Bidirectional tandem pseudoproline ligations of proline-rich helical peptides

Article abstract of DOI:10.1021/ja000128g

We have developed a bidirectional ligation strategy for preparing proline-rich peptides that couples three unprotected segments in tandem to form two pseudoproline bonds (thia- or oxaproline) without the need for a protection scheme. Ligation in the C←N d

Full text of DOI:10.1021/ja000128g

VOLUME 122, NUMBER 18
MAY 10, 2000
© Copyright 2000 by the
American Chemical Society
Bidirectional Tandem Pseudoproline Ligations of Proline-Rich Helical
Peptides
Zhenwei Miao and James P. Tam*
Contribution from the Department of Microbiology and Immunology, Vanderbilt UniVersity, MCN A5119,
NashVille, Tennessee 37232
ReceiVed January 12, 2000
Abstract: We have developed a bidirectional ligation strategy for preparing proline-rich peptides that couples
three unprotected segments in tandem to form two pseudoproline bonds (thia- or oxaproline) without the need
for a protection scheme. Ligation in the CfN direction exploits the regioselectivity of an amino terminal
(NT)-Cys in forming a thiaproline bond over an NT-Ser or NT-Thr peptide in forming an oxaproline bond
with a peptide that bears a carboxyl terminal (CT)-glycoaldehyde ester. Thus, successive ligations of three
unprotected segments in a predetermined order formed a thiaproline and then an oxaproline bond. However,
ligation through the NfC direction is flexible. An NT-Cys, NT-Ser, or NT-Thr segment bearing a CT-glycerol
ester as a masked CT-glycoaldehyde was used to form a pseudoproline bond with another CT-glycoaldehyde
ester segment. Oxidative activation of the glycerol ester product to a CT-glycoaldehyde ester effected another
round of pseudoproline ligation with an NT-Ser, NT-Thr, or NT-Cys segment. This sequential process could
be extended for ligating three or more segments. Optimized conditions for this bidirectional strategy were
applied successfully to syntheses of five analogues of a proline-rich helical antimicrobial peptide, the 59-
residue bactenecin 7 (Bac 7), using three segments containing 24, 14, and 21 amino acids, respectively. CD
spectra showed that Bac 7 and its analogues displayed typical polyproline II helical structures in phosphate
buffers. Furthermore, the ψPro-containing analogues exhibited antibacterial activity similar to Bac 7.
Introduction
helical structures.9 Collagen, the most abundant structural protein
in humans and animals, consists of three peptide chains that
form an extended triple helix.10 In each peptide chain, the
tripeptide sequence Gly-X-Y, where X and Y are often proline
or 4-hydroxyproline (Hyp), repeats about 300 times. Another
type of proline-rich sequence is found in cationic antimicrobial
peptides including RP39, mucin glycoproteins, and the bacte-
necin family.5-8 Two bactenecins, Bac 5 and Bac 7 with
approximate molecular masses of 5 and 7 kD, respectively, are
characterized by a high content of proline (>45%) and arginine
(>20%) residues. Bac 5 contains 43 amino acid residues with
a repeated sequence of Arg-Pro-Pro separated by single
Proline-rich sequences are often found as multiple tandem
repeats in proteins 1-4 and antimicrobial peptides.5-8 Structur-
ally, proline-rich peptides or proteins are known to display
* To whom correspondence should be addressed. James P. Tam,
Department of Microbiology and Immunology, Vanderbilt University, MCN
A5119, Nashville, TN 37232. Telephone: (615)-343-1465. Fax: (615)-
343-1467. E-mail: tamjp@ctrvax.vanderbilt.edu.
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10.1021/ja000128g CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/25/2000

4254 J. Am. Chem. Soc., Vol. 122, No. 18, 2000
Miao and Tam
Figure 1. Orthogonal pseudoproline ligation of a CT-glycoaldehyde segment 1 with an NT-Cys, NT-Ser, or NT-Thr segment 2a-c to form a
pseudoproline bond (ψPro) 5a, thiaproline (SPro); 5b, oxaproline (OPro); or 5c, 5-methyloxaproline (OProMe). The pseudoprolines with a
hydroxymethyl moiety at C2 are R-epimers.
hydrophobic residues.11 CD and 2D NMR studies indicate that
These peculiar effects of Pro have led to the development of
various proline analogues and pseudoprolines (ψPro) that are
intended to produce Pro-like reverse turns,24,25 to give better
control of the cis-trans ratios,26-30 and to prepare various forms
of polyproline helices.31 A popular method of ψPro synthesis
is based on cyclic condensations of Cys, Ser, Thr, Asn, and
Trp with aldehydes or ketones to form pre-made ψPro units as
building blocks in stepwise or convergent synthesis.32-35
Another method employs acid- or metal-catalyzed condensations
on small protected peptides.33c,36 However, these methods are
generally not suitable for preparing large ψPro-containing
peptides because pseudoprolines, particularly oxaprolines, are
unstable to the conditions of acidic deprotection or cleavage
steps in conventional synthetic strategies.33 To overcome this
limitation, bioincorporation of thiaproline into a protein such
as annexin V through aminoacyl-tRNA synthetases has been
developed.37 However, attempts to incorporate oxaproline or
selenaproline into annexin V were unsuccessful.
Bac 5 displays a poly-L-proline II helical structure, and
12,13
represents a new structural category
that differs from the
R-helix and â-sheet structures of antimicrobial peptides.14 Bac
7, a 59-residue peptide, contains the three tandem repeats of a
tetradecamer including several Pro-Arg-Pro triplets alternating
with single apolar residues.6 However, the structure of Bac 7
has not been studied.15
In addition to its repeated sequence, Pro is also involved in
several types of reverse turns including types I, II, IV, and
VIII.16-19 In globular and membrane proteins, Pro often occurs
as a kink in the middle of an R-helix.20,21 The cis-trans
isomerization of an Xaa-Pro bond (Xaa ) any amino acid) has
been implicated in critical roles in protein folding 22 and signal
transduction.23
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In pursuing a different strategy to form various pseudoproline
bonds, we have developed methods for orthogonal ligations
using unprotected peptide segments to form thiaproline or
oxaproline bonds.38,39 Both methods employ a CT-glycoalde-
hyde peptide segment 1 to couple an NT-dinucleophile segment
such as NT-Cys 2a, NT-Ser 2b, or NT-Thr 2c, first through an
imine 3a-c to form a thiazolidine or oxazolidine ester inter-
mediate 4a-c, and then an O,N-acyl migration to afford a
thiaproline bond SPro 5a or oxaproline bond OPro 5b or OProMe
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489. (b) Rousseau, F.; Schymkowitz, J. W.; del Pino, M.; Itzhaki, L. S. J.
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Bidirectional Tandem Ligation
J. Am. Chem. Soc., Vol. 122, No. 18, 2000 4255
the conformational and biological effects of pseudoproline
replacements in proline-rich helical peptides.
Results and Discussion
CfN Three-Segment Tandem Ligation. The strategy for
the CfN tandem ligation of three unprotected peptide segments
by a thiaproline and then an oxaproline ligation is shown in
Figure 2. For convenience, these segments are designated in
the NfC direction as an amino (N) segment bearing a CT-
glycoaldehyde, a middle (M) segment carrying both a CT-
glycoaldehyde ester and an NT-Ser or NT-Thr, and a carboxyl
(C) segment bearing an NT-Cys.
Model studies were employed to optimize the tandem ligation
conditions (data not shown). A two-stage thiaproline ligation
of the C-segment (Cys-Phe-Lys-Ile-OH) and M-segment (Ser-
Leu-Ile-Leu-Asn-Gly-OCH2CHO 10a) was found to be optimal
in two-stage aqueous buffers first at pH 5.3 for 10 h, and then
at pH 6.5 for 20 h. At pH 5.3, HPLC monitoring showed two
major intermediates as the R,S-epimers of thiazolidine-ester
which were converted via an O,N-acyl shift at pH 6.6 after 20
h in 86% yield to a single amide product, an SPro-containing
MC-segment (Ser-Leu-Ile-Leu-Asn-Gly-SPro-Phe-Lys-Ile-OH).
Adjusting the ligation condition to pH 6.6 at the second stage
accelerated the O,N-acyl rearrangement without observable side
reactions due to the thiazolidine-ester hydrolysis or random
intramolecular acyl transfer reaction that occurs at pH > 6.6.
Furthermore, under these conditions, inter-or intramolecular
oxaproline ligation of M-segment 10a forming oligomers or
cyclic peptides, respectively, was not observed.
The MC segment product was purified by HPLC and then
subjected to an oxaproline ligation with the N-segment (Asp-
Ser-Phe-Gly-OCH2CHO 9a) in a pyridine-acetic acid mixture
(1:1, mol/mol). The oxaproline ligation was completed in 35 h
to afford in 78% yield the three-segment ligated NMC product
(Asp-Ser-Phe-Gly-OPro-Leu-Ile-Leu-Asn-Gly-SPro-Phe-Lys-
Ile-OH) with two pseudoprolines. No oxazolidine-ester inter-
mediates were observed. It should be noted that the three-
segment tandem ligation scheme was simplified by the absence
of a protection or deprotection step between each ligation.
Syntheses of unprotected C-segments 12a-d were straight-
forward using conventional Boc chemistry on a commercially
available resin 11, but N- and M-segments required the use of
an acetal resin 6 (Figure 3).42,43 N-segments 9a and 9b were
synthesized using Fmoc chemistry on the cyclic acetal resin 6
as carboxyl terminal (CT)-1,2-diol precursors 7a and 7b which
were then quantitatively converted to the corresponding aldehyde
by sodium periodate in aqueous buffers at pH 4. However,
syntheses of M-segments 10a-c or any N-segments with NT-
Figure 2. CfN approach of three-segment tandem ligation. (i)
thiaproline ligation in aqueous buffers. (ii) oxaproline ligation in
pyridine-acetic mixtures. N ) Asp-Ser-Phe-Gly-OCH2CHO 9a; M )
Ser-Leu-Ile-Leu-Asn-Gly-OCH2CHO 10a; C ) Cys-Phe-Lys-Ile-OH
12a.
5c (Figure 1). The pseudoproline formed at the ligation site is
a novel proline mimetic that retains the amide peptide backbone
but contains an additional C2-hydroxymethyl moiety. The newly
created stereocenter C2 at the ψPro ring is an R-epimer due to
the A1,3-effect of chiral CR at the NT-Cys 2a, NT-Ser 2b, or
NT-Thr 2c peptide.39,40
Thiaproline ligation can be distinguished from oxazolidine
ligation through the use of different solvents and reaction
conditions. Thiaproline ligation between CT-glycoaldehyde 1
and NT-Cys 2a proceeds efficiently in both aqueous conditions
and nonaqueous pyridine-acetic acid mixtures. In contrast,
oxaproline ligation with NT-Ser 2b or NT-Thr 2c peptides is
not observed in aqueous solutions and requires the use of nearly
anhydrous conditions such as pyridine-acetic acid mixtures.39,41
Even under such nonaqueous conditions, thiaproline ligation is
>1000-fold faster than oxaproline ligation. This type of solvent-
driven regioselectivity could be exploited for a tandem ligation
strategy, in which three or more unprotected peptide segments
are coupled without a protection scheme (Figure 2). Since a
ψPro has a cis enhancement on the Xaa-ψPro bond,30,39
a
tandem ligation strategy would also enable the study of the
conformational effects of these two novel pseudoprolines in
polyproline-helical peptides such as Bac 7.
In this paper, we describe the development of a bidirectional
tandem pseudoproline ligation strategy for the synthesis of
polyproline helical peptide analogues of Bac 7 through thiapro-
line or oxaproline bonds. Furthermore, we have also determined
(29) Kern, D.; Schutkowski, M.; Drakenberg, T. J. Am. Chem. Soc. 1997,
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Delaet, N.; Toy, A.; Goodman, M.; Tancredi, T.; Motta, A.; Temussi, P.
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Rohwedder, B.; Sato. T.; Sun, X.; Mutter, M. J. Am. Chem. Soc. 1996,
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6584-6588. (c) Liu, C. F.; Chang, R.; Tam, J. P. J. Am. Chem. Soc. 1996,
118, 307-312.
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Egli, M.; Fitzi, R.; Gautschi, M.; Herrado´n, B.; Hidber, P.; Irwin, J. J.;
Locher, R.; Maestro, M.; Maetzke, T.; Mourio`o, A.; Pfammatter, E.; Plattner,
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4256 J. Am. Chem. Soc., Vol. 122, No. 18, 2000
Miao and Tam
Figure 3. Syntheses of unprotected N-, M-, and C-peptide segments. N-segments 9a,b and M-segments 8d and 10a-c were synthesized on cyclic
acetal resin 6. The C-segments 12a-d, were synthesized using the PAM resin 11.
Figure 4. CfN three-segment tandem ligation of Bac 7, affording two analogues [OPro 25, SPro 39] Bac 7 14a and [OProMe 25, SPro 39] Bac
7 14b. (i) thiaproline ligation in aqueous buffers at pH 5.2 for 10 h, then at pH 6.6 for 20 h. (ii) oxaproline ligation in pyridine-acetic acid mixture
(1:1, mol/mol) for 35 h. Here, OPro stands for OPro and OProMe
.
Ser and NT-Thr required selective oxidation because the
N-terminal 1,2 amino alcohols of NT-Ser or NT-Thr are
susceptible to periodate oxidation, particularly under neutral to
basic pH conditions.44 At pH 6.6, the major product (68.8%)
was oxidation at the NT-1,2-amino alcohol of NT-Ser 8a.
However, under strongly acidic conditions at pH < 2, selective
oxidation of CT-1,2-diol was achieved due to protonation of
the NT-amine group. The desired product 10a was obtained in
86.3% yield in 0.1 N HCl solutions. Similarly, 10b and 10c
were obtained from 8b and 8c in 84.6 and 90.9% yields,
respectively.
14b, by three N-, M-, and C segments consisting of 24, 14, and
21 amino acids, respectively. Their ligation sites were selected
at Pro25 and Pro39 where the Gly-Pro sequence offers the least
steric hindrance (Figure 4). Thiaproline ligation between the
C-segment of NT-Cys 12d, the M-segment with CT-glycoal-
dehyde and NT-Ser 10b or NT-Thr 10c was performed in the
optimized two-stage aqueous conditions. Two intermediates as
the R,S-epimers of thiazolidine-esters were observed at the pH
5.3 stage, which were converted via an O,N-acyl shift to a single
amide product of NT-Ser 13a or NT-Thr 13b at the second stage
of pH 6.2 (Figure 5). Subsequent oxaproline ligation in
pyridine-acetic acid mixtures (1:1, mol/mol) between CT-
glycoaldehyde of N-segment 9 and NT-Ser of 13a or NT-Thr
of 13b completed the syntheses of 14a and 14b. HPLC
monitoring showed that the reaction was clean and predomi-
nantly gave a single product 14a or 14b as well as the unreacted
The optimized conditions based on model peptide studies
were applied to the CfN syntheses of two Bac 7 analogues,
[OPro25, SPro39] Bac 7 14a and [OProMe25, SPro39] Bac 7
(44) Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992, 3, 138-
146.

Bidirectional Tandem Ligation
J. Am. Chem. Soc., Vol. 122, No. 18, 2000 4257
Table 1. Summary of Strategy and Yield in the Syntheses of Five
Bac 7 Analogues through CfN and NfC Three-Segment Tandem
Ligation
*Ligation sites shown in bold.
were similar to those in the CfN approach (Figure 5) and gave
the desired product as a major peak. The results are summarized
in Table 1.
The NfC approach does not require a predetermined order
as in the CfN approach because the C terminus of the
M-segment exists as a CT-glycerol ester. The thiaproline or
oxaproline ligation of the M-segment with the N-segment can
be carried out specifically in aqueous buffers or in pyridine-
acetic acid mixtures. Under these conditions, the CT-glycerol
ester is stable, and an oxidative conversion of the CT-glycerol
ester to a CT-glycoaldehyde is required to effect the subsequent
pseudoproline ligation. Thus, this approach could be expanded
to more than three segments by an iterative process to provide
a convenient method for the syntheses of peptide and proteins
containing proline-rich repeated sequences.1,2
Figure 5. HPLC profiles showing the process of CfN three-segment
tandem ligation of 10b, 12d, and 9b to the Bac7 analogue 14a. (A)
Thiaproline ligation between C-segment 12d and M-segment 10b in
aqueous buffers, first at pH 5.2 for 10 h to form thiazolidine esters
13d,e; then after O,N-acyl transfer at pH 6.6 for 20 h to form the amide
bond product 13a (hatched peak). (B) Oxaproline ligation of 13a with
an N-segment 9b in pyridine-acetic acid (1:1, mol/mol) to form 14a
(labled). *, impurities from pyridine. h, hydrolysis product of 10b to
carboxylic acid.
Confirmation of Xaa-ψPro Bond. The ligation sites, OPro25
and SPro39 of 14a,b as well as SPro25 and OPro39 of 17a-c,
were confirmed amide bonds by FT-IR which showed the
absence of the ester peaks in the region from 1710 to 1780 cm-1
.
starting materials and hydrolyzed N-segment 9b as observable
byproducts (Figure 5). The results of this synthesis of the Bac
7 analogues are summarized in Table 1.
The amide linkage was further confirmed by attempted ami-
nolysis with 1.0 M H2NOH at pH 9.1 and hydrolysis with 0.1
M LiOH for 10 h. Under these conditions, the susceptible
unrearranged ester bond would yield two cleaved segments, but
no aminolysis or hydrolysis product was detected by HPLC.
Furthermore, aldehydes have been known to form heterocycles
with an Arg side chain.45 On the basis of MS analysis, no
evidence of Arg modification by the glycoaldehyde moiety was
found.
CD Characterization. To investigate the influence of
pseudoproline replacement by OPro, OProMe, and SPro on the
structure of Bac7, CD spectroscopy was employed to character-
ize the Bac 7 structure and those of its eight analogues
containing one or two ψPro bonds (Figure 7). 14a,b and 17
a-c prepared by CfN and NfC three-segment ligation
strategies comprise two ψPro bonds, whereas [OPro25] Bac7
18a, [OProMe25] Bac 7 18b, and [SPro25] Bac 7 18c prepared
by two-segment pseudoproline ligation 43 contain a single ψPro
bond in each peptide. For each peptide, the CD spectrum was
determined in 0.02-0.2 mM sodium phosphate buffers at pH
7.2.
NfC Three-Segment Sequential Ligation. To avoid the
selective oxidation needed in the CfN approach, an NfC
three-segment ligation strategy was developed for syntheses of
three Bac 7 analogues, [SPro25, OPro39] Bac 7 17a, [SPro25,
OProMe39] Bac 7 17b and [SPro25, SPro39] Bac 7 17c (Figure
6). For comparison, the same ligation sites as those in the CfN
approach were used, but the ligation sequence was reversed,
starting with an N-segment (Figure 6).
Syntheses of N-segment 9b bearing a CT-glycoaldehyde,
M-segment 8d bearing an NT-Cys and a CT-glycerol ester, and
the C-segments bearing an NT-Ser 12b, NT-Thr 12c, and NT-
Cys 12d are shown in Figure 3.43 Similar to the CfN approach,
the thiaproline ligation between N-segment 9b and M-segment
8d was performed in two-stage aqueous buffers, in which the
1,2-diol moiety of CT-glycerol ester was not affected. The O,N-
acyl rearranged amide product 15 of thiaproline ligation was
obtained in 92.5% yield. Oxidative activation of the peptide
glycerol ester 15 by NaIO4 in aqueous buffers at pH 4 converted
the resulting peptide to a glycoaldehyde ester 16. Oxaproline
or thiaproline ligation between 16 and three C-segments, NT-
Ser 12b, NT-Thr 12c, and NT-Cys 12d, afforded the final
products 17a, 17b, or 17c, respectively. The ligation courses in
this NfC approach as monitored by HPLC (data not shown)
As shown in Figure 7, the CD spectra of Bac 7 and its eight
analogues were consistent with typical polyproline helix II
structures, exhibiting strong broad negative π-π* bands at
(45) Yamasaki, R. B.; Vega, A.; Feeney, R. E. Anal. Biochem. 1980,
109, 32-40.

4258 J. Am. Chem. Soc., Vol. 122, No. 18, 2000
Miao and Tam
Figure 6. Synthesis of Bac 7 through an NfC three-segment sequential ligation strategy affording three analogues 17a-c. Conditions for i and
ii are similar to those in Figure 4. ψPro standing for OPro, OProMe, and SPro; for structures, see Figure 1.
∼206 nm.46-48 These results indicated that these peptides could
form stable polyproline helical structures in aqueous buffers.
Furthermore, dilution experiments did not show significant
changes in CD spectra of Bac 7 and its eight analogues at
concentrations from 0.02 to 0.2 mM (data not shown), suggest-
ing these analogues largely exist in monomeric forms.
substitutions. The blue shifts were [OPro25, SPro39] Bac 7 14a
(6 nm) > [OPro25] Bac7 18a (4.6 nm), [OProMe25, SPro39]
Bac 7 14b (3.5 nm) > [OProMe25] Bac 7 18b (1.5 nm) and
[SPro25, SPro39] Bac 7 17c (0.9 nm) > [SPro25] Bac 7 18c
(0.6 nm). In addition, the substitution at position 25 is a little
more sensitive than that at position 39, so that the blue shifts
were [OPro25, SPro39] Bac 7 14a (6 nm) > [SPro25, OPro39]
Bac 7 17a (5 nm) and [OProMe25, SPro39] Bac 7 14b (3.5 nm)
> [SPro25, OProMe39] Bac 7 17b (2.5 nm).
Antibacterial Activity. To examine the effects of ψPro
replacements on antimicrobial activity, Bac 7 and five analogues
14a,b and 17a-c, derived from the bidirectional tandem ligation
strategy, were assayed against Gram-positive (Staphylococcus
aureus) and Gram-negative (Escherichia coli) bacteria as well
as a fungal strain (Candida kefyr) using the radial diffusion
method.52 The results (Table 2) showed that 14a,b and 17a-c
displayed activities similar to that of Bac 7 with minimal
inhibition concentrations (MIC) at 0.2-0.4 µM.
The replacement of ψPro caused a shift in the maximal
negative π-π* band to shorter wavelength (blue shift). Com-
pared with the maximal negative band of Bac 7 at 209 nm,
different blue shifts were observed in its analogues. For example,
a blue shift from 209 to 203 nm was observed for Bac analogue
17a (Figure 7A). This blue shift is likely caused by the presence
of a minor population of cis conformations (poly-L-proline I
helix).12,13 Proline has a significant propensity to impart the cis
isomer in peptides and proteins due to the low free energy
between the cis and trans isomers of an Xaa-Pro bond (Xaa )
any amino acid).49,50 We have previously observed that pseudopro-
lines enhance cis conformation with 40-67% cis isomers of
Xaa-ψPro bonds, in the order of OPro > OProMe >SPro.39 This
order is consistent with the trends of the blue shifts: [OPro25]
Bac7 18a (4.6 nm) > [OProMe25] Bac 7 18b (1.5 nm) >
[SPro25] Bac 7 18c (0.6 nm). The cis isomer caused a structural
conversion from polyproline helix II to I. Similar results were
also observed by Mutter et al.51 in C2-disubstitued oxaproline-
containing peptides.
Conclusions
Two-segment ligation strategies by thiaproline,38 oxaproline,39
cysteine,53,54 glycine,55 methionine,56 and histidine57 bonds have
been developed for peptide and protein syntheses. This repertoire
of orthogonal ligation methods specific for different NT-amino
acids of peptide segments permits the logical development of a
tandem ligation scheme that can successively ligate multiple
The cis effects on the polyproline helical structures were
further confirmed by the Bac 7 analogues 17a-c with two ψPro
(46) Ronish, E. W.; Krimms, S. Biopolymers. 1974, 13, 1635-1651.
(47) Loomis, R. E.; Borgey, E. J.; Levine, M. J.; Tabak, L. A. Int. J.
Pept. Protein Res. 1985, 26, 621-629.
(52) (a) Lehrer, R. I.; Rosenman, M.; Harwig, S. S.; Jackson, R.;
Eisenhauer, P. J. Immunol. Med. 1991, 137 167-173. (b)Turner, J.; Cho,
Y.; Dinh, N.; Waring, A. J.; Lehrer, R. I. Antimicrob. Agents Chemother.
1998, 42 2206-2214.
(48) Rabanal, F.; Ludivid, M. D.; Giralt, E. Biopolymers 1993, 33, 1019-
1028.
(53) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1995, 92, 12485-12489.
(49) (a) Wu¨thrich, K.; Grathwohl, C. FEBS Lett. 1974, 43, 337-346.
(b) Brandts, J. F.; Halvorson, H. R.; Brennan, M. Biochemistry 1975, 14,
4953-4963.
(54) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12485-12489.
(50) Orengo, C. A. Flores, T. P.; Taylor, W. R.; Thornton, J. M. Protein
Eng. 1993, 6, 485-500.
(55) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
118, 5891-5896.
(51) Mutter, M.; Wo¨hr, T.; Gioria, S.; Keller, M. M. Biopolymers (Peptide
Sci.) 1999, 51, 121-128.
(56) Tam, J. P.; Yu, Q. Biopolymers 1998, 46, 319-327.
(57) Zhang, L.; Tam, J. P. Tetrahedron Lett. 1997, 38, 3-6.

Bidirectional Tandem Ligation
J. Am. Chem. Soc., Vol. 122, No. 18, 2000 4259
Table 2. Comparison of Antimicrobial Activity of Bac 7 and Five
ψPro Analogues 14a,b and 17a-c from Three-Segment Ligation
position of ψPro
25 39
Pro Pro
MIC (µM)a
peptide
E. coli
S. aureus
C. kefyr
Bac 7
14a
14b
17a
17b
17c
0.24
0.28
0.22
0.26
0.21
0.22
0.23
0.35
0.37
0.20
0.28
0.31
0.21
0.22
0.20
0.24
0.22
0.40
OPro
OProMe
SPro
SPro
SPro
OPro
OProMe
SPro
SPro
SPro
a MIC, minimal inhibition concentration, was obtained by radial
diffusion assay with underlay gel containing 1% agarose in 10 mM
phosphate buffer.
ligation strategy can be used for protein conjugation and peptide
dendrimer synthesis.58,59
Experimental Section
Abbreviations. Standard abbreviations are used for the amino acids
and protecting groups (IUPAC-IUB Commission for Biochemical
Nomenclature, J. Biol. Chem., 1985, 260, 14). Other abbreviations are
as follows: CD, circular dichroism; DIEA, diisopropylethylamine;
DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; HBTU, O-benzotriazole-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole;
MALDI-MS; matrix assisted laser desorption ionization mass spec-
trometry; OPro, 2-hydroxymethyloxaproline; OProMe, 2-hydroxymethyl-
5-methyloxaproline; Pyr, pyridine; RP-HPLC, reversed phase high
performance liquid chromatography; SPro, 2-hydroxymethylthiaproline;
TFA, trifluoroacetic acid.
General. Analytical HPLC was run on a Shimadzu 10A system using
a Vydac C18 column (4.6 × 250 mm, 5 µm) with a flow rate of 1.0
mL/min, monitored at 225 nm. Preparative HPLC was performed on
Waters 600 equipment with a Vydac C18 column (22 × 250 mm). All
HPLC was carried out with a reversed phase linear gradient of buffer
A, 0.05% TFA in H2O, and buffer B, 60% CH3CN in H2O with 0.04%
TFA. Mass spectra were obtained by MALDI-TOF method on a
PerSeptive Biosystems Voyager Elite 2 instrument. FT-IR spectra were
recorded on an ATI Mattson Genesis Series FTIR spectrometer with
the sample filmed on CaF2 surface.
Peptide Segments Syntheses. a. N- and M-segments. The peptide
chain assembly was accomplished on the acetal resin 6 using Fmoc/
tBu strategy (Figure 3).43 The tethering of the first amino acid to the
acetal resin 6 was achieved using 4 equiv of (Fmoc-Gly)2O and a
catalytic amount of DMAP and HOBt in anhydrous DMF at RT for 12
h. Stepwise synthesis used HBTU/HOBt 60 as coupling agents and 20%
piperidine in DMF as deprotecting agents. In each synthesis cycle, 2.5
equiv of amino acid was used. Final cleavage of peptides from the
resin was performed with TFA-glycerol-anisole-thioanisole (90:4:
3:3, 40 mL/g resin) for 3 h. Preparative HPLC gave the purified peptide
glycerol esters 7a,b, 8a-d (60-75% yields based on the substitution
of resin 6). 8d was used directly as an M-segment in the NfC strategy
without a periodate oxidation step. 7a,b was completely converted to
N-segment 9a,b with 3 equiv of NaIO4 in aqueous buffers at pH 2 to
7 for 15-30 min. 8a-c were converted to M-segment 10a-c by
selective oxidation with 2 equiv of NaIO4 in 0.1 N HCl solution for
5-15 min. The analysis data for these segments are shown below. 7a,
Figure 7. Comparison of CD spectra of Bac 7 and eight Bac 7
analogues in phosphate buffers at pH 7.2. (A) Bac 7 with [OPro 25,
SPro 39] Bac 7 14a, [SPro 25, OPro 39] Bac 7 17a and [OPro 25] Bac
7 18a. (B) Bac 7 with [OProMe 25, SPro 39] Bac 7 14b, [SPro 25,
OProMe 39] Bac 7 17b and [OProMe 25] Bac 7 18b. (C) Bac 7 with
[SPro 25, SPro 39] Bac 7 17c and [SPro 25] Bac 7 18c.
peptide segments without the need for protection or deprotection.
The bidirectional strategy developed in this study provides an
example, validating the usefulness of orthogonal ligation. Five
Bac 7 analogues 14a,b and 17a-c with different pseudoproline
replacements are obtained in good yields and purities. CD
studies reveal that Bac 7 adopted polyproline II helical structures
in neutral aqueous solutions, whereas the replacements of the
cis-enhanced SPro and OPro in Bac 7 analogues 14a,b and
17a-c result in a minor population of polyproline I structures
in their predominant polyproline II forms. Nevertheless, the
antimicrobial activities of these ψPro analogues are similar to
that of Bac 7.
Asp-Ser-Phe- Gly-OCH2CH(OH)CH2OH, 78.8% yield from 6, tR
)
13.2 min, HRMS m/z 499.2034 (M + H+, C21H31N4O10 requires
499.2040); 7b, Arg-Arg-Ile-Arg-Pro-Arg-Pro-Pro-Arg-Leu-Pro-Arg-
Pro-Arg-Pro-Arg-Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-OCH2CH(OH)-
CH2OH, 74.4% yield based on the acetal resin 6, tR ) 17.4 min,
MALDI-MS m/z 3014.2 (M + H+, 3012.6 calcd for C136H231N51O27).
9a, Asp-Ser-Phe- Gly-OCH2CHO, 94.2% yield from 7a, tR ) 12.6 min,
A potential application of the bidirectional strategy is to
provide various protein analogues with different ψPro combina-
tions at the ligation sites for studies of protein folding pathways
and structure-function relations. Furthermore, the versatility
of a bidirectional ligation strategy makes it possible for
combinatorial synthesis of proteins with unprotected segments
from synthetic or recombinant source. Additionally, the tandem
(58) Zhang, L.; Torgerson, T. R.; Liu, X.-Y.; Timmons, S.; Colosia, A.
D.; Hawiger, J.; Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9184-
9185.
(59) Shao, J.; Tam, J. P. J. Am. Chem. Soc.. 1995, 117, 3893-3899.
(60) Dourtoglou, V.; Gross, B. Synthesis 1984, 572-574.

4260 J. Am. Chem. Soc., Vol. 122, No. 18, 2000
Miao and Tam
HRMS m/z 467.1788 (M + H+, C20H27N4O9 requires 467.1778); 9b,
Arg-Arg-Ile-Arg-Pro-Arg-Pro-Pro-Arg-Leu-Pro-Arg-Pro-Arg-Pro-Arg-
Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-OCH2CHO, 94.4% yield from 7b,
tR ) 17.4 min, MALDI-MS m/z 2980.2 (M + H+, 2980.6 calcd for
NfC Three-Segment Tandem Ligation of Bac 7. The NfC
strategy started with a thiaproline ligation between N-segment 9b (13.2
mg, 4.4 µmol) and M-segment 8d (6.7 mg, 4.0 µmol) that was carried
out at 20 °C in 1 mL of phosphate buffer, first at pH 5.3 for 10 h and
then at pH 6.6 for 20 h. The amide bond product 15 with a CT-glycerol
ester was obtained in 92.6% yield and was completely converted to
peptide aldehyde ester 16 by 3 equiv of NaIO4 at pH 4. The subsequent
pseudoproline ligations between 16 (6.4 mg, 1.4 µmol) and C-segments
12b (2.9 mg, 1.2 µmol), 12c (2.9 mg, 1.2 µmol) and 12d (3.0 mg, 1.3
µmol) were performed in 1 mL of pyridine-acetic acid mixture (1:1,
mol/mol) at 20 °C for 35 h, giving three Bac 7 analogues 17a, 17b,
and 17c in 74.2, 76.4, and 81.0% yields, respectively (Table 1). The
ligation products were confirmed by chemical analysis and MS. 15, tR
) 22.6 min (25-55% B in 30 min), MALDI-MS m/z 4640.5 (M +
H+, 4638.6 calcd for C212H349N74O42S); 16, tR ) 23.1 min, MALDI-
MS m/z 4608.7 (M + H+, 4606.6 calcd for C211H525N345O41S);
17a, tR ) 25.7 min, MALDI-MS m/z 6987.9 (M + H+, 6988.5 calcd
C135H227N51O26); 8a, Ser-Leu-Ile-Leu-Asn-Gly-OCH2CH(OH)CH2OH,
75.8% from 6, tR ) 12.4 min (20-60% B), HRMS m/z 690.4031 (M
+ H+, C30H56N7O11 requires 690.4037); 8b, Ser-Arg-Pro-Ile-Pro-Arg-
Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-OCH2CH(OH)CH2OH, 77.1% yield
based on the acetal resin 6, tR ) 19.4 min (20-60% B), MALDI-MS
m/z 1661.4 (M + H+, 1661.0 calcd for C77H125N23O18); 8c, Thr-Arg-
Pro-Ile-Pro-Arg-Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-Gly-OCH2CH(OH)-
CH2OH, 81.7% yield based on the acetal resin 6, tR ) 19.8 min,
MALDI-MS m/z 1675.8 (M + H+, 1675.1 calcd for C78H127N23O18);
8d, Cys-Arg-Pro-Ile-Pro-Arg-Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-Gly-
OCH2CH(OH)CH2OH, 71.5% yield based on the acetal resin 6, tR
)
20.7 min, MALDI-MS m/z 1678.5 (M + H+, 1677.1 calcd for
C77H125N23O17S); 10a, Ser-Leu-Ile-Leu-Asn-Gly-OCH2CHO, 86.3%
from 8a, tR ) 12.8 min (20-60% B), HRMS m/z 658.3781 (M + H+,
C29H52N7O10 requires 658.3775); 10b, Ser-Arg-Pro-Ile-Pro-Arg-Pro-
Leu-Pro-Phe-Pro-Arg-Pro-Gly-OCH2CHO, 84.3% from 8b, tR ) 19.4
min (20-60% B), MALDI-MS m/z 1630.9 (M + H+, 1628.9 calcd for
C76H121N23O17); 10c, Thr-Arg-Pro-Ile-Pro-Arg-Pro-Leu-Pro-Phe-Pro-
Arg-Pro-Gly-Gly-OCH2CH(OH)CH2OH, 90.1% from 8c, tR ) 19.8 min,
MALDI-MS m/z 1644.1 (M + H+, 1642.9 calcd for C77H123N23O17).
b. C-segments. The C-segment peptides 12a-d were synthesized
on PAM resin using Boc/Bzl and HBTU/HOBt strategy.60 The peptides
were cleaved from the resin by anhydrous HF-anisole (95:5, v/v) and
then purified by HPLC. The amino acid analysis and MS gave the
desired results. 12a, Cys-Phe-Lys-Ile-OH, tR ) 13.2 min, HRMS m/z
510.2733 (M + H+, C24H40N5O5S requires 510.2750); 12b, Ser-Arg-
Pro-Ile-Pro-Arg-Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-Pro-Arg-Pro-Ile-
Pro-Arg-Pro-OH, tR ) 22.4 min (20-60 B%), MALDI-MS m/z 2402.11
(M + H+, 2400.9 calcd for C112H182N36O23); 12c, Thr-Arg-Pro-Ile-Pro-
Arg-Pro-Leu-Pro-Phe-Pro-Arg-Pro-Gly-Pro-Arg-Pro-Ile-Pro-Arg-Pro-
OH, tR ) 22.5 min, MALDI-MS m/z 2416.4 (M + H+, 2414.9 calcd
for C113H184N36O23); 12d, Cys-Arg-Pro-Ile-Pro-Arg-Pro-Leu-Pro-Phe-
Pro-Arg-Pro-Gly-Pro-Arg-Pro-Ile-Pro-Arg-Pro-OH, tR ) 23.3 min,
MALDI-MS m/z 2419.1 (M + H+, 2416.9 calcd for C112H182N36O22S).
CfN Three-Segment Tandem Ligation of Bac 7. The CfN
strategy consisted of two orthogonal pseudoproline ligations. The
thiaproline ligation between C-segment 12d (12.1 mg, 5.0 µmol) and
M-segment 10b (8.9 mg, 5.5 µmol) was carried out initially at 20 °C
in 1 mL of phosphate buffer at pH 5.3 for 10 h. The R,S-epimers of
thiazolidine ester intermediates were observed as two major products,
which were converted to a single amide bond product 13a by adjusting
the pH to 6.6 with 1 M NaHCO3. The conversion was completed at
pH 6.6 for 20 h to yield 13a in 92.6% yield (Figure 5A, Table 1).
Similarly, the amide product 13b of thiaproline ligation between 12d
(12.1 mg, 5.0 µmol) and 10c (9.1 mg, 5.5 µmol) was obtained in 93.1%
yield. The following oxaproline ligations between N-segment 9b (4.2
mg, 2.2 µmol) and the thiaproline ligation products 13a (8.1 mg, 2.0
µmol) and 13b (8.1 mg, 2.0 µmol) were performed in 0.5 mL of
pyridine-acetic acid mixture (1:1, mol/mol) for 35 h at 20 °C,
respectively, giving two Bac 7 analogues 14a and 14b in 80.7 and
77.6% yields, respectively (Figure 5B, Table 1). The ligation products
were confirmed by chemical analysis and MS. 13a, tR ) 24.5 min (20-
50% B in 30 min), MALDI-MS m/z 4026.8 (M + H+, 4026.9 calcd
for C188H300N59O38S); 13b, tR ) 24.8 min, MALDI-MS m/z 4040.7 (M
+ H+, 4040.9 calcd for C189H302N59O38S); 14a, tR ) 25.7 min (20-
55% B in 30 min), MALDI-MS m/z 6986.7 (M + H+, 6988.5 calcd
for C323H524N110O63S); 14b, tR ) 26.2 min, MALDI-MS m/z 6999.0
(M + H+, 7002.5 calcd for C324H526N110O63S).
for
7026.1 (M + Na+, 7024.5 calcd for C324H525N110O63SNa). 17c, tR
26.6 min, MALDI-MS m/z 7029.9 (M + Na+, 7026.6 calcd for
C323H524N110O63S); 17b, tR ) 26.1 min, MALDI-MS m/z
)
C323H523N110O62SNa).
Circular dichroism (CD) Spectroscopy. CD measurements of Bac
7 and its eight analogues 14a,b, 17a-c, and 18a-c were carried out
on a Jasco 720 spectropolarimeter connected to a temperature controller
and an IBM computer. Each peptide was dissolved in phosphate buffers
at pH 7.2 in a concentration of 0.02-0.2 mM. CD spectra were recorded
at 20 °C in a quartz cell with 0.1 mm path length, using a 2.0 nm
bandwidth, a sensitivity of 50 mdeg, a time constant of 4 s, and a 50
nm/min scanning speed with 0.2 nm resolution.
Antimicrobial Assays. Gram-negative E. coli ATCC 25922 and
Gram-positive S. aureus 29213 bacteria, as well as the fungal strain C.
kefyr ATCC 37095 were used for antimicrobial assays. The strains were
incubated in trypticase soy broth (TSB) and used for experiments less
than 4 weeks after being taken from stock.
A sensitive and reproducible two-stage radial diffusion assay method
developed by Lehrer et al.52 was employed. Briefly, a (1-4) × 106
cfu/ml aliquot of a test organism was mixed with 10 mL of molten
underlay gel solution and poured into 10 × 10-cm Petri dishes to form
a uniform layer. The gel solution contained 10 mM sodium phosphate
buffer, 0.03% TSB, and 0.02% Tween 20. In high-salt assay conditions,
the gel solution contained 100 mM NaCl, whereas no NaCl was used
in the low-salt assay. After solidification, gel wells with 3-mm diameters
were made by a template in an evenly spaced array. An aliquot of 5
µL of a serial half-log dilution of testing peptides 14a,b and 17a-c at
seven concentrations was added to each well after removing the gel
plugs. The dishes were incubated at 37 °C for 3 h to allow test peptides
to diffuse into the underlay gels. Gels were overlaid with 10 mL of
1% agarose in 6% of TSB (w/v). After incubation at 37 °C for 16-24
h, the diameter of the clear zone surrounding the wells (colony-free)
was measured under the microscope. Antimicrobial activities were
expressed in units (0.1 mm ) 1 U), and the MICs were determined
from the x intercepts of the dose-response curves. The low-salt results
of activities are summarized in Table 2.
Acknowledgment. We thank Dr. Jin-Long Yang for help
with antimicrobial assays. This work was in part supported by
U.S. Public Health Service NIH GM57145, CA36544, and
AI46164.
JA000128G