Synthesis and Characterization of a Series of Orthogonally Protected l-Carnosine Derivatives

Article abstract of DOI:10.1007/s10989-018-9680-2

l-Carnosine (β-alanyl-l-histidine) is an endogenous dipeptide that has been recognized for its broad spectrum of beneficial biological activities. However, the therapeutic utility of molecule has been hampered by its instability in human plasma (half-life

Full text of DOI:10.1007/s10989-018-9680-2

International Journal of Peptide Research and Therapeutics
https://doi.org/10.1007/s10989-018-9680-2
Synthesis and Characterization of a Series of Orthogonally Protected
l-Carnosine Derivatives
Mohammad H. El‑Dakdouki1 · Nadine Daouk1 · Hiba Abdallah1
Accepted: 30 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
l-Carnosine (β-alanyl-l-histidine) is an endogenous dipeptide that has been recognized for its broad spectrum of beneficial
biological activities. However, the therapeutic utility of molecule has been hampered by its instability in human plasma
(half-life in human serum<5 min) due to the presence of carnosinase enzyme that catalyzes its hydrolysis into the respec-
tive individual amino acids. While a large number of carnosine derivatives have been synthesized to optimize its overall
pharmacokinetic profile, reports that provide molecular evidence as to how the dipeptide interacts with its biological target
are scarce. Therefore, many questions are yet to be answered concerning the pharmacophoric regions in carnosine and its
significance to the molecule’s diverse biological activities. In this study, we set out to construct a small library of carnos-
ine analogues that can be used in assessing the influence of the various functional groups of the dipeptide on its important
biological properties. Orthogonal protection/deprotection of selected functional groups led to the exposure of amino group
at the N-terminus, the carboxyl group at the C-terminus, and the imidazole ring of histidine. To examine the significance
of the imidazole group in preventing the aggregation of the β-amyloid plaques, histidine was replaced by phenylalanine
and a series of β-Ala-Phe analogues was generated. To study the influence of the length of the carbon chain in β-Ala on the
β-amyloid aggregation, a series of Gly-His analogues was synthesized. A series of Gly-Phe was also constructed and will
be used as negative control in future β-amyloid plaque assembly experiments. The synthesized carnosine derivatives were
characterized by NMR (proton, carbon, and 1H–1H COSY), and mass spectroscopy.
Keywords l-Carnosine · Orthogonal deprotection · β-Alanine · Histidine · Peptides
Introduction
peptide therapeutics market was valued at USD 18.9 billion
in 2013 and is estimated to reach USD 25 billion by 2018
(Craik et al. 2013). Of the simple peptides that has gained
much attention as a pharmaceutical agent is l-carnosine
(CAR) (Fig. 1), an endogenous dipeptide that is found pre-
dominantly in human muscles, heart, liver, brain, kidneys,
and other long-lived tissues (Boldyrev 2000; Bauer 2005).
CAR is synthesized from β-alanine and l-histidine by the
ATP-driven enzyme carnosine synthetase, and is hydrolyzed
by specific metal ion-dependent homodimeric dipeptidase
called carnosinases in the blood and other tissues (Pegova
et al. 2000; Teufel et al. 2003).
Given their attractive pharmacological profile and intrinsic
properties, peptides represent an excellent starting point for
the design of novel therapeutics, and their specificity trans-
lated into excellent safety, tolerability, and efficacy profiles
in humans (Sachdeva 2017). Peptides are characterized by
predictable metabolism, shorter time to market, lower attri-
tion rates, lower production costs, and standard synthetic
protocols (Fosgerau and Hoffmann 2015). In fact, the global
CAR possesses activities that suppress age-related dys-
functions (Hipkiss 2009). CAR is also well-documented to
exhibit antioxidant properties by scavenging reactive oxygen
species (ROS) such as hydroxyl radicals, superoxide and
singlet oxygen, as well as reactive carbonyl species (RCS).
Moreover, CAR can chelate heavy metals that cause cellu-
lar damage, thus protecting macromolecules, such as lipids,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10989-018-9680-2) contains
supplementary material, which is available to authorized users.
* Mohammad H. El-Dakdouki
m.eldakdouki@bau.edu.lb
1
Department of Chemistry, Beirut Arab University, Riad El
Solh, P.O. Box 11-5020, Beirut 11072809, Lebanon
Vol.:(0123456789)
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International Journal of Peptide Research and Therapeutics
to the nanoparticles through carefully pre-selected func-
tional groups.
The main goal of this study is to synthesize and charac-
terize a series of l-carnosine analogues with orthogonally
protected functional groups. This series can be utilized
in future studies aiming at understanding the relationship
between CAR’s functional groups and its broad spectrum of
biological activities. The functional groups of the dipeptide,
i.e. the N-terminus amino group, the C-terminal carboxyl
group, and the imidazole group of histidine, were protected.
Orthogonal deprotection procedures will selectively expose
specific functional groups leading to the assembly of a
small library of CAR analogues. To examine the signifi-
cance of the imidazole group, histidine (His) was replaced
by phenylalanine (Phe) and a series of β-Ala-Phe analogues
was generated. To study the influence of the length of the
carbon chain in β-Ala, a series of Gly-His analogues was
synthesized. Finally, a series of Gly-Phe derivatives was
synthesized and can be used as negative control in future
structure–activity relationship studies.
Fig. 1 Chemical structure of l-carnosine (CAR)
proteins and DNA, from damages that result in ageing and
age-related disease (Hipkiss et al. 2013). This multifunc-
tional profile of carnosine suggests that the molecule can
be associated with neurodegenerative diseases such as Alz-
heimer’s and Parkinson diseases. While there are no current
reports of clinical trials concerning CAR’s effects towards
clinically-defined age-related dysfunction, it is reported
that CAR improved cognition in schizophrenics. In senes-
cence-accelerated mice, a diet supplemented with carnosine
decreased the signs of ageing and increase mean lifespan by
20% (Boldyrev et al. 1999).
Experimental Section
Structure activity relationships suggested that the pres-
ence of the β-alanine residue is implicated in the antioxidant
properties while the histidine residue has properties includ-
ing the ability to bind to transition metal ions (Castelletto
et al. 2011). Another important role of the histidine residue
in carnosine is its ability to inhibit glycation-induced pro-
tein crosslinking. In addition, it has been reported that both
the amino group of the β-alanyl residue and the imidazole
ring of l-histidine act synergistically in trapping cytotoxic
α,β-unsaturated aldehydes such as 4-hydroxy-trans-2-none-
nal (HNE) (Aldini et al. 2002; Vistoli et al. 2016). Unfor-
tunately, CAR, like other naturally-occurring peptides, is
often not directly suitable for use as convenient therapeutics
because of its intrinsic weaknesses (Fosgerau and Hoffmann
2015; Hamley 2007). The main limitation for the therapeutic
use of CAR is the rapid hydrolysis mostly in human plasma
by carnosinase (Hipkiss 2009). A number of strategies have
been deployed to overcome this perceived obstacle, essen-
tially through the chemical derivatization, conjugation with
several types of potentially beneficial organic molecules,
and the synthesis of its structural analogues (Guiotto et al.
2005; Vistoli et al. 2009; Orioli et al. 2011; Bertinaria et al.
2012). In addition, CAR has been conjugated onto different
types of nanoparticles for various purposes. However, the
conjugation was non-covalent and non-specific, thus poten-
tially hindering functional groups important for the inter-
action of CAR with its intended biological target (Bellia
et al. 2013; Malkar et al. 2015; Durmus et al. 2011; Li et al.
2016; Krpetić et al. 2012; Saada et al. 2011).Therefore, it is
highly desirable that l-carnosine is controllably conjugated
Materials and Methods
Reagents were purchased from local suppliers and used
without further purification. Anhydrous dichloromethane
(DCM), anhydrous methanol (MeOH), anhydrous THF,
hexane, and silica gel for column chromatography were
purchased from Acros Organics. Ethyl acetate, DCM and
MeOH reagent grade were attained from Fischer Scientific.
All amino acids used are natural. Boc-β-Ala-OH, L-Phe-
OMe.HCl, Boc-Gly-OH, and Boc-His(Bzl)-OH were pur-
chased from Sigma–Aldrich. Triethylamine (TEA) and
diisopropylethylamine (DIPEA) were obtained from Fluka,
while trifluoroacetic acid (TFA) was purchased from Pan-
reac. Thin Layer Chromatography (polyester, silica 60, UV
254) were obtained from Albet. NMR data were recorded
with Bruker 500 MHz spectrometer and chemical shifts were
reported as ppm. Mass spectroscopy spectra were obtained
on Waters Quattro micro API LC/MS/MS in the positive
mode at Michigan State University (USA).
Synthesis of Boc-His(Bn)-OMe (2)
Boc-His(Bn)-OH 1 (1 g; 2.9 mmol) was dissolved in anhy-
drous DCM (10 ml) and CDI (0.56 g, 3.5 mmol) was added.
The reaction mixture was stirred at room temperature for
1 h after which anhydrous methanol (5 ml) was added. The
reaction was stirred for 18 h after which the volatiles were
evaporated on a rotavap. The residue was purified by column
chromatography using DCM:MeOH (9:1) as an eluent. The
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International Journal of Peptide Research and Therapeutics
desired product was collected as colorless oil in 91% yield
(0.95 g). The structure of 2 was confirmed by NMR (proton,
carbon and COSY) and MS. 1H-NMR (500 MHz, CDCl3)
δH 1.43 (s, 9H), 2.97–3.06 (m, 2H), 3.49 (s, 1H), 3.64 (s,
3H), 4.45 (d, J=10.12, 1H), 5.06 (s, 1H), 5.32 (s, 1H), 6.64
(s, 1H), 7.12 (d, J = 5.12, 2H), 7.31–7.36 (m, 3H), 7.45
(d, J = 5.22, 1H). 13C-NMR (75 MHz, CDCl3) δC 172.47,
155.57, 137.70, 137.08, 135.89, 128.97, 128.30, 127.23,
116.95, 79.55, 53.57, 52.11, 50.87, 30.16, 29.67, 28.31; MS
(ESI). Calculated for C19H25N3O4: Theoretical mass: 359.18;
Found: m/z [M+H]+: 360.1.
of the starting material. The volatiles were evaporated on a
rotavap and the residue was dried on a high vacuum pump.
The dried residue was purified by column chromatography
using DCM:MeOH (8:2) as an eluent. The desired prod-
uct was collected in 87% yield (130 mg). The structure was
confirmed by NMR (proton, carbon, COSY) and MS. 1H-
NMR (500 MHz, CD3OD) δH 2.95–3.05 (m, 2H), 3.22 (s,
2H), 3.32 (s, 3H), 4.71 (t, J=15.21, 1H), 5.16 (d, J=5.14,
1H), 6.90 (s, 1H), 7.22–7.37 (m, H), 7.42 (s, 1H). 13C-NMR
(75 MHz, CD3OD) δC 171.71, 137.16, 136.89, 128.53,
127.77, 127.19, 117.46, 52.74, 51.37, 50.21, 43.61, 41.84,
29.56. MS (ESI). Calculated for C16H20N4O3: Theoretical
mass: 316.15; Found: m/z [M+H]+: 317.2.
Synthesis of Boc-Gly-His(Bn)-OMe (5)
Boc-His (Bn)-OMe 2 (0.6 g, 1.7 mmol) was dissolved in
DCM (10 ml) and TFA (2 ml) was added. Three drops of
triisopropylsilane (TIPS) was added as a radical scavenger.
The reaction mixture was stirred at room temperature for
3 h until the disappearance of the starting material as indi-
cated by TLC. The volatiles were evaporated on a rotavap.
The resulting residue was watched with hexane (10 ml × 3)
to remove residual TFA, and dried on high vacuum pump.
The formation of the desired product [H-His(Bn)-OMe, 3]
was confirmed by was confirmed by MS: MS (ESI). Cal-
culated for C14H17N3O2: Theoretical mass: 259.13; Found:
m/z [M+H]+: 260.1. The dried residue was dissolved in
DCM (10 ml), and DIPEA (1 ml) was added. In a sepa-
rate round bottom flask, Boc-Gly-OH 4 (0.35 g, 2.0 mmol)
was dissolved in anhydrous DCM (20 ml), and CDI (0.4 g,
2.4 mmol) was added. The reaction mixture was stirred at
room temperature for 1 h after which the H-His(Bn)-OMe
3 mixture was added. The mixture was stirred at room tem-
perature for 18 h after which the volatiles were evaporated.
The resulting residue was purified by column chromatog-
raphy using DCM:MeOH (9:1) as an eluent. The desired
product was collected as colorless oil in 86% (0.6 g). The
structure was confirmed by NMR (proton, carbon, COSY)
and MS. 1H-NMR (500 MHz, CDCl3) δH 1.45 (s, 9H), 1.77
(s, 1H), 3.00–3.12 (m, 2H), 3.64 (s, 3H), 3.88 (d, J=5, 2H),
4.80–4.83 (m, 1H), 5.05 (s, 2H), 5.20 (s, 1H), 6.67 (s, 1H),
7.12–7.14 (m, 2H), 7.31–7.39 (m, 3H), 7.44 (s, 1H), 7.62
(d, J = 10, 1H). 13C-NMR (75 MHz, CDCl3) δC 171.66,
169.12, 155.73, 137.82, 137.14, 135.97, 129.01, 128.34,
127.34, 127.22, 117.02, 79.95, 53.44, 52.37, 52.26,50.83,
43.99, 29.56,28.34. MS (ESI). Calculated for C21H28N4O5:
Theoretical mass: 416.21; Found: m/z [M+H]+: 417.1.
Synthesis of Boc-Gly-His-OMe (7)
Boc-Gly-His(Bn)-OMe 5 (0.12 g, 0.29 mmol) was dissolved
in methanol (10 ml), and the mixture was cooled to 0 °C in
an ice bath. Pd/C (12 mg) was added to the reaction mixture
which was subjected to hydrogenation for 18 h. The reac-
tion mixture was then filtered on a Celite bed, and methanol
was evaporated. The resulting residue was purified by col-
umn chromatography using DCM:MeOH (9:1). The desired
product was obtained in 96% (90 mg). The structure was
confirmed by NMR (proton, carbon, COSY) and MS. 1H-
NMR (500 MHz, CDCl3) δH 1.46 (s, 9H), 3.11–3.25 (m,
2H), 3.0 (s, 1H), 3.72 (s, 3H), 3.82 (d, J =5.24, 2H), 4.82
(dd, J=15.21–3.02, 1H), 5.34 (s, 1H), 6.80 (s, 1H), 7.37 (s,
1H), 7.96 (s, 1H). 13C-NMR (75 MHz, CDCl3) δC 171.45,
169.18, 156.66, 135.35, 80.56, 53.44, 52.57, 50.79, 44.38,
28.32. MS (ESI). Calculated for C14H22N4O5: Theoretical
mass: 326.16; Found: m/z [M+H]+: 327.1.
Synthesis of Boc-β-Ala-Phe-OMe (10)
Boc-Ala-OH 8 (1 g, 5.3 mmol) was dissolved in anhydrous
dichloromethane (50 ml) and carbonyldiimidazole (CDI)
(1 g, 6.34 mmol) was added. The reaction mixture was
stirred at room temperature for 1 h. In a separate reaction
flask, L-Phe-OMe. HCl 9 (1.37 g, 6.34 mmol) is suspended
in anhydrous dichloromethane (20 ml), and DIPEA (1.23 ml)
was added. The solution was stirred at room temperature for
30 min, after which it is added to the Boc-Ala-OH mixture.
The reaction mixture was stirred at room temperature for 6 h
until TLC indicates the disappearance of starting material
and appearance of product. DCM was evaporated and result-
ing residue was dissolved in ethyl acetate. The mixture was
then washed with dilute aqueous HCl solution (0.1 M), satu-
rated aqueous. sodium bicarbonate solution, and brine. The
organic layer was collected, dried on anhydrous sodium sul-
fate, and volatiles are evaporated on RotaVap. The collected
product was purified by column chromatography using
EtOAc:Hexane (2:1). The desired product 10 was collected
Synthesis of H-Gly-His(Bn)-OMe (6)
Boc-Gly-His(Bn)-OMe 5 (0.15 g, 0.36 mmol) was dis-
solved in DCM (5 ml) and TFA (1 ml) was added. TIPS
was added as a radical scavenger. The reaction mixture was
stirred at room temperature for 3 h until the disappearance
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International Journal of Peptide Research and Therapeutics
in 94% yield (1.7 g) as colorless viscous oil. The structure of
10 was confirmed by NMR (proton, carbon, and COSY) and
MS. 1H-NMR (500 MHz, CDCl3) δH 1.43 (s, 9H), 2.37 (t,
J=10.32, 2H), 3.04–3.18 (m, 2H), 3.37 (dd, J=15.25–5.15,
2H), 3.79 (s, 3H), 3.85–3.88 (m, 1H), 5.08 (s, 1H), 6.00 (d,
J=10.05, 1H), 7.08–7.10 (m, 2H), 7.23–7.34 (m, 3H). 13C-
NMR (75 MHz, CDCl3) δC 171.90, 171.06, 156.02, 135.71,
129.17, 128.63, 127.27, 53.08, 52.41, 37.87, 36.49, 36.09,
28.39. MS (ESI). Calculated for C18H26N2O5: Theoretical
mass: 350.18; Found: m/z [M+Na]+: 373.1.
53.88, 52.50, 37.40, 36.36, 31.87. MS (ESI). Calculated
for C13H18N2O3: Theoretical mass: 250.13; Found: m/z
[M+H]+: 251.2.
Synthesis of Boc-β-Ala-His(Bn)-OMe (13)
Boc-His(Bn)-OMe 2 (0.7 g, 1.95 mmol) was dissolved in
DCM (10 ml) and TFA (2 ml) was added. The reaction
mixture was stirred at room temperature for 3 h until the
disappearance of the starting material. The formation of
the desired product 3 was confirmed by MS. The volatiles
were evaporated on a rotavap and the residue was dried on
a high vacuum pump. The dried residue was dissolved in
DCM (10 ml) and DIPEA (1 ml) was added. In a separate
round bottom flask, Boc-Ala-OH 8 (0.44 g, 2.34 mmol) was
dissolved in anhydrous DCM (20 ml), and CDI (0.46 g,
2.80 mmol) was added. The reaction mixture was stirred at
room temperature for 1 h after which the H-His(Bn)-OMe 3
mixture was added. The mixture was stirred at room temper-
ature for 18 h after which the volatiles were evaporated. The
resulting residue was purified by column chromatography
using DCM:MeOH (9:1) as an eluent. The desired product
was collected as colorless oil in 83% (0.7 g). The structure
was confirmed by NMR (proton, carbon, COSY) and MS.
1H-NMR (500 MHz, CDCl3) δH 1.42 (s, 9H), 1.65 (s, 1H),
2.43 (dd, J=15.12–5.21, 2H), 3.00–3.07 (m, 2H), 4.43 (dd,
J = 10.31–5.12, 2H), 3.63 (s, 3H), 4.80 (d, J= 5.25, 1H),
5.04 (s, 2H), 5.75 (s, 2H), 6.63 (s, 1H), 7.23 (d, J= 5.31,
1H), 7.32–7.37 (m, 3H), 7.46 (s, 1H). 13C-NMR (75 MHz,
CDCl3) δC 171.79, 137.45, 129.00, 128.35, 127.71, 116.93,
52.54, 52.24, 50.85, 36.4, 36.23, 29.39, 28.44. MS (ESI).
Calculated for C22H30N4O5: Theoretical mass: 430.22;
Found: m/z [M+H]+: 431.2.
Synthesis of Boc-β-Ala-Phe-OH (11)
Dipeptide 10 (200 mg, 0.57 mmol) was dissolved in THF
(10 ml) and the solution was cooled to 4 °C. An aqueous
solution of 1 M LiOH (3 ml) was added dropwise, and the
reaction mixture is stirred at 4 °C. The progress of the reac-
tion was followed by TLC. The pH of solution was brought
to 3–4 by the dropwise addition of 1 M aqueous HCl solu-
tion. THF was evaporated on a rotavap, the product was
extracted by ethyl acetate. The ethyl acetate layer is collected
and dried on anhydrous sodium sulfate. Ethyl acetate was
evaporated, and product was purified by column chromatog-
raphy using DCM:MeOH (9:1) as an eluent. The structure of
the desired product 11 was confirmed by NMR (proton, car-
bon, and COSY) and MS. The product was collected in 92%
yield (176 mg) as a colorless substance. 1H-NMR (500 MHz,
CDCl3) δH 1.44 (s, 9H), 2.39 (s, 2H), 3.04–3.07 (m, 1H),
3.20–3.36 (m, 4H), 4.83 (d, J=5.42, 1H), 5.17 (s, 1H), 6.59
(d, J=5.23, 1H), 7.16–7.29 (m, 4H). 13C-NMR (75 MHz,
CDCl3) δC 173.67, 171.38, 156.66, 136.06, 129.43, 128.50,
127.06, 79.93, 53.32, 37.46, 36.95, 33.61, 29.70, 28.39. MS
(ESI). Calculated for C17H24N2O5: Theoretical mass: 336.17;
Found: m/z [M+Na]+: 359.1.
Synthesis of Boc-β-Ala-His-OMe (14)
Synthesis of H- β-Ala-Phe-OMe (12)
Boc-Ala-His(Bn)-OMe 13 (0.15 g, 0.35 mmol) was dis-
solved in methanol (10 ml), and the mixture was cooled to
0 °C in an ice bath. Pd/C (15 mg) was added to the reac-
tion mixture which was subjected to hydrogenation for 18 h.
The reaction mixture was then filtered on a Celite bed, and
methanol was evaporated. The resulting residue was puri-
fied by column chromatography using DCM:MeOH (9:1).
The desired product 14 was obtained in 92% (110 mg). The
structure was confirmed by NMR (proton, carbon, COSY)
and MS. 1H-NMR (500 MHz, CDCl3) δH 1.27 (s, 9H), 2.44
(t, J=10.15, 2H), 3.12 (t, J=5.42, 2H), 3.39–3.44 (m, 2H),
3.49 (s, 1H), 3.71 (s, 3H), 3.70–3.82 (m, 1H), 5.58 (s, 1H),
6.80 (s, 1H), 7.19 (d, J=10.23, 1H), 7.56 (s, 1H). 13C-NMR
(75 MHz, CDCl3) δC 171.85, 171.58, 156.19, 135.22, 52.61,
52.44, 50.75, 36.92, 36.52, 28.93, 28.43. MS (ESI). Calcu-
lated for C15H24N4O5: Theoretical mass: 340.17; Found: m/z
[M+H]+: 341.1.
Dipeptide 10 (200 mg, 0.57 mmol) was dissolved in DCM
(10 ml) and trifluoroacetic acid (TFA) (1 ml) was added. Tri-
isopropylsilane (TIPS) (50 µl) was added as a radical scav-
enger. The reaction mixture was stirred at room temperature
for 2 h, or until the disappearance of reactants and appear-
ance of the product. The volatiles were first evaporated on a
rotavap, and then on a vacuum pump. The resulting residue
was purified by column chromatography using DCM:MeOH
(8:2) as an eluent. The desired colorless product was col-
lected in 88% yield (174 mg). The structure of the desired
product 12 was confirmed by NMR (proton, carbon, COSY)
1
and MS. H-NMR (500 MHz, CDCl3) δH 2.52–2.67 (m,
2H), 2.93–3.17 (m, 4H), 3.66 (s, 3H), 4.72–4.79 (m, 1H),
7.13–7.28 (m, 5H), 7.63 (d, J = 10.31, 1H), 8.08 (s, 3H).
13C-NMR (75 MHz, CDCl3) δC 172.59, 170.73, 1662.16,
161.88, 136.01, 129.11, 128.56, 127.10, 117.48, 114.88,
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International Journal of Peptide Research and Therapeutics
Synthesis of H-β-Ala-His(Bn)-OMe (15)
Synthesis of Boc-Gly-Phe-OH (17)
Boc-Ala-His(Bn)-OMe 13 (0.2 g, 0.46 mmol) was dis-
solved in DCM (5 ml) and TFA (1 ml) was added. TIPS
(50 µl) was added as a radical scavenger. The reaction
mixture was stirred at room temperature for 3 h till the
disappearance of the starting material. The volatiles
were evaporated on a rotavap and the residue was dried
on a high vacuum pump. The dried residue was purified
by column chromatography using DCM:MeOH (8:2)
as an eluent. The desired product was collected in 85%
yield (129 mg). The structure was confirmed by NMR
Boc-Gly-Phe-OMe 16 (200 mg, 0.6 mmol) was dissolved
in THF (10 ml) and the solution was cooled to 4 °C. An
aqueous solution of 1 M LiOH (3 ml) was added dropwise,
and the reaction mixture is stirred at 4 °C. The progress of
the reaction was followed by TLC. The pH of solution was
brought to 3–4 by the dropwise addition of 1 M aqueous
HCl solution. THF was evaporated on a rotavap, and the
product was extracted by ethyl acetate. The ethyl acetate
layer was collected and dried on anhydrous sodium sulfate.
Ethyl acetate was evaporated, and the product was purified
by column chromatography using DCM: MeOH (9:1) as an
eluent. The product was collected in 97% (190 mg) as a
colorless substance. The structure of the desired product 17
was confirmed by NMR (proton, carbon, and COSY). 1H-
NMR (500 MHz, CDCl3) δH 1.43 (s, 9H), 3.01–3.21 (m,
2H), 3.61–3.91 (m, 3H), 4.82 (s, 1H), 4.90 (s, 1H), 5.74
(s, 1H), 6.03 (s, 1H), 6.88 (s, 1H), 7.15–7.27 (m, H). 13C-
NMR (75 MHz, CDCl3) δC 173.98, 169.75, 156.35, 135.81,
129.37, 128.56, 127.07, 80.58, 53.21, 43.9, 37.39, 28.3.
1
(proton, carbon, COSY) and MS. H-NMR (500 MHz,
CD3OD) δH 2.60 (dd, J = 10.31–5.02, 2H), 3.02–3.19
(m, 4H), 3.31–3.32 (m, 1H), 4.70–4.72 (m, 1H), 5.31 (s,
2H), 7.18 (s, 1H), 7.32–742 (m, H), 8.82 (s, 1H). 13C-
NMR (75 MHz, CD3OD) δC 171.19, 170.69, 135.85,
135.41, 128.77, 128.35, 127.57, 118.72, 52.06, 51.57,
51.40, 35.41, 31.23, 27.86. MS (ESI). Calculated for
C17H22N4O3: Theoretical mass: 330.17; Found: m/z
[M+H]+: 331.1.
Synthesis of H-Gly-Phe-OMe (18)
Synthesis of Boc-Gly-Phe-OMe (16)
Dipeptide 16 (200 mg, 0.6 mmol) was dissolved in DCM
(10 ml) and trifluoroacetic acid (TFA) (1 ml) was added. Tri-
isopropylsilane (TIPS) (50 µl) was added as a radical scav-
enger. The reaction mixture was stirred at room temperature
for 2 h, or till the disappearance of reactants and appear-
ance of the product. The volatiles were first evaporated on a
rotavap, and then on a vacuum pump. The resulting residue
was purified by column chromatography using DCM:MeOH
(8:2) as an eluent. The structure of the desired product 18
was confirmed by NMR (proton, carbon, COSY) and MS.
The desired colorless product was collected in 90% yield
Boc-Gly-OH 4 (1 g, 5.7 mmol) was dissolved in anhy-
drous dichloromethane (30 ml) and carbonyl diimidazole
(CDI) (1.11 g, 6.85 mmol) was added. The reaction mix-
ture was stirred at room temperature for 1 h. In a separate
reaction flask, L-Phe-OMe. HCl 9 (1.47 g, 6.85 mmol)
is suspended in anhydrous dichloromethane (20 ml), and
DIPEA (1.4 ml) was added. The solution was stirred at
room temperature for 30 min, after which it is added to the
Boc-Gly-OH mixture. The reaction mixture was stirred at
room temperature for 6 h until TLC indicates the disap-
pearance of starting material and appearance of product.
DCM was evaporated and resulting residue was dissolved
in ethyl acetate. The mixture was then washed with dilute
aqueous HCl solution (0.1 M), saturated aqueous sodium
bicarbonate solution, and brine. The organic layer was col-
lected, dried on anhydrous sodium sulfate, and volatiles
are evaporated on RotaVap. The collected product was
purified by column chromatography using EtOAc:Hexane
(2:1). The desired product 16 was collected in 93% yield
(1.8 g) as colorless viscous oil. The structure of the (3)
was confirmed by NMR (proton, carbon, and COSY)
1
(173 mg). H-NMR (500 MHz, CDCl3) δH 2.93–3.08 (m,
2H), 3.57 (s, 3H), 3.63 (d, J=15.21, 1H), 3.79 (d, J=15.12,
1H), 4.76 (dd, J=10.32–5.01, 1H), 7.09–7.27 (m, 6H), 7.93
(d, J=10.21, 1H), 8.10 (s, 3H). 13C-NMR (75 MHz, CDCl3)
δC 172.12, 166.30, 162.24, 161.99, 135.86, 129.14, 128.52,
127.05, 54.03, 54.07, 52.41, 40.68, 37.47. MS (ESI). Cal-
culated for C12H16N2O3: Theoretical mass: 236.17; Found:
m/z [M+H]+: 237.2.
Results
1
and MS. H-NMR (500 MHz, CDCl3) δH 1.45 (s, 9H),
3.07–3.16 (m, 2H), 3.72 (s, 3H), 4.87–4.91 (m, 1H), 5.10
(s, 1H), 6.53 (d, J = 5.21, 1H), 7.1–7.15 (m, 2H), 7.2–7.31
(m, 3H). 13C-NMR (75 MHz, CDCl3) δC 171.66, 169.03,
135.58, 129.23, 128.65, 127.19, 53.04, 52.38,44.21, 37.88,
28.27. MS (ESI). Calculated for C17H24N2O5: Theoretical
mass: 336.17; Found: m/z [M+H]+: 337.12.
Synthesis of Gly‑His Series
The first synthesized series of CAR analogues was based
on the Glycine-Histidine (Gly-His) dipeptide. The overall
synthetic scheme is depicted in Scheme 1. The rationale
behind establishing this homologous series was to examine
1 3

International Journal of Peptide Research and Therapeutics
Scheme 1 Overall synthetic
route for the synthesis of Gly-
His analogues
the significance of β-Ala on CAR’s biological activity. A
glycine moiety with one methylene (CH2) group shorter
substituted β-Ala.
The amino group of compound 2 was selectively depro-
tected upon treatment with trifluoroacetic acid (TFA) in
DCM for 3 h at room temperature to produce His analogue 3
(Scheme 1). Triisopropylsilane (TIPS) was used as a radical
scavenger. The residue obtained following the evaporation
of the volatiles was washed with hexane to afford the Boc-
free His 3 in 96% yield. TLC analysis showed a smaller Rf
for 3 than 2. This increase in polarity was attributed to the
regeneration of the amino-terminal indicating a successful
reaction. The success of the reaction was further confirmed
by the mass spectroscopy where a (M+H)+ peak appeared
at 260 matching the molecular mass of 3.
Synthesis of the Gly-His series started by protecting
the carboxyl group of Boc-His(Bn)-OH (1) in the form of
methyl ester (Scheme 1). Compound 1 was coupled with
methanol using carbonyl diimidazole (CDI) as a coupling
reagent to produce the fully protected His analogue 3. The
success of the synthesis was confirmed by NMR (1H, 13C,
1
1
and H–1H COSY) and mass spectroscopy. The H-NMR
spectrum confirmed the appearance of a singlet at 3.64 ppm
integrated for three protons corresponding to the methyl
1
ester (OCH3) group. In addition, the H-NMR spectrum
The fully protected dipeptide Boc-Gly-His(Bn)-OMe 5
was next synthesized by coupling His 3 and the Boc-pro-
tected glycine (Boc-Gly-OH) 4 using CDI as a coupling
reagent (Scheme 1). NMR and mass spectroscopic data con-
firmed the successful conjugation. The 1H-NMR spectrum
showed a singlet at 1.45 ppm integrated for nine protons
corresponding to tert-butyl group of Boc, two multiplets
at 2.99–3.12 and 4.80–4.85 ppm corresponding to the CH2
and CH groups of His, respectively, a singlet at 5.05 ppm
corresponding to the CH2 group of the benzyl group, and a
doublet at 3.88 ppm corresponding to the CH2 group of Gly.
showed a singlet at 1.43 ppm integrated for the nine protons
of the tert-butyl group of Boc, a multiplet at 2.97–3.06 ppm
integrated for two protons of the CH2 group, and a multiplet
at 4.51–4.57 ppm integrated for one proton of the CH group.
13C-NMR further confirmed the success of esterification.
The connectivity of the atoms was assessed by collecting
1H–1H COSY spectrum and correlating the various peaks.
Additional supportive data was obtained from the mass spec-
troscopy spectrum that showed at peak at m/z 360 which
corresponds to (M+H)+.
1 3

International Journal of Peptide Research and Therapeutics
13C-NMR showed three peaks at 169, 171, and 173 ppm cor-
responding to the three C=O groups in the molecule. Further
confirmation for the success of the reaction was obtained
from mass spectroscopy which depicted a m/z peak at 417
corresponding to (M+H)+.
analytical sample was not obtained and therefore NMR spec-
tra were not clean.
Synthesis of β‑Ala‑Phe Series
Analogue 6, with a deprotected amino group, was
afforded upon the treatment of 5 with TFA/DCM in the
presence of triisopropylsilane (TIPS) as a radical scavenger.
The disappearance of the peak at 1.4 ppm in the 1H-NMR
spectrum and the peaks at 28 and 52.3 ppm in the 13C-NMR
spectrum suggested the selective removal of the Boc group.
Mass spectroscopy showed a peak at m/z 317 correspond-
ing to (M+H)+ of 6, thus further supporting the successful
formation of 6 (Scheme 1).
The next synthesized series of carnosine analogues was
based on β-Ala-Phe (Scheme 2). The rationale behind syn-
thesizing this series was to investigate the role of the imi-
dazole ring in CAR’s biological activity. Therefore, His
was substituted for Phe with a phenyl ring in the side chain
instead of the imidazole one.
The fully protected dipeptide 10 was synthesized as
depicted in Scheme 2. Following the activation of car-
boxyl group of Boc-β-Ala-OH 8 using CDI, H-Phe-OMe
9 was added leading to the generation of the desired prod-
uct 10. The structure of 10 was confirmed by NMR and
The orthogonal deprotection of the imidazole ring of His
was achieved by subjecting compound 5 to catalytic hydro-
genation conditions using 10% Pd/C as a catalyst (Scheme 1)
(Li et al. 2016). The progress of the reaction was traced by
TLC, and the reaction was stopped when all starting mate-
rial disappeared. The desired product 7 was obtained in 96%
yield following purification by column chromatography.
NMR spectral data confirmed the success of the chemical
transformation. In the 1H-NMR, the peaks that integrate for
the five aromatic protons in the 7–7.5 ppm region and the
singlet at 5.05 ppm that integrate for the two methylene pro-
tons of the benzyl group disappeared. This was correlated
with data from of the 13C-NMR spectrum that showed the
disappearance of the peaks between 120 and 150 ppm cor-
responding to the carbons of the phenyl group, as well as the
peak at 29.5 ppm that corresponds to the methylene group
carbon. Further support for the success of the reaction came
from mass spectroscopy that presented a peak at m/z 327
corresponding to (M+H)+.
1
mass spectroscopy. In the H-NMR, the peaks at 1.43,
2.73, and 3.36 ppm corresponded to the Boc and the two
CH2 groups of β-Ala, respectively, while the peaks at 3.15,
3.73, and 4.87 ppm corresponded to CH2, OCH3, and CH
groups of Phe, respectively. In addition, the multiplets at
7.08–7.31 ppm integrated for the five aromatic protons of
1
the phenyl group. The H–1H COSY spectrum assisted in
assigning the peaks to the respective protons. Furthermore,
the 13C-NMR provided the right number and chemical shifts
of the carbon atoms with carbonyl groups of the amide and
ester functionalities appearing at 171.06 and 171.9, respec-
tively. Finally, the mass spectroscopy spectrum collected for
10 depicted a peak at m/z 373 corresponding to (M+23)+.
Dipeptide 11 with a free carboxyl group was obtained by
base hydrolysis of the methyl ester group using an aqueous
lithium hydroxide solution in THF at 0 °C (Scheme 2). The
successful generation of compound 11 was supported by
Boc-Gly-His(Bn)-OH was approached through the
hydrolysis of the methyl ester functionality of dipeptide
5 under basic conditions. While the chemical transforma-
tion was successful as indicated by mass spectroscopy, an
1
the disappearance of the peak at 3.73 pm in the H-NMR
spectrum and the peak at 52.4 ppm in the 13C-NMR spec-
trum corresponding to CH3 of the methyl ester functionality.
Scheme 2 Overall synthetic
scheme for the preparation of
β-Ala-Phe series
1 3

International Journal of Peptide Research and Therapeutics
Furthermore, mass spectroscopy showed a peak at m/z 359
corresponding to the (M+23)+.
generation of compound 13. The connectivity of the atoms
1
was deduced from the analysis of the H–1H COSY spec-
Finally, analogue 12 with a free amino group was
obtained by reacting dipeptide 10 with TFA in the pres-
ence of triisopropylsilane (TIPS) as a radical scavenger
(Scheme 2). The product was purified by column chromatog-
raphy using DCM: MeOH (8:2) as an eluent. The disappear-
ance of the peak at 1.43 ppm in the 1H-NMR spectrum and
the peak at 28.39 ppm in the 13C-NMR spectrum provided
evidence for the success of the hydrolysis. In addition, the
mass spectrum collected for compound 12 showed a peak at
3/z 251 corresponding to (M+H)+.
trum. Additional confirmative indication for the formation
of the fully protected analogue came from the mass spectros-
copy spectrum that showed a peak at m/z 431 corresponding
to the (M+H)+.
Orthogonal deprotection procedures started by subject-
ing dipeptide 13 to catalytic hydrogenation using Pd/C as
a catalyst which led to the selective removal of the benzyl
protecting group from the imidazole ring (Scheme 3). The
crude product was purified by column chromatography to
afford the desired dipeptide 14 with an unprotected imida-
zole ring in 92% yield. The disappearance of the singlet at
5.02 ppm and the peaks in the aromatic region which corre-
spond to the methylene and phenyl groups of benzyl, respec-
tively, confirmed the successful deprotection. In 13C-NMR,
the disappearance of the four peaks in the 116–130 ppm
and the peak at 79 ppm corresponding to the carbon atoms
of the phenyl and the methylene groups of benzyl, respec-
tively, added evidence to the success of the reaction. The
persistence of the peaks at 1.44 and 3.7 ppm corresponding
to the tert-butyl group of Boc and the methyl group of the
methyl ester, respectively, reinforced the orthogonality of the
applied deprotection approach. Additional evidence was col-
lected from the mass spectroscopy which presented a peak
at m/z 341 corresponding to (M+H)+.
Synthesis of the β‑Ala‑His Series
In an attempt to investigate the pharmacophoric functional
groups of carnosine, a series of β-Ala-His analogues was
synthesized with orthogonally exposed functional groups.
The overall synthetic routes to access these analogues is
depicted in Scheme 3.
Boc-β-Ala-His(Bn)-OMe 13, the fully protected carnos-
ine analogue, was synthesized by coupling Boc-β-Ala 8 with
H-His(Bn)-OMe 3 using CDI as a coupling agent. Dipep-
tide 13 was produced in 83% yield following purification
by column chromatography using EtOAc:Hexane (9:1) as
an eluent. All collected spectroscopic data supported the
1
production of 13. In the H-NMR spectrum, the peaks at
Orthogonal deprotection procedures continued by the
selective removal of the Boc group protecting the N-ter-
minal of dipeptide 13. This was accomplished by treat-
ing compound 13 with TFA, and analogue 15 with a free
amino group was generated in 85% yield after purification
on a silica column using DCM:MeOH (8:2) as an eleuent
(Scheme 3). Analysis of the NMR spectrum showed the dis-
appearance of the peak at 1.42 ppm in the 1H-NMR spec-
trum and the peak at 28.44 ppm in the 13C-NMR spectrum
provided evidence for the removal of Boc group. In addition,
the existence of a peak at 5.29 ppm and another at 3.66 ppm
2.44 and 3.42 ppm which integrated for two protons each
correspond to the two methylene groups of Ala, while the
peaks at 3.03 and 4.79 ppm corresponds to the CH2 and CH
groups of His. In addition, the singlet at 1.42 ppm which
integrated for nine protons, the singlet at 3.43 ppm which
integrated for three protons, and the peaks in the 7–7.4 ppm
region which integrated for five protons corresponded to the
–C(CH3)3, –OCH3, and the phenyl group, respectively. The
singlet at 5.04 ppm corresponded to the methylene of the
benzyl protecting group. The 13C-NMR also supported the
Scheme 3 Overall synthesis
scheme for the preparation of
β-Ala-His series
1 3

International Journal of Peptide Research and Therapeutics
which are assigned to the CH2 of the benzyl group and the
CH3 of the methyl ester functionality, respectively, proved
the orthogonality of the deprotection reaction. The m/z peak
at 331 of the mass spectroscopy spectrum of compound 15
which corresponds to (M+H)+ further validated the pro-
posed chemical structure for 15.
Dipeptide Boc-Gly-Phe-OH 17 with a free carboxyl
group at the C-terminal was synthesized by treating com-
pound 16 with aqueous LiOH solution in THF (Scheme 4).
The consumption of reactant 16 and the formation of a more
polar compound as shown by TLC suggested a successful
chemical transformation. Purification of the crude mixture
by column chromatography yielded the desired product 17
in 97% yield. The disappearance of the peak at 3.72 ppm in
the 1H-NMR spectrum and the peak at 52 ppm of the 13C-
NMR spectrum, both of which are attributed to the methyl
protecting group, validated the success of the reaction.
The final analogue to be synthesized was H-Gly-Phe-
OMe with a free amino group at the N-terminal of the dipep-
tide. This required the selective removal of the of the Boc
group under acidic group. The anticipated product 18 was
obtained in 90% yield. Analysis of an analytical sample by
NMR and mass spectroscopy confirmed the success of the
reaction. The singlet at 1.45 ppm in the 1H-NMR spectrum
and the peak at 28 ppm in the 13C-NMR spectrum, which
correspond to the –C(CH3)3 group, disappeared. In addition,
analysis of the product by mass spectroscopy validated the
structure of 18 by showing a peak at m/z 237 which matched
(M+H)+.
Synthesis of the Gly‑Phe Series
The Glycine-Phenylalanine (Gly-Phe) series was the final
set of synthesized carnosine analogues that can be used as a
“negative control” when assessing CAR’s biological activity.
The overall synthetic scheme is shown in Scheme 4.
Boc-Gly-Phe-OMe 16 was prepared by reacting mixture
of Boc-Gly-OH 4 and H-Phe-OMe 9 in anhydrous dichlo-
romethane in the presence of CDI as a coupling reagent
(Scheme 4.18). The formation of 16 was tracked by TLC.
The product was purified by column chromatography using
EtOAc:Hexane (2:1) following the disappearance of start-
ing material and appearance of product. The desired prod-
uct was obtained in 93% yield demonstrating the efficacy
of the applied synthetic approach. The OCH3, CH2, CH,
and Ph groups from Phe produced peaks at 1.45, 3.14, 4.88,
1
and 7.0–7.3 ppm, respectively, in the H-NMR spectrum.
In addition, the tert-butyl group of Boc and the methylene
group of Gly showed peaks at 1.45 and 3.75 ppm, respec-
tively. Peaks in the 13C-NMR corroborated the production
of 16. The peaks at 169 and 171 ppm were attributed to the
carbonyl groups of the amide and the ester functionality,
respectively, while the four peaks between 127 and 135 ppm
were assigned to the phenyl group carbon atoms. On the
other hand, the peaks at 28, 37, 44, 52 and 53 ppm were
attributed to the –C(CH3)3, CH2 (Phe), CH (Phe), CH2 (Gly),
and OCH3 groups respectively. Mass spectroscopy further
confirmed the structure of 16 showing a peak at m/z 337
corresponding to (M+H)+.
Discussion
Carnosine is an endogenous dipeptide widely and abun-
dantly distributed in the muscle and nervous tissues of sev-
eral animal species. Many functions have been proposed
for this compound because of its antioxidant and metal ion-
chelator properties. However, the therapeutic uses of the
dipeptide are strongly limited by the mechanism governing
its homeostasis. This fact has been the main reason for the
synthesis of carnosine derivatives with interesting potenti-
ality, but until now, there have been very few applications.
The main goals reached by the carnosine functionalization
Scheme 4 Overall synthetic
scheme for the preparation of
the Gly-Phe series
1 3

International Journal of Peptide Research and Therapeutics
have been avoiding or, at least, reducing the carnosinase
hydrolysis, conferring a lipophilic character with the aim to
aid the BBB-crossing, enhancing or, at least, maintaining the
beneficial effects of the dipeptide, counterbalancing the side
effects of the grafted compound, and aiming at the targeted
delivery (Bellia et al. 2012).
very significant increase in comparison to l-carnosine. In
addition, the new derivative was able to cross the blood brain
barrier (BBB) and to concentrate in the rat brain after intra-
venous administration. Besides the amide derivatives, an
ethyl ester analogue of carnosine has been reported. Unlike
the amide derivatives of carnosine which formed dimeric
complexes with Cu(II), though with lower thermodynamic
stability compared to l-carnosine, the ethyl ester analogue
only formed monomeric species probably due to the lack of
carboxylate (Lanza et al. 2011; Bertinaria et al. 2011). The
series of CAR analogues prepared in the current study will
offer an opportunity to re-visit the this topic and investigate
the role that each functional group of l-carnosine plays in
complexing to metals ion such as copper and zinc ions.
Castelletto et al. studied the effect of attachment of a
bulky hydrophobic aromatic unit, namely N-(fluorenyl-
9-methoxycarbonyl) (Fmoc), on the self-assembly of Fmoc-
l-carnosine and its zinc-binding properties (Castelletto et al.
2011). The authors showed that Fmoc-l-carnosine formed
well-defined amyloid fibrils containing β sheets above a crit-
ical aggregation concentration, and concluded that modify-
ing the dipeptide by incorporation of a terminal Fmoc unit
may improve the efficiency of carnosine in biotechnology
applications, since Fmoc-l-carnosine can form fibers while
still chelating metal ions. A similar study has been pub-
lished recently assessing the ability of l-carnosine-derived
Fmoc-tripeptides to form pH-sensitive and proteolytically
stable supramolecular hydrogels (Mahapatra et al. 2017).
Therefore, it would be interesting to investigate the effect of
attaching a smaller, aliphatic, hydrophobic group like Boc on
the self-assembly of carnosine fibers and its metal ion com-
plexing capabilities. This feature is encountered in many of
the carnosine analogues prepared in this study. Furthermore,
the effect of varying the β-Ala chain length (i.e. the Gly
series), hindering the carboxyl group (i.e. the methyl ester
series) and masking the imidazole ring (i.e. the protected
His and Phe series) would provide a deeper understanding
of the factors affecting self-assembly of carnosine fibers and
its hydrogelation in the presence of metal ions.
In an attempt to elucidate the chemical constituents that
are responsible for carnosine’s anti-crosslinking activity,
Hobart et al. tested the individual amino acids in carnosine
(β-alanine and l-histidine) as well as modified forms of his-
tidine (α-acetyl-histidine, l-methyl-histidine) and methylated
carnosine (anserine) (Hobart et al. 2004). β-Alanine showed
anti-crosslinking activity but less than that of carnosine,
suggesting that the α-amino group is required in preventing
protein crosslinking. Interestingly, histidine, which has both
α-amino and imidazolium groups, was more effective than
carnosine. Acetylation of histidine’s α-amino group or meth-
ylation of its imidazolium group abolished anti-crosslinking
activity. The authors postulated that the primary amine was
crucial as evidenced by the observed anti-crosslinking activ-
ity of β-alanine and the abolished activity of acetylated his-
tidine with a blocked α-amine. The authors also suggested
that imidazole play a supportive role in preventing glyca-
tion-induced crosslinking. Methylation at the N-1 position
of imidazole of carnosine also diminished its anti-crosslink-
ing activity of carnosine (Hobart et al. 2004). Despite these
interesting findings, the study reached its conclusion based
on assessing the anti-crossing activity of individual amino
acids rather than carnosine analogues, which discards any
potential synergistic effect resulting from linking β-alanine
and histidine. Furthermore, histidine was found to be more
effective than carnosine suggesting that β-alanine might be
acting as an auxophore rather than part of the pharmacoph-
ore. Therefore, preparing a library of carnosine analogues
such as the one prepared in this study would lead to a better
understanding of the functional groups that are responsible
for carnosine’s anti-crosslinking activity.
It has been reported that the carboxylic group is important
in the recognition of carnosine by the carnosinase enzymes
(Unno et al. 2008). Thus, the conversion of carboxyl group
into a different functionality such as an amide or ester might
afford derivatives that are very stable to the carnosinase
action, yet retaining some important biological functions.
Several studies reported the synthesis of l-carnosine deriv-
atives modified at the carboxyl group (Lanza et al. 2011;
Bertinaria et al. 2011; Bellia et al. 2008). Bertinaria et al.
reported the synthesis, physicochemical characterization,
and biological activities of carnosine derivatives modified
at the carboxyl end by an amide. These derivatives proved
to be stable in human serum and thus can be useful as poten-
tial neuroprotective agents. In particular, the propyl amide
derivative of carnosine was able to protect primary hip-
pocampus neurons against HNE-induced death, showing a
The broad spectrum biological activity of carnosine con-
tinues to entice scientist to develop novel analogues with
improved pharmacokinetic profile. For example, Iacobini
et al. recently studied the effect of carnosinol, a carnosinase-
resistant and bioavailable carnosine derivative, on the onset
and progression of diabetic nephropathy in db/db mice (Iac-
obini et al. 2017). Carnosinol was found to be selective and
reactive as RCS sequestering agent. In addition, it is charac-
terized by lack of toxicity and a favorable pharmacokinetic
profile, related to recognition by human peptide transporter
1 as well as resistance to carnosinase. The molecule was
effective in preventing the onset and halting progression of
diabetic nephropathy by quenching RCS, thereby reducing
the accumulation of their protein adducts and the consequent
1 3

International Journal of Peptide Research and Therapeutics
Boldyrev AA (2000) Problems and perspectives in studying the bio-
logical role of carnosine. Biochemistry 65:751–756
inflammatory response. Therefore, a simple reduction of
the carboxyl group of l-carnosine into a primary alcohol
resulted in a novel compound that may represent a promising
advanced glycation endproducts (AGE)-reducing approach
for diabetic nephropathy therapy.
Boldyrev AA, Gallant SC, Sukhich GT (1999) Carnosine: the protec-
tive, anti-aging peptide. Mol Asp Med 19:581–587
Castelletto V, Cheng G, Greenland BW, Hamley IW, Harris PJ (2011)
Tuning the self-assembly of the bioactive dipeptide L-carnosine
by incorporation of a bulky aromatic substituent. Langmuir
27:2980–2988
Craik DJ, Fairlie DP, Liras S, Price D (2013) The future of peptide-
based drugs. Chem Biol Drug Design 81:136–147
Conclusion
Durmus Z, Kavas H, Baykal A, Sozeri H (2011) Synthesis and charac-
terization of L-carnosine coated iron oxide nanoparticles. J Alloys
Compd 509:2555–2561
It is clear that research addressing novel derivatives of car-
nosine is far from over and plenty of questions are yet to be
answered. The study presented in this study offers a feasible
synthetic approach for the preparation of new analogues with
orthogonally protected functional groups that might aid in
addressing or answering many of the mysteries associated
with the biological benefits of carnosine.
Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status
and future directions. Drug Discov Today 20:122–128
Guiotto A, Calderan A, Ruzza P, Borin G (2005) Carnosine and carnos-
ine-related antioxidants: a review. Curr Med Chem 12:2293–2315
Hamley IW (2007) Peptide fibrillization. Angew Chem Int Ed
46:8128–8147
Hipkiss AR (2009) Carnosine and its possible roles in nutrition and
health. Adv. Food Nutr Res 57:87–154
Hipkiss RA, Cartwright PC, Bromley C, Gross SR, Bill RM (2013)
Carnosine: can understanding its actions on energy metabolism
and protein homeostasis inform its therapeutic potential. Chem
Cent J 7:38–46
Acknowledgements We are grateful for Dr. Kamal Bouhadir from the
American University of Beirut for helping in NMR experiments.
Hobart LJ, Seibel I, Yeargans GS, Seidler NW (2004) Anti-crosslink-
ing properties of carnosine: significance of histidine. Life Sci
75:1379–1389
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of
Iacobini C, Menini S, Fantauzzi CB, Pesce CM, Giaccari A, Salomone
E et al (2017) FL-926-16, a novel bioavailable carnosinase-resist-
ant carnosine derivative, prevents onset and stops progression of
diabetic nephropathy in db/db mice. Br J Pharmacol. https://doi.
org/10.1111/bph.14070
interest.
Human and Animal Rights The article does not contain any studies
with human or animal subjects performed by any of the authors.
Krpetić Z, Guerrini L, Larmour IA, Reglinski J, Faulds K, Graham D
(2012) Importance of nanoparticle size in colorimetric and SERS-
based multimodal trace detection of Ni(II) ions with functional
gold nanoparticles. Small 8:707–714
Informed Consent The authors declare that there is no informed con-
sent in the article.
Lanza V, Bellia F, D’Agata R, Grasso G, Rizzarelli E, Vecchio G (2011)
New glycoside derivatives of carnosine and analogs resistant to
carnosinase hydrolysis: synthesis and characterization of their
copper(II) complexes. J Inorg Biochem 105:181–188
Li H, Chen J, Huang H, Feng J-J, Wang A-J, Shao L-X (2016) Green
and facile synthesis of L-carnosine protected fluorescent gold
nanoclusters for cellular imaging. Sens Actuators B 223:40–44
Mahapatra RD, Dey J, Weiss RG (2017) L-Carnosine-derived Fmoc-
tripeptides forming pH-sensitive and proteolytically stable supra-
molecular hydrogels. Langmuir 33:12989–12999
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