Strategies for the solid-phase diversification of poly-L-proline-type II peptide mimic scaffolds and peptide scaffolds through guanidinylation

Article abstract of DOI:10.1021/jo8012258

(Chemical Equation Presented) A strategy for the solid-phase diversification of PPII mimic scaffolds through guanidinylation is presented. The approach involves the synthesis N-Pmc-N′-alkyl thioureas as diversification reagents. Analogues of Fmoc-Orn(Mtt)-OH can be incorporated into a growing peptide chain on Wang resin. Side chain deprotection with 1% TFA/CH₂Cl₂ followed by EDCI-mediated reaction of N-Pmc-N′-alkyl thioureas with the side chain amine affords arginine analogues with modified guanidine head groups. The scope, limitations, and incidental chemistry are discussed.

Full text of DOI:10.1021/jo8012258

Strategies for the Solid-Phase Diversification of Poly-L-proline-Type
II Peptide Mimic Scaffolds and Peptide Scaffolds Through
Guanidinylation
Stevenson Flemer, Alexander Wurthmann, Ahmed Mamai, and Jose´ S. Madalengoitia*
Department of Chemistry, UniVersity of Vermont, Burlington, Vermont 05405
jose.madalengoitia@uVm.edu
ReceiVed June 6, 2008
A strategy for the solid-phase diversification of PPII mimic scaffolds through guanidinylation is presented.
The approach involves the synthesis N-Pmc-N′-alkyl thioureas as diversification reagents. Analogues of
Fmoc-Orn(Mtt)-OH can be incorporated into a growing peptide chain on Wang resin. Side chain
deprotection with 1% TFA/CH2Cl2 followed by EDCI-mediated reaction of N-Pmc-N′-alkyl thioureas
with the side chain amine affords arginine analogues with modified guanidine head groups. The scope,
limitations, and incidental chemistry are discussed.
Introduction
Within the past 15 years, poly-L-proline type II (PPII) helices
have been recognized as playing an essential role in multiple
cellular signaling pathways. For example, SH3 domains, some
SH2 domains, WW domains, EVH1 domains, MHC class II
proteins, as well as other proteins bind peptide ligands in the
PPII conformation or similar extended conformations.1 PPII
helices are predominantly located on the surface of proteins and
are characterized by an extended structure that makes them well
suited for mediating protein-protein interactions.2 Our interest
in the development of PPII mimics stems from their potential
utility in unraveling complex signaling cascades that involve
PPII recognition and binding.
Interestingly, while the name poly-L-proline type II is derived
from the conformation of polyproline, PPII helices in globular
proteins are often composed of amino acids other than proline
and often it is these nonprolyl residues that are critical for
recognition of a PPII helix by a receptor.1 Therefore, a PPII
mimic must not only adopt the peptide backbone conformation
of polyproline, it must also be able to incorporate the nonprolyl
side chain functionality required for receptor binding. Our
FIGURE 1. Design of PPII mimics from PTAAs.
strategy for mimicking the PPII secondary structure involves
first the synthesis of proline-templated amino acids (PTAAs)
in which the proline pyrrolidine ring serves as a template for
the nonprolyl amino acid side chains (Figure 1). The synthesis
of PTAA oligomers from PTAAs then affords peptides that
populate the PPII conformation in solution and possess the
* Author to whom correspondence should be addressed.
(1) (a) Kay, B. K.; Williamson, M. P.; Sudol, M. FASEB 2000, 14, 231. (b)
Siligardi, G.; Drake, A. F. Peptide Sci. 1995, 37, 281. (c) Sudol, M.; Sliwa, K.;
Russo, T. FEBS Lett. 2001, 490, 190. (d) Ball, L. J.; Jarchau, T.; Oshkinat, H.;
Walter, U. FEBS Lett. 2002, 513, 45.
(2) Adzhubei, A. A.; Sternberg, M. J. E. J. Mol. Biol. 1993, 229, 472.
10.1021/jo8012258 CCC: $40.75
Published on Web 08/28/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7593–7602 7593

Flemer et al.
most diverse collection of arginine analogues yet assembled with
which to study bioactive peptides such as the ones noted above
that possess critical arginine residue(s).
Results and Discussion
The development of a method for the diversification of PPII
mimic scaffolds through iterative guanidinylation reactions must
take careful account of a suitable protecting group strategy. As
such, we envisioned that PTAAs possessing Fmoc-N-terminal
protection and side chain Mtt (methyltrityl) protection (10)
would be compatible with Fmoc solid-phase peptide synthesis.
After incorporation of the PTAA into a growing peptide chain,
the Mtt group can be removed orthogonally with 1% TFA/
CH2Cl2 without cleavage of the peptide from Wang resin.7 Next,
we envisioned that guanidinylation of the deprotected residue
could then be accomplished with an N-alkyl-N′-Pmc thiourea
(10) and EDCI similarly to the strategy described in Scheme 1.
Although several guanidinylation protocols have been described
in the literature,8 we selected the development of N-alkyl-N′-
sulfonyl9 thioureas as guanyl-transfer agents as they would offer
a flexible and expedient diversification method. Although our
initial studies had employed a carbamoyl guanidine-protecting
group, an advantage of sulfonyl-based protecting groups (such
as Pmc) is that they sufficiently deactivate the nucleophilicity
of the guanidine group allowing clean extension of a peptide.
An additional advantage to the Pmc group is that it is stable to
Mtt deprotection conditions (1% TFA/CH2Cl2), but can be
removed with TFA-based cleavage cocktails. Thus, after
guanidinylation of a resin-bound ornithine PTAA with an
N-Pmc-N′-alkyl thiourea 9, Fmoc deprotection of the N-terminus
may then proceed followed by coupling of another Fmoc-
PTAA(Mtt)-OH residue (Scheme 2). Mtt deprotection of this
residue will then set up another round of diversification. This
strategy should therefore be adaptable to iterative rounds of
PTAA coupling and diversification affording large and complex
libraries.
Synthesis of N-Alkyl-N′-Pmc Thioureas. The synthesis of
the N-alkyl-N′-Pmc thioureas was accomplished through a
modification of the classical conditions for the synthesis of
N-sulfonyl isothiocyanates as has been previously disclosed
(Scheme 3).10,11 Commercially available PmcCl (14) was treated
with ammonia in CH2Cl2 to give the corresponding sulfonamide
15 in quantitative yield. Sulfonamide 15 was then allowed to
react with KOH and CS2 in a CS2/benzene mixture with
azeotropic removal of water to afford the intermediate 16, using
a modification of the procedure described by Barton.10 Treat-
ment of this mixture with phosgene then afforded the critical
Pmc-isothiocyanate 17 in 75% yield. Without the azeotropic
removal of water, the yield for this transformation averaged
FIGURE 2. Representative PTAAs.
nonprolyl functionality necessary for recognition by the receptor.
This design strategy has been validated by solution studies that
confirm that oligoPTAAs do indeed populate the PPII confor-
mation in water as well as organic solvents.3
Since PTAAs are essentially amino acids, it is possible that
PPII mimic libraries could be constructed from a diversity of
PTAAs through the strategies already developed for the
synthesis of peptide libraries. One potential drawback to this
goal, however, is the labor involved in the synthesis of the
PTAAs. For example, the synthesis of PTAAs 1-3 requires 11
to 13 linear steps (Figure 2).3a,b,4 More functionalized PTAAs
by necessity require more steps to synthesize. Thus, the assembly
of a large collection of PTAAs can involve a significant synthetic
effort. To minimize the labor involved in the synthesis of PPII
mimic libraries, we have introduced the concept of a “diversi-
fiable” PTAA, a PTAA that can be selectively deprotected on
a solid support and then diversified through an appropriate
reaction. Since arginine is critical to many PPII helix-receptor
interactions, our initial efforts have been directed toward
developing methods for the facile synthesis of arginine analogues.
In our first communication on this approach, we reported the
results of our initial studies on the diversification of proline-
templated ornithines through a guanidinylation reaction that then
affords arginine analogues.5 The strategy involved the incor-
poration of Boc-PTAA(Fmoc)-OH (4) into a growing peptide,
followed by deprotection of the side chain Fmoc group (Scheme
1). The side chain amine was then guanidinylated with N-alkyl-
N′-ethoxycarbonyl thioureas (RNHCdSNHCO2Et) in a reaction
promoted by EDCI. After cleavage from the solid support, the
peptides 8 were obtained in good yield and purity. These studies
showed that the chemistry was amenable to solid-phase diver-
sification, proceeded in good yield, and was open to the
incorporation of a large number of guanidine substituents.
However, the chemistry was not compatible with Fmoc solid-
phase peptide synthesis and the guanidine groups possessed a
protecting group (CO2Et) that is not easily removed. In this full
account of our work, we report the establishment of a general
strategy for the diversification of PPII mimic scaffolds through
a guanidinylation reaction that is amenable to Fmoc solid-phase
peptide synthesis and affords peptides that possess fully
deprotected guanidine groups. Furthermore, the chemistry is
equally adaptable to proline-templated ornithines as well as
ornithine itself. It is worth noting that arginine is a ubiquitous
residue that is critical to the bioactivity of numerous peptides
such as cell-penetrating peptides, HIV-Tar RNA binding pep-
tides, amphiphilic antibacterial peptides, RGD peptides, etc.6
The chemistry developed in this program allows access to the
(6) (a) Austin, R. J.; Xia, T.; Ren, J.; Takahashi, T. T.; Roberts, R. W. J. Am.
Chem. Soc. 2002, 124, 10966. (b) Ziegler, A.; Seelig, J. Biochemistry 2007, 46,
8138. (c) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.;
Steinman, L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003–8.
(d) Raguse, T. L.; Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem.
Soc. 2002, 124, 12774.
(7) Aletras, A.; Barlos, K.; Gatos, D.; Koutsogiani, S.; Mamos, P. Int. J.
Peptide Protein Res. 1995, 45, 488.
(8) (a) Powell, D. A; Ramsden, P. D; Batey, R. A. J. Org. Chem. 2003, 68,
2300. (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.
(9) (a) Zhang, Z.; Pickens, J. C.; Hol, W. G. J.; Fan, E. Org. Lett. 2004, 6,
1377. (b) Zhang, Z.; Fan, E. J. Org. Chem. 2005, 70, 8801.
(3) (a) Zhang, R.; Brownewell, F. E.; Madalengoitia, J. S. J. Am. Chem.
Soc. 1998, 120, 3894. (b) Zhang, R.; Madalengoitia, J. S. J. Org. Chem. 1999,
64, 330. (c) Mamai, A.; Zhang, R.; Natarajan, A.; Madalengoitia, J. S. J. Org.
Chem. 2001, 66, 455.
(4) Mamai, A.; Hughes, N. E.; Wurthmann, A.; Madalengoitia, J. S. J. Org.
Chem. 2001, 66, 6483.
(5) Mamai, A.; Madalengoitia, J. S. Org. Lett. 2001, 3, 561.
(10) Barton, D. H. R.; Fontana, G.; Yang, Y. Tetrahedron 1996, 52, 2705.
(11) Flemer, S.; Madalengoitia, J. S. Synthesis 2007, 13, 81.
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Solid-Phase DiVersification of PPII Mimic Scaffolds
SCHEME 1
TABLE 1. Reaction of N-Pmc Isothiocyante with Amines
SCHEME 2
SCHEME 3a
Pmc-isothiocyanate. We have found, however, that the crude
N-Pmc-isothiocyanate is homogeneous enough to be used
without further purification. The final step of the synthesis
involves reaction of N-Pmc-isothiocyanate (17) with various
amines to afford N-alkyl-N′-Pmc thioureas 18a-p in good to
excellent yields (Table 1).11
Synthesis of PTAAs. To increase the spatial diversity of PPII
mimic libraries, the synthesis of PTAAs with different side chain
orientations was desirable. Accordingly, the synthesis of several
differential substituted, appropriately protected PTAAs was
undertaken for these studies. The synthesis of PTAA 24 based
on the 3-azabicyclo[3.1.0]hexane system begins with the tricycle
19, available in multigram quantities from pyroglutamic acid
a Reagents and conditions: (a) NH3, CH2Cl2 (100%); (b) KOH, 1:4 CS2/
benzene, D.S. trap; (c) phosgene, toluene, 0 °C (75%); (d) RNH2, CH2Cl2
(58-100%).
∼54%. The TLC of the isothiocyanate revealed the presence
of a small amount of an impurity, most likely a dimer since
N-sulfonyl isothiocyanates are known to dimerize.12 An analyti-
cal sample of Pmc-isothiocyanate could be obtained by flash
chromatography on silica gel, but is significantly decreased,
which is most likely due to decomposition of the highly reactive
(13) Zhang, R.; Mamai, A.; Madalengoitia, J. S. J. Org. Chem. 1999, 64,
547.
(12) Dickore, K.; Kuhle, E. Angew. Chem., Int. Ed. 1965, 4, 430.
J. Org. Chem. Vol. 73, No. 19, 2008 7595

Flemer et al.
SCHEME 4a
SCHEME 5a
a Reagents and conditions: (a) TFA; (b) MttCl, Et3N; (c) MeOH, 50
° C.
SCHEME 6a
a Reagents and conditions: (a) LAH, THF reflux; (b) Boc2O (68% from
19); (c) (i) H2, Pd-C, (ii) FmocCl (44%); (d) TEMPO, NaClO2, NaOCl
(100%); (e) (i) TFA, (ii) MttCl, Et3N, (iii) MeOH, 55 °C (65%).
a Reagents and conditions: (a) (i) TFA, (ii) FmocCl (98%); (b) (i) H2,
Pd-C, (ii) H2, PtO2 (80%); (c) (i) MttCl, Et3N, (ii) MeOH, 50 ° C (45%).
(Scheme 4).13 Reduction of the two amides and oxazolidine
functions with LAH in THF at reflux afforded the bicycle 20.13
The resulting primary amine was subsequently protected with
Boc2O giving carbamate 21 in 68% yield from the primary
amide 19. N-Debenzylation of the tertiary amine was ac-
complished via hydrogenolysis in H2 atmosphere over Pd-C.
Reprotection of the resultant secondary amine with FmocCl in
dioxane/saturated aqueous NaHCO3 then gave the carbamate
22 in 44% yield over two steps. The primary alcohol was then
smoothly oxidized to the carboxylic acid 23 with TEMPO,
NaClO2, and bleach in quantitative yield.14 All that then
remained to complete the synthesis of PTAA 24 was Boc-
deprotection of the side chain nitrogen and reprotection of the
resultant primary amine with an Mtt group. Boc deprotection
of 23 was accomplished with neat TFA. The resulting TFA salt
was then allowed to react with excess MttCl and Et3N thus
protecting both the carboxylate and amine groups. To obtain
the side chain Mtt-protected amino acid, the Mtt ester was
selectively methanolized at 50 °C affording the Mtt-protected
PTAA 24 in 65% yield from the Boc-protected PTAA 23.7
The synthesis of the 3-substituted ornithine 27 was ac-
complished from the Boc-protected PTAA 26 that is readily
synthesized from the bicyclic lactam 25 (Scheme 5).4 As above,
the side chain Boc-group was deprotected with TFA. The
resulting TFA salt was allowed to react with excess MttCl (2.5
equiv) and Et3N (3 equiv) to protect both the side chain amine
and the carboxylic acid group. Selective methanolysis of the
Mtt ester was then accomplished at 50 °C to give the PTAA 27
in 56% yield from 26.7
FmocCl in a dioxane/saturated aqueous NaHCO3 solution
affording the Fmoc carbamate 29 in 98% yield. Next, it was
surmised that the reduction of the nitrile could be accomplished
with concomitant deprotection of the benzyl ester under standard
hydrogenolysis conditions. However, when this transformation
was attempted (H2, Pd-C, even at elevated pressures >250 psi),
the product was obtained debenzylated, but with the nitrile intact.
To reduce the nitrile, the product was resubjected to hydrogena-
tion conditions, but with PtO2 as the catalyst to obtain the
primary amine in 80% yield for both steps. Finally, Mtt
protection of the side chain nitrogen was effected through the
two-step procedure involving Mtt protection of both the primary
amine and carboxylate groups followed by selective solvolysis
of the Mtt ester as above to give the PTAA 31 in 45% yield for
both steps.
The synthesis of the other 4-substituted PTAA 37 was
accomplished from the alcohol 32 derived from 4-hydroxypro-
line.16 Tosylation of the 4-position with inversion of configu-
ration was accomplished under typical Mitsunobu conditions
with p-TsOMe, Ph3P, and DEAD in 53% yield.17 Displacement
of the tosylate with NaN3 in DMF at 60 °C was easily
accomplished to afford the azide 34 in 87% yield. Next, the
R-nitrogen was Boc deprotected with TFA and Fmoc reprotected
with FmocCl in a dioxane/saturated aqueous NaHCO3 solution
to give the product 35 in 84% yield for both steps. Reduction
of the azide to the primary amine and deprotection of the benzyl
ester was accomplished with H2, and Pd-C, furnishing the
amino acid 36, which was used without further purification. Mtt
protection of the side chain nitrogen was again accomplished
through the diprotection of the amine and carboxylate groups
with MttCl (2.5 equiv) and DIPEA (3 equiv) in DMF followed
by selective methanolysis of the Mtt ester at 50 °C to give the
Fmoc-Mtt-protected PTAA 37 in 70% yield (Scheme 7).7
The synthesis of the 4-substituted PTAA 31 begins with the
known nitrile 2815 that is suitably functionalized for transforma-
tion into 31 (Scheme 6). The first issue to be addressed was
Fmoc-protection of the R-nitrogen. Accordingly, Boc-depro-
tection of the R-nitrogen was accomplished with TFA followed
by Fmoc-reprotection of the resulting secondary amine with
(14) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tsachen, D. M.; Grabowski, E. J. J.;
Reider, P. J. J. Org. Chem. 1999, 64, 2564.
(15) Webb, T. R.; Eigenbrot, C. J. Org. Chem. 1991, 56, 3009.
(16) Williams, M. A.; Rapoport, H. J. Org. Chem. 1994, 59, 3616–3625.
(17) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron 1996, 52,
15017.
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Solid-Phase DiVersification of PPII Mimic Scaffolds
SCHEME 7a
the diversification of PTAAs 27, 31, and 37 and natural ornithine
with randomly selected N-alkyl-N′-Pmc thioureas to illustrate
the versatility of the method (entries 54-62, Scheme 8).
Beginning with Fmoc-Ala-Wang resin, the amino terminus was
deprotected with 20% piperidine/DMF. Fmoc-Ala-OH was
coupled with DIC/HOBt and the N-terminus was deprotected
to give the resin bound Ala-Ala dipeptide. Next, one of the
PTAAs 24, 27, 31, 37, or Fmoc-Orn(Mtt) was coupled to the
Ala-Ala-dipeptide giving the trimer 39 suitably protected for
diversification. It is worth noting that since the synthesis of the
PTAAs 24, 27, 31, and 37 requires 11 to 13 steps from
commercially available starting materials, the couplings were
performed with 1.3 equiv of PTAA instead of the typical 3 to
5 equiv of amino acid normally used in solid-phase peptide
synthesis. In all instances, ninhydrin and TNBS tests showed
the couplings were complete within 2 h. Next, Mtt-side chain
deprotection was accomplished with 1% TFA/CH2Cl2 to give
the side chain deprotected ornithine residue 40. The Mtt
deprotection can be visually monitored since the Mtt cation is
yellow. We have found that an average of six 5-min washes
was required to obtain a colorless deprotection solution (in
agreement with a published study).18 We next explored the
guanidinylation reaction with the N-alkyl-N′-Pmc thioureas (1.5
equiv) and EDCI (4 equiv) in DMF. A key advantage of the
a Reagents and conditions: (a) p-TsOMe, PPh3, DEAD (53%); (b) NaN3,
DMF, 60 ° C (87%); (c) (i) TFA, (ii) FmocCl, NaHCO3, dioxane/H2O
(84%); (d) H2, Pd-C, MeOH (98%); (e) (i) MttCl, (i-Pr)2NEt, DMF, (ii)
MeOH, 60 ° C (70%).
Scope of the Solid-Phase Guanidinylation. To assess the
scope and limitations of the diversification strategy, we first
investigated the diversification of a single PTAA (24) with all
thioureas (18a-p) to look for trends in the reactivity of the
thioureas (entries 43-53, Scheme 8). We then explored
SCHEME 8a
a Reagents and conditions. (a) 20% piperidine/DMF; (b) Fmoc-Ala-OH, DIC, HOBt; (c) 20% piperidine/DMF; (d) 24, 27, 31, 37, or Fmoc-Orn(Mtt)-OH,
DIC, HOBt; (e) 1% TFA/CH2Cl2; (f) 18a-p, EDCI, DMF; (g) 20% piperidine/DMF; (h) TFA.
J. Org. Chem. Vol. 73, No. 19, 2008 7597

Flemer et al.
SCHEME 9
guanidinylation reaction is that since the guanyl group is
transferred Pmc-protected, a Kaiser or TNBS resin test can be
used to monitor the reaction. Accordingly, some trends in the
reactivity of the thioureas became evident (discussed in more
detail below). Most guanidinylations were complete within 2 h,
but some required as long as 12 h. To complete the synthesis
of the model peptides, the amino terminus was deprotected with
20% piperidine/DMF and the peptides were cleaved and
deprotected with 95% TFA/H2O.
Scheme 8 demonstrates the ease and flexibility with which a
wide array of arginine analogues may be incorporated into a
peptide. Yields ranged from 34% to 90% for the eight-step
sequence. It should first be noted that the diversification
chemistry works to yield arginine PTAAs based on the 3-aza
bicyclo[3.1.0]hexane system (43-53), the 4-substituted PTAAs
(54-57), the 3-substituted PTAAs (58, 59), as well as natural
ornithine (60-62). We desired to ascertain if the diversification
chemistry afforded crude compounds in enough purity for high
throughput screening. Thus, after cleavage and deprotection, the
tripeptides were triturated with Et2O and purity was determined
by HPLC. From this simple procedure, most compounds were
obtained in >85% purity. Among the tripeptides that were not
obtained in acceptable levels of purity were 45 and 55, derived
from the t-Bu-ester thiourea 18j that was designed to undergo
t-Bu deprotection under the cleavage conditions. In addition,
tripeptides 48, 49, and 60 derived from the heterocyclic thioureas
18k and 18l were not obtained in acceptable levels of purity.
That is not to say that diversification of resin-bound PTAAs
with these thioureas does not work, but rather that peptides
derived from these PTAAs would require purification prior to
screening.
most likely proceeds through the formation of the highly reactive
N-sulfonyl carbodiimide 63 (Scheme 9). In a normal course,
the carbodiimide 63 may then react with a resin-bound PTAA
40 giving the arginine analogue 65. We believe that the high
reactivity of carbodiimide 63 facilitates attack by the pyridyl
nitrogen of another thiourea (or carbodiimide) establishing an
equilibrium with the adduct 64. The effect of the competing
reaction is to lower the concentration of carbodiimide 63, thus
slowing the rate of the solid-phase guanidinylation reaction.
Other thioureas such as 18m, 18n, 18o, and 18p failed to
give a negative ninhydrin test even after prolonged reaction
times or after the use of 4 molar excess of thiourea. In the case
of thioureas 18m and 18n, we propose that the guanidinylation
reaction failed due to intramolecular cyclization pathways
(Scheme 10). Indeed, from the attempted guanidinylation of a
resin-bound PTAA with the 2-pyridyl-derived thiourea 18m,
we isolated the cyclic guanidine 69,11 which is consistent with
attack of the pyridyl nitrogen on the carbodiimide carbon.
Similarly, from the attempted guanidinylation of a resin-bound
PTAA with the ethanolamine-derived thiourea 18n, we isolated
the cyclic isourea 67,11 which is consistent with attack of the
hydroxyl-group on the carbodiimide carbon. The reaction of
hydrazine-derived thiourea 18p with a resin-bound PTAA also
failed to give a negative ninhydrin test. In this case, we
hypothesize that intramolecular attack of the carbonyl oxygen
on the carbodiimide carbon resulted in the heterocycle 71,
although we did not isolate this product. We also investigated
solid-phase guanidinylation of PTAAs with the thiourea 18o
since this would afford after cleavage and deprotection the
arginine analogues without a guanidine substituent. However,
this reaction also failed to give a negative ninhydrin test most
likely because the in situ generated carbodiimide 72 quickly
tautomerizes to the less reactive cyanamide 73. We were,
however, unable to isolate the cyanamide 73. Interestingly, the
guanidinylation of resin-bound ornithine with the highly hin-
dered 1-adamantyl thiourea 18h did proceed to completion in
8 h. However, after cleavage and deprotection, the product 75
was obtained without the adamantyl group. Apparently, the
acidic deprotection conditions promote solvolysis of the ada-
mantyl group through the intermediacy of the tertiary carboca-
As previously noted, information about the relative rates of
the guanidinylation reactions could be obtained through a
standard ninhydrin test. Most guanidinylation reactions pro-
ceeded to completion in 2 h; however, more hindered thioureas
such as the 2-adamantyl derived 18f required 8 h. One surprising
observation was that reaction of the N-benzyl-N′-Pmc thiourea
18j required 2 h to proceed to completion, while the isosteric
pyridyl derivatives 18k and 18l required at least 12 h to give a
negative ninhydrin test. Clearly, since the thioureas 18k and
18l are isosteric with 18j the pyridyl nitrogen is involved in
retarding the rate of the guanidinylation reaction. The reaction
(18) Li, D.; Elbert, D. L. J. Pept. Res. 2002, 60, 300.
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Solid-Phase DiVersification of PPII Mimic Scaffolds
SCHEME 10
SCHEME 11a
a Reagents and conditions: (a) piperidine/DMF; (b) Fmoc-Pro-OH, DIC, HOBt; (c) piperidine/DMF; (d) Fmoc-Orn(Mtt)-OH, DIC, HOBt; (e) 1% TFA/
CH2Cl2; (f) 18b, EDCI, Et3N; (g) piperidine/DMF; (h) Fmoc-Xxx-OH, DIC, HOBt; repeat three more times; (i) piperidine/DMF; (j) 31, DIC, HOBt; (k) 1%
TFA/CH2Cl2; (l) 18f, EDCI, Et3N; (m) piperidine/DMF; (n) TFA.
tion. Thus, although not originally intended to, the thiourea 18h
serves as a surrogate for thiourea 18o in affording arginine
analogues without a guanidine substituent.
To highlight the feasibility of our approach in the synthesis
of PPII combinatorial libraries through iterative rounds of PTAA
coupling and diversification, we synthesized a peptide in which
J. Org. Chem. Vol. 73, No. 19, 2008 7599

Flemer et al.
mL). After 30 min, the reaction was allowed to rise to room
temperature. The solvent was removed in vacuo, and the crude
product was purified on silica gel with 5:45:50 MeOH/EtOAc/Hex
affording 1.21 g of carbamate 21 (68% for two steps) as a colorless
solid: mp 83-86 °C; [R]25D -2.34 (c 1.0, CH3OH); 1H NMR (500
MHz, CDCl3) δ 7.19-7.33 (m, 5H), 4.72 (br s, 1H), 3.78 (d, J )
13.6 Hz, 1H), 3.68 (d, J ) 13.6 Hz, 1H), 3.50-3.61 (m, 2H), 3.15
(dd, J ) 4.6 Hz, J ) 10.0 Hz, 1H), 2.96-3.08 (m, 3H), 2.61 (d, J
) 10.0 Hz, 1H), 1.45 (s, 9H), 1.36 (m, 1H), 1.33 (dd, J ) 3.1 Hz,
J ) 7.4 Hz, 1H), 1.00 (m, 1H); 13C NMR (125 MHz, CDCl3) δ
155.8, 139.8, 128.3, 128.1, 126.9, 79.2, 67.0, 62.9, 58.0, 55.3, 42.8,
28.4, 26.6, 25.7, 23.1 ppm; IR (film) 3348, 1693 cm-1; MS (CI)
m/z 333 (MH). HRMS calcd for C19H29N2O3 [M + H]+ 333.2178,
found 333.2171.
to positions have undergone rounds of PTAA coupling and
diversification. We selected a peptide Arg-Pro-Leu-Pro-Pro-Arg-
Pro-Ala as a model based on the peptide Arg-Lys-Leu-Pro-Pro-
Arg-Pro that has been shown by the Schreiber group to bind to
the PI3K SH3 domain in the PPII conformation.19 The synthesis
begins with Fmoc-Ala-Wang resin using a primer Ala to avoid
diketopiperazine formation (Scheme 11). Fmoc deprotection and
coupling with Fmoc-Pro-OH followed by Fmoc-deprotection
and coupling with Fmoc-Orn(Mtt)-OH affords the resin-bound
tripeptide 76. Mtt deprotection is accomplished by washing with
1% TFA (6 × 5 min) to give the tripeptide 77 with the
deprotected side chain. The EDCI-mediated reaction of the trip-
eptide with N-Pmc-N′-cyclopropyl thiourea 18b afforded the
guanidine 78. Extension of the peptide through the subsequent
coupling of Pro, Pro, Leu, Pro, and PTAA 31 gave the peptide
80. Mtt deprotection of the side chain amine was accomplished
with 1% TFA (6 5 5 min) to give the deprotected amine 81.
Reaction of the amine 81 with N-Pmc-N′-(2-adamantyl) thiourea
18f gave the diguanidine 82. Finally, Fmoc-deprotection of the
amino-terminus and cleavage and deprotection with TFA gave
the fully deprotected peptide 83. HPLC analysis of the peptide
83 revealed that 83 was obtained in 96% purity.
In summary, we have devised an approach for the facile
synthesis of arginine-rich PPII mimic libraries. The strategy
involves incorporation of ornithine analogues possessing Fmoc-
NR protection and Mtt-side chain protection into a resin-bound
peptide. After incorporation of the ornithine analogues into a
growing peptide chain, the side chain can be deprotected with
1% TFA/CH2Cl2 and diversified with N-alkyl-N′-Pmc thioureas
in a reaction promoted by EDCI. After TFA-cleavage from the
solid support, the peptides are obtained in good yields and purity.
Scheme 8 highlights the vast array of arginine analogues that
can be generated through this approach. Furthermore, our
approach is suitable for the synthesis of complex libraries
through iterative rounds of coupling and diversification as
demonstrated by the synthesis of a model peptide.
(1S,2S,5S,6R)-3-Aza-3-[(fluoren-9-ylmethyl)oxycarbonyl]-6-[((tert-
butyl)oxycarbonyl)aminomethyl]bicyclo[3.1.0]hexyl-2-methanol (22).
Compound 21 (0.80 g, 3.25 mmol) was added to a 100 mL round-
bottomed flask along with 0.17 g (5 mol %) of 10% Pd/C, a
magnetic stirring bar, and 30 mL of 2:1 AcOH/EtOAc. The reaction
flask was purged with H2 and the mixture was stirred overnight
with double balloon pressure additional H2. At the end of this time,
the reaction mixture was filtered through celite, concentrated, and
taken to pH 13 with 30% aq. KOH. This aqueous solution was
then extracted with CH2Cl2 (3 × 30 mL), dried over MgSO4, and
concentrated to afford the debenzylated product as a light yellow
oil that was carried through the Fmoc-protection step without further
purification. The debenzylated amino alcohol and 0.54 g (6.50
mmol) of NaHCO3 in 1:1 dioxane/H2O (30 mL) was cooled to 0
°C and FmocCl (0.92 g, 3.57 mmol, 1.1 equiv) was added in small
portions over 1 h. The mixture was then allowed to come to room
temperature and was stirred for an additional hour. At the end of
this time, the reaction mixture was partitioned between EtOAc (100
mL) and water (100 mL), and the organic phase was separated.
The aqueous layer was re-extracted with EtOAc (2 × 50 mL), and
the organic portions were combined, dried over MgSO4, and
concentrated. The crude 22 was purified on silica gel with 1:4:5
MeOH/EtOAc/Hex affording 0.67 g of product 22 (44% for two
steps) as a colorless foam: mp 58-62 °C; [R]25 -2.8 (c 1.05,
D
1
CH3OH); H NMR (500 MHz, CD3OD) δ 7.79 (d, J ) 7.5 Hz,
2H), 7.58 (t, J ) 7.6 Hz, 2H), 7.86-7.42 (m, 2H), 7.25-7.32 (m,
2H), 4.54 (m, 1H), 4.40 (dd, J ) 5.7 Hz, J ) 10.7 Hz, 0.5H), 4.35
(dd, J ) 6.2 Hz, J ) 10.7 Hz, 0.5H), 4.20 (q, J ) 5.6 Hz, 1H),
3.80 (m, 0.5H), 3.45-3.63 (m, 2H), 3.27-3.44 (m, 2H), 3.25 (m,
Experimental Section
0.5H), 2.90-3.02 (m, 2H), 1.27-1.60 (m, 11H), 0.53 (m, 1H); 13
C
(1S,2S,5S,6R)-3-Aza-3-benzyl-6-[((tert-butyl)oxycarbonyl)amino-
methyl]bicyclo[3.1.0]hexyl-2-methanol (21). To a 14/20 100 mL
round-bottomed flask was added powdered LiAlH4 (0.31 g, 8.0
mmol) along with anhydrous THF (25 mL) and a magnetic stirring
bar. The flask was fitted with a condenser and purged with nitrogen.
The LAH slurry was stirred vigorously while 1.38 g (5.36 mmol)
of 19 (dissolved in 10 mL of anhydrous THF) was added dropwise
through a septum at the top of the condenser. During the addition
process, the slurry began to boil. At the end of the addition process,
heat was applied to the reaction mixture, and it was allowed to stir
at reflux for 1 h. At the end of this time, the heat source was
removed, and sat. aq. Na2SO4 was added dropwise until ∼10 mL
had been added. The reaction mixture was then filtered through
celite, and the solid filtered material was washed with 4 × 20 mL
of EtOAc. The organic washes were then combined with the filtered
supernatant, and the solvent was removed in vacuo. The residue
was taken up in 100 mL of EtOAc and partitioned against 100 mL
of water. The organic portion was dried over MgSO4 and the solvent
was removed in vacuo. The resulting primary amino alcohol 20
was carried through the Boc-protection sequence without purifica-
tion. A solution of di-tert-butyl dicarbonate (1.20 g, 5.51 mmol) in
CH2Cl2 (5 mL) was added dropwise to a 0 °C solution of primary
amine LAH reduction product (1.25 g, 5.40 mmol) in DCM (30
NMR (125 MHz, CDCl3) δ 156.0, 155.8, 154.8, 143.9, 143.8, 141.3,
128.3, 127.7, 127.0, 126.8, 124.9, 124.7, 119.9, 80.7, 79.4, 67.1,
66.5, 65.1, 64.0, 61.8, 60.8, 48.3, 47.9, 47.3, 42.0, 28.4, 28.2, 24.2,
23.5, 22.3, 21.8, 21.0, 20.3 ppm; IR (film) 3355, 1689 cm-1; MS
(CI) m/z 487 (M + Na). HRMS calcd for C27H32N2O5Na [M +
Na]+ 487.2209, found 487.2192.
(1S,2S,5S,6R)-3-Aza-3-[(fluoren-9-ylmethyl)oxycarbonyl]-6-[((tert-
butyl)oxycarbonyl)aminomethyl]bicyclo[3.1.0]hexane-2-carboxy-
lic Acid (23). Starting alcohol 22 (0.63 g, 1.36 mmol) was dissolved
in acetonitrile (10 mL) and added to a 50 mL round-bottomed flask
containing 0.67 M aq. NaH2PO4 buffer (10 mL) and TEMPO (15
mg, 0.08 mmol). The reaction mixture was brought to 35 °C and
5% aq. NaOCl (40 µL diluted with an additional 2 mL of H2O)
and NaClO2 (0.31 g, 2.72 mmol, dissolved in 4 mL of H2O) were
added dropwise over 30 min. The reaction mixture was then allowed
to stir overnight at 35 °C. At the end of this time, the reaction
mixture was allowed to come to room temperature and was poured
into 20 mL of ice-cold aq. sat. Na2SO3, followed by extraction with
EtOAc (4 × 20 mL). The organic portions were combined and
extracted with 3 × 15 mL sat. aq. NaHCO3. The aqueous layers
were combined and acidified at 0 °C to pH 3 with concd HCl. The
aqueous portion was extracted with 4 × 20 mL of EtOAc, and the
organic fractions were dried over MgSO4 and concentrated in vacuo
to afford the carboxylic acid 23 (100%) as a colorless foam in 0.65 g
yield: mp 95-100 °C dec; [R]25D -3.1 (c 1.0, CH3OH); 1H NMR
(19) Yu, H.; Chen, J. K.; Feng, S.; Dalgarno, D. C.; Brauer, A. W.; Schreiber,
S. L. Cell 1994, 76, 933.
7600 J. Org. Chem. Vol. 73, No. 19, 2008

Solid-Phase DiVersification of PPII Mimic Scaffolds
(500 MHz, CDCl3) δ 7.72 (dd, J ) 7.5 Hz, J ) 19.1 Hz, 2H),
7.50-7.58 (m, 2H), 7.32-7.40 (m, 2H), 7.25-7.31 (m, 2H), 4.72
(br s, 1H), 4.38-4.50 (m, 1.5H), 4.30-4.38 (m, 1.5H), 4.22 (t, J
) 6.9 Hz, 0.5H), 4.14 (t, J ) 6.5 Hz, 0.5H), 3.70 (d, J ) 10.6 Hz,
0.5H), 3.56-3.65 (m, 1.5H), 2.96-3.20 (m, 2H), 1.69 (m, 0.5H),
1.64 (m, 0.5H), 1.38-1.55 (m, 10H), 0.87 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 175.4, 175.0, 156.0, 155.5, 154.8, 143.9, 143.7,
141.2, 127.64, 127.59, 127.0, 125.0, 119.9, 81.2, 79.6, 67.5, 61.3,
60.8, 48.6, 48.2, 47.2, 42.6, 41.7, 29.6, 28.3, 25.3, 24.3, 22.4, 21.0,
20.2 ppm; IR (film) 3356, 1689 cm-1; MS (CI) m/z 501 (M +
Na). HRMS calcd for C27H30N2O6Na [M + Na]+ 501.2002, found
501.1861.
trans-4-Cyano-N-[(fluoren-9-ylmethyl)oxycarbonyl]-L-proline Ben-
zyl Ester (29). The nitrile 2815 (2.22 g, 6.72 mmol) was dissolved
in trifluoroacetic acid (40 mL) and maintained at rt for 30 min.
The TFA was removed under reduced pressure. To this residue
was added a 1:1 solution of saturated aqueous NaHCO3 (5.00 g,
59.5 mmol) and FmocCl (2.08 g, 8.04 mmol). After 12 h, the
reaction mixture was extracted with EtOAc (3 × 250 mL).
The combined fractions were dried (MgSO4) and concentrated. The
crude oil was purified by flash column chromatography eluted with
hexanes and ethyl acetate (2:1) affording the product 29 (2.98 g,
98%) as a colorless solid. Mp 52-55 °C; [R]25 -2.76 (c 0.86,
D
CH3OH); 1H NMR (500 MHz, CDCl3) δ 7.77-7.72 (m, 2H),
7.55-7.47 (m, 2H), 7.41-7.23 (m, 9H), 5.26-4.94 (m, 2H), 4.58
(d, J ) 2.5 Hz, 0.5H), 4.46-4.40 (m, 1.5H), 4.34(d, J ) 6.4 Hz,
0.5H), 4.23 (t, J ) 6.7 Hz, 0.5H), 4.00 (t, J ) 6.3 Hz, 0.5H),
3.95-3.88 (m, 1H), 3.73 (t, J ) 9.1 Hz, 0.5H), 3.64 (t, J ) 9.4
Hz, 0.5H), 3.25-3.15 (m, 1H), 2.55-2.44 (m, 1H), 2.41-2.33 (m,
1H); 13C NMR 125 MHz, CDCl3) δ 170.9, 154.0, 153.6, 143.8,
143.7, 143.5, 143.3, 141.2, 135.0, 134.9, 129.6, 128.3, 127.8, 127.1,
124.9, 120.0, 119.9, 118.6, 67.9, 67.8, 67.4, 58.2, 57.8, 49.5, 48.9,
47.0, 34.7, 33.5, 27.0, 26.2 ppm; IR (film) 3065, 2957, 2248, 1746,
1709 cm-1; HRMS calcd for C28H24N2O4Li [M + Li]+ 459.1896,
found 459.1893.
(1S,2S,5S,6R)-3-Aza-3-[(9-fluorenylmethyl)oxycarbonyl]-6-[((4-
methylphenyl)diphenylmethyl)aminomethyl]bicyclo[3.1.0]hexane-
2-carboxylic Acid (24). N(R)-Fmoc-N(δ)-Boc-protected carboxylic
acid 23 (0.62 g, 1.30 mmol) was dissolved in CH2Cl2 (4 mL) in a
50 mL round-bottomed flask, and trifluoroacetic acid (8 mL) was
added dropwise to the resulting solution. The reaction was allowed
to stir for 30 min, at which time the solvent was removed in vacuo
to give the N(ꢀ)-Boc-deprotected intermediate that was converted
without further purification to the corresponding Mtt-protected
product. The N(δ)-Boc-deprotected residue was dissolved in 2:1
CHCl3/DMF (15 mL) in a 50 mL round-bottomed flask and brought
to 0 °C. DIPEA (0.90 mL, 5.2 mmol) was added dropwise over 15
min followed by dropwise addition of Mtt-chloride (0.84 g, 2.86
mmol) dissolved in 5 mL of CH2Cl2 over 10 min. The reaction
mixture was allowed to slowly reach room temperature overnight.
Methanol (2.5 mL) was then added, and the temperature was raised
to 50 °C for 2 additional hours. At the end of this time, the reaction
mixture was allowed to cool to room temperature, and was poured
into a 250 mL separatory funnel containing EtOAc (50 mL) and
0.5 M aq. citric acid (80 mL). Following separation of the organic
layer, the aqueous phase was extracted with additional EtOAc (2
× 50 mL). The organic fractions were combined, dried over MgSO4,
and concentrated to yield the crude product. Purification was carried
out by silica gel chromatography with 1:4:5 MeOH/EtOAc/Hex as
eluent to give 0.53 g of PTAA 24 (65% for two steps) as a colorless
foam: mp 150-160 °C dec; [R]25D -2.5 (c 0.84, CH3OH); 1H NMR
(500 MHz, CD3OD) δ 7.77 (t, J ) 8.1 Hz, 2H), 7.54-7.65 (m,
2H), 7.22-7.40 (m, 16H), 7.15 (dd, J ) 6.0 Hz, J ) 7.6 Hz, 2H),
4.26-4.38 (m, 2.5H), 4.22 (m, 1H), 4.12 (m, 0.5H), 3.58 (m, 1H),
3.50 (s, 1H), 2.56 (m, 1H), 2.44 (m, 0.5H), 2.37 (m, 0.5H), 2.30
(d, J ) 3.2 Hz, 3H), 1.56 (m, 0.5H), 1.50 (m, 0.5H), 1.37 (m, 0.5H),
1.33 (m, 0.5H), 0.77 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 175.8,
175.1, 155.6, 154.9, 144.7, 144.1, 144.0, 143.8, 141.2, 136.5, 128.7,
128.6, 128.0, 127.6, 127.1, 126.7, 125.2, 119.9, 110.5, 79.8, 71.8,
67.5, 67.4, 62.0, 61.5, 48.7, 48.4, 47.2, 45.8, 25.9, 24.8, 22.2, 21.5,
20.8, 20.6 ppm; IR (film) 3062, 1705 cm-1; MS (CI) m/z 635 (MH).
HRMS calcd for C42H39N2O4 [M + H]+ 635.2910, found 635.2898.
trans-4-[((4-Methylphenyl)diphenylmethyl)aminomethyl]-N-[(flu-
oren-9-ylmethyl)oxycarbonyl]-L-proline Benzyl Ester (31). The
nitrile 29 (9.00 g, 20.0 mmol) was dissolved in EtOH (100 mL,
dried over molecular sieves) and Pd/C (10% w/w, 2.11 g, 10 mol
%) and PtO2 (0.408 g, 10 mol%) were added. H2 (250 psi) was
applied. After 12 h the catalyst was removed by filtration through
celite. The solvent was removed under reduced pressure to afford
the amino acid 30 (5.5 g) as a colorless foam that was used without
further purification. The amino acid 30 (1.00 g, 2.73 mmol) was
dissolved in DMF (10 mL) and cooled to 0 °C and DIPEA (1.52
mL, 8.74 mmol) was added dropwise over 10 min. Mtt-Cl (2.00 g,
6.83 mmol) was then added as a solution in DMF (10 mL) and the
mixture was allowed to warm to room temperature. After 12 h,
MeOH (50 mL) was added and the reaction mixture was warmed
to 50 °C for 2 h. The product was then partitioned between water
and EtOAc (2 × 100 mL). The organic fractions were combined
and dried (MgSO4) before the solvent was removed under reduced
pressure. Purification of the residue by flash chromatography eluting
with CH2Cl2:MeOH (97:3) afforded the product as a colorless solid
(0.760 g, 45%). Mp 162-165 °C; [R]25D -4.61 (c 1.20, CH3OH);
1H NMR (500 MHz, 9:1 CD3OD:CDCl3) δ 8.71-7.66 (m, 22H),
4.51-4.00 (m, 4H), 3.71-3.53 (t, J ) 8.7 Hz, 0.5H), 3.53-3.42
(t, J ) 8.7 Hz, 0.5H), 3.02 (s, 1H), 2.97-2.85 (t, J ) 8.9 Hz,
0.5H), 2.85-2.66 (t, J ) 8.9 Hz, 0.5H), 2.07-1.82 (m, 1H), 1.71
(d, J ) 15.7 Hz, 3H), 1.54 (m, 3H), 1.43-1.26 (q, J ) 10.0 Hz,
0.5H), 1.26-1.07 (q, J ) 10.0 Hz, 0.5H); 13C NMR (125 MHz,
CDCl3) δ 176.9, 175.7, 155.8, 154.5, 145.7, 145.0, 144.2, 144.0,
143.9, 142.5, 141.8, 141.4, 136.3, 136.1, 128.7, 128.6, 128.0, 127.8,
127.6, 127.1, 126.6, 126.5, 125.1, 120.0, 119.9, 79.1, 71.4, 70.8,
67.9, 67.5, 59.7, 59.2, 51.0, 50.8, 50.7, 47.3, 46.5, 46.3, 38.6, 37.0,
35.3, 33.7, 30.4, 29.7, 21.0, 16.3 ppm. IR (film), 3061, 3021, 2949,
2246, 1684, 1598 cm-1. HRMS calcd for C41H37N2O4Li2 [M +
2Li - H]+ 635.3073, found 635.3073.
trans-3-[2-((4-Methylphenyl)diphenylmethyl)aminoethyl]-N-(9-
fluorenylmethyl)-L-proline (27). The procedure is identical with that
previously described for the protecting group conversion of 23 to
24. Starting from PTAA 26 (1.00 g, 2.08 mmol) afforded 0.75 g
of PTAA 27 (56% for two steps) as a colorless foam: mp 120-128
°C dec; [R]25D -1.2 (c 1.0, CH3OH); 1H NMR (500 MHz, CD3OD)
δ 7.59-7.65 (m, 2H), 7.50 (dd, J ) 7.5 Hz, J ) 16.8 Hz, 1H),
7.45 (dd, J ) 2.7 Hz, J ) 7.4 Hz, 1H), 7.09-7.33 (m, 16H), 6.99
(dd, J ) 3.2 Hz, J ) 8.2 Hz, 2H), 4.15-4.23 (m, 1.5H), 4.07 (t, J
) 2.7 Hz, 0.5H), 4.00 (m, 1H), 3.89 (d, J ) 4.9 Hz, 0.5H), 3.75
(d, J ) 4.9 Hz, 0.5H), 3.40 (m, 0.5H), 3.23-3.37 (m, 1.5H), 2.51
(m, 1H), 2.43 (m, 1H), 2.24 (m, 0.5H), 2.09-2.22 (m, 3.5H), 1.82
(m, 1H), 1.72 (m, 1H), 1.61 (m, 0.5H), 1.52 (m, 0.5H), 1.31 (m,
1H); 13C NMR (125 MHz, CDCl3) δ 176.1, 175.9, 155.2, 154.7,
144.8, 144.2, 144.0, 143.8, 141.2, 141.1, 136.2, 128.6, 127.9, 127.6,
127.4, 127.0, 126.6, 119.8, 119.6, 71.3, 67.5, 65.3, 64.7, 47.1, 45.9,
trans-4-Azido-N-[(fluoren-9-ylmethyl)oxycarbonyl]-L-proline Ben-
zyl Ester (35). The Boc-protected azide 3417 (6.5 g, 18.8 mmol)
was dissolved in TFA:CH2Cl2 (1:1, 100 mL) and maintained at rt
for 30 min. The solvent was then removed by rotary evaporation
and a mixture of saturated NaHCO3 (1.5 g, 17.9 mmol) and FmocCl
(5.32 g, 20.6 mmol) was added. After being stirred for 12 h, the
reaction mixture was extracted into EtOAc (2 × 250 mL). The
organic fractions were combined, dried (MgSO4), and concentrated.
The residue was purified by flash chromatography eluting with
EtOAc/hexanes (1:4) and afforded the product as a colorless oil
(7.4 g, 84%). [R]25D -4.59 (c 10.55, CH3OH); 1H NMR (500 MHz,
CDCl3) δ 7.77 (d, J ) 7.7 Hz, 2H), 7.60-7.52 (m, 2H), 7.42-7.27
(m, 9H), 5.26-5.04 (m, 2H), 4.56 (t, J ) 7.1 Hz, 0.5H), 4.48-4.09
45.6, 42.3, 41.1, 33.6, 30.1, 29.0, 20.8 ppm; IR (film) 3063 cm-1
,
1702 cm-1; MS (CI) m/z 637 (MH). HRMS calcd for C42H42N2O4
[M + H]+ 637.3066, found 637.3035.
J. Org. Chem. Vol. 73, No. 19, 2008 7601

Flemer et al.
(m, 4H), 4.02 (t, J ) 6.1 Hz, 0.5H), 3.79-3.74 (m, 1H), 3.70 (d,
J ) 10.9 Hz, 0.5H), 3.59 (d, J ) 10.8 Hz, 0.5H), 2.37 (m, 1H),
2.20 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 171.5, 154.4, 143.9,
143.8, 143.4, 141.2, 141.1, 135.3, 135.1, 128.5, 128.4, 128.0, 127.6,
127.0, 124.9, 124.8, 119.9, 119.8, 77.3, 77.0, 76.8, 67.6, 67.0, 66.9,
59.2, 58.4, 57.8, 57.5, 51.7, 51.2, 47.0, 36.3, 35.1 ppm; IR (film)
3065, 2951, 2104, 1748, 1709 cm-1. MS (CI) m/z 469.5 (MH).
Anal. Calcd for C27H24N4O4: C, 69.22; H, 5.16; N, 11.96. Found:
C, 69.52; H, 5.12; N, 11.68.
was swelled in DMF (3 mL) for 10 min. After removal of solvent,
the resin was treated with 20% piperidine in DMF (3 mL, 3 × 10
min), followed by multiple washing with DMF (3 × 3 mL). The
deprotected beads were then incubated with Fmoc-Ala-OH (3
equiv), HOBt (3 equiv), and DIC (3 equiv) for 1 h in DMF (3
mL). Following this coupling procedure, the beads were washed
with DMF (3 × 3 mL), and then Fmoc-deprotected with 20%
piperidine in DMF (3 mL, 3 × 10 min). After multiple washings
with DMF (3 × 3 mL), the resin was treated with Fmoc-
PTAA(Mtt)-OH (1.5 equiv), HOBt (1.5 equiv), and DIC (1.5 equiv)
for 4 h in DMF (3 mL). After PTAA coupling, the side chain amine
was then Mtt-deprotected with 1% TFA in CH2Cl2 (2 mL, 7 × 10
min). The resin was washed (2 × 3 mL of CH2Cl2, 1 × 3 mL of
1% piperidine/DMF, 2 × 3 mL of DMF), then was incubated with
representative N-Pmc-N′-substituted thiourea 18 (1.5 equiv) and
EDCI (4.5 equiv) for 2-3 h in DMF (3 mL) (note: thioureas 18e
and 18h required greater concentrations and longer reaction times:
typically 4 equiv of thioureas and 24 h of reaction time for
completion). The completed peptide sequence was then Fmoc-
deprotected by using 20% piperidine/DMF (3 mL, 3 × 10 min)
followed by repeated washing with CH2Cl2 (5 × 3 mL). Cleavage
of the peptide from the resin and Pmc deprotection was carried
out by stirring the resin in 100% TFA (3 mL) for 4 h. The mixture
was filtered through glass wool, and the supernatant was concen-
trated in vacuo to yield the crude peptide residue, which was
triturated with diethyl ether and carefully decanted to yield the
representative peptide as a colorless powder. 43 (13.7 mg, 52%):
1H NMR (500 MHz, CD3OD) δ 4.55 (br s, 1H), 4.33-4.40 (m,
2H), 3.49-3.60 (m, 2H), 3.27-3.39 (m, 2H), 2.95 (m, 1H), 2.13
(m, 1H), 1.86-1.95 (m, 2H), 1.84 (m, 1H), 1.72-1.80 (m, 2H),
1.62 (m, 1H), 1.24-1.46 (m, 11H), 1.20 (m, 1H); MS (ESI) m/z
423 (MH); HRMS calcd for C20H35N6O4 423.2720, found 423.2714
[M + H]+.
trans-4-[((4-Methylphenyl)diphenylmethyl)amino]-N-[(fluoren-
9-ylmethyl)oxycarbonyl]-L-proline (37). trans-4-azido-N-[(fluoren-
9-ylmethyl)oxycarbonyl]-L-proline benzyl ester (35) (17.0 g, 36.3
mmol) was dissolved in EtOH (150 mL). Pd/C (10% w/w, 4.62 g,
12 mol %) was added and H2 (350 psi) was applied. The reaction
mixture was stirred for 12 h after which time the catalyst was filtered
through celite and rinsed with MeOH (250 mL). The solvent was
removed by rotary evaporation to yield the reduced product 36 as
a colorless solid (crude yield: 12.51 g; 98%) that was used without
further purification. The amino alcohol 36 (1.00 g, 2.84 mmol) was
dissolved in DMF (15 mL) and DIPEA (1.59 mL, 9.09 mmol) was
added dropwise at 0 °C. Mtt-Cl (2.07 g, 7.10 mmol) was dissolved
in DMF (15 mL) and added dropwise to the reaction mixture. The
reaction was allowed to reach room temperature and stirred
overnight. After 12 h MeOH (50 mL) was added and the reaction
mixture was warmed to 50 °C for 2 h. The product mixture was
then partitioned between water (500 mL) and EtOAc (500 mL).
The layers were separated and the aqueous layer was washed with
EtOAc (500 mL). The combined organic fractions were washed
with water (4 × 500 mL), dried (MgSO4), and concentrated under
reduced pressure. Purification of the residue by flash chromatog-
raphy eluting with CH2Cl2:MeOH (97:3) afforded the product 37
as a colorless solid (1.20 g, 69.9%). Mp 145-149 °C; [R]25D 2.05
1
(c 0.72, CH3OH); H NMR (500 MHz, CDCl3) δ 7.99-6.94 (m,
22H), 4.36-4.02 (m, 4.5H), 3.41-3.23 (m, 1.5H), 3.22-3.01 (m,
1H), 2.90-2.78 (m, 0.5 Hz), 2.76-2.67 (t, J ) 9.4 Hz, 0.5H), 2.31
(s, 0.5H), 2.29-2.23 (m, 1.5H), 2.20 (s, 1H), 1.88-1.74 (q, J )
10.1 Hz, 0.5H), 1.73-1.65 (q, J ) 10.1 Hz, 0.5H), 1.65-1.53 (m,
0.5H), 1.43-1.32 (m, 0.5H), 1.32-1.13 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 176.1, 174.7, 155.9, 154.2, 146.5, 144.3, 144.1,
143.8, 143.3, 141.3, 137.3, 136.2, 128.8, 128.5, 128.4, 128.1, 127.9,
127.8, 127.1, 127.0, 126.6, 125.3, 120.0, 78.6, 77.3, 77.1, 76.8,
70.9, 68.1, 67.3, 62.6, 62.1, 58.2, 57.6, 52.8, 51.9, 47.3, 47.1, 38.2,
36.9, 31.6, 29.7, 20.9, 14.6, 14.4 ppm; IR (film) 3062, 2951, 2248,
1700 cm-1. HRMS calcd for C40H35N2O4Li2 [M + 2Li - H]+
621.2917, found 621.2924.
Acknowledgment. Financial Support for this work was
provided by CHE-0411831 from the NSF.
Supporting Information Available: Detailed experimental
procedures, characterization data for compounds 15, 17, 18a-p,
1
44-62, 68, and 69, copies of H spectra for all compounds,
13C spectra all compounds except 43-62, and mass spectra for
compounds 43-62 and 83. This material is available free of
charge via the Internet at http://pubs.acs.org.
General Procedure for the Preparation of Trimer Peptides
(43-62). Wang resin-Ala(Fmoc) (0.8 mmol/g, 50 mg, 0.04 mmol)
JO8012258
7602 J. Org. Chem. Vol. 73, No. 19, 2008