Synthesis of vitamin E-carnosine (VECAR): New antioxidant molecule with potential application in atherosclerosis

Article abstract of DOI:10.1080/00397911.2011.632829

Natural antioxidants such as carnosine and α-tocopherol (vitamin E) provide protection against several oxidative stress-related diseases such as atherosclerosis and Alzheimer's. The synthetic combination of α-tocopherol and carnosine can take advantage of the cellular transport mechanism of α-tocopherol by α-tocopherol transfer protein (α-TTP) to colocate α-tocopherol and carnosine at the interface between a lipophilic and a hydrophilic domain and protect both from oxidation. Successful synthesis of a novel heterodimer of α-tocopherol (vitamin E) and carnosine, VECAR was carried out in a total of nine steps. The VECAR design uses a 13-carbon phytyl-chain mimic to link carnosine to Trolox at the C2 carbon position. This design feature is anticipated to maintain binding to α-TTP, while maintaining the antioxidant activity of the two heterodimer components. Our results confirmed that there was no loss in antioxidant activity in VECAR using an in-vitro DPPH assay, versus α-tocopherol and Trolox. Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications to view the free supplemental.

Full text of DOI:10.1080/00397911.2011.632829

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Synthesis of Vitamin E-Carnosine
(VECAR): New Antioxidant Molecule with
Potential Application in Atherosclerosis
Carlos E. Astete a , Danielle Songe Meador b , David Spivak b &
Cristina Sabliov a
a Biological and Agricultural Engineering, Louisiana State University,
Baton Rouge, Louisiana, USA
b Chemistry Department, Louisiana State University, Baton Rouge,
Louisiana, USA
Accepted author version posted online: 20 Apr 2012.
To cite this article: Carlos E. Astete , Danielle Songe Meador , David Spivak & Cristina Sabliov (2013):
Synthesis of Vitamin E-Carnosine (VECAR): New Antioxidant Molecule with Potential Application in
Atherosclerosis, Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 43:9, 1299-1313
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Synthetic Communications1, 43: 1299–1313, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.632829
SYNTHESIS OF VITAMIN E-CARNOSINE (VECAR):
NEW ANTIOXIDANT MOLECULE WITH POTENTIAL
APPLICATION IN ATHEROSCLEROSIS
Carlos E. Astete,1 Danielle Songe Meador,2 David Spivak,2
and Cristina Sabliov1
1Biological and Agricultural Engineering, Louisiana State University,
Baton Rouge, Louisiana, USA
2Chemistry Department, Louisiana State University, Baton Rouge,
Louisiana, USA
GRAPHICAL ABSTRACT
Abstract Natural antioxidants such as carnosine and a-tocopherol (vitamin E) provide pro-
tection against several oxidative stress–related diseases such as atherosclerosis and Alzhei-
mer’s. The synthetic combination of a-tocopherol and carnosine can take advantage of the
cellular transport mechanism of a-tocopherol by a-tocopherol transfer protein (a-TTP) to
colocate a-tocopherol and carnosine at the interface between a lipophilic and a hydrophilic
domain and protect both from oxidation. Successful synthesis of a novel heterodimer of
a-tocopherol (vitamin E) and carnosine, VECAR was carried out in a total of nine steps.
The VECAR design uses a 13-carbon phytyl-chain mimic to link carnosine to Trolox at the
C2 carbon position. This design feature is anticipated to maintain binding to a-TTP, while
maintaining the antioxidant activity of the two heterodimer components. Our results con-
firmed that there was no loss in antioxidant activity in VECAR using an in-vitro DPPH
assay, versus a-tocopherol and Trolox.
Supplemental materials are available for this article. Go to the publisher’s online edition
of Synthetic Communications1 to view the free supplemental.
Received September 1, 2011.
Address correspondence to Cristina Sabliov, Biological and Agricultural Engineering, Louisiana
State University Agricultural Center, 149 EB Doran Building, Baton Rouge, LA 70803, USA. E-mail:
csabliov@lsu.edu; or David Spivak, Chemistry Department, 735 Choppin Hall, Louisiana State
University, Baton Rouge, LA 70803, USA. E-mail: dspivak@lsu.edu
1299

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C. E. ASTETE ET AL.
Keywords Alzheimer’s; antioxidant; atherosclerosis; carnosine; a-tocopherol; Trolox
INTRODUCTION
Antioxidant theory links oxidative stress with cardiovascular diseases and other
major diseases such as cancer, acute inflammation, Parkinson’s, and Alzheimer’s.[1–7]
In 2007, an estimated $430 billion was spent to treat approximately 80 million people
suffering from some type of cardiovascular disease.[8,9] Antioxidant agents play vital
roles in the defense system against free radicals and oxidant molecules by avoiding
radical propagation (chain reaction) via radical scavenging, metal ion chelation,
and coantioxidant action (antioxidant regeneration). The balance between oxidative
species and antioxidant systems (natural molecules, enzymes, and proteins) defines
the oxidative stress of a living system, and the oxidative stress level determines if a
biological system has a high risk for diseases.[10]
Atherosclerosis in particular has been related to the oxidative process, in that
high levels of oxidant molecules or low levels of antioxidants were shown to be
responsible for high oxidative stress, triggering the atherosclerosis process.[11–14]
The ox-LDL (i.e., the oxidized form of low-density lipoproteins, LDLs) acts at differ-
ent levels, stimulating the host response and formation of foam cells. Among the lipo-
philic antioxidants, a-tocopherol (the most bioactive isomer of vitamin E) is one of
the most potent natural antioxidants shown to protect LDLs from oxidation. To
highlight the importance of a-tocopherol in humans, it has been shown that a specific
enzyme a-tocopherol transfer protein (a-TTP) is responsible for a-tocopherol trans-
port into lipoproteins. Lipoproteins then act as the main vehicle for a-tocopherol
transport to different tissues.[15–17] Lipophilic antioxidants prevent oxidation of the
lipid fraction of LDLs, but the more hydrophilic apolipoproteins (e.g., Apo B-100)
are not protected from oxidation even in the presence of lipophilic antioxidants.[18–21]
The limitations of lipophilic natural antioxidants can be overcome with an
adequate design of an alternative ‘‘natural’’ antioxidant formed by covalently linking
the hydrophobic antioxidant with a naturally occurring hydrophilic molecule with
antioxidant properties; the new structure will ensure that the component will be loca-
lized closer to apolipoproteins, have better antioxidant performance, and have
potential synergetic behavior because of antioxidant–coantioxidant phenomena.
Hydrophilic antioxidants have the ability to protect proteins, DNA, and carbohy-
drates from oxidative molecules and to regulate oxidative molecules for signaling
purposes. Among hydrophilic molecules, vitamin C and carnosine play important
roles in the protection of proteins and DNA from oxidative molecular attacks. Other
molecules, such as flavonoids, uric acid, micronutrients, peptides, and proteins, have
been researched for their potential antioxidant actions.[22–32] The dipeptide carnosine
(b-alanyl-L-histidine) has been researched as a protective agent against aging
because of its antioxidant properties and ability to suppress protein glycation and
crosslinking. Carnosine has also been suggested as an important agent in controlling
secondary problems in diabetes.[33–41] Thus, it is not surprising that analogs combin-
ing Trolox, a substructure of vitamin E, with carnosine have been reported.[42] The
heterodimers used in these studies coupled Trolox directly to carnosine without
inclusion of the phytyl chain component in between, which is important (vide supra)
for maximizing transport to LDLs in vivo by a-TTP.

SYNTHESIS OF VITAMIN E-CARNOSINE
1301
The main goal of this project was to synthesize a next-generation antioxidant
molecule based on combining a more complete structure of a-tocopherol and
carnosine, with potential applications in prevention of oxidative stress–related dis-
eases, most notably atherosclerosis. The hypothesis is that conjugation of a vitamin
E derivative with carnosine (referred to as VECAR) by a phytyl-type tether will
result in a new molecule with antioxidant properties capable of in vivo protection
of polyunsaturated fatty acids (PUFAs) and LDL from oxidation. It has been shown
that fluorescent molecules tethered to a-tocopherol derivatives are successfully trans-
ported to LDL particles in vitro, and the similar design of VECAR is anticipated to
also be transported actively to LDL particles via endogenous a-TTP. Studies on
VECAR both in vitro and in vivo will further determine if a-tocopherol can serve
as a generic vehicle for delivery of a wide array of therapeutic compounds through
the lipoprotein transport mechanisms. This would be a powerful addition to
methods currently used to increase bioavailability of important drug compounds.
RESULTS
Molecular Design of VECAR
The molecular design of VECAR incorporated various structural components
shown to be important when modifying the structure of a-tocopherol. Several
synthetic antioxidant molecules have been derived from vitamin E (e.g., Trolox,
3-oxa-chromanol derivatives, brominated a-tocopherolmethano-dimer, raxofelast,
and vitamin EC) in an effort to improve bioavailability or antioxidant potential.
In general, these derivatives reported in the literature use the OH moiety of a-toc-
opherol for adding new functionality to the molecule, which is often accomplished
in one step or modify the chromanol ring, requiring more complex synthetic strate-
gies.[43–49] An important aspect in design of derivatives is maintaining the biotran-
sport properties that are inherent to natural a-tocopherol. There is one specific
transport protein, a-TTP, that is responsible for transport of a-tocopherol to LDLs;
other transport mechanisms are passive and related to the lipophilicity of the mol-
ecule. For this study, it was of interest to ensure that the a-tocopherol derivative
is recognized by the a-TTP for further transport to the LDL particles. The modifi-
cation of the OH or methyl groups on the chromanol ring of a-tocopherol deriva-
tives significantly reduce or inhibit uptake of these compounds by a-TTP.[50,51] On
the other hand, modification of the phytyl chain does not affect recognition of a-toc-
opherol derivatives; therefore, this was the site chosen for further modification with
additional bioactive molecules such as the hydrophilic carnosine antioxidant.[17,51–54]
Hydrophilic molecules like carnosine are not expected to enter into lipophilic
pathways in living systems, and it was of central interest to determine whether the
a-tocopherol structure could shuttle bioactive molecules into LDL particles that
ordinarily could not be localized there. It was envisioned that this could be
accomplished by tethering the hydrophilic bioactive molecule via the phytyl tail.
The phytyl tail of a-tocopherol consists of a 13-carbon chain with two chiral methyl
substituents and one achiral one; however, it has been reported that substituting a
straight chain for the phytyl tail does not affect the absorption and antioxidant
activity in rats while maintaining the lipophilicity of the molecule.[55] Therefore,

1302
C. E. ASTETE ET AL.
Figure 1. Vitamin E derivatives: 1, Trolox, (S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; 2,
Raxofelast, 2,3-dihydro-5-acetyloxy-4,6,7-trimethyl-2-benzofuranacetic acid; 3, 3-oxa-chromanol derivatives;
4, a-tocopherol succinate; 5, a-tocopherol butyric acid; 6, (E)-((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-
trimethyltridecyl)chroman-6-yl) 5-(5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yloxy)-4-
oxopent-2-enoate; and 7, (2Z,4Z,6E,8E)-((R)-2,5,7,8-tetramethyl-2-((4 R,8 R)-4,8,12-trimethyltridecyl)
chroman-6-yl) 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate.
the VECAR design for the synthesis in this study employed a 13-carbon tether with a
carboxylate terminus for further functionalization. This has been done for similar
analogs using Wittig coupling of the chromanol ring to the hydrophobic tail, and
that approach was adopted for this synthesis as well. Unique to this synthesis is
the conjugation of carnosine to the open end of the tail to provide a bioactive mol-
ecule with the antioxidant advantages of both lipophilic and hydrophilic compo-
nents. The ability of a-tocopherol to transport a conjugated fluorescent molecule
to the surface of Hep=G2 cells in culture by an a-TTP dependent pathway has been
previously reported in the literature. It is anticipated that the carnosine conjugated
molecule reported here will also be transported by an a-TTP dependent pathway to
Figure 2. Schematic structure of VECAR. There is a hydrophilic section formed by the a-tocopherolchro-
manol ring and the carnosine dipeptide. The hydrophobic section is provided by the carbon tail that links
polar heads to important sections. The circles around the chromanol ring represent the key zones for enzy-
matic recognition of VECAR by a-TTP enzyme.

SYNTHESIS OF VITAMIN E-CARNOSINE
1303
the cells and ultimately to the LDL particles in cells, which has been shown to occur
with vitamin E. Although the lipophilic vitamin E will be internalized in the LDLs,
the hydrophilic carnosine may reside more toward the polar surface of the LDL par-
ticles. The complementary positioning of carnosine in polar regions of LDL particles
may offer a unique and improved defense versus vitamin E alone toward oxidation
of LDLs, which has been postulated as an important event in preventing atheroscler-
osis. Possibly more interesting is the delivery of a hydrophilic antioxidant to LDLs
that could not otherwise locate there.
Synthesis of VECAR
Based on the molecular design of the VECAR molecule, the synthesis was car-
ried outas shown in Scheme 1, involving nine steps in the overall synthesis starting
Scheme 1. Overall synthesis of VECAR.

1304
C. E. ASTETE ET AL.
from commercially available Trolox. Several steps follow similar procedures used by
Lei and Atkinson to synthesize a vitamin E derivative with the same a-tocopherol
structural component.[56] The first step was esterification of Trolox, achieved in
92% yield using thionyl chloride to form the acid chloride in the presence of methanol.
This method was more dependable, reduced the reaction time from 18 h to less than
2 h, and resulted in a greater yield than the previous method reported that used
p-toluenesulfonic acid to catalyze Fischer esterification using methanol. The protec-
tion of the hydroxyl group present in the chromanolring of Trolox ester 9 using tert-
butyldimethylsilyl chloride (TBDMS) provided a white solid rather than a yellow oil
as reported. It should be noted that use of excess magnesium sulfate for drying the
organic extracts resulted in residual water in this product, which is difficult to remove
afterward. This was significant because poor yields were observed in the subsequent
DIBAL reduction when residual water was present in 10. The third step was the
reduction of the Trolox ester 10 to an aldehyde 11 using DIBAL at ꢀ78 ꢁC over a
span of 2 h. The purification of the aldehyde 11 posed some difficulty because of
the small Rf in separation of the unreacted ester (which can be due to water in the
reaction) with the aldehyde and required gradient elution during column chromato-
graphy. Next, the ylide (14) formed from 12-bromododecanoic acid (12), and triphe-
nyhlphosphine was tethered to 11 through a Wittig reaction to form 17. The double
bond formed from the Wittig reaction was further reduced in the presence of hydro-
gen to produce 18 with yields comparable to similar steps in the literature.[56]
The coupling reaction of carnosine was complicated by the hydrophobic=
hydrophilic behavior of the new molecule. Therefore, the carnosine was modified
to carnosine ester (16) to improve its solubility in organic solvent and more impor-
tantly to avoid reactions between the carboxylic group of carnosine and its own
amine terminus. Initially, esterification with diazomethane was investigated, but
resulted in poor yield with several side products. Another approach was successful
using thionyl chloride and methanol to form the ester without significant interference
from the terminal amine. With the ester in hand, coupling of the carnosine derivative
was investigated with commonly employed approaches such as using dicyclohexyl-
carbodiimide (DCC) with HOBt, the formation of asymmetric anhydrides, and (2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (HBTU).
The best results were achieved when HBTU was used as coupling reagent. Purifi-
˚
˚
cation was first attempted using typical 60-A and 300-A pore-size silica gel; however,
the product remained adsorbed under all mobile phases investigated. Thus, reverse-
phase silica gel, synthesized according to the literature,[57] was employed and found
to successfully separate the product from other impurities. The last step, deprotec-
tion of the silyl protecting group, was achieved using standard tetrabutylammonium
fluoride (TBAF) conditions for 1.5 h; longer reactions times gave lower yields and
produced more side products. Purification of the final product was again done using
reverse-phase silica gel to give 26% yield overall of the isolated product.
Antioxidant Activity of VECAR by DPPH
The antioxidant behavior of VECAR was studied with the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay.[58] Measurements were performed at five concentra-
tions [Eq. (1)] (Supplemental J) to determine IC50, which is the point at which

SYNTHESIS OF VITAMIN E-CARNOSINE
1305
50% of DPPH has been reduced by VECAR. The IC50 was found to be
24.9 ꢂ 1.4 mM for VECAR; and the value for a-tocopherol alone was 24.4 ꢂ 1.5 mM.
These values confirm that VECAR has antioxidant activities that are similar to
pure a-tocopherol, which is the main hydrophobic antioxidant used in the synthesis.
CONCLUSION
The successful synthesis of a novel heterodimer of a-tocopherol and carnosine
(VECAR) was carried out in a total of nine steps. The key aspect of VECAR design
was the use of a 13-hydrocarbon phytyl-chain mimic to link carnosine to Trolox at
the C2 carbon position. This design feature is anticipated to not interrupt binding to
a-TTP, while maintaining antioxidant activity of the two heterodimer components.
Our results confirmed that there was no loss in antioxidant activity in VECAR using
an in vitro DPPH assay, versus a-tocophorol and Trolox. Further studies in cell and
animal matrices need to be carried out to determining the bioavailability and efficacy
of the chimeric VECAR in vivo.
EXPERIMENTAL
The following chemicals were purchased from Acros Organics: Trolox 97%,
tert-butylchlorodimethylsilane 98%, and triethylamine 99%. Methanol anhydrous
99.8%, imidazole >99.5% (GC), dimethylformamide anhydrous 99.8%, diisobutyla-
luminum hydride (DIBAL) 1.0 M solution in hexane, 12-bromododecanoic acid
97%, acetonitrile anhydrous 99.8%, triphenylphosphine 99%, tetrahydrofuran
(THF) <99.9%, lithium bis(trimethylsilyl)-amide (LiHMDS) 1.0 M solution in
THF, palladium (Pd=C) 10 wt.% (dry basis) on activated carbon, (wet, Degussa type
E101 NE=W), L-carnosine 99%, and 4-(dimethylamino) pyridine (DMAP) 99% were
purchased from Sigma-Aldrich. Thionyl chloride 99þ% and magnesium sulfate
99.5% were purchased from Alfa Aesar. The chemicals purchased from Mallinckrodt
Chemicals were methanol, ethyl acetate, toluene, ammonium chloride, chloroform,
sodium hydroxide, and sodium bicarbonate. Dichloromethane and hexanes 95%
˚
˚
n-hexane were purchased from J.T. Baker. Silica gel (porosity: 60 A, particle size:
2
40–63 mm, surface area: 500–600 m =g, pH range: 6.5–7.5, and 300 A particle size)
was purchased from Sorbent Technologies, and O-benzotriazole-N,N,N0,N0-
tetramethyl-uronium-hexafluoro-phosphate (HBTU), 99.58%, was purchased from
ChemPep Inc. The following chemicals were purchased from Fisher Chemical:
1-propanol and sodium chloride. Sodium bicarbonate was purchased from EMD
Chemicals.
A stock solution of DPPH in methanol at 0.4 mM was prepared and kept at
ꢀ20 ꢁC in the dark prior to use. The sample was diluted with water (pH ¼ 3.5 pre-
pared by addition of acetic acid) to 1 ml. The sample was added to dilute DPPH
stock in methanol to obtain a final sample volume of 2 ml with a 0.1 mM DPPH con-
centration. The ratio between water and methanol was 1 to 1 v=v. The blank was pre-
pared with 1 ml of water (pH 3.5) and pure methanol. The controls were prepared for
each sample, with 1 ml of water (pH 3.5) and 1 ml of DPPH in methanol solution.
Absorption readings were taken after 30 min at 518 nm using a Geminys 6 spectro-
photometer (Thermo Scientific, Waltham, Mass.). The formula used to calculate the

1306
C. E. ASTETE ET AL.
rate of oxidation was
% change in activity ¼ ½ðAbscontrol ꢀ AbssampleÞ=Abscontrolꢃ ꢄ 100
ð1Þ
Reverse-Phase Silica Gel
The synthesis of reverse-phase silica gel was performed for purification of
VECAR after the coupling reaction. Normal-phase silica gel (100 g) was suspended
in toluene (500 ml), and 21 ml (0.52 mol) of octadecyltrichlorosilane (C18, ODTS)
was added dropwise. After 10 min, triethylamine (28 ml, 4 equiv.) was added to
the suspension, and the final suspension was refluxed for 24 h at 125 ꢁC. Finally,
the reverse-phase silica gel was washed with toluene (1 L), DCM (1 L), ethyl acetate
(1 L), and methanol (4 L). The product was dried under high vacuum.[57]
Synthesis of a-Tocopherol-Carnosine (VECAR)
The synthesis of a-tocopherol-carnosine (VECAR) was based on the protection of
OH group, activation of thecarbonyl group, Wittig reaction, attachment of carnosine, and
deprotection. The method is based on the work of Atkinsons and coworkers.[56,59–67]
(S)-Methyl 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (9)
Commercially available (Sigma-Aldrich) S-Trolox (1) (3.04 g, 12.1 mmol) was
dissolved in 25 mL dry methanol; 2.4 mL (17.0 mmol) triethylamine was added and
the mixture stirred at 0 ꢁC for 15 min. Thionyl chloride (1.06 mL, 14.6 mmol, 1.2
equiv.) was added to the solution and stirred at 0 ꢁC for an additional 10 min. The
solution was brought to 70 ꢁC and refluxed for 1 h, and after the reaction completed
the solution was cooled to room temperature and purged with nitrogen to remove
any remaining thionyl chloride. The solvent was evaporated and the brown solid
1
was recrystallized from 75 mL methanol to give white crystals (2.95 g, 92%). H
(CDCl3, 400 MHz) d 4.31 (br, 1H, OH), 3.69 (s, 3H, OCH3), 2.68 (m, 2H), 2.45
(m, 1 H), 2.20 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H), 1.87 (m, 1H), 1.62 (s, 3H, CH3).
13C NMR (CDCl3, 100 MHz) d 174.53, 145.52, 145.31, 122.56, 121.30, 118.45,
116.87, 77.03, 52.38, 30.63, 25.45, 20.97, 12.21, 11.84, 11.26. IR nmax 3527.61,
2989.30, 2926.84, 1738.34, 1454.75, 1193.27, 1113.77 cmꢀ1. MS (ESI-TOF): calcd.
for C15H20NaO4 (M þ Na)þ: 287.1260; found: 287.1241.
(S)-Methyl 6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethylchroman-2-
carboxylate (10)
Following previous published procedures,[56] (S)-methyl-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylate (9) (1.254 g, 4.7 mmol) was mixed with tert-butyl-
dimethylsilyl chloride (1.081 g, 7.2 mmol) and imidazole (1.339 g, 19.7 mmol); and
dry dimethylformamide (10 mL) was added. The mixture was stirred and heated in
an oil bath under an argon atmosphere at 85 ꢁC for 5 h, at which point none of
the starting material (9) was detected by TLC. The reaction mixture was poured into

SYNTHESIS OF VITAMIN E-CARNOSINE
1307
100 mL saturated aqueous NaCl, and then extracted with 4 ꢄ 50 mL ethyl acetate.
The combined organic extracts were dried over magnesium sulfate and evaporated
to give an oily sample with a light yellow color. The crude product was purified
by column chromatography on silica gel using CH2Cl2–hexane ¼ 3:1, providing a
1
white solid after evaporation with a yield of 1.62 g (91%). H (CDCl3, 400 MHz) d
ppm 3.66 (s, 3H, OCH3), 2.57 (m, 2H), 2.44 (m, 1H), 2.15 (s, 3H), 2.11 (s, 3H),
2.02 (s, 3H), 1.88 (m, 1H), 1.60 (s, 3H), 1.04 (s, 9H), 0.11 (s, 6H). 13C NMR (CDCl3,
100 MHz) d ppm 174.55, 145.90, 144.81, 126.14, 123.48, 122.70, 117.06, 77.02, 52.30,
30.58, 26.11, 25.41, 21.07, 18.62, 14.34, 13.39, 12.00, ꢀ3.28, ꢀ3.34. IR nmax 2957.55,
2928.25, 2856.92, 1746.92, 1464.01, 1260.53, 1110.79 cmꢀ1. MS (ESI-TOF): calcd.
for C21H34NaO4Si (M þ Na)þ: 401.2124; found 401.2171.
(S)-6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethylchroman-2-
carbaldehyde (11)
Similar to previous procedures,[56] compound 10 (3.282 g, 8.67 mmol) was
added to dry hexane (35 mL) and cooled in an acetone=dry ice bath to ꢀ75 ꢁC. Dii-
sobutylaluminum hydride (DIBAL-H, 1.0 M in hexane, 16 mL, 16 mmol) was added
using a syringe so as not to exceed ꢀ70 ꢁC. After 2 h the reaction was quenched with
dry methanol (25 mL) and stirred for 15 min. The solution was removed from the
acetone=dry ice bath and was allowed to warm to ꢀ28 ꢁC. Then water (15 mL) added
to the solution by syringe. The solution was then poured into 100 mL saturated aque-
ous NaCl and extracted with 4 ꢄ 100 mL hexane = ethyl acetate (2:1). The combined
organic extracts were dried over magnesium sulfate, and the solvent was evaporated.
The crude product was purified by column chromatography using CH2Cl2–hexane
1
(1:1 to 3:1) to give 2.33 g (77%) yield. H (CDCl3, 400 MHz) d ppm 9.63 (s, 1H),
2.55 (m, 2H), 2.27 (m, 1H), 2.17 (s, 3H), 2.12 (s, 3H), 2.02 (s, 3H), 1.82 (m, 1H),
1.39 (s, 3H), 1.05 (s, 3H), 0.12 (s, 6H). 13C NMR (CDCl3, 100 MHz) d ppm
204.91, 145.56, 145.13, 126.50, 123.89, 122.84, 117.62, 80.28, 27.93, 26.08, 21.62,
20.49, 18.60, 14.35, 13.38, 12.06, ꢀ3.31, ꢀ3.34. IR nmax 2928.40, 2857.11, 2799.65,
1741.47, 1463.23, 1255.44, 1101.77, 836.53 cmꢀ1. MS (ESI-TOF): calcd. for
C20H33O3Si (M þ H)þ: 349.2199; found 349.2023.
11-Carboxyundecyl)triphosphonium Bromide (14)
A solution of 12-bromododecanoic acid (1.66 g, 5.9 mmol) in dry acetonitrile
(13 mL) was heated and stirred. Triphenylphosphine (1.637 g, 6.2 mmol) in dry acet-
onitrile (13 mL) was heated, stirred, and then added to the 12-bromododecanoic sol-
ution; the solution was refluxed overnight. The solvent was removed. The crystals
were dissolved in DCM (14 mL). Toluene (40 mL) was added, and the solution
was stirred and heated to remove the DCM. The solution was cooled, and the phos-
phonium salts crystallized where removed by filtration. The solution was decanted
1
with toluene. The yield of 14 was 3.01 g (94%). H NMR (CDCl3, 400 MHz) d
ppm 7.76–7.67 (m, 9H), 7.67–7.27 (m, 6H), 3.56–3.51 (m, 2H), 2.28 (t, 2H,
J ¼ 7.42 Hz), 1.53–1.45 (m, 6H), 1.17–1.11 (m, 12H). 13C NMR (CDCl3, 100 MHz)
d ppm 177.51, 135.11, 135.09, 133.61, 133.51, 130.61, 130.48, 118.60, 117.75,
34.50, 30.37, 30.21, 29.09, 29.07, 28.97, 28.94, 28.81, 24.73, 22.88, 22.52, 22.48,

1308
C. E. ASTETE ET AL.
22.39. IR nmax 2925.16, 2848.82, 1715.11, 1436.26, 1113.18 cmꢀ1. MS (ESI-TOF):
calcd. for C30H39BrO2P (M þ H-Br)þ: 461.2831; found 461.2332.
(S,E)-13-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethylchroman-
2-yl)tridec-12-enoic Acid (17)
The suspension of phosphonium salts (14) (2.50 g, 11.5 mmol) was dissolved in
dry THF (40 mL) at room temperature under argon. A solution of LiHMDS in THF
(1 M in THF, 16.5 mL, 11.5 mmol) was added dropwise via a syringe. The red ylide
was stirred for 2 h under argon. A solution of 11 (1.45 g) in dry THF (8 mL) was
added dropwise. The color changed from red to pale yellow. The suspension was stir-
red for an additional 3 h, until 3 could not be found by TLC. The reaction was
quenched with saturated NH4Cl (80 mL) and water (80 mL) and then extracted with
ethyl acetate. The solution was dried over magnesium sulfate. After solvent removal,
trituration with cold hexane removed triphenylphosphine oxide. The hexane solution
was evaporated, and the crude product was purified by column chromatography on
1
silica gel using ethyl acetate:hexane (1:1).[56] Yield of 17 was 1.90 g (86%). H NMR
(CDCl3, 400 MHz) d ppm 5.33–5.31 (m, 2H), 2.55–2.53 (m, 2H),2.36–2.33 (m, 2H),
2.11 (s, 3H), 2.10 (s, 3H), 2.08–2.01 (m, 2H), 2.04 (s, 3H), 1.65–1.62 (m, 2H), 1.47 (s,
3H), 1.29–1.22 (m, 16H), 1.05 (s, 9 H), 0.12 (s, 6H). 13C NMR (CDCl3, 100 MHz) d
ppm 179.99, 146.06, 144.13, 133.26, 132.49, 125.77, 123.46, 122.42, 117.83, 75.52,
34.04, 33.40, 29.97, 29.54, 29.42, 29.23, 29.05, 27.97, 27.23, 26.09, 24.67, 21.27,
18.58, 14.34, 13.40, 12.17, ꢀ3.35. IR nmax 2927.02, 2855.11, 1709.96,
1462.79,1254.07, 1090.75, 837.25 cmꢀ1. MS (ESI-TOF): calcd. for C32H54O4Si
(M)þ: 530.3791; found 530.3740.
(R)-13-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethylchroman-2-
yl)tridecanoic Acid (18)
Pd=C (0.505 g) was added to a solution of 17 (0.95 g, 3.67 mmol) in ethyl acet-
ate (85 mL). A hydrogen balloon was attached to the reaction mixture and it was stir-
red for 18 h at room temperature. The mixture was filtered and evaporated to obtain
18 as oil. The crude product was purified by column chromatography using hexane–
ethyl acetate (4:1). The yield of 18 was 0.91 g (96%).[56] 1H NMR (CDCl3, 400 MHz)
d ppm 2.60 (t, 2H, J ¼ 7.40), 2.40 (t, 2H, J ¼ 6.28), 2.16 (s, 3H), 2.13 (s, 3H), 2.11 (s,
3H), 1.79–1.92 (m, 2H), 1.59–1.70 (m, 4H), 1.47–1.53 (m, 2H), 1.32 (br, 16H), 1.28 (s,
3H), 1.11 (s, 9H), 0.18 (s, 6H). 13C NMR (CDCl3, 100 MHz) d ppm 180.52, 145.97,
144.10, 125.86, 123.52, 122.72, 117.52, 77.31, 39.66, 34.18, 31.60, 30.26, 29.71, 29.68,
29.52, 29.33, 29.14, 26.18, 24.74, 23.88, 23.70, 20.97, 18.65, 14.39, 13.47, 12.01,
ꢀ3.29. IR nmax 2928.58, 2853.19, 1711.92, 1252.43, 1087.10, 836.06 cmꢀ1. MS
(ESI-TOF): calcd. for C32H56O4Si (M þ H)þ: 533.4026; found 533.4011.
Carnosine Methyl Ester Dihydrochloride (16)
Thionyl chloride (0.25 mL, 3.3 mmol) was added dropwise to a suspension of
carnosine(15) (602.9 mg, 2.7 mmol) in anhydrous methanol (24 mL) at 0 ꢁC and stir-
red for 10 min. The solution was refluxed at 75 ꢁC for 1 h. After cooling to room

SYNTHESIS OF VITAMIN E-CARNOSINE
1309
temperature, the mixture was concentrated to give a quantitative yield. The product
was used in the next step without further purification.[68] 1H NMR [(CD3)2SO] d 9.04
(s, 1H), 8.89–8.87 (d, 1 H), 8.09 (br, 3H), 7.49, (s, 1H), 4.62–4.56 (m, 1H), 3.65 (s,
3H), 3.19–3.05 (m, 2H), 2.92 (br, 2H), 2.57 (t, 2H, J ¼ 6.82). 13C NMR (DMSO,
100 MHz) d ppm 171.36, 170.15, 134.06, 129.33, 117.50, 52.71, 52.01, 35.38, 32.31,
26.23. IR nmax 3417.02, 3138.25, 1737.08, 1660.26, 1624.71 cmꢀ1. MS (ESI-TOF):
calcd. for C10H17ClN4O3 (M ꢀ H)þ: 275.0911; found 275.0912.
Coupling Carnosine Methyl Ester with Carboxylic Acid (19)
Carboxylic acid compound 18 (840 mg, 1.59 mmol), DMAP (19 mg,
0.16 mmol), carnosine–methyl ester hydrochloride (16) (662 mg, 2.39 mmol), and
triethylamine (1.67 mL, 5 equiv) were added to anhydrous DMF (13.4 ml). The sol-
ution was cooled to 0 ꢁC in an ice bath. HBTU (721 mg, 1.9 mmol) in anhydrous
DMF (3 mL) was added to the reaction mixture dropwise and stirred for an
additional 20 min at 0 ꢁC. The reaction was stirred at room temperature overnight.
Next, the solvent was evaporated and dried under high vacuum. The washing and
extraction steps were performed with chloroform and water without addition of
magnesium sulfate. The organic phase was collected and evaporated under vacuum.
The purification was performed with reverse-phase silica gel (C18). The silica gel used
˚
was 300 A pore size with a mixture of methanol–water (7:3) as eluent. The yield of 19
was 0.62 g (61%).[69–71] 1H NMR (MeOD) d 7.58 (s, 1H), 6.87 (s, 1H), 4.69–4.66 (m,
1H), 3.72 (s, 3H), 3.41–3.37 (m, 2H), 3.12–2.96 (m, 2H), 2.54 (t, 2H, J ¼ 6.62 Hz),
2.41 (t, 2H, J ¼ 6.66 Hz), 2.15 (t, 2H, J ¼ 7.60 Hz), 2.07 (s, 3H), 2.03 (s, 3H; s,
3H), 1.82–1.71 (m, 2H), 1.57 (m, 3H), 1.44 (m, 3H), 1.26 (br, 16H), 1.20 (s, 3H),
1.04 (s, 9H), 0.10 (s, 6H). 13C NMR (MeOD, 100 MHz) d ppm 176.30, 173.64,
173.46, 147.22, 145.32, 136.37, 126.56, 124.42, 123.52, 118.68, 75.51, 54.10, 52.76,
40.31, 37.11, 36.82, 36.36, 32.81, 31.20, 30.74, 30.63, 30.46, 30.33, 26.94, 26.68,
24.57, 24.22, 21.84, 19.50, 14.86, 13.91, 12.26, ꢀ3.07. IR nmax 3446.06, 2927.19,
2854.43, 1733.62, 1716.69, 1683.57, 1652.84, 1646.99, 1256.82, 1090.42, 837.37 cmꢀ1
MS (ESI-TOF): calcd. for C42H70N4O6Si (M þ H)þ: 755.5134; found 755.5127.
.
Deprotection of Hydroxyl Group of Chromanol Ring (20)
The deprotection of 19 was performed by dissolving 19 (255 mg) in dry THF
(10 ml), and the addition of TBAF (2.9 mL of a 1 M THF solution) was performed
dropwise via a syringe. The solution was stirred for 1.5 h at room temperature. The
THF was evaporated and replaced with chloroform (200 ml). The solution was
washed with water three times. The crude VECAR product was purified by flash
˚
chromatography on reverse-phase silica gel (60 A) using gradient elution, meth-
anol–water (30%) and methanol–water–ammonium hydroxide (8:1:1). The first
two fractions collected the impurities, and pure VECAR was collected in the last
1
fraction. The yield of 20 was 175 mg (81%). H (MeOD, 400 MHz) d 7.68 (s, 1H),
6.89 (s, 1H), 4.69–4.65 (m, 1H), 3.69 (s, 3H), 3.39–3.34 (m, 2H), 3.12–2.95 (m,
2H), 2.57 (t, 2H, J ¼ 6.9 Hz), 2.39 (t, 2H, J ¼ 6.7 Hz), 2.14 (t, 2H, J ¼ 3.6 Hz), 2.11
(s, 3H), 2.07 (s, 3H), 2.03 (s, 3H), 1.79–1.72 (m, 2H), 1.59–1.53 (m, 3H), 1.43–1.39
(m, 3H), 1.27–1.26 (br, 16H), 1.19 (s, 3H). 13C NMR (MeOD, 100 MHz) d ppm

1310
C. E. ASTETE ET AL.
176.43, 173.70, 173.36, 146.74, 136.25, 124.42, 122.99, 122.06, 118.26, 75.42, 53.99,
52.81, 40.22, 37.11, 36.85, 36.38, 32.99, 31.21, 30.71, 30.61,30.44, 30.31, 29.87,
26.96, 24.58, 24.15, 21.77, 12.80, 12.02, 11.83. IR nmax 3445.78, 2962.90, 2928.89,
2854.65, 1733.63, 1652.73, 1646.86, 1260.60, 1083.82, 844.71 cmꢀ1. MS (ESI-TOF):
calcd. for C36H56N4O6 (M þ H)þ: 641.4278; found 641.4293.
ACKNOWLEDGMENTS
We thank NSF (CBET-0854105) and ACS Petroleum Fund for funding this
work.
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