Synthesis of a cyclic pentapeptide mimic of the active site His-Tyr cofactor of cytochrome c oxidase

Article abstract of DOI:10.1021/jo901744y

(Chemical Equation Presented) Arylboronic acid based technology provides a mild, regioselective, and nontoxic N-arylation procedure for accessing the unusual N-arylated side chain histidine found in the active site of cytochrome c oxidase (CcO). The N-ary

Full text of DOI:10.1021/jo901744y

pubs.acs.org/joc
Synthesis of a Cyclic Pentapeptide Mimic of the Active Site His-Tyr
Cofactor of Cytochrome c Oxidase
†
Maximillian E. Mahoney, Allen Oliver, Olof Einarsdottir, and Joseph P. Konopelski*
ꢀ
€
ꢀ
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064.
†Present address: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
IN 46556-5670.
joek@chemistry.ucsc.edu
Received August 13, 2009
Arylboronic acid based technology provides a mild, regioselective, and nontoxic N-arylation procedure for
accessing the unusual N-arylated side chain histidine found in the active site of cytochrome coxidase (CcO).
The N-arylated histidine is elaborated to the complete cytochrome c oxidase cyclic pentapeptide cofactor.
Molecular modeling of the cofactor provides insight into the dynamic character of the N-aryl bond.
Introduction
residues 290 and 291, is bound to CuB, providing a rigid
binding site for the metal (Figure 1).4 A detailed under-
standing of this unusual cross-linkage is necessary to fully
define the reaction mechanism of CcO.
Of the four electrons needed in the catalytic cycle, it is
accepted that two electrons arise from the redox cycle of heme
a3 (FeII to FeIV) and one electron from the oxidation of CuB. It
has been postulated that the His-Tyr cross-link plays a role in
stabilizing either a Tyr radical or tyrosinate, allowing the
cofactor to provide a proton5-7 and the fourth electron8
Cytochrome c oxidase (CcO) is the terminal electron
acceptor of the mitochondrial electron transport chain and
is responsible for the four-electron reduction of dioxygen to
water.1 This reaction drives the translocation of four protons
across the inner mitochondrial membrane, establishing an
electrochemical gradient that drives ATP synthesis.2 The
active site of CcO consists of a binuclear center (heme a3
and CuB) and a peptide cofactor, the latter of which is of
particular interest to our research program. This cofactor
was discovered and characterized through X-ray crystal-
lography, revealing an unusual post-translational modifica-
tion: an N-arylation linking N1 of the His240 side chain to the
εC of Tyr244 (bovine heart numbering).3 The macrocyclic
pentapeptide, formed with the intervening residues Pro, Glu,
and Val, is part of an R-helix that, along with distal His
(4) PDB ID: 2zxw; Aoyama, H.; Muramoto, K.; Shinzawa-Itoh, K.;
Hirata, K.; Yamashita, E.; Tsukihara, T.; Ogura, T.; Yoshikawa, S. Proc.
Nat. Acad. Sci. U.S.A. 2009, 106, 2165–2169.
(5) Proshlyakov, D. A.; Pressler, M. A.; Babcock, G. T. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 8020–8025.
(6) Gennis, R. B. Biochim. Biophys. Acta 1998, 1365, 241–248.
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(7) Sucheta, A.; Szundi, I.; Einarsdottir, O. Biochemistry 1998, 37, 17905–
17914.
(8) (a) Blomberg, M. R. A.; Siegbahn, P. E. M.; Babcock, G. T.;
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(1) Ferguson-Miller, S.; Babcock, G. T. Chem. Rev. 1996, 96, 2889–2907.
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Wikstrom, M. K. F. J. Inorg. Biochem. 2000, 80, 261–269. (b) Svistunenko,
(2) Wikstrom, M. K. F. Nature (London) 1977, 266, 271–273.
D. A.; Wilson, M. T.; Cooper, C. E. Biochim. Biophys. Acta 2004, 1655, 372–
380. (c) Proshlyakov, D. A.; Pressler, M. A.; DeMaso, C.; Leykam, J. F.;
Dewitt, D. L.; Babcock, G. T. Science 2000, 290, 1588–1591. (d) Tomson, F.;
Bailey, J. A.; Gennis, R. B.; Unkefer, C. J.; Li, Z. H.; Silks, L. A.; Martinez,
R. A.; Donohoe, R. J.; Dyer, R. B.; Woodruff, W. H. Biochemistry 2002, 41,
14383–14390. (e) Nyquist, R. M.; Heitbrink, D.; Bolwien, C.; Gennis, R. B.;
Heberle, J. Proc. Natl. Acad. Sci. U.S.A 2003, 100, 8715–8720.
(3) (a) Yoshikawa, S.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.;
Yamashita, E.; Inoue, N.; Yao, M.; Fei, M. J.; Libeu, C. P.; Mizushima, T.;
Yamaguchi, H.; Tomizaki, T.; Tsukihara, T. Science 1998, 280, 1723–1729. (b)
Ostermeier, C.; Harrenga, A.; Ermler, U.; Michel, H. Proc. Natl. Acad. Sci. U.
S.A. 1997, 94, 10547–10553. (c) Soulimane, T.; Buse, G.; Bourenkov, G. P.;
Bartunik, H. D.; Huber, R.; Than, M. E. EMBO J. 2000, 19, 1766–1776.
8212 J. Org. Chem. 2009, 74, 8212–8218
Published on Web 10/07/2009
DOI: 10.1021/jo901744y
r
2009 American Chemical Society

Mahoney et al.
JOCArticle
SCHEME 1. Retrosynthesis for the CcO Cyclic Pentapeptide
Model
FIGURE 1. (Left) CuB of CcO bound to the peptide cofactor.
(Right) Previously synthesized cofactor mimics.
required to cleave the dioxygen bond and produce water.
Furthermore, the His-Tyr cross-link appears to create a kink
in the R-helix, placing the phenol directly at the edge of the
K-channel and, thus, facilitating proton delivery to the active
site.9
SCHEME 2. Synthesis of Tyrosine Aryllead(IV) Triacetate
Previously, this laboratory synthesized models of the CcO
active site incorporating a His-phenol cross-link and a
pyridyl appendage that, together with the histidine main
chain nitrogen, act as substitutes for the distal histidine
ligands to CuB (Figure 1).10 However, these His-phenol
mimics could not be expected to properly portray the effects,
both electronic and structural, of the entire cyclic pentapep-
tide found in the native enzyme.
Electron delocalization of a tyrosine-based radical species
onto the histidine imidazole through the unique C-N bond
of the cofactor could be attenuated by the dihedral angle
between the two aryl rings, which in turn would be dictated
by the energetics of the cyclic pentapeptide structure. In
addition, the stereochemistry imparted by the biaryl dihedral
angle is important for mimicking through-space interactions
between the tyrosine-phenol and CuB. Thus, it seemed
prudent to expand our synthetic efforts toward a cyclic
pentapeptide structure that incorporates the cofactor.
Herein is presented the synthesis of macrocyclic pentapep-
tide 1 (Scheme 1). The native Glu has been replaced by Ala
for reasons that will be discussed, and the C-terminal func-
tionality has been removed. Hence, this model system in-
corporates a tyramine unit (abbreviated “tyr” to contrast
with the standard 3-letter abbreviation “Tyr” for the amino
acid tyrosine). Macrolactamization was executed at the
His-Pro bond, utilizing the turn-inducing preference of
proline to facilitate cyclization. The linear pentapeptide
was assembled via coupling of the C-terminus of tripeptide
A to the terminal amino group on N-arylated histidine
fragment B.
substrates. Of the remaining methods, the substrate scope
rarely incorporates substituted imidazoles, which consis-
tently afford diminished yields as a result of regioselectivity
issues. This laboratory previously described the regioselec-
tive N-arylation of histidine with activated tyrosine and
phenol substrates using aryllead(IV) tricarboxylates under
mild conditions.10 We envisioned expanding this methodo-
logy to include the His-Tyr and His-tyr systems needed to
complete the synthesis of the fully formed cyclic pentapep-
tide mimic of CcO.
Commercially available Boc-Tyr-OH was monoiodi-
nated, benzyl-protected (2), and protected on the phenol
functionality to afford compound 3 (Scheme 2) by proce-
dures developed in our laboratory for another project.11
Treatment with PdCl2(PPh3)2 and hexabutylditin under mild
conditions provided stannylated compound 4. Transmetala-
tion to the aryllead(IV) tricarboxylate (5) was accomplished
under mercury catalysis at 30 °C. This material is highly
light-sensitive and must be handled under inert atmosphere;
it is stable in the dark at 0 °C for several months.
Results and Discussion
Although there are many methods in the literature for
N-arylation, nearly all involve the use of base and heat,
conditions that are expected to be unacceptable for peptide
With the suitably protected and activated tyrosine deriva-
tive prepared, N-arylations with the histidine coupling part-
ner were examined. As with previous models, we chose to
employ a histidine residue bearing both N-methyl and
N-protection groups on the main chain nitrogen. Previous
work had identified a facile route to Bn-MeHis-OMe and
(9) Proshlyakov, D. A.; Pressler, M. A.; Babcock, G. T. Proc. Natl Acad.
Sci. U.S.A. 1998, 8020–8025.
(10) (a) Elliott, G. I.; Konopelski, J. P. Org. Lett. 2000, 2, 3055–3057. (b)
Cappuccio, J. A.; Ayala, I.; Elliott, G. I.; Szundi, I.; Lewis, J.; Konopelski, J.
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P.; Barry, B. A.; Einarsdottir, O. J. Am. Chem. Soc. 2002, 124, 1750–60. (c)
ꢀ
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Landaverry, Y. R.; White, K. N.; Olmstead, M. M.; Einarsdottir, O;
Konopelski, J. P. Heterocycles 2006, 70, 147–152. (d) White, K. N.; Sen, I.;
Szundi, I.; Landaverry, Y. R.; Bria, L. E.; Konopelski, J. P.; Olmstead, M.
(11) Moore, E. M., Masters Thesis, University of California Santa Cruz,
2000.
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M.; Einarsdottir, O. Chem. Commun. 2007, 31, 3252–3254.
J. Org. Chem. Vol. 74, No. 21, 2009 8213

Mahoney et al.
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SCHEME 3. Synthesis of a Suitably Protected Histidine Cou-
pling Partner
TABLE 1. Optimizing the N-Arylation
yield
solvent product (%)a
entry tyr
R
Cu source
Cu(OAc)2
14 Cbz CuI
1
2
3
4
5
6
7
8
9
14 Bn
DCM
DCM
DCM
DCM
DMF
DMF
MeOH
15
16
16
16
16
16
16
16
16
19b
0b
14 Cbz Cu(OAc)2
14 Cbz Cu(OAc)2
12 Cbz CuI
12 Cbz CuI
18 Cbz CuI
41
25b,c
0b,d
0b,e
traceb
61f
18 Cbz [Cu(OH)TMEDA]2Cl2 DCM
19 Cbz [Cu(OH)TMEDA]2Cl2 DMSO
0f
aIsolated yield. bReaction performed under an inert atmosphere of
Ar(g) or N2(g). DBU was added to the reaction. 2-Hydroxybenzalde-
SCHEME 4. N-Arylation with Tyrosine Aryllead(IV) Triace-
tate
c
d
e
hyde phenylhydrazone added to the reaction. 2-Acetylcyclohexanone
added to the reaction. fReaction performed under an atmosphere of dry
air (drying tube with CaCO3).
N-terminal Boc group of 10. Under the more exotic ruthe-
nium conditions, the biaryl system of 10 underwent degrada-
tion.13 It has recently been shown that ruthenium can insert
into N-aryl bonds, which supports this degradation path-
way.14
To explain the low arylation yields, we postulated that the
metal binding ability of the histidine residue, bearing both
the strongly chelating imidazole ring and the main chain
alkylamine in Bn-MeHis-OMe, sequestered the reactive lead
species. Alternatively, this highly active metal binding site
could bind spent Pb(OAc)2, thus decreasing the nucleophilic
character of the imidazole and making it an unsuitable
substrate for reaction with the aryllead(IV) triacetate. Thus,
we looked to decrease the metal binding ability of this
residue by switching the benzyl protection for the electron-
withdrawing -Cbz protection group and compound 8
(Scheme 4).15 In addition, we chose to eliminate the C-
terminus of tyrosine, which would bypass the ester differ-
entiation problems and, it was hoped, improve the N-aryla-
tion yields (Table 1). To this end, aryllead(IV) tyramine
derivative 14, comparable to tyrosine derivative 5, was pre-
pared (Scheme 5).
Me-His-OMe (Scheme 3).12 The former compound easily
suffered ester exchange to afford allyl ester 6, while the latter
compound proved to be an adequate starting material for
bis-carbamate protected compound 7 and main chain car-
bamate protected 8.
The first attempts at forming the desired His-Tyr coupled
product followed our previously successful route in forming
His-phenol model systems, namely, subjecting Bn-MeHis-
OMe and aryllead(IV) reagent 5 to Cu(OAc)2 catalysis.
Unfortunately, unacceptably low yields of desired product
9 were obtained by this route, with the main byproduct being
the loss of lead from the tyrosine residue (Scheme 4).
Additionally, simple base (LiOH) hydrolysis of methyl
ester 9 surprisingly was accompanied by a more rapid (5 min
at 0 °C) benzyl ester hydrolysis, leading to an inseparable
mixture of the benzyl-deprotected N-arylated histidine and
N-arylated histidine diacid. Consequently, nonhydrolytic
methods for affecting the selective cleavage of a methyl ester
in the presence of a benzyl ester were attempted. Reaction of
the N-arylated histidine with Na2S (3.0 equiv) in DMPU,
with or without Ti(i-OPr)4, left only unreacted substrate
after 24 h. Hydrolysis attempts with the highly nucleophilic
TMSOK in either Et2O or DCM were unsuccessful. One
possible way to avoid this selective deprotection problem
involved changing the methyl ester protection to an allyl
protection on the histidine. However, the reaction of allyl
ester 6 with 5 under Cu(OAc)2 catalysis also gave low yields
of desired product 10. Allyl deprotection conditions using
the standard palladium method adventitiously removed the
Commercially available tyramine HCl was nitrogen-pro-
3
tected and iodinated to give 11 under the conditions deve-
loped for the synthesis of 3 and then subjected to phenol
protection under standard conditions to give 12.16 Stannyla-
tion (13) and subsequent plumbation yielded aryllead(IV)
coupling partner 14 (Scheme 5).
Although the tyramine aryllead(IV) triacetate could be
prepared in improved yield over the tyrosine system, the
(13) Kawasaki, T.; Enoki, H.; Matsumura, K.; Ohyama, M.; Inagawa,
M.; Sakamoto, M. Org. Lett. 2000, 2, 3027–3029.
(14) Koreeda, T.; Kochi, T.; Kakiuchi, F. J. Am. Chem. Soc. 2009, 131,
7238–7239.
ꢁ
(15) Shieh, W. -C.; Xue, S.; McKenna, J.; Prasad, K.; Repic, O.; Black-
lock, T. Tetrahedron Lett. 2006, 47, 5645–5648.
(16) Hart, M. E.; Suchland, K. L.; Miyakawa, M.; Bunzow, J. R.;
Grandy, D. K.; Scanlan, T. S. J. Med. Chem. 2006, 49, 1101–1112.
(12) White, K. N.; Konopelski, J. P. Org. Lett. 2005, 7, 4111–4112.
8214 J. Org. Chem. Vol. 74, No. 21, 2009

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SCHEME 5. Synthesis of Tyramine Aryllead(IV) Tricarboxylate
SCHEME 6. Synthesis of Tyramine Boronic Ester, Acid and
Trifluoroborate Salt
N-arylation yields were uniformly low (Table 1, entries 1-4).
Variations in copper source and the addition of DBU, which
has been used to increase yields in certain aryllead coupling
reactions,17 did not lead to increased recovery of desired
product. Therefore, more traditional methods for N-arylation,
employing aryl iodides, were explored; however, no product
was observed for any of these reactions (entries 5 and 6).18
Lam-Chan coupling procedures have been used for the
arylation of simple imidazoles with arylboronic acids.19
However, such protocols require the use of 2 equiv of
boronic acid and struggle with regioselectivity issues on
substituted imidazole substrates. Nonetheless, we next at-
tempted to produce a tyramine coupling partner functiona-
lized with a boronic acid group.
A variety of methods were screened to affect the Miyaura
reaction on iodide 12, including both microwave and con-
ventional heating as well as variations in ligand design.20-22
Optimal conditions included the use of pinacol borane
rather than the traditional bis-pinacol borane and DCBP
as a ligand to afford crystalline aryl boronic ester 17
(Scheme 6). Oxidative cleavage of the boronate ester with
NaIO4 provided arylboronic acid 18 in 88% yield.
aerobic conditions, albeit with modest regioselectivity for
substituted imidazoles. Additionally, as in the aryllead case,
a 40% excess of arylboronic acid is needed with respect to the
imidazole. However, in our hands the optimal reaction
conditions afforded 61% of the N-arylated histidine 16
(Table 1, entry 8) as a single regioisomer using a 1:1 ratio
of boronic acid to histidine.24 It should be noted that this
stoichiometery represents an unusual finding in that com-
monly a 2:1 ratio of boronic acid to histidine is optimal. The
˚
use of 4 A molecular sieves, as employed by Collman,
afforded variable yields and were, therefore, not used.
Much of the same arylboronic acid chemistry can be
achieved with the easily prepared and highly crystalline tri-
fluoroborate salts.25 Attempts to bypass the arylboronic ester
in synthesizing the trifluoroborate salt 19 from 12 failed,26 but
proceeded from 17 by treatment with KHF2.27 N-Arylation
reactions failed with the trifluoroborate salt (Table 1, entry 9).
However, 19 could be easily recrystallized and hydrolyzed to
18 for incorporation into the optimized procedure.28
With a viable His-tyr dipeptide fragment in hand (B in
Scheme 1), tripeptide Boc-Pro-Ala-Val-OH 22 (serving as
fragment A in Scheme 1) was prepared easily on a large scale
(Scheme 7). Alanine was chosen to replace the glutamic acid
residue of the mammalian CcO structure to avoid any addi-
tional problems with side chain protection groups. It was
believed that this replacement would not have an impact on
the ring conformation and C-N bond rotation energetics.
Dipeptide 20 was prepared in high yield using an HOBt/EDC
Unfortunately, Lam-Chan-type coupling with CuI in
MeOH provided no product (Table 1, entry 7). A procedure
by Collman et al. for the N-arylation of imidazoles with
arylboronic acids, originally based on the work of Lam et al.,
was adopted for our needs.23 Collman reported high yields
and mild conditions using a catalytic amount of a novel
copper system (in the form [Cu(OH)TMEDA]2Cl2) under
coupling of Boc-Ala-OH with H-Val-OMe HCl,29 which was
3
then nitrogen-deprotected under standard conditions and
coupled with Boc-Pro-OH to provide protected tripeptide 21.
Hydrolysis revealed N-protected tripeptide 22.
(17) Pinhey, J. T.; Rowe, B. A. Aust. J. Chem. 1983, 36, 789–794.
(18) (a) Shafir, A.; Buchwald, S. L. J. Am. Chem. Soc. 2006, 128, 8742–
8743. (b) Jiang, Q; Jiang, D; Jiang, Y.; Fu, H.; Zhao, Y. Synlett 2007, 1836–
1842. (c) Zhu, L.; Li, G.; Luo, L.; Guo, P.; Lan, J.; Youet, J. J. Org. Chem.
2009, 74, 2200–2202.
(19) (a) Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.; Winters, M.
P.; Chan, D. M. T.; Combs, A. Tetrahedron Lett. 1998, 39, 2941–2944. (b)
Lan, J. -B.; Chen, L.; Yu, X.-Q.; You, J.-S.; Xie, R.-G. Chem. Commun.
2004, 188–189.
(20) Gerstenberger, B. S., Ph.D. Thesis, University of California Santa
Cruz , 2007
(21) Poriel, C.; Lachia, M.; Wilson, C.; Davies, J. R.; Moody, C. J. J. Org.
Chem. 2007, 72, 2978–2987.
(22) Decicco, C. P.; Song, Y.; Evans, D. A. Org. Lett. 2001, 3, 1029–1032.
(23) Collman, J. P.; Zhong, M. Org. Lett. 2000, 2, 1233–1236.
(24) Increasing the arylboronic acid to histidine ratio to 1.4:1 does not
improve the yield.
(25) Molander, G. A.; Figueroa, R. Aldrichimica Acta 2005, 38, 49.
(26) Park, Y. H.; Ahn, H. R.; Canturk, B.; Jeon, S. I.; Lee, S.; Kang, H.;
Molander, G. A.; Ham, J. Org. Lett. 2008, 10, 1215–1218.
(27) Skaff, O.; Jolliffe, K. A.; Hutton, C. A. J. Org. Chem. 2005, 70, 7353–
7363.
(28) Yuen, A. K. L.; Hutton, C. A. Tetrahedron Lett. 2005, 46, 7899–7903.
(29) Ginisty, M.; Roy, M.-N.; Charette, A. B. J. Org. Chem. 2008, 73,
2542–2547.
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SCHEME 7. Synthesis of Tripeptide 2
FIGURE 2. Crystal structure of cyclic pentapeptide 24.32
SCHEME 8. Synthesis of Cytochrome c Oxidase Peptide
Cofactor 1
2 steps). Other methods, such as CIP31 or PyBOP/HOAt,
resulted in significant byproduct formation.
Standard LiOH hydrolysis of the methyl ester in 23 was
accomplished in high yield. This crude salt was subjected to
TFA deprotection of the remaining Boc group, again in the
presence of the MOM functionality, to provide the linear
pentapeptide as a free carboxylic acid/ammonium salt, which
was taken on without purification to the cyclization reaction.
At this stage, a variety of activation agents were screened in an
attempt to cyclize this 21-membered macrocycle. Among
them, PyBOP/HOAt and EDC/HOBt provided only minimal
product after 2 d of reaction, whereas EDC/pentafluorophe-
nol (Pfp) provided the ring-closed intermediate at an excep-
tional rate. This material was fully MOM-deprotected in high
yield, providing the free phenol macrocyclic pentapeptide 24,
as confirmed by LCMS and single crystal X-ray analysis
(Figure 2). At this point, the Cbz group was removed with
Parr hydrogenolysis to provide the complete pentapeptide 1,
which includes the cytochrome c oxidase peptide cofactor.
Modeling
Our goal was to synthesize a compound that more closely
resembled the natural peptide system in CcO as regards the
global conformation and corresponding rotational freedom
around the novel C-N bond linking the two aryl side chains
(biaryl dihedral angle, BDA), in full recognition of the
significant contributions likely made by both metals, their
corresponding ligand systems, and the polypeptide structure
of the native protein.
Of all the compounds studied in this laboratory, the crystal
structure of compound 24 exhibits a BDA magnitude most
like that of the native system. However, the sign is opposite
(-40°, synthetic; þ44°, native). It is compelling to propose
that this similarity in BDA magnitude represents the natural
tendency of the bicyclic system33 based on sound physical
Selective deprotection of the N-Boc functionality of 16 in
the presence of the -MOM group was performed (10%
impurity of MOM-deprotected material)30 prior to standard
HOBt/EDCI coupling with 22, which afforded the linear
pentapeptide 23 in 83% yield (Scheme 8, 58% overall for the
(32) Solvent and water molecules omitted for clarity.
(33) DFTcalculationson simplemodelsystems ( Pratt, D. A.; Pesavento, R.
P.; van der Donk, W. A. Org. Lett. 2005, 7, 2735–2738.) put the BDA at 42.5°,
very closetothe native enzyme BDA. Most X-ray structures, including ourown
previous work, suffer from intermolecular hydrogen bonds that likely perturb
the fundamental BDA; in our laboratory we have documented BDAs ranging
from -25.7° to -36.3°. By contrast, the structure of 24 appears devoid of
serious intermolecular interactions. Hence, this molecule affords the best
experimental evidence for the intrinsic BDA of the biaryl cofactor.
(30) Nicolaou, K. C.; Chen, D. Y.-K.; Huang, X.; Ling, T.; Bella, M.;
Snyder, S. A. J. Am. Chem. Soc. 2004, 126, 12888–12896. While this 10%
impurity of free phenol was removed to obtain a pure sample of 23, its
presence posed no problems for the production of 24.
(31) Akaji, K.; Hayashi, Y.; Kiso, Y.; Kuriyama, N. J. Org. Chem. 1999,
64, 405–411.
8216 J. Org. Chem. Vol. 74, No. 21, 2009

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TABLE 2. Duhedral Angles (j, ψ) of Native and Synthetic Peptide
Key to the synthesis was an N-arylation procedure, which
improved the stoichiometry and regiocontrol over previous
methods. The biaryl dihedral angle is found to be almost
identical in magnitude to that found in the natural system,
while opposite in sign. Continued synthetic efforts to append
a copper binding site to the histidine residue of the cyclic
pentapeptide are ongoing and will be reported in due course.
native peptidea
synthetic peptideb
j (deg)
ψ (deg)
j (deg)
ψ (deg)
His
Pro
-68
-51
-57
-69
-77
-51
-34
-39
-29
-26
-170
-73
-65
-69
77
-14
142
Glu/Ala
Val
Tyr/tyr
-37
c
c
BDAd
44
-40
Experimental Section
aData from crystal structures given in ref 4. bData from crystal
structure. cThe absence of a C-terminus on the synthetic peptide
eliminates the ability to report these angles. dBiaryl dihedral angle.
(S)-Methyl 2-((Benzyloxycarbonyl)(methyl)amino)-3-(1-(5-(2-
(tert-butoxycarbonylamino)ethyl)-2-(methoxymethoxy)phenyl)-
1H-imidazol-4-yl)propanoate (16). A solution of histidine deri-
vative 8 (0.51 g, 1.61 mmol, 1.0 equiv) in DCM (6.4 mL, 0.1 M)
was added to freshly prepared boronic acid 18 (0.52 g, 1.61
mmol, 1.0 equiv). Copper catalyst [Cu(OH)TMEDA]2Cl2
(0.074 g, 0.161 mmol, 0.1 equiv) was added to the reaction under
N2, which turned the solution dark blue. A drying tube (CaSO4)
was put in place to allow diffusion of dry air into the reaction
mixture, which turned the solution a pale aqua color. After
stirring for 36 h the reaction was concentrated to a green oil and
loaded onto a column for purification with 1:4 ethyl acetate/
hexanes followed by ethyl acetate; 0.59 g (61%) was recovered as
a clear oil. 1.0:0.9 dr (Cbz rotomers on main chain nitrogen); 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.68 (d, J = 14.0 Hz, 1H),
7.23-7.29 (m, 5H), 7.19 (dd, J = 1.0, 8.5 Hz, 1H), 7.13 (d, J =
8.5 Hz, 1H), 7.04 (d, J = 9.5 Hz, 1H), 6.978 (s, 1H), [6.88 (s,
1H)], 5.11 (s, 2H), 5.06-5.08 (m, 2H), 5.01-5.03 (m, 1H),
[4.92-4.93 (m, 1H)], 4.62 (bs, 1H), 3.74 (s, 3H), [3.64 (s, 3H)],
3.33 (s, 3H), [3.32 (s, 3H)], 3.305-3.34 (m, 2H), 3.06-3.19 (m,
2H), 2.91 (s, 3H), [2.90 (s, 3H)], 2.76 (d, J = 4.0 Hz, 2H), 1.41 (s,
9H); 13C NMR (125 MHz, CDCl3) δ (ppm) 171.4, 155.7, 137.9,
136.4, 133.4, 128.9, 128.9, 128.3, 127.7, 127.4, 125.6, 116.6,
116.5, 95.1, 79.2, 67.1, 59.5, 56.2, 52.2, 52.1, 41.5, 35.1, 28.2;
HRMS for C31H41N4O8 [M þ H] calcd, 597.2919, found,
597.3001 (error = 13.73 ppm); [R]2D8 = -36.1 (c 0.2, MeOH);
FIGURE 3. Ensemble of structures minimized about the biaryl bond.
IR (thin film) 1642, 1519, 1404, 1344, 1145, 1039, 990 cm-1
.
organic chemistry principles and that the sign is dictated by
second sphere forces (heme, metals, polypeptide backbone).
By contrast, the dihedral angles (j, ψ) defining the cyclic
peptide backbone are found to be quite different from those
of the native system (Table 2).
Cyclic Pentapeptide, N-Cbz Protection TFA Salt (24). Linear
3
pentapeptide 23 (0.23 g, 0.26 mmol, 1.0 equiv) was dissolved in a
1:1 mixture of THF/H2O (5.2 mL, 0.05 M) and cooled to 0 °C.
After the solution reached 0 °C, LiOH (0.033 g, 0.79 mmol, 3.0
equiv) was added and stirred for 1 h, at which point the ice bath
was removed and the solution was allowed to reach rt, where it
was kept for an additional 2 h. The THF was removed in vacuo,
and the aqueous mixture was loaded onto a 50 mL Sephadex
C18 solid phase extraction column (preconditioned by running 1
column volume (cv) of MeOH followed by 1 cv of H2O). The
sample was eluted with 3 cv of H2O followed by 3 cv of MeOH.
The sample eluted immediately with MeOH over ∼20 mL. The
organic layer contained only the desired acid salt as determined
by LCMS. These fractions were lyophilized to provide the linear
pentapeptide lithium carboxylate salt. This salt was dissolved in
TFA (2.6 m, 0.1 M) at rt and stirred for 4 min. At this time, 100
mL of DCM was added, and the solution was evaporated in
vacuo. The compound was evaporated two additional times
with DCM to provide the bis-TFA salt as a yellow oil. Some
(10%) of this material was free phenol (-MOM), deprotected
under these conditions and was carried through as a mixture.
For R = MOM: HRMS for C38H50N7O8 [M þ H] calcd,
732.37154, found, 732.3782 (error = 9.09 ppm). To the oil
was added DMF (6.6 mL, 0.05 M). The solution was cooled to
0 °C, and EDC (0.05 g, 0.26 mmol, 1.0 equiv) and pentafluoro-
phenol (Pfp) (0.24 g, 1.32 mmol, 5.0 equiv) in DMF (3.3 mL)
were added. This mixture was allowed to react for 30 min. At this
point the pH of the solution was increased with the addition
of DIPEA (0.44 mL, 2.64 mmol, 10.0 equiv). The reaction
was periodically monitored by LCMS, which indicated that
The barrier to rotation around the unusual C-N bond
was probed through computational studies.34 Driving the
dihedral from -40° to þ40° over 10° increments and mini-
mizing the energy at each step provided the ensemble of
structures shown in Figure 3. The barrier to rotation about
this bond was 12.6 kcal/mol, low enough so that in solution
there is no expectation of atropisomer formation. The
dynamic nature of this biaryl system has a 2-fold impact.
First, the lack of atropisomers simplifies the purification
process of the complete cyclic pentapeptide. Second, it has
been proposed that the conformational changes between the
oxidized and reduced forms of the enzyme are implicated in
proton pumping.35 A flexible biaryl system is required for
this conformational mobility.
Conclusion
Synthesis of a pentapeptide containing the unusual amino
acid cofactor of cytochrome c oxidase has been presented.
(34) Macromodel v9.0.014 was used for the calculations. The Amber*
force field was employed, dielectric = 80, with a PRCG minimization method.
(35) Qin, L.; Liu, J.; Mills, D. A.; Proshlyakov, D. A.; Hiser, C.;
Ferguson-Miller, S. Biochemistry 2009, 48, 5121–5130.
J. Org. Chem. Vol. 74, No. 21, 2009 8217

Mahoney et al.
JOCArticle
cyclization proceeded rapidly (little starting material remained
after 15 min) and was complete after 3 h. An LCMS of the crude
reaction mixture revealed only the presence of product (a
variable mixture of MOM protected and deprotected) with
Pfp as the only other identifiable product. Purification was
accomplished with a C18 Sephadex column (prepared as de-
scribed previously for isolating the carboxylate salt). The reac-
tion mixture was diluted with water (50 mL) and loaded onto the
column. This was eluted with 3 cv of H2O followed by 3 cv of
MeOH. This first separation is used to remove all DMF. After
the organics were removed a second purification was necessary.
The crude product was loaded with 20% MeOH/H2O and
eluted in 10 mL portions of 50%, 60%, 70%, 80%, 90%,
100% (20 mL) MeOH/H2O. The desired material eluted be-
tween the 80% and 90% MeOH/H2O gradient. The purity was
analyzed by LCMS analysis; see chromatograph in Supporting
Information Part 2. The macrocyclic pentapeptide (0.030 g,
0.036 mmol), as a variable mixture of MOM protected and
MOM deprotected material, was stirred in a 1:1 TFA/DCM
(1 mL) mixture. After 2 h the mixture was concentrated in
vacuo, and any excess TFA was removed by evaporation with
DCM. X-ray quality crystals were grown from a H2O/DMSO/
CH3CN solvent system, which confirmed the connectivity of
compound 24. Data for R = H: due to the multiple conforma-
tions in solution, see 1H and 13C NMR spectra for purity.
HRMS for C36H46N7O7 [M þ H], calcd, 688.3453, found,
688.3402 (error = 7.41 ppm); [R]2D8= -32.8 (c 3.3, DMSO).
Acknowledgment. We wish to thank the NIH [GM53788]
(O.E.) for partial funding of this work. Purchase of the 600
ꢀ
MHz NMR used in these studies was supported by funds
from the National Institute of Health (S10RR019918) and
National Science Foundation (CHE-0342912). Samples for
synchrotron crystallographic analysis were submitted
through the SCrALS (Service Crystallography at Advanced
Light Source) program. Crystallographic data were collected
at Beamline 11.3.1 at the Advanced Light Source (ALS),
Lawrence Berkeley National Laboratory. The ALS is sup-
ported by the U.S. Dept. of Energy, Office of Energy
Sciences, under contract DE-AC02-05CH11231. The
authors would also like to thank C. M. Binder and R. A.
Turner for their insightful conversations.
Supporting Information Available: Synthetic procedures;
complete spectroscopic data; 1H, 11B, and 13C NMR spectra for
all new compounds; and LCMS and CIF data for 24. This
material is available free of charge via the Internet at http://
pubs.acs.org.
8218 J. Org. Chem. Vol. 74, No. 21, 2009