Synthesis of tyrocidine A and its analogues by spontaneous cyclization in aqueous solution

Article abstract of DOI:10.1021/ol0263191

(figure presented) Head-to-tail cyclization of peptides is a multistep process involving tedious C-terminal activation and side chain protection. Here we report a facile, quantitative cyclization method in aqueous ammonia solution for the total syntheses of the cyclic decapeptide antibiotic Tyrocidine A and its analogues from their fully deprotected linear thioester precursors on a solid support. This novel aqueous method is conformation-dependent and may be applicable to syntheses of other natural cyclic peptides.

Full text of DOI:10.1021/ol0263191

ORGANIC
LETTERS
2
002
Vol. 4, No. 17
893-2895
Synthesis of Tyrocidine A and Its
Analogues by Spontaneous Cyclization
in Aqueous Solution
2
Xianzhang Bu, Xiaoming Wu, Guiyang Xie, and Zhihong Guo*
Department of Chemistry and Biotechnology Research Institute,
The Hong Kong UniVersity of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong SAR, China
chguo@ust.hk
Received June 7, 2002
ABSTRACT
Head-to-tail cyclization of peptides is a multistep process involving tedious C-terminal activation and side chain protection. Here we report a
facile, quantitative cyclization method in aqueous ammonia solution for the total syntheses of the cyclic decapeptide antibiotic Tyrocidine A
and its analogues from their fully deprotected linear thioester precursors on a solid support. This novel aqueous method is conformation-
dependent and may be applicable to syntheses of other natural cyclic peptides.
Tyrocidine A, similar to its well-studied relative gramicidin
S, is a cyclic decapeptide antibiotic adopting a preformed
â-pleated sheet conformation under physiological conditions.
fact that widespread antibiotic resistance has become a
serious threat to public health.
4
1
The major disadvantage of tyrocidine A and gramicidin S
is their low specificity toward microorganisms. Although
both compounds have high antimicrobial activities, they also
disrupt higher mammalian cell membranes, as indicated by
their high hemolytic activity. Generation of analogues of
these peptides for structure-activity relationship studies
aiming to find drug candidates with high antimicrobial and
low hemolytic activities has thus become an area of active
Although the mode of action of the â-sheet antimicrobial
peptides has not been elucidated, it has been suggested that
the bacterial membrane is their primary target, where their
accumulation results in disruption of its normal barrier
2
function. Development of resistance to these peptide anti-
biotics is unlikely since it requires significant alternation of
the lipid composition for the cell wall. So far, no resistance
3
has been found for these peptide antibiotics. Therefore, both
5
research. However, synthesis of these cyclic peptides is a
natural cyclic peptides represent an attractive class of
antibacterial agents for new drug discovery, in light of the
challenge. The difficulty lies in the C-terminus activation
and cyclization of the linear precursor from solid-phase
peptide synthesis. Traditionally, N-hydroxysuccinimide ester
6
7
(1) (a) Hodgkin, D. C.; Oughton, B. M. Biochem. J. 1957, 65, 752. (b)
(
ONSu) and azide methods are most often used for the
Kuo, M.-C.; Gibbons, W. A. Biochemistry 1979, 18, 5855. (c) Kuo, M.-C.;
Gibbons, W. A. J. Biol. Chem. 1979, 254, 6278.
peptide cyclization. Both of these protocols are multistep
processes suffering from low yield and racemization.8
(2) Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo, M. Synthetic
Aspects of Biologically ActiVe Cyclic Peptides - Gramicidin and Tyro-
cidines; Kodansha, Ltd., and Wiley: Tokyo, 1979.
(3) Hancock, R. E. W. Lancet 1997, 349, 418.
(4) Travis, J. Science 1994, 264, 360.
1
0.1021/ol0263191 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/30/2002

Inspired by the high-yield formation of a cyclic product in
our recent treatment of an on-resin thioester linear precursor
9
of tyrocidine A with aqueous ammonia solution, we
explored the possibility of developing this cyclization
phenomenon into a general synthetic method for tyrocidine
A and its analogues.
Using Fmoc solid-phase peptide synthesis (SPPS) and an
9
optimized Fmoc deprotection method, a fully deprotected
linear peptide thioester 2a mimicking the biosynthetic
precursor of tyrocidine A10 was synthesized on TentaGel-
OH (Scheme 1). The on-resin linear peptide thioester was
Scheme 1
Figure 1. LC-ESI-MS analysis (A) and semipreparative HPLC
chromatogram (B) of the cleavage product of the linear precursor
2
a by 7 M NH
3
2
‚H O.
Since there are two active amine groups on the fully
deblocked linear precursor 2a, the cyclic product identified
in the LC-MS analysis (Figure 1, retention time 43.10 min)
could be either the expected tyrocidine A or the aminolysis
2
product of the C-terminal thioester by δ-NH of the ornithine
subsequently treated with 7 M aqueous ammonia solution,
and the product was collected for structural characterization.
As illustrated in Figure 1A, LC-ESIMS analysis showed that
a cyclic product was formed with a measured molecular ion
in the sequence. To distinguish these two possibilities,
semipreparative reverse-phase HPLC was used to purify the
cyclic product, which (Figure 1B, retention time 36.78 min)
was verified by molecular weight determination with FAB-
+
1
of 1270.59 ([M + 1] ), consistent with the calculated
MS. The purified product was analyzed by H NMR
molecular weight of 1269.65. Furthermore, the linear hy-
drolytic or aminolytic products (4a or 5a, respectively,
Scheme 2) were not detected,11 indicating quantitative
conversion of the linear precursor to the cyclic product.
spectroscopy and found to be identical to the natural
1
b,c
tyrocidine A reported earlier. In addition, the minimum
inhibition concentration (MIC) of the purified product was
determined with a standard method for a Bacillus substilis
strain to be 12 µg/mL, consistent with that of 20 µg/mL for
(5) (a) Dathe, M.; Wieprecht, T.; Nikolenko, H.; Handel, L.; Maloy, W.
12
the wild-type tyrocidine A. On the basis of these results,
L.; MacDonald, K.; Beyermann, M.; Bienert, M. FEBS Lett. 1997, 403,
the cyclic product from the linear precursor 2a is indeed the
wild-type tyrocidine A (3a). Thus, the deblocked peptide
thioester precursor cyclized quantitatively to form the specific
head-to-tail product under treatment of 7 M aqueous am-
208-212. (b) Blondell, S. E.; Houghten, R. A. Biochemistry 1992, 31,
12688. (c) Oren, Z.; Hong, J.; Shai, Y. J. Biol. Chem. 1997, 272, 14643-
14649. (d) Shai, Y.; Oren, Z. J. Biol. Chem. 1996, 271, 7305. (e) Oren, Z.;
Shai, Y. Biochemistry 1997, 36, 1826. (f) Dathe, M.; Shumann, M.;
Wieprecht, T.; Winkler, A.; Beyermann, M.; Krause, E.; Matsuzaki, K.;
Murase, O.; Bienert, M. Biochemistry 1996, 35, 12612. (g) Kondejewski,
L. H.; Jelokhani-Niaraki, M.; Farmer, S. W.; Bruce, L.; Kay, C. M.; Sykes,
B. D.; Hancock, R. E. W.; Hodges, R. S. J. Biol. Chem. 1999, 274, 13181.
2
monia, regardless of the δ-NH on the ornithine residue.
The high specificity and quantitative yield of the cycliza-
tion reaction is likely due to the formation of a certain
conformation that favors the ring closure. Resembling the
cyclic product shown to adopt a rigid antiparallel â-pleated
sheet structure by forming four interstrand hydrogen bonds,1
the linear precursor 2a could fold into a similar conformation
(6) Matsuura, S.; Takiguchi, H.; Waki, M.; Izumiya, N. Mem. Fac. Sci.,
Kyushu UniVersity 1975, Ser. C 9, 277.
(
7) Tamaki, M. Bull. Chem. Soc. Jpn. 1984, 67, 3210.
(8) (a) Izumiya, N.; Kato, T.; Waki, M. Biopolymers 1981, 20, 1785.
(
b) Ji, A.-X.; Bodanszky, M. Int. J. Pept. Protein Res. 1983, 22, 590.
9) Bu, X.; Xie, G.; Law, C. W.; Guo, Z. Tetrahedron Lett. 2002, 43,
419.
b,c
(
2
9
(10) von D o¨ hren, H.; Keller, U.; Vater, J.; Zocher, R. Chem. ReV. 1997,
7, 2675.
(
11) The conclusion was drawn from the absence of elution peaks with
(12) (a) Okamoto, K.; Yonezawa, H.; Izumiya, N. J. Chromatogr. 1974,
92, 147. (b) Okamoto, K.; Nonaka, K.; Izumiya, N. Bull. Chem. Soc. Jpn.
1977, 50, 231.
+
2+
the molecular ions [M + 1] or [M + 2] of 1288.67 or 644.8, respectively,
for 4a and 1287.67 or 644.4, respectively, for 5a in the LC-ESI-MS analysis.
2894
Org. Lett., Vol. 4, No. 17, 2002

Table 1. Product Distribution of the Cleavage Reaction of the Linear Thioester Precursor 2a-h in 7 M NH
3
‚H
2
O Solutiona
linear productsb
cyclic product
[M + 2]2
+
[M + 1]+
rt (min)
calcd mass
[M + 1]+
rt (min)
ratio (cyclic:linear)
c
precursor
AA1
calcd mass
2
2
2
2
2
2
2
2
a (wt)
DPhe
DPhe
DPhe
DPhe
Tyr
Tyr
Tyr
Tyr
1287.67
1301.68
1271.67
1285.69
1303.66
1317.68
1287.67
1301.68
not detected
not detected
not detected
not detected
1305.49
1269.66
1283.67
1253.66
1267.68
1285.65
1299.67
1269.66
1283.67
1270.59
1284.59
1254.50
1268.59
1286.51
1300.55
1270.51
1284.57
43.10
42.78
44.14
44.04
37.05
37.02
37.97
37.64
>55:1
>46:1
>40:1
>45:1
<1:35
<1:20
<1:19
<1:30
b
c
d
e
f
653.06
660.14
644.61
652.14
9.0
9.6
10.5
9.0
1318.49
1288.51
1302.52
g
h
a
Results are derived from the LC-ESIMS analysis. Samples were run on a ThermoQuest Hypersil Elite C18 column (2.1 × 100 mm, 5 µm) coupled to
a Finnigan LCQ Classic mass spectrometer. HPLC conditions: flow rate at 0.2 mL/min; linear gradient from 20 to 30% acetonitrile in water within 5 min,
3
5
0% acetonitrile for the next 20 min, and finally a linear gradient from 30 to 80% acetonitrile within 25 min. b Hydrolytic and aminolytic products 4 and
2+
+
are inseparable under the HPLC conditions. They are different by approximately 0.5 and 1.0 in mass for the molecular ions of [M + 2] and [M + 1] ,
c
respectively. Data for the hydrolytic product 4 are given in the table above. Ratio was calculated by assuming that the detection limit of the LC-MS
analysis is 1% of the total ion intensity over the entire elution process.
that brings the aminoxy and carboxy termini into close
proximity to facilitate the cyclization reaction.
the formation of the â-sheet structures of the cyclic product
and the precursor. In addition, Phe and Lys were simulta-
7
9
On the basis of this assumption, the preference of the
ammonia cleavage reaction for the cyclic product over the
aminolytic or hydrolytic product will disappear if this
conformation is disrupted. To test this, we chose to change
2
neously introduced into the positions of Tyr (AA ) and Orn
(
AA
synthesized as shown in Scheme 1. The LC-ESIMS results
of the cleavage products from 7 M NH ‚H O treatment are
summarized in Table 1. As expected, only cyclic products
are formed when the D-configuration at position-4 (AA ) is
3
), respectively, and all eight possible sequences were
3
2
4
4
1
the D-Phe to L-Tyr at the position of AA (Scheme 1), a
â-turn residue in tyrocidine A1 believed to be crucial to
b,c
1
maintained, whereas hydrolytic and aminolytic products
dominate the reaction when the amino acid at position-4
(AA ) is changed to L-Tyr. These results show that the ring
1
Scheme 2
closure reaction is indeed dependent on formation of a
Tyrocidine A-resembling conformation of the linear precur-
sor.
In summary, we have found a quantitative cyclization
method for the total syntheses of the cyclic decapeptide
antibiotic Tyrocidine A and its analogues from their fully
deprotected linear thioester precursors on a solid support.
This novel aqueous method is conformation-dependent and
may be applicable to syntheses of other natural cyclic
peptides.
Acknowledgment. This work was supported by the
Innovation and Technology Fund (ITS/119/00) from the
Innovation and Technology Commission of the Government
of the Hong Kong Special Administrative Region and the
HKUST (DAG01/02.SC09). We also thank the Department
of Chemistry at the HKUST for providing a postdoctoral
fellowship to Dr. G. Xie and a studentship to Mr. X. Wu.
Supporting Information Available: Experimental pro-
cedures for the solid-phase peptide synthesis of 2a-h and
1
MIC determination of 3a, H NMR and FAB-MS of
tyrocidine A (3a), and LC-ESIMS results for cleavage
products of 2a-h. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL0263191
Org. Lett., Vol. 4, No. 17, 2002
2895