Introduction
============

The choice of the active acylating species used to form a peptide bond is crucial in peptide synthesis.\[[@b1]--[@b4]\] Highly reactive moieties, such as acid halides, are suitable to acylate very poor nucleophilic amines. However, they are often not the best choice for the activation of the *N^α^-*alkoxycarbonyl derivatives of amino acids \[mostly fluorenylmethoxycarbonyl (Fmoc), *tert-*butoxycarbonyl (Boc), and allyloxycarbonyl (Alloc)\] because of their marked electrophilic character, which derives in a considerable loss of configuration.\[[@b5]--[@b9]\] In contrast to acid chlorides, the corresponding active esters offer a more balanced range of reactivity, thus, circumventing most of the side reactions caused by an excessive activation.\[[@b1], [@b2], [@b9], [@b10]\] From the extensive range of these esters, *N-*hydroxylamine esters, preferably prepared in situ by the carbodiimide approach using the corresponding *N*-hydroxylamines as additives, are possibly the most widely used.\[[@b11]--[@b13]\] Among these, 1-hydroxybenzotriazole (HOBt, **1**) and its derivatives, including 1-hydroxy-7-azabenzotriazole (HOAt, **2**), have prevailed over the other carbodiimide additives during recent decades as a result of their great acylation potency (Figure [1](#fig01){ref-type="fig"}).\[[@b12], [@b14], [@b15]\] However, a few years ago, the explosive nature of benzotriazoles prompted the reevaluation of the strongly acidic oxime, ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, **3**) as a potential replacement, which represented a hallmark in the field.\[[@b16], [@b17]\]

![Structure of *N*-hydroxylamines currently available for use as additives to carbodiimides.](open0001-0147-f1){#fig01}

In contrast to standard peptide-bond formation, which occurs in common organic solvents, the acylation of peptides or proteins for bioconjugation purposes typically takes place in aqueous pH-buffered environments. Thus, stability rather than reactivity is the main desired feature of active esters generated in such a polar medium. In a similar manner, preformed amino acid active esters need to be stable enough for shelf storage of up to several weeks. Consequently, *N*-hydroxysuccinimide esters (HOSu, **4**) have found broad application in these scenarios as less reactive but more suitable alternatives to benzotriazoles (Figure [1](#fig01){ref-type="fig"}).\[[@b30]--[@b31]\] For example, HOSu derivatives **4** have been used for the preparation of alkoxycarbonyl amino acids, thereby avoiding the undesired formation of oligomers.\[[@b20]\] However, the limited reactivity of HOSu active species continues to be a decisive drawback. In order to pursue an optimal balance of reactivity for stable active esters, we focused our attention on a family of promising *N-*alkyl-cyanoacetamido oximes (**5**--**8**, Figure [2](#fig02){ref-type="fig"}). In a recent publication, Fmoc-carbonates derived from *N*-hydroxy-2-morpholino-2-oxoacetimidoyl cyanide (MorOx, **5**), *N*-hydroxy-2-oxo-2-(piperidin-1-yl) acetimidoyl cyanide (PipOx, **6**), 2-amino-*N*-hydroxy-2-oxoacetimidoyl cyanide (AmOx, **7**), and 2-(ethylamino)-*N*-hydroxy-2-oxoacetimidoyl cyanide *(N*-Oxyma, **8**) presented desirable reactivities with superior performances to Fmoc-OSu (Figure [2](#fig02){ref-type="fig"}).\[[@b21]\]

![Structure of the *N*-alkyl-cyanoacetamido oximes proposed herein.](open0001-0147-f2){#fig02}

Following previous research on Fmoc-oxime carbonates, the present study aimed at performing a screen of *N-*alkyl-cyanoacetamido oximes **5**--**8** as additives to carbodiimides in replacement of HOSu (**4**). In addition to the abovementioned set of cyanoacetamides, 2-(*N,N*-dimethylamino)-*N*-hydroxy-2-oxoacetimidoyl cyanide (DmOx, **9**) was also evaluated. This molecule differs from AmOx (**7**) in its capacity to act as a hydrogen donor and in its nonplanar structure (Figure [2](#fig02){ref-type="fig"}).\[[@b22]\] While oximes **5**--**9** have been broadly applied in organometallic chemistry, biomedicine and agrochemistry, to our knowledge, the only reports of an additive to carbodiimides presenting an *N*-alkyl-cyanoacetamido oxime scaffold were described in the 1970s, excluding the recent reevaluation of Oxyma (**3**).\[[@b17], [@b22]--[@b25]\] At the beginning of that decade, Itoh determined the acidity of Oxyma (**3**, p*K*~a~ 4.6) and AmOx (**7**, p*K*~a~ 5.2), and evaluated the racemization suppression of the former in a dipeptide model.\[[@b22]\] A few years later, Izdebski further examined the capacity of Oxyma (**3**) and AmOx (**7**), together with other less suitable oximes, to decrease epimerization in *N*,*N′*-dicyclohexylcarbodiimide (DCC)-mediated couplings using an optimized protocol.\[[@b24], [@b25]\] Nonetheless, given the preliminary character of these studies, they did not provide a conclusive overview of the performance of these oximes, although a lower reactivity of acetamido analogue **7** was envisaged.\[[@b22], [@b25]\] Here, we revised the capacity of AmOx (**7**) together with *N*-alkyl-cyanoacetamido oximes **5**--**9** to retain the optical configuration of the activated amino acid and their coupling efficiency, while comparing their performance with that of HOSu (**4**).

Results and Discussion
======================

Synthetic approach
------------------

The pool of *N*-alkyl-cyanoacetamido oximes selected for screening as additives (**5**--**9**) included several linear and cyclic *N*-alkyl moieties, which involve diverse bulkiness and the possibility to allow hydrogen-bonding interactions. In comparison with the ester functionality within Oxyma (**3**), the carboxamido group displays restricted conformational rotation around the amide bond, as shown earlier by 1H NMR studies.\[[@b22]\], [@b23b], [@b23f] This limitation could have some impact on the reactivity of the corresponding esters.

Regarding the syntheses of the proposed oximes, **5**--**8** were already reported in the evaluation of the derived Fmoc-carbonates.\[[@b21]\] Following the same methodology, DmOx (**9**) was synthesized in high yield from ethyl cyanoacetate (**10**) in only two steps by means of an amidation/nitrosation sequence (Scheme [1](#sch01){ref-type="scheme"}).\[[@b21]\] In the first step of this synthetic approach, ethyl cyanoacetate (**10**) was readily transformed into *N,N*-dimethylcyanoacetamide (**11**) after reaction with dimethylamine at 70 °C. Next, the subsequent Meyer nitrosation of intermediate **11** in a methanolic solution of sodium nitrite afforded desired oxime **9** in 76 % overall yield.\[[@b26]\] Although this reaction sequence leading to DmOx (**9**) has already been described in the literature, our optimized methodology leads to a significant improvement in yield and purity compared with former strategies, which describe an initial amidation in aqueous medium or do not generate methyl nitrite in situ.\[[@b23]\] Like cyanoacetamido oximes **5**--**8**, the high water solubility of DmOx (**9**) had a positive impact on the final aqueous work-up.\[[@b21]\]

![Two-step synthetic route to cyanooxime, DmOx (9). *Reagents and conditions:* a) ethyl cyanoacetate (10), aq Me~2~NH (40 %), 70 °C; b) Intermediate 11, NaNO~2~, MeOH/H~2~O, 2 h at RT.](open0001-0147-f5){#sch01}

Control of epimerization
------------------------

Retention of the optical configuration during amino acid coupling is central to peptide synthesis, especially when manufacturing active principle ingredients (APIs).\[[@b9], [@b27]\] The undesired loss of chirality at C~α~ is dramatic in more sensitive building blocks, such as glycopeptides.\[[@b28]\] In order to study the impact of epimerization when using *N*-alkyl-cyanoacetamido oximes **5**--**9** in solution phase, we selected peptides **12**--**14** as test models, which are a reliable platform to study the loss of configuration during stepwise and \[2+1\] fragment couplings (Figure [3](#fig03){ref-type="fig"}).\[[@b29]\] After *N*,*N′*-diisopropylcarbodiimide (DIC)-mediated coupling of equimolar amounts of acid and amine and aqueous work-up, the impact of epimerization on peptides **12**--**14** was determined by reversed-phase HPLC, using optimized conditions that achieve clear separation of the two epimers. Simultaneously, a direct comparison with HOSu (**4**) and Oxyma (**3**), the latter as the most efficient additive, was established in every model.

![Peptide models used to test the reduction of epimerization induced by *N*-alkyl-cyanoacetamido oximes.](open0001-0147-f3){#fig03}

The performances of additives **3**--**9** in a stepwise coupling was examined during the coupling of *N*-carboxybenzyl (Cbz)-protected Z-[l]{.smallcaps}-phenylglycine (Z-Phg-OH) onto [l]{.smallcaps}-prolinamide (H-Pro-NH~2~), resulting in dipeptide **12** (Table [1](#tbl1){ref-type="table"}).[@b29] The α-phenyl moiety in Phg ensured a high sensitivity towards epimerization, because of the pronounced stability of the resulting anion. In this model, HOSu (Entry 2, Table [1](#tbl1){ref-type="table"}) rendered the lowest yield of dipeptide and an unacceptable loss of configuration. The percentage of epimerization achieved (32.7 %) was even more detrimental, taking into account that the absence of preactivation in a peptide coupling usually attenuates epimerization. Remarkably, all *N*-alkyl-cyanoacetamido oximes **5**--**9** surpassed HOSu (**4**) in their capacity to maintain the desired chirality of the test dipeptide (cf. Entries 2 and 3--7, Table [1](#tbl1){ref-type="table"}). In particular, PipOx (**6**) was the least efficient in reducing epimerization, but still afforded three times higher purity than HOSu (**4**) (cf. Entry 2 and 4, Table [1](#tbl1){ref-type="table"}). In comparison with MorOx (**5**), the presence of a proton-accepting oxygen in the six-membered ring caused a considerable increase in purity of the desired peptide (cf. Entry 3 and 4, Table [1](#tbl1){ref-type="table"}).\[[@b30]\] Noteworthy, AmOx (**7**) and *N*-Oxyma (**8**) showed similar configuration retentions to Oxyma (**3**), which afforded only 1.0 % of [dl]{.smallcaps} epimer, with a simultaneous improvement in yield (cf. Entry 1 and 5, 6, Table [1](#tbl1){ref-type="table"}).\[[@b17]\]

###### 

Yield and epimerization during the formation of Z-Phg-Pro-NH~2~ (12) using additives 3--9 (solution phase synthesis)[\[a\]](#tf1-1){ref-type="table-fn"}

  Entry   Coupling Reagent        Yield \[%\]                                [dl]{.smallcaps} \[%\][\[b\]](#tf1-2){ref-type="table-fn"}
  ------- ----------------------- ------------------------------------------ ------------------------------------------------------------
  1       DIC/Oxyma (**3**)       89.9[\[c\]](#tf1-3){ref-type="table-fn"}   1.0[\[c\]](#tf1-3){ref-type="table-fn"}
  2       DIC/HOSu (**4**)        70.3                                       32.7
  3       DIC/MorOx (**5**)       88.4                                       5.7
  4       DIC/PipOx (**6**)       86.8                                       10.7
  5       DIC/AmOx (**7**)        81.4                                       3.0
  6       DIC/*N*-Oxyma (**8**)   92.1                                       3.8
  7       DIC/DmOx (**9**)        93.4                                       8.3

Couplings were performed without pre-activation in DMF at RT.

The [ll]{.smallcaps} and [dl]{.smallcaps} epimers of this peptide model have been described in the literature.[@b29] Retention times (*t*~R~) for each epimer were identified after co-injection with a pure [ll]{.smallcaps} sample.

Results are extracted from Ref. \[[@b17]\].

Once the stepwise system was evaluated, the next step in the epimerization study of *N*-alkyl-cyanoacetamido oximes **5--9** corresponded to the \[2+1\] assembly of dipeptide Z-Phe-Val-OH onto H-Pro-NH~2~, affording tripeptide **13** (Table [2](#tbl2){ref-type="table"}).[@b29b] The activation of a dipeptide acid is a very interesting scenario to test the performance of an additive, since oxazolone formation is promoted as a result of the electron-donating effect of the *N*-aminoacyl substitution.\[[@b17], [@b30]\] As observed in Table [2](#tbl2){ref-type="table"}, the percentages of unwanted epimer were considerably higher than in the previous stepwise system. Following the trend observed in the synthesis of Z-Phg-Pro-NH~2~ (**12**), most of the cyanoacetamido oximes induced an approximately twofold higher retention of configuration than HOSu (**4**). Nonetheless, this \[2+1\] system produced moderate yields (66--84 %).

###### 

Yield and epimerization during the \[2+1\] formation of Z-Phe-Val-Pro-NH2 (13) using additives 3--9 (solution phase synthesis).[\[a\]](#tf2-1){ref-type="table-fn"}

  Entry   Coupling Reagent        Yield \[%\]   [ldl]{.smallcaps} \[%\][\[b\]](#tf2-2){ref-type="table-fn"}
  ------- ----------------------- ------------- -------------------------------------------------------------
  1       DIC/Oxyma (**3**)       89.8          12.8
  2       DIC/HOSu (**4**)        73.1          24.7
  3       DIC/MorOx (**5**)       80.5          13.2
  4       DIC/PipOx (**6**)       71.5          32.6
  5       DIC/AmOx (**7**)        66.7          14.8
  6       DIC/*N*-Oxyma (**8**)   83.6          18.4
  7       DIC/DmOx (**9**)        79.5          17.0

Couplings were performed without pre-activation in DMF at RT.

The [lll]{.smallcaps} and [ldl]{.smallcaps} epimers of this tripeptide model have been described in the literature.[@b29b] Retention times (*t*~R~) for each epimer were identified after co-injection with a pure sample.

Concerning the relative performance of the *N*-alkyl-cyanoacetamido oxime series (**5**--**9**), PipOx (**6**) again showed the least efficient control of optical purity, providing an even higher percentage of [ldl]{.smallcaps} epimer than HOSu in this system (cf. Entry 2 and 4, Table [2](#tbl2){ref-type="table"}). In sharp contrast, its morpholino analogue **5** showed a similar level of performance to Oxyma (**3**), providing an almost threefold higher retention of chirality than PipOx (**6**) (cf. Entries 1 and 3, 4, Table [2](#tbl2){ref-type="table"}). Moreover, MorOx (**5**) was one of the most efficient oximes in terms of yield of target tripeptide **13**. With regard to AmOx (**7**), a remarkably low content of epimer was detected, although accompanied by one of the lowest yields in the oxime series (Entry 5, Table [2](#tbl2){ref-type="table"}). Finally, *N*-Oxyma (**8**) was again clearly less efficient than its parent Oxyma (**3**) at preventing the loss of chirality (Entry 6, Table [2](#tbl2){ref-type="table"}).

The last epimerization model used to evaluate cyanoacetamido oximes **5--9** also consisted of a \[2+1\] segment coupling, in this case rendering tripeptide Z-Gly-Phe-Pro-NH~2~ (**14**) (Table [3](#tbl3){ref-type="table"}).[@b29b] Although this system is undoubtedly less sensitive than the previous one (**13**), valuable data were gathered. Unlike the synthesis of Z-Phe-Val-Pro-NH~2~ (**13**), PipOx (**6**) was not only more efficient than HOSu in decreasing epimerization (cf. Entry 2 and 4, Table [3](#tbl3){ref-type="table"}), but also superseded the performance of morpholine derivative **5** (cf. Entry 3 and 4, Table [3](#tbl3){ref-type="table"}). Consistent with previous epimerization models, the performance of HOSu (**4**) was poorer than *N*-alkyl-cyanoacetamido oximes **5**--**9**, including the relative yields obtained (cf. Entry 2 and 3--7). Surprisingly, MorOx (**5**) afforded one of the highest obtained contents of [ldl]{.smallcaps} epimer, along with *N*-Oxyma (**8**), which offers threefold less pure crude products than Oxyma (**3**) (cf. Entries 1 and 3, 6, Table [3](#tbl3){ref-type="table"}). Finally, of the cyanoacetamido oximes tested in the stepwise and fragment couplings **12**--**14**, AmOx (**7**) regularly performed at a remarkable level, offering the closest percentages of epimer to those obtained with Oxyma (**3**). Thus, AmOX (**7**) stands out as one of the most promising derivatives to substitute HOSu (**4**) as an epimerization-suppressant additive.

###### 

Yield and epimerization during the \[2+1\] formation of Z-Gly-Phe-Pro-NH~2~ (14) using additives 3--9 (solution phase synthesis)[\[a\]](#tf3-1){ref-type="table-fn"}

  Entry   Coupling Reagent        Yield \[%\]   [ldl]{.smallcaps} \[%\]
  ------- ----------------------- ------------- -------------------------
  1       DIC/Oxyma (**3**)       90.2          2.7
  2       DIC/HOSu (**4**)        80.4          9.0
  3       DIC/MorOx (**5**)       88.3          8.0
  4       DIC/PipOx (**6**)       86.6          7.6
  5       DIC/AmOx (**7**)        88.9          4.3
  6       DIC/*N*-Oxyma (**8**)   87.6          8.3
  7       DIC/DmOx (**9**)        87.9          5.4

Couplings were conducted without pre-activation in DMF at RT.

\[b\] The [lll]{.smallcaps} and [ldl]{.smallcaps} epimers of this tripeptide model have been described in the literature.[@b29b] Retention times (*t*~R~) for each epimer were identified after co-injection with a pure sample.

Coupling potency
----------------

A key step in the assessment of the quality of cyanoacetamido oximes **5**--**9** as additives to carbodiimides was the evaluation of their acylation potency in hindered couplings. To emphasize differences in the reactivity of the various active esters, peptide models containing demanding *N*-methyl or α,α-dialkyl amino acid junctions are often used.\[[@b17], [@b30]\] Among these, those featuring two or more consecutive α-aminoisobutyric acid (Aib) residues are particularly difficult to achieve, not only because of their considerable bulkiness, but also due to the stabilization of helical structures by the Thorpe--Ingold effect.\[[@b31]\] Hence, additives **3**--**9** were used in the solid-phase assembly of the Aib-enkephaline pentapeptide (**15**, Figure [4](#fig04){ref-type="fig"}).\[[@b17], [@b30], [@b32]\]

![Aib-enkephaline pentapeptide (15) used as coupling efficiency model to test *N*-alkyl-cyanoacetamido oximes 5--9.](open0001-0147-f4){#fig04}

The demanding conditions of this efficiency model (using short coupling times) were responsible for the very high percentage of des-Aib deletion tetrapeptide H-(Tyr-Aib-Phe-Leu-NH~2~), obtained with the various additives (**3**--**9**; Table [4](#tbl4){ref-type="table"}). Consistent with the trend observed in the epimerization experiments, the coupling performance of HOSu (**4**) was surpassed by all *N*-alkyl-cyanoacetamido oximes **5**--**9** (cf. Entry 2 and 3--7, Table [4](#tbl4){ref-type="table"}). Thus, most of the crude mixture obtained when using HOSu (**4**) consisted of deletion tripeptides, and no desired pentapeptide **15** was detected (Entry 2, Table [4](#tbl4){ref-type="table"}). In contrast, oximes **5--9** produced 10--30 % of Aib-pentapeptide **15** (Entries **3**--**7**, Table [4](#tbl4){ref-type="table"}). Particularly impressive was the efficiency displayed by the DIC/DmOx (**9**) system, which yielded the highest percentage of pentapeptide **15** in the cyanoacetamido oxime series (Entry 7, Table [4](#tbl4){ref-type="table"}), an efficiency comparable to HOAt (**2**) in similar conditions and similar to that of Oxyma (**3**, Entry 1, Table [4](#tbl4){ref-type="table"}).\[[@b29]\] The Aib-enkephaline model (**15**) also highlighted the enormous difference in acylation potency between DmOx (**9**) and the rest of the cyanoacetamido oximes (Entry 7 and 3--6, Table [4](#tbl4){ref-type="table"}). In a similar fashion to the epimerization assessment, PipOx (**6**) was found to be the least potent additive in amino acid activation (Entry 4, Table [4](#tbl4){ref-type="table"}). With respect to the acetamido analogue of Oxyma (**8**), a threefold decrease in reactivity was observed in comparison with parent ester **3**.

###### 

Percentage of tetrapeptide des-Aib (H-Tyr-Aib-Phe-Leu-NH~2~) during solid-phase assembly of pentapeptide 15 (H-Tyr-Aib-Aib-Phe-Leu-NH~2~)[\[a\]](#tf4-1){ref-type="table-fn"}

  Entry   Coupling Reagent                                        **15** \[%\]   des-Aib \[%\][\[b\]](#tf4-2){ref-type="table-fn"}
  ------- ------------------------------------------------------- -------------- ---------------------------------------------------
  1       DIC/Oxyma (**3**)[\[c\]](#tf4-3){ref-type="table-fn"}   46.5           53.5
  2       DIC/HOSu (**4**)                                        0              12.2
  3       DIC/MorOx (**5**)                                       13.6           75.8
  4       DIC/PipOx (**6**)                                       10.9           78.0
  5       DIC/AmOx (**7**)                                        18.9           73.1
  6       DIC/*N*-Oxyma (**8**)                                   15.4           79.9
  7       DIC/DmOx (**9**)                                        31.3           68.7

10 min pre-activation and 30 min coupling times were generally applied, except for Aib-Aib (30 min double coupling).

Deletion tetrapeptide des-Aib was identified by peak overlap in reversed-phase HPLC with an authentic sample obtained in solid-phase.

Results are taken from Ref. \[[@b17]\].

Conclusion
==========

Here, we evaluated several *N*-alkyl-cyanoacetamido oximes (**5**--**9**) as additives to carbodiimides in substitution of HOSu (**4**) for the construction of stable amino acid esters. One of the main advantages of the proposed cyanoacetamido oximes, which can be accessed by a shown optimized amidation/nitrosation procedure, is their enhanced water solubility, which was particularly impressive in MorOx (**4**). Consequently, not only was byproduct removal increased, thereby allowing more facile work-up, but the water-soluble nature of cyanoacetamido oximes also allows their use in protein conjugation and labeling in addition to peptide synthesis.

A thorough assessment of the reduction of epimerization in various stepwise and \[2+1\] fragment models and the coupling efficiency in demanding junctions indicated that the cyanoacetamido family of additives showed a much greater capacity of configuration retention and acylation potency than *N*-hydroxysuccinimide (**4**). Among the *N*-alkyl-cyanoacetamido oximes **5--9**, PipOx (**6**) can be considered the least efficient analogue of the series. Nonetheless, the inclusion of an oxygen atom in the six-membered ring (MorOx, **5**) not only resulted in an increase in solubility but also in coupling efficiency. With regard to *N*-Oxyma (**8**), the envisaged decrease in reactivity and control of configuration by abandoning the ester moiety in Oxyma (**3**) was confirmed. Last but not least, carboxamido analogues AmOx (**7**) and DmOx (**9**) are highlighted as the most promising cyanoacetamido oxime derivatives. While the unsubstituted carboxamide (**7**) stands out in the epimerization experiments due to its marked capacity to retain chirality, DmOx (**9**) showed superiority to the rest of the oximes in the reactivity of the corresponding active ester.

Experimental Section
====================

Instruments and materials
-------------------------

All solvents used for recrystallization, extraction, HPLC analyses and TLC were of HPLC-reagent grade. They were distilled before use, and stored under dry conditions. 1 H NMR were recorded on a Mercury 400 MHz spectrometer (Varian) at RT, using tetramethylsilane (TMS) as internal standard. Chemical shifts were reported in *δ* units (parts per million) relative to TMS. Melting points were determined using a Mel-Temp apparatus (B-540, Büchi) and are uncorrected. Reaction monitoring and determination of the purity of the compounds were performed by TLC on silica gel-protected aluminium sheets (Type 60 GF254, Merck), and the spots were detected by exposure to UV light at *λ*=254 nm. Analytical separation and characterization was conducted on a reversed-phase 2695 HPLC separation module (Waters) using a SunFire C~18~ column (3.5 μm, 4.6×100 mm) and a linear gradient of CH~3~CN in H~2~O supplemented with 0.1 % trifluoroacetic acid (TFA) at a flow rate of 1.0 mL min^−1^. Detection at 220 nm was performed using a coupled 2998 PDA UV detector (Waters). Chromatograms were processed with Empower software (Pro version). Peptide mass was detected by means of an HPLC-PDA system as described above, coupled to a Micromass ZQ mass detector (Waters), using the MassLynx 4.1 software. The compounds were named using ChemDraw Ultra version 11 (CambridgeSoft Corporation).

Preparation of N*-*alkyl-cyanoacetamido oximes\[[@b21]\]
--------------------------------------------------------

Oximes **5**--**8** were synthesized in a previous related publication addressing their inclusion in Fmoc-carbonates and were used for the present study.\[[@b21]\] The procedure used is an optimization of earlier amidation/nitrosation protocols and is detailed below for the preparation of DmOx (**9**).\[[@b23]\]

**Preparation of 2-(dimethylamino)-*N*-hydroxy-2-oxoacetimidoyl cyanide (DmOx, 9)**:\[[@b21], [@b23]\] An aq solution of Me~2~NH (40 %, 40 mL, 350 mmol) was added to ethyl cyanoacetate (**10**, 22.6 g, 200 mmol) at −10 °C. The reaction mixture was stirred for 1 h at RT and then refluxed for 6 h at 70 °C. Next, the solvent was evaporated in vacuo, leaving 2-cyano-*N,N*-dimethylacetamide (**11**) as an oil, which was used in the next nitrosation step without further purification. Gaseous methyl nitrite was generated from a suspension of NaNO~2~ (8.3 g) in a mixture of MeOH (10 mL) and H~2~O (10 mL) by dropwise addition of a mixture of concd H~2~SO~4~ (5 mL) and H~2~O (10 mL) and introduced to an ice-cooled solution of intermediate **11** (100 mmol) and NaOH (4 g, 100 mmol) in MeOH (30 mL). The mixture was stirred for 2 h at RT and concentrated to dryness in vacuo. The residue was dissolved in the least amount of H~2~O (5 mL), ice-cooled, and acidified with concd HCl to induce precipitation. The product was obtained as white crystals (21.43 mg, 76 %)\[[@b23]\]: mp 138 °C; ^1^H NMR (C~3~D~6~O): *δ*=3.02, 3.18 (2s, 6 H, C*H*~3~), 13.09 ppm (s, 1 H, O*H*, D~2~O exchangeable).

General method for racemization experiments\[[@b17], [@b30]\]
-------------------------------------------------------------

Acid Z-Phg-OH, Z-Phe-Val-OH, or Z-Gly-Phe-OH (0.125 mmol), DIC (20 μL, 0.125 mmol), and additive (0.125 mmol) were dissolved in dimethylformamide (DMF; 2 mL) at 0 °C. H-Pro-NH~2~ (14.3 mg, 0.125 mmol) was added, and the reaction mixture was stirred for 1 h at 0 °C and overnight at RT. The reaction mixture was diluted with EtOAc (25 mL), extracted with 1 [n]{.smallcaps} HCl (2×5 mL), 1 [n]{.smallcaps} NaHCO~3~ (2×5 mL), and saturated NaCl (2×5 mL), dried over anhyd MgSO~4~ and filtered. The solvent was removed in vacuo, and the crude peptide was directly analyzed by reversed-phase HPLC.

**Z-Phg-Pro-NH~2~**: A linear gradient of 25--50 % CH~3~CN in H~2~O supplemented with 0.1 % TFA over 25 min was applied: *t~R~*([ll)]{.smallcaps}=9.3 min, *t~R~* ([dl)]{.smallcaps}=9.9 min.

**Z-Phe-Val-Pro-NH~2~**: A linear gradient of 20--70 % CH~3~CN in H~2~O supplemented with 0.1 % TFA over 14 min was applied: *t~R~*([lll)]{.smallcaps}=6.7 min, *t~R~*([ldl)]{.smallcaps}=7.1 min.

**Z-Gly-Phe-Pro-NH~2~**: A linear gradient of 20--70 % CH~3~CN in H~2~O supplemented with 0.1 % TFA over 14 min was applied: *t~R~*([lll)]{.smallcaps}=5.5 min, *t~R~*([ldl)]{.smallcaps}=5.8 min.

Solid phase synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH~2~ (15)
---------------------------------------------------------

Pentapeptide **15** was prepared as reported.\[[@b17], [@b30]\] Fmoc-RinkAmide-PS resin (0.7 mmol g^−1^, 100 mg) was deblocked with piperidine in DMF (20 %, 10 mL) for 10 min, and washed with DMF (2×10 mL), CH~2~Cl~2~ (2×10 mL), and DMF (2×10 mL). Fmoc-Leu-OH (3 equiv), DIC (3 equiv) and additive (3 equiv) were mixed in DMF (0.5 mL), preactivated for 10 min and then added to the resin. The resulting slurry was stirred slowly for 1 min and allowed to couple for 30 min (double coupling for Aib-Aib). The ninhydrin test was performed during the introduction of the first residue (negative). The resin was subsequently washed with DMF (2×10 mL) and Fmoc removed with piperidine in DMF (20 %, 10 mL) for 7 min. Next, the peptide resin was washed with DMF (2×10 mL), DCM (2×10 mL), and DMF (2×10 mL), and coupled with the next amino acid. Coupling and deblocking steps were repeated to provide the pentapeptide. The percentage of des-Aib (Tyr-Aib-Phe-Leu-NH~2~) during solid-phase assembly of pentapeptide **15** was confirmed by peak overlap in the presence of an authentic sample. Crude pentapeptide **15** was analyzed by reversed-phase HPLC. A linear gradient of 10--90 % CH~3~CN in H~2~O supplemented with 0.1 % TFA over 20 min was applied: *t~R~* (**15**)=7.30 min, \[*M*+H\]^+^=611.35; *t~R~* (des-Aib)=7.50 min, \[*M*+H\]^+^=526.30.

This work was partially funded by the Secretaría de Estado de Cooperación Internacional (AECI, Spain), the Comisión Interministerial de Ciencia y Tecnologia (CICYT, Spain) (CTQ2009--07758), the Generalitat de Catalunya (Spain) (2009SGR 1024), the Institute for Research in Biomedicine Barcelona (IRB Barcelona), and the Barcelona Science Park. R.S.F. thanks the Ministerio de Educación y Ciencia (Spain) for a FPU PhD fellowship. Yoav Luxembourg (Luxembourg Biotechnologies) is thanked for his support.
