Tandem ligation of unprotected peptides through thiaprolyl and cysteinyl bonds in water

Article abstract of DOI:10.1021/ja0035654

Tandem ligation for the synthesis and modification of proteins entails forming two or more regiospecific amide bonds of multiple free peptide segments without a protecting-group scheme. We here describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the N → C direction, first by an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both an NT-Cys and a CT-thioester. The newly created O-ester-linked segment with a CT-thioester was then ligated to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently, this basic condition also catalyzed the O,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides ranging from 19 to 70 amino acids, including thiaproline → proline analogues of somatostatins and two CC-chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for combinatorial segment synthesis.

Full text of DOI:10.1021/ja0035654

J. Am. Chem. Soc. 2001, 123, 2487-2494
2487
Tandem Ligation of Unprotected Peptides through Thiaprolyl and
Cysteinyl Bonds in Water†
James P. Tam,* Qitao Yu, and Jin-Long Yang
Contribution fom the Department of Microbiology and Immunology, Vanderbilt UniVersity, A5119 MCN,
NashVille, Tennessee 37232-2363
ReceiVed October 2, 2000
Abstract: Tandem ligation for the synthesis and modification of proteins entails forming two or more
regiospecific amide bonds of multiple free peptide segments without a protecting-group scheme. We here
describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain
N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-
Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo
selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the
N f C direction, first by an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a
peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both an
NT-Cys and a CT-thioester. The newly created O-ester-linked segment with a CT-thioester was then ligated
to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently,
this basic condition also catalyzed the O,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond
at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed
in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides
ranging from 19 to 70 amino acids, including thiaproline f proline analogues of somatostatins and two CC-
chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological
activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free
peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach
for combinatorial segment synthesis.
Introduction
ers.12,13 An additional advantage is their mild reaction conditions,
mostly performed in aqueous solutions that permit ligation
chemistry to modify living systems such as cell surfaces and
phage-display libraries.14,15
Chemoselective ligation methods, widely popular in protein
conjugation chemistry, generally afford a nonamide bond such
as a thioether or an oxime at the ligation site. They can be
achieved by appropriately placing a mutually reactive nucleo-
phile-electrophile pair on each peptide segment. The more
demanding and recently developed orthogonal ligation strat-
egy16-19 affords a specific amide bond, usually a backbone
Chemoselective and orthogonal methods for ligating free
peptides, proteins, and biopolymers in water have recently
attracted considerable attention because of their wide applicabil-
ity in both chemistry and biology. A major advantage of these
methods is that they are not burdened by the use of a protection
scheme or a coupling reagent. As a result, they can employ
building blocks derived from chemical, natural, or biosynthetic
sources to form macromolecules of different shapes or sizes in
aqueous solutions. Indeed, successful applications of ligation
methods have been demonstrated in semi- and total syntheses
of peptides,1-3 glycopeptides,4-6 proteins,7-11 and dendrim-
(3) Melnyk, O.; Bossus, M.; David, D.; Rommens, C.; Gras-Masse, H.
J. Pept. Res. 1998, 52, 180-184.
* To whom correspondence should be addressed. Telephone: (615) 343-
1465. Fax: (615) 343-1467. E-mail: tamjp@ctrvax.vanderbilt.edu.
†Abbreviations: Standard abbreviations are used for the amino acids
and protecting groups [IUPAC-IUB Commission for Biochemical No-
menclature (1985) J. Biol. Chem. 1985, 260, 14]. Other abbreviations are
as follows: NT, N-terminal; CT, carboxyl terminal; SPro, thiaproline; Sm-
28, somatostatin-28; N-segment, amine segment; M-segment, middle
segment; C-segment, carboxyl segment; Boc, t-butoxycarbonyl; Fmoc,
fluorenylmethoxycarbonyl; RP-HPLC, reverse phase-high performance
liquid chromatography; MALDI-MS, matrix assisted laser desorption
ionization mass spectrometry; TFA, trifluoroacetic acid; HF, hydrofluoric
acid; MBHA, methylbenzhydrylamine; MIP, macrophage inflammatory
protein; TCEP, tris(carboxyethyl)phosphine; MPA, 3-mercaptopropionic
acid; DMSO, dimethyl sulfoxide; S-tBu, sulfenyl-tert-butyl; Acm, Aceta-
midomethyl; BOP, benzotrizol-1-yl-oxy-tris(dimethylamino)phosphonium-
hexafluorophosphate; DCM, dichloromethane; DCC, N,N′-dicyclohexyl-
carbodiimide; DIC, N,N-diisopropylcarbodiimide; DIEA, N,N-diisopro-
pylethylamine; DMF, dimethylformamide; HOBt, N-hydroxybenzotriazole.
(1) Lu, Y.-A.; Clavijo, P.; Galatino, M.; Shen, Z.-Y.; Liu, W.; Tam, J.
P. Mol. Immunol. 1991, 6, 623-630.
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10.1021/ja0035654 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/24/2001

2488 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tam and et al.
peptide bond formed by a regiospecific coupling reaction
between an R-amino terminus of one peptide chain and an
R-ester terminus of another peptide chain. In this regard, the
orthogonal ligation strategy may provide a chemical model for
an abiotic approach to peptide synthesis leading to specific
formation of functional biopolymers under aqueous conditions
without the assistance of known cellular and molecular ma-
chinery.
Thus far, the abiotic concept for amide-bond ligation of two
unprotected peptide segments largely exploits the principle of
a proximity-driven acyl transfer reaction through a cascade
sequence of chemoselective capture and acyl migration.18,19,22-26
An early approach that has elegantly illustrated this principle
for peptide synthesis is the “Thiol Capture” developed by Kemp
and his co-worker.20,21 The Thiol Capture, with the aid of a
rigid tricyclic template, is regiospecific to an amino-terminal
(NT)-Cys and a carboxyl terminal (CT)-ester in the presence
of other amines, side-chain nucleophiles, and electrophiles.
Subsequent works have shown that NT-Cys-containing peptides
with a super nucleophilic thiol side chain can undergo orthogonal
ligations without the use of a template. Indeed, NT-Cys-
containing peptides have been exploited for two orthogonal
ligation strategies that include imine ligation yielding an Xaa-
ψPro (pseudoproline)24 and thioester ligation giving an Xaa-
Cys bond.18,19 Over the past 5 years, other methods based on
NT-Cys mimetics16,26,27 have also extended ligation methods
to include peptides containing NT-amino acids other than NT-
Cys.
Figure 1. A tandem ligation scheme of SPro-imine and Cys-thioester
With an expanded repertoire for ligating amide bonds, a
tandem ligation scheme of coupling multiple segments to form
two or more regiospecific amide bonds without a protection
scheme would be desirable to increase the flexibility of the
orthogonal ligation for forming complex peptides and pro-
teins.28-30 Such a strategy would be different from a tandem
three-segment ligation that combines chemoselective and or-
thogonal ligation methods to afford macromolecules with one
or two nonamide linkages at both ligation sites.25,28,29 Recently,
we reported a successful method of tandem ligation of three
unprotected peptide segments in tandem to form two pseudopro-
line bonds by imine ligation involving the orthogonality of NT-
Cys peptide and an NT-Ser/Thr peptide without a protecting-
group scheme between each ligation step.27 In this paper, we
describe the development of a semi-orthogonal tandem ligation
strategy by employing imine and thioester ligation methods
based on the chemoselectivity of NT-Cys peptides toward two
CT-electrophiles to form, in tandem, an Xaa-SPro (thiaproline)
and an Xaa-Cys bond at the ligation sites. We also optimize
ligation.
conditions to prepare six SPro f Pro peptides including
somatostatin-28 (Sm-28) and two proteins, human macrophage
inflammatory proteins MIP-1R and MIP-1â. Since thiaproline
is a proline surrogate, we show here that the biological activity
is not altered by the proline f thiaproline substitutions in these
three bioactive peptides.
Results and Discussion
Strategy for Tandem Peptide Ligation. The tandem three-
segment ligation strategy was performed in the N f C direction
(Figure 1) on three unprotected segments to form first an Xaa-
SPro (thiaproline as an Xaa-Pro bond surrogate) and then an
Xaa-Cys bond. For convenience, these three segments were also
designated in the N f C direction as the amine segment (N-
segment) 1 containing a C-terminal glycoaldehyde ester, the
middle segment (M-segment) 2 containing both a CT-thioester
and an NT-cysteine, and the carboxyl segment (C-segment) 3
also containing an NT-cysteine.
SPro-imine ligation of the N- and M-segments proceeds
through an imine capture of the CT-glycoaldehyde ester. A
thiazolidine ester intermediate 4 is formed, and then an O,N-
acyl transfer takes place to form a C2-hydroxymethyl thiaproline
5 at the ligation site through a favorable five-membered ring
intermediate (Figure 1). Recently, we have confirmed that the
imine ligation with an NT-Cys (L-configuration) segment is
stereospecific and affords an R-epimer at the newly created
stereocenter at the C2 carbon of the SPro at the ligation site.27
Intermolecular Cys-thioester ligation of the NM-segment 5
containing a CT-thioester with the C-segment 3 containing an
NT-Cys proceeds through a covalent and branched thioester 6
that undergoes a spontaneous S,N-acyl migration through a five-
membered ring to form an Xaa-Cys bond in the three-segment
product 7. Several methods for the Xaa-Cys bond formation,
including the use of a CT-thiocarboxylic with an NT-Cys to
(17) Coltart, D. M. Tetrahedron 2000, 56, 3449-3491.
(18) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-.
(19) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12485.
(20) Kemp, D. S.; Carey, R. I. J. Org. Chem. 1993, 58, 2216-2222.
(21) Kemp, D. S. Biopolymers 1981, 20, 1793-1804.
(22) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs
Ann. Chem. 1953, 583, 129-149.
(23) Wieland, T.; Schneider, G. Liebigs Ann. Chem. 1953, 583, 159.
(24) Liu, C. F.; Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6584-
6588.
(25) Muir, T.; Dawson, P. E.; Kent, S. B. H. In Methods in Enzymology;
Fields, G. B., Ed.; Academic Press: San Diego, CA, 1997; Vol. 289, pp
266-298
(26) Tam, J. P.; Yu, Q. Biopolymers 1998, 46, 319-327.
(27) Tam, J. P.; Miao, Z. J. Am. Chem. Soc. 1999, 121, 9013-9022.
(28) Baca, M.; Muir, T.; Schnolzer, M.; Kent, S. B. H. J. Am. Chem.
Soc. 1995, 117, 1881-1887.
(29) Canne, L. E.; Ferre-D’Amare, A. R.; Burley, S. K.; Kent, S. B. H.
J. Am. Chem. Soc. 1995, 117, 2998-3007.
(30) Huang, H.; Carey, R. I. J. Pept. Res. 1998, 51, 290-296.

Tandem Ligation of Unprotected Peptides
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2489
form a covalent perthioester, have also been reported.30,31
However, the intramolecular thioester ligation of a peptide such
as the M-segment 2 containing both an NT-Cys and a CT-
thioester is equally facile and has been extensively exploited
for preparing cyclic peptides.32-36 Thus, the inter- and intramo-
lecular ligation of the M-segment poses a challenge in our
synthetic scheme.
Previously, we have shown that inter- and intramolecular
thioester-cysteine ligations are best performed at basic conditions
(pH > 7),19,32 whereas SPro-imine ligation prefers acidic
conditions at pH < 5.37,38 In acidic conditions, the thiol-
thioester exchange between an NT-Cys and the CT-thioester is
slow, while the imine capture between an aldehyde and an NT-
Cys is fast. This differential reactivity of an NT-Cys peptide
under acidic and basic conditions toward two different CT-esters
provides the basis for the three-segment ligation involving two
NT-Cys to form two amide bonds in tandem.
Segment Preparation. All segments were prepared by a
stepwise solid-phase method. Both N- and M-segments contain-
ing CT-esters required specialized resin supports for their
syntheses. C-segments, however, required only an NT-Cys and
a free carboxylic acid or carboxamide at the CT and were
generated either by Boc- or Fmoc chemistry on commercially
available resins. All peptide segments were purified by HPLC
and characterized by MALDI-MS.
N-segments containing a CT-glycoaldehyde ester 1 were
prepared by Fmoc-chemistry on an acetal resin 8.38 TFA-
mediated cleavage of the assembled peptide esters anchored on
the resin supports afforded the unprotected N-segment glyceryl
ester. These masked CT-glycoaldehyde esters have the advan-
tage of being purified and stored for future use without side
reactions. Prior to the imine ligation, the 1,2-diol glyceryl moiety
was then transformed to aldehyde 1 by periodate oxidation under
aqueous conditions.
M-segments containing both an NT-Cys and a CT-thioester
were synthesized on a thioester resin 1039,40 with a bifunctional
removal linker of the 3-mercaptopropionic acid coupled to the
MBHA resin. This 3-mercaptopropionyl-MBHA resin was then
used to assemble the desired sequence. Because thioesters are
labile to piperidine treatment in Fmoc chemistry, M-segments
2 were prepared by Boc chemistry and released from the resin
supports and protecting groups by HF. Recently, several groups
have independently reported the use of a safety-catch resin or
a new deblocking reagent to prepare thioester peptides by Fmoc
chemistry.41-43
Figure 2. Possible side reactions during the ligation between N- and
M-segments.
ligation and then to attach the C-segment through Cys-thioester
ligation. A major concern of this ligation step is that the
M-segment 2 containing an NT-Cys can react competitively with
the CT-glycoaldehyde ester of the N-segment 1 or the CT-
thioester of the M-segment. As previously discussed, another
concern is that the M-segment containing both an NT-Cys and
a CT-thioester is prone to end-to-end cyclization to yield
byproduct 12 that terminates the tandem ligation scheme. In
addition, these CT-esters are sensitive to aqueous hydrolysis,
yielding byproducts 11 and 13 (Figure 2). These problems were
examined in the model peptides. The second group of problems
involved oxidation-sensitive residues such as NT-Ser, NT-Thr
and Met present in N-segments and the presence of Cys in the
M-segments also containing CT-thioesters. These problems were
examined in the syntheses of the thiaproline analogues of
somatostatin-28. The third group of problems, involving site
selection, the presence of cysteine in the N-segments, and the
influence of conformation in the ligation reactions, was exam-
ined in the syntheses of the thiaproline analogues of macrophage
inflammatory proteins, MIP-1R and MIP-1â.
1. pH Optimization and Minimization of Side Reactions
for Tandem Imine and Thioester Ligations. M-segments with
5 to 10 amino acids are prone to end-to-end cyclization and
provide suitable models for the feasibility study of the SPro-
Cys tandem ligation scheme. The rates and pH effects on imine
ligation were examined using an eight-residue N-segment GL8-
CHO 1a (tR ) 12.7 min, MH 839) to ligate a five-residue
M-segment CG5-SR 2a (tR ) 5.9 min, MH 670). When ligations
were performed between pH 5 to 7, >80% of the M-segment
2a underwent the side reaction of end-to-end backbone lactam-
ization to afford the cyclic CG5 12a (tR ) 7.9 min, MH 565)
within 3 h (Table 1). Significant hydrolysis of the unreacted
glycoaldehyde ester 1a to the free carboxylic 11a (tR ) 13.5
min, MH 797) was also observed at this pH range. At pH 4,
the cyclization rate significantly decreased, but hydrolysis of
2a to 13a (hydrolyzed product 13a, tR ) 6.3 min, MH 573)
was still observable. In contrast, when the ligation was
performed at pH 3 or lower, both side reactions attributed to
cyclization of the M-segment 2a and hydrolysis of the N-
segment 1a significantly decreased. However, lowering the
reaction pH also markedly retarded the O,N-acyl migration of
Potential Synthetic Problems. Three groups of synthetic
problems were considered during the development of the
thiaproline-cysteine tandem ligation scheme. The first involved
conditions to first ligate N-and M-segments through SPro-imine
(31) Liu, C.-F.; Rao, C.; Tam, J. P. Tetrahedron Lett. 1996, 37, 933-
936.
(32) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370.
(33) Tam, J. P.; Lu, Y. A. Tetrahedron Lett. 1997, 38, 5599-5602.
(34) Camarero, J. A.; Muir, T. W. Chem. Commun. 1997, 1369-1370.
(35) Camarero, J. A.; Pavel, J.; Muir, T. W. Angew. Chem., Int. Ed. 1998,
37, 347-349.
(36) Shao, Y.; Lu, W.; Kent, S. B. H. Tetrahedron Lett. 1998, 39, 3911-
3914.
(37) Liu, C. F.; Tam, J. P. J. Am. Chem. Soc. 1994, 116, 4149-4153.
(38) Botti, P.; Pallin, T. D.; Tam, J. P. J. Am. Chem. Soc. 1996, 118,
10018-10024.
(39) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117.
(40) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1999, 121, 3311-3320.
(41) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc.
1999, 121, 11369-11374.
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2490 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tam and et al.
Table 3. Yields of Cys-Thioester Ligation between NM- and
Table 1. Effect of pH on Side Reaction in Ligation between
GL8-OCH2CHO 1a and CG5SR 2aa
C-Segments
NMC-ligated product
N-segment
aldehyde
M-segment (%)
NM-segment
thioester
ligated
product
HPLC yieldb
(min)a (%)
MH+
(calcd)
thioester
thioester cyclized NM-ligated
C-segment
pH hydrolysis (%)b remainingc hydrolysisc productc productc
[Thz9]GG13 4a CY6c 3a
[SPro9]GY19 7a
25.9 82.0 2172.6 (2173.0)
25.5 69.0 2962.6 (2963.4)
24.7 69.0 3188.5 (3187.6)
22.8 72.4 3423.9 (3424.7)
23.5 80.6 3199.2 (3199.0)
24.2 66.3 8066.8 (8065.8)
7
6
5
4
3
>99
>99
25.5
27.8
<1
<1
5.3
3.3
41.1
63.5
<1
5.2
7.8
2.3
>99
89.5
79.4
11.1
<1
<1
<1
[Thz10]SG14 4ca CC12 3b [SPro10]SC26 7cb
[Thz10]FG14 4ba CC12 3b [SPro10]FC26 7bb
[Thz10]FG16 4bc CC12 3b [SPro10]FC28 7b
[Thz10]SG16 4c CC12 3c [SPro10]Sm-28 7c
9.3
45.3
36.0
[Thz38]AV50 4d CA20 3d Cys11,35 (Acm)
[SPro38]MIP-1R 7d
<1
[Thz21]AL34 4e CN35 3e [SPro21]MIP-1â 7e 22.6 73.6 7869.7 (7868.0)
a N- and M- segments were reacted for 3 h. b (%) based on
N-segment. c (%) based on M-segment.
a tR in min. b Yields were based on NM-segment thioesters and
calculated from area ratio corresponding to peaks of NM-segment and
NMC-ligated product. c CY6 ) CNSFRY, CC12 ) CKNFFWKT-
FTSC, CA20 ) CADPSEEWVQKYVSDLELSA, CN35 ) CSQPAV-
VFQTKRSKQVCADPSESWVQEYVYDLELN.
Table 2. Yields and Analytical Data of Thiazolidine Thioester in
the Thiaproline Ligation between N- and M-Segments
ligated NM-segment thioester
N-segment- M-segment-
HPLC yieldb
OCH2CHO
thioester
NM-thioester
(min)a
(%)
MH+ (calcd)
GL8c 1a
FK9 1b
FK9 1b
SA9 1c
SA9 1c
AK37 1d
AL20 1e
CG5 2a
CG5 2a
CG7 2c
CG5 2a
CG7 2c
CV13 2d
CL14 2e
[Thz9]GG13 4a
[Thz10]FG14 4ba
[Thz10]FG16 4bc
[Thz10]SG14 4ca
[Thz10]SG16 4c
[Thz38]AV50 4d
[Thz21]AL34 4e
14.9
17.9
13.0
17.7
13.4
22.8
22.3
79.6
80.0
69.5
70.7
86.4
91.9
93.0
1490.1 (1490.0)
1779.9 (1781.9)
2016.8 (2018.1)
1555.9 (1556.7)
1792.5 (1791.8)
5901.9 (5901.3)
4100.0 (4099.0)
a tR in min. b Yields were based on the limited M-segments and
calculated from area ratio corresponding to peaks of M- and NM-
segments. c Letters indicate the first and last amino acids and numbers
indicate the numbers of amino acids in the sequences. GL8 )
GIAYGGFL, FK9 ) FETSSQC(Acm)SK, SA9 ) SANSNPAMA,
AK37 ) ASLAADTPTAC(Acm)C(S-tBu)FSYTSRQIPQNFIADY-
FETSSQ-C(Acm)SK, AL20 ) APMGSDPPTACCFSYTARKL, CG5
) CKLYG, CG7 ) CRERKAG, CV13 ) CGVIFLTKRSRQV, CL14
) CRNFVVDYYETSSL, Thz ) -OCH2-thiazolidine.
the thiazolidine ester 4a to the Xaa-SPro bond and required
>100 h for completion. Once formed, the thiazolidine ester 4
is relatively stable, and we envisioned that the O- to N-acyl
migration could be mediated concurrently during or even after
the second ligation.19,26,32,38
Table 2 shows the ligation yields between N- and M-
segments. The imine capture forms a new stereocenter at the
C2-position of the thiazolidine ring, and the thiazolidine-ester
products eluted as doublets in HPLC profiles. NMR study
showed that the O,N-acyl migration of the C2-R epimer proceeds
faster than the C2-S epimer.27 The unreacted C2-S epimer likely
undergoes an equilibration to a mixture of R and S epimers
through ring-chain tautomerization to favor the C2-R epimer
as major product after O,N-acyl migration under basic condi-
tions.
Figure 3. A scheme of three-segment tandem ligation mediated by a
diester intermediate.
In thioester ligation, the thiol capture is a rate-limiting step,
while the S,N-acyl migration occurs spontaneously to generate
a Xaa-Cys bond at the ligation site. In contrast, the slow O,N-
acyl migration at the N-to-M-segment ligation site becomes the
rate-determining step of this three-segment tandem ligation
reaction. Because this acyl migration was often not complete
even when the thioester ligation was completed in <12 h, the
thioester ligation was prolonged >24 h. For the model peptides,
the O,N-acyl migrations were observed from changes in the
HPLC profile.
Tandem Ligation Using the Diester Intermediate. On the
basis of the above results, an optimized condition for the
thiaproline-thioester ligation in tandem was used for all subse-
quent syntheses (Figure 3). (1) The first SPro-imine ligation of
N- and M-segments (1 and 2) was performed at pH 3. Under
such a condition, the protonated nature of the NT-cysteinyl thiol
inhibits thioester ligation but permits imine capture of the CT-
glycoaldehyde ester and its tautomerization to a stable thiazo-
lidine ester 4. At pH 3, the O,N-acyl migration to form an Xaa-
NM-segment and C-segment proceeded with thioester ligation
smoothly at pH 7.2 phosphate buffer and in the presence of
excess TCEP and 3-mercaptopropionic acid (MPA). Table 3
shows the yields of model peptide and analogues. MPA, a small
thiol, is a useful additive because it is a water-soluble reducing
agent and does not have a strong stench. More importantly, it
maintains the NT-cysteinyl thiol in the reduced form critical
for the thiol capture step.19 However, it also promotes thioester
hydrolysis. This disadvantage may not be significant for small-
or middle-size peptides in the range of 30-50 residues because
the ligation is normally completed within 3 h. To minimize side
reactions, we also used other thiol catalysts such as thiophenol44
and 2-mercaptoethanesulfonic acid45 in the tandem ligation for
the synthesis of the CC-chemokines.
(44) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, B. H. J. Am.
Chem. Soc. 1997, 119, 4325-4329.
(45) Evans, J.; Benner, J.; Xu, M.-Q. Protein Sci. 1998, 7, 2256-2264.

Tandem Ligation of Unprotected Peptides
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2491
condition and found that the optimal pH was around pH 6. At
pH 6.2, treatment of the N-segment SA9-glycerylester with 5
equiv of NaIO4 in 0.5 h afforded the desired CT-aldehyde with
80% yield without affecting Met. Longer oxidation time
produced a significant amount of Met(O) and an R-oxo side
product from NT-Ser oxidation.
Imine capture between N- and M-segments (1 and 2) and
thioester ligation between NM-and C-segments (4 and 3)
proceeded smoothly under the conditions used in model
peptides. In the presence of excess thioester 4, the free cysteinyl
thiol produced under the ligation condition can react again with
the NM-segment thioester 4 yielding an N,S-diacyl byproduct.
To limit the formation of this diacyl byproduct, a slight excess
of C-segment 3 was used. After ligation, a small thiol MPA
was added to the reaction mixture to regenerate product 7 from
the N,S-diacyl byproduct.16,19
After completing the tandem ligation, the crude product was
treated with 20% DMSO in an aqueous solvent buffered at pH
6.2 for 24 h for the disulfide formation between Cys17 and Cys28.
As discussed previously, the O,N-acyl migration of the thiazo-
lidine ester at the N-to-M-segment ligation site was slow, and
therefore the period for disulfide oxidation was prolonged to
permit completion of the O,N-acyl migration. Progress of the
ligation reaction as monitored by HPLC showed that the
thiaproline bond was indeed complete after the DMSO oxida-
tion. Treatment with 1 M NH2OH at pH 9.0 for 1 h further
confirmed that the thiaproline ligation site was an amide bond
because the thiazolidine ester bond was susceptible to base
treatment and would yield two segments.24,38
In the synthesis of Sm-28 analogues, three short segments
of <10 residues are used. Since these segments can be
synthesized rapidly by solid-phase methods, the SPro-Cys
tandem ligation scheme may be useful for combinatorial
segment synthesis. At present, combinatorial peptide synthesis
by a stepwise method is not reliable for peptides >20 residues
due to unpredictable side reactions that make decoding impuri-
ties difficult. The three-segment ligation of the somatostatin
model with purified segments provides unambiguous products
and, more importantly, can be performed entirely in aqueous
conditions.
Figure 4. A tandem ligation synthetic scheme of [SPro10]Sm 28.
SPro bond is slow and incomplete but can be accomplished
during steps 2 and 3 when they are performed at pH 7 to 8. (2)
The second step is the Cys-thioester ligation of NM- and
C-segments that produces NMC-branch esters 6a (Figure 3). It
can proceed, after adjusting the pH to >7.2 with solid NaHCO3,
either directly by adding a C-segment to the reaction mixture
(one-pot reaction) or to the purified NM segment. Yields of
the Cys-thioester ligation are shown in Table 3. (3) The third
step is the disulfide formation mediated by adding 10-20%
DMSO by volume to the reaction medium at pH 6-8 of step 2
to complete the three-segment ligation in tandem.
2. Synthesis of Somatostatin-28 Analogues: Susceptibility
of NT-Ser to Oxidation and the Presence of Cysteine in the
M-Segments. Somatostatin-28 (Sm-28) contains 28 amino acid
residues and is widely distributed in central nervous system and
digestive tract tissues.46 [SPro10]Sm-28 with a thiaproline f
proline10 replacement was prepared by three-segment tandem
ligation to validate its application for a medium-length peptide
containing disulfides (Figure 4). The ligation sites were based
on the occurrence of Pro and Cys, respectively, at positions 10
and 17 of the Sm-28 sequence.
Sm-28 was also selected because it contains an NT-Ser that
presents both a potential opportunity and synthetic problem.
Recently, imine ligation has been successfully extended to
ligating segments with NT-Ser or Thr to form an oxaproline at
the ligation sites.27 Thus, the presence of NT-Ser at the
N-segment is desirable for developing a four-segment tandem
ligation using oxaproline ligation. However, NT-Ser at the
N-segment is susceptible to oxidization during the NaIO4
conversion of the CT-glyceric to its glycoaldehyde ester for
imine ligation. At pH 7, oxidation of NT-Ser to an R-oxo moiety
was 1000-fold faster than the CT-1,2-diol. At pH < 6, our model
compound showed a reverse selectivity of diol over the 1,2-
amino alcohol of NT-Ser oxidation with NaIO4. Selectivity
increased with an increase of acidity, which probably can be
attributed to the increased protonated states of the NT-Ser. At
pH 3, the CT-glyceryl ester was completely oxidized to aldehyde
within 3 min without significant oxidation of NT-Ser. Thus,
for N-segments containing NT-Ser and NT-Thr, selective
conversion of CT-glyceryl ester to its corresponding aldehyde
can be achieved at acidic pH. Under this condition the
methionine residue in the N-segment SA-9 was oxidized to Met-
(O).47 To avoid the Met oxidation, we fine-tuned the oxidation
3. Syntheses of Human MIP-1r and MIP-1â: Site Selec-
tion, Presence of Cysteines in the N-Segments, and Confor-
mational Influence. MIP-1â and MIP-1R are members of the
CC-chemokine proteins of 8-10 kD.48-50 These chemokines
are primarily involved in the chemoattractant potential of
monocytes and lymphocytes and have been implicated in a
variety of clinically important inflammatory diseases, and more
recently as inhibitors of HIV pathogenesis. Structurally, these
two chemokines display significant sequence homology that
permits comparison of ligation sites at different Pro and Cys
residues to test the conformational influence of the tandem
ligation reactions. A synthetic challenge is the presence of two
adjacent Cys at the N-terminal segments, a sequence motif of
CC-chemokines, that requires manipulations to avoid side
reactions during the oxidative transformation to the CT-
glycoaldehyde ester for the SPro-imine ligation between the N-
and M-segments.
(47) Geoghegan, K. F.; Stroh, H. G. Bioconjugate Chem. 1992, 3, 138-
146.
(48) Lodi, J.; Garrett, D. S.; Kuszewski, J.; Tsang, M. L.; Weatherbee,
J. A.; Leonard, W. J.; Gronenborn, A. M.; Clore, G. M. Science 1994, 263,
1762-1767.
(49) Clore, G. M.; Gronenborn, A. M. FASEB J. 1995, 9, 57-62.
(50) Gao, J. L.; Kuhns, D. B.; Tiffany, H. L.; McDermott, D.; Li, X.;
Francke, U.; Murphy, P. M. J. Exp. Med. 1993, 177, 1421-1427.
(46) Pradayrol, L.; Jornvall, H.; Mutt, V.; Ribet, A. FEBS Lett. 1980,
109, 55-58.

2492 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tam and et al.
MIP-1r. The 70-residue MIP-1R contains Pro at positions
21 and 38, either of which is a suitable choice as an imine
ligation site to afford a SPro f Pro analogue. Ligation at Pro38
would require an N-segment with a CT-Lys37-glycoaldehyde
ester. The participation of this CT-lysyl side-chain amine
through either intramolecular lactam or Schiff base formation
may interfere in the imine ligation. To resolve this potential
side reaction, we selected Lys37-Pro38 as the imine ligation site
and Cys52 for the thioester ligation. Thus, three segments of
37, 13, and 20 residues corresponding to the N-, M-, and
C-segments (1d, 2d, and 3d, respectively), with the N-segment
1d being the longest of the three, were prepared for the tandem
ligation via thiaproline (37 + 13 aa-segments) and thioester (50
+ 20 aa-segments) for the synthesis of [SPro38]MIP-1R 7d.
MIP-1â. The 69-residue MIP-1â also contains Pro at posi-
tions 21 and 38. To prepare [SPro21]MIP-â, Pro21 and Cys35
were selected as ligation sites and required the syntheses of three
segments 1e, 2e, and 3e containing 20, 14, and 35 amino acid
residues of which the C-segment 3e was the longest. While the
Cys-thioester ligation of MIP-1R consisted of two segments of
50 and 20 residues, the NM- and C-segments (4e and 3e), ligated
in MIP-1â were roughly equal, containing 34 and 35 residues,
respectively.
Figure 5. A tandem ligation synthetic scheme of [SPro38]MIP-1R.
Conformation Influence in the Tandem Ligation of MIP-
1r and MIP-1â. The SPro-imine ligation of N-segments 1d
and 1e of MIP-1R and MIP-1â with their corresponding
M-segments proceeded smoothly to form the NM-segments at
pH 3.0. Turbidity in the reaction solutions was observed, and
33% acetonitrile by volume was added to the aqueous solution
to improve the solubility of the N-segments. However, denatur-
ants were not required in these thiaproline ligation steps.
Furthermore, the presence of Lys37 at the CT-glycoaldehyde
ester did not pose a synthetic problem, probably due to
reversibility of the intramolecular Schiff base formation under
the aqueous acidic condition in which the SPro-imine ligation
was performed.
An unpredictable factor in ligation chemistry for protein
synthesis is the conformational influence of large peptide
segments on the ligation rate observed in the three-segment
tandem ligation of CC-chemokines MIP-1R and MIP-1â.
Although we found that the SPro-imine ligation of the N-and
M-segments proceeded smoothly under aqueous conditions, the
second ligation of the resulting larger NM segment 4d, 4e with
the C-segment 3d, 3e had to be performed under strongly
denatured conditions.
Figure 6. A tandem ligation synthetic scheme of [SPro21]MIP-1â.
1â contain three â strands from the N-terminal to the middle
of the molecule and then an R-helix beginning with Cys52 at
the C-terminus.49 Furthermore, these CC-chemokines have a
strong propensity to form dimers. Thus, a folded or partially
folded NM-segment or dimerization of the Cys52-segment may
hamper the Cys-thioester ligation. Our observation that under
nondenaturing conditions the Cys52-segment 3d or 3e rapidly
formed a covalent dimer provides experimental support for this
explanation.
Various runs of thioester ligation of both NM-segments 4d,
4e with their corresponding C-segments 3d, 3e under aqueous
conditions in pH 7.6 phosphate buffer with additional organic
cosolvents and reducing agents such as TCEP and MPA were
unsuccessful. In all cases, the expected ligated products were
not observed from the HPLC profile after 3 h. After 24 h, most
of the thioesters of the NM-segments were hydrolyzed to their
corresponding carboxylic acids, while their C-segments formed
disulfide dimers. Thus, a denatured condition using 8 M
guanidine HCl at pH 7.6 was used for ligating NM- and
C-segments of MIP-1R and MIP-1â. Thiophenol (1 vol %) was
added to maintain the reaction mixture under this reducing
condition and also as an additional denaturing reagent.18 Under
this denatured condition, thioester ligations between NM- and
C-segments of the thiaproline analogues of MIP-1R and MIP-
1â were obtained in satisfactory yields (Figure 7, Table 3).
Disulfide Formation. Both MIP-1R and MIP-1â contain four
cysteine residues with two adjacent cysteines (Cys11 and Cys12)
proximally located at the N terminus (Figures 5 and 6). During
the NaIO4 treatment for forming CT-aldehyde, the random
disulfide formation of these two free Cys in the N-segment
posed a problem that necessitated a temporary protection
scheme. We pursued two methods that employed sulfenyl-tert-
butyl (S-tBu) and Acm-protecting groups. In MIP-1â, only the
S-tBu-protecting groups were used, including both cysteines of
the N-segment. The disulfide bonds of the S-tBu-cysteinyl
residues offered protection during the NaIO4 oxidation, but they
were removed during the thioester ligation by the presence of
small thiols in the reaction mixture. Since two of the three
geometric disulfide isomers were favored to form under the
DMSO-mediated oxidation, we were able to obtain the desired
isomer after sequential RP-HPLC purification in relatively low
The difficulty in achieving the thioester ligation may due to
the conformational resistance of these two segments. Various
structural determinations have shown that MIP-1R and MIP-

Tandem Ligation of Unprotected Peptides
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2493
Figure 7. (A) HPLC profile of ligation between NM-segment 4e and
C-segment 3e to form [SPro21]MIP-1â 7e and (B) MS profiles of
[SPro21]MIP-1â 7e.
yield without optimization of the oxidative refolding of the
ligated MIP-1â product.
To avoid the random oxidation method for disulfide forma-
tion, we used an orthogonal thiol-protecting strategy of S-tBu
and Acm for Cys12,51 and Cys11,35 in the synthesis of MIP-1R,
respectively. The S-tBu-protecting group at Cys12 was removed
during the thioester ligation of the MN + C segments. DMSO-
mediated disulfide formation of the ligated MNC segments
generated the Cys12,51 disulfide pair. The Cys11,35 disulfide was
formed by treatment with I2/MeOH. This two-step oxidation
forms a single product with the desired disulfide linkages. The
stability of the S-tBu-protecting groups during the oxidation
conditions for CT-aldehyde formation of N-segments of MIP-
1R and MIP-1â was also studied. At pH 6.2 with 5 equiv of
NaIO4, the S-tBu group was found to be stable, and the desired
CT-aldehyde was formed in a 70% yield. At pH < 4.2 or with
prolonged oxidation over 30 min, a complex mixture was
obtained with observation of the CT-ester diol being overoxi-
dized and with decomposition of the S-tBu groups to form
various undetermined byproducts.
Biological Activity. The biological activities of three ligation
products, [SPro10]somatostatin 8c, [SPro38]MIP-1R 9d, and
[SPro21]MIP-1â 8e, were compared with their native products
by radioreceptor binding assay and chemotaxis assay, respec-
tively. Dose-dependent response of radioreceptor assays showed
that the activity of the native somatostatin-28 and [SPro10]-
somatostatin 8c were indistinguishable and both completely
inhibited specific receptor binding in mouse AtT-20 cells
expressing somatostatin receptors at about 10 nM. The migration
capacity of [SPro38]MIP-1R 9d and [SPro21]MIP-1â 8e counter
was compared with the native MIP-1R and MIP-1â on P4-CCR5
cells using a chemotaxis assay (Figure 8). All four compounds
showed dose-dependent responses at concentrations of 0.01-
1000 ng/mL to P4-CCR5 cells with a maximal response at 1
ng/mL. The chemotactic indexes of MIP-1R and MIP-1â at 1
ng/mL were 3.9 and 2.7, respectively. At the same concentration,
the chemotactic indexes of [SPro38]MIP-1R 9d and [SPro21]MIP-
1â 8e were 3.1 and 2.3 ng/mL, respectively. There was no
significant difference (P < 0.05) between the thiaproline and
native MIP-1R or MIP-1â chemokines.
Figure 8. Chemotaxis assays of synthetic and native (A) MIP-1R and
(B) MIP-1â. Migration of P4-CCR5 cells at the indicated chemokines
concentrations are shown as the mean ( SEM of three individual
experiments. The chemotactic index is defined as the number of cells
migrating in response to test peptides divided by the number of cells
migrating in response to medium alone.
assisted ligation for both semi- and total protein syntheses.16,51,52
A successful demonstration of an abiotic scheme may provide
clues to the formation of functional biopolymers under prebiotic
conditions.
Our report shows the potential of a tandem ligation approach
to peptide and protein synthesis by employing two ligation
methods, SPro-imine and Cys-thioester ligation, to couple three
unprotected peptides to form larger peptides and proteins in
aqueous solutions with high efficiency. With the development
of recombinant DNA methods, particularly the intein-expressed
CT-thioester as building blocks, abiotic peptide synthesis
approach through orthogonal and tandem ligation will extend
the range and the versatility for protein syntheses. These will
include many soluble proteins with interesting biological
functions such as growth factors, chemokines, cytokines, and
other regulatory proteins.
A concern for our tandem ligation scheme is the formation
of an unusual thiaprolyl bond (SPro) instead of a normal prolyl
peptide bond in peptides and proteins. Proline is a convenient
ligation site because it often occurs at reverse turns, and
thiaproline appears to be a good structural surrogate. Thus far,
we have found that substitution of proline by ψPro in selected
bioactive peptides and proteins, as demonstrated in this work
with somatostatin-28 and CC-chemokines, does not result in
loss of their bioactivities. These results are consistent with our
previous work on antimicrobial peptides and enzymes. For
example, replacement of SPro for Pro39 in HIV protease
analogues does not affect their folding, dimerization, and
biological activity.53 In addition, the 2-hydroxymethyl thiazo-
lidine ring could be considered as a chimeric amino acid, which
has found applications in the rational design of peptide
hormones.54
Conclusions
The development of tandem ligation of three peptides is of
both practical and fundamental interest. The ability to ligate
multiple peptide segments to form two or more regiospecific
amide bonds in aqueous solutions, without the use of a
protecting-group scheme and coupling reagents, may provide a
useful strategy for convergent, combinatorial, or conformation-
Recently, another imine ligation, oxaproline ligation, has been
developed in our laboratory.27 Oxazolidine ligation uses Ser or
(52) Ayers, B.; Blaschke, U. K.; Camarero, J. A.; Cotton, G. J.; Holford,
M.; Muir, T. W. Biopolymers 1999, 51, 343-354.
(53) Liu, C. F.; Rao, C.; Tam, J. P. J. Am. Chem. Soc. 1996, 118, 307-
312.
(54) Kolodzriej, S. A.; Nikiforovich G. V.; Skeean R.; Lignon, M. F.;
Martinez, J.; Marshall, G. R. J. Med. Chem. 1995, 38, 137-149.
(51) Evans, T. C., Jr.; Xu, M.-Q. Biopolymers 1999, 51, 333-342.

2494 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Tam and et al.
purified by HPLC, giving a yield of 70-90%. For MIP-1R and MIP-
1â, one-third volume of acetonitrile was added to increase the solubility
and give better results. Yields and MS data are shown in Table 2.
Thioester Ligation between NM- and C-Segments. To the solution
of NM-segment (0.1 µmol) in 100 µL of 0.2 M phosphate buffer (pH
7.2) containing 3 equiv of TCEP was added 1.1 equiv of C-segment.
After 3 h at room temperature, 1 µL of MPA was added, and the
reaction mixture was agitated for 40 min. The ligation product was
then purified by HPLC and lyophilized to dryness. For MIP-1R and
MIP-1â, 0.1 µmol of NM-segments 4d, 4e and 1.2 equiv of C-segments
3d, 3e were dissolved in 200 µL of 8 M guanidine HCl/0.2 M phosphate
buffer (50/50, v/v) pH 7.6. Thiophenol (1 vol %) was added. The
ligation mixture was shaken overnight (18-24 h). Water (400 µL) then
was added ,and the reaction mixture was extracted with 3 × 200 µL of
ether to remove the thiophenol. The aqueous phase was evaporated
under vacuum to remove ether. The solution was then applied to HPLC
to give the ligation product 7d and 7e. Yields and MS data are given
in Table 3.
Disulfide Bond Formation. The disulfide bond formation was
achieved by dissolving 1 mg of three-segment ligated products 7c, 7d,
or 7e in 1 mL of phosphate buffer (pH 6.6) with 10-20% in volume
of DMSO. The solution was stirred at room temperature for 24 h, and
the oxidized product was purified by HPLC. [SPro10]Sm28 8c: MS
3197.8 (calcd 3197.2), [Cys11,35Acm-SPro37]MIP-1R 8d: MS 8064.8
(calcd 8063.8), and [SPro21]MIP-1â 8e; MS 7867.7 (calcd 7866.0). In
the two-step disulfide formation of MIP-1R, the solution containing
8d was adjusted with acetic acid (2 M) to pH 3 and treated with 3
equiv of I2/MeOH solution to remove Acm and to form [SPro37]MIP-
1R with 15% overall yield; MH+ 7974.9 (calcd 7976.0).
Thr instead of Cys as NT-nucleophiles to react with aldehyde
to form oxaproline in the ligation site. Since the oxaproline
ligation is orthogonal to Cys-thioester ligation, a three- or four-
segment tandem ligation strategy can be envisioned which
incorporates both ligations. However, the technical aspects of
such a strategy need to be studied to arrive at a tandem ligation
for multiple segments without a protecting-group scheme.
Experimental Section
Materials and Methods. All solvents of HPLC grade were obtained
from VWR Scientific Co. and used without further purification.
Chemicals were obtained from Aldrich Chemicals (Milwaukee, MI).
Boc-amino acids and MBHA resin were purchased from Bachem,
California. Analytical RP-HPLC was performed on a Shimadzu system
with a Vydac column (0.46 cm × 25 cm, C18) at a flow rate of 1 mL/
min with a linear gradient of 10-100% buffer B (60% acetonitrile in
H2O/0.04% TFA) in buffer A (5% acetonitrile in H2O/0.045% TFA)
over 30 min. Effluent was monitored with UV at 225 nm. A preparative
HPLC equipment (Waters Associates) with a Vydac C18 reverse phase
column was used for peptide purification. Mass spectra were obtained
from a Kratos mass spectrometry instrument with positive matrix-
assisted laser desorption ionization mode (MALDI-MS).
Synthesis of Peptide-Glycoaldehyde. Fmoc-Gly-OCH2-cyclic acetal
resin was prepared according to the described procedure.38 After
cleavage using 95% TFA, the crude peptide-glyceric ester was dissolved
in a phosphate buffer (0.2 M, pH 6.2). To this solution, NaIO4 solution
in the same buffer (0.5 mmol/mL) was added. After 30 min, the peptide
aldehyde was purified by HPLC. Typically, AK37-CHO 1d was
prepared from AK37-(OH)2 (1 mg in 200 mL of buffer, 0.2 µmol) and
20 µL of NaIO4 solution (1 µmol) in a yield of 72% (yield was based
on area ratio of corresponding HPLC peaks, MH+ calcd 4326.9, found
4327.5).
Thioester Resins. Thioester resins were prepared with a modified
Hojo and Aimono’s procedure.39,40 Four equivalents each of 3-mer-
captopropionic acid, HOBt, and DIC were sequentially added to a
suspension of 4-methylbenzhydrylamine (MBHA) resin in DMF (10
mL/g resin). The mixture was shaken at room temperature until a
ninhydrin test of the resin indicated that no free amino sites were
present. The resin was washed with DMF, DCM, CH3OH, DCM, and
DMF. The resulting resin was treated with a mixture of 1 equiv each
of cysteine methyl ester hydrochloride, triphenylphosphine, and DIEA
in DMF/DCM (3:1, v/v) for 2 h. After thorough washing with DMF,
DCM, CH3OH, and DCM and drying in vacuo, the mercaptopropionyl
MBHA resin was obtained in quantitative yield (6.3 g from 6.0 g of
MBHA resin).
Boc amino acid (4 equiv) preactivated with BOP (4 equiv) and DIEA
(6 equiv) for 5 min was added to a suspension of mercaptopropionyl
MBHA resin (1 equiv) in DMF (10 mL/g resin). The mixture was
shaken at room temperature for 2 h, and free thiol groups were
monitored by Ellman’s reagent.55 The resin was washed with DMF,
DCM, CH3OH, and DCM and used for stepwise peptide synthesis.
Synthesis of Peptide Thioesters. All thioesters were prepared by
solid-phase synthesis starting from Boc amino acid thioester resin. The
solid-phase synthesis was carried out on a peptide synthesizer 430A
(Applied Biosystems Inc., Foster City, CA) using conventional Boc-
chemistry and DCC-HOBt coupling protocol. The side-chain protecting
groups of Boc-amino acid used were as follows: Arg(Tos), Asp-
(OcHex), Cys(4-MeBzl or Acm), Glu(OcHex), His(Dnp), Lys(ClZ),
Ser(Bzl), and Thr(Bzl). After peptide chain elongation, the resin was
treated with high HF procedure (90% HF). After evaporation of HF,
the residual solid was washed with ether. The crude peptide thioesters
were extracted with 50% acetic acid, purified by preparative HPLC,
and lyophilized. All synthetic peptides were characterized by MALDI-
MS.
Somatostatin Radioligand Binding Assay. The receptor-binding
assays for Sm-28 and [SPro10]Sm-28 were performed by MDS Panlabs
(Bothell, WA). Briefly, AtT-20 cells were grown in DMEM containing
10% fetal calf serum at 37 °C in a humidified atmosphere of 10% CO2-
90% air. For receptor binding assay, cells were incubated with [125I-
Tyr25]Sm-14 in the absence or presence of [SPro10]somatostatin. Cells
were pellected by centrifugation and washed with buffer. The radio-
activity of pellects was quantitated in γ-spectrometer.56
CC-Chemokine Chemotaxis Assay. The Hela-CD4/LTR-lacZ
indicator and CCR5 expression cell line P4-CCR557,58 was employed
for chemotaxis assay. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Cellgro) supplemented with 10% fetal bovine
serum (FBS, Atlanta Biologicals) and penicillin plus streptomycin as
well as hygromycin (1 µg/mL). Cell migration in response to MIP
peptides was assessed in a modified 48-well microchemotaxis chamber
(Neuro Probe Inc., Cabin John, MD). Serial dilutions (0.01-1000 ng/
mL) of SPro38MIP-1R and SPro21 MIP-1â were compared with two
recombinant human chemokines MIP-1R and MIP-1â (R and D
Systems) in 35 µL of DME medium loaded in the wells of the lower
compartment in duplicates. Medium alone without chemokine was
loaded in three wells as control. A suspension of 4 × 104 cells in 50
µL of DME medium with 10% FBS without protein was added to the
upper compartment which was separated from the lower compartment
by a collagen-coated poly(vinylpyrrolidone)-free polycarbonate mem-
brane with 5-µL diameter pores. The chambers were incubated in a
humidified atmosphere of 5% CO2 in air at 37 °C for 4-6 h. The filter
was fixed with Diff-Quik Fixative and stained in Diff-Solution I and
II (Dade Begring Inc.). The cells that migrated into lower wells were
counted under microscopy. Results were expressed as a chemotactic
index, which defined as the number of cells migrating in response to
test protein divided by the number of cells migrating in response to
medium alone. The concentration required for 50% of the maximal
response (chemotaxis EC50) was derived from the dose-response
curves.
Acknowledgment. This work was in part supported by U.S.
Public Health Service NIH Grants, CA 36544 and GM57145.
Imine Ligation between N- and M-Segment. Typically, to the
solution of 0.1 µmol N-segment dissolved in 200 µL of 0.2 M acetate
buffer pH 3.0, 1.1 equiv of M-segment was added. The ligation mixture
was kept at room-temperature overnight. The ligation product was then
JA0035654
(56) Srikant, C. B.; Heisler, S. Endocrinology 1985, 117, 271-278.
(57) Kim, S.; Byrn, R.; Groopman, J.; Baltimore, D. J. Virol. 1989, 63,
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