Introduction of histidine units using benzotriazolide activation

Article abstract of DOI:10.1002/psc.2483

N^α-Boc-N^im-(4-toluenesulfonyl-l-histidylbenzotriazole) enables convenient acylation of N-, O-, S-, and C-nucleophiles with no detectable racemization. We report efficient syntheses of novel histidine-containing di-, tri-, and tetra-p

Full text of DOI:10.1002/psc.2483

Research Article
Received: 15 August 2012
Revised: 18 December 2012
Accepted: 19 December 2012
Published online in Wiley Online Library: 16 January 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2483
Introduction of histidine units using
benzotriazolide activation
Kiran Bajaj,a Siva S. Panda,a Mohamed A. Ibrahim,a,b,c Said A. El-Fekyb
and Alan R. Katritzkya,d*
Na-Boc-Nim-(4-toluenesulfonyl-L-histidylbenzotriazole) enables convenient acylation of N-, O-, S-, and C-nucleophiles with no
detectable racemization. We report efficient syntheses of novel histidine-containing di-, tri-, and tetra-peptides and models
for the preparation of potentially biologically active histidine N-, O-, S-, and C-conjugates. Copyright © 2013 European Peptide
Society and John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: histidine; conjugates; benzotriazole; acylation
ring and an amino group has complicated the development of
synthetic methods to prepare histidine C-terminus derivatives.
Introduction
Histidine is of prime importance as an essential component of
the active site of many enzymes [1], which is widely distributed
in nature, and its derivatives exhibit diverse biological activities
[2,3]: for example, naphthalene-substituted histidine derivatives
act as useful antihypertensive agents [4], a histidine-estrone
conjugate has pronounced anti-tumor activity [5], and histidine-
tocopherol conjugates are antioxidants and prodrug delivery
vehicles [6]. Carnosine (b-alanyl-L-histidine) and carcinine
(b-alanyl-histamine) occur in the non-protein fraction of mamma-
lian tissues [7]. In proteins, histidine residues play an important
role in interactions with metal ions because of the presence of
three coordinating groups. Histidine coordinates strongly and
forms six membered rings in which a metal is attached to an
imidazole nitrogen atom and the a-amino group [8]. For
example, the N-terminus of the protein bovine serum albumin
(Asp-Thr-His-Lys-OH) specifically binds the Cu(II) ion because
of the histidine residue [9]. Histidine-containing peptides act
as catalysts in Michael addition of nitroalkanes [10], direct
asymmetric aldol reactions [11], and asymmetric synthesis of
cyanohydrins [12].
Histidine residues are often involved in neutral hydrolytic
metalloenzyme active centers and have the potential to bind
DNA. The unprotonated imidazole (pH > 7) is nucleophilic,
whereas the protonated form (pH < 7) serves as a proton donor.
Histidine is often susceptible to racemization during solid-phase
synthesis (2.1% racemization detected for the L-His-containing
peptide and 1.9% detected for the D-His-containing peptide) [13].
Ishiguro et al. reported unexpected chain-terminating side reac-
tions caused by histidine in solid-phase peptide synthesis [14].
Racemization during the synthesis of polypeptides could be a
serious problem as a high degree of steric homogeneity is usually
required for biological or physical studies. The detection of small
amounts of diastereoisomeric impurities and their removal from
synthetic polypeptide preparations are often difficult; thus,
synthetic procedures that minimize the risk of racemization are
commonly used. In a histidine unit, the presence of the imidazole
The nucleophilic imidazole ring of histidine easily undergoes
N-acylation in absence of appropriate protecting groups, which
can result in side reactions such as acyl transfer. In particular, histi-
dine derivatives are known to be prone to racemization during
activating and coupling processes involving the p-nitrogen of the
imidazole ring [15]. Thus, several protecting groups as well as
activating reagents have been introduced in attempts to solve
problems such as the regioselective protection of the p-nitrogen,
and this enables avoidance of racemization. For this purpose, the
Np-benzyloxymethyl (Bom) group has been widely employed in
Boc chemistry [16], and the Nt-Trt protecting group [17], used in
combination with Na-Fmoc protection [17,18]. Although the steric
hindrance of the Trt group may help reduce racemization to a
certain extent, Nt-protecting groups cannot inherently prevent
racemization. Other Np-protecting groups including Np-t-butoxy-
methylhistidine [His(Bum)] [19], Np-1-adamantyloxymethylhistidine
[His(1-Adom)] [20], and 4-methoxybenzyloxymethyl (MBom) [21,22]
have been introduced. The use of several activating agents has
been reported in literature including (i) solution phase synthesis
of a histidine-containing dipeptide derivative from Z-Phe-OH and
H-His-OMe using isobutyl chloroformate [12], (ii) preparation of
the histidine dipeptide (Boc-His(Tos)-Leu-OH) from Boc-His(Tos)-
*
Correspondence to: Alan R. Katritzky, Center for Heterocyclic Compounds,
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
E-mail: katritzky@chem.ufl.edu
a Center for Heterocyclic Compounds, Department of Chemistry, University of
Florida, Gainesville, FL 32611-7200, USA
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Zagazig University, Zagazig 44519, Egypt
c
Department of Organic Chemistry, College of Pharmacy, Misr University for
Science and Technology, Al-Motamayez District, P.O. Box 77, Egypt
d Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia
J. Pept. Sci. 2013; 19: 110–117
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

HISTIDINE PEPTIDES AND CONJUGATES
OSu and H-Leu-OH [23], (iii) dipeptide (Boc-Ala-His(Tos)-OMe) from
protected histidine and alanine in the presence of EDCI and Et3N
[24], and (iv) dipeptide (Boc-His(MBS)-Gly-OBzl) utilizing p-TsOH,
and diethyl phosphorocyanidate as a coupling agent [25]. Litera-
ture routes to C-terminus histidine conjugates include the use of
the coupling reagents (i) DCC with DMAP [26,27], (ii) EDCI-HOBt
[28], and (iii) isobutyl chloroformate [29]. These methods showed
good yields, but products needed column purification. Additionally,
some reports indicate that histidine derivatives racemize on activa-
tion with DCC [30].
irradiation power. Completion of the reaction was monitored by
TLC. After completion, the reaction mixture was quenched with
water. The precipitate obtained was washed with saturated solu-
tion of Na2CO3 to afford the desired products, which were further
crystallized from methylene chloride/hexane.
(S)-tert-Butyl-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-(1-tosyl-
1H-imidazol-4-yl)propan-2-yl)carbamate (Boc-His(Tos)-Bt, 3a)
White microcrystals (65%); mp 150–152 ꢀC; 1H NMR (CDCl3) d 8.18
(d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H),
7.75 (d, J = 8.4 Hz, 2H), 7.66 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz,
1H), 7.34 (d, J = 8.4 Hz, 2H), 7.01 (s, 1H), 6.12–6.06 (m, 1H),
5.96–5.84 (m, 1H), 3.40–3.22 (m, 2H), 2.44 (s, 3H), 1.44 (s, 9H);
13C NMR (CDCl3) d 170.7, 155.6, 146.5, 146.1, 139.6, 136.8, 134.9,
131.3, 130.8, 130.6, 127.5, 126.6, 120.5, 115.1, 114.6, 80.6, 54.3,
30.8, 28.5, 22.0. HRMS (+ESI-TOF): m/z for C24H26N6O5S [M + H]+
calcd 511.1758, found 511.1749.
We have extensively applied N-acylbenzotriazoles for N-acyla-
tion, C-acylation, O-acylation, and S-acylation [31]. In these reac-
tions, N-(Z-aminoacyl)benzotriazoles coupled with unprotected
amino acids to produce peptides in 85–95% yield with minimal
racemization [32]. N-(Z-a-aminoacyl)benzotriazoles prepared from
N-protected a-amino acids with unfunctionalized side chains such
as Ala, Val, and Phe coupled successfully with unprotected amino
acids (Ala, Val, Phe, Ser, Trp) in the presence of Et3N in partially
aqueous solution (CH3CN/H2O). We have also prepared N-(Z- or
Fmoc-a-aminoacyl)benzotriazoles derived from Tyr, Trp, Cys, Met,
and Gln containing unprotected side-chain functionality [33], and
successfully coupled them with a-amino acids (L-Ala, L-Phe) in
partially aqueous medium to form the expected peptides. NMR
and HPLC analysis supported the preservation of the original
chirality. These findings were further confirmed by NMR and
HPLC comparisons of the diastereomeric dipeptides prepared by
coupling N-(Z-Tyr, Trp, Cys, Met, Gln and Fmoc-Trp, Met acyl)benzo-
triazole with H-DL-Ala-OH, H-DL-Phe-OH [33]. We have also
reported successful benzotriazole activation in racemization free
tri- and tetra-peptides [31]. Herein, we report another method to
introduce histidine residues at the N-terminus in solution phase
peptide synthesis by utilization of benzotriazole-activated histidine
intermediate 3a and 3b (Scheme 1). Compound 3a was also used
to synthesize novel histidine conjugates by N-, O-, S-, and C-acyla-
tions. There is a previous reference [23] for the preparation of 5c
(Scheme 2), but no experimental details are given.
(S)-tert-Butyl-1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(1-benzyl-1H-
imidazol-4-yl)-1-oxopropan-2-ylcarbamate (Boc-His(Bzl)-Bt, 3b)
1
White microcrystals (62%); mp 85–87 ꢀC; H NMR (CDCl3) d 8.16
(d, J = 8.2 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H),
7.50–7.45 (m, 2H), 7.32–7.26 (m, 3H), 7.05–7.03 (m, 2H), 6.63
(d, J = 7.7 Hz, 1H), 6.55 (s, 1H), 5.94–5.87 (m, 1H), 4.98 (s, 2H),
3.48–3.27 (m, 2H), 1.44 (s, 9H); 13C NMR (CDCl3) d 170.9, 155.6,
145.7, 137.4, 137.0, 135.9, 131.1, 130.4, 128.8, 128.1, 127.1, 126.2,
120.0, 117.2, 114.3, 79.9, 54.8, 50.7, 30.4, 28.3.
General Procedure for the Preparation of
Histidine-containing di-, tri-, and tetra-peptides
(5a–f)
To a solution of compound 3 (1.0 eq.) in acetonitrile, a solution
of amino acid, dipeptide or tripeptide (1.0 eq.), and Et3N
(1.2 eq.) in water was added dropwise, and the reaction mixture
was stirred at room temperature for 3–4 h. After complete
consumption of compound 3 (TLC), the solution was concen-
trated under reduced pressure. The solution was neutralized
by 4N HCl and extracted with ethyl acetate, dried over anhy-
drous MgSO4, and evaporated under reduced pressure to
afford a white solid, which was either crystallized from diethyl
ether or purified by flash column chromatography with 30%
ethyl acetate–hexane solution as eluent.
Materials and Methods
Melting points were determined on a capillary point apparatus
equipped with a digital thermometer. NMR spectra were
recorded in CDCl3, DMSO-d6, or CD3OD-d4 on Mercury, Gemini,
or Varian NMR spectrometers operating at 300 MHz for 1H
(with TMS as an internal standard) and 75 MHz for 13C NMR.
Elemental analyses were performed on a Carlo Erba-EA1108
instrument. All microwave-assisted reactions were carried out
with a single mode cavity Discover Microwave Synthesizer (CEM
Corporation, NC, USA). The reaction mixtures were transferred
into a 10 mL glass pressure microwave tube equipped with a
magnetic stirrer bar. The tube was closed with a silicon septum,
and the reaction mixture was subjected to microwave irradiation
(Discover mode; run time: 60 s; PowerMax-cooling mode).
(S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-(1-tosyl-1H-imidazol-
4-yl)propanamido)-3-phenylpropanoic acid (Boc-L-His(Tos)-
L-Phe-OH, 5a)
White microcrystals (79%); mp 133–134 ꢀC; 1H NMR (CDCl3) d 7.98
(s, 1H), 7.81 (d, J = 6.9 Hz, 2H), 7.38–7.29 (m, 3H), 7.26–7.18 (m, 5H),
7.09 (s, 1H), 5.39 (d, J = 8.7 Hz, 1H), 4.76–4.68 (m, 1H), 4.45
(t, J = 9.0 Hz, 1H), 3.20–2.92 (m, 3H), 2.53–2.41 (m, 1H), 2.42
(s, 3H), 1.35 (s, 9H); 13C NMR (CDCl3) d 174.3, 170.9, 155.2, 146.9,
139.9, 136.5, 134.6, 130.7, 129.9, 128.5, 127.7, 127.1, 115.6, 79.6,
54.2, 53.7, 38.7, 32.8, 28.4, 22.0; Anal. calcd for C27H32N4O7S: C,
58.26; H, 5.79; N, 10.07; found: C, 58.15; H, 6.13; N, 10.09.
Preparation of Na-Boc-Nim-protected-L-histi-
dylbenzotriazole (3a,b)
Compounds 3a,b were synthesized by irradiating an equimolar
amount of Na-Boc-Nim-protected-L-histidine with 1-(methylsulfo-
nyl)-1H-benzo[d][1,2,3]triazole (BtSO2Me) in the presence of
1.0 eq. of Et3N in anhydrous DMF for 30 min at 50 ꢀC and 20 W
J. Pept. Sci. 2013; 19: 110–117 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BAJAJ ET AL.
2-((S)-2-((tert-Butoxycarbonyl)amino)-3-(1-tosyl-1H-imidazol-
4-yl)propanamido)-3-phenylpropanoic acid (Boc-His(Tos)-DL-
Phe-OH, 5a+5a0)
(6S,12S)-12-Isopropyl-2,2-dimethyl-4,7,10-trioxo-6-((1-tosyl-
1H-imidazol-4-yl)methyl)-3-oxa-5,8,11-triazatridecan-13-oic
acid (Boc-L-His(Tos)-Gly-L-Val-OH, 5e)
White microcrystals (72%); mp 175–177 ꢀC; 1H NMR (CDCl3) d
8.97–8.96 (d, J = 4.4 Hz, 1H), 8.16–8.07 (m, 1H), 7.50–7.47 (m, 2H),
7.27–6.98 (m, 8H), 4.51–4.43 (m, 1H), 4.32–4.25 (m, 1H), 3.19–
2.64 (m, 4H), 2.29 (s, 3H), 1.32 (s, 9H).13C NMR (CDCl3) d 172.6,
170.3, 170.1, 155.1, 145.5, 137.7, 137.3, 133.7, 129.5, 129.5, 129.2,
128.2, 128.1, 126.5, 125.5, 116.8, 116.6, 78.5, 53.3, 53.0, 36.8,
36.6, 28.1, 27.1, 20.8; Anal. calcd for C27H32N4O7S: C, 58.36; H,
5.79; N, 10.07; found: C, 57.87; H, 6.02; N, 9.83.
White microcrystals (68%); mp 113–115 ꢀC; 1H NMR (CDCl3) d 8.02–
8.00 (m, 1H), 7.87–7.74 (m, 3H), 7.45–7.14 (m, 5H), 5.95–5.87 (m, 1H),
4.49–4.39 (m, 1H), 4.11–3.78 (m, 2H), 3.08–2.93 (m, 2H), 2.42 (s, 3H),
2.42–2.13 (m, 1H), 1.33 (s, 9H), 0.94–0.90 (m, 6H); 13C NMR (CDCl3) d
174.7, 172.6, 169.8, 155.8, 146.8, 139.9, 136.8, 134.6, 130.7, 127.7,
126.0, 115.4, 80.3, 58.3, 58.1, 54.2, 43.4, 30.7, 28.4, 21.9, 19.4, 18.1.
Anal. calcd for C25H35N5O8S: C, 53.08; H, 6.24; N, 12.38; found: C,
52.68; H, 6.43; N, 12.32.
(6S,15S)-15-((S)-sec-Butyl)-2,2-dimethyl-4,7,10,13-tetraoxo-6-
((1-tosyl-1H-imidazol-4-yl)methyl)-3-oxa-5,8,11,14-tetraaza-
hexadecan-16-oic acid (Boc-L-His(Tos)-Gly-Gly-L-Ile-OH, 5f)
(S)-2-((S)-3-(1-Benzyl-1H-imidazol-4-yl)-2-(tert-butoxycarbony-
lamino)propanamido)-3-methylbutanoic acid (Boc-His(Tos)-
L-Val-OH, 5b)
1
White microcrystals (60%); mp 140–142 ꢀC; H NMR (DMSO-d6) d
Oil (72%); 1H NMR (CDCl3) d 8.04 (s, 1H), 7.82 (d, J =8.1 Hz, 2H), 7.35
(d, J = 8.1Hz, 1H), 7.11 (s, 1H), 5.52 (d, J = 8.7 Hz, 1H), 4.58–4.49
(m, 2H), 3.13 (d, J = 14.1 Hz, 1H), 2.61–2.50 (m, 1H), 2.43 (s, 3H),
2.25–2.19 (m, 1H), 1.31 (s, 9H), 0.98–0.92 (m, 6H). 13C NMR (CDCl3)
d 174.4, 171.5, 155.4, 146.9, 139.9, 136.6, 134.6, 130.8, 130.7, 127.8,
115.6, 79.7, 57.8, 53.9, 32.4, 31.8, 28.4, 22.0, 18.8, 18.1. HRMS (+ESI-
TOF): m/z for C23H32N4O5 [M+ H]+ calcd 507.1919, found 507.1926.
12.62 (s, 1H), 8.28 (s, 1H), 8.22–8.10 (m, 2H), 7.92 (d, J = 8.1 Hz,
2H), 7.49 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H),
4.23–4.16 (m, 2H), 3.79 (t, J = 5.7 Hz, 2H), 3.66 (d, J = 5.4 Hz, 2H),
2.92–2.80 (m, 1H), 2.75–2.65 (m, 1H), 2.40 (s, 3H), 1.82–1.70 (m,
1H), 1.30 (s, 9H), 1.24–1.11 (m, 2H), 0.85–0.78 (m, 6H); 13C NMR
(DMSO-d6) d 172.8, 171.5, 168.9, 168.6, 155.1, 146.3, 140.7, 136.7,
134.4, 130.6, 127.2, 114.7, 78.3, 56.2, 53.5, 42.2, 41.6, 36.4, 30.3,
28.1, 24.6, 21.2, 15.5, 11.3. Anal. calcd for C28H40N6O9S: C, 52.82;
H, 6.33; N, 13.20; found: C, 52.88; H, 6.70; N, 13.09.
2-((S)-3-(1-Benzyl-1H-imidazol-4-yl)-2-(tert-butoxycarbonyla-
mino)propanamido)-3-methylbutanoic acid (Boc-His(Tos)-DL-
Val-OH, 5b+5b0)
1
Procedure for Preparation of Compound 7
Oil (70%); H NMR (CDCl3) d 8.03 (s, 1H), 7.83 (d, J = 8.1 Hz, 2H),
7.36 (d, J = 8.1 Hz, 2H), 7.21–7.14 (m, 1H), 5.86–5.84 (m, 1H),
5.65–5.62 (m, 1H), 4.68–4.50 (m, 2H), 3.18–3.05 (m, 1H), 2.74–
2.64 (m, 1H), 2.43 (s, 3H), 2.28–2.19 (m, 1H), 1.31 (d, J = 12.0 Hz,
9H), 0.96–0.90 (m, 6H). 13C NMR (CDCl3) d 174.6, 174.3, 171.6,
171.4, 155.6, 155.4, 146.8, 139.9, 136.5, 134.6, 130.7, 127.7, 115.6,
79.8, 79.7, 66.0, 57.7, 57.3, 53.9, 31.7, 31.6, 28.4, 21.9, 18.9, 18.8,
18.0, 17.9, 15.4. HRMS (+ESI-TOF): m/z for C23H32N4O5 [M + H]+
calcd 507.1919, found 507.1932.
To a solution of compound 3b (1.0 eq.) in acetonitrile, a solution
of D-H-Phe-OMe (1.0 eq.) and Et3N (1.2 eq.) in water was added
dropwise, and the reaction mixture was stirred at room tempera-
ture for 3 h. After complete consumption of compound 3b (TLC),
the solution was concentrated under reduced pressure. The
residue was extracted with ethyl acetate, dried over anhydrous
MgSO4, and evaporated under reduced pressure to afford the
desired product as an oil.
(S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-(1-tosyl-1H-imida-
zol-4-yl)propanamido)-4-methylpentanoic acid (Boc-L-His
(Tos)-L-Leu-OH, 5c)
Methyl 2-((S)-3-(1-benzyl-1H-imidazol-4-yl)-2-(tert-butoxy-
carbonylamino)propanamido)-3-phenylpropanoate (Boc-His
(Bzl)-Phe-OMe, 7)
White microcrystals (89%); mp 121–123 ꢀC [lit. mp 146–148 ꢀC]16;
1H NMR (CDCl3) d 8.02 (s, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.75
(d, J = 7.5 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.15 (s, 1H), 5.60
(d, J = 9.0 Hz, 1H), 4.64–4.52 (m, 2H), 3.14–3.05 (m, 1H), 2.68–2.56
(m, 1H), 2.43 (s, 3H), 1.78–1.56 (m, 3H), 1.32–1.22 (m, 9H), 0.96
(d, J = 6.3 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H); 13C NMR (CDCl3) d
175.6, 171.4, 155.4, 146.8, 139.9, 136.5, 134.6, 130.7, 127.7, 115.7,
79.7, 53.7, 51.5, 42.6, 32.3, 31.8, 28.3, 25.0, 22.8, 22.6, 21.9, 14.3.
Oil (69%); 1H NMR (CDCl3) d 7.42–7.38 (m, 1H), 7.34–7.16 (m, 7H),
7.13–7.06 (m, 3H), 6.96–6.94 (m, 1H), 6.71–6.63 (m, 1H), 6.39–6.27
(m, 1H), 4.97 (s, 2H), 4.79–4.75 (m, 1H), 3.69 (s, 1H), 3.63 (s, 1H),
3.59 (s, 1H), 3.06–2.84 (m, 4H), 1.40 (d, J = 4.0 Hz, 9H). 13C NMR
(CDCl3) d 175.4, 171.6, 171.5, 171.4, 155.6, 138.5, 138.2, 136.7,
136.0, 135.8, 129.2, 128.9, 128.5, 128.4, 128.3, 128.2, 127.4, 127.3,
126.9, 126.8. HRMS (+ESI-TOF): m/z for C28H34N4O5 [M + H]+ calcd
507.2602, found 507.2604.
2-((S)-2-(tert-Butoxycarbonylamino)-3-(1-tosyl-1H-imidazol-4-
yl)propanamido)propanoic acid (Boc-L-His(Tos)-L-Ala-OH, 5d)
Procedure for Preparation of Compound 8a
White microcrystals (92%); mp 151–153 ꢀC; 1H NMR (CDCl3) d 8.02
(s, 1H), 7.84–7.82 (d, J = 8.1 Hz, 2H), 7.38–7.35 (d, J = 8.1 Hz, 2H),
7.12 (s, 1H), 5.66–5.63 (d, J = 8.2 Hz, 1H), 5.46–5.44 (d, J = 7.9 Hz,
1H), 4.63–4.50 (m, 1H), 3.19–3.10 (m, 1H), 2.70–2.48 (m, 2H), 2.44
(s, 3H), 1.51–1.27 (m, 12H); 13C NMR (CDCl3) d 157.9, 171.3,
155.3, 146.9, 139.8, 136.5, 134.5, 130.8, 127.8, 115.7, 79.7, 53.6,
49.2, 48.8, 32.5, 28.3, 22.0, 18.9. HRMS (+ESI-TOF): m/z for
C21H28N4O7S [M + Na]+ calcd 503.1571, found 503.1571.
N-(3-Aminopropyl)-imidazole (1.0eq.) was dissolved in THF and
Et3N (1.5eq.). Na-Boc-Nim-4-toluenesylfonyl-histidylbenzotriazole
3a (1.0 eq.) was added to the solution, and the mixture was stirred at
room temperature for 3 h. The mixture was acidified with 4N HCl, con-
centrated and then diluted with ethyl acetate. The organic layer was
washed with 10% Na2CO3 solution, water and dried over anhydrous
MgSO4, filtered, and evaporated to give a white solid, which was
further washed with diethyl ether to give the desired compound.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 110–117

HISTIDINE PEPTIDES AND CONJUGATES
(S)-tert-Butyl-(1-((3-(1H-imidazol-1-yl)propyl)amino)-1-oxo-3-
(1-tosyl-1H-imidazol-4-yl) propan-2-yl)carbamate (8a)
(S)-Naphthalen-2-yl 2-(tert-butoxycarbonylamino)-3-(1-tosyl-
1H-imidazol-4-yl)propanoate (8d)
1
White microcrystals (92%); mp 64–66 ꢀC; H NMR (CDCl3) d 7.93
Oil (55%). 1H NMR (CDCl3) d 8.00 ( d, J = 1.2 Hz, 1H), 7.84–7.76
(m, 5H), 7.51–7.46 (m, 3H), 7.27 (d, J = 7.8 Hz, 2H), 7.18 (s, 1H),
7.12 (dd, J = 8.9, 2.3 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 4.86–4.83
(m, 1H), 3.33–3.12 (m, 2H), 2.37 (s, 3H), 1.45 (s, 9H); 13C NMR
(CDCl3) d 170.6, 155.6, 148.3, 146.5, 140.2, 136.9, 135.0, 133.8,
131.7, 130.6, 129.6, 127.9, 127.5, 126.8, 126.0, 121.0, 118.6, 115.2,
80.3, 53.4, 30.5, 28.5, 21.9. HRMS (+ESI-TOF) m/z for C28H29N3O6S
[M + H]+ calcd 536.1850, found 536.1850.
(s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H), 7.35 (d, J = 8.1 Hz, 2H),
7.10 (s, 1H), 7.04 (s, 1H), 6.91 (br s, 2H), 5.93 (d, J = 7.2 Hz, 1H),
4.40–4.33 (m, 1H), 3.87 (t, J = 6.9 Hz, 2H), 3.14 (q, J = 6.3 Hz, 2H),
3.05 (dd, J = 14.7, 5.4 Hz, 1H), 2.90 (dd, J = 14.7, 5.4 Hz, 1H), 2.43
(s, 3H), 1.92–1.84 (m, 2H), 1.42 (s, 9H); 13C NMR (CDCl3) d 171.7,
155.9, 146.7, 140.7, 137.3, 136.3, 134.8, 130.6, 129.7, 127.6,
119.0, 115.1, 80.5, 54.4, 44.2, 36.4, 31.2, 30.3, 28.5, 21.9; HRMS
(+ESI-TOF): m/z for C24H33N6O5S [M + H]+ calcd 517.2228, found
517.2229.
General Procedure for Preparation of Com-
pound 8e,f
Procedure for Preparation of Compound 8b
The mercapto nucleophile (1.0 eq.) was dissolved in THF and Et3N
(1.5 eq.). Na-Boc-Nim-4-toluenesylfonyl-L-histidinylbenzotriazle 3a
(1.0 eq.) was added to the solution, and the mixture was stirred
at room temperature for 2 h. The mixture was concentrated
under reduced pressure, then diluted with ethyl acetate. The
organic layer was washed with 10% Na2CO3 solution, water and
dried over anhydrous MgSO4, filtered, and evaporated to give
the desired compound.
A dried heavy-walled Pyrex tube containing a small stir bar was
mixed with compound 3a (0.3 g, 0.587 mmol) and 2-aminopyri-
dine (0.056 g, 0.587 mmol) in dry DMF (5 mL) for 30 min at 50 ꢀC
and 20 W irradiation power. After completion (TLC), the reaction
mixture was quenched with water. The precipitate obtained
was washed with saturated Na2CO3 solution and water to afford
a solid, which was flash chromatograhed with 30% ethyl ace-
tate/hexane solution to give compound 8b.
(S)-S-Benzyl 2-((tert-butoxycarbonyl)amino)-3-(1-tosyl-1H-
imidazol-4-yl)propanethioate (8e)
(S)-tert-Butyl-(1-oxo-1-(pyridin-2-ylamino)-3-(1-tosyl-1H-imi-
dazol-4-yl)propan-2-yl)carbamate (8b)
1
White microcrystals (80%); mp 60–61 ꢀC; H NMR (CDCl3) d 7.91
1
White microcrystals (91%); mp 87–88 ꢀC; H NMR (CDCl3) d 9.00
(s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.28–7.13
(m, 5H), 7.05 (s, 1H), 6.10 (d, J = 8.7 Hz, 1H), 4.70–4.57 (m, 1H),
3.97 (s, 2H), 3.09 (dd, J = 15.0, 6.0 Hz, 1H), 2.95 (dd, J = 13.2,
5.1 Hz, 1H), 2.40 (s, 3H), 1.42 (s, 9H); 13C NMR (CDCl3) d 201.0,
155.3, 146.4, 139.9, 136.8, 136.5, 134.8, 130.5, 128.8, 128.6, 127.4,
127.3, 115.0, 80.3, 59.7, 33.3, 30.0, 28.4, 21.8; Anal. calcd for
C25H29N3O5S2: C, 58.23; H, 5.67; N, 8.15; found: C, 58.11; H, 6.00;
N, 8.54.
(br s, 1H), 8.24 (d, J = 4.5 Hz, 1H), 8.14 (d, J = 8.4Hz, 1H), 7.94
(s, 1H), 7.73 (d, J= 8.1Hz, 2H), 7.67 (t, J = 8.0Hz, 1H), 7.25
(d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 7.08–7.00 (m, 1H), 6.18–6.04 (m, 1H),
4.62–4.54 (m, 1H), 3.22–3.12 (m, 1H), 2.99 (dd, J = 15.0, 5.1 Hz, 1H),
2.39 (s, 3H), 1.44 (s, 9H); 13C NMR (CDCl3) d 170.3, 155.4, 151.1,
147.9, 146.4, 140.4, 138.5, 136.6, 134.9, 130.5, 127.4, 120.0, 115.1,
114.2, 80.7, 55.1, 30.2, 28.4, 21.9; HRMS (+ESI-TOF): m/z for
C23H28N5O5S for [M+ H]+ calcd 486.1806, found 486.1796.
(S)–Methyl 2-((2-((tert-butoxycarbonyl)amino)-3-(1-tosyl-1H-
imidazol-4-yl)propanoyl) thio)acetate (8f)
General Procedure for Preparation of Compound
8c,d
White microcrystals (78%); mp 110–111 ꢀC; 1H NMR (CDCl3) d 7.94
(d, J = 1.2 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.36 (dd, J = 8.1, 0.6 Hz,
2H), 7.10 (s, 1H), 6.10 (d, J = 8.7 Hz, 1H), 4.68–4.58 (m, 1H), 3.70
(s, 3H), 3.58 (d, J = 10.5 Hz, A part of AB system, 1H), 3.53
(d, J = 10.5 Hz, B part of AB system, 1H), 3.09 (dd, J = 14.7, 6.2 Hz,
1H), 2.97 (dd, J = 14.7, 4.5 Hz, 1H), 2.44 (s, 3H), 1.44 (s, 9H); 13C
NMR (CDCl3) d 200.3, 169.1, 155.4, 155.3, 146.5, 139.6, 136.5,
134.9, 130.5, 127.5, 115.3, 80.5, 59.7, 52.8, 31.2, 29.9, 28.4, 21.8;
Anal. calcd for C21H27N3O7S2: C, 50.69; H, 5.47; N, 8.44; found: C,
51.04; H, 5.89; N, 8.08.
The O-nuceophile (0.783 mmol) was added to a solution of
compound 3a and DMAP (0.05 g, 0.392 mmol) in dry THF (2 mL).
The mixture was irradiated in microwave at 60 ꢀC for 60 min
and 100 W. The solvent was evaporated, and the residue was
purified over flash chromatography to give 8c or 8d.
(S)-4-Ethylphenyl 2-(tert-butoxycarbonylamino)-3-(1-tosyl-1H-
imidazol-4-yl)propanoate (8c)
Oil (50%), 1H NMR (CDCl3) d 7.96 (d, J = 1.3 Hz, 1H), 7.79
(d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 3H),
6.87 (d, J = 8.4 Hz, 2H), 5.77 (d, J = 8.1 Hz, 1H), 4.77 (br s, 1H),
3.26–3.07 (m, 2H), 2.62 (q, J = 7.6 Hz, 2H), 2.42 (s, 3H), 1.43
(s, 9H), 1.22 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3) d 170.6, 155.6,
148.6, 146.4, 142.0, 140.2, 136.8, 135.0, 130.6, 128.8,127.5, 121.2,
115.0, 80.1, 53.2, 30.5, 30.4, 28.4, 21.9, 15.7. HRMS (+ESI-TOF)
m/z for C26H32N3O6S [M + H]+ calcd 514.2006, found 514.2025.
Procedure for Preparation of Compound 8g
To a solution of 3a (0.07 g, 0.137 mmol) and malononitrile (0.02 g,
0.274 mmol) in dry THF (1 mL), diisopropylethylamine (0.026 mL,
0.151 mmol) was added. The mixture was irradiated in microwave
at 60 ꢀC for 30 min and 100 W. The solvent was evaporated, and
the residue was purified by flash chromatography to give 8g.
J. Pept. Sci. 2013; 19: 110–117 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BAJAJ ET AL.
(S)-tert-Butyl 4,4-dicyano-3-oxo-1-(1-tosyl-1H-imidazol-4-yl)
butan-2-ylcarbamate (8g)
Results and Discussion
Histidine 1 protected by a tosyl/benzyl group on the imidazole
nitrogen and a Boc group on the primary amine, on treatment
with 1-(methylsulfonyl)-1H-benzotriazole (2) under microwave
1
Yellow microcrystal (52%), mp 75–77 ꢀC. H NMR (CD3OD-d4) d
8.05 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.23
(s, 1H), 4.61 (br s, 1H), 3.27–3.24 (m, 1H), 2.86–2.66 (m, 2H), 2.38
(s, 3H), 1.31 (s, 9H); 13C NMR (CD3OD) d 194.2, 157.2, 148.1,
141.8, 138.0, 136.2, 131.8, 128.7, 121.7, 120.5, 116.5, 80.5, 55.5,
32.7, 28.8, 21.8. HRMS (+ESI-TOF) m/z for C21H23N5O5S [M + H]+
calcd 480.1320, found: 480.1312.
irradiation for 30 min, in the presence of Et3N, gave Na-Boc-Nim
-
protected-L-histidylbenzotriazole (3a,b) in 62–65% yield
(Scheme 1), which we used for the preparation of diverse
histidine derivatives.
Na-Boc-Nim-4-toluenesylfonyl-L-histidyl-benzotriazole (3a) cou-
ples readily with free amino acids (4a–d, 4a+4a0, 4b+4b0), dipep-
tide 4e, and tripeptide 4f at 20 ꢀC in the presence of 1.2 eq. of
Et3N to give the corresponding peptides (5a–f, 5a+5a0, 5b+5b0)
(Scheme 2a and b, Table 1).
Procedure for Preparation of Compound 8h
Thiophenol (1.0 eq.) was dissolved in THF and Et3N (1.5 eq.). Na-
Boc-Nim-4-benzyl-L-histidylbenzotriazole 3b (1.0 eq.) was added
to the solution, and the mixture was stirred at room temperature
for 2 h. The mixture was concentrated under reduced pressure
and then diluted with ethyl acetate. The organic layer was
washed with 10% Na2CO3 solution, water and dried over
anhydrous MgSO4, filtered, and evaporated to give the
desired compound.
The enantiomeric purity of compound 5a was confirmed by
HPLC analysis; thus, using a Chiralcel-OD column, 5a showed a
single peak (11.36 min), whereas the racemic mixture 5a+5a0
showed two peaks (10.16 and 10.60 min), one of which had
nearly the same retention time as the single peak from 5a. To
justify the small difference in retention times, we ran the HPLC
of the 1 : 1 ratio mixture of 5a and 5a+5a0 and observed the
two peaks at 10.48 and 11.01 min in a ratio of 1 : 3, respectively,
which confirmed the previous analysis. We have also observed
duplication of peaks in 13C NMR of 5a+5a0 and 5b+5b0, whereas
in the case of 5a and 5b, extra peaks were not observed
(Figure 1a and 1b). However, duplications of peaks in 13C NMR were
observed when Na-Boc-Nim-4-benzyl-L-histidyl-benzotriazole 3b
was coupled with D-phenylalanine methyl ester (Scheme 3). This
indicates that tosyl-protected histidine is less prone to racemization
as compared with benzyl-protected histidine. Similar results were
also observed in optical activity.
(S)-S-Phenyl 3-(1-benzyl-1H-imidazol-4-yl)-2-(tert-butoxycar-
bonylamino)propanethioate (8h)
Yellow microcrystals (60%); mp 130–132 ꢀC; 1H NMR (CDCl3) d
7.55–7.51 (br s, 1H), 7.42–7.31 (m, 10H), 7.00 (s, 1H), 5.34 (br s,
2H), 4.74–4.62 (m, 1H), 4.02 (d, J = 6.3 Hz, 1H), 3.37–3.34 (m, 2H),
1.39 (s, 9H). 13C NMR (CDCl3) d 199.2, 169.7, 155.7, 134.8, 134.7,
133.2, 131.3, 129.6, 129.3, 129.1, 128.7, 127.4, 118.6, 80.4, 73.9,
53.0, 28.3, 18.9. HRMS (+ESI-TOF): m/z for C24H27N3O3S [M + H]+
calcd 438.1846, found 438.1850.
Na-Boc-Nim-4-toluenesylfonyl-L-histidyl-benzotriazole 3a and
3b were reacted with different N-, O-, S-, and C-nucleophiles of
biological importance in the presence of base to prepare the
O
O
N
N
N
DMF, Et3N
N
OH
N
N
N
HN
N
S
HN
MW 50 oC, 20 W
Pg
N
Pg
O
N
Boc
O
Boc
Me
2
1a Pg = Tosyl
1b Pg = Benzyl
3a Pg = Tosyl
3b Pg = Benzyl
Scheme 1. Synthesis of Na-Boc-Nim-protected-L-histidylbenzotriazole 3a,b.
O
R
O
N
O
H
N
R
O
H
N
CH3CN:H2O (7:3)
Et3N
N
N
OH
O
S
N
N
N
O
H
H2N
OH
N
HN
O
R1
O
HN
Boc
O
S
O
R1
N
Boc
n = 0,1,2
n = 0,1,2
3a
4
5a-f
Me
Me
R, R1= H, CH2Ph, CH(CH3)2, CH(CH3)CH2CH3, CH2CH(CH3)2, CH3
b
O
R
N
N
O
O
N
OH
H2N
CH3CN:H2O (7:3)
Et3N
N
O
S
OH
N
O
H
N
HN
Boc
+
O
HN
Boc
O
O
R
N
S
N
4a' R = CH2Ph
4b' R = CH(CH3)2
5a+5a' R = CH2Ph
5b+5b' R = CH(CH3)2
Me
3
Me
Scheme 2. (a) Syntheses of histidine-containing di-, tri- and tetra-peptides 5a–f. (b) Syntheses of histidine-containing dipeptides 5a+5a0, 5b+5b0.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 110–117

HISTIDINE PEPTIDES AND CONJUGATES
Table 1. Syntheses of histidine containing di-, tri-, and tetra-peptides 5a–f, 5a+5a0, 5b+5b0
23
Product
Reactant 4
Yield (%)
mp (ꢀC) (lit. mp)
½aꢁD
5a
H-L-Phe-OH 4a
H-DL-Phe-OH 4a0
H-L-Val-OH 4c
79
72
72
70
89
92
68
60
133–134
ꢂ11
Racemic
ꢂ44
5a+5a0
175–177
5b
Oil
Oil
5b+5b0
5c
H-DL-Val-OH 4c0
Racemic
ꢂ15
H-L-Leu-OH 4c
121–123 (146–148) [21]
151–153
5d
H-L-Ala-OH 4d
ꢂ8
5e
H-Gly-L-Val-OH 4e
H-Gly-Gly-L-Ile-OH 4f
113–115
ꢂ12
5f
140–142
ꢂ9
Figure 1. (a) 13C NMR spectra of compound 5b. (b) C NMR spectra of compound 5b+5b0.
13
J. Pept. Sci. 2013; 19: 110–117 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BAJAJ ET AL.
O
O
N
O
N
OMe
H2N
CH3CN:H2O (7:3)
Et3N
N
H
OMe
N
N
N
HN
Boc
+
HN
Boc
O
N
N
3b
6
7
Scheme 3. Syntheses of histidine-containing dipeptide 7.
O
O
References
N
N
N-, O-, S-, C-Nucleophile
N
N
Nu
N
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8c
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8e
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3a
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3a
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Ethylphenol
B
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80
78
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D
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Acknowledgements
We thank the University of Florida, the Kenan Foundation, King
Abdulaziz University, Jeddah, Saudi Arabia. We also thank Dr Hall
for English checking.
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J. Pept. Sci. 2013; 19: 110–117 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci