Oxyma: An efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion

Article abstract of DOI:10.1002/chem.200900614

Oxyma [ethyl 2-cyano-2-(hydroxyimino)acetate] has been tested as an additive for use in the carbodiimide approach for formation of peptide bonds. Its performance in relation to those of HOBt and HOAt, which have recently been reported to exhibit explosive properties, is reported. Oxyma displayed a remarkable capacity to inhibit racemization, together with im-pressive coupling efficiency in both automated and manual synthesis, superior to those of HOBt and at least comparable to those of HOAt, and surpassing the latter coupling agent in the more demanding peptide models. Stability assays showed that there was no risk of capping the resin under standard coupling conditions. Finally, calorimetry assays (DSC and ARC) showed decomposition profiles for benzotriazolebased additives that were consistent with their reported explosivities and suggested a lower risk of explosion in the case of Oxyma.

Full text of DOI:10.1002/chem.200900614

DOI: 10.1002/chem.200900614
Oxyma: An Efficient Additive for Peptide Synthesis to Replace the
Benzotriazole-Based HOBt and HOAt with a Lower Risk of Explosion[1]
Ramon Subirꢀs-Funosas,[a, b] Rafel Prohens,[c] Rafael Barbas,[c]
Ayman El-Faham,*[a, d, e] and Fernando Albericio*[a, b, f]
Abstract: Oxyma [ethyl 2-cyano-2-(hy-
droxyimino)acetate] has been tested as
an additive for use in the carbodiimide
approach for formation of peptide
bonds. Its performance in relation to
those of HOBt and HOAt, which have
recently been reported to exhibit ex-
plosive properties, is reported. Oxyma
displayed a remarkable capacity to in-
hibit racemization, together with im-
pressive coupling efficiency in both au-
tomated and manual synthesis, superior
to those of HOBt and at least compa-
rable to those of HOAt, and surpassing
the latter coupling agent in the more
demanding peptide models. Stability
assays showed that there was no risk of
capping the resin under standard cou-
pling conditions. Finally, calorimetry
assays (DSC and ARC) showed de-
composition profiles for benzotriazole-
based additives that were consistent
with their reported explosivities and
suggested a lower risk of explosion in
the case of Oxyma.
Keywords: additives · calorimetry ·
oximes · peptide synthesis · solid-
phase synthesis
Introduction
peptide chemistry. Of these compounds, the most extensive-
ly used displayed a benzotriazole core: 1-hydroxybenzotria-
zole (HOBt), probably the most common reagent found in a
peptide synthesis laboratory, was the first to be unveiled in
the early 1970s,[2] whereas later on the use of the more pow-
erful 1-hydroxy-7-azabenzotriazole (HOAt)[3] and, more re-
cently, 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt) for the
same purpose was reported.[4] The presence of any of these
compounds in the coupling medium induces the formation
of an active ester, which then undergoes aminolysis to yield
the desired peptide bond. The acidities of the additives (pKa
for HOBt: 4.60, pKa for HOAt: 3.28, and pKa for 6-Cl-
HOBt: 3.35)[5,6] are key factors with regard to the stabilities
and reactivities of their related active esters, because they
are connected with their behavior as leaving groups. In addi-
tion, the nitrogen at the 7-position in HOAt provides a clas-
sic neighboring group effect that can both increase the reac-
tivity and reduce racemization.[3]
The use of additives to support various coupling methodolo-
gies is common practice in research laboratories devoted to
[a] R. Subirꢀs-Funosas, Prof. A. El-Faham, Prof. F. Albericio
Institute for Research in Biomedicine, Barcelona Science Park
Baldiri Reixac 10, 08028 Barcelona (Spain)
Fax : (+34)93-403-71-26
E-mail: aymanel_faham@hotmail.com
albericio@irbbarcelona.org
[b] R. Subirꢀs-Funosas, Prof. F. Albericio
CIBER-BBN, Networking Centre on Bioengineering
Biomaterials and Nanomedicine, Barcelona Science Park
Baldiri Reixac 10–12, 08028 Barcelona (Spain)
[c] R. Prohens, R. Barbas
Plataforma de Polimorfisme i Calorimetria
Serveis Cientificotꢁcnics, University of Barcelona
Barcelona Science Park
Baldiri Reixac 10–12, 08028 Barcelona (Spain)
The carbodiimide approach to formation of peptide bonds
has long taken advantage of the properties of benzotriazole-
based additives, because the active esters formed are less re-
active but more stable than the O-acylisoureas. These inter-
mediate species lead to lower levels of racemization and the
suppression of other undesired side reactions such as the
formation of inactive N-acylisoureas.[7] Other coupling strat-
egies, such as the combination of base and stand-alone cou-
pling reagents, such as immonium (HATU, HBTU/TBTU,
and HCTU/TCTU) or phosphonium salts (PyAOP, PyBOP,
[d] Prof. A. El-Faham
Department of Chemistry, College of Science
King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia)
[e] Prof. A. El-Faham
Department of Chemistry, Faculty of Science
Alexandria University, Ibrahimia 21321, Alexandria (Egypt)
[f] Prof. F. Albericio
Department of Organic Chemistry, University of Barcelona
Martꢂ i Franquꢃs 1–11, 08028 Barcelona (Spain)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900614.
9394
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Chem. Eur. J. 2009, 15, 9394 – 9403

FULL PAPER
and PyClock) have also been enhanced by the use of these
additives.[8–10]
in two-phase systems),[18] although giving a higher yield of
target dipeptide than HOAt and HOBt (Table 1, entry 5 vs
entries 1 and 2), gave poorer retention of configuration
However, a potentially explosive character of HOBt and
its related additives has recently been reported.[11] This ob-
servation has led to their reclassification under a Class 1 ex-
plosive category and has consequently increased transporta-
tion difficulties. In view of the relevance of these com-
pounds in day-to-day peptide chemistry, it became evident
that there was a need for another family of safe and efficient
additives, based on a different template. This poses a diffi-
cult challenge if it is borne in mind that the performances of
additives based on other compounds, such as N-hydroxysuc-
cinimide (HOSu) or pentafluorophenol (HOPfp), are not
comparable to those of HOBt and HOAt, because active
esters of OSu or OPfp esters are less reactive than benzo-
triazole esters.[12–14]
Here we report an exhaustive study of ethyl 2-cyano-2-
(hydroxyimino)acetate, an oxime first described in the 1970s
with an acidity similar to those of the above additives
(pKa 4.60).[15] The suitability of this compound as a substi-
tute for benzotriazole-based additives is discussed in terms
of its capacity to control racemization, its effectiveness in
difficult couplings either in manual or automated synthesis,
and its stability in the presence of growing peptide chains.
We have also evaluated the safety profile of this additive by
calorimetric techniques.
Table 1. Yields and racemization during the formation of Z-Phg-Pro-
NH2 in DMF (stepwise solution-phase synthesis).[a]
Entry
Coupling reagent
Yield [%]
dl [%]
1
2
3
4
5
6
HOAt/DIC
HOBt/DIC
81.4
81.9
89.9
88.2
88.2[d]
83.4
3.3
9.3
1.0
1.1
17.4
26.1
Oxyma/DIC[b]
Oxyma/DIC[c]
HOPO/DIC
HOPO/DIC[c]
[a] Couplings were conducted without preactivation, except for entries 4
and 6. ll and dl epimers of the test dipeptide have been described else-
where.[17] The tR values of ll and dl were identified by coinjection with
pure samples of ll. [b] Unreacted Z-Phg-OH was detected. [c] A 2 min
preactivation time was used. [d] Extra peak was found at 22.4 min
(6.0%).
(Table 1), especially when a 2 min preactivation time was
used (Table 1, entry 6). In contrast, the performance of
Oxyma (Table 1, entry 3) exceeded not only that of HOPO,
but also those of HOBt and even HOAt (1.0%). Neverthe-
less, in the experiment conducted without preactivation,
some of the starting acid remained unreacted. This incon-
venience was overcome by use of a 2 min preactivation
period (Table 1, entry 4). Moreover, the outstanding lack of
racemization was maintained (1.1%).
Results and Discussion
In the case of the more racemization-prone [2+1] seg-
ment coupling (Z-Phe-Val-OH onto H-Pro-NH2), a similar
trend was observed (Table 2). HOPO was the poorest race-
mization-suppressing additive out of the compounds tested,
with a percentage of dl epimer of nearly 50% (Table 2,
entry 4). In view of the results with Oxyma in the stepwise
model, only the 2 min preactivation experiment was per-
formed. In the segment model, the degree of racemization
was clearly lower than that seen with HOBt and comparable
to that achieved with HOAt (Table 2, entry 3 vs entries 1
and 2). Moreover, the yield obtained with Oxyma was
higher (almost 90%).
In our search for a class of safe and efficient additives, we
came across a family of strongly acidic oximes that showed
properties of interest, reported by Itoh in the early 1970s
and by Izdebsky a few years later.[15,16] Out of all the oximes
tested in those studies, ethyl 2-
cyano-2-(hydroxyimino)acetate
(Figure 1, from now on referred
to as Oxyma) displayed an ap-
propriate balance of availability
Figure 1. Structure of ethyl 2-
and ease of handling. Nonethe-
less, since the publication of
those papers, which were not
conclusive about its perfor-
cyano-2-(hydroxyimino)acetate
(Oxyma).
Table 2. Yields and racemization during the formation of Z-Phe-Val-Pro-
NH2 in DMF (segment solution-phase synthesis).[a]
mance relative to those of benzotriazoles, Oxyma has not
played an active role in day-to-day peptide chemistry, nor
has it been used for general amide bond formation.
Deeper investigation into racemization and the com-
pound’s effectiveness for coupling yields were therefore car-
ried out. For study of racemization, peptide models (Z-Phg-
Pro-NH2 and Z-Phe-Val-Pro-NH2 in solution and H-Gly-
Cys-Phe-NH2 on solid-phase) more reliable than those
chosen in the previous papers were used for comparison of
HOAt, HOBt, N-hydroxy-2-pyridinone (HOPO), and
Oxyma.[17]
Entry
Coupling reagent
Yield [%]
dl [%]
1
2
3
4
HOAt/DIC
HOBt/DIC
Oxyma/DIC[b]
HOPO/DIC
86.1
78.8
89.8
88.5
2.1
8.9
3.8
45.1
[a] Couplings were conducted without preactivation, except in the case of
entry 3. lll and ldl forms have been described elsewhere[17] and were
coinjected with authentic and pure samples. [b] A 2 min preactivation
time was used.
With regard to stepwise coupling (Z-Phg-OH onto H-Pro-
NH2), the non-benzotriazole-based additive HOPO (se-
lected due to its association with low levels of racemization
Further racemization experiments were carried out with
regard to loss of configuration during elongation of Cys-con-
taining peptides in the solid-phase approach (Table 3). For
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F. Albericio, A. El-Faham et al.
that purpose, the model tripeptide H-Gly-Cys-Phe-NH2 was
assembled in stepwise fashion on a Fmoc-RinkAmide-
MBHA-PS resin (0.45 mmolgÀ1), with use of a 5 min preac-
Gly, or des-Asn) were the most common, but misincorpora-
tions of two amino acids also occurred (see Table 4). HOAt
was the most efficient additive, with more than a 70% yield
of ACP being obtained (Table 4, entry 1). Although the per-
centages of some deletion peptides were lower with HOBt,
the decapeptide was produced only in about 60% yield
(Table 4, entry 2). In contrast, the performance of Oxyma
was clearly closer to that of HOAt than to that of HOBt
(69%, entry 3, Table 4).
The performances of HOAt, HOBt, and Oxyma were fur-
ther tested in the manual synthesis of Leu-enkephalin (H-
Tyr-Gly-Gly-Phe-Leu-NH2) analogues.[10] The modifications
included the replacement of consecutive Gly amino acids by
MeGly, MeAla, or Aib residues. These N-methylated or
a,a-disubstituted residues were selected because they show
steric hindrance suitable for emphasizing differences in reac-
tivity between the active esters. The strategy followed in set-
ting up the experiment is illustrated in Scheme 1.
tivation time and CysACTHNUTRGNEUNG
(Trt) as protecting group.[19,20] Levels
of racemization were generally much lower than in the Z-
Phg-Pro-NH2 or Z-Phe-Val-Pro-NH2 models, with <1% of
dl epimer being obtained (Table 3). Comparison between
HOAt, HOBt, and Oxyma showed the same trend as ob-
served in solution phase: Oxyma performed with a level of
inhibition of racemization similar to that of HOAt (Table 3,
entry 1 vs entry 3) and superior to that of HOBt (Table 3,
entry 2 vs 3 entry ). Moreover, Oxyma afforded the tripep-
tide in a higher yield than the other additives (Table 3, en-
tries 1 and 2 vs entry 3).
Table 3. Yields and racemization during the elongation of H-Gly-Cys-
Phe-NH2 in DMF (stepwise solid-phase synthesis).[a,b]
Entry
Coupling reagent
Yield [%]
dl [%]
Resin-bound tripeptides 1a, 1b, and 1c of general struc-
ture H-aaX-Phe-Leu-resin were manually assembled on a
Fmoc-RinkAmide-AM-PS resin (0.63 mmolgÀ1) by means of
30 min couplings with use of DIC/Oxyma. A quantitative
yield was verified by use of the Kaiser test for primary
amines. There was no need for recoupling. Stepwise incorpo-
ration of the last two residues was then studied with each
additive. Amino acids were preactivated for 3 min to ensure
full formation of the active esters being compared. Coupling
1
2
3
HOAt/DIC
HOBt/DIC
Oxyma/DIC
88.4
84.1
90.8
0.1
0.2
0.1
[a] A 5 min preactivation time at room temperature was used in all ex-
periments. The tR values for ll and dl forms were identified by coinjec-
tion with authentic and pure samples. [b] An extra peak at 3.5 min, corre-
sponding to the disulfide-bonded dimer (~1%), was found in all experi-
ments.
The effectiveness of Oxyma
Table 4. Percentages of ACP (65–74) decapeptide (H-Val-Gln-Ala-Ala-Ile69-Asp-Tyr-Ile72-Asn-Gly-NH2) and
various deletion peptides obtained during automated synthesis using different additives.[a]
in terms of yields in the solid-
phase approach was tested by
the assembly of the ACP (65–
74) decapeptide (H-Val-Gln-
Ala-Ala-Ile69-Asp-Tyr-Ile72-Asn-
Gly-NH2) on a Fmoc-RinkA-
Entry Coupling des-
des-Ile- des-
Ala
[%]
des-Ile- des-
Gly
[%]
des-
Val
[%]
des-
2Ala
[%]
des-
Ala
[%]
des-
Gly
[%]
ACP des-
[%] Asn
[%]
reagent
2Ile
[%]
Ile72
[%]
Ile69
[%]
[b]
1
2
3
HOAt/
DIC
HOBt/
DIC
Oxyma/ 0.6
DIC
1.7
3.9
2.5
2.6
5.9
–
5.8
4.9
7.9
1.8
3.2
2.1
2.9
4.2
0.5
0.2
0.1
71.8 1.5
[b]
[b]
0.5
12.6
9.9
7.8[c]
–
6.0[d]
5.1
62.3
–
mide-Aminomethyl-PS
resin
(0.63 mmolgÀ1).[9c] For this pur-
pose, an ABI 433A peptide syn-
thesizer and standard Fmoc/tBu
protocols were used. The rela-
tive effectivenesses of Oxyma,
HOAt, and HOBt were tested
in terms of percentage of target
[b]
–
2.7
68.7 0.3
[a] HPLC-MS showed the right mass for the ACP decapeptide at 1062.7. [b] No misincorporation was detect-
ed. [c] The observed mass could also correspond to that of the des-Ala-Val product. [d] Along with the mass
of des-Ala, that of des-Gln was also detected.
decapeptide and deletion peptides obtained. Before compar-
ison of the additives, it was necessary to find suitable condi-
tions under which differences would be clearly detectable.
Five- or 10-fold excesses of reagents (Fmoc-amino acids,
DIC, and additive) were not satisfactory for achieving this
objective because nearly quantitative yields of ACP were
obtained with HOBt. Use of twofold excesses, however,
yielded mixtures of various deletion peptides, so relative po-
tencies were more emphasized. No problems were encoun-
tered in the preparation of a solution of Oxyma in DMF.
Several deletion peptides were detected with use of the
different additives, which made separation highly challeng-
ing. Those originating from misincorporation of a single
amino acid (i.e., des-Ile72, des-Ile69, des-Val, des-Ala, des-
Scheme 1. Strategy followed in the manual synthesis of Leu-enkephalin
analogues to test the different additives.
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Peptide Synthesis Additives
FULL PAPER
Table 6. Percentages of H-Tyr-MeAla-MeAla-Phe-Leu-NH2 (2b) and re-
lated deletion peptides obtained in solid-phase assembly with 30 min cou-
pling times with the different additives.[a]
times ranged from 5 min to 1 h depending on the Leu-enke-
phalin analogue, under conditions that afforded the best sce-
narios for comparing the additives. Pentapeptides 2a, 2b,
and 2c of general sequence H-Tyr-aaX-aaX-Phe-Leu-NH2
were obtained after cleavage from the resin with concentrat-
ed TFA. The efficiency of each additive was measured in
terms of percentages of pentapeptide and deletion tetrapep-
tides (and occasionally also starting tripeptide) obtained.
The pentapeptide model 2a (H-Tyr-MeGly-MeGly-Phe-
Leu-NH2) was, compared to 2b and 2c, the least difficult to
assemble. Therefore, it is not surprising that a 1 h couplings
for the incorporation of both MeGly and Tyr were not the
most suitable conditions to highlight the relative efficiencies
of the different additives, because quantitative yields of 2a
were obtained. Coupling times were thus shortened to 5 min
for this purpose, although even then high and similar per-
centages were detected with all three additives (see
Table 5). Nevertheless, the deletion tetrapeptides des-
Entry Coupling re- Pentapeptide des-MeAla des-Tyr
Tripeptide
[%]
agent
[%]
[%]
[%]
1
2
3
HOAt/DIC
HOBt/DIC
Oxyma/DIC 79.0
73.6
46.1
23.2
38.1
16.9
3.1
15.2
4.0
0.1
0.6
0.1
[a] HPLC-MS showed the right mass for the pentapeptide at 611.5.
was slightly better than that of HOAt (Table 6, entry 3 vs
entry 1), with the purity being increased up to almost 80%.
This superiority is attributed to the greater effectiveness of
the incorporation of the MeAla residue, whereas the incor-
poration of Tyr was more even. The high percentage of the
des-MeAla product relative to the des-Tyr product confirms
the higher complexity of the sequence in relation to that of
2a. The presence of a certain pentapeptide lacking the N-
methyl group at the MeAla residues (about 1%), regardless
of the additive used, should be mentioned.
The coupling conditions that had afforded the pentapep-
tide 2b in moderate yields were not valid for the 2c ana-
logue (H-Tyr-Aib-Aib-Phe-Leu-NH2),[9c] because the purities
were much lower (see Table 7). The enormous percentages
of the des-Aib product present in the crude mixtures con-
firm that the synthesis of 2c is the most demanding of those
of the three analogues. Consistently with the trend observed
in the previous tests, it was observed that HOBt was the
least efficient additive, capable of affording the pentapep-
tide only in extremely low purity (3%, Table 7, entry 2).
Use of its sister additive HOAt did not greatly increase the
level of purity (11%, entry 1, Table 7). The superiority of
Oxyma in this model was evident, its difference from HOAt
in terms of reactivity being greater than that seen in the syn-
thesis of the 2b analogue (28%, entry 3, Table 7). In view of
this general low purity profile, 1 h couplings were also car-
ried out. The same trend in relative effectiveness was ob-
served, although this time the percentages of pentapeptide
were enhanced. Not only did HOBt still afford the lowest
purity (9.8%, entry 5, Table 7), but it was the only reagent
that still gave some of the des-Tyr product and the starting
tripeptide under the new conditions. The relative perform-
ances of Oxyma and HOAt were maintained (Table 7,
entry 6 vs entry 4).
Table 5. Percentages of H-Tyr-MeGly-MeGly-Phe-Leu-NH2 (2a) and re-
lated deletion peptides obtained after solid-phase assembly with 5 min
coupling times with the different additives.[a]
Entry Coupling re- Pentapeptide des-MeGly des-Tyr
Tripeptide
[%]
agent
[%]
[%]
[%]
1
2
3
HOAt/DIC
HOBt/DIC
Oxyma/DIC 91.4
94.9
84.8
1.4
7.5
3.8
3.2
6.6
4.2
0.5
1.1
0.6
[a] HPLC-MS showed the right mass for the pentapeptide at 583.8.
MeGly and des-Tyr (and even starting tripeptide 1a) were
found in the crude products. The highest percentage of pen-
tapeptide was obtained in the experiment with HOAt
(94.9%, entry 1, Table 5), an excellent purity for such ex-
treme conditions. HOBt was not as effective as its aza coun-
terpart, but still provided the pentapeptide 2a in an 84.8%
yield (Table 5, entry 2). Under these conditions, the perfor-
mance of Oxyma was impressive, surpassing that of HOBt
(Table 5, entry 3), rising above 90% in percentage of penta-
peptide, and showing a similar potency to HOAt. There was
almost no starting tripeptide in the crude mixture (0.6%).
In some cases, the percentage of des-Tyr was higher than
that of des-MeGly, thereby confirming that this model pen-
tapeptide was the least demanding.
In view of our observations of the structural similarities
between pentapeptide 2b (H-Tyr-MeAla-MeAla-Phe-Leu-
NH2) and 2a, we applied the same coupling times. In this
system, however, very little pentapeptide was formed, but
surprisingly Oxyma was almost ten times more effective
than HOAt (0.6% for HOAt, 0.2% for HOBt and 5.3% for
Oxyma). When 30 min couplings were used, considerable
percentages of pentapeptide were observed and, unlike in
the previous model, more significant differences arose (see
Table 6). Synthesis with HOBt resulted in the least pure
crude mixture (below 50%, entry 2, Table 6), whereas the
other two additives afforded the pentapeptide 2b in a much
more efficient manner. Again, the performance of Oxyma
In order to improve the level of coupling of the Aib resi-
due further, double couplings of 30 min and of 1 h were per-
formed. Although use of the former set of conditions did
not result in any significant changes in the degrees of purity
obtained, use of the latter resulted in enhanced percentages
of 2c, especially in the synthesis with Oxyma (69%,
entry 12, Table 7). It is noteworthy that des-Tyr was still ob-
served even in the longest coupling when HOBt was used
(entry 11, Table 7).
Because Oxyma was clearly the additive that displayed
the highest potency in the most difficult sequence, but the
coupling to afford 2c could still not be driven to completion
under the conditions tested, a 4 h double coupling for the in-
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9397

F. Albericio, A. El-Faham et al.
Table 7. Percentages of H-Tyr-Aib-Aib-Phe-Leu-NH2 (2c) and related
deletion peptides obtained in solid-phase assembly under various sets of
coupling conditions with the different additives.[a]
resin, without the rest of the reagents that would be added
in a standard coupling, to provoke the appearance of side-
reactions. Apart from the expected byproduct (named B4),
others were also detected (see Scheme 2).
Entry Coupling
Coupling Pentapeptide des-
des-
Tyr
Tripeptide
[%]
conditions reagent
[%]
Aib
[%]
Experiments 1–3 were carried out with the H-Gly-Phe-
Val-resin model tripeptide. Experiment 1 was assisted with
microwaves for 10 min at 808C, the resin being mixed with a
solution of Oxyma in DMF. After cleavage from the resin
with 90%TFA/10% H2O, samples were analyzed by HPLC-
PDA, revealing 43.2% of the unmodified peptide (Figure 2).
The major byproduct found was B2 (47.9%), a peptide for-
mylated at the N terminus, which presumably involves the
solvent. Byproducts B1 and B3 (2.5 and 2.4%, respectively,
Path A, Scheme 2), and the predicted B4 (4.0%, Path B)
were also detected. The byproduct B1 was the product of
attack at the electrophilic carbon of the oxime group and
B3 appears to be its hydrolyzed derivative at the ethyl ester
(either during microwave irradiation or in the cleavage
step), although it should appear at a lower retention time
than B1 because of its higher polarity. The mass of B4 was
detected as two close peaks, suggesting that two isomers
were present. The UV profiles of these additive-based by-
products each show a characteristic maximum of absorbance
at 235–240 nm, like Oxyma. An unknown impurity with the
same retention time as B1 but with a +43 mu mass differ-
ence ([M+H]+ =364.3) with respect to the unmodified tri-
peptide ([M+H]+ =321.2) was present in the crude mixture,
as detected by electrospray HPLC-MS.
[%]
1
HOAt/
DIC
HOBt/
DIC
Oxyma/ 28.0
DIC
HOAt/
DIC
HOBt/
DIC
Oxyma/ 55.7
DIC
HOAt/
DIC
HOBt/
DIC
Oxyma/ 46.5
DIC
HOAt/
DIC
HOBt/
DIC
Oxyma/ 69.0
DIC
11.3
3.0
86.1
91.0
70.5
71.3
86.9
44.3
68.4
90.6
53.5
45.0
80.6
31.0
1.8
0.9
1.1
–
0.8
5.1
0.4
–
2
30 min
3
4
28.7
5
9.8
1.6
–
1.7
–
1 h
6
7
31.2
0.4
0.8
–
–
8
8.0
0.6
–
30 min[b]
9
10
11
12
55.0
–
–
18.9
0.5
–
–
1 h[b]
–
[a] HPLC-MS showed the right mass for the pentapeptide at 611.4. [b] A
double coupling was performed.
In experiment 2, the same solution of the additive in
DMF as used in experiment 1 was mixed with the resin at
room temperature and left overnight. These considerably
milder conditions afforded H-Gly-Phe-Val-NH2 in 92.3%
purity (Figure 2). Unlike in experiment 1, only the byprod-
uct B2 (2.1%), which is not additive-based, was observed,
along with a compound (5.4%) showing an unusual UV pro-
file that would suggest that it is not peptide-based. Coinjec-
tion of the products from experiment 1 and experiment 2
confirmed that this compound was not present in the prod-
ucts of experiment 1. The unknown compound with the +43
corporation of Aib and a standard 1 h coupling for introduc-
ing Tyr was conducted with this additive. A near-quantita-
tive yield (92%) was observed. It is worth noting that the
coupling effectiveness of Oxyma in these pentapeptides can
be considered similar to that of HOAt, one of the most effi-
cient additives and classed among the most powerful cou-
pling reagents.
The stability of Oxyma in relation to the N terminus of a
resin-anchored peptide was studied. Our concern mainly re-
lated to the side-reaction involving nucleophilic attack of
the N terminus amino group on
the ethyl ester of Oxyma, which
might generate a resin-bound
byproduct. This unwanted reac-
tion would lead to the termina-
tion of the peptide chain, one
of the most undesirable events
in solid-phase peptide synthesis.
To check the viability of this
side reaction, experiments in-
volving two tripeptides (H-Gly-
Phe-Val-resin and the less nu-
cleophilic H-MeGly-Phe-Leu-
resin) under various strong re-
action conditions were carried
out. In all experiments 10 equiv
of a solution of Oxyma in DMF
or NMP was mixed with the resin.
Scheme 2. Main byproducts found after cleavage of tripeptides used in Oxyma stability experiments from the
9398
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Chem. Eur. J. 2009, 15, 9394 – 9403

Peptide Synthesis Additives
FULL PAPER
mass difference with respect to the unmodified tripeptide.
As in experiment 2, no B1 was detected. In all these experi-
ments, Oxyma-based byproducts were detected only in
those carried out under the more extreme conditions
(Exps. 1 and 3).
Unlike Exps. 1–3, which were designed as the worst sce-
narios possible, including the presence of the less hindered
(and therefore more prone to react) amino acid Gly at the
N terminus, experiment 4 was conducted with the less nucle-
ophilic H-MeGly-Phe-Leu-resin (which was also used in the
coupling efficiency assays). The experiment was carried out
in DMF, the regular solvent for SPPS, although the above
results had been poorer. than those obtained in NMP, and
with 10 min irradiation in the microwave. The unmodified
tripeptide was obtained in 94.5% purity, much higher than
the 43.2% achieved with the H-Gly-Phe-Val-resin tripep-
tide. Compound B1 was the only additive-based byproduct
detected (1.5%), together with B2 (4.0%).
Having addressed the relative potencies of HOBt, HOAt,
and Oxyma and the stabilities of the proposed additives, we
next focused on the safety profiles of the additives. In view
of the recent reports relating to the potentially explosive
properties of benzotriazole-based additives, which restrict
their transportation and commercial availability, it was cru-
cial to ensure that Oxyma did not follow the classical pat-
tern observed in explosive substances: fast decomposition
with simultaneous large increase in pressure.[21]
To explore the thermal safety of the additives, dynamic
Differential Scanning Calorimetry (DSC) and Accelerating
Rate Calorimetry (ARC) assays were carried out. DSC
allows the heat released by a certain compound to be mea-
sured, by comparing it to a reference when both are exposed
to the same thermal treatment.[22] This experiment also pro-
vides valuable information about relative decomposition ki-
netics (which are connected with the explosive character of
a given compound), when heated in a closed crucible under
N2 flow, from 30 to 3008C at a constant heating rate of
108CminÀ1. As a result, diagrams displaying the heat flow as
a function of time/temperature are obtained. In addition to
displaying higher normalized exothermic DH values
(234 kJmolÀ1 for HOBt hydrate and 226 kJmolÀ1 for HOAt,
vs 125 kJmolÀ1 for Oxyma), the kinetic profiles of the ben-
zotriazole-based derivatives differed significantly from that
of Oxyma. Whereas the former compounds decomposed
quickly, the latter decomposed in a much slower and con-
stant manner (Figure 3). This decomposition profile cannot
be labeled as non-explosive per se, because the study was
not an explosivity test. However, it clearly does not coincide
with the standard thermal behavior of an explosive sub-
stance, such as those observed for HOBt hydrate and
HOAt. Interestingly, at 1158C, Oxyma, unlike HOAt and
HOBt hydrate, melted before decomposing, and this endo-
thermic phenomenon can be considered a possible safety
barrier. Although this barrier is not very precise, because
melting and decomposition processes occur almost simulta-
neously, it might moderate the exothermic released heat. In
the experiment with HOBt hydrate, another endothermic
Figure 2. HPLC-PDA and UV profiles of crude mixtures from stability
experiments: experiment 1 (above), experiment 2 (center), and coinjec-
tion of samples from experiment 1 and experiment 2 (below), showing
the tripeptide H-Gly-Phe-Val-NH2 (2.1 min, [M+H]+ =321.2) and by-
products B1 (3.9 min, [M+H]+ =463.3, experiment 1 and coinjection), B2
(4.2 min, [M+H]+ =349.2), B3 (5.1 min, [M+H]+ =435.3), and B4 (8.2
and 8.8 min, [M+H]+ =417.2). Unknown impurities are found at 3.9 min
([M+H]+ =364.3, experiments 1, 2, and coinjection) and at 4.4 min
([M+H]+ =620.4, experiment 2 and coinjection).
mass difference seen in experiment 1 was also detected
(<0.2%) in experiment 2. The low mass increase and the
different UV profile in relation to the additive-based by-
products suggests that this impurity was not generated by
Oxyma.
In view of the effect of DMF in the stability experiments,
experiment 3 was carried out in the microwave as in experi-
ment 1 but this time with a solution of Oxyma in NMP. As
expected, B2 almost disappeared completely (3.6%), with
the unmodified tripeptide being obtained in 64.0% purity.
Comparison with experiment 1, which afforded only
a
43.2% yield of tripeptide, illustrates the impact of the sol-
vent on the purity obtained, although more byproducts were
detected, including B3, B4, and the impurity with the +43
Chem. Eur. J. 2009, 15, 9394 – 9403
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9399

F. Albericio, A. El-Faham et al.
Figure 4. ARC experiments. Decomposition profiles of HOBt hydrate
~
^
(
), HOAt (&), and Oxyma ( ) showing released pressure (bar) as a
function of time (min).
decomposing. In contrast, the associated pressure detected
in the assay conducted with Oxyma was considerably lower
(61 bar).
With regard to the onset temperatures, in the case of
Oxyma decomposition began at 1248C, whereas in the ex-
periments with HOBt hydrate and HOAt it began at 1458C
and 1788C, respectively (Figure 5). In order to work under
safe conditions, it is recommended that the temperature of a
given compound be kept at values at which the time to max-
imum rate under adiabatic conditions is greater than 24 h.[24]
As a rule of thumb, this temperature value can be estimated
in many cases by subtracting 50 K from the decomposition
onset observed in an ARC experiment.[25] In the case of
Oxyma, this safety value is at a lower temperature than is
the case for HOBt hydrate and HOAt (748C vs 1288C and
958C, respectively). Nonetheless, under standard conditions
for peptide synthesis, the working temperature does not usu-
ally rise above 258C. Moreover, when stability assays were
conducted with microwave irradiation at 808C, no incident
was reported. Although in terms of thermal risk assessment
Oxyma is less thermally stable, showing a lower decomposi-
tion onset temperature, the above results (mainly the pres-
sure rise associated with decomposition) suggest that the
risk of this process ending in a thermal runaway is less likely
Figure 3. Thermograms showing heat flow versus temperature and time
for DSC experiments with A) HOBt hydrate, B) HOAt, and C) Oxyma.
process, corresponding to the desolvation heat, can be ob-
served. The approximate onset temperature at which de-
composition began was 1318C, considerably lower than in
the cases of HOBt hydrate (1908C) or HOAt (2188C).
Complementary to the DSC assays is the ARC technique,
which enables study of a given decomposition under adia-
batic conditions.[23] The associated pressure rise can be mea-
sured and the onset temperatures accurately determined,
unlike in DSC experiments, which suffer from uncertainty
because of the low amount of sample.[21] The “heat-wait-
seek” method is applied until self-heating of the sample is
detected and then the experiment is changed to adiabatic
mode. Once decomposition begins, pressure and tempera-
ture increase. The assay is stopped when the temperature
rises above 3008C.
Again, the benzotriazole-based additives showed marked-
ly different behavior from that of Oxyma in terms of the
pressure measured (Figure 4). In the case of the former
compounds, high pressures were observed (178 and 167 bar
for HOBt hydrate and HOAt, respectively). These results
are not surprising, because benzotriazoles release N2 when
Figure 5. ARC experiments. Decomposition profiles of HOBt hydrate
~
^
(
), HOAt (&), and Oxyma ( ) showing temperature (8C) as a function
of time (min).
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Chem. Eur. J. 2009, 15, 9394 – 9403

Peptide Synthesis Additives
FULL PAPER
mide-MBHA-PS-resin (0.45 mmolgÀ1, 50mg), with use of the Fmoc/tBu
and CysACTHNUGRTENUNG(Trt) protection strategy. Glycine was introduced in order to ach-
when using Oxyma than in the cases of HOBt hydrate or
HOAt.
ieve better separation of ll and dl isomers than in the case of des-Gly
dipeptides. Coupling times of 1 h were used after 5 min preactivation of a
solution of Fmoc-amino acid, Oxyma, and DIC (3 equiv excess) in DMF
(0.3m) at room temperature. Fmoc removal was carried out with piperi-
dine/DMF (1:4, 2ꢅ5 min). The peptide chain was released from the resin
by treatment with TFA/ethane-1,2-dithiol/H2O/TIS (94:2.5:2.5:1) for 1 h
at room temperature. The colorless solution was filtered and the resin
was washed with CH2Cl2 (0.5mLꢅ3). The solvent and residues from the
cleavage cocktail were concentrated under nitrogen. The crude pentapep-
tide was precipitated with cold Et2O (3 mLꢅ4) and, after being lyophi-
lized, was analyzed by reversed-phase HPLC, with use of a Waters Sun-
Fire C18 Column (3.5 mm, 4.6ꢅ100 mm), linear gradient 5 to 100% of
0.036% TFA in CH3CN/0.045% TFA in H2O over 8 min, with detection
at 220 nm. The tR values of the ll and dl epimers were 3.37 and
3.60 min, respectively.
Conclusions
To conclude, here we have extensively tested ethyl 2-cyano-
2-(hydroxyimino)acetate (Oxyma) as an additive for peptide
synthesis for use in combination with carbodiimides. Oxyma
showed clear superiority to HOBt in terms of suppression of
racemization and of coupling efficiency in all the experi-
ments conducted. In some cases, such as the racemization
assay carried out on the stepwise model or the demanding
assembly of MeAla and Aib analogues of Leu-enkephalin,
its performance was even superior to that of HOAt. The
elongation of the ACP decapeptide in an ABI 433A peptide
synthesizer also demonstrated the compatibility and cou-
pling efficiency of Oxyma in an automated synthesis. Stabili-
ty assays of Oxyma with regard to N-terminal amino groups
showed no peptide-bound byproducts when extremely
strong conditions were avoided, even with use of 10 equiv in
the absence of base, carbodiimide, and Fmoc-amino acid.
Last but not least, two calorimetry techniques, DSC and
ARC, showed decomposition profiles for HOAt and HOBt
that resemble that of a explosive substance, because of their
fast decomposition and high derived pressure rises. In con-
trast, Oxyma did not follow this pattern, and decomposed at
a slower rate, releasing only one third of the pressure ob-
served in the experiments with HOBt and HOAt. Therefore,
Oxyma can be considered a potent replacement for benzo-
triazole-based additives showing a lower thermal risk.
Solid-phase automated synthesis of ACP (65–74) (H-Val-Gln-Ala-Ala-
Ile-Asp-Tyr-Ile-Asn-Gly-NH2):[14] The ACP (65–74) decapeptide model
was
assembled
on
Fmoc-RinkAmide-Aminomethyl-PS-resin
(0.63 mmolgÀ1, 0.1 mmol) with use of an automated ABI 433A peptide
synthesizer. Twofold excesses of Fmoc-amino acids and solutions (0.2m)
of the corresponding additive and DIC in DMF were used (1 mL of the
solution was added in each coupling step: 0.2 mmol). The peptide chain
was cleaved from the resin by TFA/H2O (9:1) treatment over 2 h at room
temperature. After filtration of the solution containing the peptidic mate-
rial, the resin was washed with CH2Cl2 (1 mLꢅ2), which was removed
under nitrogen along with TFA. The resulting crude peptide was purified
with cold Et2O (2 mLꢅ3) and lyophilized. Peptide purity was analyzed
by reversed-phase HPLC, with use of a Waters Symmetry C18 column
(5 mm, 4.6ꢅ150 mm), linear gradient 12.5 to 17% of 0.036% TFA in
CH3CN/0.045%TFA in H2O over 30 min, with detection at 220 nm. Re-
tention times were: tR decapeptide=26.88 min, tR des-2Ala=24.48 min,
tR des-Ala=24.50 min, tR des-Asn=32.01 min, tR des-Gly=26.33 min, tR
des-Ile69, Ile 72 =2.97 min, tR des-Ile, Ala=4.20 min, tR des-Ile72
7.17 min, tR des-Ile69 =9.38 min, tR des-Val=20.22 min.
=
Solid-phase general synthesis of H-aaX-Phe-Leu-resin and H-Gly-Phe-
Val-resin tripeptides: Peptides were manually assembled on a Fmoc-Rin-
kAmide-Aminomethyl-PS resin (0.63 mmolgÀ1, 2 g). Coupling times were
30 min, with 3 equiv excess of each Fmoc-amino acid, Oxyma, and DIC.
During preactivation (1.5 min), a bright yellow color was observed imme-
diately after addition of DIC. Fmoc removal was carried out with piperi-
dine/DMF (1:4, 2ꢅ5 min). Tripeptide purity was checked after sample
cleavage (10 mg) from the resin with TFA/H2O (19:1) at room tempera-
ture for 1 h. The solution was filtered and the resin was washed with
CH2Cl2 (0.5 mLꢅ2). The solvent and TFA were removed under nitrogen
flow. In all cases, reversed-phase HPLC analysis showed purity above
99%.
Experimental Section
General: Oxyma (ethyl cyanoglyoxylate-2-oxime, 97%) was obtained
from commercial sources (Aldrich). DMF was used in peptide grade
purity. All peptides, des-amino acids, and byproducts were identified by
HPLC-MS electrospray mass spectroscopy. The synthesis of ACP was
carried out in an Applied Biosystems ABI 433A automated peptide syn-
thesizer by the Fmoc/tBu protection strategy. Experiments involving side
reactions related to Oxyma were conducted in a CEM Discover Micro-
wave. Differential scanning calorimetry assays were performed in a Met-
tler–Toledo DSC-30 differential scanning calorimeter, with high-pressure
crucibles of a capacity of 30 mL. In the adiabatic calorimetry experiment,
an accelerating rate calorimeter from Thermal Hazard Technology was
used, along with ARCTC-HC-MCQ (Hastelloy) test cells. The sensitivity
Solid-phase synthesis of H-Tyr-MeGly-MeGly-Phe-Leu-NH2 (2a) with
use of the different additives: The pentapeptide was manually elongated
on a pure previously assembled H-MeGly-Phe-Leu-Rinkamide-Amino-
methyl-PS-resin (0.63 mmolgÀ1, 50 mg) with a preactivation time of 3 min
and use of Fmoc-amino acids (3 equiv, excess), the corresponding addi-
tive (3 equiv), and DIC (3 equiv). The coupling time was 5 min for the in-
troduction of both MeGly and Tyr. The pentapeptide was cleaved from
the resin by treatment with TFA/H2O (9:1) for 2 h at room temperature.
The solution was then filtered and the resin was washed with CH2Cl2
(1 mLꢅ2), which was removed along with TFA under nitrogen. The
crude pentapeptide was precipitated with cold Et2O (2 mLꢅ3) and, after
being lyophilized, was analyzed by reversed-phase HPLC, with a Waters
Symmetry C18 column (5 mm, 4.6ꢅ150 mm), linear gradient 16 to 16.5%
of 0.036% TFA in CH3CN/0.045%TFA in H2O over 30 min, with detec-
tion at 220 nm. The tR value of the pentapeptide was 26.31 min, whereas
the tR values of des-MeGly, des-Tyr, and the tripeptide H-MeGly-Phe-
Leu-NH2 were 25.81, 9.62, and 8.58 min, respectively.
threshold was set at 0.028CminÀ1
.
Racemization tests with model peptides in solution phase: Test couplings
were carried out as previously described for Z-Phg-Pro-NH2 and Z-Phe-
Val-Pro-NH2.[17] Crude products were analyzed by reverse-phase HPLC
(with a Waters Symmetry C18, 5 mm, 4.6ꢅ150 mm column), linear gradi-
ent over 30 min of 20 to 50% (Z-Phg-Pro-NH2) or 20 to 80% (Z-Phe-
Val-Pro-NH2) CH3CN/0.036% TFA in H2O/0.045%TFA, detection at
220 nm. In the Z-Phg-Pro-NH2 model, the tR values of the ll and dl epi-
mers were 26.01 and 27.40 min, respectively, whereas in the Z-Phe-Val-
Pro-NH2 case, the tR values of the lll and ldl epimers were 19.98 and
21.05 min, respectively.
Solid-phase synthesis of H-Tyr-MeAla-MeAla-Phe-Leu-NH2 (2b) with
use of the different additives: The pentapeptide was manually assembled
on a pure previously synthesized H-MeAla-Phe-Leu-Rinkamide-Amino-
Study of cysteine racemization during assembly of H-Gly-Cys-Phe-NH2
on solid phase:[19, 20] Experiments consisted of the study of the stepwise
coupling of Cys and Gly residues onto previously formed H-Phe-RinkA-
Chem. Eur. J. 2009, 15, 9394 – 9403
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F. Albericio, A. El-Faham et al.
methyl-PS-resin (0.63 mmolgÀ1, 50 mg), with use of Fmoc-amino acids
(3 equiv excess), the corresponding additive, and DIC. Preactivation and
coupling times for the introduction of both MeAla and Tyr were 3 and
30 min, respectively. The peptide chain was cleaved from the resin by
treatment with TFA/H2O (9:1) over 2 h at room temperature. The solu-
tion was then filtered and the resin was washed with CH2Cl2 (1mLꢅ2),
which was removed along with TFA under nitrogen. The crude pentapep-
tide was purified with cold Et2O (2 mLꢅ3) and lyophilized. Purity was
analyzed by reversed-phase HPLC, with use of a Waters SunFire C18
Column (3.5 mm, 4.6ꢅ100 mm), linear gradient 20 to 25% of 0.036%
TFA in CH3CN/0.045%TFA in H2O over 8 min, with detection at
220 nm. The tR value of the pentapeptide was 8.21 min, whereas the tR
values of des-MeAla, des-Tyr, and the tripeptide H-MeAla-Phe-Leu-NH2
were 8.81, 4.73, and 2.34 min, respectively.
Toledo DSC-30 differential scanning calorimeter. Diagrams showing heat
flow as a function of temperature and time were obtained.
General Procedure for ARC experiments: Assays were carried out on an
Accelerating Rate Calorimeter (ARC) from Thermal Hazard Technolo-
gy, in ARCTC-HC-MCQ (Hastelloy) test cells. Samples (2.083 g of
HOBt hydrate, 1.605 g of HOAt, and 3.451 g of Oxyma) were introduced
into the calorimetric test cell at room temperature, without stirring. The
cell was heated at the initial temperature (308C) and the “heat-wait-
seek” method was applied; this consisted of heating the sample by 58C
and, after 15 min of equilibrium, measuring whether self-heating was oc-
curring at a rate higher than 0.028CminÀ1 (default sensitivity threshold).
When self-heating was detected, the system was changed to adiabatic
mode. After decomposition, the assay was stopped when the temperature
rose above 3008C. The phi factors[26] were: 2.6058 (HOBt hydrate),
3.04373 (HOAt), and 1.95557 (Oxyma).
Solid-phase synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2 (2c) with use of
the different additives:[14] The pentapeptide was manually elongated on a
pure previously synthesized H-Aib-Phe-Leu-Rinkamide-Aminomethyl-
PS-resin (0.63 mmolgÀ1, 50 mg), with use of Fmoc-amino acids (3 equiv,
excess), the corresponding additive (3 equiv), and DIC (3 equiv). Resi-
dues were preactivated for 3 min prior to addition to the resin. Coupling
times are displayed in Table 7. The peptide chain was cleaved from the
resin by treatment with TFA/H2O (9:1) for 2 h at room temperature. The
solution was then filtered and the resin was washed with CH2Cl2 (1 mLꢅ
2), which was removed along with TFA under nitrogen. The crude pep-
tide was purified with cold Et2O (2 mLꢅ3) and, after lyophilization,
purity was checked by reversed-phase HPLC, with use of a Waters Sun-
Fire C18 Column (3.5 mm, 4.6ꢅ100 mm) and a linear gradient of 20 to
35% of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min, with de-
tection at 220 nm. The tR value for the pentapeptide was 5.75 min, where-
as the tR values for des-Aib, des-Tyr, and the tripeptide H-Aib-Phe-Leu-
NH2 were 6.03, 3.93, and 2.25 min, respectively.
Acknowledgements
This work was partially supported by CICYT (CTQ2006–03794/BQU),
Luxembourg Bio Technologies, Ltd., the Generalitat de Catalunya
(2005SGR 00662), the Institute for Research in Biomedicine, and the
Barcelona Science Park. R.S.F. thanks the Ministerio de Educaciꢀn y
Ciencia for a FPU PhD fellowship. We also thank the Calorimetry Plat-
form at the Barcelona Science Park for their support in the DSC and
ARC experiments.
[1] Abbreviations not defined in text: Aib, a-aminoisobutyric acid;
ACP, acyl carrier protein decapeptide (65–74); AM-PS, aminometh-
yl-polystyrene resin; ARC, accelerating rate calorimetry; 6-Cl-
HOBt, 6-chloro-1-hydroxybenzotriazole; DIC, N,N-diisopropylcar-
bodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethyl-
formamide; DSC, Differential Scanning Calorimetry; HATU, N-
General procedure for stability assays with Oxyma: Stability experi-
ments 1–4 were conducted on two resin-bound tripeptides: H-MeGly-
Phe-Leu-resin and H-Gly-Phe-Val-resin. The resin was weighed into a
2 mL solid-phase syringe, swelled in CH2Cl2 (ꢅ5), and then conditioned
in the reaction solvent, DMF or NMP (ꢅ5). In the overnight experiment,
a solution of Oxyma in DMF or NMP (0.02m, 10 equiv) was directly
added to the resin. After 12 h at room temperature, the resin was filtered
and washed with DMF or NMP (ꢅ10) and CH2Cl2 (ꢅ10). In contrast, in
the microwave-assisted experiment, the resin was first transferred into a
suitable microwave-compatible vial. The solution of Oxyma in DMF or
NMP (0.02m, 10 equiv) was then added to the resin and the mixture was
irradiated at 808C for 10 min in a CEM Discover Microwave. The resin
was then transferred back into the syringe and washed with NMP or
DMF (ꢅ10) and CH2Cl2 (ꢅ10). In both types of experiments, the resin-
bound compounds were cleaved from the resin by treatment with TFA/
H2O (9:1) for 1 h at room temperature. The solution was filtered and the
resin was washed with CH2Cl2 (0.05mLꢅ3), which was removed together
with TFA under nitrogen. The crude peptide was purified with cold Et2O
(2mLꢅ3) and lyophilized. The byproduct content of the samples was
checked by reversed-phase HPLC. Exps. 1–3 (H-Gly-Phe-Val-resin) were
analyzed by use of a SunFire C18 Column (3.5 mm, 4.6ꢅ100 mm), linear
gradient 15 to 30% of 0.036% TFA in CH3CN/0.045%TFA in H2O over
8 min, with detection at 220 nm. Retention times were: tR unmodified tri-
peptide=2.13 min, tR byproduct B1=3.87 min, tR byproduct B2=
4.17 min, tR byproduct B3=5.09 min, tR byproduct B4=8.22 and
8.61 min, tR impurity with detected mass M+43=3.80 min, tR unknown
impurity=4.43 min. experiment 4 (H-MeGly-Phe-Val-resin) was analyzed
with use of a Waters Symmetry C18 column (5 mm, 4.6ꢅ150 mm), linear
gradient 5 to 100% of 0.036% TFA in CH3CN/0.045%TFA in H2O over
15 min, with detection at 220 nm. Retention times were: tR unmodified
tripeptide=6.25 min, tR byproduct B1=6.88 min, tR byproduct B2=
7.68 min.
[(dimethylamino)-1H-1,2,3-triazoloACTHNUTRGNEUGN[4,5-b]pyridin-1-yl-methylene)-N-
methylmethanaminium hexafluorophosphate N-oxide; HBTU, N-
[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]N-methylmetha-
naminium hexafluorophosphate N-oxide; HCTU, N-[(1H-6-chloro-
benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanami-
nium hexafluorophosphate N-oxide; HOAt, 7-aza-1-hydroxybenzo-
triazole; HOBt, 1-hydroxybenzotriazole; HODhbt, 3-hydroxy-3,4-di-
hydro-4-oxo-l,2,3-benzotriazine; HOPO, 2-hydroxypyridine-N-oxide;
HOPfp, pentafluorophenol; HOSu, N-hydroxysuccinimide; NMP, N-
methyl-2-pyrrolidinone; PyAOP, azabenzotriazol-1-yl-N-oxy-tris(pyr-
rolidino)phosphonium hexafluorophosphate; PyBOP, benzotriazol-
1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; Py-
Clock, 6-chloro-benzotriazol-1-yl-N-oxy-tris-pyrrolidino-phosphoni-
um hexafluorophosphate; TBTU, N-[(1H-benzotriazol-1-yl)(dime-
thylamino)methylene]N-methylmethanaminium
tetrafluoroborate
N-oxide; TCTU, N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)-
methylene]-N-methylmethanaminium tetrafluoroborate N-oxide;
TFA, trifluoroacetic acid; Z, benzyloxycarbonyl. Amino acids and
peptides are abbreviated and designated following the rules of the
IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol.
Chem. 1972, 247, 977).
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General Procedure for dynamic differential scanning calorimetry assays:
The thermal behavior of HOAt, HOBt hydrate, and Oxyma was tested.
Samples (1mg) were heated from 308C to 3008C at a heating rate of
108CminÀ1 in a closed high-pressure crucible with N2 flow in a Mettler–
9402
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Chem. Eur. J. 2009, 15, 9394 – 9403

Peptide Synthesis Additives
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Received: March 8, 2009
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Published online: July 2, 2009
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9403