CHAPTER 2
`
`Methods
`
`the Fmoc Group
`for Removing
`Gregg B. Fields
`
`1. Introduction
`The electron withdrawing
`fluorene ring system of the 9-fluorenyl-
`methyloxycarbonyl
`(Fmoc) group renders the lone hydrogen on the
`P-carbon very acidic and, therefore, susceptible to removal by weak
`bases (I,2). Following the abstraction of this acidic proton at the 9-posi-
`tion of the fluorene ring system, p-elimination proceeds to give a highly
`reactive dibenzofulvene
`intermediate (I-5). Dibenzofulvene can be
`trapped by excess amine cleavage agents to form stable adducts (1,2).
`The stability of the Fmoc group to a variety of bases (6-10) is reported in
`Table 1. The Fmoc group is, in general, rapidly removed by primary (i.e.,
`cyclohexylamine, ethanolamine) and some secondary (i.e., piperidine,
`piperazine) amines, and slowly removed by tertiary (i.e., triethylamine
`[EtsN], N,iV-diisopropylethylamine
`[DIEA]) amines. Removal also
`occurs more rapidly
`in a relatively polar medium (ZV,iV-dimethyl-
`formamide [DMF] or N-methylpyrrolidone
`[NMP]) compared to a rela-
`tively nonpolar one (dichloromethane
`[DCM]). During solid-phase
`peptide synthesis (SPPS), the Fmoc group is removed typically with pip-
`eridine, which in turn scavenges the liberated dibenzofulvene to form a
`fulvene-piperidine adduct. Standard conditions for removal include 30%
`piperidine-DMF
`for 10 min (II), 20% piperidine-DMF
`for 10 min
`(12,13), 55% piperidine-DMF
`for 20 min (I4), 30% piperidine in tolu-
`ene-DMF (1: 1) for 11 min (ll,15-17), 23% piperidine-NMP
`for 10 min
`(9), and 20% piperidine-NMP
`for 18 min (18). Piperidine-DCM should
`not be utilized, since an amine salt precipitates after relatively brief stand-
`
`From: Methods
`by M W Pennmgton
`
`Vol 35 PeptIde
`Brology,
`m Molecular
`and 6. M. Dunn Copyright
`Q1994
`
`Synthesis
`Humana
`
`Protocols
`Press
`Inc
`
`E&ted
`
`, Totowa,
`
`NJ
`
`17
`
`Illumina Ex. 1107
`IPR Petition - USP 10,435,742
`
`

`

`Compound
`
`Fmoc-Gly-PS
`
`Fmoc-Gly-PS
`
`Fmoc-Gly-PS
`
`Fmoc-Val
`
`Fmoc-Ala-OtBu
`
`Fmoc-Gly-PS
`
`Fmoc-Val
`
`10% Morpholine
`
`10% Morpholine
`
`50% Morpholine
`
`50% Morpholine
`
`50% Morpholine
`
`10% Piperidine
`
`20% Pipendine
`
`Fmoc-Gly-HMP-PS
`
`23% Piperidine
`
`Table 1
`Removal of the Fmoc Group
`
`Base
`
`Solvent
`
`Time, min Deprotectron, % Reference
`
`DCM
`
`DMF
`
`DCM
`
`DMF
`
`DCM
`
`DCM
`
`DMF
`
`NMP
`
`240
`
`240
`
`240
`
`1
`
`120
`
`240
`
`0.1
`
`0.25
`
`18=
`
`75”
`
`1W
`
`5ob
`
`1OOC
`
`loo”
`
`5ob
`
`5od
`
`6
`
`6
`
`6
`
`7
`
`8
`
`6
`
`7
`
`9
`
`8
`
`Fmoc-Ala-OfBu
`
`Fmoc-Val
`
`Fmoc-Ala-OrBu
`
`Fmoc-PCA
`
`Fmoc-Val
`
`Fmoc-Ala-OfBu
`
`Fmoc-Val
`
`Fmoc-Ala-OrBu
`
`Fmoc-Val
`
`50% Piperidine
`
`5% Piperazine
`
`50% Piperazine
`
`59% 1,4-bis-(3aminopropyl)prperazine
`
`50% Dicyclohexylamine
`
`50% Dicyclohexylamine
`
`50% DIEA
`
`50% DIEA
`
`10% 4-Drmethylammopyridme
`
`DCM
`
`DMF
`
`DCM
`
`CDCl,
`
`DMF
`
`DCM
`
`DMF
`
`DCM
`
`DMF
`
`<5
`
`0.33
`
`60
`
`2
`
`35
`
`>1080
`
`606
`
`>1080
`
`85
`
`1W
`
`506
`
`1W
`
`1W
`
`5ob
`
`1W
`
`506
`
`100”
`
`506
`
`7
`
`8
`
`IO
`
`7
`
`8
`
`7
`
`8
`
`7
`
`

`

`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OfBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OfBu
`
`Fmoc-Ala-OfBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-Ala-OfBu
`
`Fmoc-Ala-OtBu
`
`Fmoc-PCA
`
`50% DBU
`
`50% Pyrrolidme
`
`50% Cyclohexylamine
`
`50% Ethanolamine
`
`50% Diethylamme
`
`50% Triethylamine
`
`50% Ammonia
`
`50% Tributylamine
`
`1 .O mIt4 triethylenediamine
`
`10 m&f Hydroxylamine HCI
`
`0 5 mm01 Proton sponge
`
`2 0 mmol NaOH
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`DCM
`
`30% CHsOH-p-dioxane
`
`50% Tris(2aminoethyl)amme
`
`CDCl,
`
`<5
`
`<5
`
`<5
`
`<5
`
`180
`
`>1080
`
`~1080
`
`>1080
`
`>1080
`
`>1080
`
`>1080
`
`<5
`
`2
`
`100”
`
`100”
`
`1OOC
`
`1OOC
`
`100C
`
`100=
`
`100”
`
`1OOC
`
`100”
`
`1OOC
`
`100C
`
`100C
`
`100’
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`8
`
`IO
`
`Fmoc-PCA
`
`2
`
`1OOe
`
`IO
`
`59% 1,3-Cyclohexanebis-(methylamine)
`uDeprotection of Fmoc-Gly-PS was quantitated spectrophotometrrcally
`bDeprotectton of Fmoc-Val was quanhtated by amino acid analysis (7)
`CDeproteetron of Fmoc-Ala-0-tBu
`was quantitated by thin-layer chromatography
`dDeprotectron of Fmoc-Gly-HMP-PS
`was quantitated by mnhydrm analysts (9).
`(10). Drbenzofulvene was scavenged m
`eDeprotectron of 9-fluorenylmethyl
`N-p-chlorophenyl
`carbamate (Fmoc-PCA) was quantitated by ‘H-NMR
`2 mm by trrs(2ammoethyl)amme,
`15 mm by 1,3-cyclohexanebis-(methylamine),
`and 50 min by 1,4-bis-(3-aminopropyl)piperazine.
`
`CDCl,
`
`at 273 run (6)
`
`(8)
`
`

`

`Fields
`
`ing (II). An inexpensive alternative to piperidine for Fmoc removal is
`diethylamine, with standard conditions being 60% diethylamine-DMF
`for 180 min (19,2(I) or 10% diethylamine-ZV,N-dimethylacetamide
`(DMA) for 120 min (21,22).
`2. Monitoring
`Fmoc removal can be monitored spectrophotometrically because of
`the formation of dibenzofulvene or fulvene-piperidine adducts. Monitor-
`ing is especially valuable in “difficult” sequences, where Fmoc removal
`may be slow or incomplete (I7,23,24). Slow deprotection has been cor-
`related to a broad fulvene-piperidine peak detected at 3 12 nm (24-26).
`Monitoring of a broad fulvene-piperidine peak at 365 nm has been used
`to demonstrate slow deprotection from Fmoc-(Ala)5-Val-4-hydroxy-
`methylphenoxy (HMP)-copoly(styrene- 1 %-divinylbenzene)-resin (PS);
`in turn, detection of a narrow fulvene-piperidine peak demonstrated
`efficient deprotection of the same sequence on a different solid support
`(HMP-polyethylene glycol-PS) (27). Monitoring of fulvene-piperidine
`at 3 13 nm was utilized during the successful synthesis of the entire 76-
`residue sequence of ubiquitin (28). Dibenzofulvene formation has been
`monitored at 270 or 304 nm (29).
`
`3. Side Reactions
`Repetitive piperidine treatments can result in a number of deleterious
`side reactions, such as diketopiperazine and aspartimide formation and
`racemization of esterified Cys derivatives. Base-catalyzed cyclization of
`resin-bound dipeptides to diketopiperazines is especially prominent in
`sequences containing Pro, Gly, b-amino acids, or N-methyl amino acids.
`For continuous-flow Fmoc SPPS, diketopiperazine
`formation is sup-
`pressed by deprotecting for 1.5 min with 20% piperidine-DMF at an
`increased flow rate (15 mL/min), washing for 3 min with DMF at the
`same flow rate, and coupling the third Fmoc-amino acid in situ with
`benzotriazolyl N-oxytrisdimethylaminophosphonium
`hexafluoro-
`phosphate (BOP), 4-methylmorpholine, and 1-hydroxybenzotriazole
`(HOBt) in DMF (30). For batch-wise SPPS, rapid (a maximum of 5 mm)
`treatments by 50% piperidine-DMF should be used, followed by DMF
`washes and then in situ acylations mediated by BOP or 2-(lH-
`benzotriazole- 1 -yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate
`(HBTU)
`(31). Piperidine catalysis of aspartimide formation from side-
`
`

`

`Fmoc Removal
`
`21
`
`chain-protected Asp residues can be rapid, and is dependent on the side-
`chain-protecting group. Treatment of Asp(OBzl)-Gly, Asp(OcHex)-Gly,
`and Asp(OtBu)-Gly with 20% piperidine-DMF
`for 4 h resulted in 100,
`67.5, and 11% aspartimide formation, respectively (32), whereas treat-
`ment of Asp(OBzl)-Phe with 55% piperidine-DMF
`for 1 h resulted in
`16% aspartimide formation (33). The racemization of C-terminal-esteri-
`fied Cys derivatives by 20% piperidine-DMF
`is also problematic, with
`D-Cys
`formed to the extent of 11.8% from Cys(Trt), 9.4% from
`Cys(Acm), 5.9% from Cys(tBu), and 36.0% from Cys(StBu) after 4 h of
`treatment (34).
`Some piperidine-catalyzed side-reactions may be minimized by using
`other bases to remove the Fmoc group. Two percent 1,8-diazabi-
`cyclo[5.4.0]undec-7-ene (DBU)-DMF, at a flow rate of 3 mL/min for 10
`min, is used to minimize monodealkylation of either Tyr(POsMeJ or
`Tyr(POsBzl& (29). For example, 50% monodealkylation of Tyr(POsMe&
`occurred in 7 min with 20% piperidine-DMF,
`but required 5 h with 1M
`DBU in DMF, whereas 50% monodealkylation of Tyr(POsBzlz) occurred
`in 12 min with 20% piperidine-DMF and 14 h with 1M DBU in DMF
`(29). Racemization of esterified Cys(Trt) was reduced from 11.8% with
`20% piperidine-DMF to only 2.6% with 1% DBU-DMF after 4 h of treat-
`ment (29,34). Unfortunately, aspartimide formation of Asp(OtBu)-Asn
`is worse with DBU compared to piperidine (35). This reagent is recom-
`mended for continuous-flow syntheses only, since the dibenzofulvene
`intermediate does not form an adduct with DBU and thus must be washed
`rapidly
`from the peptide resin to avoid reattachment of dibenzoful-
`vene (29). However, a solution of DBU-piperidine-DMF (1: 1:48) is effec-
`tive for batch syntheses, since the piperidine component scavenges the
`dibenzofulvene.
`
`Synthesis
`4. Glycopeptide
`The mild conditions of Fmoc chemistry are, in general, more suited
`for glycopeptide syntheses than Boc chemistry, because repetitive acid
`treatments can be detrimental to sugar linkages (36). However, some
`researchers prefer morpholine to piperidine as an Fmoc removal agent
`during glycopeptide SPPS, because the pK, of morpholine (8.3) is lower
`than that of piperidine (11. l), and is thus less detrimental to side-chain
`glycosyls (36,37). Side-chain Ser and Thr glycosyls are stable to base
`deprotection by neat morpholine (38,39) for 30 min (40) and 50%
`
`

`

`22
`
`Fields
`
`for 20-30 min (4143). A 4-h treatment of Cys(Trt)
`morpholine-DMF
`with 50% morpholine-DMF resulted in 3.8% D-Cys, which is consider-
`ably less racemization than that seen with piperidine (34).
`5. Solution
`Syntheses
`For rapid solution-phase synthesis, it is desirable to use an Fmoc
`removal agent that forms a dibenzofulvene adduct that can be extracted
`in phosphate buffer (pH 5.5). Such an adduct is obtained when either
`4-(aminomethyl)piperidine
`(44) or tris(2-aminoethyl)amine
`is used
`for Fmoc removal (IO). Precipitates or emulsions can form during
`4-(aminomethyl)piperidine-fulvene adduct extraction from a DCM layer,
`so tris(2-aminoethyl)amine
`is preferred (10). Complete deprotection and
`scavenging of 9-fluorenylmethyl N-p-chlorophenyl carbamate (Fmoc-
`PCA) (0.14 mmol) was achieved in 2 min with 2 mL of tris(2-amino-
`ethyl)amine (100 Eq) in 2 mL CDCla (10). Polymeric-bound amines,
`such as piperazine-PS (2.4 mEq/g) (45) and a copolymer of styrene,
`2,4,5-trichlorophenyl acrylate, and N,N’-dimethyl-N,N’-bisacryloylhexa-
`methylene diamine, with subsequent replacement of activated ester
`groups by l-(2aminoethyl)piperazine
`(3.3 mEq/g) (46), also efficiently
`remove the Fmoc group in solution-phase syntheses. The use of poly-
`meric-bound amines allows for the isolation of the free amino compo-
`nent by simple filtration of the resin, since the polymer
`traps the
`dibenzofulvene (45,46).
`
`6. Notes
`1. Amine impurities that could possibly remove the Fmoc group include
`dimethylamine found m DMF (47) and methylamme found in NMP (48)
`Fmoc-Gly was found to be deprotected after 7 d m DMA, DMF, and NMP to
`the extent of 1,5, and 14%, respectively (49). Although these rates of decom-
`position are considered extremely low, it is recommended that these solvents
`be freshly purified before use (2647). The presence of HOBt (O.OOl-O.lM)
`greatly reduces the detrimental effect of methylamine (48,50) whereby
`Fmoc-Gly-HMP-PS was cl % deprotected after 20 h in NMP (48).
`2. The primary and secondary amine lability of the Fmoc group also prompted
`an mvestigation of Fmoc removal by esterrfied or resin-bound amino acids.
`Fmoc-Ala and Fmoc-Gly (m DMF) were labile to Pro-OtBu, where t,,* - 9
`and 7 h, respectively (51). Fmoc liberation was less rapid by Pro-Lys(4-
`NO,-Z)-Gly-OET
`(t,,* - 40 and 35 h for Fmoc-Ala and Fmoc-Gly, respec-
`tively, m the presence of 1 Eq DIEA), and greatly reduced by the presence
`of HOBt (1 Eq) and 2,4-dinitrophenol
`(2 Eq) (51). The Fmoc group was
`
`

`

`Fmoc Removal
`
`23
`
`(51).
`ti,, =
`(8).
`
`to primary amino acid esters, even in the presence of DIEA
`less labile
`(in DCM) was deprotected
`very slowly by Gly-PS, with
`Fmoc-Leu
`300 and 1500 h in the presence of 1.8 and 1.2 Eq of DIEA,
`respectively
`These rates of Fmoc removal by Gly-PS are msignificant
`in SPPS.
`3. There are several alternatives
`to base removal of the Fmoc group, such as
`fluoride
`ion or hydrogenation.
`Fmoc-Phe was rapidly deprotected
`(- 2 min)
`by 0.05-O.lM
`tetrabutylammonium
`fluoride
`trihydrate
`(TBAF)
`in DMF
`(52). Continuous-flow
`Fmoc SPPS of Leu-Ala-Gly-Val,
`carried out with
`20-min deprotecttons of 0.02M TBAF
`in DMF,
`resulted
`in a highly homo-
`geneous crude product
`(52). Adding
`100 Eq of MeOH
`to TBAF-DMF
`solutions
`could
`inhibit
`readdition
`of dibenzofulvene
`to the peptide
`resin
`and diketopiperazine
`formation
`(52). Succinimide
`formation
`from Asn,
`glutarimide
`formation
`from Gln, and the mstability
`of benzyl ester groups
`are potential
`problems
`of TBAF
`deprotection
`(53,54).
`Complete
`deprotection
`of Fmoc-Ala
`(in CHsOH),
`Fmoc-Gly
`(in 95% ethanol),
`and Fmoc-Leu
`(in 75% aqueous ethanol)
`by hydrogenation
`with 10%
`Pd-on-charcoal
`catalyst
`in the presence of acetic acid (two drops) occurred
`m 4, 22, and 4 h, respectively
`(55). Deprotection was solvent-dependent,
`with generation of Gly
`from Fmoc-Gly
`occurring with
`tu2 - 30 h m 20%
`acetic acid-CHsOH,
`tllz - 17 h m DMF, and tu2 - 7 h in DMF containing
`2 Eq of DIEA by hydrogenation
`with 10% Pd-on-charcoal
`catalyst
`(49).
`Fairly
`rapid Fmoc-Gly
`deprotection
`in DMF
`(t,,* - 2.5 h) was found when
`Pd(OAc)z was used as the catalyst
`instead of Pd-on-charcoal(49).
`Studies
`with Fmoc-Gly-OBzl
`showed selective
`removal of the benzyl ester in the
`presence of the Fmoc group by hydrogenation
`in CH,OH
`with 10%
`Pd-BaS04
`catalyst
`for - 1 h (56).
`References
`1. Carpino, L. A. and Han, G. Y. (1972) The 9-fluorenylmethoxycarbonyl
`protecting group. J. Org. Chem. 37,3404-3409.
`2. Carpino, L. A. (1987) The 9-fluorenylmethyloxycarbonyl
`amino-protecting groups. Act. Chem. Res. 20,401-407.
`of 9-fluorenylmethanol
`3. O’Ferrall, R. A. M. and Slae, S. (1970) b-elimination
`aqueous solution: an ElcB mechanism. J. Chem. Sot. (B), 260-268.
`4. O’Ferrall, R. A. M. (1970) p-elimination
`of 9-fluorenylmethanol
`in solutions of
`methanol and t-butyl alcohol. J. Chem. Sot. (B), 268-274.
`5. O’Ferrall, R. A. M. (1970) Relationships
`between E2 and ElcB mechanisms
`J. Chem. Sot. (B), 274-277.
`of B-elimination.
`6. Merrifield, R. B. and Bach, A E. (1978) 9-(2-Sulfo)fluorenylmethyloxycarbonyl
`chloride, a new reagent for the purification of synthetic peptides. J. Org. Chem 43,
`4808-48 16.
`7. Atherton, E., Logan, C J , and Sheppard, R. C. (1981) Peptide synthesis, part 2:
`procedures for solid-phase synthesis using ~-fluorenylmethoxycarbonylamino-
`
`ammo-
`
`family of base-sensitive
`
`in
`
`

`

`24
`
`Fields
`
`acids on polyamide supports: synthesis of substance P and of acyl carrier protem
`65-74 decapeptide. J. Chem. Sot. Perkrn Trans. I, 538-546.
`8. Chang, C-D., Waki, M., Ahmad, M , Meienhofer, J., Lundell, E. O., and Haug, J.
`D. (1980) Preparation
`and properties of N”-9-fluorenylmethyloxycarbonyl-
`amino acids bearing
`terr.-butyl
`side chain protection. Znt. J. Peptide Protein
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`9. Harrison, J. L., Petrie, G. M., Noble, R L , Beilan, H. S., McCurdy, S. N., and
`Culwell, A. R. (1989) Fmoc chemtstry’
`synthesis, kinetics,
`cleavage, and
`deprotection of arginine-containing
`peptides, in Techniques in Protein Chemistry
`(Hugli, T. E., ed.), Academic, San Diego, pp 506-516
`10. Carpino, L. A., Sadat-Aalaee, D., and Beyermann, M. (1990) Tris(2-ammo-
`ethyl)amine as a substitute for 4-(ammomethyl)piperidine
`in the FMOC/polyamine
`approach to rapid peptide synthesis. J. Org. Chem. 55, 1673-1675.
`11. Alberho, F., Kneib-Cordonier, N., Biancalana, S., Gera, L., Masada, R. I., Hudson,
`D., and Barany, G. (1990) Preparation and application of the 5-(4-(9-fluorenyl-
`methyloxycarbonyl)am~nomethyl-3,5-dimethoxyphenoxy)valeric
`acid (PAL)
`handle for the solid-phase synthesis of C-terminal peptide amides under mild con-
`ditions. J. Org. Chem. 55,373Q-3743.
`12. Atherton, E , Fox, H., Harktss, D., Logan, C. J., Sheppard, R C , and Wilhams, B.
`J. (1978) A mild procedure for sohd phase peptide synthesis: use of fluorenyl-
`methoxycarbonylamino-acids
`J. Chem. Sot., Chem Commun 537-539.
`13. Atherton, E., Fox, H , Harkiss, D , and Sheppard, R. C (1978) Application of polya-
`mide resins to polypeptide synthesis: an improved synthesis of P-endorphin using
`fluorenylmethoxycarbonylamino-acids.
`J. Chem. Sot., Chem Commun 539,540
`14. Chang, C -D., Felix, A M., Jimenez, M. H., and Meienhofer, J (1980) Solid-phase
`peptide synthesis of somatostatin using mild base cleavage of Na-fluorenyl-
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`acids. Znt. J. Peptide Protein Res
`l&485-494.
`15. Hudson, D. (1988) Methodological
`implications of simultaneous solid-phase pep-
`tide synthesis 1: comparison of different coupling procedures. J Org. Chem. 53,
`617-624
`16. Otvos, L., Jr., Urge, L , Hollosi, M , Wroblewski, K., Graczyk, G., Fasman, G. D ,
`and Thurin, J. (1990) Automated solid-phase synthesis of glycopepttdes:
`incorpo-
`ration of unprotected mono- and disaccharide units of N-glycoprotein
`antennae
`into T cell epitopic peptides. Tetrahedron Lett. 31,5889-5892.
`17. Fontenot, J. D., Ball, J M., Miller, M A., David, C. M., and Montelaro, R C
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`the fluorenylmethoxycarbonyl
`procedure. Peptide Res. 4, 19-25.
`18. Fields, G. B. and Fields, C. G (1991) Solvation effects in solid-phase peptide syn-
`thesis. J. Am. Chem. Sot 113,4202-4207.
`19 Sivanandaiah, K. M., Gurusiddappa, S., and Babu, V V. S. (1988) Peptides related
`to leucme-/methionine-enkephalinamtdes:
`synthesis and biological
`activities
`Indian J. Chem. 27B, 645-648.
`20. Slvanandaiah, K. M , Gurusiddappa, S , Channe Gowda, D , and Suresh Babu, V.
`V. (1989) Improved solid phase synthesis of lutemtzmg hormone releasing hor-
`mone analogues using 9-fluorenylmethyloxycarbonyl
`amino acid active esters and
`
`

`

`Fmoc Removal
`
`catalytic transfer hydrogenation with minimal side-chain protection and their bio-
`logical activities. J. Biosci. 14,3 11-3 17.
`load
`21 Butwell, F. G. W., Haws, E J., and Epton, R. (1988) Advances in ultra-high
`polymer supported peptide synthesis with phenolic supports 1: a selectively-labile
`C-terminal
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