Protein synthesis with conformationally constrained cyclic dipeptides

Article abstract of DOI:10.1016/j.bmc.2020.115780

We have synthesized several conformationally constrained dipeptide analogues as possible substrates for incorporation into proteins. These have included three cyclic dipeptides formed from Boc derivatives of 2,4-diaminobutyric acid, ornithine and lysine, having 5-, 6-, and 7-membered lactam rings, respectively. These dipeptides were used to activate a suppressor tRNA transcript, the latter of which had been prepared by in vitro transcription. Using modified E. coli ribosomes described previously, these activated suppressor tRNAs enabled the incorporation of the three cyclic dipeptides into dihydrofolate reductase (DHFR) at positions 18 and 49. The suppression yields increased with increasing lactam ring size and were found to proceed in suppression yields ranging from 3.4 to 8.9% at two different protein sites for the 5-, 6- and 7-membered lactam dipeptides. The greater facility of incorporation of the 7-membered lactam prompted us to prepare two 7-membered cyclic acylhydrazides (4 and 5) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-mediated cyclization of amino acids having selectively protected hydrazine functional groups in their side chains. In common with the lactam dipeptides, acylhydrazide dipeptides 4 and 5 could be used to activate the same suppressor tRNA transcript and to incorporate the cyclic dipeptides into DHFR. They were incorporated into the same two DHFR sites in suppression yields ranging from 8.3 to 11.2%.

Full text of DOI:10.1016/j.bmc.2020.115780

Journal Pre-proofs
Protein synthesis with conformationally constrained cyclic dipeptides
Chao Zhang, Xiaoguang Bai, Larisa M. Dedkova, Sidney M. Hecht
PII:
S0968-0896(20)30610-6
https://doi.org/10.1016/j.bmc.2020.115780
BMC 115780
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Bioorganic & Medicinal Chemistry
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Accepted Date:
27 July 2020
12 September 2020
16 September 2020
Please cite this article as: C. Zhang, X. Bai, L.M. Dedkova, S.M. Hecht, Protein synthesis with conformationally
constrained cyclic dipeptides, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115780
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Protein synthesis with conformationally constrained cyclic dipeptides
Chao Zhang, Xiaoguang Bai, Larisa M. Dedkova* and Sidney M. Hecht*
Biodesign Center for BioEnergetics and School of Molecular Sciences, Arizona State University,
Tempe, Arizona 85287, United States
ARTICLE INFO
Keywords:
Conformational constraint
Lactam and acyl hydrazide cassettes
Non-proteinogenic dipeptides
Protein modification
1

ABSTRACT
We have synthesized several conformationally constrained dipeptide analogues as possible
substrates for incorporation into proteins. These have included three cyclic dipeptides formed
from Boc derivatives of 2,4-diaminobutyric acid, ornithine and lysine, having 5-, 6-, and 7-
membered lactam rings, respectively. These dipeptides were used to activate a suppressor tRNA
transcript, the latter of which had been prepared by in vitro transcription. Using modified E. coli
ribosomes described previously, these activated suppressor tRNAs enabled the incorporation of
the three cyclic dipeptides into dihydrofolate reductase (DHFR) at positions 18 and 49. The
suppression yields increased with increasing lactam ring size and were found to proceed in
suppression yields ranging from 3.4 to 8.9% at two different protein sites for the 5-, 6- and 7-
membered lactam dipeptides. The greater facility of incorporation of the 7-membered lactam
prompted us to prepare two 7-membered cyclic acylhydrazides (4 and 5) by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI)-mediated cyclization of amino acids having
selectively protected hydrazine functional groups in their side chains. In common with the lactam
dipeptides, acylhydrazide dipeptides 4 and 5 could be used to activate the same suppressor tRNA
transcript and to incorporate the cyclic dipeptides into DHFR. They were incorporated into the
same two DHFR sites in suppression yields ranging from 8.3 to 11.2%.
2

1. Introduction
Nature has provided numerous examples of peptide hormones and other peptide-based mediators
of biological processes.1-5 In nature, it is not unusual for these to be employed under precise
temporal and spatial control. In contrast, the use of such compounds to control biological
processes experimentally, including during therapeutic intervention, requires consideration of the
chemical and metabolic stability of the peptide analogues employed. Frequently, it also requires
a consideration of peptide stability and conformation to optimize the desired properties of the
peptides as agonists and antagonists.6-11
Peptide stability has long been studied, e.g. by the replacement of proteinogenic amino acids in
peptides with amino acid and peptide surrogates resistant to natural peptidases, or which can help
to stabilize the native conformation.12-14 Some of these structural surrogates can also confer
useful conformational constraints, for example at peptide sites known to be conformationally
labile or important for receptor recognition.15 In recent years, however, it has become
increasingly common to control peptide conformation by creating peptides in which much of the
structure has more limited conformational freedom, e.g. by the use of cyclic peptides. These can
be made to restrain linear amino acid sequences, such as in cyclic peptides which mimic the
RGD domain of fibronectin.16-18 Modified ribosomal systems are currently also employed in
chemoenzymatic protocols to elaborate libraries of modified cyclic peptides containing non-
proteinogenic amino acids.19-21
In comparison, the modification of proteins with non-proteinogenic amino acids to confer
protein stability to cellular proteases is less common, although a number of examples may be
cited.22,23 Likewise, while many modifications undoubtedly alter protein conformation, the
3

purposeful construction of structural cassettes designed to constrain specific regions of proteins
to a subset of the conformations usually accessible has not been pursued as actively.
Recently, we have studied the use of modified bacterial ribosomes24 to introduce dipeptides and
dipeptidomimetic species into proteins.25-28 Presently, we describe the preparation of several
cyclic dipeptides that are constrained conformationally, but which can be incorporated into
proteins from an activated suppressor tRNA using modified bacterial ribosomes. In particular, E.
coli ribosomal clone 010328R225 containing a modified 23S rRNA was shown to incorporate
five conformationally constrained cyclic dipeptides (1 – 5) (Figure 1) into two different positions
of dihydrofolate reductase.
O
O
O
H2N
H2N
COOH
N
H2N
N
COOH
N
COOH
1
2
3
O
O
H2N
COOH
H2N
COOH
NCH3
N
N
NH
4
5
Figure 1. Structures of five conformationally constrained cyclic dipeptides synthesized and incorporated
into E. coli dihydrofolate reductase at positions 18 and 49.
2. Results and discussion
Compounds 1 – 3 were prepared for potential use to induce conformational constraints in
peptides in a report by Freidinger et al.29 Amblard et al.30 subsequently incorporated them in lieu
of Pro7-Phe8 in bradykinin and found that the resulting bradykinin analogues bound to the
human bradykinin B2 receptor. The preparation of dipeptide analogue 1, and the procedure used
for its activation of a suppressor tRNACUA transcript obtained by in vitro transcription, is shown
in Scheme 1. The routes used here for the preparation of compounds 1 – 3 were different from
4

those reported by Freidinger et al.,29 and there were no identical compounds due to differences in
protecting groups and the order of steps, so no comparison of properties can be made.
The synthesis of dipeptide 1 began with N-Boc (2S)-2,4-diaminobutyric acid, which was
O
O
O
H
H
N
b
H2N
c
a
N
NH
OH
Boc
N
OBn
Boc
Boc
60%
100%
HN
O
54%
O
7
6
O
O
O
d
e
OH
H2N
H2N
N
N
OBn
N
OH
N
H
100%
O
O
O
9
8
1
NH2
N
N
O
O
P
NH2
N
O
O
O
O
O
N
O
O
g
O
f
O
CN
N
N
O
P
N
N
H
7% (two steps)
O
O
9%
O
O
10
O
O
O
OH
N
NH2
N
N
H
11
N
O
O
NH2
N
tRNA-C
O
P
O
O
O
N
O
h
N
O
P
N
O
O
O
O
O
O
OH
N
N
H
O
12
Scheme 1. General procedures for the synthesis of a suppressor tRNACUA transcript activated with five-
membered lactam-constrained dipeptide analogue 1. Reagents and conditions: (a) BOP reagent, NaHCO3,
DMF, rt, 12 h; (b) NaH, benzyl bromoacetate, anh. THF, rt, 5 h; (c) 20% CF3COOH, anh. CH2Cl2, 0 oC, 1
h; (d) 10% Pd/C, H2, 95% EtOH, rt, 1h; (e) 4-pentenoyloxysuccinimide, diisopropylethylamine, anh.
DMF, rt, overnight; (f) chloroacetonitrile, Et3N, anh. DMF, rt, overnight; (g) pdCpA TBA salt, 9:1 anh.
DMF-Et3N, sonication, rt, 12 h; (h) T4 RNA ligase, tRNA-COH, ATP, 37 °C, 2 h.
converted to lactam 6 in 54% yield by treatment with the BOP reagent ((benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate). Treatment of lactam 6 with
sodium hydride and benzyl bromoacetate in THF at room temperature afforded fully protected
cyclic dipeptide 7 as a colorless oil in 60% yield after purification by silica gel chromatography.
Following deprotection in quantitative yield by successive treatments with CF3COOH and
5

hydrogenolysis over 10% palladium on carbon, free dipeptide 1 was isolated as its colorless
trifluoroacetic salt. The dipeptide was reprotected on N of the pyrrolidine moiety as its N-
pentenoyl derivative 9, and then activated as cyanomethyl ester 10 in low overall yield (7%)
following purification by column chromatography on silica gel.
In order to enable tRNA activation, activated ester 10 was combined with the
tetrabutylammonium salt of the dinucleotide pdCpA, which is a sequence common to the 3′-end
of all cytoplasmic tRNAs. The resulting pdCpA ester of 10 (compound 11) was formed in low
yield, and purified by C18 reversed phase HPLC, as were all of the pdCpA derivatives in this
study. As shown in Scheme 1, this activated dinucleotide was attached to a suppressor tRNA in
vitro transcript by enzyme mediated ligation with T4 RNA ligase.24 The N-pentenoyl protecting
group attached to N of the pyrrolidine moiety of 12 could be removed prior to the use of the
activated dipeptidyl tRNA in protein synthesis by brief treatment with dilute aqueous iodine.31,32
The synthesis of dipeptide 2 was carried out in a fashion closely analogous to the preparation
of 1, except starting with N-Boc-protected ornithine rather than N-Boc 2,4-diaminobutyric acid
(Schemes 1 and S1). The yields of the intermediates leading to the individual dipeptides 1 and 2
were also quite similar. Interestingly, while the two-step procedure leading to cyanomethyl ester
10 was very low, the analogous preparation of cyanomethyl ester 17 proceeded in 23% yield.
Likewise, while pdCpA derivative 11 was formed in 9% yield (Scheme 1), the yield of 18 was
18% (Scheme S1). The synthesis of dipeptide 3 was also carried out as for 1 and 2, but starting
with S-lysine. The yields of 3 were also quite similar to those realized in the steps leading to 1
and 2. For dipeptide 3, conversion to the fully protected cyanomethyl ester 24 proceeded in 21%
yield and the pdCpA derivative was obtained in 38% yield (Scheme S2).
The synthesis of dipeptide 4 is outlined in Scheme 2. Ethyl (2S)-2-[(benzoxycarbonyl)(tert-
6

O
O
O
O
c
a
b
BocHN
BocHN
H
O
N
O
N
O
90%
H
68%
67%
N
N
N
Boc
CBz
Boc
27
CBz
Boc
CBz
28
O
O
O
H
N
e
d
H2N
f
BocHN
CBz
N
O
NH
N
O
67%
CBz
HN
62% (two steps)
N
54%
CBz
N
CBz
CBz
Boc
29
CBz
30
31
O
O
O
O
H
O
O
H
N
H2N
O
N
g
h
i
CBz
N
N
N
OEt
OEt
CBz
OEt
NH
N
NH
62% (two steps)
90% (two steps)
32
33
34
NH2
N
N
O
O
P
NH2
N
O
O
O
O
N
N
O
CN
O
P
N
O
O
H
N
j
k
O
O
O
N
NH
O
O
15%
O
O
OH
H
N
O
35
N
O
NH
NH2
N
36
N
O
O
P
tRNA-C
NH2
N
O
O
O
N
N
O
O
P
N
O
O
O
O
O
O
OH
H
N
O
N
O
NH
37
Scheme 2. General procedures for the synthesis of a suppressor tRNACUA transcript activated with seven-
membered cyclic acylhydrazide-constrained dipeptide analogue 4. Reagents and conditions: (a) tert-butyl
carbazate, anh. THF, 0 oC, 18 h; (b) NaBH3CN, HOAc, EtOH, 0 oC, 17 h; (c) benzyl chloroformate,
diisopropylethylamine, anh. CH2Cl2, rt, overnight; (d) 37.5% CF3COOH, anh. CH2Cl2, 0 oC, 3 h; (e) (1)
3:1 THF–MeOH, LiOH, 0 oC, 3 h; (2) HOBt, EDCI, diisopropylethylamine, CH3CN, rt, 18 h; (f) NaH,
ethyl bromoacetate, anh. THF, rt, overnight; (g) 10% Pd/C, H2, 95% EtOH, rt, anh. MeOH, overnight; (h)
4-pentenoyl chloride, triethylamine, anh. CH2Cl2, rt, overnight; (i) (1) 3:1 THF–MeOH, LiOH, rt, 3 h; (2)
chloroacetonitrile, Na2CO3, anh. DMF, rt, overnight; (j) pdCpA TBA salt, 9:1 anh. DMF–Et3N, rt, 15 h;
(k) T4 RNA ligase, tRNA-COH, ATP, 37 °C, 2 h.
butoxycarbonyl)amino]-5-oxopentanoate33 was converted to hydrazone 27 in 68% yield by
treatment with tert-butyl carbazate, and then reduced to the corresponding hydrazine derivative
28 by treatment with NaBH3CN. Suitable positioning of protecting groups, and subsequent
7

cyclization afforded a 1,2-diazepane with CBz protecting groups on N-1 and the 4-NH2
substituent (31). Treatment with ethyl bromoacetate then introduced the C-atoms of glycine at
the 2-position of the diazepane. Following removal of the CBz protecting groups, the 4-NH2
substituent was protected by alkylation with 4-pentenoyloxysuccinimide, giving 34 in a yield of
62% for the two steps. Conversion of ethyl ester 34 to activated cyanomethyl ester 35 provided
an intermediate capable of effecting esterification of dinucleotide pdCpA with the modified 1,2-
O
O
O
b
c
a
BocHN
H2N
BocHN
N
O
N
OH
CBz
N
H
O
33% (three steps)
76%
N
HN
N
Boc
38
CBz
Boc
CBz
28
39
O
O
O
H
O
O
H
N
H2N
N
f
CBz
e
CBz
d
N
N
NH
N
OEt
OEt
N
N
55% (two steps)
92%
40
41
CN
O
O
O
O
H
H
N
h
76%
g
O
N
O
N
N
O
OEt
N
N
77% (two steps)
42
43
NH2
NH2
N
N
N
O
N
O
O
P
O
P
NH2
tRNA-C
NH2
N
O
O
O
O
O
O
N
N
N
N
i
N
O
O
O
N
O
P
N
O
O
P
O
O
O
O
O
O
O
O
O
OH
O
OH
H
N
H
N
O
O
N
N
O
O
N
N
44
45
Scheme 3. General procedures for the synthesis of a suppressor tRNACUA transcript activated with seven-
membered cyclic acylhydrazide-constrained dipeptide analogue 5. Reagents and conditions: (a) CH3I,
diisopropylethylamine, anh. DMF, rt, overnight; (b) (1) 50% CF3COOH, anh. CH2Cl2, 0 oC, 3 h; (2)
LiOH, 16:5:1 THF–MeOH–H2O, rt, 1 h; (c) 1:1 CH3CN–DMF, HOBt, diisopropylethylamine, EDCI, rt,
18 h, (d) NaH, anh. THF, ethyl bromoacetate, rt, overnight; (e) 10% Pd/C, anh. MeOH, rt, overnight; (f)
anh. CH2Cl2, diisopropylethylamine, 4-pentenoyl chloride, rt, overnight; (g)(1) LiOH, 3:1 THF–MeOH;
(2) anh. DMF, chloroacetonitrile, Na2CO3, rt, overnight; (h) pdCpA TBA salt, 9:1 anh. DMF–Et3N, rt, 15
h; (i) T4 RNA ligase, tRNA-COH, ATP, 37 °C, 2 h.
8

diazepine, albeit in low yield. Dinucleotide 36 was ligated to the abbreviated tRNACUA-COH
transcript, forming activated suppressor tRNA 37.
Methyl (2S)-2-[(enzoxycarbonyl)(tert-butoxycarbonyl)amino]-5-(tert
butoxycarbonylhydrazinyl)pentanoate (28), an intermediate in the synthesis of dipeptide 4, was
also used for the preparation of dipeptide 5 (Scheme 3). Following methylation of the hydrazine
moiety in 76% yield, compound 38 was treated with CF3COOH to remove the Boc protecting
groups and LiOH to hydrolyze the methyl ester. Cyclization by treatment with HOBt,
diisopropylethylamine, and EDCI then effected cyclization, affording (4S)-4-
(benzoxycarbonyl)amino-1-methyl--3-oxo-1,2-diazepane (40). Following treatment with ethyl
bromoacetate to effect substitution at position 2 of the 1,2-diazepane in 92% yield, the CBz
group at position 4 was replaced with an N-pentenoyl protecting group. Replacement of the ethyl
ester at ring position 2 with a cyanomethyl ester then enabled esterification of the dinucleotide
pdCpA with protected dipeptide 5.
The cyclic peptidyl-pdCpA derivatives (compounds 11, 18, 25, 36 and 44) were all ligated to
an abbreviated suppressor tRNACUA-COH prepared by in vitro transcription to afford the full
length activated tRNAs by the use of T4 RNA ligase, as illustrated in Scheme 1 for the
conversion of 11 → 12.31,32 The ligation of the cyclic dipeptidyl-pdCpAs to the abbreviated
tRNAs was verified by analysis on 8% polyacrylamine–7 M urea gels at pH 5.0 (Figure 2).34 As
noted in the Schemes, these tRNAs were protected with N-pentenoyl protecting groups, and were
deprotected prior to their use in a protein synthesizing system by treatment with aqueous iodine
at room temperature.31,32
9

1
2
3
4
5
6
Figure 2. Analysis of the activation of a suppressor tRNA transcript with cyclic dipeptides by
polyacrylamide gel electrophoresis under acidic conditions. Lane 1, abbreviated tRNACUA-COH (74
nucleotides). Lanes 2 – 6, ligation products of tRNACUA-COH with pdCpA derivatives 11, 18, 25, 36 and
44, respectively (76 nucleotides). The lower bands in lanes 2, 3 and 4 comigrated with the starting
material (tRNA-COH).
Because E. coli ribosomes normally incorporate only -L-amino acids, we have devised a
strategy to alter the 23S ribosomal RNA at key positions, and to select modified ribosomes
capable of recognizing activated tRNAs containing non-proteinogenic amino acids.24 These have
included D-amino acids, -amino acids, phosphorylated amino acids, dipeptides and
dipeptidomimetics.35 In the present case, we utilized ribosomal clone 010328R2, originally
selected to recognize a puromycin derivative that contained the dipeptide phenylalanylglycine.25
Because this clone was found to recognize several other dipeptides and dipeptidomimetic
species, we used it in the present study to determine whether it would incorporate
conformationally constrained dipeptides 1 ‒ 5 into the model protein E. coli dihydrofolate
reductase (DHFR).
Protein synthesis was carried out in triplicate independent experiments using an S-30
preparation with ribosomes from clone 010328R2. As shown in Figure 3, the five dipeptides
were all incorporated, although the amounts varied from one analogue to another, and seemed to
be incorporated somewhat more efficiently at position 18 than at position 49. The yields are
summarized in Table 1. As shown in the Table, at both positions of DHFR, dipeptide analogue 3
(7-membered ring) was incorporated more efficiently than analogue 2 (six-membered ring),
which was incorporated more efficiently than analogue 1 (five-membered ring). The suppression
10

A
B
1
1
2 3
4
5
5
6
7
2
3
4
6
7
Figure 3. SDS‒polyacrylamide gel electrophoretic analysis of the synthesis of proteins containing cyclic
peptides 1 – 5 at DHFR positions 18 (panel A) or 49 (panel B). (A) Comparison of wild-type DHFR (lane
1) and proteins synthesized using a mRNA with a UAG codon at position 18 in the absence of any
suppressor tRNA (lane 2) or in the presence of tRNACUA activated with dipeptide 1 (lane 3), dipeptide 2
(lane 4), dipeptide 3 (lane 5), dipeptide 4 (lane 6) and dipeptide 5 (lane 7). (B) Comparison of wild-type
DHFR (lane 1) and proteins synthesized using a mRNA with a UAG codon at position 49 in the absence
of any suppressor tRNA (lane 2) or in the presence of tRNACUA activated with dipeptide 1 (lane 3),
dipeptide 2 (lane 4), dipeptide 3 (lane 5), dipeptide 4 (lane 6) and dipeptide 5 (lane 7). In panel B, the
stronger lower bands are DHFR truncated at position 49 (no suppression). In panel A, the shorter
truncated protein ran off the gel.
yields for 7-membered ring dipeptides 4 and 5 at position 18 (11.2 ± 0.9% and 10.2 ± 0.3%,
respectively) were greater than for 7-membered ring 3 (8.9 ± 0.7%). The same relative
efficiencies of incorporation were noted at position 49, although the differences between them
was smaller (Table 1). Importantly, no significant incorporation of 1 – 5 was noted when an S-30
preparation made from wild-type ribosomes was employed (not shown). The choice of positions
18 and 49 in DHFR for replacement with non-proteinogenic amino acids was made to facilitate
characterization as described.36 Mass spectrometric data supporting the incorporation of 4 and 5
into DHFR at position 49 is illustrated in Table S1.
11

Table 1
Characterization of yield of full length DHFR during in vitro translation from modified genes in
the presence of different cyclic dipeptidyl-tRNACUA
s
Cyclic dipeptides
Amount of full length proteins from modified genes, % wt
TAG in position 18
0.6 ± 0.1
TAG in position 49
0.8 ± 0.2
-
1
2
3
4
5
4.2 ± 1.2
6.3 ± 1.1
8.9 ± 0.7
11.2 ± 0.9
10.2 ± 0.3
3.4 ± 0.8
4.8 ± 0.7
7.9 ± 2.5
8.7 ± 0.8
8.3 ± 1.2
The suppression yields in these experiments were less than what is sometimes observed for in
vitro suppression using wild-type ribosomes. Nonetheless, earlier studies from our laboratory
using DHFR37-39 have explored a range of substitutions enabling several biochemical studies to
be carried out, and the yields reported here will clearly allow such cyclic dipeptides to be used in
support of mechanistically focused mutagenesis experiments. One further issue that needs to be
addressed in future experiments is whether conformational constraint, which has been
exceptionally useful in producing peptides with improved properties, can also be exploited
productively for protein modifications. In this regard, it may be noted that nature already
employs proline to control conformation. This is accomplished both through judicious placement
of proline residues at protein sites that can benefit from conformational constraint,40 and from
cis-trans isomerization of the polypeptide backbone to effect reversible control of protein
conformation.41 It is anticipated that the conformationally constrained dipeptides described here
may find utility though the constraint of protein structure.
3. Conclusions
Five cyclic dipeptides have been prepared to enable the study of conformational constraint in
proteins, a type of modification that has proven very useful in peptides. The modifications have
included lactam-derived dipeptides having five-, six and seven-membered rings (1 – 3), and
12

seven-membered acylhydrazides (diazepanes) 4 and 5. Each of these synthetic dipeptides was
activated as a pdCpA ester and used to prepare a suppressor tRNA by the use of T4 RNA ligase.
The activated cyclic dipeptidyl-tRNAs were used to introduce the five dipeptides into positions
18 and 49 of E. coli dihydrofolate reductase. The suppression yields obtained in replicate
experiments were entirely sufficient to provide the amounts of protein required for routine
biochemical experiments.
4. Experimental section
All chemical and biochemical methods are described in detail in the Supplementary Material
sections.
Funding
This study was supported by National Institute of General Medical Sciences Grant GM12367,
National Institutes of Health.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Appendices A and B. Supplementary data
Supplementary data to this article can be found online at https://
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Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper
Graphical abstract
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