A phthalimidation protocol that follows protein defined parameters

Article abstract of DOI:10.1039/c4cc08503e

This work outlines the first phthalimidation protocol suitable for protein labeling and performed in aqueous media at room temperature and neutral pH with no catalyst or co-reagent required. The methodology is suitable for a range of amines and its efficiency was determined with chemoselective and site-selective protein labeling. This journal is

Full text of DOI:10.1039/c4cc08503e

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. Singudas, S. R.
Adusumalli, P. N. Joshi and V. Rai, Chem. Commun., 2014, DOI: 10.1039/C4CC08503E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Page 1 of 5
ChemComm
View Article Online
DOI: 10.1039/C4CC08503E
Journal Name
RSCPublishing
COMMUNICATION
A Phthalimidation Protocol That Follows Protein
Defined Parameters
Cite this: DOI: 10.1039/x0xx00000x
Rohith Singudas,a† Srinivasa Rao Adusumalli,a† Pralhad Namdev Joshi,a and Vishal
Raia*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
En route to development of a convenient methodology for protein
conjugation, it is imperative to overcome the barrier imposed by low
reaction rate of a chemical transformation at working concentration
of proteins. This necessitates the use of 10ꢀ1000 folds excess reagent
to render practically useful conversions. Our primary focus was to
enable the transformation in physiological conditions in the absence
of a catalyst or an interfering coꢀreactant. We hypothesized that a
This work outlines the first phthalimidation protocol suitable
for protein labeling and performed in aqueous medium at
room temperature and neutral pH with no obligation of
catalyst or co-reagent. The methodology is suitable for a
range of amines and its efficiency was determined with
chemoselective and site-selective labeling of a protein.
twoꢀstep oneꢀpot process
– initiated by a fast reversible
intermolecular step forming an amphoteric intermediate 2 capable of
rendering rate determining irreversible intramolecular step (Scheme
1), can prove critical.
Organic reactions with small molecules have evolved over
generations and provide access to enormous library of compounds
through simple chemical transformations. Yet, chemical
modification of protein residues have posed stark challenges owing
to unique set of protein defined parameters. Change in the landscape
of reaction conditions such as solvent, substrate concentration,
temperature and pH has proved nonꢀtrivial.1 Adhering to the
redefined conditions and achieving high reaction rate constant has
also been arduous. Additional definite challenge has been associated
to the low chemoselectivity of reactants or coꢀreactants. For
example, azides can react with cysteine thiols,2 and phosphines can
reduce disulfide bonds and result heterogeneous protein milieu in
Staudinger ligation.3 In a recent report for a protein Nꢀterminal
modification under oxidative conditions, cysteine thiols undergo side
reactions.4 Under these circumstances, only a few methodologies
qualify and find their application in protein conjugation.5 Such
chemical tools for mild and siteꢀspecific derivatization of proteins is
desired for their wide range of applications in understanding
biological interactions,6 ligand discovery,7 disease diagnosis,8 and
highꢀthroughput screening.9 These transformations can also route
access to the knowledge of encoded protein function and enhance
genetic information pool.10 Typically, these indispensable diagnostic
tools can be accessed by the derivatization of nucleophilic side
chains in case of unmodified proteins.11 However, the diversity of
reactions that can be performed with these functional groups in
physiological conditions is very limited.12 This is largely due to the
restrictions over major synthetic chemist’s tools such as catalyst, coꢀ
catalyst and reaction conditions. The task is particularly challenging
because control over reactivity of substrate is the only preferred
parameter for modulation. One of the successful example in this
perspective is showcased with Cu free click chemistry enabled by
strained cyclooctynes.13
latent nucleophile
Nu1
Nu2
E1
E2
fast
slow
E3
2
3
1
Scheme 1. Generation of an amphoteric intermediate.
Phthalic anhydride or pthalimides were identified for the generation
of amphoteric intermediate. In retrospect, the imide derivatives are
important functionalities and present number of applications in
chemistry,14 polymers,15 and chemical biology.16 Additionally,
phthalimides have served as solvatochromic fluorophore in detection
of biomolecular interactions.17 However, all the reported methods
drive on factors such as high temperature, strong bases, and coupling
reagents.14 A biomolecule compatible pthalimidation protocol has
remained elusive till day. Here, we report phthalimidation of primary
amines in aqueous buffer at ambient temperature, physiological pH
in typically 4ꢀ48 hours. The success of transformation holds in
designing the substrate for appropriate reactivity of the amphoteric
intermediate. The methodology proved efficient for labeling of
RNase A. The chemoselectivity is inherent to the methodology,
whereas the siteꢀselectivity is driven by kinetic preferences of amino
acid residue at Nꢀterminus and rendered K1 monolabeled RNase A.
The phthalimidoamine product offers an electrophilic center suitable
for secondary amine catalyzed lateꢀstage modification. The reported
protocol overcomes the drawback of instability of reagent,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 1

ChemComm
Page 2 of 5
COMMUNICATION
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4CC08503E
intermediate or product towards hydrolysis associated with several
other bioconjugation methodologies based on lysine modification.12
In a model reaction, benzylamine was stirred with phthalic
anhydride 4a and phthalimides 4bꢀe for its efficiency to undergo
transimidation and give phthalimidoamines (Scheme 2). The reaction
with 4a results in intermediate 6 (64%) with high threshold for
conversion to product 7 (entry 1, Scheme 2). Phthalimide 4b resulted
in 67% conversion and extended reaction time offered no
improvement. Diphenylphosphate derivative of phthalimide 4c was
unreactive, whereas Nꢀallyloxyphthalimide 4d gave a mixture of
intermediate 6d, product 7 and unreacted starting material 4d after
14 h. Nꢀhydroxyphthalimide 4e reacted with benzyl amine to give
the product 7 in >99% conversions.
Figure 1. Potential energy curve for phthalimidation of 5a with 4e.
Scheme 2. (a) Evaluation of reagents for pthalimidation, (b) side
products 8 and 9 in phthalimidation.
The reaction involves a oneꢀpot twoꢀstage transformation of Nꢀ
hydroxyphthalimide (NHP) 4e with primary amine 5a involving a
ring opening reaction followed by transimidation to provide
phthalimide labeled primary amine 7 (Figure 1). The intramolecular
nucleophilic addition of latent nucleophile (see intermediate 6e) to
electrophilic carbonyl is the rate determining step (rds). Design of
reagent 4 plays a crucial role in success of this transformation as Z
indirectly controls the electrophilicity of carbonyl by regulating n to
π* contributions (see second order perturbation theory analysis in
ESI for details).
Various aliphatic and aromatic amines with disparate
nucleophilicity were tested for their reactivity with NHP 4e (Scheme
3a). The transformation was efficient with range of aliphatic and
aromatic amines and products 10aꢀ10n were formed in 40ꢀ85% yield
(also see Table S1, ESI). NHP is partially soluble in phosphate
buffer at pH 7.0. A common observation includes disappearance of
NHP within 30ꢀ120 seconds as it converts to the intermediate 6. A
clear light yellow solution stirred further results in precipitated
product that can be filtered and triturated to render the analytically
pure product. Filtering the product gradually with time shows no
effect on the reaction time. There were few exceptions where amines
were insoluble in buffer e.g. tryptamine (product 10d). For such
cases, amines were dissolved in DMSO and dispensed in reaction
mixture (10% DMSO in buffer). The products soluble in water, e.g.
10g, were isolated by liquidꢀliquid extraction. Reaction with
aminoacetaldehyde diethyl acetal showcases the importance of mild
reaction conditions where the product 10h was isolated without
tracing any side product.
Scheme 3. (a) Phthalimidation of amines, (b) and (c) transformations
of phthalimidoamine 7.
The reaction with electronically diverse set of substrates underwent
smoothly and the chemoselectivity was apparent with 10i, 10j and
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 5
Journal Name
ChemComm
View Article Online
COMMUNICATION
DOI: 10.1039/C4CC08503E
10k. Aromatic amines are sparingly soluble in aqueous buffer but Scheme 4. Chemoselective and siteꢀselective phthalimidation of
they dissolve with the progress of reaction. Degassing of solvent was RNase A.
found necessary for 10k in improving the yields from 53% to 68%.
The preferential reactivity of αꢀamine with respect to εꢀamine in an
amino acid was apparent with 10l, 10m and 10n.
Parent Ion Mass : m/z 995.55 [M+H]+
MS2
The phthalimidation protocol works smoothly in the pH range
6.0ꢀ7.8 to give product 7 in >99% conversion (Table S2, ESI).
Efficiency of this transformation is not compromised at various
scales ranging from 0.05 mmol to 50 mmol where product was
isolated from 10 mg to 8.5 g (Table S3, ESI). Whereas benzyl amine
reacts with NHP in 14 h to give high yields of the product, benzyl
alcohol and benzyl thiol gave no conversions within 24 h. The
phthalimidoamine product can be conveniently converted back to
amine in presence of hydrazine (Scheme 3b).14c Treatment of the
product with Na2S or piperidine can catalyze the ring opening of
phthalimidoamine followed by hydrolysis to render aromatic
carboxylic acid (Scheme 3c).
Phthalimide
Phthalimide
y6y5 y4y3y2
MS2
K E T A A A K F
K E T A A A K F
(1-8)
a
1b2b3
b
4b5
a3c4
Figure 3. MS2 spectra of phthalimide labeled peptide fragment (K1ꢀ
F8) of RNase A.
Inꢀsolution digestion of modified RNase A by chymotrypsin resulted
in peptide fragments where mass change of single fragment (K1ꢀF8:
KETAAAKF) can be observed from 865 Da to 995 Da. Presence of
two Lys residues necessitated MS2 experiment that confirmed K1
modification by presence of b2, b3, b4, b5, y2, y3, y4, y5 among
other fragments (Figure 3). Formation of heterogeneously labeled
protein was ruled out with the support of MS and MS2 data. We
believe that αꢀamine is labeled in K1 as its inherent nucleophilicity is
higher than εꢀamine at pH 7.0. The unique reactivity of αꢀamine in
the protein backbone renders the siteꢀselectivity.4,20 The proposal is
further supported by selective labeling of αꢀamine in a tetrapeptide
GGGKꢀNH2 when it was subjected to phthalimidation and
characterized by MS and MS2 (fragments b2, b3, y1, y2, z1, z2;
Figure S11, ESI). The results were further substantiated by
experiments with HGGKꢀNH2, NGGKꢀNH2, MGKꢀNH2, and SFKꢀ
NH2.22
In conclusion, we have developed a phthalimidation protocol in
aqueous buffer that provides good yields with range of substrates.
The control over reactivity of the amphoteric intermediate was found
to be the key in regulating chemoselectivity. Analytically pure
products were isolated without column chromatography, in general.
The reaction can be scaled up to multigram scale without
compromising the reactivity and selectivity. The mild reaction
conditions allowed us to avoid side reactions involving
functionalities of other amino acid residues. RNase A gave 45ꢀ50%
conversion to homogeneously monolabeled conjugate with one
equivalent of reagent within 8 h. The transformation does not require
large excess of the reagent.21 Bimolecular protein conjugation is
generally plagued by hydrolysis that follows pseudo first order
kinetics. We have demonstrated that it can be evaded by shifting the
rateꢀdetermining step to an intramolecular process. Development of
Nꢀhydroxyphthalimide derivatives that can offer fluorescent probes
(Figures S16 and S17, Scheme S2, ESI) and precursor to the click
chemistry is currently under investigation in our laboratory.
(a)
(b)
Figure 2. (a) NMR structure of RNase A18 with highlighted ten
lysine residues. (b) Docking investigations of NHP and RNase A.
We selected RNase A as a model protein for examining the
efficiency of our methodology in protein labeling. The protein
comprises of all the natural amino acids except tryptophan. It
includes ten lysine residues where most of them are solvent exposed
(Figure 2a). Five equivalents of NHP with RNase A resulted in
monoꢀ, bisꢀ and trisꢀlabeled products within 24 h (for details, see
ESI). An explicit chemoselectivity for primary amine residue was
evident with no apparent labeling of competing functionalities. In
addition, the difference in reactivity of multiple copies of primary
amine was substantiated. The relative pKa values of αꢀamine and εꢀ
amine suggest that the prior would be more nucleophilic in the given
conditions. We argued that selectivity between ten copies of εꢀamine
can result due to binding of NHP to an energetically favorable site
where labeling would be dependent on the binding specificity.
According to this hypothesis, K41 in RNase A active site will be
labeled preferentially, whereas K66 and K7 can offer the second
choice (docking studies, Figure 2b and ESI). The alternative
rationale would allow the solvent accessible amine (K1, K66, K91)5
to be labeled under kinetic control. The mass analysis of digested
protein suggests that the three modified residues are K1 or K7, K37
or K41 and K91. This result indicates that all the three factors
discussed above might be at interplay. Realizing the potential of
methodology, we examined the reaction with equimolar NHP.
Interestingly, only monolabeled product (45ꢀ50% conversion) was
formed in 8 h (Scheme 4).19
R.S., S.R.A., and P.N.J. are recipient of research fellowship
from CSIR, India. V.R. is a Ramanujan Fellow (SERB, India).
The authors are thankful to SERB, DAE and DBT, India for
research grants.
Notes and references
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

ChemComm
Page 4 of 5
COMMUNICATION
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4CC08503E
a
Department of Chemistry, Indian Institution of Science
Education and Research (IISER) Bhopal, Indore Byꢀpass Road,
Bhauri, Bhopal, MP, India 462066. Eꢀmail: vrai@iiserb.ac.in
Chem. Soc., 2012, 134, 2589ꢀ2598. (b) H. B. F. Dixon, J.
Protein Chem., 1984, 3, 99ꢀ108.
21 Large excess of reagents (10ꢀ1000 equivalents) are typically
required for bioconjugate transformations. See reference 12
for details.
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available:
Methods, procedures, additional results and discussion, 22 For experimental details and data, see ESI (Figures S12ꢀ15).
characterization data, computation details. See
DOI: 10.1039/b000000x/
1
(a) Y. X. Chen, G. Triola and H. Waldmann, Acc. Chem.
Res., 2011, 44, 762ꢀ773. (b) M. Chilamari and V. Rai,
Indian J. Chem. Sec A., 2013 52A, 992ꢀ1003.
2
3
4
5
J. E. Reardon, R. C. Crouch and L. St John Williams, J.
Biol. Chem., 1994, 69, 15999ꢀ16008.
O. Dmitrenko, C. Thorpe and R. D. Bach, J. Org. Chem.,
2007, 72, 8298ꢀ8307.
A. C. Obermeyer, J. B. Jarman and M. B. Francis, J. Am.
Chem. Soc., 2014, 136, 9572ꢀ9579.
For kinetic labeling of protein by acylation, see: X. Chen,
K. Muthoosamy, A. Pfisterer, B. Neumann and T. Weil
Bioconjugate Chem. 2012, 23, 500ꢀ508.
6
(a) L. D. Lavis and R. T. Raines, ACS Chem. Biol., 2008,
142ꢀ155. (b) M. Pellecchia, D. S. Sem and K. Wuthrich,
Nat. Rev. Drug Discov., 2002, , 211ꢀ219.
B. Rup and D. O’Hara, AAPS J., 2007, , E148ꢀE155.
C. A. Burtis and E. R. Ashwood, Tietz Textbook of Clinical
Chemistry, 2nd Ed., (Ed.: W. B. Saunders), Philadelphia,
PA, 1994.
3,
1
7
8
9
9
P. Jonkheijm, D. Weinrich, H. Schröder, C. M. Niemeyer
and H. Waldmann, Angew. Chem. Int. Ed., 2008, 47, 9618ꢀ
9647.
10 A. H. Dent and M. Aslam, Bioconjugation
: Protein
Coupling Techniques for the Biomedical Sciences
,
Macmillan Reference Ltd., London, 1998.
11 R. L. Lundblad, Chemical Reagents for Protein
Modification, 3rd Ed., CRC Press, Boca Raton, FL, 2005.
12 G. T. Hermanson, Bioconjugate Techniques, 3rd Ed.,
Academic Press, San Diego, CA, 2013.
13 J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39
,
1272ꢀ1279.
14 For reports on phthalimidation protocols, see (a) S. N. Rao,
D. C. Mohan and S. Adimurthy, Org. Lett., 2013, 15, 1496ꢀ
1499; (b) M. E. Vázquez, J. B. Blanco and B. Imperiali, J.
Am. Chem. Soc., 2005, 127, 1300ꢀ1306; (c) M. Goodman,
Synthesis of Peptides and Peptidomimetics, E22a, Houbenꢀ
Weyl, Thieme, Stuttgart, 2004.
15 X. Guo, F. S. Kim, S. A. Jenekhe and M. D. Watson, J. Am.
Chem. Soc., 2009, 131, 7206ꢀ7207.
16 G. Loving and B. Imperiali, J. Am. Chem. Soc., 2008, 130
,
13630ꢀ13638.
17 (a) G. Loving and B. Imperiali, Bioconjugate Chem., 2009,
20, 2133ꢀ2141. (b) J. Riedl, R. Pohl, N. P. Ernsting, P.
Orsag, M. Fojta and M Hocek, Chem. Sci., 2012, 3, 2797ꢀ
2806.
18 PDB ID: 2AAS. J. Santoro, C. Gonzalez, M. Bruix, J. L.
Neira, J. L. Nieto, J. Herranz and M. Rico, J. Mol. Biol.,
1993, 229, 722ꢀ734.
19 Conversions were calculated by spectroscopic determination
of unreacted NHP. The extraction of NHP was optimized
for <5% error. For details, see supplementary information.
20 For siteꢀselective Nꢀterminal modification of proteins, also
see: (a) A. O.ꢀY. Chan, C.ꢀM. Ho, H.ꢀC. Chong, Y.ꢀC.
Leung, J.ꢀS. Huang, M.ꢀK. Wong and C.ꢀM. Che, J. Am.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C4CC08503E
O
O
N
X
N R
H2N
R
O
O
HX
At 2-5 min
4-48 h
No catalyst or co-reagent
Ambient Temp. / Aq. buffer (pH 7.0)
Reaction time: 4-48 h
Convenient purification / Scalable