Water-driven chemoselective reaction of squarate derivatives with amino acids and peptides

Article abstract of DOI:10.1021/ol702275q

(Chemical Equation Presented) Here, we report a new class of highly chemoselective reactions between squarate derivatives and the amino acid cysteine or unprotected peptides with a N-terminus cysteine that proceed most efficiently in entirely aqueous solu

Full text of DOI:10.1021/ol702275q

ORGANIC
LETTERS
2007
Vol. 9, No. 23
4897-4900
Water-Driven Chemoselective Reaction
of Squarate Derivatives with Amino
Acids and Peptides
Preeti Sejwal, Yongbin Han, Akshay Shah, and Yan-Yeung Luk*
Department of Chemistry, Syracuse UniVersity, Syracuse, New York 13244-4100
yluk@syr.edu
Received September 18, 2007
ABSTRACT
Here, we report a new class of highly chemoselective reactions between squarate derivatives and the amino acid cysteine or unprotected
peptides with a N-terminus cysteine that proceed most efficiently in entirely aqueous solution at neutral pH. Kinetic and structural studies
reveal that the presence of hydrogen bonding in water is primarily responsible for both the high yield and fast rate of the reaction.
Chemoselective transformations in aqueous environments are
the most important reactions for sustaining life. While most
organic synthesis is conducted in toxic organic solvents,
studies have shown that hydrogen bonds can catalyze or
facilitate organic reactions albeit in nonaqueous environ-
ment.1 In addition, water has been found to enhance the
reactivity of many types of organic reactions.2 Particularly,
the recently reported “on water” reactions3 appear to be
driven by the enhanced hydrogen bonding at the organic-
aqueous interface.4
unprotected peptides) in an entirely aqueous environment at
neutral pH, whereby chemoselectivity and rate enhancement
appear to be imparted by hydrogen bonding of the abiotic
reactant in an aqueous solution. We also present a kinetic
study that reveals how water promotes this reaction relative
to other protic and aprotic polar solvents, as well as how
hydrogen bonding can catalyze this reaction in organic
solvents.
Designed chemoselective reactions that can proceed in
water are relatively scarce, but have been found to bear
enormous potential for studying and interfacing with biologi-
cal systems. Past examples include synthesizing a whole
protein without protecting groups,5 orthogonal ligation
methods for peptides and proteins,6 decoration of cell surfaces
by modified Staudinger ligation,7 click chemistry,8 modifying
In this work, we report a new class of chemoselective
reactions between squarate derivatives and amino acids (or
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10.1021/ol702275q CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/12/2007

proteins by metal-catalyzed organic reactions,9 and site-
selective peptide modification.10 Furthermore, development
of chemoselective reactions in water with abiotic molecular
structures are important because new structural elements can
be incorporated into complicated living systems.
Due to the extensive π-conjugation with the carbonyl
groups and the ring strain of the cyclobutene group, esters
derived from squaric acids (squarate derivatives) are more
reactive than generic esters.11 Interestingly, substituting the
squarate derivatives with an amino group (e.g., 1a) increases
long-term stability against hydrolysis in water at pH 7.02 to
at least 7 days. Presence of esterases or cell lysates does not
promote hydrolysis either (see Supporting Information).
Examining the reactivity of the substitution reaction of
amino squarate derivatives (Figure 1) with a mixture of five
Figure 2. Plot of conversion of 1a to 2a in different solvents versus
time.
has a higher dielectric constant than methanol, the dielectric
constant or polarity of the solvent does not seem to play a
critical role in facilitating this reaction. Furthermore, as both
the amino acid and squarate derivatives are highly soluble
in water, the hydrophobic packing proposed for enhanced
Diels-Alder reaction in water does not seem to be a viable
rationale for this reaction.2e,f
Urea-Catalyzed Coupling via Hydrogen Bonding. To
further investigate the effect of hydrogen bonding on the
reaction, we measured the rate of the reaction by adding 20%
D2O or 5 M urea into DMSO-d6. In both cases, the reaction
is revitalized for both the yield and the rate of the reaction
(Figure 2 and Table 1). Interestingly, when urea is added to
Figure 1. Chemoselective reaction of squarate derivatives with
amino acids (a mixture of L-cysteine, L-serine, glycine, L-tyrosine,
L-lysine) and with peptides containing N-terminus cysteine (N′-
CAGRGDS-C′).
amino acids (L-cysteine, L-serine, glycine, L-tyrosine, and
L-lysine), however, indicates that squarate derivatives react
exclusively with cysteine in water at neutral pH. When the
pH is increased to 8.5, the L-lysine residue also starts to
substitute the squarate derivative (see Supporting Informa-
tion).
Reaction Driven by Hydrogen Bonding. Measuring the
rate of the reaction of 1a and L-cysteine ethyl ester in
different solvents (D2O, CD3OD, DMSO-d6 ,and THF-d8)
reveals that the reaction is driven by the presence of hydrogen
bonds in the solvent rather than the dielectric properties of
the solvent (Figure 2). First, this reaction does not proceed
in polar aprotic solvents (DMSO-d6 and THF-d8), but
proceeds in protic solvents. Second, this reaction proceeds
with the fastest rate and highest yield in water. As DMSO
Table 1. Rates of the Reaction of 1a and L-Cysteine Affording
2a in Different Solvents
solvent
ꢀa k (×103)b relative rate % conversionc
D2O
80.0
-
-
32.6
-
48.0
9.0
5.5
1.0
0.6
0.3
30.0
18.3
3.3
2.0
1.0
-
100 (80d)
100
43
5 M urea/D2O
5 M urea/DMSO-d6
CD3OD
20% D2O/DMSO-d6
DMSO-d6
22
15
e
-
-
a Dielectric constant at 25 °C. b Initial rate constant (see Supporting
Information). c Calculated from the 1H NMR peak integration (see Sup-
porting Information). d Isolated yield. e No detectable conversion is ob-
served.
(8) (a) Krasinski, A.; Radic, Z.; Manetsch, R.; Raushel, J.; Taylor, P.;
Sharpless, K. B.; Kolb, H. C. J. Am. Chem. Soc. 2005, 127, 6686. (b)
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K. B.; Kolb, H. C. J. Am. Chem. Soc. 2004, 126, 12809.
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126, 15942.
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Gin, D. Y. J. Am. Chem. Soc. 2004, 126, 12712. (c) Zhu, Y.; van der Donk,
W. A. Org. Lett. 2001, 8, 1189.
water, the initial rate of the reaction is slightly suppressed,
but the complete conversion of the reactant is still obtained
in the same time frame (Table 1). This result is consistent
with the notion that urea, functioning as a weaker hydrogen-
bond donor than water, promotes the reaction in DMSO but
disrupts the initial rate of the reaction in water. Urea is known
to catalyze carbon-carbon bond formation by activating
aldimines12a,b and ketoimines12c via hydrogen bonding in
Mannich12 and hydrocyanation reactions. In this work we
demonstrate that urea can also be used to catalyze the amide
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70, 10792. (b) Xie, J.; Comeau, A. B.; Seto, C. T. Org. Lett. 2004, 6, 68.
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2003, 68, 83.
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Org. Lett., Vol. 9, No. 23, 2007

formation between squarate derivatives and cysteine in
organic solvents. Although the catalysis by urea is slower
in DMSO-d6 than D2O, this result appears to support the
proposition that the electrophilicity of the squarate substrates
is enhanced by hydrogen bonds.
Table 3. Reaction of 1a with Different Nucleophiles To
Determine Functional Group Requirement of This
Chemoselective Ligation
Effects of Squarate Substituents and Leaving Groups.
Examining the reactivity of different squarate derivatives
indicates that this reaction tolerates a wide range of substi-
tutents. But the presence of both of the carbonyl groups is
necessary for facilitating this chemoselective reaction in
water at pH 7.02 (Table 2, entry 4). In our attempt to extend
Table 2. Ligation of Different Squarate Derivatives with
L-Cysteine Ethyl Ester in Phosphate Buffer (pH 7.02)
a Isolated yield after purification by filtration and washing with water.
b No detectable conversion was observed.
first reacting with the squarate derivative (1a) to form a
covalent intermediate, followed by an intramolecular dis-
placment by the amino group (S f N acyl transfer) to afford
the final product.5d
For â-mercaptoethanol, the thiol group is less acidic (pKa
9.61)13b compared to â-mercaptoethylamine and thus does
not efficiently react with squarate derivative, 1a. The low
yield (∼5%, entry 3, Table 3) of the reaction between
cystamine and 1a is consistent with the lack of the thiol group
in the solution containing disulfide amine (cystamine).
Furthermore, it is known that the substitution by an amine
on monoamine squaric acid derivative is slow.14
a Isolated yield; small amounts of hetero- and homodimer were also
isolated. b Isolated yield; the product was recovered as homodimer of
disulfide. c No detectable conversion was observed.
Together these results indicate that both the amine and
thiol groups in close proximity are required for the chemo-
selective reaction to occur in aqueous buffer at neutral pH.
For mechanistic consideration, we believed that this chemo-
selective reaction is likely enabled when the pKa of the thiol
group is lowered by having an amino group in close
proximity in an aqueous solvent, and the reaction proceeds
via an intramolecular S f N acyl transfer.5d
The initial step of the reaction involves attack of the
thiolate ion on the electrophilic carbon of the squarate to
form a thioester-type intermediate. This intermediate then
undergoes facile intramolecular S f N acyl transfer to form
a stable ligated product (Figure 3).
this reaction to peptides containing N-terminus cysteine, we
observed that the leaving group on squarate is a critical
component for ligating peptides to the squarate derivatives.
When peptides were used instead of amino acids, the ligated
product was obtained in high yield only when the phenoxy
derivative was used instead of the ethoxy derivative (Figure
1).
Requirement of the Nucleophile. To determine the
requirement for the nucleophiles, we investigated the reaction
of 1a with â-mercaptoethyl amine, â-mercaptoethanol, and
cystamine (Table 3). The reaction between 1a and â-mer-
captoethylamine resulted in the formation of ligated product
4a. When â-mercaptoethanol was used in place of â-mer-
captoethylamine, no detectable conversion was observed.
When cystamine was used as the nucleophile, only trace
amount (∼5% yield) of the product was obtained.
For â-mercaptoethylamine, the thiol group has a pKa of
8.50,13a and the ligation result is consistent with the thiolate
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Figure 3. Proposed mechanism for the chemoselective reaction
of squarate derivative with cysteine.
Org. Lett., Vol. 9, No. 23, 2007
4899

It is interesting that, while the squarate derivatives are
activated sufficiently for the nucleophilic attack by thiolate
ions in water at neutral pH, they are also stable against
hydrolysis in water.
proceeds preferentially in water and is pH dependent, the
squarate chemistry may offer further selectivity between
different thiol groups in more complex biological conditions.
This topic and the use of this reaction to synthesize new
non-peptidic mimics of integrin antagonists are the subjects
of our ongoing research.
To conclude, we have demonstrated a new chemoselective
reaction between squarate derivatives and N-terminus cys-
teine residues in water at neutral pH. The reaction is primarily
promoted by the hydrogen bonds rather than by the high
dielectric property of water. By using this chemoselective
ligation reaction we have developed a rigid linker to ligate
two unprotected peptides together. We believe that this class
of water-driven reactions has the potential for developing
biocompatible reagents for in ViVo studies, and for developing
useful nonnatural structural moieties for modifying biomol-
ecules.
Coupling Peptides Using Squarate. We explored the
utility of 3,4-diphenoxy-1,2-cyclobutenedione as a linker to
couple two peptides together. First, we coupled a peptide
containing a lysine (NH2-AKGRGDS-COOH) residue onto
the diphenoxy squarate in DMSO/Et3N to obtain a mono-
substituted product. No disubstitution was observed as the
second substitution is much slower than the first.14 After
purifying the lysine-phenoxy squarate conjugate by extrac-
tion, we coupled a second peptide containing N-terminus
cysteine (NH2-CAGRDGS-COOH) in phosphate buffer at
pH 7 (Figure 4).
Acknowledgment. We thank the Chemistry Department
of SU and the Syracuse Center for Excellence CARTI award
supported by the U.S. Environmental Protection Agency
[Grant X-83232501-0] and NSF-CMMI (Grant No. 0727491)
for partial financial support, Dr. Boddy for instrumentation
(LC/MS) and Drs. Goodisman and Sponsler for helpful
discussion.
Figure 4. Schematic representation to stitch two peptides together
using rigid squarate linker.
Supporting Information Available: Kinetics studies,
characterization of the stability of squarate derivatives in
esterase and cell lysates; experimental procedures and
spectral data for all the new compounds; MS and HPLC
traces of reactions with peptides. This material is available
free of charge via the Internet at http://pubs.acs.org.
We note that the maleimide group also offers selective
coupling with the thiol group.15 Comparing the squarate
coupling to the maleimide coupling in aqueous environment,
we did not observe a noticeable difference in the kinetics
and the concentration requirement by using alkoxy squarate
(results not included). However, because squarate coupling
OL702275Q
(14) Owen, R. M.; Carlson, C. B.; Xu, J.; Mowery, P.; Fassella, E.;
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Org. Lett., Vol. 9, No. 23, 2007