Synthetic Chemistry: Formation of the Amide Bond

Christian A. G. N. Montalbetti and Virginie M. Falque,

Evotec, United Kingdom
doi: 10.1002/9780470048672.wecb635

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Article Contents
Amide Bond Formation: Overview Amide Bond Strategies Conclusions Abbreviations Formations: Methods and

Amide bonds are found ubiquitously in natural or synthetic molecules of biologic interest. Since the early days of synthetic organic chemistry, methods for the formation of amides have been described. More recently, with the development of solid-phase chemistry and automated peptide synthesis, new strategies and reagents have been devised to overcome typical problems such as low conversion and racemization. This article provides an overview of the methodology that is available today. Depending on the nature of the synthetic target and the associated synthetic challenges, different approaches can be envisaged. Methods range from the rather straightforward use of acyl halides, anhydrides, and carbodiimides, to the more elaborate, low-racemization inducing methods that use phosphonium/uronium-based reagents. New amide bond-mediated ligation methodologies now offer new convergent strategies for the synthesis of highly functionalized molecules of biologic interest.

Amide bonds are found commonly in small or complex synthetic or natural molecules of biologic interest. Amide bonds are, for example, the structural backbone of proteins that play a crucial role in almost every biologic process. In nature, amides are formed via complex enzymatic pathways that ensure selectivity and specicity of the formed molecule. An analysis of the Comprehensive Medicinal Chemistry database revealed that more than 25% of known drugs bear amide functionality (1). For example, Taxol (pacitaxel; Bristol Myers Squibb, New York) 1 is a highly functionalized diterpenoid, which is extracted originally from the bark of the pacic yew tree. It is used to treat ovarian and breast cancers (2). Fuzeon (enfuvirtide; Roche Pharmaceuticals, Nutley, NJ) 2 is a synthetic biomimetic peptide and is the rst of a novel class of fusion inhibitor antiretroviral drugs used to treat HIV-1 infection (3). Lipitor (atorvastatin hemicalcium salt; Pzer, Inc., New York) 3 is the best-selling drug in the world, and it is used to treat high cholesterol (4). Sprycel (dasatinib; Bristol Myers Squibb) 4 is an oral dual BCR/ABL and Src family tyrosine kinase inhibitor, which is approved for use in patients with chronic myelogenous leukemia (5) (See Fig. 1). Since the early 1840s (6), amide bond formation has been a very active eld of research in organic chemistry. A multitude of synthetic methods have been elaborated and optimized (7). Relevant examples of these methods available to the bioorganic,

organic, medicinal, or combinatorial chemist are reported in this article.

Amide Bond Formation: Overview

Amide bond formation by direct condensation between an acid and an amine is not obvious and must overcome adverse thermodynamics (8). This dehydrative process can be achieved under forcing conditions such as high temperatures (160180 C), which are usually incompatible with the presence of other functionalities. Contrary to the ester formation between an acid and an alcohol, which is an equilibrium, the acid and the amine undergo rst an acidobasic reaction that yields the stable salt. Therefore, the acid must be activated by the attachment of a leaving group before being reacted with the amine (see Fig. 2). These two steps can be carried out separately with intermediate isolation of the activated species or by a one-pot synthesis with late introduction of the amine. More recent conditions allow coupling to occur with both the acid and the amine present in the reaction mixture. Often, the choice of the methodology for one specic amide formation is not only governed by the yield. Avoiding racemization at neighboring chiral centers, improving difcult isolation 1

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Synthetic Chemistry: Formation of the Amide Bond

O O O O NH O N O OH Taxol 1 HO O O H O O 1/2 Ca O Lipitor 3 O N H S N N Sprycel 4 N NH N N OH Ac-Tyr-Thr-Ser-Leu-IIe-His-Ser-Leu-IleGlu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-AsnGlu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-LysTrp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2 Fuzeon 2 O O OH OH F O OH N H

Cl

Figure 1 Examples of marketed drugs that contain amide bonds.

from reaction by-products or reducing the costs of the reagents, might be the key elements for decision.

Amide Bond Formations: Methods and Strategies

Acyl halides
Acyl or acid chlorides are used frequently in amide formation as activated forms of the corresponding carboxylic acid. A wide selection of acyl chlorides is available commercially. Otherwise, they can be prepared readily from the corresponding carboxylic acid in the presence of reagents such as thionyl chloride (9), oxalyl chloride 5 (10), phosphorus trichloride (11), and phosphorus pentachloride (12). Reactions that use oxalyl chloride or thionyl chloride are promoted by the addition of a catalytic amount of DMF (13) (see Fig. 3). Despite the high reactivity and low cost, these chlorinating reagents generate hydrogen chloride in situ and are not suitable for amide formation in the presence of acid labile groups. Hence, alternative basic conditions have been studied. For example, cyanuric chloride is used commonly to generate acyl chloride in nonacidic conditions (14). The presence of an organic or inorganic base maintains the basic pH conditions throughout the reaction. The cyanuric chloride method is also used in large-scale synthesis (15). Neutral conditions (i.e., non-acid generating) that use triphenylphosphine and a source of chloride as carbon tetrachloride (16), hexachloroacetone (17), trichloroacetonitrile (18), or trichloroisocyanuric acid (19) have been developed. Alternatively, 1-chloro-N,N ,2-trimethylpropenylamine (20) converts acids into acyl chlorides readily without HCl formation.
pKa~3-5 RCOOH + pKa~7-11 RNH2 RCOO +

The coupling reaction with the amine usually requires an additional acid scavenger (often a base like triethylamine, DIEA, or NMM) to trap the formed HCl. The reaction can also be accelerated in the presence of a catalytic amount of DMAP (21), pyridine, or metallic zinc (22). Nevertheless, acyl chlorides have some limitations. Acyl chlorides that bear -hydrogens can undergo ketene 6 formation under basic conditions (23). The subsequent amine addition occurs with potential loss of chirality and side reactions (24). Oxazolone-mediated racemization is encountered in peptide chemistry. N -protected peptidoyl chlorides yield the corresponding oxazolones 7 spontaneously. These transient species react readily with nucleophiles; but, under the standard aminolysis conditions, the -proton is acidic enough to enable acidobasic equilibrium, which compromises the chirality of the -center. Peptides are, therefore, grown usually at their N -terminus, thus avoiding the oxazolone formation. Furthermore, the acyl chloride activation of N -urethane-protected amino acid (e.g ., Boc, Fmoc, or Cbz) is unadvisable, as they react with the carbonyl of the neighboring urethane to yield the corresponding NCA 8 (25). NCA functionalities are notoriously reactive and promote many side reactions (see Fig. 3). Similar problems can also be observed with other activation methods. Alternatively, acid uorides are used to activate the acid. Acyl uorides are less sensitive to moisture and are more reactive toward primary and secondary amines than the corresponding acyl chloride. Furthermore, they are compatible with basic- (Fmoc and Cbz) or even acid- (Boc) labile amine protecting groups and less prone to promote racemization than their chlorinated homologs (26). Thus, the acid uoride method is often used in peptide synthesis (27). Cyanuric uoride 9 (28), TFFH (29), DAST (30), and deoxouor (31) are used commonly as uorinating reagents (see Fig. 4).
RNH3 H2O RNH2 Aminolysis + RCONHR

activating agent Activation

Figure 2 Activation and aminolysis process.

RCOX activated acid

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Synthetic Chemistry: Formation of the Amide Bond

Acyl chloride formation using oxalyl chloride 5 and DMF as catalyst: CO2 + CO O Cl 5 O Racemisation via ketene formation: R R H O Cl
NEt3

O Cl + H N H Cl

Cl N

RCOOH

O H N + HCl + R

O Cl

O
H2NR base

N R H

R C R 6 O

H2NR addition

R R H O

R N R + R H racemic

O N R H

Racemisation via oxazolone formation: H N O R O .. N H R Cl O O

aminolysis

O H
B

O R
BH B

H N R

O N 7

R
BH

H N O R

O N

H N O
H2NR

O N R 7

R H

H2NR

aminolysis

H N O NCA mediated side reactions: O R1O N H R2 OH O R

O N H

H N O

R O
R2 O O

H N R

O N H

H N O

SOCl2 R1OH

HN O O 8

R2 O

+ cat. Nu CO2

R2 H2N O Nu

+n x

HN O

O H2N R2 N H

R2 Nu n O

n x CO2

Polymerisation

Figure 3 Acyl chloridemediated amide coupling and potential side reactions.

Acyl azides
The acyl azide strategy was developed for peptide synthesis in the early 1900s. The original preparation of the acyl azide 10 from the corresponding methyl ester is a two-step synthesis. Displacement of the methyl ester with the hydrazine, which generates the acyl hydrazide, is followed by the nitrosation reaction to yield the acyl azide 10. Isocyanate formation, also called the Curtius rearrangement, is a possible side reaction, and ureas 11 are often observed as side products (see Fig. 4). An improvement of this method is the one-pot synthesis of the acyl azide from the carboxylic acid using DPPA 12 (32). Acyl azides are, however, potential explosives and the leaving group (free azide) is toxic, which provides some limitation to this method.

Similarly, carbonylimidazolium salts have been introduced. For example, CBMIT 15 is described as an efcient amino acylating reagent for peptide synthesis with sterically hindered amino acids (36). Recently, acyl benzotriazoles have been described as O -, N -, and S -acylating agents (37). These agents are prepared easily by reacting benzotriazole with acyl chlorides. N -protected -aminoacyl- or peptidoyl-benzotriazoles are coupled in aqueous acetonitrile solution with free amino acids or dipeptides (38). Under some specic conditions, hydroxyl carboxamides can be prepared directly from the corresponding hydroxyl acids without protection of the alcohol (39).

Anhydrides and mixed anhydrides

Anhydrides react readily with diverse nucleophiles such as amines. For example, the use of acetic anhydride was reported in the 1850s to produce acetamides (40). Symmetric anhydrides 16 can be prepared by dehydrating the corresponding acid under strong acidic conditions or at high temperatures. A more practical approach, however, consists of treating the carboxylic acid with DCC 17 (41). The anhydride is then subjected to aminolysis with the desired amine (see Fig. 5). This method is usually applicable to peptide synthesis, and, in theory, no additional base is required, as the carboxylate anion is produced in situ . Unfortunately, half of the acid is wasted during the 3

Acyl imidazoles
CDI 13 (33) allows one-pot amide bond formation and is also used for large-scale peptide chemistry (34). Initially, the mechanism may involve the formation of acyl carboxy imidazole and imidazole. Both intermediates react together to lead to the activated species as the acyl imidazole 14. Then the amine is added to undergo aminolysis (see Fig. 4). As imidazole is generated in situ , the reaction does not need an additional base and it is usually compatible with amine HCl salts (35). Incidentally, the acyl imidazole intermediate can also be isolated and stored. Some simple acyl imidazoles are even available commercially.

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Synthetic Chemistry: Formation of the Amide Bond

Acyl fluorides
F N N N 9 pyridine F

F O R N N + HF.Pyridine HO N

F N N F + R O F RNH2 base R O NHR

O R OH

O N F activated ester

Acyl azides O
H2N NH2

O
HONO

O R 10 N2 N2

RNH2 base

O R NHR

OMe

N NH2 H Curtius rearrangement

RNH2

O R N H NHR 11

R N C O Acyl midazoles O R OH O + N N 13 O N N 15 N N 2OTf N HN N N + R O O O N N CO2

O R N 14 N

RNH2

O R NHR

Figure 4 Acyl uorides, acyl azides, and acyl imidazoles.

O 2 eq R OH

DCC 17

O R O 16

O R + N H

O N H

RNH2

O R N R H2 + R

O O R

O NHR + R

O OH

Figure 5 Symmetric anhydride preparation and coupling reaction.

process, which practically limits the application of this method to cheap and commercially available symmetric anhydrides. Several mixed anhydride methods have, therefore, been developed. The acid is coupled to a second acid moiety that is considered to be disposable. This strategy relies fully on the regioselectivity of the aminolysis where the reactive center a is more reactive than b (see Fig. 6). The concept of mixed anhydrides has been extended to carbonic, boric, iso-ureas, phosphoric, and phosphinic acid-derived species. Iso-ureas and phosphorous-containing reagents will be discussed in separate articles. Mixed pivalic anhydride 18 (42) is an example of selective mixed carboxylic anhydride. Selective aminolysis could be caused by the steric hindrance of the tert -butyl group. A mixed carbonic anhydride strategy has also been studied to 4

overcome the aminolysis selectivity problem. This method exhibited excellent selectivity when ethoxycarbonyl 19 (43) or tert -butyloxycarbonyl 20 (44) anhydrides were used. The selectivity is mainly caused by the higher electrophilicity of one of the carboxylic centers toward the amine. Ethoxycarbonyl anhydrides can be prepared conveniently using ethyl chloroformate or EDDQ 21 (45). Acyl aryl boron species also react with amines to yield amides with mixed results. For example, catecholborane was used to generate lactams successfully (46). Aryl boronic acids with electron-withdrawing groups, such as 3,4,5-triuorobenzeneboronic acid and 3,5-bis-(triuoromethyl)benzeneboronic acid, can act efciently as an amidation catalyst when added to a mixture of acid and amine (47).

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Synthetic Chemistry: Formation of the Amide Bond

O R
a

O R

O
a

O O 18
b

O P OR OR b Phosphoric O R
a

N EtO O 21 OEt or tBu 20 19

OEt

Carboxylic O R OH R O
a

O O P R R b Phosphinic O R
a

O O
b

Carbonic R N H R R O O
a

N O
b

O B
b

Iso urea

Boric

Figure 6 Different types of mixed anhydrides used for amid bond formation.

O-acyl iso-ureas
O -acylisourea species 22 are generated easily by reacting carbodiimides with a mixture of the desired carboxylic acid and amine. Then, they undergo aminolysis readily to form the amide and the urea by-product (48). Often, formation of unreactive N -acylurea 23 by acetyl transfer and racemization are observed (see Fig. 7). This side reaction can be minimized substantially by reacting the acid with the carbodiimides at 0 C before adding the amine or by using DMAP or HOBt 24 as adjuvants (49). DCC 17 (50), DIC 25, EDC 26, and polymer supported PS-CC (51) 27 are available commercially. Elimination of the resulting ureas can be achieved easily by ltration or solvent wash depending on their solubility. In the case of PS-CC, the
R O R + O + N C N R

urea by product is formed advantageously on the solid support while the amide is released in solution.

Esters
Alkyl esters
Alkyl esters (e.g., methyl, ethyl, benzyl esters) are usually stable toward amines and, thus, are used as protecting groups. Some anecdotic examples of amide bond formation with alkyl esters, however, are reported in the literature. High reaction temperature, addition of a Lewis acid (52), or use of organoaluminium species generated from DIBALHH2 NR (53) can enable these reactions. Saturated ammonia in methanol can also react with
O O NH R + R NHR

RNH2 + R

O O

R NH R

Aminolysis

HN R O

RNH3

O -acylisourea 22

Acyl tranfer

R O

R NH R

N-acylurea 23

Name DCC - 17

R N C N R N C N

Comment Resulting DCU poorly soluble. Eliminated by filtration. Solution-phase chemistry. Resulting diisopropyl urea soluble in DCM. Eliminated by DCM washes. Solid-phase chemistry. N Resulting urea is water-soluble. Eliminated by aqueous work-up Solution-phase chemistry. Resulting urea immobilised on solid support Solution-phase chemistry using polymer supported reagents

DIC - 25 EDC - 26

N C N

N C N

HCl

PS-CC - 27

N C N

Figure 7 Carbodiimides. WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Synthetic Chemistry: Formation of the Amide Bond

O R + R N H OH
n

Mes N

N Mes

O R N n R + MeOH OH

OMe

IMes 5 mol% THF

Figure 8 Hetereocyclic carbene catalyzed amidation.

methyl esters at room temperature to form the primary amides (54). Recently, amidation of methyl esters with unprotected amino alcohols in the presence of a catalytic amount of IMes, a readily available carbene, was achieved in good yields (see Fig. 8). Initially, the carbene was proposed to react with the methyl ester to generate the activated C2-acylimidazolium intermediate (55). X-ray evidence, however, suggests a more complex mechanism (56).

Fig. 10) (64). Racemization during DIC- 25 / HOCt- 33 mediated coupling is negligible with all amino acids except histidine (65). Another eld of application for active esters is solid-phase synthesis. Some polymer-supported reagents are available commercially (see Fig. 9). The acid is rst immobilized on a polymer support as an active ester and the excess reagents are washed away conveniently. Finally, the amide is released by amine treatment. During the cleavage, a limited amount of amine can be used to avoid the presence of excess amine in the nal mixture. The acid is loaded onto the resin using classic ester condensation methods for TFP resin 35 (66), HOBt resin 36 (67), and oxime resin 37 (68). In the case of the triazine resin 38, the acid is loaded via an aromatic nucleophilic substitution in the presence of a base (69).

Onium salts
An elegant one-pot coupling method based on both mixed anhydride and activated ester strategy was introduced by peptide chemists in the early 1970s. These coupling reagents were produced by combining reactive onium salts, such as phosphonium, uronium, iminium, or quaternized nitrogen species, with racemization suppressants, such as HOBt 24 or HOAt 31. Most of these coupling reagents are now available commercially and are compatible with both Boc- or Fmoc-strategy peptide synthesis (70, 71), using solid-phase parallel and combinatorial techniques (72). Usually, the coupling is performed by reacting the desired amine and acid in the presence of the onium-coupling reagent and an organic base such as triethylamine, DIEA, NMM, 2,6-lutidine, or collidine. In most cases, good yields and low racemizations are reported.

Activated esters
Usually, esters of phenols are easier to hydrolyze than alkyl esters and they also react with a wide range of nucleophiles such as amines. The reactivity is increased when electron withdrawing groups are present on the phenyl ring. Activated esters can be obtained from the acid using DCC- 17 mediated coupling or via the acid chloride. They can be used immediately without additional purication or they can be stored. For example, PNP 28 esters are puried easily by recrystallization in alcoholic solvents with which they do not react. Then, aminolysis is performed at room temperature (57). Other examples of alcohols used to activate acids are represented in Fig. 9. PFP 29 esters have been recommended for the synthesis of heterocyclic amides, when DCC 17 or DIC 25 alone had failed (58). HOSu 30 is coupled to acids easily using N,N-disuccinimidyl carbonate (see chapter on DIC) and, being water soluble, it can be removed readily at the purication stage (59). PFP and OSu esters are of signicant importance, especially in the preparation of biomaterials and in bioconjugation chemistry. An increasing number of these activated esters are available now commercially. HOBt 24 is one of the most common reagents in peptide chemistry. Good yields and low racemization are observed with O -Bt esters (60). Furthermore, yields and enantiomeric purities are increased when used in conjunction with DCC 17 (61) or even HOBt-based phosphonium- or uronium-coupling reagents (see chapter on Onium salts). In 2005, HOBt monohydrate, the standard form for this reagent, was reclassied by the United Nations as a desensitized explosive. This measure limited its availability drastically (UN3380). Other efcient racemization suppressants are HOAt 31, HODhbt 32, HOCt 33, and N -hydroxytetrazole 34 (62). The lack of popularity of HODhbt 32 in peptide coupling, despite very low racemization, can be explained by the high degradation levels of the activated ester, which are observed during the aminolysis step (63). HOAt 31 has been reported to be more efcient than HOBt 24 in some difcult cases. Additional chelation or neighboring effect caused by the pyridine ring could explain this increased reactivity (see 6

Phosphonium-based coupling reagents

BOP 39, also known as Castros reagent, was the rst of the HOBt-based onium reagents (73) (see Fig. 11). The deprotonated acid reacts rst with BOP 39 to produce both the highly reactive (acyloxy)phosphonium 40 species and the OBt anion. The initial mechanism postulated the direct attack of the OBt anion on the (acyloxy)phosphonium 40, which generates the aminolysable-activated O -Bt ester 41. This process is driven by the formation of the stable phosphoric triamide 42 (74). More recent studies suggested the existence of an additional step. The (acyloxy)phosphonium intermediate 40 rst reacts with the carboxylate to form the symmetric anhydride 43. The subsequent activation with HOBt 24 is relatively slow and yields the anticipated O -Bt ester while regenerating the carboxylate. The additional step can be accelerated by the addition of HOBt 24 to the reaction mixture (75). Whether it is really necessary to use HOBt 24 in conjunction with HOBt-based uronium or phosphonium reagent is still debatable and it must be investigated on a case-by-case basis. PyBop (G.L. Biochem (Shanghia) Ltd., Shanghai, China) 44 and PyAOP 45 (76) where the dimethyl moiety was replaced were introduced to avoid the generation of toxic HMPA 42 (77). Other examples of phosphonium-coupling reagents are shown in Fig. 12. Halogenophosphonium reagents often give better yields and lower racemization than HOBt-based ones for the coupling

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Synthetic Chemistry: Formation of the Amide Bond

O R ROH : OH

ROH ester preparation

O R O R

RNH2 NR3

O R NHR + ROH

F HO NO2 HO F PNP 28 O HO N N HO N N HOBt 24 PFP 29

F F F HO

Cl Cl Cl 2,4,5-trichlorophenol

O HO N O HONB

O HO N O HOSu 30

N N

HO N

N N HO N N N

COOEt HO N

N N N

N HOAt 31

HODhbt 32 Polymer supported reagents O C F F TFP resin 35

Figure 9 Activated esters.

HOCt 33 OH

34

F F OH N H

N H

OH N N N

H N N NO2

N N Cl

Cl

HOBt resin 36

Oxime resin 37

Triazin resin 38

N N N H N N O R'' R R O O NHR'R''

phosphoric- and phosphinic-based coupling reagents are shown in Fig. 12 [DEPBT 51 (84), BDP 52 (85), BOP-Cl 53 (86)].

Uronium/guanidium-based coupling reagents

Another popular family of reagents is based on uronium species such as HBTU 54 and TBTU 55 (87, 88). These species are the hexauorophosphate and tetrauoroborate salts of the same molecule, respectively. The coupling is performed the same as the phosphonium reagents, and the nature of the non-nucleophilic counter ion has no inuence on the outcome. The driving force is, in this case, the formation of the stable tetra-methylurea 56 (see Fig. 13). In some cases, the amine can react directly with the coupling reagent to form the undesired guanidine 57. This side reaction can be suppressed by adding HOBt 24 to the reaction. Usually, these reagents are found in their more stable guanidinium form (N -form) (89, 90). The uronium species of HBTU 54 and HATU 58, however, have been isolated and are more active than the guanidinium species. Unfortunately, under standard coupling conditions, the O -form is converted quickly to the N -form (91) (see Fig. 14). Some examples of uronium-based coupling reagents are represented in Fig. 15 [e.g., BCC 59 (92), TDBTU 60, TNTU 61, TPTU 62, TSTU 63 (88), HAPyU 64 (93), TAPipU 65 (94), CIP 67 (95), BTFFH 68 (96), HOTT / TOTT 69 (97)]. As for phosphonium-based coupling reagents, poor results are observed with sterically hindered amines when HOBt 24 is present. Therefore, some alternative reagents have been designed. For 7

R'

Figure 10 Additional chelation with HOAt 31.

of N -methylated amino acids. The Bt ester is believed to be too stable to react with these hindered amines, which enables degradation or racemization to occur. Some effective reagents that eliminate the need for HOBt 24 have therefore been developed (see Fig. 12). For example, PyBrop 48 is an efcient peptide-coupling reagent for N -methylated amino esters (78). It is interesting to notice that reagents such as CloP 46 (79) and BroP 47 (80) had been considered initially as poor peptide-coupling reagents as they lead to noticeable racemization with primary amino acids. Other organo-phosphorous reagents are based on the mixed carboxylic-phosphoric or phosphinic anhydrides. Initially used to convert carboxylic acids into acyl azides, DPPA 12 has been introduced as a one-pot coupling reagent for peptide chemistry (32), and it was adapted later to solid-phase chemistry (81). The driving force of these reactions is the formation of the phosphoric or phosphinic acids and their salts. Later DPP-Cl 49 (82) and FDPP 50 were introduced. FDPP 50 has been used successfully in macro cyclizations (83). Examples of

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Synthetic Chemistry: Formation of the Amide Bond

O N PF6 + R'NH3 O RCOO

Et3N

O N + OBt N P N N 42
+ OBt

N N N BOP O P N 39 N

O OBt R activated ester 41 +

R
R'NH2

O P N N 40

NHR' + HOBt 24 + Et3NH

PF6

+ RCOO

RCOO

O N P N N 42
Figure 11 BOP 39-mediated coupling.

O +

R O R symmetric anhydride 43

Phosphonium based coupling reagents N N N N O P N N BOP 39 PF6 PF6 N N N N N O P N N AOP N PF6 N N N O P N N PyBOP 44 PF6 PF6 PF6 N N N N N O P N N PyAOP 45 PF6 PF6

N N P Cl N

N N P Br N

N N P Cl N PyCloP

N N P Br N PyBroP 48

CloP BroP 46 47 Miscellaneous organophosphorus coupling reagents N N N O P OEt O OEt N N N O BDP 52 O P Ph O P Ph DPP-Cl 49

O DEPBT 51 F F F F

O P OEt OEt

O PhO P N3 PhO DPPA 12 O O P

2

O F FDPP 50

Ph

Ph

Cl

Cl

BOP-Cl 53

Figure 12 Organophosphorous coupling reagents.

example, HATU 58 has proved to be very efcient in some sterically hindered couplings (64, 98). The superior reactivity of the At-activated ester toward amines is discussed in the Activated Esters section. TOTU 70 enables the formation of an activated acyl oxime intermediate, and low racemization in peptide couplings has been reported (99). 8

Iminium-based coupling reagents

Iminium reagents are inspired directly from the uronium family. They differ structurally by the replacement of the amino groups with a hydrogen, an alkyl, or an aryl group. For example, BOMI 71 was reported to be more reactive than the correspondent uronium reagents (100). The increased reactivity could stem

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Synthetic Chemistry: Formation of the Amide Bond

N N

PF6 BF4

HBTU - 54 or TBTU - 55

O R OH

O O R O N O N R + O N 56 N OBt
RNH2

NHR

N N O
RNH2

NEt3

+ HOBt 24

OBt

N N N OH 24 + RN

N N

57

Figure 13 HBTU 54/TBTU 55-mediated coupling.

N N
[(Me2N)2CCl] NEt3

N N

Mukaiyamas reagent
PF6 guanidinium N -Form

N O

X = C 24 X = N 31

N X N N OH
1) K2CO3 2) [(Me2N)2CCl]

X
N

NEt3

N X N O N N

PF6

uronium O -Form

Figure 14 Uronium and guanidinium forms of HBTU (X = C) 54/HATU (X = N) 58.

2-Chloro-1-methylpyridinium iodide 77, also called Mukaiyamas reagent, in the presence of a carboxylic acid and a tertiary amine yields an activated 2-acyloxy-pyridinium species 78. This intermediate reacts with a range of nucleophiles. The driving force for this reaction is the generation of a stable tautomeric pyridone. Mukaiyamas reagent has been reported in the preparation of activated esters, the formation of amides, and the conversion of -amino acids to the corresponding -lactams (104, 105). The poor solubility of pyridinium iodides in conventional solvents indicates that the reaction requires reuxing in methylene chloride. To alleviate this limitation, novel reagents have been suggested: BEMT 79 (106), BEP 80, FEP 81, BEPH 82, and FEPH 83 (107) (see Fig. 16). Tetrauoroborate and hexachloroantimonate have been adopted as counter ions. These reagents were used successfully in the synthesis of oligopeptides and in solid-phase peptide synthesis.

from the reduced number of mesomeric forms observed in the iminium species compared with the uronium forms. Representative examples of iminium reagents are shown in Fig. 15 [BOMI 71, BDMP 72 (101), BPMP 73, FOMP 74 (100)].

Isoxazolinium Salts
Woodwards reagent K or NEPIS 84 is a zwitterionic isoxazolinium that reacts with N -protected amino acids in presence of triethylamine to form the activated enol ester 85 (108) (see Fig. 16). This intermediate can be reacted without additional purication with an amine to yield the desired amide and the sulfonate by-product that can be easily removed by aqueous extraction.

Quaternized nitrogen-based coupling reagents

Triazinyl esters
Recently, DMTMM 75 has been described as an efcient, one-pot coupling reagent for ester and amide bond formation (102). This reagent rst undergoes an SN Ar reaction as in the case of cyanuric uoride 9. The activated ester then undergoes aminolysis. The in situ liberation of N -methyl morpholine avoids the use of an additional base conveniently. The triazinone 76 by-product is eliminated easily by an aqueous wash (see Fig. 16). An inexpensive substitute to DMTMM is 2-chloro-4, 6-dimethoxy-1,3,5-triazine in the presence of an additional organic base (103).

Amide bonds by chemical ligation

New ligation strategies allow the selective formation of an amide bond between two highly functionalized fragments such as unprotected peptides, glycopeptides, or other molecules of biologic interest. The convergent assembling of complex, preformed peptidic sequences overcomes the inevitable contamination issues observed during extended linear peptide synthesis (109).

Native thioligation
This original methodology (110) requires the presence of a cysteine at the N -terminal position of fragment B and the

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Synthetic Chemistry: Formation of the Amide Bond

Uronium / Guanidinium based coupling reagents N N N O BCC 59 N N N N N N N N PF6 N N N N O N O

BF4

O N O N N

BF4 N N O TPTU 62 O N

BF4

O N O O N TSTU 63 X N NC N

BF4

N N CIP 67

PF6 Cl

O TDBTU 60 N PF6 N N N N N BF4

TNTU 61

N N

PF6 F N O S

N O COOEt N N

BF4

HAPyU TAPipU 65 64 Iminium based coupling reagents N N N O N BOMI 71 SbCl6 N N N O N BDMP 72

BTFFH 68

X = PF6 : HOTT X = BF4 : TOTT 69 F N SbCl6 N N O N BPMP 73 F O N F F F

TOTU 70

SbCl6

SbCl6

FOMP 74

Figure 15 Uronium/guanidinium and iminium-based coupling reagents.

introduction of a thioester at the C-terminal position of fragment A. Regiospecic coupling of the two unprotected fragments can be achieved in a two-step process described in Fig. 17. The rst step is a chemoselective transthioesterication between the thiol functionality of the terminal cysteine and the terminal thioester to form the thioester linked intermediate. This step is called the capture reaction. The second step is the rapid, intramolecular acyl transfer from the thio- to the amino-position of the cysteine to yield the desired amide bond. Usually, no racemization is observed (111). Different Boc (112) or Fmoc (113) compatible solid-phase strategies have been devised to allow the preparation of peptides bearing a C -terminal thioester. Recently the introduction of a 2-(ethyldisulfanyl)phenol ester at the C -terminal position of fragment A has been used in an elegant solution phase approach (114) (see Fig. 18). The rst amino acid of fragment A is coupled to 2-(ethyldisulfanyl)phenol 86. The resulting phenol ester is sufciently stable to be used as a protecting group and to allow the growing fragment A to use standard Boc strategy peptide synthesis. After nal deprotection, fragment A can undergo native thioligation. First, the disulde bond is cleaved under reductive conditions or in the presence of an excess of thiol reagent. The resulting 2-mercaptophenyl ester might be in an unfavorable, yet dynamic, equilibrium with the corresponding S-2-hydroxyphenyl thioester via intramolecular O - to S -acyl transfer, which generates in situ the appropriate setup for native thioligation. 10

A similar type of native chemical ligation has been extended to B fragments that contain homocysteine (115), selenocysteine (116), and histidine (117) at their N-terminal positions.

Staudinger ligation
The Staudinger ligation (118) is a less restrictive approach that can be applied to couple unprotected peptides. First, a C -terminal phosphinomethylthioester is introduced on Fragment A 87, and the N-terminal amine of fragment B is converted to the corresponding azide 88. The two fragments are then reacted together via a Staudinger reaction (see Fig. 19), which yields the iminophosphorane 89. This intermediate undergoes intramolecular S - to N -acyl transfer readily to produce the corresponding hydrolysable amidophosphonium salt 90. In principle, this methodology is independent of the nature of amino acids present at the ligation point and can be even extended to all types of acids and amines. For example, N -glycosylated amides (119) and peptides have been prepared via stereoselective Staudinger ligation (120). Challenging, medium-sized (7- to 10-membered) lactams have been ring-closed in good yield using the Staudinger ligation sequence described in Fig. 19 (121). In this example, the phosphine reagent has been protected judiciously as a borane complex. The Staudinger ligation is then triggered by the deprotection of the phosphine under basic conditions.

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Synthetic Chemistry: Formation of the Amide Bond

DMTMM 75 mediated coupling O R + O

O N N 75 OMe N Cl OMe

O R O

OMe N N N OMe + O

H N Cl + RNH2

O R NHR O

OMe N N HN 76 OMe

RNH3 Mukayiama coupling O

NEt3

O OH
77 Cl N I

O N O Cl + Et3NHI R O 78 N Cl
RNH2

O R NHR

+ O N

+ HNEt3, Cl

S Mukayiama type reagents: N

Br BF4

Et BEMT 79 NEPIS 84 mediated coupling O

OH

SbCl6 BF4 N X X N Et Et BEP (X = Br) 80 BEPH (X = Br) 82 FEP (X = F) 81 FEPH (X = F) 83

R O NHEt
then RNH2

O3S 84

O N Et

O R

base

O3S 85

+ NHR

O3S

O NHEt O

Figure 16 Quaternized nitrogen-based reagents.

HS Fragment A O SBn + H2N Fragment B Fragment A S O H2N + HSBn

Fragment B

HS O Fragment A
Figure 17 Native thioligation.

N H

Fragment B

Enzymatic catalysis
Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospecicity of over 99%. Despite limited enzyme-substrate compatibility, specic conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using -chymotrypsin, a peptidase isolated from bovine pancreas.

Conclusions
This article presents an overview of the different amide bond formation methodologies that are available to the organic and biochemist. For nearly two centuries, the methods have evolved from the original symmetric anhydrides and acyl chlorides. When Fischer started to study peptidic couplings in the early 1900s, it became obvious that poor yields and racemization would present a major challenge. During the last three decades, the design and synthesis of new coupling reagents have been an area of intense investigation. The predominance of carbodiimide and active ester procedures have been replaced gradually 11

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Synthetic Chemistry: Formation of the Amide Bond

EtS2 1)

O BocHN R1 OH

HO

86

O H2N

EtS2 O

Multistep Boc Strategy Peptide synthesis

O Fragment A R1

EtS2 O

EDC 26, DMAP, THF

R1
intramolecular acyl transfer

2) 4N HCl Dioxane O Fragment A R1

HS

excess RSH HO S O Fragment A R1 HS O

Native thioligation
H2 N Fragment B

Figure 18 Convenient fragment A preparation.

General Principle O Fragment A S 87 PPh2 + N3 Staudinger Reaction Fragment B 88 Fragment A 89 O S PPh2 N + N2

Fragment B

Acyl transfer O Fragment A N H Fragment B

H2O

O Fragment A 90 S N Fragment B

PPh2

Non peptidic example of Staudinger ligation

BH3 Ph2P SH N3Tf, CH2Cl2 CuSO4 H2O, MeOH K2CO3

BH3 Ph2P S O
DABCO THF, H2O 99.5 : 0.5 70oC

HOOC H 2N

HOOC N3

DCC or EDC CH2Cl2, 0oC

O HN

N3 98%

99%
Figure 19 Staudinger ligation.

80%

by onium salts approaches. The introduction of racemization suppressants combined with the development of solid-phase chemistry and urethane-based protecting group have allowed high-throughput chemistry and automated peptide synthesis to be a reality. Emerging technologies such as ligation allow convergent synthesis by amide coupling between two highly functionalized molecules.

BCC BDMP

BDP BEMT BEP BEPH Boc BOMI

Abbreviations
AOP (1H -7-azabenzotriazol-1-yloxy)tris (dimethylamino)phosphonium hexauorophosphate azabenzotriazole

At 12

benzotriazolyloxy-bis (pyrrolidino)carbonium hexauorophosphate 5-(1H -benzotriazol-1-yl)-3,4-dihydro-1methyl 2H -pyrrolium hexachloroantimonate N-oxide benzotriazol-1-yl diethyl phosphate 2-bromo-3-ethyl-4-methylthiazolium tetrauoroborate 2-bromo-1-ethylpyridinium tetrauoroborate 2-bromo-1-ethylpyridinium hexachloroantimonate tert-butyloxycarbonyl N -(1H -benzotriazol-1-ylmethylene)-N methylmethanaminium hexachloroantimonate N -oxide

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Synthetic Chemistry: Formation of the Amide Bond

BOP

BPMP BOP-Cl BroP Bt BTFFH CBMIT Cbz CIP CDI CloP DAST DCC DEPBT DCU DIC DIEA DMAP DMF DMTMM DPPA DPP-Cl EDC or EDCI EDDQ FDPP FEP FEPH Fmoc FOMP

HATU

HAPyU

HBTU

(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexauorophosphate or Castros reagent 1-(1H -benzotriazol-1-yloxy)phenylmethylene pyrrolidinium hexachloroantimonate bis(2-oxo-3-oxazolidinyl)phosphinic chloride bromo-tris-(dimethylamino)phosphonium hexauorophosphate benzotriazole 1,1,3,3-bis(tetramethylene)uorouronium hexauorophosphate N ,N -carbonylbis(3-methylimidazolium) triate benzyloxycarbonyl 2-chloro-1,3-dimethylimidazolidinium hexauorophosphate carbonyl diimidazole chloro-tris-(dimethylamino)phosphonium hexauorophosphate diethylaminosulfur triuoride dicyclohexylcarbodiimide 3-(diethoxyphosphoryloxy)-1,2,3benzotriazin-4(3H )-one dicyclohexylurea diisopropylcarbodiimide N ,N -diisopropylethylamine also known as H unigs base N ,N -dimethylaminopyridine N ,N -dimethylformamide 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4methyl-morpholiniumchloride diphenylphosphoryl azide diphenylphosphinic chloride 1-ethyl-3-(3-dimethylamino)carbodiimide HCl salt, also known as Water Soluble Carbodiimide, HCl (WSCHCl) 2-ethoxy-1-ethoxycarbonyl-1,2dihydroquinoline pentauorophenyl diphenyl phosphinate 2-uoro-1-ethylpyridinium tetrauoroborate 2-uoro-1-ethylpyridinium hexachloroantimonate 9-uorenylmethoxycarbonyl 5-(pentauorophenyloxy)-3,4-dihydro-1methyl 2H -pyrrolium hexachloroantimonate O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexauorophosphate 1-(1-pyrrolidinyl-1H -1,2,3-triazolo[4,5b]pyridine-1-ylmethylene)pyrrolidinium hexauorophosphate N -oxide O-(1H -benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexauorophosphate

HMPA HOAt HOBt HOCt HODhbt HONB HOSu HOTT

IMes NCA NEPIS NMM PFP PNP PS-CC PyAOP

PyBop

PyBrop TAPipU

TBTU

TDBTU

TFFH TFP TNTU

TPTU

TOTT

TOTU

TSTU

hexamethylphosphoric triamide 1-hydroxy-7-azabenzotriazole 1-hydroxybenzotriazole ethyl-1-hydroxy-1H -1,2,3-benzotriazine 3,4-dihydro-3-hydroxy-4-oxo-1,2,3benzotriazine N -hydroxy-5-norbornene-2,3dicarboximide N -hydroxysuccinimide S -(1-oxido-2-pyridinyl)-1,1,3,3tetramethylthiouronium hexauorophosphate N ,N -bismesitylimidazolylidene N -carboxyanhydride N -ethyl-phenylisoxazolium-3-sulphonate, also known as Woodwards reagent K N -methylmorpholine pentauorophenyl para-nitrophenyl N -cyclohexylcarbodiimide-N -methyl polystyrene 7-azabenzotriazol-1-yloxy-trispyrrolidinophosphonium hexauorophosphate benzotriazol-1-yloxy-trispyrrolidinophosphonium hexauorophosphate bromotri(pyrrolidino)phosphonium hexauorophosphate O -(7-azabenzotriazol-1-yl)-1,1,3,3bis(pentamethylene)uronium tetrauoroborate O -(1H -benzotriazol-1-yl)-1,1,3,3tetramethyluronium tetrauoroborate N ,N ,N ,N -tetramethyl-O -(3,4-dihydro-4oxo-1,2,3-benzotriazin-3-yl)uronium tetrauoroborate N ,N -tetramethyluoroformamidinium hexauorophosphate tetrauorophenol 2-(5-norbornene-2,3-dicarboximido)1,1,3,3-tetramethyluronium tetrauoroborate O -(1,2-dihydro-2-oxo-pyridyl)-1,1,3,3tetramethyluronium tetrauoroborate S -(1-oxido-2-pyridinyl)-1,1,3,3tetramethylthiouronium tetrauoroborate O -((ethoxycarbonyl)cyanomethylene amino)-N ,N ,N ,N tetramethyluronium hexauorophosphate N ,N ,N ,N -tetramethyl-O -(N succinimidyl)- uronium tetrauoroborate

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Synthetic Chemistry: Formation of the Amide Bond

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Further Reading
Han S-Y, Kim Y-A. Recent development of peptide coupling reagents in organic synthesis. Tetrahedron. 2004;60:24472467. Montalbetti CAGN, Falque V. Amide bond formation and peptide coupling. Tetrahedron 2005;61:1082710852. Pearson, AJ, Roush, WR, eds. Handbook of Reagents for Organic Synthesis: Activating Agents and Protecting Groups. 1999. John Wiley & Sons, New York. Zabicky, J, ed. Synthesis of Amides. 1970. Interscience, London.

See Also
Biomolecules, Asymmetric Synthesis of Biomolecules, Enzymatic Synthesis of Chemical Ligation: Peptide Synthesis Organic Chemistry in Biology Peptide Combinatorial Synthesis Peptide Synthesis Small Molecules Synthesis, Key Reaction of

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