Water-driven ligations using cyclic amino squarates: A class of useful SN1-like reactions

Article abstract of DOI:10.1039/c0cc03989f

We report a class of water-soluble and -stable cyclic amino squarates that ligate with cysteine or lysine residues without side-products in an entirely aqueous environment. The ligations include addition-elimination reactions that are promoted by water in

Full text of DOI:10.1039/c0cc03989f

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Water-driven ligations using cyclic amino squarates: a class of useful
SN1-like reactionsw
Dawei Cui, Deepali Prashar, Preeti Sejwal and Yan-Yeung Luk*
Received 20th September 2010, Accepted 26th October 2010
DOI: 10.1039/c0cc03989f
We report a class of water-soluble and -stable cyclic amino
squarates that ligate with cysteine or lysine residues without
side-products in an entirely aqueous environment. The ligations
include addition–elimination reactions that are promoted
by water in a way similar to SN1 reactions. The structural
versatility of the reactants allows the potential recognition of
selected amino acid residues on proteins.
Organic reactions in entirely aqueous environment1 are important
Scheme 2
for green chemistry and for applications in life sciences,
facilitate the reactions with a faster rate and also enable
coupling with weaker nucleophiles, such as amino groups at
near neutral pH, which has many biological applications.
We first targeted cyclic amino squarate with a six-member
ring (Scheme 2, 2). Cyclic amino squarate 2 reacted with
amino groups in organic solvents (see ESIw), but was not
soluble in water. In addition, thiol groups are not desired in
the ligation product because of disulfide formation. We have
been unable to synthesize the corresponding cyclic amino
squarate with an oxygen bridge (X = O), probably due to
the high strain in the heteroatom ring. These observations
prompted us to hypothesize that including a seven-member
ring, as shown in molecules 3 to 7, may facilitate both
solubility and ligation in water.
including bioconjugation, protein modification or even reactions
in a living system.1c The SN1 substitution reactions typically
proceed best in water; however, these reactions are generally
not very useful. Here, we describe a new class of substitution
reactions that are driven by water in a way similar to SN1
reactions. This advance relies on the use of cyclic amino
squarates, which are both soluble and stable in water, to ligate
with thiol or amino groups under different reaction conditions
without the formation of side products (Scheme 1).
Squaric acid derivatives have enabled many studies in
bioorganic chemistry, including developments in bioconjugation
methods,2 phosphate ester mimics,3 and sensors.4 Recently, we
discovered that amino phenoxy squarates react selectively with
cysteine residues in water, but not in organic solvents.1p This
class of reactions suggests that the preference of water is not
limited to SN1 reactions, but that using water to promote other
types of reactions is feasible, probably either by activation of
the reactants or stabilization of the transition states. This class
of chemoselective reactions is relatively slow (finishes in B1 h),
but highly chemoselective, which allows surface reactions to
distinguish cysteine groups at the N-terminus positions over
other locations in a peptide.2d Here, we further report that by
introducing strain into the reactant structure, water can
Cyclic amino squarates 3–5 are readily accessible synthetically
(Scheme 3). Briefly, amine 8, which was obtained by a one-pot
reductive amination of 2-formylphenol and 4-aminomethyl-
pyridine, coupled with 3,4-dichloro-3-butene-1,2-dione5 to
afford the 7-membered ring cyclic squarate pyridine derivatives.
Treatment of 9 with organoiodides readily produced the
desired products. The pyridinium facilitates water solubility
and can be used to tether small drug molecules or ligands to
the cyclic amino squarate molecule. As oligo(ethylene glycol)
is often used as water-soluble linkers, we also synthesized
compounds 6 and 7 (see ESIw). However, these two compounds
exhibited sluggish water solubility. We note that an often
neglected issue with oligo(ethylene glycol) is its propensity to
form complexes with divalent (or higher valent) metal ions.6
For these reasons, using other moieties, such as positive
charges from pyridinium groups, to facilitate water solubility
is worth exploration.
Coupling of cysteine amino acids and peptides containing
cysteine residues to the cyclic amino squarate (Scheme 2, 3)
was about 12 times faster than with the open squarate
Scheme 1
Department of Chemistry, Syracuse University, Syracuse, NY-13244,
USA. E-mail: yluk@syr.edu
w Electronic supplementary information (ESI) available: Experimental
procedures and spectral data for all the new compounds. See DOI:
10.1039/c0cc03989f
Scheme 3
c
1348 Chem. Commun., 2011, 47, 1348–1350
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Table
1
Ligation of cyclic amino squarate derivative 3 with
unprotected thiol-containing peptides in water (Scheme 1)a
Entry
1
Reactant
Product
Time/min
5b
2
60c
3
N0-CAGRGDS-C0
180c
Scheme 4
a
b
c
Reaction conditions: H2O, r.t. Quantitative. >95% conversion.
reactivity, squarate 3 exhibited a positive solvatochromism
(red shift from 250 to 264 nm in UV absorption) when the
solvent polarity is increased from CH3CN to water (see ESIw).
This result suggests that the excited state of the molecule,
which is more polar, is better stabilized by solvation than the
ground state of the molecule in water.10
molecule, 1.2d In the presence of a b-amino group, the thiolate
attack on the squarate was followed by an intramolecular S to
N squarate transfer. Table 1 shows selected results of cysteine
coupling with 3. This class of reactions is mild, selective,
quantitative, and without side products (Table 1).
At a slightly higher pH (B7.8), the cyclic amino squarate
also ligated effectively with amino groups and with proteins in
water, albeit at a slow reaction rate (Table 3). Because
of this reactivity in water, this class of molecules provides a
bioorganic reaction tool for modifying proteins through lysine
residues.
To understand in some detail the nature of this reaction, we
measured the rate of the reaction in different deuterated
solvents, including D2O, D2O/DMSO-d6 (v/v, 1 : 4; molar
ratio, 1.05 : 1), DMSO-d6, CD3OD, D2O/CD3CN (v/v, 1 : 3;
molar ratio, 1.05 : 1) and CD3CN (Table 2). The results
showed that the ligation proceeds with the highest rate in
water, or when water is present.
To demonstrate the ability of modifying proteins, we
explored modifying lysozyme and ribonuclease A with 3–5
(Scheme 5). The proteins (1 equiv. of lysozyme or ribonuclease A)
were incubated with 3, 4 or 5 (50 equiv.) in 10 mM PBS buffer
(pH 7.75) at 28 1C for 3 days. The products were dialyzed,
As these solvent effects are almost identical to those
observed in SN1 reactions,7 this water-driven reaction appears
to be promoted by water stabilizing a charged intermediate
and polar transition states. We believe that the presence of
multiple resonance structures (as opposed to that of generic
carbonyl substitution reactions) stabilizes the intermediates
and transition states for this reaction, which is similar to the
way tertiary carbocations stabilize the intermediates of a
typical SN1 reaction. When the energy of the intermediate is
low enough, water solvation of charges becomes effective to
facilitate the chemical transformation (Scheme 4). In contrast,
the most commonly used N-hydroxysuccinimide (NHS)-
activated ester for modifying lysine residues8 primarily activates
the reactants rather than stabilizing the transition states, and
also reacts with the solvent water. In the presence of multiple
nucleophiles such as sugar moieties that can promote hydrolysis,9
the NHS ester method becomes unreliable. Parallel to chemical
Table 3 Ligation of cyclic amino squarate derivative 3 with amino
groups in water (Scheme 1)
Entry
1
Reactant
Product
Time/d
6a,b
2
3
6a,b
Table 2 Reactions of compound 3 with L-cysteine ethyl ester
dihydrochloride in different deuterated solvents
3c,d
Entry
Solvent
Timea
1
2
3
4
5
6
D2O
5 min
5 min
1 h
4
6a,b
D2O/DMSO-d6 (1/4)
DMSO-d6
CD3OD
D2O/CD3CN (1/3)
CD3CN
16 h
3 h
b
—
a
b
Reaction conditions: H2O, rt. >95% conversion. 10 mM PBS
c
d
a
b
>95% conversion. No observable conversion.
buffer, pH 7.75. B30% conversion.
c
This journal is The Royal Society of Chemistry 2011
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with small molecules through pyridinium moiety, this reaction
is useful for applications involving difficult bioconjugation.
We thank the Syracuse Center of Excellence for the CARTI
award supported by the U.S. EPA (Grant X-83232501-0), and
NSF-CAREER (Grant 0845686) for financial support.
Notes and references
Scheme 5
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Table 4 Number of lysine modifications per protein using 3, 4 or 5
determined by MALDI-TOFa
3
4
5
Lysozyme
RNase A
1b
5
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3
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2
a
b
Reaction conditions: 10 mM PBS buffer (pH 7.75), rt, 3 d. The
yield of modification increased as fluoro-substitution increases in
squarates 3 to 5.
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amino squarate, the efficiency for the modification was different
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extent of lysine modifications for RNase A decreased from
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To conclude, we have developed a new class of ligation
reactions in entirely aqueous solutions, for which the reaction
is likely promoted by stabilization of the transition states. The
thiol-containing molecules react with cyclic amino squarate
molecules significantly faster than with acyclic amino squarate.1p
We believe that the enhanced reactivity is due to the incorporated
ring strain in the 7-membered ring. Our discovery suggests that
using water to enable useful reactions in a way similar to SN1
reactions is possible.11 This work also suggests the promising
likelihood of using water to promote other types of reactions
by building reactants that fulfill certain structural requirements
such as molecular strain in the reactant structures, and resonance
stabilization in the transition states and intermediates. As the
cyclic amino squarate is stable and can be readily derivatized
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c
1350 Chem. Commun., 2011, 47, 1348–1350
This journal is The Royal Society of Chemistry 2011