Arginine thioacid in synthesis of arginine conjugates and peptides

Article abstract of DOI:10.1039/c4ra04897k

Protected arginine thioacid enables convenient N-acylation with no detectable racemization. We report efficient syntheses of potentially biologically active arginine conjugates and novel arginine-containing di-, tri- and tetrapeptides in good yields witho

Full text of DOI:10.1039/c4ra04897k

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Arginine thioacid in synthesis of arginine
conjugates and peptides†
Cite this: RSC Adv., 2014, 4, 55210
Ravil N. Khaybullin, Siva S. Panda,a Saeid Mirzai,a Ellen Toneff,a Abdullah M. Asiri,bc
a
*
C. Dennis Hall and Alan R. Katritzkyab
a
*
Received 23rd May 2014
Accepted 9th October 2014
Protected arginine thioacid enables convenient N-acylation with no detectable racemization. We report
efficient syntheses of potentially biologically active arginine conjugates and novel arginine-containing
di-, tri- and tetrapeptides in good yields without loss of chiral integrity.
DOI: 10.1039/c4ra04897k
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hinder the acylation and as a result arginine is converted to
ornithine.23 One possible way to avoid this problem could be
Introduction
prior transformation of the carboxyl group to thioacid. Indeed
during the last decade close attention has been paid to the
potential of thioacids, and these compounds have been
increasingly applied in the design of complex molecules
particularly for peptide synthesis.24 Danishefsky's group repor-
ted that thioacids can act as highly reactive acyl donors in
peptide ligation reactions25 and methods have been developed
for peptide and amide bond formation using thioacids and
azides26,27 or isonitriles.28,29 Recently, coupling procedures
between thioacids and dithiocarbamate terminal amines has
been investigated.30 In general, the mild conditions and selec-
tivity of thioacid reactions make them attractive intermediates
in bioorganic and peptide chemistry. Synthetic methods using
thioacids usually include pre-activation of the carboxyl group
with carbodiimides,31 CDI,32 or conversion to such reactive
intermediates as p-nitrophenyl esters,33 N-hydroxysuccinimide
esters,34 mixed anhydrides,35 or N-acylbenzotriazoles.36 An
alternative approach using Lawesson's reagent developed by
Danishefsky avoids the activation step but requires harsh
reaction conditions.37 Melnyk's group recently reported the
synthesis of peptide thioacids at neutral pH using bis-
(2-sulfanylethyl)amido peptide precursors in moderate yield.38
Herein, we present a novel approach to arginine-containing
compounds comprising the coupling of protected Nu-nitro-
arginine thioacid to the N-termini of amino acids, peptides and
other N-nucleophiles.
Arginine, an essential amino acid, is one of the most metabol-
ically versatile amino acids and is involved in numerous bio-
logical processes.1 Arginine is also the precursor for the
synthesis of urea, ornithine, nitric oxide (NO), polyamines,
proline, creatine, glutamate and agmatine.2,3 Synthetic arginine-
rich peptides are efficient transporters of diverse biomolecules
including nucleic acids, peptides, and proteins.4–6 Arginine
conjugates are reported to enhance gene expression in tumors7,8
and arginine peptides are known for targeted gene delivery.9,10
Arginine is the source of nitrogen in the biosynthesis of
nitric oxide (NO),2,11,12 which is important in the elimination of
numerous pathogens. The synthesis of NO requires L-arginine
(but not D-arginine) entering the binding site of NO syn-
thase.13,14 Overproduction of NO may lead to diseases like Alz-
heimer's or Parkinson,15,16 and inammatory diseases such as
arthritis17 or colitis.18 The synthesis of NO from L-arginine can
be inhibited by Nu-methyl-L-arginine and Nu-ethyl-L-arginine.19
More selective inhibition was observed with dipeptides
and dipeptide esters containing Nu-nitroarginine and
phenylalanine.20
The highly basic nature of the arginine guanidine moiety can
cause serious problems during chemical transformations. Even
protection of the guanidine moiety before chemical manipula-
tion does not completely prevent side reactions. A major
problem is lactam formation which frequently occurs during
the activation of the arginine carboxyl group.21,22 It has also been
shown that urethanes as guanidino protecting groups do not
Results and discussion
aCenter for Heterocyclic Compounds, Department of Chemistry, University of Florida,
Gainesville, FL 32611-7200, USA. E-mail: ravilx84@gmail.com; charlesdennishall@
gmail.com; Tel: +1-352-392-0554
The well-known intramolecular cyclization during carboxyl
group activation of protected arginines has caused difficulty in
the synthesis of the corresponding C-terminus derivatives. A
recently reported benzotriazole-activated arginine, Cbz-
protected L-uNO2-Arg-Bt (ref. 22) is potentially an attractive
precursor for the synthesis of novel arginine conjugates. For
this purpose, in our initial study we examined benzotriazole-
activated arginine and encountered some limitations. First,
bCenter of Excellence for Advanced Material Research, King Abdulaziz University,
Jeddah, 21589, Saudi Arabia
cDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
† Electronic supplementary information (ESI) available: Copy of 1H NMR, 13C
NMR, HRMS/elemental analysis of all the compounds. See DOI:
10.1039/c4ra04897k
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Table 1 Preparation of Cbz-protected arginine conjugates from Cbz-protected arginine benzotriazolide
N-Nucleophile
Solvent
Yield of product 2 (%)
Yield of by-product 3 (%)
n-Propylamine
THF
THF
THF
Acetonitrile–H2O
85%
83%
Traces
80%
11%
12%
90%
15%
N-(3-Aminopropyl)imidazole
Dipropylamine
Glycine
Scheme 1 Synthesis of Cbz-protected arginine conjugates from Cbz-protected arginine benzotriazolide.
Fig. 1 Fragments of 13C NMR (DMSO-d6) of Cbz-L-Arg(NO2)-SH 4 in DMSO-d6 at rt: A – freshly prepared thioacid, B – after 24 h, C – pure
protected arginine.
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partial decomposition of L-Cbz-uNO2-Arg-Bt was observed in
solution although this compound was stable in the solid state
for at least 6 months at room temperature.22 Moreover, attempts
to couple benzotriazole derivatives with a bulky amine such as
dipropylamine, led to formation of cyclic lactam 3. However,
benzotriazole-activated arginine reacted smoothly with primary
amine or amino acids to form the products shown in Table 1.
The tendency of benzotriazole-activated arginine
1 to
undergo cyclization prompted us to nd a new synthetic
derivative of arginine which could be easily obtained and
handled during synthetic manipulations (Scheme 1).
The ability to efficiently convert Cbz-L-uNO2-Arg-Bt to the
corresponding thioacid with retention of chirality36 without any
side reactions, inspired use of the arginine thioacid as a new
precursor for the synthesis of arginine C-terminus derivatives.
The thioacid of protected L-arginine, Cbz-L-uNO2-Arg-SH 4, is
a stable white crystalline compound readily soluble in EtOAc,
MeOH, THF and CH3CN. However, arginine thioacid 4 hydro-
lysed to corresponding carboxylic acid in wet-DMSO-d6 at room
temperature (Fig. 1).
Scheme 2 Synthesis of Cbz-protected arginine conjugates from Cbz-
protected arginine thioacid 4.
Table 3 Preparation of Cbz-protected arginine peptides
Product
Amino acid/peptide
Yield (%)
Mp (ꢀC)
[a]2D0a
5a
5b
5c
5d
5e
5f
D-Ala-OMe
L-Ala-OMe
DL-Ala-OMe
L-Val-OMe
L-Ala-L-Phe-OMe
Gly-L-Ala-L-Phe-OMe
55
58
56
68
71
56
149–151
158–160
140–142
138–139
152–154
103–105
+15.8
ꢁ18.3
Racemic
ꢁ25.0
ꢁ44.3
ꢁ35.2
Preparation of arginine conjugates
Recently several strategies of amide bond formation have been
developed using simple thioacid and amine as starting mate-
rials. Thus S-nitrosation of thioacids results in very reactive
S-nitrosothioacid intermediates which react with a wide range
of amines.39 Another very mild and fast method of converting
thioacids to the corresponding amides includes simple treat-
ment of a thioacid with amine in presence of Cu(II) salts.40,41
These carboxylic acid derivatives did not show any reactivity
towards hydroxyl groups.
Both methods were tried, but in our hands S-nitrosation is
more efficient (Table 2). The optimized standard conditions39
were found to be slow addition of isoamyl nitrite (2.0 equiv.) to a
solution of arginine thioacid, Cbz-L-uNO2-Arg-SH (1.0 equiv.),
and amine (1.2 equiv.) in THF at 0 ꢀC and in the dark. Although,
formation of the arginine amide products was observed
immediately, consumption of starting arginine thioacid
required 0.5–2 h depending on the amine substrate. Thus, bulky
dipropylamine reacts slowly under these conditions. In this
approach formation of the cyclic side product 3 was not
observed, and the desired arginine conjugates were easily
obtained in pure form by precipitation from ether (Scheme 2)
(Table 3).
a
c ¼ 1.0, MeOH.
Preparation of arginine di- and tri-peptides
As a further application of arginine thioacid 4 in peptide
synthesis, different esters of amino acids, dipeptides and tri-
peptides were used in the above S-nitrosation reaction resulting
in the synthesis of arginine di-, tri- and tetrapeptides in good
yields without loss of chiral integrity (Scheme 3). Arginine
containing peptides are known for their better penetration
ability and bioactivity.
Table 2 Preparation of Cbz-protected arginine conjugates from Cbz-
protected arginine thioacid
Conjugates, 2
N-Nucleophile
Yield (%)
2a
2b
2c
2d
2e
n-Propylamine
80
78
45
66
56
N-(3-Aminopropyl)imidazole
Dipropylamine
Benzylamine
Piperidine
Scheme 3 Synthesis of Cbz-protected arginine peptides.
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Fig. 2 13C NMR (DMSO-d6) of compounds 5a–c.
Fig. 3 HPLC spectra of compounds 5a–c (chiral column, 50% ACN/H2O, 1.0 mL minꢁ1).
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To prove the retention of chirality in our described method N HCl and brine. The organic layer was dried with anhydrous
we coupled the protected arginine thioacid 4 with D-Ala-OMe, MgSO4 and solvent was removed under reduced pressure.
L-Ala-OMe and dl-Ala-OMe. Compound 5c displayed duplicate Treatment of the resulting crude product with ether followed by
peaks in the NMR spectra. However, duplication of peaks in ltration and washing 2–3 times with ether afforded the desired
cases of 5a and 5b was not observed (Fig. 2). The chiral integrity product in analytical purity.
of other synthesized products was supported by NMR data. For
Benzyl-(S)-(5-(2-nitroguanidino)-1-oxo-1-(propyl amino)
further proof, HPLC analysis of 5a–c using a chiral column pentan-2-yl)carbamate (2a). White solid (316 mg, 80%); mp
showed single peak for 5a and 5b but two peaks in the case of 133–135 ꢀC; 1H NMR (300 MHz, DMSO-d6) d 8.60–7.60 (m, 4H),
5c, this indicates 5c is mixture of diastereoisomers of 5a and 5b 7.28–7.50 (br s, 5H), 5.01 (br s, 2H), 4.01–3.85 (m, 1H), 3.19–3.07
(Fig. 3).
(m, 2H), 2.93–3.05 (m, 2H), 1.69–1.43 (m, 4H), 1.44–1.31 (m,
2H), 0.82 (t, J ¼ 7.4 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) d
171.5, 155.9, 137.1, 128.5, 128.4, 127.8, 127.7, 65.5, 54.5, 39.5,
29.4, 22.3, 11.4. HRMS (ESI) calcd for C17H27N6O5 [M + H]+
Conclusion
In conclusion, Cbz-protected arginine thioacid is a convenient 395.2037, found: 395.2033, calcd for C17H26N6O5Na [M + Na]+
coupling reagent, sufficiently reactive to form amide bonds at 417.1857, found: 417.1865%.
ambient temperature. This reagent offers an efficient method to
Benzyl-(S)-(1-((3-(1H-imidazol-1-yl)propyl)amino)-5-(2-nitro-
prepare N-protected arginine conjugates and arginine peptides guanidino)-1-oxopentan-2-yl)carbamate (2b). White solid (359
in synthetically useful yields without detectable racemization or mg, 78%); mp 157–159 ꢀC; 1H NMR (300 MHz, DMSO-d6) d 8.61–
intramolecular cyclization.
8.20 (br s, 1H), 8.19–7.65 (m, 3H), 7.58 (br s, 1H), 7.42 (d, J ¼ 8.4
Hz, 1H), 7.39–7.24 (m, 5H), 7.13 (br s, 1H), 6.86 (br s, 1H), 5.01
(br s, 2H), 4.00–3.84 (m, 3H), 3.18–3.05 (m, 2H), 3.04–2.93 (m,
2H), 1.86–1.72 (m, 2H), 1.68–1.38 (m, 4H); 13C NMR (75 MHz,
DMSO-d6) d 171.8, 156.0, 137.3, 137.0, 128.4, 128.3, 127.8, 127.7,
Experimental section
General
All solvents were reagent grade. Tetrahydrofuran was distilled 119.3, 65.4, 54.5, 43.4, 35.6, 30.7, 29.1. Anal. calcd for
from sodium/benzophenone under dry nitrogen. Melting points 20H28N8O5: C, 52.17; H, 6.13; N, 24.33. Found: C, 52.25; H,
were determined on a capillary point apparatus equipped with a 6.07; N, 24.12%.
C
digital thermometer. NMR spectra were recorded in DMSO-d6
Benzyl-(S)-(1-(dipropylamino)-5-(2-nitroguanidino)-1-oxope-
on Mercury, Gemini or Varian NMR spectrometers operating at ntan-2-yl)carbamate (2c). White solid (196 mg, 45%); mp 95–
1
1
300 MHz for H and 75 MHz for 13C. Elemental analyses were 97 C; H NMR (300 MHz, DMSO-d6) d 8.66–7.55 (m, 3H), 7.48
performed on a Carlo Erba-EA1108 instrument and mass (d, J ¼ 8.3 Hz, 1H), 7.40–7.26 (m, 5H), 5.01 (br s, 2H), 4.41–4.27
spectrometry was done with electro spray ionization (ESI). HPLC (m, 1H), 3.28–3.18 (m, 2H), 3.16–3.00 (m, 4H), 1.62–1.50 (m,
spectra were recorded on a Shimadzu instrument using Supelco 4H), 1.52–1.32 (m, 4H), 0.84 (t, J ¼ 7.8 Hz, 3H), 0.78 (t, J ¼ 7.8 Hz,
ꢀ
chiral HPLC column.
3H); 13C NMR (75 MHz, DMSO-d6) d 171.6, 159.8, 156.4, 137.5,
(S)-2-(((Benzyloxy)carbonyl)amino)-5-(2-nitro guanidino) 128.8, 128.2, 128.1, 65.8, 50.9, 48.9, 47.3, 29.4, 22.5, 20.9, 11.6,
pentanethioic-S-acid (4). Thio arginine 4 was prepared by 11.4. Anal. calcd for C20H32N6O5: C, 55.03; H, 7.39; N, 19.25.
following our previous protocol36 with a modied workup Found: C, 54.65; H, 7.80; N, 19.12%.
procedure. Aer completion of the reaction, THF was evapo-
Benzyl-(S)-(1-(benzylamino)-5-(2-nitroguanidino)-1-oxopentan-
rated and residue was vigorously stirred with a mixture of 2-yl)carbamate (2d). White solid (292 mg, 66%); mp 133–135 ꢀC;
diethyl ether and 1 N HCl (2 : 1) at 0 C for 2 h. The resulting 1H NMR (300 MHz, DMSO-d6) d 8.56–7.51 (m, 3H), 7.43 (d, J ¼
ꢀ
white solid was ltered, washed with cold diethylether and 8.0 Hz, 2H), 7.36–7.17 (m, 10H), 5.02 (s, 2H), 4.31–4.20 (m, 2H),
dried under high vacuum at room teꢀmperature. White micro- 4.06–3.93 (m, 1H), 3.17–3.05 (m, 2H), 1.73–1.39 (m, 4H); 13C
crystals (344 mg, 93%); mp 153–155 C (lit36 mp 153–155 C); NMR (75 MHz, DMSO-d6) d 171.8, 159.3, 156.0, 139.3, 137.0,
ꢀ
[a]2D0 ꢁ9.0 (c ¼ 0.5, MeOH); 1H NMR (DMSO-d6, 300 MHz) d 8.47 128.4, 128.3, 127.8, 127.7, 127.1, 127.1, 126.8, 126.7, 65.5, 54.5,
(s, 1H), 8.14–7.79 (m, 3H), 7.30–7.42 (m, 5H), 5.04–5.21 (m, 2H), 42.1, 39.5, 29.2. Anal. calcd for C21H26N6O5: C, 57.00; H, 5.92; N,
4.28–4.41 (m, 1H), 3.07–3.22 (m, 2H), 1.51–1.82 (m, 4H); 13C 18.99. Found: C, 56.70; H, 6.30; N, 19.11%.
NMR (DMSO-d6, 75 MHz) d 196.3, 159.4, 156.3, 136.7, 128.5,
Benzyl-(S,E)-(5-(2-nitroguanidino)-1-oxo-1-(piperidin-1-yl)
128.0, 127.8, 66.1, 63.6, 60.9, 32.5, 28.0; anal. calcd for pentan-2-yl)carbamate (2e). White solid (235 mg, 56%); mp 86–
1
ꢀ
C
14H19N5O5S: C, 45.52; H, 5.18; N, 18.96. Found: C, 45.85; H, 88 C; H NMR (300 MHz, DMSO-d6) d 8.70–7.47 (m, 3H), 7.45
5.15; N, 18.62%.
(d, J ¼ 8.3 Hz, 1H), 7.40–7.24 (m, 5H), 5.02 (s, 2H), 4.50–4.35 (m,
General procedure for the synthesis of N-protected arginine 1H), 3.51–3.33 (m, 4H), 3.19–3.07 (m, 2H), 1.65–1.32 (m, 10H);
conjugates. To a stirred solution of arginine thioacid, Cbz- 13C NMR (75 MHz, DMSO-d6) d 169.8, 159.69, 156.3, 137.5,
L-uNO2-Arg-SH (1.0 mmol), and amine (1.2 equiv.) (or 1.2 mmol 128.8, 128.2, 128.1, 65.8, 65.4, 50.6, 46.2, 43.0, 29.1, 26.5, 25.8,
amino acid/peptide hydrochloride and 1.2 mmolꢀTEA) in 10 mL 24.5, 15.6. Anal. calcd for C19H28N6O5: C, 54.27; H, 6.71; N,
THF, isoamyl nitrite (2.0 mmol) was added at 0 C in the dark. 19.99. Found: C, 53.90; H, 7.03; N, 20.32%.
The reaction was stirred for 2 h while slowly warming to rt. The
Methyl-N2-((benzyloxy)carbonyl)-Nu0-nitro-L-arginyl-D-alani-
reaction mixture was then concentrated under reduced pres- nate (5a). White solid (241 mg, 55%); mp 149–151 ꢀC. 1H NMR
sure, diluted with ethyl acetate, and washed with sat. NaHCO3, 1 (300 MHz, DMSO-d6) d 8.56–7.53 (br s, 1H), 8.32 (d, J ¼ 7.2 Hz,
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1H), 7.43–7.25 (m, 5H), 5.03 (s, 2H), 4.27 (q, J ¼ 7.2 Hz, 1H), 48.2, 42.4, 37.0, 29.4, 25.1, 18.7, 15.7. HRMS (ESI+) calcd for
4.11–3.98 (m, 1H), 3.62 (s, 3H), 3.17–3.07 (m, 2H), 1.68–1.41 (m,
4H), 1.26 (d, J ¼ 7.2 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
d 172.8, 171.5, 159.3, 155.8, 137.0, 128.3, 127.7, 109.5, 65.4, 54.0,
51.9, 47.5, 43.7, 29.4, 17.2. Anal. calcd for C18H26N6O7: C, 49.31;
H, 5.98; N, 19.17. Found: C, 49.23; H, 6.37; N, 19.54%.
C
C
29H39N8O9 [M + H]+ 643.2835, found: 643.2830, calcd for
29H38N8O9Na [M + Na]+ 665.2654, found: 665.2658%.
Acknowledgements
Methyl-N2-((benzyloxy)carbonyl)-Nu0-nitro-L-arginyl-L-alani-
nate (5b). White solid (232 mg, 58%); mp 158–160 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d 8.56–7.53 (m, 1H), 8.35 (d, J ¼ 7.2 Hz,
1H), 7.43–7.25 (m, 5H), 5.02 (br s, 2H), 4.26 (q, J ¼ 7.2 Hz, 1H),
4.09–3.94 (m, 1H), 3.60 (s, 3H), 3.15–3.04 (m, 2H), 1.71–1.44
(s, 4H), 1.28 (d, J ¼ 7.2 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
d 173.4, 172.1, 159.1, 156.3, 137.4, 128.8, 128.2, 128.1, 65.8, 54.3,
52.3, 48.0, 29.6, 17.3. Anal. calcd for C18H26N6O7: C, 49.31; H,
5.98; N, 19.17. Found: C, 49.00; H, 6.23; N, 18.81%.
We thank the University of Florida, the Kenan Foundation for
nancial support. The work was also funded in part by support
from King Abdulaziz University under grant no. (D-006/431).
The authors, therefore, acknowledge the technical and nan-
cial support of KAU.
Notes and references
Methyl-N2-((benzyloxy)carbonyl)-Nu0-nitro-L-arginyl-dl-alani-
nate (5c). White solid (246 mg, 56%); mp 140–142 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d 8.12–7.40 (m, 3H), 8.36–8.28 (m, 1H),
7.37–7.25 (m, 5H), 5.07–4.95 (m, 2H), 4.26 (q, J ¼ 7.2 Hz, 1H),
4.10–3.95 (m, 1H), 3.59 (s, 3H), 3.15–3.04 (m, 2H), 1.70–1.44 (s,
4H), 1.31–1.20 (m, 3H); 13C NMR (75 MHz, DMSO-d6) d 173.4,
173.3, 172.1, 172.0, 159.7, 156.3, 137.4, 128.8, 128.2, 128.1,
128.1, 65.9, 65.8, 54.5, 52.3, 52.3, 48.0, 29.6, 25.3, 17.6, 17.3.
Anal. calcd for C18H26N6O7: C, 49.31; H, 5.98; N, 19.17. Found:
C, 49.27; H, 6.16; N, 19.45%.
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Methyl-N2-((benzyloxy)carbonyl)-Nu0-nitro-L-arginyl-L-vali-
nate (5d). White solid (317 mg, 68%); mp 138–139 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d 8.62–7.51 (m, 3H), 8.10 (d, J ¼ 8.1 Hz,
1H), 7.43 (d, J ¼ 7.8 Hz, 1H), 7.40–7.23 (m, 5H), 5.02 (s, 2H),
4.25–4.02 (m, 2H), 3.62 (s, 3H), 3.20–3.04 (m, 2H), 2.12–1.94 (m,
1H), 1.71–1.41 (m, 4H), 0.97–0.75 (m, 6H); 13C NMR (75 MHz,
DMSO-d6) d 172.6, 172.3, 159.7, 156.3, 137.5, 128.8, 128.8, 128.3,
128.2, 128.1, 65.8, 57.8, 54.4, 52.1, 30.4, 29.6, 25.3, 25.2, 19.4,
18.6. Anal. calcd for C20H30N6O7: C, 51.49; H, 6.48; N, 18.02.
Found: C, 51.16; H, 6.79; N, 18.09%.
Methyl-N2-((benzyloxy)carbonyl)-Nu0-nitro-L-arginyl-L-alanyl-
L-phenylalaninate (5e). White solid (416 mg, 71%); mp
152–154 ꢀC. 1H NMR (500 MHz, DMSO-d6) d 8.55–8.37 (br s, 1H), 11 J. B. Hibbs Jr, R. R. Taintor, Z. Vavrin and E. M. Rachlin,
8.31 (d, J ¼ 7.8 Hz, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz,
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