IBTM-containing gramicidin S analogues: Evidence for IBTM as a suitable type II' β-turn mimetic

Article abstract of DOI:10.1021/ja9705755

The 2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate system (IBTM) has been proposed as a dipeptide surrogate of type II' β-turns. To evaluate which of the 11bR and 11bS diastereomers of IBTM best reproduces the conformational properties of type II' β-turns, gramicidin S (GS), a cyclic antibiotic peptide that contains two such units, has been chosen as a test compound and the effect of either diastereomer on both conformation and activity of the resulting peptide analogues has been determined. A conventional approach to the cyclic peptide structure based on solution cyclization of a partially protected precursor was only practicable for the (S)IBTM diastereomer. As an alternative, a solid phase mediated cyclization approach has been devised and applied successfully to both gramicidin S and its Lys^2,2' analogue, then extended to the (R)-IBTM-containing analogues. NMR conformational analysis has clearly shown that only the (R) diastereomer of IBTM is a suitable mimic of the type II' β-turn conformation typical of GS. Differences in antibacterial activity between the (S)- and (R)-IBTM-containing GS analogues confirm the conformational results.

Full text of DOI:10.1021/ja9705755

J. Am. Chem. Soc. 1997, 119, 10579-10586
10579
IBTM-Containing Gramicidin S Analogues: Evidence for
IBTM as a Suitable Type II′ â-Turn Mimetic1,2
David Andreu,*,3a Sergi Ruiz,3a Cristina Carren˜o,3a Jordi Alsina,3a
Fernando Albericio,3a Mar´ıa AÄ ngeles Jime´nez,3b Natalia de la Figuera,3c
Rosario Herranz,3c Mar´ıa Teresa Garc´ıa-Lo´pez,3c and Rosario Gonza´lez-Mun˜iz3c
Contribution from the Department of Organic Chemistry, UniVersity of Barcelona,
E-08028 Barcelona, Spain, and Institutes of Structure of Matter and Medicinal Chemistry,
CSIC, E-28006 Madrid, Spain
ReceiVed February 24, 1997. ReVised Manuscript ReceiVed August 6, 1997X
Abstract: The 2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate system (IBTM) has been proposed as
a dipeptide surrogate of type II′ â-turns. To evaluate which of the 11bR and 11bS diastereomers of IBTM best
reproduces the conformational properties of type II′ â-turns, gramicidin S (GS), a cyclic antibiotic peptide that contains
two such units, has been chosen as a test compound and the effect of either diastereomer on both conformation and
activity of the resulting peptide analogues has been determined. A conventional approach to the cyclic peptide
structure based on solution cyclization of a partially protected precursor was only practicable for the (S)-IBTM
diastereomer. As an alternative, a solid phase mediated cyclization approach has been devised and applied successfully
to both gramicidin S and its Lys2,2′ analogue, then extended to the (R)-IBTM-containing analogues. NMR
conformational analysis has clearly shown that only the (R) diastereomer of IBTM is a suitable mimic of the type
II′ â-turn conformation typical of GS. Differences in antibacterial activity between the (S)- and (R)-IBTM-containing
GS analogues confirm the conformational results.
The â-turn motif, a segment of four amino acid residues that
reverse the direction of peptide chains, is an important structural
feature of proteins. The surface localization of turns in proteins,
and the predominance within them of residues containing
potentially critical pharmacophoric information, has led to the
hypothesis that turns play relevant roles in diverse recognition
events.4 There is substantial evidence to suggest that smaller
peptides also possess â-turns in their bioactive conformations,
and the resulting compact structures have clustered side chains
available for interaction with receptors.5 A general approach
to confirm these suggestions involves the use of non-peptide
building blocks which, when inserted into a peptide chain,
enforce or stabilize a particular type of â-turn. In this respect,
a variety of conformationally restricted compounds, mostly
lactams, have been proposed as dipeptide mimetic replacements
for the i + 1 and i + 2 residues of â-turns.6,7 Incorporation of
such â-turn surrogates into model peptides containing well-
established â-turns produces analogues whose structural analysis
indicates how well the replacements match the original â-turn
conformation. A useful candidate for this type of studies is
the antibiotic gramicidin S8 [GS, cyclo(Val1-Orn2-Leu3-D-Phe4-
Pro5)2, 1], a cyclic decapeptide9 that exists in a very stable
solution conformation with two symmetrical type II′ â-turns
(5) (a) Dyson, H. J.; Cross, K. J.; Houghten, R. A.; Wilson, I. A.; Wright,
P. E.; Lerner, R. A. Nature 1985, 318, 480-483. (b) Oka, M.; Montelione,
G. T.; Scheraga, H. A. J. Am. Chem. Soc. 1984, 106, 7959-7969. (c)
Freidinger, R. M.; Veber, D. F.; Perlow, D. S.; Brooks, J. R.; Saperstein,
R. Science 1980, 210, 656-658.
(6) For recent reviews on peptide conformation mimetics see: (a)
Holzemann, G. Kontakte 1991, 3-12, 55-63. (b) Kahn, M. Synlett 1993,
821-826. (c) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bo¨s, M.; Cook,
C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.; Madison,
V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G. P.; Voss, M.
E. J. Med. Chem. 1993, 36, 3039-3049. (d) Liskamp, R. M. J. Recl. TraV.
Chim. Pays-Bas 1994, 113, 1-19.
X Abstract published in AdVance ACS Abstracts, October 1, 1997.
(1) Abbreviations: AAA, amino acid analysis; Al, allyl; Boc, tert-
butoxycarbonyl; ATCC, American Type Culture Collection; BOP, (benzo-
triazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; COSY,
homonuclear correlated spectroscopy; DCM, dichloromethane; DIEA, N,N-
diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMAP,
N,N-dimethylaminopyridine; DMF, N,N-dimethylformamide; DMSO, di-
methyl sulfoxide; DSC, disuccinimidyl carbonate; Fmoc, 9-fluorenyl-
methoxycarbonyl; GS, gramicidin S; HATU, N-[(dimethylamino)(1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl)methylene]-N-methylmethanaminium
hexa-
fluorophosphate N-oxide; HOAc, acetic acid; HOAt, 7-azahydroxybenzo-
triazole; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid
chromatography; IBTM, 2(S)-amino-3-oxohexahydroindolizino[8,7-b]indole-
5(S)-carboxylic acid; MALDI-TOF MS, matrix-assisted laser desorption
ionization, time-of-flight mass spectroscopy; MBHA, p-methylbenzhydryl-
amine (resin); MeCN, acetonitrile; NOE, nuclear Overhauser effect; NOESY,
nuclear Overhauser enhancement spectroscopy; ODS, octadecylsilica; Pam,
4-phenylacetamidomethyl (resin); PyAOP, (7-azabenzotriazol-1-yloxy)tris-
(pyrrolidino)phosphonium hexafluorophosphate; Su, succinimidyl; TBTU,
N-[(1H-benzotriazol-1-yl)dimethylaminomethylene)-N-methylmethanamin-
ium tetrafluoroborate N-oxide; TFA, trifluoroacetic acid; THF, tetrahydro-
furan; TOCSY, total correlation spectroscopy; TSP, sodium 3-trimethylsilyl-
[2,2,3,3-2H4]propionate.
(2) Parts of this work have been previously reported in preliminary
form: (a) De la Figuera, N.; Jime´nez, M. A.; Biacs, M.; Garc´ıa-Lo´pez, M.
T.; Gonza´lez-Mun˜iz, R.; Andreu, D. In Peptides 1994: Proceedings of the
Twenty-Third European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM
Science Publishers: Leiden, The Netherlands, 1994; pp 702-703. (b)
Andreu, D.; Ruiz, S.; Carren˜o, C.; Alsina, J.; Albericio, F.; Jime´nez, M.
A.; De la Figuera, N.; Herranz, R.; Garc´ıa-Lo´pez, M. T.; Gonza´lez-Mun˜iz,
R. In Peptides 1996: Proceedings of the Twenty-Fourth European Peptide
Symposium; Ramage, R., Epton, R., Eds.; Mayflower Worldwide Ltd.:
Kingswinford, England, 1997; in press.
(3) (a) University of Barcelona. (b) Institute of Structure of Matter. (c)
Institute of Medicinal Chemistry.
(4) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem. 1985,
37, 1-109.
(7) Other representative examples of â-turn mimetics include: (a) Genin,
M. J.; Johnson, R. L. J. Am. Chem. Soc. 1992, 114, 8778-8783. (b) Genin,
M. J.; Gleason, W. B.; Johnson, R. L. J. Org. Chem. 1993, 58, 860-866.
(c) Lombart, H.-G.; Lubell, W. D. J. Org. Chem. 1994, 59, 6147-6149.
(d) Kemp, D. S.; Li, Z. Q. Tetrahedron Lett. 1995, 36, 4175-4178. (e)
Hanessian, S.; Ronan, B.; Laoui, A. Bioorg. Med. Chem. Lett. 1994, 4,
1397-1400. (f) Robl, J. A. Tetrahedron Lett. 1994, 35, 393-396. (g)
Mueller, R.; Revesz, L. Tetrahedron Lett. 1994, 35, 4091-4092. (h)
Hanessian, S.; McNaughton-Smith, G. Bioorg. Med. Chem. Lett. 1996, 6,
1567-1572. (i) Slomczynska, U.; Chalmer, D. K.; Cornille, F.; Smythe,
M. L.; Beusen, D. D.; Moeller, K. D.; Marshall, G. R. J. Org. Chem. 1996,
61, 1198-1204.
(8) Consden, R. J.; Gordon, A. H.; Martin, A. J. P.; Synge, R. D. M.
Biochem. J. 1947, 28, 596-602.
S0002-7863(97)00575-1 CCC: $14.00 © 1997 American Chemical Society

10580 J. Am. Chem. Soc., Vol. 119, No. 44, 1997
Andreu et al.
Scheme 1
connected by a short antiparallel â-sheet.10 The replacement
of the D-Phe-Pro residues in GS by rigid mimetics has become
a standard method for measuring their capacity to serve as
surrogates of the i + 1 and i + 2 residues in type II′ â-turns.11
Moreover, since the antibacterial activity of GS has been related
to its particular conformation,12 the cyclic decapeptide provides
not only structural but also functional evidence to assess the
adequacy of â-turn mimetic candidates.
IBTM4,5Lys2,2′]GS (4a), [(R)-IBTM4,5,Lys2,2′]GS (4b), and [(R)-
IBTM4,5]GS (5b), and the comparison of (S)- and (R)-IBTM-
containing peptidomimetics to GS by NMR conformational
analysis. Additional evidence on how well these dipeptide
surrogates mimic the topology of D-Phe4-Pro5 in GS has been
provided by the activity of compounds 4a, 4b, and 5b against
several bacterial strains, in comparison with those of GS and
its [Lys2,2′] analogue 2.
In a recent paper we have proposed the 2-amino-3-oxo-
hexahydroindolizino[8.7-b]indole-5-carboxylate system (H-
IBTM-OH1) as a new type of â-turn surrogate.13 Molecular
dynamics studies on model structures Ac-S-IBTM-NHMe and
Ac-R-IBTM-NHMe have revealed that, although both deriva-
tives are able to adopt conformations close to an ideal type-II′
â-turn, the diastereoisomer with the 11bR configuration is a
more effective replacement than the 11bS isomer for the i + 1
and i + 2 residues of this type of â-turn.13 In a similar way,
the temperature dependence of the NH-Me amide hydrogen
chemical shift of the 11bR isomer in DMSO (∆δ/∆T ) -2.8
ppb/K) indicated that this proton is involved in an intramolecular
hydrogen bond,14 characteristic of â-turn conformations. The
corresponding value for the S derivative (-3.9 ppb/K), however,
was within a region where it is difficult to draw concrete
conclusions with respect to hydrogen bonding.9g
Results and Discussion
Synthesis. Our initial approach to IBTM-containing GS
analogues chose a conventional synthetic strategy based on
solution cyclization of a partially protected linear precursor
(Scheme 1) assembled by solid-phase methods15 on a Pam16
resin. As in previous studies on GS analogues,11c the replace-
ment of the two Orn residues in 1 by Lys was primarily dictated
by synthetic reasons, i.e., commercial availability of the
orthogonally protected derivative Boc-Lys(Fmoc)-OH. Pro4′
was chosen as C-terminal residue to minimize the risk of
epimerization at the cyclization step, and the peptide was built
from that residue by Boc-based chemistry17 (Scheme 1). In the
case of 4a, the (S)-IBTM pseudodipeptide unit was incorporated
into the sequence as the Boc derivative 6a. HF acidolysis of
the peptide-resin gave the linear bis-Fmoc protected precursor
3a, which was purified on reverse phase, cyclized under high
dilution conditions, and deprotected to give the target compound
4a, satisfactorily characterized by AAA and MALDI-TOF MS.
A parallel synthesis with the (R)-IBTM derivative 6b readily
provided the expected H-D-Phe-Leu-Lys(Fmoc)-Val-(R)-IBTM-
Leu-Lys(Fmoc)-Val-Pro-OH intermediate. However, attempts
to cyclize this precursor using a variety of activation reagents
and conditions led invariably to very complex crudes in which
no significant levels of 4b could be detected after Fmoc
In search of a more conclusive evaluation of the fitness of
the IBTM system as a type II′ â-turn mimetic, we decided to
replace one of the D-Phe4-Pro5 dipeptide units in GS with either
S- or R-IBTM surrogates. This in turn depended on convenient
access routes to these IBTM-containing GS analogues. The
present paper reports the synthesis of three such peptides, [(S)-
(9) For recent reviews on cyclic peptides see: (a) Kopple, K. D. J. Pharm.
Sci. 1972, 61, 1345-1356. (b) Blout, E. R. Biopolymers 1981, 20, 1901-
1912. (c) Hruby, V. J. Life Sci. 1982, 31, 189-199. (d) Ovchinikov, Y.
A.; Ivanov, V. T. In The Proteins; Neurath, H., Hill, R. L., Eds.; Academic
Press: New York, 1982; pp 307-642. (e) Schmidt, U. Pure Appl. Chem.
1986, 58, 295-301. (f) Tonelli, A. E. In Cyclic Polymers; Semlyen, J. A.,
Ed.; Elsevier Applied Science: London, 1986; pp 261-284. For reviews
on cyclic peptides as models for conformational studies see: (g) Kessler,
H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512-523. (h) Rizo, J.; Gierasch,
L. M. Annu. ReV. Biochem. 1992, 61, 387-418.
(10) (a) Dygert, M.; Go, N.; Scheraga, H. A. Macromolecules 1975, 8,
750-761. (b) Huang, D.-H.; Walter, R.; Glickson, J. D.; Kirshna, N. R.
Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 672-675. (c) Krauss, E. M.; Chan,
S. I. J. Am. Chem. Soc. 1982, 104, 6953-6961.
(11) (a) Sato, K.; Nagai, U. J. Chem. Soc., Perkin Trans. 1 1986, 1231-
1234. (b) Bach, A. C.; Markwalder, J. A.; Ripka, W. C. Int. J. Peptide
Protein Res. 1991, 38, 314-323. (c) Ripka, W. C.; De Lucca, G. V.; Bach,
A. C.; Pottorf, R. S.; Blaney, J. M. Tetrahedron 1993, 49, 3609-3628. (d)
Lombart, H.-G.; Lubell, W. D. In Peptides: Chemistry, Structure and
Biology. Proceedings of the 14th American Peptide Symposium; Kaumaya,
P. T. P., Hodges, R. S., Eds.; Mayflower Scientific Ltd.: Kingswinford,
England, 1996; pp 695-696.
(12) (a) Imazu, S.; Shimohigashi, Y.; Kodama, H.; Sakaguchi, K.; Waki,
M.; Kato, T.; Izumiya, N. Int. J. Peptide Protein Res. 1988, 32, 298-306.
(b) Shimohigashi, Y.; Sakamoto, H.; Yoshikomi, H.; Waki, M.; Kawano,
K.; Ohno, M. In Peptides, Chemistry and Biology, Proceedings of the 12th
American Peptide Symposium; Smith, J. A., Rivier, J. E., Eds.; Escom:
Leiden, 1992; pp 271-272. (c) Tamaki, M.; Akabori, S.; Muramatsu, I.
Int. J. Peptide Protein Res. 1996, 47, 369-375.
(13) De la Figuera, N.; Alkorta, I.; Garc´ıa-Lo´pez, M. T.; Herranz, R.;
Gonza´lez-Mun˜iz, R. Tetrahedron 1995, 51, 7841-7856.
(14) De la Figuera, N. Unpublished results.
deprotection. In view of these difficulties, an alternative route,
involving solid-phase-mediated cyclization as the key step, was
explored. This type of cyclization has been successfully applied
(15) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
Merrifield, R. B. In Peptides. Synthesis, Structures and Applications; Gutte,
E., Ed.; Academic Press: San Diego, 1995; pp 93-169.
(16) Mitchell, A. R.; Kent, S. B. H.; Engelhard, M.; Merrifield, R. B. J.
Org. Chem. 1978, 43, 2845-2852.
(17) Barany, G.; Merrifield, R. B. In The Peptides. Analysis, Synthesis,
Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1979;
Vol. 2, pp 1-284.

EVidence for IBTM as a Suitable Type II′ â-Turn Mimetic
J. Am. Chem. Soc., Vol. 119, No. 44, 1997 10581
Scheme 2
to the synthesis of cyclic peptides in recent years.18 The original
approach used carboxyl (Asp, Glu)- or carboxamide (Asn, Gln)-
containing residues for side chain anchoring to the polymer,
with the R-COOH of those residues orthogonally protected with
respect to the chemistry used for chain elongation. Selective
removal of both N- and C-terminal protections allows cyclization
to proceed, ideally under the pseudodilution conditions provided
by the polymeric matrix.19 One of our laboratories has
developed a variation of this strategy that allows anchoring
through the side chain amino groups of Lys or Orn, via a
carbamate-functionalized resin.20 The procedure requires Fmoc
chemistry for chain elongation and uses orthogonal allyl
protection of the R-COOH of the Lys or Orn residue. We
decided to apply this method to prepare the (R)-IBTM-
containing analogues of GS that were not accessible by
conventional solution cyclization.
Figure 1. HPLC analysis of crude 1 (left panel) and 2 (right panel)
after acidolytic cleavage from resin. ODS column (0.4 × 25 cm, 5
µm). Elution by a linear 5-95% gradient of MeCN (+0.036% TFA)
into H2O (+0.045% TFA) over 30 min, followed by 5 min isocratic at
95%. UV detection at 220 nm. Retention times are indicated above
the peaks.
Trial syntheses of GS (1) and [Lys2,2′]GS (2) were first
performed (Scheme 2). The starting resin, 3-[4-(hydroxymeth-
yl)phenoxy]propionyl-Ala-MBHA21 (with Ala used as the
internal reference amino acid22), was activated with DSC/DMAP
to give a Su-carbonate resin, to which the trifluoroacetate salt
of Fmoc-Xaa-OAl (Xaa ) Orn, Lys) was anchored in the
presence of DIEA. From this point, chain assembly was
continued by Fmoc chemistry, with Boc protection for the side
chains of Orn or Lys. Deprotection of the C-terminal allyl ester,
followed by Fmoc removal at the N-terminus, allowed cycliza-
tion on the solid phase, with activation of the R-carboxyl of
Orn/Lys by means of PyAOP/HOAt/DIEA.23 Finally, acidolytic
deprotection and cleavage furnished 1 and 2 in good purity
(Figure 1), although in moderate yields (ca. 10% after purifica-
tion, based on initial resin substitution).
of the Fmoc derivative of (R)-IBTM, 7b, as replacement for
positions 4,5. Its incorporation to the peptide resin was
facilitated by the strong activating agent HATU.23 A depro-
tection and cleavage sequence similar to the ones used for 1
and 2 above provided the target compounds, albeit in much
lower yields, presumably due to the enhanced hydrophobicity
of IBTM, which substantially decreased recoveries at the
purification steps. The cyclic pseudopeptides were satisfactorily
characterized by AAA and MALDI-TOF MS.
NMR Conformational Analysis. Since type II′ â-turns
present negative φ3 angles, and since only an equatorial
disposition of the C5H proton in [(S)-IBTM4,5,Lys2,2′]GS (4a)
and [(R)-IBTM4,5,Lys2,2′]GS (4b) is compatible with negative
φ3 angles (Figure 2), NMR analysis was started by investigating
the equatorial or axial disposition of that particular proton. A
3J5,6 of ∼0 Hz is expected for the downfield C6H proton of
IBTM in an equatorial disposition, while a large value would
Extension of this approach to the (R)-IBTM-containing
mimetics 4b and 5b was essentially straightforward with use
(18) For recent examples of solid phase mediated cyclizations, see: (a)
Kates, S. A.; Sole´, N. A.; Albericio, F.; Barany, G. In Peptides: Design,
Synthesis and Biological ActiVity; Basava, C., Anantharamaiah, G. M., Eds.;
Birkha¨user: Boston, 1994; pp 39-58 and references cited therein. (b)
Valero, M. L.; Camarero, J. A.; Adeva, A.; Verdaguer, N.; Fita, I.; Mateu,
M. G.; Domingo, E.; Giralt, E.; Andreu, D. Biomed. Pept. Proteins Nucleic
Acids 1995, 1, 133-140. (c) Valero, M. L.; Giralt, E.; Andreu, D.
Tetrahedron Lett. 1996, 37, 4229-4232.
(19) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677-
683.
(20) Alsina, J.; Rabanal, F.; Giralt, E.; Albericio, F. Tetrahedron Lett.
1994, 35, 9633-9636.
(21) Albericio, F.; Barany, G. Int. J. Peptide Protein Res. 1985, 26, 92-
3
be expected for an axial disposition. The experimental J5,6
value observed for compounds 4a and 4b in aqueous solution
at pH 3.0 and in DMSO is ∼0 Hz, which confirms the equatorial
disposition of the C5H proton in both 4a and 4b. Thus, both
(S)- and (R)-IBTM skeletons prefer conformations with negative
φ3 angles, as required for the formation of a type II′ â-turn.
The NMR parameters of [(S)-IBTM4,5,Lys2,2′]GS (4a) under
both experimental conditions investigated are very different from
those previously reported for native GS.10c Differences in the
97.
(22) Matsueda, G. R.; Haber, E. Anal. Biochem. 1980, 104, 215-227.
(23) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem.
Soc., Chem. Commun. 1994, 201, 203.

10582 J. Am. Chem. Soc., Vol. 119, No. 44, 1997
Andreu et al.
Table 2. Temperature Coefficients for Amide Protons (∆δ/∆T,
ppm 103/K)
GS (1)
residue no. DMSOa
(S)-IBTM-GS (4a)
(R)-IBTM-GS (4b)
H2Ob
DMSOc
H2Ob
DMSOc
Val 1
-1.6
-5.0
-2.8
-7.4
-1.6
-5.0
-2.8
-7.4
-9.0
-8.5
-1.5
-1.0
-5.5
-9.0
-9.0
-10.5
-5.2
-2.5
-3.3
-0.6
-4.9
-4.0
-6.4
-5.4
-3.0
-9.0
-2.5
-8.5
+0.5
-8.5
-3.0
-12.0
-1.6
-5.3
-1.6
-4.7
-0.4
-5.1
-2.4
-7.6
Xaa 2d
Leu 3
Yaa 4e
Val 1′
Xaa 2′ d
Leu 3′
D-Phe 4′
a Values from ref 11b. b pH 3.0, temperature range 5-25 °C.
Figure 2. Significant dihedral angles of the â-turn-like conformations
of Ac-(S)- and Ac-(R)-IBTM-NHMe model compounds.
c Temperature range 20-50 °C. d Xaa is Orn in GS and Lys in
e
compounds 4a and 4b. D-Phe-Pro in GS, and IBTM in compounds
4a and 4b.
Table 1. Chemical Shifts of Amide and Pro CδH protons (δ, ppm
from TSP)
GS (1)
(S)-IBTM-GS (4a) (R)-IBTM-GS (4b)
residue no. H2Oa DMSOa H2Ob
DMSOc
H2Ob
DMSOc
Val 1
7.62
8.61
8.81
8.99
7.62
8.61
8.81
8.99
3.68
2.59
7.22
8.68
8.34
9.11
7.22
8.68
8.34
9.11
3.60
2.48
8.44
8.57
7.78
7.80
8.20
8.19
8.18
8.39
3.67
3.29
8.14
8.12
7.44
7.91
7.90
7.81
7.92
8.35
3.58
3.30
7.53
8.64
8.27
9.39
8.44
8.56
8.72
9.01
3.66
2.54
7.23
8.52
8.10
9.46
8.14
8.53
8.46
8.82
3.58
2.49
Xaa 2d
Leu 3
Yaa 4d
Val 1′
Xaa 2′d
Leu 3′
D-Phe 4′
Pro 5′e
a Values from ref 10c. b DMSO, 20 °C. c H2O, pH 3.0, 5 °C. d Xaa
is Orn in GS and Lys in compounds 4a and 4b; Yaa is D-Phe-Pro in
GS, and IBTM in compounds 4a and 4b. e Values corresponding to
CδH and Cδ′H protons.
δ-values of most NH and CRH protons of [(S)-IBTM4,5
,
Lys2,2′]GS (4a) with respect to GS (1) are large (Table 1). The
CRH protons of residues involved in an antiparallel â-sheet are
expected to be downfield shifted24 with respect to the random
coil values.25 As shown in Figure 3, this is the case for the
CRH protons of Val1,1′, Orn2,2′, and Leu3,3′ in GS (1), but not
for the same CRH protons in [(S)-IBTM4,5,Lys2,2′]GS (4a), which
are upfield shifted. A small absolute value for the temperature
coefficient of an amide proton indicates its protection from
solvent exchange by either involvement in an intramolecular
hydrogen bond or inaccessibility to solvent. The temperature
coefficients measured for the amide protons of [(S)-IBTM4,5
,
Lys2,2′]GS (4a) do not fit with the hydrogen-bonding pattern
expected for a GS-like structure (Table 2). If 4a were to adopt
a GS-like structure such as shown in Figure 4, one should expect
temperature coefficients for Val1,1′ and Leu3,3′ to be small (in
absolute value), as previously reported for GS (1);10c however,
Val1,1′ and Leu3′ have large coefficients, indicative of their
accessibility to bulk solvent. The pattern of 3JCRH-NH coupling
constants of 4a is also very different from that of GS (Table
3). Small values for Val1 and Lys2 may suggest the presence
of reverse turns involving these residues. The observation of
CRH(Val1)-NH(Leu3), NH(Val1)-NH(Lys2), and NH(Lys2)-
NH(Leu3) NOE connectivities is also compatible with the
presence of a type I â-turn centered at Val1-Lys2. In addition
to those NOEs, several long-range NOEs involving the IBTM
nucleus and Val1, Leu3′, and D-Phe4′ residues were found for
4a in both aqueous solution and DMSO (Table 4 and Figure
5). These nonsequential NOEs could be explained by the
Figure 3. Conformational CRH ∆δ shifts (∆δ ) δobserved - δrandom coil
,
ppm) as a function of sequence obtained for GS (∆δ was obtained
from the δ-values reported by Krauss and Chan10c) for compound 4a
and compound 4b in aqueous solution and in DMSO. IBTM and D-Phe
residues are not included as no random coil values have been reported
for them.
formation of antiparallel dimers that bring the indole ring and
the Val1, Leu3′, and D-Phe4′ residues into close proximity, or
by monomeric species with a hydrophobic cluster in which the
IBTM skeleton and the hydrophobic side chains of Leu3′ and
D-Phe4′ interact. In any event, the â-turn involving the D-Phe4′-
Pro5′ in GS residues is not detected in 4a.
In contrast to the previous data, the NMR parameters found
for [(R)-IBTM4,5,Lys2,2′]GS (4b) in both aqueous solution, pH
3.0, and DMSO clearly indicate that it adopts a GS-like structure.
The δ values of NH and CRH protons in both solvents are very
close to those reported for GS (1), while substantially different
from those of [(S)-IBTM4,5,Lys2,2′]GS (4a) (Table 1). As
(24) (a) Wishart, D. S.; Sykes, B. D. Methods Enzymol. 1994, 239, 363-
392. (b) Case, D. A.; Dyson, H. J.; Wright, P. E. Methods Enzymol. 1994,
239, 392-416.
(25) Wu¨trich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986.
expected for residues in a â-strand24 and observed in GS,10c
values of the CRH protons of Val1,1′, Lys2,2′, and Leu3,3′ are
δ

EVidence for IBTM as a Suitable Type II′ â-Turn Mimetic
Table 3. Coupling Constants of Amide Protons (3JNH-CRH, Hz)
J. Am. Chem. Soc., Vol. 119, No. 44, 1997 10583
GS (1)
(S)-IBTM-GS (4a)
(R)-IBTM-GS (4b)
residue no. DMSOa
H2Ob
DMSOc
H2Ob
DMSOc
Val 1
9.7
8.9
9.2
2.6
9.7
8.9
9.2
2.6
5.5
2.8
9.6
7.0
9.0
8.8
5.5
8.6
9.5
7.5
8.1
8.2
12.5
8.4
7.3
7.9
8.9
8.9
8.0
6.4
9.3
8.4
8.0
3.8
12.0
10.3
9.1
4.3
12.3
9.7
Xaa 2d
Leu 3
Yaa 4e
Val 1′
Xaa 2′
Leu 3′
D-Phe 4′
10.3
2.6
a 25 °C; values from ref 11b. b 5 °C, pH 3.0. c 50 °C. d,e As in Table
2.
Table 4. Long-Range NOEs observed for (S)-IBTM-GS (4a, 5
mM)
NOE intensity in
residue i
residue j
H2O, pH 3.0, 5 °C DMSO, 20 °C
NH Val 1
N11H IBTM
N11H IBTM
N11H IBTM
C10H IBTM
N11H IBTM
N11H IBTM
C10H IBTM
C11bH IBTM
weak
strong
medium
medium
strong
medium
weak
not detected
a
medium
strong
CRH Val 1
CγH3 Val 1
CγH3 Val 1
CRH Leu 3′
CδH3 Leu 3′
Cδ′H3 Leu 3′
CâH D-Phe 4′
b
mediumc
Figure 5. Selected regions of the NOESY spectrum of [(S)-
IBTM4,5,Lys2,2′]GS (4a) in H2O-D2O (9:1 v/v), pH 3.0 at 5 °C.
Nonsequential NOEs are boxed and labeled. The sequential assignment
for most residues can also be followed through CRH-NH(i,i+1) NOEs
(lower regions) and through CγH-NH(i,i+1) and CδH-NH(i,i+1)
NOEs (upper regions).
strongc
strong
medium
not detected
not detected
Câ′H D-Phe 4′ C11bH IBTM
a NOE connectivity could not be observed due to closeness to
diagonal. b NOE connectivity could not be observed due to overlapping
with other NOE crosspeaks. c CδH3 and Cδ′H3 protons of Leu 3′ have
the same δ value.
Table 5. Nonsequential NOEs Observed for (R)-IBTM-GS (4b, 5
mM)
NOE intensity in
residue i
residue j
H2O, pH 3.0, 5 °C DMSO, 20 °C
NH Val 1
CRH Lys 2
CRH Lys 2
NH Leu 3
NH Leu 3
NH Leu3′
CRH Lys 2′
NH Leu 3′
NH Val 1′
CRH Lys 2′
weak
a
b
weak
b
not detected
not detected
weak
not detected
weak
weak
strong
medium
a
medium
weak
weak
not detected
weak
Cγγ′H Val 1 NH Leu 3
CâH Val 1
NH Val 1′
CâH Val 1′
NH Leu 3′
CγH Leu 3
NH Leu 3
Cγγ′H Val 1′ NH Leu 3
medium
medium
weak
Figure 4. Schematic representation of the GS-like backbone conforma-
tion. Hydrogen bonds are indicated by arrows and the expected NOEs
by black lines.
Cγγ′H Val 1′ Cγââ′H Leu 3
medium
weak
CâH Val 1
NH Leu 3′
a NOE connectivity could not be observed due to closeness to the
diagonal. b NOE connectivity could not be observed due to overlapping
with other NOEs.
downfield shifted (Figure 3) with respect to random coil
values.25 The amide protons of residues Val1,1′ and Leu3,3′ are
hydrogen bonded, as in the GS structure (Figure 4). Their
temperature coefficients, which are small in absolute value in
both solvents (Table 2), as expected for hydrogen-bonded amide
protons, confirm that [(R)-IBTM4,5,Lys2,2′]GS (4b) adopts a GS-
like structure. The strongest evidence for the existence of such
structure comes from the analysis of NOE connectivities. The
isms. However, (R)-IBTM derivatives 4b and 5b were quite
comparable in antibiotic activity to the reference GS against
gram-positive strains, while the (S)-IBTM analogue 4a was
practically inert against these organisms. The Lys for Orn
replacement in 2 did not produce any significant difference in
antibiotic potency relative to GS.
CRH(Lys2)-CRH(Lys2′),
CRH(Lys2)-NH(Leu3′),
and
NH(Leu3)-CRH(Lys2′) NOE crosspeaks, as well as some non-
sequential i,i + 2 NOEs involving at least one side chain proton,
fully support the proposed GS-like structure (Figure 6 and Table
5). Further confirmation is obtained from the 3JCRH-NH coupling
constants of 4b (Table 3).
Conclusions
The NMR studies reported here clearly show that (R)-IBTM,
when incorporated into [Lys2,2′]GS, does not disrupt the
conformation of this peptide. Moreover, [(R)-IBTM4,5]GS and
[(R)-IBTM4,5,Lys2,2′]GS retain the biological activity of the
parent peptides. These results conclusively demonstrate that
(R)-IBTM is an effective type II′ â-turn mimetic. However,
similar studies on the (S)-IBTM-containing peptide analogue
do not support this type of conformation.
Antibacterial Activity. Minimal inhibitory concentrations
of mimetics 4a, 4b, and 5b, GS (1), and its [Lys2,2′] analogue
2 were determined against two gram-positive and two gram-
negative organisms (Table 6). The results showed again a clear
difference between the (R)- and (S)-IBTM GS analogues. As
is the case with GS, none of the IBTM-containing peptides
showed significant activity toward gram-negative microorgan-
The versatility of (R)-IBTM as a suitable dipeptide surrogate
is evidenced by its incorporation into peptide sequences by solid-

10584 J. Am. Chem. Soc., Vol. 119, No. 44, 1997
Andreu et al.
Table 6. Minimal Inhibitory Concentrations (µg/mL) of GS and Analogues
gram-positive
gram-negative
peptide
Bacillus subtilis
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
GS (1)
4
4
128
4
4
8
128
16
8
128
256
128
>64
>128
256
256
[Lys2,2′]GS (2)
[(S)-IBTM4,5,Lys2,2′]GS (4a)
[(R)-IBTM4,5,Lys2,2′]GS (4b)
[(R)-IBTM4,5]GS (5b)
>512
>64
2
>128
were of the highest purity commercially available. Solvents for peptide
synthesis (DMF and DCM) and HPLC supplies (MeCN and ODS
columns, 4 × 250 mm, 5 µm) were from Scharlau (Barcelona, Spain).
Analytical HPLC runs used linear gradients (usually 5-95%) of MeCN
(+0.036% TFA) into H2O (+0.045% TFA) over 30 min at 1 mL/min,
with UV detection at 220 nm. Amino acid analysis of peptide
hydrolysates (6 N HCl, 150 °C, 4 h) were run in a Beckman 6300
autoanalyzer. Mass spectra of peptides were acquired by the MALDI-
TOF ionization technique in a Kompact I instrument (Kratos Analytical,
Manchester, UK) with an R-cyano-4-hydroxycinnamic acid matrix.
Electrospray mass spectra were obtained in a VG Quattro instrument
(Fisons Instruments, Wythenshawe, UK) working in the positive mode,
using nitrogen as nebulizing and drying gas (10 and 450 L/h,
respectively), a source temperature of 80 °C, a capillary voltage of 3.5
kV, and a focusing voltage of 60 V. The same instrument, fitted with
a Cs gun, was used to acquire FAB spectra.
Solid-Phase Synthesis and Purification of [(S)-IBTM4,5,Lys2,2′]-
GS (4a). Boc-Pro-OCH2-Pam resin (300 mg, 0.22 mmol) was
submitted to a cycle of Boc solid-phase synthesis for each residue of
the sequence, as follows: (i) deprotection with TFA-DCM (2:3 v/v; 1
+ 20 min); (ii) washes with DCM (4 × 0.5 min) and DMF (2 × 1
min); (iii) neutralization with DIEA-DCM (1:19 v/v; 2 × 1 min); and
(iv) coupling with Boc-amino acid (0.66 mmol), TBTU (0.66 mmol),
and DIEA (1.32 mmol) in dry DMF for 60 min. All couplings were
complete after this time by the Kaiser test26 except for the (S)-IBTM
residue, which required a second coupling with 6a (0.33 mmol), HATU
(0.33 mmol), and DIEA (0.66 mmol) during 60 min for complete
acylation. The fully protected linear sequence (50 µmol, 150 mg of
resin) was cleaved from the resin and Boc-deprotected at the NR function
by acidolysis in anhydrous HF-anisole (9:1 v/v) for 1 h at 0 °C. After
evaporation, the residue was triturated with anhydrous ethyl ether,
dissolved in glacial HOAc, and lyophilized to give 37 µmol (74% yield)
of the bis-Fmoc protected linear precursor 3a. Prior to cyclization, 18
µmol of this peptide was lyophilized several times from H2O to remove
traces of HOAc and dried under vacuum over P2O5 and KOH overnight.
The dried, HOAc-free peptide was dissolved in dry DMF (45 µM final
concentration) under nitrogen, then treated with DIEA (0.36 mmol, 20
equiv) and BOP (0.18 mmol, 10 equiv) under mild stirring at 25 °C.
Once HPLC analysis showed the cyclization to be complete (ca. 6 h)
the reaction mixture was concentrated to 20 mL and treated with 100
mL of piperidine-DMF (1:4 v/v) for 2 h at 25 °C. Evaporation gave
an oil that was redissolved in glacial HOAc, lyophilized, and purified
by reverse-phase chromatography (ODS, 2 × 25 cm, 15-20 µm) with
use of a 10-70% linear gradient of MeCN into H2O over 4 h at 3
mL/min. Fractions judged to be homogeneous by HPLC were pooled
to give 4a (4 µmol; cyclization and purification yield 22%), with the
expected amino acid composition and molecular mass (MALDI-TOF;
calculated average molecular mass for C63H93N13O10 1192.52; positive
spectrum, m/z 1194.4 [MH+]).
Figure 6. Selected regions of NOESY spectra of [(R)-IBTM4,5Lys2,2′]-
GS (4b) in DMSO at 20 °C. Nonsequential NOEs are boxed and
labeled. The sequential assignment for most residues can also be
followed through CRH-NH(i,i+1) NOEs (lower regions) and through
CâH-NH(i,i+1), CγH-NH(i,i+1), and CδH-NH(i,i+1) NOEs (upper
regions). Intraresidue crosspeaks at the upper regions are not labeled.
phase synthesis, using both Boc and Fmoc chemistries. From
a structural point of view, it is interesting to note the presence
of an indole moiety in (R)-IBTM which can be considered as
the side chain of the i + 2 residue of the â-turn mimetic. This
fact could represent an advantage for mimicking â-turns with
aromatic or hydrophobic amino acids at this position, since most
of the dipeptide mimetics reported so far lack substituents for
the corner residue side chains. Finally, one can easily envisage
the extension of the solid-phase-mediated cyclization scheme
used in this work to other cyclic peptidomimetics.
Experimental Section
General. Full details on the synthesis and characterization of (R)-
and (S)-IBTM amino acids and their Boc derivatives 6a,b have appeared
previously.13 Conversion of the zwitterion H-(R)-IBTM-OH into its
Fmoc (7b) derivative is described below. Boc-protected amino acids,
BOP, TBTU, and MBHA resin were purchased from Novabiochem
(La¨ufelfingen, Switzerland). Boc-Pro-OCH2-Pam resin (0.73 mmol/
g) was from Applied Biosystems (Foster City, CA). Fmoc-protected
amino acids and HATU, HOAt, and PyAOP coupling reagents were
from PerSeptive Biosystems (Framingham, MA). All other chemicals
(26) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.

EVidence for IBTM as a Suitable Type II′ â-Turn Mimetic
J. Am. Chem. Soc., Vol. 119, No. 44, 1997 10585
Fmoc-(R)-IBTM-OH (7b). To a suspension of the zwitterion H-(R)-
IBTM-OH (400 mg, 1.4 mmol) in 10% NaHCO3-dioxane-DMSO
(20:5:1 v/v/v, 26 mL) was added Fmoc-Cl (541 mg, 2.1 mmol) in three
portions over a 30-min period, and the mixture was stirred for another
3.5 h at 25 °C. The pH was maintained at 9 throughout this period by
addition of 10% NaHCO3 as required. The reaction mixture was chilled
in an ice bath for 20 min and filtered, and the filtrate was extracted
with hexane (3 × 150 mL) and acidified to pH 3 with 1 M KHSO4,
whereupon 7b separated as an off-white solid (370 mg, yield 52%)
that was not recrystallized, given its insolubility in most solvents except
DMSO and DMF. The purity of the material was judged to be adequate
for peptide synthesis (>95%) by analytical HPLC (see above) with
use of a 0-100% MeCN (+0.036% TFA) into H2O (+0.045% TFA)
gradient over 30 min at 1 mL/min (retention time 23.5 min). 13C NMR
(50 MHz, CD3SOCD3) δ 172.4 (COOH), 171.8 (CON), 141.0 (Car),
136.5 (Car), 132.8 (Car), 127.6 (Car-H), 127.0 (Car-H), 126.6 (Car-H),
125.0 (Car-H), 121.3 (Car-H), 119.9 (Car-H), 118.8 (Car-H), 117.8 (Car-
H), 111.0 (Car-H), 104.6 (Car), 66.2 (CH2), 52.0 (CH), 50.6 (CH), 50.3
(CH), 46.8 (CH), 32.4 (CH2), 23.5 (CH2). Mass spectroscopy (FAB,
“magic bullet”, dithioerythritol-dithiothreitol, 3:2 v/v): calculated
monoisotopic mass of C30H26N3O5 507.86, positive spectrum, m/z 508.1
[MH+].
Fmoc-Orn-OAl, Trifluoroacetate Salt. A solution of Fmoc-
Orn(Boc)-OH (4 g, 8.8 mmol) in MeCN (15 mL) was treated with
allyl bromide (20 mL, 231 mmol) and DIEA (2.9 mL, 17 mmol) for 5
h at 40 °C, with magnetic stirring. The reaction mixture was diluted
with ethyl acetate (200 mL) and washed with 0.1 N HCl (4 × 125
mL), 10% NaHCO3 (4 × 125 mL), and saturated NaCl (4 × 125 mL),
and the organic phase was dried over MgSO4 and evaporated to give
Fmoc-Orn(Boc)-OAl as a white solid in quantitative yield. To this
intermediate was added dropwise 100 mL of TFA-DCM (1:1 v/v), and
the solution was stirred for 2 h at 25 °C. Solvent removal followed by
repeated evaporations from ethyl ether provided Fmoc-Orn-OAl as a
clear oil (yield 4.1 g, 92%; Rf 0.56, CHCl3/MeOH/HOAc, 75:25:2),
which was used without further purification. 1H NMR (200 MHz,
CD3SOCD3) δ 7.28-7.92 (m, 9H), 5.85 (m, 1H), 5.29 (dd, J1 ) 1.3
Hz, J2 ) 17.4 Hz, 1H), 5.19 (dd, J1 ) 1.3 Hz, J2 ) 10.4 Hz, 1H), 4.58
(d, J ) 5.1 Hz, 2H), 4.29 (t, J ) 15.1 Hz, 1H), 4.28 (d, J ) 15.1 Hz,
2H), 4.06 (m, 1H), 2.79 (m, 2H), 1.50-1.80 (m, 4H). Mass
spectroscopy (electrospray): calculated monoisotopic mass of C23H26N2O4
394.19, positive spectrum, m/z 395.1 [MH+].
Synthesis and Purification of Gramicidin S (1). Polymer (0.5 g)
loaded with Fmoc-Orn-OAl (ca. 0.22 mmol Orn) was submitted to one
cycle of Fmoc solid-phase synthesis for each residue of the sequence,
as follows: (i) deprotection with piperidine-DMF (1:4 v/v, 2 × 1 min
+ 1 × 10 min); (ii) DMF washes (5 × 0.5 min); (iii) coupling of Fmoc
amino acid (4 equiv) in the presence of TBTU (4 equiv) and DIEA (8
equiv) in DMF for 60 min; and (iv) DMF washes (5 × 0.5 min).
Couplings were monitored for completion by the Kaiser26 or chloranil27
tests; all residues were satisfactorily incorporated after the first coupling.
Once the target sequence, Fmoc-Leu-D-Phe-Pro-Val-Orn(Boc)-Leu-D-
Phe-Pro-Val-Orn(OAl)-polymer, was assembled, the peptide-resin (0.28
mmol/g, 160 mg, 44 µmol) was suspended on 4 mL of DMSO-THF-
0.5 M HCl-morpholine (2:2:1:0.1 v/v) and treated with Pd(PPh3)4 (0.44
mmol, 10 equiv) for 2.5 h under Ar. The resin was then washed with
THF (3 × 2 min), DMF (3 × 2 min), DCM (3 × 2 min), DIEA-
DCM (1:19) (3 × 2 min), DCM (3 × 2 min), sodium diethyldithio-
carbamate-DMF (5 g/L; 3 × 15 min), DMF (5 × 2 min), and DCM
(3 × 2 min). Following N-terminal deprotection with piperidine-DMF
(as above), cyclization was performed with PyAOP (229 mg, 0.44
mmol, 10 equiv), HOAt (60 mg, 10 equiv), and DIEA (153 µL, 20
equiv) in dry DMF for 2 h at 25 °C, after which ninhydrin analysis
showed no remaining free amino groups. Acidolysis with TFA-H2O
(19:1 v/v, 2 h, 25 °C) and diethyl ether precipitation furnished 11.9
mg of crude 1, ca. 90% pure by HPLC. This material was dissolved
in 3 mL of THF-H2O (1:4 v/v), loaded onto a reverse-phase column
(ODS, 2 × 25 cm, 15-20 µm) and eluted with a linear 20-50%
gradient of THF into H2O at 1.5 mL/min. Fractions judged to be
homogeneous by HPLC were pooled to give 5.2 mg (4.55 µmol,
purification yield 47%) of purified 1, with the expected amino acid
composition and molecular mass (MALDI-TOF, calculated average
mass for C60H92N12O10 1141.46; positive spectrum, m/z 1142.5 [MH+]).
Synthesis and Purification of [Lys2,2′]Gramicidin S (2). Starting
from a resin loaded with Fmoc-Lys-OAl (ca. 0.21 mmol of Lys), the
target sequence, Fmoc-Leu-D-Phe-Pro-Val-Lys(Boc)-Leu-D-Phe-Pro-
Val-Lys(OAl)-polymer, was assembled, deprotected (N- and C-termini),
and cyclized (40 µmol scale) by a procedure analogous to the one
described above for GS (1). Acidolysis yielded 9.4 mg (8 µmol, 20%
yield) of crude product that was purified as above to give 5.1 mg (yield
54%) of 2, with the expected amino acid composition and molecular
mass (MALDI-TOF, calculated average mass for C62H96N12O10 1169.53;
positive spectrum, m/z 1170.6 [MH+]).
Fmoc-Lys-OAl, Trifluoroacetate salt. Fmoc-Lys(Boc)-OH (1 g,
2.2 mmol) was treated with allyl bromide (7 mL, 89 mmol) and DIEA
(0.77 mL, 4.4 mmol), then deprotected with TFA as described above
to give the title compound as a clear oil (yield 0.99 g, 89%; Rf 0.25,
CHCl3/MeOH/HOAc, 75:25:2), which was used without further
purification. 1H NMR (200 MHz, CD3SOCD3) δ 7.28-7.93 (m, 9H),
5.87 (m, 1H), 5.29 (dd, J1 ) 1.7 Hz, J2 ) 17.2 Hz, 1 H), 5.19 (dd, J1
) 1.7 Hz, J2 ) 10.2 Hz, 1H), 4.57 (d, J ) 5.2 Hz, 2H), 4.29 (t, J )
14.7 Hz, 1H), 4.28 (d, J ) 14.7 Hz, 2H), 4.03 (m, 1H), 2.75 (m, 2H),
1.30-1.85 (m, 6H). Mass spectroscopy (electrospray): calculated
monoisotopic mass of C24H28N2O4 408.19, positive spectrum, m/z 409.1
[MH+].
Synthesis and Purification of [(R)-IBTM4,5]GS (5b). The same
protocols described for 1 and 2 were employed to prepare the target
sequence, Fmoc-Leu-D-Phe-Pro-Val-Orn(Boc)-Leu-(R)-IBTM-Val-
Orn(OAl)-polymer. Fmoc-(R)-IBTM was activated with HATU (2
equiv) and DIEA (4 equiv) in DMF and coupled for 2 h at 25 °C.
Deprotection and cyclization of a 40 µmol resin sample furnished 12.8
mg (11 µmol, yield 27.5%) of crude 5b that was purified on reverse
phase (ODS, 2 × 25 cm, 20 µm) by means of a 50-100% linear
gradient of methanol into H2O to give 1.4 mg of pure 5 (11% yield),
with the expected amino acid composition and molecular mass
(MALDI-TOF, calculated average mass for C61H89N13O10 1164.47;
positive spectrum, m/z 1165.7 [MH+]).
Side Chain Anchoring of Fmoc-Orn-OAl and Fmoc-Lys-OAl.20
MBHA resin (0.5 g, 0.56 mmol/g) was reacted with Boc-Ala-OH (265
mg, 1.4 mmol) in the presence of equimolar amounts of DIPCDI and
HOBt in DMF. The Boc-Ala-resin (Ala serving as internal reference
amino acid22) was deprotected with TFA-DCM (2:3 v/v), neutralized
with DIEA-DCM (1:19 v/v), and loaded with the bifunctional spacer
3-(4-(hydroxymethyl)phenoxy)propionic acid (165 mg, 0.84 mmol) in
the presence of equimolar amounts of DIPCDI and HOBt in DMF.
The resulting polymer was placed under an Ar stream, reacted with
DSC (2.8 mmol, 10 equiv) and DMAP (0.28 mmol, 1 equiv) in dry
DMF, and stirred mechanically for 2 h, with periodic Ar bubbling
through the suspension. After filtration and DMF washes (5 × 0.5
min), the polymer was reacted with the trifluoroacetate salt of Fmoc-
Orn-OAl or Fmoc-Lys-OAl (2.8 mmol, 10 equiv) and DIEA (5.5 mmol,
20 equiv) in dry DMF for 4 h at 25 °C, with periodic Ar bubbling.
Amino acid analysis of the polymers after 12 N HCl-propionic acid
(1:1) hydrolysis (4 h, 150 °C) showed incorporation of Orn and Lys to
be 71% and 74%, respectively, corresponding to actual substitutions
of ca. 0.45 mmol/g in both resins.
Synthesis and Purification of [(R)-IBTM4,5,Lys2,2′]GS (4b). The
same protocol described for 5b was employed to assemble the target
sequence, Fmoc-Leu-D-Phe-Pro-Val-Lys(Boc)-Leu-(R)-IBTM-Val-
Lys(OAl)-polymer. Deprotection and cyclization of a 35 µmol resin
sample provided 13.9 mg (11.6 µmol, yield 32%) of crude that was
purified as above to give 1.2 mg of pure 4b (9% yield), with the
expected AAA and mass (MALDI-TOF, calculated average mass for
C63H93N13O10 1192.52; positive spectrum, m/z 1192.4 [MH+]).
NMR Conformational Analysis. NMR samples were about 5 mM
in H2O/D2O (9:1) or in DMSO. pH of the samples in aqueous solution
was adjusted to 3.0 by addition of minute amounts of DCl or NaOD.
pH measurements were not corrected for isotope effects. Sodium
3-trimethylsilyl(2,2,3,3-2H4)propionate (TSP) was used as internal
reference. NMR experiments were performed on a Bruker AMX-600
spectrometer. All the two-dimensional spectra were acquired in the
phase sensitive mode with the use of the time proportional phase
(27) Christensen, T. Acta Chem. Scand. 1979, 33, 760-762.

10586 J. Am. Chem. Soc., Vol. 119, No. 44, 1997
Andreu et al.
incrementation (TPPI) technique28 with presaturation of the water signal.
COSY29 and NOESY30 spectra were recorded by using standard phase-
cycling sequences. Short mixing times (200 ms) were used in the
NOESY experiments in order to avoid spin-diffusion effects. TOCSY31
spectra were acquired with use of the standard MLEV17 spinlock
sequence and a 80 ms mixing time. The size of the acquisition data
matrix was 2048 × 512 words in f2 and f1, respectively, and prior to
Fourier transformation the 2D data matrix was multiplied by a phase-
shifted square-sine bell window function in both dimensions and zero-
filled to 4096 × 1024 words. The phase shift was optimized for every
spectrum.
aqueous solution and in DMSO are given in Table 1 and those for the
other protons are available as Supporting Information.
Antibiotic Activity. Minimal inhibitory concentrations of the
peptides were determined against Bacillus subtilis ATCC 6633,
Staphylococcus aureus ATCC 6538P, Pseudomonas aeruginosa ATCC
9027, and Escherichia coli ATCC 10799, using a 100 µL inoculum of
a 2 × 105 CFU/mL log phase bacterial culture. Serial dilutions of the
peptides covering a 256-0.01 µg/mL range were prepared in 10 mM
sodium phosphate buffer, pH 7.3. After incubation at 37 °C for 20 h,
inhibition of bacterial growth was determined by turbidimetry measure-
ment with a microplate reader at 650 nm.
1
Assignment of the H NMR resonances of [(S)-IBTM4,5,Lys2,2′]GS
(4a) and [(R)-IBTM4,5,Lys2,2′]GS (4b) in both aqueous solution and
DMSO was readily performed by using the sequential assignment
method.32,25 For the connection of spin systems through sequential NOE
connectivities, the C2H and C5H IBTM protons were considered as
CRH protons. Strong CRH-CδH(i,i+1) and CRH-Cδ′H(i,i+1) NOEs
permitted the connection between D-Phe 4′ and Pro 5′ and indicated
the trans conformation of the X-Pro bond in compounds 4a and 4b.
The sequential assignment of [(R)-IBTM4,5,Lys2,2′]GS (4b) in DMSO
can be followed in the NOESY spectral region shown in Figure 6. The
δ values of the NH amide protons of compounds 4a and 4b in both
Acknowledgment. We thank Dr. Irene Ferna´ndez (Mass
Spectrometry Service, University of Barcelona) for the mass
spectra. Work at the University of Barcelona was supported
by Fondo de Investigaciones Sanitarias (94/0007) and by Centre
de Refere`ncia de Biotecnologia (Generalitat de Catalunya,
Spain). J.A. is a predoctoral fellow from the Ministerio de
Investigacio´n y Ciencia, Spain. Work at the Institute of
Medicinal Chemistry was supported by CICYT (SAF94-0705).
Work at the Institute of Structure of Matter was supported by
DGICYT (PB93-0189).
(28) Marion, D.; Wu¨thrich, K. Biochim. Biophys. Res. Commun. 1983,
113, 967-974.
Supporting Information Available: Tables of 1H chemical
shifts of CRH and side chain protons of 4a and 4b (2 pages).
See any current masthead page for ordering and Internet access
instructions.
(29) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64,
2229-2246.
(30) Kumar, A.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res.
Commun. 1980, 95, 1-6.
(31) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360.
(32) Wu¨thrich, K.; Billeter, M.; Braun, W. J. Mol. Biol., 1984, 180, 715-
740.
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