Preparation of isodesmosine-KLH conjugate for ELISA system

Article abstract of DOI:10.1002/chir.23175

Chronic obstructive pulmonary disease (COPD) is a degenerative condition with limited diagnostic detection efficiency. Currently with no available cure, COPD is associated with irreversible elastic tissue degradation in lungs, which results in release of

Full text of DOI:10.1002/chir.23175

Received: 21 November 2019
DOI: 10.1002/chir.23175
Revised: 19 December 2019
Accepted: 8 January 2020
S P E C I A L I S S U E A R T I C L E
Preparation of isodesmosine-KLH conjugate for ELISA
system
Daria A. Baut | Nao Tanaka | Reiko Yokoo | Toyonobu Usuki
Department of Materials and Life
Abstract
Sciences, Faculty of Science and
Technology, Sophia University, Tokyo,
Japan
Chronic obstructive pulmonary disease (COPD) is a degenerative condition
with limited diagnostic detection efficiency. Currently with no available cure,
COPD is associated with irreversible elastic tissue degradation in lungs, which
results in release of unusual amino acids, isodesmosine and desmosine. These
biomarkers are potential key elements in enzyme-linked immunosorbent assay
(ELISA), an analytical method, which can detect certain compounds including
antigens and proteins in easy and affordable manner. In order to target a bio-
marker with ELISA, it is necessary to prepare its specific antibody, which can
be achieved by immunization of host organism with appropriate antigen con-
taining the biomarker. Although preparation of these types of conjugates has
been published, desmosine and isodesmosine used by researchers are obtained
from natural sources such as animal tissues. Here, we report the first synthetic
preparation of isodesmosine and keyhole limpet hemocyanin (KLH) conjugate
from commercially available chiral amino acids and carrier protein. Formation
of the core pyridinium of isodesmosine was achieved through key reaction—
Correspondence
Toyonobu Usuki, Department of Materials
and Life Sciences, Faculty of Science and
Technology, Sophia University 7-1
Kioicho, Chiyoda-ku, Tokyo 102-8554,
Japan.
Email: t-usuki@sophia.ac.jp
Funding information
Japan Society for the Promotion of
Science, Grant/Award Number:
KAKENHI Grant Number JP17K01953
Chichibabin pyridinium synthesis—to deliver
a 1,2,3,5-tetrasubstituted
pyridinium amino acid selectively. Further modifications involving KLH and
maleimide linker provided the target conjugate, which could potentially
invoke an immune response to produce anti-isodesmosine antibody for the
ELISA system.
K E Y W O R D S
amino acids, Chichibabin pyridinium synthesis, conjugate, COPD
1 | INTRODUCTION
mature elastin structures. According to World Health
Organization (WHO),2 COPD became the third most
A term chronic obstructive pulmonary disease (abbrevi-
ated as COPD) embraces several types of respiratory
system diseases, including chronic bronchitis and
emphysema.1 It is known to be a progressive, degenera-
tive condition characterized by limited airflow to the
lungs resulting from irreversible degeneration of their
common cause of death globally as of 2016, having
killed more than 3 million of people over a wide range
of differently developed countries. In addition to lack
of treatment, COPD may be difficult to diagnose due to
the nature of its symptoms, which initially do not indi-
cate seriousness of condition. Even at present, it tends
to be only recognized in late stages, which greatly
decreases patient's chances for survival. For that rea-
son, an alternative method for detection of early onset
Professor Koji Nakanishi contributed equally to this work.
Chirality. 2020;1–6.
wileyonlinelibrary.com/journal/chir
© 2020 Wiley Periodicals, Inc.
1

2
BAUT ET AL.
of this disease is strongly needed to date. Solution to
this problem might be found in COPD biomarkers, iso-
desmosine and desmosine (Figure 1), which are cros-
slinkers of elastin.3
2 | MATERIALS AND METHODS
All nonaqueous reactions were conducted under an
atmosphere of nitrogen with magnetic stirring unless
otherwise indicated. All reagents were obtained from
commercial suppliers and used without further purifica-
tion unless otherwise stated. Analytical thin-layer chro-
matography (TLC) was performed on silica gel 60 F254
plates produced by Merck. Column chromatography
was performed with acidic silica gel 60 (spherical, 40-
50 μm) or neutral silica gel 60N (spherical, 40-50 μm)
produced by Kanto Chemicals (Tokyo, Japan). High-
performance liquid chromatography (HPLC) purifica-
tion was performed with SHIMADZU LC-6AD,
degasser unit DGU-20A3R, refractive index detector
RID-10A, UV-Vis SPD-20A, valve unit FCV-12AH, and
fraction collector FRC-10A. Removal of small amount
of solvent was performed by Smart Evaporator CEV1-
SQ-P2, CEV1-SK-P2, and CEV1A-GR-P2 (Kanagawa,
Japan).
Optical rotations were measured on a JASCO P-2200
digital polarimeter at the sodium lamp (λ = 589 nm) D
line and are reported as follows: [α]DT (c g cm−3, solvent).
1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a JEOL JNM-ECA 500 spectrometer
(500 MHz). 1H NMR data are reported as follows: chemi-
cal shift (δ, ppm), integration, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet), coupling con-
stants (J) in hertz, assignments. 13C NMR data are
reported in terms of chemical shift (δ, ppm). Electrospray
ionization-mass spectrometer (ESI-MS) spectra were
recorded on a JEOL JMS-T100LC instrument and are
reported in mass-to-charge ratio (m/z).
With onset of the sickness, elastin structures in
lungs become increasingly damaged, and both com-
pounds are released into the patient's body, where their
rising levels can be detected in most bodily fluids like
blood, sputum, and urine by liquid chromatography-
mass spectrometry (LC-MS or LC-MS/MS) analysis.4,5
Easily accessible for analysis, these biomarkers have a
potential usage as key elements in enzyme-linked
immunosorbent assay (ELISA), a promising analytical
method, which can detect certain compounds including
antigens and proteins in an easy and affordable
manner.
Successful performance of ELISA requires an anti-
body able to detect the antigen of interest with high spec-
ificity.6 Although there have been previous reports
regarding application of ELISA for detection of COPD
biomarkers desmosines,7,8 there has been no previous
chemical synthesis of the conjugate from readily avail-
able chiral amino acid starting materials, without involv-
ing animal tissues in the process. Moreover, most of the
published studies focus their research on desmosine as
the immune response-inducing antigen component, with-
out giving much attention to other crosslinkers (despite
both biomarkers existing in vivo in approximately same
abundance).
The aim of this study is to prepare an isodesmosine
and keyhole limpet hemocyanin (KLH) conjugate-based
solely on synthetic isodesmosine, which can be formed
from chiral amino acids by lanthanide-promoted
Chichibabin pyridinium synthesis.9,10 The prepared con-
jugate can be useful as an immunogen (antigen) for prep-
aration of antibody, which could pave the way towards
ELISA-supported detection method of COPD. Mean-
while, the ability to produce an antigen conjugate purely
out of commercially available organic compounds could
open up a cleaner and more controlled formation
process.
2.1 | 1-tert-Butyl-2-N-benzyloxycarbonyl-
L-lysine (5)
60% HClO4 (0.064 mL, 0.59 mmol, 1.1 eq) has been added
into flask containing Cbz-protected lysine 6 (150 mg,
0.53 mmol, 1.0 eq). After thorough distribution of acid,
addition of tBuOAc (1.10 mL) has been done, and content
was stirred magnetically for 48 hours, after which quench
with saturated NaHCO3 was done, followed by extraction
using CH2Cl2. Crude was then checked with NMR, which
revealed no major impurities; hence, column purification
was abandoned. Product 5 (86.1 mg, 0.26 mmol, 48%)
1
was obtained as yellow oil. H NMR (500 MHz, CDCl3,
δ): 7.40-7.28 (5H, m, CH2Bn), 5.37-5.26 (1H, m, NH),
5.16-5.06 (2H, m, CH2Bn), 4.30-4.21 (1H, m, NH), 2.72-
2.63 (2H, m, CH2NH2), 1.87-1.78 (1H, m,
CHCH2CH2CH2), 1.68-1.57 (5H, m, CHCH2CH2CH2),
1.46 (9H, s, tBu).
FIGURE 1 Structures of isodesmosine and desmosine

BAUT ET AL.
3
2.2 | 2-{16-(tert-Butoxycarbonyl)-16-(S)-
[bis-(tert-butoxycarbonyl)-amino]-butyl}-
3,5-bis-{20,24-(tert-butoxycarbonyl)-20,24-
(S)-[bis-(tert-butoxycarbonyl)-amino]
propyl}-1-{11-(tert-butoxycarbonyl)-11-(S)-
[(carboxybenzyl)-amino]-pentyl}-
pyridinium (3)
1.0 eq) and maleimide linker 7 (3.3 mg, 10.21 μmol, 1.05
eq) were added into the reaction flask. Following nitro-
gen substitution, a solution of NMM in DMF (1:200,
140 μL) was injected, and reaction was stirred for 5 hours
at room temperature. Funnel separation and drying gave
crude, which was then purified with silica gel column
chromatography (hexane/EtOAc 1:1, then EtOAc, then
CH2Cl2/MeOH = 10/1). Product 2 was obtained as white
oil in two separate fraction sets, depending on counter
ion (total 10.1 mg, 60%).; Rf 0.55 (CH2Cl2/MeOH = 10/1);
1H NMR (500 MHz, CDCl3, δ): 9.85-9.76 (1H, s, H6),
7.95-7.91 (1H, s, H4), 6.69-6.66 (2H, m, H32,33), 4.96-4.65
(5H, m, H7/16/20/24), 4.31-4.25 (1H, m, H11), 3.52-3.47
(2H, m, H31), 3.20-3.06 (2H, m, H13), 3.00-2.70 (4H, m,
H18/22), 2.51-1.80 (14H, m, H8/9/10/14/15/19/23/26/27/
30), 1.68-1.54 (6H, m, H8/9/10/14/15/19/23/26/27/30),
1.53-1.48 (54H, m, Boc), 1.46-1.42 (36H, m, tBu), 1.41-
1.20 (4H, m, H28/29); 13C NMR (125 MHz, CDCl3, δ):
173.76, 171.59, 170.88, 169.19, 169.10, 169.07, 152.89,
152.77, 152.60, 152.56, 145.55, 145.34, 140.78, 140.60,
134.03, 83.48, 83.31, 82.00, 81.72, 81.53, 81.34, 58.01,
57.98, 57.86, 52.62, 37.92, 36.02, 31.98, 31.26, 30.84, 30.25,
29.84, 29.71, 29.38, 29.10, 28.76, 28.47, 28.10, 28.04, 27.93,
Pr(OTf)3 (16.5 mg, 28.0 μmol, 50 mol%) was added to
the solution of 5 (18.9 mg, 56.1 μmol, 1.0 eq) and
4 (90.1 mg, 224.4 μmol, 4.0 eq) at room temperature
and stirred for 24 hours in H2O/DMF (2:1) solvent
(0.56 mL). After 1 day, the content of flask was diluted
with EtOAc and extracted. After silica gel column chro-
matography (hexane/EtOAc = 1/1, then EtOAc, then
EtOAc/MeOH = 5/1), the product was obtained as yel-
low oil (27.5 mg, 16.8 μmol, 30%); Rf 0.50 (CH2Cl2/
MeOH = 10/1); 1H NMR (500 MHz, CDCl3, δ): 8.84-
8.81 (1H, s, H6), 7.91-7.94 (1H, s, H4), 7.39-7.26 (5H, m,
CH2Bn), 5.68-5.62 (1H, m, NH), 5.15-5.03 (2H, m,
CH2Bn), 4.72-4.36 (5H, m, H7/16/20/24), 4.22-4.14 (1H,
m, H11), 3.19-3.05 (2H, m, H13), 2.97-2.74 (4H, m,
H18/22), 2.46-2.14 (4H, m, H10/23), 2.13-1.92 (4H, m,
H15/19), 1.83-1.67 (6H, m, H8/9/14), 1.49 (54H, s, Boc),
1.43 (36H, m, tBu); 13C NMR (125 MHz, CDCl3, δ):
171.3, 169.2, 169.1, 169.1, 156.2, 153.2, 152.8, 152.6,
145.6, 144.6, 141.1, 140.5, 136.5, 128.4, 128.0, 127.9,
119.2, 83.4, 83.3, 83.3, 82.2, 82.0, 81.7, 81.42, 77.3, 77.0,
76.8, 66.8, 58.1, 57.9, 57.9, 53.7, 32.3, 31.6, 31.1, 29.9,
29.8, 29.7, 29.0, 28.5, 28.3, 28.1, 28.0, 28.0, 27.9, 22.1;
26.57, 26.20, 26.15, 25.63, 22.71, 22.51; HRMS (ESI, m/z):
[M]+ calcd for C81H133N6O23
,
1,557.9422; found
+
1,557.9390.
2.4 | 2-[16-(S)-Amino-16-carboxy-butyl]-1-
[11-(S)-(31-maleimido-260-oxoheptyl)-
amino-11-carboxy-pentyl]-3,5-bis-[20,24-(S)-
amino-20,24-carboxy-propyl]-pyridinium
(8)
high-resolution mass spectrometry (HRMS) (ESI, m/z):
[M]+ calcd for C78H126N5O22
,
1,484.8895; found
+
1,484.8910.
A solution of TFA/CH2Cl2 (95:5, 1.81 mL) was injected
into nitrogen-purged flask containing starting material
2 (13.5 mg, 7.9 mmol, 1.0 eq). Content was stirred for
2 hours at room temperature, and change of color of the
solution was observed from clear to very light pink-
orange. Evaporation of acid and solvent was then done
using rotary evaporator, and reverse phase silica column
purification (H2O, then H2O/MeOH 1:1) gave crude prod-
uct. Desired 8 (7.6 mg, 8.97 mmol, quant) was then
obtained after HPLC purification (Conditions: Cosmosil
5C18-AR-II column (10 mm × 250 mm); mobile phase:
HPLC-grade distilled H2O/MeOH (gradient, solvent A:
MeOH with 0.1% TFA; solvent B: H2O with 0.1% TFA, 0-
2 min: A/B = 2/98; 2-4 min: A/B = 2-10/98-90; 4-9 min:
A/B = 10:90; 9-30 min: A/B = 10-50/90-50; 30-35 min: A/
B = 50/50; 45-47 min: A/B = 50-98/50-2), flow rate: 1.0
2.3 | 2-{16-(tert-Butoxycarbonyl)-16-(S)-
[bis-(tert-butoxycarbonyl)-amino]-butyl}-
3,5-bis-{20,24-(tert-butoxycarbonyl)-20,24-
(S)-[bis-(tert-butoxycarbonyl)-amino]
propyl}-1-{11-(tert-butoxycarbonyl)-11-(S)-
[(31-maleimido-26’-oxoheptyl)-amino]-
pentyl}-pyridinium (2)
Pd/C (10.3 mg, 9.70 μmol, 1.1 eq) was added to solution
of 3 (14.4 mg, 8.81 μmol, 1.0 eq) in MeOH (0.39 mL).
Reaction system was subjected to hydrogen substitution,
and content was stirred at room temperature overnight.
Disappearance of Cbz group was confirmed by TLC as
well as crude NMR, and solution was filtered through cel-
ite with MeOH. Silica gel column chromatography
(EtOAc, then CH2Cl2/MeOH = 10/1) gave the product
amine as yellow oil (9.5 mg, 6.3 μmol, 72%); Rf 0.45
(CH2Cl2/MeOH = 10/1). The amine (14.6 mg, 9.72 μmol,
mL/min; detection: 278 nm; injection amount: 20 μL;
25
temperature: 40ꢀC; Rf 0.2 (H2O/MeOH = 9/1); [α]D
=
4.1 (c = 0.003 g cm−3 in MeOH); H NMR (500 MHz,
1

4
BAUT ET AL.
MeOH-d4, δ): 8.80-8.71 (1H, s, H6), 8.38-8.31 (1H, s, H4),
6.82-6.74 (2H, m, H32/33), 4.65-4.51 (2H, m, H7), 4.46-
4.39 (1H, dd, J = 9.4, 5.0, H11), 4.14-4.09 (1H, t, J = 6.1
Hz, H16/20/24), 4.09-4.05 (1H, t, J = 6.6 Hz, H16/20/24),
4.05-4.01 (1H, t, J = 6.4 Hz, H16/20/24), 3.50-3.45 (2H, t,
J = 7.1 Hz, H31), 3.23-2.92 (6H, m, H13/18/22), 2.4-1.46
(10H, m, H8/9/10/14/15/19/23/26/27/30), 1.40-1.25 (4H,
m, H28/29); 13C NMR (125 MHz, MeOH-d4, δ): 176.4,
175.3, 172.7, 172.7, 155.5, 147.4, 145.1, 142.3, 140.3, 135.4,
135.3, 59.7, 52.86, 38.5, 36.7, 32.1, 31.9, 31.8, 31.7, 31.4,
3 | RESULTS AND DISCUSSION
Overall design of suitable conjugate involved selection
of segments aimed at strengthening the immunizing
potential. Besides use of KLH (which itself is highly
immunogenic), a selective sulfhydryl-based linkage at
one amine position of isodesmosine was envisioned in
hopes it would enable full exposure of hapten.
According to retrosynthetic route (Scheme 1), target
1 could be obtained from 2 through trifluoroacetic acid
(TFA)-mediated global deprotection of acid-liable
groups, followed by Michael addition with thiolated
KLH protein. 2 on the other hand would be accessible
29.7, 29.7, 29.4, 28.7, 28.6, 27.4, 26.8, 26.8, 25.3, 23.6;
+
HRMS (ESI, m/z): [M]+ calcd for C35H53N6O11
,
733.3772; found 733.3750.
from protected isodesmosine
3
after selective
deprotection of benzyloxycarbonyl (Cbz) group and
attachment of maleimide segment. Pyridinium core of
3 would be formed through key reaction, Chichibabin
pyridinium synthesis,9,10 using 4 equivalents of alde-
hyde 4 and 1 equivalent of lysine 5. It has been previ-
ously reported that in appropriately adjusted
conditions, this reaction can yield an isodesmosine-type
compound selectively.10b
Synthesis of desired aldehyde 4 was accomplished
using foundations of the synthetic route established pre-
viously, starting from commercially available chiral glu-
tamic acid with protecting groups.10a, 11 To assemble
second key substrate 5, commercially available chiral
lysine 6 was tert-butylated in the presence of tBuOAc and
HClO4 (Scheme 2) in 48% yield.12
2.5 | Isodemosine-KLH conjugate (1)
8 (2.0 mg, 2.36 μmol) was dissolved in thiolated KLH (9)
solution (348 μL, in phosphate-buffered saline [PBS] con-
taining 1 mM EDTA) and was stirred for 2 hours at room
temperature. When the sample has completely entered
the bed, it was eluted with PBS (pH, 7.4) and the solution
was collected. The procedure of Ellman assay is as fol-
lows: blank solution for calibration was prepared in 1.5
mL of Eppendorf tube. 200 μL of distilled water and
200 μL of PBS buffer were injected into the tube using
automatic micropipette, along with 4 μL of DTNB solu-
tion. Calibration using the blank was made on JASCO V-
730BIO spectrophotometer. After that, sample solution
was prepared in another Eppendorf tube. 200 μL of dis-
tilled water, 160 μL of PBS buffer, 4 μL of DTNB, and
40 μL of sample were injected into the tube. Such pre-
pared solution was then analyzed with the spectropho-
tometer, indicating lack of compounds containing free
thiol groups.
With both 4 and 5 in hand, Chichibabin pyridinium
synthesis was conducted in the presence of 50 mol% Pr
(OTf)3 catalyst and in DMF/H2O = 1/2 solvent system for
24 hours at room temperature (Scheme 3).10b Desired
tetrasubstituted pyridinium 3 was selectively obtained in
30% yield. In further step, selective deprotection of Cbz
SCHEME 1 Retrosynthetic route of target compound 1

BAUT ET AL.
5
SCHEME 2 Synthesis of lysine segment 5
SCHEME 3 Synthetic
scheme towards 8
As the last step towards target compound, Michael
addition of 8 and thiolated KLH protein 9 was performed
in PBS (pH, 7.4).14 After stirring for 2 hours at room tem-
perature, white precipitation arose gradually, indicating
formation of conjugate. Successful conjugation was con-
firmed with Ellman assay.
4 | CONCLUSION
In summary, synthesis of target conjugate 1 has been
finalized starting from commercially available protected
glutamic acid and protected lysine 6 through use of key
reaction, Chichibabin pyridinium synthesis, which
furnished the main core of isodesmosine. Further selec-
tive deprotection revealed free amine group that reacted
with maleimide substrate to give 2. TFA-mediated global
deprotection of acid-liable groups delivered 8, which
reacted with thiolated KLH protein 9, forming desired
immunoreagent 1. As of current state, immunization
using the antigen on animal host organism is being
investigated.
SCHEME 4 Formation of conjugate 1
group was performed using Pd/C condition in hydrogen
atmosphere in 65% yield. Free amine was then reacted
with maleimide segment 7 in presence of mild base to
obtain 2 in 60% yield.13 In the next step, isodesmosine-
maleimide intermediate 2 was subjected to deprotection
of acid-liable Boc and tBu groups, and finally, 8 was puri-
ACKNOWLEDGMENTS
This paper is dedicated to Professor Koji Nakanishi. T.
U. is grateful for working with Nakanishi Sensei as one
of his last postdocs at Columbia University. This work
was supported by JSPS KAKENHI Grant Number
JP17K01953. We are grateful to Ms Aisya Syahmina
(Sophia University) for manuscript suggestions.
fied by HPLC in quantitative yield. The optical rotation
value was observed to [α]D = 4.1(c = 0.003 g cm−3
,
25
MeOH), suggesting that the obtained compound 8 was
not racemic.

6
BAUT ET AL.
standard for the degraded elastin biomarker desmosine. Org.
Biomol. Chem. 2014;12(48):9887-9894.
ORCID
Toyonobu Usuki https://orcid.org/0000-0002-0212-5371
6. An example of small molecule and career protein conjugate is
A2E and BSA, see, Abeywickrama C, Matsuda H, et al. Immu-
nochemical recognition of A2E, a pigment in the lipofuscin of
retinal pigment epithelial cells. Proc. Natl. Acad. Sci. USA.
2007;104:14610-14615.
7. Gunja-Smith Z. An enzyme-linked immunosorbent assay to
quantitate the elastin crosslink desmosine in tissue and urine
samples. Anal. Biochem. 1985;147:258-264.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
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