Secondary amine selective Petasis (SASP) bioconjugation

Article abstract of DOI:10.1039/c9sc04697f

Selective modification of proteins enables synthesis of antibody-drug conjugates, cellular drug delivery and construction of new materials. Many groups have developed methods for selective N-terminal modification without affecting the side chain of lysine by judicious pH control. This is due to lower basicity of the N-terminus relative to lysine side chains. But none of the methods are capable of selective modification of secondary amines or N-terminal proline, which has similar basicity as lysine. Here, we report a secondary amine selective Petasis (SASP) reaction for selective bioconjugation at N-terminal proline. We exploited the ability of secondary amines to form highly electrophilic iminium ions with aldehydes, which rapidly reacted with nucleophilic organoboronates, resulting in robust labeling of N-terminal proline under biocompatible conditions. This is the first time the Petasis reaction has been utilized for selective modification of secondary amines on completely unprotected peptides and proteins under physiological conditions. Peptide screening results showed that the reaction is highly selective for N-terminal proline. There are no other chemical methods reported in literature that are selective for N-terminal proline in both peptides and proteins. This is a multicomponent reaction leading to the synthesis of doubly functionalized bioconjugates in one step that can be difficult to achieve using other methods. The key advantage of the SASP reaction includes its high chemoselective and stereoselective (>99% de) nature, and it affords dual labeled proteins in one pot. The broad utility of this bioconjugation is highlighted for a variety of peptides and proteins, including aldolase and creatine kinase.

Full text of DOI:10.1039/c9sc04697f

Showcasing research from Professor Monika Raj’s
laboratory, Department of Chemistry and Biochemistry,
Auburn University, Alabama, United States.
As featured in:
Secondary amine selective Petasis (SASP) bioconjugation
Secondary amine selective Petasis (SASP) bioconjugation
for the selective labelling of peptides and proteins with
N-terminal proline is developed. This reaction exploits the
ability of secondary amines to form highly electrophilic
iminium ions with aldehydes, which rapidly react with
nucleophilic organoboronates, resulting in the robust
labelling of secondary amines under biocompatible
conditions. This is a multicomponent reaction, as doubly
functionalized secondary amines are synthesized in one step
on completely unprotected peptides and proteins under
physiological conditions. The key advantage of the SASP
reaction include its high chemoselective and stereoselective
(>99% de) nature.
See Monika Raj et al.,
Chem. Sci., 2020, 11, 53.
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Secondary amine selective Petasis (SASP)
bioconjugation†
Cite this: Chem. Sci., 2020, 11, 53
a
b
b
ac
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Yonnette E. Sim,
Mikhail Y. Reibarkh
Ogonna Nwajiobi,
Sriram Mahesh,
Ryan D. Cohen,
c
b
*
and Monika Raj
Selective modification of proteins enables synthesis of antibody-drug conjugates, cellular drug delivery and
construction of new materials. Many groups have developed methods for selective N-terminal modification
without affecting the side chain of lysine by judicious pH control. This is due to lower basicity of the N-
terminus relative to lysine side chains. But none of the methods are capable of selective modification of
secondary amines or N-terminal proline, which has similar basicity as lysine. Here, we report a secondary
amine selective Petasis (SASP) reaction for selective bioconjugation at N-terminal proline. We exploited
the ability of secondary amines to form highly electrophilic iminium ions with aldehydes, which rapidly
reacted with nucleophilic organoboronates, resulting in robust labeling of N-terminal proline under
biocompatible conditions. This is the first time the Petasis reaction has been utilized for selective
modification of secondary amines on completely unprotected peptides and proteins under physiological
conditions. Peptide screening results showed that the reaction is highly selective for N-terminal proline.
There are no other chemical methods reported in literature that are selective for N-terminal proline in
both peptides and proteins. This is a multicomponent reaction leading to the synthesis of doubly
functionalized bioconjugates in one step that can be difficult to achieve using other methods. The key
advantage of the SASP reaction includes its high chemoselective and stereoselective (>99% de) nature,
and it affords dual labeled proteins in one pot. The broad utility of this bioconjugation is highlighted for
a variety of peptides and proteins, including aldolase and creatine kinase.
Received 18th September 2019
Accepted 29th October 2019
DOI: 10.1039/c9sc04697f
rsc.li/chemical-science
developed methods for targeting the N-terminus of proteins,
while avoiding lysine modication by controlling the reaction
pH.17–19 Our group utilized a transamination approach and aldol
chemistry for selective modication of protein N-termini.20
Recently, Francis and co-workers developed a robust method for
the N-terminal modication of proteins.21 But none of these
methods are capable of labeling secondary amines or N-terminal
proline in a selective manner in both peptides and proteins.
Here, we report a multicomponent secondary amine selective
Petasis (SASP) bioconjugation method for selective labeling of
Introduction
The site-selective synthetic modication of proteins enables the
construction of biomolecular hybrids for a multitude of bio-
analytical,1 therapeutic, chemical biology2 and bioengineering
applications.3–5 The synthesis of these constructs requires che-
moselective and regioselective bioconjugation reactions that
proceed under ambient, aqueous conditions at a kinetically
favorable rate. Common methods for selective protein modi-
cation include alkylation of introduced cysteine residues,6 tar-
geting articial amino acids with distinct reactivity,7–9 native
chemical ligation,10,11 and enzymatic labeling techniques.12,13
There have been several reported methods for specic labeling
of particular amino acids at the N-terminus, such as cysteine,10
tryptophan,14 serine,15,16 and threonine.15,16 Some groups
aDepartment of Chemistry and Biochemistry, Seton Hall University, South Orange, New
Jersey 07079, USA
bDepartment of Chemistry and Biochemistry, Auburn University, Auburn, Alabama
36830, United States. E-mail: mzr0068@auburn.edu
cDepartment of Process Research and Development, Merck & Co., Inc., Rahway, New
Jersey 07065, USA
† Electronic supplementary information (ESI) available: General experimental
methods, spectroscopic data and supplementary gures (PDF). See DOI:
10.1039/c9sc04697f
Fig. 1 SASP bioconjugation reaction.
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N-terminal proline of peptides and proteins (Fig. 1). Previously, and proteins is derived from the well-known Petasis reaction,
a method for modication at N-terminal proline was attempted which forms stable C–N and C–C bonds at the site of conjuga-
through oxidative coupling with an amino phenol;22 however, it tion between amine, aldehyde and organoboron reagent. Key
leads to the modication of any amino acid at the N-terminus of features that caught our attention for choosing the Petasis
peptides. The chemoselectivity was not observed for proline over reaction for developing
a
secondary amine selective-
other amino acids in peptides. The oxidative approach also leads bioconjugation reaction are (i) the moderately high reactivity
to the modication of cysteine; thus, cysteine protection was of the Petasis reaction for secondary amines in contrast to
required for selective labeling.22 To the best of our knowledge, primary amines and ammonia; (ii) the exclusive preference of
there has been no chemical method reported for selective labeling boronic acid to attack the carbon–nitrogen double bond, over
of secondary amines, such as proline, in peptides and proteins any carbon–oxygen double bonds; (iii) the compatibility of
under physiological conditions. A major chemical challenge that Petasis reaction with water;26 (iv) stability of organoboron
accompanies the development of a reaction that selectively reagents towards air and moisture, (v) tolerance to a broad
modies proline (pKa ¼ 10.5) is the presence of competing, more range of reactive functional groups; and (vi) the multicompo-
reactive primary amino acids (pKa ¼ 9.5), and the equally reactive nent nature of the Petasis reaction to provide a method for dual
side chain of lysine (pKa ¼ 10.6).22 The secondary amine selective labeling of proteins in one-pot. In our design of the SASP
Petasis (SASP) reaction accomplishes this goal by exploiting the reaction we were guided by a thorough kinetic study of the
ability of secondary amines to form more electrophilic reactive classical Petasis reaction indicating that formation of an imine/
iminium ions (1A, Fig. 2) towards nucleophilic organoboronates as iminium ion intermediate is partially rate limiting.27 Taking
compared to imines (1B, Fig. 2) obtained from primary amines.23,24 note of the rapid rates at which amines condense with alde-
This makes the secondary amine selective Petasis (SASP) bio- hydes and ketones, we anticipated that the rate of the iminium
conjugation quite different from other methodologies attempted ion formation would be faster than the imine formation
in literature. Peptide substrates were rst screened to identify the because of the high nucleophilicity of secondary amines (N ¼
site of modication. The results showed high selectivity for N- 17) as compared to primary amines (N ¼ 12) in water24 (Fig. 2).
terminal proline over any other amino acids. We did not observe
Moreover, iminium ions obtained from secondary amines
modication of any other amino acids. The reaction was then are more electrophilic as compared to imines toward organo-
applied to protein substrates, showing similarly high conversions boronates at physiological pH 7.3. This was most likely due to
when an N-terminal proline residue was present. This multicom- the low basicity of imines (pKa ꢀ 9), which remained unproto-
ponent reaction dually labeled the peptides and proteins in one nated under physiological conditions. These points suggested
pot under physiological conditions with various cargoes. More- that the SASP reaction could be utilized for chemo- and site-
over, the SASP reaction generated a chiral centre at the site of selective labeling of N-terminal proline under physiological
conjugation with high stereoselectivity. This was most likely due to conditions. Thus this method was utilized for selective func-
the tendency of proline to induce asymmetric induction through tionalization of biological molecules containing N-terminal
the iminium ion mechanism.25 Unique selectivity, operational proline that are native or readily introduced by standard site-
simplicity, no catalyst requirement, fast reaction kinetics, ability to directed mutagenesis.28
generate a stable C–C bond, and physiological conditions are the
major strengths of the SASP bioconjugation.
Evaluating the reactivity and selectivity of the SASP reaction
on peptides
Results and discussion
Design of Petasis reaction for selective modication of
Initial experiments were designed to identify the optimal reac-
tion conditions for labeling biological molecules containing N-
terminal proline. The high chemoselectivity of the SASP reac-
tion for N-terminal proline was determined by carrying out
reactions with tripeptides XAF, where X was substituted with 19
different canonical amino acids (pKa ¼ 9.5) including amino
acids with reactive side chains such as Lys, Thr, Ser, Asn, Gln,
Glu, Asp, Tyr, Trp, Met and Cys (Fig. 3). These peptides were
synthesized on solid support using standard Fmoc synthesis,
cleaved from the resin, and puried by HPLC. Aer purication
and lyophilization, peptides were exposed to salicylaldehyde
SAL 2a and phenylboronic acid PBA 3a. The reactions were
carried out in 25 mM phosphate buffer (pH 7.3) : DMF (4 : 1) at
room temperature, and reaction mixtures were analyzed by LC-
MS (Fig. 3). DMF was used as a cosolvent due to the low solu-
bility of PBA 3a in aqueous buffer. The results showed 70%
conversion to Petasis product 4a with peptide PAF 1a containing
proline at the N-terminus (Fig. 3). The formation of the Petasis
product with peptides XAF containing primary amino acids at
secondary amines
Our design of one-step, site-selective, and dual labeling of
secondary amines/proline in completely unprotected peptides
Fig. 2 Proposed high reactivity and selectivity towards secondary
amines compared to primary amines.
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carbanion.29 Further, the high selectivity of the SASP reaction
for proline in the presence of highly reactive PMB 3b was veri-
ed by carrying out the reaction with a mixture of two peptides
PAF 1a and XAF (where, X ¼ Glu, Lys, Thr, Trp, Tyr, Leu, and
His) and SAL 2a in one pot under physiological conditions
(Fig. S3†). Again, the Petasis product 4b was observed from
peptide PAF 1a only. As further validation of the reaction site
specicity, no modication of lysine and cysteine was observed
with peptides, PFKAFV and PFCAF, containing both N-terminal
proline (pKa 10.5) and the reactive side chain of lysine (pKa ¼
10.6) and cysteine (Fig. S4†), respectively.
We also investigated SASP reactions of peptide PAF 1a and
PMB 3b with water-soluble aldehydes such as 2-pyr-
idinecarboxyaldehyde
(2PCA)
2b
and
3-hydroxy-2-
pyridinecarbaldehyde (HPCA) 2d under physiological condi-
tions. The results showed moderate conversion to Petasis
products with both the aldehydes. On detailed analysis we
found that this is due to the tendency of these aldehydes to
generate an imidazolidinone product with the N-terminus as
reported by Francis et al.21 To minimize the formation of the
imidazolidinone product and to increase the yield of the Petasis
product with aldehydes such as 2PCA 2b and HPCA 2d, we
carried out optimization studies with HPCA 2d. The reagent
ratios of aldehyde and boronic acid were varied. Peptide PAF 1a
(15 mM) was reacted with aldehyde HPCA 2d (1.5–3 equiv., 22.6–
45 mM) and PMB 3b (4–8 equiv., 60–120 mM) in 25 mM phos-
phate buffer (pH 7.3) : DMSO (4 : 1) at room temperature for 4 h
(Table S1†). It was demonstrated that conversion to Petasis
product was highest using 1.5 equiv. of HPCA 2d and 8 equiv. of
PMB 3b. We also observed the fast reaction rate under such
conditions. The Petasis product 4c of peptide PAF 1a, 2PCA 2b
and PMB 3b was then synthesized on a larger scale and char-
acterized by 2D NMR and HRMS (Fig. S5†).
Fig. 3 Chemoselectivity of SASP reaction for N-terminal proline. The
reaction showed high selectivity for N-terminal proline as analyzed by
LC/MS for a panel of XAF peptides, where X represents a variable amino
acid. Reaction conditions: 2 mg XAF (12–17 mM, SAL 2a (3 equiv., 36–
51 mM), PBA 3a (4 equiv., 48–68 mM) in buffer (pH 7.3) : DMF (4 : 1) at
room temperature for 4–24 h.
the N-terminus was not detected, thereby showing that SASP
exhibited high selectivity for secondary amines. A second
observation of this screen was the fact that lysine and cysteine
side chains were not modied under the reaction conditions.
Next, we synthesized a water-soluble analogue of SAL by
combining 4-formyl-3-hydroxybenzoicacid with propargylamine
Verication that SASP modies only N-terminal proline
The chemoselectivity of SASP reaction towards N-terminal (Fig. S6†). The resulting SAL analogue 2a0 is highly soluble in
proline was further veried by competition reactions using water. We investigated the selectivity of aldehydes such as
a mixture of two peptides, PAF 1a and XAF (X ¼ Ala, Glu, Arg, water-soluble SAL analogue 2a0, 2PCA 2b and HPCA 2d by
Val, Asn, Asp, Ser, Trp, Tyr, Leu, Lys, and Met), in one pot carrying out reactions with various N-terminal tripeptides XAF
(Fig. S1†). Different peptides were mixed together and exposed (where, X ¼ Glu, Thr, Ala, Gly, Phe, Tyr, Leu, Met, Pro and Gln)
to SAL 2a and PBA 3a in 25 mM phosphate buffer (pH 7.3) : DMF and PMB 3b under optimized conditions. The peptides XAF 1
(4 : 1) solution. Among all combinations, the Petasis product 4a (12–17 mM) were reacted with aldehydes 2a0, 2b or 2d (1.5
was observed from peptide PAF 1a only, while the other peptides equiv., 18–25.5 mM) and PMB 3b (8 equiv., 96–136 mM) in
XAF did not lead to any products under any tested conditions 25 mM phosphate buffer (pH 7.3) : DMSO (4 : 1) for 4 h at 37 ꢁC.
(Fig. S1†).
The reactions were quenched by dilution with cold water and by
ꢁ
Next, peptide PAF 1a and SAL 2a were exposed to more water freezing at ꢂ80 C followed by lyophilization. With aldehydes
soluble p-methoxyphenyl boronic acid PMB 3b and reaction was 2b and 2d, the proline terminal peptide reached the highest
carried out under physiological conditions in 25 mM phosphate levels of conversion; although, modications of other N-
buffer (pH 7.3) : DMSO (4 : 1). The results showed progressively terminal peptides were also observed under the reaction
greater reactivity as 90% conversion to Petasis product 4b was conditions (Fig. 4). Despite efforts to optimize conditions for all
achieved within 2 hours as determined by LC-MS. The product N-termini for 2PCA and HPCA mediated Petasis reaction,
4b of Petasis reaction between peptide PAF 1a, SAL 2a and PMB proline still stood out as the most reactive species. As observed
3b was synthesized on larger scale and characterized using two- earlier with SAL 2a, the Petasis reaction with water-soluble SAL
dimensional NMR and high resolution mass spectrometry analogue 2a0 was also highly chemoselective for N-terminal
HRMS (Fig. S2†). The greater reactivity of 3b is most likely due to proline. The formation of the Petasis product with peptides
the electron-donating group (EDG) at the para position that XAF containing primary amino acids at the N-terminus was not
signicantly increases nucleophilicity of the phenyl detected with water-soluble SAL analogue 2a' (Fig. 4 and S7†).
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Petasis product 4c obtained from the reaction of peptide PAF 1a
with 2-PCA 2b (with no ortho-hydroxyl group) and PMB 3b using
LC-MS (Fig. S9†). No degradation of the Petasis product 4c was
observed under any of tested conditions demonstrating the
hydrolytic stability of the product. The ability of the linkage to
withstand these conditions makes this method applicable for
the development of new materials for different biological
applications.
Effects of pH on the rate of the SASP reaction
The rate of the SASP reaction at different pH conditions ranging
from 5.5 to 9.5 was explored on the model peptide PAF 1a with
SAL 2a and PMB 3b. Although the rate is quite high at neutral
pH 7.3 with 62% conversion to the Petasis product 4b in 15 min,
the highest conversion (80% within 15 min) was observed at pH
9.5 (Fig. S10†). However, considering the biocompatibility of
our method, physiological pH is the ideal condition for
promoting this reaction with peptides and proteins. Next, we
explored the reactivity of primary amines at lower pH, as it
could be possible to protonate them and therefore add boronic
acids. We screened N-terminal primary tripeptides XAF with
SAL 2a/2PCA 2b and PMB 3b at pH 5.5. The results showed that
SAL 2a is highly selective for N-terminal proline even at low pH
5.5. The results with 2PCA 2b showed high reactivity with N-
terminal primary amines at low pH 5.5 as compared to physi-
ological pH 7.3 (Fig. S11†). It could be because imines generated
by primary amines can protonate faster at lower pH, and
therefore are more reactive towards boronic acids.
Fig.
4 Peptide modification with Petasis reaction. Peptides with
sequence XAF, where X represents a variable amino acid were
screened for Petasis reaction with PMB 3b and a variety of aldehydes
2b, 2d and water-soluble SAL analogue 2a0. % conversion to Petasis
products were determined by LC/MS. Reaction conditions: XAF (12–17
mM), RCHO 2 (1.5 equiv. 18–25.5 mM), PMB 3b (8 equiv. 96–136 mM)
in 25 mM buffer (pH 7.3) : DMSO (4 : 1) and 37 ꢁC. The samples were
incubated for 4 h.
Stereoselective nature of SASP bioconjugation
Another unique feature of the SASP reaction is its ability to
generate a new chiral centre at the site of conjugation with high
stereoselectivity. This reaction could be of a great benet in the
derivatization of peptides and proteins involved in drug
discovery since it has been reported that regioselectivity and
stereoselectivity are crucial for assuming the active binding
conformation.30
Stability studies of the SASP product.
The analysis of the SASP product 4b showed the formation of
a newly generated chiral centre with high stereoselectivity (de
>99%) and with the absolute conguration of (R) as determined
by analysis of dipolar couplings obtained from ROESY NMR
spectroscopy and by comparing the observed proton and carbon
chemical shis versus those calculated from density functional
theory (Fig. 5a and S12†). In addition, the DP4+ method,31 which
applies a Bayesian statistical treatment to the NMR data, pre-
dicted the R-conguration to be the correct stereoisomer with
greater than 99% probability. The high stereoselectivity of the
SASP bioconjugation was most likely directed by the chirality of
N-terminal proline and by the boron which guides the nucleo-
philic attack of a phenylcarbanion on the iminium ion inter-
mediate from the Re face since attack from the Si face is
hindered, instead of the electronic effects of other substitu-
ents32,33 (Fig. 5b). To further validate the role of proline for
obtaining high stereoselectivity with R absolute conguration,
the SASP reaction was performed with peptide pAF containing D-
proline under the optimized conditions. The Petasis product 4B
Given the different reactivity of aldehydes with proline, we
veried the stability of the product to a variety of conditions.
The puried Petasis product 4b obtained from peptide PAF 1a,
SAL 2a and PMB 3b was exposed to acidic and basic pH (pH 3.5–
10.5) at room temperature. The peptides were analysed aer
regular intervals by LC-MS. Over the course of 24 h, the majority
of the Petasis product 4b was stable at a pH range of 6.5 to 9.5;
whereas at low pH 3.5 to 5.5, half of the Petasis product 4b was
hydrolyzed to the starting peptide PAF 1a, as analyzed by LC-MS
(Fig. S8†). We hypothesized that Petasis product 4b becomes
protonated under acidic conditions leading to the hydrolysis of
Petasis product by nucleophilic attack from the neighboring
ortho-hydroxyl group to a newly generated chiral centre of the
Petasis product 4b. Notably, pH had a huge effect on the degree
of decomposition. Through substrate variation, it is likely that
products of increased stability can be obtained for applications.
To validate the process of hydrolysis, we analyzed the stability of
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exhibits Lewis basic groups proximal to the reactive carbonyl
carbon (Fig. 6b). Notably, Petasis products were not observed
with 4-hydroxy benzaldehyde 2e, which contains a distal Lewis
basic group, or from 2-nitro benzaldehyde 2f, which lacks Lewis
basic group (Fig. 6b). The results showed that the presence of
a Lewis base directly adjacent to the aldehyde was crucial for
this reaction. Ultimately, SAL 2a, water-soluble SAL analogue
2a0, 2PCA 2b, glyoxylic acid 2c and HPCA 2d reagents emerged
as the most effective and synthetically tractable aldehydes for
further studies and modications.
Role of boronic acids in SASP reaction
The third reagent involved in the multi-component SASP reac-
tion is organoboronic acid. Based on the initial SASP reaction
and the proposed mechanism, organoboronic acid acts as
a nucleophile. The high nucleophilicity of the organoboronic
acid signicantly accelerates the rate of the SASP reaction. With
this reactivity behavior in mind, various aryl- and alkenyl-
boronic acids (3a–i) were screened for their potential to
modify a model peptide PAF 1a with SAL 2a or 2PCA 2b under
physiological conditions (Fig. 6b and S14†). The results showed
that arylboronic acid with electron donating group EDGs such
as para-methoxyboronic acid (PMB) 3b exhibited progressively
greater reactivity (90% conversion in 2 h) as compared to aryl-
boronic acid containing electron withdrawing groups (EWGs)
such as 4-triuoromethylphenylboronic acid 3c (0% conversion
in 24 h) (Fig. 6b). Interestingly, we observed the formation of
a Petasis product with straight chain octenylboronic acids
under the reaction conditions. To conrm the structure of the
Petasis product with octenylboronic acid, we carried out NMR
on the isolated product obtained from the reaction of mor-
pholine with octylenylboronic acid and SAL 2a/2PCA 2b
(Fig. S15†). The substrate scope was further explored with
boronic acid derivatives containing an alkyne group 3j and
a dye, dansyl methylphenylboronic acid, 3k which can then be
utilized for click bioconjugation and cell imaging studies,
respectively. These derivatives of boronic acids (3j and 3k) were
synthesized from commercially available 4-(2-carboxyethyl)
benzeneboronic acid 3e and 4-(aminomethyl)phenylboronic
acid (for synthesis, see Fig. S16†)
Fig.
5 Chemo- and stereo-selective SASP bioconjuation under
physiological conditions. (a) NMR ROEs of Petasis product 4b are
consistent with R configuration from molecular mechanics Monte
Carlo. (b) Plausible pathway for high stereoselectivity.
was obtained from the reaction of peptide pAF with SAL 2a and
PMB 3b and characterized by 2D NMR (Fig. S13†). The analysis
of the Petasis product 4B showed formation of a newly gener-
ated chiral centre with (S) absolute conguration (de > 99%)
which is opposite from the product obtained from peptide PAF
with L-proline (Fig. S13†). This result showed that proline not
only functions as a site of conjugation in the SASP reaction but
also acts as a chiral asymmetric catalyst responsible for asym-
metric induction in the Petasis product 4b, 4B and 4c (Fig. 5b
and S5, S12 and S13†).
Mechanistic studies of SASP reaction: role of aldehydes.
Aldehydes, such as salicylaldehyde SAL 2a, 2PCA 2b and HPCA
2d, were used for our initial studies with the view that the
SASP reaction on biologically active peptides
hydroxyl and pyridine groups at the ortho position relative to The broad substrate scope of the SASP bioconjugation was
the aldehyde coordinates with PMB 3b by a Lewis acid–base determined by carrying out reactions with bioactive peptides
mechanism (by dative bond), which brings the organoboronic with varying amino acid composition such as angiotensin I
acid in close proximity to the reaction centre (Fig. 6a). Also, this (PDRVYIHPFHL), a peptide hormone that causes vasoconstric-
dative bond with organoboronic acid facilitates the release of tion and a subsequent increase in blood pressure, bradykinin
a carbanion to react with an electrophilic iminium ion through (PPGFSPFR), an inammatory mediator, and hyper-
a favorable ve-membered cyclic transition state, thereby trehalosemic neuropeptide (PVNFSPGWGT)29 by using SAL 2a
generating a stable C–C bond at the site of conjugation (Fig. 6a). and PMB 3b under optimized conditions. The amino acid
To test this proposal, a set of other aldehydes such as glyoxylic composition of the peptide exhibited minimal impact on the
acid 2c, 4-hydroxy benzaldehyde 2e and 2-nitro benzaldehyde 2f degree or selectivity of the modication, as observed by good to
were investigated for their potential to modify the N-terminus of excellent yields for singly modied peptides at N-terminal
a model peptide PAF 1a using PMB 3b under optimized proline in all cases (Fig. 7 and S17†). The SASP reaction was
conditions (Fig. 6b, and S14†). Glyoxylic acid 2c showed carried out on a peptide PICSLYQLENYCN derived from chain A
progressively greater reactivity for the SASP reaction since it of insulin with water-soluble SAL derivatives (Fig. 7). The results
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Fig. 6 Selective dual labeling with diverse functional groups. (a) Significance of Lewis-base near to the aldehyde. (b) Aldehyde substrate scope
and boronic acid scope with peptide PAF 1a. Reaction conditions. To a peptide PAF 1a (2 mg, 12 mM), in 0.5 mL solution of 25 mM phosphate
buffer (pH 7.3) : DMSO (4 : 1) was added SAL 2a or 2PCA 2b (1.5 equiv., 18 mM) and a variety of boronic acids 3 (8 equiv., 96 mM). The reactions
were stirred at room temperature for 2–24 h.
showed single modication to Petasis conjugate at N-terminal
proline with >80% conversion. The reaction with a correspond-
ing peptide ICSLYQLENYCN containing primary amino acid at
the N-terminus did not lead to the formation of any Petasis
product.
Protein scope for SASP reaction
The SASP reaction with N-terminal amino group was rst tested
on native proteins such as myoglobin and a-lactalbumin, which
contain several lysine residues (pKa ¼ 10.6) and lack proline at
their respective N-termini. The native proteins were reacted
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Fig. 7 Secondary amine selective Petasis reaction of bioactive peptides as analyzed by ESI-MS for angiotensin I, bradykinin, and hyper-
trehalosemic neuropeptide reactions with SAL 2a and PMB 3b. ESI-MS for insulin chain A derived peptides with water soluble SAL derivatives.
Reaction conditions. 2 mM solution of the bioactive peptide was reacted with SAL 2a (1.5 equiv., 3 mM) and PMB 3b (8 equiv., 16 mM) in 0.5 mL
25 mM phosphate buffer pH 7.3 : DMSO (4 : 1) and reactions were stirred at room temperature for 15 h.
with water-soluble 2PCA 2b and reactive PMB 3b under opti- observed with more reactive N-terminal proline containing
mized reaction conditions. We do not see any modication on proteins. Peptide screening in Fig. 4 showed that N-terminal
these proteins as analyzed by MS and gels (Fig. 8a). The protein amino acids can undergo Petasis reaction with 2PCA 2b, but
reactions were explored with higher amount of 2PCA 2b (5 the proline terminal peptide has highest conversion. Despite
equiv.) and PMB 3b (50 equiv.) but any modication on these efforts to optimize conditions for all N-termini, proline still
proteins were not observed. This could be because they have stood out as the most reactive species. When applied to protein
less reactive primary amino acids at the N-terminus.
substrates, the reaction showed a stronger requirement for the
Next, N-terminal proline containing native proteins such as N-terminal proline therefore we did not observe modication of
aldolase and creatine kinase were evaluated for the reaction myoglobin and a-lactalbumin.
with 2PCA 2b and PMB 3b. The protein reactions were carried
Aer purication, incubation of N-terminally modied
out with a slight excess of 2-PCA 2b (5 equiv.) and PMB 3b (50 creatine kinase from a pH range of 3.5 to 9.5 showed full
equiv.) in 25 mM phosphate buffer (pH 7.3) at room tempera- product stability over a 24 h period as detected by MS
ture for 16 h (Fig. 8b).
(Fig. S18†).
Analysis by MS demonstrated that creatine kinase [m ¼
43 178 (+197 Da)] and aldolase [m ¼ 39 409 (+197 Da)] were
modied by the formation of the Petasis product. We did not
observe any second modication indicating that reaction is
highly selective for N-terminal-proline. Interestingly, the Petasis
reaction with 2PCA 2b worked with a primary amino acid at the
N-terminus of peptides, but in proteins, the Petasis product was
Study of protein structure and function aer the modication
To evaluate the effect of the N-terminal modication on the
secondary and tertiary structure of aldolase, we compared the
circular dichroism (CD) of the native and the SASP modied
aldolase. The result showed no change in the secondary
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Fig. 8 Chemo- and site-selective dual labeling of proteins with 2-PCA 2b and PMB 3b. (a) No modification was observed for myoglobin and a-
lactalbumin lacking N-terminal proline. SDS PAGE images of proteins before and after SASP reaction. (b) Proteins, creatine kinase and aldolase
with N-terminal proline underwent modification to SASP product, as characterized by ESI-MS. (c) CD spectra of aldolase before and after SASP
reaction showed no change in the secondary structure of a protein. Reaction conditions: A protein (100 mM) was dissolved in 400 mL of 25 mM
phosphate buffer pH 7.3. 2PCA (0.5 mM) and PMB (5 mM) was added in the solution. The reaction was agitated at room temperature for 16 h.
structure of the modied aldolase (Fig. 8c). Currently, we are proline residues (pKa ¼ 10.5) even in the presence of free cysteine
using this dual-labeling strategy to introduce analogues of and lysine residues which is particularly challenging due to the
boronic acid and SAL, which act as IR probes or are detectable strong nucleophilicity of the thiol group and equivalent basicity of
by 19F NMR spectroscopy.34
the gꢂchain of lysine (pKa ¼ 10.6) at relevant pH values.
Furthermore, the secondary amine selective nature of this
reaction suggests that it could be very useful for selective
tagging/enriching proteins with monomethyl lysine post-
translational modications PTMs for determining their role in
regulating various cellular signaling processes.35 This work is
currently underway in our laboratory. Considering the easy
setup, high selectivity for secondary amines, dual labeling
nature and broad substrate scope with a variety of aldehydes
and organoboronic acids, we anticipate that this method will
become a highly useful tool in many scientic studies.
Conclusions
In this study, we present SASP reactions for selective modi-
cation of N-terminal proline containing peptides and native-
proteins. Proline can be introduced readily in N-terminal
positions using site-directed mutagenesis.28 The SASP
method reported here offer two distinct advantages over other
N-terminal labeling methods. First, it is highly selective for N-
terminal proline and does not lead to modication of any other
amino acids. Second, it is a multicomponent reaction and
dually labels peptides and proteins in a single step in one pot
under physiological conditions, without the use of any cata-
lysts. This reaction features excellent site specicity and
promising scale-up potential.
Conflicts of interest
There are no conicts to declare.
Moreover, the SASP reaction leads to the generation of a new
chiral centre at the site of conjugation with high stereoselectivity
(de >99%). This is due to the ability of proline to carry out
Acknowledgements
asymmetric synthesis. The results on a wide variety of peptides This research was supported by NSF CAREER Award #1752654
and proteins strongly indicate that the SASP modies N-terminal granted to M.R.
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D. J. Lindner and R. B. Pepinsky, Bioconjugate Chem., 2006,
17, 179–188.
Notes and references
18 R. Singudas, S. R. Adusumalli, P. N. Joshi and V. Rai, Chem.
Commun., 2015, 51, 473–476.
19 A. O. Chan, C. M. Ho, H. C. Chong, Y. C. Leung, J. S. Huang,
M. K. Wong and C. M. Che, J. Am. Chem. Soc., 2012, 134,
2589–2598.
20 T. S. Howard, R. D. Cohen, O. Nwajiobi, Z. P. Muneeswaran,
Y. E. Sim, N. N. Lahankar, J. T.-H. Yeh and M. Raj, Org. Lett.,
2018, 20, 5344–5347.
1 S. Zalipsky, Bioconjugate Chem., 1995, 6, 150–165.
2 J. Rao, A. Dragulescu-Andrasi and H. Yao, Curr. Opin.
Biotechnol., 2007, 18, 17–25.
3 L. S. Witus, C. Netirojjanakul, K. S. Palla, E. M. Muehl,
C.-H. Weng, A. T. Iavarone and M. B. Francis, J. Am. Chem.
Soc., 2013, 135, 17223–17229.
4 S. C. Alley, N. M. Okeley and P. D. Senter, Curr. Opin. Chem.
Biol., 2010, 14, 529–537.
21 J. I. MacDonald, H. K. Munch, T. Moore and M. B. Francis,
Nat. Chem. Biol., 2015, 11, 326–331.
5 L. S. Witus and M. B. Francis, Acc. Chem. Res., 2011, 44, 774–
783.
22 A. C. Obermeyer, J. B. Jarman and M. B. Francis, J. Am. Chem.
Soc., 2014, 136, 9572–9579.
23 N. A. Petasis and I. Akritopoulou, Tetrahedron Lett., 1993, 34,
583–586.
24 F. Brotzel, Y. C. Chu and H. Mayr, J. Org. Chem., 2007, 72,
3679–3688.
25 M. Raj and V. K. Singh, Chem. Commun., 2009, 6687–6703.
26 N. R. Candeias, L. F. Veiros, C. A. M. Afonso and
P. M. P. Gois, Eur. J. Org. Chem., 2009, 1859–1863.
27 N. Schlienger, M. R. Bryce and T. K. Hansen, Tetrahedron,
2000, 56, 10023–10030.
28 P. H. Hirel, M. J. Schmitter, P. Dessen, G. Fayat and
S. Blanquet, Proc. Natl. Acad. Sci. U. S. A., 1989, 86, 8247–
8251.
6 S. Szobota, P. Gorostiza, F. Del Bene, C. Wyart, D. L. Fortin,
K. D. Kolstad, O. Tulyatham, M. Volgraf, R. Numano,
H. L. Aaron, E. K. Scott, R. H. Kramer, J. Flannery,
H. Baier, D. Trauner and E. Y. Isacoff, Neuron, 2007, 54,
535–545.
7 V. W. Cornish, K. M. Hahn and P. G. Schultz, J. Am. Chem.
Soc., 1996, 118, 8150–8151.
8 A. Deiters, T. A. Cropp, M. Mukherji, J. W. Chin,
J. C. Anderson and P. G. Schultz, J. Am. Chem. Soc., 2003,
125, 11782–11783.
9 J. C. M. van Hest, K. L. Kiick and D. A. Tirrell, J. Am. Chem.
Soc., 2000, 122, 1282–1288.
10 P. E. Dawson, T. Muir, I. Clark-Lewis and S. Kent, Science,
1994, 266, 776–779.
´
29 F. Berree, A. Debache, Y. Marsac and B. Carboni, Tetrahedron
11 T. W. Muir, Annu. Rev. Biochem., 2003, 72, 249–289.
12 P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 3000–
3005.
Lett., 2001, 42, 3591–3594.
30 L. A. Nguyen, H. He and C. Pham-Huy, Int. J. Biomed. Sci.,
2006, 2, 85–100.
31 N. Grimblat, M. M. Zanardi and A. M. Sarotti, J. Org. Chem.,
2015, 80, 12526–12534.
32 T. J. Southwood, M. C. Curry and C. A. Hutton, Tetrahedron,
2006, 62, 236–242.
33 N. A. Petasis, A. Goodman and I. A. Zavialov, Tetrahedron,
1997, 53, 16463–16470.
34 M. A. Danielson and J. J. Falke, Annu. Rev. Biophys. Biomol.
Struct., 1996, 25, 163–195.
13 C. Uttamapinant, K. A. White, H. Baruah, S. Thompson,
´
´
M. Fernandez-Suarez, S. Puthenveetil and A. Y. Ting, Proc.
Natl. Acad. Sci. U. S. A., 2010, 107, 10914–10919.
14 X. Li, L. Zhang, S. E. Hall and J. P. Tam, Tetrahedron Lett.,
2000, 41, 4069–4073.
15 K. F. Geoghegan and J. G. Stroh, Bioconjugate Chem., 1992, 3,
138–146.
16 Y. Zhang, C. Xu, H. Y. Lam, C. L. Lee and X. Li, Proc. Natl.
Acad. Sci. U. S. A., 2013, 110, 6657–6662.
17 D. P. Baker, E. Y. Lin, K. Lin, M. Pellegrini, R. C. Petter,
L. L. Chen, R. M. Arduini, M. Brickelmaier, D. Wen,
D. M. Hess, L. Chen, D. Grant, A. Whitty, A. Gill,
35 L. E. West, S. Roy, K. Lachmi-Weiner, R. Hayashi, X. Shi,
E. Appella, T. G. Kutateladze and O. Gozani, J. Biol. Chem.,
2010, 285, 37725–37732.
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