Protein prosthesis: 1,5-Disubstituted[1,2,3]triazoles as cis-peptide bond surrogates

Article abstract of DOI:10.1021/ja075865s

Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. This attribute affects many processes, including the rate at which proteins fold, their structures, and their activities. Other aliphatic amino acids can serve as mimics for proline residues with trans-peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis-prolyl peptide bonds. Here, 1,5-disubstituted[1,2,3]triazoles were assessed as cis-peptide bond surrogates. Huisgen's 1,3-dipolar cycloaddition reaction of amino alkynes and azido acids and a Ru(II) catalyst was used to synthesize a variety of Xaa-1,5-triazole-Ala modules in moderate-to-high yields. Two of these modules, along with their 1,4-triazole regioisomers, were installed in a turn region of bovine pancreatic ribonuclease by using expressed protein ligation. The resulting semisynthetic enzymes displayed full enzymatic activity, indicating the maintenance of native structure. The 1,5-triazole surrogates instilled conformational stability that was comparable to that of Xaa-cis-Pro segments, whereas the 1,4-triazoles conferred markedly less stability. The stability conferred by both surrogates was independent of the Xaa residue, eliminating an uncertainty in protein design. We conclude that Xaa-1,5-triazole-Ala modules can serve as viable mimics of Xaa-cis-Pro segments. The possibility of synthesizing this surrogate by the ligation of fragments in situ and the emergence of biocompatible catalysts for that process portends its widespread use. Copyright

Full text of DOI:10.1021/ja075865s

Published on Web 10/03/2007
Protein Prosthesis: 1,5-Disubstituted[1,2,3]triazoles as cis-Peptide Bond
Surrogates
Annie Tam,† Ulrich Arnold,‡ Matthew B. Soellner,†,§ and Ronald T. Raines*,†,||
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706, and
Department of Biochemistry and Biotechnology, Martin-Luther UniVersity, 06099 Halle, Germany
Received August 4, 2007; E-mail: raines@biochem.wisc.edu
Scheme 1
Proline is unique among the natural amino acids in the similar
propensity of its peptide bond to be in the cis or trans conformation.1
This attribute affects many processes, including the rate at which
proteins fold,2 their structures,3 and their activities.4 Other aliphatic
amino acids can serve as mimics for proline residues with trans-
peptide bonds. In contrast, chemical synthesis is needed to create
surrogates for cis-prolyl peptide bonds.1,5-7
An effective surrogate for a cis-peptide bond must meet certain
criteria. The surrogate must be similar in size to proline, so as not
to disturb secondary and tertiary interactions within the folded
protein. The surrogate must be able to accommodate adjacent
residues, which could be important in protein structure and function.
Finally, it is desirable if the surrogate is accessible via a facile
synthetic route and amenable to solid-phase peptide synthesis.
We reasoned that 1,5-disubstituted[1,2,3]triazoles could meet all
of these criteria. In particular, we noted that an Xaa-1,5-triazole-
Ala module is a remarkable isostere of an Xaa-cis-Pro dipeptide,
Table 1. Ru(II)-Catalyzed 1,3-Dipolar Cycloaddition of Various
Amino Alkynes and Azido Acids
retaining its stereochemistry and number of non-hydrogen atoms
and maintaining similar hybridization (Scheme 1). A regioisomer,
1,4-disubstituted triazoles, had been incorporated into small
peptides,7-9 and a simple 1,5-disubstituted triazole induced peptoid
oligomers to adopt a turn.10 The synthesis and use of 1,5-
disubstituted triazoles as cis-peptide bond mimics are, however,
unknown. Likewise, neither 1,5- nor 1,4-disubstituted triazoles has
been incorporated into a protein. Herein, we report on the attainment
yield
triazole (%)
entry
R
PG1
PG2
solvent
temp
of these goals.
To synthesize the triazole, we employed Huisgen’s 1,3-dipolar
cycloaddition reaction.11 Catalysis of this reaction by Cu(I) is known
to yield exclusively a 1,4-disubstituted triazole,12 but we required
the 1,5-disubstituted regioisomer. There are reports of regioselective
formation of 1,5-disubstituted triazoles being mediated by metals13
or stereoelectronic effects,14 but only under harsh conditions.
Recently, Ru(II) catalysts had been shown to yield exclusively the
1,5-disubstituted triazole in a mild reaction,15 though this methodol-
ogy had never been applied to substrates containing amino acids.
As a model protein, we chose bovine pancreatic ribonuclease
(RNase A; 124 residues), which has been the object of much
seminal work in protein chemistry.16 In the native protein, residues
Gly112-Asn113-Pro114-Tyr115 form a Type VIb reverse turn
(Scheme 1), in which the Asn113-Pro114 peptide is in the cis
conformation. We targeted that dipeptide segment for replacement
with 1,5-triazole-Ala surrogates.
1
2
3
4
5
6
7
H (3)
Boc tBu (1) toluene rt
9
45
62
55
67
90
92
91
CH2CONH2(Trt) (4) Boc tBu (1) dioxane 60 °C 10
CH2CONH2(Trt) (4) Boc Bn (2) dioxane 60 °C 11
CH3 (5)
CH2Ph (6)
CH(CH3)2 (7)
CH(CH3)2 (8)
Boc tBu (1) toluene rt
Boc tBu (1) toluene rt
Boc tBu (1) toluene rt
Fmoc tBu (1) toluene rt
12
13
14
15
group to give 1 or 2, respectively. The route to the amino alkynes
was also general, starting from the Weinreb amide of an amino
acid, and followed by reduction to the aldehyde and conversion to
the alkyne with the Bestmann-Ohira reagent18 to give 3-8.19
The scope of the Ru(II)-mediated cycloaddition with these
R-amino acid derivatives is shown in Table 1. The cycloadditions
were effected by the catalyst Cp*RuCl(COD), which had been
reported to be effective for the cycloaddition of secondary azides.20
For substrates with a diverse set of proteinogenic functionalities,
the reaction afforded the desired regioisomers 9-15 under mild
conditions and in moderate-to-high yield. No 1,4-disubstituted
regioisomers were detected by the comparison of NMR spectra to
those of analogous 1,4-disubstituted triazoles synthesized by Cu-
(I)-mediated cycloaddition.12 Toluene was used as the solvent,
unless the substrate was found to be insoluble (entries 2 and 3).
Benzyl protection of the carboxyl group of the azido substrate was
necessary for orthogonal deprotection relative to the acid-sensitive
The chemoselectivity of the 1,3-dipolar cycloaddition enables a
convergent synthetic route to the requisite triazole (Table 1). The
synthesis of the azido acids was achieved by Cu(II)-catalyzed diazo
transfer of commercially available amino acids,17 followed by
protection of the carboxyl functionality with a t-butyl or benzyl
† Department of Chemistry, University of WisconsinsMadison.
‡ Martin-Luther University.
§ Current address: Department of Chemistry, University of California, Berkeley.
|| Department of Biochemistry, University of WisconsinsMadison.
9
12670
J. AM. CHEM. SOC. 2007, 129, 12670-12671
10.1021/ja075865s CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Properties of RNase A Variants
by an ACS Division of Organic Chemistry Fellowship, sponsored
by Abbott Laboratories. U.A. was supported by Grant 3537C/0903T
(Land Saxony-Anhalt). NMRFAM was supported by Grant
P41RR02301 (NIH).
Tm
C)b
kcat/KM
1
c
residues 113
−114
origina
(
°
(107 M-1 s-
)
Asn-Pro (16)
bovine pancreas 64.0 ( 0.1
5.7
5.0
6.1
4.5
4.2
5.6
Asn-1,5-triazole-Ala (17) semisynthesis
Asn-1,4-triazole-Ala (18) semisynthesis
60.4 ( 0.2
54.3 ( 0.1
Supporting Information Available: Procedures for the preparation
of all novel compounds and related analytical data, including a crystal
structure of triazole 12. This material is available free of charge via
the Internet at http://pubs.acs.org.
Ala-Pro (19)
Escherichia coli 61.2 ( 0.2
Ala-1,5-triazole-Ala (20) semisynthesis
Ala-1,4-triazole-Ala (21) semisynthesis
60.8 ( 0.2
54.6 ( 0.2
a All variants except 16 contain an N-terminal methionine residue.
b Values were determined by CD spectroscopy in 50 mM sodium phosphate
buffer (pH 8.0) containing NaCl (25 mM) and protein (0.5-1.0 mg/mL).
c Values were determined at 25 °C in 0.10 M MES-NaOH buffer (pH 6.0)
containing NaCl (0.10 M). Experimental error was ∼10%.
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trityl group on the asparagine side chain (entry 3). It is noteworthy
that this chemistry is compatible with Boc- and Fmoc-protecting
group strategies (entries 6 and 7), both of which are common in
solid-phase peptide synthesis. Moreover, subjecting the resulting
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piperidine in DMF) produced no epimerization detectable by NMR
spectroscopy.
Next, we used expressed protein ligation21 to replace Asn113-
Pro114 of RNase A with Asn-1,5-triazole-Ala. To probe the
generality of our approach, we also replaced the analogous turn in
the N113A variant of RNase A with Ala-1,5-triazole-Ala. In
addition, we incorporated Asn-1,4-triazole-Ala and Ala-1,4-triazole-
Ala into RNase A. To do so, the four triazole surrogates were
incorporated into peptides corresponding to residues 95-124 by
solid-phase peptide synthesis. RNase A fragment 1-94 with a
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ods.6,22 After ligation of the two fragments, the semisynthetic
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variants 17 and 20 have Tm values comparable to those of the wild-
type enzyme (16) and its N113A variant (19), respectively. The
slight decrease in the Tm value of the 1,5-triazole variants could be
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by the pyrrolidine ring of Pro114. The 1,5-regioisomers (17 and
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the cis-prolyl bond, as indicated by the lower Tm values for the
latter pair.
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alkyne 4 had 88% ee; phenylalanine alkyne 6 had 98% ee. After
cycloaddition, the desired diastereomer was isolated by flash chromatog-
raphy.
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design. The trans/cis ratio of an Xaa-Pro peptide bond depends
on the nature of Xaa. The higher Tm value of wild-type RNase A
compared to its N114A variant is likely due in part to the greater
propensity of Asn-Pro than Ala-Pro peptide bonds to assume a
cis conformation.25 In contrast, the Tm values of the semisynthetic
proteins are independent of the Xaa residue. This distinction arises
because cis-trans isomerization is not a factor with the two Xaa-
triazole-Ala surrogates.
We conclude that Xaa-1,5-triazole-Ala modules can serve as
viable mimics of Xaa-cis-Pro segments in a protein. The possibility
of synthesizing this surrogate by the ligation of fragments in situ8
and the emergence of biocompatible catalysts for that process15,20
portends its widespread use.
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Acknowledgment. We thank I. A. Guzei for X-ray crystal-
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for contributive discussions. This work was supported by Grants
GM44783 (NIH) and INT 0129163 (NSF). M.B.S. was supported
(24) See Supporting Information.
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