α-Substitution effects on the ease of S → N-Acyl transfer in aminothioesters

Article abstract of DOI:10.1111/cbdd.12092

In S-acylcysteines and homocysteines, the efficacy and rate of S → N-acyl transfer (5 and 6 cyclic TSs) vary with the size of S-acyl group. Conformational and quantum chemical calculations indicate that the spatial distance, b(N-C), between the terminal amine and the thioester carbon is shortened by α-C(O)X (X=OH, OMe, NH₂) substituents. Spatial distance, b(N-C), between the terminal amine and the thioester carbon is shortened by α-C(O)X (X=OH, OMe, NH₂) substituents.

Full text of DOI:10.1111/cbdd.12092

Chem Biol Drug Des 2013; 81: 577–582
Research Article
a-Substitution Effects on the Ease of S fi N-Acyl
Transfer in Aminothioesters
Bahaa El-Dien M. El-Gendy1,2, Ebrahim H.
vorable ring strain (13). The work of Seitz suggests that
internal-cysteine ligations of 8- and 11-membered TS can
be disfavored processes (12). Molecular dynamic simula-
Ghazvini Zadeh1, Ania C. Sotuyo1, Girinath G.
Pillai1 and Alan R. Katritzky1,3,
*
tions of these systems (12) have computed distances
between the N-terminal amino group and the sp2-carbon of
Cys thioester and indicated that the ligation rate varies
inversely with the H3N+)C(O) distance (14). A study by Seitz
used a modified NCL protocol where the reaction vessels
were incubated in a thermo mixer at 35 ꢀC (12). We showed
that NCL via 11-membered transition state was achieved
(ꢀ14%) when the reaction mixture was subjected to micro-
wave irradiation of 50 W at 50 ꢀC (15).
1Department of Chemistry, Center for Heterocyclic
Compounds, University of Florida, Gainesville, FL 32611-
7200, USA
2Department of Chemistry, Faculty of Science, Benha
University, PO Box 13511 Benha, Egypt
3Department of Chemistry, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
*Corresponding author: Alan R. Katritzky,
katritzky@chem.ufl.edu
Larger ring (14, 17, 20, 23, 26, 29, and 32-membered)
transition states ligations proceed at significant rates; thus,
Wong and coworkers reported efficient sugar-assisted O-
and N-glycopeptide ligation via 14- and 15-membered ring
transition states, respectively (16,17). Brik et al. (18) exam-
ined peptide ligation using a side chain auxiliary, which
involves a 15-membered transition state. A comprehensive
report by Seitz concludes that internal-cysteine ligations
take place via 14, 17, 20, and 23-membered cyclic transi-
tion states with the highest rate for the 17-membered TS
(12). These results support the molecular dynamic simula-
tions, which relate the rate of ligation to the +H3N)C(O)
distance (12). This depends on the ability of an optimal
conformation of the peptide that can situate the thioacyl
component in the immediate vicinity of the N-terminal
amino group.
In S-acylcysteines and homocysteines, the efficacy
and rate of S fi N-acyl transfer (5 and 6 cyclic TSs)
vary with the size of S-acyl group. Conformational and
quantum chemical calculations indicate that the spatial
distance, b(N-C), between the terminal amine and the
thioester carbon is shortened by a-C(O)X (X = OH,
OMe, NH2) substituents.
Key words: aminothioesters, conformational analysis, S fi N-
acyl transfer
Received
3 August 2012, revised 8 October 2012 and
accepted for publication 17 October 2012
The chemical synthesis of peptides through selective liga-
tions involving O fi N or S fi N-acyl transfer can circum-
vent problems during the expression and purification of
small recombinant proteins (1) that often contain ’difficult
sequences’ in which hydrophobic amino acid side chains
lead to aggregation and hence poor yields (Scheme 1) (2).
Ongoing efforts to extend the applicability of chemical liga-
tion to non-cysteine ligation sites have included the use of
The present work investigates the ease of ligations in com-
pounds with all carbon ⁄ oxygen (but no amidic group)
backbones as compared with those found in peptide
sequences with 5-, 6-, and 8-membered cyclic TSs, aim-
ing initially to examine the differential effect of cysteine-free
acid, a-ester, and a-amide groups on transition state con-
formation during the ligation process.
O-acyl isopeptides, which undergo
a pH-dependent
O fi N intramolecular acyl migration; this can facilitate the
synthesis of difficult peptide sequences including water-
soluble antitumor taxoid prodrug derivatives (3–6) and HIV-
1 protease inhibitors (7–11).
Methods and Materials
Melting points were determined on a capillary point appa-
ratus equipped with a digital thermometer and are uncor-
rected. Column chromatography was conducted on flash
silica gel (200–425 mesh). NMR spectra were recorded in
CDCl3 or DMSO-d6 with TMS for 1H (300 MHz) and 13C
(75 MHz) as an internal reference.
The ease of intramolecular acyl transfers via cyclic transition
states depends significantly on the transition state size.
Entropic forces greatly assist both 5- and 6-membered TS
ligations (12), while ligation rates can be slow for 8-, 9-, 11-,
and 12-membered ring transition states because of unfa-
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12092
577

El-Gendy et al.
Scheme 1: Synthesis of difficult sequence peptides from O- and
S-acyl isopeptides.
N-(2-Mercaptoethyl)-4-methyl benzamide (6a)
A solution of 5a (0.231 g, 1 mmol) and Et3N (0.279 mL,
2 mmol) in DCM (5 mL) was stirred at rt for 30 min. The
mixture was washed with NH4Cl, brine, dried over MgSO4,
and concentrated. The resulting solid was crystallized from
DCM ⁄ hexanes to give 6a as white needles (0.186 g,
95%); mp. 150.0–151.0 ꢀC. 1H NMR (300 MHz, CDCl3) d
7.74–7.70 (m, 2H), 7.23–7.16 (m, 2H), 7.05–6.93 (m, 1H),
3.78 (q, J = 6.2 Hz, 2H), 2.97 (t, J = 6.3 Hz, 2H), 2.39 (s,
3H), 1.70 (s, 1H). 13C NMR (75 MHz, CDCl3) d 167.9,
142.2, 131.5, 129.3, 127.2, 39.2, 38.2, 21.6. Anal. Calcd.
For C10H13NOS (195.29): C, 61.50; H, 6.71; N, 7.17.
Found: C, 61.14; H, 6.14; N, 7.01.
Scheme 2: Synthesis of 6a,b as examples for S fi N-acyl trans-
fer via 5-membered TS.
Study of S fi N-acyl transfer via a 5-membered TS
To prepare compounds 6a, b (Scheme 2), 2-aminoetha-
nethiol hydrochloride 1 was treated with Boc2O to give
compound 2, which was acylated by 1-(aroyl)-1H-benzo-
triazoles 3a, b under mild basic conditions to yield 4a,b.
Compounds 4a,b were subsequently deprotected by HCl
(4 N) in 1-4,dioxane to afford the HCl salts 5a,b. Inter-
mediates 5a,b were converted into 6a,b by Et3N in mixed
CH3CN ⁄ H2O. Interestingly, the aqueous workup of 5a
often afforded nearly 50% of the product 6a through
S fi N-acyl transfer. Thioesters 5a,b were designed to
resemble the chemical structure of S-acylated cysteine
without the a-carboxylic group.
N-(2-Mercaptoethyl)-1-naphthamide (6b)
1
Pale yellow solid (0.18 g, 78%). H NMR (300 MHz, CDCl3)
d 8.28–8.09 (m, 1H), 7.93–7.75 (m, 2H), 7.61–7.40 (m, 3H),
7.34-7.29 (m, 1H), 6.94-6.88 (m, 1H), 3.75 (qd, J = 6.3,
1.9 Hz, 2H), 2.95 (td, J = 6.4, 1.8 Hz, 2H), 1.44 (br s, 1H).
13C NMR (75 MHz, CDCl3) d 1670.0, 134.0, 133.7, 130.8,
130.1, 128.3, 127.1, 126.4, 125.4, 125.3, 124.7, 39.0,
37.9. Anal. Calcd. For C13H13NOS (231.32): C, 67.50; H,
5.66; N, 6.06. Found: C, 67.31; H, 5.31; N, 5.87.
To determine whether S fi N-acyl transfer proceeds intra-
molecularly or intermolecularly, equimolar ratios of 5a, n-
propylamine, and Et3N were mixed in CH3CN ⁄ H2O at rt
N-(3-Mercaptopropyl)benzamide (13)
A solution of 12 (0.062 g, 0.2 mmol) and Et3N in DCM
(20 mL, 1 mM) was stirred at rt for 30 min. The mixture was
washed with HCl (2 N, 3 · 15 mL), brine (15 mL), and dried
over MgSO4. The solvent was removed, and the crude solid
was recrystallized (DCM:hexanes) to afford 13 as white solid
1
for 3 h (Scheme 3). H NMR spectral analysis showed that
the major product obtained was 6a (ꢀ97%), while 7a was
not detected, indicating that S fi N tolyl transfer followed
an intramolecular pathway.
1
(0.03 g, 85%). H NMR (300 MHz, CDCl3) d 7.82–7.72 (m,
When 5b was treated with Et3N in the presence of n-pro-
2H), 7.52–7.44 (m, 1H), 7.43–7.35 (m, 2H), 6.62 (br s, 1H),
3.57 (q, J = 6.6 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.05 (p,
J = 7.0 Hz, 2H), 1.68 (s, 1H). 13C NMR (75 MHz, CDCl3) d
167.8, 134.6, 131.6, 128.7, 127.0, 38.8, 33.7, 22.4. Anal.
Calcd. For C10H13NOS (195.29): C, 61.50; H, 6.71; N, 7.17.
Found: C, 61.32; H, 6.50; N, 7.03.
pylamine,
a surprisingly different result was obtained
Results and Discussion
We examined S
fi N-acyl transfer via 5-, 6-, and 8-
Scheme 3: Competition experiment between 5a and n-propyla-
membered TSs in substrates 5a, b, 12, and 18.
mine.
578
Chem Biol Drug Des 2013; 81: 577–582

Structural Features of Aminothioesters
Scheme 4: Competition experiment between 5b and n-propyla-
mine.
(Scheme 4). 1H NMR spectra indicated that the reaction
mixture was comprised of a 2:1 of 6b and 7b. In effect,
the naphthoyl group sterically hindered the intramolecular
S fi N-acyl transfer, resulting in competitive acylation by
n-propylamine.
Study of S fi N-acyl transfer via a 6-membered TS
Aminopropanol 8 was heated under reflux with HBr (48%)
to provide derivative 9 (55%). Boc-protection of the amine
group then gave 10, which reacted with thiobenzoic acid
to afford the corresponding thioester 11 (Scheme 5). The
Boc group was removed with TFA to give 12 (97%). Inter-
mediate 12 has a structure similar to that of a decarboxy-
lated homocysteine, which could undergo transacylation
Scheme 6: Synthesis of 18 as an example for S
transfer via 8-membered TS.
fi N acyl
Study of S fi N-acyl transfer via an 8-membered
TS
through
a 6-membered TS. Homocysteine has been
employed as a ligating moiety in several studies to prepare
small proteins such as GstI protein of Rhizobium legumino-
sarum, the endogenous inhibitor of the glnII (glutamine
synthetase II) gene expression (19,20). On stirring 12 with
2 equiv. of TEA in DCM, S fi N-acyl transfer took place to
give product 13 (85%).
N-Boc protection of 2-(-aminoethoxy)ethanol 14 afforded
15 (95%) and subsequent tosylation of the hydroxyl group
furnished 16 (70–82%) (Scheme 6). Nucleophilic substitu-
tion of the OTs in 16 by thiobenzoic acid yielded thioester
17 (90%). Subsequent deprotection of the Boc group by
HCl gave 18 as a white solid (quantitative). Compound 18
has the skeletal structure of C-terminal cysteine-containing
dipeptide. Although it is reported (12) that S- fi N-acyl
transfer via 8-membered TS is not favored, we attempted
to evaluate the effect of the backbone in compound 18. We
expected that in the absence of any directing structural
motifs (turn-inducers, i.e. proline), the acyl transfer would be
random. In fact, when 18 was treated with Et3N in CH3CN,
a mixture of bis-acylated product 19 and the ligated pro-
duct 20 was obtained, indicating that the intramolecular
acylation via an 8-membered TS was not favored.
Computational assessment of substituent effects
on S- fi N-acyl transfer
A prerequisite for facile S- fi N-acyl transfer is to bring
the terminal amino group in close proximity to the thioester
carbon atom. We therefore applied techniques previously
employed (21) for similar ligation reactions including a full
conformational search followed by scoring the conformers
based on energies and spatial distances between relevant
centers (b(N-C)). To justify the spatial distance b(N-C),
quantum chemical calculations were carried out at the
DFT ⁄ 6-31G* level of theory (22) using HYPERCHEM 8.0 Soft-
ware (a). The relevant energies and b(N-C) calculated for
the different pre-organized structures are considered to
Scheme 5: Synthesis of 12 as an example for S
transfer via 6-membered TS.
fi N-acyl
Chem Biol Drug Des 2013; 81: 577–582
579

El-Gendy et al.
Table 1: Respective structure, energy, b(N-C) and AlogP of 5r, 5s, 5t, 5x, 5y, and 5z.
Entry
1
Thioester 5
Structure
Energy (kcal ⁄ mol)
)101.85
b(N-C) (Aꢀ)
ALog P
1.19
5r
2.927
S
Ph
HO
O
O
NH2
2
3
5s
5t
)59.69
)98.49
2.915
3.059
0.33
1.22
S
Ph
Ph
H2N
O
O
NH2
S
O
O
O
NH2
S
Ph
Ph
4
5
6
5x
5y
5z
)21.83
)22.86
)25.00
3.325
3.189
3.172
1.79
1.74
2.29
O
H
NH2
S
O
H3C
NH2
S
Ph
O
NH2
prioritize the conformers. Thus, the conformation with the
shortest geometrical distance between these centers (b(N-
C)) afford the pre-organized structure through which the
transfer was expected to occur.
presence of a-substituents (Thorpe-Ingold effect), b(N-C)’s
for 5y and 5z were computed. Interestingly, b(N-C)’s for
5y and 5z were considerably larger than that of 5r-t. In
addition, the energy levels of 5x-z lie significantly higher
than those of 5r-t. The results indicate that a stabilization
effect furnished by the a-carboxylic acid, ester, or amide
substituent favors intramolecular S- fi N-acyl transfer.
A full conformational search considering both rotatable
bonds and the phenyl ring of six related structures 5r, 5s,
5t, 5x, 5y, and 5z (Table 1) was implemented using the
MMX force field, in PCMODEL v. 9.3 software (b). The resul-
tant conformers were ranked in ascending order of b(N-C),
and the best pre-organized structure, with the smallest
b(N-C) values, is shown in Figure 1. In addition, the spatial
distances b(N-C) and AlogP for five structures are shown
in Table 1, respectively.
Conclusions
Competition experiments on S fi N-acyl transfer via 5-
membered TS compared with intermolecular attack
showed that the size of the acyl group can present a steric
hindrance that may slow the rate of intramolecular acyla-
tion. The results complement the findings of Dawson (23)
that explains the role which the amino acid adjacent to the
C-terminal cysteine ligation site plays in determining the
rate of ligation. Similar reasoning may be used to explain
S fi N-acyl transfer via a 6-membered TS; however,
molecules that undergo acyl transfer via an 8-membered
TS must accommodate more strain and therefore unfavor-
able intramolecular acylation as found by Seitz et al.
We compared the effect of the carboxylic acid moiety in
5r against 5x (no CO2H), 5y (+CH3), 5z (+C2H5), 5s
(+CONH2), and 5t (+CO2Me) in Figure 1. The conforma-
tional analysis shows that the presence of the a-carboxylic
acid, ester, or amide substituent brings the terminal amine
and thioester centers closer to each other compared with
the system with no a-substituents (5x). To determine
whether this result is due to the congestion offered by the
580
Chem Biol Drug Des 2013; 81: 577–582

Structural Features of Aminothioesters
5r
5s
5t
5x
5y
5z
Figure 1: Best pre-organized conformers of 5r, 5s, 5t, 5x, 5y, and 5z. Green lines show the b(N-C) in Angstrom (Aꢀ). Accelrys Discovery
Studio Visualizer 3.1(c) to generate 3D representations.
Conformational analysis and quantum chemical calcula-
tions showed that the spatial distance between the term-
inal amine and the thioester carbon b(N-C) is a central
factor in controlling reaction rates and product yields.
While the presence of either a-CO2H, CO2R, and CONH2
shortens b(N-C), this shortening was not observed when
the CO2H group was replaced by Me or Et groups, sug-
gesting that the Thorpe-Ingold effect is not pronounced in
these structures.
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Additional Supporting Information may be found in the
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