A highly efficient in situ N-acetylation approach for solid phase synthesis

Article abstract of DOI:10.1039/c3ob42096e

We describe a new general N-acetylation method for solid phase synthesis. Malonic acid is used as a precursor and the reaction proceeds by in situ formation of a reactive ketene intermediate at room temperature. We have successfully applied this methodology to peptides and non-peptidic molecules containing a variety of functional groups. The reaction gave high yields compared to known acetylation methods, irrespective of the structure, conformation and sequence of the acetylated molecule. Computational studies revealed that the concerted mechanism via the ketene intermediate is kinetically favorable and leads to a thermodynamically stable acetylated product. In conclusion, our method can be easily applied to acetylation in a wide variety of chemical reactions performed on the solid phase.

Full text of DOI:10.1039/c3ob42096e

Volume 12 Number 12 28 March 2014 Pages 1825–1996
Organic &
Biomolecular
Chemistry
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ISSN 1477-0520
PAPER
Assaf Friedler et al.
A highly efficient in situ N-acetylation approach for solid phase synthesis

Organic &
Biomolecular Chemistry
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A highly efficient in situ N-acetylation approach
for solid phase synthesis†
Cite this: Org. Biomol. Chem., 2014,
12, 1879
Koushik Chandra,a Tapta Kanchan Roy,a,b Johnny N. Naoum,a Chaim Gilon,a
R. Benny Gerbera,b,c and Assaf Friedler*a
We describe a new general N-acetylation method for solid phase synthesis. Malonic acid is used as a
precursor and the reaction proceeds by in situ formation of a reactive ketene intermediate at room temp-
erature. We have successfully applied this methodology to peptides and non-peptidic molecules contain-
ing a variety of functional groups. The reaction gave high yields compared to known acetylation methods,
irrespective of the structure, conformation and sequence of the acetylated molecule. Computational
studies revealed that the concerted mechanism via the ketene intermediate is kinetically favorable and
leads to a thermodynamically stable acetylated product. In conclusion, our method can be easily applied
to acetylation in a wide variety of chemical reactions performed on the solid phase.
Received 21st October 2013,
Accepted 21st January 2014
DOI: 10.1039/c3ob42096e
www.rsc.org/obc
a ketene intermediate using strong electron withdrawing
partners11 such as pre-synthesized phosphate and malonate
Introduction
Acetylation of amines or alcohols is a widely used organic ester derivatives was also reported. Using these catalysts
transformation.1,2 It is extremely useful in solid-phase syn- results in high yields in the cases of relatively simple, less steri-
thesis of peptides (SPPS), peptidomimetics and organic mole- cally hindered small organic molecules containing amines or
cules. Acetylation is used to determine the degree of coupling alcohols. A serious limitation of these methods is when they
in SPPS2 and is widely used for capping at the end of each are employed for rigid, more complex molecules, especially
coupling cycle.3 It is used for mimicking the peptide bond in during SPPS. This results in low yields, prolonged reaction
the native protein by eliminating the positive charge from the times and the use of excess reagents or catalysts. This, in turn,
N-termini of peptides.4 Lysine acetylation is a naturally occur- might lead to possible side reactions such as unspecific
ring post-translational modification that needs to be intro- surface binding of catalysts, early deprotection of orthogonal
duced into peptides in many cases.5,6
protecting groups, truncation of peptides or undesired peptide
Acetylation in SPPS is typically carried out by acetic anhy- epimerization.12
dride (AC2O) under basic conditions.7 However, this conven-
Here we report a novel, cost-effective, mild and highly
tional acetylation method sometimes fails to provide adequate efficient approach for the N-acetylation of peptides containing
yields8 and its yields strongly depend on the nature of the multiple functional groups in solid phase synthesis. The reac-
target peptide.9 A variety of both homogeneous and hetero- tion proceeds via a highly reactive in situ ketene intermediate
geneous catalysts are routinely used to improve the yields of along with the formation of CO2 at room temperature
acetylation reactions. These include 4-dimethyl amino pyridine (Scheme 1). Malonic acid serves as the starting material
(DMAP), iodine, p-toluene sulfonic acid, zeolite HSZ-360, for the acetylation, replacing the conventionally used Ac2O.
various metal oxides, silicate, chlorides, nitrate, triflate salts, The methodology is highly favorable both kinetically and
montmorillonit K-10 and KSF, ferric perchlorate adsorbed on thermodynamically.
silica-gel, Nafion-H, NBS, [TMBSA][HSO4] ionic liquid and
potassium dodecatungstocobaltate trihydrate.10 O-acylation via
aInstitute of Chemistry, The Hebrew University of Jerusalem, Safra Campus,
Givat Ram, Jerusalem 91904, Israel. E-mail: assaf.friedler@mail.huji.ac.il
bThe Fritz Haber Research Center, The Hebrew University of Jerusalem,
Safra Campus, Givat Ram, Jerusalem 91904, Israel
cDepartment of Chemistry, University of California, Irvine, CA 92697, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42096e
Scheme 1 The general protocol for peptide N-acetylation.
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in their sequence, length, MW, conformation (cyclic vs. linear)
and hydrophobicity. In spite of their different properties, all
Results and discussion
The N-terminal free amine of a resin bound protected peptide peptides (1–25) were chemoselectively N-acetylated with high
was treated with malonic acid that was pre-activated using yields.
O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-
In the cases of hydrophobic peptides (17, 19, 22, 23), the
phosphate (HBTU) and diisopropyl ethyl amine (DIPEA) in amine is less exposed to the solvent and thus the conventional
DMF at room temperature. The acetylation typically took acetylation fails to provide reasonable yields in many cases.
10–30 minutes. The completion of the reaction was monitored Similarly, the secondary amine of proline residues in peptides
by the Kaiser-ninhydrin and chloranil tests.13 The peptide (10, 12, 13) is usually difficult to acetylate using conventional
was cleaved from the resin using standard TFA mediated clea- approaches.16 All these limitations were successfully tackled
vage.14 MALDI-TOF MS of the resulting peptides confirmed using the technique presented herein. Even a long chain, high
the successful incorporation of the acetyl group (Table 1). All MW, rigid and polyfunctionalized linear peptide (25) was
the acetylated peptides exhibited a higher retention time com- acetylated under the same conditions. The yield was not
pared to the corresponding non-acetylated peptides in the affected by the stereochemistry (D or L) of the acetylated resi-
reverse phase HPLC, indicating increased hydrophobicity due dues. Our acetylation technique was applicable for various
to the acetylation. The addition of the acetyl group was con- coupling agents including BOP, PyBOP, HATU, HCTU, TBTU
firmed by the appearance of
a sharp singlet around and TSTU. The acetylation was also not influenced by altering
1
1.9–2.2 ppm in H NMR (Fig. 1 and ESI†). The progress of the the resin from Rink amide to Wang, polystyrene or PEG based
reaction was also reflected by the observation that the ratio resins. For example, a single phenylalanine (24) underwent
between the peak at 1.86 ppm (representing the acetyl group) successful acetylation irrespective of the nature of the resin.
and the peak at 2.7 ppm (representing the formation of tetra-
To estimate the efficiency of the new acetylation protocol,
methyl urea) versus the reaction time formed a sigmoidal curve our acetylation technique was quantitatively compared with
(Fig. 2). DIPEA and Et3N in DMF or DMSO were the optimal the conventional AC2O-mediated technique using peptide 13
conditions to obtain maximum efficiency in terms of yield and as a model. Peptide 13 was selected for N-acetylation since it
reaction time (see ESI†).
contains the less reactive amine of the proline residue at its
We have applied the methodology to numerous peptides N-terminal position. In both cases, we used the same number
(1–25) derived from various proteins studied in our laboratory of equivalents of the starting materials and the same reaction
and from other sources (Table 1). The peptides were different time. The formation of the acetylated peptide was analyzed
Table 1 The peptides acetylated in this study*
Obs.
MW
Reaction
time (min) (%)
Yield
a
b
Non-acetylated peptide sequence
MW
Acetylated product sequence
WNSLKIDNLDV,15 1
LSNWKIDNLDV, 2
1316 (Ac)-WNSLKIDNLDV-CONH2, 27
1316 (Ac)-LSNWKIDNLDV-CONH2, 28
1838 (Ac)-LMLSPDDIEQWFTED-CONH2, 29
1789 (Ac)-KILNPEEIEKYVAEI-CONH2, 30
1356 (Ac)-(Succinyl)GWSLKIDNLDV-CONH2, 31
1356 (Ac)-W(succinyl)DGSLKIDNLDV-CONH2, 32
2046 (Ac)-WHKQEVRRLKSLHEAE-CONH2, 33
1969 (Ac)-RAEREQDPRAVAPQQCN-CONH2, 34
1842 (Ac)-CYCCGLRSFRELTYQ-CONH2, 35
2799c (Ac)-PDCYWGRWCRTQVRKAHHAMKF-CONH2, 36
1802 (Ac)-WVARPLSPTRLQPALP-CONH2, 37
1573 (Ac)-QAGPPSRPPRYSSSS-CONH2, 38
1626 (Ac)-PYSPLSPKGRPSSPR- CONH2, 39
1945 (Ac)-RQAGDDFSRRYRRDF-CONH2, 40
1130 (Ac)-NSLKIDNLDV-CONH2, 41
1539 WNSLK(Ac)IDNLDV-CONH2, 42
619 (Ac)-FIVLK-CONH2, 43
775 (Ac)- WIDNLD-CONH2, 44
801 (Ac)- MLVVLIL-CONH2, 45
375 (Ac)-GGGGK-CONH2, 46
647 (Ac)-WNSLK-CONH2, 47
769 (Ac)- GFFRWG -CONH2, 48
769 (Ac)- FGRFWG -CONH2, 49
166 (Ac)-F-CONH2, 50
5857 (Ac)-WQHPEKENEGDITIFPESLQPSETLKQM-
NSMNSVGTFLDVKRLRQLPKLF-CONH2, 51
1358 10
1358 14
1882 20
1832 16
1398 22
1398 25
2089 19
2060c 16
1884 22
2841c 30
1844 15
1616 20
1668 30
1987 30
1172 20
1581 10
661 20
839c 28
843 25
417 12
689 15
811 20
811 25
208 14
5899 30
98
95
96
98
94
96
93
92
94
93
96
91
92
90
99
98
97
96
96
97
98
92
93
99
92
LMLSPDDIEQWFTED, 3
KILNPEEIEKYVAEI, 4
NH2-(Succinyl)GWSLKIDNLDV, 5
NH2-W(succinyl)DGSLKIDNLDV, 6
WHKQEVRRLKSLHEAE, 7
RAEREQDPRAVAPQQCN, 8
CYCCGLRSFRELTYQ, 9
PDCYWGRWCRTQVRKAHHAMKF 10
WVARPLSPTRLQPALP, 11
QAGPPSRPPRYSSSS, 12
PYSPLSPKGRPSSPR, 13
RQAGDDFSRRYRRDF, 14
NSLKIDNLDV, 15
FmocNH-WNSLK(NH2)IDNLDV, 16
FIVLK, 17
WIDNLD, 18
MLVVLIL, 19
GGGGK, 20
WNSLK, 21
GFFRWG, 22
FGRFWG, 23
F, 24
WQHPEKENEGDITIFPESLQPSETLKQ-
MNSMNSVGTFLDVKRLRQLPKLF, 25
*For the MS and HPLC data of all peptides see ESI Fig. S1 and S2. a Molecular weight of the unmodified peptides. b Molecular weight of the
acetylated peptides as measured by MALDI-TOF. c Molecular weight with the addition of sodium or potassium.
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Fig. 1 Monitoring the in situ acetylation kinetics of anisidine as a model compound by 1H NMR. (a) A–G represent the ketene formation; H shows
the disappearance of the ketene intermediate; I–J depict the attack by an external amine of anisidine, 53; (b) relative formation of N-acetyl anisidine
(54) vs. tetramethyl urea with time; (c) scheme of N-acetylation with anisidine.
using MALDI-TOF MS. The initial mass of the starting peptide Mechanism of acetylation
(13) was found at the calculated value of 1626 Da. Peptide 13
was acetylated using equivalent amounts of AC2O and Et3N.
The MALDI-MS taken after 30 min showed that the acetylation
was incomplete. Both the starting peptide 13 (1626 Da) and
the acetylated peptide 39 (1668 Da) were observed at a roughly
5 : 1 intensity ratio, indicating that conversion was far from
complete. The yield did not improve significantly when the
reaction was performed overnight and in the presence of 20
equivalents (excess) of AC2O. Therefore, the conventional
acetylation technique was not suitable for peptide 13. When
performing the acetylation of the same peptide 13 for
30 minutes using our malonic-acid mediated protocol, we
observed in MALDI-TOF MS that the acetylation reaction was
successfully completed. The peak of the acetylated peptide (39)
at 1668 Da was the only one observed, while no peak
corresponding to the starting peptide (13) at 1626 Da was
found. This shows that our methodology can be applied to
peptides with hindered amines where acetylation is difficult to
We performed NMR based kinetics study on the acetylation
of the simple molecule anisidine as a model (53) to under-
stand the mechanism of in situ acetylation at room tempera-
ture. Stoichiometric amounts of malonic acid (55), HBTU (57)
and DIPEA were mixed in d6-DMF in an NMR tube. The
spectra were recorded for 3 minutes each. Initially, a malonate-
TMU complex (58, δH 3.1 ppm) and a ketene (61, δH 2.6 ppm)
were formed due to the removal of HOBt (62) (Fig. 1 and
Scheme 2). The intensity of the ketene peak gradually
decreased with time and the peak completely disappeared at
24 min immediately after adding the anisidine (53). At
27 minutes, a distinct peak at δH 2.2 ppm began to appear,
indicating the N-acetylation of anisidine (54). A similar
phenomenon was also observed in d6-DMSO where anisidine
was added in the beginning of the reaction. The spectra were
recorded every 3 minutes at room temperature. The N-acetyl-
ated anisidine (54) was formed after 9 minutes and the reac-
tion was completed immediately within 12 minutes. After that,
achieve by conventional methods. In general, no epimerization, there was practically no change in intensity of the acetylated
truncation or aggregation was evident in the isolated peptides.
peak (ESI Fig. S3†). These two experiments provide direct
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dissociation to form the ketene and CO2. The feasibility of the
three different mechanisms was investigated by theoretical cal-
culations using the density functional theory based hybrid
B3LYP17 method with the 6-311++g** basis set18 applying the
Gaussian 0919 program suit. Based on the experimental con-
ditions, we have considered DMF as the solvent and the polari-
zation continuum model (PCM) with the integral equation
formalism variant (IEFPCM)20 implemented in Gaussian 09 as
the solvation method. Geometry optimization and subsequent
frequency calculations were performed to determine the
extrema for all the reactants, intermediates, transition states
and products. The zero point energy was considered for all the
species in evaluating energy profiles of the reaction mecha-
nisms (Fig. 2) shown in Scheme 2. All the transition states
were determined by a single imaginary frequency characterized
by the corresponding reaction coordinates. The calculated
energy barrier for the self-dissociation of 58 (path A) is only
10.2 kcal mol−1 (TS1) and the reaction is exothermic by
−9.2 kcal mol−1 (Fig. 2a). This shows that this reaction
pathway is kinetically and thermodynamically favorable. For
Fig. 2 Energy profiles for the three proposed reaction pathways show
that path A is favourable: (a) Path A (blue) represents the self-dissociation
of the intermediate complex 58 to form tetramethyl urea (59), CO2 and
the reactive ketene; path B (red) represents the formation of the cyclic
intermediate 64. The corresponding reaction is indicated in Scheme 2; path B, the TS energy for the formation of the cyclic intermedi-
ate 64 is 12.8 kcal mol−1 (TS2) but endothermic in nature
(Fig. 2a). We could not detect any subsequent self-dissociation
(b) Path C (green and purple) represents the formation of 63 followed by
its self-dissociation and is also shown in Scheme 2; (c) the optimized
structure for the most favorable path A. Shown are the reactant (58),
transition state (TS1) and the products (59–60–61).
of 64. Rather, it readily went back to 58 and followed Path
A. The energy profile indicates that the backward reaction is
highly favorable with a barrier of 3.3 kcal mol−1. The NMR
evidence for the spontaneous in situ formation of a ketene
intermediate (61).
studies also support this observation. If the cyclic intermediate
64 would be formed from the linear 58, we would expect a sig-
nificant shift in the position of the methylene group peak
towards a more shielding region. However, no change in the
chemical shift of the methylene peak was observed (Fig. 1). We
conclude that Path B is not feasible. For Path C, the attack by
the HOBt anion (62) to generate 63 is an exothermic and
almost instantaneous with a small barrier of only 4.6 kcal mol−1
(TS3a, Fig. 2b). However, the subsequent self-dissociation is
endothermic in nature and has a barrier of 13.6 kcal mol−1
(TS3b, Fig. 2b), which is higher than Path A. The main driving
Based on the NMR results, three different possible pathways
for the acetylation mechanism could be suggested (Scheme 2).
Initially, the malonate reacts with the HBTU to form an inter-
mediate complex (58) following the leaving of the HOBt (62).
In path A, the complex 58 undergoes self-dissociation to form
CO2 (60) and a reactive ketene (61). In path B, 58 forms a cyclic
intermediate 64, followed by self-dissociation to CO2 and
ketene. In path C, the HOBt reacts with intermediate 58 to
form a HOBt-malonate complex (63) followed by similar self-
Scheme 2 Suggested mechanisms for N-acetylation.
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force for this reaction could be the formation of the CO2 gas.
When considering the CO2 as a separate species that readily
leaves the system and the HOBt-ketene complex (61–62) as the
only remaining product in the reaction medium, the reaction
is found to be exothermic by −13.8 kcal mol−1. The overall
energy profiles lead to path A that is energetically more favor-
able than path C. To quantify this, we have further calculated
the rate of the corresponding two self-dissociation reactions.
The calculated rate for path A is 3.7 × 106 s−1 and that for path
C is 5.7 × 103 s−1. This shows that path A is approximately 650
times faster than path C and thus the self-dissociation process
follows explicitly path A.
4 (a) S. H. Chen, C. R. Chen, S. H. Chen, D. T. Li and
J. L. Hsu, J. Proteome Res., 2013, 12, 3277; (b) K. Zhang,
P. M. Yau, B. Chandrasekhar, R. New, R. Kondrat,
B. S. Imai and M. E. Bradbury, Proteomics, 2004, 4, 1.
5 (a) X. Wu, M. H. Oh, E. M. Schwarz, C. T. Larue,
M. Sivaguru, B. S. Imai, P. M. Yau, D. R. O. And and
S. C. Huber, Plant Physiol., 2011, 155, 1769; (b) J. Zhang,
R. Sprung, J. Pei, X. Tan, S. Kim, H. Zhu, C. F. Liu,
N. V. Grishin and Y. Zhao, Mol. Cell. Proteomics, 2009, 8, 215.
6 (a) E. M. Sol, S. A. Wagner, B. T. Weinert, A. Kumar,
H. S. Kim, C. X. Deng and C. Choudhary, PLoS One, 2012,
12, e50545; (b) Y. Sun, Y. Xu, K. Roy and B. D. Price, Mol.
Cell Biol., 2007, 27, 8502; (c) D. E. Sterner and S. L. Berger,
Microbiol. Mol. Biol. Rev., 2000, 64, 435.
7 R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149.
8 For conventional acetylation in SPPS, see: (a) F. A. Macías,
J. M. Aguilar, J. M. G. Molinillo, G. M. Massanet and
F. R. Fronczek, Tetrahedron, 1994, 50, 5439; (b) J. E. Baldwin,
C. N. Farthing, A. T. Russell, C. J. Schofield and A. C. Spivey,
Tetrahedron Lett., 1996, 37, 3761.
9 For reviews on SPPS, see: (a) E. Atherton and R. C. Sheppard,
Solid Phase peptide synthesis: a practical approach, IRL Press,
Oxford, England, 1989, ISBN 0199630674; (b) J. M. Stewart
and J. D. Young, Solid phase peptide synthesis, Pierce
Chemical Company, Rockford, 2nd edn, 1984, vol. 91, ISBN
0935940030.
Conclusions
We have demonstrated
a highly efficient, cost effective
approach for peptide acetylation via an in situ formation of a
ketene intermediate. Activated malonic acid is shown to be a
more potent acetylating precursor compared to Ac2O. We have
established a reaction mechanism of simultaneous in situ
ketene formation and decarboxylation at room temperature.
The process is highly favorable both kinetically and thermo-
dynamically. A combination of NMR studies and theoretical
analysis revealed the acetylation mechanism. This novel acetyl-
ation reaction should be highly applicable to solid phase
peptide and organic synthesis.
10 For representative examples of acetylation, see: (a) A. C. Spivey
and S. Arseniyadis, Angew. Chem., Int. Ed., 2004, 43, 5436;
(b) N. Deka, A. M. Mariotte and A. Boumendjel, Green
Chem., 2001, 3, 261; (c) F. Rajabi and M. R. Saidi, Synth.
Commun., 2005, 35, 483; (d) S. D. Khaja and J. Xue, Lett.
Org. Chem., 2006, 3, 554; (e) M. Guidotti, C. Canaff,
J. M. Coustard, P. Magnoux and M. Guisnet, J. Catal., 2005,
230, 375; (f) B. M. Choudary, M. Sateesh, M. Lakshmi
Kantam, K. V. S. Ranganath and K. V. Raghavan, Catal.
Lett., 2001, 76, 231; (g) H. J. Yoon, S. M. Lee, J. H. Kim,
H. J. Cho, J. W. Choi, S. H. Lee and Y. S. Lee, Tetrahedron
Lett., 2008, 49, 3165; (h) N. P. Bizier, S. R. Atkins,
L. C. Helland, S. F. Colvin, J. R. Twitchell and M. J. Cloninger,
Carbohydr. Res., 2008, 343, 1814; (i) G. K. Rawal, S. Rani,
A. Kumar and Y. D. Vankar, Tetrahedron Lett., 2006, 47, 9117;
(j) Z. Yang and W. Pan, Enzyme Microb. Technol., 2005, 37,
19; (k) E. Rafiee, S. Tangestaninejad, M. H. Habibi and
V. Mirkhani, Synth. Commun., 2004, 34, 3673.
Acknowledgements
AF was supported by a starting grant from the European
Research Council under the European Community’s Seventh
Framework Programme (FP7/2007-2013)/ERC grant agreement
no. 203413 and by the Minerva Center for Bio-Hybrid Complex
Systems. KC and TKR are supported by a PBC fellowship,
Council of Higher Education, Israel. TKR thanks the CSC-IT
Center for Science, Finland for the computational resources.
We thank Dr Norman Metanis for his critical reading of the
manuscript.
Notes and references
1 (a) D. H. Kim, J. Heterocycl. Chem., 1976, 13, 179; 11 R. Shelkov, M. Nahmany and A. Melman, J. Org. Chem.,
(b) G. E. Perlmann, J. Biol. Chem., 1966, 241, 153. 2002, 67, 8975.
2 For recent reviews on acetylation, see: (a) S. H. Chen, 12 For examples of drawbacks in conventional SPPS acetyl-
C. R. Chen, S. H. Chen, D. T. Li and J. L. Hsu, J. Proteome
Res., 2013, 12, 3277; (b) T. Arnesen, PLoS Biol., 2011, 9,
e1001074; (c) V. Schiza, D. Molina-Serrano, D. Kyriakou,
A. Hadjiantoniou and A. Kirmizis, PLoS Genet., 2013, 9,
e1001074.
3 (a) C. S. Nielsen, P. H. Hansen, A. Lihme and
P. M. H. Heegaard, J. Biochem. Biophys. Methods, 1989, 20,
69; (b) C. A. Hood, G. Fuentes, H. Patel, K. Page,
M. Menakuru and J. H. Park, J. Pept. Sci., 2008, 14, 97.
ation, see: (a) J. A. Iera, L. M. M. Jenkins, H. Kajiyama,
J. B. Kopp and D. H. Appella, Bioorg. Med. Chem. Lett.,
2010, 20, 6500; (b) K. H. Hsieh, M. M. Demaine and
S. Gurusidaiah, Int. J. Pept. Protein Res., 1996, 48, 292;
(c) A. Pessi, V. Mancini, P. Filtri and L. Chiappinelli,
Int. J. Pept. Protein Res., 1992, 39, 58; (d) I. Coin, R. Dölling,
E. Krause, M. Bienert, M. Beyermann, C. D. Sferdean and
L. A. Carpino, J. Org. Chem., 2006, 71, 6171; (e) S. J. Taylor
and J. P. Morken, Science, 1998, 280, 267.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1879–1884 | 1883

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Paper
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13 For common chemical analysis of peptide formation, see:
W. Yang and R. G. Parr, Phys. Rev. B: Condens. Matter, 1988,
37, 785.
(a) E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook,
Anal. Biochem., 1970, 34, 595; (b) T. Vojkovsky, Pept. Res., 18 For the 6-311++g** basis set, see: (a) R. Krishnan,
1995, 8, 236.
J. S. Binkley, R. Seeger and J. A. Pople, J. Chem. Phys., 1980,
72, 650; (b) T. Clark, J. Chandrasekhar, G. W. Spitznagel
and P. V. R. Schleyer, J. Comput. Chem., 1983, 4, 294.
19 M. J. Frisch, et al., Gaussian 09, Revision C.01, Gaussian,
Inc., Wallingford CT, 2010.
14 C. A. Guy and G. B. Fields, Solid-Phase Peptide Synthesis,
in Enzymology, ed. G. B. Fields, Academic Press, 1997,
vol. 289, p. 67.
15 Z. Hayouka, J. Rosenbluh, A. Levin, S. Loya, M. Lebendiker,
D. Veprintsev, M. Kotler, A. Hizi, A. Loyter and A. Friedler, 20 For the solvation model in Gaussian09, see: (a) J. Tomasi,
Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 8316.
16 E. L. Gershey, G. Vidali and V. G. Allfrey, J. Biol. Chem.,
1968, 243, 5018.
17 For the DFT based hybrid B3LYP method, see:
(a) A. D. Becke, J. Chem. Phys., 1993, 98, 5648; (b) C. Lee,
B. Mennucci and R. Cammi, Chem. Rev., 2005, 105, 2999;
(b) J. Tomasi, B. Mennucci and E. Cancès, J. Mol. Struct.,
1999, 464, 211; (c) J. Tomasi, R. Cammi, B. Mennucci,
C. Cappelli and S. Corni, Phys. Chem. Chem. Phys., 2002, 4,
5607.
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