Traceless Templated Amide-Forming Ligations

Article abstract of DOI:10.1021/jacs.9b03597

Template assistance allows organic reactions to occur under highly dilute conditions - where intermolecular reactions often fail to proceed - by bringing reactants into close spatial proximity. This strategy has been elegantly applied to numerous systems, but always with the retention of at least one of the templating groups in the product. In this report, we describe a traceless, templated amide-forming ligation that proceeds at low micromolar concentration under aqueous conditions in the presence of biomolecules. We utilized the unique features of an acylboronate-hydroxylamine ligation, in which covalent bonds are broken in each of the reactants as the new amide bond is formed. By using streptavidin as a template and acylboronates and O-acylhydroxylamines bearing desthiobiotins that are cleaved upon amide formation, we demonstrate that traceless, templated ligation occurs rapidly even at submicromolar concentrations. The requirement for a close spatial orientation of the functional groups - achieved upon binding to streptavidin - is critical for the observed enhancement in the rate and quantity of product formed.

Full text of DOI:10.1021/jacs.9b03597

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Traceless Templated Amide-Forming Ligations
Alberto Osuna Gálvez, and Jeffrey W. Bode
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 May 2019
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Traceless Templated Amide-Forming Ligations
Alberto Osuna Gálvez and Jeffrey W. Bode*
Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences,
ETH Zürich, 8093 Zürich, Switzerland
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Supporting Information Placeholder
a photocleavable PNA-templated native chemical ligation
ABSTRACT: Template assistance allows organic reactions to
occur under highly dilute conditions – where intermolecular
reactions often fail to proceed – by bringing reactants into
close spatial proximity. This strategy has been elegantly
applied to numerous systems, but always with the retention of
at least one of the templating groups in the product. In this
report, we describe a traceless, templated amide-forming
ligation that proceeds at low micromolar concentration under
aqueous conditions in the presence of biomolecules. We
utilized the unique features of an acylboronate–
hydroxylamine ligation, in which covalent bonds are broken in
each of the reactants as the new amide bond is formed. By
using streptavidin as a template and acylboronates and O–
acylhydroxylamines bearing desthiobiotins that are cleaved
upon amide formation, we demonstrate that traceless,
templated ligation occurs rapidly even at sub-micromolar
(NCL)10 that allows cleavage of the directing PNA strands
from the ligated peptide by irradiation.11 However, a traceless,
templated ligation in which the templating moieties are
cleaved concomitantly with bond formation12 has not been
successfully implemented, likely due to the lack of covalent
bond-forming reactions suitable for this purpose.
(a) Complementary template-assisted reactions without scaffold release (refs. 3,6)
Fg1 Fg2
Fg1 Fg2
Cleavage
(b) Complementary template-assisted reactions with partial scaffold relese (refs. 6,8)
Fg2
Fg2
Fg1
Fg1
concentrations. The requirement for
a close spatial
orientation of the functional groups – achieved upon binding
to streptavidin – is critical for the observed enhancement in
the rate and quantity of product formed.
(c) External template-assisted reactions without scaffold release (refs. 7,9)
Fg1
Fg2
Fg1
Fg2
Molecular
template
Templated organic reactions are widely used by biological
systems to accelerate covalent bond formation by bringing
reactants in close proximity and increasing their effective
concentration;1 well-known examples include ribosomal
peptide synthesis and DNA ligation. The same principles have
been applied in purely synthetic template-promoted
reactions2 and notable successes include DNA-and PNA-
templated ligation.3 Despite the elegance of these approaches
and applications to areas such as DNA-templated synthesis,
proximity-driven mapping of binding pockets4 and
combinatorial synthesis,5 their utility for broader synthetic
applications is limited by the persistence of at least one of the
templating groups (Figure 1a-c).3d,6,7 For example, a number
of template-assisted native chemical ligations (NCL) take
place with release of one templating scaffold, but the second
must be installed at a distal site of the other reactant (Figure
1b).8,9 To address this limitation, Diederichsen et al. disclosed
Molecular
template
Molecular
template
(d) This work: External template-assisted ligations with complete scaffold release
O
Molecular
template
N
H
Fg1
Fg2
Fg1
Fg2
Molecular
template
Molecular
template
Figure 1. Schematic representation of types of templated
reactions. (a) Complementary template-assisted reactions
without directing scaffold release. (b) Complementary template-
assisted reactions with partial directing scaffold release. (c)
External template-assisted reactions without directing scaffold
release. (d) This work: External template-assisted amide-forming
ligations with concomitant release of the directing scaffolds.
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(a) Traceless templated amide-forming ligation of hydroxylamines and acylboronates
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HO
F
O
F
B
N
NH
Me
O
F
O
B
O
O
NH
Me
O
B
O
HN
N
O
Streptavidin
N
O
N
Me
O
N
linker
N
H
linker
Templated ligation
with simultaneous
linker cleavage
linker
linker
linker
linker
Desthiobiotin
Cleaved linkers
Traceless ligation
product
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(b) Design of starting materials
O
O
O
NEt2
O
H
Quenched fluorophore
F
N
N
H
Internal
quenching
NH
O
Cleavable
hydroxylamine
O
B
Desthiobiotin
Me
O
Cleavable
acylboronate
O
N
F
N
H
N
O
N
NMe2
N
Me
O
N
N
O
S
O
NH
N
O
3
Me
HN
N
NH
N
N
DABSYL quencher
HN
O
NH
O
O
NH
3
NH
O
Desthiobiotin
1a
2a
Acyl donor
Amine ligation partner
Figure 2. (a) Streptavidin-templated ligation of acylboronates and O-acylhydroxylamines, each bearing a desthiobiotin connected to the
departing section of their respective functional groups. (b) Specific molecules used for this study (see Supporting Information for
detailed synthetic routes).
During the course of our studies on amide-forming ligations
from acylboronates,13,14 we identified variants that fulfilled the
rare criteria of simultaneous cleavage of two derivatizable
groups during an intermolecular coupling reaction,15 offering
the possibility for true traceless templated reactions. In this
manuscript, we demonstrate templated amide-formation in
water at low micromolar concentrations using desthiobiotin
as the ligand and streptavidin as the template. This work
anticipates a generic approach to conjugations that overcome
the inherent limitations in the rate of non-enzymatic coupling
reactions. Detailed kinetics analysis and modeling show that
template-induced proximity of the two reaction partners
substantially enhances product formation above the
background rate.
Importantly, it occurs under aqueous conditions without the
need for any other reagents or catalysts – making it suitable for
use in the presence of biological molecules.
As our molecular template of choice, we selected the
streptavidin-desthiobiotin pair by linking the ligating
functional groups to desthiobiotin. The approach builds on a
long history of streptavidin as a template for inducing
molecular proximity, including elegant work from Ward on
the
development
of
streptavidin-based
artificial
metalloenzymes.16 Furthermore, Winssinger has previously
demonstrated that two distinct reactants can be brought into
proximity through a similar system for the template-assisted
photocatalyzed azide reduction.17,18 Streptavidin displays a
particular arrangement of its four binding pockets, located so
that two of them are in spatial proximity to each other.19 Its use
has the disadvantage of being able to form non-productive
complexes, but considerably simplifies the synthesis of the
substrates and employs a readily available template with a low
The reaction of acylboronates bearing pyridine-derived
ligands and O-acylhydroxylamines results in the formation of
amide bonds with the simultaneous loss of both the ligated
boron and the carbamate by N–O bond cleavage (Figure
2a).13 The template binding groups can be located in regions
of the reaction partners that are cleaved during the ligation,
which would result in traceless, templated product formation.
dissociation constant (KD ~ 10–14 M for biotin and ~10–11
M
for desthiobiotin) and fast association kinetics (kon >106 M–1
s–1).20
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(a)
O
O
HN
1
2
3
4
5
6
7
N
N
O
HN
Me
N
Linker
O
N
H
O
O
F
Streptavidin
S
NH
O
O
O
H
N
HN
(S, 0-5.0 equiv)
N
NH
B
N
H
N
H
N
N
N
O
N
O
O
N
O
Linker
aqueous citrate
buffer pH 6.0,
25 ºC
F
Et2N
O
F
O
O
Me
H
N
NH
O
Me2N
O
N
Fluorescence ON
HN
N
H
N
H
Me
O
Et2N
O
2a (0-10.0 equiv)
3
1a (1.0-5.0 µM)
8
Linker
=
Linker
=
O
O
3
3
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Conditions
Conditions
1a (5.0 µM)
80
60
40
20
0
(a)-1
(a)-2
60
1a (5.0 µM)
2a (2.0 equiv)
Tetravalent S (0.5 equiv)
2a (2.0 equiv)
Tetravalent S (0.4 equiv)
1,2-Divalent S (0.8 equiv)
40
20
0
S (0 equiv)
Background rate
S (0 equiv)
Background rate
Tetravalent S (0.5 equiv)
Biotin (10 equiv)
Monovalent S (0.8 equiv)
1,3 + 1,4-Divalent S (0.8 equiv)
2a (0 equiv)
S (0 equiv)
0
20000
40000
0
20000
t (s)
40000
t (s)
Conditions
Conditions
100
80
60
40
20
0
(a)-3 40
(a)-4
1a-1d (1.0 µM)
2a-2d (10.0 equiv)
Tetravalent S (0.5 equiv)
1a (5.0 µM)
2a (2.0 equiv)
=
=
=
=
1a, 2a
Linker
Linker
Linker
Linker
O
O
O
3
30
20
10
0
1c, 2c
1b, 2b
1d, 2d
10
6
Tetravalent S
(6 h conv)
1,2-Divalent S
(6 h conv)
1,3- and 1,4-Divalent S
(6 h conv)
0
1
2
3
4
0
20000
40000
60000
80000
Equiv S
t (s)
(b)
O
HN
O
S
HN
O
2a (2.0 equiv)
1a
NH
N
3
N
O
N
N
O
aqueous citrate
buffer pH 6.0
25 ºC
aqueous citrate
buffer pH 6.0
25 ºC
N
3
Me2N
O
O
H
N
1,2-Divalent S
O
N
O
N
H
N
H
Me
100
80
O
Et2N
O
1a-S + 2a (2.0 equiv)
1a-S (5.0 µM)
60
1a-S + 2a (2.0 equiv)
Biotin (5.0 equiv)
40
A (280 nm)
A (425 nm)
A (500 nm)
A (280 nm)
A (425 nm)
A (500 nm)
1a-S + 4 (2.0 equiv)
20
0
0
O
20000
40000
t (s)
60000
80000
BF3K
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48
45
46
V (mL)
47
48
V (mL)
F
4
Figure 3. (a) Experiments performed using hydroxylamines 1a–d (1.0–5.0 µM), acylboronates 2a–d (0–10.0 equiv) and expressed
streptavidins (S, 0–4.0 equiv) in aqueous citric acid / sodium citrate buffer pH 6.0 (25 mM) at 25 °C. Product formation monitored by
fluorescence (lex = 430 nm, lem = 485 nm). All plots are mean values from three replicates (error bars are omitted for clarity). (a)-1:
Conversion plot comparing the reaction in the presence or absence of added biotin (10.0 equiv). Performed with 1a (5.0 µM), 2a
(2.0 equiv) and tetravalent S (0.5 equiv). (a)-2: Performance of different streptavidin mutants in the reaction. Performed with 1a
(5.0 µM) and 2a (2.0 equiv). (a)-3: Streptavidin loading studies. Performed with 1a (5.0 µM) and 2a (2.0 equiv). (a)-4: Effect of linker
length. Performed with 1a–1d (1.0 µM), 2a–2d (10.0 equiv) and tetravalent S (0.5 equiv). (b) Isolation of the association complex 1a–
S between 1,2-divalent streptavidin and 1a; FPLC traces of the purified protein at different wavelengths. A conversion plot over time for
the reaction of 1a–S (5.0 µM) with 2a (2.0 equiv) is shown on the bottom-right corner.
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The association of two different biotin-equipped starting
plateau, experiments were analyzed by LC-MS to confirm the
final conversion values (see the Supporting Information).
Only desired amide product 3, unreacted starting materials 1a
and 2a, and byproducts derived from the cleavage of 1a and
2a could be observed by LC-MS analysis of the mixture.
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3
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materials with streptavidin could produce a number of
different spatial combinations, most of which lead to non-
productive associations. While using a weakly associating
ligand may allow it to dissociate from unproductive
arrangements, such a system would unnecessarily complicate
the analysis of the ligation and kinetics. We therefore elected
to use desthiobiotin and adopted two approaches to minimize
non-productive binding arrangements during our validation
of a true traceless templated ligation. First, we employed a
small excess of one ligation partner to ensure that as many
productive orientations as possible were formed. Second, we
followed the convenient procedure of Howarth and co-
workers to express and separate streptavidin mutants having
different valencies and spatial arrangements of the binding
pockets.21
The reaction exhibited templated ligation only in the
presence of tetravalent and 1,2-divalent (“cis”) streptavidins;
monovalent and 1,3- or 1,4 (“trans”) streptavidins did not
show any enhancement over the background reactivity
(Figure 3a-2). Streptavidin loading studies showed that the
optimal loading is 0.5 equiv relative to 1a for tetravalent
streptavidin and 1.0 equiv for the 1,2-divalent mutant. This is
in excellent agreement with the number of possible matching
combinations of starting materials (Figure 3a-3). We
examined a variety of hydroxylamines and acylboronates
having different linker lengths between the desthiobiotin unit
and the ligating functional groups (Figure 3a-4); optimal
performance was found when a short PEG linker was
employed (1a and 2a). The use of longer PEG chains (1b, 1c
and 2b, 2c) resulted in slightly diminished performance and a
short alkyl spacer (1d and 2d) showed no acceleration over
the background rate.
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To accurately monitor the rate of ligation we designed
starting materials whose conversion could be determined at
low concentrations by real-time fluorescence measurements
(Figure 2b). Hydroxylamine 1a was equipped with
a
coumarin fluorophore attached to the N-atom and a 4-
(dimethylaminophenylazo) benzenesulfonyl (DABSYL)
quencher was connected to the cleavable part of the molecule
– the O-acyl side – along with a desthiobiotin and a
polyethylene glycol (PEG) linker. This design allows for
internal fluorescence quenching of the coumarin moiety
(FRET effect); as the reaction proceeds the fluorophore and
the quencher on 1a are separated by the N–O bond cleavage,
resulting in an increase in the fluorescence signal output
(Figure 3a). Acylboronate 2a bearing a desthiobiotin attached
to the ligand on boron via a short PEG linker could be easily
prepared in two steps from commercially available potassium
acyl trifluoroborate (KAT) reagent 4.22 As the rate of KAT
ligations are strongly influenced by pH,23 we chose aqueous
buffers at pH 6.0 – as the expected rate was low enough to
benefit from templation with a moderate background rate at
low micromolar concentrations. Streptavidin is well known to
be stable at this pH.24
To further confirm our hypothesis that streptavidin assists
the amide formation by binding both 1a and 2a, we formed a
1:1 complex (1a–S) of 1,2-divalent streptavidin and 1a and
isolated it by FPLC. Upon addition of 2.0 equiv of 2a at
5.0 µM (Figure 3b), we observed rapid formation of the amide
product and good overall conversion. Addition of biotin to
block the binding pockets again resulted in a considerable
decrease of initial rate and conversion. This result shows
clearly that productive binding of the acylboronate and
hydroxylamine in adjacent streptavidin binding sites is
responsible for the increase in the observed rate of amide
formation.
To a first approximation, the formation of amide 3 is
expected to depend on two processes: 1) template-assisted
formation of 3 from the productive association of reagents 1
and 2 into two proximal streptavidin pockets – a step
governed by the first-order rate constant kT. 2) The
background reaction of 1 and 2, represented by the second-
order rate constant kB.
With the starting materials in hand, we screened a number
of streptavidin mutants and reaction conditions. In a typical
experiment, 1a (1.0 equiv) was initially mixed with
streptavidin in aqueous citrate buffer (pH 6.0) for 5 min
followed by addition of 2a (2.0 equiv). The reaction was
monitored by fluorescence (lex = 430 nm, lem = 485 nm) over
time. The coupling of 1a and 2a in the presence of expressed
tetravalent streptavidin (Figure 3a-1, dark red dots) was
accelerated over the background (orange dots), and as
compared to a negative control experiment in which excess
biotin was added to block streptavidin binding pockets (grey
dots). The stability of reagent 1a and streptavidin during the
course of the reaction was confirmed by fluorescence
measurements (Figure 3a-1, black dots). After reaching the
To provide a mathematical model for the effect of
templation on the rate of product formation, we adopted
pseudo-first order conditions ([2]
corresponding integrated rate equation adopts
exponential form as shown in Equation 1.
≥ 10·[1]). The
a
bi-
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Streptavidin (S, 0.5 equiv or 0 equiv)
(–kB·[2]0·t )
100·[3]
[1]0
(–kT·t )
1a
2a
3
1
+
(100–F)· 1–e
% conv =
=
F · 1–e
aqueous citrate buffer pH 6.0
25 ºC
(10.0 equiv)
(0.5 – 5.0 µM)
2
3
4
5
100·[Productive complex]0
[1]0
F = initial fraction of productive complex =
100
80
(1)
kT = first order rate constant of templated amide formation (s–1
)
kB = pseudo-first order background rate constant (M–1 s–1
[2]0 >> [1]0
)
6
7
8
9
60
Assumed irreversible binding of 1 and 2 with S
40
The first exponential term corresponds to product
formation from template assistance – a parameter directly
proportional to the fraction of productive associations of
starting materials 1 and 2 to streptavidin (F parameter). The
second term expresses the conversion of product due to non-
templated, background processes. Under first-order
conditions, the background exponent contains the
concentration of reagent 2 as a constant parameter.
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0
0
20000
40000
60000
80000
t (s)
1a (5.0 µM)
2a (10.0 equiv)
1a (1.0 µM)
2a (10.0 equiv)
1a (0.5 µM)
2a (10.0 equiv)
1a (0.5 µM)
2a (10.0 equiv)
S (0 equiv)
Background reaction
F = 85%
F = 79%
F = 71%
kT = 4.8·10–5 s–1
k
T = 4.2·10–5 s–1
kT = 4.0·10–5 s–1
F = 0
To further evaluate the validity of our model, we conducted
kinetics experiments under pseudo-first order conditions
(Figure 4). We compared a number of reaction plots
conducted at different concentrations but with comparable
setup and reagent equivalencies. The conversion over time
curve at 5.0 µM was fitted to Equation 1 with 85% templation
amplitude (F factor) and a templation rate constant of
kB = 1.4 M–1 s–1
kB = 0.2 M–1 s–1
kB = 0.2 M–1 s–1
kB = 0.2 M–1 s–1
(constrained)
(constrained)
(constrained)
Figure 4. Kinetics experiments under pseudo-first order
conditions fitted to Equation 1. Dots represent mean data points
from three experiment replicates performed with 1a (0.5–
5.0 µM), 2a (10.0 equiv) and tetravalent streptavidin (S, 0–
0.5 equiv) in aqueous citrate buffer pH 6.0. Error bars are omitted
for clarity. Lines are non-linear fits from Equation 1 using the
fitted parameters inside the grey boxes (bottom part).
kT » 4 • 10–5 s–1 by constraining the background rate constant
(kB) to 0.2 M–1 s–1 (red dots).25 A slightly lower templation
amplitude and constant (kT and F) were obtained when
fitting Equation 1 to the plot measured at 1.0 and 0.5 µM
concentration (blue and green dots respectively). The
background reaction followed a monoexponential growth
(black dots). The background rate constant was lower in the
presence of streptavidin, a factor that we attributed to the
increased steric hindrance of bound reagents compared to
when they are in solution.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures,
supplementary results and spectroscopic data for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org..
AUTHOR INFORMATION
Corresponding Author
bode@org.chem.ethz.ch
In conclusion, we have established a streptavidin-templated
reaction of desthiobiotin-equipped hydroxylamines 1 and
acylboronates 2 with traceless formation of the corresponding
amide. The reaction achieves high levels of conversion, even
at 500 nM – far above the conversion expected from the
background reaction alone. The present approach to
templated-ligation establishes that successful traceless
templated reactions can provide substantial enhancement in
conversion and can be implemented with a straightforward
streptavidin–desthiobiotin binding pair. This approach will
be useful for applications in both signaling and
chemoselective conjugations under dilute aqueous
conditions.
Funding Sources
This research was supported by ETH Zürich
ACKNOWLEDGMENT
Raphael Hofmann (ETH Zürich) is acknowledged for his
guidance and support in the expression of recombinant
streptavidins. A.O.G. thanks Dino Wu, Yi-Chung Dzeng,
Anne Schuhmacher, Chalupat Jindakun and Iain Stepek
(ETH Zürich) for fruitful discussions.
REFERENCES
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(25) For comparison, a bimolecular reaction would require k ~8 M–1 s–1
to achieve 80% conv after 4·104 s uxnder the same reaction conditions
(0.50 µM, 10-fold excess of one reactant), and k ~200 M–1 s–1 when using 1:1
starting materials ratio.
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