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HCTU and TCTU:
New Coupling Reagents:
Development and Industrial Aspects.

 Oleg Marder, Youval Shvo and Fernando Albericio
1. Luxembourg Industries Ltd., 27 Hamered Street, P.O.Box 13,
    Tel Aviv, Israel.
2. Department of Organic Chemistry, University of Barcelona, 08028 -
    Barcelona, Spain.

 
Introduction

Many groups working in peptide synthesis, be it for research or for production purposes, are discovering that the limiting factor is often the small choice of available, affordable and safe coupling reagents ( 1) .

Although a large number of in situ coupling reagents have been developed for the purpose of peptide bond formation, most of them are not commercially produced and are not available to the research scientist and industry for the following reasons:
  1. Synthesis procedure requires unusual and unavailable raw material.
  2. Manufacturing process is not conventional andrequires special equipment.
  3. Coupling requires unusual conditions( low temperatures, solvents) .
  4. Potentially hazardous reagents( allergenic, unstable, explosive) .
Today, large and medium scale peptide manufacturers and university core facilities, mainly rely on the following coupling reagents:
  1. Carbodiimide coupling reagents ( DIC, DCC, EDIC) .
  2. Aminium salts of benzotriazoles ( HBTU, TBTU, HATU) .
  3. Phosphonium salts of benzotriazoles ( PyBOP, PyAOP) .
  4. Others ( DEPBT, MSNT, triazines) .
Testing of coupling reagents and research of possible new reagents, which may be implemented in industry, is therefore becoming an important research activity and results should be available to the peptide community. The main goal of our research was to investigate the influence of the electron withdrawal effect of halogen on the benzotriazole component in order to develop effective and stable coupling reagents. The research was based on previous studies reflecting the effect of CF3- substituted benzotriazole- based coupling reagents ( 2) and Cl- substituted hydroxybenzotriazoles ( 3) . 		 

Cl- HOBt was applied as a substitute for HOAt for inactivation of fluoride ion of TBAF and further coupling during one- pot method for peptide elongation. In this case, the acidity of Cl- HOBt is similar to that of HOAt ( pKa 3.35 for Cl- HOBt and pKa 3.28 of HOAt) , but without the neighboring group effect of the N of the pyridine ring ( assisted basic catalysis) . However, similar to HOAt, enhancement of coupling was observed. Later, when applied together with HBTU and DIEA, Cl- HOBt was found to be an effective additive showing comparable to HOAt effect on peptide fragment condensation and epimerization ( 4) . During our research, using a combinatorial approach, we investigated a different halogen ( Cl, Br, I) substituted hydroxybenzotriazole- based coupling reagents. Up until now 6- Cl- HOBt based aminium salts gave the most prominent results ( 5,6) .

Our philosophy for research and development of new coupling reagents:

An optimal coupling reagent should have the following properties:
  1. Effective coupling reagent.
    • Works with high efficiency for a wide variety ofpeptide sequences.
    • Has a high conversion rate at room temperature.
    • Is soluble in all the currently used solvents and can beused at high concentrations.
    • Solutions are stable for several days at roomtemperature.
    • Shows low side reactions and low racemization.
  2. Safety to the producer - non - hazardous raw materials.
  3. Safety to user - does not cause allergic reactions and irritations, non- explosive.
  4. Safety to environment – does not create waste problems.
  5. Raw materials are commercially available.
  6. Established conventional technology is available.
  7. Cost- effective coupling reagent.
Synthetic Strategy



Results:
 1. Chemical structure ( Revealed by crystal structure analysis)


HCTU

CA name:
1 H- Benzotriazolium, 1- [bis ( dimethylamino) methylene ] - - 5- chloro- , hexafluorophosphate ( 1- ) , 3- oxide
CAS number: 330645- 87- 9		  

TCTU

CA name:
CA name: 1 H- Benzotriazolium, 1- [bis ( dimethylamino) methylene ] - - 5- chloro- , tetrafluoroborate ( 1- ) , 3- oxide
CAS number: 330641- 16- 2

2.Safety coupling reagents.

2.1 T xicity and irritation study



Conclusions:LD50 is greater than 200mg/kg for both HCTU and TCTU. The reagents are not considered poisonous.HCTU and TCTU are not a dermal irritant/non-corrosive and does not require packing group assignment.
2.2 Explosivity study
HCTU and TCTU found to be non-explosive compounds:
  • No mechanical sensitivity with respect to shock ((hammer test)was observed
  • No mechanical sensitivity with respect to friction was observed
3.Stability
3.1 Stability of solid crystals (accelerated stability study) No degradation of HCTU and TCTU were observed when crystals were kept at 40 °C and 75%of relative humidity for one month.This finding gives an indication for 6-month stability at RT and ambient humidity.

3.2 Stability in DMF

Solutions (0.05M)of HCTU and TCTU were prepared in DMF in a vial with a septum (no nitrogen flow used)and the purity of the coupling reagent was checked by HPLC after different time periods.

4.Solubility study


5.Efficacy study. Two peptides -(des-pGlu1)-bombesin and (K)10-F3 -were synthesized on a ABIMED AMS-422 peptide synthesizer.HCTU,TCTU and ByBOP were compared for their efficiency as coupling agents.Fmoc-strategy and Rink resin were used.Double coupling,using a four-fold excess of amino acid and coupling agent for ByBOP and HCTU,two fold for TCTU,and a coupling time of 20 minutes were used. Peptides were isolated using standard methods.Yield is based on the amount of crude peptide obtained (mass).

Synthesis of (des-pGlu 1)-Bombesin

AAA -According to expected values

Synthesis of K-K-K-K-K-K-K-K-K-K-F-F-F

AAA -According to expected values
Conclusion:For synthesis of the two difficult-to-synthesize peptides,coupling with HCTU yielded the best results.However,it must be remembered that only a two- fold excess of amino acid and TCTU was used. The stability of the HCTU/TCTU solutions was good over the whole synthesis time (no coloration over >24 hours at room temperature),an important characteristic of a reagent in automated peptide synthesis.

HCTU and CF3-HBTU:COMPARISON ON SOLID-PHASE MODE Purity of peptides is analyzed by HPLC,and each synthesis were carried out in parallel and by duplicate ACP (65-74)H-VQAAIDYING-NH2 BAP (36-42)H-VGGVVIA-NH2 ,?-amyloide protein



Conclusion:HCTU is better than CF3-HBTU coupling reagent when applied for coupling natural amino acids containing peptides [ACP (65-74)and BAP (36-42)]

Conclusions:
1.Effective potential coupling reagents.
2.Guanidinium salt,by crystal structure analysis
3.Safety to producer,user and environment.
  • Non--toxic coupling reagents.
  • Do not cause allergic reactions and irritation.
  • Non--explosive coupling reagents.


    • Future plans:

    1.HCTU and TCTU
  • Preparation and testing of the O - derivatives..Currently,we are preparing the real uronium salt following the recent publication of Carpino et al (7).


  • Further coupling effectivity study on different sequences and difficult couplings,including unnatural amino acids.
  • Racemization and side reaction studies.
  • Stability up to 24 months will be investigated.


    • 2.Luxembourg Industries and it ’s academic partners are performing a systematic investigation into known reagents and are actively seeking new molecules with the potential to become coupling reagents or additives for peptide synthesis.

    Reference:
    1.Albericio,F.,Chinchilla,R.,Dodsworth,D.J.,and N?jera,C.Org.Prep.Proc.Int.33 :202-303 (2001).
    2.Wijkmans,J.C.H.M.,Kruijtzer,J.A.W.,van der Marel,G.A.,van Boom,J.H.,and Blomhoff W.Trav.Chim.
    Pays-Bas 113 :394-397 (1994).
    3.Ueki,M.and Yanagihira,T.(1998)In Bajusz,S.and Hudecz,F.(Eds.),Peptides 1998 (Proceedings of
    the 25 th European Peptide Symposium),Akademiai Kiado,Budapest,p.252-253
    4.Mader,C.,Young,D.,Bray,B.Peptides 1998 In Bajusz,S.and Hudecz,F.(Eds.),(Proceedings of the 25 th
    European Peptide Symposium).
    5.Israeli Patent Application No:141804
    6.Ueki,M.,Komiya,H.,Tajima,A.and Nakashima,H.“Peptide Science 2001 ” Proceedings of the 38th
    Japanese Symposium.
    7.Carpino et al.Angew.Chem.Int.Ed.41 :442-445 (2002).

    Luxembourg Industries Ltd.
    27 Hamered St. Tel Aviv 68125, Israel
    Phone: +972-3-796 4300 Fax: +972-3-510 0474
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