Solid-phase guanidinylation as a diversification strategy of poly-L-proline type II peptide mimic scaffolds.

Article abstract of DOI:10.1021/ol0069711

[reaction: see text] Solid-phase guanidinylation of proline-templated amino acids is studied as a diversification strategy of poly-L-proline type II scaffolds.

Full text of DOI:10.1021/ol0069711

ORGANIC
LETTERS
2001
Vol. 3, No. 4
561-564
Solid-Phase Guanidinylation as a
Diversification Strategy of Poly-L-proline
Type II Peptide Mimic Scaffolds
Ahmed Mamai and Jose S. Madalengoitia*
Department of Chemistry, UniVersity of Vermont, Burlington, Vermont 05405
jmadalen@zoo.uVm.edu
Received December 6, 2000
ABSTRACT
Solid-phase guanidinylation of proline-templated amino acids is studied as a diversification strategy of poly-L-proline type II scaffolds.
The poly-L-proline type II (PPII) secondary structure is
uniquely characterized by an extended left-handed helical
fold with φ ≈ -75° and ψ ≈ 145°. In the past decade, PPII
helices have been found to mediate a number of cellular
signaling pathways. For example, SH2 domains, SH3
domains, MHC class II proteins and other proteins bind
peptide ligands in the PPII conformation or in a similar
geometry.1,2 Although PPII helices derive their name from
the conformation of polyproline, short spans of PPII helices
Figure 1. PPII mimic design strategy.
in globular proteins are often composed of amino acids other
than proline. Because it is often these nonprolyl amino acids
that are critical for recognition of the PPII helix by a receptor,
we are involved in developing a program that both mimics
the PPII three-dimensional structure and incorporates the
critical nonprolyl functionality. Our strategy for mimicking
this secondary structure involves first the synthesis of what
we call proline-templated amino acids (PTAAs) and then
the synthesis of peptides from PTAAs (Figure 1).3 Oligo-
PTAAs are designed to adopt the PPII conformation in
solution since (1) φ ≈ -75° as a result of the constraints of
the PTAA pyrrolidine ring, (2) ψ is constrained to ∼145°
by pseudo-A(1,3) strain between adjacent PTAAs, and (3)
the trans amide bond conformation is favored. Our design
strategy is validated by solution studies showing that a unit
as small as a PTAA dimer is conformationally constrained
to the φ/ψ angles of a PPII helix.3 OligoPTAAs therefore
provide a conformationally defined scaffold that can vary
both the orientation of the nonprolyl side chains as well as
the nature of these side chains.
(1) For a review see: Siligardi, G.; Drake, A. F. Pept. Sci. 1995, 37,
281.
(2) (a) Raj, P. A.; Marcus, E.; Edgerton, M. Biochemistry 1996, 35, 4314.
(b) Lee, C.-H.; Saksela, K.; Mirza, U. A.; Chait, B. T.; Kuriyan, J. Cell
1996, 85, 931. (c) Peng, S.; Kasahara, C.; Rickles, R. J.; Schreiber, S. L.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12408. (d) Jardetzky, T. S.; Brown,
J. H.; Gorga, J. C.; Stern, L. J.; Urban, R. G.; Strominger, J. L.; Wiley, D.
C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 734. (e) Zeile, W. L.; Purich, D.
L.; Southwick, F. S. J. Cell Biol. 1996, 133, 49.
Because PTAAs are in essence amino acids, it is conceiv-
able that PPII mimic libraries could be generated from
(3) (a) Zhang, R.; Brownewell, F.; Madalengoitia, J. S. J. Am. Chem.
Soc. 1998, 120, 3894. (b) Zhang, R.; Madalengoitia, J. S. J. Org. Chem.
1999, 64, 330.
10.1021/ol0069711 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/27/2001

appropriately protected PTAAs as potential inhibitors of
signaling pathways that involve PPII recognition and binding.
One potential drawback to this goal, however, is the labor
required for the synthesis of a large diversity of these PTAAs.
For example, 10 synthetic steps are required for the synthesis
of the leucine PTAA 1, and 13 steps are required for the
synthesis of the arginine PTAA 2 (Figure 2).4 Highly
amines. The N-carbamoyl-N′-alkyl thioureas then serve as
guanyl transfer reagents in the presence of EDCI and DIEA
(Scheme 1).
The synthesis of the guanyl acceptor PTAAs was ac-
complished as shown in Schemes 2 and 3. The cyclopropyl-
Scheme 2a
Figure 2. Representative PTAAs.
a Reagents and conditions: (a) (i) LAH, THF, reflux; (ii) FmocCl
NaHCO3, dioxane/H2O, 76%; (b) H2, Pd-C, Boc2O, EtOAc, 95%;
(c) TEMPO, NaClO2, bleach CH3CN, 90%.
functionalized PTAAs that would be more desirable would
of course require more steps to make. In this paper we begin
to address how a large diversity of PPII mimics may be
generated without the need for a large diversity of PTAAs.
We propose to use the concept of a “diversifiable” PTAA,
i.e., a PTAA that can be deprotected on a solid support and
allowed to react with a number of reagents as a means of
diversification without the need for the solution synthesis
of all PTAAs that would eventually be generated. This
strategy could, of course, take many forms depending on
the diversification reaction chosen. Since several receptor-
bound PPII helices have been shown to incorporate arginine
residues,1 we begin by exploring the solid-phase guanidi-
nylation of two proline-templated ornithines (6 and 9) as the
means of diversification. The two ornithine analogues are
chosen to highlight that the diversity of the library can be
increased through the use of PTAAs with different side chain
orientations.
substituted PTAA 6 was synthesized from tricycle 3, which
we can make in multigram quantities.4 LAH in refluxing THF
served to reduce both amides as well as the oxazolidine ring.
Scheme 3a
a Reagents and conditions: (a) FmocCl, NaHCO3, dioxane/H2O
(70%).
A number of guanidinylation protocols have been de-
scribed in the literature.5 Of these, we decided to explore
the procedure recently described by Hamilton because it
appeared to offer an expedient and easy method for the
introduction of structural diversity (Scheme 1).5c Hamilton’s
The crude amine was then protected with FmocCl in a
saturated aqueous NaHCO3/dioxane mixture to afford 4 in
76% yield for both steps. N-debenzylation in the presence
of Boc2O afforded the N-Boc prolinol 5 in 95% yield.
Oxidation of the primary alcohol to the carboxylic acid was
accomplished with TEMPO/NaClO2/bleach to give the
PTAA 6 suitably protected for these studies.6 The second
Scheme 1
(5) (a) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org.
Chem. 1998, 63, 3804. (b) Feichtinger, K.; Sings, H. L.; Baker, T. J.;
Matthews, K.; Goodman, M. J. Org. Chem. 1998, 63, 8432. (c) Linton, B.
R.; Carr, A. J.; Orner, B. P.; Hamilton, A. D. J. Org. Chem. 2000, 65,
1566. (d) Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677. (e)
Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett. 1993, 34,
3389, (f) Poss, M. A. Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z. Tetrahedron
Lett. 1992, 33, 5933. (g) Yong, Y. F.; Kowalski, J. A.; Lipton, M. A. J.
Org. Chem. 1997, 62, 1540. (h) Robinson, S.; Roskamp, E. J. Tetrahedron
1997, 53, 6697. (i) Dodd, D. S.; Wallace, O. B. Tetrahedron Lett. 1998,
39, 5701. (j) Wilson, L. J.; Klopfenstein, S. R.; Li, M. Tetrahedron Lett.
1999, 40, 3999. (k) Dodd, D. S.; Kozikowski, A. P. Tetrahedron Lett. 1994,
35, 977.
method generates a number of N-carbamoyl-N′-alkyl thio-
ureas by reaction of N-carbamoyl isothiocyanates with
(4) Zhang, R.; Mamai, A.; Madalengoitia, J. S. J. Org. Chem. 1999, 64,
547.
562
Org. Lett., Vol. 3, No. 4, 2001

Figure 3. Reagents and conditions: (a) 20% piperidine/DMF; (b) 1.3 equiv of 6 or 9, DIC, HOBt; (c) 20% piperidine/DMF; (d) NH-
(CO2Et)CdSNHR, EDCI, DIEA; (e) 50% TFA/CH2Cl2.
PTAA 9 was obtained by the simple Fmoc-protection of the
known PTAA 8.7
substitution was chosen to reflect a number of different
properties (hydrophobicity, conformational flexibility, con-
formational restriction, etc.) An advantage of using an
N-carbamoyl thiourea is that since the guanyl group is
transferred already protected, the progress of the guanidi-
nylation reaction can monitored by a standard Kaiser or
TNBS resin test. Typically, the reactions showed a negative
Kaiser test after 24 h, but to ensure full couplings without
large excesses of N-carbamoyl-N′-alkyl thioureas, the reac-
tions were allowed to proceed for 40 h. TFA treatment of
the resin resulted in Boc-deprotection and liberation of the
dimer from the resin. We wanted to determine how cleanly
this sequence of steps proceeded, so the purity of the peptides
was examined after trituration with Et2O.8 Most of these
compounds were obtained in >90% purity as determined
Our initial studies were designed to probe the feasibility
of using the guanidinylation reaction as a means of easily
and cleanly affording the guanidine-possessing products. For
these studies, the Boc-PTAA(Fmoc)-OH amino acids were
initially coupled to H-Gly-Wang resin. Because the synthesis
of the these PTAAs still requires a number of steps, we
explored the use of less than the standard 3-5 equiv of amino
acid in the coupling reaction in order to reduce the amount
of PTAA wasted. We are pleased to report that the reaction
proceeded to completion with 1.3 equiv of Boc-PTAA-
(Fmoc)-OH under standard conditions (diisopropylcarbodi-
imide/HOBt). Side chain deprotection was then accomplished
with 20% piperidine in DMF. The guanidinylation reaction
was next explored with a number of different thioureas.
Figure 3 highlights the flexibility and ease with which a
diversity of groups can be introduced. The guanidine
1
by integration of the crude H NMR spectra. The only
compound not obtained in >90% purity was 20.9 As evidence
of purity, crude spectra are included in Supporting Informa-
tion.
(6) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tsachen, D. M.; Grabowski,
E. J. J.; Reider, P. J. J. Org. Chem. 1999, 64, 2564.
(7) Webb, T. R.; Eigenbrot, C. J. Org. Chem. 1991, 56, 3009.
(8) The yield of peptide 12 is without trituration because it was found
to be ether-soluble.
Org. Lett., Vol. 3, No. 4, 2001
563

Scheme 4a
a Reagents and conditions: (a) 1.3 eq 6, DIC, HOAt; (b) 20% piperidine/DMF; (c) NH(CO2Et)CdSNH(cyclopentyl), EDCI, DIEA; (d)
(i) 50% TFA/CH2Cl2; (ii) 30% DIEA/DMF; (iii) 1.3 eq 9, DIC, HOAt; (e) NH(CO2Et)CdSNHR (R ) t-Bu, or R ) p-C6H5Me), EDCI,
DIEA; (f) TMSOTf, TFA, thioanisole, 0 °C.
1
the H NMR spectra revealed that peptide 28 had lost the
tert-butyl group during cleavage and deprotection but was
otherwise remarkably clean. As a result of the inherent
“greasy” character of the peptides,11 the major impurity
As proof of principle that the chemistry could be used in
iterative cycles to diversify PPII scaffolds, we synthesized
two model peptides: PTAA-PTAA-Gln-Ala-Ile (28) and
PTAA-PTAA-Ala-Ala-Ile (29) on PAM resin. These model
pentapeptides are based on the sequence Arg-Arg-Asn-Ala-
Ile from the cAMP-dependent protein kinase (PKA) inhibitor
PKI that has been shown to bind to the PKA active site in
the PPII conformation. The synthesis strategy is shown in
Scheme 4. The second and third amino acids were incorpo-
rated using standard conditions (DIC/HOBt). PTAA 6 was
then introduced, and the side chain was deprotected (20%
piperidine/DMF). Guanidinylation of the side chain was
accomplished with N-ethoxycarbonyl-N′-cyclopentyl thiourea/
EDCI. The N-terminus was then deprotected (TFA), and
PTAA 9 was coupled. Side chain deprotection (20% pip-
eridine/DMF) and guanidinylation of the resultant amine with
either N-ethoxycarbonyl-N′-tert-butyl thiourea or N-ethoxy-
carbonyl-N′-p-toluidine thiourea completed the solid-phase
synthesis. Cleavage and deprotection was accomplished with
TMSOTf/TFA/thioanisole.10 Peptides were then precipitated
with ether and analyzed by 1H NMR and MS. Inspection of
1
detected in the crude H NMR spectra was thioanisole and
thioanisole byproducts. Crude spectra are included in Sup-
porting Information.
In conclusion, we have shown a novel strategy for the
synthesis of PPII mimics that should be easily applicable to
the synthesis of PPII mimic libraries.
Acknowledgment. Financial Support for this work was
provided by CHE 9870003 from the NSF.
Supporting Information Available: Experimental pro-
cedures and characterization data for 4, 5, 6, and 9 and copies
of crude 1H NMR spectra of 10-28. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0069711
(10) (a) Yajima, H.; Fujii, N.; Funakoshi, N.; Watanabe, T.; Murayama,
E.; Otaka, A. Tetrahedron 1988, 44, 805. (b) Hughes, J. L.; Leopold, E. J.
Tetrahedron Lett. 1993, 34, 7713.
(9) The synthesis of 20 was repeated, but 20 was still obtained in lower
purity than the other compounds.
(11) One of the referees asked for an estimate of the aqueous solubility
of the pentapeptides (solubility > 30 mg/mL H2O).
564
Org. Lett., Vol. 3, No. 4, 2001