Synthesis and application of unprotected cyclic peptides as building blocks for peptide dendrimers

Article abstract of DOI:10.1021/ja9621105

We describe an efficient regiospecific method for cyclization of unprotected peptide segments based on intramolecular transthioesterification of unprotected cysteinyl peptide thioesters under the control of ring-chain tautomeric equilibrium in aqueous buf

Full text of DOI:10.1021/ja9621105

J. Am. Chem. Soc. 1997, 119, 2363-2370
2363
Synthesis and Application of Unprotected Cyclic Peptides as
Building Blocks for Peptide Dendrimers
Lianshan Zhang and James P. Tam*
Contribution from the Department of Microbiology and Immunology, Vanderbilt UniVersity,
A5119 MCN, NashVille, TN 37232-2363
ReceiVed June 24, 1996X
Abstract: We describe an efficient regiospecific method for cyclization of unprotected peptide segments based on
intramolecular transthioesterification of unprotected cysteinyl peptide thioesters under the control of ring-chain
tautomeric equilibrium in aqueous buffered solutions at pH ranging from 5 to 7.5. The initial cyclization to form an
intramolecular thioester under the ring-chain tautomeric equilibrium is reversible and could be performed in relatively
high concentrations without observable oligomerization. This method overcomes the limitation of conventional
cyclization methods that require high dilutions. The reaction becomes irreversible by a subsequent, spontaneous
proximity-driven S- to N-acyl transfer to the adjacent NR-amine of cysteine to form an end-to-end cyclic peptide.
The cyclization is regioselective. No side reactions were observed with side-chain functionalities such as the Nꢀ-
amine of lysine, thiol of internal cysteine, or imidazole of histidine. Since a free thiol group was introduced to the
product after cyclization, these cyclic peptides were exploited as building blocks for synthesizing peptides with
unusual architectures such as bicyclic peptides containing end-to-end backbones and disulfide bridges as well as
cascade branched peptide dendrimers.
Introduction
is an unprotected cyclic peptide containing a pendant sulfhydryl
group that can be ligated to a branched core matrix via thiol
chemistry5a,b to form a cyclic peptide dendrimer. The use of
unprotected cyclic peptides for such an assembly has the
advantages of good aqueous solubility and accessibility to
analysis and purification by well-established methods.5c
Cyclic peptides have several advantages over their open-chain
counterparts. Cyclization of bioactive peptides generally im-
proves their biological properties, such as metabolic stability
and receptor selectivity.11,12 Cyclization imposes constraints that
enhance conformational homogeneity and facilitate conforma-
tional analysis.2,13 Consequently, stepwise structural rigidifi-
cation through cyclization may provide insights into the
biologically active conformation of linear peptides. Cyclic
peptides have also been used to mimic the surface antigens of
proteins for immunological studies.14 Thus, a cyclic peptide
dendrimer may have advantages observed in monomeric cyclic
peptides.
The design and synthesis of novel artificial peptides and
proteins with supramolecular characteristics represents an active
field of research at the interface of chemistry and medical
science.1,2 Peptide dendrimers, which are a class of biomol-
ecules with extensive uses in immunology and other biomedical
applications, can be placed in this category.3 They are
composed of a defined number of copies of a bioactive peptide
attached to a lysinyl core matrix. One example is cascade
peptide dendrimers, known as multiple antigen peptides (MAPs),
that usually contain four or eight copies of the peptide and have
been used as immunogens,4,5 diagnostic agents,6 and artificial
enzymes.7 They have been recently employed in mimicking
the pore structure of ionic channels8 and in the development of
antibiotics9 and drug delivery vehicles.10 One of the challenges
in synthesizing peptide dendrimers is the construction of
constituent molecular building blocks. A useful building block
* To whom correspondence should be addressed. Phone: (615) 343-
1465. Fax: (615) 343-1467.
A convenient method for the synthesis of a cyclic dendrimer
is through a modular scheme in which unprotected cyclic
peptides are assembled onto a dendritic core matrix through a
pendant sulfhydryl group of the cysteinyl side chain.5 Several
approaches to the synthesis of cyclic peptides have been
developed employing a variety of linkages in different forms.15
X Abstract published in AdVance ACS Abstracts, February 15, 1997.
(1) (a) Hecht, M. H.; Richardson, J. S.; Richardson, D. C.; Ogden, R. C.
Science 1990, 249, 884-891. (b) Kamtekar, S.; Schiffer, J. M.; Xiong, H.;
Babik, J. M.; Hecht, M. H. Ibid. 1993, 262, 1680-1685. (c) Regan, L.
DeGrado, W. F. Ibid. 1988, 241, 976-978. (d) Ghadiri, M. R.; Granja, J.
R.; Milligam, R. A.; McRee, D. E.; Khazanovich, N. Nature (London) 1993,
366, 324-327. (e) Sasaki, T.; Kaiser, E. T. J. Am. Chem. Soc. 1988, 111,
380-381.
(9) Yoshida, M.; Tam, J. P.; Merrifield, R. B. In Peptide Chemistry;
Proceedings of the 2nd Japan Symposium on Peptide Chemistry; Yanaihara,
N., Ed.; ESCOM, Leiden, The Netherlands 1993; pp 297-299.
(10) Sheldon, K.; Liu, D.; Ferguson, J.; Gariepy, J. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 2056-2060.
(11) (a) Hruby, V. J. Life Sci. 1982, 31, 189-199. (b) Hruby, V. J. Trends
Pharm. Sci. 1985, 6, 259-262. (c) Al-Obeidi, F.; Castrucci; A. M. del L.;
Hadley, M. E.; Hruby, V. J. J. Med. Chem. 1989, 32, 2555-2561.
(12) (a) Charpentier, B.; Dor, A.; Roy, P.; England, P.; Pham, H.;
Durieux, C.; Roques, B. P. J. Med. Chem. 1989, 32, 1184-1190. (b) Rivier,
J. E.; Rivier, C.; Vale, W.; Koerber, S.; Corigam, A.; Porter, J.; Gierasch,
L. M.; Hagler, A. T. In Peptides; Rivier, J. E., Marshall, G. R., Eds.;
ESCOM, Leiden, The Netherlands 1990; pp 33-37. (c) Terada, S.; Kato,
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Int. Ed. Engl. 1990, 29, 138-175.
(4) (a)Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5409-5413.
(b) Defoort, J. P.; Nardelli, B.; Shiu, D. R.; Ho, D.; Tam, J. P. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 3879-3883. (c) Nardelli, B., Lu, Y. A.; Shiu,
D. R.; Profy, A. T.; Tam, J. P. J. Immunol. 1992, 148, 914-920. (d) Tam,
J. P.; Clavijo, P.; Lu, Y. A.; Nussenzweig, V.; Nussenzweig, R.; Zavala. F.
J. Exp. Med. 1990, 171, 299-306.
(5) (a) Lu, Y.-A.; Clavijo, P.; Galatino, M.; Shen, Z.-Y.; Liu, W. Tam,
J. P. Mol. Immunol. 1991, 6, 623-630. (b) Defoort, J. P.; Nardelli, B .;
Huang, W.; Tam, J. P. Int. J. Peptide Protein Res. 1992, 40, 214-221. (c)
Shao, J.; Tam, J. P. J. Am. Chem. Soc. 1995, 117, 3893-3899.
(6) Tam, J. P.; Zavala. F. J. Immunol. Methods 1989, 124, 53-61.
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1547.
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Angew. Chem. 1986, 25, 997-999.
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G. J. T.; Van de Werken, G.; Poolman, J. T.; Hoogerhout, P. Int. J. Peptide
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S0002-7863(96)02110-5 CCC: $14.00 © 1997 American Chemical Society

2364 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Zhang and Tam
Nearly all are based on linear protected peptide precursors either
in solution or anchored on a resin support.15-17 Recently, we
and others have developed methods for macrocyclization of
unprotected peptide precursors.18 One particularly useful form
of macrocyclization method is to link unprotected peptides in
an end-to-end fashion, forming a peptide bond as frequently
occurs naturally.19
There are two major considerations in the macrocyclization
of unprotected peptide segments to form end-to-end cyclic
peptides. First and foremost is regioselectivity. The achieve-
ment of regioselectivity of amide bond formation without
protecting groups has been demonstrated in the intermolecular
coupling of linear peptides.20-22 A common principle in
obtaining regioselectivity of amide bond formation is entropic
activation achieved by placing the NR- and CR-termini in close
proximity to allow an intramolecular acyl shift, thus forming a
peptide bond. Brenner et al.23 advocated the template-driven
amino acyl insertion approach while Kemp et al.24 have
developed a proximity-driven acyl transfer approach that results
in highly effective molarity in amide bond formation. We have
investigated other variations and refer to these amide bond
approaches as orthogonal coupling methods.25
Orthogonal coupling is similar in concept to chemoselective
ligation based on thiol or aldehyde chemistries,26-29 but the
resulting product contains an amide bond similar to that obtained
in an amino acid coupling reaction. In end-to-end macrocy-
Figure 1. Cyclization of N-terminal cysteinyl peptides through
transthioesterification under the control of ring-chain tautomeric
equilibrium.
(15) (a) Manesis, N. J.; Goodman, M. J. Org. Chem. 1987, 52, 5331-
5341. (b) Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G.
Biopolymers 1991, 31, 745-750.
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Kates, S. A., Sole´, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.; Albericio,
F. Tetrahedron Lett. 1993, 34, 1549-1552. (c) Richter, L. S.; Tom, J. Y.
K.; Burnier, J. P. Tetrahedron Lett. 1994, 35, 5547-5550
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Protein Res. 1985, 25, 171-177. (b) Felix, A. M.; Wang, C. T.; Heimer,
E. P.; Fournier, A. J. Int. J. Peptide Protein Res. 1988, 31, 231-238.
(18) (a) Botti, P.; Pallin, D. P.; Tam, J. P. J. Am. Chem. Soc. 1996, 118,
10018-10024. (b) Jackson, D. Y.; Burnier, J. P.; Wells, J. A. J. Am. Chem.
Soc. 1995, 117, 819-820. (c) Wood, S. J.; Wetzel, R. Int. J. Peptide Protein
Res. 1992, 39, 533-539.
(19) (a) Hotchkiss, R. D.; Dubos, R. J. J. Biol. Chem. 1940, 136, 803-
804. (b) Wieland, T. Naturwissenschaften 1987, 74, 367-373. (c) Wieland,
T.; Lu¨ben, G.; Ottenheym, H. C. J.; Faesel, J.; de Vires, J. X.; Konz, W.;
Prox, A.; Schmid, J. Angew. Chem., Int. Ed. Engl. 1968, 7, 204-208.
(20) (a) Liu, C. F.; Tam, J. P. J. Am. Chem. Soc.1994, 116, 4149-4153.
(b) Liu, C. F.; Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6584-
6588.
clization, the coupling reaction must be specific between an NR-
amine and a weakly activated CR-acyl moiety and proceeds
independently of the Nꢀ-amine of the side-chain amines and
other side-chain functional groups. For our purpose of obtaining
a cyclic peptide containing a thiol moiety which can then be
used for attachment to a functionalized scaffolding, this could
be achieved by orthogonal coupling via intramolecular tran-
sthioesterification of a linear peptide precursor containing an
N-terminal cysteine and a C-terminal thioester. The thioester
intermediate would subsequently undergo a proximity-driven
S- to N-acyl transfer to give an end-to-end cyclic peptide with
a peptide backbone and would introduce a thiol moiety that is
necessary for the further assembly of peptide dendrimers. The
feasibility of intermolecular coupling of two unprotected peptide
segments was proposed 40 years ago by Wieland et al.30 and
was recently demonstrated in larger peptide segments by
Dawson et al.21 and our laboratory.22 However, the feasibility
and regioselectivity of intramolecular transthioesterification for
preparing cyclic peptides have not been determined.
The second consideration in the macrocyclization of unpro-
tected peptide segments is the avoidance of oligomerization.31
Since both ends of a cysteinyl linear thioester peptide precursor
are reactive moieties, oligomerization would result in low yields
and would require that the macrocyclization be performed in
high dilution. Recently, we have found that we could overcome
this limitation by exploiting the equilibrium of ring-chain
tautomerization deriving from the weakly activated nature of
the CR-acyl moiety used for orthogonal coupling. In ring-chain
tautomerization (Figure 1), an open-chain system (1) containing
(21) Dawson, P. E., Muir, T. W., Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779.
(22) (a) Tam, J. P., Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12485-12489. (b) Liu, C. F.; Rao, C.; Tam, J. P.
Tetrahedron Lett. 1996, 37, 933-936.
(23) (a) Brenner, M.; Zimmerman, J. P.; Wehrmu¨ller, J.; Quitt, P.;
Photaki, I. Experientia 1955, 11, 397-399. (b) Brenner, M.; Zimmerman,
J. P.; Wehrmu¨ller, J.; Quitt, P.; Schneider, W.; Hartmann, A. HelV. Chim.
Acta 1957, 40, 604-610. (c) Brenner, M.; Zimmermann, J. P.; Wehrmu¨ller,
J.; Quitt, P.; Hartmann, A.; Schneider, W.; Beglinger, U. Ibid. 1957, 40,
1497-1517. (d) Brenner, M.; Zimmermann, J. P. Ibid. 1957, 40, 1933-
1939. (e) Dahn, H.; Menasse, R.; Rosenthaler, J.; Brenner, M. Ibid. 1959,
42, 2249-2251.
(24) (a) Kemp, D. S. Biopolymers 1981, 20, 1793-1804. (b) Fotouhi,
N.; Galakatos, N.; Kemp, D. S. J. Org. Chem. 1989, 54, 2803-2817. (c)
Kemp, D. S.; Carey, R. I. Tetrahedron Lett. 1991, 32, 2845-2848. (d)
Kemp, D. S.; Carey, R. I. J. Org. Chem. 1993, 58, 2216-2222.
(25) Tam, J. P.; Liu, C. F.; Lu, Y.-A.; Shao, J.; Zhang, L.; Rao, C.;
Shin, Y. S. In Peptides Chemistry, Structure and Biology; Proceedings of
the 14nd American Peptide Symposium; Kaumaya, P. T. P., Hodges, R. S.,
Eds.; Mayflower Scientific Lit., 1996; pp 15-17.
(26) (a) Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33. (b) Dawson, P.
E.; Kent, S. B. H. J. Am. Chem. Soc. 1993, 115, 7263-7266.
(27) Wallace, C. J. A.; Clark-Lewis, I. J. Biol. Chem. 1992, 267, 3852-
3861.
(28) (a) King, T. P.; Zhao, S. W.; Lam, T. Biochemistry 1986, 25, 5774-
79. (b) Geoghegan, K. F.; Stroh, H. G. Bioconjugate Chem. 1992, 3, 138-
146. (c) Gaertner, H. F.; Rose, K.; Cotton, R.; Timms, D.; Camble, R.
Offord. R. E. Bioconjugate Chem. 1992, 3, 262-268.
(29) (a) Rose, K.; Vilaseca, L. A.; Werlen, R.; Meunier, A.; Fisch, I.;
Jones, R. M. L.; Offord, R. Bioconjugate Chem. 1991, 2, 154-159. (b)
Fisch, I.; Ku¨nzi, G; Rose, K.; Offord, R. Bioconjugate Chem. 1992, 3, 147-
153.
(30) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs
Ann. Chem. 1953, 583, 129-149.
(31) (a) McMurray, J. S.; Lewis, C. A.; Obeyesekere, N. U. Peptide Res.
1994, 7, 195-206. (b) Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345-1356.

Synthesis of Unprotected Cyclic Peptides
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2365
Table 1. Summary of Yields and MALDI-MS Data of Peptide
Thioesters and Their Cyclic Products
thioester
cyclic peptide
yieldc
(%)
sequence
linear
yielda
cyclic (%)
MWb
MWb
CGGFL (CL-5) (11)
CYGGFL (CL-6) (12)
CKYGGFL (CL-7) (13)
CKAYGGFL (CL-8) (14)
CAVSEIQFMHNLGK
(CK-14) (15)
CSNLSTCVLGKLSQEL
(CL-16) (16)
CSNLSTCVLGKLSQEL
(CL-16) (16)
c11
c12
c13
c14
c15
87
85
82
83
72
581.7
744.9
873.1
944.1
1721
92
91
93
90
90
477.6
641.0
768.9
840.0
1559
Figure 2. Preparation of peptide thioesters from stepwise solid-phase
peptide synthesis.
an NR-amine at one end and a polar multiple bond at the other
end is transformed into a cyclic intermediate (2 or 3) through
reversible intramolecular addition of the thiol group to the polar
multiple bond.32 A water, thiol, or alcohol molecule is then
eliminated to yield a stable nonisomeric heterocycle (4 or 5).
By positioning the acyl group and the amine moiety in close
proximity, intramolecular acyl migration can be effected to give
an amide bond in compound 6 or 7. This principle has been
successfully demonstrated in an open-chain system of an amino
aldehyde peptide containing an N-terminal cysteine and a
C-terminal glycoaldehyde ester (Figure 1, pathway 1). An open
chain of an N-terminal cysteinyl peptide thioester can be
considered as a variation of this system since the initial step in
transthioesterification is also a reversible reaction, particularly
in the presence of an excess of a small thiol compound such as
3-mercaptopropionic acid. Upon formation of the cyclic
intermediate 3 which is a cyclic isomer of the open-chain
compound 5, an alkanethiol leaving group is eliminated to give
a cyclic thiolactone (5) (Figure 1, pathway 2). This nonisomeric
heterocycle will subsequently rearrange into a lactam (7) through
a five-membered ring S- to N-acyl transfer. In this way,
racemization may be greatly minimized, and cyclization can
be carried out in high concentration.
c16
17
70
70
1780
1780
82
75
1677
1675
a Crude yield from solid-phase synthesis. b MW obtained by MALDI-
MS and in agreement with the calculated MW. c Isolated yield after
cyclization.
In this paper, we describe and define the scope of the
intramolecular transthioesterification for the cyclization of
unprotected cysteinyl peptide thioesters under the influence of
ring-chain tautomerization equilibrium and their application
for the synthesis of a bicyclic peptide and two cyclic peptide
dendrimers.
Figure 3. Products and a cyclic intermediate identified during the
cyclization of Cys-Gly-Gly-Phe-Leu-SCH2CH2CONH2 (11) (CL-5).
advantage over their corresponding aryl thioesters with respect
to the susceptibility to hydrolysis in aqueous solutions.
Results
Concentration-Independent Cyclization and Ring-Chain
Equilibrium. Four model peptides ranging from 5 to 8 residues
(11-14) used for cyclizations via transthioesterification were
analogs of enkephalin and contained the core sequence of Gly-
Gly-Phe-Leu (Figure 3). Cysteine was placed at the N-termini
and a thioester at the C-termini of the peptides to obtain an
end-to-end cyclic peptide. After cleavage from the resin
support, the crude unprotected cysteinyl peptide thioesters were
found to be sufficiently pure (>87%) and were used directly
for cyclization at pH g 6.4 in phosphate buffer containing a
3-5 fold excess of 3-mercaptopropionic acid (Table 1). The
roles of a thiol are to minimize polymerization and convert the
cysteinyl S-acyl byproduct to the starting material.22 In general,
cyclization occurred when the pH of the cysteinyl peptide
thioester solution was adjusted to >4. Water-soluble tris-
(carboxyethyl)phosphine (TCEP)35 was added to prevent dis-
ulfide formation and to accelerate the desired reaction.22 The
cyclization occurred cleanly and efficiently as shown by RP-
HPLC (Figure 4) in yields ranging from 78 to 92% (Table 1).
Usually, cyclization was completed within 4 h. The cyclization
could be run in moderate to high concentrations at millimolar
levels without affecting the yield. For example, cyclization of
purified CL-7 (13) was carried out in 1, 3.5, and 7 mM at pH
Synthesis of Peptide Thioesters. All peptide thioesters were
prepared directly by solid-phase peptide synthesis33 on a
methylbenzhydrylamine (MBHA) resin (8) using Boc (tert-
butyloxycarbonyl) chemistry with the exception of 15 for which
Boc-Gly-4-[[(oxymethyl)phenyl]acetamido]methyl (Boc-Gly-
Pam) resin was used (Figure 2). The thioester resin 9 was
prepared according to Aimoto et al.34 by converting the
C-terminal Boc-amino acid to its 3-mercaptopropionic acid ester
and then attaching it onto the resin by N,N′-diisopropylcarbo-
diimide (DICI)/1-hydroxybenzotriazole (HOBt). After assembly
of the sequence on the solid support by using the (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) coupling protocol, the peptide thioester 10 was cleaved
from the resin by HF/anisole (9:1, v/v) at 4 °C. Thus, the
N-terminal cysteinyl peptides obtained were alkyl thioesters 11-
16 containing 3′-propionamide (Table 1). All peptides were
purified by reversed phase HPLC (RP-HPLC) and characterized
by matrix-assisted laser desorption ionization MS (MALDI-MS).
Peptide alkyl thioesters were found to be relatively stable and
could be purified under acidic conditions. Thus, they have the
(32) Valters, R. E.; Fulop, F.; Korbonits, D. AdVances In Heterocyclic
Chemistry; Academic Press: New York, 1995, Vol. 64, pp 251-321.
(33) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
Merrifield, R. B. Science 1986, 232, 341-347.
(35) (a) Levison, M. E.; Joseph, A. S.; Kirschenbaum, D. M. Experientia
1969, 25, 126-127. (b) Burnes, J. A.; Butler, J. C.; Moran, J.; Whitesides,
G. M. J. Org. Chem. 1991, 56, 2648-2650.
(34) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117.

2366 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Zhang and Tam
Figure 5. RP-HPLC profiles of product distribution during the
cyclization of the linear precursor, Cys-Gly-Gly-Phe-Leu-SCH2CH2-
CONH2 (11): (A) starting material 11; (B) pH 4.5 after 1 h; (C) pH
4.5 after 18 h; (D) pH 6.7 after 1 h (11, starting material; 18, cyclic
thiolactone intermediate; c11, cyclic lactam product; 20, byproduct
formed by dimerization of the starting material via an amide bond; 21,
byproduct formed by dimerizatin of the product via a disulfide bridge.
Figure 4. RP-HPLC profiles of the cyclization reaction of 13 (CL-7),
Cys-Lys-Tyr-Gly-Gly-Phe-Leu-SCH2CH2CONH2.
Table 2. Cyclization Rates of N-Terminal Cysteinyl Peptide
Thioesters at Different pH Values
ring size
t1/2 (min)
peptide sequence
linear
amino pH pH pH
cyclic atoms acid 5.2 6.4 7.5
Thiolactone Intermediate. In cyclization via transthioes-
terification, the thiolactone is usually not observed when the
reactions are performed at pH 6.5-7.5. This thiolactone rapidly
rearranged through an S to N shift to the desired end-to-end
cyclic peptide. To show that the cyclization does occur through
a thiolactone intermediate and not by direct thioester displace-
ment of the NR-amine, a pentapeptide thioester, Cys-Gly-Gly-
Phe-Leu-S-CH2CH2CONH2 (11) (CL-5), was used as a model.
In this case, the thiolactone intermediate 18 is a 16-membered
ring, and a ring contraction via an S to N shift gives a 15-
membered end-to-end cyclic peptide (c11) (Figure 3). Because
of the ring strain, the contraction via S- to N-acyl shift is likely
to be slow, and it should be possible to isolate the labile
thiolactone intermediate 18. Indeed, at pH 4.5, cyclization of
this pentapeptide proceeded slowly and gave a major product
eluting between the starting material 11 and the lactam c11 in
analytical RP-HPLC (Figure 5). There were several lines of
evidence indicating that this peak was the thiolactone intermedi-
ate. These included the change of retention time in RP-HPLC
(Figure 5B), the correct molecular weight in the MALDI-MS
(found 499.5, calcd for [M + Na - H]+ 499.5), and the
susceptibility of the thiolactone intermediate to hydroxylamine
at pH 936 (Figure 3). The corresponding hydroxylamine
derivative 19 of this peptide was isolated and further confirmed
by MALDI-MS (found 532.9, calcd for [M + Na - H]+ 532.6).
In our study of the intermolecular transthioesterification of two
unprotected peptide segments, the S- to N-acyl transfer occurs
spontaneously, and thus far, the thioester intermediate has not
been observed. Therefore, it is interesting that we could confirm
the thioester intermediate using this cyclic pentapeptide model.
At pH <4, the cyclic intermediate underwent a new intermo-
lecular transthioesterification with the starting material, giving
rise to a dimer of CL-5 as a byproduct (20) (Figure 5C). The
byproduct had a molecular mass of 1082 as detected by MALDI-
MS (found 1082, calcd for [M + Na - H]+ 1082). It was
identified as CGGFLCGGFL-S-CH2CH2CONH2. This side
reaction could be avoided by performing the reaction at pH >
6 to accelerate the S to N transfer in the cyclic intermediate
(Figure 5D).
CGGFL (CL-5) (11)
CYGGFL (CL-6) (12)
CKYGGFL (CL-7) (13)
CKAYGGFL (CL-8) (14) c14
CAVSEIQFMHNLGK
(CK-14) (15)
CSNLSTCVLGKLSQEL
(CL-16) (16)
c11
c12
c13
15
18
21
24
42
5
6
7
8
14
30
10
29
4.4 1.7
1.5 0.5
5.0 0.5
30 6.5 0.6
162 40
c15
2.2
c16
48
16
198 50
3.5
7.5. Oligomerization of the peptide which is often the case in
cyclization using protected peptide segments was not detected
by RP-HPLC. In all three cases, more than 90% of cyclic CL-7
(c13) was recovered as the isolated yield after RP-HPLC
purification. Similar results were also obtained from CL-6 (12),
CK-14 (15), and CL-16 (16). In the case of 15, the reaction
could be carried out in a concentration of 20 mM peptide, which
was >200-fold higher than that of the conventional approach
using active ester and high dilution.31b Thus, in the model
studies, the cyclization appeared to be concentration-independent
and conformed with the expected equilibrium behavior of ring-
chain tautomerism.
Effect of Ring Size and pH. To determine the dependence
of the cyclization rates on ring size, a series of cyclic analogs
with 15-48-atom rings were synthesized, and their cyclization
rates were assessed (Table 2). Peptides 11-14 with 5-8
residues cyclized with t1/2 < 0.5 h at pH 5.6 while peptides 15
and 16 with large rings of 14 and 16 residues proceeded slowly
with t1/2 ) 2.7 and 3.3 h. This result is consistent with the
correlation of cyclization rates and the distance between the N-
and C-termini of peptide chains with faster rates observed in
smaller peptides.
The pH dependence of cyclization was also examined at pH
5.2, 6.4, and 7.5 (Table 2), and the cyclization rates were found
to be faster as the pH increased. In the case of CL-6 (12), the
t1/2 was 10, 1.5, and 0.5 min when cyclization was performed
at pH 5.2, 6.4, and 7.5, respectively. In general, the useful
cyclization pH range is between 6 and 7.5. Below pH 5, the
reaction is too slow, and side reactions occur at pH > 8 due to
thioester hydrolysis at a rate of about 20%/h. The enhanced
reactivity of the cyclization observed at higher pH is in line
with the expected basicity of the unprotonated cysteine amino
groups during the S to N-acyl transfer.
(36) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.

Synthesis of Unprotected Cyclic Peptides
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2367
Selectivity. Two factors affecting cyclization were examined,
the first regioselectivity and the second structural propensity.
The former involves competing side-chain functional groups
such as the amine of lysine, thiol of cysteine, and imidazole of
histidine at internal positions of the peptide sequence. To study
the effect of structural propensity on the cyclization reaction,
two groups of peptides were selected. The first group consisted
of flexible enkephalin analogs with internal glycine. The second
group, exemplified by the N-terminal peptides of human
parathyroid hormone (hPTH) and salmon calcitonin, has an
amphiphilic character and R-helix propensity.37,38
Two peptides, CL-7 13 and CL-8 14, were used to determine
the regioselectivity of the cyclization. Because the Nꢀ-amine
of lysine near the NR-amine terminus would be most likely to
interfere with the S- to N-acyl transfer, a lysine residue was
inserted near the NR-termini of the peptides to compete for the
S to N shift during cyclization. It is important to stress that
both forms of cyclic peptides (end-to-end vs end-to-side-chain)
are likely to be favorable because there is little difference in
their ring sizes. The end-to-end cyclic product of CL-7 is a
21-membered ring while the lysine side-chain cyclic product is
a 22-membered ring. Similarly, the ring sizes of both CL-8
products would differ only by 1 atom (24 vs 25). In both cases,
no lysine side-chain cyclic peptides were observed, showing
that the side-chain amino group of lysine was not involved in
the cyclization reaction.
Using CL-7 (13) as an example, the following method was
used to determine regioselectivity of the intramolecular acyla-
tion. Cyclization of CL-7 was carried out in 0.2 M phosphate
buffer (pH 7.2) in the presence of 2 equiv of TCEP. The
reaction was complete within 2 h. Only one major peak
appeared, and the yield was 95% as indicated by analytical RP-
HPLC. The cyclized product c13 was characterized by MALDI-
MS (found 770, calcd for [M + H]+ 769) and amino acid
analysis. To verify R- and ꢀ-cyclization, the peptide was
oxidized with HCO3H to convert cysteine into cysteic acid and
then treated with 2,4-dinitro-1-fluorobenzene (Sanger’s re-
agent)39 and hydrolyzed with 5.7 M HCl at 110 °C for 24 h.
RP-HPLC analysis showed that no NR-(dinitrophenyl)(DNP)-
cysteic acid was found (tR ) 4.7 min), and only the desired
Nꢀ-DNP-lysine was detected (tR ) 11.7; for HPLC conditions,
see the Experimental Section), indicating the cyclization had
occurred at the N-terminus. Enzymatic digestion of the product
further confirmed the end-to-end cyclization since treatment of
the peptide with trypsin gave a single peak with tR ) 16.6 min
on RP-HPLC (for HPLC conditions, see the Experimental
Section). The expected peptide, YGGFLCK, was further
characterized by MALDI-MS (found 788, calcd for [M + H]+
788).
Figure 6. Synthesis of bicyclic calcitonin fragment 1-16 (17).
and subsequent hydrolysis of the peptide revealed that the
cyclization had occurred in an end-to-end fashion because only
Nꢀ-DNP-lysine was obtained by analytical RP-HPLC. The
product was further confirmed by MALDI-MS (found 1559.2
( 1, calcd for [M + H]+ 1558.8). Trypsin digestion of (c15)
(tR ) 20.27 min) gave the expected linear peptide as detected
by analytical RP-HPLC (tR ) 18.47 min) and MALDI-MS
(found 1578.4, calcd for [M + H]+ 1577.8).
To determine whether the internal cysteine interferes with
the desired reaction, a 16-residue peptide (16) corresponding
to the N-terminal sequence of salmon calcitonin 1-16, Cys-
Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-
Leu-S-CH2CH2CONH2 (CL-16), was subjected to cyclization,
which was carried out in 0.2 M phosphate buffer (pH 7.2)
containing 50% DMF in the presence of 6 equiv of TCEP. N,N-
Dimethylformamide (DMF) was used as a cosolvent to increase
solubility. Analytical RP-HPLC showed the completion of the
reaction after 3 h. A small amount of byproduct (<5%) was
formed due to the hydrolysis of the peptide thioester as detected
by RP-HPLC. Similar cyclization experiments without adding
TCEP led to oligomerization of the cyclic peptide via disulfide
bond formation. Results were improved when the reaction was
performed at 4 °C overnight to suppress thioester hydrolysis.
The cyclic peptide c16 was purified by preparative RP-HPLC
and identified by amino acid analysis and MALDI-MS (found
1677 ( 1, calcd for [M + H]+ 1676.9). Treatment of c16 (tR
) 22.42 min) with trypsin yielded the expected peptide as
identified by analytical RP-HPLC (tR ) 19.24 min) and
MALDI-MS (found 1695.4, calcd for [M + H]+ 1695.0). End
group analysis using Sanger’s reagent, 2,4-dinitro-1-fluoroben-
zene, showed that no DNP-cysteic acid was found, indicating
that only cysteine was the ligation site. The results revealed
that the internal cysteine did not affect transthioesterification
and the subsequent S- to N-acyl rearrangement.
Applications. An advantage of transthioesterification is the
presence of a free thiol in the end product. Two applications
could be visualized to exploit this advantage for the formation
of peptides with unusual architectures. These included the
synthesis of two cascade peptide dendrimers as well as a bicyclic
peptide containing an end-to-end backbone and a disulfide
bridge.
1. Bicyclic Peptide. To demonstrate the feasibility of
forming bicyclic peptides, purified cyclic CL-16, cyclo(Cys-
Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Ser-Gln-Glu-
Leu) (c16), obtained as previously described was oxidized with
10% dimethyl sulfoxide (DMSO) in 0.2 phosphate buffer (pH
7.2) for 12 h (Figure 6). The bicyclic product 17, an end-to-
end cyclic peptide with a disulfide bridge, was obtained in a
yield of 82% (method B, Figure 6). This bicyclic product could
also be synthesized by a one-pot reaction, in which cyclization
and disulfide bond formation of the linear thioester peptide 16
The N-terminal sequence of human parathyroid hormone
(hPTH 1-13), Cys-Ala-Val-Ser-Glu-Ile-Gln-Phe-Met-His-Asn-
Leu-Gly-Lys-S-(CH2)2CONH-CH2CO2H (15) (CK-14) obtained
from a different thioester resin is a more stringent example for
demonstrating regioselectivity because it is rich in side-chain
functional groups, including a C-terminal lysine and an internal
histidine. Cyclization of unprotected peptides such as CK-14
could potentially generate several cyclic products due to end-
to-side-chain cyclization. The cyclization was performed es-
sentially the same as described in the case of CL-7. Only one
product (c15) was obtained when the cyclization was effected
at pH 7.2. Treatment of the cyclic product with Sanger’s reagent
(37) Barden, J. A.; Kemp, B. E. Biochemistry 1993, 32, 7126-7132.
(38) (a) Motta, A.; Morelli, M. A.; Goud, N.; Temussi, P. A. Biochemistry
1989, 28, 7996-8002. (b) Meadows, R. P.; Nikonowicz, E. P.; Jones, C.
R.; Bastian, J. W.; Gorenstein, D. G. Biochemistry 1991, 30, 1247-1254.
(39) Sanger, F. Biochem. J. 1945, 39, 507-515.

2368 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Zhang and Tam
Figure 7. RP-HPLC profiles of the cyclization progress of 16: (A)
starting material 16; (B) reaction after 1 h; (C) reaction after 8 h; (D)
purified bicyclic product 17 (16, starting material; 17, bicyclic product;
c16, monocyclic intermediate; 16a, byproduct resulting from dimer-
ization of 16 via a disulfide bond.
Figure 9. RP-HPLC profile of the conjugation of cyclic CL-14 (c15)
to the tetravalent lysinyl core peptide 22 (left) and MALDI-MS analysis
of the product 24. 25 was identified as the dimer of c15 via a disulfide
bridge.
Using this condition, cyclic CK-14 also could be assembled
to the tetravalent (chloroacetyl)lysinyl core peptide 22. The
reaction proceeded cleanly as shown in analytical RP-HPLC
(gradient 30-100% buffer B in 30 min) and was complete in
40 h (Figure 9). The product 24 was isolated from the excess
cyclic CL-14 in a yield of 80% based on the core peptide. TCEP
was added to the reaction mixture prior to isolation of the
product to convert the dimeric byproduct 25 formed via a
disulfide bridge to its monomer. The monomer c14 was
recovered from the reaction and used in a recycling manner.
The product was finally analyzed by RP-HPLC and confirmed
by MALDI-MS (found 6869.7 ( 1, calcd for [M + H]+ 6868.7).
Figure 8. Synthesis of cyclic peptide dendrimers 23 and 24 through
thioether linkage.
Discussion
were achieved sequentially without isolation of the intermediate
(method A, Figure 6). The reaction was monitored by analytical
RP-HPLC (Figure 7). Interestingly, an intermediate was found
during the reaction. This intermediate was characterized as the
monocyclic peptide bearing free thiol groups (c16) by MALDI-
MS (found 1677 ( 1, calcd for [M + H]+ 1676.9), indicating
that the amide bond formation via transthioesterification pro-
ceeded much faster than disulfide bond formation mediated by
DMSO even though intramolecular disulfide formation was
favored at this oxidative solution. After 18 h the bicyclic peptide
was purified by preparative HPLC in a yield of 75%. No free
thiol groups were present in the cyclic peptide, as detected by
Ellman’s reagent.36 The product 17 was characterized by amino
acid analysis and MALDI-MS (found 1675 ( 1, calcd for [M
+ H]+ 1674.9). The bicyclic products prepared by both methods
were identical in RP-HPLC behavior and MS.
Intermolecular transthioesterification has been successfully
applied to the formation of a peptide bond between two
unprotected peptide segments.21,22 We exploit this new chem-
istry for the cyclization of unprotected peptides. An unanswered
question is whether transthioesterification can fulfill the require-
ments for forming an end-to-end cyclic peptide from an
unprotected linear precursor with high efficiency and regiospeci-
ficity. Our results show that transthioesterification appears to
meet these requirements. The ability of the cysteinyl peptide
thioester to undergo ring-chain tautomerism may contribute
to the high efficiency of cyclization and limit competing
oligomerization. Ring-chain tautomerism is made possible by
the weakly activated CR-moiety which allows the thioester
exchange between the CR-thioester and the free thiol at the
N-terminus. In this case, intramolecular addition of a free thiol
to the carbonyl of the CR-thioester followed by the elimination
of a thiol molecule results in a nonisomeric heterocycle as a
cyclic intermediate. This reaction occurs even in the presence
of an excess of another small thiol molecule. Such a reversible
intramolecular reaction is commonly observed in the synthesis
of macrocycles and heterocyclic compounds.32 The influence
of ring-chain tautomerism is supported by evidence that the
cyclization can be performed in different concentrations without
affecting the yield. In the case of peptide CL-5, it is interesting
that the S- to N-acyl transfer of the cyclic intermediate proceeds
too slowly at pH 4, and that a cyclic thioester intermediate of
this reaction can be isolated and characterized. Usually, the S-
to N-acyl transfer occurs spontaneously at pH 7, and no cyclic
thioester is observed.
2. Cyclic Peptide Dendrimers. To demonstrate the useful-
ness of cyclic peptides as building blocks for the construction
of peptide dendrimers, we assembled cyclic CL-6 (12), an
enkephalin derivative, onto a four-branched tetravalent lysinyl
core peptide. The thiol group of the cyclic CL-6 was intended
as a linking site. Thus, a tetravalent (chloroacetyl)lysinyl core
peptide was synthesized by the solid-phase method (Figure 8).
The assembly of 12 and the core peptide 22 through thioether
formation was performed by incubation of 2.5 equiv of cyclic
CL-6 with the core peptide in 0.2 M phosphate buffer/DMF
(3:1, pH 8.5) containing 0.01 M EDTA under argon. The
reaction was checked by RP-HPLC and was complete in 48 h.
The desired tetravalent cyclic peptide dendrimer 23 was found
as a major product and confirmed by MALDI-MS (found 3128.4
( 1, calcd for [M + Na - H]+ 3127.7. The byproduct was
identified as a dimer of cyclic CL-6 via a disulfide bond.
Previous methods for cyclization of peptides in solution rely
on a combination of enthalpic activation of the CR-carboxy

Synthesis of Unprotected Cyclic Peptides
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2369
group and a protecting strategy for side chains. In addition,
cyclization reactions need to be carried out in high dilution to
minimize unwanted competing intermolecular oligomerization.
In contrast, our method uses a combination of a weakly activated
thioester and a chemoselective transthioesterification, resulting
in a proximity-driven acyl transfer that avoids the limitations
of conventional methods. Furthermore, because the intramo-
lecular transthioesterification is favored under equilibrating
conditions of ring-chain tautomerizaton, moderate to high
concentrations of peptides can be used for the cyclization
reaction.
cyclic peptides bearing a free thiol can be utilized for assembly
of cyclic peptide dendrimers or synthetic protein constructs.
Such complex constructs of defined structure are beginning to
find application as vaccine candidates, as immunodiagnostic
agents, and as drug delivery vehicles. Two tetravalent cyclic
dendrimers were synthesized as examples to demonstrate the
usefulness of this method. The cyclic peptide dendrimers
obtained in this way were of high homogeneity. A bicyclic
peptide with interesting structural complexity was also prepared.
It contained an end-to-end backbone and a disulfide bridge.
Several natural products with interesting medicinal properties
such as circulins43a and kalata43b,c belong to this category, and
our approach may be useful for their syntheses.41
The high regiospecificity of cyclization achieved through
transthioesterification is demonstrated in our model peptides in
which there are no significant side reactions in the peptides
containing side-chain functionalities such as the amine of lysine,
thiol of cysteine, guanidine of arginine, and imidazole of
histidine at internal positions of the peptide sequence. The
selectivity of thiol over amines at side chains is understandable
because the thiol group is a stronger nucleophile at nearly neutral
pH. The selectivity of the thiol at the N-terminus over the
internal cysteinyl thiol, although not readily apparent, could be
attributed to two factors. First, N-terminal cysteine contains
an R-amine in a 1,2-relationship which can serve as a general
base to facilitate transthioesterification while the internal cysteine
does not have this advantage. The general base assistance of
an NR-amine in the rate acceleration of disulfide formation on
NR-cysteinyl peptides have been shown to be 3-10-fold faster
than the corresponding disulfide formation between internal
cysteinyl thiols.40 This also provides an explanation as to the
great susceptibility of NR-cysteinyl peptide to form a disulfide
bond homodimer as compared to those peptides with an internal
cysteine residue. Second, the transthioesterification between
N-terminal cysteine and thioester will lead to a proximity-driven
S- to N-acyl transfer to form a stable peptide bond whereas
transthioesterifications between thiols of internal cysteines are
likely to be reversible. The reversibility of S-acylation of an
internal cysteine has been demonstrated and can be suppressed
with the addition of an excess of thiol compounds such as
3-mercaptopropionic acid.22
Experimental Section
Solid-Phase Peptide Synthesis. All peptide thioesters were prepared
by SPPS according to the procedure described by Aimoto et al.34 using
4-methylbenzhydrylamine (MBHA) or Boc-Gly-[[4-(oxymethyl)phenyl]-
acetamido]methyl (Boc-Gly-Pam) resin at a substitution level of 1.1
mmol/g. The first amino acid thioester was incorporated on the resin
in its propionic acid form. Typically, 0.3-0.4 g of resin was used for
each synthesis. All amino acids were protected with an NR-tert-
butoxycarbonyl (Boc) group. The side-chain protections were as
follows: Arg(Tos), Asp(OcHex), Cys(4-MeBzl), Glu(OcHex), His(Tos),
Lys(2-ClZ), Ser(Bzl), and Thr(Bzl). Each synthesis cycle consisted
of (i) a 25-min deprotection with 55% trifluoroacetic acid/CH2Cl2 and
(ii) coupling with 4 equiv each of Boc-amino acid and BOP or HBTU
in the presence of 6 equiv N,N-diisopropylethylamine (DIEA) in DMF
for 1 h. All couplings were monitored by the ninhydrin test, and a
double coupling was used with 3 equiv each of Boc-amino acid and
N,N′-diisopropylcarbodiimide (DICI)/1-hydroxy-7-azabenzotriazole
(HOAt)44 if necessary.
After assembly of the sequence using BOP/N,N-diisopropylethy-
lamine (DIEA, 1:1.5) as coupling reagent, the peptide thioesters were
cleaved from the resin by HF/anisole (9:1) at 4 °C for 1 h. After HF
was removed, the resulting residue was washed with diethyl ether and
then extracted with 60% acetonitrile in H2O containing 0.045%
trifluoroacetic acid (TFA). All crude peptides were purified on a
preparative C18 reversed-phase (RP) HPLC column (250 × 22 mm)
with a linear gradient of H2O containing 0.045% TFA and 60%
acetonitrile in H2O containing 0.039% TFA at a flow rate of 10 mL/
min. The major fractions were lyophilized, and the total yields ranged
from 70 to 87%, based on the first amino acid loading to the resin
(Table 1). In general, such peptide thioesters can be purified and stored
under appropriate conditions without reaction or loss of the thioester
moiety.
Characterization of Peptide Analogs. Amino acid analysis of each
synthetic peptide was carried out in 5.7 M HCl at 110 °C for 24 h and
was found to agree with the theoretical ratio. Analytical HPLC for all
peptides was performed on a Vydac column (250 × 4.6 mm) with a
1-min isocratic gradient of 10% buffer B and a 30-min linear gradient
of 10-100% buffer B (gradient 1) at a flow rate of 1 mL/min (buffer
A, 0.045% TFA in H2O; buffer B, 0.039% TFA in 60% CH3CN in
H2O). All synthetic peptides were finally characterized by MALDI-
MS (Table 1).
Verification of regiospecificity of cyclization reactions. I. De-
termination of Nr-DNP-cysteic Acid and NE-DNP-Lys To Verify
the Coupling Site. (i) Preparation of Nr-DNP-cysteic Acid and NE-
DNP-Lys Standards. (a) Nr-DNP-cysteic Acid. L-Cysteic acid
monohydrate (18.7 mg, 0.1 mmol) was dissolved in 0.2 M phosphate
Although peptides ranging from 5 to 16 residues in our study
are cyclized in relatively high concentrations with high yields,
the structural factor contributing to high efficiency of cyclization
has not been throughly worked out. In this study, we cyclized
without any difficulties peptides containing internal glycine,
which favors reverse turns, as well as peptides with high
R-helical propensity such as the N-terminal sequences of human
parathyroid hormone (CK-14) and salmon calcitonin (CK-16).
In a related study, we also used ring-chain tautomerism to
cyclize peptides ranging from 5 to 26 residues with a â-strand-
turn-â-strand structure18a as well as circular permutated peptides
with >30 amino acid residues.41 Small to medium flexible
peptides appear amenable to ring-chain tautomeric equilibrium
with a low risk of oligomerization. However, it remains to be
determined whether the approach is applicable to the cyclization
of larger and rigidified peptides, or whether a strong denaturing
condition, such as the presence of a high concentration of urea
or guanidine, is required to force such peptides to cyclize with
high efficiency.
(42) Kaiser, E. T.; Lawrence, D. S.; Rokita, S. E. Annu. ReV. Biochem.
1985, 54, 565-595. (b) Jankowski, K. In Chemistry and Biochemistry of
Amino acids, Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker,
Inc.: New York, 1974; pp 145-200.
Cyclic peptides with a thiol moiety are useful building blocks
for further transformations. Thiols can be transformed into other
functional groups, providing a useful entry for the synthesis of
cyclic peptides containing unusual amino acids.42 Furthermore,
(43) (a) Gustafson, K. R.; Sowder, R. C., II; Henderson, L. E.; Parsons,
I. C.; Kashman, Y.; Caedellina, J. H., II; McMahon, J. B.; Buckheit, R.
W., Jr.; Pannell, L. K.; Boyd, M. R. J. Am. Chem. Soc. 1994, 116, 9337-
9338. (b) Gran, L. Lloydia 1973, 36, 174-178, 207-208, (c) Saether, O;
Craik, D. J. Campbell, I. D.; Sletten, K.; Juul, J.; Norman, D. G.
Biochemistry 1995, 34, 4147-4158.
(40) Tam, J. P.; Wu, C.-R.; Liu; W.; Zhang, J.-W. J. Am. Chem. Soc.
1991, 113, 6657-6662.
(41) Lu, Y.-A.; Yu, Q. T.; Tam, J. P. Unpublished results.
(44) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.

2370 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Zhang and Tam
buffer (400 µL, pH 6.9). This solution was added to 2,4-dinitro-1-
fluorobenzene (18.6 mg, 0.1 mmol). The mixture was heated at 40 °C
for 2 h. After removal of the solvent in Vacuo, the residue was washed
several times with diethyl ether. The product had a tR of 4.7 min by
analytical HPLC as detected at 408 nm (gradient 2: 1 min 30% B,
isocratic; 30-100% within 30 min). The molecular mass of NR-DNP-
cysteic acid was confirmed by MALDI-MS (found 336.3, calcd for
[M + H]+ 336.2).
Synthesis of cyclo(Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-
Lys-Leu-Ser-Gln-Glu-Leu) (c16). The linear precursor, Cys-Ser-Asn-
Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu)-S-CH2CH2-
CONH2 (16) (18 mg, 10 µmol, CL-16), was dissolved in 0.2 M
phosphate buffer (pH 7.2) containing 50% DMF and 6 equiv of TCEP
in three dilutions: 0.1, 0.2, and 0.6 mM. Analytical HPLC showed
the completion of the reaction within 3 h in all three cases (tR ) 22.42
min, gradient 1). A small amount of byproduct (<5%) resulting from
hydrolysis of the peptide thioester was detected. The cyclic peptide
was finally purified by preparative HPLC and identified by MALDI-
MS (found 1677 ( 1, calcd for [M + H]+ 1676.9). Yield: 82%.
Synthesis of Bicyclic Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-
Lys-Leu-Ser-Gln-Glu-Leu (17). (i) Method A. The linear peptide,
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-
S-CH2CH2CONH2 (16) (12 mg, 6.74 µmol), was dissolved in H2O (20
mL). To this solution was added DMSO (5 mL). The pH of the
reaction was adjusted to 9.5 for 10 min using 10% Na2CO3 and
readjusted to 6.7 using acetic acid. The reaction was monitored by
analytical HPLC (Figure 7). After 18 h, the bicyclic peptide was
purified by preparative HPLC. No free thiol groups were detected by
Ellman’s reagent. The product was isolated by preparative HPLC to
give a yield of 75%. The bicyclic peptide 17 was analyzed by RP-
HPLC (tR ) 21.19 min, gradient 1) and characterized by amino acid
analysis and MALDI-MS (found 1675 ( 1, calcd for [M + H]+1674.9).
(ii) Method B. Cyclic CL-16 (c16) (3.2 mg, 0.6 µmol) was
dissolved in H2O (30 mL). The pH of this solution was adjusted to 5
with (NH4)2CO3. DMSO (3 mL) was added to the solution. The
progress of the oxidation was monitored by analytical RP-HPLC. The
reaction was finished in 6 h. The product was purified by RP-HPLC
and characterized by MALDI-MS (found 1675 ( 1, calcd for [M +
H]+ 1674.9).
Kinetic Study. Kinetic analysis was accomplished by RP-HPLC
determination of the reaction progress. All reactions were carried out
at a concentration of 2 mM in 0.2 M phosphate buffer (pH 5.2, 6.4,
and 7.5) in the presence of TCEP. Aliquots (10 µL) were withdrawn
at various time points, and CF3CO2H (5 µL of 10% solution) was added
to quench the reaction. The progress of the cyclization was analyzed
immediately by RP-HPLC. First-order plots of ln [C]/[C0] versus time
(where [C] is equal to the concentration of the linear thioester precursor
and [C0] is equal to the amount of the linear thioester precursor at time
0) were used to calculate the rate and half-life of the cyclization reaction.
Preparation of Chloroacetyl Tetravalent Lysinyl Core Peptide
22. The tetravalent lysinyl core peptide was synthesized as described
previously. The synthesis of the first level of carrier core to form Boc-
Lys(Boc)-Ala-OCH2-Pam resin was achieved using a 4-fold excess of
Boc-Lys(Boc) dicyclohexylamine salt via (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate (BOP) in dichlo-
romethane. The second level of lysine was generated by the same
protocol. After Boc deprotection, tetravalent chloroacetyl moieties were
introduced to the core peptide by using a 10-fold excess of chloroacetic
acid via DICI/HOBt activation. The tetravalent (chloroacetyl)lysinyl
core peptide 22 was cleaved from the resin by HF/anisole (9:1) and
finally purified by RP-HPLC. MALDI-MS: found 780.5, calcd for
[M + H]+ 780.5; found 802.3, calcd for [M + Na - H]+ 802.5.
Synthesis of Cyclic Peptide Dendrimers 23 and 24 through
Thioether Formation. General Procedure. Cyclic peptide (2.8 ×
10-2 mmol) was dissolved in 0.2 M phosphate buffer containing 0.01
M EDTA (1.25 mL, pH 7.4). The solution was purged with argon for
10 min. To this solution was added the tetravalent (chloroacetyl)lysinyl
core peptide (1.1 mg, 1.4 × 10-3 mmol) predissolved in DMF (0.75
mL). The mixture was allowed to vortex for 24 h. The product was
then isolated by RP-HPLC. Yields for cyclic CL-6 and cyclic CK-14
dendrimers were 3.5 and 7.7 mg, respectively. Both cyclic dendrimers
gave a single peak in analytical HPLC. The products were finally
characterized by MALDI-MS: cyclic CL-6 dendrimer 23 (found 3218.4,
calcd for [M + Na - H]+ 3218.4); cyclic CK-14 dendrimer 24 (found
6869.7, calcd for [M + H]+ 6869.9).
(b) NE-DNP-Lys. NR-Boc-Lys-OH (24.6 mg, 0.1 mmol) was
suspended in 0.2 M phosphate buffer (400 µL, pH 6.9). To the
suspension was added 2,4-dinitro-1-fluorobenzene (18.6 mg, 0.1 mmol).
The mixture was heated at 40 °C for 2 h. After removal of the solvent
in Vacuo, the residue was treated with TFA for 10 min. The product
was isolated by preparative HPLC and confirmed by MALDI-MS
(found 313.5, calcd for [M + H]+ 313.3). Nꢀ-DNP-Lys had a tR of
11.7 min (HPLC conditions as described above).
(ii) Determination of Nr-DNP-cysteic Acid and NE-DNP-Lys
through Analytical HPLC. A 100 nmol sample of cyclic peptide was
dissolved in 250 µL of 88% formic acid. The solution was cooled to
4 °C, and then 500 µL of a premade mixture of 88% formic acid and
30% hydrogen peroxide (9:1) was added. The mixture was allowed
to stand for 2 h in an ice bath. The mixture was diluted with 10 mL
of H2O and lyophilized. The sample was then treated with 2,4-dinitro-
1-fluorobenzene as described above and hydrolyzed with 5.7 M HCl
at 110 °C for 24 h. The hydrolysate was analyzed with RP-HPLC
using gradient 2. No NR-DNP-cysteic acid was detected in cyclic
peptides obtained from CL-5, CL-6, CL-7, CL-8, CK-14 and CL-16,
indicating the coupling proceeded through the R-amino terminus of
cysteine.
(II) Enzymatic Digestion of Cyclic Peptides. c13, c15, and c16.
To a solution of cyclic peptide (0.15 µmol) in 0.01 M Tris buffer (pH
7.3, 0.5 mL) was added 10 µg trypsin of dissolved in the same buffer
(2 mg/1 mL). The mixture was shaken for 3 h, and an aliquot was
taken for analytical RP-HPLC using gradient 1. As expected, all three
cyclic peptides were digested into linear peptides. MALDI-MS showed
these to be expected products corresponding to Lys-Xaa bond hydroly-
sis. Anal. RP-HPLC (tR in min): tR ) 20.98 (c13), tR ) 16.65 (c13
after digestion); tR ) 20.27 (c15), tR ) 18.47 (c15 after digestion), tR
) 22.42 (c16), tR ) 19.24 (c16 after digestion). MALDI-MS: 788.8
(calcd for YGGFLCK 786.7, tR ) 16.65), 1578.4 (calcd for
CAVSEIQFMHNLGK 1577.8, tR ) 18.47), and 1695.4 (calcd for
LSQELCSNLSTCVLGK 1695.0, tR ) 19.24).
General Procedure for the Cyclization of Enkephalin Analogs.
Linear precursors 11-16 were dissolved in 0.2 M Na2HPO4 0.1 M
citric acid buffer (pH 7.2) and diluted with the same buffer to the desired
concentration which ranged from 1 to 7 mM. A 1-2 equiv sample of
TCEP was added to the solution. Aliquots were withdrawn for
analytical HPLC. The cyclizations were usually complete within 2 h.
The cyclized peptides were isolated by preparative HPLC. Analytical
data and cyclization yields are summarized in Table 1.
Isolation and Characterization of Cyclization Intermediate 18
and Byproduct 20. Cys-Gly-Gly-Phe-Leu-S-(CH2)2CONH2 (11) (0.58
mg, 1 µmol) was dissolved in 0.02 M Na2HPO4/0.01 M citric acid
buffer (370 µL, pH 6) containing TCEP (1.2 mg, 4.2 µmol). The
reaction pH was approximately 4.5. The reaction was allowed to
proceed for 8 h. The cyclization intermediate 18 was then isolated by
preparative HPLC. MALDI-MS: 477.6 (calcd for [M + H]+ 478.6),
499.5 (calcd for [M + Na - H]+ 499.5), 515.9 (calcd for [M + K -
H]+ 516.6). 18 was treated with 1 M NH2OH (pH 9) to give Cys-
Gly-Gly-Phe-Leu-NH-OH (19). 19 was further confirmed by MALDI-
MS (found 532.9, calcd for [M + Na - H]+ 532.6). The dimeric
byproduct 20 was also isolated from the reaction and characterized by
MALDI-MS (found 1082, calcd for [M + Na - H]+ 1082).
Synthesis of cyclo(Cys-Ala-Val-Ser-Glu-Ile-Gln-Phe-Met-His-
Asn-Leu-Gly-Lys) (c15). Cys-Ala-Val-Ser-Glu-Ile-Gln-Phe-Met-His-
Asn-Leu-Gly-Lys-S(CH2)2CONHCH2CO2NH2 (15) (6 mg, 3.4 µmol)
was dissolved in 0.2 M Na2HPO4/0.1 M citric acid buffer (2 mL, pH
7.5) containing TCEP (1.04 mg, 3.6 µmol). The mixture was vortexed
for 6 h. Analytical HPLC indicated the completion of the cyclization
(tR ) 20.27 min, gradient 1). A 5 mg sample of the cyclic product
was isolated from the reaction. Yield: 88%. MALDI-MS: found 1560
( 1 (calcd for [M + H]+ 1559.8).
Acknowledgment. This work was in part supported by U.S.
Public Health Service NIH Grants AI37965 and CA35644.
JA9621105