L.; Albericio, F.; Barany, G. React. Polym.
1994, 22, 243-258.
(26) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39,
8669-8672.
J . Org. Chem, Vol. 69, No. 12, 2004 4103

Tulla-Puche and Barany
SCHEME 2. P r ep a r a tion of cyclo(Cys-Th r -Abu -Gly-Gly-Ala -Ar g-P r o-Asp -P h e)
1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-meth-
ylmethanaminium hexafluorophosphate N-oxide (HATU)/
HOBt/N,N-diisopropylethylamine (DIEA) couplings in
DMF. For the last cycle, Trt-Cys(Xan)-OH was combined
with DIPCDI/HOAt (4:4, 0.18 M), with 5 min preactiva-
tion, to minimize racemization.35 Next, the C-terminal
allyl group ester was cleaved in the presence of catalytic
Pd(0) under neutral conditions first described by Albericio
and co-workers;36 note that literature alternatives for
removal of allyl-type protection37 were found to be not
applicable to the highly acid-sensitive substrate for the
present studies (details not shown). Finally, HATU/DIEA
coupling was used to add Phe-SBzl and thereby provide
the desired resin-bound peptide thioester.15c
minal cysteine with both the NR-amine and the â-thiol
in free form. This simultaneous removal of both the N-Trt
and S-Xan protecting groups was achieved by treatment
of the peptide-resin with TFA-Et3SiH-CH2Cl2 (1.0:0.5:
98.5) for 2 h at 25 °C. There was a relatively small and
readily tolerated loss (∼7%) of chains from the support,
due to premature cleavage of the Câ-p-alkoxybenzyl ester.
Next, native chemical ligation was effected by swelling
the peptide-resin in a buffer of 0.25 M sodium phos-
phate, 6 M guanidinium hydrochloride (Gdn‚HCl), and
0.15% thiophenol, made fresh and adjusted to pH 7.5.
As is commonly done, the Gdn‚HCl was included to serve
as a denaturant; the idea was that reasonably stable
partially folded conformations where the cyclization sites
were not near to each other would be forced to equilibrate
with conformations more favorable to cyclization when
in the presence of Gdn‚HCl. Furthermore, the thiophenol
was added to promote thiol exchange, as first proposed
Solid -P h a se Liga tion /Cycliza tion . With the C-ter-
minal thioester established in the linear resin-bound
sequence, it was next necessary to establish an N-ter-
(35) (a) Han, Y.; Albericio, F.; Barany, G. J . Org. Chem. 1997, 62,
4307-4312. (b) Angell, Y. M.; Alsina, J .; Albericio, F.; Barany, G. J .
Pept. Res. 2002, 60, 292-299.
(36) Thieriet, N.; Alsina, J .; Giralt, E.; Guibe´, F.; Albericio, F.
Tetrahedron Lett. 1997, 38, 7275-7278.
(37) (a) Lloyd-Williams, P.; J ou, G.; Albericio, F.; Giralt, E. Tetra-
hedron Lett. 1991, 32, 4207-4210. (b) Kates, S. A.; Daniels, S. B.;
Albericio, F. Anal. Biochem. 1993, 212, 303-310.
4104 J . Org. Chem., Vol. 69, No. 12, 2004

Solid-Phase Native Chemical Ligation/ Cyclization
F IGURE 1. Analytical HPLC of H-Cys-Thr-Abu-Gly-Gly-Ala-
Arg-Pro-Asp-Phe-SBzl, synthesized on (A) PEGA, (B) CLEAR,
and (C) PEG-PS. HPLC was on a C-18 column, developed at
1.0 mL/min, with a linear gradient of 0.1% aqueous TFA-0.1%
TFA in CH3CN, from 9:1 to 2:3 for 30 min. The title peptide is
the main peak (tR ) 19.1 min) and the asterisk indicates the
racemized peptide: H-Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-D-
Asp-Phe-SBzl (tR ) 17.8 min).
F IGURE 2. Formation of cyclic peptide as a function of time
for each of the three resin supports: PEGA (b), CLEAR (2),
and PEG-PS (9). Peptide-resin aliquots were taken at the
indicated times, cleaved with reagent R, and studied by HPLC
(compare to Figure 3 for typical data). Percentages indicate
area of desired cyclic peptide divided by total area of all peptide
material, with no further corrections.
by Dawson and coworkers.38 Final release of the cyclic
peptide, with concomitant removal of side-chain protect-
ing groups, was accomplished by exposure of the pep-
tide-resin to TFA-thioanisole-1,2-ethanedithiol-ani-
sole (90:5:3:2) (reagent R)39 for 1 h.
Con clu sion s
A new strategy has been developed for synthesis of
cyclic peptides by on-resin native chemical ligation. Key
aspects of this strategy include Fmoc/tBu chemistry, side-
chain anchoring, allyl protection of the penultimate
residue for later introduction of the C-terminal thioester,
and a new derivative to manage N-terminal cysteine, i.e.,
Trt-Cys(Xan)-OH, that allows simultaneous, selective,
and mild removal of both protecting groups. A model pep-
tide was prepared successfully with either PEGA or
CLEAR resin supports, which have good aqueous com-
patibility properties; PEG-PS did not give as good re-
sults but the question remains open for all three of these
supports how ligation outcomes would differ with mixed
aqueous/organic or even polar organic solvent milieus.
Future work will also establish the generality of this
strategy to additional cyclic peptides, including those that
do not use a Cys residue for ligation/cyclization. In the
latter regard, further developments in the area of tem-
porary auxiliaries41 and in the applications of selenocys-
teine20 ought to be generalizable to ligation/cyclization.
Another issue that needs to be addressed relates to
whether, for some longer target sequences that could po-
tentially aggregate under aqueous conditions if still pro-
tected, all other side-chain protecting groups [not just
that on the â-thiol of N-terminal Cys] should be removed
before the ligation/cyclization step. This could be done
easily, either by changing to more acid-sensitive groups
or by using a more acid-stable (perhaps orthogonally
cleavable) anchor.
Ch em istr y a s a F u n ction of Resin Su p p or t. Puri-
ties and yields of the linear precursor H-Cys-Thr-Abu-
Gly-Gly-Ala-Arg-Pro-Asp-Phe-SBzl [analyzed after full
deprotection and cleavage from the support] were com-
parable with all three resins studied (Figure 1). In all
cases, the main byproduct observed was Cys-Thr-Abu-
Gly-Gly-Ala-Arg-Pro-D-Asp-Phe-SBzl, in 6-8% of the am-
ount of the main species. Given that a peptidyl C-ter-
minal residue (Asp) is activated when a C-terminal thio-
ester (Phe-SBzl) is installed, a certain level of racemiza-
tion is inevitable.6c,7b,40 The level observed is acceptable
since the product diastereomers are well separated, but
could probably be reduced with careful attention to
experimental details.
With essentially comparable resin-bound linear pre-
cursors, the final stages of the overall ligation/cyclization
strategy (Scheme 2) could be studied as a function of time
(Figure 2). For PEGA, reaction reached 74% completion
within a few hours. Reaction on CLEAR was several-fold
slower but reached 74% after a day. However, the reac-
tion on PEG-PS gave only 27% of the desired cyclic pep-
tide on the same time scale, with substantial levels of
unreacted starting linear peptide (∼52%). In all cases,
reaction products at the end points (Figure 2) were ana-
lyzed further by MALDI-TOF. For PEG-PS, cyclodimer
appeared after 6 h of reaction and reached the level of
11% after 24 h. For CLEAR, cyclodimerization was only
1% after 24 h, whereas for PEGA the side reaction was
detected from the beginning and reached 11% after 2 h.
Exp er im en ta l Section
Gen er a l. Materials, solvents, instrumentation, and general
methods, as well as procedures for peptide synthesis, followed
the literature from our laboratory8d,15a,c,32,35 or others.36 Fmoc-
(38) Dawson, P. E.; Churchill, M.; Ghadiri, M. R.; Kent, S. B. H. J .
Am. Chem. Soc. 1997, 119, 4325-4329.
(39) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.;
Masada, R. I.; Hudson, D.; Barany, G. J . Org. Chem. 1990, 55, 3730-
3743.
(41) (a) Canne, L. E.; Bark, S. J .; Kent, S. B. H. J . Am. Chem. Soc.
1996, 118, 5891-5896. (b) Offer, J .; Boddy, C. N. C.; Dawson, P. E. J .
Am. Chem. Soc. 2002, 124, 4642-4646.
(40) Kemp, D. S. In The Peptides; Gross, E., Meienhofer, J ., Eds.;
Academic Press: New York, 1979; Vol. 1, pp 315-383.
J . Org. Chem, Vol. 69, No. 12, 2004 4105

Tulla-Puche and Barany
1H), 3.08 (dd, J ) 14.5 and 8.0 Hz, 1H). 13C NMR (DMSO-d6)
δ 195.6, 136.3, 134.2, 129.6, 128.9, 128.5, 128.4, 127.4, 127.3,
59.2, 36.9, 32.4. ESMS: m/z calcd for C16H17NOS, 271.10;
found, 271.94 [M + H]+.
SCHEME 3. P r ep a r a tion of
Nr-Tr ityl-S-(9H-xa n th en -9-yl)-L-cystein e
HMP A-CLEAR. CLEAR resin (0.3 g, 0.45 mmol of NH2/
g) was washed thoroughly with DMF (3 × 1 min) and
(3 × 1 min). Next, a solution of HMPA (0.20 g, 1.12 mmol) in
DMF (0.7 mL) was added to the resin, followed in turn by
HOAt (0.15 g, 1.12 mmol) in DMF (0.5 mL) and then DIPCDI
(170 µL, 1.12 mmol). After 12 h of reaction, the resin was
washed with DMF (3 × 1 min) and CH2Cl2 (3 × 1 min), and
then dried in vacuo.
F m oc-Asp (HMP A-CLEAR)-OAllyl. HMPA-CLEAR (0.3
g, 0.45 mmol/g) was washed with DMF (3 × 1 min) and CH2Cl2
(3 × 1 min). Fmoc-Asp-OAllyl (0.21 g, 0.54 mmol) plus HOBt
(83 mg, 0.54 mmol) were dissolved in CH2Cl2 (0.5 mL), and
added to the resin, followed by DIPCDI (95 µL, 0.54 mmol)
and DMAP (28 mg, 0.23 mmol). Coupling was carried out for
90 min, following which the resin was washed with CH2Cl2 (3
× 2 min) and dried in vacuo.
Asp-(PAC-PEG-PS)-OAllyl, CLEAR, and HMPA-PEGA were
obtained from Applied Biosystems (Foster City, CA), Peptides
International (Louisville, KY), and Novabiochem (La¨ufelfingen,
Switzerland), respectively. Analytical HPLC was performed
on a two-pump instrument with a C18 analytical reversed-
phase column (5 µm particle size; 0.46 × 25 cm) with UV
detection at 220 nm.
S-(9H-Xa n th en -9-yl)-L-cystein e (left structure in Scheme
3). This starting material was made in 87% yield on a 10 mmol
scale, exactly following Han and Barany.32 Analytical data
matched what was published, and in addition, the following
were recorded: 13C NMR (CD3OD) δ 172.6, 154.0, 130.9, 130.6,
130.2, 130.1, 124.9, 117.6, 55.3, 43.1, 32.5. ESMS: m/z calcd
for C16H15NNaO3S, 324.1; found, 324.1 [M + Na]+.
F m oc-Asp (HMP A-P EGA)-OAllyl. HMPA-PEGA (0.3 g,
0.55 mmol/g) was washed with DMF (3 × 1 min) and CH2Cl2
(3 × 1 min). Fmoc-Asp-OAllyl (0.26 g, 0.66 mmol) plus HOBt
(100 mg, 0.66 mmol) were dissolved in CH2Cl2 (0.5 mL) and
added to the resin, followed by DIPCDI (117 µL, 0.66 mmol)
and DMAP (28 mg, 0.23 mmol). Coupling was carried out for
90 min, following which the resin was washed with CH2Cl2
(3 × 2 min) and dried in vacuo.
H-Th r (tBu )-Abu -Gly-Gly-Ala -Ar g(P m c)-P r o-Asp (P AC-
P EG-P S)-OAllyl. Automated Fmoc solid-phase synthesis was
carried out on an Applied Biosystems Pioneer synthesizer.
Side-chain protection was provided by 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl (Pmc) for Arg and tert-butyl (tBu) for Thr.
Fmoc removal was achieved with piperidine-DMF (1:4, 5 min),
and 1 h couplings of Fmoc-amino acids (4 equiv) were mediated
by N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-me-
thylmethanaminium hexafluorophosphate N-oxide (HBTU) (4
equiv)/HOBt (4 equiv)/DIEA (8 equiv) in DMF at 25 °C. When
chain assembly was complete, the fully protected peptide-
resin was dried in vacuo [and stored as such, at 4 °C]. An
aliquot of the resin (10 mg) was washed with CH2Cl2 (5 × 2
min) and with DMF (10 × 0.5 min), and the Fmoc group was
cleaved by treatment with piperidine-DMF (1:4) (3 × 1 min,
2 × 5 min, 3 × 1 min, and 2 × 5 min). After the peptide-resin
was washed with DMF (5 × 1 min) and CH2Cl2 (3 × 1 min),
freshly prepared reagent R, TFA-thioanisole-1,2-ethanedithi-
ol-anisole (90:5:3:2) was added (1 mL), and the filtrate after
1 h of cleavage was concentrated under N2. The peptide was
precipitated with cold Et2O (0.5 mL), vortexed, centrifuged,
and decanted, and the vortex/centrifugation/decantation cycle
was repeated twice more with fresh cold Et2O (0.5 mL). The
remaining ether was evaporated with a stream of N2, and the
dried peptide was dissolved in 0.01 N aqueous HCl (0.5 mL)
and lyophilized. The peptide was characterized by analytical
HPLC (tR ) 7.5 min for main peak, ∼91% of total) and MALDI-
TOF (m/z calcd for C33H55N11O12, 797.42; found, 798.43 [M +
H]+).
H -Th r (tBu )-Ab u -Gly-Gly-Ala -Ar g(P m c)-P r o-Asp (H M-
P A-P EGA)-OAllyl a n d H-Th r (tBu )-Abu -Gly-Gly-Ala -Ar g-
(P m c)-P r o-Asp (HMP A-CLEAR)-OAllyl were prepared as
described immediately above for the corresponding synthesis
on PEG-PS. Portions of the peptide-resin were cleaved and
shown to give the same major product by analytical HPLC (tR
) 7.5 min) with the following purities: 90% for CLEAR and
84% for PEGA.
Tr t-Cys(Xan )-Th r (tBu )-Abu -Gly-Gly-Ala-Ar g(P m c)-P r o-
Asp (P AC-P EG-P S)-OAllyl. Coupling of NR-trityl-S-(9H-
xanthen-9-yl)-L-cysteine was carried out for 1 h, in the presence
of DIPCDI/HOBt (4 equiv each with respect to resin, 0.18 M,
5-min preactivation) in DMF to minimize racemization.35a An
aliquot of the resin (10 mg) was cleaved as described earlier
and characterized by analytical HPLC (tR ) 8.1 min) and
Nr-Tr ityl-S-(9H-xa n th en -9-yl)-L-cystein e (right structure
in Scheme 3). In a tritylation procedure modeled on several
in the literature,33 S-(9H-xanthen-9-yl)cysteine (3.01 g, 10.0
mmol) was dissolved in CHCl3 (30 mL), and then H2O (4 mL)
and Et2NH (4 mL). were added. The heterogeneous mixture
was cooled to 4 °C (ice bath), and trityl chloride (2.93 g, 10.5
mmol) was added with magnetic stirring over 30 min. After a
further 2 h of stirring at 25 °C, the layers were separated,
and the organic phase was washed twice with 4% aqueous
Et2NH, dried (MgSO4), and evaporated to dryness. The result-
ing residue was converted to its diethylammonium salt by
refluxing in Et2NH-EtOH (1:30, 31 mL) for 10 min. The salt
was then partitioned between EtOAc (30 mL) and 5% aqueous
KHSO4 (20 mL), and the organic layer was washed with 5%
aqueous KHSO4 (20 mL) and H2O (20 mL) and then dried
(MgSO4) and concentrated to provide a white residue, which
was recrystallized from toluene-petroleum ether to give a
white crystalline solid (3.9 g, 72%). 1H NMR (CDCl3) δ 7.39
(m, 2H), 7.18-7.34 (m, 17H), 7.05-7.12 (m, 4H), 5.20 (s, 1H),
3.52 (dd, J ) 5.5 and 3.5 Hz, 1H), 2.81 (dd, J ) 13.5 and 3.5
Hz, 1H), 1.82 (dd, J ) 13.5 and 5.5 Hz, 1H). 13C NMR (CDCl3)
δ 176.3, 152.7, 152.2, 144.9, 129.9, 129.7, 129.4, 129.2, 129.0,
128.7, 128.5, 128.1, 128.0, 127.9, 127.5, 123.9, 123.7, 116.9,
67.3, 55.8, 42.2, 32.9. HR-FABMS: m/z calcd for C35H29NaO3S,
566.1766; found, 566.1781 [M + Na]+.
L-P h en yla la n in e S-Ben zyl Ester , Hyd r och lor id e Sa lt
(H-P h e-SBzl‚HCl). Following a general procedure from our
laboratory,15c a solution of Boc-Phe-OH (1.77 g, 8.0 mmol) in
CH2Cl2 (50 mL) was cooled to 0 °C and HOBt (1.23 g, 8.0
mmol), DCC (1.65 g, 8 mmol), and DIEA (2.79 mL, 16.0 mmol)
were added sequentially. After 10 min of stirring, benzyl
mercaptan (0.94 mL, 8.0 mmol) was added and the resultant
mixture was stirred for 2 h at 0 °C and then overnight at 25
°C. The reaction mixture was washed with 1 N aqueous HCl
(2 × 150 mL), 10% (w/v) aqueous Na2CO3 (2 × 150 mL), and
H2O (2 × 150 mL), dried (MgSO4), and concentrated in vacuo.
The resultant oil was taken up in 4 N HCl-dioxane and stirred
for 90 min to remove the Boc group. The mixture was
concentrated and chased with Et2O (8 × 50 mL) to obtain the
1
title product as a white solid (1.86 g, 86%). H NMR (DMSO-
d6) δ 8.84 (br s, 3H), 7.22-7.32 (m, 8H), 7.12-7.18 (m, 2H),
4.48 (br s, 1H), 3.71 (s, 2H), 3.23 (dd, J ) 14.5 and 5.5 Hz,
4106 J . Org. Chem., Vol. 69, No. 12, 2004

Solid-Phase Native Chemical Ligation/ Cyclization
MALDI-TOF (m/z calcd for C36H60N12O13S, 900.43; found, 901.4
[M + H]+). Procedures and results were the same for synthesis
on PEGA and on CLEAR.
Tr t-Cys(Xan )-Th r (tBu )-Abu -Gly-Gly-Ala-Ar g(P m c)-P r o-
Asp (P AC-P EG-P S)-OH. The fully protected nonapeptide-
resin just described (0.1 g, 23 µmol) was washed with CH2Cl2
(5 × 0.5 min) under argon. Next, a solution of PhSiH3 (68 µL,
0.55 mmol) in CH2Cl2 (0.4 mL) was added, and the peptide-
resin was stirred manually for 1 min. Following that, a solution
of Pd(PPh3)4 (2.7 mg, 2.3 µmol) in CH2Cl2 (0.2 mL) was added,
and the peptide-resin was gently agitated on a rotary mixer
for 10 min under Ar. The resin was then washed with CH2Cl2
(3 × 1 min), and the deprotection step was repeated. An aliquot
of peptide-resin was cleaved and characterized by analytical
HPLC (tR ) 3.8 min) and MALDI-TOF (m/z calcd for
C
33H56N12O13S, 860.43; found, 862.43 [M + H]+). Procedures
and results were the same for synthesis on PEGA and on
CLEAR.
Tr t-Cys(Xan )-Th r (tBu )-Abu -Gly-Gly-Ala-Ar g(P m c)-P r o-
Asp (P AC-P EG-P S)-P h e-SBzl. H-Phe-SBzl‚HCl (62 mg,
0.23 mmol) was dissolved in CH2Cl2 (1.3 mL), and DIEA (80
µL, 0.46 mmol) was added. The resultant solution was added
to the resin described above. Coupling was initiated by addition
of HATU (87 mg, 0.23 mmol) and the peptide-resin was
stirred for 30 min at 25 °C. The resin was then washed with
DMF (5 × 0.5 min) and CH2Cl2 (5 × 0.5 min), and an aliquot
was taken and cleaved to provide the desired peptide thioester
(75% cleavage yield), which was characterized by analytical
HPLC (tR ) 19.1 min, 85%) and MALDI-TOF (m/z calcd for
F IGURE 3. Analytical HPLC of cyclo(Cys-Thr-Abu-Gly-Gly-
Ala-Pro-Asp-Phe) on (A) PEGA, (B) CLEAR, and (C) PEG-
PS. HPLC was on a C-18 column, developed at 1.0 mL/min
with a linear gradient of 0.1% aqueous TFA-0.1% TFA in CH3-
CN from 9:1 to 2:3 for 30 min. The main peak (A, B) is the
title peptide (tR ) 11.5 min); (*) racemized cyclic peptide
cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Pro-D-Asp-Phe) (tR ) 10.8 min);
(§) cyclodimer cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Pro-Asp-Phe-
Cys-Thr-Abu-Gly-Gly-Ala-Pro-Asp-Phe) (tR ) 13.5 min); (+)
linear precursor H-Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-
Phe-SBzl (tR ) 17.8 min).
C
49H71N13O13S2, 1113.50; found, 1114.23 [M + H]+). The main
byproduct, H-Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-D-Asp-Phe-
SBzl, was characterized by HPLC (tR ) 17.8 min, 8%) and
MALDI-TOF. Procedures were the same for synthesis on
PEGA and on CLEAR. For PEGA, the cleavage yield was 72%
for material of 74% purity by analytical HPLC; H-Cys-Thr-
Abu-Gly-Gly-Ala-Arg-Pro-D-Asp-Phe-SBzl was present at 7%.
For CLEAR, the corresponding values were 68%, 89%, and 6%.
Cyclo(Cys-Th r -Abu -Gly-Gly-Ala -Ar g-P r o-Asp -P h e). The
resin-bound peptide thioester (Scheme 2) was washed with
CH2Cl2 (3 × 1 min) and then a mixture of CH2Cl2-Et3SiH-
TFA (98.5:0.5:1, 2 mL) was added. After 2 h, the resin was
washed with CH2Cl2 (5 × 1 min) and dried. To effect cycliza-
tion, the peptide-resin was treated with a solution containing
0.25 M sodium phosphate, 6 M Gdn‚HCl, and 1% thiophenol,
at pH 7.5, and reaction progress was followed by HPLC
(Figures 2 and 3). After the appropriate time, the peptide-
resin was washed with H2O (3 × 0.5 min) and CH2Cl2 (5 × 1
min) and dried. The peptide was cleaved as described previ-
ously and checked by analytical HPLC (tR ) 11.5 min) and by
MALDI-TOF (m/z calcd for C42H63N13O13S, 989.50; found,
990.19 [M + H]+). The following data were obtained: PEG-
PS, 27% purity, 86% cleavage yield; CLEAR, 74% purity, 76%
cleavage yield; PEGA, 74% purity, 77% cleavage yield. In all
cases the cyclodimer, cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-
Asp-Phe-Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe) was ob-
served by analytical HPLC (tR ) 13.5 min) and characterized
by MALDI-TOF (m/z calcd for C84H126N26O26S2, 1978.9; found,
1980.1 [M + H]+). The following amounts of cyclodimer were
obtained: PEG-PS, 11% after 24 h; CLEAR, 1% after 24 h;
PEGA, 11% after 2 h.
Ack n ow led gm en t. We thank Drs. Fernando Alberi-
cio and Simon K. Shannon for careful reading and
helpful suggestions on the manuscript. This work was
supported by the National Institute of Health (GM
42722 and GM 51628).
J O049839D
J . Org. Chem, Vol. 69, No. 12, 2004 4107