Oxime-Based carbonates as useful reagents for both n-protection and peptide coupling

Article abstract of DOI:10.3390/molecules171214361

We have demonstrated that oxime-based mixed carbonates are very effective reagents for both N-protection and peptide coupling.

Full text of DOI:10.3390/molecules171214361

Molecules 2012, 17, 14361-14376; doi:10.3390/molecules171214361
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Oxime-Based Carbonates as Useful Reagents for Both
N-Protection and Peptide Coupling
Yahya El-Sayed Jad 1, Sherine N. Khattab 1, Ayman El-Faham 1,2,3,* and Fernando Albericio 3,4,5,6,
*
1
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
21321 Alexandria, Egypt
2
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
11451 Riyadh, Saudi Arabia
3
Department of Chemistry and Molecular Pharmacology, Institute for Research in Biomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
4
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
5
Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11,
08028 Barcelona, Spain
6
School of Chemistry, University of KwaZulu-Natal, 4041 Durban, South Africa
* Authors to whom correspondence should be addressed;
E-Mails: aymanel_faham@hotmail.com (A.E.-F.); albericio@irbbarcelona.org (F.A.);
Tel.: +20-122-3140-924 (A.E.-F.); Fax: +20-3-5771-399 (A.E.-F.); Tel./Fax: +34-93-4037-126 (F.A.).
Received: 22 October 2012; in revised form: 23 November 2012 / Accepted: 30 November 2012 /
Published: 5 December 2012
Abstract: We have demonstrated that oxime-based mixed carbonates are very effective
reagents for both N-protection and peptide coupling.
Keywords: oxime; additives; N-protection; activation; coupling; peptide
1. Introduction
Peptides are increasingly gaining recognition as potential bioactive ingredients in the pharmaceutical
industry [1–3]. Peptide synthesis depends on the strategies used for protecting the α-amino group and
for activating the carboxylic acid group prior to peptide coupling. The two main classes [4–6] of
carboxylic acid group activation methods are: (i) those that require in situ activation of the carboxylic

Molecules 2012, 17
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acid and (ii) those that require an activated species that has previously been prepared (usually from an
in situ activation step), isolated, purified, and characterized.
The amino group is most commonly protected by preparing the corresponding carbamate derivative.
Despite the vast number of reagents reported to date for introducing the protecting group into the
N-terminal amino group, there is still no universally active species capable of providing optimal
protecting group introduction.
The traditional chloroformate strategy is an extremely powerful approach, providing fast amino
group protection [7–9]. Nevertheless, in some cases, presence of the free carboxylic acid group can
interfere with the reaction and lead to formation of byproducts such as dipeptides and even tripeptides
(Scheme 1) [10].
Scheme 1. Mechanism for the formation side products (dipeptides and tripeptides) during
the protection of amino acids with haloformates.
(a): Indicating the N-protection by chloroformate; (b): indicating the side reaction which could happen to
form the di- and tripeptide.
Since these side-reactions are associated with the quality of the leaving group, the less reactive
species such as the dicarbonates 2 (Figure 1) [11–14] and the succinimidocarbonates 3 (Figure 1) [15,16]
have previously been proposed as alternatives to the chloride 1 (Figure 1). Use of the azide derivatives
4 (Figure 1), [17,18] has also been proposed as an alternative for the chloride to prepare the
N-protection of amino acids, but the explosive nature of azides precludes their use in large-scale
synthesis. Moreover, several other approaches, based on the use of other less reactive species such as the
1,2,2,2-tetrachloroethyl [19,20], the 5-norbornene-2,3-dicarboximido [21], the pentafluorophenyl [22–24],
and the 1-hydroxybenzotriazole [14,25,26] mixed carbonates 5–8 (Figure 1), have been proposed.
Recently, ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure®, 12a) has been tested as an additive
for use in the carbodiimide approach for the formation of peptide bonds [27]. OxymaPure® and its
uronium-based phosphium coupling reagents displayed a remarkable capacity to inhibit racemization,
together with impressive coupling efficiency, in both automated and manual synthesis, superior to those
of 12d and which has recently been reported to exhibit explosive properties [26] at least comparable to
those of HOAt uronium-based phosphonium coupling reagents [28–34].

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Later, we reported a series of Fmoc/Alloc-oxime carbonate reagents which are easy to prepare,
stable, and highly reactive crystalline materials that afford nearly pure Fmoc/Alloc-amino acids in high
yields. Among the Fmoc-oxime carbonates that we evaluated for the preparation of Fmoc/Alloc-Gly-OH,
the N-hydroxypicolinimidoyl cyanide derivative 9 (Figure 1) gave the best results [35]. More recently,
our research group reported the cyanoacetamide-based oximes 10 (Figure 1), which show unusual
ability to afford Fmoc-protected amino acids in high yield, high purity and at lower cost relative to
compound 9 [36].
Figure 1. Structure of carbonates derivatives.
Herein, we extended our studies for the synthesis of a new family of carbonate derivatives based on
OxymaPure®, which are easy to prepare, stable, and have shown high efficiency in N-protection as
well as peptide coupling.
2. Results and Discussion
2.1. Preparation of Carbonate Derivatives
The carbonate derivatives 13 were readily prepared by reacting ethyloxycarbonyl chloride (11) with
an oxime (compounds 12a or 12b), N-hydroxy-2-pyridinone (12c) or benzotriazole derivatives (HOBt, 12d
or 6-Cl-HOBt, 12f) in the presence of sodium carbonate in DCM/H2O (3:2) as solvent at 0 °C, with
stirring at this temperature for 2 h (Scheme 2). After subsequent workup followed by isolation
and recrystallization from CH2Cl2/hexane, ethyl 2-cyano-2-(ethoxycarbonyloxyimino)acetate (13a),
(ethoxycarbonyloxy)carbonimidoyl dicyanide (13b), ethyl 2-oxopyridin-1(2H)-yl carbonate (13c),
1H-benzo[d][1,2,3]triazol-1-yl ethyl carbonate (13d), and 6-chloro-1H-benzo[d][1,2,3]triazol-1-yl ethyl
carbonate (13f), were prepared in 46–78% yield (Table 1).

Molecules 2012, 17
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Scheme 2. Preparation of the carbonate derivatives 13.
Table 1. Yield, m.p. and elemental analysis of the carbonate derivatives 13.
Elemental Analysis: Calculated (Found)
Compd.
Method
Yield (%)
m.p. (°C)
C
H
N
13a
13b
13c
A
A
A
A
B
A
78
69
76
52
77
76
44–45
oily
44.86 (45.08)
43.12 (43.25)
52.46 (52.21)
52.17 (51.96)
46.16 (45.88)
44.74 (44.53)
4.71 (4.63) 13.08 (13.17)
3.02 (2.89) 25.14 (25.33)
64–67
4.95 (5.16)
7.65 (7.91)
13d
13e *
13f
138–139
133–135
144–145
4.38 (4.54) 20.28 (20.49)
3.87 (4.14) 26.91 (27.19)
3.34 (3.61) 17.39 (17.18)
* 13e was prepared by reacting ethyloxycarbonyl chloride 11 with HOAt 12e in the presence of
anhydrous potassium hydroxide (1 equivalent) in acetonitrile as solvent at 0 °C.
Three different oxime carbonate derivatives: ethyl 2-cyano (isobutoxycarbonyloxyimino)acetate
(17), ethyl 2-(allyloxycarbonyloxyimino)-2-cyanoacetate (18) and ethyl 2-(benzyloxycarbonyloxy-
imino)-2-cyanoacetate (19) were prepared by reacting the corresponding chloroformates 14–16 and
OxymaPure® (12a) in the presence of sodium carbonate in DCM/H2O (3:2) as solvent at 0 °C (Scheme 3).
After subsequent workup followed by isolation and recrystallization from CH2Cl2/hexane, the
corresponding oxime carbonates 17–19 were obtained in 87–94% yield (Table 2).
Scheme 3. Preparation of oximinocarbonate derivatives.

Molecules 2012, 17
Table 2. Yield, m.p. and elemental analysis of the oximinocarbonate derivatives 17–19.
14365
Elemental Analysis: Calculated (Found)
Product
Yield (%)
m.p. (°C)
C
H
N
17
18
19
93
94
87
59–60
Oily
49.58 (49.81)
47.79 (47.93)
56.52 (56.23)
5.83 (5.57)
4.46 (4.61)
4.38 (4.62)
11.56 (11.74)
12.39 (12.58)
10.14 (10.41)
99–100
2.2. Preparation of 4-(Ethoxycarbonylamino)benzoic Acid
To study the reactivity of prepared carbonate derivatives 13a–f and their utility for the preparation
of the N-protected amino acids, we initiated our studies with 4-aminobenzoic acid (20), which on
treatment with the previously synthesized carbonate derivatives 13a–f in a homogenous acetone/aqueous
solvent mixture in the presence of sodium carbonate with stirring overnight at room temperature,
provides the product 21. Samples of 4-(ethoxycarbonylamino)benzoic acid (21) were obtained from
the different carbonate derivatives after removing the unreacted starting carbonate by extracting with
ether and acidifying the aqueous layer with 1N HCl (Scheme 4). The purity of the product 21 was
determined after injection onto reverse-phase HPLC are shown in Table 3.
Scheme 4. N-protection of 4-aminobenzoic acid using carbonate derivatives 13a–f.
O
HN
O
O
Na2CO3
O
O
R
+
H2N
O
O
CH3COCH3/H2O
OH
OH
0 C
rt
20
13 a f
( - )
21
Table 3. Yield %, m.p., purity % of 4-(ethoxycarbonylamino)benzoic acid 21.
Carbonate
13a
Yield (%)
m.p. (°C)
198–200
199–201
198–200
184–192
198–202
190–195
Purity * (%)
95.3
43
38
59
11
31
42
13b
88.5
13c
100
13d
77.1
13e
97.0
13f
83.6
* The purity was determined by HPLC using the following Conditions: detection at 220 nm (Waters 996 PDA
detector); Sunfire C18 column (3.5 µm 4.6 × 100 mm); linear gradient over 14 min (10 to 100% CH3CN in
H2O/0.1% TFA); flow rate 1.0 mL/min. tR [4-(ethoxycarbonylamino)benzoic acid] = 4.18 min.
Table 3 showed that, 1H-benzo[d][1,2,3]triazol-1-yl ethyl carbonate (13d) provided the lowest
purity (77.1%) and yield (11%) of all the ethoxycarbonyl carbonate derivatives. (Ethoxycarbonyloxy)
carbonimidoyl dicyanide (13b) and 6-chloro-1H-benzo[d][1,2,3]triazol-1-yl ethyl carbonate (13f)
provided moderate levels of purity (88.5% and 83.6%) and yield (38% and 42%). Ethyl 2-cyano-2-
(ethoxycarbonyloxyimino)acetate (13a) and 3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl ethyl carbonate
(13e) provided high purity (95.3% and 97.0%, respectively) and moderate yield (43% and 31%), while,

Molecules 2012, 17
14366
ethyl 2-oxopyridin-1(2H)-yl carbonate (13c) provided the highest yield (59%) with excellent purity
(100%) as indicated from the HPLC traces.
2.3. HPLC Study of the Rate of Formation of the Active Ester
Before attempting simultaneous protection and activation of amino acids, we tried to prepare active
esters of N-protected amino acids using the carbonate derivatives 13a, 17, 18, and 19 to ensure that
these compounds can activate carboxylic acids by forming the corresponding active ester for different
Fmoc-amino acids. The reaction of Fmoc-amino acids with oxime carbonate derivatives was monitored
by HPLC to study the rate of formation of the active ester. Aliquots (5 µL) of the reaction mixture
were taken, diluted with acetonitrile, and then analyzed by HPLC. Follow-up samples were studied at
intervals of time 30 min and 1, 2, 4 and 24 h pre-activation. This enabled us to determine the optimum
pre-activation time for each carbonate reagent, as excessively long times could lead to greater formation
of alkyl or aryl esters.
2.3.1. The Rate of Formation of the Active Ester of Fmoc-Val-OH Using Oxime Carbonate Derivatives
i
Mixing of Fmoc-Val-OH 22 with the oxime carbonate reagents EtocOXY 13a, BuocOXY 17,
AllocOXY 18 and ZOXY 19 in the presence of pyridine in DMF, we observed maximum levels of the
active ester was formed at 4 h for 13a, 2 h for 17, 30 min for 18 and 1 h for 19. Whereas the alkyl or
i
aryl esters 26 started to be formed after 2 and 1 h in case of EtocOXY 13a and BuocOXY 17,
respectively, and after half an hour in case of AllocOXY 18 and ZOXY 19 respectively. Therefore, the
optimum pre-activation time should not exceed more than 2 and 1 h in case of EtocOXY 13a and
iBuocOXY 17, respectively, and should be less than half an hour in case of both AllocOXY 18 and ZOXY
19 systems. Best results for formation of the active ester were obtained with the oxime carbonate 17
and 18, while the oxime carbonates 13a and 19 gave high yield of the alkyl ester (Tables 4–7).
Table 4. The rate of formation of the active ester of Fmoc-Val-OH 22 using ethyl 2-cyano-
2-(ethoxycarbonyloxyimino)acetate (13a).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Val-OH 22
Active ester 27 Ethyl ester
½
1
7.2
3.7
40.6
48.0
29.5
18.7
17.4
37.8
39.8
61.7
61.1
15.0
n/a
n/a
2
2.3
n/a
4
3.5
13.2
48.5
24
19.1
Table 5. The rate of formation of the active ester of Fmoc-Val-OH 22 using ethyl 2-cyano-
2-(isobutoxycarbonyloxyimino)acetate (17).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Val-OH 22 Active ester 27 Isobutyl ester
½
1
3.1
0.9
54.7
45.9
37.2
27.3
16.0
33.6
44.3
54.8
63.5
21.9
n/a
n/a
2
1.4
1.9
4
2.6
6.7
24
16.8
45.3

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Table 6. The rate of formation of the active ester of Fmoc-Val-OH 22 using ethyl
2-(allyloxycarbonyloxyimino)-2-cyanoacetate (18).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Val-OH 22
Active ester 27 Allyl ester
½
1
n/a
n/a
9.4
14.2
5.7
63.9
58.1
73.0
55.9
10.8
3.7
6.1
n/a
2
n/a
4
23.6
2.8
Table 7. The rate of formation of the active ester of Fmoc-Val-OH 22 using ethyl
2-(benzyloxycarbonyloxyimino)-2-cyanoacetate (19).
Pre-activation time (hr)
Oxyma 12a
1.5
Fmoc-Val-OH 22 Active ester 27 Benzyl ester
½
1
40.4
28.1
19.0
14.1
14.1
52.5
53.7
48.4
32.2
2.1
n/a
7.5
3.4
2
6.9
13.1
20.2
31.4
4
11.6
24
22.1
2.3.2. The Rate of Formation of the Active Ester of Fmoc-Phe-OH Using Oxime Carbonate Derivatives
Due to the best results obtained from the previous example with the oxime carbonate derivatives 17
and 18, Fmoc-Phe-OH 23 was tested with iBuocOXY 17 and AllocOXY 18 under the same conditions
used in the previous example. From the results obtained from HPLC monitoring, the maximum levels of
the active ester 27 are formed from the oxime carbonate derivatives 17 and 18 at 1 and 2 h, respectively;
while the alkyl esters 26 appeared after 30 min and 1 hour, respectively. Thus, the pre-activation time
in both systems should not exceed more than 30 min (Tables 8 and 9).
Table 8. The rate of formation of the active ester of Fmoc-Phe-OH 23 using ethyl
2-cyano-2-(isobutoxycarbonyloxyimino)acetate (17).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Phe-OH 23
Active ester 27 Isobutyl ester
½
1
n/a
0.8
0.5
0.8
3.5
28.3
15.8
19.5
16.2
21.4
43.0
72.0
58.2
63.3
52.7
14.7
8.4
2
6.1
4
1.5
24
22.0
Table 9. The rate of formation of the active ester of Fmoc-Phe-OH 23 using ethyl
2-(allyloxycarbonyloxyimino)-2-cyanoacetate (18).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Phe-OH 23
Active ester 27 Allyl ester
½
1
4.7
4.6
24.7
22.3
12.6
19.2
4.6
58.56
65.9
74.6
68.3
53.4
0.4
0.6
2
5.5
4
5.8
0.4
24
13.5
25.4

Molecules 2012, 17
14368
2.3.3. The Rate of Formation of the Active Ester of Fmoc-Pro-OH Using Oxime Carbonate Derivatives
To ensure that we will get the same results with the oxime carbonate derivatives 17 and 18, further
study was performed with the more sterically hindered amino acid Fmoc-Pro-OH 24 using the two
i
carbonate derivatives BuocOXY 17 and AllocOXY 18. From the results obtained by HPLC
monitoring, we observed the maximum levels of the active ester 27 after 30 min and 2 h, respectively;
while the alkyl esters 26 appeared after 30 min and 1 hour, respectively. Thus, the pre-activation time
in both systems should not exceed more than 30 min, which in agreement with the previous results
(Tables 10 and 11).
Table 10. The rate of formation of the active ester of Fmoc-Pro-OH 24 using ethyl
2-Cyano-2-(isobutoxycarbonyloxyimino)acetate (17).
Pre-activation time (hr)
Oxyma 12a
Fmoc-Pro-OH 24
Active ester 27 Isobutyl ester
½
1
0.2
1.6
13.4
47.4
30.7
30.8
75.9
59.8
34.1
53.9
55.8
6.3
5.0
6.7
3.1
3.8
n/a
2
1.3
4
1.6
24
17.8
Table 11. The rate of formation of the active ester of Fmoc-Pro-OH 24 using ethyl
2-(allyloxycarbonyloxyimino)-2-cyanoacetate (18).
Pre-activation time (hr)
Oxyma 12a
1.4
Fmoc-Pro-OH 24
Active ester 27 Allyl ester
½
1
13.2
13.3
15.2
58.0
46.7
65.7
70.3
70.6
24.3
18.7
n/a
2.8
1.8
2
3.0
2.7
4
13.8
3.9
24
19.4
15.2
The results obtained from Tables 4–11 may indicate that, the activation of carboxylic acid group of
the amino acid using oximinocarbonate derivatives may proceed through a mixed anhydride-type
intermediate 25, which may react with the oxyma anion to afford the active ester 27 (Scheme 5). Its
decarboxylation will afford alkyl or aryl esters 26, which is a rather slow step. All intermediate stages
of the reaction are run at low temperature to prevent side reactions.
Scheme 5. Mechanism of the formation of the oxime active ester using oxyme carbonates.

Molecules 2012, 17
14369
2.4. Synthesis of Dipeptide Fmoc-Val-Ala-OMe
As an initial model to examine the reactivity of the oxime carbonate 13a, 17, 18 and 19 as coupling
reagents, these reagents were examined in the stepwise coupling of a previously studied model system [29]
Fmoc-Val-Ala-OMe 28. The pre-activation time was determined for each of these coupling reagents
according to the previous studies obtained from HPLC for the rate of formation of active ester. All
coupling reactions were performed in the presence of 2 equiv. pyridine as a base and in DMF as
solvent. The results for each coupling reagent are given in Table 12.
Table 12. Coupling of Fmoc-Val-OH with H-Ala-OMe 28 using different oxime carbonate derivatives.
Coupling reagent
EtocOXY 13a
iBuocOXY 17
AllocOXY 18
ZOXY 19
Pre-activation time (hr)
m.p. (°C)
195–196
192–193
194–195
194–195
Yield (%)
HPLC Purity (%)
2
1
77
54
75
64
100
96.1
98.7
97.7
½
½
The purity of the dipeptide 28 was determined by HPLC, and was found to be 96.1 to 100% at
tR LL [Fmoc-Val-Ala-OMe] = 8.32 min. From Table 12, the highest yield and purity was obtained with
i
EtocOXY 13a, while the lowest yield and purity was obtained with BuocOXY 17 and ZOXY 19.
While, the AllocOXY 18 had moderate yield and purity. None of the D,L-isomer was detected by
HPLC or NMR spectra in all the cases, but the impurities were related to the alkyl ester and oxime.
3. Experimental
3.1. Materials
The solvents used were of HPLC reagent grade. Melting points were determined with a Mel-Temp
apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer 1600 series
Fourier transform instrument as KBr pellets. Nuclear Magnetic resonance spectra (1H-NMR and
13C-NMR spectra) were recorded on a JOEL 500 MHz and on a Mercury 400 MHz spectrometer with
chemical shift values reported in δ units (ppm) relative to an internal standard. Elemental analyses
were performed on Perkin-Elmer 2400 elemental analyzer, and the values found were within ±0.3% of
the theoretical values. Follow-up of the reactions and checks of the purity of the compounds was done
by TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck, Barcelona, Spain) and the
spots were detected by exposure to UV-lamp at λ 254 nm for a few seconds. The compounds were
named using ChemDraw Ultra version 11, CambridgeSoft Corporation (Cambridge, MA, USA).
3.2. General Method for Preparation of Ethyloxycarbonyl Derivatives 13(a–d, f)
A solution of ethyloxycarbonyl chloride (11, 0.95 mL, 10 mmol) in CH2Cl2 (30 mL) was added
slowly to a solution of sodium carbonate (2.12 g, 20 mmol) and 10 mmol of oximes (12a, 12b),
1-hydroxypyridin-2(1H)-one (12c), or benzotriazole derivatives (12d or 12f) in H2O (20 mL) with
stirring at 0 °C. The resulting clear mixture was stirred at 0°C for 30 min and then at room temperature
for 2 h. After dilution with CH2Cl2 (50 mL), the organic phase was collected and washed with water

Molecules 2012, 17
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and saturated aqueous NaCl (30 mL), dried over anhydrous Na2SO4 and then filtered, and the solvent
was then removed on a rotary evaporator. The residue was recrystallized from CH2Cl2/hexane to give
the ethyloxycarbonyl derivatives 13(a–d, f).
Ethyl 2-cyano-2-(ethoxycarbonyloxyimino)acetate (13a). The product was obtained as white crystals
(1.67 g; 78.17% yield) (m.p. 44–45 °C). IR (KBr): 2241 (w, CN), 1812 (s, CO), 1741 (s, CO, ester)
cm−1. 1H-NMR (CDCl3): δ 1.39–1.41 (m, 6H, 2 CH3), 4.43–4.47 (m, 4H, 2 CH2). 13C-NMR (CDCl3): δ
14.14, 14.26, 64.76, 67.33, 106.67, 131.03, 150.87, 156.81. Elemental analysis Calcd for C8H10N2O5:
C, 44.86; H, 4.71; N, 13.08. Found: C, 45.08; H, 4.63; N, 13.17.
(Ethoxycarbonyloxy)carbonimidoyl dicyanide (13b). The product was obtained as an oil at room
1
temperature (1.15 g; 68.89% yield). IR (KBr): 2248 (w, CN), 1811 (s, CO, ester) cm−1. H-NMR
(CDCl3): δ 1.43 (t, 3H, 3J = 7.2 Hz, CH3), 4.49 (q, 2H, 3J = 7.2 Hz, CH2). 13C-NMR (CDCl3): δ 14.18,
68.32, 104.81, 108.03, 114.44, 149.70. Elemental analysis: Calcd for C6H5N3O3: C, 43.12; H, 3.02; N,
25.14. Found: C, 43.25; H, 2.89; N, 25.33.
Ethyl 2-oxopyridin-1(2H)-yl carbonate (13c). The product was obtained as white crystals (1.38 g;
75.47% yield) (m.p. 64–67 °C). IR (KBr): 1792 (s, CO), 1668 (s, CO, amidic) cm−1. 1H-NMR (CDCl3): δ
3
4
1.42 (t, 3H, 3J = 7.2 Hz, CH3), 4.42 (q, 2H, J = 7.2 Hz, CH2), 6.20 (td, 1H, 3J = 6.8 Hz, J = 1.6 Hz,
Py-H), 6.72–6.74 (m, 1H, Py-H), 7.36 (td, 1H, 3J = 6.8 Hz, 4J = 2 Hz, Py-H), 7.46 (dd, 1H, 3J = 6.8 Hz,
4J = 2 Hz, Py-H). 13C-NMR (CDCl3): δ 14.25, 67.53, 105.29, 123.18, 135.14, 139.69, 152.45, 157.31.
1H-Benzo[d][1,2,3]triazol-1-yl ethyl carbonate (13d). The product was obtained as white crystals (1.07 g;
1
51.84% yield) (m.p. 138–139 °C). IR (KBr): 1751 (s, CO) cm−1. H-NMR (CDCl3): δ 1.53 (t, 3H,
3J = 7.2 Hz, CH3), 4.63 (q, 2H, 3J = 7.2 Hz, CH2), 7.56 (td, 1H, 3J = 8.4 Hz, 4J = 0.8 Hz, Ar-H), 7.78
(td, 1H, 3J = 8.4 Hz, 4J = 1.2 Hz, Ar-H), 8.00 (d, 1H, 3J = 8.4 Hz, Ar-H), 8.21 (d, 1H, 3J = 8.4 Hz, Ar-H).
13C-NMR (CDCl3): δ = 14.42, 65.67, 115.27, 115.88, 126.46, 132.91, 133.54, 147.52. Elemental
analysis: Calcd for C9H9N3O3: C, 52.17; H, 4.38; N, 20.28. Found: C, 51.96; H, 4.54; N, 20.49.
6-Chloro-1H-benzo[d][1,2,3]triazol-1-yl ethyl carbonate (13f). The product was obtained as white
crystals (1.81 g; 75.67% yield) (m.p. 144–145 °C). IR (KBr): 1743 (s, CO) cm−1. 1H-NMR (CDCl3): δ
1.53 (t, 3H, 3J = 7.2 Hz, CH3), 4.62 (q, 2H, 3J = 7.2 Hz, CH2), 7.72 (dd, 1H, 3J = 8.8 Hz, 4J = 2 Hz, Ar-H),
4
3
13
8.00 (d, 1H, J = 2 Hz, Ar-H), 8.16 (d, 1H, J = 8.8 Hz, Ar-H). C-NMR (CDCl3): δ 14.42, 65.99,
115.70, 116.32, 132.19, 132.81, 133.78, 147.32. Elemental analysis: Calcd for C9H8ClN3O3: C, 44.74;
H, 3.34; N, 17.39. Found: C, 44.53; H, 3.61; N, 17.18.
3.3. 3H-[1,2,3]Triazolo[4,5-b]pyridin-3-yl Ethyl Carbonate (13e)
A solution of HOAt (12e, 0.68 g, 5 mmol) and anhydrous potassium hydroxide (0.3 g, 5.5 mmol) in
acetonitrile (5 mL) was cooled to 0 °C. A solution of ethyloxycarbonyl chloride (11, 0.47 mL, 5 mmol)
in acetonitrile (5 mL) was slowly added dropwise for 30 min to the solution as it was stirred
magnetically. The resulting clear mixture was stirred at room temperature overnight. It was then
filtered, and the solvent was removed with a rotary evaporator. The residue was recrystallized from
CH2Cl2/hexane to give 3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl ethyl carbonate (13e). The product was

Molecules 2012, 17
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obtained as 0.8 g (77.14% yield) of white crystals (m.p. 133–135 °C). IR (KBr): 1744 (s, CO) cm−1.
1H-NMR (DMSO): δ 1.05 (t, 3H, 3J = 7.2 Hz, CH3), 3.43 (q, 2H, 3J = 7.2 Hz, CH2), 7.49–7.53 (m, 1H,
Ar-H), 8.52–8.55 (m, 1H, Ar-H), 8.75–8.77 (m, 1H, Ar-H), 13C-NMR (DMSO): δ 18.47, 55.94,
120.62, 124.09, 134.52, 139.50, 151.01. Elemental analysis: Calcd for C8H8N4O3: C, 46.16; H, 3.87;
N, 26.91. Found: C, 45.88; H, 4.14; N, 27.19.
3.4. General Method for Preparation of Oxime Carbonate Derivatives 17–19
A solution of chloroformate (10 mmol) [isobutyloxycarbonyl chloride (14), allyloxycarbonyl
chloride (15) or benzyloxycarbonyl chloride (16)] in CH2Cl2 (30 mL) was added slowly to a solution
(10 mmol) of oxima 12a and sodium carbonate (2.12 g, 20 mmol) in H2O (20 mL) with stirring at 0 °C.
The resulting clear mixture was stirred at 0 °C for 30 min and then at room temperature for 2 h. After
dilution with CH2Cl2 (50 mL), the organic phase was collected, washed with water and saturated
aqueous NaCl (30 mL), and then dried over anhydrous MgSO4. It was then filtered, and the solvent
was removed with a rotary evaporator. The residue was recrystallized from CH2Cl2/hexane to give
oxime carbonate derivatives 17–19.
Ethyl 2-cyano-2-(isobutoxycarbonyloxyimino)acetate (17). The product was obtained as a white solid
1
(2.42 g; 93% yield) (m.p. 59–60 °C). IR (KBr): 1814 (s, CO), 1758 (s, CO, ester) cm−1. H-NMR
(CDCl3): δ 1.00 (d, J = 6.8 Hz, 6H, 2 CH3), 1.42 (t, J = 7.2 Hz, 3H, CH3), 2.06–2.13 (m, 1H, CH), 4.17
13
(d, J = 6.4 Hz, 2H, CH2), 4.50 (q, J = 7.2 Hz, 2H, CH2). C-NMR (CDCl3): δ 14.16, 18.89, 27.95,
64.75, 106.70, 130.97, 151.10, 156.84. Elemental analysis: Calcd for C10H14N2O5: C, 49.58; H, 5.83;
N, 11.56. Found: C, 49.81; H, 5.57; N, 11.74. The purity of 17 was determined after injection onto
reverse-phase HPLC. Conditions: detection at 254 nm Waters 996 PDA detector, Sunfire C18 column
3.5 µm 4.6  100 mm, linear gradient over 14 min of 10 to 100% CH3CN in H2O/0.1% TFA, flow rate
1.0 mL/min. tR [ethyl 2-cyano-2-(isobutoxycarbonyloxyimino)acetate] = 7.38 min; purity 100%.
Ethyl 2-(allyloxycarbonyloxyimino)-2-cyanoacetate (18). The product was obtained as an oily
substance that solidified in the refrigerator (2.26 g; 94% yield). IR (KBr): 2211 (w, CN), 1809 (s, CO),
1758 (s, CO, ester) cm−1. 1H-NMR (CDCl3): δ 1.42 (t, 3H, 3J = 7.2 Hz, CH3), 4.48 (q, 2H, 3J = 7.2 Hz,
CH2), 4.85–4.87 (m, 2H, CH2), 5.39–5.51 (m, 2H, CH2), 5.96–6.03 (m, 1H, CH). 13C-NMR (CDCl3): δ
14.11, 64.78, 71.23, 106.62, 121.27, 130.07, 131.24, 150.74, 156.73. Elemental analysis. Calcd for
C9H10N2O5: C, 47.79; H, 4.46; N, 12.39. Found: C, 47.93; H, 4.61; N, 12.58. The purity of 18 was
determined after injection onto reverse-phase HPLC. Conditions: detection at 254 nm Waters 996 PDA
detector, Sunfire C18 column 3.5 µm 4.6  100 mm, linear gradient over 14 min of 10 to 100% CH3CN in
H2O/0.1% TFA, flow rate 1.0 mL/min. tR [ethyl 2-(allyloxycarbonyloxyimino)-2-cyanoacetate] = 6.69 min;
purity 100%.
Ethyl 2-(benzyloxycarbonyloxyimino)-2-cyanoacetate (19). The product was obtained as white crystals
1
(2.76 g; 87% yield) (m.p. 99–100 °C). IR (KBr): 1802 (s, CO), 1743 (s, CO, ester) cm−1. H-NMR
(CDCl3): δ 1.41 (t, 3H, 3J = 7.2 Hz, CH3), 4.47 (q, 2H, 3J = 7.2 Hz, CH2), 5.38 (s, 2H, CH2), 7.40–7.44
(m, 5H, Ar-H). 13C-NMR (CDCl3): δ 14.17, 64.80, 72.57, 106.63, 129.06, 129.23, 129.63, 131.23,
133.59, 151.00, 156.76. Elemental analysis: Calcd for C13H12N2O5: C, 56.52; H, 4.38; N, 10.14.

Molecules 2012, 17
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Found: C, 56.23; H, 4.62; N, 10.41. The purity of 19 was determined after injection onto reverse-phase
HPLC. Conditions: detection at 254 nm Waters 996 PDA detector, Sunfire C18 column 3.5 µm
4.6  100 mm, linear gradient over 14 min of 10 to 100% CH3CN in H2O/0.1% TFA, flow rate
1.0 mL/min. tR [ethyl 2-(benzyloxycarbonyloxyimino)-2-cyanoacetate] = 7.31 min; purity 100%.
3.5. Synthesis of 4-(Ethoxycarbonylamino)benzoic Acid (21)
A solution of ethyloxycarbonyl derivative 13(a–f) (1 mmol) in acetone (10 mL) was added
dropwise to a stirring solution of 4-aminobenzoic acid 20 (0.14 g, 1 mmol) and sodium carbonate
(0.32 g, 3 mmol) in acetone (20 mL) and H2O (10 mL). After stirring overnight, the reaction mixture
was concentrated under reduced pressure, and then extracted with CH2Cl2 (20 mL) to remove the
unreacted ethyloxycarbonyl derivatives. The reaction mixture was acidified with 1 N HCl (detected
with Congo red litmus paper) to give a white solid, which was filtered, washed with water several
times, dried and then recrystallized (ethyl acetate/n-hexane) to give a white solid. The purity of 21 was
determined by reverse-phase HPLC. Conditions: detection at 220 nm (Waters 996 PDA detector);
Sunfire C18 column (3.5 µm 4.6 × 100 mm); linear gradient over 14 min (10 to 100% CH3CN in
H2O/0.1% TFA); flow rate 1.0 mL/min. tR [4-(ethoxycarbonylamino)benzoic acid] = 4.18 min. IR (KBr):
3334 (w, NH), 3400–2500 (br, OH, acid), 1704 (s, CO, acidic), 1686 (s, CON) cm−1. 1H-NMR (DMSO):
3
3
3
δ 1.24 (t, 3H, J = 7.2 Hz, CH3), 4.13 (q, 2H, J = 7.2 Hz, CH2), 7.54 (d, 2H, J = 8.4 Hz, Ar-H), 7.82
(d, 2H, J = 8.4 Hz, Ar-H), 9.93 (s, 1H, NH). 13C-NMR (DMSO): δ 15.11, 61.11, 117.91, 124.91,
3
131.05, 144.12, 153.00, 167.63.
3.6. HPLC Study of the Rate of Formation of Active Esters
3.6.1. The Rate of Formation of the Active Ester of Fmoc-Val-OH 22 Using Oxime Carbonate
Derivatives 13a, 17–19
A solution of Fmoc-Val-OH 22 (0.0423 g, 0.125 mmol) and the oxime carbonate derivatives 13a,
17, 18 or 19 (0.125 mmol) was dissolved in DMF (2 mL) in the presence of pyridine (20 µL). The
reaction was monitored by HPLC. Aliquots (5 µL) were taken from the reaction mixture, diluted with
ACN and detected by HPLC. Follow-ups were done at 30 min and at 1, 2, 4 and 24 h pre-activation.
The percentages of OxymaPure® 12a, Fmoc-Val-OH 22, active ester 27, and alkyl or aryl ester 26
were determined by HPLC analysis of the diluted reaction mixture. Conditions: detection at 254 nm
(Waters 996 PDA detector); Sunfire C18 column (3.5 µm 4.6  100 mm); linear gradient over 14 min
(10 to 100% CH3CN in H2O/0.1% TFA); flow rate 1.0 mL/min. tR [active ester] = 8.4 min. The
percentages of OxymaPure® 12a, Fmoc-Val-OH 22, active ester 27, and alkyl or aryl esters 26 are
shown in Tables 4–7.
3.6.2. The Rate of Formation of the Active Ester of Fmoc-Phe-OH 23 Using Oxime Carbonate
Derivatives 17 or 18
A solution of Fmoc-Phe-OH 23 (0.0483 g, 0.125 mmol) and oxime carbonate derivative 17 or 18
(0.125 mmol) was dissolved in DMF (2 mL) in the presence of pyridine (20 µL). The reaction was
monitored by HPLC. Aliquots (5 µL) were taken from the reaction mixture, diluted with ACN and

Molecules 2012, 17
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detected by HPLC. Follow-ups were done at 30 min and at 1, 2, 4 and 24 h pre-activation. The
percentages of OxymaPure® (12a), Fmoc-Phe-OH 23, active ester 27 and alkyl ester 26 were
determined by HPLC analysis of the diluted reaction mixture. Conditions: detection at 254 nm (Waters
996 PDA detector); Sunfire C18 column (3.5 µm 4.6  100 mm); linear gradient over 14 min (10 to
100% CH3CN in H2O/0.1% TFA); flow rate 1.0 mL/min. tR [active ester] = 8.4 min. The percentages
of OxymaPure® (12a), Fmoc-Phe-OH 23, active ester 27 and alkyl esters 26 are shown in Tables 8 and 9.
3.6.3. The Rate of Formation of the Active Ester of Fmoc-Pro-OH 24 Using Oxime Carbonate
Derivatives 17 or 18
A solution of Fmoc-Pro-OH 24 (0.0421 g, 0.125 mmol) and oxime carbonate derivative 17 or 18
(0.125 mmol) was dissolved in DMF (2 mL) in the presence of pyridine (20 µL). The reaction was
monitored by HPLC. Aliquots (5 µL) were taken from the reaction mixture, diluted with ACN and
analyzed by HPLC. Follow-ups were done at 30 min and at 1, 2, 4 and 24 h pre-activation. The
percentages of OxymaPure® (12a), Fmoc-Pro-OH 24, active ester 27 and alkyl ester 26 were
determined by HPLC analysis of the diluted reaction mixture. Conditions: detection at 254 nm Waters
996 PDA detector, Sunfire C18 column 3.5 µm 4.6  100 mm, linear gradient over 14 min of 10 to
100% CH3CN in H2O/0.1% TFA, flow rate 1.0 mL/min. tR [active ester] = 8.2 min. The percent of
Oxyma 12a, Fmoc-Pro-OH 24, active ester 27 and alkyl esters 26 are shown in Tables 10 and 11.
3.7. General Method for the Synthesis of Dipeptide Fmoc-Val-Ala-OMe 28
A solution of Fmoc-Val-OH 22 (0.339 g, 1 mmol) and the appropriate coupling reagent (1 mmol) in
DMF (2 mL) was cooled to 0 °C and treated dropwise with pyridine (0.088 mL, 1.1 mmol). The
reaction mixture was stirred for pre-activation at different times, depending on the conditions of the
entry studied, and then treated with a solution of H-Ala-OMe.HCl (0.139 g, 1 mmol) and pyridine
(0.088 mL, 1.1 mmol) in DMF (1 mL). The reaction mixture was stirred overnight. After dilution with
25 mL of ethyl acetate, the organic phase was washed with 5% citric acid (3 × 15 mL), saturated aq.
NaHCO3 (3 × 15 mL) and saturated aq. NaCl (3 × 15 mL), and then dried over anhydrous Na2SO4 and
filtered. The solvent was removed with a rotary evaporator, and the residue was recrystallized from
CH2Cl2/hexane to give the dipeptide Fmoc-Val-Ala-OMe 28. The purity of 28 was by reverse-phase
HPLC. Conditions: detection at 220 nm (Agilent 1200 PDA detector); Eclipse plus C18 column
(3.5 µm 4.6 × 100 mm); linear gradient over 14 min (10 to 100% CH3CN in H2O/0.1% TFA); flow
rate 1.0 mL/min. tR LL [Fmoc-Val-Ala-OMe] = 8.32 min. The results of coupling of Fmoc-Val-OH
1
with H-Ala-OMe using different oximinocarbonate derivatives are shown in Table 12. H-NMR
3
(CDCl3): δ 0.94–0.98 (m, 6H, 2CH3), 1.40 (d, 3H, J = 6.9 Hz, CH3), 2.10–2.11 (m, 1H, CH), 3.73
(s, 3H, CH3), 3.90–4.00 (m, 1H, CH), 4.17–4.21 (m, 1H, CH), 4.34–4.43 (m, 2H, CH2), 4.55–4.58
(m, 1H, CH), 5.47–5.51 (m, 1H, NH), 6.42–6.45 (m, 1H, NH), 7.25–776 (m, 8H, Ar-H).
4. Conclusions
Protection of the amino group and activation of the carboxylic acid groups are the most important
steps associated with peptide synthesis. A possible strategy is to use oxime carbonate derivatives to

Molecules 2012, 17
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simultaneously protect the amino group as a carbamate derivative and activate the carboxylic acid
group as an active oxime ester was performed. A detailed study is carried out to understand the scope
and limitations of this method using different oxime carbonate derivatives. The efficiency of these
derivatives depends on the nature of oxime carbonates and also on the nature of the amino acids. From
our studies we determined that the new family of oximes are useful reagents for both N-protection and
i
activation of the protected amino acid. As a final conclusion from our studies, the BuocOXY
compound 17 and AllocOXY compound 18 both give the best results for formation of the active ester
with less alkyl ester formation, while the EtocOXY compund 13a gave the best results for the coupling
step. The ZOXY reagent 19 might be not useful in either the activation or coupling steps.
Acknowledgments
In Spain, the work was partially financed by CICYT (CTQ2009-07758), the Generalitat de
Catalunya (2009SGR 1024), the Institute for Research in Biomedicine Barcelona (IRB Barcelona) and
the Barcelona Science Park. The Science and Technology Development Fund (STDF) in Egypt is
thanked for its partial support through the Research Project TC/12/RSG/2012 (Proposal ID (4769)).
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Sample Availability: Samples of the most part of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).