Coupling Reagents

The ‘coupling reaction’ refers to the controlled formation o f a peptide/amide bond between the activated carboxyl group o f one amino acid or peptide and an amino group o f an another. The stereochemical integrity o f the carboxylic component must be preserved during this bond formation. A particular problem is that activation o f the carboxy group increases the acidity o f the a-proton; this may lead to oxazolone formation. (Figure 34). (14) R^O N . . . w R^o--- ^ ^ OH (15) (13)

Figure 34. The activated dipeptide (13) undergoes intramolecular nucleophilic attack with loss o f Y (the activating group) to form the oxazolone (14) this can enolise to give compound (15) with resulting loss o f stereochemical intregity. (R^O is an 0-alkyl moiety and R is a residue side chain).

Compared to synthesis in solution, problems with racémisation are much reduced in solid phase peptide synthesis. Alkoxyoxazolones (14) are less easily racemised and more easily aminolysed in comparison to oxazolones derived from acyl amino acids. In solid phase synthesis, amino acids are activated when N “-Fmoc- or Boc-protected, these urethane moieties prevent the tendency to form oxazolones which are then prone to racémisation. The use o f excess reagents also promotes faster coupling reactions that minimise the chances for such activated carboxy components to racemise.

In solution, the azide method is one o f the oldest and most useful coupling procedures. This method gives minimal racémisation in both peptide segment condensation and in the preparation o f cyclic peptides under high dilution conditions (Figure

NHgNHz Alkyl Nitrite Ester R^NHg

R OR R NHNH2 H * /~ 0 ° C R N3 R NHR^

(16) (17) (18) (19)

Figure 35. Hydrazinolysis o f a protected peptide or amino acid ester (16) yields a hydrazide (17) which is converted in to an azide compound (18). Reaction with the amine component o f an amino acid generates the final peptide (19).

Cyclising linear peptides in solution, after assembly on solid support with coupling agent DPPA remains popular, because o f its ability to form peptide azides in situ

with subsequent high coupling yields and minimal racémisation. 163,164 However, a direct comparison o f cyclisation o f identical linear peptides in solution with TBTU/HOBt/DIEA and DPPA in DMF indicated better overall yields with the former coupling reagents.

The main disadvantage o f using the azide method for coupling include the instability o f peptide azides at room temperature. Hence, low reaction temperatures are required for successful couplings. This introduces both problems with peptide solubility and appropriate choice o f solvent. Recently, a variation o f this methodology avoids these problems by converting the peptide azide into an active ester in the presence o f HO At or HOCt. The active peptide ester is then successfully coupled to another peptide segment in solution.

Carbodiimides have been used since the 1950’s in peptide synthesis, by generating the (9-acylisourea with a protected carboxylic acid and reacting this with an amino moiety yielding an amide bond (Figure 36).^^^

N---C— N (21) (20) 0

X

+ H U H — N C— N (22)

Figure 36. An 0-acylisourea (21) is formed from a nucleophilic attack o f a carboxylic acid on DIC (20). The final amide product is accompanied by the urea by-product (22).

If a second equivalent o f the carboxylic acid is available, a further reaction with the non-isolable (9-acylisourea can produce a symmetric anhydride. This too can react \vith an amino moiety and form an amide bond. However, to avoid racémisation activation o f protected carboxylic with carbodiimides is usually carried out in the presence o f a hydroxylamine derivative such as HOBt to form an active ester in situ

ready for coupling (Figure 37).

or

(23) N C = (24) O

II

-c-

R'NH R NHR'

Figure 37. An amide bond is formed with an activated ester (25) which is generated from HOBt (23) reacting with O-acylisourea (24), which has been made in situ.

The carbodiiraide DIC is compatible with Fmoc//-Bu solid phase peptide synthesis as the urea by-product is soluble in DMF and, therefore, ideal for both batch and continuous synthesis. DCC is ideal for Boc/Bzl strategy as the DCU urea by-product is soluble in TFA.

The phosphonium and so-called uronium salts derived from l-hydroxy-7-azabenzotriazole (HOAt) are the preferred choice o f coupling agents when compared to benzotriazole analogues, both in solution and solid phase peptide synthesis. This is attributed to higher coupling yields and minimal racémisation. The greater efficiency o f HO At relative to HOBt is governed by the formation or reactivity o f the active ester intermediate (Figure 37, similar to compound 25). HOAt esters have an electron withdrawing nitrogen atom that can stabilise the overall charge o f the leaving group, and is strategically placed at position 7 to participate in the neighbouring group effect with an incoming amino moiety (Figure 38).^^^ This has not been observed with the 4-isomer o f HOAt.

7-H O A t H O B t

R2N--

4-H O A t OH

N G P effect

A successful synthesis o f peptide fragments containing multiple Aib residues was reported with the potassium salt o f HOBt (referred to as KOBt) and Fmoc-Aib-Cl.^^® The use o f KOBt replaced the need to use HOBt and an organic base, thus, preventing oxazolone formation and allowing for extended reaction times.

Recently a novel coupling agent, ethyl 1-hydroxy-H/-l,2,3-triazole-4-carboxylate (HOCt) was introduced to enable better monitoring o f the automated coupling efficiency in solid phase peptide synthesis (Figure 39). Reliance on monitoring the absorbance o f the deprotection mixture with the Fmoc-piperidine adduct in the presence o f coupling agents HOBt and its derivatives may not always indicate real time monitoring as they also absorb at 302 nm. This could be vital in the synthesis o f long peptide chains o f over 20 residues to avoid multiple deletion sequences. Otherwise both low yields and ensuing purification problems are to be expected. The coupling agent HOCt avoids this prospect as it does not have a UV absorbance at 302 nm. HOCt is not commercially available but can be synthesised on a large scale locally.

O

EtO

OH

HOCt

Figure 39. The HOCt coupling agent.

The phosphonium salts derived from HOAt include (7-azabenzotriazol-1 -yloxy)-tris(dimethylamino)-phosphonium hexafluorophosphate (AOP) and (7-azabenzotriazol-l-yloxy)-tris(pyrrolidino)-phosphonium

hexafluorophosphate (PyAOP) (Figure Similarly, BOP and PyBOP are phosphonium derivatives o f HOBt.

MejN I NMes NM02 A O P

o

PyA O P PFf MejN I NM62 NM62 BOP PyBO P

Figure 40. The phosphonium based coupling agents.

The pyrrolidino derivatives PyBOP and PyAOP are more reactive than the dimethylamino derivatives BOP and AOP. The latter two reagents also release a known carcinogenic compound, HMPA, during the activation step.

The so-called uronium analogues o f the phosphonium salts include HATU, HAPyU and However, X-ray analysis has indicated that these analogues exist as guanidinium A-oxides or aminium salts when crystalline rather than the corresponding uronium salts (Figure 41).^^^’^^^

X = N,C H

MegN' 'NM02

So-called Uronium Derivative

HAPyU

Me,N. PFg

Me,N PF

HATU HBTU

NMC2

Figure 41. The aminium based coupling agents.

The coupling agent, 2-propanephosphonic acid anhydride (T3P) (Figure 42), has found applicability in cyclising sequences o f short sterically hindered peptides.

Although stepwise coupling o f urethane-protected activated amino acid esters in peptide synthesis occurs without loss o f configuration, for head-to-tail cyclisation o f short penta- or hexapeptides with sterically hindered amino acids, partial racémisation may occur,179

0 ^ '

T3P

Figure 42. An alkyl phosphonic anhydride that does not produce any by-product that may have solubility problems analogous to the urea by-product o f DCU.

HAPyU and a phosphonic acid-based condensation agent, T3P, were utilised as coupling reagents for the cyclisation o f increasingly sterically hindered linear

pentapeptides.^^^’^^^ The sterically hindered amino acids chosen included a,a-dialkyl- (Aib) or A-methyl-amino acids at the N-terminal position. T3P was shown to be as effective as HAPyU in cyclisations o f sterically less demanding T-pentapeptides, but superior to HAPyU for sequences that contained Aib, (N-Me)Ala or (A/-Me)Phe residues at the cyclisation site.^^^

The 7-aza-based coupling agents derived from HOAt (PyAOP, HATU and HAPyU) have been demonstrated to be superior to the HOBt-based reagents in solid phase peptide synthesis with regard to coupling efficiency and maintenance o f chiral integrity. However, aminium salts have also been known to react with an available amino moiety in a peptide chain and form a guanidino derivative that terminates any further peptide chain elongation or intra-chain cyclisation (Figure

4 3 ) 1 6 0 H2N— -Tyr-Dab-/)-Glu-Phe-Leu— LINKER NMe, MeoN

A

NH H O M02N

HBTU (as a guanidino derivative)

Tyr-HN

O

:

Phe-Leu- LINKER

Figure 43. The attempted synthesis o f cyclo[Dab(2)-Z)-GIu(3)] with HBTU results in the formation o f a tetramethylguanidium derivative at the y-amino side chain

An attempted side chain-to-side chain cyclisation between adjacent a,Y-diaminobutyric acid (Dab) and D-glutamic acid residues in a 10-membered chain yielded a monomer and dimer with a tetramethylguanidinium moiety from HBTU

attached to the side chain o f Dab. Similarly, a head-to-tail cyclisation between an Asp and D-Val residue in a pentapeptide chain with TBTU was unsuccessful due to the attachment o f a tetramethylguanidinium moiety to the free amino component o f the D-Val.^^^ Both attempted cyclisations took place over the standard two hours and have been attributed to the faster side reaction competing with an already difficult cyclisation reaction. Phosphonium reagents cannot take part in analogous phosphine imine-forming side reactions. Hence, PyAOP appears to be the ideal reagent for cyclisation on-resin due to its faster coupling times and absence o f guanidino forming side reactions.

There continues to be significant development in the variety o f coupling reagents (Figure 44). A series o f analogues o f HBTU (TBTU, TSTU, and TNTU) developed by Knorr et seem to have applicability for peptide couplings in mixed aqueous/organic media. An analogue o f HAPyU, referred to as TAPipU had comparable cyclisation efficiency accompanied by minimal racémisation in solid phase synthesis. A uronium salt, HDTU derived from HODhbt had comparable coupling efficiency to HATU in solution, but was less effective in solid phase synthesis. PyBroP and PyCloP are two halotripyrrolidinophosphonium hexafluorophosphates that efficiently couple A-methylated amino acid esters in solution when compared to PyBOP, but their applicability to solid phase synthesis is debatable. Another efficient ‘solution only’ coupling reagent is BOP-Cl, however, épimérisation at the activated site cannot be avoided. A reagent that has found use in both solution and solid phase coupling o f A-methyl amino acids is 2-chloro-1,3-dimethyl-2-imidazolinium hexafluorophosphate (CIP).^^^ Another efficient solution/solid phase coupling reagent is tetramethylfluoroformamidinium hexafluorophosphate (TFFH).^^^ This reagent helps to generate highly reactive Fmoc-protected amino acid fluorides in situ ready for coupling. A further less

familiar coupling reagent for solid phase synthesis is BOI which has comparable efficiency to NM62 TBTU NM62

<

B F f NM62 NMe, TNTU TSTU + / N O --- B F f NM62 NMe-j H OD hbt OH PyBroP, X=Br PyC loP, X=C1 +C— NMe HDTU TAPipU Me

I

-N, ©

I

Me CD* - C l 'P F f Me NMe2 F— — NMe2 PFe" TFFH

Figure 44. Other coupling agents.

PF,

Me

Peptide couplings can also occur between isolated active esters such as those derived from pentafluorophenol (HOPfp).^^^ The OPfp esters have found use in couplings that require swiftness to avoid competing side reactions and/or minimise problems associated with the standard coupling reagents, for example, guanidino derivatives with aminium salts.

In document Synthesis of stable α-helical peptides (Page 62-72)