Efficient Pictet–Spengler Bioconjugation with N-Substituted Pyrrolyl Alanine Derivatives

Article abstract of DOI:10.1002/anie.201814200

We discovered N-pyrrolyl alanine derivatives as efficient reagents for the fast and selective Pictet–Spengler reaction with aldehyde-containing biomolecules. Other aldehyde-labeling methods described so far have several drawbacks, like hydrolytic instabil

Full text of DOI:10.1002/anie.201814200

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Accepted Article
Title: Efficient Pictet Spengler Bioconjugation with N-Substituted
Pyrrolyl Alanine Derivatives
Authors: Sebastian Pomplun, Mohamed YH Mohamed, Tobias
Oelschlaegel, Christian Wellner, and Frank Bergmann
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814200
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Efficient Pictet Spengler Bioconjugation with N-Substituted
Pyrrolyl Alanine Derivatives
Sebastian Pomplun,*[a] Mohamed YH Mohamed, [a] Tobias Oelschlaegel, [a] Christian Wellner, [a] Frank
Bergmann[a]
developed by Carrico and Bertozzi.[7] In the last years many
Abstract: N-pyrrolyl alanine derivatives are described here as
techniques for labeling the protein via the aldehyde function
efficient reagents for the fast and selective Pictet Spengler reaction
have been described.[3, 8] Among these techniques there are
with aldehyde containing biomolecules. Other aldehyde labeling
for example the classical oxime or hydrazone ligation,[9] Pictet
methods described so far have several drawbacks, like hydrolytic
Spengler (PS) reaction[10] and the improved iso-Pictet
instability, slow reaction kinetics or not readily available labeling
Spengler type conjugation developed by Bertozzi and
Rabuka[11]. The oxime ligation of aldehydes is limited by the
reagents. The reaction speed in Pictet Spengler cyclizations of
pyrrolyl 2-ethylamine substituted at the pyrrole nitrogen is shown to
be significantly faster than the analogues substituted at the - and
- position and functionalized N-pyrrolyl alanine derivatives can be
easily synthesized in only 2-3 steps from commercially available
materials. The small size of the reagent, the fast reaction rate and
the easy synthesis make pyrrolyl alanine Pictet Spengler (PAPS)
an attractive choice for bioconjugation reactions. PAPS was shown
as an efficient strategy for the site selective biotinylation of an
antibody as well as for the condensation of nucleic acid derivatives,
proofing the versatility and usefulness of this novel reagent.
hydrolytic instability of the formed conjugates. Oximes formed
at -oxo aldehydes seemed to be more resistant to
hydrolysis,[12] but here the labeling position is constrained to
the N-terminus of the protein, which could result as a severe
limitation, e.g. when this causes impairment of antibody
binding performance. In the Pictet Spengler based conjugation
an irreversible covalent ring closure affords stable products.
To overcome the extremely slow reaction kinetics of
tryptamine based Pictet Spengler bioconjugation, a modified
Pictet Spengler conjugation, where a hydrazine or
hydroxylamine substituent is placed in the -position of an
indole, has been proposed [11]. The main limitation of this
technique is the multistep synthesis of the alpha-
hydrazino/hydroxylamine-indole building block (which makes
this methodology inaccessible for laboratories without a strong
synthesis background) and the relatively big size of the
tricyclic conjugation moiety.
Bioconjugation reactions are essential tools in modern
chemical biology.[1] Those reactions enable the condensation
of biomolecules, postsynthetic labeling of biomolecules with
small molecules, immobilization of biomolecules on solid
supports and more. In the field of protein conjugation early
methodologies focused on modifications based on the
reactivity of single amino acids (e.g N-hydroxysuccinimide
(NHS) conjugation to lysine, or maleimide conjugation to
cysteine), but in the last years site specific derivatizations
have emerged as preferred strategy to obtain unambiguously
predictable and defined conjugates.[2] Among several
techniques available to date, the introduction of aldehydes into
proteins has been shown as a versatile and profitable
methodology to provide site-specifically a reactive moiety
which can undergo chemoselective conjugation reactions with
nucleophiles in an orthogonal manner to all other functional
groups exposed by a protein. A variety of chemical,
enzymatic, and genetic methods have been developed to
introduce aldehydes into proteins site-specifically.[3] These
include periodate oxidation of N-terminal serine or threonine
residues,[4] pyridoxal phosphate-mediated N-terminal
transamination to yield an alpha-ketoamide or glyoxamide,[5]
genetic encoding of peptide tags that direct enzymatic ligation
of aldehyde- or ketone-bearing small molecules[6] and genetic
encoding of a site for modification by the formylglycine
generating enzyme (FGE), the "aldehyde tag" method
Figure 1. Bioconjugation reactions based on Pictet Spengler cyclization. a)
Classic Pictet Spengler using a labeled tryptophan. This reaction is limited by
extremely slow reaction kinetics. b) Hydroxylamine/hydrazino iso-Pictet
Spengler: the reaction rates are significantly higher compared to tryptophan,
while the reagent is still quite bulky and has to be prepared by a multistep
synthesis. c) N-pyrrolyl alanine Pictet Spengler (PAPS), described in this
work, enables fast bioconjuagtion, using a small and easy to synthesise
reagent.
[a]
Dr. Sebastian Pomplun*+, Dr. Mohamed Yosry Mohamed, Dr.
Tobias Oelschlaegel, Dr. Christian Wellner, Dr. Frank Bergmann
Roche Diagnostics GmbH
Nonnenwald 2, 82377 Penzberg
*E-mail: sebastian.pomplun@gmail.com
+Current address: Massachussetts Institute of Technology,
Department of Chemistry, 77 Massachusetts Ave, Cambridge, MA
02139, USA
Since Pictet Spengler reaction of histamine with acetaldehyde
under physiological conditions has been described[13] we
envisioned this natural amino acid as a promising starting
Supporting information for this article is given via a link at the end of the
document.
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10.1002/anie.201814200
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reagent. N-pyrrolyl alanine with either Fmoc or Boc protection
on the amino functionality was obtained in only one step by
Paal-Knorr pyrrole synthesis condensing dimethoxy
point for the development of a smaller and synthetically more
accessible bioconjugation reagent.
As test system containing an aldehyde, we synthesized formyl
glycine containing peptide 1[14] (Figure 2a), which was
incubated with biotinylated histidine (2) for 24 h at 37 °C and
pH = 5 and at pH 7.4 (conditions described by Ohya and
Niitsu[13]). Since in both conditions only the semistable imine
intermediate was observed (data not shown), while no Pictet
Spengler ring closure occurred, we decided to analyze other
histidine analogues for the possibility to undergo
biocompatible Pictet Spengler reaction. In particular we
envisioned to overcome the low reactivity of the electron poor
imidazole heterocycle by using electron richer variants of this
structure: 10 equivalents of each of histamine (3), 1-methyl
imidazole (4), 3-methyl imidazole (5) and −pyrrole 2-
ethylamine (6) were added to peptide 1 and incubated for 24 h
at 37 °C and pH = 5.4. While in the case of the histamine and
the methylimidazoles no ring closure occurred, the pyrrolyl
derivative (6) showed full conversion of the aldehyde into a
stable Pictet Spengler product.
Figure 3. a) Conditions: 10 µL of peptide 1 (10 mM aq. sol.) and 20 µL of
amine (50 mM aq. sol.) were added to 170 µL of acetate buffer (0.05 M, pH
= 5.4), 37 °C. b) Conversion rates of the three pyrrolyl ethylamines. Reactions
were followed by LCMS. After 24 h quantitative product formation was
observed also for 9 and 10.
c)
a)
TPSRGSLFTGR
Ac-
tetrahydrofuran with Fmoc-NH-L-Dap-OH or Boc-NH-L-Dap-
OH, respectively, under acidic conditions (Scheme 1). The
Fmoc protected pyrrolyl alanine was further transformed into
the active pentafluorophenyl ester (16), affording in overall
only two steps a practical reagent to be coupled to any amino
group in a label molecule of interest. Since unprotected
pyrrolyl alanine (14) is also commercially available, derivatives
15 and 16 could be obtained by direct protection of this
unnatural −amino acid. Although both Paal-Knorr pyrrole
synthesis and direct protection of 14 would allow chirality
conservation of a defined stereocenter, at this point of the
research we considered the stereocontrol of marginal
importance in the context of bioconconjugation and we
synthesized most of the following derivatives starting from
stereochemically undefined 14. To confirm accurately the
formation of a covalent Pictet Spengler ring closure product,
we condensed 14 with 2-(Benzyloxy)acetaldehyde (simply by
dissolving the two substrates in a 1:1 mixture of MeCN and
0.1 M sodium acetate buffer at pH = 5 and removing the
solvent after 1 h). The product NMR (Scheme 1, 18) shows
clearly the disappearing of both the aldehyde proton and of
one of the pyrrole protons. The Pictet Spengler ring closure
occurs with no stereocontrol, forming a second undefined
stereocenter.
3
4
Formylglycine containing peptide 1
5
6
b)
2
Figure 2. a) Formylglycine containing peptide (1). b) Biotinylated PEG-
histidine (2). c) Histamine analogues (3 – 6).
Ongoing from this gratifying result we decided to examine
other pyrrolyl-2-ethylamines regarding their reaction speed,
since this has been shown as the major limitation of classical
Pictet Spengler with tryptophan derivatives. While indoles
show higher reactivity at their -position in electrophilic
substitution[11a], pyrroles preferentially react at their −position,
suggesting that the 2-ethylamine moiety should either be
placed in the -position of the pyrrole ring or at the nitrogen.
To examine this hypothesis, − − and N- 2-ethylamine
substituted pyrroles (6,7 and 8) were reacted with peptide 1
and the conversion rates were observed by LCMS (Figure 3).
The reaction was performed with 5 equivalents of pyrrole at a
peptide concentration of 0.5 mM in acetate buffer at 37°C and
pH 5.4. While surprisingly no significant rate difference
between the − and the −2-ethylamine substituted pyrrole
was observed, the N-substituted pyrrole displayed a
For the synthesis of a biotin labeled pyrrolyl alanine (19),
suitable for detectable protein conjugation, 16 was coupled to
biotin-EDEA (EDEA = 2,2’-(Ethylenedioxy)bis(ethylamine) and
subsequently Fmoc deprotected. To enable the determination
of the N-pyrrolyl alanine Pictet Spengler (PAPS) reaction rate
by LCMS, we synthesized also a sulforhodamine-B-pyrrolyl
alanine derivative (21).
significantly faster conversion rate, achieving a complete
conversion of the formylglycine peptide 1 in the Pictet
Spengler product (11) within 7 hours. We therefore chose this
regioisomer for further investigations.
For straightforward installation of further moieties on the
pyrrolyl N-ethylamine core, while keeping the reagent size as
small as possible, we envisioned -(N-pyrrolyl)-alanine (14,
from here called N-pyrrolyl alanine to simplify the text) as ideal
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10.1002/anie.201814200
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steric hindrance caused by the peptide. Compared to
classical Pictet Spengler under physiological conditions (k =
6.25 x 10-4 M-1s-1 )[16] our PAPS rate constant still represents
an improvement of 2 orders of magnitude and also an
improvement compared to the hydroxylamine iso-Pictet
Spengler in a biomolecule context. Although an exact
comparison between our PAPS reaction rates (measured at
pH = 5 and room temperature) and the other published Pictet
Spengler reactions rates (measured at pH between 4 and 6
and temperatures between 25 and 60 °C) is impossible to
describe due to the different conditions used, we considered
our rate constants fast enough to try the use of PAPS
conjugation in a more complex system, such as the
conjugation to an antibody.
The aldehyde tag derived from human arylsulfatase A
(LCTPSRAALLTGR )[7b, 17] was genetically fused to the C-
terminus of a monoclonal antibody (anti-thyroid stimulating
hormone (TSH)). The antibody was co-translationally modified
by the co-expression of the human sulfatase-modifying factor
1 (SUMF1, a formylglycine-generating enzyme), which
converts the cysteine comprised in the aldehyde tag
sequence into formylglycine (LCTPSRAALLTGR ->
L*formylglycine*TPSRAALLTGR)[7a]. After purification, this
formylglycine containing antibody (AB-CHO) was conjugated
at 37 °C for 24 h with a 5-fold excess of biotinylated pyrrolyl
alanine 19 (in 100mM Na-citrate buffer with 150 mM NaCl at
pH 5; Figure 4a) affording biotinylated antibody AB(PAPS)Bi.
As control experiment, a similar antibody (AB), comprising the
sequence LATPSRAALLTGR, i.e. not containing the FGE-
modifiable cysteine, was incubated under the same conditions
with a 100-fold excess of 19 (Figure 4b). The rate of IgG
biotinylation was assessed by complex formation with
fluorescein-labeled streptavidin (SA-FLUO; absorbs at 280
and 494 nm). Successful conjugation of the formylglycine
containing antibody after 24 h with the N-pyrrolyl alanine biotin
label was observed by the presence of additional peaks at
3.981, 4.373 and 5.057 min retention time on a GFC300
analytical -column corresponding to IgG-biotin/SA-FLUO
complexes absorbing light also at 494 nm in addition to
280 nm (Figure 4b and SI Fig. 3). In constrast, the non-
formylglycine containing control IgG did not form any
Scheme 1. Reagents and conditions: a) CH3COOH, 120 °C, 10 min, 24%
(15), 53% (16a); b) Pentafluorophenol trifluoroacetate, DIPEA, CH2Cl2, 0°C,
1 h, 40 %; c) NaHCO3, Boc2O, 1,4-dioxane/H2O, r.t., 16 h, quant.; d) Na2CO3,
Fmoc-Cl, THF/H2O, 0 °C – r.t., 4 h, 57 %; e) MeCN, acetate buffer, pH = 5,
r.t., 1 h, quant. f) 16, DIPEA, DMF, 45 min, r.t.; g) Et2NH, 10 min, r.t.; h) EDEA,
MeCN, 2 h, r.t., 83 %; i) 15, HATU, DIPEA, DMF, 20 min, r.t.; j) CF3COOH +
5 % H2O, 10 min, 28 %; k) Isobutyraldehyde, acetate buffer, pH = 5, r.t. l) 1,
acetate buffer, pH = 5, r.t.. DIPEA = N,N-Diisopropylethylamine; THF =
complexes with SA-FLUO and only unconjugated IgG was
observed as a single peak on the same analytical SEC column
at a retention time of 5.348 min absorbing light only at 280 nm
but not 494 nm (Fig. 4c and SI Fig. 4). This suggests that the
N-pyrrolyl alanine biotin label has reacted specifically and
almost quantitatively with the aldehyde containing antibody.
To further prove the conjugation, we performed one more
conjugation of the same antibody with another pyrrolyl alanine
PEG biotin derivative. MALDI-TOF MS data are provided in
the Supporting Information (SI Fig.5). The functionality of the
AB(PAPS)Bi was tested in the TSH Elecsys® immunoassay
(Roche Diagnostics) on a cobas e170 module (schematic
representation of the assay setup in Figure 4c) in direct
comparison with a commercial standard TSH antibody-biotin
conjugate, biotinylated by random conjugation with NHS
esters on lysines (AB(amide)Bi) and an formylglycin
containing TSH antibody labeled at the aldehyde by oxime
ligation with an aminooxy biotin label (AB(oxime)Bi) (Fig. 4d)
Interestingly, both site-spefically labeled AB(PAPS)Bi and
AB(oxime)Bi showed higher signals than the original
commercial TSH assay. This might be caused by several
factors, including a better orientation of the site-specific
conjugates on a bead and the full integrity of all lysines in the
antibody binding site, which is not guaranteed following
random NHS conjugation. Furthermore, to assess the stability
tetrahydrofuran;
(Ethylenedioxy)bis(ethylamine).
DMF
=
dimethylformamide;
EDEA
=
2,2’-
For kinetic measurements 21 was conjugated to
isobutyraldehyde in acetate buffer at pH = 5, room
temperature and a reactant concentration of 1 mM
(stoichiometry 1:1). The reaction was followed by LCMS,
analyzing the conversion rate by integration of the peaks at
565 nm (characteristic for sulforhodamine B). The resulting
second order rate constant (k) is 0.32 M-1s-1 (SI Figure 1).
Saito et al. had shown a strong dependence of the Pictet
Spengler reaction rate depending on the nature of the
aldehyde[15] (published reaction rates for hydroxylamine iso-
Pictet Spengler with isobutyraldeyde are 2-10 M-1s-1 .The
reaction with an aldehyde containing peptide displayed the
significantly lower constant of 0.015 M-1s-1, measured at
60 °C). For this reason, we repeated the experiment
conjugating 21 to the formyl glycine containing peptide 1.
Indeed, in this experiment we obtained a lower rate constant:
k = 0.05 M-1s-1 (SI Figure 2). Aldehyde reactivity towards
nucleophilic attack is mainly controlled by steric and electronic
factors. Since both aldehydes tested here are aliphatic, one
might consider the slower reaction rate a consequence of the
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10.1002/anie.201814200
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of the N-pyrrolyl alanine biotin conjugates all immunoassay
measurements were also done after incubation of the
conjugates at 35°C for one week and for three weeks.
AB(PAPS)Bi showed only slightly decreased signals after
incubation at 35°C for one and for three weeks similar to the
original assay variant with AB(amide)Bi conjugates
suggesting that those conjugates were largely stable. In stark
contrast, signal loss for more than 80% could be measured in
the assay variant with the AB(oxime)Bi conjugates
suggesting a strong decomposition (Fig. 4d). Additionally,
complex formation AB(PAPS)Bi conjugates with SA-FLUO
have been reassessed after three weeks incubation of the
conjugate at 35°C. Analytical GFC300 SEC-profiles were
nearly indistinguishable from treated and nontreated
conjugates, indicating that there is no detectable
deconjugation (Figure 4b and SI Figure 3). To further explore
the versatility of N-pyrrolyl alanine as bioconjugation reagent
we envisioned to use PAPS as ligation reaction for nucleic
acid derivatives. We chose the condensation of a
hexaphosphate-cytidine to an oligonucleotide tag.
Hexaphosphate-oligonucleotide conjugates had been
described by Fuller et al.[19] and were obtained by copper (I)-
catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry
and used for nanopore sequencing applications. With our
model reaction we wanted to examine the possibility to
substitute the CuAAC reaction by PAPS conjugation, thus
avoiding the use of copper catalyst, which tightly binds to the
phosphate groups, and has been shown to accelerate the
decomposition of the hexaphosphate moiety, if not thoroughly
removed by additional EDTA washing steps. Amino undecanol
hexaphosphate cytidine was synthesized according to
literature procedures[19] and further functionalized with 4-
formylbenzoate NHS-ester (26). A decathymidine oligo
nucleotide with a terminal hexynyl amino linker was reacted
with 16 directly on the solid synthesis support (CPG –
controlled pore glass) and subsequently deprotected with
diethylamine and conc. ammonia, leading after dialysis to the
unprotected pyrrolyl alanine-decathymidine (Figure 5a).
Aldehyde 28 and the pyrrolyl alanine modified oligonucleotide
25 were dissolved at a concentration of 0.5 mM in acetate
buffer at pH = 5.4 and the reaction afforded smoothly the
desired Pictet Spengler ligation product 29 (Figure 5b).
Although several other methodologies for post-synthetic
nucleic acid modification[20], e.g. the significantly faster
tetrazine-cyclooctene ligation[21] or copper free strain promoted
azido alkyne cycloadditions[22] have been successfully applied,
PAPS represents a novel useful instrument in this “toolbox”,
especially appealing for the good combination of the easy
accessibility of the reactive moieties, reasonable reaction rate
and the small and hydrophilic conjugation moiety formed upon
reaction.
Figure 4. a) Scheme of the biotinylation of an TSH mouse IgG containing the
aldehyde tag (AB-CHO) peptide sequence (100 mM Na-citrate buffer, 150
mM NaCl, pH = 5, 37 °C, 24 h). b) SA-Fluo analysis of the IgG containing the
formylglycine, which shows the quantitative conversion to the biotinylated
product (black line = absorption at 280 nm; red line = absorption at 494 nm).
The double peak of the product derives from the different IgG-SA complexes,
caused by the combination of streptravidin binding sites and biotinylated-IgG.
c) Negative control: SA-Fluo analysis of the TSH mouse IgG not containing
an aldehyde (AB). After incubation with a 100-fold excess of pyrrolyl alanine
labeling reagent (same conditions as a) no reaction could be observed.
d) Schematic representation of the cobas Elecsys sandwich immunoassay.
e) Elecsys measurements using
a commercial biotinylated antibody
AB(amide)Bi (conjugated by NHS chemistry), an antibody biotinylated by
oxime formation (AB(oxime)Bi) and the PAPS biotinylated antibody
(AB(PAPS)Bi), at t = 0, after one and after three weeks incubation at 35°C.
The measurements confirm the functional integrity of the PAPS labeled
antibody and show good stability of the PAPS linkage.
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Acknowledgements
We wish to acknowledge the help provided by Bernhard
Roettig and Annika Frauenstein by establishing the aldehyde
tag methodology in our laboratories.
Keywords: protein modification • Pictet Spengler •
oligonucleotide modification • aldehydes • antibody
conjugation
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Figure 5. a) PAPS ligation of decathymidine-PyrAla with an aldehyde
functionalized hexaphosphate-2’deoxycytidine. Reagents and conditions:
a) 16, DIPEA, DMF, 1h, r.t.; b) Et2NH, MeCN, 10 min, NH3 conc. 2 h, r.t.; c)
Na-borate buffer, 0.1 M, pH = 8.5, 3 h, r.t., 46 %; PAPS conditions: pH = 5.4,
Acetate buffer 0.1 M, reagent concentration = 2 mM.. b) HPLC analysis of
the crude reaction mixture at t = 5 min and t = 120 min (after vivaspin
centrifugation).
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In summary we identified N-pyrrolyl alanine as an efficient
reagent for fast and biocompatible Pictet Spengler
conjugations. The straightforward synthesis of N-pyrrolyl
alanine is a great advantage over other bio-Pictet Spengler
reagents. Furthermore, the small size of pyrrolyl alanine and
the small resulting PAPS linkage guarantees a minimal
perturbation of the native biological system. PAPS was used
for the site selective biotinylation of a TSH antibody obtaining
quantitatively labeled antibodies with high stability, which in a
cobas Elecsys® immunoassay performed better than the
commercial product. Also, the condensation of a
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Table of Contents
COMMUNICATION
Small but mighty: the atom
efficient and easy to
synthesize reagent N-
pyrrolyl alanine is shown to
undergo fast and site
Sebastian Pomplun,*
Mohamed YH Mohamed,
Tobias Oelschlaegel,
Christian Wellner, Frank
Bergmann
selective Pictet Spengler
bioconjugation. This catalyst
free conjugation strategy
was successfully applied for
the biocompatible, site
specific and stable labeling
of aldehydes contained in
peptides, antibodies and
nucleic acids.
Page No. – Page No.
Efficient Pictet Spengler
Bioconjugation with N-
Substituted Pyrrolyl Alanine
Derivatives
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