Tandem Wittig/Diels-Alder diversification of genetically encoded peptide libraries

Article abstract of DOI:10.1039/c7ob01635b

In this paper, we developed a tandem of two carbon-carbon bond-forming reactions to chemically diversify the libraries of peptides displayed on a bacteriophage. The Wittig reaction of a biotin-ester from a stabilized phosphorane ylide with model peptides containing N-terminal glyoxal exhibits reaction rates of 0.07 to 5 M^-1 s^-1 in water at pH 6.5-8. The log(k) scaled linearly with pH from pH 6 to 8; above pH 9 the reaction is accompanied by the hydrolysis of the ester functionality. Capture of the phage displaying the biotinylated product by streptavidin beads confirmed the rate of this reaction in a library of 10⁸ peptides (k = 0.23 M^-1 s^-1 at pH = 6.5) and also confirmed the regioselectivity of this modification. Olefins introduced into the Wittig reaction can act as Michael acceptors: addition of glutathione, cysteamine, and DYKDDDDKC ( FLAG-Cys ) peptide occurs with k = 0.12-4.1 M^-1 s^-1 at pH 7.8. Analogous reactions with the DYKDDDDKC peptide take place on phage-displayed peptides modified via the Wittig reaction. This reaction is manifested as a progressive emergence of a FLAG-epitope on the phage and detected by the capture of this phage using an anti-FLAG antibody. Olefins introduced into the Wittig reaction also act as dienophiles in the Diels-Alder reaction with cyclopentadiene. The conversion of the dienophile to norbornene-like adducts on the phage was observed by monitoring the disappearance of the thiol-reactive olefin on the phage. This report broadens the reaction scope of genetically-encoded peptide libraries displayed on the phage, expanding the structural diversity of these platforms and increasing their potential to be used in screening against important protein targets. The possibility of monitoring tandem reactions by the use of different labels illustrates the feasibility of obtaining highly functionalized peptides with chemical motifs impossible to achieve using conventional translational machinery.

Full text of DOI:10.1039/c7ob01635b

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Tandem Wittig/Diels-Alder diversification of
genetically encoded peptide libraries
Vivian Triana, Ratmir Derda*
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
* Corresponding author
Abstract
In this paper, we developed a tandem of two carbon-carbon bond-forming reactions to
chemically diversify libraries of peptides displayed on bacteriophage. Wittig reaction of a biotin-
ester from a stabilized phosphorane ylide with model peptides containing N-terminal glyoxal
exhibit reaction rates of 0.07 to 5 M-1s-1 in water at pH 6.5-8. The log(k) scaled linearly with pH
from pH 6 to 8; above pH 9 the reaction is accompanied by hydrolysis of the ester functionality.
Capture of the phage displaying the biotinylated product by streptavidin beads confirmed the rate
of this reaction in a library of 108 peptides (k=0.23 M-1s-1 at pH=6.5) and also confirmed the
regioselectivity of this modification. The olefins introduced in the Wittig reaction can act as
Michael acceptors: addition of glutathione, cysteamine, and DYKDDDDKC (“FLAG-Cys”)
peptide occur with k=0.12-4.1 M-1s-1 at pH 7.8. Analogous reactions with DYKDDDDKC
peptide take place on phage-displayed peptides modified via Wittig reaction. This reaction is
manifested as a progressive emergence of FLAG-epitope on phage and detected by capture of
this phage using anti-FLAG antibody. Olefins introduced in Wittig reaction also act as
dienophiles in Diels-Alder reaction with cyclopentadiene. The conversion of the dienophile to
norbornene-like adducts on phage was observed by monitoring the disappearance of the thiol-
reactive olefin on phage. This report broadens the reaction scope for genetically-encoded peptide
libraries displayed on phage, expanding the structural diversity of these platforms and increasing
their potential to be used in screening against important protein targets. The possibility of
monitoring tandem reactions by the use of different labels illustrates the feasibility of obtaining
highly functionalized peptides with chemical motifs impossible to achieve by conventional
translational machinery.
1

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Introduction
Chemically-modified peptide libraries are a valuable source for discovery of ligands for
fundamental research, development of diagnostics and biomaterials as well as discovery of
therapeutic leads.1 Chemical post-translational modification of peptides made of 20 “natural”
amino acids is the simplest strategy for production of such libraries because it bypasses the need
of advanced constructs for incorporation of unnatural amino acids. Introduction of unnatural
fragments into genetically-encoded peptide libraries makes it possible to equip these libraries
with value-added functionality not encodable by conventional translational machinery.1–4
Although there exists a rich palette of chemical transformations for site-specific modification of
proteins made of natural amino acids, only a limited scope of chemical transformations have
been adopted for diversification of genetically-encoded peptide libraries. These reactions include
SN2,5–8 and SNAr substitution,9 Michael10 and allenamide11 addition reactions as well as tandem
elimination-addition reaction12 starting from sulfhydryl group of Cys; nucleophilic substitutions
of selenocysteine,13,14 addition of alpha-nucleophiles to aldehydes,15–18 and cycloadditions to
unnatural size chains containing azide or propargyl group19.
Several carbon-carbon bond forming strategies—based on cycloadditions and transition metal
catalyzed reactions—have been reported for modification of amino acid side chains of individual
proteins or peptides in water18,20–22. However, the only example to date of a C-C bond formation
reaction performed on the phage display context has been reported by Lin and coworkers, who
described an elegant optimization of the Sonogashira cross coupling in a subset of peptides on
phage libraries. This coupling, however, hinged on expression of an unnatural
homopropargylglycine amino acid residue via amber suppression.23
2

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Wittig olefination is one of the attractive reactions to explore for diversification of peptide
libraries on phage because it has already been employed to modify individual proteins in water
as well as amino acids ligated to DNA strands.24–26 Wittig chemistry has also been employed by
Liu and coworkers for macrocyclization of DNA-templated libraries.27 In proteins, aldehyde
handles suitable for Wittig reaction are readily introduced by mild oxidative cleavage of natural
residues that contain 1,2-aminoalcohols (N-Ser/Thr)15,32,33 or 1,2-diols (glycans);34 it can also be
incorporated via UAA-mutagenesis,35 or encoded as a L(C/A)T(P/A)S(A/R) peptide sequence
recognized by the formylglycine generating enzyme.18,36 Specifically, in peptide libraries
displayed on phage, the orthogonal aldehyde handle can be quantitatively introduced by selective
oxidation of N-terminal serine.15 Conveniently, the Wittig reaction between N-terminal glyoxy-
aldehyde and ester-stabilized ylide produces an olefin within an extended conjugation framework
(Fig. 1) that enhances its performance both as Michael acceptor and dienophile. The introduced
conjugated alkene functionality could enable survey of up to 106-109 Michael electrophiles to
identify suitable covalent or reversible covalent inhibitors.29–31 Introduction of norbornene-like
moieties via tandem Wittig-Diels Alder reaction also opens the door for robust labeling via
bioorthogonal aziridination45 or inverse electron demand Diels-Alder cycloaddition with
tetrazine46 in a phage display context. While individual aforementioned reactions are well-
established, the tandem use of such modifications in 106-109 scale genetically encoded libraries
has not been investigated or reported to date.
The Wittig reaction of stabilized ylides can yield both E and Z isomers, and the rate and
selectivity of this chemistry strongly depends on the reaction conditions.36 Similarly to reactions
in methanol, most “on-water” and “in-water” Wittig reactions of stabilized ylides with
substituted benzaldehydes, vinyl aldehydes, heterocyclic derived aldehydes or alkyl aldehydes
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exhibit an increase in rates compared to analogous reactions in aprotic solvents at the expense of
lower E selectivity.37–41 In this report, we observed a strong influence of the aldehyde nature on
the stereoselectivity of the Wittig reaction with a stabilized ylide in water. Products of N-
terminal glyoxy-aldehyde handles and 2-nitrobenzaldehyde showed significantly lower E/Z
selectivity than products for benzaldehyde at the same conditions. We invoke the dipolar
moment effect reported by Harvey and Aggarwal to explain these observations36,42 and when
possible, we determine difference in chemical reactivity towards Michael addition and Diels-
Alder of the two isomeric dienophiles.
Results
Nucleophilic addition of biotin-PEG-alcohol to Bestmann ylide yielded a carbonyl stabilized
phosphonium salt (YEB) in 65% yield. The same positively charged product was obtained in two
steps by reaction of the alcohol with bromoacetyl bromide and triphenylphosphine (Scheme 1),
although scaling up of this second strategy generated bisacetylated adducts, considerably
reducing the yield and making it less practical. To optimize the Wittig reaction in the
environment akin to that of the glyoxal on phage-displayed peptides, we employed a model
sequence o-VEKY produced by NaIO4 oxidation of the peptide SVEKY.15 The Wittig product
(YEB-VEKY) was then used for evaluation of rates for Michael addition, retro- Michael and
Diels Alder reactions.
The YEB phosphonium salt, with expected pKa of ∼10-11 in water,43 was sufficiently
deprotonated in pH 6-8 buffered solution to undergo Wittig reaction. Mixtures of E/Z isomers
were observed in reactions with benzaldehyde, 2-nitrobenzaldehyde, and the N-terminal glyoxal
in o-VEKY (Fig. 1A and S1-5). Measured rate constants (Fig. S6-S8) and E/Z isomers ratio
depended on the nature of the aldehyde. Benzaldehyde reacted with modest rate and produced
4

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high E/Z isomers selectivity (93:7 or 93% E) whereas 2-nitrobenzaldehyde reacted significantly
faster but yielded 60-70% E isomer (Fig. S3-S5). The loss in E-selectivity in o-
nitrobenzaldehyde, when compared to benzaldehyde were in accord with previous of Bergdahl
and coworkers.44 The rate of reaction and E/Z isomers selectivity (50-70 % E) of oxoacetamide
were similar to that of 2-nitrobenzaldehyde and significantly different from that of benzaldehyde.
Similar E/Z isomers ratios were observed via HPLC (Fig. S1-S2) and 1H NMR analysis of either
the crude reaction mixtures, or the purified Wittig products (Fig. S3-S5).
The rate of the Wittig reaction increased exponentially with pH: the graph for the log(k) vs.
pH for all types of aldehydes had identical slope of ~1 (Fig. 1C and Fig. S6-S8) in the pH 6-8
range. The slope suggested that the rate-determining step involves an anion. Also, lack of any
leveling of rate dependence at pH 8 suggested that these anionic species have pKa>9. This
observation is in line with previously measured pKa of an ester stabilized phosphonium salt
(10.5 in water)43 and it differs from the reported value of 8.5 in DMSO.45 While rate scaled
dramatically with pH, the E/Z selectivity changed only subtly (Fig. S3). Rate of the Wittig
reaction also modestly varied with changes in buffer concentration (Fig. S9B) and buffer
compositions (Fig. S9A).
Z-isomer produced in Wittig reaction hydrolyzed significantly faster than E-isomer (Fig.
S10). At pH 8, the half-life of hydrolysis of the E-ester group was t1/2 = 18 hours, while Z-ester
hydrolyzed completely in 7 hours. At pH 5, half-life of hydrolysis was >75 hours for both
isomers. At these pH values, the rate of hydrolysis can be readily decoupled from the rate of
Wittig reaction (Fig. S11) and at pH 5, the product can be stored for three days without any
hydrolysis. Although we have not shown it explicitly, selective hydrolysis of Z-isomer can be
used as one of the strategies to mitigate poor E/Z-selectivity in the product and yield
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predominantly E-product. Subsequent studies of Wittig reaction on peptides and phage displayed
peptide libraries were performed in conditions that yield E-Z-mixture. Where possible, we
focused on differential reactivity between the E and Z products.
To confirm the yield and specificity of Wittig reaction on phage displayed peptides, we
employed a technology based on a streptavidin pull-down assay extensively used by our group.15
Specifically, we use the phosphorane ylide containing a biotin tag (YEB) to modify a mixture of
two phage types: (i) one displaying a SVEK sequence or SX7 library of peptides displayed on
pIII protein and LacZ reporter; this phage produces blue plaques when plated on agar
supplemented with X-Gal/IPTG and (ii) phage that displays N-terminal alanine and contains no
LacZ, forming white plaques on the same plates. In regioselective functionalization only “blue”
phages should acquire biotin and this biotinylation can be detected by pull-down with
streptavidin beads. The biotin capture technique15 confirmed that the reaction of YEB with clonal
phage displaying SVEK peptide at pH 7.8 exhibited nearly 90% conversion after one hour,
whereas wild type phage in the same reaction, which do not possess the N-terminal serine,
exhibited insignificant reactivity (Fig. 2B). The modification of 100,000,000-member SX7
library of peptides displayed on phage reached 40-60% conversion in the same conditions (Fig.
2B). Incomplete conversion of Wittig reaction in phage libraries was not due to low reaction
rates, as shown by measurement of kinetics of modification of the library (Fig. 2C). The half-life
of library modification was ~20 min even at lower pH (6.5); the convergence exhibited a first
order kinetic profile and reached saturation at ~50% after one hour. The same incomplete
conversion has been observed in N-terminal modification of phage libraries.5,8 To rule out
sequence dependence of the oxidation step before Wittig reaction, SX7 library was subjected to
NaIO4 treatment for different time intervals and then reacted with YEB. Increase in oxidation
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time did not change the percentage of functionalization in the SX7 library context (Fig. S12).
Wittig reaction, thus, effectively diversified phage displayed library of 108 peptides with only a
modest impact on integrity of the library: phage “viability” decreased only to ∼50% after three
hours reaction, which is similar to viabilities observed in other reactions.5,9
The Wittig product contains a Michael acceptor that could potentially form Michael addition
products by attack of thiols. We tested glutathione, cysteamine and the peptide sequence
DYKDDDDKC, aka “FLAG-Cys” peptide, with this Michael acceptor and rates were ranging
from 0.12 to 4.1 M-1s-1 at pH 7.8 (Fig. S13). We noticed a minor difference in reactivity in
Michael addition of FLAG-Cys thiol to E and Z alkenes; (Fig. S13C) however, both isomers
were quantitatively converted to the Michael addition adducts. The Michael acceptor can form
either irreversible or reversible covalent interactions with cysteine-like nucleophiles. Therefore,
Wittig-diversified libraries can be potentially used for discovery of either irreversible or
reversible covalent inhibitors. We evaluated the efficiency of Michael warhead using the
approach of Tauton and co-workers.29 Monitoring the retro-Michael reaction in isolated addition
products of thiols with different pKa values (GSH and cysteamine) at pH ≥ 6.529,46,47 uncovered
little to no retro-Michael reaction (Fig. S14), indicating that these Michael acceptors favor
irreversible additions. Lack of reversibility of analogous Michael adducts has also been reported
by Bernardes and coworkers.48
We applied Michael addition on phage-displayed alkene-peptide obtained by Wittig
derivatization of SVEK monoclonal phage to confirm the integrity of the Wittig product on
phage. Removal of the excess reagent was critical for this reaction and we lowered the excess
YEB from initial 4 mM to a sub-nanomolar concentration via dialysis of the modified phage
against pH 5 buffer, which minimized possible hydrolysis. After 5-6 buffer exchanges were
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performed in 48 hours, we observed < 20% loss of modification (Fig. S16); the concentration of
small organic molecule reference (fluorescein) present in this condition was reduced to single
digit nM concentrations (Fig. S15C). Purified Wittig product displayed on phage (YEB-VEK
phage) reacted effectively with FLAG-Cys and were captured by anti-FLAG (Fig. 3). Neither the
WT phages nor purified YEB-VEK phage not exposed to FLAG-Cys peptide were captured in
this condition (Fig. 3). To rule out non-specific reactivity with thiol, we confirmed that the phage
that was SVEK phage not exposed to YEB did not exhibit significant reactivity in Michael
addition (Fig. 4C).
The Wittig product can be “quenched” with cyclopentadiene via the Diels-Alder reaction to
suppress any Michael addition to the olefin functionality. Aqueous Diels-Alder reaction in the
model peptide exhibited a differential reactivity of E and Z isomers: after 48 hours E-isomer
exhibited 100% conversion but only minimal conversion for Z isomer was observed (Fig. S16).
Identity of the residual Z olefin was confirmed by NMR (Fig. S16D). Addition of co-solvents
(acetonitrile or DMF) or Lewis acids (LiCl or Cu(NO3)2) known to catalyze the Diels-Alder
reaction,32,49 did not improve the reactivity of the Z isomer (Fig. S17). Changing pH had no
effect on the reaction and, therefore, Diels Alder can be effectively performed at pH 5 in
conditions that suppress hydrolysis of ester. In these conditions, the E isomer is consumed after
20 hours, while conversion of the Z isomer is negligible (Fig. S16B). Most importantly, the
olefin that did not react in Diels-Alder preserved its ability to act as Michael acceptor in reaction
with FLAG-Cys thiol (Fig. S16C). This observation was used for subsequent “pulse-chase”
quantification of the Diels-Alder reaction on phage-displayed peptides.
The Wittig modified phage can also be diversified via Diels-Alder reaction with
cyclopentadiene. To evaluate the efficiency of the Diels Alder reaction on phage, we employed
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the pulse-chase technique developed in our group.5,15 Specifically, exposure of purified YEB-
modified SVEK phage (Fig. 4B) to FLAG-Cys (pH 7.8, 2 h, RT) renders phage tagged if and
only if it has accessible Michael acceptor. As phage was “pulsed” with cyclopentadiene at pH 5
for 48 hours, the alkene was consumed in the Diels-Alder reaction and the labeling in the
“chase” reaction with FLAG-Cys peptide decreased. The pulse-chase approach suggested that 70
% of the population modified via Wittig reaction was converted to a norborene product (Fig. 4D
left bar). We recognized that the loss of Michael reactivity in “pulse chase” approach can be due
to: i) blocking of the Michael acceptor due to Diels-Alder reaction, ii) hydrolysis of the ester in
α-position to the double bond, iii) reaction with other nucleophiles present in the reaction buffer.
To rule out possibilities ii) and iii), we demonstrated that Wittig-modified phage preserved its
ability to react with FLAG-Cys peptide even after incubation with pH 5 buffer for 48 hours (Fig.
4D middle bar). We note that incomplete (70%) conversion in Diels-Alder reaction mirrors the
lack of reactivity of the Z Wittig adduct observed in reaction on synthetic peptide. While direct
structural analysis of E/Z isomers on phage-displayed peptides is presently impossible, the
results of pulse-chase study strongly suggest that: i) Wittig product is formed on phage in a
similar E/Z ratio as was observed in model synthetic peptide (~70% E), ii) Diels-Alder reaction
selectively converts phage-population displaying the E-isomer to trans-norborene products; iii)
subset of library remaining after Diels-Alder reaction is significantly enriched in Z-isomer.
Discussion
Successful implementation of chemical transformations to diversify a library of substrates
requires understanding of the mechanistic aspects of this transformation. The active species in
the Wittig reaction is the phosphorane ylide, which results after deprotonation of the
phosphonium salt. When the reaction is conducted at pH << pKa, the log(k) of such reaction
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should scale with pH and level off at pH near this pKa. This model is overly simplistic as the
rates could also be affected by variations in [H+] due to the associated changes in hydrogen
bonding, solvation effects and dipole-dipole interactions with the transition state (Fig. S9). In
peptide-aldehyde substrates, additional attenuation of the transition state can stem from
neighboring amino acid side chains. The most important conclusion emanating from the pH
profile is the invariance of E/Z selectivity at all tested pH values. Increase in proton
concentration, thus, does not significantly change the location of the transition state nor the
mechanism of the reaction; the location of the TS is only substrate dependent (Fig. S3).
Kinetically controlled formation of an O-apical oxaphosphetane via [2+2] cycloaddition
through an asynchronous transition state is also accepted for the Wittig reaction in and on water
and standard, electron rich aldehydes (reference50,51 and references within). A comparison of
transition states between the Wittig reaction of oxoacetamides and benzaldehyde can be
visualized via More O'Ferrall Jencks (MOJ) diagram (Scheme 2). Lowering of the betaine corner
due to the presence of the α-carbonyl that stabilizes the negative charge developed on the
aldehyde oxygen results in an anti-Hammond movement towards the betaine. As a result, the
TS2 for oxoacetamide should have more betaine character (or higher asynchronous character,
where C-C bond is formed while P-O bond has significant charge character)42 when compared to
the TS1 for benzaldehyde-like aldehydes. We anticipated that such a shift in the pKa of the
transition state could be manifested as a different pH-dependence of rate for different aldehydes
at the pH near the pKa of the betaine-like transition state (Scheme 2). Unfortunately, hydrolysis
of the substrate and products above pH 10 complicated such studies. We note that the location of
the transition state on MOJ plot alone does not reflect the 3D conformation of TS.50,52,36 1,2-
interactions, 1,3-interations and dipole-dipole interactions in cis and trans TSs towards
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oxaphosphetanes (OPA-TS) can influence the 3D conformation and determine the final E/Z
selectivity. The latter statement assumes irreversibility of the OPA formation, which has been
observed experimentally52,53 and suggested theoretically.36
While many mechanistic investigations of Wittig reaction were based on benzaldehyde-like
substrates, there exists a critical difference in dipole-dipole interactions between OPA-TA of
benzaldehyde and peptide-oxoacetamide substrates. The conformations of OPA-TS were
hypothesized to minimize the overall dipole moment while minimizing 1,2 and 1,3-interactions.
The TS of reaction originating from the benzaldehyde substrate towards the trans OPA was
proposed to be semi-puckered due to the generation of favorable dipole-dipole
interactions.50,51,36,54 As the additional α-amide of oxoacetamide introduces an extra dipole, we
hypothesize that the TS originating from oxoacetamide towards trans OPA cannot adopt the
same geometry as benzaldehyde TS due to incomplete cancellation of dipole moment of the
carbonyl group. In a similar analysis to the one done by Harvey and Aggarwal on α-alcoxy
aldehydes,42 we propose that the additional dipole destabilizes the puckered trans TS, making it
similar in energy to a nearly planar cis TS and thus, decreasing the expected steroeselectivity of
the Wittig reaction with stabilized ylides towards the E isomer (Scheme 3). Similar dipole-
induced de-stabilization of the trans-puckered TS could potentially explain the lower E/Z
selectivity of 2-nitrobenzaldehyde.
The mechanistic hypothesis underpinning the E/Z selectivity in peptide aldehydes can be
further attacked by profiling of reactions in various peptide substrates that contain amino-acid
side chains capable of screening or enhancing dipole-dipole interactions. Implementation of the
Wittig reaction in a population of 100 million diverse peptides open doors to numerous
mechanistic studies that involve selection of substrates of higher reactivity and even selection of
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subsets of library that yield exclusively E or Z products (e.g., using a differential reactivity of
these isomers in a Diels-Alder or ester hydrolysis reactions). We believe that fundamental
mechanistic investigations of Wittig reaction and a downstream Michael addition and Diels-
Alder reactions will serve as a robust foundation for future substrate profiling studies within a
library of genetically-encoded substrates.
In conclusion, we have successfully implemented the Wittig reaction to diversify a library
of peptides displayed on phage. The modification is highly regio- and chemo-selective and it
exhibits only a minimal effect on infectivity of phage. We further diversified the Wittig-products
displayed on phage via Michael addition and Diels-Alder reactions. The efficiency of both
transformations can be monitored in phage-displayed context; neither of these reactions have a
significantly impact on the infectivity of the virions. This report broadens the scope of reactions
compatible with genetically-encoded libraries of phage-displayed peptides and increases the
potential of this platform for identification of diverse ligands for important protein targets
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Acknowledgements
This work was supported by research grants from the National Science and Engineering
Research Council of Canada (NSERC, #492943), NSERC Accelerator Supplement, and Alberta
Glycomics Centre. Infrastructure support was provided by Canadian Foundation for Innovation
(CFI) New Leaders Opportunity. We thank Mark Miskolzie for assistance with solvent
suppression experiments. We are grateful to Chris Noren and Beth Paschal at New England
Biolabs for providing the sample of SX7 libraries and SVEK-clone.
†Electronic Supplementary Information (ESI) available: Fig. S1-S16, synthesis and
characterization of YEB and its peptide derivatives, experimental procedures for reactions on
model peptide and phage libraries. MatLab scripts for kinetic fit in Figures 1-2 and S1 See DOI:
10.1039/x0xx00000x
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Scheme 1. Synthetic pathways towards ylide ester biotin phosphonium salt precursor (YEB).
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Fig. 1 (A) Wittig reaction on model aldehydes. (B) Absorbance of reactant and product
(extracted as absolute area of the peaks in HPLC trace) of reaction between YEB and o-VEKY at
different times and pH values. The data was fit to a equation At = A(1–e-k[YEB]t), where k is the
presuto-first order rate constant, [YEB] – initial concentration of the ylide, and A is the
maximum absorbance. (C) Rate constants of reactions of three aldehydes at different pH values
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Fig. 2 (A) Quantification of yield and selectivity of Wittig reaction on phage using biotin-capture
technique. (B) Capture % of phage clones displaying a SVEK-sequence and phage displaying the
SX7 library of peptides in presence of WT phage as control at pH 7.8 after one hour reaction with
1 mM YEB. (C) Kinetics of reaction between SX7 library and YEB (4 mM) at pH 6.5. (D)
Monitoring of viability of SX7 library in the reaction described in (C).
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Fig. 3 Reaction of Wittig functionalized phage and control WT phage with DYKDDDDKC
peptide (FLAG-Cys peptide). Pull down assay with anti-FLAG immobilized on protein G
magnetic beads quantified the yield of incorporation of FLAG and confirmed that FLAG was not
incorporated into WT-phage
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Fig. 4 (A) Diels-Alder reaction on Wittig product of model peptide. E selectivity was confirmed
by analyzing the recovered unreacted starting material by NMR (Supporting Figure S16D). (B)
Wittig reaction on phage clone displaying SVEK peptide. Phage not subjected to Wittig reaction
did not exhibit any detectable capture whereas (C) alkene-functionalized phage, when reacted
with FLAG-Cys peptide captured by anti-FLAG yields 86% capture. (D) Exposure of phage for
48 h with 250 mM cyclopentadiene after which an aliquot was taken and reacted with FLAG-
Cys peptide quantified the efficiency of Diels-Alder modification. for two hours. (E) Bar graph
summary: 86% of the phage population reacted with FLAG-Cys and was captured by anti-FLAG
when no cyclopentadiene was added. After exposure to cyclopentadiene for 48 hours, only 16%
of phage remained reactive towards FLAG-Cys. These observations extrapolate a 70%
conversion for Diels-Alder reaction.
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Scheme 2. More O’Ferrall Jencks plot showing transition states of Wittig reactions that
proceed via concerted oxaphosphetane (TS1) and movement of TS1 towards asynchronous
betaine-like transition state (TS2) in response to changes in electronic properties of the
aldehyde.
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Scheme 3. (A) Transition states towards oxaphosphetane of Wittig reaction between
benzaldehyde and triphenylphosphine ester stabilized type ylides showing a conformer that
minimizes 1,2 and 1,3-interactions as well as dipole-dipole interactions. (B) Analogous transition
states towards oxaphosphetane of Wittig reaction between oxoacetamide and triphenylphosphine
ester-stabilized type ylides cannot completely cancel dipole moment in any conformation.
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79x38mm (300 x 300 DPI)