Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine-Lysine Relay

Article abstract of DOI:10.1021/acs.joc.8b00625

Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments.

Full text of DOI:10.1021/acs.joc.8b00625

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Article
Computation-guided rational design of a peptide motif that reacts
with cyanobenzothiazoles via internal cysteine-lysine relay
Samantha G. L. Keyser, Ashley Utz, and Carolyn R Bertozzi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00625 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
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Computation-guided rational design of a peptide motif that reacts with cyanobenzothiazoles via
internal cysteine-lysine relay
Samantha G. L. Keyser,†, ‡ Ashley Utz, Carolyn R. Bertozzi
‡
*,‡,§
†Department of Chemistry, University of California, Berkeley, California 94720, United States
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‡
§
Department of Chemistry and Howard Hughes Medical Institute, Stanford University, Stanford,
California 94305, United States
Corresponding Author’s Email Address: bertozzi@stanford.edu
Table of Contents/Abstract Graphic:
Abstract:
Site-selective protein modification based on covalent reactions of peptide tags and small
molecules is a key capability for basic research as well as for the development of new therapeutic
bioconjugates. Here we describe the computation-guided rational design of a cysteine- and lysine-
containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives.
Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety
to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine
residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for
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future development of peptide tags in the context of site-selective modification of lysine residues
within engineered microenvironments.
Introduction:
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Site-selective protein modification is an important tool for imaging targets in vitro and in vivo,
creating new therapeutics, and installing functionality on proteins to probe activity.1-4 However,
the limited number of proteinogenic amino acids and their natural repetition in proteins limits them
as targets for site-selective ligation. Easy access to different modifications, site selectivity, and
the potential for a large substrate scope makes covalent modification of engineered peptide tags
by small molecules an attractive and widely used platform for biotherapeutic and basic research.5
Much like an enzyme active site, these peptide tags are engineered to produce a
microenvironment that enables preferential ligation to a peptide sequence of interest, thus
allowing for selective and specific labeling of a protein. For application in biological systems, any
reaction between the peptide tag and the small molecule should proceed in aqueous conditions,
at physiological pH and temperature, and, if intended for in cellulo or in vivo studies, without
deleterious effects.
Previously, Tsien and coworkers described a foundational approach to rational design of a peptide
sequence that can be covalently modified by a small molecule.6-7 In their sequence (Cys-Cys-Pro-
Gly-Cys-Cys), four cysteine residues are pre-organized into a secondary structure that rapidly
reacts with FlAsH-EDT , a fluorescein-based diarsenical probe. The engineered peptide tag
2
consists entirely of proteinogenic amino acids, yet selective ligation is achievable –any off-target
interactions with naturally occurring peptide sequences can be reversed with addition of
ethanedithiol (EDT). As no unnatural amino acids or changes to the endogenous translational
machinery are necessary, perturbations to the cell are minimized. The early success of this
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rationally designed peptide for protein imaging inspired an ongoing wave of research into novel
peptide tags.
Recently, Pentelute and colleagues introduced the shortest small molecule-binding peptide to
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date, the ‘-clamp’ (Phe-Cys-Pro-Phe). Using a library selection method to identify peptides that
are subject to arylation reactions in water, they found that this four-residue tag provided a
favorable microenvironment for the cysteine residue to displace a fluorine substituent on a
perfluoroaromatic probe via a nucleophilic aromatic substitution. As they had previously shown
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that endogenous cysteines do not react with perfluoroaryl moieties in water, the discovery of -
clamp indicates the feasibility of designing other short natural amino acid sequences that elicit
emergent functionality.
Both the FlAsH tag and the -clamp exploit the tunability of cysteine’s reactivity when developing
new peptide conjugation methods. In addition, cysteine is the most reactive of the twenty
canonical amino acids at physiological pH and appears in proteins at a relatively low frequency,
which makes it a prime target for ligation chemistry.2-3, 5 However, cysteine plays fundamental
structural and chemical roles in proteins, so modifying or removing endogenous cysteine residues
from a protein of interest to accommodate an engineered peptide tag is not always feasible. Thus,
engineered peptide tags that enable site-selective modification of different amino acid residues
without requiring exogenous enzymes or translational machinery remain an unmet need.
Another amino acid that is nucleophilic under certain conditions is lysine. Cravatt and coworkers
recently reported the results of a global profiling experiment on lysine reactivity in the human
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proteome. They found that of the more than 9,000 lysines they quantified in the human proteome,
a few hundred had heightened reactivity, and over one hundred could be targeted by amine-
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reactive electrophilic fragments. As lysine is a common surface-exposed residue but non-
nucleophilic at physiological pH due to protonation of the side chain amine, these data suggest
peptide tags can form a microenvironment conducive to lysine modification. Further supporting
this hypothesis is the recent work of Bernardes and coworkers, in which the site-selective
modification of the single most reactive lysine on a given protein by sulfonyl acrylate reagents
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was reported. Moreover, the authors were able to computationally predict which lysine would be
modified in each of the five proteins they analyzed.
In designing a new small molecule-binding peptide tag, we took inspiration from the enhanced
amine reactivity seen in native chemical ligation (NCL) between an N-terminal cysteine and a C-
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terminal thioester (Figure 1A). During NCL, the nucleophilic N-terminal cysteine thiol undergoes
reversible transthioesterification with the C-terminal thioester. Then, the acyl group is irreversibly
transferred to the N-terminal amine, resulting in a new amide bond between the two original
peptides. The transthioesterification step increases the effective concentration of the amine,
greatly enhancing the rate of amide bond formation. Transglutaminases use a similar concept,
forming a thioester bond between an enzyme cysteine and a substrate glutamine which can then
be attacked by a second peptide’s N-terminal amine or a lysine side chain to form an isopeptide
bond.13-14 In the case of NCL, two distinct nucleophiles on the same residue are key to allowing
amide bond formation to proceed at a reasonable rate. We hypothesized that an engineered
peptide could position a cysteine residue and a lysine residue in such a way as to attain an
analogous functionality to NCL, but without the requirement of an N-terminal cysteine.
Additionally, we were drawn to the reaction between 2-cyanobenzothiazole derivatives and 1,2-
aminothiols. In the most widely used application, firefly luciferin is synthesized by reacting 6-
hydroxy-2-cyanobenzothiazole with D-cysteine in the laboratory and in nature (Figure 1B).15-16 The
cysteine thiol and the nitrile group react to form a thioimidate, which is then attacked by the lysine
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amine to form a tetrahedral intermediate. In contrast to NCL, in which the thiol “hands off” the acyl
group to the amine, the tetrahedral intermediate eliminates a molecule of ammonia to reach the
final product. Rao and coworkers found that the reaction of a 6-amino-2-cyanobenzothiazole (CBT)
-1 -1
derivative with cysteine proceeds with a second order rate constant of 9 M s in vitro at room
temperature and physiological pH.17 This feature as well as the reversibility of the reaction
between CBT and endogenous thiols allowed them to develop a method to selectively label
engineered N-terminal cysteines in vivo. Other groups have also devised clever ways to utilize
this reaction.18-20 Most recently, Lin and coworkers reported a genetically encodable CBT
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derivative-binding peptide tag discovered through a phage display experiment. As with previous
examples of engineered peptide tags, this too is a cysteine conjugation tag, and substituting
adjacent residues with lysine did not appear to change the mode or mechanism of binding.
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Figure 1. Chemoselective reactions involving aminothiols. A. Mechanism of native chemical
ligation. Transthioesterification occurs when an N-terminal cysteine on one peptide attacks a C-
terminal thioester on another. An SN acyl transfer occurs, irreversibly forming a new amide bond.
B. 6-Hydroxy-2-cyanobenzothiazole and D-cysteine react to form firefly luciferin. The cysteine
thiol attacks the nitrile group to form a thioimidate intermediate, then the amino group attacks the
thioimidate to form a tetrahedral intermediate. Finally, irreversible loss of ammonia drives the
reaction to completion. C. A proposed peptide (teal line) that uses cysteine and lysine residues to
accomplish a reaction analogous to the native chemical ligation. The cysteine attacks the
electrophile and then transfers it to the lysine residue in an irreversible reaction.
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With both NCL and the CBT reaction in mind, we focused on developing a short, genetically
encodable peptide tag that selectively and irreversibly ligates the nitrile group of a CBT derivative
under physiological conditions. To do so, we designed in silico a peptide with a lysine and a
cysteine residue oriented such that a selective reaction with the nitrile group of an N-acylated 6-
amino-2-cyanobenzothiazole would occur (Figure 1C). Herein, we show that our first-generation
engineered peptide creates a microenvironment for a “cysteine hand-off” mechanism analogous
to NCL, resulting in an irreversible, covalent ligation.
Results and Discussion:
Initially, we considered selecting candidate peptide sequences using the phage display method
reported previously Weiss, Wells, and coworkers.22-24 However, this method requires a disulfide
bond in the peptide library to restrict the conformational space during the selection process, which
complicates library generation and screening for our reaction of interest as our intended peptide
would require a reduced cysteine for reaction with CBT. Considering the knowledge that phage
display can lead to unanticipated results25 and the nontrivial nature of designing selection
conditions that would favor a final product with the CBT analog attached to the lysine rather than
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the cysteine, we alternatively pursued a rational design in silico. This approach yielded several
short peptide sequences (ten to eleven amino acids long) that efficiently bind cyanobenzothiazole
derivatives under various conditions.
A.
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Pro
N
Asp/Glu
HS
-
O
-
+
Pro
O
H N
SH
3
O
H N
N
Cys
HS
N
3
O
+
N
N
Pro
NH
NH
+
NH3
His
Lys
B.
AAAAAW GHGDGCKWGW GSGHGWAAAAA GGH PDPCPKGG
GGH PKPCPHGG
GGC PKPSPHGG
GGKPHPDPCGG
GGKPHPGPDGGa
GGKPCPHPDGG
GGRPWPKPHCGG
AAAAAW GDGCKW GWGSGHGAAAAA
GGEPCPHPKGG
GGRPWPKPHCGGG
AAAAAHDGCKGDHAAAAA
PWGDGCKWGWGSGHGWP
GGHPEPCPKGG
GGHPECPKGG
GGGGC PKPHGGGG
GGH PCPHPKGG
GGRPWPCPHKGGG
GGWPCPHPKGG
GGKPCPDPHGG
PGHDCKDHGP
GGGGGGH CD KHGGGGGGGG
GGGGGGH CD DKHGGGGGGGG
GGEPHPCPKGG
GGEPHKCGGG
GGGEHKCGGGG
GGH PCGH PKGG
GGH GC PHPKGG
GGH GC GHPKGG
GGH PCPKPHPHGG
GGH PHPCPKPHPHGG
GGH PHPCPKPHGG
GGWPHPCPKGGG
GGWPHGPCPKGG
GGWPHPGCPKGG
GGDPCPHPKGG GGCPDPHPKGG
GGGGGGH CGDKHGGGGGGGG
GGGGGGH CD GKHGGGGGGGG
GGGGGGH SDGKCGGGGGGGG
GGGEHCKGGGG
GGGFCPFGGG
AAAAAAAAFCPFAAAAAAAA
GGH CGH PKGG
GGH GC HPKGG
GGH PKPHGCGG
GGC PWPKPHGG
GGW PCPKPHGG
GGGGH PCPKPHPHGGGG
GGWPHPGKPCGG
GGHPWPGCPKGG
GGKPCGPWPHGG
GGGGGGH GDGKCGGGGGGGG
GGGGH SDGKCGGGGGG
GGGGH GDGKCGGGGGG
GGGGGGGH DGKCGGGGGGGG
GGGGGH DGKCGGGGGG
GGGGGGGD HGKCGGGGGGGG
GGHGDGKCGG
GGGGGGH GHGKCGGGGGGGG
GGGGGGGGH GKCGGGGGGGG
GGHCDGKGGG
AAAAAAAAFPCFAAAAAAAA
AAHPDPCPKAA
AAHPDPCPKPFAA
AAAAAAAAAHPDPCPKAAAAAAAAA
AAAAAAAAAHPDPCPKPFAAAAAAAAA
GGH PDPCPKPFGG
GGH PKPWPCGG
GGC PKPVPHPHGG
GGC PKPWPHGG
GGH PWPKPCGG
GGH PWPKPCGG
GGC PKPHPW GG
PPPPPPPPPP
GGAPKPHCW GG
PPPPPPPPPPPPPPPPPPPPPPPP GGW PHPKPCGG
PPPPPP
GGGGH CPKPHPHGGGG
GGH PHPCPKGG
GGKPCPHPHGG
GGKPCPWPHGG
GGH PWPCPKGG
GGWPHPCPKGG
GGH PHPKPCGG
GGH PWPKPCGG
GGKPCPGWPHGG
GGCPKPGWPHGG
GGFMGFPHPKPHPCGSGG
GGHPDCPKGG
GGHPDPCPKGG
GGHPDPCKGG
GGKPCPHPDGG
GGWPHPCPKGG
GGWPHPGCPKGG
GGFPFPKPHPCGG
GGFPFPGKPHPCGG
GGFPFPGPKPHPCGG
GGFPFPCPKPHPCGG
GGFPFPCPKPCPHGG
GGHPCPKPGPFPFGG
GGHPCPGPKPFPFGG
GGCPHPGPKPFPFGG
GGCPKPFPFPDPHGG
GGCPHPFPFPKGG
GGH PDPCPKFGG
GGH PGPDPKFGG
GGH PDPCPFPKGG
GGH PDPCPFKGG
GGH PTPCPKGG
GGC PKPTPHGG
GGH PHPKPCPDGG
GGC PTPKPHGG
GGHPHPKPCPEGG
GGH PWPKPCPDGG
GGHPWPKPCPEGG
GGC PKPYPHGG
GGHGDGCKGG
GGDGHGCKGG
GGH PDPCPYKGG
GGH PDPCPWKGG
AAAAAAAAAAPAAAAAAAAAA
GGKCPHPPPHGG
GGGC PHKPPHGG
GGKPCPHPPPHGG
AAAAAPPPAAAAA
GGC PHPHPKGG
GGC PHPWPKGG
GGC PKPEPHGG
GGHPEPCPKGG GGKPCPGWPHGG
GGHPHPKPCPEGG
GGDGHGCGKGG
GGGGH PDPCPW KGGGG
GGKGDGHGCGG
GGGGH PDPKCPWGGGG
GGHPCPFPFPKGG
GGKGDHGCGG
GGKGDHCGGG
GGDGHPCGKGG
GGDGHPCPKGG
GGGGH PCPKDW GGGG
GGGGH PCPDKW GGGG
GGGGH PCPDKPWGGGG
GGGGH WCPDKGGGG
GGSPCPFPFPKGG
GGHCPFPFPKGG
GGHPFPFPCPKGG
GGHPFPFPKPCGG
GGHPECPKGG
GGH PYPCPKGG
GGDPHPCPKGG
GGKGHPCDGG
GGDPHPCKGGG
GGGGH CPDKPW GGGG
GGGGW PCPDKHGGGG
GGGGGGGGH PDPCW KGGGGGGGG
GGCPKPDPHGG
GGC PKPHPEGG
GGC PKPHPDGG
GGC PKPHPYGG
GGKPCPHPYGG
GGC PKPRPHGG
GGHPKPFPFPCGG
GGHPKPCPFGG
GGCPHPFPKGG
GGEPHPCPKGG GGHPWPKPCPEGG
GGDGHPCKGGG
GGDPHPCKGGG
GGHPDPCKGGG
DGDPHPCKGGS
GGGGGGH CH GKGGGGGGGG
GGDPHPCGKGG
GGDPHPGCKGG
GGDPCPHGKGG
GGDPCPHPKGG
GGHPCPDGKGG
GGHPCPDPKGG
GGHPKPDPCGG
GGHPDCPKGG
GGGGGGGGH PW PDPCPKGGGGGGGG
GGGGGGGGH PDPW PCPKGGGGGGGG
GGGGGGGGH PDPCPW PKGGGGGGGG
AAAAAAAAAHPPDPCPKAAAAAAAAA
GGGGGGGGD PHPKPCPWGGGGGGGG
GGGGGGGGH PDPCPW KGGGGGGGG
GGH PPDPCPKGG
GGH PKPHPCGG
GGC PHPKPHGG
GGCPKPHPHGG
GGC PHPKPDGG
GGH PDPKPCGG
GGH PCPKPDGG
GGHPKPCPDGG
GGD PCPKPHGG
GGD PHPKPCGG
GGD PKPHPCGG
GGD PKPCPHGG
GGC PHPDPKGG
GGCPDPHPKGG
GGC PRPKPHGG
GGC PKPKPHGG
GGC PKPHPKGG
GGC PHPKPKGG
GGKPCPHPKGG
GGKPKPHPCGG
GGKPKPCPHGG
GGH PHPKPCPRGG
GGH PKPHPCPRGG
GGH PWPKPCPRGG
GGC PKPHPRGG
GGR PWPKPCPHGG
GGR PWPKPHPCGG
GGCPFPHPKGG
GGHPCPKPFGG
GGFPCPKPHGG
GGFPCPKPHFG
GGFPCPKPHFGG
GGHPKPGCGFGG
GGCPKPHPHGG
GGCPKPDPHGG GGHPKPCGFGG
GGHPKPCPDGG GGHPKPGCPFGG
GGHPKPGCPFGG
GGHPKGPCPFGG
GGHPKPGCGFGG
GGHPKPGCFGG
GGHPKPCGFGG
GGHPCPKGFGG
GGHPKPWGFGG
!
GGH PCPKPHGG
GGC PKPHHGG
GGC PKHHGG
GGC PDPKPHGG
GGKPDPHPCGG
GGKPDPCPHGG
Rational design and modeling
of peptide sequences
Synthesis of most promising
peptides
Identification of
best sequence
C.
Examples of sequences selected for synthesis
Examples of sequences not selected for synthesis
Figure 2. Rational design of a peptide that covalently binds a CBT derivative. A. The principles
behind generating peptide sequences for computational modeling. Lysine and cysteine residues
were interspersed with histidine and aspartic or glutamic acid residues to increase nucleophilicity.
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Prolines were added to lock the sequence (teal) into a favorable conformation for reaction with a
CBT derivative. B. Selection of sequences to test for reaction with a CBT derivative. 218
sequences were modeled using the program PEP-FOLD (see Supplemental Table S1 for a full
list).26-27 Of these peptides, eighteen that best fit our selection criteria were synthesized by solid-
phase peptide synthesis (SPPS) and tested in a binding assay. C. Examples demonstrating how
peptides were selected for SPPS. The peptides on the left were two of the best models, whereas
the two peptides on the right were not chosen for SPPS.
0
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0
For the in silico approach, the online computational tool PEP-FOLD26-27 was used to model short
peptides containing lysine, cysteine, histidine, aspartic or glutamic acid, and proline residues.
Inspired by enzyme-resident catalytic triads, we hypothesized that histidine and aspartic acid
would increase the nucleophilicity of cysteine and lysine residues if proline residues were
incorporated to help lock the peptide into the desired conformation (Figure 2A). In addition, the
protonated imidazole of the histidine residue could also catalyze the reaction between the
cysteine thiol and the nitrile group of a CBT derivative.28-29 Of the 218 sequences modeled
(Supplemental Table S1), eighteen were selected for synthesis via solid phase peptide synthesis
(SPPS) and further analysis (Figure 2B, C). In Figure 2C, the two peptides on the left are
representative of the best models: the lysine, histidine, aspartic acid, and cysteine residues are
all in relatively close proximity. In particular, the histidine side chain appeared poised to help
deprotonate the lysine residue. The two peptides on the right do not fit our criteria: either the
histidine residue was not in a position to deprotonate the cysteine or the lysine residue or the
cysteine and lysine residues appeared too far apart to feasibly work together in forming covalent
bonds with a CBT derivative.
13
Based on MALDI-TOF MS, many of the peptides reacted with N-(2-(cyano- C)-benzo[d]thiazol-
6-yl)acetamide (CBTNAc, compound 1) in phosphate-buffed saline (PBS) at room temperature
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over 48 hours even in the absence of reducing agents such as dithiothreitol (DTT) or tris(2-
carboxyethyl)phosphine (TCEP) (assay workflow and representative data shown in Figure 3; data
for all eighteen peptides, peptides 4-21, shown in Supplemental Figure S1). Several but not all of
the peptides also reacted with two equivalents of the CBT derivative. We hypothesized that if only
one equivalent of CBTNAc was bound, it could be attached to either the lysine or cysteine residue,
while if two were bound, they were attached to both the lysine and cysteine residues. In a follow-
up experiment, free cysteine was added to the peptide-CBTNAc adducts to trap unbound
CBTNAc as a luciferin analog. As the reaction of the peptide’s cysteine residue with the CBTNAc
nitrile group is reversible, any CBTNAc bound to the cysteine and not transferred to the lysine
residue should be in equilibrium with free peptide and CBTNAc. When free cysteine is added, it
reacts irreversibly with unbound CBTNAc to form the luciferin analog. Depletion of the unbound
CBTNAc in this manner will then drive the peptide-CBTNAc adduct toward free CBTNAc and an
unmodified cysteine residue on the peptide by Le Châtelier’s Principle. Indeed, addition of free
cysteine resulted in significant loss of CBTNAc from the peptides, with only one equivalent
remaining on some (Figure 3B, Supplemental Figure S1) and no modification remaining on others
(Figure 3C, Supplemental Figure S1). Placing the cysteine and the lysine residues adjacent to
one another resulted in minimal reaction with CBTNAc, but placing a proline in between them was
not in itself sufficient to ensure significant formation of a stable adduct (Supplemental Figure S1F,
S1O), while placing additional residues in between the cysteine and the lysine resulted in
formation of just as much or more of the stable CBTNAc adduct as some peptides with cysteine
and lysine close together (e.g., Supplemental Figure S1C, S1J, and S1Q compared to S1F, S1O).
Exchanging aspartic acid and glutamic acid altered the reactivity of a given peptide (Supplemental
Figure 1A, 1E), as did replacing an aspartic or glutamic acid residue with or adding a residue such
as histidine (Supplemental Figure S1H, S1O), tryptophan (Supplemental Figure S1L, S1M, S1N,
S1P), or phenylalanine (Supplemental Figure S1Q, S1R).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
4
5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1
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1
1
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
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3
3
3
4
4
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4
4
4
4
4
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5
5
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5
5
5
5
6
A. I
II
0
1
2
3
4
5
6
7
8
9
0
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2
3
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5
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9
0
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9
0
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2
3
4
5
6
7
8
9
0
(
1)
III
B. Peptide with extensive modification of Lys: GGHPDPCPKGG
1063.49, [M+H]+
1501.81
–
-
.5–
I
II
- III
5 –
2
.1
[M+2CBTNAc+H]+
2
1
–
-
1281.63
- [M+CBTNAc+H]
085.55, [M+Na]+
+
1
1282.17
4
3
2
1
–
1
0
0
[M+CBTNAc+H]+
107.02, [M+2Na]+
-
–
-
1
-
–
-
.9
–
-
–
–
-
–
-
.3
|
|
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|
1000
1200
1400
1600 1000
1200
1400
1600 1000
1200
1400
1600
m/z
m/z
m/z
C. Peptide with minimal modification of Lys: GGDPHPCKGG
1.0–
-
2
.5
–
I
– II
III
6
5
1229.45
-
–
-
[
M+CBTNAc
0.8–
2.0–
2Na]+
+
-
4–
-
1
.5
.0
0.6
.4
–
-
–
–
–
1
[
009.38
1447.53
M+2Na]+
3–
-
1009.88
1229.27
1009.54
[M+2CBTNAc
0
[M+2Na]+ [M+CBTNAc+2Na]
+
1
[M+2Na]+
–
-
–
+2Na]+
2
–
-
0.2
0.5
1–
-
|
|
|
|
|
|
|
|
|
|
|
|
|
|
-|
|
|
|
|
|
|
900
1100
m/z
1300
1500 900
1100
1300
1500
900
1100
1300
1500
m/z
m/z
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Figure 3. Assay for peptides that are covalently modified by CBTNAc (1) and representative
results. A. Overview of assay used to determine degree of irreversible covalent modification of
candidate peptides. Candidate N-terminal acetylated, C-terminal amidated peptides were
incubated with CBTNAc in PBS for 48 h at room temperature, then an aliquot of the reaction
mixture was analyzed by MALDI-TOF MS. The remaining solution was incubated for 48 h at room
temperature with free cysteine, with or without DTT or TCEP reducing agent, and the reaction
mixture was again analyzed by MALDI-TOF MS. B. Representative MALDI-TOF MS spectra at
each stage of the assay for a peptide with a lysine residue extensively modified by CBTNAc
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
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2
2
3
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3
3
3
4
4
4
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
peptide 4). The first spectrum (I) shows crude peptide alone, the second spectrum (II) was taken
after incubation with CBTNAc and desalting, and the third spectrum (III) followed incubation with
free cysteine. C. Representative MALDI-TOF MS spectra at each stage of the assay for a peptide
with a lysine residue not significantly modified by CBTNAc (peptide 5). The samples analyzed in
each spectrum were prepared the same way as in B. Red boxes indicate the expected locations
of masses corresponding to modified peptide.
Of the eighteen peptides tested, we proceeded with characterization of the peptide modified to
the greatest extent by CBTNAc, CBTag 1.0 (Ac-Gly-Gly-His-Pro-Asp-Pro-Cys-Pro-Lys-Gly-Gly-
NH , 4). Performing the assay in the presence of 5 mM glutathione to mimic physiological thiol
2
conditions still resulted in a significant amount of the post-cysteine treatment CBTag 1.0-CBTNAc
adduct, though less of it than when conducting the assay in the absence of glutathione
(
Supplemental Figure S2), These results indicated that some competition between endogenous
thiols and the CBTag 1.0 cysteine is probable, but, promisingly, a stable bond between CBTag
.0 and CBTNAc can still form. To determine the necessity of the cysteine and the lysine residues,
1
two modified peptides were synthesized: one with the lysine residue converted to arginine to
preserve the positive charge while decreasing nucleophilicity further (22), and one with the
cysteine residue converted to serine to decrease nucleophilicity (23). These derivatives were
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5
6
subjected to the conditions described in the previous paragraph. The lysine to arginine mutant
reacted with one equivalent of CBTNAc before cysteine treatment, likely due to the cysteine
residue in the peptide (Supplemental Figure S3A). Treatment with free cysteine abrogated binding,
resulting in free peptide, as expected. The cysteine to serine mutant did not bind any equivalents
of CBTNAc, indicating that the cysteine residue is necessary for activity (Supplemental Figure
S3B). Thus, both cysteine and lysine are necessary for irreversible covalent bond formation with
CBTNAc. Combined with the earlier observation that not all of the synthesized peptides
irreversibly reacted with CBTNAc, these data led us to propose that cysteine and lysine are
necessary but not sufficient for CBTNAc binding, indicating that conformation of the peptide is
important for the intended reactivity.
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0
To further establish that the remaining equivalent of CBTNAc was not attached to the cysteine
residue, the peptide-CBTNAc adduct was reacted with chloroacetate after treatment with free
cysteine. Chloroacetate specifically reacts with thiols, not amines, and thus should only label the
peptide-CBTNAc adduct if the CBTNAc is not bound to the cysteine residue (i.e., if the CBTNAc
is bound to lysine). Indeed, a clean mass shift indicating reaction with chloroacetate was observed
for the CBTag 1.0-CBTNAc adduct after cysteine treatment (Supplemental Figure S4A). Moreover,
when CBTag 1.0 was incubated with CBTNAc but not treated with free cysteine, chloroacetate
did not react with any peptide bound to two equivalents of CBTNAc. If the CBTag 1.0 peptide was
pretreated with chloroacetate, no binding of CBTNAc occurred, though this could be due to the
addition of a negative charge causing a conformational shift (Supplemental Figure S4B, 4C). As
a control, we confirmed that the cysteine to serine mutant did not react with chloroacetate, while
the lysine to arginine mutant did (Supplemental Figure S4D).
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Additionally, tandem mass spectrometry (MS/MS) on CBTag 1.0 following cysteine treatment
confirmed that the remaining CBTNAc was indeed bound to lysine (Figure 4). Finally, 13C NMR
spectra acquired after reaction between CBTNAc with a 13C-labeled nitrile group (13CBTNAc,
compound 3) and CBTag 1.0 did not contain a peak corresponding to a stable tetrahedral
intermediate but did contain peaks that match predicted values for the -carbon of a thioimidate
or amidine bond (CBTag 1.0-13CBTNAc amidine adduct 24, Supplemental Figure S5).
Figure 4. Tandem mass spectrometry (MS/MS) on CBTag 1.0-CBTNAc demonstrates that
1
1
1
1
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1
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0
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0
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0
Modification at Cys
Modification at Lys
y y
y y
Ac-G G H P D P C P K G G-NH
2
Ac-G G H P D P C P K G G-NH
2
b b
b b
-
-
MS/MS, 640.8 m/z, [M + CBTNAc + 2H]2+
5
4
3
2
1
–
-
775.3
-
-
-
–
-
-
2
66.1
-
-
–
575.3
-
-
-
-
–
9
87.4
-
706.3
-
-
-
167.1
506.2
–
-
-
-
0 -
–|
|
|
|
|
|
|
|
|
|
|
|
|
0
0
200
400
600
m/z
800
1000
1200
modification is on the lysine residue. Top, predicted masses for MS/MS b and y product ions if
the CBTNAc (purple star) is on the cysteine residue (left) or the lysine residue (right). Bottom,
tandem MS/MS on the [M + CBTNAc + 2H]2+ ion shows product ions matching those predicted
for lysine modification (green boxes).
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5
6
In order to quantitate relative amounts of starting materials, intermediates, and products over time,
we developed an LC-MS method to follow the course of the reaction. As expected, the lysine
adduct, the cysteine adduct, and the doubly modified peptide were resolvable using our method,
though higher concentrations of the peptide than were used in the MALDI-TOF assay were
necessary in order to clearly observe a 210 nm signal suitable for integration (Supplemental
Figure S6-S7). In order to deconvolute the data, TCEP was included in the injected samples to
remove peaks corresponding to disulfide dimers. Comparing peak areas in the chromatographs
over time, we found that at the first time point (1.75-2.8 h), the majority of the CBTNAc attached
to CBTag 1.0 at the final time point (40-45 h) was already covalently bound to either a cysteine
or a lysine residue; however, only 7-10% of this peptide-bound CBTNAc population was attached
to lysine, suggesting the thioimidate intermediate forms relatively quickly but transfer to the lysine
is slow (Supplemental Figure S8, Table S2-S4). By the end of the experiment, approximately 35-
0
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41% of peptide-bound CBTNAc was on a lysine residue whether the ratio of CBTag 1.0 to
CBTNAc was 1 to 1 (22.5 mM each), 4 to 1 (22.5 mM and 5.63 mM), or 1 to 4 (5.63 mM and 22.5
mM), while the percentage of total CBTag 1.0 that had a lysine residue modified by CBTNAc
ranged from 17 to 38% depending on the conditions (Supplemental Figure S8, Table S2-S4). The
low variation in rate of formation of lysine-bound CBTNAc despite significant changes in
concentration of the peptide also provide evidence that transfer of the CBTNAc is intramolecular,
not intermolecular. These numbers matched the approximate yields of 16-36% we obtained when
we isolated the amidine product after cysteine treatment. However, somewhat unexpectedly
based on the MALDI-TOF MS results, conversion of the free peptide to peptide modified on the
cysteine, the lysine, or both ranged from 38 to 80% - under the conditions of this assay, full
conversion to a bound state was not observed even when CBTNAc was present in excess
(
Supplemental Figure S8, Table S2-S4). We attribute this to reversion of some portion of the
cysteine monoadduct to the free peptide and unreacted CBTNAc on passage through the column
(no peaks corresponding to hydrolysis of CBTNAc were observed), unforeseen effects of
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including TCEP in the reaction, aggregation of the peptide or the CBTNAc at higher
concentrations, or differences between running the reaction in a test tube and an LC-MS vial.
Again, as a confirmation, the lysine to arginine mutant did form a peak corresponding to cysteine
modification over time, while the cysteine to serine mutant spectrum did not change
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
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3
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0
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0
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0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Supplemental Figure S9-11).
As an additional confirmation that transfer from cysteine to lysine is intramolecular and not
intermolecular, a 1:1 mixture of CBTag 1.0 (K9R) (22) and CBTag 1.0 (C7S) (23) was subjected
to our initial MALDI-TOF MS assay conditions (Supplemental Figure S12). Since CBTag 1.0 (K9R)
has a cysteine but not a lysine and reacts with one equivalent of CBTNAc prior to cysteine
treatment, while CBTag 1.0 (C7S) has a lysine but not a cysteine and does not react with CBTNAc
at all, any formation of a CBTag (C7S)-CBTNAc adduct would have to be a result of intermolecular
cysteine-lysine relay. While a mass corresponding to formation of the CBTag 1.0 (K9R)-CBTNAc
adduct was observed prior to cysteine treatment, no mass corresponding to the CBTag 1.0 (C7S)-
CBTNAc adduct appeared. Moreover, treatment with cysteine reversed the CBTag 1.0 (K9R)-
CBTNAc adduct, resulting in a MALDI-TOF-MS spectrum that showed only the unreacted
peptides. These data and the LC-MS data indicating that rate of formation of the CBTag 1.0-
CBTNAc lysine adduct is independent of peptide concentration suggest an intramolecular
cysteine-lysine transfer mechanism.
Together, our results support the hypothesis that the nucleophilic cysteine thiol rapidly reacts with
the CBTNAc to form a thioimidate adduct, which is slowly transfered to the lysine, creating a
stable amidine bond (Figure 5). To further rationalize why the tetrahedral intermediate proceeds
to the amidine product instead of the product of the condensation reaction (as in luciferin), we
used Maestro software to build both products and the integrated program MacroModel to search
for the lowest energy conformations. The optimized conformation of the amidine product had a
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significantly lower potential energy than that of the product of the condensation reaction, even
taking into account the released ammonia molecule (-488 kcal/mol and -456 kcal/mol, respectively;
a difference of 22 kcal/mol) (Supplemental Figure S13). These results are not unexpected, as the
entropic gain from the release of ammonia is unlikely to compensate for the entropic loss
associated with the increased rigidity of an already large macrocycle, especially in the absence
of conditions favoring the loss of ammonia. Additionally, the transition state for the amidine
product is likely lower in energy relative to that of the condensation product. In both transition
states, a partial positive charge builds up on a nitrogen atom, but in the transition state for the
amidine product, the partial negative charge is on the cysteine sulfur atom, whereas in the
transition state for the condensation product, the partial negative charge is on a nitrogen atom.
Sulfur atoms are more polarizable than nitrogen atoms and are better able to stabilize a negative
charge; thus, the amidine transition state is stabilized more than the condensation reaction
transition state. In sum, both the transition state and the product energies favor the amidine
product.
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Figure 5. Proposed mechanism of reaction between CBTag 1.0 and CBTNAc. First, the
nucleophilic thiol on the cysteine residue reversibly forms a thioimidate bond with CBTNAc, likely
catalyzed by the imidazole of the histidine residue.28-29 Then, the lysine amine attacks the
thioimidate, resulting in a tetrahedral intermediate. Unlike the reaction to make firefly luciferin, a
condensation step that releases ammonia does not proceed (red arrows). Instead, the end result
is a stable amidine bond between the CBTNAc and the lysine residue in a “cysteine hand-off”
mechanism analogous to that of NCL.
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Conclusion:
In conclusion, we have designed and characterized a peptide that forms an irreversible amidine
bond between a uniquely reactive lysine residue and a CBT derivative. We have demonstrated
that similar to NCL, this mechanism occurs through transfer of the electrophilic group from a
cysteine thiol to a lysine amine. These results are a promising first step toward a novel peptide
tag for site-specific lysine functionalization, though significant optimization remains to be done
before this system can be implemented as a practical tool. We are currently exploring mRNA
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30
display-based methods to select for an improved version of CBTag 1.0 as well as computational
methods to engineer a peptide sequence that strongly favors the reactive conformation of the
cysteine and lysine residues. Regardless, in using concepts that invoke themes from enzymology
to bioorthogonal chemistry, we provide here a potential template for rational design of peptide
tags with engineered “hot” lysines that can undergo site-selective covalent modification.
Experimental Section:
General. Reactions were performed in flame- or oven-dried glassware under an inert nitrogen
atmosphere unless otherwise noted. Anhydrous solvents were either purchased or obtained by
passing solvent through an activated alumina column via a Pure Process Technology Glass
Contour Solvent Purification System. All reagents and solvents were used as received unless
otherwise noted. Water was passed through a Milli-Q filtration system prior to use. Where noted,
samples were concentrated in vacuo at 40 C using a BÜCHI Rotavapor R-114 equipped with a
BÜCHI B-480 heating bath and a Welch Self-Cleaning Dry Vacuum System (Model 2025) or an
IKA RV 10 basic rotary evaporator equipped with an IKA HB 10 basic heating bath and a Welch
Self-Cleaning Dry Vacuum System (Model 2025). If necessary, compounds were then further
dried under high vacuum using an Edwards RV8 Two Stage Rotary Vane Pump or by
lyophilization in a LABCONCO FreeZone 4.5Plus.
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Thin layer chromatography was performed using SiliCycle SiliaPlate glass-backed silica gel plates
containing a fluorescent indicator (Fisher Scientific 50964470). Plates were visualized using a
UVGL-25 Compact UV Lamp, 254/365 nm, 4 W (P/N 95-0021-12). For flash column
chromatography, the stationary phase was SiliCycle SiliaFlash P60 or Fisher Silica Gel Sorbent
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(
230-400 Mesh, Grade 60) silica gel. For purifications involving preparative or semi-preparative
Reverse Phase-High Performance Liquid Chromatography (RP-HPLC), the following conditions
were used: the instrument consisted of an Agilent Technologies ProStar 325 UV-Vis detector, two
PrepStar Solvent Delivery Modules, and a 440-LC Fraction Collector; the column was either a
Varian Microsorb 100Å C18, 8 m, 21.4 x 250 mm Dynamax preparative column (R0080220C8)
equipped with a Microsorb 100Å C18, 8 m guard column (R0080220G8) or an Agilent ZORBAX
Eclipse XDB 80Å C18, 5 m, 9.4 x 250 mm semi-preparative column (990967-202). Solvent A
was 0.1% TFA in Milli-Q water; solvent B was 0.1% TFA in acetonitrile (MeCN). The UV-Vis
detector was used to monitor wavelengths at 210 and 254 nm. All pure compounds and stock
solutions were stored at -20 C.
Liquid Chromatography-Mass Spectrometry (LC-MS) experiments were performed on an Agilent
Technologies 1260 Infinity attached to a 6120 Quadrupole MS and a Peak Scientific NM32LA
nitrogen generator. An Agilent InfinityLab Poroshell 120 EC-C18, 2.7 m, 4.6 x 50 mm analytical
LC column (part number 699975-902) was used. As with the HPLC, solvent A was 0.1% TFA in
Milli-Q water; solvent B was 0.1% TFA in MeCN. Wavelengths of 210 nm, 254 nm, and 280 nm
were monitored using the diode array detector. For ESI-MS, the 105-2000 m/z range was
monitored in both positive and negative ion mode. Data was processed and analyzed using
LC/MSD ChemStation (Agilent Technologies, Rev. B.04.03[16]). High resolution mass
spectrometry (HRMS) data were acquired by ESI-LC/MS on a Waters Acquity UPLC and Thermo
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Exactive Orbitrap mass spectrometer by Dr. Theresa McLaughlin at the Stanford University Mass
Spectrometry facility.
MALDI-TOF MS spectra were obtained on either an Applied Biosystems Voyager-DE PRO
BioSpectrometry Workstation using an Applied Biosystems Sample Plate SS, Numbers & Circles
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(
P/N V700666) and the Applied Biosystems Voyager Instrument Control Panel v5.10 software
(QB3/Chemistry Mass Spectrometry Facility, UC Berkeley) or a Bruker microFlex MALDI-TOF
(S/N 256969.00028) using a microScout Target MSP 96 target polished steel BC plate (P/N
8280800) and the Bruker Daltonics FlexControl software (Vincent Coates Foundation Mass
Spectrometry Laboratory, Stanford University). In all cases, the matrix was -cyano-4-
hydroxycinnamic acid (CHCA) dissolved in 50% MeCN in 0.1% TFA. Plates were spotted with a
mixture of 0.5 L sample and 0.5 L matrix. For the Voyager, parameters were as follows:
reflectron mode, positive ion detection; pulsed nitrogen laser (337 nm, 20 Hz); accelerating
voltage 20 kV, grid voltage 94%, guide wire voltage 0.05%, delay time 270 nsec; mass range 200-
2500 Da, low mass gate 200 Da; spectrum acquisition – manual control, 1500 shots/spectrum;
manual laser intensity ~1500-2500. For the Bruker microFlex, the parameters were as follows:
reflectron mode, positive ion detection; pulsed nitrogen laser (337 nm, 3 ns pulse width, pulse
energy 150 J, 60 Hz); detector gain (reflector) 4.0x, 1810 V; sample rate 2.00 GS/s; laser power
3
4
percentage was adjusted on a sample-by-sample basis to obtain an arbitrary intensity of 10 -10
and a flat baseline; mass range 0-4000 Da; spectrum acquisition – random walk partial sample
mode, results were the sum of at least 3 x 100 laser shots; calibration of the system was
performed using the Bruker Peptide Calibration Standard mixture (part number 8206195).
Spectra were analyzed using the open-source software mMass (version 5.5.0)31-33 after data had
been exported as an ASCII file from Voyager (Applied Biosystems) or FlexAnalysis (Bruker
Daltonics).
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Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker AVIII HD 400 MHz NMR
spectrometer (UC Berkeley), a Varian Inova 500 (Stanford University), or an Agilent VNMRS 800
MHz NMR spectrometer (Stanford University). The 800 MHz NMR spectra were obtained by Dr.
Corey Liu and Dr. Stephen Lynch of the Stanford University Department of Chemistry NMR
Facility. Spectra were processed using MestReNova v10.0.2-15465 (Mestrelab Research S.L.,
2015).
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PEP-FOLD screening. PEP-FOLD26-27 was used for initial in silico screening of peptide
sequences (Supplemental Table S1). MacPyMOL Molecular Graphics System v1.4 (Schrödinger,
LLC, New York, NY, 2010) was used to visualize output.
Binding assay (adapted from reference 22). N-terminal acetylated, C-terminal amidated
peptides (36 L of a 1 mM stock solution, pH adjusted to approximately 7) were incubated with
CBTNAc (0.4 or 4 L of a 100 mM stock solution in DMF) or DMF alone (negative control) in PBS
for 48 h at room temperature. An aliquot (0.5 L) of each reaction mixture was analyzed by
®
MALDI-TOF MS either without prior purification or after desalting with a -C18 ZipTip .
Cysteine competition assay. After MALDI-TOF MS analysis, half (20 L) of the binding assay
solution was incubated for 48 h at room temperature after adding an equal volume of cysteine (10
mM or 10 equiv. relative to CBTNAc, unless cysteine would interfere with a later assay, in which
case 1 mM or 1.1 equiv. was used; pH adjusted to approximately 7) with or without DTT or TCEP
reducing agent (10-15 mM or 10-15 equiv., unless the reducing agent would interfere with a later
assay, in which case 1 mM or 1.1 equiv. was used; pH adjusted to approximately 7). The
remainder of the binding assay solution was incubated with an equal volume of PBS or an equal
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volume of DTT or TCEP reducing agent (10-15 mM or 10-15 equiv., unless the reducing agent
would interfere with a later assay, in which case 1 mM or 1.1 equiv. was used; pH adjusted to
approximately 7). The reaction mixtures were again analyzed by MALDI-TOF MS either without
®
prior purification or after desalting with a -C18 ZipTip .
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Binding assay in the presence of glutathione. CBTag 1.0 (18 L of a 1 mM stock solution in
PBS) was incubated with reduced glutathione (0.2 L of a 500 mM stock solution in PBS) and
CBTNAc (2 L of a 10 mM stock solution in DMF) or DMF alone (negative control) in PBS for 48
h at room temperature. After completion, an equal volume (20 L) of a solution of cysteine and
DTT (10 and 15 mM, respectively) in PBS was added, and the resulting solution was incubated a
further 48 h at room temperature. An aliquot (0.5 L) of each reaction mixture was analyzed by
MALDI-TOF MS without prior purification.
Chloroacetate labeling of cysteine residues. Peptide solution (0.9 mM; 0.45 mM if after
treatment with a slight excess of cysteine) was incubated with an equal volume of 30 or 500 mM
chloroacetate (pH adjusted to approximately 7) overnight at room temperature. The reaction
mixtures were analyzed by MALDI-TOF MS without prior purification.
Tandem LC-MS/MS. After the cysteine competition assay (using 1.1 equiv. cysteine), samples
were diluted 1:9 with PBS, then 1:4 with water to give final concentrations (where applicable) of
1
0 M peptide, 10 M CBTNAc, 10 M cysteine, 15 M DTT. Dr. Theresa McLaughlin of the
Stanford University Mass Spectrometry facility analyzed the samples by ESI-MS on an Agilent
260 HPLC connected to a Bruker MicroTOF-QII mass spectrometer and equipped with an
1
Agilent Pursuit 5 diphenyl 2.1 x 150 mm LC column held at 60 C. The injection volume was 10
L, the flow rate was 0.3 mL/min, and data was collected in autoMS2 mode with a mass range of
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0-2000 Da. Solvent A was 0.1% formic acid (FA) in water, solvent B was 0.1% FA in MeCN, and
the gradient was as follows: 5% B for 2 minutes, 5-95% B over 5 minutes, 95% B for 1 minute,
5-5% B over 0.1 minutes, and 5% B for 6.9 minutes. Predicted fragment ions were calculated
9
using the MS/MS Fragment Ion Calculator in the Proteomics Toolkit provided by the Institute for
Systems Biology (http://db.systemsbiology.net/proteomicsToolkit/FragIonServlet.html).
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LC-MS time-course experiment. Samples were prepared as follows: In an LC-MS vial equipped
with a 300-L insert, a solution of 22.5 or 5.63 mM peptide (CBTag 1.0, CBTag 1.0 (K9R), or
CBTag 1.0 (C7S); adjusted to pH 7 in PBS), 2 mM TCEP (adjusted to pH 7 in water), and 22.5 or
5.63 mM 13CBTNAc (3) was prepared in 5-6% DMF in PBS (total volume of 10 L) and the
starting time noted. Ratios of 1:1, 1:4, and 4:1 peptide/13CBTNAc were assessed. An LC-MS
sequence was prepared such that each sample was injected five times over approximately 45
hours, with an injection volume of 1 L each time. The LC-MS gradient was: 5% solvent B for 2
minutes, 5-75% solvent B over 15 minutes, 75-100% solvent B over 1 minute, 100% solvent B for
6
minutes, 100-5% solvent B over 1 minute, and 5% solvent B for 3 minutes, where solvent A was
.1% TFA in water and solvent B was 0.1% TFA in acetonitrile. Identification of peaks was
0
performed by comparison to the spectra of pure compounds, determining whether predicted
masses were present, and checking whether addition of an equal volume of 200 mM cysteine
(
adjusted to pH 7 in water) caused the disappearance of the peak over time. The relevant peaks
in the 210 nm and 254 nm chromatographs for two replicates were integrated manually and used
to calculate degree of 13CBTNAc modification over time. See Supporting Information Figure S6-
11, Table S2-4, and LC-MS spectra and integration results section for details.
Computational determination of lowest energy conformer for amidine and condensation
reaction products. For conformational searching and molecular dynamics calculations, Maestro
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(
Version 10.7.015, MMshare Version 3.5.015, Release 2016-3, Platform Darwin-x86_64,
Schrödinger, LLC, New York, NY, 2016) and the integrated program Macromodel (Schrödinger,
LLC, New York, NY, 2016) were used. In general, standard parameters were applied. For
calculating potential energy, an OPLS3 force field with water as the solvent was used. Structures
were minimized using the Polak-Ribiere Conjugate Gradient (PRCG) method until a gradient
below the convergence threshold of 0.05 kJ/Å-mol was reached, with a maximum of 2500
iterations. For conformational searching, a mixed torsional/low-mode sampling method (multi-
ligand) was used, with 1000 steps maximum, 100 steps per rotatable bond, and the energy
window for saving structures set to 5.02 kcal/mol. For molecular dynamics simulations, a
stochastic dynamics method was used, the simulation temperature was 300.0 K, the time step
was 1.5 fs, the equilibration time was 1.0 ps, and the simulation time was 10 ns. Each structure
was subjected to five rounds of conformational searching, with molecular dynamics simulated
after rounds 2, 3, and 4. MacPyMOL Molecular Graphics System v1.4 (Schrödinger, LLC, New
York, NY, 2010) was used to save images of the lowest energy conformer for the amidine product
and the condensation reaction product (Supporting Figure S13).
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Synthesis.
N-(2-cyanobenzo[d]thiazol-6-yl)acetamide (CBTNAc, 1). Synthetic procedures were adapted
from reference 22; NMR spectra (methanol-d and chloroform-d) were reported in references 22
4
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and 29. 6-Amino-2-cyanobenzothiazole (12 mg, 0.069 mmol) was suspended in anhydrous DCM
(
0.60 mL). Pyridine (3 drops) and acetyl chloride (0.020 mL, 0.28 mmol, 4.8 equiv.) were added,
and the reaction was allowed to stir 1 h at room temperature. The resulting clear light yellow-
orange solution was concentrated in vacuo then resuspended in ethyl acetate (5.0 mL) and
aqueous saturated sodium bicarbonate (5.0 mL). The ethyl acetate layer was separated and
concentrated in vacuo to a yellow-white solid. The crude product was purified by silica gel
chromatography (1:5 ethyl acetate/hexanes to 100% ethyl acetate). By TLC (1:1 ethyl
acetate/hexanes), the product was visible under shortwave UV light as a dark bright blue spot at
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R
f
0.145. Product-containing fractions were combined and concentrated in vacuo then lyophilized
to yield 7.87 mg (52.5%) of an off-white powder. LC-MS indicated ~96% purity.
1H NMR (500 MHz, Acetone-d
6
) δ 9.68 (br s, 1H), 8.81 (d, J = 2.0 Hz, 1H), 8.12 (d, J = 9.0 Hz,
1
1
H), 7.72 (dd, J = 9.0, 2.0 Hz, 1H), 2.15 (s, 3H). 13C{ H} NMR (126 MHz, Acetone-d
6
) δ 169.6,
+
1
49.0, 141.2, 137.9, 135.6, 125.8, 121.3, 114.1, 111.4, 24.4. HRMS (ESI) m/z: [M + H] Calcd for
10
C H
8 3
N OS 218.0383; Found 218.0381.
N-(2-chlorobenzo[d]thiazol-6-yl)acetamide (ClBTNAc, 2). Synthetic procedures were adapted
from reference 33. 6-amino-2-chlorobenzothiazole (0.66 g, 3.57 mmol) was dissolved in acetic
anhydride (6.5 mL, 68.8 mmol, 19.2 equiv.). Pyridine (1.5 mL, 19.3 mmol, 5.2 equiv.) was added
dropwise, then the light yellow-brown solution was slowly raised to boiling point (approx.140 C)
in an oil bath. After heating for 5 minutes, the dark orange-brown solution was cooled, poured into
water (20 mL), and extracted three times with 20-mL aliquots of ethyl acetate. The combined
bright yellow ethyl acetate layers were washed twice with 20-mL aliquots of brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo to an orange-brown oil. The crude
product was purified by silica gel chromatography, eluting with 2.5 column volumes of 40% ethyl
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acetate in hexanes then 5 column volumes of 50% ethyl acetate in hexanes. By TLC (1:1 ethyl
acetate/hexanes) visualized with shortwave UV, three major spots eluted. By NMR peaks and
mass spectrometry, these were identified as starting material (R
f
0.818), product (R
f
0.515), and
starting material with the chlorine atom replaced by a hydrogen atom (R
f
0.318). Removal of the
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solvent in vacuo followed by drying under high vacuum overnight yielded recovered starting
material as a pale orange-yellow powder (0.186 g, ~28.2% unreacted) and product as a pale
yellow-tan powder (0.364 g, 45% yield, 76.8% yield based on recovered starting material). LC-
MS indicated 96-98% purity.
1H NMR (400 MHz, Methanol-d
) δ 8.32 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.45 (dd, J
4
=
1
2
8.8, 2.2 Hz, 1H), 2.15 (s, 3H). 13C{ H} NMR (101 MHz, Methanol-d
) δ 171.7, 153.3, 148.3,
1
4
+
38.3, 137.9, 123.4, 120.6, 112.9, 23.9. HRMS (ESI) m/z: [M + H] Calcd for C
9
H
8
ClN OS
2
27.0040; Found 227.0038.
1
3
N-(2-(cyano- C)-benzo[d]thiazol-6-yl)acetamide (13CBTNAc, 3). Synthetic procedures were
adapted from reference 34; NMR spectra (chloroform-d) of the compound (not enriched for 13C)
were reported in references 22 and 35. [CAUTION: Potassium cyanide is extremely hazardous;
compounds dissolved in DMSO are more easily absorbed by skin. Use appropriate safety
1
3
measures.] Potassium cyanide- C purchased from Cambridge Isotope Laboratories (48.3 mg,
.71 mmol, 1.19 equiv.) in anhydrous DMSO (12.5 mL) was heated at 130 C in an oil bath until
0
it was mostly dissolved into a clear solution (15 minutes). ClBTNAc (2) (139.4 mg, 0.615 mmol)
was added, and the resulting dark orange reaction mixure was stirred 2 h at 130 C, becoming
bright red over time. The solution was allowed to stand at room temperature overnight, turning a
deep red-orange on cooling. The next day, the reaction mixture was poured into a separatory
funnel and 75 mL of 0.2 M monopotassium phosphate (KH
2
PO , pH 4-5) was added. This
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aqueous solution was extracted once with 125 mL of diethyl ether and twice with 50-mL aliquots
of diethyl ether. The combined neon yellow ether extracts were washed twice with 25-mL aliquots
of water then dried over anhydrous sodium sulfate. The solution was filtered, silica gel (1.1 g) was
added, and the solvent was removed in vacuo. The dry residue was added to a column of silica
gel (100 mL) pre-packed with petroleum ether, then product was eluted using 0-50% ethyl acetate
in petroleum ether followed by 50-100% ethyl acetate in hexanes. Pure fractions were identified
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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0
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0
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by TLC (1:1 ethyl acetate/hexanes), where the product was visible as a dark blue spot (R
f
0.26)
under short- and longwave UV light. The impurity (R 0.31) eluted just before the product.
f
Concentration of the product-containing fractions in vacuo followed by drying under high vacuum
overnight yielded 74 mg (55.1%) of product as a white to pale yellow solid. Product was ~97%
pure by LC-MS.
1H NMR (500 MHz, Acetone-d
6
) δ 9.68 (br s, 1H), 8.81 (d, J = 2.1 Hz, 1H), 8.12 (d, J = 9.0 Hz,
1
1
H), 7.72 (dd, J = 8.9, 2.1 Hz, 1H), 2.16 (s, 3H). 13C{ H} NMR (126 MHz, Acetone-d
6
) δ 169.6,
+
1
49.0, 141.1, 137.9, 135.1, 125.8, 121.3, 114.1, 111.4, 24.4. HRMS (ESI) m/z: [M + H] Calcd for
13CH
C
9
8
N OS 219.0416; Found 219.0414.
3
General peptide synthesis conditions. Peptides were made by solid-phase peptide synthesis
SPPS) either manually in a peptide synthesis vessel or using an AAPPTec Apex 396 automated
(
peptide synthesizer equipped with a 96-well reactor. Solvents were HPLC grade or peptide
synthesis grade; neither solvents nor reagents were anhydrous. Rink Amide AM resin (100-200
mesh, 0.41 mmol/g loading) from EMD Millipore (855130) was used as the solid phase unless
otherwise noted. All coupling, deprotection, and cleavage steps were performed at ambient
temperature.
General procedure for manual synthesis. A 50 mL peptide synthesis vessel (VWR 80071-382)
was equipped with a house nitrogen and a vacuum line. Rink Amide AM resin (0.05 g, 0.02 mmol)
was added and allowed to swell for 10 minutes in either dichloromethane (DCM) or N,N-
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dimethylformamide (DMF) without nitrogen agitation. The resin was drained and dried with several
cycles of vacuum and nitrogen, then the resin Fmoc protecting group was cleaved with 20%
piperidine in DMF or N-methyl-2-pyrrolidone (NMP) (5-10 mL) for 15-20 minutes with nitrogen
agitation, rinsing the sides every 5-10 minutes to ensure all beads were deprotected. Beads were
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drained and dried with several rounds of vacuum and nitrogen, then a Kaiser test was performed
on a small amount of resin to ascertain degree of completion. If deprotection was complete, the
first amino acid was coupled with monomer (0.2 mmol, 10 equiv.), coupling reagent (0.2 mmol,
10 equiv. of HATU for Fmoc-His(Trt) coupling, COMU for all others), and N,N-
diisopropylethylamine (DIPEA; 15 equiv. for Fmoc-Cys(Trt) coupling, 20 equiv. for all others) in
NMP (1.5 mL) or 1:1 DCM/NMP (1.5 mL; Fmoc-Cys(Trt) coupling only) with nitrogen agitation.
Directly before coupling, the DIPEA was added to the monomer/coupling reagent solution and the
vial gently inverted for 30-60 seconds before addition to resin. Again, sides of the vessel were
rinsed with minimal DCM or DMF 5-10 minutes into the reaction. After 15-30 minutes, the resin
was drained and washed thoroughly with DCM, then the first coupling reaction was repeated. The
Kaiser test was used to confirm completion, then any remaining free amines on the resin were
capped with a solution of 10% acetic anhydride and 5% DIPEA in DMF or NMP, agitating with
nitrogen for 15-20 minutes. After draining and washing the resin, the Fmoc group on the first
amino acid was removed with 20% piperidine in DMF or NMP as previously. Subsequent
couplings were performed only once unless the amino acid was -branched or the Kaiser test did
not show completion. After the coupling and deprotection of the final amino acid, the N-terminus
was acetylated using a solution of 10% acetic anhydride and 5% DIPEA in DMF or NMP, agitating
with nitrogen for 15-20 minutes. In preparation for deprotection and cleavage, the resin was
thoroughly washed with DCM and left under vacuum until dry (15-30 minutes). A clean
roundbottom flask was attached to the synthesis vessel and the cleavage cocktail Reagent L (for
0.02 mmol resin: 0.96 g dithiothreitol, 0.04 mL triisopropylsilane, 0.1 mL ddH O, and trifluoroacetic
2
acid (TFA) to a final volume of 2 mL) was added to cleave and deprotect the peptide. The resin
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was allowed to sit 1.5 h without nitrogen agitation; the mixture was manually stirred every 30
minutes with a glass stir rod. After draining the cleavage solution into the clean roundbottom flask,
the solvent was removed in vacuo and the resulting viscous cloudy pale yellow to pale white
solution was pipetted into ice-cold ether (45 mL). The flask was rinsed with minimal TFA and the
resulting solution was also added to the ether. The suspension was centrifuged in a Sorvall
Legend RT at 3700 x g and 4C for 8-10 minutes. The ether was decanted carefully, and the
remaining pale yellow to off-white precipitate was washed twice more with ice-cold ether (45 mL).
After the final centrifugation, the ether was carefully decanted and the precipitate allowed to dry
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15-30 minutes in a hood to remove any remaining ether. Finally, the precipitate was resuspended
in minimal ddH O and lyophilized in a pre-weighed vial using a LABCONCO FreeZone 4.5Plus.
2
Crude peptide was stored at -20 C until purification.
General procedure for automated peptide synthesis. Rink Amide AM resin (0.05 g, 0.02 mmol)
was added to a well in the AAPPTec Apex 396 automated peptide synthesizer. The instrument
was programmed to complete the following steps: NMP (1 mL) was added and the resin was
mixed for 3 minutes at 600 rpm. After draining the solvent, this step was repeated. To deprotect
the first Fmoc group, 20% piperidine in DMF (1 mL) was added and the resin mixed for 3 minutes
at 600 rpm. After draining, another aliquot of 20% piperidine in DMF (1 mL) was added and
deprotection was allowed to proceed for 20 minutes at 600 rpm. After the deprotection solution
was drained, the resin was washed three times with NMP (1 mL), MeOH (1 mL), and NMP (1 mL)
at 600 rpm for 3 minutes each. Then, 0.4 M protected monomer in NMP (0.6 mL), 0.4 M HATU or
HCTU in NMP (0.5 mL), and 2 M DIPEA in NMP (0.25 mL) were transferred to the well and mixed
for 30 minutes at 600 rpm. After draining the solution, the coupling step was repeated, then the
resin was washed twice with NMP (1 mL) for 3 minutes at 600 rpm. The deprotection and coupling
steps were repeated until the final amino acid was deprotected, then the N-terminus of the peptide
was acetylated by twice adding 11.63% acetic anhydride in DMF (1.3 mL) and 2 M DIPEA in DMF
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